Biochemical Systematics and Ecology 37 (2009) 116–119 Contents lists available at ScienceDirect Biochemical Systematics and Ecology journal homepage: www.elsevier.com/locate/biochemsyseco Chemical constituents from the root bark of Ozoroa insignis Margaret Mwihaki Ng’ang’a a, Hidayat Hussain b, Sumesh Chhabra a, **, Caroline Langat-Thoruwa a, Karsten Krohn b, * a b Department of Chemistry, Kenyatta University, P.O. Box 43844-00100, Nairobi, Kenya Department of Chemistry, Universität Paderborn, Warburger Straße 100, 33098 Paderborn, Germany a r t i c l e i n f o Article history: Received 6 May 2008 Accepted 30 November 2008 Keywords: Ozoroa insignis Anacardiaceae Triterpene Flavone 1. Subject and source The roots of Ozoroa insignis Del. (Heeria insignis Del.) (Anacardiaceae) are used as a remedy for diarrhea, venereal diseases, tapeworm and hookworm, schistosomiasis, kidney trouble, migraine, and malaria (Liu and Abreu, 2007). In previous biological screening of O. insignis extracts, anthelmintic effect, cytotoxic activity, and topoisomerase inhibition were reported (Liu and Abreu, 2006a). The root bark of O. insignis were collected in Masaai land, Rift valley province in Kenya, in January 2005 and authenticated by Simon Mathenge, of Nairobi University, Kenya. A voucher specimen (MM/08/04) is deposited in Nairobi University herbarium, Chiromo Campus. 2. Previous work Early studies regarding the chemical constituents of O. insignis revealed the presence of tirucallane triterpenes (Liu and Abreu, 2006a), alk(en)yl phenols (Liu and Abreu, 2006b; Rea et al., 2003), one macrolide (Liu and Abreu, 2007), and 6-pentadecylsalicylic acid (He et al., 2002). 3. Present study The dried and powdered root barks (1 kg) of O. insignis were exhaustively and sequentially extracted with n-hexane, CH2Cl2, EtOAc, and MeOH. Each extract was concentrated in vacuo to obtain n-hexane, CH2Cl2- and ethyl acetate-soluble fractions. The CH2Cl2 soluble extract (80 g) was subjected to VLC on silica gel using petroleum ether, petroleum ether–EtOAc, EtOAc–MeOH and finally, pure MeOH as the mobile phase and yielded 65 fractions (F1–65). Fraction F19–27 were further separated by silica gel column chromatography eluting with petroleum ether–EtOAc (9:1) to give white cotton needles of * Corresponding author. Tel.: þ49 5251 602172; fax: þ49 5251 603245. ** Corresponding author. E-mail address: k.krohn@uni-paderborn.de (K. Krohn). 0305-1978/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.bse.2008.11.019 117 M.M. Ng’ang’a et al. / Biochemical Systematics and Ecology 37 (2009) 116–119 b-amyrin (1, 20 mg) (Heupel, 2005) and fraction B. Similarly, SephadexÒ LH-20 CC of fraction B, which was eluted with MeOH:CH2Cl2 (5:5) gave four fractions (C–F). Betulonic acid (2, 70 mg) (Kuroyangi et al., 1986) was purified from fraction C by crystallization using petroleum ether–acetone (8:2). Further purification of fraction C [petroleum ether–acetone (8.5:1.5)] afforded magnificol (3, 7 mg) (Ulubelen et al., 1989). The ethyl acetate crude extract (50 g) was similarly subjected to VLC on silica gel and eluted with a gradient of petroleum ether and acetone yielding 55 fractions (F1–55). Repeated column chromatography of F33–38 using petroleum ether–acetone (7.5:2.5) afforded betulinic acid (4, 8 mg) (Ikuta et al., 1995). Similarly purification of fraction F20–25 using a SephadexÒ LH-20 column, eluted with a mixture of CH2Cl2–MeOH (100:0 to 9:1) furnished 6-tridecyl anacardic acid (5, 3.0 mg) (Li et al., 2004) and 6-[8(Z)-pentadecenyl]anacardic acid (6, 4.5 mg) (Rea et al., 2003). Fraction F38–45 on SephadexÒ LH-20 column using petroleum ether–acetone (7:3) afforded 6-[10(Z)-heptadecenyl]anacardic acid (7, 5.5 mg) (Pan et al., 2006) and 6-[nonydecyl]anacardic acid (8, 3.5 mg) (Navarrete et al., 1989). Preparative TLC of F46–55 eluted with acetone:CH2Cl2 (1:9) yielded 5,20 ,40 - trihydroxy flavone (9, 6.7 mg). The structures of known compounds (1–8) (Fig. 1) were established conclusively by UV, IR, MS and extensive 1H- and 13C NMR spectra analysis and comparison with literature data. Ozoranone (9) was obtained as a yellow powder and the UV spectrum exhibited absorption maxima at 263 and 328 nm. This was supported by IR bands at 1720 cm1 for carbonyl absorption, a broad signal at 3400 cm1 for non-chelated hydroxyl groups. Analysis of the HREIMS gave a molecular ion at m/z 270.0519 [M]þ, corresponding to the molecular formula C15H10O5, supported by the 1H NMR, 13C NMR and DEPT analysis. The 1H NMR in CDCl3 of ozoranone (9) showed signals for seven deshielded protons. From the 1H–1H-COSY, two pairs of three protons were coupling to one another while one proton appeared as a singlet, suggesting the presence of two pairs of ABX spin system of three aromatic protons each. The first ABX spin system of three aromatic protons of ring A appeared at d 7.59 (d, 2.0 Hz, H-5), 7.36 (dd, J ¼ 8.0, 2.0 Hz, H-7), 6.96 (d, J ¼ 8.0 Hz, H-8) and the ring B ABX spin system appeared at d 7.63(d, J ¼ 8.0 Hz, H-60 ), d 6.84 (d, J ¼ 2.0 Hz, H-30 ) and d 6.80 (dd, J ¼ 8.0, 2.0 Hz, H-50 ). The 1H NMR spectrum also indicated the presence of a singlet at d 6.49 and its position at C-3 was confirmed by HMBC correlation to C-2 and C-4. The 13C NMR and DEPT spectra revealed 15 carbon signals, including a carbonyl carbon at d 181.4 and fourteen aromatic carbons at d 98.5–168.0. From these data, compound 9 was considered as a 20 ,40 ,6-oxygenated flavone. The position of the hydroxyl groups at C-20 , C-40 , and C-6 along with assignments of all carbons and hydrogen were confirmed by interpretation of the cross signals in the 1H–1H COSY (Fig. 2), 1H–13C HMQC and 1H–13C HMBC spectra (Fig. 2) combined with the coupling constants of the signals in the 1H NMR spectrum. Therefore, based on the above data, the structure of compound 9 was established as 2-(2,4-dihydroxyphenyl)-6-hydroxy-4H-chromen-4-one (Fig. 1). The flavonoid (9) is new as a natural product but has been reported as a synthetic compound in a Japanese patent (Kyogoku et al., 1979). Ozoranone (9): Yellow powder. 1H NMR (500 MHz, CDCl3): d 6.49 (1H, s, H-3), 6.80 (1H, dd, J ¼ 8.0, 2.0 Hz, H-50 ), 6.84 (1H, d, J ¼ 2.0 Hz, H-30 ), 6.96 (1H, d, J ¼ 8.0 Hz, H-8), 7.36 (1H, dd, J ¼ 8.0, 2.0 Hz, H-7), 7.59 (1H, d, 2.0 Hz, H-5), 7.63 (1H, d, J ¼ 8.0 Hz, CO2H R1 R2 HO 1 2 R1, R2 = O, R3 = O 4 R1 = OH, R2 = H CO2H CO2H HO R HO 3 3' HO 8 O 7 2 6 HO 2' 1' 4 5 9 O 3 OH 5R= 4' 5' 6' 6R= 7R= 8R= * 12 * 7 5 9 5 * * 18 Fig. 1. Structures of Compounds 1–9 isolated from Ozoroa insignis. 118 M.M. Ng’ang’a et al. / Biochemical Systematics and Ecology 37 (2009) 116–119 H H OH HO H O H H HO H Ozoranone (9) O 1H-1H-COSY HMBC Fig. 2. Selected 1H–1H COSY and HMBC correlations for compounds 9. H-60 ). 13C NMR (125 MHz, CDCl3): d ¼ 98.5 (C-30 ), 111.3 (C-3), 112.1 (C-50 ), 114.1 (C-10 ), 115.6 (C-8), 117.7 (C-5), 119.9 (C-10), 124.7 (C-7), 125.6 (C-60 ), 145.2 (C-9), 146.4 (C-6), 147.3 (C-40 ), 165.7 (C-20 ), 168.0 (C-2), 181.4 (C-4). IR-nmax (CHCl3): 3400, 1720, 1590, 710 cm1. UV (CHCl3) lmax: 263 (3.10), 328 (4.10). HREIMS: m/z 270.0519 (Calcd. 270.0527 for C15H10O5). 4. Chemotaxonomic significance Alkyl phenols, alkylhexenones, tannins, triterpenes, and flavones are widely distributed in the Anacardiaceae-family (Kapche et al., 2007). The present study reports the isolation of one oleaneane(1)and three lupane type triterpenoids (2–4), four anacardic acid[alk(en)yl-phenol] derivatives (5–8), and one flavone (9) for the first time from the root bark of O. insignis. Interestingly, compounds 1, 2 and 4 were characterized for the first time from the genus Ozoroa and have been isolated from the genus Rhus of the same family (Franke et al., 2001; Gu et al., 2007; Lee et al., 2005). This finding confirms that the genera Ozoroa and Rhus are closely related taxonomically. On the other hand, compound 3 was characterized for the first time from the Anacardiaceae-family, and thus isolation of compounds 1–4 in the present investigation is a major contribution to chemotaxonomic studies of the Anacardiaceae-family. Many alk(en)yl phenol derivatives were obtained from anacardiaceous plants (Kapche et al., 2007) and recently Liu and Abreu reported 41 alk(en)yl phenols from O. insignis (Liu and Abreu, 2006b). The carbon lengths of side chains in previously known alkenylphenols and alkenylsalicylic acids from Anacardiaceae have usually been C15 or C17 (Masuda et al., 2002). Therefore compound 8 with alkyl chain of C19 is less common in the Anacardiaceae-family (Liu and Abreu, 2006b). Compound 6 has been reported from O. insignis (Rea et al., 2003) but this is the first report of compounds 5, 7 and 8 in the genus Ozoroa as well as in the Anacardiaceae-family. Interestingly, compounds 5–7 have been isolated from the genus Ginkgo of the Ginkgoaceae family (Li et al., 2004; Ni and Wu, 2006; Pan et al., 2006). Thus the isolation of the compounds 5–7 in the family Anacardiaceae is particularly interesting since this strengthens the chemotaxonomic relationship of Anacardiaceae and Ginkgoaceae. This is the first report of a flavone, ozoranone (9), from the genus Ozoroa, although flavones have been reported from other species within the family Anacardiaceae (Matsuda, 1966). Acknowledgments M. M. Ng’ang’a gratefully acknowledges in country Postgraduate Scholarship by DAAD (German Academic Exchange Services) as well as a funded research visit to University of Paderborn, Germany where all the spectroscopic work was carried out; Kenyatta University for granting her study leave during the research visit and Sigma Xi, Grant-in-Aid of research for their support. References Franke, K., Masaoud, M., Schmidt, J., 2001. Planta Med. 67, 477. Gu, Q., Wang, R.-R., Zhang, X.-M., Wang, Y.-H., Zheng, Y.-T., Zhou, J., Chen, J.-J., 2007. Planta Med. 73, 279. He, W., Van Puyvelde, L., Bosselaers, J., De Kimpe, N., Van der Flaas, M., Roymans, A., Mathenge, S.G., Mudida, F.P., Mutiso, P.B.C., 2002. Pharm. Biol. 40, 74. Heupel, R.C., 2005. Phytochemistry 24, 2929. Ikuta, A., Kamiya, K., Satakek, T., Saiki, Y., 1995. Phytochemistry 38, 1203. Kapche, G.D.W.F., Laatsch, H., Fotso, S., Kouam, S.F., Wafo, P., Ngadjui, B.T., Abegaz, B.M., 2007. Biochem. Syst. Ecol 35, 539. 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