黑豆萌芽水提物抗氧化活性研究及其机理初探
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摘要
黑豆(Glycine max var)为豆科植物大豆(Glycine max(L.)merr)的黑色种子，又名橹豆、乌豆、黑大豆、马料豆。黑豆味甘性平，具有高蛋白、低热量的特性，是一种药食同源的豆类，在安徽、东北、河南、河北、山东、陕西等省均有种植。黑豆萌芽，俗称“大豆卷”，具有补肾、利尿消肿、滋阴壮阳、助消化、降血脂和软化血管等功效。本论文以黑豆萌芽为研究对象，对其萌发过程和抗氧化活性进行了系统研究，分析了与其抗氧化活性相关基因的表达，初步探讨了黑豆萌芽水提物（Black Soybean Sprouts Extract,BSSE）抗氧化活性的产生机理。
（1）BSSE抗氧化活性研究：通过清除自由基，抑制酶活性和抗细胞氧化损伤三个方面系统研究了BSSE的抗氧化活性。结果表明：0-10cm芽长的BSSE（40mg/mL）对DPPH自由基和羟基自由基均有优异的清除作用，清除率均在90%以上，与同浓度的Vc相当，其中萌动期（0.5cm芽长）的BSSE的清除率高于其它芽长；0-10cm芽长BSSE（40mg/mL）对酪氨酸酶的抑制率可达90%左右，与同浓度的熊果苷接近，表明BSSE对酪氨酸酶有强抑制作用，其中萌动期（芽长0.5cm）BSSE活性最强；人皮肤成纤维细胞与BSSE孵育24h后，再与H2O2反应，能够显著抵抗H2O2引起的细胞损伤（p<0.05），芽长为0.5cm，1cm，2cm时，细胞的存活率较高。三个方面的研究结果都表明BSSE具有抗氧化活性，且萌动期活性优于干种子和其它芽长，说明适当的萌芽过程可以提高黑豆的抗氧化活性。
（2）基因角度的机理研究：采用基因芯片技术，研究不同芽长黑豆萌芽的基因表达差异，结果表明：0.5cm芽长与5cm芽长黑豆萌芽的基因表达存在显著差异；GO term分析结果表明：在0.5cm的黑豆萌芽中总共有918个基因上调表达，其中属于GO:0006979（氧化压力响应）的10个基因显著高表达，这些基因主要调控热激蛋白和抗氧化酶类的合成，这些酶类与保持细胞氧化还原代谢平衡有密切关系；RT-PCR验证结果证明：基因芯片数据和RT-PCR验证结果一致，GO:0006979（氧化压力响应）的10个基因在0.5cm的黑豆萌芽中确实显著高表达。研究发现：萌发过程确实影响了黑豆基因的表达，调动了与抗氧化活性相关的基因显著高表达，产生了更多、活性更强的物质，提高了0.5cm芽长黑豆萌芽的抗氧化活性，从基因层面初步阐释了黑豆萌芽抗氧化活性增强的机理。
（3）主要成分分类研究结果表明，BSSE的主要成分是蛋白质和多糖，基本不含黄酮类物质；主要成分的抗氧化活性实验表明，BSSE中的蛋白质是主要的抗氧化功效成分，多糖也具有优异的抗氧化活性；SDS-PAGE法测定蛋白质的分子量主要集中在三个范围：20.0ku以下；26.0-45.0ku；66.2-94.0ku，其中20.0ku以下的蛋白质清除自由基活性最强；MALLS/GPC法测定多糖的分子量主要分布在17.9-36.0ku之间，其中23.0ku多糖组分的抗氧化活性最优，说明低分子量的黑豆萌芽蛋白质和多糖具有更强的抗氧化活性。
（4）响应面法优化提取工艺：在单因素实验的基础上，采用响应面法研究了黑豆萌芽中抗氧化活性成分提取工艺中的主要独立变量的相互作用，获得黑豆萌芽中抗氧化物质的最佳提取工艺为：超声提取时间32.13min；提取温度30℃；液料比29.19:1。根据最优提取条件，提取液对DPPH自由基的实际清除率可达67.60%，与理论预测值66.36%非常相近，优化的提取工艺提高了水提物的抗氧化活性。本实验优化得出的黑豆萌芽提取工艺，为开发利用黑豆资源提供了基础。
（5）采用红细胞（RBC）溶血实验、鸡胚绒毛尿囊膜（CAM）实验和人体皮肤斑贴实验对BSSE的安全性进行研究，结果显示：RBC溶血率均低于10%，CAM模型未达到显著性差异（P＞0.05），人体皮肤斑贴实验全部评定为0级反应，表明BSSE对眼睛和人体皮肤都是安全无刺激的；稳定性研究的结果显示：BSSE适宜的pH环境为6~10之间，可以耐热、耐寒、耐光照，在常温和冷热交替的条件下，都可以保持理化性质、主要成分和抗氧化活性的稳定；防腐挑战实验第14天即无微生物检出，证实防腐体系稳定可靠；产品适用性研究表明，BSSE可以添加在膏霜、乳液和啫喱等剂型的化妆品中，以及50%以下浓度的酒精体系中，不建议用于水剂化妆品。研究结果表明BSSE是一款安全、稳定、有效、普适的化妆品功效添加剂。
Black soybean (Glycine max var) is the black seed of the soybean Glycine max (L.) merr,also known as the black bean. Black beans are rich in protein and mainly cultivated in theprovinces of Anhui, Dongbei, Henan, Hebei, Shandong and Shanxi for both food andmedicinal purposes. The Supplement to Compendium of Materia Medica states that blackbeans can be beneficial to sperm and bone marrow production, muscle strength, hair growth,and the immune system. Modern scientific research shows that black beans havehypolipidemic and antioxidant properties and can be used to beautify the skin.
Antioxidant Activity: Anti-oxidizing capacities of extracts were measured in vitro usingthe DPPH scavenging test, OH· scavenging test, tyrosinase inhibition test and MTTcolorimetric assay which used to study the effects of the bean sprout extracts on human skinfibroblasts damnified by H2O2. Results show that radical scavenging rates at different shootlengths were all greater than90%（40mg/mL）, while the tyrosinase inhibition capacity of theextracts reached90%. Extracts from sprouts grown to0.5cm had the highest capacity.
Analysis of Gene Expression: Microarray and GO (gene ontology) term enrichmentanalysis were used to research the expressed genes related to antioxidant activity, and theresult was confirmed by qRT-PCR. Results indicated that: sprout of0.5cm in length has thehighest antioxidant activity; Microarray analysis of expressed genes between sprouts (0.5and5cm) showed that the most up-regulated genes in0.5cm sprout belonged to GO:0006979(response to oxidative stress), in which ten genes were significant highly expressed; Theresult of qRT-PCR analyses on up-regulation genes was consistent with the microarrayanalysis, indicating that the results were biologically reproducible and believable.
Active Ingredient: Main active ingredients in black bean sprout were studied. The massconcentrations of proteins, polysaccharides and total flavonoid in water extracts of blacksoybean sprouts were measured by the Flint-phenol method, the phenol-sulfuric acid methodand AlCl3reagent color-developing method respectively. Results showed that black beansprouts contain much proteins, polysaccharides and little flavonoids. Proteins andpolysaccharides were separated using ammonium sulfate fractionation and alcoholprecipitation fractionation. Anti-oxidizing capacities of ingredients were measured byDPPH·scavenging test. The molecular weight distribution of proteins and polysaccharideswere measured by SDS polyacrylamide gel electrophoresis and multi-angle laser lightscattering, gel permeation chromatography (MALLS/GPC) respectively. Results showed thatproteins were the main antioxidant, and polysaccharides also have antioxidant activity, andproteins and polysaccharides with lower molecular weight have the strongest antioxidantactivity.
Optimization of Extraction Using RSM: Response surface methodology (RSM) usinga central composite design (CCD) was employed to optimize the conditions for extraction ofantioxidants from black soybean (Glycine max var) sprouts. Three influencing factors:liquid-solid ratio, period of ultrasonic assisted extraction and extraction temperature wereinvestigated in the ultrasonic aqueous extraction. Then Response Surface Methodology (RSM)was applied to optimize the extraction process focused on DPPH radical-scavenging capacityof the antioxidants with respect to the above influencing factors. The best combination ofeach significant factor was determined by RSM design and optimum pretreatment conditionsfor maximum radical-scavenging capacity were established to be liquid-solid ratio of29.19:1,extraction time of32.13min, and extraction temperature of30°C. Under these conditions,67.60%of DPPH radical-scavenging capacity was observed experimentally, similar to thetheoretical prediction of66.36%.
Safety and Stability: The safety of the extracts was determined using the red blood cell(RBC) test, chick chorioallantoic membrane (CAM) assay and human patch test. Hemolysisrate in all extracts were lower than10%, below the20%regulatory limit for the RBC test; TheCAM results show that the effect of sample groups on CAM was similar to normal saline(negative control) without irritation; No signs of allergic reactions were observed in thehuman patch tests; indating that black soybean sprout extract was safe. Results of stabilitystudy show that black soybean sprout extract was stable at room temperature, light, heat, cold,alternating hot and cold, or with pH stay within the range of6to10; The anticorrosion wasstable; The extract was safe, stable, active and can be added into gel, cream, emulsion andalcohol formula.
（1）BSSE抗氧化活性研究：通过清除自由基，抑制酶活性和抗细胞氧化损伤三个方面系统研究了BSSE的抗氧化活性。结果表明：0-10cm芽长的BSSE（40mg/mL）对DPPH自由基和羟基自由基均有优异的清除作用，清除率均在90%以上，与同浓度的Vc相当，其中萌动期（0.5cm芽长）的BSSE的清除率高于其它芽长；0-10cm芽长BSSE（40mg/mL）对酪氨酸酶的抑制率可达90%左右，与同浓度的熊果苷接近，表明BSSE对酪氨酸酶有强抑制作用，其中萌动期（芽长0.5cm）BSSE活性最强；人皮肤成纤维细胞与BSSE孵育24h后，再与H2O2反应，能够显著抵抗H2O2引起的细胞损伤（p<0.05），芽长为0.5cm，1cm，2cm时，细胞的存活率较高。三个方面的研究结果都表明BSSE具有抗氧化活性，且萌动期活性优于干种子和其它芽长，说明适当的萌芽过程可以提高黑豆的抗氧化活性。
（2）基因角度的机理研究：采用基因芯片技术，研究不同芽长黑豆萌芽的基因表达差异，结果表明：0.5cm芽长与5cm芽长黑豆萌芽的基因表达存在显著差异；GO term分析结果表明：在0.5cm的黑豆萌芽中总共有918个基因上调表达，其中属于GO:0006979（氧化压力响应）的10个基因显著高表达，这些基因主要调控热激蛋白和抗氧化酶类的合成，这些酶类与保持细胞氧化还原代谢平衡有密切关系；RT-PCR验证结果证明：基因芯片数据和RT-PCR验证结果一致，GO:0006979（氧化压力响应）的10个基因在0.5cm的黑豆萌芽中确实显著高表达。研究发现：萌发过程确实影响了黑豆基因的表达，调动了与抗氧化活性相关的基因显著高表达，产生了更多、活性更强的物质，提高了0.5cm芽长黑豆萌芽的抗氧化活性，从基因层面初步阐释了黑豆萌芽抗氧化活性增强的机理。
（3）主要成分分类研究结果表明，BSSE的主要成分是蛋白质和多糖，基本不含黄酮类物质；主要成分的抗氧化活性实验表明，BSSE中的蛋白质是主要的抗氧化功效成分，多糖也具有优异的抗氧化活性；SDS-PAGE法测定蛋白质的分子量主要集中在三个范围：20.0ku以下；26.0-45.0ku；66.2-94.0ku，其中20.0ku以下的蛋白质清除自由基活性最强；MALLS/GPC法测定多糖的分子量主要分布在17.9-36.0ku之间，其中23.0ku多糖组分的抗氧化活性最优，说明低分子量的黑豆萌芽蛋白质和多糖具有更强的抗氧化活性。
（4）响应面法优化提取工艺：在单因素实验的基础上，采用响应面法研究了黑豆萌芽中抗氧化活性成分提取工艺中的主要独立变量的相互作用，获得黑豆萌芽中抗氧化物质的最佳提取工艺为：超声提取时间32.13min；提取温度30℃；液料比29.19:1。根据最优提取条件，提取液对DPPH自由基的实际清除率可达67.60%，与理论预测值66.36%非常相近，优化的提取工艺提高了水提物的抗氧化活性。本实验优化得出的黑豆萌芽提取工艺，为开发利用黑豆资源提供了基础。
（5）采用红细胞（RBC）溶血实验、鸡胚绒毛尿囊膜（CAM）实验和人体皮肤斑贴实验对BSSE的安全性进行研究，结果显示：RBC溶血率均低于10%，CAM模型未达到显著性差异（P＞0.05），人体皮肤斑贴实验全部评定为0级反应，表明BSSE对眼睛和人体皮肤都是安全无刺激的；稳定性研究的结果显示：BSSE适宜的pH环境为6~10之间，可以耐热、耐寒、耐光照，在常温和冷热交替的条件下，都可以保持理化性质、主要成分和抗氧化活性的稳定；防腐挑战实验第14天即无微生物检出，证实防腐体系稳定可靠；产品适用性研究表明，BSSE可以添加在膏霜、乳液和啫喱等剂型的化妆品中，以及50%以下浓度的酒精体系中，不建议用于水剂化妆品。研究结果表明BSSE是一款安全、稳定、有效、普适的化妆品功效添加剂。
Black soybean (Glycine max var) is the black seed of the soybean Glycine max (L.) merr,also known as the black bean. Black beans are rich in protein and mainly cultivated in theprovinces of Anhui, Dongbei, Henan, Hebei, Shandong and Shanxi for both food andmedicinal purposes. The Supplement to Compendium of Materia Medica states that blackbeans can be beneficial to sperm and bone marrow production, muscle strength, hair growth,and the immune system. Modern scientific research shows that black beans havehypolipidemic and antioxidant properties and can be used to beautify the skin.
Antioxidant Activity: Anti-oxidizing capacities of extracts were measured in vitro usingthe DPPH scavenging test, OH· scavenging test, tyrosinase inhibition test and MTTcolorimetric assay which used to study the effects of the bean sprout extracts on human skinfibroblasts damnified by H2O2. Results show that radical scavenging rates at different shootlengths were all greater than90%（40mg/mL）, while the tyrosinase inhibition capacity of theextracts reached90%. Extracts from sprouts grown to0.5cm had the highest capacity.
Analysis of Gene Expression: Microarray and GO (gene ontology) term enrichmentanalysis were used to research the expressed genes related to antioxidant activity, and theresult was confirmed by qRT-PCR. Results indicated that: sprout of0.5cm in length has thehighest antioxidant activity; Microarray analysis of expressed genes between sprouts (0.5and5cm) showed that the most up-regulated genes in0.5cm sprout belonged to GO:0006979(response to oxidative stress), in which ten genes were significant highly expressed; Theresult of qRT-PCR analyses on up-regulation genes was consistent with the microarrayanalysis, indicating that the results were biologically reproducible and believable.
Active Ingredient: Main active ingredients in black bean sprout were studied. The massconcentrations of proteins, polysaccharides and total flavonoid in water extracts of blacksoybean sprouts were measured by the Flint-phenol method, the phenol-sulfuric acid methodand AlCl3reagent color-developing method respectively. Results showed that black beansprouts contain much proteins, polysaccharides and little flavonoids. Proteins andpolysaccharides were separated using ammonium sulfate fractionation and alcoholprecipitation fractionation. Anti-oxidizing capacities of ingredients were measured byDPPH·scavenging test. The molecular weight distribution of proteins and polysaccharideswere measured by SDS polyacrylamide gel electrophoresis and multi-angle laser lightscattering, gel permeation chromatography (MALLS/GPC) respectively. Results showed thatproteins were the main antioxidant, and polysaccharides also have antioxidant activity, andproteins and polysaccharides with lower molecular weight have the strongest antioxidantactivity.
Optimization of Extraction Using RSM: Response surface methodology (RSM) usinga central composite design (CCD) was employed to optimize the conditions for extraction ofantioxidants from black soybean (Glycine max var) sprouts. Three influencing factors:liquid-solid ratio, period of ultrasonic assisted extraction and extraction temperature wereinvestigated in the ultrasonic aqueous extraction. Then Response Surface Methodology (RSM)was applied to optimize the extraction process focused on DPPH radical-scavenging capacityof the antioxidants with respect to the above influencing factors. The best combination ofeach significant factor was determined by RSM design and optimum pretreatment conditionsfor maximum radical-scavenging capacity were established to be liquid-solid ratio of29.19:1,extraction time of32.13min, and extraction temperature of30°C. Under these conditions,67.60%of DPPH radical-scavenging capacity was observed experimentally, similar to thetheoretical prediction of66.36%.
Safety and Stability: The safety of the extracts was determined using the red blood cell(RBC) test, chick chorioallantoic membrane (CAM) assay and human patch test. Hemolysisrate in all extracts were lower than10%, below the20%regulatory limit for the RBC test; TheCAM results show that the effect of sample groups on CAM was similar to normal saline(negative control) without irritation; No signs of allergic reactions were observed in thehuman patch tests; indating that black soybean sprout extract was safe. Results of stabilitystudy show that black soybean sprout extract was stable at room temperature, light, heat, cold,alternating hot and cold, or with pH stay within the range of6to10; The anticorrosion wasstable; The extract was safe, stable, active and can be added into gel, cream, emulsion andalcohol formula.
引文
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