Eliza et al. ISBN 978-979-18962-0-7 Proceeding of The International Seminar on Chemistry 2008 (pp. 265-268) Jatinangor, 30-31 October 2008 Three flavanoid derivates from leaves of Artocarpus Lanceifolius Eliza, Yana M. Syah, Lia D. Juliawaty, Sjamsul A. Achmad, Lukman Makmur, Euis H. Hakim* Natural Product Chemistry Research Group, Department of Chemistry, Institut Teknologi Bandung, Jalan Ganesha 10, Bandung 40132, Indonesia *e-mail: [email protected], Fax: 62-22-2504154 Abstract The roots and stems, but not the leaves, of Artocarpus lanceifolius (Moraceae) have been reported to produce a number of prenylated flavonoids. As part of chemical investigation on this species, we have examined the chemical constituents of the leaves of this plant which led to the isolation and identification of three flavanoid derivatives, namely 7,4’-di-O-methylnaringenin (1), 8-prenyl-4’-Omethyl-naringenin (2) and 3’-geranylnaringenin (3). In this report, the activities of the isolated compound against P-388 leukemia murine cells will also be described. Keywords: Moraceae, A. lanceifolius, flavanones OMe Introduction 4' MeO Artocarpus comprises 45 species and distributed from Sri Lanka to South China and through to Solomon Islands and Australia. Artocarpus species are elements of forest in ever wet climates or where there is a short dry seasons, mostly below 1000 m. Some species can be found at altitudes up to 1500 or to 1800 m. This genus is known as a source of edible fruits, such as A. heterophyllus (Jackfruit), A. integer (Chempedak), A. rigidus (Monkeyjack) (Verheij & Coronel 1991). The lightweight hardwood of several Artocarpus species is attractive to be used for several purposes, such as for construction, veneer and various utensils (Djarwaningsih, 1995). Several Artocarpus species also produce exudates in the form of latex (Boer and Ella 2000) to be used as birdlime or as medicines or stimulants (Van der Vossen & Wessel 2000). A. lanceifolius Roxb. is a rare species endemic to the province of West Sumatera, Indonesia, and locally known as ‘keledang’. Some prenylated flavonoids with interesting biological activities have been isolated from the roots and stems of this plant (Hakim, 2006), but no work has been done to the leaves part of this plant. This paper reports the isolation and structural elucidation of three flavanoids derivatives from the leaves of A. lanceifolius characterized as 7,4’-di-O-methylnaringenin (1), 8prenyl-4’-O-methyl-naringenin (2) and 3’geranylnaringenin (3). The structure of these compounds was elucidated based on spectroscopic data and by comparison with literature data. The occurrence of these compounds in Moraceae family is for the first time. O 7 2 2' 3 5 OH O 1 OMe 4' HO O 7 2 2' 3 5 OH1 O 2 OH 4' HO O 7 1" 2 3 2' 5" 2" 3" 5 OH O 4" 6" 10 " 7" 8" 3 9" Material and Methods General Melting points were determined on a micromelting point apparatus and are uncorrected. UV and IR spectra were measured with a Varian Conc.100 instrument and Perkin Elmer Spectrum One FTIR spectrometer, respectively. 1H and 13C NMR spectra were recorded with Jeol ECP500 operating at 500 265 Eliza et al. Proceeding of The International Seminar on Chemistry 2008 (pp. 265-268) Jatinangor, 30-31 October 2008 (1H) and 125 (13C) MHz, using residual and deuterated solvent peaks as reference standards. VLC (vacuum liquid chromatography) used Merck silica gel 60G, radial chromatography was carried out by using Merck silica gel PF254 and for TLC analysis, precoated silica gel plates (Merck Kieselgel 60 GF254, 0.25 mm thickness) were used, while other column chromatography used sephadex LH-20. 2856, 1631, 1506, 1440, 1373. 13C-NMR (d6-acetone3, 125 Hz) see Table.1. Results and Discussion Methanol extract of leaves of A lanceifolius Roxb was partitioned by VLC using increasing polarity of eluent (N-hexane: EtOAc) and subsequented by radial chromatography repeatly to obtain compound 1-3. Compounds 1-3 were identified as 7,4’-di-Omethylnaringenin (1), 8-prenyl-4’-O-methylnaringenin (2) and 3’-geranylnaringenin (3), respectively. Compound 1, assigned as 7,4’-di-Omethylnaringenin, was obtained as white needles crystals. The IR spectrum showed strong absorptions at 1631 cm-1 (chelated C=O group) and 3300 cm-1 (OH). The characteristic UV absorptions bands (MeOH) at 215 and 287 nm suggested a flavanone skeletone, which was corroborated by the observation of 1H-NMR signals (CDCl3, 500 Hz) characteristic for this flavonoid type at δH 5.34 (1H, dd, J = 3.1, 13.1 Hz, C-2), 3.10 (1H, dd, J = 13.1, 17.7 Hz, C-3a), 2.73 (1H, dd, J = 3.1 & 17.7 Hz, C-3b) attributed to the ring C protons. The 13C-NMR spectrum (APT) of 1 exhibited 15 resonances for 17 carbon atoms distributed into 13 C-sp2, including a conjugated carbonyl and four oxyaryl carbon atoms, and 4 C-sp2, two of which were assignable to two methoxyl carbon atoms. Thus, this compound was assumed to be a dimethyl derivative of 5,7,4’-trihydroxyflavanone. Consistent with this assumption, the aromatic proton signals exhibited a pair of meta-coupled signals at δH 6.02 (1H, d, J = 2.5 Hz), 6.04 (1H, d, J = 2.5 Hz) for H-6 and H-8, and a pair of ortho-coupled signals of 1,4-disubstituted benzene moiety at δH 7.37 (2H, d, J = 7.6 Hz) and 6.35 (2H, d, J = 7.6 Hz). Furthermore, in the 1H-NMR two methoxyl proton signals also appreared at δH 3.79 and 3.82 and their positions were suggested at C-7 and C-4’ due to the presence of the phenolic –OH at δH 12.04. From these spectroscopic data the structure of 7,4’-di-O-methylnaringenin was assigned for compound 1. Plant Material Leaves of A. lanceifolius were collected in March 2007 from Bukit Tambun Tulang Padang Pariaman District Indonesia. The plant was identified at the Herbarium Anda, Biology Department, Andalas University Padang and Herbarium Bogoriense, Bogor Botanical Garden, Bogor, Indonesia, and a voucher specimen was deposited at both herbariums. Extraction and Isolation The powdered leaves of A. lanceifolius (2.3 kg) were extracted with MeOH (3 x 11 L) at room temperature for each 24 hours. The combined MeOH extracts were filtered and evaporated in vacuo to give residue 170 g. A portion of this extract (40 g) was fractioned by VLC using increasing polarity of eluents (n-hexane:EtOAc) to give seven major fractions (A-G). Recrystalization of fraction C (158 mg) and D (81 mg) with acetone yielded 1 (143 mg), while with the same methods from fraction E (853 mg) gave 2 (26 mg). Then, fraction F were subjected to a sephadex LH-20 column (MeOH) repeatly and then purified with radial chromatography to afford 3. 7,4’-Di-O-methylnaringenin (1): white needles crystals, mp. 119-120oC and [λ]D25 = -11.19 (CHCl3, 0.042), UV (MeOH); λmax (nm) 215, 287 nm, UV (MeOH+NaOH) λmax (nm); 203, 287, UV (MeOH+AlCl3) λmax (nm); 378, 310, 225, 202, UV (MeOH+AlCl3+HCl) λmax (nm); 378, 309, 224, 202, UV (MeOH+NaOAc) λmax (nm); 287, 213, 204. The IR (KBr) υmax (cm-1): 3444, 3004, 2912, 2842, 1631, 1577, 1519, 1461, 1434. 13C-NMR (CDCl3, 125 Hz) see Table 1. Compound 2 was obtained as white needles crystals and identificated as 8-prenyl-4’-O-methylnaringenin. The IR and UV spectra of spectrum of 2 were essentially similar to those 1. The 1H-NMR spectrum (CDCl3, 500 Hz) showed proton signals at δH 5.35, (1H, dd, J=3.1, 12.8 Hz, C-2), 3.05 (1H, dd, J=12.8&17.1 Hz, C-3a) and 2.80 (1H, dd, J = 17.1& 3.1 Hz, C-3b) that comfirmed to the flavanone Cring. The two signals of four aromatic protons at δH 7.37 (2H, d, J = 8.6, C-2’/6’) and 6.94 (2H, d J = 8.6 Hz, C-3’/5’) attributed monosubtituted phenyl of Bring and one signal at δH 6.02 (1H, s, C-6) indicated four substituted of phenyl A-ring. Moreover, the 1HNMR data indicated the presence of two methyl groups at δH 1.69 and 1.71(each 3H, s) and one 8-Prenyl-4’-O-methylnaringenin (2): white needles crystals, mp. 165-167oC, [λ]D25 = -13.28 (CHCl3, 0.007), UV (MeOH) λmax (nm); 225, 287; UV (MeOH+NaOH) λmax (nm) 287; UV (MeOH+AlCl3) λmax (nm) 309, 224 and UV (MeOH+NaOAc) λmax (nm) 202, 288. IR (KBr) υmax (cm-1) 3250, 2991-2854, 1635, 1600, 1517, 1438, 1355. 13C-NMR (CDCl3, 125 Hz) see Table.1. 3’-Geranylnaringenin (3) yellow gum, UV (MeOH) λmax (nm) 217, 287; UV (MeOH+NaOH) λmax (nm) 325, 322, 246, 217; UV (MeOH+AlCl3) λmax (nm) 216, 287, UV (MeOH+ NaOAc) λmax (nm) 322, 287, 216. IR (KBr) υmax (cm-1) 3427, 3035, 2970, 2922, 266 Eliza et al. Proceeding of The International Seminar on Chemistry 2008 (pp. 265-268) Jatinangor, 30-31 October 2008 previous method (Alley,1988). Two of them, 2 and 3, exhibited cytotoxic activity with IC50 3.4 and 3.2 µg/mL, respectively, while 1 showed was not active (IC50 41.5 µg/ml). olefinic proton at δH 5.19 (1H, t, J=7.3 Hz) and 3.28 (2H, d, J= 6.8 Hz), attributed to a 2,2-dimethylalyl (or prenyl) group. Since the 1H-NMR spectrum compound 2 showed aromatic signals consistent with a monosubstituted phenyl group of the ring B, the prenyl group must be located at ring A. In addition, 1 H-NMR spectrum also showed the signal a methoxy proton at δH 3.79 (3H, s). The 13C-NMR data of 2 showed 21 carbon atoms that were classified as two methyl carbons (C-4” and C-5”), two methylenes carbon (C-3 and C-1”), seven methines carbons (C-2, C-6, C-2’, C-3’, C-5’, C-6’ and C-2”), three quartener carbons (C-4a, C-1’ and C-3”) and four hydroxylated carbons (C-5, C-7, C-8a and C-4’) and one carbonyl carbon (C-4) from APT experiment and HMQC spectral analysis. From the 1H-NMR and 13C-NMR data compound 2 must be a flavanone substituted with one prenyl and one methoxyl groups. The location of prenyl and methoxy was confirmed by the HMBC experiments. Long-range correlations were observed between the following proton and carbons: H-1” was correlated with C-7, C-2”, C-3” confirming that a prenyl unit was located at C-8 and correlation between the methoxyl proton with C-4’ indicated that this group should be attached at C-4’. From these spectroscopic data l was assigned as 8-prenyl-4’methoxynarigenin. Compound 3 was obtained as a yellow gum designated as 3’-geranylnaringenin. The IR and UV spectra of 3 also very similar with those compounds 1 and 2, and together with proton signals (d6-acetone, 500Hz) at δH 5.36 (1H, dd, J = 13.1, 3.1 Hz), 3.13 (1H, dd, J = 17.1, 3.1 Hz), and 2.67 (1H, dd, J = 17.1, 3.1 Hz), suggested a flavanone structure. The presence of a geranyl group was obvious from the signals of three methyl singlets at δH 1.53, 1.59 and 1.69 and two olefinic protons at δH 5.08 (1H, t, J = 7.3 Hz) and 5.35 (1H, t, J = 7.9 Hz) and three methylenes proton at δH 3.34 (2H, d, J = 6.7 Hz), 2.07 (2H, m), 2.01(2H, m) in the 1H-NMR spectrum. The coupling ABX patterns of the B-ring protons, δH 7.26 (d, J= 2.5), 6.86 (d, J=8.0), 7.17 (dd, J=2.5 & 8.0)), and HMBC correlation data showed that there were longrange correlations between H-1” with C-4’, C-2’ establishing that the geranyl is located at C-3’. The proton chemical shifts of H-6 and H-8 in acetone-d6 solution gave the same value (δH 5.92) and the coupling of these protons was not observed. The 13CNMR (APT and HMQC) spectrum were identified 25 carbon atoms that classified as three methyl carbons (C-4”, C-9”, C-10”), four methylenes carbons (C-3, C-2”, C-5”, C-6”), eight methines carbons (C-2, C-6, C-8, C-2’, C-5’, C6’, C2”, C-7”), five quartener carbons (C-4a, C-1’, C-3’, C-3”, C-8”), four hydroxylated carbons (C-5, C-7, C-8a, C-4’) and one carbonyl carbon. Based on all of spectroscopic data, compound 3 was assigned as 3’-geranyl-4’,5,7trihydroxyflavanone or 3’-geranylnaringenin. The cytotoxicity of compounds 1 - 3 against P-388 murine cells were evaluated according to the Table 1 13C NMR of compound 1-3 Carbon Number 2 3 4 4a 5 6 δC (ppm) 1 78.9 43.1 196.0 103.1 164.0 94.9 7 167.9 8 94.1 8a 162.8 1’ 130.3 2’ 127.6 3’ 114.1 4’ 159.9 5’ 114.1 6’ 127.6 1” 2” 3” 4” 5” 6” 7” 8” 9” 10” 7-MeO 55.6 4’-MeO 55.3 2 78.7 43.0 196.6 103.0 162.0 96.7 163.8 106.5 159.7 130.7 127.5 114.0 159.8 114.0 127.5 21.7 121.7 134.3 17.8 25.8 - 3 80.6 43.4 197.8 103.0 165.4 97.0 163.4 96.2 164.3 130.9 128.9 129.5 156.7 115.7 126.2 29.1 123.7 137.0 40.8 27.7 125.4 132.2 25.9 16.2 17. 8 Conclusions The present study has successfully isolated and identified three flavanones derivatives, compounds 1 – 3, from the leaves of A. lanceifolius. All these compounds are the first time to be reported from Artocarpus plant. Compound 2 and 3 are active against murine leukemia P-388 cells with IC50 3.4 and 3.2 µg/mL, respectively. Acknowledgements This work has been partially supported by the University Research for Graduate Education Project, Ministry of National Education, Republic of Indonesia. We are grateful also to the Herbarium Anda, Padang and Herbarium Bogoriense, Bogor, Indonesia for the identification of the plant specimen. We also thank to Indonesia Institute Sciences Research Centre for NMR facility. 267 Eliza et al. Proceeding of The International Seminar on Chemistry 2008 (pp. 265-268) Jatinangor, 30-31 October 2008 Djarwaningsih. T., D.S. Alonso, S. Sudo & M.S.M. Sosef, 1995, Plant resources South-east Asia 5, 2, Timber trees: Minor commercial timbers, p. 5971. Hakim E.H., S.A. Achmad, L.D. Juliawaty, L. Makmur, Y.M. Syah, N. Aimi, M. Kitajima, H. Takayama & E.L. Ghisalberti, 2006. Prenylated flavonoids and related compounds of the Indonesian Artocarpus (Moraceae). J. Nat. Med., 60:161-184. Van Der Vossen H.A.M & M.Vessel, 2000, Plant resources of South-east Asia 16, Stimulant. 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