CN112402415B - 一种火麻仁低聚肽的应用 - Google Patents
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Abstract
本发明涉及一种火麻仁低聚肽应用,包括在制备抗氧化保健品、药品,及在制备改善便秘有关的保健品、药品中的应用。本发明提供的火麻仁低聚肽分子含量为20~30%谷氨酸、30~40%组氨酸和10~30%精氨酸,其在具有抗氧化能力的同时,还为进一步作为改善便秘有关的保健品和药品的制备提供了物质基础。
Description
技术领域
本发明涉及火麻仁应用技术领域,尤其涉及一种火麻仁低聚肽的应 用。
背景技术
作为中国的传统中药材和食材的火麻仁虽然拥有多种生理作用, 市场潜力巨大,但由于对其研究还不够深入,限制了火麻仁在食品、 保健食品或医药领域中的应用。
发明内容
有鉴于此,有必要提供一种火麻仁低聚肽的应用,发掘其低聚肽新 的应用领域。
本发明提供一种火麻仁低聚肽的应用,包括在制备抗氧化保健品、 药品,及在制备改善便秘有关的保健品、药品中的应用。
其中,所述火麻仁低聚肽分子中包含分子含量为20~30%谷氨酸、 30~40%组氨酸和10~30%精氨酸,所述火麻仁低聚肽分子量为461Da。
进一步的,所述火麻仁低聚肽的制备方法包括以下步骤:
将火麻仁的原料经浸泡、脱糖和变性后,进行酶解,灭酶后将获得 的酶解液经过滤,取滤液浓缩,干燥即可得所述火麻仁低聚肽。
具体的,所述浸泡步骤中按料液比1:(10~20)。
具体的,所述脱糖步骤采用淀粉酶水解处理1~2h。
具体的,所述酶解步骤中采用碱性蛋白酶处理,温度为50~60℃,pH=8.5~9.0,处理时间为5~6h。
有益效果:
本发明提供的火麻仁低聚肽分子含量为20~30%谷氨酸、30~40%组 氨酸和10~30%精氨酸,其在具有抗氧化能力的同时,还为进一步作为改 善便秘有关的保健品和药品的制备提供了物质基础。
附图说明
图1为本发明实施例提供的火麻仁低聚条DPPH清除能力图。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白,以下结合附 图及实施例,对本发明进行进一步详细说明。应当理解,此处所描述的 具体实施例仅仅用以解释本发明,并不用于限定本发明。
火麻仁低聚肽及其制备方法
本发明实施例提供的火麻仁低聚肽,其分子中包含分子含量为 20~30%谷氨酸(Glu)、30~40%组氨酸(His)和10~30%精氨酸(Arg), 其分子量为461Da。该火麻仁低聚肽具有抗氧化和改善便秘作用。
具体的,该火麻仁低聚肽的制备方法包括以下步骤:
将火麻仁的原料经浸泡、脱糖和变性后,进行酶解,灭酶后将获得 的酶解液经过滤,取滤液浓缩,干燥即可得所述火麻仁低聚肽。
具体的,浸泡步骤中按料液比1:(10~20)。
具体的,脱糖步骤采用淀粉酶水解处理1~2h。
具体的,酶解步骤中采用碱性蛋白酶处理,温度为50~60℃, pH=8.5~9.0,处理时间为5~6h。
为进行探索该火麻仁低聚肽的制备方法,主要考察其浸泡、脱糖和 酶解步骤。
取火麻仁药材,粉粹,取若干份,每份5g,精密称定后,置于100ml 锥形瓶中,按照料液比1:(10~20)投料加水(如加水50ml,75ml,100ml), 搅拌浸泡,浸泡时使物料与水充分混合。然后进行脱糖处理,采用淀粉 酶进行水解脱糖,将其降解成小分子寡糖,便于后续处理。然后升温至 100℃条件下,使得淀粉酶变性2~3h,使其失去活性。再采用降温至 50~60℃,pH=8.5~9.0,处理时间为5~6h,降解火麻仁内部蛋白成低聚肽, 后续在经过灭酶、过滤、浓缩和干燥就可得到火麻仁低聚肽。
表1将火麻仁制备过程中的实施例进行统计,考察其制备过程条件 对其应用的影响。
表1
实施例 | 料液比 | 糖解 | 酶解 |
实施例1 | 1:10 | 1h | 50℃,pH=8.5~9.0,5~6h |
实施例2 | 1:15 | 1h | 54℃,pH=8.5~9.0,5~6h |
实施例3 | 1:20 | 1h | 56℃,pH=8.5~9.0,5~6h |
实施例4 | 1:20 | 2h | 60℃,pH=8.5~9.0,5~6h |
对比例1 | 1:8 | 1h | 45℃,pH=8.5~9.0,5~6h |
对比例2 | 1:22 | 1h | 65℃,pH=8.5~9.0,5~6h |
对比例3 | 1:10 | 0.5h | 45℃,pH=8.5~9.0,5~6h |
对比例4 | 1:10 | 2.5h | 65℃,pH=8.5~9.0,5~6h |
对比例5 | 1:10 | 1h | 40℃,pH=2.5~3.5,5~6h(酸性蛋白酶) |
火麻仁低聚肽抗氧化能力
试验方法:
将1.5mg样品加入1.5mL 0.1mmol/L DPPH(95%乙醇)中,混匀并 在25℃保温30min。在517nm处测量吸光度。Vc溶液用作对照。DPPH 清除能力W(%)计算如下:
W(%)=[1-(A1-A2)/A0]×100%
其中A0是1.5mL蒸馏水和1.5mL含0.1mmol/L DPPH的95%乙醇 的吸光度值,A1是含有0.1mmol/L DPPH·的1.5mL水解产物的吸光度 值,A2是吸光度值1.5mL水解产物和1.5ml95%乙醇。
图1示出了不同浓度火麻仁低聚肽的DPPH自由基清除能力,可知。 火麻低聚肽对DPPH自由基具有较为明显的清除作用,同时其自由基清 除率呈现剂量依赖性。当火麻低聚肽浓度为6.0mg/ml时,清除DPPH自 由基为92.88%。通过计算得到火麻仁低聚肽的IC50为1.72mg/ml。说明 火麻低聚肽中有给出质子,稳定DPPH自由基的能力,有较强的抗氧化 能力。
而对于不同的实施例和对比例,其抗氧化能力必然不同。如表2可 知,实施例1-4的抗氧化能力均高于对比例1-5。表2给出了当各实施例 和对比例制备的不同火麻仁低聚肽的浓度能够得到的最大的W(%),对 应的IC50及制备的火麻仁低聚条的氨基酸组成。由表2可知:实施例1-4 的最大DPPH·清除能力均大于对比例,且其IC50均低于对比例;实施例 1-4的氨基酸组成为20~30%谷氨酸(Glu)、30~40%组氨酸(His)和10~30% 精氨酸(Arg),而对比例不在此范围内。
表2
实施例 | 最大的W(%) | IC50 | 氨基酸组成 |
实施例1 | 90.11% | 2.05mg/ml | Glu 25.3%,His 35.8%,Arg 11.5% |
实施例2 | 90.32% | 1.99mg/ml | Glu 27.3%,His 38.2%,Arg 13.2% |
实施例3 | 91.62% | 1.87mg/ml | Glu 28.6%,His 38.3%,Arg 15.4% |
实施例4 | 92.88% | 1.72mg/ml | Glu 29.1%,His 39.1%,Arg 18.2% |
对比例1 | 75.22% | 4.33mg/ml | Glu 13.6%,His 32.3%,Arg 9.4% |
对比例2 | 61.21% | 6.22mg/ml | Glu 15.3%,His 28.4%,Arg 11.1% |
对比例3 | 59.44% | 7.35mg/ml | Glu 17.6%,His 27.8%,Arg 9.6% |
对比例4 | 62.25% | 8.26mg/ml | Glu 16.4%,His 29.5%,Arg 8.1% |
对比例5 | 63.32% | 9.71mg/ml | Glu 18.2%,His 26.3%,Arg 7.2% |
火麻仁低聚肽改善便秘作用
1、实验动物
健康雄性昆明小鼠若干(体重29-32g),八周龄,购自某动物实验中心。 同笼饲养,饲养温度为25℃左右,湿度为50%左右,小鼠自由饮水进食。 实验开始前,小鼠适应环境一天。
2、分组和造模
小鼠适应环境一天后,随机分组。其中,试验品为上述实施例和对 比例制备的火麻仁低聚肽。
对照组:
给予小鼠灌胃等量重蒸水15天,每天1次,每次1ml。
模型组:
给予小鼠灌胃等量重蒸水15天,每天1次,每次1ml。
治疗组:
给予小鼠灌胃等量上述实施例提供的火麻仁低聚肽的试验品,每只 小鼠灌胃剂量为500mg/kg,每天1次,灌胃15天。
3、小肠运动实验
取复方地芬诺酯25mg,用研钵研碎呈粉末后加水至100m1,临用前 配制,需不断振荡以保持其浓度均一。
取印度墨水2m1,加入98m1重蒸馏水配制成2%墨汁,灌胃剂量为 0.lml/(l0g·bw)。用墨汁与各试验品水溶液勾兑,灌胃总量保持lml。
从动物房将干预7天后禁食不禁水16h的小鼠取出,模型组和治疗 组用复方地芬诺酯溶液灌胃(5mg/(kg·bw),空白对照组用蒸馏水灌胃;30 分钟后,治疗组用含有相应量试验品水溶液的墨汁灌胃,空白组和模型 组用纯墨汁灌胃;25分钟后,立即脱颈椎处死动物,打开腹腔分离肠系 膜,剪取上端自幽门、下端至回盲的肠管,轻轻将小肠拉成直线,测量 肠管长度为“小肠总长度”,从幽门至墨汁前沿为“墨汁推进长度”。
墨汁推进率(P)=墨汁推进长度/小肠总长度。
在进行数据转换后,先对各组X值进行方差齐性检验,若满足方差 齐性则进行方差分析,不满足方差齐性则进行秩和检验。若P<0.05则表 明各组墨汁推进率之间存在显著差异,然后再在多个实验组和一个对照 组之间进行两两q检验。受试样品组小鼠X值显著高于模型对照组X值 时,可判定受试样品具有润肠通便功能。
4、排便时间和重量测定实验
取复方地芬诺酯25mg,用研钵研碎呈粉末后加水至50m1,临用前 配制,需不断振荡以保持其浓度均一。
取品红染料1g,加入49m1重蒸馏水配制成2%品红溶液,灌胃剂量 为0.lml/l0g·bw。用品红溶液与试验品的水溶液勾兑,灌胃总量保持lml。
从动物房将干预7天后禁食不禁水16h的小鼠取出,模型组和治疗 组用复方地芬诺酯溶液灌胃(l0mg/kg·bw),空白对照组用蒸馏水灌胃;30 分钟后,治疗组用含有相应量的试验品的品红溶液灌胃,空白组和模型 组用纯品红溶液灌胃;从灌品红溶液开始,记录协只动物首粒排红色粪 便的时间和排便重量。
先对各组数据进行方差齐性检验,若满足方差齐性则进行方差分析, 不满足方差齐性则进行秩和检验。若P<0.05则表明各组墨汁推进率之间 存在显著差异,然后再在多个实验组和一个对照组间进行两两q检验。 6h内排便时间(即6h内已经排便)、排便重量及小肠运动试验任一项结 果阳性,可判定该受试物具有润肠通便功能。
5、测试结果
由表3可知:
1、模型对照组小鼠的墨汁推进率显著低于正常对照组(P<0.01),表 示成功建立便秘模型。治疗组中实施例1-4的小鼠墨汁推进率均高于模型 对照组,而治疗组中对比例1-5的小鼠墨汁推进率甚至低于模型对照组。
2、模型对照组的小鼠首便时间大于正常对照组(P<0.01),而粪便的 粒数小于正常对照组(P<0.01),说明便秘模型成立。治疗组的首便时间比 便秘模型对照组的缩短,其中实施例1-4还显著低于对比例1-5(P<0.01), 对比例1-5显著低于模型组(P<0.01)。治疗组中实施例1-4的粪便量均大 于便秘模型对照组和正常组。而治疗组中对比例1-5的粪便量均低于对照 组(P<0.01),与模型组相当。以上结果表明,治疗组中实施例1-4的受试 样品能明显缩短便秘小鼠的首便时间,并增加其所排便数量及重量,而 对比例1-5组效果则不明显。
3、另外,各组小鼠在实验前和实验结束时的体重与阴性对照组及模 型对照组比较,均无显著性差异,表明该样品对小鼠体重增长无显著影 响。
综上表明,火麻仁低聚肽具有良好的润肠通便效果。将功能物质加 入饲料中给药的方式符合动物生理过程,但灌胃给药的方法剂量控制更 加精确,且不受动物摄食量和其他因素的影响。因此,本实验的结果具 有更高的可信度。本试验采用的主要方法来自于《保健食品功能检测规 范》(2003版)中“通便功能检验方法”。在实验过程中,我们发现染成红颜色的粪便相对于染成黑色的粪便而言更容易观察,有助于精确确定首 粒粪便的排出时间。
表3
以上所述,仅为本发明较佳的具体实施方式,但本发明的保护范围 并不局限于此,任何熟悉本技术领域的技术人员在本发明揭露的技术范 围内,可轻易想到的变化或替换,都应涵盖在本发明的保护范围之内。
Claims (2)
1.一种火麻仁低聚肽在制备抗氧化药品中的应用,其特征在于,所述火麻仁低聚肽分子中包含分子含量为20~30%谷氨酸、30~40%组氨酸和10~30%精氨酸,所述火麻仁低聚肽分子量为461Da;
所述火麻仁低聚肽的制备方法包括以下步骤:
将火麻仁的原料经浸泡、脱糖和变性后,进行酶解,灭酶后将获得的酶解液经过滤,取滤液浓缩,干燥即可得所述火麻仁低聚肽;
所述浸泡步骤中按料液比1:(10~20)投料加水;
所述脱糖步骤采用淀粉酶水解处理1~2h;
所述酶解步骤中采用碱性蛋白酶处理,温度为50~60℃,pH=8.5~9.0,处理时间为5~6h。
2.一种火麻仁低聚肽在制备改善便秘的药品中的应用,其特征在于,所述火麻仁低聚肽分子中包含分子含量为20~30%谷氨酸、30~40%组氨酸和10~30%精氨酸,所述火麻仁低聚肽分子量为461Da;
所述火麻仁低聚肽的制备方法包括以下步骤:
将火麻仁的原料经浸泡、脱糖和变性后,进行酶解,灭酶后将获得的酶解液经过滤,取滤液浓缩,干燥即可得所述火麻仁低聚肽;
所述浸泡步骤中按料液比1:(10~20)投料加水;
所述脱糖步骤采用淀粉酶水解处理1~2h;
所述酶解步骤中采用碱性蛋白酶处理,温度为50~60℃,pH=8.5~9.0,处理时间为5~6h。
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