CHEMISTRY OF NATURAL PRODUCTS
THESIS SUBMITTED FOR THE DEGREE OF Boctor of ^hilo^ophp IN CHEMISTRY
NAZNEEN PARVEEN
DEPARTVENT OF CHEMISTRY ALICARH MUSLIM UNIVERSITY ALIO ARM (INDIA) JUNE 1987 M
T3445
CKED-2002
^Q "% ^. ^-
This is to certify that the work discussed in this thesis is an original contribution of the can didate and is suitable for submission for the partial fulfilment for the award of Ph.D. degree.
•fNizam Ud-din Khan) Supervisor ACKNOWLEDGEMENT
I place on record my deep sense of gratitude to Dr. 'lizam- '::
Khan who not only guided me but inspired me at all st^i^es. With out his keen interest affectionate supervision and constant help thro\ighout, it would have been impossible for rae to complete this work.
I express my sincere thanlcs to Prof. M.S. Ahmad, Ghaiman,
Department of Chemistry'-, Aligarh Muslim University, Aligarh for providing necessary facilities.
The continuous help and cooperation of Ms. Fe)imeeda Khatoon is gratefully acknowledged. Thanks are also due to other labo ratory collegues for their help throughout the work.
( NA^EEN PARVEEN ) GO II TENTS
Pa^ e^JAi o,
CHAPTjiR - 1
INTKODUCTION 1
1. Gneraistry of Biflavonoids 1
(i) Natural Siflavonoids 1 (A) Biaryl type of Biflavonoid 4
The Amentoflavone group 4 The Agathisflavone grout) 10 The Gupressuflavone group 11 The Robustaflavone group 13 The SuGcedaneaflavonoid group 14 The Strychnobiflavone group 15 (J3) Garcinia and Taiwania Biflavonoids 15
Garcinia biflavonoid 15 Taiwania biflavonoid 17 (G) Biaryl ether type of biflavonoid 18
The Hinokiflavone group 18 The Ochnaflavone group 20
(ii) Extraction and Purification 21
(iii) Identification and Structure Elucida tion 21 Ultraviolet spectroscopy 22 Infrared Spectscopy 24 Huclear Magnetic Resonance Spectro scopy 25 "^^G iniR 34 ilass Spectroscopy 5e Pa^e Mo, Constituent of the gen.is Araucaria 52 Terpenoids 5? Lignins 66 Biflavonoids 68 I'liQcellaneous compounds 69 Dis emission 70 Experimental 77 References 84
CHAPTJII - 2
Constituents of genus Taxus 95 Alkaloids a^id other Taxane derivatives 93 Jicdysones and Triterpenoids 103 [jignins 104 Glycosides 106 Flavonoids 108 Kiscellaneous compounds 109 Discussion 110 Bxnerimental 116 References 122
CHAPTER - 3
The glycoside 128 Constituents of the genus G-elonium 138 Discussion 138 Experimental 145 Referecnes 150
CHAPTER - 4
Constituents of genus Acer 152 Flavones 152 Pa^e No.
Terpenoids and steroids 153 Glycosides 154 Tannins 158 Acids 159 Miscellaneous compounds 162 Discussion 165 Experimental 175 References 182
CHAPTER - 5
Constituents of genus Rhus 188 Flavonoids 188 (a) Plavones 183 (b) Biflavanones 191 (c) Biflavones 193 (d) Flavonoidic glycosides 195 Miscellaneous comnounds 198 (a) Aromatic acid 198 b) Aliphatic acid 200 c) Tannins 201 Discussion 204 Experimental 211 References 218
CHAPTER - 6
Constituents of Homonoia 223 Discussion 224 Experimental 230 References 234 GHAPTJiR - 7
Antileukemic activity of a biflavonoid isolated from the leaves of Thu.j a oriontalis 255 Introduction 235 Discussion 236 Experimental 237 References 241
List of oul3lic,?.tj.onc 2^^2 ABBRBVIATI0N3
1 "^ 13 G NMR G Nuclear magnetic resonance ?ig. Figure H MR n Uuclear magnetic resonance IR Infrared spectroscopy MS Mass spectrum m.p. melting point ra.m.p, mixed melting poi^^t nm nanometre PC Ppper chromatography PLC Preparative layer chromatography
% TLG Jhin layer diromatography UV UltraTiolet spectroscopy Chapter 1 Phytochemistry, developed as a distinct discipline, deals with chemical structure, biosynthesis, natural distribution and biological function of org-mic substances accumulated by plants. Investigation of drag plants used in indigeneous medicine in India was started in the early part of the present century. Since 1940s onward new vegetable driags cane into prominence so much so that approximately one third of pharma ceuticals are of plant origin. The much greater emphasis on the exr)loitation of plants as source of chemicals can be expected in future, consequently, tnere has been a return to natural Droducts as sources of inspiration for organic chemists.
Althought our country abounds in medicinal herbaceous flora, very few indigenous plants have been subjected to phytoc lemic^J. study for the characterization of active principles. This inspired us to investigate some medicinally important plaints for the presence of acti/e compounds of possible therapeutic values. The flavonoids, a comparatively neglected class of natural metabolites, have recently attracted attention due to their physiological importance.
The term flavonoid covers a large group of naturally occurring com-oounds in which t\ra benzene rings are linke by a - 2 - propane bridge i.e. having (G/:-G^-G^) carbon skeleton except in isoflavone in which the arrangement is (G/--0-G-G), however, the "6 flavojaoid coranounds are now tiLon to include not only those substances having the true flavonoid structure but also such closely related classes of compounds such as chalcone, isoflavones, aurones, the stilbenes and th*^ cinnamic acids and counarins, which are demonstrably associn-ted with the true flavonoids being formed in plants by a single synthetic Pathway .
The potent uses of flavonoids may be listed as contraceptive 2 3 4 drugs , heart stimulants , coronary vasodxalators , antiviral 5 6 effect , analgesic and bronchodilator activity , spasmolytic and 7 8 9 antihistamine activity , oesterogenic activity ' , anthelmintic activity , treatment of allergic diseases , antitumor effect and effective inhibitors of blood cell aggregation 13 . GertaJn flavonoids influence the metabolism of blood vessel walls and cause an increase in capillary resistance. The most important flavonoids that produce a normalization of pathologically reduced capillary resistance are rutin derivatives, Citrus flavonoids, catechins and some flavones and isofalvones, such as taxifolin, diosmetin or sar)horicogide. The chief theraDeutic areas are haemorrhagic diathesis, diabetes, hypertension and arterios- chlerosis. - 3 -
The flavonoida are of commercial interest as antioxidants for fats and oils ' .
The hiflavonoids which are dimer of monoflavonoids have lately been given more attention because of their physiological properties. - 4 -
I, CHEMISTRY OF BIFLAV0N0ID3
(i) NATURAL BIFLAVONOIDS
Siflavonoids may be classified into three catagories:
(A) Biaryl type
(B) G-arcinia and Taiwania "biflavonoids
(C) Biaryl ether type.
The formation of all tyxDe of naturally occ"urring bifla- vonoidsmay be explained in terms of oxidative coupling of two chalkone units 15
(A) BIARYL TYPE 0? BIFLAVOITOIDS; In this category of bifla- vanoids, two raonoflavonoid units are linked thro\igh A or B ring.
The amontoflavone grouT): This group is represented by (l-3",II-8) linked biflavones (I to IV), flavanone-flavone (V), biflavanones (VI to VIII). - 5
WO OM
OR3 OR^O
0R3
( I )
1 R R' R- R'
, 4 (>T 16,17 (la; Ii • H H H H IS— '^C/ do. Saqaoiaflavone GH^ H H ii H (ic: Bilobetln H H H H J 2? (Id: yotet'3uCl xvone""' H Oli^r H d H o-^ (le; Podocarpa'jilavone-A.'' H H H H ^H ,. , ,.21,24 (If 0, i.-^- H H H
(ig: loOt^inkf^etin ' '^ H !I H Gli^ CH
"J -^ (Ih, Podocarrjuotlavone-B CHv H II il OH
1-4',II-7-Di-O-nethyl-
(ii; 1 T a^ientoflavone 2-^ H H ra. ) I-7,II-7-Dl-0-methyl- (ij amentoflavone" ^ GIL, CH., H H H 11-7,11-4'-Di-0-methy] 27 araentoflavone H CH- H H CH. HeveaflavonTT -f>T e 28a', "b (ii: CH 3 CH. H H CH- (im; Xayaflavone'^^'^'^ H GH; H GH rn. - 6 -
r R' r R'
(In) Sciadopitysin ' CH, H H JH. GH, do) I_4«^I_7,IT-7-'rri-0- 26a methylamentoflavone GH, GH. II GH. H 1-4',11-4 M-7,II-7-Tetra- 29 30 0-methylamentoflavone * CH. GH, H OH. OH, 31 GH. GH (Iq) Hexa-0-raethylaraentoflavone GH, 3 GH, CH,
HO
(II) 1-5' Methoxy bilobetin^^
(III) Biluteolin^^ OH 0
(IV) 1-4•,II-4',1-5,II-5,II-7-Pent ahydroxy,I-7-O-methyl, I_6-G-methyl[l-3',II-8]biflavone^^.
F\'0
R-*- R^ R^ R^
(Va) 2,3-Dihydroamen-toflavone^'^'^^ H H H H (Vb) II-4',II-7-Di-0-methyl-2,3~ 27 dihyd ro ament of la vo ne H GH^ H CH, (Vc) 1-4',11-4',I-7-Tri-0-methyl- 27 2,3-dihydroamentoflavone GH.'T H GH-r CH-7 8 -
OH 0
OH 0
(VI) Tetrahydroamentoflavone 36
HoCO
OCH,
R (Vila) I-7,l-4',II-4'-Tri-0-methyl-I-5,II-5,II-3'' Trihydroxy[l-3',II-8]biflavanone^ H (VIlb) 1-7,1-4',11-4',II-3•-Tetra-O-methyl-I-5, II-5-dihydroxy[l-3',II-8]biflavanone^'^ CH, - 9 -
(VIII)
R (Villa) Semecarpusflavanone H (Vlllb) Galluflavanone OH lu
The Agathisflavone .fi:roup: Thase are (1-6,11-8) iinkea biflava- noids comprising biflavones (IXa--^), flavanonyl-flavone-rhu.^fla- vone (X) md bifiavanones-rhusLlavanone (XI).
OW/ \VOR3
OH 0
(IX)
R^ K^ R-^ R4
40 (IXa) AgatUisriavone H H H 50 (IXb) I-V-O-Methylagathisflavone CH^ H H (IXc) 1-7, II-7-Di-0-metlijrlajathi3- flavone CH^ GH^ H H (iXd) 11-4 ' ,I-7-Di-0-methylagathi3- flavone OiU H E JH (iXe) II-4',I-7,II-7-Tri-0-methyl- ?6a CH.^ GiL agathisflavono" ' (iXf) I-4',II-4M-7,II-7-.etra-0-
metayla^athijflavon.' Gil;, :H , G^i, ^r - 11 -
OH 0
(X) Rh\isflavone'^5
(XI) Hhusflavanone^^
The Gupressuflavono .f^roupsrThls jroup comprises [l-8,Il-8] linked loiflavone (Xlla-g), flavanone-flavone (XIII) and ciflavaiione (XIV). - 12 -
OH 0
(XII 1 R- R^ R' R 4
45 (Xlla) Cupressuflavone H H H H 46 (Xllb) 1-4'-0-raethylcupressuflavone H n G?I^ H
(XIIc) I-7-O-raethylcupressuflavone CH, H H H (Xlld) 1-7, II-7-Di-0-meth.ylcut)ressu- PT 40 flavone GH, GH- H II (Xlle) I-4'/lI-4',I-7/lI-7-Di-0- methylcupressuflavone •^b 3 J:l CH^ H (Xllf) 1-4 '/II-4 ' , 1-7, II-7-'rri-0- methylcupressuflavone" GH^ GH3 H GH.^ H Oil 47 (XII^) Tetra-O-methylcupressuflavone GH, GH, GH^ Gil - 13 -
OH 0
OH HO
OH 0 OH 0 (XIII) Mesuaferrone-B,4' 8 (XIV) Mesuaferrone-A^^ The RobustafIavone i^roup; This groups is represented by robusta- flavone (XV), a [l-3',II-6] linlced biflavone and its methyl ethers which are not completely characterized.
(XV) Robust aflavone"^^ - 14 -
(XVI) Tetrahydrorobustaflavone 56a
H3CO
OCH:
(r/II) Atiesin 50
The succea~ineaflav'o:iold prruur): A 11-6, II-6]biflavanone, succeda- neaflavanone (XVIII) is the only member of this i^^roup isolnted so far. - 15 -
OH 0
(XVIII) SuGcedaneaflavanone 51
The Strychnobiflavone .s;roup; A [l-2 • ,II-8]biflavone.
OH 0
(XIX) strychnobiflavone32' (B) &ARCINIA AND TAIWANIA BIFLAVONOIDS; Garoinia biflavonoid; First isolated from Grarcinia morella , this group comprises [l-3,II~8] linked flavanonyl-flavone3(XXa-c), biflavanones (XXIa-f) and flavanone-ch2X)mone (XXII). - 16 -
(XX) R R" (XXa) Volkensiflavone^^"^^ H H (XXb) (=BGH-III-talbotaflavone) Fukugetin^^"^^ H OH (XXc) (=BGH-II-morelloflavone) H OCH II-3'
(XXI) R-* R R^ R" R^
'XXIa) G3-I a 59 H H H H H (XXIb) GB-I•- 59 H H H H OH 59 (XXIc) GB-2a H H OH H H (XXId) GB-259 H H OH H OH (XXIe) II_4 t _o-methyl-GB-2 bO H H OH GH-, H (kolailavanone) 61 (XXIf) Marmiflavanone OH K OH H OH - 17 -
(xra) 1-4',1-5,11-5,1-7,II-7-Pentahydroxyflavanone [l-3,II-8] chromone
Taiwanla Bifla\ o'loids: These are [l-3,II-3'J linlced biflavones (XXIIIa-c) .
R'O
(XXIII)
1 9 R^ R" 63 (XXIIIa) Taiwanial'lavone H - 13 -
R^ R'^ A^
63 (XXIIIb) I-7-C-raethyltaiwaniaflc.voiie OIU H H or
II-7-0-ne1:hyltaiw'---ni'^fl'^vone GH^ H (XXIIIc) II-4 ' jI-Y-Di-O-me^hvit-tiw^nia-
o-lavono CH^ H \^1 1 3 or II-4',II-7-Di-0-rTiethyltaiwania- flavone H Cli, CH^
( C) ^^lAIiYL ETIiaK TYPS OF BI.i'LAVONOlO_S:
Jhe :iino!<:iilavono grouo: I'liese Diiiavonoids have [l-/-'-u~lI-6 j diaryl ethor link, several biflavone (XXIVa-h) of this grovp and one flav;anonyl-flavone (XXV) occur in nature.
(ZXI/)
R R R
(XXlVa) linokin-T—-^"Tone ^ H :i H
(XXIVb) 1, eocryDtonerin H 11 R - 19 -
H^ H^ H^ R4 R5
(XXIVc) I so cryoto me r•m 65 H H H Me II 66 (XXIVd) Gryptonerin-A H H H H Me 67 (XXIVe) Ghamae cry pari n n Me II Me H (XXIVf) I-7,II-4'-Di-0-methyl- hinokiflavone H Me H H Me GG (XXIV^) Gryptomerin-B H H H Me Me (XXIVh) I-7,II-7,II-4'-Tri-0- 68 methylhinokiflavone H Me H Me Me
(XXY) 1-2,3-Dihydrohinokiflavone^'^ - 20 -
The Ochnailavone /^roup; Kerabers of this group are [l-3 •-0-II-4' ] linked diaryl ether [XXVIa-d] aiid constitute the first example of naturally occurring biflavanoids in wiiich neither of A-ring is involve! in the interflavonyl lin^v.
OR3
0 OR4 (XXVI)
R1 R2 ^^3 ^A R^
69 (XXVI a) Ochnaflavone H H H H (XXVIb) I-4'-0-methylochna- flavone H i'le H H (XXVIc) II-7-O-methylochna- f~-t -» flavone H H H H He (XXVId) I-7,I-4'-Di-0-methyl- 69 OGhnaflavone(=03lII) H Me Me H H or
1-4',II-7-Di-O-methyl- ochnaflavone (=:.03III) ^'^ H He H He - 21 -
(ii) IXTRAOTICN AND PURIFICA^"ION
Acetone aiid trichloroethylene have been used for the extrac tion of the biaryl and biaryl ether type of biflavonoids ' from defatted material. Chloroform has been used for the extrac- tion of Garcinia biflavonoids '^'^^. Crude crystals of biflavonoids may be obtained by solvent treatment or by extraction with dilute 25 alkali and subsequent acidification
For the purification of crude biflavonoids column ohroma- to^^raphy on silica gel and magnesium silicate have been used with greater ouccess ''^ » -. G-ood separation on polyamide and polyvinyl pyrrolidone hav3 been reported 27 *3 1 . For monitoring the separation on columns of adsorbent, TIC is employed. Two most widely used developing systems for TLC on silica gel are toluene/ethyl formate/formic acid (5:4:1) and benzene/pyridine/ formic acid (36:9:5)7 3 . 'biflavonoids can be separated in larger 27 40 59 amount using preparative TLC" '' * . Visualization of biflavo noids is effected either by spray reagent or in UV light.
Counter current distribution has been used for the separa tion of isomeric biflavones in two phase systems
(ill) iD::::TiriGATiv3:: MAU siituCTuaj ELUCIDAIIU.;
Methyl ethers and acetates are usually prepared from the parent and partially methylated natural biflavonoids. Character- - 22 -
istic w^hade in uv light and R^ values on TLG provide fairly
accurate method of identification of the type of biflavonoids 74
The final identification ±a done Py comparison of the compound
•;\ath authentic somnles.
Co-chromato£;raiohy, mix'-^d m.r)3. of the substance and its
derivative, UV, IR, NMR and Mass spectrometry are employed for
the structure elucidation. Stracture can finally be supported
by synthesis.
Ultraviolet Spectroscopy
The ultraviolet spectra of different flavoxioids are very 75 75-77 characteristic and along with colour reactions , have been
used extensively to distinguish the various group of this class
of compounds. The absorption maxima of flavones have been correlated to the oresence of a cinnamoyl (XXVII) and benzoyl no (XXVIII) grouping , the former giving rise to the high wavelength band at 320 to 350 ran and the leter to the wavelength band at 240-270 mii. On the basis of this generalization, important deductions nave been made about the location of substituent in the two rings. - 25 -
Substitution in the B ring specially at 4' stabilipos the cinnamoyl chromo^hore resulting in a bathochronic shift of band I where as substitution in the A rin/^ has a similar effect on the position of band II. Gom^o^^nds having a free 5-hydroxyl absorb at highe-^" wav^le igtli and methylTtion of this hydroxyl brings about a hyosochromic shift of 10 to l'-5 nu of both maxiran. The presence of a hydroxyl at this nosihion is routinely ost.ablished by nieasuring the spectrum in nresf^noe of '\1C1. 79 . Hydroxyl groups ^t '', " ' rr^-^ •^^^.^ nci'^io than otners a:.T.i 3 batliochroraic shift of band I or II on addition of fus^^d sodium LcetaLe is a ^ood indication of the presence of OH gi'oaos it th^se Dositions but the results of tneTe raeasaremenl ha/e to De inierpreced with caution ana requires furtnor confirmation by de^radalion. This for example lucidm , ice CO sin' ' and sciposin'^'^ fiiiled to give a batho chromic shift v/xth sodLj.ra acetate though they \7ere definitely shown to possess a '^-OH group.
In flavanones absence of cinnamoyl chromonhore has tne effect at sucressing the hi/h wavelength hand which is either tot-illy a:)sent or Dresent only as an inflection. The snoot ca of iso'lTvone ir? f s3 mar'cpd ^)y th ^ absence of the high wavelength bfoid, biochanir A, irlge^in aad omiferin absorb only betw^^^n 61 to ?7b m|i . '''hus it is iil'^'icult to distinguish betx/ccn flavanones and i :;o"lavones '/it}i the aelp of uv spectrin"! ilcnie.
The altr..vjolpt t'r)^c'f"> o" biflavonyls ar^^ ver";, simiLir to thTt of monof-Lavo Toid u, in . • th the only difference that - 24 - the molecular extinction coefficient of the biflavone is aToroxi- mately double as compared to that of corre;-iDondin/^ raonoflnvo loii. unit. This demon-trat as the presence of two isolated chromorihores of fla^'o loids pel moleculo of hifl -Qioid.
Infrared 3pe^trosfopy
The 11^ spectrum of flav \none showi the c^rbonyl absorption r-t 16 30 cm~", the standard value for aromatic ketones. The shift of the carbonyl b;uid to 1620 cia~ in 5-OH flavanones is lari^^fly due to electron donating tenaency of the orthohydroxyl groun, coupled ^d-th chel'itlon. Consoqut^ntly, methyl"tion of tie 5-UH produces only a srrriLl hyosocnromic .^>hift of 10 cm . A similar shift toward long wavelength of 4' substituted flavanone is i.wever, attributed to iniiexiaoleGular hydrogen bonding 27 . fjie IK spectrum of flavone shows the carbonyl band at 1660 era" C/dng to conjugation with the olefinic double bond. Introduction of a hydroxyl at 5-position does not alter the band position ai^nre- ciably, luteolin .-md anigenin show the carbonyl band at 1655 and -1 85 XODO cm ,respectively . The IR spectra of isoflavones are similar to those of flavonos. Chelation of the 5-OH in all cases has the effect of broadening the 0-H stretching oand to a 'ooint where it car no longer be made out. The aromatic region is not of any great usefulness as no reliable ^reiictioa about the suDstitution pattern can be made on the basis of absorption bands - 25 - in this region. Tlie infrared lipectra of alkylated flavo loids i'jive some indication of the presence or abv^ence of gem dimethyl f'^rouDs and of epoxide linkage but these pointn can .lOv/ be better
u T uanlxsii.f'ja .fjx on one uClo ol ,I,..L . .' c o T'-,
Nuclear MaP'netie l^esonence Spectro^cop.y
The foregoing discussion of the IR and UV spectra of fl avortoids illustrates the usefulness of these two techniques in determining the structare of unknown flavoloids. It is clear that although much useful information regarding the oxygenation pattern of a flavo loid ccai be obtained in this way, it falls short of aroviding complete and un'^ribiguous evidence for or against a ^jresumed structure. Thus the infrared spectrum does not go beyond distinguishing between the y-oyrone system of flavo loids and the -r-pyrone system of coumarins besides indicating the Dresence of a chelated 5-hydroxyl. The 'JV snectrum is much more informative and distinguished clearly bet-jeen flavones, isoflavones and coumarins.
The aoalication of }Mtl spool t^oscopy has aroved to be rest powerful tool in the stracturp determination of flavonoids. oy the use of IIHR studies of silyl dprivntives , double irradintion techniques'^^, solvent iniuoi shift studies"^^''^'-''^'^, lanth-nide induced shift , nuclear overhausor effect (NOi]) and C-rillii - 26 - svectroscooy Q6 , on':i can come to structure of flavonoids occurring even in uiinor quantities wLt.'LOut tedious and time consurain,-^ chemical degradation and syrthesis.
The trimethyloilyl dfjcivatives caii "be conveniently orepared by treatment of the compouni uith hexajnethyl disilazone and trimethylsilyl chloride in ^;yridine and the spectrum is then measured in carbon tetrachloride with tetramethylsilane as external or internal reference.
Table - 1 .-^^ives the chemical shifts of different protons of certain representative oi' flavones and isoflavones. The most detailed and systematic stu.. les of the ITHR spectra of flavo.ioids 97 98 ^9 lOU are due to I-Iabry ' , Batterliain and Highet , Clark Lev/is , luassicot , Ivawano , and Pelter and Haliman" ' "* . These .itudi^s have simplified the task of determining the substitution pat^tern of flavonoids with the help of ilIK spectroscopy. Aa obvious advan tage of this technique in itT application to flavimones is that ^hey can be readily distinguished not only from flavones fuid isoflavones but MISO from t^ie isomeric chalcones which in vi-^w of the extreme ea3e of the i ^Oiaeri ^.ation nrocr-s, is often lot ')D33lble \TLth other raethodr;. H - 27 -
O o C\J II I rt oi in •. to ro om II ? to H H Pi in 00 o u H o 0) CO to II •H tn ^ W II -p O I in II X 0) il X II II X H tn to II >i rt CO II ^
PH W in 0) I tn to to o •H KH 1-1-4 O W II •H I >^ C-- II W II CM • • II 0) f- II II 3^ '^ n -P II ^•^ to • u II ai fc! to M II O II II The moat co-riuionly occurring tiydroxylation pattern in natural ilavoioicis is 4 ' , T , 7-trih,/iro^y (XXXI) system. The cheraical shifts of the protons of rinj A and li prove to be independent of each other but ar ; effected by the nature of rin:;; G. OH 0 (XXXI) Ring A: The two A rin^ protons of flavo loids with 5,7-hydroxy- lation pattern ^3ive rise to tvro doublets (J = P.'jHz) between 6 6.00-6.66 frorp tetramethyi ailane. There p.re hov?ever sn J-L but predictable varintion in the chemjLcal shifts of the C-6 and G-8 proton si£:nals depending on the ^)- am^ 7- substituents. In flavanoniis the 6,8-protons i,ive a si,;nal peak near 6 3.95, vri-th the addition of a 3-hydroxy group (flavanols) the cheraical shift of these protons are sli^jhtly altered and the pattern changes to very strongly couoled pair of doublets. The presence of double bond in ring G of flavones and flavanols causes a marked downfield shift of these peaiis again "^reducing the two doublet pattern out of 6 and 8 protons, the Ip-^er aonears downfield. - 29 - ainf. 3: A.li 3 rin^ protons appea^' around 6 S.1-1.1 a region separate from the usual A rinf; protons. The signals from the aroratitic protons of a 3-ring in a flavone appear as a hroad peak centred at about 6 l.Ab. I'he presence of G-rin^'-^ double bond causes a shift of 2', 6'-protons and the spectrum shows two broad 10? peaks one centred ^t & a.Ou (?',6') and other at 6 7 .b( 3 ' , 4 ' , 5 ' ) . V/ith the introductioa of 4'-hydroxyl group the 3-ring protons appear effectively as a four pealc r)attern. This is cali led ApBp pattern. Introduction of one more substituent to ring 3 glares the normal A3'J pattern, the hydroxyl group increases the sheildLng on the adjacent 3', 5'-i^rotons and their peak moves substantially upfield* The 2 * , 6 ' —proton'": of fl^vanoncs give signal "• '-•orit. "»^<^'^ '"•t ab out 6 7.35. Ring G: Considerable variations are generally found for the chemical shifts of G-ring protons among the several flavonoid classes. For example, the G-3 proton in flavones gives a sharp singlet near 6 6.3, "the G-2 proton of isoflavones is normally observed at about 6 7.7, while the G-2 proton in flavanone is split by C-3 protons into a doublet of doublet (J . =: 5Hz, J,TJ^ ran ^ s = llHz) and occur near 6 5.2. The two G-3 protons o^cur fls two qaartets (J---j,_^u =" 17Hz) -lear 5 7.7. Ilov/ever, they often apoear as two doublets sinc^ two signjils of each qua.rtet ace of low intensity. The G-2 proton in dihydroflavonols appear near - 30 - 6 4.9 as a 'loahlc t (j = llK'/.i co-'j^^led to the C-3 proton winch coraes at about 6 A.? as doublet"^ . In the sf^mctare elucidrtion of biflavoioids certain useful inforin'ition can be obt.dn.d by GOUITD irison of their lIMit 3^)6ctra wi bh those of tlieir correo")onding monomers. Such a choice, however, is compellin^^i b ^ t by no means in falliable, c'wap^rison of the hllR spectra of methyl and acyi derivative^-, of a biflavonoid witu those of oifla^'O-ioids of the sj^e series as well as with those of bifltivxioids of other series in which at least one monoflavcnoid imit is similarly constituted is irery helpnul in assi/^nxn,: each and individual proton ind the position of rr 3thox;y ,crroups. The problen of int erflavo ioi ; al link-age has been successfully solved by solvent induced shift studies of methoxy resonances'' * and Ifanth^nide induced shift studies. In biohenyl type biflavones such as amentoflavone, cupressuflavone, agathin^'lavone etc., the peaks of ring ^rot:ins involved in int erf Lavo loid linkage art^ear at somewhat lov^er field ('^0.5 pom) as compared vri.th the joakj of the same protons m monomer due x,o conjugation. It has been observed'^ both in biphenyl as well as in biphcnyx etlicr" type biflavonoids that the 'j-OIIe groun of a -i linked laonofla^ o loid unit in a biflavoioid shows uo below - 31 - 6 4.00 in deuterochloroform in all the cases examined so far (Table Z). This obsarvation may be explained on the basis of extended conjio^ation. 5-Methoxy grout) of a 8 linked monofl*^- voiioid unit in biflavonoids of BGH-series 54 *5 5 *5 7 , UH~-eries^^'^'^'^'^'^'''^^ and Gd-series^^'^^"^® does not show ' briiow 6 4.0U as the linkof^e is through heterocyclic ring. i'A3LE - 2 Hethoxy -orotons (6 valuer) of fully methylated biflavoioius Biflax^onoid I-5-0!Ie II-5-OMe Juprossuflavone [l-S,Il-3j 4.15 4.15 Anentoflavone [l~j',i C-8] 3.97 4.16 Agathiaflavone [l6,II-8] 3.86 4.15 [I_.i -_o_ll-8j 4.00 4.U8 I- ^, 3-dJ.hydroarnGnboflavone _ 4.05 The H miR dat:a of some biflavonoids are give: in Tables 5,4. TO I I Hi m :> 7 - L'^ -- c CT C3 r to 73 tr- ^ a ^£1 O r t- CO » » e^Q^ -5 C TJ ID •- UL. V cj r-i M) II CC o t- - t-J 1 (\j 1 a 5 '! w >-5 ") en C 1 O II •p 1 O 1 n n r V o. i N^ -- i i It X V-1 II H I t9 T3 en m m X T3 •o •c •J) U) N CO II (D X CO (N in in •*• II 1 •*• IT 03 O o i^ If o in il II 1II1 «.0 X) \r VJ3 «£> ^o VC to to r~ 11 11 ^ « 11 II 11 II 1 II 1 •* fA K> 1 CNJ 1^ 1 1 11 ~- <^ X IIII r! in in --< 1 1 " « o rj •0 ^n^ HM , . «5- Ci I -o.> h c- J"\ r-l O —. -- O N • •»J LfN• I ;z t-3 o H •• :; I O •- O Ti T3 J • - • • C> ^ o CM —r<-N^ -1 -J- r-4 e Tl 11 • 'T 3- N X M -r" N I H -<•- r-J K rH K -•^ -o o a; TJ Td . T^ . •n*> "-T ^ CM (X•, c •—' r-1 H H —o ' H H H 'ti t^ II r- I' jA II II ll •t-j • •-i • 1^ o 'T T;* . r& O I O r- x^ •D II •^ O —•' m o > r I I I Lfr^ 0) V O_^ I c\ f N *--. ir •X (^ w TJ*. t?» O T)* U2• I I 0—.^0 -— -;r ' o • 1 OCi I I O n ll EH "-3 • • t-• «x>• r U o 0) J' •p rH H K-, cS IJ2 •s •• •L T ••^ Ln• r>• 1^• -F^ o o I -P o o I o 1 1 m •r) -7 C LTN Q T! H H ^ I 1 X - C-MMR spectrosccpy ol' biflavonoids A namber of papers demostrating the utility of 13 G-IIMR foi. the structure elucidation of the biflavonoids have appeared recently^^'^^^"'^. Wagner et al.-*-^^^ have discussed the "^^G-MR spectra of various 0-0 linked biflavonoids. The assignments of 13 the signals in the G-MR spectra of biflavonoids have been achieved on the basis of off- resonance, proton coupled spectra and by analogy with published data of compounds with similar functional grouns on the basis of chemical shift3 of the signals and their multiplicities in the off-resonance npp>ctrp, it \vas possible to determine the interflavonoid linkage in biflavonoids. This method pixjved equally important for the location of methoxy substitution directly in a naturally occurring methylate' bifla- vonoids. 13 G-MR data of some biflavonoids of different G-G linkage are given in Table-5. 1 U 1 '•5^- 1 1 1 , IT * r • i-n L~- c 13 3J Jl c • r- * 1 1 CM— OJ C\ :(\ j rvi rvj CJ r» r\ 1 1 H r-* r-\l r-l r-< r-( ^-J —1 t—1 r-l 1 1 II ^ '^ — -- 1 1 1 1 ^i- II ^ O ^ -H (X) rH 'ch ••A D - •^ 11 L • • • n 1 1 r-i Xi vD O D D LTi r s 1 II C3 1 r ^r-i r-^ r-l -J OJ H rH '~^ ^ r- r-l r- 1 II •H r-J r-l H r-l —1 ,—_l , --J 1 1 1 ^ "^ ' - 1 r- rr-^ II 1 0 1-1 r-' r-f (M -H t^ O -J r II 1 ^ 1 II 1 II ^ r-l ,-< r-J r-J r-J rr r-J r ^ ^ L n r VP -AJ a ir £) f r / 1 r-' r-{ ^ r-l H' \ H r 1 —-1 J rH r-l —J I/I II II ^ 1 II 1 1 1 O H H H l-^ X! fT II 1 •* > "'— -* ^ *^-' 1 II 1 1 ^C U ^n £) r 0 D m J-N lA LA rN 11 ,-j ^ ^ ii O 1 r-J r-l r-J r f-J rH rJ H r-l r-l rH r-J r-J 1 1 1 ^-J r-l —I r-J r-l H H ~1 -1 rH r-l rH II 1 " '- II 1 1 1 1 O f cr T 0 O lA r-l r-l r-l rH N-N K\ 1 O 1 r- 1 1 r-J (r •- T" ^ r-J CD CO CO cn C" cr 0? 1 O 1 lA t\ rv Cv r OJ lA OJ (M r 0 r r O 1 1 1 r-l r-J M r-^ r-l r-l H H r-( rH H 1 1 ^ ( 1 1 JJ 1 1 II 1 1 1 1 1 H 1 FA t<^ r- ir •^ >XI PJ H F^ K V 1 1 II ^ ^ ^ II c-> 1 H c r-l r-J ^ r-l H H cn -^ cr r-J rr> CT' 1 1 1 CM r o (M (\J fv '\J o r\ r r^ 1 (^ (^ ^ 1 r-J i-J H r-l r-( r- r-t r-l r-l r-l — r-J r-l —1 1 1 "x; r N-^ t<^ o lA vx> vO N" cr. rr- 1 II 1 — ^ ^ 1 a 1 t^ MASS SPBGTROMi^::TRY Recently mass spectrometry has been successfully employed for the structure elucidation of iiioiio- and biflavonoids * * and conclusive structure fragmentation pattern relationship estab lished. Since most of the naturally occurring flavonoids and biflavonoids possess at least 5,7,4'-hydroxylation pattern. The present discussion is mainly centred at such compounds. Flavones: The principal mode of fragmentation in flavones involve (i) fission of the heterocyclic ring via reverse Mels-Alder reaction (ii) loss of carbon monoxide from molecular or fragment ion. This is illustrated in Scheme (I) for an unsubstituted flavone"^"^^ (XXXII). Apigenin ^ (XXXIII) has the parent molecu3ar ion as base peak which loses a molecule of carbon monoxide to give a major fragment ion m/e 242 (XXXIV). 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 a highly oxygenated flavone (apigenin) it is observed that fragmentation via RDA reaction is less favoured in the later. This is due to the stabilization of the initially produced ion radical by mesomerism over a number of oxygen atoms. These minor - 37 - CM X I CM O CM CM CM 01 i M M + . X >< 0) 5 >< 0 0) CO I CM ft) - 38 - en •^ I r s 00 ? hH (L M CO E M fM (H X! ^ X a ^ CO O o I CM i - 39 - 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 trimethyl ether (XXXV)-''"'-^"-'--^'^ the molecular ion appears as the base peak. Further fragmentation of the molecular ion via RDA process yields the ketone m/e 180 (XXXVI) and the acetylene m/e 132 (XXXVII) (Route-I) and the carbonyl ion, m/e 135 (XXXVIII) (Route-II) Scheme III. Flavanones: In the case of flavanones (XXXIX) fragmentation by pa'h A (RDA fission of heterocyclic ring) and path B are of great importance as they lead to clear out, characteristic spectra 111 Another method of break: down that helps to characterise the flavanone is the loss of either a hydrogen atom (XL) or an airyl radical at C-2 (XLI) from the molecular ion to give even electron fragments. 40 - m E X <^z:o o o v>v CM M M > o > = o o» cn E hz/ i / \ u «> o -o 2 CO / io % rO o I OD f^ ;^ 9—\ M ^—A X 2? Q-8 1 g^ —> "°l o io > o X x^ _ AT - (XXXIX) (XLI) These fragmentation process are illustrated in the case of 4'-metiioxy flavanone (>ZLII) (SGhome-IY)o The fragment with methox;/l group takes nearly all the charge. A further peak is at m/e 108 (XLEII) arising from a hydrogen transfer reaction. _y-ocH3 OCH, ^IQ 108 (10) (XLIl) (XLIII) - 42 - £2 O O I o n CO I + 0 m CM e o o M I E O CO T- O C5 I IN. GO + 0 O E E - 43 - The presence of a hydroxyl (XIIV) or methoxyl group at C-4' position of ring B facilitates, by enhanced stabilization of the resulting fragment ion, the fragmentation of p-hydroxyl benzyl (XLV) or p-methoxyl benzyl ion respectively (or their equivalent tropolium ions). These ions appear as peaks of significant 55,106 inten ,'ity in the mass spectrum of naringenin/its methyl ether OH — (XIY) The mass spectrum of 5, 5,7-tin.hyd2XDxy-4'-methoxy falvanone (XLVI) 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 -1 -I O is usually the base peak . Apart from the fragmentation process mentioned for apigenln trimethyl ether, these compounds also tmdergo (i) fission of the C-C or C-O-C linkage between the aromatic residues (ii) elimination of CO (28) or CHO (?Q) from the biphenyl ether and (iii) rearrangements involving - 44 - I O I + 0 I I in o 2 I o 04 > I AI II I X o CO (n O in c\ /; 0) - 45 - condensation between 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 observed. II II II ^ II II II II II - 46 - II E^ II If II II II II II 11 II II II 11 EH II II 11 li It u 1! II II 11 II II CTi (NJ UD H •'~- a\ cv en II II « Pi (1 t<-\ H f<-N H co H rv' CM II II II -^ "-' "— '— ^ ' "*^ ^ ' II O 11 1 1 1 II II > E-^ II CD C- tA OO CM in H t- II II ^ p£) II O o CT\ r~ (<> ^c^ H HoCO CH Tni« 132^14) Tn(« 576(4) H3CO 0 n^ * [W-I]*^ mie 621(38) OCH3 H3CO -) [M-IS]*^ m/e 607(8) H3CO OCH3 i [M-30]-. ml9 592(18) H3CO 0 M-^mie 622(100) H3CO ^3^° °TP/« 245(11) (490 appears at m/« 245) Scheme - VI; MS frac^ents for cupressuflavone peimethyl ether (Me-ether of Xlla)'^^. - 48 - H3CO -\' ""XXc III H3C0-V_ CH mle 132 (3) 0 OCH3 m/e 576(10) "^/« 621(31) ocHaO^^^o 4^-'5]*TT,/,607(33) OCH3O M*, m/e 622(100) 1 ["-30]%/, 592(8) n^ HfO • II H3CO 0 TTi/e I35(i6) mle 180 (3) m/e 31! Scheme - VII: MS fragments from amentoflavone permethyl ether (I ^,31 - 49 - n• + 0CH3 OCH3O "^I« 245 (22) (49d*appears at mU 245) n- 0CH3 OCH3O M* Tn/e 622(90) OCH3 OCH- OCH3O mie 135 (65) Tn|« 311 (100) Scheme - VIII: MS fragments from agath-isfavone permethyl ether (Me-ether of IXa)"^^. 50 - OCH OCH3O OCH3O m/e 281 (22) m/e 327(23) H3CO 0 tn/e 296(75) OCH3O OCH3O M*, m/tt 608(39) OCH. OCH3O O mie 431(7) cC 0 H3CO OCH3 OCH3O OCH3O m|«297(29)-^mle296(75) ^,^ 31,(22) , mle 312 (22) 1 *°Ta"°a^'°-o: — mJc 313 (100) •OH m/el35(l9) m/e)32r8^ ^^ , ^ r \ [M]- J mj^j 579(11) , Tn/c 607 (12), m/g 593 (36), Tn/e578 (ll) Scheme - IX: MS fragments for hinokiflavone perraethyl ether (Me-ether of XXIIIa)^"^. - 51 - ^6H HaCO OH n^/e 167(7) OH 0 Tn/e 446(100) OH 0 ^\ "i/e 612 (59) Tin/e 2S4(5) -H H^CO Tn/e 107 (18) Tr»/c 283 (7) Scheme - X: MS fragments for morelloflavone tetramethyl ether (Me-ether of XXO)^® - 52 - 2. CONSTITUENTS OP THE GENUS ARAUGABIA A genuvs of ever green trees including about 15 species distributed in Australia and South America. Most of the Araucaria plants constitute biflavonoids and diterpenoids. Some plants contain monoterpenes, sesquiterpenes, triterpenes and lignins. Terpenoids Terpenoids are the most frequently isolated natural products in the essential oil and oleoresin of Araucaria plants. Monoterpenes 1iQ T PD A. araucgqia " ^ and A. bidvrilli constitutes only limonene (XLVII) and Geraniolene. (XLVII) Limonene Sesquiterpenes Sesquiterpenes (XIVIII, XLIX) are found only in A. araucana"'''^^ - 53 - HO* .» i H (XLVIII) (+) -Y-cadinene11 9 (XLIX) (-) -a-cadinol11 9 Diterpenes Diterpenea are more common than monoterpenes and sesquiter penes. The diterpenes with labdane skeleton more frequently occur than those with other skeleton. Almost all the Araucaria plants examined so far contain bicydLic labdane derivatives. Tricyclic and tetracyclic diterpenes have also been isolated from some species. Bicyclic diterpenes Bicyclic diterpenes with labdane (L) skeleton are more 1 PI 1 op "1 p"^ frequently found in A. an^ustifolia and A. arauoana ' a R' (La) Imbricatolic acid"^^^*-'-^^ CO2H CH2OH "1 p P (lb) 15-Acetoxy imbricatolic acid GOjH CH2OAC - 54 - R R' (Lc) Imbricatadiol ' CH20H CH2OH (Ld) Imbricatolal-^^^*"^^^ CH20H GHO (Le) 15-Acetoxyimbricatolal 122* 123 CH20AC GHO (Lf) 15-AGetoxylabd 8(17)-en-'-^^ CH^OAc Me (Lg) Me-15-hydroxylabd-8(l7)-en-19 GH^- oat4.e 123 GH2OH G = 0 GH, (Lh) Me-15-acetoxylabd 8(17)-en- OH, 1 3 19 oate"'-^^ CH-0/T GOOMe (Li) 15-hydroxyladb-8(l7)-ene-^^^ GH2OH Me Diterpenes with 8(17), 13-labdadiene skeleton 123 A. araucaaa contain Me, ^-aGetoxylabda-8(17),-14-diene-19- oate. The Diterpenes with 8(17), 13-labdadiene skeleton (LI-LXI) occur in A. angustifolia A. bidwilli * , A. cooki "" , , , , ..126 , , . ..128 , . T 129 A. Gunnin^hajnii , A. hunstenix and A. excelsa R R' (Lla) Agathic acid''-^'^ GO^QXHX V-»VGOo/ Q H - 55 - H R' 124 (Lib Dimethylagathate C02Me C02Me (Lie A^atholic acid " CO2H CH2OH 124 (Lid Methylagatholate G02Me GH,OH 124 (Lie Acetylagathoiat e G02Me GH2OAC 125 (Lif Agathadiol GH2OH GH2OH (Lig Acetyl isocupressic acid GH2OAC GOjH (Lih Methyl isocupressate-'-^^'''"^ GH2OH G02Me 126 (Lli Methyl-acetyl isocupressate CH2OAG G02Me (LIj Methylagathalate"^^"^ G02Me CHO (LII) R (Llla) Methylent ap-hydroxylabd-E-13-en-15- oate 127 G02Me (Lllb) Ent-8f3,15-labd-E-13-en-diol-^^'^'-^^^ GH^OH 56 (LIIE) R 127 (LIII) Methylent-8a-hydroxylabd-E-15-en-15 oate G02Me (LIV) 127 (LIVa) Ent-.15-acetoxylabd-8E-13-diene CH2OAC (LIVb) Ent-labd-8E-13-diene-15 ol"'-^'^ CH2OH R o R (LVa) Manool OH Me lie - 57 - 1 R R- R' (LVb) Torulosol-^^'^ OH Me CH2OH (LVc) ToirulosaL"'-^^'-'-^^ Me OH GHO (LVd) Guprossic acid"^^^'-^^^ OH Me CO2H (LVe) Methyl-13(p) hydroxy-8(17)~ 14-labdadiene-19-oate-^'^^'-'-^^ Me OH C02Me (LVf) 13p-Acetoxy-8(l7),14~labda- 125 19-oic acid Me COgAc GO2H (LVg) Methyl 13-acetox5'labd 8(17), 14-diene-19-oj 123 OAc Me GH2-Me (LVh) epi-torul03ol Me OH CH2OH (LVI) R R^ (LVIa) 15-Hyd~8,E~labdadiene-19- oxc acid GH2OH CO2H (LVIb) Methyl-15-acetoyy-8,E,13- labdadiene-19~oate"'-'^^ GH2OAG GO2 (LVIc) Methyl-19-hydroxy-8,^,13- 1?S labdadiene 15 oate G02Me GH2OH ^n R R-^ (LVId) 8,E-13-labdadien-15,19~ diol-^^^ CHoOH CHoOH ^2^.. v.x.2^ (LVIe) 15-lcetoxy-8,E-la'bdadiene- 19-01-^^^ GHoOA^2"^"c GHoOW.X2V H (LVIf) 8,E-13-labdadiene-15,i9- diacetate-^^^ OE^OkQ GHoOAc ^2-^^ v...2^ (LVIg) 15-nydroxy-8,E-labdadiene- ig-al^^*^ GH^OH GHO '2' (LVIh) 15-Acetoxy-8,E-labdadiene- ig-al-*-^^ GH2OAC GHO CH2OAC Me02C (LVII) (LVII) 7-Oxo-acetoxyester-^^^ - 53 (L7III) R (LVIIIa) MGthyl-13(p)-hydroxy-8,14-labdadiene-19- , 125 G02Me 125 .; ; Illb) 13j3-Hydroxy-8,14-labdadiene-19-al G02Me 125 (LVIIIc) 8,14-Labdadiene-13p-l9-diol CH2OH C02Mt (LIX) (LIX) ent~labdane ester (LX) R R R (LXa) Methyl-ent-Ba-hydroxylabda-E- 13-en-19-oate-^2'^ OH Me G02Me - 60 - R R R' (LXb) Methyl-ent-8p-hydroxylabda~E-13- en-15 oate Me OH C02Me 124 (LXc) Ent-8p,15-labda-E-13-en-diol Me OH CH2OH (LXI) R (LXIa) Ent-labda-8, E-13-dien-l5-ol-'-^^ GH2OH 124 (LXIb) Ent-15-acetoxylabda-8,E-13-diene CHjOAc Unusual alcohols with norlabdane skeleton These occur in A. excelsa'12 9 HO '' (LXII) 18-norlabda-8(17)» (LXIII) 19-norlabda-8(lYj,i4-dien I4-dien-4a,13-diol ^^^ 4(^,13-diol^^^ - 61 Labdatriene TOO T O ^ A. excelaa"" and A. cunnin^hamli ""' constitute labdatriene. (LXIV) R r (IXlVa) Trans-commxuaic acid ^ CO2H (LXIVb) Methyl-trans communate C02Me Clerodanes 124- 1 Pft Clerodanes are found in A. bidwllli and A. hunstenii , C02Me C02M< (LXY) ent-4(l8),E-13-clerodadien- (LXVI) ent-3,E-13-clerodadien- 15-oate-'-^'^ 15~oate-^^'^ - 62 - (LXYII) ent-4(18)-E-13-clerdadione-15-ol 128 Tricyclic diterpene Tricyclic diterpene such as abietaaes (LXVIII, LX.IX) and 1 PI 1 PR pimaranes (LXX) are found in A. angustifolia , A. cookii 129 and A. excelsa' (LXVIII) ent-4(18)-E-l3-clerodadien-15-ol 128 - 63 - Pi P? (LXIX) R R" R" (LXIXa) Abietenal-^^^'-^^^ H Me CHO (LXIXb) Abietic acid"^^^*"^^^ H Me COOH R (LXXa) Sandaracopimaric acid 300H (LXXb) Sandaracopimaradienol^^ CH2OH -. 64 - Tetracyclic diterpenes 119 I'-^O These occur xn A., araucana and A. rulei . 19 '18 19 '18 (LXXI) (-)-Atisirene^^-'^ (LXXII) Isoitisirene-'-^'^ .9' '"18 (LXXIIIa)(-)-Tetracyclobine-^^'^ (LXXIV)(-)-Kaurene 119 (LXXIIIb) (-)-Hibaene-'--^^ (LXXY) Isokaurene^-"-^ (LXXVI) Phyllo cladene^^^ - 65 - Triterpenes In Araucarrla species the triterpenes are investigated only from A, an/mstifolia 131 . (LXXVIl) R (LXXVIIa) Sitosterol"'-^-'- Me (LXXVIIb) Garapesterol 1^1 H (LXXVIII) Cholesterol^^l - 66 - Lignins are distributed in different parts of A. angustifoli,.„-,. a 131-154 . OMtt / \V0R' OMe (Lx:xix) R .1 (LXXIZa) Pinoresinol-^^-^'-^^^ rf H (LXXIXb) (+)-Pinoresinol monoraethyl ether 131 11 Me Or Me H (LXXIX) ( + )-Pinoresinol dimethyl ether"^^"^'^^'^ Me Me - 67 - M«0 pio-^ r^OM« (LXXX) R R1 (LXXXa) Lariciresinol-^^^'-^^^ 'H H (LXXXb) Lariciresinol-4-methyl ether 154 Me (LXXXI) 1 ? 3 R H R R'' (LXXXIa) Isolariciresinol"^^-^'-^^^ H H H H (LXXXIb) Isolariciresinol-4-methyl etherl53,134 H H H Mo (LXXXIc) .rso.lariciresinol-4 '-methyl ether-"-^^ H n Me H ''^3 - (LXXXII) ,1 R R" (LXXXIIa) Secoisolariciresinol-^^-'-'-^^^ H H (LXXXIIb) Secoisolariciresxnol monomethyl ether '~* (+)-Hinokiresinol H OMe 0M« (LXXXIII) Gyciogalg ravin'13 4 Biflavonoids Several biflavonoida 'i.ve been isolated f"n.:.m different T . , T 26a , , . .,, .26a,40,41 , , ..30b AiTaucaria plants. A. excels a , A. b] d\»alli * ' , A. cnokxx and A. cunninghaiiii '30 a constitute amentofiavone rnd their raethvl - o9 - ethers (la, c, d,i, j ,m-p). A. excelsa ^, A. bidvalli ^' ' , A. cunninghamii-^ > 40 > 4 ^ ^^ rulei , A. cookii-^ and Araucaria species constitute agathisflavone and their partial methyl ethers (IXa-e), cupressuflavone and their partial methyl ethero (Xlla, Xlld-g). Hinokiflavone (XXIVa) was only isolated from 7-3 A. cookii . Miscellaneous compounds A. ailgustlfolia »--''"» ^ constitute palmitic, linolenic, oleic and stearic acid, a-(p-hydroxystyryl) C=havicol and Me^C = CHGHoCCH^-GMe = CHCH^)^A (A-hydroxyl or acyloxy group), CHpCMe = GH-GHp = transisoprene Oti2-CMe=GH-CH2-cisigoprene. A. araucana constitute acetovanillone, p-hydroxybenzaldehyde, syringic aldehyde, vanillin, proanthocyanidin, 3,4-dimethoxy- 141,142 benzoic acid. Tannin and C-glycosyflavone from Araucaria species A. bidwilli constitute arabinofuranose, Rhamnopyranose, galactopyranose glucuronic acid, sequence, 0-rhamnopyranosyl (1-4), glucopyranosyluronic acid and (1-6) galactopyranose. A. cookii constitute D-galactose, L-Rhamnose, L-arabinose, L-Glucoronic acid, on hydrolysis it yield 2 oligosaccharide (Gal - (1-2) Rhg. and Galp - (I-6) - Galp). Discussion - 70 - FLAVQ.TOIDIC CONSTITUENT FROM THE LEAVES OF ARAUCARIA ARACJCANA From the acetone extract of Araucaria araucana (Araucariaceae) mono-O-methylagathisflavone, mon-O-methylamentoflavone, di-O-methyl agathisflavone, di-O-methylaraentoflavone and oupressuflavone, tri-O- methylagathisflavone, tri-O-methylamentoflavone and cupressuflavone, tetra-O-methylament of lavone and cupressuflavone have been isola,ted. Mono-O-methylagathisflavone, mono-O-methylamentoflavone and di-O- methyl cupressuflavone were characterized by spectral studies and other constituents were identified by TLG (i.e. comparison of parent biflavones and their methylated derivatives with the authentic sample A number of biflavones have been reported from the leaves of Araucaria bidwilli^ '40,41^ Araucaria cooki^^^. Araucaria cunninghamii ' * , Araucaria excelsa ^ and Araucaria rulei^" 7 The chemotaxonomic significance of the biflavones in genus Araucaria prompted us to investigate Araucaria araucana. The coarsely powdered fresh leaves of Araucaria araucana collected from Lylod Botanical Garden, Darjeeling were extracted with acetone. The acetone extracts were purified by solvent treatment and then treated with water, a yellow solid mass was obtained which responded to the usual flavonoid colour test. Thin layer chromatographic examination (silica gel, benzene-pyridine formic acid, 36:9:5 developing solvent system) of the flavonoidic - 71 - mixture revealed the presence of seven bands labelled as AA--I(R^ 0.4), AA-II(R^ 0.51), AA-III(R^ 0.60), AA-IV(R^ 0.72), AA-V(R^ 0.80), AA-VI(R^ 0.84),and AA-VII(R^ 0.87). The flavonoidic mixture was then subjected to preparative layer chrouiatography (silica gelj 3PF, 36:9:5). After establishing the homogeneity by chromatography m different developers, such as benzene-pyridine-formic acid (36:9:5), toluene-ethylformate-formic acid (5:4:1) and toluene-pyridine-acetic acid (10:1:1), each fraction was methylated, separately. AA-I, AA-III and AA-V gave the same methyl ether which was identical with authentic sample of agathis- flavone hexamethyl ether (R^ values, m.p., mmp. characteristic fluorescent in UV light). AA-II, on methylation, gave hexamethyl ether of amentoflavone. AA-IV, AA-VI and AA-VII, on methylation, gave a mixture of hexamethyl ether of amentoflavone and cupressu- flavone. Characterization of AA-I AA-I was comparable with the authentic sample of I-7-O-methyl- agathisflavone AA-I, on acetylation with acetic anhydride and pyridine, gave AA-IA which was crystallized from CHCl,-EtOH, m.p. 136°C. The result of •'"H-NMR spectrum of AA-IA is given in Table-6 (Fig.l), - 72 - TABLE - 6 Chemical shifts of protons of AA-IA (6 value) Assignment AA-IA (acetate of AA-I) H-I-8 6.94 (s,lH) H-II-6 6.95 (s,lH) H-I-3,II-5 6.52, 6.58 (s,lH each) H-I-3',5' 7.25 (d, J = 9Hz, 2H) H-I-2',6' 7.88 (d, J = 9Hz, 2H) H-II-2',6' 7.44 (d, J = 9Hz, 2H) ^.-11-3',5' 7.01 (d, J = 9Hz, 2H) 'OMe/OAc 1-7,11-7 3.74, 2.05 (s,3H each) 1-5,11-5 2.10, 2.40 (s,3H each) I-4',II-4' 2.30, 2.20 (s,3H each) s = singlet, d = doublet, spectrum run in ODCl, at 60 MHz, trimethyl- silane (TM3) as internal standard. 73 - The NMR spectrum of AA-IA was found to be identical with that o: 1-5,11-5,1-4' ,II-4?II-7-pentaacetoxy, I-7-O-methylagathisflavone . Therefore, AA-I was characterized as I-7-O-methylagathisflavone (iXh). ^^ ^ (iXb)AA-I R-H (LXXXIV)A./V-IA R=Ac Characterization of AA-II AA-II was comparable with the authentic sample of II-7-O-methyl- . ^-, 20b amentoflavone AA-II, on acetylation with acetic anhydride and pyridine gave AA-IIA, m.p. 155^0, which was characterized as 1-5,1-7,11-5,1-4', II-4'-pentaacetoxy, II-7-O-methylamentoflavone by comparison of its NMR data ( Table - 7 ) with that of authentic sample^^^. The result of "^H-NMR of AA-IIA is given in Table-7 (Fig. 2 ). 74 - OR 0 OR 0 (Id) AA-II B;=H (LXXXV) AA-IIA R-Ac TABLE - 7 Chemical shift of protons of AA-IIA Assignment AA-IIA H-I-8 7.20 (d, J = 3Hz, IH) H-I-6 6.75 (d, J = 3Hz, IH) H-I-3,II-3 6.51, 6.60 (s, IH each) H-II-6 6.68 (s,lH) H-I-2' 7.87 (d, J = 3Hz, IH) H-I-6' 7.86 (dd, J = 8Hz, 3Hz, IH) H-I-5• 7.34 (d, J = 8Hz, IH) H-II-2',6' 7.43 (d, J = 8Hz, 2H) H-II-3',5' 6,96 (d, J = 8Hz, 2H) 0\tcVOAc 1-7,11-7 2.25, 3.80 (s,3H) 1-5,11-5 2.36, 2.45 (s,3n) I-4',II-4' 1.93, 2.20 (s,3H) s = singlet, d = doublet, dd = double doublet, spectrum run in CDCl, at 60 MHz. TM3 as internal standard. - 75 - AA-III, on raethylation, gave a permethyl ether which was found to be identical with hexamethyl ether of agathisflavone 7'5 . The R^ value of the parent compound was comparable with I-7,II-7-di~0- 41 methylagathisflavone AA-IV was separated into AA-IVa and AA-IVb by preparative tic (silica gel, benzen-pyridine-formic acid, 40:10:2). AA-IVa, Identified as di-0-methylamentoflavone, compared with 1-4',11-7- v^-methylamentoflavone 73 and its permethyl ether was identical with amentoflavone hexamethyl ether . AA-IVb was acetylated with acetic anhydride and pyridine and was found to be 1-5,11-5,1-4',11-4'-tetraacetoxy, I-7,II-7-di-0~ methylcupressuflavone (AA-IVbA), m.p. 275-80 C. It was characterized by comparison of its NMR data (Table-8) with that of authentic sample'^°''^^(Pig.3). TABLE - 8 Chemical shift of protons of AA-IVbA Assignment AA-IVbA H-I-3,II-3 6,54 (s,2H) H-I-6,II-6 6.81 (3,2H) H-I-3',5',II-3',5' 7.05 (d, J = 8.8Hz, 4H) H-I-2',6',II-2',6' 7.34 (d, J = 8.8Hz, 4H) OMe/OAc I-4MI-4' 2.26 (3,6H) 1-5,11-5 2.51 (s,6H) 1-7,11-7 3.84 (s,6H) s = singlet, d = doublet, spectrum run in CDCl^ at 60 MHz, TMS as internal standard. - 76 - OR 0 OP^ 0 (XIId) AA-lVb R^H (LXXXVI)AA~IVbA ItAc AA-V was comparable with 1-4'fl-Ttll-T-tri-O-methylagathis- pfia flavone and its permethylated derivative was identicaQ. with the agathisflavone hexamethyl ether. It was partially identified as tri-0-methylagathisflavone. AA-VI, an inseparable isomeric mixture of the trtmethyl ethers of amentoflavone and cupressuflavone, was comparable with 73 kayaflavone and 1-4*,I-7,II-7-tri-0-methylcupres3uflavone and on methylation, it gave a mixture of hexamethyl ether of amento- flavone and cupressuflavone 73 . AA-VII was partially characterized as the mixture of tetra methyl ethers of amentoflavone and cupressuflavone by comparison of its permethylated derivative with authentic samples 73 . Experimental - 77 - EXTRACTION OP PLAVONOIDS PROM THE LEAVES OP ARAUCARIA ARAGUAI-IA (ARAUGARIAGSAE) Dried and powdered leaves of Araucaria araucana (1 kg) were completely exhausted with light petrol (40-60). The petrol extr act gave negative test for flavonoids. The petrol treated leaves were completely exhausted with boiling acetone till the extract was almost colourless. The acetone extract was concentrated first at atmospheric pressure and then binder reduced pressure, a dark green mass so obtained was refluxed with light petrol (40-60°), benzene, and chloroform, till the solvent in each case was almost colourless. The residue left behind was then treated with boiling water and filtered, A yellow solid (4 g) thus obtained responded to the colour test with Zn-HCl. Separation of Biflavonoids by Preparative Layer Chromatography Glass plates (40x200 cm) were coated with a well stirred suspension of silica gel using a thin layer spreader (Desaga, Heidelberg) (35 g in 70 ml of water for two plates) to give a layer approximately 0.5 mm in thickness. After drying for 2 hours at room temperature, the plates were activated at 110-120° for 1 hour. ^ ^V,;, ^^y '•^^- - 78 ~ The complexity of the biflavojioid mixture obtained after purification by coloumn chromatography was examined by TLG using the following solvent systems: (a) Benzene-pyridi-K-^-formic acid (BPF, 56:9:5) (b) Toluene-ethylformate-formic acid (TEF, 5:4:1) (c) Toluene-pyridine-acetic acid (TPA, 10:1:1). In solvent system 'a' the spots were compact and the difference in R^ values were so marked as to make it the dove- loping solvent system of choice for preparative TLG. Solution of biflavonyl mixture in pyridine was applied to plates with the help of mechanical applicator (Desaga, Heidelberg), 2 cm from the lower edge of the plates. The plates mounted on a stainless steel frame were plated in a Desaga glass chamber (45x22x25 cm) containing 500 ml of the developing solvent (BPP 36:9:5)« When the solvent front had travelled 16 cm from the starting line, the plates were taken out and dried at room tempe rature. The position of the bands were marked in UV light. The marked pigment zones were scrapped as separate bands and eluted with dry acetone. The eluent in each case was distilled off and on treatment with water yielded yellow precipitate. It was filtered, washed with water and dried. The homogeneity of the pigments was again checked by TLG using the different solvent systems. The components were labelled as AA-I(R-. = 0.47), AA-II (R^ = 0.51), AA-III(R^ = 0.60), AA-IV(R^ = 0.72), AA-V(R^ ^. 0.80), - 7C) - AA-Vl(Rf = 0.84) and AA-VII(R^ = 0.87). AA-I(Rj = 0.47) compared with I-7-O-methylagathisflavone'^ . Methylation of AA-I AA-I was methylated with dimethyl sulphate and anhydrous potassium carbonate in dry acetone on water bath for 6 hours. Refluxing was continued until it gave a negative ale. FeCl, test. It was filtered and the residue evaporated to dryness. The yellow residue left behind was treated with petroleum ether and then dissolved In chloroform and washed with water. The methylated product was found to be agathisflavone hexamethyl ether (R^ and characteristic shade in irv light). I-4'.II-4M--3.II-5.II-7-Pentaacetoxv. I-7-Ometh.vl ri-6.II--8l biflavone (AA-IA) A mixture of AA-I (55 mg), pyridine (1 ml) and acetic anhydride (2 ml) was heated on water bath for 2 hours to give an acetate which slowly crystallized from GHGl^-BtOH as colour less needles (20 mg) m.p. 136°G. H-NMR (GDCl,):Values on 6 scale 2.05(3,3H, OAc-II-7). 2.10(s»3H, OAc-I-5), 2.20( 3,3H,0Ac-II-4'i 2.30(s,3H, OAc-I-4'), 2.40(s,3H, OAc-II-5), 3.74(s,3H, OMe-I-7), 6.52 and 6.58(s,lH each, H-I-3,II-3), 6.94(s,lH, H-I-8),6.95(s,lH, H-II-6), 7.01(d,2H, J = 9Hz, H-II-3',5'), 7.44(d,2H, J = 9Hz, - 80 H-.II-2?,6'), 7.88(d,2H, J = 9Hz, H-I-2',6'). AA-II AA-II (R» = 0.51) was compared with II-7,0-methylamento- ilavone AA-II (10 mg) was methylated by usual method and identified as amentoflavone hexamethyl ether (R^ and characteristic shade in UV light). 1-5.11-5.1-7.1-4'.II-4'-Pent aacetoxy.II--7-0-ffiethvlfI~3'.II--8lbiflavone AA-II (50 mg) was acetylated with pyridine (2 ml) and acetic anhydride (4 ml). The acetate was crystallized with CHGl^-EtOH as colourless needles, m.p. 155°C. •^H-NMR (CDGl,) rvalues on 6 scale 1.93(s,5H, 0AC-.I-4'), 2.20(s,3H, OAc-II-4'), 2.25(s,3H, OAc-I-7), 2.36(3,3H, OAc-I-5), 2.45(3,3H, OAc-II-5), 3.80(s,3H, OMe-II-7), 6.51 and 6.60(s,lH, each, H-I-3,II-3), 6.68(s,lH, H-II-6), 6.75(d,lH, J = 3Hz, H-I-6), 6.96(d,2H, J = 8Hz, H-II-3',5'), 7.20 (d,lH, J = 3Hz, H-I-8), 7.34(d,lH, J = 8Hz, H-I-.5'), 7.43(d,2H, J = BHz, H-II-2',6'), 7.86(dd, IH, J = 8Hz, 3Hz, H-I-6'), 7.87 (d, IH, J = 3Hz, H-I-2'). AA-III AA-III(10 mg) was methylated as usual sind TLO examination of the methylated product was found to be hexamethyl ether of - 81 ~ agathisflavone on comparison with that of authentic sample. The R^ value of AA-III was comparable with I-7,II-7-di-0-methyl- A T agathisflavone but the material was in minor amo\uit therefore AA-IIIwas partiall^^ identified as di-0-methylagathigflavone. AA-IY AA-IV (10 mg) was methylated as usual and TLG examination of the permethylated derivative showed the presence of hexa-0- methylamentoflavone and cupressuflavone. AA-IV was again subjected to preparative TLC in benzen- pyridine-formic acid (40:10:2) arid it was separated into AA-IVa and AA-IVb. AA-IVa AA-IVa (8 mg) was methylated as usual and was partially identified as di-0-methylamentoflavone 73 by comparing it and its -oermethyl ether with authentic sample. AA-IVb AA-IVb was comparable with 1-7,II-7-di-0-methylcupre3Su- flavone'^O''^^ AA-IVb (10 mg) was methylated and the permethyl ether so obtained was identical with cupressuflavone hezamethyl ether. ~ 8? 1-3.II-3.1-4',II-4'-Tetraacetoxy-I-T,II-T-dl-O-methylcupressu- flavpne AA-IVb (30 mg), pyridine (l ml) and acetic anhydride (2 ml) were mixed and heated on water bath for 2 hours to yield an acetate which slowly crystallized from CHCi^-EtOH as colourless needle (20 mg), m.p. 275-80^0. •'•H'NMR (CDGl,):Values on 6 scale 2.26(s,6H, 0Ac-I-4',II-4'), 2.5l(s,6ll, OAc-I-5,II~5), 3.84 {--•,'oE, 0Me-I-7,II-7), 6.54(s,2H, H-I-3,II-3), 6.81(s,2H, H-I-6,II-6), 7.05(d,4H, J = BHz, H-I-3 •S/I-3 ', 5 '), 7.34(d,4H, J = 8.8Hz, II-I-2', 6',II-2',6'). AA-Y AA-V (10 mg) was methylated with dimethyl sulphate and the permethyl ether so obtained was identical with agathisflavone hexamethyl ether. liA-V was therefore partially identified as tri-0-methylaigathisflavone 26a AA-VI AA-VI (5 mg) was methylated m.th. dimethyl sulphate and anhydrous potassium carbonate. The methyl ether was found to be mixture of hexa-0-methylamentoflavone and cupressuflavone. The R^ value of AA-VI was comp^arable with kayaflavone and 1-7,11-7, 83 - I-4'--tri-0-methylcfupressuflavone . Therefore AA-VI WTS partially- identified as a mixture of tri-O-methylamentoflavone and cupressu- flavone. AA-VII AA-VII (10 mg) was methylated as usual. The methyl ether was fotmd to be identical with hexa-0-methyl ether of amento- flavone and cupressuflavone. So AA-VII was a mixture of tetra-0- methylamentoflavone and cupressuflavone Refrcnces - "^4 1. Swain. T.(Ed); Chemical Jr'iant Taxonomy Academic Press, London (1963). 2. 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No. Vol. 100, 13544 9g (1983). 141. Doat, J., Jios, For. Trop 1^, 37-54 (1973). 142. Lebreton, P., Boulard, B., Thivend, S., C.R.Hebd. Seance Acad. Sci. Ser D 287(13) , 1255-8 (1978). 143. Ghosn, T.K., Rao, C.K.V. , Indian J. Cherr. 233 (10) , 958-61 (1984) . Chapter 2 CONSTITUENTS OF GENUS TAXUS (TAXACEAE) Taxus is a member of Taxaceae, a family of 5 genera: Amentotaxas, Pilger, Austrotaxus compton, Pseudotaxus Cheng, Taxus Linnaeus and Torrya am 1 * 2 . The chemistry of Taxus has special significance because of the ocairrence of antitumor taxanes ' , molting hormones ' , ry O alkaloids and other physiologically important compounds in its various species. Although there are scattered reports'^ about various types of natural organic compounds by different groups, yet no attempt has been made for the extensive study of Taxus constituent. Alkaloids , diterpenoids ~ vd.th taxane skeleton, lingnins, biflavojioids and cyanoglucosides are commonly found to occur in 9 Taxus, ^. baccata has been most extensively studied and reviewed amongst the seven species of Taxus. Alkaloids and other Taxane derivatives The naturally occurring oxitanes with the taxane skeleton are the typical constituents of Taxus. In 1980, Miller revievied Tajcus alkaloids and gave comprehensive account of the work done 7 in this area upto 1979 . In the present survey an upto date - 94 - account of naturally occurring taxane derivatives from Taxus species follows. Tlie niimberin^ of taxane compounds is based on lUPAC nomenclatui^e given in structure I ' In 1856, Lucas reported the presence of an alkaloid called taxine from T. bacoata. In the following years, taxine, vAiich " 13,14 produced taxinol anii "taxiTilne, was also louTid In %, cuspid at a Taxine was reported to be a mixture of taxine A, taxine B and T q If. 17—35 Ldf 10—2.0 taxine C ' . After many investigations ^ on T. baccata T. cuspidata'^-^*'^^*^ , !• canadensis^, T, speciosa-^ and T. brevifolia for more than a century, the taxinol(Il) and taxinine (III) were identified in 1963 . In 1954, Bourbeau named a CT^Hrr^N-O-, ,^ compound as taxinine which was isolated from 3( J I y 10 H (ii^i Taxinol T. canadensis^^. Taxine-I, an ester of tajcicin-l (Illb) was isolated from T. baccata as major alkaloid and taxine-II as minor alkaloid 25 *3 7 • 38 39 The heart wood and roots of T. baccata were found to contain baccatin which was identified partially. On further invesliigatioTLS ol "ttie Vieaxiiwood of ^. baccai&a, Halsall and coworkers discovered baccatin-I (iVa) ' , 5-deacetyl baccatin-I (iVb)'^^, l(3-hydroxybaccatin-I (iVc)'^^, baccatin-Il'''^, baccatin-III (Vd)'^°''^^'^^, baccatin-IV (Vla)'^^'^^, 1-dehydroxy- baccatin-IV (Vlb)^^, baccatin-V (Va)"^^, baccatin-VI (VIc)'^^, baccatin-VII (Vld)^^, tetraol (Vh)^^, Taxa-4(20), ll-diene-5T, ga,10p,13a-tetraol-9,10-diacetate (Vlle)'^'^, Taxa-4( 20) ,ll-diene- 5a,7p,9a,10p,13a-pentaol pentaacetate (Vllf)^-^, Taxa-4( 20) ,11- diene-2a,5a,9a,10p,13a-pentaol pentaacetate (Vllg) , Taxa-4(20), 11-diene, 2a, 5a,7(3,9a,10p,13a-hexaol hexaacetate /(Vllh N4) 1 , Taxa-4(20),ll-diene-2a,5cc,7p,10p-tetraol-5,7,10-triacetate-2a- methyl butyrate (VJlIa)^-'", Taxa-4( 20) ,ll-diene-2a, 5a, 7p ,9a:,10p- pentaol-7,9,10-triacetate (Vlllb)^-^, Taxa-4(20) ,ll-diene-2a, 5a, 7p,9a,10i3-pentaol-5,7,9,10-tetraacetate (VIIIc) and taxusin (Vlld) . Taxusin, first isolated from the heartwood of T. brevifolia , T. cuspida'*'a has also been found in T. floridana^'^ and T. mairej (Lemec and Le'Veille) 3.Y. Hu^^. / x47 The hydrolysis of taxusin gave tetraol (Vlllh) . _T. cusp id at a also constitute taxinine (Ilia) , taxinine-A (IIIc) , taxinine-B (IIIe)^°, taxinine-3 (Vllb)^^, taxinine-H (llld)^^. - 96 - taxinine-J (VIIc)^°, taxinine-K (IXa)"^ and taxinine-L (IXb)"^^. Caatellano and Holder determined the crystal and molecular 51 structure of baccatin V(Va) . Taxinine (Ilia) and taxinine-A 52 (IIIc) were also isolated from T. chinensis . Taxine was also found in the leaves of ^. baccata 53 54 and the needles of T. hrevifolia^-'-. In 1971, Wani et al. 55 isolated an antitumor taxane named taxol (Vb) from the stem bark of T. brevifolia, T. baccata and T, cuspidata. In 1981, Ghauviere et al. 56 reported the presence of taxol (Vb), 10-deacetyltaxol (Vc), cephalomanine (Vf), 10- deacetyl cephalomanine (Vg) in trunk, and taxigifine (Xa), tetraol (Vila) and 19-hydroxybaccatin (Ve) in the leaves of _T. baccata. •3 4 In the same year. Miller and coworkers isolated 19-hydroxy- baccatin III (Ve) , 10-deacetylcephalomanine (Vg) , 10-deacetyl taxol (Vc) , taxol (Vb) and cephalomanine (Vf) from T.wallichiana In 1982, Ghauviere et al. 57 isolated another new taxane-taxigifin 58 (Xb). In the same year, Kingston et al. reported two new taxanes, taxa-4( 20) ,ll-diene-2a, 5oc, 7p ,9a,10p,13a-hexaol penta- acetate (Vlli) and Taxol (Vb) from T. brevifolia. In 1984, Long et al. 59 reported the occurrence of taxinine (Ilia), taxusin (Vlld) and Taxa-4(10),ll-diene-5a,9a,10p,13a-tetraol-9a,10p- diacetate (Vile) from the heartwood of T. mairei. while Senilh fin et al. isolated taxol A and taxol B, two new analogs of taxol from T. baccata. - 97 - '--n-"^o •^ R3 0 ^S-OE^OIlFh (III) R1 R^ R^ R'^ R5 R Ta^ininel5,14,49,52 * (Ilia) H Ac H Ac Ac (Illb) Taxicin-I^^»2^»^'^ OH H H H H H 4Q 52 (IIIc) Taxinine-A^^'-^ H Ac H H Ac Ac r49 (Illd) Taxinine-H H Ac Ac H Ac Ac 50 (Ille) Taxinine-B H Ac * OAc Ac Ac 0 II 0-Cv ,CH3l CH3 HaCv//'"' 0 if H3C-C-O'' o=c 'CH3 (IV) R^ R (IVa) Baccatin-l'^^'^^ H Ac - 98 - (iVb) 5-Deacetyl baccatin-i'^^ H H (IVc) ip-Hydroxy baccatin-I OH Ac 0 OH Ph 0 ^ =0-C-CH-GH-lIH-C-Ph 0 0 He Me 4.«^ =0-G-Cn-CH-ITH-G-C=G^ (V) OH Ph H R1 R5 R R^ R (Va) Baccatin-V^^ OH H OH H OAc H (Vb) Taxol4'55,56,58 H OH H OAc H (Vc) 10-Deacetyl Taxol^^ H OH If H OH H (Yd) Baccatin-IIl40'42,45 H OH OH H OAc H (Ve) 19-Hydroxybaccatin^'^^ H OH OH H OAc OH - 99 - R R2 R5 R H5 (Vf) Cephalomannine''''^^ H OH H OAc OH (Vg) 10-Deacetylcephalo- mannme' H OH H OH K (Vh) Tetraol 40 H OH OH H OH H 9,^ H3C'' (VI) R-' R' R' (via) 3aGcatin-IV'^°»'^5 OH Ac Ac (VIb) l-Dehydroxybaccatin-IV^^ H Ac Ac (Vic) Baccatin-Vl'^^ OH COPh Ac (VId) 3accatin-VII 45 CH CO Ac - 100 •-Yr-o-R? 0 Ik = -G-GH=:GH-Ph (VII) 4 Br R' R- K R5 R (Vila) Tetraol (Taxa-4(20), 11-diene-5a, 9a, 10(3, 13a-tetraol)^-'-'^'^ H H H H H H (Vllb) Taxinine-E 41 OAc •f H Ac Ac Ac (VIIc) Taxinine-J 41 OAc * OAc Ac Ac Ac (Vlld) ^ . 41,46-48,59 H Ac H Ac Ac Ac (Vlle) Taxa-4(20),11-diane- 5a, 9a, 10(3,13a-tetraol- 9,10-diacetate'^"'-'^^ H H H Ac Ac H (Vllf) Taxa-4(20),ll-diene- 5a,7(3,9a,lOp,13a- ^^ pentaol pentaacetate H Ac OAc Ac Ac Ac (Vllg) Taxa-4(20),11-diene- 2a,5a,9a,10(3,13a- ^^ pentaol pentaacetate OAc Ac H Ac Ac Ac - 101 - R-, x R R^ R4 R' R (Vllh) Taxa-4(20),ll-diene- ?cc,5a,7p,9a,10p,13a- hexaol hexaacetate OAc Ac H Ac Ac Ac (Vlli) Taxa-4(20),ll-diene- 2a, 5a,7(3, 9a:,lOp, 13a- C Q hexaol pentaacetate OAc H OAc Ac Ac Ac ^o r-C-H CH2CH3 (VIII) R-" R'^ (Villa) Taxa-4(20),ll-diene-2a,5a,7p, lOp-tetraol-5,7,10-triacetate- 2a-methyl butyrate Ac H - 10? - R R (Vlllb) Taxa-4(20),ll-diene-2a,5a,7fi, 9a,10p-pentaol-7,9,10-tria- acetate H OAc (VIIIc) Taxa-4(20) ,ll-diene-2a,5a,7;3, 9a,10p-pentaol-5,7,9,10-tetra- acetate Ac OAc 0. 4 0 o (IX) R 49 (iXa) Taxinine-K H (iXb) Taxinine-L 49 Ac - 103 - 0 O-C-CH3 0-C-CH3 ^^^fl^***0'C-CH=CHPh C OAc (X) 1 R- r 56 (Xa) Taxigifine H Me (Xb) Taxigifine 57 Me Me Ecdysones and Triterpenoid3 Phytoecdysteroids, well knovm molting hormones are another important class of naturally occurring organic compoxinds isolated from Taxus species. Taxus baccata * ' , T. chinensis and T. cuspidata 64' contain ecdysterone (Xi) vmile taxisterone 5 /(XII) , ponasterone A and makisterone A were also isolated from . _54 T. cuspidata. |3-Sitosterol was isolated from T. chinensis and T. mairei 59 • - 104 - (XI) Ecdysterone^'^-'-"^^ (XII) Taxisterone Lifinins *70 A V AQ The lignins are distributed in the roots , heartwood * , leaves and needles * of Taxus species; Taxiresinol (XIII), 105 - isotaxiresinol (XIV) and 3»4-divanillyl tetrahydrofuran (XY) were isolated from Taxus oaccata ' , while roots, stems and needles of Taxus wallichiana 70 contain a different lignin, isoliovil (XVI). T. cuspidata 71 containslignin such as component A, component B (Isotaxiresinol) and component D (Isolariciresinol). The heartwood of T. mairei 59 contains (-)-3ecoisolariciresinol, meso-secoisolaricireainol and isotaxiresinol. (XIII) Taxiresinol^'^*^^ (XIV) Isotaxiresinol*^^'^^ - 106 - •^aC^N^^^^V^'^^ :xT"v xr OH (XV) 3,4-Divanillyl tetrahydrofuran * OH I CH "'X^\x:xxOH (XVI) Isoliovil^'^'^^ Glycosides A novel glycoside, taxi cat in (XVII) was isolated from 72-74- 75 the leaves of T. baccata and T. brevifolia . Its structure as 3,5-dimethoxyphenol glucoside was confirmed by 75 its synthesis from 3,5-dimethoxyphenol and a-acetobromoglucose Another new glycoside which was also named as taxicatine ^^13^22*^7"*"'^^2^^ (XVII) was isolated from T. baccata.'^ . H2OH H HO^^^! /o—^\\ OCH3 (XVII) Taxi cat in ~ 107 - Epiraoric cyanoglucosides(R)-taxiphylline (XVIIIa) and (S)-dhurrine (XVIIIb) along with triglochinine (XlXa) and isotriglochinine (XlXb) were quantitatively determined from T. "baccata 77 using high pressure liquid chromatography. Stereochemical aspects of the biosynthesis of these glucosides no in T. cuspidata have been discussed by Rosen et al. (1975). CH2OH H OH (XVIIIa) (R)-taxiphyllin^'^ (XVIIIb) ( S)-dhurrin''"^ HO2C 0-giu HO2C (XlXa) Triglochinine'''^ (XlXb) Isotriglochinine 77 - 108 - Flavanoid.3 Taxu3 constitutes the biflavanoids of amentoflavone series only. Sotetsuflavone {TL\y)^"^" ^'''" ^'^ gink^^etin (XXl)^'^ and sciadopitysin (XXe) ' were isolated from the leaves of T. baccata ~ and T. cuspid at a • Latter plant also contains PiA. kayaflavone (XXf) . Sequoiaflavone (XXc) was also isolated from T. baccata . The leaf of T. wallichjana contains amentofavone (XXa) mon-0-methyl-amentoflavone, di-0-methyl- amentoflavone and sciadopitysin (XXe). 09? 0 (XX) 1 R- a' R- BP R' OCT (XXa) Amentoflavone -^ H H H H H (XXb) Sotetsuflavone^*^'®^'®^ H CH^ H H H (XXc) Sequoiaflavone GH^ H H H H (XXd) Ginkgetin'^^ GH^ H H CII3 H (XXe) Sciadopitysin'^5'^° GH, H H GH^ GH 84 (XXf) Kayaflavone H GH, H GH, CH - 109 - Miscellaneous Compounds From T. baccata ~ abscisic acid, shikmic acid, quinic acid and hydrocyanic acid were isolated. In 1959 90 Hegnauer reported the presence of hydrocyanic acid in ^. media. T. canadensis 92 contains the.fatty acids such as palmitic, oleic, lenoleic and linolenic, palmitoleic, steric and myristic acid.. Carotenoids were isolated from the leaves of T_. baccat9a3 . Vanillin, COnifereldehyde,a-conidendrin and new biphynyls were isolated from To mairei 59 . Betuloside was isolated by Khan et al. 79 from T. baccata. Discussion - 110 - CONSTITUEIOTS OF TAXUS WALLIGHIANA From the acetone extract of the leaves of Taxus wallichiana Zucc.(Taxaceae), amentoflavone, mono-0-methylamentoflavone, di-O-methylamentoflavone (two derivatives) and sciadopitysin have been isolated. Amentoflavone and sciadopitysin were characterized by spectral studieo and other constituents were identified by TLG (i.e. comparison of parent biflavones and their methylated derivatives with the authentic samples). The occurrence of antitumor taxanes ' , molting ho.rmones * , alkaloids and other physiologically important compounds in Taxus stimulated our interest in Taxus species. A number of biflavones of amentoflavone series have been reported from m V 4. 7^-82,84 , ,-n . , . 84 ^. , ^ ^ Taxus baccata ' ' ana Taxus cusplaaoa Sxeb and Zucc. Taxanes * and lignins have been isolated from Taxus wallichiana Zucc,(Himalayan Yew) and there is no report of biflavonoid constituents. Taxus wallichiana leaves were investigated for biflavonoids and amentoflavone, mono-0-methyl and di-O-methyl amentoflavone and sciadopitysin were isolated and characterized. The coarsely povrdered fresh leaves of Taxus wallichiana Zucc. collected from Royal Botanical Garden, Godawari, Lalitriur (Nepal) were extracted with acetone. The acetone extracts were •^'^'x^. Ill - purified by solvent treatment followed by coloumn chromatography (silica gel) and a yellow solid mass was obtained which responded to the usual flavojioid color test. Thin layer chromatographic examination (using silica gel, BPF, 36:9:5 developing solvent system) of the flavonoidic mixture revealed the presence of four bands labelled as TW-I (R^ 0.18), TW-II (R^ 0.34), TW-III (a-R^ 0.55, b-Rp 0.58) and TW-IV (R^ 0.73) corresponding to amentoflavone, mono-, di- and tri-0-methylamentoflavone, respectively. The flavoxLOidic mixture was then subjected to coloumn chromatography usiiig silica gel and eluted with benzene, benzene ethylacetate (9:1, 8:2, 7:3, 1:1) and then finally with ethyl- acetate. TW-IV was obtained in pure form with benzene-ethylacetate (9:1) as eluent. Rest of the bands,TW-I to TW-III, were present in the fractions benzene-ethylacetate (8:2, 7:3, 1:1) and ethylacetate. These fractions were combined and each band was separated in pure form by preparative TLC (silica gel, BPF, 36:9:5). After establishing the homogeneity by chromatography in different de'•elopers, such as benzene-pyridine-formic acid (36:9:5), toluene-ethylformate-formic acid (TEF, 5:4:1), and toluene-pyridine-acetic acid (TPA, 10:1:1), each fraction was methylated separately. All of them gave same methyl ether which was identical with authentic sample of amentoflavone hexamethyl ether (R^ values, m.p., m.m.p. characteristic fluorescence in uv light). H- CN r (^ h E ex a i>0 U5 h oo - 112 - TW-I was acetylated with pyridine and acetic anhydride to give TW-IA as colourless needles (mop. 241-43 C). The result of H NMR spectrum of TW-IA are given in Table-1.(Fig.l). TABLE - 1 Chemical shift of protons of TW-IA (6 scale) Assignment Chemical shift of protons H-I-8 7.26(d, IH, J = 2.5Hz) H-I-6 6.84(d, IH, J = 2.5Hz) H-II-6 7.01(1H, s) H-I-6' 7.98(q, IH, J-L = 3Hz, J^ = 9Hz) H-I-2' 8.03(d, IH, J = 3Hz) H-I-5* 7.46(d, IH, J = 9Hz) H-II-2',6' 7.49(d, 2H, J = 9Hz) H-II-3',5' 7.06(d, 2H, J = 9Hz) H-I-3,II-3 6.68, 6.65(s, IH each) OAc/l-5,II-5 2.45, 2.4l(s, 3H each) 1-7,11-7 2.05. 2.01(s, 3H each) I-4',II-4' . 2.28, 2.23(s, 3H each) s = singlet, d = doublet, q = quartet, spectrum run in CDCl^ at 60 MHz, TMS as internal standard. - 113 - In the M-1R spectrum of TW-IA, there were evidenced ABX and ApBp systems, associated with ring I-B and II-B, res pectively. Thus ring 1-3 and II-A of the biflavone seemed to be involved in interflavonoid linkage. Thus the structure of TW-IA was assigned as 1-5,11-5,1-7,11-7,1-4',11-4'-hexa- acetoxy[l-3',II-8]biflavone. OR 0 (XXa) TW-I R = H (XXI) TW-IA R = Ac The examination of TW-IV and its methyl ether indicated it to be tri-0-methyl amentoflavone. It was then acetylated with pyridine and acetic anhydride. On usual work up and crysta llization of acetylated product (TW-IVA)^ ojlourless needle like crystals (m.p. 240-41 C) were obtained. The H iH-IR spectrum of TW-IVA integrated for protons of the three methoxy groups and three acetoxy groups. The H NMR data of TV/-IVA and sciadoni- tysin triacetate are given in Table-2. (Fig. 2). - 114 - TABLE - 2 Chemical shift of protons of TW-IVA tind sci ado pity sin triacetate (6 scale) Assignment TW-IVA Sciadopitysin '^'^s.ceta.te H-I-8 6.82(d, IH, J=2.5Hz) 6.85(d, IH, J=2.5Hz) H-I-6 6.62(d, IH, J=2.5Hz) 6.56(d, IH, J=2.5Hz) H-II-6 7.00(s, IH) 6.96(s, IH) H-I-2',6' 8.03-7.93(q, 2H) 7.96-7.80(q, 2H) H-I-5' 6.95(d, IH, J=9Hz) 7.15(d, IH, J=9Hz) H-II-2',6' 7.43(d, 2H, J=9Hz) 7.39(d, 2H, J=9Hz) H-II-.3',5' 6.82(d, 2H, J=9Hz) 6.77(d, 2H, J=9Iiz) H-I-3,II-3 6.63, 6.60(s, 2H) 6.78, 6.57(s, 2H) OMe/OAc I-4MI-4' 3.75, 3.85(s, 3H each) 3.73, 3.83(s, 3H each) 1-7,11-7 3.80, 2.03(s, 3H each) 3.78, 2.03(s, 3H each) 1-5,11-5 2.45, 2.4l(s, 3H each) 2.46, 2.4l(s, 3n each) s = singlet, d = doublet, q = quartet, spectrum run in GDGl^ at 60 MHz, TMS as internal standard. - 115 - The NI^ll spectram of TW-IVA was identical vd.th sciado pity sin triacetate. Thus the structure of TW-IV was assinged as 1-5,11-5 II-7-trihydroxy,I-4',11-4',I-7-tri-0-methylamentoflavone (Sciadopitysin). OCH3 (XXe) TW-IV R = H (XXII) TW-IVA R = Ac Experimental - 116 - EXTRACTION OF BIfLAVONOlDS x''ROM LEAVES OF TAXUS WALLIGHIANA ZUGC (TAXAGEAE) Dried and powdered leaves of Taxus wallichiana Zucc (1 kg) were completely exhausted with petroleum ether (40-60 ). The extracts were concentrated first at atmospheric pressure and then under diminished pressure. An oily green residue left behind gave negative test for flavonoids. The petrol treated leaves were completely exhausted with boiling acetone till the extract was almost colourless. The combined acetone extract were concentrated, a daric green gummy mass obtained was refluxed with petroleum ether (40-60 ), benzene chl02X)form, successively till the solvait in each case was almost colourless. The redisue left behind was then treated with boiling water. The insoluble mass was dissoved in alcohol and dried under reduced pressure. A yellow solid residue (2 g) thus obtained responded to usual flavonoid colour test. Purification of biflavonoid mixture by coloumn chromatography A well stirred suspension of silica gel (100 g) in dry petroleum ether (40-60°) was poured into a coloumn (100 cm long and 50 mm in diameter). When the adsorbent was well settled, the excess petroleum ether was allowed to pass through the col'«.umn. - 117 - The yellow solid residue (2 g) adsorbed on silica gel was added to the colCumn. The coirumn was eluted with organic solvents successively in the order of increasing order of polarity (Table-3). TABLE-3 Solvent Nature of Product 1. Petroleum ether (40-60°) Greenish gummy mass 2. Benzene Green waxy product 3. Benzene-Ethylacetate (9:1) Yellow solid 4. Benzene-Ethylacetate ' Yellow solid (8:2, 7:3 and 1:1) 5. Ethylacetate Brownish yellow solid Fractions 3-5 gave positive colour test for flavanoids. They were checked by TIG, 3rd was in pure fomn v^ile 4th and 5th were mixtures and they were combined. Separation of biflavanoid mixture by preparative layer chromato graphy Using a thin layer spreader (Desaga, Heidelberg), glass plates (40x200 cm) were coated with a well stirred suspension of silica gel (35 gm in 70 ml of water for two plates) to give - 118 - a layer approximately 0.5 mm In thickness after drying for 2 hrs at room temperature, the plates were activated at 110-120 for 1 hour. The complexity of the biflavonoid mixture obtained after purification by coloumn chromatography was examined by TLG using the following solvent systems; (a) Benzene-Pyridine-Forraic acid (BPF, 36:9:5) (b) Toluene-Ethylformate-Fonnic acid (TEF, 5:4:1) (c) Toluene-Pyridine-Acetic acid (TPA, 10:1:1). In solvent system 'a' the spots were compact and the diffe rence in Rf. values were so marked as to make it the developing solvent system of choice for preparative TLG. Solution of biflavouyl mixture (fractions 4bh arid 5th) in pyridine was applied to plates with the help of mechanical applicator (Desaga Heidelberg), 2 cm from the lower edge of the plates. The plates mounted on a steel frame were plated in a Desaga glass chamber (45x22x25 cm) containing 500 ml of develo ping solvent (BPF, 36:9:5) when the solvent front had travelled 16 cm from the starting line, the plates were taken out and dried at room temperatiire. The position of the band mariced in uv light. The marked pigment zones were scrapped as separate bands and eluted mth dry acetone. The eluent in each case was distilled off and on treatment with water yielded yellow precipitate. It - 119 - was filtered, washed with water and dried. The homogeneity of pigments was again checked by TLC using the different solvent system. The components were labelled as TW-I (R^ 0.18, 50 mg), TW-II (R^ 0.34), TW-III (a-R^ 0.55, b-R^ 0.58) and TV-IV (R^ 0.73, 200 mg). TW-I, Methylation TW-I was methylated with dimethylsulphate and anhydiX)U3 potassium carbonate in dry acetone on water bath for 6 hrs. Refluxing continued until it gave a negative ale. FeCl-z test. It was filtered and the residue evaporated to dryness. The yellow residue left behind was treated with petroleum ether and then dissolved in chloroform and washed with water. On TLC examination, the methylated product was found to be ainentoflavone hexamethyl ether. 1-5.11-5.1-7^11-7,1-4 Ml-4'-hexaacetoxyri-3MI--8lbiflavone (TW-IA) A mixture of TW-I (30 mg), pyridine (1 ml) and acetic anhydride (2 ml) was heated on water bath for 2 hours to give an acetate which slowly crystallized from CHCl^-EtOH as colour less needles (20 mg), m.p. 242-43°. "'"H-NMR(GDGl^) : Values on 6 scale 7.26(d, IH, J = 2.5Hz, H-I-8), 6.84(d, IH, J = 2.5Hz, H-I-6), - 120 - 7.01(s, IH, H-II-6), 7.98(q, IH, J-^ = 3Hz, J^ = 9Hz, fI-I-6'), 8.03(d, IH, J = '5Hz, H-I-2'), 7.46(d, IH, J = 9Hz, H-I-5' ), 7.49(d, 2H, J = 9Hz, H-II-2',6'), 7.06(d, 2H, J = 9Hz, H-II-3',5'), 6.68, 6.65(s, IH each H-I-3,II-3), 2.28, 2.23, 2.05, 2.01, 2.45, 2.41(s, 3H each, OAc-I-4',II-4',1-7,11-7,1^5,11-5, respectively). TV/-II and TW-III TW-II and TW-III (minor fractions) were methylated by asual method and identified as amentoflavone hexamethyl ether m.p. 226-27°, Rf Oo40, fluorescent yellow in UV light. TW-IV This fraction was methylated as usual and TLC examination showed it to be hexamethyl ether of amentoflavone. I~5,II-5.II-7-Triacetoxy . I-4MI-4M-7-tri-0-methyiri-3 ' ,11-8] biflavone (TW-IVA) TW-IV (50 mg) was acetylated with pyridine and acetic anhydride and an acetate (TW-IVA) was obtained which was crysta llized from CHCl^-EtOH as colourless needles (40 mg), m.p.240-41°. •4l-NMR( GDGl^) : Values on 6 scale 6.82(d, IH, J =2.5Hz, H-I-S), 6.62(d, IH, J = 2.5Hz, H-I-6), 7.00(s, IH, H-II-6), 8.03-7.93(q, 2H, J-|_ = 5Hz, J^ = 9Hz, H-I-2',6') 6.95(d, IH, J = 9Hz, H-I-5'), 7.43(d, 2H, J = 9Hz, H-II-2',6'), - 121 - 6.82(d, 2H, J = 9Hz, H-II-3',5'), 6.63, 6.60(s, IH, each H-I-3, II-3), 3.85, 3.75, 3.80(s, 3H each, OMe-I-4',11-4,1-7), 2.45, 2i41, 2o03(s, 3H each, OAc-I,II-5,II-7). ftcfrcnces - 122 - 1. Lawrence, H.M.G., "Taxonomy of vascular plants". The Macmillan Co., New York 360 (1962). 2. Willis, J.C, "A Dictionary of the Flowering Plants and Ferns", 7th Ed., H.K.A. Shaw, ed., Cambridge University Press, Cambridge, 1102 (1966). 3. Mclaughlin, J.L,, ^4iller, R.W., Powel, R.G., Smith, C.R. Jr., J. Nat. 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Delia Casa de Marcano, D.F., Halsall, T.G. and ''ornby, G.K., Chem. Commun 216 (1970). 43. Delia Casa de KarcanD, D.F. and halsall, T.G., Chem. Corrp un 1381, (1970). 44. Delia Casa de Marcano, D.P., Halsall, T.G., Castellano, 3 and Hodder, O.J.R., Chem. Comrnun. 1382(1970). 45. Delia Casa de Marcano and Halsall, T.G., Chen. Corrrrun, 365 (1975) . 46. Miyazaki, M., Kazumasa, S., Mishima, H and Kurabayashi, M., Chem. Pharm. 3ull (Tokyo) 16(3) , 546(1968). 47. Srdtman, H., Tsuno, K., Ph/tocnemistry 8 (5), 931(196S). 48. Lee, C.L., Hirosc, Y and Nakatsuka, T., M^kazai Gakkais-^i 2J^, 249 (1975). 49. Chiang, H.C., vnods, M.C., Nakadaira, Y and Nakanis-ii, K., Chem. Commun. 23_, 1201 (1967). 50. W^ods, M.C., Chiang, H.C., Nakadaira, Y and Nakanishi, K., J. Am. Chem. Soc, _92' ^^^ (1968). 51. Castellano, £!.£•, Hodder, O.J.R., Acta Crvstallogr Sect 3 29 (Ft 11) 2566 (1973) 52. Cniang, H.C., Shih Ta -isuch Pao 20_, 147 (1975). 53. Ki'-zoev, Kh. 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Chapter 3 THE GLYCOSIDES (xlycosides are organic compoimds in which there is usually a semi acetal linkage between the reducing group of a sugar and an alcoholic or phenolic hydroxyl group of a second molecule called an aglycone. This link being effected through oxygen, gives rise tq the O-glycosides which are most common in plants. These compounds are easily hydrolysed to the parent sugar and the aglycone by either acid or enzyme. There is a group of compounds known as G-glycoside which resist normal hydrolysis but have IR spectra and yield alkaline degradation products that indicate the presence of sugar like chain. The flavonoid glycosides also occur in acylated form with acid such as p-coumaric, caffeic, sinapic, ferulic, gallic, benzoic, p-hydroxybenzoic, acetic and malonic acids of these, the most frequently found are p-coumaric and ferulic acids. Acy lated glycosides may be recognized by their high chromatographic mobility on paper in solvents such as 15*/ acetic acid and phenol and low mobility in water when compared with the corresponding unacylated glycosides. - 129 - Acylated glycosides have also distinctive spectral proper ties those acylated with aromatic acids are readily distinguished by UV spectroscopy, since the aromatic acid absorption superimposed on the normal flavonoidic spectral bands. The acyl group can then be removed by mild alkaline hydrolysis and the acid present is recovered and identified by standard procedure. In acylated glycosides, there is usually only one acyl group and this is almost invariably attached to one of the sugar hydroxyls and is not directly linked to the flavonoid skeleton. !• Quercetin-Galact oside-Grallat e Quercetin-3-p-D-galactopyranoside-2"-gallate (I) and quercetin- 3-p-D-galactopyranoside-6"-gallate (II) have been isolated from Eaphorbiacean verrucosa and B. platiphyllos.respectively. (I) - 130 - (II) 2. l3orhamnetin~3-0-[6"-0-acet.yll^lucoside The flavonol-0-acyl glucoside (III) has been isolated 2 from Salix viminalis . HoC C-O-CH2 H OH (III) 131 - 3. Naringenin-7-0-r6'-0-P-coumaryl]-B-D-^luco3ide 3 Rahman et al. have isolated an aqylated flavonone glycoside (IV) from nutshell of Anacardium occidentale. O C CH2 HC=HC HO HO (IV) OH O 4. Acylated Aliose-containing S-hydroxyflavone glycoside A novel flavone glycoside has been isolated from the whole plant of Vernonica filiformis and identified as isocutellarein-4'' methyl ether 7-0-p-(6'"-0-acetyl-2''-0-p-allogyl glucoside). OCH3 R = 6-0-acetyl-|3-D-allosyl-p-D-gluGOsyl (v; - 132 - 5. Narin^enin.-T~0-B--D(6"~0--^allo.yl)-^lucop.yranoalcLe Plavanone-0-acylglycoside (VI) h.as been isolated from pods of Acacia fernesiana. H OH 6. Linarin--0-2-methylbutyrate This has recently been isolated from from Valeriana wallichi. CH2 HO-J—"T""^ OH ^ OCH3 (VII) OH O - 133 - (Vila) R^ = H, R^ = Et(Me) GH-CO (Vllb) R^ = H, R-'- = Et(Me) CH-GO Two acylated flavauone glycoside namely Pinocemberin 7-0-p(3'*' -0-acetyl) neohesperidoside (Villa) and Pinocemberin- 7-0-p(6"-0-acetyl) neohesperidoside (Vlllb) have been isolated from Nierembergia hippomonica 7 (VIII) G-glycosides from Swertla .laponica Q Komatsu et al. have reported two G-glycosides (IX) 6-G-p- D-glycopyranosyl genkwanin (Swertisin) and (X) 6-G-p-D-glyGO- pyranosyl luteolin-7-methyl ether (Swertla .japonin) from the herb of Swertla .1aponica» - 134 - V-OH (IX) ?"H^O (X) Acylated kaempferol Glycosides from Aconitum Pour acylated kaempferol glycosides have been isolated from Aconitum noveboracense and A. columbianum, ( i ) Kaempferol-3-(caffeyl glucoside)-7-gluco3ide ( ii ) Kaempferol-3-gentiobio3ide-7-(cafferylarabinosylrhamnoside) (iii) Kaempferol-3-glucoside -7-(P-coumaryl glucoside) ( iv ) Kaempferol-3-(P-coumarylrutinoside)-7-glucoside, ~ 135 - Biflavonold Glycosides M. Konoshima et al.-"-^• •'"•^ have isolated fukugiside (XI) and spicataside (XII) from Garcinia spicata and xanthochyrnuside (XIII) from Garcinia xanthochymus. (XI) Gukugiside p-D-glucose OH (XII) Spicataside p-D-glucose H (XIII) Xanthochymuside p-D-glucose - 136 - ri~3.II-8l-Binarinfienin-.II-7-0-6-Glycoside 12 A new biflavanone glucoaide (XIV) has very recently been isolated from Qarcinia multiflora Amentoflavone-0-filycosides "* Wallace and Markham have reported a series of amentoflavone- 0-glycosides from three species of psilotales, an order that slriows one of the most primitive organization of any living vasealar plant. However, these authors could not establish the glycosyl pattern in the compound and suggested the structure as mono-,di- tri- and tetra-0-glucosides of amentoflavone. Tetrahydro hinokiflavone-Q-glycoside 14 Murthy et al. have recently isolated occidentoside a biflavonoid-C-glycoside in whidi a flavanone unit is linked with a chalcone unit via C-O-C linkage from defatted nuts of - 137 - Anacardium occldentale linn. This constitutes the first example of biphenyl glycoside with reduced heterocyclic ring system. Discussion - 138 - CONSTITUENTS OF THE GENUS GELQNIUM (EUPHORBIAQEAB) The genus Gelonium, a small tree or shrubs distributed in the tropical and sub-tropical parts of Asia and Africa. Three species occur in India. The various part of this genus is well known for its medicinal properties 15 . Since no more work has been done on the genus Gelonium. A mxiltiflorenol (C^QHCQO), hydrocarbon (G-^QH.Q), and metalo- protein has been isolated from Gelonium multiflorum "] ft* n 7 Misra has been isolated steroid from G. bifarium . This is the first report on flavonoid constituent from the genus gelonium. The defatted and crushed leaves of Gelonium multiflorum (procured from PRI Dehradun) were extracted with methanol. The methanol extract was dried and treated with solvent in the order of increasing polarity. The ethyl acetate fraction gave a yellow compound vdiich was further purified by column chromatography over silica gel. Elution with ethyl acetate gave a pure compound which was further crystallized with ethyl acetate-acetone as yellow neddles, mp^300 G. It was named as GM-I. 139 - GM-I; The purity of the compound was checked on TLG (silica gel, polyamide) and paper chromatography using the following solvent system. a. Benzene-pyridine-formic acid (36:9:5) b. Toluene-Ethyl formate-formic acid (5:4:1) c. Chloroform-methanol (8:2, 1:1) d. Acetic acid-water (15:85). The glycosidic nature of the product was evidenced by positive MoUisch test obtained after hydrolysis and by the formation of osazone. It gave pink color with Mg/HGl and the appearance of a maxima at 335 rm and 285 nin in UV spectrum indicated the compound to be a flavone glycoside. GM-I gave green color with Fed, and the presence of a chelated hydroxyl group is further indicated by the band at 3400 cm" in its infrared spectrum. The IR spectrum further showed an a,p-unsaturated ketone at 1650, 1601 cm" and an aromatic ring 900, 850, 770 cm" . The mass spectrum of the glycoside showed base peak at (M-glucose) 314 M"*". The Ti-MR spectrum of GM-I in DMSO-d^ is given in Table-1 (Pig. l). - 140 - TABLE ~ 1 Chemical shifts of protons of GM-I (6 scale) Assignment GM-I H-8 7.03 (d, J = 2Hz, IH) H-6 6.60 (d, J = 2Hz, IH) H-3 6.90 (s, IH) H-2' 7.63 (d, J = 8Hz, IH) H-6' 7.85 (d, J = 8Hz, 2Hz, IH) H-5* 7.68 (d, J = 9Hz, IH) OCH^-7,4' 3.87, 3.90 (s, 3H each) 6 glucosyl protons 3.30-4.0 s = singlet, d = doublet, spectrum run in EMSO-d^ at 60 MHz TMS as internal standard. The H-NMR spectrtun showed an AMX syston in the B ring at 6 7.63 (H-2'), 7.85(H-6') and 7.68(H-5'). The two aromatic methoxyl appear at 6 3.87 3.90 (OCH,-7,4'). The A-ring protons showed an AB system of two aromatic protons (6 7.03, H-8; 6 6.60 H-6). The H-3 proton appear as a singlet at 6 6.90. The anomeric proton, H-1" of the glucose appeared as a doublet at 6 5.69 (J = 7Hz). The chemical shift showed the direct attachment to - 141 - the a^lycone and the diaLXial coupling (J = 7Hz) between H-1" and H-2'' indiGated the p-Gonfiguration. The UV spectra of GM-I with shift reagent is giren in Table-2. TABLE - 2 MeOH X nm: 285, 335 max + AlCl^ 303, 365 + AlCiyHGl 303, 365 + NaOAc 288, 335 + NaOAc/H^BO^ 288, 335 The UV spectram of GM-I showed a bathochromio shift only upon the addition of AlGl^ which indicated the free hydroxyl at 5-position. The mass spectrum of the glycoside showed the base peak at m/z 314 (M-glucose) and other peaks at m/z 167(A-, f H) 148(B2) , 285, 271, 138 and 133. The mass spectrum is fully in agreement with one methoxyl and one hydroxyl in both ring A and B of the aglycone molecule. - 142 - Hydrolysis of GM-I with 6/ HCl gave an aglycone GM-IA, m.p. 236, M"*" 314 (calcd for G, 65.0; H, 4.49). The UV spectrum is given in Table-3. TABLE - 3 MeOH A ^• 285, 335 , max + AlCl^ 303, 365 + AlCl^/HCl 303, 365 + NaOAc 288, 335 + NaOAc/H^BO^ 288, 335 + NaOMe 292, 330 GM-IA was acetylated with pyridine and acetic anhydride to give an acetate GM-IAA, m.p. 185°. The result of •'•R-NMR spectra is given in Table-4. TABLE - 4 Chemical shift of protons of GM-IAA (6 scale) Assignment GM-IAA H-8 6.83 (d, J = 2Hz, IH) H-6 6.58 {d, J = 2Hz, IH) H-3 6.47 (s, IH) - 145 - (Pable-4 continued Assignment GM-IAA H-5' 7.02 (d, J = 8Hz, IH) H-2' 7.54 (d, J = 2Hz, IH) H-6' 7.69 (dd, J = 8Hz, 2H2, IH) OAc-5,3' 2.35, 2.42 (s, 3H each) OMe-7,4' 3.78 (a, 2 OGH^) s = singlet, d = doublet, dd = double doublet spectrum rum in CDGl^ at 60 MHz, TMS as internal standard. The NMR data confirmed the presence of two acetoxy and two methoxy groups in the aglycone molecule. The glycoside GM~I was methylated and then hydrolysed (the methylated product) with 6/^ HGl to give an aglycone characterized as 3'-0H, 5,7,4'-trimethoxy flavone m.p. 220°. The UV spectrum is given in Table-5. - 144 - TABLE - 5 . MeOH X nm: 285, 335 max + AICI3 285, 335 + AlCiyHGl 287, 335 + NaOAc 285, 335 + NaOAc/H^BO^ 285, 335 + NaOMe 290, 335 Prom UV data it was confirmed that the sugar was attached with 3'-position of the aglycone molecule. The identity of the svigar was confirmed as glucose by co-paper chromatography with an authentic sample. From these data it was confirmed that GM-I is a new flavone glycoside (XV). CH2OH H3C0 OCH3 Experimental - 145 - EXTRACTION OF THE LEAVES OF GELONIUM MJLTIFLORUM (EUPHORBIACEAE) Air dried, powdered leaves (500 gm) were exhaustively extracted by refluxing with methanol. The methaaol concentrate was successively treated with petroleum ether (60-80 ), benzene and chloroform. A brown semi solid mass was left behind. It was subjected to coloumn chromatography over silica gel. Purification of flavonoidic mixture - column chromatography A well stirred suspension of silica gel (50 gm) in petroleiim ether (60-80 ) was poured into a column (100 cm long and 50 mm in diameter). When the adsorbent was well settled, the excess petroleum ether was allowed to pass through the column. The semi solid mass dissolved in methanol was adsorbed on silica gel in a beaker. The excess solvent was evaporated and the dry residue was transferred over to the column. The coltimn was successively eluted with petroleum ether (60-80°), benzene and ethyl acetate. The ethyl acetate fraction on concentration gave yellow solid, which was further crystallized with ethyl acetate-acetone as pale yellow needles, m,p. ^300*^0 and it was labelled as GM-I. It gave orange color with Mg/HCl and green color with FeCl,, In UV light it appear as a brown spot. - 146 - GM-I UV .MeOH 285, 335 nm; (MeOH-AlCl3)-296, 355 nm; (MeOH-AlGl,-HCl) max -296, 355 nm; (MeOH-NaOAc)-288, 335 nm; (Me0H-Na0Ac-H^B0^)-288, 335 nm; (MeOH-NaOMe)-290, 335 nm. •^H-NMR (DMSO-dg): values on 6 scale 6.60, 7.03(d, IH each, J = 2Hz, H-6, H-8), 6.90 (s, IH, H-3), 7.68(d, IH, J = 8Hz, H-5'), 7.85(dd, IH, J = 2Hz, 9Hz, H-6'), 7.63(d, IH, J = 2Hz, H-2'), 2.87, 3.90(3H each, 0^^-7,4'), 3.30-4.0(6, glucosyl protons). IR VSx ^'^"•^^ 5400, 1650, 900, 850, 770 Mass MS: (M-glucose)"*" 314(M'^), 285(M'^-29), 271(M'*"-43) , 268(M'*"-46), 167. Hydrolysis of GM-I The anhydrous glycoside (35 mg) was hydrolysed by refluxing with 50 ml of 0.6N HCl at 100°C. The hydrolysis appeared to be completed within 30 minutes. The refluxing was continued for two hours to ensure complete hydrolysis. After leaving overnight. - 147 - the yellow aglycone thus separated out was filtered, washed well with water and dried. The crude product crystallized from methanol in yellow needles of GM-IA m,p. 236 . Anal. calcd. for 0-^rjE^^O^: C, 65.0; H, 4.49, Poxind: C, 64.60; H, 4o76, Characterization of the aglycone _ w KBr _-! The IR spectra of aglycone was found, to have Vj^^^^^ cm -^, 3400(OH), 1650(G=0). UV MeOH X 285, 335 mn; (MeOH-AlCl3)-303, 365 nm; (MeOH-AlGl^-HCl)- max 303, 365 nm; (MeOH-NaOAc)-288, 335; (MeOH-NaOAc-H^BO^)- 288, 335; (MeOH-NaOMe)-292, 330 nm. Acetylation The aglycone (20 mg) was heated under reflux with acetic anhydride (2 ml) and pyridine (1 ml) over a water bath for two hours. It was poured onto crushed ice, scrated and left over night, on crystallization from ethanol. It gave colorless needels of GM-IAA, m.p. 185°. -'•H-NMR (CDCl^)-.Values on 6 scale 6.83(d, IH, J = 2Hz, H-8), 5.58(d, IH, J = 2Hz, H-6), 6.47(s, IH, H-3), 7.02(d, IH, J = 8Hz, H-5'), 7.54(d, IH, - 148 J = 2Hz, H-2'), 7.69(dd, IH, J = 8Hz, 2Hz, H-6'), 2o35, 2.42 (s, 3H each, OAc-5,3'), 3.78(s, 6H, 20CH^). Methylation The glycoside (10 n^) was methylated vd.th dimethyl sulphate and anhydrous potassium carbonate in dry acetone for 5 hours. The methylated product was hydrolysed with 6/[ HGl to give an aglycone vdaich was characterized as 3'-0H, 5,7-4'- trimethoxy flavone,m,p, 220°. UV ^ MeOH X 285, 335 nm; (MeOH-AlCl3)-287, 335 nm; (MeOH-AlGl,-HCl)- max 287, 335 nm; (MeOH-NaOAc)-285, 335 nm; (MeOH-NaOAc-H^BO^). 285, 335 nm; (MeOH-NaOMe)-290, 335 nm. From these data it was confirmed that the sTXgar moiety was present at 3'-position. Chromatographic identification of sugar The acidic filtrate left after filtering the aglycone was extracted with ether and then with ethyl acetate 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 - 149 - paper. The syrup was chromatographed on Whatman No.l filter paper using butanol-acetic acid-water (4:1:5), employing the descending technique. After rtmning for 24 hours and drying at room temperature in fume cupboard, paper was sprayed with aniline hydrogen phthalate. The chromatogram on drying at 100-105° showed the presence of glucose only. Estimation of sugar The quantitative estimation of sugar by Samogyi'a copper micro method gave the value (0.44 ml) which correspond to 1 mole of sugar per mole of aglycone. Refrences 150 - 1. Nahrstedt, A., Dumkow, K., Janistyn, B, and Phol, R., Tetrahedron Letters, 559 (1974). 2. Karl, G,, Pederson, P. A. and Schhwarz, C,, Phyto ohemistry 16, 1117 (1977). 3. Rahman, W., Ishratullah, Kh., Wagner, H., Seligmann, 0., Ghari, V.M. and Osterdahl, B.G., Phyto chemistry, 1/7, 1064 (1978). 4. Ghari, V.M., Grayer-Berkmeijer'Harbome, J.B. and Osterdahl, R.G., Phytochemistry, 20, 1977 (1981). 5. Blsissi, H.I., Saleh, N.A.M., Elnegoumy, S.D,, Wagner, H., Iyengar, M.A. and Seligmann, 0., Phytoch«nistry, 13, 2843 (1974). 6. Ghari, V.M., Jordon, M., Wagner, H. and Thies, P.W., Phytochemistry, 16, 1110 (1977). 7. Ganzalez, M.D. and Pomilio, A.B., Phytochemistry, 21, 757 (1982). 8a. Komatsu, M., Tomimori. T. and Michiko Ito, Ghem. Pharm. Bull. (Tokyo), 15, (3), 263-69 (1967). b. Komatsu, M., Tomimori, T. and Michiko Ito, Chem, Pharm. Bull. (Tokyo), 11, (10,^, 1567-72 (1967). 9. Young. D.A. and Stener, R.W., Phytochemistry, 20, 2055 (1981). 10. Konoshima, M., Ikeshiro, Y,, Tetrahedron Letters, 20, 1717 (1970). 11. Konoshima, M., Ikeshiro, Y. and Miyahara, S., Tetrahedron Letters, 48, 4203 (1970). 12. Ghing-Fa, Meeilin-yuh and Jen-Chin Hung, Phytochemistry, 14, 818 (1975). - 151 - 13. Wallace, J.W. and Markham, K.R., Phytochemistry, 17, 1313 (1978). 14-a. Murthy, S.S., Anjaneyulu, A.S.R., Row, L.H., Pelter, A. and Ward, R.S, Abstract, 68th Session of the Inuian Science Congress, Varanasi, p. 67 (1981). b. Rahman, W.,Unpublished results. 15. The wealth of India Ed. Sastri, 3.N., 1956, GSIR, New Delhi, pp. 122. 16. Khastgir, H.N. and Sengupta, P., Chem. Ind., 945 (1961). 17. Montanaro, L., Sperti, S., Zamboni, M., Ital J. Biochem 34(1). 1-10 (1985). 18. Misra, D.R., Naskar, D.B., Ray, T.K. and Khastgir, H.N., Phytochemistry, 12(7). 1819 (1973). 19. Somogyi, M., J. Biol. Chem., 195, 19 (1952). Chapter 4 CONSTITUENTS OP GENUS ACER (ACERACEAE) Acer is a large genus consisting of about 150 species. Most of them are trees and a few are shrubs, chiefly distri buted in the north temperate region. They are commonly found in eastern parts of North Aiaerica and East Asia. About 15 species occur in the Himalayas . The chemical examination of 2-15 Acer species revealed the presence of flavones , terpe noid s""-^"-^^, glucosides^^"^"^, tannin^^"^^, aromatic acids^^"^^ 67-99 and some other compounds . Flavones Michiyo first isolated the quercetin from the leaves of 2 "^ A. aizuense . It was also found in the leaves of A, marmoratum , 3 5 3 4- A, carpinifolium * , A. diabolicum , A. pseudoplatanus and in 6 7 Acer . Kaempferol was isolated from the leaves of A. negundo and A. pseudoplatanus . A. pseudoplatanus constitute luteolin. Plavonoids were also isolated from the leaves of Acer8 '9 , 10 11 12 9 A. saccharum , A. negundo and A. campestre . A. saccharum constitute another flavonoid 3-methoxy quercetin. - 153 - Terpenoids and steroids Kupehan et al. isolated the novel triterpene aGerotin(Ia) and acerocin(Ib) from the tumor inhibitory saponins of A. negvmdo. Acerogenin (II) was isolated from the heartwood of A. caesium . From A. saccharum , isoprene and monoterpene 17 were isolated. In 1983, Svoboda et al. isolated 24-methylene- 7 7 cholesterol, sitosterol, -stigmasten-3p-ol, , 24(28)- campestadien-3p-ol from the pollen of Maple. From the leaves -I O and stem bark of A. nikoense ^ p-amyrin, p-sitoterol, campes- terol, stigmasterol and p-amyrin acetate were isolated. 19 59 p-Sitosterol was also isolated from A. ginnala and A. negundo. MQ Me Et (M^CH C(0)" Mc P^^ Et(M«)CH R-^ R^ R^ (la) Acerotin X Ac Me (lb) Acerocin Z Ac Me - 154 - Me Me M«Ma (II) Acerogenin 17 Glycosides 1 ft Tl PT The novel compounds aceroside I (^25^'52*^8^ (Ilia) »'- » , aceroside IV (Illb)^^, epi-rhododendrin (G-L5H24O7) (IV)^^»^*^ and arbutin (V) 20 were isolated from leaves and stem bark of TO pA PI A. nikoense * * . Besides these, p-sitosterol glucoside and quercitrin were also isolated ^^ \6 15 (III) (Ilia) Aceroside I 20-22a p-D-glucose i •'"OH (Illb) Aceroside IV 20 p-D-glucose 0 - 155 - H HO--^^^"""^^ OR R (IV) Bpirhododendrin^®•^° p-D-glucose H( '/ \^ -p-D-glucopyrano syl (V) Arbutin^O 22 In 1983 Nagai et al. isolated aceroside III ('^•50^40^12^ (VI), aceroside VI (C25H,208*-^/-^2^^ (VII), apiosyl epirhodo- dendrin (VIII) and apiin (IX) from stem bark of A. nikoense. CH2OH (VI) Aceroside III 22 - 156 (VII) Aceroside VI 22 H p-D-glucopyranosyl H I HO '/ \N CH2-CH2- C —CH3 OR* R (VIII) Apiosyl epirhododendri._2n 2 p-D-apiofuranosyl (1 —> 6)-f3-D-gluGopyranosyl CH2OH OH OH (IX) Apiin22 157 - Afzelin was isolated from the leaves of A. carpinifolium 3' 5 •:? -7 (T A. diabolicum and A.'marmoratum . A.carpinifolium also consti- 3 tute trifolin. Hyperin was found in the leaves of A. negimdo . Vitexin, saponaretin and homoorientin were isolated from the leaves of A. palmatujfl" ' while hyperoside was isolated from A. pseudoplatanus and heteroside from A. negundo and 3 A. pseudoplatanus . Two antibacterial glycosides ginnalin B(X) and ginnalin C (XI) were isolated from the leaves of A. ginnala which show the activities in vitro towards Shigella flexneri said Z. sonnei, O A etc. Geranin was isolated from A. palmatum . CH OH (X) Ginnalin B 19 (XI) Ginnalin G"^^ 158 - Tannins Crystalline tannins which were called as acertannin were isolated from A. js^innala * and A. spicatum" Its structure 27 (XII) was established by Klaus et al. in 1980. Aceritannin (XIII) was isolated by Jingyun et al. which shows the bacterio 29 static principles. Tannin was also isolated from A.mandschurictun CHjO-gaiioYi HON / OH o-gaiioyi O2C—// ^V-OH (XII) Aceritannin27 (XIII) Aceritannin Tsujimura^'-^*^ isolated the Maple tannin (XIV) from the leaves of A. aizuense. A. nefiundo 31 also constitute the tannin. Grallotannins were found in A. platanoid and A. compestre . The leaves of A. saccharum 35 also constitute the tannin. - 159 - lll-GO H G (XIV) Maple tannin^*^° glucose Acids A. negundo ~ constitutes oleanolic acid , butelinic acid , ursolic acid , ammo acid , allantoic acid * , •7 Q "2 0 octadecatrienoic acid include 9-octadecenoic , 11-eicosenoic , 13-decosenoic , 15-tetracosenoic and 9,12-octadecadienoic 38 39 39 39 acid . It also constitute palmitic , oleic , linoleic , 39 39 39 39 39 ferulic , vanillic , p-hydroxybenzoic , syringic , sinapic 39 39 "^9 p-coumaric (XV), p~hydroxyphenyl acetic , caffeic , 39 39 39 protocatechuic , quinic , shikmic and 2,6-dihydroxybenzoic acid^^. CH=CK-COOH R (XV) P-coumaric acid H 160 - _A. saccharinum '" constitutes abscisic acid where as A. saccharum also constitutes abscisic acid * along with gallic acid (X¥I), gibberellic acid (XVII), p-coumaric , and ferulic acid 47 . Anisic acid, vanillic acid, gentisic acid, caffeic acid and sinapic acid were also isolated from g A. saccharum . .0 H HOOC COOH 44 (r/I) GsULlic acid (XVII) Gibberellic acid^^ A. pseudo plat anus """ constitutes allantoic acid , abscisic 49.51 50 52 53 Amino acid ' , gallic acid , amino acids and fatty acid , acidsand gibberelic acid were fovmd in A, tataricum' 55 . A. penta- 56 pomicum constitutes gallic acid and ester of gallic acid (Me,3,4,5~trihydroxy ben^oate, B,3,4,5-trihydroxy benzoate). 58 59 A. oircinatum constitute syringic acid where as A. spicatum constitute inositol hexaphospheric acid, gallic acid, ellagic acid (XVIII). Shikmic acid were found in the leaves of A. buer^eranixm ' • A. plataiioid constitutes linolenio acid^ -61 palmitic acid and linoleic acid where as the fatty acid ester of lutein such as 3-nionopalmitate, 3'-nionopalmitate, 3-monolino- lenate, 3'-cjonolinolenate, 3,3'-dipalmitate, 3-palraitate 3'-linolenate, 3-linolenate 3''-palmitate, 3,3'-dilinolenate and acid we 63 63 linolenic acid were found in A. platanoid and A. campesure , ..64,65 A« platanoid also constitutes free gallic acid and ascorbic acid. Ascorbic acid was fo\ind in A. ne^tmdo 64 0,C -0 OH HO-// \\_<^^oH b-cb (XVIII) Ellagic acid^^'^-^ Acerogenic acid (XlXa) and 24-hydroxyacerogenic acid (XlXb) 14 were isolated from A. negundo . Me M« Me R-' (XlXa) Acerogenic acid H H Me (XlXb) 24-Hydroxyacerogenic acid 14 H H GHjOH - 16?. - Miscellaneous compoimd In 1978 Inoue et al."^21 isolated scopoletin, (+)-rhododendrol, (+-)-catechin and epi-rhododendrin (Cj^^Hp.Or,) from stem bark of A* nikoense. A. ne^undo constitute p-phenylethylamine, tysamine, histamine, acetylcholine, propinyl choline and choline. A. saccharum * constitutes catechol and kinetin (XX) in the wood tissues and bark. Catechol was also found in A. rubrum while kinetin was isolated from A. tataricum 54 NHCH2 fl N—k^^ (XX) Kinetin44'54 66 20,75 fflobotannin was isolated fx'om A. plataiioid , A. nikoense constitute acerogenin A(XXIa), acerogenin B(XXIb) and rhododendrol (XXII). - 165 - 1 R R' (XXIa) Acerogenin A 20 H •'OH H (XXIb) Acerogenin B^^*"^^ H < OH H '/ \^ HO 2 2 I 9 OR R 20 (XXII) Rhododendrol H Saponins were isolated from the leaves and bark of fi7 fi7 A. platanoid and A, saccharinuni . Lignins were also isolated es from different Acer species. A. platanoida * constitut carotenoids such as a-carotene, p-carotene, xanthophyll, violaxanthin and neoxanthin...7.0 Lutein 5-linolenate was also isolated from A. platanoid Anthocyanins were isolated from 7T 71 '7 f\ A. platanoid , A. pal mat urn , A. negunda and some other 72 '^3 angiosperms . A_, saccharum' contain some catechols such as - 164 (-) epicatechol, (-) gallocatechol, (-) epigallocatechol and epig alio catechol gallate. Y-gl^"tamyl peptide and p-fluorophenyl- alanine were isolated from A. pseTxdo plat anus ' . 2,6-Dimethoxy p-benzoquinone (XXIII) was isolated from the genus Acer which show an anti-inflammatory action. M«0 (XXIII) 2,6-dimethoxy-p-benzoquinone VQ RO The leaves of A. platanold * contain liposol. Carbo- hydrate, lignin and polyphenols were isolated from A. negendo 81 , A. saccharum , A. rub rum and A. pensylvanicxun . A. saccharum also constitute 1-triacontanol. Polygalitol was isolated from 83 Q4 the leaves of A. ginnala . Hydrocarbans were found in A.tataricum. Gsirbohydrates were isolated from A. pseudoplatanus , 95 96 97 98 A. saccharum '^*^ , A. negundo and A. tataricvun^ . Discussion - 165 - CONSTITUENTS OP ACER OBLONGOM (ACERAGEAE) From the methanol extract of the leaves (2 kg) of Acer oblongom (Aceraceae) p-amyrin, p-sitosterol, apigenin, kaempferol, quercetin, ethyl gallate and D-2-O-methyl-^Mro-inositol were isolated. p-Amyrin, p-sitosterol, quercetin, ethyl gallate and D-2-0--methyl-chiro-inositol were characterized by spectral studies and other constituents were identified by TLG and m.p. (i.e. comparison with the authentic sample). The coarsely powdered fresh leaves (2 kg) of A. oblongom collected from Royal Botanical Garden, Godawari, Lailitpur (Nepal) were extracted with hot methanol. The methanol extract were concentrated first at atmospheric pressure and then under reduced pressure. A dark green gximmy mass was obtained this was treated with petrol (60-80 ), benzene, ethyl acetate and then with methanol. The petrol fraction and benzene fraction were mixed and vras purified by column chromatography and by preparative layer chromatography. Two compo\ands were separated out by crysta llization. - 166 - AO-I m.p. 258 was not completely identified. AO-II AO-II, m.p. 198°G analysed for G^QH^QO (M"^ 426). The re sill t of hi NMR spectrum of AO-II is given in Table-1. TABLE - 1 Chemical shift of protons of AO-II (6 scale) Assignment Chemical shifts of protons 8CH, 0.78(s, 3H), 0.83(s, 3H), 0.88(s, 6H), 0.95(s, 3H), 0.98(s, 3H), 1.0 (s, 3H), 1.14(s, 3H) -CHp and -CH protons 1.08-2.01, 3.21(dd, J = 9Hz and 7Hz, of cyclic system and IH) side chain -OH proton 4.88(s,br, IH) Olefinic proton 5.2l(m, IH) s = singlet, dd = double doublet, br = broad, m = multiplet, spectrum run in CDCl^ at 60MHz, TMS as internal standard. - 167 - In the mass spectrum it gave the molecular ion at M 426 and IR spectrum showed the; bands at 3360, 2960, 2880, 1650, 1465, 1040 and 890 cm" . Prom these data and the direct comparison with authentic sample it was characterized as p-amyrin CXXIV). H3C ,CH3 (XXIV) AO-III m.p. 136-7°, soluble in OH CI,. The resiat of -^H NMR is given in Table - 2. TABLE - 2 Chemical shift of protons of AO-III (6 scale) Assignment Chemical shift of protons 18-Me 0.68(3, 3H) 28-Me 0.74(d, J = 6.SHz, 3H) 26,27-Me 0.84(d, J = 6.5Hz, 6H) - 168 - Table-2 continued Assignment Chemical shift of protons 21-Me 0.92(d, J = 6.5Hz, 3H) 19-Me 1.0l(s, 3H) 3-ax-H 3.52(m, IH) -OH proton 5.15Cm, IH) Olefinic proton 5.39(m, IH) -GH2 and -GH 1.07-2.34 protons of cyclic system and side chain s = singlet, d = doublet, m = multiplet, spectrum run in CDGl^ at 60 MHz, TMS as internal standard. The mass spectrum shows the molecular ion at (M 414) and IR spectrum exhibit bands at 3400, 2900, 2830, I64O, 1470, 1380, 1370, 1360, 1065 and 810. From these data and the direct comparison with authentic sample it was characterized as p-sitosterol (XXV). - 169 - (XXV) The ethyl acetate fraction was purified by column chroma tography and different constituents were separated out. AO~IV Yellow crystals from benzene-acetone, m.p. 346 G, R^ = 0.55 36:9:5). It was comparable with the authentic sample of 5,7,4'-trihydroxyflavone (apigenin). AO-V Yellow crystals from benzene-mathanol m.p. 276-8°C, lU = 0.53 (BPP, 36:9:5). It was comparable with the authentic sample of 5»7,3',4'-tetrahydroxyflavone (kaempferol). AO-VI Yellow solid from methanol, m.p. 155*^, R^ = 0.50 (BPF, 36:9:5) The result of -"-H NMR is given in Table - 3 (Fig. 1). - 170 - The triplet at 6 1.316 and a quartet at 6 4.24 were assigned to methyl and methylene protons, respectively. A singlet appea ring at 6 7.11 integrating for two protons is attributable to H-2,6. TABLE - 3 Chemical shift of protons of AO-VI (6 scale) Assignment Chemical shift of protons CH^ 1.3l6(t, J = 7Hz, 3H) CH2 4.24(q, J = 7Hz, 2H) H-2,6 7.11(s, 2H) s = singlet, t = triplet, q = quartet, spectrum run in DMS0-d6 at lOOMHz, TMS as internal standard. 1"^ The result of "^C MR is given in Table - 4 (Figs. 2,3). The signal at 6 6.955(t) and the signal for identical carbons at position 2 and 6 appeared at 6 109.98(d). The signal for C-3 and C-5 appeared at 6 145.96(s). The signals at 6 122.2(s) and 6 138.53(s) were assigned to C-1 and C-4, respectively. The signal for the highly deshielded carbonyl carbon appeared at 6 I66.84(s). "• Ilirjl ™}! !;' ^:ii:'': :;;• i'i.i ^' " "'•]". i ^ ! 'Ti •1 : I , r^-rr-1—! ••V"|'"l.T TTTFiil ... ^ ... , 1 (-•• •:i i • i 1 . 1 ••r:^:iii ^^1 •• ;• .. 1 : ! •;•' ! 1 1 : j 4 i : i j i i ! y • j ! iii? 1 ... ; •• • -, „__ •••;•• : ;;•! ; :i • ; ...j • 1 ' ••:•; 1 i. • i ; !, j... \ 'i • •i I- 1 ! -i .i r :••• •:ii ••.: ;':!;|i! ii' i'" m 1 : i i ! 1 i :::?• ^i.i i i ,... ::;CN:;: 1 •::^r ,;: -1- ••• • 1 -f-j V ;::: ::;: ::>^;" ;;: — ;;;;; 1 " 'T -=J r = •i'ii.i ilii iiii = =i= : •1 ,• iii; .;,, ::;: iii: •:..: . i ...: . 3^ •:'•' :ii;i ;';• il •; ;•:;• :'.'i iii: ,i:: 'i^i iii'iiii iiiii :; ;;;' ..'.. .:.. •'4 ••-• :- rifij 1; >;•• il^i ••" •";" ii' liH .::: •ii: :;;:.::. r::b fi:: .... , , • . ,1 ''•'i'i i •:;• ;:; :: :^^':^1 i • : ,1 ii^^ ~ ii-|r- :;:: :::: ;:;:; 1 ;;.; ii\v}}. .ii; i::; ::i iiii' •: : :.., 'A •ii' Ii ;,:;..: t" "'i! :|ii :::i 5-: i"i :.• -•i:'i,:.iiii iiii •-? ;i-' .... _i. ^i •:'! 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'.'.',', 1 :;;• M ^::;. ii ;:;; iii iliiji •=i: ii: is iiii iiii^l :ii iii' 1 1 I i 1 rrrf iii" . •'. i'l iii iii ;:• :•:; ^i'-ii iiii i:i •::i i::: iiii r' iii . ;; ; 1 1 iliiitiiii % iiii • :i iii: ::::[., :!:: Ii! iiii^ iii; iiiii""; '"i ! ::;, I :;; •::: i! iiii iii ii^: 1:: : :i;' iiii ::: iii iiii !i:ii ,:;;i;iu rrr:: ;::. ii;iii ;;:: hi- iiii jiiii ••ii 1' "'iiii: liiii| ij'ii'ii ;;;• lii B •••• ii^- :~ '•i ;.! ::;. •• i' • 1If '. % ;^ .:l iiii ii'i iiii iiii iiii i iii li^^iii x>:: •rr*T- -r ^•i ; iiii r ' !•* !':i.:iiii::: lii. ; ' ;::. ii: :;,: :;ii ;!;; ii :::. '•ii ;:: :i-Tj :;! iiii •iiiiii 1 \^ i!i: \ :r i:;i 11 ii.^ ;ii i i 1 i;; '!•: :.; ::• if :., 1 ^:i iiii •:: liiiii^i "I iii^ iiiii^iiii iiiii :;. ;.: .;'(.<; 1 'ii: III :'. :.i. :;• ^;i Hi- :i i:::|i' iiii iiii! ii:;: ;:: iii f. ' 'iii :;!!::iii!: i;|,:;::: ::••.: :,i :ii. .ii. i ;' ;:; li-il •ii i'iiiii^iii' ;: :,;• : iiJi: : ;•. :i; :i iii .... •rr-T r: a..;...... ;li, — I'' •:;• ••:• r •; .;; i.ii. ::i!i iiiii: :'•: •ii 1" •ii 'i'; n % > ii ii, iii ". tr;J . iili'il iiii M iiiii' ^ :;• 1 • , r i' :'!• ::• '','•'• ••"ry m '••••• '^A ii'i ::" 1 1 . i'ii iii. :i;i I i •I ':••. :: iii: ii'i ::: i'-ir^i- ipi- ilii I'ii 1 :;:, ;•;, :;,: , 1 'ii'iii iiii i:i [ -.. ... iiiii: ::: liiiiriuiuj •Iff ^r- -1— 1„. ii^* rrr iiii iii 11,1 Ilii -li'lj;':! ^'"' i l.ii !':: : li;!iiii fir 11 1 1^ \A P ill ii- iii iiii iii iiii ill i'-; iii: :il: ill iiii1 Iiii i i'*t iiii iiii li:iiiiliii; .i Uli!^ 1 INTENSITY ca Q CS3 Q I I I I I I I I I I tn I tn cs- ro : en - C2 -3 S" I I 1 \ \ LL> ;^' mo • C3 CS) - 171 - TABLE -• 4 •^^0 NMR data of AO-YI Assignment Chemical shift GH^ 15.09(q) CH^ 60.955(t) G-2,6 109.98(d) G-1 122.207(3) G-4 138.53(s) G-3,5 145.960(3) G=0 I66.84(s) s = singlet, d = doublet, t = triplet, q = quartet, spectrum run in DMS0-d6 at 47.3MHz. The MS of AO-VI (Fig.4) showed molecular ion at m/z 198.0529 and a base peak at 153.0181(G,yHt-0.). The diagnostic fragment ion at m/z 183(M-GH^), 170(M-C2H5) and 125(M-37) further support the structure of AO-VI as ethyl gallate (XXVI). (XXVI) - 172 - AO-VII Yellow needles from benzene-methanol, m.p. 315 G, R^ = 0.22 (BPF, 36:9:5). The TJV spectra with shift reagents is given in Table - 5. TABLE - 5 ^ MeOH A„„^ nm : 256, 268 sh, 370 max +NaOMe 248 sh, 322 (decomp). + AlCl, 371, 303 sh, 332, 457 + A1C1,/HG1 266, 358, 429 + NaOAc 256 sh, 275, 328, 391 (decomp) + NaOAc/H,BO, 262, 304 sh, 387 From UV data, m.p. and the direct comparison with authentic sample AO-VII was characterized as quercetin (XXYII). HO OH O ( XXVII) - 173 ~ The methanol fraction was purified by celliilose column and AO-VIII was separated out. AO-VIII White crystals from methanol-water, m.p, 183 > R- = 0.09 (TEP, 5:4:1), [a]p2^ + 60. The result of -^H MR is given in Table - 6 (Figs. 5,6). It showed signals at 6 3.35 for methoxy protons and 6 3.15-3.9 for six methine protons. Hydroxy signals were observed between 6 4.26-4.63. TABLE - 6 Chemical shift of protons of AO-VIII (6 scale) Assignment Chemical shift of protons OMe 3.35(s, 3H) -GH protons 3.15-3.9 (6H) OH protons exchangeable 4.26, 4.31, 4.43(q), 4.63(q) with D^O s = singlet, q = quartet, spectirura run in DM30-d6 at 10014Hz TMS as inteimal standard. The results of -"-^C NMR of AO-VIII is given in Table - 7 (Pigs. 7,8). The signals of G OT-IR were assigned by comDarison INTENSITY -1 I I I I I I L_l I I en CS Q C3 5 + en mo ' « C3 - 174 - of the "'"^G NMR data of L-2-0-methyl-chiro-inositol(. XXIX)-^^^ and AO-VIII was characterized as D-2-O-methyl-chiro-inositol ( XXVIII). TABLE - "7 •"•^G NMR data of AO-VIII and L-2-0-methyl-chiro-iositol-"-°°(6 scale) ^^J^°^ Chemical shift of AO-VIII L-2-O-methyl-chiro-inositol 0H3 56.99(t) 56.8(t) G-1 68.002(d) 67.2(d) C-5 70.476(d) 70.4(d) C-6 71.903(d) 71.3(d) G-3 72.131(d) 71.9(d) C-4 73.269(d) 72.8(d) G-2 80.180(d) 80.1(d) d = doublet, t = triplet, spectrum run in DMS0-d6 at 25MHz. The MS spectrum (Fig. 9) showed the moleciolar ion at 195. OH OH HO OH (JCXVIII) (XXIX) Experimental - 175 - EXTRACTION OP THE CONSTITUENTS PROM THE LEAVES OP ACER OBLONGOM (ACERACBAE) Dried and powdered leaves of A. oblonfiom (2 kg) collected from Royal Botanical Garden, Godawari, Lalitpur, Nepal were completely exhausted with hot methanol and the methanol extracts were concentrated to yield dark green gvunmy mass. This was treated with (A) petroleum ether (60-80°) and benzene, (B) ethyl acetate and then with (C) methanol. (A) Separation of nonflavonoidic fraction by column chromatography The petrol and benzene fractions were mixed (5 g) and adsorbed on silica gel (8 g) and transferred over a silica gel (150 g) column set with petroleum ether. The column was eluted with petrol. Petrol-benzene (1:1) and then with benzene. The fraction which were eluted with petrol-benze .€ (i; .; were found to be a mixture of two components. They were separated by preparative TLG [silica gel (BDH), petrol:benzene(P:B), 3:7] and labelled as AO-I and AO-II. AO-I Colourless needles from GHCl,-EtOH, mo p. 238° R^ = Oo50 (P:B, 3:7), an unidentified triterpene. - 176 - AO-II Colourless needles from GHCl,-EtOH, m.p. 198°, H^ = 0.63 (P:B, 3:7), gave positive Liebermann burchard test. It was identified as p-amyrin. •"•H-NMR (60MHz, CDCl^) : Values on 6 scale 0.78(3H, s), 0.83(3H, s), 0.88(6H, s), 0.95(3H, s), 0.98 (3H, s), 1.0(3H, s), 1.14(3H, s), 1.08-2.01(-GH2 and -GH protons of cyclic system and side chain), 3.21(lH, dd, J = 9Hz and 7Hz), 4.88 (a broad singlet, IH, OH proton), 5.21(1H, olefinic proton). MS ; m/z 426 (M"^) IR y^ cm"-'- : 3360, 2960, 2880, 1650, 1465, 1040 and 890 cm"''- AO-III The elution of the column with benzene afforded a solid which on repeated crystallization from CHCl^-BtOH gave white needles, m.p. 136-7 0. It was identified as p-sitosterol (m.p., m.mop., CO-TLC). - 177 - ''"H-MR (eoifflZjGDCl^) : Values on 6 scale 0o68(5H, s, 18-Me), Oo74(3H, d, J = 6.aHz, 28-Me), 0.84(6H, d, J = 6.5Hz, 26, 27-Me), 0.92(3H, d, J = 6,5Hz, 21-Me), 1.01(3H, s, 19-Me), 3.52(1H, m, 3-ax), 5.15(1H, m, hydroxyl proton), 5o39(lH, m, olefinic protons), 1,07-2.34(-CH2 and -CH protons of cyclic system and side chain). MS : m/z 414(M"*') IRIT??^ cm"-'- : 3400, 2900, 2830, 1640, 1470, 1380, 1370, 1360, 1065 and 810. (B) Purification of ethyl acetate fraction The ethyl acetate fraction (10 g) from the crude extract gave positive colour with Zn/HCl (orange). It was adsorbed on silica gel (15 gm) and transferred over a column containing silica gel (100 gm) set with benzene. The column was eluted with benzene, benzene-ethyl acetate (9:1, 8:2, 1:1) and then with ethyl acetate. The fraction which was eluted with benzene was crystallized with benzene-acetone to yield chromatographically homogeneous compound, AO-IV. - 178 - AO-IV Yellow crystals, m.p. 346 C, R^ = 0,55 (silica gel, benzene- pyridine-formic acid, 36:9:5). It was comparable with 5,7,4'- trihydroxyflavone. The fraction, which was eluted with benzene-ethyl acetate (9:1, 8:2) showed the presence of two components on TLG (BPF, 36:9:5) which were labelled as AO-V and AO-VI. They were separated by preparative TLG (silica gel, BPF, 36:9:5). AO-V Yellow crystals from benzene-methanol, m.p. 276-78°C, H^ = 0.53 (BPF, 36:9:5) was comparable with 5,7,3',4'-tetra- hydroxyflavone (kaempferol). AO-YI Yellow solid from methanol, m.p. 155°, R^ = 0.50 (BPF) analysed for CQH,QOC(M'*' 198). It gave brown colour in uv light and blue colour with ale. FeCl,. It was identified as ethyl gallate. -4l-MR (lOOMHz. I3MSO-d6):Values on 6 scale 1.3l6(t, 3H, J = 7Hz, GH^), 4.24(q, 2H, J = 7Hz, GH2) and 7.11(3, 2H, H-2,6). - 179 - •^^C-MR (47.3 Ifflz, 6 scale) 15.09(q, CH^), 60.955(t,CH2), 109.98(d, C-2,6), 122.207 (s, C-1), 138.53(s, G-4), 145-960(s, G-3,5) and l66.84(s, C=0). MS : m/z 198.0529(45, M"^, GgH-LQO^), 183(8), 170(20), 153.0181(100), G^H^O^), 125(20), 107(4), 79(9) and 51(6). AO-VII Elution with benzene-ethyl acetate (1:1) yielded AO-VII. It gave orange colour with Zn/HGl. It was ciystallized from benzene-methanol as yellow needles, m.p. 315°G, R^ = 0.22 (BPF) UV Spectral data with shift reagent AmS" ^ 256, 268 sh, 370, + UaOMe 248 sh, 322 (decomp), 405, + AlCl^ 271, 303 sh, 332, 457 + AlGl^ + HGl 266, 358, 429 + NaOAc 256 sh, 275, 328, 391(decomp) + NaOAc + H^BO^ 262, 304 sh, 387. From UV spectral data and R^ value it was characterized as 5,7,3,3*,4'-pentahydroxyflavone (quercetin). - 180 - (0) Purification of methanol fraction The methanol fraction was dried and then treated with water. The water soluhle fraction was purified by cellulose column. The water soluble fraction was transferred over a column containing cellulose powder settled with distilled water. The column was eluted with water, water-methanol (1:1) and then with methanol. From water-methanol fraction (1:1) a white solid was separated out which was filtered off and washed with methanol. AO~VIII Irfhite crystals from methanol-water. Soluble in water, m.p. 183°C, R^ = 0.09(TEP, 5:4:1), M^'^^ + 60, analysed for ^17^14^6 ^^^ 195). It was identified as D~2-0~methyl-chiro- inositol. •Sl-NMR (lOOMHz. .IMSO-df;);Values on 6 scale 3.35(s, 3H, OMe), 3.15-3.9(6H, -CH protons), 4.26, 4.31, 4.43(q), 4.63(q) (OH protons exchangeable with DpO). •'-^C-MR (25MHz, DMSO-dfi): Values on 5 scale 56.99(t, CH^), 68.002(d, C-l), 70.476(d, C-5), 71.903(d,C-6), 72.13l(d, C-3), 73.269(d, G-4), 80.180(d, C-2). - 181 - MS ; m/z 195(M+1, 100), 194(M'^, C^H^^Og), 177(10), 159(15), 127(30), 109(25), 86(15). la y^ cm"-'- : 3300(br, OH), 2900, 1500, 1440, 1380, 1355, 1330, 1270, 1225,'ll90, 1140, 1105, 1050(br), 1020, 950, 910, 865, 760, 720, 660. Refrence^ - 182 1. The Wealth of India Ravr Material, CSIR, XlJ., 21 (1948). 2. Tsujimoto, M., Nat. Sci Rept., Ochanomizu Univ (Tokyo), 2, 138-41 (1951). 3. Aritomi, M., Yakugaku Zasshi 84(4). 360-2 (1964). 4. Karatodorov, K, and Kolarova, R., Izu-Durzh. Inst. Kontrol Lek. 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Food Res., 19, 597-602 (1954). 100. Dorman, D.B., Angyal, 3.J. and Roberts, J.D., J. Am. Ghera. Soc, 92, 1351 (1970). Chapter 5 CONSTITUENTS OP GENUS HHUS (ANACARDIACEAE) The genus Rhus belongs to the family Anaoardiacea which consists of 73 genera comprising about 600 species . The genus Rhus, one of the largest genera of trees, shirubs and climbers, is chiefly distributed in the warm temperate regions of both hemisphere extending into the tropical and cold temperate region. About a dozen Rhus species occur in India. The chemical exami- 2-17 nation of the Rhus species has revealed the presence of flavones biflavanones"'-^*^-'-*^^"^'^, biflavones''-^'^^'^^, flavonoidic glycoside5'4'7,9,10,28-34^ aromatic acids^'^-lO, 12,14, 31, 32,35-41,48^ waxes42-45 ^^ tannins'^''^'^^"^'7. Flavonoids (a) Flavones; Ahmad and coworkers reported the presence of pongapin (I), tetramethoxyfisetin (II) and dimethoxy kanugin (III) in the heartwood of R. chinensis. ON 0M9 Ox (I) (II) (III) Pongapin' Tetramethoxy fisetin' Dimethoxy kanugin' - 189 - 4,5,7 Quercetin (IVa) was afforded by stem and root of R, chinensia , 3 5 6 leaves of R. mysureasls , R. salioifolia , R. lancea , 7 o q '\ o R. samlalata . R. coriaria , R. parvlflora , R. typhlna and 10 5 R. aromatica . R. salicifolia also afforded ombuin (IVb) rhamnetin (IVc), europetin (IVd), syringetin (IVe), myricetin(IVf) and kaempferol(IVg). Myricetin (IVf) was also isolated from the 3 4 6 7,8,33 leaves of R. mysurensis , R. chinensis , R. lancea , R.coriaria , R, aromatic7 a1 0 7,4'-Dimethy7 l myiricetin 9 (IVh) was isolate10 d from R. glabra , R. copallina , R. parvLflora , R. typhina and R. lancea . Kaempferol (IVg) was found to occur in the leaves •z R A A of R, mysurensis . R. salicifolia , R. lancea , R. coriaria , R. typhina and R. aromatica Quercetin (IVa) kaempferol (IVg) and myricetin (IVf) were also isolated from the leaves 61,64 R-" R' R' R*^ (IVa) Quercetin^-lO'^^'^"^ OH H OH OH (IVb) Ombuin^ OMe H OMe OH (IVc) Rhamnetin^ OMe H OH OH (IVd) Europetin^ OMe OH OH OH - 190 - R1 R2 R5 R4 (IVe) Syringetin^ OH OMe OH OMe (IVf) Myricetin^"^^*^^'^!*^'^ OH OH OH OH (lYg) Kaempferol^'^'^'^'^^'^^'^"^ OH H OH H (IVh) 7,4'-Dimethyl myricetin^ OMe OH OMe OH Pisetin (V) was present in the leaves of R. typhina 7« 7 7 11 l"? R. glabra , R. copillana . R. cotinua and R. ooriarla ^. R. coriaria and R. rhodanthma afforded fisetin (V) in wood, The stem and roots of R. chinensia 17 also have fisetin. 14,62 R. succedanea constituted the fisetin in the heartwood." HO (V) Pisetin'^*^^*^^*^^'!'^ Pustin (VI) was found to occur in the leaves of R. glabra1 5 and R. trichocarpa 12 . It is also present in roots and stem of R, chinensi3 and in the heartwood of R. succedanea 0 (VI) Pustin^2,l4,15,l6,62 - 191 - The roots of R. undulata 17 constitute 5-hydroxy-4',7- 65 dimethoxy flavone (VII). R. insifinis constitute 3,7,3',4'- tetrahydroxyflavanone, 5,7,3',4'-pentahydroxyflavanone and 5,7,4'-trih.ydroxyflavanone. H3CO OCH3 OH 0 (VII) 5-Hydroxy 4', 7-dimeth.oxy flavone 17 (b) Bifalvanoneg ; Mesuaferron-A (Villa) and B-(neorhus- flavanone) (Vlllb) were reported from R. suocedanea ' OH 0 HO HO OH 0 (Villa) Mesuaferone-A"*-®*^-^ 2\ 3" -sat (Vlllb) Mesuaferone-B"'-^'^-'- 2", 3" -unsat - 192 - Seed kernal and drups of R. auccedanea constitute rhusflavone, rhusflavanone and succedaneaflavanone. OH O 26b (IXa) Rhusflavone (IXb) Bhusflavanone 23-26 HO (X) Succedanea flavanone23-26 - 193 - 21 A I-3',II-8-binaringenin (XI) was reported from R. toxicodendron . OH 0 (XI) Binaringenin27 18 IQ 22 20 (c) Blflavones: R. guccedanea * * and R. punjabnesis constitute amentoflavone (XII), agathisflavone (XIII), robustaflavone (XIV) and hinokiflavone (XV). Cupressuflavone (X7I) was present only in R. succedanea * . Amentoflavon.61 e was also isolated from the leaves of R. wallichi OH 0 (XII) Amentoflavone^®-2°'22'^^ Agathisflavone (XIII) was also isolated from the leaves 63 of R, semialata . - 194 - OH 0 (XIII) Agathisflavone^^^^^»22,63 (XIV) Robuataflavone-''®"^^*^^ OH O OH O 18-20,22 (XV) Hinokiflavone - 195 - OH 0 °" ^ 18 iq (XVI) Cupressuflavone '^ (d) Plavonoidic glycosides; Chxysanthemin (XYII) and peonidin- 3-nionoglucoside were isolated from the leaves of R. succedanea (XVII) Chrysanthemin 28 Rhoifolin (XVIII) was isolated from the leaves of R. succedanea "* , R. trichocairpa . R. ambigua and 4 R. sylvestris . Rham-O OH O 4,29,30 (XVIII) Rhoifolin - 196 - Myidcetrin (XIXa)wa3 isolated from the leaves of R. chinensls , fi. aromatica , R. ambigua . R. coriarla Q Q and R. parvlflora^. The leaves of R. parvlflora^ also afforded afzelin (XIXlj). OH 0 (XIX) R1 R2 R5 R4 (XlXa) Myricetrin''^*^'^^'^,10,3^1 Rham OH OH OH (XlXb) Afzelin^ Rham H OH H The leaves of R. typhina and R. aromatica constitute 7 myricetin 3-a-L-rhamnofuranoside vdaere as leaves of R. lancea constitute myricetin 3-galactoside and leaves of R. mysurensis constitute myricetin 3-0-rhamnoside. The leaves of R. .javanica^^ and R. chinensis"^ afforded R^ R** OH OH - IP? - Leaves of R. typhlna and R. aromatica cjonstitute quercetir 3-p-D-glycopyranoside. Quercetin 3-0-rhamnoside was reported from R. mysurenais . Leaves of R, ooriaria constitute quercetin 33 33 3-0-a-L-rhamno-furanoside , avicularin (quercetin 3-0-a-L 31 arabinofxixanoside) and isoquercitrin (XXI). OH O R1 R2 R5 R^ (XXI) Isoquercitrin^-'- Glu H OH OH Leaves of R, typhina and R. aromatica srfforded kaempferol 3-p-D-glucopyranoside whereas kaempferol 3-0- •3 rhamnoside was reported from the leaves of R, mysurensia . Astrigalin (kaempferol 3-O-p-D-glucopyranoside) was found in 33 the leaves of R. coriaria . Isorhamnetin 3-a-L-arahino3ide was reported from the leaves and stem of R, parviflora » Leaves of R, wallichi * ^ constitute querecetin 3-O-ara- binopyranoside, quercetin-3-O-xyloside -3-0-glactoside and -3-0- glucoside and p-sitosterol glucoside. - 198 - Miscellaneous compounda (a) Aromatic acid: p-Goiuaaric (XXIIa) and caffeic acid(XXIIb) 9 were isolated from R. parviflora . CH=CH-COOH OH (XXII) ^ (XXIIa) p-Coumaric acid^ H (XXIIb) Gaffeic acid^ OH Leaves of R. coriaria^'^-^. R. filabra^^, R. typhina-^^*^'^"^^. R. aromatica , R. samialata and R. trichocarpa afforded gallic acid (IXIII). Grallic acid was also found in the leaves emd heartwood of R. succedanea * » • HOOC (XXJlDOallic acid7'8»10,12,14,31,35-39,62 - 199 - Leaves of R. coriaria ' also constitute m-digallic acid Methygallate (XXIVa) and ethylgallate (XXIVb) and ellagic acid (XXV). Methyl gallate was also reported from the leaves "52 of R, .iavanlca . Ellagic acid was reported in the leaves of 39 48 R. typhina , R. semialata and in the heartwood of R, succedanea * , PkOOC R (XXIVa) Me-gallate®'^-"-*^^ Me (XXIVb) Et-gallate^'^-^ Bt OC-0 0-CO (XXV) Bllagic acid^^'^^'^^*"^^ - 200 - (b) Aliphatic acid; Maleic acid was reported from the leaves of R. glabra and R. typhina . Leaves of Ho typhina , R. cariaria and R. trichocarpa also afforded palmitic acid (CH,(CHp)-, .COOH). Where as stearic, arachidic, behanic, oleic, elaidic, linolenic acid, tetracosanolic (Gp/iH.gO,), decosanolic (CPQH./O,), eicosanolic (GPQH^^QO^), octadecanolic (C^gH^gO,) were found in the fruit wall of Ro typhina » Leaves of Ro trichocarpa afforded tricocarpinic acid (G-j_gH,gO,). Two dibasic aliphatic acids, HOOC( 0112)20 COOH 41 HOOC(CHp),gCOOH were found in R. trichocarpa and R. succedanea , (b) Waxes: The urushi and haze waxes of Rhus were analysed by Kawaguchi and coworkers (1978). (-f-)-Gallo catechol was reported from R. aromatica 43a . Some other catechol derivatives were isolated from R. striata . Whereas urushiol and dimethyl 44 45a-f urushiol were from R, t oxicodendron and Ro vernicifera and a compound 3,5-dimethyl~3-pentadecyl catechol (XXVI) was reported from poison ivy . Poison ivy also afforded urushiol, cardol and cardsuiol. Me ^'^"3. (XXVI) 4,5-Diraethyl 3-pentadecyl catechol^^ - 201 - (c) Tannins: Tanninahave been reported from leaves of 4 7 48 49,51,68 AQ «50 R. succedanea . R. samialata * , R. typhina ' , R. cotinus * C"l Co CO f,Q R. corlarik and R. glabra *. .It was also reported from the wood, root and bark of R. oxyacanthone 53 . A tetraflavonoid condensed tannin(XXVII)was also reported from the leaves of 54 R. Isoicea M«0 OMe MeO OMe OMe OMe (XXVII) Tetraflavonoid condenced tannin 54 - 202 - Leaves of R. semialata , R. coriaria"^-^, R. chinensis * afforded gallotannin. It was also reported from the heartwood of R. .lavanloa 56 A few other components like p-sitosterol was reported 57 from stem and root of R. chinensis and an indol 2,2'-bis- 59 (3-indolyl) indoxyl (II)(7XVIII)was reported from R. trichooarpa. 59 (XXVIII) Indol 2,2'-bis-(3-indolyl) indoxyl Rhus lactone (XXIX) was reported from bark of R, .javanica 58 C=CH2 Me Me 58 (XXIX) Rhus lactone - 203 - OYalitenone (Dibenzoylmethane) ( XXX ) was reported from 2 the heartwood of R, diinensis . O O ( XXX ) Ovalitenone^ Sulfuretin aad 2-benzyl-2,6,3' ,4'-tetrgLliydroxy coumarin- 3-one were isolated from heartwood of R, succedanea Polyphenols were isolated from R, glabra * , R. coriaria 68 and R. typhina . A new triterpene semimoronic acid (XXXI) was isolated from R. semialata 69 . MftM« COOH (XXXI) Discussion - 204 - THE CONSTITUENTS OP RHUS PUNJABlfESIS AND BHUS SEMIALATA The cardiotonic activity of Chinese syrup "Shu Guang Tong" "] ft 17 prepared from the extract of R. chinensis * and isolation of allergens from R, toxicodendron 71 prompted us to investigate other species of Rhus. A number of chemotaxonomically significant 1 R 1 Q ^O O'^ Of\ biflavones have been isolated from R. succedanea * ' ' , R, toxicodendron , R. pun.jabnesis , R. mysurensis and R, alata 72 . A major component of the sap of Japanese lac trees (Rhus vemicifera) has been extensively investigated by liquid chromatography -^ and high performance liquid chromatography^-'®' '^. 1. Constituents of the leaves of R. pun.jabnesis The petrol fraction of the leaves of R. pun.labnesis. after coliunn chromatography over silica gel (BDH) gave fractions labelled as RP-I to RP-VI RP-I was found to be simple hydrocarbon by IR, "TI-NMR and MS. The molecular ion appeared at m/z 35l(M -H, ^25^51^ ^^ *^® MS. The IR spectrum of RP-II indicate it to be hydrocarbon, while its MS shows traces of urushiol as well. ~1 ""•1 -= -1- - -t - -T- _ '•! n- t- l-.J - '"•J - i-'J - C'J ^ t o ij, -•::. 1- - CO " 1 C' r _ij CM T—1 o '::> =r -•fi C'"| r- = T •r-l I", _ -1- _- T-t C''i Q T^ )---[ 1 CO ——TT - I"'J - C'J t—1 l"' 1 . I c-j CO o ^ __~ l-»-l ^ —= f-, "~[;", -£ •:_ r- - Cn — t iTi C'J -^ -:? - CO • CM C'J VH - r- - r- ^ C'J •:::• - '£l - ITLl.i C'J ._ -=. C'J — - 1 ' 1 ' 1 ' [ -I— 1 1 1 f ' T - 1— 1 • 1 ' 1 1 ri t'"! c> o '_i' •j.' C' ("0 ' 0 'f ''•' Fir '^' '-'-' •L< t C'J T-H Mo-" y% ^9 s Uo - 205 - RP-III constitutes urushiol and a minor impurity showing molecular ion at m/z 446 in MS (Pig. 1). The urushiol is nor mally a mixture of more than one compound. In RP-III the compound with highest molec-ular weight shows molecular ion at m/z 348 in MS: The presence of phenolic group is indicated by positive test with ferric chloride. The ii-NMR spectrum (Pig. 2) of RP-III is typical of catechol substituted with aliphatic hydrocarbon with 17 carbons. It showed signals at 6 1.30 for methylene protons (approximately 30 protons), a triplet at 6 3.0 integrating for two protons (approximately) for benzylic protons. The aromatic protons approximate for three protons are of ABX type. No olefinic proton appeared in tl NMR. Therefore, RP-III may be a mixture of catechol derivatives substituted at 0-4 of benzene ring with a saturated hydrocarbon chain of varying length. The component appearing at m/z 348 be compound (XXXII). CH2(CH2)|5CH3 (XXXII) -1- o f'-l r- - i-j CM __ fi - : r- - o r- (••J r-- "T. u-1 r- r- t- cn I 1"'J i^j o o O o i"'l rr T'-l EJfr'^ If I'J -^ . ' ' r- r'j '•'J r- r- - 1"''-*J' I'M _ • - r- 5r iTt I I-'J 1—••—r Ci O o o cz< • o 1 J f- r- •'•J • i"'j 1"J - -t "•J tn I Oi I '""^T ' T O O C r-1 E, s ro oj- 00 © t© Ln • • t^ -^- 00 00 • Lr>• m o LT•v « •^ •^ H CO t~- CVi -2o6- V_^ V ' VD t •-_-f- • —' .,.^r~- —' s_^ t^ c- r rv a L'-\ to '\D—^ VX) , ^ CO LT ^ r-l ^ ":} C •J- t~- r^ ^ cc KA cr- (\J (T> T- r-J in c H vo H Xi> r~-^ f-l t<^ r<^ H •^ H rA >D r CT t\l li- C^. t^j H Lr> ir> o in in v:\ IJ^ fNJ ^^ (~ rv rH ^ H -\ 1—' -•D <\, 0^ m o (M CO ^o H ^ rA rH ^ (A —N^ ' —r-\ ' %in^ ^^ ^ O r— '-0 O t^ ^ij tNJ H "^1 H H '^j H o '^ H t-- • .-—. o v-D CT^ o r-^ t^ CM H rH H H H in H •—-r r in ffT •«—t- ' m. —* cr "•^ o X t-- r- c: o H H (M r-l Ho fvl O^ o o •M C\l CM o o 0) O tu —I CI CI W O o o CO o Q. O o O o o O ^ 00 CM CO 00 > l-H > I I - 207 - RP-IV, RP-V and SP-VI showed moleciilar ions at m/z 348 (38.1), m/z 348 (53.9) and m/z 348 (58.2), respectively in MS (Table I, Figg«3-5). These samples of urushiol show identical base peak at m/z 108 and identical fragment ions. Their IR spectirum is also identical. Therefore compound I, detected in RP-III is common in RP-IV to RP-YI. However, without the use of HPLC, it is difficult to comment as to how many other compo nents with molecular weight less than 348 are present in RP-III, RP-IV, RP-V and RP-VI. Further analysis of these samples by HPLC will be studied. 2. Constituents of the leaves of Rhus semialata The leaves of R. semialata were extracted with light petrol, benezene and acetone. Petrol extract, mainly constituting urushiol has been retained for HPLC. The benzene fraction constituting terpenes was mainly dominated by chlorophyll and could not investigated completely. The acetone fraction responded to flavonoidic colour, test with Zn/HCl and it was chromatographed over a silica gel column. The column was eluted with benzene, benzene-ethyl acetate (1:1), ethyl acetate,ethyl acetate-acetone (8:2) and then with ethyl acetate-methanol (9:1). The compoimds isolated were labelled as RS-I—RS-IV. I/) on a CD— SJ t/}— I U. . G3 Vi^Jl^^t: en c 1 "T— 1 ea ca 40 za - 208 - RS~I Crystallized from ethyl acetate-acetone as light green solid, mop.,155°C. It analysed for GgH^QO^ (M"^ 198) and was characterized as ethyl gallate(XXIVb) by direct comparison with the authentic sample of ethyl gallate. MS (Pig.6) showed molecular ion at m/z 198 and the base peak appeared at m/z 153 which further supports the structure of RS-I as ethyl gallate (XXIVb). HO HO-^ y—C00CH2CH3 HO (XXIVb) RS-II It was comparable with parent agathisflavone 75 . It gave positive colour with Zn/HGl. On methylation with dimethyl sulphate and anhydrous potassium carbonate it was found a mixture of agathisflavone hexamethyl ether (RS-IIMI) and robustaflavone hexamethyl ether (RS-IIMII) (R^, fluorescence in uv light with authentic sample). - 209 - R3-III 75 R3-III was comparable with parent amentoflavone and on methylation it was found to be amentoflavone hexamethyl ether (R^ value and fluorescence in UV light with authentic sample). R3-IY Crystallized from methanol as pale yellow needles, m.p. 358-59°C. It gave orange colour with Mg/HCl indicating it to be flavonoid. Brown shade in UV light and positive colour with alcoholic feriric chloride support the presence of phenolic group in R3-IV. The UV spectral data with shift reagent (Table-2) and hi NMR of its acetate (Table-3, Fig.7) support structure IVf for RS-IV. hi NMR spectinm R3-IV acetate (RS-IVA) showed two meta coupled doublet at 6 6,86 and 7.33 for H-6 and H-8, respec tively. A singlet integrating for two protons appearing at 6 7.61 may be assigned to H-2',6'. Singlets at 6 2.31 and 2.42 were assigned to acetoxy groups. TABLE - 2 ^ MeOH ^max. ^ = 255, 372 + AICI5 272, 449 + AICI3+HCI 268, 429 + NaOAc 269, 339 (dec) + HaOAc/H^BO^ 259, 393 - 210 - TABLE - 3 Chemical shifts of protons of RS-IVA (6 scale) Assignment Chemical shift of protons (RS-IVA) H-8 7.33 (d, J = 2.5Hz, IH) H-6 6.86 (d, J = 2o5Hz, IH) H-2',6' 7.61 (s, 2H) OAc/7,3',4',5' 2.31 (s, 12H) 3,5 2.42 (s, 6H) s = singlet, d = doublet, spectrum run in CDGl, at 90 MHz. TMS as internal standard. From these data RS-IV was characterized as 5, 7, 3,3', 4',5'• hexahydroxyflavone (myricetin). OR 0 (iVf) R = H (XXXin) R = CH,CO Experimental - 211 - EXTRACTION OP THE LEAVES OP RHUS PUNJABNESIS Air dried powdered leaves of Rhus pun.jabnesis (400 gm) were exhaustively extracted with petroleum ether (60-80 ), benzene and ethyl acetate. Purification of the petrol fraction The crude petrol extract (12 g) was adsorbed on silica gel (20 g) and transferred over a coliimn of silica gel (200 g) set with petroleum ether (60-80°). The column was eluted with petrol, petrol-benzene (1:1, 3:7, 2:8, 1:9), benzene-ethyl acetate (9:1, 1:1, 7:5, 2:3, 1:9). The fractions were labelled as RP-I (R^ = 0.97, n-Hexane), RP-II (R^ = 0.61, CHCl^ : MeOH, 20:1), RP-III (R^ = 0.74, CHGl^ : MeOH, 20:1), RP-IV (R^ = 0.78, CHCl^ : MeOH, 20:1), RP-V (R^ = 0.80, GHGl, : MeOH, 20:1), RP-VI (R^ = 0.91, GHGl^ : MeOH,12:1). RP-I Eluted with petrol, crystallized from EtOAc, m.p. 65^0, R^ = 0.97 (Hexane) UV inactive, 025^52 (Hydrocarbon). MS:m/z 351 (M^, 0.9), 323(0.9), 309(0.9), 295(0.9), 281(1), 267(0.9), 253(1.1), - 212 - 239(1.2), 225(1.2), 211(1.5), 197(1.5), 183(1.7), 169(2.1), 155(3), 141(3.9), 127(5.6), 113(8.7), 99(14.2), 85(39.8), 71(67.7), 57(100). -^H-NMH (60MHz, CDGl,)^Values on 6 scale : 0.80(t, 6H), 1.30(br, 46H, methylene protons). RP-II Bluted with petrol-benzene (1:1) crystallized from EtOAc, m.p. 83°C R^ = 0.6l(C!HCl^ - MeOH, 20:1). MS : m/z 474(0.8), 446(3.2), 4.18(2.1), 390(1), 362(0.9), 348(1.1), 334(1.2), 306(1.6), 278(1.4), 250(2.2), 208(1.9), 167(3.6), 139(8.2), 125(19), 111(37.7), 97(67), 83(69.2), 69(55.7), 57(100). RP-III Eluted with petrol-benzene (3:7), crystallized from petrol- EtOAc, m.p. 78°G, R^ = 0.74(GHC1, : MeOH, 20:1). It gave brown colour with alcoholic FeCl^. MS : m/z 446(3.1), 348(37.1), 330(18), 312(12), 301(9.7), 287(13.1), 273(12.3), 259(8.3), 245(4.6), 227(4.9), 213(4.3), 199(10.6), 189(7.4), 185(12), 175(14.9), 161(54.6), 152(32.3), 147(62.0). 134(47.7), 133(27.1), 121(17.1), 120(11.1), 108(70), 107(46.9), 105(39.7), 99(13.4), 97(12.6), 91(16.9), 85(42), 83(17.4), 77(20.9), 71(56.9), 70(11.1), 69(29.7), 57(100), 56(17.7) and 55(62). •Sl-NMR(60MHz, CDGl^) j Values on 6 scale : 0.90(br, 3H, OH,), 1.30(br, 30H, CE^ proton), 3.0(t, J = 8Hz, 2H, GH2 —pH). Aromatic protons : 6.9(dd, J = 9Hz and 2Hz), 6.8(dd, J = 9Ha and 2Hz), 7.42(d, J = 9Hz). - 21-5 - Integration of aromatic protons approximately three but it indicate that RP-III is a mixture of more than one compound. IR : yf^l cm"-'- : 2900, 2840, 1660, 1645, 1610, 1465, 1450, 1320, 1300, 1220, 1170, 950, 900, 820, 750, 720, 700. RP-IV Eluted with petrol-henzene, 2:8, white crystals from petrol-EtOAc, m.po 80 G, fluorescene in UV light, brown with alcoholic PeCl,, R^ = 0.78 (GHCl, : MeOH, 20:1). MS : m/z 348 (38.1), 330(19.9), 312(14.6), 304(12.6), 301(13.3), 287(16.4), 273(16.1), 259(11.8), 245(6), 241(5.1), 231(3.7), 227(6.7), 213 (6.5), 199(13.4), 189(9.7), 185(16), 175(19), 173(10.2), 162(18.9), 161(62.1), 152(35.3), 151(19.3), 149(14.4), 148(32.1), 147(62.1), 146(30.7), 145(16.9), 135(13.9), 134(52.2), 133(31.3), 121(19.5), 120(13.3), 109(10.6), 108(100), 107(58.8), 105(39.9), 91(18.1), 77(25), 57(24.7), 55(35.7). IR : V^ cm"^ : 2920, 2850, 1660, 1605, 1460, 1445, 1305, 1245, 1225, 1170, 1060, 900, 820, 720. RP-V Bluted with petrol-benzene (1:9), crystallized from EtOAc - petrol as vdaite crystalline solidj m.p. 82°G. It gave blue fluorescene in UV light and brown colour with alcoholic FeCl^, R^ = 0.80(CHC1^ : MeOH, 20:1). MS : m/z 348(53.9), 330(22.7), 312(15.1), 304(20.6), 301(13.2), 287(17.5), 273(15.8), - 214 - 259(11.2), 245(6.6), 227(5.8), 213(5.8), 199(12.8), 189(8.6), 185(13.9), 175(17.1), l62(19o3), l6l(60.7), 160(13.6), 159(11), 152(36.9), 151(17.8), 149(14.&), 148(30.3), 147(55.6), 146(27.8), 145(14.9), 135(13), 134(48.3), 133(25.8), 121(18.4), 120(10.8), 108(100), 107(52.7), 105(32.7), 91(11.9), 77(18.4), 57(18.2), and 55(27o5). IR : U^ °ni~^ • 2905, 2850, 1655, 1605, 1462, 1445, 1310, 1250, 1220, 1210, 890, 815, 725, 710, 640. RP-VI Eluted with benzene - ethyl acetate (9:1). It was crysta llized from CHGl, - oetrol as white solid, m.p. 84 C, fluorescence in UV light, brown with ale. PeCl,, R^ = 0.91(CHC1^ : MeOH, 12:1). MS : m/z 348(58.2), 330(26.1), 312(17.2), 304(15.6), 301(16.2), 287(19), 273(18.1), 259(13.5), 245(8), 241(6.5), 227(8.3), 213(8.4), 203(6.1), 200r7.7), 199(17.7), 189(12.3), 188(6.5), 187(7.7), 186(10), 185(20.8), 176(9.4), 175(22.2), 174(10.5), 173(13.8), 172(11.4), 171(8.8), 162(32), l6l(83.2), 160(21.2), 159(17,7), 158(12.9), 157(10.4), 152(61.4), 151(27.8), 149(20.8), 148(46.3), 147(93.7), 146(46.9), 145(22.4), 135(20.1), 134(74.6), 133(37.3), 132(13.6), 121(22.9), 120(15.4), 180(100), 107(67.7), 105(46.9), 95(10.1), 91(21), 79(12.2), 77(27.8), 57(25.8) and 55(51.5). - 215 - EXTRACTION OF THE LEAVES OF RHUS SEMIALATA Leaves of R. aemlalata (^ kg) procured from Royal Botanical Garden Godawari, Lalitpur, Nepal were extracted with light petrol (60-80°), benzene and acetone till the solvent in each case was almost colourless. Petrol and benzene fractions were mainly, urushiol, and terpenes respectively they were kept for future investigation, acetone extract gave positive colour test with Zn/HCl indicating the presence of flavonoid. Purification of the acetone soluble fraction by column chromato graphy The crude mixture (3 g) was adsorbed on silica gel (5 gm) and transferred to a column of silica gel (80 gm) set with benzene. The column was eluted with benzene, ethyl acetate,EtOAc-acetone (8:2) and then with ethyl acetate-methanol (9:1). RS-I was separated from ethyl acetate, ethyl acetate-acetone (8:2) fraction was found to be the mixture of biflavones (RS-II, RS-III) and ethyl acetate-methanol gave RS-IV. F -I Eluted with ethyl acetate and crystallized from EtOAc- acetone as greenish solid, m.p. 155 G. It gave brown colour in - 216 - UV light and blue colour with ale. FeCl,, R^ = 0.18(BPP). It was characterized as ethyl gallate by direct comparison with our authentic sample. MS : m/z 198(85), 183(15), 170(50), 153(100), 125(50), 113(10), 108(5), 107(15), 79(60), 53(25), 39(55) and 27(45). The fraction eluted with EtOAc-acetone (8:1) was foxmd to be a mixture of two component in BPP (36:9:5). They were further separated by preparative TLC (BPP, 36:9:5) as RS-II (Rp = 0„15) and RS-III (R^ = 0.18). RS-II It was methylated as usual. The methyl ether was found to be a mixture of two components RS,IIMI, RS-IIMII. They were comparable with the authentic sample of agathisflavone hexa- methyl ether and robustaflavone hexamethyl ether (R^ value, mop. and fluorescence in UV light). RS-III RS-III on methylation gave amentoflavone hexamethyl ether (R^ value, m.p. and characteristic fluorescence in UV light). Elution of the column with BtOAc : MeOH (9:1) gave the positive colour test with Mg/HGl. It was recrystallized from MeOH as yellow crystalls. - 217 - 5.7.3.3',4',5'-Hexahydroxyflavone (Myricetin. RS-IV) It was crystallized from HeOH as pale yellow needles (30 mg), R^ = 0.12 (BPP, 36:9:5), m.p. 358-59°C. Brown colour in UV and green colour with ale. PeOl,. UV MeOH ^mlx ^^^» ^'^^ ^» (MeOH-AlCl5)-272, 449 nm; (MeOH-AlGly HGl)-268, 429 nm; (MeOH-NaOAc)-269, 339 nm (dec); (MeOH- NaOAc-.H^BO^)-259, 393 nm. 3»5,7,3',4',5' —Hexaacetoxjrfiavone (RS-IVA) RS-IV (25 mg) was heated with pyridine (0.5 ml) and acetic anhydride (1 ml) on a water bath for 2 hrs. worked up as usual and crystallized from GHGl,-EtOH as colourless needles (20 mg), m.p. 218-19°C. -^H-ITMR (gOMIz, GDGl^ j Values on 6 scale : 6.86 (d, IH, J = 2.5Hz, H-6), 7.33(d, IH, J = 2„5Hz, H-8), 7.6l(s, 2H, H-2',6'), 2o3l(s, 12H, OAc-7,3',4',5'), 2.42(s, 6H, OAc-3,5). Refrcnces - 218 - 1. ••The Wealth of India, Raw Material"* CSIR Publication New Delhi India, Vol.IX pp 18-20 (1972). 2. Ahmad, J., Khan, H, and Shamsuddin, K.M,, Indian J. Chem., Sect B. 19B(5) 420-21 (1980). 3. Sarada, M. and Adinarayana, D., J. Ind. Chem. Soc, 61(7). 649-50 (1984). 4. Hiroaki, M., Chem. Pharm. Bull (Tokyo), 14(8). 877-82 (1966). 5. Wollenweber, E., Z. Naturforsch, Teil C 29(9110). 526-8 (1974). 6. Nalr. A.G.R., Kotiyal, J.P. and Bhardwaj, D.K., Phytochemistry 22(1). 318-9 (1983). 7. Adam, M., Acta Polon Pharm, 11, 263-7 (1954). 8. Sissi, H.I. El., Saleh, N.A.M. and Abd. El. Wahid, M.S., PlantaMed., 14(2). 222-31 (1966). 9. Nair, A.G.R., Kotiyal, J.P. and Sankarasubramanian, S. Curr. Sci., 46(13). 448-9 (1977). 10. Buziashvili, I. sh., Komisaarenko, N.P. and Kolesnikov, D.G., Khim Prir Soedin, 9(4). 555-6 (1973). 11. Chas, B.S. and Bartlett, H.H., Am. J. Botany, ^, 112-9 (1918). 12. Masaichi, Y. and Yoahishige, K,, Yakugaku Zasshi, 77, 1045-47 (1957). •"" 13. Josef, K., Sitzb Akad.Wiaa.132(1). 19-23 (1923). 14. Hillis, W.B. and Inoue, T., Phytochemistry, 5(3). 483-90 (1966). -^-^ 15. Keppler, H.H., J. Chem. Soc, 2721-4 (1957). 16. Ronghua, Z., Ji, Lianfang, Yaoxue, Tongbao, 17(4). 216-8 (1982). 17. Fouire, T.G. and Synckers, F.O., J. Nat. Prod. 47(6). 1057-8 (1984). - 219 - 18. Chen, Pa-Ching, Tai-wan K'O Hsuch, 26(3-4), 100-3 (1963). 19. Lin, y.M. and Chen, F.G., Hua Hsuch 14-16 (1974). 20. Kamil, M., Ahmad, I. and Ilyas, M., J. Ind. Chem. Soc., 61(4). 375 (1984). 21. Chen, P.O., Lin, Y.M. and Lin, Y.C., Heterocycles 9(5), 663-8 (1978). 22a. Lin, Y.M. and Chen, F.G., Phytochemistry 13(8), 1617-19(1974). 22b. Lin, Y.M. and Chen, P.O., Phyto chemist ry 13(3). 657-8 (1974). 23. Chen, P.O. and Lin, Y.M., J. Chem. Soc. Perkin Trans 1(1), 98-101 (1976). 24. Lin, Y.M. aad Chen, P.C, Tetrahedron Lett., (48). 4747-50 (1973). 25. Chen, P.C, Lin, Y.M., Shue, Y.K. and Ueng, T., Heterocycels, 3(7). 529-32 (1975). 26a. Chen, PoC. and Lin, Y.M. Phytochemistry, 14(7). 1644-7 (1975). b. Chen, P.C, Lin, Y.M. and Wu, J.C, Phyto chemistry, 13(8). 1571-4 (1974). c. Chen, P.C, Lin, Y.M, and Liang, CM., Phyto chemistry, 13 276(1974). 27. Elsohly, M.A., Craig, J.C, Waller, CW. and Turner, CE., Phytochemistry, 17(12). 2140-1 (1978). 28. Ishikura, N., Phytochemistry, 11(8). 2555-58 (1972). 29. Shizuo, H. and Hiraoki, M., Science (Japan) 21, 643 (1951). 30. Wagner, H., Aumhammer, G., Hoerhammer, L. and Parkas, L., Chem. Ber., 102(6). 2083-8 (1969). 31. Sissi, El, H.I., Ishak, M.S., Abd El. Wahid, A.E., Planta Med. 21(1). 67-71 (1972). 32. Aritomi, M, Miyazaki, K. and Mazaki, T., Yakugaku Zasshi 84(9). 894-95 (1964). 33. Buziashvili, I. sh, Komissarenko, N.P. and Kolesnikov, D.G., Khim, Prir Soedin, 6(5). 627 (1970). 34. Bhakuni, D.S., Satish. S., Shukla, Y.N. and Tondon, J.S. Phytochemistry, 10(11). 2829-31 (1971). 35. Sando, CE. and Bertlet, H.H., J. Agr, Research, 22, 221-9 (1921). "^ - 220 - 36. Nariyiki, I., Shunzo, H, and Kiyoshi, T,, Bot. Mag., 97(1047). 355-67 (1984). 37. Comthwaite, D. and Haalam, B., J. Chem. Soc. 3008-11 (1965). 38. Zenk, M.H., Z. Naturforsch, 19b(l), 83-4 (1964). 39. Tiacher, J. Pharmazie, 15., 83-9 (I960). 40. Chotaro, T., J. Pharm. Soc. Japan, 6g, 375-7 (1942). 41. Shichiro, S., J. Soc. Chem. Ind. Japan, 49, 18-20 (1946). 42. Kawaguchi, T., Asano, S. and Tats\ik:a, K., Kanzei Chao Bunsekishosho, 18, 105 (1978). 43a. Zaprometov M.N. and Biikhlaeva, V.Ya, Penol'nye Soedin, Ikh Biol, Punkts, Mater, vses Simp, Is* 236-8 (1966), (Pub. 1968). b. Nakano, T. Medina, J.D. and Hurdata, I. Planta Med. 18(3). 260-5 (1970). 44. Baer, H., Hootan, M,. Pales, H., Wu, A. and Schaub, P., Phytochemistry, 19(5). 799-802 (1980). 45a. Yamauchi, Y., Murakami, T. and Kumamotani, Ju, J. Chromatogr., 214(3). 243-8 (1981). b. Yiunin. Du; Oshima, R. and Kximanotani, Ju,,J. Chromatogr., 284(2). 463-73 (1984). c. Yumin, Du; Oshima, R., Iwatsuki, H.,and K\amanotani, Ju., J. Chromatogr, 299(1). 179-86 (1984). d. Yamauchi, Yoshio; Oshima, R. and Kumanotani, Ju., J. Chromatogr., 243(1). 71-84 (1982).(e) 49, 198 (1980). f. Parveen, N., Naqvi, S.W.I, and Khan, N.U., Unpublished result. 46. Joseph, S., Byck ahd Charles, R.D., J. Org. Chem. 32(4). 1084-8 (1967). 47. Symes, W.F. and Dawson, C.R., Nature, 171, 841-2 (1953). 48. Kursanov, A.L. and Zaprometov, M.N., Biokhimiya, 14, 467-75 (1949). ~~ - 221 - 49. Zielinska, R., Dissertationers Pharm., 2f 35-41 (1957). 50. Singh, P., J. Soc. (Jhem. Ind., 25, 39-40 (1917). 51. Leonard, W., Haddaway Anal. Chem. 28, 1624-25 (1956). 52. J.C. and Berthal. Deg. Pecock; Am. J. Pharm, 97, 463-71 (1925). 53. Bravo, G.A., Attiufficalali assoc ital Chum. tech. Conciarla, 2, 98, 100 (1940). 54. Ferreira, D., Hindt, H.K.L. and Roux, D.G., J. Chem. Soc. D, (20). 1257-9 (1971). 55. Buziashvili, I. Sh., Komissarenko, N.P,, Kovalev, I.P., Oordienko, V.G. and Kolesnikov, D.G., Khim. Prir. Soedin •(6). 789-93 (1973). 56. Kitao, K. and Araki, M. Makuzai, Kenkyu, No.34, 57-61 (1965). 57. Chen-LingjZhongcaogao, 12(9). 391 (1981). 58. Shung, O.K., Akiyama, T., Sankawa, U., Litaka, Y. and Han, Dac-Suk., J. Chem. Soc. Chem. Commiimn. (19). 909-10 (1980). 59. Medvedev, V.A., Korshikov, I.I., Bashkatov. V.G. and Tarabrin, V.P., Piziol Rest (Moscow), 24(4). 858-60 (1977). 60. Al, H.R. .and Osman, P., J. Iraqi Chem. Soc, 2(1) 87-93 (1977). "^ 61. Khatoon, P., Khabir, M. and Ansari, W.H., J. Indian Chem. Soc, 62(7) 560-1 (1985). 62. Kondo, R. and Imamura, H., Makuzai Gakkaishi 31(11). 927-34 (1985). 63. Bagchi, A., Sahai, M. Ray, AnilB., Plant a Med. (5), 467-8 (1985). 64. Sinha, S.C, Sahai, M. and Ray, Anil, B., J. Nat. Prod. 49(3). 546 (1986). 65. Ansari, P.R., Khatoon, P., Ansari, W.H., and Rahman, ¥., J. Indian Chem, Soc, 62(6). 487-8 (1985). 66. Verzele, M., Delahaye, P., and Van Damme, P., J. Chromatogr. 362(3). 363-4 (1986). - 222 - 67. Campbell, T.A. and Grasse, K.A., Biomass 9(3). 187-94 (1981). 68. Karimdzhanov, A.K,, Islambekov sh, yu, Mavlyanov, 3.M, and Isamilov, A.I., KMm Prir Soedin Xll» 386-7 (1986). 69. Bagchi, A., Sahai, M. Sinha, S.C., Ray Anil. B. Oshima, Y. and Hikino, H.S., J. Chem. Rea., Synop. (12). 398-9 (1985). 70. Chen, Y.Y., Ghting Tsao Yao, 12(-1). 21 (1981). 71. Watson, B.S., Murphy, J.C, and Elsohly, M.A., J. Invert. Dermcitol, 80(3). 149 (1983). 72. Parveen, M. and Khan, N.U., unpublished result. 73. Sunthanker, S.V. and Dawson, C.R., J. Amer. Chem, Soc., 76, 5070 (1954). 74. Ma, C,R., Blsohly, M.A. and Baker, J.K,, J. Chromatogr,, 62, 200 (1980). 75. Khan, N.U., Ansfari, W.H., Usmani, J,N., Ilya4, M. and Rahman, W., Phytochemistry, 10, 2129 (1971). Chapter 6 GONSTITUaiTS OF SEMIS HQMONOIA (EUPHORBIAGEAE) A small genus of shrubs and small trees distributed from India to New Guinea. Four species occ\ir in India. H. reparia, an erect rigid ever green dioecious shrub found in North, East and Central India, usually inhabiting rocky river beds. The various part of this genus is important for their medicinal value . The root is laxative, diuretic and emetic. A decoction of the root is given in piles, stone in the bladder, gonarrhoea, syphilis and chest pain. In philippnes it is used as a mouth wash for tooth ache. No work on the chemical constituent of genus Homonoia has been reported. Discussion :Nr[MGiTY INTFNGIT'i •s GJ c^j en I I 1 ' I I <5r J' ^- "1' (O \ '5^-3 1- O - 'S5 L- — 4 H ^ J .3 -< b7 V V - 224 - CONSTITUENTS OP HQMONOIA RIPAHIA Since no work has been reported from this genus, the present work constitutes the first example of chemical investigation on the genus Homonoia. The coarsely powdered aerial part (^ kg) of Homonotl. reparia (procured from Molim, Goa, India) were extracted with methanol. The methanol extract was adsorbed on silica gel and was purified by coliimn chromatography. The column was eluted with petrol, petrol-benzene (1:1), benzene, ethyl acetate and then with methanol. Three compounds were isolated from different fractions. HR-I HR-I was eluted with petrol-benzene (1:1) and crystallized from GHCl^-BtOH as colourless needles, m.p. 240°G.It was analysed for G^QH^QO (M"*" 424). The IR spectrum showed a strong wide carbonyl band at 1690-1700 cm" and mass shows (Pig. 1) the base peak at m/z 300 and molecular ion at m/z 424. The result of H NMR is given in Table-I and Pig. 2). The H NMR showed five singlet attributable to the protons of eight methyl groups at 6 0.83(3H), 0.93(6H), 0.97(3H), 1.06(9H) - 225 - and 1,12(3H). Methylene protons appeared as multiplet at 6 1.32 and 6 1.5-1.9. The multiplet at 6 2,28-2.52 integrating for 2 protons will be assigned to methylene proton at G-2 which is adjacent to carbonyl group at 0-3. A double doublet at 6, 5.56 integrating for 1 proton is attributable the olefinic proton. These data oo!spare well with taraxerone and. srzpport Its stiructure.(l). (I) TABLE - I Chemical shift of prtons of HR-I (6 scale) Assignment Chemical shift of prtons (HR-I) CH^ 0.83 (s, 3H) 2 X GH, 0.93 (s, 6H) CH^ 0.97 (s,.3H) 3 X CH, 1.06 (s, 9H) - 226 - Table I continued Assignment Chemical shift of protones (HR-I) GH^ 1.12 (s, 3H) methylene proton 1.32(m), l,5-1.9(m) methylene proton 2.28-2.52(m, 2H) at C-2 olefinic proton 5.56 (dd, IH) s = singlet, dd = double doublet, m = multiplet spectrum run in GDGl, at 60MHz, TMS as internal standard. HR-II Eluted with benzene, crystallized with benzene-acetone, m.p. 253 C, it was comparable with the authentic samr)le of gallic acid. HR-III [Quercetin 3~0(6"-0-a-L-rhamnopyrano3yl)-3-D-glucopyrano- side] Pale yellow solid, m.p. 187-188^0, gave positive Shinoda and Molisch tests. The chromatographic spot appeared deep purple under UV light which turned yellow with ammonia indicating it to be a flavonoid glycoside. UV spectral shifts with diag nostic reagent (Table-2) indicated the presence of 7-OH, 5-OH and 3",4'-orthodihydroxyl groups in HR-III. - 227 - TABLE ^MeOH ^^ . 258, 265 sh, 298sh, 359 + NaOMe 272, 328, 407 -I- AlOl^ 276, 432 + AlGl^HCl 270, 330 sh, 401 + NaOAc 272, 323, 389 + NaOAc/H^BO^ 263, 301, 383 On complete hydrolysis "with 8'/^ ale. HCl it gave quercetin glucose and rhamnose. The sugars were identified by paper chromatography and quercetin by m.m.p., comparison of its R^ value and UV spectral data with authentic sample. Partial hydrolysis of HR-III with l'/, HpSO- for 1 hr. yielded a glycoside HR-IV and rhamnose, HR-IV on further hydrolysis (acidic and enzymatic) afforded quercetin and D-glucose. D-glucose was identified by PC. The UV spectrum of the aglycone (HR-IIIH) (quercetin) was almost similar to that of HR-III and HR-IV except for the presence of free 3-hyd2X>xyl (bathochromic shift in Band I acid stable complex formation with AlGl^). Which must therefore be glycosylated in HR-III, HR-IV was thus identi fied as quercetin 3-0-p-D-glucoside. The above treatment also showed that the two sugars must be in the disaccharide form - 228 - linked to only one position of the aglycon (G-3 of quercetin) with rhamnose as the terminal sugar. Acetylation of HR-III with ACpO and pyridine afforded an acetate HR-IIIA, ni.p.l35 C, which was comparable with the authentic sample of quercetin 3- 0-rutinoside because in the sugar region a multiplet at 6 0.91 and a doublet at 4.52(J = 2Hz) were assigned to rhamnose methyl and rhamnose G-1 proton, which indicate that rhamnose should be the second moiety of a rhamnosyl-glucosyl disaccharide. The anomeric proton (H-1") of glucose appeared as a doublet at 6 5.35(J = 7Hz). This confirmed the direct attachment of the glucose to the aglycon and diaxial coupling between H-1" and H-2" indicated p-configuration and the rhamnose methyl appeared as multiplet at 6 0.91. From these data it was identified as rutinose moiety. The mass spectrum show the molecular ion at [M-sugars] 302 which further support the aglycon as quercetin. HR-III on methylation by usual method followed by hydro lysis afforded quercetin 5,7,3',4*-tetramethyl ether, 2,3,4- tri-0-methyl-L-rhamnose and 2,3,4-tri-O-methyl-D-glucose. The partially methylated sugars were identified by comparison with authentic samples (R^ value and shade on TLC). On the basis of above facts HR-III was partially charac terized as quercetin -3-0-p-D-glucopyranosyl[l—> 6]-0-.aJ. rhamnoside (II). - 229 - OH ^^H (II) Experimental - 230 - EXTRACTION OF THE CONSTITUENTS FROM THE LEAVES OF HOMONOIA REPARIA (EUPHORBIACEAE) The dried and crushed aerial part (-^ kg) of H, reparia were extracted with methanol. The whole methanol extract was purified by coltimn chromatography (silica gel, BDH). The column was eluted with petrol, petrol-benzene (1:1), benzene, ethylacetate and then with methanol. Taraxerone (HR-I) The fraction eluted with petrol-benzene (1:1) crystallized from GHCl^-EtOH as colourless needles, m.p. 240 G. It turns red on addition of cone. HpSO.^ followed by acetic anhydride (Liebermann Burchard test). R^ = 0.25(Benzene). IR -^f^l cm"-'- : 1690-1700(0=0) V mamaxx MS : m/z 424(37), 409(18.3), 300(100), 285(45.4), 257(9), 232(7.3), 218(16.5), 205(62.6), 204(95), 189(10), 149(10) 133(25), 121(12), 119(10), 109(15), 107(10), 95(17), 93(10), 91(8), 67(15), 57(15), 43(12). - 231 - •'•H-NMR (60MHz, GDGl,) : Values on 6 scale 0.83(s, 3H, Me), 0.93(s, 6H, 2Me), 0.97(s, 3H, Me), 1.06(s, 9H, 3Me), 1.12(s, 3H, Me), 1.32(m), 1.5-1.91(m, methylene proton), 2.28-2.52(m, 2H, methylene proton at 0-2), 5.56(dd, IH, olefinic proton). HR-II It was eluted with benzene and crystallized from benzene- acetone, m.p. 253°C (decomp). On TLG examination it was found to be comparable with the authentic sample of gallic acid. Quercetin-3-O--(6"-O-a-L-rhamnopyrano3yl-0-D-glucopyrano3ide) (HR-III) Blution of the column with ethyl acetate afforded yellow solid which was further crystallized from EtOAc-acetone as yellow crystalls (25 mg), R^ = Oo46(BAW), m.p. 187-88^0. It was comparable with authentic sample of quercetin3-0-rutinoside ^MeOH 256, 265sh, 298sh, 359nm; (MeOH-NaOMe)--272, 328, 407 nm; (MeOH-AlGl,) 276, 432nm; (MeOH-AlGl^-HGl) 270, 330sh, 401nm; (MeOH-NaOAc)'-272, 323, 389nm; (MeOH-MaOAc-H^BO^)-263, 301, 383nm. The acetate was comparable with the authentic sample. - 232 - Hydrolysis of HR-III An alcoholic solution of HR-III (5 rag) was heated with • 8/ aqueous hydrochloric acid on a water bath. Heating was continued for 2 hours to ensure complete hydrolysis. The yellow solid separated from the aqueous hydrolysate was filtered off and was crystallized from methanol to give yellow needles of the aglycone (HR-IIIH), m.p. 315°C showed no depre ssion in m.p. on admixture with authentic sample of quercetin. Chromatographic identification of sugars The filtrate was neutrallized with BaCOH)^ and was examined by paper chromatography (3AW). The chromatograms were developed by spraying with aniline hydrogen phthalate and heating at llO^C for 5 minutes. The sugars were identi fied as D-gl\icose (R^ 0.18) and L-rhamnose (Rp 0.36) by comparison with authentic samples of siigars. Partial hydrolysis of HR-III The glycoside (10 mg) was hydrolysed by refluxing with IX HpSO,. Aliquates were taken out at different intervals and examined by paper chromatography (BAW). After one hour it yielded a glycosidic compound HR-IV and a sugar L-rhamnose. HR-IV was crystallized from methanol, m.p. 235-37°G, UV spectral data are same as HR-III. - 233 - Acidic hydrolysis of HR-IV HR-IY (5 mg) on hydrolysis with 8/f HCl for two hours yielded an a^lycone identical with HR-IIIH in m.p. R» and UV spectral data. The sugar was identified as glucose. Permethylation of HR-III followed by hydrolysis HR-III was methylated with dimethyl sulphate and anhydrous potassium carbonate which on hydrolysis with HCl gave quercetin 5,7,3',4'-tetramethyl ether (HR-III HM) m.p. 194-196°C, 2,3,4-tri-0-methyl-3-glycose (R^ = 0.54) and 2,3,4-tri-O-methyl-L-rhamnose (R^ = 0.75). UV spectral data of HR~IIIHM -^lyieOH 252, 359nm; (MeOH-AlGl,), 261, 419nm; (MeOH-AlGl,- *^max HCl), 260, 419nm; (MeOH-NaOAc) 253, 362nm; (MeOH-NaOAc-H^BO^) 251, 360nm. Refrences - 234 - 1. "The Wealth of India, Raw Material", C3IR Publication New Delhi India, Vol.V, pp. 114-15 (1959). 2a. Tolloch, A.P., Lipids 12, 233 (1977). b. DJerassi, 0., Budzikiewicz, H. and Wilson, J.M,, Tetra hedron Letters, 7, 263 (1962). 3. Hattori, S., in The Chemistry of Plavonoid Gompoimds (ed. T.A. Geissman) Pergajnon Press, Oxford (1962). 4. Harbome, J.B.,'in Biochemistry of Phenolic Gompoimds' Harbome, J.B. Ed., Academic Press Inc. New York, N.Y., 133 (1964). Chapter 7 - 235 - Og,A SIFLAVONOID ISOLMBD gR01LJHB.JaiMI aj"!! rf w Several flavonoids are moderately effective against labora tory cultures of malignant cells. Kupchan and coworkers have shown that eupatin (I) , eupetoretin (II) and centaureidin ^III) are moderately effective agaiiast a caroiiioma froa nasopharynx. OMe 0M« OR O OH O (I) Eupatin (R = H) (III) Centaureidin (II) Bupatcretin (R = Me) Quercetin and its glycosides were weakly inhibitory to human brain tumour cells . Wattenberg and Leong found that quercetin pentamethyl ether and rutin were strongly and moderately effective inhibitors, respectively, of benzo (a) pyrene induced pulmonary adenoma in mice. However, no work has been reported on the anticancer activity of biflavonoids. This stimulated us to study the biflavonoids. - 236 - DISCUSSION 5 The biflavonoids have been tested for spasmolysis , inhi- g bition of cyclic AMP phosphodiesterase and cyclic GMP phospho- 7 fl diesterase and inhibition of hepatoma cells , The present study deals with antileukemic activity of benzyl drivative of natural biflavonoid, amentoflavone. Amentoflavone was isolated from the leaves of Thu.ja q orientalis by the method of Pelter et al. . The racemic mixture of hexa-0-benzylamentoflavone (NSC No.367782) was prepared by the reaction of amentoflavone with benzyl chloride in presence of dimethyl formamide and anhydrous potassium carbonate. The testing of hexa-0-benzylamentoflavone was done in P 338 leukemic tumor system. Treatment was as a single intraperitonial injection on day 1 post tumor implant. Doses tested were 400, 200 and 100 mg/kg/injection. The P 388 tujnor inocul-um was 1x10 injected intraperitoneally. The doses of hexa-0-benzylamentoflavone of 400 and 200 mg/kg/ injection were not active (T/G<127, T/C '(, : ratio of survival in days of test animals and control animals) and the doses of 100 mg/kg/injection was toxic (T/C <86 is non toxic). The screening data are the result of screening performed under the auspices of the Developmental Therapeutics Program, Division of Cancer Treatment, National Cancer Institute, Bethedsa. - 237 - EXPERIMENTAL ISOLATION A3^TD IPBNTIPICATION OF ISOMBinQ BIHiAVONES FROM THE LEAVES OF THUJA ORIENTALIS (CUPRESSACBAB) Thu;1 a orlentalis Linn (cupressaceae) was procured from the Department of Botany, A.M.U,, Aligarh, India. The dried and powdered leaves (5 Kg) were completely exhausted with hot methanol and the methanol extracts were concentrated first at atmospheric pressure and then under reduced pressure. A gummy dark green mass obtained. This was treated with petroleum ether (60-80°) and benzene till the solvent in each case was almost colourless, to remove nonflavonoidic and resinous matter. The gummy mass was then refluxed with ethyl acetate for 24 hrs and filter. The filterate was evaporated to dryness and the residue treated with hot water. The water insoluble mass was dissolved in methanol and dried under reduced pressure to give a dark green residue (10 g) which responded to the usual colour test for flavonoids. The crude flavonoidic mixture (10 gm) was adsorbed on silica gel (50 g) and transferred over a column of silica gel (200 g) set with petroleum ether (60-80°). The column was eluted successively with petroleum ether, benzene and benzene- ethyacetate (9:1, 8:2 and 1:1). The fraction eluted with - 238 - benzene was found to "be in pure form. It was comparable with 5,7,4'-trihydroxyflavone (apigenin) (R^ = 0.54). The fraction eluted with benzen-ethyl acetate were combined and solvent distilled off, A yellowish brown solid (3.5 g) thus obtained, on TLC examination (BPP, 36:9:5) showed the presence of five compact spots. It was therefore subjected to preparative layer chromatography (silica gel BDH, BPP 36:9:5) and the five bands were separated and labelled as TO-I (Rp = 0.12, 100 mg) (myricetin), TO-II (R^ = 0.17, 600 mg) (mixture of amentoflavone and cupressuflavone), TO-III (R^ = 0.22, 75 mg) (quercetin), TO-IV (R- = 0.36, 100 mg) (mixture of hinokiflavone and mono methyl ether of amentoflavone), TO-V (R^^ = 0.52, 60 mg) (kaempferol). TO-II TO-II was comparable with amentoflavone. On methylation with dimethyl sulphate and anhydrous potassitun carbonate in dry acetone it was found to be a mixture of amentoflavone and cupressuflavone hexamethyl ether. Benzylation Biflavone mixture (500 mg) was benzylated with benzyl chloride (2 ml) in dry DMP (100 ml) and anhydrous potassium carbonate (10 gm). The reaction mixture was refluxed on a heating mentle for about 24 hours and then the reaction - 259 - mixture was poured into water. White ppt were separated out which was filtered washed with water and dried. On TLC examination (silica gel, Benzen-Ethyl acetate-Formic acid, 45:5:1) it showed two fluorescent spots, A(Rf. = 0.5) and B(R^ = 0.71) in uv light. The major product was purified by- column chromatography (silica gel) using chloroform, chloroform- ethyl acetate (8:2) as the eluent. The chloroform fraction yielded compound A which was cryststLlized with ethyl acetate. Compound A (1-4'.II-4M-5. II-5,1-7.II-7-Hexa-O-benzyir 1-3'. II-81 biflavonej It was crystallized from ethyl acetate as colourless needles (300 mg), m.p. 185-86^ mol.wt 1078 (mass). •hi NMR(CDCl^) : Values on 6 scale 7.94(d, J = 9Hz, 2H, H-I-2',6'); 7.65(d, J = 9Hz, 2H, H-II-2',6'); 7.30-7.50(m, 20 H, phenyl-I-4',11-4',1-7,11-7)j 7.15-7.28(m, lOH, phenyl-I-5,II-5); 7.05(dd, J = 9Hz, 3Hz, IH, H-I-5'); 6.85(d, J = 9Hz, 2H, H-II-3',5'); 6.70(s, IH, H-II-6); 6.64(s, IH, H-II-3/H-I-3); 6.58(d, J = 3Hz, s(merged), 2H, H-I-8, H-I-3/H-II-3); 6.49(d, J = 3Hz, IH, H-I-6); 5.20(s, 2H, -OCH2-II-5); 5.19(s, 2H, -OCH2-I-5); 4.90(s, 8H, -0CH2-I-4', II-4', 1-7,11-7). - 240 - Anticancer Testing P 388 leukemic tumor system was used for testing. Tumor inoculum was 1x10 injected intraperitoneally. The treatment was as a single intraperitonial injection on day 1 post tiimor implant. Doses tested were 400, 200 and 100 mg/kg/injection, T/C i is the ratio of survival in days of test animals and control animals. 241 - 1. Kupchan, S.M., Siegel, C.W., Knox, J.R. and Udayamurthy, M.S., J. Org. Chem. 2i» 1460 (1969). 2. Kupchan, S.M. and Bauerschmidt, E., Phytochemistry 10, 664 (1971). 3. Dittman, J.H., Herrmann, D. and Palleske, H., Arzneim. Porsch 21, 1999 (1972). 4. Wattenberg, L.W. and Leong, J.L., Cancer Res. ^, 1922(1970). 5. Horhammer, L., Wagner, H. and Reinhardt, H., Naturforsch, 22B, 768 (1967). 6. Ruckstulin, M., Beretz, A., Anton. R. and Landry, Y., Biochem. Pharmacol. 28, 535 (1979). 7. Beretz, A., Joly, M., Stociet, J.C. and Anton, R., Planta Med., 26, 193 (1979). 8. Joly, M., Beck, J.P., Haag-Bermier, M. and Anton, R., Planta Med.,^22» 230 (1980). 9. Pelter, A., Warren, R., Hameed, N., Khan, N.U., Ilyas, M. and Rahman, W., Phytochemistry, ^, 1897 (1970). - 2!+2 - LIST OF PUBLICATIMS 1. Biflavones from the leaves of Himalayan Yew, Taxus wall!chiana Zucc., Nazneen Parveen : H.M. Taufeeq, N.U. Khan; J. Nat. Prod,, 48(6), 944 (1986). 2. Biflavones from the leaves of Araucaria araucana, Nazneen Parveen, H.M. Taufeeq, N.U. Khan, J. Nat. Prod. 50(2-4) Mar-April (1987). 3. Luteolin 7,4'-dimethyl ether, 3'-0-p-D-glucoside from G-eloniimi multiflon;un, Nazneen Parveen and Nizam U. Khan Phytochemistry 26(7). 2130-31 (1987). 4. Biflavones from the leaves of Fitzroya patagonica Hook F, S.1V.1. Naqvi, N. Parveen, M, Parveen and N.U. Khan, Curr. Sci., 56(10), 480 (1987). 5. Constituents of genus Taxus, Nizam U. Khan and Nazneen Parveen, J. Sci. Ind. Res. 1987 in press. 6. The constituents of the Himalayan Yew Taxus wallichiana Zucc. N. Parveen, H.M. Taufeeq and N.U. Khan, 1st Annual Conference ISSAC-I, April 28-29, 1984, Calcutta. - 2^3 - 7. Structure and anticancer activity of biflavonoids, N.U. Khan, N. Parveen, M. Parve^n and H.M. Taufeeq, Flavone Sympoaiura of Medical Foundation of Buffalo Inc., Buffalo New York, Jyly 22-26, 1985. 8. Biflavones from the leaves of Araucaria araucana, Nazneen Pairveen and Nizam U. Khan, 22nd Annual Convention of Chemist, Indian Chemical Society, Sep. 3» 1985, Calcutta. Papers Under Preparation 1. Chemical constituents from the leaves oi Homonbidreparia. 2. Phenolic constituents from Rhus pun.jabnesis and Rhus semi alat a. 3. Constituents from the leaves of Acer oblongom. CHEMISTRY OF NATURAL PRODUCTS SUMMARY THESIS SUBMITTED FOR THE DECREE OF ©ottor of ^Ijilosiopljp IN CHEMISTRY NAZNEEN PARVEEN ^^AAC DEPARTMENT OF CHEMISTRY ALIGARH MUSLIM UNIVERSITY ALICARH (INDIA) JUNE 1987 THE CHEMISTRY OF NATURAL PRODUCT SUl^IMARY The thesis constitutes seven chapters. Chapter - 1 and 2 deal with the isolation and characterization of biflavones from the leaves of Araucaria araucana and Taxus wallichiana, respec tively. Chapter - 3 includes the isolation of a new flavonoid glycoside from Gelonium multiflorum. In chapter 4 to 6, consti- tuents, such as, phenols, flavonoidg^nd terpenes are being reported for the' first time from Acer oblong tun, Rhus pun.jabnesis Rhus semialata and Homonoia reparia. 'Chapter - 7 constitutes antileukemic activity of a«entoflavone derivative isolated from Thu.1 a orient alls. Bach chapter includes a comprehensive review on the genus investigated for the phytoconstituents and also highlights the recent advances in the respective areas. Chapter - 1: Flavonoidic constituents from the leaves of Araucaria araucana (Araucariaceae) Powdered leaves of Araucaria araucana were exhaustively extracted with acetone. The acetone concentrate after purifi cation was subjected to preparative layer chromatography (benzene-pyridine-formic acid; 36:9:5) and seven fractions were - 2 - isolated. The structure of I-7-O-methyl agathisflavone (AA-I), II_7_0-methyl amentoflavone (AA-II) and I-7,II-7-di-0-methyl cupressuflavone (AA-IVb) were established by "li-NIffi and other biflavones such as dimethyl ether of agathisflavones(AA-III), dimethyl ether of amentoflavone (AA-IVa), 1-4',I-7,II-7-tri-0- methyl agathisflavone (AA-V), trimethyl ether of amentoflavone, -cupressuflavone (AA-VI), tetramethyl ethers of amentoflavone and cupressuflavone were partially identified by comparison with authentic samples© Chapter - 2 : Constituents of Taxus wallichiana (Taxaceae)- The phenolic extractives of Taxus wallichiana on solvent fractionation and column chromatography followed by preparative layer chromatography (silica gel; benzene-pyridine-formic acid, 36:9:5) gave four homogeneous fractions. Amentoflavone (TW-I) and sciadopitysin (TW-IV) were characterized by TI-NMR and other biflavones such as monomethyl ether of amentoflavone (TW-II) and two isomers of dimethyl ether of amentoflavone (TW-III) were partially identified by comparison with authentic sample. Chapter - 3 : Constituents from the leaves of Gelonium multiflorum (Euphorbiaceae) The methanolic extract of Gelonium multiflorum after solvent fractionation and column chromatography yielded GM-I - 3 - which was characterized by chemical and spectral methods as luteolin-7,4'-dimethyl ether 3'-0-p-D-glucogide.This glycoside is being reported for the first time. Chapter - 4 : Constituent of Acer oblongom (Aceraceae) The methanolic extract of the leaves of Acer oblongom on solvent fractionation and coliunn chromatography (silica gel, cellulose) followed by preparative chromatography (silica gel, petrol-benzene, 3:7; benzene-pyridine-formic acid, 36J9:5) gave eight components labelled as AO-I—AO-VIII. p-Amyrin (AO-II), p-sitosterol (AO-III), ethyl gallate (AO-VI), quercetin(AO-VII) and D-2-0-chiro-inositol (AO-VIII) were characterized by chemical and spectral methods. Other constituents such as a triterpene (AO-I), apigenin (AO-IV) and kaempferol (AO-V) were partially identified. Chapter - 5 : The constituents of Rhus pun.i'abnesis and Rhus semialata (Anacardiaceae) The phenolic extractive of the coarsely powdered leaves of Rhus punjabnesis on solvent fractionation and column chromato graphy gave six components labelled as RP-I to RP-VI, RP-I is a single hydrocarbon and RP-III—RP-VI are urushiols with different degree of purity. The characterization of urushiol - 4 - mixture is based on H-NMR and MS data. The phenolic extractive of Bhus semialata yielded five compounds. Isolation and characterization of ethyl gallate (RS-I) and myrcetin (RS-IV) is based on spectral and chemical data. The biflavones were partially identified. Chapter - 6 : Constituents of Homonoia reparia (Euphorbiaceae) The methanolic extract of the crushed aerial part of Homonoia reparia on pixrification by column chromatography yielded three compounds. Taraxerone (HR~I) and quercetin 3-0--rutinoside (HR-III) were characterized by "TI-MR, MS and chemical methods. ^I^xi^f: o^'-^d (HR-II) was identified by m.p. and comparison with the authentic sample. This is the first report on the chemical examination of the genus Homonoia. Chapter - 7 : Antileukemic activity of a biflavonoid isolated from the leaves of Thu.la orientalis (Cupressaceae) Amentoflavone was isolated from the leaves of Thu.ja orientalis and its hexabenzyl ether was prepared. 1-7,11-7,1-5, 11-5,1-4',11-4-'-hexa-0-benzyl amentoflavone shows anticancer activity. This is the first report of anticancer activity of a biflavonoid. - 4k.