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(295 mg) containing fatty substances [2], the third and most polar (564 mg) containing steroidal substances. The second fr. (41 mg) was submitted to repeated CC and prep. TLC on silica gel with hexane-CH,Cl, (2: 1, several developments) to afford two mixts. Further purification on reverse phase silica gel TLC (RP 8) with MeOH-H,O (8: 1, several developments) yielded 8, 10 and 12 mg of ferulic esters l-3, respectively, which all exhibited identical spectra. UV lE’oHnm: 205,219sh, 235sh, 330; (NaOH) 210, 390, IRvKB’cm-r: 3400 (OH), 1708 (CO), 1628, 975, EIMS, ‘H and 13CNMR: see Tables 1 and 2. Hydrolysis ofesters. Hydrolysis of compounds l-3 was carried out by refluxing for 30 min at loo” with 5% NaOH in MeOH. The neutral portion was taken up in CHCI, (3 x 50 ml). Removal of solvent afforded octadecanol, icosanol and docosanol for compounds 1-3, respectively. ‘H NMR (CDCI,); 60.88 (Me), 1.25 [(CH,),], 1.56 (CH,), 3.63 (OCH,). 13CNMR: 614.07 (Me), 22.65,25.75,29.66,31.88, 32.82 [CH,).], 64.29 (-OCH,-). EIMS m/z 270,298 and 326 [M] + for compounds l-3, respectively. The aq. portion obtained after removal of the neutral fr. was acifidied with dil H,SO,, coned in uacuo and extracted with CHCI, (3 x 50 ml). On removal of solvent, ferulic acid was obtained. ‘HNMR (CDsOD); 63.80 (s, OMe), 6.34 (d, J= 15.9 Hz, H-8), 6.77, 7.08 (m. H-5, H-6), 7.26 (m, H-2), 7.48 (d, J = 15.9 Hz, H-7).

REFERENCES 1. Bald&,A. M., Van Hoof, L., Pieters, L., Vanden Berghe, D. A. and Vlietinck, A. J. (1990) Phytother. Res. (in press). 2. Bald&, A. M., Janssens, J. J., Esmans, E., Pieters, L. A. and Vlietinck, A. J. (1990) J. Ethnopharmacology (submitted). 3. Nair, M G. and Burke, B. A. (1988) Phytochemistry 27,3169. 4. Sakushima, A., Hisada, S., Nishibe, S. and Brandenberger, H. (1985) Phytochemistry 24, 325. 5. Timmermann, B. N., Hofmann, J. J., Jolad, S. D., Schram, K. H., Klenck, R. E. and Bates, R. B. (1983) J. Nat. Prod. 46, 365.

6. Cheminat, A., Zawatzky, R., Becker, H. and Broullard, R. (1988) Phytochemistry 27, 2787. 7. Bengsch, E., Perby, B., Deleuze, C. and Valero, A. (1986) J. Magn. Res. 68, 1. 8. Morishita, H., Iwahashi, H., Osaka, N. and Kido, R. (1984) J. Chromatogr. 315, 253. 9. Harborne, J. B. (1980) in Secondary Plant Products, New Series Vol. 8, p. 329. Springer, Berlin. 10. Chatterjee, A., Dhara, K. P., Rej, N. R. and Ghosh, P. C.

(1977)Phytochemistry 16, 397. 11. Joshi, K. C., Sharma, A. K. and Singh, P. (1985) Planta Med. 71.

Phytochemistry,

Vol.30, No. 3, pp. 1026-1029,1991 Printed in Great Britain.

A BUTYROLACTONE

0031.-9422/91 $3.Oo+o.M)

PergamonPressplc

LIGNAN DISACCHARIDE

FLACOURTIA

FROM

RAMONTCHI

V. SATYANARAYANA,G. L. DAVID KRUPADANAM and G. SRIMANNARAYANA* Department of Chemistry, Osmania University, Hyderabad 500 007, India (Received in revised form 17 August 1990)

Key Word Index--Flacourtin ramontchi; Flacourtiaceae; pyranosyl(l~4)]-/?-2,3-di-O-methyl-o-xylopyranoside.

heartwood;

ramontoside;

diphyllin-4-0-[B-D-gluco-

Abstract-fi-Sitosterol, /3-sitosterol-/I-D-glucopyranoside and a butyrolactone lignan disaccharide, ramontoside, were isolated from the heartwood of Flacourtia ramontchi. The structure of ramontoside was determined as diphyllin-4-O[~-D-glucopyranosyl(l-+4)]-~-2,3-di-0-methyl-D-xylopyranoside by hydrolysis and spectral data.

INTRODUCTION

Plants belonging to the family Flacourtiaceae are well known for their physiological and medicinal properties [l-3]. The seeds of shrubs and trees of this family contain

*Author to whom correspondence should be addressed.

oils, called ‘Chaulmoogra oils’, which have been in use in the treatment of leprosy for over a thousand years [4]. The fruits of F. rnmontchi are sweet, appetizing and digestible and are used to treat jaundice and enlarged spleen rl]. Its gum is administered along with other ingredients in cholera [ 11. The bark is considered astringent, diuretic and used as a tanning material [S]. We deal with the isolation and characterization of a steroid, its glucopyranoside and a butyrolactone lignan disaccharide (I).

Short Reports

,.A,.

-

1 2

R’= H R’ = AC

Ha

3

R=H

RESULTS AND DISCUSSION

The dried and powdered heartwood of F. ramontchi yielded three crystalline compounds, two of which were identified as fl-sitosterol [S] and j?-SitOSterOl-/&D-&COpyranoside [6-S]. The third compound, ramontoside (I), had the molecular formula C,,H,,O,, ([Ml’ 702 by FABMS). It was insoluble in 10% aq. NaHCO, but was slowly soluble in dilute aq. NaOH to give a pale yellow colour, indicating the presence of lactone ring system (IR vkti 1755 cm-‘). It gave a positive Molisch test showing it to be a glycoside. It also gave a positive labat test for a methylenedioxy group. Its UV data, LE::FHnm (log E): 265 (5.10), 290 (sh) (4.46), 320 (4.42), 355 (4.05), was similar to that of diphyllin glycosides [9]. Ramontoside (1) formed a tetraacetate (2) indicating the presence of four alcoholic hydroxyl groups. Hydrolysis of 1 by methanolic H2S0, yielded a compound which was identified as diphyllin (3) [9, lo] by its spectral data (IR, UV, ‘H and ’ 3C NMR and MS) [9-l l] (Tables 1 and 2) and direct comparison with an authentic sample. The two different monoses isolated from hydrolysate were identified by PC as b-D-glucopyranose [8] and

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2,3-di-O-methyl-/I-D-xylopyranose [12]. In the ‘H NMR of 1 the two doublets at 65.22 (J= 7 Hz) and 4.84 (J =8 Hz) are characteristic of anomeric protons of B-Dglucopyranose and 2,3-di-O-methyl-/I-D-xylopyranose respectively [13, 141. In the “CNMR spectrum of 1 the two anomeric sugar carbons appeared at 102.91 (C-l of 2,3-di-0-methyl-j-D-xylopyranose) and 103.80 ppm (C-l of B-D-ghICOpyranOSC) indicating the sugars are linked with /?-configuration [15] (Table 2). The sequence of sugars in 1 was determined by the FAB mass spectrum. Compound 1 showed prominent ions at m/z 725 [M +Na]+, 703 [M+H]+ (which are quasi molecular ions), 641,571,541, 523,409,381,321 and 293. The intense ion at m/z 541 arises due to the cleavage of the terminal /3-D-glucopyranose. The base peak at m/z 381 arises due to the cleavage of the disaccharide unit. Therefore, 1 is diphyllin-4-0-[B-D-glucopyranosyl (l-*4)]-/I-2,3-di-0-methyl-D-xylopyranoside. A few diphyllin glycosides were isolated from Cleistanthus spp. (Euphorbiaceae), i.e. cleistanthins A [9], B [16], C [13], D and E [ll], and cleistanthosides A [14] and B [17]. Ramontoside (1) is isomeric to cleistanthin C (diphyllin-4-O-[/?-2,3di-O-methyl-D-xylopyranosyl (l-+4)]-/I-D-glucopyranoside) and differs in the sequence of sugars. As cleistanthin A, which is structurally related to 1 showed anti-cancer activity [ 18],1 may have similar biological activity. The isolation of 1 from F. ramontchi constitutes the first report of a butyrolactone lignan glycoside from Flacourtiaceae. This observation is interesting as Flacourtiaceae is taxonomically related to Euphorbiaceae [2], several species of which are known to elaborate butyrolactone lignan glycosides [9, 14, 171. The present investigation of F. ramontchi shows the chemotaxonomic relationship between Flacourtiaceae and Euphorbiaceae.

Table 1. ‘H NMR data for compounds l-3 H

1

2

3

5

8.00 s

8 2’,5’,6 MeO-6,7 O-CH,-O

7.11 s 6.77-7.11 m 4.06 s 3.75 s 6.11 s

7.70 s 7.05 s 6.83-6.97 m 3.99 s 3.71 s 6.09 s

Fknzylic methylene

5.51 br s

B-D-Glucopyranose, H-l

5.22 d (7)

7.82 s 7.08 s 6.75-6.95 m 4.13 s 3.80 s 6.03 d (1.5) 6.08 d (1.5) 5.40 d (15) 5.52 d (15) Merged with other sugar protons 4.92 d (7.17) 3.05-5.10 m 3.61 s 3.46 s 1.80 s 2.00 s 2.03 s 2.11 s

2,3-di-0-Methyl-fi-n-xylopyranose, Other sugar protons (-H, -OH) Sugar methoxyls Acetoxyls of sugar

H-l

4.84 d (8) 3.24-4.35 m 3.46 s 3.68 s -

5.36 s

-

Compound 1 90 MHz in acetone-d,; 3 300 MHz in acetone-d, + DMSO-d,; 2 300 MHz in chloroform-d. TMS (6 = 0) as int. standard. Coupling constants in Hz are given in parentheses.

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Table C

1

2

3

1

120.20

119.50

119.26

124.60 128.60 129.88 130.34 131.51 151.72 153.22 103.82 { 111.88 145.48 148.41 148.54 105.12 107.01 124.20 68.00 170.11

123.66 126.94 128.45 130.65 130.86 150.32 151.90 101.42 111.60 144.05 147.55

121.05 123.96 128.82 129.98 130.08 150.02 151.68 100.87 111.28 145.12 146.09 146.81 105.90 107.62 123.94 67.06 169.73

2 3 4 9 10 627 538 1’ 3’ 4 2’,5’ 6 CH, c=o

( x 2)

106.35 108.19 123.00 67.35 169.69

2. 13CNMR

data for compounds

C 2,3-di-O-methyl-/&Dxylopyranosyl 1 2 3 4 5

1

2

102.91 79.30 80.20 84.40 62.90

101.87 80.02 80.36 84.65 63.09

103.80 75.60 77.70 71.70 77.90 60.40 102.20 55.99 56.64 58.27 56.97

103.34 72.16 73.16 71.98 76.52 61.47 101.21 55.85 55.94 58.36 56.52 20.80 20.56 20.50 20.22 170.28 170.25 169.69 169.28

3

/?-D-ghCOpyranOSyl

1 2 3 4 5 6 OCH,O OMe

OAc(Me)

OAc(C=O)

Compound

l-3

1 22.63 MHz; 2 and 3 75.47 MHz. Acetone-d,

EXPERIMENTAL

The heartwood of Flacourtin ramontchi L’Herit was collected from the Eturunagram forest of Andhra Pradesh, India. Compounds were detected by observing fluorescence under UV light or by spraying with 10% alcoholic H,SO, followed by heating at 100” for 24 min. PC (descending) of the sugars was performed on Whatman No. 1 paper. TMG (2,3,4,6-tetra-O-methyl-P_Dglucopyranose) was used as reference sugar. The R,,,/R, values were calculated by the standard procedure [8]. Spray reagent: aniline hydrogen phthalate, followed by heating the paper chromatogram at 95-105” for 10 min. FABMS: the sample was dissolved in a glycerol matrix. The powdered heartwood (10 kg) was extracted with MeOH in a Soxhlet apparatus and concentrated in ~acuo to give a brown gummy residue (500 g). This residue was redissolved in minimum quantity of MeOH (1 1) and adsorbed over extracted plant material (1.5 kg), air-dried overnight until all the solvent was completely removed and then extracted successively with petrol (@SO’), CHCI,, EtOAc, Me&O and MeOH, each 3 1. Evaporation of the CHCI, extract in uucuo gave a brown semi-solid (10 g) which was subjected to CC over silica gel. Isolation of @itosterol. The petrolC,H, (9: 1) elute gave a solid (0.3 g) identified as /Lsitosterol, mp 137-138” (lit. [S] mp 136137”) (mmp, superimposable IR with authentic sample). Isolation of jhitosterol-/l-D-ghcopyranoside. The C,H,EtOAc (3:7) elute gave a solid. Crystallization from EtOH afforded p-sitosterol-p-D-glucopyranoside, as needles (0.3 g), mp 28&282” (dec) (lit. [6] mp 280-283” (dec)), [alo -42.13” (pyri-

100.29 55.93 55.30

solns at 368 K using TMS (6 =0) as int. standard.

dine; c 0.57) (lit. [7] [a],, -42.1” (pyridine; c 0.57)). Tetraacetate crystallized from MeOH as needles (90 mg), mp 168-169” (lit. [7] mp 169-170”); 13CNMR (22.63 MHz, CDCl,): 637.43 (C-l), 29.83 (C-2), 80.26 (C-3), 39.12 (C-4), 140.70 (C-5), 122.31 (C-6), 29.89 (C-7), 32.10 (C-8), 50.49 (C-9), 36.91 (C-lo), 22.33 (C-11), 40.03 (C-12), 42.56 (C-13), 57.06 (C-14), 26.51 (C-15), 29.5O(C-16), 56.34(C-17), 12.15 (C-18), 19.49 (C-19), 36.32 (C-20), 18.99 (C-21), 34.24 (C-22), 28.40 (C-23), 46.14 (C-24), 29.63 (C-25), 19.23 (C-26), 18.97 (C-27), 24.43 (C-28), 12.02 (C-29), 21.25, 20.79, 20.66, 19.95 (C-2’,3’,4’,6’ AC-Me), 99.88 (C-l’), 71.87 (C-2’), 72.00 (C-3’), 69.01 (C-4’), 73.24 (C-5’), 62.45 (C-6’), 170.79, 170.46, 169.55, 169.42 (C-2’,3’,4’,6’ Ac-C=O). 13CNMR data for tetraacetate was not reported earlier. Isolation of ramontoside (1). The CHCI,-Me&O (5: 6) elute gave a solid. Crystallization from CHCl,-Me&O mixt. afforded ramontoside (1) as shiny crystals (0.15 g), mp 14&142” (dec). IR 11::; cm _ I: 3500 (br) (OH), 1755 (lactone carbonyl), 1620 (aromatic), 920 (methylenedioxy); EIMS (70 eV) m/z (rel. int.): 380 [M of aglycone] ’ (lOO),321(20), 293 (48), 175 (20), 180 (20), 163 (20), 161 (20), 160 (90), 129 (25); FABMS (positive) m/z (rel. int.): 725 [M+Na]+ (lo), 703 [M+H]+ (lOO), 641 (lo), 571 (30), 541 (45), 523(10),409(12),381 (100),321 (15),293(15);FABMS(negative) m/z (rel. int.): 793 [M+glycerol-HI(15), 701 [M-H](lo), 379 (50); [Found: C, 58.11; H, 5.4. CSdHJBO,e requires: C, 58.12; H, 5.41%]. Acetylation of l(lO0 mg) using Ac,O-pyridine at room temp. for 24 hr gave compound 2, which crystallized from a CsH,-CHCl, mixt. as needles (90 mg), mp 215-220” (dec), [alA

Short Reports - 17.30” (CHCl,; c 0.231); UV 1:::” nm (log s): 261 (4.92), 292 (sh) (4.20), 320 (4.32), 356 (4.01); IR v:z cm-‘: 1755 (lactone carbonyl), 1740 (acetate carbonyl), 1620 (aromatic), 930 (methylenedioxy); EIMS (70 eV) m/z (rel. int.): 380 (lOO), 348 (15), 331 (40), 321 (15), 293 (20), 161 (22); (Found: C, 57.91; H, 5.27. C,,H,,O,, requires: C, 57.93; H, 5.29%). Hydrolysis of 1. Compound 1 (100 mg) was dissolved in 8% methanolic H,S04 (10 ml) and refluxed for 4 hr. Work-up gave 3 (diphyllin) which crystallized from MeOH as needles (50 mg), mp 291” (lit. [9, lo] mp 291”). UV A:::” nm (log E):360 (3.90), 330 (4.01), 295 (4.66), 260 (4.90), 235 (4.46) (lit. [lo]); IR vi:; cm-‘: 3200 (br) (OH), 1730 (lactone carbonyl), 1610 (aromatic), 930 (methylenedioxy) (lit. [lo]); HRMS: CZ1Hi607 (theor. 380.0894) gave 380.0895. EIMS m/z (rel. int.): 380 [M of aglycone]+ (loo), 321 (20), 293 (48), 175 (20), (lit. [ll]). After filtration of 3 the hydrolysate was neutralized with BaCO, and the pptd BaSO, was filtered off. The clear aq. hydrolysate was repeatedly extracted with CHCl, (150 ml) and upon concn in uucuo gave 2,3di-O-methyl-B-o-xylopyranose as a solid (20 mg), mp 80-81” (lit. [13] mp 80-81”), [alo +22.01” (H,O; c 1) (lit. [13], [a], +22” (H,O; c 1)). R,, 0.74 (lit. [12] 0.74) in n-BuOH-EtOH-H,O (5: 1:4). The remaining aq. hydrolysate upon concn in uacuo gave p-o-glucopyranose as a solid (22 mg), mp 147-150” (lit. [19] mp 148-150”). R,, 0.1 (lit. [8] 0.1) in n-BuOH-EtOH-H,O (5 : 1: 4). The identities of these sugars was confirmed by direct comparison with the authentic samples on PC.

REFERENCES 1.

2.

3. 4. 5. 6. 7. 8. 9. 10. 11.

12. 13. 14. 15.

Acknowledgements-We thank Dr S. T. Ramachandra Chary (Govt. B.R.R. Degree College, Jadcherla) for identifying the plant, Prof. K. Nakanishi and Dr B. N. Rao (Columbia University, New York) for recording the FABMS and providing 2,3-di-0-methyl-/I-D-xylopyranose respectively and also Dr M. Vairamani (IICT, Hyderabad, India) for recording the high resolution MS. We also thank Mr K. V. S. Babu (Principal Chief Conservator, Forest Department, Government of Andhra Pradesh) for the kind supply of plant material.

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16. 17. 18. 19.

Nadkami, A. K. (1976) Indian Materia Medica Vol. 1, p. 555. Popular Book Depot, Bombay. Nayar, M. P. (1984) Flora ofIndia (Series IV), Key Words to the Taxonomy of Flowering Plants of India, Vol. 2, p. 183. Bot. Surv. India, Howrah, India. The Wealth of India, Raw Materi&, (1956) Vol. 4, p. 43. Council of Scientific and Industrial Research, New Delhi. Mangold, H. K. and Lundberg, W. 0. (1980) J. Oil Technul. Assoc. 12, 108. Buckingham, J. (1982) Dictionary oforganic Compounds 5th Edn, Vol. 5, p. 5009. Chapman & Hall, New York. Buckingham, J. (1982) Dictionary of Organic Compounds 5th Edn, Vol. 5, p. 5010. Chapman & Hall, London. Ramaiah, P. A., Lavie, D., Budhiraja, R. D., Sudhir, S. and Garg, K. N. (1984) Phytochemistry 20,143. Heftmann, E. (1961) Chromatography, p. 509. Reinhold, New York. Govindachari, T. R., Sathe, S. S., Vishwanathan, N., Pai, B. R. and Srinivasan, M. (1969) Tetrahedron 25, 2815. Govindachari, T. R., Sathe, S. S., Vishwanathan, N., Pai, B. R. and Srinivasan, M. (1967) Tetrahedron Letters 3517. Anjaneyulu, A. S. R., Atchuta Ramaiah, P., Ramachandra Row, L., Venkateswarlu, R., Pelter, A. and Ward, R. S. (1981) Tetrahedron 37, 3641. Meek, E. G. (1956) J. Chem. Sot. 219. Anjaneyulu, A. S. R., Atchuta Ramaiah, P. and Ramachandra Row, L. (1975) Phytochemistry 14, 1875. Sastry, K. V. and Venkata Rao, E. (1983) Planta Med. 47, 227. Bock, K., Pedersen, C. and Pedersen, H. (1984) in Adoances in Carbohydrate Chemistry and Biochemistry Vol. 42 (Tipson, R. S. and Horton, D., eds), p. 193. Academic Press, New York. Lakshmi, T. G., Srimannarayana, G. and Subba Rao, N. V. (1970) Curr. Sci. 39, 395. Sastry, K. V., Venkata Rao, E., Buchanan, J. G. and Sturgeon, R. J. (1987) Phytochemisny 26, 1153. Rao, R. R. and Nair, T. B. (1970) Zndian J. Pharmac. 2, 80. Buckingham, J. (1982) Dictionary of Organic Compounds 5th Edn, Vol. 3, p. 2761. Chapman & Hall, New York.