芦荟汁中胶原酶抑制成分的分离纯化与抑制作用研究
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摘要
本论文主要对芦荟汁中胶原酶活性抑制成分进行了研究,包括胶原酶活性和抑制剂抑制效果测定方法、芦荟汁干粉中有效抑制成分的分离纯化、结构鉴定与抑制效果测定,初步探讨了可能的抑制机理并进一步寻找到其它具有抑制作用的植物提取物。
实验首先研究了羟脯氨酸化学比色法的影响因素,确定实验条件:1.0mL待测溶液中加入2.0mL异丙醇,1.0mL氧化剂氧化5min,再加入2.0mL显色剂,60℃加热显色20min,冰浴冷却5min后测定561nm处的吸光度,根据标准曲线法计算羟脯氨酸质量。
以羟脯氨酸含量测定为检测手段,初步研究了胶原酶对胶原蛋白的酶解反应过程,并在此基础上设计了胶原酶活性及抑制剂抑制效果测定方法:胶原蛋白10mg,胶原酶液浓度0.1mg/mL,反应温度37.0℃;Tris-HCl(三羟甲基氨基甲烷-盐酸)缓冲溶液中胶原酶催化降解固态不溶性胶原蛋白分解为可溶性肽进入溶液,反应终止后滤除未反应胶原蛋白,滤液加浓盐酸密封加热酸解后测定其中游离羟脯氨酸的质量;抑制剂与空白实验的羟脯氨酸质量差值体现出抑制剂的抑制效果。
实验发现芦荟汁干粉中蒽醌苷类化合物具有明显的胶原酶抑制效果;芦荟汁干粉先用热水煎煮,滤液经乙酸乙酯萃取,除去溶剂后得粗提取物;该粗提取物再经硅胶柱层析分离多次,得到含有两种主要物质的混合物;最后经半制备高效液相色谱分离出两种纯物质;经定性实验和HPLC、UV、IR、MS、1H NMR谱图鉴定化学结构,分别确定为芦荟素和异芦荟素。抑制效果测定结果表明两者具有相似的抑制效果并随质量浓度的增大而近似线性升高,1mg/mL时百分抑制率分别达到42.4%和35.0%,低浓度下的抑制效果高于常见抑制剂四环素。
根据文献中胶原酶抑制机理——Zn2+螯合理论,初步分析认为芦荟素、异芦荟素可能也符合该理论,抑制作用可能与其分子结构有关;后续实验还发现芍药、川芎提取物也具有胶原酶抑制效果。
In this paper, we mainly studied the collagenase inhibitory ingredients from the sap of Aloe, including the method of collagenase activity and inhibition test, the extraction and purification of useful ingredients, chemical structure identify, inhibition ratio test, primarily discussed possible inhibitory mechanism and other inhibitory plants extracts in further study.
First of all, we studied the effective factors of Hydroxyproline photocolorimetric method, confirm the test condition as: 1.0mL sample add 2.0mL Isopropanol, 1.0mL oxidation reagent and oxidated 5min, then add 2.0mL color reagent, cooled in icy water 5min after heated 20min at 60℃; Test its UV absorption at 561nm, calculate the Hydroxyproline mass by its standard curve.
Use the Hydroxyproline photocolorimetric method, we studied the interaction between collagenase and collagen, then designed the method of collagenase activity and inhibition test: 10mg collagen with 0.1mg/mL collagenase solution at 37.0℃in Tris-HCl buffer, collagenase degraded the insoluble collagen to soluble protein into the buffer, Filtrated the undegraded collagen and add HCl heated in a sealed tube. Then test the Hydroxyproline’s mass in the tube by Hydroxyproline photocolorimetric method, collaterally calculated the collagen’s mass. The difference between blank sample and inhibition sample indicated the effect of inhibitor.
Inhibitory test found the Anthraquinones glycoside materials in Aloe sap have obviously collagenase inhibition; Aloe Power was decocted by hot water, extracted by ethyl acetate and got crude extract after dissolvent; This crude material was separated several times by silica column and got an extract mainly contain two ingredients, then isolated to two pure ingredients by Semi- Preparative HPLC. UV, IR, MS & 1H NMR tested their chemical structure, confirmed as Barbaloin & Isobarbaloin respectively. Inhibition ratio test found they have similar and dose dependent inhibitory activity, respective inhibition ratio was 42.4% & 35.0% at 1mg/mL, higher inhibition ratio in low concentration compared with tetracycline.
According to the collagenase inhibitory mechanism of Zn2+ chelating theory, we primarily considered Barbaloin & Isobarbaloin also follow this theory, the inhibition maybe due to their Special chemical structure in the molecule. Further studies found Paeonia lactiflora & Ligusticum chuanxing extraction also have collagenase inhibition.
实验首先研究了羟脯氨酸化学比色法的影响因素,确定实验条件:1.0mL待测溶液中加入2.0mL异丙醇,1.0mL氧化剂氧化5min,再加入2.0mL显色剂,60℃加热显色20min,冰浴冷却5min后测定561nm处的吸光度,根据标准曲线法计算羟脯氨酸质量。
以羟脯氨酸含量测定为检测手段,初步研究了胶原酶对胶原蛋白的酶解反应过程,并在此基础上设计了胶原酶活性及抑制剂抑制效果测定方法:胶原蛋白10mg,胶原酶液浓度0.1mg/mL,反应温度37.0℃;Tris-HCl(三羟甲基氨基甲烷-盐酸)缓冲溶液中胶原酶催化降解固态不溶性胶原蛋白分解为可溶性肽进入溶液,反应终止后滤除未反应胶原蛋白,滤液加浓盐酸密封加热酸解后测定其中游离羟脯氨酸的质量;抑制剂与空白实验的羟脯氨酸质量差值体现出抑制剂的抑制效果。
实验发现芦荟汁干粉中蒽醌苷类化合物具有明显的胶原酶抑制效果;芦荟汁干粉先用热水煎煮,滤液经乙酸乙酯萃取,除去溶剂后得粗提取物;该粗提取物再经硅胶柱层析分离多次,得到含有两种主要物质的混合物;最后经半制备高效液相色谱分离出两种纯物质;经定性实验和HPLC、UV、IR、MS、1H NMR谱图鉴定化学结构,分别确定为芦荟素和异芦荟素。抑制效果测定结果表明两者具有相似的抑制效果并随质量浓度的增大而近似线性升高,1mg/mL时百分抑制率分别达到42.4%和35.0%,低浓度下的抑制效果高于常见抑制剂四环素。
根据文献中胶原酶抑制机理——Zn2+螯合理论,初步分析认为芦荟素、异芦荟素可能也符合该理论,抑制作用可能与其分子结构有关;后续实验还发现芍药、川芎提取物也具有胶原酶抑制效果。
In this paper, we mainly studied the collagenase inhibitory ingredients from the sap of Aloe, including the method of collagenase activity and inhibition test, the extraction and purification of useful ingredients, chemical structure identify, inhibition ratio test, primarily discussed possible inhibitory mechanism and other inhibitory plants extracts in further study.
First of all, we studied the effective factors of Hydroxyproline photocolorimetric method, confirm the test condition as: 1.0mL sample add 2.0mL Isopropanol, 1.0mL oxidation reagent and oxidated 5min, then add 2.0mL color reagent, cooled in icy water 5min after heated 20min at 60℃; Test its UV absorption at 561nm, calculate the Hydroxyproline mass by its standard curve.
Use the Hydroxyproline photocolorimetric method, we studied the interaction between collagenase and collagen, then designed the method of collagenase activity and inhibition test: 10mg collagen with 0.1mg/mL collagenase solution at 37.0℃in Tris-HCl buffer, collagenase degraded the insoluble collagen to soluble protein into the buffer, Filtrated the undegraded collagen and add HCl heated in a sealed tube. Then test the Hydroxyproline’s mass in the tube by Hydroxyproline photocolorimetric method, collaterally calculated the collagen’s mass. The difference between blank sample and inhibition sample indicated the effect of inhibitor.
Inhibitory test found the Anthraquinones glycoside materials in Aloe sap have obviously collagenase inhibition; Aloe Power was decocted by hot water, extracted by ethyl acetate and got crude extract after dissolvent; This crude material was separated several times by silica column and got an extract mainly contain two ingredients, then isolated to two pure ingredients by Semi- Preparative HPLC. UV, IR, MS & 1H NMR tested their chemical structure, confirmed as Barbaloin & Isobarbaloin respectively. Inhibition ratio test found they have similar and dose dependent inhibitory activity, respective inhibition ratio was 42.4% & 35.0% at 1mg/mL, higher inhibition ratio in low concentration compared with tetracycline.
According to the collagenase inhibitory mechanism of Zn2+ chelating theory, we primarily considered Barbaloin & Isobarbaloin also follow this theory, the inhibition maybe due to their Special chemical structure in the molecule. Further studies found Paeonia lactiflora & Ligusticum chuanxing extraction also have collagenase inhibition.
引文
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