CHAPTER IV

TRUE STRUCTURE OF FLAVONOlDS FROM

Limnophila gratissima

4.1 4.2
4.3

Introduction Present work Results and discuss~on Experimental Figures and Tables

4.4

4.5

References

112

TRUE STRUCTURE OF FLAVONOIDS FROM LIMNOPHILA GRATISSIMA

4.1 INTRODUCTION

The Scrophulariaceae is quite a big family embracing about 210 genera and 3000 species' mostly of herbaceous plants distributed throughout the world, The family is represented by about 275 species in India'. Mostly herbs or small shrubs, a few are chlorophyll-less parasites and others are chlorophyll contaming parasites or saprophytes. The common Indian genera include Antrrrhrum cronrlunl and A.majus found all over India. The Scrophulariaceae are not a particularly important family and except for the the plants are valued primarily as garden ornamentals. drug plant D~girulls, Aurones have been identified from many species of Anrrrrhrum," from 14 species of L~nurla', from flowers of Calceolaria chelidonoides'

an aurone like substance was

reported by Harborne but its structure defied analysis. Similar behaviour was noted with a compound from an unidentified Calecolorracultivar by Bohm and Saleh.bOther genera In the fam~lytested were negative. Anrrrrhlum mujus' is found to contain naringenin-7glucoside. Scrophulariaceae are also found to contain C-glycosyl flavonoidss and 6hydroxyluteolinq and methylated derivative^.^ L~mnophlla R.Brl"', is a genus of herbs found throughout Africa. Asia and Australia. About 20 species occur in India. Some are strongly aromatic and are ofien used as pot-herb or as a flavouring agent.

Some of the chemical constituents identified from Limnophila include a new mterpene", 5-hydroxy,7,8,2',4'-tetramethoxy flavone, ursolic acid" and 5,2'-dihydroxy 7,8,4'-trimethoxy flavone" from L.he~erophylla, betulin and betulinic acid1*,another new triterpene," 7-methoxy,5,2',4'-trihydroxyflavone (artocarpetin)16 and 5-hydroxy.72',4'-trimethoxy flavone" from L.mgosa. Essential oil of L . r u g o ~ aand ' ~ L.conferral' and some of their constituents exhibited significant antibacterial and antifungal activity. Also a comparison of anti-inflammatory activ~ty of certain methoxy flavonoids from L.conferta" like wogonin, nevadensln and quercetln pentamethyl ether shoued an methoxylation. Chemical and phannacologlcal

increase in activity with increased lnvestigatlons of L.conferla"

and L h e r e r ~ p h ~ l i a 'have ' shown that its essential oil

contalning 15 compounds has antifungal activity and nevadensin is found to be antiinflammatory, cytotoxic and anti tubercular. Limnoph~laa r o m a r r ~ a '(Lam) ~ Merill Syn L gratissrma Blume," ( known as kutna in Hindi and manganari in malayalam) is a'stout aromatic herb 30-50 cm high found in South Bihar. Onssa, Sundarbans, Ala hills (Assam). Deccan and Western parts of South Indla, upto 600m in damp places, margins of ponds and backwaters It is a

much branched decumbent aromatic herb (odour of turpentine), copiously rooting at the lower nodes, leaves sessile, opposite or in whorls of 3, h e a r oblong or lanceolate, sharply serrate, flowers purplish in axillary and terminal racemes, pedicels long slender and glandular, fruits small obovoid- oblong capsules covered by the striate calyx. The plant possesses the odour of turpentine and yields 0.13% of an essential oil containing d-limonene and d-periallaldehyde as the principal constituents. It is used as spinach in Java and eaten raw or steamed. Leaves are applied as poultice for sore on legs.

The juice of the plant is given in fever and to nursing mothers when the milk is sour. The plant is s o d 1 , slightly bitter, refrigerant, emollient, antiseptic, galactogogue, aperient, appetiser, digestive, carminative, anthelminthic, anti-inflammatory, febrifuge. diuretic and

It is useful in vitiated conditions of pitta, foul ulcers, agalactia, galactic

impurities, anorexia, dyspepsia, helminthiasis, constipation, strangury, fever and pharyngitis. A colour photograph of the plant is given in Fig 4.1. A rare methylated flavonol, 7-desmethyl artemetin, was reported" from

L.grarissima. In view of the rare occurrence of flavonols in the family Scrophulariaceae

along with the doubt on the reported spectral and chromatographic data on the compound, and in continuation of the work on flavonoids of Scrophulariaceae by our group''?O it was considered des~rable to re-examine this plant. Adopting standard procedures two

flavones nevadensin (5.7-dihydroxy 6,8,4'-trimethoxy flavone) and salvigenin(5-hydroxy 6,7,4'-trimethoxy flavone) and two phenolic acids (caffeic acid and chlorogenic acid) were isolated and characterised. 7-desmethyl artemetin reported was thus found'to be an intimate mixture of these two methylated flavones. The details of the investigation* are presented in t h ~ s chapter.

*~~-account

of the work was presented at UGC-DRS National Symposium in

Madurai Kamaraj University, Madurai, April 22-24, 1998, and later published in Indian

J. Chem., (1999). 37B,----.

Fig. 4.1 Colorr Photograph of Lgrntirrima

4.2 PRESENT WORK

The isolation of methylated flavonoli4 from L.gralissima [Scrophulariaceae, known to biosynthesise flavonols very rarely] and the non-identity of spectral / chromatographic data of 7-desmethyl artemetin with that isolated in our laboratory," prompted re-examination of the plant. leading to the isolation of two methoxylated flavones and two phenolic acids. The identity of all the compounds have been

established by chemical and spectral evidence and confinned by direct comparison with authentic samples. The plant material (2kg) was refluxed with 95% bolllng EtOH and e upon fractionat~onwith benzene, concentrated in vacuo. The aqueous c ~ d extract dichloromethane and ether, gave residues of which the latter two fractions showed posithe test for polyphenolics. The CH,CI, extract was concentrated and subjected to column chromatography (Si0?,100-200 mesh) with different proponions of hexane.

C,fl,, CHCI, and their binary mixtures in increasing order of polarity and 15 fractions of
200 ml each collected. Fractions 11-13 indicated the presence of two W active

compounds which was funher separated by preparative polyamide TLC with C,H, : pet ether : MEK : MeOH (5:5:1:1) as developing solvent. The eluate from the separated zones gave compound A (HR, 15. major) and compound B (HR, 37. minor) as yellow residues. The residue from the ether extract was subjected to preparative PC (hlatman
3, 15% HOAC) when two bands seperated. The eluates from them gave compound C

(HR, 15 ) and compound D (HR(73).

4.2.1 Characterlsation of compound A

(5,7-dihydroxy 6,8,4'-trimethoxy flavone - nevadensin) Compound A, C,,H,,O,, mp. 186-lag0, gave yellow colour with alkalis, green with Fe"' and red with Mg-HCI, showing it to be a flavonoid. It was purple under UV and W M H , and its R, values (Table 4.1) were characteristic of a flavone. Thls was h,,~ ~ (MeOH) '~ 282,296sh, 329 nm. The presence of a supported by the W s p e ~ t r u m ~

m in band 1 of AICI, MCI free 5-OH was indicated by the bathochromic shift of 20 n
~pectrum'~ (compared to band 1 of its MeOH spectrum). The absence of any

bathochromic shlft in band I1 of NaOAc spectrum indicated the absence of a free 7-OH or free 7-OH with 6 - 0 substitution." The longer J.,, (band I) of KaOMe compared to

NaOAc showed the presence of 7-OH. The mass spectrum showed the molecular ion peak at m/z 344 (M'.72) and base peak at 329. The characterist~cfragment ions at 197 (A-CH,,30) and 169 (A-CH,C0,26) indicated the presence of two OMe groups in A ring and the other characteristic f r a b e n t ions at 135(B,,20) and 132(B,,17) gave evidence1' for the presence of one OMe group in B ring. Its 'H NMR spectrum (DMSO-d,) accounted for the aromatic protons at 3,2',3',5',6' and the protons of three OMe groups at 6,8,4'. The compound on acetylation yielded a diacetate mp. 172-174'. The 'H NMR spectrum of the diacetate gave signals for the 2 acetyl groups at 6 2 . 4 % ~ and ) S 2.37(s). Compound A was thus charactensed as nevadensin" (5.7-dihydroxy 6,8,4'-trimethoxy flavone) (Fig 4.2) which was further confirmed by m.mp and co-chromatography with an authentic sample from Acnnthospermum hispidum."

4.2.2 Characterlsation of compound B

( 5-hydroxy 6,7,4'-trimethoxy flavone salvigenin)

Compound B, C,,H,,O,, mp. 185-186' gave yellow colour w ~ t h alkali, green with Fel* and red with Mg-HCI showing it to be a flavonoid. It was purple under UV and

WNH, and 11s R, values (Table 4.1) were characteristic of a flavone.

This was

supported by its W spectrum in MeOH which showed peaks at 282, 296sh and 329 nm A bathochromic shift of 24 nm in band I of AIC1,MCI spectrum compared to band I of MeOH spectrum Indicated the presence of a free 5-OH.'" The absence of any shift in band I1 of NaOAc showed the absence of free 7-OH "" Its MS showed the molecular Ion peaks at mlz 328 (M'.100) and other charactenstic peaks at 181 (A-CH,,23), I53 (181-C0.31). 135 (B?,l5) and 132 (B.10) indtcating the presence of two OMe groups in A ring and one OMe group In B ring. The 'H ?iMR spectrum of the compound had signals accounting for the aromatic protons at 3,8,2',3',5' and 6 ' and protons of three OMe groups. The compound on methylation yielded tetra 0-inethyl scutellarein and on demethylation 11gave scutellarein (5,6,7,4'-tetrahydroxy flavone). These data along with the shifl In the AICI, spectrum showed the compound to be 5.6.7.4'-oxygenated flavone with one OH at C, and three OMe at C,,C, and C, . The compound was thus identified as 5-hydroxy 6,7,4'-tr~methoxy flavone (salvigenin)" (Fig 4.3) and the ~dentttyconfinned by m.mp and co-chromatography with an authentic sample fiom Angelonla grand~flora.'"

4.2.3 Characterisadon of compound C (3,4-dihydroxy E-cimarnic acid - caffeic acid) Compound C, C&,O,, mp. 210-212", gave light yellow colour with hX,, blue under UV changing to bright blue under UVINH,. It did not answer Shinoda's test but gave bnsk effervescence with NaHCO, and green colour with Fe'-. It also decolounsed Br, water. The W spectrum showed

A , ,

(MeOH) 245, 325 nm. and had R, (table 4.1)

typical of a phenyl propanoic acid. Acetylation of this compound yielded a diacetate mp. for 1,3,4-trisubstituted benzene derivative (6 201-203". 'H NMR spectrum gave s~gnals 7.16, d, JZ2.5Hz, lH, H-I; 7.03, dd, J=8.3Hz & 2.3Hz. lH, H-6; 6 86, d, J=8.3Hz, IH, H-

5). The trans stereochemistry was deduced from the peaks at 6 7.53 and 6.26 (d,
J=15.9Hz). The mass spectrum having molecular ion peaks at miz 180 along w ~ t h other character~sticpeaks at 163, 136, 134, 69, 57, 55 and 42 indicated the presence of a benzene nng contaming two OH groups and the side chain -CH=CH-COOH. It had IR bands ar 3440, 1172 (OH of COOH and phenolic OH), 1640 ( G O ) , 1505 ( k ) , 1212, 11 18, 972, 899, 849 and 812 cm-1 (substituted benzene). Based on these compound C was characterised as (E)-3.4-dihydroxy crnnamlc acid, [(E)-caffe~c ac~d] (Fig 5.4) whose identity was confirmed by co-PC and superimposable IR spectra with an authentic sample from Berberls o r ~ s l a t u . ~ ~

4.2.4 Characterisation of compound D

(3-0-caffeoyl quinic acid - chlorogenic acid) Colourless needles, C,,H,,O,, mp.199-200, gave deep blue with Fe'., rosy red with phenol red reagent and blue UV fluorescence changing to yellow green with

WNH,. It had A,,

(MeOH) 243, 305sh and 329 tun and R, (table 4. I) suggesting it to

be a phenyl propanoid. Alkali hydrolysis yielded caffeic acid along with an aliphatic acid. ElMS exhibited molecular ion peaks at m/z 354 (5%) along with other fragment ions at m/z 336 (32), 180(60) and 163(100%). The peak at m/z 180 was due to caffeic acid thereby suggesting
11

to be a caffeoyl ester of quin~cacid and the fragmentation

pattern found to be in close agreement with the reported values." The occurrence of M.(5%) and M--18(32) were due to the formation of lactone In the quinic acid moiety and the peak at m'z 163 (100%) was that of caffeoyl pan.'' The quinic acid moiety was further supported by HPLC. 'H and "C NMR spectra. 'H h'MR spectrum of compound D gave further evidence by exhibiting signals at aromatlc region due to caffeoyl protons. The trans-stereochemistry was deduced from the peaks at 6 7.39 and 6.15 (d, J=16Hz). The signals for hydroxyl protons at 6 9.84, 9.22, 4.95, 4.79 and 3.92 disappeared on addition of DIO. The aliphatic regions showed slgnals for three hydroxyls(l.4 and 5-OH) and two -CH?- groups (2,6-CH,-) and a multiplet (2H. H - 4 , s ) . The singlet at 6 5.05 (m,lH) was in agreement with 3-0-caffeoyl quinic acid? F~nally"C NMR supported

the characterisation of the compound D to be 3-0-caffeoyl quinic acid (chlorogenic acid)" (Fig 4.5) and the identity was further confirmed by co-PC with an authentic sample From Berberi. a r i ~ r a t a . ' ~

STRUCTURES OF COMPOUNDS FROM

L.gratissima

Fig. 4.2

nevadensin

Fig. 4.3 sabigenin

Fig. 4.4 caffeic acid

Fig. 4.5

chlomgenk acid

4.3 RESULTS AND DISCUSSION

Re-examination of the aerial parts of L.grutissima resulted in the identification of two methylated flavones and two phenyl propanoic ac~ds. 7-desmethyl artemetin could not be detected in the present examination. A sample of 7-desmethyl artemetin" isolated and klndly supplied by one of the authors was found to be a mixture of from L.gratiss~ma the same two flavones nevadensin and salvigenln isolated now. In addition two phenyl propanoic acid
-

caffeic acid and chlorogenic acid have also been isolated and

charactensed. This is the first repon of isolation of all these four compounds from this plant. Funher, nevadensin has been subsequently Isolated from a slster specles

L.conferra'" justifying its presence In the genus L~mnoph~lu and thus strengthen~ngthe

present finding.
A probable reason for misidentifying the mixture of nevadensin and salv~genin as

7-desmethyl artemetin in L.grarissima by earlier workers is the great difficulty in effecting separation of the components of the mixture. In the present study, separation of the components as homogenous species has been achieved by the use of elaborate column chromatography over fine grain S10: followed by preparative TLC on polyamide. Once the separation was completed the identification became a comparat~velyeasy task. The ready availability of samples of all the four compounds also made the characterisa~ion and differentiation less difficult. Of the 9 L~mnophila species" available In India, 6 have been examined for flavonoids. All of them were found to contain unusually oxygenated and methoxylated flavones. Only L.confertaZo was reported to contain a flavonol
-

quercetin and its pcnta

methyl ether.

Nevadensin has been reported from L.confertaz0 and L.helerophylla"

earlier and L.gmtissima studied now. Thus, this rare flavone appears to be a common flavone of the genus Limnophrla. Further careful analysis of the other species is required to establish its status as a chemotaxonomic marker of this genus. Nevadensin with its reported anti inflammatory, anti tubercular and cytotoxic properties and present in a number of Limnophila species may contribute to the medicinal importance of this genus.

4.4 EXPERIMENTAL

Fresh aerial parts ofL.gratissima (2kg) collected from Mahe region, South India, were refluxed with 95% boiling EtOH and concentrated under reduced pressure. The aqueous crude extract was fractionated into benzene,

dichloromethane and ether solubles of which the latter two tested positive for polyphenolics. The dichloromethane extract was concentrated and subjected to column chromatography (SiO,, 100-200 mesh) with different proponions of Hexane. C,H,, CHCI, and thelr binary mixtures in increasing order of polarity. 15 fractions of 250 ml each were collected of which fractions 11-13 indicated the presence of two UV active compounds. Further separation of these two

compounds (0.25g) was carried out by preparative polyamide TLC with C,H,:Petether:MEK:MeOH ( 5 : S : l : l ) as the developing solvent. The eluate from the

separated zones, HR, 15 (major, designated. A) and 37 (minor, des~gnated,B) were concentrated to yield yellow residues. Recrystallisation from MeOH yielded pure compounds A and B The residue frcm the ether extract (100mg) was subjected to preparative PC (whatman No.3, 15% HOAc) when two bands separated. The eluates from them. HR,. 15, designated, C and 73, designated, D were concentrated and residue recrystallised from MeOH to yield pure compounds C and D.

4.4.1 Compound A

(nevadensin) Light yelllow needles, C,,H,,O,, mp. 186-88'. It gave yellow colour with

NH,, red with Mg-HC1 and green with Fe3'. It was purple under UV and WMH,
(Found C, 62.51; H,4.59; Calc C.62.77; H,4.69 %).

U V (A,,.,
MeOH NaOMe NaOAc

nm)
: 282,296sh. 329 : 284,304sh. 378
: 282, 304sh, 376
:

NaOAc/H,BO, AICI, AICI,MCI

286,330
: 264,290,3 10,354,412sh

261.288sh, 308,349,412sh.

R,. Table 4.1

IR (v,,,

CHCI,. c m ' ) (Fig 4.6)

3450,2923, 1754, 1657, 1592, 1496.1401. 1238, 1123, 1028,824

'HNMR ( 350MHz. DMSO-d,, 6, TMS) (Fig 4.7)

8.02(d, J=9.2Hz, 2H, H-2',63 7.15(d,J=9.2Hz, 2H. H-3'5') 6.84(s, IH, H-3) 3.88, 3.87.3.78 (s each. 3H each. 3-OMe)

MS ( EIMS, mlz, re1 intensity as %) (Fig 4.8)

344(M,72) 329 (M*-15,100) 31 1 (329-18, 15) 301 (329-28, 16) 197 (A-15, 30) 169 (197-28. 26) 135 (B,, 20) and 132 (B,, 17).

(nevadens~n diacnate) Compound A (25mg) dissolved in C,H,N (0.51111) was treated with acetlc anhydride (0.5ml) and left at RT for 24hr. On usual working up, it yielded colourless needles from MeOH, mp. 172-174".

'H NMR ofdiacetate (400 MHz, DMSO-d,, 6, TMS) (Fig 4.9)

8.02(d3J=9Hz.2H,H-2'6'), 7.14(d,J=9Hz,ZHJ-3 ',5 '), 6.82($,1HH-3), 4.00,3.86.3.77(~ each. 3H each. 3 OMe). 2.45 &2.37(s each 3H each, 2 OCOCH,).

4.4.2 Compound B

(saivigenin) Light yellow solid, C,,H,,O,, mp.185-186. It gave yellow colour wlth hX,, green with Fe)., red with Mg-HCI. It was purple under UV and UVlTU'H,. (Found C65.80, H 4.98; cald C, 65.83, H, 4.92)

UV (I,,., m)

MeOH
NaOMe

: 276,329

: 297,372sh, 396sh

NaOAc NaOAclH,BO, AICI, AICI,MCI

: 276,329 : 276,330 : 262,290,299,354 : 262,290,299,353.

R : Table 4.1

IR (v,,,

CHCI,, c m ) (Fig 4.10)

3019,2361, 1605, 1495, 1358, 1215, 1030.756

'HNMR (400 MHz, DMSO-d,, 8, TMS) (Fig 4.1 1)

8.07(d,J=9Hz,ZH,H-2:67,7.13(d, J=9Hz,2H, H-3'.5")66.6(~,IH, H-8).6.91 (8, lH,
H-3), 3.94,3.88,3.75 (s each, 3H each, 3-OMe).

MS (EIMS,m/z, re1 intensity as %)(Fig 4.12)

328 (M', loo), 314 (M--14. 14), 31 3 (M--15,85), 299 (M-CH0.25). 181 (A-1 5, 231, 153 (181-CO, 31), 135 (B:. 15). and 132(B,, 10).

(salvigenin monoacetate) Compound B (25mg) dissolved in pyridine (0.5ml) was treated with acetic anhydride (0.5ml) and left at RT for 24hr. On usual working up. it yielded colourless needles from MeOH mp.166-67'.

(tetra-0-methyl scutellarein) Compound B (25mg)was methylated uslng Me,SO, (O.5ml) and anhyd K,CO, (lg) in dry acetone (201111) medium by refluxing at 70' for 48 hr. The methyl ether after usual working up and crystallisation (MeOH) came out as colourless needles, mp.16062".

(scutellare~n) To a solution of compound B (25mg) In AC,O (2ml) HI (2ml) was added slowly w~th st~mng after adding each drop. It was refluxed at 170-180' for 2hr. The reaction product was cooled, poured into a saturated solution of NaHCO, and extracted with ether. The residue from the dried eather extract on crystallisation (MeOH) came out as yellow needles. not melting below 300'. The demethylated product was identified as scutellarein by co-chromatography.

4.4.3 Compound C

((E)-caffeic acid ) Crystallised from MeOH as pale yellow needles, C,H,O,, mp.210-212", gave bnsk effervescence with saturated NaHCO, solution, light blue with FeJ' and decolourised Br? water. It was blue under UV and deep blue under UVMH,.

UV (Lm,.,nm)
MeOH NaOH AICI, AICI,MCI
: 245,325
: 306sh, 334

: 234sh, 265,320,360 : 234sh. 300sh, 320.

R : Table 4.1

'HNMR spectrum (350 MHz. DMSO-d, 6, TMS) ((Fig 4.13)

7.53(d,J-l 5.9Hz, l H, a-H),7. I6 (6, J=2 3Hz, l H, H-Z), 7.03 6),6.86(d, J=8.3Hz, lH, H-5),6.26(d,J=15.9Hz, 1H.fi.W.

(a, J=8.3 & 2.3Hz. IH, H-

MS (EIMS.m1z. re1 intensity as 96) (Fig 4.14)

180 (M., 100) 163 (M'-17, 30) 136 ( X - 4 4 . 84) 134 (35) 69 (20) 57 (30) 55(20) and 44 (42).

(caffeic acid diacetate) Compound C (25mg) in C,H,N (0.5ml) and Ac,O (0.5ml) left at RT for 24hr yielded colourless needles ( EtOAc-peml) mp. 201-203~mmp.unaltered.

4.4.4 Compound

(chlorogenic acid) Colourless needles (Me,CO), C,,H,,O,, mp.199-200, [a]," -35.2 (H,O) deep blue with ~ e ' * rosy , red with phenol red reagent, brisk effervescence with NaHCO,, blue fluorescence under W chang~ng to yellow green with Wt'NH,.

UV

( A , , . . nm)
243. 305sh, 329 265sh, 300sh, 380
: 260sh. 3 10,358 : 240,300sh. 327.

MeOH NaOH AICI, AICI,MCI

& : Table 4.1

'HNMR (350MHz. DMSO-d, 6, ppm) (Fig 4 . 1 9

9.649.22 (each

b, each 1H.7 ,8'-OH), 7.41 (P.J=16Hz, IH, H-3'1, 7.39 (d. J=2Hz, IH,

H- 9 1 , 7.00 (d, J=8 & ZHz, I H, H-5'), 6.65 (4. J=SHz, I H, H-6 1 . 6.1 5 (d, J=16Hz, IH,

H - 2 3 , 5.05

(m, IH, H-3), 4.95,4.79.3.92 (each b s ,each 1H. 1.4.5-OH).3.9-3.6 (m, 2H,

H-4,5), 2.05- 1.73 (m, 4H, 2.6-CH2-)

"C NMR (broad band decoupled, 67.89 MHz)(Fig 4 . U )

175.OO(C-7). 166.13(C-l'), 148.21(C-7'), 145.92(C-6'), 145.08(C-3'), 125.68(C-4'), 121.62(C-9'), 115.82(C-2'), 114.49(C-5'/8'), 114.32(C-5'/8'), 73.72(C-I), 70 74(C-3), 70.62(C-4), 68.4qC-5), 36.95(C-2), 36.45(C-6).

MS (ElMS,m/z, intensity as %)
354 (M*.5), 336 (M-H,O, 32), 180 (caffeic a c i r , 60), 163 (caffeoyl-, 100). 162 (180-H,O,
23).

HPLC :
Retention time (R,, min) was determined on phenyl bondapak column, gradlent elution with 5% HOAc-MeOH at 25'. The acid as well as authent~c chlorogenic acid had R,-16.16.

v
(caffeic acid) Compound D (IOmg) was treated with alkal~ (5% NaOH. 80'. Ih) neutralised with IN HCI and extracted with ether. Purification by PC ( 3 0 ' 1 0 HOAc) and recrystall~satlon from MeOH yielded light yellow needles, identical with cornpound C.

Fig.4.8 MS spectrum of aevadensin (see page 125)

TABLE 4.1
Rr values of the polyphenolics from L.gratisslrna

Rr x 100 (Whatman No.1, ascending, 28 i 2 ' ~ )

' Compound

/
1

Hz0

Nevadens~n Salvlgen~n Scutellare~n Caffe~c ac~d

15% HOAC 0 I 20 0 I I2 1 6 62 1 50

50% HOAc 79 60
47 74

BAW I PhOH 95 91 80 86 95 95 72 54

Forestal 93 94 58

/
I

t-BAW 94 92 67 85

78

BAW

. n-BuOH
: H0.4~

Forestal Seikal tBAW PhOH

: HOAc . HIO

: H20
: Conc HCI

(4:l:S) top layer (30:10:3)

(1:l)

. 27% HOAc , n-BuOH : 1-BuOH : HOAc : HzO

(3:I 13)

Phenol saturated with water

131

4.5
1.

REFERENCES
Lawrence, H.M.G. (1951) "Taxonomy o f ~ a s c u l plantsn, a oxford & IBH Publishing Co., New Delhi, p.695.

2.

Vasishta, P.C. (1974) "Taxonomy of Angiosperms", ~ . c h a n & d c o , , N~~ ~ ~ l h i , p.605.

3. 4.
5.

Harbome, J.B. (1963) Phpochemrstry, 2, 327. Harbome, J.B. (1966) ibrd, 5, 1l l Valdes. V. (1970) rbrd, 9, 1253. Bohrn. B.A. (1975) in "The Flavono~ds"(Harborne, J B., Mabry, T.J & Mabry, H.

6.

Eds) Chapman & Hall, London, p.487.

7. 8. Seikel, M.K. (1955) J A m Chem.Soc.,77,5685. Borodin, L.I., Litvinenko, V.I. & Kunmaya, N.V. (1970) Khrm. Prir. Soedrn., 6, 19.
9

Harbome, J.B. (1975) In "The Flavono~ds"(Harbome, J.B.. Mabry, T.J. & Mabry, H. Eds) Chapman and Hall. London, p. 1061.

10.

Anonymous (1988) "The Wealth of India - Raw Materials". Vol 6. C.S.I.R.. New Delhi, p.115.

11.

G. & Manna. T.K. (1995) Ph~tochemrstq~. 38, Mukheqee. K.S.. Brahmachar~,

1273.

12.

M u k h e j ~ K.S., , Manna. T.K., Laha, S. & Brahmachari, G. (1994)

Soc., 71.

Indian Chem

13.

hK.s., ~ Brahmachari, j ~ ~ , G., Manna, T.K. & Mukhejee, P.(1998) lbjd. 75,

260.

14.

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