接骨木根皮促进骨折愈合有效部位化学成分和药理作用研究
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摘要
接骨木(Sambucus williamsii Hance)为忍冬科接骨木属植物接骨木干燥的根皮,又名续骨木、大接骨丹、公道老、扦扦活、马尿骚等。性味甘、苦、平,无毒;具有接骨续筋、活血止痛、祛风利湿之功效。近年来临床应用接骨木根皮治疗骨折取得了显著的治疗效果,但对接骨木根皮促进骨折愈合的活性成分及药理作用的研究鲜有报道。为了确定接骨木根皮促进骨折愈合的有效部位,系统地阐明该有效部位促进骨折愈合的作用机理,并深入阐明有效部位的活性成分,对接骨木根皮的50%EtOH提取组分进行了药理学和化学方面的研究。
首先,在确定接骨木根皮促进骨折愈合有效部位的基础上,结合药理学的方法,进行了促进骨折愈合、血液生化学、抗炎、镇痛等方面的研究。结果表明,接骨木对骨折家兔一些相关生化指标具有明显改善的作用;可加速骨折的愈合;促进骨折后期骨基质的成熟和钙化;促进胶原合成和钙盐的沉积;提高骨痂质量。另外还具有抗炎、镇痛的作用。
其次,对接骨木根皮促进骨折愈合的有效部位进行了系统的提取分离和化学结构鉴定研究。通过硅胶柱色谱、ODS柱色谱、HPLC等分离方法,采用~1H-NMR、~(13)C-NMR、~1H-~1H COSY、HSQC和HMBC等1D、2D-NMR波谱学测试手段,结合ESI-MS质谱,分离并鉴定了23个化合物的结构。其中12个环烯醚萜类化合物、10个木脂素类化合物和1个苯丙醇类化合物。在这23个化合物中,有5个环烯醚萜类化合物和4个木脂素类化合物为新化合物,另有12个化合物在该植物中首次发现。9个新化合物分别被命名为接骨木苷A(7β-O-ethylepimorroniside)、接骨木苷B(7β-O-ethylmorroniside-(6′-O-7″)-β-morroniside)、接骨木苷C(α-D-glucopyranosyl(1→2)-D-fructofuranosyl(4→6)-β-morroniside)、接骨木苷D(α-D-apiofuranosyl(1″→6′)-7-deoxyloganin)、接骨木苷E(α-D-apiofuranosyl(1″→6′)-loganin)、接骨木苷F((7R,8R)-4,9-dihydroxy-3-methoxy-4′:7,5′:8-benzodioxanelignan-9′-O-β-D-glucopyranoside)、接骨木苷G((7S,8R)-4,9-dihydroxy-3,3′-dimethoxy-7,8dihydrobenzofuran-1′-propylneolignan-9′-O-[α-D-apiofuranosyl(1→6)]-β-D-glucopyranoside)、接骨木苷H(4,7,9,3′-tetrahydroxy-3-methoxy-8-O-4′
Sambbucus williamsii Hance belongs to the genus Sambbucus chinenis. As a species of the genus, Sambbucus williamsii Hance is also called Xu-gu-mu, Da-jie-gu-dan, Gong-lao-dao, etc. Sambbucus williamsii Hance is a folk medicine with a long history of safe use for treatment of fracture and joint disease in China. However, the active constituent of Sambbucus williamsii Hance and the effects of Sambbucus williamsii Hance on bone fracture remain largely unknown. In this study, the components obtained from the stems of Sambbucus williamsii were elucidated. Besides, the effects of the components on bone fracture and their mechanisms of action were examined in vivo and in vitro.At first, we identified the bioactive constituents from the 50% ethanol extract of the stems of Sambbucus williamsii. Then, the effects of isolated compounds on bone fracture, hemorrheology, blood biochemistry, anti-inflammation and analgesia were evaluated. The results showed that the active components markedly improving bone fracture recovery. Treatment with isolated compounds exerted a significant elevation in serum level of AIP and BGP and they showed marked anti-infflamatory effect, analgesic effect as well. Administration of the active components markedly ameliorated hemorheology when compared with model.In this study, isolation procedure was carried out by column chromatography and HPLC;structures of isolated compounds were identified by ~1H-NMR, ~(13)C-NMR, ~1H-~1HCOSY, HSQC, HMBC and ESI-MS. Isolation of the 50% ethanol extract of the stems of Sambbucus williamsi afforded 23 constituents, including 12 iridoids compounds, 10 lignans compounds, 1 phenylpropanol compound, in which 5 iridoids compounds and 4 lignans compounds were novel compounds and 12 compounds were firstly found in Sambucus williamsii Hance . The structures of the nine novel compounds were identified as Williamsoside A(7- β -O-Ethylepimo-rroniside),Williamsoside B (7β -O-ethylmorroniside-(6'-O-7")- β -morroniside), Williamsoside C ( α -D-glucopyranosyl(1→2)-D-fructofuranosyl(4→6)- β -morroniside ),Williamsoside D ( α -D-apiofuranosyl (1"→6' )-7-deoxyloganin),Williamsoside E ( α-D-apiofuranosyl(1"→6')-loganin) ,Williamsoside F ((7R,8R)-4,9-
dihydroxy-3-methoxy-4'.- 7, 5': 8-benzodioxanelignan-9'-O-￡-D-glucopyrano-side) ,Williamsoside G ((7S,8R)-4,9-dihydroxy-3,3'-dimethoxy-7,8dihydrobenzo-furan-1' -propylneolignan-9' -O-[ a -D-apiofuranosyl (1—6)]- ￡ -D- glucopyrano-side) ,Williamsoside H (4,7,9,3' -tetrahydroxy-3- methoxy-8-0-4' isolignan-9' -O-0 -D- glucopyranoside (thero)) and Williamsoside I.Then in vitro experiments were performed to determine the effects of morroniside on proliferation, differentiation and mineralization of MC3T3-E1. Results suggested morroniside markedly improved the proliferation, differentiation and mineralization of MC3T3-E1. At the same time, the effects of morroniside on the level of Ca2+ in cell were investigated by confocal laser scanning microscope. Results showed that morroniside significantly increased the level of Ca2+ in cell. In conclusion, this study identified the bioactive components of Sambbucus williamsii Hance and investigated the effects of the bioactive components on bone fracture and the mechanisms involved in it. It is undoubted that this study not only affords an evidence to discover a new folk medicine to treat bone fracture but also improve the cognition for bone fracture recovery from pharmacological point.
首先,在确定接骨木根皮促进骨折愈合有效部位的基础上,结合药理学的方法,进行了促进骨折愈合、血液生化学、抗炎、镇痛等方面的研究。结果表明,接骨木对骨折家兔一些相关生化指标具有明显改善的作用;可加速骨折的愈合;促进骨折后期骨基质的成熟和钙化;促进胶原合成和钙盐的沉积;提高骨痂质量。另外还具有抗炎、镇痛的作用。
其次,对接骨木根皮促进骨折愈合的有效部位进行了系统的提取分离和化学结构鉴定研究。通过硅胶柱色谱、ODS柱色谱、HPLC等分离方法,采用~1H-NMR、~(13)C-NMR、~1H-~1H COSY、HSQC和HMBC等1D、2D-NMR波谱学测试手段,结合ESI-MS质谱,分离并鉴定了23个化合物的结构。其中12个环烯醚萜类化合物、10个木脂素类化合物和1个苯丙醇类化合物。在这23个化合物中,有5个环烯醚萜类化合物和4个木脂素类化合物为新化合物,另有12个化合物在该植物中首次发现。9个新化合物分别被命名为接骨木苷A(7β-O-ethylepimorroniside)、接骨木苷B(7β-O-ethylmorroniside-(6′-O-7″)-β-morroniside)、接骨木苷C(α-D-glucopyranosyl(1→2)-D-fructofuranosyl(4→6)-β-morroniside)、接骨木苷D(α-D-apiofuranosyl(1″→6′)-7-deoxyloganin)、接骨木苷E(α-D-apiofuranosyl(1″→6′)-loganin)、接骨木苷F((7R,8R)-4,9-dihydroxy-3-methoxy-4′:7,5′:8-benzodioxanelignan-9′-O-β-D-glucopyranoside)、接骨木苷G((7S,8R)-4,9-dihydroxy-3,3′-dimethoxy-7,8dihydrobenzofuran-1′-propylneolignan-9′-O-[α-D-apiofuranosyl(1→6)]-β-D-glucopyranoside)、接骨木苷H(4,7,9,3′-tetrahydroxy-3-methoxy-8-O-4′
Sambbucus williamsii Hance belongs to the genus Sambbucus chinenis. As a species of the genus, Sambbucus williamsii Hance is also called Xu-gu-mu, Da-jie-gu-dan, Gong-lao-dao, etc. Sambbucus williamsii Hance is a folk medicine with a long history of safe use for treatment of fracture and joint disease in China. However, the active constituent of Sambbucus williamsii Hance and the effects of Sambbucus williamsii Hance on bone fracture remain largely unknown. In this study, the components obtained from the stems of Sambbucus williamsii were elucidated. Besides, the effects of the components on bone fracture and their mechanisms of action were examined in vivo and in vitro.At first, we identified the bioactive constituents from the 50% ethanol extract of the stems of Sambbucus williamsii. Then, the effects of isolated compounds on bone fracture, hemorrheology, blood biochemistry, anti-inflammation and analgesia were evaluated. The results showed that the active components markedly improving bone fracture recovery. Treatment with isolated compounds exerted a significant elevation in serum level of AIP and BGP and they showed marked anti-infflamatory effect, analgesic effect as well. Administration of the active components markedly ameliorated hemorheology when compared with model.In this study, isolation procedure was carried out by column chromatography and HPLC;structures of isolated compounds were identified by ~1H-NMR, ~(13)C-NMR, ~1H-~1HCOSY, HSQC, HMBC and ESI-MS. Isolation of the 50% ethanol extract of the stems of Sambbucus williamsi afforded 23 constituents, including 12 iridoids compounds, 10 lignans compounds, 1 phenylpropanol compound, in which 5 iridoids compounds and 4 lignans compounds were novel compounds and 12 compounds were firstly found in Sambucus williamsii Hance . The structures of the nine novel compounds were identified as Williamsoside A(7- β -O-Ethylepimo-rroniside),Williamsoside B (7β -O-ethylmorroniside-(6'-O-7")- β -morroniside), Williamsoside C ( α -D-glucopyranosyl(1→2)-D-fructofuranosyl(4→6)- β -morroniside ),Williamsoside D ( α -D-apiofuranosyl (1"→6' )-7-deoxyloganin),Williamsoside E ( α-D-apiofuranosyl(1"→6')-loganin) ,Williamsoside F ((7R,8R)-4,9-
dihydroxy-3-methoxy-4'.- 7, 5': 8-benzodioxanelignan-9'-O-￡-D-glucopyrano-side) ,Williamsoside G ((7S,8R)-4,9-dihydroxy-3,3'-dimethoxy-7,8dihydrobenzo-furan-1' -propylneolignan-9' -O-[ a -D-apiofuranosyl (1—6)]- ￡ -D- glucopyrano-side) ,Williamsoside H (4,7,9,3' -tetrahydroxy-3- methoxy-8-0-4' isolignan-9' -O-0 -D- glucopyranoside (thero)) and Williamsoside I.Then in vitro experiments were performed to determine the effects of morroniside on proliferation, differentiation and mineralization of MC3T3-E1. Results suggested morroniside markedly improved the proliferation, differentiation and mineralization of MC3T3-E1. At the same time, the effects of morroniside on the level of Ca2+ in cell were investigated by confocal laser scanning microscope. Results showed that morroniside significantly increased the level of Ca2+ in cell. In conclusion, this study identified the bioactive components of Sambbucus williamsii Hance and investigated the effects of the bioactive components on bone fracture and the mechanisms involved in it. It is undoubted that this study not only affords an evidence to discover a new folk medicine to treat bone fracture but also improve the cognition for bone fracture recovery from pharmacological point.
引文
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