玉米须功能因子活性评价及其降血糖机理研究
摘要
玉米须是中国传统草药，在很多中医药典籍中均有所记载，如《滇南本草》和《中药大辞典》。记录表明，玉米须无毒，具有利尿、利胆等功效，可以在临床用于治疗糖尿病、肾炎、高血压等多种疾病。玉米须的多种功能活性引起了世界各地研究者的广泛关注，经研究表明玉米须中除了含有大量的多糖类化合物、多酚类化合物以外，还含有甾醇、有机酸和皂苷等，日本研究者发现玉米须中还含有维生素、生物碱、肌醇、隐黄质、尿囊素等多种功能因子。玉米须生物活性与药用价值主要体现在其具有降血糖、降血压、降血脂作用、免疫调节作用、缓解疲劳、抗肿瘤、抗衰老、清热利胆作用、利尿、通便，抑制结石功效。通过大量的临床试验表明，玉米须提取物对高血压人群、糖尿病人群及高血脂人群作用比较显著。中国传统医药著作《中药大辞典》中对玉米须的安全性有所记载，确认其低毒，此观点被美国食品药物管理局得到证实，记载玉米须是安全、无毒的，由玉米须所提取的功能因子制得的药物为非处方药，可以在药店自行销售。
因此，本课题在前期研究基础上提出以玉米须为原料，对玉米须的多糖类化合物、黄酮类化合物及甾醇类化合物进行提取，优化最佳提取工艺参数。考察三种功能活性成分的降血糖、抗氧化、抗凝血、降血压及抗痛风的活性。其中以对α-葡萄糖苷酶和α-淀粉酶的抑制活性为指标，筛选玉米须体外降血糖高活性组分，经色谱纯化富集活性物质，灌胃高血糖小鼠测定体内降血糖活性，通过考察饮水量、进食量、体重、血糖变化和最大糖耐受量的变化等指标阐述玉米须提取物质降血糖的作用机理。课题研究目的明确，符合吉林省经济发展的需要。
首先在建立了玉米须β-谷甾醇、多糖类化合物和黄酮类化合物的测定方法的基础上，开展了玉米须功能因子的提取。通过优化单因素试验，响应面设计法建立了玉米须β-谷甾醇、多糖类和黄酮类化合物的提取参数模型。通过方差分析，对各回归模型进行了拟合度和显著性检验，根据多元函数极值理论，确定在最优提取工艺参数下最大提取得率。在最优的提取条件下进行验证试验，得到玉米须β-谷甾醇、多糖类化合物和黄酮类化合物最优提取工艺参数为：提取温度48.8(℃)，提取时间87.1(min)，液固比42.1(ml/g)和乙醇浓度为84.3(%)，在此条件下，β-谷甾醇的得率为1.47%；提取温度82.6(℃)，提取时间77.3(min)和液固比87.4(ml/g)，在此条件下，多糖的得率为6.60%；乙醇浓度为74(%)，提取时间3.96(h)和液固比54.4(ml/g)。在此条件下，玉米须黄酮的得率为17.15mg/g。
此外，通过三氯化铝显色法和红外光谱法测定了玉米须乙醇提取物，结果表明，乙醇提取物含有黄酮类化合物特征官能团，表明存在黄酮类化合物；通过苯酚-硫酸显色法和红外光谱法测定了玉米须水提取物，结果表明，水提取物含有C-H伸缩振动的吸收峰和C-O伸缩振动的吸收峰等多糖类物质特征吸收峰。
通过高通量体外酶法筛选平台，进行玉米须功能因子的活性筛选，依次考察了玉米须粗甾醇、粗黄酮和粗多糖类化合物对凝血酶活性、血管紧张素转化酶（ACE）、α-葡萄糖苷酶、α-淀粉酶和黄嘌呤氧化酶的抑制能力及对自由基清除能力、还原力和金属离子螯合性能力。试验结果表明：玉米须三种提取物的抗凝血活性均呈现整体上升趋势，但甾醇的凝血酶抑制率最低。多糖化合物的体外抗凝血活性较高；玉米须粗提物对ACE酶的抑制活性不明显，表明玉米须提取物调节血压并不是通过抑制血管紧张素转化酶ACE的活性来实现的；玉米须粗多糖，粗黄酮和粗甾醇对α-葡萄糖苷酶和α-淀粉酶抑制能力依次为：粗多糖＞粗黄酮＞粗甾醇；对黄嘌呤氧化酶的抑制能力依次为：粗黄酮＞粗多糖＞粗甾醇。玉米须三种提取物均具有一定的抗氧化活性。其中自由基清除能力依次为： Vc对照品＞粗多糖＞粗黄酮＞粗甾醇提取物。还原力的大小顺序依次为：Vc对照品＞粗多糖＞粗黄酮＞粗甾醇。金属离子螯合能力依次为：EDTA对照品＞粗多糖＞粗黄酮＞粗甾醇。
运用柱色谱法分离纯化高活性组分并富集，通过对聚酰胺色谱纯化玉米须粗多糖的单因素试验（上样量、洗脱体积、洗脱液种类和吸附时间）的筛查，以多糖保留率和蛋白质脱除率为考察指标，确定最优纯化工艺参数：上样量为8ml、吸附时间为4h，洗脱体积为100ml时，多糖保留率为98.37%，蛋白质脱除率为70.22%。此外，对纯化得到的玉米须多糖类化合物依次进行了分子量的测定，红外光谱的结构预测及纯化的多糖类化合物α-葡萄糖苷酶和α-淀粉酶的抑制率的测定。
同时在体外降血糖活性的筛选前提下，对聚酰胺纯化组分进行了高血糖模型鼠的体内灌胃试验。试验结果表明，玉米须多糖纯化组分能够有效降低高血糖小鼠的血糖水平，并且在第四周呈现显著的降血糖作用，效果与二甲双胍相近，同时，玉米须多糖纯化组分能够显著降低高血糖小鼠总胆固醇的水平。此外，高剂量的纯化玉米须多糖在改善高血糖小鼠血糖及糖耐受能力同时，还能有利提高其抗疲劳能力和改善抑郁行为。
本项目研究旨在通过生物酶技术、色谱技术，从功能性食品开发的角度研究玉米须功能活性成分，筛选生物活性，具有降血糖的活性成分纯化富集，动物实验验证。若将本课题研究成果进行实际应用和产业转化，不但能够将废弃物玉米须变成有用的资源，而且通过高附加值食品的开发而具有明显的经济效益，还能够有效解决玉米精深加工企业的废弃物玉米须渣体所造成的环境污染问题，具有极为广阔的市场前景，显著的经济效益和社会效益。
Corn silk is a well-known traditional Chinese medicine that has been widely usedfor treatment of some diseases. the " Southern Yunnan Materia Medica "," ChineseDictionary "and other classics of Chinese medicine have been documented, for thetreatment of nephritis, gallstones, diabetes, jaundice, measles, chylous urine,vaginal bleeding disorders. Modern nutrition analysis shows Corn containspolysaccharides, flavonoids, sterols, organic acids, vitamins, alkaloids, saponins,inositol, cryptoxanthin, and allantoin. The bioactive and medicinal value of CornSilk have been showed, such as anti-diabetic, adjusting blood pressure, immuneregulation, relieve fatigue, anti-tumor, anti-aging. On the clinical applicationshowed that Corn silk can be used for hypertension, diabetes and other diseases Inaddition, the "Chinese Dictionary " records the toxicity, the U.S. Food and DrugAdministration confirmed that corn silk to be safe.Previous research showed thatphenolic acids, favonoid, aglycons and related monosaccharides from corn silkpossessed anti-oxidative activities,and its polysaccharides could lead to weight lossand improve gastrointestinal movement. On the Other hand, the phenolic acids andfavonoids in corn silk have been well investigated, including extraction, structuralidentifcation, bioactivities and the relationship between antioxidant activity andcontents of various polyphenol classes.
Therefore, on the basis of preliminary studies, polysaccharide compounds,flavonoids and sterols from Corn silk were extracted to optimize the best extractionprocess parameters. The anti-oxidation, anti-coagulation, anti-gout, α-glucosidaseand α-amylase inhibitory activity and hypotensive activity were screened invitro.The active substance was purified by chromatography and was fed a highblood sugar mice, by examining water intake, food intake, body weight, bloodglucose, and maximum tolerated dose of sugar and other indicators. Research clarityof purpose, to meet the needs of economic development in Jilin Province.
First, the basic measurement method of a β-sitosterol, polysaccharide andflavonoids from corn silk, they were extracted and optimized by response surfacemethodology(RSM), based on a Box–Behnken design (BBD). By analysis ofvariance, regression model for each of the goodness of fit and significance tests,according to the multi-function extreme value theory to determine the optimal valueof each index under optimum conditions. The experimental data were ftted to asecond-order polynomial equation and also profled into the corresponding3-Dcontour plots. The β-sitosterol, polysaccharides compounds and flavonoids ofoptimal extraction conditions were as follows: extraction temperature48.8(℃),extraction time87.1(min), liquid-solid ratio42.1(ml/g) and ethanol concentrationwas84.3(%), in this condition, β-sitosterol yield of1.47%; extractiontemperature82.6(℃), extraction time77.3(min) and liquid-solid ratio of87.4(ml/g), under this condition, the yield was6.60%polysaccharides; ethanol concentration of74(%), extraction time3.96(h) and liquid-solid ratio of54.4(ml/g). Under these conditions, corn yield of flavonoids17.15mg/g.
By the in vitro enzymatic screening platform, the functional activity of Corn silkwas screened. The sterols, flavonoids and crude polysaccharide compounds for theactivity of thrombin, angiotensin converting enzyme (ACE), α-glucosidase, α-amylase and xanthine oxidase inhibitory ability and free radical scavenging capacity,reducing power and metal ion chelating capabilities were investigated. The resultsshowed: Corn silk extracts had anticoagulant actives,and showed overall upwardtrend.The polysaccharide have higher anticoagulant activity in vitro; Corn silkcrude extracts on ACE inhibitory activity was not obvious, indicating that corn silkextracts can been used to regulates blood pressure not by inhibiting the activity ofangiotensin-converting enzyme ACE achieved; Corn silk polysaccharides, crudeflavonoids and crude sterols for α-glucosidase and α-amylase inhibitory abilitywere: crude polysaccharide> crude flavonoids> crude sterols; against xanthineoxidase inhibitory capacity as follows: crude flavonoids> polysaccharide> Thecrude sterols.Corn silk crud extracts have certain antioxidant activity, the radicalscavenging capacity were: Vc> polysaccharide> crude flavonoids> crude sterolextract. Restore the order of the size of the force: Vc> polysaccharide> crudeflavonoids> crude sterols. Metal ion chelating ability were: EDTA reference>polysaccharide> crude flavonoids> crude sterols.
The highly active ingredients were separated, purified and enriched by usingcolumn chromatography. The single factor (sample volume, elution volume,elution and adsorption time) of purification by chromatography were screened toenrich polysaccharide. The optimal process parameters showed: the sample volumewas8ml, adsorption time was4h, the elution volume of100ml, polysaccharideretention rate was98.37%, protein removal rate was70.22%. In addition to thepurified polysaccharide compound from corn silk were measured sequentiallydetermined molecular weight and structure prediction by the infrared spectrum, andthe α-glucosidase and α-amylase inhibition rate.
Meanwhile, the hypoglycemic activity in vitro of the polyamide purifiedfractions were screened by administering the high blood sugar mouse model. Theresults showed that the purified polysaccharide component from corn silk caneffectively reduce glucose levels in mice, and showed a significant hypoglycemiceffect in the fourth week, the effect is similar with metformin, while purifiedpolysaccharide component can significantly reduce the total blood cholesterol levelsin mice. In addition, high doses of purified polysaccharide can be used to improvingblood sugar and glucose tolerance, while also enabling improved resistance tofatigue and improve their depressive behavior.
The aims of this project to study the screening of biological activity,hypoglycemic active ingredient purified and enriched, animal experiments bybiological enzyme technology, chromatographic techniques developed for theperspective of functional food research. If this research results can be application andindustrial transformation, not only the waste corn silk can be turned into a usefulresource, but also through the development of high value-added food products and has obvious economic benefits, has a very broad market prospects and significanteconomic and social benefits.
因此，本课题在前期研究基础上提出以玉米须为原料，对玉米须的多糖类化合物、黄酮类化合物及甾醇类化合物进行提取，优化最佳提取工艺参数。考察三种功能活性成分的降血糖、抗氧化、抗凝血、降血压及抗痛风的活性。其中以对α-葡萄糖苷酶和α-淀粉酶的抑制活性为指标，筛选玉米须体外降血糖高活性组分，经色谱纯化富集活性物质，灌胃高血糖小鼠测定体内降血糖活性，通过考察饮水量、进食量、体重、血糖变化和最大糖耐受量的变化等指标阐述玉米须提取物质降血糖的作用机理。课题研究目的明确，符合吉林省经济发展的需要。
首先在建立了玉米须β-谷甾醇、多糖类化合物和黄酮类化合物的测定方法的基础上，开展了玉米须功能因子的提取。通过优化单因素试验，响应面设计法建立了玉米须β-谷甾醇、多糖类和黄酮类化合物的提取参数模型。通过方差分析，对各回归模型进行了拟合度和显著性检验，根据多元函数极值理论，确定在最优提取工艺参数下最大提取得率。在最优的提取条件下进行验证试验，得到玉米须β-谷甾醇、多糖类化合物和黄酮类化合物最优提取工艺参数为：提取温度48.8(℃)，提取时间87.1(min)，液固比42.1(ml/g)和乙醇浓度为84.3(%)，在此条件下，β-谷甾醇的得率为1.47%；提取温度82.6(℃)，提取时间77.3(min)和液固比87.4(ml/g)，在此条件下，多糖的得率为6.60%；乙醇浓度为74(%)，提取时间3.96(h)和液固比54.4(ml/g)。在此条件下，玉米须黄酮的得率为17.15mg/g。
此外，通过三氯化铝显色法和红外光谱法测定了玉米须乙醇提取物，结果表明，乙醇提取物含有黄酮类化合物特征官能团，表明存在黄酮类化合物；通过苯酚-硫酸显色法和红外光谱法测定了玉米须水提取物，结果表明，水提取物含有C-H伸缩振动的吸收峰和C-O伸缩振动的吸收峰等多糖类物质特征吸收峰。
通过高通量体外酶法筛选平台，进行玉米须功能因子的活性筛选，依次考察了玉米须粗甾醇、粗黄酮和粗多糖类化合物对凝血酶活性、血管紧张素转化酶（ACE）、α-葡萄糖苷酶、α-淀粉酶和黄嘌呤氧化酶的抑制能力及对自由基清除能力、还原力和金属离子螯合性能力。试验结果表明：玉米须三种提取物的抗凝血活性均呈现整体上升趋势，但甾醇的凝血酶抑制率最低。多糖化合物的体外抗凝血活性较高；玉米须粗提物对ACE酶的抑制活性不明显，表明玉米须提取物调节血压并不是通过抑制血管紧张素转化酶ACE的活性来实现的；玉米须粗多糖，粗黄酮和粗甾醇对α-葡萄糖苷酶和α-淀粉酶抑制能力依次为：粗多糖＞粗黄酮＞粗甾醇；对黄嘌呤氧化酶的抑制能力依次为：粗黄酮＞粗多糖＞粗甾醇。玉米须三种提取物均具有一定的抗氧化活性。其中自由基清除能力依次为： Vc对照品＞粗多糖＞粗黄酮＞粗甾醇提取物。还原力的大小顺序依次为：Vc对照品＞粗多糖＞粗黄酮＞粗甾醇。金属离子螯合能力依次为：EDTA对照品＞粗多糖＞粗黄酮＞粗甾醇。
运用柱色谱法分离纯化高活性组分并富集，通过对聚酰胺色谱纯化玉米须粗多糖的单因素试验（上样量、洗脱体积、洗脱液种类和吸附时间）的筛查，以多糖保留率和蛋白质脱除率为考察指标，确定最优纯化工艺参数：上样量为8ml、吸附时间为4h，洗脱体积为100ml时，多糖保留率为98.37%，蛋白质脱除率为70.22%。此外，对纯化得到的玉米须多糖类化合物依次进行了分子量的测定，红外光谱的结构预测及纯化的多糖类化合物α-葡萄糖苷酶和α-淀粉酶的抑制率的测定。
同时在体外降血糖活性的筛选前提下，对聚酰胺纯化组分进行了高血糖模型鼠的体内灌胃试验。试验结果表明，玉米须多糖纯化组分能够有效降低高血糖小鼠的血糖水平，并且在第四周呈现显著的降血糖作用，效果与二甲双胍相近，同时，玉米须多糖纯化组分能够显著降低高血糖小鼠总胆固醇的水平。此外，高剂量的纯化玉米须多糖在改善高血糖小鼠血糖及糖耐受能力同时，还能有利提高其抗疲劳能力和改善抑郁行为。
本项目研究旨在通过生物酶技术、色谱技术，从功能性食品开发的角度研究玉米须功能活性成分，筛选生物活性，具有降血糖的活性成分纯化富集，动物实验验证。若将本课题研究成果进行实际应用和产业转化，不但能够将废弃物玉米须变成有用的资源，而且通过高附加值食品的开发而具有明显的经济效益，还能够有效解决玉米精深加工企业的废弃物玉米须渣体所造成的环境污染问题，具有极为广阔的市场前景，显著的经济效益和社会效益。
Corn silk is a well-known traditional Chinese medicine that has been widely usedfor treatment of some diseases. the " Southern Yunnan Materia Medica "," ChineseDictionary "and other classics of Chinese medicine have been documented, for thetreatment of nephritis, gallstones, diabetes, jaundice, measles, chylous urine,vaginal bleeding disorders. Modern nutrition analysis shows Corn containspolysaccharides, flavonoids, sterols, organic acids, vitamins, alkaloids, saponins,inositol, cryptoxanthin, and allantoin. The bioactive and medicinal value of CornSilk have been showed, such as anti-diabetic, adjusting blood pressure, immuneregulation, relieve fatigue, anti-tumor, anti-aging. On the clinical applicationshowed that Corn silk can be used for hypertension, diabetes and other diseases Inaddition, the "Chinese Dictionary " records the toxicity, the U.S. Food and DrugAdministration confirmed that corn silk to be safe.Previous research showed thatphenolic acids, favonoid, aglycons and related monosaccharides from corn silkpossessed anti-oxidative activities,and its polysaccharides could lead to weight lossand improve gastrointestinal movement. On the Other hand, the phenolic acids andfavonoids in corn silk have been well investigated, including extraction, structuralidentifcation, bioactivities and the relationship between antioxidant activity andcontents of various polyphenol classes.
Therefore, on the basis of preliminary studies, polysaccharide compounds,flavonoids and sterols from Corn silk were extracted to optimize the best extractionprocess parameters. The anti-oxidation, anti-coagulation, anti-gout, α-glucosidaseand α-amylase inhibitory activity and hypotensive activity were screened invitro.The active substance was purified by chromatography and was fed a highblood sugar mice, by examining water intake, food intake, body weight, bloodglucose, and maximum tolerated dose of sugar and other indicators. Research clarityof purpose, to meet the needs of economic development in Jilin Province.
First, the basic measurement method of a β-sitosterol, polysaccharide andflavonoids from corn silk, they were extracted and optimized by response surfacemethodology(RSM), based on a Box–Behnken design (BBD). By analysis ofvariance, regression model for each of the goodness of fit and significance tests,according to the multi-function extreme value theory to determine the optimal valueof each index under optimum conditions. The experimental data were ftted to asecond-order polynomial equation and also profled into the corresponding3-Dcontour plots. The β-sitosterol, polysaccharides compounds and flavonoids ofoptimal extraction conditions were as follows: extraction temperature48.8(℃),extraction time87.1(min), liquid-solid ratio42.1(ml/g) and ethanol concentrationwas84.3(%), in this condition, β-sitosterol yield of1.47%; extractiontemperature82.6(℃), extraction time77.3(min) and liquid-solid ratio of87.4(ml/g), under this condition, the yield was6.60%polysaccharides; ethanol concentration of74(%), extraction time3.96(h) and liquid-solid ratio of54.4(ml/g). Under these conditions, corn yield of flavonoids17.15mg/g.
By the in vitro enzymatic screening platform, the functional activity of Corn silkwas screened. The sterols, flavonoids and crude polysaccharide compounds for theactivity of thrombin, angiotensin converting enzyme (ACE), α-glucosidase, α-amylase and xanthine oxidase inhibitory ability and free radical scavenging capacity,reducing power and metal ion chelating capabilities were investigated. The resultsshowed: Corn silk extracts had anticoagulant actives,and showed overall upwardtrend.The polysaccharide have higher anticoagulant activity in vitro; Corn silkcrude extracts on ACE inhibitory activity was not obvious, indicating that corn silkextracts can been used to regulates blood pressure not by inhibiting the activity ofangiotensin-converting enzyme ACE achieved; Corn silk polysaccharides, crudeflavonoids and crude sterols for α-glucosidase and α-amylase inhibitory abilitywere: crude polysaccharide> crude flavonoids> crude sterols; against xanthineoxidase inhibitory capacity as follows: crude flavonoids> polysaccharide> Thecrude sterols.Corn silk crud extracts have certain antioxidant activity, the radicalscavenging capacity were: Vc> polysaccharide> crude flavonoids> crude sterolextract. Restore the order of the size of the force: Vc> polysaccharide> crudeflavonoids> crude sterols. Metal ion chelating ability were: EDTA reference>polysaccharide> crude flavonoids> crude sterols.
The highly active ingredients were separated, purified and enriched by usingcolumn chromatography. The single factor (sample volume, elution volume,elution and adsorption time) of purification by chromatography were screened toenrich polysaccharide. The optimal process parameters showed: the sample volumewas8ml, adsorption time was4h, the elution volume of100ml, polysaccharideretention rate was98.37%, protein removal rate was70.22%. In addition to thepurified polysaccharide compound from corn silk were measured sequentiallydetermined molecular weight and structure prediction by the infrared spectrum, andthe α-glucosidase and α-amylase inhibition rate.
Meanwhile, the hypoglycemic activity in vitro of the polyamide purifiedfractions were screened by administering the high blood sugar mouse model. Theresults showed that the purified polysaccharide component from corn silk caneffectively reduce glucose levels in mice, and showed a significant hypoglycemiceffect in the fourth week, the effect is similar with metformin, while purifiedpolysaccharide component can significantly reduce the total blood cholesterol levelsin mice. In addition, high doses of purified polysaccharide can be used to improvingblood sugar and glucose tolerance, while also enabling improved resistance tofatigue and improve their depressive behavior.
The aims of this project to study the screening of biological activity,hypoglycemic active ingredient purified and enriched, animal experiments bybiological enzyme technology, chromatographic techniques developed for theperspective of functional food research. If this research results can be application andindustrial transformation, not only the waste corn silk can be turned into a usefulresource, but also through the development of high value-added food products and has obvious economic benefits, has a very broad market prospects and significanteconomic and social benefits.
引文
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