CN104257767B - 一种藏边大黄有效成分的提取方法及其用途 - Google Patents
一种藏边大黄有效成分的提取方法及其用途 Download PDFInfo
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Abstract
本发明公开了一种藏边大黄有效成分的提取方法及其用途。包括清洗、筛选、打浆、酶解、过滤、浸提、均质、干燥、筛分、灭菌等步骤。本发明采用酶法提取藏边大黄中的多种有效成份,更好地保留其生物活性,使藏边大黄抑真菌、收敛、止泻、降低血脂和轻度致泻等作用更有效地、充分地保留;本发明酶法提取藏边大黄中的各种有效成分使有效成分更多地提取出来,减少高温提取对功能性成分的破坏,更好地发挥藏边大黄的有益功效。本发明提取方法采用复合酶法提取藏边大黄中的有效成分,缩短了提取时间,提高了藏边大黄提取物的得率。
Description
技术领域
本发明属于植物有效成分提取方法及其提取物的用途技术领域,特别是涉及一种藏边大黄有效成分的提取方法及其用途。
背景技术
藏边大黄系蓼科大黄属波叶组植物藏边大黄RheumemodiWall的根与根茎,味酸、苦,性寒;主要分布在西藏中东部、青海、四川、云南等省区,多为野生,西藏部分地区有种植;藏、蒙医用于治疗胃肠炎、肾炎,外用止血、治疮、消炎、愈伤口等。藏边大黄的化学成分主要有蒽醌类、二苯乙烯类和鞣质类化合物。其中,二苯乙烯类化合物含量较高,特别是Piceatannol-4′-O-β-D-吡喃葡萄糖苷(藏黄苷A,PGP)的含量平均达7.5%,具有抗氧化、改善脑功能、改善心血管、降血脂和降血糖等作用。
藏边大黄含有丰富的蒽醌类化合物,如大黄素、大黄素甲醚和大黄酚,其具有明显的抗菌消炎、免疫调节、抗衰老以及抗肿瘤等作用。
藏边大黄中含有丰富的芪类化合物,目前有文献报道其具有较好的降血糖作用,已成为研究热点。芪类(stilbene)是具有二苯乙烯母核或其聚合物的一类物质总称,它具有广泛的生物活性,除早已知道的抗菌作用,近年来又发现有降血脂,降血糖,保肝、扩张毛细血管改善微循环、扩张冠状血管及降压等作用。藏边大黄中含有较多的鞣质,有多种药理功效,能改善肾功能、抗病毒、抗凝血,能治疗精神病、治疗胃出血等疾病。
藏边大黄根及根茎含总蒽醌5.94%,其中以大黄素、芦荟大黄素及大黄酚为甙元的结合型蒽醌为5.66%,游离型仅为0.28%,还含上大黄甙及多量鞣质。藏医用于治疗胃肠炎症,外用止血消炎。据卫生部药检所动物实验表明,本品无下泄作用。
藏边大黄与大黄(又称正品大黄、生品大黄或掌叶组大黄,是指药典收载的植物来源为掌叶大黄、药用大黄和唐古特大黄的干燥根茎和根)具有明显不同药性,后者主要药效成分为蒽醌类化合物,包括有强烈致泄作用的双蒽醌,如有番泻苷A、B、C、D、E、F等;前者含有土大黄音、波叶大黄多糖、大黄酚、大黄素甲醚与微量的大黄素,并含少量的芦荟大黄素,有抑真菌、收敛、止泻、降低血脂和轻度致泻等作用,其中所含的波叶大黄多糖有抗癌、抗辐射、抗炎、抗凝血、抗血栓、强心、降低血压、降低血脂、降低血糖、增强机体免疫功能和核酸、蛋白质合成及抗衰老的作用,其与正品大黄显著不同的是所含蒽醌中没有强烈致泄作用的双蒽醌类。
目前,藏边大黄主要用于保健品及一些医药原材料中。对于藏边大黄的加工方法均存在有不同程度的问题,如中国专利申请201110124226.9一种藏边大黄提取物及其制备方法提供了一种蓼科大黄属波叶组植物藏边大黄的提取物,是藏边大黄依次经乙醇渗漉浸提与乙醇洗脱的提取物。此方法的提取时间长,提取率低,有效成分比例及吸收降低等问题。
发明内容
本发明根据现有技术的不足公开了一种藏边大黄有效成分的提取方法及其用途。本发明的目的是提供一种资源高效利用,提取率高,提取时间短,提取成分易被人体吸收,采用生物酶解方法提取藏边大黄中有效成分的方法及其提取物的应用。
本发明通过以下技术方案实现:
藏边大黄有效成分的提取方法,依次包括以下步骤:
清洗:筛选无损坏的新鲜藏边大黄,用水清洗干净;
打浆:将清洗干净的藏边大黄与水按1:1的重量比例混合打浆,使藏边大黄的颗粒大小均匀;
酶解:
(1)在上述打浆得到的原料中加入65IU/100ml、2万U/g的纤维素酶,温度控制在40~55℃,PH控制在3.5~6.5,酶解时间1~2h;
(2)将上述(1)中的酶解液制成16~17°Bé的淀粉浆,调pH至6~7,加入按原料重量计0.2%CaCl2,将α-淀粉酶调浆后加入淀粉浆中,每克原料用酶6u,搅拌,60~70℃液化20min;
(3)将上述(2)中的酶解液冷却至40~50℃左右,加入120u/g复合酶,用量在0.2~0.8g/kg,PH控制在5.0~8.0,酶解1~2h;
过滤:将酶解后的酶解液过滤;
浸提:将过滤后的滤渣用纯水浸提;
均质:将浸提液用均质机均匀混合;
干燥:将过滤后的酶解溶液和浸提得到的浸提液通过高速离心喷雾干燥机干燥;
筛分:将干燥后的藏边大黄提取物通过80-120目的筛孔进行分筛,使颗粒大小均匀;
灭菌:将筛分后的藏边大黄提取物粉末进行灭菌,形成成品。
所述过滤是将酶解后的酶解液经过硅藻土过滤器过滤,过滤条件包括过滤面积15.9㎡,理论流量以白酒为单位13-15T/H。
所述浸提是将过滤后的滤渣进行浸提处理,在原料中加入体积比为1-1.5倍的纯水,混合搅拌20-40分钟,然后通过离心机离心,得到浸提溶液,重复两次,收集浸提酶解溶液;将浸提后的干渣部分加入体积比为4-6倍、浓度为50%的乙醇溶液浸提,浸提时控制温度50-70℃,时间1-2小时,不断搅拌,干渣浸提2次,离心收集浸提液。
所述干燥是将过滤后的酶解溶液通过高速离心喷雾干燥机进行干燥,干燥条件:入口温度140-350℃,出口温度80-90℃,水份蒸发量25kg/h,雾化器转速25000rmp,得到藏边大黄提取物;另外由干渣浸提得到的浸提液,通过喷雾干燥进行干燥,回收乙醇溶液,得到的乙醇浸提物。
本发明上述提取方法中利用的各种酶:纤维素酶、α-淀粉酶、复合酶均有市售。
本发明上述藏边大黄有效成分提取方法得到的提取物可用于医药、食品、日化产品的制备,各制成品可充分利用提取物具有的抑真菌、收敛、止泻、降低血脂,还有抗癌、抗辐射、抗炎、抗凝血、抗血栓、强心、降低血压、降低血脂、降低血糖、增强机体免疫功能和核酸、蛋白质合成及抗衰老的作用。
本发明有益性:本发明采用酶法提取藏边大黄中的多种有效成份,更好地保留其生物活性,使藏边大黄抑真菌、收敛、止泻、降低血脂和轻度致泻等作用更有效地、充分地保留;本发明酶法提取藏边大黄中的各种有效成分,使有效成分更多地提取出来,减少高温提取对功能性成分的破坏,更好地发挥藏边大黄的有益功效。本发明提取方法采用复合酶法提取藏边大黄中的有效成分,缩短了提取时间,提高了藏边大黄提取物的得率。
附图说明
图1是本发明提取工艺流程示意图;
图2是本发明检测蒽醌类化合物测定的标准曲线;
图3是本发明检测例二苯乙烯类化合物测定的标准曲线;
图4是本发明鞣质类化合物测定的标准曲线。
具体实施方式
下面通过实施例对本发明进行进一步的描述,实施例只用于对本发明进行进一步的说明,但不能理解为对本发明保护范围的限制,本领域的技术人员可以根据上述本发明的内容作出一些非本质的改进和调整属于本发明保护的范围。
实施例1
藏边大黄有效成分的提取方法
本方法从资源高效利用,提取率高,提取时间短,提取成分易被人体吸收等方面考虑,采用生物酶解的方法提取藏边大黄中的有效成分。
结合图1。
1、清洗:筛选无损坏的新鲜藏边大黄,清洗干净;
2、打浆:将清洗干净的藏边大黄进行打浆,使藏边大黄的颗粒大小均匀,利于酶解提取;
3、酶解:
(1)在原料中加入65IU/100ml 2万U/g的纤维素酶,温度控制在40~55℃,PH控制在3.5~6.5,酶解时间1~2h;
(2)将上述(1)中的酶解液制成16~17°Bé的淀粉浆,调pH至6~7,加入按原料重量计0.2%CaCl2,将α-淀粉酶调浆后加入淀粉浆中,每克原料用酶6u,搅拌,60~70℃液化20min左右;
(3)将上述(2)中的酶解液冷却至40~50℃左右,加入120u/g复合酶,用量在0.2~0.8g/kg,PH控制在5.0~8.0,酶解1~2h;
4、过滤:将酶解后的酶解液经过硅藻土过滤器过滤,过滤条件:过滤面积15.9㎡,理论流量以白酒为单位13-15T/H;
5、浸提:将过滤后的滤渣进行浸提处理,在原料中加入体积比为1-1.5倍的纯水溶液,混合搅拌20-40分钟,然后通过离心机离心,分离水解溶液,重复两次,收集酶解溶液。再收集干渣部分,然后加入4-6倍浓度为50%的乙醇溶液浸提干渣部分,浸提时控制温度50-70℃,搅拌浸提时间1-2小时,反复将干渣浸提2次,以便更多的提出藏边大黄中的有效成分,最后离心收集浸提液;
6、均质:将步骤4中滤液和步骤5中浸提液通过均质机,使物料能更均匀的相互混合;
7、干燥:将过滤后的酶解溶液通过高速离心喷雾干燥机进行干燥,干燥条件:入口温度140-350℃,出口温度80-90℃,水份蒸发量25kg/h,雾化器转速25000rmp,得到藏边大黄提取物;另外由干渣浸提得到的浸提液,也通过喷雾干燥进行干燥,回收乙醇溶液,得到的乙醇浸提物用于其他食品的开发;
8、筛分:将干燥后的藏边大黄提取物通过80-120目的筛孔进行分筛,使颗粒大小均匀;
9、灭菌成品:将筛分后的藏边大黄提取物粉末进行灭菌,形成成品。
实施例2
提取方法对比实验
将物料平均分成6组,分别为①、②、③、④、⑤、⑥,每组10kg,①、②、③组以本发明方法提取,具体实施方法按实施例1试验步骤进行;④、⑤、⑥为一般提取法,以物料:水=1:8的比例进行提取,提取8小时,过滤,干燥,称量。
一般水提法和本发明酶法提取藏边大黄有效成分的得率比较,采用酶法提取藏边大黄比采用一般提取法得到的得率要高,从提取的时间上,酶法也大大缩短了提取的时间,所以,采用本发明方法提取的藏边大黄在时间和提取得率上都具有明显的改进。
实施例3
检测实验
将本发明所得到提取物进行蒽醌类、二苯乙烯类和鞣质类化合物的测定。
一、蒽醌类化合物的测定
蒽醌类化合物主要检测化合物中的总蒽醌含量,主要检测方法如下:
1.1仪器与药品
紫外分光光度计、电子分析天平、本发明所得提取物M1、一般水提法所得提取物M2、1,8-二羟基蒽醌、醋酸镁、甲醇等,所用试剂均为分析纯。
1.2实验方法
1.2.1显色剂的配制
称取0.4g醋酸镁,用甲醇溶解配制成0.6%的醋酸镁甲醇溶液,充分摇匀待用。
1.2.2标准曲线的制备
精密称取1,8-二羟基蒽醌10.7mg,用甲醇溶解并定容为25ml的标准液。精密吸取标准液20、40、80、160、200、300、400μL于10mL容量瓶中,加入0.6%醋酸镁甲醇溶液定容至10mL,充分摇匀后与510nm处测定吸收度。以0.6%醋酸镁甲醇液做空白对照,测定结果见表2。
表2:标准曲线的制备
标准液μL | 0 | 20 | 40 | 80 | 160 | 200 | 300 | 400 |
浓度(mg/L) | 0 | 0.748 | 1.6549 | 3.282 | 6.317 | 8.631 | 12.526 | 16.281 |
吸收度A | 0 | 0.032 | 0.071 | 0.144 | 0.293 | 0.371 | 0.560 | 0.700 |
以浓度为纵坐标,吸光度为横坐标制作得到图2。
1.2.3总蒽醌含量的测定
精密称取本发明提取物(M1)以及一般水提法提取物(M2)50mg与烧瓶中,同时加30mL氯仿和15mL2.5mol/L硫酸,沸水浴回流水解2小时,分出氯仿层。称取适量无水Na2SO4加入氯仿中脱水后回收氯仿,惨渣加甲醇溶解并定容为10mL。精密吸取1mL与10mL容量瓶中,加0.6%醋酸镁甲醇溶液并定容,摇匀后与510nm处测定吸收度,将M1稀释3倍,得到吸光度A1为0.803;将M2稀释3倍,测定吸光度A2为0.657,。测定值代入回归方程,计算含量得:
C1=18.47×3=55.41mg/L,得到本发明提取物中总蒽醌含量为5.6%;
C2=15.28×2=30.59mg/L,得到一般水提法提取物总蒽醌含量为3.06%。二、二苯乙烯类化合物的测定
二苯乙烯类化合物主要测定二苯乙烯苷的含量,主要检测方法如下:
2.1仪器和材料
高效液相色谱、电子分析天平、本发明所得提取物M1、一般水提法所得提取物M2、2,3,5,4'-四羟基二苯乙烯-2-O-β-D-葡萄糖苷对照品、乙腈为分析纯、乙醇。
2.2二苯乙烯苷含量测定
2.2.1色谱条件
C18色谱柱,流动相乙腈-水(21:79),检测波长320nm,流速1.0mL/min,柱温30℃。
2.2.2标准曲线的制备
精密称取2.32mg2,3,5,4'-四羟基二苯乙烯-2-O-β-D-葡萄糖苷对照品置25mL容量瓶中,用50%乙醇溶解并定容,摇匀,用0.45μm微孔滤膜滤过,得对照品溶液,精密吸取对照品溶液0,2.5,5.0,7.5,10.0,12.5μL进样,测得峰面积,计算色谱峰面积(Y)与样品量(X)如下表3:
样品量(X) | 2.5μL | 5.0μL | 7.5μL | 10.0μL | 12.5μL |
峰面积(Y) | 45163 | 93675 | 131188 | 180708 | 217212 |
测得的标准曲线及回归方程式如图3:图中横坐标是样品量,纵坐标是峰面积。
2.2.3样品含量的测定
精密称取M1、M25mg,置于25mL容量瓶中,用50%乙醇溶解并定容,摇匀,0.45μm微孔滤膜滤过,分别进样10μL,测定峰面积。计算含量:
M1峰面积为87592,M2峰面积为53249;分别得到M1和M2的含量分别为48.33%,28.41%。
三、鞣质类化合物的测定
鞣质类化合物主要测定鞣质的含量,主要检测方法如下:
3.1仪器及药品
紫外可见分光光度计、没食子酸、磷钼钨酸、本发明所得提取物M1、一般水提法所得提取物M2、干酪素、碳酸钠。
3.2对照品溶液的制备
精密称取没食子酸对照品50mg,置100ml棕色量瓶中,加水溶液并稀释至刻度,精密量取5ml,置50ml棕色量瓶中,用水稀释至刻度,摇匀,即得(每1ml中含没食子酸0.05mg)。3.3标准曲线的制备精密量取对照品溶液0.5ml、1.0ml、2.0ml、3.0ml、4.0ml、5.0ml,分别置25ml棕色量瓶中,各加入磷钼钨酸试液1ml,分别加水11.5ml、11ml、10ml、9ml、8ml、7ml,用29%碳酸钠溶液稀释至刻度,摇匀,放置30分钟以相应的试剂为空白,照紫外可见分光光度法,在760nm的波长处测定吸光度,以吸光度为纵坐标,浓度为横坐标,绘制标准曲线表4。
浓度mg/L | 1 | 2 | 4 | 6 | 8 | 10 |
吸光度A | 0.0513 | 0.1653 | 0.4633 | 0.6913 | 0.9893 | 1.1573 |
得出标准曲线以及线性回归方程如图4,图中横坐标是浓度mg/L,纵坐标是吸光度A。
3.4供试品溶液的制备
取M1、M2,精密称定2g,置250ml棕色量瓶中,加水150ml,放置过夜,超声处理10分钟,放冷,用水稀释至刻度,摇匀,静置(使固体物沉淀),滤过,弃去初滤液50ml,精密量取续滤液20ml,置100ml棕色量瓶中,用水稀释至刻度,摇匀,即得。
3.5测定法
3.5.1总酚的测定
精密量取供试品溶液2ml,置25ml棕色量瓶中,照标准曲线的制备项下的方法,自“加入磷钼钨酸试液1ml”起,加水10ml,依法测定吸光度,从标准曲线中读出供试品溶液中没食子酸的量(mg),计算,即得。
A1=0.681,A2=0.355;
C1=5.94mg/L,C2=3.35mg/L。
3.5.2不被吸附的多酚的测定
精密量取供试品溶液25ml,加至已盛有干酪素0.6g的100ml具塞锥形瓶中,密塞,置30℃水浴中保温1小时,时时振摇,取出,放冷,摇匀,滤过,弃去初滤液,精密量取续滤液2ml,置25ml棕色量瓶中,照标准曲线的制备项下的方法,自“加入磷钼钨酸试液1ml”,加水10ml,依法测定吸光度,从标准曲线中读出供试品溶液中没食子酸的量(mg),计算,即得。
A3=0.358,A4=0.154;
C3=3.73mg/L,C4=1.75mg/L。
按下式计算鞣质的含量:鞣质含量=总酚量-不被吸附的多酚量
测定得M1、M2鞣质的含量为:
M1=C1-C3=2.21mg/L;M2=C2-C4=1.6mg/L
由上述一系列得到的数据对比,可以看出,本发明提取物中含有蒽醌类、二苯乙烯类和鞣质类化合物等有效物质,同时,上述数据也证明了采用本发明所得到的提取物中的有效物质含量较一般水提法提取出来的有效物质含量高,因此,本发明是可行的。
Claims (3)
1.一种藏边大黄有效成分的提取方法,其特征是依次包括以下步骤:
清洗:筛选无损坏的新鲜藏边大黄,用水清洗干净;
打浆:将清洗干净的藏边大黄与水按1:1的重量比例混合打浆,使藏边大黄的颗粒大小均匀;
酶解:
(1)在上述打浆得到的原料中按每100ml加入65IU、规格为2万U/g的纤维素酶,温度控制在40~55℃,PH控制在3.5~6.5,酶解时间1~2h;
(2)将上述(1)中的酶解液制成16~17°Bé的淀粉浆,调pH至6~7,加入按原料重量计0.2%CaCl2,将α-淀粉酶调浆后加入淀粉浆中,每克原料用酶6u,搅拌,60~70℃液化20min;
(3)将上述(2)中的酶解液冷却至40~50℃,加入120u/g复合酶,用量在0.2~0.8g/kg,PH控制在5.0~8.0,酶解1~2h;
过滤:将酶解后的酶解液过滤;
浸提:将过滤后的滤渣用纯水浸提;
均质:将浸提液用均质机均匀混合;
干燥:将过滤后的酶解溶液和浸提得到的浸提液通过高速离心喷雾干燥机干燥;
筛分:将干燥后的藏边大黄提取物通过80-120目的筛孔进行分筛,使颗粒大小均匀;
灭菌:将筛分后的藏边大黄提取物粉末进行灭菌,形成成品。
2.根据权利要求1所述的藏边大黄有效成分的提取方法,其特征是:所述过滤是将酶解后的酶解液经过硅藻土过滤器过滤,过滤条件为过滤面积15.9m2,流量以白酒为单位13-15吨/小时。
3.根据权利要求1所述的藏边大黄有效成分的提取方法,其特征是:所述浸提是将过滤后的滤渣进行浸提处理,在原料中加入体积比为1-1.5倍的纯水,混合搅拌20-40分钟,然后通过离心机离心,得到浸提溶液,重复两次,收集浸提酶解溶液;将浸提后的干渣部分加入体积比为4-6倍、浓度为50%的乙醇溶液浸提,浸提时控制温度50-70℃,时间1-2小时,不断搅拌,干渣浸提2次,离心收集浸提液。
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