- Email: [email protected]
Chemical constituents from the leaves of Ilex urceolatus
Biochemical Systematics and Ecology 54 (2014) 92–95
Contents lists available at ScienceDirect
Biochemical Systematics and Ecology journal homepage: www.elsevier.com/locate/biochemsyseco
Chemical constituents from the leaves of Ilex urceolatus Yi Wu a, *, Xue-Cong Zhang a, Jian-Bo Sun b, Xin Wu c, You-Ying Li a, Wei Gu a, De-Yun Wang a, Jia-Guo Liu a, Yuan-Liang Hu a, * a
Institute of Traditional Chinese Veterinary Medicine, College of Veterinary Medicine, Nanjing Agricultural University, #1 Weigang, Nanjing 210095, Jiangsu Province, PR China b Department of Natural Medicinal Chemistry, China Pharmaceutical University, #24 Tongjiaxiang, Nanjing 210009, PR China c Guangdong Key Laboratory for Research and Development of Natural Drugs, Department of Pharmacology, Guangdong Medical College, Zhanjiang 524023, Guangdong, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 December 2013 Accepted 11 January 2014 Available online
Eighteen compounds, including four hemiterpene glycosides (1–4), three triterpenoid saponins (5–7), four triterpenes (8–11), five sterols (12-16) and two monoterpene glucosides (17 and 18), were isolated from the leaves of Ilex urceolatus C. B. Shang, K. S. Tang et D. Q. Du, which was identified as a new species belonging to the genus Ilex. Among them, compounds 14–18 were firstly isolated from the genus Ilex, others were obtained from I. urceolatus for the first time. This work represented the initially phytochemical study on this plant. The isolated compounds have significant chemotaxonomic characteristics with the other species from this genus. Crown Copyright Ó 2014 Published by Elsevier Ltd. All rights reserved.
Keywords: Ilex urceolatus Hemiterpene glycosides Triterpenoid saponins Triterpenes Sterols
1. Subject and source The genus Ilex (Aqifolilceae) contains more than 400 species worldwide and is mainly distributed over tropical, subtropical and temperate regions, particularly in East Asia, Southeast Asia and Central America. There are approximately 200 species growing in South and East China (Chen, 1999), some of which have a variety of traditional uses by the aborigines including treatment of inflammation, cardiovascular diseases, obesity and bacteria infections (Song et al., 2013; Li et al., 2013). Ilex urceolatus C. B. Shang, K. S. Tang et D. Q. Du (I. urceolatus), as a new species of the genus Ilex, is an evergreen arbor tree morphologically characterized by the waved margin with 10–19 pairs of sharp spines of leaves and urceolate or subglobose fruit (Shang et al., 2010). The leaves of I. urceolatus were collected from the countryside of Lianyuan City, Hunan Province of China, in July 2012, and were identified by Prof. Deyun Wang (College of Veterinary Medicine, Nanjing Agriculture University, Nanjing 210095, P. R. China). The voucher specimen (No. 20120703) was deposited in Nanjing Agricultural University. 2. Previous work Previous phytochemical and biological investigations on the genus Ilex have indicated the presence of triterpene saponins (Zhang et al., 2010), flavonoids (Del Pero Martinez et al., 1997), hemiterpene glycosides (Kim et al., 2012; Fuchino et al., 1997;
* Corresponding authors. Tel.: þ86 25 84395203; fax: þ86 25 84398669. E-mail addresses: [email protected], [email protected] (Y. Wu), [email protected], [email protected] (Y.-L. Hu). 0305-1978/$ – see front matter Crown Copyright Ó 2014 Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bse.2014.01.002
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Jiang et al., 2005), phenylpropanoids (Wen and Chen, 1996), sterols (Liu et al., 2003; Li et al., 2013) and tannins (Nakatani et al., 1988). However, to the best of our knowledge, the chemistry of I. urceolatus has not been reported. 3. Present work The air dried leaves of I. urceolatus (8 kg) were extracted with EtOH (85%,v/v) under refluxed three times. The combined solvent was then concentrated under reduced pressure to yield a brown crude extract (1.02 kg). The extract was then dissolved in water and partitioned sequentially with petroleum ether (PE, boiling point 60–90 C), EtOAc and n-BuOH to give 160 g, 225 g and 360 g of the corresponding extracts after solvent recovery, respectively. The n-BuOH fraction was subjected to a macroporous resin D101 column eluted by water-EtOH (100:0, 70:30, 50:50, 30:70 and 0:100, v/v) to give five fractions (Fr. C1-Fr. C5). The Fr. C2 (30% EtOH extract, v/v, 56 g) was performed on a silica gel (100–200 mesh, inactivated) column with a step gradient of CH2Cl2–MeOH–H2O (from 9:1:0.1 to 1:1:0.1), collecting 180 fractions of 500 mL. On the basis of TLC detection, the fractions were pooled into five groups (Fr. C2a-Fr. C2e). Fr. C2a was applied to ODS column eluted with aqueous MeOH (20%/80%, v/v) to give five fractions (Fr. C2a1-Fr. C2a5). Fr. C2a2 was further isolated by preparative HPLC (column Kromasil 250 10 mm, 5 mm, solvent 28% aqueous MeOH, v/v) and purified by Sephadex LH-20 column with MeOH–H2O (1:1, v/v) to afford 3 (8 mg) and 4 (11 mg). Fr. C2b was chromatographed over a silica gel (100–200 mesh, inactivated) column eluted with CH2Cl2–MeOH–H2O (from 9:1:0.1 to 1:1:0.1) to give four fractions (Fr. C2b1-Fr. C2b4). Fr. C2b3 was further applied to a ODS column eluted with MeOH–H2O (1:9, 3:7, 5:5 and 7:3, v/v) and further isolated by preparative HPLC (column Kromasil 250 10 mm, 5 mm, solvent 35% aqueous MeOH, v/v) to obtain 1 (5 mg), 2 (9 mg), 17 (10 mg) and 18 (15 mg). The Fr. C4 (70% EtOH extract, v/v, 42 g) was performed on a silica gel (100–200 mesh, inactivated) column with a step gradient of CH2Cl2– MeOH–H2O (from 49:1:0.1 to 1:1:0.1) to give six fractions (Fr. C4a-C4f) by TLC analysis. Fr. C4c was subjected to ODS column eluted with aqueous MeOH (10% / 90%, v/v) to give four fractions (Fr. C4c1-Fr. C4c4). Fr. C4c3 was further isolated by preparative HPLC (column Kromasil 250 10 mm, 5 mm, solvent 27–32% aqueous MeOH, v/v) and purified by Sephadex LH-20 column with MeOH–H2O (1:1, v/v) to afford 5 (17 mg), 6 (29 mg) and 7 (23 mg). Fr. C4a was applied to silica gel column eluted with CH2Cl2–MeOH (from 30:1 to 3:1, v/v) to obtain three fractions (Fr. C4a1-Fr. C4a3). Fr. C4a1 was chromatographed over a silica gel (200–300 mesh) column eluted with PE-EtOAc (30:1/10:1) to give 8 (47 mg) and 9 (60 mg) by recrystallization from MeOH. Fr. C4a2 was further isolated by preparative TLC (silica gel H, solvent PE-EtOAc and PE-Acetone) and purified by Sephadex LH-20 column with CH2Cl2–MeOH (1:1, v/v) to afford 10 (19 mg) and 11 (23 mg). The PE fraction was chromatographed on a silica gel (100–200 mesh) column eluted with PE-EtOAc (49:1, 19:1, 9:1, 3:1, and 0:1, v/v) to obtain five fractions (Fr. A1-Fr. A5) by TLC analysis. Fr. A2 was divided by silica gel (200–300 mesh) columns, eluting with PE-EtOAc (20:1 to 3:1, v/ v) to yield 12 (52 mg). Fr. A3 was performed on silica gel (200–300 mesh) columns, eluting with PE-EtOAc (from 15:1 to 1:1, v/ v) to give four fractions (Fr. A3a-Fr. A3d). Fr. A3a was rechromatographed by preparative TLC (silica gel H, PE-Acetone and PEEtOAc as eluents) repeatedly and subjected further on Sephadex LH-20 columns with CH2Cl2–MeOH (1:1, v/v) as solvent to give 14 (28 mg), 15 (34 mg) and 16 (23 mg). Fr. A4 was fractionated by silica gel (200–300 mesh) columns, eluting with PEEtOAc (12:1 to 0:1, v/v) and CH2Cl2–MeOH (50:1, 20:1 and 8:1, v/v) successively to give three fractions (Fr. A4a-Fr. A4c). Fr. A4b was separated by preparative TLC (silica gel H, PE-Acetone and PE-EtOAc as eluents) repeatedly and purified further on Sephadex LH-20 columns with CH2Cl2–MeOH (1:1, v/v) to obtain 13 (57 mg). The obtained compounds were identified on the basis of their 1H NMR, 13C NMR, MS, and IR spectra by comparison with those reported data in the related references as follows: rotundarpenoside A (1) (Kim et al., 2012), rotundarpenoside B (2) (Kim et al., 2012), aohada-glucoside C (Fuchino et al., 1997) (3), pubescenosides B (Jiang et al., 2005) (4), ilexsaponin B1 (Zhang et al., 2010) (5), ilexsaponin B2 (Zhang et al., 2010) (6), mussaendoside R (Zhang et al., 2010) (7), oleanolic acid (Mousa et al., 1994) (8), ursolic acid (Mukherjee et al., 1982) (9), siaresinolic acid (Zhou et al., 2013) (10); pomolic acid (Zhou et al., 2013; Xie et al., 2007) (11), b-sitosterol (Liu et al., 2003; Li et al., 2013) (12), daucosterol (Liu et al., 2003) (13), stigmast-7, 22-ene-3b-ol (Jahan et al., 1995) (14), stigmast-5, 22-ene-3b, 7a-diol (15) (Jahan et al., 1995), stigmast-7, 22-ene3b, 5a, 6b-triol (16) (Zhang and Lu, 2006), (Yaoita et al., 1999), itoside O (17) (Chai et al., 2008) and betulalbuside A (18) (Morikawa et al., 2004)(Fig. 1). 4. Chemotaxonomic significance The present phytochemical study reports on the isolation and identification of eighteen compounds, including four hemiterpene glycosides (1-4), three triterpenoid saponins (5-7), four pentacyclic triterpenes (8–11), five sterols (12–16) and two monoterpene glucosides (17 and 18) from Ilex urceolatus. Compounds 1–4 are hemiterpene glycosides which are rarely reported from plants. Compound 1 and 2 were initially obtained from I. rotunda (Kim et al., 2012), and compound 3 was isolated from Ilex macropoda (Fuchino et al., 1997), while compound 4 was reported from Ilex pubescens (Jiang et al., 2005). The isolation of compounds 1–4 from I. urceolatus reveals a close relationship among the four species of Ilex (Aquifoliaceae). The variations in the hemiterpene glycosides present in the different species suggest that they could have chemotaxonomic significances and further research on these compounds in justified. Compounds 5–7 are triterpenoid saponins isolated from I. pubescens (Zhang et al., 2010). The presence of these triterpenoid saponins in I. urceolatus is in agreement with the previous reports for I. pubescens indicating the chemotaxonomic relationship that I. urceolatus has with other species of Ilex. Compounds 8–11 are reported in a diversity of families including the quifoliaceae. Among the four pentacyclic triterpenes 8–11, compound 8 has also been isolated from Ilex
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Y. Wu et al. / Biochemical Systematics and Ecology 54 (2014) 92–95
Fig. 1. Structures of compounds 1–18.
centrochinensis (Li et al., 2013) and Ilex cornuta (Zhou et al., 2013), and compound 9 has been obtained from Ilex kudingcha (Liu et al., 2003), Ilex pernyi (Xie et al., 2007) and Ilex Merr (Wen et al., 1999). Compound 10 has been reported from I. cornuta (Zhou et al., 2013) and Ilex latifolia (Ouyang et al., 1998), while compound 11 has been isolated from I. cornuta (Zhou et al., 2013) and I. pernyi (Xie et al., 2007). These pentacyclic triterpenes confirm the relationship between I. urceolatus and other species of Ilex. Pentacyclic triterpenes could like the hemiterpene glycosides be used to differentiate among the species of Ilex. The sterols 12 and 13 have been isolated from species of Ilex plants (Li et al., 2013; Liu et al., 2003; Wen et al., 1999), whereas the sterols 14–16 have not previously been reported from the Ilex. Additionally, the two monoterpene glucosides (compounds 17
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and 18) are reported from the genus Ilex for the first time. Compound 17 has previously been isolated from Itoa orientalis (Flacourtiaceae) (Chai et al., 2008) and compound 18 is originally obtained from Cunila spicata (Lamiaceae) (Morikawa et al., 2004). Whether these compounds have systematic importance needs further evaluation. The phytochemical study suggests that there is a close chemotaxonomic relationship between I. urceolatus and other species of Ilex. Acknowledgments This work was financially supported by National Natural Science Foundation of China (NSFC, Grant No. 31302135), Natural Science Foundation of Jiangsu Province of China (Grant No. BK20130678), A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), the China Postdoctoral Science Foundation (Grant No. 2013M531375), Specialized Research Fund for the Doctoral Program of Higher Education (SRFDP, Grant No. 20130097120026) and the Fundamental Research Funds for the Central Universities-Nanjing Agricultural University Youth Science and Technology Innovation Fund (Grant No. KJ2012015). References Chai, X.Y., Xu, Z.R., Tang, L.Y., Chen, Y.P., Bai, C.C., Zhou, F.R., Tu, P.F., 2008. J. Chin. Pharm. Sci. 17, 79. Chen, S.K., 1999. Zhongguo Zhiwu Zhi (Flora of China), vol. 45(2). Science Press, Beijing, p. 2. Del Pero Martinez, M.A., Pelotto, J.P., Basualdo, N., 1997. Biochem. Syst. Ecol. 25, 619. Fuchino, H., Tachibana, H., Tanaka, N., 1997. Chem. Pharm. Bull. 45, 1533. Jahan, N., Ahmed, W., Malik, A., 1995. J. Nat. Prod. 58, 1244. Jiang, Z.H., Wang, J.R., Li, M., Liu, Z.Q., Wang, H.B., Chau, K.Y., Zhao, C., Liu, L., 2005. J. Nat. Prod. 68, 397. Kim, M.H., Park, K.H., Oh, M.H., Kim, H.H., Choe, K.I., Park, S.H., Lee, M.W., 2012. Arch. Pharm. Res. 35, 1779. Li, J.J., Yang, J., Cui, W.X., Chen, X.Q., Chen, G.L., Wen, X.D., Wang, Q., 2013. Evid. Based Complement. Altern. Med. 2013, 451975. Li, L.J., Du, P., Sun, K.H., Cao, M.Q., Wu, Z.Z., 2013. Zhongguo Zhongyao Zazhi 38, 354. Liu, S., Qin, Y., Du, F.L., 2003. Zhongguo Zhongyao Zazhi 28, 834. Morikawa, H., Kasai, R., Otsuka, H., Hirata, E., Shinzato, T., Aramoto, M., Takeda, Y., 2004. Chem. Pharm. Bull. 52, 1086. Mousa, O., Vuorela, P., Kiviranta, J., Wahab, S.A., Hiltohen, R., Vuorela, H., 1994. J. Ethnopharmacol. 41, 71. Mukherjee, K.S., Bhattacharya, M.K., Ghosh, P.K., 1982. Phytochemistry 21, 2416. Nakatani, M., Urata, Y., Hase, T., 1988. Rep. Fac. Sci. Kagoshima Univ. Math. Phys. Chem.) 21, 127. Ouyang, M.A., Liu, Y.Q., Wang, H.Q., Yang, C.R., 1998. Phytochemistry 49, 2483. Shang, C.B., Tang, K.S., Du, D.Q., 2010. Bull. Bot. Res. 30, 645. Song, J.L., Qian, Y., Li, G.J., Zhao, X., 2013. Mol. Med. Rep. 8, 1256. Wen, D., Zheng, X., Inoue, K., 1999. Zhongguo Zhongyao Zazhi 24, 223. Wen, D.X., Chen, Z.L., 1996. Phytochemistry 41, 657. Xie, G.B., Zhou, S.X., Lei, L.D., Tu, P.F., 2007. Zhongguo Zhongyao Zazhi 32, 1890. Yaoita, Y., Endo, M., Tani, Y., Machida, K., Amemiya, K., Furumura, K., Kikuchi, M., 1999. Chem. Pharm. Bull. 47, 847. Zhang, C.X., Lin, C.Z., Xiong, T.Q., Zhu, C.C., Yang, J.Y., Zhao, Z.X., 2010. Fitoterapia 81, 788. Zhang, J., Lu, W.G., 2006. Nat. Prod. Res. Dev. 18, 883. Zhou, X.X., Xu, Q.M., Zhou, Y., Li, X.R., Yang, S.L., 2013. J. Zhongyaocai 36, 233.
Contents lists available at ScienceDirect
Biochemical Systematics and Ecology journal homepage: www.elsevier.com/locate/biochemsyseco
Chemical constituents from the leaves of Ilex urceolatus Yi Wu a, *, Xue-Cong Zhang a, Jian-Bo Sun b, Xin Wu c, You-Ying Li a, Wei Gu a, De-Yun Wang a, Jia-Guo Liu a, Yuan-Liang Hu a, * a
Institute of Traditional Chinese Veterinary Medicine, College of Veterinary Medicine, Nanjing Agricultural University, #1 Weigang, Nanjing 210095, Jiangsu Province, PR China b Department of Natural Medicinal Chemistry, China Pharmaceutical University, #24 Tongjiaxiang, Nanjing 210009, PR China c Guangdong Key Laboratory for Research and Development of Natural Drugs, Department of Pharmacology, Guangdong Medical College, Zhanjiang 524023, Guangdong, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 December 2013 Accepted 11 January 2014 Available online
Eighteen compounds, including four hemiterpene glycosides (1–4), three triterpenoid saponins (5–7), four triterpenes (8–11), five sterols (12-16) and two monoterpene glucosides (17 and 18), were isolated from the leaves of Ilex urceolatus C. B. Shang, K. S. Tang et D. Q. Du, which was identified as a new species belonging to the genus Ilex. Among them, compounds 14–18 were firstly isolated from the genus Ilex, others were obtained from I. urceolatus for the first time. This work represented the initially phytochemical study on this plant. The isolated compounds have significant chemotaxonomic characteristics with the other species from this genus. Crown Copyright Ó 2014 Published by Elsevier Ltd. All rights reserved.
Keywords: Ilex urceolatus Hemiterpene glycosides Triterpenoid saponins Triterpenes Sterols
1. Subject and source The genus Ilex (Aqifolilceae) contains more than 400 species worldwide and is mainly distributed over tropical, subtropical and temperate regions, particularly in East Asia, Southeast Asia and Central America. There are approximately 200 species growing in South and East China (Chen, 1999), some of which have a variety of traditional uses by the aborigines including treatment of inflammation, cardiovascular diseases, obesity and bacteria infections (Song et al., 2013; Li et al., 2013). Ilex urceolatus C. B. Shang, K. S. Tang et D. Q. Du (I. urceolatus), as a new species of the genus Ilex, is an evergreen arbor tree morphologically characterized by the waved margin with 10–19 pairs of sharp spines of leaves and urceolate or subglobose fruit (Shang et al., 2010). The leaves of I. urceolatus were collected from the countryside of Lianyuan City, Hunan Province of China, in July 2012, and were identified by Prof. Deyun Wang (College of Veterinary Medicine, Nanjing Agriculture University, Nanjing 210095, P. R. China). The voucher specimen (No. 20120703) was deposited in Nanjing Agricultural University. 2. Previous work Previous phytochemical and biological investigations on the genus Ilex have indicated the presence of triterpene saponins (Zhang et al., 2010), flavonoids (Del Pero Martinez et al., 1997), hemiterpene glycosides (Kim et al., 2012; Fuchino et al., 1997;
* Corresponding authors. Tel.: þ86 25 84395203; fax: þ86 25 84398669. E-mail addresses: [email protected], [email protected] (Y. Wu), [email protected], [email protected] (Y.-L. Hu). 0305-1978/$ – see front matter Crown Copyright Ó 2014 Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bse.2014.01.002
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Jiang et al., 2005), phenylpropanoids (Wen and Chen, 1996), sterols (Liu et al., 2003; Li et al., 2013) and tannins (Nakatani et al., 1988). However, to the best of our knowledge, the chemistry of I. urceolatus has not been reported. 3. Present work The air dried leaves of I. urceolatus (8 kg) were extracted with EtOH (85%,v/v) under refluxed three times. The combined solvent was then concentrated under reduced pressure to yield a brown crude extract (1.02 kg). The extract was then dissolved in water and partitioned sequentially with petroleum ether (PE, boiling point 60–90 C), EtOAc and n-BuOH to give 160 g, 225 g and 360 g of the corresponding extracts after solvent recovery, respectively. The n-BuOH fraction was subjected to a macroporous resin D101 column eluted by water-EtOH (100:0, 70:30, 50:50, 30:70 and 0:100, v/v) to give five fractions (Fr. C1-Fr. C5). The Fr. C2 (30% EtOH extract, v/v, 56 g) was performed on a silica gel (100–200 mesh, inactivated) column with a step gradient of CH2Cl2–MeOH–H2O (from 9:1:0.1 to 1:1:0.1), collecting 180 fractions of 500 mL. On the basis of TLC detection, the fractions were pooled into five groups (Fr. C2a-Fr. C2e). Fr. C2a was applied to ODS column eluted with aqueous MeOH (20%/80%, v/v) to give five fractions (Fr. C2a1-Fr. C2a5). Fr. C2a2 was further isolated by preparative HPLC (column Kromasil 250 10 mm, 5 mm, solvent 28% aqueous MeOH, v/v) and purified by Sephadex LH-20 column with MeOH–H2O (1:1, v/v) to afford 3 (8 mg) and 4 (11 mg). Fr. C2b was chromatographed over a silica gel (100–200 mesh, inactivated) column eluted with CH2Cl2–MeOH–H2O (from 9:1:0.1 to 1:1:0.1) to give four fractions (Fr. C2b1-Fr. C2b4). Fr. C2b3 was further applied to a ODS column eluted with MeOH–H2O (1:9, 3:7, 5:5 and 7:3, v/v) and further isolated by preparative HPLC (column Kromasil 250 10 mm, 5 mm, solvent 35% aqueous MeOH, v/v) to obtain 1 (5 mg), 2 (9 mg), 17 (10 mg) and 18 (15 mg). The Fr. C4 (70% EtOH extract, v/v, 42 g) was performed on a silica gel (100–200 mesh, inactivated) column with a step gradient of CH2Cl2– MeOH–H2O (from 49:1:0.1 to 1:1:0.1) to give six fractions (Fr. C4a-C4f) by TLC analysis. Fr. C4c was subjected to ODS column eluted with aqueous MeOH (10% / 90%, v/v) to give four fractions (Fr. C4c1-Fr. C4c4). Fr. C4c3 was further isolated by preparative HPLC (column Kromasil 250 10 mm, 5 mm, solvent 27–32% aqueous MeOH, v/v) and purified by Sephadex LH-20 column with MeOH–H2O (1:1, v/v) to afford 5 (17 mg), 6 (29 mg) and 7 (23 mg). Fr. C4a was applied to silica gel column eluted with CH2Cl2–MeOH (from 30:1 to 3:1, v/v) to obtain three fractions (Fr. C4a1-Fr. C4a3). Fr. C4a1 was chromatographed over a silica gel (200–300 mesh) column eluted with PE-EtOAc (30:1/10:1) to give 8 (47 mg) and 9 (60 mg) by recrystallization from MeOH. Fr. C4a2 was further isolated by preparative TLC (silica gel H, solvent PE-EtOAc and PE-Acetone) and purified by Sephadex LH-20 column with CH2Cl2–MeOH (1:1, v/v) to afford 10 (19 mg) and 11 (23 mg). The PE fraction was chromatographed on a silica gel (100–200 mesh) column eluted with PE-EtOAc (49:1, 19:1, 9:1, 3:1, and 0:1, v/v) to obtain five fractions (Fr. A1-Fr. A5) by TLC analysis. Fr. A2 was divided by silica gel (200–300 mesh) columns, eluting with PE-EtOAc (20:1 to 3:1, v/ v) to yield 12 (52 mg). Fr. A3 was performed on silica gel (200–300 mesh) columns, eluting with PE-EtOAc (from 15:1 to 1:1, v/ v) to give four fractions (Fr. A3a-Fr. A3d). Fr. A3a was rechromatographed by preparative TLC (silica gel H, PE-Acetone and PEEtOAc as eluents) repeatedly and subjected further on Sephadex LH-20 columns with CH2Cl2–MeOH (1:1, v/v) as solvent to give 14 (28 mg), 15 (34 mg) and 16 (23 mg). Fr. A4 was fractionated by silica gel (200–300 mesh) columns, eluting with PEEtOAc (12:1 to 0:1, v/v) and CH2Cl2–MeOH (50:1, 20:1 and 8:1, v/v) successively to give three fractions (Fr. A4a-Fr. A4c). Fr. A4b was separated by preparative TLC (silica gel H, PE-Acetone and PE-EtOAc as eluents) repeatedly and purified further on Sephadex LH-20 columns with CH2Cl2–MeOH (1:1, v/v) to obtain 13 (57 mg). The obtained compounds were identified on the basis of their 1H NMR, 13C NMR, MS, and IR spectra by comparison with those reported data in the related references as follows: rotundarpenoside A (1) (Kim et al., 2012), rotundarpenoside B (2) (Kim et al., 2012), aohada-glucoside C (Fuchino et al., 1997) (3), pubescenosides B (Jiang et al., 2005) (4), ilexsaponin B1 (Zhang et al., 2010) (5), ilexsaponin B2 (Zhang et al., 2010) (6), mussaendoside R (Zhang et al., 2010) (7), oleanolic acid (Mousa et al., 1994) (8), ursolic acid (Mukherjee et al., 1982) (9), siaresinolic acid (Zhou et al., 2013) (10); pomolic acid (Zhou et al., 2013; Xie et al., 2007) (11), b-sitosterol (Liu et al., 2003; Li et al., 2013) (12), daucosterol (Liu et al., 2003) (13), stigmast-7, 22-ene-3b-ol (Jahan et al., 1995) (14), stigmast-5, 22-ene-3b, 7a-diol (15) (Jahan et al., 1995), stigmast-7, 22-ene3b, 5a, 6b-triol (16) (Zhang and Lu, 2006), (Yaoita et al., 1999), itoside O (17) (Chai et al., 2008) and betulalbuside A (18) (Morikawa et al., 2004)(Fig. 1). 4. Chemotaxonomic significance The present phytochemical study reports on the isolation and identification of eighteen compounds, including four hemiterpene glycosides (1-4), three triterpenoid saponins (5-7), four pentacyclic triterpenes (8–11), five sterols (12–16) and two monoterpene glucosides (17 and 18) from Ilex urceolatus. Compounds 1–4 are hemiterpene glycosides which are rarely reported from plants. Compound 1 and 2 were initially obtained from I. rotunda (Kim et al., 2012), and compound 3 was isolated from Ilex macropoda (Fuchino et al., 1997), while compound 4 was reported from Ilex pubescens (Jiang et al., 2005). The isolation of compounds 1–4 from I. urceolatus reveals a close relationship among the four species of Ilex (Aquifoliaceae). The variations in the hemiterpene glycosides present in the different species suggest that they could have chemotaxonomic significances and further research on these compounds in justified. Compounds 5–7 are triterpenoid saponins isolated from I. pubescens (Zhang et al., 2010). The presence of these triterpenoid saponins in I. urceolatus is in agreement with the previous reports for I. pubescens indicating the chemotaxonomic relationship that I. urceolatus has with other species of Ilex. Compounds 8–11 are reported in a diversity of families including the quifoliaceae. Among the four pentacyclic triterpenes 8–11, compound 8 has also been isolated from Ilex
94
Y. Wu et al. / Biochemical Systematics and Ecology 54 (2014) 92–95
Fig. 1. Structures of compounds 1–18.
centrochinensis (Li et al., 2013) and Ilex cornuta (Zhou et al., 2013), and compound 9 has been obtained from Ilex kudingcha (Liu et al., 2003), Ilex pernyi (Xie et al., 2007) and Ilex Merr (Wen et al., 1999). Compound 10 has been reported from I. cornuta (Zhou et al., 2013) and Ilex latifolia (Ouyang et al., 1998), while compound 11 has been isolated from I. cornuta (Zhou et al., 2013) and I. pernyi (Xie et al., 2007). These pentacyclic triterpenes confirm the relationship between I. urceolatus and other species of Ilex. Pentacyclic triterpenes could like the hemiterpene glycosides be used to differentiate among the species of Ilex. The sterols 12 and 13 have been isolated from species of Ilex plants (Li et al., 2013; Liu et al., 2003; Wen et al., 1999), whereas the sterols 14–16 have not previously been reported from the Ilex. Additionally, the two monoterpene glucosides (compounds 17
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and 18) are reported from the genus Ilex for the first time. Compound 17 has previously been isolated from Itoa orientalis (Flacourtiaceae) (Chai et al., 2008) and compound 18 is originally obtained from Cunila spicata (Lamiaceae) (Morikawa et al., 2004). Whether these compounds have systematic importance needs further evaluation. The phytochemical study suggests that there is a close chemotaxonomic relationship between I. urceolatus and other species of Ilex. Acknowledgments This work was financially supported by National Natural Science Foundation of China (NSFC, Grant No. 31302135), Natural Science Foundation of Jiangsu Province of China (Grant No. BK20130678), A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), the China Postdoctoral Science Foundation (Grant No. 2013M531375), Specialized Research Fund for the Doctoral Program of Higher Education (SRFDP, Grant No. 20130097120026) and the Fundamental Research Funds for the Central Universities-Nanjing Agricultural University Youth Science and Technology Innovation Fund (Grant No. KJ2012015). References Chai, X.Y., Xu, Z.R., Tang, L.Y., Chen, Y.P., Bai, C.C., Zhou, F.R., Tu, P.F., 2008. J. Chin. Pharm. Sci. 17, 79. Chen, S.K., 1999. Zhongguo Zhiwu Zhi (Flora of China), vol. 45(2). Science Press, Beijing, p. 2. Del Pero Martinez, M.A., Pelotto, J.P., Basualdo, N., 1997. Biochem. Syst. Ecol. 25, 619. Fuchino, H., Tachibana, H., Tanaka, N., 1997. Chem. Pharm. Bull. 45, 1533. Jahan, N., Ahmed, W., Malik, A., 1995. J. Nat. Prod. 58, 1244. Jiang, Z.H., Wang, J.R., Li, M., Liu, Z.Q., Wang, H.B., Chau, K.Y., Zhao, C., Liu, L., 2005. J. Nat. Prod. 68, 397. Kim, M.H., Park, K.H., Oh, M.H., Kim, H.H., Choe, K.I., Park, S.H., Lee, M.W., 2012. Arch. Pharm. Res. 35, 1779. Li, J.J., Yang, J., Cui, W.X., Chen, X.Q., Chen, G.L., Wen, X.D., Wang, Q., 2013. Evid. Based Complement. Altern. Med. 2013, 451975. Li, L.J., Du, P., Sun, K.H., Cao, M.Q., Wu, Z.Z., 2013. Zhongguo Zhongyao Zazhi 38, 354. Liu, S., Qin, Y., Du, F.L., 2003. Zhongguo Zhongyao Zazhi 28, 834. Morikawa, H., Kasai, R., Otsuka, H., Hirata, E., Shinzato, T., Aramoto, M., Takeda, Y., 2004. Chem. Pharm. Bull. 52, 1086. Mousa, O., Vuorela, P., Kiviranta, J., Wahab, S.A., Hiltohen, R., Vuorela, H., 1994. J. Ethnopharmacol. 41, 71. Mukherjee, K.S., Bhattacharya, M.K., Ghosh, P.K., 1982. Phytochemistry 21, 2416. Nakatani, M., Urata, Y., Hase, T., 1988. Rep. Fac. Sci. Kagoshima Univ. Math. Phys. Chem.) 21, 127. Ouyang, M.A., Liu, Y.Q., Wang, H.Q., Yang, C.R., 1998. Phytochemistry 49, 2483. Shang, C.B., Tang, K.S., Du, D.Q., 2010. Bull. Bot. Res. 30, 645. Song, J.L., Qian, Y., Li, G.J., Zhao, X., 2013. Mol. Med. Rep. 8, 1256. Wen, D., Zheng, X., Inoue, K., 1999. Zhongguo Zhongyao Zazhi 24, 223. Wen, D.X., Chen, Z.L., 1996. Phytochemistry 41, 657. Xie, G.B., Zhou, S.X., Lei, L.D., Tu, P.F., 2007. Zhongguo Zhongyao Zazhi 32, 1890. Yaoita, Y., Endo, M., Tani, Y., Machida, K., Amemiya, K., Furumura, K., Kikuchi, M., 1999. Chem. Pharm. Bull. 47, 847. Zhang, C.X., Lin, C.Z., Xiong, T.Q., Zhu, C.C., Yang, J.Y., Zhao, Z.X., 2010. Fitoterapia 81, 788. Zhang, J., Lu, W.G., 2006. Nat. Prod. Res. Dev. 18, 883. Zhou, X.X., Xu, Q.M., Zhou, Y., Li, X.R., Yang, S.L., 2013. J. Zhongyaocai 36, 233.