刺楸皂苷结构解析及其结构与活性关系研究
摘要
本文对刺楸树皮进行了系统的化学成分研究,以醋酸扭体法扭体反应抑制率、延长扭体潜伏期、降低醋酸致小鼠腹腔毛细血管通透性和提高小鼠热板痛反应阈为活性筛选指标,考察了刺楸树皮75%乙醇提取物经萃取分离得到的乙醚层、氯仿层、醋酸乙酯层、正丁醇层及水层等五个提取部位的药理活性,结果显示刺楸氯仿层、醋酸乙酯层和正丁醇层是主要活性部位。根据活性筛选的结果,从不同活性部位分离得到25 个单体化合物,经光谱技术和理化方法鉴定了20 个单体成分,其中6 个为新化合物,6 个为首次从该植物中分离得到的化合物。本文采用高效液相色谱-蒸发光散射检测器联用技术首次对刺楸皂苷酸水解物中单糖种类和数量进行了分析,并采用分子力学力场推测刺楸皂苷分子的空间构型变化,探讨了皂苷分子糖链数以及糖基数对刺楸皂苷的空间结构、1H NMR 以及药理活性的影响。药理活性研究表明刺楸皂苷类成分的结构与活性之间存在一定相关性。
Saponins are abundantly distributed throughout the nature, which are composed of two main moieties: aglycone and sugar chains. Aglycones are triterpenes, steroids, and steroid alkaloids, while sugar chains are more complexity consisting of mono-saccharides that are quite simple. Because of little heed to the regulating effects on the activities of aglycone, many researches of pharmacological activities mainly focus on the aglycones rather than sugar chains over a long period of time, but numerous facts have led to a conclusion that the sugar chains of saponins play much more important roles in regulating the saponin or the aglycone in recent years. The genus Kalopanax (Araliaceae) comprises 1 species of deciduous, small to medium-sized tree that is native to China, Japan, eastern Russia, and Korea, while in China the plant was authenticated as Kalopanax septemlobus (Thunb.) Koidz.. The dried bark has been used as a traditional chinese medicine for expelling so-called wind-evil, eradicating wetness, mediating meridian, destroying intestinal parasites, also clinically been treated with paralysis caused by wind and damp, pain along the knee and spine. Korean scientists studied the cytotoxicity activities of hederagenin, δ-hederin, kalopanaxsaponin A, kalopanaxsaponin I, and sapindoside C
in the bark of K. pictus, meanwhile their extracts are consists of aglycone, monosaccharide triterpenoid saponin, bisaccharide triterpenoid saponin, trisaccharide triterpenoid saponin, and quadsaccharide triterpenoid saponin, respectively. The results show that the cytotoxicity activity of the aglycone is low, otherwise the activities of kalopanaxsaponin A, kalopanaxsaponin I, and sapindoside C are relatively high, while the monosaccharide saponin of δ-hederin has no avail. In the in vivo experiments, kalopanaxsaponin A apparently increased the life span of mice bearing Colon 26 and 3LL Lewis lung carcinoma, those results indicated that the number of sugars linked to the aglycone is much more important in pharmacological effects. In our investigation of antiinflammatory activity fractions in the bark of K. septemlobus, 25 compounds were isolated with solvent extraction, silica gel, Sephadex LH-20, reverse phase C-18, and semipreparative HPLC, etc. Among them, 20 compounds were elucidated on the basis of chemical and spectral methods such as 1H NMR, 13C NMR, DQF-COSY, DEPT, HMQC, HMBC, TOCSY, NOESY, ESI-MS, HRESI-MS. In the dissertation , 6 new triterpenoid saponins were named as septemloside Ⅰ, septemloside Ⅱ, septemloside Ⅲ, septemloside A, septemloside B, and septemloside C; along with 6 well-known compounds: β-sitosterol , gallic acid, syringin, phenylacetic acid, p-cresol, sinapyl alcohol were separated and characterized respectively for the first time.
Because of the difficulty in crystalization of triterpenoid saponins, the steric conformation of triterpenoid saponins cannot be determined by method of using X-ray diffraction. Our further explorations in the steric conformation of kalopanaxsaponins lies on the molecular mechanics force field (MM2). As result, the predicted geometries by MM2 for the most
stable conformers of the kalopanaxsaponins were studied through initial molecular dynamics. Two main stable conformers were named as twist style and extension style, for two-sugar-chain kalopanaxsaponin. The twist style is the conformation that two sugar chains of C-3 and C-28 in the molecule are close to each other, while the extension style is the conformation that the two sugar chains are apart from each other on the contrary. Along with the number of saccharides increases, the two sugar chains in kalopanaxsaponins tend to transform from extension style to twist style, which is affected by the molecular concentration. The NOE was determined betweenδ6.71 of C2-OH in the terminal D-xylose at C-3 sugar chain and δ6.63 of C2-OH in the terminal D-xylose at C-28 sugar chain, but no NOE was detected among any hydroxyl bwteen the terminal D-xylose and L-rhamnose at C-28 sugar chain in septemloside Ⅲ, these suggest that the branched sugar chains at C-28 are apart from each other whereas the C-3 sugar chain is close to the C-28 sugar chain. The NOESY and COSY spectra confirmed septemloside Ⅲexist a groove which is built by C2-OH of terminal D-xylose at C-28 sugar chain with which hydroxyls close to in the space. The groove explains the reason of so low magnetic field of hydroxyl owing to the oxygen’s paramagnetic effect.
Kalopanaxsaponins have variable steric conformations which can be transformed by changing their concentration, which was confirmed by the 1H NMR spectra. Singal can not be detected among δ6~8 at the examine concentration of 20 mg·mL-1 in pyridine-d5, while some were found among δ6~8 at the examine concentration of 50 mg·mL-1 in pyridine-d5, these phenomenon suggest the steric conformation of septemloside Ⅲmight transform to other as examine concentration changed, i.e.
Saponins are abundantly distributed throughout the nature, which are composed of two main moieties: aglycone and sugar chains. Aglycones are triterpenes, steroids, and steroid alkaloids, while sugar chains are more complexity consisting of mono-saccharides that are quite simple. Because of little heed to the regulating effects on the activities of aglycone, many researches of pharmacological activities mainly focus on the aglycones rather than sugar chains over a long period of time, but numerous facts have led to a conclusion that the sugar chains of saponins play much more important roles in regulating the saponin or the aglycone in recent years. The genus Kalopanax (Araliaceae) comprises 1 species of deciduous, small to medium-sized tree that is native to China, Japan, eastern Russia, and Korea, while in China the plant was authenticated as Kalopanax septemlobus (Thunb.) Koidz.. The dried bark has been used as a traditional chinese medicine for expelling so-called wind-evil, eradicating wetness, mediating meridian, destroying intestinal parasites, also clinically been treated with paralysis caused by wind and damp, pain along the knee and spine. Korean scientists studied the cytotoxicity activities of hederagenin, δ-hederin, kalopanaxsaponin A, kalopanaxsaponin I, and sapindoside C
in the bark of K. pictus, meanwhile their extracts are consists of aglycone, monosaccharide triterpenoid saponin, bisaccharide triterpenoid saponin, trisaccharide triterpenoid saponin, and quadsaccharide triterpenoid saponin, respectively. The results show that the cytotoxicity activity of the aglycone is low, otherwise the activities of kalopanaxsaponin A, kalopanaxsaponin I, and sapindoside C are relatively high, while the monosaccharide saponin of δ-hederin has no avail. In the in vivo experiments, kalopanaxsaponin A apparently increased the life span of mice bearing Colon 26 and 3LL Lewis lung carcinoma, those results indicated that the number of sugars linked to the aglycone is much more important in pharmacological effects. In our investigation of antiinflammatory activity fractions in the bark of K. septemlobus, 25 compounds were isolated with solvent extraction, silica gel, Sephadex LH-20, reverse phase C-18, and semipreparative HPLC, etc. Among them, 20 compounds were elucidated on the basis of chemical and spectral methods such as 1H NMR, 13C NMR, DQF-COSY, DEPT, HMQC, HMBC, TOCSY, NOESY, ESI-MS, HRESI-MS. In the dissertation , 6 new triterpenoid saponins were named as septemloside Ⅰ, septemloside Ⅱ, septemloside Ⅲ, septemloside A, septemloside B, and septemloside C; along with 6 well-known compounds: β-sitosterol , gallic acid, syringin, phenylacetic acid, p-cresol, sinapyl alcohol were separated and characterized respectively for the first time.
Because of the difficulty in crystalization of triterpenoid saponins, the steric conformation of triterpenoid saponins cannot be determined by method of using X-ray diffraction. Our further explorations in the steric conformation of kalopanaxsaponins lies on the molecular mechanics force field (MM2). As result, the predicted geometries by MM2 for the most
stable conformers of the kalopanaxsaponins were studied through initial molecular dynamics. Two main stable conformers were named as twist style and extension style, for two-sugar-chain kalopanaxsaponin. The twist style is the conformation that two sugar chains of C-3 and C-28 in the molecule are close to each other, while the extension style is the conformation that the two sugar chains are apart from each other on the contrary. Along with the number of saccharides increases, the two sugar chains in kalopanaxsaponins tend to transform from extension style to twist style, which is affected by the molecular concentration. The NOE was determined betweenδ6.71 of C2-OH in the terminal D-xylose at C-3 sugar chain and δ6.63 of C2-OH in the terminal D-xylose at C-28 sugar chain, but no NOE was detected among any hydroxyl bwteen the terminal D-xylose and L-rhamnose at C-28 sugar chain in septemloside Ⅲ, these suggest that the branched sugar chains at C-28 are apart from each other whereas the C-3 sugar chain is close to the C-28 sugar chain. The NOESY and COSY spectra confirmed septemloside Ⅲexist a groove which is built by C2-OH of terminal D-xylose at C-28 sugar chain with which hydroxyls close to in the space. The groove explains the reason of so low magnetic field of hydroxyl owing to the oxygen’s paramagnetic effect.
Kalopanaxsaponins have variable steric conformations which can be transformed by changing their concentration, which was confirmed by the 1H NMR spectra. Singal can not be detected among δ6~8 at the examine concentration of 20 mg·mL-1 in pyridine-d5, while some were found among δ6~8 at the examine concentration of 50 mg·mL-1 in pyridine-d5, these phenomenon suggest the steric conformation of septemloside Ⅲmight transform to other as examine concentration changed, i.e.
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