CHEMISTRY of NATURAL PRODUCTS Boctor

Home , Amentoflavone, Flavanonol, Zapotin

CHEMISTRY OF NATURAL PRODUCTS

THESIS SUBMITTED FOR THE DEGREE OF Boctor lof $||Uos;op|)p IN CHEMISTRY

MEHTAB PARVEEN

DEPARTMENT OF CHEMISTRY AUGARH MUSUM UNIVERSITY: AUGARH (INDIA) JUNE 1981 343?

T3497 (i)

CHEMISTRY OF NATURAL PRODUCTS II .ii.! •• • •• • • m I ii ,.•>.•> •••••••••••.•I ,1 • 1 iM aMxtaiMiMii.

s u M M A R Y

The thesis comprises four chapters. Chapter-I deals with the isolation of methylated flavones from Ardisia floribunda. Chapter-II and III constitute isolation and characterization of biflavones from Cupressus funebris,

Arancaria cooXii and Rhus alata, respectively. Chapter-II also includes antibacterial activity of biflavonoids.

Chapter-IV deals with the isolation and characterization of triterpenoids and xanthones from Garcinia mangostana.

Each chapter includes an upto date introductory review of the area covered in the discussion.

Two new flavones, two new isoprenylated xanthones and rare cycloartenol derivatives have been isolated and characterized in addition to several known biflavones,

Chapter-I i Two new flavones from Ardisia floribunda

Two new flavones were isolated in the pure form from the twigs of Ardisia ^oribunda. They were characterized by UV, ^H NMR and mass spectroscopy as

(a) 5,6,3 ',4 '-tetramethoxyflavone

(b) 5 ,6,2 ',3',4 '-pentamethoxy flavone (ii)

A mixture of ^-sitosterol and campesterol were also isolated and identified.

Chapter-il :

1. Biflavones from Cupressus funebris

The biflavones from the leaves of Cupressus funebris were isolated and characterized by chemical and spectral techniques as

(a) Sequoiaflavone

(b) Robustaflavone

(d) Di-O-methylrobustaflavone

Biflavonoids were tested for antibacterial activity

(in vitro) and were found active against Stephylococcus aureus and pseudomonas aeruginosa.

purified amentoflavone isolated from C. funebris was benzylated and mixed with the same compound isolated from

Thuja orientalis. it was tested in the P3 88 leukemic tumor system and it was found toxic. (iii)

2, Biflavones from Ara34caria CooXii

The biflavones from the leaves of Araucaria cookll were reinvestigated and amentoflavone, cupressuflavone, agathisflavone, robustaflavone, mono-O-methylagathisflavone

& mono-0-methyl robustaflavone were detected by TLC and preparation of permethylated derivative.

Antibacterial activity of the biflavonoidic fraction was also studied and it was found to be active against

E«eoli t tgalmonella euteriditis and pseudomonas aeruginosa.

Chapter-III i Biflavones from Rhus alata

Biflavones from the leaves of Rhus alata were

Isolated and characterized by chemical and spectral methods.

Following biflavonoids were found to occxir in the leaves of

Rhus alata.

(a) Agathisflavone

(b) Amentoflavone

Chapter-IV : Constituents of Garcinia mangostana

Several triterpenes were isolated from the petrol fraction of the leaves of G.mangostana. They were 'character ized by IR, ^H NMR and mass spectroscopy as

(a) Cycloartenol

(b) Friedeleln (iv)

(d) Betulin

(e) 9, 19-cyclolanost-24-en-3, 26-dlol

(f) Manglfrollc acid.

The leaves of G,mangostana were investigated for / the occunance of xanthones and triterpenoids. Two new xanthones alongwith a known xanthone were isolated.

They were characterized by UV, IR, ^H NMR and mass spectroscopy as

(a) 1 ,5 , 8-trihydro-2 [^3-inethyl-2-butenylJ xanthone (b) 1 ,6-dihydroxy-3-raethoxy-2 ^3-n'.ethyl-2-buteny2j xanthone. (c) Gartanin

a and b have been reported for the first time. PHONE-Office : 5515 DEPARTMENT OF CHEMISTRY ALIGARH MUSLIM UNIVERSITY ALIGARH, U. P.. INDIA

Ref. No. Date

Tlifti IB to cortlQr tbrnt voflt dlsoussed

tR thjjB is th« orlglnaS. oontrllMjfelcsQ of th®

Qsn^dftto is sultsLblB for sutsmlssiofi for the

Partial ftilfljteient of tbo 'Iegp«« of Doctor of

T / '( Him Sh^ ) Acknowledgement

I wish to express my deep sense of gratitude to Dr. Nizam Ud-Din Khan, Reader, Department of

Chemistry, Aligarh Muslim University, Aligarh whose consistant guidance and encouragement led to the development of the work embodied in this thesis.

I owe an enormous debt to him for his sincere advice and useful suggestions.

I extend my heartfelt thanks to Prof. M.S.Ahmad,

Chairman, Department of Chemistry, Aligarh Muslim

University, Aligarh for providing necessary research facilities.

I am highly indebted to my other laboratory colleagues for continuous help and encouragement,

I express my sincere regards to my revered parents for their sacred love and good wishes which has been a great source of inspiration to me,

( Mehtab parveen )

M.Sc., M.Phil. Dedicated to my Late Father Contents

CHAPTER - I

INTRODUCTION Page

1 . CHEMICAL CONSTITUENTS OP ARDISIA 1 Patty Acids 1

Terpenes 1

Phenolic Compounds 4

Quinones 6

Miscellaneous 8

2. NATURAL DISTRIBUTION OF PLAVONOIDS 9

(i) Naturally occurring tetramethoxy flavones 9

(ii) Naturally occurring pentamethoxy flavones 10

3. IDENTIFICATION OF FLAVONES 1 2

Ultraviolet and visible spectroscopy 1 2

proton magnetic resonance spectroscopy 14

(i) Solvent 14

(ii)PMR spectral analysis 14

Mass spectroscopy of flavonoids 1 8

DISCUSSION 22

Characterization of B-1 23

Characterization of C-1 27

EXPERIMENTAL 32

isolation of Constituents from Ardisia floribunda 32

Fraction - A 32 Fraction - B 33

Fraction - C 34

Fraction - D 35

REFERENCES 36

CHAPTER - II

INTRODUCTION 40

1 . CHEMICAL CONSTITUENTS OF CUPRESSUS 40

Tropolones 40

Monoterpenes 44

Sesquiterpenes 48

Tricyclic sesquiterpenes 49

Diterpenes 53

Flavonoids 58

Cupressus flavonoids 58

(i) C-C linked biflavonoids 58

(ii) C-O-C linked biflavonoids 60

2o CHEMICAL CONSTITUENTS OF ARAUCARIA 61

3. NATURAL DISTRIBUTION OF BIFLAVONOIDS 67

Identification of biflavonoids 70

"" H NMR spectroscopy 70

use of solvent induced shift for establishing interflavonoid linkage 77

Lanthanide induced shift 79

"" ^C M^R spectroscopy 83

Mass spectroscopy of biflavonoids 86 DISCUSSION

1 . BIFLAVONES FROM CUPRESSUS FUNEBRIS 91

Characterization of CF-1 92

,, »» CP-2 94

ft f * CF- 3 95

» > » ) CF-4 96

CF-5 99

Antibacterial activity 101

Anticancer activity 101

2. BIFLAVONES FROM ARAUCARIA COOKII 102

Isolation of biflavonoids from the leaves of Araucaria Cookii 1 02

Characterization of aC-1 1 04

» » », AC-2 104

f > ,, AC-3 105

> > ,, AC-4-AC-9 105

Antibacterial activity of biflavo noidic fraction 106

EXPERIMENTAL 107

1. ISOLATION ^ND CHARACTERIZATION OF BIFLAVONES FROM C. FUNEBRIS 107

Extraction 107

Purification of biflavonoidic mixture Column chromatography 107

Separation of biflavonoidifi mixture- 1 08 PTliC

CF-1 109

CF-2 110 CF-3 110

CF-4 111

CF-5 112

Antibacterial activity of biflavonoidic fraction of C.funebris 113

Anticancer activity of biflavonoidic fraction of C,funebris 114

2o ISOLATION AND CHARACTERIZATION OF BIFLAVONOIDS FROM ARAUCARIA COOKII 115

Methylation of AC-1 116

AC-2 11 6

^c-3 117

AC-4 117

AC-5 117

AC-7 117

AC-8 118

AC-9 119

Antibacterial activity of biflavonoidic fraction of A.cookii 119

REFERENCES 1 20 CHAPTER-III

INTRODUCTION 130

CHEMICAL CONSTITUENTS OF RHUS 1 30

Terpenoids 1 30

Simple Phenols 1 31

Plavonoids 1 32

Biflavonoids 133

Flavonoid glycosides 138

Miscellaneous 139

DISCUSSION 142

Characterization of RA-1 142

Characterization of RA-2 146

EXPERIMENT Ali 149

Isolation and Characterization of biflavonoids from the leaves of Rhus alata 149

RA-1 149

RA-2 1 51

REFERENCES 152-54

CHAPTER-IV

INTRODUCTION 1 55

1. CHEMICAL CONSTITUENTS OF GARCINIA 1 55

Terpenoids 155

Xanthones 1 61 - Dioxygenated xantbones 1 61

- Trioxygenated xantnones 1 62

- Tetraoxygenated xanthones 1 63

- isoprenylated xantt»ones i 64

- Geranylated and prenyiated 1 65 Xanthones

- Xanthones with cyclized isoprenoid 1 70

Benzophenones 1 74

Flavonold 177

Monoflavonoids 177

Biflavonoids 178

Miscellaneous 1 86

2. IDENTIFICATION OF ISOPRENYLATED XANTHONES 192

^H mR spectroscopy 1 92

IR, UV and MS spectroscopy 1 99

3. TERPENOIDS AND THEIR IDENTIFICATION 205

DISCUSSION 21 2

EXPERIMENTAL 2S9

REFERENCES 248 LIST OF ABBREVIATIONS

TLC = Thin layer chromatography

PTLC s preparative thin layer chromatography

UV B Ultraviolet

IR = Infrared

NMR sz Nuclear magnetic resonance

PMR = proton magnetic resonance

MS s Mass spectroscopy

Rf = Mobility relative to front nm a Nanometer

BPF Benzene, Pyridine: Famic acid (3 6:9:5) Chapter-I Introduction : 1 :

The genus Axd is la] belongs to the family Myrsinaceae, constituting 32 genia and about 1000 species, with Southern limits in New Zealand and South Africa and Northern limit in Japan, Mexico and Ploxida. Ajd isia is one of the largest genus of family

Myrs inaceae and constitutes 250 species,

1. CHEMICAL CONSTITUENTS OF ARDISIA;

The compounds most frequently encountered in the genus Ard isia are fatty acid^, terpenoids^"^, phenols^'''"" , acids^' ''' quinones^'^'^''^'^'^^""'^, flavans"*® and glycosides^^'^''.

Fatty acids: Plant source e Oleic acid, Linoleic acid, Ardisia cornudentata'

Stearic acid & Palmitic acid

Tercenes:

Cl) (I) Menthol A. cornudentata'

CHaR

(II) R (Ila) -sitosterol, CH. A.cornudentata ,

A. iia-pQ-nica^

(lib) Campesterol, H • A. .janon ica^ ! 2 !

(Ill)

(III) Stigmasterol A .cornudentata'

(ro Cholesterol A. .japon ica 3

4 3 ^ (V) oC--4myrln A. solanacea, _A. .janon lea ' " ! ? !

( n) ^ -Hmvr m A. solanceal A. jgooTi Ica^'^

COOH

3 S (VII) Hederas-en in A. .janon lea '

CH2OH

VIII)

(VIII) Betulin A. ianon ica '''® ' 4 J

COOH

(IX)

(IX) Ursolic acid A. .janon ica 3,8 X.

COOH

(X) Oleanolic acid 3 Q A. ,iat)onica * Sangu isor bigen in _A. .lapon ica' >3,8 Ilexol (.Probably a A.ja Don ica 5,6 7 9 niix_ of baurenol & A.colorata > A. sielbod ii^ bauex ad ienol) 4 9 Baurenol A.solanacea , A.sielbod ii q Bausiad ie::ol A. sielbod ii^

PHiDNOLIC COMPOUNDS;

e -hV^V(€H2)7--CH =CHBu

H VIII) 2-:i]ethyl-::!-(c is-tx idec-S-enyl) A. iaDon ica^*""^ -21-d ihydr oxy benzene 5-(c is-tr idec-3-enyi) .iagon ica^ -'Ti-d ihydroxy benzene A. OH ! 5 :

H O^^^^^CH2)7-CH = CH (All) fi 11 (XII) Aid is in ol-1 A. japon ica '

—CHrrrCH—(CH2)3Me (XIII)

6 11 (XIII) Aid isinoI-II A . .-ianon lea '

y VoH

OH 0 (XIV)

(XIV) wuercet in A. j a 13 on ic a

A.hortorum 12

(KV) (XV) Ardisic acid-B A. .ja-pon ica^'^

1 ? 1 ^ (Bergen in) A.hortoruni ' ^ : 6 :

Heptahydr oxyfl-Hvan &

3,4,5,7,3',/, ^'-heptahydroxy-

flavan A.^acrocarna 16-1B

QUINOl'J.giS:

p-Benzoqu inone A. cornudentata'

(CH2)io—CH3 0 (XVI) 14 (X/I) Enbelin A. .japonica^'^ Ardisia specjg 15 A.macrocarDa

Maesaqu in one .4cetylrnaesaqu In one 14 2-hydxoxy-5-a^'tboxy-3- Ardisia scecies pentadecenyl-(tri-decenyl- and tridecyl-) benzoquinone 0 0 MeOv^A^ (CH2)7-CH =C H - (CH2)7 ^^JK^^I

Si '' 0 (XVITc-o) H R. «2 1 4 (XvIIa) Ardisiaquinone-A H ri CH, Ardisia species, q A. seilbod ii"^ 14 (XIID) iixd isiaquinon0-B H H H Ardisia species,

A. se ilbodii*^

14 (XIIc) -^rd i&iaquinons-C CH,CO H H Ardisia species. Q or H OH^CO H A. seilbod ii-^

or H H Crl^CO : 7 :

0 HO. Ci.13"2H 7

V^OH 0 (XVIII) 7 (XVIII) RapanoT^e A. col ox at a. 14 Ardlsia species, 15-13 A.niacrocarna, 1 A.Dolycephala ' riomoxapanoTie, Homoembel in A.macrocarpa 1 5 : a : "iGCGllc.ne ouG:

0 Me

9 Me (XIX) Plant source 2 (XIX) Theophylline A.cornudentata

Heptacosane, triacontane Sr, hutanol A.cornudenta ta'

3-Methylcaidol A. napon ica IB Leucoanthocyan id ins A.macrocajpa

Cyclamiratin A & D A.alyyiafolia^^

Delphinidin, Malvinidin

i-eonidin, Pelazgonidin & A.crenata 21

±"6 tun id in glycoside : 9 :

2, Matural Distribution Of Flavonolds

beveral natuxally occutriing flavones and theix detivatives 22 23 have been xepoxted. ' More than 475 natuxally occuxing

hydxoxy-, methoxy-, methylenedioxy-, isopxenyl and C-methyl 22 flavones and flavonols have been xepoxted sofax. Some

natuxally occurring tetxamethoxy and pentamethoxy flavones are

listed below for refexence in discussion. (i) Natuxally Occuc.!dn,g Tetxametboxyflavones

OMe (XX^XXII) a. H. R. Plant source •1 '2 (XX) 5,6,7,S-Tetxa 24 methoxyflavone OMe OMe H Helichrysun. berbaceum, Lindera lucida. Zelvhera tuberclosa^^ 2^ (XXI) Scutellare in- OMe H OMe Calicarpa nap on ica/'k ickxia 29 tetxamethyl- Ion Ig-era, Colebrook ia e t be r opoositIfolia^'^ Cbrornolaena odorata?^ Oitxus cult^^ Kickxia spuxia?^ c itrus aur'^nt ium?^ Mar rub lum peri.er inum 35,3' 6

37 (XXII) Isoscutellarein- H OPIe 0'4e Citrus cultp C. ret iculata; 30 tetxametbylether c. sinensis. C. parad isii : 10 :

MeO Of^e 0 (XXIII, XXIV) R R 1 (XXIII) Zapotin OMe H Casimiroa edul

(XXIV) Cerosilin H OMe Casigjioa edulisj^ 45 Sarffentia gregji

OMe 0 (xxr,xxvi)

(XXV) 5,7,2', 4'-Tetra- 46 methoxyflavoTie OMe H Tgjqltialia arluna.

47 (XXVI) 5,7,3,4-Teti:a- H OMe Merillia caloxylon

methoxyflavone"

(ii)- Naturally Occurrinr:?entatBethoxyflavones R

OMe

OMe 0 (XXVII^ XXVIII) R

48 (XXVII) Sinensitin H OMe OrthosLpbon stamlneus Citrus sinensis and 34 AO-.51 citrus auxantium ' : 11 :

H R 1 40 (XXVIII) Tangeretin OMe H Citrus soecies

.OMe

Me.0 / "^OMe

OMe 0 (XXIX, XXX)

R R^ 47 (XXIX) 5,7,3,4,5-Penta- H OMe Mer illia caloxylon

rnetb oxyflavone

(XXX) Hypolaetix] Penta OMe H C Itrus aurant iutn^'^O jtrus 3S 52 met hylethex sinensis. Citrus parad isii

MeO

(XXXI a & h)

R R 1 53 (XXXia) Cerrosillln-B OMe H Sar,?eTitia .gregi i

54 (XXXI-b) Proroger in-0 H OMe ProsQ-pia spicjgeria : 12 :

IDilHTIFIC ATION OF FL

(a). ULTRa/IOLjST AICD VISIBLE SPECTROSCOPY

5 Ro U? spectxoscopy-^-^'^ has become the majoi technique for the

structure analysis of the flavonoid for two main reasons. The

first is that only small amount of the pure material is required.

The second is that the amount of the structural information

gained from the UV spectrum is considerably enhanced by the use

of specific reagents which react with one or more functional

groups on the flavonoid nucleus. The addition of each of these

reagents separately to an alcoholic solution of the flavonoid

induces structually significant shifts in the UV spectrum. Shifts

of this type are commonly induced by the addition of the sodium

methoxide (NaOMe), sodium acetate (NaoAc), sodium acetate/ijor ic

acid (NaO-Ac/H^JtiO^), aluminium chloride (AlCl^) and aluminium

chloride/Hydrochloric acid (AlCl^/HCl).

UV spectra of most flavonoids consists of two ma;ior-

absorption maxima, one of which occurs in the range 240-285 nm

(band II) and the other in the range 300-400 nm (band I).

In general terms the band II absorption may be considered as

having originated from the A-r ing benzoyl system and band I from

the B-ring cinnamoyl system. : 31 :

(1 , Benzoyl 1 cinnanioyl)

In flavones, band I absorption (304-350 ntn) provides a guide to the type of the flavonoid being examined. Further more, highly oxygenated flavones tend to absorb at larger wave lengths than those with fewer oxygen substituents. Band II is effected less by t I changes in B-ring oxygenation, although 3,4-d ihydroxylated flavones generallI y show two peaks (or one peak with shoulder) in this region, while 4-hydroxylated flavones show only one. On the other hand, band II is significantly affected by changes in A-ring oxygenation pattern, and increases from 250 nm in flavone itself to 252 nm in

7-bydroxyflavone , 263 nm in 5-hydr oxyflavone and 5,7-d ihyd roxyflavon^

274 nm in 5,6,7-trihydroxyflavone and 231 nm in 5,7,3-trihydroxyflavone. The absence of hydroxyl group in either ring is usually evidenced by relatively weak intensity of the relevant band.

xicetylation of flavonoid compound tends to nullify the effect of the phenolic hydroxyl groups on the spectra. Thus, acetylation can be a valuable technique .for locating alkoxy groups. For example, hydroxyflavones methylated at 4-position (e.g. acacetion) t after acetylation have UV spectra approximation that of 4-methoxyflavone (max 320 nm). : 14 :

(-b) pfiOTON MAGNETIC I^ESONANCE SPBOTEOSOOPY (pm)

PI® spectroscopy^ is now well established as a method of

flavonoid structure analysis."

(i) Solvent

Although solvents such as CCl^, CDGi^, benzene (or CgDg),

Pyridine, D2O & mixtures of these have been used for Pl^©

v/ork on flavonoids, DMS0-d6 is the most generally useful

solvent for underivatized flavonoids. Normally tbe use

of tr imethylsilylether derivatives of flavonoids with ,56 b 001^ as solvent is preferred'

( ii) PMR Spectral Analysis

PMH in trimethylsilylated flavonoids normally occurs

between ^ oc 9pp7i. 'IMS is used as standard reference,

chemical shift values are quoted in parts per million

(ppm) on ^-scale . : 15 :

A-Hin^ protons

In 5,7-d ihydroxyflavonoids protens at C-6 & "^-S appear sepaxatly as doublets (d,J=2.5 H ) in the range 5.7-6.9 pp^n. z The H-6 doublet consistently occurs at higher field than the H-8.

In 7-hydroxyflavonoids, the additional proton at C-5 is strongly deshidded by 4-keto group and appear near S.O ppm, as a doublet ;,d,J=ca SB. )due to ortho coupling with H-6. Signals for Z ""

H-6 (a quartet, q,or=9 and 2.5 H^) and H-3 (a doublet the 5,7- dihydroxyflavouoids and may even reverse their positions relative to one another.

B-i^ing protons

l' 3'

-OR

In 4-oxygenated flavonoids protons at C-2,3,5, and due to free rotation of B-r ing appear as two pairs of ortho coupled doublets (d) J=ca. S'.5H in the range 6.5-7.9 (i.e. some what z down field from the A-ring protons). The H-3', 5' d oublet always : 16 :

I t occurs upfield at 6.5-7.1 ppa fron the H-2,6 doublet due to

sbieldlng effect of the oxygen substituent and to the deshielding t I influence of C-ring functions at H-2 & H-6. The position of the I I H-2,6 doublet is dependent upon the level of- oxidation of C-ring

and occurs in the range 7.7-7.9 4-oxygenated^lavonoids. I » t In 3,4- dioxygenated flavones the C-5 protons appears as a

doublet (d,J=S.5H ) in the region 6.7-7.1 ppm.

The signals of the protons at 0-2* (d, J=2.5H ) and C-6'

(q,J=2.5 & S.5H^) which often overlap usually occur between 7.2 I and 7.9 ppm. From the relative chemical shifts of the C-2 and • I t 6 protons, methylation of the 3 - hydroxyl can be d istr inguished t » from that at the 4 - hydroxyl. For example, H-2 is usually at t t I a higher field than H-6 in the 4-methoxy-3- hydroxyflavones I t whereas 3 - methoxy-4- hydroxyflavones of the position is reversed

III ? t In 3, 4 . 5-tr ihydroxylated flavonoids, H-2 and H-6 are

equivalent and appear as a two proton sinelet in the range » 1 6.5-7.5 ppm. Methylation of the 3 or 5 - hydroxy may lead to the t I nonequ ivalence of H-2 and H-6 with the consequence that these

protons appear as distinct Doublets (J=ca. 2H ) I 17 !

C-Rins DZotons

The C-5 proton in flavone usually appears as sharp singlet near 6.3 ppm. As such, it can be confused with C-6 or C-S proton signals in 5,6,7-, 5,7,B-or 5,6,7,3- oxygenated flavones.

It has been suggested that a distinction may be made between the

H-6 & H-3 signals be means of signal intensity?^^ In S-methoxy- flavones, long range coupling of H-6 with the 8-methoxylprotons causes the H-6 signal to be slightly broadened, and hence of lower intensity. It has been found that selective detrimethyl- silylation of the 5-hydroxyl group affected the chemical shift of the C-6, C-S & C-3 proton signals in different ways. The H-3 signal shifts downfield by at least 0.15 ppm. and H-6 remain virtually unaffected?^^

Aromatic acetyl and methyl protons in 'Sins' A,B & C.

Aromatic acetyl signals appear at 2 .30-2.50 ppm. Methoxyl proton signals with few exception appear in the range 3.5-4.1 ppm when solvents such as CCl^, CDC13 & DMS0-d6 are used 55b t 18 :

Electron impact mass spectrometry of flavonoid^^ aglycones

serves as one of the valuable aids in determining their

structures.

Most flavonoid aglycones yield intense peaks for the molecular

ions (M^) and indeed this is often the base peak. In addition to

the molecular ion, flavonoid aglycones usually afford major peaks

for M-H and when methoxylated, M-CH- The most useful

fragmentation in terms of flavonoid identification are those which

involve cleavage of intact A-and B-ring fragments, which are

designated here as A^jA^ and ^^ respectively; sorne 56 a of these ions are derived by retro-Diels-Alder (BDA) Process.

Two common fragmentations, designated as pathway I & pathway II

are given below. Pathway I represents the retro-Diels-Alder cleavage

of r ing-C of the flavonoid.

Pathway I

]-I-Transfer

• H : 19 t

i ithw^ay IT

0 Ci

Fl-avones with four or more hydroxyl or methoxyl ffroups such as luteolin and its derivatives ?ive moderatly intense A^. and i3|. fragments (see Fig.1), indeed these are usually the most 56 b structuaiiy diagnostic ions in the spectra.

t r Luteolin-3,4-d imethy iether ( see Fig.1 ) gives a ion at m/e 162 which clearly indiote^ the presence of two methyl groups in the B-ring. The m/e values ( but not the relative

intensities) for the A^"^. and [k+'r^'^ ions at m^e and 153

respectively, are the .same a^ those observed for the correspond inp-

ions from otner flavone.- with the ''-d i hyd r oxy su bst i tut i on pattern ( e.g. apigenin arjd acacetin, pee fiT.?)5. 6 b : ?0

HO 0 Pathway I M'^'m/z 314 (100) with II Transfer Pathway I

,OMe

HC=C VoMe

Bf • m/s 16 2 (12) jV"n/z 15 2 (4)

-Me

^-15 n/s 147 (10)

+ 0H

A + Hn- f n/s 155 (25)

Ficure 1. Selected L'S fracnents fron lute olin-p ' , 4 '-dine thyl ether. 21 1

T+

/ \VoR

_M-287 , E=H,m/s 242(19) - CE5 HO E-Me.m/s 256(15)

/•n-17 /H-437T - E=H,R/Z 269(13) R=Ke,n/z 241(7) ErIIe,R/z 285(9)

tir, r.rH,n/z 270(100) R=IJe,m/z 284(100) Pathway-I Pathway-I with PathTTay-ir H-transfer

' f

HO 0 + 0 t A, R=H.ni/z 152(16) R=H,m/z Il8(l4) ^2, ?;=n ,ia/zl21(6) 152(6) R-ne,n/z 152(15)

- CO

/"A-28/ ,E =H ,n/z 124(18 ) >/"A-»2^ , E =H ,n/ Z 125 (10) II HO tOH E=H,n/z 155( 22) R = ne,:./s 155(1)

Pig-are-2. I'.S fragEent ions frora apigenin and its 4'-r.ethyl ether acace tin. Discussion : 22 :

Tbe isolation of tv/o antituberculax phenols, ardisinol-I and ardisinol-II from Ardisia .jaoonica^ and earlier reports on the constituents of Chinese drug KAi-HO-CHEIN (Ard isia hortorum) prompted us to reinvestigate other species of Ard is ia. Isolation of quinones"^'^'^^''"'^'''^, flavan"''^, triterpenoids^'^''^and phenols^'''® have been reported from different Ard isia species. "We now report the isolation of two new flavones from Ard isia fl or i bund a.

The dried twigs of Ardisia floribunda (Ikg) were cut into pieces and extracted with ethanol. The crude product (0.700 gm) on solvent fractionation and column chromatography over silica gel yielded the fractions A,B and C in benzene. The elution of the column with benzene-ethylacetate (9:1,S:2) gave the fraction D.

The fraction A, on further purification by preparative thin layer chromatography (PTLG) and crystallization from chloroform- methanol gave the component 4-1 (60 mg), Rf 0.34 (silica gel, benzene: chloroform,1:1) A-1 was characterized as the mixture of

^-sitosterol and campesterol, by comparison of its mp, Rf value,

Mass, ""mmR, ^^GNMR spectra with authentic sample.

i^'ract ion .B, on purification by PTLG (silica gel) gave the tit component B-1 which was characterized as 5,6 ,2 , 3,4-I'entametboxy- flavone by Mb, IR, "" HNMR and ^ ^GNMR spectra.

Fraction C on rechromatography on silica gel column gave the component G-l and an unidentified component 0-2. C-.^ was t t characterized as 5,6,3,4~Tetramethoxyflavone. J 25 t

Fraction D on cxystall izat ion from chloxoform cietbanol gave the component D-1 which was partially identified.

Characterization of B-1

B-l,mp 124-25°C, crystallized from chloroform- metbanol and gave orange colour with Mg-HCl and Zn-HGi and negetive test with alcoholic ferric chloride, indicating it to be methoxyflavone. It has UV absorption maxima at 270 and 330nra in chloroform.

The IR spectrum showed a strong band for charbonyl ^roup at 1645 cm''.

The PI© spectrum (Figure-3) of B-1 integrated for five aromatic protons and fifteen methoxy protons (3X50ife) which is given in

Table-1. Two ortbo coupled doublets atS'6.7S (J=9Hz) and 7.50

(J=9Hz) were assigned to H-5'' and H-6'', respectively. The singlet at S6.B3 was assigned to H-3 and another singlet integrating for two protons appearing at S 7.26 was assigned to H-7,S on the basis of data given in Table-2. ^C NMH (33-3HZ, FX-90Q) ^-scale (Pig-4) showed five carbons at 5 56 .1 , 57. 3, 61.0, 61 .2 and 61.9 and fifteen SP^ carbons at 07,4,111,6, 113.2, 119.1, 123.9, 14^.?,

150.5, 155.4, 156.1, 159.7, 163.9, 163.7, 173.7 and 178.25. ^S (Pig.5) shows molecular ion at m/z 372 and diagnostic fragments at m/z 357

(i.l_i 5), 342 (i'I-30), 327 (M-45), 312 (M-30-30), 299, 234, 256 , 177 I t f and 149. Therefore, B-1 was characterized as 5,6,2,3,4-pentamethoxyflavone (XXXlla) which has been isolated for the first time from the natural source. Further work is in progress for the synthesis of

B-1 .

o rts I : 2<- :

xxr. iij 1-H

OMe -15 m/^ 557(55) OMe 6MeO OMe 1,1 , n/s 572(40) - CO

"7+ n/s 542(6)

OMe

MeO OMe OMe OMeni/s 344(5) MeO

or -

n/a"^ 299(5)

- CO

0-Me n/s 284; 4) MeO + Mea OMe n/s- 177(8) -CO

- CO e/C 256(6)

VMe )V 0-M+ e

n/s 149 (85) 7iGurc-5. i:r,cs frc,-neatation.- pr.t'hera of 5 ,6 , 2', 5 ' »4*-pentaric tho::7f Ir voae(£ 1-25:

Table - 1

Chemical shifts (S-scale) of protons of B-1

UI^ Signals Number of J value in Ass ignment Pr ot on s Hz

3.90 (s) 3 5,6 ,2,3U'-Methoxyl

3.93 6 groups

3.94 3

3.96 (s) 3 b.7B (d) 1 9 H-5' 6.S3 (s) 1 3 H-3

7.26 (s) 2 H-7,S

7.50 1 9 H-6'

s. =sing let, d=doublet, spectrun run in CDCl^ at lOOMHz,

TMS as internal standard. t 26

Table - 2

Gheniical shift values (S-scale) of A- and B-ring jrTotons of 5,6- and 5,7- substituted Plavoue in CDCl^.

Methoxyfl avones Chemical Shift (^,pp!!i)

H-6 H-8 H-7,S H-3 (d)(j=2Hz) (d)(J=2Hz) (s) (s)

Zapotin (XXXIlIb) I I 5,6,3,5-Tetxamethoxy- flavone^^ 7.40 6.65

5.6, J-Trirnethoxy- flavone (XXXIIIa)^^ 7.30 6.63

B-1 (XXXIIa) 7.26 6.S3

C-1 (XXXIIb) 7.30 6.61

5.7, 3,4-Tetramethoxy- flavone (XXXIVa)^® 6.35 6.5B 6.58

5 ,7,2 ,4-Tetxarnethoxy- flavone (XXXiVb)^^ 6.36 6.50 6.95

I I > 5,7,3,4, 5-i'enta^ethoxy- 6.56 6.72 flavone (XaxiVc)^® 6.33 : 27 :

MeO

OMe (XXXII)

( a) 0M8

(b) H

OhaiacterIzation of C-1

C-1, mp 171-72°c, M"^=542, crystallized from cbloroforn-methanol and gave orange colour with Zn-HCi and i-'Ig-HCl. It has ultraviolet

(Uv)' absor_L.tion ^axinuni at 243.7 3nd'^27.4n3] in chloroform. In the infrared (W) spectrum, a strong band appears at 1645 cm for the carbonyl gro'jp. In the PMR of C_i (Fig-6, Table-3), the singlets at

S3.93 (3H), 3.95 (3H) and 3.96 (6H) were assigned to methoxyl I I protons at 5,6,3 and 4 position of the flavone. Aromatic region showed a singlet for H-3 at 6.61 (Table-2), an ortho couplet doublet t (J=9Hz) for H-5 at 7.0'J, a metacoupled doublet (J=9 & 2.5 Hz) for I I H-6 at 7.56 v/hich is comparable with B-r ing protone of 5,7,3,4-

Tetramethoxyflavone (XXXiVa)^® The protons at C_7 and C-S appeared < Tl

FtG.g, ''HNMR OF C-1

1 r 3-83 7-5 7 6'5 t 28 s

Table - 3

Cheuical shift (^-scale) of protons of C-1

K!F.yjR Number of J value in Aoo,-rrv,,r,o-K,+ i^JIffi bignals photons Hz Assignment

3.93 (s) 3 -

3.95 (s) 3 - 5,6,3,4-Methoxyl

3.96 (s) 6 - groups.

6.61 is) 1 - H-3 ( 7.00 (d) 1 9 H-5

7.30 (s) 2 - H-7,S

7.36 (d) 1 2.5 H-2'

7.56 (dd) 1 9 & 3 H-6

a .= singlet, d=doublet, dd=doublet of doublet. Spectrum

run in CDCl^ at 60 MHz, TMS as internal standard. I 29 «

as singlet atS7.:50 which is conr-arable with other 5,6-substituted favories^^, such as zapotion (XXXIVb) and 5,6,3 - tr i^iethoxyflavone

OMe

MeO

t I (XXXIIIa), and 5,7,'3',4'--tetra"ietboxyflavoT^6 (XXXIVa), 5,7,2,4- tetxanethoxyflavone (XXXIVb) and 5,7 , 3,4*, 5-pentaTiethoxyflavone

(XXXIVc) (Table-2).

Rl R

OMe

OMe (XXXIV)

(a) OHe H H

(b) H OMi H

The MS (-^ig-^) shows a tiolecular ion at -^/z 342, and the diagnostic fragments at n/z 527 (M-15), 312 (H-30), 297 (H-30-15),

2B2 (i'I-2HcH0 ox M-4Me), 165 (M-136-41) and 137 (M-16 5-41.

Therefore, 0-1 was assigned as 5,6,3',4-tetjcafnethoxyflavone (XXXITb) I 50 :

XXX lib

MeO" y Y OMe MeO ^ 0 li' , n/z 542

MeO" Y ^Jf MeO 0

-15

n/s- 23 2

n/^ 131

n/z 165

Figure-7. I'ass fragneatation pattern of 5 » 6 ,5' »4'-tetrane thorcyf lo.vonfi

( Ci ). t 51 5

which as been isolated fro-n the natural source fox the first tine,

However, a synthetic compound with sa-ne structure and having -.td

179-31° has been reported?'^ Experimental : 52 :

-.11 '^siting points aie uncorrected, analytical and preparative

TLC /ere perfornied on silica gel G (Sisco). PIIR spectra were taken in CDCl^ on a Yarian D-60 and JE0L-4H100 instrument using Tt^IS as internal standard. Mass spectra (MS) v^?ere determined on a JEOL-

OSIG double focussing high resolution mass spectrometer.

Isolation of the constituents of Ardisia floribunda

The dried twigs of .Ard isia flor ibunda (1kg) were collected from the Forest. Research Institute, Dehradun, U.P ( India ).

The ethanol extract of snail pieces of dried twigs of

Ard isia flor ibunda on evaporation and defatting with petrol yielded the crude product (0.700 gm) which gave positive colour test with

Wg-HCl. The chloroform extract (0.40u gm) of the crude product was subjected to column chromatography over silica gel (3 f^") . The colu-nn v/as eluted with organic solvents in order of increasing polarity, (petrol, petrol:benzene, 9:1,3:2,7-3,1:1) and benzene.

Petrol and petrol: benzene fractions contained chlorophyll and hydrocarbon and were rejected. On elution with benzene fraction

A, B and G were obtained.

Fract ion-A

The initial fraction A showed one major band alonp-v/ith chlorophyll was separated through preparative thin layer cnromatography (pTLC, benzene: chloroform, 1:1) which gave A-1

(.60 mg) crystallized trom chloroform-etbanol (mp123-50°, t^f 0.3^, silica gel, benzene: chloroform_, 1:1 ) . A-1 v/as found to be the S 35

mixtuie of/B_sitosterol and ca'inpeste rol on corriparison of its mps,

Rf value, Mass, ''mMR and "" ^GNMR spectx a with authentic sample.

It also gave positive Leiberman Burchard test.

MS data: 414(45,M"^), 400(30), 396(10), 337(5), 382(8), 367(4),

329(8), 315(9), 303(7), 289(8), 273(10), 255(11 ), 231 (10), 213(11 ),

185(3), 173(10), 159(18), 149(23), 121(20) , 105(26), 95(38), 81(53),

69(78), 53178), 55(100).

''hNMR (($'-scale): 5.31(d,=CH), 2.31,2.21 ,2.08,1.92,

1.79, 1.65, 1.43, 1.26, 1.18, 1.00, 0.95, 0.85, 0.80, 0.78, 0.77, and 0.68 (CH^ and OE^ protons)

140.76(5), 121 .69(6), 71 .79(3), 56 .78( 14), 56 .08( 17),

50.17(9), 45.89(4), 42,32(16,24), 39.82, 37,28(1 ), 36.5(10),

36.14(22), 35.86(20), 33.98, 33.8, 31.91(2,7), 31.7(8), 29.7,

29.2, 28.23(12 and 25), 26.17, 24.33(15), 23.08(23), 21.08(27),

19.78(26), '19.40(11), 19.07(19), 18.80(21), 11.47(18 & 18).

Pract ion-B

The fraction B showed one major component at Rf 0.83 on TLC

(silica gel,chloroform: etbylacetate, 9:1) alongwith the minor bands of fraction A. The major yellow fluorescence spot at Rf

0.83 was separated through prepara-tive thin layer chromatography

(irTLC, chloroform: ethylacetate^ 9:1) to give component B-^ . On crystallization of component B-^ in chloroform-methanol, cream t t 1 coloured graunular grystals of 5,6,2,3,4-pentamethoxyflavone

(XXXIIa) (3omg,mp 124-5°, Rf 0.83, silica gel, chloroform-. ethylacetate, 9:1 ) v/ere obtained. It gave orange colour with Mg-HOl and Zn-HCl 5.6 .2'. 3'.4Lnenta'Tiethn-srvfl avone f XXXIIa ^ . analvsed ! 54 J for CJQH^^O^ (:•:", 372), MS m/z 372 (M^,40), 357(b5), 3^2(6 ), 344(5),

327(7), 314(5), 312(4),299(3), 284(4), 2BO(5), 279(20), 256(6),

177(3), 167(30), 165(15), 149(35),137(20), 109(15), 95(19), 31(45),

69(100). TR^l^i ctn"\ 2900, 1645, I60O, 1530, 1430, 1420, 1 365, tiiax 1320, 1275, 1210, 1170, 1120, 1090, 1065, 1045, 1015, 960, 900,

SBO, 315; 300, 745, 710, 690.

(100 MHz,g-scale): Five aethoxyl groups, 3.90(S,3H), 3.93(S,6H),

3.94(S,3H), 3.96(S,3H), aromatic Protons 6.78 (d,J=:9Hz, 1H,H_5'),

6.S3(S,1H,H-3), 7.26(S,2H,H-7,B), 7.50(d,J=9HZ,1 H,H-6') .

"^^CNMR (35.3HZ,FX-90Q): Five Sp^C at 56.1, 57.3, 61.0, 61.2 an^

61.9, fifteen Sp^C at 107.4, 111 .6, 1 1 3.2, 1 19.1 , 123.9, 149.3, ^CKCl:.

150.?, 1 55.4, 156.1 , 1 59.7, 163.9, I6S.7, 173.7 and 178.2 5 U'^-max ^

appeared at 270 and 330 rni.

FRACTION C

The fraction G was gum'Tiy and showed two niajox spots and some

minor impurities on TLG. Therefore, it was chromatographed over

silica gel column and the column was eluted with petrol and benzene,

successively. The fraction from benzene-ether and after repeated

crystallization, two components C-^ and C-^ separated out.

Component C-^ was crystallized from chloroform-methanol as white

gramnular- crystals of 5,0,3,4-Tetramethoxyflavone (XXXIIb) 50 mg,

m.p. 171-72°, '^f 0.81, yellowish blue fluoreOscence in UV light

and gave orange colour with Mg-HCl and Zn-HCl. C-^ was characterrezed

as 5,6,3',4-Tetramethoxyflavone. C^gH^gOg (M^342), t-is: m/z 342

(1^1^96.3), 340(93), 327(63), 325(68.3), 312(25), 310(83), 297( 59), : :

232(100), 231(95), 237(30), 210(30), 208(30), 166(60), 165(76),

149176), 137(66.5), 119(73) and 77(59.6).

cm'' 2900, 1645, 1600, 15B0, 1520, 14B0, 1420, 1370,1340, UIAX 1290, 1270, 1255, 1200, 1170, 1140, 1030, 1060, 1030, 1015, 950,

335, 360, 840, 310, 800, 770, 750 and 680.

Pl^ffi (60MHz,^-scale): Four metboxyl protons 3.93 (S,3H), 3.95(S,3H),

3.96(6,6H), aromatic protons 6 .61 (S,1H,H-3), 7.00 (d , J=9Hz,1H,H-5'

7.30(S,2H,H-7,8) , 7.36 (d , J=2 . 5Hz, 1H, H-2' ) , 7.56(dd,J=9 & 3Hz,1H,H-6')

UV ^ CHClj 248.7 and 327.4 nra. ^ \ nax o component C-2 (10 mg, mp 64 c, Rf 0.30, silica gel, chloroform: ethylacetate 9:1) gave orange colour with Dragon Dorffs' Reagent and is uvinactive. It does not gave any colour with FeCl^ and does not show any colour reaction Zn-HCl and Mg-HCl.

Fraction D

On further eluting the column with benzene: ethylacetate (9:1

& 8:2) gave the fraction D which showed on -najor blue fluorescent spot which crystallized from chloroform-'iiethanol as white fine o needles of unidentified co-npound labelled as D-1 (10 mg,Tnp M-176-8 c,

Rf,0.20, benzene: acetone 3:1). It showed negative test with Mg-HCl and Zn-HCl and does not gave any colour with FeCl^. It gave Orange colour with Dragon Dorffs' Reagent.

The quantity of the component C-2 and D-1 was not sufficient for further studies. REFERENCES : :

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124:^ (1 .79). Chaptcr-ll Introduction : A-C s

Cupr e 5 p u s , a member of subfamily cupresscldeae (f amii ly cupressaceae), constitutes tv/enty species, largely distributed in Northern Hemisphere'''^.

AraUcaria belongs to the family Araticariaceae v/hich 2 comprises two genera of about 32 species .

I. CHEMICAL CONSTITUENTS OF CUPRESSUS

The compounds most frequently encountered in the genus

6-17 ^ 8-10,1 8-33 . Cupressus are tropolones , terpenoids and flavonoide29>34-41^ ^^^ scarcity of alkaloids in cSifer is y's unfortunate since these compounds are physiologically important 3 and also have taxonomic value •

Tropolones

All tropolones so far isolated from Cupressus exhibit distinct structural relations to terpenes.

The thujaplicins exhibited puzzling properties and appeared to be of aromatic nature some v;hat resembling sali- cylaldehyde. However, they show no sign of carbonyl groups and were stronger acids (pka 7) than normal monophenols. Furthernox^ 4 these compounds showed characteristic spectral proDerties .

° OH

(I) (I) isC-Thuj aplicin C .macngibi ana^' C.macrocarpa'^ ,

C. torulosa 8 • /I

(II) 6 7 T (II) /S-Thujaplicin G.macnabia-na,' C.macxocarpa, C. torulosa^ C.arizonica^ C. Stephen son li'*'^

G.abiamslana^ G..goweniana^ G. sargent ii^

C. sempejvlven^ G.pygmaeal 12 G.duprezlana.

(III)

(III) G. macnabiana^ G. ax Izonica'^ G. stephenson ii"!'^ G. lusltan lea"! ^ 1 2 G.dupreziana.

(IV)

(IV) -KT-Thugaplic inol G.abramsiana^ G.pygmaea"! ^

G ./?owen Ian a6 ! 4-2 J

(v) ^-Thujaplicinol C.axizoT] ica^ C. sajgent ii^ Q C.torulosa

7/ \

CVI) Debydrothuja- 0.abxamslana, pl ic in- ^ - C. p;owen iana^ dolabrin C. pygmeea! 0 • sar,geTit

cm)

CVII) ^-Dolabxinol C. abramslana^

C.gowenlana^ 11 0. o.ygmaea. 0 HOvJ

(VIII)

(VIII) Pygmaein Oxgo^Men iana^ C. pygmaea^

(IX) 6 Q 6 (IX) Nootkat in ax izon ica, * ^ G .abiamsiana . 6 15 C^gowetiiana, G. macnabiana,

G > macrocajpa7 , G.pygmaea11,

G, sargent ii^ G. sempervixeri^

torulosa^,^G. stepbensonii'*

(X)

(X) Nootkatinol -^IlMleii]'^ G .duprez lapa'' ^ OMe

(XI)

g q 6 15 (XI) Garvacrol/ Q. aiizonica. * C.^nacTiablana.' Caxvacxol 1 8 methyl ether O«macrocarpa( G.torulosa, Monoterpene s 1 Pi ^ C. Stephenson 11, C. sergpervlren,

C.dupxeziana,1 2

(XII)

1S 1S (XII) ^-Terpinene Q.abramslana, C.^ov/en iana, IS 1S C.macjocarpa, C.pygmaea, HQ -| p C. sai^entll. O.lusltanlea.

(XIII)

13 IS (XIII) Tllerpinene C.abramslana, G.gpwenlana, 13 G.macrocarpa,1 3 G.pygmaea, 1 S _Q. sargent 11. (XIY)

1 R 1 R (XIV) x;-Pinene C.abiamslana! C.jgowe-nlana 13 1B C.macrocarna, C.pjgmaea 18 1S 9.. sargent 11, C. lusltan lea 0. stephensoplj.l'^ Q.funebj Is^^ 20 21 C.sempeivlrens. ^.dupjezlana

(XV)

1 Q 1 R (XV) yS-PI'ineni e C.funebr_is, C. macjocar pa,

C.sgr^ent11 1 8

^OH

(XVI) n n (XVI) ^-Texplnol C.duprezlana ! 46 J

(XVII) (XVII) p-Cymene C.dupreziana^^ C.funebris"'^ 1 s C.lusitan ica

(XVIII)

22 1Q (XVIII) Camphene O.dupreziana, C.funebris

C.lusitanica 18

(XIX) (XIX) Ocioiene C.dupreziana^^ C.funebris'' ^

• (XX) 22 1S (XX) y^-Phelland rene C.dupjeziana, C.sargent il 1S 18 C. abramslana, C.lusitan ica : 75:

(XXI)

22 1S (XXI) Limonene C.dupreziana. C.futtebris •j) g g U. abxamsiana, C. saxgent ii 22 jene C. duDrez iana

(XXII) 1 q -] Q (XXII) Myxcene C.funebxIs, C.saxgent il

C. lus itan ica 1 B

( XXIII)

18 (XXIII) ( Caxene C. saxgent li IB 13 3 - Oarene Q . go'..-en lana, C .-pygvuaea IS 21 C.lusitanica. C. sempervixen IB • 13 oe b inene C .lusitan ica , C . saxgent ii ^ Q -1 Q 3.macxocaxpa;^ C. Dv^maea : :

^sesquiter pene s

22 2 3 o

(XXIV)

Q g Humulens G. ar Iz on ica, 0. t orulosa

(XX^)

22 22 (XXV) f-Elumene C. semperviren. C .du preziana

22 23 ^-and ^ -selinenes G. semperviren; ^ .dupreziana

21 Selinene -11-0-4f O.aupreziana

AT."

XXVI) 21 (XX\^I) CostoI,(3esqu iben ihiol) C.dupreziana : 9 4 t

(XXVII)

(XXVII) Copaene C.duorez iana 22

(XXVITI) 22 iXXVIII) E-Gadinane C .durireziana

(XXIX)

(XXIX) Calanene ^.seuperviren,2 2 G.duprez iana 22

( D) Tricyclic sesquiterpene

,XXX) ;; y 2/ (Xaa) uongifolene G,dupreziana7" G. -riacrocarpa I 50 t

(XXXI)

22 24 (XXXI) Junipexol C .duTpreziana, U .macjocaxpa

(XXXII)

9 (XXXII) •C-Cedzene U.arizon ica

22 1,7-Diepi- <^-ce5rene G.semperviren 22 1 ,7-Diepi- -cedrens G.duprez iana

•(XXXIII)

2 5 Q (XXXIII) cedrol C.dupreziana , C. ar izon ica^

2 S 0. se-^oerv iren : 9 4 t

(XXXI?)

q 3 (XXXIV) Thujopseni C. ar IzoTiica, C. tojulosa 22 C.duprezlana

q 3 (XXXV) widdXDl C. ar izon lea. C, t orulosa 22 0.duDreziana

(XXXVI)

o 22 (XXXVI) Cuparene Me Q. torulosa, C.duprezlana 22 C. setrnDerviren

(XXXVII) P7 (XXXVII) •^-^une brine U.funebris. 0.duoreziana-' : 9 4 t

p^ p "7 (Xia\riII) ^-dupzezianene Q.funebjis, C.duprezlana '

22 ?2 Prezianene C.sempervlren, C.duprez iana"

22 22 ^yS-Acoxad iene C.sempejviren, C.duprezlana

22 22 Y-Ac or ad iene C.sempervirep, C.duprezlana

(XXXIX)

(XXXIX) Boxpeol G. duprez iapa 21

(XL)

(XL) -4laskadiep - 4,11-ol-14 C .duorez iapa2 1 HO

(XLI) •

(XLI) Alaskad ien-4,7ol-14 C. auoreziana 21

(XLII) 21 (XLII) Alaskadien-^, 11-01-14 C.duDxeziana

Piterpenes

tieveral bicyclic, tricyclic and tetracyclic diterpenes have been repoted from Cupressus species. )H

(XLIi:a-e) R (XLIII a) Manool S 23 Ms _•torulosa, G.dupreziana ^•semperviren^^

(XLIII bJ 'lorulosoi OH^OH C. torulosa? C.dupreziana^^ S 2 S lXx.111 c) Torulosal oHO ^• torulo^, 0. duorez iana CX-blll d) lorulosyl 2 5 23 •acetate CH^OAO 0.senperviren. 0.duprez iana (XLIII ei Cupressic ac id COOH J. torul osa^^ ^ • semoe rvir en^^ ! J

Me COOH (XL IV)

(XLIV) Neocupxessic acid Q. sempervijen^^

CH2OH

HOOC (XLV)

(XLV) Isocupressic acid 0n , sem-oervire. n 51

ROOC (XLVIa-b)

32 33 (aLVI a)' ^oraraunic acid C.torulosa, U.semperviren H (XL\^I b) Methyl( + ) 32 -c ornmunate Me U.toruiosa 32 ( + )-Se?npexvir ol C.torulosa : !

HOOC (XLVIIa-b)

(XLVII a) Imbricatolic acid C.torulosa^^

(XL\AII b) (+)-Acetyliriibri- 2y catolic acid CH^OAG 0. torulosa

(XLVIII)

32 (XLVIII) Ferxuginol C.torulosa

H (XL IX) 32 (XLIX) (+)-Tot3rol torulosa : 9 4 t

(L)

3 28 (L) (+)-Hinokiol O.tojulosa, C.dunreziana

(LI)

p 00 {LI) (+)-Hinokione C. torulosa, C.duoreziana

ILII) Totaroione 0 .duTDieziana 23 '. rJ; 7I •.

R R (LIII a) Sandaxacopimar ic 1 ac id CooH H C.torulosa 29

(LIII b) Sandaxacopitnar-S, 2S 15-dien-2 ^-ol Me OH C.duDxeziana

2S Piraax in ol C .duTDXez iana

(LIV)

(LIV) (+)-Hibaene G.macn x ocarna 24

(Lv)

(LV) (+)-PhyIlocladene C .rnacr ocaxD32a : :

ZM^O^OIDS:

29 J!'lavoTioids are found to co-occur alongv/ith procyanidin .

In C.goweniana^^ and C.anstialls^^ apigenin and its ditner are found to CO-occur.

Qupressus .Biflavonoids:

Biflavonoids with C-C and C-O-G linkages have been isolated 36 from several Oupressus plants.

(a) C-C Linked i^iflavonoids

H 0

/ VOH

OH 0 (L7Ia-b) (Lv'I a) CupxessuiTavone C . funebr is^'^' . gpwen iana H 31 M 37 0.semoerv iren, '0,torulosa 4Q J-austratis, S.luti tanica ^^^

34 40 (LVI b) 1-4 -0-aethyl- I-Ie U . '^owen iana, C . Lusitan ica . ro • • ^ -- •

OH 0 (LVIIa-b)

R (LVII a) imentoflavone C.fuTiebjis?'^ O.gQweTiiaTia^^'^'^ H G.austiatls?^ C.lusitanlca^^,'

'J. semperviren 41

(LVII d) Podocarpus- 4n 41 f 1 av on e Me C. lusitan ica '^ C. seglT)erv^re^

Mono-O-methyl

araentoflavone 0.australis^^

iVIII) rioDustal'iavone C.australis^^ : 9 4 t

C-O-C Linked Jiiflavonoids

OH 0

(LIXa-b)

5Q 57 (LIX a) Hinokil'lavone ^ C .ar izon ica, C . funebxis '

0 . torulosa?'^ u. setmpeivlren^'^

C.goweniar.a"^'^'^

(LiX b) Isocryptomex in Me G.gQwenlana?^'^^ C.funebrls' 54 C. seniDexviren

Mono-U-aethyl G.austalis^^ C.lusitanica^'^ bin ok iflavone :6l:

CHEMICAL CONSTITUENTS OF ARAUCARIA

Araucaria, belongs to the family Araucarir.ceae, and 2 comprises about 32 species ,

Most of the Araucaria plants investigated so far, 49 44 —"^"a R—A'S constitute diterpenoids and biflavonoids ° ° .

Some Araucaria species (Araucaria= A.) also contain monoter- 4 2,43 42,44 . 54 ^ ,, . 54-57 penes , sesquiterpenes , traterpe nes and lignins

as well. Diturpenes with labdane skeleton are more frequent

Araucaria than those with other skeleton. Almost all the

species of Araucaria examined so far contain bicyclic labdane ^ , , 45-52 . 45,49,52 ^ , 42-52 derivatives . Tricyclic and tetracyclic

diterpenes have also been isolated from some species, Tri- 54 terpenes were reported only from A.angustifalia . Some of

the miscellaneous compounds include palmitic, linolenic and 54 stearic acid from A. angustifolia , acetovanilJ one, p-

hydroxybenzaldehyde, syringic aldehyde and Vanillin from

b.' a^aucana^^, proanthocyanidins from A. araucana^*^ , eO- (p-

hydroxystyryl )Chavicol from A.angustifoli a and 3,4-dimethoxy-

benzoic acid and 4,5-dimethoxyisopthalic acid from A.araucana^ polymer of cis and trans isoprene was isolated from A . angustifplia 69 , while Ar?uc^ri? species was found to contain

tannis"^^ and C-glycosylflavcne"^! Some polysaccharide have 72 73 been reported from A. bidwilli and A. cookii

Several Araucaria plants have been investigated after

the first report of biflavone, WB^ (LVlg) from the leaves of . ^ - ..58 A. cunninghamii :62:

The distrioution of bif .lavonoids is incluaed here as our investigation narnly dealswith bifiavono- idic fr-action.

7-0-methyl-(LVIC) and 7,V-di-o-methylcupressu- 60 , 51 flavone(LVId) occur in the leaves of Ao bidwi1li and rulei ^^. A. cunninghamii ^^ ^nd A. cookii^^ constitute dimethyl ethers LVid ^nd LVie ^^longwith trimethyl ether LVIf and tetrarnethyl ether LVig. a, 59 bidwiHi constitutes amentoflavone(LVlla) ^nd bi lobetin ( LVlic ) ^^ ' and A. cookii ^^ constit\ites all the four partial methyl ethers LVIld, LViie, LVllg, 5 9 LVIIh and LVIIj. A. excelsa constitutes LVlla, LVIIf, 6 3 LVIIi and LVIIj while A. cunninghamli contains LVIIe,

LVIIh ^nd LVIIj. 7-0-raethylao--^thisf l=ivone ( Lxb) -nd 7,7"- di-o-methy lag athi sf lavone ( LXc ) v/ere rei^or-ced from Ao bidwi 111^'^ ^ ^^ while in _Ac cun ni ng hami i " ^ ' , LXb exists 62- alongv/ith LXd. ConpouncLs LXd v/as isolated from A.excelsa and A. rulei ^^. Tri-o-methylagathlsfLavone(LXe5 9 ) could be identified only in Ao exct. Isa although it h^s been

-letected alongv/ith agathisf L =vone in other species ;5S we Eiphenyl ether ty;;ie or bifLavono, hi noki f lavone has been reoor-ced from A. cookii^^- :65

OR2

OH 0 (LVia, c-g)

R1. 2R _ R3- R4 (liVIa) Cupressuf lavone H H H H A. excelsa » 59 A. bidwilli

(LVIc) 7-0-rr.ethyl cupressuf lavone H H Me H Ac bidwillif'^' ^^ ZT A. rule!

(LVid) 7,7"-D±-c-methyl -cupressuflavone H H Me Me A. bidwilli^®, —r2" _A . rulei , _A.cunn jgi nghanii , A.cookij

(LVie) 7-Di-C- methyl cupressu- . 63 flavone Me H Me H A. cunninghamii r62r- or H Me Me H A. cookii

(LVIf) 4 ',7',7"-Tri-o- methyl Me H Me Me A. cunninqhami. 63> 64 A. cookii

(LVIg) 4',4'", 7,7"- 63 Tetra-o-methyl- Me Me Me Me A. cunnlnghainll, 64 ~ A. cookii :64

59 (LVlIa) Amentoflavone A. excelsa , 59 A. bidwilli

60,61 (LViic) Bilobetin Me H H H A. bidwilli

64 (LVlId) 7"-C-methyl- H H H Me A. cookii

64 (LVlIe) 4 • ,7"-Bi-C-ir.ethyl-Me H . . 63 •Lnghamix

(LVIIf) 7,7"-Di-0-methyl- K H Me Me A. excelsa 59

64 (LVllg) Kayaflavone Me Me H Me A. cookii

64 (LVllh) Sciadopitysin Me Me Me H A. cookii ,A.cunn B3 inghamii

(LVIIi) 4 •,7,7"-Tri-0- 59 r^.ethyl- Me H Me Me A. excelsa

(LVII j ) 4 • ,4'»» ,7 ,7" - 64 Tetra-C-rr.ethyl- Me Me Me Me A.cookii , 59 A.excelsa 63 A.cunninghamli :65.

y \v-o OH 0

HO 0 (LIX)

£5 (LIX) Hinokiflavone A. cookii :66:

0 OH (LXa - e)

5 9 (LXa) Agathisflavone H H H H A. excelsa ^,

h' bidwllli^^, 65 Araucaria species

(LXb) 7-0-ir.ethyl- 60,61 ag athi af1avone H H Me H A. bidwilli A. cunninghamii '

(LXC) 7,7'*-Di-G-methyl- H H Me Me A, bldwllli^^ * ^^ —————— A. excelsa

(LXd) ,7-Dl-O-methyl- H Me Me H A. cunningh^ll^^ * ^ A. rule2

(LXe) 4"', 7,7"-Tri- 59 C-methyl- H Me Me Me A. excelsa ••67 :

3. N^TUR^L DISTRIBUTION OF BIFLAVONOIDS

Flavonoids belong to an important group of naturally occurring constituents in which two benzene rings are linked through a propane bridge (C.-C-C-C-C ) except in isoflavones 6 6 where the arrangement is (C^-C-C-C) e.g. chalcone (LXI) , 5 I =6 flavone(LXII), isoflavone(LXIII), flavonol(LXIV), flavanone

(LXV), isoflavanone(LXVI), flavanonol(LXVII) and aurone(LXVlil

(LXIII) (LXIV) (LXV)

(LXVIII) • Do •

Biflai'onoids are recognized by having tv/o flavonoia units.

They have mostly, been isolated from Gymnosperms^ Among 74 angiosperms some plants belonging to Guttiferae , Eupho- 75 7 9 80 rbiaceae , Caprifoliaceae , Anacardiaceae and some 8 "1 82 ferns belonging to selaginallaceae and psilotales have been found to contain biflavonoids.

Several naturally occurring biflavonoids (Biaryl type, Garcinia and Taiwania biflavonoids^and Biarylether ti'pe) and their derivatives reported till 1 982 have been 83 84 reviewed ' . Nev/ biflavonoids which have been reported recently have only been mentioned here. In 1984, Nicoletti et.al reported the presence of strychnobif lavone ( LXIX) , wh-i le in the same year Chatterji 85 b et.al, reported Abie sin(LXX) from Abies webbiana . Murthy reported the presence of LXXIa-c, jeediflavanone( LXXId), semicarpuflavanone(LXXle) ana galluflavanone(LXXIf) from ^ . ;• 86 Semicarpus sn^^rdium

(LXIX) Me \0H

Plant source SJ (LXIX) Strychnobiflavone ^rychnos pseuuequir o cu ••69 :

85 b

(LXXIa-f)

(LXXIa) R S

OH H OH H OH H OH

(LXXIb) OH H OH OH H H OH

(LXXIC) OH H H OH H H OH

(LXXId) OH H OH OH OH H OH

(LXXl^e) OH H H OH OH OH OH

(LXXIf) OH OH H OH OH OH H ••70 :

IDENTIFICATION OF BIFLAVONOIDS

Apart from colour re?>ctions, the physical methods generally employed in the identification and structural 87 88 89 studies of biflavonoids are chromatography , \JV , IR , 90 91-93 NMR and mass spectral studies , and degradation and . 109-111 synthesis O Q QQ

The use of UV and IR for the diagnosis of the functionalities of flavonoids has been reviewed. The carbonyl stretching frequency provides useful clues for the structural elucidation of the flavones. The frequency is effected by the presence of functional groups in the flavone skeleton. For example, carbonyl frequencies of \ KBr ~1 monohydroxyflavone varies from "\J 1 620-1 653 cm depend- " max ing upon the position of hydroxy substituent. Carbonyl > frequency of m.ononethoxyflavone varies from 1 637-1 649 max cm"' with the position of methoxyl substituent and carbonyl \ KBr frequency of monoacetoxyflavone varies from ""nI 1750-1759 / max cm" with the position of acetoxy substituent.

^H Neuclear Magnetic Resonance Spectroscopy

1 90 On the basis of H Nr>1R studies of silyl derivatives , 64 ^^ double irradiation technique > solvent induced shift studies 95 lanth^niJe induced shift studies , nuclear overhauser effect 13^ 07 and t: rl-IR specroscopy" , it has been made possible to elucidate the structure of flavonoids. • 7TJ - •

Use of NI'IR studies for the structure determination of monof levonoicis has been discussed in Part-I (Page - 14 ) of this thesis.

Comparison of ""h NMR spectra of bif lavonoids with the corresponding monomers, provides useful method for their structural determination. Comparison of spectra of methyl and acetyl derivatives of a biflavonoid with those of biflavonoids of the same series as well as of other

series where one mo noflavonoid unit is similarly constituted is very useful in assigning the protons and locating the methoxyl groups.

The interflavonoid linkage is established with the 94 help of solvent-induced shift and lanthanide induced

shift studies 95

In biphenyl type biflavones, such as amento-, cuoressu- and agathisflavones, the protons of the ring involved in interflevonoid linkage appear at somewhat lower^ ,

((90.5 ppm) as compared to monomer due to extended conjugation .

In biphenyl and biphenylether types of biflavonoids, the 5-methoxy group of an 8-linked monoflavonoid unit appear? above ^4.00 ppm in CDCI3 in all cases examined^ so far

(Table-I). It is again due to extended conjugation. The

5 - me thox:"-protons of the 8-linked monof lavonoid unit of BGH-,

V7GH- and GE- series do not appear above 6^4.00 ppm as the linkage is through heterocyclic ring. : 72-'

6 3 Table - I : oMe proton shifts (^-sc-le) of bifl^vonoids

per methyl ethers of 5-OMe 5"-OMe

Cupressuflavone jl-8 , 11-^ 4.15 4.15

Amentof lavone ^1-3 , II-sj 3.87 4 .06

Agathisflavone jl-e , ll-Sj 3.59 4 o05

1-4 ' 0-II-8 biflavone (Synthetic) 4.00 4.08

Aromatic protons show consistency in cupressu-, amento-, agathis-and hinokiflavone series. The protons of ring I-B in agathisf lavone appear at lov/er field, than 63 protons of ring II-B

The protons at C-8 in the ring II-A in penta-o-methyl

'-O-II-eJ biflavone (LXXV) and at C-8 in ring I-A in

hexa-o-methyl^I-6, II-^ biflavone(LXXIV) appear at except- ionally lowfield, ^7.05 ppm and 6.91 ppm, respectively.

This may he diagnostic of H-8 of a 6-linked biphenyl and biphenyl ether type of biflavones.

The PMR data of some representative biflavones of different groups are given' in Tables Ila-IId. : 75:

Table_- lla; pmr data of amentofl^vone hexamethyl ether 64 (LXXI1)

Assigned position Chemical shifts of protons ( - scale )

H-6 • 7 .90 (d ,d ,J ,-9K Z, J2=3H2)

H-2 ' 7 . 84 (d,J=3H2)

H- 2, 6-' 7 .40 (d , J=9Hi3)

H-5 ' 7 .12 (d , J=9K2)

H-3'", 5-' 6 .76 (d ,J=9:-2)

H- 6" 6 . 62 (s) IT H-3 ,3 6 .58 (s) 6.52 (S)

H-8 6 .48 (s)

H- 6 6 .34 (d,J=3H'z)

one - 5 ,5" 4 .06 , 3.92 (s,3H each)

OHe-7 3 . 8P) 1.7 2 (S,3H each)

OMe-4 3 .75-, 3.7 3 (s,3H each) s= sing let, d = doublet. Spectrum run in CJCl at 60MC, TMS O as internal standard. ••74 :

Table - lib: P?:R data of cupressuflavone hexarnethyl ether

(LXXIII ) MeO

OMfr

OMe.

(LXXIII)

Assigned positions Chemical shifts of protons( 5-scale)

H-2 ' ,6 ' 6'" 7o30 (d,J=9Hz)

H-3 • ,5 • ,3'", 5../ 6.77 (d,J=9Hg)

H-&,6" ,3,3" 6.'57 , 6.5 9 (s, 2H each)

OMe-5 ,5'* 4.12 (s,6H)

owe-7,7" 3. 86 (P,6H)

OMe-4 • ,4'" 3c77 (3,6H)

3= Singlet, d = doublet, Spectrum run in CDCl^ at 60 MC, TMS as internal standard. 75--

Table - llc; PKP. dPtP of l^vone hexanethyl ether

(LXXIV)^^'^^

Me 0

(LXXIV)

Assigned position Chemical shift of protons( S"scale)

H-2 • , 6 ' 7. 88 (d ,J=9H z)

H-2"' ,6'" 7.37 (d ,J=9Hz)

H- 3 ' , 5 ' 7 .01 (d ,J=9H'z)

H- S 6.91 (s)

H-3"' 6.78 (d ,J=9KZ) H- 6" 6.64 (5) H-3,3" 6.53--5.51 (s,IH e ach)

OMe-5" 4 .05 (s, 3H)

one-7 ,7" 3. 88 ,3.86 (s,3H e ach)

OMe-4 ' ,4'" 3.78 ,3.76 (.s, 3H e ach)

cue-5 3.59 (S, 3H)

s = singlet, d = ocublet, Spectrum run in CLJCI at 60 nC, TMS as internal standard. .'To :

Table-Ild: PMR data of hinokif 1 avone pentar;.e-:ir.vl ether " Q p (LXXV):"

OMe

MeO

Assigned position Chemical shifts of protons( scale)

H- 2 ' , 6 • 7 .88 (d ,J=9Kz)

H-2'" , 6'" 7 .80 (d ,J=9Wz)

H-8" 7 .05 (s)

H- 3 ' , 5 • 7o02 (d, J=9Hz)

H-3'" 6.94 (d ,J=9H.^)

H- 3 , 3" 6.62 i 6.59 (s, IH each)

H- S 6.5 5 (d,J=2H2)

H- 6 6.37 (d,J=2Hz)

CMe-5 ,5" ,7,7" ,4'" 3.91- 3.94

s = singlet, d = doublet, Spectrum run in CDCl at 100 iClz, O TMS as internal standard. ••77 :

Oii use Of Solvent Induced Shift"' ' for Establishing Inter- flavonold Linkage.

9 9 It has been observed that the methoxyl groups at

C-5, C-7, C-2• and C-4' exhibit large positive A value

( SCDCl- 0.5-0.8) in the absence of methoxyls 3 5 5 or hydroxyls ortho to these groups i.e. they move upfield in benzene relative to CDCl^-

The methoxyl groups have ability to conjugate with the electron pulling carbonyl group thereby decreasing electron density on the oxygen atoms. This result in association of benzene at these electron deficient sites

and as a consequence, increases the shielding effect. The

C-3 methoxyl, in contrast shov;s des'nielding or only slight shielding ( -0.07 to + 0.34 ppm) in benzene, suggesting that C-3 methoxyl groups, in general, prefer the conformation

LXXVI .

Similarly, a C-5 methoxyl group in presence of 6- substituent, shows small positive or negative solvent shift in benzene presuiiiably diae to higher population of the conformer LXXVII.

(Lxx\a) (LXX\^II) ••78 :

OMe

(LXXVIII) OMe

In hexa-O-methylamentoflavone (LXXII), hexa-0-methyl-

cupressuflavone (LXXIII) and linked hlnokifla-

vone methylether (LXXVIII), all the methoxyl groups move

upfield (50-60 cps) on change of solvent from CDCl^ to C^H^

indicating that every methoxyl group has at least one ortho

proton and therefore a C-8 rather than C-5 linkage is

established. In agathisflavone methyl ether(LXXIV) only

five of the six methoxyl groups show large upfield shifts.

One of the methoxyl groups was unique in that upto 50/o

dilution with benzene no shift was observed and then a

strong downfield shift was evidenced. it is reasonable to

assume that the methoxyl group in question was the one at

C-5 flanked by ring-A(II-A) of second biflavone unit and

carbonyl group. Similarly, in hinokiflavone methyl ether

(LXXV) only four methoxyl groups move upfield and C-5 methoxyl groups show dov;nfield shift. The benzene induced

solvent shifts are appreciably enh;=*nced by the addition of

small quantity (3?4, v/v) of trif luoroacetic acid (TFA) . ••79-

This helps to distinguish 'toetv/een methoxyl groups which

CR.n conjugate with the carbonyl group and those which can not conjugate in ground state.

•a Me - 0 — C- C= CO' Me - O == C-C=C-0

Me - 0 - C - C=0 • •

The TFA induced solvent shift of 5-methoxyl group has a relatively large negative value (-0.36 to 0.44) which distinguishes it from other methoxyl groups. It is due to hydrogen bonding between protonated carbonyl group and oxygen of 5- methoxyl group. The protonation of carbonyl group (LXXIX) is more in TFA than in benzene containing TFA.

d P

(LXXIX)

95 Lanthanide Induced Shift

In the recent past lanthanide shift reagents have extensively been employed''for the structural and conformational studies of organic natural products. The introduction of these reagents has greatly enhanced the importance of H iXTlR spectroscopy. •• 80:

The addition of certair. lanthanide conplexes (shift

reagents) to a solution of compounds having appropriate

lone pair of electrons causes the proton resonances to

spread out, the effect being greatest at the protons nearest

to the site of coordination. The shift reagent coordinates

with electro-negative atom in the substrate and thus modifies the magnetic field experienced by the neighbouring protons, thereby simplifying the spectra. Since the strengtll

of this field varies with the distance from the paramagnetic

source, the chemical shift of each proton is modified to a different extent, coupling constants appear virtually

unaffected 1 01 „ The most commonly used shift reagent for the

structural elucidation of biflavones is tris-i, 1,1,2,3,3-

heptafluoro-7,7-dimethyl-octan-4,6- dionato europium^ 1 02 EuCfod)^ . The application of lanthanide shift reagents for structure elucidation of flavones and biflavones has 95 been reported recently

Seven fully miethylated biflavones, narriely hexa-O- m.ethylamentof lavone (LXXII) , hexa-C-m.ethylcupressuf lavone

(LXXIII) , hexa-C-methylagathisf lavor)e (LXXIV) , hexa-C-methylrobustaflavone(LXXX), penta-0-methylhinokflavone(LXXV), hepta-C-methyl ^- C-1i- S^ biapigenin(LXXVI11) have been studied using EuCfod)^ as a shift reagent. The^-values are 95b recorded in Table-lII :81:

OMe 0

(LXXX)

(LXXXI)

95b Table-III : 6-value of fully methylated biflavones with Eu(fod) J*

Compounds protons (LXXII) (LXXIII) (LXXIV) (LXXX) (LXXV) (LXXXI) (LXX ______

Me 0- 5 6.12 7 .34 2.14 10.58 10o02 5c1 3 6o88

_ RII 8.78 - 11.16 2 o 64 4.38 1 1 .90 6. 60

-7 Oo36 0.72 0.44 0.74 0o80 0o46 0.82

-7" 1 .06 - 2.04 0.56 0.58 2.50 0 = 52

• 0o1 2 --Q.06 0.02 Oo32 - -0.50 -

>«i -0.08 - -0.08 -0.06 -0.06 0 -0.10

H -3 OcC2 0oi a 0.06 0.30 0.36 - 0.14

_ -3 -0.16 - 0.28 0.16 -0.06 -0.44 -0.2 6 :8 2 :

Table-III (continued)

Compounds Protons (LXXII) (LXXIII) (LXXIV) (LXXX) (L>:xv) (LXXXI) (LXX- 111)

-6 2.76 3.66 - 4 .84 4 .80 2 .20 3.36

-6" 4 .24 - 5.80 - - 6.10 3c52

-8 0o50 0.64 1 .20 1 .14 0.50 0.72

-8" - - - 0.50 0.74 - -

-2 ' 0.36 0.56 0.08 1 .42 0.04 0o40 -0.02

-6 • 0 - - 0o36 - - -

-0.12 - 0.52 Co08 - 0.14 0c20 0

- - - - - 0c20 -

-3 ' - 0.24 0.02 - 2 oOO -0.30 -0.10

- 5 ' 0.10 - - 0.52 - - * _ OKI -0.08 - 0.06 0.06 - 0.08 0.34Me 0.1 8

- 5 - - - - - 0.03 -

- * Tentative assignments

The result of the table may be summarized as :

(a.) The smallest 5*-value for the protons at C-6 in ring

I-A or II-A is still larger than the largest ($'-value

for the proton at C-8 in ring I-A or II-A which decide

the interflavonoidic linkage through C-6 or C-8 in

biflevones.

(b) 5"-valine for the proton at C-3 in the rings I-C or II-C

are so small that these protons are indistinguishable

from the protons at C-8 in ring I-A or II-A in the

same flavone nucleus" • ••109 :

although H-3 and H-5 of ring I-B of hinokifl^vone-

pentamethylether (LIXXV) show a significantly large

shift (2.00 ppm) than those of other compounds because

the phenyl group is attached to C-6 of the second

flavone nucleus.

1 3 C NMR Spectroscopy

In ''H NMR spectroscopy involving shifts of the methoxyl signals in the spectrum of permethyl ethers, progressive 103 addition of S^D^ has bee n used for establishing inter- 63,98 flavonoadic linkage . However, in hepta-O-methylsahar- anflavone(LXXXI), one methoxy signal does not shift at all on addition of C^^D^, supporting a j^l - , 11-^ linkage inspite of the fact that linkage was established by synthesis 109-111

1 3 A technique of wider scope, C M4R, has proved more useful for establishing the interfavonyl linkage in bifla- 1 3 vones. The assignments of signals in C NMR spectra of biflavonoids was achieved on the basis of off resononce and proton coupled spectra and also by analogy v/ith the 1 04 values for monomers. This technique obviates the necessity of preparing permethylethers and as a consequence, helps in location of methoxyl substitution in naturally occuring partially methylated biflavonoids. : 84 r

Linkage involving ring-A

1 3 C-6 and c-8 signals in C NMR spectra of 5,7-dihydro- xyflavones are distinguished on the basis of their rrrulti- plicities in proton coupled spectra and specific proton 104 decoupling. The resonances for these C-atoms are found betv/een 5^0.0 ppm and ^^lOO.O ppm. The signal for C-6 is at lower field as compared to C-8 in a variety of flavonoids

(Table-IV).

1 3

In the C NMR spectrum of cupressuflavone(LVIa) only•

13 resonances are there due to symmetry of the molecule.

The signal of 1-6 and II-6 carbons appear at (^99.0 ppm where as the signal for 1-8 and ii-8 caroons shift downfield to 5*98.7 ppm due to substitution effect of the interflavonoidic linkage. In the hexa-0-methylcupressuf 1 avone ( LXXIIt) the signal for 1-8 and ii-8 carbons appears downfield, compared to C-8 carbons of tri-O-methylapigenin(LXXXII) at

5'101.2 ppm whore as 1-5 and II-6 carbons are not appreciabCj shifted.

OMe

MeO (LXXXII)

The spectrum of agathisf lavone (LXa) shovvs eight distinct resonances in the region $"93.0 ppm to 104.0 pom I-S and II-& carbon signals appear at expected values ^^ 93.7 pprn and 98.9 ppm, ".vhile I-& and II-8 carbons appear at

^103.6 pprn and 99-4 ppm. The downfield shift experienced by later- carbons is due to substitution affect of the interflavonoidic linkage. The other four signals between

(5102o8 ppm and 104.0 ppm are assigned to 1-3, II-3, 1-10 and 11-10 carbon atoms, respectively. liinkage involving ring-A and B

The three unsubstituted aromatic carbons, II-8, 1-5 and 1-8 of robustaf lavone (LVm) are assigned the signals at (^9 3.9 ppm, 99.0 ppm and 94.0 ppm, respectively. The signal for II-6 appears at 103.5 ppm. The spectral interval.

5*116.0 ppm to 131.0 ppm consist of eight resonances of carbon atoms. This pattern is almost, identical with spectra?, region of amentoflavone(LVIla) and signals are assigned on the basis of shifts experienced for aryl substitution at

1-3' carbon of resonance spectrum. 1-6, I-S and 11-5 carborj appear at(S'89.9 ppm, 94.2 ppm and 99.1 ppm, respectively, whereas Ii-S carbon signal appears . downfield ((5'l04.1 pprr.y

Location of methoxyl groups

The position of methoxyl substituent in a biflavonoid is'determined on the basis of. chemical shifts caused by steric crov;ding. G6

Steric crowding, as in -he case of 5,6,7-cri-0-methyl- flavone is responsible for a aownfield shift of methoxyl carbons by 6.0 ppm, whereas in a 5-hydroxy-5,7-aimethoxyl derivative, the shift is only of the order of 3.0 ppm.o

The dov/nfield shift of the signal for the ortho carbon atom to the oxygenated carbon is more reliable, thus enabling an 1 05 indirect determination of the site of O-methylation

Mass Spectroscopy of Biflavonoids

Mongwith UV, IR and NMR spectroscopy, mass spectrometry has become an indispensable tool for structure determination of biflavonoidso

Flavones with fewer than four hydroxyl groups do not readily fragment, due to stability of their molecular ion, but tend to undergo retro DielsAlder decomposition'"^^.

Whemi heavily substituted with OH or OKe, the spectrum is dominated by the molecular ion and ions at M-15, M-2S and 107 M-4 3= Also, doubly charged ions are frequently"observed

A specific study of mass fragmentation pattern of permethyl ethers of amento-, cupressu-and hi nokif lavones- 108 has been made . In biflavones, molecul=»r ion is usually the base peako They undergo (a) fission of the C-C or

C-C-C linkage between the aromatic residues (b) elimination of CO and CHO from the biphenyl ethers (c) rearrangement involving condensation between phenyl rings. Steric factors : G7 !

also pl=y their part in the mode of fragment?tion =nd internal condensations. The mass spectra of amentoflavone hexamethyl ether (LXXII) and cuxDressuf 1 avone hexamethyl ether(LXXIII) are similar, molecular ion being the base peak in each case. Difference lies in the intensities of corresponding peaks due to variation in substitution pattern and steric disposition of one flavone unit relative to the other.

The main peaks alongwith their intensities are given in the schemes- I - III.

The mode of fragmentation of hinokiflavone pentamethylether (CVI) is considerably different from amento-.cupressu- and agathisflavone hexamethyl ethers. The base peak appears at m/z 313 and molecular ion (m/z 608) is 39% of base peak.

It may be due to easy rupture of biphenyl bridge; hydrogen transfer then leads to m/z 313 fragments

The fission of ether bridge may occur in two v/ays.

Route-I affords the ion at m/z 297 and m/z 311, while route-M leads to the ion at m/z 2 81 and m/z 327o The observation that the m/z 313 ion peak is most intense, suggests route-I i.e. the bond between the oxygen bridge and highly oxygenateci. phenyl ring breaks preferably.

The structure determination of biflavonoids can also be supported by synthesis. For example, cupressuflavone hexamethyl ether amentoflavone hexamethyl ether, hinokiflavone 109-111 pent=methyl ether have teen synthesized OMe

(l-^-^, I.t/2) n/2 311{5)

247) (49 0'^'V2) (5) 511(5)

Schei.-e-I. TasG frr^r.icn-brtion pattern of r,r.cntofIr.vono hevancthyl

e-cner ( L::::iT MeO 0 II C

n/s 152(14) I 1

0 MeO n/c 245 C4}0''"72)(11)

4- n/s 155 (26)

OMe

n/s 511 (14)

OMe

MeO

, ni/a 6 22(100)

/"n-iyt n/s 621(53) /KI-15ytn/c607(S) /M-467 . r./s 576(4)

/yi-^q/'^, r./s 592(18)

ScIier.:o-iT. race; "raciientati on. pattci-ii of cuTsresEuf Ir.vonB

hc:.;E.r.cthyl other ( Lx:;iII MeO

^p./c 607 (11)

n/:: 504(2), M+f

MeO 0 n/s 2C1(22) OMe 0 OHe n/2 527 (2?) n/z ICl (11) Schci.e-j.ll. i:r,ss frr.rrentc.ti0.1 pr.ttern of hino::iflr,vone peiitanethyl a on e-^ner ( L,.,-. v) Discussion ••91 :

1. BIFLAVOriES PROM C'JPRESSUS FUNEBRIS

A number of cupressus species have been examined

for blflavones. Amentoflavone and cupressuflavone were

reported as the characteristic biflavones of the genus 34-4 Q

Cupressus which may serve as the useful taxonomic marker

Amentoflavone, cupressuflavone, hinokiflavone and isocrypto- ir.eria have been detected by thin layer chromatography in 37 the leaves of Cupressus funebris we now report the presence of sequoif1avone and three partially identified

biflavones(parent, mono- and di-c-methylrobustaflavone) from

the leaves of C. 37funebri s along with the four biflavones reported earlier . Detection of robustaflavone derivative

9»f P"gbris. shovjs its affinity with the memter of 112 Araucariales »

The dried twigs of C.funebris(2.5 kg) were exhaus-

tively treated with ethanol. The ethanol extract v;as dried under reduced pressure and treated with ethylacetate, The ethylacetate soluble fraction responded to Shinoda test for 113

flavone . After solvent fractionation and column chromatography of ethylacetate fraction, yellow solid, CF(1.2 gm) was obtained. CF yielded five chromatographically homoge-

neous fractions, after preparative thin layer chromatography

(TLC) silica gel, benzene-pyridine-formic acid(BPF),^36 ;9 which v/ere labelled CF-1 - CF-5. : 92 :

Characterization of CF-1 :

CF-1 (800 mg) (Rf 0.14, silica gel, BPF) was compa-

rable with inseparable mixture of amentoflavone and cupre-

ssuflavone ,

CF-1(50 mg) was methylated with dimethylsulfate in

presence of potassiiim carbonate (1 gm) in dry acetone (100 ml)

by the method of Khan et.al^^. The crude permethylated

derivative on TLC examination showed the presence of three

compounds CF-1M-1(Rf 0.40, silica gel, BPF), CF-1M-2(Rf 0.43_,

silica gel, EPF) and CF-1M-3(Rf 0.49, silica gel, BPF) bright

yellow, orange and blue shade, respectively, in UV light.

CF-lM-1 and CF-1M-2 were found to be identical with amento-

flavone hexamethylether and cupressuflavone hexamethylether, 3 7 as reported earlier , with respect to Rf and characteristic 87 shade in UV light ' . CF-lM-3 was identical with robustn-

flavone hexamethy lether v/ith respect to Rf value & shade in 11 2 UV light' ^c

CF-1(100 mg) was acetylated with ^c^O-pyridine to

yield the crude acetylated product v/hich on crystallization

with chloroform-ethanol gave CF-lAc-1(80 mg, mp 250°C) as major fraction and -was characterised as cui^ressuflavone 1 hexaacetate by comparing its H M-lR (Table-1 ) with authentic f^ 1 114

sample" ' . The crystallization of amentof lavone hexa-

acetate and robustaflavone hexaacetate from the mother llqucv wss unsuccesfulc : 9 4 t

Table-I : Chemical shift value ( £-scaie) of CF-IAc-

M^IR signal NO. Of j value in Assignment protons H2

2.03 (s) 6 - OAc-7,7"

2.16 (s) 6 - OAC-4 ,4"'

2c43 (s) 6 - OAc-5,5"

6o4 8 (s) 2 - H-3,3"

6o92 (d) 4 9 H- 3 • , 5 • & H-3'" ,5"'

6.96 (s) 2 - H- 6 , 6"

7.21 (d) 4 9 H-2 • ,6 • & H-2"" ,6"'

NMR run at 60 MHz in CDCl , TMS as internal standard, s = singlet, d = doublet. : 9 4 t

CF-1AC-1 was assigned as cupressufiavone hexaacetate

(LVIh) on the basis of H NMR data (Table-1).

QAc 0

OAc

OAc 0

(LVIh)

(LVIh) Cupressuflavone hexaacetate.

Characterization of CF-2

CF-2(15 mg), a minor band(Rf 0.25, silica gel, BPF) 112 compared with robustaflavone monomethylether

On methylation, CF-2(5 mg) gave the permethylated derivative, CF-2M comparable v;ith authentic robustaflavon112 e hexamethylether(Rf 0o49, blue shade in UV light)

Therefore, CF-2 was partially characterised as robustflavone monomethyl ether. Position of the methoxyl group could not be assigned as the sample v/as insufficient for preparing acetate » : 95 :

Characterization of CF-1 :

CF-3(150 mg , Rf 0.34 , silica gel, BPF) compared with sequaoiaflavone

CF-3(15 mg) on methylation revealed the presence of two compound CF-3M-1(Rf 0.40, silica gel, BPF, bright yellow fluorescence in UV light) and CF-3M-2(Rf 0<,48, silica gel, EPF, blue shade in UV light). CF-3M-1 and

CF-3M-2 were found to be identical with amentoflavone hexamethylether and hinokiflavone pentamethyl ether, respectively, with respect to Rf and characteristic shade in

UV light^®.

CF-3(110 mg), on acetylation with Ac^o-pyridine gave white solid which shov/ed the presence of two component,

CF-3AC-1 and CF-3Ao2. On crystallization with chloroform- ethanol it gave CF-SAc-l (40 mg, mp 196-9°C) as the major component which was characterized as sequoiaflavone pentaacetate (LViik) by comparison of its Rf value, mp and H r^lR

(Table-2) with authentic sample"^ ^^ ^. The ^C NMR(Table-3) of CF-3 acetate is recorded here for the first time which showed the presence of 5 carbonyl carbon of OAc at 167.9,

168c4 , 1 68.8, 1 69.4 , 1 69.5 and carbonyl carbon of C-4,4" at 2 176,2. Assignments of aromatic carbons and other sp carbons is given in Table-3. I 96 :

OAc

AcO ^

(LVIIk)

(LVlik) Sequoiaflavone pentaacetate

Characterization of CF-4

CF-4(5 mg), was comparable with di-o-methylrobustaflavone''''^.

The methylation of CF-4(5 mg) was done by the method of Khan et.al^^. The robustaflavone hexamethylether CF-4m

(Rf 0.49, silica gel, BPF, blue shade in UV light) was obtained as product wliich was identical with authentic 112 sample . The position of methoxyl group could not be assigned due to lack of quantity to prepare acetate for

R study. : 9 4 t

Table-2 Chemical shift value ( 6"-scale) of CF-3AC-1

NMR signal No. of J value in Assignment proton HZ

2o05 (s) 3 - OAC-4 •

2o09 (s) 3 - OAC-7"

2.26 (S) 3 - OAC-4"'

2,44 (S) 3 - OAC-5

2.49 (S) 3 - 0 AC- 5 "

3.85 (S) 3 - OMe-7

6.61 (d) 1 3 H-6

6.65 & 2 - H- 3 ,3" 6o66 (S)

6.80 (d) 1 3 H-8

7o0l (s) 1 ^ H-6"

7.05 (d) 2 9 H-3'" ,5'"

7o45 (d) 1 9 H-5 '

7o51 (d) 2 9 H-2'" ,6'"

7o98(dd) 1 3 & 9 H-6 '

8.06 (d) 1 3 H-2 •

NMR run at in CDCl^> TMS as internal standard, s = singlet, d = doublet, dd = double doublet. : yo {

Table-3: ''^c NMR of CF-3AC-1

Assigned Assigned position of position of carbons carbon

20.6 5 Me of OAc 128o1 2 ' 21 o1 129c1 6 •

56.0 OMe 129o8 2'" ,6"'

98.7 8 149=9 4 •

1 08.2 6 150.5 5'»

1 08c7 3.3" 151 .1 4 <«(

1 O.'B. 9 8" 1 51 „9 5

1 14 „8 6'« 153.3 7"

1 22c4 1,1'" , 3 • 158o8 7

1 23.9 1 0 1 60o3 9 1 61 .6 g.l 1 27.3 5 ',5'" ,3'" 1 63c7 2 ,2" 1 27o7 1 0" 1 67.9 1 68o4 168.8' 5 of 1 1 69.4 1 69.5 1 76.2 4 ,4" 5 9? :

Characterization of CF-5

C?-5(150 mg) (R£ 0.55, silica gel BPF) was comparable with isocryptomerln.3' 7

The fraction CF-5(10 mg) was methylated by the 65 method of Khan et.al The permethyl ether CF-5M('Rf 0.4 8 silica gel, BPF, blue shade in UV light) was identical with 3 7 authentic sample of hinokiflavone

CF-5(120 mg) was acetylated with AC20-pyridine gave the compound CF-5Ac(50 mg, mp 207-9°), crystallized from chloroform-ethanol. CF-5Ac was identified as isocryptomerin •1 tetraacetate (LIXc) by comparing its mp Rf and H Nt^R(Table-4) 11 7 v;ith authentic sample

OAc

OAc 0

(LIXc) isocryptomerin tetraacetate : ICO :.

Table-4 : Chemical Shift ( ^'-scale) of CF-5AC

mR signal NO. Of J value i n Assig nment protons HZ

2o30 (s) 3 - 0AC-4«»»

2o33 (s) 6 - OAc-5,7

2c40 (S) 3 - OAC-5"

3,88 (S) 3 - OMe- 7"

6o55 (S) 1 - H-3"

6o56 (S) 1 H-3

60 80 (d) 1 2.5 H-6

6o98 (d) 2 9 H- 3 ' ,5 •

7o2 3 (d) 2 9 H-3'" ,5'"

7.23 is) 1 - H-S"

7.30 (d) 1 2 c5 H-S

7.70 (d) 2 9 H- 2 ' ,6 '

7.82 2 9 H-2'" ,6'"

NMR run at 60 MHz in CDCl^j TMS as internal standard, s = singlet, d = doublet. : 101 :

Antibacterial activity;

The crude mixture of biflavonoids (CF) was tested for antibacterial activity in vitro. It was active against Staphylococcus aureus (zone of inhibition 0.7 cm) and pseudomonas aeruginosa(zone of inhibition 0.8 cm), while it was inactive against E.coli and Salmonella enteriditis at 0.5 mg/ml concentration on the nutrient agar media follov/ing the Kirby-Bauer fitter paper disc method

The antileukemic activity of amentoflavone derivative

The racemic mixture of hexa-0-benzyl amentoflavone

(50 mg) obtained from the leaves of Cupressus funebris was mixed with the same compound(150 mg) isolated from Thuj a

Orient alls by similar procedure. The sample was tested in the PSSSi^^kemic tunor system and it was found toxic.

The doses of hexa-0-benzylamentof1avone of 400 ^nd 200 mg/kg/injection were not active and the doses of 100 mg/ kg/injection were toxic. : 1C2 :

2.Eiflavones from Araucaria Cookii

119 The biflavonoids have been tested for spasmolysis »

peripheral vasodit ation^ ^^, antibr adyki nin activity''^^, 1 20 antispasmogenic action against prostaglandin PGE^ , inhibi- 12 122 tion of cyclic AMP and cyclic GMP "phosphodiesterase, 12 3 124 inhibition of hepatoma cells and anticancer activity .

This prompted us to investigate antibacterial activity of

biflavonoidic fraction of leaf extract of Araucaria cookii.

The active constiuents were isolated by the method of Khan 65

etc el > and amentoflavone, cupressuflavone, agathisflavone,

robustaflavone, mono-o-methylagathisflavone and mono-o-

methylrobustaflavone were identified by comparison of the

parent compounds and their permethylated derivatives with

authentic samples in addition to other biflavones reported 64 ,11 6 ,11 7 ,1 2 5 earlier ' .

isolation of biflavonoids from the leaves of Araucaria

Cookii R.Br.FxoD.Don

The leaves of Araucaria copkii were procured from

Vice Chancellor's Lodge, Aligarh Muslim University, Aligarh,

India. Extraction of the fresh leaves followed by solvent

fractionation and preparative TLC gave nine fractions

labelled as AC-1-AC-9. The usual colour reaction and other

diagnostic test indicated all of them to be flavonoidic. : 103 :

The complexities of different fractions were studied by thin layer chrornetographic examination of their methylated products o

The isolation and characterization of 7,7"-di-0- methylcupressuf lavone (LVid) , Kayaf lavone (LVIIg ) , 4 ' ,4'*' ,

7,7'«-tetra-0-methylhamentof lavone (LVlij) , and 4 * ,4'*' ,7,7"- tetra-C-methylcupressuflavone(LVlg) as major constituents

64 of leaf extract of A.cookii have been reported . The isolation and characterization of some minor constituents, such as hinoKiflavone(LIX) and 7"-C-methyl amentoflavone

(LVIId) ,4'" ^7-di-O-methylogathis f lavone ( LXd) , 4',7"-di-0- ir.ethylamentof lavone (LVile) , sci adopitysin ( LVIIh) , and 4 ',7,

7"-tri-O-methylcupressuf lavone (LVIf) were also reported^''^

The present discussion deals with the systematic identification of some minor biflavones in addition to ten biflavones reported earlier^'^ '"" ^ ' ^'''''^^ . The reinvestigation of the leaf extracts of A.cookii revealed the presence of agathisflavone(LXa), cupressuflavone(LVia) , amentoflavone (LViia), robustaflavone(LVIII) , agathisflavone monomethylether and robustaflavone monomethylether which were isolated and identified on the 12basi 5 s of Rf values and characteristic shade in UV light

The TLC examination of purified product revealed the presence of nine fractions which v/ere labelled as

AC-1 (Rf C.I 6), AC-2(Rf 0.17), JvC-3(Rf 0.26), AC-4 (Rf 0.35),

AC-5(Rf 0.44), AC-6(Rf 0.51), AC-7(Rf 0,70) , AC-S(Rf 0.77)

AC-9(Rf 0.78)o : 1C< :

The bands AC-1 , AC-3, AC-5 correspond to the 3gathj.s- flavone and its mono- and dimethylethers and AC-2,AC-4,AC-6 and AC-7 correspond to the amentoflavone, its mono-, di- and trimethyl ethers; the band AC-8 & AC-9 correspond to the tetrainethylethers of amentof lavone and cupressuf 1 avone , respectively. Each band was separated and purified by column chromatography followed by preparative-layer-chromatography using benzene/pyridine^formic acid (BPF) (36 :9 :5) as the developing solvent system.

Characterization of AC-1

This fraction has same Rf value and shade as that of parent agathisflavone and robustaflavone. It was methylated with dimethyl sulfate in dry acetone. TLC examination of methylated product revealed the presence of agathisflavone hexamethylether and robustaflavone hexamethylether(Rf value and characteristic shades in UV light). Therefore, AC-1, was identified as the mixture of parent agathisflavone and robust aflavone.

Characterization of AC-2

This fraction resembles parent amentoflavone and cupressuf lavone on TLC • On methylation and TLC examinatior. of the methylethers, amentoflavone hexamethylether and cupressuflavone hexamethyl ether were detected (comparison of Rf value of authentic samples and characteristic shades 65 in UV light) . Therefore, AC-1, is an isomeric mixture of amentoflavone and cupressuflavone. : 105 :

Characterization of AC-3

It resembles monomethylether of agathisfiavone on

TLC(Rf value and shade). On methylation, it gave agathis-

flavone hexantethyl ether and robustafiavone hexamethylether

which were identified by comparison with the authentic

samples (Rf value and shades in UV light). AC-3 is, therefore,

a mixture of agathisfIavone monomethylether and robusta-

flavone monp-methylether.

Characterization of AC-4, AC-5, AC-5, AC-7, AC-8, and AC-9

Fraction of AC-3-AC-9 were compared with the authentic

samples isolated earlier . Their permethylated derivatives were also prepared by reaction with dimethylsulfate in acetone and the methylethers were compared with the

authentic permethylated C-C linked biflavones. The results were in conformity with the earlier characterization''''^"^o

The isomeric mixtures identified in fraction AC-1,

AC-2 and AC-3 are under investigation by HPIC. : 106 :

Antibacterial activity of biflavonoidic fraction:

The biflavonoidic fraction, AC tested for antibacterial activity, was found to be active against E»coll

(standard and K^^ strains), Salmonella euteriditis, and pseudomonas aeruj1nosa(Table-1).

Table-1 : Antibacterial activity of crude mixture of biflavonoids Isolated from A»cookii against various bacteria.

Bacteria Minimum inhibitory Diameter of concentration mg/ lr}hibition ml (M.I.C.)

1. Esch. coll Oo5 mg/ml 1.1 cm (standard strain)

Esch.coll » » 0, 85 cm strain)

Staphylococcus »> aureus (Oxford strain)

pseudomonas, > » 1 cm aerujlnosa (standard strain)

Salmonella 0o9 cm euteriditis Experimental . _1 ^ I •.

CupresGus funebris was collected from Kathmandu

valley, Nepal and identified by Dr. K.R. Rajbhandari,

Taxonomist, Royal Botanical Garden, Godawari, Lalitpur,

Nepal.

The H NMR spectra were run on Varian D-60 and FX-90

Jeol 100 Mz and IR spectra on Shimadzu IR-J^08.

1 ,Isolation and-Characterization of Biflavonoids from

Cupressus funebris

Extraction

The dried leaves(2.5 kg) were exhaustively treated

with ethanol. The ethanol extract was dried under reduced

pressure and treated with ethylacetateo The ethylacetate

soluble fraction responded to the colour test with Mg-HCl

indicating the presence of flavone. The ethylacetate v/as

removed under reduced pressure. The gunmy greenish mass

(5 gm) so obtained, was refluxed with petroleum ether(40-&0°J

and benzene successively to remove the nonphenolic compo-

nents. The residue was then refluxed with ethylacetate

which responded to the usual colour reaction for flavonoids.

Purification of Biflavonoid Mixture-Column Chromatography

A well stirred suspension of silica-gel(200 gm) in

dry petroleum ether(40-60°) was poured into a column(150 cm

long and 50 mm in diameter)® When the adsorbent was well

settled, the petroleum ether was allov/ed to pass through the -Ou :

coliimn. The crude mixture of bif lavonoid (2 gm) adsorbed

on silica gel was added to the column. The column v/as

successively eluted with petroleum ether, benzene, benzene- ethylacetate (9:1 , 8:2, 7:3, 6:4, 1:1), ethylacetate and

acetone. Benzene-ethylacetate fraction(9 :1, 8:2, 7:3, 6:4,

1:1) gave the yellow crude biflavonoids(1 .5 gm) which revealeel the presence of five bands on TLC ^silica-gel, benzene- pyridine-formic acid(BPF), 36:9:5j . The bands were labelled

as CP-1(Rf 0.14), CF-2(Rf 0o25), CF-3(Rf 0o34), CF-4(Rf 0,44)

and CF-5(Rf 0o55 ) .

Separation of Biflavonoid mixture-preparative Thin Layer

Chromatography

The glass plate (20x20 cm) were coated with a well stirred suspension of silica-gel (35 gm, 70 ml water). After drying the plate for 2 hrs at room temperature, the plates were activated at 110-120^ for 1 hr. The flavonoidic fraction were then subjected to preparative thin layer chromatography using BPF(36:9:5) as the developing solvent.

The position of the bands were marked in UV light. The marked pigment zones were scrapped with the help of spatulla and eluted in separate columns with dry acetone. The solvent was recovered till the eluant was reduced to 20-30 ml. The addition of water and HCl yielded yellov/ precipitate in each case. The precipitate was filtered, washed with water and dried. Homogeniety of the bands was checked by TLC. The components were labelled as CF-1., CF-2, CF-3, CF-4 and CF-5. : 109 :

Each component v/as checked by methylation for accurate

identification.

CF"1 :

CF-1 (Rf 0o14, silica gel, BPF) was comparable with the isomeric mixture of amentoflavone and cupressuflavone.

Methylation of CF-1

CF-1 (150 mg), anhydrous potassium carbonated gm) dimethylsulfate(0.5 ml) and dried acetone(100 ml) were refluxed on waterbath for 6 hrs. A small portion of the reaction mixture was taken out and tested for alcoholic

Feci, reaction. Refluxing continued until it gave a negative FeCl_ test. It was then filtered and evoporated to drynesso The yellow oily mass left behind was treated with petroleum ether and the residue was then dissolved in chloroform. The chloroform soluble portion was washed with water, dried over anhydrous sodium sulfate and concentrated to give a crude solid. The methylated prod\j,ct by TLC examination showed the presence of three methylethers, amentoflavone hexamethylether, CF-lM-1(Rf 0t,40, silica-gel BPF, bright yellow shade in UV light), cupressuflavone hexamethylether,

CF-1M-2(Rf 0.43, silica gel, EPF, orange shade in UV light) and robustaflavone hexamethylether, CF-1M-3(Rf 0.49, silica gel, EPF).

CF-1 acetate

CF-1 (100 mg) was acetylated with pyridine(0.03 ml) and acetic anhydride(0.09 ml) to give the crude solid which : 110 :

on fractional crystallization xvith chloroform-ethanol gave cupressuflavone hexaacetate, C?-1 AC-1 (SO mg, mp 250°).

''H NMR(CDC1 ,5*-scale), 2.03(6H,S, OAC-7,7"), 2 .1 6 (6H ,S ,OAc-

4,4"/), 2.43(6H,S ,OAC-5,5" ) , 6.4 8 (2H, S ,H-3 , 3" ) , 6.92(4H,d,

J=9H2) H-3',5' & H-3'" ), 6.96(2H,S, H-6,6"), 7.21 (4H,d, A ICRy — -1 J=9H2, H-2',6' & H-2'" ,6'" ), IR"^ em 1 775 , 1 655 , 1 600, max 1505, 1420, 1410, 1370, 1290, 11 95, 1 1 65, 1 100, 1080, 1015,

905 , 84 5 ,

The crystallization of amentoflavone hexaacetate and robustaflavone hexaacetate in pure form from mother liquor was unsuccessful.

CF-2 ;

CF-2 (Rf 0o25, silica gel, BPF) was comparable with the authentic mono-0-methylrobustsflavone.

CF-2 permethylated derivative

On methylation, CF-2 gave robustaf1avone hexamethylether, CF-2M(Rf 0.4 9, silica gel, EPF, blue shade in UV light^ identified by comparison with authentic sample.

CF-3 :

CF-3(Rf 0.34, silica gel, EP?) was comparable v/ith sequoiafLavone pentaacetate.

C F- 3 p e rme t hy 1 ate j ^ j g ^ i y at iy e s

The fraction CF-3 on methylation, followed by TLC examination, revealed the presence of amentoflavone hexa- : 111 :

metbylether, CF-3K-1(Rf 0o40, silica gel, BPF), brieht yellow shade in UV light) and hinoJciflavone pentamethylether, CF-3M-2(Rf 0.4 8, silica gel BPF, dark blue shade in UV light)«

CF- 3 Acet ate

CF-3(110 mg) was acetylated with pyridine(0.03 ml) and acetic anhydride (0.09 ml)o Fractional crystallization from ethanol gave sequoiaflavone pentaacetate(CF-3Ac-1 ), m.p 1 96-99'^> ""h NMR in CDCl^ ( scale) 2.05, 2.09, 2.26,

2.44 & 2.49(3H each, OAc-4 ', 7",4'" ,5,5") and 3.85(3H, OMe-7)

6.61 (1H, d,J=3Hz, H-6), 6 = 65 and 6.66(4Heach, S, H-3,3"),

6.80(1H, d, J=3Hz, H-8) , 7 o 01 (1 H, S , H-6" ) , 7 .05 (2 H ,d , J=9HZ ,

H-3'" ,5"' ), 7.45 (1 H,d ,J=9Hz , H-5 ') , 7.51 (2 H ,d , J= 9HZ , H-2'" ,6'*')

7.98(1 H,dd ,J=3HZ and J=9Hz,H-6'), 8 .06 (1 H ,d^=3Hz , H-2 ') "" ^C

NMR ( scale) , 20.6, 21.1 (5 CH of OAc); 5 6.0(OMe), 98.7(8),

108.2 (6), 108.7(3,3"), 108.9(8"), 114.8(6'M, 122=4(1,1'" ,3')»

123.9 (1 0), 1 27.3 (5 ,5"' ,3"' ), 127.7(10"), 128.1(2'), 129.1(6'),

129.8(2'" ,6"' ), 149.9(4'), 150.5(5"), 151.1(4"' ), 151.9(5),

153.3(7"), 158.8(7), 160.3(9), 161.6(9"), 163.7(2,2"), 167.9,

1 68.4, 1 68c8, 1 69.4, 1 69.5 (5>00 of OAc), 176.2(4,4").

The quantity of minor fraction, probably hinokiflavone pentaacetate(CF-oAc-2) was not enough for spectral studies.

CF-4 ;

CF-4(Rf 0.44, Silica gel, BPF) was comparable with di-G-methylrobust sflavone. : 112 :

CP-4 permetbylated derivative

The metbylotion of CF""^ (5 mQ) Vv'bs done as in the

case of CF-1 . The robustoflavone hexamethylether CF-4m

(Rf 0o49, silica gel, BPF, blue shade in UV light) was

obtained as the product which was compared with authentic

sample.

CF-5 ;

CF-5 (Pvf 0.55, silica gel, BPF) was compared with authentic isocryptomerin.

CF-5 permethylated derivative

The fraction CF-5(10 mg) was methylated as in the case of CF-1 . The hinokiflavone pentamethylether CF-5M

(Rf 0.4 8, silica gel, BPF) was the only product which was identified by comparison with authentic sample.

CF-5 acetate

CF-5 (120 mg) was acetylated v/ith pyridine (0.03 ml) and acetic anhydride(0.09). Isocryptomerin tetraacetate,

CF-5AC(50 mg, mp 207-9^) was obtained by crystallization in CHCl - EtOH, and v/as characterized by H NMR in CDCl

( (^'-scale) 2.30(3H,S, 0Ac-4'»' ), 2.33(6H,S, OAC-7,5), 2.40

(3H ,S ,0Ac-5" ) , 3.88(3H,S, OMe-7'» ) » 6 .55 (1 H ,S ,H-3" ) , 6.56

(1H,S, H-3), 6.80 (1H,d,J=2.5Kz , H-6 ) , 6.98 (2H,d , J=9Kz , H-3',

5'), 7.2 3 (2H, d,m,J=9Hz , K-3"/ ,5'"), 7.2 3 (1 H , S ,H-8" ) , 7.30

(1H,d,J=2.5 HZ, H-8), 7. 70 (2 H ,d , J= 9Kz , H-2',6'), 7.82(2H, d, J=9HZ, H-.2'" ,6"' ).. : 11? :

\ KRr - -I IRy cm ' 1 775, 1 640, 1 605, 1 500, 1460, 1 375, 1 360, max

1300, 1275, 1240, 1190, 1140, 1120, 1090, 1035, 1020, 910,

84 5 .

Antibacterial activity of biflavonoidic fraction of

Cupressus funebris

The crude mixture of biflavonoids(CF) was tested against antibacterial activity. Antibacterial activity 1 8 v/as done by disc diffusion method . The indicator bacteria used were 1. Staphlococcus aureus, 2o pseudomonas aerujnosa

3o Salmonella euteriditus, and 4. Esch. Coli• A lawn of the bacteria was made on nutrient agar plate and after drying, dry sterile 6 mm diameter filter paper discs were placed on the lawn. Then 0.01 ml of the chemical under test (1 .5 mg/ml) was placed on the disc and the plates incubated at 37°C for 18 hrs. The zone of inhibition was then measured in centimeters. The crude m.ixture of biflavonoid(CF) was found to be active against Staphlococcus aureus (zone of inhibition 0.7 cm) and pseudomonas aerujinosa

(zone of inhibition 0.8 cm) while in active against

Salmonella and Esch.coli. ! IK-

The antileukemic activity of amento-flavone derivative:

The mixture of amentoflavone and CupressufIcvone

(CF-1) isolated from the leaves.of Cupressus funebris was benzylated by its reaction with benzyl chloride in presence of potassium carbonatec The hexa-0-benzyl amentoflavone was separated from hexa-O-benzylcupressu- flavone by PTLC(silica gel, B:P:F 36:9 :5)„

The testing of hexa-O-benzyl amentof lavone v;as done in the P3 88 leukemic tumer system. Treatment was as a single intraperitoneal injection on day 1 p©st tumar implant. Doses tested were 4 00, 200 and 100 mg/ kg/injection.

The antileukemic screening data are the result of screening performed under the auspice^-s of the develop- mental therapeutic prograrT.ie division of cancrr treatment .

National Cancer Institute,Betheddsa. : li:- !

2 oisolation and Characterization of Blflavonolds from

Araucaria cookii.

leaves of Araucaria cookii were procured from the

University campus, Aligarh Muslim University and identified

by Dr. w. Husain, Department of Botany, A.M.U., Aligarh.

The dried leaves of Araucaria cookii (0.5 kg) were

extracted with ethanol, the ethanol extracts were dried

first at atmospheric pressure, then under reduced pressure.

The dried mass was refluxed with petrolevmi ether (4 0-50°),

benzene, chloroform, ethylacetate and acetone successively

till the solvent in each case was almost colourless. The

chloroform and ethylacetate extracts were mixed together

which on concentration gave a dark brown solid which

responded to the usual flavonoidic colour test.

The biflavonoidic mixture was separated by column &

preparative layer chromatography by the procedure described

for Cupressus funebrls. Homogenlety of the Individual

components were checked by TLC silica gel, benzene-pyri-

dlne-formic acid(BP?), 36:9:5 . The components were

labelled as AC-1 - AC-9, AC-1(Rf 0.16), AC-2(Rf 0.17), AC-3

(Rf 0o26), AC-4(Rf 0o35), AC-5(Rf 0.44), AC-6(Rf 0.51),

AC-7(Rf 0.70), AC-SC^f 0.77 and AC-9(Rf 0.78).

The complexities of different fractions were studied

by TLC examination of their fully methylated products. { 116 :

Methylation of AC-1

AC-1 , a minor constituent, was methylated by the method of Khan et.a'l^^ using appropriate quantity of dimethylsulfate and freshly ignited potassium carbonate in dry acetone. It was refluxed on water bath for about 6 hrs. A small portion of the reaction mixture was taken out and tested for alcoholic ferric chloride reaction.

Refluxing continued until it gave a negative ferric chloride test. It was then filtered and evaporated to dryness. The yellow solid mass left behind was treated with petroleum ether and then dissolved in chlorofonn.

The chloroform soluble portion was washed with water, dried over anhydrous sodium sulfate and concentrated to give a crude solid. The methylated mixture, on TLC exam.ination

(silica gel, BPF, 36:9:5), showed the presence of agathisflavone hexamethylether(Rf 0o45) and robustaflavone hexa- m.ethylether (Rf 0o49) which were identified by comparison with authentic samples and characteristic shades in UV light

AC-2 : 65 It was methylated by the procedure of Khan et.al.

The methylated product was examined on thin layer chromatography using silica gel as absorbent and benzene:pyridine: formic acid(36:9:5) as developer and compared with the authentic samples(Rf values and characteristic fluorescence in UV light) o TLC of the m'e'thylated product revealed, the { 117 :

presence of amentof le.vone hexamethylether (Rf 0.40) and cupressuflavone hexamethylether(Rf

AC- 3 :

It was methylated by the procedure of Khan et.al^^.

TLC of the methylated product was performed on silica gel

using benzene:pyridine :formic acid (36:9:5) as developer

and compared with authentic samples (Rf values and character-

istic shades in UV light)o TiC of the methylated product

revealed the presence of agathisf1avone hexamethylether

(Rf 0o45) and robustaflavone hexamethyl ether(Rf 0o49).

AC-4 ;

The methylated product, obtained by the methylation

of AC-4 using the procedure similar to AC-1, was examined

on thin layer chromatography using silica-gel as absorbent

and benzene:pyridine :formic acid(3 6:9:5) as developer and

compared v/ith authentic samples. TLC of the methylated product indicated the presence of cupressuflavone hexa- m.ethylether (Rf 0.43), amentof lavone hexame thy lether (Rf 0e40)

snd hinokif lavone pentamethyl ether (Rf 0o4 8)<.

AC- 5 :

This fraction was methylated as in the case of AC-1 .

TLC of the methylated product was performed on silica-gel

using benzene :pyridine :formic acid (36:9:5) as developer and

compared with authentic sample(Rf value and characteristic

shades in UV light) which revealed the presence of only

agathisflavone hexamethylether(Rf 0o45). : 118 :

AC- 6 ;

It was methylated by the procedure mentioned in the

case of AC-1. The methylated product was examined on thin

layer chromatography using silica-gel as absorbent and

benzenerpyridine:formic acid (36:9:5) as developer and

compared with the authentic samples, TLC of the methylated

product revealed the presence of amentoflavone hexamethyl

ether(Rf 0o40) and cupressuflavone hexamethylether(Rf 0=43),

AC- 7 :

This fraction was methylated as in the case of AC-1.

The TLC of the methylated product was performed on silica- gel using benzene:pyridine:fonnic acid(36:9:5) as developer

and compared with authentic samples (Rf values and character-

istic fluorescence in UV light). Amentoflavone hexamethylether (Rf 0.40) and cupressuflavone hexamethylether(Rf 0o43) were identified in the permethylated product of AC-7.

AC- 8:

It was methylated by the procedure mentioned as in the case of AC-1. The methylated product was examined on thin layer chromatography using silica-gel as absorbent and benzenes :pyridine :formic acid(36:9:5) as developer and compared with authentic samples. TLC of the methylated product revealed the presence of only amentoflavone hexamethylether (Rf 0o40). t 11

AC- 9 ;

It was methylated as in the case of AC-1 . The TLC

of methylated product was performed on silica-gel using

benzene :pyridine:formic acid(36:9;5) as developer and

compared with authentic sample. TL.C of methylated product

indicated the presence of only cupressuflavone hexamethyl-

ether.

Antibacterial activity of Biflavonoidic Fraction of

Araucaria cookii :

The crude mixture of biflavonoids AC was tested

against antibacterial activity as in the case of cupressus

funebris. It was found to be active against Esch.coli

(standard strain) (zone of inhibition 1.1 cm) Esch.coli

strain) (zone of inhibition 0.85 cm). pseudomonas

aerujino sa(standard strain) (zone of inhibition, 1cm) and

Salmonella euterlditis (zone of inhibition 0.9 cm) and inactive against staphylococcus aureus. REFERENCES : 12 'J !

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126o Indian Science Congress Association, 73rd Session

Jan 3-S, Late Abstract Pc 1 9 (1 986) » Chapter-Ill Introduction : x: :

The ,^enus Hbu s belongs to the i-^Tiily .^ngcard iaceae, v/'r. icb

insists ox geneza co-npiisins about 600 species'.

Chemical constituents of Rhus

The compounds most frequently isolated in the genus Bhus are terpenoids^ ' Urushiols'^~^ , flavonoid s"', flavonoid glycosldes!1>^2,14,29-35

leroenoids

Plant source

(I) ^-Sitosterol . ty-ph ina^

(II)

(II) rJ. tyohina^ ! i:>l :

(III)

(III) y^ - -Amy r in . tyDh ina^ 2 A n\amber of undetected . typb in a tr iterpenes

iter 0 idfcy/tr iterpe n oId s ^hus sr^ecies c ir oten o id s

Simole ohenols

Urushiols ^. V9 r"n ic lera R. t ox i c od e n d r on '', ^ h]; s s p_e c i "•5

heutadecartr ienyl) c -atecbol o^.vsrriciiGr . a 6

'I-VB' Z, 11 ' 13,1 3' Z-pents-

cecartx Lenyl)cate c h ol r?. vern icifera" ^-(lo'z,13'z,l6' -hettr^aca- rtrienyl) c-ateohol ^ . ve '•"n i c if e r a"

Z,1 1 ' ~iy2vAa- jrc jrtrienyl)cgtechol H , verr. ic ifera • I -) p ' • — J «

- _ VOL0las

irlant source 5-nyaroxy-/-, 7- 10 d imethoxyflavone ^.undul3ta

(Va-h)

. 11 ^ n 12 (Va) I'lyx ice t in OH OH OH OH i- rnysurensis, n.lancea

( ^-b) 7,4'_Ji_o- Tiethyl^yr- 1 ? icet in c::e riT Oj'lO CH lancea

. 11 ( V-c) >-tuerect in TJ QTT Qi-T Tivsu ren 1 s. H. 1 • 1 ^ 1 4 i- c*" inensLS, R . pa rv if lor a

- iv sal ic LI ol la 1 5 (vd) O'nbu in O'-e n Oi-Ie OTI I' silicifolia 1 5 (ve) Rhannet in c::e H OH I- s-^l ic if ol i ca ( v-f) '--yr in ?et in C':e oae S ^ T C 11 ol ] 4 5 C^i) jiij r opet in die CH O^T CH salic1folia' . 11 12 ( v^'^) Kae^fex Oi. CH tj OH -•ysurensis. T. 1nncea ! -L;

Plant source (VI)

(71) tongapim H.chlTiensis"^^

^ . . .16 (VII) Xetia^iethoxyf isittn H .c ninens IS

^ (VIII)

.16 (VIII) L'e'Tiethoxykanu r'irj .cr.ineriE is

Bifl'jvorjoids

HO -OH

(IX) OH 17 (K) l^e ox hucilav an one r!. £uc oed '^nea ! :

OH 0

Plant source

1 S 'I Q (X) --^husf lavanone H.succedanea ' ^

HO-V/ \ / W-OH

(XI)

(XI) . uc , i.. ••.neal'lavanone rxVoH

HO- V (XII)

riant source

21,22 (XII) Rhus flavone ^ . succed

(XIII)

, . 22 (XI,I) -.ecu'jfei o.:e j-.-i -1 • succec anea : 1 !

Plant source HO 0 (^iv) 22 (XIV) He suaferones-B R.succed anea

(XV)

(XV) Oui-ressuf lavone ^ . succed anea^^'^ ^

(>:vi)

;xvi) ^^lent of lavor.e . Gucceo, anea 22,2' 4 9 5 .toxicodendr om"

li. •a uben s i-2s 6 ! i:i :

(XVII)

Plant Source

(XVII) Agatbisflavone Ori . succedanea^ .22,2 ' 3 T?.t)u.'n.iab9"nsis"'

(XVIII) / WoH

(xviil) aobustaflavone E. succed anea^"^,

H. ouri.iabe'ns is^^

(XIX)

3 ^ 22,24,27,23 (XIX) H in ok if 1 a v on e .1. succedanea ' 3 . , .26 _.punnacensiF,

-i."iVEurensis I3B t

Flavonoid .~lvcosi6ec

HO^. / VoH

Plant source

29 (XX) Jhrysantheniin 3.succedanea

(XXI)

30 (XXI) Shoiiol in rE. succed Tnea

te on iu in-':3-r.Tor. 0- 9Q ?lucos ide . succed ar.e

OMe

AH 0 (XXII) 1 2 (AX[[) OG ids of . lance a 7,4 -d i-C-metr.^^l- '^yxicetin : 135 !

(XxiIIa-d)

a a, h^ Plant source

14 3' (XXII la) I'iyricitin Rham OH OH OH R.carviflora, R.chin en sis

R.coriaria,3 2 R.mysurensis 11

(XXIIIb) v.uercetin Rba^ H OH OH R ,chinqns is,3 1 R . javan icus3 3

R . Tvsurens is"!''

(AAIIIC) Isoquezc- etin 31 u H OH OH R.coriaria 32

(XXiIId) -Hiselin Rhan H OH OH R.carviflora 1 /I

11 Kae^nfer ol-3-O-r ha:::moside R. 'fnvsuren s is r n

I'IIDC-ijjjj AL £0'" S

CH=CH-C00H

(XXIVa-b)

(XXIVa) -u-Cou-nar ic and ri R .Tparviil ora

(XXl'^b) Gafferic acid OH . -o^rv iilor a ! l-^-O :

(XXVa-b) R

(XXVa) Gallic acid H 52 R,cor iar ia, R . succedanes

R. cemiliata^^ (XXVb) R. .lavan icus 33

R .c or iar ia 32

0 (XXVI)

(^Vl) -illagic acid 32 34 R.coriaxa, R.guccedanee J-Shikki-nic acid R. succedanea^ ^ rolypxenois 36 ^. typhina'

(XXVII) rs (XXv'lI) Ovaliten'one ^.chinensi, . .s l^'l

37 Tannins R. sei 1 iata;"" 'R,cor iara;

^ • sue ced ane

G-aliotannins R .c or iar la , .cblnensls "

Bl-and tr iflavonoid s 'R.leptod Ictyaf^ H .lancea^'^

Galloylesteis 3 . ser ruolata'^'' Discussion : 142 !

The aried leaves of Rhus a lata (1 kg) procure-i from

Aiz aul (Mizor ain ) were extracted with ethanol. Ethanol extract on solvent fractionation and column- chromatogra- phy over silica gel yielded a flavonoidic mixture which responded to positive colour test with Mg-HCl and alcoholic ferric chloride,, The crude product was examined by thin layer chromatography(TLC) over silica gel using benzene- pyridine-formic acid (BPP) , 36:9:5. TLC sho\i?ed the presence of three components which were labelled as RA-1 (Rf 0.15) and RA-2(Rf 0o34). RA-1 & RA-2 were separated from the mixture by preparative TLC(Silica gel, BPP, 36:9:5)o

Characterization of RA-1

RA-1 was comparable with authentic sample of agathis- 42 flavone and amentoflavone o The mixture was methylated using methyl sulfate to yield three compounds RA-lM-1,

RA-1M-2 and RA-1M-3 which were separated by FTLC(Silica gel,

EPF, 3 6:9:5).

RA-lM-1,mop. 162-6O4°C, was characterized as agathisflavone hexamethyl ether (i) by comparison of H M-IR data 43 (Table-1) with the authentic sample. All the signals in

''H NNIR are in conformity with ag at hi sf lavone hexamethyl ether except two singlets at $"6.94 and 6o80 which have been tentatively assigned to H-3" H-3/H-3" protons, respectively. Actually, H-6" and H-3/H-3" in agathisflavone hexamethyl ether appear about 0.3 ppm dov/nfield from the : l43 :

Table-1 : Chemical shif t s ( 5* - sc a le ) of pro-cons of RA-li'!-1

PMR Signals Number of J values Assignment protons in HZ XVII XXVIII

3.61 (s) 3 - OMe-5 ayie-5

3o7S(s) 3 - OM e- 4 avie-4'"

3.82 (s) 3 - OMe-4 ' OMe-4 •

3.91(s) 6 - OMe- 7,7''* OMe- 7, 6"

4.08(S) 1 - H- 3/H- 3*' H-3/H-3'*

6.55 (s) 1 - H- 3/H- 3" * H-3/K-3"

6.92(s) 1 - H- 8 H- S

6.94(s) 1 - H- 6" * H- 7" *

6.81 (d) 2 9 H- 3'", 5"^ T4ii _ wO It,/ ^ tW

7.05(d) 2 9 H- 3 ' , 5 ' H- 3 ',5 '

7„41 (d) 2 9 H-2'", 6'" H-2'", 6"'

7<,91 (d) 2 9 H-2 6 ' H-2 ' , 6 '

* Tentative assicnments, s= singlets, d=doutlets, TMS used

?s internal standard, spectrum run in CDCl^. : 144 !

values noted here. Therefore, RA-1M-1 may have the structure XX^'III instead of . Hov/ever, further studies are in progress to characterize RA-1M-1. f

(XVIZ) I W (a) R = H 0F\ O (a) R = H (b) R = Me (b) R = Me RA-1M-2 was comparable with robustaflavone hexa- 44 methyl ether • Hov;ever, the quantity was not enough for spectral Studie-So

R1-1-3, mP. 2 2 5*^0 was comparable with amentof lavone hexamethyl ether"^^. Its H NMR data is given in Table-2,

Therfore, RA-1 may be characterised as a mixture of agathisf lavone (tentative) (XVIIa) and amentof lavone (X\'I) » : 145 !

Table-2 : Chemical shift dat a ( -S" sc ale ) of RA-1M-3.

PMR Signals Number of J values Assignments protons in HZ

3.80(s) 6 - OMe-4 4"i

3.86(s) 3 - OMe- 7

3., 94 (s) 3 - . OMe-7"

3c96 (S) 3 - OMe-5

4 0 (s) 3 - OMe-5"

6o40 (d) 1 3 H-6

6c54 (d) 1 3 H-8

6c54 (s) 1 - H-6"

6.56 (s) 2 - H-3,3"

6o SO (d) 2 9 H-3'",

7o1 8(d) 1 9 H-5 •

7.44 (d) 2 9 H-2'" ,6'"

7.90(d) 1 • 3 H-2 •

SoC4 (d,d) 1 3,9 H- 6 '

s= singJ.et, d= doub.lrt, d,d= double doublet, TMS

as internal standard, spectrum run in CDCl^o : 146 :

Characteriz?(tion of R^-2

RA-2, m.p. >350°C, was comparable with hinokifla- 45 vone and its permethylation yielded hinokiflavone pentamethyl ether on acetylation, it gave a white crystals of

RA-2AC, m.p.207°C from chloroform-methanol. The ''H NMR data of RA-2AC is given in Table-3. It was characterized

at 5,7,4",7" -pentaacetoxyhinokiflavone(XlXb) by comparison

OR 0

(XIX) (a) R = H (b) R = Me

1 4 5 of its H NT'iR data with authen.tic sample RA'2 , is therefore, hinokiflavone (XlXa). I4-7 :

TaLle-3: Cher.iical shift data( g'-scale) of RA-2AC.

PMR Signals Number of J value Assignraent protons in Hz

2»12(s) 3 - OAC- 7'*

2.24(s) 3 - 0Ac-4'»'

2o35 (s) 6 - OAC-7,5

2o45 (S) 3 - OAc- 5"

60 60(s ) 1 - H-3 ,3"

6oi6(s) 1 - H-3 ,3"

60 86(d) 1 2 o5 H-6

7o05 (d) 2 9 H-3 ' ,5 '

7 = 34 (d ) 1 2 c5 H-S

7C47(S) 1 - H-8"

7 . 83 (d ) 2 9 H- 2 • ,6 '

7=91(d) 2 9 H-2'" ,6'"

s = sing Jet , d= doublet, TMS used es interne 1 «"L nc; rd : 14

On the basis of chernicel, chromatographic (Rf and characteristic shade in lA^ light for different type of linkages in biflavones) and ^H NMR data(permethylated or acetylated derivatives), RA-1 and RA-2 rnay be characterized as agathisflavone, amentoflavone and hinokiflavone, respectively. Experimental : :

~~Z^ll melting points are uncorrected. ''h M^IR spectra are~~

~~taken in CDCl^, on 100 MHz JEOL F>:90Q and 400 MHz Bruker~~

~~MSii 400.~~

~~isolation and Characterization of Biflavonoids from the~~

~~leaves of Rhus alata~~

~~Leaves of Rhus alata were procured from Pacchunga~~

~~University College, Aizaul, Mizoram.~~

~~The dried leaves of Rhus alata (1 kg) were extracted~~

~~with ethanol. The ethanol extract was dried under reduced~~

~~pressure. The dried mass was treated with petrol, benzene~~

~~and acetone. The acetone extract which responded to the~~

~~flavonoidic colour test and FeClg test was chro.riatographed~~

~~over silica gel column and the biflavonoidic mixture was~~

~~checked on TLC over silica gel(benzene-pyridine-formic acid,~~

~~36:9:5). The TLC showed presence of two components RA-1~~

~~(Rf 0.16) and R!\-2(Rf 0.34). Preparative thin layer chro-~~

~~matography was used to separate biflavonoidic mixture into two components RA-1 and RA-2.~~

~~The complexities of the above components were checked~~

~~by TLC examination of fully methylated derivatives to~~

~~ascertain the linkage of biflavones. Major constituents~~

~~were characterized by ''H MAR.~~

~~RA-1~~

~~The crude mixture of RA-1(0.16) was comparable with~~

~~authentic agathisflavone and amentoflavone. : 150 :~~

~~Methylation of RA-1~~

RA-1(50 mg) was methylated with anhydrous potassium carbonated gm) and dimethyl sulfate (0.5 ml) in dried 42 acetone by the method of Khan et.al . The crude methylated product revealed the presence of three bands RA-1M-1

~~(Rf 0.45, silica-gel, BPF, yellow fluorescence in UV light),~~

RA-1M-2(Rf 0.49, Silica gel, BPF, blue fluorescence in UV light) and RA-lM-3(Rf 0.40, silica gel, EPF, yellow fluorescence in UV light). RA-IM-i was comparable with agathisflavone hexamethyl ether and was characterized with the help of H NMR( S'-scale, CDCl^) , 3.61 (s, 3H, OMe-5), 3.7S(s, 3H,

~~OMe-4"") , 3.82 (s, 3H, OMe-4'), 3.91 (s, 6H, OMe-7 , 7'S tent ative~~

~~4.08 (s, 3H, OMe-5'* )» 6.55 (s, 1H, OH-3,3")» 6.80(s, 1H, H-3,~~

~~3", tentative), 6.92 (s, 1H, H-8), 6.94(s, 1H, H-6",tentative'~~

~~6.81 (d, 2H, J=9Hz, H-3'",5'"), 7.05 (d, 2H, J=9Hz, H-3',5'),~~

~~7.41 (d, 2H, J=9HZ, H-2'",6'"), 7.91 (d, 2H, J=9HZ , H-2',5'). 4 3 RA-1M-2 was comparable with robustaflavone hexamethyl ether~~

~~(characteristic fluorescence in UV light and Rf value).~~

~~RA-1M-3 was comparable with authentic amentoflavone hexamethyl ether(characteristic fluorescence in UV light and~~

~~Rf value) and was characterized by H NMR( iS'-scale, CDCl^),~~

~~3.80(5, 6H, OMe-4 ',4'"), 4.86(s, 3H, OMe-7), 3.94 (s, 3H,~~

~~OMe-7"), 3.96(s, 3H, QMe-5), 4.l0(s, 3H, OMe-5"), 6.40(d,~~

~~1H, J=3HZ, H-6), 6.54 (d, 1H, J=3Hz, H-S) , 6c54(s, 1H, H-6" ) ,~~

~~6.56(S, 2H, H-3, 3" ) i 7.18(d, 1H, J=9Hz, H-3'",5'"), 7.l8(d, : 184 !~~

~~1H, J=9Hz, H-5')» 7.44 (d, 2H, J=9Hz , H-2'",6'"), 7.90(d,1H,~~

~~J=3HZ, H-2'), 8.04 (d, d, 1H, J=9H2 & 3Hz , H-5').~~

~~RA-2~~

~~RA-2(30 mg, ^^f 0.34), mp )350°C, was comparable 42 with hinokiflavone~~

~~•Methylatlon of RA-2~~

RA-2 was methylated by the method of Khan et.al using appropriate quantity of potassium carbonate and dimethyl sulfate. The methylated product revealed the presence of one band RA-2M(Rf 0.48, daik blue fluorescence in UV light). RA-2M was comparable with hinokiflavone pentamethyAcetylationl oetherf RA-. 2 RA-2(20 mg) was acetylated using acetic anhydride and pyridine to yield RA-2Ac,mp 207^^ (r^crted mp 242°C) which was characterized as hinokiflavone pentaacetate by

~~''H NMR ( S-scale, CDCl ) , 2o12(s, 3H, OAc-7" ) , 2.24 (s, 3H~~

~~0AC-4'»'), 2.35 (s, 6H, OAc-7,5), 2.4 5 (s, 3H, 0Ac-5'»), 6.60~~

~~(H-3,3'»), 6.86(d, 1H, J=2.5 Hz, H-6) , 7.05 (d, 2H, J=9Hz,~~

~~H-3',5'), 7.34 (d, 2H, J= 9Hz , H-3'", 5'"), 7. 35 (d, 1H, J=2.5~~

~~HZ, H-8), 7.47(S, 1H, H-8'< ) » 7.83(d, 2H,- J=9Hz, H-2',6') and 7.91 (d, 2H, J=9Hz, H-2'", 6'"). REFERENCES ! 152 :~~

~~Re fe i-ji: ze s~~

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~~17. Jnen, Lin, Y-.:. and -.in, Y.C., "letexoc.^c!es 9(5)~~

~~653 (1975).~~

~~1B. Oher), F-0. and Lin, Y-H, J.Ohen. Soc, Perkin Tzans~~

~~I, 93(1976).~~

~~19. Lin, Y-ii. and Chen, P-C. , Tetxahed jon Lett 48, 4747-50~~

~~( 1975).~~

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~~(1975).~~

~~21. Chen, -f-C. , Lin, Y-M. and V/u, J.O., Phyt oc hem istry 13,~~

~~1571 (1974).~~

~~22. Chen, F-C., Taiwan K'0 Hsueh 26 , 100 (1975).~~

~~25. Lin, Y-i-I. and Chen, ?-C., Hua Hsueh (_1), 14-16 (1974).~~

~~24. Chen, P-C., Lin, Y-.I a:jd Liang, C.M., Phytoche-iistry 13,~~

~~276 (1 974).~~

~~25. ilisohly, H.^., Craig, J.C., '••/aller, O.W., and Tumor, C.3.,~~

~~rhrtochenistiy 17(12), 2140-1 (1973).~~

~~26. Kamil, .1., Iqbal, il. and Ilyas, 11., Indian. J. Che':;. ~oc.~~

~~61(4), 575 (1954).~~

~~27. Lin, Y-i: and Chen, F-C. Phytoche-iistry 15(S), I6I7 (19^74).~~

~~26. Chen, P-C., Lin, Y-A. and Liang, C-H. , Phytocheistry 1 5{ 1 ) ,~~

~~276-3(1^74).~~

~~29. l£hil:uxa, Ij., Phy t ochemistry 11(^). 2 5 1^.~~

~~50. Hutxcri, 3. and IJatsuda, ^ (Ja-oan) 21,~~

~~645 (1951).~~

~~51. -.atsuda, H., Che-^. l-har-. Pull. 14(3^, ^'77 (1?56).~~

~~52. Jlsissi, H.I., l£ha>, ::.:;. and Vahid, -A. 5. Plinta. ''ed.~~

~~21(n, 67 (1972). : 154- :~~

~~x^rito-ij . riyazaki, K. and ^^azaki, T., Yaku ?aku Za "sb i~~

~~6.111, : J:;o4}.~~

~~•jL. Ishikuxa, . , Hayashida, i:. and ^azaki, X., Bp-t .;-:a-?.~~

~~97 (1047). 555-67 (19S4)-~~

~~55. Uota, H. and Nisida, K., J. Aer. Cbeqi.Soc. Japan 17, 512~~

~~(1941).~~

~~36. Cbojnacki, T. and Vogtaan, T., Acta. Blochia. Pol. 31 (1) ,~~

~~115-26 (19B4).~~

~~37. Buziashvilli, I.S., Koaissarenko, N.F. and Kolesnikov, O.G.,~~

~~Hast. Resur 6(2), 237 (1972).~~

~~33. Hishizawa, M., Yamagishi, T., I'onaka, G., and Ilishioka, I.,~~

~~J.Ghea. aoc. Perkln Txans-1, (12), 296 3-3 (1932).~~

~~39. Chen, X., Sen, X., wunkai, M., Line ban Huaxue. Y"u. Songye~~

~~3(1), 40-2 (ch)(l933).~~

~~40. Vivers, P.II., Kolodziej, TJ. ^ Young, D.A., Ferreira,D. and~~

~~Houx, D. S., J. Chetn.oQC.Perkln Trans-1 , (IQ) , 2555-62 (1933).~~

~~41. Haddock, ^.A., Gapta, Alsbafi, S.'MC. , Hasla-, and~~

~~i-langoiata, D., J.Cbe-^. Soc. Perkln -^rans-l . (11) 2"'~~

~~(1932).~~

~~42. i\ban, K.U., .-insaii, i.'.H., UsTani, J.-T., Ilyas, H. and~~

~~rtabnan,;/., rhytochemistry ^0,2129 (1971).~~

~~43. Khan, IT.IT., Ilyas, "I., Rabnan, , '-asbi-na, T., Oigav/a,~~

~~and Kav/ano. i:., letrabedron. 23, 5639 (1972).~~

~~44. Vaxsbney, i-i.k., ^icull, 'A., Rabnan, Oklgav/a, i;. and Kav/ano,~~

~~1. , -c-bytocberr^lstj- 12 , 1 yjl (1 973).~~

~~45. xetrex, .4., ./arren, R., Ut^iani, J.'-'., and~~

~~Rab":an, ./,, Tetrahedron Letters £2, ?53 (1^59). Chapter-lV Introduction ! 135 !~~

~~The genus Garclnla belongs to Guttifereae, a family~~

~~almost tropical in distribution and composed of about 32 genera and 400 species,~~

~~Garcinia are cultivated for ornamental or for edible fruit. The mangosteen(Garcinia mangostana) is one of the most highly priced edible fruit of species'* ,~~

~~CHEMICAL CONSTITUENT OF GARCINIA~~

~~The compounds mostly isolated from the genus Garcinia are terpenoids^" ^ , xanthones^ , polyisoprenoid xantho-~~

~~= 14 ,1 5,19,55-64 , .. 3,5,7-^14,8 noads , benzophenones ' ' and flavonoads~~

~~20,58,65-74~~

~~(A) Terpenoids Plant source~~

~~(I)~~

~~(I) »C-Terpinol G.mangostana~~

~~(II)~~

~~(II) Copaene G .mangostana s 156 :~~

~~(IIX)~~

~~(III) (+)-Cadlnene G.mangostana~~

~~(IV)~~

~~(IV) (+)- ^-Cadinene G.mangostana~~

~~(V)~~

~~(V) oC-Bisabolene G .mangostana'~~

~~(Vla-b)~~

~~3 4 (VI a) Friedlein O Me ' G.oyallfolia~~

~~(VI b) Friedlein- oCH CH2r.H G.spicata" -3j3,28-diol ^cH : 131 :~~

~~H 0 (VII)~~

~~3 4 (VII) Sitosterol G.splcata » G.ovallfolla ,~~

~~G.thawaitesli^, G.kola^ >~~

~~7 G.terpnophylla » G.echlno-~~

~~7 8 carpa , G.mannl~~

~~(VIII)~~

~~8 (VIII) Stlgmasterol G.mannii ! 158 :~~

~~(IX)~~

~~(IX) ^-Amyrin G.thawaitesii"~~

~~(X)~~

~~(X) Tirucallol G .thawatesii^~~

~~(XI) Cycloartenol G.heckel" : :~~

~~(XII)~~

~~(XII) 24-Me-Cycloartenol G.heckel'~~

~~(XIII)~~

~~(XIII) flC-Spinosterol G.quaesita^ ^~~

~~R = CH2-CH=CMe2~~

~~(XIV)~~

~~(XIV) Garcinol G.lndica 11 1-160 I~~

~~R=CH -CH=CMe 2 2~~

~~(XV)~~

~~11 (XV) isogarcinol G.indica~~

~~CMe^^Ca~~

~~R=CH2-CH=CMe2~~

~~(XVI)~~

~~(XVI) Camboginol G.indica^'' , G.cambogig 12 ! 161 :~~

~~(B) XANTHONES;~~

~~A large number of xanthones have been isolated from~~

~~Garclnla species.~~

~~Pioxygenated xanthones~~

~~^^ (XVII a-'b) R 1 3 (XVIIa) 1 ,5-Dihydroxyxanthone H G.buchanani^ i , G.echinocarpa , G,terpnophylla , ^ jj 1 5 G»xanthochymus * 1 3 (XVlIb) 1-Hydroxy-5-rr,ethoxy- OMe G.buchananii xanthone )H HO.~~

~~(XVIII) 14 15 G.xanthochymus * , (XVIII) 1,7-Dihydroxy-xanthone L— (Euxanthone} G.eugenifolia , G.hermonli Golndica 1 8~~

~~(XlXa-d)~~

~~1 3 (XlXa) 1,6-Dihydroxyxanthone H H G.buchananii 1 3 (XlXb) 1-Methoxy-e-hydroxy Me H G.buchananii xanthone 162~~

~~13 (XIXC) 1 , e-Dimethoxyxanthcine Me Me 2. • fa^chananil~~

~~(XlXd) 1-Hydroxy-e-methoxy- H Me G.buchananil 13 xanthone~~

~~Trioxygenated xanthones~~

~~0 OH~~

~~OHe (XX)~~

~~1 3 (XX) Buchananaxanthone G.buchananil~~

~~(XXI)~~

~~1 6 (XXI) Gentisin G.eugenifolia~~

~~(XXII)~~

~~(XXII) 1 ,5,6-Trihydroxy- G.eugenifolia 1 6 xanthone OR ao~~

~~(XXIIIavbJ* ^ 1 6 (XXIII a) 1 ,6,7-Trihydroxy- h G.eugenifolia xanthone ! 165 :~~

~~1 6 (XXIllb) 1,6,7-Trimethoxy- Me G.eugenifolia xanthone~~

~~Tetraoxygenated xanthones~~

~~0 Of^~~

~~OK (XXlVa-b) R 1 6 (XXIVa) 1 ,4jT-Trihydroxy-S- H G.eugenifolia methoxyxanthone~~

~~1 6 (XXIVb) 1 ,3,4,7-Tetramethoxy - Me G.eugenifolia xanthone~~

~~(XXV) 19 (XXV) 1 ,3,5,7-Tetrahydroxy- G.pundunculata xanthone~~

~~(XXVI)~~

~~(XXVI) 1 ,3,6,7-Tetrahydroxy- G.echinocarpa, xanthone G.hermonii'' 19 G. punduncul at a Gomultiflora : 164 :~~

~~0 OMe~~

~~MeO~~

~~(XXVII)~~

~~(XXVII) 2,5-Dihydroxy.1, G.thawaitesii" 6-ciimethoxyxanfhone isoprenylated xanthones~~

~~(XXVIII)~~

~~1 7 (XXVIII) 1,3,7-Trihydroxy-2 3-methyl H G.hermonii (3-methylbut-2-enyl) but-2- -xanthone enyl~~

~~R. R "2 3 33 (XXIXa) 1,5-Dihydroxy-2 H OH H G .mangostana (3-niethylbut-2-enyl)- -3-methoxyxanthone : 165 :~~

~~(XXIXb) 1-Hydroxy-2 O-methyl H OMe H G.mangostana 21 but-2-enyl)- 3,5- dimethoxyxanthone 21 (XXIXc) 1 ,3,5-Triinethoxy-2 Me OMe H G.mangostana (3-methylbut-2-enyl) xanthone~~

~~21 (XXIXd) 1 ,3,7-Triinethoxy-2 Me H OMe G.mangostana (3-rnethyl but-2- enyl) xanthone~~

~~21 (XXIXe) 1,7-Dihydroxy-2(3- H H OH Gomangostana methyl but-2-enyl) 3-me thoxyx a ntho ne~~

~~Geranylated and prenylated xanthone~~

~~ch^H~~

~~(XXX) 22 (XXX) Cowanol G.cowa s 166 :~~

~~MeO~~

~~(XXXIa-d) R R^~~

~~(XXXIa) Rubraxanthone H geranyl~~

~~(XXXIb) Cowaxanthone geranyl H G.cowa 22,23~~

~~(XXXIc) Cowanin geranyl prenyl G.cowa 22,23~~

~~22,23 (XXXId) Mangostin prenyl prenyl G.cowa G.mangostana 24-27~~

~~(XXXIIa-b) R 2 3 25 ,28 (XXXIla) ^-Mangostin Me H Me G.mangostana~~

~~25,27 (XXXIlb) "/^Mangostin h H H G.mangostana~~

~~(Normangostln) 29,30 I 167 :~~

~~(XXXIIIa-b) R~~

~~29,30 (XXXIIIa) Gartanind ,3,5,8- OH G.mangostana Trihydroxy-2,4-di 3,3 '-dimethylallyl xanthone)~~

~~29-31 (XXXIIIb) 8-Deoxygartanin H G.mangostana (1,3,5-Trihydroxy- 2,4-bis(3-methyl- but-2-eny1)xanthone)~~

~~(XXXIV) R 32 (XXXIV) 1,3,5-Trihydroxy- H CH„ — CH G.quadrifaria II 4,8-di(3,3 •-dimethyl- CMe, allyl) xanthone : 204:.0 :~~

~~(XXXVg)~~

~~27 (XXXVa) 1 ,3,6-Trihydroxy-2 ,4- G »mangostana bis(methylbut-2-enyl)- - 9H-xanthen-9-one (Garcinone-A)~~

~~(XXXVI)~~

~~27 (XXXVI) 1,3,6,7-Tetrahydroxy-2 G.mangostana~~

~~(3-methylbut-2-enyl)~~

~~- 8(3-hydroxy-3-methyl~~

~~butanyl)-9H-xanthen-9-~~

~~one(Garcinone-C) : 169 :~~

~~M^O~~

~~(XXXVII) 27 (XXXVII) 1,7-Dihydroxy-3,6- G.mangostana dimethoxy-2, 8-di [3 • ,3 '-DMA} -9H- xanthe n-9-one~~

~~(XXXVIII)~~

~~33 (XXXVIII) 3,6-Dihydroxy-&,7-di G.mangostang [3' ,3 '-DMJ^-SH-xanthen- 9-one~~

~~(XL) 33 (XL) 1 ,3,6,7-Trihydroxy-2, 8-di G.mangostana (3 ,3 '-dimethylallyDxanthone 170 !•~~

~~Xanthones with cyclized isoprenoid~~

~~31 (XL1 ) 1 ,5-Dihydroxy-6 ' , 6 '-dimethyl- G.densivenia -2K-pyrano(2 ' ,3 • :3,2)-6", 6"- diinethyl-2K-pyrano(2" ,3"-6,7) xanthone(pyranojacacreubin)~~

~~(XLII) 31 (XLII) 1 ,5,6-Trihydroxy-6•,6 G.densivenia dimethyl-2K-pyrano(2',2 ' :3 ,2)-7 (3 ,3-diinethylprop-2-enyl)-xanthone (7-prenyljacareubin)~~

~~(XLIII)~~

~~31 (XLIII) Rheediaxanthone-A <^ensivenia » G.staudii^^ : 171 :~~

~~(XLIV)~~

~~(XLIV) 5 ,9-Dihydroxy-8-it\ethoxy-.2 , G.mangostana'^c^ 2-dimethyl-7(3-methylbut-2" enyl)-2K,6H-pyrano(3,2-b)- xanthen-6-one )H~~

~~HO OH 34 (XLV) Jacareubin G.densivenia~~

~~(XLVI) 27 (XLVI) 5,9,11-Trlhydroxy-10- G.mangostana (3-rTiethylbut-2-enyl)-3 , 3-dlmethyl-3H,12H-pyrano (3,2-a) xanthen-12-cne (Garcinone-B) s 172 :~~

~~(?CLVII)~~

~~(XLVII) Macluraxanthone G.ovalifolia~~

~~(XLViiia-g)~~

~~(XLViiia) Morellin Me CHO Me G.more 11 a^^"^""~~

~~(XLViiit)) Isomorellin CHO Me Me~~

Me Me (XLVIIlc) De oxyisomere- Me llin 36,38 Me G.morella (XLVIIId) Me CHO llin(no.d.b In cyclohexane ring) 36 (XLVIIle) Morellie acid cooH Me Me G .inorella 36 (XLVIIlf) Isomorellic Me COOH Me G.morella acid 47,52 (XLVllig) Gambogic acid COOH Me CH^-C^.^ GG.morell a 36 ,53,54 CH G.hanburyi II CMe, ,^•36,40 .90 :

~~54 G.hanburyii~~

38 (L) Ethoxydihydroisomorellin G.morella 40 D ihydromorellln, Tetrahydro- G. more 11 a' morellin, Octahydromorellin he xahydromorellin 44 ,45 Morellin-L G.morella * 45 Morellin-M G.morella I, 44,45 Moi-ellin-T G.morella 37,49,50 Neomorellin G.morella 84 isomorellin G.morella : 174 :

~~OC^H^~~

~~37,48,51 ethanol CHO Me G.morella adduct of morellin 48 (Lib) Isomoreo'i Me CHO G.morella llin~~

~~(C) Benzophenones~~

~~OH CJ (LII) 1 9 (LID 2,4,6,3•,5'-penta- G.pundunculata hydroxy benzophenone '55 G.xanthochymus (maclurln)~~

~~Prenylated benzophenones~~

~~(LIII) 57 (LIII) Kolanone G.kola :: 175 :~~

~~(LIV)~~

~~14 15 (LIV) Xanthochymol G.xanthochymus * ^6,5 8- 60 Gc staudi^^ G.homobronlana^^~~

~~(LV)~~

~~14 15 (LV) isoxanthochymol G.xanthochymus * : llC :~~

~~(LVI)~~

~~(LVI) Cambogin G. cambogla'' '~~

~~Me »I R= CH„ - CH= C- CH„ - CH= CM e 2 2 2~~

~~61, G.homobroni ana .64~~

~~(LVIIb)~~

~~64 (LVllb) Bromianone(revised G.homobroni ana structure in light of structure of xanthochyraol) : 177 :~~

~~(D) Flavonoids~~

~~Monoflavonolds~~

~~HO 0~~

~~(LVIII)~~

~~5 8,65 (LVlii) Eriodictyol G.conrauana~~

~~(LIXa-c) R 1 (LIXa) Apigenin OH OH G.multlflora^°, G.buchanani G.eugeni foil a 66 67 (LIXb) Apigenin-5, OMe OMe G.kola 7,4-trimethyl ether 67 (Lixc) Apigenin-4 OH OMe Gokola methyl ether : 170 :~~

~~67 (LX) Fisetin Gokola~~

~~Biflavonoids~~

~~(LXIa-rr.)~~

~~R 8,5fi (LXIa) Manniflavone H OH OH OH OH G.mannl~~

~~(LXIb) Nonamethyl- Me OMe OMe OMe OMe G.manni 8 manniflavone~~

~~(LXIc) Octamethylma- Me OH OMe OMe OMe G.manni' nniflavone ! 194 :~~

~~3 (LXId) G B. H OH H OH H G. splcata , I " 5 G.thawaitesii ,~~

~~7 G»terpnophylla , 14 G•xanthochymus~~

~~G.mannll~~

~~G.multiflora^®~~

~~Go buchananil^^~~

~~G.eugenifolia^^ 9 G,heckel 3 (LXIe) G B^ ^^^ H H H OH H G. splcata ,G. terpno 7 phy11a , G.xanthochymus 14, G•punduncu 19 8 '^fl lata , G.mannli~~

~~G.rnultif lora 20 jG.kc^t ^ , , ..66,70 G. buchananij. ,~~

~~eugenlfolla^^,~~

~~• G.hecKel^ 7 (LXIf) G B H OH H OH OH G.terpnophy 11 a , 73 G.th aw alte si1^, 8 5 8 ^^ G.mannli ' ,G .kola.~~

~~^ . . 66,70 G.buchananii ,~~

~~G_.eugenifoli a^^, ' 9 Gcheckel ,~~

~~G o xanthochymus : 180 :~~

~~(LXIg) G B_ , V H H H OH OH G.-spicata^ ,G,m^nnij ^ C'~~

~~G.multiflora" ,~~

~~G.buchananll^^~~

~~G.eugenifolia^^,~~

~~G.dulcis'^''~~

~~8 (LXIh) Kolaf lavanone H OH H OMe OH Gomanii^, G.kola^'^* ^^ 9 G.heckel'~~

~~58 (LXi) H OH OH OH OH G.mannii~~

~~(LXIj) 1-4 ',11-4 ',1-5, H H H OH H G.thawaltesii^~~

~~11-5,1-7,11-7~~

~~Hexahydroxy-~~

~~(1-3,11-8)~~

~~biflavanone~~

~~(LXIk) 1-4 ',11-3H h" H OH OH Gothawaitesii^~~

~~11-4 ',1-5,11-5,~~

~~I-!7,11-7-~~

~~Keptahydroxy (1 - 3 ,~~

~~II-8) biflavanone~~

~~7 (LXIl) Fullymethylated Me OH H OMe OMe G.terpnophyl-1 a~~

~~33 (LXIm) Partially Me OH H OMe OMe Goterpnophylla~~

~~methylated : I8l :~~

~~OMe~~

~~H ^OMe MeO~~

~~(LXII) g (LXII) Chalcone G.mannii~~

~~,OMe~~

~~OMe~~

~~(LXIII) 71 (LXIII) Chalcone G.dulcis : 18:~~

~~(LXIVa-d)~~

~~32 (LXIVa) 0-Methylfukugetin H OMe G.quadrlfarla , 34 G.desnivenia 58 G .concrauana t~~

~~G.splcata^^''^^~~

~~(LXIVb) Morelloflavone H OH (fukugetin) 7 t'8 ^crhinoc arpa, indie a 14 15 GoXanthochymus ' , 32 f Goquadrifaria ,G.densi .34 5 8 venx a , G.conr auapa 2n Gomultiflora ^,G<.dulciS~~

~~(LXlvc) Volkensiflavone H H Goechinocarpa ,G.xantho ~ T:?~T5 ~ TR' chymus ' ,G. indie a. ; Gc spicata^^,G.iTulti~ flora^O" 71 (LXlvd) Volkensiflavone Me H G.dulcis hexamethylether : 183 :~~

~~1 £ TaIbotaf1avone G.pundunculata~~

~~5 ,7,4 • ,3'» ,5" ,7" ,4" 2. • a n t hochymus~~

~~Heptahydroxy(3,8")~~

~~biflavanone~~

~~15 5 ,7,4 ' GoXanthochymus~~

~~Heptahydroxy(3,8")~~

~~biflavanone~~

~~(LXV)~~

~~(LXV) Airientof 1 avone G.kola^"^, G.dulcis"'' 73 G.rrultif lora ! 194 :~~

~~(LXVI)~~

~~73 (LXVI) CXipressuflavone G.multiflora~~

~~h6 0~~

~~(LXVI I)~~

~~73 (LXVII) Agathisflavone G.multiflora 185 i~~

~~(i^iiia-b)~~

~~R (LXVlila) 1-41-5,11-5,1-7,11-7-penta- H Gcdulcis 71~~

~~hy d r o xy f 1 a V a no ne c hr om o ne~~

~~71 (LXViilb) 1-4',1-5,11-5,1-7,11-7-penta Me G.dulcis~~

~~me t hy 1 f 1 av a no ne c hr omo ne~~

~~1 8 Dimethylterepthalate G.indica 69 Kolaviron(mixture of bifla- G.kola vanone)~~

~~(LXIX)~~

~~74 (liXIX) FR-9C019 8 G.spicata : 1S6 ;~~

~~MISCELLANEOUS~~

~~I ^ H (LXX)~~

~~(LXX) Guainene G.mangostana'~~

~~(LXXI)~~

~~(LXXI) 5 ,7-Dihydroxychronione G.concrauana^®*^^~~

~~DMA=3,3-Dimethylallyl 6 .Ci^H^j group~~

~~(LXXIX a-e)~~

~~5 8,65 (LXXIIa) Conrauanalactone H OH H G.conrauana~~

~~58 (LXXIIb) 4 - Hydroxy-3 (3'S3CH - CH OH H G.conrauana DMA) - 6-pentadec- CMe. pyran-2-one OR 3-(3",3"-dimethyl allyl) : 18 7 :~~

~~(LXXIIc) 4-iiydroxy-5- (3- ,3"- H OH CK -CK G .conrauana^®~~

~~2 II — DMA)-6-pentadecpyran- ^^^ 2-one ^ (LXXIId) 4-Methoxy-3-(3",3"- CH - CH OMe H G.conrauana^® z „ _ DMA)-6-pentadec- ^^ pyran-2-onc ^~~

~~(LXXIIe) 4-Methoxy-5-(3" ,3''- H OMe CH^-CH G.conrauana^® It DMA) -6-pentadecpyran- CMe 2-one~~

~~(LXXIIIa-b)~~

~~C, (LXXIIIa) 3-Methoxy-3- (3" ,3"- CH - CH H G.conr^^na^ 8 A „ — • - DM -&-pent adecpyr an- ^ ^ CMe 4-one~~

~~(LXXIIb) 2-Methoxy-5- (3",3"- H CH - CH G.conrauana^^ 2 i» ~ DMA)-6-pentadecpyran- CM_ e 2-one ^ H~~

~~(LXXIV)~~

~~(LXXIV) 3-X2'-Hydroxy-5"-(hepta- G .mannii'"^ dec-S '-enyl)-tetrahydro- furan-2-one : lafi :~~

~~'lo'^n~~

~~(LXXV)~~

~~10 (LXXV) Quaesitol G.quaesita — Acetoxyoleanic acid G.quesita 1 0~~

~~MeO OMe~~

~~10 T (LXXVia) Hermonionic acid G.quaesita ' " S S "9~~

~~10,7S (LXXVlb) Decarboxylated G.quaesita product of Hermo- nionic acid~~

~~C^ Hg = -CH^- CH = Oie^~~

~~S 0 - CH^ - CH = aie (Cri^ ) - CH= CMe ^ : 1S9 !~~

~~R- / VoR~~

~~(I.XXVIIa-b) (R=H, AC,Me) R, R R"1. 58 (LXXVIIa) Fukugiside H /3-D-glc OH~~

~~G.conrauana ,~~

~~77 G.multiflor68a (LXXVllb) Spicatoside H p-D-qlo H G.spicata , 77 Gomultiflora~~

~~HO (LJ:XVIIIa-b)~~

~~63 (LXXVIIIa) Xanthochymuside /^-J-glc OH G o X a n thochym u s G .multif lor a"^"^ .90 :~~

~~(LXXVliib) 3,8"-Binaringe- f> -J-glc H Gomultiflora'^'^ nin-7"-0- f> - glucose~~

~~7 8 Cyanidin-3- G.lndlca mango "is glucoside tana~~

~~79 Cyanidin-3- G.mangostana sophoroside~~

~~Cyanidin-3- G.indica 78 sambubioside~~

~~79 Hydroxycitric acid G.indica , 80 and lactone of GoCambogia citric acid~~

~~Oleic, Palmitic, Gckola",~~

~~Palmitooleic, G .cochinensis 81 Linoleic and stearic acid~~

~~Myristic acid Gokola~~

~~Guttiferins~~

~~, , 49,50,51 , -Guttiferin G.morelJ^ ^82-34 , , 49 , 82 -Guttiferin Gomorella » 49 -Guttiferin Gomorella 82 ,84 -Guttiferinic acid G.morella 84 '^-Guttiferic acid G.morella s 191 :~~

~~,82- -Guttiferin('~~

~~y-Guttiferinic acid~~

~~^-Guttiferinic acid ,84 ,83, X-Guttiferin ,83, Y-Guttiferln G.morella^^'^®^~~

~~y,-Guttiferin G.morella'^^'®^ 83 72.-Guttiferin G.morella n 82 Y-Guttiferinic acid G.morella : 1S9 !~~

~~IDENTIFICATION O' ISOPRSNYL^TED Xa^NTHONES~~

~~The structure of x??nthones c^.n be determined v/ith -j the help of H 1^3:R , IR, UV and mass spectrosoopyo~~

~~H NMR spectroscopy ; ''h l^iR spectroscopy provides a clue for structure elucidation. H NMR of some important xanthones are given below in Table 1-7o 0~~

~~(XXlXa,e)~~

~~R^ ^2~~

~~(XXIXa) OH H (XXIXe) H OH~~

~~21 Table-1 : H OT-IR of isopreny1ated xanthones • Chemical shi ft ( S-scale) Assianrnent XXI Xa XXI Xe~~

~~1 c66(s,3H) 1 c53 (s,3H) Me protons of 1 „76(s,3H) 1 „73 (s,3H) isoprene lanit 3.2-3 o 6(d,2H,marked 3o2-3o4 (d,2H,marked -CH - by H^ 0- J:; SO sig na 1) by K;,0-DM30 signal)~~

~~3 »99(s,3H) 3.91(s,3H) OMe =) 2 (t , 1 H, J= 6 „ 5 HZ ) 5.^ 6(t,1H,J=6 = 5Hs) - CH= 6o75 (3,1 H) 6o1 7 (s,1K) H-4 7c35 (m,2H) - H-5,7~~

~~- 7.3-7c e(m,3H,ALC H-5,6 , S system)~~

~~7.65 ((5,1 H) - H-S 1 3»1 0 (s ,1 K) 1 3ol4 (s,1 H) 1 - OH~~

~~s = singlet, d = doublet, t = triplet, q = quartet, m = r.-iulti-pent , spectrum run in at SC VAlz , TM S •=s interriol st^ndard^ : 229:.0 :~~

~~KoO (XXXIIa-b,d)~~

~~(XXXld) Mangostin 2 (XXXIIa) ^-Mangostin H Me~~

~~(XXXIlb) •y-Mangostin Me Me (Normangostin) H H~~

~~Table-2 : H M^IR of i soprenylated xanthones^^.~~

~~Chemical shift (^'scale ) Assignraent b (XXI Xd) (XXXIIa) (XXXIlb)~~

~~1,68 (s) 1 c 64 (s) 1.68(3) Methyl protons of 1 o73(s) 1oS5 (d) 1 c77 (s) i soprene 1 o 80(s) 1c 68(s) unit~~

3,4C (d ,J=6K2) 3«91 (c^ J=6Hz ) 3c3P(C,> J=6H2) 4,17 (d , J=6K2 ) 4.00(d,J=6HZ) 4.22 (d J=6Hz) J 3 <. 84 (£ ) 3.74(s) 01 ie 3„9-i (s) / 5 »33 (t , J=6KS ) 5.20 (t, J=6K2 ) 6c43(s) 6c52 (s) 6c45 (s) H-4 f.83 (s) 6o 83 (s) C c 87 (s) 11-5

1 3o6C (s) 1 2 o7C (s) n.rr . CK 4 cCC (o) 5c3C(b) 3.95(b) s = singlet, d= doublet, t=triplot, b= breed , n.r,-. .= no iT>easurer,ent . spcctru; run ^ in at v^rien £0, Ti^S int«rn^l star.u;?rd. ! 194 :

~~(XXXIIIA-B)~~

~~R (XXXIIIa) Gartanin OH~~

~~OOCXIIIb) 8-Deoxy-g art ani n H Table-3 : ''^ ^^ isoprenylated xanthone"" •~~

~~Chemical shift ( seale) Assignment XXXIIIa XXXIIlb~~

~~1 o 80, 1 o70(s,s,l2H) 1o82 & 1 o66 (s,s,12H) Me Protons of isoprene unit~~

~~3 O 65-3O35(br,4K) 3. 60-3o40 (br ,4K)~~

~~5 o2 5 (br,2H) 5o24(br,2K) Vinylic orotons (-CH=)~~

~~6.52 (d,1 H,J=9Hz) H- 6~~

~~7 o22 (d,1H,J=9Hz) H-7~~

~~7.3G-7.C5 (rr. ,2H) H-6 S- K-7~~

~~7 c 5 S (q , 1 H) H- 8~~

~~9o?3(s,1H) So75(s,1H), 9.Co 3-OH ^nd 5-OK (s,1 H) 1 1 c33 (s,1H) £- CH~~

~~1 2o1 6 (s ,1H) 13.1 6 (s , 1 H) 1 - OK~~

~~s = singlet, d = doub.'.et, b = broad, rn ?? multiplGt, spectrum run in CJCl^ at 6C Mc, TMS -s internal standard 195~~

~~(XXXIV)~~

~~(XXXIV) 1 ,3,5-Trihydroxy- 4,8-di-(3 ',3 '-dimethyl allyl) Xanthone~~

~~1 32 Table-4 • H N>IR of isoprenylated xanthone~~

~~Chemical shift (S -scale) Assignment XXXIV~~

~~1 .63 (s,3H) Me proton of both isoprene 1 .72 (s , 6H) units. 1 o82 (s,3H)~~

~~3c59 (d,2H,J=1OHZ) H-1 ' 3 ' ,3 '-dimethyl 4,00 (d ,2H,J=10K2) H- 1 allyl 5 <,4 0 (t ,2r], J=1 GHZ ) unit~~

~~6.36 (s,1H) H-2~~

~~7.09, 7o23 (^xEq,2H, J=9'-:z:) K- 6 ? H~7j~~

~~'lOcBO and 9.31 3-OK & 5-OK~~

~~11o73 (s,i:-i) 1 - OK s = singlet, d = doublet, t = triplet, AEq « ABquartet, spectrum rnn in He2C0-d^. "t 90 rlHz , TI-IS as internal standard. : 196. :~~

~~0 QAc~~

~~AcO~~

~~(XXXVb)~~

~~(XXX\/b) Garcinone-A triacetate~~

~~Table-5 : H NMR of isoprenylated xanthone .~~

~~Chenical shift ( -scale) Assiannent X>CXVb~~

~~1 .60(s, 3H) Me protons o£ 1 .66 (s, 3H) both isoprene~~

~~1 o75 (S, 3H) units~~

~~1 o99(S, 3H) J~~

~~3 . 31 (d , 4H, J=6HZ ) -CK2 -~~

~~5oC6(t, 2H, J=6H2) - CH=~~

~~7 o2.5 - 7 O45 (IT ,2K) H-7 and H-5~~

~~8.15 (dd, J=9HZ) H- 8~~

~~s = singJet, d = doi: t ..-t , dd. = double doublet, t = triplet spectriOT run in CJCl^ 9C l-jHz , TM S as internal standard. : 197~~

~~(XXXVI)~~

~~(XXXVI) Garcinone-C~~

~~1 2 7 Table-6: " of isoprenylated xantbone .~~

~~Chenicp.l shift scale) Ji.ssignment XXXVI~~

~~1O20 (S, 6H) Methyl protons~~

~~1 O 62 (S , 3H) of isoprene and butane unit 1O72 (S, 3K)~~

~~3o27 (4H, masked by H2 O- Bensylic protons signal) of both the side chains.~~

~~5.18 (t,LH, J=6 = 5KZ) -CK=~~

~~6O33 (S,1H) H-4~~

~~60 74 (£,1H) n- -J~~

~~13O98(s,1H) 1 - OH~~

~~s = singlet, t = triplet, spectrum run in at 59.8 MHZ, TMS as internal standard,, : 18 7 :~~

~~(LXXIX)~~

~~(LXXIX) Celebixanthone~~

~~Table-7 : ''^ NMR of isopreny 1 ated xanthone®^,~~

~~Chemical shift ( -scale) Assiqnmenfc LXXIX~~

~~1 o 67 (S , 3H) Me Proton of 2.83 (s, 3K) isoprene unit~~

~~3c97 (d,2H,J=6Kz) —CH^ proton~~

~~3c7 6 CMe~~

~~5o20 (t,1H,J=6Hz) -CH=~~

~~r-o41 (t,1H,J=8Hz)~~

~~6o51 (d,1H,J=SK2) H-5, H-G & H-7 •~~

~~6o75 (t ,1 H, J=SHZ )_~~

~~11.2 8 (v.iro?d, 2M) 3-CH, 4-OH~~

~~13o'i6 {s'narp, 1H) o - CH , s t r o ng 1 y hy i r oi^ e n bonded~~

~~s = singlet, d = doublet? t = triplet, spectrur, run in acetone A-L 66 l>c, TM5 R-^s iriter;?! standard. ! 199 :~~

~~The IR , UV and mass spectroscopy of isopreny1ated xanthones are given below(Table 8-13).~~

~~Table - 8~~

~~IR, UV & MS data of isoprenylated xanthones~~

~~21 21 (XXIXa) (XXIXe)~~

~~•^Nujol IR 3350 (chelated OH) 3250 (chelated OH) max 1 650 ( T^-pyrone , 1 64 2 ( 7^-pyrone ,~~

~~^OO). >C=0)o~~

~~y^^EtOH nni(iQg^) 245 (4 ^53), 256 240(4,10), 265 (4„32), •Am ax (4o48), 312 (4.21 ), 309 (4 .15), 378(3o90).~~

~~375 SH(3c51.)O~~

~~Mass m/2 326 (27 , M ) 326(20, M )~~

~~31 1 (25 ,M'*'-1 5) 31 1 (1 8,m'^-1 5)~~

~~283 (63,m'^-43) 283 (50,m'^-43)~~

~~271 (100,M"^-55) 271 (100,M'^-55)~~

~~253(6)~~

~~241 (7) t 200 !~~

~~Table - 9~~

~~IR, UV & MS data of isoprenylated xanthones~~

~~25 25 (XXXid?' (xxxila) (XXXIIb)~~

~~^^ ,, Nujol -1 IR )J cm 1 625 ,1595,1450 1 645 ,1 598, 1 3 60,1 610, •I max 1 4 60 ,1 4 3 3 1 5 80,14 50~~

~~212 (4c42) , 247 (4 ,43) ,261 imax 244 (4 .53) , (4.47), 314~~

~~259(4.47) , (3.34),3.64~~

~~315 (4 .36) , (3.90)~~

~~34 9 (3.97)~~

~~(bathochromic shift v;ith~~

~~NaOAC+NaOH)~~

~~mass m/z 410 (M ) , (XXXIla- 396(N/) acetate ) 393 4 66(M'^) ,~~

~~367 423~~

~~355 410~~

~~354 395~~

~~339 381 2o:~~

~~Table-10~~

~~IR , UV & MS data of isoprenylated xanthones~~

~~(XXXIIIa)^^ (XXXIIIb)^^~~

~~IR^ 3420,3200,1 630, 3540,3190,1 650, max 1585,810 1 620,1380,850~~

~~EtOH 259,284,325(SH), 244,2 60,324,375 m^ max 351 4„30, (log £ , 4.47,4 .37,~~

~~4.38,3.87,4»05) 4.17, 3o55)~~

~~. EtOH-NaOAC 240,284 ,380 m/^ max (log ^ , 4 „48,4 ,4C ,~~

~~4 .34)~~

~~EtOH-AlCl^ 269,299,330(SH), A max 3.88 m^(log6 ,4,36,~~

~~4 c42,3.94 ,4.07)~~

~~Mass m/z 396 (M*^) 380 (M*^)~~

~~3 81 3 65~~

~~379 363~~

~~353 337~~

~~341 (m'^-55 ) 325~~

~~325 309~~

~~297 281 (100)~~

~~285 (100,341 -56)~~

~~In the mass spectrum of (XXXIlia)^®, the major fragments at 341 (k"*"-55) , 285 (100, 341-56) appear due to cleavage of two prenyl groups. : 202 :~~

~~Table - 11~~

~~IR, UV & MS data of isoprenylated xanthones~~

~~(XXXIV~~

~~IrV^^^ cm""' 3380, 2920, 1 635, 1580, 1410 max~~

~~nm(log^ ) 251(4,43), 2 60 SH(4„39), 273 Sh(4.3i), ""/I max 330(4.10)( 4 AlClg) 258, 273 Sh, 295,~~

~~258.~~

~~Mass m/z 380(59, M*^)~~

~~337(100, M"^-4 3) (M'^-CgH^)~~

~~.325 (12 , M'^-55) (M'^-C^H^)~~

~~2 69(17, M'^-111) o I D~~

~~32 In the mass spectrmi of (XXXIV) ) the fragments at~~

~~(m''"-55) and (m"^-!!!) are due to the presence o£ two~~

~~noncyclic prenyl units linked directly to the xanthone. ! 205~~

~~Table - 12~~

~~IR , UV & MS data of isoprenylated xanthones~~

~~(XXXVfe?~~

~~IRy^^J^^ cm"'' 3380 (chelated OH), 1 635 ( T^pyrone , y 1ma x >C=0>~~

~~y^\EtOH nm(iog^) 245 (4 o5), 2 60(4 .0), 2 89(4 .0), 323 max (4,1 ) 370 (3o5)~~

~~,^EtOH-Na^c 244 (4.4 ), 295 (3.9), 326(4,0), 369 /imax ^ ' (4o3)~~

~~Mass m/z 380 (25 , m'*")~~

~~337 (5, m'^-4 3)~~

~~324 (7, m'^-5 6)~~

~~323 (5)~~

~~311 (63, M"^-69)~~

~~295 (1 7)~~

~~283 (5)~~

~~269 (1 6)~~

~~257 (100) : 204 :~~

~~Table - 11~~

~~IR, UV & MS data of isoprenylated xanthones~~

~~(XXXVI~~

~~3550(OH), 3440 and 3150(chelated OH) max 1 640( >C=0)~~

~~\ EtOH , ^ ^ . UV \ nm (log fc ) 243 (4o5) , 260 (4 „4) , 370 (4 o3) /\ max~~

~~Mass m/z 414 (10, M"^)~~

~~396(80, M'^-18)~~

~~381 (15, M'*'-1 8-1 5 )~~

~~379(30)~~

~~353(70, M'^-51 )~~

~~341 (100, M"^-! S-55)~~

~~340(50, M'^-18-56)~~

~~325(90)~~

~~299(30)~~

~~297 (92 )~~

~~285 (52)~~

~~In the IR Spectrum the ^C=0 Str. frequency for pyrone appears in the region 1 610-1650 cm" o O05 '~~

~~TERPENOIDS ANP THE IP IDE NTI J C r.T I ON~~

~~Terpenoids are all based on the isoprene molecule~~

CH2=C(CH2)- CHsCH^ and their carbon skeleton are built up from the union of two or more of these C^ units. in this chapter, we have discussed isolation and characterization of some triterpenes. Therefore, a brief introduction of triterpenoids, natural compounds with a carbon skeleton based on six isoprene unit is given.

~~First comprehensive survey of important advances on naturally occurring tetracyclic triterpene was made by 86 G. Ourisson, P. Grabbe and 0. Rodig in early sixties .~~

~~The classification of different .types of tetra- and penta- cyclic triterpenes is described by J.B. Harborne®"^, while their reaction and biosynthesis is given in the book by~~

~~Pridhan®®.~~

~~All types of triterpenoias are separated by very similar procedures, mainly by TLC and GLC. Identities are confirmed by melting point, rotation, GC-MS, IR and 87 KMR spectroscopy : 18 7 :~~

~~The S2DectrRl datp of some repr'v r .-rt ~rive triterpGro::ds p.rc civt-r; fcelov; in Tables 14-1".~~

~~(LXXX)~~

~~.'H~~

~~(LXXX) Cyclolsudenol \ OH~~

~~89 Table-14: ''^ cyclclaudenol~~

~~Chernicp.l shift -scale) Assionment LXXX~~

~~0O4 8 & C.47 (AEquartet, 2H, J=5HZ) Cyclopropane ring~~

~~CO 82 - 1 . 65 (21 H) 7xMe~~

~~4o7 (s,2K) >C =-CH,~~

~~s = si.'iclet; spuctrurn run in CJCL^, TMS as internal stand arc. : 18 7 :~~

~~(LXXXI)~~

~~H R = (LXi'CXI) 3-Epilaudenol'~~

~~1 89 TaLl6-15: H M-IR of 3-Epi laudenol~~

~~Chemical shift ( (S^-scale) Assignment~~

~~..XXXI~~

~~Co4 3 & 0.47 (AB quartet, 2H,J=5Hz) Cycloprop a ne ring~~

~~Oo 87 - 1 . 65 (21 H) 7xrie~~

~~4.7 (s,2K) —-2~~

~~s = singlet, spectrun run in CDCI3, TIIS as internal StcHu =ru . :: 207 !~~

~~(LXXXI)~~

~~H R = (LXXXI) 3-Epilaudenol'~~

~~Tabl6-15: ''H :.3-;R of 3-Epi laudenol ^^ .~~

~~Chemical shift ( scale) Assignment~~

~~LXXXI~~

~~Co4S & 0.4 7 (AB quartet, 2H,J=5Hz) . Cyc loprop ne ring~~

~~Co 87 - 1 . 65 (21 H) 7:~~

~~4o7 (s,2H)~~

~~s = singlet, spectrum run in CJOl^, Ty'iS as internal standard. ! 245 :~~

~~(XI)~~

~~H ( XI ) Cycloartenol \ OH Table-IG: H M^'IR of Cycloartenol®^.~~

~~Chemical shift ((^'-scale) Assio nraent XI~~

~~0o4 3 & Go 65 (ABquartet 2H, J=5Hz) Cyc loprop pne ri net~~

~~CO 37 - 1 O 6 (21 H) / x;: e~~

~~Spectrum run in CJCl^ • Tl.'S interr.;=l stanu^ra, : 18 7 :~~

~~(VIb)~~

~~(VIb) Friedelan-3/S , 28 diol R^ = oC-H, p-CK, R2 = CH20H~~

~~Table-17: ''H mm of Friedelan-S^ y 28, diol^.~~

~~Chemical shift (^-scale) Assig nnient VIb~~

~~Oc85 (s, 3H) He~~

~~Go 96 (s, 6H) Me~~

~~1.06 (s, 6K) ke~~

~~1c10 (s, 3H) Me~~

~~2 o 0 8 (rn , 1 H)~~

~~3 <,50 (br.s,2H) CH^OK~~

~~s= singlet, br= bro-^^. m= nf'.uItiplet , spoctrun run,_in CDCl, at 60 MH2 , TilS =s intern.-=l st^nd^rdo IR and mass spectra of sone important triterpenoid~~

~~are given belovj(Table 16-19).~~

~~89 Table-ISj IR and MS data of Triterpenes~~

~~89 89 (LXXX) (LXXXI)~~

~~IR^ Nujol^^-1 3450(OH,3050(cyclo- 3430 (OH), 1 370, 1 360 max propane ring), 1370, (geminal dimethyl) 1360 (gemlnal di- 3050 (cyclopropane methyl), 880(termi- ring ) nal methylene group)~~

~~Mass m/z 440 (85, M*) 440 (85 , M^)~~

~~425 (42, M"^-Me) 425 (42, M'^-Me)~~

~~422 (86, M"^- H^O) 422 (86, M'^-K20)~~

~~407 (56) 407 (56)~~

~~379 (24) 379 (24)~~

~~353 (27) 353 (27)~~

~~315 (22) 31 5 (22)~~

~~300(100) 300(100)~~

~~175 (76) 175 (76) : 18 7 :~~

~~Table-19: IR and MS data of Triterpenes.~~

~~89 (VIb) (XI. )~~

~~,. KBr -1 IR^j cm 3400(OH, 1375, 1360 3555, 2592, 1380 max (geminal dimethyl), 1120.~~

~~3040 (cyclopropane~~

~~ri ng) .~~

~~Mass m/2 426 (55 , m"^) 427 (100, m"^)~~

~~411 (53, M'*'-Me) 410 (41)~~

~~408 (72) 383 (36)~~

~~393 (60) 2 89 (64 )~~

~~365 (24) 272 (1 8)~~

~~339 (25) 245 (86)~~

~~315 (9) 227 (22)~~

~~286 (57)~~

~~1 75 (44) Discussion : 18 7 :~~

~~Chemical Constituents of Garcinia liapo^^tana Linn,~~

A number of Garcinia species have been studied 2-12 IS-""^ extensively for the presence of terpenoids ,xanthones, 25—54 141519 56-64 polyisoprenylated xanthonoids , benzophenones * ' ' , .. 3,5 ,7-D,14 ,19,20,58,65-74 and flavonoids

Isolation and characterization of several xanthones have been reported from heartwood and fruit of Garcinia 21 24-31 33 35 mangostana ' ^^ ^ ^^^ present discussion deals with the isolation and characterization of xanthones and triterpenes from the leaves of Garcinia mangostana Linn.

~~The dried leaves of G. mangostana(4 kg) were extracted with petrol, and then with ethanol.~~

The petrol extract was concentrated and purified by column chromatography on silica gel. Column was eluted with solvents with increasing polarity. Elution with petrol gave simple hydrocarbons which were not further investigated. Elution with petrol-benzene (1 :1) gave GM-1 89 which was characterized as cycloartenol(XI) • Further elution with petrol-benzene(1:1•3) o0yielde 0-] d GM-2 which was characterized as friedJelji (Via) " '" .The elution of the column v/ith benzene gave GK-3 v;hich was comparable v;ith /S- sitosterol and was not investigated further. Elution with benzene-ethyl acetate (9 :1 ) gave GM--^ and its characterization is still under investigation. Further elution with I, 215 :

~~benzene-ethyl acetate (9 :1) yielded GM-5, which was characterized as 2 5-dehydro-3/S , 24 6 -dihydroxycycloartane~~

~~(LXXXII)^^The recrystallization of the mother liquor~~

~~•of GM-5 yielded CM-6, characterized as betulin (IJXXXIII )~~

~~On further elution of coliimn with benzene-ethylacetate (9 j1 )-~~

~~yielded GM-7 which was characterized as 9,19-cyclolanost-24- 95 en-3, 2 6-diol (LXXXIV) , which has been isolated for the~~

~~95 first time by Anjaneyulu et al • Further elution with~~

~~benzene-ethyl acetate(8:2) yielded GM-8 after removing a~~

~~minor impurity by preparative thin layer chromatography~~

~~(silica gel, benzene-acetone, 8:2). It was characterized~~

~~as mangiferolic acid (LXXXV)^^'^*^.~~

~~Ethanol extract was concentrated and treated with~~

~~benzene. Benzene soluble fraction was adsorbed on silica~~

~~gel and subjected to column chromatography. Initial~~

~~elution with benzene yielded GM-9 and GM-lO which were~~

~~comparable with ^-sitosterol on TLC(silica gel, benzene-~~

~~chloroform, 1 ) but had different melting points. GM-9 and GM-10 are under investigation. Further elution with~~

~~benzene yielded GM-11, which was characterized as gartanin 29~~

~~(XXXIIIa) . Elution of column with benaene-ethyl acetate~~

~~(9:1) gave GM-12 which was found be a mixture of two -~~

~~compounds, GM-12 was crystallized from benzene-petrol to~~

~~yield a new compound, GM-12A which was characterized as~~

~~1 ,5 , 8-trihydroxy-3-methoxy-2 1^3-methyl buteny^xanthone~~

~~(tXXXVl). From the mother liquor GM-12 B could not be~~

~~Isolated in pure state. Therefore, mother liquor was acetylated and the acet?^tes of GM12 A & GM12 B were~~

~~separated by PTLC. On the basis of the ''H NMR of GM12 B-~~

~~acetate, it has been tentatively identified as a new xanthone, 1 , 6-dihydroxy-3-methoxy-2£3-inethyl bute ny]J xanthone(LXXXVII) .~~

~~Cycloart^nolj GM-1 )~~

~~GM-1 crystallized from chloroform-cthanol as white crystals(i50 mg), mp VS-e'^c:, analysed for C^qH^^O (M^ 426),~~

~~Rf 0.41 ^ (silica gel, petrol-benzene 1 :1 ). It was characterized as cycloartenol by comparison of its NMR 89 (Table-20), IR and mass spectra with authentic sample~~

~~H 0~~

~~(XI) Cycloartenol~~

~~Table-2C: Chemical shift values ( S"-scale) of protons of Cycloartenol(XI)~~

~~NMR signals Niimber of J value Assignment of GM-1 protons in Hz~~

~~0.32 Cyclopropane ring (ABq) 0.56~~

~~0,80 - 1.68 21 7 x Me~~

~~3.20 - 3.40(q) 1 6.5 -OH~~

~~5.10 (br, t) 6 = CH~~

~~q=quartet, ABq= ABquartet , br,t - broad trivet spectrum run in CDCl^ atlOO, MHz ,TMS as internal standard. : 18 7 :~~

~~Fried lein (Ca^l-Z )~~

01-1-2 was crystallized from chloroform-acetone as white needle shaped crystals(100 mg) m.p.2 63- It analysed for C3QH5QO (m"^ 406) [Rf 0.73 (silica-gel, petrol- benzeno, 1 :1 )J o It was characterized as friedlein (Via) by comparison of its ""h NMR (Table-21 ) , IR and mass spectra 3,90,91 with authentic sample

~~(Via) Friedlein GM-3~~

~~GM-3 was crystallized from chloroform-ethanol as white crystalline solid(40 mg ) m .p . 140-1 |^Rf 0.32, silica-gel, benzene-chloroform(1and was comparable v;ith ^-sitosterol.~~

~~GM-4 was crystallized from chloroform-methanol as white crystals(lO mg) m.p.l4i°C^Rf 0.70, silica-gel, benzene-acetone (8o Its characterization is under progress. : 21:.0 :~~

~~Table-21 : Chemical shift values( scale) of protons of friedlein(Vla)?°~~

~~NMR Signals Number of J value Assignment of GM-2 protons in HZ~~

~~0.70 3 - Me~~

~~0.84 (s) 3 - Me~~

~~0.85 (s) 3 - Me~~

~~0.92 (s) 3 - Me~~

~~0.98 (s) 6 - 2xMe~~

~~1 .02 (s) 3 - Me~~

~~1.16 (s) 3 - Me~~

~~1.26''~~

~~1 .36 22 - CH^ protons 1 o42~~

~~1 o52_^~~

~~2o20 - 2.34 (m) 3 - C^-C}^ and C^-CH~~

~~s = singlet, m = multiplet, spectrum run in CDCI3 at~~

~~100 MHz, TMS as internal standard. . OTJ . . c ) .~~

~~25-dehydro-3 , 24 £-dlhydroxycycloartane (GM-5)~~

~~GM-5 was crystallized from ethyl acetate as white~~

~~amorphous powder (30 mg) , m.p. ^Rf 0.69 (silica gel,~~

~~benzene-acetone, (8:2 ) and analysed for C3QH5QO2 (M^ ) .~~

~~Its IR spectrum showed*]) at 3360 (OH group), 3030 max (cyclopropanering) , 1 640 (C=C), 1450, 1 3 65 (gem dimethyl) 1 89 and 885 cm" ( .~~

~~""H NMR spectrum (Table-22 , Fig. 1 ) shows ^B~~

~~quartet (J=5H2) at 6^0.32 and 0.54 for the methylene protons~~

~~of the cyclopropane ring, a quartet (J=6Hz and 9Hz) at ^3.26~~

~~for methine proton at C-3 & a broad triplet(J=6Hz) at ^4.00~~

~~for a proton attached to a secondary carbon bonded to OH group. A doublet(J=1OHz) at ^4.85 attributable to olefinic protons indicate the presence =CH2 group.~~

~~MS (Chart-1 ) shows moleculax ion at m/z 442 and base peak~~

~~at m/z 43. The diagnostic fragment ions at m/z 315~~

~~(H-CgH^^O), 313(M-side chain-2H), 302 (M-140) and 297 (31 5-~~

~~H2O) support the structure LXXXII for GM-5. Therefore,~~

GM-5 was characterized as 25-dehydro-3^ , 2-56 -dihyiroxy- cycloartane (LXXXII) . Its isomer with OH group ;=t C-24 has different melting point and spectral data^^' ^^and its 9 acetate has been assigned 24»C configuration . Therefore,

~~24 p configuration may be speculated for GM-5. Further studies are in progress to confirm the stereochemistry of C-24, I o~~

~~LU O~~

~~\ !~~

~~I I~~

~~. I- 'til c?> I I 1 1 I : 18 7 :~~

~~(KXXII)~~

~~- e~~

~~n/z 427(5) ^^^ ^~~

~~II , n/z 442(6.25) n/s 515(6.25) - K2O - H2O n/2 424(12.5)~~

~~> n/2 177 (15.75)~~

~~502 ;:i5.7:':) GG f rr.:;neatr.tio-a pr-t'.ern, of GM-5 . ! 219- I~~

~~Table-22: Chernical shift vplves( S^-sc^lo) of protons oi 23-AEHYARO-3/3 , 246 -dihydroxi^cycioairtane (LX:'C~~

~~NMR Signal Niomber of J values Assignment of GM-5 protons ie HZ~~

~~Oo32 (ABq) 2 5 Cyclopropane 0.54. j~~

~~0.7S (s)" 0c85 (S) 1 5 - b X Me 0.92 (si~~

~~1 .68 3 - CH3 - c = C~~

~~3.2 6 (q) 1 \\0 H~~

~~4o0 (br,t) 1 6 c/" \0H~~

~~4 0 85 (d) 2 10 = CH^~~

~~q = quartet, ABq = ABquartet, d = doublet, brt • broad triplet, spectrum run in CDC 13 at 100 MHZ , ms as internal standard.~~

~~Betulin (GM-6)~~

~~GM-6, on crystallization with chloroform-methanol~~

afforded white crystals(15 mg) , M.P.227-S°C, j^Rf 0.69 benzeno-acetone |[8:2 )J analysed for C^qH^qO (M*^ 2 ) . It was characterized as betuli n ("LXXXI11) on comparison of its H jNT-iP (Table-23), IR and mass spectra with authentic 94 sample : 18 7 :

~~Table-23: Chemical shift values ( S-scale) of protons 94 of betulin(LXXXIII)~~

~~NMR Signal Number of J value Assignment of GM-5 proton in Hz~~

~~0.75 (s)~~

~~18 - Methyl groups 0.91 (s) 1.0 (s)~~

~~1.20-1,65 - CH2 protons~~

~~1.63 (s) 3 - .~~

~~3.20 (q) 1 9,6 3, ^CH-OH~~

~~3o30 - CH2OH~~

~~4.61 (d) 2 10 HZ = CH 2~~

~~s=singlet, t=triplet, spectrum run in CDCl^ at 100 MHz, TMS as internal standard^~~

~~9 ,1 9-Cyclolanost-24~en-3 , 26-Diol (GM-7)~~

~~GM-7 was crystallized from chloroform-methanol as white needles(25 mg), m.p. i44-45°> Rf 0.68(silica gel, benzene-acetone, (8:2) . It analysed for C3QH5QO2 (M"*" 442)~~

~~and showed the presence of bands in the IR spectrum at~~

~~3365 (OH group) and 2960 cm""" (cyclopropane ring). In the~~

~~^H NMR spectrum(Table-24), two doublets integrating for one proton each at ^0.33 and 0.5 8 were characteristic to methylene protons in the cyclopropane ring. : ci^l~~

~~(LXXXII)~~

~~(liXXXII) 25-dehydro-3/S-24£ -dihydroxycycloartane,~~

~~^CHzOH~~

~~(LXXXIII)~~

~~(LXXXIII) Betulin ! 222 :~~

~~Singlets So. 30, 0.90 snd 0.96 integrating for 15 protons were assigned to five tertiary methyl groups while~~

~~a broad singlet at ^1.66 integrating for 3 protons is~~

~~attributable to the methyl group attached to the carbon-~~

~~carbon double bond. a triplet at 5*3.2 was assigned to~~

~~methine proton at C-3 and a quartet at ^5,45 was assigned to vinylic proton at C-24 of the cycloartane skeleton.~~

~~The methylene protons at C-26 appeared at ^4.00 as a~~

~~broad singlet.~~

~~In mass spectrum (chart-2), molecular ion appeared at.n?/z442~~

~~and the base peak appeared at m/z 28 . The appearance of the diagnostic peaks at m/z 42 7 (M-1 5), 424 (m-18), 315 (M-~~

~~side chain), 313(M-sidechain-2H), 302(M-140) and 297(315-~~

~~H„0) support structure LXXXIV for GM-7. Therefore, GM-7 ^ 95- was characterized as 9,19-cyclolanost-24-en-3, 26-diol(LXXXIV}~~

~~CH2OH~~

~~(LXXXIV) 9 ,1 9-Cyclolpnost-24-en-3 ,26-did : 18 7 :~~

~~CH2OH~~

~~(LXXXIT)~~

-e

~~,ch2 oh~~

~~r./2 427 (1?) r./z 424 (55)~~

~~Chrrt-2. frc.^^e^.t^.^^ion pri-he::n. of GM-7. : 224 :~~

~~Table-24 • Cheraical shift values ( S-scale) of protons of 9,19-cyclolanost-24-en-3, 26-diol(LXXXIV) 9 5~~

~~NMR Signals Number of J values Assignment of GM-7 protons in Hz~~

~~0o33" (ABq) CU^ in cyclopropane 0o58, ring~~

~~0.80 (s) 3~~

~~0.90 (s) 3 Five tertiary methyl groups 0.96 (s)~~

~~1.20 - 1.51 CH2 protons~~

~~1.66 (s) Me-CH=CH2~~

~~3.25 (q) 1 9,5 CHOH~~

~~4.00 (s) 2 q^OH~~

~~5o45 (t) = CH d = doublet, s= singlet, q = quartet. Spectrum run in CDClg at 60 MHz, TMS used as internal standardo~~

~~Mangiferolic acid (GM-8)~~

CM-8, crystallized fi'om chloroform-methanol as white solid(80 mg), m.p.159-61°C and analysed for C3QH42O3 (m"^ 451) [nf 0.4 8 (silica gel, benz ene-acetone (3:2)J , It was characterized as mangiferolic acid(LXXXV) by comparison of its ''H (Table-25 ) IR and mass spectra with authentic , 96,97 sample : 225

~~COOH~~

~~(LXXXV)~~

~~Table-25 : Chemical shift values(5-scale) of proton of mangiferolic acid ( LXXXV)~~

~~NMR Signal Number of J values Assignment XDrotons in H2~~

~~0.30' (ABq) Cyclopropane CH2 0.54,~~

~~0o78 (s) 3 Me~~

~~0c94 (s) 3 Me~~

~~1 .1 -1 . 84 CH2 protons~~

~~1 . 84 (s ) 3 Me-~~

~~3.26 (q) 1 9,6 3 CHOH~~

~~6o92 (t,br) 7 H d = doublet, s= singlet, q = quartet, t,br = broad triplet, spectrum run in CDCI3 at 100 MHz. TMS as internal standard. : 18 7 :~~

~~GM- 9~~

~~GM-9, on crystallization from chloroform-ethanol~~

~~yielded white needle shaped crystals (SO mg), m.p.150-1°C~~

~~I^Rf 0. 32 (silica-gel, benzene-chlorof orm, (1 :1 comparable~~

~~with -sitosterol on TLC~~

~~GM-1 0~~

~~GM-10, on crystallization with chloroform-ethanol~~

~~afforded brown crystals (40 mg) , m.p . 147-51 °C) j^Rf 0.32,~~

~~silica-gel, benzene-chloroform, (1 comparable with ^ -~~

~~sitosterol on TDCr.~~

~~Since GM-9 and GM-10 have different, melting points~~

~~and different nature from sitosterol, therefore, their~~

~~characterization is under investigation.~~

~~Gartanin (GM-11)~~

~~GM-11, on cryst;=^llization with benzene-petrol~~

~~yielded yellow crystalsd gm), m.p. 163-5°C^Rf 0o38, silica-~~

~~gel, benzene-chloroform, (1 :1)J and analysed for~~

~~(M"*" 396). It v/as characterized as Gartanin by comparing~~

~~its H M>IR (Table-26,27)1 R and mass spectra with authentic~~

~~sample 2 9~~

~~R R 1 (XXXIIla) H H~~

~~(xxxiiic) o^c H : 18 7 :~~

~~Table-2 6: Chemical shift ( S*-scale) of protons of 99 Gartanin (x::::iITr )~~

~~NMR Signals Number of J values Assignments of GM-11 protons in Hz~~

~~1 .75 (s) 6 - 4xMe protons 1 c81 (S) 6~~

~~3.50 (br) 4 - 2XCH2~~

~~5 .23 (br) 2 - 2 X = CH -~~

~~606O (d) 1 8. 8 H-6~~

~~7o24 (d) 1 8. 8 H-7~~

~~9„51 (br) 2 - 2xOH~~

~~11.19 (br) 1 - OH~~

~~1 2<,20 (br) 1 - OH~~

~~d = doublet, br = broad, run in DMSO-d^, at Spectrum Q~~

~~12c MHZ, TMS as internal st andard • : 267:.0 :~~

~~Table-27: Chep.ical shift value ( (5*-scale) of protons of~~

~~1-hydroxy-3,5 , 8-tri qcetoxy-2)4-ai /s ,3 '-dimethyl-~~

~~allylj xanthone(GM11 ^c, XXXIIIc),~~

~~NMR Signals Number of J values ^ssignne of GM-1 1 AC protons in HZ~~

~~1 o70 (s) 12 - 4 X Me~~

~~2.47"~~

~~2o4 8 '(s) 9 - O^c~~

~~2.5 6^~~

~~3.32 (br) 4 - 2 xyTCH^~~

~~5o1 7 (br) 2 - 2 X =CH~~

~~6.90 (d) 1 So 8 H- 6~~

~~7.42 (d) 1 8. 8 H- 7~~

~~s = singlet, d = doublet, br = bro;=d, spectrum run in~~

~~CDClg at 60 MHZ, TMS as internal st-nd^rd. ! 229 :~~

~~GLI-'. 2 (GM,1 „ ^ RND GI:. I„ 2- ^ :~~

~~The crystallization of GM-12 from benzene-petrol yielded yellow amorphous powder(90 mg) of mopo193-5°C,~~

~~Rf Oo25 (silica gel, benzene-chloroform, 1:1) and Rf 0.44~~

~~(silica gel, benzene-ethylacet ate , 9:1). gave green colour with alcoholic ferric chloride and its ethanolic~~

~~solution g a\ e red colour with p-benzoCquinone (- gossypetone~~

~~9.8 reaction)"" 3hcv.-inc the presence of ^uinol moity. It~~

~~analysed for C^ ^H^ gO^ (M342) and in the infra_.red spectrum,~~

~~3400 (phenolic OH ), 1775 (carbonyl group), 1 650 and 1600 cm"'ma' x (aromatic system) v/ere noted ,~~

~~The ^H KFiR spectrum of CM^ 2 ^ (Table-2e, ?ig . 2 ) showed singlets at and 1.72. e-^ch intogr^tirg for three ore.tons for ger.inal methyl croups) a bro?d signal~~

S3O25 for r..ethy Ic.r_-rouors, a singlet at(^3.92 foi* the three methoxy protcais C: a bropc triplet inteigrating for one prcron at^S.GS for ciefinjc prcron. A pair of ortho couple-. dGuh:etE(J = S. 8 Hz) Pt &C.5S and 7.24 v/ere attributable to

~~I--6 and H-7 of the xe-xhone nucleus. ^ sir-,glet at 6* 60 64 integrating -^or one _rcton --ssicred to by comparison~~

~~\:ith other x-rtho:'? ~ ' " unsvbstit u ed at position~~

~~Hydroxy protons sho\/ei broad sicn=?ls pt 5'''0.91 , 11c02 ^nd~~

~~11.03 o~~

~~: 2:.0 :~~

~~Table-2 S: Chemical shift value ( g-scale) of pre.tons of~~

~~1 ,5,8-trihydroxy-3-nethoxy-2 ^3-methyIbuteny~~

~~xanthone(LXXXVI).~~

~~NMR Signals Number of J values A.ssig nme nt GM-12 A protons in Hz~~

~~1 .6 (s) -] 3 Me protons~~

~~1 .72 3 - cJ 3.25 (br) 2 CH^~~

~~3o92 (s) 3 OMe~~

~~5c0 8 (br)_ 1 = CH~~

~~6o58 (s) H-6~~

~~6. 64 (£) 1 H-4~~

~~7.24 (s) 1 H- 7~~

~~10.91 (br) 1 5 - OH~~

~~1 1 .02 (br) 1 1 - OK~~

~~11.03 (br) 1 8- OH~~

~~s = singlet, d = doublets, br = broad, spectrum run in~~

~~DMSO-dg at Sc MKZ, TKS as internal standard. •C\J~~

~~h ro~~

~~< cJ I Z o o — LO~~

~~CO I CJ3 r~~

~~-7 r-- : 18 7 :~~

~~- CK- ^ 527 (61.1) LXXXYIa • " ^ • » •OMe OH U"^,' m/z 542(85.3) - isor)reiie-2H~~

~~- 55 n/z 271 (50.9)~~

~~OH 0 QH~~

~~OH EI/z 28 7 (100)~~

~~^n/s 271(5 0.9)~~

~~E/S 29? (90.?)~~

~~Chc:rt-5. I'S -rrrC"-er.tr tion of 1, 5 »£ -trihrdr o::y_5-no th o::y-2/~5-:-.o thyl-~~

~~2-ou.tcr.yiy::r.n tl. one . : 2:.0 :~~

~~:.rj-29 : Ch'f ical sp.ift v P. : ae s ( ^ - sc ^ 1 - ) of pro-cons~~

~~Qf 1 - pyrirc;cy- , 8-uj ^cetorc^'-o-r c ht~~

~~3-methyl-2-buteny^ xanthone( LXXX"/) .~~

~~H NMR Signals Number of J values Assignment of GM-, ^^ protons IN HZ~~

~~1 c 69 (s) 3 - lie protons~~

~~1 o75 (S) 3 - Me Protons~~

~~2.^1 (S) 3 - OAC~~

~~2 .4 5 (S) 3 - OAC~~

~~3o30 (br) 2 - CH2~~

~~3,93 (s) 3 - OMe~~

~~T o1 0 (br,t) 1 - = CH~~

~~6c33 (s) 1 - H-4~~

~~6.97 (d) 1 8. B H- 6~~

~~7c43 (d) 1 So 8 H-7~~

~~s = singlet, doufolet, br = broad, spectrur run in CJCI3 at , Ti>;3 =.s internal standard. :• 2;.4 :~~

~~The mother liquor of QH^^K was acetylated and~~

~~examined on TLC(silica gel, benz ene-chlorcf orrn, 1 :1 ) . It'~~

~~shov/ed presence of two compounds, ^^^ 0o2 6) and~~

~~0<,40) which were separated on PTJX: (Silica gel,~~

~~benzene-chloroform, 1 :1 ) . identical with the~~

~~acetate of obtained by first crystallization.~~

~~characterized by ''H ivMR (Table-30 , Fig . 4 )~~

~~and MS data.~~

~~Table-30: Chemical shift value ( ^-scale) of protons of 1 - hydroxy- 6- acetoxy- 3-methoxy-2 3-methyl-2- butenyl^ xanthone ( LXXXVIIb) .~~

~~"'H NMR Signals Number of J values Assignment of CM 1 protons in Hz~~

~~1 o70 (s) 3 - Me Protons~~

~~1 o80 (s) 3 - Me Protons~~

~~2c4 8 (S) 3 - OAc 3.39 (d) 2 6 CH2~~

~~3o98 (3) 3 - ME~~

~~(br) 1 - «CH~~

~~6o43 (s) 1 - H-4 7o40 (d) 1 9 H-8 7o48 (d) 1 3 H-5 8o3 8 (d,d) 1 9 and 3 H-7~~

~~s = singlet d = doublet, br = brpad. Spectrum run in CDCl^ at 60 MHz, TMo as internal standards U )~~

~~C\1~~

~~h-ro~~

~~o cc~~

~~2 h VD X~~

~~»< r o~~

~~LO : 18 7 :~~

~~'^he ' snectruip, of I z AC shovrcd Zets~~

~~-•^t SlclC =nd 1.30 for re-.i n J r^t'iyl rroups. a sir.-_let~~

~~atS2o48 for acetoxy protons, a doublet (J= 6H2 ) atS'3o39~~

~~for methylene protons, a singlet at S'3.98 for met boxy~~

~~XDrotons and a broad triplet at S 5.20 for a olefinic~~

~~proton. The singlet appearing atS6.43 integrating for~~

~~one proton v/as assigned to H-4 . Aromatic region shows~~

~~AEX pattern supporting the oxygenation at 6 position.~~

~~Tho doul-lot ) :^.tS"7.40 for H-8, doublet (J= 3Hs ) at~~

~~g7o48 for K-5 and a double doublet (J=9Hz) atgS.SB for~~

~~H-7 siipport the structure of ^^ 1-hydroxy-6-~~

~~acetoxy-3-methoxy-2 ^3-methy1-2-buteny^ xanthone(LXXXVIlb)»~~

~~COCHo-~~

~~Th'- TuPSs Sp'^ctrurn of GM^ ^ E Ac(QT.art.-4 ) shows molecular jon~~

~~RT rn/2 3:8(S8c7j) ==nd a b-nse pc^'?. m/s 313(i-j-55)c The~~

~~aiegncsric fr^^grner.t ions ^'.t m/s 3r3(34'i;, M-CH3 ), 325~~

~~(S.;, i>CCCH,)j 283 (50;., M-15-69-H) and 27-i(5i;o', 11-43-55+H)~~

~~fur-ch.er support s-cructuro LXX'XVxlb for Grl^QLAc1 2^- .~~

~~The aeacetylp-cion of a Sir,all apnount of GH^ yjfel^.fc..', parent co-pouni, Gij-i2S. rc.pss spcctrun of~~

~~sho-'S moJeculs: ioa at m/z 326(52 « > .') = pe^JN at r,/z 2 71 (n-c5) ^lo xj'v.'it-h -che frT^v^renr. tcrs : 18 7 :~~

~~- 15 ^n/z 355 (54)~~

~~H , m/z 568 (88.7)~~

~~-e - 69-H - 55 EXXXVIIb~~

~~- 45 H/Z 285 (50)~~

~~OMe n/z 515 (100)~~

~~H/Z 325 (8 0)~~

~~H* -h~~

~~Q PH OH~~

~~^CHz - 55~~

~~^OMe,~~

~~n/z 271 (51)~~

~~Chart-4. frasnentation pattern of 1-hydroxy-6-ace•tozy-5• ne th oxy-2/5-rie thyl-2-l3U tenylj:-"-anth onef.LZXX VIII)). :• 237 :~~

~~- 15 311 (14)~~

~~M^, m/z 526 (52.5) -e - 69-H KXXVIIa i~~

~~- 55 lu/z 241 (10)~~

~~OMe~~

~~n/z 271 (100)~~

~~Chart-5. I.'ass fracEeatatioa pattern of l,6-dih7dro>:y-3-ne tho.\'y-2~~

~~/3-nethy-2-bu i-.enyl/x ant hone ( LXXXVIIa ) . : 18 7 :~~

~~311(14FO' , M-CHG) and FI-I5-£9-H), Therefore, the parent xanthone, GH^^B, may be characterized as 1,6- dihydroxy-3-methoxy-2 ^3-methyl-2-butenyl^ Xanthone~~

~~(LXXXVlla) which is a nevj compound.~~

~~Antibacterial activity of GM-11 (Gartanin)~~

~~CM-11 was tested for antibacterial activity and~~

~~was found to be active against Staphylococcus aureus (.zone~~

~~of inhibition 0.7 cm/ 0,5 mg/mlj. Experimental ! 239 :~~

~~All melting points are uncorrected, ^H NMR spectra are taken in CDCl^ and DMSO-d, on Varian D-60 or 100 MHz 3 c and IR spectra are taken on Shimadzu IR-4 0 8o~~

~~Isolation of the constituents of leaves of Garcinia mangostana~~

~~Dried leaves(4.0 kg) of Garcinia mangostana were collected from Berliar (lower flops of Nilgri Hills, India),~~

The dried leaves were extracted with petrol and then with ethanol. The petrol extract(7oO gm) was adsorbed on silica-gel (25.2 gm) and poured over a glass column (160 cm long and 30 mm in diameter) containing silica gel(BDH, 140 gm) as an adsorbent in n-hexane. After development of the bands it was eluted with organic solvents with increasing polarity. The fraction obtained on elution with petrol was crystallized with chloroform-acetone which afforded white crystals of a hydrocarbon (60 mg) , m.p.61*^C. On comparison with IR & NMR, it v/as found to be a long chain hydrocarbon and it was not investigated further.

The fractions obtained from petrol-benzene(1 ;1 ) gave the oily mass which showed the presence of one major band. Crystallization from chloroform-ethanol afforded cycloartenol-GM-1 (150 mg) m.p. 75-Rf 0.41 (silica gel, petrol-benzene, 1 :1 ) » characterized by comparison of ''H NMR,

~~IR and mass spectra with the authentic sample. : 18 7 :~~

~~''h NMR( 5-scale, CDCI3), 0.34, 0.5 6 (cyclopropane ring, ABq, 2H, J=5Hz), 0,80- 1.68(7xMe, 21H), 3.20-3.40~~

~~(q, 1H, J^e.S HZ, OH), 5.lO(br t, J=6HZ, =CH).~~

~~IR-p cm"', 3300(br, OH), 302 5 (cyclopropane ring), 2900(br, C-H str), 1625, 1450, 1365, 1350, 1325,~~

~~1275, 1090, 1035, 1015, 985, 880, 805, 710, MS m/z 426,~~

~~M'^(11), 41 1 (M-xMe)'•'(1 1 ) , 408(M-H20) *(14.3) , 393 (M-Me-H2 0)~~

~~(17.6), 365(6.6), 339(8.8), 286(M-I 40) (20.9 ) , 271 (12.1),~~

~~205 (22), 203(23.1 ), 201 (16.5), 1 91 (11 ), 189 (18.7), 187(17.6),~~

~~1 77(5.5), 175 (36.3), 173(29.7), 1 67 (9.9), 1 65 (9.9), 163(23.1 ),~~

~~1 62(13.2), 161 (30.8), 160(13.2), 159(23.1 ), 150(18.7),~~

~~148(26.4), 147(45.1 ), 145 (24.2), I4l(i5.4), 1 37(24.2),~~

~~1 36(18.7), 1 35 (56.1 ), 1 34 (34 .1 ), 1 33 (46.2), 131 (22 ), 123~~

~~(45.1 ), 122 (29.7), 1 21 (67 . 1 ) , 1 20 (20 . 9) , 11 9(52.8), 117(17.6),~~

~~111 (17.6), 110(16.5), 109(78.1), 108(25.3), 1 07 (71 . 5 ) , 106 (22 ) ,~~

~~105 (8.2), 97 (26.4), 96(23.1 ), 95 (89.1 ), 94 (26.4), 93(69.3),~~

~~92 (1 8.7), 91 (55), 83(49.5), 82 (37.4), 81 (78.1 ), 79(56.1 ),~~

~~77(33), 71 (34.1 ), 70(25.3), 69(100), 68(17.6), 67(60o5),~~

~~65 (1 6.5), 57(52.8), 56(26.4), 55 (84 .7), 53 (29.7), 44 (61 .6),~~

~~43(79.2 ), 42 (1 8.7), 41 (83.6), 39(25.3).~~

Further elution with petrol-benzene(1:1) gave the oily mass showing the presence of one major band which was crystallized from chloroform-acetone into white needle shaped crystals of GM-2(100 mg), m.p.263-4°, Rf 0.70 (silica gel, petrol-benzene, 1:1), characterized as friedlein by comparison of its ^H NMR, IR and mass spect&a with authentic Sample : 18 7 :

~~"•H NMR( G'-scale, CDCI3) C 0.70(S, 3H, CH^) , 0O84~~

~~(s, 3H, CH3), Oc85(s,3H,CH3), 0.92(s, 3H, CH3), 0.98(s,3H~~

~~2XCH3) , 1O02(s, 3H,CH3), 1O16(S, 3H, CH3),NO26, 1O36,~~

~~1O42, 1O52(CH2 protons), 2o20-2 .34 (m, C^-CH^ and s, 1H,~~

~~C4-CH) .~~

~~cm"'' 2900 (br, OH), 1705, 1455 , 1 380, 1 370, max 1355, 1305, 1295, 1275, 1240, 1215, 1200, 1185, 1170, 1070,~~

~~1015, 1000, 975, 715. MS m/2 426, M*" (92) , 411 (40) , 341 (32) , 303 (70) , 273 (100).~~

Elution of the colxjmn with benzene gave the oily mass which was crystallized from CHClg-EtOH as white crystilline solid.of GM-3 (4 0 mg ) , m.p. 140-1'^C, Rf 0.32 (silica gel, benzene-chloroform, 1 :1). It also gave Leiberman-

~~Eurchard test and was comparable with authentic sample of~~

~~sitosterolo~~

The fraction obtained on elution with benzene-ethyl acetate (9:1) was crystallized with CHCl3-MeOH as white crystals of GM-4(10 mg), m.p.141,Rf 0o70 (silica gel, benzene-acetone, 8:2). It is under investigation.

Further elution of the column with benzene-ethyl acetate (9:1) gave the greenish mass which on crystallization with ethyl acetate yielded white amorphous powder of a new compound characterized as 25-dehydro- 3/5 , 24£ -di- o hydiDxycycloartane-CM-S (30 mg), m.p.140 > Rf 0. 69 (si lica-gel benzene-acetone, 8:-2). t 242 f

~~1 H NMR( <^-scale, CDCI3) : 0.32, 0.54 (?VBq, 2H, J=5Hz , cyclo-~~

~~propane ring), 0.78, 0.85, 0.92(15H, 5xCH ), 1.68(s, 3H,~~

~~CH« -OC), 3.26(atC^- ), 4.0(brt, C. - , J»6Hz), 4.85 (d, ^ NQH OH J=6H2, "CH2).~~

~~jj^^Nujol . 3360 (br,M-OH), 3030 (cyclopropane max~~

~~ring) 2900(br, C-H, str)1640 (C=C) , l450, 1405, 1365, 1355,~~

~~1300, 12 75, 1215, 1090, 1055, 1040, 1020, 1000, 985, 945,~~

~~885( =CH2), 795.~~

~~MS m/z 442, M*(6.25), 427(5), 424 (12.5), 409(13.75), 393(3.75~~

~~391 (3.75), 381 (5), 355 (3.75), 31 5 (6. 25 ) (M*^ - CgH^ 5O) 313(M*-~~

~~Sidechain-2H) (3.75) , 302 (M*-1 ^0) (1 3 . 75 ) , 297 (31 5-H2O) (7. 50) ,~~

~~223(11 .25), 205 (13.75), 203(18.75), 201 (10, 197(12.5), 189~~

~~(1 3.75), 187(12.5), 177(1 3.75), 175 (27.5), 173(15), 1 63(15),~~

~~1 6 (20), 159(15), 155 (10), 150(25), 147(26.25), I45(l5),~~

~~141 (1 3.75), 137(13.5), 1 36(1 1 .25), 1 35 (41 .23), 1 34 (21 .25)~~

~~1 33 (28.75), 1 31 (12.5), 127(11 .25), 125 (12.5), 123 (26.25),~~

~~122 (20) , 121 (43.75) , 120(15) , 11 9(32 .50) , 111 (12.5) , 109~~

~~(51 .25), 108(1 6.25), 107(48.75), 105 (41 .23), 104 (15),~~

~~97(1 8.75), 95 (71 .25), 94 (16,25), 93 (50), 91 (33.75), 85 (15),~~

~~83(28.75), 82(13.75), 81 (62.75), 79(31 .25), 77 (28.75),~~

~~71 (43.75), 69(67.50), 67(38.75), 65 (26.25), 57(65), 56(42.50)~~

~~55(83.75), 44(72.50), 43(100), 41(96.35), 39(27.50), 36(20).~~

~~The mother liquor on crystallization with CHCI3-~~

~~MeCH afforded white crystals of GM-6(15 mg), m.p.227-8°C,~~

~~Rf 0.69 (silica gel, benzene-acetone,8 :2), characterized~~

~~as betulin.~~

~~''H NMR( S'-scale, CDCI3): 0.75, 0.80, 0.91, 1.0 (singlets~~

~~1 8H, methyl groups), 1 .20-1 .65 (CH2protons), 1.63(s,3H, 24 5 1~~

~~3.20(q, 3 ^CH-OH), S.SOCCH^OH), 4 . 81 (d, .~~

~~jj^-^KBr 2370 (OH), 2930, 2860, 1 680, 1 630, 1450, max 1380, 1370, 1350, 1180, 1110, 1630(br), 880.~~

~~Continuous elution of the column with benzene-ethyl-~~

~~acetate (9:1) gave the fraction which was crystallized with~~

~~chloroform-methanol as white needles of mangiferadiol-GM-7~~

~~(25 mg), m.p.144-45°, Rf 0.48 (silica gel, benzene-acetone,~~

~~8:2). It was characterized as 9,19-cyclolanost-24-en-3,~~

~~26-diol(mangifera diol). . .~~

~~^H NMR( S-scale, CDClg) : 0.33, 0o58(d, iHeach,~~

~~cyclopropane CH2), 0.80, 0.90, 0.96(s, 15H, Me), 1.20-1.51~~

~~(CH^ protons), 1.66(s, 3H, Me), 3.2 (q, 3/3 CHOH) , 400 (2H,~~

~~CH2OH) and 5.4 5 (1H, = CCT^~~

~~IR^^®^ cm'"': 3365 (OH) , 2960 (cyclopropane) , 2920, 2850, max 1450 (br), 1365, 1195, 1090, 1065, 1035, 1015.~~

~~MS: m/2 442 (17.5), 427(15), 424 (35), 407(35), 405 (15),~~

~~381 (12.5), 355 (10), 353(7.5), 352 (7.5), 350 (25), 342 (7.5),~~

~~339(7.5), 315(7.5), 313(7.5), 302(20), 300(12.5), 327(17.5),~~

~~297(10), 223(20), 205 (22.5), 203(35), 189(10), 187(10),~~

~~1 35 (47.5), 1 33(32.5), 113 (25), 111 (32.5), 148(25), 95 (80),~~

~~85 (12.5), 83(27.5), 81 (40), 44 (95), 28(100).~~

~~Elution of the column with benzene-ethylacetate(8:2)~~

~~gave the fraction which showed one major spot, GM-8,~~

~~alongwith minor impurities which was separated through~~

~~preparative their layer chromatography (FTLC)(Silica gel,~~

~~benzene-acetone, 8:2). It was crystallized with chloroform-~~

~~methanol as white solid of GM-8(80 mg) , m .p . 1 5 9-61 : 18 7 :~~

~~Rf 0.4 8(silica gel, benzene-ace-cone, 8:2). It was 94 95 characterized as mangifrolic acid' ' .~~

~~"'h Nr4R( scale, CDC1_) : 0.30, 0.54 (d, 1H each, cyclo- 3 propane CH2), 0.78(s, 3H, Me), OcS6(s, 3H, Me), 0.94(s,~~

~~3H, Me), 1.1-1.84 (CH2 protons), 1.84 (s, 3H, Me ),~~

~~3.26(1, 3/^CHOH), 6.92 (t, br, . MS; m/z 456 (3),~~

~~441 (4 ), 438(5) (M-H2O, 8), 395 (4 ), 369 (4 ), 31 6 (3), 315 (1 ),~~

~~301 (12), 297 (8), 278(2), 205 (12), 148(90), 95 (20), 85 (IOO) ,~~

~~83 (100) c~~

~~Ethanol extract was concentrated and treated with~~

~~benzene. Benzene soluble fraction (2 <>5 gm) was adsorbed~~

~~on silica-gel (9 gm) and poured over a glass column(105 cm~~

~~long and 25 mm in diameter) containing silica gel(BDH, 60 gm)~~

~~in petroleum ether. After the development of the bands the column was eluted with organic solvents in order of~~

~~increasing polarity. The fraction obtained on elution~~

~~with benzene gave the oily mass which was crystallized with~~

~~chloroform-ethanol and yielded white needle shaped and~~

~~brown crystals of compounds GM-9 and GM-10, respectively.~~

~~White crystals, GM-9, 80 mg, mop. 150-1°C, Rf 0»32 (benzene-~~

~~chloroform, 1 :1); Brown crystals, GM-10, 40 mg m.p.147-1°C)~~

~~Rf 0.32(benzene:chloroform 1 :1 )» Compounds GM-9 and GM-10 were comparable with sitosterol. Since their melting points are different from /^-sistosterol. It is difficult~~

~~to comment on their identity. : 18 7 :~~

On further elution of the column with bensene, the oily mass so obtained shov/ed the presence of one major band on TIC (brown in UV light) which appeared to be yellow with nacked eye. On crystallization with benzene-petrol it gave yellow crystals of gartanin-GM-11

~~(1 gm),mp 163-5°C, Rf 0,38(silica gel, benzene-chloroform,~~

~~1:1). It was characterized by comparing its ''H NMR, IR and mass spectra with the authentic sample.~~

~~''h NMR(DMSO-d^, g-scale) : 1 o 81 , 1 , 75 (sing let s, Me protons)~~

~~5„23(2H, br,2K. = fl[,-H) , 3.50(br, 1H, 2 ACH2) , 6.60(d, 1H,~~

~~J=8.8 Hz, H-6), 7o24(d, 1H, J=8.8 Hz, H-7), 12o20(br, 1H,~~

~~OH), 11o19(br, 1H, OH), 9.51 (br, 2H, OH).~~

~~IR^^^^cm'"' 3200, 1 630, 1590, 1490, 12 65, 1225, 11 80, max 1100, 1005, 810, 730.~~

~~MS m/z 39 6, m"^ (4 8.5) , 3 . 81 (9 o 2 ) (M -15) , 353 (11 ,9) , 34 1~~

~~(47o 8), 325 (52.6), 297(25<.8), 2 85 (100), 2 84 ( 35 . 8), 2 73 ( 7<,8)o~~

~~1-Hydroxy-3,5, 8-triacetoxy-2,4-di 3,3 '-dimethyl allyl xanthone: GM-II Ac~~

1 ,3,5, 8-Trihydroxy-2 ,4-di^ ,3 '-dimethyl ally^ xanthone(50 mg) was acetylated with pyridine(1.0 ml) and acetic anhydride(0.2 ml) by heating in a round bottom flask fitted with a CaCl2 tube on a waterbath for 4 hrs. it was then poured on crushed ice, the separated solid was filtered and washed with H2O. It was dried and crystallized with chloroform-ethanol, light yellow coloured crystals of 1 -hydroxy-3 ,5, 8-triacetoxy-2 ,4-di^,3 '-dimethylallylj t 246 !

~~xanthoneOO mg) , m.p.125°C was obtained „ ^H NMRCCDCl^,~~

~~^-scale) :1.70(s, 12H, 4xMe), 2=47, 2.48, 2.56(s, each,~~

~~GH, OAC) , 3c32(br, 1H, 2 -CH2), 5„17(br, 2H, 2x =C-H) ,~~

~~6.90(d, 1H, J=8.8 HZ, H-6), 7.42(d, 1H, J= 8. 8 HZ, H-7)c~~

~~Elution of the column with benzene-ethyl acetate~~

~~(9:1) gave oily mass which was crystallized from benzene- petrol to yield yellow crystals of GM-12A,1,5,8-trihydroxy-~~

~~3-methoxy-2 |^3-methyl butenyl^ xanthone(100 mg), m.p.193-5°~~

~~Rf 0o25 (silica-gel, benzene-chloroform 1:1). Rf 0o44~~

~~(silica gel, benzene-ethylacetate, 9:1). It gave green colour with alcoholic FeCl3 and red colour with p-benzoquinone (gossypet-one reaction). It appeared yellow with~~

~~nacked eye and v/as characterized by its ''H NMR, IR and mass~~

~~spectra. ''H NMR (DMSO-d^, ^-scale): 1 .62 , 1 .72 (s,s,Me proton), 3o2(br, 2H,-CH2), 3»92(3H, s,CMe), 5.08(1H, br,1~~

~~=C-H), 6.58(S, 1H, H-6), 6o64(s,1H, H-4), 7.24(s, 1H, H-7),~~

~~10.91 (br, 1H, 5-OH), 11o02(br, 11H, 1-)H), 11.03(br, 1H,8-0H)-~~

~~IR^ cm"'' J 3400, 1 775, 1 650, 1 600, 14 85 , 1440, 1315, 1230, max~~

~~1 1 85 , 1 155, 1095, 1075, 810, 7 80.~~

~~MS:m/z 343 (17.7), 34 2 ( 83 » 3 ) , 327 (61 c1 ), 326(31 .7), 299 (90o4),~~

~~287 (10(J, 286(1 62), 285 (1 3.9), 2 74 (1 7. 3 ) , 271 (50 . 9 ) .~~

Compound GM-12,on"aoetylation and-repe^ted crystallization from CHClg-EtOH, afforded acetate , m.p.172-5°C, which gave positive test with alcoholic FeQl^ indicating the presence of phenolic group resitant to pcetylation.

~~The ''H NT'IR spectrum of acetate (II)(60 MHz , 5"-sc=5le, solvent .J 247 :~~

~~CDCl^) showed signals at 5 1 •69 and 1.75(s,s,3H each,~~

~~=C(CH-)_ ), 2o4l(s, 3H, OAC), 2.45(s,5H,OAC), 3.30(br, o z~~

~~2H, CH2), 3.93(s,3H,OMe), 5.1 0(br,t,1H,C=CH), 6.33(S,1H,~~

~~H-4), 6O97(d,1 H,J=8.8 HZ, H-6) and 7.43(d,1H,J=808 Hz,H-7).~~

~~The mother liquor of 0412A was acetylated to yield~~

~~GM-12AAC & GM-12BAC which were separated on PTLC(silica gel, Benzene-CHCl3 ,1 :1 ). GM12-A-AC was identical with the acetate of GM12A obtained by first crystallization. 2~~

~~BAc(i5 mg mp 162°) was characterized as 1,6j-dihydroxy-S- methoxy-2 methyl buteny^ xanthone by H NMR.~~

~~""h NMR (60 MHZ scale, CDCI3 ) : 1 . 70 (s ,3H, & 1 . 80 (s,3H, gem dimethyl group), 2.48(s,3H,OAc), 3,39(d,J=6Hz, CH2),~~

~~3.98(s,3H,OMe) , 5 . 2 0 (br , t , = CH) , 6.4 3 (s , 1 H, H-4 ) , 7.40(d,lH,~~

~~J=9H2, H-8), 7.48(d,1H,J=3H2, H-5), 8.3 8 (d ,d , 1 H, J=9Hz ,H-7) .~~

~~MS: m/z 368(M"^, 88.7), 353 (34), 325 (80), 313 (100), 283 (50) and 271 (51~~

~~The deacetylation of a small quantity of GM1.2BAC yielded the parent compound GM12B, (&€i mg_, mp above 240°).~~

~~MS: m/z 32 6 (M*^, 52<,5) , 31 1 (14) , 2 71 (100) and 241 (10).~~

~~Antibacterial activity of GM-ll~~

~~Gartanin GM-11 was tested for antibacterial activity by the method described in Chapter-2 (page II5) and found to be active against Staphylococcus aureus~~

~~(zone of inhibition 0.7 cm/0o5 mg/ml). REFERENCES : 18 7 :~~

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~~1. A new isoprenylated xanthone from G.mangostana Linn., Mahtab Parveen and Nizam Ud-Din Khan, Chemistry and Industry 1987 (in press).~~

~~2. Two new flavones from Ardisia floribunda, Mahtab parveen and Nizam Ud-Din Khan, Indian J. Chemistry 1987 (in press).~~

~~3. Biflavones from the leaves of Fitzroya patagonica Hook F., S.W.I. Naqvi, N. Parveen, M. Parveen and N.U. Khan, Curr Sci , 56(10), 480 (1987).~~

~~4. Biflavones from Rhus alata, M. Parveen and N.U. Khan, Curr. Sci. 1987 (in press)~~

5. Biflavonoids from the leaves of Araucaria cookil, Mahtab Parveen and Nizam Ud-Din Khan, Proceedings of the Seventy Third Session of the Indian Science Congress, Delhi. Part IV (Late abstracts) 1986, p. NO. 1 9.

~~6. Two flavones from Ardisia floribunda, Mahtab Parveen and Nizam Ud-Din Khan, proceedings of the Seventy Fourth Session of Indian Science Congress Bangalore 1987, p.n0.l2 8.~~

~~7. The constituents of Cupressus funebris(Cupressaceae), M.Parveen, H.M. Taufeeq & N.U. Khan, 1st Annual Conference, ISSAC-I, April 28-29, 1984.~~

~~P ap er c omrnu ni c at e d ;~~

8. Antileukemic activity of Biflavone, N.U. Khan, N. Parveen, M. Parveen and H.M. Taufeeq, Curr. Sci. 9. Xanthones from the leaves of Garclr.i 3 mangostans Linn, M. Parveen and N.U. Khan, Phytochemi stry,

~~Papers under preparation:~~

~~10. Chemical constituents of Cupressus.~~

~~11. Chemical constituents of Garclnla.~~

~~12. Trlterpenolds from G. mangostana.~~