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Phytochemical Studies on cashmiriana

1SADIA KHAN , 1MEHDI HASSAN KAZMI*, 2EJAZ AHMED AND ABDUL MALIK 1Department of Applied Chemistry and Chemical Technology University of Karachi, Karachi, Pakistan. 2International Centre for Chemical and Biological Sciences, H.E.J. Research Institute of Chemistry University of Karachi, Karachi- 75270, Pakistan. [email protected]*

(Received on 16th November 2011, accepted in revised form 27th July 2012)

Summary: Eleven compounds have been isolated for the first time from Sorbus cashmiriana namely ursolic acid (1), stigmasterol (2), myricadiol (3), taraxerol (4), 5α , 8α –epidioxyergosta –6, 22- diene-3 β –ol (5), 3β , 5α –dihydroxy –6β − methoxyergosta – 7,22 – diene (6), betulonic acid (7), betulinic acid (8), β –sitosteryl acetate (9), 5α , 8α –epidioxyergosta-6, 9 (11), 22– trien –3β –ol (10) stigmasterol 3-O- β -D-glucopyranoside (11), respectively. Their structures have elucidated by spectroscopy techniques.

Introduction

Sorbus cashmiriana Hedlund, Monog is a of two seasons, one in the spring with lovely Results and Discussion pink – tinted and one in the autumn when the are gone and glorious white shine out. It The methanolic extract of the whole is distributed in Kashmir and the western Himalayas. was divided into n-hexane, chloroform, ethyl acetate, There are about one hundred under genus n-butanol and water soluble fractions. The ethyl Sorbus and seven are native to the Indo-Pakistan acetate soluble fraction was subjected to column subcontinent. The tea made from its bark is used to chromatography over silica gel eluting with n– treat nausea and to cleanse the blood. A bark hexane–ethyl acetate, ethyl acetate and ethyl acetate - preparation is also used against heart disease. The methanol in increasing order of polarity to obtain berries are rich in vitamin C and used to cure scurvy eleven compounds, reported for the first time from [1–5]. The ethanopharmacological and this specie. These could be identified as ursolic acid chemotaxonomic importance of the genus sorbus led (1), stigmasterol (2) myricadiol (3), taraxerol (4), 5α , us to investigate the chemical constituents of sorbus 8α –epidioxyergosta -6, 22– dien 3 β –ol (5), 3 β, 5α cashmiriana. dihydroxy – 6 β methoxy ergosta – 7–22 diene (6),

The methanolic extract of sorbus betulonic acid (7), betulinic acid (8), β –sitosteryl cashmiriana showed strong toxicity in a brine shrimp acetate (9), 5α , 8α epidioxy ergosta- 6, 9 (11), 22– lethality test [6, 7]. On further fractionation a major trien–3 β–ol (10) and stigmasterol 3–0 –β–D–gluco toxicity was observed in the ethyl acetate soluble pyranoside (11) respectively on the basis of their fraction. Pharmacological screening of the ethyl spectral data. acetate soluble fraction showed strong inhibition against Lipoxygenase enzyme using method Experimental developed by Tappel (1962) [8, 9]. This prompted us to carry out bioassay directed isolation studies of the General active constituents. Herein we report the isolation and structure elucidation of ursolic acid (1), stigmasterol Column Chromatography was carried out (2), myricadiol (3), taraxerol (4), 5α , 8α – using silica gel (E-Merck, 230-400 mesh). TLC was epidioxyergosta -6, 22–diene-3 β –ol (5), 3β , 5α – performed over precoated silica gel G60-F254 preparative plates (20x20cm, 0.5mm thick, E Merck) dihydroxy – 6β -methoxy ergosta – 7,22 – diene (6), were used to check the purity of the compounds and betulonic acid (7), betulinic acid (8), β -sitosteryl were visualized under UV light of (254 nm and 366 acetate (9), 5α , 8α –epidioxy ergosta–6, 9 (11), 22 nm) and followed by ceric sulfate as Spraying trien 3β –ol (10) stigmasterol 3–O– β –D– reagent in 10% H2SO4. Melting point was determined glucopyranoside (11), respectively from the ethyl by Gallenkemp apparatus. Optical rotations were acetate fraction. measured on a Jasco DIP-360 digital polarimeter. UV

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and IR spectra were recorded on Hitachi UV-3200 afford β-sitosteryl acetate (9) and 5α , 8α – and Jasco-320A spectrometers respectively. El and Epidioxy ergosta-6, 9 (11), 22 trien 3β–ol (10). The HR-El-MS were measured in an electron impact fraction obtained from the ethyl acetate : methanol mode on Finnigan MAT 12 or MAT 312 (9.0 : 1.0) were rechromatographed over silica gel spectrometers and ions are given in m/z (%).1H and 13 eluting with ethyl acetate - methanol in increasing C –NMR spectra was recorded on Bruker AMX- order of polarity and final purification by PTLC 400 MHz spectrometer with TMS as an internal using solvent system ethyl acetate - methanol (9.5: standard. 0.5) to yield stigmasterol 3-O–β–D–glucopyranoside (11). Plant Material Ursolic acid (1) The whole plant (20 Kg) Sorbus cashmiriana Hedlund, Monog was collected in the Crystallized from EtOH (25 mg), m.p. 283- month of July and August 2004 from Kashmir 20 285 °C; [α]D + 62.5° (c = 0.2, CHCl3); IR (CHCl3) (Pakistan) and identified by Dr. Surraiya Khatoon, -1 1 νmax cm : 3510, 3050, 1697, 1635, 820; H-NMR Plant Taxonomist, Department of Botany, University (CDCl , 400 MHz) δ: 5.11 (1H, m, H-12), 3.19 (1H, of Karachi, Karachi, Pakistan where a voucher 3 dd Jax,ax = 10.0 Hz, Jax,eq = 4.5 Hz, H-3), 1.20 (3H, s, specimen has (No. KUH73/67760) been deposited. Me-27), 1.07 (3H, s, Me-23), 0.94 (3H, s, Me-25), 0.91 (3H, d, J = 6.6 Hz, Me-30), 0.86 (3H, s, Me-26), Isolation 0.81 (3H, s, Me-24), 0.80 (3H, d, J = 6.8 Hz, Me-29). 13 C-NMR (CDCl3, 100 MHz) δ: 176.2 (C-28), 138.7 The freshly collected whole plant material (C-13), 125.8 (C-12), 79.1 (C-3), 55.2 (C-18), 52.4 (20 kg) was cut into small pieces and extracted with (C-5), 47.9 (C-17), 47.4 (C-9), 42.0 (C-14), 39.6 (C- methanol (3 x 30 L). The combined methanolic 8), 38.5 (C-1), 37.0 (C-22), 37.1 (C-10), 33.2 (C-7), extract was evaporated under reduced pressure to 30.5 (C-19), 30.3 (C-20), 29.4 (C-15), 27.5 (C-21), yield a residue (900 g), which was partitioned 24.5 (C-27), 27.4 (C-2), 24.0 (C-23, C-30), 23.9 (C- between n-hexane and water. The aqueous fraction 11), 23.5 (C-16), 22.4 (C-29), 18.3 (C-6), 17.2 (C- was successively partitioned with chloroform (95 26), 15.9 (C-25) and 15.4 (C-24) (Fig. 1) gm), ethyl acetate (180 gm), n-butanol (65 gm), and water (48 gm). The ethyl acetate soluble fraction (180 30 gm) was subjected to column chromatography over silica gel eluting with n-hexane, n-hexane-ethyl 29 20 19 acetate, ethyl acetate and ethyl acetate- methanol in 21 increasing order of polarity to obtain 6 major 12 18 22 13 17 fractions CA-CF. The Fraction obtained from n- 11 28 25 26 hexane- ethyl acetate (7.0: 3.0) were combined and 1 HCOOH 9 14 rechromatographed over silica gel using solvent 16 2 10 8 15 system n-hexane- ethyl acetate (4.0: 6.0) and finally H 5 27 purified by PTLC solvent system (6.5 : 3.5) to afford 7 3 4 ursolic acid (1), stigmasterol (2) and myricadiol (3) HO 6 respectively. The fraction obtained from n-hexane- ethyl acetate (5.0 : 5.0) was further purified on PTLC 23 24 with solvent system (4.0 : 6.0) offered taraxerol (4) , Fig. 1: Structure of Ursolic acid (1). 5α , 8α Epidioxyergosta-6, 22-diene 3 -ol (5) and

3 , 5 dihydroxy-6 β –methoxy ergosta-7, 22-diene The physical and spectral data were in (6) respectively. The fraction obtained from the n- agreement with those in literature [10-12] hexane- ethyl acetate (4.0: 6.0) gave two spots on TLC which was further rechromatography on PTLC Stigmasterol (2) under the solvent system (3.2: 6.8) to give betulonic acid (7) and betulinic acid (8) respectively. The Colorless crystallized solid (5 mg), m.p. 25 fraction obtained from the n-hexane- ethyl acetate 170-171 °C; [α]D -51.5˚ (c = 0.28, CHCl3); IR -1 1 (3.0 : 7.0) were combined and rechromatographed (CHCl3) νmax cm : 3432, 1648; H-NMR (CDCl3, over silica gel using solvent system n-hexane- ethyl 400 MHz), δ: 5.33 (1H, m, H-6), 5.15 (1H, dd, J = acetate (2.0 : 8.0) and finally purified by PTLC 15.2, 8.4 Hz, H-22), 5.02 (1H, dd, J = 15.2, 8.6 Hz, solvent system n-hexane- ethyl acetate (2.5 : 7.5) H-23), 3.28 (1H, m, H-3), 0.90 (3H, d, J = 6.5 Hz,

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Me-21), 0.83 (3H, d, J = 6.6 Hz, Me-26), 0.84 (3H, t, J = 7.0 Hz, Me-29), 0.81 (3H, d, J = 6.5 Hz, Me-27), 0.80 (3H, s, Me-19), 0.65 (3H, s, Me-18). 13C-NMR (CDCl3, 100 MHz) δ: 140.9 (C-5), 138.40 (C-22), 129.48 (C-23), 121.78 ( C-6), 71.92 (C-3), 57.0 (C- 14), 56.0 (C-17), 51.31 (C-24), 50.37 (C-9), 42.50 (C-13), 42.20 (C-4), 40.50 (C-20), 39.73 (C-12), 37.42 (C-1), 36.61 (C-10), 31.89 (C-8), 32.0 (C-25), 31.91 (C-7), 31.81 (C-2), 28.90 (C-16), 25.41 (C-28), 24.41 (C-15), 21.20 (C-27), 21.12 (C-21), 21.01 (C- 11), 19.40 (C-19), 19.0 (C-26), 12.43 (C-18) and 12.0 (C-29) (Fig. 2).

The physical and spectral data corresponded to the reported values [13-15]

29 Fig. 3: Structure of Myricadiol.(3). 28 21 22 26 Taraxerol (4) 24 18 20 25 23 Crystallize with chloroform (30 mg), m.p. 12 17 o 20 27 278- 279 C; [α]D : + 0.72 (c = 0.972 in CHCl3), IR 11 16 –1 1 19 13 (KBr) max cm : 358, 305, 163, 815; H-NMR 1 9 14 15 (CDCl3, 300 MHz)δ 0.81, 0.91, 0.95 (6H, s, 6xCH3), 2 10 8 0.98 and 1.10 (3H, s, 2x CH3), 3.17 (1H, m, H-3 ), HH 13 7 5.53(1H, dd, J = 4.0, 7.0 Hz H-15); C-NMR (CDCl3 HO 3 5 75 MHz) δ 38.2 (C-1), 27.3 (C-2), 79.3 (C-3), 39.2 4 6 (C-4), 55.7 (C-5), 19.0 (C-6), 35.3 (C-7), 38.9 (C-8), Fig. 2: Structure of Stigmasterol (2). 48.9 (C-9), 37.9 (C-10), 17.7 (C-11), 36.0 (C-12), 37.7 (C-13), 158.2 (C-14), 117.1 (C-15), 36.9 (C-16), Myricadiol (3) 38.1 (C-17), 49.4 (C-18), 41.4 (C-19), 29.0 (C-20), 33.9 (C-21), 33.2 (C-22),28.1 (C-23), 15.6 (C-24), Crystallize with methanol ( 20 mg), m.p. 15.6 (C-25),30.1 (C-26), 26.0 (C-27),30.1 (C-28), o 25 33.6 (C-29), 21.5(C-30) (Fig. 4). 269-271 C ; []α D = 7.5 (c = 0.8, CHCl3); IR max (KBr) cm-1: 3550, 3050, 1640, 815; 1H-NMR The physical and spectral data were in

(CDCl3, 300 MHz) δ : 0.80 (3H, s, H-24), 0.90 (3H, agreement with those in literature [16, 17] s, H-23), 0.92 (3H, s, H-29), 0.95 (3H, s, H-25), 0.97 (3H, s, H-30), 0.98 (3H, s, H-27), 1.08 (3H, s , H-26), 3.18 (1H, m, H-3a), 3.72 (2H, s, H-28), 5.45 (1H, dd, 13 J = 4.0, 7.0 Hz, H-15). C-NMR (CDCl3, 75MHz) : 37.9 (C-1), 27.8 (C-2), 78.4 (C-3), 41.2 (C-4), 55.9 (C-5), 19.2 (C-6), 36.3 (C-7), 39.2 (C-8), 45.9 (C-9), 37.9 (C-10), 17.7 (C-11), 32.0 (C-12), 38.3 (C- 13), 158.7 (C-14), 11.9 (C-15), 32.9 (C-16), 38.1 (C- 17), 49.5 (C-18), 41.5 (C-19), 28.8 (C-20), 33.9 (C- 21), 28.7 (C-22), 28.4 (C-23), 16.6 (C-24), 15.6 (C- 25), 30.1 (C-26), 26.1 (C-27), 64.1 (C-28), 33.7 (C- 29), 22.1 (C-30).

The physical and spectral data showed complete agreement with those reported in the literature [16] (Fig. 3). Fig. 4: Structure of Taraxerol (4).

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5 , 8 -Epidioxyergosta-6, 22-diene-3 -ol (5) The physical and spectral data showed complete resemblance with those reported in the Crystallize with methanol ( 25 mg ),m.p. literature [20] o 25 o 176-178 C; [ ]D : -31 (c = 0.90, CHCl3); IR (KBr) -1 1 max cm , 3400, 3021, 1650 ; H-NMR (CDCl3, 400 MHz), 6.51 (1H, d, J = 8.5 Hz, H-7), 6.25 (1H, d, J = 8.5 Hz, H-6), 5.24 (1H, dd, J = 15.3, 8.1 Hz, H-22), 5.15 (1H, dd, J = 15.3, 8.3 Hz, H-23), 3.98 (1H, m, H-3), 1.00 (3H, d,J= 7.0 Hz, H3-21), 0.92 (3H, d, J = 6.8 Hz, H3-28), 0.89 (3H, s, H3-19), 0.84 (3H, d, J=6.7 Hz, H3-27), 0.62 (3H, s, H3-18) and 0.82 (3H, 13 d, J = 6.5 Hz, H3-26). C-NMR (CDCl3, 100 MHz), 34.7 (C-1),30.1(C-2), 66.5(C-3), 37.0(C-4), 82.1(C-5), 135.6(C-6), 130.8(C-7), 79.5(C-8),

51.1(C-9), 36.9(C-10), 20.7(C-11), 39.3(C- Fig. 6: Structure of 3β, 5α-dihydroxy-6β- 12),44.5(C-13), 51.7(C-14), 23.4(C-15), 28.6(C-16), methoxyergosta-7, 22-diene (6). 56.2(C-17), 12.9(C-18), 18.2(C-19), 39.8(C-20), 20.9(C-21), 135.4(C-22), 132.30(C-23),42.7(C-24), Betulonic acid (7) 33.0(C-25), 19.6(C-26), 20.0(C-27), and17.5(C-28) Crystallized from methanol (15 mg),m.p.

o 25 o The physical and spectral data showed 257-258 C ; [α]D : + 30 (c = 0.32, CHCl3) IR (KBr) -1 1 complete resemblance with those reported in the max cm : 3020, 1700, 1640, 880; H-NMR (CD Cl3, literature [18, 19] (Fig. 5). 500 MHz): 4.74 and 4.61 (1H, br, s, H-29), 1.69 (3H, s, H-30), 1.44, 1.08, 1.01, 0.99, 0.97, (3H, s) 13 C-NMR (CDCl3, 125 MHz): 40.3 (C-1), 34.8 (C- 2), 218.7 (C-3), 48.0 (C-4), 55.6 (C-5), 20.3 (C-6), 34.3 (C-7), 41.3 (C-8), 50.5 (C-9), 37.7 (C-10), 22.0 (C-11), 26.2 (C-12), 39.2 (C-13), 43.2 (C-14), 31.2 (C-15), 32.8 (C-16), 57.0(C-17), 49.9 (C-18), 47.5 (C-19), 150.9 (C-20), 30.3 (C-21), 37.6 (C-22), 27.3 (C-23), 21.6(C-24), 16.6 (C-25), 16.5 (C-26), 15.3 (C-27), 182.1 (C-28), 110.4 (C-29), 20.0 (C-30).

The physical and spectral data showed complete agreement with those reported in the Fig. 5: Stucture of 5 , 8 -Epidioxyergosta-6, 22- literature [21-23] (Fig. 7). diene-3 -ol (5).

3 ,5 -dihydroxy-6 -methoxyergosta-7,22-diene (6)

Crystallize with methanol (20 mg),m.p.173- o 25 o 176 C; [ ]D - 30.5 (c = 0.90, CHCl3) IR (KBr) -1 1 max cm 3400, 1640 ; H-NMR (CDCl3, 300 MHz) 0.58 (3H, s, H-18), 0.91 (3H, d, J = 7.0 Hz, H-26), 0.82 (3H, d, J = 7.0 Hz, H-27), 0.89 (3H, d, J = 6.5 Hz, H-28), 0.95 (3H, s, H-19), 0.97 (3H, s, OMe), 4.1 (1H, m, H-3), 5.15 (2H, dd, J = 8.0, 16 Hz, H-22), 5.25 (2H, dd, J = 7.5, 16 Hz, H-23), 5.40 (1H, m, H- 13 7). C-NMR (CDCl3, 75MHz): 32.2 (C-1), 30.6 (C-2), 68.0 (C-3), 39.6 (C-4), 78.0 (C-5), 82.5 (C-6),

114.5 (C-7), 144.2 (C-8), 44.5 (C-9), 38.1 (C-10), 22.3 (C-11), 39.6 (C-12), 44.5 (C-13), 55.5 (C-14), Fig. 7: Structure of Betulonic acid (7). 23.3 (C-15), 27.8 (C-16), 56.3 (C-17), 14.2 (C-18), Betulinic Acid (8) 18.6 (C-19), 40.7 (C-20), 20.2 (C-21), 132.0 (C-22), 135.5 (C-23), 43.2 (C-24), 33.2 (C-25), 20.1 (C-26), Crystallize from methanol (20 mg), m.p. o 25 o 21.8 (C-27), 18.3 (C-28), 58.3 (OMe) (Fig. 6). 280-282 C; [α]D + 27 (c = 0.32 CHCl3) IR (KBr)

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-1 1 max cm , 3020, 1697; H-NMR (CDCl3, 500 MHz): 4.94 (1H, d, J = 2.0 Hz), 4.77 (1H, s), 3.55 (1H, m, H-3), 1.80, 1.23, 1.08, 1.07, 1.02 and 0.83 (each 3H, 13 S, 6 x CH3). C-NMR (CDCl3, 125 MHz) : 38.7 (C-1) 27.4 (C-2), 80.5 (C-3), 38.8 (C-4), 57.4 (C-5), 18.3 (C-6), 34.3 (C-7), 42.0 (C-8),. 51.5 (C-9), 37.2 (C-10), 20.8 (C-11), 25.5 (C-12), 38.4 (C-13), 43.6 (C-14), 30.5 (C-15), 32.1 (C-16), 58.1 (C-17), 48.5 (C-18), 50.4 (C-19), 152.1 (C-20), 29.7 (C-21), 37.0 (C-22), 27.9 (C-23), 17.1 (C-24), 17.2 (C-25), 16.1 (C-26), 15.6 (C-27), 17.6 (C-28), 110.5 (C-29), 19.4 (C-30) (Fig. 8).

The physical and spectral data correspond to the reported values [23, 24] Fig. 9: Structure of β-Sitosteryl acetate (9).

5α,8α-epidioxyergosta-6,9(11),22-trien-3β-ol(10)

Gummy solid (24 mg); IR (KBr) V max cm- l I : 3440, 3024 and 1640. H-NMR (CDCI3, 500 MHz), δ: 6.61 (IH, d, J = 8.5 Hz, H-7), 6.30 (lH, d, J = 8.5 Hz, H-6), 5.43 (lH, dd, J= 6.0,1.8 Hz, H-11), 5.24 (IH, dd, J= 15.1, 8.0 Hz, H-22), 5.17 (IH, dd, J = 15.1, 8.5 Hz, H-23), 3.98 (IH, m, H-3), 1.09 (3H, s, H3-19), 1.01 (3H, d, J= 6.6 Hz, H3-21), 0.92 (3H, d, J= 7.2 Hz, H3-28), 0.84 (3H, d, J= 6.7 Hz, H-3-27), 0.83 (3H, d, J= 6.5 Hz, H3-26) and 0.75 (3H, s, H3- I3 18). C-NMR (CDCl3, 125 MHz), δ: 32.8 (C-1), 30.8 (C-2), 66.4 (C-3), 36.2 (C-4), 83.0 (C-5), 135.2 (C-6), 135.8 (C-7), 78.5 (C-8), 142.8 (C-9), 38.0 (C -10),

119.8 (C-11), 41.3 (C-12), 43.7 (C-13), 48.3 (C-14), Fig. 8: Structure of Betulinic Acid (8). 22.8 (C-15), 28.7 (C-16), 56.0 (C-17), 12.8 (C-18),

25.5 (C-19), 39.9 (C-20),20.9 (C-21), 135.5 (C-22), β-Sitosteryl acetate (9) 132.5 (C-23), 42.8 (C-24), 33.1 (C-25), 19.7 (C-26),

20.0 (C-27) and 17.5 (C-28) (Fig. 10). Crystallized from methanol (40 mg), m.p. 25 119-121 °C; [α]D –38.5° (c = 0.10, CHCl3), IR The physical and spectral data showed -1 1 (CHCl3) νmax cm , 3050, 1730, 1650 and 815. H- complete resemblance with those reported in the NMR (CDCl3, 400 MHz), δ: 3.55 (lH, m, H-3), 5.23 literature [19] (lH, m, H-6), 0.68 (3H, s,M e-18), 1.01 (3H, s, Me- 19), 0.92 (3H, d, J = 6.2Hz, Me-21), 0.83 (3H, d, J = 6.5 Hz, Me-26), 0.81 (3H, d, J = 6.5Hz, Me-27), 0.84 (3H, t, J = 7.0 Hz, Me-29) and 2.05 (3H, s, CH3- 13 COO), C-NMR (CDCl3, 100 MHz), δ: 37.1 (C-l), 31.8 (C-2), 77.2 (C-3), 42.4 (C-4), 139.8 (C-5), 122.6 (C-6), 32.0 (C-7), 32.0 (C-8), 50.1 (C-9), 36.6 (C-10), 21.1 (C-11), 40.0 (C-12), 42.6 (C-13), 56.7 (C-14), 24.3 (C-15), 28.2 (C-16), 56.2 (C-17), 11.9 (C-18), 19.4 (C-19), 36.2 (C-20), 19.1 (C-21), 34.0 (C-22), 29.3 (C-23), 45.8 (C-24), 26.2 (C-25), 18.8 (C-26),

19.8 (C-27), 23.1 (C-28), 11.9 (C-29), 20.1 (CH3CO) Fig. 10: Structure of 5α,8α-epidioxyergosta-6,9 (11), and 171.2 (CO) (Fig. 9). 22-trien-3β-ol (10).

The physical and spectral data showed Stigmasterol 3-O-β-D-glucopyranoside (11) complete agreement with those reported in the Crystallized from methanol (30 mg), m.p. literature [25, 26] o 25 289-290 C [α]D –51.5° (c = 0.22, CH3OH), IR 1 1 (KBr) νmax cm 3454, 3024, 1646. H-NMR (CD3OD,

5 Uncorrected Proof

400 MHz) δ: 5.23 (1H, br d, J = 5.4 Hz, H-6), 5.14 Chemistry, Vol. Elsevier: Amsterdam, p.383 (1H, dd, J= 15.2, 8.4 Hz, H-22), 5.02 (1H, dd, J= (1997). 15.2, 8.6 Hz, H-23), 4.78 (1H, d, J= 7.4 Hz, H-1′), 7. B. N Meyer, N. R Ferrigni, J. E. Putnam, L. B 3.83 (1H, m, H-3), 3.84-4.44 (m, Glc-H′), 1.01 (3H, Jacobsen, D. E. Nichols and J. L McLaughlin, s, Me-19), 0.90 (3H, d, J = 6.2 Hz, Me-21), 0.83 (3H, Planta Medica, 45, 31 (1982). d, J = 6.5 Hz, Me-26), 0.82 (3H, t, J = 7.0 Hz, Me- 8. A. L. Tuppel, Methods in Enzymology, vol. 5, 29), 0.80 (3H, d, J = 6.5 Hz, Me-27), 0.67 (3H, s, Academic Press New York p 539 (1962). 13 9. D. M Barrett and G. E Arithon, Journal of Me-18). C-NMR (CD3OD, 125 MHz) δ 141.5 (C- 5), 138.9 (C-22), 129.1 (C-23), 121.1 (C-6), 102.8 Agriculture Food Chemistry, 49, 32 (2001). (C-1'), 79.8 (C-3), 76.9 (C-3'), 76.7 (C-5'), 74.2 (C- 10. H. Budzikiewicz, J. M. Wilson and C. Djerassi, 2'), 70.6 (C-4'),62.2 (C-6'), 57.0 (C-14), 56.1 (C-17), Journal of American Chemical Society, 85, 3688 52.1 (C-24), 50.8 (C-9), 43.9 (C-4), 43.1 (C-13), 40.5 (1963). (C-20), 39.9 (C-12), 37.8 (C-l), 36.9 (C-10), 32.9 (C- 11. K. Hiller, H. D. Woitke and G. Lehmann, 25), 32.8 (C2), 31.9 (C-7), 31.7 (C-8), 28.9 (C-16), Pharmazie, 28, 391 (1973). 25.6 (C-28), 24.5 (C-15), 21.9 (C-21), 21.7 (C-27), 12. K. Junghyun, S. J. Dae, K. Hyojun and S. K. Jin, 21.5 (C-11), 19.5 (C-19), 19.1 (C-26), 12.6 (C-18), Archives Of Pharmacia Research, 32, 983 12.1 (C-29) (Fig. 11). (2009). 13. S. G. Wyllie, B. A. Amos and L. Tokes, Journal The physical and spectral data correspond to of Organic Chemistry, 42, 725 (1977). the reported values [13, 14, 27, 28] 14. I. Rubinstein, L. J. Goad, A. D. H. Clague and J. L. Mulheirn, Phytochemistry, 15, 195 (1976). 15. K. M. M. S. Prakash, P. V. S. Naidu and P. Muralidhar, International Journal of Pharmacy and Technology, 3, 2868 (2011). 16. N. Sakurai, Y. Yaguchi and T. Inoue, Photochemistry, 26, 217 (1987). 17. K. Tiwatt, S. Rutt, P. Thitima and R. Nijsiri, Journal of Health Research, 23, 175 (2009). 18. A. A. C. Gunatilaka, Journal of Organic Chemistry, 46, 3860 (1981). 19. M. D. Greca, L. Mangoni, A. Molinaro and L. Previtea, Gazzeta Chimica Italiana, 120, 391 (1990). [Fig. 11: Structure of Stigmasterol 3-O-β-D- 20. H. C. Kwon, S. D. Zee, S. Y. Cho, S. U. Choi glucopyranoside (11). and K. R. Lee, Archives of Pharmacal Research, References 25, 851 (2002). 21. S. B. Mahato and A. P. Kundu, Photochemistry, 1. S. K. Bhattacharjee, Hand Book of Medicinal 37, 1517 (1994). , Pointer Publisher; Jaipur, India, p 327 22. A. G. Gonzalez, J. Amaro, B. M. Fraga and J. (2004). Luis, Photochemistry, 22, 1828 (1983). 2. L. M. Perry and J. Metzger, Medicinal Plants of 23. Z. C. Sang, C. Y. Min, U. C. Sang and R. L. East and South Asia, The MIT Press: Cambridge Kang, Archives of Pharmacal Research, 29, 203 England, p. 341 (1980). (2006). 3. A. Krishna, The Wealth of India, Vol. IX. 24. H. Kojima, H. Tominaga, S. Sato and H. Ogura, Council of Scientific and Industrial Research: Phytochemistry, 26, 1107 (1987). New Delhi, p. 435 (1972). 25. K. Aizawa, S. Yoshida and T. Takahashi, 4. D. M. A. Jayaweera, Medicinal Plants, Vol. IV, Organic Mass spectrum, 9, 470 (1974). Published by N. S. C of Srilanka 4715 Maitland 26. P. Monaco and L. Previtera, Journal Of Natural Place Colombo, p. 257 (1982). Products, 4, 673 (1964). 5. A. C. Krachmal, A Guide to the Medicinal 27. H. L. Holland, P. R. P. Diakow and G. J. Taylor, Plants of the United States, Quadrangle New Canadian Journal of Chemistry, 56, 3121 York Times Book Co: New york, 9, p. 207 (1978). (1980). 28. M. Ibrahim, M. Imran, A. Hussain, M. Aslam, F. 6. J. L. McLaughlin, C. J Chang , D. L Smith , A. S. Rehmain, B. Ali, H. Nawaz and A. Malik, U. Rahman. Studies in Natural Product Pakistan Journal of Chemistry, 2 (2012).

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