CN112326567A - 一种西葫芦抗氧化活性成分的优化提取方法及抗氧化活性的验证方法 - Google Patents
一种西葫芦抗氧化活性成分的优化提取方法及抗氧化活性的验证方法 Download PDFInfo
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Abstract
本发明提供了一种西葫芦中抗氧化活性成分的优化提取方法及抗氧化活性的验证方法。以西葫芦作为原料,方法包括:(1)样品前处理;(2)优化提取方法;(3)测定样品中总多酚、总黄酮、总三萜和抗坏血酸的含量;(4)利用DPPH自由基清除能力、ABTS自由基清除能力、FRAP法对西葫芦的抗氧化活性进行验证。本发明优点是本发明中抗氧化活性物质的优化提取及抗氧化验证方法,是以优化西葫芦中抗氧化活性物质的最佳提取条件最优提取溶剂,并测定体外抗氧化能力为目的,结合水浴加热提取方法,以达到最佳提取效果,并进一步有效验证抗氧化效果。本发明成本低、操作简便,耗时少,效率较高,具有良好的应用前景。通过本方法为西葫芦中抗氧化活性成分的筛选提供理论依据,并且结合西葫芦中抗氧化活性的验证方法为开发西葫芦相关的产品提供一定的参考。
Description
技术领域
本发明涉及抗氧化活性评价方法领域,具体为一种西葫芦抗氧化活性成分的优化提取及抗氧化活性的验证方法。
背景技术
西葫芦(CucurbitapepoL)又叫西葫、熊(雄)瓜、白瓜、菜瓜和荨瓜等,西葫芦果实部分皮薄、果肉较多、水分含量较高,所以它普遍作为蔬菜食用,既可以炒菜也用来包馅,并且嫩西葫芦和老西葫芦都可用来食用,是人们在日常生活中仅次于黄瓜的一种瓜类蔬菜。
西葫芦中富含维生素C、葡萄糖、淀粉、钙类等营养成分,因此具有一定的营养价值,它还具有除烦止渴、润肺止咳、清热利尿、消肿散结的功效,帮助调节人体代谢;对烦渴、水肿腹胀、疮毒以及肾炎、肝硬化腹水等症状具有辅助治疗的作用;能够增强免疫力,发挥抗病毒和抗肿瘤的作用;还可以促进人体胰岛素分泌,在一定程度上有效防治糖尿病,预防肝肾病变,增强肝肾细胞的再生能力等。
西葫芦中含有比较丰富的维生素C、维生素E和一些微量元素,它们具有较强的抗氧化作用。西葫芦内部许多成分都具有抗氧化能力,但由于西葫芦不同部位的化学成分导致抗氧化成分不同,故其抗氧化能力也不同。有研究表明西葫芦皮中包括碳水化合物、胡萝卜素、脂肪、蛋白质、植酸、氨基酸、丙氨酸、精氨酸、胱氨酸、谷氨酸、甘氨酸、羟脯氨酸、亮氨酸、丝氨酸、色氨酸和果酸等物质。其籽粒中含有丰富的蛋白质和脂肪(分别占39%和44%),远远高于许多植物种子的蛋白质和脂肪含量。葫芦籽的脂肪酸组成表明,油酸甘油酯和亚油酸甘油酯占籽粒油总量的68%。此外,西葫芦种子也被发现是某些氨基酸、磷、铁和镁的良好来源。
控制糖尿病的基本途径是降低血糖,糖尿病患者由于自身代谢功能紊乱,葡萄糖不能被正常吸收导致血糖上升。西葫芦能够促进人体内胰岛素的分泌,有助于降低血糖。
现代药理研究表明,清热解毒类药物具有抗炎、抗菌作用。葫芦科植物化学成分复杂,入药部位广泛,主要有甾醇类、三萜类、脂肪酸类、氨基酸类等成分,部分有黄酮、木质素及酚类。苦味西葫芦果实提取物对金黄色葡萄球菌、大肠杆菌及猪霍乱沙门氏菌都有一定的抑制作用。因此对西葫芦抗氧化活性成分的优化提取及抗氧化活性的验证方法进行研究,可为西葫芦中主要抗氧化活性成分的进一步筛选提供一定的理论依据。
本发明以西葫芦为原料,结合水浴加热提取方式,通过测定4种抗氧化成分,并进一步有效验证西葫芦的抗氧化效果。
发明内容
有鉴于此,本发明创造旨在提供一一种西葫芦抗氧化活性成分的优化提取方法及抗氧化活性的验证方法。
为达到上述目的,本发明创造的技术方案是这样实现的:
一种西葫芦抗氧化活性成分的优化提取方法及抗氧化活性的验证方法,包括如下步骤:
(1)西葫芦样品前处理:
将新鲜的西葫芦切成厚薄均匀的条状后,自然干燥时间为10~24h后用粉碎机粉碎,过40~100目筛后装入自封袋中备用;
(2)西葫芦粗提液的制备:
准确称取经前处理后的西葫芦样品各2.5g于500mL回流瓶中,按照液料比50分别加入溶剂蒸馏水、无水乙醇或甲醇,进行水浴加热辅助提取,提取温度为60~80℃,提取时间为2~4h,过滤后取上清液,滤渣以同样的方式进行水浴提取,过滤后取上清液,将两次上清液混合均匀后,经真空旋转蒸发仪浓缩,真空旋转蒸发仪浓缩温度为40~60℃,压强为0.1~0.15MPa,最后用相应的溶剂定容于100mL容量瓶中;
(3)西葫芦活性成分含量的测定:
总多酚物质含量的测定:以没食子酸为标准品,采用福林酚比色法,在碱性条件下对西葫芦粗提液中的总多酚含量进行测定;吸取1.0mL滤液于10mL比色管中,加入2.5mL福林酚试剂,摇匀,加入2.5mL的15%Na2CO3溶液,加水定容至刻度后摇匀。在40℃水浴60min,静置冷却20min,在778nm处测定其OD值;
总黄酮含量的测定:以芦丁为标准品,采用分光光度法,在弱碱性条件下对西葫芦粗提液中的总黄酮含量进行测定;吸取提取液0.2mL到10mL比色管中,加入0.4mL的三氯化铝溶液,混匀后再加入0.4mL的醋酸钾溶液,再次混匀后加入30%的乙醇溶液定容到刻度,混匀后静置,在30min内以零管调节零点后在415nm处测定OD值;
总三萜含量的测定:以齐敦果酸为标准品,采用香草醛-冰醋酸比色法,在高氯酸环境下对西葫芦粗提液中总三萜含量进行测定;不同溶剂提取液各1.0mL分别置于10mL的比色管中,经氮吹仪氮吹后加入5%(m/V)香草醛-冰醋酸0.5mL,再分别加入高氯酸1mL,摇匀后在70℃水浴20min。取出后在冰水浴中冷却,用乙醇稀释至刻度,摇匀后静置5min,以试剂空白为基线,在460nm下测定OD值;
抗坏血酸含量的测定:参照抗坏血酸测定试剂盒进行测定抗坏血酸含量;
(4)抗氧化活性的比较评价:分别采用DPPH、ABTS、FRAP法对西葫芦样品的抗氧化活性进行综合对比评价;
DPPH自由基清除能力的测定:吸取0.5mL的西葫芦粗提液,0.5mL的DPPH混合均匀,在室温下避光反应20min,以无水乙醇调零,以抗坏血酸代替粗提液作为阳性对照,测定517nm处的OD值;
ABTS自由基清除能力的测定:取6个2mL的离心管,依次加入各浓度的粗提液0.5mL,再加入稀释后的ABTS和过硫酸钾混合液0.5mL,混合均匀后,避光放置6min,以抗坏血酸代替粗提液作为阳性对照,在734nm处测定OD值;
FRAP还原能力测定:分别吸取浓度为25mg/mL的西葫芦提取液和不同浓度的FeSO4溶液各150μL,加入4.5mL的FRAP工作液,37℃下反应10min,在593nm下测定OD值;以FeSO4浓度为横坐标,吸光度值为纵坐标绘制标准曲线,将上述测得的样品吸光度值代入标准曲线计算出相应的FeSO4浓度,还原力用相应的FeSO4浓度(μmol/L)表示。
优选的,步骤(1)中西葫芦干燥时间为18h,过80目筛。
优选的,步骤(2)中水浴加热回流提取的温度为70℃,辅助提取时间3h,真空旋转蒸发仪浓缩温度为50℃,压强为0.13MPa,最终的提取液浓度为25mg/mL。
优选的,步骤(2)中优化的溶剂是甲醇。
优选的,步骤(3)中总多酚含量的测定中:吸取提取液为1.0mL,测定波长为778nm;总黄酮含量的测定中:吸取提取液为0.2mL,测定波长为415nm;总三萜含量的测定中:吸取提取液为1.0mL,
测定波长为460nm;抗坏血酸含量的测定中:测定波长为536nm。
优选的,步骤(4)中分别在波长为517nm、734nm和460nm处测定OD值,计算DPPH自由基清除率、ABTS自由基清除率和FRAP值。
按照上述方法,本发明提供了一种西葫芦抗氧化活性成分的优化提取及抗氧化活性的验证方法。
与现有技术对比,本发明创造所述的一种西葫芦抗氧化活性成分的优化提取及抗氧化活性的验证方法在抗氧化活性的优化提取和抗氧化活性的验证方法中具有以下优点和积极效果:
本发明首次对西葫芦中抗氧化活性物质的优化提取及抗氧化验证方法进行研究,以优化西葫芦中抗氧化活性物质的提取条件和提取溶剂,并测定体外抗氧化能力为目的,结合水浴加热提取方法,以达到最佳提取效果,并进一步有效验证抗氧化效果。经过试验表明西葫芦中抗氧化活性物质的水浴加热提取方法最佳提取条件为:提取时间为2h,料液比为1:50,提取温度70℃,最佳提取溶剂为甲醇。甲醇提取物中DPPH自由基清除能力和FRAP自由基清除能力分别为76.32%和85.46%,FRAP值为315.37μmol/L。本发明不但可以为西葫芦中抗氧化活性成分的进一步筛选和抗氧化活性的比较评价方法提供理论依据,还可以为关于开发西葫芦相关的产品提供一定的参考依据。本发明成本低、操作简便,耗时少,效率较高,具有良好的应用前景。
附图说明
构成本发明创造的一部分的附图用来提供对本发明创造的进一步理解,本发明创造的示意性实施例及其说明用于解释本发明创造,并不构成对本发明创造的不当限定。在附图中:
图1为芦丁标准曲线
图2为没食子酸标准曲线
图3为齐墩果酸标准曲线
图4为西葫芦粗提液中总黄酮、总多酚、总三萜和抗坏血酸的含量对比
图5为DPPH自由基清除能力
图6为ABTS自由基清除能力
图7为FeSO4标准曲线
图8为FRAP值
具体实施方式
下面结合实施方法和附图对本发明的技术方案做进一步说明,但本发明并不限于这些实施案例。
以下所述3个实施例所采用的西葫芦提取液样品分别为水提液、乙醇提取液和甲醇提取液。
实施例1
(1)西葫芦样品前处理:将新鲜的西葫芦切成厚薄均匀的条状后,经晒干后用粉碎机粉碎,过筛后装入自封袋中备用;
(2)西葫芦粗提液的制备:准确称取步骤(1)所得的西葫芦样品2.5g于500mL回流瓶中,按照料液比1g(干样):50mL分别加入蒸馏水,进行水浴加热辅助提取,提取温度为75℃,提取时间为2h,过滤后取上清液,滤渣以同样的方式进行水浴提取,将两次上清液混合均匀后,上清液经真空旋转蒸发仪浓缩,真空旋转蒸发仪浓缩温度为60℃,压强为0.1MPa,最后用相应的溶剂定容于100mL容量瓶中;
(3)西葫芦活性成分含量的测定:经上述操作制得的水提液经过进一步处理后,分别在波长为778nm、415nm、460nm和536nm处测定OD值,计算总多酚、总黄酮、总三萜和抗坏血酸的含量。水提液中芦丁、没食子酸和齐墩果酸的标准曲线分别见图1、图2和图3;总黄酮、总多酚、总三萜和抗坏血酸的含量见图4;
总多酚物质含量的测定:以没食子酸为标准品,采用福林酚比色法,在碱性条件下对西葫芦粗提液中的总多酚含量进行测定。吸取1.0mL滤液于10mL比色管中,加入2.5mL福林酚试剂,摇匀,加入2.5mL15%的Na2CO3溶液,加水定容至刻度后摇匀。在40℃水浴60min,静置冷却20min,在778nm处测定其OD值。
总黄酮含量的测定:以芦丁为标准品,采用分光光度法,在弱碱性条件下对西葫芦粗提液中的总黄酮含量进行测定。吸取提取液0.2mL到10mL比色管中,加入0.4mL的三氯化铝溶液,混匀后再加入0.4mL的醋酸钾溶液,再次混匀后加入30%的乙醇溶液定容到刻度,混匀后静置,在30min内以零管调节零点后在415nm处测定OD值。
总三萜含量的测定:以齐敦果酸为标准品,采用香草醛-冰醋酸比色法,在高氯酸环境下对西葫芦粗提液中总三萜含量进行测定。不同溶剂提取液各1.0mL,分别置于10mL的比色管中,经氮吹仪氮吹后加入5%(m/V)香草醛-冰醋酸0.5mL,再分别加入高氯酸1mL,摇匀后在70℃水浴20min。取出后在冰水浴中冷却,用乙醇稀释至刻度,摇匀后静置5min,以试剂空白为基线,在460nm下测定OD值;
抗坏血酸含量的测定:参照抗坏血酸测定试剂盒进行测定抗坏血酸含量;
(4)抗氧化活性的比较评价:分别在波长为517nm、734nm和460nm处测定OD值,计算DPPH自由基清除率、ABTS自由基清除率和FRAP值,对西葫芦粗提液的抗氧化活性进行比较评价;水提液中的抗氧化活性评价分别见图5、图6、图7和图8;
DPPH自由基清除能力的测定:吸取0.5mL的西葫芦粗提液,0.5mL的DPPH混合均匀,在室温下避光反应20min,以无水乙醇调零,以抗坏血酸代替粗提液作为阳性对照,测定517nm处的OD值;
ABTS自由基清除能力的测定:取6个2mL的离心管,依次加入各浓度的粗提液0.5mL,再加入稀释后的ABTS和过硫酸钾混合液0.5mL,混合均匀后,避光放置6min,以抗坏血酸代替粗提液作为阳性对照,在734nm处测定OD值;
FRAP还原能力测定:分别吸取浓度为25mg/mL的西葫芦提取液和不同浓度的FeSO4溶液各150μL,加入4.5mL的FRAP工作液,37℃下反应10min,在593nm下测定OD值。以FeSO4浓度为横坐标,吸光度值为纵坐标绘制标准曲线,将上述测得的样品吸光度值代入标准曲线计算出相应的FeSO4浓度,还原力用相应的FeSO4浓度(μmol/L)表示。
实施例2
(1)西葫芦样品前处理同实施例1;
(2)西葫芦粗提液的制备:准确称取步骤(1)所得的西葫芦样品2.5g于500mL回流瓶中,按照料液比1g(干样):50mL分别加入无水乙醇,进行水浴加热辅助提取,提取温度为70℃,提取时间为2h,过滤后取上清液,滤渣以同样的方式进行水浴提取,将两次上清液混合均匀后,上清液经真空旋转蒸发仪浓缩,真空旋转蒸发仪浓缩温度为60℃,压强为0.1MPa,最后用相应的溶剂定容于100mL容量瓶中;
(3)西葫芦活性成分含量的测定同实施例1:将制得的乙醇提取液经过进一步处理后,分别在波长为778nm、415nm、460nm和536nm处测定OD值,计算总多酚、总黄酮、总三萜和抗坏血酸的含量。乙醇提取液中芦丁、没食子酸和齐墩果酸的标准曲线分别见图1、图2和图3;总黄酮、总多酚、总三萜和抗坏血酸的含量见图4;
(4)抗氧化活性的比较评价同实施例1中步骤(4),乙醇提取液中的抗氧化活性评价分别见图5、图6、图7和图8。
实施例3
(1)西葫芦样品前处理同实施例1;
(2)西葫芦粗提液的制备:准确称取步骤(1)所得的西葫芦样品2.5g于500mL回流瓶中,按照料液比1g(干样):50mL分别加入溶剂,进行水浴加热辅助提取,提取温度为70℃,提取时间为2h,过滤后取上清液,滤渣以同样的方式进行水浴提取,将两次上清液混合均匀后,上清液经真空旋转蒸发仪浓缩,真空旋转蒸发仪浓缩温度为40℃,压强为0.1MPa,最后用相应的溶剂定容于100mL容量瓶中;
(3)西葫芦活性成分含量的测定同实施例1:将制得的甲醇提取液经过进一步处理后,分别在波长为778nm、415nm、460nm和536nm处测定OD值,计算总多酚、总黄酮、总三萜和抗坏血酸的含量。甲醇提取液中芦丁、没食子酸和齐墩果酸的标准曲线分别见图1、图2和图3;总黄酮、总多酚、总三萜和抗坏血酸的含量见图4;
(4)抗氧化活性的比较评价同实施例1中步骤(4),甲醇提取液中的抗氧化活性评价分别见图5、图6、图7和图8。
甲醇提取物中DPPH自由基清除能力和FRAP自由基清除能力分别为76.32%和85.46%,FRAP值为315.37μmol/L。其他指标相比甲醇提取物也优于其他两种溶剂,因此用甲醇做溶剂效果最好。
以上所述仅为本发明创造的较佳实施例而已,并不用以限制本发明创造,凡在本发明创造的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明创造的保护范围之内。
Claims (6)
1.一种西葫芦抗氧化活性成分的优化提取方法及抗氧化活性的验证方法,其特征在于,包括如下步骤:
(1)西葫芦样品前处理:
将新鲜的西葫芦切成厚薄均匀的条状后,自然干燥时间为10~24h后用粉碎机粉碎,过40~100目筛后装入自封袋中备用;
(2)西葫芦粗提液的制备:
准确称取经前处理后的西葫芦样品各2.5g于500mL回流瓶中,按照液料比50分别加入溶剂蒸馏水、无水乙醇或甲醇,进行水浴加热辅助提取,提取温度为60~80℃,提取时间为2~4h,过滤后取上清液,滤渣以同样的方式进行水浴提取,过滤后取上清液,将两次上清液混合均匀后,经真空旋转蒸发仪浓缩,真空旋转蒸发仪浓缩温度为40~60℃,压强为0.1~0.15MPa,最后用相应的溶剂定容于100mL容量瓶中;
(3)西葫芦活性成分含量的测定:
总多酚物质含量的测定:以没食子酸为标准品,采用福林酚比色法,在碱性条件下对西葫芦粗提液中的总多酚含量进行测定;吸取1.0mL滤液于10mL比色管中,加入2.5mL福林酚试剂,摇匀,加入2.5mL的15%Na2CO3溶液,加水定容至刻度后摇匀。在40℃水浴60min,静置冷却20min,在778nm处测定其OD值;
总黄酮含量的测定:以芦丁为标准品,采用分光光度法,在弱碱性条件下对西葫芦粗提液中的总黄酮含量进行测定;吸取提取液0.2mL到10mL比色管中,加入0.4mL的三氯化铝溶液,混匀后再加入0.4mL的醋酸钾溶液,再次混匀后加入30%的乙醇溶液定容到刻度,混匀后静置,在30min内以零管调节零点后在415nm处测定OD值;
总三萜含量的测定:以齐敦果酸为标准品,采用香草醛-冰醋酸比色法,在高氯酸环境下对西葫芦粗提液中总三萜含量进行测定;不同溶剂提取液各1.0mL分别置于10mL的比色管中,经氮吹仪氮吹后加入5%(m/V)香草醛-冰醋酸0.5mL,再分别加入高氯酸1mL,摇匀后在70℃水浴20min。取出后在冰水浴中冷却,用乙醇稀释至刻度,摇匀后静置5min,以试剂空白为基线,在460nm下测定OD值;
抗坏血酸含量的测定:参照抗坏血酸测定试剂盒进行测定抗坏血酸含量;
(4)抗氧化活性的比较评价:分别采用DPPH、ABTS、FRAP法对西葫芦样品的抗氧化活性进行综合对比评价;
DPPH自由基清除能力的测定:吸取0.5mL的西葫芦粗提液,0.5mL的DPPH混合均匀,在室温下避光反应20min,以无水乙醇调零,以抗坏血酸代替粗提液作为阳性对照,测定517nm处的OD值;
ABTS自由基清除能力的测定:取6个2mL的离心管,依次加入各浓度的粗提液0.5mL,再加入稀释后的ABTS和过硫酸钾混合液0.5mL,混合均匀后,避光放置6min,以抗坏血酸代替粗提液作为阳性对照,在734nm处测定OD值;
FRAP还原能力测定:分别吸取浓度为25mg/mL的西葫芦提取液和不同浓度的FeSO4溶液各150μL,加入4.5mL的FRAP工作液,37℃下反应10min,在593nm下测定OD值;以FeSO4浓度为横坐标,吸光度值为纵坐标绘制标准曲线,将上述测得的样品吸光度值代入标准曲线计算出相应的FeSO4浓度,还原力用相应的FeSO4浓度(μmol/L)表示。
2.根据权利要求1所述的一种西葫芦抗氧化活性成分的优化提取方法及抗氧化活性的验证方法,其特征在于:步骤(1)中西葫芦干燥时间为18h,过80目筛。
3.根据权利要求1所述的一种西葫芦抗氧化活性成分的优化提取方法及抗氧化活性的验证方法,其特征在于:步骤(2)中水浴加热回流提取的温度为70℃,辅助提取时间3h,真空旋转蒸发仪浓缩温度为50℃,压强为0.13MPa,最终的提取液浓度为25mg/mL。
4.根据权利要求1所述的一种西葫芦抗氧化活性成分的优化提取方法及抗氧化活性的验证方法,其特征在于:步骤(2)中优化的溶剂是甲醇。
5.根据权利要求1所述的一种西葫芦抗氧化活性成分的优化提取方法及抗氧化活性的验证方法,其特征在于:步骤(3)中总多酚含量的测定中:吸取提取液为1.0mL,测定波长为778nm;总黄酮含量的测定中:吸取提取液为0.2mL,测定波长为415nm;总三萜含量的测定中:吸取提取液为1.0mL,测定波长为460nm;抗坏血酸含量的测定中:测定波长为536nm。
6.根据权利要求1所述的一种西葫芦抗氧化活性成分的优化提取方法及抗氧化活性的验证方法,其特征在于:步骤(4)中分别在波长为517nm、734nm和460nm处测定OD值,计算DPPH自由基清除率、ABTS自由基清除率和FRAP值。
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