Isoflavones from Garcinia nervosa

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ISOFLAVONES MOHD. Section

of Natural

FROM

Phymhmiary.

Vol. 34, No. 3. pp 807_809,19~l Ebena SciMce Lid Rinlcd in Glut nlitain an-942zpr s7.m+o.m

GARCZNIA NER VOSA

ILYAS,*MOHD. KAMIL, MEHTAB PARVEENand MOHD. SOHRABKHAN Products,

Department

of Chemistry,

Aligarh

Muslim

University,

Aligarh-202

002, India

(Received in reuisedjorm 23 Nowmber 1993)

Key Word Index-Garcinia neruosa; Clausiaceae; oxyisoflavone; irigenin; 7-methyltectorigenin.

isoflavones;

5,7,4’-trihydroxy-2’,3’,6’-trimeth-

Abstract-A new isoflavone, 5,7,4’-trihydroxy-2’,3’,6’-trimethoxyisoflavone, nervosin, along with two known isoflavones, irigenin (5,7,3’-trihydroxy-6,4’,5’-trimethoxyisoflavone) and 7-methyltectorigenin(5,4’-dihydroxy_6,7dimethoxyisoflavone) have been isolated from the leaves of Garcinia neruosa. Their structures have been elucidated on the basis of chemical and spectral evidence.

INTRODUCDON The genus Garcinia comprises 180 species, which are well known for their antiinflammatory [ 11, antimmunosupp msive Cl] and antimicrobial [2] properties and for the healing of skin infections and wounds [3]. We therefore carried out a comprehensive investigation of leaves of G. neruosa (syn G. andersonii). Earlier investigation from this plant reports the isolation of nervosaxanthone [4] and a flavanoylflavone [S]. We now report the isolation and characterization of a novel isoflavone named nervosin (la) along with two known isoflavones, irigenin and 7methyltectorigenin not reported so far from G. nervosa.

“%OR OMe 1 R

a

H

b

AC

C

MC

RESULTSANDDISCUSSION The chloroform-soluble fraction of the alcoholic extract of air-dried leaves of G. neruosa afforded GN-1, GN2 and GN-3. The compounds GN-2 and GN-3 were characterized as irigenin (5,7,3’-trihydroxy-6,4’,5’trimethoxyisoflavone) 163 and 7-methyltectorigenin (5,4’dihydroxy-6,7-dimethoxyisoflavone) [7], while GN-1 was identified as a novel compound nervosin (5,7,4’trihydroxy-2’,3’,6’-trimethoxyisoflavone). GN-1 (nervosin, la), (M’, 360, C,sH,,OJ, yellow needles from CHCI,-MeOH mp 240” [8]. Its UV spectrum showed maximum absorption at 262 nm and an inflection at 329 nm. The Micro-Ziesel determination showed the presence of three methoxyl groups while the active hydrogen estimation showed the presence of three hydroxyl groups. This was confirmed by the formation of triacetate (lb) and hexamethyl ether (1~). The IR spectrum showed the characteristic bands at 3310(OH), 1665(C==O), 163O(aromatic, C=C). A bathochromic shift of 12 nm with AlCI, showed the presence of a chelated 5hydroxyl group which was further confirmed by the

*Author

to whom correspondence

should

be addressed.

signal at 6 13.5 in its ‘H NMR spectrum. Furthermore, bathochromic shift of 10 nm with NaOAc indicated the phenolic hydroxyl at the 7position [9, lo]. The methanolic solution of the compound was not oxidized by pentamine cobaltrichloride, indicating the absence of adjacent phenolic hydroxyl groups, this eliminates the possibility of a third hydroxyl group in the same ring [8]. In the ‘H NMR spectrum, the isoflavone nucleus was evidenced by the presence of a sharp singlet for a C-2 proton at 67.86. A pair of meta-coupled doublets at 66.62 (J = 2.5 Hz) and 66.66 (J = 2.5 Hz) each integrating for one proton were attributed to C-6 and C-8 protons, respectively. A sharp upfield singlet of one proton at 66.79 could be assigned to the C-S proton shielded by two ortho and one pma oxygens. The hexamethyl ether of GN-1 (lc) furnished 2,3,4,6-tetramethoxybenzoic acid when subjected to oxidative degradation with alkaline H,Or [ 111. The identity of 2,3,4,6_tetramethoxybenzoic acid was confirmed by Co-TLC and mmp with an authentic sample. The presence of three methoxyls was indicated through three singlets at 63.84, 3.85 and 4.01, each integrating for three protons. The remaining hydroxyl group may be placed at either C-3’ or C-4’.

M.

808

ILYAS et a/.

However, the comparison of 13C NMR spectra of la with that of its acetate (lb; Table 1) ruled out the possibility of a hydroxyl group at C-3’. The signal of C-4’ of the acetate lb moved upfield by 7.7 ppm, while the signals due to Cl’, C-3’ and C-5’ moved downfield by 6.1.8.8 and 8.1 ppm, respectively. The upheld shift of C-4’ of the acetate and downfield shift of carbons ortho and para to it confirmed the position of the hydroxyl group at C-4’ [12, 133. The signals due to meta-carbons (2’6’) remained almost unchanged in the acetate (lb). The above

assigned

structure

was further

supported

by

of la. It showed Mf at m/z 360. The fragment ions at m/z 345, 330 and 315 were due to [M - 1S] *, [M - 30) + and [M - 453 +, corresponding to the successive loss of three methyl groups. The RDA cleavage representing ring-A at m/z 153, 152 and ring-B at m/z 208, 207 and 178 further supported the presence of two hydroxyls in ring-A and one hydroxyl and three methoxyls in ring-B. The significant fragment at m/z 329 [M -OMe] + indicated the presence of a 2’-methoxy [ 143. Thus compound GN-1 is 5,7,4’-trihydroxy-2’,3’,6’trimethoxyisoflavone, named as nervosin (la), which is being reported for the first time.

the mass

spectrum

EXPERIMEh’TAL

All mps were uncorr. ‘H and 13CNMR spectra were recorded in CDCI,, chemical shifts were given on the ppm scale with TMS as int. standard. The MS were recorded by direct inlet method at 70eV ionization potential on JEOL JMS-300 mass spectrometer. Silica gel (120- 160 mesh) was used for CC and Merck’s Kieselgel G was used for TLC. P/ant material. Leaves of Garcinia nerc’osu were procured from Zaria, Nigeria. A voucher specimen is deposited at the herbarium of Department of Botany, A.M.U., Aligarh-202 002. Extraction and isolation. Air-dried leaves of G. neruosu (2 kg) were exhaustively extracted with EtOH. The EtOH extract was coned under vacuum. The brown gummy mass left behind on evapn of EtOH was treated with petrol (bp 40-W) and chloroform, respectively. The CHCl,-soluble portion was subjected to CC over silica gel. Elution of the column with &H,-CHC13 mixts (9: l-l: 1) followed by crystallization with CHCl,MeOH afford 3 light yellow crystalline compounds labelled as GN-1, GN-2 and GN-3. Nervosin (la). Light yellow needles (200 mg), mp 240’ (found C 60.2, H 4.5; C, *H ,60, required C 60.0, H 4.4%). It gave a greenish-brown colour with FeCl,, no colour with Mg/HCl and a red colour with Na/Hg-HCI. UV i!$T” nm; 262, 329 sh: +AlCI,, 274, 328 sh, 372; AlCI,/HCl, 275, 328 sh, 373; +NaOAc, 272, 326; +NaOMe, 268, 272, 332 sh. ‘HNMR (CDCI,): 63.84 (3H, s, OMe), 3.85 (3H, s, OMe), 4.01 (3H, s, OMe), 6.62 (lH,d,J=2.5Hz.H-6),6.66(lH,d,J=2.5Hz.H-8),6.79 (lH, s, H-5’), 7.86 (IH, s, H-2), 13.4 (lH, s, 5-OH), 10.51 (lH, s, 7-OH), 9.41 (lH, s, 4’-OH). 13CNMR (CDCI,): 655.7 (OMe), 55.8 (OMe), 55.2 (OMe), 94.0 (C-8), 100.0 (C-6), 105.0 (C-IO), 118.1 (C-5’). 122.5 (C-l’), 127.0 (C-3).

Table

spectral data of new isotlavone

I. “CNMR

Chemical shift In

lb

153.1 127.0 177.3 159.2 100.0 162.1 94.0 156.6 105.0 122.5 146.0 148.8 148.5

153.3 126.5 178.0 151.5 113.4 152.9 110.2 151.9 117.5 128.0 146.1 157.6 140.8 126.2 14X.3

C 2 3 4 5 6 7 8 9 10 I’ 2 3’ 4 5’ 6’ OMe

3xc=o of OAc

3 x Me groups of OAc

118.1 148.2

56. I 56.8 55.5 168.2 168.5 169.4 20.6 20.9 21.1

146.0 (C-2’), 148.2 (C-6’), 148.5 (C-4’), 148.8 (C-3’), 153.1 (C-2). 156.6 (C-9), 159.2 (C-5), 162.1 (C-7), 177.3 (C-4). MS m/z (rel. int.): 360 [M] + (80), 345 [M - Me] + (42). 342 [M -H,O]+ (IS), 332 [M-Co]+ (7.1) 330[M-CH,O] or [M-2Me]’ (9). 329 [M-OMe]’ (15), 327 [M-Me -H,O]+ (11.9). 315 [M-3Me]’ (20) 317 [M-3Me +H]’ (26) RDA fragments, ring-A 153 [A,+H]’ (13). 152 [A,]‘ (14.5) 151 [A,-H]’ (8.4): ring-B 208 [Bi]’ (12.8), 207 [B,-H]’ (IO), 178 [B,-CH,O]+ (12.5). Acetate lb, needles from CHCl,-MeOH mp 132”. It showed ‘H NMR (CDCI,): 62.31 (3H, s, OAc), 2.39 (3H, s, OAc) 3.85 (9H, s, 3 x OMe), 6.72 (IH, d, J=2.5 Hz, H-6), 6.97(1H,d,J=2.5 Hz, H-8). 7.19(lH,s, H-5’),7.85(1H,s, H-2). 13CNMR (CDCI,): 620.6, 20.9, 21.1 (3 x Me of OAc) 55.5 (OMe), 56.1 (OMe), 56.8 (OMe), 110.2 (C-8), 113.4 (C-6). 117.5 (C-lo), 126.2 (C-5’), 126.5 (C-3), 128.6 (C-l’), 140.8 (C-4’), 146.1 (C-2’) 148.3 (C-6’), 151.5 (C-5), 151.9 (C-9), 152.9 (C-7). 153.3 (C-2). 157.6 (C-3’), 168.2, 168.5, 169.4 (3 x >C=O of OAc), 178.0 (C-4). MS m/z (rel. int.): 486 [Ml: (47). Hexamethyl ether (1~). plates, mp 156”. ‘H NMR (CDCI,): 63.85-3.96 (18H, s, 6 x OMe), 6.66(1H,d,J=2.5Hz,H-6),6.75(lH,d,J=2.5Hz,H-8), 7.23 (lH, s, H-5’), 7.85 (lH, s, H-2); MS m/z (rel. int.): 402

WI: (12). Oxidation oflc with alkaline H,O,. GN-1 (20 mg) was treated with 5% ale. KOH (aq.) (10 ml) followed by 30% H,O, (1 ml); the mixt. was kept at 45” for 2 hr, cooled,

lsoflavones from poured into ice-cold Hz0 (10 ml), acidified with cold cont. HCI and extracted with Et,O. The Et,0 soln was washed with H,O and then shaken with satd NaHCO, soln. The bicarbonate fr. was acidified and extracted with EtlO. Evapn of the solvent gave a crystalline solid (5 mg) mp 184”. It was found to be identical with 2,3,4,6trimethoxybenzoic acid by mmp and co-chromatography. Irigenin (GN-2). Yellow needles (CHCI,-MeOH) (130mg), mp 189” (found C 60.1, H 4.3; C,sH,,Os required C 60.0, H 4.4%). UV, IR, MS, ‘HNMR and 13CNMR spectral data identical to lit. values. Acetate, mp 127-128”. 7-Methyltectorigenin (GN-3). Yellow needles (CHCl,MeOH) (140 mg), mp 237” (found C 64.95, H 4.45; C17H,406 required C 64.96, H 4.46%). UV, IR, MS, *H NMR and 13CNMR spectral data identical to lit. values. Acetate, mp 183”. Acknowledgement-We are thankful to Dr Shaheer H. Khan, Department of Chemistry, University of Alberta, Edmonton for spectral analysis. One of us (M.P.) is thankful to ICMR for financial assistance.

REFERENCES

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Garcinia

nefuosa

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H., eds) pp. 48-61. Chapman & Hall, London. 11. Adinarayanan, D. and Rao, J. R. (1972) Tetrahedron 28, 5377. K. R. and Chari, V. M. (1982) in The 12. Markham, Flavonoids. Advances in Research (Harbome, J. B. and Mabry, T. J., eds), Chapter 2, pp. 23-24. 13. Jha, H. C., Zilliken, F. and Breitmaier, E. (1980) Can. J. Chem. 58, 1211. 14. Campbell, R. N. M., Harper, S. H. and Kemp, A. D. (1969) J. Chem. Sac. (C) 1696.