鸢尾苷的提取分离及其与透明质酸酶作用研究
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摘要
本课题主要研究从射干中提取分离鸢尾苷的条件,并对提取物鸢尾苷进行表征;研究鸢尾苷与透明质酸酶之间的作用,同时探究鸢尾苷的抗氧化性能;利用荧光光谱、同步荧光光谱、圆二色谱等仪器分析方法对鸢尾苷抑制透明质酸酶的作用机理进行了探索。
鸢尾苷最佳提取条件为乙醇浓度70%,固液比1:8(w/v),每次提取30 min,该条件下得到纯度70.1%的鸢尾苷,得率为1.333%。经AB-8型大孔吸附树脂和硅胶进行柱层析分离得到纯度96.8%的鸢尾苷,得率为0.653%。使用熔点、紫外、红外、核磁和液质等方法检测提取物,并与标准品比对,确定提取物是鸢尾苷。
提取分离得到的鸢尾苷具有良好的抑制透明质酸酶作用,并且抑制率随着鸢尾苷浓度的增大而增大,抑制率最高达到81.2%。同时,清除过氧化氢(H_2O_2)实验、清除羟自由基(OH·)实验和清除DPPH·实验的结果表明鸢尾苷提取物具有良好的抗氧化性能,对三种自由基最大清除率分别为68.5%、80.7%、39.6%。证明鸢尾苷具有良好的抗致敏性能。
透明质酸酶在347nm附近出现最强发射峰,透明质酸酶的荧光强度随着鸢尾苷浓度的增大逐渐降低。透明质酸酶的猝灭方式为静态猝灭,结合常数KA为0.613×10~3L·mol~(-1)(296K),1.643×10~3L·mol~(-1)(303K),2.441×10~3L·mol~(-1)(310K),结合位点数n≈1,鸢尾苷对透明质酸酶的作用过程是一个自发过程,而鸢尾苷和HAase体系中△H~0=78.53kJ·mol~(-1)大于0,同时△S~0=319.04J·mol~(-1)·K~(-1)大于0,表明鸢尾苷和HAase之间的键合方式主要是疏水作用力。同步荧光光谱(△λ=60nm)和圆二色谱分析表明鸢尾苷使透明质酸酶α-螺旋含量的降低。
The main research contents are as follows: Optimizing the conditions for the extraction of tectoridin, which was characterized by spectroscopic instrument; The inhibition of hyaluronidase by tectoridin, and its antioxidative ability; The interaction mechanism between tectoridin and hyaluronidase by fluorescence spectrum, cirular dichroism, etc.
Results show that the optimum extraction conditions are as follows: ethanol concentration is 70%, the ratio of solid to liquid is 1:8(w/v), extracting 30 min each time, and under this condition the purity of tectoridin is 70.1%, the extraction rate is 1.333%. Through AB-8 macroporous resin and silica gel separation, the purity of tectoridin is 96.8%,and the extraction rate is 0.653%. Extract was tested by m.p,IR, UV,LC-MS and 1H-NMR,and compared with the standard, we ascertain that the extract is tectoridin.
The isolated tectoridin has an active effect on the inhibition of hyaluronidase. Its inhibiton rate are able to reach 81.2% with the increase of tectoridin’s intensity. At the same time, by the eliminate experiment of H_2O_2, OH·and DPPH·, the extract displays reliable ability in antioxidant. The maximum clearance rate against the 3 kinds of free radical are 68.5%, 80.7%, 39.6%, respectively. The research shows that tectoridin has good anti-allergenic.
The result of Fluorescence spectrumshows us that Hyaluronidase appeared the strongest emission peak and the fluoresecence intensity decreased as tectoridin's concentration increased.The way of Hyaluronidase is static quenching,binding constants is 0.613×10~3L·mol~(-1)(296K),1.643×10~3L·mol~(-1)(303K),2.441×10~3L·mol~(-1)and binding number≈1.At the same time, the procedure of tectoridin’s effect on Hyaluronidase happens spontaneously.The bonding construction between tectoridin and Hyaluronidase is lyophobic. The synchronous fluorescence spectra (△λ=60nm) and circular dichroism spectra analysis results shows that tectoridin cause the reduction of theα-helix content of hyaluronidase.
鸢尾苷最佳提取条件为乙醇浓度70%,固液比1:8(w/v),每次提取30 min,该条件下得到纯度70.1%的鸢尾苷,得率为1.333%。经AB-8型大孔吸附树脂和硅胶进行柱层析分离得到纯度96.8%的鸢尾苷,得率为0.653%。使用熔点、紫外、红外、核磁和液质等方法检测提取物,并与标准品比对,确定提取物是鸢尾苷。
提取分离得到的鸢尾苷具有良好的抑制透明质酸酶作用,并且抑制率随着鸢尾苷浓度的增大而增大,抑制率最高达到81.2%。同时,清除过氧化氢(H_2O_2)实验、清除羟自由基(OH·)实验和清除DPPH·实验的结果表明鸢尾苷提取物具有良好的抗氧化性能,对三种自由基最大清除率分别为68.5%、80.7%、39.6%。证明鸢尾苷具有良好的抗致敏性能。
透明质酸酶在347nm附近出现最强发射峰,透明质酸酶的荧光强度随着鸢尾苷浓度的增大逐渐降低。透明质酸酶的猝灭方式为静态猝灭,结合常数KA为0.613×10~3L·mol~(-1)(296K),1.643×10~3L·mol~(-1)(303K),2.441×10~3L·mol~(-1)(310K),结合位点数n≈1,鸢尾苷对透明质酸酶的作用过程是一个自发过程,而鸢尾苷和HAase体系中△H~0=78.53kJ·mol~(-1)大于0,同时△S~0=319.04J·mol~(-1)·K~(-1)大于0,表明鸢尾苷和HAase之间的键合方式主要是疏水作用力。同步荧光光谱(△λ=60nm)和圆二色谱分析表明鸢尾苷使透明质酸酶α-螺旋含量的降低。
The main research contents are as follows: Optimizing the conditions for the extraction of tectoridin, which was characterized by spectroscopic instrument; The inhibition of hyaluronidase by tectoridin, and its antioxidative ability; The interaction mechanism between tectoridin and hyaluronidase by fluorescence spectrum, cirular dichroism, etc.
Results show that the optimum extraction conditions are as follows: ethanol concentration is 70%, the ratio of solid to liquid is 1:8(w/v), extracting 30 min each time, and under this condition the purity of tectoridin is 70.1%, the extraction rate is 1.333%. Through AB-8 macroporous resin and silica gel separation, the purity of tectoridin is 96.8%,and the extraction rate is 0.653%. Extract was tested by m.p,IR, UV,LC-MS and 1H-NMR,and compared with the standard, we ascertain that the extract is tectoridin.
The isolated tectoridin has an active effect on the inhibition of hyaluronidase. Its inhibiton rate are able to reach 81.2% with the increase of tectoridin’s intensity. At the same time, by the eliminate experiment of H_2O_2, OH·and DPPH·, the extract displays reliable ability in antioxidant. The maximum clearance rate against the 3 kinds of free radical are 68.5%, 80.7%, 39.6%, respectively. The research shows that tectoridin has good anti-allergenic.
The result of Fluorescence spectrumshows us that Hyaluronidase appeared the strongest emission peak and the fluoresecence intensity decreased as tectoridin's concentration increased.The way of Hyaluronidase is static quenching,binding constants is 0.613×10~3L·mol~(-1)(296K),1.643×10~3L·mol~(-1)(303K),2.441×10~3L·mol~(-1)and binding number≈1.At the same time, the procedure of tectoridin’s effect on Hyaluronidase happens spontaneously.The bonding construction between tectoridin and Hyaluronidase is lyophobic. The synchronous fluorescence spectra (△λ=60nm) and circular dichroism spectra analysis results shows that tectoridin cause the reduction of theα-helix content of hyaluronidase.
引文
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[4] Shawl AS, Kumar T. Isoflavonoids from Iris Croceo[J]. Phytochemistry, 1992, 31: 1399-1401.
[5] Fujita M, Inoue T. Studies on the constituents of Iris florentina II C-glycosides of xanthones and flavones from the leaves of Iris florentina[J]. Chem Pharm Bull, 1982, 30: 2342-2348.
[6]吉文亮,秦民坚,王峥涛.射干的化学成分研究(I)[J].中国药科大学学报, 2001, 32(3): 197-199.
[7] Ito H, Onoue S, Yoshida T. Isoflavonoids from Belamcanda chinensis[J]. Chem Pharm Bull, 2001, 49(9): 1229-1231.
[8]周立新,林茂,赫兰峰.射干的化学成分研究(I)[J].中草药, 1996, 27(1): 8-10, 59.
[9] Yamaki M, Kato T, Kashihara M, et al. Isoflavones of Belamcanda chinensis[J]. Planta Med, 1990, 56(3): 335.
[10]余亚纲,汪聪慧,刘岱,等.中药射干亲脂中性成分研究[J].药学学报, 1983 , 18(12): 969-972.
[11]秦民坚,吉文亮,王峥涛.射干的化学成分研究(Ⅱ) [J].中草药, 2004, 35(5): 487-489.
[12] Zhou Li-xin, Lin Mao. Studies on chemical constituents of Belamcanda chinensis(L.)DC. (Ⅱ)[J]. Chin Chem Lett, 1997, 8(2): 133-134.
[13] Jin Li, Chen Hai-sheng, Xiang Zhao-bao, et al. New flavone and isoflavone glycoside from Belamcanda chinensis[J]. Chin Chem Lett, 2007, 18(2): 158-160.
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