Research Journal of Chemical Sciences ______________________________________________ ISSN 2231-606X Vol. 4(9), 7-11, September (2014) Res. J. Chem. Sci. International Science Congress Association 7 Phytochemical investigation Affordedanovel Cycloartanetriterpenoid from PiperthomsoniGoswami Rajeev and Jain S.C.Department of Chemistry, University of Delhi, Delhi-110 007, INDIAAvailable online at: www.isca.in, www.isca.me Received 3rd August 2014, revised 30th August 2014, accepted 10th September 2014Abstract A Novel alkenyl phenol, 4-(7E-dodecenyl)phenol () and triterpenoid cycloart-23-en-3-one () and also first time from Piper genus 3-22-dihydroxylanosta-8,24-dien-26-oicacid--lactone () were isolated from the leaves and stems DCM:MeOH (1:1) extracts of Piper thomsoni. All isolated compounds structures were definedusing modern spectral techniques. Keywords: Piper thomsoni; Piperaceae; cycloart-23-en-3-one; 4-(7E-dodecenyl)phenol; anticancer.Introduction Since ancient time, plants have been the source of medicines and the plants belong to the genus Piper are known for their medicinal importance1,2. Piper species are geographically disseminated in the tropical and subtropical climatic zones and are explored as folk medicines in several ways. P. nigrumripened fruit is used as white pepper, where as black pepper is sourced from its unripe fruit. Isobutyl amides isolated from P. nigrum fruits showed larvicidal effecton Aedesaegypti, A. togoi and Culexpipienspallens larvae. P. amalagois used as anti-inflammatory agent for alleviating chest pain. P. aborescens stems chloroform extract exhibited good activity against P-388 lymphocytic leukemia cells and KB cell. P. cubeba has long been a source of folk and herbal medicine. The P. longum fruit has also been source of Indigenous medicines, which includes Indian Ayurvedic medicine, to treat bronchitis, diarrhea, malaria, viral hepatitis and tumors. An amide, separated from the fruit of P. longum L, displayed inhibitory activity against the fourth-instar larvae of Aedesaegypti. Alkaloids isolated from CHOH extract of P. lolot exhibited good inhibition of thrombocyte clustering caused by polyunsaturated -6 fatty acid (arachidonic acid) and PAF-acether. Rats treated with pipermethystine, abundant in Piper methysticum, showed increased cytosolic superoxide dismutase, cachectin mRNA expression, hepatic glutathione, CYP 1A2 and 2E1 which suggested adaptive feature to induce oxidative stress and likely drug-drug interactions10. Piper thomsoni is one of the forty-five species of the family Piperaceae and is being used as traditional and folk medicines. It is well documented in the Indian Ayurvedic system of medicine. P. thomsoni leaves are the source of Pan, which also applied to wounds and swellings; whereas its aqueous root extracts is used as diuretic. Previously P. thomsoniphyto chemical explorations has resulted in the isolation and characterization of several alkaloids and terpenoids11,12. Here, we describe the isolation and characterization of bioactive secondary metabolites from the leaves and stems combined extract of this species, which afforded two new compounds an alkenyl phenol and one cycloartaneterpenoid, additionally seven earlier reported compounds (figure-1). However, out of seven known compounds, lanostanetriterpenoid () is first time reported from the Piper genus. Herein, extraction, compounds isolation and their detailed characterization are discussed. Material and MethodsSolvents used for column chromatography (CC) were procured from Merck and used after distilling them. 60-120 mesh silica-gel was used for CC from Merck. Melting points were checked using Fischer Johns equipment and are uncorrected. The H, 13C NMR and 2D NMR spectra were recorded withte tramethylsilane as an internal standard on a Bruker-300 Spectrometer in deuterated solvents as required. Perkin-Elmer Infra-red Spectrometer was used to record IR spectra either as KBr pellets or film. The ESI mass spectra were determined using Jeol Spectrophotometer and elemental analysis was done on GmbH Vario EL V3.00 elemental analyzer. Plant Material: Piper thomsonileaves and stems (650g) were collated from the Botanical Survey of India, Shillong forests and identified by Dr. B.M. Wadhwa.Extraction and isolation: Leaves and stems were dried and their powdered mixture (500 g) was extracted with cold CHCl:MeOH with a Soxhlet apparatus. Solvent was evaporated from the extract under vacuum and thus obtained residue (8.0 g) was purified by CC using silica-gel. Elutions were made exploiting a linear gradient of hexane-EtOAc-MeOH and totally 101 fractions (400 ml each) were eluted and as per their TLC pattern combined to make 14 fractions (F1-F14). F1 was composed of waxy material. Compound 2 (15 mg; colourless oil) and 4(25 mg; white amorphous powder) were obtained from fractions F2 and F4, respectively. F8 gave Research Journal of Chemical Sciences ___________________________________________________________ ISSN 2231-606XVol. 4(9), 7-11, September (2014) Res. J. Chem. Sci. International Science Congress Association 8 compound 8as a white amorphous powder (15 mg). Additionally, six other known compounds viz., dotriacontanol (1), octadec-10Z-enoic acid (3), octacosanoic acid (5), stigmast-6-en-3-ol (6), (2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)-1-(piperidin-1-yl)penta-2,4-dien-1-one (7) and 7-methyl-5H-[1,3] dioxolo [4',5':4,5] benzo [1,2,3-de] benzo [g] quinoline-5, 6(7H)- dione (9) were also isolated from other fractions.)-4-(dodec-7-en-1-yl)phenol (2): Colourless oil; [ –24.1 (c 0.40, CHCl); UV(MeOH) max: 274 nm; IR max (film) cm-1: 3550, 2919, 2850, 2359, 1638, 1462, 1377, 1265, 1028, 930, 708; H NMR (CDCl): ppm 0.88 (t, 3H, J = 6.2 Hz, H-12), 1.28 (brs, 6H, 3x-CH-), 1.63 (m, 6H, H-2, H-5& H-10), 1.98 (m, 4H, H-6 and H-9), 2.53 (t, 2H, J = 7.6 Hz, H-1), 5.1 (brs, 1H, -OH), 5.42 (m, 2H, H-7 and H-8), 6.74 (d, 2H, J = 8.5 Hz, H-2 and H-6), 7.03 (d, 2H, J = 8.5 Hz, H-3 and H-5); 13C NMR (CDCl): ppm 14.6 (C-12), 22.5-31.5 (C-2 to C-5, C-10 and C-11), 34.6 (C-6and C-9), 34.9 (C-1), 130.1 (C-2 and C-6), 131.3 and 131.4 (C- and C-8), 132.9 (C-3 and C-5), 138.1 (C-4), 153.5 (C-1); EIMS (m/z): (M, 260), 242 (M-HO), 232, 203 (M-C), 177 (M-C11), 167, 149, 135, 120, 106, 95, 85, 83, 74, 57, 43. Cycloart-23-en-3-one (4): White amorphous powder; m.p. 118-120C; [ + 38.1 (c 0.41, CHCl); Elemental analysis: C 84.83%, H 11.52%, O 3.65% (required C 84.87%, H 11.49%, O 3.65%); IR max (KBr) cm-1: 2927, 1712, 1451, 1377, 1113, 757; H and 13C NMR (CDCl): detected via HMBC correlations, see table 1; EIMS (m/z): 438(M), 423 (M-CH), 410 (M-CO), 395 (M-CH-CO), 381, 329, 313 (M-C17), 298 (M-C17CH), 286, 270 (M-C17-CH-CO), 243, 255, 231, 213, 138, 121, 107, 95, 81, 43. bb-22-Dihydroxylanosta-8,24-diene-26-oicacid-dd-lactone (8):White amorphous powder; m.p. 286-288C; [ + 27.3 (c 0.32, CHCl); UV (MeOH) max: 203, 252 nm; IR max (KBr) cm-1: 3531, 2930, 2862, 1709, 1457, 1373, 1261, 1141, 1091, 1036, 855, 803; Elemental analysis: C 79.22%, H 10.23%, O 10.55% (required C 79.25%, H 10.20%, O 10.56%); H and 13C NMR (CDCl): detected via HMBC correlations, see table 1; EIMS (m/z): 454 [M], 439 (M-CH), 421 (M-CH-HO), 315, 311, 299, 281, 253, 241, 227, 215, 201, 139 (cleavage of C17-C20bond), 127, 111 (139-CO), 95, 74.Results and Discussion The leaves and stems DCM:MeOH (1:1) extract of P. thomsoni was subjected to column chromatography on silica-gel and eluted with different polarity solvent combinations, yielded two new compounds, which include an alkenyl phenol () and acycloartaneterpenoid (), and seven known compounds (1,3, 5). Figure-1 Structures of compounds 1–9 Research Journal of Chemical Sciences ___________________________________________________________ ISSN 2231-606XVol. 4(9), 7-11, September (2014) Res. J. Chem. Sci. International Science Congress Association 9 Table-1H and 13C NMR and HMBC spectral data for compounds 4 and 8 HMBC 1 HMBC 1 H 33.2 5 191.52 (m), 1.81 (m)34.4, , 191.71 (m), 1.26 (m) 37.52.68 (dt, J = 14.0, 6.2 Hz), 2.35 (ddd, J = 13.9, 4.3, 2.3 Hz)30.7, 1.50 (m), 1.68 (m) 216.5- -79.3, , , 29, 303.23 (dd, J = 11.3, 4.5 Hz) 50.6- -37.8 49.1 6 7 191.66 (m)49.2, , 19, 29, 301.64 (m) 22.3 7 81.01 (m)18.0, 1.97 (m), 1.32 (m) 22.5 6 81.25 (m)25.3, 2.15 (m), 1.35 (m) 43.1 7 11 15 19 311.58 (m)134.9- - 36.7- -134.4- - 1036.0- -35.9- - 1134.4 12 191.39 (m)19.9122.26 (m), 1.53 (m) 1227.111 17 181.34 (m)29.711, 181.47 (m) 1345.7- -43.4- - 1446.9- -48.8- - 1533.816 17 311.61 (m)29.816, 171.58 (m) 1634.215 17 202.02 (m)26.615, 17, 202.04 (m) 1750.615 1618 20 21221.34 (m)44.71215, 16, 18, 20, 21, 221.66 (m) 1818.412 170.85 (s)14.312, 170.70 (s) 1929.9 5 110.78 (d, J = 4.2 Hz), 0.58 (d, J = 4.2 Hz)17.1, 0.91 (s) 2048.216 17 21 221.46 (m)39.316, 17, 21, 22, 231.88 (m) 2118.617 20 220.93 (d, J = 6.7 Hz)12.217, 20, 221.04 (d, J = 6.6 Hz) 2231.617 20 21 252.20 (m)80.717, 20, 21, 244.47 (m) 23157.228.620, 22, 242.02 (m), 2.56 (m) 24106.322 254.67 (d, J = 15.4 Hz)139.922, 23, 276.59 (dd, J = 6.4, 3.2 Hz) 2530.122 24 26 27 281.95 (m)128.6- - 2637.825 27 281.42 (m)166.9- - 2726.225 26 281.10 (d, J = 5.8 Hz)16.0241.91 (s) 2826.225 26 271.10 (d, J = 5.8 Hz)14.5150.98 (s) 2921.1 300.98 (s)23.3, , 300.81 (s) 3022.2 291.04 (s)26.8, , 291.00 (s) 3119.6 150.95 (s) Compound , obtained as oil, gave a molecular ion peak [M] at m/z 260 in its EIMS and coupling this information with C and H count in its NMR suggested it to have molecular formula 1828O. In its 13C NMR spectrum,the appearance of resonances for eight sp hybridized carbons suggested one aromatic ring and one carbon-carbon double bond. Only twenty-seven hydrogens could be characterized through the H and DEPT spectra, which indicatedthat left out one hydrogen could be in the form of a hydroxyl group. The initial assignments of two partial skeletons weredone on the bases of H NMR and H-H COSY spectral data. Two aromatic resonances at 6.74 (d, 2H, J = 8.5 Hz) and 7.03 (d, 2H, J = 8.5 Hz) suggested that a distinctive 1,4-substitution pattern is present in the aromatic ring. The one substituent in the aromatic ring was in the form of a phenolic hydroxyl group as was indicated by its H NMR which displayed an exchangeable signal with DO at 5.1 and also supported by its 13C NMR showed a peak at 153.5, characteristic for a carbon bearing hydroxyl group in the benzene ring. H NMR when coupled with its mass spectrum indicated that second substituent to be an alkenyl chain attached to the 4th position of the aromatic ring. The two proton multiplet at 5.42 suggested a disubstitutedtrans double bond in the side chain which was confirmed by its 13C NMR spectrum in which sp carbons appeared at 131.3 and 131.4 and also by its IR spectrum as it exhibited a band at 930 cm-1. Double bond position in the chain was assigned by mass fragmentation, in which two allylic cleavages were observed as a result of a peak at m/z 203 after the loss of C9 group and at m/z 177 after the Research Journal of Chemical Sciences ___________________________________________________________ ISSN 2231-606XVol. 4(9), 7-11, September (2014) Res. J. Chem. Sci. International Science Congress Association 10 loss of C11. This suggested that double bond placed at C-7 in the alkenyl chain. The peak at m/z 167 confirmed twelve carbon atoms in the side chain. Hence compound was characterized as (E)-4-(dodec-7-en-1-yl)phenol. Compound , obtained as a white amorphous solid, was assigned the chemical formula using elemental analysis as 3150O, which was supported by its molecular ion peak m/z438 in its EIMS. It passes Liebermann Burchard test and yellow colour with tetranitromethane, characteristic for a tetracyclic triterpenoid. ItsH NMR showed two doublets at 0.58 ( = 4.2 Hz) and 0.78 ( = 4.2 Hz) and characterized for C-9& C-10 cyclopropyl methylene group of a cycloart-3-one triterpenoid13. The HMBC correlation studies confirmed that the protons at 0.58 and 0.78 were cyclopropyl methylene protons and attached to carbons at 36.7 () and 36.0 () and the carbon at 36.7 was also attached with the carbons at 33.2 (-CH) and 37.5 (CH). Absorption band in its IR spectrum displayed at 1710 cm-1 suggested the presence of a carbonyl group, which was confirmed by its 13C NMR spectrum which displayed a peak at 216.5. The HMBC correlation of –C=O group with carbons at 33.2 (-CH), 37.5 (-CH), 50.6 () and 49.1 (�CH-) confirmed the presence of carbonyl group at C-3, which was also akin to the basic unit of cycloart-3-one13,14. The mass fragmentation displayed a prominent peak at m/z 313 (M+-C17) due to the loss of 125 amu thereby showing one site of unsaturation in the side chain. Further its 13C NMR and DEPT spectrum showed two sp carbons at 157.2 (q) and 106.3 (-CH) andindicated the presence of an exocyclic methylene group which was confirmed by its H NMR spectrum as a characteristic doublet appeared at 4.67 ( = 15.4 Hz). This exocyclic double bondwas fixed at C-23 because this was the only best suited position available in the side chain which was also supported by its McLafferty-type fragmentation15(figure-2), in the absence of other olefinic proton and the vinylic methyl in the molecule. Figure-2 McLafferty-type rearrangement of the side chain of compound 4 The H NMR spectral pattern as well as the mass fragmentation of was compared to those reported for cycloart-25-en-3-one isolated from Polypodium formosanumfern16. The HMBC spectrum correlations (figure-2) were deduced to cycloart-23-en-3-one, the constitution assigned for compound . HMBC spectra finally assisted in the assignment of the protonated carbons (table-1). On the basis of spectral discussion, compound was defined as (2a, 3, 5a, 5b, 7a, 11a, 12a)-2a, 5a, 8, 8-tetramethyl-3- (()-6-methyl-4-methyleneheptan-2-yl)tetradecahydro-9,12-cyclopenta [] cyclopropa [] phenanthren-9-one (which shortly termed as cycloart-23-en-3-one). Figure-2 HMBC (HC) correlation of compounds 4 and 8 Compound was purified as a white amorphous solid and its chemical formula was confirmed as C3046 by its elemental analysis and EIMS. Its DEPT and H-13C COSY spectrum showed signals for seven methyl, nine methylene and five methine groups. The H NMR spectrum displayed among other resonances, the two low field protons attached to carbons bearing oxygen at 3.23 (dd, = 5.1 Hz and 11.3 Hz) and at 4.47 (m). The presence of 3-hydroxy was confirmed by a peak at 79.3 for C-3 in 13C NMR and also by a characteristic double doublet at 3.23 for 3-hydrogen. The IR absorption at 1707 cm-1 and UV absorption at 252 nm indicated the presence of a six membered , - unsaturated -lactone ring which was supported by its 13C NMR spectrum which displayed characteristic resonances at 167.13, 140.29, 127.96 and 80.51. The HMBC correlation (figure 2 and table-1) with respect to –CH group at 1.91 (s) showed correlation at 140.29 (), 167.13 () and 127.96 (=CH-) carbons suggesting it attached to lactone ring at C-25 position. HMBC correlation suggested that –CH-O- group at 4.47 (m) correlations with 167.13 (�C=O), 127.96 (=CH-), 39.3 (�CH-), 28.6 (�CH), 12.2 (-CH) carbons clearly suggested that it is C-22 carbon from lactone ring and confirmed the presence of six-membered -unsaturated lactone ring and is attached at 39.3 (C-20) carbon with lanostane unit. The 13C NMR spectra also revealed tetrasubstituted olefinic carbons at 134.1 and 134.4 positioned between C-8 and C-9. By comparison of these spectral features with steroids containing -unsaturated -lactone indicated that has lanostane skeleton which was further confirmed by the HMBC spectrum (figure-2) and its Mass fragmentation pattern. Also -lactone carbon resonances of was identified with those reported for steroids containing -lactones17,18. Based on that we have assigned the streochemistry as 22. On the basis of above spectral analysis and also comparing with reported data19, Research Journal of Chemical Sciences ___________________________________________________________ ISSN 2231-606XVol. 4(9), 7-11, September (2014) Res. J. Chem. Sci. International Science Congress Association 11 compound was assigned as 3-22-dihydroxylanosta-8,24-dien-26-oicacid--lactone. Conclusion We have performed a phytochemical exploration of Piper thomsoni and isolated two novel secondary metabolites of (E)-4-(dodec-7-en-1-yl) phenol () and cycloart-23-en-3-one (). However, 3-22-dihydroxylanosta-8, 24-dien-26-oicacid-lactone () was first time reported from the Piper genus. AcknowledgementWe thank Danish International Development Agency (DANIDA), Denmark for the financial assistance. 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