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To cite this article: Reiko Inatani, Nobuji Nakatani & Hidetsugu Fuwa (1983) Antioxidative Effect
of the Constituents of Rosemary (Rosmarinus officinalis L.) and Their Derivatives, Agricultural and
Biological Chemistry, 47:3, 521-528
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Agric. Bioi. Chem., 47 (3),521-528,1983 521
Antioxidative activity was measured on extracts of rosemary (Rosmarinus officinalis L.), one of
the herb spices belongs to the Family Labiatae, using different solvents. Strong activity was
observed in the weakly acidic fraction of the n-hexane extract, which was further fractionated and
purified to afford several active compounds. An ordorless and colorless compound, named
rosmanol, showed high antioxidative activity in both lard and linoleic acid, and particularly in lard,
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was about four times more active than synthetic antioxidants, such as BRT and BRA.
Furthermore, the antioxidant activities of the derivatives of rosmanol and carnosol were measured
by the ferric thiocyanate method and the TBA method, to observe the correlation between chemical
structure and activity as an antioxidant.
Various efforts have been made to preserve especially strong antioxidative activity. In
the quality of foods. In particular to depress 1969, Brieskorn et al. S ) presumed that the
rancidity of fats and oils, synthetic antioxi- antioxidative effect of rosemary was based on
dants such as tert- butyl-4-hydroxyanisol (BHA) carnosic acid and carnosol. However, Chang
and tert-butyl-4-hydroxytoluene (BHT), and et al. 6 ) have recently suggested that other
natural antioxidants such as tocopherols, are antioxidants exist in rosemary and sage, but
widely used. However, the use of BHA and they have not yet reported the isolation and
BHT as food additives is restricted in several structural determination of the active com-
countries, because these compounds seem to ponents. Very recently we found a new anti-
cause an undesirable effect on the enzymes of oxidative compound, named rosmanol, from
liver and lung. Consequently, we have directed rosemary and reported its structure. 7 ) We de-
our attention towards edible plants as re- scribe here the antioxidative effect of rosma-
sources of safer and more effective natural nol (1), carnosol (2) and their derivatives.
antioxidants, and have especially investigated Three methods were adopted for the anti-
the anti oxidative effect and chemical structure oxidative assay. The ferric thiocyanate and
of the components of spices. thiobarbituric acid methods represent respec-
Spices are generally used to flavor and sea- tively the amount of hydroperoxide of linoleic
son foods. In addition some spices have been acid at an early stage, and the production of
applied for their antiseptic activity and have carbonyl compounds degraded from the per-
been utilized in the storage of meat from early oxide at a later stage in an aqueous ethanolic
times. Chipault et al. l ,2) and Palitzsch et al. 3 ,4) solution. The third method was used the active
measured the antioxidative effect of many oxygen method, which indicates the peroxide
kinds of spices, and reported that the spices value in an oil system.
belonging to the Family Labiatae have an
t This study was presented at the Annual Meeting of the Agricultural Chemical Society of Japan, Tokyo, April
1982.
* Address correspondence to this author.
522 R. INATANI, N. NAKATANI and H. FUWA
OH OH
2 3
CH30~OCH3 CH31A-rr---<C-O
W'l::::::../H
OH OH
4 5
MATERIALS AND METHODS after the addition of O.l ml of 2 x 10- 2 M ferrous chloride
in 3.5% hydrochloric acid to the reaction mixture, the
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Extraction of rosemary. Dried and ground (20-60 absorbance of the red color developed was measured at
mesh) leaves of rosemary (500 g) from France were extract- 500nm.
ed with n-hexane (1200ml) under stirring at room tem- 2. Thiobarbituric acid method (TBA).!!) To 2ml of the
perature. This procedure was repeated three times. The sample solution, which was prepared and incubated as
combined extracts were freed from the solvent to yield up discribed above, was added 2 ml of 20% trichloroacetic
to 19.7 g from the n-hexane extract. The residual leaves acid aq. solution and 1 ml of 0.67% thiobarbituric acid aq.
were successively extracted with CH 2Ci2 four times solution. This mixture was placed in a boiling water bath
(1500ml each) and then EtOH twice (1500ml each) to give for 10 min and, after cooling, was centrifugated at 3000
36.8 g and 60.7 g, respectively. Both the hexane and the rpm for 10 min. The absorbance of the supernatant was
CH2Cl2 extracts were separately steam distilled to give the measured at 532 nm.
volatile and the non-volatile fractions. Each non-volatile 3. Active oxygen method (AOM). The antioxidative
fraction was fractionated into the basic, the strongly effect in lard was determined by the Tentative Method Cd
acidic, the weakly acidic and the neutral fractions in the 12-57, of the American Oil Chemists' Society (A.O.C.S.).
usual way.8) The EtOH extract was divided into the Lard (20 g) and the sample in a test tube (25 x 200 mm)
benzene soluble and insoluble fractions. were kept at 98C in an oil bath under airation (2 ml/sec),
and the peroxide values were measured by the A.O.C.S.
Chromatographic fractionation of the weakly acidic com- Official Method Cd 8-53.
ponents. The weakly acidic components of the n-hexane
extract were subjected to chromatography on a silica gel Samples for antioxidative assay.
(Merck Kiselgel 60) column with benzene-acetone as the 1. Rosmanol (1). Rosmanol was obtained from fraction
elution solvent system to separate into twelve fractions. Nos. 8 and 9 by repeated column chromatography on
The antioxidative activity of these fractions was measured silica gel as previously reported,7) mp 241C.
by the ferric thiocyanate method and thiobarbituric acid 2. Carnosol (2). Carnosol was isolated from fraction
(TBA) method. The antioxidative activity was also as- Nos. 6 and 7 by recrystallization from acetic acid, mp
sayed by the active oxygen method (AOM) on five groups, 233C. 7 )
e.g. groups I, II, III, IV and V comprising the chromatog- 3. Rosmadial (3). Rosmadial was obtained from frac-
raphed fraction Nos. 1-5, 6-9, 10, 11 and 12, tion No.4 by repeated chromatography and recrystallized
respectively. from benzene, mp 225C.!2)
4. Flavone I (5-hydroxy-7,4'-dimethoxy flavone) (4).
Antioxidative assay. The fraction Nos. 3 and 4 were rechromatographed on a
1. Ferric thiocyanate method. The methods of Mitsuda et silica gel column using dichloromethane as the elution
al. 9 ) and Osawa et al.lD) were slightly modified. Briefly, a solvent to afford pale yellow crystals of flavone I, which
mixture of a 2ml sample in 99.5% ethanol, 2.052ml of were recrystallized from benzene, mp 172C. MS m/z 298
2.51 % linoleic acid in 99.5% ethanol, 4ml of 0.05 M (M+, base), 269, 255,166,135,132.
phosphate buffer (pH 7.0) and l.948 ml of water was 5. Flavone II (5,4'-dihydroxy-7-methoxy flavone) (5).
placed in a vial (4)=38, h=75mm) with a screw cap and The fraction No.8 was triturated with a large amount of
placed in a oven at 40C in the dark. To 0.1 ml of this acetone, and the mixture was filtered. The insoluble matter
sample solution was added 9.7 ml of 75% ethanol and was recrystallized from DMF to give pale yellow needles,
0.1 ml of 30% ammonium thiocyanate ..Precisely 3 min mp 273C. MS m/z 284 (M+, base), 255, 241, 167, 166,
Antioxidative Effect of the Constituents of Rosemary 523
121, 118. Nos. 32~45 and 54-75 gave compounds 13 and 14,
6. Diacetyl carnosol (6). To a solution of carnosol (2) respectively. Compound 13: mp 180C (isopropyl ether),
(70 mg) in pyridine (0.5 ml), acetic anhydride (0.5 ml) was UV Amax (EtOH) nm (loge): 209 (4.43),227 sh. (4.12), 287
added and the mixture was allowed to stand at 38C (3.38). IR Vmax (Nujol) cm- I : 3400, 1745, 1250, 1220, 1200,
overnight. The reaction mixture was worked up in 1170,1158,1110,1025. NMR (CDCI3 ).5: 0.95 (3H, s), 1.04
the usual manner to afford diacetyl carnosol (87 mg), (3H, s), 1.21 (3H, d, J=7.2Hz), 1.25 (3H, d, J=7.2Hz),
crystallized from n-hexane as colorless prisms, mp 1.4-2.3 (5H, m), 2.15 (lH, s), 2.8-3.5 (3H), 3.80 (3H, s),
158C. 4.57 (lH, d, J= 3.6 Hz), 4.75 (lH, br d, J=3.6Hz), 5.98
7. Methyl derivatives (7, 8, 9 and 10) of carnosol.I 2 ) To a (1H, s), 7.08 (lH, s). MS m/z 360 (M+, base), 316, 301,
cooled solution at 5C of carnosol (2) (1.32 g) in dry 287, 273, 259, 245. Compound 14: mp 196C (isopropyl
acetone (50ml) were added methyl iodide (IOml) and ether). UV Amax (EtOH) nm (loge): 208 (4.51), 226 sh
anhydrous K 2C0 3 (3 g). This mixture was stirred for 6 hr (4.18),289 (3.47). IR Vmax (Nujol) cm- I : 3500, 3450,1760,
at 7 - 10C. After filtration of this reaction mixture, the 1250, 1220, 1172, 1120, 1078, 1048, 1000. NMR (CDCI3 )
filtrate was concentrated in vacuo. Ether and H 20 were 15: 0.93 (3H, s), 1.02 (3H, s), 1.21 (6H, d, J= 6.6 Hz),
added to the residue and the mixture was shaken. The 1.4-2.5 (2H), 2.22 (lH, s), 3.0-3.5 (3H), 3.74 (3H, s),
organic layer was dried over anhydrous MgS04 and 4.55 (lH, d, J=3.6Hz), 4.75 (lH, br d, J=3.6Hz), 6.12
concentrated to dryness. The product (1.4 g) was purified (1H, s, D 20 exchangeable), 6.87 (IH, s). MS (m/z): 360
by chromatography on a silica gel column using n-hexane- (M+, base), 316, 301, 287, 273, 259, 245.
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>-
o
"-
..... WA fro (non V.).
insol. fro
25 50 5 25 50 25 50 hr
FIG. 1. Antioxidative Effect from Extracts of Rosemary with Different Solvents. (AOM)
Cont., control (without additives); SA fr., strongly acidic fraction (0.01%); WA fr., weakly acidic fraction
(0.01 %); Neut. fr., neutral fraction (0.01 %); non V., non-volatile fraction.
524 R. INATANI, N. NAKATANI and H. FUWA
tration of 0.01 % into fresh lard. As shown in extract. As shown in Fig. 2, the antioxidative
Fig. 1, the volatile fraction, the strongly acidic activities of groups I, II and III were higher
and neutral fractions of non-volatile com- than that of a-tocopherol when measured by
ponents from the n-hexane and dichloro- AOM. In particular a remarkable activity was
methane extracts had no activity, and neither observed in group II. In addition, the activity
had the ethanol extract. On the other hand, the of the fractionated weakly acidic components
weakly acidic fraction of the n-hexane extract (fraction Nos. 1 ~ 12) was examined by the
showed a strong antioxidative activity and that ferric thiocyanate method, as shown in Fig. 3.
of the dichloromethane extract showed a slight Fraction No. 1 had no activity, but the other
activity. fractions were effective. Especially, strong ac-
tivity was observed in the fraction Nos. 2, 3, 4,
Antioxidative effect of fractionated weakly aci- 6, 8, 9, 11 and 12. Also similar results were
dic components n-hexane extract obtained when the TBA values were measured
The groups I, II, III, IV and V were ob- on the 7th day (Fig. 4). This weakly acidic
tained by chromatography from the non- fraction may have contained several com-
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V\ 1/ ;-TOC. Cont.
\
5: Cont I 50
~
'-
0-
W
E
I
>
o
"-
"'-II
50 hr 25 50 hr
FIG. 2. Antioxidative Effect of Fractionated Weakly Acidic Components of n-Hexane Extract. (AOM)
0.0.
2.0 0'41L- Cont. / Fr. 5
~
A/Fr.3
-==-=-_Fr.4
o~!:::~~~~~=:! - Fr. 2
2 4 6
...- Fr. 7
~:~~: 8,9
6
1.0
$
10
12
BHA 11
~~~~e~8! BHT
2 4 6 day
FIG. 3. Antioxidative Effect of Fractionated Weakly Acidic Components of n-Hexane Extract. (Ferric
Thiocyanate Method)
Each fractionated sample, conc. 0.02%; BHA, BHT and a-Toc., conc. 0.01%.
Antioxidative Effect of the Constituents of Rosemary 525
Antioxidant effect of constituents of rosemary TBA methods. From the results of measure-
From the fraction Nos. 6 and 7, carnosol (2) ment by AOM at a concentration of 0.02%
was isolated as the main compound of the shown in Fig. 5, the antioxidant activities of
weakly acidic fraction. Rosmanol (1) was ob- carnosol and rosmanol are much higher than
tained from the fraction Nos. 8 and 9, and those of (X-tocopherol, BRA and BRT. At a
rosmadial (3) from the fraction Nos. 3 and 4. lower concentration (0.01 %), carnosol showed
Furthermore, the fraction Nos. 3", 4 and 8 '" 9 an activity comparable with that of BRA
afforded flavone I (4) and flavone II (5), (0.02%). Rosmanol was more active than car-
respectively. The activities of these compounds nosol and the synthetic antioxidants. The ac-
were measured by the AOM, thiocyanate and tivity of rosmanol at a concentration of
0.005% was still stronger than that of the
0.02% concentration of BRA and BRT. By a
BHT
calculation simply based on the concentration,
BHA
rosmanol has an antioxidative activity four
'" -Toc. times more strong than that of BRA. On the
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o>
Co.
0.02%
5 25 50 25 50 25
-Flavone I
1.0
__ Rosmadial
a-Toe.
::::: Rosmanol
I~~~~~~~~~~t- Carnosol
o f = - - - BHT
2 4 6 day
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BHT
a- Toe.
Carnosol
Rosmanol
Rosmadial
F1a von e :':':':':':':'.:.:.:.:.
Flavone II ~:.:~.:.:~.:.:.~:.:.~:.:.~:.:.:~.:.:~.:.:~.:.:.~:.:.;:':'~:':':~':':~':':~':':';:':'~: : :~: :'~:':'~:':~J
Control ~.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:':':':':':':::1
FIG. 6. Antioxidative Effect of Constituents of Rosemary. (Ferric Thiocyanate Method and TBA)
Isolated compounds, cone. 0.02%; BHT and ex-Toe., cone. 0.01%.
oxidative activity, while the O-monosubstituted adjacent to an isopropyl group, had a higher
derivatives (D and F) were as strong as car- value than that of E'.
nosol (A) at a concentration of 0.02%. There
was no difference in the activity between the
CONCLUSION
compound with a lactone ring in the molecule
and that without it. Especially, compound F Antioxidative activity was measured for the
was particularly effective. extracts of rosemary with different solvents.
Strong activity was observed in the weakly
Antioxidative effect of rosmanol derivatives acidic fraction of the n-hexane extract, which
As shown in Fig. 8, triacetyl (B') and di- was further fractionated and purified by col-
methylrosmanol (C') showed weak activity umn chromatography on silica gel to afford
and the monomethylated compounds (D' and several active compounds. Amongst these, car-
E') were as strong as rosmanol (A') at a nosol and the new antioxidant rosmanol show-
concentration of 0.02%. However, at a 0.01 % ed remarkable activity. Rosmanol especially
concentration, the activities of D' and E' demonstrated an activity more than four times
decreased to one third that of A'. When the higher than BHA and BHT by AOM. This
effectiveness of D' was compared with that of compound is odorless and tasteless. The anti-
E', D', in which a methoxy group is located oxidative effect of several derivatives of these
Antioxidative Effect of the Constituents of Rosemary 527
1.0 0.02%
A P
c :::.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.
D~
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0.5 E ::::::.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:
F b
cant ..:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:
o'--.......- -.......----r- o 05 1.0 O.D.
3 5 day
0.01%
AI2J
B :.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.::::::::::.:.:.:.:.:.:.:.:.:.:.:
C :.:.:....:..:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.::::3
D.:.:.:.:.:.:.:.:.:.......
0.5
E ..:.:.:.....:.:....:.:...:.:.:.:.:.:.:.:.:.:.:.:.:.::::::::::::i
_F F m
~::::"---==::::::=:=--"""A cont.i:o:.::r.'.:.::::::::.:::::::::::::::::::::::::::::::.:.:::::::.:::::.:.:::::.:.:::::.:.:""'.:.:""'.:.:"'.:.:"'.:.:"'.:.:"'.:.:""'.:.
o~ .......---~---~- o 0.5 1.0
3 5 day 0.0.
(Ferric Thiocy~nate Method) (TBA, 6th day)
diterpenes was also measured in the aqueous O. Lundberg, Food Res., 17, 46 (1952).
ethanolic solution. The fully O-substituted de- 2) J. R. ChipauJt, G. R. Mizuno and W. O. Lundberg,
Food Technol., 10, 209 (1956).
rivatives showed negligible activity, while the
3) A. Palitzsch, H. Schulze, F. Metzl und H. Bass, Die
activity of the mono-substituted ones was as Fleischwirtshaft, 49, 1349 (1969).
strong as that of original compounds, ros- 4) A. Palitzsch, H. Schulze, G. Lotter und A. Steichele,
manol and carnosol. Die Fleischwirtschaft, 54, 63 (1974).
5) C. H. Brieskorn und H. J. Domling, Z. Lebensmitt-
Acknowledgments. We are grateful to Dr. T. Osawa, Untersuch, 141, 10 (1969).
Nagoya University, for valuable suggestions on the 6) S. S. Chang, B. O. Matijasevic, O. A. L. Hsieh and C.
measurement of antioxidant activity. We are indebted to L. Huang, J. Food Sci., 42, 1102 (1977).
Dr. T. Yoshida and Mr. H. Kanno, Takasago Perfumery 7) R. Inatani, N. Nakatani, H. Fuwa and H. Seto,
Co. Ltd., for technical co-operation on AOM measure- Agric. Bioi. Chern., 46, 1661 (1982). The stereochem-
ments and for suppling rosemary. istry of the OH group on C-7 of rosmanol has been
revealed to be an ex-configuration as the structure 1
REFERENCES on the basis of a reexamination of the NOE analysis.
This result will be reported soon in this journal.
1) J. R. Chipault, G. R. Mizuno, J. M. Hawkins and W. 8) N. Nakatani, R. Inatani and H. Fuwa, Agric. Bioi.
528 R. INATANI, N. NAKATANI and H. FUWA
0.0.
0.02%
1.0
A' ~
B' .;.;.;.;.;.;...;.;.;.;.;.;.;.
C' .;.;.;.;.;.;.;.;.;.;.;.;.;.;.;.;.;.;.;.;.;.;.;.;.;.;.;.;
0.5
0' ~
_ E' E' ~
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0.0. 0.01%
1.0
-E' A' ~
-D'
B' ;...;.;.;.;.;.;.;...;........................................................;.........;.;.....
C' .;.;.;.;.;.;.;.;.;.;.;.;.;.;.;.;.;.;.;.;.;.;.;.;.;.;.;.;.;.;.;.;.;.;.;.;.;.;.;.;.;.;.;.;.;.
0.5
E'.
o/~
o 3 day
o 0.5 1.0 0.0.
Chern., 44, 2831 (1980). 11) A. Ottolenghi, Arch. Biochern. Biophys., 79, 355
9) H. Mitsuda, K. Yasumoto and K. Iwami, Eiyo to (1959).
Shokuryo, 19,210 (1967). 12) N. Nakatani and R. Inatani, Agric. BioI. Chern.,
10) T. Osawa and M. Namiki, Agric. BioI. Chern., 45, 735 47, 353 (1983).
(1981).