我国常见保健食品原料抗氧化活性的研究
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摘要
随着经济的发展和人们生活水平的提高,食物消费正从温饱型向小康型过渡,人们对食品的质量提出了更高的要求,保健食品由此应运而生。中国的保健食品受传统药食同源思想和中医理论的影响,其原料多以中草药为主；中草药中富含具有一定抗氧化活性的功效成分,且目前国家已批准的保健食品功能中的降血糖、降血压、增加免疫力、抗衰老等功能均与抗氧化活性有关,因此,以抗氧化活性为共性指标评价对中草药为主的保健食品原料进行评价,以现代医学和营养学的角度对其进行解释,有助于保健食品原料更加适应现代社会的需要。
一、目的
1.通过对常见保健食品原料营养成分、功效成分的测定获取相关数据,为今后保健食品原料的抗氧化研究做出基础性工作。
2.通过体外抗氧化测定方法、以抗氧化活性为共性指标对常见保健食品原料抗氧化活性做出评价。
3.研究营养成分、功效成分对抗氧化活性的影响,为保健食品功能声称评价提供科学基础。
4.筛选出具有较高抗氧化活性和抑制a-葡萄糖苷酶的活性的保健食品原料,为今后保健食品原料的使用做出指导。
二、材料与方法
结合中国传统医学药食同源思想和中医理论,以《卫生部关于进一步规范保健食品原料管理的通知》(卫法监发2002(51)号文件,以下简称51号文件)为依据选择常见保健食品原料做为研究对象。
1.常见保健食品原料基本营养成分的研究
采用国家标准方法对170种保健食品常见原料进行营养成分的测定,其中水分采用直接干燥法(GB/T5009.3-2003)、蛋白质采用凯氏微量法(GB/T5009-2003)、灰分采用(GB/T5009.4-2003)方法、镁采用GB/T5009.90-2003方法、钾采用GB/T5009.91-2003方法、钠采用GB/T5009.91-2003方法测定,磷采用分光光度法(GB/T5009.87-2003)测定,铁采用GB/T5009.90-2003测定,钙采用原子吸收分光光度法(GB/T5009.92-2003)进行测定,脂肪采用索氏抽提法(GB/T5009.6—2003),维生素AGB14750-1993、维生素E采用高效液相色谱法(GB/T5009.82-2003)、维生素C荧光法(GB/T5009.86-2003)、维生素B1采用(GB/T5009.84-2003)、维生素B2采用荧光法(GB/T5009.85-2003)。
2.常见保健食品原料主要功效成分的研究
分别采用铝盐比色法、香草醛-高氯酸法和酸性染料比色法测定原料的总黄酮、总皂苷和总生物碱的含量。
3.常见保健食品原料抗氧化活性的评价研究
选择经典的DPPH·自由基清除法和目前国际上最先进的ORAC方法同时对常见保健食品原料进行抗氧化活性评价。采用p-NPG法、以对硝基苯基-α-D-吡喃葡萄糖苷(p-NPG)为底物、利用全自动生化分析仪对常用保健食品原料进行α-葡萄糖苷酶抑制活性的高通量测定作为抗氧化活性的辅助评价,筛选出具有高抗氧化能力与高抑制α--葡萄糖苷酶活性的原料。将传统中医理论与现代营养学结合,对常用保健食品原料的ORAC值按照“四性”、“五味”和“阴阳”进行分类,运用统计学方法分析、观察不同类别的原料其ORAC值的高低和差异。
4.营养成分、功效成分与抗氧化活性的相关性研究
调用SPSS软件中的Correlation模块对原料的营养成分、功效成分和抗氧化活性进行相关性研究,观察不同成分对抗氧化活性的影响；调用SPSS软件中Classify Hierarchical Cluster模块进行聚类分析,根据抗氧化能力指数对常用保健保健食品原料进行聚类,以类别界限值作为今后抗氧化能力高低评判的标准。
三、研究结果
按照51号文件共购得170种常见保健食品原料做为研究对象,大部分经过专业炮制后为干品,少数为鲜品。
1.常见保健食品原料基本营养成分研究
宏量营养成分中,蛋白质在动物性原料中含量普遍较高,植物性原料中以“籽”、“仁”等名称的植物性样品蛋白质含量较高。动物性原料普遍含有脂肪,但含量低于紫苏子、苦杏仁等植物性原料,脂肪含量最低的原料是竹茹、灯心草、穿心莲、玫瑰花、人参、太子参、玫瑰茄,含量均为0.09g/100g。原料中碳水化合物含量最大的原料是竹茹,灯心草等植物性原料也较高,碳水化合物含量最小的原料是刺猬皮,大部分原料的碳水化合物介于60～80g/100g之间；能量最高的原料为紫苏子,其次是苦杏仁和郁李仁等“仁”、“子”类原料,动物性原料中阿胶的能量最高(393kcal)。能量最低的原料为石决明和珍珠；大部分原料的能量值在300-400kcal之间；植物性原料除种子类原料的能量平均值最高,其它部位能量分布比较均匀,而动物和其它类原料的能量值比较接近。动物性原料中的矿物质含量普遍高于植物性原料,其中,有9种原料的磷含量大于1000mg/100g,植物的种子和花部位磷含量较高；钠含量最多的原料分别是海参(3643mg/100g)、制肉苁蓉(1995.11mg/100g)和燕窝(1041.1mg/100g),均大于1000mg/100g,植物性原料的茎、叶部位其钠含量较高；钾含量最高的前5位原料分别是墨旱莲、豆蔻、茵陈、姜黄和积雪草,在动物性原料中普遍含量不高；钙含量最高的原料为海螵蛸(30187.5mg/100g),其次是石决明、马鹿胎和乌梢蛇,植物性原料中钙含量最高的为首乌藤(6735.9mg/100g)；镁含量最高的原料为乌梢蛇和刺猬皮,分别高达2636.7,1680.0mg/100g,其次是蓼大青叶、蒲黄等植物性原料；蝉蜕、地龙、茜草、半边莲和茵陈是铁最高的前5位原料,尤其是蝉蜕(蝉的幼虫变成成虫时蜕下的壳),铁含量高达1634.7mg/100g；维生素A、维生素C和维生素E在植物性原料中含量很低,几乎损失殆尽；维生素B1主要存在于植物的果实、种子和花中,火麻仁、藏红花、麸炒神曲、紫苏子和白芥子是含量高的前5位；维生素B2主要存在于植物和花部和叶部,含量排名前5名的是藏红花、地龙、团螵蛸和水蛭。就原料的不同药用部位来看,动物性原料中普遍含有蛋白质、钠；植物性原料的种子、果实富含蛋白质和脂肪；植物的茎部位其磷含量较高；植物的茎、叶部位其钠含量较高；钾、钙、镁和铁富集于植物的整株、花和叶部位。
2.常见保健食品原料功效成分研究
建立了精密度、稳定性、回收率均良好的总黄酮、总皂苷和总生物碱测定方法。通过对保健食品常用原料的功效成分测定和分析发现,原料中总黄酮、总皂苷在不同分析部位间含量差异较大,变化范围也较大,总生物碱含量普遍偏低。共有100个原料检出总黄酮含量,大部分原料的黄酮含量在500mg/100g以下,花、叶类原料中富含黄酮类物质,尤其在花中普遍总黄酮含量较高,平均含量高达1.42%；含量最高的原料为槐米(6690mg/100g),其它较高的的原料依次为槐米、槐角、厚朴、刺五加、紫苏子、月季花、合欢花、香薷、荷叶和诃子；共有151个原料检出了总皂苷含量,占到了总样品量的88%。多数原料的皂苷含量不高于3000mg/100g；叶、果实和花类的皂苷含量较为接近,叶和果实是皂苷含量较高的一类,以叶中含量较为丰富,平均含量达到了1.65%；总皂苷含量排名前10位的样品依次为苦丁茶、大皂角、知母、怀牛膝、酸枣仁、覆盆子、西洋参、吴茱萸、连翘和益智；大部分原料的生物碱含量为125mg/100g以下,生物碱含量普遍偏低,除了个别含量较高的原料外,其余原料的生物碱含量变化差异性较小,根据分析部位来看,生物碱在根及根茎中平均含量相对较高,其它部位中的生物碱含量都较为接近。总生物碱含量排名前10位的样品依次为防己、黄连、芦荟、香加皮、西洋参、大皂角、麻黄、罗布麻花、川芎和柴胡。黄酮、皂苷和生物碱一般不存在于动物性样品中。
3.常见保健食品原料抗氧化活性研究
通过DPPH·自由基清除实验共筛选出147种原料不同程度地均有一定的清除自由基活性,23个原料基本无清除自由基活性,有16种原料活性高于阳性对照维生素C,按照活性由高到低的顺序排列它们分别是淫羊藿、桂枝、石斛、首乌藤、制肉苁蓉、吴茱萸、荷叶、罗布麻花、连翘、诃子、团螵蛸、玫瑰花、槐角、金荞麦、芦荟和积雪草,所有原料中清除活性最弱的为淡豆豉；通过数学运算将清除自由基活性的IC50值转换成TAC值(总抗氧化能力),既避免了自由基清除活性IC50值比较时的困难,也消除了原料浓度对清除效果表达的影响。
ORAC实验是目前国际上推荐的新型抗氧化评价方法。经过对常见保健食品原料的水溶性ORAC值测定后发现：所有原料的ORAC值介于0.27～3579μmolTE/g之间；ORAC值高于阳性对照品维生素C的为苦丁茶、芦荟和槐米；其中,ORAC值最高的为苦丁茶(3579μmol TE/g),最低的为珍珠(0.27μmol TE/g)其它较高的样品有芦荟、槐米、吴茱萸、槐角、丹参等；而海螵蛸、石决明、燕窝、海参、芡实、猪苓和马鹿胎则为ORAC值很低的原料,多为动物性样品。调用SPSS软件中的Correlation模块对二种抗氧化评价方法得到的数据进行统计学上的相关性分析,发现清除DPPH·自由基法得到的结果与ORAC法测得的结果相关性较强,表明二种方法在抗氧化活性评价时有相同的趋势。从分析部位来看,自由基清除效果较好的原料主要是花类的原料,其次为叶类；ORAC值按其药用部位归类,发现茎、叶类原料虽然数量不多但ORAC值却是最高的。
以拜糖平为阳性对照物,采用经典的p-NPG方法对常见保健食品原料进行α-葡萄糖苷酶抑制活性高通量筛选。结果表明共有84种原料有一定的抑制活性,其中有25种的抑制活性强于阳性对照物拜糖平。
在抑制α-葡萄糖苷酶活性高的前20位原料中,有9种原料既有很强的抑制a-葡萄糖苷酶活性,同时又具有很强的ORAC值,它们分别是玫瑰花、诃子、金荞麦、淫羊藿、首乌藤、罗布麻叶、虎杖、月季花和赤芍。有8种原料既有很强的抑制α-葡萄糖苷酶活性,同时又具有很强的TAC值,它们分别是淫羊藿、首乌藤、罗布麻花、诃子、玫瑰花、金荞麦、赤芍和罗布麻叶。有8种原料既有较高的ORAC值,又有较高的TAC值,分别是玫瑰花、诃子、金荞麦、淫羊藿、首乌藤、罗布麻叶、首乌藤和芦荟；三者之间的互相比较表明高抗氧化活性原料同时也有较高的抑制α-葡萄糖苷酶活性。
抑制α-葡萄糖苷酶活性的IC50值与TAC值、ORAC值间的相关性分析结果显示：抑制α-葡萄糖苷酶活性的IC50值与ORAC值和TAC值均呈现负相关关系,且有一定的统计学意义,表明原料抑制α-葡萄糖苷酶活性越强,其IC50值越小,抗氧化能力随之越强,为今后α-葡萄糖苷酶抑制活性与抗氧化活性的协同性研究提供了有益的资料。
4.常见保健食品原料抗氧化活性与中医理论的探讨
中医理论认为物有“四性”即“寒”、“热”、“温”、“凉”和“五昧”即“辛“、“甘”、“酸”、“苦”、“咸”,并同时常常用“阴”、“阳”来对保健食品原料进行评价。本研究中的保健食品原料多以中草药为主,因此,将它们的ORAC值根据“四性”、“五味”和“阴阳”分别归类后发现,通过“四性”分类的原料其ORAC值组间无显著性差异(p>0.05)；按照“五味”分组的原料其ORAC值有显著性差异(p<0.05)；阴类原料和阳类原料的ORAC值有显著性差异(p<0.05)
5.常见保健食品原料营养成分、功效成分与抗氧化活性的相关性研究和抗氧化能力指数的聚类研究
调用SPSS软件中的Correlation模块、Classify模块和Regression模块分别将营养成分、功效成分与ORAC值、TAC值进行相关性分析、聚类分析和线性回归分析。结果表明：170种原料的蛋白质、脂肪和能量均与抗氧化能力指数无相关性(p>0.05),碳水化合物与抗氧化能力有一定的相关性,但相关系数很小(p<0.05)。与抗氧化能力指数呈现负相关关系的元素有钠和磷,而钾、钙、镁和维生素B2与抗氧化能力指数有一定的正相关性且有统计学意义(p<0.05),维生素B1和铁与抗氧化能力指数的相关性无统计学意义(p>0.05)。宏量营养素与自由基清除能力无相关关系(p>0.05)。钙和维生素B2与TAC值有一定的正相关性(p<0.05),磷和维生素B1与抗氧化能力呈现负相关关系,其它元素与TAC值无相关(p>0.05)。当消除ORAC值的影响时,α-葡萄糖苷酶抑制活性与自由基清除能力呈现负相关关系但无统计学意义(p>0.05)；当消除TAC值的影响时,α-葡萄糖苷酶抑制活性与抗氧化能力指数也呈现负相关关系,但无统计学意义(p>0.05)。清除自由基能力与抗氧化能力指数有较为显著的相关性(p<0.05),表明二种方法在进行抗氧化评价时,有一致的趋势。功效成分含量与抗氧化活性间相关性研究结果显示总黄酮、总皂苷与ORAC值有一定的相关性,相关系数分别为0.500、0.288(p<0.01)。TAC值与总黄酮、总皂苷成分也有一定的相关,相关系数分别为0.314、0.208(p<0.01)。
总黄酮、总皂苷含量与抑制α-葡萄糖苷酶活性的IC50值(mg/mL)呈现负相关但无统计学意义(p>0.05)；生物碱与抑制α-葡萄糖苷酶活性的IC50值(mg/mL)相关性不明显(p>0.05)
调用SPSS软件中的Classify-Hierarchical cluster模块对170种原料的抗氧化能力指数共分为6类,其中,苦丁茶因ORAC值最高而自动归为第1类,定义为“极强抗氧化能力”级；槐米和芦荟(ORAC平均值为2667μmol TE/g)的2个原料自动归为第2类,定义为“强抗氧化能力”级；以香薷为代表的15种原料(ORAC平均值为1260μmol TE/g)归为第3类,定义为“高抗氧化能力”；以牡丹皮为代表的25类原料归为第4类,ORAC平均值为723μmol TE/g,定义为“中抗氧化能力”；以木香为代表的47类原料归为第5类,ORAC平均值为314μmol TE/g,定义为“弱抗氧化能力”；第6类为珍珠、西洋参等原料,共计80种,ORAC平均值为40.5μmol TE/g,定义为“极弱抗氧化能力”。
四、结论
1.常见保健食品原料营养成分研究
170种常见保健食品原料的宏量营养成分中,植物性原料中以“籽”、“仁”等名称的植物性样品蛋白质含量较高。动物性原料脂肪普遍低于个别植物性原料。大部分原料的碳水化合物介于60～80g/100g之间。植物性原料除种子类原料的能量平均值最高,其它部位能量分布比较均匀,而动物和其它类原料的能量值比较接近。动物性原料中的矿物质含量普遍高于植物性原料,主要集中在植物的根部和动物类、其它类原料中,植物的种子和花部位磷含量较高；钠含量最多的原料为动物性原料,植物性原料的茎、叶部位其钠含量较高；钾含量最高的原料全部是植物性原料,在动物性原料中普遍含量不高；钙多富集于动物性原料中；镁多存在于动物性原料中,蓼大青叶、蒲黄等植物性原料含量也较高；铁普遍存在于植物的地上部分,蝉蜕(蝉的幼虫变成成虫时蜕下的壳)中铁含量最高；维生素A、维生素C和维生素E在植物性原料中含量很低,几乎损失殆尽；维生素B1含量最高的前5位原料分别是火麻仁、藏红花、麸炒神曲、紫苏子和白芥子；维生素B2主要存在于植物的花、叶中。就原料的不同药用部位来看,植物性原料的种子、果实富含蛋白质、脂肪和维生素B1；植物的茎部位其磷含量较高；植物的茎、叶部位其钠含量较高；钾、钙、镁和铁富集于植物的整株、花和叶部位。
2.常见保健食品原料主要功效成分研究
共有100个原料检出总黄酮含量,共有151个原料分别检出了总皂苷和总生物碱含量。大部分原料的黄酮含量在500mg/100g以下,花、叶类原料中富含黄酮类物质；多数原料的皂苷含量不高于3000mg/100g；叶、果实和花类的皂苷含量较为接近,叶和果实是皂苷含量较高的一类；大部分原料的生物碱含量为125mg/100g以下,生物碱含量普遍偏低,除了个别含量较高的原料外,其余原料的生物碱含量变化差异性较小,根据分析部位来看,生物碱在根及根茎中平均含量相对较高,其它部位中的生物碱含量都较为接近。3.常见保健食品原料抗氧化活性研究
共筛选出147个原料均有一定的清除自由基能力,且有16种原料活性高于阳性对照维生素C,表明常见保健食品原料对以氮为中心的有机自由基具有较普遍的清除作用；ORAC方法测定结果显示原料的抗氧化能力指数差异较大,表明常见保健食品原料对氧自由基具有一定的清除作用,但因样品不同而差异较大；ORAC值最高的前10名原料分别是苦丁茶、芦荟、槐米、吴茱萸、槐角、丹参、玳玳花、罗巴斯塔咖啡豆(烤前)、虎杖和丹参。中医传统理论“四性”、“五味”和“阴阳”与抗氧化能力指数进行组间方差分析,结果表明：依照“四性”不同,其原料组间ORAC值无显著差异(p>0.05),显示按照中医传统理论的“四性”分类,难以对原料的抗氧化能力进行区分；“苦”和“苦”类原料组间抗氧化能力有显著差异间差异(p<0.05),表明依照“五味”划分抗氧化能力有一定的作用；“阴”和“阳”、“中”类原料组间抗氧化能力有显著差异间差异(p<0.05),表明依照“阴阳”划分抗氧化能力有一定的作用。
共筛选出84种有α-葡萄糖苷酶抑制活性的原料,其中有25种的抑制活性强于阳性对照物拜糖平。常用保健食品原料对α-葡萄糖苷酶的抑制具有较大的差异性,IC5α值变化范围较大,有些原料的抑制酶活性很强,有些则很弱,超过三分之一的原料不仅有抑制活性,还具有较强的活性。中草药的主要部位都对α-葡萄糖苷酶有抑制作用,活性较强的部位有花、果、根茎等部位。抑制α-葡萄糖苷酶活性较高的前20位原料中,有9种原料既有很强的抑制α-葡萄糖苷酶活性同时又具有很强的ORAC值；有8种原料既有很强的抑制α-葡萄糖苷酶活性同时又具有很强的TAC值,为今后抗氧化活性和抑制α-葡萄糖苷酶活性的协同研究提供了有价值的参考依据。
碳水化合物、能量、钾、钙、镁和维生素B2与抗氧化能力指数值有一定的正相关性且有统计学意义(p<0.05)；碳水化合物、钙和维生素B2与自由基清除能力有一定的正相关性；总黄酮、总皂苷百分含量与抗氧化能力指数呈一定的相关性,相关系数分别为0.500、0.288(p<0.01)；黄酮、皂苷成分与清除自由基活性也有一定的相关性,而生物碱含量则与TAC值无相关性。
抑制α-葡萄糖苷酶活性的IC50值与ORAC值和TAC值均呈现负相关关系(p<0.05),表明抑制α-葡萄糖苷酶活性越强,其ICso值越小,抗氧化能力随之越强；通过相关性分析表明清除自由基能力与抗氧化能力指数有较为显著的相关性,显示二种方法在进行抗氧化评价时,有一致的趋势；通过聚类分析对170种原料的抗氧化能力指数共分为6类,苦丁茶为第1类,定义为“极强抗氧化能力”级；槐米和芦荟归为第2类,定义为“强抗氧化能力”级；以丹参、葛根为代表的15种原料归为第3类,定义为“高抗氧化能力”；以月季花、枳壳为代表的25类原料归为第4类,定义为“中抗氧化能力”；以番泻叶、木香为代表的47类原料归为第5类,定义为“弱抗氧化能力”；第6类为珍珠、木贼等原料,共计80种,定义为“极弱抗氧化能力”。
五、创新之处
1.首次对170种常见保健食品原料进行了营养成分和功效成分的测定,为今后保健食品原料的使用提供数据支持和指导。
2.首次通过抗氧化活性的高低对保健食品原料性质进行系统评价和聚类分析,并探讨了成分和活性间的关系。
3.首次将以物质为基础的营养学理论和现代医学中的共性指标—抗氧化活性来阐述传统中医学理论用“四性”、“五味”和“阴阳”对中草药原料的整体性评价。
With the development of economy and people's living standards, food consumption is moving from subsistence to a comfortable level, people put a higher quality of food requirements; functional food thus came into being.
Regulatory function of the functional food depends on its raw material and ingredients, but the functional food raw materials has been the lack of systematic research.
China's functional food is functionalse on the traditional Chinese medicine and Medicinal and Edible theories, the raw materials mainly are Chinese herfunctionall medicine.
Function of claims is still the traditional Chinese medicine formulation, lack combination with modern Western medicine and the nutrition. For example, the function of antioxidant, the functional food ingredients seldom have antioxidant system evaluation, especially litter research about the relationship among the antioxidant functional food claims and the raw materials and components.
As China's most authoritative food composition datafunctionalse-China food composition is also lack of this part of the data. Therefore, the research about the raw materials often used in China functional food, especially its nutritional content,the functional components and the relationship between composition and antioxidant activity is under the circumstances.
Objectives
1. Add data to Chinese food composition table, fill data gaps of functional food nutritional ingredients, functional components.
2. Using the in vitro antioxidant evaluation method to evaluate the antioxidant activity of common raw materials in functional food, while make aid evaluation for antioxidant activity evaluation to common raw materials about functional food by a-glucosidase inhibitory activity.
3. Observe the relationship and effectiveness between the nutrients, functional factors and antioxidant activity, provide the scientific functionalsis for functional food claim.
4. Selecting the high antioxidant activity of functional food raw materials make the guidance for using of functional food raw materials in future
5. Apply the statistical methods to construct regression model between functional components and the in vitro antioxidant activity
Material and Methods
On the functionalsis of the traditional Chinese medicine and Medicinal and Edible theories, according to the inform of "to further standardize the management of raw materials to functional food of Ministry of Functional "(Document No.51), we selected170species common raw materials including plants, animals, involving a total of65subjects in our study.30species are belong to Document No.51annex I,85species belonging to Annex Ⅱ, and the remaining55species belong to other common functional food raw materials, mainly are traditional Chinese herfunctionall medicine.
1. Analysis of the functionalsic nutrients
Adopt national standards to analyze nutrition Determination of170kinds of common raw materials of functional food. Water used the method of GB/T5009.3-2003, protein is GB/T5009-2003)、ash is GB/T5009.4-2003, magnesium used GB/T5009.90-2003, potassium used GB/T5009.91-2003, sodium used GB/T5009.91-2003, phosphorous used GB/T5009.87-2003, iron used GB/T5009.90-2003, Calcium used Atomic absorption spectrophotometry(GB/T5009.92-2003), fat used Soxhlet extractor method(GB/T5009.6-2003), vitamin A used GB14750-1993, vitamin E used HPLC method(GB/T5009.82-2003), vitamin C used fluorometric method (GB/T5009.86-2003), thiamine used GB/T5009.84-2003, Riboflavin used fluorometric method (GB/T5009.85-2003)。
2. Analysis of functional components
Total flavonoids determination is Aluminum chloride colorimetric assay, total saponins used Vanillin perchloric acid assay and total alkaloids used Acid dye colorimetry.According different plant medicinal part, classify the raw materials and discussed the distribution of nutrients, medicinal ingredients in different parts
3. Antioxidant activity evaluation and high-throughput screening of a-glucosidase inhibited activity
Respectively adopting the most advanced in vitro antioxidant evaluation method-ORAC method and the classical DPPH· radical scavenging method for the common functional fodd raw material. We reformed the DPPH· radical scavenging method by the automatic biochemical analyzer to improve experimental conditions to achieve a high throughput screening. DPPH· free radical removal efficiency is expressed by the concentration of IC50before, and now, after the mathematical conversion, the total antioxidant capacity (TAC value) is the express result,easy to understand and more clear and intuitive, using p-NPG method, p-NPG as substrate to screen a-glucosidase inhibit activity of170kinds of functional food raw materials, using automatic biochemical Analyzer to achieve high-throughput screening, a-glucosidase inhibition efficiency is expressed by50%inhibitory ration needed the raw materials concentration (IC50value). According to different medicinal parts, observe the antioxidant activity level in different medicinal parts. Screening the raw material with high Antioxidant capacity and high α-glucosidase inhibitory activity. According to traditional Chinese medicine theory, classify the common functional food raw materials in accordance with the four properties, five flavors and yin and yang, to observe the different categories of the level of antioxidant activity.
4. Correlation of Nutritional composition, functional components and antioxidant activity
Correlation research is developed by SPSS software, spearman method for the nutritional content, functional and antioxidant activity components of170species of raw materials, results showed that antioxidant activity and flavonoids are most closely. Using SPSS software Classify Hierarchical Cluster modules, clustering principle indicator cluster (R type) in the correlation coefficient distance (Pearson correlation), methods for the class on average chain method (Between-groups linkage) clustering of all raw materials analysis, comparing the degree of similarity between different indicators. According to cluster analysis and correlation analysis results, further use of SPSS software, the Regression module, to establish the regression equation, flavonoids as independent variables and ORAC value as dependent variable.
Research result
1. Basic nutrition ingredients Determination of170kinds of common functional food raw materials
Following on Determination of170kinds of common functional food nutrients ingredients, we found that nutrients are abundant and widely distributed. Among Macronutrients, the water content distribution is in the0.5g-69.5g/100g, with a maximum water content of fresh Rhizoma Imperatae and royal jelly; Protein in the name of "benevolence","seed" category of raw material were higher, mostly distributed in fruits, seeds. Ash mainly exist in animal raw materials; iron is distributed in different parts of plant, To free oneself (cicada larvae into adults under the shell when the slough) was highest (1634.7mg/100g), is the lowest of the royal jelly of1256times. More magnesium present in the raw materials of animal and plant part of above ground, between the range of3.26-2636.7mg/100g. Potassium is mainly distributed in the ground part of the raw materials; Compositae Eclipta has the highest concentrations. Sodium mainly exist in animal samples, the highest concentrations is sea cucumber. Phosphorus is distributed in the roots to the highest content of Rhodiola and Siberian, and their analysis parts are the roots. The highest calcium content of raw materials is the Cuttlebone (dried squid squid related species within the shell), and in the plant raw material is more at leaf. Vegetable fats are generally essential fatty acids, linoleic acid and linolenic acid, after Soxhlet extraction method and we found that170kinds of raw materials contain a certain amount of fat, changes in range between0.09-53.7g/100g, more present in the plant fruit, seeds, such as perilla seed, hemp seed, and some plant material in the fat content even higher than the animal samples. After determination of vitamin analysis we found that subjects have generally low levels of vitamin, especially vitamin A, vitamin C and vitamin E, in most of the raw materials or even not detected, which may be due to raw materials are processed professionally in the sun, washing and bring loss.
2. Determination results of functional ingredients
Flavonoids, saponins are the most frequent claim components in functional food. Therefore, in this study, they are chosen as the main target, and on this basis, select the alkaloids of this ubiquitous component of Chinese herbs while a total of three indicators170kinds of raw materials for the determination of functional components. After the analysis, the percentage of total flavonoids, total saponins and total alkaloids of170kinds of functional food showed:Most of the material in the total flavonoids, saponins were significantly different and varied greatly. The top10highest total flavonoid content of raw materials were FlosSophorae Sophoricoside, Magnolia, Acanthopanax, perilla, Chinese rose, Albizia julibrissin, Hong Ru, the leaves and myrobalan; The total saponin content of samples of the top10were Kudingcha, Anemarrhenae, Achyranthes bidentata, Suanzaoren, raspberries, American ginseng, Evodia, forsythia and Fructus Alpiniae Oxyphyllae. In Weight loss function food, total alkaloids are major components, through the determination of the levels found generally low, and little difference in content; top10samples were anti has, berberine, aloe, Hong Gapi, American ginseng, fruit of Chinese honeylocust, ephedra, cannabis Rob, and Bupleurum Chuanxiong
After the analysis of the raw material in accordance with the medical site classification and then to observe the different parts of the content of functional components and we found:a high flavonoid content in plants generally flower parts, fruits and leaves, flowers, high content of total flavonoids in general, the average content of1.42%; saponins are secondary metabolites of plants to the leaves of more abundant, the average content was1.65%; roots and rhizomes of the class highest alkaloid content. Flavonoids, saponins and alkaloids in general does not exist in animal samples.
3. Antioxidant activity and inhibition of a-glucosidase activity of raw materials in China Functional food
Antioxidant function is one of the important functions of functional food. Evaluation of antioxidant activity often choose two kinds of methods or more were determined simultaneously. Our study chose traditional DPPH· radical scavenging method and the current world's most advanced ORAC method to analyze for170kinds of commonly used functional food raw materials. DPPH· radical is a stable chemical radicals, at the presence of antioxidants, antioxidants and their single-electron pair and its absorption gradually fade, and their acceptance of its fading into the quantitative relationship between the number of electrons, with spectrophotometric changes in absorbance calculated by free radical scavenging activity of samples, that is antioxidant activity. In our study, with the automatic biochemical analyzer, DPPH· method of traditional experimental conditions were improved to achieve a high throughput screening, and to avoid the free radical damage to human body. By measuring,23kinds of raw materials basically no free radical scavenging activity, and the remaining raw materials are some free radical scavenging activity, and there are16kinds of raw materials more active than the positive control of vitamin C, according to the order of their activity from high to low are epimedium, Cinnamomum Ramulus, Dendrobium, vine of multiflower knotweed, system Cistanche (system large Yun), Evodia, lotus leaf, Rob cannabis, forsythia, Terminalia, group cuttlebone, roses, Sophoricoside, wild buckwheat, Aloe vera and Centella asiatica, the weakest scavenging activity of all raw materials is the semen sojae praeparatum; through mathematics the IC50values of free radical scavenging activity converted to TAC value, which avoids the radical scavenging activity (IC50values) comparison difficult, but also eliminates the effect of raw material concentration on the expression of clear. ORAC assay is recommended by the international evaluation of new anti-oxidation. After ORAC analyze, the170kinds of raw materials, water-soluble ORAC value are between0.27～3579μmol TE/g. the highest ORAC value is Kudingcha, the lowest is the pearl, the other hith values samples are aloe, FlosSophorae Evodia, Sophoricoside, Danshen; the low values samples are cuttlebone, abalone, bird's nest, sea cucumber (Gypsophila), Gorgon, Polyporus, Red Deer fetus etc. for the ORAC value of the minimum of raw materials, and more for the animal samples. We used SPSS software Correlation module to develop statistical correlation analysis, we found clear DPPH·free radical method the results obtained with the results measured by ORAC method has some relevance, that means these two kinds of method in the evaluation of antioxidant activity have the same trend. From the analysis of different parts, we found that free radical scavenging effect of better raw materials are mainly raw materials like flowers, with an average IC50value of0.99mg/mL, followed by leaf; ORAC value are classified according to their medicinal part, found that stem, leaves material although a small number, but with the highest ORAC average values. α-glucosidase is a very important enzymes, through the hydrolysis of a-1,4glycosidic bond from the non-reducing end of polysaccharide glucose cut. The body of the starch, dextrin, sucrose and other carbohydrates in the use of absorption depends on the intestinal brush border on the enzyme activity, control of diabetes and the need to reduce the activity of the enzyme, a-glucosidase inhibitors by competitive inhibition of a-glucosidase activity, can block two-sugar sugar solution into a single delay the absorption of sugar, so blood sugar steady and slow to maintain a certain level. In our study, acarbose is used as the positive control, using the classic method of p-NPG for170kinds of raw materials on the a-glucosidase inhibitory activity screening test, the use of automatic biochemical analyzer to achieve high-throughput screening and more efficient than96microplates. The results showed that a total of84raw materials has some inhibitory activity, of which25species of inhibitory activity was stronger than positive control acarbose. Overall, Chinese herbal medicine as an α-glucosidase inhibitor, the more complex compositions lead to mechanism needs further study.
About inhibiting a-glucosidase activity, in the the top20raw material, there are9kinds of raw materials both strong suppression of α-glucosidase activity and the high ORAC value. They were roses, Terminalia, Fagopyrum, Epimedium, vine of multiflower knotweed, Apocynum leaves, cuspidatum, Chinese rose with Radix. There are8kinds of raw materials both strong suppression of a-glucosidase activity and strong TAC value. They are epimedium, vine of multiflower knotweed, Rob cannabis, Terminalia, roses, wild buckwheat, red Shao and Apocynum leaves.8both have a high ORAC value and a highTAC values were roses, Terminalia, wild buckwheat, Epimedium, vine of multiflower knotweed, Apocynum leaves, vines,vine of multiflower knotweed and aloe. In the future, these materials need more research and application to play important role.
4.Antioxidant activity relationship with the theory of Traditional Chinese Medicine
TCM theory holds that objects have "four properties" of the cold, hot, warm, cool and "five flavors " that is acrid, sweet, sour, bitter, salty, and also often use the "Yin","Yang" to evaluate the effects of Chinese herbs. In this study, the functional food are mainly herbal-based raw materials, so with four properties, five flavors and yin and yang were classified, we listed each type of material corresponding ORAC value. After research, we found:Cold material compared to the hot, warm and cool materials, the ORAC value of the highest, but the quantity is not much; bitterness of raw materials compare to the acrid, sweet, sour, salty class of its ORAC value not only high, quantity is greater; yin materials with high ORAC value and the number is greater. After further analysis of variance, we found that sour, bitter class materials ORAC value have significantly different (p<0.05), but by four properties classification, the raw materials of their ORAC values were no significant differences (p>0.05); yin yang kind of raw materials and class materials ORAC values significantly different (p<0.05), showed that the antioxidant activity by Yin Yang is more reliable classification.
3.1The correlation analysis, cluster analysis regression equation
SPSS software Correlation modules, Classify module and Regression modules respectively are used in nutrition, functional components and ORAC value, TAC values of the correlation analysis, cluster analysis and linear regression analysis. The results showed that:ORAC value and total flavonoids, total saponin content have some correlation, correlation coefficients were0.500、0.288(p<0.01), while the alkaloid content was almost no correlation with the ORAC value. TAC values and flavonoids, saponins also have some correlation coefficients were0.314、0.208(p <0.01), total flavonoids, total saponin and inhibited α-glucosidase activity of the IC50value (mg/mL) showed a negative correlation, shows that flavonoids, saponin content is more, the inhibition of α-glucosidase activity is stronger, and the IC50value is more lower, which is consistent with theory, though not statistically significant.
Cluster analysis is based on characteristics of the thing itself, individual classification is based on the principle of feather flock together something categories. Used SPSS software Classify-Hierarchical cluster module cluster to analyze the different indicators and we found that flavonoids and ORAC values of the short distance in the statistical relationship between the tree, therefore, the flavonoid content can be regarded as the main ORAC value effect factors, other factors are in turn based on distance close to the level of saponin, TAC values and alkaloids; α-glucosidase inhibitory activity with IC50value of total alkaloids and a tree closest relationship, followed by the flavonoids and saponins.
Using SPSS software Classify-Hierarchical cluster module to classify170kinds of raw materials based on antioxidant capacity index, then they are divided into six categories, which, because of ORAC value of the highest Folium llicis Latifoliae automatically classified as Class1, is defined as "very strong antioxidant "class; Flos Sophorae Immaturus and Aloe (ORAC mean2667μmol TE/g) of the two raw materials automatically classified as category2, defined as" the strong antioxidant capacity "level; to Salvia, Pueraria, represented by15kinds of raw materials (ORAC average1260μmol TE/g) classified as Category3, defined as "high antioxidant capacity"; to Chinese rose, Citrus aurantium, represented by25types of raw materials classified as Category4, ORAC mean723μmol TE/g, defined as "antioxidant capacity"; to senna, Saussurea costus represented47categories classified as Category5, ORAC mean314μmol TE/g, is defined as "weak antioxidant capacity";6category pearl, Radix Aucklandiae and other raw materials, a total of80species, ORAC mean40.5μmol TE/g, is defined as "a very weak antioxidant activity."
Conclusion
China's functional food raw material is widely used, evolved many families and distribution. Many kinds of medicinal parts of more nutritious ingredients, big difference in the effectiveness of ingredients, different parts of the nutrients, functional components were significant differences in content, but a distribution of components in the same position with convergence, such as moisture, protein, ash, mainly in the animal raw materials, flavonoid, saponins, alkaloids mainly present in plant material
After the traditional method of DPPH free radical scavenging screening, we found among170kinds of common functional food raw materials, there are152have scavenging activity and there are16kinds of raw materials higher than the positive control activity of vitamin C;170kinds of raw materials ORAC value is quite different, ORAC value material difference between the highest and lowest13255times. Free radical scavenging method and the ORAC method were significantly correlated, indicating that the same trend in two ways. There were selected from84kinds of α-glucosidase inhibitory activity of raw materials, including25species of inhibitory activity was stronger than positive control acarbose. Chinese herbal medicine are all major parts of α-glucosidase inhibition, Chinese herbal medicine as an α-glucosidase inhibitor mechanism of the onset is more complex, needs further study, α-glucosidase inhibition activity is higher in the top20raw materials, raw materials are9strong inhibition of both α-glucosidase activity was also highly ORAC value; are eight kinds of raw materials both strong a-glucosidase inhibitory activity, while TAC has a strong value.
According to the Chinese "four properties","five flavors ","Yin Yang" Theory and Comparative antioxidant activity of the classified study found cold, cool, negative raw materials usually have high ORAC value. After analysis of variance showed that by " four properties " of classified material whose ORAC value is no significant difference (p>0.05), through the "five flavors" for the raw materials of its ORAC value groups were significantly different (p<0.05), that by " four properties "to distinguish between antioxidant activity is open to question;"yin "type of raw materials and" yang "type of raw materials ORAC values significantly different (p <0.05), showed that the antioxidant activity through the yin and yang is a more reliable classification.
After statistical correlation analysis, the result showed that the total flavonoids, total saponin content and ORAC value of percentage of a certain correlation (p<0.01), in particular flavonoids and a high correlation coefficient; alkaloid content and ORAC value is the basic no correlation. Flavonoids, saponins and free radical scavenging activity has some correlation (p<0.01), total flavonoids, saponins and inhibition of a-glucosidase activity of the IC50value (mg/mL) showed a negative correlation, indicating that flavonoids, saponins with the higher content, the inhibitory activity is more stronger and the IC50value is more smaller; and alkaloid content was almost no correlation with the activity. After statistical clustering analysis showed that flavonoids and ORAC values in the statistical relationship between the short distance and have the tree, therefore, affect the flavonoid anti-ORAC value may be regarded as a major factor, other factors are in turn based on distance close to the level of saponin, TAC value, and alkaloids; α-glucosidase inhibitory activity with IC50value of alkaloids and a tree closest relationship, followed by the composition of flavonoids and saponins.
170kinds of raw materials based on antioxidant capacity index, using SPSS soft ware, then they are divided into six categories, which, because of ORAC value of the highest Folium llicis Latifoliae automatically classified as Class1, is defined as "very strong antioxidant "class; Flos Sophorae Immaturus and Aloe (ORAC mean2667μmol TE/g) of the two raw materials automatically classified as category2, defined as" the strong antioxidant capacity "level; to Salvia, Pueraria, represented by15kinds of raw materials (ORAC average1260μmol TE/g) classified as Category3, defined as "high antioxidant capacity"; to Chinese rose, Citrus aurantium, represented by25types of raw materials classified as Category4, ORAC mean723μmol TE/g, defined as "antioxidant capacity"; to senna, Saussurea costus represented47categories classified as Category5, ORAC mean314μmol TE/g, is defined as "weak antioxidant capacity";6category pearl, Radix Aucklandiae and other raw materials, a total of80species, ORAC mean40.5μmol TE/g g, is defined as "a very weak antioxidant activity."
Innovations
First time, we analyzed the functional food raw materials about their nutrients and functional components at large extent for the Chinese food composition and meanwhile, we fill the gaps in this part of the data, providing the basic data for future use.
For the first time by multi-functional microplate reader and automatic biochemical analyzer respectively, used ORAC test and high-throughput DPPH· radical scavenging for herbs by applying complex evaluation of antioxidant activity of raw materials, the first time the use of automatic biochemical analyzer to the a-glucosidase inhibitors in high throughput screening.
The first time to compare high antioxidation ability and high a-glucosidase inhibitory activity of samples.
The first time, applied the cluster analysis and evaluation model for in vitro antioxidant acitivity and composition, providing reference to applications functional food raw materials in future.
一、目的
1.通过对常见保健食品原料营养成分、功效成分的测定获取相关数据,为今后保健食品原料的抗氧化研究做出基础性工作。
2.通过体外抗氧化测定方法、以抗氧化活性为共性指标对常见保健食品原料抗氧化活性做出评价。
3.研究营养成分、功效成分对抗氧化活性的影响,为保健食品功能声称评价提供科学基础。
4.筛选出具有较高抗氧化活性和抑制a-葡萄糖苷酶的活性的保健食品原料,为今后保健食品原料的使用做出指导。
二、材料与方法
结合中国传统医学药食同源思想和中医理论,以《卫生部关于进一步规范保健食品原料管理的通知》(卫法监发2002(51)号文件,以下简称51号文件)为依据选择常见保健食品原料做为研究对象。
1.常见保健食品原料基本营养成分的研究
采用国家标准方法对170种保健食品常见原料进行营养成分的测定,其中水分采用直接干燥法(GB/T5009.3-2003)、蛋白质采用凯氏微量法(GB/T5009-2003)、灰分采用(GB/T5009.4-2003)方法、镁采用GB/T5009.90-2003方法、钾采用GB/T5009.91-2003方法、钠采用GB/T5009.91-2003方法测定,磷采用分光光度法(GB/T5009.87-2003)测定,铁采用GB/T5009.90-2003测定,钙采用原子吸收分光光度法(GB/T5009.92-2003)进行测定,脂肪采用索氏抽提法(GB/T5009.6—2003),维生素AGB14750-1993、维生素E采用高效液相色谱法(GB/T5009.82-2003)、维生素C荧光法(GB/T5009.86-2003)、维生素B1采用(GB/T5009.84-2003)、维生素B2采用荧光法(GB/T5009.85-2003)。
2.常见保健食品原料主要功效成分的研究
分别采用铝盐比色法、香草醛-高氯酸法和酸性染料比色法测定原料的总黄酮、总皂苷和总生物碱的含量。
3.常见保健食品原料抗氧化活性的评价研究
选择经典的DPPH·自由基清除法和目前国际上最先进的ORAC方法同时对常见保健食品原料进行抗氧化活性评价。采用p-NPG法、以对硝基苯基-α-D-吡喃葡萄糖苷(p-NPG)为底物、利用全自动生化分析仪对常用保健食品原料进行α-葡萄糖苷酶抑制活性的高通量测定作为抗氧化活性的辅助评价,筛选出具有高抗氧化能力与高抑制α--葡萄糖苷酶活性的原料。将传统中医理论与现代营养学结合,对常用保健食品原料的ORAC值按照“四性”、“五味”和“阴阳”进行分类,运用统计学方法分析、观察不同类别的原料其ORAC值的高低和差异。
4.营养成分、功效成分与抗氧化活性的相关性研究
调用SPSS软件中的Correlation模块对原料的营养成分、功效成分和抗氧化活性进行相关性研究,观察不同成分对抗氧化活性的影响；调用SPSS软件中Classify Hierarchical Cluster模块进行聚类分析,根据抗氧化能力指数对常用保健保健食品原料进行聚类,以类别界限值作为今后抗氧化能力高低评判的标准。
三、研究结果
按照51号文件共购得170种常见保健食品原料做为研究对象,大部分经过专业炮制后为干品,少数为鲜品。
1.常见保健食品原料基本营养成分研究
宏量营养成分中,蛋白质在动物性原料中含量普遍较高,植物性原料中以“籽”、“仁”等名称的植物性样品蛋白质含量较高。动物性原料普遍含有脂肪,但含量低于紫苏子、苦杏仁等植物性原料,脂肪含量最低的原料是竹茹、灯心草、穿心莲、玫瑰花、人参、太子参、玫瑰茄,含量均为0.09g/100g。原料中碳水化合物含量最大的原料是竹茹,灯心草等植物性原料也较高,碳水化合物含量最小的原料是刺猬皮,大部分原料的碳水化合物介于60～80g/100g之间；能量最高的原料为紫苏子,其次是苦杏仁和郁李仁等“仁”、“子”类原料,动物性原料中阿胶的能量最高(393kcal)。能量最低的原料为石决明和珍珠；大部分原料的能量值在300-400kcal之间；植物性原料除种子类原料的能量平均值最高,其它部位能量分布比较均匀,而动物和其它类原料的能量值比较接近。动物性原料中的矿物质含量普遍高于植物性原料,其中,有9种原料的磷含量大于1000mg/100g,植物的种子和花部位磷含量较高；钠含量最多的原料分别是海参(3643mg/100g)、制肉苁蓉(1995.11mg/100g)和燕窝(1041.1mg/100g),均大于1000mg/100g,植物性原料的茎、叶部位其钠含量较高；钾含量最高的前5位原料分别是墨旱莲、豆蔻、茵陈、姜黄和积雪草,在动物性原料中普遍含量不高；钙含量最高的原料为海螵蛸(30187.5mg/100g),其次是石决明、马鹿胎和乌梢蛇,植物性原料中钙含量最高的为首乌藤(6735.9mg/100g)；镁含量最高的原料为乌梢蛇和刺猬皮,分别高达2636.7,1680.0mg/100g,其次是蓼大青叶、蒲黄等植物性原料；蝉蜕、地龙、茜草、半边莲和茵陈是铁最高的前5位原料,尤其是蝉蜕(蝉的幼虫变成成虫时蜕下的壳),铁含量高达1634.7mg/100g；维生素A、维生素C和维生素E在植物性原料中含量很低,几乎损失殆尽；维生素B1主要存在于植物的果实、种子和花中,火麻仁、藏红花、麸炒神曲、紫苏子和白芥子是含量高的前5位；维生素B2主要存在于植物和花部和叶部,含量排名前5名的是藏红花、地龙、团螵蛸和水蛭。就原料的不同药用部位来看,动物性原料中普遍含有蛋白质、钠；植物性原料的种子、果实富含蛋白质和脂肪；植物的茎部位其磷含量较高；植物的茎、叶部位其钠含量较高；钾、钙、镁和铁富集于植物的整株、花和叶部位。
2.常见保健食品原料功效成分研究
建立了精密度、稳定性、回收率均良好的总黄酮、总皂苷和总生物碱测定方法。通过对保健食品常用原料的功效成分测定和分析发现,原料中总黄酮、总皂苷在不同分析部位间含量差异较大,变化范围也较大,总生物碱含量普遍偏低。共有100个原料检出总黄酮含量,大部分原料的黄酮含量在500mg/100g以下,花、叶类原料中富含黄酮类物质,尤其在花中普遍总黄酮含量较高,平均含量高达1.42%；含量最高的原料为槐米(6690mg/100g),其它较高的的原料依次为槐米、槐角、厚朴、刺五加、紫苏子、月季花、合欢花、香薷、荷叶和诃子；共有151个原料检出了总皂苷含量,占到了总样品量的88%。多数原料的皂苷含量不高于3000mg/100g；叶、果实和花类的皂苷含量较为接近,叶和果实是皂苷含量较高的一类,以叶中含量较为丰富,平均含量达到了1.65%；总皂苷含量排名前10位的样品依次为苦丁茶、大皂角、知母、怀牛膝、酸枣仁、覆盆子、西洋参、吴茱萸、连翘和益智；大部分原料的生物碱含量为125mg/100g以下,生物碱含量普遍偏低,除了个别含量较高的原料外,其余原料的生物碱含量变化差异性较小,根据分析部位来看,生物碱在根及根茎中平均含量相对较高,其它部位中的生物碱含量都较为接近。总生物碱含量排名前10位的样品依次为防己、黄连、芦荟、香加皮、西洋参、大皂角、麻黄、罗布麻花、川芎和柴胡。黄酮、皂苷和生物碱一般不存在于动物性样品中。
3.常见保健食品原料抗氧化活性研究
通过DPPH·自由基清除实验共筛选出147种原料不同程度地均有一定的清除自由基活性,23个原料基本无清除自由基活性,有16种原料活性高于阳性对照维生素C,按照活性由高到低的顺序排列它们分别是淫羊藿、桂枝、石斛、首乌藤、制肉苁蓉、吴茱萸、荷叶、罗布麻花、连翘、诃子、团螵蛸、玫瑰花、槐角、金荞麦、芦荟和积雪草,所有原料中清除活性最弱的为淡豆豉；通过数学运算将清除自由基活性的IC50值转换成TAC值(总抗氧化能力),既避免了自由基清除活性IC50值比较时的困难,也消除了原料浓度对清除效果表达的影响。
ORAC实验是目前国际上推荐的新型抗氧化评价方法。经过对常见保健食品原料的水溶性ORAC值测定后发现：所有原料的ORAC值介于0.27～3579μmolTE/g之间；ORAC值高于阳性对照品维生素C的为苦丁茶、芦荟和槐米；其中,ORAC值最高的为苦丁茶(3579μmol TE/g),最低的为珍珠(0.27μmol TE/g)其它较高的样品有芦荟、槐米、吴茱萸、槐角、丹参等；而海螵蛸、石决明、燕窝、海参、芡实、猪苓和马鹿胎则为ORAC值很低的原料,多为动物性样品。调用SPSS软件中的Correlation模块对二种抗氧化评价方法得到的数据进行统计学上的相关性分析,发现清除DPPH·自由基法得到的结果与ORAC法测得的结果相关性较强,表明二种方法在抗氧化活性评价时有相同的趋势。从分析部位来看,自由基清除效果较好的原料主要是花类的原料,其次为叶类；ORAC值按其药用部位归类,发现茎、叶类原料虽然数量不多但ORAC值却是最高的。
以拜糖平为阳性对照物,采用经典的p-NPG方法对常见保健食品原料进行α-葡萄糖苷酶抑制活性高通量筛选。结果表明共有84种原料有一定的抑制活性,其中有25种的抑制活性强于阳性对照物拜糖平。
在抑制α-葡萄糖苷酶活性高的前20位原料中,有9种原料既有很强的抑制a-葡萄糖苷酶活性,同时又具有很强的ORAC值,它们分别是玫瑰花、诃子、金荞麦、淫羊藿、首乌藤、罗布麻叶、虎杖、月季花和赤芍。有8种原料既有很强的抑制α-葡萄糖苷酶活性,同时又具有很强的TAC值,它们分别是淫羊藿、首乌藤、罗布麻花、诃子、玫瑰花、金荞麦、赤芍和罗布麻叶。有8种原料既有较高的ORAC值,又有较高的TAC值,分别是玫瑰花、诃子、金荞麦、淫羊藿、首乌藤、罗布麻叶、首乌藤和芦荟；三者之间的互相比较表明高抗氧化活性原料同时也有较高的抑制α-葡萄糖苷酶活性。
抑制α-葡萄糖苷酶活性的IC50值与TAC值、ORAC值间的相关性分析结果显示：抑制α-葡萄糖苷酶活性的IC50值与ORAC值和TAC值均呈现负相关关系,且有一定的统计学意义,表明原料抑制α-葡萄糖苷酶活性越强,其IC50值越小,抗氧化能力随之越强,为今后α-葡萄糖苷酶抑制活性与抗氧化活性的协同性研究提供了有益的资料。
4.常见保健食品原料抗氧化活性与中医理论的探讨
中医理论认为物有“四性”即“寒”、“热”、“温”、“凉”和“五昧”即“辛“、“甘”、“酸”、“苦”、“咸”,并同时常常用“阴”、“阳”来对保健食品原料进行评价。本研究中的保健食品原料多以中草药为主,因此,将它们的ORAC值根据“四性”、“五味”和“阴阳”分别归类后发现,通过“四性”分类的原料其ORAC值组间无显著性差异(p>0.05)；按照“五味”分组的原料其ORAC值有显著性差异(p<0.05)；阴类原料和阳类原料的ORAC值有显著性差异(p<0.05)
5.常见保健食品原料营养成分、功效成分与抗氧化活性的相关性研究和抗氧化能力指数的聚类研究
调用SPSS软件中的Correlation模块、Classify模块和Regression模块分别将营养成分、功效成分与ORAC值、TAC值进行相关性分析、聚类分析和线性回归分析。结果表明：170种原料的蛋白质、脂肪和能量均与抗氧化能力指数无相关性(p>0.05),碳水化合物与抗氧化能力有一定的相关性,但相关系数很小(p<0.05)。与抗氧化能力指数呈现负相关关系的元素有钠和磷,而钾、钙、镁和维生素B2与抗氧化能力指数有一定的正相关性且有统计学意义(p<0.05),维生素B1和铁与抗氧化能力指数的相关性无统计学意义(p>0.05)。宏量营养素与自由基清除能力无相关关系(p>0.05)。钙和维生素B2与TAC值有一定的正相关性(p<0.05),磷和维生素B1与抗氧化能力呈现负相关关系,其它元素与TAC值无相关(p>0.05)。当消除ORAC值的影响时,α-葡萄糖苷酶抑制活性与自由基清除能力呈现负相关关系但无统计学意义(p>0.05)；当消除TAC值的影响时,α-葡萄糖苷酶抑制活性与抗氧化能力指数也呈现负相关关系,但无统计学意义(p>0.05)。清除自由基能力与抗氧化能力指数有较为显著的相关性(p<0.05),表明二种方法在进行抗氧化评价时,有一致的趋势。功效成分含量与抗氧化活性间相关性研究结果显示总黄酮、总皂苷与ORAC值有一定的相关性,相关系数分别为0.500、0.288(p<0.01)。TAC值与总黄酮、总皂苷成分也有一定的相关,相关系数分别为0.314、0.208(p<0.01)。
总黄酮、总皂苷含量与抑制α-葡萄糖苷酶活性的IC50值(mg/mL)呈现负相关但无统计学意义(p>0.05)；生物碱与抑制α-葡萄糖苷酶活性的IC50值(mg/mL)相关性不明显(p>0.05)
调用SPSS软件中的Classify-Hierarchical cluster模块对170种原料的抗氧化能力指数共分为6类,其中,苦丁茶因ORAC值最高而自动归为第1类,定义为“极强抗氧化能力”级；槐米和芦荟(ORAC平均值为2667μmol TE/g)的2个原料自动归为第2类,定义为“强抗氧化能力”级；以香薷为代表的15种原料(ORAC平均值为1260μmol TE/g)归为第3类,定义为“高抗氧化能力”；以牡丹皮为代表的25类原料归为第4类,ORAC平均值为723μmol TE/g,定义为“中抗氧化能力”；以木香为代表的47类原料归为第5类,ORAC平均值为314μmol TE/g,定义为“弱抗氧化能力”；第6类为珍珠、西洋参等原料,共计80种,ORAC平均值为40.5μmol TE/g,定义为“极弱抗氧化能力”。
四、结论
1.常见保健食品原料营养成分研究
170种常见保健食品原料的宏量营养成分中,植物性原料中以“籽”、“仁”等名称的植物性样品蛋白质含量较高。动物性原料脂肪普遍低于个别植物性原料。大部分原料的碳水化合物介于60～80g/100g之间。植物性原料除种子类原料的能量平均值最高,其它部位能量分布比较均匀,而动物和其它类原料的能量值比较接近。动物性原料中的矿物质含量普遍高于植物性原料,主要集中在植物的根部和动物类、其它类原料中,植物的种子和花部位磷含量较高；钠含量最多的原料为动物性原料,植物性原料的茎、叶部位其钠含量较高；钾含量最高的原料全部是植物性原料,在动物性原料中普遍含量不高；钙多富集于动物性原料中；镁多存在于动物性原料中,蓼大青叶、蒲黄等植物性原料含量也较高；铁普遍存在于植物的地上部分,蝉蜕(蝉的幼虫变成成虫时蜕下的壳)中铁含量最高；维生素A、维生素C和维生素E在植物性原料中含量很低,几乎损失殆尽；维生素B1含量最高的前5位原料分别是火麻仁、藏红花、麸炒神曲、紫苏子和白芥子；维生素B2主要存在于植物的花、叶中。就原料的不同药用部位来看,植物性原料的种子、果实富含蛋白质、脂肪和维生素B1；植物的茎部位其磷含量较高；植物的茎、叶部位其钠含量较高；钾、钙、镁和铁富集于植物的整株、花和叶部位。
2.常见保健食品原料主要功效成分研究
共有100个原料检出总黄酮含量,共有151个原料分别检出了总皂苷和总生物碱含量。大部分原料的黄酮含量在500mg/100g以下,花、叶类原料中富含黄酮类物质；多数原料的皂苷含量不高于3000mg/100g；叶、果实和花类的皂苷含量较为接近,叶和果实是皂苷含量较高的一类；大部分原料的生物碱含量为125mg/100g以下,生物碱含量普遍偏低,除了个别含量较高的原料外,其余原料的生物碱含量变化差异性较小,根据分析部位来看,生物碱在根及根茎中平均含量相对较高,其它部位中的生物碱含量都较为接近。3.常见保健食品原料抗氧化活性研究
共筛选出147个原料均有一定的清除自由基能力,且有16种原料活性高于阳性对照维生素C,表明常见保健食品原料对以氮为中心的有机自由基具有较普遍的清除作用；ORAC方法测定结果显示原料的抗氧化能力指数差异较大,表明常见保健食品原料对氧自由基具有一定的清除作用,但因样品不同而差异较大；ORAC值最高的前10名原料分别是苦丁茶、芦荟、槐米、吴茱萸、槐角、丹参、玳玳花、罗巴斯塔咖啡豆(烤前)、虎杖和丹参。中医传统理论“四性”、“五味”和“阴阳”与抗氧化能力指数进行组间方差分析,结果表明：依照“四性”不同,其原料组间ORAC值无显著差异(p>0.05),显示按照中医传统理论的“四性”分类,难以对原料的抗氧化能力进行区分；“苦”和“苦”类原料组间抗氧化能力有显著差异间差异(p<0.05),表明依照“五味”划分抗氧化能力有一定的作用；“阴”和“阳”、“中”类原料组间抗氧化能力有显著差异间差异(p<0.05),表明依照“阴阳”划分抗氧化能力有一定的作用。
共筛选出84种有α-葡萄糖苷酶抑制活性的原料,其中有25种的抑制活性强于阳性对照物拜糖平。常用保健食品原料对α-葡萄糖苷酶的抑制具有较大的差异性,IC5α值变化范围较大,有些原料的抑制酶活性很强,有些则很弱,超过三分之一的原料不仅有抑制活性,还具有较强的活性。中草药的主要部位都对α-葡萄糖苷酶有抑制作用,活性较强的部位有花、果、根茎等部位。抑制α-葡萄糖苷酶活性较高的前20位原料中,有9种原料既有很强的抑制α-葡萄糖苷酶活性同时又具有很强的ORAC值；有8种原料既有很强的抑制α-葡萄糖苷酶活性同时又具有很强的TAC值,为今后抗氧化活性和抑制α-葡萄糖苷酶活性的协同研究提供了有价值的参考依据。
碳水化合物、能量、钾、钙、镁和维生素B2与抗氧化能力指数值有一定的正相关性且有统计学意义(p<0.05)；碳水化合物、钙和维生素B2与自由基清除能力有一定的正相关性；总黄酮、总皂苷百分含量与抗氧化能力指数呈一定的相关性,相关系数分别为0.500、0.288(p<0.01)；黄酮、皂苷成分与清除自由基活性也有一定的相关性,而生物碱含量则与TAC值无相关性。
抑制α-葡萄糖苷酶活性的IC50值与ORAC值和TAC值均呈现负相关关系(p<0.05),表明抑制α-葡萄糖苷酶活性越强,其ICso值越小,抗氧化能力随之越强；通过相关性分析表明清除自由基能力与抗氧化能力指数有较为显著的相关性,显示二种方法在进行抗氧化评价时,有一致的趋势；通过聚类分析对170种原料的抗氧化能力指数共分为6类,苦丁茶为第1类,定义为“极强抗氧化能力”级；槐米和芦荟归为第2类,定义为“强抗氧化能力”级；以丹参、葛根为代表的15种原料归为第3类,定义为“高抗氧化能力”；以月季花、枳壳为代表的25类原料归为第4类,定义为“中抗氧化能力”；以番泻叶、木香为代表的47类原料归为第5类,定义为“弱抗氧化能力”；第6类为珍珠、木贼等原料,共计80种,定义为“极弱抗氧化能力”。
五、创新之处
1.首次对170种常见保健食品原料进行了营养成分和功效成分的测定,为今后保健食品原料的使用提供数据支持和指导。
2.首次通过抗氧化活性的高低对保健食品原料性质进行系统评价和聚类分析,并探讨了成分和活性间的关系。
3.首次将以物质为基础的营养学理论和现代医学中的共性指标—抗氧化活性来阐述传统中医学理论用“四性”、“五味”和“阴阳”对中草药原料的整体性评价。
With the development of economy and people's living standards, food consumption is moving from subsistence to a comfortable level, people put a higher quality of food requirements; functional food thus came into being.
Regulatory function of the functional food depends on its raw material and ingredients, but the functional food raw materials has been the lack of systematic research.
China's functional food is functionalse on the traditional Chinese medicine and Medicinal and Edible theories, the raw materials mainly are Chinese herfunctionall medicine.
Function of claims is still the traditional Chinese medicine formulation, lack combination with modern Western medicine and the nutrition. For example, the function of antioxidant, the functional food ingredients seldom have antioxidant system evaluation, especially litter research about the relationship among the antioxidant functional food claims and the raw materials and components.
As China's most authoritative food composition datafunctionalse-China food composition is also lack of this part of the data. Therefore, the research about the raw materials often used in China functional food, especially its nutritional content,the functional components and the relationship between composition and antioxidant activity is under the circumstances.
Objectives
1. Add data to Chinese food composition table, fill data gaps of functional food nutritional ingredients, functional components.
2. Using the in vitro antioxidant evaluation method to evaluate the antioxidant activity of common raw materials in functional food, while make aid evaluation for antioxidant activity evaluation to common raw materials about functional food by a-glucosidase inhibitory activity.
3. Observe the relationship and effectiveness between the nutrients, functional factors and antioxidant activity, provide the scientific functionalsis for functional food claim.
4. Selecting the high antioxidant activity of functional food raw materials make the guidance for using of functional food raw materials in future
5. Apply the statistical methods to construct regression model between functional components and the in vitro antioxidant activity
Material and Methods
On the functionalsis of the traditional Chinese medicine and Medicinal and Edible theories, according to the inform of "to further standardize the management of raw materials to functional food of Ministry of Functional "(Document No.51), we selected170species common raw materials including plants, animals, involving a total of65subjects in our study.30species are belong to Document No.51annex I,85species belonging to Annex Ⅱ, and the remaining55species belong to other common functional food raw materials, mainly are traditional Chinese herfunctionall medicine.
1. Analysis of the functionalsic nutrients
Adopt national standards to analyze nutrition Determination of170kinds of common raw materials of functional food. Water used the method of GB/T5009.3-2003, protein is GB/T5009-2003)、ash is GB/T5009.4-2003, magnesium used GB/T5009.90-2003, potassium used GB/T5009.91-2003, sodium used GB/T5009.91-2003, phosphorous used GB/T5009.87-2003, iron used GB/T5009.90-2003, Calcium used Atomic absorption spectrophotometry(GB/T5009.92-2003), fat used Soxhlet extractor method(GB/T5009.6-2003), vitamin A used GB14750-1993, vitamin E used HPLC method(GB/T5009.82-2003), vitamin C used fluorometric method (GB/T5009.86-2003), thiamine used GB/T5009.84-2003, Riboflavin used fluorometric method (GB/T5009.85-2003)。
2. Analysis of functional components
Total flavonoids determination is Aluminum chloride colorimetric assay, total saponins used Vanillin perchloric acid assay and total alkaloids used Acid dye colorimetry.According different plant medicinal part, classify the raw materials and discussed the distribution of nutrients, medicinal ingredients in different parts
3. Antioxidant activity evaluation and high-throughput screening of a-glucosidase inhibited activity
Respectively adopting the most advanced in vitro antioxidant evaluation method-ORAC method and the classical DPPH· radical scavenging method for the common functional fodd raw material. We reformed the DPPH· radical scavenging method by the automatic biochemical analyzer to improve experimental conditions to achieve a high throughput screening. DPPH· free radical removal efficiency is expressed by the concentration of IC50before, and now, after the mathematical conversion, the total antioxidant capacity (TAC value) is the express result,easy to understand and more clear and intuitive, using p-NPG method, p-NPG as substrate to screen a-glucosidase inhibit activity of170kinds of functional food raw materials, using automatic biochemical Analyzer to achieve high-throughput screening, a-glucosidase inhibition efficiency is expressed by50%inhibitory ration needed the raw materials concentration (IC50value). According to different medicinal parts, observe the antioxidant activity level in different medicinal parts. Screening the raw material with high Antioxidant capacity and high α-glucosidase inhibitory activity. According to traditional Chinese medicine theory, classify the common functional food raw materials in accordance with the four properties, five flavors and yin and yang, to observe the different categories of the level of antioxidant activity.
4. Correlation of Nutritional composition, functional components and antioxidant activity
Correlation research is developed by SPSS software, spearman method for the nutritional content, functional and antioxidant activity components of170species of raw materials, results showed that antioxidant activity and flavonoids are most closely. Using SPSS software Classify Hierarchical Cluster modules, clustering principle indicator cluster (R type) in the correlation coefficient distance (Pearson correlation), methods for the class on average chain method (Between-groups linkage) clustering of all raw materials analysis, comparing the degree of similarity between different indicators. According to cluster analysis and correlation analysis results, further use of SPSS software, the Regression module, to establish the regression equation, flavonoids as independent variables and ORAC value as dependent variable.
Research result
1. Basic nutrition ingredients Determination of170kinds of common functional food raw materials
Following on Determination of170kinds of common functional food nutrients ingredients, we found that nutrients are abundant and widely distributed. Among Macronutrients, the water content distribution is in the0.5g-69.5g/100g, with a maximum water content of fresh Rhizoma Imperatae and royal jelly; Protein in the name of "benevolence","seed" category of raw material were higher, mostly distributed in fruits, seeds. Ash mainly exist in animal raw materials; iron is distributed in different parts of plant, To free oneself (cicada larvae into adults under the shell when the slough) was highest (1634.7mg/100g), is the lowest of the royal jelly of1256times. More magnesium present in the raw materials of animal and plant part of above ground, between the range of3.26-2636.7mg/100g. Potassium is mainly distributed in the ground part of the raw materials; Compositae Eclipta has the highest concentrations. Sodium mainly exist in animal samples, the highest concentrations is sea cucumber. Phosphorus is distributed in the roots to the highest content of Rhodiola and Siberian, and their analysis parts are the roots. The highest calcium content of raw materials is the Cuttlebone (dried squid squid related species within the shell), and in the plant raw material is more at leaf. Vegetable fats are generally essential fatty acids, linoleic acid and linolenic acid, after Soxhlet extraction method and we found that170kinds of raw materials contain a certain amount of fat, changes in range between0.09-53.7g/100g, more present in the plant fruit, seeds, such as perilla seed, hemp seed, and some plant material in the fat content even higher than the animal samples. After determination of vitamin analysis we found that subjects have generally low levels of vitamin, especially vitamin A, vitamin C and vitamin E, in most of the raw materials or even not detected, which may be due to raw materials are processed professionally in the sun, washing and bring loss.
2. Determination results of functional ingredients
Flavonoids, saponins are the most frequent claim components in functional food. Therefore, in this study, they are chosen as the main target, and on this basis, select the alkaloids of this ubiquitous component of Chinese herbs while a total of three indicators170kinds of raw materials for the determination of functional components. After the analysis, the percentage of total flavonoids, total saponins and total alkaloids of170kinds of functional food showed:Most of the material in the total flavonoids, saponins were significantly different and varied greatly. The top10highest total flavonoid content of raw materials were FlosSophorae Sophoricoside, Magnolia, Acanthopanax, perilla, Chinese rose, Albizia julibrissin, Hong Ru, the leaves and myrobalan; The total saponin content of samples of the top10were Kudingcha, Anemarrhenae, Achyranthes bidentata, Suanzaoren, raspberries, American ginseng, Evodia, forsythia and Fructus Alpiniae Oxyphyllae. In Weight loss function food, total alkaloids are major components, through the determination of the levels found generally low, and little difference in content; top10samples were anti has, berberine, aloe, Hong Gapi, American ginseng, fruit of Chinese honeylocust, ephedra, cannabis Rob, and Bupleurum Chuanxiong
After the analysis of the raw material in accordance with the medical site classification and then to observe the different parts of the content of functional components and we found:a high flavonoid content in plants generally flower parts, fruits and leaves, flowers, high content of total flavonoids in general, the average content of1.42%; saponins are secondary metabolites of plants to the leaves of more abundant, the average content was1.65%; roots and rhizomes of the class highest alkaloid content. Flavonoids, saponins and alkaloids in general does not exist in animal samples.
3. Antioxidant activity and inhibition of a-glucosidase activity of raw materials in China Functional food
Antioxidant function is one of the important functions of functional food. Evaluation of antioxidant activity often choose two kinds of methods or more were determined simultaneously. Our study chose traditional DPPH· radical scavenging method and the current world's most advanced ORAC method to analyze for170kinds of commonly used functional food raw materials. DPPH· radical is a stable chemical radicals, at the presence of antioxidants, antioxidants and their single-electron pair and its absorption gradually fade, and their acceptance of its fading into the quantitative relationship between the number of electrons, with spectrophotometric changes in absorbance calculated by free radical scavenging activity of samples, that is antioxidant activity. In our study, with the automatic biochemical analyzer, DPPH· method of traditional experimental conditions were improved to achieve a high throughput screening, and to avoid the free radical damage to human body. By measuring,23kinds of raw materials basically no free radical scavenging activity, and the remaining raw materials are some free radical scavenging activity, and there are16kinds of raw materials more active than the positive control of vitamin C, according to the order of their activity from high to low are epimedium, Cinnamomum Ramulus, Dendrobium, vine of multiflower knotweed, system Cistanche (system large Yun), Evodia, lotus leaf, Rob cannabis, forsythia, Terminalia, group cuttlebone, roses, Sophoricoside, wild buckwheat, Aloe vera and Centella asiatica, the weakest scavenging activity of all raw materials is the semen sojae praeparatum; through mathematics the IC50values of free radical scavenging activity converted to TAC value, which avoids the radical scavenging activity (IC50values) comparison difficult, but also eliminates the effect of raw material concentration on the expression of clear. ORAC assay is recommended by the international evaluation of new anti-oxidation. After ORAC analyze, the170kinds of raw materials, water-soluble ORAC value are between0.27～3579μmol TE/g. the highest ORAC value is Kudingcha, the lowest is the pearl, the other hith values samples are aloe, FlosSophorae Evodia, Sophoricoside, Danshen; the low values samples are cuttlebone, abalone, bird's nest, sea cucumber (Gypsophila), Gorgon, Polyporus, Red Deer fetus etc. for the ORAC value of the minimum of raw materials, and more for the animal samples. We used SPSS software Correlation module to develop statistical correlation analysis, we found clear DPPH·free radical method the results obtained with the results measured by ORAC method has some relevance, that means these two kinds of method in the evaluation of antioxidant activity have the same trend. From the analysis of different parts, we found that free radical scavenging effect of better raw materials are mainly raw materials like flowers, with an average IC50value of0.99mg/mL, followed by leaf; ORAC value are classified according to their medicinal part, found that stem, leaves material although a small number, but with the highest ORAC average values. α-glucosidase is a very important enzymes, through the hydrolysis of a-1,4glycosidic bond from the non-reducing end of polysaccharide glucose cut. The body of the starch, dextrin, sucrose and other carbohydrates in the use of absorption depends on the intestinal brush border on the enzyme activity, control of diabetes and the need to reduce the activity of the enzyme, a-glucosidase inhibitors by competitive inhibition of a-glucosidase activity, can block two-sugar sugar solution into a single delay the absorption of sugar, so blood sugar steady and slow to maintain a certain level. In our study, acarbose is used as the positive control, using the classic method of p-NPG for170kinds of raw materials on the a-glucosidase inhibitory activity screening test, the use of automatic biochemical analyzer to achieve high-throughput screening and more efficient than96microplates. The results showed that a total of84raw materials has some inhibitory activity, of which25species of inhibitory activity was stronger than positive control acarbose. Overall, Chinese herbal medicine as an α-glucosidase inhibitor, the more complex compositions lead to mechanism needs further study.
About inhibiting a-glucosidase activity, in the the top20raw material, there are9kinds of raw materials both strong suppression of α-glucosidase activity and the high ORAC value. They were roses, Terminalia, Fagopyrum, Epimedium, vine of multiflower knotweed, Apocynum leaves, cuspidatum, Chinese rose with Radix. There are8kinds of raw materials both strong suppression of a-glucosidase activity and strong TAC value. They are epimedium, vine of multiflower knotweed, Rob cannabis, Terminalia, roses, wild buckwheat, red Shao and Apocynum leaves.8both have a high ORAC value and a highTAC values were roses, Terminalia, wild buckwheat, Epimedium, vine of multiflower knotweed, Apocynum leaves, vines,vine of multiflower knotweed and aloe. In the future, these materials need more research and application to play important role.
4.Antioxidant activity relationship with the theory of Traditional Chinese Medicine
TCM theory holds that objects have "four properties" of the cold, hot, warm, cool and "five flavors " that is acrid, sweet, sour, bitter, salty, and also often use the "Yin","Yang" to evaluate the effects of Chinese herbs. In this study, the functional food are mainly herbal-based raw materials, so with four properties, five flavors and yin and yang were classified, we listed each type of material corresponding ORAC value. After research, we found:Cold material compared to the hot, warm and cool materials, the ORAC value of the highest, but the quantity is not much; bitterness of raw materials compare to the acrid, sweet, sour, salty class of its ORAC value not only high, quantity is greater; yin materials with high ORAC value and the number is greater. After further analysis of variance, we found that sour, bitter class materials ORAC value have significantly different (p<0.05), but by four properties classification, the raw materials of their ORAC values were no significant differences (p>0.05); yin yang kind of raw materials and class materials ORAC values significantly different (p<0.05), showed that the antioxidant activity by Yin Yang is more reliable classification.
3.1The correlation analysis, cluster analysis regression equation
SPSS software Correlation modules, Classify module and Regression modules respectively are used in nutrition, functional components and ORAC value, TAC values of the correlation analysis, cluster analysis and linear regression analysis. The results showed that:ORAC value and total flavonoids, total saponin content have some correlation, correlation coefficients were0.500、0.288(p<0.01), while the alkaloid content was almost no correlation with the ORAC value. TAC values and flavonoids, saponins also have some correlation coefficients were0.314、0.208(p <0.01), total flavonoids, total saponin and inhibited α-glucosidase activity of the IC50value (mg/mL) showed a negative correlation, shows that flavonoids, saponin content is more, the inhibition of α-glucosidase activity is stronger, and the IC50value is more lower, which is consistent with theory, though not statistically significant.
Cluster analysis is based on characteristics of the thing itself, individual classification is based on the principle of feather flock together something categories. Used SPSS software Classify-Hierarchical cluster module cluster to analyze the different indicators and we found that flavonoids and ORAC values of the short distance in the statistical relationship between the tree, therefore, the flavonoid content can be regarded as the main ORAC value effect factors, other factors are in turn based on distance close to the level of saponin, TAC values and alkaloids; α-glucosidase inhibitory activity with IC50value of total alkaloids and a tree closest relationship, followed by the flavonoids and saponins.
Using SPSS software Classify-Hierarchical cluster module to classify170kinds of raw materials based on antioxidant capacity index, then they are divided into six categories, which, because of ORAC value of the highest Folium llicis Latifoliae automatically classified as Class1, is defined as "very strong antioxidant "class; Flos Sophorae Immaturus and Aloe (ORAC mean2667μmol TE/g) of the two raw materials automatically classified as category2, defined as" the strong antioxidant capacity "level; to Salvia, Pueraria, represented by15kinds of raw materials (ORAC average1260μmol TE/g) classified as Category3, defined as "high antioxidant capacity"; to Chinese rose, Citrus aurantium, represented by25types of raw materials classified as Category4, ORAC mean723μmol TE/g, defined as "antioxidant capacity"; to senna, Saussurea costus represented47categories classified as Category5, ORAC mean314μmol TE/g, is defined as "weak antioxidant capacity";6category pearl, Radix Aucklandiae and other raw materials, a total of80species, ORAC mean40.5μmol TE/g, is defined as "a very weak antioxidant activity."
Conclusion
China's functional food raw material is widely used, evolved many families and distribution. Many kinds of medicinal parts of more nutritious ingredients, big difference in the effectiveness of ingredients, different parts of the nutrients, functional components were significant differences in content, but a distribution of components in the same position with convergence, such as moisture, protein, ash, mainly in the animal raw materials, flavonoid, saponins, alkaloids mainly present in plant material
After the traditional method of DPPH free radical scavenging screening, we found among170kinds of common functional food raw materials, there are152have scavenging activity and there are16kinds of raw materials higher than the positive control activity of vitamin C;170kinds of raw materials ORAC value is quite different, ORAC value material difference between the highest and lowest13255times. Free radical scavenging method and the ORAC method were significantly correlated, indicating that the same trend in two ways. There were selected from84kinds of α-glucosidase inhibitory activity of raw materials, including25species of inhibitory activity was stronger than positive control acarbose. Chinese herbal medicine are all major parts of α-glucosidase inhibition, Chinese herbal medicine as an α-glucosidase inhibitor mechanism of the onset is more complex, needs further study, α-glucosidase inhibition activity is higher in the top20raw materials, raw materials are9strong inhibition of both α-glucosidase activity was also highly ORAC value; are eight kinds of raw materials both strong a-glucosidase inhibitory activity, while TAC has a strong value.
According to the Chinese "four properties","five flavors ","Yin Yang" Theory and Comparative antioxidant activity of the classified study found cold, cool, negative raw materials usually have high ORAC value. After analysis of variance showed that by " four properties " of classified material whose ORAC value is no significant difference (p>0.05), through the "five flavors" for the raw materials of its ORAC value groups were significantly different (p<0.05), that by " four properties "to distinguish between antioxidant activity is open to question;"yin "type of raw materials and" yang "type of raw materials ORAC values significantly different (p <0.05), showed that the antioxidant activity through the yin and yang is a more reliable classification.
After statistical correlation analysis, the result showed that the total flavonoids, total saponin content and ORAC value of percentage of a certain correlation (p<0.01), in particular flavonoids and a high correlation coefficient; alkaloid content and ORAC value is the basic no correlation. Flavonoids, saponins and free radical scavenging activity has some correlation (p<0.01), total flavonoids, saponins and inhibition of a-glucosidase activity of the IC50value (mg/mL) showed a negative correlation, indicating that flavonoids, saponins with the higher content, the inhibitory activity is more stronger and the IC50value is more smaller; and alkaloid content was almost no correlation with the activity. After statistical clustering analysis showed that flavonoids and ORAC values in the statistical relationship between the short distance and have the tree, therefore, affect the flavonoid anti-ORAC value may be regarded as a major factor, other factors are in turn based on distance close to the level of saponin, TAC value, and alkaloids; α-glucosidase inhibitory activity with IC50value of alkaloids and a tree closest relationship, followed by the composition of flavonoids and saponins.
170kinds of raw materials based on antioxidant capacity index, using SPSS soft ware, then they are divided into six categories, which, because of ORAC value of the highest Folium llicis Latifoliae automatically classified as Class1, is defined as "very strong antioxidant "class; Flos Sophorae Immaturus and Aloe (ORAC mean2667μmol TE/g) of the two raw materials automatically classified as category2, defined as" the strong antioxidant capacity "level; to Salvia, Pueraria, represented by15kinds of raw materials (ORAC average1260μmol TE/g) classified as Category3, defined as "high antioxidant capacity"; to Chinese rose, Citrus aurantium, represented by25types of raw materials classified as Category4, ORAC mean723μmol TE/g, defined as "antioxidant capacity"; to senna, Saussurea costus represented47categories classified as Category5, ORAC mean314μmol TE/g, is defined as "weak antioxidant capacity";6category pearl, Radix Aucklandiae and other raw materials, a total of80species, ORAC mean40.5μmol TE/g g, is defined as "a very weak antioxidant activity."
Innovations
First time, we analyzed the functional food raw materials about their nutrients and functional components at large extent for the Chinese food composition and meanwhile, we fill the gaps in this part of the data, providing the basic data for future use.
For the first time by multi-functional microplate reader and automatic biochemical analyzer respectively, used ORAC test and high-throughput DPPH· radical scavenging for herbs by applying complex evaluation of antioxidant activity of raw materials, the first time the use of automatic biochemical analyzer to the a-glucosidase inhibitors in high throughput screening.
The first time to compare high antioxidation ability and high a-glucosidase inhibitory activity of samples.
The first time, applied the cluster analysis and evaluation model for in vitro antioxidant acitivity and composition, providing reference to applications functional food raw materials in future.
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