AnamSHORT Sajid et al., COMMUNICATION J.Chem.Soc.Pak., Vol. 40, No. 01, 2018 249 Bioassay Directed Isolation Studies on Hypericum oblongifolium 1Anam Sajid, 1Ejaz Ahmed*, 1Ahsan Sharif, 1Faiza Arshed, 1Muhammad Arshad, 2Muhammad Sher 3Arfaa Sajid and 1Sumra Amanat 1Institute of Chemistry, University of The Punjab, Quaid-e-Azam Campus, Lahore, 54590, Pakistan. 2Department of Chemistry, Allama Iqbal Open University, Islamabad, 44000, Pakistan. 3Department of Chemistry, Government College Woman University, Faisalabad, Pakistan. [email protected]* (Received on 1st August 2017, accepted in revised form 20th October 2017) Summary: One new taraxastane type triterpene (1) along with eight known compounds have been isolated from chloroform soluble fraction of Hypericum oblongifolium. The structures of the isolated compounds were elucidated on the basis of modern sophisticated 1D and 2D-NMR and mass spectrometric techniques. The known compounds were recognized as 4,4-dimethyl cholesterol (2), lupeol (3), taraxerol (4), 4,4-dimethylergosta-8,14,24(28)-triene-3β,12β,17α-triol (5), oleanolic acid (6) erectasteroid D (7), (S)-4',5-dihydroxy-7-methoxyflavanone (8), β-sitosterol-3-O-β-D- glucopyranoside (9). All the compounds were evaluated for their lipoxygenase inhibitory potential. Keywords: Hypericum oblongifolium, Taraxastane, 1D, 2D-NMR and MS techniques, Lipoxygenase enzyme Introduction Hypericum oblongifolium is a flowering Experimental plant in the Hypericaceae family, which comprises 9 genera and 540 species. It is a herbaceous plant and Column chromatography (CC): silica gel generally 612 meters in height. The leaves are 0.060-0.200 mm, 60 A. TLC: pre-coated silica gel 60 yellowish green in color and 12 cm long. The plant F254 plates. UV: detection at 254 nm and/or by using is considered as a native flowering plant of Eurasia. ceric sulphate reagent. Optical rotations: Jasco-DIP- Normally it is found at an altitude of 40006000 360 digital polarimeter. IR spectra: Hitachi-UV- 3200 meter, especially in Himalaya, northern parts of and Jasco-302-A spectrophotometers respectively. 1 13 Pakistan and in China [1]. The genus Hypericum is H- and C-NMR spectra: Bruker spectrometers represented in Pakistan by nine species [2]. operating at 500 MHz and 125 MHz respectively, Traditionally H. oblongifolium has been used for chemical shifts (δ) in ppm relative to treatment of hepatitis, microbial infections, swellings, tetramethylsilane as international standard and J 2 1 inflammations, and nasal hemorrhage. It was value in Hz. D-NMR (HMBC, HMQC, H- 1 considered as a remedy for dog and sting bites. In HCOSY, NOESY) Bruker spectrometers operating recent years the plant has gained media attentions due at 500 MHz. EIMS, HR-EIMS: Jeol JMS-HX-110 to its uses in alternative medicines. Now a days it has and JMS-DA-500 MS, m/z: (relative intensity). For been proved to be anti-ulcer, anti-proliferative and determination of antimicrobial and enzyme inhibition anti-inflammatory agent. During pharmacological activities (lipoxygenase), all chemicals and enzymes evaluation, this plant was reported to have anti- were purchased from Sigma (St. Louis, MO, USA) spasmodic, bronchodilator, hypotensive and cardiovascular inhibitory activity [3-6]. In the present Plant material study, one new taraxastane type triterpene, 3-oxo- 20(30)-taraxastene-28,13β-olide (1) was isolated The whole plant of Hypericum along with eight known compounds (2-9). The oblongifolium (10 Kg) was collected from Swat, structures of the known compounds were elucidated Malakand, in April 2013 by Dr. Mumtaz Ali from as 4,4-dimethyl cholesterol (2), lupeol (3), taraxerol Department of Chemistry, University of Malakand, (4), 4,4-dimethylergosta-8,14,24(28)-triene- KPK, Pakistan. The plant was identified from Botany 3β,12β,17α-triol (5), oleanolic acid (6) erectasteroid Department, University of Malakand, KPK, Pakistan. D (7), (S)-4',5-dihydroxy-7-methoxyflavanone (8), and β-sitosterol-3-O-β-D-glucopyranoside (9). All the Isolation compounds were evaluated for lipoxygenase inhibitory activity against lipoxygenase enzyme. The dried plant material (10 Kg) was crushed, ground, and extracted with methanol. The methanolic extract was evaporated under reduced *To whom all correspondence should be addressed. Anam Sajid et al., J.Chem.Soc.Pak., Vol. 40, No. 01, 2018 250 pressure and the gummy material (0.7 Kg), was 22.3 (C-23), 20.7 (C-24), 14.2 (C-25), 16.2 (C-26), partitioned between water, n-butanol, ethyl acetate 17.2 (C-27), 178.4 (C-28), 14.4 (C-29), 106.8 (C-30). chloroform, and n-hexane soluble fractions. The chloroform fraction was chromatographed over silica 4,4-Dimethyl cholesterol (2) gel in a column eluting with n-hexane-chloroform Colorless crystalline solid; m.p. 109-110 °C; and chloroform-methanol mixtures in increasing [α] 20 + 20.5 (c = 0.50, CHCl ); IR (KBr) ν cm1 order of polarity to obtain 12 major fractions labelled D 3 max 3437, 2950, 2825, 1610, 1485, 1038, 812; HREIMS, as A-L. The fraction B eluting with n-hexane- + [M ] at m/z 414.3857 (calcd. For C29H50O, chloroform (4:1) was subjected to column + 414.3862); EIMS, [M ] m/z (rel. int. %) 414 (45), 413 chromatography again and obtained several semi (30), 301 (67), 283 (50), 275 (23), 203 (63), 175 (42), pure fractions. The fraction at 7.8:2.2 (n-hexane- 148 (21), 135 (59), 57 (100). chloroform) was subjected to Preparative TLC using solvent system n-hexane-acetone (4:1) to afford 2 (33 Lupeol (3) mg), 3 (19 mg) and 4 (14 mg). Similarly, the fraction 20 C (n-hexane-chloroform; 7:3) was again Colorless crystals; m.p. 215216 °C; [α]D 1 chromatographed over silica gel column +26.9 (c = 0.11, CHCl3); IR (CHCl3) νmax cm 3475, chromatography eluting with n-hexane-acetone in 3081, 1645, 873; HREIMS, [M+] at m/z 426.3835 + increasing order of polarity to give 4 sub-fractions (calcd. for C30H50O, 426.3861); EIMS, [M ] m/z (rel. C1-C4. The fraction C-4 was subjected to Preparative int. %) 426 (20), 411 (25), 408 (30), 393 (35), 385 silica gel TLC using solvent system n-hexane- (15), 220 (80), 218 (55), 207 (25), 189 (100), 139 acetone (3:1) to afford compound 1, (13 mg), 5 (15 (70). mg), 6 (40 mg) and 7 (11 mg). The fraction H (n- hexane-chloroform, 1:4) was again column Taraxerol (4) chromatographed over silica gel using solvent system 20 n-hexane-ethyl acetate in increasing order of polarity. White crystals; m.p. 275277 °C; [α]D + 1 Repeated column chromatography by same solvent 0.72 (c = 0.972, CHCl3); IR (KBR) νmax cm 3584, + system (n-hexane-ethyl acetate, 1:4) to give 3052, 1638, 818; HREIMS, [M ] at m/z 426.3826 + compound 8 (10 mg). The fraction K (chloroform- (calcd. for C30H50O, 426.3846); EIMS, [M ] m/z (rel. methanol, 9:1) was further purified over silica gel int. %): 426 (5), 408 (10), 393 (15), 363 (20), 348 column chromatography eluting with chloroform- (17). methanol (9.7:0.3) to afford compound 9 (26 mg). 4,4-Dimethylergosta-8,14,24(28)-triene-3β,12β,17α- triol (5) 3-Oxo-20(30)-taraxastene-28,13β-olide (1) 23 Colorless powder; [α]D 15.2 (c = 2.8, Colorless needles; m.p. 246248 °C; [α] 20 MeOH); UV λmax (MeOH) 247 (ε = 15, 160); IR D 1 1 (CHCl3) νmax cm 3472, 3039, 1631, 864; HREIMS, +55.5 (c = 1.08, CHCl3); IR (KBr) νmax cm 2950, + 1755, 1695, 1650, 895; HREIMS, [M+] at m/z [M ] at m/z 456.3632 (calcd. for C30H48O3, 456.3603); EIMS, [M+] m/z (rel. int. %): 456 (72), 452.3265 (calcd. for C30H44O3 452.3290); EIMS m/z 1 438 (34) 420 (20), 397 (12), 394 (19), 379 (31), 369 438, 424, 409,394, 379; H-NMR (CDCl3, 500 MHz) δ: 1.52, 1.30 (1H each, m, H-1), 2.10 (2H, m, H-2), (33), 327 (60), 301 (15), 300 (72), 273 (24), 271 (29). 1.01 (1H, m, H-5), 1.58, 1.67 (1H each, m, H-6), Oleanolic acid (6) 1.18, 1.75 (1H each, m, H-7), 1.30 (1H, dd, J = 11.6, Colorless needles (MeOH); m.p. 305306 4.8 Hz, H-9), 1.27, 1.09 (1H each, m, H-11), 1.68, 20 1.84 (1H each, m, H-12), 1.87, 2.11 (1H each, m, H- °C; [α]D + 78.9 (c = 0.07, CHCl3); IR (KBr) νmax 1 + 15), 1.30, 2.14 (1H each, m, H-16), 2.22 (1H, d, J = cm 3420, 1707, 1637, 815; HREIMS, [M ] at m/z 4.6 Hz, H-18), 2.02 (1H, dq, J = 6.9, 4.4, H-19), 2.15 456.3610 (calcd. for C30H48O3, 456.3603); EIMS, m/z + (2H each, m, H-21), 1.58, 1.68 (1H each, m, H-22), (rel. int. %) [M ] 456 (4), 248 (98), 208 (12), 203 0.76 (3H, s, Me-23), 1.03 (3H, s, Me-24), 0.94 (3H, s, (60), 133 (53). Me-25), 0.95 (3H, s, Me-26), 0.97 (3H, s, Me-27), Erectasteroid D (7) 1.10 (3H, d, J = 6.9 Hz, Me-29), 5.10, 4.66 (1H each, 13 24 s, H-30); C-NMR (CDCl3, 125 Hz) δ: 36.1 (C-1), Gummy solid; [α]D + 29.0 (c = 0.4, 1 29.4 (C-2), 216.5 (C-3), 40.6 (C-4), 53.0 (C-5), 16.5 CHCl3); IR (CHCl3) νmax cm 3414, 2938, 1725, (C-6), 57.8 (C-7), 42.7 (C-8), 51.3 (C-9), 33.9 (C-10), 1665, 1454, 1381, 1249, 1037, 787; HREIMS, [M+] 15.7 (C-11), 29.6 (C-12), 89.9 (C-13), 40.6 (C-14), at m/z 458.3396 (calcd.