1 Phytochemical Studies on Sorbus Cashmiriana
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Uncorrected Proof Phytochemical Studies on Sorbus 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 tree of two seasons, one in the spring with lovely Results and Discussion pink – tinted flowers and one in the autumn when the leaves are gone and glorious white fruits shine out. It The methanolic extract of the whole plant is distributed in Kashmir and the western Himalayas. was divided into n-hexane, chloroform, ethyl acetate, There are about one hundred species 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 1 Uncorrected Proof 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, 2 Uncorrected Proof 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.