CN105769656B - 一种藏药材组合萃取物及其用途 - Google Patents
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Abstract
本发明公开了一种藏药材组合萃取物及其用途，该藏药材组合萃取物按重量份比，包括30‑50份藏药材黑木耳萃取液、10‑30份藏药材灵芝萃取液以及10‑30份藏药材天麻萃取液，该藏药材组合萃取物可作为抗氧化成分添加于皮肤保养品、养生健康品中。
Description
技术领域
本发明涉及一种以藏药材黑木耳、灵芝、天麻为主要成分的藏药材组合萃取物及其用途。
背景技术
青藏高原特殊的地理位置、生态环境和气候条件造就了品种独特且丰富的动植物资源。西藏黑木耳、灵芝、天麻具有独特的理化特性和药效作用，人类却对藏药材缺乏系统的深入研究。藏药原料产地经济落后，交通不便，且缺乏先进技术的支持，所以藏药材多以原材料的形式对外销售，无法创造更大的经济效益。如何把藏药材的研究与开发从简单的直接粗加工向精加工转化，提高其质量和药效；如何在现有工艺基础上大量引入高科技技术，开发新剂型、新产品以适应临床和市场需求是当前最亟待解决的问题。
黑木耳(Auricularia auricular)来源于真菌门，担子菌纲，木耳目，其子实体除含有丰富的蛋白质、维生素、钙、磷、铜等物质外，还含有大量的具有生物活性的多糖，是一种重要的药食兼用真菌。大量研究表明黑木耳作为生物应答效应物(Biological ResponseModifier，BRM)具有多种生理功能，而这些重要的生理功能与其多糖组分密切相关。已证实黑木耳多糖具有降血脂、抗凝血、抑制肿瘤、降血脂、降血糖等生物活性，现已成为食品、医疗、保健等领域的研究热点。
灵芝，又名灵芝草、神芝、芝草、仙草、瑞草，为多孔菌科真菌赤芝或紫芝的干燥子实体，在我国许多省区均有分布，主要生长在栎树或其他种类的阔叶树木桩上，野生居多，两者均有人工栽培。野生品于夏、秋季采收，栽培品多于子实体成熟孢子散出后采收，药材外形呈伞状。在《本草纲目》中被列为一种不可多得的药食两用珍品。据《中国药典》2010年版记载，其性平味甘，归心、肺、肝、肾经。补气安神、止咳平喘，用于心神不宁，失眠心悸，肺虚咳喘、虚劳短气、不思饮食。现代研究证明：灵芝含有多种生物活性成分，如多糖类、三萜类、甾醇类、核苷类、生物碱类等[3]。其中，灵芝多糖(GLP)是从灵芝中提取的主要功效成分，与灵芝的多种药理活性有关，可以提高机体免疫能力、抗肿瘤、抗衰老、抗炎、调节血脂、提高机体耐缺氧能力等。
天麻，原名赤箭，《神农本草经》中作为上品，是我国四大名贵中药(冬虫夏草、人参、鹿茸)之一，具有平肝熄风、祛风止痛之功效，广泛用于头痛、眩晕、肢体麻木、半身不遂等。天麻主要活性成分为天麻素、氨基酸和总黄酮等。天麻素具有滋肾补脑、提神益气、调节心脑血管功能、镇惊、抗癫痫、镇静、安眠和增强机体免疫能力等功效。氨基酸是人体必需的营养成分，并且具有特殊的药理作用。黄酮类化合物具有抗炎和抗变态反应、提高机体免疫力等功效。
发明内容
本发明的目的在于提供一种藏药材组合萃取物。
本发明提供的技术方案如下：
一种藏药材组合萃取物，其特征在于：按重量份比，包括30-50份藏药材黑木耳萃取液、10-30份藏药材灵芝萃取液以及10-30份藏药材天麻萃取液。
其中，所述的藏药材黑木耳萃取液、藏药材灵芝萃取液以及藏药材天麻萃取液都为乙醇提取液。
所述的乙醇为1％-100％乙醇。
作为本发明的优选，所述的乙醇为50％-90％乙醇。
作为本发明的进一步优选，所述的乙醇为70％-85％乙醇。
本发明的组合萃取物的制备方法优选如下：
三种藏药材黑木耳、灵芝、天麻分别经过干燥，粉碎，过筛后，称取三种藏药材，分别加入乙醇，超声细胞破碎提取2-3次，合并超声提取液，用乙醇定容即为各藏药材的萃取液；然后按重量比，将30-50份藏药材黑木耳萃取液、10-30份藏药材灵芝萃取液以及10-30份藏药材天麻萃取液混合即为组合萃取液。
其中，每种藏药材加入的乙醇总量为固液比(W/V)1：50-200。
其中，所述的过筛优选为过40目筛。
其中，所述的定容优选为用乙醇定容至0.5-2g藏药材黑木耳、灵芝或天麻/100ml乙醇。
本发明的组合萃取液可作为抗氧化成分添加于皮肤保养品、养生健康品中。
本发明提供了一种藏药材组合萃取物，它作为抗氧化成分，具有更佳的协同效应，能够比比单一的成分起到更佳的抗氧化效果。
具体实施方式
采用75％乙醇作为溶剂，超声细胞破碎仪对三种藏药材进行提取，抗氧化能力的测定使用分光光度计，应用DPPH、ABTS、脂质过氧化效能、细胞内活性氧(ROS)生成量评估实验进行测定。通过测定三种藏药材乙醇提取液及组合物对DPPH自由基的清除能力和FRAP值，表征其抗氧化能力。结果表明:组合物30％-50％体积分数的黑木耳、10％-30％体积分数的灵芝、10％-30％体积分数的天麻在10mg/mL的浓度下，对DPPH自由基的清除率超过70％，总抗氧化能力的FRAP值＞200，抗氧化能力较强。此藏药材组合萃取物可作为功效成分添加于皮肤保养品、养生健康品中，发挥抗氧化效能。
实施例1 提取液制备：
分别将三种藏药材黑木耳、灵芝、天麻经过干燥，粉碎，过40目筛，粉末经恒温(60℃)干燥24h后，精密称取三种藏药材各0.5g，分别置50mL的三角瓶中，每个三角瓶分三次加入75％的乙醇(20、15、15mL)，超声细胞破碎提取3次，每次3min(每次辐射时间10s，超声功率350Hz，间隙10s，工作18次)，合并3次的超声提取液，用75％乙醇于容量瓶定容至50mL，即为各藏药材的萃取液(浓度为10mg/ml)。
藏药材组合萃取物则含有40％体积分数的黑木耳萃取液、30％体积分数的灵芝萃取液、30％体积分数的天麻萃取液。
实施例2 抗氧化活性的测定:
◆清除自由基能力的测定(DPPH值测定)
取各萃取液，稀释3倍后移液器取100μL，加入2mL 0.2mmol/L DPPH的乙醇溶液，混合均匀，黑暗下室温避光反应20min，在517nm处测定吸光度，同时以2mL DPPH+100μl磷酸盐缓冲溶液混合后的吸光度为空白组。结果显示各个萃取液对DPPH均呈现不同程度的抑制作用，而藏药材组合萃取物对DPPH自由基的清除率超过70％，具有较强的抗氧化效能。
◆清除自由基能力的测定(ABTS)
将7mmol/L ABTS+和2.45mmol/L的过硫酸钾(终浓度)混合，在室温避光条件下静置过夜，将生成的ABTS+溶液用水稀释，使其在30℃，734nm波长下的吸光度为0.7±0.02，即得到ABTS+工作液。取三种藏药材萃取液及组合萃取液各1.5mL加入试管中，再分别加入1.5mL的ABTS+溶液，空白管用蒸馏水代替样品溶液，对照管用ABTS+工作液，并做重复试验，室温避光放置6min，于波长734nm下测定其吸光度。每份样品平行操作3次，计算公式为：清除率(％)＝[A空白-(A样品-A对照)]/A空白×100
结果显示藏药材组合萃取物对ABTS自由基的清除率超过50％，具有较强的抗氧化效能。
◆脂质过氧化效能评估
亚麻油酸样品混合液的配置步骤如下：
1)将样品及标准品分别配置成浓度为4、2、1、0.5、0.25mg/ml的乙醇溶液。
2)各取样品或标准品0.5ml。而对照组为等量的乙醇0.5ml(即样品浓度为0)，再依序分别加入0.02M的亚麻油酸2.5ml及0.05M磷酸盐缓冲液(PH7.0)2ml。
3)放入有盖的指形瓶中混合均匀后，置于37℃的恒温培养箱内，每隔24h取出做测试。
实验步骤：
1)取上述的混合液5μl，再依序加入75％的乙醇溶液235μl，30％的硫氰酸铵5μl，及0.02M的溶在3.5％HCL的氯化亚铁四水合物5μl。
2)于96孔盘中震荡均匀3min后，以ELISA测其在500nm波长下的吸光值。
当置于37℃恒温培养箱时间越长，脂质过氧化程度就越高，氢过氧化物的生成量也越多，呈色也跟着加深。不过加入抗氧化剂时会抑制氢过氧化物的产生，使呈色速度降低，吸光值也越低，表示加入抗氧化剂的抗氧化能力越强，借此比较抗氧化剂的抗氧化能力；而抗氧化能力越高表示油脂越安定，加入样品的抗氧化性越强。
亚麻油酸(PH7.0)的配制方法：于50ml的0.02M磷酸盐缓冲液(PH7.0)中加入0.2804g的亚油酸及0.2804的吐温20，以磁石搅拌成乳状液即可，此乳状液必须新鲜配制。
结果显示藏药材组合萃取物抑制脂质过氧化物率超过70％，具有较强的抗氧化效能。
◆抑制细胞内活性氧(ROS)生成量的评估
将1X105cell/ml的细胞数培养在96孔盘中，并在37℃、5％CO2培养箱中生长24小时以上，再加入不同浓度萃取液。达反应时间后，移除上清液，以PBS清洗，加入含有0.1mM H2O2的新鲜培养液作用一小时，接着移除培养液，加入含有10μM2',7'-二氯荧光黄双乙酸盐(DCFH2DA)的新鲜培养液避光作用一小时后，移除培养液，加入定量PBS，于激发光502nm/散色光524nm下测其吸光值。
结果显示藏药材组合萃取物对细胞内活性氧(ROS)生成量抑制程度较高，具有较强的抗氧化效能。
Claims (8)
1.一种藏药材组合萃取物的制备方法，所述的藏药材组合萃取物，按重量份比，包括30-50份藏药材黑木耳萃取液、10-30份藏药材灵芝萃取液以及10-30份藏药材天麻萃取液；所述的藏药材黑木耳萃取液、藏药材灵芝萃取液以及藏药材天麻萃取液都为乙醇提取液；方法包括如下步骤：
三种藏药材黑木耳、灵芝、天麻分别经过干燥，粉碎，过筛后，称取三种藏药材，分别加入乙醇，超声细胞破碎提取2-3次，合并超声提取液，用乙醇定容即为各藏药材的萃取液；然后按重量比，将30-50份藏药材黑木耳萃取液、10-30份藏药材灵芝萃取液以及10-30份藏药材天麻萃取液混合即为组合萃取液。
2.如权利要求1所述的一种藏药材组合萃取物的制备方法，其特征在于：所述的乙醇为1％-100％乙醇。
3.如权利要求1所述的一种藏药材组合萃取物的制备方法，其特征在于：所述的乙醇为50％-90％乙醇。
4.如权利要求1所述的一种藏药材组合萃取物的制备方法，其特征在于：所述的乙醇为70％-85％乙醇。
5.如权利要求1所述的一种藏药材组合萃取物的制备方法，包括如下步骤：每种藏药材加入的乙醇总量为固液的质量体积比1：50-200。
6.如权利要求1所述的一种藏药材组合萃取物的制备方法，其特征在于：所述的过筛为过40目筛。
7.如权利要求1所述的一种藏药材组合萃取物的制备方法，其特征在于：所述的定容为用乙醇定容至0.5-2g藏药材黑木耳、灵芝或天麻/100ml乙醇。
8.一种藏药材组合萃取物的用途，其特征在于：作为抗氧化成分添加于皮肤保养品、养生健康品中，所述的藏药材组合萃取物，按重量份比，包括30-50份藏药材黑木耳萃取液、10-30份藏药材灵芝萃取液以及10-30份藏药材天麻萃取液。
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