CN110090246A - 一种食叶草提取物及其在制备拮抗内毒素的药物和保健品中的应用 - Google Patents
一种食叶草提取物及其在制备拮抗内毒素的药物和保健品中的应用 Download PDFInfo
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- CN110090246A CN110090246A CN201910322066.5A CN201910322066A CN110090246A CN 110090246 A CN110090246 A CN 110090246A CN 201910322066 A CN201910322066 A CN 201910322066A CN 110090246 A CN110090246 A CN 110090246A
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Abstract
本发明公开了一种食叶草提取物及其在制备拮抗内毒素的药物和保健品中的应用，本发明提供了食叶草提取物的制备方法，并且通过实验确定了提取的试剂和萃取试剂，且提供了食叶草提取物在制备拮抗内毒素中的应用，本发明的食叶草提取物用于拮抗内毒素的应用明显降低了使用名贵中药材缓解内毒素引起身体机能下降的成本，也使食叶草运用于食品领域提供了充足的实践证明，具有良好的市场应用前景。
Description
技术领域
本发明属于医药技术领域，具体涉及一种食叶草提取物及其在制备拮抗内毒素的药物和保健品中的应用。
背景技术
食叶草(Rumex hanus by.)，又名蛋白草、氨基酸草，属多年生宿根药食两用草本植物，蓼科酸模属，是将鲁梅克斯酸模与皱叶酸模生物杂交而获得的新品种，生长适应能力强，产量高，近10年来开始在我国试推广人工种植与开发，广泛分布于我国各个省区，产量高。食叶草营养极其丰富，富含多种氨基酸、膳食纤维、维生素、矿物质(钾、硒、锌、铁、钙)等营养成分。其中VC、胡萝卜素、大黄素、大黄酚(蒽醌类)、苷类、超氧化物歧化酶(SOD)含量较为丰富，蛋白质含量高达30％以上。因此食叶草能构建细胞组织、调节新陈代谢、增强免疫力、促进调节身体各种机能，同时能促进肠胃蠕动、保持神经肌肉兴奋，具有降血脂、清热泻火、解毒止血、抗衰老、抗氧化、抑菌等功效。
内毒素(Lipopolysaccharide，LPS)是由肠道革兰氏阴性菌降解产生的一类脂多糖。正常人体血液内的内毒素含量极少，但是由于各种不规律作息习惯或者应激反应使得人体肠道菌群紊乱，其中厚壁菌等有益菌的比例下降，杆菌和疣微菌比例升高，肠道菌群移位的同时会产生大量内毒素，进入门静脉和体循环后首先与内毒素受体——脂多糖结合蛋白(Lipopolysaccharide binding-protein，LBP)结合，形成LPS-LBP复合物，此复合物与肝Kupffer细胞表面的一种糖蛋白受体CD14结合，通过跨膜蛋白Toll样受体(Toll-likereceptors，TLRs)把胞外信号转导至胞内，下游炎性因子大量产生诱导了炎症反应，反过来又会影响蛋白质合成受阻、肝脏和血液循环异常、肠上皮细胞绒毛减少变短，淋巴细胞和分泌型免疫球蛋白A的数量质量下降，使得肠黏膜的免疫、机械以及生物屏障再次遭到破坏，持续恶性循环加剧肝脏损伤。
研究发现中国传统中药葛根、葛花、枳椇子、大黄、决明子等都可以通过降低肝组织和血液内毒素浓度、TLR4及CD14表达量达到缓和肝脏损伤。国外深入研究关于肠源性内毒素肝损伤的保护机制，研究指出从植物提取出的皂苷、多酚类、异黄酮类、抗坏血酸、锌以及海洋生物螺旋藻等对酒精性肝损伤都有显著保护作用。
目前，国内人们对食叶草的整体认识和研究开发利用还处于初级阶段，由于食叶草本身酸甜适口，蛋白含量丰富，生长适应能力强，将食叶草加工开发为家畜家禽的饲料是大多开发者的首选。研究开发人员将食叶草引种于大庆地区，并对食叶草最佳种植条件和做为饲料应具备的各项条件进行摸索探究，发现大庆地区适合引种食叶草作为奶牛饲料，这对于当今中国草场退化起到了缓解作用。再者食叶草中蛋白质含量极高，可以充分替代动物蛋白和谷物蛋白，避免了由动物谷物蛋白引起过敏反应的同时也提高了饮用品的适口性。除此之外，食叶草另外一个最大的优势是胆固醇含量极少，并且含有VC、芦丁、单宁以及蒽醌类等药用功效成分，所以食叶草在人类食品中的应用近年来逐渐被开发，食叶草植物奶、食叶草蛋白饮料、食叶草功能性茶叶、食叶草面食品、食叶草营养大米等相继出现，其相应的工艺已经得到一定程度的完善。
相比较国内来说，国外并未发现单独研究食叶草的文献，但是对于酸模属植物有着深入广泛的研究。酸模属植物的研究主要集中于化学物质的分离鉴定及其功效性能方面，酸模属植物的根部、叶部中都含有大量功能性成分——酚酸类、蒽醌类、黄烷醇类、黄酮类、酸模素、多糖等，利用乙醇水提取后再利用不同极性溶剂萃取，分离纯化后得到显著抑制疫霉属孢子移动、登革热2型病毒以及具有杀虫活性化合物，值得一提的是科学家们对酸模属植物中特有的酸模素研究得出酸模素通过抑制破骨细胞分化从而达到降低骨质疏松症的疗效，同时还对糖尿病有一定的改善作用。
酸模属植物作为中药性成分本身就具备一定的研究价值，其特有的解毒功效得到了一定程度的认可，对由四氯化碳引起的肝脏毒性，庆大霉素和重铬酸钾引起的肾脏毒性都有显著的保护作用，作用机理与其中的功能性成分抑制丙二醛的积累和增强抗氧化酶活性有关，而其具备的良好抗氧化活性也已经得到证实，酸模属植物乙酸乙酯提取物有较强的DPPH·清除能力，其IC50为20.74±0.6g/mL。酸模属植物作为一种天然植物，对生态环境也做出了一些贡献，酸模与其他植物间作可有效修复被铅锌等金属污染的土壤。之所以说国外对酸模属植物研究较为深入，是因为研究人员根据其本身含有的二苯乙烯类等强抗氧化物质将其与银制成纳米颗粒，对革兰氏阳性菌有着广谱抑制作用。还有研究从基因层面入手得到在金属胁迫下酸模属植物中编码酸性转化酶的基因转录水平显著高于无金属状态下的水平，对于酸模属植物改善重金属污染土地提供理论基础。从食品方面来说，酸模属植物酸甜适口且具有丰富的营养物质，通过离子束抑制钝叶酸模中有毒代谢产物-可溶性草酸盐，完全可以做成蔬菜沙拉成为婴幼儿辅食，这又对酸模属植物的研发提供了一种新思路。
虽然众多食叶草相关食品相继研发，但是关于食叶草这种杂交品种的深入理论研究匮乏，同时食叶草结合了酸模属中众多优良特点，含有大量活性成分，其抗氧化、抑菌等功能性都有初步研究。
发明内容
针对现有技术，本发明提供了一种食叶草提取物及其在制备拮抗内毒素的药物和保健品中的应用，本发明通过实验证明所述的食叶草提取物具有拮抗内毒素的功能。
为了达到解决上述技术问题的目的，本发明采用以下技术方案予以实现：
本发明提供了一种食叶草提取物，所述食叶草提取物的提取步骤为：
(1)将食叶草去根洗净，冷冻干燥后制得食叶草粉末；
(2)称取食叶草粉末，加入溶剂，超声碾碎提取并过滤；反复提取三次，合并滤液，旋转蒸发浓缩后冷冻干燥得到初级提取物；
(3)将初级提取物均匀分散在水中，使用有机溶剂萃取，得到食叶草提取物。
进一步的：所述步骤(2)中的溶剂为水、甲醇、丙酮、乙醇或乙酸乙酯中的至少一种。
进一步的：所述步骤(2)中的溶剂为体积比为70％的乙醇。
进一步的：所述步骤(2)中食叶草粉末和溶剂的料液比为1∶8～12。
进一步的：所述步骤(2)中超声条件为：温度为50～55℃，功率为100～120W，时间为30～40min，
进一步的：所述步骤(3)中的有机溶剂为石油醚、乙酸乙酯、正丁醇或水中的至少一种。
进一步的：所述步骤(3)中的有机溶剂为乙酸乙酯。
进一步的：所述步骤(3)中有机溶剂萃取次数为8～12次。
本发明还提供了所述的食叶草提取物在制备拮抗内毒素的药物和保健品中的应用。
进一步的：所述食叶草提取物的使用浓度范围为1-5mg/mL。
与现有技术相比，本发明的优点和积极效果是：本发明通过实验确定食叶草的活性成份对内毒素拮抗作用是本发明的创新之处，可以将所述食叶草提取物用于制备拮抗内毒素的药物或者保健品，能够明显降低使用名贵中药材缓解内毒素引起身体机能下降的成本，也使食叶草运用于食品领域提供了充足的理论支撑和实验证明，具有良好的市场应用前景。
附图说明
图1为内毒素标准曲线；
图2为食叶草的水提取物、甲醇提取物、丙酮提取物、70％乙醇提取物、乙酸乙酯提取物和多黏菌素B对内毒素的抑制率；
图3为30％乙醇提取物、50％乙醇提取物、70％乙醇提取物、95％乙醇提取物和多粘菌素B的对内毒素的抑制率；
图4为石油醚、乙酸乙酯、正丁醇萃取物、剩余的水相和多黏菌素B对内毒素的抑制率。
图5为食叶草不同极性萃取相羟自由基清除率。
图6为食叶草不同极性萃取相超氧阴离子自由基清除率。
图7为食叶草不同极性萃取相DPPH·清除率。
图8为食叶草不同极性萃取相还原能力。
具体实施方式
下面结合具体实施方式对本发明的技术方案进一步详细说明。
实施例1
本实施例中食叶草提取物的提取方法如下：
1、原材料预处理
食叶草去根清洗干净，擦拭干净，冷冻干燥后碾碎制得食叶草粉末，密封保存备用。
2、不同溶剂提取
称取一定量的所述食叶草粉末，按料液比1∶10加入乙酸乙酯，超声波50℃、100W提取30min，八层纱布过滤，再按照相同料液比加入乙酸乙酯相同条件下提取，反复提取3次，收集合并滤液后，滤纸过滤，收集合并滤液，50℃旋转蒸发浓缩后冷冻干燥得乙酸乙酯提取物。同理，把提取试剂换成丙酮、甲醇、30％乙醇、50％乙醇、70％乙醇、95％乙醇、水，即可得丙酮、甲醇、30％乙醇、50％乙醇、70％乙醇、95％乙醇、水提取物。将各种提取物配制成20mg/mL备用。
3、不同极性有机溶剂萃取
以所述几种食叶草提取物为样品进行拮抗内毒素活性测定，选取拮抗内毒素活性最好的提取物进行下一步萃取。将拮抗内毒素活性最好的食叶草提取物均匀的分散在水中，按照沸程为60-90℃石油醚、乙酸乙酯、正丁醇的顺序进行萃取分层，每次有机相反复萃取10次，收集各有机相及剩余水相。进行50℃旋转蒸发浓缩后冷冻干燥得石油醚、乙酸乙酯、正丁醇萃取物以及剩余水相。
4、拮抗内毒素活性检测
4.1内毒素标准溶液配制
标准曲线所采用的的内毒素浓度为0.01，0.025，0.05，0.1Eu/mL。稀释方法如下：取10Eu/瓶细菌内毒素标准品1瓶，加入1mL细菌内毒素检查用水，置旋涡混匀器上剧烈振摇15min，注意振摇过程中不得溅出，取上述已经配制好的10Eu/mL内毒素标准溶液0.2mL加入1.8mL内毒素检查用水，即配成1.0Eu/mL内毒素标准溶液，以1.0Eu/mL内毒素标准溶液为母液按照表1稀释成0.01，0.025，0.05，0.1Eu/mL的浓度梯度。每稀释一步均应在旋涡混匀器上剧烈振摇30s。稀释的内毒素溶液静置时间超过10min，用前需要在旋涡混匀器上剧烈振摇1min，配制好的内毒素标准溶液应在4h内用完。
表1内毒素标准溶液配制
4.2拮抗内毒素标准曲线
取2组除热源试管，按照表2的顺序加入试剂，并按照表中的反应条件顺序进行反应，每次做三组平行，重复3次试验，结果见图1。
表2拮抗内毒素标准曲线制作
4.3体外拮抗内毒素活性测定
取3组除热源试管，按照表3的顺序加入相应的试剂，并按照表中的反应条件顺序进行反应，每次做三组平行，重复3次试验。
表3体外拮抗内毒素活性测定
实验结果见图2，经过实验得出食叶草的水提取物、甲醇提取物、内酮提取物、70％乙醇提取物、乙酸乙酯提取物都具有拮抗内毒素的作用。抗生素多粘菌素B通常作为治疗杆菌感染的药物，在本次实验中多粘菌素B作为阳性对照也具有拮抗内毒素的活性。食叶草的水、甲醇、丙酮、70％乙醇、乙酸乙酯提取物以及多粘菌素B对内毒素的抑制率分别为76.44％、82.54％、59.29％、90.22％、81.68％、39.60％。其中食叶草提取物都显著好于阳性对照药物多粘菌素B的拮抗内毒素活性，而在食叶草提取物物中70％乙醇提取物拮抗内毒素活性显著好于其食叶草他试剂提取物，70％乙醇、甲醇和乙酸乙酯提取物的拮抗内毒素活性也高于其他溶剂提取物。
由图2的实验结果可得到食叶草70％乙醇提取物活性最好，所以选择不同比例的水-乙醇溶剂继续探究。实验结果如图3所示，以多粘菌素B作为阳性对照，其抑制率为44.84％，其他不同食叶草的水-乙醇提取物也都具有拮抗内毒素的活性并且都显著好于多粘菌素B。其30％、50％、70％、95％乙醇提取物的抑制率分别为33.02％、81.75％、88.94％、88.90％。70％乙醇与95％乙醇提取物无显著性差异，并且显著好于30％和50％乙醇提取物。因为部分水能有效渗入植物细胞间隙，这样能充分发挥出乙醇这种大极性的提取溶剂的优点，能最大效率的提取出有效活性成分。对于拮抗内毒素活性而言，效果最好的是体积比为70％、95％的乙醇提取物。
图4是食叶草70％乙醇提取物冷冻干燥粉末分散于水溶液后利用不同极性的有机试剂继续萃取的拮抗内毒素活性结果。由图4得石油醚、乙酸乙酯、正丁醇萃取物以及剩余的水相都有拮抗内毒素活性，其抑制率分别为78.34％、93.51％、88.40％、85.20％，都显著高于多粘菌素B抑制率43.00％。其中乙酸乙酯萃取物的活性最好，显著高于其它萃取物的活性。乙酸乙酯萃取物的活性最好，是因为食叶草中具有拮抗内毒素的活性成分大部分溶于中等极性的乙酸乙酯中。
实施例2：抗氧化活性测定
1、样品制备：
准确称取所述60.00g食叶草干燥粉末，按照料液比为1∶10(w∶v)加入蒸馏水，于200W下超声波辅助提取50min，八层纱布过滤，收集滤液；滤渣按上述步骤反复提取3次，合并滤液。将滤液在55℃条件下蒸发浓缩后蒸馏水定容至100mL，即为食叶草粗提取物。
将100mL食叶草粗提取物转移至分液漏斗中，按体积比1∶1加入三氯甲烷，摇匀，静置至完全分层，将水层与三氯甲烷层分离，连续萃取3次，将3次三氯甲烷层合并，在50℃条件下蒸发浓缩，得到食叶草的三氯甲烷相萃取物。采取同样方法将剩余水溶液依次用乙酸乙酯、正丁醇萃取，即可分别得到食叶草三氯甲烷、乙酸乙酯、正丁醇相萃取物，将最初的水相以及不同极性相萃取物进行浓缩，将浓缩液冷冻干燥后得不同极性萃取物粉末。
2、食叶草不同极性萃取相的抗氧化活性测定：
取5组试管，按照表4的顺序加入相应的试剂，并按照表中的反应条件顺序进行反应测定羟自由基清除能力，每次做三组平行，重复3次试验。
表4食叶草不同极性相萃取物的羟自由基清除能力测定
按照以下公式进行计算：
取4组试管，按照表5的顺序加入相应的试剂，并按照表中的反应条件顺序进行反应测定超氧阴离子自由基清除能力，每次做三组平行，重复3次试验。
表5食叶草不同极性相萃取物的超氧阴离子自由基清除能力测定
按照以下公式进行计算：
取4组试管，按照表6的顺序加入相应的试剂，并按照表中的反应条件顺序进行反应测定DPPH·清除能力，室温充分混匀，黑暗处静置30min，在517nm处测定吸光值每次做三组平行，重复3次试验。
表6食叶草不同极性相萃取物的DPPH·清除能力测定
按照以下公式进行计算：
取2组试管，按照表7的顺序加入相应的试剂，并按照表中的反应条件顺序进行反应测定还原能力，每次做三组平行，重复3次试验。
表7食叶草不同极性相萃取物的还原能力测定
3、食叶草不同极性萃取相的抗氧化活性结果与分析：
表8食叶草不同极性相抗氧化能力的半抑制质量浓度
抗氧化能力的半抑制质量浓度(IC50)是指当抗氧化能力达到半值时所需样品的质量浓度。由表8可知在清除羟自由基方面，乙酸乙酯相的IC50低于正丁醇相和三氯甲烷相，分别为4.41、5.85、7.53mg/mL；在清除超氧阴离子自由基方面，乙酸乙酯相的IC50低于正丁醇相和三氯甲烷相，分别为3.70、7.19、8.09mg/mL；在清除DPPH·方面，乙酸乙酯相的IC50低于正丁醇相和三氯甲烷相，分别为0.99、1.91、2.55mg/mL；在还原能力方面，乙酸乙酯相的IC50低于正丁醇相和三氯甲烷相，分别为0.92、1.84、2.95mg/mL。乙酸乙酯相的总体抗氧化能力强于其他两相，从纵向比较看，四种抗氧化能力评价中乙酸乙酯相和正丁醇相的还原能力强于其他三种活性氧自由基清除能力，其还原能力IC50分别为0.92、1.84mg/mL，而三氯甲烷相清除DPPH·能力较强，IC50为2.55mg/mL。
由图5可知在质量浓度为1-5mg/mL之间，食叶草乙酸乙酯相、正丁醇相和三氯甲烷相都有一定的羟自由基清除能力且随着样品质量浓度的不断增加，清除羟自由基的能力越强。在1-5mg/mL质量浓度之间，乙酸乙酯相的羟自由基清除能力强于正丁醇相强于三氯甲烷相，在样品质量浓度5mg/mL时，食叶草乙酸乙酯相的羟自由基清除率为56.03±2.82％，正丁醇相为47.23±2.68％，三氯甲烷相为34.09±2.22％。食叶草乙酸乙酯相中主要含有蒽醌类黄酮类等活性物质，都具有很强的抗氧化能力，所以相对于正丁醇和三氯甲烷两相，食叶草的乙酸乙酯相羟自由基清除能力较强。
由图6可知在质量浓度为1-5mg/mL之间，食叶草乙酸乙酯相、正丁醇相和三氯甲烷相都有一定的超氧阴离子自由基清除能力且随着样品质量浓度的不断增加，清除超氧阴离子自由基的能力越强。在1-5mg/mL质量浓度之间，乙酸乙酯相的超氧阴离子自由基清除能力强于正丁醇相强于三氯甲烷相，在样品质量浓度5mg/mL时，食叶草乙酸乙酯相的清除率为63.05±2.32％，正丁醇相为38.28±1.13％，三氯甲烷相为33.54±0.17％。具有强抗氧化的活性物质大多存在于乙酸乙酯相中，类似于黄酮类和蒽醌类，所以超氧阴离子自由基清除能力乙酸乙酯相较强。
由图7可知在质量浓度为1～5mg/mL之间，食叶草乙酸乙酯相、正丁醇相和三氯甲烷相都有一定的DPPH·清除能力且随着样品质量浓度的不断增加，清除DPPH·的能力越强。在1～5mg/mL质量浓度之间，乙酸乙酯相的DPPH·清除能力强于正丁醇相强于三氯甲烷相，在样品质量浓度5mg/mL时，食叶草乙酸乙酯相的清除率为95.84±3.21％，正丁醇相为92.33±2.92％，三氯甲烷相为87.56±4.44％。DPPH·是一种很稳定的以氮为中心的自由基，二苯乙烯类物质、黄酮类以及蒽醌类物质都可以清除DPPH·，这些物质大多被乙酸乙酯相萃取出来，所以乙酸乙酯相清除DPPH·能力最强。
由图8可知在质量浓度为1-5mg/mL之间，食叶草乙酸乙酯相、正丁醇相和三氯甲烷相都具有还原能力且随着样品质量浓度的不断增加，在700nm处的吸光值越大，还原能力越强。在1-5mg/mL质量浓度之间，乙酸乙酯相的还原能力强于正丁醇相强于三氯甲烷相，在样品质量浓度5mg/mL时，食叶草乙酸乙酯相的吸光值为0.947±0.243，正丁醇相为0.738±0.239，三氯甲烷相为0.654±0.211。在食叶草的叶和茎浸膏悬浮于水用乙酸乙酯萃取后经过鉴定所含主要成分为大黄酚、大黄素等蒽醌类物质，可有效还原三价铁离子，所以乙酸乙酯相还原能力优于其他两相。
以上所述实施例仅表达了本发明的优选实施方式，其描述较为具体和详细，但并不能因此而理解为对本发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形、改进及替代，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。
Claims (10)
1.一种食叶草提取物，其特征在于：所述食叶草提取物的提取步骤为：
（1）将食叶草去根洗净，冷冻干燥后制得食叶草粉末；
（2）称取食叶草粉末，加入溶剂，超声碾碎提取并过滤；反复提取三次，合并滤液，旋转蒸发浓缩后冷冻干燥得到初级提取物；
（3）将初级提取物均匀分散在水中，使用有机溶剂萃取，得到食叶草提取物。
2.根据权利要求1所述的食叶草提取物，其特征在于：所述步骤（2）中的溶剂为水、甲醇、丙酮、乙醇或乙酸乙酯中的至少一种。
3.根据权利要求2所述的食叶草提取物，其特征在于：所述步骤（2）中的溶剂为体积比为70%的乙醇。
4.根据权利要求1所述的食叶草提取物，其特征在于：所述步骤（2）中食叶草粉末和溶剂的料液比为1：8~12。
5. 根据权利要求1所述的食叶草提取物，其特征在于：所述步骤（2）中超声条件为：温度为50 ~55℃，功率为100~120 W，时间为30~40 min。
6.根据权利要求1所述的食叶草提取物，其特征在于：所述步骤（3）中的有机溶剂为石油醚、乙酸乙酯、正丁醇或水中的至少一种。
7.根据权利要求6所述的食叶草提取物，其特征在于：所述步骤（3）中的有机溶剂为乙酸乙酯。
8.根据权利要求1所述的食叶草提取物，其特征在于：所述步骤（3）中有机溶剂萃取次数为8~12次。
9.权利要求1-8任一项所述的食叶草提取物在制备拮抗内毒素的药物和保健品中的应用。
10.根据权利要求9所述的应用，其特征在于：所述食叶草提取物的使用浓度范围为1-5mg/mL。
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