AnamSHORT Sajid et al., COMMUNICATION J.Chem.Soc.Pak., Vol. 40, No. 01, 2018 249

Bioassay Directed Isolation Studies on 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 in the family, which comprises 9 genera and 540 species. It is a herbaceous plant and Column chromatography (CC): silica gel generally 612 meters in height. The leaves are 0.060-0.200 mm, 60 A. TLC: pre-coated silica gel 60 yellowish green in color and 12 cm long. The plant F254 plates. UV: detection at 254 nm and/or by using is considered as a native of Eurasia. ceric sulphate reagent. Optical rotations: Jasco-DIP- Normally it is found at an altitude of 40006000 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 cm1 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. 215216 °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. 275277 °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. 246248 °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. 305306 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. for C29H46O4, 458.3398); 23.4 (C-15), 19.4 (C-16), 42.1 (C-17), 53.2 (C-18), EIMS, m/z [M+] 458 (10), 399 (34), 381 (64), 363 32.8 (C-19), 151.9 (C-20), 30.6 (C-21), 28.1 (C-22), (39), 288 (56), 252 (45), 57 (100) Anam Sajid et al., J.Chem.Soc.Pak., Vol. 40, No. 01, 2018 251

30

21 29 19 20 20 22 O 21 18 O 12 24 12 28 22 17 23 18 11 11 19 13 26 25 26 13 17 16 25 1 9 1 9 14 16 14 27 2 2 10 8 15 10 8 15 27 7 7 3 3 4 5 HO 5 O 6 2 6 4 23 24 1 28 29

30 29 30

22 20 29 19 21 20 27 19 12 12 21 22 18 17 18 17 11 11 25 26 13 25 13 26 28 28 1 9 1 9 14 16 16 14 2 2 10 8 10 8 15 15 27 7 4 7 3 4 5 HO 3 5 HO 6 6 23 24 3 23 24 4

28 29 21 28 20 OH 20 22 19 21 18 12 18 17 OH 22 12 23 24 17 11 11 19 13 26 25 26 13 16 25 CO2H 1 1 9 30 9 14 16 14 27 2 2 10 8 15 10 8 15 27 7 4 7 3 4 5 HO 3 5 HO 6 6 5 30 29 23 24 6

21

20 22 18 12 24 17 23 3' HO 11 OH 13 2' 19 16 25 26 4' 1 9 14 8 1 2 27 H3CO O 5' 10 8 15 9 1' 7 2 6' 7 3 5 HO O 6 3 4 6 10 4 5 28 O 29 7 OH O 8 Anam Sajid et al., J.Chem.Soc.Pak., Vol. 40, No. 01, 2018 252

21 28 20 22 29 18 12 17 24 11 23 19 13 26 16 25 1 9 14 27 2 15 OH 10 8 6' 7 3 5 5' O O 4 6 4' 1' 9 HO 3' 2' HO OH

Fig. 1: Structures of compounds 1-9

(S)-4',5-dihydroxy-7-methoxyflavanone (8) Enzyme kinetics software (Perella Scientific Inc. Amherst, USA). 20 Colorless needles; m.p. 152154 °C; [α]D 1 8 (MeOH); IR (KBr) νmax cm 3434, 1685, 1705; Results and Discussion + HREIMS, [M ] at m/z 286.0980 (calcd. for C16H14O5, 286.0961); EIMS, m/z [M+] 286 (45), 254 (29), 237 Column chromatography of the chloroform (55), 193 (48), 143 (73), 93 (100). and ethyl acetate fractions of whole plant of H. oblongifolium led to the isolation of one new and β-Sitosterol-3-O-β-D-glucopyranoside (9) eight known compounds.

25 White solid; m.p. 279280 °C; [α]D 14.5 Compound 1, colorless needles, from the 1 chloroform soluble fraction of H. oblongifolium, (c = 0.3, CHCl3); IR (KBr) νmax cm 3442, 3038, 1649, 1598; HRFABMS, [M+] at m/z 576.4379 behaved positively against Liebermann-Burchard test (calcd. for C H O , 576.4389); EIMS, m/z (rel. int. for triterpenes [8]. The IR spectrum showed 35 60 6 1 %) [M+] 414 (16), 396 (13), 399 (15), 381 (75), 329 absorption bands at 2950 cm (CH- stretching), 1755 (31), 275 (11), 273 (21), 255 (41). cm1 (γ-lactone) 1695 cm1 (C=O), and 1650 cm1 (terminal double bond). The molecular formula Lipoxygenase Inhibitory Assay C30H44O3 was determined by HREIMS, which produced the molecular ion peak [M+] at m/z Lipoxygenase inhibiting assay was 452.3265 (calcd. for C30H44O3 452.3290), with nine 1 performed according to the method developed by degrees of unsaturation. Inspection of the H-NMR Tappel [7] with slight, modifications. 150μL sodium indicated signals of six methyls; among them five phosphate buffer, (100 mM, pH 8.0) was taken in appeared separately as singlets at δ 0.76, 0.94, 0.95, which 10μL test compound was added along with 0.97 and 1.04 (3H each) and one as doublet at δ 1.10 15μL purified lipoxygenase enzyme and mixed well. (3H, J = 6.9). Two characteristic signals at δ 4.66 The mixture was incubated at 25°C for 10 minutes (1H, s) and δ 5.10 (1H, s) were observed, which and absorption was measured at 234 nm. Now 25μL indicated the presence of terminal olefinic moiety in 13 linoleic acid (substrate) solution was added to initiate the molecule [9-11]. The BB and DEPT C-NMR the reaction and change in absorbance was detected spectra displayed thirty carbon signals out of which after 6 min at, 234 nm. Test compounds and the six were methyl, eleven methylene, four methine and control were dissolved in methanol. All reactions nine quaternary carbons. A signal at δ 216.5 depicted were performed in triplicates in 96-well micro-plate the presence of keto group while a signal at δ 178.4 in Spectra Max 340 (Molecular Devices, USA). indicated the presence of γ-lactone moiety in the Baicalein was used as a reference standard. IC50 molecule. The olefinic carbons resonated at δ 151.9 values of compounds were calculated, using EZ-Fit and δ 106.8 and an oxygenated carbon appeared at δ 89.9. All above spectral data revealed that it was a Anam Sajid et al., J.Chem.Soc.Pak., Vol. 40, No. 01, 2018 253

tarxastane type triterpene having a lactone, a ketone dimethylergosta-8,14,24(28)-triene-3β,12β,17α-triol group and a terminal olefinic moiety. Further (5) [17], oleanolic acid (6) [18], erectasteroid D (7) confirmation of the structure was done by the [19], (S)-4',5-dihydroxy-7-methoxyflavanone (8) [20] inspection of HMQC and HMBC experiments. The and β-sitosterol-3-O-β-D-glucopyranoside (9) [21]. proton of C-18 position resonated at δ 2.22 (1H, J = 4.6 Hz) and the lower magnitude of coupling constant pointed towards the β configuration of this proton. It also confirmed the existence of equatorial and β configuration of neighboring proton at C-19 (δ 2.02), hence proved the axial and α configuration of the secondary methyl group attached to C-19 [12]. In HMBC correlations (Fig. 2) H-23 (δ 0.76) and H-24 (δ 1.03) showed J2 correlation with C-4 (δ 40.6), and J3 correlations with C-3 (δ 216.49) and C-5 (53.0) allowing to place the oxo moiety at C-3, which also supported by the biogenetic analogy. The carbonyl carbon of γ-lactone at δ 178.4 showed correlations with H-16 (δ 1.30, 2.14) and H-22 (δ 1.68, 1.58) while carbon bearing lactone oxygen at δ 89.9 correlated with H-15 (δ 1.87, 2.11), H-18 (δ 2.22), and H-27 (δ 0.99). This data indicated that the carbonyl and tertiary oxygen bond of a lactone were connected at C-17 and C-13 position, respectively, in the taraxastene type terpenoid skeleton. The olefinic Fig. 2: Important HMBC correlation in 1. protons δ 5.10. 4.66 (H-30) showed J2 correlation with C-20 (δ 151.9) and J3 correlations with C-19 (δ Conclusion 32.8) and C-21 (δ 30.6) establishing the presence of terminal double bond at C-20. On the basis of these From this research work it was concluded evidences and comparison with the spectral data of that Hypericum oblongifolium contain potent already reported compounds in the literature, [13] the lipoxygenase inhibitory constituents which can be compound was considered as a new taraxastene type taken up by the local herbal pharmaceutical industry triterpene and hence the structure of 1 was assigned for further drug designing. as 3-oxo-20(30)-taraxastene-28,13β-olide. The enzyme inhibitory activity of 1-9 against Acknowledgments lipoxygenase was determined using the method developed by Tappel [7]. The IC50 values were found We are grateful to International Center of between 31.0 µM and 83.0 µM against IC50 value of Chemical and Biological Sciences, Karachi 22.0 µM observed for Baicalein as a positive control University, Karachi for NMR and MS data (Table 1). acquisition. We also want to thank HEC (Higher Education Commission), Pakistan for financial Table-1: In vitro quantitative inhibition of support. The authors declare no conflict of interest. lipoxygenase enzyme by compound 1 - 9. a Compound IC50 ± SME ) [µM] References 1 48.7 ± 0.10 2 68.5 ± 0.10 3 61.5 ± 0.10 1. S. Ferheen, E. Ahmed, A. Malik, N. Afza, M. A. 4 83.0 ± 0.10 Lodhi, M. I. Choudhary. Hyperinol A and B, 5 71.0 ± 0.10 6 68.5 ± 0.10 Chymotrypsin Inhibiting triterpenes from 7 31.0 ± 0.10 Hypericum oblongifolium. Chem. & Pharm. 8 31.8 ± 0.10 9 39.3 ± 0.10 Bull, 54, 1088 (2006). Baicaleinb 22.0 ± 0.04 2. M. Ali, M. Arfan, H. Ahmad, K. Zaman, F. a) Standard mean error (SME) of three experimental determinations Khan, R. Amarovicz. Comparative antioxidant b) Positive control used in assays and antimicrobial activities phenolic compounds Depending upon the comparison of physical extracted from five Hypericum species. Food and spectral data of known compounds they were Tech. and Biotech, 49, 205 (2011). recognized as 4,4-dimethyl cholesterol (2) [14], 3. M. Ali, A. Latif, K. Zaman, M. Arfan, D. lupeol (3) [15], taraxerol (4) [16], 4,4- Maitand, H. Ahmad and M. Ahmad. Anti-ulcer Anam Sajid et al., J.Chem.Soc.Pak., Vol. 40, No. 01, 2018 254

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