Chemistry of Oxygen Heterocycles- Flavones and Biflavones

& ^

CHEMISTRY OF OXYGEN HETEROCYCLES- FLAVONES AND BIFLAVONES

(SUMMARY)

THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN CHEMISTRY

U&tch, 1974 NAJMA HAMEED T»31 Fee", to C'^'vtu

ALIGARH MUSLIM UNIVERSITY ALIGARH SUMMARY

CHEMISTRY OF OXJGM H£TEftO-,CYCL£S FLATONJ5S AMP BIFLAVONES

The leaf extracts of a number of Gymnosperms were purified by solvent fractionation and column chromatography on magnesium trisilicate

(Woelm). The biflavone mixtures thus obtained were separated by preparative layer chromatography on silica get G (E.Merck) using benzene : pyridine : formic acid (36:9:5). The homogeneity of each component was established by a number of TLG systems, counter current distribution method and methylation followed by TLC. In cases where it was not possible to separate the mixture into parent forms, they were separated as methyl ethers. Each pure component was fully characterized by the prepration of derivatives, co-chromatography with authentic samples and spectral methods. The minor components were only detected (TLC). The plants investigated and the biflavones characterized/detected are shown below:

1. JUNIPBRUS CH3UENSIS LINNAEUS

(a) Amentoflavone

(b) Hinokiflavone

(a) Amentoflavone

(b) Hinokiflavone

(d) Sciadopitysin

3. JUNIPERUS VIRGINIAHA LDIMAEUS

(a) Amentoflavone

(b) Cupressuflavone (c) Robustaflavone (d) Hinokiflavone

4. JUNIPERUS RECURVA BUCHHAM

(a) Amentoflavone

(b) Cupressuflavone

(d) Cupressuflavone monorasthyl ether

(ej Hinokiflavone

(f) Amentoflavone dimethyl ether

(g) 1-7,II-7-Di-O-methyl cupressuflavone

The presence of members of cupressuflavone and robustaflavone in Juniperus species is reported for the first time.

5. THUJA ORIEMTALIS LTOAEUS

(a) Amentoflavone

(b) Hinokiflavone 3

6. THUJA PLICATA D.DON

(a) Amentoflavone (b) Hinokiflavone

7. THUJA JAFONIQA MkXIMDWlCZ

(a) Amentoflavone (b) Cupressuflavone

(d) Hinokiflavone (e) Sequoiaflavone (f) Hinokiflavone monomethyl ether

(g) I~7>II-7-Di-O-methylamentoflavone (h) Hinokiflavone dimethyl ether

(i) Sciadopitysin

Cupressuflavone and robustaflavone have been reported for the

first time in Thu.ja species. A new biflavone 1-7,II-7-Di-O-methylamentoflavone

is also reported.

8. P0D0CARPU3 TAXIFQLIA KONTH .

(a) Amentoflavone

(b) Hinokiflavone

(d) Bilobetin

(e) Sequoiaflavone 4

The presence of three monomethyl ethers of one single series is noteworthy. They have been separated by CCD in pure forms and authenticated as podocarpusflavone A, bilobetin and sequoiaflavone.

9. ARAITGAR1A EXGSLSA LAMB

# (a) Amentoflavone

«•(b ) Cupressuflavone

(d) Agathisflavone monomethyl ether

(e) 11-7-0-Met hylamento flavone

(f) 1-7,II~7-Di-0-methylagathi3flavone

(g) 1-7, II-7-Di-O-methylamento flavone

(h) 1-7,1-4' or lI-4'~Di-0-methylcupressuflavone

(i) TI-4!,1-7,II-7-Tri-O-methylagathisflavone

(j) Cupressuflavone trimethyl ether

(fc) 1-4', 1-7, II-7-Tri-O-methylamentoflavone

(1) 1-4',11-4',1-7,II-7-Tetra-O-methylamentoflavone

(m I-41, II-4', 1-7, II-7-Tetra-O-methylcupressuflavone.

Three new biflavones have been characterized as 1-7,1-4' or

II-4'-di-O-methylcupressuflavone, II-4',1-7, II-7-triTO-methylagathisflavone and 1-4', 1-7,II-7-tri-O-methylamentoflavone.

Note: Those marked with aestricks are only detected. 10. PHLOX DRUMMQMDII HOOK

The flower extract of P.drummondii yielded the following two new

C-glycosides.

(a) 0-F hamno ayl-6-C-xylosylluteolin

(b) 0-Rhamnosyl-6-C-xylo3ylapigenin. CHEMISTRY OF OXYGEN HETEROCYCLES FLAVONES AND BIFLAVONES

THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN CHEMISTRY

March, 1974 NAJMA HAMEED

ALIGARH MUSLIM UNIVERSITY AL1GARH f— < ,u

\>/-t?v

T1376

TV376

s i 6 JUM ^

TV4 tn Cc^'X'UCS

1996-$ COCKED >

ACKNOWLEDGEMENT

The author wishes to express her high sense of gratitude to Prof. W.'Rahman who directed these researches, for his unstinted help and encouragement throughout, to Prof. S.M. Fazlur Rahman for providing the facilities necessary for execution of the work and to the University Grants Commission and Council of Scientific and Industrial

Research for the. award of fellowship.

The author would like to express her appreciation to

Prof. K. Kawano (University of Nagasaki, Japan) and to Prof. T.J. Mabry

(University of Texas, U.S.A.) for their help in spectra], studies. CONTENT S Page

1. THEORETICAL ...... 1-122

2. DISCUSSION ...... 123-187

3. CONCLUSIONS ...... 188-191

4. EXPERIMENTAL ...... 192-244

5. BIBLIOGRAPHI ... '.... ' 245-254 THEORET ICAL BIFLAVANOIDS The flavanoids belong to an important group of naturally

occurring organic compounds and are among the earliest dye stuff

known. They have attracted attention of the plant chemists for centuries.

During the last few years there has been a revival of interest in the

chemistry, biogenesis and physiological studies of flavanoids. The term "flavanoid" covers a large group of naturally occurring compounds

in which two benzene rings are linked by a propane bridge (C^-C-C-C-Cx)

except in the isoflavones in which the arrangement is (C,-C-9~C). " i °6

The flavanoids include chalcones, dihydrochalcones, aurones,

fLavanones, flavones, flavanonols, flavonols, flavan-3, Vdiols

(leucoanthocyanidins), anthocyanidins, proanthocyanins and catechins.

The carbon skeleton of flavanoid compounds occurs with variation

in the oxidation level of C_ portion of the molecule (Table-I)« The range

of oxidation level extends from the highly reduced catechin (I) to th©

highly oxidized flavonol (XI). 2

A '/Bv

VII VIII kr^°>-A cH^S>

XI T A B L E - I

Fig.No. Compound Type Oxidation State of C

I Catechols A-CH2-CH0H-CH0H-B

II Dihydrochalcoi A-C0-CH2-CH2-B

III Chalcones A-CO-CHcCH-B

IV Flavanones A-C0-CH2-CH0H-B

V Isoflavanones A-CO-CH-CHOH i B

VI KLavanonols A-CO-CHOH-CHOE-B

VII Flavones A-C0-CH2-C0-B

VIII Iso flavones A-CO-CH-CO t B

IX Anthocyanins A-CH2-C0-C0-B

Aurones A-C0-C0-CH2-B

XI Flavonols A-CO-CO-CHOH-B Numerous physiological activities have been attributed to

1 2 flavanoids ' . The potent uses of flavanoids may be listed as vitamin P activity (i.e. the property of reducing the capillary fragility and permeability) , diuretic action , treatment of allergy, protection

7 8—10 against X-rays and other radiation injuries , cure of frost-bite ,

11 12 13 :,^+„14-l6 antibacterial activity , prophylactic action ' , oestrogenie activity 17 and anti-tumour effects . They are also used as antioxidants.

Recent addition to this class is Mbiflavanoids". The biflavanoids are derived from two Havone or fLavanone or fLavanone-flavone units and have been mostly isolated from Gymnosperms. Among the angiosperms, some

18 19 20 plants belonging to Guttiferae , Euphorbiaceae , Caprifoliaceae , 21 22 23 1L.

Archegoniateae , Selaginellaceae , Anacardiaceae and Ochnaceae ^ have been found to contain biflavanoids.

NOMENCLATURE FOR BIFLAVANOIDS2^;

After consideration of the views of professors W.D.Ollis, N.Kawano,

T.R.Seshadri and Drs. A.Palter, R.S.Chan and L.C.Cross, the following systematic nomenclature for flavanoid polymers has been evolved. In this nomenclature the generic term "biflavanoid" has been

adopted in preference to biflavanoid or biflavonyl since in general

the saturated system is regarded as the parent for the nomenclature.

The ending 'oid' may then be modified to cover specific types of fLavanoid dimers such as biflavanone, biflavone, biflavan etc. and for mixed

systems flavanone-flavone. This system follows general IUPAC policyj the unmodified nomenclature is utilized as a generic term in the naming of dimeric, trimeric, tetramsric etc. derivatives by insertion of the

appropriate Greek prefices bi-,ter-,quater etc. giving biflavanoid, terflavanoid, quaterflavanoid etc.

To identify specific ring positions in fLavanoids and their polymeric derivatives, the present long accepted system (exemplified in (XII) for naringenin) is retained, extending it in the case of polymeric fLavanoid by assigning to each monomer unit a Roman numeral

I, II, III, etc, running in the sequence from one end of the molecule to another. The points of linkage between neighbouring fLavanoid units are identified by a combination of a Roman numeral (to identify the flavanoid unit) and an Arabic numeral (to identify the position of the inter flavanoid linkage), the two numbers being coupled with a hyphen and enclosed within square brackets. This has been demonstrated in the case of amentoflavone (XIII).

(XII) (XIII)

1-4«, II-4«, 1-5, II-5, 1-7

II-7-Hexahydroxy- C1-3', II-8J bifLavone (Amentoflavone).

All the biflavanoids known to date may be classified into two main groups:-

(I) C-C linked biflavanoids, and (II) C-O-C linked biflavanoids.

(I) C-C LINKED HEFLAVANOIDS:

Depending upon the nature of the constituent monomeric units and of the position of linkage we have different series. (a) Cupressuflavone Series:

These are. derived from two apigenin units with C1-8, II-8 2

linkage and are represented by six members. OupressufLavone (XTVa) is the

parent compound while the other five are its partial methyl ethers.

0R3 0

(XIV)

R1 R2 R3 R4 R5 R6 (a) Cupressuflavone ' H H H H H H

27 28 (b) I-7-O-Methyl ' Me H H H H H

27 29 (c) 1-7, II-7-Di-O-methyl ' Me Me H H H H

30 31 (d) I-4«, 1-7, II-7-Tri-O-methyl ' Me Me H H Me H

(e) 1-4', 11-4', 1-7, II-7-Tetra-O-mathyl29 Me Me H H Me Me

*(f) I-4,,II-4'»I-5,I-7,II-7-Penta-0-methyl32 Me Me Me H Me Me

* (Synthetic) (b) Amentoflavone Series:

These are derived from two apigenin units with D-3% II-8 ZJ linkage, and are represented by sixteen members with amentoflavone (XVa) as the parent compound.

0R3 0

OR/, i

(XT)

R1 R2 R3 \ R5 R6 33-36 H H H H H H (a) Amentoflavone (b) t-7-O-Methyl37-39 Me H H H H H (Sequoiaflavone)

(d) II-7-O-Methyl (Sotetsuflavone)3^'40 H Me H H H H (e) II-4'-0-Methyl (Podocarpusflavone A)33'^1H H H H H Me

(f) I-^I-V-Di-O-Methyl35'36'42'43 Me H H H Me H (Ginkgetin) R1 R2 R3 R4 R5 R6 (g) I-41, II^'-Di-O-methyl41'43'44 H H H H Me Me (Isoginkgetin)

(h) 1-7, II-4'-Di-0-M9thyl41 Me H H H H Me

(Podocarpusflavone B)

(i) I-4S II-7-Di-O-methyl31 H Me H H Me H

(j) 1-7, II-7-Di-O-methyl45 Me Me H H H H (k) 1-7, II-7, II-4' -Tri-0-methyl46 Me Me H H H Me (Heveaflavone) (1) 1-4', II-4*, I-7-Tri-O-methyl38'39'41'47 Me H H H Me Me (Sciadopitysin) (a) 1-V,*1-V, II-7-Tri-O-methyl44'4*'49 H Me H H Me Me (Kayaflavone)

(n) 1-4', 1-7, I I-7-Tri-O-methyl45 Me Me H H Me H

(o) 1-4', II-4', 1-7, II-7-Tetra-O-methyl50 Me Me H H Me Me

(p) I-4», II-41, 1-5, 11-5, 1-7, Me Me Me Me Me Me II-7-Hexe-0-methyl51

These are derived from two apigenin units with C1-6, 11-8 3 linkage. This class has recently been recognised and includes only

five members with agathisflavone (XVIa) as the parent compound 10

(XVI)

R1 R2 R3 R4 R5 R6

(a) Agathisflavone H H H H H H

(b) I-7-0-Metbyl27'2B>52 Me H H H H H

PA Me Me H H H H (c) 1-7, II-7-Di-O-methyl^5 Me H H H H Me (d) 1-7, II-4,-Di-0-methyl27,52 Me Me H H H Me (e) 1-7, II-7, II-4'-Tri-0-methyl' (D) Robustaflavone Series;

^his class has been recognized very recently and is represented

JC only by robustaflavone (XVII) as the parent compound and its mono and dimethyl ethers 53 , characterized only as their complete methyl ethers.

These are derived from two apigenin units with C1-3% 11-63 linkage.

(XVII)

(E) 2.3-Dihydroamentoflavone Series ^" ;

This series has also been very recently recognized and is represented by 2,3-dihydroamentofLavone (XVIIIa) (1-4% H-4% 1-5,

II-5, 1-7, Il-y-^exahydroxy flavanone Cl-3% II-8D flavone) as the parent compound and its two partial methyl ethers. The constituent monomeric units are a naringenin and an apigenin.

Ofy 0

(XVIII)

R1 R2 R3 R4 R5 R6

(a) 2,3-Dihydroamentoflavone H H H H H H

(b) II-4-S II-7-Di-O-methyl H Me H H H Me

(F) BGH Series:

These are derived from a naringenin and an apigenin or luteolin unit with CI-3,3lI-3 linkage and are represented by BGH-II (XlXa) and

BGH-III (XlXg) as the parent compounds, respectively. 1

R*0

(XIX)

R R.. R„ R.5 R R- R 4 6 (a) BGH-II (Morelloflavone/fhkugetin)57'58 OH H H H H H H

(b) lI-3'-0-Methyl59 OMe H H H H H H

(d) II-3 •, 1-4', II-4', H-5,1-7, II-7- OMe H Me Me Me Me Me 59 Hexa-O-methyl^7 h (e) II-3«, 1-4', H-4', 1-7, II-7- OMe H H Me Me Me Me Penta-O-methyl59

*(f) 11-3',11-4,1-7,11-7-Tetra-O-methyl59 OMe H H Me Me H Me

(g) BGH-IIlCTalbotaflavone/VolkensifLavone) 'H1 H H H H H H

*( Synthetic) (G) WGH Series ;

Two new biflavones of this series have recently been synthesised by dehydrogenation of BGH-II and BGH-III,

(a) R - OHj H-31, 1-4*, II-4S 1-5, II-5, 1-7, II-7-Heptahydroxy - CI-3, 11-81- biflavone (WGH-II or Saharanflavone).

(b) R « H, 1^4', H-4S 1-5, II-5, 1-7, II-7-Hexahydroxy - Ll-3, II-8D- biflavone (WGH-III).

(H) GB Series62"64:

These are derived from naringenin linked with a naringenin or aromadendrin or toxifolin through CIT3, H-&3 linkage. Four members are reported to occur in nature. J

OH 0

(xn)

R1 R2 (a) GB-I OH H

(b) GB-Ia H H

(d) GB-IIa OH OH

(I) 1-4', H-4S 1-5,11-5,1-7,II-7-HexahydroxyC 1-3, II-3J biflavone (Ml)65:

The series comprising of only one member has recently been synthesised by oxidative coupling of apigenin, HW°vV/ WOH

(mi) 16

(J) I-4SII-4S 1-5,11-5,1-7,II-7-Hexahydroxy CI-3,H-3D biflavone(XXIII)65:

The sole member of the series has also been obtained during oxidative coupling of apigenin.

0 OH (XXIII)

(K)CI-3', 11-811- biflavanone 23J>..

This class is represented by only three members, characterised as their partial/complete methyl ethers.

0M<2 R1 R2 R3 R4

(a) 1-4' ,11-4',1-7-Tri-O- methyl OH OH OH H

(b) n-3', I-4S H-4,-I-7-Tetra-0-methyl OH OH OMe H

2. C-O-C LINKED KEELAVANOIDS:

(a) Hinokiflavone Series;

These are derived from two apigenin units with CI-4'-0-11-63 linkage, Hinokiflavone (XXVa) is the parent compound with six others as its partial methyl ethers.

OR-! 0

(XXV) R1 R2 R3 R4 R5

(a) Hinokiflavone36'67*69 H H H H H

(b) I-7-O-Methyl (Neocryptomerin)^1'70 H H Me H H

(d) II-4'-0-Methyl (Cryptomerin-A)70'73 H H H H Me 70 (e) 1-7, II-7-Di-O-methyl (Chamaecyparin) H H Me Me H

*(f) II-4',II-7-Di-0-methyl(Cryptoinerin-By0"7:3 H H Me Me

*(g) 11-4',1-7, II-7-Tri-O-nethyl70'73 H H Me Me Me

*(Synthetic)

Previously naturally occurring hinokiflavone and its derivatives

were assigned C1-4'-0-II-83linkage on the basis of spectral and

degradative evidence, which has later been revised to Cl-4,-0-II-6D.

TheCI-4,-0-II-8I] linked hinokiflavone pentamethyl ether (XXVI) has also

been synthesdsed .

MeQ

OMe 0 OMe

OMe 0 5 56 (b) 2.3-Dihydrohinokiflavone (XXVII) ^ ;

The sole member of this series has recently been isolated from the leaves of Metasequoia glyptostroboides and Cycas species. The con stituent monomsric units are a naringenin and an apigenin linked through

Cl-4*-0-II-63.

OH 0 OH 0

(XXVII)

This class has recently been recognized and includes only three members with ochnaflavone (XXVIIIa) as the parent member. These are derived from two apigenin units with CI-3*-0-11-4*3 linkage.

RAO- 0R5

Rsa^v^N // \

1 0 (XXVIII ) R1 R2 R3 R4 R5

(a) Ochnaflavone H H H H H

(b) I-V-O-Methyl H H H H Me

'(d) I-4\ 1-7, II-7-Tri-O-methyl H H Me Me Me

*(Synthetic)

Biflavanoid Glycosidess:

M.Konoshima et al. hgtve recently isolated fukugiside (XXIXa)

and spicatoside (XXEXb) from Garcinia spicata and xanthochymiside (XXX)

from Garcinia xanthocjtiyinis.

(XXIX) 59 (a) Rikugiside 7 R« - OHj R = f3-&-gluc.

(b) Spicatoside7 5 R' = H; R =(3-D-gLuc, 21

OH o

( XXX )

Xanthochymuside 75 R =[3-D-glue.

OPTICAL ACTIVITY IN BIFLAVANOIDS;

76 Carl Djerassi examined the rotatory dispersion characteristic of two members of the biphenyl type biflavanoids, isoginkgetin and sciadopitysin in the range of 390-700 nm , but found no optical activity. The argument, however, used for optical inactivity in the series was that all the ortho positions of the biphenyl residue were not substituted. An interesting coiranent on the optical activity of the biphenyl type biflavanoids was made by

25 Seshadri et al on cupressuflavone. This biflavone incorporates a biphenyl system in which all the ortho positions are substituted by oxygen atoms.

However, no optical activity could be detected either in the natural pigment or any of its derivatives. Recently a large number of naturally occurring optically active biphenyl type biflavanoids have been reported (Table-II).

The optically active biflavanoids belonging to amentoflavone, cupressu- fLavone and agathisflavone series incorporate a biphenyl system in which at least three out of four ortho positions are substituted. These ortho substituents interfere with one another in coplaner position and are com fortable only in non-planer positions. Complete rotation is, therefore, prevented and optical resolution becomes possible. This phenomenon of restricted rotation leading optical activity in biphenyl type systems, is known as "atropisomerism". However, in fukugetin and xanthochymuside, the optical activity may either be due to the asymmetric centre (CL) alone or to both the asymmetric centre and restricted rotation. 23

T A B L E - II

OPTICALLY ACTIVE BIFLAVANOIDS

a, b, c, d, e Biflavanoids D( pyridine) Source

Amentoflavone ( XVa) +9a Podocarpus gracillior.4' 4 Cupressuflavone (XlVa) +1001 Tfaqa orient alia,4 9 c 45 +63 Cupressus torulosa 29 I-7,II-7-Di-0-methylcupressuflavone(XI?c) +65b Araucaria Cookii ' <45 +37+37., 5 Arauearia cunninghamii 29 1-4',II-4',1-7,II-7-Tetra-O-methyl- +30c Araucaria cookii ' cupressfuflavone ( XlVe) „ »50 1-4', II-4«, 1-7, II-7-Tetra-O-methyl- +41' amentoflavone (XVo)

1 ,50 Kayaflavone (XVm) +18 58 Pukugetin (XlXa) +17CT Garcinia spicata' Podocarpusflavone A (XVa) -6a Podocarpus gracilior4 4 52 1-7,11-4* -Di-O-methyl agathisflavone(XVIa) -55° Agathis palmerstonii I-7-O-Kethylagathisflavone (XVIb) -50c

1-7,II-7-Di-O- methylagathis flavone(XCIc) -12.5 Araucaria bidwilli 75 Xanthochyimiside (XXX) -40^e Garcinia xanthochynaia ,45 II-7-0-methylamentoflavone (XVd) -18.2 Araucaria cunninghamii' 1-4' ,II-7-Di-0-methylamentoflavone (Xvi) +22.7

a = 40°, b = 34°, c « 29°, d = 25° and e = 20° 24

STRUCTURE DETERMINATION OF HTFLAVANOIBS;

The problem of structure determination of biflavanoids is a complex one because of (a) occurrence of more than one biflavanoid in chromatographically homogeneous fractions with the consequent difficulty in their isolation in pure form (b) insolubility in the usual organic solvents and (c) the intricate problem of establishing the fLavanoid linkage.

The various methods generally used for structural determination may be classified as under:-

1. Colour reactions

2. Physical method

(a) Chromatography. (b) U.V.spectroscopy. (c) I.R,spectroscopy. (d) NMR spectroscopy. (e) Mass spectroscopy.

3. Degradation

4. Synthesis

1. COLOUR REACTIONS;

The usual reagents for detecting the presence of fLavanoid 77-79

(monomers) in plant materials have been found generally useful in biflavanoids 25

The only exception is that unlike monomers all kinds of biflavanoids give positive test with zinc and hydrochloric acid, a test characteristic of flavanonols * The observation is under investigation .

2. PHYSICAL METHODS;

The physical methods generally employed in structure elucidation are chromatography, U.V., I.R. NMR and mass spectroscopy,

(a) Chromatographic Methods;

A number of papers and review articles have appeared on the separa tion and identification of flavanoid pigments specially by paper chromatography in aqueous and alcoholic solvent systems . Recently extensive thin-layer chromatographic studies of biflavanoids, their partially and fully methylated derivatives have been carried out in our laboratories .

Benzene-pyridine: formic acid (36:9:5) and benzene-pyridine-ethyl formate- dioxan (5:1:2:2) have been found as the most satisfactory developing solvent systems both for qualitative as well as quantitative purposes. Further,

the relative differences in Rf values of the complete methyl ethers (even in traces) coupled with the characteristic fluorescence in U.V.light 2 6

(benzene-pyridine-formic acid) were found to be of some help in their identification.

Baker et al and Kawano et al have used counter current dis tribution between ethyl methyl ketone and a borate or phosphate buffer

(definite pH) for the separation of individual biflavanoids from isomeric

i mixtures as well as from mixtures of biflavanoids of different series.

(b) Ultraviolet Spectrascopy;

Spectral method have been applied in the identification and structure analysis of plant pigments. But in the case of biflavanoids ultra violet spectra has been found extremely helpful and as an important tool in elucidating the structure of these compounds.

Gomparision of the ultraviolet spectra (Table-Ill) of ginkgetin, isoginkgetin and their tetraacetates and tetramethyl ethers with those of apigenin (XXXIa), acacetin (XXXEb) and genkewanin (XXXIc) and their deriva tives suggest very strongly that ginkgetin and isoginkgetin are derivatives of 5,7,4'-trihydroxy flavone (XXXIa). Although the position of the maximal absorptions are -scery similar, the molecular extinction coefficients of ginkgetin, isoginkgeting and their derivatives are approximately double those of corresponding simple flavones. This demonstrates the presence of two isolated chromophores of flavones per molecule in ginkgetin and i so ginkgetin.

R R1 (a) H H

7 \VOR- (b) H Me

ULTRAVIOLEI SPECTRA OF ELAVOMES AND BIFLAVONES A MAX IN NM; FIGURES IN PARENTHESIS ARE £

No. Band I Band II

I.Ginkgetin (XVf) 271.5 I[42,200 ) 335 (40,000)

2,Isoginkgetin (XVg) 271.5 [42,000) 330 (36,500) 3.Apigenin (XXXEa) 269 I[18,800 ) 340 (20,900)

4.Acacetin (XXXIb) 269 I[20,300 ) 330 (20,800)

5.Ginkwanin (XXXIc) 269 I[17,000 ) 337 (19,600) 6.Ginkgetin tetraacetate 24B-258 ([34,500 ) 317 (47,000)

7.Isoginkgetin tetraacetate 250 ([41,000 ) 314 (50,500) S.Acaeetin diacetate Z5B I[13,300 ) 325 (26,300)

9.Ginkgetin tetrarosthjrlether 267 <[48,000 ) 328 (45,500)

lO.Apigen trimethyl-ether 265 ([21,200 ) 325 (24,000)

H.Sciadopitysin (XVj) 271.5 <[37,600 ) 330 (35,000)

12.Kayaflavone (XVm) 271.5 <[44,100 ) 329 (41,000) 13.Bilobetin (XVc) 272 I[44,500 ) 337 (35,200) N/50-Ethanolic NaOEt N/500-Eth .ic NaOEt N/5000-Bthanolic NaOEt

No. Band I Band II Band I Band II Band I Band II

1. 284 (46,500) 397 (30,800) 271 (38,500) 340 (30,000) 270 (38,8000) 343 (32,000)

2. 280 (53,500) 376.5(24,300) 279.5(45,000) 378 (45,000) 274 (45,000) 345 (29,000)

3. - 277 (21,900) 400 (31,700)

4. 279 (31,300) 371 (13,300) 278 (32,600) 376 (14,200) 278 (26,600) 347 (14,500)

5. - 269 (13,600) 397 (23,800)

11. 287 (50,800) 378 (16,000) 273*(34,000) 348 (26,200) 272 (35,000) 317 (30,000)

12. 281 (58,000) 378 (20,800) 276 (51,600) 353 (24,200) 274 (48,000) 308 (32,000)

13. 278.5 (49,400) 395 (36,200) 278.5(50,400) 395 (36,300) 279 (57,500) 395 (36,6oo)

* Point of inflection.

t\3 C£2 30

76 Baker et al proposed structure (XXXIIa) or (XXXIIb) for ginkgetin on the basis of spectroscopic evidence and degradation products.

7 \VOR"

HO 0

(XXXII) R R» R» (a) Me H H (b) H H Me

A study of base upon the ultravoilet spectra of the biflavones

(Table-Ill) allowed a decision to be made between the two structures

(XXXIIa) and (XXXIIb) for ginkgetin. The effect of base upon the spectra of simple hydroxyHavone ig already known8 6 ' 87 and following are the results with biflavanoids and some model hydroxyflavones. These show certain common features which Here of value in the structure determination of the biflavanoids. 3

The spectra in neutral ethanol showed two maxinra ., band I

(240-270 ran) and band II (330-340 nm). The effect of base upon the positions and intensities of these two bands is as follows: -

(a) When a T-hydroxyl group is present (XXXIII), band I shifts (270*280 nm) with greatly increased intensity, (b) When a 4'-hydroxyl group is present

(XXXIV), band I shifts (270+280 nm) with reduced intensity and band II

shifts (330-400 nm) with increased intensity.

The effect of base upon 7 - or 4'-bydroxyflavone is to generate the mesomeric anions (XXXIII) and (XXXIV). When a flavone contains both

7- and 4' hydroxyl groups, the effect of weak base is predominantly to remove a proton from the more acidic 7-hydroxyl group. Nevertheless, in

a 7,4'-dihydroxyflavone even with the weak base some effect (XXXIIb) due to ionization of the 4'-bydroxyl group should also be apparent, because

an equilibrium will be established involving both types (XXXIII) and (XXXIV) of anions. In 5-hydroxyflavone the effect associated with ionisation of this

* Band I for benzoyl grouping and band II for cinnamoyl grouping. 88 group will be small , because it is internally hydrogen bonded.

(XXXIII) (XXHV)

The effect of base upon the ultraviolet spectra of three of the

biflavanoids, isoginkgetin, sciadipitysin and ginkgetin recorded in the table has been discussed in details by Baker et al .

bands at 1660 cm" as do those of mono-5-hydroxyflavanoids. This band

is characteristic of 5-hydroxyflavones (XXXV) and although this hydroxyl

group is internally hydrogen-bonded the effect of 5-0-alkylation and

5-0-acylation is opposite to that shown in the case of single 0-hydroxy- ketones. Because of internal hydrogen bonding in O-hydroxy.ketones, the carbonyl bands of these conpounds show a shift to higher frequencies on 33

either O-alkylation or O-acylation. However, a similar comparison of the infrared spectra of 5-hydroxyflavones and 5-hydroxy chromones with the spectra of their 5-0-alkyl and 5-0-acyl derivatives shows a shift

in the opposite direction^ that is to lower frequencies. The reason of the anomaly has been discussed along with monoflavanoids. In practice this effect is very useful in diagnosing the presence of a 5-hydroxyflavone

structure.

OH 0

(XXXV)

(d) Nuclear Magnetic Resonance Spectroscopy;

The application of n.m.r. spectroscopy has proved to be the most powerful tool in the structure determination of bifLavanoids. By the use

go QQ irt of silyl derivatives, ' double irradiation technique , solvent induced

91-94 95

shift studies , and very recently introduced lanthanide shift studies ,

one can come to the structure without tedious and time consuming chemical

degradation and synthesis. The valuable contributions in this field have been made by a number of workers 18' 70 ' 96-99 .

The most commonly occurring hydroxylation pattern in natural

fLavanoids is 4',5,7-trihydroxy system (XXXVI),

(XXXVI)

The chemical shifts of the protons of rings A and B prove to be independent of each other, but are affected by the nature of the C ring. In flavanones,

6,8-protons give a single peak nearT4»05. With the introduction of a

3-hydroxy group (flavanonols), the chemical shifts of these protons are

slightly altered and the pattern changes to a strongly coupled pair of doublets. The presence of a double bond in the ring G of flavones and

CLavonols causes a marked downfield shift of these peaks, again producing the two doublet pattern (T3.3-4.0, J , = 2.5 cps). Out of 6 and 8 protons, the latter appears downfield.

All B ring protons appear around T 2.3-3.3, a region separate

from the usual A ring protons. The signals from the aromatic protons of an unsubstituted B ring in a flavone appear as a broad peak centered at about T 2,55» In flavones, the presence of C-ring double bond causes a downfield shift of 2',6' protons and the spectrum shows two broad peaks, one centered at T 2.00 (2«,6») and the other atT2.4 (3,,4',51)96-

With the introduction of 4f-hydroxyl group, the B-ring protons appear effectively as a four peak pattern. The hydroxy group increases the shielding on the adjacent 3%5! protons, and the peaks move substantially upfield. The 2',6' protons of flavanones give signals at about T 2.65, The introduction of 2,3 double bond (flavones and flavonols) again causes these protons to resonate at much lower field. Such a four peak pattern is called

Ar>, B„ pattern. Introduction of one more substituent in ring B gives a normal AB3 pattern.

The olefenic protons (ring C) of flavones and isofLavones of normal structures give rise to signals nearT3,2 andT 1.7, respectively.

Their position, however, is affected by the substitution in A or B ring, the electron donating groups causing upfield shifts and electron withdrawing groups downfield shifts. The spectra of flavanones contain typical ABX 36 -36-

multiplets arising from a 2-proton and two 3-protons. The 2-proton is

generally a double doublet nearT4.5 i^cia~ 5 cps, J. = 11 cps) the pracise position depending on the substitution in ring B. The two 3-protons

= C S give rise to multiplets of eight lines nearT7«0 (JH o H-3h ^ P )»

However, they often sppear as two doublets since two signals of each quartet are of low intensity. 3-Hydroxyflavones give rise to a doublet

(J = 11 cps) nearT5.1 for C-2 proton and another doublet at aboutT5.8

for C-3 protons. The configuration, therefore, is assigned as trans

(diequitorial).

The proton of a 5-hydroxyl group to a C,-carbonyl of a fLavanoid gives rise to a sharp signal at a very low field (T-3.00) consistent with the strong hydrogen bonding between the two groups. Methylation of a hydroxy! group commonly produces an upfield shift (^0.2 ppm) of the signals of ortho protons with a somewhat smaller effect on those of para protons and little or no effect on the meta protons. Acetylation of a hydroxyl group as expected, causes downfield shift of the ring protons.

In the structure elucidation of biflavanoids certain useflil information can be obtained by comparison of their n.m.r. spectra with 37

those of their corresponding monomers. Such a choice, however, is compelling but by no means infallible.

In biphenyl type of biflavones such as amentoflavone, cupressuflavone and agathisflavone, the peaks of ring protons involved in interfLavanoid linkage appear at somewhat lower field (~ 0.5 ppm) as compared to the peaks of the same protons in monomer due to extended conjugation.

It has been observed both in biphenyl as well as in biphenyl ether type biflavanoids that the 5-methoxy group of an 8-linked monoflavanoid unit in a bifLavanoid shows up belowT6,00 in deuterochloroform, in all the cases examined so far (Table-IV). This observation may be explained on the basis of extended conjugation, 5-Methoxy group of an 8-linked mono flavanoid unit of a biflavanoid of BGE-series WGH-series and GB-series does not show up belowT6.00 as the linkage is through heterocyclic ring. 3

1 A B L E - IV

METHOXY PROTON SHIFTS (T values) OF FOLLY METHYLATED HLFLAVANOIDS

Biflavanoid I-5-OMe II-5-OMe

Cupressuflavone (1-8,11-8) 5.85 5.85

Ament o flavone (1-3',11-8) 6.13 5.94

Agathisflavone (1-6,11-8) 6.H 5.95 Hinokiflavone (I-41 -0-II-8) 6.00 5.92

2,3-Dihydroamentoflavone (1-3* ,11--8) - 5.95

Aromatic protons are completely self consistent in cupressu

flavone, amentoflavone, agathisflavone and hinokiflavone series. The

protons of ring I^B appear consistantly lower than those of ring II-B.

The protons at II-8 in hinokiflavone and at 1-8 in agathis

flavone methyl ether appear at exceptionally low positions,T 2.95 and

T3.09, respectively. Thi3 may be diagnostic of H-8 of a ©-substituted

ring in biflavanoid methyl ether both of biphenyl and biphenyl ether type.

The methoxy group at C-5 agathisflavone methyl ether (T6.41)

and one methoxy in chalcone flavone corresponding to BGH-III methyl ether

(T6.80) and WGH-II methyl ether (T6.56) showed up at exceptionally high

field than the other methoxy groups. This shielding effect is also evident 3

in the case of chalcones BGH-IIl heptaacetate and BGH-II octaacetate

in which the proton of one acetoxy group appear atT8.08 whereas those of others atT7.26-7.80.

The dependency of H-6 of ring II-A upon its mode of bonding

with the other half of the biflavanoid has been observed.

Biflavanoid methyl ether H-6 (ring II-A) C (ring II-A) bonded to '8ft BGH-IIl T3.82 Reduced heterocyclic ring

BGH-II T3.74

WGH-III T3.55 Heterocyclic ring

WGH-II T3.49

Oupressuflavone T3.41, 3.42 1-8, II-8

Amento flavone T3.38 1-3', 11-8

Agathi s flavone T3.36 1-6, II-8

Mass Spectroscopy:

The mass spectra of a wide variety of organic natural products

have been studied only during the last few years. The inlet system

suitable for volatilization of high molecular weight (M+, 300-1200) organic materials has increased the utility of mass spectroscopy.

Generally fragmentation pattern is related to the structures of the intact molecule. Recently a number of papers on the evaluation of

structure-fragmentation pattern relationship in mono-and biflavanoid have appeared 100-103

Flavones;

Unsubstituted flavone (XXXVII) gives the molecular ion as the base peak at m/e 222(100). The base peak shows subsequent loss of one hydrogen to give exi ion, m/e 221 (33) of doubtful structure, while elimirjation of CO gives ion m/e 194 (52) (Chart-I route A). The fission of heterocyclic ring in flavone, gives two ions, one with a quinonoid, m/e 120 (80) and the other a phenylacetylene m/e 102 (12) structures.

m/e 221

(CHART —I) The ion at V© 120 may lose CO to give the ion at, m/e 92 (metastable

peak at 70.5) (Chart-I route B).

Apigenin (XXXVIII) has the parent molecular ion as base peak,

which loses a molecule of carbon monoxide to give a major fragment ion

m/e 242. Fragment ion of much less abundance correspond? to RDA fission in

the heterocyclic ring.

-r-J-

OH 0 OH 0 M+m/e 270 (100) (XXXVIII)

-CO

HOS^^^N^O

HO y

m/e 152 m/e 118 m/e 242 (major)

(CHART- II ) The discrepancy between the results obtained lies in the importance assumed by breakdown via RDA reaction in the natural products as compared with the parent flavone. In the highly oxygenated natural products this fragmentation is minor (15-16$ of molecular ion) whilst in flavone itself peak due to species with a quinonoid structure is 80% of the intensity of the molecular ion. It appears, therefore, that oxygenation of nucleus profoundly influences the breakdown observed.

Presumably, if the initially produced ion radical can be stablized by mesomerism over a number of oxygen atoms, then breakdown via RDA is strongly diminished. These minor breakdowns may still prove to be of diagnostic value as they frequently represent the only even numbered peaks in their particular region and hence"are readily distinguished.

In case of apigenin triraethyl ether (XXXIX), the molecular ion appears as the base peak. This is commonly represented either without precisely defining the location of the charge or with the charge localized on the heterocyclic oxygen. Further fragmentation of the molecular ion by the retero-Diels-Alder process yields the ketone

(m/e 180), the carbonyl ion (m/e 135) and the acetylene (m/e 132) via 43

routes 1,2 and 3 respectively. (Chart-III).

-OCH3-

L H3CO 0 m/e311 0CH3 0 (XXXIX)

c H3CO 0 H3CO d 11 IRDA m/el80 H3C0 0 I ROUTE -II m/e 181 c 0CH3 ^,^VOCH- 3 ^ ~°" ^ CH \=/ &-u_m/<2 135 CH r^^N-°' 3 H C / RDA & 3 ^V°W \V5CH3 > . I • I II IJ \=_/ R0UTE-1II iff ^^ m/e 132 H3C0 y (CHART-HI) Flavanones:

In case of reduced fLavanoids, in which the heterocyclic ring

is no longer aromatic, breakdown by paths A and B are of great

103 importance as they lead to clean cut, characteristic spectra (Chart-IV) .

Path-A >N C II HC = CH 0 2

+CH

( CHART -IV) 103 In the mass spectrum of 5, 7-dimethoxy flavanone (XL) the major

pathway involves breakdown by mode A to give the fragments of w/e

180 and m/e 104, the former containing two methoxy groups taking

most of the charge. This species loses carbon monoxide to give

fragment at m/e 152, the series being terminated by the loss of

methyl radical to give the even electron species at m/e 137.

HoCO HoC

0CH3 ° OCH3 °

m 113.8

H3CO H3ca 0 v^\ A;0 ^> + f\ c LJJ m/e 137 OCH3 0CH3y +CH I

(20.0) m/e 152 m/e180 .CH? (21. 0) (100) m/e j 04(8.2) 4 3

Another method of breakdown that helps to characterise the

fLavanones is the loss of either a hydrogen atom or an aryl radical

from the molecular ion to give even electron fragments.

Ho CO.

OCH3 OH m/e 283 ( 90)

OCH3 OH m/e 207 (1A-3) A very similar breakdown pattern is found for 4'-methozyflavanone

(XLI), once more the fragment with methoxyl group taking nearly all the

charge. Path B is more noticeable, the breakdown scheme being as shown

below (Chart-V). +. +•.

HC->\^ I 0" CH2 H2C M*m/e 254 m/e!2 0 m/e 134 m/«119 (3.5) (100) (13.2)

-CO

^0 OCH3 'H

II OH OH m/

'/ \\&„3_^ ^Y0C"3

m/e108 (10) (XL1)

The presence of a hydroxyl/methoxyl group at C-4 position of ring B facilitates, by enhanced resonance stabilization of the resulting fragment ion, the formation of p-hydroxy benzyl/p-methoxy benzyl fragment

(or its tropolium ion). p-Hydroxy/p-methoxy"benzyl ion appears as peak of significant intensity in the mass spectrum of naringenin/its trimethyl ether (XIII)103.

OH 0 CH2

( XLII ) 4 7

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

is of great interest as it was the first reduced fLavanoid encountered

in which the base peak is neither the molecular ion nor a fragment arising

from breakdown via path A. However, this type of breakdown as well as

path B, are found as shown below:

H0 CH=CH PATH^ T^f°" + 0Hm»12l.5 f^f m<'74. 5 l^ C H3CO+ OH b OH 0 + OH m/e 135(60.9) m/e 302(75.0),M m/e 152 (12.0) m/e 150(89.0) PATH-B m*85.0

^ VCH-CH OH m/e 107 (19.0) m/e 153(65.0) The loss of a hydrogen atom followed by the loss of methyl

radical is of importance, but the base peak is found at m/e 137. 48

OCH3

OH OH m/e 301 (26.0)

OH OH m'e 286 ,95) The metaslable peak at m/e 62.2 indicates that this fragment is found

directly from the molecular ion. Several processes can give rise to

this species.

0 II • HO C- CH CH I rj^rCV Oh C CHOH *C HCO-^^ OH IK 0H 1 ° m/el6.5(1.1) o m/e 137 (TOO) (XLllI)

^W^ OCH3 —> m/e 13 7(100) 4-

0'4° 49

In the case of 2'-hydroxy flavanoids strong intramolecular interactions occur and the breakdown pattern becomes so profoundly modified that it is frequently difficult to classify the substance by

10Z. reference to standard breakdown patterns .

If)/

2'-Hydroxy flavanone (XLIV) showed breakdown patterns A and B as well as the loss of phenyl or hydrogen radical from C-2 to give even electron species, but the base peak was at M -18 and the third largest peak at M 17-19. It has been proposed that these peaks arise by ring opening of the molecular ion followed by ring closure on to the

2'-hydroxy group as shown below, (Chart-VI). J(CHART-VI) m/c22K60 m/e 222(100) 51

Biflavones:

The interpretation of the mass spectra of biflavones should take into consideration the fragmentation modes of apigenin trimethyl ether 62a ' 10' and of biphenyls106*'107'108, or of biphenyl ethers106b'c'108.

102 Seshadri et al have reported the fragmentation pattern in biphenyl and biphenyl ether type biflavones. Molecular ion is usually the base peak. Apart from fragmentation processes mentioned for apigenin trimethyl ether, these compounds also undergo (i) fission of G-G or the

C-O-C linkage between the aromatic residues (ii) elimination of CO (28.0) or CHO (29.0) from the biphenyl ethers and (iii) rearrangements involving condensation between the phenyl rings. Steric factors seem to play an important role in influencing the breakdown mode and internal condensa tion. Formation of doubly charged ions is frequently observed. These factors become so much dominant in agathisflavone hexamethyl ether, that the ion at m/e 311 appears as base peak instead of molecular ion, m/e

622 (90). Main peaks in their mass spectra are given below. Cupressuflavone hexamethyl ether (XLV);

0CH3 6 (XLV)

Main peaks appear at : 622 (100); 621(38); 607(8), 592(18);

576(4); 312(7); 311(H); 245(11); 135(26) and 132(14) (Chart-VIl).

Since cupressuflavone hexamethyl ether (XLV) possesses two apigenin units it is to be expected that the doubly charged molecular ion (M ) vdll be of eonsiderable intensity with one +ve charge located on one oxygen atom in each of the flavone units. However, the singly charged molecular ion (M ,m/e 622) appears as the base peak.

It is surprising that ketone fragment at m/e 180 which should be expected as a result of the EDA fission of apigenin trimetbyl ether unit) is practically absent in the spectrum of cupressuflavone hexamethyl ether . Amentoflavone hexamethyl ether (XL VI);

H3CO

OCH3

OCH3 (XLVI)

Main peaks appear at : 622(100); 621(31)j 607(33); 592(8); 576(10);

312(2); 311(5); 245(5); 135(16) and 132(8)(Chart-VIII).

Tte peaks at m/e 607 and 592 obviously arise by the loss of methyl

groups. The peak at m/e 576 has been assigned the structure (B), a condensation product. Such a condensation product has been reported .' to be formed when amentoflavone is heated with zinc dust. An important reason for a difference in the intensities of such ions in the spectra of cupressuflavone hexamethyl ether (U%) and amentoflavone hexamethyl ether (10$) is that the former is a symmetric type. This results in differences in the steric disposition of flavone unit relative to the other, thus hindering or favouring condensation between the phenyl 5

102 rings . The peaks at m/e 135, 132 and 245 (doubly charged C) arise via RDA fission of C or/and F rings.

The main differences in the fragmentation pattern of amentoflavone hexamethyl ether and cupressuflavone hexamethyl ether are in the intensities of the corresponding peaks due to variations in the substitution patterns and steric factors*

The difference in the relative abundance of the ion at m/e 311

(M , M /2) in the spectra of cupreasufLavone hexamethyl ether (XLVII)

(14$) and amentoflavone hexamethyl ether ( XLVIIl) (5%) are of significance and could be explained as follows:-

The variation in thy oxygenation pattern of the biphenyl residues in these compounds is responsible for differences in the labile nature of the interapigeninyl bond which consequently leads to

10? differences in the relative intensities of the ion at m/e 311 .

Another explanation is also possible for the origin of the peak at m/e 311 in the case of(XLVIII). Double RDA fission of (XLVIIl) yields the .fragment (XLIX) m/e 310 (3) which accepts a hydrogen atom leading to the ion (XLIXa) having m/e 311« Since this ion has the

same elemental make up (C-igH-icOr) as {W%) it would not be possible

to distinguish between them by accurate mass determination.

0CH3 0

OCH3

OCH3 0

(XLVH) M* m/e 622 (100.0)

H3CO 0 H3CO °

7 \Voc H3

OCH3 OCH3

H3CO 0 OCH3 0 ++ m/e 245 (49.0 ) C m/e 576 (A)B

(CHART-VIU 56

°>-^VWH3 0=C-/ E/V0CH3

m/e 135 (16) OCH3

m/e 311 (M>T/2 ) Mt*\/e 6 22 (100) (XLVIII) c 11 0 m/c 180(3)

OCH3

H3C6 y 0CH-,

m/c 2 45 (A90+'f) OCH3 0

m/c576 (10)B (XLIXa)

OCH3 HC5C\>

m/e 311

(CHART VIII) ^

0CH30" m/c 310(3) (XLIX) 57

AgathisfLavone hexamethyl ether ( L );

0 OCH3 v u '

Main peaks appear at: 622(89); 607(54); 591(98);573(24); 561(15);

521(12); 497(24); 325(20); 311(100); 231(12); 245(22) and 135(65).

Steric factors become so much dominant in agathisfLavone hexamethyl ether that the ion at w/e 311 appears as base peak instead of molecular ion m/e 622(90).

Hinokiflavone pentaroethyl ether ( LI ):

X==/ ^J\S 6cH3 8 0CH3 ° (LI) 5

Main peaks appear at : 608(39); 607(12); 593(36); 579(11); 431(7);

327(27); 313(100); 312(22); 311(22); 297'(29); 296(75); 281(22); 181(3);

135(19) and 132(18). Chart-IX),

102

The mode of fragmentation of hinokiflavone pentamethyl ether

(LII) which contains a biphenyl ether system, is considerably different from those of biphenyl type biflavones described above.

The base peak appears at m/e 313 and the molecular ion m/e 608 amounts to 39% of this peak. This could be attributed to the easy rupture of biplzenyl ether bridge followed by hydrogen transfer. Ions at m/e 593 and 578 arise by the loss of methyl groups, m/e 580 and m/e 579 by the loss of CO and CH0 respectively, and m/e 576 by internal condensation.

The ions due to loss of CO and CH0 are not found in the spectra of biphenyl type biflavones. 59

0CH3

H3CO 0 m/e 297(29) kg \ m/. 311(22) »'/«S«('00)

Route I

t H3COv^\

OCH3

OCH3 0 OCH3 0 m/c 281 (22) m/e 327(23) ( C HART-IX) 60

BifLavanones:

Jackson et al 62a ' c have successfully applied mass spectroscopy to elucidate the structure of biflavanoids of GB-series containing two flavanone units linked through CI-3, 11-83 .

GHC - R = OH, R2 m E

GHEa - a « H, R2 = H

GB2 - R1 = H, R2 = OH

GB2a - R1 = R2 = OH

All the features observed in the mass spectra of biflavanoids of

GB-series and their methyl ethers have analogies with similarly substituted monoflavanones. The mass spectrum of GB-I (LIII) heptamethyl ether showed the presence of ions at m/e 121, 154* 181, 312 and 476. The presence of

ions at m/e 154 and 181, consistant with the fragmentsCC/H_(0Me)20H 3

and CC,H9(OMe)pOHCO 3 respectively, supported the presence of 61

phloroglucinol ring system derived from a 5»7-dihydroxy fLavanone

system. An idea about the nature of linkage could also be derived since the fragmentation of molecular ion at m/e 6$6 can be rationalized by

RDA reaction of fLavanones first at ring C to give a fragment ion at m/e 476 followed by a similar fragmentation at ring F to give an ion at m/e 312. (This two stage breakdown pattern is fully substentiated by the presence of metastable peaks). These results can only be accommodated by a linkage from the oxygen heterocyclic ring C to the phloroglucinol ring D, The fragmentation pattern of GB-I heptamethyl ether is shown below (Chart-X). 62

OCH3

H3CO

'OCH3 OCH3 OCH3 0 m/e 476

Mtm/e(656) (LIII)

OCH3 m/e 154 H3CO

Y m/e 312

m/e 121 6

The production of dimethyl pb2 oroglucinol ion at m/e 154 is probably of thermal origin. In fact, phloroglucinol is so readily lost from GB biflavanones that if the temperature of the ion chamber in the mass spectrometer much exceeds the minimum (~220 ) for evaporation of the sample, there is difficulty in detecting the molecular ion. The thermal instability of GB-I, was established by heating it in a tube at

280 and from the pyrolysis products phloroglucinol was isolated and characterized.

As suggested by Pelter , the ion at m/e 312 cannot be due to the formation of apigenin trimethyl ether because fragmentation pattern below m/e 312 of GB-I bears no resemblance to that of apigenin trimethyl ether.

The ions, m/e 1S0 and m/e 132, which could arise by EDA reaction of apigenin trimethyl ether, are entirely absent from the spectra of GB biflavanone methyl ethers.

Flavanone-Flavone type biflavanoid3: cry £{\ A-1

Venkataraman et al , Jackson et al and Vishwanathan et al have reported the mass spectral studies of C1-3, H-83 linked flavanone- flavone type of bifLavanoids. These combine the characteristic fragmenta tion patterns of a flavanone and flavone. The mass spectrum of 6

talbotaflavone hexamethyl ether (LIV) shows fragmentation as shown

in (Chart-Xl).

H3CO H **TO*°^CO ^•m/e152->m/el37(l2) K^C H3CO HO H3C0 0 H3O m/el80 (15) 0CH 3 OCH3

m/e 517(5>(E) +CH2m/e 121 ( 100)

OCH3

H3CO

H3CO.

E ,>-0CH3

H3CO 0 \

M?m/e 624 (38 ) ( LIV )

C = 0 m/c 135(22) (D)

^ccy^ OHT H3cov^r^ OH

H CN „CH2 ^ H3CO JCH3 H3C6 •» H^O^» m/e15A(36) m/c195(92) m/.,8l<8A, ^ 3ll°( ,5)(H ) I (CHART-XI) 65

The mass spectral fragmentations of morellofLavone heptamethyl

except for the increase of 30 mass units in the ions C (3%), D(10#),

E(2#), F(46#) and G(22$) due to an extra methoxyl group in E ring. The

intensities of other ions are A (32$), B(32#), H(13#) and 1(100$).

Thus mass spectral studies of the biflavanoids isolated from natural sources reveal that their fragmentation patterns depend not only

on constituent monomaric fLavanoid units but also on the nature and the position of interflavanoid linkages, while the cracking patterns of

simpler flavanoids are less complex, in application of these concepts to biflavanoids one has to take into consideration the influence of the

additional structural and steric factors.

3. Degradation;

Degradation of flavanoids and biflavanoids can be brought about

either by alkaline hydrolysis or oxidation with alkaline hydrogen peroxide.

Alkaline hydrolysis: In general, a fLavone ( LV) gives four products which arise by

opening of pyrone ring followed by the fission of the intermediate O-hydroxy-n-diketone by two different paths (a) and (b).

0 (LVI )

OH + 0 | B OH \i C II I 0 CH3

The intermediate (LVl) can be isolated if cold ethanolic solution of caustic soda is used.

In the case of biflavones, "ketoflavones" are characteristic 67

degradation products of alkaline hydrolysis. Hydrolysis of ginkgetin

(LVIl) ' gave p-hydroxy acetophenone, 2, 3-dihydroxy-4-methoxy aceto-

phenone and a ketoflavone whose structure was established as (LVIII).

Alkaline hydrolysis of both isoginkgetin and sciadopitysin (LIX) gave

(LVII)

r HaCO^^.OH o l^jJ^^CH3 V^ CH3 HO HO 0 (LVIII)

the same ketoflavone (LX), thus supporting the structure proposed for

110 these biflavanoids

OCH3

H = H, isoginkgetin (LX ) R = Me, sciadopitysin 68

Oxidation with Alkaline hydrogen per oxide:

Alkaline hydrogen peroxide oxidation has been very helpful in the determination of interfLavanoid linkage. Ginkgetin (LVII) tetramathyl ether on oxidation with alkaline hydrogen peroxide 111 gave anisic acid,

2-hydroxy-4-6-dimethoxy benzoic acid and a compound ^JEL^Og.

COOH COOH

^ II + LI] ^yHsOfl Ginkgetin tetramethyl ether.

OCH3 OCH3 The compound ^X/0- was shown to be a diearba^ylic acid, containing three methoxyl groups and one hydroxyl group. To fit in all the spectral data, two structures A and B were proposed for the dicarboxylic acid.

H3CO

(B) 69

These facts proved that biphenyl residue mu3t exist in ginkgetin molecule and that interfLavanoid linkage must involve position V-of one flavanoid residue and 6 or 8 of the other. The linkage Cl-3% 11-63was considered unlikely since II-5 hydroxyl group in a compound with this structure would be severely hindered and there was no evidence that this hydroxyl group in ginkgetin was exceptionally difficult to methylate. The structure with CI-3, 11-83 linkage was, therefore, proposed for ginkgetin tetramethyl ether and structure A for the dicarboxylic acid.

SYNTHESIS;

(A) Ullmann Coupling:

A number of biflavanoids have been synthesised by the application

112 of Ullmann reaction. Nakazawa accomplished the synthesis of amentoflavone hexamethyl ether by mixed Ullmann reaction between 3'-iodo-4%

5,7-tri-0-methyl flavone (LXI) and 8-iodo-4S5,7-tri-0-methylflavone

(LXII). Cupressuflavone hexamethyl ether was obtained as a b%>roduct 70

and was found identical with the one obtained from natural sources.

25 Later on, Seshadri et al have also synthesised cupressuflavone

hexamethyl ether from 8-iodo-4% 5,7-tri-O-methylflavone under modified conditions of Ullmannreaction. Recently CI-4'-0-II-83and Cl-4»-0-II-6D

113 linked hinokiflavone have been synthesised by Nakazawa in his elegent

seven steps synthesis. 71

The permethylated 3,-nitrobi£Lav-anoids (LXEV & LXVl), the key- intermediates were obtained by condensation of 3,-nitro-4l-iodo-5,

7-di-O-iaethylflavone (LXIII) and 8 - or 6 - hydroxy-A'^T-tri-O- methylflavone (LXV). The nitro ethers were reduced by NagS-O- in aqueous DMP, diazotized and decomposed with 50% ELPO3 to give pentamethyl ethers of hinokiflavones. A new route has been very recently reported for the synthesis of

1 "1 / "11^ cupressuflavone and agathisflavone hexamethyl ethers. This involves Ullmann coupling between two molecules of I-iodo-2,4> 6-tri- methoxy benzene (LXVIl) to form a biphenyl system (LXVIll) as the first step. Subsequent Friedal Craft's acylation, partial demethylation and condensation with anisaldehyde leads to a biehalcone. Oxidative cyclisation of this bichalcone by SeOg gives the biflavone hexamethyl ether (CHART-XII).

•1

(+) Hepta-O-methylfukugetin and hepta-O-methyl-saharanflavone have been synthesised in the following manner. Luteolin tetramethyl ether (LXIX) was chloromethylated with paraformaldehyde and hydrogen chloride to give a 8-chlorometbyl compound (LXX), Treatment of (LXX) with KCK and subsequent hydrolysis yielded an acid (LXXI) which was converted into the acid chloride (LXXII). Esterification (LXXII) with phloroglucinol dimethyl ether gave (LXXIII) which underwent the

Fries rearrangement in the presence of titanium tetrachloride in nitrobenzene at room temperature to give the required ketoflavone(LXXIV).

The ketoflavone was condensed with anisaldehyde in the presence of 50$

KOH in pyridine to give a chalcone (LXXV), Finally this was cyclized in boiling 2,5% aethanolic sulphuric acid to give a biflavanoid (LXXVI).

This was proved to be identical in the (+) hepta-O-methylfukugetin.

Cyclization of (LXXV) with anisic anhydride and sodium anisate

(Allan-Robinson method) afforded the required biflavanoid (LXXVIl) which was also obtained by the oxidation of (LXXVI) with iodine pot, acetate in acetic acid. The synthetic biflavanoid was identical in the hepta-0- methylsaharanflavone. 74 OCH3

OCH3 OCH3 CH2.Cl H3CO OCH3—> OCH3

H H3CO 0 (LXX) 3P0 0(LXIX) CH2COOH CH2CN OCH3 OCH3 H3CQ^\/0 OCH3

H3C0 d (L xxn

CH2C00R 0CH3 CH2COCI 0CH3

H3CO 0 (LXXIII) H^CO 0 (LXXII) OCH3 R^ J/ V H3C0 OCH3

(B) Oxidative Coupling:

117 Recently , attention of organic chemists has focused on

the synthesis of natural products by procedures which simulate certain

steps of a proposed biosynthetic pathway, rather that discovering new

reagents and reactions. The application of phenol oxidation to synthetic

chemistry has, therefore, been extensively studied.

It has been experimentally established that in the phenol oxida tion mechanism, the phenolate ion is oxidized, by an electron oxidant like ferric chloride or potassium ferricyanide, to a phenoxy radical.

The free electron in the phenoxy radical may be shown at various places

by mesomeric effect* The free radicals are than coupled rapidly and

irreversibly to dimeric and polymeric products under kinetic control.

It is reasonable to assame that coupling occurs fastest at the positions of highest density of the free electronsexcept where there is steric hinderance of approach.

Dimerization of phenols may involve carbon-carbon, carbon-

oxygen or oxygen-oxygen bonding as illustrated in the following simple

examples. (a) Carbon-carbon coupling; Para-^ara

»°\j- *•

Ortho-ortho

OCH3 H3CO OCH3

H3CO" H3CO OH QH 0CH3 OH

Ortho-pars 78

(b) Cjarbon-oxygen coupling;

R I COOH •^ 0=/ Vo-/ YfCH^COOH

H 0 0-U /A \\ /V(CH2)ACOOH+(CH3)2 = Cri2

R= -(CH3)3 R I

0— 0

A large number of exanples of biogenetic type synthesis involving

C-b, C-0 and 0-0 coupling have been reported in lieterature. These include

bisnaphthols, bisterpenoids, bisanthraquinones and bisflavanoids. The parent 79

biflavones, amentoflavone, cupressuflavone, agathisflavone, and

binokiflavone together with various O-methyl ethers exhibit either

C-C or C-0 linkage between the flavanoid units which might be expected to arise through oxidative coupling of an apigenin derived radical

(LXXVII or LXXVIII) by three of the many modes of dimerization theoritically possible, as shown below:

ir\\ //-°'

OH 0 t

HO 0 1 HO o HO 0

(LXXVII) (LXXVIII) 80

Mblyneux et al have reported that the oxidation of apigenin with alkaline potassium ferricyanide gave two novel dimers, namely, CI-3, II-3J

(LXHX) and CI-3, 11-3*3 (LXXX) biapigeninyls, but no naturally occurring biapigeninyl was formed

(LXXIX) (LXXX)

The synthetic compounds (LXXIX) and (LXXX) appear to arise by oxidative coupling of the radical (LXXVII) although none of the symmetrical

CI-3', II-3'D linked dimer, which might also be expected to be formed, could be isolated. These observations are consistent with the findings of Khunle

118 et al who studied the EPR spectra of flavanoid anion radicals (derived from polyhydroxyflavones and having a 5-hydroxyfunction) and concluded that 81

the electron density is mostly located in the 3-position of the heterocyclic ring and in the side phenyl positions of the heterocyclic ring. Moleneux et al 65 , have, therefore, suggested that biosynthesis of natural bifLavanoids does not involve radical coupling of apigenin but probably takes place by the electrc- philic attack of an apigeninyl radical on the 6 or & positions of another molecule of apigenin,

119 Pelter et al have tried oxidative coupling of V-nydroxy-T-methoxy- flavone using potassium ferricyanide and got back the starting material.

120 Seshadri et al have recently carried out oxidative coupling of apigenin

7,4*-dimethyl ether with ferric chloride and isolated dimer in 6% yield. The structure of its methyl ether has been established as CI-8, II-8 D biapigeninyl hexamethyl ether by NMR studies. However, a direct comparlsion of the dimer and its methyl ether with authentic cupressuflavone tetra and hexamethyl ether showed that they are not identical. It appears to be definite that cupressuflavone hexamethyl ether and the dimer methyl ether are isomers with the synthetic ones. Studies are underway to settle this problem. On the basis of these findings, Seshadri et al have suggested that when hyddoxyl groups are protected eg. by methylation (leaving only the 5-CH free), dimerization takes place through 6 or 8-positions of the

A rings. It is reasonable to expect that in nature adequate mechanisms are available for protecting the hydroxyl groups and bringing about the coupling through the positions in A ring. C-GLICOSYLFLAVONES 83

The term glycoside was adopted to embrace a large and remarkably varied group of organic compounds which on hydrolysis yield, in addition to sugar other substances, frequently of aromatic nature* The non sugar part

(aglycone) may include a wide variety of compounds occurring in nature* In the case of flavanoid glycosides this moiety is generally a phenolic compound.

A large number of glycosides which were earlier isolated from natural sources and studied were O-linked glycosides. In these the sugar portion is linked to the aglycone through the oxygen of a phenolic or alcoholic hydroxyl group* A noTrel and stable type of glycosides has : been discovered and these are distinguished by a direct C-C link between the sugar and non sugar part.

The relationship between O-and C- glycosides is indicated below.

- 0-H H - 0 - C6Hl105 - 0 - C6H1l0? 0 - glycoside

^ OH H-0-C6Hl105 frC-C^O C - glycoside Some of the C-glycosides have been known for over a hundred years in crystalline form, but it is only during past 14 years that their special nature has been understood. They are rapidly increasing in number and they seem to occur widely. The aglyeone involved belongs to different types, and so far, representatives have been found in the groups cf enthrones, anthra- quinones, flavones, flavonols, flavonones, dihydrochalconas, isoflavones, xanthones and iso-coumarins. 85

CLASSIFICATION: 6 (a) Flavones and Flavonols:

Compound IH, \n0 HUM R« T~R $R i Reference J. V v 7 ViterLn H OH H H H glu H 121 Isovitexin H OH H H glu H H 122,123 Orientin H OH H OH H glu H 124,125-132 Isoorientin H OH H OH glu H H 123,130,132 Parkinsonin A H OCH- H OH H glu H 131

Parkinsonin B OCRj OCH3 H OH H glu H 131 Lucenin-1 H OH H OH glu glu H 133 Violathin H OH H H hexosyl hyxosyl HH 134 Swertisin CH, OH H H H glu H 132 I so swert isin CR. OH H H glu H H 132 Swertiajaponin CE. OH H OH H glu H 132 Isoswertiajaponin CH. OH H OH glu H H 132 Bayin H H OH H H glu H 135 Cratenacin H OH H H H glu-0-glu H 136 8-C-p-D-glucopyranosyldiosmetin H OH CiL OH H glu H 137 6-C-p-D-glucopyranosyldiosmetin H OH CH_ OH glu H H 137 O-Rhamnosyl-6-C-xylosylapigenin H OH H xyl-,0-rham H H 138 0-Rhanmosyl-6-C*xylosylluteolin H OH OH xyl-0-rham H H 138 Saponarin glu OH H glu H H 139-142 Scoparin H OH H OCH- H glu H 143 Lutonarin glu OH H OH glu H H 127-129 Cytisoside H OH CH OH H glu H 144 Keyakinin Ctt, OH H H glu H OH 145,146 6,8-Di-C-p-D-glucogy^ano^yl- H OH H glu glu H 147 86

Re n (b) Elavanone: N 1 ft VoR3

"sVV ** 0R2 0

Compound \ R1 J Rg I R3 I R^ J R5 j R6 j R? j Reference"

Hemiphloin H H H H gLu H H 148 Isohemiphloin H H H H H glu H 148 Keyakinol CR, H H H glu H OH 149

Puerarin H H OH glu H 149 Di-O-acetylpurarin H H OAc H glu 150 Paniculatin H H OH glu glu 151 (d) Dihydrochaleone derivatives;

Aspalathin (LXXXI) is the only known C-glycoside and occurs in

Aspalathin lenearis ' .

"O^^OH CH? CH?

(LXXXJ)

(e) Kanthone derivatives;

Mangiferin (Hedysaride) (LXXXII) was isolated from Mangifera indica ,

•ice "\Kf\ Selicia prinoids and Hedysarum abscurum , while isomangeferin (LXXXIII)

157 was isolated from Anemarhena aaphodelaides . CH2OH

G OH (Lxxni) (LXXXIII)

(f) laocoumarin derivatives

Bargenin (LXXHV) is the only known C-glycoside which was isolated from Bergerea cilliata, B.strechi , Connarus monocarpus , Vateria

160 1Al indica and Berginia crassifolia .

(LXXXIV) (g) Anthracene derivatives

162 Barbaloin (LXXXV) was isolated from Rhemnus purshiana

OH 0 OH

CH20H

HOHpc/ /

HO OH (LXXXV)

(h) Anthraouinone derivatives

Carminic acid (LXXXVl) differs from other C-gLycosyl compounds is being an animal product and not a plant product as is usually the case. It is obtained from the insect Dactylopiuscoccus costa ' .

OCH3 0 HOOC

(LXXXVl) 90

(i) Pyrimidine derivatives

Pseudouridine is a C-gLycoside of uracil present in transfer

ribonucleic acid and is represented as (LXXXVII).

NH NH

H0H2HC^ OH (LXXXVII)

BIOSYNTHESIS OF C-GLYCOSIDES;

The formation of a C-glycosyl compound seems to involve a C-glycosylation process analogousto C-alkylation and can occur either before or after

synthesis of the aglycone, the former route would involve substitution in a poly-fi -ketone precursor. Although no strong argument in favour of one or other route is available at present, the latter route i.e. C-glycosylation of the aglycone seems more probable.

The sugar residue of these substances are attached to highly anionoid centres in the aglycone, most probably as an ultimate step in the biosynthesis* 91

The anion of the phenolic aglycone by interaction with a derivative of a

1-phosphorylated sugar would furnish the 0-and C-glycosides respectively in a manner exactly analogous to 0- and C-alkylation.

(LXXXVIII)

On analogy with other G-C bond forniing reactions in organic synthesis it is conceivable that the glycosylation might under physiological conditions, be directed towards an anionoid carbon atom as much as towards an oxygen atom.

In this connection it is of biogenetic interest to note the co-occurrence in plants of many C-glycosyl derivatives with their 0-glycosides.

The O-glycosides undergo rapid hydrolysis in the digestive system of animals and their medicinal properties are, therefore, considerably affected. They have, therefore, to be administered by injection. This dis ability does not exist in the cases of C-glycosides. They are generally stable to the action of digestive juices and can function as such in the system. 92

THE ISOLATION OF C-GLTCOSYL FLAVANOIDS;

The isolation of C-glycosyl compounds from plant material involves two main operations (a) solvent extraction (b) purification from column

chromatography.

(a) Solvent Extraction:

Any single solvent extraction is not suited to all the plant materials.

In general the fLavanoid compounds of fresh or desiccated plant material can be completely extracted by means of ethyl alcohol, but it is often advantageous especially, when dried material is used, to carry out a systematic series of extractions with the use of three or four solvents of increasing polarity. In order to achieve a complete extraction generally a soxhlet extractor is used.

(b) Purification;

Chromatographic Methods;

During the past two decades considerable improvements have been made in the methods for the purification and characterization of flavanoids by the use of chromatographic and partition techniques. The C-glycosides by 93

virtue of inherent property of developing colours in the visible and ultraviolet light, are ideally suited for chromatographic analysis. It is the method of choice for separating mixtures especially when working with small quantities. In recent years chromatographic techniques have

been found to be so versatile that they have completely replaced the classical methods. The usefulness of chromatographic methods is enhanced by the fact that they can be employed for characterization purposes,

(i) Column Chromatography;

In the study of C-g3ycosyl compounds the first method to be used was that of column chromatography. The adsorbant used were magnesol , polyamide , silica gel , cellulose podder and ion exchange resin ' ' ' .

(ii) Paper Chromatography:

Among the absorption methods, paper chromatography has been extensively used by various workers for the isolation and identification of flavanoid glycosides.

The paper chromatograpic methods of analysis involve a number of techniques. One dimensional paper chromatography is used frequently for 94

preliminary quantitative study. The two dimensional paper chromatography using two different solvents in succession is particularly suitable for the study of C-glysides in plants. The organic solvents generally used are polar in character and water is always one of the constituents. Butanol:acetic acid: water mixtures are the most common solvents used in the study of these pigments.

The spots or bands obtained after the development on paper chromatograms are observed either in ordinary light or under ultra violet light. Spraying or

170 fuming is generally done to obtain more deeply coloured spots .

(iii) Thin-layer Chromatography:

Thin layer chromatography on polyamide has been used by many workers ' for the identification of fLavanoid glycosides by comparison of R„ values with authentic samples. Methanol:water:glacial acetic acid (90:5J5) and ethyl methyl ketone: toluene: glacial acetic acid: methanol: water (80:10:2:5:6) have been found as the most satisfactory developing solvent systems both for quantitative as well as qualitative purposes. '/ 95

CHARACTERIZATION:

The chief perplexities encountered in the identification and the determination of the structure of C-glycosyl compounds are those to be expected in the handling of polyhydroxy compounds, namely difficulties in purifying the starting material and uncertainties in the determination of the degree of hydration and the number of hydroxyl groups. For the latter, complete methylation and subsequent methoxyl determination seems to provide a most reliable method. The recognition that the compound has a C-glycosyl structure generally comes from the following evidences:

The compound is polyhydroxylic, but gives no sugar on the attempted acidic or enzymic hydrolysis, yet it does give a sugar on mild oxidative

17Z. degradation with ozone or aqueous ferric chloride. Bate-Smith and Swain ^ have described the paper chromatographic behaviour of two substances which occur in acid hydrolyzates of the leaves of many monocotyledonous plants, especially in the Liliaceae and the genus potentilla (Rosaceae) and Lathyrus

(Leguminaceae) and have suggested on the basis of this behaviour that these 96

substances are C-glycosyl derivatives of the flavonols (LXXXIX)

Kaempferol (R=H) and quercetin (R=PH) .

Prolonged treatment of C-glycosyl compounds although fails to release any sugar, but leads instead to a slow partial conversion into a different C-g3ycosyl derivative, inalogous Wesseley-Moser interconversion have been observed e.g. vitexin (XC) to isovitexin (XCl) orientin (XCII) to iso-orientin130(XCIIl).

(LXXXIX)

'/ VOH

OH OH (XCl) 97

OH 0

(xcri) (XC1II)

Various methods generally used for structure determination of

C-giycosides may be classified as under:

(1) Colour reactions (2) Degradation (3) Physical methods

(1) COLOUR REACTIONS:

C-glycosides give the same type of colour reactions as are reported

earlier for bi flavanoids.

(2) DEGRADATION:

Degradation of flavanoid can be brought about by alkaline hydrolysis or by alkali fusion. 98

(a) Alkaline Hydrolysis;

In general a fLavone (XGIV) gives four products which arise by

opening of the pyrone ring followed by the fission of the intermediate

O-hydroxy-(3-diketone (XCV) by two different paths (a) and (b).

Vo? \ O-Hydroxy- p-diketone ( XCV ) fY o* ^VCH» or.C-OH -CH3 ID ©I II OH 0 Acetophenone O-Hydroxy benzoic acid O-Hydroxy acetophenone benzoic acid

The intermediate can be isolated if cold ethanolic solution of caustic

soda is used. If on the other 4iand, a normal solution of barium hydroxide is used as the degradating agent, then the products are usually salicylic acid and acetophenone.

On treatment with acetic anhydride and sodium acetate at 100°C, vitexin (XCVI) gives a hepta - acetate which shows no hydroxyl absorption band in its infrared spectrum and gives no ferric reaction* With acetic anhydride and pyridine at 100 , vitexin gives a penta-acetate (XCVII) which shows a positive ferric reaction and which, with methyl iodide and potassium carbonate ia boiling acetone (twice) affords tetra-O-acetyl- tri-O-methyl vitexin (XCVIII) which also shows no hydroxyl absorption band in the infrared region. Deacetylation of the tetra-acetate gives tri-0- methyl vitexin (XCIX) which gives no ferric reaction.

On treatment of tri-O-methyl vitexin with hot sodium hydroxide

(aqueous) gives p-methoxyacotophenone (C) and p-anisic acid (CI), the side chain being removed from the aromatic nucleus, but with boiling aqueous barium hydroxide, an additional product, di-O-methyl-apovitexin (CII) is obtained. Thi9 ether, when treated with an excess of aqueous periodic acid, gives 3-formyl- 4, 6-di-0-methylphloroacetophenone (QIII), identical to

with a synthetic sample. Oxidation of tri-O-methylvitexin (XCIX) with lead tetra-acetate in acetic acid at room temperature for 5 days, or

with dilute nitiic acid at 100 for 15 hours, yield, 8-formyl-tri-0-methyl

apegenin (cIV). These reactions which lead to the partial structure for vitexin are summerized below.

The nature of the CJH_(0H). side chain was deduced mainly from the results of periodate oxidation studies. Direct oxidation of vitexin in aqueous solution with either periodic acid or sodium metaperiodate gives variable results. These indicate an initial, rapid uptake of 1.5 to

2 moles of oxidant per mole, apparently without formation of formic acid.

It was also noted that small proportions of volatile acid are formed on further oxidation. Oxidation of a methanolic solution of vitexin with sodium metaperiodate results in the precipitation of a compound (yield 50#) whose molecular formula, Cb-| Ho8°10' corresPonds *° tne oxidation of a cyclic 1,2-glycol system and which was accordingly named dehydro-secovitexin. 10

C6H70(OH)4

H0 0v V^>^ h—^M-OH AC2 O/NaOAc /100° epta acetate

OH 0 (XCVI) vitexin

AC2O/Pyridine/100c

C H 0(OAc) C6H70(OAc)A 6 7 A 0 MeO ^ // W OAcMeI/K2Co3 OMe in boiling acetone OMe 0

(XCVI1I) NH3/MeOH

V 0=CH C6H7(OH)4 MeO, 0Me Pb(OAc) MeO A OMe acetic acid

OMe OMe 0 (XCIX)

8- Formyl - tri- O-methyl apigenin ( CIV ) Ba(OH)2/H2O/100° NaOH

0=CH CeH 0(OH)A OH 7 MeOYAv J1I04 MeV^V°H IPX** D-TV OMe H3 I V—/ II l^ C-CH3 CH3 OH OMe 0 OMe jj p-Anisic acid (CUD p-Methoxy Di-O-methyl acetophenone ( CI) 3-Formyl-A,6di- apovitexin -O-methyl phloro- (C ) acetophenone (err) t02

Unlike vitexin, dehydro-secovitexin is unstable to acid. Since

vitexin contains no C-methyl group, this is an artefact, and it was shown

that dehydro-secovitexin gives D-glyceraldehyde dimethyl acetal. This acetal

gives the pyruvaldehyde derivative on treatment with (2, 4-dinitrophenyl

hydrazine).

On being heated x-jdth methanolic sulphuric acid, dehydro-secovitexin

gives the three products: D-glyceraldehyde dimethyl acetal and two isomeric,

optically active, crystalline substances, A, C ^H O,(0Me)?m.p.36O°, and

B,dec. 188-190°.

Compounds A and B contain three hydroxy! groups, two of which are phenolic,

177 Robertson et al suggested that the two compounds have the hemiacetal

structure (CV), the two isomers differing only in their configuration,

CHOMe MeCH ^9H0H •o [

OH 0 103

about the starred carbon atom. Both A and B were considered to be derived

from (CVI), itself formed by the msthanolysis of (CVII).

0=CH 0=CHH-C = 0 MeO-CH I i i HO.CH CH.O.CH.CH2OH CHOH

(CVI)

This evidence leads to the full structure (CVIII) for vitexin.

HO-CH CH.OH I I CH CH.CH2.OH 0 CHOH 04

173 Rao and Venkateswarlu have re-examined the periodate oxidation of vitexin. They claimed that the initial uptake of meta- periodates is two moles per mole and that this is accompanied by the formation of between 0.64 and 0*82 mole of an acid which they supposed to be formic acid, Briggs and Cambie 1779 had already shown that an acidic product, reported by Robertson and coworkers 177 as being formed during the periodate oxidation of vitexin, is, in fact, formic acid, but the proportions formed were stated to be small and were thought to arise

178 from over oxidation. Rao and Venkateswarlu do not share this view, they believe that their results point to the presence of a -CHOH-CHOH-CHOH system in vitexin. This led them to propose the C-glycosyl structure

(CIX) for vitexin.

CH (CH0H)3 CH-CH20H 105

The only evidence advanced in favour of these reformulations was garnered from elementary analysis, and, since the differences in the theoretical, analytical figures are not great, the case for the revised structure can hardly be said to have been proved. However, it is of

180 considerable interest that Koeppen has recently shown that the tetramethyl ether of orientin consumes only 2.0 moles of periodate per mole, with the formation of 1 mole of formic acid when the oxidation is carried out in aqueous solution with only a three fold excess of periodate, and it may well be that a careful study of the periodate oxidation of tri- o-methylvitexin under these new conditions will resolve the present situation.

Unpublished work on a small sample of vitexin has shown that, on vigorous treatment with acid it forms D-glucose, and this discovery establishes the configuration of the side chain.

(b) Alkali Fusion;

Flavones undergo hydrolytic fission by fusion with caustic potash or by heating in sealed tube with 30% alcoholic solution of NaOH 106

or by refluxing for a few hours with 30-50$ KOH solutiion. This method has been widely used for establishing the structure of various polyhydroxy aglycones.

OH 0

Chrysin Phloroglucinol Benzoic acid

The above method is not suitable for determining the structure of methylated aglycones, because it has been reported that methoxyl group is sometimes knocked out during the process leading to demethylated product.

Thus acacetin (8X) gives p-hydroxy benzoic acid (CXI) and phloroglucinol (CXIl) by alkali fusion. Bat on the other hand, if the degradation is carried out by refluxing with concentrated KOH solution

(25$) the products are phloroglucinol (CXIII) and anisic acid (CXIV). H)7

o HO OH C-(J~\

(CXI) OH (CXII )

0 cY/__\yocH3 HO ^ '

(CXIV ) 3. PHYSICAL METHODS:

Chromatographic and spectral methods (i.e. UV,IR and NMR spectroscopy) have been applied in the identification and structural analysis of plant pigments. They provide an excellent tool in the hands of organic chemist for elucidation of structure of even minor constituents.

(a) Chromatographic Methods;

175 Recently paper chromatography and paper electrophoresis have been used in distinguishing between 6-and 8-C-glycosylflavones. 10

6-C-Glycosylflavones on paper chromatography in aqueous solvent and on paper electrophoresis in aqueous sodium borate migrate faster than

8-C-glycosylflavones (Table-V).

(b) ultra.violet Spectroscopy;

Flavones and flavonols have generally been found to eadiibit high intensity absorption in the region 320-380 nm (Band I) and 240-270 nm

(Band II). As the introduction of hydroxyl or methoxyl groups in the molecule of a flLavanoid produces bathocbromic shifts in the absorption bands, the determination of U.V. spectra has been of considerable use in the detection and location of these groups in flavanoids. Band II of

flavones and flavonols which contain only 4'-substituents in the ring B e.g. apigenia or kaempferol has a well defined peak. For flavones and flavonol which have -OH or -0CH- substituents in both 3' and 4' -positions eg. luteolin or quercetin, band II shows two definite peaks or one peak and profound inflecfcidn ..

The identification of completely alkylated polyhydroxy flavones and flavonols is based on the spectral data of these compounds 109

in neutral solvents, because these compounds are devoid of ionisable or chelatogenic groups in their molecules. The spectrum of acetylacted polyhydroxyflavone or flayonol has been found to be similar to that of flavone itself e.g. quercetin penta-acetate (> max. 300, 252 nm), flavone

("X max 297, 250 nm)« It is because the absorption due to phenolic hydroxyl groups is nullified by the introduction of acetyl groups.

182 It has been observed that the use of reagents like A1C1 ' sodium ethylate , fused soditmyacetate ** and boric acid -sodium acetate can be of considerable help in the characterization of plant pigments.

These reagents produce shifts in the absorption maxima depending upon the location of the functional groups in a flavanoid molecule. The flavones and flavonols which do not contain free hydroxyl groups in

5- or 3- positions do not form complexes, with aluminium-chloride.

Consequently there is not much difference in the absorption spectra of these compounds. 5-hydroxyflavones and 5-hydroxyflavonols in which

3-hydroxyl group is protected from aluminium complexes with aluminium chloride resulting in bathochromic shifts of absorption bands. Sodium 1

acetate can ionize hydroxy! groups located at positions 7-,3-and 4'-of the flavone nucleus. The ionization of 3-and 4' hydroxyl groups by sodium acetate produces bathochromic shifts of band J,but the position of band II remains unaffected. As the ionization of 7-hydroxyl group produces a pro nounced bathochromic shift of band II, the flavones and fLavonols possessing a free hydroxyl group at this position are detected by the 8-20 nm batho chromic shift of the low wave length band.

Similar bathochromic shifts caused by the action of reagents, sodium ethylate and boric acid-sodium acetate on Havanoids have been reported and found helpful in obtaining structural information. Flavanones are colourless compounds and absorb at comparatively short wave lengths.

Since hydroxylation in the 2-aryl group has very little influence on the position of maximum absorption of flavanones, the use of absorption spectra in this class of compounds is largely limited to classification of types and, therefore, cannot be successfully applied to structural analysis. 11

T A B L E - V

MIGRATION OF FLAVONES AND C-GLYCOSYLFLAVONES ON PAPER CHORMATOGRAPHY & ELECTROPHORESIS

- . |10# HOAC I Rf value in{HJOH:HOAC:H JEtoHiH.CO H:H J Electrophoresis ompoun g pp^HOAC } (a0:6.15) * j (10i2;3)a ^f migration

8-C- p-D-glucopyrano- 0.13 0.42 0.58 0.39 0.48 syldiosmetin

6-C-/3 - D- glue opyr ano- 0.30 0.61 0.68 0.47 0.75 syldiosmetin

B 2 -0- prD-xylosylvite- 0.65 0.74 0.62 0.39 0.86 xin

6,8-Di-C-glygosylapi- 0.50 0.68 0.55 0.15 1.8a. genin

6, 8-Di-C-glyeosyldiosme-0.39 0.64 0.52 0.13 1.35 tin

Apigenin 0.03 0.28 0.94 0.87 0.30

Diosmetin 0.02 0.23 0.90 0.86 0.08

Vitexin 0.20 0.46 0.63 0.43 1.00

Isovitexin 0.20 0.63 0.72 0.71 1.31

Orientin 0.12 0.38 0.42 0.28 1.15

Isoorientin 0.27 0.56 0.55 0.38 1.50

(a) Upper Phase (b) In 0.1 M aqueous aodium borate, ca,9.00 v, 20-40 m. A,, ca. 3 hrs. Whatmann No.1.paper. The migrations are given relative to vitexin * 1.00. 112 en 8 8 en in to CM O sO OS to CM u\ UN to " UN s? UN UN H en en *en 8 (A en en $ O 8* 3* «* * •> sO UN o> o o CM sO UN sQ. NT UN "1 8? en en en en £ & en Cn o sO UN •» * CO•« to« * UN«\ u\ UN en Q O en o O O en en $ « IS- CM CM .«» 8 8 en en ON ON CM UN $3 •» * T*«»» «0•» 5 UN u\ so Cs. en seOn sO C» CM CM & n CM CM CM CM CM CM CM CM O

UN. o -4- vO O CM 3 5 s UN UN CM en 8 o 9 CN, m en 8 ef « en en en en en •V en 8•f c •I •k o r- en •* _•» en * •> vO sO 28 0 & 8 $ CM & CM CM 27 1 28 1 CM CM eOnN sO UN UN UN -** NO O o f}? m en $ en CM ON sO to en CD •> en en•V 8 «0 -t UN •* _* * * •» •* IA^ 1— »A O *— r— £ r- CM CM o o en £ C£M o o c^ en •t en en•s m en R £ o r- ITV ^ CN.%. ON * •» * * c^ sO to to o o CA CM cxT c^ to CM n fc fc CM n O U\ en >ft vfl tO >t 2 3 ON -5- O >T o CM en en en en en en en ^ *v •* ^ CM os CM o en v\ «o NT W\ *p o o ON to to CM S r S N en en o t- OS 8^ so N sO SO »• en 33 6 CM en CM en CM SM 33 3 en en en en CM CM CM CM CM en w en to CM -4- to £>- t> ON UN CM Q CM U\ * v \ so$ VS UN v\ vrs VN so UN UN UN "««• UN UN 5f

CM CM CM CM c\i ?M CM CM CM CM CM CM CM CM CM CM 270 , CM 5 113

186 Hergent and Kunth have measured the infra-red frequencies of a series of fLavones and related compounds in nujol or perfLurokerosene mull. It has been observed that the unsubstituted flavonea show an absorp tion band at 1680 cm-'. Introduction of hydro:xyl groups in the 3'-and

41-positions causes a carbonyi frequency shift to 1655 cm . Acetylation

of these groups causes a shift back to 1680 cm , a frequency identical with that of unsubstituted fLavones.

The flavone derivatives do not show marked lowering of carbonyi frequency when a hydroxyl group is present in the 5-position.

Acetylation of hydroxyl groups decreases rather then increases the carbonyi frequency. The 5-hydroxyl group is involved in chelation is apparent since the OH band is absent in 5-hydro3qr-3,3,,4l,7-tetra- methoxy flavone. Introduction of a methoxyl group in the 5-position

-1 ' causes a shift to 1627 cm for3,3>5>4%7-pentamethoxyflavone. This

_-) value is 22 cm lower than that of the corresponding flavone derivative.

The lowering is due, at least partially, to increase conjugation which 1

is not possible in the flavanones.

(d) NMR Spectra of C-glycosides;

Although C-glycosylflavanoids occur widely in nature, progress with the elucidation of their structures has been slow and few have been completely worked out. These substances yield very small amount of sugar when hydrolysed or degraded. Consequently the nature of the sugar moiety and its location have been largely deduced indirectly and conclusions have been dependent on several uncertainties, such as isomerization, which can occur during examination of these relatively unstable compounds.

Accordingly, the nor study has been made of a number cf these substances and related model compounds to provide more direct information about the structure of this class of compounds.

The C-glyco3yl flavanoid acetate can be prepared under mild conditions and are easily soluble in deuterechloroform unlike the parent compound which can only be examined satisfactorily in deuterodimethyl sulphoxide or deuteiopyridine -,. However, as the acetates are of much higher molecular weight, there is some loss of sensitivty but as will ii5

be seen, the position of the signals from protons of acetyl groups provide useful information.

The aromatic and acetyl signals of C-glycosylfLavanoid acetates

fall conveniently in different ranges of the nmr spectra, the former between S>2.30 andS2.40 and the latter between 61.67 andS-2.09. Massicot

187 et al have shown that the signals of the 4' sad 7-acetoxyl protons in simple flavone acetates are in the range 82.30-S 2.35, while those of 5-acetoxyl protons are at S 2.45. In the spectra of C-glycosylflavanoid acetates such as bayin acetate (Table VII) which have 4' and 7-acetoxyl groups but lack a 5-acetoxyl group the signal found at 6 2.37 is assigned to the 4'-acetyl, and that at8 2.45 to 7-acetyl, since the latter only is likely to be shifted by the presence of the sugar ring.

The remaining structural questions have to be dealt with the identification and point of attachment of the glycosyl residue. In case where the identity of the sugar residue of C-glycosyl compounds has been established, it has been found to be fi -glucosyl. 11

(Table VIII) catalogues the band positions of the aliphatic acetyl methyl groups of various acetylated 8- and 6-C-glycosylflavone

(group A and B respectively) and of a number of miscellaneous acetylated

C-glucosyl compounds (group C). The acetyl bands of 8-substituted fLavones form a pattern quite distinct from that of the acetyl bands of the 6-substituted and miscellaneous groups. Thus, in 8-C-glucosylflavones both the

2«-0-aeetyl band (51.70 -51.73) and the 6n-0-acetyl band (S 1.90-1.95) occur at consistently higher field than they do in the other groups of compounds (S1.77-1.83) for the 2"-0-acetyl in groups B and C and (61.98-2.04) for the 6rt-0-acetyl in (groups B and G). There is, in fact, essentially no difference in the spectra of the group B and C compounds in spite of wide variations in structure. The distinct character of the spectra of the group A compounds must be due to some special structural feature, mo3t likely the proximity of the glucosyl residue to the B ring of the flavone.

The unusually high field position of the 2"-0-acetyl band in the Various compounds listed in (Table VII) is due primarily to shielding 1

by the aromatic A ring to which the glycosyl residue is linked, since this acetyl is situated mainly in the diamagnetic region above or below the plane of the A ring (CX7)188*189'190. The slightly greater shielding of the 2tt-0-acetyl in group A compounds is of some diagnostic value and can be explained on the assumption that the 2"-0-acetyl in group A is shielded not only by the A ring of the fLavone but to some extent by the B ring.

OrC

The greater shielding of the 6"-0-aeetyl in group A can be accounted for on the assumption that it lies in the diamagnetic region of the B ring of the flavone nucleus. Models show that (CXV) is to be a likely conformation. Since any similar shielding can obviously be ruled lis

out in group B and C, the 6"-0-acetyl bands of these compounds occur at the normal position (S 1.93-2.04) i«e. the same position as the

6-0-acetyl band of simple glucose derivative such as Q-glucose-

2,3,4,6-tetraacetate (S 2.03).

A consequence of the interaction of the 2B-and 6"-0-acetyl groups with the B ring and C-7 substituent in 8-substituted flavones is that these compounds exhibit hindered rotation about the C-1"-C-8 bend.

Even at temperature as high as 30-40 they appear to exist largely in two principal rotational conformations. The existence of the conformers, which is markedly temperature dependent, is evidenced in the nmr spectrum at

35 by the splitting or broadening of these bands of certain aromatic protons and acetyl groups, particularly the 6w-0-acetyl group (Table VII).

Since the glycosyl portion of the groups B and C compounds has a smaller rotational barrier, their spectra are generally sharp in all regions, with the possible exception of the slightly broadened 2"-0-acetyl band. A further point of difference between the spectra of the acetylated 8-C-glucosylflavones and the other acetylated C-gLucosyl compounds lies in the band 1

positions of the 3" and 4n-0-acetyl groups. In the 8-substituted flavones these bands occur in two discrete ranges,^ 2.01-2.03 and6 2.08-2.10, res pectively, while in the 6-substituted and miscellaneous compounds they overlap or occur very close together in the range 82.03-208. Since the

4n-0-acetyl lies near the axis of rotation of the glucosyl ring, it should be relatively insensitive to the rotational conformation of the molecula.

In the 8-substituted flavones the signal at62.08-2.10 is a sharp singlet and is, therefore, assigned to the 4M-0-acetyl. The band atS 2.01-2.03 is usually broadened or split and is assigned to the 3"-0-acetyl. T A B L E - VII

CHEMICAL SHIFTS OF ACETYL METHYL AND BENZYLIC (H-1w)PROTONS OF ACETYLATED C-GLYCOSYL COMPOUNDS IN DEDTERIOCHLOROFORMa

Chemical shift and coupling ppm (J,HZ) Compound 4"-0Ac 3"-OAc 6"-0Ac 2n-0Ac H-1" Reference

8 - Substituted Flavones (Group A) Vitexin heptaacetate 2.09 2.01 1.91° 1.73 4.98 86 Cytisoside hexaacetate 2.08 2.01 1.92 1.73 d 87

5,7,4'-Tri-0-vitexin tetraacetate 2.09 1.98 1.93° 1.71 5.25(10) Mil 5*7,4',3M, 4", 6n-Hexa-0-methylvitexin acetate - - - 1.72 5.06(10) 86 1 B 5,7,4 , 2 , 3 ", 4*-Hexa-0-methylvitexin acetate - M» 1.93 - 5.02(10) 86 Orient in acetate 2.09 2.02 1.95e 1.72 b - Bayin hexaacetate 2.10 2.02 1.90 1.72 d 84,85 , 7,4 -Di-0-n»8thylbayin tetraacetate 2.10 2.01 1.91 1.70 d 84,85 e 8-C-B-D-Glucopyranosyldiosmetin heptaacetate 2.08 2.02 1.94 1.71 f «M Range for Group A i.02-2.10) (1.98-2.02) (1.90-1.95) (1.70-1.73)

6 - Substituted Flavoma s (Grout) B)

Isovitexin heptaacetate 2.08 2.08 2.04 1.83 4.91(10) (•* C-B-D-Glucosyldi-O-methylphloroacetophenone 2.07 2.05 2.02 1.77 5.14(10) - pentaacetate

Iso-orientin actaacetate 2.08 2.08 2.02 1.82 4.85(9.5) T Keyakinin heptaacetate 2.05 2.05 2.01 1.30 4.25 84 6-C-B-D-Glucopyranosyldiosmetin heptaacetate 2.07 2.07 2.02 1.81 4.86(10) — TABLE -VII (CONTD)

Chemical shift and coupling ppm (J, HZ ) Compound W 4"-0AC 3 -0AC 6"-0AC 2"-0AC H-1" Reference Miscellaneous C-G£Lucosyl Compounds ( Group C ) C-B-D-Glucosylbenzene tetraacetate 2.04 2.03 1.98 1.78 4.36 84 C-B-B-Glucosyl-di-O-methylphloroacetophenone pentaacetate 2.07 2.07 2.04 1.80 d 89 Mangiferin octaacetate 2.07 2.05 2.01 1.79 4.92 Aspalathin nonaacetate 2.06 2.03 2.0(D 1.78 4.72(9-10) 48,90 3,4>2*,4* >6t-Penta-0-methylaspalathin tetraacetate 2.09 2.06 2.03 1.78 5.34(9-10) 48,90 Isohemiphloin heptaacetate 2.03 2.03 2.00 1.80 $•04 84 Hemiphloin heptaacetate 2.05 2.05 2.03 1.83 d 84 Combined ranges for groups B and C (2.03-2.08) (2.03-2.07) (1.98-2.04) (1.77-1.83) Other Compounds D-Glucose 2,3,4»6-tetraacetate 2.10 2.10 2.03 2.02 Puerarin hexaacetate 2.07 2.07 2.C5 1.72 84 a. Spectra were determined at 30-35 • Tetramethylsilane was used as internal standard. b. Numbers are text references. c. This band is split, the other branch occurring at 2.03. d. Not reported. e. This band is split, but the other branch was not clearly discernible at this temperature. f. Insufficient sample was available to discern this proton clearly. 122

TABLE- VIII

CHEMICAL SHIFTS OF AROMATIC AND METHOXYL PROTONS OF ACETXLATED FLAVONES IN DEUTERIOCHLOROFORM

Chemical shift and coupling ppm (J,HZ) r Compound H-3 H-6 H-8 H-2» H-6' H-3 H-5' CH30

Apigenin triacetate 6.61 6.86(2.5) 7.35(2.5) 7.87(8.5) 7.87(8.5) 7.26(8.5) 7.26(8.5) - Acacetin diacetate 6.55 6.84(2.5) 7.31(2.5) 7.80(9.0) 7.00(9.0) 7.00(9.0) 7.00(9.0) 3.88

Luteolin tetraacetate 6.60 6.86(2.5) 7.30(2.5) 7.73(2.5) 7.78(2.5,9) - 7.36(9.0) mm

Chrysoerial triacetate 6.58 6.83(2.5) 7.33(2.5) 7.38(2.5) 7.45(2.5,9) - 7.13(9.0) 3.90

Diosmetin triacetate 6.55 6.83(2.5) 7.33(2.5) 7.56(2.5) 7.71(2,5,8) - 7.05(8.0) 3.90

6-C-B-D-Glucosyldiosmetin 6.54 - 7.33 7.53(2.0) 7.71(2,8.5) mm 7.06(8.5) 3.90 heptaacetate

Vitexin heptaacetate 6.70 6.84 - 8.11,7.95(9) 8.11,7.75(9) 7.40(9) 7.40(9) -

Isovitexin heptaacetate 6.65 mm 7.38 7.90(9) 7.90(9) 7.30(9) 7.30(9) - DISCUSSION BIFLAVANOIDS 12

HIELAVOMES FHOM JUHIPERUS SPECIES;

1Q? "iA. Kariyone and Hsii in their studies on the distribution of biflavanoids in gymnosperms reported the presence of amentoflavone, hinokiilavone, kayaflavone and sciadopitysin in the leaves of Jimiperus conferta, J.formosana, J.procumbens, J.sargentii, J.squamata and J.utilis.

More recently Kawano et al 195 found that J.rigida contained amentoflavone and podocarpus-flavone A.

The present discussion deals with the study of complex mixture of biflavanoids in the leaf extract of (a) Juniperus chinensis

(b) J.hmrizontalis (c) J.recurva and (d) J.virginiana. The biflavanoid constituents identified in each Juniperus species ax>& recorded in

(Chart-XIl). C H A R T - XII

Distribution of biflavanoids in Juniperus species

Band Biflavanoids ABCD B FGHIJK

Ag a=Amento fl avone a Am + + + b=6upressuflavone e=RobustafLavone Cu + Ro

d d d d d d Hi + + + + + + d = HinokifLavone + + + + e II M-Am + + e = Podocarpusfl avone M-Cu

M-Hi + f = 1-7,11-7-di-O-meth III f cupressuflavone * D = Cu +

g h h h h h h IV T-Am . + g = Sciadopitysin + + + + + + * h = Kayaflavone

A = Juniperus chinensis, B = J.horizontalis, C = J.virginiana, D = recurva, S = J.utilis, F = J.coferta, G = J. formosana, H = J .procumbens, I = J.sargentii, J = J.squamata, K = J.rigida, Am = Amentoflavone, Cu=cupressuflavone, Ro = Robustaflavone, H = HinokifLavone, M = Mono, D = Di, T = Tri, + = detected, + with superscript = fully characterized.

4>« 12 3

BIFLAVONES FROM JDNIPERUS CHINENSIS LIMNAEUS

Leaves of Juniperus chinensis were procured from Forest Research

Institute Dehradun, India. The phenolic extractives of the coarsely powdered

leaves by solvent fractionation, column chromatography (magnesium silicate)

followed by thin layer chromatography (silica gel) yielded three components.

After establishing the homogeniety (TLC), they were obtained in fine crys

talline forms and labelled as JC.., J'C_ and JG_. The usual colour reactions

and OV spectra indicated them to be flavanoids.

JC1 and JC„ were characterised as amentoflavone (XVa) and

hinokiflavone (XXVa) respectively by m.ps,, m.m.ps and by NMR spectra of

their acetates and methyl ethers. JC_ being the minor component was detected

(TLC) as monomethyl ether of hinokiflavone.

I-4,,II-4,lI-5,II-5,I-7,II-7-Hexahydroxy_CI-3,II-Sa -biflavone (JC-,) m.p. „ Rf Mol.wt.

+ JC1 (parent) 250°C ot 3g-30° 0.173 538(M )

+ JC1A(acetate 228-30° - 790(M )

+ JC1M(methyl ether) 217-20° 0.40 622(M ) 126

The results of NMR spectrum of JC M are given in (Ta.ble-IX)

T A B L E -IX Chemical shifts o f prontons of JC M

Signal No .of protons Assignment

3.52(d) H-I-8 3.66(d) H-I-6

3.38(d) H-II-6

2.10(q) H-I-6

2.88(d) H-I-5 2.16(d) H-I-2' 2.62(d) 2 H-II-2;iI-6/

3.24(d) 2 H-II-3',11-5'

3.48(e)

3.42(a) 2 H-I-3,II-3 5.94,6.08 6 OMe-I-5,II-5

6.12,6.18 6 OMe-I-4^11-4' 6.25,6.27 6 OMe-I-7,11-7

8 = singlet, d « doublet, q = quartet.

* Spectrum run in CDC1_ at 100 Mc/S. TMS as internal standard = T 10.00.

Associated with rings I-B and II-B there were evidenced ABX and

AgR* systems. Thus I-B and II-A of the biflavone seemed to be involved in interflavanoid linkage. In particular the T values shovred that C-I-3 is linked to either C-II-6 or C-II-8. The observation that in flavones, having aromatic substituent at C-I-8 the I-5-0Me group generally appears belowT 6.0 (Table-X), led us to believe that substituent (flavone unit) in JC M was located at C-II-8 and not at C-II-6.

TABLE - X

Biflavanoid I-5-0Me II-5-0Me

1. Cupressuflavone hexamethyl ether T 5*88 T 5*88

2. Amentoflavone hexamethyl ether T6.13 T5.94

3. Agathisflavone hexamethyl ether T6.41 T5.95 4. Hinokiflavone pentamethyl ether ^6.00 1*5.92 (C-I-4-0-C-II-8) 5. JC^M T6.08 T5.94 6. Hinokiflavone pentamethyl ether ^6.06-6.09 (C-I-4^0-C-II-8)

Further all methoxy groups on change of solvent from deuterochloroform to benzene moved upfield as cupressuflavone hexamethyl ether showing that every methoxy group had at least one ortho proton and, therefore, aC -11-8 rather than C-II-6 linkage was confirmed.

An authentic sample of + amentaflavone hexamethyl ether was shown to give an NMR identical with JC M in all respects including solvent dependent methoxy shifts. The IR, UV and mass spectra were also identical. 128

The results of NMR studies on hexaacetate of JC are shown in (Table-Xl).

T A B L E -XI

Chemical shifts of protons of JC. hexaacetate

Signal No.of Protons Assignment

2.73 (d) H-I-8

3,31 (d) H-I-6 2.97 (q) H-II-6

1.99 (q) H-I-6' 2.48 (d) H-I-5' 1.94 (d) H-I-2'

2.92 (d) 2 H-II-3^11-5'

2.50 (d) 2 H-II-2,'lI-6' 3.30 (s) ( ) 3.30 (s) 2 H-I-3,II-3

7.50,7.54 6 0AC-I-5,II-5

7.89,7.93 6 OAc-I-4^11-4'

7.67,7.72 6 0Ac-I-7,II-7 s = Singlet, d = doublet, q = quartet.

•Spectrum run in CDCI3 at 100 MC/S, TMS as internal standard =T10.00

JC1 was, thus, assigned the structure 1-4,11-4^1-5,11-5, 1-7, II-7-hexahydroxy- Cl-3,11-83 bifLavone. 12

1 II-4J1-5, H-5,1-7, II-7-Pentahydroxy- CI-4 0-H-63-biflavone (JCg)

m.p Rf Mol.wt JCg (parent) 335° 0.32 538 (M+)

JCgA (acetate) 237-40° - 74S(M+) + JC5M(methyl ether) 260° 0.52 608(M )

The TLC examination of JC_ and its methyl ether and mass spectrum of JC„M (m/e 6o8,M ) indicated that JC„ may be hinokiflavone

(m/e 538,M+).

By comparison of NMR data (Table-XIl) of JC A and JCJM with the chemical shifts of authentic samples, JC_ was assigned the structure of II-4J 1-5, II-5, 1-7, II-7-pentahydroxy-CI-4-0-lI-6a-biflavone.

T A B L E - XII

NMR signals (Tscale) of methyl and acetyl protons in pyridine solution Assigned position in biflavone nucleus Compound II-4' 1-5 II-5 1-7 II-7

Hinokiflavone 7.70 7.65 7.55 7.76 7.88 pentaacet ate

JC2A 7.71 7.60 7.50 7.71 7.89

Hinokiflavone (6.23) (6.19) (5.92) (6.21) (6.14) pentamethyl ethsr JC-M ' (6*19> <6'12) (5.88) (6.19) (6.16) 2

Figures in parentheses show chemical shifts of methoxy protons 1

BIFLAVONES FROM JUKIPKRUS HORIZoNTALIS MOENCH

The phenolic extractives of dried leaves of Juniperus

horizantalis (procured from Lyod Botanical Garden, fiarjeeling) after

similar treatment as with J.chinensis yielded four components. After

establishing homogeneity (TLC), they were labelled as JH , JEL, JH

and JH.,. The usual colour reactions and UV spectra indicated them to be

flavanoids.

JH.., JH? and JH. were characterised as amentoflavone (XVa),

hinokiflavone (XXVa) and aciadopitysin (XVe), respectively by m.ps., m.m.ps. and by NMR spectra of their acetates and methyl ethers. JR,

being the minor component was detected (TLC) as monomethyl ether of hinokiflavone.

I-4,,II-4,,I-5,H-5,I-7,II-7-Hexahydroxy-D:-3,,II-S; J -biflavone (JH )

m.p. Rf Mol.wt. JH.. (parent) 340° 0.17 538 (M+)

+ JH..A (acetate) 242-44° » 790 (M )

JH..M (methyl ether) 227° 0.40 622 (M+) 131.

II-^I-5,H-5,I-7,II-7-Pentahydroxy-Cl-4-0-II-61 -biflavone (JHg)

m.p. Rf Mol.wt

JH^ (parent) 395° 0.32 538(M+)

+ JH2A (acetate) 240° - 74S(M )

+ JH2M(methyl ether) 260° 0.52 608(M )

1-5, H-5, II-7-Trihydroxy-I-4; H-£ I-7-tri-0-methyl- CI-3', 1L-B3 -biflavone(JH )

m.p. R~ Mol.wt

JH.'(parent) 295° 0.613 580(M+) 4 /

JH/A (acetate) 2$0° - 706(M+)

JH;M (methyl ether) 212° 0.40 622(M+)

TLC examination of JH./ and mass spectrum of its acetate

(m/e 706,M ) indicated that JH,,was tri-O-methyl derivative of amentoflavone. The structure of JH,/was elucidated by comparison of methoxy and acetoxy resonances of JH;A with those of authentic samples (Table-XIIl).

TABLE- XIII Chemical shifts (f scale) of methyl and acetyl protons in pyridine Assigned position in biflavone nucleus Compound I=£ II-4' 1-5 II-5 1-7 II-7 JH^A 6.22 6.43 (7.52) (7.50) 6.26 (7.91)

Sciddopitysin 6.24 6.42 (7.50) (7.47) 6.27 (7.90) triacetate Kayaflavone 6.27 6.40 (7.53) (7.42) t?.76) 6.20 triacetate Figures in parentheses show the chemical shifts of acetyl protons. 1

NMR spectrum of JH / was identical with that of sciadopitysin

39 triacetate . JH.,was, therefore, assigned the structure 1-5, H-5, II-7- 4- trihydroxy-I-4.; II-4J I-7-tri-O-methyl- C1-3', II-8 2 -biflavone.

BIFLAVONES FROM JUNIPERUS VIRGINIANA LINNAEUS

The leaf extracts of Juniperus virginiana (procured from Lyod

Botanical Garden, Darjeeling) after a similar treatment as in J.chinensis yielded two components. After establishing homogeneity (TLC), they were

labelled as JV1 and JV„. The usual colour reactions and UV spectra indicated thsm to be filavanoids.

JV was methylated and the methyl ethers JV.A, JV B, JV C and

JV.D were separated by TLC. JV I, JV.B and JV..C were characterised as

(a) amento .flavone hexamethyl ether - (b) cupressuflavone hexamethyl ether and (c) robustaflavone hexamethyl ether, by mps., m.m.ps. and by NMR spectra. JV..D was detected by TLC examination and characeristic fluorescence in UV light as agathisflavone hexamethyl ether. JV_ was characterised as hinokiflavone (XXVa) by m.ps., m.pps. and by NMR spectra of its acetate and methyl ether. J 33

1 I-4«,II-4», 1-5,11-5,1-7,II-7-Hexa-0-methyl- Cl-3 ,11-83 -biflavone(JV1A), (CXVII)

m.p. Rf Mol. wt. + JV.,A 228 0.40 622 (M )

MeO.

OMe

MeO 0

(GXVII)

I^SII-^SI^^l-SjI^II^-Hexa-Omethyl-Cl-S^I-SD-biflavoneCJV^)

The results of NMR spectrum of JV B are shown in (Table-XIV). 134

TABLE *-HV

Chemical shifts of protons of JV-B

Signal No.of protons Assignment

3.41 (s) 2 H-I-3, II-3

3.43 (s) 2 H-I-6; II-6'

2.70 (d) 4 H-I-2,' 1-6; II-2.IW

3.23 (d) 4 H-I-3; 1-5', II-5;iI-5'

5.88 6 0Me-I-5,II-5

6.14 ) 6 ) 6.22 ) 6 ) OMe-1-4; II-4; 1-7,11-7

S = Singlet, d = doublet

* Spectrum run in CDCL, at 100 MC/S,

TMS as internal standard =T10.00

NMR spectrum of JV..B suggested that molecule had an axis of symmetry. H-I-3, H-3 and H-I-6, II-6 were distinguished, the former (T3.41) appearing at the characteristic position for the H-I-3 of a flavone. It was immediately obvious that rings I-B and II-B were substituted only

1-4; II-4' methoxy groups as there was a clear AgBp pattern of protons of 135

rings I-S and II-B. Two methoxy groups had values belowT6.0 (caT5.88).

These were the I-5> H-5 methoxy groups which were present in two different

monoflavanoid units of the biflavone. This may perhaps be the characteristic

of such groups in 1-4, 11-8 linked biflavones or in an 8f-linked monoflavanoid unit of a biflavone. This observation was supported by the studies both in biphenyl as well as biphenyl ether type series of biflavanoids (Table-X).

There was a singlet representing two protons around T3.4. This was assigned to aromatic protons at H-I-6, II-6. Its high position indicated that it must be between two oxygen atoms and ring I-A, therefore, be meta

substituted by oxygen. To meet the requirements of symmetry and NMR spectrum

the two possible structures for the fraction JV..B may be represented by

(CXVIII) and (CXIX). 0M<2 0

7 VS-OMe

MeO OMe

(CXVIII) (C-I-8,II-8 linked) 136

(CXIX) (C-I-6, II-6 linked)

The final decision between C-I-8, II-& and C-I-6, II-6 linkage was taken by benzene induced solvent shift studies of methoxy resonances.

The shifts in methoxyl resonances as a result of change of solvent from deuterochloroform to benzene are shown in (Table-XV).

T A B L E - XV

Position of Signals in Signals in Shifts in c/s OMe CDC1- c/s C^ c/s

C-5 412 356 +56

C-4 386 329 +59

C-7 371 302 +75 13 7

Thus all the three methoxy groups shifted upfield as expected

for a C-I-8,11-8 linkage.

JV..B, therefore, has been assigned the structure 1-4,11-4,'

1-5,11-5, 1-7, II-7-nexa-0-methyl-CI-8,n-8D-biflavone (CXVIII).

0M<2 0

MeQ OMe

OMe 0

(CXVIII)

1-4; n-4i 1-5,11-5,1-7,II-7-Hexa-0-methyl-CI-3^II-6D-biflavone (JV C)

m.p. Mol. wt,

JV.,0 316C 622 (H+)

The results of NMR studies of JV C are shown in (CXX) and

(Table - XVI). 138

^wn-VjeW -TH^wS^Y^Uw**

(CXX) 139

T A B L E*- XVI

Chemical shifts of protons of JV.C

Signal No.of protons Assignment

3.5? (d) 2 H-I-3,II-3

3.65 (d) 1 H-I-6

2.19 (d)

( ) 2 H-1-2,1-6 3.15 (q)

(3.13)(d) 2 H-II-2,II-6

3.91 (d) 1 H-I-5

(2.98)(d) 2 H-I-3,H-5

6.07,6.39 - OMe-I-5,H-5

(6.12,6.18) - 0Me-I-7,II-7

6.14,6.12 — 0Me-I-4,II-4 s = singlet, d = doublet, q= quartet

* Alternative assignment is possible for figures with an aestrik as well as for those in parentheses

* Spectrum run in CDC1„, TMS as an internal standard =T10.00

The mode of interflavanoid linkage as C-I-3-C-II-6 in JV C was established from benzene induced solvent shift studies of methoxy resonance- 140

On change of solvent from CDC1„ to C/.H,., a methoxy group atT 6.39 moved downfield, whereas the other five showed upfield shifts like penta-O-methyl hinokiflavone and hexa-O-methylagathisflavone . This finding is compatible with the structure of JV..C because XH--II-5 in this case is located in similar environments as OCH--I-5 of penta-O-methylhijiokiflavone and OCH -1-5 of hexa-O-methylagathisflavone. The above mode of linkage gains further support from mass spectral studies of JV..C. The presence of m/e 311 as a major peak in JV..C and hexa-O-methylagathisflavone bat very minor one in hexa-O-methylamentoflavone is of considerable significance. This may be attributed to facile carbon-carbon cleavage in JV.C and hexa-O-methyl agathisflavone due to steric reasons.

JV..C, therefore, has been assigned the structure I-4.,H-4»'l-5»

II-5,I-7,II-7-hexa-0-methyl- Cl-3',11-63 -biflavone (CXXE). 141

The structure of JV C gains further support from lanthanide

induced shift studies by EU (F0D)o carried out in our laboratories.

11-4)1-5, 11-5,1-7, II-7-Pentahydroxy- CI-4-0-II-6Z] -biflavone (JVg).

m.p. Ef Mol.wt. JVg (parent) 355° 0.32 538 (M+)

+ JVgA (acetate) 238-39° - 748 (M )

+ JV2M (acetate) 260° 0.52 608 (M )

EIFLAVONES FROM JUNIPERUS RECURVA BUCHHAM

The phenolic extractives of dried leaves of Juniperus recurva

(procured from Lyod Botanical Garden, Darjeeling) after similar treatment as in J.chinensis yielded three components. After establishing homogeneity

(TLC), they were labelled as JR.., JRg and JR_. The usual colour reactions and UV spectra indicated them to be flavanoids.

JR1 and JR? were minor constituents and were detected by TLC and characteristic fluorescence in UV light as mixtures of (a) amentoflavone and cupressuflavone (b) hinokiflavone and monomethyl ethers of amentoflavone 142

and cupressuflavone. The fraction JR.- although homogeneous in chromatographic

behaviour was separated into two components JR~X and JR~Y by CCD between ethyl methyl ketone and barate buffer (pH 9.8). JR_X was characterised as

I-7,II-7-di-0-methyl cupressuflavone (XIVc). JR^Y being a minor component

could not be identified.

I-4;iI-4;i-5,II-5,Tetrahydro3{y-I-7, II-7-di-0-methyl- CI-8,11-83 -biflavone(JR X)

m.p. Rf Mol.wt. JR_X (parent) 300° 0.54 566 (M+)

JR.JCA (acetate) 273-74° 734 (M+)

JR-XM (methyl ether) 298° 0.43 622 (M+).

The mass spectrum showed a dominant peak at m/e 566 the other

significant peaks were at m/e 565 and m/e 283, the latter showing a symmetrical cleavage of the stable molecule, the pattern suggesting a biflavone breaking down to its units. The acetate has the molecular ion at m/e 734, losing mass unit of CHLC =0 to give ultimately the base peak at m/e 556 as in the parent

compound. The rest of the spectrum was as before for the parent compound

showing it to be a tetraacetate of JR~X. Methyl ether (JR-XM) has the base 143

peak at m/e 622 with accompanying minor breakdowns as in hexa-O-methylcupre ssu flavone.

The results of NMR spectra of JR-XA and JR XM are given in (Table

XVII and XVIII).

TABLE*- XVII

Chemical shifts of prontons in JR-XA

Signal No.of protons Assignment

3.45 (s) 2 H-l-3,II-3

3.20 (s) 2 H-I-6,II-6

2.66(d) 4 H-I-2',I-6,'lI-2,'lI-6'

2.95 (d) 4 H-I-3',I-5',II-3,'lI-5

6#13 6 0Me-I-7, II-7

7.49,7.74 12 OAC-I-4,' 11-4^1-5,11-5

s = singlet, d = doublet * Spectrum run in CDC1- at 100 tec/s, TMS as an internal standard = 10.00 J44

TABLE- XVIII

Chemical shifts of protons in JR~X M

Signal No.of protons Assignment

3.41 (s) 2 H-I-3,II-3

3.43 (s) 2 H-I-6,II-6

2.70 (d) 4 H-I-2^I-6',II-2,'lI-6'

3.23 (d) 4 H-I-3;i-5;iI-3,'U-5'

5.&S 6 OMe-I-5, H-5

6.H) ) 12 0Me-I-4,'lI-4,' 1-7, H-7 6.22)

s = singlet, d <= doublet.

» Spectrum run in CDC1_ at 100 Mc/s., TMS as an internal standard = T10.00.

B2 of both A?B2 pairs in JRJCA are moved fromT3.l6 (Jftj X) and 3.23 (JR^XM) toT2.95 (JRjXA). The downfield shift in acetate confirms the presence of OAb-1-4'and OAc-II-4.' The two methoxy groups may not be assigned to 1-5 and II-5 positions. If this would have been the case, the signals for methoxy groups in the parent compound would have been shown up Ho

belowf6.00 and not atT6.19. The signal belowT6.00 (T5.88) appears only after methylation indicating thereby that the parent compound contains hydroxy groups at 1-5 and II-5. With the assignment of methoxy groups at

1-7 and II-7, the protons at 1-6 and II-6 (T3.20) compare very well under similar environment with those of diacetate of 1-4/ II-4, 1-5, II-5-tetra-

O-methylcupressuflavone.

Thus JR_X has been assigned the structure I-4>H-4>'I-5,II-5- tetrahydroxy - 1-7,II-7-di-O-methyl cupressuflavone.

In addition to the presence of amentoflavone and hinokiflavone a number of their partial methyl ethers have been isolated and characterised.

The members belonging to cupressuflavone and robust aflavone series are being reported in Juniperus species for the first time. These finding may be of some interest to chemotaxonomists. 146

HEFLAVONES FROM THUJA SPSCIBS

191

Sawada in his studies on the distribution of biflavanoids reported the presence of hinoVdflavones in Thuja standishi and T. occidentalis.

The present discussion deals with the study of biflavanoids in the leaf extracts of Thuja orientalis, T.plicata and T.japonica. The biflavanoid constituents identified in each T. species are recorded in (Chart-XIII).

CHART -XIII

Distribution of biflavanoids in Thuja species

Band Biflavanoids ABODE Remarks

A J) &. I Am + + + a = Amentoflavone

Gu + b = Cupressuflavone

Ro + c = Robustaflavone

II M-Am +4 d = Sequoiaflavone

e e e e Hi + + + + e = Hinokiflavone

f III D-Am* + f « l-7,II-7-Di-0-methylamentoflavone

IV T-Am' +« g = Sciadopitysin

A = Thuja Orientalis, B= T.Plicata, C=T.Japonica, D=T. Standishi, E=T.Occidentalis, Am=Amentoflavone, Cu=Cupressuflavone, Ro=Robustoflavone, Hi=Hinokiflavone,M=Mono- D=Di-, T=Tri-, + = detected, + with superscript = fully characterized. U7

BIFLAVONES EKOM THUJA ORIENTALIS LINNAEUS

Leaves of Thuja orientalis were procured from Lyod Botanical

Garden Darjeeling, India. The phenalic extractives of the coarsely powdered leaves by solvent fractionation, column chromatography (magnesium silicate) followed by thin layer chromatography (silica gel) yielded two components.

After establishing homogeneity (TLC), they were obtained in fine crystalline forms and labelled as TO., and TO,. The usual colour reactions and UV spectra indicated them to be flavanoids.

T01 and T0? were characterised as amentoflavone (XVa), the first optically active biflavone of amentoflavone series and hinokiflavone (XXVa) respectively by m.ps., m.m.ps. and by NMR spectra of their acetates and methyl ethers.

I-4;il-4j 1-5,11-5,1-7,II-7-Hexahydroxy- CI-3',11-81 -biflavone (TO )

ttt.p1. Rj Mol.wt. + T01 (parent) 250-55COC3Q°+100 0.173 538(M )

TCjA (acetate) 225° 790 (M+)

TCLM (methyl ether) 170-71° 0.40 6Z2 (M+) 148

II-4', 1-5, II-5,1-7, IJ-7-Pentahydroxy- I-4'-0-II-6 -biflavone (TOg)

m.p. R_ Mol.wt.

+ T02* (parent) 345-4$° 0.32 538 (M )

+ T02A (acetate) 240-41° - 748 (M )

TOgM (methyl ether) 268-70° 0.52 608 (M+)

BIFLATONES FROM THUJA PLICATA D.DON

The phenolic extractives of dried leaves of Thuja plicata

(procured from Lyod Botanical Garden, Darjeeling) after similar treatment

as in T.orientalis yielded two components. After establishing homogeneity

(TLC), they were labelled as TP.. and TP_. The usual colour reactions and

UV spectra indicated them to be flavanodds.

TP and TF_ were characterised as amentoflavone (XVa) and hinokiflavone (XXVa) respectively by m.ps., m.m.ps and by NMR spectra of their acetates and methyl ethers.

1-4',H-4SI-5,H-5,1-7,II-7-Hexahydroxy- 1-3',H-8 -biflavone (TP )

m.p. R f Mol.wt. + TP1 (parent) 340° 0.17 538 (M )

TP.jA (acetate) 240° - 790 (M+)

+ TP1 (methyl ether) 228° 0.40 622 (M ) 149

II-4.; 1-5,11-5,1-7, II-7-Pentahydroxy- C 1-4^0-11-63 -biflavone (TPg)

m.p. E'1 Mol.wt. f

+ TP2 (parent) 345-46° 0.32 538 (M )

+ TP2A (acetate) 240° - 748 (M )

+ TP2M (methyl ether) 228° 0.40 622 (M )

BIFLAVONES FROM THUJA JAPONICA MAXIMOWICZ

The phenolic extractives of dried leaves of Thuja japonica

(procured from Government garden Ooty, India) after similar treatment as

in T. orientalis, yielded four components. After establishing homogeneity

(TLC), they were labelled as TJ., TJ ,TJ_ and TJ.. The usual colour reactions and UV spectra indicated them to be flavanoids,

TJ1 was methylated and the methyl ethers TJ^, TJ B and TJ^ were characterized as (a) hexa-0-methyl amentoflavone (b) hexa-O-methyl cupressuflavone and (c) hexa-O-methyl robustaflavone respectively by m.ps., m.m.ps and MR studies of their methyl ethers. The fraction TJ , although homogenous in chromatographic behaviour was separated into two components,

TJpX and TJ_Y by CCD separation between ethyl methyl ketone and borate buffer (pH 9.8), and were characterized as hinokiflavone (XXVa) and

I-7-O-methyl-amentoflavone (XVTD) by m.ps., m.jn.ps. and MMR spectra of

their acetate? JThe fraction TJ- was also separated by CCD into two

components TJ-X and TJ,Y. TJ-X was characterized as 1-7,II-7-di-O-methyl-

atnentoflavone a new bifLavone by NMR spectrum of its acetate. TJ_Y being

a minor component and could not be identified. TJ. was also separated by

CGD in a major component TJ,X and TJ.Y (minor). TJ X was identified as

sciadopytisin (XVI) by m.ps., m.m.ps. and NMR spectrum of its acetate.

TJ .Y could not be identified. 4

1-4^11-4; 1-5,11-5,1-7,II-7-Hexa-O-methyl LI-3,II-BJ -bifLavone (TJ^)

TJ^, nup. 228°, Mol. wt. 622 (M+).

1-4',II-4.;i-5, H-5,1-7,II-7-Hexa-O-methylC1-8,11-83 -biflavone (TJ B)

TJ^, m.p. 296°, Mol. wt. 622 (M+ )

I-4iII-4', 1-5,11-5, 1-7, II-7-Hexa-O-methylC 1-3',II-6D-biflavone (TJ^)

TJ^, m.p. 316° Mol. wt. 622 (M+) 15!

1-4*1-5, H-5,1-7,H-7-Pentahydroxy-Cl-410-II-6D -biflavone (TJ X)

m.p. Rf Mol.wt. TJ X (parent) 345° 0.32 538 (M+)

TJgXA (acetate) 240° - 748 (M+)

TJgXM (methyl ether) 228° 0.40 622 (M+)

I-4i H-4i 1-5, H-5, II-7-Pent ahydroxy-I-7-0-methyl- Cl-3, II-8 3 - biflavone (TJgl)

m.p. Rf' Mol.wt.

+ TJ2Y (parent) < 300° 0.37 552 (M )

TJgYA (acetate) 245° - 762 (M+)

TJgYM (methyl ether) 212° 0.40 622 (M+)

TIC examination of the parent compound and its complete methyl

ether and mass spectrum of TJ?YA m/e 762 (M ) showed it to be a monomethoxy- pentaacetate of amentoflavone.

The structure of TJ„Y was further elucidated by comparision of methoxy and acetoxy resonances of TJgYA (acetate) with those of the authentic samples (Table-XIX). J c) **

T A B L £ - XIX

Chemical shifts ( scale) of methyl and acetyl protons in pyridine •

Q J Assigned position in biflavone nucleus 1-4' II-4* 1-5 H-5 1-7 H-7

TJ2YA (7.96) (7.87) (7.54) (7.49) 6.28 (7.90)

Sequoiaflavone (7.95) (7.85) (7.53) (7.49) 6.28 (7.90) pentaacetate

Bilobetin 6.28 (7.85) (7.53) (7.49) (7.76) (7.90) pentaacetate

II-7-O-methyl (7.95) (7.85) (7.53) (7.48) (7.77) 6.28 pentaacetate

Numbers in parentheses represent acetoxy groups.

The NMR spectrum of TJ„YA was found to be identical with that of sequoiaflavone pentaacetate. IJpI was, therefore, assigned the structure

1-4', II-41,1-5, II-5, II-7-pentahydroxy-I-7-0-methylamento flavone. 1-4' .11-4' .1-5.11-5. TetrRhvdroxv-I-7.II-7-di-0-methvl- CI-3'.II-83 -biflavone (TJJO m.p. R Mol.wt.

TJ X (parent) - 0.54 566 (M+)

TJ XA (acetate) - - 734 (M+)

TJ XM (methyl ether) 228° 0.4 622 (M+) 153

R„ value, fluorescence in UV light mass and NMR spectrum

of methyl ether (TJ_XM) obtained from TJJC was identical with that of

authentic amentoflavone hexamethyl ether in all respects.

The mass spectrum of TJ^X acetate (TJ_XA), (m/e 734>M ) indicated

it to be a dimehtoxy-tetraacetyl amentoflavone*

Although the methyl ether of TJ_X was identical in all respects

with the authentic amentoflavone hexamethyl ether, the parent compound

and its acetate (TJ_XA) were not comparable with any of the known dimethyl ethers of amentoflavone. The NMR spectrum of TJ-XA and the NMR data of

TJ-XA and other members of amentoflavone series are given in (SXXII) and

(Table-XX).

PPM(6l

(CXXII) T A B L B - XX Chemical shifts of protons (Tscale)

Compound 1-8 1-6 II-6 I-2«,6» 1-5' H-2',6' H-3',5" 1-3,11-3 1-4',11-4' 1-5,11-5-' 1-9,11-7

TJ.XA 3.22 3.40 3.25 2.03 2.58 2.50 2.97 3.47 8.01 7.51 6.14 (H,d) (H,d) (H,s) (H,d) (H,d) (2H,d) (2H,d) (2H,d) 7.75 7.59 6.17 2.08 (6H) (6H) (6H) (H.q) Sequoiaflavone- 3.22 3.43 3.01 1.97 2.57 2.52 2.98 3.36 7.96 7.54 6.28 pentaacetate (H,d) (H,d)(H,s) (H,d) (H,d) (2H,d) (2H,d) (2H) 7.87 7.49 7.90 2.06 (6H) (6H) (6H) (H.q) II-7-O-methyl- 2.78 3.18 3.25 2.02 2.56 2.51 2.96 3.36 7.62 7.44 7.96 ament oflavone (H,d) (H,d) (H,s) (2H, q) (H,d) (2H,d) (2H,d) (2H) 7.68 7.50 6.09 (6H) (60 (6H) Podoc arpu sfl avone B 3.21 3.39 3.01 1.92 2.52 2.57 3.21 3.38 7.94 7.52 7.94 diacetate (H,d) (H,d) (H,s) (H,d) (H,d) ($H,d) (2H,d) 3;40" 6.45 7.46 6.45 2.02 (2H) (6H) (6H) (6H) (H,q) I-4»,II-7-di-0- 2.73 3.20 3.25 2.0 2.87 2.56 2.97 3.47 6.22 7.50 6.13 methyl-amento- (H,d) (H,d) (H,s) (H,d) (2H,d) (2H,d) (2H) " 7.73 7.56 $.78 flavone (6H) (6H) (6H)

Sciadopitysin 3.22 3.44 3.04 2.09 2.85 2.61 3.22 3.22 6.27 7.54 6.22 triacetate (H,d) (H,d) (H,s) (2H,q) (H,d) (2H,d) (2H,d) 3.43 6.17 7.59 7.97 (2H) (6H) (6H) (6H)

Kayaflavone 2.73 3.22 3.2 2.10 2.86 2.61 3.22 3.43 6.22 7.54 7.59 triacetate (H,d) (H,d) (H,s) (H,d) (H,d) (2H,d) (2H,d) (2H) 6.24 7.58 6.24 • 2.04 (6H) (6H) (6H) LHi^} Numbers in parentheses represent methoxy groups. 1 0 0

The new dimethyl ether of amentofLavone is assigned the structure (CXXCII) by the comparison of NMR spectrum of its acetate with the spectra of acetates of sequoiaflavone and II-7-O-methyl-amentofLavone.

The proton signals of I-A and II-A rings are also comparable with those of similarly constituted I-A and II-A rings of acetates of (a) podocarpusflavone B

$b) 1-4, II-7-di-0-methyl amentoflavone (c) Sciadopitysin and (d) kayafLavone.

TJJf was, therefore, assigned the structure 1-4^11-4,1-5,11-5- tetrahydroxy-I-7, II-7-di-O-methyl- amentofLavone.

MeQ

OH o

(CXXIII)

1-5, H-5, II-7-Trihydroxy-I-4;iI-4,' I-7-tri-0-methyl-CJ-3; H-8D -biflavone(TJ ,X)

m.p. Rp Mol.wt.

TJ^X (parent) 295° 0.613 580 (M+)

TJ.XA (acetate) 270 706 (M+)

TJ,XM (methyl ether) 218C 0.40 622 (M+) 156

BIFLAVOKES FROM PODOCARPACEAE

Kawano et al in their studies on the distribution of biflavanoids

in gymnosperms reported the presence of hinokiflavone, • neocryptomerin, podocarpusflavone A, podocarpusflavone B and sciadopitysin in the leaves of podocarpus macrophylla and amentoflavone, podocarpusflavone A and

isoginkgetin in the leaves of podocarpus nagi. Later on Rahman et al

found that podocarpus graciliar contained amentoflavone, podocarpusflavone A,

isoginkgetin and kayaflavone and podocarpus latifolius contained amento

flavone, bilobetin, isoginkgetin and kayaflavone.

The present discussion deals with the study of complex mixture of biflavanoids in the leaf extract of podocarpus taxifolia. The biflavanoid constituents identified in each podocarpus species are recorded in

(Chart-XXIV). C H A B T - XI?

Distribution of biflavanoids in Podocarpus species

Band Biflavanoids A B C D' D Remark

a a a a I Am + + + a= Amentoflavone +

II Hi +b +b b= Hinokiflavone c c c d c M-Am + + + + + c= Podocarpusflavone A +d d= Bilobetin + e= Sequoiaflavone III H-Hi +x f= Neocryptomerin D-Am +g + + + g= Podocarpusflavone B h= Isoginkgetin

T-Am +X +J +*1 i= Sciadopitysin j= Kayaflavone

A= Podocarpus macrophylla, B= P.nagi, C= P.gracilior, D = P.latifolins, Ess P.taxifolie, Am= Amentoflavone, Hi= Hinokiflavone, M= Mono, D= Bi, T= Tri, + = detected, + with superscript = fully characterized. 158

BIFLAVONES FROM PODOCARPUS TAXEFOLIA KUMTH

Leaves of Podocarpus taxifolia were procured from Lyod Botanical

Garden, Darjeeling, India. The phenolic extractives of the coarsely powdered leaves by solvent fractionation, column chromatography (magnesium silicate) followed by TLG (silica gel) yielded two components. After establishing homogeneity (TLC), they were obtained in fine crystalline forms and labelled

as PT1 and PT„. The usual colour reactions and UV spectra indicated them to be flavanoids.

PT1 was characterized as amentoflavone (XVa) by m.ps., rn.rn.ps., and by KMR spectra of its acetate and methyl ether. The fraction PT although homogenous in chromatographic behaviour was separated into four

components PTgW, PTgX, PTy and PTgZ by CCD separation between ethyl methyl ketone and barate buffer (pH 9.8), and were characterized as (a) hinokiflavone (XVa), (b) bilobetin (XVc), (c) podocarpus-flavone A (XVe) and

(d) sequoiaflavone (XVb) by m.ps., m.ro-ps and NMR spectra of their acetates. 1 5 9

1-4-', II-4', 1-5, II-5,1-7, II-7-Hexahydroxy- CI-31,11-83 -biflavone (PT )

m.p, R_ Mol.wt.

+ PT1 (parent) 340° 0.17 538 (M )

PT A (acetate) 240° - 790

FT M (methyl ether) 228° 0.40 622 (M+)

II-4', 1-5, H-5,1-7, II-7-pentahydroxy- CI-4* ,0-11-6 3 biflavone (PTgW)

m.p. R„ Mol. wt.

+ PTgW (parent) 346° 0.32 538 (M )

PTgWA (acetate) 240° - 748 (M+)

PTgWM (methyl ether) 228° 0.40 622 (M+)

II-4', 1-5,11-5,1-7, Il^-Pentahydroxy-I^' -O-methyl- C1-3', 11-83 -biflavone(PT X)

m.p. Hf Mol.wt. + PT2X (parent) 300° 0.37 522 (M )

+ PTgXA (acetate) 183-84° - 762 (M )

PTgXM (methyl ether) 212° 0.40 622 (M+)

TLC examination of the parent compound. (IT X) and its methyl

ether and mass spectrum of PT?XA(acetate) m/e 762 (M ) showed it to be 160

•v EtOH a monomethoxy - pentaacetate of amentoflavone. The A ,. appeared at

275nm (band I) and 332 nm (band II). Addition of N/50 NaOEt caused a bathochromic shift of band I with an increased intensity and of Band II with a moderate decrease in intensity, thus indicating that methoxy group was present at C-I-7 or C-II-7. The structure of PT X was further elucidated by comparision of methoxy and acetoxy resonances of PT_XA with those of authentic samples, (Table XXI).

TABLE -XXI

Chemical shifts of methyl and acetyl protons in Pyridine solution

Assigned position in biflavone nucleus 1^4' II-V 1-5 H-5 1-7 II-7

PT2XA 6.28 (7.87) (7.54) (7.47) (7.78) (7.91)

Bilobetin acetate 6.28 (7.85) (7.53) (7.49) (7.76) (7.90)

Podocarpusflavone A (7.95) !6.44 (7.56) (7.48) (7.78) (7.84) acetate

Sequoiaflavone acetate(7.95) (7.85) (7.53) (7.49) 6.28 (7.90)

II-7-O-methyl-amento- (7.95) (7.85) (7.53) (7.48) (7.77) 6.28 flavone

Number in parentheses show the chemical shifts of acetyl protons. 16

The NMR spectrum of PT?XA was found to be identical with that of bilobetin pentaacetate. PTgX was, therefore, assigned the structure

I1-41,1-5,H-5,1-7,11-7-pentahydroxy-I-4«-0-methylamentoflavone.

1-4', 1-5,H-5,1-7, II-7-pentahydroxy-II-4"-O-methyl-Q-3', II-8D -biflavone(PT2Y)

m.p. Rf Mol.wt

+ PT2I (parent) 266-68° 0.37 552 (M )

PTglA (acetate) 254° - 762 (M+)

PTgYM (methyl ether) 212° 0.40 622 (M+)

TLC examination of the parent compound (PT_Y) a*1** its methyl ether and mass spectrum of FTpYA (acetate) n/e 762 (M ) showed it to be a monomethoxy-pentaacetate of amentoflavone. The \ appeared at max

275 nm (Band I) and 335 nm (Band II). Addition of N/50 NaOBt caused a bathochromic shift of band I with an increase in intensity and of band II with a moderate decrease in intensity. Thus indicating that no methoxy group was present at C-I-7 or C-II-7. The structure of PT„Y was further elucidated by comparison of methoxy and acetoxy resonances of PT IA with those of authentic samples (Table-XXII). 162

TABLE- XXII

Chemical shifts of methyl and acetyl protons in pyridine solution

Assigned position in biflavone necleus 1-4' 11-4' 1-5 H-5 1-7 II-7

PT2YA (7.68) 6.35 (7.51) 7.56) (7.90) <7.94)

Podocarpusflavone A (7.95) 6.44 (7.56) (7.48) (7.78) (7.84) acetate

Bilobetin acetate 6.28 (7.85) (7.53) (7.49) (7.76) (7.90)

Sequoiaflavone acetate (7.95) (7.85) (7.53) (7.49) 6.28 (7.90)

II-7-0-nBthyl (8.95) (7.85) (7.53) (7.48) (7.77) 6.28 amentoflavone

Numbers in parentheses show the chemical shifts of acetyl protons.

The EMR spectrum of PT„YA was found to be identical with that of

Y was podocarpusflavone A. PT2 > therefore, assigned the structure 1-4',1-5-

II-5,1-7, II-7-Pentahydro3ty-II-4'-O-methylamentoflavone.

<^-> I-41,11-4', 1-5, H-5, II-7-Pentahydroxy-I-7-0-rnethyl- CI-3,'lI-83 -biflavone(PTgZ)

m.p. . Rf Mol.wt. + PTgZ (parent) 300° 0.37 552 (M )

+ PTgZA (acetate) 245° - 762 (M )

PTgZM (methyl ether) 212° 0.40 622 (M+) 163

EIELAVONES FROM ARAUCARIA SPECIES

29 31 193 Rahman et al ' ' in their studies on the distribution of biflavanoids in Araucariales have reported the presence of the members belonging to amentoflavone, cupressuflavone, agathifLavone and hinokiflavone in Araucari cookii, A' cunninghamii and A.bidwilli. The remarkable features of this order are (i) the presence of biflavcnes belonging to all the important series of biflavanoids and (ii) optical activity due to restricted rotation as shown by some of the members. The above facts prompted us to investigate the. biflavanoid contents from Araucaria excelsa.

The biflavanoid constituents identified in each Araucaria species are recorded in (Chart - XV). C H A R T - XV Distribution of biflavanoids in Araucaria species

Band Biflavanoids A B C D Remarks

I Ag + + Am + + Cu + +

a a II M-Ag + + + (a) I-7-O-Methylagathisflavone

III M-Am + (b) II-7-O-Methylamenfcoflavone M-Cu + + (c) Bilobetin Hi + (d) I-7-0-methylcupressuflavone (e) Hinokiflavone

f f f f IV + (f) I-7,II-7-Di-0-methylagathisflavone D-Ag + + + h (g) I-4iH-7-lJi-0-methylamentoflavone V D-Am + . + . (h) I-7,II-7-Di-0-methylamentoflavone D-Cu (i) 1-7,II-7-Di-O-methylcupre ssuflavone M-Hi (j) 1-7,1-4'or 11-4^-Di-O-methylcupressuflavone

VI T-Ag (k) 1-7,11-4; II-7-Tri-0-Hi3thylagatbisflavone :l- —-ar ~TT (l) Kayaflavone VII T-Ag + + + T-Cu + + (m) Sciadopitysin (n) 1-4; 1-7, II-7-Tri-O-methylamentofLavone (o) I-4i1-7,II-7-Tri-O-methylcupressuflavone

VIII Te-Am J> 4P (p) I-4;II-4',I-7,II-7-Tetra-O-methylamentoflavone

IX Te-Cu A A A (Q) 1-4',11-4',1-7,11-7-Tetra-O-methylcupressuflavone A= Araucaria cookii, 3= A.cunningiiamii, C= A.bidwiUi, D= A.sxcelsa, Ag= Agathisflavone, Am= AmentofLavone, Cu= cupressuflavone, Hi= Hinokiflavone, M= Mono, D= Di, T= Tri, Te= Tetra, + = detected, + with superscript = fully characterized.

4^- 165

KEFLAVONBS EROM ARAUCARIA SXCELSA LAMB

Leaves of Araucaria excelsa were procured from Government

Garden, Obty, India. The phenolic extractives of the coarsely powdered leaves by solvent fractionation, column chromatography (magnesium silicate)

followed by TLC yielded nine components. After establishing homogeneity (TLC), they were obtained in crystalline form and labelled as AE., AE_, AE_, AE,, 1

~~AE5,AE^,AE_,AEg and AE,., The usual colour reactions UV spectra in ethanol indicated them to be flavanoids.~~

~~AIL and AE- were minor constituents and were detected by TLC examination and characteristic fluorescene in UV light as mixtures of~~

(a) amentoflavone, cupressuflavone and agathisfl&vone, and (b) monomethyl ether of agathisflavone. AE., and AE. were characterised as II-7-O-methylamentoflavone (XVd) and I-7,II-7-di-0-m3thylagathisflavone (XVIc) respectively.

The fraction AE,., although homogenous in chromatographic behaviour wa3 separated into two components AE^X and AE-Y by CGD separation between ethyl methyl ketone and borate buffer (pH 9.8) and characterized as I-7,II-7-di-0- methylamsntoflavone (CXXIII) and 1-4'or II- 4^-7-3.'i-OL-methyl cupressuflavone. 166

~~AE/- was characterised as II-4I,I~7,II-7-tri-0-methylagathisflavone. The~~

fraction AE7 although homogenous in chromatographic behaviour was also separated into two components AE„X and AE~Y by GGD between ethyl methyl ketone and barate buffer (pH 9.8). AE_X was characterized as 1-4',1-7, II-7- tri-O-methylamentoflavono. AE„Y being a minor component and could not be

~~1 identified. AEft and AEQ were characterized as I-4 , II-4', I-7-H-7-tetra-0- methylamentoflavone (XVc) and 1-4', H-4,1-7, II-7-tetra-O-methylcupressuflavone~~

~~(XlVe) respectively.~~

~~The structures of different constituents have been fully elucidated by UV, IR, NMR and mass studies.~~

~~I-4«, II-4,1-5, H-5,1-7-Pentahydroxy-II-7-0-methyl-CI-3', 11-83 -biflavone(AE3)~~

~~m.p. Rf Mol.wt, AE_ (parent) 300° 0.37 522 (Vt)~~

~~AEJL (acetate) 165° - 762 (M+)~~

~~AE_M (methyl ether) 212° 0.40 622 (M+)~~

~~TLC examination of the parent compound and its complete methyl ether and mass spectrum of AE- A, m/e 762 (M ) showed it to be a 16~~

~~monomethgrxy-penta-acetate of amentoflavone.~~

~~The strucufce of Aft, was further elucidated by comparison of methoxy and acetoxy resonances of AE_A with those of the authentic samples~~

~~(Table-XXIIl).~~

~~TABLE- XXIII~~

~~Chemical shifts (Tscale) of methyl and acetyl protons in pyridine~~

~~C a nd Assigned position in biflavone nucleus 1-4' II-4' 1-5 H-5 1-7 11-7~~

~~AE3A (7.62) (7.68) (7.44) (7.50) (7.95) 6.09~~

~~II-7-O-methyl (7.63) (7.69) (7.42) (7.50) (7.94) 6.09 amentoflavone~~

~~Sequoiaflavone ($.95) (7.35) (7.53) (7.49) 6.28 (7.90) pentaacetate~~

~~Bilobetin 6.28 (7.85) (7.53) (7.49) (7.76) (7.90) pentaacetate~~

~~Numbers in parentheses represent acetoxy groups.~~

~~The NMR spectrum of AE_A was found to be identical with that of~~

~~II-7-0-methylamentoflavone. AEL was, therefore, assigned the structure~~

~~I-4,'lI-4^ 1-5, II-5,1-7-penta-hydroxy-II-7-0-methylamentoflavone. 168~~

~~1-4,' II-4il-5, II-5-Tetrahydroxy-I-7, II-7-di-O-methyl- C 1-6,11-83 -Biflavone(AE ) ....^______«__«_«____,«»______._^______^-—»-———-—.—_—_ — 4-~~

~~m.p. Hi, Molwt.~~

~~AE, (parent) 308° 0.43 566 (M+)~~

~~AE.A (acetate) 174° - 734 (M+)~~

~~AE.M (methyl ether) 160° 0.45 622 (M+) 4~~

~~R^ value, fluorescence in UV light, mass and NMR spectra of the methyl ether (AE.M) obtained from AE, was identical with that of authentic agathisflavone hexamethyl ether in all respects.~~

~~The mass spectrum of AE,A (m/e 734>M ) showed it to be dimethoxy- 4 \ etracytylagathisflavone.~~

~~The results of NMR studies of AE, and AE,A are shown in (Table-XHV).~~

TABLE4- XXIV Chemical shifts of protons Assigned positions AE.A *&L H-I-2;i-6' 1.99 (2H,d) 2.02 H-1-3,'1-5' 2.41 (2H,d) 2.70 H-II-2JII-6' 2.96 (2H,d) 2.46 H-II-3;iI-5' 3.18 (2H,d) 2.91 I-I-8 3.06 (H,s) 2.96 H-II-6 3.27 (H,s) 3.23 H-I-3,11-3 3*38 /R \ 3.31 3.47 {ii,3) 3.42 1-4' 0.7-1.0 7.88 II-4' 7.76 1-5 -3.07 7.67 II-5 -3.30 7.53 1-7 6.13 (6H,s) 6.19 II-7 6.17 s= singlet, d= doublet * spectrum in CDC1- at 100 MC; TMS as internal standard = T10.00 figures in parentheses show the cnemical shifts of methoxyl protons. 169

The two sets of A„B2 type doublets of AE, acetate suggest that no methoxy group was present at either of the I-4'and II-4 positions. This was also supported by the following observations. B„ of both AgB- pairs

~~(rings I-B and II-B) in AE,A, moved down field as compared to the AE. and~~

~~AE.M (Table-XXV) 4- T A B L E - XXV~~

~~Compound II-I-3', 1-5' B-II-3,'lI-5 '~~

~~AE^ 2.41 3.18~~

~~AE.M 2.99 3.22 A-~~

~~AE,A 2.70 2.91~~

In the parent compound (AE/, Table-XXIV) there are two hydrogen bonded hydroxy groups atT-3.07 andT-3.30 for C-I-5 and C-II-5 positions. The methoxy groups were not located at C-I-4'and C-II-4'as shown earlier. This left the only possibility of the assignment of two methoxy groups at C-I-7 and C-II-7. 170

~~AE, was, therefore, assigned the structure 1-4', 11-4% 1-5,11-5- 4-~~

~~tetrahydroxy-I-7, II-7-di-O-methylagat hisflavone.~~

~~1-4' .11-4' .1-5.II-5-Tetrahvdroxv-I-7.Il-7-di-0-methyl C1-3'.II-8U- biflavone (AE5X) m.p. R„ Mol. wt.~~

~~AE. ( parent) - 0.54 566 (M+)~~

~~AE A( acetate) - - 734 (M*)~~

~~ASM methyl ether) 228° 0,4 622 (M+)~~

~~I-4'or II-4'.I-5.II-5.II-7-Tetrahvdroxv-I-A' or II-4'.I-7-di-0-methvl- E. 1-8,11-83 -biflavone (AE.Y) m.p. R„ Mol.wt~~

~~+ AE5(parent) - 0.54 566 (M )~~

~~AE.A (acetate) - - 734 (M*)~~

~~+ AE5M (methyl ether) 228° 0.4 622 (M )~~

~~• ' E „ value fLuoresence in UV light mass and NMR spectra of methyl ether~~

~~(AE-YM) obtained from AE^Y was identical with that of the authentic cupressu-~~

~~flavone hexamethyl eth^r in all respects. 171~~

~~The mass spectrum of AE^Y acetate (m/e, 734 M ) indicated it to be a dimethoxy-tetraacetyl cupressuflavone.~~

Although the methyl ether of AE~Y was identical in all respects with the authentic cupressuflavone hexamethyl ether, the parent compound and its acetate were not comparable with any of the known dimethyl ethers of cupressuflavone. The NMR spectrum of AE^YA and the NMR data of AEL YA and other members of the series are given in (CXXIV) and (Table-XXVl).

~~(CXX1V) T A B L E - XXVI Chemical shifts of protons ( Tscale) .~~

~~Compound 1-6,11-6 1-3,11-3 I-2',6',II-2' ,6' i-a; 5, n-3,5' l-4'-H-4' 1-5,11-5 1-7,11-7~~

~~AECYA 3.20 3.48 2.70 2.92 7.76 7.52 6.16 5 (H,S) (H,S) (4H,d) (2H,S) C6.22J (6H) 7.98 3.00 3.52 3.28 (6H) (6H) (H,S) (H,S) (2H,S)~~

~~I-7-0-Methylcupressuflavone 3.21 3.49 2.67 2.97 7.73 7.50 C6.15D (H,S) (H,S) (H,d) (H,d) (6H) (6H) 7.94 2.91 (H,S) 3.44 (6H) (H,S)~~

~~1-7,II-7-Di-O-methyl- 3.20 3.47 2.69 2.95 7.71 7.47 C6.14D cupressuflavone (2H,S) (2H,S) (H,d) (H,d) (6H) (6H)~~

~~I^'-H^SI^^I^-Tetra- 3.19 3.45 2.75 3.22 C6.223 7.51 C6.20ZI O-methylcupressufLavone (2H,S) (2H,3) (4H,S) (4H,d) (6H) (6H) (6H)~~

~~Numbers in parentheses represent methoxy groups. 173~~

~~The new dimethyl ether of cupressuflavone is assigned the structure (CXXV) by the comparison of NMR spectrum of its acetate~~

~~(AEJCA) with the spectra of acetates of I-7-O-methylcupressuflavone,~~

~~1-7,II-7-di-O-methylcupressuflavone and 1-4',II-4',1-5,II-5-tetra-~~

~~O-methylcupressuflavone (Table-XXVl).~~

~~AEJf was, therefore, assigned the structure I-41 or II-4',~~

~~1-5,II-5,II-7-tetrahydroxy-I-4' or II-4', I-7-di-O-methylcupressuflavone~~

~~(CXXV),~~

~~MeO~~

~~/ \VoMe~~

~~OH 0~~

~~(CXXV) I-2J f I-5.II-5-Trihydroxy-lI-4' .1-7. II-7-tri-O-methyl-C 1-6.11-83 biflavone (AE^)~~

~~m.p. Rf Mol.wt.~~

~~AE, (parent) 300° 0.613 580 (M+)~~

~~AE,A (acetate) 185 706 (M+)~~

~~AE^M (methyl ether) 242° 0.45 622 (M+)~~

~~TLC examination of AE, and mass spectrum of its acetate~~

~~(m/e 706,M ) indicated that AE, was tri-0-methyl derivative of~~

~~agathisflavone. The NMR spectrum AE,A and NMR data of AE,A and~~

~~other members of the series are given in (CXXVI) and (Table-XXVIl)~~

~~PPM (6)~~

~~(CXXVI) TABLE- XXVII~~

~~Chemical shifts of protons ( scale)~~

~~Compound 1-8 II-6 1-3,11-3 1-2',6' 1-3',5' II-2',6' II-3S5' 1-4', II-41 1-5,H-5 1-7,11-7~~

~~AE,A 2.97 3.26 3.33 2.05 2.72 2.57 3.13 7.88 7.67 L 6.193 (H,S) (H,S) 3.49 (2H,d) (2H,d) (2,H,d) (2H,d) C 6.263 7.54 C 6.223 (6H) (6H) (6H)~~

~~1-7, II-7-Di-O- methyl- 2.97 3.35 3.47 2.07 2.72 2.50 2.94 7.85 7.50 C6.173 agathisflavone (H,S) (H,S) 3.40 (2H,d) (2H,d) (2H,d) (2H,d) 7.73 7.64 C6.153 (6H) (6H) (6H)~~

~~11-4',1-7-Di-O-nethyl- 3.01 3.02 3.38 2.08 2.73 2.60 3.19 7.86 7.67 C6.213 agathisflavone (H,S) (H,S) 3.46 (2H,d) (2H,d) (2H,d) (2H,d) C6.243 7.56 7.91 (6H) (6H) (6H)~~

~~Numbers in parantheses represent mathoxy groups~~

~~Of 176~~

~~By coinparasion of the WMR spectrum of the acetate of (AB,) with the spectra of the acetates of 1-7,II-7-di-O-methyl-agathisflavone and~~

11-4% I-7-di-0-methyl_ agathisflavone (Table-XXVIl), the new methyl ether is assigned the structure, I-41,1-5,II-5-trihydroxy-II-4', 1-7,11-7-tri-0- methyl.agathisflavone (GXXVIl). The acetoxyl, methoxyi and proton signals in all the three bifLavones are comparable. The chemical shifts of H-3',51 protons (T3.18) in trimethyl ether, as expected, is comparable with that of II-4', I-7-di-O- methyl -agathisflavone (T3.19) but show an upfield shift in contrast to I-7,H-7,-di-0-methylagathisflavone (*Y2.94).

~~OH 0~~

~~(CXXVII) 17?~~

~~1-4', 1-5, II-5-Trihydroxy, I- 4*, 1-7, II-7-tri-O-methyl- CI-3',11-83 -biflavoneCA^X)~~

~~m.p. Rf: Mol.wt.~~

~~AE„X (parent) 0.613 580 (M+)~~

~~AE„XA (acetate 706 (M+)~~

~~AE^XM (methyl ether) 298 0.40 622 (M+)~~

TLC examination of AE„X and mass spectrum of its acetate (m/e 706,M ) ijndicated that AELX was tri-0-methyl derivative of amentofLavone. The NMR spectrum of AE„X and NMR data of AE~XA and other members of the series are given in (CXXVIII) and (Table-XXVIIl).

~~t#ik4i#U#mv»W' w* vitiftfHim^mmLuuuUu J t^V%w~~

~~' 6 5 ' J 2 PPM (6)~~

~~(CXXVIII) TABLE- XXVIII Chemical shifts of protons (T scale)~~

~~1 n * T O T ^ TT ^ 1-2' T Cf H-2 TT 1 • I~3 I"4' I"5 I "7 Compound 1-8 1-6 II-6 ^ 1-5 II-6, 11-3^ II-3 n_4, I;D_5 II-7~~

AE-XA 3.19 3.44 3.25 2.14 2.88 2.57 2.98 3.44 C6.23H 7.51 C6.13Z] ' (H,d) (H,d) (H,S) (H,d) (H,d) (2H,d) (2H,d) (2H,S) 7.75 7.58. C6.17D 2.11 (6H) (6H) (6H) (H,q) I-7,II-7-Di-0-methyl- 3.22 3.40 3.25 2.03 2.58 2.50 2.97 3.43 8.01 7.51 C6.143 amentoflavone (H,d) (H,d) (H,S) (H,d) (H,d) $2H,d) (2H,d) (2H,S) 7.75 7.59 C6.173 2 08 (6H) (6H) (6H) (H, q) Bilobetin 2.75 3.19 3.05 3.13 2.84 2.53 2.95 3.36 C6.22D 7.56 7.73 penta.acetate (H,d) (H,d) (H,S) (H,d) (H,d)($H,d) (H,d) (2H,S) 7.79 7.60 7.98 2.08 (6H) (6H) (6H) (H,q) Sciadopitysin 3.22 3.44 3.04 2.09 2.85 2.61 3.22 3.22 C6.273 7.54 C6.22D triacetate (H,d) (H,d) (H,S) (H,q) (H,d) (2H,d) (2H,d) 3^43 C$.43D 7.59 7.97 (2H) (6H) (6H) (6H)

Kayaflavone 2.73, 3.22 3.2 2.10 2.86 2.61 3.22 3.43 C6.213 7.54 7.96 triacetate (H,d) (H,d) (H,S) (H,d) (H,d) (2H,d) (2H,d) (2H) C6.24D 7.58 C6.24D 2.04 (6H) (6H) (H^q) Figures in parentheses represent methoxy groups The comparison, of NMR spectrum of acetate of (AELX) with the spectra of the acetates of (a) I-7,II-7-di-0-amentoflavone for I-A, II-A and II-B rings and (b) biolobetin for I-B ring supports structure (CXXIX) for the new tri-O-methyl-amentofLavone. Further support to structure

~~(CXXIX) is given by comparing the NMR spectra of its acetate with those of the known tri-O-methyl ethers of amentoflavone (Table-XXVIII).~~

~~(CXXIX) 180~~

~~1-5. II-5-Dihvdroxy-I-A1. II-A'. 1-7. II-7-tetra-O-methyl- 1-3«. II-8 - biflavone (AEg)~~

~~m.p, Mol.wt.~~

~~+ AE8 (parent 275° 594 (M )~~

~~+ AEgA (acetate) • 225° 678 (M )~~

~~+ AEgM (methyl-ether) 212° 622 (M )~~

~~The usual colour reactions and UV spectra in neutral as well as with diagnostic reagents indicated that the compound was flavanoid~~

~~in nature,~~

~~TLC examination of the parent compound and its complete methyl ether~~

~~and mass spectrum of AE^A, m/e 678 (M ) showed it to be a tetra-methoxy diacetate of amentoflavone.~~

~~The structure of AE^ was further elucidated by comparison of methoxy and acetoxy, resonances of AE^A with those of the authentic samples~~

~~(Table-XXIX). 18~~

~~TABLE- XXIX~~

~~Chemical shifts ( scale) of methyl and acetyl protons~~

~~« J Assigneb d Bpositio n in biflavone nucleus Compound - I-41 H-41 1-5 II-5 1-7 Ifc-7~~

~~AE8A 6.25 6.16 (7.52) (7.59) 6.15 6.28~~

~~1-4',II-4',1-7,11-7- 6.26 6.15 (7.52)(7.59) 6.15 6.26 Tetra-O-methylamentoflavone~~

~~Sciadopitysin 6.27 6.17 (7.54) (7.59) 6.22 (7.97) triacetate~~

~~Kayaflavone 6.21 6.24 (7.54) (7.58) (7.96) 6.24 triacetate~~

~~Numbers in parentheses represent acetoxy groups.~~

~~The NMR spectrum of AEUA was found identical with that of~~

~~1-4',II-4',1-7,II-7-tetra-0-methyl amentoflavone. AE- was, therefore, assigned the structurel-5-,11 r5-dihydroxy-I-4',II-4',I-7,H-7-tetra-0- methylamento flavone. 182~~

~~I-5.II-3-Dihv-droxy-I-4.' .11-4.' .1-7.11-3-tetra-O-methyl- CI-8.I1-8J - biflavone (AEg)~~

~~m.p. Mol.wt.~~

~~25 + AE9 (parent) 150° +30° 594 (M )~~

~~+ Afi9A (acetate) 155° 678 (M )~~

~~AE^M (methyl ether) 160° 622 (M+)~~

~~The green ferric reaction, the behaviour with Alcl- in UV, the carbonyl absorption at 1655 cm were characteristic of 5-hydroxyl group chelated with carbonyl group. The shift of the carbonyl frequency~~

~~-1 —1 to 1650 cm on acetylation and 1640 cm on methylation further supported the presence of chelated carbonyl function. The foregoing evidence suggesting~~

~~5,7,A-1 -oxygenation pattern.~~

TLC examination of the parent compound and its complete methyl ether and mass spectrum of AHLA, m/e 678 (M+) showed it to be a tetra- methoxy-diacetate of cupressuflavone. The structure of AEU was further elucidated by comparison of methoxy and acetoxy resonances of AEQA with those of the authentic samples

~~(Table-XXX). 1~~

~~T A B L E - XIX~~

~~Chemical shifts (Tscale)of methyl and acetyl protons~~

~~Assigned position in biflavone nucleus ComPound I-4',II-4' 1-5,11-5 1-7,11-7~~

~~AE9A 6.25 (7.52) 6.22~~

~~1-4',11-4',1-7,11-7-Tetra- 6.26 (7.51) 6.20 0-met hylcupre ssu flavone~~

~~Cupressuflavone hexaacetate (7-75) (7.52) (7.92)~~

~~Numbers in parentheses represent acetoxy groups.~~

~~The KMR spectrum of AELA was found identical with that of~~

~~1-4', II-4', 1-7, II-7-tetra-O-methylcupressuflavone. AErt was, therefore, assigned the structure 1-5,II-5-dihydroxy-I-4',II-4',I-7,II-7-tetra-0- methylcupre ssuflavone. C-GLICOSYLFLAVONES 184~~

~~C-GLYCOSYLFLAVONES FROM PHLOX DRUMMONDII HOOK (POLEMONIACBAE'):~~

The white flowers of Phlox drummondii were collected from the campus of Aligarh Muslim University (Aligarh). The ethanol extracts of the flowers gave three homogenous components by paper chromatography on

~~Whatman No.3 filter paper followed by column chromatography on polyamide~~

~~(Woelm). After establishing the homogeneity (TLC), they were obtained in fine crystalline forms and labelled as I,II and III. The usual colour reactions and UV spectra indicated them to be flavanoids.~~

~~The twomjor components I, m.p. 198 and III m.p. 210 have been characterized as O-Rhamnosyl-l-6-C-xylosylluteolin (CXXXI) and O-Rhamonsyl-~~

~~I-6-C-xylosylapigenin (CXXXIII) respectively. Both the C-glycosylflavones constitute new compounds.~~

~~By ferric chloride oxidation both I and III gave the same mixture~~

~~of sugars which were authenticated as rhamnose and xylose by paper~~

~~chromatography. The aglycones obtained by HI treatment of I and III were~~

~~characterized as luteolin and apigenin respectively by the preparation of~~

~~acetates and methyl ethers as well as by UV (six sets), (Table-XXXI), IR~~

~~and NMR spectra. TABLE- XXXI~~

~~Ultraviolet absorption spectra( ^ max in run)~~

~~Luteolin Isocrientin Apigenin Isovitexin~~

~~MeOH 242 sh,253,267,291sh, 349 242sh, 255,271,349 267,296sh,336 271,336~~

~~NaOMe 266sh,329sh,401 267,278sh, 337sh, 406 275,324,392 278,329,398~~

~~A1C13 274,300sh,328,426 278,302sh,332,429 276,301,348,384 262sh, 278,304,352,382~~

~~AlciyHcl 266sh, 275,294sh, 355,385 265sh,279,296sh, 361,384 276,299,340,381 260sh,280,302,344,380~~

~~NaOAC 269,326sh,384 276,323,393 274,301,376 279,303,385~~

~~KaOAC/H3B03 259,301 sh, 370,430sh 265,377,429sh 268,302 sh,338 274,346,Z08sh~~

~~QO Of J86~~

~~Acid hydrolysis of I and III gave rhamnoae as the common sugar~~

~~in addition to substances II m.p, 260 and IV m.p. 228 respectively.~~

~~The compound II was identified by co-chromatography in TBA and HOAC,~~

~~m.p. m.m.ps; IR and mass spectra corresponds to synthetic I-6-C-xylosyl-~~

~~luteolin (CXXXJl) since the UV spectra of both I and II were essentially~~

~~identical, the rharanose must be attached to the c-xylosyl moiety; the~~

~~exact position of which has not yet been determined. This is further~~

~~supported by the identity of UV spectra of I and II with those of luteolin~~

~~and isocrientin (Tahle-XXXT). Structures I and II have also been confirmed~~

~~by NMR spectra of trimethylsilyl ether and acetate of I.~~

~~The structures III and IV were also confirmed on similar grounds~~

~~as described for those of I and II.~~

~~(CXXX) (I) R1 e O-rhamnosyl-C-xylosyl, Rg = OH~~

~~(II) R1 = C-xylosyl, Rg » OH~~

~~(ITL^ R1 = O-rhamnosyl-C-xylosyl, Rg = H~~

~~(IV) R1 = C-xylosyl, Rg = H. CONCLUSIONS 18S~~

~~CONCLUSIONS~~

The leaf extracts of a number of Gymnosperms have been investigated for biflavone contents. They have been isolated in pure forms and fully characterized. Those marked with aestrick are only detected (TLC).

~~1. JUNIPBRUS CHINENSIS LINNAEUS~~

~~(a) Amentoflavone~~

~~(b) Hinokiflavone~~

~~•st* (c) Hinokiflavone monomethyl ether.~~

~~2. JUNIPEftUS H0RIZANTALI3 M3ENCH~~

~~(a) Amentoflavone~~

~~(b) Hinokiflavone (c) Hinokiflavone monomethyl ether~~

~~(d) Sciadopitysin~~

~~3. JTJNlPcKUS VIRGINIAN A LINNAEUS~~

~~(a) Amentoflavone~~

~~(b) Cupressuflavone~~

~~(c) Robustaflavone~~

~~(d) Hinokiflavone 4. JT]N3PgRUS R3CURVA BUCHHAM~~

~~(a) Amentoflavone~~

~~(b) Cupressuflavone (c) Amentoflavone monomethyl ether~~

~~(d) Cupressuflavone monomethyl ether~~

~~•>$• (e) Hinokiflavone (f) Amentoflavone dimethyl ether~~

~~(g) 1-7,II-7-Di-O-methyl cupressuflavone~~

~~The presence of members of cupressuflavone and robustaflavone in Juniperus species is reported for the first time.~~

~~5. THUJA ORIfiNTALIS LBMAflUS~~

~~(a) Amentoflavone~~

~~(b) Hinokiflavone~~

~~6. THUJA PLICATA D.DON~~

~~(a) Amentoflavone (b) Hinokiflavone~~

~~7. THUJA JAPONICA MAXIMOWICZ~~

~~(a) Amentoflavone~~

~~(b) Cupressuflavone~~

~~(c) Robustaflavone~~

~~(d) Hinokiflavone 190~~

~~(e) Sequoiaflavone~~

~~(f) Hinokiflavone monomethyl ether (g) 1-7, II-7-l>i-0-methylamentoflavone (h) Hinokiflavone dimethyl ether~~

~~(i) Sciadopitysin~~

~~Cupressuflavone and robustaflavone have been reported for the first time in Thu.ia species. A* new biflavone 1-7, H-7-Di-O-methylamentoflavone is also reported.~~

~~8. PODOCARPUS TAXIFOLIA KUNTH~~

~~(a) Amentoflavone (b) Hinokiflavone (c) Podocarpusflavone A (d) Bilobetin (e) Sequoiaflavone~~

~~The presence of three monomethyl ethers of one single series is noteworthy. They have been separated by CCD in pure forms and authenticated as podocarpusflavone A, bilobetin and sequoiaflavone.~~

~~9. ARAUCARIA EXCBLSA LAMB~~

~~(a) Amentoflavone~~

~~(b) Cupressuflavone~~

~~(c) Agathisflavone J9i~~

~~(d) Agathisflavone monomethyl ether (e) II-7-O-Methylamento flavone~~

~~(f) 1-7,II-7-Di-0-methylag8thi9flavone (g) 1-7, IT-7-Di-O-methylamento flavone~~

~~(h) 1-7,1-4' or II-4' -Di-O-msthylcupressuflavone~~

~~(i) II-4',1-7,II-7-Tri-O-methylagathisflavone~~

~~(j) Cupressuflavone tri-methyl ether~~

~~(k) 1-4',1-7, II-7-Tri-O-methylamentoflavone~~

~~(1) I-4«,II-4', 1-7, II-7-T etra-O-methylamentoflavone~~

~~(m) I-4<, II-4', 1-7, II-7-Tetra-O-metharlcupreasuflavone~~

~~Three new bifLavones ha.ve been characterised as 1-7,1-4' or~~

~~H_4i -di-O-methylcupressuflavone, II-4', 1-7, II-7-tri-O-methylagathisflavone and 1-4', 1-7, II-7-tri-O-methylamentoflavone.~~

~~10. PHLOX DRUMMONDII HOOK~~

~~The flower extract of P.drummondii yielded the following two new C-glycosides.~~

~~(a) O-Rhamnosyl-6-C-xylosylluteolin (b) O-Rhamnosyl-6-C-xylosylapigenin. EXPERIMENTAL 192~~

~~The following plants were investigated for the extraction and isolation of biflavanoida. l) Juniperus chinensis Linnaeus Cupressaceae~~

~~2) Juniperus horizontalis Moench~~

~~3) Juniperus virginiana Linnaeus~~

~~4) Juniperus recurva Buchham~~

~~5) Thuja orientalis Linnaeus~~

~~6) Thuja plicata D.Don~~

~~7) Thuja j'aponica Maximowicz~~

~~8) Podocarpus taxi folia Kunth Podocarpaceae~~

~~9) Araucaria excelsa Lamb Araucariaceae~~

~~A typical procedure as used in the case of Juniperus chinensis is being described.~~

~~1. JUNIPERUS CHENMSIS LIMAEUES~~

~~Dried and powdered leaves of Juniperus chinensis (1.5 kg) were completely exhausted with petroleum ether (-40-60 ). The petrol extracts 193~~

~~were concentrated first at atmospheric pressure and then under diminished~~

~~pressure. An oily green residue left behind gave negative test for flavanoids~~

~~and was rejected.~~

~~The petrol treated leaves were completely dried and exhausted with~~

~~boiling acetone till the extract was almost colourless. The combined acetone~~

~~extracts were concentrated first at atmospheric pressure and then under reduced~~

~~, pressure. A gummy dark green mass was obtained. This was refLuxed \d.th~~

~~petroleum ether ( 40-60 ), benzene and chloroform successively till the~~

~~solvent in each case was almost colourless. The residue left behind was~~

~~then treated with boiling water. The insoluble mass was dissolved in alcohol~~

~~and dried under reduced pressure. A solid green residue (4 gms) thus obtained,~~

~~responded to usual flavanoid colour tests.~~

~~PURIFICATION OF BIFLAVAflOID MIXTURE COLUMN CHROMATOGRAPHY:~~

~~A well stirred suspension of magnesium silicate (Woelm; 200 g) 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 excess petroleum 194~~

ether was allowed to pass through the column. The crude mixture of biflavanoids (4 g) was dissolved in dry acetone (75 ml) and was added to the column. After development of the column a circular filter paper was placed on the top of the adsorbent. The column was run in with organic solvents in the increasing order of polarity. The results are given in

~~(Table-XXXCl)~~

~~TABLE -XXXII~~

~~Solvent Nature of the product~~

~~1, Petroleum ether (40-60°) greenish gummy mass~~

~~2. Benzene green waxy product~~

~~3. Chloroform green oily product~~

~~4. Ethyl acetate yellow solid (1.5 g)~~

~~5. Acetone brown solid (350 mg)~~

~~6. Ethyl acetate (saturated xri.th water) yellow solid (7.5 mg)~~

~~7. Ethyl alcohol brownish gummy mass.~~

~~The fractions obtained with ethyl acetate, acetone and ethyl acetate (saturated with water) gave usual colour tests for flavones. J 95~~

~~COLOUR TESTS:~~

~~(1) Magnesium + hydrochloric acid orange~~

~~(2) Alcoholic ferric chloride dark green~~

~~(3) Zinc + hydrochloric acid red~~

~~(4) Sodium amalgam + hydrochloric acid pinkish violet.~~

~~SEPARATION OF KEPLAVANOID MIXTURE PREPARATIVE THIN LAYER CHROMATOGRAPHY;~~

~~Using thin layer spreader, (Desaga-Heidelberg) glass plates~~

~~(20 x 20 cm) were coated with a well stirred suspension of silica gel G~~

~~(E, Merck) to give a layer approximately 0-5 mm in thickness, after drying~~

~~for 2 hxs at room temperature, the plates were activated at 120 for 1 hr~~

~~and preserved in a desiccator until required.~~

~~Thin layer chromatographic examination of the yellow pigments obtained as a result of alution with ethyl acetate, acetone and fethyl~~

~~acetate (saturated with water) in eight solvent systems indicated below indicated the presence of three compounds in each fraction. These three~~

~~fractions were combined (2.5 g) and subjected to separation by preparative thin layer chormatography. 196~~

~~SOLVENT SYSTEMS USED:~~

~~(a) Benzen:pyridine: formic acid (BPF) 36:9:5~~

~~(b) Toluene:pyridine:acetic acid 5:4:1~~

~~(c) Toluene:pyridine:acetic acid 10:1:1~~

~~(d) Benzene:ethyl acetate:acetic acid 8:5'-2~~

~~(e) Benzene:ethyl formate:acetic acid 5:2:1:2~~

~~(f) Benzene:acetone 7:3~~

~~(g) Benzene: acetone 5:5~~

~~(h) Chloroform: ethyl acetate 1:1~~

~~In solvent system (a) the spots were compact and the difference in~~

~~R„ values were so marked as to make it the developing system of choice for preparative thin layer chromatography. This solvent system was used for all the subsequent separations of bifLavanoids.~~

Solution of bifLavanoid mixture (5%) in pyridine was applied to plates with the help of mechanical applicator (Desaga,Heidelberg) 2 cm from the lower edge of the plates. The plates mounted on stainless steel frames were placed in a Desaga glass chamber (45 x 22 x 25 cm) containing 500 ml 197

of the developing solvent (benzene:pyridine: formic acid, 36:9:5). When the solvent front travelled 15 cm from the starting line the development was interrupted and the plates were dried at room temperature. The positions of the bands were marked in UV light. The marked pigment zones were scraped with the help of a spatulla and eluted in separated columns with dry acetone.

The solvent was recovered till the eluents were reduced to 20-30 ml. The addition of water yielded yellow precipitate in each case. The precipitate was filtered, washed with water.and dried. Homogeneity of the pigments was checked by TLC using eight solvent systems already listed. The components were labelled as JC , JCg and JC-.

~~I-4',II-V,I-5,II~5,I-7,II-7Hexahydro2y -CI-3',H-8a -biflavone (JC^~~

~~Crystallized as yellow needles (150 mg) from CHCI_-Me0H; m.p.250 ,~~

~~Rf 0.173, CoG - 30 (pyridine-methanol, 1 mg/ml), }\ 272 nm, 330 nm, D max Mol.wt. 538 (M+).~~

~~I.R :~~

~~"\rKBr3027, 1648, 1604, 1568, 1499, 1425, 1358, 1285, 1285, 1237,1172,1160, ^ max 1107, 1047, 1027, 996, 984, 940, 910, 835, 767, 752, 735. ns~~

~~1-4',II-4',I-*5,II-5,1-7,II-7-Hexaacetoxy- CI-3',II-S3 -biflavone (JC^):~~

~~A mixture of JO. (50 mg), pyridine (1 ml) and acetic anydride (1ml) was heated on a water bath for 2 f.-.hrs, cooled and poured 6-nto crushed ice.~~

~~A white solid was obtained which crystallized from CHCI„-EtOH in the form of colourless needles,(35 mg), m.p. 228-230°, Mol.wt. 790 (M+).~~

~~MMR values (^scale'):~~

~~7.97 (3H, OAc-I-41); 7.86 (3H, 0Ac-II-4')j7.55 (3H,OAc-I-5); 7.48~~

~~(3H,OAc-II-5); 7.78 (3H,0Ac-I-7) and 7.89 (3H,0Ac-II-7).~~

~~I-4,,II-4,,I-5,II-5,I-7,II-7-Hexa-0-methyl-Cl-3f,II-83 -biflavone (JC^M):~~

A mixture of JC1 ( 60 mg ), potassium carbonate ( 2 g) and dimethyl sulphate (1 ml), in dry acetone ( 200 ml) was refluxed on a water bath for about 8 hrs. It was filtered and the residue washed several times with hot acetone. The filtrate and washings were combined and evaporated to dryness. The yellow residue washed 2-3 times with petroleum ether and then taken up in CHCI. (100 ml) and washed several times with water in a separatory 199

~~funnel. The chloroform solution was concentrated and purified by- preparative TLC. The solid crystallized from CHCI„-MeOH as colourless needles (40 mg), m.p. 217-20°. Mol.vt. 622 (M+).~~

~~I.R :~~

~~KBr 3082, 3000, 2945, 2852, 1648, 1600, 1572, 1505, 1495, 1460, 1420,~~

~~"^ 1385, 1338, 1298, 1254, 1213, 1175, 1156, 1115, 1066, 1057, 1026,~~

~~980, 952, 932, 911, 832, 822, 807, 722.~~

~~KMR Values ( TScale):~~

~~6.25 (6H,0Me-I-4',I-7); 6.41 (3H,0Me-II-4')j 6.13 (3H,0Me-I-5); 5.94~~

~~(3H,0Me-II-5); 6.18 (3H,OMe-II-7).~~

~~II-4', 1-5, II-5,I-7,H-7-Pentahydroxy- Cl-4'-0-lI-6 ZJ-biflavone (JCg):~~

~~Crystallized as yellow needles (200 mg) from CHCI„-MeOH, m.p. 355°.~~

~~II-4', I-5,H-5,1-7, II-7-Pentsacetoxy- C1-4' -0-11-6D-bifLavone (JC A):~~

~~,.. •„,..,..— ..*, », » •,. •• . ••• - ,i. rim., •.H—,-,,— ,....,..^,., -,,,. i!..... ^ in~~

~~JCp (75 mg) was acetylated with pyridine (1.5 ml) and acetic anhydride (2 ml). After usual work up the acetate crystallized from~~

~~CHCT--EtOH as colourless needles m.p. 237-40°. 200~~

~~NMR. Values (Tscale):~~

~~7.50 (3H,0Ac-ll-5)j 7.60 (3H,0Ac-I-5); 7.71 (6H,0Ac-l-.7,I-4');~~

~~7.85 (3H,0Ac-II-7).~~

~~II-4',I-5,lI-5,I-7,IJ-7-Penta-0-methyl- CI-4'-0-Il-6j]-biaavone (JC M): „ ...Hi.,- — ,. .,_».„&—~~

A mixture of JCp (50 mg), potassium carbonate ( 1 g) and dimethyl sulphate ( 1 ml) in dry acetone (200 ml) was refLuxed for 8 '. hrs. After usual work up it crystallized from CHCI^-MeOH as colourless needles (35 mg) m.p. 260 .

~~NMR. Values (Tscale):~~

~~5.SB (3H,0Me-II-5); 6.12 (3H,0Me-I-5); 6.16 (3H,0Me-Il-7); 6.19~~

~~(6H,0Me-II-4', 1-7).~~

~~JC3~~

~~JCL (20 mg) was methylated using dimethyl sulphate and potassium carbonate in dry acetone. The methylated mixture by TLC examination showed the presence of monomethyl ether of hinokiflavone. 201~~

~~2. JANIPERUS HOR1ZANTALIS MOJENCH;~~

~~The following homogeneous components were obtained and labelled as JEL, JHg, JH_ and JH,.~~

~~1-4',II-41,1-5,H-5,1-7,II-7-Hexahydroxy.CI-31,11-83 -biflavone (JEj):~~

~~Crystallized as yellow needles (150 mg) from CHCI3-MeOH, m.p. 340°,~~

~~+ Rf 0.17, \ ^^ 272 nm, 330 nm, Mol. wt. 538 (M ). '' max~~

~~I.R. KBr 3027, 1643, 1604, 1568, 1499, 1425, 1358, 1285, 1237, 1172, V "^ 1160, 1107, 1047, 1027, 966,984, 940, 910, 835, 767, 752, 735.~~

~~1-4',II-41,I-5,n-5,I-7,II-7-Hexaacetoxy-CI-3',11-83-biflavone (JH A):~~

~~JH. (70 mg) was acetylated with pyridine (2 ml) and acetic anhydride~~

~~(2 ml) . After usual work up the acetate crystallized from CHCl„-Et0H as colourless needles m.p. 242-44 . Mol. wt. 790 (M+).~~

~~MMR values (T scale):~~

~~7.97 (3H,0Ac-l-4f); 7.86 (3H,0Ac-lI-4')j 7.55 (3H,0Ac-I-5); 7.48~~

~~(3H, OAc-11-5); 7.78 (3H,0Ac-I-7) and 7.89 (3H,0Ac-II-7). 202~~

~~I-4,,II-4l,I-5,H-5,I-7,II-7-Hexa-0-niethyl-i; 1-3',11-8^-biflavone (JILM):~~

A mixture of JIL (60 mg) potassium carbonate ( 1 g) and dimethyl sulphate ( 1 ml) in dry acetone (200 ml) was refluxed for 8 hrs. After usual work up it crystallized from CHCI^-MeOH as colourless needles (40 mg) m.p. 227°, Mol. wt. 622 (M+).

~~I.R. :~~

~~KBr 3082, 3000, 2945, 2852, 1648, 1600, 1572, 1505, 1495, 1560, 1420,~~

~~maX 1385, 1338, 1298, 1254, 1213, 1175, 1156, 1115, 1066, 1057, 1026,~~

~~980, 952, 932, 911, 932, 822, 807, 722.~~

~~NMRg_ value s (T sc ale):~~

~~6.25 (6H, OMe-I-4',1-7); 6.41 (3H,0Me-II-4*)j 6.13 (3H,OMe-I-5)j~~

~~5.94 (3H,0Me-II-5); 6.18 (3H, 0Me-lI-7).~~

~~II-41,1-5,H-5,1-7, II-7-Pentahydroxy- C I-4'-0-lI-6D-biflavone (JHg):~~

~~Crystallized as yellow needles (100 mg) from CHCI -MeOH, m.p.355°.~~

~~1 II-4 ,1-5,11-5,1-7,II-7-Pentaacetoxy-C 1-4'-0-ll-6j-biflavone (JHgA):~~

~~JH? (50 mg) was acetylated with pyridine (1.5 ml) and acetic 203~~

~~anhydride (2 ml). After usual work up the acetate crystallized from~~

~~CHCI„-EtOH as colourless needles m.p. 240°. 5~~

~~MMR. values (TScale):~~

~~7.50 (3H,0Ao-II-5); 7.66 (3H,OAc-I-5); 7.71 (3H,0Ac-I-7,II-4');~~

~~7.85 (3H,0Ac-II-7).~~

~~II-41,1-5, n-5,1-7, II-7-Penta-0-methyl-CI-4" -0-11-6J-biflavone (JHgM);~~

~~A mixture of JH? ( 50 mg), potassium carbonate ( 2g) and dimethyl~~

~~sulphate ( 1 ml) in dry acetone { 200 ml ) was refLuxed for 8 • hrs. After~~

~~usual work up it crystallized from CHCI^-MeOH as colourless needles (30 mg) m.p. 259-60°.~~

~~NMR values (Tscale);~~

~~5.88 (3H, OMB-II-5); 6.12 (3H, 0Me-I-5); 6.16 (3H,0Me-Il-7); 6.19~~

~~(6H, OMe-II-4', 1-7).~~

~~JH_ (15 mg) was methylated using deimethyl sulphate and potassium~~

~~carbonate in dry acetone. The methylated mixture by TLC examination showed~~

~~the presence of monomethyl ether of hinokiflavone. (R„ values and character~~

~~istic fluorescence in UV light). 204~~

~~5t=~~

JH. was methylated using dimethyl sulphate and potassium carbonate in dry acetone. The methylated mixture by TLC examination showed the presence of hinokiflavone dimethyl ether and amentoflavone trimethyl ether. JH, on fractional recrystallization gave a major component labelled as JH./. 4

~~1-5. II-5. II-7-Trihvdroxy-I-4'. II-4'. II-7-tri-O-methyl- C1-3'.II-83-biflavone-~~

~~Crystallized as yellow needles (100 mg) from pyridine-MeOH,~~

~~Et0H + m.p. 295°j Rf0.6lj "\ 276 nm, 335 nm; Mol. wt. 580 (M ). max~~

~~1-5,II-5,II-7-Triacetoxy-I-4',H-4',I^-tri-D-methyl-Cl^',II-8ZJ-biflavone(JH, $~~

~~JH,/(40 mg) was acetylated with pyridine (1 ml) and acetic anhydride ( 2ml). After usual work up the acetate crystallized from~~

~~CHCI--EtCH as colourless needles m.p. 270°, Mol. wt. 706 (M+) .~~

~~NMR values (Tscale):~~

~~6.26 (3H, OMe-I-4'); 6.43 (3H,0Me-II-4')j 6.28 (3H,0Me-I-7);~~

~~7.52 (3H,0Ac-I-5); 7.50 (3H,0Ac-II-5); 7-91 (3H,0Ac-II-7). 205~~

~~I-41,U-4', 1-5, II-5,1-7, II-7-Hexa-O-methyl- C1-3', II-SD-biflavone (JH^M) :~~

A mixture of JH.,(40 mg), potassium carbonate ( 1 g ) and dimethyl sulphate ( 1 ml) ±n dry acetone ( 200 ml ) was re fluxed for 6 - b-rs. After usual work up it crystallised from CHCI„-Me0H as colourless needles (30 mg), m.p. 228°, Mol.wt. 622 (M+).

~~NMRr values (Tscale);~~

~~6.25 (6H,0Me-I-4*,I-7); 6.41 (3H, OMe-II-4'); 6.13 (3H,0Me-I-5);~~

~~5.94 (3H,0Me-Il"-5)and 6.18 (3H,0Me-lI-7).~~

~~3. JUKIPERUS VIRGINIANA LIMNAEUBS;~~

~~The following homogeneous components were obtained and labelled as~~

~~JVV J72 and JV3.~~

~~JV (150 mg) was methylated using dimethyl sulphate, potassium carbonate in dry acetone. The methylated mixture showed the presence of four components (TLC).~~

~~The mixture was separated by preparative layer chromatography. The 206~~

~~homogeneous components were labelled as JV..A, JV..B, JXC and JXD.~~

~~I-4« ,11-4' ,1-5,11-5,1-7, n-7-Hexa-O-nBthyl- C I-3f,II-8D -biflavone (JV A):~~

~~Crystallized from CHCI -MeOH as colourless needles (40 mg) m.p. 228°, Mol.wt. 622 (M+).~~

~~NMR. values (Tscale):~~

~~6.25 (6H,0Me-I-4',I-7); 6.41 (3B,0Me-II-4')j 6.13 (3H,OMe-I-5);~~

~~5.94 (3H,OMe-II-5); 6.18 (3H,OMe-II-7).~~

~~1-4',H-41,1-5,11-5,1-7,11 -7-Hexa-O-methyl-Cl-8,II-8D -biflavone (JV^):~~

~~Crystallized from CHCl_-MeOH as colourless needles (45 mg) m.p. 298°, Mol.wt. 622 (M+).~~

~~NMR values (Tscale):~~

~~6^23 (6H,0Me-I-4I,II-4'),- 6.14 (6H,OMe-I-5,II-5); 5.88 (6H,0Me- < 1-7,11-7).~~

~~1-4', H-4', 1-5,H-5,1-7, II-7-Hexa-O-methyl- Cl-3', II-6 J -biflavone (J VJS):~~

~~Crystallized from CHCl„-MeOH as light yellow needles (30 mg) m.p. 316°, Mol.wt. 622 (M+). 207~~

~~MMR values (T scale):~~

~~3.15 (d,2H,H-I-2,,I-6l); 3.91 (d,H,H-I-5')j 3.13 (d,2H.,H-II-2-II-6');~~

~~2.98 (d,2H,H-II-3t,H-5'); 3.65 (d,H,H-I-6); 3.42,3.12 (d,q,4H,H-I-8,II-8);~~

~~2.98 (d,2H,H-I-3,II-3); 6.14,6.12 (3H each, OMe-I-4',11-4')J 6.07,6.39~~

~~(3H each, QMe-I-5,II-5)j 6.12,6.18 (3H each, 1-7,11-7).~~

~~JV D (10 mg) showed the presence of agathisflavone (R„ values and characteristic fluroescence in UV light).~~

~~II-4* ,1-5,11-5,1-7, II-7-Pentahydroxy- CI-4'-0-II-6D -biflavone( JVg) :~~

~~Crystallized as yellow needles (10 mg) from CHClo-Me0H, 3 m.p. 355 ,~~

~~l II-4 ,I-5,II-5,I-7,II-7-Pentaacetoxy-CI-4'-0-II-63-biflavone (JV£A):~~

~~JVg (50 mg) was acetylated with pyridine (1.5 ml) and acetic anhydride (2 ml). After usual work up the acetate crystallized from~~

~~CHCl_,Et0H, as colourless needles m.p. 238-39°. 20S~~

~~NMR, value ( scale):~~

~~7.50 (3H,OAc-II-5); 7.60 (3H, 0Ac-I-5); 7.71 (3H,0Ac-I-4SH-4');~~

~~7.85 (3H,CAc-II-7).~~

~~II-4',1-5,11-5,1-7,II-7-Penta-0-methyl-Cr-4'-O-II-oJ.biflavone (JV M):~~

A mixture of J?2 (50 mg), potassium carbonate ( 1 g ) and dimethyl sulphate (1 ml) in dry acetone (200 ml) was refluxed for 8 i hrs. After usual work up it crystallized from CHCl_-MeOH as colourless needles (35 mg) m.p.260°.

~~NMR values ( scale);~~

~~5.88 (3H,0Me-II-5); 6.12 (3H,0Me-I-5); 6.16 (3H,0Me-II-7 )j 6.19~~

~~(6H-0Me-II-4«,I-7).~~

~~4. JUKIFERUS RflCURVA HJCHHAAM:~~

~~The following homogeneous components were obtained and labelled as JR.., JRp and JR„.~~

~~JR1 :~~

~~JR^ (25 mg) was methylated using dimethyl sulphate, potassium carbonate in dry acetone. The methylated mixture by TLC examination showed 209~~

~~the presence of ametoflavone and cupressuflavone (R„ value and characteristic fluorescence in UV light).~~

~~JRg:~~

JR_ (40 mg) was methylated using dimethyl sulphate, potassium carbonate in dry acetone, The methylated mixture by TLC examination showed the presence of amentoflavone monomethyl ether, cupressuflavone monomethyl ether and hinokiflavone (Rf values and characteristic fluorescence in UV light). fr

JR~ was methylated using dimethyl sulphate and potassium carbonate in dry acetone. The methylated mixture by TLC examination showed the presence of amentoflavone dimethyl ether and cupressuflavone dimethyl ether (R„ value and characteristic fluorescence in UV light).

~~The CCD separation of JR.- (65 mg) between ethyl methyl ketone and borate buffer (pH 9.5, 350 transfers) gave the following two fractions. JR-X~~

~~(40 mg),(101-150 transfers) and JR-I (10 mg),(61-95 transfers). 210~~

~~1 1-4', H-4 ,1-5,II-5-Tetraacetoxy-I-7, II-7-di-O-methyl- CI-8, II-8;>biflavone(JR3XA):~~

~~JRJ( (40 mg) was acetylated with pyridine (1.5 ml) and acetic anhydride (2 ml). After usual work up the acetate crystallized from CHCl_-MeOH as colourless needles (30 mg), m.p. 273-74°.~~

~~NMR. values ( scale):~~

~~6.13 (6H,OMe-l-7,II-7); 7.49 (6H,0Ac-I-5,H-5); 7.74 (6H,0Ac-I-4',H-4^~~

~~JR^Y :~~

JR~Y (10 mg) was methylated using dimethyl sulphate-potassium carbonate in dry acetone. The methylated mixture by TLC examination, showed the presence of amentoflavone dimethylether (R„ values and characteristic fluorescence in UV light).

~~5. THUJA 0RL5NTALIS LINNAEUS:~~

~~The following homogenous components were obtained and labelled~~

~~as TO and T02«~~

~~I-4,,II-4t,I-5,H-5,I-7,II-7-Hexahydroxy- CI-3',11-83 -biflavone (TO^:~~

~~Crystallized as yellow needles (150 mg) from CHCl3-MeOH, m.p. 250-55°,~~

~~30 Et0H + Rf 0.173, CoCD + 100°, \ 272 nm, 330 nm, Mol.wt. 538 (M ). D '' max 211~~

~~1-41, II-4' ,I-5,H-5,I-7,Il-7-Hexaacetoxy- CI-3JII-8:] -biflavone (TC^A):~~

~~T01 (60 mg) was acetylated with pyridine (1.5 ml) and acetic anhydride ( 3ml) . The acatate after usual work up crystallized from~~

~~CHCU-iStOH as colourless needles (4-5 mg), m.p. 225°, Mol. wt. 790 (M+).~~

~~MMR values (Tscale);~~

~~797 (3H,0Ac-I-4«); 7.86 (3H,0Ac-II-4!)j 7.55 (3H,OAc-I-5);~~

~~7.48 (3H,OAc-II-5)j 7.78 (3H,0Ac-I-7)j 7.89 (3H,0Ac-H-7).~~

~~1-4', II-4' j-I-5,11-5,1-7,II-7-Hexa-0-m3thyl-LI-3', II-8>biflavone (TO^l) :~~

A mixture of TO. (50 mg), potassium carbonate (1 g) and dimethyl sulphate (1.5 ml) in dry acetone (200 ml) was refluxed for 12 .h-trs. After usual work up it crystallized from CHCL_-MeOH as colourless needles (40 mg),

~~+ m.p. 170-71°, Rf 0.40, Mol. wt. 622 (M ).~~

~~KMR values (Tscale);~~

~~6.25 (6H,0Me-I-4',I-7); 6.41 (3H,0Me-II-4«); 6.13 (3H,0Me-I-5);~~

~~5.94 (2H,0Me-II-5); 6.18 (3H,0Me-II-7). 212~~

~~11-4' ,1-5,II-5,I-7,II-7-Pentahydroxy- C 1-4-' -0-II-6U -biflavone (T0g) :~~

~~Crystallized as yellow needles (200 mg) from pyridine -MeOH,~~

~~m.p. 34-5-46 •~~

~~! II-4' ,1-5,11-5,1-7,Il-7-pentaacetoxy- D-4 -0-II-6D -biflavone (TOgA):~~

~~TO^, (50 mg) was acetylated with pyridine (1 ml) and acetic~~

~~anhydride (1.5 ml). The acetate after usual work up crystallized from~~

~~CKCl~-MeOH as colourless needles (40 mg), m.p. 240-41 .~~

~~MMR values ( Tscale);~~

~~7.50 (3H,OAc-13.-5); 7.60 (3H,OAc-I-5); 7.71 (6H,0Ac-lI-4',I-7);~~

~~7.S5 (3H,0Ac-lI-7).~~

~~1 II-4 ,1-5,II-5,1-7,II-7-renta-O-methyl-El-4>-0-11-63-biflavone (TOgM):~~

~~A mixture of T0? (50 mg), potassium carbonate ( 1g) and dimethyl~~

~~sulphate (1.5 ml) in dry acetone (150 ml) was re fluxed for 10 * hrs. After usual work up it crystallized from CHCl--Me0H as colourless needles (40 mg), m.p. 268-70°. NMR values (Tscale):~~

~~5.88 (3H,OMe-II-5); 6.12 (3H,OMe~I-5); 6.16 (3H,0Me-II-7);~~

~~6.19 (6H,0Me-II-4',I-7).~~

~~6. THUJA PLLCATA D.DON:~~

~~The following homogenous components were obtained and labelled~~

~~as TP1 and TPg.~~

~~1-4' ,11-4',1-5,11-5,1-7,II-7-Hexahydrpxy- C1-3',II-3D-biflavone (TPj:~~

~~Crystallized as yellow needles (200 mg) from pyridine - MeOH,~~

~~Bt0H + m.p. 340°, Rf 0.17, ~\ 272 nm, 330 nm, Mol.wt. 538 (M ). max~~

~~1-4',11-4',1-5,11-5,1-7,11-7-Hexaacetoxy- Cl-3,H-8D-biaavone (TP A) :~~

~~TP (75 mg) was acetylated with pyridine (1.5 ml) and acetic~~

~~anhydride (2 ml). The acetate after usual work up crystallized from~~

~~CHCl--MeOH as colourless needles (60 mg), m.p. 240°.~~

~~NMR values (Tscale):~~

~~7.97 (3H,0Ac-I-4')j 7.86 (3H,0Ac-Il-4»); 7.55 (3H,OAc-I-5);~~

~~7.48 (3H,OAc-II-5); 7.78 (3H,0Ac-I-7); 7.89 (3H,0Ac-II-7). 214~~

~~I-41,11-4',1-5,11-5,1-7,U-7-Hexa-0-methyl-C 1-3',11-83-biflavone (TPJ4) :~~

~~A mixture of TP. ( 50 mg), potassium carbonate (1 g) and dimethyl sulphate (1 ml) in dry acetone (150 ml) was re fluxed for 10 i hars.~~

~~After usual work up it crystallized from CHCl_,-Me0H as colourless needles~~

~~+ (45 mg). m.p. 228°, Rf 0.40, Mol.wt. 622 (M ).~~

~~NMR, values (T scale):~~

~~6.25 (6H, 0Me-I-4',I-7); 6.41 (3H,0Me-II-4'); 6.13 (3H,0Me-I-5)j~~

~~5.94 (3H,0Me-II-5); 6.18 (3H,0Me-II-7).~~

~~Crystallized as yellow needles (150 mg) from pyridine-MeOH, m.p.345-46 .~~

~~II-4', 1*5,II-5,1-7, II-7-Pentaacetoxy- CI-4-1-0-II-6D -biflavone (TPgA):~~

~~TPg (50 mg) was acetylated with pyridine (1 ml), and acetic anhydride (1.5 ml). After usual mork up the acetate crystallized from~~

~~CHCl_-Me0H as colourless needles (40 mg) m.p. 240-41°. 215~~

~~NMR values ( scale);~~

~~7.50 (3H,OAc-II-5); 7.60 (3H,0Ac-I-5); 7.71 (6H,0Ac-I-7,II-4');~~

~~7.85 (3H,0Ac-II-7).~~

~~II-V ,1-5,11-5,1-7,II^-Penta-O-methyl-Ll-V -0-II-6D -biflavone (TP2M):~~

A mixture of TPg (50 mg), potassium carbonate (1 g) and dimethyl sulphate (1.5 ml) in dry acetone (150 ml) was refluxed for 10 --.hrs. After usual work up it crystallized from CHCl--Me0H as colourless needles (40 mg), m.p. 268 .

~~MR values (Tscale):~~

~~5.BB (3H, GMe-II-5); 6.12 (3H,OMe-I-5); 6.16 (3H,0Me-II-7);~~

~~6.19 (6H,0Me-II-4,,I-7).~~

~~7. THUJA JAPOMICA MAHM0WICZ;~~

~~The following homogenous components were obtained and labelled~~

~~as TJ1, TJg,. TJ3 and TJ .~~

~~TJ.,:~~

~~^J (120 mg) was methylated using dimethyl sulphate, potassium~~

~~carbonate in dry acetone. The methylated mixture showed the presence of 2H~~

~~three components (TLC).~~

~~The methylated mixture was separated by preparative layer chromatography. The homogeneous components were labelled as TJ A,~~

~~T^B and T^C.~~

~~I-4«, II-4',1-5,H-5,1-7,II-7-Hexa-0-methyl- C I-3',H-83-biflavone (T^A):~~

~~Crystallized from CHCl^-MeOH as colourless needles (40 mg) m.p. 228°, Mol. wt. 622 (M+). fiiMR value (Tscale);~~

~~6.25 (6H,0Me-I-4',I-7)j 6.41 (3H,QMe-II-4«)j 6.13 (3H,OMe-I-5);~~

~~5.94 (3H,OMe-II-5); 6.18 (3H,0Ke-II-7)j~~

~~1-4',II-4', 1-5,11 -5,1-7,n-7-Hexa-O-methyl-C 1-8,II-83-biflavone. (TJ B):~~

~~Crystallized from CHCl_-MeOH as colourless needles (45 mg) m.p. 296°, Mol.wt. 622 (M+).~~

~~MMR values (Tscale):~~

~~6.23 (6H,0Me-I-4',lI-4'); 5.88 (6H,0Me-I-5,H-5); 6.14 (6H,0Me-~~

~~1-7,11-7). 217~~

~~I^SH-V,1-5,H-5,1-7,II-7-Hexa-0-methyl-Cl-3l,II-6D-biflLavone (TJ^C):~~

~~Crystallized from GHCl„-MeOH as colourless needles (25 mg) m.p. 316°, Mol.wt 622 (M+).~~

~~NMR values (Tscale);~~

~~3.15 (d,2H,H-I-2', 1-6'); 3.91 (d,H,II-5»); 3.13 (d,2H,H-Il-2«,Il-6');~~

~~2.98 (d,2H,H-II-3',II-5'); 3.65 (d,H,H-I-6); 3.42, 3.12 (d,q,4H,H-I-8,II-8) j~~

~~2.98 (d,2H,H-I-3,II-3); 6.14, 6.12 (3H each, OMe-I-41,11-40J 6.07, 6.39~~

~~(3H each, 0Me-I-5, H-5) J 6.12, 6.18 (3H each, OMe-I-7,II-7).~~

~~TJ was methylated using dimethyl sulphate and potassium carbonate~~

~~in dry acetone. The methylated mixture by TLG examination showed the presence~~

~~of hinokiflavone pentamethyl ether and amentoflavone hexamethyl ether (R„ value and characteristic fluorescence in UV light).~~

~~The CCD separation of TJg (200 mg) between ethyl methyl, ketone~~

~~and borate buffer (pH 9.4, 145 transfers) gave the following two fractions.~~

~~TJgX (130 mg), (21-50, transfers) and TJgY (45 mg), (121-140 transfers). 218~~

~~II-4', 1-5,II-5,I-7,II-7-Pentaacetoxy-C1-4'-O-H-63 -biflavone (TJgXA) :~~

~~TJ„X (125 mg) was acetylated with pyridine (1.5 ml) and acetic~~

~~anhydride (2 mi). The acetate after usual work up crystallized from~~

~~+ CHCl3-EtOH as colourless needles (120 mg), m.p. 240°, Mol. wt. 748(M ).~~

~~NMR values (Tscale) :~~

~~7.50 (3H,OAc-II-5); 7.60 (3H,0Ac-I-5); 7.71 (6H,0Ac-II-4',I-7);~~

~~7.85 (3H, OAc-II-7).~~

~~1-4.'.II-4' .1-5.11-5.II-7-Pentaacetoxy-I-7-0-met hvl- L1-3*. 11-83 -biflavone ' (TJgYA):~~

~~TJpY (35 nig), was acetylated with pyridine (1 nil) and acetic anhydride (1.5 ml). The acetate after usual work up crystallized from~~

~~CHCLj-MeOH as colourless needles (30 mg) m.pj. 245°.~~

~~NMR values (Tscale);~~

~~6.28 (3H,0Me-I-7); 7.96 (3H,0Ac-I-4')J 7.84 (3H,0Ao-II-4!)i~~

~~7.54 (3H, OAc-1-5); 7.49 (3H,OAc-II-5); 7.90 (3H,0Ac-II-7),~~

~~TJ3 :~~

~~TJ- was methylated using dimethyl sulphate and potassium carbonate in dry acetone. The methylated mixture by TLG examination showed the 219~~

~~presence of amentoflavone dimethyl ether and hinokiflavone monomethyl ether (R „ value and characteristic fluorescence in UV light).~~

~~The CCD separation of TJ~ (20 mg) between ethyl methyl ketone and borate buffer (pH 9.8, 103 transfers) gave the following two fractions.~~

~~TJ„X (13 mg), (81-100 transfers) and TJ3Y (2 mg), (61-75 transfers). l-4VMI-4.M-5.II-5-Tetraacetoxy-I-7.II-7-di-0-methvl-Cl-3MI-8 3 -biflavone (TJ-X&):~~

~~TJ-X (13 mg) was acetylated with pyridine (1.5 ml) and acetic anhydride (2 ml). After usual workup the acetate crystallized from~~

~~CHCl~-MeOH as colourless needles (11 mg).~~

~~NMR values (Tscale);~~

~~2.03, 2'.08 (d,q,2H,I-2',I-6»); 2.58 (d,H,I-5); 2.50 (d,2R,H-II-2«,~~

~~II-6')j 2.97 (d,2H,II-3;iI-5)j 3.22 (d,H,I-S); 2.40 (d,H,I-6); 3.25 (s,H,~~

~~II-6); 3.47 (s,H, 1-3,11-3); 8.01, 7.75 (3H each, OAc-I-4*,II-V); 7.51,~~

~~7.59 (3H each, OAc-I-5,II-5); 6.14, 6.17 (3H, each, 0Me-J-7,-II;-7) •~~

~~TJ4 :~~

~~TJ was methylated using dimethyl sulphate and potassium 220~~

~~carbonate in dry acetone. The roethylsted mixture by TLC examination showed the presence of anientofLavone trimethyl ether and hinokifLavone~~

~~dimethyl ether (Rf value and characteristic fluorescence in UV light).~~

~~The CCD separation of TJ. (47 mg) between ethyl methyl ketone *+ and borate buffer (pH 9-8, 30 transfers) gave major fraction TJ ,X (30 mg),~~

~~(17-30 transfers).~~

~~I-5.II-5.II-7-TriacetoxY-I-4'.II-4' .I-7ftri-0-methyl- Cl-3'.11-83 -biflavone (TJ.XA):~~

~~TJ X (30'mg) was acetylated with pyridine (1 ml) and acetic anhydride (2 ml). The acetate after usual work up crystallized from~~

~~CHCl~-Et0H as colourless needles (25 mg), m.p. 270 .~~

~~NMR values ( scale1):~~

~~6.26 (3H, 0Me-I-V); 6.43 (3H,0Me-II-4»); 6.28 (3H,0Me-I-7);~~

~~7.52 (3H, 0Ac-I-5); 7.50 (3H,0Ac-II-5); 7-91 (3H,0Ac-II-7).~~

~~8. P0D0CARFU5 TAXCFOLIA KUKTH;~~

~~The following homogenous components were obtained and labelled~~

~~as PT1 and FT£. 221~~

~~I-41, II-4*, 1-5, II-5,1-7, II-7-Hexahjdroxy_ CI-3 •, II-Qj-biflavone (FT^ :~~

~~Crystallized as yellow needles (1C0 mg ), from pyridine -MeOH,~~

~~+ m.p. 340°, Hf 0.17, Mol.wt. 53$ (M ).~~

~~I-4«,II-4', 1-5,11-5,1-7,II-7-Hexaacetoxy- CI-3', 11-83 -biflavone(PT A):~~

~~PT1 (40 mg) was acetylated with pyridine (1 ml) and acetic anhydride (1.5 ml). The acetate after usual work up crystallized from~~

~~CHC1 rMe0H as colourless needles (30 mg), m.p. 240°.~~

~~NMR values ( Tscale);~~

~~7.97 (3H,0Ac-I-4')j 7.86 (3H,0Ac-II-4'); 7.55 (3H,0Ac-I-5);~~

~~7.48 (3H,OAc-II-5); 7.78 (3H,0Ac-l-7); 7.89 (3H, 0Ac-II-7).~~

~~1-4», II-4*,I-5,H-5,1-7, II-7-Hexa-0-methylCI-3', II-8D -biflavone (FT M):~~

~~A mixture of PI (40 mg), potassium carbonate (1 g) and dimethyl sulphate (1 ml) in dry acetone (150 ml) was refluxed for 10 hours. After~~

~~usual work up it crystallized from CHClo-Me0H as colourless needles~~

~~(30 mg), m.p. 228°, Mol.wt. 622 (M+). 222~~

~~NMR values ( scale);~~

~~6.25 (6H,0Me-I-4',I-7); 6.41 (3H,0Me-II-4')j 6.13 (3H,0Me-I-5);~~

~~5.94 (3H,OMe-Il-5); 6.18 (3H, OMe-II-7).~~

~~^2j~~

~~. PT2 was methylated using dimethyl sulphate and potassium carbonate in dry acetone. The methylated mixture by Ti£ examination showed the presence of hinokiflavone and amentoflavone monomethyl ethers~~

~~(E-f value, and characteristic fluorescence in UV light).~~

~~The CCD separation of PTp (150 mg) between ethyl methyl ketone and borate buffer (pH 9.5, 130 transfers) gave following four fractions.~~

~~PT2W (25 mg), PTgX (30 mg,, 16-50 transfers},PTgY (55 mg, 61-90 transfers)~~

~~and PTgZ (40 mg, 96-120 transfers).~~

~~H-4«,I-5,II-5,I-7,II-7-Pentaacetoxy-C I-4'-0-II-63-biflavone (PT„WA):~~

~~• ft~~

~~PT W (25 mg) was scetylated with pyridine (1.5 ml) and acetic anhydride (2 ml). After usual work up the acetate crystallized from CHCl -~~

~~£t6H as colourless needles (20 mg), m.p. 240 . NMR values (Tscale):~~

~~7.50 (3H,0Ac-II-5); 7.60 (3H,OAc-I-5), 7.71 (6H,0Ac-I-4',I-7);~~

~~7.85 (3H,0Ac-II-7). lI-4'.I-5.II-5.I-7.Il-7-Pentaacetoxy- .'. 1-4'-0-methvl- CI-*'. 11-83 -bifLavone (PTgXA):~~

~~PTpX (30 rag) was acetylated with pyridine (1.5 ml) and acetic anhydride (2 ml). After usual work up the acetate crystallized from~~

~~CHC13-E#0H as colourless needles (25 mg), m.p. 183-84 .~~

~~NMR values (Tscale);~~

~~6.28 (3H,0Me-l-4,)j 7.87 (3H,0Ac-II-4'): 7.54 (3H,OAc-I-5) j~~

~~7.47 (3H,OAc-II-5); 7.78 (3H,0Ao-I-7); 7.91 (3H,0Ao-II-7).~~

~~I-4,.I-5.II-5.I-7.II-7-Fentaacetoxv~Il-4,-0-methvl-Cl-3'rII-8J -biflavone (PTgYA):~~

~~PTgY (50 mg) was acetylated with pyridine (1 ml) and acetic anhydride (1.5 ml). The acetate after usual work up crystallized from~~

~~CHCl~-.E)tOH as colourless needles (40 mg), m.p. 254°, Mol.wt. 762 (M+). MR values ( Tscale) :~~

~~6.47 (3H,0Me-II-4')j 8.0 (3H,0Ac-I-4')j 7.59 (3H,OAc-I-5);~~

~~7.50 (3H,OAo-Il-5)j 7.80 (3H,0Ac-I-7); 7.88 (3H,0Ac-II-7).~~

~~1-4' .11-4' .1-5.11-5. II-7-Pentaacetoxy-I-7-0-methyl- C1-3' .11-8 J -biflavone (PTgZA):~~

~~PT2Z (40 mg), was acetylated with pyridine (1 ml) and acetic anhydride (1.5 ml). The acetate after usual work up crystallized from~~

~~CHCL-3-EtOH as colourless needles (30 rag), m.p. 245° Mol.wt 762 (M+).~~

~~NMR values ( Tscale):~~

~~6.28 (3H,0Me-I-7); 7.96 (3H, OAc-I-4'); 7.84 (3H,0Ac-Il-4') j~~

~~7.54 (3H,QAc-I-5); 7.49 (3H,OAc-II-5); 7.90 (3H,0Ac-II-7). 225~~

~~9. BIELAVONfiS FROM ARUCARIA EXGELSA LAMB:~~

~~The following homogenous components were obtained and labelled~~

~~as AE.J, AB2,AE3,AE.,AE5>AE6,AE7,AEg and AE„.~~

~~AB1 :~~

AE1 (30 mg), was methylated using dimethyl sulphate and potassium carbonate in dry acetone. The methylated mixture by TLC examination showed the presence of amentoflavone, cupressuflavone and agathisflavone hexamethyl ethers.

~~AEp (10 mg) was methylated using dimethyl sulphate and potassium carbonate in dry acetone. The methylated mixture by TLC examination showed the presence of agathisflavone monomethyl ether.~~

~~1-4.' .H-4.M-5. JI-5.I-7-rPentahydroxv-II-7-0-methyl~ Cl-3' .11-83 -biflavone (AEL):~~

~~Crystallized as yellow needles (100 mg); from CHCl„-MeOH,~~

~~m.p. 300°, Rf 0.37. 226~~

~~I-JJ .11-4' .I-5.II-5.I-7-Pentaacetoxv-II-7~0-methvl-C 1-3' .11-8 J -biflavone (AE~A):~~

~~AE_(50 mg), was acetylated with pyridine (2 ml) and acetic anhydride (2.5 ml). After usual work up the acetate crystallized from~~

~~CHCl~-Et0H as colourless needles (40 mg), m.p. 165 .~~

~~NMR values (Tscale):~~

~~1.99 (q,1H,H-I-6»); 1.99 (d,1H,H-I-2'); 2.48 (d,2H,H-lI-2',II-6');~~

~~2.53 (d,1H,H-I-5'); 2.74 (d,lH,H-I-8)j 2.95 (d^H-II^SII^'); 3.16 (d,1H,~~

~~H-I-6); 2.23 (s,1H,H-II-6); 3.41, 3.33 (s,2H,H-I-3,II-3); 6.09 (s,3H,0Me-~~

~~11-7)j 7.44 (s,6H,OAc-I-5,H-5)j 7.68 (s,6H,0Ac-I-4',II-4'); 7.95 (s,3H,~~

~~OMe-I-7);~~

~~1-4',H-4', 1-5,H-5,1-7,11-7-Hexa-O-methyl- CI-3',H-83 -biflavone (AS3M) ;~~

~~A mixture of AEL (40 mg), potassium carbonate (1 g) and dimethyl sulphate (1 ml) in dry acetone (150 ml) was refluxed for 8 ,hrs.~~

~~After usual work up it crystallized from CHCl_-MeOH as colourless needles~~

~~(30 mg), m.p. 212°. Mol. wt. 622 (M+). 227~~

~~NMR values (Tscale);~~

~~6.25 (s,6H,0Me-I-4',I-7); 6.41 (s,3H,0Me-II-4'); 6.13 (s,3H,OMe-I-5);~~

~~5.94 (s,3H,0Me-II-5); 6.1S (s,3H,0Me-II-7).~~

~~I-4MI-4'.I-5.II-5T Tetrahydroxv-I-7.II-7-di-0-methyl-C I-6.II-83 -biflavone (AE.):~~

~~Crystallized as yellow needles (75 nig), from CHCl„-MeOH,~~

~~m.p. 308°, Rf 0.43.~~

I-4MI-4'.I-5.II-5-Tetraacetoxy-I-7.II-7-di-0-methyl-Cl-6.II-8J -biflavone (AE.A): a AE,(30 mg), was acetylated with pyridine (1 ml) and acetic 4 anhydride (1.5 ml). After usual work up the acetate crystallized from

~~CHCl3-EtOH as colourless needles (25 mg), m.p. 174°.~~

~~NMr values (T scale);~~

~~6.17 (s,3H,0Me-II-7); 6.19 (s,3H,0Me-I-7); 7.53 (s,3H-OAc-H-5);~~

~~7.67 (s,3H,OAc-I-5); 7.76 (s,3H,0Ac-II-4')j 7.88 (s,3H,0AC-I-4*).~~

~~1-4', 1I-41,1-5,11-5,1-7,II-7-Hexa-0-methyl- 1-6,11-8 -biflavone (AE.M): __ £b~~

~~A mixture of AS, (40 mg), potassium carbonate (1 g) and~~

~~dimethyl sulphate (1 ml) in dry acetone (150 ml) was refluxed for 10 hrs. 228~~

~~After usual work up it crystallized from CHCl_-MeOH as colourless needles (30 mg), m.p. 160°, Mol. wt. 622 (M+).~~

~~NM8 values (Tscale);~~

~~2.12 (d,2H,H-I-2',I-60; 2.99 (d, 2H,H-I-3',I-5); 2.63 (d,2H,~~

~~H-II-2', 11-60; 3.22 (d, 2H, H-II-3',11-5*); 3.09 (s, 1H,H-I-8)j 3.36 (s,~~

~~1H,H-II-6)j 3.47, 3.49 (s,2H,H-I-3,II-3); 6.26 (s,3H,0Me-I-4')j 6.22 (s,3H,~~

~~OMe-II-4')} 6.14 (s,3H,0Me-I-7); 6.12 (s,3H,0Me-II-7); 6.41 (s,3H,0Me-~~

~~1-5); 5.95 (s,3H,0Me-II-5).~~

~~AEL :~~

~~AEc was methylated using dimethyl sulphate, potassium carbonate in dry acetone. The methylated mixture by TLC examination showed the~~

~~presence of amentoflavone and cupressufLavone dimethyl ethers (Rf value and characteristic fluorescence in UV light).~~

~~The CGD separation of AE_ (125 mg), between ethyl mathyl ketone and borate buffer (pH 9.8, 140 transfers) gave following two fractions.~~

~~AEJC (100 mg), (81-115 transfers) and AELY (10 mg), (131-140 transfers). 2 2,9~~

~~I-4MI-41.1-5.II-5-Tetraacetoxy-l-7.II-7-di-0-Kiethyl- C 1-3' .11-8 3 -biflavone (AEJCA):~~

~~AEJC (60 mg), was acetylated with pyridine (1.5 ml) and~~

~~acetic anhydride (2 ml). After usual work up the acetate crystallized~~

~~from CHC1 -EtOH as colourless needles (50 rag), Mol. wt. 734 (M+).~~

~~NMR values (Tscale):~~

~~8.01 (a,3H,0Ac-I-4«); 7.75 (s,3H-0Ac-II-4*); 6.14 (s,3H,OMe-I-7);~~

~~6.17 (s,3H-0M&-1I-7); 7.51 (a,3H,OAc-I-5)j 7.59 (s,3H,OAc-II-5).~~

~~1-4' or II-4t.I-5.II-5.II-7-Tetrahydroxy-I-4' or II-4M-7-di-0-nieth.vl- C1-8,II-8 3-biflavone (AfLYA):~~

~~AE-Y (10 mg), was acetylated with pyridine (1 ml) and acetic~~

~~anhydride (1.5 ml). After usual work up the acetate crystallized from~~

~~+ CHCl3-EtOH as colourless needles ( 9 mg). Mol.wt. 734 (M ).~~

~~MR values (Tscale):~~

~~7.76 (s,3H,0Ac-I-4f or 11-4*); 6.22 (s, 3H,OMe-I-4* or II-41);~~

~~7.52 (s,6H, OAc-I-5,II-5); 6.16 (s,3H,0Me-I-7); 7.98 (s,3H,0Ac-II-7). 230~~

~~1-4' .I-5.II-5TTrihydroxy-lI-A' .I-7.II-7-tri-0-methyl-C I-6.II-83 -biflavone (AE^):~~

~~Crystallized as yellow needles (60 mg), from CHCl^-MeOn, m.p. 300,~~

~~Rf 0.613.~~

~~I-4M-5.II-5-Triacetoxy-II-^M-7.II-7-tri-0-methvl- Cl-6.11-8 3 -biflavone (AELA.):~~

~~AE, (30 mg), was acetylated with pyridine (1 ml) and acetic anhydride (1.5 ml). After usual work up the acetate crystallized from~~

~~CHCl_-etOH as colourless needles (25 mg), m.p. 185°.~~

~~NMR values ( Tscale):~~

~~2.05 (d,2H,H-I-2',l-6'); 2.72 (d,2H,H-I-3',I-5'); 2.57 (d,2H,H-II-2«,~~

~~II-6'); 3.18 (d,2H,H-Il-3',II-5')j 2.97 (s,1H,H-I-8); 3.28 (s, 1H, H-Il-6);~~

~~3.33, 3.49 (2H,H-I-3,H-3)j 7.88 (s,3H,0Ac-I-4!); 6.26 (s,3H,0Me-II-4');~~

~~7.67 (s,3H,OAc-l-5); 7.54 (s,3H,OAc-II-5); 6.19 (s,3H,0Me-I-7); 6.22 (s,3H,~~

~~OMe-n-7).~~

~~1-4',II-4', 1-5,11-5,1-7, II-7-Hexa-O-methyl- C 1-6,11-8 3 -biflavone( AS6M):~~

~~A mixture of AS^ (25 mg), potassium carbonate (1 g) and dimethyl sulphate ( 1ml) in dry acetone (150 ml), was refluxed for 10 hrs. After 231~~

~~usual work up it crystallized from CHCl_-MeOH as colourless needles~~

~~(20 mg), m.p. 242°. Mol. wt. 622 (M+).~~

~~NMR values (Tscale);~~

~~6.26 (s,3H,0Me-I-4'); 6.22 (s^HjOMe-II-V); 6.14 (s,3H,0Me-~~

~~1-7); 6.12 (s,3H,0Me-II-7); 6.41 (s,3H,OMe-I-5); 5.95 (s,3H,OMe-II-5).~~

~~AE? : •~~

~~AB„ was methylated using dimethyl sulphate, potassium carbonate in dry acetone. The methylated mixture by TLC examination showed the~~

~~presence of amentoflavone and cupressuflavone trimethyl ether (Rf value and characteristic fluorescence in W light).~~

~~The CCJ3 separation of AEL (40 mg), between ethyl methyl ketone and borate buffer (pH 10.2 ' . , 92 transfers) gave the fraction AELX (20 mg),~~

~~(81-91 transfers).~~

~~II-A' .1-5.II-5-Triacetoxy-I-4'. I-7.II-7-tri-0-methyl- CI-31.II-83 -biflavone (AEJCA):~~

~~AE„X (30 mg), was acetylated with pyridine (1 ml) and acetic anhydride (1.5 ml). After usual work the acetate crystallized from CHG1_-~~

~~EtOH as colourless needles (25 mg). /• 0 4~~

~~NKR value (Tscale):~~

~~2.14 (d,1H,H-I-2»); 2.11 (q,1H,H-I-6«); 2.88 (d,1H,H-I-5');~~

~~2.57 (d,2H,H-II-2',II-6')j 2.98 (d, 2H,H-II-3^II-5,) j 3.19 (d,1H,H-I-3);~~

~~3.44 (d,1H,H-I-6)j 3.25 (s,1H,H-II-6)> 3.44 (s,2H,H-I-3,II-3)r6»23 (s,3H,~~

~~1 l OMe-I-4 ); 7.75 (s>3H,0Ac-II-4 ); 7.51 (s,3H,0Ac-l-5); 7.58 (s,3H,0Ac-II-5);~~

~~6.13 (s,3H,0Me-I-7); 6.17 (s,3H,0M&-II-7).~~

~~I~5.II~5rPi»h.vdroxy-I-4t .II-4'.1-7.11-7-tetra-O-methyl-C 1-3' .11-83 -biflavone (AE-):~~

~~Crystallized as yellow ijeedles (too mg), from CflCl -MeOH, m.p. 275°.~~

~~1-5.II-5-Diaceto:>ty-I-4' .11-4' .I-7.II-7-tetra-0-methyl- Cl-31.11-83 -biflavone (A3^A):~~

~~AEg (50 mg), was acetylated with-pyridine (1 ml) and acetic anhydride (2 ml). After usual work up the acetate crystallised from~~

~~CHCl.-iStOH as colourless needles (40 mg), m.p. 225°.~~

~~NMR values (Tscale):~~

~~6.26 (s,3H,OMe-I-4'); 6.15 (s,3H,0Me-II-4'); 7.52 (s,3H,OAc-I-5)j~~

~~7.59 (s,3H,OAc-II-5); 6.15 (s,3H,OMe-I-7); 6.26 (s,3H,0Me-II-7). 233~~

~~1 1 I-4 ,11-4' ,I-5,H-5,1-7,11 -7-Hexa-O-methyl- C I-3 ,11-8D -biflavone (AEgM):~~

A mixture of AEg (40 mg), potassium carbonate (1 g) and dimethyl sulphate (1 ml) in dry acetone (150 ml) was refluxed for 8 • hrs. After usual work up it crystallized from CKCl_-Me0H as colourless needles (35 mg), m.p. 212°. Mol. wt. 622 (M+)

~~NMR values (7scale):~~

~~6.25 (s,6H,0Me-I-4',I-7); 6.41 (S,3H,0Me-II-4' )j 6.13 (s,3H,~~

~~0Me-I-5); 5.94 (s,3H,OMe-II-5)j 6.18 (s,3H,0Me-II-7).~~

~~I-5.II-5-Dihvdroxy-I-4' .1I-41.I-7.II-7-tetra-0-methyl- CI-8.II-8 ZJ -biflavone (AEQ):~~

~~Crystallized as yellow needles (150 mg), from CHCl^-MeOH,~~

~~25 . m.p. 150 , Cod + 30 . D~~

~~-1-5.II-5-Diacetoxy-I-4f .11-4'.I-7.Il-7-tetra-0-methyl-C 1-8.11-83 -biflavone (AEQA): .—- 2__~~

~~ASQ (80 mg), was acetylated with pyridine (2 ml) and acetic anhydride (2.5 ml). After usual work up the acetate crystallized from~~

~~CHCl„-St0H as colourless needles (70 mg), m.p. 155 . 23 4~~

~~NMR values ( Tscale):~~

~~6.25 (s, 6H,0Me-I-4',II-4'); 7.52 (s,6H,OAc-I-5,II-5);~~

~~6.22 (s,6H,OAc-I-7,II-7).~~

~~I-4»,II-4',I-5,H-5,1-7,II-7-Hexa-O-methyl-C 1-8,11-33-Biflavone (AE M): , . iL_n _~~

~~A mixture of AEQ (50 mg), potassium carbonate ( 1 g) and dimethyl sulphate ( 1 ml) in dry acetone (150 ml), was refluxed for~~

~~6 hrs. After usual work up it crystallized from CHGl„-MeOH as colourless need les (45 mg), m.p. 160 .~~

~~Mffi values (Tscale):~~

~~6.14 (s^OKe-I^SH-^); 5.88 (s,6H,0Me-I-5,H-5); 6.22~~

~~(s,6H,0Me-I-7,II-7). 235~~

~~C-GLrcOSILFLAVONES FROM PHLOX DRUMKONDII HOOK (PQLBMQNIAGSAE):~~

~~Dried and coarserly powdered white flowers of Phlox drummondii~~

(500 g) were refluxed with petroleum ether (40-60 ) for 6 hrs. The extract was decanted off and then treated with fresh quantity of petroleum ether and refluxed again for 6 hrs. The petroleum ether treated flowers were completely dried and exhausted with ethanol till the extract was almost colourless. The combined ethanol extracts were concentrated on a jaater bath whereby a highly viscous brown mass was left behind. The concentrate was taken into hot water (900 ml) and filtered. The isoluble residue gave no test for flavanoids and was, therefore, discarded. The aqueous solution was extracted with n-butanal. The process was repeated four times till the aqueous solution was almost colourless. The n-butanol extracts were combined and the solvent was recovered under diminished pressure. The semi solid mass left behind was marked as PD.

~~CHROMATOGRAPHIC EXAMINATION OF PD;~~

~~The pyridine solution of PD was subjected to chromatographic analysis on Whatman iio.1 filter paper employing both the ascending and 236~~

~~the descending techniques. The following solvent mixtures were used:~~

~~1. Butanol : acetic acid : water (4 : 1 : 5 ) 2. Butanol : acetic acid : water (6:1:2)~~

~~3. Butanol : acetic acid : water (8:2:5)~~

~~4. Acetic acid : water (60 : 40 ) 5. Acetic acid : water (20 : 80 )~~

~~6. Benzene : pyridine : water (100:1:100 )~~

The chromatograms were run for 12 hrs. After drying at room temperature the chromatograms on examination under UV light revealed only two spots except in solvent system number 2, where the presence of three spots were noted.

Thin layer chromatographic plates (5 x 20 cms) of 0.5 mm thickness were prepared by the usual methods with the help of the spreader using polyamide (Waelm). The spot of 0.1$ of PD in pyridine was applied to the starting line and the plate was run with the solvent system methanol : acetic acid : water (90:5:5). The plate was run to the distance of 15 cm, removed from the tank and dried. On examination under UV light the chromatogram showed the presence of three spots, two major and one minor. 237

~~PURIFICATION OF PD (COLUMN CHROMATOGRAPHY);~~

~~Water (400 ml) was added to polyamide (50 gms) and the mixture stirred to give a thin slurry. The slurry was atonce added to a column~~

(15 mm in diameter), and the sides of the column were rinsed down with water. When the adsorbent settled, the excess of water was run off, leav ing a thin layer of water above the surface of the adsorbent. An aqeous solution of the semi solid PD (1 gm) was added. After the solution had passed into the column the column was developed with water, 5% methanol and methanol respectively. On chromatographic examination (TLC) all the three fractions were found to be the mixtures and were therefore combined.

~~PURIFICATION OF PD (PRSPARATIVfi PAPER CHROMATOGRAPHY);~~

A pyridine solution of PD (1$) was applied as a streak from a wide band pipette on the whatman 3 MM chromatographic paper. A hair drier was used for the solvent evaporation between repeated application of the solvent to the paper. The chromatograms were developed with n-butanols acetic acid:water (6:1:2) for 12 hrs. After drying the bands were marked under UV light with the help of the lead pencil. The encircled 23S

pigment zones were labelled as I,II and III. The bands marked I and III were carefully cut and extracted separately by refluxing them with 10$ methanol. On recovery of the solvent a small amount of residue was left in each case. The fractions thus separated were tested for homogeneity by

~~TLG on polyamide by using the following solvent systems.~~

~~1. Methanol:acetic acid:water (90:5:5)~~

~~2. Ethyl methyl ketone:toluene:acetic acid:methanol:water(80:10:2:5:6)~~

~~3. Ethyl methyl ketone:ethyl:acetate:formic acid:water (3:5:1:5)~~

~~4. Sthanol:water (3:2)~~

~~5. Water:ethanol:ethyl methyl ketone:acetyl acetone (15:3:3:1)~~

~~The homogeneity of each fraction was further established by two dimensional paper chromatography using t-butanol:acetic acid:water~~

~~(3:1:1) as first solvent and aq. acetic acid (15$) as the second solvent, fractions I and III gave the usual tests for flavanoids.~~

~~COLOUR TESTS:~~

~~1. Magnassium + hyrochloric acid orange 2. Zinc + hydrochloric acid dark red~~

~~3. Sodium amalgam + hydrochloric acid Pinkish violet~~

~~0~Rhamnosyl-I-6-C-xylosvlluteolin (I).~~

~~Crystallized from 10$ MeOH as yellow needles (200 mg), 239~~

~~aup. 198°, Hf (TBA) = 0.29, (HOAC) » 0.52.~~

~~NMR of TMS ether of I in CC1, (Tscale):~~

~~2.70 (m,2H,H-I-2',I-6»); 3.20 (d,1H,H-I-5')j 3.65 (s,1H,H-I-8);~~

~~3.78 (s,1H,H-I-3); rhamnose-H-I, centered at 5.40; sugar signals, 5.60-7.10; rhamnose-CH„ 9.25.~~

~~Acetate of I:~~

~~A mixture of I (50 mg), pyridine (1 ml) and acetic anhydride~~

~~(1.5 ml) was heated on water bath for 2 hrs, cooled and poured onto crush ice. A white solid was obtained which was filtered and dried.~~

~~NMR in CDCL„ (Tscale): 2.~~

~~2.30 (m,2H,H-l-2«,II-6'); 2.65 (s,H,H-I-8); 2.65 (d,1H,H-I-5');~~

~~3.40 (s,1H,H-I-3); sugar range, 4.20-6.00; aromatic acetyl range 7.40-7.80; sugar acetyl range 7.9-8.2; rhamnose-CH_-9.4.~~

~~1-3',1-4' .I-5.I-7-Tetrahvdroxv-flavone;~~

To the suspension of 1 (75 mg), in phenol (0»5 g), HI (1 ml), was added gradually with cooling. The mixture was gently refluxed (135-37?) for 6 hrs and poured into aq. NaHSO,. and filtered. Repeated crystallisation 24 0

~~of the product from MeOH gave yellow needles (65 mg), m.p. 330~~

~~-v MeOH /\ 242sh nm, 253 nm, 267 nm, 291 sh nm, 349 nm max~~

~~Acetylation of the aglycone (50 mg), using acetic anhydride and pyridine and crystallisation of the acetate from CHCl^-MeOH afforded colourless needles (40 mg), m.p. and m.m.p.222-24 •~~

~~I-6-C-xylosylluteolin(lI);~~

The anhydrous glycoside I (60 mg), was hydrolysed by re fluxing with aq. hydrochloric acid (10 ml). The refluxing was continued for two hrs to ensure complete hydrolysis. After leaving" for 1 hr at room tempera ture, the yellow solid II, thus separated out was filtered washed well with water and dried. The crude product crystallised from 10$ methanol as

~~yellow needles (40 mg), m.p. 260°, R£ (15$ HOAc) 0.23.~~

~~UV spectra ( 7s max nm );~~

~~MeOH 242 sh, 255, 271, 349~~

~~NaOMe 267,278 sh, 337 sh, 406~~

~~A1G13 278, 302 sh, 332, 429 A1C1-/HC1 265 sh, 279, 296 sh, 361, 384 241~~

~~NaOAc 276, 323, 393~~

~~HaOAc/H BO„ 265, 377, 429 sh~~

~~CHROMATOGRAPHIC IDENTIFICATION OF SUGAR;~~

~~The filtrate from which the compound II was removed was~~

~~neutralized by passing it over a column of cation exchange resin~~

~~(Amberlite IR-120). It was concentrated under reduced pressure to a~~

~~syrup. The sugar was identified by paper chromatography on Whatman no.1~~

~~filter paper using butanol; acetic acid:water (40:10:50) with authentic~~

~~sugars as checks. Aniline phthalate and p-anisidine phosphate solutions were used as spray reagents. The chromatograms on drying at 100-105~~

~~showed the presence of rhamnose only.~~

~~FERRIC CHLORIDE OXIDATION:~~

~~The compound I (75 mg), was treated with aq. FeCl» (1 g),10 ml~~

~~water) yielding a dark coloured complex which was refluxed at 115° for~~

~~15 min and then at 125 for 6 hrs. The mixture was diluted with water~~

~~and the dark coloured complex filtered off. The filtrate was neutralized~~

~~as described earlier. The sugars were identified by paper chromatography 242~~

~~as rhamnose and xylose.~~

~~O-Rhamnosyl-I-6-G-xylosylapigenin (III);~~

~~Crystallised from 10$ MeOH as yellow needles (250 mg), m.p. 210 .~~

~~NMR of TM3 ether of III in CDC1 (Tscale):~~

~~2.35 (d,2H,H-I-2',I-6«); 3.20 (d,2H,H-I-3',I-5'); 3.65 (s,1H,H-I-8);~~

~~3.75 (s,1H,H-I-3); H-I-rhamnose, 5.1; H-I xylose, centered at 5.4; sugar signals, 6.00-7.20, rhamnose-CH_, 9.15.~~

~~Adetate of III :~~

~~A mixture of III (75 mg), pyridine (1 ml) and acetic anhydride~~

~~(1.5 ml) was heated on water bath for 2 hrs. After usual work up a white solid (60 mg), was obtained which was dried.~~

~~KMR in CDC13 ( scale):~~

~~2.2 (d,2H,H-l-2«,I-6'); 2.8 (d,2H,H-I-3*, 1-5'); 2.75 (s,JH,H-I-8);~~

~~3.44 (s,1H,H-I-3); sugar signals 4.50-6.70; I-4NI-5,I-7-aromatic acetyl,~~

~~7.44, 7.60, 7.70; sugar acetyls, 7.92, 7.99 (two), 8.1,8.14, rhamnose-CH -9.4- 243~~

~~l^.'.I-5.I-7-Trihydroxy flavone;~~

~~To the suspension of III (75 mg), in phenol (0.5 g), HI (1 ml)~~

~~was added gradually with cooling. The mixture was gently refluxed (135-137°)~~

~~for 6 hrs and poured into aq. NaHS0_ and filtered. Repeated crystallization~~

~~of the product from MeOH gave yellow needles (50 mg), m.p. 347°~~

~~-v MeOMeOHH /\ 267 nm, 296 sh nm, 336 nm max~~

~~Acgtylation of the aglycone using acetic acid and pyridine and~~

~~the crystallization of the acetate from CHCl„-MeOH afforded colourless~~

~~needles m.p. and m.m.p ig^g^P, -. ' .-•~~

~~I-6-C-xylosylapigenin(IV);~~

~~The anhydrous glycoside III (75 mg), was hydrolysed by re fluxing~~

~~with aq. hydrochloric acid (20 ml). After usual work the crude product was crystallized from 10$ methanol as yellow needles (60 mg), m.p. 228°.~~

~~Rf (15J8 HOAc) 0.37.~~

~~UV spectra (7\ max nm);~~

~~MeOH 271, 336 NaOMe 278, 329, 398~~

~~A1C13 262 sh, 278, 304, 352, 382 244~~

~~A1C1VHC1 260 ah, 280, 302, 344, 380~~

~~NaOAc 279, 303, 385~~

~~Na0Ac/H3B03 274, 346, 408 sh.~~

~~CHROMATOGRAPHIC IDENTIFICATION OF SUGAR:~~

~~The filtrate from which the compound IV was removed worked up as usual. The chromatogram showed the presence of rhamnose only.~~

~~FERRIC CHLORIDE OXIDATION:~~

~~The'component III (75 mg), was treated with aq. FeCl„ (1 g, 10 ml water). After usual work up the chromatogram showed the presence rhamnose and xylose only. BIBLIOGRAFHI 245~~

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~~1. Flower Pigments: Flavanoid glycosides from Peltophorum inernis, Jour. Indian Chem. Soc, 3_, 276 (1969)~~

~~8. Flavanoids from the flowers of Acacia farnesiana Willd, Jour. Indian Chem. Soc, 2_, 183 (1970).~~

~~3. Biflavonyl pigments from Thuja orientalis, Phytochemistry, % 1897 (1970).~~

~~4. New C-glycosides from the flowers of Phlox drummondii, Phytochemistry, 10, 677 (1971).~~

~~5. Biflavonyl pigments from Juniperus chinensis, Jour. Indian Chem. Soc, 2, 204 (1971).~~

~~6. Biflavonyls from Thuja plicata, Jour. Indian Chem. Soc, % 917 (1972).~~

~~7. Biflavones in the leaves of two Juniperus plants, Phytochemistry, 12, 1494 (1973).~~

~~8. Biflavones in the leaves of Podocarpus taxifolia, Phytochemistry, 1J2, 1497 (1973).~~