Thesis Submitted

For

The Fulfillment of the Degree of

DOCTOR OF PHILOSOPHY By

IJAZ AHMAD

H. E. J. Research Institute of Chemistry International Center for Chemical Sciences University of Karachi, Karachi-75270, Pakistan

2006

1

" I i

In tfte name of

I

I

fJJie most Compassionate, fJJie most merciful

DEDICATION

They fed me when I was hungry, gave strength when weak protected me when in danger, taught me to walk on my feet, nurse me when hurt, encouraged when dejected and helped me to live honorably in this world

I dedicated this work to my parents

with love and gratitude

Contents

CONTENTS

ACKNOWLEDGMENTS i

SUMMARY iii

KHULLASA , ix

01. INTRODUCTION (ONOSl~1A HISPIDA) 1

1.1 BORAGINACEAE 2

1.1.1 Economic Uses of the Family Boraginaceae.................... 3

1.2 Genus Onosma 4

1.2.1 Botanical Description of the Genus Onosma 4

1.2.2 Biological Significance of Genus Onosma 6

1.2.3 Onosma hispida Wall. 6

1.3 Literature Survey of Genus Onosma 7

1.4 FLAVONOIDS 16

1.4.1 Introduction , 16

1.4.2 Biological Properties of Flavonoids 21

1.4.3 Biosynthsis of Flavonoids 23

1.4.3.1 General Aspects 23

1.4.3.2 Individual Steps to Flavonoids Classes 28

1.4.3.2.1 Flavanone Formation 28

1.4.3.2.2 Isollavone Formation 29

1.4.3.2.3 Flavone Formation 31

1.4.3.2.4 Flavonol Formation 32

1.4.3.2.5 Glycosylation 33

1.4.3.2.6 Methylation 34

02. RESULTS AND DISCUSSION 36

2.1 New Compounds from Onosma hispida 3 7

2.1.1 Structural Elucidation of Hispidone (43) 37

2.1.2 Structural Elucidation of On os min A (44) 41

2.1.3 Structural Elucidation of Onosmin B (45) 44

2.2 Structural Elucidation of Known Compounds 47

2.2.1 5,2' -Dihydroxy-7,5' -dimethoxyflavone (46) .4 7

2.2.2 Apigenin (47) 48

2.2.3 3,5,7 - Trihydroxy-6,4' -dimethoxyflavone (48) 49

2.2.4 3,5A' - Trihydroxy-6,7-dimethoxyflavone (49) 50

2.2.5 Apigenin 7-0-P-D-glucoside (50) 51

2.2.6 Benzoic Acid (51) .. " 52

2.2.7 4-Hydroxybenzoic Acid (52) 53

2.2.8 5-Hydroxy-3,6,7,4' -tetramethoxyflavone (53) 54

2.2.9 5,4'-Dihydroxy-3.6,7-trimethoxyflavone (54) 55

2.2.10 Methyl 4-methyltetradec-9-enoate (55) 56

2.2.11 Methyl 4-methyltetradeca-9, 12-dienoate (56) 57

2.3 Cholinesterase Inhibitory Activity.i.... 58

Contents

2.3.1 Cholinesterase Inhibitory Activity of 43 and 46........ 58

2.4 Lipoxygenase Inhibitory Activity 59

2.4.1 Lipoxygenase Inhibitory Activity of 44 and 45 59

03. EXPERIMENTAL 62

3.1 General Experimental Conditions 63

3.2 Plant Material , 65

3.3 Extraction and Isolation 65

3.4 Enzyme Inhibition Assays 70

3.5.1 Characterization of Hispidone (43) 72

3.5.2 Characterization of On os min A (44) 73

3.5.3 Characterization of On os min B (45) 74

3.5.4 Characterization of 5,2' -Dihydroxy-r.S"-

dimethoxyflavone (46) 75

3.5.5 Characterization of Apigenin (47) 76

3.5.6 Characterization of 3,5,7 - Trihydroxy-6,4' -

dimethoxyflavone (48) 77

3.5.7 Characterization of 3,5,4' - Trihydroxy-o, 7-

dimethoxyflavone (49) 78

3.5.8 Characterization of Apigenin 7-0-~-D-glucoside (50) 79

3.5.9 Characterization of Benzoic Acid (51) 80

3.5.10 Characterization of 4-Hydroxybenzoic Acid (52) 81

3.5.11 Characterization of 5-Hydroxy-3,6,7,4'-

tetramethoxyflavone (53) 82

3.5.12 Characterization of 5,4' -Dihydroxy-3,6, 7-

trimethoxyflavone (54) 83

3.5.13 Characterization of Methyl 4-methyltetradec-9-enoate (55) 84

3.5.14 Characterization of MethyI4-methy1tetradeca-9,

12-dienoate (56) 85

04. REFERENCES 86

05. INTRODUCTION (BUDDLEJA CRlSPA) 95

5.1 BUDDLE.JACEAE 96

5.2 Genus Buddleja 96

5.2.1 Pharmacology of genus Buddleja 96

5.2.3 Buddleja crispa 97

5.3 Litrature Survey on Budd/eja species 97

5.4 Literature Review on Iridoids I 16

5.4.1 General Introduction 116

5.4.2 Structural Classification 117

5.4.3 Biological Activity of lridoids 1 19

5.4.4 Biosynthesis ofIridoids 122

5.5 Literature Review on Sesquiterpenes 125

5.5.1 General Introduction 125

5.5.2 Biological Properties of Sesquiterpenoids 126

5.5.3 Biosynthesis ofSesquiterpenes 127

Contents

5.6 Literature Review on Steroids 132

5.6.1 General Introduction 132

5.6.2 Biosynthesis of Steroids 132

5.7 Cinnamic Acids 145

5.7.1 Biosynthesis ofCinnamic acid Derivatives......................... 145

5.7.2 Biosynthesis of Sinapic Acid 145

5.8 Benzoic acid Derivatives 149

5.8.1 Biosynthesis of Benzoic acid Derivatives 149

5.8.2 Biosynthesis of Gallic acid 149

06. RESULTS AND DISCUSSION 152

6.1 New Compounds from Buddleja crispa 15 3

6.1.1 Structure Elucidation of Buddlejoside A (267)................ 153

6.1.2 Structure Elucidation of Buddlejoside B (268) 15 7

6.1.3 Structure Elucidation of Buddlejoside C (269) 160

6.1.4 Structure Elucidation of Buddlejone (270) 164

6.1.5 Structure Elucidation of (22R)-stigmasta-7, 9(11 )-dien-

22a-ol 3 ~-O·p-D-galactopyranoside (271) 167

6.1.6 Structure Elucidation of Compound 272 172

6.1.7 Structure Elucidation of Hexyl p-hydroxycinamate (273) 175

6.1.8 Structure Elucidation of Nonyl benzoate (274).......... 177

6.2 Structural Elucidation of Known Compounds 179

6.2.1 P-Sitosterol (275) 179

6.2.2 Ursolic acid (276) 180

6.2.3 Genipin (277) 181

6.2.4 ~-Gardiol (278)............ 182

6.2.5 Buddlejoside A2 (279) 183

6.2.6 Buddlejoside As (280) 184

6.2.7 I-Heptacosanol (281) 185

6.2.8 Methyl benzoate (282) 186

6.3 Antioxidant activity 187

6.3.1 Antioxidant activity of Buddlejosides 267-269 187

6.4 Cholinesterase and Lipoxygenases Inhibitory Activity 188

6.4.1 Cholinesterase and Lipoxygenases Inhibitory Activities

of Compounds 270-274 189

07. EXPERIMENTAL 190

7.1 General Experimental Conditions 191

7.2 Plant Material 193

7.3 Extraction and Isolation 193

7.4 Antioxidant Activity , '" , 199

7.5 Enzyme Inhibition Assays 199

7.6.1 Characterization of Buddlejoside A (267) 201

7.6.2 Characterization of Buddlejoside B (268) 202

7.6.3 Characterization of Buddlejoside C (269) 203

7.6.4 7.6.5

7.6.6 7.6.7 7.6.8 7.6.9

7.6.10 7.6.11 7.6.12 7.6.13 7.6.14 7.6.15 7.6.16

Contents

Characterization of Buddlejone (270) 204

Characterization of (22R)-stigmasta-7, 9( 11 )-dien-22a-ol

3 P-O-p-o-galactopyranoside (271) 205

Characterization of Compound 272 206

Characterization of Hexyl p-hydroxycinamate (273) 207

Characterization ofNonyl benzoate (274) 208

Characterization of P-Sitosterol (275) 209

Characterization of U rsolic acid (276).... .. .. 210

Characterization of Genipin (277)............................... 211

Characterization of p-Gardiol (278)............ 212

Characterization of Buddlejoside A2 (279) 213

Characterization of Buddlejoside As (280) 214

Characterization of l-Heptacosanol (281), 215

Characterization of Methyl benzoate (282) 216

08. RERERENCES 217

LIST OF PUBLICATIONS 226

Acknowledgments

ACKNOWLEDGMENTS

.:. First of jf[{ I 60'W down my Iiead' to the Omnific, Omniscient, Omnipotent and Omnipresent )fL-9rf.U;;'H!1!Y.ft.L£.ftJ{ who provided me opportunity of expfon'ng texture of Iiis naturat beauties at the molecular Ieoel. ../4nd' a[[ respects for the JfoCy fPropliet Jfazrat 9dufiamttuuf (Peace 6e upon him) for enfightening our conscious unt Ii essence of faith in jUL;4Jf, cooerinq a{[ J-l1S Rjru[ness and mercy upon liim .

.:. I O'We a deep deptli of lie a rtiest regardS to my research. superoisorlProj 'Dr. )f.6tful9rf.~ 5.1., wliose excellent supervision, oaiuabie suggestiollS, constant encouragement and' precious attentions througliout the course of these investigations resulted in tlie successful' completion of this research wor~

.:. 'The wliofe wort( 'was carried out anti completed' witli the excellent faciiities made accessible at 'Jf.r£.J. CRssearcli Institute of Chemistry, due to dedication and continuous efforts of lProf. 'Dr. .fttta-Ut'..lJ?p/iman, :N. I" H. I., S. I., 'I. I. (Director of the IrIS tit ute .

• > I am also gratefll{ to (prof. 'Dr. 9d. Iq6aC Clioutf/io.ry, 5.1., 'II. (j)irector (Jktingj anti his students 'Dr. Sarfraz )t !Nawaz, !M.s. )fsma fEjaz and 9rf.r. 9rf. • .ftrif £otfIii for their coffa60ration in determination of tfw 6iofogica{ activities of my compounds .

.:. I am also tfiank/u{ to lPro! 'Dr. o/iqar VUtn)l/inuuJ; 'Jf.1., 5.1. <Prof. 'Dr. <Bina S.

Sidirtqui, 5. I., T. I. anti ([)r. 'l(fiafi.tf!.M. '1(/ian, if I. for their cooperation in recording the 13C-J'lfI},1(j{spectra, cfiecRJng of this doctoral tfiesis ana teaching of the prescribed' courses of the institute.

<* I afro fee{ pleasure to record' my appreciations to (])r. 9rf.. S!ilJiq.Jl/i, ([)r. Zalieer )JJtmetf and (])r. 9rf.. ~a Sliali for their precious attention tfirougfiout the course of this study.

Acknowledgments

.:. lowe my deepest gratitwie to CDr. N"Wliat .J8afta, 7:1. (pcsJ<J( 'Karachi} for her many hefpful comments and suggestions .

• :. ~My special tlianf<J are extended to my friends especia{[y CDr. Zia v~ tDr. )tznar-~ Haq. !Mr. Slier (]ja/Uufar '1(/ian, !Mr. 'Wahi6 Noor and!Mr. Jlyaz Jt{am for pfO'vitfing me vafua6fe assistance, moral supports ana smifing moments durinq my stay in mJ,

.:. J would [iK.! to express my sincere gratituae to my fa6 coffeagues tDr. Itrat Jlnis, CDr. tErum Jlkjer, !Ms. Itrat Patima, !Ms. Jltia, !Mr. Irfan ami !Mr. Jlman for their cooperation,

.:. I am fiigfify tlianifuf to fProj. CDr. ~ (]j. q'aretn and!Mr. !Manzoor jlf""mfwho fiefped us in tfie collection of plants material.

.:. I am thall!ifu[ to a[f tlie technicai and non-technical staff of tfie institute especially !Mr. }lsif !Mefl1nooa tJ?pja and!Mr. ?d. IsCam who hefped me in tlie completion of my researcli ~wor~

(+ I wouid' [iK.! to express my fieartiest gratitude ami regards to my motliet; fatliet; 6rotliers) sisters and otlier family mem6ers for tlieir love, prayers and encouraqement particularly my twin brothers !Mr. Nuar )fiimati and!Mr. Nafee.s)fiimati wfio constantly supported me during my studies.

IJAZAHMAD

11

Summary

Summary

The present Ph. D. thesis comprises two parts, Part A and Part B. The Part A describes the isolation and structural elucidation of a new flavonoid and two new natural arnines

from the whole plant of Onosma hispida Wall. In addition to this eleven known compounds have also been isolated for the first time from this species.

New compounds isolated from Onosma hispida 1. Hispidone (43)

8

HOt(

.)

r

••• ,\\'\1. OCH)

2

-

3 OCH)

OH °

Chem. Pharm. Bull .. 51,412.2003

2. Onosmin A (44)

6'

Chern. Pharm. Bull., 53,907.2005

111

3. Onosmin B (45)

3'

Chern. Pharm. Bull., 53,907,2005

Summary

CH,

Known compounds isolated for the first time from Onosma hispida

1. 5, 2'-Dihydroxy-7, 5'-dirnethoxyflavone (46)

2. Apigenin (47)

3. 3,5, 7-Trihydroxy-6,4' -dirnethoxyflavone (48)

4, 3,5,4' - Trihydroxy-o, 7 -dimethoxyflavone (49)

5. Apigenin 7-0-p-o-glucoside (50)

6. Benzoic acid (51)

7. 4-Hydroxybenzoic acid (52)

8, 5.4' -Dihydroxy-3,6, 7 -trimethoxyflavone (53)

9, 5-Hydroxy-3,6,7,4'-tetramethoxyflavone (54)

10. Methyl 4-methyltetradec-9-enoate (55)

11. Methyl 4-methyltetradeca-9, 12-dienoate (56)

The compounds isolated from Onosma hispida were screened against various clinically important enzymes studied in the enzymology section of our Institute, including acetyland butyryl cholinesterase and lipoxygenase. SAR studies were also carried out in case of the enzyme lipoxygenase inhibitors. The results are described in Tables-13 and -14.

IV

Summary

The Part B describes the isolation and structural elucidation of three new iridoids, a new sesquiterpene, a new steroidal galactoside and three new long chain aryl esters from the whole plant of Buddleja crispa Benth. In addition to this eight known compounds have also been isolated for the first time from this species.

1. Buddlejoside A (267)

OH

Heterocycles, 63, 1875, 2004

2. Buddlejoside B (268)

OH

Heterocycles, 63, 1875, 2004

v

3. Buddlejoside C (269)

4. Buddlejone (270)

OH

Chem. Pharm. Bull., (in press)

Heterocycles, 63, 1875, 2004

5. (22R)-Stigmasta-7, 9(11 )-dien-22a-ol 3 B-O-B-D-galactopyranoside (271)

Z Nalwforsch., 60b, 341, 2005

VI

Summary

6. Compound 272

Chem. Pharm. Bull.. (in press)

7. Hexyl p-hydroxycinamate (273)

I" 3' 5"

~6"

0.,. CH,

~ 4" -

3

z. Naturforsch., 60b, 341, 2005

8. Nonyl benzoate (274)

o

'~o

4V~

3

Z. Naturforsch. , 60b, 341, 2005

VB

Summary

Summary

Compounds isolated for the first time from Buddleja crispa

1. B-Sitosterol (275)
2. Ursolic acid (276)
3. Genipin (277)
4. p-Gardiol (278)
5. Buddlejoside A2 (279)
6. Buddlejoside A5 (280)
7. l-Heptacosanol (281)
8. Methylbenzoate (282) Different compounds isolated from Buddleja crispa were screened against DPPH (I, 1- diphenyl-Z-picryl hydrazyl) free radical scavenging activity and inhibitory activities against the enzymes such as acetyl- and butyryl cholinesterase and lipoxygenase. The results are summarized in Tables-32 and -33.

V III

JIJ.L..( ... S~ UJJi JlJJJjl;)\J)~ ,.:;:...~ ~ Uy-V'jJ).f.,.JlP(j'; ~JJY--(.;

••• " *: , • to ~

J t:/)SdvJ~ S-:.-V/~~J"'l...~UjJ?~I)Jf 'f-V\!~~/~Jr~J~

-.:;:...tSL.fJ~f/u~)~LJli~ b;

,

'\.f?,-f)i." \~ L...... ~ )\_'f-~ ~ (Onosma hispida) \~ L.... ~ )\,...v~ 'f-t'fiLJi.J/.jk'JI(f~q)~(Vf )J''f-r£~~~'~(;(BoraginaCeae) '))JI Flavlonoid ~~J. -~.{.~L-:.-V/LJ~i;;L~rJ){Vf . -LJ1 v(...Lf.::.-V/ '~~ ;"~)~~0}VI~J~Lvl -u.rJ~Aromatic amines (Cholinesterase) ~~J ,j.1)Jf (Upoxygenase);Cf (y L .::.-V/ yj ex: v! ~I

-SJi~J~-J~LU)/(;

"#~''f.f.''f-~):;( (Buddleja crispa) '--r-- ~~~u:.e'-/J) .tJ("'~V/Ldju:.?L~'-)?V'-f-c:£~~dJ~(;(BUddlejaCeae) ,(Iridoid 9IycOSideS).A:vfL{.A:f~)'Ju:. d' -IS r);,'{.;--;J J dvJdl)JI~

._1 . ~/ .r.gJ~/ ~ ,......;_~ ~~ ,......; V'- )Jf (Steroidal qlactosidej> vKt ).~~f ( (Sesquiterpene)I;I_'" r H ~r

S df)JI~~ ~Llf-:.-V/ ;;~;)r~,-){ V' ~J~L ~'-u.r J~;,r.:'JJ)1 ~~I JI", (Upoxygenase) fl~~ L -:.-V/ jI~ u:.l:J1-J S ~~ .::.-~,-J ~ VI J!.f L .::.-V/~ -f? JJi~ J~-J~L UJ/(; (Chol inesterase)

-JJi ~Lf~,.J (Antioxidant)

IX

PART-A

Phytochemical Investigations on Onosma hispida Wall.

Onosma hispida Wall.

Introduction

01. INTRODUCTION

Onosma hispida

1.1 Family Boraginaceae (Forget-me-not family)

The family Boraginaceae consists of 100 genera and 200 species distributed in temperate, especially Mediterranean and tropical regions. In Pakistan it is represented by 32 genera and 135 species. The plants belonging to family Boraginaceae are herbs. shrubs or trees that have flowers in helicoids cymes and often have herbage that is coarsely hairy. The leaves are simple, mostly entire and alternate; stipules are lacking. The flowers are nearly always bisexual and actinomorphic. The fruits consist of 4-1- seeded nutlets or 1-4-seeded nut or drupe fleshy or non-fleshy; when dry, dehiscent or indehiscent or a schizocarp [Ali & Nasir, 1989]. It is one of the most important family from medicinal point of view. The roots of the family are known to be used not only for treatment of burns and skin deseases, but also as red-dye in the drugs, cosmetics and textile industries [Papageorgiou, 1980]. The natural products obtained from the Boraginaceae members exhibit many other interesting biological effects including anticancer [Shukla et al., 1971]. anti-AIDS [Kashiwada et al., 1995], anti-bacterial [Funsun et al., 1983], anti-fungal [Papageorgiou, 1980], anti-inflammatory [Takana et al., 1986], anti-tumor [Ahm et al.. 1995], analgesic [Hayashi, 1977], anti-pyretic [Hayashi, 1977] and immunostimulatory activities [Wagner et al.. 1988]. In the last two decades, research pertaining to their chemical and biological properties and transformations, as well as pharmacology and formulations has increased dramatically.

A red substance (alkannin and shikonin) is present in the roots of many members of the family. The pigment always arises in the cells in which it occurs, and does not diffuse through the cell walls. In the root of the seeding plant, the red pigment first appears in the epidermal cells and root hairs. The epidermis and cortex are cast off during the subsequent development of the root, after which the outermost of the remaining layers of cells becomes suberized, and pigment is formed in the cells. It is stated, however, that the suberized pigment cells are not part of the normal cork, the latter being produced on the inside of the pigmented layer after the colouring matter has been formed. The pigment also arises in association with splits along the medullary rays and other pathological cavities in the root. It is believed that the pigment formation is a wound reaction [Metcalfe el aI., 1950). In Pakistan the family Boraginaceae is represented by 32 genera (Table-l).

2

Introduction

s. No.

Table 1: Genera of Boraginaceae family found in Pakistan

Genera

2

Lindelofia

17

Ehretia

Geaera

Onosma

Coldenia

Arnebia

4

Bugglossoides

20

21

22

23

24

25

26

27

29

Cordially

18

Heliotropium

5

Echiochilon

19

Lithospermum

6

Anchusa

Echium

Nonea

Caccinea

Bothrisopermum

Trichodesma

Asperugo

Anoplocaryum

Hackelia

28

Rochelia

7

Gas I rocotyl e

Paracaryum

30

Heterocaryum

8

Sericostoma

31

Solenanthus

9

Eritrichium

32

1.1.1 Economic uses of the family Boraginaceae

10

Myosotis

The economic uses of plants of family boraginaceae are as ornamentals, pot herbs. dyes for wood, stone, medicine, wine, cosmetics and some important honey plants [Ali & Nasir, 1989]. The most important timber-producing genus is Cordia, with woods varying from light to hard and heavy and from pale to dark brown. The pale-coloured woods are usually general utility timbers, e.g. the Indian C. myxa Linn.; the dark-colored ones are often handsomely streaked and are primarly cabinet woods, e.g. the Bocote of Mexico [Metcalfe et al., 1950].

1 1

Psyudomertensia

12

Lappula

13

Cynoglossum

14

Mattiastrum

15

Lapechiniealla

16

3

Onosma hispida

1.2 Gen us Onosma

The genus Onosma consists of 85 species, occurring mainly in Iran and westwards to Syria, Turkey and Europe. It is represented in Pakistan by 8 species [Ali & Nasir, 1989] (Table-2).

Table 2: Species of Onosma genus found in Pakistan

3 Onosma hispida Wall.

Species

S.No.

Onosma Limitaneum

2 Onosma chitralieum

4 Onosma dichroantha

5 Onosma khyberianum

6 i Onosma hypoleucum

7 Onosma thomsonoii

8 Onosma griffithii

1.2.1 Botanical description

These plants are perennial or biennial suffruticose herbs with hispid parts. Hairs setose, usually arising from a tubercled base; the base sometimes setulose. Flowers bracteate, in terminal or both terminal and lateral cymes, nodding. Calyx 5-sect or sometimes shortly tubular, usually accrescent. Corolla tubular, gradually broadening upwards, yellow, blue white or crearnish; lobes short, patent or not. Nectariferous glands sometimes present. Filaments adenate to corolla tube in lower half. Anthers usually adenate to one another basaly or sometimes laterally, included in the corolla tube or sometimes exserted. Stigmas 2, bilobed or capitate. Nutlets 4 or less usually somooth [Ali & Nasir, 1989]. The botanical description of the eight Pakistani species is given in Table-

3.

4

Table 3: Characteristics of 8 Pakistani snecies of genus, Onosma

Introduction

S. No ~flara(t(eristi£s S~~s

1 I Anthers free, united only at the base. 0. limitaneum

2 Anthers united. 0. chitralicum

3 Corolls 7-8 mm long. Anthers 3-7 mm 0. ichroantha long. Nutlets c. 4.5 mm long.

4 Corolls 25-30 (-35) mm long. Anthers 8- 0. limitaneum 11 mm long. Nutlets c. 3-3.5 mm long.

5 Anthers ± included in corolla 0. hispida Wall.

6 Anthers exserted to more than half of 0. hyberianum corolla.

7 Filaments ± 2.5 mm long. 0. chitralicum

8 Filaments 5-10 mm long. 0. hypoleucum

9 Plants 10- 70 em or more tall. Basal 0. hispida Wall. leaves 9-35 em long.

10 Plants up to 30(-35) em tall. Basal leaves 0. thomsonii up to 10 em long.

II Bracts attenuate 10 upper part and 0. khyberianum acuminate. Corolla 30 mm or more 3 10

length. Fi laments 10-12 mm long.

12 Bracts not as above. Corolla less than 30 0. griffithii mm long. Filaments up to 9 mm long.

13

Caulin

leaves

10-30 mm long; 0. hypoleucum

undersurface with both spreading and appressed hairs. Corolla tube antrorsely pubescent externally and internally villous towards the base.

14 Caluline leaves 50-80 mm long, 0. thomosonii undersurface with spreading hairs. Corolla

tube retrorsely pubescent externally and

internally glabrous.

5

Onosma hispida

1.2.2 Biological significance of genus Onosma

The plants of the genus has a cooling, laxative, anthelminitic, alexipharmic effects and are effective in the diseases of the eye, derangements of the blood, bronchitis, abdominal pain, stangury, thirst, itch, leucoderma, fevers, wounds, piles and in urinary calculi [Kirtikar & Basu, 1994]. It is used in the Tibetan system of medicine [Bhartacharjee, 1998]. Bruished root is used as an application to eruption [Kirtikar & Basu, 1994]. Leaves possess alternative properties, and powder is given to children as purgative [Kirtikar & Basu, 1994]. Flowers are prescribed as a stimulant and cardiac tonic in rheumatism and diseases of the heart [George, 1972].

Rat growth studies on vanaspati colored with turmeric extract or Onosma echioides extract using an adequate diet showed no toxic effect on liver and is thus suitable for coloring vanaspati [Bhuraneswaran et al., 1963]. Pharmacological study of Onosma bracteatum extract showed that it lowers the blood pressure, depresses the heart and causes vasoconstriction. It also relaxes the small intestine and blocks the stimulating action of acetylcholine [Dandy & Arora, 1957]. Biological study of shikonin showed that in concentration of 20-30 ug/ml, it had a bactericidal effect of lactic acid bacteria [Aivazyan, 1975]. Onosma echioides extract has anti-inflammatory and anti-bacterial properties [George, 1985]. The roots of Onosma heterophylla are of particular importance in the biosynthesis of fatty substances [Kehui et aI., 1989]. Studies have shown that fungal elicitor changed slightly the relative content of each of the shikonin derivatives [Ning et al., 1996].CuCb stumulates the biosynthesis of shikonin derivatives [Zhang et al, 1997].

1.2.3 Onosma hispida Wall.

Onosma hispida Wall. occurs in western Himalaya from Kashmir to Kuman. It is widely distributed from Siberia and Kabul to France [George, 1972]. In Pakistan it is very common in Baluchistan, Landi Kotal, Chitral, Swat, Kaghan and Kashmir [Nasir & Ali, 1972].

Onosma hispida Wall. is known as Cerinth, Melinet in French, Ratanjot in Hindi, Cerinta in Italian, Newarmaharangi in Nepal. Koame, Laljari, Maharangi and Ratanjot in Punjab. It is one of the most important species of the genus, Onosma. The plant has a

6

Introduction

bitter and sharp last [Kirtikar & Basu, 1994], a perennial herb up to 70 em tall with a prominent tap root. Stem (-s) many, mostly simple, hollow, densely hairy with long spreading hairs 3.5-5 mm long. having tuberculate bases, intermixed with shorter thinner hairs. Basal leaves 90-350 x 5-10 mm, linear to oblanceolate, middle cauline leaves often larger, uppermost smaller. Inflorescence a terminal cyme, dense at anthesis, elongating to 12 em in fruit bracts lanccolate, leaf like. but smaller. Pedicel short, densely hairy. upto 10 mm in fruit. Calyx I 1- 13 mm long, up to 20 mm in fruit, densely hairy, lobes lanceolate. Corolla cream ish white to light yellow, tubular-cornpanulate, 20-30 nun long, puberulous to the outside; lobes 1.5-1.8 nun long, deflexed. Filaments attached 7-12 rnm from corolla base, 5-9 mm long. flattened, the decurrent base 4-5 mm long. Anthers united laterally to form a tube upto 1.6 mm long, sterile tips 1.5-2 mm long. Nectary forming an annular collar, 0.2-0.3 mrn high, glabrous. Style up to 20 mm long, glabrous. Nutlets 5-6 mm long, shiny. Flowering period is May-June [Nasir & Ali, 1972].

1.3 Literature survey of genus Onosma

A variety of compounds have so far been reported from genus Onosma and theses are described in Table-4.

7

Onosma hispida

Table 4: Compounds Isolated from the plants of the genus Onosma

n- I ' Alkannin (1)

MP. 116-1170

[ '0

.a] Hg-

]57

,_, •• ',, __ ........•.. __ .. ' ••... __ :1 _._._, .. _ .. -<<;~H{J)_._ .. _ ._. _ _

C16H1604 272 MP 0. heterophylla .

94-95° Millidis et al., 1987

0. heterophylla Millidis et al.. 1987

__ ,.,., ... __ ~ L_' ~ • ~ T~ ~ .. ~ _

._ _ _.. _,"" __ _ - - ~ __ - .. _ _ .. n _ _ _ .. _ ~ .. ~ _ ~ _ .. _ _ ,., .. _ .. __ .. _. .. .." r __ ., ~

Sp-Acetoxy . C23H260g 430 a 0. paniculatum

isovalerylalkannin(5) Zhu et al., 1990

......... , .. - ..... - ~ ~ • _. __ •• , ~ r ~ __ ."

*

5, 8-Dihydroxy-2-( 1 -p,p C23H260g dimethylacryloyloxy -4- methyljpentyl-l A-

. _. 1!~J?ht.~~I~I1~~J~n~ {~L _. _ .. .. __ . __ . __ . __ .. _. __ .. _ .. . _ .. . __ . _. " .... ... _._

7 Shikonnin (7) C'6H160S 288

MP 0. Caucasicum

144-45° Romanova et

. _ __ .. . . _ .. . __ . . .. '.. __ . __ . ,.' __ ' .. _ .. _ . __ . _ .. , _ __ . __ . _ _ al. 1 .! 5??.7 _ _ .,_,

S. ,(;ompounil N"ame

No ..

4

{3, P-

Dimethylacrylshikonnin (10)

Mot FOFDlula

Mol~ We~t

370

372

Phy~iCaI Data

MP 116-117°

MP 94-95°

Souree.It r.efel1enee

0. heterophylla Millidis et al .. 1987

0. heterophylla Millidis et al., 1987

2 Deoxyalkannin (2)

P, p-Dimethylacryl . alkannin (3)

Isovalerylalkannin (4)

6

8

Deoxyshikonnin (8)

430

272

320

a*

MP 85-86°

0. heterophylla Millidis et al .. 1987

0. confrtum Kehui et al., 1989

9

Acetylshikonnin (9)

0. stosum

Kagramanyan

- -- - .. _ . . _ .. - - - - __ .. _ .. - . _ .. _ _ _ _ _ .. _ _. .. _ . . __ . _ .. . .. _ .. _ _ _.. .. _ . _ _ . _ .. _ .. __ . ~!_ ~': c _ L~ ~.~ .

MP 0.

111-114 ° zerizaminum

[0.]60022 Traeeva et al.,

+222 1971.

(EtOH) 0. confrtum

Kehui et al.. 1989

- :if·-· 'H~i i~tr(di~~ -{i iY······· --~·c~t·Yliio~ ·--·'f . i 5-S·· - ... "- . - .. -. ~-* -. - - ... 0: -h~i~~~phj!iYa-

10

370

8

Introduction

I

*

Millidis et al., 1988

a" 0. heterophylla Millidis et al., 1988

-----~-- ------- --------------~----------- -,--------~---.-,---- -,---.-.-~.-.--.- .. -- ----._------ --.,.,.~-'- ... ------~-----,-.

12

13

14

I -Ethylene pyrrolizidine C9H13NO

(12)

151

Ursolic acid (13)

I-Methyl-2- phenylethylisoferulate (14)

MP 291 [a]o25 +66 (Et20)

0. hispidium Sbamshad et al., 1990

0. heterophylla Millidis et al .. 1993

456

312

________ L ~ • ~L ,, , •• , '" u. •• _'. ~ _

IS I~Methyl-3-(3'-methoxy- C2oH2206 358 * 0. heterophylla .

4 -hydroxyphenyl)- Millidis et al..

_ .. p~~px!_c~K(~~!~_L15t , J?~~ _

16 Apigenin (16) C1SHIOOS 270 MP 0. heterophylla

348-350° Millidis et al., 1993

17

18

~9

Luteolin (17)

Chrysoeriol (18)

Quercetin (19)

286

MP 328-330°

0. heterophylla Millidis et al., 1993

0. heterophylla Millidis et al., 1993

300

MP 330-331°

0. heterophylla Millidis et al.. 1993

20 Intermedine (20) Cl5H2SNOs 299 MP 0. alborosea. 140-142 0 are naris

[U]02S 0.

+7.8 sanguinolenta

(c 1.49 Roeder et al ..

_, _. , • ~ ,& ~ ~_t9I-{L ,J?~~ . _

21 . Lycopsamine (21) CISH25NOs 299 a" 0. alborosea. 0. are naris

o. sanguinolenta Roeder et al.. 1993

22

Acetylintermedine (22)

302

MP 313~314°

O. alborose, 0. arenaris, 0. sanguinolenta Roeder et al..

a*

9

Onosma hispida

0. alborosea,

, 0. are naris

0. sanguinolenta Roeder et al., 1993

--.--.-,- ------.--------------- -------.~-- .. --.------------- ~---:-------- ------2S----~-- ----------------------

24 Leptanthine (24) C15H25N06 31) [a]o -2.0 0. lepthantha

(c 0.20, Kretsi 0_ et al.,

____ ." _ __ _ _ _ _ ~ ~ ~. _~ ~.1_e~~L '?_Q~3 _ _ _ _ _ _ _ _ _

25 Leptanthine-N-oxide (25) . CIsH2SN07 331 ,[a]o . 0. lepthantha

+16.5 (c Kretsi et al.,

0.20 2003

MeOH)

~-----.-.-------------.--------------- ----- --""-------------- - -------- -- ------20------~ ----------------- ----.

26 Echihumiline (26) C.20H31N07 397 [a]o +10 0. lepthantha

(c 0.1, Kretsi et al.,

____ ..... ~ .__ __ .. ?_tQI:-!L_ _'?_Q9~ "

>I<

0. lepthantha Kretsi et al .. 2003

- - - - - - - - - - • - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -. ".-. - - - - - - - - . - - - -. -- --- - - - - " - - -. - - - - - - 2-0- .. - - - - . - - . - - - - - . - - . - - - - -~ - - -

28 3 -0- C22l-h3N09 4)5 [a][) 0. lepthantha

acetylyechihumiline-N- +35.0 (c Kretsi et al.,

oxide (28) 0.20 2003

_____ . __ ._. . 1 . . _~,tt:Q!-J) __ . . . .

>;<

Acetyllycopsamine (23) C17HnN06 341

23

n .. Decatriane (30)

27

Echihumiline-N-oxide (27)

29

n-Dodecane (29)

30

32 Methyl tetradecanoate (32)

35

i70

184

10

a*

1993

0. heterophylla Millidis et al., 1987

--------- ,-------- --- -~--- --- ---

>I<

0. heterophylla Millidis et al .. 1987

0. heterophylla Millidis et al ..

I 1987

0. heterophylla Millidis et al .. 1987

- ._, .. ~ __ ~ ~ ~ n -.' __ "" ... ~ __ ~ __ ._,

31 Methyl dodecanoate (31) C13H2602 214 *

. 33- - ---Methyf~4~meihyi~ - - - - - - - ---- -C;~H~i); - ----'. -252 - -- - -- -" -- - - -* - - --- -0. -h-~i~~opf~~vii"d-

tetradeca-v.l 2-dieneoate Millidis et al.,

, (?~2. , . .----- ._ .. 1 J-?~?----- ._ " _

34 Methyl-4-methyltetradec- C16H3002 254 * 0. heterophylla

9-eneoate (34) Millidis et al., 1987

, - - - - - - ~ - ~ - - - .. _ .. - - ,~- - - - - - - - -.- - - ,-, - _.- - - - - - - -"- ~~- .. - - - ~ - - - - - - - - - - - - - - - - -

Methyl hexadec-9- C 17H3202 268 * 0. heterophylla

eneoate (35) Millidis et al., 1987

36-- -iVieth;l-hexad~cano~te- --- -\C;;H;~O;------i70-- ---. &.-- ---*--- -- -0: -h~i~;·o£.hYii"d-

--- -_ .. "-- - - - - - - -- - - - .... _.- -,-,- -- ~ - - - -'.'- - _ .. - - - - - -- ~ - - - - - - - ~ - - --

Cl5H3002 242 *

Introduction

__ .. _., .. ;=. ••• ~ • ,., __ , ,. • __ J _ •. .,, r _

*

37

38

39

40

41

(36)

i

Ethyl hexadecanoate (37) CUH3602

Isopropyl hexadecanoate (38)

Methyloctadeca-9, 12,15- Cg)H3202 trieneoate (39)

__ ••• __ ... _ ••• _ _ _ _ _ .. n _ _ _ _ _ _ _ _ _ ., _ L _ po ~ _ _ _ _ _ _ _ _.. _ ~ ....... ~ _ _ _ _ _ _~ __ ~ ~ L ••• _ •

*

, T r 0 OR_OR __ • ~ ,. • • _

*

- - •. R_" .. ~... ~ ~ _ _ _ _ _ _ _ _ _ _ _ •• _ .... _ .. .. . '''' ' ..... __ .. ._ ,~_ ......... .. __ .. _ _ _ - __ .. __ .. - - _ .. ,. ~ _

*

0. heterophylla Millidis et al.,

- 42~ --- -Methyi -oct~dec~;ri~~te-- -""- -C;~H~~02 -,,-- -298-- -- -- - + -- -- --* -- -- -- - g.~:i;~~PhYii~;-

(42) 'I Millidis et ai ..

I 1987

Methyl octadeca-9, 12- dieneoate (40)

Methyl octadec-9- eneoate (41)

• * Not reported

• a' The refrence not availble locally.

1 1

284

298

292

294

296

Miilidis et al., 1987

a. heterophylla Millidis et al., 1987

0. heterophylla Millidis et al., 1987

0. heterophylla Millidis et al .. 1987

0. heterophylla Millidis et at" 1987

Onosma hispida

Naphthaquinones:

OH 0

NO. ,\ COMPOUND RI R2
t, Alkannin H OH
2. Deoxyalkannin H H
3. p,p-Dimethylacryl- H OCOCH=C(CH3h
alkannin
4. lsovalerylalkannin H OCOCH2CH (CI-h)2
5. ~-Acetoxy-isovaleryl- H OCOCH2C(OCOCH3)(CH3)2
alkannin
7. Shikonnin OH H
8. Deoxyshikonnin H H
9. Acetylshikonnin OCOCH3 H
.10. p,p-Dimethylacryl- OCOCH=C(CH3)2 H
shikonnin OH a
0y
a
~
OH a ~yy 5, 8-Dihydroxy-2-( 1 -~, ~ dimethylacryloyloxy-4-methyl)pentyl-1 ,4-naphthalenedione (6)

Figure-I: Structures of reported compounds from genus Onosma

12

Introduction

rJ-I-

~ ~

14. l-Methyl-z-phenylethy I isoferulate

13. Lrsolic acid

HO

OH 0 16. Apigenin

15. 1-:-'1ethyl-3-(}' -methoxv-a -r-ydroxvphcnv] )-propyl isofcrulate

HO

I-lO

Of-!

o

OH

o

17. Leuteolin

18. Chrysoeriol

IIO

Oli 0

19. Quercetin

Fi~ure-l (cont.): Structures of report ed compounds from genus Onosma

13

Onosma hispida

Pyrrolizidine Alkaloids:

RI ~ OR2
~
:\ 11. Heliotridine, RI = H R2=H

24. Leptanthine, R = H

26.EclUhwn;t;",. R= ~C~ CI-~

Figure-I (COOL): Structures of reported compounds from genus Onosma

CH,

l4

Introduction

No,. Compound Name Structural formula
29 n-Dodecane CH3(CH2)lOCH3
I 30 n-Decatriane CH3(CH2)IICH3
3.1 Methyl dodecanoate CH3(CH2)lOC02CH3
32 Methyl tetradecanoate CH3(CH2)12C02CH3
33 Methyl-4-methyl- CH3
I
tetradeca-v.l z-dieneoate CH3CI-I=CHCH2CH=CH(CH2)4CH(CHhC01CHJ
34 Methyl-4-methyltetradec- CH3
I
9-eneoate CH3(CH2)3CH=CH(CH2)4CH(CHhC02CH3
35 Methyl hexadec-9-eneoate CH3(CH2)SCH=CH(CH2hC02CH3
36, Methyl hexadecanoate CH3(CH2)14C02CH3
37 Ethyl hexadecanoate CH3(CI-I2)14C02C2H;
38 Isopropyl hexadecanoate CH3(CH2)I4C02CH(CH3h
39 Meiliyloctadeca-9, I L 15- CI-l3CI-l2CH =CI-lCH]C1--1 =CI-lCH 2CH=CH(CH2hCOzCH 3
trieneoate
40 Methyloctadeca-9. 12- CH3.(CH2)4CH=CHCH2CH=CH(CI-I2hC02CH]
dieneoate
41 Methyloctadec-9-eneoate CH3(CH2hCH=CH(CH2hC02CH3
42 Methyl octadecanoate CH3(CH2)16C02CH3 Figure-I (cont.): Structures of reported compounds from genus Onosma

15

Onosma hispida

As flavonoids form main subject matter of this part of thesis, an account of their types and biosynthesis is presented in the proceeding section.

1.4 FLA VONOIDS

1.4.1 INTRODUCTION

The flavonoids, one of the most diverse and widespread group of natural products, occupy a prominent position among the natural phenols. The name "flavonoid" is derived from Greek word "flavus' (yellow). These are important biologically active plant ingredients and about 2% of all the carbon photosynthesized is converted into flavonoids [Harbone, 1988].

o Chalconoids

Flavanoids

(a)

Auronoids

o

Flavonoids

Figure-2: Flavonoids having 1 ,3-diphenylpropane skeleton

Flavonoids are the colouring co-pigment of the plants [Zechmeister, 1957L which are also called anthoxanthins. The wide range of colours and shades in flowers are principally due to the presence of flavonoids. These usually occur as pigments in the cells of flower tissues [Manitto & Sammes, 1981].

16

Introduction

Flavonoids occur in a variety of structural forms, All contain fifteen carbon atoms In their parent nucleus and share the common structural feature of two phenyl rings linked by a three-carbon chain (diphenyl propane derivatives). The compounds possessing a l , 3-diphenylpropane (Figure-2) skeletons are regarded as chalconoids.

(a)

Isoflavanoids

Isoflavonoids

3-P heny Icoumarin s

Neoflavonoids

(b)

Figure-3: Flavonoids having 1, 2 and 1, 1 diphenylpropane skeleton

The three-carbon chain may be formed into a third (five or six membered) ring through an oxygen on one of these phenyl rings generating a tricyclic system. The tricyclic compounds possessing a five membered heterocyclic ring are referred to auronoids, whereas those possessing a six membered heterocyclic ring are designated as flavonoids (Figurc-2). The tricyclic compounds derived from 1, 2-diphenylpropane (Figure-3a) system are known as isoflavonoids and 3-phenylcoumarins (Figure-3). whereas those derived from 1,2-diphenylpropane (Figure-3b) are called neoflavonoids (Figure-3).

17

Onosma hispida

7

Flavan-3-ol

4'

3'

2'

6

5

o

Flavanone

Flavanonol

o

Anthocynanidin

Flavone

Isoflavanone 0

o

Flavonol

Isoflavone 0

o

3-Methoxyflavone

Isoflavan

o

3-Phenylcoumarin Figure-4: Basic skeleton and numbering pattern in flavonoids

18

Introduction

4
4' 3
3' Chalcone Chalcan-1,3-dione
2' p-
o
7
'·0
6 '\ ~ 5'
5 l' I
0 2' fi 4'
p'-Chalcanol Aurone
iSH 3' !3'-Chalcanone

Aurononol 0

o

a-Chalcanol

Auranol

o

Chalcone

o

o

1-Phenylallylbenzene

0-Chalcanol

o

Figure-4 (continue)

19

Onosma hispida

11 2'

7

6

o

o

Homoflavone

Homoisoflavone

5'

Homoisoflavan

Rotenoid

o Homoisoflavanone

Dehydrorotenoid

Figure-5: Basic skeleton and numbering pattern in homoflavonoids

In tricyclic compounds of the flavonoid, auronoid and isoflavonoid types, rings are labeled as A, B, C and the individual carbon atoms are referred to by a numbering system which utilizes ordinary numerals for the A and C rings and primed numerals for the Bring (Figure-4). Some authors refer to carbon 9 and 10 in flavonoids as 8a and 4a, respectively, In chalcones the A ring, normally written to the left, is given primed numbers, while the B ring carbon are given ordinary numbers. Exocyclic carbons are designated as a and ~ relative to the carbonyl function (Figure-2),

Natural flavonoinds and isoflavonoids are usually oxygenated and bear hydroxyl and/or methoxy substituents. The structure and numbering of some common naturally occurring classes of monomeric flavonoids are illustrated in figure-4.

Hornoflavonoids (Figure-S) are the class of flavonoids, which contain an additional carbon in their skeleton. This additional carbon is designated as C-II. A large number of flavonoids occur as O-glycosides in which one or more of the hydroxyl groups of the flavonoid are bound to a sugar or sugars via an acid labile hemiacetal bond. In flavonoid with C-glycosides [He liar & Fork mann, 1980. Harbone & Mabry, 1982]. the sugar is C-linked and this linkage is acid resistant. The effect of glycosylation renders the flavonoid less reactive and more water-soluble.

20

Introduction

1.4.2 Biological Properties of Flavonoids

Various classes of flavonoids have been investigated tor different physiological activities. No universal function for the tlavones and/or tlavonols in plants has yet been established, in spite of the fact that these are the most conunon and widely distributed. However. many functions in individual plants or plant groups have either been demonstrated or proposed [Harborne & Mabry, 1982].

Willaman has reviewed the biological effects of the flavonoids and listed thirtythree different manifestations of activity under the heading "Bioflavonoids" [Willaman . 1981]. Rutin and hesperdin, also called vitamin P or permeability factors, are used in the treatment of various diseases, I ike capillary bleeding, increased capillary fragility, diabetes, allergic manifestation and hypertention. Claims have also been advanced for the citrus bioflavonoids in the treatment of symptoms of common cold [Tyler et al., 1988]. A number of flavonoids and chalcones have anti-protozoal activities [Wright & Phyllipson, 1998]. Phenolic substances are well known to have anti-inflammatory activity. Some flavonoids like myrcetin and kaempferol-3-glucoside have an anti HIV -] Potency at non toxic concentration [Hostettrnan et al., 1995].

The position of the substitution also affects the properties. The tlavonols containing two ortho or para hydroxyl in the 2-phenyl ring have anti-oxidant properties, while free hydroxyl at the 5,7-positions have a pro-oxidant effect [Zecluneister, 1957]. Apigenin and genkwanin, which are present in the Chinese, drug "Yuen-hua" are believed to have diuretic and anthelmintic properties. These actions become more pronounced with an increasing number of hydroxyl groups. Fluka has reported the cardiac stimulation and vasoconstriction of the flavones and their glycosides [Zechrneister, 1957].

Some flavones have the properties of prevention from anaphylactic shock, protection against X-rays and cure from frostbite [Willarnan, 1981]. The simple isoflavone and coumestans have oesterogenic activities, rotenoids have insecticide properties while isoflavonoid phytoalexins have anti-fungal and anti-bacterial activities [Ingham, 1983].

Some of the minor flavonoids have very interesting activities. They have antimicrobial, anti-fungal and cytotoxic properties [Harborne & Baxter, 1999]. Tricin has

21

Onosma hispida

smooth muscle movements activity [Ferguson et al., 1950]. Anthocyanin peonin and isorhamnetin of the algae Chlamydomonas are highly potent sex determining hormone [Kuhn et al., 1994]. Due to the lack of toxicity, flavones are prone to further pharmacological investigation and as a result an increase in its therapeutic applications.

The flavonoid glycosides have also a vital pharmacological role. DeRodriguez et. al., reported an anti-viral activity for the 5-0-methylgenistein-7-0-I3-D-glucopyranoside. This isoflavonide glycoside was isolated from the Ulex europaeus L. [DeRodriguez et al., 1990], Similarly, prenylflavonol glycoside, epimedokoreanoside isolated from the Epimedium koreamum (Berberidaceae) showed a hypotensive activity [Pachaly et al., 1990].

Anthocyanins have wide applications in the food industry. It helps in determining wine quality. It is used as natural colourant to replace synthetic red dyes [Barbone, 1988]. Anthocyanins are also used for the inhibition of larval growth in insects. Anthocyanidin (aglycones of anthocyanins) have been investigated for biological activity. It was found that delphindin chloride has a microvascular protective activity [Gabetta et al., 1990].

Chalcones have been thoroughly investigated by G. Michiro and it was found that these have anti-microbial activity, which is enhanced by the bromination. Dihydroxychalcones, which is a usual class of flavonoids are generally considered physiologically inactive but 2,6-dihydroxy-4-methoxy-3,5-dimethylhydrochalcone has anti-microbial and lipoxygenase inhibiting activity [Michihiro el al., 1990]. C-mehtylated dihydrochalcones are very rare as natural products. The exudates of Myrica gale fruits are a rich source of C~methylated dinydrochalcones [Yoshiaka et al., 1973]. One of the function of flavonoids in plants is to protect them from the diseases caused by microorganisms. Uvaritin and isouvaritin have anti-microbial and cytotoxic activities [Malterud et al., 1990, Alcoraz et al., 1990, Ruzicka, 1953]. Some dihydrochalcones have uncoupling and inhibitory activities on isolated mitochondria [Harbome & Mabry, 1982].

Some flavonoids and dihydroxychalcones have taste properties. Their different tastes normally depend upon the point of attachment of the substituent. Isoflavonoids act as venom antidote. Some flavonoids are used as inhibitors of CC14 induced cytotoxicity in isolated hepatocytes. The isoflavones genistein has oesterogenic activities, which is important for sheep breeding in the dairy areas of western Australia. The biosynthesis of

22

Introduction

coumesterol in leaves is induced by UV light, so its level is used for the indication of damage of UV to the plant system [Harborne & Mabry, 1982, Ruzicka, 1953].

Bioflavonoids functions as UV filters. These functions as guard on the leaf from insect and microbial attack. These inhibit adhesion of blood platelets and blocking of inflammatory effects. These also act as heart stimulant (Ruzicka. 1953].

1.4.3 BIOSYNTHESIS OF FLA VONOIDS 1.4.3.1 General Aspects

The biosynthesis of all flavonoids involve the formation of central C-15 intermediate known as chalcone. Two main biosynthetic pathways contribute to the biosynthesis of flavonoids, the shikimate and the acetate pathways and they are responsible for the formation of chalcones, the basic precursors to all flavonoids.

The central role of chalcones in flavonoid biosynthesis has been attested in a number of investigations [Grisebach, 1965, Wong, 1965]. The formation of chalcone is catalyzed by chalcone synthase, which is the key enzyme of flavonoid biosynthesis. The precursors for chalcone formation (Scheme-L) are malonyl-CoA and 4-coumaroyl-CoA (hydroxycinnamic acid CoA ester). Both flavonoid precursors are derived from carbohydrates. Malonyl-CnA is synthesized from the glycolysis intermediate acetyl-CnA and carbon dioxide. the reaction being catalyzed by acetyl-Co A carboxylase. The synthesis of 4-coumaroyl-CoA is more complex and involves the shikimate pathway (Scheme-2), which is the main route to aromatic amino acid, phenylalanine and tyrosine in higher plants [Jensen, 1965]. The biosynthesis of shikimic acid (Scheme-L) begins with the condensation of D-erythrose-4-phosphate and phosphoenolpyruvic acid [Manirto & Sanunes, 1981]. The enzymes leading to various flavonoid classes are listed in Table-5.

23

Onosma hispida

f S.No.

Table 5: List of enzymes leading to various flavonoids classes

Classe4'

Acetyl-CoA carboxylase

2 Phenylalanine ammonia-lyase II

3 Cinnamate 4-hydroxylase II

4 4-Coumarate-CoA ligase II

5 Chalcone synthase Flavonoids

6 Chalcone isomerase II

7 2-Hydroxyisoflavone synthase II

8 Flavone synthase II

9 (2S)-Flavanone 3-hydroxylase 1/

10 Flavonol synthase 1/

u Dihydroflavonol 4-reductase /1

24

Non-flavonoids

Introduction

D-Glucose + CO2

I

I

Pentose phosphate Glycoslysis

r ~ r-.

po::l)(c3 :'0

HO H ~~

H Phosphoenol pyruvic acid (PEP)

D-Ep/throse-4-phosphate

!-:

CO2 PO

HO ~ OH OH

OH

Syn elimination ~ - H,O

OH

Shikimate

Scheme-1

25

Onosma hispida

A ATP

HO""~OH OH

Shikimate

6C02 p00J.:EP- Eicon H

• cO2 ~ ~

PO\\'·. OH pO ~ 0 CO2

OH Pi OH _ j

Pi. __ ,

-

O'XH2COC02 .. cb~

u WCo,

OH

OH rNArt ,\a... NADH

Chorismate

-CO2 ,..

OH
+NH3
-<C02H
H2C
NH2
-NH2 ..
..
OH OH OH
p-hydroxy coumaric acid
Scheme-2 26

Introduction

CARBOHYDRATES
, OH
0
II
CH3-C-SCoA
Acetyl-CoA HO
t o 4-hydroxy coumaric acid
3 [ nSCOA 1 CoASH
Hooe -H2O
0
Malonyl-CoA OH
t
t
0 OH

Chalcone Synthase +

OH

HO

Chalcone

OH 0

Scheme-3

27

Onosma hispida

1.4.3.2 INDIVIDUAL STEPS TO FLA VONOIDS CLASSES

1.4.3.2.1 Flavanone Formation

Flavanones are formed from chalcones by isomerization. There is a good evidence for the in vitro and in vivo existence of equilibrium between flavanones and the corresponding chalcones [Manitto & Sammes, 1981]. The interconversion between chalcones and flavanones is catalyzed in vivo by enzyme known as chalcone isomerase. The stereospecificity of this enzymatic reaction is apparent in the (S) chirality of C-2 in flavanone derivative.

OH aD"
H H ........ \\~
- BH
..
k
H-OH
OH OH ~H') \]
H--A
V
Chalcone ;/ A
o
H .,~'t,""\"
·""'1/ *
H
OH 0
Flavanone
Scheme-4 Therefore, it is not accidental that all the flavanones found in nature have the (S) configuration at C-2 and are levorotatory. With chalcones having at least two free hydroxyl groups at C-2 and C-6, the equilibrium in an aqueous solution is shifted completely and rapidly to the flavanone (Scheme-4). The stabilization energy of the strong hydrogen bond between the carbonyl group and the ortho-phenolic hydroxyl group

28

Introduction

greatly influence the position of equilibrium and the interconversion rate. When only one hydroxyl is available. either for the cyclization or for hydrogen bonding, the system ends to remain in the open form (chalcone form).

1.4.3.2.2 Isoflavone Formation

The key step in isoflavone formation is the 2,3-migration of the aryl side chain of a flavanone chalcone intermediate. An enzyme activity catalyzing this transformation was recently found in microsomal preparations from elicitor-challenged soybean cell suspension cultures. It transforms (2S)-naringenin (flavanone) into genistein [Hagmann et al., 1984] (Scheme-S).

It was found that two enzymatic steps are involved in this transformation. The first step comprises of oxidation and rearrangement of naringenin to 2-hydroxy-2. 3- dihydrogenistein. This step is strictly dependent on NADPH and molecular oxygen. The second enzyme which catalyses the elimination of water from the 2-hydroxyisoflavanone is identified but has not yet been characterized [Kochs & Grisebach, 1986].

For the following reasons, (2S)-flavanones and not the chalcones are very probably the actual substrates: (a) a stereospecific incorporation of (2S)-naringenin into biochanin A (5,7-dihydroxy-4'-methoxyisoflavone) [Patschke et al., 1966]. (b) Only the (2S) but only the (2R) enantiomer acts as a substrate in vitro [Kochs & Grisebach, 1986]. (c) The equilibrium of 4,2',4',6'-tetrahydroxychalcone is at least 1000: I in favour of the flavanone [Boland & Wong, 1975]. The equilibrium of 4,2',4',6'-tetrahydroxycha1cone is flavanone to isoflavone (Scheme-S) is consistent with the participation of NADPH and molecular oxygen.

29

Onosma hispida

(Y0H

I HOW· ""

"":::: .. "

Ih

OH 0

(Y0H

I

HO~OI"""'\

yY

OH OH

Naringenin

C:B-Enz H

JJ

HO

HO

OH

2-Hydroxydihydrogenistein

Genistein (Isoflavone)

Scheme-S

The sequence of events may be initiated by an epoxidation step. Protonation and subsequent cleavage of the epoxide would render a positive charge to the B-ring. Ketoenol tautomerism, as indicated by proton exchange at C-3 in flavanones [Grisebach & Zilg, 1968], allows homoallylic interaction between C-3 and C-I', and rearrangement of the structure then takes place. Addition of hydroxyl ion to C-2 leads to the 2- hydroxyisoflavanone intermediate, which is transformed to the isoflavone by elimination of water molecule.

30

Introduction

1.4.3.2.3 Flavone Formation

The in vitro conversion of flavanones to flavones was first observed in parsley plants [Sutter et al., 1975]. The reaction has been studied in more detail in parsley cell suspension cultures [Britsch et al .. 1981] and in Antirrhinum flowers [Stotz & Forkrnann, 1981] .

The parsley enzyme requires 2-oxoglutarate, Fe2T and possibly ascorbate as cofactors. This co-factor requirement would classify it as belonging to the 2-oxoglutarate dependent dioxogenase. Ascorbate stimulates this and other 2-oxoglutarate dependent dioxygenases involved in the flavonoid pathway and, in addition. exhibits a remarkable stabilizing effect on the enzyme activity [Britsch & Grisebach, 1986].

In Antirrhinum [Stotz & F orkmann, 1981] and in flavone producing flowers of a range of other plants including Verbena [Stotz et al., 1975] Dahlia, Streptocarpus and Zinnia [Forkrnann & Sratz, 1984], reduction of flavanone to flavone is catalyzed by a microsomal enzyme requiring NADPH as co-factor. Although mutants recessive with respect to falvone formation are not known, yet, evidence for the enzyme being involved in flavone formation is provided by the fact that flowers of plants that naturally lack flavones are also devoid of this NADPH dependent microsomal enzyme activity.

Both the parsley and the flower enzyme catalyzed the reaction from (2S)naringenin (flavanone) to apigenin (flavone) (Schcme-6). The mechanism of double bond formation is still unclear. It has been suggested that 2-hydroxyflavanone is formed in the first step, and water is then eliminated via a dehydratase [Britsch et al., 1981, Stotz & Forkmann, 1981]. However, no such 2-hydroxy intermediate has yet been observed even with a nearly homogenous enzyme protein [Britsch & Grisebach, 1986]. On the other hand, 2-hydroxyflavones certainly exist as plant metabolites and they are indeed, the substrates in C-glycosylflavones formation [Kerscher & Franz, 1987].

31

Onosma hispida

HO

HO

[OJ

HO 0 HO 0
Naringenin ~ /",0
OH
HO HO 0 Apigenin (Flavone)

Scheme-6

1.4.3.2.4 Flavonol Formation

Enzymatic conversion of dihydroflavonols to flavonols was first observed with enzyme preparations from parsley cell suspension cultures [Britsch et al., 1981]. Synthesis of flavonols was found to be catalyzed by a soluble 2-oxoglutarate dependent oxygenase. Flavonol synthesis, most probably, proceeds via a 2-hydroxy intermediate such as 2-hydroxydihydrokaempferol with subsequent dehydration, giving rise to the respective flavonols [Hauteville et al.. 1979] (Scheme-7). Flavonol synthase has also been demonstrated in flower extract from Matthiola (Spribille & Forkmann, 1984] and Petunia [Forkmann & Sratz, 1984]. As in parsley, flavonol formation in these flowers is catalyzed by a soluble 2-oxoglutarate dependent dioxygenase.

32

Introduction

HO

101

HO

o

HO

o

Naringenin

Dihydrokaempferol

~IOI

OH

HO

HO

OH

HO

o

Kaempferol (Flavonol)

2 -H ydroxyd i hydrokaem pfero I

Scheme-7

1.4.3.2.5 Glycosylation

A vast number of flavonoid glycosides, found in nature, suggests the occurance of a range of glycosyltransferases with varying substrate specificities [Harbome, 1975, 1982, Stumpf & Conn, 1981].

Novel flavonol O-glycosyltransferases were demonstrated in enzyme preparations from tulip anthers [Kleinehollenhorst et al., 1982]. Chrysosplenium shoots [Bajaj et al .. 1983, Khouri et al.. 1986] and Anethum cell cultures [Mohle et al., 1985]. These enzymes exhibit a pronounced specificity with regard to the substrate, the position and the sugar transferred. The isoflavone 7-0-glucosyltransferase isolated from Cicer shows a similar high specificity [Koster & Barz, 1981]. Two enzymes isolated from Chrysosplenium extracts glycosylate the B-ring of highly methoxylated flavonols in the 2' and 5' positions [Ibrahim, 1986]. A particular interesting situation has been found during extensive genetic and biochemical studies of the glycosylation of isovitexin (6-C-glucosylapigenin)

33

Onosma hispida

in Silene paratensis and Silene dioica [Steyns et al., 1984]. Eleven functional alleles, spread over six loci. have now been identified. These codes for different glycosyltransferases which catalyzed glucosylation, galactosylation and xylosylation of the 7-hydroxyl group or giucosylation, rhamnosylation, xyiosylation and arabinosylation of the 2-hydroxyl group of the carbon bound glucose of isovitexin. Different glycosyltransferases and their sources are listed in Table-6.

1.4.3.2.6 Methylation

Many enzymes catalyzing a methyl transfer from S-adenosylmethionine to the various hydroxyl groups of flavonoid substrates [Harborne & Mabry, 1982J. Some of them require Mg +2 as an obligatory co-factor. S-andenosylhomocysteine formed in the reaction is an inhibitor of these enzymes. An 8-hydroxyflavonol-8-0-methyltransferase was reported from Lotus corniculatus flowers [Jay et al., 1983. 1985]. Lotus flowers also contain 3- and 3'-O-methyltransferases, which have been shown to methylate the flavone (luteolin) and flavonols (quercetin, myricetin and isorhamnetin) in the relevant positions [Jay et al., 1983].

34

Introduction

Table 6: A list of glycosyltransferases and their sources

&.No.. SOUKe ~,: ' - Enqme
S:., iNo~
I I I: I I
1 Silene paratensis a 2" -0- X Y los y ltransferase
(Petals, green parts) b 7 -O-Galactosyltranferase
c 7-0-Glucosyltranferase
d 2"-0-Rhamnoside
glucosyltransferase
------- ------_ .... -- .. ---- ...... --------- --------- ------------------- ...... ------
2 Cicer arietinum a 7-0-Glucosyltransferase
(Roots)
--- ...... --- ... --------------------------- --------- ---------------------------
3 Chrysosplenium a Flavonol 2' and
americanum (shoots) 5'-O-Glucosyltransferase
-------- .. - .. ------ -- .................... ------- -- .. - .. ---- ----------------- .. ---------
4 Anethum graveolens a 3-0-Glucuronosyltranseferase
(Cell cultures)
-------- ---------------------------- -- .... _--- ...... ----------------------- .... ---
5 Pisum sativum a 3-0-Glucosyltranferase
(Flowers) b 3-0-Glucoside
c glucosyltransferase
3-0-Digl ucoside
glucosyltransferase
-------- ---------------------------- --------- --------------------- - ... - ---
6 Tulipa (Anthers) a 3-0-Glucosyltransferase
b 3 -a-Glucoside
c rharnnosy ltransferase
3 -a-Glucoside
xylosyltransferase
-------- ------------------------- .. _- --------- ---------------------------
7 Matthiola incana a 3-0-Glucosyltransferase
(Flowers) b 3-0-Glucoside
c xylosyltransferase
3-0-Glycoside 5-0-
glucosyltransferase 35

Results & Discussion

02. RESULTS AND DISCUSSION

36

Onosma hispida

Our preliminary pharmacological screening of the ethanolic extract revealed inhibitory activity against cholinesterase and lipoxygenase enzymes. This prompted us to carry out phytochemical studies on this plant.The shade-dried whole plant material (10 kg) was extracted three limes with ethanol at room temperature. The combined ethanolic extract was evaporated under reduced pressure to afford a dark-greenish residue which was suspended in water and successively extracted with n-hexane, CHCh, EtOAc and nBuOH. The ElOAc soluable-fraction (lOg) was remixed with the ClIClj-soluble fraction on the basis of samilar TLC profile. The ClICh-soluble fraction was subjected to column chromatography using hexane-Cl+CI, in increasing order of polarity to give six subfractions. The n-hexane fraction was subjected to column chromatography eluting with nhexane-Cl+Cl, in increasing order of polarity to provide four fractions. Their further separation resulting in the isolation of fourteen compounds included one new flavonoid (43), two new aryl amines (44, 45) and eleven known compounds (46-56).

2.1 New Compounds from Onosma hispida 2.1.1 Structure Elucidation of hispidone (43)

OCH3

1-10

OH 0

Hispidone 43

Hispidone (43) was isolated as colorless crystalline solid. The molecular formula was assigned to be C1sH1S07 by high-resolution electron-impact (HR-EI)-MS showing an [Mr ion at m/z 346.1050 (caled. for C1sH1807, 346.1052). The IR spectrum revealed the presence of hydroxyl groups (3452 cm'), a methoxyl group (1588,1112 em"), and an a. ~-unsaturated carbonyl group (1666 ern"). The UV absorption band at i.max 287 nm suggested it to be a flavanone [Mabry et al., 1972],

37

Results & Discussion

The IH-NMR spectrum provided signals of functional groups including a chelated hydroxyl group (& 12.09, IH. s), methoxyl groups [& 3.78 (3H, s, MeO-7), 3.81 (3H, s, MeO-5'), 3.83 (3H, s, MeO-4')], and a dihydropyrone moiety in the flavanone structure showing a characteristic ABX-type coupling pattern [& 2.S\ (I H, dd, J = 17.2, 3.2 Hz) 3.20 (1 H, dd, J = 17.2, 13.4 Hz) (l-b-3), 5.72 (l H, dd, J = 13.4, 3.2 Hz, H-2)] and two superimposable signals at 0 6.05 (d, J"" 2.0 Hz, H-S) and 6.07 (d, J = 2.0 Hz, H-6) of meta-substituted ring A. The two aromatic proton singlets of ring B appeared at 8 6.53 (H-3 ') and 3 7.21 (H-6 ') on a 2',4',5' -tri-oxygenated substituted ring B [Iinuma et al., 1993].

The broad band and distortionless enhancement by polarization transfer 13C-NMR spectra of 43 corroborated the presence of three methyl, one methylene, five methine, and nine quaternary carbons. The signals at (3 75.S and (3 43.2 were typical of C-2 and C-3 carbons of the flavanone skeleton.

EI-MS gave a distinct peak at mlz 346 [M]" and further two fragment ions were observed at m/z 167 ([A+Hf) and m/z 180 ([B3r) which were caused by retro-DielsAlder fragmentation, confirming the one hydroxyl and methoxyl group in ring A, and one hydroxyl and two rnethoxyl groups in ring B of the flavanone skeleton [Iinuma et at" 1993] (Scheme-8).

The position of the hydroxyl and methoxyl groups was further confirmed by heteronuclear multiple-quantum coherence (HMBC) and nuclear Overhauser effect correlations [Iinuma et al., 1993] (Figure-e).

38

Onosma hispida

OCH1· OCH,l co ~.:.....- .. ~

H;CO

Oo-g·

OCHJ

OH 0

OH

m= 318

1.\11+ m/: J~6

H;COvOf

~~,

OH 0

m: 167

m: 180

Scheme-8: Mass fragmentation pattern of 43

Figure-6:

~ I-IMBC correlations of H ;". ", NOE interactions of H

Dihydroflavanones are reported to have the S configuration at C-2 [8aruah et al., 1979 ] which could be confirmed by the CD spectrum which showed similar Cotton effects as reported in the literature [Baruah et al., 1979 and Iinuma et al., 1992]. On the basis of the above evidence, the structure of 43 was determined to be 2S-(S,2' -dihydroxy- 7,4' ,5' -trimethoxyflavanone.

39

Table 7:

13C_NMR (CD30D, 100 m u It i p Ii cit i e S 0 f 43

MHz)

Results & Discussion

chemical shifts and

Muldpli4:ity llC'..:NM_R

(DEPT) (i)

..

1 '

Multi'plieity IJC",NMR

(DEPT ,~)

2'

3'

4'

5'

6'

OMe-7

OMe-4'

OMe-5'

II

3

4

4a

5

6

7

8

8a

-c-

-c-

-c-

CH

-C-

CH

-c-

43.2

197.6

103.3

164.4

95.4

167.5

94.8

163.1

-c-,

-c-

CH

-c-

-c-

CH

147.2

104.9

150.2

144.6

113.6

55.2

56.7

57.3

Table 8: IH-NMR and One-bond HMQC interactions of 43

IH-NMR ~Je .. COJNo IH-NM&
C:,N~ a(.f.: Ih) NMR &~.i~Hz)
(5~
2 I 5.'12\ {dd, IjA, )1.2) 7'S.8 r 3

4

4a

5

6

7

8

8a

3.20 (dd, 17.2, 13.4)

2.81 (dd, 17.2,3.2)

6.07 (d, 2.0)

6.05 (d, 2.0)

43.2

197.6

103.3

164.4

95.4

167.5

94.8

163.1

2'

3'

6.53 (s)

4'

5'

6'

7.21 (s)

OMe-7 3.78(s)

OMe-4' 3.83 (s)

OMe-5' 3.81 (s)

40

r rz.z

147.2

104.9

150.2

144.6

113.6

52.2

56.7

57.3

Onosma hispida

2.1.2 Structure Elucidation of Onosmin A (44)

6 /H
0
6
2' I ~
Onosmin A44 CH,
3' Onosmin A (44) was isolated as a white amorphous solid. The molecular ion peak at mJz 241.l100 in the high resolution electron-impact (BR-EI)-MS indicated its molecular formula to be Cl5BISN02 (caled. 241.1103). The UV spectrum in MeOB showed ;"ma~ at 343 run (log E 3.78). Absorption bands in the IR spectrum of 44 suggested the presence of a carboxylic acid (1680 cm'), amine (3450 ern") and aromatic ring system (1610, 1500, 1460 em"), It gave effervescence with dilute sodium carbonate solution confirming the presence of carboxylic acid group.

Distortionless enhancement by polarization transfer (DEPT) experiments revealed the presence of one methyl, one methylene, six methine and five non-protonated carbons. The down field signal at 8 168.7 could be assigned to the acid carbonyl carbon. The methyl and methylene carbons were observed at 8 20.3 and 3 49.1, respectively. The signals at 3 148.9, 138.5, 135.9. 133.7, 130.3, 130.3 x 2, 127.2 x 2, 116.9. 1 13.5 and l l 1.2 showed the presence of aromatic rings.

In the IH-NMR there were eight aromatic protons which could be assigned to 1,2- and 1,4-disubstituted phenyl rings on the basis of I B-1 B COSY spectrum and the coupling patterns. The 1 H NMR displayed a pair of ortho-coupled A2B2 type signals at 8 7.04 and 0 6.29 (each 2B, d, J = 8. I I-Iz), indicating the presence of a I,4-disubstituted benzene ring. It also showed a dd at (5 6.90 (.J = 7.9, 1,6 Hz, H-6), dd at 8 8.0 (J = 8.6, 1.5 Hz, H-3), a pair of ddd at 8 7.27 (.J = 8.6, 7.2, 1.5 Hz, H-4) and 8 6.67 (.J = 7.9, 7.2, 1.0 Hz, H-5), respectively, confirming the presence of I,2-disubstituted benzene ring. The signals at (5 2.28 (31-1, s), 4.46 (2H, brs) and 8.30 (lH, brs) could be assignable to

41

Results & Discussion

aromatic methyl, benzylic protons and an amine. respectively. The mass fragmentation pattern of 44 is shown in in Scheme-9.

Scheme-s

mlf- 2J1:\f •

miz=196 .. · .. · .... ·• .. ·· .. Jlo ... · .. ·· .... - .... m/~=105

'\ ,. ....

'i.. .~,.

A .. 01-1/ - 45

...:···~·r·~.::i'· .... · ...... · .. · .. · .... mlz=

" .. NH : I "=:

" :

'121 .. • .. • .... • .. ·.... ; /:

~z- . ~

; en,

. -

m/z - 136 :

The heteronuclear multiple-bond coherence (HMDC) spectrum (Figure-Z), the benzylic protons (H- T, 04.46) showed 2 J correlation with C-l' (0 138.S) and 3J correlations with C-2', C-6' (8 127.2 x 2) and C-2 (8 148.9), thus confirming that the secondary amine nitrogen combined with benzyl group and a phenyl ring. The H-6 at 8 6.90 showed 3J correlations with C-2 (8 148.9), C-4 (8 133.7) and C-7 (8 168.7). The methyl protons at 82.28 showed 2J correlation with C-4' (0 135.9) and 3 J correlations with C-3', C-S' (0130.3 x 2), respectively. On the basis of these evidences. onosmin A (44) was assigned the structure as 2-[(4-methylbenzyl)amino]benzoic acid.

Fii:,ure·7: lmpertant 1-[~mC Corrclanons of ~~

42

Onosma hispida

Table 9: 13C-NMR (CDCIJ, 100 MHz) chemical shifts and multi plicities of 44

C.No.,

Mulfipliciqr uC .. NMR, C. NG!

CDEPT) (i)

Multiplkity IJc-~M.R

(DEPT (I]

I r

2

3

4

5

6

7

7'

-c- I In .L \ I~

-C-

CH

CH

CH

CH

-c-

148.9

130.0

133.7

116.9

113.5

168.7

49.1

2'

3'

4'

5'

6'

MeA'

-c- ' 138.5

CH

CH

-c-

CH

CH

127.2

130.3

135.9

130.3

127.2

20.3

Table 10: IH-NMR and One-bond HMQC interactions of 44

IH .. NMR: 1(.1= lIZ)

IJ(Z"" NMR l6,

C.No

2

3

4

5

6

7

7'

8.0 (dd, 8.6, 1.5)

7.27 (ddd, 8.6, 7.2, 1.5)

6.67 (ddd, 7.9, 7.2, 1.5)

6.90 (dd, 7.9, 1.5)

4.46 (brs)

148.9

tt 1.2 r: 138.5

127.2

130.0

133.7

116.9

113.5

168.7

49.1

MeA' 2.28 (s)

2'

3'

4'

5'

6'

NH

7.04 (d, 8.1)

6.29 (d, 8.1)

6.29 (d, 8.1)

7.04 (d, 8.1)

8.30 (brs)

130.3

135.9

130.3

127.2

20.3

43

Results & Discussion

2.1.3 Structure Elucidation of Onosmine B (45)

~l

3"

o

6

4

Onosmin B (45) was also obtained as a white amorphous solid. It was assigned the molecular formula Cl6HI7N02 (m/z 255.1255, calcd. 255.1259) by HR-EI-MS. The UV spectrum in MeOH showed I.ma>; at 353 run (log E 4.11). It showed the presence of ester (1720 em"), amine (3470 em") and aromatic ring system (1600,1510,1455 ern") in its IR spectrum.

The 'u and 13C_ NMR spectra were found to be similar to those of 44 (Table-9, 10), except for an additional signal due to a methoxyl group at OH 3.90 and at Oc 51.6, respectively. The absence of free carboxylic group indicated that 45 is the corresponding methyl ester of 44. The mass fragmentation pattern of 45 is ShOW11 in in Scheme-lO.

mr: = 196 ~ ..

o

mif-255 ,( •

. m/: =105

~ , OM(!.··· "

! ; m/: = 59

h-/NHf~' ~

.' : !

. .

. .

m/: = 134 ~............... : h-

: CHJ

m/: = 150 :

Scheme- to

44

Onosma hispida

Conclusive evidence was provided by HMBC experiments. Besides the interactions previously described for 44, a 3 J correlation was observed by carbornethoxy protons at 0 3.90 and C-7 (8 169.1). Thus, onosmin B (45) was assigned the structure methyl 2-[(4-methylbenzyl)amino]benzoate. Although 44 and 45 are a rare class of compounds which are not previously been isolated from genus Onosma but related compounds have been reported from natural resources [Onoda et al., 1989, Niemann & Versluis, 1990, Qu et al., 2003]. Moreover, these are genuine natural products as their presence has been confirmed in two further batches of fresh plant material.

45

Results & Discussion

Table 11: I3C-NMR (CDCI3, 100 MHz) chemical shifts and multi plic i ties of 45

MlIltipllei'1 (DE~P1)

M.ltiplitily uC-NMR

(QEPT (5)

2

3

4

5

6

7

7'

-c-

-C-

CH

CH

CH

CH

-C-

!'I 0.7

149.4

131.4

134.1

1\6.0

113.6

\69.1

48.9

I I

2'

3'

4'

5'

6'

MeA'

-c-

CH

CH

-c-

CH

CH

138.7

126.5

130.7

135.3

130.7

126.5

20.3

51.6

Table 12: lH-NMR and One-bond HMQC interactions of 45

C. No. IH .. NMR lJ~~ ~.No t· ~ l~C-~MR
II-NMR
3(.1'= Ib:) NMR '(/alk) (~)
(3)
'f IT 0.7 I 7.06 (d, 8.0) 126.5
2'
------- ------------- - .... - --------- -------- ---------------- -----------
2 149.4 3' 6.26 Cd, 8.0) 130.7
------- ... _--------------- --------- -------- ---------------- -----------
3 7.90 (dd, 8.0, 1.6) 131.4 4' 135.3
------- ----------------- --------- ... ------ ... ---------------- -----------
4 7.24 (ddd, 8.0, 7.6, 134.2 5' 6.26 (d,8.0) 130.7
1.6)
------- ----------------- --------- --- ... _--- --_ .... ------------ -----------
5 6.65 (ddd, 7.9, 7.6, 116.0 6' 7.06 (d, 8.0) 126.5
1.6)
------- ------------_ ..... -- --- .. _---- -------- ---------------- -----------
6 6.70 (dd,7.9, 1.6) 113.6 Me-4' 2.30 (s) 20.3
------- -- - -------------- --------- -------- - .. _----------- -- -----------
7 169.1 OCH3 3.90 (s) 51.6
------- -------- - .. - - - - - - ... --------- -------- ---------------- -----------
7' 4.48 (brs) 48.9 NH 8.\ 0 (brs)
-_ .. ---- - ---------------- --------- ---- ... _-- -- - .. - - - - -- ------ -----------
I' 138.7 46

Onosma hispida

2.2 Structural Elucidation of Known Compounds

2.2.1 Structure elucidation of 5,2' -dihydroxy-7,5' -dimethoxyflavone (46)

Compound 46 showed the UV absorption at i.max 323 and 283 nm indicating it to be a flavonoid. The IR spectrum displayed absorption bands at 3433 and 1710 cm·1 due to the hydroxyl and ketonic functions. The molecular formula was confirmed as C!9H1S07, through molecular ion peak in HR-EIMS at m/z 316.0939 (caled. for C!7HI606, 316.0946).

The 'n NMR data showed the signals of two methoxyls and five protons in the aromatic region. Out of the five aromatic protons, two appeared as a doublet at 06.48 (d, 2.0) and 6.11 (d, 2.0) typical for meta-substitution of ring A, and other three proton of ring B showed an ABX system showing signals at 0 7.50 (2H, dd, J = 8.4, 1.8 Hz), 7.30 (l H, d, J = 1.8 Hz) and 6.81 (1 H, d, J = 8.4 Hz). There was a downfield singlet at 12.10 due to chelated hydroxyl group at C-5.

In the mass spectrum peak at m/z 166, 159 after retro Diel Alder fragmentation, confirming the presence of the one methoxyls and one hydroxyl group in ring A. and one methoxyls and one hydroxyl in ring B [Dawson et al., 1966]. The !3C NMR spectrum (BB and DEPT) of compound 46 corroborated the presence of two methyl, six rnethine and eight quaternary carbons. The physical and spectral data of 46 was in complete agreement to those reported in literature for 5,2' -dihydroxy-7,5' -dimethoxyflavone [Hai el al., 1995].

H,CO

OH

o

Compound 46

47

Results & Discussion

2.2.2 Structure elucidation of Apigenin (47)

The absorption at 3490 and 1670 cm-1 in the IR spectrum of 47 was indicative of hydroxyl and carbonyl functions respectively. The UV spectrum showed absorptions at 336, 296 and 267 nm. The EI MS of 33 showed the molecular ion peak at m/z 270. The M+ in HREI MS, established the molecular formula C1sHlO05• The other prominent fragments were observed at mlz 242, 152 and 118.

The lH-NMR showed two doublets each integrating for two protons with same coupling constants (8.6 Hz), showed an AA BB coupling pattern at 8 7.19 (H-3 , H-5 ) and 7.90 (H-i, 1-1-6'). Two more artomatic methines showed their precence in the 'n. NMR spectrum at 8 6.42 (H-6) and 6.80 (H-8) as a pair of broad singlets. The broadness of both singlets revealed that the protons were oriented meta to each other.

The 13C-NMR of 47 showed 13 signals, which were resolved through DEPT experiments as five methine and eight quaternary carbons. The downfeild signals at 0 161.7 and 159.1 revealed their linkage with oxygen atoms and were assigned to C-5 and C-7, respectively [Nikolov et al.. 1982, Escober et al., 1981, Chopin et al., 1977].

With these observations and comparison with the cited data [Agrawal & Schneider, 1982, Lao et al., 1986], the structure of 47 was elucidated as 5, 7, 4- trihydroxyflavone, which is commonly know as apigenin.

1-10

01-1

OJ-[ 0

Api~eDin 47

48

Onosma hispida

2.2.3 Structure elucidation of 3,S,7-trihydroxy-6,4' -dimethoxyflavone

(48)

The HRMS showed molecular ion peak at m/z 330.0785 corresponding to the molecular formula C'7H'407 (caled. for C17I-I'407 330.0739). The UV spectrum displayed maxima at 271 and 341 nm. These values indicated that compound 48 was a 3- hydroxy flavone [Voim, 1983]. The IR spectrum showed bands at 3310 (OH), 1642 (a, punsaturated ketone), 1603 (aromatic).

In 'H-NMR spectrum two methoxyl singlets were present at 6 3.75 and 3.92. A singlet at 0 6.69 was due to H-S. The doublets at 8 8.12, 6.95, showing only orthocoupling (1 H, J = 8.9 Hz) were due to H-2', 6' and H-3', 5', respectively.

In the 13C-NMR spectrum the signals at 0 158.9 (C-2), 136.5 (C-3), 175.9 (C=O) confirmed that 48 was a 3-substituted flavone. The chemical shift of C-5 (8 152.5) supported the presence of a phenolic group at this position [Agarwal, 1989].

The spectral data was in complete agreement to those of reported in literature for

3,5,7-trihydroxy-6,4' -dimethoxytlavone [Wollenweber, 1971, Wollenweber et al.,

1985].

OCH,

H,(O

OH 0

Compound ..\8

49

Results & Discussion

2.2.4 Structure elucidation of 3,5,4' -trihydroxy-6,7-dimethoxyflavone

(49)

Compound 49 showed the UV absorption at ;'ma, 369 and 266 run characteristics of flavonoid. The IR spectrum displayed absorption bands at 3270 and 1640 cm-] due to the hydroxyl and ketonic functions. The molecular formula was confirmed through HRElMS as C17H]407 showing M- peak at mlz 330.0735 Cealed. for C17H1407, 330.0739).

The IH NMR data showed the presence of two methoxyl signals and five protons in the aromatic region. Out of the five aromatic protons, the one which appeared as a singlet at & 6.97 was typical for 5, 6, 7-oxygenated substitution pattern, and other four protons showed signals at 0 7.08 (2R d, J = 8.9 Hz) and 8.25 (2H, d, J = 8.9, Hz), indicating the placement of one methoxyl group at C-4·.

In the mass spectrum the appearance of [A 1 r peak at m/z 196 confirmed the presence of two methoxyls and one hydroxyl group in ring A. The observation of a [B2r fragment at mlz at 121 confirmed the placement of one hydroxyl group in ring B [Mabry & Markham, 1975]. The lJC NMR spectrum (BB and DEPT) of compound 49 corroborated the presence of two methyl, five methine and ten quaternary carbons. The physical and spectral data of 49 was in complete agreement with those reported in literature for 3,5,4' -trihydroxy-o, 7-dimethoxyflavone [Quijano et al., 1970].

01-]

OH 0

Compound 49

50

Onosma hispida

2.2.5 Structure elucidation of apigenin 7-0-P-D-glucoside (50)

The positive FAB mass spectrum exhibited the molecular ion peak at m/z 433.1129 (M-Hr corresponding to the molecular formula C21H200 (caled. for C21H200, 433.112S) consistent with twelve degree of unsaturation. The IR spectrum of 50 showed the absorption bands at 3370 and 1660 em" due to hydroxyl and carbonyl functions in the molecule.

The 19 signals appearing in the broad-band spectrum of 88 were resolved with the help of DEPT experiment, into a methylene, ten methine and eight quaternary carbons. The signal at 94.9 corresponded to C-8, while the signal at 103.0 was due to C-3. The signals at 100.3,77.0,76.4, 73.7, 71.2 and 61.9 were due to sugar moiety.

The I H NMR spectrum showed a singlet at (5 6.75 corresponding to the H-8 position, while the doublets at (5 7.86 (2H, J= 9.0 Hz) and 6.92 (2H, J= 9.0 Hz) were due to H-2', H-6', H-3' an H-S'. respectively. The anomeric proton signal appeared as a doublet at 5.04 with a coupling constant of 7.5 Hz.

The sugar unit was assigned as glucose by comparing the NMR chemical shifts values with the reported data (Sadikum et al., 1996. Redaelli et al., 1980] and after acid hydrolysis of 50, followi ng co- TLC with authentic sample.

By comparison with the data published in literature [Redaelli et al., 1980], 50 was identified as apigenin 7-0-p-o-glucoside.

OH

OH Compound SO

51

Results & Discussion

2.2.6 Structure elucidation of benzoic Acid (51)

The IR spectrum of compound 51 displayed absorption bands at 3270, 2605, 1710 and 1615 ern" due to the hydroxyl, acid carbonyl and aromatic functions. The molecular formula was confirmed as C7H602. through molecular ion peak in HR-EIMS at m/z 122.0364 (caled. for C7H602, 122.0360).

The IH NMR spectrum of 51 displayed three resonances in the aromatic region at 8 8.30 (2H, d, .J = 8.6 Hz, H-2. I-I-6), 7.95 (1 H, t, J = 8.6 Hz, I-I-4) and 7.41 (2H, L J = 8.6 Hz, H-3, H-S), and a carboxylic proton at 0 12.01 (II-l, brs).

The l3C NMR spectrum (BB and DEPT) of compound 51 corroborated the presence of five carbon signals for three methine and two quaternary carbons. The downfield signal at 8 179.7 could be assigned to acid carbonyl where as other signals in the aromatic region at 8 133.9, 130.5, 129.7 and 131.1 were assigned to the aromatic methines and quaternary carbon atoms.

The physical and spectral data of 51 coincided with those described in the literature for benzoic acid [Aldrich Library. 1992].

o

OH

Benzoic acid (51)

52

Onosma hispida

2.2.7 Structure elucidation of 4-bydroxybenzoic Acid (52)

The IR spectrum of compound 52 displayed absorption bands at 3354, 3100, 1715 and 1620 em -I due to the hydroxyl, acid carbonyl and aromatic functions. The molecular formula was confirmed as C7H603, through molecular ion peak in HR-EIMS at m/z 138.0313 (caled. for C7H603, 138.0309).

The 1 H NMR data showed two resonances in the aromatic region at 0 6.70 (2H, d, J = 8.4 Hz, H-2, 6) and 8 7.90 (2H, d, J = 8.4 Hz, H-3, 5) and a carboxylic proton at <5 12.10 (1 H, brs).

The 13C l\TMR spectrum (BB and DEPT) of compound 52 disclosed the presence of five carbon signals for two methine and three quaternary carbons including signal carbonyl group at 0 180.3.

The physical and spectral data of 52 was in complete agreement with those reported in literature for -l-hydroxybenzoic acid [Aldrich Library, 1992, Scott, 1972].

OH

-t-Hydroxybenzoic acid (52)

53

Results & Discussion

2.2.8 Structure elucidation of 5-hydroxy-3,6,7,4' -tetramethoxyflavone

(53)

Compound 53 showed the UV absorption at ;.max 355 and 271 nm indicating it to be a flavonoid. The IR spectrum displayed absorption bands at 3510 and 1730 cm'l due to the hydroxyl and ketonic functions. The molecular formula was confirmed as Cl9Hl807, through molecular ion peak in HR-EIMS at m/z 358.1059 (caled. for CI9H1S07, 358.1052).

The 1 H NMR data showed the signals of four methoxyls and five protons in the aromatic region. Out of the five aromatic protons, one appeared as a singlet at <5 6.48 typical for 5,6,7-oxygenated substitution pattern, and other four showed disubstituted phenyl ring (AB system) showing signals at <5 7.01 (2H, d, J= 9.1 Hz) and 8.06 (2H, d, J = 9.1 Hz), allowing the placement of one methoxyl group at C-4'. There was a downfield singlet at 12.60 due to chelated hydroxyl group at C-5.

In the mass spectrum peak at mlz 196 after retro Diel Alder fragmentation, confirming the presence of the two methoxyls and one hydroxyl group in ring A. and one methoxyl in ring B [Dawson et al., 1966].

The l3C NMR spectrum (BB and DEPT) of compound 53 corroborated the presence of four methyl. five methine and ten quaternary carbons. The physical and spectral data of 53 was in complete agreement to those reported in literature for 5-hydroxy-3,6, 7 A'tetramethoxyflavone [Dawson et al., 1966 and Markham, 1989].

ocu,

OJ-! 0

Compound 53

54

Onosma hispida

2.2.9 Structure elucidation of 5,4' -dihydroxy-3,6,7-trimethoxyflavone

(54)

The HR-EIMS showed the molecular ion peak at m/z 344.0862 corresponding to molecular formula CIgHl607 (calcd. for C1sH1607, 344.0896). The UV spectrum displayed the maxima at 357 and 272 run. These values indicated that compound 54 is a flavone being substituted at C-3 [Voirn, 1983]. The IR spectrum showed the absorption bands at 3335,1650, 1600, 1370 and 888 em-I.

The I H NMR spectrum showed a singlet at 8 6.49 corresponding to the H-8 position, while the doublets at <') 7.09 (2H, .J = 8.7 Hz) and 8.03 C2H, .J = 8.7 Hz) were due to H-2', H-6', H-3' and H-5', respectively.

The DC NMR spectrum CBB and DEPT) of compound 54 corroborated the presence of three methyl, five methine and ten quaternary carbons. The signal at 94.6 corresponded to C-8, while the signal at 138.1 was due to C-3. The signals at 60.3,60.5 and 55.9 were due to three methoxyl groups.

By comparison with the data published in literature [Flores & Herran, 1958], 54 was identi fied as 5,4' -hydroxy-3,6, 7 -trimethoxyflavone, reported previously from Brickellia pendula.

OH

0]-] 0

Compound 54

55

Results & Discussion

2.2.10 Structure elucidation of methyI4-methyltetradec-9-enoate (55)

The IR spectnun of compound 55 displayed absorption bands at, 1720 and 1665 cm-l due to the ester carbonyl and double bond. The molecular formula was confirmed as C16H3002, through molecular ion peak in HR-EIMS at mlz 254.0311 (caled. for C 16H3002, 254.0309).

The lH NMR data showed resonances for double bond at 8 5.30 (2H, m, H-9, 10), 3.60 (s, OCH3), C-4 and terminal methyl were observed at 00.90 (3H, s) and 8 0.88 (l H, t, J = 6.7 Hz). The signals between 8 1.07-2.0 were indicative of long chain hydrocarbon.

The 13C NMR spectrum (BB and DEPT) of compound 55 disclosed the presence of three methyls, nine methylenes, three methine and one quaternary carbons. The 13C NMR spectrum showed peaks for carbonyl group at 175.6 and signals for olefinic carbons at \30.7 and 130.1. The methoxy group resonated at 8 51.7. The signals between 32.0 and 22.5 (all methylene in DEPT) were indicative of long chain hydrocarbon.

The physical and spectral data of 55 was in complete agreement to those reported in literature for methyI4-methyltetradec-9-enoate (Mellidis & Papageorgiou, 1987].

Compound 55

1-13C............ ~ ..... (CH)lY<Cl!l):yO

(Cl-Lh/ ~ 4 J ""

- . 9 CH)

o

56

Onosma hispida

2.2.11 Structure elucidation of methyl 4-methyltetradeca-9, 12-dienoate

(56)

The IR spectrum of compound 56 displayed absorption bands at, 1715 and 1660 em" due to the ester carbonyl and double bond. The molecular formula was confirmed as Cl6H2802, through molecular ion peak in HR-EIMS at m/z 252.0311 (caled. for C16H2S02, 252.0309).

The 1 H NMR data showed resonances for double bonds at 0 5.34 (2H, m, H-Il, 12), 5.20 (2H, m, H-9, 10) 3.64 (s, OCH3), C-4 and terminal methyl were observed at (5 0.96 (3H, s) and at () 1.67 (I H, d, J = 6.7 Hz). The signals between 0 1.09-2.68 were indicative of long chain hydrocarbon.

The l3C NMR spectrum (BB and DEPT) of compound 56 corroborated the presence of three methyl, seven methylene, five methine and one quaternary carbons. The l3C NMR spectrum showed peaks for carbonyl group at () 176.4 and signals for olefinic carbons at 0 131.6, 130.3, 129.3 and 125.0. The signals between 0 36.4 and 29.5 (all methylene in DEPT) were indicative of long chain hydrocarbon. The methoxy group resonated at IS 51.4.

The physical and spectral data of 56 was in complete agreement to those reported in literature for methyl 4-methyltetradeca-9, 12-dienoate [Mellidis & Papageorgiou, 1987].

Compound 56

He 12 :---..

, ~(CI-[2)~y(CHJ:'yo"""

9 CI-L,

o

57

Results & Discussion

2.3 Cholinesterase inhibitory activity

According to the cholinergic hypothesis, memory impairment 111 patients with senile dementia of the Alzheimer type results from a deficiency in cholinergic function in the brain [Perry, 1986, Bartus et al., 1982]. Hence the most promising therapeutic strategy for activating central cholinergic function has been the use of cholinergomimetic agents. The enzyme acetyl cholinesterase (AChE) has long been an attractive target for rational drug design and the discovery of mechanism-based inhibitors for the treatment of Alzheimer disease (AD). The aim of administering acetylcholinesterase inhibitors is to boost the endogenous levels of' acetylcholine in the brains of AD patients, thereby increasing cholinergic neurotransmission. Recently, it has also been found that butyryl cholinesterase (BChE) inhibition may be an effective tool for the treatment of AD and related derneruias [Yu et a!., 1999]

2.3.1 Cholinesterase inhibitory activity of Hispidone (43) and 46

In the course of this investigation we evaluated compounds 43 and 46 to determine their enzyme inhibitory activity against AChE and BChE, at various concentrations. Both compounds were found to be potent cholinesterase inhibitors and inhibited enzymes in a concentration-dependent manner with the ICso values 11.6 and 28.0 11M against AChE and 15.7 and 7.9 !lM against BChE, respectively. Compound 46 was selective for BChE, whereas compound 43 showed significant inhibitory action against AChE but the galanthamine positive control was approximately 5 times more potent against AChE (Table-B).

Table 13: In vitro quantitative inhibition of AChE and BChE by compounds 43 and 46

S.,Ne. Compound. cAChE BebB
1e:$O (p.M) ~ (pM)
1 43 11.6 ± 0.6 15.7±2.0
---- ... _------ - - - - - - - - - - - - - _ ... - - - - .. ------------------- ---------------------
2 46 28.0 ± 5.0 7.9 ± 1.6
------------ --- --_ .......................... - .... ---------------- ---------------------
3 Galanthamine 32.2 ± 2.5 163.0± 5.0 ICjo values are the mean ± standard mean (SEM) error of three assays

58

Onosma hispida

2.4 Lipoxegenase inhibitory activity

Lipoxygenases (LOX, EC l.13.11.12) constitute a family of non-haem iron containing dioxygenases that are widely distributed in animals and plants, The non-haem iron serves as a catalytic center for the stereo- and regia-specific dioxygenation of select carbon atoms in polyunsaturated fatty acids containing a 1 A-pentadiene motif. Linoleic acid is the primary substrate of the plant LOXs while the mammalian isozymes mainly catalyze the metabolism of arachidonic acid. In mammalian cells LOX are key enzymes in the biosynthesis of many bioregulatory compounds such as hydroxyeicosatetraenoic acids (HETEs), leukotrienes, lipoxins and hepoxylines.

It has been found that these LOX products playa role in a variety of disorders such as bronchial asthma, inflammation [Steinhilber, 1999] and also profound influence on the development of several human cancers [Ding et al., 2001]. LOX are therefore potential target for the rational drug design and discovery of mechanism-based inhibitors for the treatment of bronchial asthma, inflammation. cancer and autoimmune diseases.

2.4.1 Lipoxegenase inhibitory activity of Onosmin A (44) and B (45)

Compounds 44 and 45 inhibited LOX enzyme in a concentration-dependent manner with K, values 22.0 pM and 31.1 ,liM and IC50 values 24.2 ,liM and 36.0 ,liM, respectively (Table-14). K, values were calculated in three ways; first, the slopes of each line in the Lineweaver-Burk plot were plotted against different concentrations of 44 and 45. Secondly the lIV'maxapp was calculated by plotting different fixed concentrations of linoleic acid versus 6.V in presence of different fixed concentration of 44 and 45, and then K, was calculated as an intercept on the x-axis, by plotting different concentrations of 44 and 45 versus lIVma"app. In third method, K, was directly measured from Dixon plot as an intercept on x-axis. Determination of the inhibition type is critical for the identification of mechanism of inhibition and the sites of inhibitor binding. Lineweaver-Burk and Dixon plots and their replots indicated pure non-competitive type of inhibition of 44 and 45 against LOX enzyme. In other words we can say that 44 and 45 and linoleic acid bind randomly and independently at the different sites of LOX. It indicates that inhibition depends only on the concentrations of 44 and 45 and dissociation constant (Ki).

59

Results & Discussion

From results (Table-14), it is clear that 44 containing benzoic acid pharmachophore posseses strong (K, = 22.0 pM) binding strength against LOX than 45 containing methyl benzoate moiety (K, = 31.1 pM). The higher inhibitory potential of 44 may also be due to the presence of -COOH, which has strong electron withdrawing effect than -COaCH] that can convert active state Fe-2 of LOX into inactive Fe"3 more efficiently.

Table 14: In vitro quantitative inbibition of lipoxygenase by compounds 44 and 45

S~Ntw IC~ :I:: Ii a) :I:: T~ ot IDltibition
-I
Cbmpounds SEM'b)IluMI SEMLuMt
1 I 44 24.2 ± 0.54 22.0 ± 0.1 I Non-competitive
------- --------- - - -- .. - _ .. - - - - - ------- - .... ---------- ---------------------
2 45 36.0 ± 0.03 31.1± 0.05 Non-cornpetiti ve
------- - -B~~~I-ei~cf -- ------------ ...... - -------- - - - -- -------------.-------
3 22.0 ± 0.05 18.0 ± 0.02 Mixed-type
.. ,) K, (dissociation constant or inhibition constant) was determined from nonlinear regression analysis by Dixon plot and secondary Lineweaver-Burk plot at various concentrations of 44, 45. (Each point in Lineweaver-Burk and Dixon plots and their replots represents the mean of three determinations).

b) Standard mean error of 3-5 assays, c) Positive control used in assays

60

-

'c=

.= 3

""'"

'::1

(I
-2 (I 2 4 6 8 leD
»s (rn ~1)
5

- 4
"'
..§
....: 3
-
c
E 2
.3-
- I
-4 0 4 8 12
i.s (mM) --:_ 4

:::1

Onosma hispida

0.6-.-------.--------,

'-' c,

....

o: 04 c:.

c,

,

~ 02

~ Slope C

·20 (I 20 ·10 GO 0 +-r-F'-r +r+r- +-r---,--"T---,---y-,--,--,--,-!

Cone.ofcompoWld 44 < .".11.1")

61

-25

(I

25

50

·25 (I 25 50 Core. ofcompound 45 ( ."\\)

Cone. of compound 44 ( 1'~1)

1.6-.-------.------."

i!.

~ 12

CJ:

Co. <:.

~ 0.8 ::

F

0.4

-30 -[5 0 IS 30 45

Cone.ofcompOlllld 45 (.u\1)

Figure-8: Steady-state inhibition of lipoxygenase enzyme by compounds 44-45_ (A) is the Lineweaver-Burk plot of reciprocal of initial velocities versus reciprocal of four fixed linoleic acid (substrate) concentrations in absence (_) and presence of 10 JIM C.-), 20 JIM (e), 40 JIM (.6.) of compound 44, (8) is the Dixon plot at tour fixed linoleic acid concentrations, (_) 0.2 mM, ( ... ) 0.1 mM, (e) 0.066 mM and (.6.) 0.05 mM. (C) is the secondary replots of the Lineweaver-Burk plot, 11 Vma'iapp or slope versus various concentrations of compound 44. (D) is the Lineweaver-Burk plot of reciprocal of initial velocities versus reciprocal of four fixed linoleic acid (substrate) concentrations in absence (_) and presence of 10 JIM ( ... ), 20 .uM (e), 40 .uM (.6.) of compound 45, (E) is the Dixon plot at four fixed linoleic acid concentrations, (_) 0.2 mM, ( ... ) 0.1 mM, (e) 0.066 mM and (.6.) 0_05 mM. (F) is the secondary replots of the Lineweaver-Burk plot, lIVma.\app or slope versus various concentrations of compound 45. Correlation coefficient for all the lines of all the graphs was> 0.99, each point in the graphs represents the mean of three experiments.

Experemintal

03. EXPERIMENTAL

. I

I I

62

Onosma hispida

3.1 General Experimental Conditions 3.1.1 Physical Constants

3.1.1.1 Melting points: Melting points were determined In glass capillaries using Buchi 535 melting point apparatus and are uncorrected.

3.1.1.2. Optical rotations: Optical rotations were measured on JASCO DIP-360 digital polarimeter.

3.1.1.3 Solvents: All commercial grade organic solvents were doubly distilled before use. In some cases analytical grade solvents (Merck) have also been used.

3.1.2 Spectroscopy

3.1.2.1 UV Spectra: Ultraviolet CUY) spectra were recorded in methanol on Hitachi U- 3200 spectrophotometer.

3.1.2.2 IR Spectra: Infrared (IR) spectra were measured as a KSr pellet or In chloroform on JASCO 302-A Infrared Spectrometer.

3.1.2.3 Mass Spectra: Low-resolution electron impact mass spectra were recorded on a MAT 311 A mass spectrophotometer or FilU1iganMA T 312 double-focusing mass spectrophotometer, coupled with PDP 11/34 computer system. Peak matching, field desorption (FD) and field ionization (FI) were performed on the Finnigan MAT 312 mass spectrometer. High resolution mass measurements and fast atom bombardment (F AS) mass measurement were carried out on Jeol JMS HX 110 mass spectrometer: F AB source using glycerol or thioglycerol as the matric while cesium iodide (CsJ) was used as internal standard for accurate mass measurements.

3.1.2.4 Nuclear magnetic resonance (NMR) Studies: 'J-J-NMR spectra were recorded at 300, 400 and 500 MHz on Bruker AM-300, At\1-400 or AMX-500 nuclear magnetic resonance spectrometers using TMS as an internal reference. The I3C-NMR

63

Experemintal

spectra were performed in the same instruments at 75. 100 and 125 MHz, respectively. The chemical shift (is) values were reported in ppm and coupling constants (J) were measured in Hz.

20 NMR spectra were recorded on a Bruker AMX 500 NMR spectrometer. The heteronuclear 20 IH_I3C chemical shift correlation experiments were carried out at 500 MHz with a sweep width of 12820 Hz (2K data points) in 0)2 and 1024 Hz (256 tl values zero-filled to 2K) in co I. In both the 2D experiments, a 2 sec. relaxation delay was used and 16 transients were performed for each tl value.

For NOE difference measurements, the sample was frozen under liquid nitrogen and degassed. A lower decoupler power of 0.2 watt with 35 attenuation in dbs was used. The pre-irradiated time was II sec; which is the sum of three delays as used in the NOE difference programme of Bruker. The impulse length of 100 microsecond was maintained to avoid saturation.

3.1.3 Technique employed for the purification of compounds

3.1.3.1 Column chromatography: Column chromatography (CC) was carried out using silica gel (Si 60, 70-230 mesh, E. Merck) as stationary phase and organic solvents as mobile phase.

3.1.3.2 Flash column chromatography: Flash column chromatography (FCC) was performed on Eyela Flash Chromatograph model EF-IO, using silica gel (Si 60, 230-400 mesh, E. Merck) as adsorbent.

3.1.3.3 Thin layer chromatography: Precoated silica gel GF254 preparative plates (20x20, 0.5 mm thick) (E. Merck) were used for preparative layer chromatography. Purity of the samples was also checked on the same precoated plates.

3.1.3.4 Vacuum liquid chromatography: VLC was performed on silica gel (Si 60, E. Merck).

64

Onosma hispida

3.1.3.5 Gas chromatography mass spectrometry: The GC was performed on a shirnadzu gas chromatograph (GC-9A) (3% OV -1 silanized chromosorb W, column temperature 180°C, injection port and detector temperature 275-300°C, flow rate 35 ml/min, flame-ionization detector).

3.1.4 Spray reagents for visualization of spot

3.1.4.1 eerie sulphate reagent: Ceric sulphate (0.1 g) and trichloroacetic acid (I g) were dissolved in 4 ml distilled water. The solution was boiled and cone. H2S04 was added drop wise until the disappearance of turbidity. The TLC plates were visualized by spraying this reagent.

3.1.4.2 Iodine: A few iodine crystals were placed in a TLC tank and warmed for few minutes on a water bath (40-50°C). Spots appeared on the TLC plate when placed in the tank for one minute.

3.2 Plant Material

Onosma hispida Wall. (Boraginaceae), whole plant was collected from Swat (Pakistan) in 2002 and identified by Dr. Jahander Shah, Plant Taxonomist, Islamia College Peshawar. A voucher specimen (No.BPU-55) has been deposited in the herbarium of the University of Peshawar.

3.3 Extraction and Isolation

The dried whole plant material (10 kg) was extracted three times with ethanol at room temperature. The ethanol extract was evaporated under reduced pressure to afford a dark-greenish residue which was suspended in water and successively extracted with nhexane, CHCb, EtOAc and n-BuOH (Scheme-ll). The CHC13 fraction was subjected to VLC over silica gel, successively eluting with n-hexane- CHCh in increasing order of polarity to obtain six fractions (A-F) (Scheme-12). While the n-hexane fraction on column chromatography eluting with n-hexane-CHCb in increasing order of polarity provided four fractions (G-J) (Scheme-13).

65

Experemintal

The Fraction A obtained from n-hexane-CHCb-(8:2) was a mixture of two components and was subjected to FCC and eluted with hexane-EtOAc (9.5:0.5 and 9: 1) as the eluent and the compounds 51 (20 mg) and 52 (16 mg) were obtained.

The fraction B obtained from hexane-ClICl, (6:4) was further purified by colwnn chromatography over silica gel using hexane-Cl+Cl, (7:3) as solvent system to afford compound 46 (14 mg).

The fraction C obtained from hexane-Cl-lCl, (1 :1) was subjected to column chromatography on silica gel using hexane-ClICl, (6:4) to obtain crystals of compound 43 (8 mg), which were finally washed with hexane to remove colored impurities.

The fraction D which eluted with n-hexane- CHCb (3:7) showed two major and two minor spots on TLC. It was further subjected to flash column chromatography using n-hexane-CHCh (l: 1) to afford compound 47 (12 rng) and n-hexane-chloroform (4:6) to obtain compound 48 (IS mg),

The fraction E which eluted with n-hexane-CHClJ (1 :9) showed two major spots on TLC. It was further subjected to column chromatography using n-hexane-CHCb (3 :7) as eluent to afford compound 49 (14 mg) and n-hexane-CHCI3 (1 :9) to provide 50 (18 rng), respectively.

The Fraction G obtained from n-hexane-CHCI3 (8:2) was a mixture of two components, which were separated by flash column chromatography using solvent system n-hexane-CHCh (9.5:0.5) and n-hexane-CHCh (9: 1) to afford compounds methyl-l,4-methyl tetradec-9-eneoate (55) (30 mg), methyl-4-methyl tetradec-9. 12- dieneoate (56) (27mg), respectively.

The fraction H obtained from n-hexane-CHCb (6:4) was flash chromatographed using n-hexane-CHCl3 (9:1-1:1) as a solvent system to give two successive fractions, The second fraction afforded onosmin A (44) (10 mg) by silica gel column chromatography using n-hexane-CI-ICb (8:2) as eluent. The first fraction which was purified through column chromatography over silica gel using n-hexane-CHCI3 (9: 1) as eluent to afford onosmin B (45) (13 mg).

The Fraction I obtained from n-hexane-Cl+Cl, (3:7) showed two major spots and was flash chromatographed using hexane-Cl+Cl, (7:3 and 4:6) as eluent to afford the

66

Onosma hispida

compounds 53 (18 mg) and 54 (18 mg), respectively. The Fractions F and J could not be worked-up due to paucity of material.

Extraction and Fractionation of Onosma hispida

Onosma hispida
10 Kg
Powdered. extracted with ethanol
and concentrated on rotary evapora
I Ethanolic extract I
500 g
n-hexane ~ water
1
luble fraction I I Hexane soluble fraction I
30 g ter

Hexane inso

Extraction with chloroform and ethyl acetate

1\
.~
I Chloroform soluble I [ Ethyl acetate soluble Insoluble fraction I
I fraction 40 g fraction 109
Partitioned between
n-butanol and water
1
Butanol fraction I Aqueous fraction
160 g
Scheme-Ll

67

Experemintal

Isolation Scheme of Chloroform Soluble Fraction


I Chloroform Soluble Fraction I
Subjected 10 VLC
by using n-hcxanc:CIICl) in increasing
order of polarity
1 1 1
Hl:xam;:CHC1, Hexanc:CHCI] l-Iexane:CHC1] Hcxanc:CHCIj Hexanc:CHC13 CHCI] I
(8:2) (6:4) (5:5) (3:7) (1:9)
Fracuon A Fracuorr B Fracuon C fraction D Fracnon E Fracuon f
Further CC on L(
I T\\o major spots I sillc~:i)~1 ull:inil; I Mixture 0 f t\\ 0 !
Hex . CH'CI! components I
I I
Two major
Further FCC on flJ.!1ti~r CC on Further FCC on spots
slll<;,.a~l using s.1~~~ ~d using Silica gel usmg
Hex CHCll Hex (,Hel) Hex. cnrr,
Hexane:CHC1]
r 1
(7:3)
I Hcxanc:CHCl3 Hexanc;c"IC'31
(5:5) (4:6) Further CC on
46 srlrca gel uSing
- Hcxanl::CHCIJ Hex' C!-ICI)
(6:4)
47 48
43
II Hcxane:CI:Clj !HCxam::CI-ICIJI ~
(95:0,,) (9:1)
I HcxaJ1c:CHOI
I HexaJ1c:CHCl
(3:7) (1:9) II
51 52
49 50
Scbeme-J2 68

Onosma hispida

Isolation Scheme of Hexane Soluble Fraction

I Hexane Soluble Fraction I

Subjected to column
chromatography
1 1 1
Hcxanc:CHCl3 Hcxanc:CHCI, Hexanc:C1-1CI, . CHCI)
(8:2) ( 6:4) (3:7)
fraction CO Fraction H Fraction I Fraction J
~ Further FCC on
silica gel using
Hex' CHel)

I Two major I
spots
I I 1
I Two major I

spots Further FCC on . Hcxanc:CHCI IHexanC:CHCI1
silica gel using (7:3 ) (6:4)
l-urthcr FCC on Hex: cucr, j
silica gel using I I I
Hex: CHC1, Fraction 1 Fraction 2
54 53

Hcxanc.C I-[C I, (9.5:0.5)

I-kxanc:CHC1, (8:2)

, 56

, 45

44

55

Scheme-IS

69

Experemintal

3.4 Enzyme Inhibition Assays

3.4.1 In vitro cholinesterase inhibition assay

AChE~ and BChE~jnhibiting activities were measured by slightly modifying the spectrophotometric method developed by Ellman et. al., 1961. Electric-eel AChE (type VI-S, Sigma), and horse-serum BChE (Sigma) were used, while acetylthiocholine iodide and butyrylthiocholine chloride (Sigma), respectively, were used as substrates of the reaction. 5,5' -Dithiobis [2-nitrobenzoic acid] (OTNB. Sigma) was used for the measurement of cholinesterase activity. A mixture of 140 I.d of 0.1 mM sodium phosphate buffer (pH 8.0), 1 0 ~l of OTNB, 20 ~I of test compound solution, and 20 ~l of AChE or BChE solution was incubated for 5 min at 25°C. The reaction was then initiated by the addition of 10 fll of acetylthiocholine or butyrylthiocholine, respectively. The hydrolysis of acetylthiocholine and butyrylthiocholine was monitored by the formation of yellow 5-thio-2-nitrobenzoate anion as the result of the reaction of OTNB with thiocholine, released by the enzymatic hydrolysis of acetylthiocholine and butyrylthiocholine, respectively, at a wavelength of 412 nm. Test compounds and the control were dissolved in 5% EtOH. All the reactions were performed in triplicate.

3.4.1.1 Estimation of ICso values

The concentrations of test compounds that inhibited the hydrolysis of substrates (acetylthiocholine and butyrylthiocholine) by 50% (lCso) were determined by monitoring the effect of increasing concentrations of these compounds in assays of the inhibition values. The ICso values were then calculated using the EZ-Fit Enzyme Kinetics program (Perrella Scientific Inc., Amherst. MA, USA).

3.4.2 In vitro Lipoxygenase inhibition assay

LOX inhibiting activity was measured by modifying the spectrophotometric method developed by Tappel, 1962. Lipoxygenase (1.13.11.12) type I-B (Soybean) and linoleic acid was purchased from Sigma (St. Louis, MO, USA). All other chemicals "· .. 'ere of analytical grade. The assay conditions and protocol was the same as described in our

70

Onosma hispida

previous article [Mukhtar et al., 2004). All the kinetic study was performed in 96-well micro-titre plates by using SpectraMax 384plus (Molecular Devices, CA USA). The fC50 values were then calculated using the EZ-Fit Enzyme kinetics program (Perrella Scientific Inc., Amherst, USA). The percentage (%) inhibition was calculated as follows (E - S)/ E x 100, where E is the activity of the enzyme without test compound and S is the activity of enzyme with test compound.

3.4.2.1 Determination of kinetic parameters

Dissociation constant/inhibition constant (K,) was determined by the interpretation of Dixon plot [Dixon, 1953], Lineweaver-Burk plot [Segel, 19931, and their secondary replots using initial velocities obtained over a substrate (linoleic acid) concentration range between 0.05-0.2 mM. The dependence of Vma, / «; and ~na, on 44- 45 [I] is given by:

KI

-- ~ K[ = K[ + [ I ]

K[ + [ I ]

3.4.2.2 Statistical analysis

Graphs were planed using GraFit program [Leatherbarrow, 1999] Values of the correlation coefficients, slopes, intercepts and their standard errors were obtained by the linear regression analysis using the same program. The correlation of for all the lines of all graphs was found >0.99. Each point in the constructed graphs represents the mean of three experiments.

71

3.5.1 Characterization of hispidone (43)

Physical Data

Colorless crystalline solid (yield 8 mg). MP: 192-193°C

[a]D25: -8.5° (c 0.11, MeOH) UV "-max (log s) (MeOH) nm:

287 (4.31) and 340 (3.82).

IR (KBr) vmax em":

3452, 2932, 1666. 1638, 1588 and 1112.

IH NMR (CD30D, 400 MHz): see Table-8. I3C NMR (CD30D, 100 MHz): see Table-7. HR-EIMS m/z:

346.1050 (caled for C1sH1s07, 346.1052).

ElMS m/; (rei. into 0/0):

[Mf 346 (70), 315 (8), 180 (64) and 167 (100).

OH 0

72

Experemintal

HO

.. ",KOCH'

'2~

3 OCH3

43

Onosma hispida

3.5.2 Characterization of Onosmin A (44)

Physical Data

White amorphous solid (yield 10 mg). MP: 185-187°C

UV Amax (log s) (MeOH) om: 343 (3.01) and 284 (2.51).

IR (KBr) vma:\ em":

3450, 1680, 1610, 1500 and 1460.

1" NMR (CDCI3, 400 MHz): see Table-IO. BC NMR (CDCI3, 100 MHz): see Table-9. HR-EIMS m/z:

241.1100 (ealed for CI5HISN02, 241.1103).

ElMS m/z (rei. into 0/0):

[Mf 241 (15), 196 (30),136 (40),121 (l00) and 105 (45).

o

73

3.5.3 Characterization of Onosmin B (45)

Expereminial

o

Physical Data

White amorphous solid (yield 13 mg). MP: I37-140°C

UV Amax (log E) (MeGH) om: 353 (2.90) and 275 (2.45).

IR (KBr) vrnax em-I:

3470,1720,1600,1510 and 1455.

IH NMR (CDC)3, 400 MHz): see Table-12. 13C NMR (CDCI3, 100 MHz): see Table-II. HR-EIMS m/z:

255. I 255 (ealed for CI6H17N02, 255.1259).

ElMS m/z (rei. int.):

[Mf 255 (12), 196 (23), 150 (34), 134 (50), and 105 (100).

6

45

74

:r

Onosma hispida

3.5.4 Characterization of of 5,2' -dihydroxy-7, 5' -dimethoxyflavone (46)

I-IO

f[,c0'f'Y0'l",,"Q

yy OCI-!,

011 0

Physical Data

Crystalline solid (yield 14 mg). MP. : 192-193 °c

[aJ])25: -8.5° (c 0.11, MeOH). UV Amax (log E) (MeOH) om:

283 (3.28) and 323 (4.10).

IR (KBr) vmax em-I:

3433 (OH), 1587, 1110 (OCH3), 1710 and 1628 (0:, p-unsaturated C=O).

IH NMR (CD30D, 400 MHz):

46

s 12.10 (chelated hydroxyl group at C-5), 7.50 (2H, dd, J = 8.4, 1.8 Hz, H-4 '), 7.30 (1 I-I, d, J = 1.8 Hz, H -6 .), 6.81 (1 H, d, J = 8.4 Hz, H-3 '), 6.48 (llt d, J = 2.0, H-6), 6.11 (l H, d, J= 2.0 Hz, H-8), 5.70 (lH, d, J= 13.1,3.3 Hz, H-2), 3.71 (3H. s, OCH)-7), 3.79 (3R s, OCH3-5'), 3.15 (IH, dd, J = 16.9, 13.1 Hz, 1-1-3) and 2.78 (IH, dd,J= 16.9,3.3 Hz, H-3).

DC NMR (CD30D, 100 MHz):

s 197.8 (C-4), 167.9 (C-7), 164.2 (C-6), 153.2 (C-5'), 146.0 (C-2'), 126.3 (C-1'), 115.2 (C-3'), 113.5 (C-4'), 111.8 (C-6'), 103.3 (C-4a), 95.1 (e-6), 94.2 (C-8). 76.2 (C-2), 55.6 (OCH3-7), 55.3 (OCH)-5 ') and 44.1 (C-3).

HREIMSmll:

316.0939 (calcd. for C 17H 1606, 316.0946).

ElMS mil (reI. int.):

[Mf 316 (100).166 (60).159 (45).

75

Physical Data

Yellowish powder (yield 12 mg). laJ])25: -58.6° (c 0.14. MeOl-I). UV Amax (log e) (MeOH) om:

336 (0.92), 296 (0.68) and 267 (1.00).

IR (KBr) vmax cm': 3490, 1670. 1610, 1505 and 1360.

IH NMR (CDCl3, 400 MHz):

s 7.90 (2H, d,.J = 8.9 Hz, H-2' and H-6'), 7.19 (2H, d,.J = 8.9 Hz, H-3 'and H-5')~ 6.89 (IH, s, H-3), 6.80 (lH, s, 1-1-8) and 6.42 (tH, s, H-6).

13C NMR (CDCl3, 100 MHz):

<5 181.9 (C-4), 165.5 (C-2), 162,9 (C-4'), 161.7 (C-5), 159.1 (C-7), 158.0 (C-9),

3.5.5 Characterization of apigenin (47)

Experemintal

OH

HO

OH

o

·41

129.2 x 2 (C-2' & 6'),123.5 (C-I0), 117.1 x 2 (C-3' & C-5'), 105.3 (C-I0). 104.3 (C-8) and 103.5 (C-3).

HR-EI-MS m/z:

270.0521 (ealed. for CI5H1oOs, 270.0528).

ElMS m/z (reI. int. 0/0):

[Mr 270 (100), 248 (38), 152 (34), 151 (38), 124 (25), 121 (50) and 118 (31),

76

Onosma hispida

3.5.6 Characterization of 3,5,7 -trihyd roxy-6,4' -dimetboxyflavone (48)

Physical Data

Amorphous light yellow powder (yield 30 mg). UV Am,'I\ (log e) (MeOH) om:

271 (3.71) and 341 (2.3 I).

IR (KBr) vrnax cm': 3310,1642,1603, 1350 and 860.

IH NMR (CD30D, 300 MHz):

08.12 (2H, d, J= 8.9 Hz. H-2' and H-6'), 6.95 (2H, d,.J = 8.9 Hz, H-3 'and H-S').

HO

01-1

48

6.69 (1 H, s, H-8), 3.92 (3H. s, OMe-6) and 3.75 (3H, s, OMe-4).

DC NMR (CD30D, 100 MHz):

o 17S.9 (C-4), 158.9 (C-2), 157.5 (C-4'), 152.5 (C-5), 151.5 (C-7), 149.2 (C-9), 136.5 (C-3), 131.5 (C-6), 130.1 (C-2 & C-6'). 121.8 (C-1'), 115.4 (C-3'), 115.4 (C-5'). 105.1 (C-lO), 91.8 (C-8), 56.8 and 55.4 (MeO-3 & MeO-4').

HREIMSmi'z:

330.0755 (calcd. for C17H1407, 330.0739).

ElMS mil. (rei. int. 0/0):

[Mr 330 (100), 315 (18), 318 (5),183 (25) and 135 (32).

77

Experemintal

3.5.7 Characterization of 3,5,4' -trihydroxy-6,7-dimethoxyflavone (49)

Physical Data

Amorphous light yellow powder (yield 28 mg). MP: 289-292 -c.

UV A.uax (log E) (MeOH) nm: 266 (4.06) and 369 (3.90).

IR (KBr) Vmax em-I: 3270,1640,1603,1350 and 860.

'H NMR (CD]OD, 300 MHz):

08.25 (2H, d, J= 8.9 Hz, H-2'and H-6'), 7.08 (2H, d. J= 8.9 Hz, H-3' and H-5'),

01-1

J-I,CO

H3CO

01-1 0

49

6.69 (1 H, s, H-8), 3.92 (3H, s, OMe-6) and 3.75 (3H, s, OMe-4).

BC NMR (CD30D, 100 MHz):

s 175.9 (C-4), 158.9 (C-2), 157.5 (C-4'), 152.5 (C-5), 151.5 (C-7), 149.2 (C-9), 136.5 (C-3), 131.5 (C-6), 130.13 (C-2' & C-6'), 121.8 (C-l'), 115,4 (C-3'), 115,4 (C-5'), 105.1 (C-IO), 91.8 (C-8), 56.8 and 55,4 (MeO-3 & MeO-4').

HR-EIMS m/z:

330,0747 (ealed. for C17H1407, 330.0739).

EI-MS m/z (reI. int.):

[Mf 330 (100), 315 (30).312 (6).196 (21) and 121 (33).

78

Onosma hispida

3.5.8 Characterization of apigenin 7-0-fJ-D-glucoside (50)

Physical Data

Amorphous powder (yield 12 mg). [a]D 25: -64.0° (c 0.14, MeOH). UV Amax (log E) (MeOH) om:

369 (4.42) and 269 (3.03).

IR (KBr) Vmax em": 3370 and 1660.

IH NMR (CD)OD, 400 MHz):

07.86 (2H. d, J= 9.0 Hz. H-2' and 1-1-6'), 6,92 (2H, d,.1= 9.0 Hz, 1-1-3 'and H-5'), 6.75 (tH, d, J = 3.0 Hz, H-8), 6.68 (lH, s, H-3), 6.43 (tH, d,.1 = 3.0 Hz, H-6),

OH

01-1

so

5.04 (lH, d, .1 = 7.5, H-I "),4.52 (rn, H-4"), 3.84 (rn, H-2"), 3.78 (rn, H-6"), 3.59 (rn, H-3 ") and 3.38 (rn, H-5 ").

BC NMR (CD)OD, 100 MHz):

(5 181.6 (C-4), 164.2 (C-2). 162.8 (C-7), 161.0 (C-5), 160.9 (C-4'), ,156.8 (C-9), 128.1 x 2 (C-2' & 6'),121.1 (C-l '),115.8 x 2 (C-3' & C-5'), 105.3 (C-I0), 103.5 (C-3), tOO.3 (C-1 "),99.6 (C-6), 94.9 (C-8), 77,0 (C-3"), 76.4 (C-5"), 73.1 rc- 2"),69.9 (C-4") and 60.8 (C-6").

HR-FAB-MS (+ve) m/z:

433.1135 (ealed. for C2IH2101O, 433.1135) [M+H]'.

ElMS m/z (reI. into %):

[Mf 432 (30), 414 (12). 169 (59), 167 (12), 81 (100) and 71 (80).

79

Experemintal

Acid hydrolysis of compound 50:

A solution of compound 50 (10 mg) in MeOH (5 ml.) containing 1 N HCI (4 mL) was refluxed for 4 h, and diluted with H20 (8 mL). It was extracted with ElOAc. The aqueous phase was concentrated and D-glucose was identified by the sign of its optical rotation ([a]D20 +52). It was also confirmed based on the retention time of its TMS ether (a-anomer 4.1 min, B-anomer 7.8 min) with a standard.

3.5.9 Characterization of benzoic acid (51)

Physical Data

Crystalline solid from hexane (28 mg) MP: 122-123 °c

UV A..nax (log E) (MeOH) om:

228 (3.62), 272 (3.77) and 300 (4.01).

IR (KBr) Vrnax em": 3260-2610 (COOH) and 1705 (C=O).

IH NMR (CDCI3, 400 MHz):

8 12.01 (1 H, s, O-H), 8.12 (2I-I, t, J = 8.5 Hz, H-2 and H-6), 7.60 (l H, t, J = 8.5

OH

o

51

Hz, H-4), 7.46 (21--1, L J= 8.5 Hz, H-3 and H-5).

DC NMR (CDCI3, 100 MHz):

5 179.9 (C-7), 133.9 (C-4). 131.1 (C-l), 130.Sx2(C-2,6), 129.7x2 (C-3, 5).

HR-EI-MS m/z: 122.0367(ca1cd.forC7Hs02,122.0360)

80

Onosma hispida

3.5.10 Characterization of 4-hydroxybenzoic acid (52)

OH

Physical Data

Crystalline solid from EtOAe (37 mg) MP: 213-214 "c

UV Amax (log E) (MeOH) nm: 222 (3.88), and 310 (3.92).

IR (KBr) Vmax em":

3510 (OH), 3330-2720 (COOH) and 1705 (C=O).

IH NMR (CDCI3, 400 MHz):

o 12.10 (lH, s, O-H), 7.92 (2H. t,J= 8.5 Hz, H-2 and H-6), 6.74 (2H, t, J= 8.5

o

Hz, H-3and H-5).

BC NMR (CDCI3, 100 MHz):

o 180.3 (C-7), 161.3 (C-4), 131.5 x 2 (C-2, 6). 122.4 (C-l), 116.5 x 2 (C-3, 5).

HR-EI-MS m/z:

138.0313 (ealed. forC7Hs03, 138.0309)

81

Experemintal

3.5.11 Characterization of 5,4' -dihydroxy-3,6,7-trimethoxyflavone (53)

Physical Data

Crystallization from MeOH afforded yellow crystals (yield: 35 mg).

MP: 221-222 -c.

IR (KBr) vrnax em-I: 3335,1650,1600,1370 and 888.

UV A.nax (logs) (MeOH) urn:

357 (3.21) and 272 (2.71).

IH NMR (CDCI3, 400 MHz):

88.03 (2H. d. J= 8.7 Hz, H-2'and H-6'), 7.09 (2H, d,.1= 8.7 Hz, H-3'and H-

01-1

[[3CO

01-1 0

53

5'),6.49 (II-I, s, H-8), 3.87, 3.68 and 3.60 (3H each, 3xOCH3).

BC NMR (CDCI3, 100 MHz):

8178.7 (C-4), 157.8 (C-4'), 155.1 (C-5), 154.1 (C-7). 152.5 (C-9), 152.1 (C- 2), 138.1 (C-3), 132.1 (C-6), 131.1 (C-2' and 6'), 122.3 (C-I '), 116.4 (C-3' and 5'), 106.1 (C-10), 94.6 (C-8), 60.3, 60.5 and 55.9 (3 x OCR,).

HR-EIMS m/z (reI. int.):

344.0862 (ealed. for ClsH1607. 344.0896).

EI-MS m/z (rel, int.):

[Mr344(lOO),329(32), 196(24) and 121 (33).

82

Onosma hispida

3.5.12 Characterization of 5-hydroxy-3,6,7,4' -tetramethoxyflavone (54)

Physical Data

Amorphous light yellow powder (yield 20 mg). MP: 173-174 -c.

UV Amax (log e) (MeOH) urn:

355 (3.84) and 271 (4.01).

IR (KBr) vmax em": 3510,1730,1603,1355 and 865.

IH NMR (CDCI3, 300 MHz):

s 12.60 (IH, s, H-5), 8.06 (2H, d,J= 9.1 Hz, H-2' and 6'), 7.01 (2H, d,.1=

01-1

54

9.1 Hz, H-3' and 5'), 6.48 (lH, s, H-8), 4.01,3.98,3.94,3.82 (3H each, s, MeO at C-6, C-4', C-7 and C-3).

13C NMR (CDCI3, 100 MHz):

o 179.2 (C-4), 161.1 (C-7), 158.2 (C-5), 156.0 (C-4'), 152.4 (C-9), 152.3 (C- 2),138.7 (C-3), 132.7 (C-6), 130.1 (C-2' and C-6'), 122.8 (C-l '),114.1 (C-3' and 5 '), 106.6 (C-I0), 90.3 (C-8), 60.8, 60.12 (MeO-6 and MeO-3), 56.3 and 55.4 (MeO-7 & MeOA ').

HR-EIMS m/z:

358.1059 (ealed. for C19H1807, 358.1052).

EI-MS m/z (reI. int.):

[Mt 358 (100), 357 (44), 343 (72), 340 (29),329 (5),327 (4.6), 196 (8), 135 (30) and 133 (25).

83