Coumarin and steroid compound from stem bark of Kleinhovia ...

N.H. Soekamto et al. Proceeding of The International Seminar on Chemistry 2008 (pp. 231-234)ISBN 978-979-18962-0-7Jatinangor, 30-31 October 2008Coumarin and steroid compound from stem bark ofKleinhovia hospita LinnNunuk H. Soekamto 1 *, Alfian Noor 1 , Iwan Dini 2 , Rudiyansyah 3 , Marry Garson 41 Chemistry Department, Hasanuddin University2 Chemistry Department, Makassar State University3 Chemistry Department, Tanjungpura University4 School of Molecular and Microdial Science, Quensland University*e-mail: Noek_1512@yahoo.comAbstractA coumarin, 7-hidroxy-6-methoxy coumarin (1) together with β-sitosterol (2) had been isolated forthe first time from the chloroform extract of stem bark of Kleinhovia hospita L. (Paliasa). Thestructure of the compound not only were determined on the basis of spectroscopic evidence includingIR, 1 H NMR and 13 C NMR spectra but also compare with known compound.Keywords: Kleinhovia hospita L., Sterculiaceae, 6-hidroxy-7-methoxy coumarin, β-sitosterolIntroductionKleinhovia hospita Linn. is a species ofSterculiaceae. This species can be found in Indonesia,especially in eastern part of Indonesia i.e. in the islandof Sulawesi, Maluku, and Irian, (Heyne, 1987). Thisplant is known locally as Paliasa. It have been usedfor long time in the indigenous system of medicine ofIndonesia (especially South Sulawesi) for liver,hepatitis, cholesterol and hypertensive. It had beenreported that leave of Kleinhovia hospita can be usedfor eye wash and hair perfume. (Heyne, 1987). InPapua New Gunea and Solomon Island, cambium ofKleinhovia hospita can be used as pneumoniamedicine and to kill hairlouse. Leave of Kleinhoviahospita extract showed antitumor activities in mouseas animal test (Latif, 1997). Beside that, people useKleinhovia hospita plant as construction material.Only few informations on chemical constituentsof Kleinhovia hospita have been known, however, theprevious research identified that there were alkaloid,flavonoid and sianogenic compounds in Kleinhoviahospita which can kill off ectoparasite such as louse(Latif, A., 1997).This paper report the isolation of compounds of stembark Kleinhovia hospita i.e. 7-hidroxy-6-methoxycoumarin (1) together with β-sitosterol (2) andbiological test fractions and compounds againstArtemia salina.Material and MethodsGeneral Experimental Procedures. Melting pointswere determined on an uncalibrated micro–meltingpoint apparatus. UV and IR spectra were measuredwith Cary 100 Conc and Perkin Elmer FT–IRspectrophotometers, respectively. VLC was carriedout using Merck silica gel 60 GF 254 , CC with Mercksilica gel 60 (70 and 230–400 mesh) and TLC onprecoated silica gel plates (Merck Kieselgel 60 GF 254 ,0.25 mm).Plant Material. Samples of K. hospita were collectedin January 2005 from Tidung Village, RappociniDistrict, Makassar, South Sulawesi, Indonesia. Theplant was identified by the staff of the HerbariumBogoriense, Bogor Botanical Garden, Bogor,Indonesia and a voucher specimen had been depositedat the Herbarium.Extraction and Isolation. The dried powdered leave(4.6 kg) of K. hospita was macerated with MeOH, andthe MeOH extract (5.2 kg), was partitionedsequentially into n-hexane (7.6 g), CHCl 3 (13,1 g) andEtOAc-soluble (2.0 g) fractions. The three fractionswere separated by Vacum Liquid Chromatography(VLC) into several fractions. The bioactivity of thefractions was tested using brine shrimp (A. salina)(Meyer et. al., 1982) and the active fractions weresubjected to VLC and repurified using the samemethod yielding two pure compounds (1 and 2) fromCHCl 3 fraction. Each compound was tested for itsactivity to brine shrimp (Artemia salina).Compounds 1 and 2 from CHCl 3 fraction wereidentified. Result showed that compounds 1 was 7-hidroxy-6-methoxy coumarin and 2 was β-sitosterol(Fig. 1). Characteristic of the compounds aredescribed as follows:Compound 1 was isolated as white powder with m.p.of 182-184 o C. UV (MeOH) λ max (log ε): 228 (3.98),344 (4.40) nm; IR (KBr) υ max : 3338 (OH), 2924, and2853 (C-H aliphatic), 1701 (C=O), 1608, 1566, 1508,1464 (C=C aromatic), 1376 (CH 3 ), 1291 (C-O) cm -1 ;1 H NMR (Chloroform-D 6 , 500.2 MHz) δ: 6.27 (1H, d,231

N.H. Soekamto et al. Proceeding of The International Seminar on Chemistry 2008 (pp. 231-234)Jatinangor, 30-31 October 2008J = 9.2 Hz, H-3), 7.60 (1H, d, J = 9.2 Hz, H-4), 6.84(1H, s, H-5), 6.92 (1H, s, H-8), 3.95 (3H, s, (OCH 3) );13 C NMR (Chloroform-D 6 , 500.2 MHz) δ: 161.67 (C-2), 150.41 (C-4’), 149.84 (C-6), 144.16 (C-7), 143.51(C-4), 113.58 (C-3), 111.66 (C-8’), 107.63 (C-5),103.35 (C-8), 56.57 (-OCH 3 ).Compound 2 was isolated as needles white crystal,with m.p. of 126 o C. The compound did not givephosphorescent under the UV lamp, The IR spectrumof 2 exhibited absorptions aliphatic (2956, 2918, 2848cm -1 ), -CH 2 (1462 cm -1 ) and -CH 3 (1377cm -1 )groups. The data indicated that compound 2 belong toterpenoid derivative. The sugestion was suported bynegatif result in the steroid test. 1 H NMR (CDCl 3 ,500.2 MHz) δ: 1.15 ppm (2H, dt, J =11.0; 3.65 Hz, H-1 eq ), 1.08, 1.84, 1.54, 3.51, 2.28, 2.22, 3.51, 1.98,1.94, 1.49, 0.91, 1.48, 2.00, 0.98, 1.58, 1.82, 1.07,1.34, 0.89, 1.65 ppm respectively (1H, m, for H-1 ax ,H-2eq, H-2 ax , H-3, H-4 eq , H-4 ax , H-5, 7eq and ax, 8, 9,11, 12eq, H-14, 15 eq , 16, 17, 20, 22 eq , 24, and 25 eq ),5.34 ppm (3H, d, J = 4.90 Hz, H-6), 0.67 and 1.00ppm (3H, s, H-18 and 19), 0.91 ppm (3H, d, J=6.1Hz), 1.14 ppm (2H, m, H-23), 0.82 and 0.80 ppmrespectively (3H, d, J = 6.7 Hz, for H-26 and 27),1.23 ppm (2H, m, H-28), 0.83 ppm (3H, t; J = 6.7 Hz,H-29); 13 C NMR (CDCl 3 , 500.2 MHz) δ: 140.8 (C-3),121.8 (C-6), 71.9 (C-2), 56.8 (C-15), 56.1 (C-17),50.2 (C-9), 45.9 (C-24), 42.4 (2C C-4 dan 14), 39.8(C-12), 37.3 (C-1), 36.6 (C-20), 36.2 (C-10),34.0 (C-22), 32.0 (2C, C-7 dan 8) 31.7 (C-2), 29.2 (C-25),28.4 (C-16), 26.1 (C-23), 24.4 (C-15), 23.1 (C-28),21.2 (C-11), 19.9 (C-26), 19.5 (C-27), 19.1 (C-19),18.9 (C-21), 12.1 (C-18), 12.0 (C-29).HOH 3 COOOHOH 3 CCH 3CH 3(1) (2)Figure 1 Scopoletin (1) and β-sitosterol (2)Results and DiscussionA coumarine derivative compound which hadbeen isolated for the first time From CHCl 3 fractionwas 7-hidroxy-6-methoxy coumarin (1). Thiscompound was purified by coulumn chromatographyand recrystallization and their structure wereH 3 CCH 3CH 3determined by spectral methods and comparison withthose reported in the literature.7-hidroxy-6-methoxy coumarin (1) was a whitepowder compound with m.p. of 182-184 o C. The UVspectrum of 1 showed absorption maxima at 228(3.98), 344 (4.40) nm, indicating the presence of anextended benzene chromophore. The IR spectrum of 1exhibited absorptions for hydroxyl (3338 cm -1 ),aliphatic (2924, and 2853 cm -1 ) and aromatic (, 1608,1566, 1508, 1464 cm -1 ) groups., -C=O (1701 cm -1 ),CH 3 (1376 cm -1 ), C-O (1291 cm -1 ).UV and IR data indicated that compound 1 iscoumarine derivative (Murray, et. al., 1982). The 1 HNMR spectrum of 1 showed two vinilic proton signalsat δ 6.27 ppm (1H, d, J = 9.2, H-3) and 7.60 ppm (1H,d, J = 9.2, H-4). Beside that 1 H NMR spectrum toshow two aromatic proton signals at δ 6.84 and 6.92ppm each 1H with multiplicity singlet assignable totetrasubstituted benzena moiety. Another informationin the 1 H NMR spectrum was the signal at δ 3.95 ppm(3H, s) for methyl –OCH 3 .The 13 C NMR spectrum of 1, showed that thereare ten signals including C carbonyl (C-2) at δ 161.67ppm, three oxycarbons at δ 144.16, 149.84 and 150.41ppm for C-6, C-7, and C-8a. And then other carbonatoms i.e. quarterner carbon C-4a, 6, 7, and 8a at δ111.66, 144.16, 149.84, and 150. 41 respectivelyassigned with the aid of HMBC measurements. It canbe proposed from these data that the structure of 1 is7-hidroxy-6-methoxy coumarin or scopoletin (Fig.2). The HMBC spectrum of 1, in particular, disclosedlong-range correlations between four the methynesignals at δ 6.27 ppm (H-3) with carbon signals at δ161.66 ppm (C-2), δ 7.60 ppm (H-4) with carbonsignals at δ 161.66 (C-2), 150.41 ppm (C-8a), δ 6.84ppm (H-5) with carbon signals at δ 149.84 (C-7),150.41 ppm (C-8a), δ 6.92 ppm (H-8) with carbonsignals at δ 144.16 (C-6), 149.84 (C-7), and δ 3.95ppm (H-9) with carbon signals at δ 144.16 (C-6) (Fig.3). Further support for structure 1 came fromcomparison of the NMR data with that of relatedcompound (Soekamto et.al., 2001), isolated fromMorus species (Table 1).Bioassay analysis to the compound 1 showmoderate activity against Artemia salina with LC 50 =569,64 µg/mL. Scopoletin was also isolated fromMorinda citrifolia L. and it can be used asantihypertensive, antiinflamatory and antiallergenic(Rosa et.al., 2003), and can also be used as inhibitorprostaglandin synthetase (MH Farah and Sauelsson G,1992).232

N.H. Soekamto et al. Proceeding of The International Seminar on Chemistry 2008 (pp. 231-234)Jatinangor, 30-31 October 2008H 3 CO3.95 (s)9HO676.84 (s)586.92 (s)4a8a7.60 (d)J = 9.5 Hz4O326.27 (d)J = 9.5 HzOH 3 COHO56.57149.84107.6 143.51144.16 111.66150.41103.35O113.58161.66O(A)(B)Figure 2 Proton signals (A) and carbon signals (B) of scopoletinHHH 3 COHHOOOHFigure 3 HMBC correlation of scopoletinTable 1 1 H-NMR and 13 C-NMR data of compound (1) in CDCl 3 and umbeliferon in DMSO (Soekamto, et. al.,2001)No δ C (1) δ C (umbelliferon) δ H (1) δ H (umbelliferon)C1 - - - -2 161.66 160.4 - -3 113.58 111.4 6.27(1H, d, J=9.5)6.27 (1H, d, J=9.5)4 143.51 144.5 7.60 (1H, d, J=9.5) 7.92 (1H, d, J=9.5)4a 111.66 111.2 - -5 107.6 129.7 6.84 (1H, s) 7.51 (1H, d, J=8.5)6 144.16 113.1 - 6.77 (1H, dd, J=2.2& 8.5)7 149.84 161.2 - -8 103.35 102.1 6.92 (1H, s) 6.70 (1H, d, J=2.2)8a 150.41 155.4 - -9 56.57 - 3.95 (3H, s)ConclusionsIn the present investigation 2 compounds,scopoletin and β-sitosterol had been isolated for thefirst time from the chloroform extract of stem bark ofKleinhovia hospita L. (Paliasa).Scopoletin showed moderate activity to Artemiasalina with LC 50 of 569,64 µg/mL. Scopoletin wascan also be used as antihypertensive, antiinflamatory,antiallergenic, and inhibitor prostaglandin synthetase.AcknowledgementWe thank LIPI Serpong Indonesia for measurementNMR data. We also thank the Herbarium Bogoriense,Bogor, Indonesia, for identification of the plantspeciment.233

N.H. Soekamto et al. Proceeding of The International Seminar on Chemistry 2008 (pp. 231-234)Jatinangor, 30-31 October 2008ReferenceHeyne, K., (1987), “Tumbuhan Berguna IndonesiaII”, Badan Litbang Kehutanan, Jakarta, hal.659.Latif. A (1997), Kleinhovia hospita Linn., In: FaridaHanum & L.J.G. Van Der Maesen. PlantResources in Southeast Asia, No. 11, TheNetherlandsMeyer, N., N.R. Ferrigini, J.E. Putnam, D.E.Jacobsen, D.E. Nichols, andJ.L.McLaughlin (1982), Brine shrimp: A convenientgeneral bioassay for active plant constituens,Planta Med. 45, 31.MH. Farah and G. Samuelsson, 1992,Pharmacologicallly active phenylpropanoidsfrom Senra incana, (online), diakses 30 April2005, 11.30 AM.Murray, R.D.H., Mendez, J., and Brown, S.A.,(1982), “The Natural Coumarins: Occurrence,Chemistry and Biochemistry”, John Willey &Sons Ltd.Rosa, Resa dan Wiwit, 2003, Noni dan Royal Jelly,(online),(http://www.pikiranrakyat.com/cetak/1003/18/hikmah/lainnya03htm,diakses 30 April 2005, 11.00 AM).Soekamto, N.H., S.A. Achmad, E.H. Hakim, Y.M.Syah, N. Aimi, M. Kitajima, dan H. Takayama(2001), Umbeliferon senyawa antifungal daritumbuhan Andalas Morus macroura, Bull. Soc.Nat. Prod. Chem. (Indonesia), 1, 13-19.234