I ndian Journal of Chemistry Vol. 40B, December 200 1 pp. 1 282- 1 284

Note

A new sterol from Holarrhena antidysenterica seeds

Ajay Kumar & Mohammed Ali*

Department of Pharmacognosy & Phytochemistry, Faculty of Pharmacy. Jamia Hamdard ( Hamdard University),

P.O. Hamdard Nagar, New Delhi 1 10 062, India. FAX No. 6088874 E-mai l : Illohllld_al i@usa.net,

lll.al i@mail .lIsa.com

Received 10 Novelllber 1 999; accepted (revised) 2 January 2001

A new sterol, named holarrhenosterol, isolated from the seeds of Holarrflena antidY.I'enterica along with f)-sitosterol, has been characterized as 5a-stigmasta-9( l 1 ),20(2 1 )-dien-3f)-01 on the basis of spectral data analysis and chemical means.

Ho/arrhena alllidysenterica (Linn.) Wall (Apocynaceae), commercially known as Kurchi, is a deciduous laticiferous shrub, about 1 0 m high, occurs almost throughout India up to an altitude of 1 300 m. All parts of this plant are being used as drug by tribles and in different systems of medicine. The drug has astringent. antidysenteric. anthelmintic. stomachic and fabrifugal properties I . All the parts of this plant contain pregnene alkaloids with one or two nitrogen atoms in the molecule. maximum ( 1 .82%) being in seeds. These alkaloids are produced from the initially formed phytosterol by oxidative degradation and subsequent amination by different plant enzymes. It was. therefore. considered worthwhile to examine the neutral portion of the extract of the kurchi seeds for pregnene or its precursors. Two pentacyclic triterpenes2.3 and sitosta-5.23-dien-3f3-of have been reported as non-alkaloidal constituents of its bark. This paper describes the isolation and structural elucidation of a new phytosterol from the seeds of the plant.

Compound 1. named holarrhenosterol. was obtained as colourless amorphous powder from pet. ether-chloroform ( 1 : 1 ) eluents. The compound responded positively to Liebermann-Burchard test for steroids. Its IR spectrum showed absorption bands for hydroxyl group (3420 cm-I ) and unsaturation ( 1 620 cm- I ) . Its mass spectrum had a molecular ion peak at rnJz 4 12 corresponding to a steroidal formula. C29H480. It indicated the six double bond equivalents. four of them were adjusted in the steroidal carbon

H 1 . R= a-H. �-OH 2 . R= a-H. �-OAc 3 . R= O

skeleton and two in olefinic linkages. The other diagnostically important peaks were observed at rnJz 397 [M-Met. 394 [M-H20t. 382 [M-2 x Met. 379 [394-Met. 273 [M-CIOH39. side chaint. 27 1 [273-2Ht. 255 [273-H20t. 2 1 3 [255-ring D fissiont. 198 [2 1 3-Met. These fragments suggested that it was C-29 sterol possessing one double bond. a C- I0 unsaturated side chain and a hydroxyl group in the carboxylic framework4-6. The ion peaks at rnJz 201 [C I . 1 0-C4•5 fission-side chaint. 302. 1 1 0 [C5.6-C9. IO fissiont. 1 24 [C6,rC9. IO fissiont. 149 [M- 1 24-side chaint. 274 [C7,g-C9. IO fissiont. 120 [M-274-H20t. 7 1 [C2,3-C5, IO-C6,7 fissiont. 57 [7 1 -CH2t. 55 [7 1 -H20t. 1 63 [302-side chaint. 9 1 [ 1 1O-H20t. 106 [ 1 24-H20t and 138 [M-274-H20t indicated the saturated nature of ring A and ring B and the presence of hydroxyl group in ring A which was placed at C-3 on biogenetic consideration. The location of the olefinic linkage at C-9 ( 1 1 ) was inferred from the prominent ion fragments at rnJz 234 and 178 generated due to Retro-Diels-Alder fragmentations of ring C and at m/z 1 74 [Cg,WCI2. 13 fission-H20t. 20 1 [M-side chain-ring At. 1 59 [20 1 -ring Dt and 8 1 [M­side chain-Ring ct. The ion fragments at rnJz 43 [C24-C25 fissiont and 85 [C23-C24 fissiont left room for the presence of exocyclic vinylic linkage at C-20 and an ethyl group in the side chain at C-23.

The IH NMR spectrum of 1 exhibited three one­proton each downfield doublets at 8 5 .35 (J = 5.2 Hz). 5 . 1 4 (J = 8.4 Hz) and 5 .02 (J = 8.4 Hz) assigned to H-1 1 . H-2 1 a and H21 b. respectively. A broad multiplet at 8 3.52 with Wl/2 1 5.6 Hz showed the presence of 3a.-methine proton (axial). Two doublets. integrating three protons each. at 8 0.86 (J = 6.5 Hz) and 0.80 (J = 6.5 Hz) were due to secondary methyls at C-25. The remaining two tertiary methyls resonated as three-

NOTES 1283

proton singlets at 8 1 .00 (Me- I9) and 0.69 (Me- I 8). The appearance of the methyls in the region 8 1 .00-0.69 suggested that these were attached to saturated carbons. The methyl resonance of 24(R)-ethyl (8 0.83) is more shielded compared to 24(S) (8 0.86)7 . The remaining methylene and methine protons appeared in the region 8 2.27- 1 .03. The 'H NMR data were compared with other related sterolss, lO• In the I 3C NMR spectrum, the olefinic carbons appeared at 8 140.78 (C-9), 120.8 1 (C- 1 1 ), 1 38.23 (C-20) and 109.59 (C-2 I ) . The I3C NMR data were compared with �-sitosterol " , lawsaritol '2 and phyllanfraternusterol 1 3 . Acetylation of 1 with acetic anhydride-pyridine yielded a monoacetyl derivative 2 confirming the presence of one acetylable hydroxyl group. Oxidation of 1 with Jones reagent formed 3-oxo derivative 3 which gave positive test with Z· '4 + Immermann reagent lor 3-oxo sterol. On the basis of these findings the structure of the new sterol 1 was established as 5a-stigmasta-9( 1 1 ),20(2 1 )-dien-3�-ol.

Compound 2, from pet. ether-chloroform ( l : 1 ) eluents, was identified as �-sitosterol on the basis of m.p., m.m.p. and spectral data.

Experimental Section General. Melting points were determined in

capillaries on a Perfit melting point apparatus and are uncorrected. Optical rotation [aJD 22 was measured on a Abes Polarimeter. UV spectra (Am.x) were recorded on Beckman DU-64 Spectrophotometer in MeOH. FT IR spectra ( vmax) were recorded on Jasco FT IR-5000 Spectrophotometer using Nujol, (vm•x values are given in cm· ' ) . 'H NMR spectra were screened on advanced DRY 400, Bruker Spectrospin 400-MHz instrument using CDCI, as solvent and TMS as an internal standard. 1 3C FT NMR spectra were recorded on advanced DRY 400, Bruker Spectrospin 1 00-MHz with TMS as internal standard in 5 mm spinning tubes. ElMS (70 eV) were recorded on JEOL-JMS­OX 303 instrument, equipped with direct inlet probe system. TLC were performed on silica gel G and iodine vapours, ceric ammonium sulphate and UV light were used for visualization of TLC spots.

Plant material. The seeds of H. antidysenterica were procured from a local market and authenticated by Dr M P Sharma, Reader, Botany Department of this University.

Extraction. The seeds were dried in an oven at a temperature below 50° C for 2-3 days and coarsely powdered. The ground seeds (4.5 kg) were extracted

exhaustively with pet. ether (60-80° C) in a Soxhlet and concentrated to yield (750 mL) reddish-brown oil. The marc was re-extracted exhaustively with ethanol (95%) and concentrated to yield (280 g) of dark brown viscous mass. The extract was analyzed chemically for determining the presence of different chemical constituents.

Isolation. The alcoholic extract was partitioned into HCI-soluble and HCI-insoluble portions. The HCI-insoluble non-alkaloidal portion was dried, dissolved in minimum amount of methanol and adsorbed on silica gel (60-80 mesh) for preparation of slurry . It was dried in air and subjected to silica gel column packed in pet. ether. The column was eluted with pet. ether, chloroform and methanol in order of increasing polarity.

Holarrhenosterol 1. Elution of column with pet. ether-chloroform ( 1 : 1 ) furnished colourless amorphous powder of 1, recrystallised from pet. ether-acetone (3:2), 102 mg (0.0026 % yield); mp 106- 108°; Rr O. 1 6 (pet. ether-chloroform, 1 : 1 ); [a]D22 _ 1 1 .54° (CHCI3, 0. 1 ); UV Am.x (MeOH) 208 nm (log E 4.3); IR (KBr) 3420, 2940, 2855, 1620, 1465, 1 375, 1060, 8 1 5 cm· l ; IH NMR: 8 5 .35 ( l H, d, J = 5.2 Hz, H- l 1 ) , 5 . 1 4 ( l H, d, J = 8.4 Hz, H-2 1 a), 5 .02 ( l H, d, J = 8.4 Hz, H-2 1 �), 3 .52 ( l H, m, J = 4.5, 4.4, 9.5, 9.9, 6. 1 , 4.6 Hz, WI/2 1 5 .6 Hz, H-3a), 2.27 ( l H, dd, J = 4.6, 9.5 Hz, H-17a), 2 .22 ( l H, ddd, J = 5.3, 9.8, 4.6 Hz, H- l �), 2.03 ( l H, m, H- l a), 2.00 ( l H, dddd, J = 2.9, 6. 1 , 2.7, 8.5 Hz, H-7a), 1 .98 ( l H, dddd, J = 6. 1 , 4.5, 8 .5 , 10.5 Hz, H-7�), 1 .87 ( l H, m, H-2a), 1 .82 (2H, m, H- I2a, H-22a), 1 .86 ( l H, m, H-2a), 1 .68 ( l H, d, J = 9.5 Hz, H- 12a), 1 .59 (2H, m, H- 12�, 22�), 1 .54 ( I H, ddd, J = 1 6. 1 , 9.7, 5 .3 Hz, H-4�), 1 .5 1 (2H, m, H- 16a, H-24a), 1 .49 (2H, m, H- 16�, H-5a), 1 .47 ( l H, m, H-6�), 1 .44 ( I H, m, H- 1 4a), 1 . 16 (2H, m, H-6a, H- I5�), 1 . 1 2 (2H, m, H-23a, H-8�), 1 . 1 1 ( l H, m, H-23�), 1 .08 ( l H, m, H-4a), 1 .06 (2H, m, H- I5a, H-25), 1 .03 (2H, m, H-28), 1 .00 (3H, br s, Me- 19), 0.83 (3H, t, J = 5.3 Hz, Me-29), 0.80 (3H, d, J = 6.5 Hz, Me-27), 0.69 (3H, br s, Me- I 8) ; ElMS mlz (reI. int.) 4 12 [Mt (C29H4SO) (46.4), 397 ( 10.6), 394 (33.9), 382 (8 .9), 379 ( l 0.4), 350 ( l 2. 1 ), 302 ( 16. 1 ), 274 (9. 1 ), 273 (26.0), 27 1 (4 1 . 1 ), 255 (42.4), 234 (88.6), 2 1 3 (26.9), 201 (9.3), 198 ( 13 .0), 1 87 ( 17.3), 178 ( 1 1 .2), 1 74 ( 13 .6), 163 ( 16.2), 160 (26.2), 1 59 (39.2), 149 (28.4), 1 24 (26.5) , 1 20 (39.8), 1 10 (3 1 .3), 106 (60.3), 95 (58 . 1 ), 9 1 (60.4), 85 ( 1 8 .2), 7 1 (27.5), 69 (88. 1 ), 57 (38 . 1 ), 55 (97.5), 43 ( 1 00). I 3C NMR: 8 37.3 (C- l ), 3 1 .6 (C-2), 70.2 (C-3), 42.2 (C-4), 45 . 1

1284 INDIAN J. CHEM. SEC B, DECEMBER 2001

(C-5), 22.3 (C-6), 3 1 .9 (C-7), 30.8 (C-8), 1 40.7 (C-9), 36.8 (C- l O), 120.8 (C- l 1 ), 42. 1 (C- 1 2), 39.9 (C- 1 3), 55.2 (C- 14), 23.9 (C- 1 5), 28.2 (C- 16), 55.9 (C- 17), 1 1 .8 (C- 1 8), 19.5 (C- 19), 1 38.2 (C-20), 1 09.6 (C-2 1 ), 39.3 (C-22), 26.2 (C-23), 45.8 (C-24), 30. 1 (C-25), 19.7 (C-26), 19.2 (C-27), 22.9 (C-28), 1 1 . 1 (C-29).

Acetylation of 1 with acetic anhydride and pyridine yielded a monoacetyl product 2, mp 10 1 - 1020; IR: 1725 em" . ElMS (reI. int.): rnIz 454 [Mt (C3 ,H5002) (7. 1 ), 439 (3. 1 ) , 4 1 1 ( 17.5), 394 ( 1 1 .6), 379 ( 10. 1 ), 3 1 5 (2.7), 272 (4.6), 255 (4 1 .6), 240 (25 .3), 2 1 3 (5. 1 ), 198 (3.6), 1 10 (3 1 .2), 95 (43 .2), 83 (45 .2), 69 (6 1 .0), 55 (8 1 .2), 43 ( 100). Oxidation of 1 with Jones reagent in acetone produced a 3-oxo derivative 3; IR: 1705 em" .

Acknowledgement The authors are thankful to Dr Jagannathan, Head,

NMR Laboratory, AIIMS, New Delhi for recording NMR spectra and to the Head, RSIC, CDRI, Lucknow for scanning mass spectra.

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