core.ac.uk · 1 Since times immemorial human mind has been curious about nature and this led to a zest and quest to understand the intricacies of Nature. Plants and plant products

ISOLATION AND CHARACTERIZATION OF NATURALLY OCCURRING COMPOUNDS

FROM INDIAN MEDICINAL PLANTS

SUMMARY THESIS

SUBMITTED FOR THE AWARD OF THE DEGREE OF

IN

CHEMISTRY

BY

RAKESH KUMAR

DEPARTMENT OF CHEMISTRY ALIGARH MUSLIM UNIVERSITY

ALIGARH (INDIA)

2003

1

Since times immemorial human mind has been curious about nature and

this led to a zest and quest to understand the intricacies of Nature. Plants

and plant products have an important place in medicinal chemistry. Since

more than 80% of the world's population use plants as their primary source

of medicinal agents, therefore medicinal plants, their extracts or pure

produts are used as main therapeutic agent for treating wide variety of

diseases and represent a rapidly expanding area. In spite of revolutionary

advances in synthetic drugs and research in that field, phytocemicals are still

widely used as drugs which are believed to be free from toxic side effects

and are, therefore, yet to be replaced by synthetics. Thus with this

philosophy there is an ongoing search for bioactive phytochemicals the need

for which is felt badly today to combat the threat posed to mankind and

civilization by infectious and non infectious diseases.

There exists an ever increasing importance of pure substances or

purified and standardized extracts which persist better analytical

characterization and so meet the quality, efficacy and safety requirements

with which every modern drug whether natural or synthetic must comply. In

any case, many traditional remedies are still in therapeutic use including

drugs as such or extracts prepared in accordance with the pharmacopoeia for

use in the the pharmaceutical forms for which they are intended.

The present thesis entitled "Isolation and characterization of

Naturally Occurring Compounds from Indian Medicinal Plants", is

divided into five chapters. The chapter first includes a critical review of the

chemistry of natural products with special reference to flavonoidic and

triterpenic compounds and highlights the recent advances in the analytical

techniques applied in the natural products isolation and structure

elucidation.

The chapters second to fifth comprise with the isolation and

identification of the naturally occurring compounds from the following four

indigenous medicinally important plants. Overall twenty nine constituents

have been isolated, out of which five are being reported for the first time.

Their structures have been established by chemical and physical data (IR,

'H-NMR, '-^C-NMR, UV and MS spectra).

A Ficus lyrata {Moraceae)

B. Cassia nodosa (Leguminoseac)

C. Hetcrophragma adenophyllwn (Bignoniaceae)

D. Diospyms montana {Ebenaceae)

Chapter Second : Chemical constituents from the leaves oi Ficus lyrata.

Following eleven compounds have been isolated and characterized from

Ficus lyrata.

Fl-1 : Stigmast-l,5-dien-3P-oI (new compound)

M.F. C29H48O

M.P. 126-28 °C

M.W. 412 HC

Crystallized from chlorofonn-acetone as white crystals.

Strucure elucidation is based on the basis of spectral studies, IR, UV,

'H-NMR, '^C-NMR and MS.

FI-2 : P-Sitosterol-D-glucoside

M.F.

M.P.

M.W.

Crystallized from chloroform-methanol as colourless crystals.

Acetylated with Ac20/pyridine (mild) - tetra-acetate

M.P. 166°C.

Structure elucidated by chemical reaction and spectral studies, IR, 'H-NMR

and MS.

FI-3 : 5,6-Dihydroxy-2-methyl chromone (new compound)

M.F. C,„H804

HO' ^

OH O

M.R 172-74 °C

M.W. 192

Crystallized from chloroform-methanol as light cream needles.

Structure elucidation is based on the basis of UV, IR, 'H-NMR, '^C-NMR

and MS spectral studies.

FI-4 : 5-Hydroxy-3,7,3',4'-tetramethoxy flavone OMe

M.F. C,9H,A

M.R 160-62 °C

M.W. 358

MeO. OMe

OH 0

Crystallized from chloroform-methanol as yellow ciystals.

Stiucture elucidated by spectral studies, IR, UV, 'H-NMR and MS.

FI-5 : 5,4'-Dihydroxy-6,7,8-trimethoxy flavone (Xanthomicrol)

OMe

M.F.

M.P.

M.W.

C18H16O7

224-25 "C

344

MeO,

MeO'

OH 0

Crystallized from chloroform-methanol as yellow crystalline solid.

Structure elucidation is based on the basis of physical studies (IR, UV,

'H-NMR and MS).

Fl-6 : 5,4'-Dihydroxy-7,8-diniethoxyfIavone (Bucegin)

M.F. C|7H,405

M.P. 290-92 "C

M.W. 314

OMe

MeO 0^

<5X

/ \ OH

OH O

Crystallized from chloroform-methanol as yellow crystals.

Stmcture elucidated by means of spectral studies, IR, UV, *H-NMR and MS.

Fi-7 : 4-Methoxy chalcone

M.F.

M.P.

M.W.

C15H14O2

77-80 °C

238

^ ^ ^ ^ ^

0

Crystallized from chloroform-methanol

as light cream ciystals.

Stmcture elucidated by means of spectral studies, UV, IR, ^H-NMR and MS.

FI-8 : 7,4'-Dimethoxy apigenin

.F.

P.

.W.

C17H14O5

185-87 "C

298

HiCX). OCH,

OH O

Crystallized from chloroform-methanol as white crystals.

Acetylated with Ac20/pyridine->mono-acetate.

M.P. 153 °C

Structure elucidation is based on the basis of chemical reaction and spectral

studies, UV, IR, 'H-NMR and MS.

Fl-9 : 5,7,4'-Trihydroxy-2'3',6'-trimethoxyisoflavone

M.F. C,sH,60s

M.P. 198-200 °C

M.W. 360

MeO OMe

OMe OH O

Ciystallized from chloroform-methanol as pale yellow plates.

Acetylated with Ac20/pyridine (mild) -> tri-actate

M.R 132T

Methylated with Me2S04/acetone (mild) -> hexamethyl ether

M.R 156°C

Structure elucidated by means of chemical reactions and spectral studies,

UV, IR, 'H-NMR and MS.

FI-10 : Acacetin-7-glucoside

M.F. C22H220,o

M.R 255

M.W. 446

CH,OH

OCH,

OH O

Crystallized from chioroform-methanol as cream coloured crystals.

Acetylated v ith Ac20/pyridine (mild) -> penta-acetate

M.R 204«C


studies IR, UV, 'H-NMR and MS.

Fl-11 : Acacetin-7-O-neohesperidoside

CHjOH

C28H32O14 \ ^ ^ o

H O '

M.F.

M.R

M.W.

266-68 "C

592 H.C

OCIl,

OH O

Crystallized from chioroform-methanol as white crystals.

Acetylated with Ac20/pyridine (mild) -» hepta-acetate.

M.R 216-20 °C

Structure elucidated by means of chemical reaction and spectral studies, IR,

UV, 'H-NMR and MS.

Chapter Third : Flavonoidic constituents from the leaves of Cassia nodosa

Following five compounds have been isolated from the leaves of

Cassia nodosa

Cn-1 : Unsubstituted flavone

M.F C , 5 H K A

M.R 96-97 °C

M.W. 222

Crystallized from benzene-acetone as cream coloured solid.

Stiucture elucidated by means of physical data (IR, UV, 'H-NMR and MS).

Cn-2 : 5,4'-Dihydroxy-7-methyl-3-benzyl chromone (new compound)

M.F.

M.P.

M.W.

C17H14O4

290-92 °C

282

Ciystallized from chloroform-methanol as pale yellow granular crystals.

Structure elucidation is based on the basis of spectral studies, IR, UV,

'H-NMR, '^C-NMR and MS.

Cn-3 : Kaempferol 3-0-rhamnoside

HO M.F. C2()H2oO|o

M.R 177-78°C

M.W. 420

Crystallized from chloroform-methanol as light OH OH

yellow granular crystals.

Acetylated with Ac20/pyridine (mild) -> hexa-acetate

M.R 159-60 °C

Structure of Cn-3 established by means of chemical reaction and spectral

studies, IR, UV, 'H-NMR.

Cn-4 : Quercetin 3-arabinoside

M.F. C2„H„0,|

M.P, 256 "C

M.W. 434 OH 0

8

Ciystallized from methanol as yellow needles.

Acetyiated with Ac20/pyridine (mild) -> hepta-acetate.

M.P. 302-03 °C

Methylated with Me2S04/acetone (followed by hydrolysis)->'tetramethylether

M.P. 193 °C

Structure elucidation is based on the basis of chemical reactions and

spectral studies, UV and 'H-NMR.

Cn-5 :

M.F.

M.P.

M.W.

Tamarixetin 3-0-arabinoside (new compound)

HO^

QnHioOii

204-05°C

448 OH 0

Crystallized from methanol-chloroform as

yellowish solid.

Structure elucidated by means of spectral studies, IR, UV, 'H-NMR and MS.

Chapter Fourth : Chemical constituents from the leaves of Heterophragma adenophyllum.

Following seven compounds have been isolated from Heterophragma

adenophyllum.

Ha-1 : Dimethyl ester of terephthalic acid oxxn,

M.F.

M.R

M.W.

C|()Hio04

138 °C

194 COOCH,

Ciystallized from chloroform-methanol as white needles.

Structure eludiction is based on the basis of spectral studies, IR, UV,

'H-NMR and MS.

Ha-2 : 24-Cyclohexyl-n-tetracosane

M.F. C:,oH6,) / - - \ / ( C H 2 ) - C H 3

M.R 71-72°C

M.W. 420

Crystallized from methanol as colourless solid.

Stmcture of Ha-2 elucidated by spectral studies, IR, 'H-NMR and MS.

Ha-3 : Friedelin

M.F.

M.R

M.W.

C,-!{iH5()0

262-64 "C

426

Crystallized from chloroform-methanol as white needles.

Stmcture elucidated by spectral studies, IR, 'H-NMR and MS.

Ha-4 : a-Amyrin acetate

M.R

M.R

M.W.

C32H52O2

93-95 "C

468 AcO

Crystallized from methanol as colourless crystals.

Structure elucidation is based on the basis of spectral studies, IR, UV,

'H-NMR, '- C-NMR and MS.

10

Ha-5 : Ursolic acid

M.F.

M.P.

M.W.

C3(|H4oO;

286-87 °C

456

COOH

Crystallized from chloroform-methanol as

shinning needles.

Acetylated with Ac20/pyridine (mild) -> mono-acetate

M.P. 292 °C.

Methylated with diazomthane/ether (mild) -^ monoethyl ether.

M.P. 210«C

Structure elucidated by chemical reactions and spectral studies, IR,

'H-NMR, i- C-NMR and MS.

Ha-6 : Resveratrol

M.F.

M.P

M.W.

C 14^1203

256-57 "C

228

Crystallized from aquous ethanol as

colourless plates.

Acetylated with Ac20/pyridine (mild) -> tri-acetate

M.P 118-19 °C


studies, IR, UV, 'H-NMR '^C-NMR and MS.

11

Ha-7 : 2,3,6,7,8,9-hexahydroxynaphthalene-2-rhamnoside (new compound)

OH

M.F. C,6H,80,o

M.P. 272-75 °C

M.W. 370

Ciystallized from chlroform-methanol as yellow crystals.

Structure elucidated by means of spectral studies, IR, UV, 'H-NMR,

'-^C-NMR and MS.

Chapter Fifth : Flavonoidic constituents from the leaves of Diospyros montana.

Following SIX compounds have been isolated from the leaves

Diospyros montana.

Dm-1 : 3'-Methoxy 3,5,7,4'-tetrahydroxyflavone (Isorhamnetin)

M.F. C,6H,207

M.R 291-92 °C

M.W. 3 16

OCH,

OH o

Ciystallized from benzene-methanol as yellow needles.

Structure elucidated by spectral studies, UV and 'H-NMR

Dni-2. : 5,7,4'-Trihydroxy flavanone (Naringenin)

M.F.

M.R

M.W.

C15H12O5

248-50°C

252

12

Ci"ystallized from ethyl acetate-acetone as light yellow needles.

Stmcture elucidation is based on the basis of spectral studies, UV and 'H-NMR.

Dm-3 : 3,5,7,3'4',5'-Hexahydroxyflavone (Myricetin)

M.F.

M.P.

M.W.

CisHioOg

358-59 °C

318 OH 0

Crystallized from chloroform-methanol as yellow crystals.

Acetylated with Ac20/pyridine (mild) -» Hexa-acetate

M.P. 218-19 °C.

Stmcture elucidated by means of spectral studies, UV, IR and 'H-NMR.

Dm-4

M.F.

M.P.

M.W.

: Quercetin 3-O-glucoside (Isoquercetin)

HO C2iH2()Oi2

218-19°C

464

HOH2C Crystallized from CHClj-MeOH as

yellow granular crystals.

Acetylated with Ac20/pyridine (mild) -> Octa-acetate

M.R 162-63 °C.

Methylated with Me2S04/acetone (mild) followed by hydrolysis ->

monohydroxy tetramethyl ether.

M.R 153-54 °C.

Structure of Dm-4 elucidated by chemical reactions and spectral studies, IR,

UV and'H-NMR.

13

Dm-5 : Myricetin 3-0-rhamnoside

HO,

OH OH

M.F. C2,H2oO,2

M.P. 196-98 °C

M.W. 464

Crystallized v/ith chloroform-methanol

as pale yellow needles.

Acetylaed with Ac20/pyridine (mild) -> Octa-acetate

M.P. 141-42 °C

Permethylated with NaH/DMSO -> Permetylated glycoside (M.P. 136-38°C)

followed by hydrolysis -> pentamethyl ether

M.P. 224-26 °C.

Structure elucidation is based on the basis of chemical reactions and

spectral studies, UV and 'H-NMR.

Dm-6 : Daidzein 7-0-glucoside

M.F. C21H20O,) »

M.P 234-35 °C

M.W. 436

Crystallized from benzene-acetone as

yellow solid.

Acetylated with Ac20/pyridine (mild) -> penta-acetate

M.P. 155-56 °C

Structure of Dm-6 elucidated by chemical reaction and spectral studies, IR,

UV 'H-NMR.

ISOLATION AND CHARACTERIZATION OF NATURALLY OCCURRING COMPOUNDS

FROM INDIAN MEDICINAL PLANTS

THESIS SUBMITTED FOR THE AWARD OF THE DEGREE OF

IN

CHEMISTRY

BY

RAKESH KUMAR

DEPARTWIENT OF CHEMISTRY ALIGARH MUSLIM UNIVERSITY

ALIGARH (INDIA)

2003

T6153

dedicated to

Worthy parents Jlatt Sister...

=J

êsyecUd

^roj. J\/i. Dlyas...

I

;

:

1

i

• !

and Respected

^ r . iJHehtab ârveen

^

DEPARTMENT OF CHEMISTRY ALIGARH MUSLIM UNIVERSITY A L I G A R H —202 002 (U. P.) I N D I A

„, Êxt. (0571)7 0 3 5 1 ^ '^^""'' Jlnt. : 17. 318

Dated..

(Unixtxtuit

This is to certify that the work described in the thesis

entitled "Isolation and Characterization of Naturally

Occurring Compounds from Indian Medicinal Plants*' is

the original work carried out by Mr. Rakesh Kumar under

my supervision and is suitable for submission for the award

of Ph.D. degree in Chemistry.

^ •

(DR. MEHTAB PARVEEN) Supervisor

J}n. the name, of LJOO, who j-auourea me to acnieue tni'i arduoui goaf, which

J/ tkootgkt unattainaote, to whom oelon^S might ana majeit^^ J Lina ail tke

praiiei ana commendation tor prouiaing me itrenqtk during the period of mu

reiearck work which helped me to overcome the trials and tribulations in tke

toilsome journeu and who Supported me whenet/er J/ declined in vigor or uitalitu.

[- raise atwaus be to him.

J/ don t find wordi eloquent enouqh to express mu deep sense of gratitude

to esteemed f- rof. frl. ^tijas, ^rormer airman, 2)eptt. of Cliemistr^,

^<l. irl. lA-, ^y^tianrh, who has been a Sole source of- inspiration and

encouragement to me throughout the worh. yynce again, J/ am immenietij

grateful and indebted to him. for his keen interest, excellent guidance, int/aluable

Suggestions, meticulous corrections and discerning Judgement. Jle was

instrumental in teaching me more than what whole of ma previous education

could not. .Jjefinitetu, he has a unique personalitu. Jf am. proud to have worked

wi tk k im and want to express mu humble gratefulness for nil that he has done for

me.

_/ feel deepiu obliged to express mu profound nratitude to mu esteemed

Supervisor .Ujr. Ifflissi inehtab [ârveen, .Jjeptt. of K^hemistru, ^yQ. nl. [/(.,

.^^ligarh, for her Scholastic guidance, persistent inspiration, vital encouragement

throughout the course of this research work. ^J4er correct advice ana timelu kelp

kave been invaluable at everu stage o I tk is work. .Jike alwaus wiltinglu spared

muck Of ker precious time to discuss mu problems. lA/ords are inadequate to

express m.u indebtedness to her, wko made this intricate ana difficult work, eaSu

and fruitful for me. J^he has indeed been a source of inspiration to me. J ' shall

alwaus remain thankful to her.

J/ take tliii opportunltu to record mu gratitude and pau mi4 iincereit thank

to rrof. ^s.irl. J^ltamiuddln, for hid ti'meCu co-operation, encouragement and

moil uatuaole iuqqeitioni.

Jj am alio tliannfut to [ rof. l(l. irluikfiq for kii periiitent inspiration

and encouraaement during the course of m.u work.

a matter of- gratincation to express mu indebtedness to (chairman,

.uJeptt. of L^hemistru and Ujr. ydamai .^hmed, ^^sstt. ^Jjirector, 2)eptt. of

Keiearch in Ljnani rrledicine for proi'idinq the necessaru research, taboratoru

facilities and co-operation in alt respects.

Jj qratejuUu offer mu sincere thanks to .Jjr. J^kaJiullak for his

gratuituous co-operation, constructive suqqestions and personal interest in tke

aduancement of mu research work and Jj was uerij muck stimulated bu kii

encouracjing attitude towards mu problems.

^y am thankful to I rof. lA/azahat ^J4usain, .uJeptt. of lOotanu,

-y^. frl. bj., .^ligarh for identification of the plants and also to ,Jjr. ilaseem

_vv. _y\han, .superintendent vSotanicai Ljarden, _vv. / / / . ij., ^^ligarh for his

co-operating attitude in. providing neceSSaru plant male rial.

.J am highlu indebted to Jjr. A . J^waroop, .uJ.J^c., f\eader, ^Jjeptt. of

rtxusics, <J-).-J). L..oi(ege, ^yvCigarh, for his significant aduice and

encouragement. Jy am also thankful to alJr. I\am. rjii/aS (^handel for his

constant and sincere help.

m.u priuilege to place on record m-u humble Sentiments of gratitude to

<UJr. Iff Id. Âlsif Irlusharraf, .Jjr. /4amai .jriroz, <Jjr. âmal ^^. ^\han for

their brotherlu treatment, moral Support and encouraging responses.

.y am eSpeciallu thankful to mu lab cotleagues <J.Jr. vjtner ^vv. lOaSudan,

:br. Jjasan W . J / . Wui aisen for their kind of unique helping attitude and

also to iiiiss Lfutrana yshuwaja, rfliss Wandana rl'iishra for their dau to dau

help. J/ am also thankful to other research scholars, who remained close to me.

/ / /M jfiends eipeciallu .Jjr. 0oaeili ^\iim.ar, ^Jr. S^.j-^. Jînak, ff/r.

Lfoqeih C^h. C/upin, /\a!eik [-^. Jînnn ana mu uncle ,Jjr. Jâumendra

^J\umar, irlr. ^ . / ^ . dSadoni aalu deierue mu nenrtfelt tkanhi for tkeir

cooperation, coordination and encouragement during tke critical Juncture of- tkis

work.

^ am veru muck grateful and alio offer mu m-oit iincere tkanki to oil tke

members of tke familiei of rr/r. Kamoaou .înak, I fir. L/kamandi Jîngk

jespeciallu fr/r. Kajkumarj, Ûr. S^.,Jj. i^kandola leipeciallij to female61 and

frir. _Ji[ak Jîngk for tneir kind bekauiour, co-operation an j/o r treating me a6

a famliu member.

Witk L it regardi, .J fcel skort of wordi to express mij deep feelinnS to mu

ujortku parents, fatker frlr. Uikam -Jîngk lex-l-^rincipall, mo tke r /r/rs. ^Jdans

J(u mari and mu brotkerS 11 jr. f- ramod ^\umar, ulr. /ditendra .J'\umar, wko

deserve jull credit for kindling tke candle of mu career. Jkeu kaue been aiwauS

til me, mentailu, emotionaiiu in giuing et/eru Lind oj support and s^mpatL^

during tke entire period of m,u researck work. Wnce again, rti« words fails to

express mu kumbte gratefulness to mu parents witkout wkoSe constant inspiration

and Support it wou idk at/e not been possible for me to be wkere .J am at present.

cJiast but definiteiu not tke least, the painstaking job of tuping bu ilrlr.

J-^radeep ^J\umar ILômputer i-^rofessionatl is kigklu appreciated.

financial assistance from CCÛW, flew "ibelki is gratefuilu

acknowledged.

(RAKESH KUMAR)

CONTENTS

Page. No.

Chapter - I: Theoretical 1-37

Introduction 1-28

References 29-37

Chapter - U : Ficus lyrata 38-110

Discussion 38-85

Experimental 86-107

References 108-110

Chapter-Ill: Cassia nodosa 111-142

Discussion 111-128

Experimental 129-140

References 141-142

Chapter-IV : Heterofragma adenophyllum 143-179

Discussion 143-167


References 178-179

Chapter-V : Diospyros montana 180-208

Discussion 180-195


References 207-208

%t

CHAPTER - I

THEORETICAL

1

The use of plants for medicinal or tonic properties goes back to

prehistoric times and has attracted the interest of scientists for centuries.

The Vedas give the earliest written record about the science of healing. The

reference to medicinal plants is also found in Ebbers Papyrus (16th century

B.C.) which lists in detail over 7000 herbal remedies, for example - poppy,

castor oil, caraway, etc. The Indians were the first to use 'Chaulmoogra'

fruits for its antileprotic activity. The Brazillians employed 'Ipecacuanha'

for the treatment of dysentery and diarrhoea. The roots of Rauwolfia

serpentina, an indigenous plant, has since time immemorial been widely used

in India and Malaysia as an antidote for insect and snake bites and in mental

derangement,

The utility of plants as therapeutic agents in traditional medicinal

system is still prevalent today, for example, the middle eastern civilisation

developed the Greco-Arabic system of medicine (Unani system of

medicine) which is practised in the Indian subcontinent. Similarly, the

Chinese race developed the Chinese system of medicine largely based on its

unique system of theories including the concept of Yen and theory of

influence imparted from nature. The Ayurveda and Sidha systems of

medicine were contributed by Indians. All these systems procure more than

80% of their medicaments from plants. Till recently these medicines used

to be prepared by the practising physician himself for the use of his patients.

In the absence of any scientific method of identification and standardisation,

these plant based medicines were more often than not, in a state of

uncertainty about the identity, use and efficacy of the drug.

However, Natural Product Chemistry has undergone an explosive

growth during the later half of the 20th century. This has been brought about

by a number of factors. One of these has been drawing number of substances

from natural sources which display interesting pharmacological activity.

Another factor has been the improvement made in the technology of

isolation process which includes High Performance Liquid Chromatography.

The techniques have allowed rapid isolation of substances previously

difficult to obtain by classical procedures. The most important factor has

been development of new spectroscopic techniques which have opened up

whole new vistas in this exciting field. Prominent in these advances have

been 'H-NMR, '''C-NMR, two dimensional NMR spectroscopy and mass

spectrometry.

Today the importance of cmde dmgs in therapeutics is much more than

it was 25 years ago. The preference to drugs, perfumes, cosmetics, dyes,

flavours and food colours obtained from plants sources has been increasing

day by day. To examplify, one of the important plant product is artemisin

which is obtained from the flowering tops of the Chinese plant Quinghao

(Artemisia annua). Preliminary clinical trials of a derivative of artemisin

have shown that it is effective against strains of Plasmodium falsiparum,

where synthetic anti-malarials have failed to cure the disease. It is useful for

treating cerebral malaria'. Taxol^ is proving to be a life saving drug against

the cancer ailment of breast and ovary. It has a unique mode of action, being

an anticancer agent which enhances both the rate and yield of microtubules

assembly and apparently has no significant effects on DNA, RNA or protein

synthesis.

In most developing countries where coverage by health services is

limited, the treatment of the sick is largely based on medicinal plants. Early

in 20th century, the greater part of medicinal therapy in the industrialised

3

countries was dependent on medicinal plants but with the growth of

pharmaceutical industries their use fell out of favour^ However, now the

value of medicinal plants is receiving distinguished global attention. Due to

the wide spread reports of toxicity and health hazards associated with

indiscriminate use of synthetic drugs and antibiotics, accompained by rising

cost of organic chemicals and reagents, the basic thinking and attitude of the

modern society has been changing since last 20 years.

A large number of plants used for health care are rich in polyphenolic

compounds'*. Various types of plant phenols have been shown to possess

antioxidant activity based on free radical scavenging activity. The activity

may underlie various effects of polyphenols in plant tissues and also their

medicinal effects, such as those related to inhibition of lipid peroxidation

and tumor promotion\ Natural polyphenols are widely used to delay/prevent

the oxidation of fats and oils in variety of food. Plants polyphenols

comprise flavonoids, alkaloids, coumarins, terpenes and steroids.

The flavonoids, one of the most numerous and wide spread group of

natural products, which are very much important to man not only because

they contribute to plant colour but also due to many members (e.g. rotenone,

phloridzin and coumestrol) are physiologically active^. Flavonoids are

distributed universally among vascular plants and found practically in all

parts of plants. Gabor^ has reviewed trends in research on the

pharmacodynamic effects of flavonoids, mostly rutin and its derivatives.

With the extensive screening programmes of plant products for anticancer

drugs^"'^, it has been reported that flavonoids may contribute to or be

effective in combating certain types of cancer^-^-'- . The effect of flavonoids

on cancer cells in various pharmacokinetics of a flavonolignan couple has

also been studied'''. A recent survey of literature showed that the flavonoid

field is still veiy important to Chemists and their interest is increasing in

screening the new flavonoids and their physiological activities. A large

number of naturally occuring flavonoids and novel flavonoids are added to

literature every year' " * .

In the present study, we have tried to carry out systematic chemical

investigation of important indegenous medicinal plants with a view to

characterise their chemical components preferably flavonoids, which could

be starting point for the chemists who are mainly concerned with

pharmacological and clinical aspects of these herbal drugs.

Since mainly the spectroscopic techniques, UV, IR, 'H-NMR, '- C-NMR

and Mass have been used in the identification and structure elucidation of

the products isolated from different plants during the course of the present

work, a brief review of each technique has been discussed here :

Ultra-Violet Spectroscopy :

The ultra-violet spectra of different flavonoids are very

characteristic-''' and along with colour reactions^^"^'' have been used

extensively to distinguish the various groups of this class of compounds.

The absorption maxima of flavones have been correlated to the presence of

a cinnamoy] (I) and benzoyl (II) groupings^^, the former giving rise to the

high wave length band at 320 to 350 nm and the latter to the low wave length

band at 240-270 nm. On the basis of this generalisation, important

deductions have been made about the location of substituents in the two

rings.

Substitution in the B-ring specially at 4'-position stabilizes the

cinnamoyl chromophore resulting in a bathochromic shift of band I whereas

substitution in the A-ring has a similar effect on the position of band II.

Compounds having a free 5-hydroxyl group absorb at higher wave lengths

and methylation of this hydroxyl brings about a hypsochromic shift of 10 to

13 nm of both the maxima. The presence of a hydroxyl group at this position

is routinely established by measuring the spectrum in presence of AlCl3^^.

Hydroxyl groups at 7,4' positions are more acidic than others and a

bathochromic shift of band 1 or II on addition of fused sodium acetate is a

good indication of the presence of free OH group at these positions^^, but

the results of these measurements have to be interpreted with caution and

require further confirmation by other physical and chemical methods. Thus,

for example, lucidin^^, acerosin^^ and scaposin^^ failed to give a

bathochromic shift with sodium acetate though they were definitely shown

to possess a 7-OH group.

In flavanones, absence of cinnamoyl chromophore has the effect of

suppressing the high wave length band which is either totally absent or

present only as an inflection. The spectra of isoflavones are also marked by

the absence of the high wave length band, biochanin A, irigenin and

pomiferin absorb only between 261-276 nm^'. Thus it is difficult to

distinguish between flavanones and isoflavones with the help of

UV-spectruiii alone.

Infrared Spectroscopy :

IR spectrum of flavanone shows the carbonyl absorption at 1680 cm"',

the standard value for aromatic ketones. The shift of the carbonyl band to

1620 cm"' in 5-OH flavanones is largely due to electron donation by the

ortho hydroxyl group, coupled with chelation. Consequently methylation of

the 5-OH group produces only a small hypsochromic shift of 10 cm"'. A

similar shift towards long wave length of 4' substituted flavanone is however,

attributed to inter molecular hydrogen bonding^'. The IR spectrum of flavone

shows the carbonyl band at 1660 cm"' ^ , owing to conjugation with the

olefmic double bond. Introduction of a hydroxyl at 5-position does not alter

the band position appreciably. Luteolin and apigenin show the cabronyl

band at 1655 and 1660 cm"' respectively^^ The IR spectra of isoflavones

are similar to those of flavones. Chelation of the 5-OH group in all cases

has the effect of broadening the 0-H stretching band to a point where it can

no longer be made out. The aromatic region is not of any great usefulness as

no reliable prediction about the substitution pattern can be made on the basis

of absorption bands in this region. The infrared spectra of alkylated

flavonoids give some indication of the presence or absence of gem dimethyl

groups and an epoxide linkage but these points can now be better established

with the help of NMR spectia.

Nuclear Magnetic Resonance Spectroscopy :

The foregoing discussion of the IR and UV spectra of flavonoid

illustrates the usefulness of these two techniques in determining the

structure of unknown flavonoids. It is clear that although much useful

information regarding the oxygenation pattern of a flavonoid can be obtained

in this way, it falls short of providing complete and unambiguous evidence

for or against a presumed structure. Thus the infrared spectrum does not go

beyond distinguishing between the y-pyrone system of flavonoids and the

a-pyrone system of coumarins besides indicating the presence of a chelated

5-hydroxyl group. The UV spectrum is much more informative and

distinguishes clearly between flavones, isoflavones and coumarins.

7

The application of NMR spectroscopy has proved to be most powerful

tool in the structure determination of flavonoids. By the use of NMR studies

of silyl derivatives^'' double irradiation techniques*^^^ solvent induced shift

studies'' ' '*^^ lanthanide induced shift ' ''''*' nuclear overhauser effect^^ (NOE)

and '-^C-NMR''" spectroscopy, one can come to structure of flavonoids

occuring even in minor quantities without tedious and time consuming

chemical degradation and synthesis.

The trimethylsilyl derivatives can be conveniently prepared by

treatment of the compound with hexamethyl disilazone and trimethylsilyl

chloride in pyridine and the spectrum is then measured in carbon

tetrachloride with tetramethylsilane as external or internal reference.

Table (1) gives the chemical shifts of different protons of certain

representatives of flavones and isoflavones. The most detailed and

systematic studies of the 'H-NMR spectra of flavonoids are due to

Mabry^'-^^, Batterham and Highet''^ Clark-Lewis^"*, Massicot^-, Kawano^^ '*',

and Pelter and Rahman^^"^ . These studies have simplified the task of

determining the substitution pattern of flavonoids with the help of NMR-

spectroscopy. An obvious advantage of this technique in its application to

flavanones is that they can be readily distinguished not only from flavones

and isoflavones but also from the isomeric chalcones which in view of the

extreme ease of the isomerisation process, is often not possible with other

methods.

The most commonly occuring hydroxylation pattern in natural

* - '° /TV. c

•CM

OH O (ffl)

TABLE - 1

^H-NMR spectral data of flavones and isoflavones, values on 5-scale

Assignment

H-2

H-3

H-5

H-6

H-8

H-2'.6'

H-5'

H-3'.5'

5-OMe

Apigenin

6.35(s)

6.15 (d. J==2.5Hz)

6.5 (d.J==2.5Hz)

7.7 (d. J==9.0Hz)

6.85 (d. J==9.t)Hz)

Luteolin

6.3(s)

6.14 (d. J=2.5Hz)

6.5 (d, J=2.5Hz)

7.35 (dd, J=8.5,2.5Hz)

6.85 (d. J=8.5Hz)

Robinetin

7.95 (d, J=8.5Hz)

6.75 (dd, J=8.5,2.0Hz)

6.18 (d, J=2.0Hz)

7.2 (d, J=2.0Hz)

Genistein 5-methyl ether

7.6 (s)

6.20 (d, J=2.2Hz)

6.24 (d. J=2.2Hz)

7.3 (d, J=8.5Hz)

6.75 (d. J=8.5 Hz)

3.8 (s)

Orobol

7.7 (s)

6,18 (d. J=2.5Hz)

6.39 (d. J=2.5Hz)

7.01 (dd,J=2.5, 9.0Hz)

6.92 (d, J=9.0Hz)

s = singlet, d = doublet, dd = double doublet

Hs

H(X J-^ .CX .H,

Hfi'

/ \ -OH

OR o m' •H,'

Apigenin - R, = OH. R:=R3=R4=H Luteolin - R|=R:=OH. R,=R4=H Robinetin - R,=H. R3=R,=R4=OH

Genistein 5-methyl ether - R'=CH3, R=H Orobol - R'=H, R=OH

flavonoids is 4', 5, 7-trihydroxy(lII) system. The chemical shifts of the

protons of ring-A and B prove to be independent of each other but are

affected by the nature of ring-C.

Ring-A protons :

The two ring-A protons of flavonoids with 5,7-dihydroxylation pattern

give rise to two doublets (J=2.5Hz) between 6 6.66-6.0 from tetra-

methylsilane. There are, however, small but predictable variation in the

chemical shfits of the C-6 and C-8 proton signals depending on the 5-and

7-substituents. In flavanones the 6,8-protons give a signal near 5 5.95, with

the addition of a 3-hydroxy group (flavonols) the chemical shift of these

protons are slightly altered and the pattern changes to veiy strongly coupled

pair of doublets. The presence of double bond in ring-C of flavones and

flavonols causes a marked downfield shift of these peaks again producing

the two doublet pattern out of 6- and 8-protons, the latter appears downfield.

Ring-B protons :

All ring-B protons appear around 5 7.7-6.7, a region separate from the

usual ring-A protons. The signals from the aromatic protons of a substituted

ring-B in a flavone appear as a broad peak centred at about 6 7.45. The

presence of ring-C double bond causes a shift of 2',6'-protons and the

spectrum shows two broad peaks one centred at S 7.35 (H-2',6') and the

other at 5 6.85 (H-3',4',5')^l

With the introduction of 4'-hydroxyl group the ring-B protons appear

effectively as a four peak pattern, A2B2 pattern. Introduction of one more

substituent to ring-B gives the normal ABC pattern, the hydroxyl group

increases the shielding on the adjacent 3',5'-protons and the signal moves

10

substantially upfield. The 2',6'-protons of flavanones give signals centred at

about 5 7.35.

Ring-C protons :

Considerable variations are generally found for the chemical shifts of

ring-C protons among the several flavonoid classes. For example, the C-3

proton in flavones gives a sharp singlet near 8 6.3, the C-2 proton of

isoflavones is normally observed at about 5 7.7, while the C-2 proton in

flavanones is split by C-3 proton into a doublet of Jcis ^ 5 Hz, Jtrans = H Hz

and occurs near 55.2. The two C-3 protons occur as two quartets

(JH3a.3b = 17 Hz) near 6 2.3. However^they often appear as two doublets

since two signals of each quartet are of low intensity. The C-2 proton in

dihydroflavonols appears near 5 4.9 as a doublet (J = 11 Hz) coupled to the

C-3 proton which comes at about 5 4.2 as doublet * .

In the structure elucidation of biflavonoids certain useful information

can be obtained by comparison of their NMR spectra with those of their

corresponding mionomers. Such a choice, however, is compelling but by no

means infalliable. Comparison of the NMR-spectra of methyl and acyl

derivatives of a biflavonoid with those of biflavonoids of the same series as

well as with those of biflavonoids of other series in which at least one

monoflavonoid unit is similarly constituted is very helpful in assigning each

and every individual proton and the position of methoxy groups. The problem

of interflavonoidal linkage has been successfully solved by solvent induced

shift studies of methoxy resonance^ ' ^^-^^ and lanthanide induced shift '' "*

studies.

In biphenyl type biflavones such as amentoflavones, cupressuflavone,

agathisflavone etc., the peaks of ring protons which are involved in

11

interflavonoid linkage, appear at somewhat lower field (-0.5) as compared

with the peaks of the same protons in monomer due to extended

conjugation.

It has been observed^^^ both in biphenyl as well as in biphenyl ether

type biflavonoids that the 5-0-Me group of a 8-linked monoflavonoid unit

in a biflavonoid shows up below 5 4.00 in deuteriochloroform in all the

cases examined so far (Table-Il). This observation may be explained

on the basis of extended conjugation. 5-Methoxyl group of a 8-linked

monoflavonoid unit in biflavonoid of BGH-series^^"'^', WGH-series^^-' ^ and

GB-series' ''''-^ does not show up below 5 4.00 as the linkage is through

heterocyclic ring.

^•^C-Nuclear Magnetic Resonance Spectroscopy :

A nucleus can have a net magnetic moment only when the concerned

nucleus has non-zero spin quantum number. The abundant '^C isotope of

carbon, with both atomic and mass numbers even, has zero spin quantum

number and hence caimot give rise to nuclear magnetic resonance. The

'^C-isotope of carbon has a very low natural abundance (1.108%) and

because of its lower gyromagnetic ratio compared to that of proton gives

rise to week NMR signals. The technique was therefore applied only in work

of biological importance employing enrichment of '''C-isotope. The lack of

sensitivity leads to high signal to noise ratio but accumulation of spectra in

digital computer involving averaging of noise, computer averaged transiets

(CAT) produce interpretable spectra. But the most economical and efficient

method of sensitivity enhancement in '-^C-NMR is the Pulse Fourier

Transform (PFT) technique which in combination with decoupling methods

such as proton broad band and off resonance decoupling^'' becomes a very

powerful tool of structure analysis.

12

Table II

Methoxy protons (6-values) of fully methylated biflavonoids

Biflavonoid I-5-OMe II-5-OMe

Cupressuflavone 4.15 4.15 [1-8, 11-8)

Amentoflavone 3.87 4.06 [1-3', 11-8]

Agathisflavone 3.86 4.05 [1-6, 11-8]

*Hinokiflavone 4.00 4.08 [1-4', 11-8]

2,3-Dihydroamentoflavone - 4.08 [1-3', 11-8]

*Synthetic

13

In conventional NMR spectroscopy the radio frequency power is kept

low to avoid saturation and the spectrum is obtained by sweeping the Rf field

in the range of Larmor frequencies of the observed nuclei. This method is

applicable to nuclei which have sensitivity of the order of protons and is

described as the static absorption or continuous wave (CW) NMR

spectroscopy. With less sensitive nuclei the signal to noise ratio is high and

useful spectra were only obtained when a new technique "The Pulse Fourier

Transform" was developed. The complete understanding of this new

technique requires considerable command over the principles of quantum

mechanical energy exchange but the underlying principle can be discussed

in general terms.

In PFT technique all nuclei of the sample are elevated to excited state

through irradiation by short intense Rf pulses. During the free induction

decay of the high energy state the transverse magnetisation vector has a

phase shift of TC/2 relative to Rf field H}. Owing to appropriate experimental

arrangement induction current stemming from the decay of this

magnetisation vector is built up in receiver coil following the Rf pulse. Now

if the Larmor frequency of the nuclei differs from the Rf, the magnetisation

vector periodically rephases and dephase with applied magnetic field

resulting in a pulse interferogram which is then analysed through Fourier

transform. When one compares the CW and FT spectra of the same sample

at low concentration, the difference is striking.

In '-^C-NMR spectroscopy coupling of the protons to '- C nuclei gives

rise to compliczited spectra which are simplified through double resonance

which involves irradiatin of the sample not only by Rf frequency Hi, at

resonance with nuclei to be observed, but additionally with a second

14

alternating field H2, at resonance with the nuclei to be decoupled. If H2 is

such that it covers the whole range of protons, the spectrum is described as

proton broad band or noise decoupled. In it the resonances of individual

carbons appear as singlets, the position of which i.e. their chemical shift,

depends on their environment. No information is, however, available from

such spectra on whether the carbon atom is primary, secondary or tertiary.

In order to know this single frequency off-resonance decoupling is applied.

In it the perturbing field H2 is not at resonance with the nculei X of an A-X

system but is so adjusted that the coupling constant JAX is larger than zero.

By proper adjustment of H2 the multiplets are so narrowed that no or only

slight overlapping occurs. Such spectra are normally obtained along with

broad band spectra to facilitate assignments.

Chemical Shifts :

The factors which influence '^C chemical shifts are not always the

same as for protons though a broad correspondence does exist. In this

context one might usefully compare the effect of ring current, electron

withdrawing and electron donating substituents on neighbouring protons and

carbons. It is well known that ring current, as in aromatic compounds, and

circulating Ti-electrons, such as in acetylene and carbonyl compounds,

generates magnetic fields which oppose or enhance the applied field in

different regions of the molecule. Due to this interaction acetylenic

hydrogens are shielded where as the aldehydic and benzenoid hydrogens are

deshielded.

Ring current is not of much importance in determining '''C chemical

shifts and benzylic methyl is only slightly deshielded as evident from

comparison of methyl cyclohexene (IV) with toluene (V). Similarly the

15

methylene-carbon facing the ring in ansa compounds is sliifted upfield by

only 50.7 ppm. The acetylenic carbons appear, however, upfield (563-88 ppm)

from olefinic carbons and the methyl carbons of ketones are also at about

the same value as the olefinic methyl.

23.4 CHj

'134.3

21.3 CHj

31.5

24.5

138,3

122.3

26.7

128.9

129.6

(ly)

126.1

(V)

It is thus apparent that '-'C spectra can not unequivocally differentiate

between allylic and ordinary carbons but electron withdrawl as expected

leads to deshielding of a-carbons. This is borne out by measurement of

shfits of halogen substituted alkanes. It is surprising, however, that the effect

is not transmitted to P, y and 5 positions with decreasing intensity, as one

would expect, instead the y-carbon is shielded. Probably factors other than

inductive effect are involved.

Crowding by alkyl substituents also causes deshielding. The methyl

carbons of ethane resonating at 5,7, the methylene carbons of isopropane at

H3C + CH3

OH CH,

-C, H3C " ^ C H j

330ppm

(VI)

+

HjC-^ ^ C H 3

248 ppm

(Via)

1K1.3

(VII)

168.5

(VIII)

o=c=o 132.0

(IX)

256 ppm

(VIb)

H 3 C — C = N

117.7

(X)

16

15.4, the methine carbon of neopentane at 24.3 and the totally substituted

carbon of tetramethyl methane at 31.4 ppm. The carbon of carbonium ions

are the most deshielded, trimethyl carbonium ion (VI) resonating at 330

ppm and the resonance stabilised carbocations (Via) and (VIb) at 248 and

256 respectively. Much use of this has been made in work on controversial

non-classical carbonium ion.

Unshared electrons, as in carbon monoxide (VII) and nitrile ion (VIII),

bring out deshielding as shown by comparisons of carbondioxide (IX) with

carbon monoxide and of methylcyanide (X) with HCN. Mesomeric effect

operates in the same way as in the case of protons and the effect of

introducing electron donating and electron withdrawing substituents on

various sites of the benzene molecule is given below :

128

iNH,

147.7

119

116.1

129.8

NO2

149.1

134.7

(XI)

124.2

129.8

Consideration of the chemical shifts given above shows that benzenoid

carbon canying substituent is the most effected and that this cannot be

attributed to inductive effect alone, is evident from the chemical shift of

17

C-1 of toluene (V). The ortho and para effects are not sharp as they are in

the case of protons and the importance of electric fields in the vicinity of

the carbon atom is shown by shielding as against the expected deshielding

of C-2 of nitrobenzene (XI). Most important from structural point of view

is the extreme deshielding of carbonyl carbon. In simple ketones (XII) it

appears at 5 201.5 ppm, introduction of a, P-unsaturation leads to shielding

by about 5 10 ppm (XIII) and hydrogen bonding brings a further shift of

about 5 5-8 ppm (XIV, XV).

201.5

(xn)

o

192.4

(XIU)

0

(X\0

180.1 0

OH

(XVI)

It is upsetting to find the carbonyl carbon at 180.1 ppm in carboxylic

acids (XVI) as one would have expected the predominant inductive effect

of the hydroxy oxygen to lead to further deshielding. The olefinic cabrons

are deshielded through conjugation but a and p-positions remain

undifferentiated whereas in PMR spectra the a-proton suffers much greater

deshielding.

Coupling Constants :

Because of the low natural abundance of '^C carbon-carbon coupling is

not detectable in the spectra i.e. it is obliterated by noise. Carbon-hydrogen

18

couplings of the A-X type depend on bond hybridisation. For Sp- hybridised

carbon it is 125 Hz, for Sp^ 156 Hz and for Sp 249 Hz. In short, the greater

the 'S character of carbon' the larger the coupling constant. Coupling

constants are also effected by the nature of substituents and the degree of

substitution. Thus C-H coupling in CH3X is about 140 Hz and in CHX3 it is

180 Hz, for X=0CH3. The effect is more pronounced in fluorides i.e. CH3X,

it is 150 Hz and CHX3 240 Hz for X=F.

The above coupling constants are not to be confused with the coupling

constants in off resonance spectra which depend on the Rf employed to

bring about decoupling. This Rf is so adjusted as to provide the best

resolution and the residual coupling is of value only in determining the

number of hydrogen atoms.

Mass Spectroscopy :

Recently mass spectrometry has been successfully employed for the

structure elucidation of flavonoids and their glycosides' *'-' ^"'*" and a

conclusive structure fragmentation pattern relationship established.

Since most of the naturally occurring flavonoids posses at least

5,7,4'-hydroxylation pattern, the present discussion is mainly centred on

such compounds.

Flavones :

The principal modes of fragmentation in flavones involve (i) fission of

the heterocyclic ring via reverse Diels-Alder reaction (ii) loss of carbon

monoxide from molecular or fragment ion. This is illustrated in Scheme (I)

for an unsubstituted flavone'"'' (XVII). Apigenin'"" (XVIll) has the parent

molecule ion as base peak which loses a molecule of carbon monoxide to

19

.0. ^^TA

o

- 0 .

(XVII)

-e

/ \ M+- nyz222(I00)

/

0 ,RDA

,CX / \ r^°

k;.- .====^

^ ,

in/zl94(52)

0

nVz 120 (80) iiVz 102 (12)

0 = 0 +.

mlz91

Scheme -1

20

irVz270(I00)

(XVni)

RDA

HO,. .0

OH O

nVz 152

H0«.

in'zllS

Scheme - II

21

give a major fragment ion m/z 242 (XIX). Fragment ions in much less

abundance correspond to RDA fission in the heterocyclic ring (Scheme II).

By comparison of the mass spectrum of an unsubstituted flavone with

highly oxygenated flavone (apigenin), it is observed that fragmentation via

RDA reaction is less favoured in the latter. This is due to stabilization of the

initially produced ion radical by mesomerism over a number of oxygen

atoms. These minor break downs may still prove to be of diagnostic value as

they frequently represent the only even numbered peaks in their particular

region and hence are readily distinguished.

In case of apigenin trimethylether (XX)'"^"'"^\ the molecular ion

appears as the base peak. Further fragmentation of the molecular ion via

RDA process yields the ketone m/z 180 (XXI) and the acetylene m/z 132

(XXII) (Route 1) and the carbonyl ion, m/z 135 (XXIIl) (Route II) Scheme ffl.

Flavanones:

In the case of flavanone (XXIV), fragmentation by path-A (RDA fission

r^^^^N^

II 0

..^^^^

CH2 +

r^^'^'N^ O

c III 0

,==x

+.CH2

22

H,CO^

H,CO^ . 0 , - ^ A - O C H ,

H.CO O M". iiVz312(I0())

Route-2

0 / Route-1

.,0

C

H3CO 0 iiv'z 180

(XXI)

H,CO^ +

, 0 — H

: =o H,CO

Iiv'z 181

+. .OCH, Q

/ '^ OCH,

nVz 132

(XXII)

O nVz 176

\v/z 135

(XXIU)

Scheme - III

23

of the heterocyclic ring) and path-B are of great importance as they lead to

clean out, characteristic spectra""^ Another method of break down that

helps to characterise the flavanone is the loss of either a hydrogen atom

(XXV) or an aryl radical at C-2 (XXVI) from the molecular ion to give even

electron fragments.

(XXVI)

These fragmentation processes are illustrated in the case of

4'-methoxy flavanone (XXVII) (Scheme-IV). The fragment with methoxyl

group takes nearly all the charge. A further peak is at m/z 108 (XXVIII)

arising from a hydrogen transfer reaction.

^ r^ScH,

ni/z 108 (10)

(XXVffl)

The presence of a hydroxyl (XXIX) or methoxyl group at C-4 position

of ring-B facilitates, by enhanced resonance stabilisation of the resulting

fragment ion, the formation of P-hydroxyl benzyl (XXX) or |3-methoxy

benzyl ion respectively (or their equivalent tropolium ions). These ions

24

. 0 . / ^ OCH,

O iiVz254

(XXW)

RDA

r^^^^^^V^ O

C H

OH CH

iiv'zl21(30)

I H.C

0

OH

iii/z 147 (7)

OCHj

-CO H C = / J

+ 11 2 91 (13)

Scheme - IV

25

appear as peaks of significant intensity in the mass spectrum of

naringenin/its trimethyl ether^^'"'".

HO—I! \—CH2

wti. 107

(XXX)

The mass spectrum of 3,5,7-trihydroxy-4'-methoxy flavanone (XXXI)

is of particular interest, as the base peak is neither the molecular ion nor a

fragment arising from break down via path A (Scheme-V).

Biflavones :

In biphenyl ether type biflavones, molecular ion is usually the base

peak.'-" Apart from the fragmentation processes mentioned for apigenin

trimethyl ether, these compounds also undergo (i) fission of the C-C or

C-O-C linkage between the aromatic residues (ii) elimination of CO (28) or

CHO (29) from the biphenyl ethers and (iii) rearrangements involving

condensation betwen the phenyl rings. Steric factors seem to play an

important role in influencing the break down mode and internal

condensations. Formation of doubly charged ions is frequently obsei-ved.

The mass spectra of amentoflavone hexamethyl ether and

cupressuflavone hexamethyl ether are similar, molecular ion being the base

peak in each case. Difference lies in the intensities of the corresponding

peaks due to variation in substitution and steric factors. The main peaks in

the mass spectrum of amentoflavone hexamethyl ether (XXXII) are given

below (Scheme-VI).

26

/"VoCH,

OH

'^H 0 ny2 302(75)

•FT

^

w/ \ -(XH,

OH Oi-1

{.)H

iiVzjOlCIo)

H( -O

CÂ

OH (.)H

nyz28('i(9)

OH

nV/J5.' (l(X))

+. 152(12)

-CO

'

ni/z 124(21)

•

nil 1.5(

nfz

'

•CO

HCd

r HOHC

-CO / " -< HOHC

+

: 0 1 i-J \ -(XH,

nVz 135(60) nyzl21(12) nyzl(T7(|9)

Scheme - V

27

OCH,

OCH,

H,CO

H3CO O

in/z 180 (3)

OCH3

iiv'z245 + + 490

Scheme - VI

28

Amentoflavone hexamethyl ether (XXXII) :

622(100), 621(13), 607 (33), 592 (8), 576 (10), 312 (2), 311 (5),

245(5), 181 (2), 180 (3), 155 (16) and 132 (3).

The mass spectral studies of the biflavonoids isolated from natmal

sources reveal that their fragmentation patterns depend not only on

constituent monomeric flavonoid units but also on the nature and the

position of interflavonoidal linkage.'°^

29

1. A. Hussain, Present and Future of Green Medicine in the World, J.

Inst. Chemists (India), 60, 37-42 (1988).

2. Journal of Medicinal and Aromatic Plant Sciences, 19, 361-65

(1997).

3. N.R. Fornsworth, How can the Well be Diy When it is Filled with

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CHAPTER - II

Ficus lyrata

Chemical Constituents of Ficus lyrata (Moraceae)

Ficus is a large genus of trees, shrubs and often climbers, with a

milky juice. It is widely distributed throughout the tropics of both

hemispheres but particularly abundant in South-East Asia and Polynesia.

About 65 species are found in India. All species of Ficus yield latex

containing caoutchouc'. Many species of Ficus are medicinally

important^. Ficus lyrata is used for the treatment of hypothermic, diuretic

and CNS-diseases\ Medicinal importance and scanty of work on this

plant encourged us to carry out the comprehensive investigation of leaves

of Ficus lyrata.

The present discussion deals with the isolation and characterization

of the following compounds from the leaves of Ficus lyrata.

(1) Stigmast-l,5-dien-3(3-ol (new)

(2) P-Sitosterol D-glucoside

(3) 5,6-Dihydroxy-2-methyl chromone (new)

(4) 5-Hydroxy-7,3,3',4'- tetramethoxy flavone

(5) 5,4'- Dihydroxy-6,7,8-trimethoxy flavone (Xanthomicrol)

(6) 5,4' - Dihydroxy-7,8,-dimethoxy flavone (Bucegin)

(7) 4- Methoxy chalcone

(8) 7,4' - Dimethoxy apigenin

(9) 5,7,4'- Trihydroxy -2',3',6'-trimethoxy isoflavone

(10) Acacetin-7- glucoside

(11) Acacetin-7-O-neohesperidoside

39

Leaves of Ficus lyrata (3.0 kg) were collected from A.M.U. Fort,

Aligarh, India. They were dried under shade and powdered. After being

defatted with light petroleum ether, the powdered leaves were thoroughly

extracted with chloroform (fraction 'A'), and finallly with hot methanol

(fraction 'B').

The fraction 'A' a gummy greenish mass (160 gms) responded

positively for the colour reactions of flavonoids''. TLC examination in

different solvent systems [benzene-pyridine-formic acid (36:9:5), toluene-

ethylformate-formic acid (5:4:1)] showed it to be a complex mixture.

Therefore, it was chromatographed over silica gel column using

successively petroleum ether, petroleum ether-benzene (9:1,1:1), benzene,

benzene-ethylacetate mixture, ethylacetate and methanol as eluting

solvents. Appropriate fractions on the basis of IR spectra and TLC were

combined. Repeated column chromatography of the fractions followed by

fractional crystallization afforded TLC homogeneous substances labelled

as Fl-1, Fl-2, Fl-3, Fl-4, Fl-5, Fl-6, Fl-7, Fl-8 and Fl-9.

FI-1

Fl-1 was isolated from petroleum ether-benzene (1:1) eluate which

on crystallization with CHCl^-acetone gave white crystals (70 mg), m.p

126-28 °C. It gave positive Leibermann-Burchard's test for triterpenoids^.

The IR spectrum showed absorption bands for hydroxyl group (3450 cm'^),

unsaturation (1640 cm"') and gem-dimethyl, isopropyl group (1380, 1308,

1080 cm''). The EIMS of Fl-1 exhibited molecular ion peak at m/z 412

coiTesponding to the molecular formula as C^^H^gO. It indicated six

degrees of unsaturation, four of which fully adjusted to the four rings of

the carbocyclic nucleus and remaining two of the unsaturated bonds.

40

The Electron Impact Mass Spectrum (Fig. I, Scheme-I) exhibited

diagnostically important fragmentations at m/z 397 [M-Me]"^, 394

[M-H20r, 379[394-Me]^ 369[M-C3H7f, 351[369-H20f, 321[M-C^n^^y,

300[M-C^H,7f, 273[M-side chain +2H]+, 271[M-side chain]^ 229(271

Ring C fission]^ and 213[229-Me]"^. The fragment ions at m/z 226, 197

were due to C^/Ci^ and C,j/C,2 fission and the ions at 248 and 164 were

due to Cg/Cj^ and C^/C^^ fission. These fragments suggested that the

compound Fl-lwas a C29-steroi possessing one hydroxyl group at C-3, a

C-10 saturated side chain and a diunsaturated skeleton, of which one of

the unsaturation was at C-2. The presence of a prominent peak at m/z 135

and 362 were suggestive for the existence of one of the double bond at

C-5 and confirmation of the existence of the hydroxyl group at C-3 in the

ring.

The 'H-NMR spectrum of Fl-1 (Fig. Ila, lib. Table-1) showed the

presence of three downfield signals as one proton doublet at 65.35 (J=5.1

Hz) assigned to H-6, a one proton doublet at 55.14 was attributed to H-1

and another one proton double doublet at 55.02 (J,=8.4Hz and J2=8.4 Hz)

was associated with H-2. A broad multiplet at 53.52 (Wy2=15.6 Hz)

integrated for one proton was assigned to H-3 axial due to biogenetic

consideration. Four doublets integrating for three protons each at 60.97

(J=6.60 Hz), 0.84 (J=6.0 Hz), 0.82 (J=6.0 Hz) and 0.88 (J=6.50 Hz) were

due to methyl functionalities at C-21, C-26, C-27 and C-29 respectively.

The remaining two methyls resonated as broad singlets at 60.68 and 1.00

and were correspondingly assigned to two tertiary methyls at C-18 and

C-19. The appearance of all the methyls in the region 50.68-1.00

suggested that these groups were attached to saturated carbons. The

methylene and methine groups appeared in the region 52.28-1.05.

41

iiVz 135 (22.0)

4

C7H10O

ITVZ 110 (26.6)

CuHifiO

nVzl64(17.I)

* . iiVz394 (33.7)

| - M e

nVz 379 (18,0)

iiVz 397 (23.1)

uVz 327 (25.7)

j . nVz 271(9.7)

H2O

irVz 146 (27.0)

n>'z 230 (14,4) nVz 253 (26.7)

j-RingD

nyz212(8.2)

| - M e

m'zl97(8.8)

-Me

ivVz 149 (6.0)

Scheme - I

C O

^^=-

no

CO

CO

c:)

c-4

-<r

C J

.

^

- ^ ~

'.~1: •

to

^ ' " L I . J

1 ^ 7 5 J

^

o -on

C-4

42

Table-1

'H-NMR spectral data of FI-1, values on 6-scale

Assignments No. of Protons Signals

H-6 1 5.35(d, J=5.1 Hz)

H-l 1 5.14(d, J=8.4 Hz)

H-2 1 5.02 (IH, dd, J=8.4 Hz and 8.4 Hz)

H-3a I 3.52(bnn, W'/2, J=15.6 Hz)

Me-19 3 1.00 (brs)

Me-21 3 0.97 (d, J=6.6 Hz)

Me-29 3 0.88 (d, J=6.5 Hz)

Me-26 3 0.84 (d, J=6.0 Hz)

Me-27 3 0.82 (d, J=6.0 Hz)

Me-18 3 0.68 (brs)

brs= broad singlet, d = doublet, dd == double doublet, brm= broad

multiplet, spectrum run at 300 MHz in CDCI3, using TMS as internal

standard.

4-II .J I

r

C i ! 1. i; ! r

•^[ •-•<f , -^

LD

-CO

OJ

c:

1

o

r Q.

o

i n

.J

'J

o in

.^

Q. Q.

43

The '^C-NMR spectrum of Fl-1 (Fig.Ill, Table-2) indicated the

presence of 29 carbon atoms. Signals at 5140.8, 121.8, 129.3 and 138.41

were assigned to olefmic carbons at C-5, C-6, C-2 & C-1, respectively.

The carbinol carbon resonated at 571.84. The assignment of carbon

chemical shifts was made by comparison of 5-values in the corresponding

carbon atoms in structurally similar sterol*'. The 24R-configuration of the

ethyl group was confirmed by comparison of the chemical shifts of

carbons and protons of the side chain ^ C and 'H-NMR spectra of Fl-1

with a series of sterols having similar configuration of C-24 (645.9)

particularly sitosterol, stigmast-5, ll(12)-dien-3p-ol and stigmast-4-en-

6p-ol-3-one.

On the basis of the these findings, Fl-l was identified as stigmast-

l,5-dien-3p-ol (I), which is being reported for the first time.

(I)

FI-2

The compound Fl-2 was eluted from the column by benzene and

crystallized from chloroform-methanol as colourless crystals (150 mg),

m.p. 282°C. It gave positive Leibermann-Burchard^ and Molisch test^. The

elemental analysis agreed with the molecular formula as C^^H^QO^.

44

Table-2

'^C-NMR spectral data of FI-1, values on 5-scaIe

Carbon numbers Chemical shifts

C-1 138.41 C-2 129.3 C-3 71.84 C-4 42.35 C-5 140.8 C-6 121.8 C-7 29.76 C-8 31.71 C-9 50.21

C-10 36.21 C-11 21.15 C-12 37.33 C-13 39.85 C-14 56.93 C-15 24.34 C-16 28.31 C-17 56.13 C-18 11.92 C-I9 19.45 C-20 34.01 C-21 18.85 C-22 31.97 C-23 26.16 C-24 45.90 C-25 29.23 C-26 19.11 C-27 19.88 C-28 23.18 C-29 12.04

Spectrum run at 400 MHz, in CDCI3, using TMS as internal standard.

i o in

O

45

The 'H-NMR of FI-2 (Table-3) showed the presence of six methyl

groups in the range of 50.70 to 51.01. The signal at 55.2 indicated the

characteristic olefinic proton of steroid. The protons appearing as

multiplet in the range of 54.0-5.1 were assigned to sugar protons. An

anomeric proton appeared at 54.2 as a doublet (J=10.0 Hz). Hence, it was

concluded that the compound Fl-2 is a monoglycoside of steroid.

Acylation of Fl-2 with acetic anhydride and pyridine gave a

colourless acetyl derivative FI-2 (Ac), m.p. 166 °C. 'H-NMR spectrum of

Fl-2 (Ac) (Table 4) showed six methyl groups in the range of 50.71-1.04.

The four independent singlets of three protons each at 51.95, 1.99, 2.02

and 52.04 supported the presence of four aliphatic acetoxyl groups of

monoglycosidic acetate. There was a signal centred at 55.4 characteristic

of an olefinic proton of steroids. It showed a group of multiplets from

54.2-5.3 for protons a- to acetoxyl reminiscent of monoglycosidic acetate.

From these data it could be concluded that the parent compound is a

monoglycoside of sterol. Similar conclusions were drawn from the mass

spectral data too. The mass spectrum of the glycoside acetate Fl-2 (Ac)

(Scheme-II) showed the fragmentation pattern characteristic of

(3-sitosterol.'^'''

Hydrolysis of FI-2 by Killiani's mixture yielded glucose and an

aglycone Fl-2 (Agl). The aglycone having m.p. 137 °C was identified as

P-sitosterol by m.p., m.m.p., co-TLC and comparison of the spectral data

(IR, 'H-NMR and MS) with those of an authentic sample.

46

TabIe-3

^H-NMR spectral data of Fl-2, values on 5-scale

Assignments No. of Protons Signals 0.70 (s) •

0.73 (s) 0.75 (s) 0.78 (s) 1.01 (s) 1.10-1.23 (m) 4.2 (d, J=10.0 Hz) 4.0-5.1 (m) 5.2 (m)

s= singlet, d=doublet and m=multiplet, spectrum run at 300 MHz in CDCI3, using IMS as internal standard.

CH^ CH, 2x CH^ CH, CH, CHj-protons H-r(anomeric) H-r,2',3',4',5',6' Olefinic proton

3 3 6 0 J

3 2 1 7 I

Table-4

^H-NMR spectral data of Fl-2 (Ac), values on 6-scale

Assignments

6xCH^

H-6(01etinic proton)

Sugar acetoxyls 4 X Acetoxyl

H-r,2',3',4',5',6'

No. of Protons

18

1

12

7

Signals

0.71-1.04

5.4 (m)

1.95(3H,s), 1.99(3H,s), 2.02(3H,s), and 2.04 (3H, s)

4.2-5.3 (m)

s=singlet, m=multiplet, spetrum run at 300 MHz in CDCl^, using TMS as internal standard.

47

(M]"^ absent

•glucose treat meat

iiVz 255

Scheme - II

48

On the basis of above results, the compound Fl-2 was characterized

as f3-sitosterol~D-glucoside'^(Il).

HO ^ Q

FI-3

It was eluted from the column with benzene-ethyl acetate (10:1) and

cr^'stallized from chloroform-methanol as light cream needles shaped

crystals (65 mg) m.p. 172-74 °C. It gave greenish brown colour with

alcoholic ferric chloride which indicated the presence of phenolic

hydroxyl group. It responded negatively to Shinoda's test"* indicating the

absence of flavone nucleus. Elemental analysis alongwith the molecular

ion peak at m/z 192 agreed with the molecular formula as C,(jHg04. The

characteristic bands in IR spectrum showed the presence of chelated OH

group at 3244 cm"' and a carbonyl group at 1648 cm"'. Its UV spectrum

(Table-5) displayed the maximum absorption at 220, 261 and 320 nm

corroborating the presense of chromone nucleus^'^"^'". A bathochromic

shift of 20nm with AICI3 indicated the presence of a hydroxyl group at

5-position, while the hypsochromic shift of 22 nm with AlCl^/HCl pointed

the presence of ortho-dihydroxyl grouping. Thus it clearly indicated the

presence of OH groups at 5 and 6-positions of chromone nucleus.

The 'H-NMR spectrum'^'' of Fl-3 (Fig.IV, Table-6) showed a sharp

singlet of three protons at 52.35, assigned to CH^ group, while a pair of

ortho coupled doublets indicating for one proton each at 66.79 (J=9.0 Hz)

Table-5

UV spectral data of FI-3 with shift reagents

49

Reagents nm.

MeOH

NaOMe

AICI3

A1C17HC1

NaOAc

NaOAc/H,BO,

220, 261, 320, 323(sh)

219,262, 320, 322(sh)

220, 270, 340, 362(sh)

219, 259, 318, 321(sh)

220, 262, 319, 323(sh)

219, 269, 327

(sh = shoulder)

Table-6

^H-NMR spectral data of Fl-3, values on 5-scale

Assignments

CH,

H-3

H-7

H-8

5-OH

6-OH

No. of Protons Signals

2.35 (s)

6.12 (s)

6.79 (d, J=9.0 Hz)

7.10(d, J=9.0Hz)

ll.O(s)

9.57 (brs)

s = singlet, d= doublet, brs= broad singlet, spectrum run at 300 MHz in DMSO-d(, , TMS as internal standard.

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and 57.10 (J=9.0 Hz) were ascribed to H-7 and H-8 respectively. The

remaining singlet of one proton at 56.12 may be assigned to C-2 or C-3

proton. The possibility for H-2 proton was ruled out as it appeared at

higher field'^. Thus the CH3 group can only be at C-2 position. It was

further confirmed by '''C-NMR spectrum (Table-7, Fig. V). The

assignments of corresponding carbons are given in (Table-7)'\

The mass spectrum (Fig.VI, Scheme-Ill), showed the molecular ion

peak at m/z 192. The fragment ions'"* are rationalized from the scheme.

On the basis of above evidences, the compound Fl-3 was

characterized as 5,6-dihydroxy-2-methyl benzopyrone(Ill) which is being

reported for the first time.

HO

OH 0

(ni

FI-4

It was obtained from the column with benzene-ethyl acetate (9:1) and

crystallized from chloroform-methanol as yellow crystals (60 mg), m.p.

160-62°C. It responded positively to Shinoda's tesf* and gave red colour

on reduction with sodium amalgam followed by acidification'''''' indicating

it to be a flavanone or 3-substituted flavonol. The possibility of its being

a flavanone was ruled out as it gave yellow colour with Wilson boric acid

reagent'^. A greenish brown colour with alcoholic ferric chloride,

indicated the presence of phenolic hydroxyl group/s. Elemental analysis

alongwith the molecular ion peak at m/z 358 corresponded to the

Table-7

^^C-NMR spectral values of Fl-3 on 5-scale

No. of Carbons

C-2

C-3

C-4

C-5

C-6

C-7

C-8

C-9

C-10

CH,

Assignments

160.26

112.14

171.10

153.98

149.44

132.19

110.19

143.34

115.51

18.26

Spectrum run in DMSO-d. at 300 MHz

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m/z 164(12)

OH O

C|()H804

[M]"^nV2 192 (42)

HO' II

OH O

C7H4O4

ITVZ 152 (60)

-CH, iiVz 177(15)

H2O

uVz 174 (10.2)

CO

+ H"

HO

• • iiVz 153

c=o

OH

iiVz 126 (9.5)

Scheme - III

m « " i

lo t) lO

lO b) •-1

Wl t) t^

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Ln t>) o

to to to

to 14 c

tq

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53

molecular formula as C^gH^Ôj. The IR spectrum revealed the presence of

phenolic hydroxyl at 3100-3150 cm"', and a carbonyl group at 1665 cm"'.

The UV spectrum (Table-8) displayed a maximum absorption at 278 &

341 nm. A bathochromic shift of 13 nm with AICI^ in band II indicated

the presence of a free hydroxyl group at 5-position'^ which was further

confirmed by the signal at 512.96 in 'H-NMR spectrum.

The 'H-NMR spectrum of Fl-4 (Table-9, Fig. Vll) showed a pair of

meta-coupled doublets of one proton each at 56.99 (J=2.0 Hz) and 7.05

(J=2.0 Hz) attributed to H-6 and H-8 protons respectively. The B-ring

protons exhibited an ABX pattern as it showed an ortho-coupled doublet

at 67.13 (J=9.0 Hz) integrating for one proton, ascribed to H-5', a meta

coupled doublet at 57.61 (J=1.8 Hz) corresponded to H-2' proton and a

double doublet at 57.72 (J, = 1.8 Hz, J^"^ 9.0 Hz) was attributed to H-6'

proton. The remaining four independent singlets of three protons each at

63.74, 3.86, 3.89 & 3.94 were assigned to four methoxyl groups located

at 3,7;3' and 4' positions.

The above assigned structural data was further supported by the mass

spectrum (Fig. VIII, Scheme-IV). The molecular ion peak appeared at

m/z 358. The fragment ions at m/z 166 & m/z 180 resulted by the retero

Diel's-Alder cleavage of the flavone nucleus, thereby indicating the

presence of one hydroxyl and one methoxyl in ring-A and two methoxyl

in ring-B and one methoxyl in ring-C. The other fragments are

rationalized from the Scheme-IV.

On the basis of above forgoing discussion including chemical and

54

Table-8

IJV spectral data of FI-4 with shift reagents

Reagents max

MeOH

NaOAc

NaOAc/H3B03

AlCl,

AICI3/HCI

NaOMe

217,278, 341

218,278, 341

218, 277, 341

219, 291, 362, 374(sh)

219, 292, 361

219, 291, 340

(sh = shoulder)

TabIe-9

^H-NMR spectral data of Fl-4 (5-scale)

Assignments

H-6

H-8

H-5'

H-2'

H-6'

OMe-3

OMe-7

OMe-3'

OMe-4'

OH-5

No. of Protons

3

3

3

3

1

Signals

6.99 (d, J=2.0 Hz)

7.05 (d, J=2.0 Hz)

7.13 (d, J=9.0 Hz)

7.61 (d, J=1.8 Hz)

7.72(dd, J|=1.8 Hz, J2=9.0 Hz)

3.74(s)

3.86(s)

3.89(s)

3.94(s)

12.90(s)

s= singlet, d= doublet, dd= double doublet, spectrum run in DMSO-d^ at

300 MHz, IMS as internal standard.

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iiyz343(62)

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Scheme - IV

•H'

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OCH,

H3CO—C=C f 7 OCH3

C11H12O3

iiVz 192 (48)

-Me

nVz 177(21)

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56

spectral evidences, the compound Fl-4 was characterized as 5-hydroxy-

7,3,3',4'-tetramethoxy flavone (IV)'l

MeO.

OH O

OMe

OMe

(IV)

FI-5

The compound Fl-5 was eluted from the column with benzene-

ethylacetate (9:1) and was crystallized with chloroform-methanol as

yellow crystalline solid (150 mg) m.p. 224-25 °C. Elemental analysis

agreed to the molecular formula CjgH,(30-,. It gave a greenish brown

colour with alcoholic FeCl^, pink colour with Zn/HCl and responded

positively to Shinoda's tesf* suggestive of a flavone nucleus. The flavone

nucleus was further evidenced by the UV spectrum (Table-10) which

showed the absorption maxima at 296 and 333 nm. Analysis with shift

reagents''' gave diagnostic shifts of 25 nm in band-11 with AICI3 and of 34

nm in band-1 with NaOMe, pointing the presence of free hydroxyls at

5- and 4'-positions respectively. IR spectrum exhibited the presence of

carbonyl group at 1660 cm'' and OH group at 3350 cm"'.

The 'H-NMR spectrum (Table-11, Fig. IX), showed a characteristic

A2B2 pattern due to the presence of a pair of ortho-coupled doublets

integrating for two protons each at 56.95 (J=9.0 Hz) and 7.94 (J=9.0 Hz),

assigned to H-3',5' and H-2',6' protons respectively. A one proton singlet

at 56.89 was ascribed to H-3 proton''^ further confiiTning the flavone

57

Table-10

UV-spectral data of FI-5 with shift reagents

Reagents

MeOH

NaOAc

NaOAc/H^BO^

AlCl^

AlCl^/HC]

NaOMe

^mav"m

209, 296, 333

215, 293, 335, 396

213, 294, 334

213, 298, 355

214, 233, 298, 354

216,291,367,400

Table-U

' H - N M R spectral data of FI-5, values on 5-scale

Assignments

OMe

OMe

OMe

H-3

H-3',5'

H-2',6'

OH-5

OH-4'

No. of Protons

3

3

3

1

2

2

1

1

Signals

3.73(s)

3.81(s)

3.91(s)

6.89(s)

6.95 (d, J=9.0 Hz)

7.94 (d, J=9.0 Hz)

12.78(s)

10.45(s)

s=singlet, d= doublet, spectrum run in DMSO-d^^ at 300 MHz using TMS as internal standard.

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nucleus. Three independent singlets of three protons each at 53.73, 3.81

and 3.91 were attributed to the methoxyl groups centred at 6,7 and

8-positions.

The mass spectrum (Scheme-V, Fig. X), was in full agrreement with

the above assigned structural data. It showed a molecular ion peak at m/z

344. The substitution pattern in A and B-ring was further confirmed by

the retero Diels-Alder fragment ions at m/z 226 [A]^ 118 [B,]^ 121 [82^,

other fragments are discussed in Scheme-V.

On the basis of above results the compound Fl-5 was identified as

5,4'-dihydroxy-6,7,8-trimethoxy flavone (Xanthomicrol) (V) '*.

OMe

MeO. J^ . 0 . / ~ \ OH

MeO"

OH 0 (V)

FI-6

Fl-6 was obtained from benzene-ethyl acetate (8:2) fraction. It was

crystallized from chloroform-methanol as yellow crystals (78 mg), m.p.

290-92 °C. Elemental analysis agreed with the molecular formula

Cj^HjÔg. It gave greenish brown colour with alcoholic FeCl^.

Flavonoidic nucleus was evidenced by positive Shinoda's test'', a pink

colour with Zn/HCl and red colour with Na-Hg/HCl'^, further confirmed

by the UV spectrum which showed the characteristic bands at 280 and

334 nm. Analysis with shift reagents showed a bathochromic shift of 20

nm in band-Il with AlCl^ and 36 nm in band-1 with NaOMe indicating the

59

oa^

m/z316 (28)

Hi

RO

-CHj

niz2l\(LS)

-CH,

nYzl%(2())

-CHj

nfzl81(5)

QSHKA

nfz329(55) ' > nizmQS)

V

iir'z289(12)

OCH,

II OH O

Q0H10Q5 ufz226(2g)

-00

QH,o iii'zll8(32)

i\izm(26)

iifel';8(10) •'"" -'» n/z 168(18) . ^ 2 1 ^ nrzl53(8)

Scheme - V

tq 0\

to W o

IN . . I . . . . 1 .

cn r-i— r i

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cr> 00-

X I

o

^-

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S-r-i in

60

presence of hydroxyl groups at 5 and 4'-positions of flavone nucleus.

Functional group analysis in IR spectrum showed the characteristic

absorption bands at 3210 (OH) and 1655 (C=0) cm' .

The 'H-NMR spectmm (Table-12, Fig. XI) showed two independent

singlets of three protons each at 63.84 and 4.04, assigned to methoxyl

groups at 6,7 or 7,8 positions. A sharp one proton singlet at 56.97 was

ascribed to H-3 proton. A pair of ortho-coupled doublets integrating for

two protons each at 57.05 (J=9.0 Hz) and 68.07 (J=9.0 Hz) were attributed

to 3',5' and 2',6'-protons of B-ring constituting A2B2 pattern. The

remaining one proton singlet at 67.03 could be assigned to either H-6 or

H-8 proton. The possibility of H-8 proton was ruled out as it gave a

negative Gibb's test^' which indicated that carbon para to the hydroxyl

group is substituted. Thus the two methoxyl groups are placed at 7 and 8

positions respectively.

The above assignments were further supported by the mass spectrum

(Fig. XII, Scheme VI), which showed the molecular ion peak at m/z 314.

The retero-Diels Alder fragments at m/z 196 [A]+- and 118 [BJ^- and 121

[B2]" * further confirmed the substitution pattern of A-ring and B-ring.

In the light of above discussion the compound Fl-6 was assigned as

5,4'-dihydroxy~7,8-dimethoxy flavone (Bucegin) (Vl)^^.

OMe

OH

OH O

(VI)

Table-12

^H-NMR spectral data of FI-6 (5-scale)

Assignments

OMe

OMe

H-3

H-6

H-3',5'

H-2',6'

4'-0H

5-OH

No. of Protons

3 o J

I

1

2

2

1

I

Signals

3.84(s)

4.04(s)

6.97(s)

7.03(s)

7.05 (d, J=9.0 Hz)

8.07 (d, J=9.0 Hz)

I0.50(s)

I3.04(s)

s= singlet, d= doublet, spectrum run in DMSO-d^ , at 300 MHz, TMS used

as internal standard.

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Scheme - VI

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63

FI-7

Fl-7 was eluted from the column with benzene-ethylacetate (8:2)

mixture and crystallized with chloroform-methanol as light cream

coloured crystals (165 mg), m.p. 77-80°C. The elemental analysis agreed

with the molecular formula Cj^HjÔj. A red colour with cone. HjSO^ and

orange to red colour with aq.NaOH suggested it to be a Chalcone'^'^\ The

IR spectrum displayed the characteristic bands at 1661 cm"' (C=0) and

1475 cm"' (C=^C). Its UV spectrum showed the maximum absorption at

345 nm and minimum absorption at 245 nm. Analysis with the diagnostic

shift reagents'^^ gave no shift in X^^^^^ indicating the absence of free

hydroxyl group/s in the chalcone nucleus, further supported by negative

ferric chloride test.

The 'H-NMR spectrum of Fl-7 (Table-13, Fig. XIII) showed a singlet

of three protons at 53.87 assigned to a methoxyl group. A pair of ortho-

coupled doublets at 56.92 (J=8.7 Hz) and 57.59 (J=8.7 Hz) integrating for

two protons each were attributed to H-3,5 and H-2,6 protons respectively.

Another pair of doublets at 57.39 (J=15.0 Hz) and 57.76 (J=15.0 Hz) were

ascribed to a and (3 protons of chalcone. The 2',6' protons appeared as an

ortho-coupled doublet at 67.99 (J=8.4 Hz) while 3',4',5' protons were

resonating as a inultiplet in the range of 67.52-7.63.

The above assigned structural data were further supported by the

mass spectrum (Scheme-VII, Fig. XIV) which showed the molecular ion

peak at m/z 238. The fragment ions at m/z 161 and 134 supported the

presence of p-methoxyphenyl ring. The other fragments were rationalized

from the Scheme-VII.

64

Table-13

'H-NMR spectral data for FI-7, values on 5-scale

Assignments

OCH3

H-3,5

H-2,6

H-a

H-P

H-2',6'

H-3',4',5'

No. of protons

3

2

2

1

1

2

3

Signals

3.87(s)

6.92 (d, J=8.7 Hz)

7.59 (d, J=8.7 Hz)

7.39(d, J=15.0 Hz)

7.76(d, J=15.0 Hz)

7.99 (d, J=8.4 Hz)

7.52-7.63 (m)

s= singlet, d= doublet, m= multiplet, spectrum run at 300 MHz in CDGI3,

using TMS as internal standard.

00000 0

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nVz210(18.18) -CO . ^ ^ ^ ^

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-H'

iii/z77 (1,5)

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r^ +.

OCH,,

-CH, iiv'z 223 (10.60)

0

Cl6H,402

[Mr:it>'z238(100)

CioHr)02 nVz 161 (49.8)

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CH,oO nyzl34(15.15)

•H*

nyzl33(1.5)

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Scheme-VII

m c. y-

C E N ^ K A L . 1)I<IIG K-rSL'ni^CII INSTITUTE 1 1 - 2 2 - 2 0 0 0

I0'>.

12 i<'\=- 1:21 Ko.\ou'..^- I-IB BaGe= £ 9 . 8 7 . r T I C = 1 7 7 7 3 l

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FIG. - XIV

66

On the basis of above results, Fl-7 was characterized as

4-methoxy chalcone( Vll)^''.

.OCH,

.^^^^^

o

v^ (vn)

Fl-8

The compound Fl-8 was obtained from the column by benzene-ethyl

acetate (7:3) mixture. It was crystallized from chloroform-methanol as

white crystals (180 mg), m.p. 185-87 °C. The elemental analysis and the

molecular ion peak at m/z 298 agreed with the molecular formula

Ci^HjÔj. The flavonoidic nucleus was evidenced by positive Shinoda's

tesf*. It gave greenish brown colour with FeCl^ solution. The UV-

spectrum showed the absorption maxima at 244 nm (band-II) and 369.5

nm (band-I). Analysis with diagnostic reagents' *^ gave a red shift of 32.5

nm with AlCl^ solution indicating the presence of free hydroxyl group at

5-position.

IR spectrum of Fl-8 showed a carbonyl band at 1669 cm"', phenolic

hydroxyl group at 3429 cm"' and complex aromatic substitution pattern at

1507, 1464, 1383, 1270, 1162, 833, 818 cm' along with two strong peaks

at 2920 and 2850 cm"'. The 'H-NMR spectrum of Fl-8 (Table-14) showed

the presence of seven aromatic protons. A sharp singlet at 56.58 indicating

the presence of C-3 proton of y-pyrone-ring. Two meta-coupled doublets

at 66.36 (J=3.0 Hz) and 56.48 (J=3.0 Hz) integrating for one proton each

were assigned to H-6 and H-8 respectively, two ortho-coupled doublets at

67

Table-14

'H-NMR spectral data of Fl-8, values on 6 scale

Assignm

OH-5

2xOCH,

H-3

H-6

H-8

H-3',5'

H-2',6'

ents No.of "protons

1

6

1

I

1

2

2

Signals

12.81(s)

3.88 (s)

6.58(s)

6.36 (d, J=3.0 Hz)

6.48 (d, J=3.0 Hz)

7.00 (d, J=8.7 Hz)

7.83 (d, J=9.0 Hz)

s^ singlet, d= doublet, spectrum run at 300 MHz in CDCI,, TMS as

internal standard.

68

57.00 (J=8,7 Hz) and 57.83 (J=9.0 Hz) integrating for two protons each

corresponding to H-3'.5' and 2',6' protons respectively. A sharp one proton

singlet at 612.81 was assigned to a hydroxyl group at 5-position. Another

a sharp singlet for six protons at 63.88 was ascribed to two methoxyl

groups.

Acetylation of Fl-8 with acetic anhydride and dry pyridine gave a

monoacetate Fl-8 (Ac) (Vlllb), m.p. 153 "C. The 'H-NMR spectrum of

the acetate showed a sharp singlet at 62.45, integrating for three protons,

was assigned to the acetoxyl group at C-5 position.

The assignment of the functional groups was further supported by the

mass spectrum of Fl-8, which showed the molecular ion peak [M" -] at

m/z 298. The fragment ions at m/z 283, 268 corresponding to the loss of

methyl groups from the molecular ion peak and from the fragment

m/z 283. The fragment at m/z 270 showed the loss of carbonyl group

from [M" '].

On the basis of the above results, the compound Fl-8 was

characterized as 7,4'-dimethoxy apigenin (Vllla)^^.

H3C0,

^

/ Vo™,

OH 0 (Vin)

(a)R = H (b)R = Ac

69

FI-9

FI-9 was obtained by elution of column with benzene-ethyl acetate

(7:3) and crystallized with chloroform-methanol as pale yellow plates

(180 mg), m.p. 198-200 °C. The elemental analysis agreed with the

molecular formula as C,gH](Ôjj. The mass spectrum showed the

molecular ion peak at m/z 360 in agreement with the molecular formula

assigned to it. Active hydrogen determination showed the presence of

three hydroxyl groups and methoxyl groups estimation showed the

presence of three methoxyl groups. This was confirmed by the formation

of a triacetate and hexamethyl ether.

A negative Durham test and absence of any colouration with Mg/

HCl"* ruled out flavanone or flavone nucleus for Fl-9. On the other hand,

treatment with sodium amalgam followed by acidification resulted in pink

colouration; suggesting an isoflavone nucleus'^, further supported by U.V.

absorption at 262 nm and an inflection at 329 nm. The colour reaction,

UV spectral studies and a sharp singlet of one proton at 58.36 in 'H-NMR

spectrum of Fl-9 suggested it to be an isoflavone having three methoxyl

and three hydroxyl groups.

The compound gave a dark green colour with ferric chloride, the

presence of a chelated hydroxyl group was also evidenced by the band at

3433 cm"' in its IR spectrum (Fig. XV). A red shift of 12 nm was also

observed in UV spectrum on addition of anhydrous AlCl^. The presence

of chelated 5-hydroxyI was further confirmed by the signal at 613.01 in

the 'H-NMR spectrum of Fl-9. Furthermore, a red shift of 10 nm with

NaOAc and 'H-NMR signal at 510.32 indicated the phenolic hydroxyl at

7-position'^^'^^\ The methanolic solution of the compound was not

o o in

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i i h ) i i H | i ! i i | i ! i i [ i i n | i i i i | i i i i | i i M | i i i i | i i i i | ! i i i [ ; i i i ! i i i i | i i i n i i i ; [ i i ; i | n i i | i i i i | i i i i i i i r i | i i i i nrr-o o

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70

oxidised by pentamine cobalt trichloride'^^, indicating the absence of

adjacent phenolic hydroxyl groups, thus eliminating the possibility of the

third hydroxyl in the same ring.

The 'H-NMR spectrum of Fl-9 (Fig. XVI, Table-15) showd a sharp

singlet at 68.36 indicating the presence of C-2 proton for y-pyrone

nucleus. A pair of meta-coupled doublets at 56.63 (J=3.0 Hz) and 6.68

(J=3.0 Hz) each integrating for one proton were assigned to C-6 and C-8

protons respectively. The C-6 proton was shielded by two ortho and one

para oxygen. A sharp upfield singlet of one proton at 56.48 could be

assigned to either C-3' or C-5' proton. The possibility of its being at any

other position in B-ring was ruled out as the hexamethyl ether of Fl-9

furnished 2,3,4,6-tetramethoxy benzoic acid when subjected to oxidative

degradation with alkaline W^'^^^''. The identity of 2,3,4,6-tetramethoxy

benzoic acid was confirmed by co-TLC and m.m.p with an authentic

sample. Thus the singlet at 56.48 was assigned to C-5' proton which was

shielded by tv/o ortho and one para oxygens. The presence of three

methoxyl groups were indicated through three singlets at 63.67, 3.73 and

3.76 each integrating for three protons. The remaining hydroxyl group

exhibited by a signal at 69.33 may be placed at C-3' or C-4'. However, the

comparison of the '^C-NMR of the parent compound (IXa) with that of its

acetate (IXb) (Table-16) ruled out the possibility of the hydroxyl group at

C-3' position. The signal of C-4' caron of the acetate (IXb) moved upfield

by 12.0 ppm, while the signal due to C-T, C-3' and C-5' moved downfield

by 6.0 ppm, 8.3 ppm and 8.4 ppm respectively. The upfield shift of C-4'

carbon of the acetate and downfield shift of carbons ortho and para to it

confirmed the presence of hydroxyl group at C-4' 23b,28 j ^ ^ signals due to

meta carbons namely C-2' and C-6' remained almost unchanged in the

71

TabIe-15

^H-NMR spectral data of FI-9 (5-scale)

Assignments

H-2

H-8

H-6

H-5'

OCH^-2'

OCH^-6'

OCH3-3'

OH-4'

OH-7

OH-5

No. of protons

I

1

1

1 ^ J

1

1

1

Signals

8.36(s)

6.68 (d, J=3.0 Hz)

6.63 (d, J=3.0 Hz)

6.48(s)

3.67(s)

3.73(s)

3.76(s)

9.33(s)

10.32(s)

13.01(s)

s=singlet, d= doublet, spectrum run in DMSO-d ^ at 300 MHz using TMS

as internal standard.

a, X Q I a x o ^ ( U O l D o ( ^ J ^ — 0 3 0 0 0 0 — x c D O U J u - ^ n i o o o o o c ^ f f i M i D L T ) — f ^ i f l C T i r u O o ' O » — C D o n m a a r ^ u J i f o t n o < D ( 0

r ^ i f\( — ry — ^ ' c n o en e o o L f \ j O J C D I D O i D O l O _* t o r n o i f O U J O O •-• O i O I / l

ta t n CM i n — Ol <D O

itli

SIS

: 3 Q o z I ( 2 r a: tij t w « *- S S > (

^ 6 t O O _ J _ _ J I I

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00 31 (J

o o y

Q. a X u o x — — <\»ryQ.iv; — ( J l i . U - U w U . C L I

8f 2

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http://Jli.U-UwU.CLI

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72

acetate (IXb). This fixed up the position of the hydroxyl group at

C-4'-position.

The '^C-NMR spectrum of Fl-9 (Fig. XVII, Table-16) showed the

C-2 carbon of the y-pyrone at 153.27 ppm and C-3 at 126.09 ppm. In

flavones the corresponding values are 163.2 and 103.1 ppm. As expected,

the values for ring-A and ring-B carbons are about the same for flavones

and isoflavones and chemical shifts depend upon only on site and degree

of oxygenation of rings ^* .

The mass spectrum of Fi-9 (Fig. XVIll, Scheme-VIII) showed M"*"* at

m/z 360 and M-15, M-30, M-45 fragment ions corresponding to the

successive loss of three methyl groups at m/z 345, 330 and 315

respectively. The RDA cleavage was facile and lead to the fragments of

mass m/z 153, 152 and 151 of ring-A and m/z 208, 207 and 178 of ring-B

which were indicative of the presence of two hydroxyls in ring-A and one

hydroxyl and three methoxyls in ring-B. The significant fragment at m/z

329 [M-31, OMe] supported the presence of isoflavone with 2'-0Me^^.

The other fragments were rationalized from Scheme-Vlll.

On the basis of above results, the compound Fl-9, was assigned as

5,7,4'-trihydroxy-2',3',6'-trimethoxyisoflavone (IXa) **.

Q MeO OMe

OMe (IXa)

a) R = H b) R = Ac c) R = Me

73

Table-16 i^C-NMR spectral data of Fl-9 and FI-9Ac, (5-scale)

Carbon No.

2 3 4 5 6 7 8 9 10 r 2' 3' 4' 5' 6'

OCH,

3xC=Oof OAc

3XCH3 ofOAc

Spectrum run at 300 MHz in CDCI3, using IMS as internal standard.

Chem

Fl-9

153.27 126.09 180.27 154.74 100.05 157.68 93.94 154.27 104.56 121.75 150.25 153.27 152.86 119.82 152.66 59.92 55.82 55.10

ical Shifts

FI-9AC

153.3 126.5 178.0 151.5 113.4 152.9 110.2 151.9 117.4 128.6 146.1 161.6 140.8 128.2 148.3 56.1 56.8 55.5 168.2 168.5 169.4 20.60 20.90 21.10

X X 3 X 3 3 X ex X a. X

II tCi <:> <ji o - ^ o n o < ^ c j p j i \ * o c 3 o o o » o o i * i x ^ o ^ o — * ( J O

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UJ Z O ( V

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74

m/z 327 (15.1) k

-H2O

iiVz 345 (45.4)

•CH,

nVz 330 (22,1)

•CH,

nVz315(58.8)

iiVz 342 (35.9)

-H,0

OCH3 OCH3

^ -OCH^ nVz 329 (10.5)

fM]' ',iiVz36fl(100) [M|"îyzl80(4.4)

OH O

iiVz 152 (4.0) OCH3

ITVZ208(8.0)

itVz314(7.1)

-CH3

m/z 299 (7.1)

Scheme - VIII

0~)

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u CI) Ul C£

i n ~J iii

a ...1 <r. Q:

r; O O (•••i

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T- f

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• i.' 0 LL

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II (J H

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75

Fraction B

The methanolic extract (fraction 'B') also gave positive tests for

flavonoids''. TLC examination of the extract in different solvent systems

showed the presence of several compounds. However we could isolate

only two pure compounds Fl-10 and Fl-Il even after repeated column

chromatography over silica gel and fractional crystallizations.

FI-10

The compound Fl-10 was eluted from the column by benzene-ethyl

acetate (1:1) mixture. It was crystallized from chloroform-methanol as

cream coloured crystals (150 mg), m.p. 255°C. The elemental analysis

agreed with the molecular formula as C22H220,Q. The glycosidic nature of

the compound was evidenced by positive Molisch test^. It gave greenish

colour with FeCl^. The flavonoidic nucleus was evidenced by positive

Shinoda's test"* and UV spectrum X^^^^ at 268 nm (band II) and 321 nm

(band I)). Analysis with diagnostic reagents' "^ gave a bathochromic shift

of 32 nm with AlCl, indicating the presence of free hydroxyl group at

5-position. The possibility of its being a flavanone glycoside was ruled

out as it gave yellow colour with Wilson-Boric acid reagent"'. Its IR

spectrum showed a carbonyl group at 1656 cm' , phenolic hydroxyl group

at 3399 cm'' and a complex aromatic substitution pattern at 1615, 1498,

1304, 1173, 1081 and 832 cm-'.

The 'H-NMR of Fl-10 in DMSO-dg (Table-17), showed the presence

of seven aromatic protons. A sharp singlet at 66.96 indicating the presence

of C-3 proton of y-pyrone ring. Two meta-coupled doublets at 56.45

(J=3.0 Hz) and 56.85 (J=3.0 Hz) integrating for one proton each were

assigned to H-6 and H-8 respectively. Two ortho-coupled doublets at

76

Table-17

'H-NMR spectral data of Fl-10, values on 5-scaIe

Assignments

H-3 H-6 H-8 H-3',5' H-2',6'

Sugar Protons : H-l"(anomeric) H-1", 2",3",4",5",6"' OMe OH-5

No. of protons

1 1 I 2 2

1 7 o J

1

Signals

6.96(s) 6.45 (d, J=3.0 Hz) 6.85 (d, J=3.0 Hz) 7.12(d, J=9.0Hz) 8.05 (d, J=9.0 Hz)

5.06 (d, J=8.0 Hz) 5.06-5.41 (m) 3.87 (s) 12.41 (s)

s= Singlet, d= doublet, m= multiplet, spectrum run in DMSO-d^ at 300 MHz, using TMS as internal standard.

77

57.12 (J-9.0 Hz) and 58.05 (J= 9.0 Hz) integrating for two protons each

corresponding to H-3',5' and H-2',6' respectively and a one proton singlet

at 512.41 for hydroxyl group at 5-position. The sugar protons appeared in

the region of 55.06-5.41 and a sharp three protons singlet at 63.87 was

assigned to methoxyl group. The H-1" proton of sugar appeared as a

doublet at 55.06 (J= 8.0 Hz) which is of glucose.

The total hydrolysis of Fl-IO with 6% hydrochloric acid yielded

glucose ai\d au aglycone Fl-lO Ag. The sugar was ideuUfted as glucose by

co-paper chromatography with an authentic sample.

The quantitative estimation of sugar by Somogyi's copper-micro

method^-', indicated the presence of one mole of sugar per mole of

glucoside.

The UV spectrum of the aglycone (Table-18) showed a bathochromic

shift of 28 nm in band-ll with NaOAc which was absent in the glycoside,

thus, suggesting that 7-position is involved in glycosylation''''^.

The acylation of the aglycone Fl-lO Ag with acetic anhydride and

pyridine gave a diacetate Fl-lO (Ac). TLC examination and 'H-NMR

spectrum indicated it to be acacetin. The 'H-NMR spectrum of Fl-lO (Ac)

(Table-19) showed one methoxyl group at 53.88. The UV spectrum of

Fl-lO(Ag) is comparable with the spectrum of acacetin (Table-18). The

'H-NMR values of Fl-lO(Ac) and acacetin diacetate are recorded in

(Table-19).

UV spectrum of Fl-lO Ag gave A,, ^ at 269 nm (Band II) and a

pronounced inflection at 327 nm. AICI3 produced a 55 nm shift of band-I

showing thereby a free 5-hydroxyl group. A shift of 31 nm in band-ll with

78

Table-18

UV spectral data of Fl-10 (Agl) and acacetin, values in nm

Reagents

MeOH

NaOAc

NaOAc/H,BO.^

AICI3

AlCyHCl

NaOMe

FI-10 Ag

269, 303sh, 327

276, 297sh, 358

269,309sh, 331

259sh, 277, 292sh,

302, 344, 382

260sh, 279, 294sh,

300, 338, 379

276, 295sh, 383

Acacetin

270, 303sh, 328

276, 298sh, 357

269, 309sh, 331

260sh, 277, 291sh,

301,344,381

260sh, 280, 294sh,

301, 337, 380

275, 295sh, 383

sh= shoulder

Table-19

*H-NMR spectral data of Fl-lO(Ac) and acacetin diacetate (5-scale)

Assignments

H-8 H-6 H-3 H-2',6' H-3',5'

OMe/OAc : -4' -5 -7

FI-lb(Ac)

7.83 (lH,d,J=4.0Hz) 6.74(lH,d,J=3.0Hz) 6.53 (lH,s) 7.78 (2H,d,J=9.0 Hz) 6.96(2H,d,J=9.0 Hz)

3.88 (3H,s) 2.38 (3H,s) 2.32 (3H,s)

Acacetin diacetate

6.55 (IH, d, J=3.0Hz) 6.20 (IH, d, J=2.5 Hz) 6.37 (IH, s) 7.80 (2H, d, J=9.0 Hz) 6.95 (2H, d, J=9.0 Hz)

3.86 {3H,s) 2.43 (3H,s) 2.33 (3H,s)

s=singlet, d= doublet, spectrum run in CDCl^ at 300 MHz, using TMS as internal standard.

79

sodium acetate indicated the presence of a free 7-hydroxyl group' * . With

NaOAc-H3B03 the spectrum of Fl-10 Ag showed a hypsochromic shift of

9 nm in band-1 relative to apigenin with a decrease in its relative intensity,

showing thereby a protected 4'-hydroxyl group.

M.P., UV, and 'H-NMR spectra of Fl-10 (Ac) were found to be

identical with those of acacetin diacetate (Table-19).

Fl-10 was, therefore, assigned the structure as acacetin 7-0-

glucoside(X)-".

CH2OH

H O "

H O - ^ OCH3

OH 0

(X)

FI-11

It was eluted from the column by ethylacetate and crystallized from

chloroform-methanol as white crystals (270 mg), m.p. 266-68 °C. The

elemental analysis agreed with the molecular formula C2gH320|4. The

glycosidic nature of Fl-11 was evidenced by positive Molisch test^. It gave

greenish-brown colour with FeClj. The flavonoidic nucleus was evidenced

by positive Shinoda's test"* and UV spectrum which showed X^^^^ at 269

and 325 nm. Analysis with diagnostic reagents"^'^ gave bathochromic shift

of 32 nm with AlCl^ indicating the presence of free hydroxyl group at

5-position. The possibility of its being a flavanone glycoside was ruled

out as it gave yellow colour with Wilson-Boric acid reagent'^. Its IR

80

spectrum displayed a carbonyl group at 1660 cm"', phenolic hydroxyl

group at 3438 cm' and complex substitution pattern at 1578, 1496, 1300,

1245, 1184, 1070, 1033, 835 and 771 cm''.

Total hydrolysis of Fl-11 with 6% HCl yielded equimolar mixture of

rhamnose, glucose and an aglycone Fl-11 Ag, m.p. 230 °C. The aglycone

was characterized as acacetin by spectral data, m.m.p and co-chromatography

with an authentic sample.

The UV spectra of the aglycone and the glycoside were almost

similar except that the aglycone gave a bathochromic shift of 20 nm in

band-11 with NaOAc (absent in glycoside), thus suggesting that the

position-7 is involved in glycosylation. Partial hydrolysis of the glycoside

(XIa) with 1% H2SO4 yielded L-rhamnose (identified by PC, co-

chromatography and GLC) and a partial glycoside (XII) indicating

rhamnose to be the terminal sugar. Partial glycoside on complete

hydrolysis with almond emulsion yielded D-glucose. Quantitative

estimation of the sugars by Somogyi's copper micro method-'' and

periodate oxidation-^^ of the glycoside further confirmed that the sugar

moiety is a disaccharide and both the sugars to be in pyranose form.

'H-NMR spectrum of Fl-11 in DMSO-d^ (Table-20) showed the

presence of seven aromatic protons. A sharp singlet at 56.45 indicated the

presence of C-3 proton, of y-pyrone ring. Two meta-coupled singlets at

66.79 and 56.95 integrating for one proton each were assigned to H-6 and

H-8 respectively. Two ortho-coupled doublets at 57.14 (J=9.0 Hz) and

68.04 (J=9.0 Hz) integrating for two protons each corresponded to H-3',5'

and H-2',6' protons respectively. A sharp three protons singlet at 53.86

was assigned to methoxyl group. A doublt at 51.07 (J=6 Hz) was ascribed

81

Table-20

'H-NMR spectral data of Fl-11, values on 8-scale

Assignments No. of protons Signals

CH, (rhm)

OCH3

H-l"'(rhm)

H-l"(glu)

Sugar proton

H-l",2",3",4",5",6",

r",2"',3"',4"',5"'

H-3

H-6

H-8

H-3',5'

H-2',6'

HO-5

3

3

1

1

12

1

1

1

2

2

1

1.07(d, J=6.0Hz)

3.86(s)

4.55 (d, J=2.5 Hz)

5.05 (d, J=6.5 Hz)

4.45-5.43 (m)

6.45 (s)

6.79 (s)

6.95 (s)

7.14(d, J=9.0Hz)

8.04 (d, J=9.0 Hz)

12.91 (s)

s= singlet, d= doublet, m= multiplet, spectrum run in DMSO-d^ at 300

MHz, using TMS as internal standard.

82

to rhamnosyl methyl. The anomeric proton of glucose H-1" appeared as a

doublet at 65.05 (J=11.0 Hz) while the anomeric proton of rhamnose H-

1'" as a doublet at 54.55 (J=2.5 Hz). The other sugar protons appeared in

the range of 54.45-5.43.

Acetylation of Fl-11 with acetic anhydride and dry pyridine gave a

hepta-acetate Fl-ll(Ac), m.p. 216-20 °C. The 'H-NMR spectrum of the

acetate (Table-21) exhibited a multiplet at 51.60-2.02 integrating for

eighteen protons assigned to aliphatic acetoxyls while the solitary

aromatic acetoxyl appeared as a singlet of three protons at 62.45. A

singlet at 63.90 integrating for three protons were assigned to methoxyl

group. It established A2B2 pattern of B-ring protons. 2',6' and 3',5' protons

appeared as ortho-coupled doublets (J=9.0 Hz to each) at 67.81 and 7.09

respectively. The C-6 and C-8 protons of A-ring appeared as meta-coupled

doublets (J=2.5 Hz to each) centred at 56.60 and 6.72. A singlet at 56.25

of one proton was assigned to H-3 proton. The anomeric protons of

rhamnose (H-1'") and glucose (H-1") were centred at 64.98 (d, J=2.0 Hz)

and 5.25 (d, J=11.0 Hz) respectively. The coupling constant of anomeric

protons of rhamnose and glucose suggested a-L-rhamnose and P-D-

glucose. The signals over the range of 53.35-5.26 account for twelve

protons of glucose and rhamnose residue. The position of anomeric

protons of rhamnosyl, glycosyl moities and that of rhamnosyl methyl'^^

and the absence of characteristic signal for 2"-0-acetyl-^^ at 51.70

suggested (l->2) inter sugar linkage (attachment of rhamnosyl residue at

2"-position of glucose, neohesperidoside). This was further confirmed by

the identification of methylated sugars. Hydrolysis of the methylated

glycoside (XIc) with 0.2 N HCl gave 2,3,4-tri-O-methyl rhamnose and

3,4,6-tri-O-methyl glucose (identified by SiOj-TLC and paper

83

Table-21

'H-NMR spectral data of FI-11 (Ac), values on 6-scale

Assignments

CH3(rhm)

0CH,-4'

Aliphatic acetoxvls

6x OAc

Aromatic acetoxvls

5-OAc

H-l"(glu)

H-r"(rhm)

Sugar protons

H-r',2",3",4",5",6"

]'", 2"', 3"',4'",5'"

Aromatic protons

H-3

H-6

H-8

H-2',6'

H-3',5'

No. of protons

3

3

18

3

1

1

12

1

1

1

2

2

Signals

1.15 (d, J=6.0Hz)

3.90 (s)

1.60-2.02 (m)

2.45 (s)

5.25(d, J=11.0Hz)

4.98 (d, J=2.0 Hz)

3.35-5.26 (m)

6.25 (s)

6.60 (d, J=2.5 Hz)

6.72 (d, J=2.5 Hz)

7.81 (d, J=9.0Hz)

7.09 (d, J=9.0 Hz)

s=singlet, d= doublet, m=multiplet, spectrum run at 300 MHz in CDCI3,


84

chromatography according to Petek' '*) and partially methylated aglycone

(Xlllb) which was characterized as 7-hydroxy-5,4'-dimethoxy flavone.

The above assigned structure was further supported by the mass

spectrum of acetylated glycoside (Xlb). The molecular ion peak as

expected v/as not observed. The presence of an acetylated

deoxyhexopyranoside and a hexopyranoside were evidenced by the

presence of fragment ios at m/z 237 and 289. The fragment ion at m/z 284

corresponded to aglycone. The retero Diels-Alder cleavage resulted in the

formation of ions at m/z 152 due to A-ring and at m/z 132 and 135 due to

ring-B.

On the basis of above results, the compound Fl-11 was identified as

acacetin-7-O-neohesperidoside-'^ (XIa).

HjC'

OR OR

OCH,

(X I )

a)R = H b)R = Ac c)R = Me

CH,OH

OR 0

OH 0

85

OCH,

(XII)

p ^ ^ ^ O C H ,

(XID)

(a)R=H (b)R = Me

86

The melting points were taken on a Kofler block and are uncorrected.

Ultra violet spectra were measured on Beckmann Model DU and Pye

Unicam PU-8800 spectrophotometers in methanol / chloroform. IR spectra

were taken on Shimadzu lR-408 Perkin Elmer 1800 (FTIR). The mass

spectra were measured in both, EI mode on Jeol D-300 and FAB mode on

Jeol SX 102/DA-6000 mass spectrophotometers. The 'H-NMR and

'^C-NMR spectra were recorded on Varian EM-360 L (60 MHz), 290

MHz, Jeol 4H-100MHZ, Perkin Elmer R-32 (90 MHz), Brucker dpx 200

MHz, DRX 300 MHz and WM 400 MHz in CDC^VDMSO-dg, (CD3)2CO,


The silica gel used for different chromatographic purposes, was

obtained from E. Merk (India) and E. Merk (Germany), SRL (India). TLC

solvent systems used were benzene : pyridine : formic acid (BPF, 36:9:5),

toluene : ethylformate : formic acid (TEF, 5:4:1), ethylacetate : ethyl

methyl ketone : acetic acid : water (EtOAc : EtMeCO : AcOH : H2O,

20:3:1:1, 5:3:1:1) ethylacetate : methanol : water (8:1:1), petroleum ether:

benzene (6:4), benzene: chloroform (9:1), chloroform : methanol (5:1,

3:1), benzene : acetone (5:1, 4:1), chloroform : methanol : water

(35:13:2), hexane : acetone (4:1), petroleum ether : toluene : ethyl acetate

(10:5:3), ethylacetate : pyridine : water (2:1:2), petroleum ether : ethyl

formate : formic acid (93:7:0.7), n-butanol : acetic acid : water (RAW,

4:1:5), n-butanol : ethanol : water (BEW, 60:16.5:28.5).

Alcoholic ferric chloride, iodine vapours, ammonia vapours, aniline

hydrogen phthalate and p-anisidine phosphate solutions were used as

spray reagents for visualization of spots on TLC and paper

chromatograms.

87

Extraction of the leaves oi Ficus lyrata (Moraceae)

The leaves of Ficus lyrata were procured from the Fort, A.M.U.,

Aligarh, India and identified by Prof. Wazahat Hussain, Department of

Botany, A.M.U., Aligarh. The air dried powdered leaves (3.0 kg) of Ficus

lyrata after being defatted with petroleum ether were then extracted with

hot chloroform. Finally, the leaves were extracted exhaustively with

methanol. The chloroform extract, after complete removal of solvent

under reduced pressure gave a greenish gummy mass (160 gm) (Fraction

'A'). The fraction 'A' responded to usual colour tests for flavonoids and

revealed a complex mixture of several compounds on TLC plates coated

with silica gel, in the following solvent systems - (i). Petroleum ether :

Benzene (6:4), (ii). Benzene : Chlorofonn (9:1), (iii). Benzene : Acetone (5:1).

The extract was, therefore, subjected to column chromatography over

silica gel. The column was eluted with petroleum ether-benzene mixture

in different ratio (9:1-2:8), benzene and benzene-ethylacetate (9:1-1:1),

ethylacetate and methanol as eluting solvents. The fractions showing the

similar behaviour on TLC examination and same IR spectra were mixed

together. Repeated column chromatography followed by fractional

crystallization afforded nine crystalline compounds, marked as Fl-1, Fl-2,

Fl-3, Fl-4, Fl-5, Fl-6, Fl-7, Fl-8 and Fl-9.

Methanolic extract (fraction 'B') also responded positively to colour

tests of flavonoids''. TLC examination of extract in a number of solvent

systems viz benzene-pyridine-formic acid (36:9:5), toluene-ethylformate-

formic acid (5:4:1), ethylacetate-ethyl methyl ketone-acetic acid-water

(5:3:1:1, 20:3:1:1), chloroform-methanol-water (35:13:2) showed the

88

presence of several compounds. Repeated column chromatography over

silica gel followed by fractional crystallization afforded two crystalline

compounds, labelled as Fl-10 and Fl-11.

FI-1

Elution of the column with petroleum ether-benzene (1:1) gave the

fractions which on ciystallization with chloroform-acetone afforded white

crystalline solid (40mg) m.p. 126-28 °C, R,- 0.52 [Hexane-acetone(4:l)].

Analysed for C29H^gO :

Cald. C, 84.46; H, 11.65%

Observed : C, 84.43; H, 11.61%

UV-Xâx (CHCI3) : 248nm (log e 6.7).

IR I) cnr^ : max

3450, 2957, 2918, 2850, 1640, 1463, 1380, 1308, 1080, 1035,

795 cm-i.

'H-NMR-data (300MHz, CDCI3), values on 8-scale :

5.35 (lH,d, J=5.1 Hz, H-6), 5.14 (lH,d,J=8.4 Hz, H-1), 5.02 (lH,dd,

J=8.4 & 8.4Hz, H-2), 3.52 (IH, brm, w'/2, J=15.6Hz, H-3a), 1.00 (3H, brs,

Me-19), 0.97 (3H,d,J=6.6 Hz, Me-21), 0.88 (3H, d, J=6.5Hz, Me-29), 0.84

(3H, d, J=6.0 Hz, Me-26), 0.82 (3H, d, J=6.0 Hz, Me-27), 0.68(3H, brs,

Me-18).

i^C-NMR-data (400MHz, CDCI3), values on 5-scaIe :

138.41 (C-1), 129.3 (C-2), 71.84 (C-3), 42.35 (C-4), 140.8 (C-5),

121.8 (C-6), 29.76 (C-7), 31.71 (C-8), 50.21 (C-9), 36.21 (C-IO), 21.15

89

(C-U), 37.33 (C-12), 39.85 (C-13), 56.93 (C-14), 24.34 (C-15), 28.31 (C-

16), 56.13 (C-17), 11.92 (C-18), 19.45 (C-19), 34.01 (C-20), 18.85 (C-

21), 31.97 (C-22), 26.16 (C-23), 45.90 (C-24), 29.23 (C-25), 19.11 (C-

26), 19.88 (C-27), 23.18 (C-28), 12.04 (C-29).

Mass m/z (rel. Intensity) EIMS :

m/z 412 [M]+-(C29H4gO) (100), 397 (23.1), 394 (33.7), 379 (18.0),

362 (8.0), 327 (25.7), 303 (27.8), 271 (19.6), 253 (26.7), 230 (14.4), 212

(21.1), 197 (8.8), 176 (10.5), 162 (17.1), 160 (19.5), 158 (22.9), 147

(19.0), 144 (27.0), 135(22.0), 134 (16.5), 132 (23.3), 130 (13.5), 122

(13.5), 120 (18.3), 118 (21.6), 109 (26.6), 94 (27.2), 81 (33.5), 71 (23.6),

55 (70.3).

Fl-2

The compound Fl-2 was obtained from the column by benzene only.

It was ciystallized from chloroform-methanol as colourless compound

(150mg), m.p 282'^C. Its gave positive Leibermanm-Burchard test and

Molisch test.

Analysed for C^^H^^Ô^ :

Calcd. : C,72.91 ; H, 10.41%

Obsei-ved : C, 72.87; H, 10.34 %

*H-NMR data (300 MHz, DMSO-d ,), values on 8-scale :

0.70 (3H, s), 0.73 (3H, s), 0.75 (6H, s), 0.78 (3H, s), 1.01 (3H, s),

1.10-1.23 (2H, m, CH^-protons), 4.2 (IH,d,J=10.0Hz, H-1'), 4.0-5.1 (7H,

m, H-r,2',3',4',5' and 6'). 5.20 (IH, m, olefinic proton).

90

Killiani's hydrolysis of FI-2 :

The compound FL-2(30mg) was taken in a thick walled test tube and

2.0 ml of Killiani's mixture (glacial acetic acid-cone, hydrochloric acid-

water 7:2:1) was added to it. The reaction mixture were heated on a

boiling water bath for 3 hours, after tightly fixing a cork on the mouth of

the test tube and tightening it further with the help of a thread. The

reaction mixture on dilution with water gave a precipitate which was

extracted with ether three times (3x25ml) in a separating funnel. All the

ethereal extracts were combined together and washed well with water to

remove acid. The ethereal extract was then dried over anhydrous sodium

sulfate and filtered to remove inorganic salts. Recovery of ether gave a

residue, which on crystallization with methanol gave a colourless

compound (10 mg), m.p. 137 °C. By co-TLC, petroleum ether : toluene :

ethylacetate (10:5:5) with an authentic sample of P-sitosterol, it was

found to be identical with it, m.p. of FL-2 when mixed with an authentic

sample of P-sitosterol did not show any depression (m.m.p. 137 °C).

Analysed for CjgH^QO :

Calcd. : C, 84.05 ;H, 12.07%

Observed : C, 84.02 ; H, 12.04%

Identification of sugar :

The acidic filtrate left after removing the aglycone was extracted

with ethylacetate to ensure the complete removal of any residual aglycone.

The filtrate was neutrallized by concentrating it to a syrup in vacuum

dessicator over KOH pellets. The hydrolysate was chromatographed on

Whatmann No.l paper using n-BuOH-AcOH-H20 (5:4:1) and

91

ethylacetate-pyridine-water (2:1:2) as developing solvents alongwith

authentic sugars as check. The chromatograms were then sprayed

separately with aniline phthalate and p-anisidine phosphate solutions and

dried at 100-05 °C in an oven for fifteen mintutes. One coloured spot was

developed on the chromatograms which showed the presence of one

sugar. The Rj-value of sugar was identical with that of glucose.

Acetylation of FI-2 :

Crystalline Fl-2(40mg) was acetylated by heating it with acetic

anhydride (2.0 ml) and diy pyridiene (1.0 ml) and allowed to stand

overnight at room temperature and then heated on water bath for two

hours. The reaction mixture was cooled at room temperature and poured

on crushed ice. The solid separated was collected, washed well with water

and dried. Which on crystallization from chloroform-methanol gave

colourless crystals (30mg), m.p. 166' C.

iR-NMR data (300MHz, CDCI3), values on 5-scale :

0.71-1.04 (18H, s, 6 X CH3), 1.95 (3H, s, OCOCH3), 1.99 (3H,

s,0C0CH3), 2.02 (3H, s, OCOCH3), 2.04 (3H, s, OCOCH,), 4.20-5.30

(7H, m, protons-a to acctoxyl), 5.40 (IH, m, H-6).

Mass m/z (rel. intensity) :

[M -] absent, 396, 255.

FI-3

Elution of the column with benzene-ethylacetate (10:1) gave the

fractions which on crystallization with chloroform-methanol afforded light

cream needle shaped crystals (65mg) m.p. 172-74 T . It gave greenish

brown colour with alcoholic FeCl^.

92

Analysed for C,„HgOj:

Calcd. C-62.50%, H.4.16%

Observed: C-62.48%, H.4.14%

UV data with shift reagents X „„, nm : o max

MeOH 220, 261, 320, 323(sh)

NaOMe 219,262,320,322(sh)

AICI3 220, 270, 340, 362(sh)

AICI3/HCI 219,259,3 18,321(sh)

NaOAc 220, 262, 319, 323 (sh)

NaOAc/H^BO^ 219,269,327

rw KBr , IR U cnT^ :

max

3244(OH), 1648(C=0), 1600, 1512,1384,1307

'H-NMR (300 MHz, DMSO-dJ, values on 5-scale :

2.35 (3H, s, CH3), 6.12 (IH, s, H-3), 6.79 (IH, d, J=9.0Hz, H-7),

7.10 (IH, d, J=9Hz, H-8), 11.0 (IH, s, 5-OH), 9.57 (IH, brs, 6-OH).

'^C-NMR (300 MHz, DMSO-d^), values on 5-scale :

160.26 (C-2), 112.14 (C-3), 171.1 (C-4), 153.98 (C-5), 149.44 (C-6),

132.19 (C-7), 110.19 (C-8), 143.34 (C-9), 115.51 (C-10), 18.26 (CH^).

Mass m/z (rel.intesity) :

192[M-*--] (42), 177 (M'-'-CH,) (15), 174 (M '-HÔ) (20), 164 (M-^-CO)

(12), RDA-fragments, 152 (60), 126 (9.5).

93

Fl-4

The compound Fl-4 was eluted from the column with benzene-

ethylacetate (9:1). Purification by repeated crystallization from benzene-

acetone gave yellow crystals (60 mg) m.p. 160-62°C. It gave red colour

with Mg/HCl and Na-Hg/HCl and a greenish brown colour with alcoholic

FeCl3.

Analysed for CjgHjgO^:

Calcd. C 63.68, H-5.02%

Observed : C 63.66, H-5.00%

UV spectral values with shift reagents A- jj nm :

MeOH 217,278,341

NaOAc 218,278,341

NaOAc/H,BO, 218,277,341

AICI3 219, 291, 362, 374(sh)

AICI3/HCI 219,292,361

NaOMe 219,291,340

WW, K B r , IR U c m ' : max

3100-3150 (OH), 1665 (C=0), 1480 (C=C).

'H-NMR (300 MHz, DMSO-d^), values on 8-scale :

3.74 (3H, s, -OCH3), 3.86 (3H, s, OCH3), 3.89 (3H, s, OCH3), 3.94

(3H, s, OCH3), 6.99 (IH, d, J=2.0 Hz, H-6), 7.05 (IH, d, J=2.0 Hz, H-8),

7.13(1H, d, J=9 Hz, H-5'), 7.61 (IH, d, J=1.8 Hz, H-2'), 7.72 (IH, dd,

J,=1.8 Hz, J2=9.0 Hz, H-6'), 12.90 (IH, s, OH-5).

94


358 [M^'](27), 330 (M+-CO)(45), 343 (M+-CH3)(62), 328 (343-CH3)

(47), 313 (328-Me) (20), 298 (313-Me) (30), RDA fragments, 166[Ar

(61), 180[Br(48).

FI-5

Fl-5 was eluted from the column with benzene-ethylacetate (9:1) and

was crystallized with chloroform-methanol as yellow crystals (150 mg)

m.p. 224-25 °C.

Analysed for CjgH^Ô^:

Calcd. C 62.79, H 4.65%

Observed : C 62.77, H 4.63%

UV data A, , nm :

MeOH

NaOAc

NaOAc/H^BO^

AICI3

AICI3/HCI

NaOMe

__, KBr I IR D cm-i :

max

209, 296,

215, 293,

213, 294,

213, 298,

214, 233,

216, 291,

333

335,

334

355

298,

367,

396

354

400

3350 (OH), 1660 (C=0), 1600, 1480 cm-'


3.73 (3H, s, OMe), 3.81 (3H, s, OMe), 3.91 (3H, s, OMe), 6.89 (IH,

s, H-3), 6.95 (2H, d, J=9.0 Hz, H-3',5'), 7.94 (2H, d, J=9.0 Hz, H-2',6'),

10.45 (IH, s, 4'-0H), 12.78 (IH, s, 5-OH).

95

Ms data m/z (rel. intensity) :

344 (M+-) (35), 329(M+-Me) (55), 289 (329-2XCH3) (12), RDA

fragments, 226[A]+ (28), 118 [B,]+ (32), 121 [B^V (26).

Fl-6

Fl-6 obtained from the column by benzene-ethyl acetate (8:2) and

crystallized from chloroform-methanol as yellow crystals (78 mg) m.p.

290-92 °C. It gave greenish brown colour with alcoholic FeCl3 and pink

colour with Zn/HCl.

Analysed for Cj Hj O^ :

Calcd. C, 64.96; H, 4.45%

Observed : C, 64.93; H, 4.43%

UV spectral data with shift reagents A, g, nm :

MeOH 280, 320, 334

NaOAc 275,334,391

NaOAc/H^BO, 276, 335

AICI3 300, 340, 384 AICI3/HCI 300, 339

NaOMe 275, 370, 400

IR U cm-1 : nil ax

3210 (OH), 1655 (C=0), 1600, 1475, 1365 cm"'

^H-NMR (300 MHz, DMSO-d^), values on 5-scale :

3.84 (3H, s, OMe), 4.04 (3H, s, OMe), 6.97 (IH, s, H-3), 7.03 (IH,

s, H-6), 7.05 (2H, d, J=9.0 Hz, H-3',5'), 8.07 (2H, d, J=9.0 Hz, H-2',6'),

10.50 (IH, s, 4'-0H), 13.04 (IH, s, 5-OH).

96


314 (M+-) (100), 299 (M+-Me) (37), 286 [M-*--CO] (25), 284 [299-

CH3], (15) RDA fragments, 196[Ar(51), 118[B,]+(19), 121[B2r(28).

FI-7

Fl-7 was obtained from the column with benzene-ethylacetate (8:2)

mixture and crystallized from chloroform-methanol as light cream

coloured crystals (165 mg), m.p. 77-80 °C.

Analysed for Cj^HjÔj :

Calcd. C, 80.67; H, 5.88%

observed : C, 80.63; H, 5.85%

IR U cm-i : max

1661 (C=0), 1475 (C=C)

UV data with shift reagents A, j,, nm :

MeOH

NaOAc

NaOAc/H^BO^

AlCl,

A1C1,/HC1

NaOMe

245, 345

245, 345

245, 345

245, 345

245, 345

245, 345

^H-NMR data (300 MHz, CDCi3), values on 5-scale

3.87 (3H, s, OCH3), 6.92 (2H, d, J=8.7 Hz, H-3,5), 7.59 (2H, d,

J=8.7 Hz, H-2,6), 7.39 (IH, d, J=15.0 Hz, H-a), 7.76 (IH, d, J=15.0 Hz,

H-(3), 7.99 (2H, d, J=8.4 Hz, H-2',6'), 7.52-7.63 (3H, m, H-3',4',5').

97

Mass m/z (rel. int.) :

238[M+-] (100), 239 [M++1] (25.75), 223 [M+'-CH,] (10.60), 210

[M+--CO] (18.18), RDA fragments, 161 (49.80), 134 (15.15), 133 [134-H+]

(1.50), 105 (22.70), 78 (40.90), 77 [78-H+] (1.50).

Fl-8

The compound Fl-8 was eluted from the column by benzene-

ethylacetate (7:3) mixture. It was crystallized from chloroform-methanol

as white crystals (180 mg), m.p. 185-87 ''C. It gave greenish-brown colour

with ferric chloride solution.

Analysed for Cj^HjÔ. :

Calcd. : C, 68.45; H, 4.69%

Observed: C, 68.41; H, 4.65%

UV data with shift reagents A, j, nm :

MeOH 244, 369.5

NaOAc 249, 367.5

NaOAc/H^BO^ 251.5,366.5

AICI3 276.5, 300.5, 329.5, 360

AICI3/HCI 277, 300.5, 330, 372.5

NaOMe 269, 298

, „ KBr , IR U cm* :

max

3429(OH), 2920, 2850, 1669 (C=0), 1507, 1464, 1383, 1270, 1162,

833, 818.

98

^H-NMR data (300 MHz, CDCI3), values on 5-scale :

3.88 (6H, s, 2 X OCH,), 6.36 (IH, d, J=3.0 Hz, H-6), 6.48 (IH, d,

J=3.0 Hz, H-8), 6.58 (IH, s, H-3), 7.00 (2H, d, J=8.7 Hz, H-3',5'), 7.83

(2H, d, J=9.0 Hz, H-2',6'), 12.81 (IH, s, OH-5).


298[M+-] (43.0), 283 [M+'-Me] (4.0), 268 [M+--2 x Me or 283-Me]

(5.4), 270 [M+--CO] (3.7), RDA fragments, 166 [Aj^] (10.2), 151 [A+,-Me]

(10.0), 123[151-CO] (12.0), 138 [A+,-CO] (9.0), 127 [B+,-CH3] (2.7).

FI-9

It was obtained by eluting the column with benzene-ethylacetate

(7:3) mixture and crystallized with chloroform-methanol as pale yellow

pellets (180 mg), m.p. 198-200°C. It gave a dark green colour with ferric

chloride.

Analysed for Cj H^ Og :

Calcd. : C, 60.00; H, 4.44%

Observed : C, 59.96, H, 4.41%

IR D cnr^ : max

3433 (OH), 1662 (>C=0), 1622 (aromatic)

UV data X^ nm :

MeOH 262, 329(sh.)

AICI3 274, 328(sh), 372

NaOAc 272, 326

NaOMe 268, 272, 332 sh.

99

^H-NMR (300 MHz, DMSO-d^), values on 5-scale :

3.67 (3H, s, OMe), 3.73 (3H, s, OMe), 3.76 (3H, s, OMe), 6.63 (IH,

d, J=3.0 Hz, H-6), 6.68 (IH, d, J=3.0 Hz, H-8), 8.36 (IH, s, H-2), 6.48

(IH, s, H-5'), 9.33 (IH, s, OH-4'), 10.32 (IH, s, OH-7), 13.01 (IH, s, OH-5).

Mass m/z :

360[M+-] (100%), 345[M+'-Me] (45.4), 342 [M+--H20] (35.9), 332

[M+--CO] (4.0), 330 [M+--CH20 or M+--2 x Me] (22.1), 329 [M+--OMe]

(10.5), 327 [M'^'-Me-H20) (15.1), 315 [MM x Me] (58.8), 317 [MM x

Me+2H+] (40.9), 314 [M+--C0-H20) (7.1), 299 [314-CH3] (7.1), 180 [M++]

(4.4), RDA fragments, 152[A+,] (4.0), 153 [A,+H^](4.4), 151 [A'-.-H^]

(4.4), 208 [B+ (8.0), 207[B+-H+] (7.5), 178 [B+-CH20](4.1).

i^C-NMR (300 MHz DMSO-d^), values on 5-scale :

55.82, 55.10, 59.92 (3xOMe), 93.94 (C-8), 100.05 (C-6), 104.56

(C-10), 153.27 (C-2), 126.09 (C-3), 180.27 (C-4), 154.74 (C-5), 157.68

(C-7), 154.27 (C-9), 121.75 (C-T), 150.25 (C-2'), 153.27 (C-3'), 152.86

(C-4'), 119.82 (C-5'), 152.66 (C-6').

Acetylation of FI-9 :

Ciystalline Fl-9 (50 mg), acetic anhydride (1.0 ml) and dry pyridine

(0.5 ml) were heated over a boiling water bath for three hours. After usual

work-up, as described earlier, the solid obtained, on several crystallization

from aq. ethanol, gave colourless needles, m.p. 132"C.

Analysed for CjjHjjO,, :

Calcd. ; C, 59.25; H, 4.52%

Observed: C, 59.19; H, 4.49%

100

^H-NMR (300 MHz, CDCI3), values on 5-scale :

2.31 (3H, s, OAc), 2.35 (3H, s, OAc), 2.39 (3H, s, OAc), 3.85 (9H,

s, 3 X OMe), 6.72 (IH, d, J=2.5Hz, H-6'), 6.97 (IH, d, J=2.5 Hz, H-8),

7.19 (IH, s, H-3'),7.85(1H, s, H-2).

i^C-NMR (400 MHz, CDCI3), values on 6-scale :

21.10, 20.60, 20.90 (3 x OCOCH3), 55.5, 56.1, 56.8 (3 x OMe),

110.20 (C-8), 113.40 (C-6), 117.40 (C-IO), 128.20 (C-5'), 126.50 (C-3),

128.6 (C-r) , 140.80 (C-4'), 146.10(C-2'), 148.30 (C-6'), 151.50 (C-5),

151.90 (C-9), 153.30 (C-2), 161.60 (C-3'), 168.20, 168.50, 169.40

(3 X C=0 of OAc), 178.0 (C-4).


486 [M^-], 444 [M+'-CH2=C=0], 402 [M+--2 x CH2=C=0)], 360

[M+--3 X CH2=C=0], 345 [360-Me], 330 [36O-CH2O or 2xMe], 315

[360-3 X Me], RDA fragments, 153 [A,+H+], 178 [B^-CUp].

Methylation of FI-9 :

Crystalline Fl-9 (50mg), dry acetone (50 ml), dimethyl sulphate

(1.0 ml) and anhydrous potassium carbonate (0.3g) were refluxed for 24

hours.The reaction mixture was filtered and the inorganic residue washed

several times with hot acetone. On distilling off the solvent, a brown semi

solid mass was left behind. It was washed with hot petroleum ether to

remove the excess of dimethyl sulphate. The solid residue on

crystallization from ethyl acetate gave colourless shining pellets (28 mg),

m.p. 156 °C.

101

Analysed for C2iH220g :

Calcd. C, 62.68; H, 5.47%

Observed : C, 62.65; H, 5.45%

iR-NMR (300 MHz, CDCI3), values on 5-scale :

3.85-3.96 (18 H, s, 6 x OMe), 6.66 (IH, d, J-2.5 Hz, H-6), 6.75 (IH,

d, J=2.5 Hz, H-8), 7.23 (IH, s, H-3'), 7.85 (IH, s, H-2).


402 [M+-], 387 [M^-15], 372 [M+-CH2O or M+-2 x Me], 357 [M+-3 x

Me], 371 [M^--OMe], RDA fragments, 167[A,-Me-2H^], 179 [B,-Me-CO].

Oxidative degradation of the methylether with alkaline HjOj :

A solution of methyl ether of FI-9 (20 mg) in acetone (50 ml) was

treated with 5% ale. KOH (10 ml) followed by 30%, H2O2 (1.0 ml), the

mixture was kept at 45°C for two hours. It was cooled, poured into ice-

cold water (10 ml), acidified with cold cone. HCl and extracted with ether.

The ether solution was washed with water and shaken with saturated

NaHCO^ solution, the bicarbonate fraction was acidified and extracted

with ether. Evaporation of ether followed by micro vacuum sublimation

gave a colourless crystalline solid, m.p. 185-86°C. It was identified as

2,3,4,6-tetramethoxy benzoic acid, by m.p., m.m.p. and co-chromatography

with an authentic sample.

FI-10

The compound Fl-10 was eluted from the column by benzene-

ethylacetate (1:1) mixture. It was crystallized from chloroform-methanol

as cream coloured crystals (150 mg), m.p. 255 °C. It gave greenish colour

with ferric chloride solution.

102

Analysed for CjjHjjOjj, :

Calcd. : C, 59.19; H, 4.93%

Observed : C, 59.16; H, 4.89%

UV data with shift reagents X nm :

" max

MeOH 268,321

NaOAc 268,318 NaOAc/H3B03 268, 324

AICI3 277,300,331

AICI3/HCI 277,300,331

NaOMe 253, 295, 382 sh

wr. KBr ,

IR D cm* : max

1656 (C=0), 3399 (OH), 1615, 1498, 1304, 1173, 1081 and 832.


3.87(3H, s, OMe), 5.06(1H, d, J=8.0, H-l" (glu)), 5.06-5.41 (7H, m),

6.45(1H, d, J=3.0 Hz, H-6), 6.85(1H, d, J=3.0 Hz, H-8), 6.96(1H, s, H-3),

7.12 (2H, d, J=9.0 Hz, H-3',5'), 8.05 (2H, d, J=9.0 Hz, H-2',6'), 12.41

(IH, s, 5-OH).

Hydrolysis of Fl-10 :

The glycoside Fl-10 (80 mg) was dissolved in water and acidified

with 6% hydrochloric acid. The reaction mixture was refluxed over water

bath for four hours and left overnight at room temperature. The separated

aglycone was filtered, washed well with water and dried. It was

crystallized from ehloroform-methanol as light yellow crystals (46 mg),

m.p. 261-63 °C.

103

Identification of Sugar :

The acidic filtrate left after removing the aglycone, was extracted

with ethylacetate to ensure the complete removal of aglycone. After usual

work-up as described earlier one coloured spot was developed on

chromatograms which showed the presence of one sugar. The R,-- value

of sugar was identical with that of glucose.

UV data of FI-10 (Ag) with shift reagents X^^^ nm :

MeOH 269, 303sh, 327

NaOAc 276, 297sh, 358

NaOAc/H3B03 269, 309sh, 33 1

AICI3 259sh, 277, 292sh, 302, 344, 382

AICI3/HCI 260sh, 279, 294sh, 300, 338, 379

NaOMe 276, 295sh, 383

Acetylation of FI-10 (Ag) :

Crystalline FI-10 (Agl) (40 mg) was acetylated by heating it with

acetic anhydride (2.0 ml) and pyridine (1.0 ml) and allowed to stand

overnight at room temperature and then heated on water bath for two

hours. As described earlier the separated solid was crystallized from

chloroform-methanol as colourless needles (30 mg), m.p. 204 °C.

^H-NMR data (300 MHz, CDCI3), values on 8-scale :

7.83 (IH, d, J=4.0 Hz, H-8), 6.74 (IH, d, J=3.0 Hz, H-6), 6.53

(IH, s, H-3), 7.78 (2H, d, J=9.0 Hz, H-2',6'), 6.96 (2H, d, J=9.0 Hz,

H-3',5'), 3.88 (3H, s, OMe-4'), 2.38 (3H, s, OAc-5), 2.32 (3H, s, OAc-7).

104

FI-11

The compound Fl-11 was eluted from the column by ethylacetate. It

was crystallized as white crystals from chloroform-methanol (270 mg),

m.p. 266-68 °C. It gave greenish-brown colour with FeCl3 solution.

Analysed for CjgHjjO,^ :

Calcd. : C, 56.75; H, 5.40%

Observed: C, 56.71; H, 5.35%

UV data with shift reagents X,„„.nm :

=> m a x

MeOH 269, 325

NaOAc 269, 326

NaOAc/H3B03 270, 328

AICI3 278,301,344,384

AICI3/HCI 279, 302, 340, 380

NaOMe 245sh, 289, 360 TT. KBr , IR U cm" :

max

3438 (OH), 1660 (C=0), 1605, 1578, 1496, 1300, 1245, 1184, 1070,

1033, 835, 771.

^H-NMR data (300 MHz, DMSO-d^), values on 8-scale :

1.07 (3H, d, J=6.0 Hz, CH3-rhm), 3.86 (3H, s, OCH3), 4.45-5.43

(I2H, m, sugar protons-H-l", H-2", H-3", H-4", H-5", H-6", H-1'", H-2'",

H-3'", H-4'", H-5'"), 4.55(1H, d, J=2.5 Hz, H-r"(rhm)), 6.45 (IH, s, H-3),

6.79 (IH, s, H-6), 6.95 (IH, s, H-8), 7.14 (2H, d, j=9.0 Hz, H-3',5'), 8.04

(2H, d, J=9.0 Hz, H-2',6'), 12.91 (IH, s, 5-OH).

105

Hydrolysis of Fl-11 :

The glycoside Fl-11 (40 mg) was dissolved in water and acidified

with 6% HCl. The mixture was refluxed over water bath for four hours

and then left overnight at room temperature. After usual work-up, the

aglycone separated, was filtered, washed well with water and dried. The

aglycone was crystalUzed from chloroform-methanol as light yellow

crystals (15 mg), m.p. 230 °C.

Analysed for CjgHjjO^ :

Calcd. : C, 67.60; H, 4.22%

Observed: C, 67.54; H, 4.19%

UV data with shift reagents )\. .„nm : " max

MeOH 268, 304sh, 328

NaOAc 277, 298sh, 360

NaOAc/H^BO, 270, 310sh, 333

AICI3 258sh, 279, 292sh, 301, 344, 385

AICI3/HCI 216sh, 280, 295sh, 303, 339, 380

NaOMe 277, 293sh, 366


The acidic filtrate left after removing the aglycone, was extracted

with ethylacetate to ensure the complete removal of residual aglycone.

The filtrate was concentrated to a syrup in vacuum dessicator over KOH

pellets. The syrup was co-chromatographed on Whatmann No. 1 paper

using BuOH-AcOH-HjO (5:4:1) as developing solvent alongwith

106

authentic sugars. After usual work-up as described earlier, the sugars were

identified as glucose and rhamnose.

Partial hydrolysis :

Fl-11 (40 mg) was hydrolysed by heating it with 1% HjSo^. Aliquots

were taken at different intervals and examined by paper chromatography

(EPW, 2:1:2). After one hour, it gave a partial glucoside labelled as Fl-11

(Pg) and sugar. The sugar was identified by usual methed as rhamnose.

Acetylation of Fl-11 :

Crystalline Fl-11 (40 mg) was acetylated by heating with acetic

anhydride (2.0 ml) and pyridine (1.0 ml) and allowed to stand overnight

at room temperature. After usual work-up the acetylated FL-ll(Ac) was

obtained and crystallized from methanol as light cream crystals (25 mg),

m.p. 216-20 "C.

Periodate Oxidation :

The glycoside methyl ether (15 mg) was dissolved in methanol (10

ml) and aq. NalO^ (0.47 N, 15 ml) was added to it. The mixture was

allowed to stand at 20 °C in dark for 24 hours followed by the addition of

NaHS03 (0.05N, 25 ml). The resultant mixture was titerated against I2

using starch as indicator. One mole of methyl ether consumed 3.14 mole

of periodate with liberation of 1.12 mole of formic acid.

'H-NMR data (300 MHz, CDCI3), values on 5-scale :

1.15 (3H, d, J-6.0 Hz, CH^-rhm), 3.90 (3H, s, 4'-OCH3), 1.60-2.02

(18H, m, 6-aliphatic acetoxyls), 2.45 [3H, s, OAc-5(aromatic acetoxyls)].

107

4.98 (IH, d, J=2.0 Hz, rhm-l"'), 5.25 (IH, d, J=I1.0 Hz, glu H-l"), 3.35-

5.26 (12H, m, sugar protons H-l", 2", 3", 4", 5", 6", H-l"', 2"', 3"', 4"',

5"'), 6.25 (IH, s, H-3), 6.60 (IH, d, J=2.5 Hz, H-6), 6.72 (IH, d, J=2.5

Hz, H-8), 7.09 (2H, d, J=9.0 Hz, H-3',5'), 7.81 (2H, d, J=9.0 Hz, H-2',6').


[M *] absent, 289 [glu (Ac)^ (33.2), 273 [rhm (Ac),] (32.5), 284

[aglycone] (95.7), RDA fragments, 152 [(A^,) (18.0), 132[B+|] (36.1), 135

[B\] (5.3).

108

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Nagai, Yakiigakee Zasshi, 98, 41-46 (1978).

9. Alan R. Ketritzky and Charles W. Rees, "Comprehensive

Heterocyclic Chemistry". (Ed. A. John Boulton and Alksander.

Mc Killop (Pergamon Press, Oxford, New York) (1984).

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109

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(Spectra No. 122-125) (1982).

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and Industry", 498-99 (1966).

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T.A. Geissman), Pergamon Press, London, (a) p. 72, (b) p. 75 (1962).

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no

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Ben, 102, 2083 (1969)

CHAPTER - III

Cassia nodosa

^ J

Flavonoidic Constituents of Cassia nodosa (Leguminoseae)

The family leguminoseae consists of about 482 genera and 1200

species of evergreen trees, herbs and shrubs. The genus Cassia comprises

about 580 species distributed mostly in the tropical and subtropical region

of the World'. About 20 species are found in India. Cassia nodosa is

extensively used in folk medicines for the treatment of cheloid tumor, ring

worm, insect bite and rheumatism^'\ Medicinal importance and scarcity of

work on this plant accelerates our interest to carryout the comprehensive

investigation of the plant Cassia nodosa.

Earlier, we have reported the isolation and characterization of

kaempferol 3-0-rhamnoside'', 2,3-dihydrokaempferol 3-0-rhamnoside'',

quercetin 3-0-rhamnoside'', nodosin^, 8-C-glucosyl genistein^ and dalpanitin

from the flowers of Cassia nodosa. Further investigation was carried out on

the leaves of the same plant and the following compounds have been

isolated.

1. Unsubstituted flavone

2. 5,4'-Dihydroxy-7-methyl-3-ben2yl chromone

3. Kaempferol 3-0-rhamnoside

4. Quercetin 3-arabinoside

5. Tamarixetin 3-0-arabinoside.

The air dried and crushed leaves (2.5 kg) of Cassia nodosa (procured

from the Fort of A.M.U., Aligarh, India) after being defatted with hot

petroleum ether were exhaustively extracted with boiling methanol. The

process was repeated twice. The two methanol extracts were combined

together and concentrated under reduced pressure. A reddish brown gummy

112

mass was obtained. Petroleum ether concentrate was found to contain

mainly oil of fatty nature and hence, was not further examined.

The methanolic concentrate reddish brown gummy mass (150 gm)

responded positively to flavonoidic tests^. TLC examination of the

concentrate in different solvent systems [benzene : pyridine : formic acid

(36:9:5), and toluene : ethyl formate : formic acid (5:4:1)] showed it to be

a mixture of several compounds. The intricate mixture was separated by

repeated column chromatography over silica gel column using successively

benzene, benzene-ethylacetate mixtures, ethylacetate, ethylacetate-methanol

mixture as eluting solvents. Appropriate fractions (IR spectra and TLC) were

combined. Repeated column chromatography of the fractions followed by

fractional crystallization afforded five pure crystalline compounds, marked

as, Cn-1, Cn-2, Cn-3, Cn-4 and Cn-5.

Cn-1

Cn-1 was obtained by elution of the column with benzene-ethyl acetate

(9:1) mixture. On crystallization from benzene-acetone it gave cream

coloured solid (50 mg), m.p. 96-97 "C. Elemental analysis agreed with the

molecular formula as C15H10O2. A pink colour with Zn/HCl and yellowish

red colour with Mg/HCl, suggested a flavone nucleus^. Absence of any

colour with alcoholic ferric chloride indicated the absence of phenolic

hydroxyl group. Its IR spectrum showed the characteristic band at 1646 cm"'

(a,P-unsaturated >C=0) besides the strong bands for aromatic system at

1605, 1568, 1465 cm"'. The UV spectrum displayed the absorption maxima

at X ax 235 and 3 10 nm, which remain unaffected when analysing with shift

reagents^. The molecular formula alongwith its UV spectrum showed it to be

an unsubstituted parent flavone.

113

The 'H-NMR spectrum (Table-1) exhibited a sharp singlet of one

proton at 57.01, assinged to H-3 proton while the A-ring and B-ring protons

appeared as multiplet in the range of 67.49-8.13. Its mass spectrum showed

the molecular ion peak at m/z 222 and RDA fragments representing ring-A

and ring-B were observed at m/z 120 [Ai]" and at m/z 102 [BJ^ respectively.

In the light of above discussion the compound Cn-1 was characterized

as unsubstituted flavone^ (I).

Cn-2

Benzene-ethylacetate (8:2) eluate of the column gave the fraction

which on crystallization with chloroform-methanol gave pale yellow

granular crystals (35 mg), m.p. 290-92 °C. It was analysed as C17H14O4

which was in agreement with the molecular ion peak at m/z 282. It gave a

greenish brown colour with FeCl3 supporting the presence of phenolic

hydroxyl group/s. Functional group analysis displayed a broad band at

3100-3300 cm"' which revealed the presence of chelated hydroxyl and a

sharp band at 1655 cm'' indicating the a,p-unsaturated carbonyl group. A

negative Shinoda's test' ruled out the possibility of flavone nucleus. The

ultra violet spectrum (Table-2) exhibited absorption maxima at 235 and an

inflection at 340 nm corresponding to homoisoflavone structure'^. Analysis

with shift reagent gave a bathochromic shift of 30 nm in band II pointing out

the presence of chelated hydroxyl group which was further confirmed by the

signal at 512.46 in 'H-NMR spectrum.

114

Table-1

^H-NMR spectral data of Cn-1, values on 5-scale


H-3 1 7.0I(s)

H-5,6,7,8,2',3',4',5',6' 9 7.49-8.13 (m)

s = singlet, m = multiplet, spectrum mn at 300 MHz in CDCI3, using TMS as

internal standard.

115

The 'H-NMR spectrum of Cn-2 (Table-3, Fig. I) revealed the presence

of an aromatic methyl by the three proton singlet at 62.27 and a benzyl

methylene by two proton signlet at 53.16. A pair of meta coupled doublets

resonated at 56.20 (J=2.0Hz) and 66.45 (J=2.0Hz) were ascribed to H-6 and

H-8 protons respectively. Another pair of ortho-coupled doublets at 56.94

(J=9.0Hz) and 67.76 (J=9.0Hz) were attributed to H-3',5' and H-2',6'

protons. The olefinic proton appearing at 58.02 was assinged to H-2 proton

and the hydroxyl protons at 512.46 and 610.13 were attributed to 5-OH and

4'-0H groups.

The above assignments were further confirmed by '^C-NMR spectrum

(Table-4, Fig. II), in which the methyl carbon appeared at 618.16, the

benzylmethylene carbon at 629.0 and carbonyl carbon at 5121.06

respectively while the assignment of other carbons are given in Table-4.

The mass spectrum (Scheme I, Fig. Ill) showed the molecular ion peak

at m/z 282 and the fragment ion at m/z 267 corresponding to loss of methyl

group. The retero-Diel's Alder cleavage was facile and led to the fragments

of mass at m/z 150, 151 due to ring-A and m/z 132, 107 due to ring-B

further confirming the substitution pattern of ring-A and ring-B.

On the basis of above results the compound Cn-2 was characterized as

a novel homoisoflavone named as 5,4'-dihydroxy-7-methyl-3-benzyl

chromone(Il).

^ \ - OH

116

Table - 2

UV spectral data of Cn-2 with shift reagents

Reagents A.max"'"

MeOH 235, 285, 340 (inf.)

AICI3 265, 306, 355 (inf.)

AICI3/HCI 264, 306, 355

NaOMe 236, 286, 340

NaOAc 235, 284, 338

inf. = inflection

Table - 3

'H-NMR spectral data of Cn-2, values on 6-scale

Assignments

-CH3

Ar-CH2 (benzyl methylene)

H-6

H-8

H-3',5'

H-2',6'

H-2

OH-4'

OH-5

No. of Protons

3

2

1

1

2

2

1

1

1

Signals

2.27 (s)

3.16 (s)

6.20 (d, J=2.0Hz)

6.45 (d, J=2.0Hz)

6.94 (d, J=9.0Hz)

7.76 (d,J-9.0Hz)

8.02 (s)

10.13 (s)

12.46 (s)

s = singlet, d = doublet, spectrum run at 300 MHz in DMS0-d6, using TMS as internal standard.

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Table - 4

'^C-NMR spectral data of Cn-2, values on 8-scale

Carbon Numbers Assignments

CH-, 18.16

Ar-CH2(C-9) 29.0

C-6 98.01

C-8 93.02

C-3',5' 115.21

C-2',6' 128.96

C-2 151.20

C-3 121.06

C-5 156.10

C-7 159.30

C-10 162.01

C-11 117.0

C-4 180.0

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119

Cn-3

The compound Cn-3 was eluted from the column with ethyl acetate and

recovery of the solvent left a solid mass, which on repeated crystallization

from chloroform-methanol yielded light yellow granular crystals (110 mg),

m.p. 177-78°. Positive Molisch test, and the formation of osazone after

hydrolysis confirmed its glycosidic nature. The glycoside gave red colour

with Mg/HCl^ and pink colour on reduction with sodium amalgam followed

by acidification indicating it to be a flavanone or 3-substituted flavonol".

The possibility of its being a flavanone glycoside was ruled out as it gave a

yellow colour with Wilson boric acid reagent'^. The spot of the compound

appeared deep purple under UV light which turned yellow on exposing to

ammonia vapours, further supporting the 3-substituted nature of the flavonol.

The analysis of functional groups revealed the presence of a,P-

unsaturated carbonyl (1650 cm"'), phenolic OH (3420 cm"') and a complex

aromatic substitution pattern, 1620, 1570, 1370, 1140, 800 cm"', besides a

strong band at 2950 cm"'. The UV spectrum showed >.,„ax at 244 (sh), 265

and 350 nm. The bathochromic shifts of 15 nm with NaOAc and 50 nm with

AICI3 in band-Il and 42 nm with NaOMe in band-1 (without any decrease in

intensity) indicated the presence of 5,7 and 4'-hydroxyls.

Hydrolysis of the glycoside with 6% HCl gave an aglycone and a sugar.

The aglycone was characterized as kaempferol by m.p. 280-81°C and mixed

melting point with an authentic sample. Further confirmation of the identity

of aglycone was furnished by co-TLC and spectral data' \ The sugar was

identified as rhamnose by co-chromatography with an authentic sample.

Acetylation of Cn-3 with acetic anhydride and pyridine afforded

hexaacetate. The 'H-NMR spectrum of the acetate (Table-5) exhibited two

120

meta-coupled-doublets at 56.40 (J=2.5Hz) and 56.21 (J=2.5Hz), each

integrating for one proton assigned to H-8 and H-6 respectively. It showed

A2B2 pattern of B-ring protons, ortho-coupled doublets at 57.80 (J=9.0Hz)

and 56.89 (J=9.0Hz) were assignable to H-2',6' and H-3'5' protons

respectively. Total number of 18 protons were observed over the range of

51.9 -2.44 attributed to six acetoxyls, comprising three aromatic acetoxyls

and three aliphatic acetoxyls. The sugar protons observed in the range of

53.25-5.10 accounted for four protons. The anomeric proton of rhamnose

appeared at 55.20 (d, J=2.0Hz) and rhamnosyl methyl appeared at 50.85

(d,J=6.0Hz).

The partial methyl ether obtained on hydrolysis of the methylated

glycoside was characterized by melting and mixed melting points as 5,7,4'-

trimethoxy kaempferol. The formation of the above trimethyl ether of

kaempferol proved the attachment of the sugar residue at C-3 position of the

aglycone. The quantitative estimation of sugar by Somogyi's copper micro

method'"* showed the presence of one mole of rhamnose per mole of

aglycone.

On the basis of above results, Cn-3 was characterized as kaempferol

S-O-rhamnoside'-"^ (III).

121

Table - 5

'H-NMR spectral data of Cn-3Ac, values on 6-scale


H-2',6'

H-3',5'

H-8

H-6

H-r'(rhanmosyl)

Sugar protons

Aliphatic acetoxyf

3xOAc 9 1.9-2.12 (m)

Aromatic acetoxyl

3x -0Ac 9 2.32,2.34,2.44

(each singlet)

Rhamnosyl methyl 3 0.85 (d, J=6.0Hz)

s = singlet, d = doublet, m = multiplet, spectrum run in CDCI3 at 200 MHz,

TMS being an internal standard.

2

2

1

1

1

4

7.80 (d, J=9.0Hz)

6.89 (d, J=9.0Hz)

6.40 (d, J=2.5Hz)

6.21 (d, J=2.5Hz)

5.20 (d, J=2.0Hz)

3.25-5.10 (m)

122

Cn-4

The compound Cii-4 was obtained from the column with ethylacetate-

methanol (9:1) eluate. It was crystallized by methanol as yellow needles

(120 mg), m.p. 256 °C. The glycosidic nature of the compound Cn-4 was

evidenced by positive Molisch test. Acetylation of Cn-4 with acetic

anhydride and pyridine afforded hepta-acetate (Cn-4Ac). The 'H-NMR

spectrum of Cn-4Ac (Table-6) showed four aromatic acetoxyls at 62.52-

2.90, three aliphatic acetoxyls at 51.9-2.21 and an anomeric proton at 55.24

(d, J=7.5Hz) indicating it to be a arabinoside. The glycoside gave positive

test with Mg/HCl and with sodium amalgam followed by acidification,

yellow colour with Wilson boric acid reagent'^ indicated it to be a

3-substituted flavonol". Cn-4 on hydrolysis gave an aglycone Cn-4 Ag,

m.p. 312-14 °C which was characterized as quercetin by co-TLC with an

authentic sample of quercetin and mixed melting point. The sugar was found

to be arabinose by Rf-value, co-paper chromatography [(B:A:W (4:1:5)] with

authentic sugar samples.

The position of the sugar residue in the glycoside was determined by

the hydrolysis of the methylated glycoside. The partial methyl ether

obtained, was characterized by melting point and mixed melting point with

an authentic sample (m.p. 193°) as 5,7,3',4'-tetramethoxy quercetin (IVa).

The formation of tetramethylether of quercetin proved the attachment of the

sugar residue at C-3 position of the aglycone.

The quantitative estimation of sugar by Somogyi's'"* copper micro

method showed the presence of one mole of arabinose per mole of aglycone.

123

Table - 6

^H-NMR spectral data of Cn-4Ac, values on 5-scale

Assignments

H-6'

H-2'

H-5'

H-8

H-6

H-1 (anomeric proton)

No. of Protons

Signals

7.54 (q,J=2.1 Hz and 9.0Hz)

7.47 (d, J=2.5Hz)

6.86 (d, J=9.0Hz)

6.40(d, J=2.1Hz)

6.20(d, J=2.1Hz)

5.24 (d, J=7.5Hz)

Sugar.protons (H-r',2",3",4",5") 3.26-5.5 l(m)

4x Aromatic acetoxyls (3',4",5,7) 12 2.52-2.90 (m)

3x Aliphatic acetoxyls 1.90-2.21 (m)

d = doublet, m = multiplet, spectrum run in CDCI3 at 300 MHz and TMS

being as internal standard

124

On the basis of foregoing discussion, Cn-4 was identified as quercetin

3-0-arabinoside"^(IV).

H3CO.

OCH3

OCH, 0

(IVa)

(IV)

Cn-5

Cn-5 was obtained from ethylacetate-metahnol (8:2) eluate and

crystallized from methanol-chloroform as yellowish solid, (160mg),

m.p. 204-05°C. Elemental analysis agreed to the molecular formula as

C2iH2nO||. It responded positively to Molisch test, Shinoda's test' and a red

colour on reduction with sodium-amalgam followed by acidification*^

indicating its flavanone or 3-substituted flavone nature. The possibility of its

being a flavanone glycoside was ruled out as it gave a yellow colour with

Wilson boric acid reagent'^. Further, the chromatographic spot appeared

deep purple under UV-light which turned yellow with ammonia indicating it

to be a 3-substituted glycoside. It gave a greenish brown colour with

alcoholic FeCl^. The IR spectrum revealed the presence of phenolic

hydroxyl (3400-3500 cm"') besides a carbonyl group at 1650 cm' . In its

UV-spectrum (Table-7), Cn-5 gave a bathochromic shift with NaOAc and

125

AICI3, in band-II pointing the presence of free hydroxyl groups at 5 and

7-positions, while the absence of any shift in band-I with NaOMe indicated

the absence of a free hydroxyl group at 4'-position.

On complete hydrolysis with 5% aquous HCl, it gave an aglycone and a

sugar. The sugar was identified as arabinose by its m.p., co-paper

chromatography (BAW, 4:1:5; BEW, 60:16.5:25.8) and GLC with the

standard sample, whereas, the aglycone was identified as tamarixetin by

comparing its spectral data with those of an authentic sample of

tamarixetin'^. The UV spectrum of the aglycone was almost similar with that

of glycoside except that it showed a bathochromic shift with AICl^ in band-1

(absent in glycoside). This indicated the absence of a free hydroxyl group at

C-3 position of the aglycone.

The 'H-NMR spectrum (Table-8, Fig. IV) of the glycoside (Cn-5)

showed a singlet integrating for three protons at 53.94 assigned to aromatic

methoxyl. A pair of meta-coupled doublets of one proton each at 56.40

(J=2.24 Hz) and 6.21 (J=2.20 Hz) were attributed to H-6 and H-8

respectively. An ortho-coupled doublet at 57.05 (J=8.79Hz) and a meta-

coupled doublet at 57.51(J=1.83Hz) integrating for one proton each were

assigned to H-5' and H-2' protons respectively. The remaining double

doublet of one proton at 57.90 (J|=8.79 and J2=1.8Hz) was ascribed to

H-6' proton. The absence of any signal for C-3 proton of the aglycone

indicated that C-3 position is substituted. The anomeric proton H-l"

appeared as a doublet at 55.23 (J=8.0Hz). The coupling constant indicated

p-configuration. The other sugar protons appeared in the range of 53.10-5.51.

The above assignments were further supported by the mass spectrum

(Fig. V, Scheme II). The molecular ion peak as expected was not observed.

126

Table - 7

UV spectral data of Cn-5 with shift reagents

Reagents ^maxom

MeOH

NaOMe

AICI3

AlCiyHCl

NaOAc

NaOAc/H3B03

230, 256.5, 265, 353

235.5, 273.5, 397.5

226, 269.5, 295.5 (sh), 404.4

225, 268, 294(sh), 402

233,275.5, 377(sh), 388.5

228, 273, 378(sh), 387.5

sh = shoulder

Table - 8

'H-NMR spectral data of Cn-5, values on 5-scale

Assignments

H-6'

H-2'

H-5'

H-6

H-8

H-1" (Anomeric proton of arabinose)

Sugar protons H-l",2",3",4",5"

Methoxyl-4'

No. of Protons

1

1

1

1

1

1

6

3

Signals

7.90(dd,J,=8.79andJ2=

7.51 (d, J=1.83Hz)

7.05 (d, J=8.79Hz)

6.40 (d, J=2.24Hz)

6.21 (d, J=2.20Hz)

5.23 (d, J=8.0Hz)

3.10-5.51 (m)

3.94 (s)

= 1.8Hz)

s = singlet, d = doublet, m = multiplet, dd = double doublet, spectrum run in DMS0-d6 at 400 MHz and 100 MHz, TMS as internal standard.

127

HO.

Arab. nVz 133

HO^ .<r \ ^ O

C II

OH 0

HVZ 152 (20.6)

^

a J y OMe

0

OH 0 H

0- H

\M]\ absent OH

OH

OH

C^C ^ ^^ OMe HC=C f V—OMe

OH

nVz 151 (16.6)

OH

iiVz 148 (23.2)

Scheme - II

4£ 10(1 I !

58

77 SI

,i liMJIg :|,

121130

107 US

ido

/ 24- 285

219 255 270

150 20C I m j |BU, II t iiji iliit^—ml

2M

298

4-. 300

-JL 3 0

FIG. V

128

The fragment ion at m/z 316 corresponded to aglycone which was further

supported by the fragment ions at m/z 152 [A J ^ 151 [Bj]^ and 148 [Bj]^

resulted by the retero-Diels Alder cleavage of the aglycone, thereby

indicating the presence of two hydroxy! groups in ring-A and one hydroxyl

and one methoxyl in ring-B.

On the basis of above chemical and spectral evidences, the compound

Cn-5 was characterized as tamarixetin 3-0-arabinoside (V) which is being

reported for the first time.

(V)

129

Extraction of leaves of Cassia nodosa (Leguminoseae)

Fresh leaves of Cassia nodosa collected from the Fort of A.M.U.,

Ahgarh, India and identified by Prof. Wazahat Hussain, Dept. of Botany,

A.M.U., Aligarh, India, were dried (2.5 kg) under shade, powdered and

defatted with petroleum ether to remove chlorophyll and fatty matter. The

defatted leves were refluxed exhuastively with methanol. The methanolic

extract was concentrated under reduced pressure over a water bath. A reddish

brown gummy mass (150 gm) was obtained, which gave usual colour tests

for flavonoids. TLC examination of the concentrate in the following solvent

systems showed the complexity of the flavonoidic mixture :

1. Toluene : Ethylformate : Formic acid (TEF, 5:4:1).

2. Benzene : Pyridine : Formic acid (BPF, 36:9:5).

3. Ethyl acetate : Ethylmethyl ketone : Acetic acid : Water (EtOAc :

EtMeCO : AcOH : H2O, 5:3:1:1, 20:3:1:1).

The concentrate was, therefore, subjected to column chromatography

over silical gel. The column was eluted with benzene-ethylacetate mixture

in different ratio (9:1-1:1), and ethyl acetate only and ethyl acetate

methanol mixture (9:1-3:7). Fractions 5-10 ml were collected in conical

flasks and monitored by TLC. Repeated column chromatography over silica

gel column followed by fractional crystallization afforded five pure

compounds, marked as Cn-1, Cn-2, Cn-3, Cn-4 and Cn-5.

Cn-1

The fraction obtained by the elution of the column with benzene-

ethylacetate (9:1) mixture was ciystallized with benzene-acetone as cream

coloured solid (50 mg) m.p. 96-97 °C.

130

Analysed for C15HJ0O2 :

Calcd. : C, 81.08; H, 4.50%

Found : C, 81.05; H, 4.47%

IR U c m ' : max

1646 ( > C = 0 ) , 1605, 1568, 1465, 1375, 1225, 1128.

UV X„^^ nm :

MeOH 235,310

NaOAc 235,310

NaOAc/H-iBO.-, 235,310

A1C1-, 235,310

AlCiyHCl 235,310

NaOMe 235,310

'H-NMR (300MHz, CDCI3), values on 5-scale :

7.01 (IH, s, H-3), 7.49-8.13 (9H, m, H-5,6,7,8,2',3',4',5',6').

Ms m/z (rel. intensity) :

222 [M+-] (100), 221 (18.0), 120 [A,]+(70.0), 102 (23.0), 97 (15.0),

92 (30.0), 77 (6.0).

Cn-2

On elution of the column with benzene-ethyl acetate (8:2) a fraction

was obtained, which on crystallization from chloroform-methanol gave pale

yellow granular crystals (35 mg), m.p. 290-92 °C.

131

Analysed for C17H14O4:

Calcd. : C, 72.34; H, 4.96%

Found C, 72.31; H, 4.92%

UV data with shift reagents A-ma nm :

MeOH

AICI3

AICI3/HCI

NaOMe

NaOAc

IR U cm- :

235, 285, 340 (inf)

265, 306, 355 (inf)

264, 306, 355

236, 286, 340

235, 284, 338

max

3100-3300 (OH), 1655 (C=0)

iH-NMR (300 MHz, DMSO-dg), values on 5-scaIe :

2.27 (3H, s, CH3), 3.16 [2H, s, Ar-CH2 (benzyl methylene)], 6.20 (IH,

d, J=2.0Hz, H-6), 6.45 (IH, d, J=2.0Hz, H-8), 6.94 (2H, d, J=9.0Hz,

H-3',5'), 7.76 (2H, d, J=9.0Hz, H-2',6'), 8.02 (IH, s, H-2), 10.13 (IH, s,

OH-4'), 12.46 (IH, s, OH-5).

i^C-NMR (400 MHz, DMSO-df,), values on 5-scale :

18.16 (CH3), 29.0 [A1-CH2 (C-9)], 98.02 (C-6), 93.02 (C-8), 115.21

(C-3',5'), 128.96 (C-2',6'), 151.20 (C-2), 121.06 (C-3), 156.10 (C-5),

159.30 (C-7), 162.01 (C-10), 117.0 (C-11), 180.0 (C-4).


282 [M+-] (100), 267 [M+-- Me] (11.2), RDA fragments, 150 [Ai+]

(19.1), 151 [A,+H'-](9.1), 132 [B|+] (14.6), 107 [OH-Ar-CHj] (10).

132

Cn-3

Cn-3 was eluted from the column with ethylacetate and crystallized

from chloroform-methanol as yellow granular crystals (110 mg), m.p.

177-78 °C. It gave positive Molisch test and brown colour with alcoholic

ferric chloride.

Analysed for C21H20O10 :

Calcd. : C, 58.33, H, 4.62%

Found C, 58.30; H, 4.58%

UV data with shift reagents A,,„axn"i •

MeOH 244sh, 265, 350

NaOMe 279, 300, 392

AICI3 265, 386sh, 392

AICI3/HCI 266, 386, 400

NaOAc 259, 369

NaOAc/H-,BO., 255, 311 sh, 368

wm. KBr IRU cm max

1

3420 (OH), 2950, 1650 (C=0), 1620, 1570, 1370, 1140, 800 comlex

aromatic substitution pattern.

Acetyiation of Cn-3 :

Acetic anhydride (3 ml) was added to pyridine (1.5 ml) solution of

Cn-3 (20 mg). The mixture was left over for 24 hours at room temperature

and then heated on a steam bath for two hours. After usual work up, the solid

133

was crystallized from methanol-chloroform mixture as colourless needles

(15 mg), m.p. 159-60°C.

^H-NMR (CDCI3, 200MHz) values on 5-scale :

7.80 (2H, d, J-9.0 Hz, H-2', 6'), 6.89 (2H, d, J=9.0Hz, H-3', 5'), 6.40

(IH, d, J=2.5Hz, H-8), 6.21 (IH, d, J=2.5Hz, H-6), 5.20 (IH, d, J=2.0Hz,

H-1" rhamnosyl), 3.25-5.10 (4H, m, sugar protons), 2.32, 2.34, 2.44 (9H,

each singlet, 3 x aromatic acetoxyls), 1.9-2.12 (9H, m, 3 x aliphatic

acetoxyls), 0.85 (3H, d, J=6.0Hz, rhamnosyl methyl).

Acid hydrolysis of Cn-3 :

The glycoside Cn-3 (50 mg) was hydrolysed by heating it with 8% HCl

on a water bath for 2 hours. The mixture was left over night. The aglycone

thus separated out was filtered washed well with distilled water and dried.

The crude product on crystallization from methanol gave yellow needles

(24 mg), m.p. 280-8 PC.

Analysed for CisHioOî:

Calcd. C, 62.93; H, 3.49%

Found C, 62.91; H, 3.45%

UV data with shift reagents X.maxnm •

MeOH

NaOMe

AICI3

AICI3/HCI

NaOAc

NaOAc/H3B03

250sh, 266, 365

278, 320, 420 (dec)

265, 270, 345, 428

269, 270, 345, 428

274, 303, 387

269, 297, 320sh, 385

134

Acetylation of the aglycone :

The aglycone (30 mg) was heated with pyridine (1.0 ml) and AC2O

(2.0 ml) on a water bath for 2 horns. After usual work up it gave cream

coloured needles (24 mg), m.p. 180-82 °C.

Analysed for C23HJ8O10 :

Calcd. : C, 60.79; H, 3.96%

Found : C, 60.75; H, 3.93%

'H-NMR of the acetate (CDCI3, 100 MHz), values of 5-scale :

8.25 (2H, d, J=9.0Hz, H-2'6'), 7.19 (2H, d, J=9.0Hz, H-3',5'), 6.85

(IH, d, J=2.5Hz, H-8), 6.62 (IH, d, J=2.5Hz, H-6), 2.34-2.45 (12H, m,

OAc-5, 7, 3,4').

Identification of the sugar :

The acidic filtrate, left after filtering the aglycone was extracted with

ether and then with ethylacetate to ensure the complete removal of any

residual aglycone. The solution was concentrated to a syrup in vacuum over

NaOH pellets. The concentration was continued till the syrup was neutral to

litmus paper. The syrup was chromatographed on Whatman No. 1 filter paper

using n-butanol : acetic acid : water (4:1:5) and n-butanol : ethanol : water

(60 : 16.5 : 28.5) as solvent systems, employing descending technique.

Authentic sugars were used as checks. The chromatograms were run for 24

hours and after drying, were sprayed with aniline phthalate and p-anisidine

phosphate solutions. The chromatograms on drying at 100-105° showed the

presence of rhamnose only.

135

The quantitative estimation of sugar by Somogyi's copper micro

method gave the value (0.44 ml) which corresponded to 1.0 mole of sugar

per mole of aglycone.

Location of the sugar moiety :

Glycoside (40 mg) was dissolved in dry acetone and refluxed with

dimethyl sulphate (1.0 ml) and ignited potassium carbonate (1.0 gm) for 36

hours on a water bath. The mixture was cooled and filtered, the residue was

washed with hot acetone. The washing and the filterate were combined and

evaporated to dryness. The reddish brown oily residue left behind was

washed by hot petrol to remove the excess of dimethyl sulphate. Repeated

attempts to crystallize the semi-solid mass proved futile. It was therefore,

directly hydrolysed by heating with 7.0% H2SO4 for two hours on a steam

bath. The reaction mixture was left over night. A fainty yellowish solid

separated out. It was cry'stallized from methanol as very pale yellow needles,

m.p. 159 "C.

Cn-4

Fraction was obtained from the column by ethyl acetate-methanol

(9:1), which on several crystallisation from methanol gave yellow needles

(120 mg), m.p. 256 "C.


Calcd. : C, 55.29; H, 4.14%

Found : C, 55.24; H, 4.12%

136

UV data with shift reagents A- axini :

MeOH 256, 264sh, 300sh, 355

AICI3 279, 303sh, 335, 430

AICI3/HCI 274, 309sh, 360

NaOAc 278, 305sh, 370

NaOAc/H^BO-, 259, 301 sh, 367

Hydrolysis of Cn-4 :

The glycoside, Cn-4 (50 mg) was hydrolysed by heating it with 12.5 ml

of 0.6 N-hydrochloric acid on a water bath. The hydrolysis appeared to be

completed within a few minutes. The heating was continued for two hours,

the yellow aglycone thus separated out was filtered, washed well with water

and dried. The crude product crystallized from methanol as yellow neeldes

(20 mg), m.p. 312-14 °C. It showed no depression in melting point on

admixture with an authentic sample of quercetin. Its identity as quercetin

was further confirmed by co-cliromatography, colour reactions, UV, 'H-

NMR spectra.

Analysed for CisHjoOy :

Calcd. ; C, 59.60; H, 3.31%

Found C, 59.54; H, 3.29%

Acetylation of Cn-4 :

Cn-4 (40 mg) was acetylated by heating it with pyridine (1.0 ml) and

acetic ahydride (2.0 ml). After usual workup, it was crystallized from

chloroform-methanol as colourless needles (35 mg), m.p. 302-303 °C.

137

'H-NMR data (CDCI3, 300 MHz), values on 5-scale :

7.54 (IH, q, J=2.1Hz and 9.0Hz, H-6'), 7.47 (IH, d, J=2.5Hz, H-2'),

6.86 (IH, d, J-9.0HZ, H-5'), 6.40 (IH, d, J-2.1Hz, H-8), 6.20 (IH, d,

J=2.1Hz, H-6), 5.24 (IH, d, J=7.5Hz, H-l"), 3.26-5.51 (sugar protons, m,

H-2",3",4",5"), 2.52-2.90 (12H, m, OAc-3',4',5,7), 1.90-2.21 (9H, m, OAc-

aliphatic).


The neutral aquous hydrolysate was examined by paper chromatography

employing n-butanol : AcOH : H2O (4:1:5) as developing solvent and using

authentic sugars as checks. The chroniatograms were sprayed with aniline

phthalate and p-anisidine phosphate solutions. The chromatograms on diying

at 100-105°, showed the presence of arabinose.

Somogyi's copper micro method gave the value (0.44 ml) which

corresponded to one mole of sugar per mole of the aglycone.

Location of the sugar moiety :

A suspension of finely powdered glycoside (20 mg) in anhydrous

acetone (40 ml) with dimethyl sulphate (1.0 ml) and ignited potassium

carbonate (0.5 gm) was refluxed, for 48 hours with frequent shaking. After

usual workup, a light yellow solid was obtained which could not be

crystallized. It was directly hydrolysed by refluxing with 7% aq. H2SO4. The

reaction mixture was cooled in an ice bath when a solid separtaed out. It was

crystallized from methanol as light yellow needles (10 mg), m.p. 193 °C.

Which on admixture with 3,5,3',4'-tetramethyl quercetin showed no

depression in melting point.

138

Analysed for Cj9H,807:

Calcd. : C, 63.68; H, 5.02%

Found C, 63.59; H, 4.98%

Cn-5

Cn-5 was obtained from the column by ethyl acetate-methanol (8:2)

mixtue and was crystallized from methanol-chloroform as yellow crystals

(160 mg), m.p. 2,04-05 "C.


Calcd. C, 56.25; H, 4.46%

Found C, 56.23; H, 4.44%

UV with shift reagents X„,,jnm

MeOH 230, 256.5, 265, 353

NaOMe 235.5, 273.5, 397.5

AICI3 226, 269.5, 295.5 (sh), 404.4

AICI3/HCI 225, 268, 294(sh), 402

NaOAc 233, 275.5, 377 (sh), 388.5

NaOAc/H^BO, 228, 273, 378(sh), 387.5

IR U cnr' : max

3400-3500 (OH), 1650 (C=0)

^H-NMR (400 MHz, DMSO-dr,), values on 5-scale

3.94 (3H, s, -OCH3), 3.10-5.51 (6H, m, sugar protons), 5.23 (IH, d.

139

J=8.0Hz, anomeric proton of arabinose), 6.21 (IH, d, J=2.2Hz, H-8), 6.40

(IH, d, J=2.24Hz, H-6), 7.05 (IH, d, J=8.79Hz, H-5'), 7.51 (IH, d,

J=1.83Hz, H-2'), 7.90 (IH, dd, J,=8.79 and J2=1.8Hz, H-6').


M""-absent, 316 [M'^-sugar moiety] (aglycone) (29.2), RDA, 152 [A,]^

(20.6), 151 [B , r (16.6) and 148 [B2r(23.2).

Hydrolysis of Cn-5 :

An alcoholic solution of Cn-5 (80 mg) was refluxed with 5% aq. HCI

on a water bath for 2 hours. The hydrolysate was diluted with water and

extracted with ethyl acetate. The ethylacetate extract was concentrated to

dryness and the solid left behind was crystallized from methanol-

chloroform, labelled as Cn-5 Ag (40 mg), m.p. 260 °C.

Analysed for Cj,sHi207 :

Calcd. : C, 60.75; H, 3.79%

Found C, 60.72; H, 3.75%

UV data with shift reagents, Xmaxi' i >

MeOH 233, 258.2, 268, 365

NaOMe 240,271.6,400.2

AICI3 235, 270.5, 298, 429.3

AICI3/HCI 239, 269.5, 300, 427.5

NaOAc 238,280.2,381.4,390.2

NaOAc/H-,B03 234, 278, 379, 388

140


The aquous hydrolysate, was concentrated to a syrup and

chromatographed on Whatman No. 1 filter sheet, in the descending mode

alongwith standard samples of sugars. The chromatograms were run in

n-BuOH-AcOH-HjO (4:1:5) and n-BuOH-EtOH-HjO (60:16.5:25.8)

separately for 24 hours. The chromatograms were dried and sprayed with

aniline phthalate and p-anisidine phosphate solutions respectively. The

chromatogrmas on drying at 100-105 °C for 30 minutes revealed the

presence of arabinose only [Rf = 0.17, n-BuOH-AcOH-H20 and 0.21,

n-BuOH-EtOH-HjO]. It was further identified by its m.p 160 °C, [ah in

H2O : + 105 °C.

141

1. '•'•Wealth of India (CSIR)"", A dictionary of Indian raw materials and

Industrial products. Vol. II, p. 93 (1963).

2. K.M. Nandkarni, '•''The Indian Materia Medica", (published by A.K.

Nandkami and Co.) Bombay, p. 76-183 (1976).

3. D.K. Pandey, H. Chandra, H.N. Tripathi, Phytopathoi, Z, 105, 175-82

(1984).

4. M. llyas, M. Parveen, M.S. Khan, Fitoterapia, LXVl, N. 3, 277 (1995).

5. M. llyas, M. Parveen, M.S. Khan and M. Kamil, J. Chem. Reserach(s),

88(M), 0601-0617 (1994).

6. V. Venkataraman in "77?^ Chemistry of Flavonuid Compounds'' (edited

by T.A. Geissman), p. 72, Pergamon Press, New York (1962).

7. T.J. Mabry, K.R. Markham, M.B. Thomas, "'The systematic

identification offlavonoids". Springer-Verlag, Berlin (1970).

8. P.W. Freeman, S.T. Murphy, J.E. Neworin and W.C. Taylor, Aus. J.

Chem., 1779-84 (1981).

9. J. Shinoda, J. Chem. Pharm. Soc. Japan, 48, 214 (1928).

10. A. Tada, R. Kasai, T. Saitoh and J. Shoji, Chem. pharm. Bull., 28, 1477

(1980).

11. L.H. Bridge and R.H. Locker, J. Chem. Soc, 2158 (1949).

12. W. Wilson, J. Am. Chem. Soc, 61, 2303 (1939).

13. A. Tsushi Hiraoka and Masquakris Maede, Chem. Pharm. Bull, 27,

3130 (1979).

14. Somogyi,./. Biol. Chem., 195, 19 (1952).

142

15. K. Tominaga and Yoshimura, J. Pharm. Soc. Japan, 79, 555 (1959).

16. T. Ohta, M. Miyazaki, J. Pharm. Soc. Japan, 79, 986 (1959).

17. G. Chakrabarty, S.R. Gupta and T.R. Seshadri, Indian J. Chem., 3, 171

(1965).

CHAPTER - IV

Heterophragma adenophyllum

^

Chemical investigation of the leaves of Heterophragma adenophyllum

(Bignoniaceae)

Heterophragma is a small genus of trees distributed in South-East Asia

and Africa. Two species occur in India. The extract of the wood is used for

skin diseases.''^ Heterophragma adenophyUum syn. Haplophragma

adenophyllum is a handsome tree, 30-50 ft. in height, found in the forests

of Assam and Andaman, and often cultivated in Indian gardens.

Earlier work on this plant is the isolation of p-amyrin, ursolic acid,

oleanoic acid"'. The present discussion deals with the isolation and

characterization of the following compounds from the leaves of

Heterophragma adenophyllum.

1. Dimethyl ester of terephtlialic acid

2. 24-Cyclohexyl-n-tetracosane

3. Friedelin

4. a-Amyrin acetate

5. Ursolic acid

6. Resveratrol

7. 2,3,6,7,8,9-Hexahydroxynaphthalene-2-rhamnoside (new).

Fresh leaves of Heterophragma adenophyllum{3.0 kg) procured from

the Botanical Garden of A.M.U., Aligarh, India, were air dried and extracted

exhaustively with ethanol. The EtOH extract was concentrated under reduced

pressure and the residue was extracted successively with petrol, benzene,

chloroform, acetone and methanol respectively. Petrol,benzene and

144

chloroform extracts showed similar behaviour on TLC examination, hence

were mixed together. The mixed concentrate (60 gm) was chromatographed

over silica gel column. Elution of the column in different solvent systems

[petrol, petrol-benzene (9:1-1:1), benzene, benzene-ethylacetate (9:1-6:4)]

afforded seven compounds marked as Ha-1, Ha-2, Ha-3, Ha-4, Ha-5, Ha-6

and Ha-7.

Ha-1

Ha-1 was eluted from the column with petroleum ether-benzene (9:1)

mixture. It was crystallized from chloroform-petrol as white needles

(100 mg), m.p. 138 ''C. The elemental analysis agreed with molecular

formula C|()Hio04, further supported by the molecular ion peak [M" '] at

m/z 194.

The IR spectrum of the compound Ha-1 showed a carbonyl band at

1720 cm"' along with bands at 1500, 1435 cm"' characteristic of an

aromatic compound. The UV spectrum showed maximum absorption at 265

nm and a weak absoiption at 340 nm.

The 'H-NMR spectrum showed a sharp singlet at 5 3.88 integrating for

six protons, corresponded to two methoxyl groups. A sharp signlet at 6 8.10

integrated for four protons was assigned to H-2, H-3, H-5 and H-6 of

phthalic acid.

The mass spectrum was quite typical for aromatic methyl esters. The

base peak was observed at m/z 163[M"' -0CH3] and other fragments were

observed at m/z 179 [M'-'-CH,], 164 [M-'--2xCH3], 135 [M '-COOCH^] and 76

[M+--2xCOOCH3] respectively.

145

On the basis of above results, Ha-1 was identified as dimethyl ester of

terephthalic acid (1)

COOCH,

COOCH3

(D

Ha-2

Ha-2 was obtained from the column from petroleum ether-benzene

(8:2) fraction. It was crystallized from methanol as colourless solid

(110 mg), m.p. 71-72 °C. Its IR spectrum demonstrated the presence of

absorption bands at 793 and 725 cm"' for long aliphatic chain. The mass

spectrum (Scheme-1, Fig, I) showed a molecular ion peak at m/z 420,

coiTesponding to molecular formula as C30H60, which indicated one-double

bond equivalent adjustable to terminal cyclohexane ring. It exhibited large

number of fragmented ions with a uniform difference of 14 mass unit,

thereby, confirming the presence of long aliphatic chain in Ha-2. The more

prominent peak corresponding to CnH2n was observed at m/z 83. The

fragment ion peaks at m/z 56, 71, 97, 111, 125 and 139 also supported the

presence of a cyclohexyl ring at one of the terminals.

The 'H-NMR spectrum of Ha-2 (Table-1, Fig. II) exhibited a three

protons triplet at 5 0.91 (J=6.5 Hz) assigned to C-1 primary methyl protons.

A one proton broad singlet at 51.50 was ascribed to C-25 methine proton

(Fig. 11). The remaining methylene protons resonated at 5 1.19 (56H,

28 X CH2). The absence of any signal beyond 5 1.49 in the 'H-NMR

.(CH2)-CH3 23

+

[ivr.],nyz420(4.0)

146

+ (CH2)-CH3 '23

+

nVz 339 (18.0)

Scheme -1

o_

CD

CI

\ri

o

o

ci4

2 i s

t o en

_ ^ ^ ^ '

147

Table - 1

'H-NMR spectral data of Ha-2, values on 5-scale


H-25 1 1.50(1H, brs)

28xCH2 56 1.19 (56H, brs)

-CH-, 3 0.91 (3H, t, J=6.5 Hz)

brs - broad singlet, t = triplet, spectrum run at 300 MHz in CDC^, TMS as

internal standard.

I MM

LI

1

u

148

and negative tests with tetranitiomethane and iodine suggested the saturated

nature of the molecule.

Thus, on the basis of above results, Ha-2 was characterized as 24-

cyclohexyl-n-tetracosane (II)

.(CI-M —CH, •'2?

an Ha-3

Ha-3 was obtained as a white solid from petroleum ether-benzene (8:2)

fraction and crystallized with chloroform-methanol as white needle shaped

ciystals (150 mg), m.p. 262-64 °C, [ajo - - 29.4" (CDCI3). It gave positve

Leibermann-Burchard'' and Nollers tests indicating Ha-3 to be a triterpene.

The elemental analysis and molecular ion peak at m/z 426 agreed with

molecular formula CôHsoO. Its identity as friedelin (111) was established by

comparison of its spectral data including ('H-NMR (Table-2), IR and mass)

and co-chromatography, m.p. and m.m.p. with that of an authentic sample*'"^

of friedelin.

an)

149

Table-2



-CH3 3 0.72 (3H,s)

-CH-, 3 0.87 (3H, s)

-CH3 3 0.89 (3H,s)

-CH3 3 0.92 (3H, s)

2 X CH3 6 0.95 (6H, s)

-CH3 3 1.05 (3H,s)

-CH3 3 1.18 (3H,s)

-CH2 and-CH protons 23 1.25, 1.34, 1.45, 1.52, 1.58 (m, 23H)

C2-2H and C4-1H 3 2.26 - 2.41 (3H, m)

s = singlet, m = multiplet, spectrum run at 200 MHz in CDCI3, using TMS as

internal standard.

150

Ha-4

Compound Ha-4 was obtained from the column with petroleum ether-

benzene (7:3) mixture. It was ciystallized from methanol as colourless

crystals (120 mg), m.p. 93-95 °C. It responded postiively to Leibermann-

Burchard test"*. Its IR spectrum demonstrated the presence of ester group

(1732 cm''). The mass spectrum (Scheme-II) showed a molecular ion peak

at m/z 468 corresponding to a pentacyclic triterpene C32H52O2. The mass

spectrum showed diagnostically important ion peaks at m/z 453 [M-Me]"^,

408 (M-HOAc)^ 393[408-Me)^ 250 [ion af, 190 [ion a - AcOH]+, 175

[190-Me]"^ and 160[]75-Me]"^ and thereby suggesting the presence of the

acetoxy group in ring A/B. The peak at m/z 218 [ion b]^ and 134 [ion

b - C5H|2]"^ supported the presence of a tetrasubstituted linkage in ring D.

The olefmic lingkage was placed at V'-' '** in the oleane/ursane skeleton on

the basis of the appearance of the base peak generated due to cleavage of

Cg.ij-Cg bonds with charge retention on either fragment'^ Besides the usual

cleavage of the ring-C, the V''' "^) bond also, migrated under electron impact

to V'^ which then undergoes retro Diel's-Alder fragmentation leading to

m/z 203"^ fragment ion.

The 'H-NMR spectrum of Ha-4 (TabIe-3) exhibited two one proton

double doublets at 5 5.13 (J=5.2 Hz and 5.3 Hz) and 5 4.50 (J=9.18 Hz and

5.46 Hz) assigned to equatorial H-I2 and H-3a respectively. A three proton

signal at 5 2.03 accounted for the acetyl group. The tertiary methyl signals

appeared at 5 1.08(Me-23), I.02(J=3.I5 Hz) at Me-29, 0.97 (Me-27), 0.95

(Me-24), 0.91 (J=7.98Hz, Me-30), 0.88 (Me-25, 28), 0.79 (Me-26). The

resonance of all the methyl groups in the range of 5 1.06-0.79 indicated the

attachment of these groups to saturated carbons.

15]

AcO

niyzn4(32.6) „y^218(l(X))

-Mc

iiVz 203 (28.8)

C32H52O2

IM'-l.m'z^jS (10.30)

AcO'

Cir,H2602

iiyz250(9.2)

-Ac

iiyz207(9.7)

-AcOH

nVz 190 (64.0)

Scheme - II

152

Table-3

^NMR spectral data of Ha-4, values on 8-scale


H-12 1 5.13(1H, dd, J=5.2Hzand5.3Hz)

H-3a I 4.50(1H, dd, J=9.18and5.46Hz)

OAc

Me-23

Me-29

Me-27

Me-24

Me-30

Me-25, Me-28

Me-26

3

3

3

3

3

3

3

3

2.03 (3H, brs)

1.08 (3H, brs)

1.02(3H, d, J=3.15 Hz)

0.97 (3H, brs)

0.95 (3H, brs)

0.91 (3H, d, J=7.98 Hz)

0.88 (3H, brs)

0.79 (3H, brs)

dd = double doublet, brs = broad singlet, d = doublet, spectrum run at 290

MHz in CDCI3, IMS as internal standard.

153

The '•'C-NMR spectrum of Ha-4 (Table-4) showed signals at 5 139.46

and 124.17, attributed to the unsaturated carbons at C-13 and C-12 while the

eight methyl carbons resonated at 5 27.19, 16.39, 16.73, 17.30, 23.28,

26.01, 17.38, and 21.13 were assigned to C-23, C-24, C-25, C-26, C-27,

C-28, C-29 and C-30 respectively.

On the basis of the above findings, the compound Ha-4 was identified

as a-amyrin acetate" (IV)

AcO

(IV)

Ha-5

Ha-5 was eluted from the column by petroleum ether-benzene (7:3)

fraction and crystallized from chloroform-methanol as shinning needles

(150mg), m.p. 286-87 °C. It responded positively to Leibermann-Burchard

test"* (red colour). Elemental analysis alongwith molecular ion peak at m/z

456 agreed with the molecular formula C3oH4gOv Infrared spectrum

displayed the characteristic bands at 3150 cm"' (OH), 2980, 1725 (C=0),

1710, 1470 (C=C) 1345, 1210 cm"'. It was acetylated with acetic anhydride

and pyridine. The 'H-NMR spectrum of acetate Ha-5 Ac (Table - 5) showed

seven methyl functions in the range of 5 0.7 - 1.0 and one acetoxyl function

as a singlet at 6 2.0. In addition to the above signals there was a multiplet

centred at 5 4.46 for a proton a to the acetoxyl and a signal at 5 5.26

characteristic of the olefinic protons.

154

Table -4

'^C-NMR spectral data of Ha-4, values on 5-scaIe

Carbon No. Chemical Shift

38.33 26.47 80.82 38.33 55.12 18.10 32.73 41.40 47.50 36.75 23.22 124.17 139.46 41.40 28.62 25.62 33.59 58.91 39.88 39.48 31.11 41.40 27.19 16.39 16.73 17.30 23.28 26.01 17.38 21.13 170.86

C-1 C-2 C-3 C-4 C-5 C-6 C-7 C-8 C-9

C-IO C-11 C-i2 C-13 C-14 C-15 C-16 C-17 C-18 C-19 C-20 C-21 C-22 C-23 C-24 C-25 C-26 C-27 C-28 C-29 C-30 -OAc

Spectrum run at 400 MHz in CDCI3, using TMS as internal standard.

155

On inethylation with diazomethane the acetate gave an acetyl methyl

ester Ha-5 AcM (30 mg), m.p. 210 °C. Its 'H-NMR spectrum (Table-6)

showed seven methyl functions at 50.72-1.02 and a singlet for the ester

methoxyl function at 5 3.6.

The spectral data mentioned above for the acetate and acetyl methyl

ester showed that the parent compound is a monohydroxy monocarboxylic

acid of triterpene. This was further confinned by the mass spectral data. The

mass spectrum of the acetate and its methyl ester showed the M^-peaks at

m/z 498 and 512 respectively. In the triterpenes, V'^ compounds undergo

predominantly retero Diels-Alder fragmentation. Thus the peaks at m/z 249

and m/z 248 in the acetyl derivative (Ha-5Ac) are due to fragments (I) &

(11), the fragment (1) arising from rings A/B and fragment (11) arising from

D/E rings. The Scheme Ilia and Illb showed the fragmentation pattern of the

acetyl derivative (Ha-5 Ac) and its acetyl methyl ester (Ha-5 AcM)

respectively.

A final identity of the parent compound as ursolic acid was established

by direct comparison on TLC examination in the solvent system, petroleum

ether-ethylformate-formic acid (93:7:0.7), which is specific for distinction

between ursolic acid and oleanolic acid'^, alongwith samples of ursolic acid

and oleanolic acid, it was found to be identical with ursolic acid (V).

CXX)H

(V)

156

Table - 5

^H-NMR spectral data of Ha-5 Ac, values on 5-scale


H-12 1 5.26 (lH,m)

H-3 1 4.46 (lH,m)

-OCOCH3 3 2.0 (3H, s)

7xCH-, 21 0.7-1.0

s = singlet, m = multiplet, spectrum run in CDCI3 at 290 MHz using TMS as

internal standard.

Table-6

^H-NMR spectral data of acetyl methyl ester Ha-5 AcM, values on 5-scale


H-12 1 5.20 (lH,m)

H-3 I 4.40(IH, m)

-COOCH3 3 3.60 (3H, s)

TxCHi 21 0.72-1.02

s = singlet, m = multiplet, spectrum run in CDCI3 at 290 MHz, TMS as

internal standard.

157

H,COCO'

H^CXXX)'

iiVz 249 (-«).())

(I)

-CH;,COOH

iiVz 189 (35.0)

COOH

nVz 24X02.0)

(D)

-COOH

COOH

nVz 20.3 (27.0)

Scheme - Ilia

158

HjCXXX)'

HjCOCO (XXXH.,

CXXXH3

nVz 249 (42.0)

-CH,CXX)H

iiVz 189 (19.0)

nVz 262 (37.0)

-COOCH,

iiVz 203 (21.0)

Scheme - Illb

159

Ha-6

Ha-6 was obtained from the column by using benzene-ethyl acetate

(8:2) mixure. It was crystallized from aq. ethanol to yield colourless pellets

(100 mg), m.p. 256-57 °C (decomp.). The elemental analysis agreed with

molecular formula C14H12OV The mass spectrum showed molecular ion peak

at m/z 228. It gave a reddish violet colouration with ferric chloride. The UV

spectrum of Ha-6 showed a weak band at 235 nm and strong bands at 305

and 318 nm. Trans stilbenes generally display a weak band at 229, strong

bands at 295, 308 and a shoulder at 320 nm. Whereas cis stilbenes show a

weak band at 283 nm and strong band at 222 nm'''•'''. The UV bands of Ha-6

are quite comparable to that of trans stilbenes, thereby showing that Ha-6

has a trans disposition. IR spectrum of the Ha-6 displayed a band at

3230 cm"' indicating the presence of phenolic functions.

'H-NMR spectrum of Ha-6 (Table-7) revealed a double doublet at 5 6.0

(J=2.5 Hz and 2.8 Hz) for one proton assigned to C-4. A doublet integrating

for two protons at 5 6.30 (J = 2.0 Hz) is attributed to identical protons at

C-2 and C-6. Another doublet appearing at 5 6.56 (J==9.0 Hz) for two

protons C-3' and C-5'. A doublet for two protons at 6.66 (J=2.0 Hz)

represents ethylenic protons in conjugation with the two aromatic nuclei of

stilbene. Again a doublet appearing at 5 7.16 (J = 9.0 Hz) integrating for two

protons C-2' and C-6'.

Ha-6 on acetylation with acetic anhydride and dry pyridine furnished a

triacetate Ha-6Ac, crystallized from methanol to yield colourless needles,

m.p. 118-19°C'\ From 'H-NMR spectrum (Table-8), it was evident that

Ha-6Ac is a triacetate, showing two overlapped singlets at 6 2.33 for nine

protons. A doublet centred at 5 7.66 (J=9.0 Hz), located for C-3' and C-5'

160

Table-7



H-4 1 6.00(1H, dd, J,=2.5Hz

andJ2=2.8Hz)

H-2, 6 2 6.30 (2H, d, J=2.0 Hz)

H-3',5' 2 6.56 (2H,d, J=9.0 Hz)

Olefinic protons 2 6.66 (2H, d, J = 2.0 Hz)

H-2 ' , 6' 2 7.16(2H, d, J=9.0Hz)

d = doublet, dd = double doublet, spectrum run at 90 MHz in CDCI3, TMS as

internal standard.

Table - 8

*H-NMR spectral data of Ha-6 Ac, values on 5-scaIe


H-3',5' 2 7.66 (2H, d, J=9.0 Hz)

Olefinic protons and H-2,6 4 7.28 (4H, m)

H-2',6' 2 7.20 (2H, d, J=9.0 Hz)

H-4 1 6.93(1H, d, J=2.5Hz)

3 X Ar - OCOCH3 9 2.33 (9H, overlapped singlets)

d = doublet, m = multiplet, spectrum run at 90 MHz in CDCI3 using TMS as

internal standard.

protons. A multiplet at 5 7.28 for four protons, attributed to C-2 and C-6

aromatic protons and two ethylenic protons of stilbene. A doublet at 6 7.20

(J=9.0 Hz) integrating for two protons, located for C-2' and C-6'. Another

doublet at 5 6.93 (J=2.5 Hz) for one proton, assigned to C-4 proton.

The above assigned structural data was further supported by '•'C-NMR

spectrum of Ha-6Ac (Table-9).

On the basis of these results, the compound Ha-6 has been identified

as resveratrol (3,5,4'-trihydroxy stilbene) (Vl)" '.

(VI)

.OCOCH,

H,COCO^

OCOCH,

(Via)

Ha-7

Ha-7 was obtained from the column by benzene-ethylacetate (7:3)

mixture. It was ciystallized from chloroform-methanol as yellow crystalline

solid (150 mg), m.p. 272-75 "C. The TLC plate gave blue purple colour when

sprayed with 5% FeCl-; reagent. Its IR spectrum showed absorption bands

162

Table -9

'^C-NMR spectral data of Ha-6Ac, values on 5-scale

Carbon No. Chemical shift

C-2',6' 127.623

C-3',5' 121.861

C-3 150.410

C-V 134.439

C-7 129.623

C-8 129.613

C-1 139.499

C-2,6 116.888

C-3,5 151.288

C-4 114.373

>C = 0(C-9) 169.336

>C = 0 (C- I1 , 13) 168.926

3X-COCH3 21.090

Spectrum run in CDCI3 at 300 MHz, IMS as internal standard.

163

due to hydroxyl group (3500, 3355), an aromatic moiety at 1665, 1561 cm"'.

The UV spectrum of Ha-7 in methanol exhibited absorption maxima at 251

and 354 nm. Elemental analysis showed the molecular formula as Ci^HigOio

on the basis of EIMS, FAB MASS, and '^C-DEPT. The EIMS (Fig. Ilia) and

FABMS (Fig. lllb) of HA-7 showed a very weak molecular ion peak at m/z

370, while FAB MS showed a prominent fragment ion at m/z 207

corresponding to the loss of rhamnosyl sugar unit. Its EIMS exhibited

absolute base peak at 314 [370-CH3=C-OH] due to fragmentation of

rhamnosyl unit (Scheme-IV).

The 'H-NMR spectrum of Ha-7 (Table-10, Fig. IV) in DMSO-dg

exhibited two one proton downfield signals at 5 7.71 and 7.51 assigned to

H-1 and H-4 respectively. It also revealed proton signals due to sugar moiety

between 5 1.12-4.78, indicating an anomeric proton at 6 4.87 (IH, d,

J=5.49 Hz, H-l'). The sugar was identified as rhamnoside in accordance with

the 'H-NMR and '^C-NMR data'^. The sugar was further identified as

rhamnose by comparison with an authentic sample.

'^C-NMR (Fig. V, Table-ll) and DEPT spectra of Ha-7 showed 16

carbon resonances appearing as eight quaternary, seven methine and one

methyl carbon peak. Among these six phenolic carbons could be attributed

correspondingly to C-2, C-3, C-6, C-7, C-8 and C-9 at 6 138.37, 159.22,

154.13, 147.36, 140.54 and 140.08 respectively. The characteristic

anomeric carbon (C-T) of the sugar appeared at 6 100.07, while the four

aliphatic methine peaks for C-2', C-3', C-4' and C-5' appeared at 5 69.76,

61.34, 69.72 and 70.67 respectively. The solitary methyl carbon peak of

C-6' appeared at 617.57. The connectivity between the protons on the

naphthalene ring (H-1 and H-4) were apparent from the cosy (Fig. VI).

164

Cif.HiijOio

|M".|. nVz37()(3.2)

:C—OH

CH,,

(luu, 56)

CH,OH

C13H14O9

nv/z314(10())

Scheme - IV

If-}

X

o- v o X

\ >

o X

X

-o

no

o

•3>

i n

(X)

LO

i n

CD

cn

CT)

n.

(3

ro

CD

en.

en OJ ,

C3 OJ ro

fN,

ru,

un OJ

CM

O J ,

rv OJ

in G)

1 ^

IT)

CD r«j

CD OJ

C5D

OJ

OJ i \ j

CD

OJ

(S

Table 10

'H-NMR spectral data of Ha-7, values of 5-scale

165

Assignments No. of Protons Chemical shifts

H-1

H-4

H-1

H-2

H-3

H-4

H-5

H-6 3

7.71 (s)

7.51 (s)

4.87(d, J=5.49Hz)

4.03 (dd, J=4.03, 5.49 Hz)

3.83 (dd, J=4.03, 3.46 Hz)

3.55(dd, J=3.46, 5.49Hz)

4.63 (d, J=5.49 Hz)

I.12(d, J=6.18Hz)

s=singlet, d = doublet, dd = double doviblet, spectrum run at 400 MHz in

DMSO-d^, using TMS as internal standard.

G:ao t: ••

^0'. C—2-

X 0-

/ = i / o X

= \ i \ o X

X -0

ssaG 0 90G 0 -•

C5I0 I

0200 I '-

0000 • '. "-

QO'.Q 0( _

oe: \.,_

CJ:O-

CD

166

Table- 11

'^C-NMR spectral data of Ha-7, values of 5-scaIe

Carbon No. Chemical Shifts

C-1 113.81

C-2 138.37

C-3 159.22

C-4 113.23

C-5 136.52

C-6 154.13

C-7 147.36

C-8 140.54

C-9 140.08

C-10 116.29

C-V 100.07

C-2' 69.76

C-3' 61.34

C-4' 69.72

C-5' 70.67

C-6^ 17.57

Spectrum run at 400 MHz in DMSO-d(s, using TMS as internal standard.

o u.

U3 •o I

O to X Q

a i

1

<

"Dp'n I 1 T t 1

. - ^ " a

f , I" 1 • : _ 1 1 t , I

" ~ : i i t u - -= [ I T I-t-t-i-• , , i i t I

_J T '^ IX 4 —^ t 'ia i

"^^ T ^ : _ : :

J : : . — J -5 - , n - -< '"r 1

I

i'" -." . ---

,' uj 1

t

i. - IJI-J

t- Ol -

. 1 -. = = =1= = =

^. JjjuL 1 • ' ' ' 1 , . . . 1 , , , nr

? 3 - 5

' r ' o • ; 1 ' 1 L

- - 4_' , . U 1 : _ 1 : J ^

1 (_ , . - j _ .. -f. 1

' t 1 •" i 1

5 1 ; • 1 " • • ! ! J

1 1 " ~ ' "" ••• "" 1 i

C C O ! ' S . _ _ n fJ 1

1 1 _,

t i .'"!"• __:::"± * ' " ' ! 6 ' r a

1 1

_ _ : r.... I

__± .L..I.. + : __:jr _f- ^ j 1 ..| 1 i ! • : 1 ! i 1 1

- T r • ' . ^ --t- -^ , . , . , . -j 1 1 M • • 1 1 .

1 I 1 1 ' i 1 i j 1 ' ; ; i ' 1 • 4

-1- , 1 ! ' ; ] " i - i f ' 1 1 1 • ; ! ! 1 1

" .'i j ;•' , • 7 1 ! i : . i

1 • . . • ! J ' • ; , , 1 • - ,

• 1 1 I I r\ • , — r 1 ; 1 1 . , 1

J I • 1

u _t__ 1 •.-]_! - L _1J ' -

" • n - | ^ . 1 1 • 1 1 •

1 . 1 • • 1 .

1 .

1 • • • : •

6 7 e 0

. ; ; , ! ; ; , , ; . . : . _ . | - ^ . N - | | ; , I , . . .

1 1 1 1 : 1 1 . _^ ,J . L . ! ; ' ! .

1 1 ' ' 1

i ' • • ' 1 1 1

1 I " t

L_ _ .. I p . 1

- T X - - T-• 1

. . " t - . ^ - . 4 - - - L . 1 1 ' 1 1 1 i ' 1 ' 1 ' 1 ' ' M 11

1 I 1 1 ' 1

1 1 i 1 1

! i ' 1 1 ' : ! 1

- f - f f 1 1 ' • \

T - ; F T T > 1 1 i 1

1 ; 1 , 1 1 , : , . - . . ' 1 .

1 • 1 ' ' ' • ^

! 1 • 1 : • . i • — J 1 1 ' 1 ; 1 • . ! 1

I ' ' ) I 1 i ' • • • • 1 1 1 • • . • •

_.;.,.:_.; - IT:. - T ~ " T T ' ;

• 1 1 M i i

4 4 - U -iTrr^: JS] ' .

1 1 1 ; .

V - i i-^ I I I '

1 1 1 X ' T ' -r - I 1-S. 1 1 :

1

1 1 1 ' 1 . • 1 ; 1 .

: ; Uj. ; . . , .

> ' 1 •

f i • • •

' 1 ' 1 ' • • . ' 1 : • \ '• '• . • • ' I •

! ! ' ; , i • i • •

• ! • • • • . • f>

' 1

1

1 , . 1 : , 1 , : • . : ! i 1 1 1 1 . • ; 1 • ' i 1

:::::±±I::+L 1 , i -Hi--

h 1 ; "^ f ' ^ 1 ! 1 I ' i : ! 1 1

._|__L,..j..-j-^4..j-...

1 f i l i , : , ,

< - . " 1 "T . : ' •

Lt.J-.i.!; j 1 . 1 •

1 M • : i i 1 • ^

. ; . . ; : . h i i ; . ! • i 1 T, • :

> I

o

167

Thus, on the basis of chemical and spectral analysis the structure of

Ha-7 was determined to be 2,3,6,7,8,9-hexahydroxy naphthalene-2-

rhamnoside (Vll).

HO OH

(VII)

168

Extraction of the leaves of Heierophragma adenophyllum

(Bignoniaceae)

Fresh leaves (3.0 kg) of Heierophragma adenophyllum collected

from Botanical Garden of A.M.U., Aligarh, India, were dried under shade.

The dried and powdered leaves were exhaustively extracted with ethanol. The

EtOH extract was concentrated under reduced pressure. The dark green

viscous mass (60 gms) left behind was extracted successively with petrol,

benzene, chloroform, acetone and methanol respectively. TLC examination

of petroleum ether, benzene and chloroform extracts revealed similar

behaviour on TLC plates and hence mixed together. The combined

concentrate was then subjected to column chromatoraphy over silica gel.

The column was eluted with different solvent systems petrol-benzene (9:1-

1:1), benzene, benzene-ethyl acetate (9:1-6:4). By repeated fractional

crystallization seven crystalline, compounds, labelled as Ha-1, Ha-2, Ha-3,

Ha-4, Ha-5, Ha-6 and Ha-7 were obtained.

Ha-1

The compound Ha-1 was obtained by elution of the column with

petroleum ether-benzene (9:1) mixture and crystallized from chloroform-

petrol as white needles (100 mg), m.p. 138 "C.

Analysed for Ci((H,„04 :

Calcd. C, 61.85; H, 5.15%

Found : C, 61.79, H, 5.12%

UV data, ?i„ax (CHCI3) nm :

265, 340

169

IR U cm' : max

1720, 1500, 1435

iH-NMR (100 MHz, CDCI3), values on 5-scale :

3.88(6H, s, 2 X OCH3), 8.10 (4H, s, Ar-H)

Mass m/z :

194 [M^-], 163[M+--OCH,] 100%, 179[M+-CH3], 164 [M' '-2 x CH,],

135[M+--COOCH3], 76[M+'-2 x COOCH3].

Ha-2


mixture and was crystallized from methanol as colourless solid (110 mg)

m.p. 71-72 °C.

Analysed for CjoH o :

Calcd. : C, 85.71; H, 14.28%

Found : C, 85.67; H, 14.25%

IR U cm' : max

2918, 2854, 2360, 793, 725

'H-NMR (CDCI3, 300 MHz), values on 5-scale :

1.50 (IH, brs, H-25), 1.19 (56H, brs, 28 x CHj), 0.91 (3H, t, J=6.5

Hz, Me-1).

Mass (rel. int.) m/z :

420 [M+-] (4.0), 405 (2.7), 377 (2.5), 349 (2.6), 339 (18.0), 321 (2.7),

293 (2.2), 265 (3.15), 251 (2.8), 237 (2.9), 223 (3.2), 209(3.6), 195 (4.0),

170

181 (4.4), 167 (5.0), 153 (5.9), 139 (6.9), 125 (8.6), 11 1 (9.7), 97 (11.3),

83 (36.0), 71 (67.2), 56 (100).

Ha-3

Ha-3 was obtained from the column with petroleum ether-benzene

(8:2) eluate and crystallized from chloroform-methanol as white needle-

shaped crystals (150 mg), m.p. 262-64 °C.

Analysed for C3oH5()0 :

Calcd. C, 84.50; H, 11.73%

Found : C, 84.47; H, 11.69%

IR U cm' : max

2900 (CH, str.), 1705 (> C=0), 1455, 1385, 1355, 1170, 1070.

'H-NMR (200 MHz, CDCI3), values on 5-scale :

0.72 (3H, s, CH3), 0.87 (3H, s, CH-,), 0.89 (3H, s, CH3), 0.92 (3H, s

CH3), 0.95 (6H, s, 2 X CHO, 1-05 (3H, s, CH3), 1.18 (3H, s, CH3), 1.25,

1.34, 1.45, 1.52, 1.58 (23H, m, -CUj and CH-protons), 2.26-2.41 [3H, m

(C2-2HandC4-lH)].


426 [M+'] (92.0), 411 (40.0), 411 (40.0), 341 (32.1), 3.03 (70.0), 273

(100.0).

Ha-4

The compound, Ha-4 was obtained from the column with petroleum

ether-benzene (7:3), It was crystallized from MeOH as colourless crystals

(120 mg), m.p. 93-95".

171


Calcd. : C, 82.05; H, 11.11%

Found C, 82.02: H, 11.07%

IR I) cm"* : max

2949, 2827, 1732, 1649, 1457, 1367, 1245, 1027, 986, 789.

UV l„,, (CHCI3) nm :

246

iR-NMR (290 MHz, CDCI3), values on 6-scale :

5.13 (IH, dd, J=5.2 Hz and 5.3 Hz, H-12), 4.50 (IH, dd, J=9.18 Hz and

5.46 Hz, H-3a), 2.03 (3H,brs, OAc), 1.08 (3H, brs, Me-23), 1.02 (3H, d,

J=3.15Hz, Me-29), 0.97 (3H bis, Me-27), 0.95 (3H, brs, Me-24), 0.91 (3H,

d, J=7.98 Hz, Me-30), 0.88 (3H, brs, IVle-25, Me-28), 0.79 (3H,brs,

Me-26).

i^C-NMR (400 MHz, CDCI3), values on 8-scale :

38.33 (C-1), 26.47 (C-2), 80.82 (C-3), 38.33 (C-4), 55.12 (C-5),

18.10 (C-6), 32.73 (C-7), 41.40 (C-8), 47.50 (C-9), 36.75 (C-10), 23.22

(C-11), 124.17 (C-12), 139.46 (C-13), 41.40 (C-14), 28.62 (C-15), 25.62

(C-16), 33.59 (C-17), 58.91 (C-18), 39.83 (C-19), 39.48 (C-20), 31.11

(C-21), 41.40 (C-22), 27.19 (C-23), 16.39 (C-24), 16.73 (C-25), 17.73

(C-26), 23.28 (C-27), 26.01 (C-28), 17.38 (C-29), 21.13 (C-30), 170.86

(OAc).


468[M+-] (10.3), 453(5.9), 408 (4.9), 250 (9.2), 218 (100), 203 (28.8),

190 (64.0), 175 (14.7), 162 (7.8), 150 (14.2), 136 (23.9), 134 (32.6), 122

(20.4), 107(30.5), 95(41.3), 69(42.2).

172

Ha-5


and crystallized from chloroform-methanol as shinning crystals (150 mg),

m.p. 286-87 °C.


Calcd. : C, 78.94; H, 10.52%

Found : C, 78.89; H, 10.49%

Acetylation of Ha-5 :

The compound Ha-5 (90 mg) was dissolved in dry pyridine (2.0 ml) and

acetic anhydride (4.0 ml) was added to it. The mixture was heated on a

boiling water bath for two hours and left overnight at room temperature.

After usual work up it gave a crude product which was dissolved in methanol

and treated with activated animal charcol. A clear light brown filtrate so

obtained was concentrated to about 25 ml and left at room temperature but

it did not crystallize out. Filtrate was evaporated to dryness and dissolved in

ethanol and extracted three times with hot petroleum ether. Petrol extract

was evaporated to diyness and again dissolved in ethanol and extracted three

times with hot petroleum ether. Petrol extract was evaporated to dryness to

give a colourless semi-solid mass, which crystallized from methanol as

colourless ciystals Ha-5Ac (75 mg) m.p. 292 "C.

• H - N M R (CDCI3, 290MHz), values on 5-scale :

0.7-1.0 (7 X CH3), 2.0 (3H, s, -OCOCH3), 4.46 (IH, m, H-3), 5.26

(IH, m, H-12).

173

Mass (m/z) :

498 (M -) (22.0), 438 (35.0), 249 (40.0), 248 (32.0), 203 (27.0), 189

(39.0).

Methylation of Ha-5Ac :

The acetate Ha-5Ac (40 ing) was treated with an excess of ethereal

solution of diazomethane and left overnight at room temperature. It was

filtered next morning and the ethereal solution was evaporated to dryness.

The residue on crystallization with methanol gave a colourless crystalline

compound Ha-5AcM (30 mg), m.p. 210 °C.

iH-NMR (CDCI3, 290 MHz), values on 8-scale :

0.72-1.02 (7 X CH3), 2.0 (3H, s, -OCOCH3), 3.60 (3H, s, -COOCH3),

4.40 (IH, m, H-3), 5.20 (IH, m, H-12).

Mass (m/z) :

512 [M+-] (18.0), 452 (31.0), 262 (37.0), 249 (42.0), 203 (21.0), 189

(19.0).

Deacetylation of the acetae (Ha-5Ac) :

The acetate (Ha-5Ac) (35 mg) was refluxed for four hours with

methanolic sodium hydroxide (2.5 ml, 4%) over a water bath. After

evaporating half of the solvent, the contents were diluted with water

(50 ml) which did not give any insoluble product. It was therefore acidified

with HCl to give colourless precipitate, which was filtered, washed with

water till free from acid and dried. The filtrate was extracted with ether

(2 X 50 ml) in a separating funnel. The two ethereal extracts were combined

together, washed with water to remove acid and dried over anhydrous sodium

174

sulphate. After filtering off the inorganic salt, ether was distilled off A

residue in very small quantity so obtained after TLC check up was combined

together with the precipitate obtained. On crstallization from methanol it

gave pure colourless crystalline compound (20 mg) m.p. 286-87°C. TLC

examination in petroleum ether-ethyl formate-formic acid (93:7:0.7)

alongside with an authentic samples of ursolic acid and oleanolic acid, it was

found to be identical with ursolic acid (Rf 0.19), m.m.p. 286' 'C.

Ha-6

Ha-6 was obtained from the column by benzene-ethylacetate (8:2)

mixture. It was crystallized from aq. ethanol as colourless pellets, m.p.

256-57*^0 (decomp.). It gave a reddish-violet colouration with ferric

chloride.


Calcd. : C, 73.68; H, 5.26%

Found C, 73.64; H, 5.22%

UV data (in MeOH) >.„iaxnni :

318, 305, 235

r « KBr , IR U cm' :

max

3230 (OH), 1605, 1585, 1510, 1460, 1437, 1380, 1145, 1007, 985,

965, 830

'H-NMR (90MHz, CDCI3), values on 5-scale :

6.00 (IH, dd, J,=2.5Hz and J2=2.8Hz, H-4), 6.30 (2H, d, J=2.0Hz,

H-2,6), 6.56 (2H,d, J=9.0 Hz, H-3',5'), 6.66 (2H, d, J=2.0 Hz, Olefinic

protons), 7.16 (2H, d, J=9.0 Hz, H-2',6').

175

Acetylation of Ha-6 :

Ha-6 (20mg), was acetylated (Ac20/pyridine) to yield a tri-acetate.

Ha-6Ac, as colourless needles from methanol (15 mg), m.p. 118-19 °C.

'H-NMR of Ha-6Ac (90MHz, CDCI3), values on 5-scale :

7.66 (2H, d, J=9.0Hz, H-3',5'), 7.28 (4H, m, olefinic protons, H-2,6),

7.20 (2H, d, J=9.0Hz, H-2',6'), 6.93 (IH, d, J=2.5Hz, H-4), 2.33 (9H,

overlapped singlets, 3 x Ar-0C0CH3).

^^C-NMR of Ha-6 Ac (300 MHz, CDCI3), values on 5-scaIe :

127.623 (C-2',6'), 121.861 (C-3',5'), 150.410 (C-3), 134.439 (C-T),

129.623 (C-7), 129.613 (C-8), 139.499 (C-1), 116.888 (C-2,6), 151.288

(C-3,5), 114.373 (C-4), 169.336 (>C=0, C-9), 168.926 (>C=0, C-11, 13),

21.090(3 X-COCH3).

Mass (rel.int.) m/z :

[M+-] 354, 312, 270, 229, 228, 219, 135, 125

Ha-7

Ha-7 was obtained from the column by benzene-ethyl acetate (7:3)

mixture. It was crystallized by chloroform-methanol as yellow crystals

(150 mg), m.p. 272-75 "C.

Analysed for CigHigOio:

Calcd. C, 51.89; H, 4.86%

Found C, 51.86; H, 4.84%

UV data of Ha-7, Ji y nm :

251, 354

176

IR U cm' : max

3500, 3355 (OH), 1655, 1561, 1058, 756

'H-NMR (400 MHz, DMSO-dfi), values on 6-scaIe :

7.71 (IH, s, H-1), 7.5! (IH, s, H-4), 4.87 (IH, d, J = 5.49 Hz, H-T),

4.03 (IH, dd, J=4.03 Hz and 5.49 Hz,H-2'), 3.83 (IH, dd, J=4.03 Hz and

3.46 Hz, H-3'), 3.55 (IH, dd, J = 3.46 Hz and 5.49 Hz, H-4'), 4.63 (IH, d,

J = 5.49 Hz, H-5'), 1.12(3H, d, J = 6.18 Hz, H-6').

'^C-NMR (400 MHz, DMSO-dg), values on 5-scaie :

113.81 (C-1), 138.37 (C-2), 159.22 (C-3), 113.23 (C-4), 136.52

(C-5), 154.I3(C-6), 147.36(C-7), 140.54 (C-8), 140.08 (C-9), 116.29

(C-10), 100.07 (C-r), 69.76 (C-2'), 61.34 (C-3'), 69.72 (C-4'), 70.67

(C-5'), 17.57 (C-6').


370[M+-] (3.2), 328 (16.9), 314 (100), 300 (34.2), 298 (22.2), 271

(11.0), 243 (8.5), 215 (7.4), 187 (9.7), 160 (8.8), 96 (9.8), 82 (22.6), 71

(25.1), 69 (30.5), 56 (23.7), 54 (21.8), 43 (33.5).

Hydrolysis of Ha-7 :

The compound Ha-7 (30 mg) was hydrolysed with 10% HCl to get the

aglycone (15 mg). The sugar in aqueous layer was characterized as rhamnose

with co-paper chromatography comparison with an authentic sample. The

ratio of aglycone to glycoside was found to be about 60% indicating only

one sugar unit per molecule.

177

Paper chromatography of sugar

Aquous layer after hydrolysis was neutralised by concentrating under

vacuum over KOH pellets. The neutral residue was subjected to paper

chromatography with an authentic sample of rhamnose, Rr=0.37, using

n-butanol-acetic acid-water (4:1:5) as solvent system. The spot of sugar was

visualized as brown in colour with aniline phthalate reagent which showed

only one sugar unit.

178

1. ''The Wealth of India'\ Raw Materials, CSIR, New Delhi, Vol. V,

p. 41-42(1954).

2. Berkill, 1., 1143; Pearson and Brown, II, 1070, Howard, 247; Rodger 132.

3. Kanchan Agrawal, R.A. Vishwakarma, S.P. Popli, Fllolerapia, 62,

459-60 (1991).

4. A.R. Irvine, " Wooding Plants ofGhana'\ p. 362 (1961).

5. C.R. Nollers, R.A. South, G.H. Harris and J.W. Walker, J. Am. Chem.

Soc, 64, 3027(1962).

6. A.A.L. Geirasatilika, Y.M.A. De-J., Silva S. Sotheeswaran, S.

Balasubramaniam and W.W. Wazeer, I'hytochcmistry, 23, 323-28

(1984).

7. M. Crawford, S.W. Hasan and M.E.S. Kokai, Tetrahedron letters, 3099

(1975).

8. H. Budzikisweiz, J.M. Wilson, C. Djerassi, ./. Am. Chem. Soc, 85,

3688 (1963).

9. Budziwickz, H. Wilson, J. Am. Chem. Soc., 85, 3688-99 (1963).

10. K.G. Das, E.P. James, ''Organic Mass Spectrometry'', Oxford and

IBM, New Delhi (1976).

11. B.S. Mahto, P.A. Kundu, Phylochemistry, 37, 1517 (1994).

12. J.B. Harborne, Phylochemical Methods, Chapman and Hall, London

(1973).

13. J.R. Dyer, "Applications of Absorption Spectroscopy of Organic

Compounds'', p. 20, Prentice-Hall of India Pvt. Ltd., New Delhi (1971).

79

14. R.M. Silverstein and G.C. Bassler, '\Speclromctric Identification of

Organic Compounds'', p. 165, John Wiley and Sons Inc., New York,

London (1967).

15. ''Dictionary of Organic Compounds'', 4th ed. Edited by Ian Heilbron

(Eyre and Spottiswoode Publishers Ltd., London) (1967).

16. Spath Kromp, Ber., 74, 867 (1941).

17. T. Honda, T. Murae, Bull. Cham. Soc. Japan, 49, 3213-18 (1976).

CHAPTER - V

Diospyros montana

J

Flavonoidic Constituents oiDiospyros montana (Ebenaceae)

The genus Diospyros is one of the largest genera (about 350 species)

of family Ebenaceae which consists of trees and shrubs and is widely

distributed in both the hemispheres''^. About 41 species occur in India

mostly in evergreen forests of Deccan, Assam and Bengal and a few are in

North India- '"*. Diospyros species are extensively used in folk medicines as

antifungal, antihelmentic, astringent and chewing stick^. Diosyros montana

is used for treatment of tuberculosis, diarrhoea^ and also as antibacterial^,

antiinflammatory^, antipyretic^ and analgesic^. Wide ranging medicinal

importance and scanty of work on this plant accelerated our interest to carry

out the comprehensive investigation of the plant Diospyros montana.

Earlier report from this plant is the isolation and characterization of

hentriacontane, hentriacontanol, |3-sitosterol, a-amyrin, lupeol, taraxerol

ursolic acid'" and yeninquinone (I)".

The present discussion includes the isolation and characterization of

the following compounds from the leaves of Diospyros montana.

1. 3'-Methoxy 3,5,7,4'-tetrahydroxyflavone (Isorhamnetin)

2. 5,7,4'-Trihydroxy flavanone (Naringenin)

3. 3,5,7,3',4'5'-Hexahydroxy flavone (Myricetin)

4. Quercetin 3-0-glucoside (Isoquercetin)

5. Myricetin 3-0-rhamnoside

6. Daidzein 7-0-glucoside

181

The leaves of Diospyros nionlana were collected from village Rampur

(Narora, U.P.), India. The air dried and powdered leaves (3.5 kg) were

thoroughly extracted with petroleum ether, benzene and methanol

successively. The petroleum ether and benzene concentrates were found to

contain mainly fatty and resinous material and, therefore, were not further

examined.

The methanol extract responded positively to flavonoidic tests'^"'^

The TLC examination of methanol concentrate in different solvent systems

revealed the presence of several compounds and was, therefore,

chromatographed over silica gel column using different solvent systems

namely benzene, benzene-ethyl acetate (9:1 - 1:8), ethyl acetate and

methanol as eluting solvents respectively. Column chromatography followed

by fractional crystallization with different solvents gave six pure

homogeneous compounds, marked as Dm-1, Dm-2, Dm-3, Dm-4, Dm-5

and Dm-6.

Dm-1

The fraction obtained by the elution of the column with benzene-ethyl

acetate (9:1) mixture, gave pale yellow coloured solid which was

crystallized from benzene-methanol as yellow needle shape crystals

(50 mg), m.p. 291-92 "C. It was found to be isorhamnetin by direct

comparison with an authentic sample of isorhamnetin (m.p, m.m.p, Rf-value,

co-chromatography). It was further confirmed by spectral studies, UV

(Table-1) and 'H-NMR (Table-2) data.

182

Table - 1

UV spectral data of Dm-I with shift reagents, A- ax""!

Reagents Dm-1 isorhamnetin

MeOH

NaOMe

AICI3

AICI3/HCI

NaOAc

NaOAc/H^BO,

271, 304sh, 321, 374 253, 267sh, 306sh, 326sh, 370

283, 330, 432 (dec) 240sh, 271, 328, 435 (dec)

264, 304sh, 369sh, 428 264, 304sh, 361sh, 431

271, 303sh, 358, 420 242, 272, 302sh, 357, 428

274, 324, 389(dec) 260sh, 274, 320, 393 (dec)

271sh, 330sh, 384 255, 270sh, 306sh, 326sh, 377

sh = shoulder

Table-2

'H-NMR spectral data of Dm-1 and Isorhamnetin, values on 5-scale

Assignments

H-2'

H-6'

H-5'

H-8

H-6

3'-OCH3

No. Prot(

1

1

1

1

1 n ^

of )ns

Isorhamnetin

7.83 (d, J=2.5Hz)

7.68(dd, J|=9.0Hz,

J2=2.5Hz)

6.95 (d, J=9.0Hz)

6.58 (d, J=2.5Hz)

6.23 (d, J-2.5HZ)

3.57 (s)

Dm-1

7.80 (d, J=2.5Hz)

7.65 (dd, J 1=9.0 Hz,

J2=2.5Hz)

6.93 (d, J=9.0Hz)

6.64 (d, J=2.5Hz)

6.26 (d, J=2.5Hz)

3.52 (s)

s = singlet, d = doublet, dd = double doublet, spectrum run in CDCI3 at 90MHz, TMS being used as an intemal standard.

183

On the basis of above findings Dm-1, was therefore, assinged the

structure as 3'-methoxy, 3,5,7,4'-tetrahydroxy flavone named as

isorhamnetin"^ (I).

OCH,

OH 0

(I)

Dm-2

The compound Dm-2 was obtained by eluting the column by benzene-

ethyl acetate (8:2) mixture, it was ciystallized from ethyl acetate-acetone as

light yellow needles (60 mg), m.p. 248-50 °C and was identified as

naringenin (11) by direct comparison with an authentic sample (Rf- value,

m.p, m.m.p, co-chromatography, and 'H-NMR spectral data). The result of

' H - N M R spectrum are recorded in (Table-3).

Dm-2 was, therefore, assigned the structure as 5,7,4'-trihydroxy

flavanone (naringenin)'^ (II).

OH o

(II)

84

Table-3

'H-NMR spectral data of Dm-2, values on 6-scale

Assignm

H-2',6'

H-3',5'

H-6

H-8

H-2

H-3,3

lents No. of Protons

2

2

1

1

1

2

Signals

7.39 (d, J-9.0HZ)

6.97 (d, J=9.0Hz)

6.66 (d, J=2.5Hz)

6.80 (d, J=2.5Hz)

5.20(q, J| = 12.0Hz, J2=4.0Hz)

2.78-2.99 (m,J| = 12.0Hz,

J2=4.0Hz, J3=I7.0Hz)

s = singlet, d = doublet, m=muitiplet, q = quartet, spectrum run in (CD3)2CO at 90MHz, TMS as internal standard.

185

Dm-3

The benzene-ethylacetate (7:3) eluate of the column on TLC

examination showed a single compound (Dm-3). It was crystallized from

chloroform-methanol as yellow crystals (40 mg), m.p. 358-59 °C. Elemental

analysis agreed with the molecular formula as CisHioOg. It responded

positively to Shinoda's test and gave greenish colour with FeCI^ It was

characterized as myricetin (Ilia) by m.p. spectral studies and further

comparing these data with an authentic sample of myricetin. The UV of

Dm-3 and 'H-NMR spectral data of its acetate (Dm-3Ac) are recorded in

(Table-4) and (TabIe-5) respectively.

Dm-3 was therefore, assigned the structure as 3,5,7,3',4',

5'-hexahydroxy flavone (myricetin)'** (llla).

OR 0

(HI)

(a) R = H (b) R = Ac

Dm-4

Dm-4 was obtained from the column by benzene-ethyl acetate (1:5)

mixture. It was crystallized from chloroform-methanol as yellow granular

crystals (210 mg), m.p. 218-19 T (anhydrous -252 "C). Elemental analysis

agreed with the molecular formula as C2iH2()0|2. It responded positively to

Molisch test and gave pink colour with Mg/HCl and red colour with sodium

amalgam followed by acidification and yellow colour with Wilson Boric

186

Table - 4

UV spectral data of Dm-3 with shift reagents

Reagents Xâ^nm

MeOH 255, 372

AICI3 270, 448

AICI3/HCI 268, 428

NaOAc 269, 339 (dec)

NaOAc/H3B03 259, 392

NaOMe 264, 284sh

sh = shoulder

Table - 5

' H - N M R spectral data of Dni-3 Ac, values on 5-scale

Assignments

H-8

H-6

H-2',6'

OAc-7,3'.

OAc-3,5

4' 5'

No. of Protons

1

1

2

12

6

Signals

7.30(d, J=2.5Hz)

6.81 (d, J=2.5Hz)

7.41 (s)

2.32 (s)

2.41 (s)

s = singlet, d = doublet, spectrum run in CDCl^ at 90 MHz, IMS as internal standard.

187

acid reagent''^ thus indicating a 3-substituted flavonol'^. The

chromatographic spot on paper appeared as deep purple under UV light and

turned yellow on fuming with ammonia vapours, further supporting the

3-substituted nature of the flavonal.

Hydrolysis with 6% aq.HCl yielded a sugar and an aglycone. The

aglycone was characterized as quercetin by m.p., R,- value, UV and 'H-NMR

spectral data and also by comparison with an authentic sample. The sugar was

identified as glucose by Revalue and co-chromatography with authentic

sugars.

'H-NMR spectrum of the acetate Dm-4Ac (Table-6) of the glycoside

(IVb) showed ABX pattern due to B-ring, doublets at 67.76 (J=2.5Hz) and at

67.35 (J=9.0Hz) and a quartet at 67.78 (J=9.0Hz and J=2.5Hz) integrating

for one proton each assinged to H-2',5' and H-6' respectively. The A-ring

protons signal appeared as meta-coupled doublets at 66.78 (J=2.5Hz) and

7.02 (J=2.5Hz) integrating for one proton each were ascribed to C-6 and

C-8 protons respectively. The 'H-NMR spectrum showed four aliphatic

acetoxyls at 51.90 (3H, s, OAc), 1.93 (3H, s, OAc), 2.02 (6H, s, 2 x OAc)

and four aromatic acetoxyls at 62.31 (3H, s, OAc), 2.34 (6H, s, 2 x OAc)

and 2.44 (3H,s, OAc). The anomeric proton appearing at 65.61 with a

coupling constant of (9.9Hz) showed P-linkage of glucose to the aglycone.

The glycoside on methylation with dimethyl sulphate followed by

hydrolysis gave straw coloured solid which on crystallization from ethanol

separated into straw coloured needles, m.p. 152-54T. It was characterized

as 3-hydroxy-5,7,3'4'-tetraniethoxy flavone as it showed no depression in

the melting point on admixture with an authentic sample of 3-hydroxy,

5,7,3',4'-tetramethoxy flavone. In UV-spectrum it showed a bathochromic

shift of 64nm in band-I with AlClv The above experiment, colour test and

Table-6

'H-NMR spectral data of Dni-4Ac, values on 5-scale

188

Assignments No. of Protons

Signals

>

Aliphatic acetoxyls

-OAc

-OAc

-OAc

-OAc

Aromatic acetoxyls

-OAc

-OAc-i

-OAc J -OAc

H-5"

H-2",3",4"

H-l"

H-6

H-8

H-5'

H-2'

H-6'

1.90 (s)

1.93 (s)

2.02 (s)

2.31 (s)

2.34 (s)

2.44 (s)

3.26-3.46 (m)

4.89-5.34 (m)

5.61 (d, J=9.9Hz)

6.78 (d, J=2.5Hz)

7.02 (d, J=2.5Hz)

7.35 (d, J=9.0Hz)

7.76 (d, J=2.5Hz)

7.78 (q, J,=9.0Hz and J2=2.5Hz)

s = singlet, d = doublet, m = multiplet, q = quarted, spectrum run at 90MHz in CDCl-t, TMS as internal standard.

189

spectral data established the attachment of glucose at 3-position of the

agiycone.

The quantitative estimation of the sugar by Somogyi's copper micro

method^" showed the presence of one mole of glucose per mole of the

agiycone.

Dm-4 was, therefore, identified as quercetin 3-0-glucoside^' (IVa).

0 \

ROH2C

(IV) (a) R = H (b) R = Ac

•~~~0R

L-OR

Dm-5

Dm-5 was obtained from the column with benzene-ethylacetate (1:8)

eluate and crystallized with chloroform-methanol as pale yellow needles,

(160 mg), m.p. 196-98 "C. Elemental analysis agreed with the molecular

formula C2|H2()0|2. It responded positively to Shinoda's^^ and Molisch tests.

A dark green colour with FeCl-t indicated the presence of free hydroxyl

groups.

Hydrolysis of glycoside (Dm-5) with 8% aquous HCl gave an agiycone

and a sugar. The sugar was identified as rhamnose by its m.p and co-paper

chromatography (BAW, 4:1:5) with an authentic sample; whereas the

agiycone was identified as myricetin by comparing its spectral data with an

190

authentic sample. On acetylation with acetic anhydride and pyridine Dm-5

afforded an acetate (Dm-5Ac), in.p. 14l-42°C.

The 'H-NMR spectrum of Dm-5 Ac (Table-7) established a

trisubstituted ring-B as it showed a singlet at 57.74 integrating for two

protons assigned to H-2', H-6'. The two doublets at 66.74 and 67.21 each

with a meta coupling, were ascribed to C-6 and C-8 protons respectively.

The 'H-NMR spectrum also showed three alcoholic acetoxyls between

61.97-2.15 and a rhamnosyl methyl doublet at 60.91 (J=6.0Hz). The

anomeric proton of rhamnose (H-1") appeared as a doublet at 65.60 with a

coupling constant of 2.0Hz, due to diequatorial coupling with H-2. This

suggested the a-linkage of rhamnose with the aglycone^-*.

The position of the sugar residue in the aglycone was confirmed by the

hydrolysis of the methylated glycoside. The partial methyl ether was

characterized as myricetin-5,7,3',4',5'-pentamethyl ether (Vb) m.p.

224-26 °C, by UV spectral studies and comparing with an authentic sample.

The methylated sugar was identified as 2,3,4-tri-O-methyl-L-rhamnose

suggesting the sugar to be in pyranose fonn. Quantitative estimation of sugar

by Somogyi's copper micro method^ ' showed 1 mole of sugar per mole of

aglycone.

On the basis of above findings, Dm-5 was identified as myricetin 3-0-

rhamnoside (Va).

OCH,

OCH,

OCH,

(Va) OH OH (M))

191

Table - 7

' H - N M R spectral data of Dm-5 Ac, values on 5-scale


H-6 1 6.74 (d, J=2.5Hz)

H-8 1 7.21 (d, J=2.5Hz)

H-2',6' 2 7.74 (s)

Anomeric proton (H-1") 1 5.60 (d, J-2.0Hz)

H-5" 1 3.62-4.01 (m)

H-2",3",4" 3 4.50-5.40 (m)

Rhamnosyl methyl 3 0.91 (d, J=6.0Hz)

Alcoholic acetoxyls

3xOAc 9 1.97-2.15 (s)

Phenolic acetoxyls

5xOAc 15 2.28-2.48 (s)

s = singlet, d = doublet, m = multiplet; spectrum run in CDCI3 at 60 MHz, TMS as internal standard.

192

Dm-6

The fraction obtained by eluting the column with ethyl acetate and

crystallized from benzene-acetone as yellow solid Dm-6 (100 mg), m.p

234-35 °C. Elemental analysis agreed with the molecular formula C21H20O9

which was in agreement with the molecular ion peak at m/z 416 in the mass

spectrum. It gave a dark green colour with FeCl3 and a positive Molisch test

supporting the glycosidic nature of the compound. Absence of any

colouration with Mg/HCl^^ and Zn/HCl ruled out the possibility of

flavanone, flavanol or flavone nucleus. On the other hand treatment with

sodium amalgam followed by the addition of excess of HCl resulted in pink

colouration, suggesting the isoflavone nucleus^"*, further supported by the

UV absorption at 258nm and an inflection at 314nm and a singlet at 58.01

for H-2 proton in its 'H-NMR spectrum. Analysis with diagnostic shift

reagents- - '- ^ showed no change in band-II (Table-8) pomtnig out the absence

of free hydroxyl groups. The infra red spectrum showed the presence of

hydroxyl group at 3400 cm"' and the unsaturated carbonyl group at 1653 cm"'.

Hydrolysis with 6% aqueous HCl gave a sugar and an aglycone which

was characterized as daidzein^*^ (Dm-6 Ag) (Vic), by m.p., its Rf-value, UV

(Table-8) and 'H-NMR spectral data and also by direct comparison with an

authentic sample. The aglycone showed a bathochromic shift of 15 nm with

NaOAc in Band-II (absent in glycoside) supporting the presence of free

hydroxyl group at 7th position, thus showing that the sugar is linked at 7th

position of the aglycone. The sugar was identified as glucose by Rf-value,

co-paper chromatography with an authentic sample and by the formation of

osazone.

193

Table - 8

UV spectral data of Dm-6 and Dm-6 Ag with shift reagents, AmaxOm

Reagents Dm-6 Dm-6 Ag

MeOH

NaOMe

AICI3

AICI3/HCI

NaOAc

NaOAc/H.BO^

258, 314sh

257, 270sh, 319sh

262, 308sh

262, 308sh, 260sh

258, 315sh

258, 314sh

240, 246, 258sh, 305sh

259, 290sh, 329

242sh, 248, 261sh, 301sh

242sh, 248, 262sh, 302sh

255, 270sh, 311, 328sh

258sh, 305

sh = shoulder

194

Acylation of Dm-6 with Ac20/Pyridine gave pentaacetate derivative

Dm-6Ac (VIb), m.p. 155-56°C. The 'H-NMR spectrum of Dm-6 Ac (Table-9)

showed a typical ABX pattern of A-ring protons, including the doublets at

66.87 (J=9.0Hz) and 56.79 (J=2.5 Hz) and a double doublet at 56.62

(J=9.0Hz and 2.5Hz) integrating for one proton each assigned to H-5, H-8

and H-6 protons respectively. The B-ring protons exhibiting A2B2 pattern as

it showed a pair of ortho-coupled doublets integrating for two protons each

at 57.01 (J=9.0Hz) and 57.78 (J=9.0Hz) were ascribed to H-3',5' and H-2',6'

protons. While the H-2 proton appeared as a singlet at 58.01. The sugar

protons appeared in the range of 63.48-5.44, while the anomeric proton was

observed at 55.40 (J=8.8Hz). The aliphatic acetoxyls appeared as multiplet

in the range of 51.82-2.02 and the aromatic acetoxyls was centred at 52.33.

On the basis of above discussion, the compound Dm-6 was identified

as daidzein 7-0-glucoside^^ (Via).

(Vic)

195

Table - 9

'H-NMR spectral data of Dm-6 Ac, values on 6-scale

Assignments

H-5

H-6

H-8

H-3',5'

H-2',6'

H-2

Sugar protons

H-1" (anomeric)

H-r',2",3' ',4",5",6"

Aliphatic acetoxyls

4xOAc

Aromatic

IxOAc

acetoxyl

No. of Protons

1

1

1

2

2

1

1

7

12

3

Signals

6.87 (d, J=9.0Hz)

6.62(dd, J,=9.0Hz& J2=

6.79 (d, J=2.5Hz)

7.01 (d,J=9.0Hz)

7.78 (d, J=9.0Hz)

8.01 (s)

5.40 (J=8.8Hz)

3.48-5.44

1.82-2.02 (m)

2.33 (s)

= 2.5Hz)

s = singlet, dd = double doublet, m = multiplet, spectrum run in CDCI3 at 300 MHz, IMS as internal standard.

196

Extraction of the leaves of Diospyros montana \Ebenaceaey

Fresh leaves of Diospyros montana were procured from village

Rampur (Narora, U.P.), India and identified by Prof. Wazahat Hussain,

Department of Botany, A.M.U., Aligarh, India and were dried under shade.

The dried and powdered leaves (3.5 kg) were exhaustively extracted with

petroleum ether, benzene and methanol successively. The petroleum ether

and benzene extracts concentrated under reduced pressure over water bath,

were found to contain mainly fatty and resinous material and, therefore, were

not further examined.

The methanolic extract was concentrated under reduced pressure on

water bath. A dark reddish brown gummy mass was obtained which responded

positively to flavonoidic tests. TLC examination of methanol concentrate in

different solvent systems viz.; [Chloroform-methanol (5:1, 3:1), benzene-

pyridine-formic acid (36:9:5), toluene-ethylformate-formic acid (5:4:1),

ethylacetate - ethylmethyl ketone - acetic acid - water [EtOAc-EtMeCO-

ACOH-H2O (20:3:1:1)], ethylacetate-methanol-water (8:1:1) revealed the

presence of several compounds and was, therefore, chromatographed over

silica gel column, using different solvents namely benzene, benzene-ethyl

acetate (9:1-1:8), ethylacetate and methanol as elating solvents respectively.

Column chromatography followed by fractional crystallization with

different solvents gave six pure crystalline compounds, labelled as Dm-1,

Dm-2, Dm-3, Dm-4, Dm-5 and Dm-6.

Dm-1

The compound Dm-1 was obtained by the elution of the column with

benzene-ethylacetate (9:1) mixture. It was crystallized from benzene-

methanol as sharp yellow needles (50mg), m.p. 291-92 °C.

197

Analysed for Ci6Hi207

Calcd.

Found

C, 60.75; H, 3.79%

C, 60.71: H, 3.76%

UV spectral data with shift reagents, X-nij nm

MeOH

NaOMe

AICI3

AICI3/HCI

NaOAc

NaOAc/H-,BO,

271, 304sh, 321, 374

283, 330, 432 (dec)

264, 304sh, 369sh, 428

271, 303sh, 358, 420

274, 324, 389(dec)

271sh, 330sh, 384

iH-NMR (CDCI3, 90MHz), values on 5-scaIe :

7.80 (IH, d, J=2.5Hz, H-2'), 7.65 (IH, dd, J|=9.0Hz, J2=2.5Hz, H-6'),

6.93 (IH, d, J=9.0Hz, H-5'), 6.64 (IH, d, J=2.5Hz, H-8), 6.26 (IH, d,

J=2.5Hz, H-6), 3.52 (3H, s, 3'-OCH3).

Din-2

Dm-2 was eluted from the column with benzene-ethylacetate (8:2)

mixture. The solvent was recovered by distillation under reduced pressure

on water bath. The residue on crystallization from ethylacetate-acetone gave

yellow needles (60mg), m.p. 248-50 "C.

Analysed for C,5Hi205 :

Calcd. C, 66.17; H, 4.41%

Found C, 66.15; H, 4.38%

198

UV spectral data with shift reagents, A-nia nm :

MeOH 287, 326sh

AICI3 311,382

AICI3/HCI 311,382

NaOAc 283, 330

NaOAc/H-,B03 285, 330

iH-NMR ((CD3)2CO, 90MHzl, values on 6-scale :

7.39 (2H, d, J=9.0Hz, H-2',6'), 6.97 (2H, d, J=9.0Hz, H-3',5'), 6.66

(IH, d, J=2.5Hz, H-6), 6.80 (IH, d, J=2.5Hz, H-8), 5.20 (IH, q, J, = 12.0Hz,

J2=4.0Hz, H-2), 2.78-2.99 (2H, m, J| = 12.0H, J2=4.0Hz, J3=17.0Hz, H-3,3).

Dm-3

The benzene-ethylacetate (7:3) fraction of the column on TLC

examination under UV light showed the presence of the prominent spot

alongwith some minor impurities of nonflavonoidic nature which were

removed by repeated crystallization with chloroform-methanol as yellow

crystals (40mg), m.p. 358-59 °C.

Analysed for CisHjoO^ :

Calcd. : C, 56.2; H, 3.14%

Found : C, 55.9; H, 3.12%

UV data; ^.â^nni :

MeOH 255, 372

AICI3 270, 448

199

AlCiyHCl 268, 428

NaOAc 269, 339 (dec)

NaOAc/H-,B03 259, 392

NaOMe 264, 284sh

IR U cm' : max

3355 (OH), 1655 (C=0)

Acetylation of Dm-3 :

Dm-3 (25 mg) was heated with AC2O (2.0 ml) and pyridine (1.0 ml) on

water bath for 2 hours. After cooling, the reaction mixture was poured over

crushed ice and left over night. The solid was collected, washed with water

and dried. On crystallization from CHCl3-MeOH it gave colourless needles

(I8mg), m.p. 218-19 "C.

'H-NMR (CDCI3, 90 MHz), values on 5-scale :

7.30 (IH, d, J=2.5Hz, H-8), 6.81 (IH, d, J=2.5Hz, H-6), 7.41 (2H, s,

H-2',6'), 2.32 (12H, s, OAc-7,3',4',5'), 2.41 (6H, s, OAc-3,5).

Dm-4

Dm-4 was obtained from the column by benzene-ethylacete (1:5)

mixture and ciystallized from chioroform-methanol as yellow granular

crystals (210mg), m.p. 218-19 °C (anhydrous-253 °C).


Calcd. C, 54.31; H, 4.31%

Found C, 54.27; H, 4.28%

200

UV data with shift reagents, Xâx""! •

MeOH 255, 265, 299sh, 358

AICI3 281, 302sh, 336, 432

AlCVHCl 275,310,358

NaOAc 278, 313sh, 373

NaOAc/HjBO, 260, 301sh, 368

Acetylation of Dni-4 :

Dm-4 (30 mg) was heated with pyridine (2.0inl) and AC2O (4.0ml) on a

water bath for three hours. After usual workup, it gave white crystals

(15mg), m.p. 162-63 °C.


Calcd. : C, 55.50; H, 4.50%

Found C, 55.46; H, 4.48%

^H-NMR (90MHz, CDCI3), values on 6-scale :

1.90 (3H, s, aliphatic - OAc), 1.93 (3H, s, aliphatic-OAc), 2.02 (6H, s,

aliphatic - 2 x OAc), 2.31 (3H, s, aromatic - OAc), 2.34 (6H, s, aromatic -

2 X OAc), 2.44 (3H, s, aromatic-OAc), 3.26-3.46 (IH, m, H-5"), 4.89-5.34

(3H, m, H-2",3",4"), 5.61 (IH, d, J=9.9Hz, H-1" anomeric), 6.78 (IH, d,

J-2.5HZ, H-6), 7.02 (IH, d, J=2.5Hz, H-8), 7.35 (IH, d, J=9.0Hz, H-5'),

7.76 (IH, d, J=2.5Hz, H-2'), 7.78 (IH, q, J|=9.0Hz; J2=2.5Hz, H-6').

Methylation of Dm-4 :

The glycoside (80 mg) in dry acetone (30 ml) was refluxed with

(CHi)2S04 (1.0 ml) and fused potassium carbonate (0.5g) for 30 hours. After

201

usual workup, followed by crystallization from ethanol, it afforded cream

coloured substance (70mg), which was hydrolysed by heating it with 6%

aquous HCl on water bath. The heating was continued for 2 hours to ensure

complete hydrolysis. The mixture was left overnight. After usual workup, it

was crystallized from ethanol as straw coloured neeldes (45 mg) m.p.

153-54 °C.

Acetylation of aglycone :

The aglycone (30 mg) was acetylated with acetic anhydride (4.0 ml)

and pyridine (2.0 ml). The product (20 mg), m.p. 195-97 °C was crystallized

from chloroform-methanol as colourless needles.


The hydrolysate was concentrated under vacuum over KOH pellets and

chromatographed on Whatman NO. 1 paper using the solvent system

n-butanol-acetic acid-water (4:1:5), employing the descending technique.

Authentic sugars were used as checks. The chromatogram was sprayed with

aniline hydrogen phthalate solution and heated at 110°C for 5 mintues. The

sugar was identified as D-glucose (Rf-0.18). The quantitative estimation of

the sugar by Somogyi's copper micro method showed the presence of one

mole of glucose per mole of aglycone.

Dm-5

It was obtained from the column by benzene-ethyl acetate (1:8)

mixture and ciystallized from chloroform-methanol as yellow needles

(160 mg), m.p. 196-98 °C.


Calcd. : C, 54.3 1; H, 4.31%

Found C, 54.29; H, 4.27%

202


The glycoside Dm-5 (35mg) was acetylated with acetic anhydride

(1.0 ml) and pyridine (0.5ml) by heating over water bath for two hours. On

usual work up, it gave colourless needles (15 mg), m.p. 141-42 °C.

^H-NMR (601VIHZ, CDCI3), values on 5-scale :

6.74 (IH, d, J-2.5HZ, H-6), 7.21 (IH, d, J=2.5Hz, H-8), 7.74 (2H, s,

H-2',6'), 5.60 (IH, d, J-2.0Hz, H-1"), 3.62-4.01 (IH, m, H-5"); 4.50-5.40

(3H, m, H-2",3",4"), 0.91 (3H, d, J=6.0Hz, rhamnosyl-CHO, 1.97-2.15 (9H,

s, 3 X OAc), 2.28-2.48 (15H, s, 5 x COCH3).

Hydrolysis of Dm-5 :

An alcoholic solution of Dm-5 (50mg) was heated with 8% aquous HCl

on a water bath. The heating was continued for 3 hours to ensure complete

hydrolysis. The yellow solid, thus separated out was filtered, washed with

water and dried. It was ciystallized from methanol as yellow needles

(20mg), m.p. 355-56 °C.

Analysed for CISHJOOH :

Calcd. : C, 56.60; H, 3.41%

Found : C, 56.58; H, 3.36%

Acetylation of the aglycone :

The aglycone (15 mg) was acetylated with AC2O (1.0 ml) and pyridine

(0.5ml) and worked up as described earlier. The solid on crystallization from

ethanol gave colourless needles (10 mg), m.p. 217-19 "C.

Analysed for C27H220,4 :

Calcd. : C, 57.00; H, 3.87%

Found : C, 56.60; H, 3.85%

203

rdentification of sugar :

The neutral aquous hydrosylate on examination by the method

described earlier showed the presence of only rhamnose.

Permethylation of Dm-5 (Hakomoris method) :

NaH (250 mg) was stirred with DMSO (15 ml) at 80 °C for 30 minutes

under N2 gas. To this reagent, solution of the glycoside (50 mg) in DMSO

(10 ml) was added and the reaction mixture was stirred for 1.0 hour at room

temperature under N2-gas. Mel (5.0 ml) was added and the reaction mixture

was further stirred for 4 hours at room temperature. The mixture was poured

into ice-water and extracted with EtOAc, washed with water, and dried. An

oily product was obtained. It was purified by TLC using C5H5-Me2CO (4:1)

as the developing solvent to afford the permethylated glycoside (28 mg),

m.p. 136-38 "C.

Location of sugar moiety :

Dm-5 (40 mg) was permethylated by Hakomoris method. The

methylated product, m.p. 136-38 °C. On hydrolysis with ACOH-HCI-H2O

(7:3:10) gave myricetin 5,7,3',4',5'-pentamethyl ether, m.p. 224-26 °C and

2,3,4-tri-O-methyl-L-rhamnose.

UV spectral data of myricetin pentamethylether : A-maxT"

MeOH 256,315,355

AICI3 260,410

AICIVHCI 260,412

NaOAc 257, 355

NaOAc/H3B03 257, 302, 357

204

Acetylation of myricetin pentamethyl ether :

The partial methyl ether (20 mg) was acetylated with acetic anhydride

(1.0 ml) and pyridine (0.5 ml) by heating over water bath for 2 hours. Work

up in usual manner gave a monoacetyl derivative (16 mg), m.p. 195-96 °C.

Dm-6

Dm-6 was obtained from the ethylacetate eluate of the column and was

crystallized with benzene-acetone as yellow solid (100 mg), m.p. 234-35 °C.

Analysed for C2iH2oOy :

Calcd.

Found

C, 60.57; H, 4.80%

C, 60.52; H, 4.76%

UV spectral data, A-maxini

MeOH

NaOMe

AICI3

AICI3/HCI

NaOAc

NaOAc/H3B03

IR U cm' : max

258, 314sh

257, 270sh, 319sh

262, 308sh

262, 308sh, 260sh

258, 315sh

258, 314sh

3400 (OH), 1653 (C=0), 1610, 1472, 1372

Hydrolysis of Dm-6 :

An alcoholic solution of Dm-6 (30 mg) was heated with 10% aquous

HCl on a water bath. Heating was continued for 2 hours to ensure complete

205

hydrolysis. The yellow solid separated out from the aquous hydrolysate was

filtered and crystallized from methanol to gave yellow needles of Dm-6 Ag

(15mg), m.p. 315-17 °C.

Analysed for C15H1QO4 :

Calcd.

Found

C, 70.86; H, 3.93%

C, 70.81; H, 3.87%

UV data A-m. nni :

MeOH 240, 246, 258sh, 305sh

259, 290sh, 329

242sh, 248, 261sh, 301sh

242sh, 248, 262sh, 302sh

255, 270sh, 311,328sh

258sh, 305

NaOMe

AICI3

AICI3/HCI

NaOAc

NaOAc/H,B03


The neutral aquous hydrolysate was examined by paper chromatography

employing n-butanol - acetic acid - water (4; 1:5) as the developing solvent

and using authentic sugars as check. The chromatogram was sprayed with

aniline hydrogen phthalate and heated at 110 "C for 5 minutes. The sugar was

identified as D-glucose (Rf-0.18).


Dm-6 (40mg) was acetylated with AcÔ/Pyridine (2.0/1.0 ml) and

worked up by usual procedure. It was crystallized from chloroform-

methanoi as colourless neeldes (30 mg), m.p. 155-56 °C.

206

'H-NMR (CDCIj, 300 MHz), values on 6-scale :

6.87 (IH, d, J=9.0Hz, H-5), 6.62 (IH, dd, J,=9.0Hz and J2=2.5Hz,

H-6), 6.79 (IH, d, J=2.5H2,H-8), 7.01 (2H, d, J=9.0Hz, H-3',5'), 7.78 (2H,

d, J=9.0Hz, H-2',6'), 8.01 (IH, s, H-2), 5.40 (IH, J=8.8Hz, anomeric

proton, H-l"), 3.48-5.44 (7H, H-r',2",3",4",5",6"-sugar protons), 1.82-2.02

(12H, m, 4 X OAc - aliphatic acetoxyls), 2.33 (3H, s, -OCHi-aromatic

acetoxyl).

207

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4. M.B. George Watt, A Dictionary of the Economic Products of India,

Vol. Ill, Cosmo Publication, New Delhi, India, p. 136 (1952).

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