CHELJICXL CONSTITUESTS O F LEAVES ,4SD ROOTS O F G YAIISOSPORIA - 1 f O S T A S A
Gymiiosporia moiitaiia (Benth.) is used in indigenous medicine as a cure for ulcers. gastrointestinal troub1e.q. dysentery, snuke bite, etc. (1. 2 ) . Joshi e / a l . ( 3 ) reported the isolation of tingenone, 3-O-acet yloleanolic acid, hesacor-ane, betulin, /3-amyrin, hesacosanol and +sitosterol from the stem bark, root bark, and leaves of G. moiitaiia. hnjaneyulu et a l . (4) isolated p-amyrin from the stem of this plant; from the roots of the same plant, a-amyrin and dukidol were isolated by Satyanarayana and llishra ( 5 ) . S o w , n-e report the isolation of p-amyrin and 12-triacontanol from the leaves of G. m o ~ t a i i a(Benth.) and ( -)epigallocatechin from the roots. Previously, ( - )epigallocatechin was isolated from a very limited number of plants (6-13). Since this is the first, report of the isolation of ( - ) epigallocatechin from the Gymiiosporia species, it may be chemotaxonomically significant. Further, the isolation of iz-triacontanol in high yields (O.lOiyc) is significant since it was reported to possess a plant growth promoting property (14). p-Am!-rin n-as identified by the preparation of its acetate and the comparison (mp, mmp and super-imposable ir) of both the compounds with authentic samples of 8-amyrin and its acetate, available in our laboratories. 72-Triacontanol n-as first isolated by crystallization. Since it is likely that fz-triacontanol generally co-occurs n-ith its homologous alkanols, the product was further purified by chromatography over silica gel. The acetate of n-triacont'anol was prepared. The mass spectrum of ntriacontanol was recorded and found 626
to be similar to the mass spectrum reported by Rice et al. (14). l'ragment ions lI-H,O, lI-CH,OH and the consecutive loss of methylene units from the parent molecular ion \\-ere observed (14) in the nis of titriacontanol. Ti-Triacontanol and its acetate n-ere found to be identical (mp. mmp, ir) with authentic samples. The structure of i-)epigallocatecliin n-as established by the preparation of the methyl ether, the meth>-lether acetate, and the peracetate arid comparison of their analytical and spectral data with those reported in literature (1.5, 16). However, an authentic sample of (-)epigallocatechin could not be procured. The mass spectrum of (-)epigallocatechin pentamethyl ether n-as recorded; its mass spectral fragmentation (3TG (43)lIf., 35s (T), 210 (92), 1S1 (76), 167 (IOO), 15%(15) ) is in agreement n-it,li those reported for catechin ( G ) .
EXPERIJ1EST;IL monfann (Benth.) is a much-branched spinescent shrub occurring throughout t h e drier p a r t s of India. T h e plant material was collected locally in September, 1976. PL.4ST
\iaTERIAL.-G'nttlospori(L
1SOLATIOS .4 S D I D E S T I F I C I T I O S OF 0-A >ITR I N A S D ?I-TRIACOSTASOL FROM T H E LEAVES OF Gymnosporie montann (Benth).-The shadedried leaves (1.5 kg) were extracted with petroleum ether (GO-SO") (5 litersj in a Soxhlet extractor, and t h e extract was concentrated in 'i'ncuo. T h e resulting gummy mass (G.0 g) was treated with boiling methanol ( 2 liters), and then t h e extract was chilled. T h e waxy material t h a t separated out was filtered and crystallized from a benzene-ethyl acetate (8:2) mixture as colorless needles of n-triacontanol. T h e product was further purified by chromatography over silica gel (70 g, .4cme's 200 mesh). T h e details of t h e eluent fractions collected a r e given in table 1. T h e pure n-triacontanol obtained from t h e fractions
626
Journal of Natural Products
[Vol. 44, KO. 5
TABLE 1. T h e eluent fractions collected from column chromatography of n-triacontanol.
Fraction numbers
1-olume of each fraction (ml)
s1. KO.
Eluent System
1.
Petroleum ether (60-80")
1-5
50
2.
Petroleum e t h e r (60-80")-benzene (8:2) Petroleum ether (60-8O0)-benzene (7:3) Petroleum e t h e r (60-80")-benzene (1:l)
6-10
50
I1
11-15
50
n
16 B: 17
50
3. 4.
5.
18-20
50
6.
Petroleum e t h e r (6&8O0)-benzene (1:l) Benzene
21-25
50
7.
Benzene-chloroform (8:2)
26-30
50
8. 9. 10.
Benzene-chloroform (7:3) Benzene-chloroform il:1) Chloroform
31-35 36-40 41-46
50 50 50
18 t o 25 was again recrystallized from a benzene-ethyl acetate (8:2) mixture. It crystallized a s colorless needles (1.6 g; O . l O i C - , ) , mp 86" [lit. (17) mp 86"], hl438(3). I n t h e ms of n-triacontanol the following prominent ions were observed: wile 420(33) (hl-HZ0); 407(3) (>I-CH*OH); 393(5) (LI-CH2OH-CH2) and ions due t o consecutive loss of fourteen mass units (loss of CH? fragments) from t n / e 393 ion. .-inal. Calcd: fo; C30H620:C, 82.19, H, 14.15. Found: C, 82.02; H, 14.23. n-Triacontanol was acetylated with acetic anhydride and pyridine a t room temperature. The acetate derivative crystallized from ethanol as colorless needles, mp 68.5' [lit. (17) 68.5"]. n-Triacontanol and its acetate were found t o be identical (mp, mmp, tlc and superimposable ir) with respective authentic samples. Anal. Calcd. for C~~Hbd02: C, 80.00; H, 13.34. Found: C, 79.92; H, 13.39. T h e methanol solution (2 liters) was concentrated t o 250 ml under reduced pressure and left for two days. T h e brown solid t h a t separated out was filtered and crystallized from a chloroform-methanol (7:3) misture as colorless needles of 3-amyrin (3.0 g, 0 . 2 5 ) mp 195" [lit. (18) mp 195'1. ilnal. Calcd. for CsnHi,O: C, 84.50; H , 11.74. Found: C, 84.39; H, 11.79, I t was acetylated with acetic anhydride and pyridine at room temperature. T h e acetate derivative crystallized from alcohol as colorless needles, mp 238", [ l i t . (18) 238"l. T h e compounds
Remarks Yielded negligible amount of silky oily m a t t e r .
Yielded negligible amount of n-triacontanol along with silky oily m a t t e r . Yielded pure n-triacontanol (0.6 g) Yielded pure n-triacontanol (1.1 g ) Yielded neglibile amount of s i l k y oily matter. 11
II I1
were identical (mp, mmp, tlc, i r ) with t h e corresponding authentic samples of P-amyrin and its acetate derivative. iinal. Calcd. : 82.12; H, 11.11. Found: C, for C 3 1 H 5 2 0 2C, 82.00; H, 11.16. ISOL.1TIOS A N D IDEXTIFICATION OF (-)EPIGALLOCATECHIS FROM THE ROOTS OF Gymno-
spuria aiunfnna (Benth.).-The method of Seshadri et a / . (19) was adopted for t h e isolation of (-)epigallocatechin. The shade-dried roots (0.5 kg) were defatted with petroleum ether (69-80") and then extracted with acetone (3.0 liters) in a Soxhlet extractor. T h e acetone extract was concentrated in i'ncuu, and the resulting mass (2.0 g) was dissolved in water (250 ml) and filtered. The aqueous solution was extracted with ether (2 x 200 ml) and than with ethyl acetate (2 x 200 ml). The combined ethyl acetate extract was dried and concentrated under reduced pressure t o a small volume (10.0 m l ) . T o this extract 4 ml of petroleum ether (60-80") wis added with stirring; a brown gummy mass separated out. FVhen t h e clear solution was decanted into another flask and 4 ml of petroleum ether (60-80°) was added, a solid again separated out. This process of fractional precipitation was repeated eight more times. T h e colorless solid fractions obtained in the last three operations were found t o be identical in nature when subjected t o paper chromatography: Whatman S o . I chromatographic paper, solvent
Sep-Oct 19811 Kumar and Shimannarayana : Gymnosporia moiztaiza
627
TABLE 2. The eluent fractions collected from column chromatography of pentamethyl e t h e r of ( - ) epitallocatechin. S1.
Eluent System
so. 1.
Benzene
1
Fraction numbers
1-olume of each fraction (ml)
I
1-5
50
Remarks
Yielded negligible amount of mixture of compounds. I1
50 3
50
Tielded pure pentamethyl e t h e r of ( - ) epigallocatechin. Tic 'ed negligible amount of mixture of compounds.
50
system butanol-acetic acid-water (4:1:5 upper layer, spray reagent alcoholic ferric chloride, R f 0.52). Therefore, they were mised and dissolved in e t h y l acetate (8.0 m l ) . T h e process of fractional precipitation with d r y petroleum e t h e r (60-80") was repeated until almost colorless (-)epigallocatechin was obtained (1.5 g; 0.3$), m p 216-18" ( d ) [lit. (15, 16) mp 218-19'1; uv X max (MeOH), nm (loge): 224, 271, 224 (4.36), 271 13.184) [lit. (15. 16) 1. ilnal. Calcd. for C I S H I ~ O ; C, : 58.83; H,> 4.58. Found: C, 5 8 . i 2 ; H , 4.64. PESTAMETHYL
ETHER
OF
(-)EPIGALLO-
-(-)Epigallocatechin was metht h dimethylsulfate and potassium carbonate in d r y acetone. T h e methyl e t h e r was chromatographed over silica gel (Acme's 200 mesh). T h e details of t h e eluent fractions are given in table 2. Pure pentamethyl e t h e r obtained from t h e fractions 11 t o 15 was recrystallized from benzene-ethyl acetate (8:2) mixture a s colorless crystals, m p 157-59" [lit. (15, 16) mp 1586 3 7 ; uv X m a s (LIeOH) nm 272 (log e ) (3.60); i r v m a s ( K B r ) 3 4 3 cm-1 (hydrosyl); [a]2SD-16.50 acetone [lit. (15, 16) [aI2jo -15.2" acetone]; ms 376 (43) hl-. 358 ( i ) , 210 (92), 181 ( X ) , 167 (loo), 152 (15). Anal. Calcd. for C21,H210i:C, 63.83: H , 6.38; OCH,, 41.22. Found: C, 63.T4; H , 6.39, O C H B ,41.02.
PETTAMETHYL ETHER MOSOACETATE OF (-)EPIG.~LLoC.~TECHIs.-The pentamethyl e t h e r of (- )epigallocatechin was acetylated v.-ith acetic anhydride and pyridine at room temperature. T h e acetate derivative crystallized from benzene-ethyl acetate (8:2) misture a s colorless needles, mp 179-80" [lit. (15, 16) mp 181-83"; uv X m a r (AIeOH) nm (log E ) 272 (3.30): ir v m a s ( K B r ) 1750 em-' (acetate carbonyl). .4nal. Calcd. for CZZHZ608: C, 63.16; H , 6.22. Found: C, 63.05; H, 6.28.
n
HEXAACET.4TE OF (- )€PIGALLOCATECHIS,(-)Epigallocatechin was acetylated with acetic anhydride and pyridine a t room temperature. The acetate derivative crystallized from a benzene-ethyl acetate (8:2) mixture a s colorless needles, mp 190-92" [lit. (15, 16) mp 191-93"]; uv X m a s (LIeOH) nm (log e) 272 (3.54); i r Y m a s 1750 cm-1 ~ acetone (acetate carbonyl); [ a ] * j -14.00" ~ acetone]. Anal. [lit. (15, 16) [ a I z S -14.00" Calcd. for C Z ; H Z ~ O IC, ~ : 58.06; H , 4.66. Found: C , 57.95; H , 4.71. ACKXOWLEDGLIENTS We acknow-ledge D r . h l . P r a b h a k a r , Department of Botany, Osmania University, Hyderabad-500 007, for t h e identification of plant material and Prof. Lalith Prakash, Department of Chemistry, University of Rajastan, Jaipur, India, for kindly sparing the authentic sample of ntriacontanol. One of us ( K A K ) acknowledges with thanks t h e encouragement given b y the 1.ivek 1-ardhini Education Society.
Receir'ed 21 .April 1980. L I T E R A T V R E CITED 1. K . R . K i r t i k a r and B . D . Basu, "Indian Medicinal Plants", 1-01. I, P u b . Lalit Mohan Basu, Allahabad, India, 1933, p. 577; A. K . Xadkarni, "Indian Materia Medica", 1-01. I , 3rd ed, Popular Book Depot., Bombay, 1954, 7,. 606. 2. T h e Wealth of India, Rawhhlaterials", 1-01, IT, C.S.I.R., New Delhi, 1956, p. 277. 3. C. K . Joshi, R . K . Bansal and P . Renuka, Planfa X e d . , 34, 211 (1978). 4 . B . Anjaneyulu, 1-. Babu Rao, A . K . Ganguly, T. R . Govindachari, B . S. Joshi, I-.K.K a m a t , A. H . Manmade, P. A . Mohammed, A . D . Rahimatulla, A . K . Saksena, D. S. Varde and N.
Journal of Satural Products
628
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[Vol. 44, s o . 5
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