Chemistry of Natural Products

IHMMR

CHEMISTRY OF NATURAL PRODUCTS

DISSERTATION SUBMITTED IN PARTIAL FULFILMENT FOR THE DEGREE

MASTER OF PHILOSOPHY

CHEMISTRY

ALIGARH MUSLIM UNIVERSITY, ALIGARH

1987

KALIM JAVED

INSTITUTE OF HISTORY OF MEDICINE AND MEDICAL RESEARCH NEW DELHI (INDIA) ^ 2 m 1918

x^^ Cottvo

.<> -^y .-0^

DS1127 643 9686 Phones: 643 9690 643 3685 Grams : EOURES IHMMR INSTITUTE OF HISTORY OF MEDICINE & MEDICAL RESEARCH P.O. HAMDARD NAGAR. NEW DELHI-110062

Ref. No Dated....^.:..'.f...... (1.^.7.

Chis is to certifa that the dissertation entitled

'Chanistry o£ Natural Products' is the original i»ork

of the candidate and is suitable for partial fulfil

ment of the requirements for the degree of Master of

Philosophy in Chemistry.

PRCF. ni.^.y. KHAN (Supervisor) ACKNOWLEDGEMENT

It g-ivei, me a Qfiaat ptta^aKd to ficcofid my deep 4en4C of^ g^'icLt-ttudc to P^o^. M.S. y. Khan andeA MhoAZ ahtd quidcuict and i,apQJw-ii>-ijon, I could be, abln to COAAIJ out alZ tivu n.zi>zaAch wo^fe.

I condtdM. it a Qficat phlvtttge to e.xpn.eM my pfio{,oand 4ett6e o;$ Qfiatitudu and tnde.btQjdn(ii>6 tjo kthaj HofexLm Abdul Hamttd Salitb, Pfiei-Ldcnt o;5 tiê. Institute 0|) HtitoAy of^ Me.dtctm and Me.dA,cal ReêoAc/t, Hamda/id Maga/L, New Vtlht ôn lu,i> alZ encou-iagemdnt and gQ,nQAoai> pfio\)iîon oi all avoAJiabtn {âcAlyitiz^ ioK. tko, smooth pn.ogfi(iAi> o{, thU, Mohk and awoAd o{^ a êZton)'i>lu.p. H-oi, constant tnteAz^t tn tlvu> wonk woi alixaip a .iou,ice oi iiUp-iAatJjon.

^\y itnceAt tiianki, a/it due, to Vfi. S.R.H. 'Rizvi., ReodeA VzpaAtinznt 0|)' CkejriAj,t/iy, A.M.(J. Atlga/ih {ôfi hi^ feeen InteAOJit and hoXpiut îuygei-t- toYib oivd ^ncou/iagme-nt AJX the. pfiogh.zi)6 o^ tkii iMonk. I am aZi>o tiiank^ûl to Viol. S.M, O^man, Head Ve.pa/itrmnt o^ Chemi^tJiy, A.M.U. KligaJih atvd to V-i. M, llijaA, ReadeA, Ve.paAtme.nt oi ChejnvUtAy^, A.M.U. ktigoAh iôx theAA tncouAagement duAtng the. wofik o^ theê itudilti,.

I am kighZy tnde.bte.d to ?fLO{^. M.R. PaAtka6aAthy, UnlveA^tty o{. Velivi (^01 kLi> vajjuiahte, ûggeJ^tioni, and {^fuiit^âZ di^cU'Siton duAing the couAê, oi tkzi,e. ^txxdieM.

My thanki) oAe. aZ^o due to MA. A.A. K^ctcoi and VKO^. M.E. Harrda^d iofi thexA bte.i6tng-is and good Mcihes.

I u)tih to expAe44 my d^^p -ien^e oi gfuitUude to VK. M. Kajntl Re^zoAch OiiiceA, V.S.R.U. AligoAh (C.C.R.U.M.) ion. kii> tnteAeJ>t.

Thanki, oAe. aJU,o due to ouA tabofiatoKy iitaH MA. Ah-*>aiiul Haqae and MA. RahmanuZ Haquz ion. thztA a6-i>-i^tajice.

KALTM JA(/EP The work described in this dissertation was carried out in the Department of Medicinal Chemistry of the Institute of History of Medicine and Medical Research, Hamdard Nagar,

New Delhi under the direct supervision and able guidance of

Prof, M.S.Y. Khan, Officer-in-charge of the Research Division of the Institute and Professor and Head, Department of

Pharmaceutical Chemistry, Hamdard College of Pharmacy, New

Delhi.

1 am highly grateful to Prof. M.S.Y. Khan for his valuable guidance and constant help and encouragement extended throughout these studies.

( KALIM JAVED ) CONTENTS Page No.

1. INTRODUCTION 1-47

2. STRUCTURE DETERMINATION 48-73

3. PRESENT WORK A. Discussion 74-83 B. Experimental 84-89

4. REFERENCES 90-101 INTRODUCTION FLAVONOIDS J

Geisman et al (1952) have applied the term flavonoid

to embrace all compounds whose structure is based on C,-C^-

C, skeleton. The flavonoids include, chaleones, dihydro- chalcones, aurones, flavones, flavanones, isoflavones,

flavonols, & flavanonols.

Flavonoids are widely distributed in plants as water

soluble glycosides. E. Wollenweber and Volker H.Dietz have 2 3 reviewed that out of 2000 flavonoids reported only 462 were, found in free state and most of them from the family

Astroceae. The family Leguminosae produces mostly flavonols while in Rutaceae flavones and flavonol are found

in abundance with remarkable number of methylated flavonoids.

In woody plants heart woods are rich sources of

flavonoids eg. Pinus (many dihydroflavonols and C-methylated compounds); Prunus (Flavanones and chalcones); in Leguminosae

like Acacia (flavonols, chalcones), Apuleia (flavonols),

Robinia and others. Bud excretions eg. populus Sp. and external deposits on plant leaves eg. farina on Gymnogramnoid ferns are also remarkable source of free flavonoids.

The structure and features of flavonoids considered to be of importance in biological function are (i) the presence of an extended conjugated resonating system with a carbonyl chromophore (ii) the presence of aromatic hydroxy groups which lead to the ability of flavonoids to interact with certain enz5mie systems, and (iii) the molecular shape which is responsible for their physiological activities due to their similarity in structure to the animal harmones. The biological functions of this group of compounds in man and animal was first suggested by Bents'ath, Rusznyak and Szent-Gybrgyi in 1936^^'^ who reported that the flavonoids present in the citrus peel were effective in preventing capillary bleeding and fragility associated with scurvy & termed vitamin P. The term vitamin P' included certain mixtures of flavones and flavonone glycosides widely distributed in many fruits; these were first isolated from paprika and lemon (citrus). The term 'citrin' was used for the flavonoid mixture from lemon which was later shown to consist of hesperidin, eriodictin and quercitrin. A review of the numerous communications in this area which appeared in 1968 established that flavonoids containing free hydroxyls at 3,3',4' positions exert beneficial physiological effects [gossypetin being highly active] on capillaries through the following ways: (a) chelate formation with metals, thereby inhibiting oxidation of ascorbic acid,

(b) protection of epinephrine by inhibiting oxidation of the 0-methyl transferose enzyme, which is said to be respon sible for maintaining capillaries in satisfactory condition and (c) stimulation of the pituitary-adrenal axis.

The action of natural flavonoids on heart was demons- trated as early as 1936. The unsubstituted parent compound, flavone, exerts coronary dilatory activity and is commerci ally available under the name 'Chromocor'; its combination with rutin and isoquercetin is also a commercial preparation under the name 'Flavo Ce', useful in the treatment of arteriosclerosis. Several studies on the antiinflammatory activity in

flavonoid have been conducted. A stable pharmaceutical

composition consisting of neomycin and a topical antiinfl

ammatory flavonoid in a suitable carrier has been patented Q for the treatment of acne . Another patent was taken on

xanthorhamnin as an antiinflammatory agent. It was isolated

from seeds of Rhamnus infectoria and recommended for use in

opthalmology, particularly for topical treatment of collyria

and in rheumatoid conditions.

The diuretic effect of myricetin, morin and kaempferol

in rabbits was observed as early as 1931; the potency incr- 9 eased with increasing number of hydroxyl groups . Flavonoidal

glycosides eg. quercitrin, rutin, kaempferol-3-rhamnoglucoside

and myricetin, luteolin, also were found to exhibit diuretic

activit,. .,y 10

Spasmolytic effects of 30 flavonoids were studied on

rat small intestine previously treated with BaCl^ as

spasmogen . As a rule, the highest activity was shown by

aglycones; the activity increased with increasing number

of hydroxyl groups. They were most active if the hydroxyls were in positions 7 and 4' in flavones; 3,7 and 4' in

flavonols; 4 and 4' in chalcones; and 7 in flavonones. In

glycosides, the spasmolytic activity also depended on the position and nature of moiety 12 . In a patent, luteolin

(5,7,3',4'-tetrahydroxy flavone) is characterized as a nontoxic compound with hypochloretic and spasmolytic . . 13 activity The antiabacterial activity of many naturally occuring

flavonoids was determined and quercetin was shown to compl

etely inhibit the growth of staphylococcus aureus at a 14 concentration of 0.1 mg/ml . Fisetin completely inhibited S. albus at a concentration of lOug/ml and S. aureus at

4 0 ug/ml =in liquid broth media. Thus, the antibacterial

effect of fisetin was abolished when the double bond between

C-2 and C-3 or the OH group on C-5 was removed chlorofl-

avonin is the first chlorine containing flavonoid type

antifungal antibiotic reported to be produced by strain of

Asperigillus candidus . Several flavonoids were found to

exhibit prophylactic action against fixed rabies virus in

mice. Of these quercetin and quercitrin showed significant

and rutin promising activities . Quercetin was viricidal

at a concentration of 100 ug/ml to human and procine strains

of herpesvirus and parainfluenze virus, as measured by

subsequent infectivity in human cell lines [He La cells].

Several flavonoids have been found to be moderately

active against laboratory cultures of malignant cells. 18 Eupatin, eupatoretin centaureidin and 6-demethoxy 19 centaureidin are moderately effective against carcinoma of nasopharynx (KB). An antitumorgenic flavonone isolated from the roots of Sophora Sp. has been patented 20 . The

effects of flavonoids have been claimed in the treatment of radiation disease ,as goiterogenic ' and antiulcer

The flavonoids are of commercial interest as antioxi dant for fats and oils ' . The antioxidant properties have been studied. Robinetin and Gossypetin v^ere claimed as the most potent and of economic importance in the tanning of leather, the fermentation of tea, the manufacture of cocoa and in the flavours qualities of good stuff 26a,b

The flavonoids are 0,^- phenolic compounds in which two benzene rings (A= & B) are linked by propane bridge in the fashion A-C 3 -C 2-C-B(I ) except in isoflavonoids & neoflavonoids which the arrangements are in the form of

A-C-^-C^-C-^(lI) and A-C^-C^-C-*-(IID respectively.

B B

(I) (II)

(III) The basic skeleton patterns of the largest group of the flavonoids depends upon the various levels of satura tion and oxidation of 1-Benzo-pyran which occurs when the cyclisation takes place between the third carbon of the chain on OH of the ring A ortho to this chain.

Chroman 2H Chromene 4H Chromene

4H Chromanone Chromone A-chromanone substituted by an aryl ring at C-2 gives rise flavanones and dihyroflavonols and similar substitution of chromone by aryl ring at C-2 leads to flavones and flavonols

Together these constitute the flavonoids groups of natural products. When the chroman nucleus is substituted by aryl ring at C-2, it leads to the 'flavanoid group' which is less commonly found in nature as compared to the flavonoids.

The first flavone to be isolated in the pure form was chrysin (IV) from the buds of the North American Populus monilefera balsamifera.

OH 0

(IV) Later studies on plant colouring matter led to the isolation, structure elucidation and synthesis of large number of flavonoids. In these compounds the level of oxidation varies in the C- bridge between two benzene rings. It is lowest in catechins (Flavan-3-ols) (V) to the highest in flavonols

(VI). Flavones, anthocyanidins and aurones, inspite of their vastly different structures have the same level of oxidation. (Table I) . C o in 4J in CO - - v£) -3- -

C c •H 4J o C QJ c !-l •H 4:: B 0 T3 o 0 I-H O •H o (U C x: •r-l C XJ •H a 0 4J C •H CO 4-J >^ CU CJ CO rH o X U QJ X C bO •H o CU O 0 ^i 0 •H C c -a o o cu (U rH 0 u 0) •H •H O o 4-1 i-H <—1 TS 4-1 u 4-1 -H CJ CO CO ^ >. ;3 CO P U (U O 0 CU K pq 2 PQ W (U

I CO -o 0 0 CO >, (U CO T-l > CO > u cu CO rH PSr-) .-1 >^ a O C CO .-I 4-1 rH 0 ^ CO CO CO -H O CJ cfl ,-( O CO l^ 1 •H4: J^ rH Q > 3 >^CJ-I -H CO Q 0 0 [iH ^^ CO Q> O ^T3 o -^ i-J •H

CO •«(, O 4-) CO c w O •H PQ 4J 0 < cd -a cn •H 1 X u X r«j o . X X 0 X X \X X x/ o CO 0—0 ( o—u s ^4^ V^ • /^w / \ >^-^ g O iJ ox u-o u-o C o o 01 (U •u >=< r= ( / >=< CO o M" •u cu en ^

4-1 o 0) PQ PQ I 4-J p:: PQ X O o pq I o :3 X 1 X v^ o CN PQ o jj 1 X 1 o I en ffi u X o 1 0 X X X CSJ II X X I o 1 0 CM CM 1 1 nc o 3: 0 0 u I o u CJ I X 1 o o <1: <1; CJ) a I I < < c o •H •U CO i-i >^ X

in Lo LO in Lo in in

TcO :3 o a 6 C o T3 •H u •H X) •H O C •H cu •H o •H rH OJ 4-1 ;j o •H en 0 <+-! QJ QJ 0) o U-l CU 0 0 0) CO C ex a, CU 4J •H E >-i •H 4-J .-I CO i-H CO X QJ QJ !-l a 0) CO CO 0 >. PH CJ Q H ^ cy IS

CO CO

I 0 <4-l CO c 0 CO 0 ^-1 '-' O w c (U C X) CO O CO en o CO >M-I O o c 0c >^ 0 . > i-i 4:: -H J-l CO -H C CO o d --< Q 0 < PL, W >

X X X o o o II r x/ / >^\ ux O CfcO o 0=0 0=0 >=< U t-l

U •U c o

a) PQ 4:: I 4-) PQ P3 PQ PQ o I I I I O O o u o O o I

.^^^ r^^^^

(V) (VI)

27 It has also been asserted that the minimum state of oxida tion (A-CH^-CH^-CHOH-B) corresponding to the flavanoids of the structure <.VII) are unknown in nature.

(VII)

In majority of flavonoids the ring A is either meta-dihydro- xylated (resorcinol type) or meta trihydroxylated (phloro- glucinol type). Except for chalcones eg. buteins (VIII), one of the hydroxyls of ring A is combined in an oxygen hetrocycle of five (IX) or six (X,XI,XII) atoms.

Ring B is monohydroxylated (XI), orthodihydroxylated 11

(VIII,IX,X), or vic-trihydroxylated (XII). In addition the different - OH groups may be methylated or glycosylated.

C=C=r.J/H ^ -OH

(VIII) (IX)

(X) (XI)

(XII) 12

FLAVONES

The flavones are derivative of flavone (2-phenyl-4-chromone) in which position 3 remains unsubstituted (XIII). The parent unsubstituted flavone is of rare occurance has been isolated 28 from the farina on species of Primula and Dionysis

. 3

(XIII)

In flavones the substitution like hydroxy, methoxy, C-methyl, C-prenyl, additional rings and glycosides format ion are very common. Methylene-dioxy group is also found.

Hydroxy/Methoxy Flavones : It was found that in hydroxy/methoxy flavones the position 5 and 7 are hydroxylated and frequently one or more of position 3',4' and 5' methylated. Position 5,7 and 4' are generally unmethyalated, but 3' and 5 are often methylated 29 13

Mono to penta hydroxy flavones and their methyl ethers

are reported but no hexa, or heptahydroxy flavone has been

isolated though methyl ethers of 5,7,8,2',3',4' and 5,6,7,

3',4'-hexa flavone and 5,6,7,8,3',4',5' hepta hydroxy

flavone are known

Among the most common members Apigenin (XlVa) and

Luteolin (XlVb) free or as glycosides, have widely distri

bution in angiosperms while Tricin (XIVc) is common in

grasses onl1y 31

R_ R^

a- Apigenin H H

b- Luteolin OH H

c- Tricin OCH^ OCH«

d- Trie itin OH OH (XIV)

2'Hydroxy flavone and 5,2'-dihydroxy flavone have been detected in the secretion of the glandular cells of Primula 32 floridae flower. The flavones with vie-trihydroxy substi tution pattern eg. 5,7,3',4',5' penta hydroxy flavone

tricetin (XlVd) has been isolated from Lathyrus pratensis 31

Some new flavones characterized from natural sources during last few years are presented in Table-2. 14

Table - 2

S.No. Substituents Plant Source Reference

OH 0CH3

1. 5 7,4' Saliva vesbenaca L. (33)

2. 5 6,7.3;4' Mentha pipertia var . Kuben^aya (34) 3. 7 5.8 Scutellaria rivularis (35)

4. 5.6 7,8.4' Thymus piperella (36)

5. 5.6,4' 7.3' I Thymbra spieata (37)

6. 5.6 7.3'.4'J

7. 5.7 8,2' Scutellaria rivilaris (35)

8. 5.7 8.4' Galeopsis angustifolia (38)

9. 5.7,3' 5'.5' Pao hyecu (39)

10. 5,7.4' 6,3' i Artemisia adamsii (30) I 11. 5.7,3' .4' 6 I 12. 5.7.2' 8.6' Scutellaria baicalensis georgi (41)

13. 5,7,2' 6 i - do - (42) i 14. 5.2' 6.7.81 15. 5,2',5 1 6,7.8 - do - (41) 16. 5.7,2' .5' 8,6' - do - (42)

17. 5,6' 6.7,2;3;4' Brickellia veronicaefolia (43)

18. 5.7.3' 6' 8,2' Scutellaria rehderiana (44) 19. Nil 5,6.7,3J4.'5' Bauhina chanpioniii Benth (45) 15

Hydroxy flavones and their methyl and methylenedioxy ether: 46 Linderoflavone A and B are very well known example of this class containing methylene dioxy group 5,7-(OH)^-6,

8-(OMe)2-3',4' - O2CH2 and 5,6,7.8 (OMe)^-3',4' - O2CH2

respectively. Two new flavone of this group have recently been isolated 5,6,7,5' (OCH^),-3',4' -methylene dioxy flavone

(XV) and 5,7,5' (OCH^)3-3 ' ,4 ' -methylene dioxy flavone (XVI) 45 from Bauhinia championii

CH30 OCH-

CH3O OCH3

(XV)

CH-,0

(XVI) 16

C-Methyl Flavones : Very few members of C-methyl flavones have been found as natural products. In this class methyl group is directly linked with carbon atom of the flavones eg. strobochrysin (6-methyl chrysin) (XVII) which was isolated from the heart wood of Pinus strobus and other species of pinus

5,7.dihydroxy 6,methyl flavone

(XVII) C-Prenyl flavones : In this class C-Prenyl group is linked with flavone. Prenyl is used as equivalent to Tf-dimethyl allyl (Me2C=CH- CH^) and isoprenoid as a more general term for a group derived from isoprene or modified group eg. Artocarpesin (XVIIl) and oxydihydroartocarpesin (XIX) were isolated from the heart-wood of Artocarpus - hetrophyllus

(XVIII) (XIX) 17

Isoprene modified group containing compounds have recently been isolated from the aerial parts of Achyrocline flaceida 49 and pods of Tephrosia falvinervis elucidated as 4' hydroxy-5-methoxy-7 (3-methyl 2-3-epoxy butoxy) flavone (XX) fulvinervin B(XXI) respectively.

(XX)

(XXI)

0. 0 \ H' Flavones with Additional Ring System :

A-Benzofuran derivative : The parent furanoflavone. Lanceolatin B (XXII a) was isolated from Tephrosia lanceolata Grab . Pinnatin (XXIIb) and gamatin (3'4' methylene dioxy pinnatin) XXIII occurs 52 in the root of Pongamia pinnata . 18

OCH3 0

(XXII) (XXIII)

(a) R=H (b) R=0CH2

Arthraxin : Arthraxin (XXIV) along with its 7-glucoside was isolated from Arthraxon hispidus 53 '5 4 which is derived from -<, Luteolin by the attachment of three acetate units (CH^CO- CHoCO-CH^O-) and subsequent reduction hydroxylation and cyclisation.

Alkaloids : The first flavonoidal alkaloids, ficine (XXV) along with its minor 6-isomer, isoficine were isolated from Ficus Pantoniana

OH HO

(XXV) 19

FLAVONOLS

Flavonols are simply flavones which bear an -OH group

at position 3. This is the only non-phenolic hydroxyl group in the molecule which alongwith other position may be methoxylated or glycosylated their skeletal structure is

(XXVI).

(XXVI)

Flavonols are unbiquitous in woody angiosperms, appear less frequently in harbaceus angiosperms and are exceptional in lower plants. In lower plants chloroflavonin (XXVII) was isolated from a strain of the fungus Aspergillus candidus

H3CO

(XXVII) 20

Among the flavonols of higher plants, kaempferol (XXVIIIa) and quercetin (XXVIIIb) free or as glycoside, have most wide distribution, less frequently is myricetin (XXVIII,c) and comparatively rarer is galangin (XXXVIII d). Together they form the fundamental structural types from which all the natural flavonols may be considered to be derived

R, Ro R. J2 _2 _I3 a- Kaempferol H OH H b- Quercetin OH OH H c- Myricetin OH OH OH d- Galangin H H H e- Isorhamnetin OCH, OH H

From monohydroxy to heptahydroxy derivatives are reported Glycosylation and methylation is also common. The best known 0-methyl derivative of common occurance is Isorhamnetin ' ((XXXVIIIe) 3,5 dimethoxy(XXIX) and 3,7 dimethoxy 5,4' dihydroxy - flavone (XXX) have recently been isolated from Linaria dalmalica^O

(XXX) 21

Flavonols encounter a number of exceptional structures such as :

6-Hydroxy flavonol 3,5,7 trihydroxy-6,3',4' trimethoxy flavone (XXXI) a new flavonoid of this group has recently fi 1 been isolated from Arnica Chamissonis

OCH-

(XXXI)

8-hydroxy flavonol - Gnaphatiin 5,7-dihydroxy-3,8-dimethoxy- flavone (XXXII) was isolated from Gnaphalium Optusifotium 62.

OCH r^^^

(XXXII) 22

6,8-dihydroxy flavonol : Eriostemin 3,8, dihydroxy 5,6,7,4' tetramethoxy flavone (XXXIII) has been isolated from Eriostemon hispidulus and E. buxifolius sub. sp. buxifolius 63

OCH3

H3CO

(XXXIII)

2'-hydroxy flavonol : Apuleidin which is 5,2',3' trihydroxy-

3,7,4' trimethoxy flavone (XXXIV) has been isolated from 64 Apuleia leiocarpa It possesses the unusual 2' hydroxy substitution.

(XXXIV) 23

5-deoxy flavonol : A new flavone of this group 3,7-dihydroxy•

8-methoxy flavone (XXXV) (lack of hydroxyl group at positi on 5) has recently been isolated from Zuecagnia punctata 65

(XXXV)

7-deoxy flavonol : Gardenin isolated from an exudate of

Gardenin iucida was proposed the structure 5-hydroxy-

3,6,8,3',4'5'-hexa methoxy flavone (XXXVI).

OCH- OCH'

OCH-

(XXXVI) Ik

Methylenedioxy flavonols - These are infrequent in nature 5,3', 4' - trihydroxy-3-methoxy-6,7 methylene-dioxy flavone has been reported in spinach chloroplast fi 7

C-Prenylated flavonols : Few new flavonoids of this group eg. Aspletin,[5,7,3',4',5' penta hydroxy-3-(3-methyl butyl) 68 flavone"! (XXXVII) from Launae a asplenifolia ^ 5 , 7-dihydroxy-3 ' -(3-hydroxymethyl butyl)- 3,6,4' trimethoxy flavone (XXXVIII), Aliarin, [5,7,4 trihydroxy-3'-(3-hydroxy methyl butyl)-3,6- methoxy flavonel (XXXIX) from Dodonaea viscosa Linn ' and other isoprene modified group containing compound 5-hydroxy 3-8-dimethoxy 7-(3methyl 2-3 epoxy butoxy) flavone (XLi from Achyrodine: flaccida have recently been isolated.

OCH3

(XXXVII) (XXXVIII) 25

(XXXIX) (XL) Aliarin Isoprene modified compound 26

Flavanone (Dihydroflavone)

(XLI) Flavanones are based upon structure of 2-phenyl benzopyrano A-one (XLI). Flavanones have a centre of a symmetry at C-2 so that naturally occurring are often optically active. Most of the naturally occurring flavanones were laevoro tatory and thus all belong to the same configuration serie. s 71-77

Though fairly widely distributed they have less common occurence than flavones or flavonols. Flavanones have fungistatic and wood preservative properties.

Flavanones are classified according to B-ring hydroxylation pattern into the following categories.

Flavanones lacking B-ring OH group : The simplest member 7-hydroxy-flavanone has been isolated from two legumes 78 79 80 platymiscium ' and Flemingia

Four other compounds of this class Falciformin [7- methexy-8-(3-hydroxy-3 methyl-but 1-enyl)flavanone] (XLII) from Tephrosia - falciformis 81^ 5-hydroxy-7-(3(methyl 2-3 epoxy butoxy) flavanone (XLIII) from Achyrocline flaccida , 27

6,7 ,2 ,2-diinethyl chromeno-8 "f "T-dimethylallyl flavanone (XLIV) from Lannea acida, Fulvinervin A (XLV) from o o Tephrosia fulvinervis have recently been isolated.

(XLII)

r<:^^

H-jC

(XLV)

(XLIV) 28

Flavanones having one B-rjng hydroxy1 : The simplest member is 7,4' dihydrcxy flavanone, liquiritigenin (XLVI, R-H) first isolated from Glycyrrhiza glabra^^. 5,7,4' -trihydroxy flavanone, (XLVI, R=OH), is the most widely distributed member.

Liquiritigenin H Naringenin OH (XLVI)

A new isoprenylated flavanone has recently been isolated Q c from the leaves of Azadirachta indiea and characterized as 8,3' di-isoprenyl-5,7-dihydroxy-4'-methoxy flavanone (XLVII).

/;K^0CH3 29

Flavanones having two B-rin^ hydroxy Is :

7,3',4' -Trihydroxy flavanone, butin which is simplest member has been found in Butea, Mangifera, Macherium and

Acacia while 5,6,7,8,3*4' -hexa methoxy flavanone is one

among the most highly methoxylated flavanone of this class

eg. Filifolin (5,7,3',4' -tetrahydroxy-6-methoxy flavanone

(XLVIII) from Filifolium sibiricum (L) ketam , 5,3-dihyd-

roxy-7,8,4' trimethoxy flavanone (ILa) and 5,3'-dihydroxy

6,7, 4' trimethoxy-flavanone (ILb) from the leaves of Vitex 87 negundo , 3'-hydroxy 5,7,4' -trimethoxy, 8-methyl flavano- 88 ne(L) from the heart wood of Adina cordifolia cudraflava- 89 none A(LI) from the root bark of cudrania tricuspidata

have recently been isolated

OCHt

H3CO OH 0 OH 0

(XLVIII) (IL) a-R-^ = OCH-, R2 = H b-R^ = H, R2 = OCH^ 30

(L) (LI)

Flavanone have three B-ring hydroxyl : No flavanones having three B-ring hydroxyl group are known to occur naturally by 1975 90 . Three flavanone have been isolated however, which have three oxygen in the B-ring, two of which are ketonic (benzoquinones type) from the members of the cyperaceae 91

A new flavanone 7,2' dimethoxy 4',5' methylene dioxy from the leaves of Lannea aceda (Rich) 92 have recently been isolated and can be listed in the same group on the basis of that B-ring have three oxygen, two of which are methy- ledene dioxy group form other one methoxy form.

H3CO

H3CO 31

FLAVANONOLS (DIHYDROFIAVQNOLS)

Flavanonols are based upon the structure of 2-phenyl

3-hydroxybenzo-pyran-4-ones(LIII)

(LIII) These are classified according to B-ring hydroxylation pattern into the following categories.

(a) Dihydroflavonols lacking B-ring hydroxyls : The simplest member 7-hydroxy dihydroflavono1 (LIV, R=R=H) has been isolated from platymiscli\jm Praecox 78 , pinobanksin (LlVa) and its 6-C-methyl derivative, strobobanksin (LIVb), are widely distributed in pinus 93 .

R R'

Pinobanksin H OH <<^^^^ Strobobanksin Me OH

(LIV) 32

Dihydroflavonols having one B-ring hydroxyls :

7,4' -dihydroxy dihydroflavonol, Garbanzol (LV.a) was

first isolated from Cicer arietium 94 . Dihydrokaempferol (LV,b) is one among the most widely distributed members.

R a) Garbanzol H b) Dihydrokaempferol OH

'OH R 0 (LV) Dihydroflavonols having two B-ring hydroxyls :

The most common example is dihydroquercetin (LVI,

R=H,R' =0H) or taxifolin, Dihydromorin (LVI, R=OH,R'=H)

isolated from Morus and Artica:ij'pus has 5,7,2',4' tetra hydroxylation pattern. 3-0-acetyl padmatin (LVII) from 95 the aerial parts of Inula viscosa has recently been isolated.

H3CO

^^y^'^^^^^ 0 c oc H 3

OH 0 OH 0

(LVI) (LVII) 33

Dihydroflavonol having three B-ring hydroxyls : Dihydrorobinetin (LVIII) 7,3', 4',5' tetra hydroxy flavoDDnol was first isolated from Robiana pseudoacacia 96 97 and later from Adenanthera pavonina . A new dihydroflavo nol, pallasii was isolated from the bark of Rhamus 98 pallasii and its structure elucidated as 2,3-dihydromy- ricetin 4-0-methyl ether (LIX).

OCH3

(LIX) 3A

CHALCONES AND AURONES

These are yellow pigments found together in the petals 99 of number in the compositea . The yellow colour is to red orange, when exposed to ammonia.

In these compounds the central chain of three carbon atoms which unites benzene rings is not in form of an oxygen hetrocylic of six atoms. In chalcones eg. butein

(LX, R=H) and oxanin (LX,R=CH) it is in the form of a linear chain i.e. jt, B unsaturated carbonyl system.

A new chalcone purpurenone (LXI) has recently been isolated from Tephrosia purpurea

0 OH

(LX) (LXI) 35

Whereas in aurones eg. sulphuretin (LXIIa), aureusidin

(LXIIb) and Leptosidin (LXlIc), it is in the form of a pentacyclic ring.

R R'

Sulphuretin H H Aureusidin OH H

Leptosidin H OCH

(LXII) 36

ISOFLAVONOIDS

Isoflavonoids embrace all compounds whose structure is based on 3-phenyl 4-chromone (LXIII). The first natural

isoflavonoid to be examined was iridin, a glucoside of

irigenin (LXIV) which was isolated from the rhizomes of Iris „ ^. 101 florentma

(LXIII)

It is noteworthy that most likely sites for oxygenation

in isoflavonoids are at 4,7 and 4', with a somewhat less

frequent occurence at 5,2' and 5'. Several isoflavonoids

have been isolated which are in a different oxidation state

from that of isoflavones; they include the isoflavanones

(LXV), the coumaranochromans (LXVI) , the X-methyldeoxyben-

zoin angolensian, (LXVII) and the isoflavan equol (LXVIII)

(LXV) 37

^^^^:^.-'^OCH.

(LXVII) (LXVIII)

5,6,7,4' -tetrahydroxy-8-methoxy isoflavone (LXIX) and 5,6,7,4' -tetrahydroxy-3' -methoxy isoflavone (LXX) from rhizomes of Iris melesii 102 ' 103 ,5,7-dihydroxy-6,2'-dime- 104 thoxy isoflavone (LXXI) from rhizomes of Iris supria 7,2',4' -trihydroxy-3'-methoxy isoflavone (LXXII) from Zollernia paraensis have recently been isolated.

OCH

OCH3

(LXIX) (LXX) 38

(LXXI) (LXXII) 39

FLAVONOIDAL GLYCOSIDES

Flavonoids mostly occur in the form of glycosides in nature. Structural variation among these flavonoid glycosides is considerable, both in the nature of the sugar residue and the position of attachment through hydroxyl groups to the flavonoid nucleus.

Six monosaccharides are commonly found in -0-glycosidic combination with flavonoids glucose, galactose, glucuronic acid, xylose, rhamnose, arabinose. These monosaccharides are generally present in the expected pyranose form, although occasionally the less stable furanose forms have been reported. Glucose, galactose, glucuronic acid, xylose are

D-sugars and are usually B linked to the aglycone hydroxyl; rhamnose and arabinose are L-sugars and normally X-linked.

The sugar moieties are almost exclusively aldoses. The presence of a ketose (e.g. D-fructose) is quite exceptional.

Apiose, a branched chain pentose is rare sugar specially associated with flavones. Two other monosaccharides newly reported are mannose and galacturonic acid

Of the disaccharides based on glucose sophrose (2-0-p-D-Glucoseyl-D-glucose) is undoubtedly the most common. Disaccharides with two galactose, two arabinose or two glucuronic acid residue are also known flavone or flavonol attachments, but general such sugars have been less well characterized. The disaccharides which contain two differ ent sugars by far the most frequent is rutinose (6-0-/-L- AO

Rhainnosyl-D-glucose) . Neohesperidose ( 2-0-X-L-Rhainnosyl-D- glucose) is less common. A new isomer of rutinose and neo hesperidose has been reported as th*^ kaempferol derivative in flowers of Rungia repens. This is rhamnosyl-«C -1 -3glucose, called rungiose by the authors 109 . Another interesting u -J ^ -1 ., ... 110 dxsaccharide of cyanogenic glycoside vicianm

Trisaccharides derivative of flavonoids have rarely been examined in detail, but atleast seven of those associated with flavonoids have been fully characterized.

These are fall into two groups according to whether the sugar is linear or branched.

Linear : Gentiotriose= O-R-Glucosyl-(1—• 6)-O-B-glucosyl-(1—• 6)-glucose Sophorotriose=0-B-Glucosyl-(1—> 2)-B-glucosyl-(1—* 2) glucose Sorborose= G-j^-Glucosyl-(1—>• 6)-0- /-glucosyl-(1—» 4)-glucose Rhamninose= 0-Rhamnosyl-(1—* 4)-O-rhamnosyl-(1—» 6)-galactose Sugar of alaternin=0-Rhamnosyl-(1—^ 3)-0-rhamnosyl-( 1—^6) galactose

Branched : Q 2 -Glucosylrutinose=0-JB-Glucosyl-(1—» 2)-0-((/-rhamnosyl- (1 —» 6) -glucose Q 3 -Glucosyl neohesperidose=0-B-Glucosyl-(1—» 3)-0-(-^-rhamnosyl-(l—» 2)-glucose

Flavone and Flavanone glycosides : In the case of flavones and flavonones in which there is no -OH group at position 3, the principal known glycosides have a sugar residue at position 7 (although a number of 5-glycosides are also known) eg. flavone glycoside, Luteolin- 41

-7-glucoside; (LXXIII) and flavanone glycoside, hesperidin (LXXIV), (-)-hesperelin 7-B rutinoside. Luteolin 7-glucos ide is known to be so wide spread in their occurence that their absence from a particular group of plant is often more significant than their presence.

CH2OH

(LKXIII)

OH 0 CH CH3

HO OH

HO 0

(LXXIV)

Several new flavone and flavanone glycoside have recently been isolated which are listed in table No. 3 and 4 respectively. O

C I—1 CNl fo —< 1—1 .—i .—1 U .—1 I—1 I—1 I—1 f—1 1—1 I—1 .—1 I—I i—i

<4-l

e QJ ^-N iJ 4-) CO ^-v ^~^ O —/ tn T3 o >-i QJ Pi C QJ QJ -~^ 5 CO O HJ r-( -"-N c fe B CO CO •cH ^^ QJ I—1 C hJ e;3 4-1 .—1 •H I-l cn CO B •r-l XI x: r-l 0 CO V 3 QJ X) CO o > 0 >-l QJ >-l C CO x: to CO to 0 to CO C >-i & >. r-t 1 QJ I—1 4-1 CO to J-l o I-l o U dc I-l 0 CJ TJ 0) •r^ -o XI •H •H QJ Q) en to D. 1 0 & •^H 3 ts] QJ to u g to CO u to 5-1 o to 0 CO 0) CO 0 •3H to •H o to d ft g to U •H iJ > xto cto 0 O O >^ c )-i S-i >^ 1 O 0 CO >, .—1 QJ :3 Oc QJ iH 1 CL bO -a ^ I-l I-l X) to 0 1 •H ^-N bO to •H B 1 Q to 1—1 1 E CO 1 vX) 1 O >^ Q 1 O QJ z 1 CQ- O 1 ^ O X) ^ QJ 1 tco U pa-, v^ :3 •H 1 c 0 ;-l to 1 1 I-l CO QJ 0 >% iH QJ bO 0 13 > a e:3 >> 13 1 C •H to o o C •H Q CO CO I-l •u o o CO 1 u Q) 0 M-( 1 o 1 I-l -o pa.. >, •T3 0 CO to Pi tjj O 1 (X •r^ 3 >. 1 I—1 1 Q 3 <3> 0 CO 1—1 X QJ to o 1 OJ rH 1 iH 0 bO 0 c W) 1 O X) bO r^ >, •H 1 rC 0 1 - 1 -H 1 1 X JQ Q 4-) > Q vO vC C O C 1 0 1 QJ to 1 ^ QJ w O 1 •iH Q •^^ pa. 6 r-l 1 -a 1 ^1 •^ I-l •u 1 U-( O -H O 13 1* O «-. C 0 - ?^ 1 CO 1 o 1 CJ o ^ 1 - o - 3 1 4J 0 bO r~^ X u-i 0 1 to bO 1 iJ in r^ C ?^ •r-l C c u C 1 C 1 •H X U •H •H •H >, •rH Q •H &• C PI 4J 0 4J m C C 1 I-l •rl •H QJ u o QcJ Oa CO CO . >% >^ CO >. •^ •H •rH :3 •H O •U 4: M u 0 X r- t-H a CUr-l ex. 1 ^1 1 x; •M 1 . <: < bO r- 00 CT^ rH (U u 43 yv ^ -'—V c(U CTi O o CM VH 1-H CN crsi CN CM CM Q) I—1 i—l I-H m ^^^ N-^ •—•

•"N ••*« •u •u •U o O o o o o o pd Pi CO ^-/^ ^-^ •H 0) U VJ CO Cd 0) CO o CO Cd I-l c •H u X .-1 o Cd cd cd I-H I-H 14-1 CO n3 3 O •H Cd OJ 1 •H m cC o o o X) o X3 CO XI C 1 I o cd CO m •H •I-l CO (1) 0) M p. o o « > o O ^1 !-i ^ o 4-3 CO (U 3 •P >- CO O O O iH tH U CO <: o PLI

I c: o o c I (X y^ O -H 0) Cd o , i-l :3 4-> u CO >> rH cd I o XI o CO bO >o c O •H Cd I 4-) X) •H X5 .-I r- >-H in C « to 1 •- •* cd vD O o t^ >-i . CO 1 c C ^ •-' 1-1 O o a. o rH QJ Q> •H XI :3 -ci o XI o O bO M V4 (U >^rH 4J 4-1 a 3 cu >> •H •H •U X) 0) O o x) ^ •U 4J I -rH •H I g Cd ex bO •H CJ •H •H - -CO XI Q •r4 rH CO I W •H )-l )-( . •H cr cr n s-i

o :z • • • • w i-H CM CO in vO 44

Flavonol and Flavanonol glycosides :

In the case of flavonol and flavanonol glycosides the sugar is usually attached at position 3 and the second sugar residue if present, usually occupies the position 7. Glycosy- lation at position 5 is extremely rare. Quercetin-3- rutinoside,Rutin (LXXV) a flavonol glycoside is known to be so wide spread in their occurence that their absence.

Rutinose

(LXXV)

Some new flavonol glycosides characterized from natural sources during last few years are presented in table 5. o C A5 in CX3 m o CM Csl u OJ CNl CM eg CM CO CO CM CO m cu

CO CO OJ CO •H CO QJ U CO > > QJ QJ •r^ CO CO 1-1 QJ CO N-^ QJ 0 M-l CO CO CO QJ •U •H •H CO CO CO I-H M .-I CO j-J CO .-I 5-1 0 QJ CO !-i CO o C4H > I •u •H SJ CO •u •H •r-l o ^. O QJ CO 4-J 04 x: CO O CM CO -o I •H e CO o CO o CO •u 0 4J •u CO 5 cu •H •rrl CO o c^ CO O o CO Xl (U •U o QJ •H CO CO CO >-l o Si CO en •H O CO u •H ;3 CO •H 0 0 o c M •H •u •H QJ C/3 pq O CO > C/J

I C •H I •P o O o QJ QJ >i QJ OJ QJ :3 Q) 13 0) T3 U •H CO CH •W •H QJ I bO-H W P. CO O CO :3 CO I CO I O O 6 o O cr O Q P CU •H O O I O CO :3 i-H :3 PQ_cO O bO i-H X I VH PI CJO o 0] r-( >^ 1—( CO !>> QJ •H O I >. CX >s bO X O O 4J CO O •H o u I QJ CO S ' O CO V^ I CO s C 4-> I o I >^ CO CO > ;3 o O o P-c QJ •H CO I CO 60 CJ O bO CO a (X C I o CO CO CO •H u CO u I I o ^3 QJ Q I U I o Q Q C Q o cr CO I I CO o I u I QJ I—t pa-. OJ Vi QJ o CO '^ t3 t^ I QJ c bO •H in ex a, o -a I CO I CN CM W o I •p C3 o O o •U CO e CO CO QJ CO I I I O I C CO o E CO CO CO CO o 1—1 I CO CO !-i CO o -^ O I C r^ O h >^ C o u •H CO I •M CX •H

CO CO

c CN eg on in en en CO CO CO CO CO CO QJ

0) Pi

CO en CO 0) CO 13 cu QJ i-l •H o B QJ u r-l o CO o CO N 14-1 •H •^^ •rl U 43 CO CO Xi CO 5-1 •H (U o 1-4 ?> I •H •H I CO CO CO I o C J3 Qi O CO I -^ •H CM CO 13 4-1 O 4-) I CM •H o O I CO O CO CO CO 5-4 H I-I I QJ o O CO & CO •H CO CO CO >- ;3 r-4 CO cu •H o U CO o •H p. O CO •I-I 13 •H u > X) CU CO a cO o 43 CO o QJ N P- CO •H I C/2 C/3 X) W C_3 •H IS) CO O C (U CO ^3 cu QJ 5-1 •H t3 I CU 13 >s W •H i-J 13 •H Cu O CO I 'I-I 1 CO O O o O I-I e (3 '^ O 1 a >> CO CO CO CO X U I CO 1 u I Vt I cu 0) >. Q I a I-I >, cu T3 (3 a •P o >~. (X X) -H O o C I PQ 13 o I I 13 (U O (3 CO O CO •I-I o .-I CU r-l 13 C 6 O C QJ I-I 43 I >^ 13 bO •H CO CO O -H 13 M-l CO i-i CO •H CO ^43 :3 4J •H U CO O O >>U r-l 13 CO >^ CO o O I 13 O. I b0 5-i O I O C PQ Xi CO O I I O o CO i •H U O o :3 X CO U o CO >^ iH 4-1 c2 CO I O a i-H CO (30 QJ ;3 O r- o M m •H 5-1 c c I u •^ o a 5-1 B B Q cu J-l •1-1 CO CM (U CO CO I Xi C 4-1 p. I I J3 43 43 PQ •H I 5-1 4-1 CO Q 4-1 I O 3) I o •rl o cr Q CO -o I (U •u I >^ PQ T) CO I CO 43 vO CO I 'H O 43 4-1 m CO 4-t I 1 QJ r^ cu CM O 0) O o B o 1 -o C X o I I I 1 >s •H I CO I I I r-- CX3 r^ X CO CO U CO CO o O >^ I C C 13 >-l O d a I c •H •iH •H 13 •i-i a 4-J o •1-4 4-1 4-J f^ 4-) •r^ •r-l Q) c 4-1 cu (U QJ X CJ) •U -i >-l >-l V^ ^1 in d CO 0) CU 0) QJ •. PC CO

o CM CO m VO C» CTi o CO i—l (N A7

A new flavanonol glycoside 3,7-dihydroxy flavan-4-One-

5-0-B-D-galactopyranosy (1-4)-B-D-glucopyranoside has been 124 isolated from the root of Andansonia digitata

Isoflavone, Chalcone, aurone glycosides :

In isoflavones usually the sugar residue is attached at position 7. In chalcone it is commonly at the corres ponding position which is numbered 4', and in aurones also the corresponding position which is numbered 6, is the one most often occupied.

Three new isoflavone glycosides viz, retusin 7-glucoside, irisolidone 7-rhamnoside and 5,7-dihydroxy-6-methoxy isoflavone 7-rhamnoside from the heart wood of pterocarpus 1 OQ marsupium and a new aurone glycoside viz Leptosidin-6-0-1 pyranosicfe from the leaves of cyperus scariosus 139 have recently been isolated.

C-glycosides C-glycosides are rare in distribution. Here the sugar residue is attached through it's carbon atom 1 with the carbon atom of an aglycone, usually a flavone, forming a C-C bond eg. vitexin viz. 8-C-glucosylapigenin, and orientin viz. 8-C-glucosyl-luteolin. STRUCTURE DETERMINATION 48

Isolation and identification of flavonoids : For the isolation of flavonoids, in general, rapidly dried and coarsely powdered plant material is extracted preferably first with nonpolar solvents followed by methanol, ethanol or water. Primary extraction with aqua distillata is preferred when it is to be followed by liquid-liquid extraction for instance with ethyl acetate or diethyl ether, before or after hydrolysis

Before chromatographic separation the plant extract is purified by various ways 140 like sequential solvent extraction with solvents of varying polarity 141 , precipi- tat ion with lead acetate , charcoal powder treatment , use of n-propanol/NaCl (wherein the aquous extract is mixed v/ith equal volume of n-propanol and than saturated with NaCl whence the propanol containing glycosides, is salted out which is separated, dried in a current of air and the residue is dissolved in non-aquous solvent like ethanol to remove small amount of NaCl (present) etc.

Sufficiently pure material so obtained, is subjected , ^ , . ^pl40,143 ppl43,144 p.rl^^ ^mrl^S to chromatographic CC .PC , GLC , HPLC separation.

Among the various methods used for structure determi nation of flavonoids such as colour reactions, degradation, synthesis and spectroscopy, the later, particularly, UV, IR, NMR and mass spectroscopies are of key importance being fast, handy and accurate. 49

(a) Ultraviolet spectroscopy The UV spectra of flavonoids have been thoroughly studied and reviewed by L-Jurd ' and T.J. Mabry Flavones and flavonols generally exhibit high intensity absorption in the 300-380rni region (Band I) and 240-270 ™i (Band II). The position and intensity of the max. of each of these varies with the relative resonance contri- but ion of the benzoyl (LXXVI,a) cinnainoyl (LXXVI,b) and pyrone ring (LXXVI,c) groupings to the total resonance of the flavone molecule.

-f-

(LXXVI.a) (LXXVI.b)

(LXXVI,c) 50

Although these groupings interact, the spectra of substituted flavones and flavonols in the neutral and alkaline solution suggest that Band I is associated chiefly with absorption in the cinnamoyl groupings

(LXXVIjb) and Band II with absorption in the benzoyl 149 (LXXVI.a) groupings

UV spectra of Aluminium complex-location of 5 and 3- hydroxyl grou^

5-Hydroxyflavone and 5-hydroxy flavonols in which the 3-hydroxyl is protected form yellow complexes of the type (LXXVII) which result in the considerable shift of Band I and II. In flavone this shift is of the order of 20-45 nm.

(IX(VII) 51

3-Hydroxy flavones readily form aluminium complexes which are stable even in presence of dilute hydrochloric acid. As a result of complex formation, flavonols produce a flavylium structure (LXXVIII) which is greatly stabilised by its quasiaroraatic character.

+ M+ 4-

-H^

-t.O-"-M

(LXXVIII)

The bathochromic shift of the flavonol Band I to the complex Band I is consistently in the order of 60 nm . A shift of this magnitude is, therefore, reliable evidence for the presence of a free 3-hydroxyl group. 52

UV Spectra in Alcoholic sodivun acetate- location of 7,3 or 4'-hydroxyl s^^oup

Sodium acetate is sufficiently basic to ionize hydroxyl groups located at position 7,3 and 4' of the flavone nucleus. Hydroxyls at other position are unaffected. Ionization of 3 and A' iiydroxyls produces bathochromic shifts of Band I, but does not affect the position in A ring ionization of a 7-OH group results in a pronounced bathochromic shift of this band. Flavones and flavonols which contain a free 7-hydroxyl group, may, therefore, be detected by the 8-20 nm bathochromic shift of the low wavelength band on the addition of a little fused sodium acetate 149

UV spectra in sodium-ethoxide-detection of a 3,4'-Di-hydroxyl grouping in flavonols Jurd and Horowitz 149 found that flavonols in which the hydroxyl group at either C-3 or C-4 is protected by methylation or glycosidation are stable in sodium ethoxide and that their stability is not appreciably influenced by other hydroxyl groups i.e., the long wavelength band shifts from 340-380 nm is ethanol to 380-420 in sodium- ethoxide .

UV spectra in sodium acetate and boric acid- detection of 0-dihydroxyl groups in flavonols

Boric acid, in presence of sodium acetate forms chelates with phenolic compound containing 0-dihydroxyl 53

groups. Thus the max. of Band I in luteolin undergoes bathochromic shift of 15-30 nm on addition of a mixture of boric acid and sodium acetate . The spectra of compounds which do not contain and 0-dihydroxyl groups are not appreciably affected .(LXXtX).

Boric Acid NaoAc

(LXXIX)

UV spectra in AlCl^ and HCl

Flavones and flavonols containing 5 or 3 hydroxy1 groups form aluminium complexes by chelation of AlCl^ with 0-dihydroxyl group. The aluminium complex (A) is not stable in presence of HCl, while aluminium complexes of flavones (B and C) having 3 or 5 hydroxyl groups formed by the chelation of A1C1„ with keto 3 or 5 hydroxyl group of flavones, are stable in presence of dilute HCl. Hence, when shift is taken with AlCl^ only, it results in a 54 bathochromic shift but when two or three drops of HCl are added in the sample a hypochromic shift is observed which indicates the presence of 0-dihydroxyl group along with hydroxyl groups at C-5 or C-3 position of flavone nucleus.(LXXX), (LXXXI), (LXXXII).

0— M

(LXXXI) 55

0—M 0 M

M = Al HCl

(LXXXII) 56

(b) Infra-red spectroscopy : -I/O IRO IQ'3 Infra red spectroscopy of flavonoids ' ' plays a great role in distinguishing the closely related compounds and comparing the sample spectra with the reference spectra for the purpose of differentiation/identification.

Some observations drawn from IR spectra of flavonoids are as follows :

a) The free OH groups are responsible for the absorption in the region of 3,300 cm . When they are hydrogen bonded ie are in position 3 or 5, the frequency of this band is lowered to 3100 cm"-^.

b) Absorption due to CH- group occurs at 2,930 cm It is often obscured by the absorption due to OH groups (a).

c) Absorption due to CO group of the heterocycle occures at 1,685 cm in case of flavanones where the carbonyl group is conjugated with the A ring. This is lowered to 2,65 0 cm in the case if 5-hydroxy flavanones. In the case of flavones, the carbonyl group is conjuga ted with both A and B rings. The absorption due to CO group in 5,7-dihydroxyflavone is at 1,655 cm , and in 7-hydroxyflavones at 1,63 0 cm . In the case of flavonol, the 57

carbonyl group absorption is lowered about 30 cm" as compared with that of homologous flavone.

d) Aromatic double bonds absorb between 1,500 and 1,610 cm , a second peak is present at 1,583 cm when the double=bond is conjugated with a benzene ring.

e) Phenolic hydroxyl groups have a strong absorp tion at about 1,3 60 cm , with a secondary peak at 1,200 cm

f) Due to me ta-hydroxy substitution ( 5 , 7-dihydroxy) a peak at 1,165 cm is found.

g) It is difficult to interprete the spectrum below 1,000 cm . However at 800 cm , absorption due to hydrogen atoms of benzene rings may be observed.

h) A comparison of the infra red spectra of 5 hydroxy flavones and 5-hydroxychromones with the spectra of their 5-0-alkyl and 5-0 -acyl derivatives show a shift in the opposite direction, that is to lower frequencies. In practice this is very useful in diagnosing the presence of 5-hydroxyflavone structure.

(c) Nuclear magnetic resonance spectroscopy The application of nmr spectrscopy has proved to be much diagnostic in structure determination of flavonoids. 58

By the use of nmr studies of silyl derivatives , double irradiation technique ' , solvent induced shift studies"^^^^' 157,158^ lanthanide induced shift studies"^^^, ^^ ^160 , 13^ ^ 161 nuclear overhauser effect , and C-nmr spectroscopy the structure may easily be established.

DMSO-d^ is by far the most generally used solvent

for underivatized flavonoids though CCl,, CDCl^, benzene or C^D,, pyridine, D^O or a mixture of these are also ,158 used' The use of TMS ether derivative of flavonoids with CCl, as solvent has achieved widespread importance 162

The most commonly occuring hydroxylation pattern in natural flavanoids is 4', 5,7-trihydroxy system (LXXXIII).

The chemical shifts of the protons of ring A and ring B

prove to be independent of each other but are affected by

the nature of ring C.

(LXXXIII)

RING A The two ring-A protons of flavanoids with 5,7- hydroxylation pattern give rise to two doublet (J=2.5H ) 59 between "r 3.3-4.0 from tetramethyl-silane. There are however small but predictable variation in the chemical shifts 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 peak near T 4.05, with the addition of a 3-hydroxyl group (flavanonols) the chemical shifts of these protons are slightly altered and the pattern changes to a very 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 All B-ring protons appear around ^ 2.3-3.3 a region separate from the usual A-ring protons. The signals from the aromatic protons of an unsubstituted B-ring in a flavanone appear as a broad peak centred at about ^2.55. In flavones, the presence of C-ring double bond causes a shift of the 2',6' protons and the spectrum shows two broad peaks one centred at T 2.00 (2',6') and the other at T 2.4 (3' ,4' .S')"^^"^.

With the introduction of a 4'-hydroxyl group the B-ring protons appear effectively as a four peak pattern called as Ar,B^ 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 their peaks move substantially upfield. The 2,6' pro tons of flavanones give signals centred at about ^2.65. 60

RING C Considerable variations are generally found for the chemical shifts of the C-ring protons among the several flavonoid classes. For example, the C3 proton in flavones gives a shart singlet near'TTS.?. The C„ proton of isoflavones is normally observed at aboutT2.3 while the C„ proton in flavanones is split by C^ protons into a doublet of doublet (J . =5H , J. =11H ^ and occurs CIS z trans z; near ^4.8. The two C„ protons occur as two quarters

H3a-3o o,=17b H z ) near T 7.0. However theyJ often appeaI r r as two doublets since two signals of each quartet are of low intensity. The C^ proton is dihydroflavonols appears near T 5.1 as a doublet (J=ll H ) coupled to the C-3 proton which comes at about 5.8 1 ft"? as doublets.

Benzens induced shifts When the nmr spectra are measured first in CDC1„ and 1 to then in benzene it has been observed that the size of benzene-induced shift (^^) of certain methoxyl signals is indicative of the position of methoxyl group on the flavone nucleus as presented in table-6.

TABLE -6 values ( CDC1-- ^^^A p.p.ni-)(in absence of orthosubstitution

Methoxyl at C-3 -0.07 to +0.34 ppm C-5 + 0.A3 to +0.58 ppm C-7 + 0.54 to +0.76 ppm C-2' +0.46 to +0.53 ppm C-A' + 0.54 to +0.71 ppm 61

16A It has further been observed that by the addition of trifluoroacetic acid (T.F.A.) to benzene increases the association of benzene with more basic methoxyl groups eg.

the very small benzene induced shifts of the methoxyls given below were observed as :

Shifts ( SC H, - ^C^H^ + TFA) of + 0.18 to + 0.34 ppm in the signals of C-5, C-7, C-2' and C-4' methoxyl groups flanked by two oxygen substituents; + 0-26 ppm for an 8- methoxyl in the presence of a methoxyl at C-7 and +0.34 ppm for a 5-metho^l flanked by a methoxyl at C-6.

The problem of solubility of naturally occuring flavonoids (when not methylated) in solvents has largely been 15 8 overcome by the use of trimethylsilyl ethers (TMS-ethers)

Using these derivatives, benzene induced shifts of both methoxyl and the TMS-ether signals may be observed giving

simultaneous information about methoxyl and hydroxyl 158 substitution in the flavonoid as has been shown in Table-7

TABLE lvalues ( SCCL,- SC,D^) for methoxyl signals in the

spectra of flavone TMS-ether derivatives

Methoxyl at C-3;4' 2',7 + 0.35 to + 0..7 0 ppm C-3,6,8 0.00 to + 0. .18 ppm

TMS-ether at C-5 0.14 to - 0..2 0 ppm C-3.6,7,8, 2,3',4' - 0.05 to + 0. .12 ppm Glycosyl 0.00 to + 0..1 0 ppm 62

Benzene induced shift data have extensively been used for the structure determination ' ~ of flavones, flavans, flavonols and biflavonoids. d) Mass spectrometry

Instead of attempting a panzer of chemical reactions to conclude a structure it is much convivial that even the elution of a single spot from TLC followed by microderivat- ization (and removal of excess of reagent) yields a residue suitable for mass spectrometry.

There are much data on electron impact mass spectrometry of flavonoids and their fragmentation . The mass spectral fragmentations follow known pathways. Routine methods have been developed for the analysis of flavonoid glycosides and aglycones in the form of their permethyl and perdeuteromethyl ^, 168-171 „. , , , ^. 172 ^. 1 . . . ^. 173 ethers . Field-desorption , field-ionisation , chemical ionisation mass spectrometry and FDMS-pyrolysis

(pyrolysis of compounds directly on the emitter surface in mass spectrometer) have proven to be supplemental structural tools in the study.

All the flavonoids are derivatives of 2-phenylchroman

(LXXXIV).

(LXXXIV) 63 and are divided into different classes due to the differential level of oxidation of central heterocycle ring (ring C). This C-ring is the weak link in C,c chain and it is this ring which fragments first under the electron impact in mass spectrometry. In general, all the flavonoids are capable of undergoing RDA fission with and without hydrogen transfer (ie. RDA + H and

A) Conjugated flavonoids In conjugated flavonoids ring A, B and C are conjugated with one another. They include flavones, isoflavones, flavonols, isoflavonols,, flavylium compounds and flavenes.

Flavones and flavone glycosides: Flavones are fully conjugated and do not posses sites for facile bond rupture. This is evidenced by the strong molecular ion peak of the simple members of this class. 177 + 'Flavone (LXXXV) itself has the M as the base peak and undergoes RDA fission to give ions at m/e 120 and m/e 102 respectively (scheme 1). The ion (LXXXV,a) which originate from ring A is far more abundant than ion (LXXXV,b) originating from ring B. The other fragments of importance are ion (LXXXV,c) at m/e 194 which arises from the loss of CO from the molecular ion; and ion (LXXXV, d) at m/e 92 arising from the loss of CO from RDA ion (LXXXV,a).

In oxygenated flavones, conversely, the B ring fragment may have as high abundance as 65%. Flavones having a 64 methoxyl or hydroxyl group at C-6 position shows an "atypical" 179 behaviour differing from normal RDA fission. Generally these compounds have a pronounced molecular ion which ejected radicals (H,CH- or Et) from several of the oxygen substituents to form stable cations eg. for a typical quercetagetin derivative (LXXXVI) the fragmentation given in scheme-2 ensues. Quite often these cations constitute the base peak. 18 0 In glycosides in general (0-glycosides the base peak correspond to the basic flavone structure after the thermal loss of sugar unit. SCHEME - 1

-h

0 CH (a) m/e 120(80.0) (b) m/e 102 (12.0) (LXXXV) M 222(100.0) C^rO •CO .-^^^

SCHEME - 2

OMe

OH 0 (LXXXCI) M 388 (100.0) CO m/e 373(85.0) m/e 345 (15.0) 65

Isoflavones : These compounds also undergo RDA fission to give the peaks corresponding to ions (LXXXVIIa) and (LXXXII,b). The intensity of these peaks is strongly influenced by substitu tion pattern of the aromatic rings. In contract with the flavones, the isoflavones show (i) only a weak (M-28) peak and (ii) have very prominent doubly charged molecule eg. 181 fragmentation of 5,7-dihydroxyisoflavone (LXXXVII) shown TOO 1QT in scheme 3. Certain workers ' observed (M-1) as being the base peak in isoflavone and 7-methoxyisoflavone.

-CO

M 254(95.0) m/e 226 (LXXXVII) Very Weak

RDA

CH 111 -V C M m/e 127 (10.0)

(b) m/e 102 (10.0) (a) m/e 152 (61.0)

m/e 124 (17.0)

SCHEME - 3 66

Flavonols : Flavonols being flavones having C-3-0H group show analogous fragmentation pattern. Subsequent fragmentation results in certain variation eg. fragmentation of quercetin 181 (LXXXVIII) 5,7, 3',4'-tetrahydroxylflavonol, has been shown in scheme-4. Here the first step is thought to involve rearrangement to the diketo structure, which then undergoes (RDA+H) fragmentation to yield peak (LXXXVIIIa) at m/e 153. The ion (LXXXVIII,b) at m/e 137 emanating from ring B is not found in flavones.

OH 0 OH 0 (LXXXVIII) M"^302 (100.0) RDA+H

,,^;^-V-OH

HO N^^^'N^^ M

OH 0 OH 0 m/e 153 (14.0)

SCHEME -4 m/e 137 (17.0) 67

Compounds bearing mechoxyl substituent in the C-3 position with an alkoxy group at C-6 e.g. triethyl oxyayanin B (LXXXIX) show an intense peak at (M-alkyl). If the alkoxy group is replaced by an- OH group eg. oxyayanin B (XC) an abundant (M-1) mass peak (66%) is found 179

A methoxyl group is position C-3 shows a= characteristic mode of breakdown by the concerted loss of an acetyl rad ical"^ ''^' •'•^^ (Me + CO = 43) from the molecular ion eg. (XC.a) in the scheme-5.

OMe

MeO

EtO EtO (LXXXIX)

MeO

(XC)

SCHEME - 5 68

Considering the mass spectra of a number of compounds following conclusions may be drawn.

a) (M-OH) : Loss of 17 mass units occurs for 2'-OH group. b) (M-H): in flavonols which contain free - OH group at C-3 or C-6 position, the loss of hydrogen from molecular ion occurs forming an stable quinonoid ion. The same loss occurs in flavone methyl ethers which have an -OMe group at either C-3 or C-5 positions. If a C-6 or C-8 methoxy group is also present (M-CH„) peak completely overshadows the (M-1) peak.

c) (M-OCHo): Loss of 43 mass units has been observed in 6- or 8-methoxyflavones. With regard to the loss of COCHo it can be concluded that an intense (M-43) peak is diagnostic for 3,6, or 8 substituted flavones (weak peak, inconclusive).

d) (Aroyl cation) : This fragment ion is formed from ring B to which is attached a keto group from ring C. The possible mass numbers at which this may occur are at M/e 105,135,165 or 195 from B ring containing 0,1,2 St 3 methoxyl groups respectively. The substi tution in B ring may thus be deduced.

For every acetyl group loss of aketene, COCH^ (42 mass units) from the pehnolic actoxyls occurs. This provides a simple method for estimating the number of hydroxyl groups and differentiating between aromatic and aliphatic hydroxyls. The loss of CH-CO (43 mass units) is generally associated 69

with an aliphatic acetyl derivative.

e) (M-CH^) : 19 m.u. loss occurs only in flavones having methoxyl groups in both the C-3 and C-5 position.

B) Reduced Flavonoids In reduced flavonoids heterocyclic ring C is no more aromatic and hence the -OH group attached hereto are also typically aliphatic. The loss of aromaticity yields a ring which is more susceptible to fragmentation by electron bombordment therefore RDA and (RDA+H) fissions assume more important propertions and the intensity of molecular ion peak is greatly reduced.

Flavanones : In flavanones eg. 5,7-dihydroxy flavanone (XCI) the major decomposition mode is RDA fission to give ions^*' (XCIa) and XCIb) at m/e 152 and m/e 104 respectively. Most of the charge is retained by the more highly hydroxylated ring A. Ion (XCIa) may further lose CO to give peak (XCI,c) at m/e 12A.

A further characteristic fragmentation is the fission of the bond to the heterocycle oxygen possibly because of nonaromatic ring C. Loss of H yields the peak (XCI,d) at m/e (M-1) and the loss of phenyl radical gives ion (XCI e) at m/e 179. This peak at m/e 179 is of high intensity. This fragmentation has been shown in scheme-6.

In 4' methoxyflavanone (RDA+H) peak is more pronounced and nearly entire charge of RDA fission resides with B ring 70

which bears the methoxyl group. When both A & B rings bear the methoxyl group (eg. in 5-hydroxy-7,4' dimethoxyflavanone) 1 Q C the charge again resides almost entirely with the B ring

The ion (XCI,b) increases with increasing substitution in ring B.

In acetylflavanones, ion (M-acetyl) fragments analogous to 5,7-dihydroxy flavanone (XCI, scheme - 6) , after the initial loss of acetyl group.

In 2' hydroxy flavanones, along with the normal frag mentation pattern, a base peak at (M-18) and the third 186 largest peak at (M-19) is observed

The flavanones show a notable lack of a fragment aris ing from the heterocyclic ring prior to the fission.

3-Hydroxyflavanones (dihydroflavonols) : In this class the character of heterocyclic ring is somewhere between a flavanone and a flavonol. Therefore the mass spectrum differs from both in many respects. The two basic modes of fragmentation of dihydrokaempferol trimethylether (XCII), the most common member of the class, are shown in scheme-7.

In RDA and RDA+H fissions, the presence of a hydroxyl groups in the parent compound makes possible the loss of a formyl radical (CHO) from ion (XCII, b) to give ion (XCII.c ) at m/e 121.

The second mode yields ion (XCII,a) at m/e 301 through loss of CHO from the parent ion. Ion (XCII, d) then gives 71 ion (XCII.e) at m/e 193 through ejection of anisole.

For (RDA + H) fragmentation to give peak ion (XCII.a) at m/e 181 the hydrogen atom involved is probably transferred 187 from C-3 hydroxyl group as the compounds acetylated in this position do not give an (RDA+H) peak at m/e 181.

Another important mode of breakdown for most of the dihydroflavonols is the loss of 29 mass unit from the molecular ion peak (ion (XCII,d) in scheme 7).

1 8R It has been suggested that this key fragment occurs by the loss of (R-CO) radical where R is the substituent other than hydroxyl, at C-3 eg. (XCIII, scheme 8).

..^^^

(XCl)M 256(100)

(a) m/e 152(47) (b) m/e 124(25) CX ^. OH 0 I-co

.OH

m/e 124(25) m/e 153(16) 72

r^^ OH OH H (d) m/e 255 (70.0) HO- •0'

OH 0 -6^. HO (XCI) M'''256 (100.0)

HO OH (e) m/e 179 (52.0)

SCHEME - 6

MeO Me

HG^ (b) ra/e 150 MeO OH (34.0) (XCII) M,+ 330(3.) (a) m/el8l (100.0)

OMe /v^OMe -CHO

HoC

.OMe OMe

MeO

(XCll) M 330(3.0)

MeO

OMe

m/e 193(16.0)

SCHEME - 7

0-H

(XCIII) M''"(29.0) m /e 211 (100.0)

SCHEME - 8 DISCUSSION Ik

CORONOPUS DIDYMUS

STUDY OF THE WHOLE PLANT OF CORONOPUS DIDYMUS (N.O. CRUCIFERAE)

Coronopus didymus is commonly known as 'Jangli hala' or 'panacholi'. It is a prostrate or an ascending, branching leafy, rather hispid herb often forming a rosette. Leaves pinnatifid or pinnati-partite; lobes spreading. Flowers pale green, small, sometimes apetalous, diandrous, pods 1x2 mm; separating into 2; indeshiscent, reticulate lobes. Seeds about 1x1 mm, brown.

It is a winter season weed, highly variable in its size and appearance, found in fields and open places on moist sandy soil. It is abundantly available in winter and is rare in summer and early rainy season. The plants that survive till early summer become somewhat woody 189

A survey of literature showed that its seeds contain 190 191 volatile isothiocyanate ' (benzyl isothiocyanate, 192 benzyl cyanide), fatty acids (Palmitic acid, oleic acid, linoleic, linolenic acids). Leaves on preliminary exam ination have been reported to give positive tests for 194 flavonoids and alkaloids only . However, no systematic and detailed work seems to have been done on these consti tuents and hence the present studies were undertaken

The plant material was collected from the I.H.M.M.R. campus and dried under shade. The dried material was exhaustively extracted with 95% ethanol. Recovery of solvent left a greenish viscous mass, which was treated with 75 petroleum ether (60-80°C) and CHC1„ inorder to remove petrol and chloroform soluble products. The residue was extracted five times with solvent ether. All the ethereal extracts were combined together. Recovery of ether left a brown semisolid mass which gave a pink colour with Mg-HCl and a green colour with ferric chloride and an intense yellow colour on exposure to ammonia vapours. All these tests indicated the presence of flavonoids. The brown semi solid mass on crystallization with methanol gave a yellow crysta lline compound m.p. "> 330, which on T.L.C. examination gave a single brown spot under U.V. light. On acetylation with pyridine and acetic anhydride it gave a colourless acetyl derivative m.p. 220-21°C.

The U.V. Spectrum of the parent compound m.p. > 330°C showed two maxima at 268 nm (band II) and 344 nm (band I) with an inflexion around 295 nm. Addition of sodium acetate shifted band II to 276 nm, thus suggesting free 7-OH group. In NaOAc/HoBO„, the spectriim reverted to the parent compound spectrum in having the maxima at 269 and 347 nm, thus suggesting the absence of any catecholic group.

Addition of A1C1„ replaced band I with a double maxima at 360 and 388 nm, thus suggesting the presence of a free 5-OH group. Addition of HCl to AlCl- containing solution did not produce any drastic change which also supported the absence of catechol system. The above data led to the con clusion that the compound is a 5,7 - dihydroxy flavonoid which does not have free catechol unit in the side phenyl. 76

However, the possibility of protected catecholic system is not ruled out.

The N.M.R. Spectrum of the above acetate showed a

singlet for two acetoxyls at S 2.5 and another singlet for

the third acetoxyl at%2.6. There was a methoxyl singlet at^A.O. In the aromatic region the H-3 appeared as a singlet at ^6.7 and H-6 appeared as a doublet (J=2H ) at

^6.95. The remaining protons namely H-8, H-2', 6',5' all

appeared as a multiplet between S7.3- $>7.6. Thus the compound appears to be a monomethyl ether of tetrahydroxy flavonoid. The presence of H-6 appearing as a meta coupled doublet indicated that the compound carries 5,7 - dioxy-

genation. The chemical shifts of the H-6 and H-8 are more

closely resembling 5-7 - diacetoxy flavonoids rather thafl

5- acetoxy, 7- methoxy systems. Hence it appears that

the methoxyl group is present in the side phenyl. The probable structure could be luteolin 3'-methyl ether

(Chrysoeriol) or 4' methyl ether (Diosmetin).

A comparison of physical data of chrysoeriol (5,7,4'-

trihydroxy 3'-methoxy flavone) with this compound confirmed

it to be chrysoeriol which has the following structure.

OCH-

CHRYSOERIOL 77

A further study to the above findings was obtained by the mass spectral studies (Chart I). In the mass spectrum, the molecular ion peak was observed at m/e 300 suggesting the mol formula C, ^H-,20^- Loss of CO from the molecular ion peak typical of flavonoids was observed in this case leading to a peak at m/e 27 2. The RDA fragmentation gave two peaks at m/e 152 originating from ring A and at m/e 148 originating from ring B. These suggest that the compound must be carrying two hydroxyls in ring A and one hydroxyl and one methoxyl in ring B. Consistent with these inferences the substituted benzoyl ion originating from ring B was observed at m/e 151. These studies also led to the conclusion that this compound is chrysoeriol.

CHRYSOERIOL

m/e 148

m/e 152 DODONAEA VISCOSA

STUDY OF THE FLOWERS OF DODONAEA VISCOSA FAMILY SAPINOACEAE

Dodonaea Viscosa is commonly known as 'Aliar'. It is considered to be an important drug in the indigenous system of treatment 195 and its leaves are used as febrifuge and sudorific and have been used for the treatment of gout, rheumatism, healing of wounds, burns and swellings. The bark of the plant is used in astringent baths and fomentat ions. The whole plant has been mentioned to have fish poison properties 196 . Its leaves have recently been found to possess hypoglycemic activity

A survey of the literature showed that its leaves, flowers bark and seeds have been chemically examined by 1 197-204 various workers

As this plant is quite interesting and of value in indigenous medicine, it was considered worthwhile to re-examine this plant more systematically. And in an effort in this direction, a re-examination of its flowers was undertaken.

In the present study the fresh flowers of p_^ Viscosa were collected which is grown as a hedge in the I.H.M.M.R. campus. The flowers were extracted with ethanol (95%). Recovery of the solvent left a brown syrupy mass. Which was extracted with petroleum ether (60-80°C), chloroform, acetone and MeOH, successively. The acetone fraction gave a pink colour with Mg/HCl indicating the presence of 79

flavonoids. This fraction was dissolved in water and filtered, The filtrate was subjected to acid hydrolysis. A blackish solid separated out, which was filtered washed with water and dried. It was then exhaustively extracted with ether. The ethereal concentrate was subjected to column chromatography on silica gel-G. The fraction eluted with Benzene and ethyl acetate mixture (4:1) on T.L.C. examination showed two brown spots (Rf 0.61 and 0.63) under U.V. light. This mixture was separated by preparative T.L.C. on silica gel-G plates into two pure fraction. The pure compounds so separated were labelled as 'A' (m.p. 305-306°C)and 'B' (m.p. 313-314°C). The compound 'A' on acetylation with pyridine/acetic anhy dride gave an acetyl derivative m.p. 202-203°C.

• * Characterisation of Compound A (m.p. 305-306°C) The compound 'A' was obtained as yellow solid, m.p. 305-306°C. It gave a positive ferric colour reaction and a pink colour with Mg/HCl characteristic of flavonoids.

The UV spectrum of the compound showed two maxima at 255.2 ran (Band II) and 37 3.2 nm (Band I) with two inflexions at 267.38 nm and 326.75 nm. Addition of sodium acetate shifted Band II to 274.5 nm thus suggesting free 7-OH group. In NaOAc/H«BO^ the spectrum shifted to the parent compound spectrum in having the maxima at 254.4 nm and 371.2 nm, thus suggesting the absence of any catecholic group.

Addition of A1C1„ shifted band I to 429 nm. This suggested the presence of a free hydroxyl group at the 5 80 position. The location of band I at 373.2 nm suggested that there is an -OH group at 3 position also. Addition of HCl to AlCl^ containing solution did not produce any drastic change which also supported our findings.

The above data suggested hydroxyl groups at 3,5,7 and 4' positions. The position of band I at 373.2 nm is also indicative of the presence of 3 and 4' -hydroxyl group.

Comparison of the spectral data with those of kaempferol and isorhamnetin which have the above hydroxylation pattern suggested that the present compound is not kaempferol but may be isorhamnetin.

Further, proof for its identity as isorhamnetin was obtained by N.M.R. spectral studies. The N.M.R. spectrum of the compound showed one methoxyl group as a singlet at ^3.80 and two meta coupled 1- proton doublets at <^6.2 and 86.3 for H-6 and H-8 of the A ring. Further downfield there was a doublet for 1-proton at ^7.0 (J = 9H ) a two proton multiplet centred atS7.75. These can be assigned to H-5' and H-2', 6' respectively. These data are consistent with the compound being monomethyl ether of quercetin i.e. isorhamnetin.

A further support to this compound, being isorhamnetin, was obtained by mass spectral studies. The mass spectrum (Chart II) showed the molecular ion peak at m/e 316 consis tent with the molecular formula C, ^H-, 2O-7. There was a peak 81 at m/e 288 for a loss of CO from the molecular ion as is normally observed in the mass spectra of flavonoids. There was also a peak of low abundance corresponding to M-15 arising by loss of a methyl possibly from methoxyl group. A T.L.C. comparison of the compound with an authentic sample of isorhamnetin 205 established its identify as isorhamnetin.

14-

ISORHAMNETIN

m/e 273

CHART Characterisation of compound 'B' (m.p. 313-314°C) The compound 'B' also gave a positive ferric colour reaction and pink colour with Mg/HCl characteristic of the flavonoids. 82

The U.V. spectrum showed two maxima at 256 nm (Band II) and 374.2 nm (Band I) with an inflexion at 325 nm. By adding sodium acetate, band II shifted to 272.5 nm, thus suggesting the presence of a hydroxyl group at 7-position. In NaOAc/H«BO« the band I showed a bathochromic shift of 12 nm, suggesting the presence of a catecholic unit in the side phenyl.

Addition of A1C1„ shifted band I to 446.2 nm. This suggested the presence of an -OH group at 5-position. The location of band I at 374.2 nm suggested the presence of an OH group at 3-position also. Addition of HCl to A1C1« brought the long-wave length maxima (Band I) to 42 4 nm. This is known in the case of catecholic systems in which the corres ponding boric acid complex breaks down with HCl. Thus the compound appears to contain hydroxyls at 3,5,7,3", 4' systems. Comparison with the spectral data and the shape of the spectra with those of quercetin led to the conclusion that the compound is quercetin. Further proof for its identity as quercetin was obtained by mass spectral analysis.

In the mass spectrum it showed the following RDA fragmentation pattern which was characteristic for quercetin (chart-Ill). Mass ion peak was at M 302 and the peak at m/e 274 was for ion (a) the peak at m/e 152 was for ion (b) and the peak at m/e 137 was for ion (c). These data accounted for the structure of the compound as quercetin. 83

QUERCETIN

HO 0

M"*" m/e 302

-CO

-V OH

HO. OH

OH r 0 0+

(a) m/e 274 (b) m/e 152 (c) m/e 137

CHART - III EXPERIMENTAL CORONOPUS DIDYMUS

STUDY OF THE WHOLE PLANT OF CORONOPUS DIDYMUS (N.O. CRUCIFERAE)

Shade dried whole plant material (1 Kg) in coarsely powdered form was exhaustively extracted with ethanol (95%). Recovery of solvent left a viscous greenish mass (150 gms), which was treat ed successively with petroleum ether (60-80°C) and CHCl^ under boiling conditions in order to remove petrol and chloroform soluble products. The petrol and chloroform insoluble residue (80 gms) was extracted with ether five times. All the ethereal extracts were combined together. Recovery of ether left a brown semi solid proudct (10 gms) which gave a pink colour with Mg/HCl, a green colour with ferric chloride and an intense yellow colour on exposure to ammonia vapours. On several crystallisations with methanol it gave a yellow microfine crystalline compound m.p. "^ 330°C. On T.L.C. examination it showed a single brown spot under U.V. light.

ACETYLATION : The above compound (100 mg) was dissolved in pyridine (3 ml) and acetic anhydride (1.5 ml) was added to it. The contents were left overnight at room temperature and then poured dropwise into cold water with constant stirring. A precipitate separated out, which was filtered, washed with a large quantity of water in order to remove acetic acid and pyridine and dried. On crystallization from methanol it gave a colourless compound (75 mg.) m.p. 220-21°C. It was T.L.C. pure in T:E:F and B:P:F solvent system. 85

SPECTRAL DATA

UV (MeOH)

MeOH 268, 295 (inf.) , 344 nm max

NaOAc -7o, 29S (inr.) 344 nm max

NaOAc/Boric Acid 269, 295 (inf. ) , 347 nm max

AlCl^ 268, 295 (inf.), 360, 388 nm, max

vAlCl^/HCl 268, 295 (inf.) , 360, 388 nm, max

NMR (CDClj) <^ 2.5(s), 2.6(s), 4.0(s), 6.7(s) , 6 . 95 (d^ J = 2H^ ) 7.3 - 7.6 (m) MASS (m/e) 300 (M '*') 111, 152, 151, 148, 124

On the basis of its physical constants it was identified as chrysoeriol. 86

DODONAEA VISCOSA

STUDY OF THE FLOWERS OF DODONAEA VISCOSA FAMILY SAPINOACEAE

EXTRACTION The fresh flowers of Dodonaea Viscosa (900 gms) were collected from I.H.M.M.R. campus and exhaustively extracted with ethanol (95%) several times. All the ethanolic extracts were combined together and the solvent recovered under reduced pressure. The concentrate (65 gms) was successively extracted with petroleum ether (60-80°C), chloroform, acetone and metha nol. The acetone extract gave a pink colour with Mg/HCl indicating the presence of flavonoids. Acetone was distilled off and the residue left was taken up in water. It was filt ered to give-water soluble and water insoluble fraction.

WATER SOLUBLE FRACTION The filtrate was subjected to acid hydrolysis with 4% v/v con. H„SO,. A blackish precipitate (3.0 gm) was obtained which was filtered washed with water several times till free from acid and dried. It was exhaustively extracted with ether. Recovery of ether gave a residue consisting of crude aglycone (1.5 gm).

ISOLATION OF PURE AGLYCONES The above crude aglycones(1.5 gms) was subjected to column chromatography on silica gel-G and the column was eluted with benzene - ethyl acetate mixture (4:1) 5 00 ml. It was evaporated to dryness to give a residue (800 mg) and examined on T.L.C. silica gel-G plates in T:E.F.' solvent system. Under U.V, light, it showed two brown spots (Rf. 0.61 and 0.63). This mixture was separated by preparative T.L.C. on silica gel-G plates using T.E.F. as the solvent system. About one hundred silica gel-G plates (size 20 cm x 20 cm) were run. The two bands were carefully separated from plates, and eluted with methanol. Methanol was recovered and the two fractions obtained in this way were crystallised from methanol to give two different compounds m.p. 305-306°C (30 mg) marked as 'A' and m.p. 313-14°C (15 mg) marked as 'B'. The compound 'A' was identified on the basis of U.V. NMR and mass spectral data as isorhamnetin. Its final identity was confirmed through m.m.p. and CO T.L.C. alongwith an authentic sample of isorhamnetin.

ACETYLATION OF 'Ac! The compound 'A; (25 mg) was dissolved in pyridine 0.5 ml and acetic anhydride (0.3 ml) was added to it. The contents were left over night and then poured drop wise into ice cold water with constant stirring, when a precipitate was obtained. It was filtered washed with a large quantity of water in order to remove acetic acid and pyridine and dried. On crystallization from methanol it gave a colourless crystalline compound (17 mg) m.p. 202-203.

SPECTRAL DATA UV (MeOH)

MeOH 255.2, 267.38 (inf.),

max 326.75(inf.) 373.2 nm.

NaOAc 274.5, 326.75 (Inf.) 373.2 nm max NaOAc/Boric Acid 254.4, 267.38(inf.) 326.75(inf.), 371.2 run. max

AlCl^ 255.2, 267.38(inf.), 326.75(inf.) max 429 nm

AlCl^/HCl 255.2, 267.38(inf.). 326.75(inf.) max 429 nm.

NMR (CDCl^)

"^3.80(S), 6.2(d), 6.3(d), 7.0(d, J = 9H^) , 7.75(m)

MASS m/e), 316 (M"*"), 288, 273

IDENTIFICATION OF 'B The compound 'B' was identified on the basis of U.V. and mass spectral data as quercetin. Further identity was established by m.m.p and Co T.L.C. alongwith an authentic sample of quercetin.

Due to paucity of material its acetyl derivative could not be prepared.

SPECTRAL DATA U.V.(MeOH) 89

MeOH 256, 325(inf.), 374.2 nm max

NaOAc

272.5, 325(inf.), 374.2 nm max

NaOAc/Boric Acid 256, 325 (inf.), 386.2 nm max

AlCl^ 256, 325(inf.) 446.2 nm max

AlCl^/HCl 256, 325 (inf.) 424 nm max

MASS

(m/e) 302 (M"*") , 274, 152, 137 REFERENCES 90

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