CN111504997B - 玉蜀黍须和秸秆成分提取及各成分体外降糖活性检验方法 - Google Patents
玉蜀黍须和秸秆成分提取及各成分体外降糖活性检验方法 Download PDFInfo
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Abstract
玉蜀黍须和秸秆成分提取及各成分体外降糖活性检验方法属于植物药提取技术领域。本发明重点研究玉蜀黎的须、秸秆皮以及秸秆芯三个部位中总黄酮对α‑葡萄糖苷酶和α‑淀粉酶活性的抑制作用。经皮尔逊相关性分析玉蜀黍不同部位总黄酮含量与α‑葡萄糖苷酶IC50成显著的负相关性,并且在一定浓度范围内对α‑淀粉酶有抑制作用。这一结果表明,可以充分利用玉蜀黍须、秸秆中的有效成分,加大秸秆资源技术的开发利用,不仅可以提高玉米秸秆等作物的总体的利用率,解决玉蜀黍须、秸秆闲置、焚烧、生活燃烧等粗放的利用方式问题,还可对推动我国玉蜀黍资源重组乃至农业经济的快速发展,具有重要的意义。
Description
技术领域
本发明属于植物药提取技术领域,特别是涉及到一种玉蜀黍须和秸秆成分提取及各成分体外降糖活性检验方法。
背景技术
近年来,我国慢性病的发病率和死亡率不断升高,口服药物的副作用也日益严重,药食同源植物受到广泛的重视,将植物中的活性成分作为天然产物来预防和辅助治疗慢性疾病,受到了人们的广泛关注。
亚洲人群的饮食结构主要以淀粉类为主,因此α-葡萄糖苷酶(α-glucosidase)和α-淀粉酶成为治疗糖尿病的重要的作用靶点。α-葡萄糖苷酶抑制剂与小肠上皮细胞刷状边缘上的糖分子可逆地竞争α-葡萄糖苷酶结合位点,从而推迟多糖和寡糖转化为可吸收的单糖,降低餐后高血糖。但是,目前临床实践中使用以阿卡波糖为主要的α-葡萄糖苷酶抑制剂会引起腹胀和肠鸣等不良反应。α-淀粉酶抑制剂可通过抑制α-淀粉酶的活性将糖尿病患者的血糖降至理想状态。据报道,玉米须乙酸乙酯的萃取物可明显的降低大鼠胰腺β细胞的活力,抑制细胞增殖,改善胰岛素和葡萄糖激酶,增强葡萄糖以刺激胰岛素分泌,从而调节血糖。目前报道的天然植物降糖药物已有数百种,它们可避免口服药物对人体的副作用。
玉蜀黍(Zeamays L.)为禾本科植物,据《本草纲目》记载:玉蜀黍最早出于西土,甘平无毒,能调中开胃。玉蜀黍含有大量维生素E和黄酮,经常食用玉米产品不仅可延缓衰老,还可增强人的体力和耐力,它对心血管疾病的治疗有辅助作用。玉蜀黍采摘后,玉蜀黍须、秸秆等大部分被直接当作废物处理,附加值低;黄酮类化合物是玉蜀黍中的主要活性成分之一,具有抗氧化、抗糖尿病、抗肿瘤及抗肥胖作用等药理活性。目前还没有以玉蜀黍须、秸秆皮、秸秆芯为研究对象,以α-葡萄糖苷酶和α-淀粉酶为靶点,筛选其中有降血糖作用的有效成分的报道。
因此现有技术当中亟需要一种新型的技术方案来解决这一问题。
发明内容
本发明所要解决的技术问题是:提供一种玉蜀黍须和秸秆成分提取及各成分体外降糖活性检验方法用于解决目前还没有以玉蜀黍须、秸秆皮、秸秆芯为研究对象,以α-葡萄糖苷酶和α-淀粉酶为靶点,筛选其中有降血糖作用的有效成分报道的技术问题。
玉蜀黍须和秸秆成分提取及各成分体外降糖活性检验方法,包括以下步骤,并且以下步骤顺次进行,
步骤一、获得玉蜀黍的须、秸秆皮以及秸秆芯三个部位的总黄酮和玉蜀黍的须、秸秆皮以及秸秆芯三个部位的总皂苷
①取玉蜀黍的须、秸秆皮以及秸秆芯三个部位阴干至含水量低于20%,粉碎过3号筛,分别称取50g过筛后的粉末,
须粉末按照质量比1:15加入质量含量为80%的乙醇溶液,秸秆皮粉末按照质量比1:15加入质量含量为80%的乙醇溶液,秸秆芯粉末按照质量比1:45加入质量含量为80%的乙醇溶液,获得三种溶液;
②将上述三种溶液分别40℃超声提取1小时~3小时,将提取液于40℃水浴锅上水浴至乙醇挥尽,加氨水至溶液为碱性,用乙酸乙酯萃取3次,取上层萃取液同时收集相应的下层萃取液,将上层萃取液浓缩至干膏,获得玉蜀黍的须、秸秆皮以及秸秆芯三个部位的总黄酮;
③分别在各下层萃取液中加入盐酸调pH值至1~2,放置4℃冰箱12小时~24小时,使其充分沉淀,以4000r/min速度离心30min,取沉淀,干燥至恒重,获得玉蜀黍的须、秸秆皮以及秸秆芯三个部位的总皂苷;
步骤二:采用亚硝酸钠-硝酸铝法以及总黄酮含量计算公式测定并获得步骤一中获得的各部位的总黄酮含量,采用香草醛-冰醋酸-高氯酸紫外显色法以及总皂苷含量计算公式测定并获得步骤一中获得的各部位的总皂苷含量,并绘制反应曲线;
步骤三:获得玉蜀黍的须、秸秆皮以及秸秆芯三个部位的总多糖冻干粉
①取玉蜀黍的须、秸秆皮以及秸秆芯三个部位阴干至含水量低于20%,粉碎过3号筛,分别称取50g过筛后的粉末,
须粉末按照质量比1:15加入蒸馏水,秸秆皮粉末按照质量比1:15加入蒸馏水,秸秆芯粉末按照质量比1:45加入蒸馏水,获得三种悬浊液;
②将上述三种悬浊液分别在40℃~60℃条件下超声提取1小时,3000r/min离心5min后,将收集的上清液于60℃旋蒸浓缩,收集浓缩液,加入无水乙醇,使其无水乙醇最终质量浓度达到75%,保鲜膜封口,放于4℃冰箱过夜,4000r/min离心30min,取沉淀,冷冻干燥,获得玉蜀黍的须、秸秆皮以及秸秆芯三个部位的总多糖冻干粉;
步骤四:采用苯酚-硫酸法以及总多糖含量计算公式分别测定并获得步骤三中获得的各部位的总多糖含量,并绘制反应曲线;
步骤五:获得玉蜀黍的须、秸秆皮以及秸秆芯三个部位的总蛋白质冻干粉
①取玉蜀黍的须、秸秆皮以及秸秆芯三个部位阴干至含水量低于20%,粉碎过3号筛,分别称取50g过筛后的粉末,
须粉末按照质量比1:15加入Tris-HCl缓冲液,秸秆皮粉末按照质量比1:15加入Tris-HCl缓冲液,秸秆芯粉末按照质量比1:45加入Tris-HCl缓冲液,获得三种悬浊液;
②将上述三种悬浊液分别用搅拌器搅拌4小时~6小时,4000r/min离心30min,取上清液,加入硫酸铵,使其硫酸铵最终质量浓度达到50%,静置12小时~24小时后,4000r/min离心30min,取沉淀物,加50%~80%蒸馏水溶解,经透析袋透析后,冷冻干燥,获得玉蜀黍的须、秸秆皮以及秸秆芯三个部位的总蛋白质冻干粉;
步骤六:采用考马斯亮蓝G250法以及总蛋白质含量的计算公式测定并获得步骤五中获得的各部位的总蛋白质含量,并绘制反应曲线;
步骤七:分别取玉蜀黍的须、秸秆皮以及秸秆芯三个部位的总黄酮、总皂苷、总多糖冻干粉、总蛋白质冻干粉,用千分之一DMSO的磷酸盐缓冲溶液溶解样品,配置成不同浓度的样品溶液,备用;
步骤八:采用酶底物反应法测定玉蜀黍的须、秸秆皮以及秸秆芯三个部位的总黄酮、总皂苷、总多糖冻干粉、总蛋白质冻干粉提取物对α-葡萄糖苷酶抑制活性
取3份pH值为6.8的0.1mol/L磷酸盐缓冲液80μL,加入20μL含千分之一DMSO的磷酸盐缓冲溶液,再加入100μL 0.5U/mL的α-葡萄糖苷酶溶液,加入在37℃摇床中孵育10min后,加入5mmol/L的PNPG溶液(4-硝基苯基-α-D-吡喃葡萄糖苷)50μL,并用酶标仪于0min和5min时刻在405nm处测定吸光度作为吸光度对照基准;
取180份pH值为6.8的0.1mol/L磷酸盐缓冲液80μL,分别加入20μL步骤七中获得的样品溶液,再加入100μL 0.5U/mL的α-葡萄糖苷酶溶液,在37℃摇床中孵育10min后,加入5mmol/L的PNPG溶液50μL,并用酶标仪于0min和5min时刻在405nm处测定吸光度,根据吸光度对照基准,利用SPSS17.0软件查找相应的半数抑制浓度,获得玉蜀黍的须、秸秆皮以及秸秆芯三个部位的总黄酮、总皂苷、总多糖冻干粉、总蛋白质冻干粉提取物对α-葡萄糖苷酶抑制活性,并通过α-葡萄糖苷酶抑制率公式分别计算并获得抑制率;
步骤九:采用SPSS17.0软件进行皮尔逊相关性分析,玉蜀黍各部位总黄酮对α-葡萄糖苷酶对抑制率测定结果以平均值±标准差表示,以皮尔逊相关系数P<0.05作为差异显著性判断的标准;
步骤十:采用3,5-二硝基水杨酸比色法测定玉蜀黍的须、秸秆皮以及秸秆芯三个部位的总黄酮、总皂苷、总多糖冻干粉、总蛋白质冻干粉提取物对α-淀粉酶抑制活性
取3个试管,分别在试管中加入含千分之一DMSO(二甲基亚砜)的磷酸盐缓冲溶液200μL,再加入288μL蒸馏水,在37℃摇床中孵育5min后,加入质量浓度0.5%淀粉溶液320μL,于37℃摇床中反应3min后,从中取出200μL溶液于试管中,并加入100μL二硝基水杨酸(DNS)终止液,沸水浴15min,取出冷却后,加入900μL蒸馏水,混合并取出200μL于96孔板中,在540nm下测定吸光度,作为空白吸光度值;
取3个试管,分别在试管中加入含千分之一DMSO(二甲基亚砜)的磷酸盐缓冲溶液200μL,再加入128μL蒸馏水,160μL1U/mL的α-淀粉酶溶液,在37℃摇床中孵育5min后,加入质量浓度0.5%淀粉溶液320μL,于37℃摇床中反应3min后,从中取出200μL溶液于试管中,并加入100μL二硝基水杨酸(DNS)终止液,沸水浴15min,取出冷却后,加入900μL蒸馏水,混合并取出200μL于96孔板中,在540nm下测定吸光度,作为对照的吸光度值;
取180个试管,分别在试管中加入一种样品溶液200μL,再加入128μL蒸馏水,160μL1U/mL的α-淀粉酶溶液,在37℃摇床中孵育5min后,加入质量浓度0.5%淀粉溶液320μL,于37℃摇床中反应3min后,从中取出200μL溶液于试管中,并加入100μL二硝基水杨酸(DNS)终止液,沸水浴15min,取出冷却后,加入900μL蒸馏水,混合并取出200μL于96孔板中,在540nm下测定吸光度,根据空白吸光度值和对照的吸光度值获得玉蜀黍的须、秸秆皮以及秸秆芯三个部位的总黄酮、总皂苷、总多糖冻干粉、总蛋白质冻干粉提取物对α-淀粉酶抑制活性,并通过α-淀粉酶抑制率公式分别计算并获得抑制率。
所述步骤二中总黄酮含量计算公式为:
其中,C黄酮为样品溶液中的总黄酮浓度,单位为mg/mL;D黄酮为样品溶液的稀释倍数;W黄酮样品为样品质量,单位为mg。
所述步骤二中总皂苷含量计算公式为:
其中,C皂苷为样品溶液中的总皂苷浓度,单位为mg/mL;D皂苷为样品溶液的稀释倍数;W皂苷样品为样品质量,单位为mg。
所述步骤四中总多糖含量计算公式为:
其中,C多糖为样品溶液中的总多糖浓度,单位为mg/mL;D多糖为样品溶液的稀释倍数;W多糖样品为样品质量,单位为mg。
所述步骤六中总蛋白质含量的计算公式为:
其中,C蛋白质为样品溶液中的总蛋白质浓度,单位为mg/mL;D蛋白质为样品溶液的稀释倍数;W蛋白质样品为样品质量,单位为mg。
所述步骤八中α-葡萄糖苷酶抑制率公式为:
其中,(A5min-A0min)对照为酶标仪分别于0min和5min时刻,在405nm处测定对照品的吸光度A值;(A5min-A0min)样品为酶标仪分别于0min和5min时刻,在405nm处测定样品的吸光度A值。
所述步骤十中α-淀粉酶抑制率公式为:
其中,A样品为酶标仪在540nm处测定样品的吸光度A样品值,A空白为酶标仪在540nm处空白的吸光度A空白值,、A对照为酶标仪在540nm处对照品的吸光度A对照值。
通过上述设计方案,本发明可以带来如下有益效果:
本发明重点研究玉蜀黍的须、秸秆皮以及秸秆芯三个部位中总黄酮对α-葡萄糖苷酶和α-淀粉酶活性的抑制作用。经皮尔逊相关性分析玉蜀黍不同部位总黄酮含量与α-葡萄糖苷酶IC50成显著的负相关性,并且在一定浓度范围内对α-淀粉酶有抑制作用。这一结果表明,可以充分利用玉蜀黍须、秸秆中的有效成分,加大秸秆资源技术的开发利用,不仅可以提高玉米秸秆等作物的总体的利用率,解决玉蜀黍须、秸秆闲置、焚烧、生活燃烧等粗放的利用方式问题,还可对推动我国玉蜀黍资源重组乃至农业经济的快速发展,具有重要的意义。同时为α-葡萄糖苷酶、α-淀粉酶抑制剂的开发和利用提供了新的路径,但玉蜀黍不同部位各成分中组分较复杂,对α-葡萄糖苷酶、α-淀粉酶起抑制作用的具体物质结构和作用机理还有待于进一步解析。
附图说明
以下结合附图和具体实施方式对本发明作进一步的说明:
图1为本发明玉蜀黍须和秸秆成分提取及各成分体外降糖活性检验方法的实施例中芦丁标准曲线图。
图2为本发明玉蜀黍须和秸秆成分提取及各成分体外降糖活性检验方法的实施例中齐墩果酸标准曲线图。
图3为本发明玉蜀黍须和秸秆成分提取及各成分体外降糖活性检验方法的实施例中葡萄糖标准曲线图。
图4为本发明玉蜀黍须和秸秆成分提取及各成分体外降糖活性检验方法的实施例中牛血清白蛋白标准曲线图。
图5为本发明玉蜀黍须和秸秆成分提取及各成分体外降糖活性检验方法的实施例中玉蜀黍不同部位总黄酮提取物对α-葡萄糖苷酶抑制率示意图。
图6为本发明玉蜀黍须和秸秆成分提取及各成分体外降糖活性检验方法的实施例中玉蜀黍不同部位总皂苷提取物对α-葡萄糖苷酶抑制率示意图。
图7为本发明玉蜀黍须和秸秆成分提取及各成分体外降糖活性检验方法的实施例中玉蜀黍不同部位总多糖提取物对α-葡萄糖苷酶抑制率示意图。
图8为本发明玉蜀黍须和秸秆成分提取及各成分体外降糖活性检验方法的实施例中玉蜀黍不同部位总蛋白提取物对α-葡萄糖苷酶抑制率示意图。
图9为本发明玉蜀黍须和秸秆成分提取及各成分体外降糖活性检验方法的实施例中表示玉蜀黍不同部位总黄酮提取物对α-淀粉酶抑制率示意图。
图10为本发明玉蜀黍须和秸秆成分提取及各成分体外降糖活性检验方法的实施例中玉蜀黍不同部位总皂苷提取物对α-淀粉酶抑制率示意图。
图11为本发明玉蜀黍须和秸秆成分提取及各成分体外降糖活性检验方法的实施例中玉蜀黍不同部位总多糖提取物对α-淀粉酶抑制率示意图。
图12为本发明玉蜀黍须和秸秆成分提取及各成分体外降糖活性检验方法的实施例中玉蜀黍不同部位总蛋白提取物对α-淀粉酶抑制率示意图。
具体实施方式
以下结合附图对本发明的优选实施例进行说明,应当理解,此处所述的优选实例仅用于说明和解释本发明,并不用于限定本发明。
玉蜀黍须和秸秆成分提取及各成分体外降糖活性检验方法,包括以下步骤,并且以下步骤顺次进行,
步骤一、获得玉蜀黍的须、秸秆皮以及秸秆芯三个部位的总黄酮和玉蜀黍的须、秸秆皮以及秸秆芯三个部位的总皂苷
①取玉蜀黍的须、秸秆皮以及秸秆芯三个部位阴干至含水量低于20%,粉碎过3号筛,分别称取50g过筛后的粉末,
须粉末按照质量比1:15加入质量含量为80%的乙醇溶液,秸秆皮粉末按照质量比1:15加入质量含量为80%的乙醇溶液,秸秆芯粉末按照质量比1:45加入质量含量为80%的乙醇溶液,获得三种溶液;
②将上述三种溶液分别40℃超声提取1小时~3小时,将提取液于40℃水浴锅上水浴至乙醇挥尽,加氨水至溶液为碱性,用乙酸乙酯萃取3次,取上层萃取液同时收集相应的下层萃取液,将上层萃取液浓缩至干膏,获得玉蜀黍的须、秸秆皮以及秸秆芯三个部位的总黄酮;
③分别在各下层萃取液中加入盐酸调pH值至1~2,放置4℃冰箱12小时~24小时,使其充分沉淀,以4000r/min速度离心30min,取沉淀,干燥至恒重,获得玉蜀黍的须、秸秆皮以及秸秆芯三个部位的总皂苷;
步骤二:采用亚硝酸钠-硝酸铝法和香草醛-冰醋酸-高氯酸紫外显色法分别测定步骤一中获得的各部位的总黄酮和总皂苷含量,并绘制反应曲线;按照公式(1)计算总黄酮含量,按照公式(2)计算总皂苷含量。
其中,公式(1)中C黄酮为样品溶液中的总黄酮浓度(mg/mL);D黄酮为样品溶液的稀释倍数;W黄酮样品为样品质量(mg)。
公式(2)中C皂苷为样品溶液中的总皂苷浓度(mg/mL);D皂苷为样品溶液的稀释倍数;W皂苷样品为样品质量(mg)。
步骤三:获得玉蜀黍的须、秸秆皮以及秸秆芯三个部位的总多糖冻干粉
①取玉蜀黍的须、秸秆皮以及秸秆芯三个部位阴干至含水量低于20%,粉碎过3号筛,分别称取50g过筛后的粉末,
须粉末按照质量比1:15加入蒸馏水,秸秆皮粉末按照质量比1:15加入蒸馏水,秸秆芯粉末按照质量比1:45加入蒸馏水,获得三种悬浊液;
②将上述三种悬浊液分别在40℃~60℃条件下超声提取1小时,3000r/min离心5min后,将收集的上清液于60℃旋蒸浓缩,收集浓缩液,加入无水乙醇,使其无水乙醇最终质量浓度达到75%,保鲜膜封口,放于4℃冰箱过夜,4000r/min离心30min,取沉淀,冷冻干燥,获得玉蜀黍的须、秸秆皮以及秸秆芯三个部位的总多糖冻干粉;
步骤四:采用苯酚-硫酸法分别测定步骤三中获得的各部位总多糖冻干粉的总多糖含量,并绘制反应曲线;按照公式(3)计算总多糖含量。
其中,C多糖为样品溶液中的总多糖浓度(mg/mL);D多糖为样品溶液的稀释倍数;W多糖样品为样品质量(mg)。
步骤五:获得玉蜀黍的须、秸秆皮以及秸秆芯三个部位的总蛋白质冻干粉
①取玉蜀黍的须、秸秆皮以及秸秆芯三个部位阴干至含水量低于20%,粉碎过3号筛,分别称取50g过筛后的粉末,
须粉末按照质量比1:15加入Tris-HCl缓冲液,秸秆皮粉末按照质量比1:15加入Tris-HCl缓冲液,秸秆芯粉末按照质量比1:45加入Tris-HCl缓冲液,获得三种悬浊液;
②将上述三种悬浊液分别用搅拌器搅拌4小时~6小时,4000r/min离心30min,取上清液,加入硫酸铵,使其硫酸铵最终质量浓度达到50%,静置12小时~24小时后,4000r/min离心30min,取沉淀物,加50%~80%蒸馏水溶解,经透析袋透析后,冷冻干燥,获得玉蜀黍的须、秸秆皮以及秸秆芯三个部位的总蛋白质冻干粉;
步骤六:采用考马斯亮蓝G250法测定步骤五中获得的各部位总蛋白质冻干粉的蛋白含量,并绘制反应曲线;按照公式(4)计算总蛋白质含量。
其中,C蛋白质为样品溶液中的总蛋白质浓度(mg/mL);D蛋白质为样品溶液的稀释倍数;W蛋白质样品为样品质量(mg)。
步骤七:分别取玉蜀黍的须、秸秆皮以及秸秆芯三个部位的总黄酮、总皂苷、总多糖冻干粉、总蛋白质冻干粉,用千分之一DMSO的磷酸盐缓冲溶液溶解样品,配置成不同浓度的样品溶液,备用;
步骤八:采用酶底物反应法测定玉蜀黍的须、秸秆皮以及秸秆芯三个部位的总黄酮、总皂苷、总多糖冻干粉、总蛋白质冻干粉提取物对α-葡萄糖苷酶抑制活性
取3份pH值为6.8的0.1mol/L磷酸盐缓冲液80μL,加入20μL含千分之一DMSO的磷酸盐缓冲溶液,再加入100μL 0.5U/mL的α-葡萄糖苷酶溶液,加入在37℃摇床中孵育10min后,加入5mmol/L的PNPG溶液(4-硝基苯基-α-D-吡喃葡萄糖苷)50μL,并用酶标仪于0min和5min时刻在405nm处测定吸光度作为吸光度对照基准;
取180份pH值为6.8的0.1mol/L磷酸盐缓冲液80μL,分别加入20μL步骤七中获得的样品溶液,再加入100μL 0.5U/mL的α-葡萄糖苷酶溶液,在37℃摇床中孵育10min后,加入5mmol/L的PNPG溶液(4-硝基苯基-α-D-吡喃葡萄糖苷)50μL,并用酶标仪于0min和5min时刻在405nm处测定吸光度,根据吸光度对照基准,利用SPSS17.0软件查找相应的半数抑制浓度,获得玉蜀黍的须、秸秆皮以及秸秆芯三个部位的总黄酮、总皂苷、总多糖冻干粉、总蛋白质冻干粉提取物对α-葡萄糖苷酶抑制活性,通过α-葡萄糖苷酶抑制率公式计算并获得抑制率,α-葡萄糖苷酶抑制率公式如下:
其中,(A5min-A0min)对照为酶标仪分别于0min和5min时刻,在405nm处测定对照品的吸光度A值;(A5min-A0min)样品为酶标仪分别于0min和5min时刻,在405nm处测定样品的吸光度A值。
步骤九:采用SPSS21.0软件进行皮尔逊相关性分析,玉蜀黍各部位总黄酮对α-葡萄糖苷酶对抑制率测定结果以平均值±标准差表示,以皮尔逊相关系数P<0.05作为差异显著性判断的标准;
步骤十:采用3,5-二硝基水杨酸比色法测定玉蜀黍的须、秸秆皮以及秸秆芯三个部位的总黄酮、总皂苷、总多糖冻干粉、总蛋白质冻干粉提取物对α-淀粉酶抑制活性
取3个试管,分别在试管中加入含千分之一DMSO的磷酸盐缓冲溶液200μL,再加入288μL蒸馏水,在37℃摇床中孵育5min后,加入质量浓度0.5%淀粉溶液320μL,于37℃摇床中反应3min后,从中取出200μL溶液于试管中,并加入100μL二硝基水杨酸(DNS)终止液,沸水浴15min,取出冷却后,加入900μL蒸馏水,混合并取出200μL于96孔板中,在540nm下测定吸光度,作为空白吸光度值;
取3个试管,分别在试管中加入含千分之一DMSO的磷酸盐缓冲溶液200μL,再加入128μL蒸馏水,160μL1U/mL的α-淀粉酶溶液,在37℃摇床中孵育5min后,加入质量浓度0.5%淀粉溶液320μL,于37℃摇床中反应3min后,从中取出200μL溶液于试管中,并加入100μL二硝基水杨酸(DNS)终止液,沸水浴15min,取出冷却后,加入900μL蒸馏水,混合并取出200μL于96孔板中,在540nm下测定吸光度,作为对照的吸光度值;
取180个试管,分别在试管中加入一种样品溶液200μL,再加入128μL蒸馏水,160μL1U/mL的α-淀粉酶溶液,在37℃摇床中孵育5min后,加入质量浓度0.5%淀粉溶液320μL,于37℃摇床中反应3min后,从中取出200μL溶液于试管中,并加入100μL二硝基水杨酸(DNS)终止液,沸水浴15min,取出冷却后,加入900μL蒸馏水,混合并取出200μL于96孔板中,在540nm下测定吸光度,根据空白和对照的吸光度值获得玉蜀黍的须、秸秆皮以及秸秆芯三个部位的总黄酮、总皂苷、总多糖冻干粉、总蛋白质冻干粉提取物对α-淀粉酶抑制活性,通过α-淀粉酶抑制率公式计算并获得抑制率,α-淀粉酶抑制率公式如下:
其中,A样品为酶标仪在540nm处测定样品的吸光度A样品值,A空白为酶标仪在540nm处空白的吸光度A空白值,、A对照为酶标仪在540nm处对照品的吸光度A对照值。
实施例一:
(1)芦丁标准曲线的测定
分别量取芦丁标准溶液(1mg/mL)10μL、20μL、40μL、60μL、80μL、100μL于试管中,补加蒸馏水至1mL,加入0.3mL的5%亚硝酸钠溶液,混合均匀后静置6min,再加0.3mL的10%硝酸铝溶液,混合均匀后,静置6min,加入4mL的4%氢氧化钠溶液,用蒸馏水补足至10mL,混合均匀,静置15min后,在510nm处测吸光度A值。以芦丁标准溶液质量浓度(mg/mL)作为横坐标(X),A510nm作为纵坐标(Y),绘制芦丁标准曲线如图1所示。
(2)玉蜀黍不同部位总黄酮样品溶液的测定
取适量总黄酮样品,加入乙醇配制成1mg/mL的总黄酮溶液,取样品溶液1mL,加入0.3mL的5%亚硝酸钠溶液,混合均匀,并静置6min,再加入0.3mL的10%硝酸铝溶液,混合均匀并静置6min后,加入4mL的4%氢氧化钠溶液,用蒸馏水补足至10mL,混合均匀,再静置15min后,在510nm处测吸光度A值。玉蜀黍秸秆皮、须、秸秆芯总黄酮含量分别为:18.37%、9.5%、6.05%。
(3)齐墩果酸标准曲线的测定
分别量取10μL、20μL、40μL、60μL、80μL、100μL齐墩果酸标准溶液(1mg/mL)于试管中,补加蒸馏水至1mL,蒸干后,加入0.2mL的5%香草醛-冰醋酸溶液、0.8mL的高氯酸溶液,混合均匀,置60℃水浴中,反应15mi后,快速冷却至室温,再加入5mL冰醋酸,混合均匀后,在575nm处测吸光度A值。以齐墩果酸标准溶液质量浓度(mg/mL)作为横坐标(X),以A575 nm为纵坐标(Y),绘制齐墩果酸标准曲线,如图2所示。
(4)玉蜀黍不同部位总皂苷样品溶液的测定
取适量总皂苷样品,加入乙醇制备1mg/mL总皂苷溶液,取样品溶液1mL,蒸干,加入0.2mL的5%香草醛-冰醋酸溶液、0.8mL的高氯酸溶液,混合均匀后,置60℃水浴中,反应15min后,用冰水浴快速冷却至室温,再加入5mL冰醋酸,混合均匀后,在575nm处测吸光度A值。
(5)葡萄糖标准曲线的测定
分别量取葡萄糖溶液(1mg/mL)10μL、20μL、40μL、60μL、80μL、100μL置于试管中,补加蒸馏水至1mL,加入1mL的5%苯酚溶液,5mL浓硫酸溶液,混合均匀,沸水浴30min,待冷却至室温后,在483nm处吸光度A值。将葡萄糖溶液质量浓度(mg/mL)作为横坐标(X),将A483nm作为纵坐标(Y),绘制葡萄糖标准曲线如图3所示。
(6)玉蜀黍不同部位总多糖样品溶液的测定
取适量总多糖冻干粉,加蒸馏水配制成质量浓度为1mg/mL样品溶液,吸取1mL样品溶液,加入1mL的5%苯酚溶液,浓硫酸溶液5mL,混合均匀后,沸水浴30min,待冷却至室温后,在483nm处测A值。玉蜀黍秸秆芯、秸秆皮、须总多糖含量分别为:31.94%、31.61%、24.16%。
(7)牛血清白蛋白标准曲线的测定
考马斯亮蓝G-250溶液的配制:准确称取100mg考马斯亮蓝G-250溶于50mL 95%乙醇溶液中,加入100mL 85%磷酸溶液混合摇匀,用蒸馏水补充至1000mL,混匀,过滤,备用;
蛋白质的标准曲线的建立:分别精密量取蛋白质标准溶液(1mg/mL)10、20、40、60、80、100μL置于试管中,用蒸馏水补足至1mL,加入5mL考马斯亮蓝溶液,混匀,室温静置5min。并设置空白对照(以蒸馏水作为空白对照),测定595nm处吸光度A值。将蛋白质溶液质量浓度(μg/mL)作为横坐标(X),将A595nm作为纵坐标(Y),绘制蛋白质的标准曲线如图4所示。
(8)玉蜀黍不同部位总蛋白样品溶液的测定
取适量总蛋白质冻干粉,加入蒸馏水配制成质量浓度为1mg/mL样品溶液,吸取1mL样品溶液,加入5mL考马斯亮蓝溶液,混合均匀后,室温反应5min,在595nm处测A值。玉蜀黍秸秆皮、须、秸秆芯总蛋白质含量分别为:13.13%、10.47%、9.37%。
(9)实验设计和统计分析:
一、标准曲线
a.黄酮标准曲线
按1.2.2项下方法进行标准曲线绘制见图1,拟合得线性方程y=0.9976x-0.0036,R2=0.9998,结果表明质量浓度为10~100μg/mL范围内时,黄酮标准溶液的质量浓度与吸光度有良好线性关系。
b.皂苷标准曲线
按1.2.3项下方法进行标准曲线绘制见图2,拟合得线性方程y=3.8028x+0.0097,R2=0.9993,结果表明质量浓度为10~100μg/mL范围内时,皂苷标准溶液的质量浓度与吸光度有良好线性关系。
c.多糖标准曲线
按1.2.5项下方法进行标准曲线绘制见图3,拟合得线性方程y=9.1386x+0.0057,R2=0.9989,结果表明质量浓度为10~100μg/mL范围内时,多糖标准溶液的质量浓度与吸光度有良好线性关系。
d.蛋白质标准曲线
按1.2.7项下方法进行标准曲线绘制见图4,拟合得线性方程y=6.1987x+0.0156,R2=0.9989,结果表明质量浓度为10~100μg/mL范围内时,蛋白质标准溶液的质量浓度与吸光度有良好线性关系。
二、玉蜀黍不同部位各成分含量;
玉蜀黍不同部位各成分含量见表1,可知玉蜀黍不同部位中总黄酮、总皂苷、总多糖、总蛋白质含量最高部位分别为:秸秆皮、须、秸秆芯、秸秆皮。
表1玉蜀黍不同部位各成分含量
三、玉蜀黍不同部位各成分对α-葡萄糖苷酶的抑制作用;
玉蜀黍不同部位各成分对α-葡萄糖苷酶的抑制作用由图5所示。从图5可看出,玉蜀黍不同部位总黄酮在0.125~2mg/mL的质量浓度范围内,对α-葡萄糖苷酶的抑制率随着样品浓度的增大而升高,呈现出浓度依赖关系。由表2结果显示,玉蜀黍不同部位总黄酮成分对α-葡萄糖苷酶均有不同程度抑制作用,对α-葡萄糖苷酶抑制作用强弱依次为:秸秆皮>秸秆芯>须,与空白组比较,各给药组都可明显抑制α-葡萄糖苷酶,差异均具有统计学意义(P<0.05)。综上所述,玉蜀黍秸秆皮中总黄酮对α-葡萄糖苷酶的抑制作用最强。但玉蜀黍不同部位总皂苷、总多糖、总蛋白质成分对α-葡萄糖苷酶的抑制作用较差,结果见下图6、图7、图8。
注:同一指标不同组别间,若字母相同,则代表差异无统计学意义(P>0.05);若字母不同,则代表差异有统计学意义(P<0.05)
四、玉蜀黍不同部位总黄酮对α-糖苷酶抑制活性相关性分析
玉蜀黍秸秆皮总黄酮含量相对较高(总黄酮182.3mg/g,总黄酮得率为1.823%),对α-葡萄糖苷酶抑制活性最强IC50为0.131mg/mL,玉蜀黍秸秆芯总黄酮含量较低(总黄酮58mg/g,总黄酮得率为0.812%),对α-葡萄糖苷酶抑制活性较强IC50为0.348mg/mL,玉蜀黍须总黄酮含量相对低(总黄酮80mg/g,总黄酮得率为0.45%),与常波等研究玉蜀黍须总黄酮得率为0.4229%实验测量结果一致,对α-葡萄糖苷酶抑制活性较弱IC50为0.63mg/mL。
经皮尔逊相关性分析(Pearson correlation analysis),IC50与玉蜀黍不同部位总黄酮含量呈现较强的负相关性,皮尔逊相关系数为r=-0.997,P=0.048,相关性显著。由此可见,玉蜀黍不同部位中总黄酮是抑制α-葡萄糖苷酶的活性成分。
五、玉蜀黍不同部位各成分对α-淀粉酶的抑制作用
玉蜀黍不同部位各成分对α-淀粉酶抑制作用见图9,从9图可知,玉蜀黍须、秸秆皮、秸秆芯总黄酮在质量浓度为1mg/mL、0.5mg/mL、0.125mg/mL时对α-淀粉酶的抑制活性强分别为:24.73%、21.46%、14.63%。由表3结果显示,玉蜀黍不同部位总黄酮成分对α-淀粉酶的活性都有一定的抑制作用,对α-淀粉酶抑制作用强弱小依次为:须>秸秆皮>秸秆芯,与对照组相比,各给药组都可明显抑制α-淀粉酶,差异均具有统计学意义(P<0.05)。而玉蜀黍不同部位总皂苷、总多糖、总蛋白质成分对α-淀粉酶的抑制作用较差,结果见图10、11、12。
注:同一指标不同组别间,若字母相同,则代表差异无统计学意义(P>0.05);若字母不同,则代表差异有统计学意义(P<0.05)。
Claims (5)
1.玉蜀黍须和秸秆成分提取及各成分体外降糖活性检验方法,其特征是:包括以下步骤,并且以下步骤顺次进行,
步骤一、获得玉蜀黍的须、秸秆皮以及秸秆芯三个部位的总黄酮和玉蜀黍的须、秸秆皮以及秸秆芯三个部位的总皂苷
①取玉蜀黍的须、秸秆皮以及秸秆芯三个部位阴干至含水量低于20%,粉碎过3号筛,分别称取50g过筛后的粉末,
须粉末按照质量比1:15加入质量含量为80%的乙醇溶液,秸秆皮粉末按照质量比1:15加入质量含量为80%的乙醇溶液,秸秆芯粉末按照质量比1:45加入质量含量为80%的乙醇溶液,获得三种溶液;
②将上述三种溶液分别40℃超声提取1小时~3小时,将提取液于40℃水浴锅上水浴至乙醇挥尽,加氨水至溶液为碱性,用乙酸乙酯萃取3次,取上层萃取液同时收集相应的下层萃取液,将上层萃取液浓缩至干膏,获得玉蜀黍的须、秸秆皮以及秸秆芯三个部位的总黄酮;
③分别在各下层萃取液中加入盐酸调pH值至1~2,放置4℃冰箱12小时~24小时,使其充分沉淀,以4000r/min速度离心30min,取沉淀,干燥至恒重,获得玉蜀黍的须、秸秆皮以及秸秆芯三个部位的总皂苷;
步骤二:采用亚硝酸钠-硝酸铝法以及总黄酮含量计算公式测定并获得步骤一中获得的各部位的总黄酮含量,采用香草醛-冰醋酸-高氯酸紫外显色法以及总皂苷含量计算公式测定并获得步骤一中获得的各部位的总皂苷含量,并绘制反应曲线;
步骤三:获得玉蜀黍的须、秸秆皮以及秸秆芯三个部位的总多糖冻干粉
①取玉蜀黍的须、秸秆皮以及秸秆芯三个部位阴干至含水量低于20%,粉碎过3号筛,分别称取50g过筛后的粉末,
须粉末按照质量比1:15加入蒸馏水,秸秆皮粉末按照质量比1:15加入蒸馏水,秸秆芯粉末按照质量比1:45加入蒸馏水,获得三种悬浊液;
②将上述三种悬浊液分别在40℃~60℃条件下超声提取1小时,3000r/min离心5min后,将收集的上清液于60℃旋蒸浓缩,收集浓缩液,加入无水乙醇,使其无水乙醇最终质量浓度达到75%,保鲜膜封口,放于4℃冰箱过夜,4000r/min离心30min,取沉淀,冷冻干燥,获得玉蜀黍的须、秸秆皮以及秸秆芯三个部位的总多糖冻干粉;
步骤四:采用苯酚-硫酸法以及总多糖含量计算公式分别测定并获得步骤三中获得的各部位的总多糖含量,并绘制反应曲线;
步骤五:获得玉蜀黍的须、秸秆皮以及秸秆芯三个部位的总蛋白质冻干粉
①取玉蜀黍的须、秸秆皮以及秸秆芯三个部位阴干至含水量低于20%,粉碎过3号筛,分别称取50g过筛后的粉末,
须粉末按照质量比1:15加入Tris-HCl缓冲液,秸秆皮粉末按照质量比1:15加入Tris-HCl缓冲液,秸秆芯粉末按照质量比1:45加入Tris-HCl缓冲液,获得三种悬浊液;
②将上述三种悬浊液分别用搅拌器搅拌4小时~6小时,4000r/min离心30min,取上清液,加入硫酸铵,使其硫酸铵最终质量浓度达到50%,静置12小时~24小时后,4000r/min离心30min,取沉淀物,加50%~80%蒸馏水溶解,经透析袋透析后,冷冻干燥,获得玉蜀黍的须、秸秆皮以及秸秆芯三个部位的总蛋白质冻干粉;
步骤六:采用考马斯亮蓝G250法以及总蛋白质含量的计算公式测定并获得步骤五中获得的各部位的总蛋白质含量,并绘制反应曲线;
步骤七:分别取玉蜀黍的须、秸秆皮以及秸秆芯三个部位的总黄酮、总皂苷、总多糖冻干粉、总蛋白质冻干粉,用千分之一DMSO的磷酸盐缓冲溶液溶解样品,配置成不同浓度的样品溶液,备用;
步骤八:采用酶底物反应法测定玉蜀黍的须、秸秆皮以及秸秆芯三个部位的总黄酮、总皂苷、总多糖冻干粉、总蛋白质冻干粉提取物对α-葡萄糖苷酶抑制活性
取3份pH值为6.8的0.1mol/L磷酸盐缓冲液80μL,加入20μL含千分之一DMSO的磷酸盐缓冲溶液,再加入100μL 0.5U/mL的α-葡萄糖苷酶溶液,加入在37℃摇床中孵育10min后,加入5mmol/L的PNPG溶液(4-硝基苯基-α-D-吡喃葡萄糖苷)50μL,并用酶标仪于0min和5min时刻在405nm处测定吸光度作为吸光度对照基准;
取180份pH值为6.8的0.1mol/L磷酸盐缓冲液80μL,分别加入20μL步骤七中获得的样品溶液,再加入100μL 0.5U/mL的α-葡萄糖苷酶溶液,在37℃摇床中孵育10min后,加入5mmol/L的PNPG溶液50μL,并用酶标仪于0min和5min时刻在405nm处测定吸光度,根据吸光度对照基准,利用SPSS17.0软件查找相应的半数抑制浓度,获得玉蜀黍的须、秸秆皮以及秸秆芯三个部位的总黄酮、总皂苷、总多糖冻干粉、总蛋白质冻干粉提取物对α-葡萄糖苷酶抑制活性,并通过α-葡萄糖苷酶抑制率公式分别计算并获得抑制率;
所述α-葡萄糖苷酶抑制率公式为:
其中,(A5min-A0min)对照为酶标仪分别于0min和5min时刻,在405nm处测定对照品的吸光度A值;(A5min-A0min)样品为酶标仪分别于0min和5min时刻,在405nm处测定样品的吸光度A值;
步骤九:采用SPSS17.0软件进行皮尔逊相关性分析,玉蜀黍各部位总黄酮对α-葡萄糖苷酶对抑制率测定结果以平均值±标准差表示,以皮尔逊相关系数P<0.05作为差异显著性判断的标准;
步骤十:采用3,5-二硝基水杨酸比色法测定玉蜀黍的须、秸秆皮以及秸秆芯三个部位的总黄酮、总皂苷、总多糖冻干粉、总蛋白质冻干粉提取物对α-淀粉酶抑制活性
取3个试管,分别在试管中加入含千分之一DMSO(二甲基亚砜)的磷酸盐缓冲溶液200μL,再加入288μL蒸馏水,在37℃摇床中孵育5min后,加入质量浓度0.5%淀粉溶液320μL,于37℃摇床中反应3min后,从中取出200μL溶液于试管中,并加入100μL二硝基水杨酸(DNS)终止液,沸水浴15min,取出冷却后,加入900μL蒸馏水,混合并取出200μL于96孔板中,在540nm下测定吸光度,作为空白吸光度值;
取3个试管,分别在试管中加入含千分之一DMSO(二甲基亚砜)的磷酸盐缓冲溶液200μL,再加入128μL蒸馏水,160μL1U/mL的α-淀粉酶溶液,在37℃摇床中孵育5min后,加入质量浓度0.5%淀粉溶液320μL,于37℃摇床中反应3min后,从中取出200μL溶液于试管中,并加入100μL二硝基水杨酸(DNS)终止液,沸水浴15min,取出冷却后,加入900μL蒸馏水,混合并取出200μL于96孔板中,在540nm下测定吸光度,作为对照的吸光度值;
取180个试管,分别在试管中加入一种样品溶液200μL,再加入128μL蒸馏水,160μL1U/mL的α-淀粉酶溶液,在37℃摇床中孵育5min后,加入质量浓度0.5%淀粉溶液320μL,于37℃摇床中反应3min后,从中取出200μL溶液于试管中,并加入100μL二硝基水杨酸(DNS)终止液,沸水浴15min,取出冷却后,加入900μL蒸馏水,混合并取出200μL于96孔板中,在540nm下测定吸光度,根据空白吸光度值和对照的吸光度值获得玉蜀黍的须、秸秆皮以及秸秆芯三个部位的总黄酮、总皂苷、总多糖冻干粉、总蛋白质冻干粉提取物对α-淀粉酶抑制活性,并通过α-淀粉酶抑制率公式分别计算并获得抑制率;
所述α-淀粉酶抑制率公式为:
其中,A样品为酶标仪在540nm处测定样品的吸光度A样品值,A空白为酶标仪在540nm处空白的吸光度A空白值,、A对照为酶标仪在540nm处对照品的吸光度A对照值。
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