Professional Documents
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Biotechnology in
Agriculture and Forestry 41
Medicinal and Aromatic Plants X
Edited by Y.P.S. Bajaj
Springer
ISSN 0934-943X
ISBN 978-3-642-63748-3
Library of Congress Cataloging-in-Publication Data. Medicinal and aromatic plants. (Biotechnology
in agriculture and forestry; 4-). Includes bibliographies and index. 1. Medicinal plants Biotecnology. 2. Aromatic plants - Biotechnology. 3. Plant cell culture. 4. Materia medica,
Vegetable. I. Bajaj, Y.P.S., 1936- . II. Series. TP248.27.P55M43 1988 660'.62 88-3059.
ISBN 978-3-642-63748-3
ISBN 978-3-642-58833-4 (eBook)
DOI 10.1007/978-3-642-58833-4
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Springer-Verlag Berlin Heidelberg 1998
Originally published by Springer-Verlag Berlin Heidelberg New York 1998
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Dedicated to
Dhanmeet and Paramjit Soin
Preface
Contents
1
5
9
11
14
17
25
26
28
30
42
42
43
45
49
62
Contents
4 Protocol
References .................................................. .
63
64
67
69
76
77
77
79
81
85
93
93
97
99
107
110
110
113
114
115
128
128
129
Contents
XI
132
135
138
138
142
145
146
148
150
151
154
157
167
187
189
194
195
195
233
236
238
241
257
257
XII
Contents
General Account. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
In Vitro Culture Studies .....................................
Micropropagation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
In Vitro Production of Secondary Metabolites in Marus alba .....
Extraction and Structure of Intermolecular Diels-Alder-Type
Adducts of Prenylchalcone and Prenylated 2-Arylbenzofuran .....
6 Summary and Conclusion ....................................
7 Protocol for Micropropagation ...............................
References ..................................................
261
264
265
271
279
281
281
282
286
288
293
299
300
300
305
307
308
318
319
320
322
329
330
332
Contents
XIII
Introduction ...............................................
In Vitro Culture Studies .....................................
Phytoecdysteroid Content in In Vitro Cultures . . . . . . . . . . . . . . . . . .
Biosynthetic Studies of Ecdysteroids in In Vitro Cultures ........
Summary and Conclusion ....................................
Protocol for Extraction and Quantification of Ecdysteroids in
P. vulgare . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
333
335
338
341
344
345
346
349
353
355
363
364
366
373
389
389
394
399
409
409
411
XIV
Contents
415
419
437
438
443
446
453
454
Subject Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
457
List of Contributors
XVI
List of Contributors
List of Contributors
XVII
XVIII
List of Contributors
List of Contributors
XIX
xx
List of Contributors
1 Introduction
1.1 Botanical Aspects
Y. Shoyama et al.
fruit gall, and finally sleep. The fruit galls contain cyclopentanoid
monoterpenes like iridomyrmecin, which is the most active stimulant
for cats (Sakan et al. 1960). In 1949, Pavan reported that iridomyrmecin
isolated from the anal glands of the Argentine ant, Iridomymex humilis,
possessed insecticidal activity. The workers of 1. humilis use this secretion
for attack and defense against their insect enemies, and presumably this
ability to wage chemical warfare is one of the factors responsible for the
expansion of the species. The occurrence of similar defensive substances
which may protect the plant against phytophagous insects has been noted.
Sakan et al. studied the plant and isolated a number of other monoterpenoids.
Figure 2 shows the biologically active monoterpenoids of A. polygama. They
consist of four groups, aldehydes (iridodial, dehydroiridodial), alcohols
(iridodiol, dehydroiridodiol, and 5-hydroxyiridodiol: strongly attractive
for Chyrysopa septempunctata and C. japana), lactones (iridomyrmecine,
dihydronepetalactone, nepetalactone, neonep etalactone: attractive to cats),
and an artificial compound (actinidine: attractive to cats). Alcohols
are specifically attractive to Felidae species. Aldehydes were reported to
be a bitter principle. It is most remarkable that cyclopentanoid monoterpenes were found to strongly attract only the male adults of Chrysopa
septempunctata (lacewing, stink flies) and C. japana in the amount of 1O- 6!lg of
neomatatabiol and isoneomatatabiol and 1O-3!lg of dehydroiridodiol, as
indicated in Table 1.
In addition, the fruit galls of A. polygama contain several triterpenoids
(Sashida et al. 1992). Some types of terpenoids have long been recognized
as carriers of important physiological functions and they are also substances
of vital importance for living organisms, i.e., some steroids, carotenoids,
ecdysone, and, in plants, phytol, which is an essential component of the chlorophyll molecule. Furthermore, triterpenoids constitute one of the largest
groups with highly diversified biological activities including antiinflammatory,
antineoplast, antibacterial, antifungal activities, diuretic, antidiabetic, and
R1
iridodiol
iridodial
R2
CH 2 0H CH 2 0H
CHO
CHO
actlnidine
5-hydroxymatatabiether
~
-;/'
matatabiol
dehydroindodiol
dehydroiridodial
HO ,I,
7-hydroxymatatabiethe r
cQ
OH
[~
ailomatatabibiol
R1
neomatatablol
isoneomatatabiol
R2
R1
R2
H
CH 3
CH 3
H
Fig. 2A,B. Structures of biologically active monoterpenoids from Actinidia polygama. (Sakan
et al. 1964)
Y. Shoyama et al.
nepetalactone
iridomyrmecin
B isoiridomyrmecin
Rl
H
CH3
neonepetalactone
dihydronepetalactone
isodihydronepetalactone
Rl
H
CH3
Compound
Iridodiol
5-H ydroxymatatabiether
7-Hydroxymatatabiether
Allomatatabiol
Matatabiol
Dehydroiridodiol
Neomatatabiol
Isoneomatatabiol
Active amount
(ug)
1
1
1
1
10- 3
10- 3
10- 6
10- 6
Callus was induced from fruit galls on the BS medium supplemented with
NAA, BAP, and kinetin (1 mg/l each) or on the LS medium containing lOmg/
I NAA and 1 mg/l kinetin. Callus growth was better on the BS than on LS
medium, as indicated in Table 2. When callus was subcultured on the MS
medium containing IBA, growth was favored. The addition of 3 mg/l IBA
favored callus growth better than the addition of a higher concentration
(Table 2), thus this medium was routinely used in the callus propagation
stage (Fig. 3a).
Y. Shoyama et al.
Fig. 3a-d. Propagation system of Actinidia polygama by callus culture (Y. Shoyama et al..
unpubl.). a Callus culture induced from fruit gall segment on MS medium supplemented
with IBA (3mg/I). b Formation of shoot primordia-like bud after three subcultures on BS
medium supplemented with NAA, BAP, and kinetin (1 mg/I each). c Shoot formation on
LS medium supplemented with 2,4-D (O.S mg/I each). d Plantlet formation on hormone-free MS
medium
Table 2. Propagation of callus induced from fruit gall of A. polygama on different media.
(Shoyama et al. 1989; Sashida et al. 1994)
Basal media
Hormone
(mg 1-1)
Fresh weight
(mg culture -1)
Gamborg BS
Linsmaier-Skoog
NAA-BAP-Kin
(1:1:1)
NAA-Kin
(10:1)
730
240
Murashige-Skoog
270
IBA
3
970
390
Culture conditions: 2S :+: 1C, 16h light, 1 month. NAA: I-naphthaleneacetic acid, BAP:
benzylaminopurine, Kin: kinetin, IBA: indol-3-butyric acid.
When the callus induced by the medium supplemented with NAA, BAP,
and kinetin (1 mg/l each) was subcultured on the same medium for three
generations, shoot primordia and adventitious roots appeared on its surface
(Fig. 3b). Some of the shoot buds were transferred to LS medium supplemented with 2,4-D and BAP (0.5 mg/l each), resulting in shoot and root
regeneration (Fig. 3c). Shoots were subcultured on MS medium supplemented
with NAA (1 mg/l) , or on hormone-free medium to induce root formation
(Fig. 3d).
Chromosome numbers (2n = 58) in the root tip of the plantlet regenerated
from callus were the same as in the mother plants (Kitamura and Murata
1979).
2.2 Micropropagation
Y. Shoyama et al.
The callus was extracted with hot EtOH several times, and the EtOH
solution was evaporated in vacuo. The extract was suspended in H 2 0.
The suspension was extracted with EtOAc and BuOH, successively. The
EtOAc soluble part was repeatedly chromatographed on a silica gel column
(CHCI3-Me2CO and CHCI3-EtOAc system) to distinguish individual
triterpenoids.
Structures were determined for these triterpenoids for their lR, ElMS,
FABMS, lH_, and 13C-NMR data (Cheung and Tokes 1968; Sakakibara and
Kaiya 1983; Kikuchi et al. 1984; Kojima and Ogura 1986; Kojima et al. 1987;
Bhandari et al. 1990; Sashida et al. 1994).
2.3.2 Results
Figure 4 shows the GC-MS spectrum of the crude terpenoid fraction
obtained from callus culture. Although the GC spectrum indicated that many
volatile compounds were contained in the crude terpenoid fraction, only
dihyronepetalactone (or isodihydronepetalactone), which is a major constituent (Fig. 4, arrow), was determined. The stereochemistry of this compound is
still unknown.
Figure 5 shows the structure of triterpenoids isolated from callus culture
and fruit gall. Table 3 shows the distribution of triterpenoids in callus tissue,
regenerated plantlets, in vivo plants, and fruit galls reported previously
(Sashida et al. 1992). It became clear that the triterpenoid contents in both in
vivo and regenerated plants are the same, in agreement with the fact that they
have the same chromosome number (2n = 58) (Kitamura and Murata 1979),
indicating that no variation occurred in callus culture. 3j3,24-dihydroxyurs12-en-28-oic acid and 2a,3j3,24-trihydroxyurs-12-en-28-oic acid are found only
in callus tissue, and this is the first example isolated from natural sources.
3j3,24-dihydroxyurs-12-en-28-oic acid may be an important product in the
oxidation process between delta-12 and delta-ll triterpenoid, because
hydroxylation of C-13 may occur through the epoxide on C-12 and C-13.
This introduction of a hydroxyl group on C-12 of triterpenoids is variable
for the transformation of various triterpenoids. 2a,3j3,24-trihydroxyurs-11-en13j3,28-0Iide, 3j3,24-dihydroxyurs-12-en-28-oic acid, 2a,3a,24-trihydroxyurs12-en-28-oic acid, 2a,3j3,24-trihydroxyurs-12-en-28-oic acid, and 3j3-( transp-coumaroyloxy)-2a,24-dihydroxyurs-12-en-28-oic acid, which are contained
in callus tissue, have the 4-j3-CH2 0H group. On the other hand, 2a,3a,23trihydroxyurs-12-en -28-oic acid, 2a,3j3 ,23-trihydroxyurs-12-en -28-oic acid,
and 3j3-(trans-p-coumaroyloxy)-2a,23-dihydroxyurs-12-en-28-oic acid having
the 4-CH2 0H group are not found in callus tissue. This clearly shows that
the hydroxylation ability of the C-4 dimethyl group in natural plants, regenerated plants, and fruit galls is nonspecific, but that in callus tissue is
specific. Therefore, it is speculated that the selective hydroxylation ability
of callus tissue can be used for the selective biotransformation of the
cyclohexane ring having the C-4 dimethyl group. The result that fruit galls
indicated the intermediate triterpenoid pattern between the in vivo plants
GC-MS
c/o OV-17(1.1 m x 2.6 mm),
15
10
min
0
250C
II
Ii
50
III
70eV
I
.1
I.
1111
1.11
'
iI
15(1
'
iIi
170
and callus tissue shows that fruit galls may possess greater metabolic ability
than callus tissue.
3 Conclusion
The procedure described here can be used as a simple method of
micropropagation, thereby helping to produce rapid strains of A. polygama.
Moreover, since the chromosome numbers of A. polygama in the root tip of
plantlets regenerated by callus culture were the same as those of the mother
plants (Kitamura and Murata 1979), this system can be utilized to supply a
homogeneous population of this plant. Although the sex of regenerated
plantlets has not been determined, it may be possible to propagate either
female or male plantlets by this system.
R4
24
727 15
'0
""
""
a-OH
CHzOH CH 3
!3-0H
CHzOH CH 3
!3 ~p--Coumaroyloxy CHzOH CH 3
OH
OH
OH
R4
CH 3
!3~OAc
CH 3
!3-0H
CH 3
!3-0H
CH 3
ex-OH
CH 3
!3-0H
CH 3
!3 -p--Coumaroyloxy CH 3
CH 3
CH 3
CH 3
CHzOH
CHzOH
CHzOH
CHzOH
!3~OH
OH
H
OH
H
OH
OH
OH
2 ex, 3 ex,
Rz
R3
"
HO""
HO"
R,
22
21
24~trihydroxyurs~
OH
--;/'
11 ~en~ 13 !3,
28~olide
Fig. 5. Structures of triterpenoids isolated from Actinidia polygama callus. (Reprinted from Sashida et a!., copyright 1994, Phytochemistry 35:377-380,
with kind permission from Elsevier Sciences Ltd, The Boulevard, Langford Lane, Kidlington OX5 1GB, UK)
3~O-acetylursolic acid
2 ex, :l!3 ~dihydroxyurs~ 1 2~en~28~oic acid
313, 24-dihydroxyurs-12-en-28-oic acid
2 ex, 3 IX, 24-trihydroyurs-12-en-28-oic acid
2 IX, 313, 24-trihydroyurs~ 12-en-28-oic acid
313 - (trans-p--coumaroyloxy) -2 ex, 24-dihydroxyurs-12-en-28-oic acid
2 a, 3 a, 23-trihydroyurs-12-en-28-oic acid
2 a, 3!3, 23-trihydroyurs-12~en~28-oic acid
3/3- (trans-p--coumaroyloxy) ~2 IX, 23-dihydroxy~
urs~ 12~en~28-olc acid
ursolic acid
25
:: 20
30
'a"
'~"
'<
::r
VJ
!-<
>-'
11
Table 3. Distribution of the triterpenoids in callus, regenerated plants, in vivo plants, and galls of
A. polygama. (Sashida et al. 1994)
Compound
Ursolic acid
3-0-acetylursolic acid
2a, 3j3-dihydroxyurs-12-en-28-oic acid
2a, 3a, 24-trihydroxyurs-ll-en-13 {3, 28-olide
3{3, 24-dihydroxyurs-12-en-28-oic acid
2a, 3a, 24-trihydroyurs-12-en-28-oic acid
2a, 3{3, 24-trihydroyurs-12-en-28-oic acid
3{3-(trans-p-coumaroyloxy)-2a, 24-dihydroxy
-urs-12-en-28-oic acid
2a, 3a, 23-trihydroyurs-12-en-28-oic acid
2a, 313, 23-trihydroyurs-12-en-28-oic acid
3{3-(trans-p-coumaroyloxy)-2a.23-dihydroxy
-urs-12-en-28-oic acid
Callus
Whole plant
Fruit gall
Regenerated'
Natural'
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
References
Bhandari H, Garg HS, Agrawal PK, Bhakuni DS (1990) Ursane triterpenoids from Nepeta
eriostachia. Phytochemistry 29:3956-3958
Cai OG, Oian YO, Ke SO, He ZC (1993) Regeneration of plants from protoplasts of kiwifruit
(Actinidia deliciosa). In: Bajaj YPS (ed) Biotechnology in agriculture and forestry, vol
23. Plant protoplasts and genetic engineering IV. Springer, Berlin Heidelberg New York,
pp 3-17
Cheung H, Tokes L (1968) Oxygenated derivatives of asiatic acid from Dryobalanops aromatica.
Tetrahedron Lett 41:4363-4366
Gamborg OL, Miller RA, Ojima K (1968) Nutrient requirements of suspension cultures of
soybean root cells. Exp Cell Res 50: 151-158
Hazama N (1942) Felidae species and Actinidia polygama. Shizen 6:55-59
Huang ZG, Tan CY (1988) Chinese gooseberry, Kiwifruit (Actinidia spp). In: Bajaj YPS (ed)
Biotechnology in agriculture and forestry, vol 6. Crops II. Springer, Berlin Heidelberg New
York, pp 166-180
Y. Shoyama et al.
12
Hyeon SB, Isoe S, Sakan T (1968) The structure of neomatatabiol, the potent attractant for
Chrysopa from Actinidia polygama. Tetrahedron Lett 5325-5326
Isoe S, Hyeon SB, Katsumura S, Sakan T (1969) Photo-oxygenation of carotenoids. II.
The absolute configuration of laliolide and dihydroactinidiolide. Tetrahedron Lett 25172520
Kapoor VK, Chawla AS (1986) Biological significance of triterpenoids. J Sci Ind Res 92:503511
Kikuchi T, Matsuda S, Kadota S, Sakai Y, Namba T, Watanabe K, Dissanayake DMRB (1984)
Studies on the constituents of medicinal and related plants in Sri Lanka. I. New triterpenes
from Hedyotis awsoniae. Chern Pharm Bull 32:3906-3911
Kitamura S, Murata G (1979) Coloured illustration of herbaceous plants of Japan II
(Choripetalae), Hoikusha, Osaka, Japan, 201 pp
Kojima H, Ogura H (1986) Triterpenoids from Prunella vulgaris. Phytochemistry 25:729-733
Kojima H, Tominaga H, Sato S, Ogura H (1987) Pentacyciic triterpenoids from Prunella vulgaris.
Phytochemistry 26:1107-1111
Konoshima T, Takasaki M, Kozuka M, Tokuda H (1987) Studies on inhibitors of skin tumor
promotion. I. Inhibitory effects of triterpenes from Euptelea polyandra on Epstein-Barr virus
activation. J Nat Prod 50:1167-1170
Konoshima T, Kokumai M, Kozuka M, Tokuda H, Nishino H, Iwashima A (1992) Anti-tumorpromoting activities of afromosin and soyasaponin I isolated from Wistaria brachybotrys. J Nat
Prod 55:1776-1778
Konoshima T, Takasaki M, Tatsumoto T, Kozuka M, Kasai R, Tanaka 0, Rui-Lin Nie, Tokuda H,
Nishino H, Iwashima A (1994) Inhibitory effects of cucurbitane triterpenoids on EpsteinBarr virus activation and two stage carcinogenesis of skin tumor. Bioi Pharm Bull 17:668671
Linsmaier EM, Skoog F (1965) Organic growth factor requirements of tobacco tissue cultures.
Physiol Plant 18:100-127
Monette PL (1986) Micropropagation of kiwifruit using non-axenic shoot tips. Plant Cell Tissue
Organ Cult 6:73-82
Monette PL (1995) Conservation of germplasm of kiwifruit (Actinidia species). In: Bajaj YPS (ed)
Biotechnology in agriculture and forestry, vol 32. Cryopreservation of plant germplasm I.
Springer, Berlin Heidelberg New York, pp 321-331
Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco
tissue cultures. Physiol Plant 15:473-497
Oliveira MM, Barroso JG, Martins M, Pais MS (1994) Genetic transformation in Actinidia
deliciosa (Kiwifruit). In: Bajaj YPS (ed) Biotechnology in agriculture and forestry, vol 29.
Plant protoplasts and genetic engineering V. Springer, Berlin Heidelberg New York, pp 193214
Pavan M (1949) Antibiotics of animal origin. Ricerca Sci 19:1011-1017
Padmaja V, Thankamany V, Hisham A (1993) Antibacterial, antifungal and anthelmintic
activities of root barks of Uvaria hookeri and Uvaria narum. J Ethanopharmacol 40:181186
Sakakibara J, Kaiya T (1983) Terpenoids of Rhododendron aponicum. Phytochemistry 22:25472552
Sakan T (1961) Study on active components of Actinidia polygama. Kagaku to Kogyo 14:10101018
Sakan T, Fujino A, Murai F (1960) Study on components of Actinidia polygama (1-3). Nippon
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Sakan T, Isoe S, Hyeon SB, Ono T, Takagi I (1964) Iridodiols, the effective components of
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Sakan T, Isoe S, Hyeon SB, Katsumura R, Maeda T, Wolinsky J, Dickerson D, Slabaugh M,
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polygama. Phytochemistry 31:2801-2804
13
1 Introduction
1.1 The Plant
The genus Alkanna (family Boraginaceae) consists of 25 species widely distributed in the Mediterranean regions and Asia. The species Alkanna tinctoria
(L.) Taush (2n = 30) also has a wide geographical distribution; in particular, it
grows wild in arid maritime areas of southern Europe. The plants are perennial herbs with prostrate bushy stems, blooming between March and May with
small (6-7mm) blue flowers (Fig. 1). The propagation of the plant occurs
naturally from seeds that are included in monospermic achenes. The percentage of seed germination is very low, which is true also for other Boraginaceae
species (Qi et al. 1993). Alkanna tinctoria has been known since ancient times
for the presence in its root of the red pigment alkannin, used since antiquity
for its therapeutical properties and as a natural dye.
1.2 Alkannin and Other Secondary Metabolites in Intact Plants
In intact plants, most alkannin derivatives are alkannin esters. The principal
alkannin esters identified are /3,/3-dimethylacryl ester and /3-acetoxy isovaleric
ester (Papageorgiou et al. 1979). The healing effect of alkannin esters has been
demonstrated on patients suffering from ulcus cruris, where after 5-6 weeks of
treatment, complete healing was observed (Papageorgiou 1978). Furthermore,
the antimicrobial activity against S. aureus and S. epidermidis was also demonstrated (Papageorgiou et al. 1979).
Alkannin has a naphtoquinonic structure and is the enantiomeric form of
another well-known important industrial pigment, shikonin, occurring naturally in the root of Lithospermum erytrhrorizon (Tabata and Fujita. 1985;
Fujita 1988; Fig. 2). A comparative analysis of the therapeutical properties of
both alkannin and shikonin performed by Tanaka et al. (1986) showed that the
antiinflammatory activities of the two compounds are identical.
[ Istituto di Rieerea sulle Bioteenologie Agroalimentari, CNR, Via Monteroni, 73100 Leece, Italy
Dipartimento di Fisiologia Vegetale, Universita di Leece, Via Monteroni, 73100 Leece, Italy
15
Fig. lA, 8. Alkanna tinctoria (L.) Tausch plant in bloom (A) and flowers (8)
C. Gerardi et al.
16
ALKANNIN
W
0
OH
,;P'
~
OH
,'pH
/ CH,
=C"
CH,
SHIKONIN
Table 1. Alkannin derivatives (mg/g dry weight) extracted from
stem and root tissues. (De Leo et al. 1992)
Plant tissue
Proximal stem
Middle stem
Distal stem
Root-suberized periderm
Secondary root
Alkannin
o
o
8.73 1.04
21.74 2.20
0.30 0.09
In a previous study, we (De Leo et al. 1992) analyzed the localization and
temporal accumulation of the pigment in plant tissues. The results revealed
that the highest accumulation of alkannin occurs during seed ripening, in
the periderm of pluriennal roots, and in the distal part of suberized stems
(Table 1).
Alkannin derivatives can be obtained by homogenization of root tissues in
chloroform. Crude extracts are purified on a Sigel column, using chloroform as
eluant, before preparative thin layer chromatography (TLC) (De Leo et al.
1992). When compared with pure alkannin, the majority of the pigments
showed a different R[, but after hydrolysis with KOH, a large fraction of the
pigments revealed the same R[ as pure alkannin. This is due to the fact that, as
has already been demonstrated (Papageorgiou and Digenis 1980), the pigments extracted from the roots contain esterified forms of alkannin. We performed gas chromatography-mass spectrometry (GC-MS) analyses of the
partially purified fractions, after KOH hydrolysis and derivatization of free
acids. The results revealed that our alkannin fractions were constituted by the
alkannin esters shown in Fig. 3.
17
COMPOUNDS
STRUCTURE
DIHYDROXYNAPHTHOQUINONIC
COMPOUNDS
(ALKANNIN AND
ITS ESTERS)
ALKANNIN
100
R=OH
N.D.
ACETYL-ALKANNIN
58.3 0.6
2-METHYL-BVTANOYLALKANNIN
ISOVALERYL-ALKANNIN
7.3 0.8
R = O-C-CH 2CH(CH,h
~
..
(Z)-2-METHYL2-BUTENOYL-ALKANNIN
(ANGELIL-ALKANNIN)
IS.9 0.1
18.6 02
Fig.3. Structure and quantitative composition (% mol) of alkannin derivatives present in purified
alkannin fraction of intact roots. (De Leo et al. 1996)
Due to the difficulties in plant cultivation and the harvesting costs, the
production of the pigment from intact roots cannot be considered a convenient
approach. Furthermore, the massive utilization of natural sources could result
in the exhaustion of wild plants, as has already happened for Lithospermum
erytrhorizon growing wild in Japan, and now almost extinct (Fujita 1988).
2 In Vitro Approaches
In vitro cultures of Alkanna tinctoria were set up to study:
1. in vitro production of regenerated plants,
2. production of alkannin from suspension cells and root cultures.
18
C. Gerardi et al.
This chapter is primarily based on our published work (De Leo et al. 1992,
1996; Mita et al. 1994).
2.1 Establishment of Cultures
We obtained two different root cultures (ATS23R and ATR1) that have
different origins. The A TS23R root culture was induced by inoculating 0.5 ml
of ATS23 cell suspension (growing in MSA) in 50ml of MS medium without
growth regulators (Mita et al. 1994). After induction, this root line was
sub cultivated in RC medium (Shimomura et al. 1991). ATR1 was obtained
from a root-induced callus growing in MG5liquid medium (Tabata and Fujita
1985).
2.2 Regeneration of Plants/Micropropagation
19
callus induction
Medium
6BAP CuM)
NAA CuM)
2,4-D CuM)
MSI
MS2
MS3
1.25
2.50
1.25
2.50
5.00
/
/
/
0.54
0.54
/
MS4
MS5
MS6
MS7
MS8
/
/
/
/
/
/
/
/
0.45
0.90
2.26
.-,
regeneration was obtained only when calli were incubated in the light. One out
of eight lines (E4) was able to produce several shoots (about 20 shootslg of
callus) in MS4 medium (Fig. 4). Continuous subcultivations in MS4 medium
resulted in a decreased regeneration ability (to 0.5 shootslg of callus). Recovery of regeneration ability was obtained by alternate sub cultivations of E4 calli
in MS4 and MS- media. Shoots were isolated to induce rooting. Several media
were tested to consider also the role of IAA, NAA, indole-3-butyric acid
(IBA), gibberellic acid (GA 3), and charcoal at different concentrations. Best
results (56% rooting) were obtained for E4 shoots incubated inMS- medium
(Fig. 5); ATF2 shoots were able to produce roots (15 to 40% of rooting) in
modified MS medium containing half-strength macronutrients, 15 gil charcoal
and 2.46 or 4.90.uM IBA. In the same medium containing IAA in place of
IBA, rooting was reduced to 20%.
20
C. Gerardi et al.
Most of the induced suspension cultures, as well as root cultures, are able to
grow very well in the growth media but are unable to produce alkannin. The
observation that actively proliferating cells are not able to produce the desired
secondary metabolites has already been reported in other species, whereas it
has been reported that stress factors (both nutrient and enviromental stresses)
induce or enhance plant secondary metabolism (Mizukami et al. 1978; Parr
1989). We tested different parameters, in order to identify the experimental
conditions useful for alkannin production in suspension cultures.
2.3.1 Effect of Growth Regulators
21
Table 3. Pigment production (;1g/g fresh weight) obtained from ATS23 and ATS23A cell lines in
MSA or MSB medium. (Mit a et al. 1994)
Cell line
Preculture
medium
Culture
medium
Alkannin
derivatives
MSA
MSA
0.64
MSA
MSA
MSB
MSA
24.08
MSA
MSB
108.20
ATS23
ATS23A
The values refer to the alkannin derivatives obtained after 15 days of preculture, followed by
further 20 days of incubation in culture medium.
MSB medium has the same composition as MSA but contains 1 JiM IAA instead of 2,4-D.
, :F/i.~~J~<.:\ . . . .. . . . .. ..
u .4000 .-----r-----.-----~----.------r-----r----~----~----~----~
[A b s 1
:
\
........................ ,........................... ,............................\ ......... ....... ..... .....
~
.
'\
,
~ '.
Yava l ano th (n .l
700. 0
Fig. 6. A TLC of KOHhydrolyzed pigments obtained from: intact roots (lane 1); cells (lane 2);
medium (lane 3). Lane 4 is pure alkannin (Roth Gmbh, Karlsruhe, Germany). B Spectrum of
alkannin derivatives obtained from E4 cell line. (Mita et al. 1994)
22
C. Gerardi et al.
Table 4. Role of agar on cell growth and production of alkannin derivatives (pg/g fresh weight) in
Alkanna tinctoria suspension cultures
Agar(%)
Cell growth'
Alkannin derivatives
0
0.01
0.05
0.10
0.20
9.34
8.10
11.36
10.44
12.10
38.05
25.62
18.62
25.43
15.46
, The values refer to the increase in fresh weight (g) of 2.5 g starting material after 20 days in
culture.
Table 5. Role of ammonium salt on cell growth and the production of alkannin derivatives (pg/g
fresh weight) in Alkanna tinctoria suspension cultures
1
5
10
20
Cell growth'
Alkannin derivatives
6.40
10.53
10.58
8.10
7.92
132.65
13.21
21.18
23.49
25.78
, The values refer to the increase in fresh weight (g) of 2.5 g starting material.
Alkannin production was stimulated when the cells were incubated in media
relatively poor in nutrients. However, in these media, cell growth is limited, so
that a convenient method is to utilize a two-stage culture system, in which two
different media are used; in the first medium (culture medium), cell growth is
stimulated, while the second medium (induction medium) promotes alkannin
production.
The presence of agar in the culture medium has been reported to be of
some importance for shikonin production in cell cultures of Lithospermum
erythrorizon (Fukui et al. 1983). We observed that the presence of agar in the
medium at a concentration of 0.2% stimulated cell growth, but had a negative
influence on alkannin production. The same effect (although more dramatic)
was observed when ammonium salts were present in the medium: in the
presence of ammonium at concentrations as low as 1 mM, the production of
alkannin was reduced by about 90% while cell growth was clearly stimulated
(Tables 4, 5). These results confirm those obtained for shikonin production
(Tabata and Fujita 1985).
2.3.3 Cell Selection
23
600
500
400
Alkannin
derivatives
300
200
100
o
o
10
15
20
25
Days
Fig. 7. Production of alkannin derivatives (j.1g/g fresh weight) from ATS23A cell line after different times of incubation in induction medium (Rel)
24
C. Gerardi et a1.
,
Fig. 8. E4 callus accumulating alkannin derivatives
Medium
Root
Culture medium
RCI
RC2
289.73
61.54
30.19
196.17
The two different lines of root cultures obtained showed different responses:
ATS23R was able to grow and produce alkannin derivatives in RC1 or
RC2 medium (Mit a et al. 1994), whereas ATR1 needs a two-stage culture
system, consisting of a culture medium (MG-5) and an induction medium
(RC1) (Table 6). In fact, ATR1 roots, when incubated in RC1 medium, were
able to produce alkannin derivatives, but their growth was completely
blocked. Interesting results were obtained with ATS23R root culture; in fact,
this line was able to release a good amount of pigments in the culture medium
25
Fig. 9A-D. Alkanna tinctoria protoplast-derived clones. A Cultured cells of A. tinctoria. B Isolated protoplasts. C Clusters derived from protoplasts after 3 weeks of culture. D Callus colonies
visible in the Petri dish after 4 weeks of culture
3 Conclusion
Alkanna tinctoria seems to be very promising both for alkannin production
from suspension cell culture and for in vitro regeneration of whole plants. The
production of alkannin from suspension cultures can hopefully be considered
for potential industrial applications. For this purpose, however, it is important
C. Gerardi et al.
26
Table 7. Pigment production and root growth in A TRI line
Culture
medium
Induction
medium
Root growth'
Alkannin
derivatives
MG5
MGS
MG5
RCI
7.3
0.1
0.0
149.5
References
De Leo P, Miceli A, Gerardi C (1992) Extraction and purification of alkannin from Alkanna
tinctoria Tausch: its location in the plant and time course accumulation in the tissues. Agric
Med 122:334-339
De Leo P, Miceli A, Ronzini L, Sgarra R (1996) Chemical characterization of alkannin esterified
derivatives. AIHTEI 7:23-25
Fujita Y (1988) Shikonin: production by plant (Lithospermum erythrorhizon) cell cultures. In:
Bajaj YPS (ed) Biotechnology in agriculture and forestry, vol4. Medicinal and aromatic plants
4. Springer, Berlin Heidelberg New York, pp 225-236
Fukui H, Yoshikawa N, Tabata M (1983) Induction of shikonin formation by agar in
Lithospermum erythrorhizon cell suspension cultures. Phytochemistry 22:2451-2453
Mita G, Gerardi C, Miceli A, Bollini R, De Leo P (1994) Pigment production from in vitro
cultures of Alkanna tinctoria Tausch. Plant Cell Rep 13:406-410
Mizukami H, Konoshima M, Tabata M (1978) Variation in pigment production in Lithospermum
erythrorhizon callus cultures. Phytochmistry 17:95-97
Murashige T, Skoog F (1962) A revised medium for rapid growth and bio-assays with tobacco
tissue cultures. Physiol Plant 15:473-497
Papageorgiou VP (1978) Wound healing properties of naphthaquinone pigments from Alkanna
tinctoria. Experientia 34:1499-1501
27
Papageorgiou VP, Digenis GA (1980) Isolation of two new alkannin esters from Alkanna
tinetoria. Planta Med 39:81-84
Papageorgiou VP, Winkler A, Sagredos AN, Digenis GA (1979) Studies on the relationship of
structure to antimicrobial properties of naphthaquinones and other constituents of Alkanna
tinetoria. Planta Med 35:56-60
Parr AJ (1989) The production of secondary metabolites by plant cell cultures. J Biotechnoll0:l26
Qi MQ, Upadhyaya MK, Furness NH, Ellis BE (1993) Mechanism of seed dormancy in
Cynoglossum olfieinale L. J. Plant Physiol 142:325-330
Shimomura K, Sudo H, Sag H, Kamada H (1991) Shikonin production and secretion by hairy root
cultures of Lithospermum erythrorhizon. Plant Cell Rep 10:282-285
Tabata M, Fujita Y (1985) Production of shikonin by plant cell cultures. In: Day P, Zaitlin M,
Hollander A (eds) Biotechnology in plant science. Academic Press, New York, pp 207-218
Tanaka S, Tajama M, Tsukada M, Tabata M (1986) A comparative study on anti-inflammatory
activities of the enantiomers, shikonin and alkannin. J Nat Prod 49:466-469
ZAKHLENJUK
and V.A.
KUNAKH
1 Introduction
1.1 Distribution and Importance of the Plant
Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine, Kiev
252143, Ukraine
Biotechnology in Agriculture and Foresty, Vol. 41
Medicinal and Aromatic Plants X (ed. by y.P.S. Bajaj)
Springer-Verlag Berlin Heidelberg 1998
Arnebia euchroma
29
Fig. 1. Arnebia euchroma (Royle) Jonst. 1 General appearance of the plant; 2 lower part of the petal; 3 fruit
nutlet
30
Naphthoquinones:
~
YV~
OH
DS
(R=H)
(R=OH)
AS
(R=OCOCH3)
IBS
(R=OCOCH(CH312)
DMAS
(R=OCOCH=C(CH312)
HIVS
(R=OCOCHzC(OH)(CH312)
Benzoquinones:
R
Echinofuran B
(R=H)
Echinofuran C
(R=OCOCH=C(CH312)
31
Arnebia euchroma
4,5
4,5
10
10
Fig. 3a,b. Densitograms of shikonin and its derivatives isolated from intact root (a) and tissue
culture (b) of A.euchroma. 1 Shikonin; 2 deoxyshikonin; 3 acetylshikonin; 4 dimethylacrylshikonin; 5 isobutylshikonin. Along the horizontal line time of scanning, min
H}BO} 6.2, KI 0.S3, CuS0 4 5H20 0.3, NaEDTA2H2 0 37.3, FeS0 4 7H20 27.S,
thiamine 0.4, nicotinic acid 0.5, pyridoxine 0.5, mesoinositol SO, indole acetic
acid 0.3, kinetin 1, and sucrose 3%.
Rabinovich and Davydenkov kindly provided several laboratories
with cell lines of established suspension tissue culture of A.euchroma
(strains 75 and 80) to carry out investigations. Independently, A.euchroma
suspension tissue culture was obtained only in China (Dong et al. 1993).
Various results of in vitro studies conducted on A.euchroma are summarized
in Table 1.
2.2 Secondary Metabolites in Amebia euchroma Tissue Culture
Cells of suspension culture after 1 year of passaging built up greater amounts
of shikonin, deoxyshikonin, and acetylshikonin as compared to intact
plant, while the accumulation of dimethylacrylshikonin and isobutylshikonin progressed less extensively than in plants (Fig. 3b; Davydenkov
et al. 1991). In addition to the above-mentioned shikonin derivatives, an
insignificant amount of hydroxyisovalerylshikonin was later identified in
the A.euchroma cell line SO-2 (Zakhlenjuk et al. 1993). Besides naphthoquinones, the cultured cells accumulated yellow pigments identified as
benzoquinones (echinofuranes Band C; Fig. 2; Davydenkov et al. 1991).
Accumulation of these six naphthoquinones, as well as of two forms
32
Reference
Zakhlenjuk (1992)
Dong et al. (1993)
Zakhlenjuk (1994)
Sokha (1996)
Arnebia euchroma
33
used as a criterion the general culture coloring, namely, the coloring of both
tissue and culture medium. For further subculturing, the flasks with culture
(here designated subcultures) showing the most intensive coloring were
picked out. However, the experience of several years of culturing the initial
strains 75, 80, and some variants derived from them had shown that this
method of selection, in spite of temporary positive results, finally proved
ineffective. As is seen from Fig. 4, the productivity of strain 80 remained
insignificant and clones generated from it exhibited a progressive decline in
productivity despite consistent screening of the most intensely colored samples of subcultures for transfers.
The failure of this selection technique was also confirmed by the results of
two alternative, simultaneously performed for 10 passages, selections: one for
increased, the other for reduced shikonin content. The results for first three
passages summarized in Table 2 reveal great differences in the shikonin content in "offspring" of the subculture selected for transfer in each passage.
Average shikonin content between all subcultures in each passage during
positive selection exceeded that for negative selection, but this difference
disappeared after four to six passages. Negative selection appeared to be even
more effective than positive, as it is interesting that by constant selection of
the least colored subcultures "offsprings" emerge that exceed not only the
original culture but the variants of positive selection as well in productivity.
34
50
I :::~~
_--3 I
40
~d'
30
~
'"
20
10
A
- -.- - -. -
-+- --.
10
15
L-~~~~L-~~~~L-~~~~L-~~L-~
20
Passage number
Fig. 4. Shikonin content in the course of sequential passages of A.euchroma tissue culture. 1
Clone 80-1; 2 clone 80-2; 3 original strain 80. Each point represents average of three to five
75
Starting population of
subcultures"
127.5/'
85.0
67.5
37.0 \,
(79.2)
(+ )
(-)
80-2
(+)
260.0/'
200.0
173.0
(211.3)\,
(- )
2nd passage
3rd passage
111.0
109.0
103.0
47.0
(92.5)
132.5
110.2
81.2
67.0
(97.7)
90.0
85.0
52.0
45.0
(68.0)
34.0
33.5
27.0
26.0
(30.1)
217.5
170.0
165.0
(184.2)
45.2
43.7
39.5
32.5
(40.2)
186.2
175.0
131.2
(164.2)
51.0
42.0
40.0
30.0
(40.7)
255.0
190.0
175.0
(206.6)
223.7
155.0
111.9
(163.5)
138.7
131.2
103.7
(124.6)
185.0
165.0
120.0
(156.6)
" Positive (+) and negative (-) selection. The material of subcultures that are underlined was used entirely for subsequent
subculturing. Parentheses indicate mean shikonin content for all
subcultures in each passage.
Arnebia euchroma
35
Such results might reveal wide range of cell chemotype variability in the
culture.
The results indicate the necessity of searching for additional selection
criteria during mass cUlturing. It was suggested that the selection of small
uniform cell aggregates might be useful for this purpose. The approximately
equal size of cell aggregates may be a decisive factor which possibly indicates
similar or relatively more synchronized cell differentiation processes within
tissue structures. Brought together, such cell aggregates may undergo more
coordinated development, thus approximating the cell culture to the status of
intact plant tissue. This suggestion is substantiated by our results (see Sect.
2.3.3).
2.3.2 Cell Aggregate Cloning
For cloning the cell aggregates of two cultured strains, 75 and 80, we used cell
clumps of 1-2 mm in size, thus obviating the need for application of feeder
layers or conditioned media. Due to the peculiarities of the cloning technique
(aggregate maintenance in drops of nutrient medium supplemented with mineral oil), the variability of aggregates in pigment accumulation manifests itself
already at the first step of cloning, thus making possible early rejection of
undesirable variants which make the oil yellow or orange, or fail to color it at
all. The latter account for 25%. The aggregates which color oil light pink make
up about 50% with the remaining ones deep pink and red. More productive
clones were obtained only from the strain 80, which, in contrast to strain 75,
was characterized by greater tissue density and less vigorous growth, as well
as by the availability of a large number of small, dense globular aggregates
that were used for cloning. Cell aggregates of strain 75 proved friable and
predominantly of lamella structure. The results indicate that dense, small
cell aggregates are more effective for cloning. It is also obvious that the
cloning procedure may be effective if the initial size of cell aggregates reaches
1-2mm.
Subsequent long-term selection of the most intensively colored subcultures of new clones failed to promote stability in increased productivity level
(Fig. 4).
2.3.3 Selection of Different Fractions of Tissue Culture
36
25
20
10
20
40
60
80
100
120
140
160
180
Shikonin. mg / L
Fig.5. Distribution of individual subcultures in terms of shikonin content in A.euchroma cell line
80-2 during long-term selection by most intensive coloring. N N umber of subcultures; V index of
variation; M mean value of shikonin content (57 mg/I)
37
Arnebia euchroma
35
30
N=37
V=13%
25
eI:
'"
20
~
;:;
15
B
:;
en
10
0
100
200
300
400
500
600
Shikonin, mgIL
Fig. 6. Distribution of individual sub-:ultures in terms of shikonin content in A.euchroma cell line
80-2 during alternation of selection by the most intensive coloring with selection of fraction of
small colored cell aggregates. N Number of subcultures; V index of variation; M mean value of
shikonin content (349 mg/I)
38
700
600
='S
]
:E
CI)
500
400
300
200
100
0
450
400
350
0:2OIl
8
300
='S
250
200
CI)
150
100
50
10
15
20
25
Passage number
Fig.7a,b. Shikonin content in A.euchroma tissue culture. a Strain 75. b Cell line 80-2. 1 Control;
2 cell lines resulting from the selection of cells and cell aggregates adhering to the flask walls. Each
point represents average of three replicates
the increased level for a year (Table 3). Microscopic monitoring failed to
reveal yellow crystals in the culture fluid. Currently, productivity in the best
subcultures may reach 1 gil for 14 days or 71.4mg/l per day. This result is
comparable to that reported for another A.euchroma suspension culture
(Dong et al. 1993). Authors claimed that the shikonin yield for 30 days of
culture growth in bioreactor was estimated to be 1.93 gil, which is equal to
64.3 mgll per day.
Thus, selection by color tint proved more effective than selection
only by the most intensive general coloring. Such selection seems to be
more useful in rejecting those subculture samples where biosynthesis of the
yellow pigments or other metabolites competing with naphthoquinones IS
enhanced.
Arnebia euchroma
39
Table 3. Shikonin content in A. euchroma tissue culture during selection of subcultures by tint of
coloring
1995
(year
quarter)
I
II
III
IV
Shikonin
content
(mg/I)"
390.8
672.5
940.0
688.8
56.2
40.0
36.0
44.3
Range
of variationb
Shikonin
content per
dry weight (%)
Biomass
dry weight
yield (g/I)
295.8
525.0
904.0
568.0
2.9
5.5
7.7
6.3
13.5
12.2
12.2
11.1
468.0
702.0
976.0
813.7
40
"0
l:l
c:
120
100
100
80
80
60
60
40
40
20
20
""'0
<i<
.5c:
0
~
CIl
Fig. Sa,b. Shikonin content in A.euchroma cell line 80-2 (a) and its PFP-resistant variant (b). J
Control medium; 2,4 medium containing indole butyric acid (1 mg/l); 3 control medium, containing PFP (lOOmg/l)
Arnebia euchroma
41
150
150
"8
100 - ----
,--..-0.:11 .,...,--------
---
100 ]
---------
c:
''"'E""
"""o
<ff:<:
o
:.0
'2
o
....
,....
cs
50
50
KIN
BAP
(mgfL)
(mgfL)
0.1
1.0
KIN
(mgfL)
0.1
:.2
(Il
1.0
BAP
(mgfL)
Fig. 9. Effect of kinetin (KIN), 6-benzylaminopurine (BAP), and indole acetic acid (fAA) on the
increase in biomass and shikonin content in A.euchroma cell line 80-2. K Control. Shikonin
content was estimated in culture fluid
NaZS04 500, MgS0 47HzO 370. After transfer of cell line 80-2 into the medium
with this composition of macroelements, increased culture productivity was
registered. Shikonin content in the first two passages comprised 842.5 and
814.4mg/l, respectively, the control, however, as little as 688.8 and 453.1 mg/l,
respectively.
Optimized macro element composition, proposed by Sokha (1996) for
medium used to cultivate cell strain 75, was as follows (mg/l, lower level):
KN0 3 3750, Ca(N03 )z 500, KCI 175, KH zP0 4 110, KzHP0 4 70, MgS0 4 6HzO
470. This medium, supplemented with 4.5% sucrose, was reported to increase
shikonin accumulation by 50%.
2.5 Differentiation
42
o.v. Zakhlenjuk
similar conditions. Root formation in cell line 80-2 did not appear to correlate
with shikonin content increase (see Fig. 8).
4 Protocol
4.1 Maintenance of Tissue Culture
Tissue cultures of A.euchroma (strains 7S, 80, and resultant variants) were maintained in modified
Linsmaier-Skoog medium (Davydenkov et al. 1991) in 2S0-ml Erlenmeyer flasks containing SOml
43
Arnebia euchroma
of medium, with agitation on shaker (80-100 rpm), in the dark at 24
cycle was 14 days.
:!:
References
Bychkova TP, Nanenina EV, Bersin VB, Miroshnikov AI (1993) Influence of jasmonic acid and
12-oxo-phytodienoic acid on the biosynthesis of shikonin in the Arnebia euchroma cell culture.
Bioorg Chern 19:1008-1012 (in Russian)
Cherepanov SK (1981) Vascular plants in the USSR. Nauka, Leningrad (in Russian)
Chukavina AP (ed) (1984) Flora of Tajik SSR, vol 7. Nauka, Leningrad (in
Russian)
Davydenkov VN, Patudin AV, Popov YuG, Rabinovich SA, Miroshnikov AI (1991) Cell culture
of Arnebia euchroma (Royle) Jonst. - novel source of shikonin production. Him Farm Zh
1:53-55 (in Russian)
Dong J, Ye H, Wu X, Li G, Wu Z, Chen J (1993) Studies of suspension culture and fermentable
culture of Arnebia euchroma. Acta Bot Sin 35:57-61 (in Chinese)
Fujita Y (1988) Shikonin: Production by plant (Lithospermum erythrorhizon) cell cultures. In:
Bajaj YPS (ed) Biotechnology in agriculture and forestry, vol 4. Medicinal and aromatic plants
I. Springer, Berlin Heidelberg New York, pp 225-236
Kozlovtseva LV, Zaitseva GV, Strogov SE (1994) Suspension culturing of macrotomia Arnebia
euchroma cells. Optimization of conditions for shikonin production. Biotechnologia 3:24-26
(in Russian)
Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays in tobacco tissue
cultures. Physiol Plant 15:473-497
44
Pimenova ME, Tareeva NV (1980) Shikonin content variability in the underground organs of
macrotomia dyeing. Rastit Resur 16:82-86 (in Russian)
Shepel LI, Erokhina LV, Strogov SE, Zaitseva GV, Rabinovich SA, Davydenkov VN (1991)
Suspension culturing of macrotomia dyeing cells. In: Progressive industrial methods for the
medical industry. Part 2, NPO "BIOMASH", Moscow, pp 23-25 (in Russian)
Sokha VI (1996) Shikonin sources in tissue culture. PhD Thesis, Chemical Pharmaceutical
Institute of St. Petersburg (in Russian)
Terada A, Tanoue Y, Taniguchi H (1990) Chemistry of shikonin, ancient purple pigment and its
derivatives. J Syn Org Chern Jpn 48:866-875 (in Japanese)
Zakhlenjuk OV (1992) Correlation between nucleolar number, nucleolar size and productivity of
macrotomia Arnebia euchroma cultured cells. Cytologia 34:69 (in Russian)
Zakhlenjuk OV (1994) Some peculiarities of p-fiuorophenylalanin-resistant macrotomia dyeing
cell culture. Biopolymery i Kletka 10:32-37 (in Russian)
Zakhlenjuk OV, Vidmachenko TV, Kunakh VA (1991) Shikonin accumulation dynamics during
long-term selection of Arnebia euchroma suspension culture. Abstr 1st All-Uinion Symp New
Method Plant Biotech, Pushchino, pp 61-62 (in Russian)
Zakhlenjuk OV, Vidmachenko TV, Kunakh VA (1992) Effect of some abiotic elicitors on the
shikonin accumulation in macrotomia suspension culture. Abstr 3rd Ukr Confr Med Bot, Kiev,
p 62 (in Russian)
Zakhlenjuk OV, Vidmachenko TV, Kunakh VA, Rabinovich SA, Davydenkov VN (1993) Comparative characteristics of shikonin accumulating Arnebia euchroma suspension culture and its
p-fiuorophenylalanine-resistant variant. Acta Hortic 330:293-298
1 Introduction
1.1 Distribution and Importance of the Plant
1 Research Group for Plant Breeding and Propagation, Department of Ornamentals. Danish
Institute of Plant and Soil Science, Kirstinebjergvej 10, DK-5792 Arslev, Denmark
2 Department of Applied Biological Sciences, Faculty of Agriculture, Saga University. 1 Honjo,
Saga 840 Japan
46
The plants used in folk medicine were collected from nature. Drugs were made
from roots or from the above-ground parts of flowering plants, that were dried
and prepared as broths or infusions (Gastaldo 1978; Barnaulov et al. 1983).
The roots and leaves of one species, C. rapunculoides, can be used as a
vegetable (Becker-Dillingen 1950). No information is available regarding
commercial production of drugs from Campanula. A number of species
have been investigated for their contents of metabolites (Table 1) of possible
pharmacological importance. In addition, one paper states the isolation
of two alkaloids, campedin and (-)lobeline, from C. medium (Dapke and
Fritsch 1970).
In many of these investigations the plant material consisted of mixtures of
plant organs, often all epigeal parts of flowering plants. However, at least the
contents of flavonoids, including anthocyanins, and phenolic acid derivatives
show dramatic differences when comparing leaves, stems, developing flower
buds, and petals (Fig. 2). Also for inulins and polyacetylenes, differences were
found when more than one plant part was tested (Pollard and Amuti 1981;
Tada et al. 1995a). Therefore, some investigations should be considered only
as a guideline to the compounds present in all tissues of the plant, since precise
information on the relative proportions of flowers, leaves, stems, and flower
buds is not available.
Campanula species contain many of the secondary compounds that are
the active principles in plants used for medicinal purposes. Generally, the
47
Anthocyanins ~ Flavonoids
i3 Phenolic acids I
0000
0000
~:i!.
1000
100
40
.~.
""
'"
<lJ
:>
ell
<lJ
....l
l!
'"
S
.....<lJ
Vl
Ii
'"
'"d
;:j
..0
...,
<lJ
C. isophylla
'"
~
.....
<lJ
p.,
I ri
'"
<lJ
:>
ell
<lJ
....l
'"
S
<lJ
.....
Vl
'"
'"d
;:j
..0
...,
.$
.....ell<lJ
Il.;
<lJ
C. rotundifolia
compounds shown in Table 1 or closely related ones are also found in genera
of more commonly utilized medicinal plants such as Platycodon, Codonopsis,
and Adenophora, (e.g., Pollard and Amuti 1981; Goto et al. 1983; Inada et al.
1992; Tada et al. 1995a; Wang et al. 1995). Campanula is thus a genus with a
possible unexploited potential as source of medicinal secondary metabolites,
as pointed out by Barnaulov et al. (1983) and Tada et al. (1996). It may be
fruitful in a survey among Campanula species to search specifically for additional compounds known to occur in, e.g., Platy codon grandiflorum (Inada et
al. 1992; Tada et al. 1975). Platycodon grandiflorum is grown on a commercial
scale in China for production of the traditional drug Jiejeng, in Japan (Kikyo)
and in Korea (Doraji) (Hosoda et al. 1992; Kim et al. 1995).
Commercial propagation of Campanula for ornamental purposes is by
seed, division, or cuttings. Biannual species, such as Campanula medium and
C. pyramidalis, are seed-propagated, since the plant usually dies after
flowering. In addition to seeds, the perennial species, e.g., C. isophylla and C.
carpatica, are propagated by division or cuttings depending on the growth
habit of each species.
Although micropropagation of Campanula is relatively easy, it is used
only to a small extent, since this propagation method is economically competitive only in special cases, e.g., to propagate disease-free elite stock plants,
initial multiplication of new cultivars, or clonal propagation of biannual
species (Brandt 1997).
48
Reference
Flavone"
Coumarin 3a
Sterol"
Anthoc. 7d
Inulin 6,
Polyac. 8b
Flavonol"
Triterpene"
C. alliariifolia
C. bayerniana
C. betulaefolia
c. bononiensis
Q
Q
C. choziatowskyi
C. carpatica
C. cochleariifolia
C.
C.
C.
C.
C.
C.
C.
C.
C.
moesica
oblongifolia
ochroleuca
ossetica
patula
C. persicifolia
C,M,B'
+
C
+
M,C,B'
Q
Q
+
C
+
+
+
+
+
+
+
C,R
+
+
+
+Q,K f
C. punctata
+
V
C. rapunculoides
+
+
+
C. phyctidocalyx
C. poscharskyana
C. raddeana
C. kirpichnikovii
C. kolenatiana
C. latif/ora
lactiloba
leskovii
letschschumensis
makaschvilii
maleevii
medium
hypopolia
isophylla
istriaca
kemulariae
C.
C.
C.
C.
C.
C.
crystallocalyx
dolomitica
elegantissima
glome rata
C. grossekii
C.
C.
C.
C.
+
Q
+
+
+
M
49
Table 1. Continued
Species
C. rotundifolia
C.
C.
C.
C.
takhtadzhianii
thyrsoides
trachelium
vidalii
Flavone l '
Coumarin"
Sterols"
Anthoc. 7d
Reference
Flavonol"
Triterpene'"
Inulin 6'
Polyac Sb
+ K
+ M
+
+
+
Luteolin glycosides; 2 Quercetin (Q) and kaempferol (K) glycosides; , Fraxetol and its glycosides;
Ursolic acid; .\ Sterols; 6 Inulin oligomers; 7 Anthocyanins: bisdeacylplatyconin (B), campanin (C),
monodeacy1campanin (M), rubrocampanin (R), violdelphin (V); 8 polyacetylenes.
Notes:
, In epigeal parts of flowering plants.
h In leaves.
, In stems.
J In flowers.
, Different chemotypes.
f Compounds found together in one sample.
1
2 In Vitro Approaches
2.1 Review of Tissue Culture Studies
There are only a few reports on tissue culture of Campanula (Table 2).
Micropropagation of Campanula using shoot clusters was reviewed by Brandt
(1997), based on earlier work (Brandt 1992, 1994), and here flower bud culture
was mentioned as a possible tool for studies of anthocyanin accumulation.
Production of secondary metabolites (the polyacetylene lobetyol and its
glucosides) from hairy root cultures of C. medium has been reported recently
(Tada et al. 1996).
Since Campanula is not an economically important medicinal plant,
both known cases of in vitro production of secondary metabolites are
described by the respective workers as model systems for biosynthetic
studies. The contents of secondary metabolites differ among various kinds
of plant tissue (Fig. 2), so it is important to be able to grow several plant
organs in vitro in order to investigate all the processes taking place in the
plant.
2.2 Establishment of Tissue Cultures and Micropropagation
50
Species
Purpose of study
Observationslremarks
Reference
C. isophylla
Development
of methods for
micropropagation
Brandt
Analysis of
genetic variation
in formation of
shoots and roots
Brandt
Polyacetylene
production in
hairy root
cultures
Tada et al.
Review of
published studies
and other
observations
C. isophylla
C. medium
C. isophylla
C. carpatica
C. pyramidalis
(1992)
(1994)
(1996)
Brandt
(1997)
Fig. 3. Shoot culture of C. isophylla after six subcultures on MS with 1 mg/l BA. (Photograph K.
Brandt)
Shoot cultures are initiated from vegetative buds (Fig. 3). The best explant
type is a 6-mm stem segment with a 1-2-mm axillary bud, taken from a position
not in direct contact with the soil surface. The stem segment is surfacesterilized in two separate steps, alternating with trimming of the cut ends
51
(Brandt 1992). The final explant, retaining about 2mm of the stem, is cultured
on MS medium (Murashige and Skoog 1962) with the macronutrients reduced
to 50%, 30gl- 1 sucrose, 4.4,uM benzyladenine (BA), and 6gl- 1 agar (Difco
Bacto). This procedure was used for C. isophylla and C. pyramidalis. For C.
carpatica 12 g 1-1 agar was used, and a photoperiod of 12 h day -I. After 5 weeks
on initiation medium, the explants were moved to a propagation medium with
full-strength MS salts, but otherwise identical to the initiation medium. Subsequently, every 5 weeks, all shoots were excised 3mm above the basal part of
the shoot cluster, using the basal parts for further culture. The shoot tips are
discarded, or they can be transferred to rooting medium (hormone-free, halfstrength MS) for production of plantlets.
Shoot cultures have not been used for deliberate production of secondary
compounds. However, it was observed that if the shoot cultures are grown on
media with high concentrations of sucrose (to facilitate root formation; Brandt
1997), the leaf color became more reddish, indicating an enhanced accumulation of anthocyanin.
2.2.2 Flower Bud Cultures
The procedure is similar to shoot cultures, but the explant is instead taken
from a generative bud (Fig. 4). On a flowering plant, the buds will be more or
Fig. 4. Flower bud culture of C. isophylla 2 months after initiation. Generative explants were
grown on MS without hormones, with 2% sucrose and an irradiance of 35 ,umol m -2 S-I. (Photograph K. Henriksen)
52
less generative according to the position on a shoot; the closer to the apex, the
more generative are the buds. Positions where the meristems are generative
are characterized by small, lanceolate sepal-like leaves, readily distinguishable
from the larger, usually rounded leaves on vegetative parts of the plant (refer
to Fig. 1). Explants were subcultured when the petals of the first visible flower
bud reached a length of 2 mm.
Using this procedure similar results are obtained in C. isophylla and in C.
carpatica. In C. isophylla, in vitro flowering has been obtained only using this
procedure, while in C. carpatica formation of flower buds was observed also in
previously vegetative shoot cultures cultured in vitro in continuous light (K.
Brandt, unpubl.). The development of the flower buds depends on both media
and irradiation. Even with optimal combinations of the factors tested so
far, most explants still revert to vegetative growth. However, the experimental
system is now so well developed that it can be used to study the synthesis of flower compounds in an isolated system with a highly controlled
environment.
2.2.2.1 Growth Regulators
The effects of adding the growth regulators BA and gibberellic acid (GA3) to
bud cultures of C. carpatica were investigated (Fig. 5). It appears that both
growth regulators depress the percentage of buds that develop into flowers, so
subsequent experiments were done without growth regulators.
2.2.2.2 Sucrose and Irradiance
The effects of different sucrose concentration in the medium and the irradiance given to flower bud cultures of C. isophylla differed during the progression through the culture period. The media were prepared with four levels of
40,~
~30
o
'-'
[fJ
20
(~
510
\)
o - ~
o 1
234
BA concentration, mg/l
Fig. 5. Effects of BA (0.5--4 mg/I) and GA3 (0 or 0.5 mg/I) on in vitro flower development in C.
carpatica. (J.M. Lanka, unpubl.)
53
sucrose: 0, 2, 4, and 6%. Mannitol was added to some of the media to compensate for the differences in osmolality, thus the concentrations of mannitol were
respectively 3.15, 2.1, 1.05, and 0%. In the initial stages, only sucrose concentration affected the survival of the explants (Fig. 6). The experiment showed a
well-defined optimum at 2% sucrose in the medium, irrespective of the irradiance. The effect of sucrose in the medium was highly significant (p < 0.0001),
while the irradiance had little effect on explant survival.
For the subsequent development of the explants into flowering or
nonflowering shoots, an interesting interaction between sucrose and irradiance appeared (Fig. 7).
The lower the irradiance, the more sucrose was required in the medium to
support the continued development of the meristem into a flower. No flowers
developed without sucrose, irrespective of the irradiance. It is known that light
can promote flower formation, but this has usually been attributed to effects
54
Fig. 8. Hairy root induction of C. medium, infected with Agrobacterium rhizogenes AI3 by the
coculture method. (Photograph K. Ishimaru)
55
peR
analysis
tor detecting rol
and
GUS
genes
225 0 b
1 1 05b
M PC PC C C T T M
I
ra l
CUS
'I'
CUS
M
; 1 00 ba se I adder lIa rker
C - ro l ;Positive contr o l
PC - GUS;P osit i ve contro l
C - ro I
;Non -t ransforllant
C - GUS
; Non-transtorlla nt
T
;Haory roots
p
Fig. 9. peR analysis for detecting rol and GUS genes. (Photograph H. Tada)
Fresh hairy roots (ca. 200 mg) were inoculated into the three media (Fig.
10). The hairy roots were harvested weekly for 8 weeks and the dry weights
recorded. MS supported the best growth, mostly because the cultures started
to grow soon after transfer.
The dry weight of the roots rapidly increased until the end of the culture
time (8 weeks) to reach the maximum amount (562.3mg per flask) (Fig. 11).
In contrast, a lag time of more than 5 weeks was observed in the two other
media. The maximum root mass in B5 and WP, at week 8, were almost half of
that in MS. The formation of polyacetylenes in hairy roots is discussed in
Section 2.3.3.
2.2.4 Callus and Suspension Cultures
56
Fig. 10. C. medium hairy roots cultured in hormone-free MS (left) , B5 (center) , and WP (right)
liquid media for 4 weeks in the dark. (Photograph K. Ishimaru)
600
~ 500
- 0-
5...
400
.c
!)I)
.Qi
~
t'
MS
B5
WP
300
200
100
0
4 5
Weeks
57
58
Bisdeacylplatyconin
Violdelphin
ID~
~O
1D~~o
OIl
OIl
ID~-=-O
ID~O
OH
ID
Monodea:rlcampanin
1D~~o
OH
1D~~o
OIl
OIl
ID~-=-O
ID~O
OIl
ID
Fig. U. The major anthocyanins found in Campanula and the proposed biosynthetic sequence of
the three compounds found as major anthocyanins in wild-type flowers (cf. Table 1).
Bisdeacy1campanin is a minor component in petals of all genotypes analyzed, and is the major
anthocyanin in light-blue-flowering mutants of C. isophyUa and C. carpatica. Rubrocampanin has
been found only in red-flowering mutants of C. medium. (After Brandt et al. 1993, with kind
permission from Elsevier Science Ltd., The Boulevard, Langford Lane, Kindlington OXS 1GB,
UK; and Terahara et al. 1990)
acylated anthcyanins is relevant for the quality of pot plants: a pale color in the
new flowers formed after a pot plant is taken from the nursery to a shop or the
home of the customer is a well-known problem for retailers and growers of pot
plants. This is a question of both quantity and quality of the anthocyanins: not
only is the total concentration of anthocyanins in the flowers reduced, also the
proportion of bisdeacylplatyconin relative to the acylated anthocyanins is
59
Campanin
OH
Rubrocampanin
greater in plants grown with insufficient light (Brandt 1990). Experiments with
intact plants showed that while the total concentration of anthocyanin was the
same, flowers of type B (with the diacylated monodeacylcampanin as the end
product) accumulated less bisdeacylplatyconin during development and processed it faster into the acylated compounds than type C flowers (able to
accumulate the triacylated campanin) (Justesen et al. 1997). This indicates that
the relative composition of anthocyanin (conversion of one anthocyanin into
another) may be easier to manipulate than the total concentration (the
number of anthocyanin molecules produced).
Efforts to improve the quality require a thorough understanding of the
effects of insufficient light on anthocyanin biosynthesis. It is particularly important to understand if it is due to a direct influence of light on the developing
bud, e.g., on the induction of acylating enzymes, or if it is an indirect effect, due
to starvation for carbohydrates, the products of photosynthesis. The in vitro
flower bud cultivation system described above was established in order to
make this kind of investigation possible. The idea was to develop an experimental system where the effects on anthocyanin accumulation of light and of
availability of photosynthate could be varied independently.
60
~ 15,,~--.------.----~
'-'
'2
8 1O +----'~+---f------l
Type B
o TypeC
....n:l>.
0..
~ 5 +---~~-r--~
v
n:l
<J.)
'"0
Cfl
Q5
20
40
60
Fig. 14. In flowers of the chemotypes Band C, containing monodeacylcampanin and campanin as
the end product of biosynthesis,
respectively. (K. Brandt, unpub!.)
An experiment was done with genotypes of C. isophylla that were seedlings from the same cross, segregating for the genes that control the acylation
of monodeacylcampanin to campanin. The seedlings were classified according
to the presence or absence of campanin as major anthocyanin in flowers, into
the phenotypes type C and type B. (Fig. 12). One result of the first experiment
is shown in Figs. 13 and 14.
Both irradiance and sucrose availability influence the acylation of
the anthocyanins; however, to some extent they appear to be able to substitute
for each other: variation in irradiance has greatest effect when sucrose is low,
and vice versa. Sucrose concentrations of 4 and 2 % are not so low that
the effect of light can be explained simply as the result of photosynthesis.
Rather, the results indicate a general requirement for light, but that this
requirement disappears when the sucrose supply is superoptimal. Corresponding to the situation in vivo, the percentage of bisdeacylplatyconin in
61
I
I
Me-C=C-C=C-C=C-CH-CH-C=C-CH CH CH OH
I
I I
I
2
OH OR
lobetyol : R=-H
lobetyolin : R=-gIc
lobetyolinin : R=_glc 6 _lgIc
Fig. 15. Structures of polyacetylenes. (Tada et al. 1995a)
62
-0-
lobetyol
4.0
-0-
3.5
--_D
lobetyolin
lobetyolinin
3.5
- - lobetyolin
---fr- lobetyolinin
~3.0
.~ 2.5
~2.0
~1.5
e}'.
1.0
0.5
-=
O!)
1:
O!)
4.0
-0-
3.5
--
3.0
.~ 2.5
-0
2.0
~ 1.5
1.0
0.5
O~
lobetyol
345
Weeks
678
f.l
f.!
2
Z!
3
f.:-=f,l
J~!
Weeks
."7
"
8
lobetyol
3.0
.~ 2.5
C 2.0
-0
~ 1.5
e}'.
1.0
0.5
O+-~~~~~~~~~~~
234
Weeks
678
Fig. 16. Contents of polyacetylenes in hairy root cultures of C. medium cultured in different
media. (Tad a et al. 1995a)
63
tissue culture and the physiology of intact plants. The genus is relatively easy
to cultivate in vitro.
4 Protocol
4.1 Flower Bud Culture
1. Culture Initiation. From the generative part of a flowering stem. a 6-mm segment with a 1-2mm axillary bud is surface-sterilized with Korsolin or NaOCI solution in two separate steps.
alternating with trimming of the cut ends to a final length of about 2 mm. For each species, the
same salts, vitamins, and gelling agent as for micropropagation are used (Brandt 1997), and no
growth regulators. Photoperiod must be 16 h day-lor longer. For initiation of bud growth 2-3%
sucrose is optimal, but 4-6% sucrose results in a larger percentage of flowering explants.
2. Production of Flower Buds. Explants may be subcultured one or more times. Most primary
flowers are produced 6 to 10 weeks after culture initiation.
3. Anthocyanin Analysis. Flowers are extracted by homogenizing with 2ml cold MeOH-H 20HC0 2H (10:7: 3) plus 4ml cold MeOH-H20-HCO,H (10:3: 3) per g frozen plant material. HPLC
analysis is carried out on a RP-1S Licrospher column (4.6mm i.d. X 250mm) with solvent
A = H 2 0-HC0 2 H (19: 1), B = MeOH-H2 0-HOAc (10: 7: 3). The column is equilibrated with
solvent A for 10 min before injection of up to 100,u1 extract. Elution is on a gradient with 10% B
at 3 min, 25% B at Smin, 60% B at 22 min, and 100% B at 25 min, continuing for 10min. Column
temperature 35 DC. Anthocyanins are detected at 535 nm. R, (min): bisdeacylplatyconin to.S,
monodeacylcampanin 14.2, camp an in 17.S, and violdelphin IS.7.
2. Production of Hairy Roots. The bacteria are eliminated with 0.5 g 1-1 Claforan, the hairy roots
transferred to liquid MS without hormones (50ml per 100-ml Erlenmeyer flask) and maintained
in the dark on a rotary shaker (100 rpm). Transformation is confirmed by detection of NPT II and
GUS genes by PCR.
Transformed cultures are inoculated in hormone-free MS medium or other media and grown
as before for 4-S weeks (on MS 6-week growth periods give the highest yield per flask). At
subculture, fresh hairy roots (ca. 200 mg) are inoculated into, each flask.
3. Polyacetylene Analysis. Lyophilized hairy roots (20-30mg) were crushed by pestle and
extracted with MeOH (2ml) for I5h at room temperature. After filtration with MiIIipore filter,
the extract was injected (3 Ill) to HPLC: column, Inertsil ODS (4.6mm i.d. X 250mm); mobile
phase, MeCN-H 20 (1:4 ---7 9:1, linear gradient in 30 min); flow rate, 0.6Sml/min; detection
270 nm (UV); column temperature 40 DC; R, (min): lobetyolinin 15.9, lobetyolin 19.5, and lobetyol
23.9.
Acknowledgments. This work was supported in part by the Danish Agricultural and Veterinary
Research Council (grant no. 13-4559) The authors acknowledge the contributions from the
64
students H. Tada. H. Justesen and J.M. Lonka, and wish to thank K. Henriksen and M. Hansen for
expert technical assistance.
References
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66
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1 Introduction
Catharanthus rose us (family Apocynaceae) is grown as an ornamental plant in
many countries, although it originated from Madagascar. It is also known as
Madagascar periwinkle or Cape periwinkle. This plant was used traditionally
as a crude medicine for diabetes and other ailments. It has also been used as
a substitute for hops in brewing beer. Now, however, C. roseus is most useful
as a source of various alkaloids; approximately 90 indole alkaloids have been
isolated from it, the most valuable being the dimeric alkaloids vinblastine and
vincristine, which show antitumor activity. They are very similar in chemical
structure, but their activity spectra and side effects are extremely different:
vinblastine is effective against Hodgkin's disease, choriocarcinoma, and the
like, while vincristine is mainly employed to treat childhood acute leukemia.
Vinblastin shows bone marrow toxicity, whereas vincristine is toxic to the
nervous system. Due to the very low yields of these dime ric indole alkaloids in
the plant (approx. 0.0005%), attempts have been made to produce alkaloid
and other secondary metabolites in cell and tissue cultures. General reviews of
work in this field have been published (see Heijden et al. 1989; David and
Tempe 1993; Hirata et al. 1994; Sakurai and Fujioka 1996).
In this chapter, attention is focused on another attribute of C. roseus cells
in culture, i.e., the ability to biotransform exogenously added hydroquinone
(HQ) to arbutin. We have extensively examined the feasibility of efficient
production of arbutin using C. roseus cell culture (Yokoyama et al. 1990, 1996;
Inomata et al. 1991).
Plant cell cultures catalyze a wide range of biotransformations, such as
glucosylation, glucosyl esterification, hydroxylation, oxidation-reduction
between alcohols and ketones, reduction of carbon double bonds (C=C),
hydrolysis, isomerization, epoxidation, dehydrogenation, methylation,
demethylation, and others (Furuya 1988; Suga and Hirata 1990; Yokoyama
1996). However, major biotransformations by plant cell cultures are
hydroxylation and glucose conjugation involving glucosylation and glucosyl
esterification; these reactions are the subject of 45 and 41 % of published
Pharmaco Science Research Laboratories, Shiseido Research Center, 1050 Nippa-cho, Kohokuku, Yokohama 223, Japan
68
M. Yokoyama
OH
OH
Product
Substrate
Type of
reaction
Plant species
Yield
(gil)
Reference
Arbutin
Hydroquinone
Glucosylation
Rauwolfia
serpentina
18
Catharanthus
roseus
Rauwolfia
serpentina
Peganum
harmala
Datura
innoxia
9.2
Lutterbach and
StOckigt
(1992)
Inomata et al.
(1991)
Lutterbach et
al. (1993)
Courtois et al.
(1988)
Umetani et al.
(1982)
p-Hydroxyphenyl- Hydroquinone
O-primeveroside
Serotonin
Tryptamine
Hydroxylation
U mbelliferone
Glucosylation
Skimmin
Glucosylation
5.8
2.5
1.6
69
70
M. Yokoyama
B
Fig.2A.B. Catharanthus roseus cells cultured for 4 days. A CrA strain; B CrB strain. (Inomata et
al. 1994)
71
30
T
11--------....
T
>,
"'-'
'5
'';::;
3:
ra
"-
Q)
OJ
"~1
Q)
(f)
,,
,,
20
,,
l-
(f)
"-
;///1
30
"'-'
.L
"'-'
:;:
I-
"OJ
20
"-
OJ
:::'
:t:: x
>, E
0. 10
0
10
'';::;
::J
(f)
U
::J
-e
<t
OJ
cJ
25
50
75
100
sucrose
- 15
101-
- 10
C
:J
:J
a 10 -
.0
.0
en
glucose
/0
0 a
15
10
L-
en
/0
o~~~~~~~~o
01234567
Sugar added ("10)
Fig. 4. Effects of sucrose and glucose on the production of arbutin in cell culture of Catharanthus roseus.
HQ was added to a final concentration of 4mM on
the 7th, 10th, and 11th days of CrA-suspension culture, and production of arbutin was evaluated on the
14th day. Sugars at the indicated concentrations were
supplied once only, on the 7th day. (Yokoyama et al.
1990)
M. Yokoyama
72
0-
::J
QJ
01
'c
'0
E
QJ
'-
'-
d
01
::J
if)
7
6
5
I.;
3
2
1
0
7
6
5
4
G1Ucose/~,:::
/;/
..... _._.-.e---.-.-._.-. _.
234567
Sugar added (%)
73
150
,;a
~
....u
~
t:
'Cl
100
\'1
ell
=
=
.t::J.
:0
50
'"
c<l
ell
10
Days
Fig. 7. Change in the ability to produce arbutin during the culture of Catharanthus roseus (CrA).
Cells from early log phase to stationary phase were collected on a 148-mm nylon mesh. Aliquots
of cells (fresh wt.) were suspended in 20ml offresh medium (e) or the original medium (0) from
which the cells had been removed. Every day, 0.2ml of200mM hydroquinone was added until the
cells died. After incubation for the biotransformation, the cell suspensions were homogenized and
arbutin in the supernatant after centrifugation was analyzed by HPLC. Each value represents an
average of three determinations, with SD. (Yokoyama et al. 1996)
stages, but in the original medium they cannot manifest their full ability owing
to an insufficient amount of some component(s) in the medium in the later
stages.
Therefore, the amounts of UDPG were assayed in cells from 3-day and 6day cultures. The control level, before HQ addition, was very high in 3-day
74
M. Yokoyama
6'2-
~ 100
'"
~
0
q
0
50
Ec;:'"
C3
~
Before HQ
addition
16
18
20
22
24
26
16
18
20
22
24
26
150
B
6'2~
~
C3
100
q
0
<.J
;>
50
.....c;:
C)
Bcfore HQ
addition
Fig. SA.B. Change in the UDPG content in cells which had been cultured for 3 (A) or 6 (B) days.
Catharanthus roseus cells were collected after culture for 3 or 6 days and resuspended in fresh
medium by the same method as mentioned in the legend to Fig. 7. HQ was added at 1 mM. UDPG
content in the cells was measured before and at various times after addition of HQ (e). Closed
columns at the left represent original UDPG content in 3-day (A) or 6-day (B) cells before
addition of HQ. UDPG content is represented relative to the original content in 3-day cells, taken
as 100%. (Yokoyama et al. 1996)
cells while in 6-day cells UDPG was hardly detected (Fig. 8), in accordance
with a previous report (Sasamoto and Ashihara 1988). This suggests that
younger cells require more UDPG, presumably for synthesizing the cell wall.
The control level of UDPG does not necessarily reflect the amount which is
available for bioconversion to arbutin. Added HQ may induce new UDPG
formation. Therefore, we assayed the amount of UDPG just after added HQ
(1 mM) had been completely consumed. That amount was expected to reflect
not only the control level, but also the level induced by adding HQ. Figure 8
shows that UDPG was clearly induced in the 6-day cells; the total supplied
amount was larger in 6-day cells than in 3-day cells. In 3-day cells, the supplied
UDPG may be used competitively both for the glucosylation of hydro quinone
75
and for synthesizing cell wall polysaccharides. On the other hand, the activity
for glucosylation of hydro quinone was essentially the same in cells from 3-day,
6-day, and 8-day cultures (data not shown).
The cell volume of erA was estimated to be 11.3mm3 X 10-6 for 3-day
cells and 16.1 mm 3 X 10-6 for 8-day cells. The increase is considered to reflect
the increase in vacuole volume. It is concluded that older cells of strain A
can accumulate arbutin in larger amounts owing to their greater vacuole
volume.
In conclusion, older cells have a greater ability to produce arbutin by
biotransformation because of the larger amount of induced UDPG which is
available for biotransformation and the greater vacuole in which produced
arbutin may be accumulated.
2.4 High-Level Production of Arbutin
76
M. Yokoyama
abc
6g
~10
~
4~
~
.i6
g~4
.c
2 'E
;;l
o~2
x .~
.J-J
B
."
o ~0. 90 a
~ ~ 'E60
e~
<
~
oo'----'---L-J'-'--6-~...........L.....J....---'--~.........
20 1 2340 1 2345
~30
~ ~ - 0r-----+-----------~--------~ 10
1
2
4~
.ie 6
CJ
;;l
~
c
2~
8114
a ~2
~~
OL.....J.....L.....J..--L..---'--'----'--.l.......L-L..L-4---+-4<==L.....LJO
0 2 0 1 234 50 1 234
Days after the start of HQ addition
Fig. 9A.B. Effects of intermittent or continuous supply of HQ on arbutin yield. CrB strain of
Catharanthus roseus was used in these experiments. HQ was added repeatedly (A) or continuously (B) to a jar fermenter as illustrated in the figure, after the cell density had increased to over
250 g fresh weight/I. Glucose was added twice to a concentration of 55 mM, at the starting point of
HQ addition and on the second day thereafter. (Inomata et al. 1991)
(9.1 g/I) and per cell fresh weight (34 mg/g fr. wt,), were essentially the same as
in the 5-1 jar fermenter.
3 Summary
The results of the biotransformation of HQ to arbutin by C. roseus cell culture
may be summarized as follows.
1. Selection of a superior cell strain was important for efficient production of
arbutin. The cell line CrB, with well developed vacuoles, produced much
more arbutin than CrA.
2. Sugars improved the cell viability. The production of arbutin was
greatly enhanced by sucrose or glucose at concentrations of up to 6%, with
77
4 Prospects
Arbutin production using C. roseus cell culture has the advantage that
there is no significant competing reaction; the conversion ratio from added
HQ to arbutin was 98%. It may be pointed out that our process is a direct
conversion of HQ and not a de novo synthesis from remote precursors. We
have developed a simple method of purification of arbutin that includes
the use of C l8 columns and recrystallization from organic solvents as the
main steps. The total period of culture is around 18 days; 2 weeks for highdensity cell culture of competent cells, and 3 or 4 days for the biotransformation; this period is relatively short for a plant cell culture process. Pure
arbutin could be produced at a cost of $300-$1000/kg by this method. The cost
range is large because the cost would depend on whether or not
the construction of new facilities for cell culture or for purification of arbutin
is necessary.
On the other hand, glucosylation of HQ by chemical means is not a simple
task; it requires several steps, including blocking the hydroxyl moieties of
glucose via acetylation, conjugation of such OH-protected glucose to HQ, and
saponification for deacetylation. The conjugating reaction requires a high
temperature. The cost is in the same range as that of the biotransformation.
Thus, production of arbutin by biotransformation could substitute for the
chemical process.
5 Protocol
5.1 Cell Culture
Cell suspensions of C. raseus (L.) G. Don cell strain A (CrA) and strain B (CrB), which had been
established at Tohoku University, were subcultured weekly in LS liquid medium (Linsmaier and
Skoog 1965), supplemented with 30g/J of sucrose and 2.2.uM 2,4-dichlorophenoxyacetic acid. Jar
fermenter culture was performed with 5-1 or 20-1 vessels equipped with modified paddle-type
impellers (Tanaka 1982) and spargers of porous sintered metal (40.urn).
78
M. Yokoyama
79
References
Akiu S, Suzuki Y, Fujinuma Y, Asahara T, Fukuda M (1988) Inhibitory effect of arbutin on
melanogenesis: biochemical study in cultured B 16 melanoma cells and effect on the UVinduced pigmentation in human skin. Proc Jpn Soc DermatoI12:138-139
Ashihara H, Mitsui K, Ukaji J (1987) A simple analysis of purine and pyrimidine nucleotides in
plant cells by high-performance liquid chromatography. Z Naturforsch 42c:297-299
Beauchamp C, Fridovich I (1970) A mechanism for the production of ethylene from methional.
The generation of the hydroxyl radical by xanthine oxidase. J Bioi Chern 25:4641-4646
Courtois D, Yvernel D, Florin B, Petiard V (1988) Conversion of tryptamine to serotonin by cell
suspension cultures of Peganum harmala. Phytochemistry 27:3137-3142
David C, Tempe J (1993) Transformation in Catharanthus species (Madagascar periwinkle). In:
Bajaj YPS (ed) Biotechnology in agriculture and forestry, vol 22. Plant protoplasts and genetic
engineering III. Springer, Berlin Heidelberg New York, pp 144-156
Furuya T (198g) Production of useful compounds by plant cell cultures - de novo synthesis and
biotransformation. J Pharm Soc Jpn (Yakugaku Zasshi) 108:675-696
Hassan HM, Fridovich I (1979) Intracellular production of superoxide radical and hydrogen
peroxide by redox active compounds. Arch Biochem Biophys 196:385-395
Heijden Rvd, Verpoorte R, Hoopen HJGT (1989) Cell and tissue cultures of Catharanthus roseus
(L.) G. Don: a literature survey. Plant Cell Tissue Organ Cult 18:231-280
Hirata K, Miyamoto K, Miura Y (1994) Catharanthus roseus L. (Periwinkle): Production of
vindoline and catharanthine in multiple shoot cultures. In: Bajaj YPS (ed) Biotechnology in
agriculture and forestry, vol 26. Medicinal and aromatic plants VI. Springer, Berlin Heidelberg
New York, pp 46-55
Inomata S, Yokoyama M, Seto S, Yanagi M (1991) High-level production of arbutin from
hydroquinone in suspension cultures of Catharanthus roseus plant cells. Appl Microbiol
BiotechnoI36:315-319
Inomata S, Yokoyama M, Wachi Y (1994) Differences in glucosylation of exogenous
hydroquinone by two morphologically different lines of Catharanthus roseus cells. Plant Tissue
Cult Lett 11:211-217
Itabashi M, Aihara H, Inoue T, Yamate J, Sanni S, Tajima M, Tanaka C, Wakisaka Y (1988)
Reproduction study of arbutin in rats by subcutaneous adiministration. Iyakuhinn Kenkyu
19:282-297
Kellogg EW, Fridovich I (1975) Superoxide, hydrogen peroxide, and singlet oxygen in lipid
peroxidation by a xanthinbe oxidase system. J Bioi Chern 250:8812-8817
Linsmaier EM, Skoog F (1965) Organic growth factor requirements of tobacco tissue cultures.
Physiol Plant 18:100-127
Lutterbach R, SWckigt J (1992) High-yield formation of arbutin from hydro quinone by cellsuspension cultures of Rauwolfia serpentina. Helv Chim Acta 75:2009
Lutterbach R, SWckigt J, Kolshorn H (1993) p-Hydroxyphenyl-O-f3-D-primeveroside, a novel
glycoside formed from hydroquinone by cell suspension cultrues of Rauwolfia serpentina. J Nat
Prod 56:1421
Parr AJ (1989) The production of secondary metabolites by plant cell cultures. J BiotechnoI1O:126
Petersen M, Seitz HU (1985) Cytochrome p-450 dependent digitoxin 12 j3-hydroxylase from cell
cultures of Digitalis lanata. FEBS Lett 188:11
Sakurai A, Fujioka S (1996) Catharanthus roseus (Vinca rosea): In vitro production of
brassinosteroids. In: Bajaj YPS (ed) Biotechnology in agriculture and forestry, vol 37. Medicinal and aromatic plants IX. Springer, Berlin Heidelberg New York pp 87-96
Sasamoto H, Ashihara H (1988) Biosynthesis of pyrimidine nucleotides and arginine in a suspension culture of Catharanthus roseus. Int J Biochem 20:87-92
Suga T, Hirata T (1990) Biotrasformation of exogenous substrates by plant cell cultures.
Phytochemistry 29:2393
Suzuki K, Amino S, Takeuchi Y, Komamine A (1990) Differences in the composition of the cell
walls of two morphologically different lines of suspension-cultured Catharanthus roseus cells.
Plant Cell Physiol 31:7-14
80
1 Introduction
1.1 The Plant
Centella asiatica (L.) Urban, synonym C. coriacea Nannfd, previously also
named Hydrocotyle asiatica L. or H. lunata Lam, of the family Apiaceae, has
numerous common names in various languages: pennywort, marsh
pepperwort, Indian waternavelwort (English); asiatisches Wassernabelkraut
(German); b6vilaque, coquelariat, violette marron (French); gotu kola (SriLanka); brahmi, brahmanduki, karivana, mandookaparni, babassa, thankuni,
vellari, vallarai (Indian); talapetraka, anamanitra, korokorona, silabola
(Malagasy); bodila-ba-dinku, tabao en Amhara (African); luo de da, ji xue cao
(Chinese). The large number of native names, especially in India, also shows
its popularity in the Ayurvedic system of medicine (Dandouau 1910; Boiteau
and Chanez 1967).
C. asiatica (Figs. 1,2) is a slender tropical herbaceous plant with crawling
stems, propagating vegetatively by runners (stolons), with entire kidneyshaped leaves (1-3 cm long; 2-4 cm wide) bearing a crenate margin at the tip of
long petioles (5 to 10 times the leaf length). In sunny places, the petioles are
shorter (only 2x the leaf), and petioles and leaves become red due to important anthocyane production. Leaves and short peduncled (2-4-cm) inflorescences arise from a rosette near the ground. The umbels are reduced and
contain mostly two to four white or pink-purple uniform small flowers (2mm)
consisting of five petals, five free stamens, a greatly reduced calyx, an inferior
ovary with two carpels and a stylopodium supporting two styles. The fruit is a
dry, flattened schizocarp with two single-seeded mericarps each with prominent ridges (Boiteau and Ratsimamanga 1956; Chadefaud and Emberger 1960;
Debray et al. 1971; Medicinal Plants of India 1976).
C. asiatica generally grows in damp, shadowed, or swampy areas, but also
in savanna and secondary forest clearings under warm climates of both the
northern and southern hemispheres. It is native to Asia and mainly found in
82
India, Pakistan, and Madagascar; but the plant also grows in tropical and
equatorial Africa, America, and the tropical regions of the New World.
Three varieties are described in relation to geographic origin which are
correlated with some leaf morphology and chemical composition.
- C. asiatica L. var. typica has weakly hairy, typically kidney-shaped leaves
with well-crenulated margins, and is !ound in southern Asia, as far as Madagascar. All references in traditional medicinal concern this variety.
83
gins, quite hairy leaves, and is present in tropical and equatorial Africa.
- C. asiatica L. var. floridana with leaves longer than wide in shape, is found
The drug is used in both Africa and Asia as a remedy for leprosy and other
skin diseases (Boiteau and Ratsimamanga 1956; Oliver-Bever 1986). Some
tribes use the leaves as a vegetable (in curries or salads). In Chinese traditional
medicine, the plant is used as an antipyretic, detoxicant (blood purifier), and
diuretic. It has also been widely sold as a body strengthener and revitalizer that
can promote longevity. High doses may have sedative or narcotic effects
(Tyler et al. 1988). Triterpenic titrated extract (in French ETCA) has shown
excellent tolerance to short-as well as long-term applications (localized or
administered generally). The drug is also a weak sensitizer (Hausen 1993). In
European pharmacopoeia, indications are cutaneous affections, skin wounds
such as chapping, grazing, insect stings, sun burn, and other light, small burns
(Dalloz-Bourguignon 1975; Poizot and Dumez 1978; Fleurentin 1991), leprous
and other ulcers (Louvet et al. 1976; Babu et al. 1995), and cicatrization after
surgery in gynecology (Basset and Eberst 1980), ophtalmology (cornea lesions), or ORL (eardrum lesions)(Leguay and Greco 1979; Vogel et al. 1990).
It is also indicated in phlebology in the case of veinous diseases (Maggiori
1992). In some recent work, an antiulcer activity of an ethanol extract of C.
asiatica roots has been reported, without establishing any chemical identity
(Sarma et al. 1995).
Several saponins containing the ursane ring system have been isolated
in Centella asiatica. Asiatic acid, made cas sic acid, madasiatic acid, and
asiaticoside are the major terpenoids found in the typica variety. Asiatic acid
(C30H4s0s) is the 2,3,23-trihydroxyurs-12-en-28-oic acid (Fig. 3; Boiteau et al.
1949). Madecassic acid is the 6-hydroxylated asiatic acid (Pinhas et al. 1967).
Asiaticoside (Bontems 1941; Boiteau et al. 1949) is trisaccharide [O-a-Lrhamnopyranosyl (1 ~4) O-fJ- D-glucopyranosyl (1 ~6)] 0-[3-D-glucopyranose
ester of asiatic acid (Polonsky 1949, 1952). Other triterpenes have also
been described, such as madecassoside (the same trisaccharide as in
asiaticoside esterifying madecassic acid), madasiatic acid (a 2,3,6trihydroxylated isomer; Boiteau and Chanez 1967; Pinhas 1969), brahmic acid
(a 2,3,6,23-tetrahydroxy isomer of madecassic acid), brahmoside, brahminoside (respectively a glycoside and an ester glycoside of brahmic acid both
with a rhamnosyl glucosyl arabinosyl-trisaccharide; Rastogi et al. 1960;
Rastogi and Dhar 1963), thankuniside, isothankunic acid, isothankuniside
84
R1
R2
R3
CH20H
Madecassic acid
OH
CH20H
Madasiatic acid
OH
CH3
CH20H
glc-glc-rhm
CH20H
glc-glc-rhm
Asiatic acid
Asiaticoside
Madecassoside
-~~-~---
--~-,
OH
. ._---------------,._.
--.~
----------
85
(Weber 1992). Such a role is also proposed for some triterpene derivatives of
licorice root, i.e., glycyrrhetinic acid (Amagaya et al. 1984) and carbenoxolone
(Sloan and Weaver 1968). The pathogenetic mechanism responsible for these
properties and the signal transduction pathway involved in the response remain to be elucidated.
2 In Vitro Approaches
2.1 Tissue and Cell Cultures
Centella asiatica callus and cell suspension cultures were initiated and further
subcultured in Professor Guignard's laboratory (Bister-Miel 1987). To our
knowledge, it is the only cell suspension of this plant still existing. In the past,
some cultures were initiated in Professor Henry's laboratory (Faculty of Pharmacy, Toulouse, France); however they did not provide enough saponin derivatives for industrial application (pers. comm.).
Cell suspensions were obtained after two steps: first, callus cultures initiation on solid medium and then tissue transfer to liquid medium. Seeds were
aseptically germinated and plantlet (stem) fragments were cut into small
Table 1. Centella asiatica callus initiation, callus, and cell suspension growing media
Kinetin
6-Benzyl-aminopurine
86
~-------I
;;:;
eIJ
.~
:::
....
I....
""
"" "
"
InoLululll
"
..c
"
(~/l)
DW Othday)
Dilution at
Week I: lI2
Week 2: lO/66
Week 3-4: 10/41
Week 5-6: 10150
Week 7-11: lI8
Week 12 ->: 1111
"""" "
O+-~-'~--'-~'---~----r---~
10
15
20
Age (weeks)
25
30
DW S7rpm
---0---
10
-eL
:c
,.;:;
eL
....
8
6
,;
~
::::;
10
12
14
16
18
20
Age (days)
pieces and placed on modified Nitsch and Nitsch medium (1956) (Table la).
Callus was first subcultured every 4 to 6 weeks on modified MS medium
(Murashige and Skoog 1962) (Table Ib) containing adenine, kinetin, 2,4-D,
and glucose. Insufficient growth led to modification of the 2,4-D concentration
and replacement of kinetin by 6-benzyl-aminopurine (Table lc).
One to 3 months after initiation, when callus had grown enough, it was
transferred to liquid growing medium, where it dissociated spontaneously and
easily, and subcultured on modified MS medium (Table Id). Optimized
growth conditions were rapidly obtained (12 weeks) with appropriate dilution
rate (Fig. 4). The suspension cultures are then maintained by subculturing
87
CHjO
------~
"-""-,
/-:--.,
I'
OCH]
Papaverine
CH10/
~ ~ /~
~
'y
)
O/~(~
-"'::i
~;::J~OCH
'~Cfy ~"
~
I
.....
OCH 3
OCH 3
Pa pa veraldine
88
100
~
"'e'"'
::
a'"'
80
.......
0
It<
60
40
:c
:::
20
101)
.~
.......
200
400
600
800
89
RIO Rp
4
~
;/'
NHCOCH 3
20--..
I
OCH 3
~8
~
II
10
R1
R2
thiocolchicine
CH3
CH3
2-demethylthiocolchicine
CH3
3-demethylthiocolchicine
CH3
2-0-glucosylthiocolchicine
CH3
glucose
3-0-glucosylthiocolchicine
glucose
CH3
(= thiocolchicoside)
Fig. 8. Structures of thiocolchicine, de methylated products (2- and 3-demethylthiocolchicine),
and glucosylated products (2- and 3-0-glucosylthiocolchicine). (Solet 1993)
90
91
14
12
10
~
EIOL
::::
Ii
....
.:ji
....
2
0
200
400
600
800
1000
10
::t
'":l
"0
e
Co
"0
OJ
';
...
..c:
'"
0
0
-'
.'i
10 II
12
92
~
2-
'"
Q,I
<Il
...0
,.-=
'-'
2
I
,./
0
0
10
II
12
glucosylated products. The direct addition to cell suspension culture of 3-0demethylthiocolchicine confirmed the capacity of these cultures to glucosylate
this intermediate into thiocolchicoside.
Demethylation and glucosylation activity of thiocQlchicine detected in cell
suspension led us to study the enzymes involved. First, crude extract of 14-dayold C. asiatica cells was incubated with thiocolchicine. Formation of 3demethylthiocolchicine (2.5 nmollh/g moist cells) was observed. The boiled
extract did not allow the appearance of metabolite. When NADPH, FAD, and
FMN were added as cofactors to the crude extract, demethylation reached
10nmol/h/g moist cells (8% of added substrate). Neither NADPH alone, nor
FAD and FMN together allowed de methylation. The low activities of all
tested subcellular fractions were probably due to the low abundance of
enzyme in cell suspension. We also noted an increase in microsome bulk
(100000 g pellet) in cells previously incubated with thiocolchicine as compared
to the control. In these subcellular fractions, spectrophotometric measurements showed similar amounts of cytochrome P-450 and P-420 (250-300pmoll
mg microsomal protein). According to subcellular localization and cofactor
requirements, the de methylating enzyme may belong to the cytochrome P-450
monoxigenase family. Research is in progress to identify the oxidizing enzymes which are involved in the oxidative process of papaverine and
thiocolchicine.
Low activity of glucosylation of 3-0-demethylthiocolchicine was detected
in the crude cell suspension extract. Glucosyltransferase was partially purified
by ammonium sulfate fractionation (30-70% salt saturation) followed by gel
filtration on a Sephadex G-lOO column. Active fractions were pooled and
chromatographed on a DEAE Trisacryl column. This protocol resulted in an
increase in the specific activity of the glucosyltransferase by 690-fold as compared with the crude extract (Solet 1993).
93
References
Amagaya S, Sugishita E, Ogihara Y, Ogawa S, Okada K, Aizawa T (1984) Comparative studies of
the stereoisomers of glycyrrhetinic acid on anti-inflammatory activities. J Pharmacobio-Dyn 7
(12) :923-928
94
Babu TD, Kuttan G, Padikkala J (1995) Cytotoxic and antitumour properties of certain taxa of
Umbellliferae with special reference to Centella asiatica (L.) Urban. J EthnopharmacoI48:5357
Basset A, Eberst E (1980) Action therapeutique de I'extrait titre de Centella asiatica sur Ie lichen
sclero-atrophique cutaneo-genital. Gaz Med Fr 87 (13):1621-1623
Bellet P (1952) Le colchicoside. Ann Pharm Fr 10:81-88
Bhattacharyya SC (1956a) Constituents of Centella asiatica. Part I. Examination of the Ceylanese
variety. J Indian Chern Soc 33 (8):579-586
Bhattacharyya SC (1956b) Constituents of Centella asiatica. Part II. Structure of the triterpene
acids. J Indian Chern Soc 33 (9):630-634
Bhattacharyya SC (1956c) Constituents of Centella asiatica. Part III. Examination of the Indian
variety. J Indian Chern Soc 33 (12):893-898
Bister-Miel F (1987) Biotransformation de la papaverine, isopapaverine et de leurs analogues
par des suspensions cellulaires vegetales non productrices d'alcaloldes. These Doct, Univ
Paris-Sud
Bister-Miel F, Agier C, Bury M, Viel C, Guignard JL (1988) Biotransformations comparees de
benzylisoquinoleines par des suspensions cellulaires vegetales non productrices d'alcaloldes.
Bull Soc Bot Fr 135 (1):57-70
Boiteau P, Chanez M (1967) Isolement d'un nouvel acide triterpenique de Centella asiatica
L. (Urb) de Madagascar: I'acide madecassique. CR Acad Sci Paris SerD 264 (2):407410
Boiteau P, Ratsimamanga AR (1956) L'asiaticoside extrait de Centella asiatica et ses emplois
therapeutiques dans la cicatrisation des plaies experimentales et rebelles (lepre, tuberculose
cutanee et lupus). Therapie 11 (1):125-149
Boiteau P, Buzas A, Lederer E, Polonsky J (1949) Derivatives of Centella asiatica used against
leprosy: chemical constitution of asiaticoside. Nature 163:258
Bonte F, Dumas M, Chaudagne C, Meybeck A (1994) Influence of asiatic acid, madecassic acid
and asiaticoside on human collagen I synthesis. Planta Med 60:133-135
Bonte F, Dumas M, Chaudagne C, Meybeck A (1995) Activite comparee de l'asiaticoside et du
madecassoside sur la synthese des collagenes I et III par des fibroblastes humains en culture.
Ann Pharm Fr 53 (1):38-42
Bontems JE (1941) Sur un heteroside nouveau, l'asiaticoside isole a partir de I'Hydrocotyle
asiatica L. (Ombelliferes). Bull Sci PharmacoI49:186-191
Chadefaud M, Emberger L (1960) Traite de botanique systematique tome II. Les vegetaux
vasculaires. Masson et Cie, Paris
Christinaki H, Bister-Miel F, Hammoumi A, Bury M, Guignard JL, Viel C (1987) Identification of
papaverine metabolites after biotransformation by Silene alba cell suspensions.
Phytochemistry 23:2991-2994
Dalloz-Bourguignon A (1975) Etude de I'action de I'extrait titre de Centella asiatica. Gaz Med Fr
82 (38):4579-4583
Dandouau A (1910) Catalogue alphabetique des noms malgaches des vegetaux de Tananarive.
Imprimerie officielle, Bull Ecom Madagascar, 2eme annee
Debray M, Jacquemin H, Razafindrambao R (1971) Contribution a I'inventaire des plantes
medicinales de Madagascar. Trav Doc Orstom 8:1-149
Diamond J (1978) Role of cyclic nucleotides in control of smooth muscle contraction. Adv Cyclic
Nucleiotide Res 9:327-340
Dorisse P, Gleye J, Loiseau P, Puig P, Edy AM, Henry M (1988) Papaverine biotransformation in
plant cell culture. J Nat Prod 51:532-536
Dutta T, Basu UP (1962) Triterpenoids. Part I. Thankuniside and isothankunic acid. A new
triterpene glycoside and acid from Centella asiatica Linn (Urb). J Sci Ind Res 21 (B)5:239240
Dutta T, Basu UP (1968) Terpenoids: Part III - Isolation of isothankuniside and constitution of
isothankunic acid from Centella asiatica Linn (Urb). Indian J Chern 6 (4):543-545
Fleurentin J (1991) Les pi antes medicinales de la pharmacopee franc;aise, Encycl Med Nat Editions techniques, Paris, Phytotherapie, Aromatherapie, D, pp 1-10
Hausen BM (1993) Centella asiatica (Indian pennywort), an effective therapeutic but weak
sensitiver. Contact Dermatitis 29 (4):175-179
95
96
1 Introduction
1.1 Botanical Aspects
The name Chenopodium is derived from the Greek words chenos (goose) and
podos (foot), because the leaves often resemble goose feet. This genus consists
of ca. 120 species, widely distributed over the world, 45 of which have been
described in India.
The family Chenopodiaceae comprises annual plants, with greenish or
reddish leaves, which may be glabrous or hairy, but often floury (Loste 1937).
Several species are known to possess medicinal properties, such as e.
ambrosioides and e. anthelminticum, used as antihelmintic, and which contain
some triterpenoid saponins and flavonol glycosides (Chirva et al. 1971;
Bogacheva et al. 1972; Jain et al. 1990). e. quinoa, growing on Peruvian
tablelands and cultivated by the ancient Incas, also contains flavonol
glycosides, which contribute to its unpleasant taste (De Simone et al. 1990).
However, the seeds are widely used by Andine people in cakes, bread, or
drinks, and are also given to animals, because of their high protein content and
good nutritional value: in the grain crop proteins represent about 20% of the
dry weight (Weber 1978). Several other members of this family have been or
are still used for alimentary purposes: in particular, Beta vulgaris (beetroot)
and Spinacia oleracea (spinach) belong to this group.
e. album, also named fat hen, a common species growing in various parts
of the world, is also used as a leaf vegetable. This plant has received much
attention due to its high protein content in seeds and has been selected for
commercial production (Weber 1978). Moreover, the ability of e. album to fix
nitrogen by association with nitrogen-fixing bacteria in its roots seems interesting (Mukherjee et al. 1985). The plant is medium-sized (Fig. 1) and our
specimens were grown in pots in a greenhouse with a 16-h light period, at 18C
during the night and 25 C during the day.
98
Because of their nutritional value, the Chenopodium species have been extensively cultured in vitro, in order to select clones and to allow their maintenance
for breeding purposes (Conger 1978). As it appeared interesting to eliminate
the saponins (triterpenic glycosides) responsible for the bitter taste in C.
quinoa seeds, the saponin content of callus and bud cultures has been investigated: sapogenins, such as oleanolic acic, were found in in vitro cultures, as in
roots and in fruits (Burnouf-Radosevich and Paupardin 1983).
To stimulate the production of multiple shoots by in vitro vegetative
propagation, different media have been tested, and the best result was obtained with modified B5 medium (Gamborg et al. 1968), decreasing sucrose,
and increasing nitrate and phosphate salts (Burnouf-Radosevich and
Paupardin 1985).
It has also been possible to establish photo autotrophic and heterotrophic
cell suspension culture (Husemann and Barz 1977; Husemann et al. 1980).
Heterotrophic callus cultures were obtained on MS medium (Murashige and
Skoog 1962) supplemented with 2% sucrose in the dark. In order to establish
green phototrophic cell suspensions, heterotrophic calli were kept on the same
medium under continuous light, and the green mixotrophic calli obtained were
transferred into sugar-free MS medium with CO 2 as carbon source. Such
cultures allowed the comparison of lipid composition: photo autotrophic cell
99
In Angiosperms, 24-alkylsterols with 5(6)-unsaturation are generally the major 4-desmethylsterols (Benveniste 1986). However, numerous species have
been reported to deviate from this main line. In particular, Caryophyllalles
(consisting of 12 families with approximatively 10000 species, including
Chenopodiaceae), often synthetize a mixture of L17 -sterols and L1 5-sterols (e.g.,
in Chenopodiaceae; Xu et al. 1990; Salt et al. 1991; Corio-Costet et al. 1993a).
A likely role for such phytosterols is to represent the precursors of particularly
interesting steroids, the ecdysteroids, which have been frequently described in
Caryophyllalles (reviews in Horn and Bergamasco 1985; Lafont and Horn
1989; Camps 1991) and which, in particular, are present in high concentrations
in many species of the Chenopodiaceae family (Toth et al. 1981; Bathory et al.
1982; Dinan et al. 1991; Grebebok and Adler 1991; Grebebok et al. 1991;
Dinan 1992a,b; Corio-Costet et al. 1993a). Initially identified as the molting
hormones of arthropods, ecdysteroids have also been found in numerous plant
species, where they are generally supposed to playa defensive role against
phytophagous invertebrates. Other possible roles for phytoecdysteroids in
plant physiology still remain conjectural (Bergamasco and Horn 1983; Lafont
et al. 1991), but these molecules, which may be relatively abundant in our food,
also have interesting pharmacological effects on mammals (reviews in Simon
and KooIman 1989; Camps 1991).
The in vitro techniques applied to various plant systems producing
ecdysteroids have been considered by several investigators as potential tools
for studying the biosynthesis of these molecules and their possible significance
in plant physiology (e.g., Hikino et al. 1971; McMorris and Voeller 1971;
Ravishankar and Mehta 1979; Camps et al. 1990; Vanek et al. 1990; CorioCostet et al. 1993a,b; Tomas et al. 1993; Svatos and Macek 1994).
We have thus explored in C. album the ability to carry out the production
of such sterols and phytoecdysteroids in vitro, in order to obtain a possible tool
for further studies on the biosynthesis, and the roles of these molecules.
The seeds, after appropriate surface sterilization (70% ethanol for 5 min; 5 %
calcium hypochlorite for 20min and, finally, two washes in sterile distilled
water), were sown in glass tubes on a solid medium containing half-strength
100
MS macro- and micronutrients (Murashige and Skoog 1962) and 0.7% agar,
without hormones, and kept at 25C under continuous light (20.uEm-2s-1).
Hypocotyl segments, leaflet pieces, and shoot apices, taken from seedlings as
explant sources, were placed in Petri dishes containing MS medium with 2,4dichlorophenoxyacetic acid (2,4-D, O.OSmg/l) and kinetin (0.05mg/l) until callus induction. Calli were then transferred into 100-ml Erlenmeyer flasks
containing liquid MS medium (without agar, pH 5.9, with 2,4-D 0.165mg/l and
kinetin 0.107mg/l), incubated on a shaker (ca. 120 rpm) under similar light and
temperature conditions, and subcultured every month. Under these conditions, calli were firm, cream-colored, fast-growing, with a fresh weight increasing from five- to sevenfold at every month of subculture.
Cell suspensions were easily obtained from friable calli, transferred to
Erlenmeyer flasks containing 100mi of the same liquid medium, under similar
light and temperature conditions. Incubations were carried out in an orbital
shaker, and subcultures were made up every month in fresh medium. The cell
suspensions obtained were yellow, fine, and homogeneous: cells became very
round, sometimes forming microcalli. During the first incubation week, the
biomass production was very slow, but increased thereafter more rapidly.
After several subcultures, the biomass maximum was generally obtained in
10 days.
2.2 Ecdysteroids and Sterols in Cell Cultures
Leaves
Roots
Cells
Ecdysteroids
Total sterols
Total of sterols and steroids
175
1470
1645
377
1370
1747
10
1980
1990
101
HPLC analysis (Fig. 2A,B) confirmed the presence of 20hydroxyecdysone (20E) as the main ecdysteroid in plants, together with
polypodine B (PoB) and other minor compounds, which is consistent with
previous reports (Toth et al. 1981; Bathory et al. 1982; Dinan 1992a). The cells
containing much lower amounts of ecdysteroids appeared difficult to analyze
by HPLC alone, but enzyme immunoassay (EIA) on HPLC fractions (Fig. 2B)
indicated the presence of several immunoreactive compounds, the two main
ones migrating respectively like PoB and 20E in this NP-HPLC system (11 and
16min retention time).
Analyses of free and esterified sterols from C. album indicated some
differences between whole plant organs and in vitro cultures. Table 2 shows
that, though free sterols were predominant in all cases, the plant contained a
higher quantity of steryl esters than cell cultures (two to three times more).
Interestingly, the highest number of esterified sterols was found in roots (more
than 30%). Although 4-desmethylsterols were the major components in all
cases (more than 55% of total sterols), they were particularly abundant in cell
cultures (more than 95% of total sterols), to the detriment of sterol
biosynthesis intermediates as 4,4-dimethylsterols and 4a-methylsterols (less
than 3 and 2% of total sterol in cells, respectively). These intermediates were
generally more abundant in the esterified fraction, except in roots, where they
represented nearly 30% of the free fraction.
The analysis of 4-desmethylsterols showed the presence of more than 15
different compounds, most of them containing a ~5- or ~7-double bond
(Tables 3, 4). However, the presence of stanols (saturated sterols, ~O) has also
been observed, particularly in roots (more than 4 %). In a gas chromatography
profile of acetate derivatives of a 4-desmethylsterol fraction (Figs. 3, 4), compounds 1,2,5,7,13, and 16 were identified as ~5-sterols and compounds 4, 9,
12, 17, and 18 as ~7-sterols. As indicated in Table 3, ~7-sterols were always
more abundant (>60%) than ~5-sterols 40%), but the extent of these two
Free sterols'
Esterified sterols'
Free sterols
4,4-Dimethylsterols b
4a-Methylsterols b
4-Desmethylsterolsh
Esterified sterols
4,4-Dimethylsterols h
4a-Methylsterols h
4-Desmethylsterolsh
Leaves
Roots
Cells
74.9
25.1
68.6
31.4
88.1
11.9
2.1
2.7
95.2
22.2
7.6
70.2
0.9
1.6
97.5
32.5
12.4
55.1
19.2
6.6
74.2
16.1
3.5
80.4
20E
20
time (min)
10
B
R
2000
1500
1000
500
0
5
10
15
20
time (min)
103
Leaves
Roots
Cells
t..5-Sterols"
Ml-Sterols
t.. 7 -Sterols
t..5,7-Sterols
t.. 7I t..5
36.3
1.2
61.5
1.0
1.7
Percentage
30.3
4.2
65.0
0.5
2.2
15.1
1.8
81.5
1.6
5.5
Free sterols
t..5-Sterols
t..7-Sterols
t.. 71 t..5
42.5
56.1
1.3
32.4
62.7
1.9
14.7
82.3
5.5
Esterified sterols
t..5-Sterols
t..7-Sterols
t..71t..5
17.7
77.3
4.6
19.2
75.6
4
17.4
76.6
5
Table 4. Composition of 4-desmethylsterols in the free and esterified fraction isolated from plant
and cell cultures of C. album. F, free sterol fraction; ES, esterified sterol fraction
4-Desmethylsterols
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
Cholesterol
Cholestanol
Lathosterol
Campesterol
Campestanol
Stigmasterol
Stigmastenol
t.. 7-Campestenol
t..5,7-Stigmastenol
**
Spinasterol
Sitosterol
Stigmastanol
t..5,7 Sitostenol
Isofucosterol
t.. 7-Stigmastenol
t..7 -A venasterol
mg of total desmethylsterols
Free form
Esterified form
RRT
1.12
1.17
1.18
1.23
1.30
1.32
1.35
1.36
1.37
1.40
1.41
1.42
1.43
1.44
1.46
1.47
1.51
1.52
Leaves (%)
Roots (%)
Cells (%)
ES
0.7
2.0
0.2
1.6
1.5
0.1
8.7
0.5
3.4
0.4
0.2
15.8
24.6
0.5
4.0
0.3
5.5
0.2
1.6
1.1
2.3
0.3
10.0
1.0
2.5
0.1
0.5
27.2
16.9
3.0
1.0
2.3
1.0
8.0
9.1
1.5
2.2
2.0
51.0
10.5
5.0
34.9
0.4
1.25
1.05
0.19
ES
0.6
3.0
0.1
0.4
1.1
0.5
1.8
0.8
4.5
0.3
0.4
14.5
9.0
2.2
0.9
3.7
45.7
10.5
1.4
31.3
1.7
0.98
0.66
0.32
ES
0.1
0.5
0.2
3.0
0.1
0.4
0.5
4.0
0.2
2.7
3.2
2.5
0.2
0.3
0.3
33.1
7.0
1.2
1.4
2.6
45.6
0.9
25.1
7.2
1.0
2.1
3.3
49.0
2.1
l.89
1.69
0.20
Unidentified compound, but seems to be desmosterol isomer with characteristic ions at m/e: 366
(M+-OAc).
** Unidentified compound.
, Relative percentage of total free or esterified sterols.
h Undetectable.
104
12
18
13
CHOL
16
14
categories is noticeable because only a few plant species have been reported to
have such a peculiarity: indeed, plants rich in ,17-sterols are generally reported
to contain almost exclusively such compounds (Xu et al. 1990).
Interestingly, ,17-sterols were found to be much more abundant in cell
cultures (more than 80%) than in plants, where the ,17/,15 ratio was not very
different in leaves and in roots. Considering free and esterified fractions, the
level of ,17-sterols was almost similar in the two fractions of cell cultures,
105
HO
Fig.4. Structure of 4-desmethylsterols found in C. album. 1 ~()-sterols or stanols; 2 ~5-sterols; 3 ~7sterols; 4 ~5.7-sterols; R structure of the side chain. [1] isomer of desmosterol (1A); [2] cholesterol
(cholest-5-en-3/3-01) (2B); [3] cholestanol (cholestan-3/3-01) (J B); [4]lathosterol (cholest-7-en-3/301) (3B); [5] campesterol [(24R)-24-methylcholest-5-cn-3j3-01] (2C); [6] campestanol [(24R)-24methylcholestan-3J3-01] (1 C); [7] stigmasterol [(22E)-(24S)-24-ethylcholesta-5,22-dien-3/3-ol]
(2E); [8] stigmastenol (24-ethylcholestan-22-en-3J3-ol) (1 E); [9] ~7-campestenol (24methylcholest-7-en-3/3-01) (3C); [10] ~5,7-stigmastenol (24-ethylcholesta-5,7,22-trien-3/3-01) (4E);
[12] spinasterol (24-ethylcholesta-7,22-dien-3/3-01) (3E); [13] sitosterol [(24R)-24-ethylcholest-5en-3j3-ol] (2D); [14] stigmastanol (24-ethylcholestan-3j3-0J) (1 D); [15] ~5,7-sitostenol, 24ethylcholesta-5,7 -dien-3/3-ol (4D); [16] isofucosterol [(24Z)24-ethylcholesta-5,24(24 1)-dien-3J3-01]
(2F); [17] ~7-stigmastenol, dihydrospinasterol (24-ethylcholest-7-en-3J3-ol) (3D); [18] ~7avenasterol, 24-ethylcholesta-7.24(24 1)-dien-3/3-01 (3F)
106
whereas it was in favor of the esterified fraction in leaves and roots, which
indicated a preferential esterification of .-17-sterols in intact plants, but not in
cells in vitro.
From a qualitative viewpoint, major .-17-sterols were .-17-stigmastenol (17,
see Table 4) and spinasterol (12). Sitosterol (13) and stigmasterol (7) were the
major .-15-sterol, whereas cholesterol (2), lathosterol (4), stanols (3, 6, 8, 14)
and .-15,7-sterols (10, 15) were minor compounds. The distribution of sterols in
free or esterified fractions showed some differences between cells in vitro and
plants, in particular of free sterols, for which higher levels of .-17 -sterols (12, 17)
and lower quantities of stigmasterol (7) or sitosterol (13) were found in cells
compared to plants.
2.3 In Vitro Incorporation of Radiolabeled Mevalonate
Sterol and steroid biosynthesis were investigated in isolated cells of
Chenopodium album after incubation of radiolabeled 14C mevalonic acid
(5.uCi/flask) for up to 7 days and analyzed as previously described (Delbecque
et al. 1995). After cell extraction, a significant incorporation of radioactivity
was obtained into sterols, more than 12% of the incorporated mevalonate
after 7 days of culture, as shown by radio-TLC analysis (Fig. 5, Table 5).
Table 5. Radioactivity recovered after 3 and 7 days of incubation of DL-[2-14C] mevalonic acid
sodium salt (5,uCilfiask; AS: 49.4mCi/mmol) in sterol and ecdysteroid fraction. L. (Chapuis and
M.-F. Corio-Costet, unpub\.)
Radioactivity recovered (%)
3 days'
7 days'
Incorporated in cells
Incorporated in medium
57.5
42.5
59.4
40.6
l1.1 b
97.8'
0.8'
1.4'
10.9b
92.8'
3.2'
3.9'
Esterified sterols
4-Desmethylsterols
4a-Methylsterols
4,4-Dimethylsterols
1.78b
59'
6.2'
34.8'
1.3 b
66.3'
6.1'
27.6'
86.1'
79.4'
4.2'
5.9'
3.8'
6.7'
97.7'
76.9'
4.9'
8.5'
5.5'
4.2c
107
'"
,.,
'"
:a
;;;
::i'
1?\
0>
8
....
:x
'"'
s
'"
::i
0
E~
'"
~
:~
~
Fig. 5. Radio-TLC-analysis of purified extract from C. album cells radio labeled with mevalonic
acid after 7 days. Extract was chromatographied on silica plates (0.2 mm) with dichloromethane as
eluting solvent. A Baseline; B 4-desmethylsterol fraction; C 4a-methylsterol fraction; D 4,4dimethyl sterol fraction; E esterified sterol fraction. (M.-F. Corio-Costet and L. Chapuis, unpub!.)
'"
E
.~
I-
108
:::l
o
U
20E
1000
~~--------------~----------------~r-------~~
10
20
time
109
(21 %) 4,4-DIMETHYLSTEROLS
STEROLS
78.3%
(7%) 4a-METHYLSTEROLS
(72%) 4-DESMETHYLSTEROLS
t.5-sterols (30%)
t.7-sterols (65 % )
t.5-7-sterols(1%) ~
stanols(4%)
HO~------'
Lathosterol (0.13%)
""
""
"
HO
7-Dehydrocholesterol (trace)
~~ /
Cholesterol (2%)
~------------~"
tOROR
ECDYSTEROIDES
21.7%
~
''''''''
HO
OR
:.
UII
JlO
ID
20-Hydroxyecdysone
OR
-~
oo~
Polypodine B
Fig. 7. Proposed scheme of sterol biosynthesis for ccdysteroid production in Chenopodium album. The various percentages are the percentage of total sterols of three sterol classes (underlined) and of the most important sterols. (M.-F. Corio-Coster, unpubl.)
110
1990). L\7-Sterols are, indeed, frequently interpreted as biosynthethic intermediates during the isomerization of ,18- to L\5-sterols. It has therefore been
proposed that high levels of L\7(8)-sterols result from a genetic block or from
a defective control of the enzyme catalyzing 5(6)-sterol synthesis (Nes 1977;
Caputo et al. 1983). This hypothesis is reinforced by a recent work on
Arabidopsis mutants, deficient in sterol biosynthesis, which accumulate ,17and L\5-sterols (Gachotte et al. 1995). The unusual ratio of L\7/L\5-sterols in C.
album plants and cells may thus reflect an intermediate regulation of the kinetic
steps, such as the ,15 -desaturase, an enzyme of the sterol biosynthesis.
Thus, from our results on C. album, a scheme of the flux from sterol to
ecdysteroid biosynthesis (Fig. 7) is proposed, where the presence of cholesterol derivatives such as ,17 (lathosterol) and ,15-7 derivatives most likely plays
an important role in ecdysteroid biosynthesis. A careful examination of the
kinetics of these molecules in plant and cell cultures at various stages may
undoubtedly improve our understanding of the biogenesis of ecdysteroids in
C. album in the future.
4 Protocols
4.1 Isolation and Analysis of Ecdysteroids
Plant material was extracted as described previously (Corio-Costet et al. 1993a). The eluted
fractions of ecdysteroids were evaporated and submitted to HPLC (high performance liquid
chromatography in reverse or normal phases, i.e., RP or NP-HPLC) and/or enzyme immunoassay
(EIA), as described by Corio-Costet et al. (1993b).
References
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plants. Lipids 30:257-262
111
Corio-Costet MF, Chapuis L, Delbecque JP (1996) Serratula tinctoria (L.) (Dyer's savory): in vitro
culture and the production of ecdysteroids and other secondary metabolites. In: Bajaj YPS
(ed) Biotechnolog in agriculture and forestry, vol 37. Medicinal and aromatic plants IX.
Springer, Berlin Heidelberg New York, pp 384--401
Co stet MF, El Achouri M, Charlet M, Lanot R, Benveniste P, Hoffmann J (1987) Ecdysteroid
biosynthesis and embryonic development are disturbed in insects (Locusta migratoria) reared
on plant diet (Triticum sativum) with a selectively modified sterol profile. Proc Nat! Acad Sci
USA 84:643~647
Co stet-Corio MF, Benveniste P (1988) Sterol metabolism in wheat treated by N-substituted
morpholines. Pestic Sci 22:243~247
Davies TG, Lockley WI, Boid R, Rees HH, Goodwin TW (1980) Mechanism of formation of the
AlB cis ring junction of ecdysteroids in Polyp odium vulgare. Biochem J 190:537~544
Delbecque JP, Beydon P, Chapuis L, Corio-Costet MF (1995) In vitro incorporation of
radiolabelled cholesterol and mevalonic acid into ecdysteroids by hairy root cultures of a plant,
Serratula tinctoria. Eur J Entomol 92:301~307
De Simone F, Dini A, Pizza C, Saturnino P, Schettino 0 (1990) Two flavonol glycosides from
Chenopodium quinua. Phytochemistry 29:3690~3692
Dinan L (1992a) The analysis of phytoecdysteroids in single (preflowering stage) specimens of fat
hen, Chenopodium album. Phytochem AnaI3:132~138
Dinan L (1992b) The association of phytoecdysteroids with flowering in fat hen, C. album, and
other members of the Chenopodiaceae. Experientia 48:305~308
Dinan L, Riseborough S, Brading M, Clement CY, Witts DJ, Smith J, Colombe S, Pettitt V,
Wheeler DA, Greenwood DR (1991) Phytoecdysteroids in the Chenopodiaceae. In: Proc Conf
Inscet Chern Eco!, Tabor 1990. Acad Prague and SPB Acad Publ, The Hague, pp 215~220
112
1 Introduction
The genus Comus (family Cornaceae) consists of about 40 species, nearly
all of which are native to the northern hemisphere. The name dogwood is
a corruption of dagwood or dagger wood because the daggers (for skewering meat) were made from the wood of some sorts (Everett 1981; another
tradition also exists that the extract of the barks was used for the treatment
of skin disease in dogs). Comus plants, with flower clusters surrounded
by large, spreading, petal-like bracts, have great decorative merit for
garden and landscape trees. C. florida (eastern flowering dogwood) and C.
nuttallii (western flowering dogwood) are especially popular trees in North
America.
Comus kousa of Japan and Korea is also often used as a garden tree; the
clusters of flowers (Fig. 1) and the brilliant red leaves in autumn look attractive
under city conditions. The varieties C. kousa chinensis (from China), which is
not botanically very distinct from the typical species, and C. kousa Milky Way,
which blooms unusually freely, occur in Japan. C. capitata, whose four bracts
turn yellow, often decorate the mountainside (evergreen forests) in the
Himalayas.
For medicinal use, C. officinalis Sieb. et Zucc., native to China, is often
employed. The extract of the dried fruits, containing organic acids (malic
acid, tartalic acid, gallic acid, etc.) and fatty oils (palmitic acid, oleic acid,
etc.) is a popular tonic, an astringent, and a hemostatic in East Asian
countries (Mitsuhashi 1988). The fruits (comus fruits) have been often one of
the ingredients in traditional prescriptions for preventing and improving
symptoms of aging, including pollakiuria and cataract (Yazaki and Okuda
1993).
Some other dogwood species provide food for wildlife (Eyde 1988); for
example, the fruits of C. mas L. can be used to make jelly. The bark of some
114
K. Ishimaru et al.
Fig. 1. Plant of Comus kousa grown at Saga University. (Photograph Ishimaru, July, 1995)
species also yields a compound that can be substituted for quinine (Brinkman
1974).
Comus plants are also rich in tannins, which can be classified into two
large groups, i.e., hydrolyzable and condensed tannins. Some chemical investigations, particularly on this medicinal plant (Okuda et al. 1981, 1984; Hatano
et al. 1989a,b; Lee et al. 1989) have clarified the major polyphenol constituents
of the plant to be hydrolyzable tannins, the metabolites of gallic acid.
115
OH
OH
.OC-Q-OH
G:
HOOC-Q-OH
OH
OH
OC
:;;-.
HOW":::'"
:::".1
HO
1 OH
OH
OH
OH
6c
HOW":::'"
:;;-.
(+ )-catechin (7)
:::".1
HO
0H
1 OH
OH
(+)-gallocatechin (8)
OC
OC
OH
HOW":::'"
:;;-.
1 OH
:::".1
OH
HO:
HOycx=
0 ..,:::".
:;;-:::".1
HO
OH
1 OH
OH
(min): 2 (6.3), 1 (8.5), 8 (11.2), 9 (16.1), 7 (17.1), 3 (19.2), 4 (20.5), 5 (22.8) and
6 (23.8).
In all plants, the major tannin was 6 (0.13~ 1.46% as dry weight; Fig. 3). In
C. capitata Mountain Moon the content of 7 was characteristically high
(0.36%, as dry wt.).
3 In Vitro Approaches
Yazaki and Okuda (1989, 1993) raised callus and cell suspension cultures of C.
officina lis and studied their tannin production. The callus and suspension
cultures, in LS medium (Linsmaier and Skoog 1965) with auxins [2,4dichlorophenoxyacetic acid (2,4-D), naphthalene acetic acid (NAA), or
indole-3-acetic acid (IAA)] and cytokinins [benzyladenine (BA) or kinetin],
produced much higher contents of trio, tetra- and penta-galloylglucoses
compared to those of the intact plant. C. stolonifera callus has been used
for physiological studies (Niki et al. 1978, 1979; Yoshida and Tagawa 1979).
Successful micropropagation by shoot cultures from apical buds of C.
florida (Coker 1982) was achieved showing nondormant bud proliferation
to be best on Knop's medium supplemented with either 1 or 2mg/l BA. In
an in vitro proliferation system for Cornus species, Trigiano et al. (1989,
1992) also studied somatic embryogenesis from immature zygotic embryos of
C. florida, and indirectly from embryogenic calli derived from zygotic em-
K. Ishimaru et al.
116
1.6
...
_ IA
~
1.6
...
'0; 1.2
'0; 1.2
:t
~ 1.0
C' 1.0
a 0.8
"CI
; o.~
t\!
06
OA
0. 2
0.0
2
1.6
...
1.4
OIl
'w 1.2
0.6
OA
0. 2
0.0
6
1.6
t '1.0
~ 1.0
"CI
; 0.8
t\!
t\!
0.6
U.8
0.6
0.4
OA
0. 2
0.2
,=..,
0.0
2
~
:t
'w l.2
0.0
6
~
&~
~ 1.0
1.6
1.4
1.2
F
Cornus capitata
'Mountain Moon '
~ 1.0
"CI
"CI
; 0.8
~ UJ~
t\!
'":t 1.2
:t
_ 1.4
1.4
"CI
1.6
:t
"CI
t\!
_ 1.4
S:?
0.6
0.6
0.4
0 .4
0.2
0. 2
0.0
9
0.0
4
..
1.6
_ 1.4
.=
'"
1.2
~
117
1.6
Comus drummodi;
'Eddie 's White Wonder'
i:' 1.0
."
-:: 1.4
c;,
H
Cornus officinaJis
'"
1.2
~
i:' 1.0
."
~ 0.8
~ 08
~
0.6
0.6
0.4
0. 4
0.2
0.2
0.0 ..I-_ _ _....r::~='_'_..L_ _ __
4
118
K. Ishimaru et al.
119
(Fig. 3A,B,F), the main component observed in in vitro plants was lower
molecular weight phenolic 2 (1.38-2.70%, as dry wt.). These observations
indicated that tannin metabolism (especially the gallic acid metabolism) in
these plants might vary with aging, seasonal change, and culture conditions.
3.2 Callus and Cell Suspension Cultures of C. kousa
Calli of C. kousa were derived from leaf segments of the parent plant. For the
induction of the calli, ten types of MS solid media (Murashige and Skoog 1962)
supplemented with various combinations of 2,4-D, NAA, IAA, and BA
(Table 1) were used. The addition of 0.1 mg/l BA to the medium evidently
promoted callus formation. Particularly, on the medium with NAA (0.5 or
2mg/l) the existence of BA was essential for callus induction. Among ten
media, that containing 0.1 mg/12,4-D and 0.1 mg/l BA (medium A) was best for
the induction of the callus.
3.2.2 Isolation of Tannins
From the calli cultured on medium A for 5 weeks in the dark, four phenolic
compounds 2 and 7-9 were isolated. Among them, 2, a monogalloyl ester of
0.0
0.4
0.8
1.2
1.6
2.0
Ii
='
0.0 1
0.4
0.8
1.2
'--3
4
=:==='
~
1\
'-=='
7
I""';;:::'"
If
-=
'"eo
-=
'"eo
to
1.6
stem
to
:r;
'0;
2.0
0.0
0.4
0.8
1.2
1.6
2.0
'i
Ii
root
leaf
,;:
Fig. 7. Polyphenol contents in in vitro plant of Comus kousa var. chinensis. (K. Ishimaru et aI., unpubL)
If
'"eo
-=
to
:r;
'0;
,;:
P"
0>
rg..
......
..tv
2.8
2.0
5 Ii
7
R
9
0.0
0.0
3
0.4
2
0.8
1.2
1.6
2.0
2.4
2.8
0.4
.
..'"
ts'?
.;:;
til)
:c
0.8
1.2
leaf
3.2
23456789
stem
Fig. 8. Polyphenol contents in an in vitro plant of Comus kousa Milky Way. (K. Ishimaru et aI. , unpubl.)
ts'?
~ 16
to
.~ 2.4
:c
3.2,
6l
;:;
......
'"'
E::
o
o
:e
'"
B
o
~
1:;
'"
:::
1.6
1.4
0.8
1.0
R
9
0.0
0.0
4
0.2
0.2
3
0.4
~ 06
1.0
0.4
~ 0.6
"0
t'
_ 1.4
..:
.~ 1.2
~ 0.8
1.0
stem
0.8
"0
t'
:t
.~ 1.2
_
..:
1.6
1.6
1.4
root
leaf
1.8
1.8
Fig. 9. Polyphenol contents in an in vitro plant of Comus capitata Mountain Moon. (K. Ishimaru et aI., unpubl.)
0.0
0.2
0.4
~ 0.6
"0
t'
:t
.~ 1.2
..:
1.8
...::r
~
0>
.,'"'
V>
?"
'--'
I::l
123
Table 1. Effects of growth regulators on callus formation on leaf
segments of Comus kousa cultured on MS solid medium for 8
weeks. (Ishimaru et al. 1993)
2,4-D
NAA
IAA
BA
0
0.1
0
0.1
0
0.1
0
0.1
0
0.1
50.9
240.4
137.9
179.5
(mg/l)
0.1
0.1
1.0
1.0
0.5
0.5
2.0
2.0
3.0
3.0
126.7
0
213
25.1
229.4
124
K. Ishimaru et al.
200
------
:2 175
--u-
=r..~ 150
callus on medium A
callus on medium B
callus on medium C
cells in medium A
-{J--
1:l
125
.a
-- 100
75
'ijl
~
50
t:25
O+---~~---r--'---~~---r~
Weeks
300
----A--
--
---0-
--0---
~ 250
.t:J.
.a
200
~
<
8
9
---0-
.a 200
-a 150
=
=
E
<100
50
50
--0---
250
100
----A--
.t:J.
!~~---"
-a 150
=
=
E
--
300
2
7
Weeks
300
-----A--
---0-
Qi
.t:J.
~
~
250
--0---
200
8
9
---0-
.,
--0---
80
II
c:
~60
~
...
-a 150
=
=
E
<
--2
--
100
2
7
8
9
/~~
2
7
Weeks
2
7
8
9
540
100
.---.
50
<
20
0
0
Weeks
0
6
Weeks
Fig. llA-D. Polyphenol production in calli (A-C) and cell suspension (D) of Comus kousa in the
dark at 25C. A On medium A. B On medium B. C On medium C. D On medium A. (Ishimaru
et al. 1993)
125
The calli cultured on medium B also produced similar phenolics (2 and 79) (Fig. lIB). In this culture, the amount of 7 increased from the early stage (1
week) and later reached the maximum level (130.3 Jig / tube) at week 4; it
rapidly decreased until the end (week 8) of the culture. The level of 9 appeared
remained steady (ca. 45 Jig / tube) during the first 4 weeks and after week 4 it
began to decrease. Compound 8, after showing its highest level (19.1 Jig / tube)
at week 2, also decreased continuously. At the end of the culture time (at week
8), these phenolics were not detected in the calli. Therefore, the combination
of NAA-BA was unsuitable for the production of phenolics in C. kousa callus
cultures.
In the calli cultured on medium C, the amounts of 2 and 7-9 gradually
increased from the early period of the culture to show their highest amounts
(2: 5.7 Jig, 7: 275.1 Jig, 8: 55 Jig and 9: 130.9 Jig / tube) at week 7 (Fig. 11 C). At
the last stage (7-8 weeks) of the culture, the simultaneous decrease of these
compounds was observed.
3.2.4 Suspension Cultures
C. kousa calli were also transferred into liquid medium A to establish cell
suspension culture. The growth rate of the cells is shown in Fig. 10. From the
early period of the culture the amount of the cells gradually increased and
after week 5 the increase was accelerated. The cells also produced phenolics
similar to those observed in the callus cultures, but their contents were fairly
low (Fig. lID). Compounds 7 and 9, which could be detected early in the
culture, suddenly decreased before week 3. Halfway (3-6 weeks) through the
culture, polyphenols did not appear at any detectable level. After week 6
the amounts of 7 and 9 increased slightly (9:28.4 Jig / flask at week 7, 7:15.3 Jig
/ flask at week 8), but, taking into account the good growth during this period,
the contents of these compounds in the cells were concluded to be very low.
Therefore, cell suspension culture of C. kousa in medium A was unsuitable for
the production of phenolics.
3.2.5 Effects of 2,4-D and BA Contents and Illumination on Growth and
Tannin Production of C. kousa Callus
To determine the effects of several combinations of 2,4-D-BA and illumination on the growth and tannin production of C. kousa calli, nine media, A
and D-K (Fig. 12), were prepared. On these media the illumination showed
no significant effect on callus growth except on media A, F, and K (Fig.
12A).
The production of phenolics (2 and 7-9) in calli cultured on these media
(A and D-K) under light or dark condition is shown in Fig. 13. In the light (Fig.
13A) the calli produced a sufficient level (over 30 Jig / tube of total amount of
2 and 7-9) of phenolics on four media F and I-K. The major compound
observed on these media was 9. On the other four media, D, E, G, and H, the
126
K. Ishimaru et al.
70
~
60
,Q
..
dark
30
.~ 30
20
t:
..
....
light
dark
r-
,Q
:: 40
60
--g!!
!>Il
..
70
0 light
50
50
!>Il
:c
40
~
~
10
20
10
0
A
medium
A
D
E
F
H
I
G
H
Medium
2,4-D
BA
0.1
0.5
1.0
0.1
0.5
1.0
0.1
0.5
1.0
0.1
0.1
0.1
0.5
0.5
0.5
1.0
1.0
1.0
G
H
Medium
(mg /I)
Fig. 12A-c' Growth of Comus kousa calli cultured on MS solid media with 2,4-D and BA for 5
weeks at 25C. A Basal MS. B Minus NH4N0 3 MS. (Ishimaru et al. 1993)
80
80
122 9
[Zl 9
70
G 8
.. 2
50
~60
~
!! 50
~40
~40
<:
<:
70
~60
,Q
!!
~ 8
o
..
7
2
=r o
~ 30
20
10
20
10
Medium
Medium
Fig. 13A,B. Polyphenol production in Comus kousa calli cultured on MS solid media with 2,4D
and BA for 5 weeks at 25C. A In the light B In the dark. (Ishimaru et al. 1993)
127
amount of 2 and 7-9 was fairly low. In the light, the addition of a high content
(1 mg/l) of BA (media I-K) seemed to be good for phenolic production. On
the other hand, in the dark (Fig. 13B), the calli produced high amounts of
phenolics on media A and F. The level of the total amount of 2 and 7-9 (ca.
34 j1g / tube) appearing on medium F was almost half that obtained on the
same medium under light. It was noteworthy that the amount of 9, observed in
high level on media I-K in the light, was very small on the same media in the
dark. This result suggested that illumination promoted the biosynthetic polymerization of 7 into 9 on these media (I-K). Except on the media with a low
content (O.lmg/l) of BA (media A and D-E), the light condition seemed to
have the greatest effect on polyphenol production in C. kousa calli.
3.2.6 Effects of NH4 N01 on Growth and Tannin Production of
C. kousa Callus
Nitrogen sources in culture medium are important factors for the production
of polyphenols in plant tissue cultures (Ishimaru and Shimomura 1991;
Ishimaru et al. 1992; Neera et al. 1992). In this experiment the effects of
NH 4N0 3 on the growth and polyphenol production of C. kousa calli were
determined. The growth of the calli cultured on nine media, A and D-K,
without NH 4 N0 3 is shown in Fig. 12B. In both light and dark conditions, callus
growth was much greater than that observed on the same media containing
NH4N0 3 (Fig. 12A). Especially, the amounts of the calli in the light were
almost 2.5 (on medium F) to 13 (on medium G) times larger than those on the
same media containing NH4 N0 3 . It was interesting that callus growth observed on the media without NH4NO} in the light was enhanced in proportion
to the concentration of 2,4-D (Fig. 12B).
The polyphenol production in the calli cultured on media A and D-K
(minus NH 4N0 3 ) was also observed to have considerably high level (Fig. 14).
1600
1400
~1 2oo
o
o
1600
1400
II 2
~1 2oo
'"
'"
fZl 9
D 8
0 7
II 2
.e woo
.&>
.e woo
.&>
~ 800
"5 600
e=
400
-==
o
e
200
200
0
A
F
G
Medium
Medium
Fig. 14A,B. Polyphenol production in Comus kousa calli cultured on MS solid media (minus
NH4 N0 3 ) with 2.4-D and BA for 5 weeks at 25C. A In the light. B In the dark. (Ishimaru et al.
1993)
128
K. Ishimaru et al.
5 Protocol
5.1 Shoot Cultures
Young branches were collected in summer and sterilized [70% ethanol (1 min), 1 % NaOel
(15 min), sterile water (rinse 2X)]. The axillary buds and shoot apices were cut off and aseptically
placed on BW medium under illumination (16-h photoperiod I a day, 3000 Ix). At 2-3-month
intervals, the shoots were subcultured on the same medium. For rooting, shoots (3-5 cm) were
transferred (after dipping of the cut ends into 70% EtOH with 20mM IBA for 1 s) on BW medium
containing 0.2mg/l NAA, 0.6mgll IBA, and 0.2% activated charcoal.
129
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Coker AL (1982) In vitro culture of flowering dogwood, Comus florida L. MS Thesis, Univ of
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K. Ishimaru et al.
Everett TH (1981) CORNUS, The New York Botanical Garden illustrated encyclopedia of
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1 Introduction
Flowers of Cynara cardunculus flavescens (cardoon) have been traditionally
used in Portugal and Spain for many centuries to produce artisanal cheeses.
One of the first reports on this use by farmers was made by Brotero (1804) in
Flora Lusitanica. Today, the Serpa and Serra cheeses are some examples of
typical and highly appreciated products of Portugal.
1.1 Taxonomy and Geographic Distribution
133
Cynara algerbiensis
Cynara comigera
. . . - - - - - - Cynara baetica
, - - - - - Cynara syriaca
. . . - - - Cynara auranitica
Cynara cardunculus subsp. cardunculus
Cynara cardunculus subsp. f1avescens - - [
cultivar Cardoon
cultivar Artichoke
Fig. 1. Tree of the genus Cynara with Cirsium vulgare as outgroup (After Wiklund 1992)
to produce cheese. Many other species in over 13 genera are also commonly
referred to as cardo (= thistle) in Portugal (Pereira Coutinho 1939; Franco
1984).
Various cheeses made with flower extracts are still being produced in
Portugal and Spain by farmers or cheese-makers. There are two main types of
cheese in Portugal; the Serra cheese, made in the region of Serra da Estrela in
the center of the country, and the Serpa cheese made in Serpa, a village
134
situated in the District of Beja, in the south. The cheeses from Azeitao and
Castelo Branco also share characteristics with these cheeses. They are all
made with the flowers of cardoon, and with ewe's milk, only. The skills in
making these cheeses have been inherited over generations within the same
families. In Portugal, many families make their own cheeses for home consumption. Also in Spain various cheeses are made with extracts from Cynara
flowers. C. humilis is used to make many types of cheeses some examples are
Serena, Torta del Casar, Pedroches, and Grazalema.
Even though the cheeses are generally made in the same way, they show
distinct characteristics and differences in taste, depending on the region where
they are made. This is due to a number of factors that influence the ripening
process. The Serra cheese is a soft, creamy cheese and exhibits a very slight
and characteristic bitter or peppery flavor. The Serpa cheese shows a higher
curd firmness and has a stronger flavor.
Traditionally, the cheeses are made only with ewe's milk. The proteinases
are extracted from the flowers in two main ways: dried styles from flowers are
ground with salt in a mortar and added to the milk; or the styles are extracted
with lukewarm water (around 30C).
Farmers collect the mature flowers or the upper part of styles during June
and July and store them in dry places to be used for clotting of milk during the
following autumn and winter, when ewe's milk can be obtained.
When the cardoon flowers are collected in the fields, other flowers of
similar color and shape are also collected (e.g., Centaurea calcitrapa, Silybum
marianum, and other species of the genus Cynara). Although some of these
flowers also have some clotting activity, the proteolytic enzymes that are
present do not have the same properties as the cardoon proteases. Therefore,
they give rise to a different proteolysis of milk proteins, which results in
different characteristics and taste in the cheeses. Generally, the quality of
these cheeses will be reduced. Also, the stage of development at which the
flowers are collected will significantly influence cheese characteristics.
Another factor that may influence the standardization of cheeses is the
enzyme variation between populations of cardoon. At least three forms of the
Table 1. Review of in vitro culture studies on Cynara cardunculus
Study conducted
Remarks/observations
Reference
Cell cultures
Somatic embryogenesis
135
136
Two other lines of cell cultures were obtained from tissues of young flowers
(unpubl.). Both these cultures were grown in B5 medium using hormone
concentrations similar to those given above. Western blot analysis and clotting
experiments have shown that these cell lines also do not synthesize cyprosins
under the conditions used (V. Carocha, M.e. Cordeiro, and M.S. Pais, unpubl.).
Cyprosins are present mostly at later stages of flower development, and
accumulate in the styles of these flowers, indicating that the synthesis of the
enzymes is related to differentiated tissues and cells. This is not the case with
cells in suspension cultures, which generally are undifferentiated.
All these cell suspension cultures may thus be used as recipient cells for
the genes encoding the cyprosins. Such transgenic cell cultures will be of great
importance in studies on posttranslational modifications of cyprosins and for
the production of cyprosins on a large scale.
2.2 Transient Gene Expression in Cardoon Protoplasts
Cell suspension cultures derived from young flower tissues were used to isolate
protoplasts. Conditions for cultured protoplasts to regenerate cell walls and
callus cultures have been established (unpubl.). Initial studies have been performed to obtain appropriate conditions for gene transfer into protoplasts.
Direct gene transfer was attempted using electroporation and polyethylene
glycol 4000 (PEG), and using the pDW2 plasmid (Pietrzak et al. 1986). This
plasmid contains the CAT (chloramphenicol acetyl transferase) reporter gene
and the 35S cauliflower mosaic virus promoter. Under the conditions studied,
no CAT activity was detected when electroporation was tested. Transient gene
expression was obtained when using 30% PEG and without the heat-shock
treatment prior to the addition of DNA and PEG (Carocha et al. 1994). These
studies suggest that direct gene transfer is possible in C. cardunculus and will
be continued to establish stable transformation using cyprosin genes.
2.3 Somatic Embryogenesis in C. cardunculus
Somatic embryos were produced from cotyledons and first leaves of in vitrogrown seedlings of C. cardunculus (Miguel and Pais 1993). Embryogenic calli
were obtained after 4-5 weeks of culture on B5 solid medium. Cotyledon
explants presented a higher percentage of embryogenic induction than leaf
explants. In the presence of 1 mg/l 2,4-D and 0.1 mg/l kinetin, initial stages of
somatic embryo development were formed on the surface of embryogenic
calli. Transfer of these cultures to B5 liquid medium and a different hormone
concentration (zeatin 1 mg/l and 2,4-D 0.1 mg/l) allowed the induction of more
embryos and their further development (Fig. 3). Mature embryos, when transferred to the same liquid medium without growth regulators, showed root
apex growth and greening of cotyledons (Fig. 3). However, the germination of
the embryos was still only occasional and plantlets obtained showed arrest of
growth before development of leaves (Miguel and Pais 1993).
137
Fig. 3A-E. Embryonic cell cultures of Cynara cardunculus. A Group of somatic embryos at
various developmental stages, including early cotyledonary stages, formed on embryogenic callus.
8-E Cultures in liquid medium. 8 Embryoids that remained attached to the cotyledon explant. C
Torpedo stage. D Initiation of root apex growth . E Mature embryos with large cotyledons. Bars
1 mm. (Miguel and Pais 1993)
138
tion and low cell growth on the walls of the fermenter were advantageous
properties of C. cardunculus cell suspensions. Proteases (not characterized)
were produced in B5 medium. In continuous culture, a higher protease level
corresponded to a higher specific growth rate of the cells, which showed that
the synthesis of proteases is linked to primary metabolism (Costa 1994).
139
kDa
94
67
43
30
'.
20
14.4
Isoelectric Points. Electrophoretically pure cyprosin 2 and 3 were run on twodimensional electrophoresis gels (IEF and SDS-PAGE) and were both separated into three isozymes with very close isoelectric points, i.e., 3.S5, 4.00, and
4.15 (Cordeiro et al. 1994b).
Preparative isoelectric focusing under nondenaturing conditions was used
to purify the three isozymes of cyprosin 3 (the most active isozyme) for further
studies of this isozyme (Cordeiro 1993).
Peptide Mapping and Protein Microsequencing. From the results summarized
above, it may be concluded that the natural population of cyprosins in flowers
of C. cardunculus is very complex. They vary in subunit size and they exist in
different isoforms. Electrophoretically (SDS-PAGE) purified subunits of
cyprosin 2 and 3 could not be directly sequenced due to this heterogeneity
(Cordeiro et al. 1994b). In an attempt to overcome this difficulty, peptides of
the subunits of these isozymes were produced by enzymatic (trypsin) or chemical (BrCN) cleavage, and electrophoretically purified.
The results obtained indicate that the various forms of cyprosins present
in flowers of cardoon have common structural features and that they may be
derived from common procyprosins. The occurrence of heterodimeric forms
of the enzyme containing subunits of different sizes may be due to proteolytic
cleavage of a procyprosin at different sites originating a group of closely
related enzymes. The occurrence of isozymes may be due to the expression of
a gene family, as discussed below (Sects. 5.1 and 5.4).
An internal partial N-terminal sequence was obtained from a BrCNpeptide obtained by cleavage of the large subunit of cyprosin 2 (Cordeiro et al.
1994b). This internal sequence (met-Ieu-asn-gln-gly-Ieu-val-gln-glu) has been
of great importance for the identification of a cDNA clone coding for cyprosin
(Cordeiro et al. 1994a).
Also an N-terminal sequence of purified cyprosin 1 has been obtained.
This sequence indicates that the N-terminus of the mature protein is asp-ser-
140
asp-leu -ala -val-val-ala -leu -thr -asn -asp-X -asp-thr -X -tyr-phe-gly -X -ile-gly
(unpubl.).
Internal sequences of cardosin A (ser-glu-glu-Ieu-gln-val-asp-asn-asn-thrleu-ser-X-met-pro) and B (ser-ala-glu-asp-ile-val-asn-asn-asn-gly-ile-ser-Xmet-pro) have been reported (Faro et al. 1993). These sequences cannot be
found in the deduced amino acid sequences of the two cyprosin cDNA clones
isolated in our laboratories (see below). This is a strong indication that the
cyprosins and cardosins are isolated from different Cynara species or cultivars.
Glycosylation. Endoglucosidase H treatment of cyprosins and subsequent
SDS-PAGE has shown that all three forms of the enzyme contain high
mannose-type glycans (Heimgartner et al. 1990). We also have indications that
a second glucan not removed by endoglucosidase H is bound to the cyprosins.
This assumption is also supported by the fact that the deduced amino acid
sequence of two cyprosin cDNAs each contain two putative glycosylation sites
(Cordeiro et al. 1994a; M. Pietrzak et al., unpubl.).
pH Optimum of Cyprosins. A synthetic peptide (lys-pro-Ieu-gln-Ieu-nph-argleu) was used to estimate the pH optimum of the three cyprosin 3 isozymes.
The pH optimum (4.1) was the same for the three isozymes (Brodelius et al.
1995).
Catalytic Properties and Substrate Specificity. Initially, the proteolytic activity
of various enzyme preparations was determined with a standard flu oro metric
proteolytic assay based on the release of TCA soluble FITC-Iabeled peptides
from casein (Heimgartner et al. 1990). Using this assay we could establish that
cyprosin 3 showed the highest specific activity and cyprosin 1 the lowest.
Furthermore, we could establish that the cyprosins belong to the aspartic
proteinase family. Addition of pepstatin A to the assay mixture inhibited the
activity of all three cyprosins almost completely, while other protease inhibitors such as leupeptin, aprotenin, cystatin, and iodoacetamide did not affect
the activity to any great extent.
A "library" of related chromophoric octa-peptide substrates with systematic variation in the amino acid residues has been used to study the proteolytic
activity of three cyprosins (pI 3.85, 4.00 and 4.15; Cordeiro et al. 1997). The
rate of hydrolysis of 46 peptides was determined to investigate the effects of
various substitutions in the PS-P j and the PZ'-P3 ' positions on enzyme activity.
From these experiments it is evident that cyprosin, like other aspartic
proteinases, preferentially cleaves peptide bonds between two hydrophobic
amino acids.
Four peptides were selected for detailed kinetic measurements. The
lowest Km values (15-25 mM) were obtained with lys-pro-ile-val-phe-nph-argleu while the highest Kcat values (34-85 S-I) were obtained with lys-pro-ile-Ieuphe-nph-arg-Ieu. The Kj-values for pepstatin A were below 0.1 nM for the
three isozymes.
141
80
70
60
~
c:
E 50
:::E
i=
z 40
2
....
CD
:::J
30
20
10
Fig. 5. Formagram from clotting assays. AlO Curd firmness; r clotting time; r + 10 clotting time +
lOmin. Samples were: A and B cyprosin 3 (60,ug/assay); C and D chymosin (94,ug/assay); E
cyprosin 2 (53,ug/assay); F cyprosin 2 (106,ug/assay); G cyprosin 1 (160,ug/assay); H cyprosin 1
(320,ug/assay). (Cordeiro et al. 1992)
142
460
440
420
S
Q.,
Q.,
400
380
'-'
Z
~
Z
,,
360
chymosin
400
340
360
320
320
300
--
280
280
0
200
10
400
20
30
600
40
800
1000
1200
A cDNA library was constructed from mRNA isolated from young flowers of
cardoon. Six clones were selected in the first screening using cyprosin antibodies and were named cyprol to cypro6. Cypro4 and cypro6, giving the
strongest signal in the screening, were sequenced but no homology to other
aspartic acid proteinases could be established. Clone cyprol actually contained two EcoRI fragments (2.0 and 1.7kb). The subclone containing the 1.7kb insert was called cyprols and sequenced. The deduced amino acid sequence
of cyprols contained the internal peptide sequence obtained by sequencing a
BrCN-fragment of cyprosin 2, showing that this cDNA clone encodes a
cyprosin.
In a second screening of the cDNA library using cyprols as probe, five
new clones named cypro7 to cyproll were selected. Cyproll (1.8kb) was
selected for sequencing.
The nucleotide sequence of clone cyprols contains a 1422-bp open reading frame coding for 474 amino acids including a putative full-length mature
protein (440 amino acids) and a partial prosequence (34 amino acids). By
PCR, a fragment corresponding to the missing 5' -end of this clone could be
143
isolated from the cDNA library and sequenced. The full-length gene product
contains 505 amino acids. The nucleotide sequence of clone cyproll contains
a 1527-bp open reading frame coding for 509 amino acids, including a putative
full-length mature protein (441 amino acids) and a prosequence (68 amino
acids).
5.2 Comparison of Deduced Amino Acid Sequence of Cyprosin to Other
Aspartic Proteinases
The deduced amino acid sequences of the two cyprosin cDNA clones are
shown in Fig. 7 in comparison with other plant aspartic proteinases. They are
called cyprosin I (cyprols) and II (cyprolJ), since it has not yet been firmly
established which form of the enzyme corresponds to which cDNA clone.
They show a relatively high homology to each other and to the other plant
aspartic proteinases, as indicated by the consensus sequence. Compared to
other aspartic proteinases, the plant enzymes contain an insert of around 100
amino acids (plant-specific insert; italics in Fig. 7) in the C-terminal part of the
protein molecule. Omitting this insert, the homology to aspartic proteinases
from other phyla is relatively high. For instance, the mature cyprosin I shows
34, 43, and 55% identity to rhizopuspepsin, bovine chymosin b, and human
cathepsin D, respectively.
Two putative active-site aspartic acid residues (DTG and DSG) have been
identified in each isozyme (bold and italics in Fig. 7). Furthermore, two putative glycosylation sites have been found in each cyprosin isozyme (underlined
in Fig. 7). One of these glycosylation sites, present in the plant-specific insert,
is conserved in all plant enzymes. It is well established that the cyprosins are
glycoproteins (Heimgartner et al. 1990). Furthermore, 12 conserved cysteins
are found in the plant aspartic proteinases (marked with * in Fig. 7).
A consensus sequence based on 30 aspartic proteinases (not shown) shows
that the mature aspartic proteinases are highly conserved proteins. In particular, the sequences around and at the active-site aspartyl residues are very
conserved. An exception to this is observed for the plant enzymes in which the
threonine of the second active site has been substituted for by serine. However, in spite of this substitution, the residues around the aspartyl residues of
the active site are conserved in the plant enzymes.
From a phylogenetic analysis (not shown), it may be concluded that the
plant enzymes are most closely related to the cathepsin D enzymes of various
animals.
5.3 The Plant-Specific Insert
hDeMCtmCqM
hDeMCtfCEM
aDpMCsaCEM
gDaaCsaCEM
sDaMCsvCEM
sgpMCnaCEM
-D-MC--CEM
GPLWILGDVF
GPLWILGDVF
GPLWILGDVF
GPLWILGDVF
GPLWILGDVF
GPLWILGDVF
GPLWILGDVF
kskGK.Ssgl
ennGKSSsgv
dEpvKSnglr
kEnaK1Sngv
kEn ..... 1g
dEaGeSng1q
-E-GKSS---
401
FTAMDvaPPh
FTAMDvaPPR
FTAMDipPPR
FiAlDvaPPR
FmAfDipPPR
FTAMDipPPR
FTAMD--PPR
GFCAsGCAAI
GFCsdGCAAI
GFCAgGCAAI
GyCesGCsAI
GFCAkGCAAI
GFCAsGCsAI
GFCA-GCAAI
201
GDVLIGdKtT
GDVLledKtT
GDVLvGGKsT
GDVLIGGapT
GDlLldGhsT
GDVLIGGKtT
GDVLIGGK-T
EPGITFlaAK
EPGITFlaAK
EPGITFlvAK
EPGITFvlAK
EtsvTFiigK
EPGlTFmvAK
EPGITF--AK
101
KeQdFIEATK
KeQdFIEATK
KdQeFIEATK
KdQeFIEATK
KnQkFIEATr
KdQeFIEATK
K-Q-FIEATK
301
NYMdAQYfGE
NYMdAQYyGE
NYMnAQYfGE
NYldAQYyGE
dYlntQYyGv
NYMnAQYfGE
NYM-AQY-GE
1
dsdgeliALK
dsgsDliALK
seeegDIVALK
sgdaDIVtLK
ssdsDpVpLv
ggegDIVALK
-----DIVALK
MGqYHTVFDY
MGrYHTVFDY
MGpYHTVFDY
MGkYHTVFDf
MGaYHTVFDf
MGaYHTVFDY
MG-YHTVFDY
AVVWMQNQir
AVVWMQNQik
AVVWMQNQLa
AvvwiQsQLr
AVVWieNQLr
AVVWMQNQLa
AVVWMQNQL-
ADSGISLLAG
ADSGISLLAG
ADSGISLLAG
ADSGISLLAG
vDSGISLLAG
ADSGISLLAG
ADSGISLLAG
FDGILGLGFQ
FDGILGLGFQ
FDGILGLGFk
FDGILGLGFQ
FDGILGLGyp
FDGILGLGFQ
FDGILGLGFQ
IGiGTPPQKF
IGiGsPPQKF
IGvGTPPQKF
IaiGTPPQKF
IG1GsPPQnF
IGvGTPPQKF
IG-GTPPQKF
YVdkLCeRLP
YVNeLCdRLP
YVNqLCnRLP
YVNeLCrRip
YaNqLCeRLP
YiNqLCdkLP
YVN-LC-RLP
AIGAaGVmSQ
AIGAkGVmSQ
kIGAaGVvSQ
AIGAsGVaSQ
AIGAeGiiSt
kIGAtGVvSQ
AIGA-GV-SQ
WYtMlnQGLV
WYnMvnQGLV
WYkMieQGLV
WYnMlkQGLy
WqsMqeQeLl
WYkMveQGLV
WY-M--QGLV
LWVPSsKCYF
LWVPSAKCYF
LWVPSAKCYF
LWVPSsKCYF
LWVPSAKCYF
LWVPSAKCYF
LWVPSAKCYF
441
GnlRVGFAeA A
GK1RVGFAeA A
GK1RiGFAkA A
GKaqVGFAeA A
GKdRiGFAks A
GKmRVGFAks A
GK-RVGFA-A A
rNeTedn1iN
QNkTqd11Ld
QNmTqer1Ld
eNkTke1ILN
QNkTqd11LN
QN-T---ILN
Q~een1iN
tTtlvTqINq
PTAIITeINh
PTAIITeINe
PTtIITmINh
PTAlvaqvNh
PTAIITeINe
PTAIIT-IN-
EISVGdAVPV
EISVGksVPl
EISVGkAVPV
EISVGnAaPV
EISVGkApPi
EISVGdAVPV
EISVG-AVPV
TVIFDTGSSN
TVIFDTGSSN
TVIFDTGSSN
TVvFDTGSSN
TVIFDTGSSN
TVIFDTGSSN
TVIFDTGSSN
SPMGESAVDC
SPMGESAVDC
SPMGESAVDC
SPMGESAVDC
SPnGEStVsC
SPMGESsVDC
SPMGESAVDC
qCKs1VdQYG
qCKT1VSQYG
eCKTiVSQYG
qCKTvVdQYG
eCKevVSeYG
eCKTvVSQYG
-CKT-VSQYG
qEPVFSFWLN
qEPVFSFWfN
sdPVFSFWLN
kEPVFSFWLN
addVFSFWLN
sEPVFSFWfN
-EPVFSFWLN
SvAC1FHSkY
SvAClFHSkY
SIACylHSrY
SIAC1FHSkY
SIACylHSrY
SIACfFHSrY
SIAC-FHS-Y
sSLSSMPnla
nSLSSMPnla
gSLgSMPdle
aqLStMPtvs
hqiSkMPnla
gSLaSMPeIs
-SLSSMP-I-
ksmiemLLsE
ktmiemLLsE
qq1LdLLLaE
qt1LdLLLsE
em1LnLLiaq
qq1LdLLLaE
--1L-LLL-E
RnaDEqEGGE
RnaDEeEGGE
RhvDEgEGGE
RnaDdeEGGE
RdpDassGGE
RhsDEgEGGE
R--DE-EGGE
rStdStTYKK
KSshSSTYKK
KagaSSTYKK
KSsrSSTYeK
nSkkSSsYKa
KSgqSSTYqK
KS--SSTYKK
FTvGGKtFnL
FTIGGKvFeL
FTIGGKkFaL
lTIGGKvFdL
FTlanKtFiL
FTIGaKkFaL
FTIGGK-F-L
eQPeK1CSQm
aQPdK1CSQm
TQPkK1CSQv
TQPkK1CSQi
TdPqKvCSQv
TQPsK1CSQv
TQP-K1CSQ-
LVFGGvDPnH
LVFGGvDPnH
iiFGGmDPkH
LVFGGvDPkH
LVFGGmDPkH
iVFGGmDPsH
LVFGG-DP-H
NGKsAAIQYG
NGtsAAIQYG
NGKpAAIQYG
NGKsAAlhYG
dGetckltYG
NGKpAAIQYG
NGK-AAIQYG
sPEqYvLKVG
cPEqYILKiG
kPEeYILKVG
aPheYvLKVG
tPEqYlvKle
kPEeYILKVG
-PE-YILKVG
kLCsFDGshd
kLCTFDGaRd
GLCTFDGtRg
GLCTFDGkRg
GLCmFDGkRs
GLCTFDGkhg
GLCTFDG-R-
fKGeHTYVPV
fKGkHTYVPV
yvGeHTYVPV
fKGqHiYVPV
yKGdHTYVPV
yKGnHTYVPV
-KG-HTYVPV
TGSIsGFFSq
TGSIsGFvSq
TGSlaGyFSe
TGalaGFFSn
sGaIsGFFSk
TGSlaGFFSe
TGSI-GFFS-
400
EGAtAQCISG
EGeAAQCISG
EGAAAQCISG
EGAAAQCISG
qGgqtvCISG
EGAAAQCISG
EGAAAQCISG
tSmi1ESVVD
aSsi1ESVVD
VSaG1rSVVD
VSmGIESVVD
VSnG1ESVVD
VSaG1kSVVD
VS-GIESVVD
300
200
TQKGYWQFeM
TeKGYWQFdM
TQKGYWQFdM
TQKGYWQFdM
srKGYWQFnM
sQKGYWQFeM
TQKGYWQF-M
100
DSVklGDL1V
DSVklGDLVV
DSVtVGDLVV
DaVtVGDLVV
DnV1VGDqVV
DSVtVGDLVV
DSV-VGDLVV
Fig. 7. Alignment of deduced amino acid sequences of six plant aspartic proteinases. The putative mature proteins are shown. The consensus sequence is
based on identical amino acid residues (upper case) in at least four of the six enzymes. Amino acid residues identical for all six plant aspartic proteinases
are shown in bold. Putative active site aspartic acids are in italic and bold. Putative glycosylation sites are underlined. The plant-specific insert is shown in
italic. Conserved cysteine residues are marked*. (EMBL Data Bank: cyprosin I: X69193; cyprosin II: X81984; barley AP: X56136; brassica AP: X77260; rice
Consensus
cyprosin I
cyprosin II
barley AP
bras sica AP
rice 1
rice 2
Consensus
cyprosin I
cyprosin II
barley AP
bras sica AP
rice 1
rice 2
Consensus
cyprosin I
cyprosin II
barley AP
brassica AP
rice 1
rice 2
cyprosin I
cyprosin II
barley AP
brassica AP
rice 1
rice 2
Consensus
Consensus
cyprosin I
cyprosin II
barley AP
brassica AP
rice 1
rice 2
?'-
(1)
::;.
0-
...,0
.j>.
.....
.j>.
145
From the consensus sequence in Fig. 7, it may be calculated that the plantspecific insert shows 73% conserved residues, indicating that this region has
similar characteristics. It is interesting to note that the six cysteine residues
present in the plant-specific region are all conserved in the six enzymes,
indicating a possible conserved tertiary structure of this region through
disulfide bridges. Furthermore, a putative glycosylation site (underlined in Fig.
7) is conserved in the plant-specific region in all six enzymes. Due to these
conserved features of the plant-specific insert, it may be assumed that this
additional part of the protein has a specific function in the plant. At present,
we can only speculate about this function.
5.4 Genomic Organization of Cyprosin Genes
146
Fig. 8. Schematic representation showing the overall "fold" of cyprosin, generated using the
program 0 (Jones et al. 1991). Helices are shown as cylinders and sheets as flat arrows .
The remaining polypeptide chain is shown as a rope which represents the turn/coil regions in the
molecule. Numbers of amino acid residues are according to Fig. 6. The location of the plantspecific insert is indicated by a gap between G246 and S351. The catalytic aspartate side chains
(D35 and D222) and conserved cysteins participating in disulfide bridges (C48-C54, C213-C217,
C360-C397) are also shown and labeled. (Cordeiro et al. 1994c)
sin. A schematic representation of the model showing the overall fold and the
probable site of the large insert is presented in Fig. 8.
In order to correlate observations from kinetics with structural features
in the enzyme, we constructed models of cyprosin complexed with ligands in
the active site to mimic the transition-state analog. Most of the pockets in
cyprosin are very similar to those in the barley-grain aspartic proteinase
(Guruprasad et al. 1994); the only difference is near the S2' and S3' pockets.
This difference is due to His295 in cyprosin compared with Arg295 in the
barley enzyme.
147
E
E
E E
E E
E
E
E
E
E
E
E
E
E
E
E
E
~
.....
If)
<"J
(//
(//
(//
(/)
(//
<"J
(/)
If)
N
(//
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large subunit
cyprosln 3
31 .0 kOa
large subunit
cyprosin 1 and 2
14.4 kOa
Fig. 9. Immunostained Western blot following SDS-PAGE gel of crude extracts from various
organs and tissues of Cynara cardunculus. The samples (to ~g of protein per lane) were flower
buds and flowers at various stages of development, corollas and styles from mature flowers, seeds,
leaves, and midribs. (Cordeiro et al. 1994b)
148
Total RNA, isolated from different organs, including several stages of flower
development, styles from mature flowers, bract, and leaf, was separated by
agarose gel electrophoresis, blotted onto a GeneScreen membrane and hybridized with the 32P-Iabeled EcoRI 1.7-kb insert of cyprols. The cDNA clone
hybridized to a 1.8-kbmRNA from flower and bract tissues, but there was no
detectable hybridization to mRNA from leaves (Cordeiro et al. 1994a).
The intensity of hybridizing transcripts increased from early stages of
floral development (flowers 6-10mm in length) to later stages of floral development (flowers up to 40mm in length), while in the later stages of floral
development (flowers 50mm in length and styles from open flowers) no hybridization signal was visualized, indicating that the synthesis of mRNA encoding the cyprosin starts in early stages of floral development and switches off
at maturation of the flower. These findings are basically in agreement with the
enzyme activity measured in extracts made from flowers at different stages of
development and also with the organ-specific expression of cyprosins.
In conclusion, the genes coding for the cyprosins are expressed during
early stages of flower development (up to 40mm long). Furthermore, the
expression of the cyprosin genes seems to be specific for organs present in
flowers and bracts.
149
Fig. lOA-C. Localization of cyprosins in styles of Cynara cardunculus. Visualization of immunogold labeling by the silver enhancement technique. A Transversell section of style showing
presence of cyprosins in the epidermal cell layer. B Amplified view of cells of the epidermal layer
of styles. C Control treated with BSA, invertase and rabbit preimmune serum. C Corolla; Cu
cuticule; Ep epidermal layer; PC parenchymatous cortex; TT transmitting tissue; VB vascular
bundle. Bars (Cordeiro et al. 1994b)
150
151
The expression of cyprosin mRNA is similar to the expression of genes controlling flower development. Examples are the agamous and plenna genes
responsible for the c function during floral organ development (Cohen 1992).
The c function in floral meristem is responsible for the development of fertile
organs (stamens and carpels). The agamous and plenna mRNAs are first
detected in initial stages of organ development at a region destined to form
stamen and carpel primordia. The expression continues further and until
stages close to mature organs. Control of flower development is performed in
a synergistic and/or antagonistic interaction between genes involved at each
stage.
The expression of cyprosin mRNA has been detected at early stages
of flower development and is switched off at later stages and in mature
styles.
Based on this general knowledge about expression of genes involved in
flower development, we can assign a hypothetical role for cyprosins and
cyprosin genes in the control of flower development and organ differentiation.
They may act directly by controlling the differentiation of one specific organ,
or by preventing the accumulation of other flower gene products that could
give rise to phenotype alterations.
Acknowledgments. This work was supported by grants from the Swedish Natural Science Research Council, the Swedish Council for Forestry, and Agricultural Research to PEB.
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1 Introduction
Botanically, Ephedra (Fig. 1) is a member of the smallest and most problematic division of flowering plants, the Gnetopsida, and major questions remain
unanswered about the taxonomy of the Gnetopsida and the evolutionary
relationships of the different genera within the division. Ephedra is the largest
and most widely distributed genus in the Gnetopsida, a subgroup of the
gymnosperms. Many anatomical and reproductive characters of Ephedra are
angiosperm-like (Alosi and Alfieri 1972; Friedman 1990; Carlquist 1992;
Simcha 1994). Recent molecular and chemical studies support the view that
the Gnetopsida are the closest living relatives of the angiosperms but that the
angiosperms are not derived from them (Martin and Dowd 1986; Chase et al.
1993; Doyle et al. 1994; Nickrent and Soltis 1995).
Pharmacologically, Ephedra has been the main botanical source of the
active alkaloids I-ephedrine (E) and d-pseudoephedrine (PE) for thousands of
years, with records of its medicinal use dating to 5000 years B.P. (Evans 1989).
The alkaloids E and PE remain important drugs today - the current world
consumption of d-pseudoephedrine salts (PE-sulphate and PE-hydrochloride)
stands at 1000-2000 tonnes per annum with a value of approximately $100-200
million (c. O'Brien, pers. comm.).
Powdered Ephedra stems are used in traditional herbal medicines as a
hypertensive aid to treat asthma, nose and lung congestion, hay fever, and
several other ailments. E. sinica and E. equisitina are the two main species
exploited. The role of I-ephedrine and d-pseudoephedrine in modern medicine has been changing slowly as new applications for these alkaloids are
realised. Acting as potent vasoconstrictors, they can be used to elevate blood
pressure and increase heart and respiratory rate (Gilman et al. 1990). They are
very close to adrenaline in structure and pharmaceutical activity, but have the
advantage that they can be administered orally (Fig. 2). Uses for the Ephedra
alkaloids continue to expand. L-ephedrine (E) is currently under investigation
155
Ephedra Species
Fig. 1. An Ephedra minima plant with ripe female cones (megasporangiate strobili on which the
bracts have become swollen). Ephedra species produce one to two seeds per cone
cgf,
cY$cr
n
,
o XlQJ
",01-1
NHCH
rn
NHCH l
I-ephedri ne
d-pseudoephedrine
I-norephedrine
d-norpseudoephedrine
HO
HO
HO
~
o X
",H
ol'
."
GI
NHCH
adrenaline
(epinephrine)
J
J
156
as a therapeutic agent for the treatment of obesity (Zgourides et al. 1989). Lephedrine has been linked with toxic psychosis (Whitehouse and Duncan
1987) with the result that the demand is increasing for the stereoisomer, dpseudoephedrine (PE), which has fewer side effects than I-ephedrine and a
weaker, longer-lasting effect upon the central nervous and cardiac systems. A
recent development is the introduction by Schering-Plough of a new combination product containing pseudoephedrine sulphate and Loratadine, a nonsedating antihistamine (Sanchez Garcia and Trejo Tapia 1993).
Two current methods of producing E and PE are outlined in Fig. 3. China
and Pakistan are the main producers. Attempts have been made to cultivate
fermentable sugar
(molasses)
CHO
benzaldehyde
"Saccharomyces cervisiae
'\
~III
lff-C--CH
OH 0
phenylacetylcarbinol (PAC)
L-1-phenyl-2-oxo-propanol
CH 3 NH 2
methylamine
I catalytic reduction
OH H
h-b-CH
IQ\
~I
I
H
NHCH 3
l-ephedrine
Qllnd
main sources
Pakistan
E. sinica
E. equisitina
E. intermedia
[Hu, 1969,
Yin, 1994
Man & Yang, 1995)
E. gerardiana
E. nebroidensis
chemical
conversion
'0'h- b-- CH
~II
OH NHCH 3
d-pseudoephedrine
Fig. 3. Current processes employed in the manufacture of I-ephedrine and d-pseudoephedrine
Ephedra Species
157
Ephedra in Australia, England, Kenya and the United States, but these ventures failed, largely for economic reasons (Morton 1977).
Although it is currently possible to manufacture these compounds from
benzaldehyde via a yeast fermentation and catalytic reduction, several species
of Ephedra continue to be exploited for these high-demand pharmaceutically
active compounds (Fig. 3). Therefore, the continued importance of these wellknown Ephedra alkaloids appears assured for some time to come, and there is
the prospect that other pharmacologically active agents may be found in
Ephedra and other members of the Gnetopsida.
158
Table 1. Reported alkaloid content of different Ephedra species and Ephedrae herbae. Alkaloid
Alkaloid content
Reference
E. americana var.
0.02% E; 0.01 % PE
Q'Dowd (1991)
Q'Dowd (1991)
andina
E. andina
E. alata Decne
E. alenda (Stapf)
Andreanszky
E. altissima Desf.
E. americana Humb.
& Bonpl.
E. andina Poepp.
E. californica S. Wats.
E. ciliata Fisch et Mey
E. dis tach ya"
E. equisitina Bunge
E. foliata Boiss
E. distachya
(continued)
E. fragilis Desf.
E. fragilis ssp.
camplyopoda
E. gerardiana Wall b
E and PE present
E present
E present
E and PE present
Afghanistan. No reference
to alkaloid but used locally
as antiasthma tic
E and PE both present
Total alkaloid: 0.29% in
nodes and 0.69% in
internodes. E, NE, PE, ME
and MPE present
0.07% E; 0.2% PE
0.04% E; 0.05% PE; 0.02%
NE; 0.05% NPE
0.75-1.0% E with variable PE
1.75% total alkaloid, 1.58% E
2.09% total alkaloid, 1.0% E,
0.05% NE, 0.4% PE, 0.47%
NPE, 0.05% ME, trace MPE
Mongolia. 1.72% total alkaloid
0.8% E; 0.5% PE
Iraqi species pharmacologically
active
Devoid of E
India (Rajasthan) during
different seasons - 0.01 % E
Thar Desert, India; 0.0% trace alkaloid
E and PE present
Menorca; 0.06% E; 0.03% PE;
0.05% NE; 0.09% NPE
0.14% E; 0.51 % PE
Total alkaloid - 0.8-1.4% of
which ~50% E: PE, ME,
NE, MPE, NPE also present
Rajasthan, India. 1.7% E
Japan. 0.03% E and 0.97-1.07%
PE. NE and ME not detected
Smith (1977)
Willaman and Schubert
(1961)
Willaman and Schubert
(1961)
Willaman and Schubert
(1961)
Smith (1977)
Willaman and Schubert
(1961 )
Younos et al. (1987)
Yamasaki et al. (1973)
Kasahara et al. (1986)
Q'Dowd (1991)
Q'Dowd (1991)
Windhotz et al. (1983)
Morton (1977)
Jian et al. (1988)
Gazaliev et al. (1988)
Q'Dowd (1991)
Al Sarraj et al. (1985)
Chaudhri (1957)
Yamasaki et al. (1973)
Chopra et al. (1956)
Smith (1977)
Previously unpubl.
Q'Dowd (1991)
Morton (1977); Duke
(1986)
Ramawat and Arya
(1979a)
Yamasaki et al. (1974)
Ephedra Species
159
Table 1. Continued
Species
Alkaloid content
Reference
Chaudhri (1957)
E. gerardiana vaL
sikkimensis
E. gracilis R. Phil
E. intermedia'
E. helba
E. major
Q'Dowd (1991)
Yamasaki et al. (1974)
Willaman and Schubert
(1961)
Akiba et al. (1979)
Willaman and Schubert
(1961)
Morton (1977);
Nishimoto (1980)
Jian et al. (1988)
Chaudhri (1957)
Yamasaki et al. (1974)
Q'Dowd (1991)
Q'Dowd (1991)
Smith (1977)
E and PE present
Material from Pakistan reported
to contain 1.3 % total alkaloid
Baluchistan. 0.57-1.12% E.
Smith (1977)
Shah and Shah (1966)
Devoid of alkaloid
E and PE present
Total alkaloid ~1.4%
Duke (1986)
Smith (1977)
Pelt et al. (1967)
Devoid of alkaloid
Trace E; 0.01 % PE
1-2.5% total alkaloid
1.38% total alkaloid of which
0.77% E, 0.05% NE, 0.31 %
PE, 0.14% NPE, 0.09% ME,
0.12% MPE
Q'Dowd (1991)
Q'Dowd (1991)
Hu (1969)
]ian et al. (1988)
160
Table 1. Continued
Species
Alkaloid content
Reference
Liu et al. (1993)
E. triandra Tul.
E. tweediana c.A.
Mey
E. viridis Coville
E. vulgaris L.c. Rich.
(see E. distachya)
Chinese Ephedrae
Herba
Pakistani Ephedra
Herba
Ephedrae Herba
Taiwan market
Major ephedrine
yielders
Contains E
E and PE present
0.0% E; 0.05% PE
Contains E, PE, NE and ME
30 mixed samples over 5-year
period: 0.8-1.8% total
alkaloid
30 mixed samples over 5-year
period: 2.5 % total alkaloid
0.9-2.3% total alkaloid of which
0.4-1.6% E, 0.2-0.8% PE,
also ME, MPE, NE and NPE
E. sinica Stapf. (China)
E. equisitina Bunge. (China)
E. gerardiana Wall. (India and
Pakistan)
Ephedra Species
161
Plant part
Dry weight
I-ephedrine (%)
Dry weight
d-pseudoephedrine (%)
o
o
0.17
0.04
0.16
0.05
0.02
0.16
0.06
0.17
0.04
0.02
0.01
0.01
0.01
OJn
0.01
0.01
Table 3. Compounds isolated from Ephedra subsequent to those listed in Gottleib and Kubitzki's
Compound
Reference
E. sinica
Mahuannin D
Catechins
Alkaloids
(- )-Ephedrine, (- )-norephedrine,
(- )-N-methylephedrine, (+ )-Wephedrine, ( + )-nor-W-ephedrine, (+ )N-methyl-W-ephedrine
Ephedralone
Lignans
( )-Syringaresinol
E. andina,
E. breana and
E. frustillata,
Various - see
Refs.
E. alata
E. alata
Glycan~
E. distach ya
E. gerardiana
E. gerardiana
E. alata
Ephedran A B, C, D and E
Saponin
Tannins
Gallic and elJagic acids
Digalloyl-glucoside
Nilocitin
In summary, there is strong evidence that the age and type of tissue
sampled, environmental growth conditions and the method of analysis used all
strongly influence the alkaloid content measured for that species. These factors are very important to keep in mind, especially when analysing small
quantities of plant tissue, and also make it very difficult to assess much of the
older work shown in Table 1.
Two methods of analysis were used to determine alkaloid presence and concentration in plant tissues; high performance liquid chromatography (HPLC)
and thin layer chromatographic analysis (TLC).
Sample Preparation. A known quantity of tissue (0.1-0.2 g parent plant or 0.51 g cultured cells) was ground in liquid nitrogen and extracted by 30-min
sonication in a known quantity (0.5-1 ml) of ethanol or 50mM Tris, pH 8.
Samples were spun and the supernatant pipetted off.
Chromatographic Conditions. Adapted from Barkan et al. (1981). HPLC was
carried out on a Spectra-Physics Model 8000 HPLC equipped with an SP 8000
integrator and Spectra Physics Model 770 UV-visible detector set at 21Onm. A
NOV A-PAK phenyl (reverse phase) column was used (IO.um particle size;
15cm X 3.9mm internal diameter). A guard column was employed. Mobile
phase was 1 % acetonitrile in 50mM monobasic sodium phosphate, flow rate
Imlmin- 1, 20.uM injection loop.
For TLC several systems were tested, including normal and reverse phase
(O'Dowd 1991). Best results were achieved with aluminium-backed 0.2-mmthick silica gel 60F254 stationary phase and butan-l-ol: acetic acid: water
(4: 1 : 1) mobile phase, localisation agent ninhydrin.
2.2 Biosynthesis of Ephedra Alkaloids
163
Ephedra Species
if'
1r.>:
:1
il
il
!I
II
I)
!
!
I
:1
;. ~:.1
"."!
(""!
,: ...!
I
t
Iijj
1,.1
~.
':'!
,
~
"I
rt' :,
~
"
"1
,I
,:,.1
I ..
f:!
,,f' Lf")
'
.:-.. j
.J'
(\1
Fig. SA-Co HPLC chromatograms of E. minima stem methanol extracts. A Top 2 em of shoots
contain no alkaloid, B Tertiary stems contain small quantities ofE (0.04% dry wt.) and PE (0.05%
dry wt.), C Primary stems contain highest levels with 0,18 and 0.19% dry wt. E and PE, respectively. Retention times vary between samples, Confirmation by spiking with E and PE standards
and TLC
found in other plant species as well, for example E and/or PE have been found
in Sida cordi/olia (Ghosal et al. 1975), Catha edulis (Kalix 1991), Roemeria
re/racta (Southon and Buckingham 1989), Pinella ternata tubers (Oshio et al.
1978), Taxus baccata, Aconitum napellus (Duke 1986) and Hamelia patens
164
(Chaudhri and Thakur 1991). The fact that they appear in such a broad
spectrum of plants suggests that they could be derived from the same primary
metabolite, e.g. amino acid or the same breakdown product of a primary
metabolic pathway, but, in fact, little has been known until recently about the
biosynthesis of the ephedrines and it is not known if these compounds are
synthesised by the same biosynthetic route in the different genera. Table 4 lists
synonyms commonly used in reference to this group of compounds. It has not
been easy to establish the biosynthetic pathway for the ephedrines (Fig. 6).
Efforts have been directed at finding the main sources of the C6 ring, the C3
side chain, the amino group and the N-methyl group.
Phenylalanine was an obvious potential precursor - it is a C6-C3 compound
with an amino group on the C2 carbon of the C3 side chain, as in the ephedrines,
but in some ways this similarity may have been misleading certainly, it has been
established that in Ephedra and Catha edulis, label from [14C] phenylalanine
can be found in I-ephedrine (Shibata and Imaseki 1956; Yamasaki et al. 1973;
Cordell 1981, Grue-Sji'lrensen and Spenser 1988, 1989). Phenylalanine was
reported to be an efficient precursor of d-norpseudoephedrine (cathine) in the
latter species (Shibata and Imaseki 1953). Therefore, it seems clear that the
ring can be derived from phenylalanine, although it cannot be assumed to be
the only source. In Ephedra, phenylalanine was found only to provide a C6-C1
unit (Yamasaki et al. 1973) and not the complete aminophenylpropanoid
system of E as originally thought.
Phenalanine ammonia lyase (PAL) catalyses the conversion of 1phenylalanine to trans-cinnamic acid by elimination of ammonia. Cinnamic
acid, in turn, can be hydrolysed by f3-oxidation into benzoic acid and acetic
acid. Although neither of these steps has been demonstrated in Ephedra, they
are generally common within the higher plant kingdom and are assumed to be
present in the Ephedra genus. Feeding experiments using [7- 14 C]-benzoic acid
confirmed that benzoic acid was incorporated with high efficiency into E in
Ephedra (Yamasaki et al. 1973). Since the incorporation of [14C]-benzoic acid
was much higher than that of p4C]-phenylalanine, Yamasaki et al. (1973)
suggested that benzoic acid may be derived by an alternative route, for example through shikimic acid. Therefore, benzoic acid could provide a C6-C 1 unit
which is subsequently condensed with a C2 unit to form the carbon backbone
ofNE/E.
Table 4. Names and synonyms of main ephedrine alkaloids
Common name
Ephedrine
Pseudoephedrine
Norephedrine
Norpseudoephedrine
Cathinone
Synonyms
(lR,2S)-( - )-ephedrine
(lS,2S)-( +)-pseudoephedrine
(lR,2S)-( - )-norephedrine
(lS,2S)-( + )-norpseudoephedrine
cathine
S,S( + ) phenylpropanolamine
(S)-( - )-2-amino-l-phenylpropanone-1one
S( - )alpha-aminopropiophenone
Ephedra Species
165
~CH2-TH-COOH
~j
NH3
t
~
PHENYLALANINE
NH2
phenylalanine ammonia lyase
@- ~-C-CH
II
PHENYLPROPANEDIONE
3
@-~-TH-CHJ
.
reductlOn
CATIllNONE
~H2 NADH+H+/NADPH+W
NAD+ / NADP
(cofactors
?)
OH
NOREPHEDRINE
OR
CH -CH-CH 3
S-adenosylmethionine
methylation
@-~:-TH-CHJ
NHCH 3
EPHEDRINE
OR
PSEUDOEPHEDRINE
Fig. 6. Proposed biosynthetic pathway for the Ephedra alkaloids, I-ephedrine and dpseudoephedrine. (After Grue-Sorenson and Spenser 1993)
166
The identity of the Cz unit which condensed with benzoic acid to complete
the three-carbon chain remained unknown until the discovery by GrueS~rensen and Spenser (1988) that it was provided by pyruvic acid. Thus, the
carbon skeleton of the Ephedra alkaloids is generated from two fragments, a
C6-C1unit from benzoic acid and a CH3CO moiety from pyruvate. The labelled
nitrogen from [15 N]-phenylalanine was reported to be incorporated into E
(Shibata and Imaseki 1953). The transfer of 15N from phenylalanine would
appear to occur either by direct transamination from phenylalanine or another
amino acid (Fig. 6) to the intermediate precursor of NE/NPE of from free
rSN]-ammonia liberated by the action of PAL. The methyl group on the
nitrogen is derived from active methionine (Shibata et al. 1958).
Efforts to establish other biosynthetic intermediates between benzoic
acid and the ephedrines have not been successful until quite recently.
Aminoacetophenone, an intermediate suggested by Yamasaki et al. (1973),
was found not to be directly incorporated into E. An alternative structure, 1hydroxy-1-phenylpropan-2-one proposed by Grue-S~rensen and Spenser
(1988) was neither isolated nor demonstrated to provide whole or partial
labelling of E from [14C]-labelled samples of this compound. Grue-S~rensen
and Spenser (1993), working with growing stems of mature E. gerardiana
plants, have recently reported that 1-phenylpropane-1,2-dione was incorporated into the alkaloids, and claim this structure to be a hitherto unknown and
critical intermediate in the biosynthetic pathway. This compound first undergoes transamination at the Cz carboxyl group to produce cathinone. Reduction
of cathinone at C1 leads to the production of NE or NPE, which are subsequently methylated to give E/PE (Grue-S~rensen and Spenser 1993). Thus the
biosynthesis of E/PE is envisaged by Grue-S~rensen and Spenser to follow the
Table 5. L-ephedrine and d-pseudoephedrine content (% dry weight) of parent plant stem, callus
and suspension cultures of several Ephedra species (O'Dowd 1991). All cultures were derived
from stem explants and maintained on MS basal medium supplemented with 5,uM auxin and
0.25,uM kinetin
Auxin
Species
E. andina
E. equisitina
E. intermedia
E. major var.
procera
E. minima
E. fragilis ssp.
camplyopoda
2,4-D
2,4-D
2,4-D
NAA
2,4-D
NAA
Dry wt.
I-ephedrine (%)
Dry wt.
d-pseudoephedrine (%)
Trace
Trace
0.00
0.80
Trace
Trace
0.06
Trace
0.27
Trace-0.005
0.00-0.004
0.50
Trace
0.14
Trace
0.00
0.13
0.08
0.00
0.50
0.01
Trace
0.15
Trace
0.75
Trace-0.04
Trace-0.14
1.04
0.003
0.51
0.01-0.06
Trace-O.01
Ephedra Species
167
pathway outlined in Fig. 6. This pathway may not be complete, since the
condensation of the pyruvic acid and benzoic acid to produce 1phenylpropane-1,2-dione appears to be a complex reaction. It will be extremely interesting to identify the enzyme( s) and cofactor( s) involved. Further
evidence to support this pathway comes from reports that cathinone is believed to be enzymatically reduced to cathine (NPE) and NE in Catha edulis
(Kalix 1991).
In our studies, [14C)-benzoic acid was incorporated into E and PE in whole
plants of E. minima. Suspension cultures of several Ephedra species failed to
produce E and PE or synthesised only trace amounts of these alkaloids (Table
5). However, in these cultures r4C)-phenylalanine and r 4C)-benzoic acid were
incorporated into compounds (also found in parent plants) which themselves
were metabolised further but could not be isolated in sufficient quantity for
structural analysis. In a recent report Song et al. (1994a) isolated and identified
a benzoic acid derivative (6-methyl-1-heptene-5-yl-4' -benzoic acid) and a
number of p-coumaroylamino acids (Song et al. 1994b, 1995) from yeastelicited E. distachya cultures, but these are unlikely candidates for
biosynthetic precursor to NE/E.
168
Straus and Gerding (1963), and Konar (1963) were the first to report in vitro
culture of Ephedra. The former group isolated and maintained callus tissues of
an unidentified Ephedra species on modified White's medium (White 1963)
supplemented with 20% coconut milk and 9.uM 2,4-D. Exogenous auxin (2,4D or NAA) was required for callus growth but the auxin IAA failed to support
growth, either alone or in conjunction with a cytokinin, an observation supported in later studies (Ramawat and Arya 1976; O'Dowd et al. 1993).
O'Dowd et al. (1993) found that IBA was similarly ineffective in the initiation
of callus from stem explants of nine species of Ephedra but that it could be
used to maintain established cultures when substituted for 2,4-D or NAA. It
also proved to be the only auxin capable of supporting healthy, friable E.
minima Hao callus growth without inducing morphogenesis. It may be that
this species is more auxin-sensitive or contains higher endogenous auxin levels
than other species examined.
General conclusions concerning the in vitro growth characteristics of the
genus are difficult to draw from the literature, as the source explants vary and
the media used were often not fully defined, frequently being supplemented
with coconut milk. Table 6 summarises the in vitro culture of Ephedra by other
workers. Unless otherwise stated, Murashige and Skoog (1962) basal MS
medium was used in all studies mentioned.
One of the most -studied Ephedra species in vitro is E. Joliata, commonly
occurring in India and containing trace or no alkaloids (Table 1). Sankhla et al.
(1967a,b) examined the development of callus from mature embryos and the
subsequent production of "embryo-like structures" in some cultures. Further
work on E. Joliata is described in Section 3.5.
Production of callus from several other Ephedra species has been
achieved using stem explants as the source material for culture initiation.
Ramawat and Arya (1977, 1979c,d) examined medium composition and its
effect on callus growth of E. Joliata and E. gerardiana, a high alkaloid-yielding
species (Table 1). They tested the growth and total carbohydrate content of
callus on a range of sugars (1-6%) and found that sucrose gave highest growth
rates followed by glucose, maltose and fructose (Ramawat and Arya 1977).
Arabinose, lactose, mannitol, sorbose, soluble starch and xylose failed to
support growth. E. Joliata grew better on all treatments than E. gerardiana.
Following this, they examined callus growth and protein content on different
nitrogen sources (Ramawat and Arya 1979d). A combination of nitrate and
ammonium salts was necessary for growth (optimally 4: 1 at 400mg NI- I ). E.
gerardiana contained almost four times as much protein as E. Joliata callus on
all treatments.
Of the 18 species, subspecies or varieties of Ephedra examined by our
group, it was possible to initiate callus cultures from all of them with varying
degrees of success (O'Dowd 1991). E. altissima, E. andina, E. americana var
andina, E. distachya, E. distachya ssp helvetica, E. equisitina, E. Jragilis, E.
Jragilis var camplyopoda, E. gerardiana, E. gerardiana var. sikkimensis, E.
intermedia, E. major, E. major ssp. procera, E. minima, E. saxatilis, E. scoparia,
2. Seeds
1. Stem internodes
4. Stem internodes
3. Female
gametophyte
embryos
2. Pollen
I. Near-mature
E. foliata
E. fragilis
I. Stem internode
E. distachya,
E. equisitina
1. Stem internode
E. andina
Medium'
Explant
Species
Adventitious
buds
Adventitious
buds
Callus
Callus
Shoot
regeneration
"Embryo-like
structures ..
Suspension
culture
Haploid
plantlets
Callus from
root
Haploid tissue
mass
Callus and roots
Shoot buds
Callus
Suspension
culture
Callus
Result
Konar (1963)
O'Oowd et al (1993)
O'Oowd et al (1993)
Reference
a,
'D
~.
(t)
"0
C/J
<:l
I'l..
~
;:,-
1. Stem internodes
1. Stem internode
Adventitious
buds
Suspension
culture
Callus
Callus
Shoot
regeneration
Shoot
regeneration
O'Dowd (1991)
Callus
Callus
Reference
Result
Medium a
E. intermedia,
E. major ssp.
procera, E.
saxatilisa
E. saxatilis
1. Stem internodes
E. gerardiana
Explant
Species
Table 6. Continued
f-'
-.J
0-
::;
;>o
d
o
:z
Ephedra Species
171
E. sinica and E. viridis were initiated into culture. Also two unidentified
species from Kew and Marburg University Botanic Gardens. Species producing large quantities of phenolics, especially E. andina, E. distachya and E.
gerardiana var. sikkimensis required movement to fresh medium every 24-48h
until darkening of the medium ceased. Callus growth rates were recorded on
MS medium supplemented with 1.2.uM kin and 27.uM NAA or 2,4-D. Growth
rates and callus quality varied with species. Over 14-day subculture periods,
the highest relative growth rate (Singer 1986) of 0.22 0.01 gig/day was
recorded for E. major ssp. procera (O'Dowd 1991).
172
Callus cultures from seven of our Ephedra species yielded E and/or PE but
these quantities were low compared to the parent plant (Table 5) and diminished to zero with successive subcultures over 3-5 months (O'Dowd et al.
1993). It is possible that alkaloid present in callus tissues in the early stages of
the culture could be residual from the parent plant and that we were looking
at a dilution effect over successive subcultures. Nevertheless, we did record
several cases in which callus showed steady alkaloid levels over two to three
subcultures or an increase in alkaloid level compared to the previous subculture (unpubl.), but this production could not be sustained or repeated. It is
possible that some cultures possessed the ability to produce alkaloid but the
growth conditions were not conducive. We examined heat and cold shock,
light quality and the abiotic "elicitor" vanadyl sulphate, but could not claim
convincing evidence of net synthesis of E, PE, NE or NPE (unpubl.).
3.1.3 Cell Suspension Culture Growth Studies
Strategies for the production of plant natural products using cell suspension
cultures have been widely discussed since they offer the potential for commercial exploitation. The first reports of the establishment of cell suspension
cultures of E. foliata were by Khanna and Uddin (1976) and Uddin (1977).
However, there are no descriptions of the morphology, growth kinetics or
alkaloid production of Ephedra cell suspensions apart from O'Dowd (1991)
and O'Dowd et al. (1993).
Suspension cultures have been established from a range of different
Ephedra species (Khanna and Uddin 1976; Uddin 1977; O'Dowd 1991,
O'Dowd et al. 1993). In all cases, the medium used was MS supplemented with
either an auxin (2,4-D) or an auxin (2,4-D or NAA) and cytokinin (kin). The
procedures used for formation of a suspension culture are relatively routine. A
period of growth on solid (agar) medium followed by selection of a friable
sector of callus as starter inoculum in liquid culture. In a comprehensive survey
of 12 Ephedra species, O'Dowd (1991) found that friable callus could be
encouraged by more frequent subculture prior to inoculation into liquid medium. For the initial formation of suspension cultures, a relatively high
inoculum density was preferable (up to 140g1- 1) since it was found that below
60 gl-l viable cultures could not be obtained. After inoculation (using 50ml
medium in 250ml Erlenmeyer flasks), the lumps of callus were broken apart
with a sterile spatula. Most of the remaining lumps either separated during
culture on an orbital shaker (120rpm) or were removed later by filtration.
Cultures which survived were subcultured three to four times at a high
inoculum density (80g1- 1) which was later reduced to 50gl- 1 for routine culture. All cultures were maintained at 24 1C; 16-h day at 60 1O,umolm2
(PAR).
It is clear that some species are more amenable than others to growth in
suspension culture (Table 7). Whereas callus from E. andina, E. fragilis var.
camplyopoda, E. intermedia, E. major ssp. procera and E. saxatilis callus
readily adapted to growth in liquid medium, E. fragilis, E. sinica, E. distachya,
Ephedra Species
173
No. of
cultures
initiated
No. of
cultures
surviving
1 st passage
Average
duration of
survlvmg
cultures
Culture appearance
Auxin used
(5,uM)
E. andina
32
2:50
passages
2,4-D
E. distachya
3 passages
E. equisitina
E. tragilis
E. tragilis var.
camplyopoda
12
4 passages
E. gerardiana
3 passages
E. intermedia
14
2:14
passages
E. major
ssp. procera
2:12
passages
E. minima
2 passages
E. saxatilis
2:6
passages
E. sinica
E. sp.
(Cyprus)
4
3
0
2
4 passages
1 passage
Green, vigorous
cultures with large
aggregates (:55 mm
diameter). Often
die on sub-culturing
Cultures gradually turn
pale brown and die
Vigorous, medium
green culture, large
aggregates (:55 mm
diameter)
Yellow to pale green
with large aggregates
(:54mm diameter)
Fine, pale green culture
becoming oatmealcoloured before dying
Fine, medium green
culture
Cultures rapidly
becoming brown and
dying
NAA
NAA
NAA and
2,4-D
NAA and
2,4-D
2,4-D
NAA
NAA
NAA,2,4D and
IAA
2,4-D
2,4-D
NAA and
2,4-D
In general, suspension cultures established from green callus had a greater rate
of survival. In callus culture E. fragilis grew almost as well as E. saxatilis, with
similar growth rates, colour and friability (O'Dowd et al. 1993), yet E. fragilis
could not be established in suspension culture. Therefore there appears to be
no clear relationship between growth performance in callus culture and adaptation to suspension culture.
As can readily be seen from Table 7, there is considerable variation in
culture morphology, such as in colour, cell aggregation and cell shape, be-
174
Fig.7A,B. Suspension cultured cells of Ephedra andina showing filmentous growth. A Characteristic branching of filaments. B Up to 1% of cells in aggregated cultures demonstrated sclariform
or spiral secondary thickening (Toluidine Blue O-stained)
Ephedra Species
175
vessels. It therefore appears that greater cell aggregation was found in the
faster-growing suspensions.
At the cellular level, Ephedra cell suspensions show variations in cell
shape. Two distinct morphologies were observed, those cultures consisting
predominantly of tangled, occasionally branching, filaments of elongated cells
(Fig. 7) and those consisting mainly of clustered spherical cells. Interestingly,
these characteristics were not species-specific; although cultures derived from
E. saxatilis and E. minima were the cell cluster type, both culture types were
found within the nine cell lines derived from E. andina. The origins and
stability of this culture variation can only be speculated upon at present but
presumably relate to some preexisting variation in the original explant or
in the selection of callus from which the suspension was derived. In the
filamentous form of Ephedra suspension cultures, the filaments became so
entangled that, at the end of the growth cycle, the cells could be picked up en
masse with a pair of forceps (O'Dowd 1991). This growth characteristic raised
difficulties for assessing culture biomass by cell counting because the cells
could not be separated satisfactorily.
Batch cultures of cell suspensions of E. andina were cultivated over a 21day growth cycle. Inoculated initially at 20g 1-1 fresh wt. (1.3 gl-1 dry wt.), the
cultures grew approximately tenfold to attain a final biomass of approx. 190282g1- 1 fresh wt. (10g1- 1 dry wt.) depending on the cell line (Table 8). The
growth rate was cell line-dependent. Uddin (1977), using a medium without a
cytokinin, reported a maximum growth index (final fresh wt. of tissue minus
initial fresh wt.) of 8 after a period of 6 weeks for E. [oliata. This growth rate
is substantially slower than those obtained by O'Dowd (1991).
Some growth kinetics of cell line E. andina (S31) over a 19-day growth
cycle are shown in Fig. 8A. The increase in both fresh weight and dry weight
follows a typical S-shaped growth curve, although there is no clear period of
constant exponential growth. An initial induction phase of about 2-3 days is
followed by a period of growth of about 7 days. The cells then enter a stationary phase about 9-10 days after inoculation. This interpretation is reinforced
by the data on mitotic index (Fig. 8B) which show a marked increase from 1 to
3.5-4% during the growth phase, subsequently declining sharply to 0.5% or
less by day 13. Cell viability (assessed by staining with fluorescein diacetate) is
uniformly high, 85-90% up to the end of the growth phase at about day 9 (Fig.
8C). However, during the stationary phase viability declines quite steeply to
Maximum biomass
(Fresh wt. gl-l)
S 31
S 21
S 10
2.5
4.6
6.0
190 10
282 10
190 20
176
....,
,-.
12
180
160
eil
'-'
140
.,.;
10
,-.
'-'
120
..c
.::'"
-----
80
Q,j
....=
\.0
:;
60
40
eil
.,.;
100
Q,j
....,
20
0
0
10
....
\.0
'CI
4
Culture fresh wt.
Q,j
....=
:;
\.0
2 U
0
12
14
16
18
20
Time (days)
12
B
10
....
,-.
..!.
4
,-.
8
3
eil
'-'
.,.;
:=
"'"'
....
\.0
Q,j
'CI
2 .~
....
....0
~
2
0
2
10
12
14
16
18
20
Time (days)
Fig. SA,B. Growth of E. andina cell suspension cultures grown in batch culture over 20 days. A
Culture fresh wt. and dry wt. (gl-l). B Mitotic index (%). C Cell viability (%). D Medium pH
177
Ephedra Species
12
100
---...... 10
~
...
,.>.
90
80
70
60
",.
...=
:;
50
40
Qj
----
--.--
2
0
0
30
--~
>.
::=
:E
.;:<:\I
4l
20 U
10
0
10
12
14
16
18
20
Time (days)
6
12
0
10
::c
---.
...
Q.
:e=
Qj
,.
...:;=
Qj
----e--
--.--
~
~
,.>.
p-l
0
0
10
12
14
16
18
20
Time (days)
Fig. 8C,D. Continued
178
Medium Inlet
Filter
sterilised
air
Line
bubbled
through
2'10 "Savlon n
solution
'"c
---.-
Batch culture
-<>--
Semicontinuous culture
;:
eu
""C
:z
10
12
14
16
18
20
22
Time (days)
Fig. 10. Growth rate of E. andina (cell line S23) in batch culture and semicontinuous culture in an
air-lift bubble reactor
Ephedra Species
179
problems with mixing, overall, these bioreactor studies showed that it was
possible to grow a relatively large and metabolically uniform biomass of cell
suspension suitable for precursor feeding experiments.
3.1.4 Cell Suspension Culture Alkaloid Production
By comparison with intact plant tissues, levels of ephedrine and pseudophedrine were relatively low in callus and suspension cultures (Table 5).
Ephedrine and psuedoephedrine levels in suspension cultures decreased over
time and were no longer detectable after four to six passages (15-20 days per
passage). Immobilisation of E. andina cell suspensions in sodium alginate
beads did not lead to slowing of growth rate or to alkaloid production. In fact,
growth was slightly accelerated and the supernatant yielded a fine suspension
of single cells (unpubl.).
There has been little investigation of the effects of different plant growth
regulators or other culture conditions on ephedrine metabolism in cell cultures. Optimising the concentration of the components in a medium is generally an effective way to improve the production of secondary metabolites as
well as to select a highly productive cell line (Fujita 1988). An alternative
explanation for the lack of secondary metabolite accumulation in Ephedra
cultures could be due to the fact that the tissues were cultured as
180
undifferentiated callus or cell suspensions. This approach may fail to elicit the
complex and highly regulated patterns of gene expression found in intact
tissues and may have resulted in cultures that were neither genetically, biochemically nor physiologically competent for high-level synthesis of secondary
metabolites.
3.2 Infection of Ephedra with Agrobacterium rhizogenes
Ephedra sp.
E. andina
E. distachya
E. equisitina'
No reaction
No reaction
85%
E. tragi/is
E. gerardiana'
No reaction
No reaction
85%. Produced from tumours
10%. Produced directly from
wound site. 4-15 roots per site
or tumour
21 %. Produced from tumours;
1-2 roots per site. Some roots
up to 4cm long
13%. Produced from tumours
E. gerardiana var
sikkimensis
E. minima
E. minima hybrid
No reaction
5%. Produced from tumours;
roots spindly, <1 cm long.
9%. Produced from tumours
Ephedra Species
181
Fig. 12A-D. Production of tumours and/or roots on Ephedra following inoculation with
Agrobacterium rhizogenes. A One-year-old E. fragilis seedlings 35 days after injection with A.
rhizogenes strain Ar2629; X indicates tumours and roots at wound sites. B One-year-old E. fragilis
seedlings 35 days after injection with heat-killed A. rhizogenes strain Ar2629; arrows indicate
wound sites. C Typical dark orange tumour on 3-year-old E. fragilis plant following inoculation with
A. rhizogenes strain Ar1000. D Roots developing on the surface of internodal explants of E. fragilis
90 days after inoculation with A. rhizogenes strain Ar2629 on growth regulator-free medium
182
semisolid nor in liquid media. Two lines of E. fragilis tumorous tissue cultured
in the absence of growth regulators were regularly analysed for E, NE, PE and
NPE over a 2-year period. The faster-growing, friable line contained no alkaloid, but the slow-growing, compact line contained 0.01% E. Similarly,
Ar2629-induced E. minima tumorous tissues contained traces of PE (unpubl.).
Tumours formed on plants in the greenhouse could not be initiated into
culture, but they were analysed for alkaloid and compared to the stem from
which they derived. In general, tumours produced in vivo on the stems of
E. fragilis and E. minima plants contained levels of I-ephedrine similar to
those found in nontransformed stem tissue, but differed in that while
d-pseudoephedrine was detected in stem extracts, it could not be detected
in tumour extracts.
Studies involving the infection of Ephedra with Agrobacterium look promising. Tumour-derived tissues thus produced may provide a model system in
which alkaloid production could be investigated.
3.3 Organogenesis
Ephedra Species
183
Fig. 13. A Formation of adventitious shoot buds on E. gerardiana callus after culturing for 8
months on 0.05)1M N AA + 0.03)1 M kin. B The same callus after 2 months on growth
regulator-free medium showin g full development and branching of shoots
184
BAP
3.4 Micropropagation
Ephedra is traditionally propagated by seed or vegetatively by the division of
older plants (Krishnamurthy et al. 1965; Morton 1977). Rooting of conventional cuttings has a low success rate. For example, Sanaenosuke and Kurihara
(1967) reported 20 and 3% rooting of cuttings from 4-year-old plants of E.
altissima and E. distachya, respectively. An efficient micropropagation system
offers the opportunity to rapidly increase elite strains (e.g. high alkaloidyielding) and to speed up the production of select Ephedra plants from conventional hybridisation or mutagenesis. Prior to our own studies (O'Dowd and
Richardson 1993a), only two Ephedra species, E. foliata and E. gerardiana,
had been micropropagated in vitro and rooting had been problematic
(Sankhla et al. 1967b; Ramawat and Arya 1976; Konar and Singh 1979;
Bhatnagar and Singh 1984; Arya and Ramawat 1988).
O'Dowd and Richardson (1993a) studied ten Ephedra species in an attempt to develop a general micropropagation protocol for the genus. There
were differences in performance between species but in several cases some
species were represented by only one parent individual. In preliminary studies,
nodal stem explants of E. fragilis were cultured on MS medium supplemented
with 3% sucrose, 0.05,uM IBA and 0-5,uM BAP, kin or zea. Typically, with
increasing cytokinin levels the number of shoots per explant increased while
shoot length decreased (Fig. 15). An optimal cytokinin concentration of
0.05,uM kin was chosen, giving a micro propagation coefficient of 5 per 30-40
days while maintaining shoot quality. Nine other Ephedra species were similarly grown on this medium and the results are summarised in Table 10.
Alkaloids were not found in shoot cultures from any of the alkaloid-yielding
Ephedra species (O'Dowd 1991).
(n = 76)
E. scoparia
(n = 79)
E. viridis
(n = 35)
E. saxatilis
(n = 98)
E. minima
E. andina
(n = 102)
E. equisitina
(n = 153)
E. fragilis var
camplyopoda
(n. ::':: 54)
E. gerardiana
(n = 69)
E. intermedia
(n = 49)
E. major
(n = 54)
E. major ssp.
procera
(n = 51)
Species
33
24
29
13
11
27
21
21
47
100
40
60
72
28
29
6.2::':: 5.3
27.9::':: 8.6
68
58
87
60
43
33
17.5::':: 6.5
42
50
Explants with
3-4 shoots (%)
56
Explants with
2 shoots (%)
Averafte
shoot ength
32
13
13
40
57
48
20
17
13
29
44
Explants with
1 shoot (%)
58
12
Non-growin,
explants (%
58
14
Dead
explants (%)
Additional notes
Table 10. Shoot production from stem nodal explants of ten species of Ephedra after 40 days on MS medium supplemented with 0.05}1M lEA and 0.05}1M
kin. n = No. of explants cultured
00
U1
f-'
V>
(D'
(1)
'"'"'
C/l
.,.'"
~
~
186
30
E
-5
.s
---
20
01)
s::
,j,l
00
..c:
10
CI'J
1.0
2.0
3.0
4.0
5.0
---
30
---0--
0.. 20
BAP
KIN
~
<l)
...
<l)
0..
~0
..c:
</J
10
0
0
1.0
2.0
3.0
4.0
5.0
Ephedra Species
187
The production of haploid tissues and plants from Ephedra has been reported.
For example, Konar (1963) cultured haploid tissues in vitro from E. foliata
pollen. The production of haploid callus from the E. foliata female
gametophyte, and the subsequent regeneration of haploid plantlets was reported by Singh et al. (1981) and Bhatnagar and Singh (1984). They found that
the female gametophyte was more easily manipulated in vitro than the male
gametophyte. Ovules were harvested when the pollination drop was present,
as this indicates that the archegonia are mature. Singh et al. (1981) described
optimal conditions for callus and root production from mature archegonia
stage ovules on 9 flM 2,4-D or 22 flM N AA. They also demonstrated the ability
of these tissues to regenerate shoots. Shoot bud formation was stimulated by
the addition of BAP (36 flM) or a combination of 2,4-D (9 flM) and kin (528flM) to the medium. Subsequent transfer to growth regulator-free medium
was essential to allow further bud development. Observation of chromosome
numbers showed these plants to be haploid (n = 7).
These techniques for the production of haploid Ephedra plants could be
used to produce mutants which, for example, overproduce alkaloid, or have a
different ratio of alkaloids, or in which the biosynthetic pathway has been
disrupted. Such mutants might provide useful information on the secondary
metabolite biosynthetic pathway of the genus.
188
Ephedra Species
189
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1 Introduction
Euglena was first discovered by Antony van Leeuwenhoek in 1675 and is
called Midori mushi in Japanese. The name Euglena means beautiful (Eu) eye
(glena). Euglena is classified in both the animal and the plant kingdoms. In the
animal kingdom Euglena belongs to the Protozoa, whereas in the plant kingdom Euglena belongs to Euglenophyta (Inoki 1981; Yamada et al. 1983).
There are more than 60 species belonging to the Euglena genus, of which
about 10 are well known (Johnson 1968; Mizuno 1976; Inoki 1981). Euglena is
considered as having stable heredity because it has no sex and no reduction
division. Cell sizes of Euglena vary from ca. lO.um (E. min uta ) to ca. 500.um
(E. oxyuris). The cell shape is very variable due to a metabolism called
euglenoid movement; being spindle-shaped and spherical, the cells may
change their shape under changing conditions. Euglena gracilis, especially
strain Z and var. bacillaris, are widely used for investigations in physiology and
biochemistry. Euglena gracilis is a unicellular microorganism (ca. 50.um in
length x ca. lO.um in width), which has chloroplasts, mitocondria, and two
flagella of different lengths, an eye spot containing carotenoid and flavin, and
other organelles such as Golgi bodies. Euglena can move by swimming, or
contracting to changing shapes and crawling and sliding; most of the species
have a forward rotary movement by swimming spirally. Euglena has the
paramylon, f3-1,3-glucan, as one of the storage substances. At first, only few
biologists were interested in Euglena as a rare microorganism. However, since
the utilization of Euglena as a test microorganism both for the measurement of
vitamin BI2 biologically in 1949 and the establishment of the Calvin-Benson
cycle in photosynthesis in the 1950s, it has become as a very important microorganism for the investigation of photosynthesis and chloroplast. Now, many
biologists, physiologists, and biochemists are interested in Euglena regarding
its utilization in the investigation of physiological and biochemical phenomena
as a small animal and a small plant. Furthermore, Euglena has characteristic
features in the TCA cycle, wax ester fermentation, and de novo fatty acid
Euglena gracilis Z
195
synthesis by adverse j3-oxidation. In industry, Euglena is used not only for cells
as nutrients (Hosoya and Kitaoka 1977; Kitaoka and Hoyoya 1977) and soluble paramyron for its anticancer effect (Yokota, unpubl), but also for commercial manufacture of torehalose from glucose by trehalose phosphorylase
(Murao et al. 1985), arachidonic acid (Sudate and Goto 1986), wax ester (Tani
et al. 1987), and vitamin E production (Tani and Tsumura 1989). The biochemistry, physiology, subcellular biochemistry, and molecular biology of Euglena
has been collected in The Biology of Euglena (Buetow 1968, 1982, 1989).
2 Cultivation
Euglena is widely distributed worldwide and lives in nonflowing water such as
ponds and swamps. It moves between the surface and the bottom of the ponds,
in search of the most comfortable living conditions. Euglena is also capable of
living in natural environments with a wide range of light, temperature, pH,
soluble Oz, and soluble COz; it is also found in animal- and plant-derived
sewage, wastewater from food processing factories, and strong acidic
wastewater from mines. Several media such as Cramer-Myers, Hutner, and
Koren-Hutner, are used for the cultivation of Euglena (Kitaoka 1989). In our
studies, Hutner medium was used (Schiff et al. 1971). Euglena gracilis Z (wildtype) was cultivated under stationary conditions at 2S C for 7-10 days under
both photoheterotrophical and photoautotrophical conditions (light illumination ca. 2000-3000Ix) in SOOml of Hutner medium in an Erlenmeyer flask
containing KH zP0 4 (O.4g), (NH 4 )zHP04 (0.2g), MgS0 47HzO (O.Sg), CaC0 3
(0.2g), DL-malate (2g), Na-glutamate (S g), EDT A 2Na (SOmg), ZnS0 47H20
(22mg), MnS0 42H20 (S.8mg). FeS0 4 (NH4)2S046HzO (S.7mg), Na 2Mo0 4
H 20 (l.Smg), CuS0 4SH20 (1.6mg), CoS0 47H zO (l.Smg), H3B04 (11.4mg),
vitamin B) (2.5mg) and vitamin Bl2 (O.02mg) in 11 distilled water (pH 3.3,
adjusted with HCI). Euglena gracilis SM-ZK, a streptomycin-bleached mutant
derived from strain Z. was also cultivated under stationary conditions and
heterotrophically in the dark. Euglena gracilis Z grows even under
photoautotrophical, photoheterotrophical, and heterotrophical conditions.
196
(average 100mg/l) for 1-30 days under the same conditions as described
above. For time course changes, aliquots (3 ml) of the cultured broth were
taken and put on the an Extrelut-column and extracted with 6ml of Et 20.
Each ether extract was analyzed by GC-MS. For aquisition of metabolites,
large-scale culture was carried out repeatedly, and the broth was extracted
with Et 20 after the removal of Euglena by centrifugation. The total recovery
ratios of metabolic products were more than 90%. The metabolites were
separated and purified by a combination of CC on silica gel, preparative GC,
Sephadex LH-20, and/or prep. HPLC. The products were identified by comparison with GC (capillary column, 30m X 0.25mm i.d., CDX-B, 13cyclodextrin/O V -1701) retention times, mass spectrometry, IR, 1H -( 400 MHz),
and 13C-NMR (100MHz) spectra with those of authentic specimens.
Hydrocarbons
Terpene aldehydes
Terpene ketones
Terpene epoxides
100
.~
'">
.~
50
o;,~-Unsaturated
ketones
abc d e f g h
k I mn
Saturated
ket('nes
p q r s t u v wx Y zo;
Compounds
Fig. I. Relationship between structure of terpenoids and survival of Euglena gracilis Z after 1 day.
(Noma et al. 1991b)
Hydrocarbons: a d-limonene(136'); b p-menthane; c a-pinene; d j3-pinene; e longifolene;
f caryophyllene
Terpene aldehydes: g d-citronellal(38); h l-citronellal(39); i dl-citronellal(38 and 39); j citral
[geranial(30) and neral(31)]; k l-perillaldehyde(7a); I l-phellandral(18); m
trans- and cis-1,2-dihydroperillaldehydes(14 and 15); n cumin aldehyde(27);
o myrtenal(l)
Other aldehydes: p cinnamic aldehyde(96)
Terpene ketones:
a,j3-Unsaturated ketones: q l-carvone(123); r d-carvone(123'); s dl-piperitone;
t isopiperitenone; u n-piperitenone; v verbenone
Saturated ketones: w dl-isomenthone; x I-menthone; y mixture of d-dihydrocarvone(124)
and isodihydrocarvone(12S); z mixture of l-dihydrocarvone(124') and
isodihydrocarvone(12S')
Terpene epoxides: a l,4-cineole; f31,8-cineole; ytrans- and cis-shisool-8,9-epoxides
Euglena gracilis Z
197
The relationship between 29 terpenoids and the survival of Euglena cultured photo heterotrophically for 1 day was investigated by measuring absorption at 640nm as turbidity; the result is shown in Fig. 1 (Noma et al. 1991b).
Most terpene aldehydes, aromatic aldehyde, and terpene ketones showed
strong inhibition for the survival of Euglena et ca. 200ppm. Table 1 and Fig. 2
Table 1. Summary of biotransformation of mono terpene aldehydes by Euglena. (Noma et al.
1991b)
Substrates
Myrtenal (1)
(lS)-Myrtanal (5a)
(lR)-Myrtanal (5b)
I-Perillaldehyde (7a)
I-Phellandral (18)
I-Citronellal (39)
D.C.
E.g.
120
E.a.
100
50
50
40
50
50
60
70
50
30
100
100
110
50
50
40
70
80
100
180
40
150
180
190
180
180
130
160
GHO
G-- G
~'
GOOH
GHO
~- ~
3
~'
--+'
4a
5a
6a
* liJ GJ -- GJ
gH 20H
GOOH
gOOH
,
~HO
4b
5b
8'
6 GHO
14
GOOH
2~
13b
2 2'
GHO
7b
8b
)(OH
11
6 - 6 6'
-0
A
A
GHO
yH 20H
13a
7a
8a
GHO
0
A
15
16
GOOH
cS' 0'
A
"-
6b
OH
12
10
---
0'
A
"-
0'
A
17
199
Euglena gracilis Z
(s~~
...............
20
2)
21
23
~
25
18
2
COOH
--
29
28
27
~w
I
31
~ooc
37
Fig. 2. Continued
33
35
CHO
--z
OH
OH
39
45
::
0~
COOH
41
n"'OH
XOH
44
200
201
Euglena gracilis Z
Substrates
Major products
I-Perillyl alcohol(8a)
dl-Perillyl alcohol(8a and b)
I-Phellandrol(19)
trans-Shisool(9)
tralls-Shisool(9)
trans-Tetrahydroperillyl alcohol(21)
lsopropenyl
Isopropenyl
Isopropyl
Fig. 3. Selective hydrogenation of I-perillyl alcohol(8a). dl-perillyl alcohol(8a and 8b), and 1phellandrol(19) by Euglena gracilis Z. (Noma et al. 1991b)
202
cis-4-heptenal (103), trans-2-decenal (104), 2,4-hexadienal (105), trans, trans2,4-heptadienal (106) and trans, trans-2,4-nonadienal (107) is summarized in
Table 2 together with the substrate concentration for the survival of the
phytofiagellate (Noma et al. 1991a). The biotransformation yield of aromatic
aldehydes and related aldehydes was ca. 100% after 1 or 2 days. Most of the
aromatic, aliphatic, and related aldehydes were transformed to the corresponding primary alcohols as major products. However, some of them gave
the corresponding acids as major or minor products. m-Chlorobenzaldehyde
(54) was transformed to m-chlorobenzyl alcohol (108) as the major product
and m-chlorobenzoic acid (109) as the minor product. However, the content of
108 decreased gradually with time and eventually 109 became predominant. pChlorobenzaldehyde (55) was strongly phytotoxic at a concentration of
100 ppm. However, it was slightly transformed to p-chlorobenzyl alcohol as the
major product and p-chlorobenzoic acid (110) as the minor product. After the
death of the Euglena cells, the predominant product became 110. The order of
preference for the formation of chlorobenzoic acids from 53-55 by Euglena
was 54 and 55. Compound 53 was not oxidized to the acid. 2Cyanobenzaldehyde (56) was transformed first to 2-cyanobenzyl alcohol,
which was further transformed via imine to phthalide (111) as the final product. On the other hand, o-phthalaldehydic acid (65) and o-phthalaldehyde (67)
were also transformed to 111. 0-, m-, and p-Tolualdehyde (62-64) gave the
corresponding alcohols as the major products together with the formation of
small amounts of toluic acid in the cases of 63 and 64. The reduction of iso- and
terephthalaldehyde (68 and 69) was carried out stepwise with the corresponding aldehydic alcohols as intermediates and iso- and terephthalalcohol as the
final products. When ca. 100mg of compounds 70-76 were added into 500ml
of cultured broth day by day, final concentrations of 70, and 72-76 were 0.12,
0.26,0.40,0.44,0.12, and 0.13%, respectively, at which Euglena died; they were
completely transformed to the corresponding alcohols (112, 114-118) (Fig. 4).
On the other hand, although compound 71 was very phytotoxic, it was transformed to 113 at low concentration, i.e., below 100mg/l. The velocity of
biotransformation for 72 and 75 to 114 and 117, respectively, in the light
(ca. 3000 Ix) by Euglena cultured photoheterotrophically was faster than in
the dark (Fig. 5). Furthermore, compound 72 was easily biotransformed to
114 in the cell-free extract of Euglena. Compounds 93--97 were also transformed to the corresponding primary alcohols. In the cases of the unsaturated
alcohols such as cinnamyl alcohol (119) and 2-methylcinnamyl alcohol (121),
the C = C double bond was hydrogenated to give the corresponding saturated alcohol (120 and 122). 3-Phenylpropanol (120) was subsequently
oxidized to 3-phenylpropionic acid via 3-phenylpropionaldehyde (95).
p-Acetylamidebenzaldehyde (92) and nicotin aldehyde (98) as nitrogencontaining aromatic aldehydes were also reduced to the corresponding
alcohols. However, compound 98 was extremely phytotoxic at ca. 100ppm. In
the present method only one container is used and has a short time reaction;
the preparation is very simple. On the basis of the above results, we consider
that Euglena is a good bioreactor for the reduction of aldehydes to the corresponding primary alcohols.
203
Euglena gracilis Z
'E.g.
fE.a. gE.w.
60
10 30
80
10 30
114
273
80
40
40
30
205
240
190
200
190
90
30
30
60
30
170
10 30
190
20
80
101
30
20
40
82
141
265
110
60
10
20
40
40
30
60
20
234
265
36
600
132
21
242
287
222
120
34
240
400
(600)
20
(100)
21
(1200) 241
(2000) 233
50
20
26
20
130
31
204
Table 2. Continued
3,4-Dihydroxybenzaldehyde (SO,
Protocatechualdehyde)
2,3-Dimethoxybenzaldehyde (Sl)
2,3-Dimethoxybenzyl
alcohol (100%, 1 day)
2,4-Dimethoxybenzaldehyde (S2) 2,4-Dimethoxybenzyl
alcohol (100%, 1 day)
2,5-Dimethoxybenzaldehyde (S3) 2,5-Dimethoxybenzyl
alcohol (100%, 1 day)
3,5-Dimethoxybenzaldehyde (S4) 3,5-Dimethoxybenzyl
alcohol (100%, 1 day)
2,3-Dichlorobenzyl
2,3-Dichlorobenzaldehyde (S5)
alcohol (100%, 1 day)
2,4-Dichlorobenzyl
2,4-Dichlorobenzaldehyde (S6)
alcohol (100%, 1 day)
2,6-Dichlorobenzaldehyde (S7)
2,6-Dichlorobenzyl
alcohol (100%, 1 day)
3,4-Dichlorobenzyl
3,4-Dichlorobenzaldehyde (SS)
alcohol (100%, 1 day)
3,5-Dichlorobenzyl
3,5-Dichlorobenzaldehyde (S9)
alcohol (100%, 1 day)
2,3,4-Trimethoxybenzaldehyde (90) 2,3,4-Trimethoxybenzyl
alcohol (100%, 1 day)
3,4,5-Trimethoxybenzaldehyde (91) 3,4,5-Dimethoxybenzyl
alcohol (100%, 1 day)
p-Acetylamidebenzaldehyde (92) p-Acetylamidebenzyl
alcohol (100%, 1 day)
tl-Phenylethanol (M)
Phenylacetaldehyde (93)
2-Phenylpropionaldehyde (94)
2-Phenylpropanol (M)
2-Phenylpropionic acid (m)
3-Phenylpropanol (120, M)
3-Phenylpropionaldehyde (95)
3-Phenylpropionic acid (m)
Cinnamyl alcohol (119, M)
Cinnamic aldehyde (96)
3-Phenylpropanol (120, M)
Cinnamic acid (m)
a-Methylcinnamic aldehyde (97)
2-Methylcinnamyl alcohol
(121, M)
2-Methyl-3-phenylpropanol
(122, M)
Nicotin aldehyde (9S)
Nicotin alcohol (M)
n-Heptylaldehyde (99, nn-Hexanol (100%, 2 day)
Enanthaldehyde, n- Heptanal)
Pelargonaldehyde (100,
n-Nonanol (41 %,2 days)
C9-aldehyde, Nonanal)
n-Nonanoic acid (41 %,2 days)
trans-2-Hexenal (101)
trans-2-Hexen-1-ol(60%,
1 day)
n-Hexanol (36%, 1 day)
trans-2-Heptenal (102)
trans-2-Hepten-1-ol (24%,
1 days)
n-Heptanol (19%, 1 day)
cis-4-Heptenal (103)
cis-4-Hepten-1-ol (82%, 3 days)
trans-2-Decenal (104)
trans-2-Decen-1-o1 (3%, 3 days)
n-Decanol (12%, 3 day)
2,4-Hexadienal (105)
2,4-Hexadien-l-01 (45%, 1 day)
4-Hexen-1-o1 (24%, 1 day)
1444
273
140
112
90
10
90
64
70
50
10
10
20
10
10
130
113
160
214
270
339
110
90
150
90
20
20
20
20
30
20
10
10
10
10
30
10
10
10
30
105
35
35
10
Table 2. Continued
trans,trans-2,4-Heptadien-l-01
(51 %,2 days)
trans-4-Hepten-l-01 (19%,
2, days)
trans,trans-2,4-Nonadien-l01 (34%, 1 day)
trans-4-Nonen-l-01 (66%, 1 day)
trans,trans-2,4-Heptadienal (106)
10
65
10
2200
74(116)
2000
S..
:a
e
5
1500
0
0
on
bb
..
::c
'0
'0
"
~
~"
1000
..
..Q
rJJ
500
400
300
200
100
10
15
20
25
28
Time (days)
206
vanillin
vanillyl alcohol(114)
100 .
light
ethylvanillyl alcohol(117)
100
light
80
80
'"
U
::I
't:O
...
dark
60
60
dark
40
40
20
20
O~--~--~----~--~--~
time(days)
OQC--~--~--------~--~
time(days)
Fig. 5. Effect of light illumination (ca. 3000 Ix) on the biotransformation of vanillin(72) and
ethylvanillin(75) by E. gracilis Z. (Y. Noma, unpubJ.)
1993, 1994a, 1995; Noma and Asakawa 1992). l-Carvone (123) was
stereospecifically transformed via d-dihydrocarvone (124) to dneodihydrocarveol (126), which was further transformed to 8-hydroxy-dneodihydrocarveol (130), namely (lR, 2S, 4R)-p-menthane-2,8-diol. l-n-,
d-iso-, and d-neoisodihydrocarveol (127-129) were also hydroxylated to give
8-hydroxydihydrocarveol (131-133), respectively (Fig. 6). On the other
hand, d-carvone (123') was also transformed mainly via l-isodihydrocarvone
(125') to l-isodihydrocarveol (128') as the major product and 1neoisodihydrocarveol (129'), together with l-dihydrocarvone (124') and 1neodihydrocarveol (126'), of which compounds 126', 128', and 129' were
further hydroxylated at the C-8 position to give the corresponding 8hydroxydihydrocarveols (130', 132', and 133'). d-Dihydrocarveol (127') was
also hydroxylated to give 8-hydroxy-d-dihydrocarveol (131') (Fig. 7). Hydrogenation of C = C double bond, reduction of C = 0 group and hydroxylation
at the C-8 position in the biotransformation of carvone occur under both light
and dark conditions. 1-Aminobenzotriazole used as a cytochrome P-450
inhibitor did not affect the inhibition of hydroxylation of dihydrocarveols
by Euglena (Noma et al. 1994a). l-Carvotanacetone (143) was also hydrogenated to d-neocarvomenthol (145) via d-carvomenthone (144). On the other
hand, the d-isomer (143') was also transformed to l-iso-(147) and 1neoisocarvomenthol (148) via l-isocarvomenthone (146) as the major products
(Fig. 8; Noma et al. 1989). (4R, 8RIS)-I-Carvone-8,9-epoxides (149a and 149b)
as the metabolites of l-carvone (123) by Streptomyces bottropensis, SY-2-1
(Noma and Nishimura 1982) were transformed via d-dihydrocarvone-8,9epoxides (150a and 150b) to d-neodihydrocarveol-8,9-epoxides (ISla and
Euglena gracilis Z
207
(y0R
/'"
137
138
(y0H
8a
&0
./
/'"
124
kI
-)I'
Ho"",6
./"
142
"6 ~
"""OH
/'"
135
/128
"
6" ,
OR
208
Substrates
I-Carvone (123)
d-Carvone (123')
l-Carvotanacetone (143)
d-Carvotanacetone (143')
dl-Carvotanacetone
(143 and 143')
I-Carvone-8,9-epoxides
(149a and b, 4R, 8RIS)
d-Carvone-8,9-epoxide (149a'
and b', 4S, 8RIS)
2-Methyl-2-cyc1ohexenone (153)
2-Cyclohexenone (156)
dl-Menthenone (159)
dl-Piperitone (160)
dl-Isopiperitenone (161)
209
Euglena gracilis Z
Table 3. Continued
n-Piperitenone (162)
d-Pulegone (163)
d-Verbenone (164)
3-Methyl-2-cyclohexenone (165)
Isophorone(3,5,5-trimethyl-2cyclohexenone)
4-0xoisophorone
d-Dihydrocarvone and disodihydrocarvone (124 and
125, 80: 20 peak area in GC)
d- Dihydrocarvone-8, 9-epoxides
(150a and b)
l-Dihydrocarvone-8,9-epoxides
(150a' and b')
d-Carvomenthone (144)
l-Isocarvomenthone (146)
dl-Menthone
I-Camphor (250')
d-Camphor (250)
dl-Camphor (250 and 250')
dl-2-Methylcyclohexanone
dl-3-Methylcyclohexanone
dl-4- Methylcyclohexanone
Cyclobutanone
Cyclopentanone
Cyclohexanone (157)
Cycloheptanone
Cyclooctanone
Cyclodecanone
Cyclododecanone
Not
Not
Not
Not
Not
transformed
transformed
transformed
transformed
transformed
4-0xodihydroisophorone (M)
3,5,5-Trimethyl-4-oxocyclohexanol (M)
d-Neodihydrocarveol (126, M)
d-Isodihydrocarveol (128, m)
d-Neoisodihydrocarveol (129, m)
d-8-Hydroxyneodihydrocarveol (130, M)
d-8-Hydroxyisodihydrocarveol (132, M)
d-8-Hydroxyneoisodihydrocarveol (133, M)
l-Neodihydrocarveol (126', M)
l-Isodihydrocarveol (128', m)
I-Neoisodihydrocarveol (129', m)
1-8- Hydroxyneodihydrocarveol
(130, M)
1-8-Hydroxyisodihydrocarveol (132', M)
1-8-Hydroxyneoisodihydrocarveol (133', M)
d- Neodihydrocarveol-8, 9-epoxide
(151a, and b, M)
Not transformed
d-Neocarvomenthol (145, M)
d-Isocarvomenthol (147, m)
d-Neoisocarvomenthol (148, m)
Not transformed
d-Isoborneol (249', 22%, 14 days)
d-Borneol (248, 18%, 14 days)
d-Isoborneol (249', 16%, 14 days)
d-Borneol (248, 5%, 14 days)
cis- and trans-2-Methylcyclohexanol (46: 39,
peak area in GC)
cis- and trans-3-Methylcyclohexanol (80: 20,
peak area in GC)
cis- and trans-4-Methylcyclohexanol (25: 75,
peak area in GC)
Cyclobutanol(80%, 1 day)
Not transformed
Cyclohexanol (158, 100%, 11 days)
Cycloheptanol (76%, 6 days)
Cyclooctanol (38%, 6 days)
Cyclodecanol (16%, 10 days)
Cyclododecanol (83%, 10 days)
2,
142'
136'
2
,
\
./
123'
'~a
./
125'
~r"
137'
2/0R
134'
124'
2;OH * '" ~O
135'
qOH
129'
7" -
oJr-.
./
127'
~H
126'
130'
9~OH
132'
9:0H
133'
OH
131'
_ ~OH
~tH -
128'
" 2~H -
~OH
H01
141'
Ho/2 -
8b
CH 20H
139'
qOR
to
"""'
'"
V>
'"
::>
'0-"
Euglena gracilis Z
211
Oo~
-
144
143
143'
145
146
2"'""
,/
147
'"
~OH
14S
0 0
0
(y0H
~
149a
0" 0
)<}
}<J
149b
150b
151b
oo~
153
0 ---
0 0 ---
154
157
~" ~o
159
160
/~~r *
8R
(y0H
8~
156
ISla
150a
~c
"-
149b'
2" *
150b'
(f0H
()OH
155
~'0
~c,~
152a
8S
149a'
ISS
"-
2;" *
150a'
~ ~o ~o
161
152b
162
163
~o
164
(\0
165
212
Although men then one (159) was slightly reduced to menthone, piperitone
(160), isopiperitenone (161), n-piperitenone (162), pulegone (163), verbenone
(164), and 3-methyl-2-cyclohexenone (165) were not transformed at all (Fig. 8;
Noma et al. 1989). Although isophorone and dihydroisophorone were not
transformed at all, 4-oxoisophorone was easily transformed to 4oxodihydroisophorone and 3,3,5-trimethyl-4-oxocyclohexanol. The efficient
formation of saturated ketones such as 124, 125',144,146, 150a and 150b, 152a
and 152b, and 154 from the corresponding a,,B-unsaturated ketones (123, 123' ,
143, 143', 149a, 149b, 149a', 149b', and 153) suggested that the C = C double
bond may be hydrogenated from behind (Si-face), if the compounds possess-
OH
C6 re face
attack
H sisi-re
-'c
.,
..
re-re-si
H I-
z~
C6 si face
R'('R2
attack
I-Carvone(123)
Isopropenyl
d-Carvone(123')
I-Carvotanacelone(143)
Isopropyl
d-Carvotanacetone(143')
I-Carvone-8,9-epoxide
(1493 and b)
Epoxyisopropenyl
d-Carvone-8,9-epoxide
(1493' and b')
2-Methyl-2-cyclohexenone
(153)
C6 si face
attack
RI
C I si face and C Z
re face attack
Substrate
C6 re face
attack
Rz
Main Products
d-Dihydrocarvone(124)
d-Neodihydrocarveol(126)
Isopropenyl I-Isodihydrocarvone(125')
I-Isodihydrocarveol (128')
H
d-Carvomenthone(144)
d-Neocarvomenthol(145)
Isopropyl
1-Isocarvomenthone(146)
d-Neoisocarvomenthol(148)
H
d-Dihydrocarvone-8,9- epoxide(1503 and b)
d-Neodihydrocarveol-8,9-epoxide(1513 and b)
H
Epoxyisopropenyl
H
I-Isodihydrocarvone-8,9-epoxide(1523 and b)
1-2-Methylcyclohexanone(154)
d-2-Methylcyclohexanol(155)
213
Euglena gracilis Z
100
123'
80
o~~~--~~~~~~~~~~
50 100 150 200 250 300 350 400 450 SOD (ppm)
Concentration
Fig. 10. Effect of carvone concentration(123, 123', and equal mixture of 123 and 123') on
biotransformation by E. gracilis Z. (Y. Noma, unpub!.)
214
E.w.d
E.a.d.
E.g.d.
5
Time(days)
Time(days)
Time(days)
/;r,oo-?--%
Time (days)
10
11
12
22
32
~.~rl~GOO~
"
"
'0
"e
01
50
~
.S
;;;
Concentration (ppm)
Fig. 12. Effect of l-carvone(123) concentration(50-400mg/l. 50mg/l interval) on the growth of E. gracilis Z and biotransformation. (Y. Noma.
unpub\.)
l.
'-~k:~~;/ 'i
5~~
:9
100
f-'
Uo
~
N
~.
f'"
216
1.0
0.9
100
,...... 0.8
S
c:
'<t
\0
'i;:j
ci
0
;;::
0.7
0.6
0.5
(,)
0.4
...
0.3
<t:
0.2
.D
0
.D
'"
40
o--f5~
20
0.1
0
0
>.
OJ
e.
~
bJ)
:a
.s
..2
~
5 ..9~
... o:i
0
>~
'0
...
0
0<:
bJ)
u
'"'" 5
-;::: ;
~
..90.
o '"
u
!:l
Fig. 13. Effect of light color on growth and biotransformation of l-carvone(123) by Euglena
gracilis Z under light illumination at ca. 3000 Ix. (Y. Noma. unpubl.)
Euglena gracilis Z
217
8------
100
10--
50
50
7~
12
0
100
'"I:::
Q,)
8<
(TJ
~
0
50
(ij
100
0
~
50
()
>.
()
>.
100
5/
12
(ij
'"
"0
\00
15 (days)
0
y-l~
50
50
0,
,
2
,6
7_
12::::::0-..
,
,
100
5 6
7 (days)
Time
Fig. 14. Biotransformation of cycloalkanones and cycloalkanols by Euglena gracilis Z under light
illumination at ca. 3000 Ix. (Noma et al. 1992b )
""'
-0
-0
::c
o ,....
10
......
12
14
Ti me (m ; n. )
,co
o....;
::c
o
1~
.0
U
~
18
20
CsC=O
CsOH
22
ClOC=O
24
Fig. 15. Gas chromatogram for biotransformation of cycloalkanones(C6, C7, and C12) and cycloalkanols(C4, C5, C8, and CIO) mixture by Euglena gracilis
Z under light illumination at ca. 3000lx. (Y. Noma, unpubl.)
u
..;t
u
u
u
::c
u ,....
::c
o CO
;I>
p..
po
N
.....
00
p-Methoxyacetophenone (183)
m-Methoxyacetophenone (181)
o-Hydroxyacetophenone (176)
m-Hydroxyacetophenone (177)
p-Hydroxyacetophenone (178)
o-Methoxyacetophenone (179)
Acetophenone (174)
2-Hydroxychalcone (170)
2' -Hydroxychalcone (171)
3-Hepten-2-one (172)
Benzalacetone (168)
f3-Ionone (167)
Substrates
dl-a-Ionone (166)
E.w.l.
Table 4. Summary of biotransformation of a- and f3-ionones, acetophenone, and related compounds by Euglena. (Noma et al. 1992a, 1994b)
67
E.w.d
~
N
~Q.
i"
t>J
Isobutyrophenone (214)
Valerophenone (215, nPentanophenone)
o-Hydroxypropiophenone (210)
p-Hydroxypropiophenone (211)
n-Butyrophenone (212)
2-Hydroxy-5-methylacetophenone (195)
2-Hydroxy-4-methoxyacetophenone (196)
2-Hydroxy-5-methoxyacetophenone (197)
4-Hydroxy-3-methoxyacetophenone
(198, Acetovanillone)
4-Hydroxy-3-methylacetophenone (199)
2,4-Dihydroxyacetophenone (200)
2,5-Dihydroxyacetophenone (201)
2,6-Dihydroxyacetophenone (202)
3,4-Dihydroxyacetophenone (203)
3,5-Dihydroxyacetophenone (204)
2,4,6-Trihydroxyacetophenone (205)
3,4,5-Trimethoxyacetophenone (206)
2,4,6-Trimethylacetophenone (207)
Propiophenone (208)
p-n-Heptylacetophenone (191)
3,4-Dimethoxyacetophenone (192)
2,5-Dimethylacetophenone (193)
p-Methylacetophenone (189)
rn-Methylacetophenone (187)
o-Methylacetophenone (185)
Table 4. Continued
Not transformed
Not transformed
Not transformed
Not transformed
Not transformed
Not transformed
Not transformed
1-(3,4,5-Trimethoxyphenyl)-l-ethanol (10%, 3 days)
Not transformed
I-Phenyl-l-propanol (209a(R) and b(S), 100%,
7 days, R:S = 21 :79, [al D = -28.2)
Not transformed
Not transformed
I-Phenyl-l-butanol (213a(R) and b(S), 94%,
5 days, R:S = 42:58, [al D = -7, (16%ee
I-Phenyl-l-isobutanol (86%, 10 days)
I-Phenyl-l-pentanol (33%, 2 days, dead)
20
10
30
20
40
20
20
10
10
156
20
10
10
30
11
10
10
10
50
20
50
30
60
50
10
10
10
30
20
40
40
30
40
III
::;!
III
III
'"
~
)-
p,.
III
III
tv
tv
0
2-Aeetylcyclopentanone (228)
2-Butanone (229)
2-Pentanone (230, Methyl-npropyl ketone)
2-Hexanone (231, Methyl-nbutyl ketone)
2-Heptanone (232, Methyl-npentyl ketone)
2-0etanone (233, Methyl-nhexyl ketone)
2-Nonanone (234, Methyl-nheptyl ketone)
2-Deeanone (235, Methyl-noetyl ketone)
2-Undecanone (236, Methyl-nnoryl ketone)
2-Dodeeanone (237, Methyl-ndeeanyl ketone)
4-Methyl-2-pentanone (238)
Ethyl-n-butyl ketone (239)
2-Aeetylcylcohexanone (227)
n-Hexanophenone (216)
n-Heptanophenone (217)
p-Hydroxy-n-heptanophenone (218)
n-Octanophenone (219)
n-Nonanophenone (220)
n-Deeanophenone (221)
4-(p-Hydroxyphenyl)-2-butanone (222)
I-Phenyl-3-hydroxy-5-hexanone (223)
4-Phenyl-3-butanone (224,
I-Phenyl-2-butanone)
5-Aeetyl-2-norbornene (225)
Benzoyl acetone (226)
20
11O/500ml
930
20
2-Nonanol (94%)
170
560
10
10
80
10
10
10
10
20
40
110
33
4-(p-Hydroxyphenyl)-2-butanol
l-Phenylhexa-3,5-diol
4-Phenyl-3-butanol (100%, 4 days)
10
20
:::l.
~
N
:l
<>to
;:
"'"
tl']
;::
<>to
Benzophenone(246)
3,3-Dimethyl-2-butanone (247, Pinacolin)
4-0xodihydroisophorone
Table 4. Continued
1920
160
260
760
460
810
"''~""
'"
0-
'"
Z
o
!::S
tv
Euglena gracilis Z
223
OH
RI~R3
R2
Rl~R3
R2
Substrates
Rl
R2
R3
a-Ionone(166)
Me
~- lonone(167)
Me
Benzaldehyde(168)
Me
Benzalacetophenone(169)
2-Hydroxychalcone(170)
Cl
2'-Hydroxychalcone(171 )
"JGJ"
Cinnamic aldehyde(96)
a-Methylcinnamic
aldchyde(97)
3- Hepten-2 -one(172)
Me
C)H7
Me
3-Buten-2-one( 173)
Me
....
Oll
Products
a-Ionol
7,8-Dihydro-a-ionol
7,8-Dihydro-a-ionone
p-Ionol
7,8-Dihydro-p-ionol
7 ,8-Dihydro-~-ionone
7,8-Di hydrobenzalacetone
4-Phenyl-2-butanone
7,8- Dihydrobenzalacetophenone
1,3-Diphenylpropanol
Not transformed
2'-Hydroxydihydrochalcone
Cinnamyl alcohol(119)
3-Phenylpropanol( 120)
2-Methylcinnamyl alcohol(121)
2-Methyl-3-phenylpropanol( 122)
2 -Heptanone(232)
2-Heptanol
2-Butanone (229)
2-Butanol
Fig. 16. Metabolic pathways of a-(I66) and f3-ionones(167) and related compounds by Euglena
gracilis Z, (Noma et aL 1992b)
trimethoxyacetophenones (206) were also reduced to the corresponding secondary alcohols. The order of preference for the reduction was 181 > 187 >
174> 189 > 179 > 185 > 191 > 177 > 183 > 192 = 193> 198. lIowever,oand p-hydroxyacetophenone (176 and 178), 2-hydroxy-5-methyl-, 2-hydroxy-
224
4-methoxy- and 2-hydroxy-5-methoxyacetophenones (195-197), 4-hydroxy-3methyl-, 2,4-, 2,5-, 2,6-, 3,4-, and 3,5-dihydroxyacetophenones (199-204), and
2,4,6-trihydroxy-, and 2,4,6-trimethylacetophenones (205 and 207) were not
transformed at all (Fig. 17; Noma et al. 1994b). Enanti()selectivity in the
biotransformation of compounds 174, 177, 179-181, 189, 193 and 208 by
Euglena was investigated using an optical isomer separating capillary column
(CDX-B, j3-cyclodextrin); the result is shown in Fig. 18. In the case of compound 174, first (lS)-1-phenyl-1-ethanol (175b) was predominantly formed [RI
S ratio = 25:75; 50% enantiomer excess(ee)]. However, with time, (lR-)l-
225
Euglena gracilis Z
Time (days)
"
(l~
O:vte
179
.
~
~
en
/~
ti
::s
'd
....0
,,0
/~
/~----- - - j\ - - - -..1-
llll)
jY/
~en
180b --'"
<~
/4~.~~-
0..
I ~2b
182a and b
nHl
ti
::s
'd
....
- jf .
'iO
_1r-
0..
;t,
-'
O.j<C-~~~-~~---~~----~~J~
11
'6
Time (days)
10
I)
V __ ~_~-Q--~~--.--!,\~~
1112),5678
Time (days)
27
209a and b
IUU
189
~
en
ti
::s
'd
0
....
00
0..
Time (days)
10
Time (days)
Fig. 18. Enantioselectivity in the biotransformation of acetophenone(174), ()- and mmethoxyacetophenones(179 and 181), p-methylacetophenone(189), and n-propiophenone(208)
by Euglena gracilis Z. Compounds as in Table 4. (Noma et al. 1994b)
226
phenyl-I-ethanol (175a) gradually increased, and finally the RIS ratio became
45:55 (10% ee) (Noma et al. 1994b). Compounds 175a and 175b were
dehydrogenated to give 174 in a small amount. In the case of compound 179,
the RIS ratio of 180a and 180b was nearly equal. In compound 181, when the
biotransformation was finished, the RIS ratio of 182a and 182b changed to
32:68 (36% ee). However, when the cultured medium including 181 was
allowed to stand for a long time (22 days) at room temperature, the RIS ratio
reached equal again. In compound 189, although the RIS ratio of 190a and
190b became 20: 47 (27% ee) in the course of the reaction, it was equal by the
end. Propiophenone (208) was transformed predominantly to (IS)-I-phenyl-lpropanols (209b, 59% ee). n-Butyrophenone (212) was transformed predominantly to (IS)-I-phenyl-l-butanol (213b, 16% ee). The results of the
biotransformations of n-propio-, n-butyro-, isobutyro-, n-heptano-, n-octano-,
n-nonano-, and n-decanophenones (187-193) and the substrate concentration
of the survival of Euglena are shown in Fig. 17 and Table 4 (Noma et al. 1992a,
1994b). Compounds 187-192 were also transformed to the corresponding
secondary alcohols. However, 0- and p-hydroxypropiophenones (210 and 211)
and p-hydroxyheptanophenone (218) were not transformed at all (Table 4). In
the biotransformation of aliphatic methyl ketones such as 2-butanone (229),2pentanone (230), 2-hexanone (231), 2-heptanone (232), 2-octanone (233), 2nonanone (234), 2-decanone (235), 2-undecanone (236), and 2-dodecanone
(237), all compounds were reduced to the corresponding alcohols and the
order of preference for the reduction was 235 > 234 > 236 > 233 > 237 > 232
> 231 > 229 > 230 (Fig. 19). On the basis of the above results, it is suggested
that the longer side chain of aliphatic methyl ketones (229-237) increases
reactivity for the reduction of carbonyl group. 4-Methyl-2-pentanone (238),
ethyl-n-butyl ketone (239), di-n-propyl ketone (240), acetyl acetone (242), 5methyl-3-heptanone (244), and 4-(p-hydroxyphenyl)-2-butanone (222), 1phenyl-2-butanone (224), pinacolin (247, 3,3-dimethyl-2-butanone), and
benzophenone (246) were also transformed easily to the corresponding
alcohols. Further, benzoylacetone (226) was transformed to 4-phenyl-4hydroxy-2-butanone, 4-phenyl-2-hydroxy-4-butanone, and 4-phenylbutane2,4-diol. However, acetonylacetone (243) was not transformed at all.
Biotransformations of terpene alcohols and related alcohols by Euglena
are summarized in Table 5 (Noma et al. 1990, 1991b, 1992b, 1993, 1994a; Noma
and Asakawa 1992). The time courses and metabolic pathways for the
biotransformations of l-trans- and cis-carveols (134 and 135), d-trans- and ciscarveols (134' and 135') and dl-trans-carveol (equal mixture of 134 and 134')
and dl-cis-carveol (equal mixture of 135 and 135') by E. gracilis Z are shown
in Figs. 6, 7, and 20. Compound 134 was transformed stereospecifically via
123 and 124 to 126 as the major product. However, compound 135 was not
transformed (Figs. 6, 20). On the other hand, compound 135' was
diastereoselectively transformed, mainly to give 123', 125', and 128' as major
product, and compound 134' was not transformed at all (Figs. 7, 20).
Enantioselectivities for an equal mixture of 134 and 134' and 135 and 135' and
the diastereoselectivities for the mixture of 134 and 135 and 134' and 135' were
observed (Noma and Asakawa 1992). Furthermore, in the biotransformation
Euglena gracilis Z
227
100
50
2345678
Time(days)
Fig. 19. Biotransformation of aliphatic ketones(229-237) by Euglena gracilis Z. Compounds as in
Table 4. (Noma et al. 1994b)
of l-trans- and cis-carveyl acetates (137 and 139) and d-trans- and cis-carveyl
acetates (137' and 139') and I-trans- and cis-carveyl propionates (138 and 140),
Euglena easily hydrolyzed the esters to give 134, 135, 134' , and 135'. Although
the alcohols 134' and 135 were not transformed at all and accumulated in the
medium, compounds 134 and 135' were further metabolized to carvones (123
and 123') and other related metabolites as described above (Figs. 6, 7). The
preferential hydrolyses for cis-forms were observed. Furthermore, in the
biotransformations of d-, 1-, and dl-borneols (248, 248', and equal mixture of
248 and 248') and d-, 1-, and dl-isoborneols (249',249, and equal mixture of 249'
and 249), the enantio- and diastereoselective dehydrogenation for 249' was
observed and I-camphor (250') was obtained at ca. 50% yield (Fig. 21; Noma
et al. 1992b). The conversion ratio was ca. 50% even at the different kind of
concentration of 249' (Fig. 22). When compound 250' was used as a substrate,
it was also converted to 249' in 22% yield for 14 days. Furthermore, compound
d-camphor (250) was also reduced to 248 in 4 and 18% yield for 7 and 14 days,
respectively. Cycloalkanols such as cyclobutanol, cyclopentanol, cyclooctanol,
and cyclodecanol were easily dehydrogenated to the corresponding ketones, as
described above. The order of preference for the dehydrogenation of the
cycloalkanols was C5 > C4 > ClO > C8 > C7 > C12 (Fig. 14, Table 5).
228
Substrates
I-trans-Carveol (134)
I-cis-Carveol (135)
l-trans-Carveyl propionate (13S)
d-trans-Carveol (134')
d-cis-Carveol (135')
229
Euglena gracilis Z
Table 5. Continued
The mixture of trans- and cis-Shisools
(9: 10 = 62: 38 peak area in GC)
l-Phellandrol (18)
Geraniol (32)
Nerol (33)
Geranyl acetate
d-Borneol (248)
I-Borneol (248')
dl-Borneol (248 and 248')
1- Isoborneol (249)
d-Isoborneol (249')
dl- Isoborneol (249' and 249)
Myrtenol (2)
Myrtanol (4a and b)
Thymol
Cumin alcohol (28)
l-Acetoxy-p-menthane
Cinnamyl alcohol (119)
2-Methylcinnamyl alcohol (121)
Eugenol
(lR)-l-Phenyl-l-ethanol (175a, R:S = 100:0)
(IS)-I-Phenyl-l-ethanol (175b, R:S
dl-l-Phenyl-l-ethanol (R:S
0:100)
50:50)
I-Buten-3-ol
2-Methylcyclohexanol(trans and cis
Cyclobutanol
Cyclopentanol
Cyclohexanol (158)
Cycioheptanol
Cyclooctanol
Cyclodecanol
Cyclododecanol
20: 80)
Cinnamyl alcohol (119) and 2-methylcinnamyl alcohol (121) were hydrogenated to 3-phenylpropanol (120) and 2-methyl-3-phenylpropanol (122), respectively (Table 5). When either (1R)- or (lS)-l-phenyl-l-ethanol (175a or 175b)
was transformed, acetophenone (174) was obtained in a small amount and the
RIS ratio of 175a and 175b became 93: 7 and 11: 87, respectively (Table 5).
230
135
100
134
~
:!l
u
50
::l
-0
123
0
I)
(,
126
1() II
Time (days)
::l
I)
Time (days)
135'
~
-0
134'
I(X)
:!l
u
Ii
50
0..
129'
I::=._.-----L-.
..
2
4
6 8
8-----8
10
10
Time (days)
Time (days)
100
~
:!l
u
::l
-0
::
0..
50
L-..L
10 12 14 16 0
Time (days)
10 II
Time (days)
Fig. 20. Enantioselectivity in biotransformation of l-trans- and cis-carveol (134 and 135), d-transand cis-carveol(l34' and 135'), and dl-trans- and cis-carveol(l34 and 134' and 135 and 135') by
Euglena gracilis Z. Compounds as in Table 3 and Figs. 9 and 10. (Noma and Asakawa 1992)
1- Acetoxy-p-menthane and geranyl acetate were also hydrolyzed to pmenthan-1-01 and geraniol (30), respectively, as well as carveyl acetates,
carveyl propionate, etc. Compound 30 was isomerized to nerol (31). Terpene
alcohols containing isopropenyl group such as dihydrocarveol and shisool
----
O~
50
,
8
"',:,u
Time(days)
/'
-"""""""M"'.:
12
0
~,
,
4
8
Time(days)
'].."J~
"-
~<49'
248'
12
250'
8
Time(days)
12
Fig. 21. Enantio- and diastereoselectivity in the biotransformation of borneols by Euglena gracilis Z. Compounds: 248' I-borneol;
249 and 249' 1- and d-isoborneol, respectively; 250' I-camphor. (Noma et al. 1992b)
Q..
L..
"0
=>
tl
249
100~
f-'
'"
(")
;:,
232
,OH
100
249'
:.~ 100ppm
~~~~.
---0-.
~
'-'
"011)
E
0
..0
0
10
50
til
......
"I!:s
---Q
Irl
~
....0
'-'
..c:::
50
0..
-.!.
100
Time(days)
Fig. 22. Effect of substrate concentration on biotransformation of d-isoborneol by Euglena
gracilis Z. Compounds: 249' d-isoborneol; 250' I-camphor. (Y. Noma et at. 1992b)
233
Euglena gracilis Z
Substrate
d-Limonene (136')
/-Limonene (136)
I-Methyl-l-cydohexene
Cyclohexene
l,4-Cineole
1,8-Cineole
~]-Carene
p-Menthane
Products
d-trans-Isopiperitenol (141')
d-cis-Isopiperitenol (142')
d-trans-Carveol (134')
d-cis-Carveol (135')
d- Perillyl alcohol (Sb)
d-Carvone (123')
I-Neodihydrocarveol (126')
/-trans- Isopiperitenol (141)
/-cis- Isopiperitenol (142)
/-trans-Carveol (134)
/-cis-Carveol (135)
I-Carvone (123)
/-Perillyl alcohol (Sa)
d-Neodihydrocarveol (126)
d-Isodihydrocarveol (12S)
3-Methyl-2-cydohexenone (165)
2-Cyclohexenol
Cyclohexanol (ISS)
Not transformed
Not transformed
Not transformed
Not transformed
234
Table 7. Microbiological reduction, oxidation, and hydrolysis pattern for terpenoids and related
compounds by Euglena gracilis Z. (Y. Noma, unpubl. data)
A. Microbiolgical reduction
1. Reduction of aldehydes to alcohols
a) Reduction of terpene aldehydes to terpene alcohols
Myrtenal (1) Myrtanal (5) l-Perillaldehyde (7a) dl-Perillaldehyde (7a and 7b)
trans- & cis-l,2-Dihydroperillaldehydes (14 and 15) l-Phellandral (18)
trans- & cis-Tetrahydroperillaldehydes (23 and 24) Cuminaldehyde (27)
Citral (30 and 31) d- and l-Citronellal (38 and 39) dl-Citronellal (38 and 39)
b) Reduction of aromatic and related aldehydes to alcohols
Benzaldehyde (46) 0-, m- & p-Hydroxybenzaldehydes (47-49)
0-, m- & p-Anisaldehydes (50-52)
0-, m- & p-Chlorobenzaldehydes (53-55)
2-,3-, & 4-Cyanobenzaldehydes (56-58) 0-, m- & p-Nitrobenzaldehydes (59-61)
0-, m- & p-Tolualdehydes (62-64)
o-Phthalaldehydic acid (65)
Terephthalaldehydic acid (66) o-Phthalaldehyde (67) Isophthalaldehyde (68)
Terephthalaldehyde (69) o-Vanillin (70) 3-Nitrosalicylaldehyde (71)
Vanillin (72) Veratraldehyde (73) Isovanillin (74) Ethylvanillin (75)
2-Hydroxy-5-methoxybenzalsehyde (76) 2,4-Dimethylbenzaldehyde (77)
2,3-,2,4-,2,5-, and 3,5-Dimethoxybenzaldehydes (81-84)
2,3,- 2,4-, 2,6-, 3,4-, and 3,5-Dichlorobenzaldehydes (85-89)
2,3,4-, and 3,4,5-Trimethoxybenzaldehydes (90 and 91)
p-Acetylamidebenzaldehyde (92) Phenyl acetaldehyde (93)
2-, and 3-Phenylpropionaldehydes (94 and 95) Cinnamic aldehyde (96)
a-Methylcinnamic aldehyde (97) Nicotin aldehyde (98)
c) Reduction of aliphatic aldehydes
n-Heptylaldehyde (99) Pelargonaldehyde (100) trans-2-Hexenal (101)
trans-2-Heptenal (102) cis-4-Heptenal (103) trans-2-Decenal (104)
2,4-Hexadienal (105) trans,trans-2,4-Heptadienal (106)
trans,trans-2,4-Nonadienal (107)
2. Reduction of ketones to alcohols
a) Reduction of saturated ketones
d-Dihydrocarvone (U4) I-Dihydrocarvone (124') d-Isodihydrocarvone (U5)
I-Isodihydrocarvone (U5') d-Carvomenthone (144) I-Carvomenthone (144')
I-Isocarvomenthone (146) d-Camphor (250) I-Camphor (250')
dl-Camphor (250 and 250') 2-,3-, and 4-Methylcyclohexanones
Cyclobutanone Cyclohexanone (157) Cycloheptanone
Cyclooctanone Cyclodecanone Cyclododecanone
d-Dihydrocarvone-8,9-epoxides (150a and 150b) d-Isodihydrocarvone )-8,9-epoxides
I-Dihydrocarvone-8,9-epoxides (150a' and 150b')
2-Butanone (229)
2-Pentanone (230) 2-Hexanone (231) 2-Heptanone (232)
2-0ctanone (233) 2-Nonanone (234) 2-Decanone (235)
2-Undecanone (236) 2-Dodecanone (237)
7,8-Dihydrobenzalacetone 7,8-Dihydrobenzalacetophenone
4-(p-Hydroxyphenyl)-2-butanone (222) I-Phenyl-3-hydroxyhexa-5-one (223)
Benzoylacetone (226) 4-0xodihydroisophorone
b) Reduction of a/i-unsaturated ketones
a-Ionone (166) j3-Ionone (167) Acetophenone (174)
0-, m- & p-Hydroxyacetophenones (176-178)
0-, m- & p-Methoxyacetophenones (179, 181, and 183)
p-Methylacetophenone (189) Acetovanillone (198)
2,5-Dimethylacetophenone (193) 3,4-Dimethoxyacetophenone (192)
Propiophenone (208) Butyrophenone (2U)
3. Hydrogenation of olefins
a) Hydrogenation of olefin conjugated with carbonyl group
I-Carvone (U3) d-Carvone (U3') I-Carvotanacetone (143)
Euglena gracilis Z
235
Table 7. Continued
B.
C.
D.
E.
236
> C12 > C8 > ClO, whereas that for the dehydrogenation of the cycloalkanols
was C5 > C4 > C10 > C8 > C7 > C12. When both cyclopentanol and
cyclohexanone were added to the cultured broth at the same time, both
dehydrogenation and reduction were observed at the same time. For dl-aionone, and j3-ionone, Euglena preferred reduction of the carbonyl group to
hydrogenation of the C = C double bond. In the case of cinnamic aldehyde
and a-methylcinnamic aldehyde, the reduction of the aldehyde group occurred predominantly, and then the resulting alcohols were further hydrogenated to the corresponding saturated alcohols. However, in the case of
benzalacetone and benzalacetophenone, hydrogenation of the C = C double
bond in the side chain occurred predominantly rather than the reduction of the
carbonyl group. Acetophenone and related ketones were also reduced
nons electively to the corresponding secondary alcohols. Aliphatic methyl
ketones were also reduced. l-trans- and d-cis-Carveols and d-isoborneol were
enantio- and diastereoselectively transformed. Terpene esters such as carveyl
acetates and carveyl propionates were hydrolyzed. Terpene hydrocarbons and
related hydrocarbons such as limonene, l-methyl-l-cyclohexene, and
cyclohexene were also biotransformed to give the hydroxylated compounds.
Euglena is considered a good bioreactor to prepare primary alcohol and
secondary alcohol from aldehydes and ketones, respectively. Among the
metabolites, very important fragrant components such as shisool, etc. have
been obtained. Microbiological reduction and oxidation pattern for terpenoids
and related compounds by Euglena gracilis Z is summarized in Table 7.
Acknowledgements. The authors would like to thank Professor Emeritus Shozaburo Kitaoka and
Professor Nagahisa Nakano (University of Osaka Prefecture), Dr. Toshihiro Hashimoto and Dr.
Hironobu Takahashi (Tokushima Bunri University), and Professor Toshifumi Hirata (Hiroshima
University) for useful discussions, and Ms. Maoko Miki, Akiko Sogo, Sachiko Fujii, Yasuko
Wakita, and Hitomi Sakamoto for technical assistance.
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and aromatics of Japan, Takamatsu, pp 250-252
Noma Y, Okajima Y, Takahashi H, Asakawa Y (1991a) Biotransformation of aromatic aldehydes
and related compounds by Euglena gracilis Z. Phytochemistry 30(9):2969-2972
Noma Y, Takahashi H, Asakawa Y (1991b) Biotransformation of terpene aldehydes by Euglena
gracilis Z. Phytochemistry 30(4):1147-1151
Noma Y, Hashimoto T, Miki N, Asakawa Y (1992a) Microbiological transformation of a-ionone
and related compounds. 36th Symp Chemistry of terpenes, essential oils and aromatics of
Japan, Nishinomiya, pp 202-204
Noma Y, Sogo A, Fujii S, Miki N, Hashimoto T, Asakawa Y (1992b) Biotransformation of
terpenoids and related compounds. 36th Symp Chemistry of terpencs, essential oils and
aromatics of Japan, Nishinomiya, pp 199-201
Noma Y, Takahashi H, Asakawa Y (1993) Formation of p-menthane-2,8-diol from carvone by
Euglena. 37th Symp Chemistry ofterpenes, essential oils and aromatics of Japan, Okinawa, pp
23-25
Noma Y, Takahashi H, Asakawa Y (1994a) Hydroxylation of dihydrocarveols and related compounds; carvone metabolism in Euglena(2). 10th Annual Meet Euglena Res Assoc, Osaka,
Abstr Pap, p 10
Noma Y, Wakita Y, Asakawa Y (1994b) Biotransformation of acetophenone and related compounds by Euglena. Annu Meet Japan Soc Bioscience, Biotechnology and Agrochemistry,
Tokyo, Abstr Pap, p 128
Noma Y, Higata T, Hirata T, Tanaka Y, Hashimoto T, Asakawa Y (1995) Biotransformation of
[2-'H]-( - )-carvone by Asp. niger, Euglena and Dunaliella. 39th Symp Chemistry of terpenes,
essential oils and aromatics of Japan, Utsunomiya, pp 367-368
Schiff JA, Lyman H, Russell GK (1971) [12] Isolation of mutants from Euglena gracilis. Methods
EnzymoI23:143-162
Sudate Y, Goto M (1986) Production of arachidoenic acid by Euglena. Proc 2nd Annu Meet
Euglena Res Assoc, Osaka, Abstr Pap, pp 17-18
Tani Y, Tsumura H (1989) Screeing for tocopherol-producing microorganisms and a-tocopherol
production by Euglena gracilis Z. Agric Bioi Chern 53:305-312
Tani Y, Okumura M, Li S (1987) Liquid wax ester production by Euglena gracilis. Agric Bioi
Chern 51:225-230
Yamada T, Maekawa F, Egami F, Yasugi R (eds) (1983) Dictionary of biology, 3rd edn. Iwanami
shoten (Tokyo)
INNOCENTI2 ,
R.
CANIAT0 1,
R.
FIUPPINI\
1 Introduction
1.1 Classification, Distribntion, and Importance of the Plant
The genus Haplophyllum belongs to the family Rutaceae. More than 70 species, growing from the Mediterranean region to eastern Siberia (most of them
in western and central Asia), are assigned to this genus (Everett 1981). Only
eight species can be found in Europe (Townsend 1968); H. patavinum (L.) G.
Don fil. is the only species occurring in Italy (Townsend 1968; Pignatti 1982).
H. patavinum was first collected on the Euganean Hills (NE Italy), probably in 1722, by the Italian botanist Micheli, who described it as Pseudo-Ruta
patavina (Micheli 1729) and stressed the differences from the genus Ruta
("plantae genus a Ruta diversum"). This species, described by Zanichelli
(1730) as Pseudoruta Micheli, was then included by Linnaeus (1753) in the
genus Ruta (Ruta patavina L.). De lussieu (1825) split the genus Ruta into
two genera, Ruta and Aplophyllum; the plant species from the Euganean Hills
was assigned to the latter genus by Don (1831). Spach (1849) changed
Aplophyllum into Haplophyllum.
At present, there is general agreement regarding Haplophyllum as a genus
distinct from Ruta, on the basis of morphological characters and chemical
evidence (Waterman 1975).
H. patavinum (Fig. 1) belongs to the section Oligoon of the genus
Haplophyllum (dehiscent fruit with five carpels and two ovules in each
loculus) according to Vvedensky (1949). It is a perennial herb with yellow
flowers in dense cymose inflorescences; leaves crowded, basal leaves simple,
middle leaves 3-sect to the base, uppermost leaves linear, simple, or 3-sect.
The leaf morphology and also the leaf width are very varied.
Two varieties of H. patavinum were described. The variety albanicum of
Baldacci (1901) later described as a separate species, H. albanicum (Bald.)
Bornm., is now regarded as a synonym of H. boissieranum Vis. & Pancic
(Townsend 1968). According to Beguinot (1905), the wide variability in leaf
width and the occurrence of many intermediate forms do not support the
variety angustifolia (Ruta patavina L. var. angustifolia Nob.).
1
2
Department of Biology, University of Padua, Via U. Bassi 58/B, 35131 Padua, Italy
Department of Pharmaceutical Sciences, University of Padua, Via Marzolo 5, 35123 Padua, Italy
239
240
241
Slightly greater amounts of viable seeds are produced by specimens transplanted to different soil (unfortunately, the transplanted specimens do not
survive for long), which supports the hypothesis put forward by Cappelletti
(1929) of sterility of mycotic origin. In plants of H. patavinum cultivated in the
Botanical Garden of Paduva, vegetative propagation occurs frequently, but
only one seedling was observed.
1.3 Productiou of Coumariu Compounds
2 In Vitro Approaches
2.1 In Vitro Culture Studies
Angustifolin
Herniarin
Obtusifolin
Tenuidin
Ramosinin
Ramosin
HC~
H?CY
OCH3
OH
OCH!
OH
OCH3
o~
II
0
c~
V~
V~
V~
H. glaberrimum
H. thesioides
H. obtusifolium
H. tenue
H. ramosissimum
H. ramosissimum
H. versicolor
Versicolin
Gashimov and
Orazmukhamedova (1978)
Reference
Species
H. hispanicum
R,
Auraptene
R4
H. bungei
R3
Osthol
R2
H. hispanicum
H. patavinum
RJ
Umbelliferone
Monoxygenated
R1
g.
'"0
'"0
0>
(')
tr1
tv
t!3
Pediccllone
6-Methoxymarmin
acetonide
6-Methoxymarmin
Geranylscopoletin
6-Methoxy-7-dimethyl
allyloxycoumarin
5,7Dihydroxycoumarin
[soscopoletin
5-Hydroxy-7methoxycoumarin
Scopoletin
Dioxygcnated
Table 1. Continued
R,
O~H
O~
0+
O~OH
OCH,
OCH
OCH,
OCH,
O~
OH
OCH
o~
OH
OCH.,
OH
R,
OH
OCH,
R,
OH
OH
OCH,
R,
Rj
cappadocicum
dauricum
hi'panicum
putavinum
pedicel/atum
ramosissimum
dauricum
H. pedicel/a tum
H. pedicel/atum
Gashimov and
Orazmukhamedova (1978)
Matkarimov et al.
Gashimov ct al. (1979a)
Gashimov and
Orazmukhamedova (1978)
G(\zler et al. (1992)
Batirov et aJ. (1984)
Gonzalez et al. (1973)
Filippini et aJ. (submitted)
Kagramanov et aJ. (1979)
Bessonova et al. (1989)
Batsuren et aJ. (1982)
Reference
ll. hispanicum
H. obtusifo/ium
1-1. ramosissimum
ll. bunRei
H. bungei
H.
1I.
H.
II.
H.
H.
H.
II. bunRei
Species
P-
VJ
...
...,
c
:>
OJ
.::0
OJ
:!:
:>
CJ
;'!
;:::
S
'"
!:i'
'"
;'!
~
:::::
;:::
~c
OH
OH
OH
R.
R,
H. tenue
H. villosum
H. mirtifolium
H. ramosissimum
H. bungei
H. ramosissimum
H. ramosissimum
Reference
H. obtusifolium
H. obtusifolium
H. albert-regelli
Species
H. dauricum
C-~-D-g1ucosyl
OCH3
OCH3
OCH3
OCH3
OCH3
R3
Dauroside D
OCH3
OCH,
OCH,
O~H
OH
Jyc-o.gIUC05Y1
OH
JyOH
OH
O~OH
O~
R,
H. pedicellatum
H'~
OH
~~
aCH3
RJ
6-Geranyloxy-7 methoxycoumarin
Tenudiol
Villosin
Scoparon
Bungeidiol
Obtusoside
Obtusinin
Obtusinol
Collinin
Table L Continued
a.
(t
't:l
't:l
QO
(l
tTl
OH
OH
Obtusiprenol
Obtusidin
OH
OH
Capensin
Obtusiprenin
OH
OH
Obtusitsin
Fraxetin-7 -O-B-Dl-glucopyranoside
OH
OH
R,
Haptusinol
Obtusin
Obtusifol
5.6-Dimethoxy
auraptene
3-Methoxy-R-hydroxy
umbelliferone
Trioxygenated
Table 1. Continued
OCHo
OCH,
o~
OH
OCH
OCH,
OCH,
OCH
OCH
OCH,
OCH
OCH,
R,
O-J1.-D-glucosyl
OH
OH
o~
OH
O~
OH
o~
OH
R,
OH
H'~
HL
OCH,
R,
\/
c---.,:9
OCH,
R,
11. obtusifolium
11. obtusifolium
H. obtusifolium
H. obtusifolium
H. obtusifolium
H. obtusij()lium
H. obtusij()lium
H. obtusifolium
H. obtusifolium
H. hispanicum
H. schelkovnikovii
Species
Batirov et aL (1982)
Matkarimov et aL (1981)
Reference
.(:>.
U,
Q..
'C"
::>
'"
...,
'0
::>
:E
S
'"
:::
2l
'"
""'"
:::
:::::
2l
'<
.g;::-
::t:
-
Thesiolen
Furocoumarins
Marmesin
Ptilin
Ptilastin
Ptilastol
Dihydrofurocoumarins
H3C _
C9
CH ,
,..
~' I
OCH
sO
OR
C~'--<=C(\
I
H2C-Y
HO-c
R=
R=
R=
;:-.... I
eM,
C-l,
H3
Sese lin
000
Xanthyletin
Pyranocoumarins
Table 1. Continued
If. thesioides
If. glaberrimum
H. ptilostylum
If. ptilostylum
If. ptilostylum
H. dubium
If. dshungaricum
If. multicaule
If. per!oratum
If. thesioides
If. cappadocicum
If. dshungaricum
If. multicaule
Species
Reference
"""
?'-
2.
CD
-0
-0
~
'"
!Tl
247
248
Fig. 3A,B. H. patavinum. A Calli 2 years old obtained from native plants. B Calli 4 months
old obtained from in vitro-grown seedling, grown on BS medium supplemented with 2,4-D
(2mg/I), NAA (O.Smg/I), IAA (0.5 mg/l) , kin (0.2mg/I); 3% sucrose. (E.M. Cappelletti et a!.,
unpub!.)
249
10
15
20
25
30
35
40
days
Fig. 4. The growth curves of 4-month suspension cultures subcultured in B5 medium supplemented with 2,4-D (2 mg/I), NAA (0.5 mg/I), IAA (0.5 mg/I), kin (0.2 mg/I); 3 % sucrose calculated
after cell fresh weight (-D-), cell dry weight (-0-), and cell volume after sedimentation (-6-).
(E.M. Cappelletti et aI., unpubl.)
(w/v) Gelrite for developing the plants. After 2 weeks, every seed was
wetted with 20,u1 of kinetin (1 mg/ml). All seeds germinated within 4 weeks.
Therefore, the in vitro germination of seeds from plants growing in
the natural habitat of the Euganean Hills was also investigated. The very
scarce seed production was the main obstacle to this in vitro germination
research. In fact, the small amount of available seeds severely limited the
number of culture media tested and prevented the statistical evaluation of
the results. The effect of scarification on seed germination was first investigated. To evaluate the effect of scarification (cone. H 2S04 treatment) on
seed germination, scarified and nonscarified seeds were sown on B5 medium
without sucrose and hormones. Scarification proved to be a basic requirement for successful seed germination; in fact the nonscarified seeds failed to
germinate.
The effects of sucrose (3%) addition to the B5 medium, and kinetin and
gibberellic acid (20,u1 of a I-mg/ml solution) treatments were also investigated
using scarified seeds. The results are quoted in Table 2. Germination was not
influenced by sucrose or hormone addition to the medium.
250
seed germination
Sucrose
Kinetin
+
+
+
GA3
Seed
germination( % )
65
50
60
55
50
50
Regeneration from nodal shoot segments has been described (Filippini et al.
1994). The source of nodal shoot explants was in vitro-raised seedlings, 10-12
weeks old. The most abundant axillary shoot proliferation was induced by
cultivating the explants (0.5-1.5 cm long) on MS (Murashige and Skoog 1962)
with 3% sucrose, 0.8% agar, 1mg/12,4-D, and 0.2mg/1 kin, pH 5.7 at 25C in
a 12-h photoperiod. A much lower incidence of shoot formation was observed
when 0.1 mg/l kinetin was added and when B5 medium supplemented with the
same hormone amounts was used. Shoots were subcultured into fresh medium
after 6 weeks for further proliferation of axillary shoots. Rooting was achieved
by transferring the isolated shoots on MS supplemented with 3% sucrose,
0.8% agar, 0.5mg/1 BAP, and 1mg/1 IAA at 25C in the dark for 12h and then
under a 12-h photoperiod. After 2 weeks they were transferred to halfstrength MS. About 70% of the isolated shoots rooted on this medium (Fig. 5);
the percentage root induction was low on the other media. Hardening of plants
before transplantation was found to be essential (Filippini et al. 1994).
2.3.3 Micropropagation through Callus Organogenesis
Micropropagation through organogenesis from callus has also been investigated; this procedure involves three stages, namely culture initiation, adventitious shoot formation, and rooting of the adventitious shoots.
Strain A and strain B calli were used for inducing adventitious shoot
formation on different media and under light condition (12-h photoperiod).
251
Fig. SA,B. In vitro regeneration of H. pata vinum from nodal shoot segments. A Axillary shoot
development. B Rooting of the axillary shoots. (Filippini et al. 1994)
Shoot formation was obtained only from the dark yellow hard calli (strain B)
(Table 3). The best results (low induction time, high shoot number, high
growth rate) were obtained on MS medium supplemented with IAA (3mg/l)
and kin (1 mg/I).
When the stems of the adventitious shoots had reached a length of about
5 mm, they were more or less dissected from the callus and transferred individually to a series of substrates with different hormone combinations for
shoot rooting (Table 4). For each substrate, two series of tests were carried
out: one directly exposed to a 12-h photoperiod, the other kept in the dark for
24h before light exposure (12-h photoperiod) to prevent possible auxin degradation. In any case, no root formation was observed, but only shoot proliferation and callus growth.
2.3.4 Somatic Embryogenesis
Both callus types (strains A and B) were cultivated on either solid or liquid MS
media with different growth regulator contents under a 12-h photoperiod at
25C to promote somatic embryogenesis (Table 5). The suspension cultures
252
Table 3. Growth media, auxin/cytokinin combinations and their effect on adventitious shoot
formation in two cell lines of H. patavinum
Cell line
Medium
Auxin
(mg/I)
Kinetin
(mg/I)
Strain A
B5
2,4-D (0.5)
NAA (0.5)
IAA (3)
2,4-D (0.5)
NAA (0.5)
IAA (3)
(0.5)
B5
MS
MS
MS
'MS 1/5 Ca'+
Strain B
B5
B5
MS
MS
MS
"MS 1/5 Ca2+
2,4-D (0.5)
NAA (0.5)
IAA (3)
2,4-D (0.5)
NAA (0.5)
IAA (3)
Shoot
induction
(1)
(0.5)
(1)
(0.5)
(1)
(0.5)
(1 )
++
+
+
were kept on a rotary shaker (80rpm). After 2 months, calli and suspension
cultures were transferred to solid or liquid hormone-free media, respectively,
to stimulate embryo development. Calli were also transferred to liquid media
and suspensions cultured to solid media. All these experiments failed to induce somatic embryogenesis.
Fairly high-frequency embryoids were produced in suspension cultures of
calli originated from in vitro-induced shoots as starting material. The abovementioned calli were obtained on hormone-free solid MS medium in the dark.
Suspension cultures were initiated, after three callus subcultures, in liquid
hormone-free MS medium under a 12-h photoperiod. Somatic embryos were
heart-shaped (Fig. 6).
Somatic embryogenesis was obtained in complete absence of exogenous
plant growth regulators; an analogous result was previously reported for another plant belonging to the same family, Citrus sinensis (L.) Osbeck (Vardi et
al. 1975).
2.4 Extraction and Structure of Coumarin Compounds
2.4.1 Extraction
Extractions of calli and suspension culture cells were carried out taking into
account the fact that coumarin compounds may be present in both free and
253
Medium
Auxin
(mg/I)
Cytokinin
(mg/I)
MS (P)
MS (P)
MS (P)
MS (P)
MS (P)
MS (P)
MS (P)
MS (P)
MS (P)
MS (P)
MS (P)
B5 (dilution 1: 10) (P)
B5 (dilution 1: 10) (P)
B5 (dilution 1: 10) (P)
B5 (dilution 1: 10) (P)
Water (A)
Water (A)
Water (A)
Water (A)
MS (dilution 1: 2) (P)
MS (dilution 1:2) (P)
MS (dilution 1: 2) (P)
MS (dilution 1: 2) (P)
Water (A)
Water (A)
Water (A)
Water (A)
IAA (1)
IBA (0.2)
IBA (1)
IBA (3)
IBA (5)
IBA (0.2)
IBA (1)
IBA (3)
IBA (5)
IAA"
IBN
lBA (1.5)
IBN
IBA (1.5)
IBN
IBA (1.5)
IBA"
IBA (1.5)
IBA"
lBA (1)
IAA (1)
IBA (1)
IAA (1)
lBA (1)
IAA (1)
lBA (1)
IAA (1)
BAP (0.5)
BAP
BAP
BAP
BAP
Gibberellin
(mg/I)
Sucrose
(mg/I)
GA,
GA,
GA,
GA,
GA,
GA,
GA,
GA,
30
30
30
30
30
30
30
30
30
30
30
30
30
0
0
30
30
0
0
30
30
30
30
30
30
30
30
(0.5)
(0.5)
(0.5)
(0.5)
BAP (0.5)
BAP (0.5)
BAP (0.5)
BAP (0.5)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
, Shoots excised were treated directly with IAA or IBA powder (Sigma). (P)
(A) = Agar.
Table 5. Auxin/cytokinin
embryogenesis
combinations
tested
Phytagel.
to
induce
Auxin
(mg/I)
Cytokinin
(mg/I)
2,4-D (5)
NAA (0.5)
2,4-D (2) + NAA (2)
2,4-D (1)
2,4-D (0, 11)
IAA (1)
IAA (0.5)
2,4-D (1)
IAA (3)
Kin (0.5)
BAP (0.5)
Kin (2)
Kin (1)
Kin (1)
Kin (1)
254
Ho~oAo
255
---.
o
Umbelliferone (I)
Osthenol (II)
Angelicin (IV)
Columbianetin (III)
The methanolic solutions were analyzed by TLC on analytical silica gel plates
(Merck, Cat. no. 5715) using CHCl3 or EtOAc-cyclohexane in different ratios
(3:1,2:1,1:1; v/v) as eluents.
Several coumarin compounds were isolated; five of them were identified
by their UV and MS spectra, and by comparison with authentic samples
(supplied by the Department of Pharmaceutical Sciences, University of
Padua). The purity of the compounds was controlled by HPLC under the
following conditions: column LiChrosorb RP 8 7 f1 (Merck), mobile phase
AcCN:H 20 (25:75; v/v); flow rate 1.5mllmin.
Two furocoumarins, columbianetin and angelicin (Fig. 7, III and IV), and
three simple coumarins, umbelliferone (7-hydroxycoumarin), osthenol (7hydroxy-8-prenylcoumarin) (Fig. 7, I and II) and 7-prenyloxycoumarin were
identified.
Umbelliferone: UV max (EtOH) 248, 258, 327nm; MS 70eV at M+ (m/z
162); prominent fragment ions at mlz 134 [M-COt, 105 [M-CHOt, 106 [MCOlo (relative abundance 55, 95, 70, and 60%).
7-prenyloxycoumarin: UV max (EtOH) 203, 249, 259, 322nm; MS 70eV at
M+ (mlz 230): prominent ions at 215 [M-CH3t, 213, 202 [M-COr, 187, 175,
162 [M-CsHs accompanied by hydrogen migrationt (relative abundance 88,
62, 50, 43, 80, and 90%).
256
Strain A
Strain A
Dark
Dark
Dark
Light
Callus
Umbelliferone
Osthenol
7-Prenyloxycoumarin
Columbianetin
Angelicin
Suspension cultures
Strain A
Medium
Callus
Medium
+
+
+
+
257
3 Conclusion
In vitro culture of H. patavinum has to be considered as a useful tool for the
conservation of this rare and endangered plant. Seed germination, which
occurs very rarely under natural conditions, is considerably enhanced in vitro,
allowing successful establishment of plants. This fact is particularly important
when the very low viable seed production from the native populations in the
Euganean Hills is considered. Moreover, seeds are preferred to vegetative
material as the source of propagation material because a wider genetic base
can be maintained (Fay 1992).
H. patavinum can be multiplied in vitro from nodal shoot segments, a
protocol which involves regeneration from existing meristems.
In comparison with other Haplophyllum species, H. patavinum is not
rich in coumarin compounds in vivo. However, a selected cell strain exhibited in vitro coumarin biogenetic potentialities stronger than in vivo.
In fact, new structures, not yet isolated in intact plants, are produced by
calli and excreted into the solid medium, from which all the intermediate
products of the biogenetic pathway of the angular furocoumarin, have been
isolated.
Although other angular furocoumarins have been detected from species
of the genus Haplophyllum (Table 1), the occurrence of the angular
furocoumarins columbianetin and angelicin, and the prenylated coumarin
osthenol had not previously reported.
Investigations on the biosynthesis of other classes of metabolites could
lead to the detection of other pharmaceutically active compounds, since preliminary unpublished data have shown the production of lignanolide compounds by H. patavinum in vitro.
References
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Khim Prir Soedin 3:403-404 (Chern Abstr 1980. 92:41854z)
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1 General Account
1.1 Botany, Distribution, and Importance of Morus Species
The genus Morus (family Moraceae) covers the numerous mulberry species
grown and cultured in various continents, mainly in Asia and Europe.
Koidzumi (1923) described 37 species, of which some common ones are given
in Table 1. The typical representative of diploid species (2n=28) is Morus
alba (white mulberry), which has long been cultivated in China, Korea, and
Japan for feeding silkworm (Bombyx mori L.). Cultivation of mulberry trees
is especially popular in the Jiangsu, Zhejiang, Fujian, and Sichuan districts
in China (Hotta 1951; Hotta et al. 1989). Another useful species, Morus
nigra (black mulberry), is a polyploid with 308 or 330 chromosomes (Agaev
1984).
Mulberries are anemophilous trees or bushes of vigorous branches and
spread crowns (Fig. 1). The trunk of the tree reaches 10m. The dentate leaves
are deciduous, light to dark green in color. Their inflorescences are represented by the male (Fig. 2A) and female (Fig. 2B) catkins which are
monoecious or dioecious. The female flowers form a thick multiple fruit (Fig.
2e). The actual fruits, little achenes or nutlets, are surrounded by the fleshy
sepals and grouped together with the fleshy axis to form the so-called syncarp
(Hill 1937). The white color of the unripe fruit changes to pink, red, or dark
red.
Mulberries are pomologically interesting for their edible sweet fruit.
In silviculture, although artificial diets for silkworms have been improved
(Ito et al. 1974, 1975), mulberry leaves are still essential. In Chinese
medicine, young twigs are applied for rheumatism and neuralgia. The leaves
are antifebrile. The fruits are nutritive and used in tonics. The root bark
is an antiinflammatory diuretic, a cough medicine, and a blood pressurelowering agent (Hotta et al. 1989). Examination of the biological activity
of Diels-Alder type morin, a light yellowish pigment in mulberry wood,
262
Origin
Somatic
chromosomes
(2n)
Reference
Australia
Central China
Japan
India
Japan
Japan, Korea
Persia
28
28
54,84, 112
28
28
28
308,330
M. tiliaefolia M.
M. rubra L.
Japan
North America
84
28
Katsumata (1979)
Bolkhovskikh et al. (1969)
Bolkhovskikh et al. (1969)
Bolkhovskikh et al. (1969)
Koidzumi (1923)
Bolkhovskikh et al. (1969)
Darlington and Wylie (1961);
Agaev (1984)
Bolkhovskikh et al. (1969)
Darlington and Wylie (1961)
Fig. 1. A MOTUS alba tree 20 years after differentiation from callus tissue induced from seedlings
(Kyoto University)
Fig. 2A-C. Reproductive organs of mulberries. A Staminate flowers. B Pistillate flowers. C Ripening fruits
264
265
kuwanon G
kuwanon J
chalcomoracin
kuwanon X
3 Micropropagation
Mulberry trees are conventionally propagated by grafting and cutting
(Honda 1972). Other propagation methods are seedling and layering.
Layering is carried out by pressing the twigs down into the soil to induce
adventitious root formation. The rooted twigs are then cut off from the
main stem (Hotta 1951). Although most of the Morus species display tremendously strong rooting ability, so that planting stem cuttings directly in the
field suffices to establish a mulberry plantation (Omura 1973), micropropagation has its own advantages (see Bajaj 1992, 1996). Some of the positive
points for raising plants through micropropagation are: large-scale production of true-to-type clones of mulberry, conservation of elite and endangered
germplasm, and overcoming problems of difficult-to-root varieties and
speCIes.
266
Table 2. Survey of regenerationimicropagation studies on mulberries. (See also Oka and Ohyama
Explant
Aim of study
Reference
Micropropagation and
preservation
Enomoto (1987)
M. alba. M. bombycis.
M. latifolia,
M. kagayame
Roots. hypocotyls.
young stems, winter
buds, leaves and
shoot tips
M. alba
Cotyledons, leaves,
hypocotyls, shoot
tips of embryos
Cloning of genotypes
M. alba
Micropropagation
M. alba
Nodal segments
Micropropagation and
cold storage
M. alba
Encapsulated adventitious
buds of leaves
Regeneration
of synthetic
seeds
M. bombycis
Plant regeneration
M. bombycis cvs.
Kenmochi, Akagi,
Shimizuwase, Ichinose
Meristems of
cryopreserved winter
buds
M. bombycis cv.
Kenmochi
Plant regeneration
long-term preservation,
cryopreservation
M. bombycis cv.
Kenmochi; M. indica
cvs. Kanva-2, S-1635,
S-36; M. multicaulis
cv. Goshoerami
Axillary vegetative
buds in calcium alginate
beads
Plant regeneration
M. indica
Micropropagation
M. indica
Plant regeneration
M. indica
Micropropagation
M. kagayamae
Shoot tip
Plant regeneration
M. laevigata
Nodal explants
M. latifolia
M.lhou
M. nigra
Winter buds
Winter bud tips
shoot tips
Plant regeneration
Plant regeneration
Rapid propagation of
local varieties
M. nigra
Micropropagation
267
Table 3. Various compounds isolated/biosynthesised in cell cultures of Morus alba
Compound
Reference
b-sitosterol
Kuwanon 1
Mulberrofuran E, kuwanon Q,R,Y
Kuwanon J,Q,R,V
Mulberrofuran T, kuwanol E
Chalcomoracin, kuwanon J
Artanin
Chalcomaracin, b-sitasterol
Hermiterpene
Prenylchalcanes
As explants, we used 1-2-mm apices isolated from shoot tips of actively growing shoots of young self-rooted plant of a local variety of Marus nigra L. The
tips were washed in distilled water and, after removing as many leaves as
possible, they were sterilized in 0.1 % HgCl z for 5 min followed by three washes
in sterile distilled water for 15 min. They were then cultured in test tubes on an
agar solidified medium composed of Knop's macroelements, MS (Murashige
and Shoog 1965) microelements with (mg/l): 0.4 thiamine hydrochloride, 100
mesoinositol, 0.5 BA, 30 g sucrose at pH 5.5. The cultures were incubated at
25C, illuminated by cool white fluorescent tubes at 55 W m -2 with a 16-h day
length.
After 1 month of culture, several small leaves and visible adventitious
buds were formed (Fig. 4A). The regenerated cultures were subcultured on
the same medium but supplemented with 0.1 mg/l GA3 to support adventitious
shoot growth. A little soft, light brown callus was always formed on the basal
cut end of the culture, but it was removed on each transfer to the fresh
medium.
3.2 Shoot Multiplication
268
Quoirin et al. (1977), but with the formation of thinner and fragile shoots.
However, firm shoots were formed on Snir (1982) medium, probably as a
result of GA3 (0.5 mg/l) in the medium.
3.3 Rooting of Shoots
Plantlets (more than 2cm long) were transferred singly to MS medium with
half-concentration macro elements and full-strength microelements, and supplemented with several combinations of growth hormones. Their effects on
rooting are summarized in Table 4.
Fig. 4A-D. Micropropagation of Moru-s nigra. A Established culture of shoot apex after 1 month
of cultivation in modified Knop's medium. B Multiplied shoots after 1 month on modified MS
medium. C Rooted shoots after 1 month on modidied MS medium with different levels of auxins
fmg/lf. From left to right 1 IBA; 0.2 IBA; 0.2 NAA; 0.2 IBA + 0.2 NAA. D Well-rooted plants 2
months after potting. (Ivanicka 1987)
MaTUS
Species (Mulberry)
269
The data show favorable rooting ability in shoots. The first roots
occurred in the 2nd week of culture, and grew without auxin; however,
its presence stimulated root quality and quantity (Fig. 4C). Within the
combinations of auxins tested, O.Smg/l IBA was the best. At a higher IBA
content, callus growth was increased on the basal parts of cultures. A combination of O.2mg IBA and O.2mg/l NAA supported thicker and shorter roots
which were less suitable for transfer, causing a lower survival of shoots in
the soil.
270
Table 4. Effects of hormones on the rooting of shoot" of Morus nigra. (1. Ivanicka)
Hormone
(mg/I)
Rooted shoots/
total shoots
Rooting
(%)
Roots per
shoot
No hormones
0.2 IBA
0.5 IBA
1.0 IBA
0.2NAA
0.2 IBA + 0.2 NAA
14/40
35/59
58/62
32/50
37/54
46/54
35.0
59.3
93.5
64.0
68.5
85.2
2-6
3-5
5-7
3-9
3-7
5-8
Survival in
soil (%)
88.2
After 1 month on the rooting medium, the plantlets were set into 80-mm
diameter pots filled with a damp mixture composed of steam-sterilized peat
and agroperlite in the ratio 2: 1. To prevent wilting, each plant was covered
with a glass jar for 10 days. Plants were acclimated for 10 days by lowering
humidity, and finally grown without glasscovers. The hardened plants were
fertilized twice a month with a liquid fertilizer. After 2 months, well-growing
plants were transferred into larger containers filled with a light substrate of
compost, peat, and agroperlite (1: 1 : 1). Survival of potted plants was high, up
to 90%. After 4 months of growth in the greenhouse they were 25cm in length
with true-to-type entire leaves (Fig. 4D).
The leaves of black mulberry varied in shape from entire to lobed, but in
the greenhouse the newly formed leaves of stems grown in the same year were
all entire and true-to-type. However, later, the same plants in the 2nd growth
year were expressively lobed. The lobed leaves persisted on the tree for about
4 years, but then when a tree entered into the mature phase, the entire leaves
began to prevail. The formation of lobed leaves on a tree in vitro is comparable to the same blade shapes on suckers or on summer shoots after thicker
branches have been cut off in an in vivo tree. Therefore, the lobed leaves on an
in vitro tree are not abnormalities, but belong to the usual symptoms of the
juvenile phase of a mulberry tree which persist for some restricted period.
Lobed leaves were also observed on in vitro field plants of M. indica (Patel et
al. 1983).
Semihardwood summer cuttings taken from young in vitro trees of black
mulberry had much higher rooting abilities, up to 90%, in comparison with
those from grafted trees with only 30% rooting. Some observations of the
morphological characteristics of M. nigra in vitro and a grafted tree are given
in Table 5.
In Morus alba after long-term (150 days) incubation of callus tissue
under illumination of about 6000 Ix resulted, shoot formation was observed.
This shoot was transferred to a hormone-free MS agar medium to promote
root growth. When the roots were about 1 cm long, the rooted shoots were
transferred to pots containing vermiculite and sand. Plants bearing at least two
intact leaves were transferred to soil mixed with peat moss and sand. One of
271
In vitro tree
Grafted tree
230
5.1
127
123
2.8
1.9
5.2
278
7.1
138
135
3.1
2.1
90
30
6.1
the plants grown well was planted at the Herbal Garden of Kyoto University.
This micropropagated tree (Fig. 1) has now grown to a height of about
10m, and various secondary metabolites have been studied in it. (Hano et al.
1989c).
272
07
HO
71
R ~
OHO
R = H: 2,2',4' -trihydroxy4- methoxychalcone
R = prenyl : 2,2',4' - trihydroxy4-methoxy-3' -prenylchalcone
vlf
07
H.
HO
711
CH.
~~Yq;::cPC:
?j IIOR
RO
'.;:
~~JCO.
H~.Vo~~ u "~OH
H
2' - hydroxy- 2,4,4' - trimethoxy- 3' - prenylchalcone
r'l
CH.O
"
q
OH
;"
~I
OH
18" - Q- methylchalcomoracin
Fig. 5. Aberrant metabolism of O-methylated precursors in Marus alba cell cultures
'><
j
300
350
...,..
250
(a)
400
EtOH
II1II
-s
de.
250
r-'}
300
il.
(b)
350
c....
.
...,?t-'"
400
EtOH
p'
450
DIll
-10
o I
+5
+10
o I
+10
.1
+20
+30
+40
-.l
W
:::s
"...,...,
0'
'"
~
,:::
""(ii'
""0
C/)
274
W
I O~
HO r
... ~I
0 orOH
~I
OHO
artocarpesin
HO
ctY'~O
r
OH
OR
artonin I
tetrahydroxynylchalcone
Fig.7. Determination of the structure of artonin I with the aid of an enzyme system of Morus alba
cell cultures. (Hano et al. 1992a)
/0
(6)
[~? 1 , HO~:
:/"1
co,
SEnz
}
OH
OH
275
regular incorporation of the DC-labeling into the two cinnamoyltriketidederived moieties. Contrary to the satisfactory incorporation of [2-13C]mevalonate into ,B-sitosterol, the BC label from the same precursor was not
incorporated into CAL. [2- 13 C]L-Leucine, a candidate for precursor of
mevalonate, was also not incorporated into the hemiterpene moieties of
CAL, whereas aromatic carbons at 3', 5', 10", 2", and 14", corresponding to
the triketide-derived moieties, were enriched. The labeling pattern from
[2- 13 C]L-Ieucine was the same as that from [l-l3C]acetate. [2-BC]L-Leucine was
thus considered to be metabolized in M. alba cell cultures to [l- 13 C]acetyl
CoA, which subsequently participates in the triketide synthesis (Hano et al.
1992b).
The 100.4 MHz 13C NMR spectrum of CAL obtained by administering [U13C6]D-glucose to M. alba cell cultures demonstrates a satisfactory incorporation of the BC label into all carbons of the molecule. Analysis of the satellite
peaks due to l3e- DC coupling between the component carbons disclosed the
13C-Iabeling pattern in CAL shown in Fig. 9, indicating that this compound
consists of two cinnamoyltriketide-isoprenoid units. However, two independent ways of 13C-labeling were observed in the aromatic rings A and F, which
are formed through the shikimate pathway (Fig. 9). Further analysis of the DC
NMR signals for the shikimate-derived A and F ring carbons revealed that
about 50% of the C-l aldehyde carbon and the penultimate carbon corresponding to erythrose 4-phosphate (E-4P) is disconnected. This C3 + C 1 type
disconnectivity in E-4P-derived moieties may arise through glucose metabolism via two triose phosphates, glyceraldehyde 3-phosphate (GAP) and
dihydroxyacetone phosphate (DHAP), which are equilibrated by triose phosphate isomerase. Both triose phosphates formed from D-[U- 13 Co]glucose
participate in the formation of fructose 1,6-diphosphate (F-1,6P), which
enters into the pentose phosphate cycle via F-6P. glucose 6-P, and then
phosphogluconate. Sedoheptulose-7-phosphate occurring in the cycle affords
E-4P with the 13C-labeling described above. This type of disconnection in the
E-4P-derived moieties was also reported in both shikimate-derived aromatic
rings of the antibiotic, obafluorin (Herbert and Knaggs 1992). The two 13C_
labeling patterns in the A and F rings can be attributed to oxidation of
two different isotopic labeled carbons, pointing to a symmetrical phydroxycinnamoyl intermediate. l3C Label from D-[U-1 3C6]glucose was extensively incorporated into the two hemiterpene moieties (Fig. 9). Further
administration of [1,3- J3 C2]- and [2-J3C]glycerol to the cell cultures revealed a
unique 13C-Iabeling pattern in CAL, as shown in Fig. 9, which suggests a novel
hemiterpene biosynthesis. In the case of the formation of an acetate unit from
the exogenous glycerol by way of glycolysis via GAP and DHAP, [1,3- 13 C2]and [2-J3C]glycerol are converted to [2- 13 C]acetate and [l-l3C]acetate, respectively. The experiment with [1 ,3_13C 2]glycerol revealed the expected enhancements at C-6" and C-24" in the starter acetate units, but the 13C labeling in the
second and third acetate units was reversed, appearing at carbons C-3" and C21". The BC NMR spectrum of CAL obtained from this experiment exhibited
13C_l3C coupling between C-3" and C-4' (Jee = 47Hz) as well as between C-21"
and C-ll" (Jee = 51.4Hz). along with long-range coupling between C-3" and C-
Hhl
and
OH
{'o
{:k:i
OH
Fig. 9. BC-Labeling pattern in CAL from D- [U- 13C6l glucose: .... acetate; CJ-EH) (G-l), pyruvate; _ - - _
erythrose 4-phosphate with a 50% disconnection between the terminal carbon and the penultimate carbon
HO
chalcomoracin
24"
and
t:l
.E .
.E.
V'
to
0\
277
4
6
. r - ...... r---OH
17"
OH
(a)
21"
(b)
Fig. 10. a 13C-Labeling patterns in CAL from [1,3- IJC,l glycerol. b 13C-Labeling patterns in CAL
from [2- 13C] glycerol
7" (lee = 4.8Hz) as well as between C-21" and C-25" (lee = 4.8Hz). The 13C
enrichments at C-7" and C-25" might be explained by transfer of 13C between
the cis-methyl and the trans-methyl carbons involving the reversible
diene formation. Pulse-feeding experiments with [2- 13 C]acetate which resulted
in a higher incorporation of 13C label into CAL revealed that the transfer
of 13C takes place not only in the isoprenyl unit at C-4' participating in the
[4 + 2]cycloaddition reaction, but also in the other unit remaining intact at C11" (Hano et al. 1992b). A similar phenomenon was also observed in the
experiment with [2-!3C]glycerol (Fig. 10). The !3C NMR spectrum of CAL
obtained in this case exhibited 13e-13C coupling between C-1" and C-2" (lee =
73.4Hz), as well as between C-22" and C-23" (lee = 74.8Hz). These reversed
ways of 13C-Iabeling at the second and third acetate carbons in both experiments imply the participation of the pentose phosphate cycle in the
hemiterpene biosynthesis. The acetyl CoA derived from the resultant GAP
via phosphoglycerate and pyruvate takes part in the hemiterpene biosynthesis as the second and third acetate units. Regarding the origin of the acetate
units participating in the hemiterpene biosynthesis for CAL, it was concluded that the starter acetate unit for the mevalonate synthesis is of
glycolytic origin, while the second and third acetate units originate from
the pentose phosphate cycle (Fig. 11). On the other hand, j3-sitosterol
cooccurring with CAL was biosynthesized in the conventional way (Hano et
al. 1994a,b).
Both [3- 13 C]L-phenylalanine and [3-!3C]L-tyrosine were incorporated intact into the shikimate-derived moities of CAL and KUJ and M. alba cell
cultures (Hano et al. 1994d). Further experiments administering [l-13C]Lphenylalanine and [3-13C]L-tyrosine simultaneously to the cell cultures revealed that the 13C enrichment of a pair of carbons, C-l' and C-8", of CAL
originating from [l- 13 C]phenylalanine and another pair of carbons, C-3 and C-
278
glucose
glycolysis
A.A
PO~OH
. /A
H3c....11 COOH
~
~~
~
CH 3 COSCoA
~ f or
1 t acetate
the s
L----
A
CH 3 COSCoA
HO&CHO
A
Fig. 11. Biosynthetic route to the hemiterpene moieties of CAL in Morus alba cell cultures: .&.
labeling from (1,3- l3C,l glycerol; labeling from [2- l3C] glycerol
CAL
KUJ
Fig. 12. 13C-Labeling patterns of CAL and KUJ in the simultaneous administration experiment
with [1- 13C]-L-phenylalanine (.) and [3- J3C]-L-tyrosine (.&.)
5", originating from [3-13C]L-tyrosine, were 17 and 4%, respectively. In the case
of KUJ, the 13C enrichment of a pair of carbons, C-8" and the chalcone
carbonyl carbon originating from [l- 13 C]L-phenylalanine, and another pair of
carbons, C-/3 and C-5", originating from [3- 13 C]L-tyrosine, were 6 and 1.5%,
respectively. The predominant contribution of [l- 13 C]L-phenylalanine over
that of [3- 13 C]L-tyrosine suggests that both aromatic amino acids contribute in
a parallel way for the biosynthesis of the prenylchalcone derivatives in M. alba
cell cultures and that direct conversion of L-phenylalanine to L-tyrosine is
unlikely (Fig. 12).
Studies were also conducted on dried root bark taken from a
micropropagated mulberry tree, and a new isoprenoid-substituted flavanone,
279
H
kuwanol C
kuwanol D
Fig. 13. Two new phenolic compounds from the root bark of a mulberry tree redifferentlated
from callus tissues. (Hano et al. 19H9c)
kuwanol C, and a new geranyl-substituted chalcone, kuwanol D, were obtained along with eight known phenolic compounds, morusin, kuwanons U, S,
morachalcone A, 2,2', 4,4' -tetrahydroxychalcone, moracins M, 0, and P
(Hano et al. 1989C; Fig. 13).
\l
OH
"I:
r\l
-
"I:
Q: R,
J (KUJ): R, = R2 = OH
= OH, R2 = H
R: R, = H, R2 = OH
V: R, = R2 = H
OH
OHO
morachalcone B
kuwanon
kuwanon
kuwanon
kuwanon
'JO
~~I
R2
H~W
Fig. 14. Diels-Alder-type adducts and related monomeric compounds in Morus alba cell cultures
morachalcone A
OH
chalcomoracin (CAL) : R, = H, R2 = OH
mulberrofuran T: R, = prenyl, R2 = OH
mulberrofuran E : R, = Rz = H
HO
~"O
~71
R2
OH
H
H~~
OH
moracin C
kuwanol E
00
!'?-
~.
~.
Y'
to
281
Apices (1-2mm) dissected from shoot trips of actively growing shoots were
the best for establishing culture.
282
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WES~OWSKA
1 Introduction
1.1 The Plant
287
288
L. Skrzypczak et al.
acid. It was observed that after the application of the same doses of the oils,
the highest concentration of final metabolites in the blood of rats (e.g.,
prostaglandin PGE j ) appeared after using evening primrose oil.
The medical importance of evening primrose oil, especially of y-linolenic
acid, was described by Horrobin (1990). Positive results are obtained in the
treatment of artheriosclerosis, cardiovascular diseases, atopic eczema, schizophrenia, diabetic neuropathy, multiple sclerosis, Alzheimer's and Raynaud's
diseases, and others. Besides numerous papers on the therapeutic effects of
this oil, some publications report negative results of clinical research (e.g.,
Biagi et al. 1994; Chenoy et al. 1994; Kleijnen 1994).
The aerial part of Oenothera species may also be a source of other biologically active compounds, especially phenolics. Flavonoids with different structures have multidirectional effects on mammals (Middleton and Kandaswami
1994). Other phenol compounds, like phenolic acids (Masquelier and
Delaunay 1965; Wagner 1985) and ellagotannins, exhibit pharmacological
activity. The hydrolyzable tannins show antitumor activity (Miyamoto et al.
1987, 1993a,b; Motoyama et al. 1988; Harborne and Baxer 1993), and also
inhibit HIV replication (As an aka et al. 1988; Nakashima et al. 1992) and
Herpes simplex HSV viruses (Fukuchi et al. 1989). These compounds are
enzyme inhibitors (Kakiuchi et al. 1985; Nishizawa et al. 1989; Kadota et al.
1990; Nonaka et al. 1990) and antioxidants (Su et al. 1988; Okuda et al. 1989),
and they also have antiviral properties (Corthout et al. 1991).
Among the first reports on flavonoid compounds was one on the presence of
the myricetin 3-,B-galactoside in the whole of the plant o. lavandulaefolia T.
and G. (Kagan 1967), and another on the glucosides of kaempferol and
quercetin found in the flowers and leaves of O. biennis L. (Kowalewski et al.
1968). Comprehensive studies of the American species of the genus Oenothera
were made by Averett et al. (1987, 1988). In the leaves of species from
five sections of the genus: Gauropsis, Hartmannia, Kneiffa, Paradoxus, and
Xylopleurium, they found a total of 33 flavonoid compounds, mostly
glucosides, derivatives of kaempferol, quercetin, isorhamnetin, and myricetin
(Fig. 2). They were the first to discover in the genus Oenothera flavonoid
C-glycosyls and, with the exception of O. speciosa (Averett et al. 1987),
glucuronids and flavonoid sulfates. The major flavonoid of 0. speciosa is
myricetin glucoside (Howard and Mabry 1970).
In the majority of the compounds, the substituents are in the C-3. These
results allowed the authors to engage in chemotaxonomic discussions, supported by their knowledge of the distribution of the species under study
throughout North America. Research was also carried out on Oenothera spe-
289
cies of the section Megapterium (Howard et al. 1972; Averett et al. 1991).
Unlike the others, they were found to lack flavonoid sulfates, but, instead, they
had a flavone compound, luteolin 7-0-rutinoside. Chromatographic methods
showed leaves to contain phenolic acids and flavonoids (Szepczyriska and
Wolbis 1992). Other authors identified 16 phenolic acids in 4 species of
Oenothera (Krzaczek and Bogucka-Kocka 1994; Krzaczek et al. 1995).
On the basis of the above works and those by Zinsmeister et al.
(Zinsmeister and Barti 1971; Zinsmeister and Biering 1973; Zinsmeister and
Schels 1975; Zinsmeister et al. 1977), it can be concluded that some 55
Oenothera species have been tested for the presence of phenolic compounds
so far. The biosynthesis of the flavonoids was controlled in seedlings of the
/ \CH/
\CH/ \ (CH )---C~
0 0
2
2 4
2
2 4
'OH
CH- (CH )
3
i-linolenic acid
HO
R
OH
Kaempferol
Quercetin
Isorhamnetin
Myricetin
Luteolin
H
OH
OH
OH
OH
H
H
CH 3
H
H
H
H
H
OH
H
OH
OH
OH
OH
H
R2
OH
Rl
R2
R3
R4
Vitexin
OH
glucosyl
Isovitexin
OH
glucosyl OH
Orientin
OH
OH
Isoorientin
OH
OH
glucosyl H
C-glycosylflavones
Fig. 2. Main compounds in Oenothera species
glucosyl
290
L. Skrzypczak et al.
HO~
co___o~O--co:g:~::
HO~
&
O-CO
00
HO'"
~OOH
1 CO--:::-<.
""'CO
HO
~
o
HO
'"
..... 1
OH
HO HO
00
OH
O-----Co
"'I
.....
OH
_ _ _ _ CO
OH
CO ~O
.... 0
0
0 __------C
HO
OH
O-CO
OH
OH
OH
:H OH
Oenothein
*OH OH
O~OH
CO/"
OH
~I
CO
'1
'"
~g . .
_____
OH
HOYOH
OH
OH
HO
HO
HO
CO
r&
o
HO
HO
...
/0
-?I
Co~O,
OH
/O~OH
/
CO
0-
HO....OH
OH
HO
~O
0
'\.
/'"
CO----- 0
I",
OH
-CO
OH
OH
*0
CO
............... CO
0,
,
....
...
'"
'"
~:H
Oenothein
OH
OH
OH
OH
HO~OH
OH
Hydrolyzable tannins
Fig. 2. Continued
291
The calculated amounts of tannins were 0.9-7.86% for stems and 3.75-17% for
leaves (Zinsmeister et al. 1970; Bartl 1975). Of the four tannin groups
(Hegnauer 1986), noteworthy are the ellagitannins called oenotheins. From
among more than 150 oligomeric hydrolyzable tannins known so far,
oenothein A and B (Fig. 2) have now been isolated as major polyphenols
from the leaves of 0. erythrosepala (Asanaka et al. 1988; Hatano et al. 1989),
O. biennis (Yoshida et al. 1991), and O. laciniata (Yoshida et al. 1995).
Oenothein B was also found in other species from the Onagraceae, Epilobium spp., and Chamaenerion angustifolium (Okuda et al. 1993). These
compounds show biological effects such as antitumor activity (Miyamoto
et al. 1987; Motoyama et al. 1988; Harborne and Baxer 1993). Oenothein
B, with its unique macrocyclic structure, was also produced by partial hydrolysis of cornusiin A (Okuda et al. 1984). From roots and stems
of O. laciniata three new tannins, oenotheins D, F, and G, together
with oenotheins A and B, have been isolated (Yoshida et al. 1995). A minor
trimar, oenothein E, was also isolated. The classification of oligomeric
hydrolyzable tannins and their occurrence in plants was published by Okuda et
al. (1993).
2.2 Oil, Fatty Acids and Other Compounds
L. Skrzypczak et al.
292
Explant
source
Medium MS Growth
regulators (mg/I)
Response
Reference
O. acerviphilla
RostaIiski
(nomen
provis.)
Seedling
parts
Shoot
tips
2,4-D(I)+ BA(2)
Callus
BA(I)+IAA(I)
Multiple
shoots
0. biennis L.
Seedling
parts
Seedling
parts
Seedling
parts
Seedling
parts
2,4-D(I)+kin(0.1)
NAA(2)+kin(0.1)
2,4-D+kin(unpub!.)
Callus
Callus
BA(I)+ IAA(O.I)
BA(I)+NAA(I)
2,4-D(0.25)+ BA(I)
Multiple
shoots
Callus
Shoot
tips
Shoot
tips
Callus
NAA(0.2) + BA(0.2)
NAA(0.2)+ BA(2)
IAA(O.I)+ BA(I)
IAA(I) + BA(I)
2,4-D(2), BA(O.5)
Multiple
shoots
Multiple
shoots
Cell
suspension
Shoot
apex
Not mentioned
O. erythrosepala
Borbas
0. hookeri
Torr. et Gray
0. hookeri
Anthers
2,4-D(2)+ NAA(2)
2,4-D(2)+ BA(2)
Callus
Callus
Seedling
parts
Leaves
2,4-D(0.5), NAA(4),
BA(I)'
NAA(4)+BA(I),
BA(2)"
Callus
Shoot
tips
Seedling
parts
Callus
IAA(O.I) + BA(I)
IAA(I)+ BA(I)
2,4-D(2)+ BA(0.5)
Kin(I)+2,4-D(0.5)
BA(0.5)+2,4-D(2)
Multiple
shoots
Callus
Cell
suspension
Shoot tips
BA(I)+ IAA(I)
Seedling
parts
2,4-D(2) + BA(0.5)
Multiple
shoots
Callus
Shoot tips
BA(I)+NAA(I)
BA(2)
BA(I)+ IAA(I)
MUltiple shoots
Multiple shoots
Multiple shoots
Seedling
parts
BA(0.5)+2,4-D(2)
Kin(I)+2,4-D(0.5)
Callus
O. silesiaca
Renner
Shoot tips
Seedling
parts
BA(I) + IAA(I)
2,4-0(0.5) + Kin(l)
Multiple shoots
Callus
O. rubricaulis
Kleb.
Seedling
parts
BA(I)+ IAA(I)
De Vries
O. hookeri
O. paradoxa
Hudziok
O. ammophila
Focke
0. fallax
Renner em
RostaIiski
Plantlets
Shoots
293
3 In Vitro Approaches
3.1 Review
Few studies have been published so far on in vitro cultures of plants whose oil
contains y-linolenic acid (Table 1). Among the first were reports on the formation of organized multicellular structures with 0. coronifera Renner (Jean
et al. 1976), the genome/plastome hybrids from Oenothera (Stubbe and
Herrmann 1982), and patents of Japanese authors (Osamu and Tadashi 1987;
Takeo et al. 1987), who showed that in o. biennis the biosynthesis of this acid
took place already at the stage of callus tissue formation. They grew cultures
in darkness, on MS (Murashige and Skoog 1962) medium enriched with 2,4-D
and kinetin or NAA and kinetin (Table 1). The y-linolenic acid content was
6.7% (Osamu and Tadashi 1987). The stimulus for the initiation and development of the callus tissue from leaves of seedlings of o. johansen L. was
provided by the products of the conjugates of IAA with alanine and IAA with
lysine (Magnus et al. 1992). Suzuki et al. (1990) devoted their studies to in vitro
cultures of O. erythrosepala Borbas, a plant containing oenothein B (2.246.67%), which has antitumor effects. They initiated cultures from shoot tips
on MS media enriched with various concentrations of NAA and BA. The
most numerous buds were observed to develop on media with 0.2 mg/l NAA
and 0.2mg/1 BA, or 0.2mg/1 NAA and 2mg/1 BA. Shoots were rooted on
media enriched with 2mg/1 NAA. The shoot tip cultures proved to be a fast
method of propagation of plants with a high content of active oenothein
B. Wolfson and Sears (1989) also derived cultures from shoot tips of
294
L. Skrzypczak et al.
Table 2. Content of oil and fatty acids in seeds of Oenothera biennis L. (Skrzypczak et al. 1994)
Species
O. biennis L.
from tissue culture plants
O. biennis L.
from in vivo plants
Oil content
(%)
Stearic
Oleic
Linolic
y-Linolenic
24.0
6.59
1.73
10.06
71.92
8.15
22.5
6.54
1.70
10.84
71.53
7.72
O. hookeri, but they did not publish their full results. The report of Martinez
,and Noher de Halac (1995) was the first on in vitro plants developed
from anthers of the genus Oenothera. Research was also carried out on
the micropropagation of O. biennis, with simultaneous control of the oil
content in seeds and fatty acids in the oil (Skrzypczak et al. 1994). Cultures
were initiated from shoot tips or nodal segments on a medium enriched
with various growth regulator concentrations. The most numerous buds
were produced on MS media with BA (1 mg/l) and IAA (0.1 mg/l), or BA
(1 mg/l) and NAA (1 mg/l). On MS medium without growth regulators the
buds developed into shoots after 2-4 weeks; the shoots rooted after 4-7 days
on MS medium containing lEA (0.5mg/I). The culture time of 0. biennis
was about 4 months from seed placement to the transfer of plants into soil.
Callus culture was obtained on MS medium with BA (1 mg/l) and 2,4-D
(0.25 mg/l). The light green callus tissue, often with a pink hue, grew slowly and
did not differentiate. When transferred to cultivation plots, rooted plants
produced a rosette of leaves in the first year, and in the second, bloomed and
produced seeds. Using the gas chromatography method, the oil content in
seeds was determined, and in the oil, the content of fatty acids (Table 2). At
the Department of Pharmaceutical Botany, we have been engaged in the in
vitro culture of O. paradoxa (Table 1) and of other Oenothera species (Thiem
et al. 1994).
3.2 Establishment of Tissue Cultures and Plant Regeneration
In vitro cultures were initiated from the seed collection of the Chair of Biology
and Botany of the Medical Academy in Wroctaw. The seeds of the following
species: O. ammophila Focke, 0. biennis L., 0. erythrosepala Borbas, O. fallax
Renner em Rostanski, 0. paradoxa Hodziok, 0. rubricaulis Kleb, O.
salicifolia Desf ex G. Don, and O. silesiaca Renner, were used.
Special care had to be taken with seed sterilization. The best results were
obtained using a 0.2% sublimate solution (6-7 min) with an addition of Tween80. Seeds free from primary infections and properly sterilized often germinated 100% after 5-14 days.
From 350 to 500 whole, undamaged seeds of each species were placed
on MS medium. The seedlings were used for micropropagation, which
was initiated from shoot tips (6-8mm long) under conditions described pre-
295
viously (Skrzypczak et al. 1988). The MS medium was enriched with various
growth regulators. The media used in shoot multiplication and in the induction
and growth of the callus are listed in Table 1. The medium containing IAA
(1 mg/l) was suitable for rooting. Plantlets derived through micropropagation
were transferred into pots, and then to experimental plots. No significant
differences were observed in the morphology and yields of plants derived from
in vitro cultures and regular field cultures. Figures 3 and 4 show some stages in
the micropropagation of the Oenothera species.
L. Skrzypczak et al.
296
O.fM.
297
Table 3. Fatty acids in seeds of Oenothera species obtained from culture in vitro
Palmitic
C 16 : 1
Oleic
Cl8o '
Linoleic
C IU
y-Linolenic
C,u
0. paradoxa Hudziok
1993/94
4.9
5.9
74.8
10.1
1994/95
5.2
6.3
76.9
9.0
1994/95
5.1
5.6
77.4
9.0
1995
O. ammophilla Focke
1995
0. erythrosepala Barbas
1994/95
O. fallax Renner
em Rostariski 1993/94
6.2
4.6
75.9
11.3
6.5
5.3
74.4
11.2
5.1
12.5
71.9
7.7
5.4
10.4
70.1
9.6
O. paradoxa
O. paradoxa
O. paradoxa
To determine the oil contents, and its fatty acids, dry seeds of O. biennis were
used that had been collected from plants grown in vitro. As Table 2 shows, the
results were compared with those obtained for seeds of soil plants cultivated in
the same localities, where plants from in vitro cultures had been grown.
298
L. Skrzypczak et al.
Fig. 5. Chromatogram of butanolic fraction from O. erythrosepala Borbas and standard compounds. G Gallic acid; E Ellagic acid; Cellulose plate, developing phases: I 15% OHAc; II
BuOH(2)-OHAc-H20 14: 1: 5, UV254 after spraying with solution of f3-ethanolamine
diphenylboric acid ester
299
The material was extracted with methanol three times for 1 h. The extracts
were combined and reduced to dryness under lower pressure at 40C.
Sediments were dissolved in hot water, cooled, and shaken with chloroform
and then butanol. Butanol fractions were reduced to dryness and dissolved
in a few cm3 of methanol. These solutions were subjected to two-dimensional
chromatography (2D-TLC) on Cellulose plates (DC Merck) in phases: I
first direction 15% OHAc, II second direction BuOH(2)-OHAc-HzO
14:1:5 (Bartl 1975). Spots of compounds were analyzed under UV-365
and UV-254 light before and after spraying the plates with 1 % ethanol
solution of aluminum chloride, or 0.1 % ethanol solution of f3-ethanolamine
diphenylboric acid ester (Naturstoffreagenz A, Roth), and a solution of
KIO).
A sample arrangement of the compounds is presented in the
chromatogram (Fig. 5) in leaves of O. erythrosepala from in vitro culture.
The results indicate that the synthesis of phenolic compounds takes place at
the stage of callus and multiple shoots. Only some spots of ftavonoids were
observed on chromatograms of the extracts of callus tissues, whereas other
phenolic compounds also appeared. The extracts from multiple shoots and
leaves showed more than ten ftavonoids on the plates.
4 Conclusion
Seeds of Oenothera species are a source of bio-oil of consumer and medicinal
significance. As a result of our studies with eight Oenothera species, it was
found that micropropagation from both shoot tips and nodal segments follows
a similar pattern. Eight to 14 shoots can be obtained from a single explant. The
cloning of the Oenothera species may find practical application in the case of
some particularly interesting varieties.
The oil synthesized in the seeds of 0. biennis plants derived from
in vitro cultures contains quantities of fatty acids similar to those in the
seeds of soil plants (Table 2). The results are especially interesting for
the contents of the C-18: 3 acid in the seeds of 1-year-old plants of O.
paradoxa and 0. ammophila in comparison with its contents in the
seeds of 2-year-old plants (Table 3). Osamu and Tadashi (1987) had earlier
reported y-linolenic acid (6.7%) in the callus tissue of the species
mentioned.
Apart from y-linolenic acid with its diverse pharmacological effects, noteworthy is the occurrence of ftavonoids and oenotheins belonging to the
ellagotannin group with anticarcinogenic and antiviral properties which have
been found in the species 0. erythrosepala, 0. biennis, and 0. laciniata. Preliminary analyses using 2D-TLC indicate the presence of ftavonoids, ellagic
and gallic acids, and probably their derivatives (Fig. 5) in callus, multiple
shoots, and leaves.
The results of our studies and those of others show that the method of
micropropagation from existing meristems can be used in mass production of
300
L. Skrzypczak et al.
the Oenothera species while preserving their genetic stability (Bajaj et al.
1988).
5 Protocol
5.1 Micropropagation
Stratified seeds, surface sterilized (6-7 min) in 0.2% HgCl, with two drops of Tween-80.
Explants: Shoot tips (6-8mm long) of seedlings (30 days old).
Medium: Shoot multiplication on MS medium with IAA and BA (Table 1).
Culture conditions: 16-h photo periods (2000 Ix) at 25C, relative humidity 60-80%.
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1 Introduction
The genus Otacanthus (family Scrophulariaceae) comprises six species that are
distributed in east Brazil. They are half-shrubs with stems lignified at the basis,
and opposite, lanceolate to obovate leaves; the inflorescence is a terminal
spike of solitary flowers with a green calyx and blue to purple, bilabiate corolla
with a white spot on the lower lip. All species possess ornamental value due to
their showy flowers that last for several weeks, especially Otacanthus coeruleus
Lind., which has the largest flowers (Fig. 1). This species originates from east
Brazil, in the states Rio de Janeiro and Espirito Santo, where it is sometimes
called incenso, which means incense. It is also found cultivated or naturalized
in north Brazil and on the Mascarenes and Seychelles (Ronse 1993). It was
cultivated as an ornamental in Belgium and France during the last century
(Van Houtte 1862), and is now again being cultivated as a pot plant in several
countries. It has also potential as a cut flower crop (Geertsen 1990).
The aerial parts of O. coeruleus contain considerable amounts of an
essential oil that consists of mono- and sesquiterpenoids (De Pooter et al.
1989). The composition of this oil is interesting from a phytochemical point of
view, since j3-copaene-4a-ol, a sesquiterpenoid that was identified for the first
time, is one of its major components (Fig. 2). Another important component
is a-copaene, which is used as a feromone in the control of Ceratitis capitata,
the Mediterranean fruitfly (Jacobson et al. 1987). It constitutes 3 to 5% of the
essential oil.
Other species of Otacanthus were found to contain essential oils consisting
of mono- and sesquiterpenoids in relatively large amounts as well. The species
that were analyzed are 0. coeruleus, O. jluminensis, 0. platychilus, 0. villosus
(three morphologically and chemically different forms of the latter), an unnamed species, and intermediate taxa between these species. Some of them
contained large amounts of unidentified sesquiterpenoids (Ae. Ronse et al.
I Dedicated to the memory of Prof. Dr. H. De Pooter, who passed away in December 1995
, National Botanic Garden. Domein van Boechout, B-1860 Meise, Belgium
3 Formerly: Laboratory for Organic Chemistry. Faculty of Agronomy, University of Gent.
Coupure Links 653. B 9000 Gent. Belgium
4 Laboratory for Plant Physiology, Faculty of Agronomy. Catholic University of Leuven, De
Croylaan 42, B 3001 Heverlee, Belgium
306
1997a, in prep.). The content and composition of these essential oils is of value
for the taxonomy within the genus, especially for the identification of taxa with
intermediate characters, that are probably hybrids. Furthermore, the presence
of significant amounts of essential oils within several genera of the Scrophulariaceae gives indications for the classification at subfamily level (Ronse
1993).
Conventional propagation of Otacanthus can be done either by taking
cuttings or by sowing. Stem cuttings taken in February and treated with a
commercial rooting preparation (0.5% IAA) all rooted when placed in peat on
307
Otacanthus Species
The structures that secrete essential oils in vegetative parts of plants can be
either trichomes, oil cells, or cavities and ducts (Fahn 1979). In order to find
out the parts of Otacanthus where essential oil is located, freeze-microtome
sections were stained with Sudan Black B and viewed with a light microscope
308
(Ronse 1993). This showed the essential oils to be situated in three types of
glandular trichomes at the surface of the aerial plant parts. Photographs of
these trichomes taken with scanning electron microscopy are shown in Fig. 3.
The first type is a capitate glandular hair with a short unicellular stalk bearing
a round head of four cells (Fig. 3, photo 1); it has a length of 48 pm. The second
type (Fig. 3, photo 2) is very similar but has a head consisting of two cells, and
an average length of 34 pm. The third type (Fig. 3, photo 3) has one or two
stalk cells, one neck cell, and one head cell, with a total length of 77 pm on
average, which becomes three times as large on adult leaves. Trichomes of
type 1 are present only on stems and leaves, those of type 2 on leaves and the
calyx, and type 3 occurs on stems, leaves, the calyx, and the outer surface of the
corolla.
2.2 Content and Composition of the Essential Oil
The essential oil content of the aerial parts of Otacanthus coeruleus amounts to
0.2 to 0.3% in fresh weight (De Pooter et al. 1989). The bulk of this is produced
by the leaves, but the stems have a considerably lower content (0.02 %); some
oil is also produced by the flowers, especially the calyx (0.19%, vs. 0.03% in the
corolla; Ronse 1993).
The volatile oil is a complex mixture of up to 64 mono- and sesquiterpenoid compounds. The composition of the essential oil found in intact
plants at different stages of maturity is shown in Table 1. Compounds that
amount to more than 1 % of the oil are listed here, yielding a total of 25
constituents. The sesquiterpenoids with Kovats index larger than 1580 were
found only in nonflowering shoots of older plants, albeit in small amounts. Of
the 28 identified monoterpenoids, 50% are oxygenated compounds, while of
the 12 identified sesquiterpenoids only 17% are oxygenated. The quantitatively most important compounds are the monoterpenoids trans-pinocarveol,
pinocarvone, myrtenal, and myrtenol, all belonging to the pinane type, and the
sesquiterpenoids f3-copaen-4a-ol and a-humulene; together they constitute
about 50% of the total oil.
Otacanthus Species
309
Table 1. Composition of the essential oil of aerial parts of
Otacanthus coeruleus
Peak
no.
Ip"
2
4
5
11
17
18
929
965
969
1021
1086
1093
19
20
1108
1126
21
22
23
24
25
26
27
28
29
34
37
47
48
49
52
54
61
1131
1141
1155
1166
1173
1176
1182
1200
1207
1374
1451
1577
1582
1594
1617
1656
1756
Compound
a-Pinene
Sabinene
,B-Pinene
Limonene
Linalo01
tr-Sabinene
hydr.
Monoterpenoid
Transpinocarveol
Camphor
Pinocarvone
Isopinocamphone
Terpinen-4-01
Myrtenal
a-Terpineol
Myrtenol
Cis-carveol
Trans-carveol
a-Copaene
a-Humulene
~-Copaen-4a-ol
Sesquiterpenoid
Sesquiterpenoid
Sesquiterpenoid
Sesquiterpenoid
Sesquiterpenoid
4.6
4.0
2.8
0.9
1.0
1.8
4.4
1.8
2.3
0.9
1.0
1.3
4.2
2.6
2.7
0.9
1.8
1.6
1.9
1.3
1.0
0.8
1.5
1.5
11.6
1.6
13.4
1.5
13.1
1.0
13.7
0.4
11.3
0.6
1.1
7.3
1.0
4.3
0.1
3.0
4.6
7.3
8.3
0.3
12.3
0.7
1.7
7.2
0.8
6.0
1.0
1.6
12.1
1.2
1.8
6.7
1.4
5.8
1.7
1.7
2.8
5.2
6.0
1.0
6.5
0.1
0.9
5.8
1.1
3.2
6.6
7.7
1.1
6.0
1.0
1.2
3.8
6.7
13.0
2.5
3.6
4.5
2.5
2.1
310
Table 2. Organogenic response of nodal stem segments of Otacanthus coeruleus to various types
and concentrations of plant growth regulators (in % of explants). (Ronse and De Proft 1992)
Plant growth
regulators
Callus
formation
CuM)
R1
NAAO.5
NAA 0.5
NAA 0.5
NAA 0.5
NAA 5.0
BA2.2
BA 13.2
2,4-D 0.5
2,4-D 2.5
0
0
43
6
100
88
0
0
37
0
+ BA 0.44
+ BA 2.2
+ BA 4.4
+ BA 2.2
Outgrowth of
nodal buds
Shoot
proliferation'
R2
R1
R2
R1
R2
0
50
86
50
17
100
43
0
43
100
9
43
43
24
0
0
0
0
5
0
12
75
86
20
0
0
43
0
43
0
0
0
43
35
0
0
0
0
0
0
0
25
86
80
67
29
57
0
29
0
The results of two replications are given, consisting of 12 and 10 explants, respectively.
R1 and R2: percentage of explants with a particular response in replication 1 and 2, respectively.
" Shoots formed from adventitious buds.
A,B
Fig. 4A-D. Nodal stem segments grown for 3 months on the basal medium supplemented with
different plant growth regulators (in .uM). A 2.2 BA + 0.5 NAA. B 0.5 NAA. C 0.5 2,4-D. D 0.44
BA + 0.5 NAA
with 0.5,uM NAA + 0.44 to 4.4,uM BA. The most rapid proliferation was
observed with 0.5,uM NAA + 2.2,uM BA, yielding a proliferation rate of 6
every 2 weeks. For a rapid micropropagation of 0. coeruleus, this treatment
was used to produce shoots that were subsequently rooted on hormone-free
Otacanthus Species
311
medium with 1 gil charcoal. Shoots could also be obtained from calli placed on
Monnier's medium (Monnier 1976). In vitro-produced plantlets were transplanted in the greenhouse with 80% survival rate, and flowered 10 months
later.
Several environmental factors were found to influence the growth velocity
and the morphology of the shoot cultures of O. coeruleus (Ronse et al. 1997c).
The factors tested were the temperature (day/night 25120 resp. 20/15 0C), the
irradiance level (either 30 or 55 pmollm2/s at plant level), the agar concentration (from 0 to 10 gil), the macrosalt concentration (from 114 to 2 times the MS
concentration) and sucrose concentration (from 5 to 50 gil); the sucrose treatments were given either without or with mannitol addition so as to obtain an
osmotic pressure of -3.5 bar.
Growth increased both with temperature and irradiance, and was optimal
with 6 gil agar, with between 20 and 30 gil sucrose, and at a macrosalt concentration equal to 1 or 1.5 times that of Murashige and Skoog medium. Significant mortality occurred at the lowest and highest sucrose concentrations,
except when mannitol was added. The addition of mannitol also enhanced
growth at most sucrose levels. The morphology and appearance of the cultures
was not influenced at the tested temperatures and irradiance levels, but it was
clearly influenced by the agar and by the sucrose concentration, and especially
by the macrosalt concentration. Agar concentrations under 6 gil yielded fastgrowing, thin, and vitrified shoots. A similar but stronger effect was observed
at the two lowest macrosalt concentrations, while the remaining macrosalt
treatments yielded slower proliferating shoots with better-developed leaves
(Fig. 5). Low sucrose concentrations, either without or with mannitol, also
gave vitrified, but highly proliferating explants.
Two other species of Otacanthus were also propagated in vitro.
Otacanthus platychilus was micropropagated successfully from nodal stem
segments that formed adventitious buds on the basal medium supplemented
with 0.5 pM NAA + either 2.2 or 4.4 pM BA. The highest cytokinin concentration gave the fastest proliferation of the explants, but many were vitrified and
died. With 2.2 pM BA, only a fourfold multiplication of the explants after 2
months was found, but these were healthy. Leaves of this species only responded to a hormone treatment of 5 pM NAA + 2.2 pM BA, which yielded
callus. Shoots were rooted with 100% success after a few weeks on hormonefree medium with 1 gil charcoal. Plantlets were transplanted to the greenhouse, where they began flowering after 16 weeks.
Nodal stem segments of Otacanthus villosus died on the basal medium
supplemented with 0.5 pM NAA + 4.4 pM BA, but they formed adventitious
buds and proliferating shoots with 0.5 pM NAA + 2.2 pM BA; the latter
treatment gave 3.5 times the number of initial explants after 5 weeks. Shoots
were better rooted on hormone-free basal medium without charcoal, than with
1 gil charcoal.
312
Fig. SA-D. Proliferating shoot cultures of O. coeruleus on macrosalt concentration 25-200% MS;
(see text)
Essential oils were extracted from plant tissue cultures and intact plants by
means of a microsteam distillation apparatus (Chrompack), according to the
method of Likens and Nickerson (1964), and adapted to small quantities (170 g) of plant material. The plant material was cut and placed in the flask with
distilled water and boiling chips for regular boiling. The solvent used was
dichloromethane. The water flask was surrounded by an oil bath with a temperature of 120-150 C, while the solvent flask was put in water of about 70C.
The extractions were effected during 2h. After drying the extract over MgS0 4,
the solvent was evaporated from the extract in a light vacuum. The extracts
were kept in a deep freezer under nitrogen atmosphere.
The extraction of essential oils from nutrient media was effected by mixing the nutrient media with distilled water, and then shaking this mixture in
a separating funnel with a mixture of two thirds pentane and one third
dichloromethane. The extraction was repeated four times consecutively with
each nutrient medium, and the obtained solvent fractions were put together.
This fraction was washed with a 5% NaCl solution. After drying over MgS04
for several hours and after filtration, the solvent was evaporated in a light
vacuum.
The analysis of the components of the essential oils was made primarily
by gas chromatography with a Varian 3700 instrument equipped either with
Otacanthus Species
313
home-made SE-30 glass capillary columns (40 or 60m X 0.5mm i.d.; coating
thickness 0.75 ,urn) or with a FSOT RSL-150 capillary column (25 m X 0.53 mm
i.d.; coating thickness 1.2,um), and an FID. Injector and detector temperatures: 220C; oven temperature: programmed from 30 to 220 C at 3C/min;
sample: 0.5,u1 of a solution of 0.5,u1 oil in 50,u1 pentane; on-column injection;
carrier gas: 3.65 ml He/min. Peak areas were calculated by a PDP 11/34
computer. Kovats indices (Ips) were obtained by coinjection of a solution of
the homologous hydrocarbon series C6-C18 with the sample solution in a
temperature-programmed run, and linear interpolation of the location of
the peaks.
Mass spectra were obtained after gas chromatography in a Varian 2700
instrument equipped with an SE-30 FSOT capillary column (25m X 0.53mm
i.d.; coating thickness 1.2,um), coupled to a Varian MAT 112 mass spectrometer by means of a platinum capillary (i.d. 0.133 mm), or with an HP 5890 gas
chromatograph equipped with an 80-m FSOT capillary column (50m SE-30 on
the injector side + 30m SE-52 X 0.2mm i.d.; coating thickness 0.2,um) and an
HP 5970A mass selective detector.
Substances were identified on the basis of their Ips and mass spectra,
which were compared with those of reference compounds. These were purchased, isolated from or identified in essential oils of known composition.
Components with a difference of Ip of 4 can be distinguished from each other.
The mass spectra are compared with known spectra of compounds by an HP
9133 workstation with GC-MS-MSD operating software HP 59974A (series
200-GCMS Master). The program uses the ten peaks with the highest intensity, and weights them by using the product of the intensity times the mass, so
that ions with a high mass become more important. For identification, we
accepted a correlation down to 98%, but in all cases the mass spectra were
controlled visually with those from the reference library. Moreover, the Ip was
used as control for the identity of the peak. Sometimes, mass spectra with
correlations as low as 75% turned out to be well identified.
3.3 Volatile Components in the Cultures of Otacanthus coeruleus
3.3.1 Oil Content
We found that all types of plant tissue cultures of 0. coeruleus contained an
essential oil under all environmental conditions, though their content varied
strongly, from 0.001 to 0.063% in fresh weight. The main factor influencing the
oil content proved to be the type of culture, as well as its growth rate (Table 3).
The growth rate, as indicated in this table, reflects the average total weight and
proliferation rate of the explants after 3 months, the latter being defined as the
number of explants obtained from one original explant. The highest essential
oil content was found in cultures that produce both callus and shoots, and
amounts to about 10% of the content in intact plants. Furthermore, a high
growth rate influenced the oil content negatively, at least in proliferating shoot
cultures.
314
Table 3. Essential oil content and number of constituents of the oil in tissue cultures of different
type and growth rate
PGR treatment
CuM)
Culture
type"
C
2.52,4-D
5.0 NAA
0.5NAA
0.5 NAA
0.5 NAA
0.52,4-D
0.5 NAA
2.2BA
+ 2.2 BA
+ 4.4 BA
+ 2.2 BA
+ 0.44 BA
+
+
+
+
+
Growth
rateb
Oil
content
(%)
No. of
components
S
F
S
F
FF
S
S
S
0.004
0.009
0.014
0.007
0.001
0.026
0.028
0.022
43
42
43
35
44
23
56
51
+
+
+
+
+
+
The culture type influenced the composition of the essential oil, as indicated by
the number of components of the essential oil (Table 3). This number lay
between 23 and 56, which is smaller than in the intact plant. Analysis of the
identity of the compounds showed that the essential oils that most resemble
that of the intact plant were found in the shoot cultures and in some of the
mixed cultures (cultures with shoots and callus). The callus cultures and some
of the mixed cultures produced oils that are less similar to that of the intact
plant, as illustrated in Fig. 6. Callus obtained with 2.5 11M 2,4-D contained the
same constituents as in the intact plant, but in different percentages, e.g., it
contained more sesquiterpenoids.
In all types of cultures, the otherwise important sequiterpenoids
,B-copaen-4a-ol and a-humulene were lacking or were present only in
small amounts. Other compounds occurred in completely different
amounts than in the intact plants under several PGR treatments. The
Otacanthus Species
315
47
A
37
27
34
2 4
I ~ II 17 It
_J._JLlLJ.~~
! 25.
L~
~ ~W
27
n.......-rrrn,.-".-"
II
37
20
17
22
52
48
54
Time (min)
I I I I
I I I
I I I I
10
I I I I
20
I I I I I I I I I
30
I I I I
I I I I
40
I I I I I I
50
Fig. 6. Gas chromatogram of the essential oil of nonflowering shoots of O. coeruleus (A) and
of a callus culture obtained with 2.5,uM 2,4-D (8). Numbering refers to the peak numbers in
Table 1
I I I I I
316
in important amounts (up to 39%) when only auxins, either 2,4-D or NAA,
were added to the cultures.
The sucrose, macrosalt, and agar concentration of the medium influenced
the relative content of the constituents. Some substances, such as valencene,
showed a large variation without apparent relation with the factors studied.
Other substances, such as the monoterpenoids of the pinane type, were
present in constant quantities after one subculture, but after five subcultures of
3 weeks, their content had significantly decreased at low sucrose and low
macrosalt concentrations, and increased at standard and high sucrose concentrations. High osmotic pressure obtained by mannitol addition, on the contrary, caused an increase in these compounds at low sucrose concentration
after five subcultures.
3.4 Volatile Components in the Nutrient Media
I-
0.'
10
20.
10
20.
I I I
30.
40.
I I I
I I I
I I I
50.
I I I
~
-I
MEDIUM
PLANT
2.5
3.0
"'a
-0
CL
Qj
l'
co
0.
0.5
1.0
1.5
l' 2.0
:c
OJ
"E
0,
3.5
5.
20..
30..
40..
sucrose concentration (in gill
10.
50..
Fig.7A,B. Essential oil content of in vitro cultures and nutrient media in relation to sucrose treatment. A Oil per treatment (in
mg)_ B Total oil content per fresh weight (in mg/g)
u
0
C 30.
0
<D
S 40.
0,
E
50.
60.
70.
----l
D:
o.
(0
VJ
'0
1:;
s:.
;,
Ei
318
Table 4. Composition of the essential oil extracted from a nutrient medium with proliferating
shoots of 0. coeruleus after 31 days of culture
Ip
Compound
Ip
1131
1140
1157
1172
1277
1351
1388
1400
1420
1459
1488
Menthon
Isomenthon
Menthol
Isomenthol
Menthylacetate
0.8
0.2
0.3
2.3
0.7
3.9
0.2
12.0
0.7
0.2
0.2
1511
1549
1580
1600
1655
1700
1825
1914
1932
1955
j3-Bourbonene
j3-Caryophyllene
Valencene
Compound
j3-Copaene-4a-ol
1.7
1.1
0.3
1.7
1.8
1.0
14.6
30.8
1.2
4.0
Otacanthus Species
319
References
De Pooter H, De Buyck LF, Schamp NM, Billiet F, De Bruyn A (1989) The volatile fraction of
Otacanthus coeruleus Lind!. containing the new copaene derivative ,B-copaen-4a-o!. Flavour
Fragrance J 4:47-51
Fahn A (1979) Secretory tissues in plants. Academic Press, London, 302 pp
Geertsen V (1990) The keeping quality of Otacanthus coeruleus - a potential new cut flower. Sci
Hortic 43:145-153
Jacobson M, Uebel EC, Lusby WR, Waters RM (1987) Optical isomers of a-copaene derived
from several plant sources. J Agric Food Chern 35:798-800
Likens ST, Nickerson GW (1964) Detection of certain hop oil constituents in brewing products.
Am Soc Brew Chern Proc 5-13
Monnier M (1976) Culture in vitro de l'embryon immature de CapseUa bursa-pastoris Moench.
Rev Cytol Bioi Veg 39:1-120
Murashige T, Skoog F (1962) A revised medium for rapid growth and bio-assays with tobacco
tissue cultures. Physiol Plant 15:473-497
Nitsch JP, Nitsch C (1969) Haploid plants from pollen grains. Science 163:85-87
Ronse A (1993) Otacanthus (Scrophulariaceae) and its essential oils: a multidisciplinary approach.
PhD Thesis no 228, Faculty of Agricultural Sciences, KUL, Leuven, Belgium
Ronse AC, De Proft MP (1992) In vitro propagation of Otacanthus coeruleus Lind. Plant Cell
Tissue Organ Cult 30:243-245
Ronse A, De Pooter H, De Proft M (1997a) Essential oils of Otacanthus (Scrophulariaceae).
Phytochemistry, in preparation
Ronse AC, De Proft MP, De Pooter H (1997b) Micropropagation of Otacanthus species. In: Bajaj
YPS (ed) Biotechnology in agriculture and forestry, vol 40. High-Tech and micropropagation
VI. Springer, Berlin Heidelberg New York, pp 252-263
Ronse A, Van de Vyver A, De Proft M (1997c) Effect of environmental factors on in vitro growth
and morphology of Otacanthus coeruleus (Scrophulariaceae). Belg J Bot 130 (1) (in press)
Van Houtte L (1962) Otacanthus coeruleus. Flore Serres Jard Eur 15:53-54
VAN STADEN
1 Introduction
The genus Oxalis L. is the largest of the Oxalidaceae (Cronquist 1981;
Mabberley 1987), and is represented by some 500 species worldwide. While
being cosmopolitan, the main centres of diversity for Oxalis are in South
America and South Africa. The South Western Cape of the latter region is
particularly rich in Oxalis species; Salter (1944) reported 208 species. These
dicotyledonous plants exhibit a wide range of growth habits (herbs, many
geophytes with bulbs or tubers, undershrubs) and inflorescence structures
(Salter 1944). Their leaves are alternate, usually compound, digitate or pinnate. Most species exhibit nastic movement. In addition to having attractive
shapes, most spectacular during rainy months, leaves of some species also have
attractive spotting. Flowers are regular, bisexual with a pentamerous perianth.
Under bright light conditions, the flowers of Oxalis open in a wide range of
hues, varying from white through yellow to scarlet. Bicoloured tubular or bellshaped flowers occur, mostly with yellow throats (Salter 1944). Many Oxalis
species are geophytes and bear a variety of perennating organs, including root
tubers, stolons, bulbili and bulbs. Aerial bulbili are also produced by some
species. Seeds are small, endospermous and are produced in a fruit (capsule)
the dehiscence of which is explosive (Knuth 1930).
The genus is of considerable economic importance and this will increase
with realization of the ornamental potential of many of the small herbaceous
species (Table 1). Their flowering characteristics (Fig. 1) make them suitable
for horticultural development and introduction as small bedding plants. One
problem that may be experienced in this process is the fact that some Oxalis
species have become naturalized outside their normal habitat. As a result of
their propensity for bulbil formation, some species have become troublesome
weeds (Holt 1987 Marshall 1987) which may harbour inoculum for diseases
(Roos and Hattingh 1986). However, many species have a highly localized
distribution, suggesting that not all taxa have this weedy tendency. Some
species, such as 0. cernua, contain oxalate in levels toxic to livestock (Rekhis
1994). 0. erosa has been mentioned as a medicinal species (Arenas 1981). In
Natal University Research Unit for Plant Growth and Development, Department of Botany,
University of Natal Pietermaritzburg, Private Bag X01, Scottsville 3209, South Africa
Biotechnology in Agriculture and Forestry, Vol. 41
Medicinal and Aromatic Plants X (ed. by Y.P.S. Bajaj)
Springer-Verlag Berlin Heidelberg 1998
Oxalis Species
321
Reason studied
Reference
O. cernua Thunb.
O. corniculata L.
O. dispar N .E. SR.
Rekhis (1994)
Holt (1987)
Sunderland (1966)
Sunderland and Wells (1968)
Ochatt and De Azkue (1984)
O. glaucifolia Knuth.
O. gracilis Jacq.
Oxalate poisoning
Weed, germination
Plastid and pigment
development
Ornamental and medicinal
plant
Ornamental plant
Ornamental plant
0. hedysaroides H.B.K.
O. helicoides Salter
Ornamental plant
Ornamental plant
O. latifolia H.B.K.
0. linearis Jacq.
0. pes-cap rae L.
Weed
Anthocyanin production
Source of inoculum for
bacterial canker
Weed
Anthocyanin production
Ornamental plant
O. erosa Knuth.
O. reclinata Jacq.
O.
O.
0.
O.
regnellii Miq.
rhombeo-ovata A. St. Hil.
stricta L.
tuberosa Mol.
O. variifolia Steud.
Fig.I. Dense-flowering habit of oxalis helicoides in winter while growing in a greenhouse (Crouch
and of Van Staden 1994)
322
1. Van Staden
0. glaucifolia
0. g/aucifolia
0('
4. Red callus
pH 5.7
0.1 NAA + 1 kinetin
+1 GA,
Complete plants
Red callus
MS.
a.Bult) agar.
3. Cell colonies
Cell colonies
B5 liquid or
0.5%
agarose
2. Protoplasts from
internodal callus
1. Stem internodes
Callus
Rooted plantlcts
Shoots
Callus
Rooted plantlcts
Shoots with
expanded k'ave:-.
and elongated
internodes
Adventitious shoot
huds
pH 5.7
16/8 h light/dark.
irradiance I.B Wm
24 + 1 0 ( ,
25
Green. nodular
callus
Response
MS
O.H% agar.
pH 5.7
2.4lyo NaOCl+O.1 %
Tween-20. 10 min
Liquid MS on
Heller
hridges.
3. Shoots
pH 5.7
MS.
O.B% agar.
2. Callus
1. Stem
internodes
0.005 IBA
Li4Uid 1/2
MS
4. Elongated
shoots
O.9GA,
5.~
16/8 h light/dark.
irradiance I.B Wm
3. Adventitious
shoot huds
pH
MS.
O.B% agar.
Culture conditions
Growth regulators
(mg/I)
1.2% NaOCI+II.I%
Triton X-IOO.
ISmin
I. Shoot tips
O. erosa
Basal medium
2. Nodular callus
Sterilization
Explant source
Oxalis species
Ochatt et al.
(1989)
Ochatt el al.
(1988)
Ochalt and
De Azkue (1984)
Reference
W
N
W
(')
'~."
."
'"Vl
~;::.,
Stem internodes
1. Stem nodes
O. gracilis
O. hedysaroides
MS,
0.8% agar.
pH 5.7
1.75% NaOCI,
4 min
Stem internodes
O. reclinata
5 NAA + 0.5 BA
MS,
0.8% agar.
pH 5.7
1.75% NaOCl,
4min
Stem internodes
O. reclinata
MS,
0.2 % Gelrite,
pH 5.7
0.2% HgCI"
10 min
Stem internodes
O. linearis
2 NAA + (U kinetin
or
5 NAA + 0.5 BA
MS,
0.8% agar,
pH 5.7
1.75% NaOCI,
4 min
Stem internodes
3 IAA + 3 kinetin
MS,
0.6% agar,
pH 5,8
Alcohol dip,
0.5% HgCl z, 3 min,
8% NaOCI+
Teepo!, 15 min
0,5 kinetin + 0,5 BA
MS,
0.8% agar,
pH 5,7
I. 75 % NaOCI,
4min
Growth regulators
(mgll)
Basal medium
Sterilization
O. helicoides
Explant source
Oxalis species
Table 2. Continued
16/8 h light/dark,
irradiance 44.5 .umol
m-1s 1.25 3e
or
21.1 {fmol m- 2 s- 1
10 2C
16/8 h light/dark,
irradiance 44.5 .umol
m-1s 1,25:!::3C
+ roots +
+ roots +
+ roots +
Callus
shoots
Shoots
bulbs
Red callus,
anthocyanin
production
Red callus.
anthocyanin
production
Shoots
bulbs
or
21.1.u mol m- 2 s l ,
10 :!: 2C
Callus + roots
Shoot elongation,
2,5 em long
rootable cuttings.
shoots
Growth of existing
buds and axillary
branching of new
shoots
Continuous light
5000 Ix
+ roots
Shoots + roots +
bulbs
Callus
Response
16/8 h light/dark,
irradiance 44.5 .umal
m-: S-I, 25 3 C
Continuous light.
21,umolm 2S"I,
23 :!: 2C
16/8 h light/dark,
irradiance 44.5 ,umol
m- 2 s- l ; 25 3C
or
21.1,umolm 2S"I,
10 2C
Culture conditions
Crouch and
Van Staden (1994)
Crouch ct al.
(1993)
Meyer and
Van Staden (1995)
Crouch and
Van Staden (1994)
Maene and
Debergh (1981)
Crouch and
Van Staden (1994)
Reference
V-J
'"
(1)
Q..
[/)
'Oi"
,...,
.j:>.
Tuber cores
Stern internodes
0. variifolia
2. Regenerated
shoots
Apical stem
internodes
4. Protoplastderived callu~
1.75% NaOCI.
4min
Tuber peeled,
sterile cork horer
0.8% agar.
pH 5.7
MS.
pH 5.8
MS.
0.8% agar,
MS.
0.8% agar,
pH 5.7
3. Cell colonies
0.6% NaOCI.
20min
agar,
pH 5.7
MS.
O.~%
B5 liquid or
0.5%
ag,uose
2.4% NaOel,
0.1 (Yo Tween-20.
lO min
2. Protoplasts from
internodal callus
1. Stem internodes
Liquid MS on
Heller
bridges.
pH 5.7
2 NAA
or
5 J';AA
+ 0.5 BA
+ 0.2 kinetin
3 NAA + 3 zeatin
or
3 NAA + 3 BA
21.1umolm -s
]():': 2C
or
16/8 h light/dark.
irradiance 44.5 pmol
m ~ s I, 25 ::!::: :; C
Callus + shoots
3 NAA + 3 zeatin
or
3 NAA + 3 BA
Shoots
bulbs
roots +
Callus + roots
Callus
Rooted plantlets
Vascular nodules
Protoplast -derived
callus
Callus
5 NAA + 0.5 BA
BA
Rooted plantlets
Shoob
Callus
Cell colonies
+ 0.5
161X h light/dark.
5 NAA
OJ GA, filter
sterilized or
aUloc!aveu
5 NAA + 0.5 BA
MS.
O.K% agar,
pH 5.7
2. Callus
10 min
2.4% NaOCI+
D,l % Tween-20.
3. Shoots
1. Stem internodes
0. tllberosa
O. tllberosa
O. rhombeo-ov{tfa
0. rhomheo-ovata
Table 2. Continued
Crouch and
Van Staden (1994)
(19RR)
Ochatt et al.
W
N
Ul
OJ>
(')
'"
r;;'
-0
(jJ
'~"
;?
326
1. Van Staden
However, bulbil production varies greatly within the genus and many species
are threatened in the wild (Crouch and Van Staden 1994). In O. hedysaroides,
an attractive pot plant, regeneration by cuttings is low due to the poor regeneration capacity of mother plants (Maene and Debergh 1981). Thus tissue
culture holds potential for rapid multiplication of economically desirable
species.
In developing the food potential of Oxalis species it will be necessary to select for reduced oxalate content, tubers with high specific
gravity and increased frost tolerance (Khan et al. 1988). In vitro techniques could help in understanding the morphogenetic potential of
various tissue explants and in identifying conditions for shoot regeneration. Khan et al. (1988) reported morphogenetic (vigour, leaf shape, tuber
shape and size, pigmentation, internode length) and compositional
(chromosome number, sugar and oxalic acid content) variation in in
vitro-generated O. tuberosa plants. This suggests a considerable degree
of somaclonal variation. While undesirable in the micro propagation
process, such somaclonal variation may be beneficial in plant improvement
programmes.
Indications are that Oxalis species are infected with viruses (Coyier 1981),
mycoplasm-like bodies (Atkey and Brunt 1982), and that they serve as a
potential source of inoculum for bacterial canker of stone fruit (Roos and
Hattingh 1986). In vitro culture of small meristematic regions or shoot tips
may help in eradication and control of various diseases, thus improving plant
quality.
2.1.1 Axillary Bud Proliferation
Bud proliferation was studied using stem node explants of O. hedysaroides (Maene and Debergh 1981) and I-mm shoot tips (comprising
the meristematic dome + three leaf primordia) of O. erosa (Ochatt and
De Azkue 1984). Stock plants for these studies were grown in a greenhouse.
With 0. hedysaroides the existing buds elongated and branched optimally
on an IAA- and kinetin-containing medium (Maene and Debergh 1981).
Using a combination of 0.5 mg/l kinetin and 0.5 mg/l BA, shoot proliferation and elongation was achieved to produce rootable cuttings (2.5 cm
long).
The shoot-tip procedure used for O. erosa was more elaborate and timeconsuming (Ochatt and De Azkue 1984). The axillary buds did not elongate
but produced a green nodular callus. Nodular calli were induced to develop
adventitious shoot buds using BA and gibberellic acid (GA3). Application of
0.9mg/C GA3 stimulated internode elongation and leaf expansion. The individual shoots were rooted in half-strength MS containing 0.005 mg/l indole
butyric acid (IBA).
Oxalis Species
327
The majority of tissue culture systems reported for Oxalis spp. used stem
internodes for the initiation of callus, subsequently manipulated in a series of
steps to produce plantlets (Table 2). In the case of 0. tuberosa, callus was
produced from tuber sections but no organogenesis was observed (Khan et al.
1988). Ochatt et al. (1988) developed a three-phase system for 0. glaucifolia
and 0. rhombeo-ovata where internodal callus generated on NAA and
cytokinins was subsequently induced to form shoots using the cytokinins
kinetin or BA plus GA,. The shoots were successfully rooted using halfstrength liquid MS medium containing 0.1 mg/l GA 3 In O. glaucifolia the
callus changed colour during subculture and turned red. Organized structures
were only produced consistently from green callus. Regeneration potential
diminished and was erratic with heterogeneously coloured or red callus
(Ochatt et al. 1988).
Limited callus formation followed by organogenesis was observed in other
studies (Khan et al. 1988; Crouch and Van Staden 1994). A microscopic study
using O. tube rosa indicated that calli first gave rise to thick roots. Swellings
arose at the base of these roots, which may well have been root tubers, as
they were tapered in shape. These swellings then developed into single or
multiple shoots (Khan et al. 1988). Combinations of 3 mg/l NAA with 3 mg/l
BA or zeatin gave best shoot formation, the latter cytokinin being most
effective.
In four South African species with great horticultural potential, callus
formation was rapid and 70% of all internodal explants produced callus within
Fig.2. Organogenesis in O. reclinata following transfer of cultures from 25 to 10C under constant
cool white fluorescent light (21 pmal m- 2 Sl) Arrows indicate bulbs. (Crouch et al. 1993)
328
1. Van Staden
21 days (Crouch and Van Staden 1994). As observed for O. tuberosa (Khan et
al. 1988), root formation initially was more prolific than shoot production.
Transfer of callus of all four species from a growth condition at 25C to a cold
room at 10 2C and a lower light intensity (21,umolm- 2 S-I) resulted in
extensive organogenesis. Shoots, roots, and, most importantly, bulbs developed (Fig. 2). Roots again developed before shoots. Both auxins and
cytokinins were necessary for organ development (Table 2). At lOoC and a
low light intensity, the vitality of developing plantlets improved. After repeated subculture flowering occurred under these growth conditions (Crouch
and Van Staden 1994; Fig. 3).
2.1.3 Plant Regeneration via Pro top lasts
Fig.3. In vitro flowering (arrows) of O. helicoides at lOoC in constant cool white fluorescent light
(21.umol m- 2 S-I). Cultures were maintained on 5 mg I-I NAA and 0.5 mg r l BA. (Crouch and
Van Staden 1994)
Oxalis Species
329
plantiets were regenerated from the red callus produced from the protoplasts. In other Oxalis callus systems the morphogenetic potential was
lost when the callus became pigmented, even in the presence of auxins
and cytokinins (Ochatt and De Azkue 1984; Ochatt et al. 1988; Meyer and
Van Staden 1995). Why the protoplast-derived red callus behaved differently is unknown. In the case of 0. rhombeo-ovata, protoplasts produced only a nodular green callus that did not develop into plant lets.
The availability of a protoplast technique will be useful for somatic hybridization and in overcoming natural barriers for sexual crossing within the
genus.
3 Anthocyanin Production
In a number of instances, Oxalis cultures turned red during culture (Khan et al.
1988; Ochatt et al. 1988, 1989; Crouch et al. 1993). The pigments were isolated
by crushing callus and extracting three times with 3% TFA-MeOH for 24h at
5C. The MeOH concentrate was dissolved in H 2 0 and partitioned against
EtOAC. The aqueous phase was extracted with n-butanol, which was concentrated to yield the crude extract. The crude extract and pigment were never
heated above 30C. The major pigment in 0. reclinata internode callus cultures (Fig. 4) was identified by NMR and GC-MS as cyanidin-3-glucoside
Fig.4. Anthocyanin-rich callus line of O. reclinata, maintained at 25C in the light (16h) provided
by cool white florescent tubes (44.5 pmol m -2 S-'). Cultures were subcultured every 3 weeks on 2%
Gelrite. (Crouch et at. 1993)
J. Van Staden
330
5'
HO
OH
OH
OH
Fig. 5. Structure of cyanidin-3-g1ucoside.
(Crouch et al. 1993; Fig. 5). Anthocyanin production was more prolific at 25
than at 10 C, which favoured organogenesis. It is also produced in suspension
cultures (Crouch et al. 1993). As this anthocyanin could serve as a natural
colorant its production in vitro was examined in stem internodal callus of O.
linearis (Meyer and Van Staden 1995). Anthocyanin-containing callus can
grow in the absence of cytokinins, but not without auxins. Callus growth was
improved with the addition of 8 or 32,uM NAA. The addition of 8,uM BA to
media containing 8 or 32,uM NAA inhibited callus growth. Growth regulators
had a different effect on anthocyanin production. Highest anthocyanin production occurred at a combination of 8,uM BA and 2,uM NAA (Table 3). Of
various cytokinins tested, iso-pentenyladenine (iP), significantly stimulated
callus growth. Pigment production was increased by BA, thidiazuron (TZ) and
zeatin (Table 4). Elevated levels of sucrose (300 to 360mM) stimulated pigment production but inhibited callus growth. Callus growth was best at 60mM
sucrose (Table 5).
4 Protocol
Surface sterilize stem internodal explants with sodium hypochlorite. Place
explants on MS medium containing 2-5 mg/l NAA and 0.1 mg/l kinetin or
0.5 mg/l BA solidified with 0.8% agar. Maintain cultures at 25C under cool
white fluorescent tubes under a 16-h light 8-h dark cycle. Transfer to 10C
induces shoots, roots and bulbs.
Oxalis Species
331
NAA
0
0
0
0
k
k
k
k
0
2
k
32
0
2
k
32
Growth
index
Specific
absorbance
0.1
1.2
6.Ob"
6.4b
0.5
0.7
4.la
3.9a
1.71
U5a
0.64a
0.42a
2.47b
2.k6c
2.26b
2.13b
Growth
index
Specific
absorbance
Control
BA
Kinetin
iP
TZ
Zeatin
5.9
2.6b"
4.6
7.7a
3.6
1.9b
1.24
2.19a
1.65
1.13
2.l0a
2.95a
Growth
index
Specific
absorbance
60
120
IkO
240
300
360
4.4
3.5
3.3
3.0
2.0a
1.5a
O.k2
1.36"
1.56a
1.7ka
3.03b
3.2kb
332
References
Arenas P (1981) Etnobotanica Lengua, Maskoy-FECIC, Buenos Aires
Atkey PT, Brunt AA (1982) The occurrence of mycoplasma-like bodies in severely diseased oca
(Oxalis tuberosa) plants from Bolivia. Phytopathol Z 103:294-300
Brummitt RK (1992) Vascular plant families and genera. Royal Botanic Gardens, Kew
Coyier DL (1981) Chlorotic ringspot and decline of ornamental shamock (Oxalis regnellii). Plant
Dis 65:275-276
Cronquist A (1981) An integrated system of classification of flowering plants. Columbia University Press, New York, pp 826-828
Crouch NR, Van Staden J (1994) In vitro propagation of a number of South African Oxalis
species. S Afr J Bot 60:134-135
Crouch NR, Van Staden LF, Van Staden J, Drewes FE, Drewes SE, Meyer HJ (1993) Accumulation of cyanidin-3-glucoside in callus and cell cultures of Oxalis reclinata. J Plant Physiol
142:109-111
Holt JS (1987) Factors affecting germination in greenhouse-produced seeds of Oxalis corniculata,
a perennial weed. Am J Bot 74:429-436
Khan MRI, Heyes JK, Cohen D (1988) Plant regeneration from oca (Oxalis tuberosa M.): the
effect of explant type and culture media. Plant Cell Tissue Organ Cult 14:41-50
Knuth R (1930) Oxalidaceae. In: Engler A (ed) Das Pflanzenreich 95. Facsimile Edition (1966)
Engelmann-Cramer, Weinheim, pp 1-389
MabberJey DJ (1987) The plant book. Cambridge University Press (1993 reprint), pp 420-421
Maene L, Debergh P (1981) In vitro propagation and culture of Oxalis hedysaroides H.B.K. cv.
Fire Tree. Med Fac Landbouw Rijskuniv Gent 46/4:1201-1203
Marshall G (1987) A review of the biology and control of selected weed species in the genus
Oxalis: O. stricta L., O. lati/olia H.B.K. and O. pes-caprae L. Crop Protection 6:355-364
Meyer HJ, Van Staden J (1995) The in vitro production of a anthocyanin from callus culture of
Oxalis linearis. Plant Cell Tissue Organ Cult 40:55-58
Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco
tissue cultures. Physiol Plant 15:473-497
Ochatt SJ, De Azkue D (1984) Callus proliferation and plant recovery with Oxalis erosa Knuth
shoot tip cultures. J Plant Physiol117:143-146
Ochatt SJ, Ciai AA, Caso OH (1986) Tissue culture techniques applied to American crops:
micropropagation of Oca (Oxalis tuberosa Mol.), an Andean tuber-bearing species. Turrialba
36:187-190
Ochatt SJ, Stampacchio ML, Escandon AS, Martinez AJ (1988) Plant regeneration from callus of
two shrubby Oxalis species from South America. Phyton 48:21-26
Ochatt SJ, Escandon AS, Martinez AJ (1989) Isolation, culture, and plant regeneration of
pro top lasts of two shrubby Oxalis species from South America. J Exp Bot 40:493-496
Rea J, Morales D (1980) Catalogo de Tuberculos Andinos. Inst Boliviano de Tecnologfa
Agropecuaria, Bogota
Rekhis J (1994) The poisonous plant Oxalis cernua. Vet Hum Toxicol 36:23
Roos IMM, Hattingh MJ (1986) Weeds in orchards as potential source of inoculum for bacterial
canker in stone fruit. Phytophylactica 18:5-8
Salter TM (1944) The genus Oxalis in South Africa. A taxonomic revision. J S Afr Bot Supp Vol
1
Sunderland N (1966) Pigmented plant tissues in culture. I. Auxins and pigmentation in
chlorophyllous tissues. Ann Bot 30:353-368
Sunderland N (1967) Pigmented plant tissues in culture. II. Growth, development, and decline of
chlorophyllous tissues. Ann Bot 31:573-591
Sunderland N, Wells B (1968) Plastid structure and development in green callus tissues of Oxalis
dispar. Ann Bot 32:327-346
1 Introduction
The wood fern Polyp odium vulgare L. (Fig. 1) belongs to the family Polypodiaceae, and is the most widely distributed fern species in the world, appearing mostly in warm, humid forests (Bonnier 1934). The name Polyp odium
arises from the particular shape of its rhizomes, branching like feet. The plant
size ranges from 10 to 50cm; the fronds have a very long petiole and the blade
is deeply divided in 20 to 40 alternate lobules. The large sori are present almost
through entire year and are placed on two parallel lines among the median
vein on the underside of the lobes. The penducled sporangia open transversely
throughout a longitudinal ring called the annulus (Strasburber et aL 1983). It
is a very popular fern in Europe and some purgative and vermifugal properties
are attributed to its rhizomes (Vol1ik et aL 1990).
Ecdysteroids (Fig. 2) are the molting hormones of insects and crustaceans. Since ponasterone A was discovered in plants in 1966 by Nakanishi
et aI., over 100 ecdysteroid structures (named phytoecdysteroids) have
been identified in plants, with 20-hydroxyecdysone (20E) being the most
common one reported (Horn and Bergamasco 1985; Lafont and Horn
1989). The biological activity of some phytoecdysteroids has been tested by
a variety of insect bioassays, with many of these structures demonstrating molting hormone activity (Bergamasco and Horn 1980; Kubo and
Klocke 1983; Mele et aL 1992; Slama et aL 1993). In addition, it has been
shown that ecdysteroids exhibit interesting pharmacological effects on mammals, including the stimulation of protein synthesis and the reduction of blood
glucose and cholesterol levels (Camps 1991, and references cited therein).
These compounds have been found in various plant structures, and recent
studies have demonstrated that active biosynthesis of phytoecdysteroids takes
place in young developing tissues and they are then transported to older ones
(Grebenok and Adler 1991; Tomas et aL 1993; Grebenok et aL 1994). The
biosynthesis and distribution of phytoecdysteroids have been recently re-
Department of Plant Genetics, IRTA, Centre de Cabrils, Ctra. de Cabrils sin, 08348 Cabrils,
Spain
2 Department of Biological Organic Chemistry, CID (CSIC), J. Girona, 18, 08034 Barcelona,
Spain
1
334
J. Messeguer et al.
Fig. 1. Plant of Polypodium vulgare growing under greenhouse conditions, showing sori
viewed by Camps (1991), Macek and Vanek (1994), and Adler and Grebenok
(1995).
The phytoecdysteroid content of P. vulgare has been previously reported
by several authors (Heinrich and Hoffmeister 1967, 1968; Jizba and Herout
1967; Jizba et al. 1967a,b; Herout 1970). The most abundant ecdysteroid
isolated from rhizomes is 20-hydroxyecdysone (20E) but minor amounts of
other phytoecdysteroids such as polypodine B (5,20E) and the parent
ecdysone (E) have also been found. Nevertheless, the contents reported are
variable, and, apparently, among other factors, one should consider the possible influence of microclimate due to geographic location as well as the season
of collection. Also, the part of the plant examined and, very likely, the age of
the field-collected material could give rise to such differences.
In a preliminary study carried out in our laboratory, the rhizome content
in 20E ranged from 0.4% of the dry weight for plants collected in January to
0.04% for those collected in October. These results prompted us to consider
the possible application of in vitro culture for mass production of 20E, in which
the seasonal and other variation factors could be avoided.
335
HO
HO
Ecdysone (E)
20-Hydroxyecdysone (20E)
Polypodine B (5,20E)
Abutasterone (20,24E)
5-Hydroxyabutasterone (5,20,24E)
24-Hydroxyecdysone (24E)
PteroSIerone (25d20,24E)
Inokosterone (25d20,26E)
R}
R2
R3
R4
Rs
H
H
OH
H
OH
H
H
H
H
OH
OH
OH
OH
H
OH
OH
H
H
H
OH
OH
OH
OH
H
OH
OH
OH
OH
OH
OH
H
H
H
H
H
H
H
H
H
OH
Rhizomes were collected in the field and cultivated under greenhouse conditions until plants were well established. Mature spores were placed inside filter
paper bags and surface disinfected (5% aq. NaCIO containing a few drops of
Tween-20) for lOmin, followed by washing with sterile distilled water (3 X
lOmin). Using a sterile paintbrush, a few spores were placed on the surface of
half-strength MS culture medium (Murashige and Skoog 1962) supplemented
with sucrose (30g/l) and Bacto Difco agar (8g/l), pH adjusted to 5.7 before
autoclaving at 1 atm for 20min. After 8-10 weeks the small prothallus clusters
(Fig. 3) were transferred into 500-ml culture vessels containing 60ml of the
same culture medium. The most vigorous clusters were selected and prothallus
lines were established by systematically subculturing them each 6 weeks, dividing into three to four sections. All cultures were incubated at 25 2 DC under
16-h light (Sylvania cool white, 37,uM/sm2 ).
After some subcultures, the growth pattern of prothallus-selected lines
was analyzed. As is shown in Fig. 4, during the first 6 weeks there was a parallel
increase between fresh and dry weight of prothallus clusters; then the dry
weight increased very slowly, whereas the fresh weight maintained its rate of
increase; after 60 days, both rates had almost leveled off and finally the fresh
weight even showed a small decrease, which was rationalized as the early
1. Messeguer et al.
336
400
r----------------,
40
at
200
20
-<
-& fr. wt
"'dry wt
0'----------------10
o
~
00
1~
Time (days)
337
Fig. 5. Callus cultures obtained from P. vulgare pro thalli, after 6 weeks in culture on MS basal
medium supplemented with 2 mg/l 2,4-0
338
1. Messeguer et al.
0.02
2.70
4.23
0.06
0.09
0.03
0.03
Fronds
Sporophyte
in vitro
0.08 0.021
1.15 0.347
1.03
1.81
0.71
0.08
0.07
0.51
0.04
0.65
0.58
0.03
0.05
0.01
0.05
0.14 0.029
0.17
0.14
0.08
0.02
0.06
0.11
Prothalli
in vitro
0.34
1.00
5.98
1.07
0.19
0.29
0.67
0.02
0.05
0.26
0.03
0.01
0.01
0.03
339
most abundant ecdysteroids in the wild plant, the 20E: 5,20E ratio in
the in vitro-cultured sporophyte (1.76) is closely related to that obtained
considering together all parts of the wild sporophyte (1.86). This fact suggests
that the biosynthesis of ecdysteroids in the in vitro-cultured sporophyte
could be comparable to that of the wild plant in terms of balance of
compounds and total activity (4.42 vs. 4.77 mg ecdysteroids/g dry weight,
respectively).
Establised callus lines derived from prothallus cultures did not produce
any phytoecdysteroid. Nevertheless, during the process of callus formation,
prothallus samples were analyzed every 3 days and it was found that, as
pro thalli increase their thickness and begin to produce callus cell clusters,
the phytoecdysteroid content strongly decreased (data not shown). Thus, the
biosynthetic ability for secondary metabolites has been lost during the
dedifferentiation process as reported for many other species (Banthorpe
1994).
As had been previously described by Camps et al. (1990a), the
highest production of ecdysteroids was achieved in the in vitromicropropagated prothalli. The ecdysteroid profile of prothalli could be
considered similar to that obtained from wild fronds: both tissues produce
the highest proportion of 20E in comparison with the contents of the
other ecdysteroids. The relative amounts of E and especially 25d20,26E, in
both in vitro sporophyte and prothallus cultures, are remarkably high when
compared with the corresponding contents in the wild plant (Reixach et al.
1996).
Prothalli derived from spores collected in different European locations
[Ahrensburg (Germany), Espoo (Finland) and Montseny (Spain)] have been
micropropagated in vitro for 6-8 years. The fact that the growth ratio and the
shape of the prothallus cultures had been maintained for more than 60 subcultures indicated that these cultures could be considered morphologically stable
for all three sources tested. The total ecdysteroid production of prothalli
from different origins is summarized in Table 2 (Reixach et al. 1996). The
phytoecdysteroid production also appeared to be stable, since it was maintained at high levels during the years analyzed. The observed differences
Table 2. Total ecdysteroid content in prothalli derived from spores of P. vulgare from different
geographical origin micropropagated in vitro for 4 consecutive years. Data are given in mg
ecdysteroid/g dry wt (mean, n = 6). (Reixach et al. 1996)
1990
1991
1992
1993
Average
Ahrensburg
(Germany)
Espoo
(Finland)
Montseny
(Spain)
Average
5.85
8.01
6.45
6.83
6.84b"
9.76
10.39
8.99
10.58
9.93c
9.14
9.40
9.55
9.20
9.44c
8.25a
9.27a
8.33a
9.11a
1. Messeguer et al.
340
Ahrensburg
6
Montseny
Espoo
10
3 4
II
;;;;;;~ 2
III
20E
25d20,24E ~ 24E
[3 5,20E
20,24E
25d20,26E
Fig. 6. Relative content of ecdysteroids in cultured prothalli derived from spores of P. vulgare
from different geographical origin. (Reixach et al. 1996)
were not significant and were probably due to variations in the subculturing
process, which was established for prothallus production every 6 weeks,
approximately. Nevertheless, the ANOV A analysis of these data detected
significant differences depending on the origin; thus, the phytoecdysteroid
productions of pro thalli derived from Espoo or Montseny spores were
similar, whereas pro thalli derived from spores from Ahrensburg were less
productive.
The separate phytoecdysteroid content for 4 consecutive years in P.
vulgare prothalli derived from spores collected in the above-mentioned
areas was also analyzed. No significant differences for each ecdysteroid production were detected among the different years analyzed (Reixach et al.
1996). In relation to the produced phytoecdysteroids (Fig. 6), although
20E is the major component, ranging from 66-70% for all the sources
studied, the percentage of the other phytoecdysteroids is distinctive for
each origin and can be used as a fingerprint character. Thus, ecdysteroid
production in prothalli generated from Ahrensburg spores shows a higher
proportion of E and 24-hydroxylated derivatives (25d20,24E; 24E; and
20,24E) and lower contents of 5,20E and 25d20,26E. Conversely, prothalli
derived from Montseny spores present an inverted profile related to the
former with higher proportion of 5,20E and 25d20,26E and lower one for
24-hydroxylated derivatives. Finally, ecdysteroid production in pro thalli
generated from Espoo spores exhibits a higher abundance of polar
ecdysteroids (20,24E and 5,20E) as well as E, and very low amounts of all
other compounds.
These differences in the biosynthetical pattern of in vitro prothallus culture from different sources and the fact that pro thalli have a simple morphology with natural laminar growth pattern, prompted us to use these cultures for
biosynthetic studies.
341
The particularly simple morphology of prothalli (see Sect. 2.1, and Fig. 3) and
its natural laminar growth allows easy access to enzymatic systems for the
biosynthetic precursors artificially supplied. Putative precursors could be topically applied with a microsyringe in water or water: acetone solution.
From the biosynthetic point of view, ecdysteroids present in P. vulgare can
be separated in two main groups: 25-hydroxyecdysteroids (E; 20E; 24E;
20,24E, and 5,20E) and 25-deoxyecdysteroids (25d20,24E and 25d20,26E; see
Fig. 2).
In order to study the relationship between 25-hydroxyecdysteroids and its
plausible immediate precursor E, [23,24-'H]-ecdysone was applied to P.
vulgare prothalli derived from spores collected in Montseny and Ahrensburg
(Table 3; Reixach et ai. 1996). After 1 week, prothalli derived from Montseny
spores metabolized a 38% of [23,24- 3H]-ecdysone to ['H]-20E, and a small
amount (3 %) of [3 H ]-5,20E was also formed. The biotransformation of
the precursor increased up to 61 % after 3 weeks and, at that moment,
radiolabeled 20,24E (3%) was also present. The fact that [3H]-24E had not
been detected may suggest that 20,24E is biosynthesized, at least in part, via a
24-hydroxylation of 20E and not by the 20-hydroxylation of 24E. For elucidating the biogenesis of24E, prothalli derived from Ahrensburg spores, which are
a good source of 24-hydroxylated ecdysteroids (see Fig. 6), were treated with
[23,24}H]-ecdysone. As shown in Table 3, after 3 weeks, all detected 25hydroxyecdysteroids, including 24E (2%), showed 3H incorporation. Nevertheless, the relative amount of labeled 20,24E with respect to control (2: 10)
was the lowest when compared to the relative ratios of the other labeled
ecdysteroids (2: 6 as minimum). This fact suggests that ecdysteroids other than
342
J. Messeguer et al.
5,20E
20E
24E
76
38
48
54
8
3
4
4
7
59
48
39
11
2
3
1
69
55
6
2
11
40
24E and 20E may be the precursor(s) for 20,24E. On the other hand, as
expected, the 25-deoxyecdysteroids were not labeled when [23,24)H]ecdysone was used as precursor.
As a means to study the biogenesis of all ecdysteroids present in P.
vulgare, an early biosynthetic radiolabeled precursor as [2- 14 C]mevalonic acid
(applied as a salt) was incorporated to in vitro-cultured prothalli (Reixach et
al. 1996). As shown in Fig. 7A, all identified phytoecdysteroids showed 14C
incorporation after 1 week in culture. Concerning the formation of 25deoxyecdysteroids, C4C]-25d20,26E was formed at the same rate as the
unlabeled one. Conversely, [14C]-25d20,24E was present in a higher amount
than in the control after the 1st week, but its contents decreased after 3 weeks
to become similar to those of the unlabeled analog. On the other hand, a
possible precursor of 25-deoxyecdysteroids (ponasterone A, 25d20E) was
tentatively identified in very low amount (1 %) in these assays. The fact that
this phytoecdysteroid has not been detected before in prothallus cultures
may suggest that it undergoes a rapid conversion. With respect to 25hydroxyecdysteroids, P4C]-E was present in high amounts during all the assay,
and although its relative contents decreased when the culture periods were
prolonged, the value after the 3-week period did not reach that of the control.
This relative decrease was associated with a relative increase of labeled polar
ecdysteroids, predominantly [14C]-20E.
When [4- 14 C]-cholesterol was fed as precursor, incorporation of radioactivity to all reported ecdysteroids was also observed (Fig. 7B; Reixach
et al. 1996). The relative incorporation profiles were similar to those obtained
with mevalonate, but with a lower proportion of E. This fact may indicate
that, although cholesterol is transformed into ecdysteroids, it may follow a
different biosynthetic pathway from that of the C27-sterol produced from
mevalonate, which should be the authentic biosynthetic precursor. In this
respect, Grebenok and Adler (1993) reported that although cholesterol
may be incorporated to ecdysteroids in Spinacia oleracea, lathosterol appeared to be the endogenous biosynthetic precursor of ecdysteroids in this
plant.
343
CONTROL
6
0
0..
20E
II
5,20E
lSI
24E
E:J
20.24E
25d20.24E
25d20.26E
1 week
2 week
3 week
1. Messeguer et a!.
344
100
(:I;l
'i
75
oS.,
=
f
50
:E
~
25
Time (days)
Fig. 8. Time course biotransformation of E in P. vulgare callus culture. (Unpub!')
345
___ 20E
E--...,. 24E
5,20E
~O,24E
",/ 25d20,24E /
25d20E
"'" 25d20,26E
346
1. Messeguer et al.
(50 mg) were extracted with MeOH (4 X 5 ml) and the combined extracts were evaporated. The
residue was dissolved in 2 X 5 ml of H,O and loaded onto a CI8 reversed-phase Sep-Pak cartridge.
After washing with H 20 (lOml) and 15:85 MeOH:H,O (lOml), ecdysteroids were eluted
with 85: 15 MeOH: H 20 (5 ml). An aliquot of this eluate was mixed with 7-ethoxycoumarin
(used as internal standard for quantification) and injected onto the HPLC system (Reixach et al.
1996).
References
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Banthorpe DV (1994) Secondary metabolism in plant tissue cultures: scope and limitations. Nat
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Bergamasco R, Horn DHS (1980) The biological activity of ecdysteroids and ecdysteroid
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Nagakari M, Kushiro T, Yagi T, Tanaka N, Matsumoto T, Kakinuma K, Fujimoto Y (1994b) 3f3Hydroxy-5f3-cholest-7-en-6-one as an intermediate of 20-hydroxyecdysone biosynthesis in a
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1 General Account
1.1 Importance of Rosemary
Rosemary (Rosmarinus officinalis L., family Labiatae), a plant native to Mediterranean countries, is an evergreen shrub (Fig. 1) commonly grown for extraction of its aromatic oils by steam distillation. About 100 metric tons of its oil are
produced each year, mainly in Spain, Italy, France, Yugoslavia, the former
USSR, Tunisia, Morocco, and the Middle East (Browse 1986). Spain is the
largest producer of rosemary. Several species of the genus Rosmarinus with
a number of varieties are commonly known (Guenther 1949). The oil yield
is much higher during summer than in winter. In summer, 12 arrobas
(1 arroba = 11.5 kg or about 25Ib.) of plant materials yield about one kg of oil,
while in winter 20 arrobas are required. The properties of rosemary oil vary
according to several environmental factors associated with the producing region, e.g., soil, climate, and season of harvesting (Guenther 1949). Sovoboda
and Deans (1992) reported that the anti oxidative properties of various samples
collected from different dealers and markets varied according to geographical
location and type of processing. The aromatic oil is used widely in the perfume
industry. Rosemary is also used in food processing as a preservative. Its
essential oils have been shown to inhibit several Gram-positive and -negative
bacteria (Narasimha Rao and Nigam 1970). Generally, Gram-positive bacteria
are more sensitive to rosemary and sage oils than Gram-negative ones. A
relationship between the chemical structures of the most abundant components in the essential oils and the antimicrobial activity was found (Farag et al.
1989). Shelef et al. (1980) reported that the inhibitory effect of rosemary and
sage was attributed to their terpene fraction, which was comprised of borneole,
cineole, pinene, camphene, and camphor for rosemary, and thujone for sage.
The antioxidative activity of rosemary has been shown by other authors.
Nakatani and Inatani (1984) isolated two diterpenes that showed four times
higher antioxidative activity in lard than synthetic antioxidants such as
Horticulture Department, College of Agriculture, Assiut University. Assiut 71526 Egypt
Horticulture Department, University of Nebraska, Lincoln, Nebraska 168583-0724,
USA
3 Department of Food Sciences and Technology, University of Nebraska, Lincoln Nebraska
168583-0919, USA
1
350
Fig.i. Two cultivars of rosemary, Rosmarinus officinalis L., used in this study: left Prostratus; right
Lockwood de Forest
351
The rosemary flavor is due to a mixture of compounds in its essential oil. The
oil contains mainly monoterpenes, some diterpenes, and sesquiterpenes. The
group of structurally related 10 carbon compounds, called monoterpenes, is
usually constituted of lower boiling fractions of essential oils (Croteau 1980).
Another group of volatile compounds called sesquiterpenes, with higher boiling fractions and 15 carbon atoms, contributes to the odor and flavor of
essential oils. Higher terpenes such as diterpenes (C-20) are readily eliminated
by distillation. Sesquiterpenoids are often found as components of volatile oils
extracted by steam distillation. Essential oils of three species of rosemary (R.
officinalis, R. eriocalyx, and R. tomentosus) were analyzed by gas chromatography (GC) and mass spectrometery (MS) (Rosua and Gareia-Grandos 1987).
These authors found 15 components (13 monoterpenes and 2 sesquiterpenes)
and the most abundant components of oil from the three species were apinene, camphor, 1,8-cineole and a-terpineol. Tucker and Maciarello (1986)
reported that 23 cultivars of the rosemary, Rosmarinus officinalis, representing
five botanical varieties, showed a wide range in the leading components:
0.6-57.45% a-pinene, 3.55-42.69% 1,8-cineole, 0.20-56.45% camphor, 0.6621.03% bornyl acetate, and 0.40-14.69% borneol. Ten monoterpenes have
been identified in the oil of both rosemary's genotypes R. officinalis L.
Prostratus and R.o. Lockwood de Forest using GC/MS (Tawfik 1992): apinene, j3-pinene, camphene, p-cymene, 1,8-cineole, limonene, linalool, camphor, borneol, and bornyl acetate (Fig. 3). The chemical structure of these
monoterpenes is shown in Fig. 2.
1.3 Extraction and Separation of Rosemary Oil
The basic method that is commercially used for obtaining the aromatic oils
from rosemary plants is steam distillation. However, many techniques have
been employed for essential oil extraction, such as organic solvent extraction.
The extraction method of choice depends on the nature of the compound of
interest and the amount of plant material used for extraction. The oil is a
colorless or pale yellow liquid having the characteristic odor of rosemary and
a camphoraceous taste.
352
pinene <alpha>
6J
ex 2
Camphor
1,S-clneole
pinene <beta>
Camphene
Borneol
Limonene
OH
Y'
o
I
C}l"OCCH,
~
Bornyl acetate
p-cymene
Llnalool
353
I a-pinene
10
2 Camphene
3 Ll-pinene
4 p-cymene
5 I,S-cineole
6 Limonene
7 Linalool
8 Camphor
9 Borneol
10 Bornyl acetate
\
\
I
354
Rosmarinus officinalis L. Prostratus and R.o. Lockwood de Forest (Fig. 1). All
explants taken from R.o. Lockwood de Forest induced dark green compact
calli when cultured on MS medium (Murashige and Skoog 1962) with the right
growth regulator. For the other genotype, Prostratus, shoot tips were the
better explant and induced excellent callus for plant regeneration on the same
medium.
A protocol to induce organogenic callus and to produce plantlets via
callus is employed (Tawfik 1992). Thidiazuron (TDZ) was better than 2,4dichlorophenoxyacetic acid (2,4-D) to induce organogenic calli. High concentrations of TDZ (up to 2mg/l) either alone or plus 0.5mg/l indole acetic acid
(IAA) induced organogenic callus.
2. Shoot- Tip Proliferation. In both the genotypes, the callus was formed on
the bases of the shoot tips cultured on MS medium supplemented with TDZ
alone or with IAA. The interaction of IAA by TDZ on the proliferated
explant fresh and dry weight was significant. The highest fresh and dry weight
of callus was obtained when 1.5 mg TDZ/I plus 0.5 mg IAA/I were applied to
the medium.
3. Stem Segment Proliferation. The stem segments of Lockwood de Forest
were excellent explants for producing large masses of organogenic callus when
cultured on the designated medium. No callus was formed on TDZ-free medium, and the presence of IAA in the callus induction medium (elM) was
essential to produce the largest mass of dark green organogenic callus. The
TDZ
0.0
0.5
1.5
mg/I
0.0
IAA
0.5
Callus from stem segment
Fig. 4. Callus proliferation from stem segments of Rosmarinus officinalis Lockwood de Forest 5
weeks after culture as affected by TDZ and IAA added to the culture medium
355
highest fresh weight was obtained from stem segments cultured on medium
supplemented with 2mg TDZ/I plus 0.5mg IAA/I (Fig. 4).
4. Leaf Segment Proliferation. The same response of TDZ and IAA, when
applied to MS medium for stem segments cultured in vitro, was observed for
the leaf segments (Fig. 5). The callus was formed on the abaxial side of the leaf
segment. After 4-6 weeks, compact dark green organogenic calli were ready
to transfer to the regeneration medium.
TDZ (mg/l)
,.....
0.5
.,.
0.5
1.5
5. Plant Regeneration. When the compact dark green healthy callus from
both genotypes was transferred to a medium supplemented with different
concentrations of benzyl adenine (BA) (0, 2, 4, 6, and Smg BAil), multiple
shoots were produced (Fig. 6). The best concentrations were 4 and 6mg
BAIL
356
Fig. 6. Shoot regeneration of rosemary Rosmarinus officinalis L. Prostratus from callus 6 weeks
after culture as affected by BA concentrations added to the culture medium
The growth responses to sucrose as a source of carbohydrate differed, depending on the genotype used. Increasing sucrose concentrations in the medium
resulted in an increase in fresh and dry weight of the proliferated explant
(shoot tips) taken from Prostratus (Table 1). However, a high concentration of
sucrose (40mg/l) resulted in decreasing the fresh and dry weight of the two
different explants (leaf segments and shoot tips) taken from R.o.L. Lockwood
de Forest (Table 2). Maretzki et a1. (1974) reported that the effect of carbohyTable 1. Effect of sucrose on the proliferated explants of
Rosmarinus officinalis Prostratus 5 weeks after culture in vitro
Sucrose
conc. (gil)
Fresh weight
(g)
Dry weight
(g)
Bornyl acetate
(%)'
10
20
30
40
0.11
0.30
0.68
1.11
0.030
0.047
0.066
0.084
3.7
4.3
7.5
7.2
Contrast b
L**
L**
L*
357
Sucrose
conc. (gil)
CFW'(g)
CDW (g)
STFW (g)
STDW (g)
j3-Pinene
10
20
30
40
0.99
1.26
1.60
0.90
0.036
0.080
0.092
0.050
0.38
0.49
0.37
0.15
0.035
0.048
0.053
0.026
6.1
7.3
6.0
3.4
6.9
7.6
6.0
4.0
Contrast'
Q*
Q**
Q**
Q**
Q**
L*
Borneol
(%)b
, CFW = callus fresh weight; CDW = callus dry weight; STFW = shoot tip fresh weight, STDW
= shoot tip dry weight.
b % of the total monoterpene in the essential oil extracted from the proliferated explants (shoot
tips). To convert to Ilglg fresh wt., multiply by (5 x 10- 4 ).
, L = linear, Q = quadratic. Contrasts are significant at P < 0.05 (*) or P < 0.01 (**), respectively.
drate on growth differed, depending on the species or clone, but not on the
tissue from which the explant was isolated.
The effect of sucrose concentrations applied to the culture medium on the
levels of the monoterpenes identified in the extract from the proliferated
explant of R.o.L. Prostratus was reported for bornyl acetate (Table 1). Increasing sucrose concentrations led to an increase in the level of bornyl acetate.
Calvo and Sanchez-Grass (1993) studied the accumulation of monoterpenes in
proliferated shoots of Lavandula latifolia Med, and found that increasing the
medium osmolarity by addition of mannitol to the culture medium resulted in
a significant increase in monoterpene accumulation in the regenerated shoots.
In R.o.L. Lockwood de Forest, the level of j3-pinene decreased when high
concentrations of sucrose were used (Table 2). Sucrose had a significant quadratic effect (P < 0.01) on the j3-pinene level. Based on the quadratic regression
line, the highest level of this monoterpene was estimated to be obtained when
approximately 199 sucrose!l was added to the culture medium (Neter and
Wasserman 1974). Norton et al. (1991) reported that rubber synthesis in
guayule (Parthenium argentarum), was stimulated by increasing the sucrose
concentration up to 7% in the medium, but higher concentrations were inhibitory. On the other hand, increasing sucrose concentration in the medium up to
40 g sucrose/l inhibited the borneol level in the proliferated explant of R.o.
Lockwood de Forest cultured in vitro in our studies (Table 2).
3.2 Effect of Calcium Ion
To study the effect of Ca2+ ion on callus induction and monoterpene levels of
Lockwood de Forest, leaf segments were used as explants and cultured on
callus induction medium (CIM) supplemented with six concentrations of Ca2+
ion (0.99, 1.99, 2.99, 3.99, 4.99, 5.99mMol!I). It was obvious that Ca 2+ ion
supplementation affected the texture of rosemary callus. The lowest concentration (0.99mMol Ca 2+/l) produced dark green and compact callus. The highest concentration (5.99mMol Ca2 + II) produced light green friable callus. The
358
3
--~
J:
Cl
';
2.9
2.8
2.7
III
::l
2.6
2.5 0.99
1.99
2.99
3.99
4.99
5.99
ct + cone. mMol/L
0.17 r - - - - - - - - - - - - - - - - - - - - - ,
................................................... .
1:
.~ 0.155
~
~ 0.15 .
III
::l
'iii
()
0.145
0.14
0.135
0.99
1.99
2.99
3.99
4.99
5.99
2+
Ca cone. mMol/L
Fig. 7A,B. Effect of Ca2 + concentration on the fresh weight (A) and the dry weight (B) of
Rosmarinus officinalis Lockwood de Forest callus proliferated in vitro
effect of calcium ion on fresh and dry weight is shown in Fig. 7A, B. The higher
concentration produced high fresh weight but low dry weight because of the
loss of moisture. However, no significant effect was found on both fresh and
dry weight. Murashige and Skoog (1962) observed that Ca2+ ion concentration
in the range of 1.5 to 6mMolli did not affect the fresh weight of tobacco pith
explants. Likewise, Gamborg et al. (1968) reported that Ca2 + ion concentrations from 1 to 4 mMolll did not affect the dry weight of soybean suspension
cultures. However, Mizukami et al. (1977) reported that increasing Ca2 + ion
concentration above 3 mMolll decreased growth in Lithospermum callus
cultures.
The calcium ion significantly affected the oil yield (P = 0.01) extracted
from the proliferated callus of rosemary. The oil yield curve, expressed
as a response to calcium ion concentrations, was cubic (Fig. 8). The highest yield of oil measured per g fresh weight was obtained from calli grown
359
0.15
-~
0.14
Y = 0.34 - 0.27 X
0.13
R = 0.75
+ 0.086 X - 0.008 X
0.12
LL 0.11
1:1
0.1
#.
0.09
0.08
0.07
0.06
0.05
0.04
0.03
0.99
1.99
2.99
3.99
4.99
5.99
2+
Ca cone. mMoI/L
Fig. 8. Effect of calcium ion on oil yield extracted from proliferated callus of Rosmarinus
officinalis Lockwood de Forest
Table 3. Effect of calcium ion on oil costituents (%) extracted from Rosmarinus officinalis
Lockwood de Forest proliferated callus 6 weeks after culture in vitro
Ca2 + (mMol/l)
1,S-Cineole
Linalool
Bornyl acetate
0.99
1.99
2.99
3.99
4.99
5.99
6.S
3.4
3.1
3.0
5.5
5.2
20.9
24.5
24.0
27.0
21.5
lS.2
2.22
2.32
1.26
2.23
2.25
2.74
O.SO
0.75
1.45
O.SO
1.20
0.44
Contrast b
C**
Q**
Q*
C*
, % of the total mono terpene in the essential oil extracted from the proliferated callus. To convert
to flg/g fresh wt., multiply by (5 X 10- 4).
b Q = quadratic; C = cubic. Contrasts are significant at P < 0.05 (*) or P < 0.01 (**), respectively.
360
Callus induction and plant regeneration of rosemary from all types of explants
using auxin and cytokinin have been described. For inducing organogenic
callus, a combination ofthidiazuron (TDZ) and 3-indoleacetic acid (IAA) was
applied to the culture medium. For plant regeneration, callus was transferred
to a medium supplemented with benzyladenine (BA).
61
'b~><
9
8
7
8
(0)
(A)
A'"
~~
0.5
",
-0.0
TOZ mg/l
~0.5
1.5
2.0
"'.
mg/I
2.0
~0.5 m<>,
- _.'"
IAA---O.0
mg,'
1.0
TDZ
10
.
IAA
'.~~~
.--~
- --- ... - - - --~
-
23
::0-
51
0.0
~4
Q.
.c:
g 6'
III
~ I
-
lOj (0)
,i
or
54
0.5
.
2;0
~O
0.5 mg/I
1.5
IAA~O.O ~
1.0
TDZ mg/l
- '-
~- - -/-- /
1.0' "
1'5
TDZ m g / I '
"
,,,
'
..- -- - - - . -'- - - - - . -
05
.
A
.'
22
c: 21
.- 20
24
~ 25
~~
~~j
30j (8)
Fig.9A-G. Effect of TDZ and IAA on the mono terpene level (%) in rosemary shoot tips cultured in vitro. A
Camphor. B I.S-Cineole. C Borneol. D Camphene. E Bornyl acetate. F Limonene. G Linalool
~l~-------,
i
()
"5. 10
.12
5 11
-13
14
16
15
(.;.l
2i
:3-'"
(1)
C/O
~
o
(::)
<"l
'"
~
s
5"
;:
(::)
:;.;,
o
362
7
!
QI
(E)
,,
,,
,,
>. 3
E
dl
"
1M
~ 0.0 ~O.5
mg/l
1.
1.0
2.0
TDZ mg/I
QI
c
QI
c
:::J
1.04 (F)
1.00 ' ,
,
0.96
,
,
0.92
0.88
0.84
0.80
0.76
0.72
0.68
0.64
0.60
0.56
0.0
1M n-+o().O
0.5
~ 0.5
.. "
,
,
,,
1.0
mg/I
"~
....
,
,,
,,
1.5
2.0
TDZ mg/I
4
(0)
'\
1.~~~~~~~~~~~~~~~
0.0
0.5
1.0
TDZ mg/l
Fig. 9E-G. Continued
1.5
2.0
363
Table 4. Effect of BA on the oil constituents of Rosmarinus officinalis Prostratus plants regenerated from callus
BA (mg!l)
Oil constituents (% Y
a-Pinene
Camphene
{:i-Pinene
1,8-Cineole
Camphor
Bornyl acetate
0
2
4
6
8
8.0
8.2
8.1
7.0
7.1
4.4
4.5
4.2
3.6
3.4
4.5
4.7
5.1
4.8
4.2
23.5
24.1
23.2
26.0
26.1
18.0
16.9
16.0
16.3
18.7
6.7
7.3
8.1
7.7
6.4
Contrast"
L*
L*
Q*
L*
Q**
Q**
, % of the total mono terpene in the essential oil extracted from the regenerant plants. To convert
to JIg!g fresh wt., multiply by (5 X 10 4).
b L = linear, Q = quadratic. Contrasts are significant at P < 0.05 (*) or P < 0.01 (**), respectively.
It is clear that the production of monoterpene constituents from proliferated explants cultured in vitro and from regenerated plants was influenced by
the components of the culture media, such as plant growth regulators. These
results partially agree with the results reported for Mentha spicata (Hirata et
al. 1990) and Zingiber officinale (Sakamura et al. 1986). The effects of plant
growth regulators on the yield of some secondary products in plant cell cultures have been investigated by several groups (see Bajaj 1996). The effects
varied greatly, depending on the kinds of metabolites being produced and on
the type of auxin and cytokinin added to the cultured medium. Based on the
study conducted by Tawfik (1992), increasing the cytokinin (TDZ) in the
culture medium increased the camphor level and decreased the bornyl acetate
in the proliferated explant. Also, increasing BA in the regeneration medium
increased the camphor level and decreased the bornyllevel in the regenerant
plants. The changes in the proportion of the monoterpenes caused by TDZ
and BA suggested that the cytokinin may have an effect on the biosynthesis of
camphor. Since camphor can be derived from borneol (Croteau and Karp
1976), the cytokinin may affect the enzymes responsible for the conversion of
borneol or borneol acetate to camphor. Tawfik et a1. (1992a) reported that
increasing BA in the shoot-tip culture medium of Salvia officinalis L. increased camphor and decreased borneol. On the other hand, Drawert (1988)
reported the biotransformation of terpenes such as borneol to camphor and
citronellal to citonellol in cell suspension culture of Salvia officinalis and
Melissa sp., respectively. The rate and conversion time of one terpene to
another depended on the kind of monoterpene.
4 Summary
1. A protocol was established to induce organogenic callus of two genotypes
of rosemary (Rosmarinus officinalis) using thidiazuron (TDZ) alone or
with indoleacetic acid (IAA) at 0.5 mg/l.
364
2. The use of IAA plus TDZ was essential to produce large masses of
organogenic callus from all types of explant used in the study.
3. The effect of TDZ varied depending upon the genotype.
4. Explants taken from the CV. Lockwood de Forest induced excellent callus,
but for Prostratus, shoot tips were the best explant to induce callus for plant
regeneration.
5. For shoot regeneration from callus, benzyladenine (BA) at 4mg/1 was the
best cytokinin treatment.
6. A significant effect of the plant growth regulators used in this study on the
monoterpenes identified in rosemary was observed.
7. Sucrose concentrations not only affected the growth of rosemary
cultures in vitro but also affected some oil constituents in both
genotypes.
8. Calcium chloride had a noticeable effect on the texture of the callus. Low
concentration of Ca2 + (O.99mMol) produced dark green and compact callus, while a higher concentration (S.99mMol/l) produced light green friable
callus.
9. Ca2 + ion significantly affected the oil yield and four of the monoterpenes
identified in the callus extract.
References
AQel MB (1991) Relaxant effect of the volatile oil of Rosmarinus officinalis on tracheal smooth
muscle. J Ethnopharmacol 33:57-62
Bajaj YPS (1996) Biotechnology in agriculture and forestry, vol 37. Medicinal and aromatic plants
IX. Springer, Berlin Heidelberg New York
Banthorpe DV, Bilyard HO, Brown GD (1989) Enol esters of caffeic acid in serveral genera of
Labiatae. Phytochemistry 28:2109-2113
Browse PM (1986) Rosemaries. Pac-hortic. San Francisco: Pacific Horticultural Foundation (Fall
1986) 47:40-46
Calvo MC, Sanchez-Grass MC (1993) Accumulation of monoterpenes in shoot-proliferation
cultures of Lavandula latifolia Med. Plant Sci 91(2):207-212
Croteau R (1980) The biosynthesis of terpene compounds. In: Croteau R, Pattensen W
(eds) Fragrance and flavor substances. Springer Berlin Heidelberg New York, pp 13-36
Croteau R, Karp F (1976) Enzymatic synthesis of camphor from neryl pyrophosphate by
soluble preparation from sage (Salvia officinalis). Biochem Biophys Res Commun 72:440447
Cuppett SL, Birt DF, Lawson T, Wheeler DDS, Hall CA (1992) Antioxidant, antimutagenic
and anti-cancer (promoting) activities of rosmariquinone, a compound found in rosemary
(Rosmarinus officinalis). (pers comm.)
Cuppett SL, Hall C, Conway H, Birt DF, Lawson T (1995) Inhibition of Mutagenicity of
Alkylating Agents by Rosmariquinone, a compound of Rosmarinus officina lis L. Cancer Prevo
Inter. 2:25-31
Drawert F (1988) Bioflavor - what does it mean? In: Schreier P (ed) Bioflafour'87 analysis.
Biochemistry Biotechnology. Walter de Gruyter Berlin, pp 3-32
Farag RS, Daw ZY, Hewedi FM, El-Baroty GSA (1989) Antimicrobial activity of some Egyptian
spice essential oils. J Food Protect 52:665-667
Gamborg OL, Miller RA, Ojima K (1968) Nutrient requirements of suspension cultures of
soybean root cells. Exp Cell Res 50:151-158
365
Guenther E (1949) The essential oils, vol III. Individual essential oils of the plant families
Rutaceae and Labiatae. Van Nostrand, New York, pp 695-710
Hayashi T, Arisawa M, Bandome T, Namose Y, Shimizu M, Suzuki S, Yoshizaki M, Berganze LH,
Ferro E, Basualdo I (1987) Studies on medicinal plants in Parganze. Studies on Romero. Plant
Med J Med Plant Res 53(4):394
Hirata T, Murakami S, Ogihara K, Suga T (1990) Volatile monoterpenoid constituents of
plantIets of Mentha spicata produced by shoot tip culture. Phytochemistry 29(2):493495
Hoefler C, Fleurentin J, Mortier F, Pelt JM, Guillema J (1987) Comparative choleretic and
hepatoprotective properties of young sprouts and total plant extracts of Rosmarinus officinalis
in rats. J Ethnopharmacol 19(2):133-143; Hort Abst 1988 vol 58 Abst 3107
Maretzki A, Thorn M, Nickell LG (1974) Utilization and metabolism of carbohydrates in cell and
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pp 329-361
Mizukami H, Konoshima M, Tabata M (1977) Effect of nutritional factors on shikonin derivative
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Nakatani N, Inatani R (1984) Two antioxidative diterpenes from rosemary (Rosmarinus officinalis
L.) and a revised structure for rosmanol. Agric Bioi Chern 48(8):2081-2085
Narasimha Rao GV, Nigam SS (1970) The in vitro antimicrobial efficiency of some essential oils.
Flavour Ind 1:725-729
Neter J, Wasserman W (1974) Applied linear statistical models. Richard D. Irwin, Homewood,
Illinois 292pp
Norton RA, Radin DN, Rodriguez (1991) Environmental and chemical effects on growth, resin
and rubber production in guayule tissue cultures. Phytochemistry 30(8):2615-2618
Rosua JL, Garcia-Granados A (1987) Analysis of essential oils species of the genus Rosmarinus
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Abst 1988 vol 58 Abst 1074
Sakamura F, Ogihara K, Sugs T, Taniguchi K, Tanaka T (1986) Volatile constituents of Zingiber
officinale rhizomes produced by in vitro shoot tip culture. Phytochemistry 25(6):1333-1335
Shelef LA, Naglik OA. Bogen DW (1980) Sensitivity of some common food-borne bacteria to the
spices sage, rosemary, and allspice. J Food Sci 45:1042-1044
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Tawfik AA (1992) Factors affecting proliferation, essential oil yield, and monoterpenoid constituents of rosemary Rosmarinus officinalis and sage Salvia officinalis cultured in vitro. PhD
Thesis, University of Nebraska, Lincoln
Tawfik AA, Read PE, Cuppett SE (1992a) Stimulation of growth and mono terpene production of
Salvia officinalis by benzyl adenine in vitro. Plant Growth Regul Soc Am 20(4):200-206
Tawfik AA, Read PE, Cuppett SE (1992b) Effect of some nutritional factors on mono terpene
synthesis in Rosmarinlls officinalis cultured in vitro. Acta Hortic 319:189-194
Tucker AO, Maciarello MJ (1986) The essential oils of some rosemary cultivars. Flavour Fragrance J 4/5:137-142
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1 Introduction
1.1 Distribution and Importance of Sesame
367
Fig. 1. Sesamum indicum plants at flowering stage in a pot on the roof of the OP corporation
building, Tokyo
such as asthma, in Ayurveda since ancient times. It is well known that sesame
has nutritive, laxative, demulcent, emollient, diuretic, and lactagogue properties (Tyler et al. 1988). The oil appears in the pharmacopoeia of many countries. It may also be employed in the preparation of liniments, plasters,
ointments, and soaps (Weiss 1971). Little work has been carried out on the
pharmacological activities of sesame other than lignan derivatives. Gilani and
Aftab (1992) reported the existence of acetylcholine-like substances in
alcholic extracts of seeds of S. indicum. This finding may explain its use in folk
medicine.
1.2 Secondary Metabolites in Sesame
368
T. Ogasawara et al.
et al. 1992), and flavonoids (Morita 1960; Krishnaswamy et al. 1970; Jain 1981)
have been isolated from the seed oil or seeds of S. indicum.
The presence of saponins has also been reported (Fenwick and Oakenfull
1983). Triterpene acids have been isolated from the aerial parts of S.
laciniatum (Krishnaswamy et al. 1991).
Various lignans in sesame have been isolated. They show biological qualities such as antitumor, antimitotic, and antiviral activities as well as the inhibition of some enzymes. The lignan skeleton is classified into four types; i.e.,
butane derivatives, butanolide derivatives, tetrahydrofuran derivatives, and
3,7-dioxabicyclo(3,3,0)-octane derivatives. Moreover, there are two types of
cyclolignans, tetrahydronaphthalene and naphthalene derivatives (MacRae
and Towers 1984). Lignans isolated from sesame have the structure of 3,7dioxabicyclo(3,3,0)-octane derivatives. Sesamin 6 and sesamolin 7 were isolated from S. indicum (for a review see Budowski and Markley 1951). The
content of sesamin 6 was significantly different in white and black seed strains,
but there was less variation in the content of sesamolin 7 in both kinds of seeds
(Tashiro et al. 1990). The four stereochemical structures of sesaminol 8 have
been established (Nagata et al. 1987). The isomers were artificially produced
after the intermolecular transformation of sesamolin 7 by the presence of acid
clay during the decolorization process used to commercially produce sesame
oil. Sesangolin 9 was isolated from S. angolense (Jones et al. 1962).
The sesamin-type lignans are known as active insecticidal synergists for
pyrethrins and rotenons (Budowski 1964), and their many physiological functions have been investigated recently (Sugano and Akimoto 1993). Sesamin 6,
sesamolin 7, and sesaminol 8 inhibited A5-desaturase at the metabolism of
linoleic acid in fungi and animals (Shimizu et al. 1991). Moreover, sesamin 6
inhibited absorption and synthesis of cholesterol in rats (Hirose et al. 1991). In
addition, it prevented breast cancer induced by a chemical carcinogen in the
rat. Antioxidative activity was also observed in vivo and it seems to participate
{?
1,&
H3C
2 sesamol
1 tocopherol
OH
H3CoAOCH3
COOH
3 syringic acid
OH
OH
r'r
0CH3
CH=CHCOOH
4 ferulic acid
sesamol dimer
Fig. 2. Continued
6 sesamin
10 Pl
sesamolin
11 sesamolinol
0
-1
'0'2:
HO
sesaminol
OH
OCH3
0-tp
12 pinoresinol
OCH3
OH
OCH3
<.>.l
0\
'D
po
c;:;
::i
.,~
::i
.,[;;
.,::i
v,
t;
lid
CH.OH 0
Fig. 2. Continued
(1 ~2)- f3 -D-glucopyranoside
H3 CO
HO
e-q
I
-? . . . . . ~OCH3
( 1~6)- f3 -D-glucopyranoside
H3 CO
t;
ellj
~I
-? . . . . . ~OCH3
- f3 -D-glucopyranoside
H3 CO
po
po
e;
O<l
:-l
-.J
'"o
Fig. 2. Continued
16 sesaminoI2'-0-/3-0-glucopyranoside
17
sesaminoI2'-0-/3-0-glucopyranosyl
( 1->2 )-0- /3 -O-glucopyranoside
ff
F~~
(1->2)-0-[ /3 -O-glucopyranosyl
( 1->6)]- /3 -O-glucopyranoside
18 sesaminoI2'-0-/3-0-glucopyranosyl
tj
ectJ
-c-
372
oH
CH20H
o -CH2-CH
0
o~
HO-Q-CH=CH-BOO
T. Ogasawara et a"1
0 OH
OH
CH3
19 34-d"h
OH OH
,
I ydrox - {3
( 1-3 )-4- 0-caffeoyl
y -phenethyl0- ex -L-rhamn opyranosyl
_{3 -glucopyranoside
OH
CH20H CH20HO
OO-CHCO
HO
O~
HOOCH=CH-BOO
0 OH
OH
CH3
OH OH
20 acteoside derivative
~00-CH2-CHCO F\
0~
CH.OH
O~
HO
CH=CH-BOO
o
0 OH
CH3
OH
OH
~OH
OH OH
21 acteoside derivative
HO
HO
000C-CH2-CH"'0 F\
~OH
CH.OH
~H
CH=CH-BOO
OH
0 OH
CH3
OH OH
"g. 2.
ontmued
y,,",,;d.
oopynmo,yl
W
'A
373
HO
OH
23 2-isopropenylnaphthazarin-2.3-epoxide
o
24 2{ 4-methyl-l.3-pentadienyl)anthraquinone
25 2 (4-methyl-3-pentenyl)anthraquinone
2 In Vitro Approaches
Callus cultures and cell suspension cultures from leaf, hypocotyl, cotyledon,
and seed have been established. There are few reports about the genetic
374
T. Ogasawara et a1.
Culture type
Result
Reference
Hairy root
Callus/
suspension/
regeneration
Anther
Protoplast
375
Media (mg/l)
Growth response
Reference
Anther
MS+KN (0.5-1)
Haploid
Shoot tip
MS+KN(2)
MS+ NAA(O.S)
MS+NAA(0.5) + KN(l)
MS+ NAA(O.S)+ BA(l)
MS+NAA(O.I)+ IAA(O.S)+
KN(2)
MS+NAA+KN-->MS+
IAA+KN-->II2MS+CH
MS+CY=(BA or ZEA or
2-iP or KN) (1)
MS+AU= (lAA or IBA or
NAA or NOA) (1)
MS+2,4-D(l)
MS+NAA(O.2)+CY=(BA
or ZEA or 2-iP or KN) (I)
MS+2,4-D(2)+CM(IS% )-->
MS+NAA(O.1)+BA(I)
MS+ BA(OJ,2)
MS+BA(4,8)
Shoot
Whole
Whole
Whole
Whole
Leaf segment
Hypocotyl and
cotyledon
Shoot tip
Shoot tip from
pretreated
seeds
MS+BA (O,U)
MS+BA(4,8)
MS+KN (8)
MS+ZEA(8)
MS+Z-iP(8)
Shoot isolated
from the explant
Cotyledon and
hypocotyl and root
Anther
MS+NAA(I)+AC(O.1 %)
plant
plant
plant
plant
Callus -->adventitious
shoot -->rooting
No callus formation
Rooting
Necrosed or callus
Callus
Excellent callus-->
embryo-like structure
Single plantlet
Multiple shoot buds
(5-6)
Single plantlet
Multiple shoot buds
(8-1S)
Single plantlet
Multiple shoot buds
(10-12)
Multiple shoot buds
(8-10)
Rooting-->plant
Callus
Callus
Protoplas-->callus
Protoplast -->callus
Multiple shoot buds-->
rooting-->plant
Adventitous shoot
Adventitous shoot
No adventitous shoot
No adventitous shoot
Callus-->adventitous
shoot
Rooting
No callus formation
Anther
MS+2,4-D(I--4) or
NAA(I--4)+ KN(l--4)
MS+2,4-D(Z)+ NAA(1)+
KN(Z)
MS+ NAA(O.3)+ BA(3)
MS + NAA(1)+-BA(l)
MS+ BA(8)-->MS+ BA(I)-->
1/2MS+NA(O.I)
MS+IAA(I)+BA(lO)
MS+NAA(l)+BA(I)
MS+IAA+BA
MS+ IAA+ BA+ABA(O.I)
MS+ lAA( 1)+ BA(l,lO)+
ABA(I)
MS+ IAA(1-3)
MS+ 2,4-0(20)
Cal1us
Anther
Hypocotyl and
cotyledon
Hypocotyl
MS+2,4-D(10)+ IAA(2)+
BA(2)
MS+2.4-D(1O)+ BA(l)
MS+2,4-D(15)+ BA(I)
MS+ IAA(S)+ BA(3)
MS+2,4-D(O.5-2)
MS+NAA(1-2)+
BA(0.2-0.6)
MS+2,4-D(I)
Hypocotyl
Cal1us
Shoot tip
Hypocotyl
Cotyledon
Callus
Callus
Callus
Callus
Callus
Callus-->embryolike structure
Masuda (1989)
Ranaweera and
Pathirana (1992)
T. Ogasawara et al.
376
Table 2. Continued
Inoculum
Media (mg/l)
Growth response
Hypocotyl callus
MS + CH(lOO0-2000) +
NAA(O.l)+BA(l-4)
1/2MS+ NAA(O.S)
MS+2,4-D(O.018)+ KN(2.3)
Callus-->adventitous
shoot
Root formation
Callus
Regenerated shoot
Seedling
Reference
Takebayashi et al.
(1994)
Abbreviations:
Basal media: MS = Murashige and Skoog (1962).
Supplements: AU = auxin, 2,4-D = 2,4-dichlorophenoxyacetic acid, IAA = indole-3-acetic acid, IBA = 3indolebutyric acid, NAA = napthalene acetic acid, NOA = naphthoxy acetic acid, CY = cytokinin, ZEA =
zeatin, BA = 6-benzylaminopurine, KN = kinetin,2iP = isopentenylaminopurine, ABA = abscisic acid, CM =
coconut milk, AC = activated charcoal, CH = casein hydrolysate.
Datta and Biswas (1986) induced calli from cotyledons of 3-4-day seedlings by
combining NAA and kinetin. When they were transferred to the medium
containing IAA 2mg/1 and kin O.5mg/l, the highest frequency of adventitious
shoot formation was 22.5%. On transferring the shoots to medium containing
casein hydrolysate, 200mg/1 roots were formed. The plantlet, a shoot with
roots, shown in their report did not resemble a complete plant.
George et al. (1987) showed callus induction from hypocotyl and
cotyledon, and multiple shoot formation from shoot tips. Hypoctyls were
more responsive than cotyledons. Root formations were observed from
explants in various auxins 1 mg/l, but the explants of cotyledon were necrosed
in 2,4-D 1 mg/l. Calli were induced in media including NAA 0.02mg/1 and
several kinds of cytokinin 1 mg/l. After seeds were soaked in MS (Murashige
and Skoog 1962) containing BA 8mg/1 for 72h, they were germinated on MS
and MS containing BA 1, 2, 4, or 8mg/l. Shoot tips which were excised from
seedlings with each treatment were then cultured. Effective regeneration and
the formation of 12-15 shoot buds per explant were observed on MS containing BA 8 mg/l, as shown in Table 2. The frequency of bud formation was 6570%. The shoots, when transferred to MS containing NAA 1 mg/l and
activated charcoal 0.1 %, resulted in rooting in 50% of the cultures. This
method is useful for the propagation of sesame. It might be necessary to select
the genotype, because the response of tissue to phytohormones differs
from that of the genotype. Calli were induced from hypocotyl with extensive
growth in the MS media containing 2,4-D 2mg/1 and coconut milk 15% (v/v).
When the calli were transferred to medium supplemented with NAA
0.1 mg/l and BA 1 mg/l, they grew vigorously and produced embryo-like structures. In contrast, plant regeneration from callus was not successful. Multiple
shoot buds were observed in shoot-tip cultures of seven cultivars. Shoot tips
excised from seeds pretreated with cytokinin showed increased frequency in
induction of multiple shoot buds. Rooted plantlets from isolated shoot buds
377
were established in soil, and the plants matured and flowered (George et al.
1989).
Lee et al. (1985) induced calli and organogenesis from shoot tips with a
single treatment of NAA and IAA. NAA was better for shoot differentiation
than IAA, but IAA was better for root differentiation. Kinetin 2mg/l was
found to be best in shoot differentiation. Whole-plant induction percentages
were 86 and 29% in combinations of NAA 0.5 mg/l + kin 1 mg/l and NAA
0.5 mg/l + BA lOmg/l, respectively. Increase in NAA reduced shoot differentiation but did not significantly influence root differentiation. The most desirable medium was MS containing N AA 0.1 mg/l + IAA 0.5 mg/l + kin 2 mg/l, in
which whole-plant induction was 93%.
Lee et al. (1988a) induced calli from cotyledon, hypocotyl, and root sections, hypocotyl showing the best reaction in callus induction. The range of 2,4D or NAA 1-4mg/l with kin 1-4mg/l was effective for callus formation and
NAA was more effective than 2,4-D. Cytokinins at high concentrations inhibited root development but promoted green part formation. Zeatin was the
most effective among the cytokinins tested, but shoots were not formed from
calli on any regeneration media.
Masuda (1989) also reported callus induction and adventitious shoot formation. Adventitious shoot formation from hypocotyls was very frequent in
two cases of BA 10mg/l + IAA 1 mg/l and BA 1 mg/l + IAA 1 mg/l. Further,
shoot formation from cotyledons was very frequent in IAA 1 mg/l + BA 1 or
10mg/l + ABA 1 mg/l. Rooting from cotyledons was induced in MS containing
only IAA 0.1-3mg/l.
Kwon et al. (1993) indicated that a combination of NAA 1-2mg/l and
BAP 0.2-0.6mg/l was efficient for formation of calli. Embryo-like structures
were formed in hypocotyl-derived calli on medium containing only 2,4-D 1 mgl
1. The addition of casein hydrolysate (1000-2000mg/l) to the regeneration
media containing NAA 0.1 mg/l and BAP 1-4 mg/l effectively increased the
rate of adventitious shoot formation from hypocotyl-derived calli. High concentrations of BAP 3-4mg/l in combination with casein hydrolysate increased
the formation of multiple shoots, while low concentrations of BAP 1-2 mg/l
induced the formation of a single shoot. Even in cases of low BAP, however,
the addition of a high concentration of casein hydrolysate tended to increase
multiple shoot formation. Regenerated shoots formed roots on half-strength
MS media containing NAA 0.5mg/1.
Govil and Singh (1982) reported induction of haploids in anther culture.
The anthers responded well on MS media containing phytohormones and 3 %
sucrose when they were cultured in the early stage of flowering in S. indicum
L. var. T -4. The pre chilling treatment did not affect the production of haploids,
while high kin 0.5-1 mg/l did. Production of haploids was enhanced by the
addition of activated charcoal.
Lee et a1. (1988b) used four cultivars of S. indicum L. to induce calli from
anthers. The most effective concentrations of growth regulators were 2,4-D
2mg/l, NAA Img/l, and kin 2mg/l, with callus induction frequency 55%. The
highest rate of callus induction was 80.2 % in cold pretreatment at 10 C for 6
days. Callus was induced mainly from tapetum tissue, anther wall, and the cut
378
T. Ogasawara et al.
end of the filament. Root regeneration was induced easily, but shoot formation
was not observed.
Ranaweera and Pathirana (1992) induced callus cultures from anthers of
S. indicum L. cultivar MI 3. The anthers from flower buds 36-48h before
anthesis were used to induce calli. Pre chilling treatment at 8C for 24 h affected callus production. The highest rate of callus induction (46 % ) was shown
in MSS media containing 2,4-D 10mg/l, IAA 2mg/l, and BA 2mg/1 within 2-3
weeks of plating.
Ryu et al. (1992) induced calli from anthers of S. indicum L. cultivar
Danbaek. Anthers (3-4mm), after cold pretreatment at 4C for 48h, were
cultured on MS medium containing 2,4-D, which was the most effective auxin
for callus induction. After 20 days, root and green tip were formed in calli
cultured on a revised MS medium supplemented with inositol.
Shoji et al. (1988) isolated protoplasts from hypocotyl tissues treated with
enzyme solution containing 2% Cellulase Onozuka R-10, 0.4% Macerozyme
R-10, 0.7M Mannitol and 0.5% potassium dextran sulfate in CPW inorganic
media. Ninety-five % of isolated protoplasts demonstrated esterase activity as
evidence of their viability. The protoplasts regenerated cell wall within 72h
and formed small clusters in MS containing casein hydrolysate 2 gil, sucrose
20g/l, mannitoI109g/l, NAA 0.3mg/l, and BA 3mg/l. Only about 1% of the
protoplasts developed to small cell colonies. Fusion of relative DNA contents
was measured to investigate whether nuclear fusion occurred by fusion of
protoplasts. The fluorescent dye DAPI( 4' ,6-diamidino-2-phenylindole) bound
specifically with DNA was used for the determination of relative DNA content. Approximately 7.4% of calli indicated tetraploid nuclear DNA content
compared with the haploid after S weeks of culture (Shoji 1989).
Bapat et al. (1989) isolated calli and cell suspensions using an enzyme
mixture consisting of 3% Cellulase, 1.S% Macerozyme, and 1.S% Hemicellulase. They were cultured on a modified MS medium supplemented with
0.6 M sorbitol, NAA 1 mg/l, BA 1 mg/l. Regeneration of cell walls occurred
72 h after culture, and multicellular colonies were obtained by subsequent
divisions.
2.1.2 Secondary Metabolites in Callus and Suspension Cultures
Khanna and Jain (1973b) established callus cultures from seedlings and also
showed production of amino acids (Khanna and Jain 1973a), sterol (Jain and
Khanna 1973), and sesamin (Khanna and Jain 1973b). Calli were induced on
revised MS media containing 2,4-D 1 mg/l and maintained for a period of
36 months by subculturing at intervals of 6-7 weeks. Thus, the biosynthesis
of calli may be possible even after cultivation for long periods. Jain
(1981) reported production of pedaliin (6-hydroxy-Iuteiolin-7-methylether-6glucoside) from callus cultures isolated from the leaves of S. indicum. The
callus cultures contained O.S% pedaliin.
Mimura et al. (1987a,b,c,d) isolated sesamin and sesamolin from calli.
They studied callus formation by using various phytohormones in MS media.
379
Table 3. Induction of callus from sections of Sesamum indicum seedling. (Mimura and Osawa
1989)
Cytokinin
BA
1 x 1O- 5 M
BA
1
1O- 5 M
Auxin
Callus induction
Cytokinin
NAA
50 x 10 5 M
5
1
0.2
0.04
0.008
0
2,4-D
5 X 1O- 5 M
1
0.2
0.04
0.008
0
KN
I X 1O- 5 M
+++
+++
++
+
KN
1
1O- 5 M
+
+
++
+++
Auxin
Callus induction
NAA
50 x 1O- 5 M
5
1
0.2
0.04
0.008
0
+++
+++
++
+
2,4-D
5 X lO-'M
+
++
++
+++
I
0.2
0.04
0.008
0
Tocopherol
Sesamol
Sesaminol Sesamolinol
460
950
nd
nd
nd
63
2125
330
273
1522
260
nd
nd
nd
nd
nd
4-11
3-5
0.002
0.2
Sesamolin
nd
nd
205
80
0.2
0.3-0.5
The calli were cultured on solid media in the dark for 21 days. The results are
shown in Table 3. Mimura and colleagues used calli induced by MS media with
NAA 9.3mg/1 (5 x lO- S M) and BA 2.3mgll (1 x 10- S M). The production of
lignans from various tissues is shown in Table 4 (Mimura 1991).
Acteoside 19 (Mimura et al. 1990a) was produced by callus cultured for 10
days at 35-36 C under illumination. From a 500-g callus, crude 19 (11.4 g) was
obtained by ethanol extraction and crude 19 (5 g) was subjected to chromatography on Amberlite XAD-2 resin to yield purified 19 (21 mg). New polyphenol
glucosides 20, 21, and 22 (Mimura et al. 1990b, 1991) were isolated in suspension cultures. From a 500-g callus, 6,13, and 18mg of20, 21, and 22 compounds,
respectively, were prepared by ethanol extraction, Amberlite XAD-2 chromatography, and HPLC. This glucoside compound 22 might be used in cosmetics
to prevent skin damage by photooxidation as an antioxidant (Sakamoto et al.
1991).
380
T. Ogasawara et al.
Triterpene acids, esculentic acid, and 3f3-(trans-p-coumaroyloxy)-2a,23dihydroxyurs-12-en-28-oic acid, have been isolated from calli of S. indicum.
Esculentic acid (102.2mg) and the latter acid (1O.6mg) were obtained from a
23.3-kg callus (Okada et al. 1994).
2.2 Hairy Root Culture and Secondary Metabolites
381
E
.2-
7.0
-0- 20 "C
6.0
----25"C
--O-30"C
--+-35"C
-I:r-40"C
UI
'00 5.0
~
'0
c
4.0
im
3.0
c
0
iii
2.0
1.0
0
Time(day)
382
T. Ogasawara et al.
Fig.4A,B. Hairy root culture in a flask (A) and 51 jarfermenter (B) (T. Ogasawara et aI. , unpubl.)
383
12
1:
10
CI
'i
.c::
UI
~
S
.c::
"
Time (day)
384
T. Ogasawara et al.
1200
1000
800
600
400
200
o
Hairy root
A-1
Fig. 6. Contents of naphthoquinone 23 in hairy roots and mother plants. (Ogasawara et al. 1993)
385
1000
1:
Cl 800
'i
~
.r:.
UI
..:!. 600
gJl
CD
I:
I:
IT
-=
400
.r:.
c.
III
I:
'0
'C
.l!!
I: 200
0
()
o
o
20
10
30
Time (day)
Fig. 7. Production of naphthoquinone 23 in the hairy roots of S. indicllm. (Ogasawara et al. 1993)
1988). In this case, chrysophanol and 8-0-methylchrysophanol showed maximum contents in the early stage of the stationary growth phase. Our studies
are in agreement with this finding. Secondary metabolites in dedifferentiated
cells were generally produced during the stationary phase but not during
vigorous growth in the log phase. However, it has been reported that hairy
roots grow at higher rates and show stable production of secondary
metabolites parallel with the results of the present experiment (Flores et al.
1987).
Naphthoquinone and anthraquinone in S. indicum had not been reported
thus far. However, quinones have been investigated actively, and Tabata
(1988) reviewed naphthoquinone production in cell cultures. Quinones
contain the same basic chromophore, which consists of two carbonyl groups
in conjugation with two carbon-carbon double bonds. Quinones can be
conveniently divided into three groups of skeletal type: benzoquinones,
naphthoquinones, and anthraquinones. There are many lipophilic forms
alkylated or substituted by the isoprenyl moiety. Others are hydroxylated
or combined with sugars. Naphthoquinones of plants are biosynthesized
by the following distinct routes: (1) ortho-succinylbenzoic acid; (2) parahydroxybenzoic acid-mevalonic acid(MV A); (3) homogentisic acid-MVA
386
T. Ogasawara et al.
80
tf
.J:.
.J:.
c;
c;
a>
60
a>
.. ~..
~
.J:.
70
~
.J:.
50
....a> ....a>
a>
a>
~I
~
'0
:t
'0
C
!!
I:
0
0
:t
!!
I:
40
30
0
0
20
10
0
0
10
20
30
Time (day)
Fig. 8. Production of anthraquinones 24 and 25 in the hairy roots of S. indicum (Ogasawara et al.
1993)
(Inouye and Leistner 1988; Tabata 1988); (4) acetic acid-MVA; (5)
MVA(Tabata 1988). The pathways of routes (1) and (4) are also utilized for
the synthesis of anthraquinones.
2.2.5 Biological Activity of Naphthoquinone
Antimicrobial activity of naphthoquinone epoxide isolated and purified from
the hairy roots of S. indicum is shown in Table 5. Its antimicrobial activity
seems to result from the presence of an OR in peri position to a carbonyl
moiety in the molecule. Since the compound has two structures in one molecule, antimicrobial activity is relatively higher. The compound also showed
cytotoxicity towards colon tumor cells (Potterat et al. 1987). Juglone is known
to show antimicrobial activity and also to inhibit germination as an allelopathic
agent of walnut tree (Clark et al. 1990; Rarborne and Baxter 1993). Its structure is similar to that of naphthoquinone epoxide 23. We determined the
difference in efficacy between naphthoquinone epoxide 23 and juglone, and
compared the activities of naphthoquinone epoxide with that of juglone in a
387
Microbe
Strain no.
Growth
Salmonella senftenberg
Bacillus cereus
Bacillus subtilis
Micrococcus luteus
Staphylococcus au reus
Lactobacillus plantarum
Pseudomonas aeruginosa
Escherichia coli
Saccharomyces cerevisiae
ATCC
ATCC
ATCC
IFO
lFO
ATCC
ATCC
IFO
ATCC
8400
11778
6633
3333
13276
14917
7700
12689
12041
+
+
+
+, Growth; -, no growth.
Table 6. Effects of juglone and naphthoquinone 23 on germination and growth in lettuce seeds.
(T. Ogasawara et aI., unpubl.)
mg/disk
Control
0.0
Juglone
0.1
1.0
Naphthoquinone 23
0.1
1.0
Germination
(%)
50/50
(100)
3/49
(6)
0/52
(0)
50/50
(100)
40/47
(85)
Growth (mm)
Hypocotyl
Radicle
Callus
Callus
Callus
Callus
Callus
Callus
Callus
Callus
Hairy root
Seedling
Seedling
Growth response
MS+2,4-D
MS+2,4-D
MS+2,4-D
MS+2,4-D
MS+2,4-D
MS+NAA
MS+NAA
?
MS
(1)
(1)
(1)
(0.02, 43)+KN (2)
(0.02, 43)+BA (2)
(2, 9)+KN (2)
(2, 9)+BA (2)
Media mg/l
Inoculum
Seeds
Seedling
Radicle
Hypocotyl and
cotyledon and
root
Triterpenes
Naphthoquinone,
Anthraquinones
Reference
Khanna and Jain (1973b)
Jain and Khanna (1973)
Jain (1981)
Mimura et a1. (1987d)
Mimura et a1. (1990a)
Mimura et a1. (1990b, 1991)
Compound isolated
Sesamin
Sterols
Pedaliin
Sesamin, sesamolin
Acteoside
Polyphenol glucosides
f:?-
'...."
'~"
e;
(JQ
co
co
389
3 Summary
In vitro production of secondary metabolites in Sesamum indicum is summarized in Table 7. It appears that the production of secondary metabolites is
lower in callus cultures than in the mother plants, except for hairy root culture.
Hairy root productivity of naphthoquinone was higher than that of the mother
plant. The secondary metabolism of hairy root culture appears to be different
from that of the mother plant. Hairy root, callus, cell suspension, and multiple
shoots in sesame might produce new compounds which explain some of its
biological activities as a medicinal plant and provide good material to study
new routes of secondary metabolites.
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Oritani T, Yamashita K (1974) Syntheses and biological activities of analogs of abscisic acid. Agric
Bioi Chern 38:801-808
Osawa T (1993) Inactivation of tumor initiators by food components. Nippon Nogeikagaku Kaishi
67(1):27-30
Osawa T, Nagata M, Namiki M, Fukuda Y (1985) Sesamolinol, a novel antioxidant isolated from
sesame seed. Agric Bioi Chern 49:3351-3352
Osawa T, Namiki M, Kawakishi S (1988) Role of dietary antioxidants in protection against
oxidative damage. In: Kuroda Y, Shankel DM, Waters MD (eds) Antimutagenesis and
anticarcinogenesis mechanisms. Plenum, New York, pp 139-153
Potterat 0, Stoeckli-Evans H, Msonthi lD, Hostettmann K (1987) Two new antifungal
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Ryu JH, Doo HS, Kwon TH (1992) Induction of haploid plants by anther culture in sesame
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Sakamoto 0, Kono Y, Kojima K, Mimura A, Takebayashi K, Takahara Y, Osawa T (1991)
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1 Introduction
1.1 Biology and Distribution
395
Fig. lA-E. S. mammosum, general appearance of plant with mature fruits (A); pear-shaped
mature fruit with mammillaries (8); plant with flowers and immature fruits (C); mature leaf and
flowers (D); long section of the mature fruit and seeds (E)
a pear-shaped fruit which has no mammilaries. A third form has sphericalshaped fruits. The fruits of different varieties do not differ significantly in their
sola so dine content (Telek et al. 1977). The seed develops from an anatropous
ovule and has an incumbent type of cotyledon arrangement. The coiled embryo is completely enclosed by a copious fleshy endosperm. The mature seeds
are oval-semi orbicular in outline, flattened laterally, with the hilar end often
slightly more flattened, aprroximately 3-3.5mm wide, 4-4.5mm long, and
1mm thick. They are nonglossy, reddish brown to dark brown in color, with a
finely reticulate surface (Miller 1969; see Fig. 1E). The plant is tolerant of
many soil types, including heavy clays. It resists drought, excessive rainfall, and
mild inundation, and is fairly immune to insect attacks (Telek et al. 1977).
396
G. Indrayanto et al.
Solasodine
Diosgenin
Tomatidine
Tomatidinol
R
Sterol
Cholesterol
R=
~
~
yvt(
yvt(
A
Campesterol
Sitosterol
Stigmasterol
Fig. 2. Structure of solasodine and its epimer, diosgenin, and sterols of the callus cultures of
s.m.
397
Glycoside
Sugar moiety
p-Solamargine
Rhamnose ~
Solamargine
(1--*4~ Glucose
Rhamnose ~ (1--*2);>
Rhamnose ~ (1--*4
Solasonine
p (1--*3~ R
Glucose
P(1--*3 ~ R
Rhamnose ~ (1--*>2)
Glucose
P(1--*3)
alactose P (1--*3~ R
R = Solasodine
Fig. 3. Structures of steroidal glycoalkaloids that accumulated in the fruits of S.m.
398
G. Indrayanto et al.
1.0
0.8
;;i
~
C
Q) 0.6
C
0
()
Q)
c 0.4
'5
0
rJ)
co
(5
C/)
0.2
0~-----Y~----~~--~~-----4--
Green
Yellow Green
Ye llow
____~
Orange
Fruit Color
Fig. 4. Effect of the degree of maturity (color) of S.m. fruit on its solasodine content (% dry wt.).
(Data from Telek et al. 1977)
1.3.1 Solasodine
Steroidal glycoalkaloids and saponins that are widely distributed in
Solanum spp. are thought to be very important in the natural defense of plants
against microorganisms and/or predators (Paczokowski and Wojciechowski
1993).
A tumor-inhibiting activity of the glycoalkaloid ,B-solamargine was reported by Kupchan et al. (1965). Cham et al. (1987) showed that the
glycoalkaloids solasonine, solamargine, and another glycoside containing
solasodine-aglycone had antineoplastic activity against Sarcoma 180 in mice.
Solasodine also showed growth inhibition activity to some fungal strains,
although its activity was less compared with solaftoridine and verazine
(Kusano et al. 1987a). Some steroidal alkaloids, including solasodine, showed
an inhibitory effect on the enzymatic conversion of dihydrolanosterol into
cholesterol (Kusano et al. 1987b). Roddick et al. (1990) reported that
solasonine and solamargine had the membrane-disrupting properties of
phosphatydy1choline/cholesterol at a concentration above 50,uM. Strong
antiviral activity of the steroidal glycoalkaloid solasonine, tomatine, and
chaconine was shown by Thorne et al. (1985) by using Herpes simplex infection of in vitro-cultured cells. Potatoes containing more than 0.02% steroidal
glycoalkaloids are considered to be toxic to man. The majority of steroidal
glycoalkaloids in potatoes are derived from solanidine and tomatidenol (Kuc
1984). Acute toxicity was observed after oral/intraperitonia/intravenous
administration of a-tomatine in rabbit and rat. The steroidal glycoalkaloid a-
399
Thewles et al. (1993) reported that biliary cholesterol output in rats was
stimulated more than threefold by feeding with diosgenin, whereas biliary
outputs of phospholipid and bile salts were not changed by diosgenin feeding.
Takechi et al. (1992) showed that some synthetic diosgenyl f3-D-glucosides
have hemolytic and antifungal activities.
Miles et al. (1994a,b) demonstrated that geeldikkop (plant-induced
hepatogenous photosensitization diseases) could be induced in sheep by oral
administration of crude saponins/extracts of Tribulus terrestris. The administered saponin was found to contain steroidal sapogenin diosgenin, yamogenin,
tigogenin, gitogenin and neotigogenin.
1.3.3 Sterols
Sterols and their derivatives promote and maintain growth and development
in plant and fungi by acting as membrane constituents. Experimental data
showing that sterol acts as a hormone in plants, are scarce (Grunwald 1975;
Burden et al. 1989).
Sitosterol is used as a hypolipidemic agent, in conjunction with dietary
modification (usual dose 2-6 g orally). It is also used in prostate disorders
(Reynolds 1993). Recent studies by Santos et al. (1995) showed that
sterols (stigmasterol and sitosterol) had anti nociceptive action in mice. Given
orally, stigmasterol and its acetate derivate exhibit significant though less
potent analgesic action against both acetic acid- and formalin-induced nociceptive in mice. Stigmasterol, stigmasterol acetate, and sitosterol, given
intraperitoneally, inhibited acetic acid-induced abdominal constriction in mice.
400
G. Indrayanto et al.
In our laboratory, the callus cultures of S.m. (cell line sm) was first initiated
by Isnaeni (1986) from young leaf petioles of a 6-month old plant, collected
on a mountain in Nongkojajar Pasuruan East Java. The explants were
cultivated on modified MS (Murashige and Skoog 1962) medium with the
addition of 2mg/1 kinetin and 1 mg/12,4-D. Callus was formed within 7-10 days
of incubation. From the various combinations of kinetin and 2,4-D that
have been tested for these cultures, 2 mg/l kinetin and 0.5 mg/l of 2,4-D
(medium K2D o.s) gave the best growth rate. All the calli were maintained
in continuous light (ca. 8001x) at 25 1C and subcultured every 3 weeks
(Fig. 5A,B). Suharno (1986) reported that the callus cultures of S.m.
(cell line sm) could also grow well on modified MS medium with the addition of 2mg/1 kinetin and 0.5mg/1 NAA (medium K2D o.s) After 1 year of
subculturing, the cultures exhibited friable callus with pale yellow to
cream (on medium K2DOS) or pale green (on medium K2NOS) color. These
calli are still growing well after 10 years of subculturing; however, these
callus cultures have a shorter lag phase in their growth curve compared to the
1-year-old calli. The calli of cell line sm could also grow well in the darkness
(Fig. 6).
Using 3% sucrose, glucose, or lactose in the media, Wijono (1987) reported that sucrose was the best carbohydrate source for the growth of callus
cultures of S.m. (cell line sm). Whereas Susilowati (1987) showed that the
optimum concentration of sucrose was 3% (see Figs. 7, 8), Sarwetini (1988)
demonstrated that the addition of banana powder could significantly increase
the growth rate of these callus cultures.
Callus cultures of S.m. could also be initiated by using shoots from shoot
cultures as explants. We recently initiated some new cell lines of the callus
cultures (code: sm-1, sm-12, sm-23) by using shoots from shoot cultures (cell
line sm-1, sm-12, sm-23) on medium K2D o.5' Figure 6 shows that the calli ofthe
new cell line sm-1 (800Ix, 25 1C) have relatively lower growth rate compared to the sm cell line.
Suspension cultures of s.m. (cell line sm) used for biotransformation
studies (see Sect. 2.5) could be initiated by inoculating ca. 5-10g friable calli in
50ml medium K2No.5 in a 250-ml Erlenmeyer flask. After 10-15 subcultures,
cell aggregates 3-7mm in diameter were formed (see Fig. 5C,D). However, the
cell aggregate suspension cultures have a very low growth rate compared to
the fine cell suspension. The cells appeared to be self-immobilized by
subculturing (Fig. 9). The formation of cell aggregates (self-immobilization) in
suspension cultures of Solanum aviculare was also demonstrated (Tsoulpha
and Doran 1991).
401
Fig. SA-D. Callus cultures of S.m. cell line sm (AI, B), sm-l (A2) cultivated on medium K2NUS;
Cell aggregate suspension cultures of cell line sm (C, D). All the cultures were maintained under
continuous light (ca. 800 Ix) at 25 ::':: 1 C
By using young shoots bearing three to four axillary buds from a 7-month-old
S. mammosum plant collected at Purwodadi Botanical Garden Malang
as explants, Isfidiati (1988) initiated shoot cultures of cell line sm-p for
micropropagation. After surface sterilization with 2% NaOel solution, the
402
G. Indrayanto et al.
14
12
--
"'-
.- 10
Ol
.c.
Ol
.c.
(/)
LL
.~
....
1'~
(J)
----'I
----~ -----}:-----
-:1J;- - - - __
:t:
0
0
10
20
30
40
50
Days
Fig. 6. Growth curve of callus cultures of S.m. cultivated on medium K2D o.5' The calli were
maintained in continuous light at light intensity of ca. 800 Ix [sm(L) and sm-l] or in darkness (D)
at 25 1C. [Data of cell line sm(L), 1985 from Isnaeni 1986]
-.-Sucrose
-.-Glucose
-A-Lactose
(J)
"0
c 4
.c.
~ 3
0
....
(9
Weeks
Fig. 7. Effect of some carbohydrate sources (3 %) in medium K2Do.5 on the growth rate of callus
cultures of cell line sm. (Data from Wijono 1987)
403
6
5
><
Q)
-g
c'5
- . - Sucrose
- . - Sucrose
- A - Sucrose
- , , - Sucrose
----+--- Sucrose
1%
3%
5%
7%
10%
---~~--'.
Weeks
Fig. 8. Effect of sucrose concentration in medium K,Dos on the growth rate of callus cultures of
cell line sm. (Data from Susilowati 1987)
90
Q)
E
::J
Fine cells
Cell aggregates
(5
>
(j)
70
or
()
..x:: 60
0
(II
0'0::f!. 50
35
'.
:.'
30
25
..x::
(/)
(II
u::
....
Cl
'(jj
---:-i_A
--.
. / /-'-A
.... ...
/'.
/.
140
.r:
80
/--:--'. ..--------.
-1'
20
:;:
(/)
Q)
....
. ,
15 LL.
---
.'
'
10
~I~~'
...:..-
30
0
10
12
Days
Fig. 9. Effect of self-immobilization of the cells on the growth rate of suspenson cultures of S.m.
(cell line sm). Data represent mean of three replicates
explants were cut into one to two nodal pieces (ca. 0.5-1 cm long) and cultivated on modified MS media with 32 combinations of phytohormones. Shoots
were formed in 2-4 weeks in ten hormone combinations (see Table 1). The
optimal shoot formation was obtained by using a modified MS medium with
404
G. Indrayanto et al.
Table 1. Effect of hormone combination on the number of shoots formed on the inoculated shoot
of S.m. (cell line sm-p). (Data from Isfidiata 1988)
Hormone (mg/l)
Kinetin
BAP
4
4
0.5
0.5
0.5
0.5
2
4
4
4
0.1
0.5
NAA
IAA
2,4-D
No.
of shoots'
GA3
+
+
2
4
0.5
Callus
formation
1.5
++
0.5
0.1
0.5
= 2-3;
+ +,
= 4-6;
+++
+
+++
+
+
+
+
Root
formation
+
+
+
+
= 7-10.
Days
4
8
12
17
23
lEA
2.5mg/1 ('Yo)
5mg/1 ('Yo)
0
42
67
91
100
0
57
91
100
100
the addition of 4mg/1 kinetin as the phytohormone. Our recent studies showed
that shoot multiplication of the shoot cultures of s.m. (cell lines sm-1, sm-12,
and sm-23) could also be achieved by using a modified MS medium with the
addition of2mg/1 BAP as the phytohormone (see Fig. 10A,B). In this case, we
used sterile-seedling hypocotyls as explants for initiating shoot cultures.
Pranachita (1992) reported that induction of root formation on the inoculated shoots of cell line sm-p could be achieved by using a modified MS
medium with the addition of 0.5 mg/l IAA as phytohormone. She demonstrated that 90% root formation on the inoculated shoots resulted within 2-3
weeks.
Our recent studies showed that root induction of the cell line sm-1 shoot
cultures was also successful on using a modified MS medium with the addition
of 2.5 mg/l or 5 mg/l IBA as hormone; 100% root formation on the inoculated
shoots occurred within 14-16 days (Table 2, Fig. lOD).
About 85% of the plantlets of cell line sm-1 survived and grew well after
transplanting to common trays containing a sterilized mixture of sand and
humus (1: 1) (Fig. 10C). After acclimatization for 3-4 weeks, the plants can be
cultivated in the field.
405
Fig. lOA-D. Shoot cultures of cell line sm-l on modified MS medium with the addition of 2mg!
I BAP. One-week (A) and 4-week (8) cultures after subculturing. Young plant of SM (cell line sm1),3 weeks after transplanting onto a glass tray containing a mixture of sand and humus (1: 1) for
acclimatization (C). Root formation on the inoculated shoot of cell line sm-l after 3 weeks of
cultivation on modified MS medium with the addition of 2.5 mg!1 IBA (D)
Indrayanto et al. (1986) reported that callus cultures of cell line sm cultivated
on modified MS medium (K2DOS) contained only cholesterol, campesterol,
stigmasterol, and sitosterol, whilst solasodine and diosgenin could not be
detected. Our unpublished results also showed that our new cell line of callus
cultures, sm-l, sm-12, and sm-23, cultivated on media K2No.s, also could not
produce solasodine. The absence of solasodine in undifferentiated cells is
reported in many publications, e.g., in calli of Solanum laciniatum, S. wrightii,
S. khasianum, S. aviculare. S. aculeatissimum, and S. aculeastrum (Carle 1979;
406
G. Indrayanto et al.
Indrayanto et al. 1983; Galanes et al. 1984; Nabeta 1993; Drewes and Staden
1995).
Attempts to induce solasodine formation in these callus cultures by addition of various concentrations of yeast extracts (Mufidah 1988) and Rhizopus
arrhizus (Karsana 1988) as bioelicitors failed. In the last two experiments, the
phytosterol contents were also not changed significantly. UV irradiation
(21 W/m 2, 24h) of cell line sm-1 calli also could not stimulate the production of
solasodine, although the same UV radiation could double the hecogenin content in the callus cultures of Agave amaniensis (Rusli 1996). The phytosterol
(mostly sitosteol) content in callus cultures of cell lines sm and sm-1 was 0.40.7 mg/g drywt.
Although solasodine was not detected in callus cultures of Solanum
laciniatum, as described above, Indrayanto et al. (1995) showed that its shoot
cultures could produce solasodine, so it seemed that solasodine production
was correlated with the availability of chlorophyll and cell differentiation, as
reported in many publications (e.g., Conner 1987; Ehmke and Eilert 1993).
However, our recent studies showed that in all of our shoot cultures (cell lines
sm-p, sm-1, sm-12, sm-23) of S.m., solasodine was not detected. These studies
showed that the production of solasodine in the plant cells was not dependent
on the cell differentiation or the availability of chlorophyll in the cells. A
recent publication by Ripperger (1995) reported that solasodine could also be
isolated from roots of various Solanum spp. According to Subroto and Doran
(1994), the hairy root cultures of Solanum aviculare produced solasodine
at a concentration of 29-32mg/g dry wt. These results confirmed that the
biosynthesis of solasodine might not be correlated to the availability of chlorophyll in some Solanum species.
Recently, cell line sm also produced betulin, a lupane (triterpenoid) derivative (identified by TLC, MS). Now we are in the process of identifying
three other triterpenoids that are isolated from the chloroform extract of these
calli. The production of some lupane derivatives (betulinic acid, betulin,
lupeol, and lupeol aldehyde) in callus cultures of Solanum laciniatum, S.
wrightii (Indrayanto et al. 1983) and S. aviculare (Vanek et al. 1985) was also
reported (see Fig. 11). Betulinic acid concentration in calli of Solanum
aviculare was relatively very high (up to 3% dry wt.).
2.4 Biotransformation by Using Suspension Cultures
Using callus cultures of cell line sm, Sondakh (1989) reported that progesterone added as substrate into the media could be transformed to 5a-pregnan-3,
20 dione (Fig. 12). The transformation of progesterone to 5a-pregnan-3,20
dione was also reported from various tissue cultures systems (Furuya et al.;
1971. Stohs and Rosenberg, 1975).
Our recent studies showed that suspension cultures of cell line sm could
also transform some salicylate derivatives (salicyl alcohol, salicylic acid, and
salicylamide into their mono glucoside (see Fig. 12). A new bioconversion
product, salicylamide 2-0-f3-D-glucopyranoside, was isolated from the cell
407
Lupane - derivative
Lupeol
Betulin
Betulin aldehyde
Betulinic acid
Cn3
Cn20n
cno
coon
Rahayu (1988) showed that the petroleum ether (40-60 0C) extract (1 mg
extract equivalent with 48mg dried calli) of callus cultures of cell line sm given
orally, have a significant antifertility effect in mice (Table 3). The extract (14mg/30g mice) did not appear to affect the behavior or activity ofthe treated
mice.
The acetone extract of the same calli (1 mg extract equivalent with 46.5 mg
dried calli) also exhibited a significant antifertility effect in mice at a dose of
2-4mg/30g mice (Table 3; Samesti 1988).
408
G. Indrayanto et al.
cm
1
C=O
Progesterone
5 -pregnan-3. 20-dionc
-Salicylamide
Salicylamide 2-0-11-glucollyranoside
Salicin
Salicyl alcohol
l~H
rQJ ~
Salicylic acid
hJH
1"0-(
if
C(x)H
CH20H
Fig. 12. Biotransformation of progesterone, salicylamide, salicyl alcohol, and salicylic acid by
409
Group
Extract
No. of
females
Total of
litters
Average of
litters/mice
Dose of extracts
(mg)'
Control
T1
T2
T3
18
18
18
18
215
199
59
16
12
11
3
1
Petroleum
ether"
Control
T5
T6
Acetone"
6
6
6
56
8
0
9
1
0
0
2
4
2
4
It was postulated that the triterpene content of these calli might cause the
antifertility effect in these experiments. It is well known that many triterpenes
have some cytotoxic activities (Das and Mahato 1983).
3 Conclusion
In vitro cultures of Solanum mammosum were initiated on modified MS
medium with the addition of 2 mg/l kinetin and 0.5 mg/l 2,4-D or 2 mg/l kinetin
and 0.5mg/1 NAA (for callus cultures), 2mg/1 BAP (for shoot cultures); 100%
root formation occurred on the inoculated shoots by using a modified MS
medium with the addition of 2.5 or 5 mg/l BAP.
Although callus cultures could produce only some sterols and lupane
derivatives, these cultures could transform progesteron and some salicylatederivatives which were added as substrates. Solasodine was not detected in all
tissue cultures of S.m.
The petroleum ether and acetone extract of callus cultures showed a
significant antifertility effect in the treated mice. This activity might be due to
the triterpene content of the calli.
410
G. Indrayanto et al.
Method 2. Sterile shoots of the shoot cultures were cut ca. 0.5-1 cm long, and placed on the
medium K2D o.5 Incubated as in method l.
The established callus cultures must be subcultured every 3-4 weeks.
411
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WICKREMESINHE
and R.N.
ARTECA
1 Introduction
1.1 Botany and Distribution
Yews are usually dioecious, evergreen (gymnosperm) trees or shrubs (Fig. 1a).
The leaves are simple, flat, linear, often falcate, with distinct but short petioles,
arranged spirally though often appearing two-ranked (Fig. 1b). They have a
single unbranched midrib and two broad stomate bands on the underside of
the leaves. The bark is reddish or purplish to dark chestnut brown, scaly or
exfoliating from the trunk and larger branches in thin flakes or long strips. The
wood is hard, dense, flexible, elastic, and fine-grained without resin ducts. The
winter buds are ovate, axillary, or terminal, with imbricate scales. The flowers
are small, solitary, or occasionally twinned, axillary. The female flower resembles an axillary vegetative bud, but is usually decurved or pendent and is easily
recognized on close inspection by the micropyle opening in the exposed ovule.
The male flower or pollen cone has several sterile scales at the base, with a
stalked globose head of 6 to 14 scales, each with 5 to 9 microsporangia or
pollen sacs. The solitary seed (which matures in late summer to fall), sometimes called a single-seeded berry or berry-like fruit (Fig. 1b), is surrounded by
a fleshy mucilaginous, scarlet outer seed coat or aril (Chadwick and Keen
1976; Silba 1986).
The genus Taxus is divided into eight species and two hybrids (Keen 1975;
Silba 1986; Hartzell 1991). The distribution of these eight species is summarized in Table 1. The two hybrids are T. x media (cross between T. baccata and
T. cuspidata) and T. x hunnewelliana (cross between T. canadensis and T.
cuspidata).
1.2 Importance
Since the commencement of civilization, the yews have been known for their
poisonous nature and the special quality of their wood for weapons such as
bows. It is well documented that primitive and early historical cultures used
Department of Horticulture. The Pennsylvania State University, University Park, Pennsylvania
16802, USA
Biotechnology in Agriculture and Forestry, Vol. 41
Medicinal and Aromatic Plants X (ed. by y.P.S. Bajaj)
Springer-Verlag Berlin Heidelberg 1998
416
Fig. I. a T. x media cv. Hicksii tree growing on the Pennsylvania State University, University Park
Campus (Pennsylvania, USA), approximately 30 years old. b A closeup of a branch showing the
arrangement of needles and single seed covered by its scarlet outer seed coat
yew for fish and animal poisons as well as arrow poison (Hartzell 1991 ). Greek,
Roman, and European archers are supposed to have made their strongest
bows with yew wood.
In more recent cultures it has been used as an abotifacient, as a cure for
hydrophobia, heart ailments, rheumatism, malaria, epilepsy, and also as
a laxative (Beal 1975; Hartzell 1991). There have also been many well-
417
Table 1. Distribution of Taxus species and their elevations. (Silba 1986; Hartzell 1991)
Taxus species
Common names
Distribution
baccata
canadensis
cuspidata
English or European
yew
Western or Pacific
yew
Canadian yew
Japanese yew
floridana
globosa
Florida yew
Mexican yew
mairei
Chinese yew
wallichiana
Himalayan yew
brevi/olia
Elevation
(m)
100-2000
0-2134
365-415
500-2400
0-30
1690-3333
457-2450
1600-3300
The demand for taxol escalated as a result of the excellent activity shown
during clinical trials in the treatment of advanced, progressive, and drug-
418
o
"
~:
Taxol
Baccatin III
.
~
xu
Fig. 2. Structure of taxol and some of the most abundant taxanes encountered in Taxus extracts
refractory ovarian cancer (McGuire et al. 1989; Einzig et al. 1991; Markman
1991), and metastatic breast cancer (Holmes et al. 1991). Most recently, taxol
has also demonstrated impressive clinical antitumor activity in patients with
lung (nonsmall cell and small cell), head and neck, and myelogenous leukemia
(DeLaPena and Pyron 1993; Rowinsky et al. 1993). Final approval (by the
419
Food and Drug Administration, USA) for the clinical use of taxol was granted
in December, 1992, for the treatment of advanced ovarian cancer (Cragg et al.
1993) and subsequently for the treatment of advanced breast cancer.
Large-scale clinical trials with taxol have been hampered for a long time
by a limiting supply of taxol (Blume 1991). The supply of this drug has been a
key issue because until early 1994, the only "approved" source of taxol (as
specified by the Food and Drug Administration, USA) was extraction from
the bark of T. brevifalia, a source that has become scarce due to its extremely
slow growth and the fact that it is predominantly found as an understory
growth in dispersed microsites within the larger older-growth ecosystem.
Total synthesis of taxol was achieved in 1994 (Holton et al. 1994; Nicolaou
et al. 1994b). However, the process for total synthesis is not expected to be
commercially feasible due to the lengthy protocol (requiring at least 28 chemical steps) and the low yield (Flam 1994; Mann 1994).
Based on the current bark-extraction procedures, 5000 to 6000 kg of bark
are needed to produce 1 kg of taxol. In the USA, over 700000 kg of yew
bark were stripped from trees and collected during 1992 to produce 130kg of
taxol, and the projected amount for 1993 was 230kg (Cragg et al. 1993; Joyce
1993).
To date, the most promising long-term sources for the large-scale production of taxol seem to be semisynthesis and plant cell culture (Nicolaou et al.
1994a). Although taxol was initially isolated from T. brevifalia, it is also found
in all trees belonging to the genus Taxus (Wani et al. 1971; Vidensek et al.
1990; Witherup et al. 1990; Mattina and Paiva 1992; Wickremesinhe 1992;
Wheeler et al. 1992; Choi et al. 1994; Wickremesinhe and Arteca 1994b; Kwak
et al. 1995). Currently, ornamental yews are being grown in commercial nurseries to collect needles and stem clippings, as an alternative source of taxol
(Joyce 1993; Wheeler and Hehnen 1993), and it is anticipated that the use of
T. brevifalia bark will be totally eliminated by 1996 (Cragg et al. 1993).
Suffness (1995b) noted that great progress has been made in research on taxol
production and that there is a strong long-term potential in: (1) plantations of
either T. baccata or several ornamental Taxus cultivars, for either direct production of taxol or production of its precursor, (2) plant cell culture, especially
utilizing advances in biosynthetic understanding and genetic engineering, and
(3) semisynthesis of taxol from its precursors.
2 In Vitro Approaches
2.1 Review of Tissue Culture/Biotechnology Studies
The work on in vitro culture of Taxus was initiated in the 1950s (LaRue 1953;
Tulecke 1959). At present, the main emphasis of tissue culture/biotechnology
has been to establish cell cultures capable of producing taxol. The overall
objective has been to establish fast-growing cultures that produce high levels
420
Table 2. Taxus species from which callus and/or cell suspension cultures have been established
Taxus species
Reference
brevifolia
baccata
canadensis
cuspidata
floridana
Salandy (1993)
x media
421
Table 3. Amount of taxol produced by tissue cultures derived from different Taxus species
Taxus species
Source
Amount
Reference
Callus
7.0::':: 2.5mg/kg
Callus
0.00059%
Medium
Medium
Medium
Cells + medium
1 to 3mg/l
3.9mg/l
1.43mg/l
620 fig/kg cells a
Shoot-tip culture
Callus
0.03 - 0.46mglkg'
2.4 ::':: 1.4 mg/kg
Callus
0.00021 %
Callus
Callus
Medium
Plantlets
0.04%b
7.83 mg/100 gb
1.5 mg/l
0.01 to 0.l0%'
canadensis
Plantlets
0.11 to 0.36%'
cuspidata
Callus
Callus
Callus
0.02%
0.02%
14.2 ::':: 2.4mg/kg
Callus
0.00109%
Cell
Cell
Cell
Cell
Cell
0.012%
0.012%
2-lOmg/kg
14mg/kg
10-14mg/kg
brevifolia
baccata
x media
cv. Hicksii
suspensions
suspensions
suspensions
suspensions
suspensions
Cell suspensions
Medium
Medium
Medium
3.4mgll
O.4mg/l
12.2mg/l
0.3-0.7mg/l
Callus
0.0131 %
Callus
Callus
0.00089%
Callus
1.1-2.5 mg/kg
Medium
3-5 mg/l
422
Table 3. Continued
Taxus species
Source
Amount
Reference
x media
cv. Densiformis
Callus
Callus
0.00078%
analytical protocol that would be suitable for use with milligram quantities of
cells. Therefore, major emphasis was also placed on the development of an
efficient extraction and analytical method (Ketchum and Gibson 1993;
Wickremesinhe and Arteca 1993b). Although HPLC analysis has been
routinely used as the initial screening tool for taxol, the necessity to confirm
the presence of taxol using additional analytical techniques beside HPLC has
become necessary due to the presence of a "putative taxol" peak observed in
some cell cultures (Wickremesinhe and Arteca 1994a).
The microtubule-stabilizing bioassay has been successfully used to confirm the presence of taxol-like activity in semipurified and purified extracts of
cell and callus cultures (Wickremesinhe 1992; Wickremesinhe and Arteca
1993a). Enzyme-linked immunosorbent assays (ELISA) have also been developed to detect and quantify the presence of taxol, 10-deacetylbaccatin III, and
related taxanes in Taxus plant extracts (Jaziri et al. 1991; Grothaus et al. 1993;
1995), and has been successfully applied to evaluate tissue culture extracts for
the presence of taxol and related taxanes (Jaziri et al. 1991; Guo et al. 1994;
Zhiri et al. 1994; 1995b).
In addition, tandem mass spectrometry (Hoke et al. 1992), high-speed
counter current chromatography (Vanhaelen-Fastre et al. 1992), liquid
chromatography-thermos pray mass spectrometry (Auriol a et al. 1992), and
LC/MS/MS (Kerns et al. 1994) are some of the other techniques that have
been used to analyze and quantify taxol and related taxanes in Taxus sp. plant
extracts. Fast-atom bombardment mass spectrometry and nuclear magnetic
resonance spectrometry has also been used to confirm the presence of taxol in
callus and cell culture (Wickremesinhe and Arteca 1993a, 1994b; Falzone et al.
1992).
The production of taxol in T . cuspidata callus and cell cultures was
reported in 1992 by Fett-Neto et al. They have subsequently reported the
stimulatory effect of gibberellic acid on callus growth and the enhancement of
taxol accumulation when treated with phenylalanine and benzoic acid (FettNeto et al. 1993, 1994a). Taxol accumulation in cell cultures has been reported
to be nongrowth-related, and the highest levels of taxol have been found in
cells during the stationary phase, while the highest levels in the medium were
423
during the early parts of the growth cycle (Fett-Neto et al. 1994b, 1995).
Supplementing the growth medium with fructose (Kim et al. 1995; Mirjalili
and Linden 1995), manipulating the gas composition of the head space
(Mirjalili and Linden 1995), and the addition of methyl jasmonate (Mirjalili
and Linden 1996) have been reported to significantly influence taxol
production.
Many studies have also been conducted on embryo culture of Taxus
species, in order to overcome their lengthy dormancy requirement (Flores et
al. 1993; Chee 1994; Zhiri et al. 1994). A method for multiple shoot and
plantlet formation from zygotic embryos of T. brevifolia has also been reported (Chee 1995).
Although many researchers have been working on cell cultures of Taxus,
most findings have not been made public due to proprietary reasons, especially
those conducted in commercial establishments throughout the world.
2.2 Establishment of Callus Cultures
Table 4. Rating of callus induction from Taxus explants (derived from immature stems).
placed on BS salts supplemented with 2x BS vitamins, 20 gil sucrose and either 2,4dichlorophenoxyacetic acid (2,4-D), indole-3-butyric acid (IBA), or a-naphthalene acetic acid
(NAA) ranging from 0.2 to 1O.Omg/l in combination with 0.2mg/16-furfurylaminopurine (kinetin)
and solidified with 2 gil Gelrite. Each observation is based on SO to 200 explants incubated in total
darkness for 4 weeks. (Wickremesinhe and Arteca 1993a)
2,4-D
Taxus species
brevifolia
baccata
cv. Repandens
cuspidata
x media
cv. Densiformis
x media
cv. Hicksii
NAA (mg/l)
2
0.2
O.S
2
1
3
3
3
2
0
0
0
1
1
1
0
0
0
0.5
0
0
2
0
0
1
2
3
3
IBA
5
10
10
1
1
1
0
0
1
3
3
2
1
0
1
1
0
0
o = no callus, 1 = less than 30% of the explants produced callus, 2 = 30 to 75% of the explants
produced callus, 3
424
Fig. 3. a Callus induction on stem explants of T. x media cv. Hicksii placed on B5 salts supplemented with 2x B5 vitamins, 20 gil sucrose 1 mgll 2,4-D, and 0.2 mg/l kinetin. b Callus cultures
growing on membrane rafts after 3 (left) and 8 (right) weeks in culture
an area within the stem, which resulted in splitting and peeling of the epidermis and related stem tissues due to the growing callus. Overall, 2,4-D was the
better source of auxin compared to NAA and IBA for both T. brevifalia and
T. baccata cv. Repandens, while NAA was better for both T. cuspidata, and T.
x media cv. Densiformis. However, with T. x media cv. Hicksii, NAA and 2,4D exhibited similar rates of success, and was better than IBA.
Although callus induction occurred when incubated under a l6-h light/8h dark day/night regime, the amount of callus produced and the overall success
rate was higher on explants incubated in total darkness. Callus cultures initiated and maintained in darkness were generally pale yellow to light brown,
and produced masses of friable callus necessitating subculture within 3 to 4
weeks. The production of "red/brown" exudates (considered to be phenolic
compounds), which eventually led to a decline in callus growth and death, was
controlled by the inclusion of 5 to 109/1 of polyvinylpyrrolidone (PVPlO).
However, almost all the callus cultures exhibited a marked decline in growth
rates following the initial subculture, resulting in very-slow-growing clumps of
callus (Wickremesinhe and Arteca 1993a). Some of these callus cultures produced "globules" of callus after long periods (from 1 to 2 years) of very slow
or no growth, while others produced "faster-growing" callus that led to the
425
production of large amounts of calli upon subculture (Fig. 3b). A similar rapid
decline in callus growth was also noted by Zhiri et al. (1995b) with light-grown
T. baccata callus cultures.
The use of membrane rafts (Sigma Chemical Co., St. Louis, Missouri,
USA) was beneficial for callus cultures because the calli could be maintained
longer without the need of subculture, and because the medium could be
changed by merely pipetting out the used medium and adding fresh medium.
This process also facilitated the evaluation of callus growth over a longer
period of time without having to physically disturb the cultures. There were no
significant differences in the growth rates when callus grown on membrane
rafts were compared to callus grown on 100 ml of solidified media in Magenta
GA7 vessels. However, the callus could be maintained only 7 to 8 weeks
without subculture on solidified medium in Magenta GA 7 vessels, compared
to 10 to 14 weeks on membrane rafts.
Research in our laboratory has led to the establishment of a habituated
callus line derived from T. x media cv. Hicksii. Supplementing the culture
Non-habituated callus
Habituated callus
d
initial weight
final weight
r-..
'"
OIl
....
'-'
ib
'a)
~
..c::
'"~
::t::
lI"l
t:Q
.....
::t::
U
::t::
lI"l
t:Q
.....
lI"l
t:Q
lI"l
t:Q
.....
::t::
U
lI"l
t:Q
!I!
~
lI"l
t:Q
lI"l
t:Q
lI"l
t:Q
Media formulation
Fig. 4. Effect of casein hydrolysate, arginine, and glutamine on T. x media cv. Hicksii habituated
callus and non habituated callus. The experiment was conducted in Petri dishes and data collected
after 5 weeks in culture. The weights represent the mean of ten replications:+:: SE. 85 Gamborg's
B5 basal medium; CHt and CH21 and 0.1 mg/l casein hydrolysate. respectively; AAt and AA21
and 0.1 mM of both arginine and glutamine, respectively; H 1 mg/l 2,4-D and 0.2 mg/l kinetin.
(Wickremesinhe and Arteca 1993a)
426
medium with 1 gil casein hydrolysate significantly increased the growth rate of
this callus, while the addition of 0.1 gil casein hydrolysate and either 0.1 or
1 mM of both arginine and glutamine had no significant effect on callus growth
(Fig. 4). Surprisingly, none of these supplements has an effect on callus cultures maintained in the presence of plant growth regulators (Fig. 4). Also,
none of the cultures showed any significant differences in the growth rates
when placed on medium adjusted to pH values ranging from 4 to 7
(Wickremesinhe and Arteca 1991, 1993a). Zhiri et al. (1995b) also reported
the formation of T. baccata callus in hormone-free medium and attributed the
phenomenon to be due to the cell proliferation properties of casein
hydrolysate.
Sucrose was significantly better than both glucose and fructose for, habituated callus growth. Supplementing sucrose (20 gil) with both glucose and
fructose (2.5 gil each) significantly increased the fresh and dry weight of the
callus, compared to sucrose alone. Growth curves for this particular callus line
and the effect of different basal salt formulations are given in Fig. 5. B5
medium and MS medium (Murashige and Skoog 1962) were superior to the
McCown's woody plant medium (Lloyd and McCown 1981). The average
callus doubling times were 13 and 14 days on the B5 and MS medium, respectively (Wickremesinhe and Arteca 1993a). Following a similar media optimization study, Ketchum et al. (1995) reported a doubling time of 3.5 to 5.6 days
for T. brevifalia callus, using a B5 medium supplemented with fructose, sucrose, a complex vitamin mixture, and a combination of Picloram, kinetin,
GA 3, and ABA (the amounts of taxol produced by the callus were not
reported).
2.3 Establishment of Cell Suspension Cultures
Cell suspension cultures were established by placing chunks of friable
callus (approximately 10 to 15 g fresh mass) in 300-ml conical flasks containing 100 ml of liquid medium, and incubating on a gyratory shaker
at 145 rpm. Cultures were periodically passed through a stainless steel sieve
(0.76 x 0.76mm) for up to about 1 year, in order to obtain a fine cell
suspension culture (Wickremesinhe 1992; Wickremesinhe and Arteca
1994a).
The cells achieved specific doubling times (based on dry weight) ranging
from 12 to 28 days (Fig. 6). Cell suspension cultures have exhibited the need of
a minimum density at subculture, in order to continue growth. In general, this
criterion has been satisfied by maintaining a density of 109 of cells (fresh
weight) per 100ml of medium, at subculture.
Interestingly, the cells were capable of hydrolyzing sucrose extracellularly,
and preferentially utilized glucose over fructose (Fig. 7). Cells were successfully grown on sucrose concentrations ranging from 20 to 80 gil, on combinations of sucrose and fructose, and on fructose alone; however, fructose
was the sole sugar being utilized during the log phase of the growth cycle
(Wickremesinhe 1992; Wickremesinhe and Arteca 1994a). Similar sucrose
427
60
2.0
-----
60
Dry weight
-;;;- 50
!9 40
~
50
.29
~ 30
tl: 20
10
14
28 42
Time (days)
20
-e10
B5
- - - - MS
O+---.---r--.r--.---.---r---r--'---.-~
14
21
28
35
Time (days)
42
49
56
63
70
Fig. 5. Growth curves for, T. x media cv. Hicksii callus cultured on 85 medium (B5), Murashige
and Skoog medium (MS), and McCown's woody plant medium (WP) supplemented with 2X 85
vitamins, 20 gil sucrose, 2.5 gil each of glucose and fructose, and 1.0 gil casein hydrolysate. Initially
3 g of callus were placed per membrane raft, and the rafts were aseptically weighed each week.
Each data point represents the mean of five rafts :+: SE; inset growth curves for same callus
cultured on B5 medium, based on fresh and dry weight. (Wickremesinhe and Arteca 1993a)
428
300 -r--------------=----..26
24
250
]:
22
.......
bO
S
oW
:fp
200
3)
~
u
..c:
C/l
18
150
J:
16
]:
.......
~
~
~
~
u
100
14
12
16
20
24
28
32
Time (days)
Fig. 6. Growth curves for T. cuspidata cell suspension cultures grown on B5 salts supplemented
with 2 X B5 vitamins, 20 gil each of fructose and glucose, 5 gil PVPI0, 2.5 mg/l L-ascorbic acid,
3.75 mg/l citric acid, 2.5 mg/l NAA, and 0.2 mg/l kinetin. Cells were grown in 125-ml flasks and
harvested in triplicate. Each data point represents the mean ::t: SE
The addition of 2.5 mg/l L-ascorbic acid and 3.75 mg/l citric acid to the
culture medium helped minimize the production of phenolic and other "redcolored" exudates which have been known to be detrimental for cell growth,
and thereby significantly increased the number of successful cultures established. Cell lines were selected based on the criteria of faster-growth and the
lack of production of red-colored exudates. Both taxol-producing and
nonproducing cell lines have been maintained. The best cell lines achieved
specific dry weight doubling times ranging from 8 to 18 days (E.R.M.
Wickremesinhe and R.N. Arteca, unpubl.). Polyvinyipolypyrrrrolidine (PVP)
and activated charcoal have also been used to minimize the production of
phenolics in Taxus cultures (Fett-Neto et al. 1992).
Extensive studies conducted in our laboratory have shown that these cells
are extremely sensitive to cryopreservation (Wickremesinhe 1992); however,
we have been able to significantly slow down the growth rates of callus and
cells by incubating them at lower temperatures. The average doubling times
increased linearly, from 22 to 29 to 44 to 69 days, when cultures were incubated
at 25, 22, 14, and 4C, respectively (Fig. 8).
Initial attempts to scale up cell suspension cultures using 1-1 stirred jars
(Fig. 9a) were unsuccessful (Wickremesinhe 1992). However, Srinivasan et al.
429
20
80
""" 15
60
~
b.()
'-'
talO
S8
40
b.()
;::l
en
20
21
40O---------------~
40.----------------,
S2F2
10
o+----.----.---~
14
Time (days)
21
Time (days)
Fig. 7. Depletion of sugars in the culture medium containing different sugar regimes, during the
growth of T. x media cv. Hicksii cells (-0- sucrose; -e- glucose; -0- fructose). S2FG 20 gil sucrose
and 2.5 gil of both fructose and glucose; S8 80 gil sucrose; F4 40 gil fructose; S2F2 20 gil each of
sucrose and fructose. Each data point represents the mean of three replications :+: SE. Day 0
represents the analysis of medium after autoclaving. (Wickremesinhe and Arteca 1994a)
430
---.
.~
'"
.0
.<::00
60
.~
:3
'">-.
50
'<:l
.S
(I)
.
00
40
.S
:g
0
'<:l
(I)
00
30
(I)
~
20+-~~~~~~-.~~~~-r~~~~-r~~~~
10
15
20
25
30
The cells reached a final biomass of 22.3 gil on a dry cell mass basis with a
specific doubling time of 8 days, during the log-growth phase in the bioreactor.
However, there was a long lag period before the cells reached the log-grwth
phase. The log-growth phase was also signified by a rapid depletion of dissolved oxygen levels in the medium (E.R.M. Wickremesinhe and R.N. Arteca,
unpubl. data). Although we have not monitored the gas composition inside the
Celligen bioreactor, the combination of 10% (v/v) oxygen, 0.5% (v/v), carbon
dioxide, and 5 ppm ethylene has been reported to be the most effective for
taxol production in T. brevifalia suspension cultures (Mirjalili and Linden
1995).
2.4 Extraction and Analysis of Taxol and Related Taxanes from Callus
and Cell Cultures
Freeze-dried callus or suspension cells (50mg) were extracted in 2ml methanol and fractionated using Sep-pak C18 cartridges (Millipore Corp., Milford,
Massachusetts, USA) as previously described (Wickremesinhe and Arteca
1993b). Media samples (in aliquots of 25 to 50ml per sample) were filtered
through a Whatman no. 4 filter to remove cells and their debris and then
431
Fig. 9. a T. x media cv. Hicksii cells grown in a 1-1 stirred jar. b T. cuspidata cells grown in a 5-1
Celligen bioreactor
432
.-..
0.08
r-
C'l
C'l
0.06
'-'
(\)
Co)
:::
eI:I
-e
0.04
0
rJ)
.D
-<
0.02
15
30
45
Time (minutes)
Fig. 10. Separation of lO-deacetyltaxol (1), cephalomannine (2), and taxal (3) from cell suspension extracts of T. cuspidata. Analysis was performed on a 4Jl Curosil G column (4.6 x 250mm)
using a mobile phase consisting of lOmM acetate buffer (pH 4): acetonitrile (56: 44), at a flow rate
of 0.6 mllmin. The compounds were detected by monitoring absorbance at 227 nm
mass). The levels of cephalomannine also followed a similar pattern, but the
levels were always lower than those of taxol (Fig. 12). The amounts of taxol
and cephalomannine secreted into the cell culture medium ranged from 23 to
70llg/1 and 30 to 117Ilg/l, respectively (E.R.M. Wickremesinhe and R.N.
Arteca, unpubl.).
Some cell suspension cultures have exhibited a putative taxol peak corresponding to levels as high as 2S.3 mg/kg of taxol, based on HPLC analysis.
However, in reality, the actual amount of taxol represented by this putative
taxol peak seems to be only a fraction of that amount, based on analysis by
fast atom bombardment mass spectrometry (Wickremesinhe and Arteca
1994a).
Fett-Neto et al. (1995) reported that cell suspensions grown in the dark
accumulated the highest amount of taxol during the stationary phase (14mg/
kg) and the highest amount found in the medium was 0.3 mg/l, compared to
cells grown in light (6.2mg/kg in the cells and O.OSmg/1 in the medium).
They also reported that the peak of baccatin III accumulation (1S0mg/kg)
preceded the taxol peak and that the content of this taxoid decreased as taxol
increased.
Neither growing cells at lower temperatures (4 and 14C), nor the addition of fungal elicitors (Verticillium dahliae and V. albo-atrum) and heavy
433
,.....,
.~
'"o:s
.D
0
12
l:bO
10
e;.
.~
"0
'-'
bIl
S
'0
Eo-
0
0
0
10
11
12
13
14
Time (weeks)
Fig. 11. Scatter diagram depicting the variation in taxollevels observed in T. x media cv. Hicksii
callus cultured on membrane rafts. B5 medium supplement with 2x B5 vitamins, 20g/1 sucrose,
2.5 gil of both fructose and glucose, and 1 gil of casein hydrolysate was used as the culture medium.
Each week an entire membrane raft was harvested and a representative sample of that callus was
used for analysis. Data from at least five membrane rafts are represented for each weekly time
point. (Wickremesinhe and Arteca 1993a)
metals (salts of lead, mercury, copper, and vanadium), had any stimulatory
effect on the levels of taxol, cephalomannine, or any of eight related taxanes
(Wickremesinhe 1992; E.R.M. Wickremesinhe and R.N. Arteca, unpubl.).
However, Ciddi et al. (1995) reported that the addition of fungal elicitors and
arachidonic acid increased taxol production by 150%. They also noted that the
addition of copper and vanadium salts had no effect on taxol. An increase in
taxol accumulation was also reported by Fett-Neto et al. (1994a) when T.
cuspidata cell cultures were supplemented with phenylalanine and benzoic
acid.
Supplementing the culture medium with fructose and a low head-space
oxygen concentration (10% v/v) has been reported to significantly increase
taxol production (12.2 mg/l), while high carbon dioxide concentrations (10%
v/v) have been shown to inhibit taxol production (Mirjalili and Linden 1995).
Srinivasan et al. (1995) demonstrated that a five-fold increase in taxol production (1.5 mg/l) can be achieved by growing cells in 1-1 pneumatically stirred
bioreactors, compared to cells grown in 1-1 stirred tank bioreactors and 250-ml
Erlenmeyer flasks.
2.5 Purification and Identification of Taxol from Callus and Cell Cultures
Taxol was purified from callus and cell suspension cultures using a
65% methanol: chloroform partitioning followed by fractionating on CIS
434
15~--------------------------------------~
---0--
Taxo!
Cephalomarmine
12
16
20
24
28
32
Time (days)
Fig. U. Amounts of taxol and cephalomannine produced by T. cuspidata cell suspension cultures,
over a growth cycle of 32 days. The data represent the mean:!:: standard error from three 125-ml
flasks harvested at each data point. Cells were grown on B5 salts supplemented with 2x B5
vitamins, 20 gil each of fructose and glucose, 5 gil PVPI0,2.5 mg/l L-ascorbic acid, 3.75 mg/l citric
acid, 2.5 mg/l N AA, and 0.2 mg/l kinetin.
435
Cells/callus
(freeze-dried)
Extract in methanol
(homogenize and sonicate)
436
........
~
0
>.
.....
wC
80
70
Q)
.....
50
Q)
40
C!l
30
a:
20
>
'';::
Q5
569
60
10
0
Mass-to-charge ratio
Fig. 14. Fast atom bombardment mass spectra of taxol purified from T. x media cv. Hicksii callus
cultures, showing the molecular ion of taxol (M + Hr at a mass-to-charge ratio of 854
437
Fig. 15. Formation of hairy-root-like adventitious roots on T. cuspidata callus cultures grown on
B5 salts supplemented with 2x B5 vitamins, 30 gil sucrose, 5 mg/l NAA, and 0.2 mg/l kinetin. The
cultures were approximately 2 years old and maintained in total darkness
threefold higher than the levels found in the stem-bark or needles, and the
roots grow at a much faster rate compared to the above-ground parts
(Wickremesinhe and Arteca 1994b; 1996b). This, therefore, appears to be a
better alternative than collecting stem and needle clippings.
438
Fig. 16. a Cuttings of T. x media cv. Hicksii rooted hydroponically b After 22 weeks in 1120
strength Johnson's solution. (Wickremesinhe and Arteca 1994b)
have been made by both organizations; however, to date, there has been
no official statement to the effect that actual commercial production has
commenced.
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442
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1 Introduction
Rotenone and its derivatives are well known for their insecticidal properties.
They occur naturally as constituents of the roots, stems, and leaves of many
leguminous species of the genera Derris, Lonchocarpus, Tephrosia, and
Amorpha (Harborne 1971; Menichini et a1. 1982). For several centuries, these
plants have been used to prepare hunting and fishing poisons. More recently,
rotenoids have become of much interest because of their selectivity and low
environmental hazard. They are highly toxic to insects and lower animal
forms; but relatively nontoxic to plants and mammals. They offer a distinct
advantage over synthetic insecticides because they are biologically active at
basic metabolic levels, less likely to lose their effectiveness through the development of tolerance, and are readily degraded in the environment to nontoxic
organic acids (Moring and McChesnay 1979).
At present, rotenone is also used in laboratory experiments on animal and
plant mitochondria (Ravanel et a1. 1984) because of its inhibitory properties
on complex I.
Tephrosia vogelii Hook f., a tropical member of the Papilionaceae originally from Angola (Fig. 1), has been traditionnally cultivated in West Africa
for its ichtyotoxic and parasiticidal activities (Kerharo and Bouquet 1950). It is
a shrub, branched from the basis, perennial, between 2m (the 1st year) to 3m
(2nd and 3rd years after cutback). The alternate leaves, between 20 to 25 cm,
are imparipennate with about ten pairs of leaflets. The white, sometimes
purple, flowers are grouped in terminal racemes, and the flat pods (between 10
to 12 cm) contain elliptical, black grains.
This plant produces a great variety of rotenoids which can reach 4 % of the
dry matter (Irvine and Freyre 1959). Rotenone and deguelin are the major
compounds present (Fig. 2).
It constitutes a promising potential source of rotenoids and should enable
developing countries to become self-sufficient in biodegradable insecticides
Laboratoire de Physiologie et Biotechnologie Vegetales. ORSTOM-IIRSDA, BP V51, Abidjan,
Cote d'Ivoire
2 Present address: Analytical Product Division, Millipore S.A., BP 307, 78054 Saint Quentin-enYvelines Cedex, France
.l Laboratoire de Ressources Genetiques et Amelioration des Plantes Tropicales, ORSTOM, BP
5045, 34032 Montpellier, France
I
444
N. Lambert et al.
"c
~i
(3)Rotenone
11
OCHJ
OCH3
OCH1
0T~ I
OCH J
~ I
OH0
(l)Rotenolone
11
CH J
CH
y
0
OH
(4) Deguelin
(2) Tephrosin
Fig. 2. Structure of the major rotenoids isolated from Tephrosia vogelii leaves
CH"
C,
,f'
CH2
CH2
CH J
Y-OCH 3
OCH J
OCH3
~ Y-OCH3
-0
u.
'""
;.;-
o
o
E:
::r::
'"
E;'
-e
is
~
;:,-
446
N. Lambert et al.
2 In Vitro Approaches
The production of rotenoids from tissue cultures of Tephrosia vogelii was first
reported by Sharma and Khanna in 1975. They established callus and cell
suspension cultures on revised MS medium (Murashige and Skoog 1962)
supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D). Total rotenoid
content was maximum in 4-week-old static cultures, and gradually decreased
in 6- and 8-week-old tissues. Rotenoid contents reached 2.8% in cell suspension culture. Presence of deguelin, elliptone, and tephrosin was confirmed by
thin layer chromatography (Sharma and Khanna 1975).
2.1 Establishment of the Cell Lines
Fig. 3A,B. Two-week-old chlorophyll-containing (A) and heterotrophic (B) callus cultures on
modified MS medium
447
448
N. Lambert et al.
sC
0
00
2!
(J:l
()
...
..,
(/J
10
Time (min)
20
10
20
Time (min)
Fig. 4. HPLC analysis of leaf extract (left chromatogram) and callus cultures (right
chromatogram) from Tephrosia vogelii. Each peak number corresponds to its compound number
the leaves (Fig. 4). However, rotenone appeared to be the main rotenoid
throughout the culture cycle (Fig. SB). Its production rate was approximately
four- to sixfold higher than that of the other rotenoid compounds. Its highest
concentration was observed after 20 days of culture (S70.ugg-ldrywt.).
Rotenolone and tephrosin were produced in lower amounts, their concentration being always lower than SO.ugg -1 drywt.
In the heterotrophic cell suspension cultures (Fig. 6A), deguelin was the
major rotenoid produced, as in the heterotrophic callus cultures. However,
tephrosin was also found in large amounts, its concentration being equivalent
to deguelin. Here also, rotenolone was not detected, although it was the major
rotenoid produced by the photomixotrophic cell suspension culture (Fig. 6B).
Rotenone was also actively synthetized by these cells. For both cell lines grown
449
Rotenolone (1)
Tephrosin (2)
V Rotenone (3)
T Deguelin (4)
600
600
A
500
500
400
400
300
300
200
200
'cD
........
~
....C
....c
Q)
(.)
~C
....
V-V
Q)
100
100
-T-T-T
10
20
30
Time (days)
40
10
20
30
40
TIme (days)
Fig. SA,B. Rotenoid production in heterotrophic (A) and chlorophyll-containing callus (B)
cultures
in liquid medium, rotenoid synthesis was increased during the growth stage
with maximum concentrations of the compounds appearing at the end of the
phase of growth. The rotenoid content was also determined in the culture
medium of the two cell lines (data not shown). The rotenoids were poorly
released and only deguelin was found in large amounts.
The rotenoid profile of the chlorophyll-containing cultures was compared
to the leaf production. Although leaves accumulated rotenone, deguelin,
and their derivatives, heterotrophic cell cultures were able to produce only
deguelin and tephrosin, while photomixotrophic cell cultures accumulated
preferentially rotenone. It seems that the biosynthesis of rotenone and
rotenolone is stimulated by photomixotrophic conditions. It is an open question whether light or photosynthesis plays a role in directing the biosynthesis
pathway towards rotenone or rotenolone, rather than towards tephrosin and
deguelin. The common precursor to deguelin and rotenone is rot-2'-enonic
acid (Crombie et al. 1982,1986). Two different enzymes are responsible for the
cyclization process which produces rotenone and deguelin. Rotenolone is an
oxydation product of natural rotenone while tephrosin is derived from
deguelin. It is possible that the relative importance of the second pathway
may vary with culture conditions. There is another possibility that the lack of
N. Lambert et al.
450
o Rotenolone (1)
TephroS1n (2)
V Rotenone (3)
'f' Deguelin (4)
40
40
0\
gj
~30
30
~
.....
t::
Q)
.....
t::
I e-:
'f'
820
1.\
'0
"0
",'v
20
'f'/\ \
t::
Q)
.....
0
0:: 10
-.
~I'I
-'f'/
10
".~
~V-V-V-
10
12
Time (days)
10
12
Time (days)
Fig.6A,B. Rotenoid production in heterotrophic (A) and photomixotrophic (B) cell suspension
cultures
451
centrifuged at 20000g for lOmin and the resulting supernatant was used as
enzyme preparation. All steps were performed at 2 0c. PAL activity was
determined spectrophotometrically according to published methods (Bolwell
et al. 1985). Protein was determined by the method of Sedmack and Grossberg
(1977) .
2.5 Biotic Stress
to
D E I (0 Jil)
4H
'? 2
ea.
. iii
24 H
48 H
1\ EI (100 !-II)
EI (500 !-II)
00
E 1.5
452
N. Lambert et al.
accumulation in the cell was monitored for 2 days (Fig. 7A). In the
treated cells, rotenoid content increased after a lag period of 4h and
reached a maximum 48h after treatment. The extent of the increase in
the rotenoid content was about threefold, relative to the content of the
controlled cells. About 60-80% of the rotenoid contents was found in
the medium of treated cells compared to 10% for the control (data not
shown). Some workers recorded drastic changes in the cell wall permeability
after elicitation (Darvill and Alberscheim 1984). The activity of PAL, the
entry point enzyme of the phenylpropanoid pathway, rapidly and transiently
increased after the treatment (Fig. 7B). The maximum was detected 4h
after addition of autoclaved mycelia, which correlated well with the accumulation of rotenoids in the elicitor-treated cells. The extent of the increase
in PAL activity was about fourfold higher in the treated cells than in the
control. Increase of PAL activity after elicitor treatment with subsequent
phytoalexin synthesis has been notably reported in soybean cells (Ebel et al.
1986).
2.6 Abiotic Stresses
453
on
on
2:
g
<.)
-0
'0
c
<>
'0
a::
o Control
O pH 5.0
4H
24 H
48 H
pH 5.5
.pH 6.0
,.-..
'0;
a.
~
-;
'E
E
,:q
>
.~
'"
....J
c..
4H
24H
48H
3 Conclusions
Heterotrophic and photomixotrophic cell suspension cultures of Tephrosia
vogelii are both able to produce rotenoids, but a specific production is observed in each culture, The photomixotrophic condition of the culture (light
essentially) promotes the production of rotenoids, and notably rotenone,
which are of economic interest.
The present investigation also provides another example where the spectra of secondary constituents of photomixotrophic and heterotrophic cell
cultures are significantly different. Therefore, depending on the type of com-
454
N. Lambert et al.
References
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Crombie L, Holden I, Kilbee GW, Whiting DA (1982) Biosynthesis of rotenoids by Amorpha
fruticosa: sequence, specificity and stereochemistry in the development of the hemiterpenoid
segment. J Chern Soc Perkin Trans 1:789-797
Crombie L, Rossiter J, Whiting DA (1986) Biosynthesis origin of the 2,2-dimethylchromen ring:
formation of deguelin by a cyclase enzyme from Tephrosia vogelii. J Chern Soc Chern Commun
352-353
Darvill AG, Alberscheim P (1984) Phytoalexins and their elicitors. A defence against microbial
infections in plants. Annu Rev Plant Physiol 35:243-275
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phenylalanine ammonia-lyase and chalcone synthase mRNAs correlation with phytoalexin
accumulation. Arch Biochem Biophys 232:240-248
Hahlbrock K, Grisebach H (1979) Enzymatic controls in the biosynthesis of flavonoids and lignin.
Annu Rev Plant Physiol 30:105-130
Hanson KR, Havir EA (1981) Phenylalanine ammonia-lyase. In: Stumpf PK, Conn EE (eds) The
biochemistry of plants. A comprehensive treatrise, vol 7. Academic Press, New York, pp 577625
Harborne JB (1971) Isoflavones and isoflavonoids. In: Harborne JB, Boulter D, Turner BL (eds)
Chamotaxonomy of the Leguminosae. Academic Press, London, pp 31-71
Hardy T, Chaumount D, Brunei L, Gudin C (1987) Photo autotrophic suspension cultures. I.
Obtention of photo autotrophic cultures from Euphorbia characias L. J Plant PhysioI128:1119
Hattori T, Ohta Y (1985) Induction of phenylalanine ammonia-lyase activation and isotlavone
glucoside accumulation in suspension-cultured cells of red-bean, Vigna angularis, by
phytoalexin elicitors, vanadate and elevation of medium pH. Plant Cell Physiol 26:11011110
Irvine JE, Freyre RH (1959) Occurrence of rotenoids in some species of the genus Tephrosia.
J Agric Food Chern 7:106-107
Jones DH (1984) Phenylalanine ammonia-lyase: regulation of its induction, and its role in plant
development. Phytochemistry 23:1349-1359
Kerharo J, Bouquet A (1950) Plantes medicinales et toxiques de la Cote d'Ivoire-Haute Volta.
Vigot freres, Paris, pp 2-4
455
Subject Index
~-copaen-4a-ol
306
6-benzylaminopurine
41
antineoplastic 398
antipyretic I, 28, 83
antitermite 387
antitumor 288,291,368,418
antiulcer 83
antiviral 368,387,398
aphrodisiac 240
arbutin 67,75
Arnebia euchroma 28 pp.
aromatic aldehyde 197
artocarpesin 272
artonin 272
asiatic acid 84
asiaticoside 84
asthma 367
astringent I
atherosclerosis 366
baccatin 418
batch culture 178
Bellflower 45
benzaldehyde 203
benzoquinones 30
bioreactor 134, 137, 176,232,429
biotic stress 451
biotransformation 67,81,88,194,341,
406
Boraginaceae 14,28
borneol 349,357
bracts 113
breast cancer 368,418
bud culture 63
callus 5
culture 18,119,247,337,400,
423
Campanulaceae 45
campanin 57
Campanula 45 pp.
campedin 46
camphene 349
~
Subject Index
458
camphor 349
carcinogenesis 350
cardoon 132
carotenoids 2
carvone 213
carvotanacetone 211
casein hydrolysate 425
Catharanthus 67
cathinone 164
cell
- culture 77, 100, 134
- selection 22
- suspension 99,119,135,137,172,247,
297,421
Centella asiatica 81 pp.
cepha10mannine 418
chalcone 279
charcoal 19,118,311,376,428
cheese-making 132
Chenopodiaceae 97
Chenopodium album 97 pp.
cholesterol 106,342,396
chrysophano1 384
clonal propagation 47
cloning 35,43,266
conventional propagation 240
Comaceae 113
Cornus kousa 113
coumarin 33,48,238
cryopreservation 266
culture 30
- studies 5
Cynara cardunculus 132 pp.
cyanidin-3-glucoside 330
cyclopentanoid monoterpenes 4
cynarin 135
cyprosin 135, 138, 142
cytokinin 331
d-carvone 210
d-limonene 210
d-pseudoephedrine 155
deguelin 443,445
de1phinidin 57
Diels-Alder-type adducts
differentiation 7, 41
dihyronepeta1actone 8
diosgenin 396
diuretic 1, 261, 367
Dogwood 113
ecdysone 3, 344
ecdysteroids 97,99, 107
279
effect
- ofBA 360
- of growth regulators 360
- of sucrose 356
- TDZ 360
e1ectroporation 136
ELISA 422
embryogenesis 134,253
emodin 384
enantiose1ectivity 225
Ephedra 154
ephedrine 154
epigenetic 42
epilepsy 45,416
essential oil 305, 345
ethy1vanillin 206
Euglena gracilis 194
Evening Primrose 286
Fat Hen 97
fatty acids 291
fermenter 382
feromone 305
flavones 57
flavonoids 286
flower bud cultures 51
formagram 141
fruit gall 6
fungal elicitors 433
fungicidal 350
furocoumarins 257
Fusarium solani 264
gallic acid 113
gene expression 134, 136
genetic
- stability 300
- transformation 54
- variation 50
growth regulators 20,40,52, 123,331
GUS genes 55
hairy root 54,374
- culture 50,54,63374,380
haploids 187,374
Haplophyllum patavinum 238
headache 45
hemiterpene 278
hemostatic 113
heterotrophic 453
HPLC 57,101,157,162,255,279,346,
381,422,434
hydrophobia 416
hypertension 366
Subject Index
immobilisation 179
immunological properties 138
in vitro 14, 67
- approaches 49,85,115,241,293
- culture 28,45,113,135,154,261,305,
320,415,443
- flowering 328
- propagation 248, 309
- studies 241,264,308,322,335,353,
399
inflammation 45
inokosterone 338
insecticidal 443
iridomyrmecin 2
juglone
386
kaempferol 288
kuwanol 261,279
kuwanon 264
Labiatae 349
lactones 2
lathosterol 342
leprosy 83
lignans 379
linalool 351, 359
linolenic acid 289
lobelin 46
lobetyol 61
lobetyolin 61
lobetyolinin 61
lupane 410
madasiatic acid 84
madecassic acid 84
madecassoside 84
malaria 416
mammilaries 395
mass culturing 33
Matatabi 1
matatabiol 3
media 252, 316
medicinal importance 83
micropropagation 1,7,18,184,250,261,
265,281,294,300,320,322,401
milk-clotting enzymes 132
molting hormones 99,333
monoterpenes 1,352
- aldehydes 197
- ketones 208
monoterpenoids 3
Moraceae 261
459
moracin 279
morin 261
Morus 261 pp.
Morus alba 270
Morus nigra 270
mulberrin 264
mulberrofuran 261,279
Mulberry 261
multiple sclerosis 288
multiple shoots 98, 292, 296,
myrtenal 200
naphthoquinone
15,29,366,381,
obesity 156
oenothein 290, 293
Oenothera species 286 pp.
oeparol 287
oil 291
- cells 307
- content 313
organogenesis 182,250,327
osthenol 255
Otacanthus species 305
ovarian cancer 418
Oxalidaceae 320
Oxalis species 320
Paduan rue 238
PAL 164,447
papaverine 87
Papilionaceae 443
papveraldine 93
parasiticidal 443
peR analysis 55
Pedaliaceae 366
pedaliin 378
Pennyworth 81
Periwinkle 67
PFP-resistant cells 39
phenolic acids 288
phenolic compounds 113,288
photomixotrophic 453
phylloquinone 387
phytoalexin 264
phytoecdysteroids 333
phytol 3
phytosteroid 397, 405
phytosterol 97
picloram 426
pigment production 21
pinoresinol 370
plant growth regulators 310
460
plant regeneration 294,328
platyconin 57
plumbagin 389
polyacetylene 45,61
polyphenol 120, 127
Polypodiaceae 333 pp.
polypodine 101,338
Polypodium vulgare 333
progesterone 406
prothallus 325,328,335,374
pterosterone 338
quinine 114
quinoline 240
retenoids 443
rheumatism 45,416
root cultures 18, 24
rooting 268
Rosemary 349
Rosmarinus officinalis 349 pp.
rosmariquinone 350
rotenone 443,445
rubrocampanin 57,59
Rutaceae 238
rutin 264
saponin 98
scanning electron micrographs 307
scopoletin 241
Scrophulariaceae 305
secondary mataboites 14,28,45,57,97,
99,135,264,271,286,366,376,394,443
Sesame 366
sesamin 378
sesaminol 368
sesamol 368
Sesamum indicum 366
sesangolin 368
sesquiterpenoids 305
shikimate pathway 275
shikonin 14,28
shisool 232
shoot 250
- buds 184
- cultures 50,117,128,401,410
- differentiation 7
- multiplication 267
- regeneration 327
- -tip culture 421
silkworm 261
sitosterol 399
Subject Index
sodium alginate beads 179
Solanaceae 394
Solanum mammosum 394
solasodine 395
somaclonal variation 326
somatic embryogenesis 56,115,136,251
sporophyte 337
steroidal alkaloids 394
steroids 2
sterol 48, 99
stigmastenol 106,399
sucrose 52
sugars 70
suspension cultures 55, 125,247,376,
406,410
syprosins 132
tannins 113,288
taxanes 418
taxol 415
Taxus species 415
Tephrosia vogelii 443 pp.
tephrosin 445
terpene
- aldehydes 196
- epoxides 196
- hydrocarbon 233
- ketones 196
terpenoids 81, 194
thidiazuron 330, 354
thiocolchicine 89
tissue culture 256
tissue culture studies 49
TLC 106, 157,255,279
tocopherol 368, 379
tomatidinol 396
torehalose 195
transformation 374
triterpene 48, 83
triterpenoid 1, 405
Umbelliferone
241,255
valencene 316
vanillin 206
violdelphin 57
vitamin E 195,373
volatile components 313
volatile oil 308
WoodFem
Yew
415
333