CN116327830B - 一种抗氧化、调节肠道菌群的中药组合物及其制备方法 - Google Patents
一种抗氧化、调节肠道菌群的中药组合物及其制备方法 Download PDFInfo
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Abstract
本发明公开了一种抗氧化、调节肠道菌群的中药组合物的制备方法,中药组合物包括质量比为1.8‑2.2:1的辣蓼和黄连,通过特定的制备方法提取辣蓼和黄连有效部位,使用本发明制得的辣蓼和黄连提取物后肠道微生物群的结构和组成发生明显变化,乳酸杆菌和阿克曼菌等益生菌群的丰度显著上调,而棱状芽孢杆菌、颤螺菌等有害菌群的丰度明显下调,并调节酪氨酸和类固醇激素在内的代谢产物水平。
Description
技术领域
本发明涉及抗氧化、调节肠道菌群的中药组合物领域,特别一种涉及抗氧化、调节肠道菌群的中药组合物的制备方法。
背景技术
肠道微生物的动态平衡与人体健康密切相关,肠道微生物群可以调节胃肠道稳态、宿主的新陈代谢和免疫***以及各种疾病的发展。肠道微生物群失调以及代谢表型改变与各种疾病的病理生理学密切相关,如GUs和炎症性肠病(IBD)等。中药或药膳通过调节肠道菌群失调在保持肠道功能完整性和减轻粘膜损伤方面具有有益作用。中药中含有的多种化合物在口服后将不可避免地与肠道微生物群相互作用,并通过调节肠道微生物群的群落结构和代谢,恢复肠道微生物群稳态,缓解病理和临床症状,从而达到其治疗效果。同时,肠道微生物群可以代谢或转化中医药的活性物质,从而改变中医药的疗效,削弱有毒成分对身体的损害,避免药物对身体的不良反应,从而使中医药在预防和治疗疾病方面发挥更好的作用。
不同的肠道微生物对宿主健康产生不同的影响。乳酸杆菌属和阿克曼菌属于有益细菌。乳酸杆菌可以缓解炎症,防止病原体入侵和艰难梭菌感染,激活巨噬细胞的吞噬作用,增强免疫力。阿克曼菌的药理作用包括缓解肠道炎症、延缓衰老、降低血脂和血糖、提高免疫力、调节神经***功能以及抗炎和抗癌。其作用机制是改善肠粘液的厚度,保持肠屏障的完整性,防止外来病原体或有毒物质的入侵。中医药通过靶向肠道菌群在多种疾病中发挥关键作用。乳酸杆菌和AKK细菌可能是中药的主要靶点之一,在用中药治疗后,AKK细菌丰度的显著增加是一个有力的证据,这种现象可能意味着过多的乳杆菌和阿克曼菌会对身体健康产生有益影响。
发明内容
鉴以此,本发明提出一种涉及抗氧化、调节肠道菌群的的制备方法,解决上述问题。
本发明的技术方案是这样实现的:
一种抗氧化、调节肠道菌群的中药组合物,中药组合物包括质量比为1.8-2.2:1的辣蓼和黄连。
进一步的,所述的抗氧化、调节肠道菌群的中药组合物的制备方法,包括以下步骤:
(1)将辣蓼全草干燥粉碎,制得辣蓼粉末,将辣蓼粉末采用蒸馏法进行处理,蒸馏后的辣蓼粉末采用乙醇溶液浸泡,得到提取液,浓缩,得到辣蓼浸膏;
(2)将辣蓼浸膏依次采用石油醚、乙酸乙酯萃取,得到乙酸乙酯部位;
(3)乙酸乙酯部位采用大孔树脂吸附,用乙醇溶剂做洗脱剂,得到辣蓼有效部位;
(4)将黄连粉碎,得到黄连粉末,得到黄连粉末,采用亚临界水为溶剂对黄连粉末进行提取,得到黄连提取液;
(5)将黄连提取液采用大孔树脂吸附,依次采用水、浓度为80-90wt%的乙醇溶液、浓度为50-60wt%的乙醇溶液和浓度为10-20wt%的乙醇溶液洗脱,收集洗脱液,干燥,得到黄连有效部位。
进一步的,步骤(1)中,所述蒸馏法蒸馏温度为55-65℃,蒸馏时间为30-40min.
进一步的,步骤(1)中,所述辣蓼粉末和乙醇溶液的质量比为1:8-10,浸泡时间为15-18h,浸泡次数为2-3次,乙醇溶液浓度为75-85wt%,所述浓缩至含水量为30-32%。
进一步的,步骤(2)中,所述辣蓼浸膏、石油醚、乙酸乙酯质量比为1:3-5:8-10。
进一步的,步骤(3)中,所述黄连浸膏和大孔树脂重量比为1:5-10,用3-5倍量柱体积的乙醇溶液洗脱,所述乙醇溶液浓度为30-90wt%。
进一步的,步骤(4)中,所述亚临界水温度为150-170℃,提取压力为10-13MPa,流速为15-18L/h,提取时间为140-160min。
进一步的,步骤(5)中,所述黄连浸膏和大孔树脂质量比为1:7-9,水的用量为3-5倍量柱体积,浓度为80-90wt%的乙醇溶液用量为7-9倍量柱体积,浓度为50-60wt%的乙醇溶液用量为1-2倍量柱体积,浓度为10-20wt%的乙醇溶液用量为3-4倍量柱体积。
进一步的,步骤(3)中,所述大孔树脂的型号为AB-8,步骤(5)中,所述大孔树脂的型号为聚苯乙烯型大孔树脂。
本发明的有益效果:
本发明中药提取物由辣蓼和黄连组成,本发明中采用特定的提取方法提取辣蓼和黄连中有效成分,使用本发明制得的辣蓼和黄连提取物后肠道微生物群的结构和组成发生明显变化,乳酸杆菌和阿克曼菌等益生菌群的丰度显著上调,而棱状芽孢杆菌、颤螺菌等有害菌群的丰度明显下调,并调节酪氨酸和类固醇激素在内的代谢产物水平。
经药效学研究表明,该组合物对肠道菌群紊乱具有显著改善作用,并提高抗氧化酶的表达,增强抗氧化能力。
附图说明
图1SOD活性对比图
图2实施例1组合物对肠道微生物多样性影响
图3实施例1组合物对肠道微生物组成影响
图4-6实施例1组合物本组合物对肠道微生物群落的影响
图7实施例1组合物对肠道乳酸杆菌组成和丰度的影响
图8实施例1组合物重要代谢通路及差异代谢物的影响
图9实施例1组合物对正常GES-1细胞增殖的影响对比图
图10实施例1组合物对对H2O2诱导GES-1细胞损伤的影响
具体实施方式
为了更好理解本发明技术内容,下面提供具体实施例,对本发明做进一步的说明。
实施例1
称取质量比为2:1的辣蓼和黄连。
(1)将辣蓼全草干燥粉碎,制得辣蓼粉末,将辣蓼粉末采用55-65℃蒸馏30-40min进行处理,蒸馏后的辣蓼粉末采用浓度为75-85wt%乙醇溶液浸泡15-18h,浸泡次数为2-3次,得到提取液,浓缩至含水量为30-32%,得到辣蓼浸膏;
(2)将辣蓼浸膏依次采用石油醚、乙酸乙酯萃取,辣蓼浸膏、石油醚、乙酸乙酯质量比为1:3-5:8-10,得到乙酸乙酯部位;
(3)乙酸乙酯部位采用AB-8大孔树脂吸附,黄连浸膏和HPD-100大孔树脂重量比为1:5-10,用3-5倍量柱体积的浓度为60wt%乙醇溶液洗脱,得到辣蓼有效部位;
(4)将黄连粉碎,得到黄连粉末,黄连粉末采用亚临界水为溶剂提取,亚临界水温度为150-170℃,提取压力为10-13MPa,流速为15-18L/h,提取时间为140-160min,得到黄连提取液;
(5)将黄连提取液采用聚苯乙烯型大孔树脂吸附,黄连浸膏和聚苯乙烯型大孔树脂质量比为1:8,依次采用4倍量柱体积的水,8倍量柱体积的浓度为85wt%的乙醇溶液,1.5倍量柱体积的浓度为55wt%的乙醇溶液,3.5倍量柱体积的浓度为15wt%的乙醇溶液进行洗脱,收集洗脱液,干燥,得到黄连有效部位。
(6)合并辣蓼有效部位、黄连有效部位,制得中药组合物。
试验例1
本发明试验肠道菌群紊乱为束缚水浸应激诱导
一、试验材料:
试剂:Pro Kit,(QIAGEN,美国);Fastpfu DNA Polymerase(TransGen,中国);AxyPrepDNA凝胶回收试剂盒(Axygen,美国);NEXTFLEX·Rapid DNA-SeqKit(Bioo Scientific,美国);MiSeq Reagent Kit v3(Illumina,美国)
仪器:电子分析天平(德国Sartorius赛多利斯);冷冻研磨仪(德国Retsch莱驰);低温离心机(德国Eppendorf艾本德);旋涡混合器(美国Crystal精骐);PCR仪(ABI9700型);测序仪(Illumina Miseq,美国)。
二、试验方法:
SD大鼠随机分为3组:正常组、模型组、本组合物组,每组10只。正常组和模型组给与蒸馏水,给药组每天给药1次,连续给药6天;造模前,禁食不禁水24h,末次给药后,将除正常组外,其余大鼠放入自制的鼠笼中,头部朝上,固定四肢,然后将鼠笼垂直浸于恒温水槽中(19±1℃),水位到达大鼠剑突位置,持续7h后造模结束。造模结束后,用10%水合氯醛腹腔麻醉,腹主动脉采集血样,离心,取血清。然后,从结肠中取粪便3颗,放入液氮中,再转入-80℃保存。
血清SOD活力测定:先进行小样本预实验,用生理盐水稀释血清原液至5~10倍后,按照SOD测定(WST-1法)试剂盒的说明书完成所有实验操作,检测微孔板OD值,计算SOD抑制率,分析样本的最佳稀释比例,使每组样品的SOD抑制率尽可能控制在40~60%之间,随后再进行大批量正式试验,计算方法参照说明书。
测序实验流程:总DNA提取与检测——PCR扩增——PCR产物鉴定、纯化及定量——构建PE文库——Illumina测序。
生物信息学分析:MiSeq测序得到的PE reads首先根据overlap关系进行拼接,同时对序列质量进行质控和过滤,区分样本后进行OTU聚类分析和物种分类学分析。基于OTU聚类分析结果,可以进行多种多样性指数分析,以及对测序深度进行检测;基于分类学信息,可以在各个分类水平上进行群落结构的统计。在上述分析的基础上,可以进行一系列群落结构和***发育等深入的统计学和可视化分析。
三、实验结果
1、参见图1,SOD结果显示,模型组SOD活性较正常组显著降低,与模型组相比,本发明组合物预处理后,可显著提高SOD活性(P<0.01)。
2、本发明中黄连和辣蓼提取物中有效成分对肠道微生物多样性影响:基于97%的相似性水平,对16S rRNA测序结果进行了α多样性分析。α多样性指数的统计分析表明,模型组的多样性与对照组没有差异,表明应激刺激对肠道微生物多样性的影响很小,而本组合物处理显著增加肠道微生物群落的多样性。随着采样序列数量的增加,稀疏曲线趋于平坦,表明测序深度足以覆盖大多数微生物物种的信息,结果见图2。
3、本发明中黄连和辣蓼提取物中有效成分对肠道微生物组成影响:运用PLS-DA对模型组、对照组和本组合物组之间肠道微生物群的组成概况以及本发明中黄连和辣蓼提取物中有效成分对肠道微生物群结构的影响进行分析。如3图所示,模型组与对照组完全分离,且分布于不同象限,存在显著差异,表明应激导致大鼠肠道菌群结构发生显著变化,而本组合物处理组与模型组完全分离,表明本组合物对肠道微生物群结构显示出显著影响。
4、本发明中黄连和辣蓼提取物中有效成分对肠道微生物群落的影响:本组合物处理对肠道微生物在属水平的改变比门水平的改变更明显。在门水平,各组中优势细菌的组成基本相似,两个优势门是厚壁菌门和拟杆菌门。与正常组相比,模型组中Verrucomicrobiota的丰度显著降低,而在本组合物处理后,Verrucomicobiota的丰富度发生了逆转,与模型组相比厚壁菌门的丰度显著增加。在属水平,三组中相对丰度最高5个属分别是乳杆菌属、Rombousia属、norank_f__Muribaculaceae属、Alloprevotella属和Akkermansia属。与正常组相比,模型组中乳杆菌和阿克曼菌的丰度显著降低,而Clostridia_UCG-014和Eubacterium_coprostanoligenes的丰度增加。本组合物处理后乳杆菌和阿克曼菌的丰度显著增加,而Clostridia_UCG-014和Eubacterium_coprostanoligenes的丰度显著降低。这些反应表明本发明中黄连和辣蓼提取物中有效成分对肠道微生物群具有明显的调节作用,见图4-6。
5、本组合物对肠道乳酸杆菌组成和丰度的影响:进一步分析了肠道微生物中乳酸杆菌的组成和变化,结果显示,本组合物对乳杆菌有显著影响,本组合物处理后乳酸杆菌的丰度显著增加,主要包括lactobacillus_murinus、Lacillus intestinalis、lacobacillusjohnsonii、Lactobacillus reuteri、gut_metagenome_g__Lactobacillus、uncultured_bacterium_g__Lactobacillus和unclassified_g__Lactobacillus。表明本发明中黄连和辣蓼提取物中有效成分能显著逆转应激诱导大鼠肠道微生物失调,见图7。
6、本发明中黄连和辣蓼提取物中有效成分对重要代谢通路及差异代谢物的影响:有两条重要的代谢途径为酪氨酸代谢和类固醇激素生物合成。这两种代谢途径中显著差异代谢产物包括L-酪氨酸、L-多巴等酪氨酸代谢物及21-脱氧皮质醇、二氢皮质醇、四氢化可的松等类固醇激素生物合成物质。本组合物能降低L-酪氨酸和L-多巴水平及21-脱氧皮质醇、二氢皮质醇和四氢化可的松水平,进而改善酪氨酸代谢和HPA轴紊乱,见图8。
7、肠道微生物群与粪便代谢产物的相关性分析:在属水平上,阿克曼菌和乳杆菌的丰度变化与酪氨酸、21-羟基孕烯醇酮、D-半乳糖和胆酸等变化呈负相关。这些差异代谢产物是调节阿克曼菌和乳杆菌的关键粪便代谢产物。本发明中黄连和辣蓼提取物中有效成分通过改善肠道微生物群落组成来调节粪便代谢产物变化。
试验例2细胞实验
1、对正常GES-1细胞增殖的影响
取对数生长期的GES-1细胞,加入0.25%胰蛋白酶1mL,室温消化1min,加入1640培养液终止消化,并制备浓度为2×105个/mL的单细胞悬液,按细胞液100μL/孔接种在48孔板上,待细胞贴壁后分别加入不同浓度的实施例1组合物(低浓度0.5mg/mL,中浓度为1mg/mL,高浓度为5mg、mL),每组样本设6个平行复孔,另设模型对照组和空白对照组,37℃、5%CO2培养24h后,弃去原液,每孔加入10ul的CC-K8试剂和90ul培养基,培养3h后,用酶标仪在450nm波长测其吸光度值。CCK-8法测定细胞存活率。
2、对H2O2诱导GES-1细胞损伤的影响
取对数生长期的GES-1细胞,加入0.25%胰蛋白酶1mL,室温消化1min,加入1640培养液终止消化,并制备浓度为2×105个/mL的单细胞悬液,按细胞液100μL/孔接种在96孔板上,待细胞贴壁后分别加入提取物,每组样本设6个平行复孔,另设阴性对照组和不含细胞的空白组,37℃、5%CO2培养24h后,弃去原液,加入200uM H2O2损伤4h,然后,吸弃培养液,每孔加入10ul的CC-K8试剂和90ul培养基,培养3h后,用酶标仪在450nm波长测其吸光度值。
参见图9,实施例1制得的中药组合物不同浓度能明显促进正常GES-1细胞增殖,并呈明显的剂量依赖性关系。
参见图10,GES-1细胞经200uM的H2O2诱导损伤4小时后,模型组细胞存活率较正常组显著降低;与模型组相比,给予本发明组合物中、低、高剂量预处理12小时后,均可明显提高细胞存活率(p<0.05)。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (8)
1.一种抗氧化、调节肠道菌群的中药组合物,其特征在于,中药组合物由质量比为1.8-2.2:1的辣蓼和黄连组成;
抗氧化、调节肠道菌群的中药组合物的制备方法包括以下步骤:
(1)将辣蓼全草干燥粉碎,制得辣蓼粉末,将辣蓼粉末采用蒸馏法进行处理,蒸馏后的辣蓼粉末采用乙醇溶液浸泡,得到提取液,浓缩,得到辣蓼浸膏;
(2)将辣蓼浸膏依次采用石油醚、乙酸乙酯萃取,得到乙酸乙酯部位;
(3)乙酸乙酯部位采用大孔树脂吸附,用乙醇溶剂做洗脱剂,得到辣蓼有效部位;
(4)将黄连粉碎,得到黄连粉末,采用亚临界水为溶剂对黄连粉末进行提取,得到黄连提取液;
(5)将黄连提取液采用大孔树脂吸附,依次采用水、浓度为80-90wt%的乙醇溶液、浓度为50-60wt%的乙醇溶液和浓度为10-20wt%的乙醇溶液洗脱,收集洗脱液,干燥,得到黄连有效部位;
(6)合并辣蓼有效部位、黄连有效部位,制得中药组合物。
2.如权利要求1所述的抗氧化、调节肠道菌群的中药组合物,其特征在于,步骤(1)中,所述蒸馏法蒸馏温度为55-65℃,蒸馏时间为30-40min。
3.权利要求1所述的抗氧化、调节肠道菌群的中药组合物,其特征在于,步骤(1)中,所述辣蓼粉末和乙醇溶液的质量比为1:8-10,浸泡时间为15-18h,浸泡次数为2-3次,乙醇溶液浓度为75-85wt%,所述浓缩至含水量为30-32%。
4.如权利要求1所述的抗氧化、调节肠道菌群的中药组合物,其特征在于,步骤(2)中,所述辣蓼浸膏、石油醚、乙酸乙酯质量比为1:3-5:8-10。
5.如权利要求1所述的抗氧化、调节肠道菌群的中药组合物,其特征在于,步骤(3)中,所述辣蓼浸膏和大孔树脂重量比为1:5-10,用3-5倍量柱体积的乙醇溶液洗脱,所述乙醇溶液浓度为30-90wt%。
6.如权利要求1所述的抗氧化、调节肠道菌群的中药组合物,其特征在于,步骤(4)中,所述亚临界水温度为150-170℃,提取压力为10-13MPa,流速为15-18L/h,提取时间为140-160min。
7.如权利要求1所述的抗氧化、调节肠道菌群的中药组合物,其特征在于,步骤(5)中,所述黄连提取液和大孔树脂质量比为1:7-9,水的用量为3-5倍量柱体积,浓度为80-90wt%的乙醇溶液用量为7-9倍量柱体积,浓度为50-60wt%的乙醇溶液用量为1-2倍量柱体积,浓度为10-20wt%的乙醇溶液用量为3-4倍量柱体积。
8.如权利要求1所述的抗氧化、调节肠道菌群的中药组合物,其特征在于,步骤(3)中,所述大孔树脂的型号为AB-8,步骤(5)中,所述大孔树脂的型号为聚苯乙烯型大孔树脂。
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