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Chemical constituents from Cynanchum paniculatum (Bunge) Kitag
Biochemical Systematics and Ecology 61 (2015) 139e142
Contents lists available at ScienceDirect
Biochemical Systematics and Ecology journal homepage: www.elsevier.com/locate/biochemsyseco
Chemical constituents from Cynanchum paniculatum (Bunge) Kitag Ya-Le Niu a, 1, Xi Chen a, 1, Yi Wu a, *, Hai-Qiang Jiang b, Xue-Lan Zhang b, En-Tao Li a, You-Ying Li a, Hong-Lei Zhou b, Jia-Guo Liu a, De-Yun Wang a, ** a
Institute of Traditional Chinese Veterinary Medicine, College of Veterinary Medicine, Nanjing Agricultural University, #1 Weigang, Nanjing 210095, Jiangsu Province, PR China b College of Pharmacy, Shandong University of Traditional Chinese Medicine, #4655 Daxue Road, Jinan 250355, Shandong Province, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 March 2015 Received in revised form 13 May 2015 Accepted 13 June 2015 Available online xxx
Twenty-nine compounds, including five acetophenone derivatives (1e5), three phenanthroindolizidine alkaloids (12e14), seven pentacyclic triterpenoids (15e21) and five C21 steroidal sapogenins (22e26), were isolated from the root of Cynanchum paniculatum (Bunge) Kitag. Their structures were determined by spectroscopic methods and comparison with reported data. Moreover, the chemotaxonomic relationships were also discussed. As a result, acetophenone derivatives, pentacyclic triterpenoids and C21 pregnane sapogenins can be recognized as chemotaxonomic markers for Cynanchum genus, and C. paniculatum has close relationships with some species of genus Cynanchum. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Cynanchum paniculatum (Bunge) Kitag Steroidal sapogenins Acetophenone derivatives Pentacyclic triterpenoids Chemotaxonomic relationships
1. 1. Subject and source The genus Cynanchum (Asclepiadaceae family) consists of nearly 200 species which distributes in east Africa, the Mediterranean regions, as well as the tropical, subtropical and temperate areas of Europe and Asia (Jiang and Li, 1977). Cynanchum paniculatum (Bunge) Kitag, a vivacious herb growing in China, South Korea and Japan, is used as a traditional Chinese medicine to treat snake bites, hives, and chronic tracheitis (Li et al., 2004; Huang et al., 2009). The dried roots of C. paniculatum were collected in Pingyi county, Shandong Province of China, in September 2012, and were identified by Dr. Yi Wu, College of Veterinary Medicine, Nanjing Agricultural University, where a voucher specimen (No. XCQ201209) was deposited. 2. 2. Previous work Previous phytochemical investigations on C. paniculatum have revealed the presence of C21 steroidal glycosides (Li et al., 2004; Dou et al., 2007), phenolic derivatives (Kim et al., 2013), alkaloids (Lee et al., 2003) and polysaccharides (Zhao et al., 2008). However, to the best of our knowledge, no systematical studies have discussed the chemotaxonomic relationships between this plant and other species of genus Cynanchum.
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected], [email protected] (Y. Wu), [email protected] (D.-Y. Wang). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.bse.2015.06.018 0305-1978/© 2015 Elsevier Ltd. All rights reserved.
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3. 3. Present study The air-dried roots of C. paniculatum (20 kg) were pulverized and extracted with 95% ethanol (v/v, 80 L 2) and 80% ethanol (v/v, 80 L 1) under reflux, each time for 2 h. The combined solvent was filtered and concentrated after removal of solvent under reduced pressure to give a brown residue (900 g). The residue was suspended in water and partitioned with petroleum ether (PE, 60e90 C), ethyl acetate (EtOAC) and n-butanol (n-BuOH), sequentially. The organic layers were concentrated in vacuum to give PE (45 g), EtOAc (105 g) and n-BuOH (260 g) extracts, respectively. The PE fraction was performed on a silica gel open column chromatography (CC) eluted with PE-EtOAc (gradient, 100:0 to 0:100, v/v). The eluents were combined into five fractions (P1eP5) by TLC detection. Fraction P1 was subjected to a silica gel column (100e200 mesh) eluted with a gradient of PE-EtOAc (80:1 to 2:1, v/v) to obtain six sub-fractions (1A-1F). Sub-fraction 1A was performed on a silica gel CC with PE-dichloromethane (CH2Cl2) (30:1 to 10:1, v/v) to give compounds 7 (50 mg) and 9 (48 mg). Sub-fraction 1C was chromatographed on a silica gel CC with PE-acetone (50:1 to 10:1, v/v) to give compounds 6 (27 mg) and 8 (34 mg). Fraction P2 was purified on a silica gel (100e200 mesh) CC eluted with a gradient of cyclohexaneEtOAc (20:1 to 2:1, v/v) to yield five sub-fractions (2A-2E). Sub-fraction 2B was isolated on a silica gel CC with PE-acetone (20:1 to 8:1, v/v) to give compounds 1 (80 mg), 2 (32 mg) and 10 (112 mg). Sub-fraction 2C was separated over a silica gel CC with n-hexane-acetone (15:1 to 10:1, v/v) to provide compounds 3 (27 mg) and 4 (41 mg). Sub-fraction 2D was fractioned by a silica gel CC with cyclohexane-acetone (12:1 to 6:1, v/v) to yield compound 5 (16 mg). Fraction P3 was subjected to a silica gel (100e200 mesh) CC with PE-EtOAc (gradient, 30:1 to 3:1, v/v) to give three sub-fractions (3A-3C). Sub-fraction 3A was performed on a silica gel CC with PE-acetone (12:1 to 6:1, v/v) to obtain compounds 15 (25 mg) and 16 (36 mg). Sub-fraction 3C was purified by a silica gel CC with PE-acetone (10:1 to 8:1, v/v) to give compounds 17 (19 mg) and 18 (11 mg). Fraction P5 was chromatographed on a silica gel (100e200 mesh) CC with PE-EtOAc (15:1 to 1:1, v/v) to give four sub-fractions (5A-5D). Sub-fraction 5B was further separated by a silica gel CC with PE-acetone (12:1 to 6:1, v/v) to yield compounds 19 (38 mg), 20 (45 mg) and 21 (17 mg). Sub-fraction 5D was performed on a silica gel CC with CH2Cl2-methanol (MeOH) (50:1, v/v) to obtain compound 11 (54 mg). The EtOAc fraction was subjected to a silica gel CC with CH2Cl2eMeOH gradient mixtures (100:1 to 0:1, v/v) to yield seven sub-fractions (E1-E7) by TLC detection. Fraction E1 was then loaded on a silica gel column and eluted with a gradient of CH2Cl2-acetone (80:1 to 1:1, v/v) to obtain three sub-fractions (1a-1c). Sub-fraction 1a was then isolated by repeated preparative TLC (gradient PE-CH2Cl2eMeOH) to achieve compounds 12 (15 mg) and 14 (17 mg). Compound 13 (23 mg) were purified from sub-fraction 1b by repeated preparative TLC (gradient PE-CH2Cl2eMeOH). Fraction E2 was processed by a silica gel CC with mobile phase of PE-acetone (20:1 to 0:1, v/v) to give three sub-fractions (2a-2c). Sub-fraction 2a was then purified by repeated preparative TLC (gradient n-hexane-CH2Cl2-acetone, 15:3:1 to 9:3:2), then decolorized by Sephadex LH-20CC (CH2Cl2eMeOH 1:1, v/v) to yield compounds 22 (13 mg), 23 (8 mg) and 24 (17 mg), respectively. Sub-fraction 2c was chromatographed by repeated preparative TLC (gradient CH2Cl2-Acetone, 7:1 to 7:3), then purified respectively by Sephadex LH-20 CC (CH2Cl2eMeOH 1:1, v/v) to yield compounds 25 (19 mg) and 26 (15 mg). Fraction E6 was fractionated by a silica gel (200e300 mesh) CC eluted with CH2Cl2eMeOH (step gradient, 30:1 to 1:1, v/v) to yield five sub-fractions (6a-6f). Compounds 27 (46 mg), 28 (33 mg) and 29 (62 mg) were isolated on silica gel (200e300 mesh) CC from sub-fraction 6f with CH2Cl2eMeOHeH2O (gradient, 49:1:0.1 to 10:1:0.1, v/v) repeatedly and Sephadex LH-20 CC CC (MeOHeH2O 1:1, v/v), respectively. The structures of the isolated compounds were elucidated according to the spectroscopic data (MS, 1H NMR and 13C NMR) and comparison with data from literature. They were identified as paeonol (1) (Deng et al., 2013), isopaeonol (2) (Lou et al., 1989), 4-hydroxyacetophenone (3) (Lin, 2005), acetovanillone (4) (Yuan, 2007), 2-hydroxy-5-methoxyacetophenone (5) (Kim et al., 2013), eicosanol (6) (Luo et al., 2010), eicosanoic acid (7) (Jia et al., 2013), hexacosanol (8) (Xu et al., 2010), hexacosanoic acid (9) (Yao et al., 1998), b-sitosterol (10) (Chen et al., 2008), daucosterol (11) (Chen et al., 2008), antofine (12) (Lou et al., 1995), 14-hydroxyantofine (13) (Zhang et al., 1991), 5-O-demethylantofine (14) (Lou et al., 1995), b-amyrin (15) (Wang et al., 2007), a-amyrin (16) (Wang et al., 2007), lupeol (17) (Pang, 2008; Ma, 2008), taraxasterol (18) (Gan, 2009), ursolic acid (19) (Shan, 2008), oleanolic acid (Wang et al., 2007) (20), maslinic acid (21) (Li, 2008), caudatin (22) (Chen, 2009), kidjolanin (23) (Gong and Bai, 2009), deacylmetaplexigenin (24) (Shan, 2008), glaucogenin C (25) (Tan, 2003), neocynapanogenin F (26) (Tan, 2003), succinic acid (27) (Yin et al., 2007), octanedioic acid (28) (Niu et al., 2006) and mannotol (29) (Jiang et al., 2008), respectively (Fig. 1). 4. 4. Chemotaxonomic significance The present study reported the isolation of twenty-nine compounds from the root of Cynanchum paniculatum, including acetophenone derivates (1e5), phenanthroindolizidine alkaloids (12e14), pentacyclic triterpenoids (15e21), C21 steroidal sapogenins (22e26), as well as some saturated hydrocarbons (6e9, 27e29). To the best of our knowledge, compounds 6, 8, 21, 28 and 29 were isolated from Asclepiadaceae family for the first time. Besides, this is the first report of compounds 9 and 18 from the genus Cynanchum, and compounds 7, 15e17, 19, 20 and 27 were obtained from C. paniculatum for the first time. The acetophenone derivatives (1e5) were reported from other species of genus Cynanchum. For example, compound 1 was previously reported from Cynanchum auriculatum Royle ex Wight (Deng et al., 2013) and Cynanchum komarovii Al. Iljinski (Yao et al., 1998), compound 3 was obtained from Cynanchum bungei Decne (Lin, 2005), while compounds 4 and 5 were isolated from Cynanchum atratum Bunge (Yuan et al., 2007; Yuan, 2007). The acquisition of 1e5 strongly supported the taxonomic
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Fig. 1. Structures of compounds 1e29.
position of C. paniculatum under the genus Cynanchum. Three phenanthroindolizidine alkaloids (12e14) were yielded from species of genus Cynanchum, such as Cynanchum hancockianum (Lou et al., 1995) (12 and 14) and C. komarovii (Zhang et al., 1991) (13). Thus, it can be confirmed that C. paniculatum is on intimate relations with C. hancockianum and C. komarovii. As to the C21 pregnane sapogenins (22e24), compound 22 was isolated from Cynanchum wallichi Wight. (Chen, 2009) and C. bungei (Gong and Bai, 2009), compound 23 was given from C. auriculatum (Yin et al., 2007) and Cynanchum wilfordii (Xing, 2008), while compound 24 was obtained from C. auriculatum (Shan, 2008). As a disecopregnane sapogenin, compound 25 was once yielded from C. atratum (Zhang et al., 1985). These evidences indicate that these species of genus Cynanchum may have similar biosynthesis system and genetic relationship with each other, and C21 sapogenins could be useful chemotaxonomic markers for genus Cynanchum. Seven pentacyclic triterpenoids (15e21), except for 18 and 21, have been once reported from the genus Cynanchum. Compounds 15, 16 and 20 were found in Cynanchum thesioides (Freyn) K. Schum (Wang et al., 2007), compound 17 in C. bungei (Pang, 2008) and Cynanchum chinense R. Br. (Ma, 2008), while compound 19 was isolated from C. auriculatum (Shan, 2008). These findings have exhibited the chemotaxonomic relationships between C. paniculatum and genus Cynanchum. However, because these compounds are very common in many plants of different genus, more powerful evidences should be found to confirm that pentacyclic triterpenoids can represent as biomarkers for genus Cynanchum. Additionally, compound 18 was previously reported from Periploca forrestii Schlechter (Asclepiadaceae) (Gan, 2009) and compound 24 was also isolated from Asclepias incarnata (Asclepiadaceae) (Warashina and Noro, 2000), which may indicate that C. paniculatum belongs to Asclepiadaceae family. Furthermore, to our knowledge, compounds 2 and 26 were only obtained from C. paniculatum. Thus, they may be regarded as fingerprints for this species to differentiate it from other species of genus Cynanchum. The saturated hydrocarbons, compounds 6e11 and 27e29, were obtained from different species in many families. They were reported from Ainsliaea spicata (Asteraceae, 6) (Luo et al., 2010), Dregea sinensis Hemsl (Asclepiadaceae, 7) (Jia et al., 2013), Kalimeris indica (Asteraceae, 8) (Xu et al., 2010), C. komarovii (Asclepiadaceae,9) (Yao et al., 1998), Cynanchum amplexicaule Sieb. et Zucc. (Asclepiadaceae, 10 and 11) (Chen et al., 2008), C. auriculatum (Asclepiadaceae, 27) (Yin et al., 2007), Semiaquilegia adoxoides (Ranunculaceae, 28) (Niu et al., 2006), as well as Bidens bipinnata L. (Asteraceae, 29) (Jiang et al., 2008), respectively. Thus, the isolation of 6e11 and 27e29 in current research could hardly provide essential chemical evidences for exploring the chemotaxonomic significances among these mentioned species. The study indicates that C. paniculatum shares some compounds with other species of genus Cynanchum but the profile of compounds in this species also differs from others. From a chemotaxonomic point of view, acetophenone derivatives and C21
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pregnane sapogenins may be recognized as markers for Cynanchum genus. Additionally, as exclusively isolated from this plant, isopaeonol and neocynapanogenin F can be served as fingerprints for C. paniculatum. Acknowledgments This work was financially supported by A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). References Chen, G., 2009. Shenyang Pharm. Univ. 56. Chen, H., Yao, Y., Qiao, L., Zhou, Y.Z., Hua, H.M., Pei, Y.H., 2008. Chin. J. Med. Chem. 18, 51. Deng, Y., He, J.B., Guan, K.Y., Zhu, H.J., 2013. Nat. Prod. Res. Dev. 25, 729. Dou, J., Li, P., Bi, Z.M., Zhou, J.L., 2007. Chin. Chem. Lett. 18, 300. Gan, X.H., 2009. Guizhou Univ. 35. Gong, S.L., Bai, Y.P., 2009. Strait Pharm. J. 21, 84. Huang, X., Tan, A.M., Yang, S.B., Zhang, A.Y., Zhang, H., 2009. Helvetica Chim. Acta 92, 937. Jia, S.H., Liu, F.L., Lv, F., Chen, Y., Dai, R.J., Meng, W.W., Deng, Y.L., 2013. Nat. Prod. Res. Dev. 25, 631. Jiang, H.Q., Wang, J.P., Liu, Y.H., Gong, L.L., Liu, H.Y., 2008. Food drug 10, 15. Jiang, Y., Li, B.T., 1977. Flora of China, vol. 63. Science Press, Beijing, p. 309. Kim, M.G., Yang, J.Y., Lee, H.S., 2013. J. Agric. Food Chem. 61, 7568. Lee, S.K., Nam, K.A., Heo, Y.H., 2003. Planta Med. 69, 21. Li, S.L., Tan, H., Shen, Y.M., Kawazoe, K., Hao, X.J., 2004. J. Nat. Prod. 67, 82. Li, Y.W., 2008. Beijing Univ. Tradit. Chin. Med. 22. Lin, A.Q., 2005. Shandong Univ. Traditional Chin. Med. 5. Lou, F.C., Li, X., Ma, Y.Q., Meng, Y., Wu, Q., 1989. J. China Pharm. Univ. 20, 167. Lou, H.X., Li, X., Chu, X.J., Jiang, Z.M., Zhao, Y.W., 1995. Acta Acad. Med. Shandong 33, 158. Luo, Y.P., Zhao, X.T., Zhao, J.F., Yang, X.D., Li, L., 2010. China Pharm. 21, 1396. Ma, Y., 2008. Shandong Univ. Traditional Chin. Med. 17. Niu, F., Xie, G.B., Cui, Z., Chen, F.K., Tu, P.F., 2006. J. Chin. Pharm. Sci. 15, 251. Pang, Y.J., 2008. Shandong Univ. Traditional Chin. Med. 15. Shan, L., 2008. Second Mil. Med. Univ. 71. Tan, H., 2003. Nanjing Agric. Univ. 28. Wang, D., Chen, G., Qiao, L., Zhang, N., Lv, A.L., Dang, Q., Pei, Y., 2007. Chin. J. Med. Chem. 17, 101. Warashina, T., Noro, T., 2000. Phytochemistry 53, 485. Xing, A.T., 2008. Second Mil. Med. Univ. 37. Xu, W.Q., Gong, X.J., Zhou, X., Mei, X.P., Chen, B.B., 2010. Zhongyaocai 41, 1056. Yao, Y.C., Li, G.R., Zhang, X., Wang, Y.D., Cui, X.L., Lin, M.L., Huang, R.Q., 1998. J. Inn. Mong. Polytech. Univ. 17, 1. Yin, M., Chen, Y., Wang, M., Dong, Y.F., Xia, B., Feng, X., 2007. Zhongyaocai 30, 1245. Yuan, Y., 2007. Shanghai Jiao Tong Univ. 43. Yuan, Y., Zhang, W.D., Zhang, C., Liu, R.H., Su, J., Jin, H.Z., 2007. Zhongguo Zhongyao Zazhi 32, 1895. Zhang, R., Fang, S.D., Chen, Y., Lv, S.X., 1991. Acta Bot. Sin. 33, 870. Zhang, Z.X., Zhou, J., Hayashi, K., Mitsuhashi, H., 1985. Chem. Pharm. Bull. 33, 4188. Zhao, Y.B., Fan, Q.S., Xu, G.L., Feng, Z.L., Hao, X.J., 2008. J. Carbohyd. Chem. 27, 401.
Contents lists available at ScienceDirect
Biochemical Systematics and Ecology journal homepage: www.elsevier.com/locate/biochemsyseco
Chemical constituents from Cynanchum paniculatum (Bunge) Kitag Ya-Le Niu a, 1, Xi Chen a, 1, Yi Wu a, *, Hai-Qiang Jiang b, Xue-Lan Zhang b, En-Tao Li a, You-Ying Li a, Hong-Lei Zhou b, Jia-Guo Liu a, De-Yun Wang a, ** a
Institute of Traditional Chinese Veterinary Medicine, College of Veterinary Medicine, Nanjing Agricultural University, #1 Weigang, Nanjing 210095, Jiangsu Province, PR China b College of Pharmacy, Shandong University of Traditional Chinese Medicine, #4655 Daxue Road, Jinan 250355, Shandong Province, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 March 2015 Received in revised form 13 May 2015 Accepted 13 June 2015 Available online xxx
Twenty-nine compounds, including five acetophenone derivatives (1e5), three phenanthroindolizidine alkaloids (12e14), seven pentacyclic triterpenoids (15e21) and five C21 steroidal sapogenins (22e26), were isolated from the root of Cynanchum paniculatum (Bunge) Kitag. Their structures were determined by spectroscopic methods and comparison with reported data. Moreover, the chemotaxonomic relationships were also discussed. As a result, acetophenone derivatives, pentacyclic triterpenoids and C21 pregnane sapogenins can be recognized as chemotaxonomic markers for Cynanchum genus, and C. paniculatum has close relationships with some species of genus Cynanchum. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Cynanchum paniculatum (Bunge) Kitag Steroidal sapogenins Acetophenone derivatives Pentacyclic triterpenoids Chemotaxonomic relationships
1. 1. Subject and source The genus Cynanchum (Asclepiadaceae family) consists of nearly 200 species which distributes in east Africa, the Mediterranean regions, as well as the tropical, subtropical and temperate areas of Europe and Asia (Jiang and Li, 1977). Cynanchum paniculatum (Bunge) Kitag, a vivacious herb growing in China, South Korea and Japan, is used as a traditional Chinese medicine to treat snake bites, hives, and chronic tracheitis (Li et al., 2004; Huang et al., 2009). The dried roots of C. paniculatum were collected in Pingyi county, Shandong Province of China, in September 2012, and were identified by Dr. Yi Wu, College of Veterinary Medicine, Nanjing Agricultural University, where a voucher specimen (No. XCQ201209) was deposited. 2. 2. Previous work Previous phytochemical investigations on C. paniculatum have revealed the presence of C21 steroidal glycosides (Li et al., 2004; Dou et al., 2007), phenolic derivatives (Kim et al., 2013), alkaloids (Lee et al., 2003) and polysaccharides (Zhao et al., 2008). However, to the best of our knowledge, no systematical studies have discussed the chemotaxonomic relationships between this plant and other species of genus Cynanchum.
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected], [email protected] (Y. Wu), [email protected] (D.-Y. Wang). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.bse.2015.06.018 0305-1978/© 2015 Elsevier Ltd. All rights reserved.
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3. 3. Present study The air-dried roots of C. paniculatum (20 kg) were pulverized and extracted with 95% ethanol (v/v, 80 L 2) and 80% ethanol (v/v, 80 L 1) under reflux, each time for 2 h. The combined solvent was filtered and concentrated after removal of solvent under reduced pressure to give a brown residue (900 g). The residue was suspended in water and partitioned with petroleum ether (PE, 60e90 C), ethyl acetate (EtOAC) and n-butanol (n-BuOH), sequentially. The organic layers were concentrated in vacuum to give PE (45 g), EtOAc (105 g) and n-BuOH (260 g) extracts, respectively. The PE fraction was performed on a silica gel open column chromatography (CC) eluted with PE-EtOAc (gradient, 100:0 to 0:100, v/v). The eluents were combined into five fractions (P1eP5) by TLC detection. Fraction P1 was subjected to a silica gel column (100e200 mesh) eluted with a gradient of PE-EtOAc (80:1 to 2:1, v/v) to obtain six sub-fractions (1A-1F). Sub-fraction 1A was performed on a silica gel CC with PE-dichloromethane (CH2Cl2) (30:1 to 10:1, v/v) to give compounds 7 (50 mg) and 9 (48 mg). Sub-fraction 1C was chromatographed on a silica gel CC with PE-acetone (50:1 to 10:1, v/v) to give compounds 6 (27 mg) and 8 (34 mg). Fraction P2 was purified on a silica gel (100e200 mesh) CC eluted with a gradient of cyclohexaneEtOAc (20:1 to 2:1, v/v) to yield five sub-fractions (2A-2E). Sub-fraction 2B was isolated on a silica gel CC with PE-acetone (20:1 to 8:1, v/v) to give compounds 1 (80 mg), 2 (32 mg) and 10 (112 mg). Sub-fraction 2C was separated over a silica gel CC with n-hexane-acetone (15:1 to 10:1, v/v) to provide compounds 3 (27 mg) and 4 (41 mg). Sub-fraction 2D was fractioned by a silica gel CC with cyclohexane-acetone (12:1 to 6:1, v/v) to yield compound 5 (16 mg). Fraction P3 was subjected to a silica gel (100e200 mesh) CC with PE-EtOAc (gradient, 30:1 to 3:1, v/v) to give three sub-fractions (3A-3C). Sub-fraction 3A was performed on a silica gel CC with PE-acetone (12:1 to 6:1, v/v) to obtain compounds 15 (25 mg) and 16 (36 mg). Sub-fraction 3C was purified by a silica gel CC with PE-acetone (10:1 to 8:1, v/v) to give compounds 17 (19 mg) and 18 (11 mg). Fraction P5 was chromatographed on a silica gel (100e200 mesh) CC with PE-EtOAc (15:1 to 1:1, v/v) to give four sub-fractions (5A-5D). Sub-fraction 5B was further separated by a silica gel CC with PE-acetone (12:1 to 6:1, v/v) to yield compounds 19 (38 mg), 20 (45 mg) and 21 (17 mg). Sub-fraction 5D was performed on a silica gel CC with CH2Cl2-methanol (MeOH) (50:1, v/v) to obtain compound 11 (54 mg). The EtOAc fraction was subjected to a silica gel CC with CH2Cl2eMeOH gradient mixtures (100:1 to 0:1, v/v) to yield seven sub-fractions (E1-E7) by TLC detection. Fraction E1 was then loaded on a silica gel column and eluted with a gradient of CH2Cl2-acetone (80:1 to 1:1, v/v) to obtain three sub-fractions (1a-1c). Sub-fraction 1a was then isolated by repeated preparative TLC (gradient PE-CH2Cl2eMeOH) to achieve compounds 12 (15 mg) and 14 (17 mg). Compound 13 (23 mg) were purified from sub-fraction 1b by repeated preparative TLC (gradient PE-CH2Cl2eMeOH). Fraction E2 was processed by a silica gel CC with mobile phase of PE-acetone (20:1 to 0:1, v/v) to give three sub-fractions (2a-2c). Sub-fraction 2a was then purified by repeated preparative TLC (gradient n-hexane-CH2Cl2-acetone, 15:3:1 to 9:3:2), then decolorized by Sephadex LH-20CC (CH2Cl2eMeOH 1:1, v/v) to yield compounds 22 (13 mg), 23 (8 mg) and 24 (17 mg), respectively. Sub-fraction 2c was chromatographed by repeated preparative TLC (gradient CH2Cl2-Acetone, 7:1 to 7:3), then purified respectively by Sephadex LH-20 CC (CH2Cl2eMeOH 1:1, v/v) to yield compounds 25 (19 mg) and 26 (15 mg). Fraction E6 was fractionated by a silica gel (200e300 mesh) CC eluted with CH2Cl2eMeOH (step gradient, 30:1 to 1:1, v/v) to yield five sub-fractions (6a-6f). Compounds 27 (46 mg), 28 (33 mg) and 29 (62 mg) were isolated on silica gel (200e300 mesh) CC from sub-fraction 6f with CH2Cl2eMeOHeH2O (gradient, 49:1:0.1 to 10:1:0.1, v/v) repeatedly and Sephadex LH-20 CC CC (MeOHeH2O 1:1, v/v), respectively. The structures of the isolated compounds were elucidated according to the spectroscopic data (MS, 1H NMR and 13C NMR) and comparison with data from literature. They were identified as paeonol (1) (Deng et al., 2013), isopaeonol (2) (Lou et al., 1989), 4-hydroxyacetophenone (3) (Lin, 2005), acetovanillone (4) (Yuan, 2007), 2-hydroxy-5-methoxyacetophenone (5) (Kim et al., 2013), eicosanol (6) (Luo et al., 2010), eicosanoic acid (7) (Jia et al., 2013), hexacosanol (8) (Xu et al., 2010), hexacosanoic acid (9) (Yao et al., 1998), b-sitosterol (10) (Chen et al., 2008), daucosterol (11) (Chen et al., 2008), antofine (12) (Lou et al., 1995), 14-hydroxyantofine (13) (Zhang et al., 1991), 5-O-demethylantofine (14) (Lou et al., 1995), b-amyrin (15) (Wang et al., 2007), a-amyrin (16) (Wang et al., 2007), lupeol (17) (Pang, 2008; Ma, 2008), taraxasterol (18) (Gan, 2009), ursolic acid (19) (Shan, 2008), oleanolic acid (Wang et al., 2007) (20), maslinic acid (21) (Li, 2008), caudatin (22) (Chen, 2009), kidjolanin (23) (Gong and Bai, 2009), deacylmetaplexigenin (24) (Shan, 2008), glaucogenin C (25) (Tan, 2003), neocynapanogenin F (26) (Tan, 2003), succinic acid (27) (Yin et al., 2007), octanedioic acid (28) (Niu et al., 2006) and mannotol (29) (Jiang et al., 2008), respectively (Fig. 1). 4. 4. Chemotaxonomic significance The present study reported the isolation of twenty-nine compounds from the root of Cynanchum paniculatum, including acetophenone derivates (1e5), phenanthroindolizidine alkaloids (12e14), pentacyclic triterpenoids (15e21), C21 steroidal sapogenins (22e26), as well as some saturated hydrocarbons (6e9, 27e29). To the best of our knowledge, compounds 6, 8, 21, 28 and 29 were isolated from Asclepiadaceae family for the first time. Besides, this is the first report of compounds 9 and 18 from the genus Cynanchum, and compounds 7, 15e17, 19, 20 and 27 were obtained from C. paniculatum for the first time. The acetophenone derivatives (1e5) were reported from other species of genus Cynanchum. For example, compound 1 was previously reported from Cynanchum auriculatum Royle ex Wight (Deng et al., 2013) and Cynanchum komarovii Al. Iljinski (Yao et al., 1998), compound 3 was obtained from Cynanchum bungei Decne (Lin, 2005), while compounds 4 and 5 were isolated from Cynanchum atratum Bunge (Yuan et al., 2007; Yuan, 2007). The acquisition of 1e5 strongly supported the taxonomic
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Fig. 1. Structures of compounds 1e29.
position of C. paniculatum under the genus Cynanchum. Three phenanthroindolizidine alkaloids (12e14) were yielded from species of genus Cynanchum, such as Cynanchum hancockianum (Lou et al., 1995) (12 and 14) and C. komarovii (Zhang et al., 1991) (13). Thus, it can be confirmed that C. paniculatum is on intimate relations with C. hancockianum and C. komarovii. As to the C21 pregnane sapogenins (22e24), compound 22 was isolated from Cynanchum wallichi Wight. (Chen, 2009) and C. bungei (Gong and Bai, 2009), compound 23 was given from C. auriculatum (Yin et al., 2007) and Cynanchum wilfordii (Xing, 2008), while compound 24 was obtained from C. auriculatum (Shan, 2008). As a disecopregnane sapogenin, compound 25 was once yielded from C. atratum (Zhang et al., 1985). These evidences indicate that these species of genus Cynanchum may have similar biosynthesis system and genetic relationship with each other, and C21 sapogenins could be useful chemotaxonomic markers for genus Cynanchum. Seven pentacyclic triterpenoids (15e21), except for 18 and 21, have been once reported from the genus Cynanchum. Compounds 15, 16 and 20 were found in Cynanchum thesioides (Freyn) K. Schum (Wang et al., 2007), compound 17 in C. bungei (Pang, 2008) and Cynanchum chinense R. Br. (Ma, 2008), while compound 19 was isolated from C. auriculatum (Shan, 2008). These findings have exhibited the chemotaxonomic relationships between C. paniculatum and genus Cynanchum. However, because these compounds are very common in many plants of different genus, more powerful evidences should be found to confirm that pentacyclic triterpenoids can represent as biomarkers for genus Cynanchum. Additionally, compound 18 was previously reported from Periploca forrestii Schlechter (Asclepiadaceae) (Gan, 2009) and compound 24 was also isolated from Asclepias incarnata (Asclepiadaceae) (Warashina and Noro, 2000), which may indicate that C. paniculatum belongs to Asclepiadaceae family. Furthermore, to our knowledge, compounds 2 and 26 were only obtained from C. paniculatum. Thus, they may be regarded as fingerprints for this species to differentiate it from other species of genus Cynanchum. The saturated hydrocarbons, compounds 6e11 and 27e29, were obtained from different species in many families. They were reported from Ainsliaea spicata (Asteraceae, 6) (Luo et al., 2010), Dregea sinensis Hemsl (Asclepiadaceae, 7) (Jia et al., 2013), Kalimeris indica (Asteraceae, 8) (Xu et al., 2010), C. komarovii (Asclepiadaceae,9) (Yao et al., 1998), Cynanchum amplexicaule Sieb. et Zucc. (Asclepiadaceae, 10 and 11) (Chen et al., 2008), C. auriculatum (Asclepiadaceae, 27) (Yin et al., 2007), Semiaquilegia adoxoides (Ranunculaceae, 28) (Niu et al., 2006), as well as Bidens bipinnata L. (Asteraceae, 29) (Jiang et al., 2008), respectively. Thus, the isolation of 6e11 and 27e29 in current research could hardly provide essential chemical evidences for exploring the chemotaxonomic significances among these mentioned species. The study indicates that C. paniculatum shares some compounds with other species of genus Cynanchum but the profile of compounds in this species also differs from others. From a chemotaxonomic point of view, acetophenone derivatives and C21
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