CN116508987A - 陈化三年广陈皮青皮提取物在制备调节脾虚导致的肠道菌群失调产品中的应用 - Google Patents
陈化三年广陈皮青皮提取物在制备调节脾虚导致的肠道菌群失调产品中的应用 Download PDFInfo
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Abstract
本发明提供了一种陈化三年广陈皮青皮提取物在制备调节脾虚导致的肠道菌群失调产品中的应用,属于医药/功能性食品技术领域。使用本发明制成的陈化三年广陈皮青皮提取物能有效提升脾虚小鼠肠道菌群的物种丰富度和多样性,恢复拟杆菌门与厚壁菌门的比例,促进Muribaculaceae属、毛螺菌科中NK4A136_group和Ileibacterium属等益生菌的恢复,减少变形菌门、疣微菌门和放线菌门中副沙门氏菌属和异杆菌属等条件致病菌的增殖,使得脾虚小鼠失调的肠道菌群基本恢复正常,且其效果优于自然恢复、参苓白术散、陈化三年广陈皮红皮以及陈化一年的青皮和红皮组。
Description
技术领域
本发明属于医药/功能性食品技术领域,尤其涉及一种陈化三年广陈皮青皮提取物在制备调节脾虚导致的肠道菌群失调产品中的应用。
背景技术
陈皮(Pericarpium Citri Reticulatae)是芸香科柑橘属植物橘(CitrusreticulataBlanco)以及其栽培变种的干燥成熟果皮,通常分为“陈皮”和“广陈皮”。广陈皮为广东新会一带所产,由橘的栽培变种茶枝柑(Citrus reticulata'Chachiensis')果皮制成。是“广东三宝”之首以及“广东十大中药材”之一。陈皮有理气健脾、燥湿化痰的功效,用于治疗咳嗽痰多、食少吐泻、脘腹胀满等症。陈皮中黄酮类化合物、生物碱、挥发油、多糖、柠檬苦素等活性成分,具有抗氧化,抗炎抗菌,抗癌,抗病毒,同时能保护心脑血管以及神经***,改善血糖、脂质代谢,促进新陈代谢,调节胃肠功能以及免疫调节功能等。广陈皮根据采收时期的不同,主要分为青皮和红皮两种。采收时期和陈化年份的不同会影响陈皮中的活性成分,产生不同的效果。
脾虚证泛指因脾气虚损引起的一系列脾脏生理功能失常的病理现象及病证,主要表现为脘腹胀满、大便溏薄、神倦乏力等,包括脾气虚、脾阳虚、中气下陷、脾不统血等,通常由于饮食不节、劳累过度、久病体虚所造成。脾有运化食物中的营养物质和输布水液以及统摄血液等作用,脾虚则运化失常,导致营养障碍,失血等症状。而肠道菌群的失调导致的胃肠动力紊乱很有可能是脾虚的原因。
肠道菌群包括人体肠道中所有寄居的微生物,约有十万亿个,其包含约3千万个基因,其所编码的基因数量是人类基因组的150倍。肠道菌群种类繁多,根据其对宿主代谢的影响,大致可以分为3类:中性菌、有害菌和有益菌,他们之间相互依存和制约,与宿主一起形成互利共生的生态。肠道菌群的失调往往导致疾病的出现,所以保证肠道菌群稳态在调节机体免疫、维持肠道正常生理功能及拮抗病原微生物等方面有重要的生理意义。
发明内容
为了解决上述技术问题,本发明提供了一种陈化三年广陈皮青皮提取物在制备调节脾虚导致的肠道菌群失调产品中的应用。使用本发明制成的陈化三年广陈皮青皮提取物能有效提升脾虚小鼠肠道菌群的物种丰富度和多样性,恢复拟杆菌门与厚壁菌门的比例,促进Muribaculaceae属、毛螺菌科中NK4A136_group和Ileibacterium属等益生菌的恢复,减少变形菌门、疣微菌门和放线菌门中副沙门氏菌属和异杆菌属等条件致病菌的增殖,使得脾虚小鼠失调的肠道菌群基本恢复正常,且其效果优于自然恢复、参苓白术散、陈化三年广陈皮红皮以及陈化一年的青皮和红皮组。
为了实现上述目的,本发明采用了以下技术方案:
本发明提供了一种陈化三年广陈皮青皮提取物在制备肠道菌群失调产品中的应用。
优选的,所述应用具体为在制备调节脾虚导致的肠道菌群失调产品中的应用。
本发明还提供了一种所述应用中陈化三年广陈皮青皮提取物的制备方法,包括以下步骤:
(1)取陈化三年广陈皮青皮,加5~7倍水浸泡25~35min;
(2)将浸泡后的陈化三年广陈皮青皮煎煮25~35min并过滤;
(3)过滤后的残渣加入7~9倍水,再次煎煮25~35min并过滤;
(4)合并两次煎煮滤液,浓缩至浓度为0.4~0.6g/ml,所述浓缩液即为所述陈化三年广陈皮青皮提取物。
优选的,所述步骤(1)中取陈化三年广陈皮青皮,加6倍水浸泡30min。
优选的,所述步骤(2)中将浸泡后的陈化三年广陈皮青皮煎煮30min并过滤。
优选的,所述步骤(3)中过滤后的残渣加入8倍水,再次煎煮30min并过滤。
优选的,所述步骤(4)中将滤液浓缩至浓度为0.5g/ml。
本发明还提供了一种使用所述制备方法制成的陈化三年广陈皮青皮提取物。
与现有技术相比,本发明具有如下技术效果:
使用本发明制成的陈化三年广陈皮青皮提取物能有效提升脾虚小鼠肠道菌群的物种丰富度和多样性,恢复拟杆菌门与厚壁菌门的比例,促进Muribaculaceae属、毛螺菌科中NK4A136_group和Ileibacterium属等益生菌的恢复,减少变形菌门、疣微菌门和放线菌门中副沙门氏菌属和异杆菌属等条件致病菌的增殖,使得脾虚小鼠失调的肠道菌群基本恢复正常,且其效果优于自然恢复、参苓白术散、陈化三年广陈皮红皮以及陈化一年的青皮和红皮组,在脾虚导致的肠道菌群失调等相关疾病的治疗中意义重大。
附图说明
图1为本发明实施例5中的门水平ASV表;
图2为本发明实施例5中的属水平ASV表;
图3为本发明实施例5中门水平的相对丰度柱状图;
图4为本发明实施例5中属水平的相对丰度柱状图;
图5为本发明实施例5中的Alpha分析原始数据;
图6~图9为根据本发明实施例5中的Alpha分析原始数据所制成的箱型图,其中:与Control组比较,##P<0.01,#P<0.05;与Model组比较,**P<0.01,*P<0.05;
图10为本发明实施例5中的PCA分析原始数据;
图11~12为本发明实施例5中的PCA分析的样本二维排序图,其中图中的每个点代表一个样本,不同颜色的点代表不同组别。
具体实施方式
以下实施例用于说明本发明,但不用来限制本发明的范围。在不背离本发明精神和本质的情况下,对本发明方法、步骤或条件所作的修改或替换,均属于本发明的范围。以下实施例中使用的试剂、试剂盒和仪器均可由市售获得,实施例中使用的方法如无特别说明,与常规使用的方法一致。
下面结合实施例,对本发明的技术方案进行进一步详细阐述。
实施例1
称取大黄药材300g,加6倍水浸泡30min,煎煮30min,滤过,再加8倍水,煎煮30min,滤过,合并两次滤液,浓缩得到1g/ml的大黄水煎液,冷藏,备用,用于脾虚型小鼠模型的建立。
实施例2
分别称取不同年份不同采收期的广陈皮500g,加6倍水浸泡30min,煎煮30min,滤过,再加8倍水,煎煮30min,合并两次滤液,浓缩得到0.5g/ml的水煎液,冷藏,备用。其中2021年11月采收的为一年红皮组,2021年9月采收的为一年青皮组,2019年11月采收的为三年红皮组,2019年9月采收的为三年青皮组。
实施例3
取64只6周龄SPF级雄性ICR小鼠,由湖南斯莱克景达实验动物有限公司购买,许可证号SCXK(湘)2019-0004。随机分为8组,分别是空白对照组、长期模型组、自然恢复组、参苓白术组、一年红皮组、一年青皮组、三年红皮组、三年青皮组,采用实施例1中制备的大黄水煎液(1g/ml)进行脾虚模型的建立,灌胃量为10ml/kg。空白对照组灌胃等量生理盐水。造模时每天灌胃1次,连续14天。造模后,各组小鼠灌胃给药14天,分组及剂量如下:
1.空白对照组(简写为:Control):灌胃10ml/kg生理盐水,每日一次。
2.长期模型组(简写为:Model):灌胃10ml/kg大黄水煎液,每日一次。
3.自然恢复组(简写为:Recovery)灌胃10ml/kg生理盐水,每日一次。
4.参苓白术组(简写为:Positive,剂量200mg/kg)灌胃10ml/kg参苓白术散冲剂(河北扁鹊制药有限公司,货号:220409),每日一次。
5.一年红皮组(简写为:H-NEW)灌胃10ml/kg陈化一年广陈皮红皮的水煎液,每日一次。
6.一年青皮组(简写为:Q-NEW)灌胃10ml/kg陈化一年广陈皮青皮的水煎液,每日一次。
7.三年红皮组(简写为:H-3)灌胃10ml/kg陈化三年广陈皮红皮的水煎液,每日一次。
8.三年青皮组(简写为:Q-3)灌胃10ml/kg陈化三年广陈皮青皮的水煎液,每日一次。
末次给药后,小鼠禁食不禁水12h。次日麻醉并解剖小鼠,快速无菌取出小鼠结肠粪便,并装无菌管中,迅速放于液氮中,再-80℃保存。
实施例4
使用16SrRNA高通量测序技术对实施例3中收集到的各组样品进行测序。
1.仪器与试剂
提取实施例3中各组样品的DNA,提取后的总DNA采用Nanodrop(型号ND-1000)进行定量,并使用1-1.5%的琼脂糖凝胶进行DNA完整性等质控。具体仪器试剂如表1所示。
表1仪器/试剂
仪器/试剂 | 用途 | 品牌 | 型号 |
Nanodrop | 紫外定量 | NanoDro pTechnologies | NanoDrop ND-1000 |
电泳仪 | 琼脂糖凝胶电泳 | Bio-Rad | PowerPac Universal |
凝胶成像*** | 凝胶成像 | Invitrogen | E-gel Safe Imager |
Agarose | 琼脂糖凝胶试剂 | Biowest | Agarose |
Marker | 衡量核酸分子量大小 | Trans | Trans2KPlusDNAMarker |
2.PCR体系与条件
使用特异性引物对各组样本16S rRNA的v3 v4区域进行PCR扩增,以获得原核生物16S rDNA的可变区,构建高通量文库。引物序列如表2所示,PCR扩增体系及条件分别如表3-4所示。
表2引物详情
primer | forward(5'-3')(SEQ ID No.1) | reverse(5'-3')(SEQ ID No.2) |
BacterialV3/V4(341F+805R) | CCTACGGGNGGCWGCAG | GACTACHVGGGTATCTAATCC |
表3 PCR扩增体系
Reagent | Volume |
Phusion enzyme | 0.5ul |
5xphusion buffer | 5ul |
2.5mM dNTP | 2ul |
F Primer | 1ul |
R Primer | 1ul |
dsDNA | 1ul |
Nuclease-Free Water | 14.5ul |
注:1)25ul体系,DNA起始量10-20ng;2)部分扩增比较困难的样品,根据实际情况对扩增体系与条件进行微调;3)Phusion酶(APExBIO K1031)。
表4PCR扩增条件
Step | Condition(25-30Cycles) | ||
1 | Initial denaturation | 98C | 1min |
2 | Denaturation | 98C | 15s |
3 | Annealing | 58C | 15s |
4 | Extension | 72C | 40s |
5 | Final extension | 72C | 2min |
6 | Hold | 4C | ---- |
3.样本建库结果
使用特异性引物扩增后,对PCR产物进行琼脂糖凝胶验证,合格的样本条带应清晰无杂带,所处位置在marker条带的500-750bp之间;同时确认扩增产物的条带大小及质量情况,如无误,使用磁珠纯化特异性引物扩增出的片段后,加入测序接头进行第二轮扩增,构建高通量文库,用于后续的数据分析。
实施例5
对实施例4中的测序结果进行分析。
1.信息分析流程
采用Qiime 2分析流程,调用DADA2对Raw Data进行去噪,相当于DNA序列以100%的相似度聚类,仅对低质量序列进行去除和校正、算法识别去嵌合等;去噪的序列直接去冗余,获得Feature(特征,是OTU、ASV等的统称)信息。对每个ASV序列做物种注释,得到对应的物种信息和基于物种的丰度分布情况(即ASV_table)。接着,基于ASV_table进行相对丰度、Alpha多样性、Beta多样性、组间差异等分析,以得到样本内物种丰富度和均匀度信息、不同样本或分组间的共有和特有ASVs信息等,以及不同样本和分组的群落结构差异分析,通过PCoA和PCA、NMDS等降维图和样本聚类树进行展示。为进一步挖掘分组样本间的群落结构差异,选用Adonis、Anosim等统计分析方法对分组样本的物种组成和群落结构进行差异显著性检验,基于非参数检验对α多样性指数进行组间差异分析,基于wald检验进行差异菌筛选。此外,还包括LEfSe分析、PICRUSt2功能预测以及Krona物种组成等其他分析结果。
2.分析软件与方法简介
序列数据分析主要使用Qiime 2(Qiime2-2020.2)和R软件(4.0.2)。
使用Qiime 2软件计算各样本的Alpha多样性指数(包括Chao1、ACE、Shannon、Simpso均匀度指数等)。
使用R软件绘制物种相对丰度柱状图(R ggplot2包)PCA图。
2.1测序数据的质量控制
测序得到的原始数据(Raw Data)会存在一定比例的低质量数据,为了保证后续信息分析结果的准确可靠,首先要对原始数据进行过滤处理,得到有效数据(Clean Data)。具体处理步骤如下:
1)去除所含低质量碱基(质量值≤15)超过一定比例(默认设为40bp)的reads;
2)去除N碱基达到一定比例的reads(默认设为10bp);
3)去除与Adapter之间overlap超过一定阈值(默认设为10bp)的reads;
测序错误率分布检查用于检测在测序长度范围内,有无异常的碱基位置存在高错误率,例如如果中间位置的碱基测序错误率显著高于其他位置,则可能存在异常碱基。一般情况下,每个碱基位置的测序错误率都应该低于1%。
测序数据的质量主要分布在Q30(≥80%)以上,这样才能保证后续分析的正常进行。根据测序技术的特点,测序片段末端的碱基质量一般会比前端的低。
2.2ASV分析和物种注释
为了研究样本的物种多样性,通过DADA2生成Feature信息;为获得物种分类信息,使用Qiime2根据silva-132-99数据库,v3-v4区扩增引物,将参考基因组Silva13299%Features,修剪为v3-v4区序列并进行训练,获得分类器,之后用训练好的naive Bayesclassifier对Feature进行分类,获得对应的物种分类信息,从而得到每个样本的群落组成。
分析软件:Qiime 2
分析方法:使用naive Bayes classifier和q2-feature-classifier plugin对Feature序列进行分类学分析,并在各个水平统计每个样本的群落组成,数据库为silva-132-99。为了方便理解与绘图,将在本项目中获得的每一个Feature分别定义为一种ASV,获得ASV表,其中门水平的ASV表如图1所示,属水平的ASV表如图2所示。
根据ASV表,可以作出展示各样本中ASV相对丰度的相对丰度柱状图,其中门水平的如图3所示,属水平的如图4所示。
结果显示,Model组的拟杆菌门(Bacteroidota)丰度有显著性下降(P<0.05),同时放线菌门(Actinobacteriota)、变形菌门(Proteobacteria)以及疣微菌门(Verrucomicrobiota)显著性增长(P<0.05)。在恢复治疗后,各陈皮组中小鼠的肠道菌群相较于Model组,拟杆菌门(Bacteroidota)丰度有所增加,同时放线菌门(Actinobacteriota)、变形菌门(Proteobacteria)以及疣微菌门(Verrucomicrobiota)明显减少,都有着程度不一的恢复。其中三年青皮在恢复拟杆菌门与厚壁菌门的比例,减少变形菌门、疣微菌门和放线菌门的增殖中表现出比其他陈皮组更好的效果。通过图4可以发现,从属水平上分析,Model组Muribaculaceae属、毛螺菌科中的NK4A136_group和Ileibacterium属等益生菌明显减少,同时副沙门氏菌属(Parasutterella)和异杆菌属(Allobaculum)等条件致病菌明显增加,可见脾虚小鼠肠道菌群明显失调。经过恢复治疗,各陈皮组的副沙门氏菌属(Parasutterella)和异杆菌属(Allobaculum)均有减少,毛螺菌科中的NK4A136_group属有所回升,Muribaculaceae则恢复到了Control组的水平。而与其他恢复组相比,Q-3组的菌群比例更为接近于Control组,可见陈皮能有效调节脾虚小鼠失调的肠道菌群,且三年青皮对肠道菌群稳态的恢复效果更为全面,优于其他陈皮组。
2.3Alpha多样性分析
Alpha Diversity用于分析样本内的微生物群落多样性,通过单样本的多样性分析(Alpha多样性)可以反映样本内的微生物群落的丰富度和多样性。
分析软件:Qiime 2
分析方法:采用对序列进行随机抽样的方法,以抽到的有效序列数进行ASV的分析,并分别计算各α多样性指数,检验结果以箱形图的方式展现,该图可直观的反映组内物种多样性的中位数、离散程度、最大值、最小值、异常值。
原始数据如图5所示,箱形图如图6~图9所示。
结果显示,脾虚小鼠的Ace指数、Chao指数以及Shannon指数下降极为显著,Simpson指数略为下降,可见脾虚小鼠相较于正常小鼠肠道菌群的丰富度和多样性下降极为明显。而在进行恢复治疗后,各组小鼠的Ace指数和Chao指数Shannon指数以及Simpson指数都有着明显升高,可见在经过广陈皮喂养的脾虚小鼠其肠道菌群丰富度和多样性得到了恢复。其中三年青皮恢复效果相较于其他陈皮更为优秀。
2.4PCA分析
组间的物种丰度分布差异程度可通过统计学中的距离进行量化分析,此次分析中共设计4种距离分析方式,分别由Bray-Curtis,Jaccard,Unweighted_unifrac,Weighted_unifrac其中的PCA分析(Principal Component Analysis),即主成分分析,是一种对数据进行简化分析的技术,这种方法可以有效地找出数据中最“主要”的特征值。通过分析不同样本的菌种群落分布,可以反映出样本间的相似度和差异,运用方差分解,将多组数据的差异反映在二维坐标图上。图中,PC1、PC2分别代表第一和第二主成分,主成分后的百分比代表此成分对样本组成差异的解释度。不同颜色的点代表不同分组,两样本点越接近,表明两样本物种组成越相似。X轴和Y轴的刻度是相对距离,无实际意义。算法计算距离,获得距离矩阵,进一步用于beta多样性分析和可视化。分析结果如图10~图12所示。
结果显示,通过分析发现,Model组样本点与包括其他各组的样本点明显处于不同的区域,表明Model组与其余各组间在肠道菌群组成方面存在显著差异。而喂养广陈皮水煎液的各组样本区域明显与Control组有重叠,样本点更为接近。其中Q-3组则与Control组最为接近,基本都在相同的区域内,可以见得这三年青皮相较于其他陈皮组对脾虚小鼠肠道菌群丰富度多样性和物种结构的恢复最为有效。
以上所述的实施例仅是对本发明的优选方式进行描述,并非对本发明的范围进行限定,在不脱离本发明设计精神的前提下,本领域普通技术人员对本发明的技术方案做出的各种变形和改进,均应落入本发明权利要求书确定的保护范围内。
Claims (8)
1.一种陈化三年广陈皮青皮提取物在制备肠道菌群失调产品中的应用。
2.根据权利要求1所述的应用,其特征在于,所述应用具体为在制备调节脾虚导致的肠道菌群失调产品中的应用。
3.权利要求2所述的应用中陈化三年广陈皮青皮提取物的制备方法,其特征在于,包括以下步骤:
(1)取陈化三年广陈皮青皮,加5~7倍水浸泡25~35min;
(2)将浸泡后的陈化三年广陈皮青皮煎煮25~35min并过滤;
(3)过滤后的残渣加入7~9倍水,再次煎煮25~35min并过滤;
(4)合并两次煎煮滤液,浓缩至浓度为0.4~0.6g/ml,所述浓缩液即为所述陈化三年广陈皮青皮提取物。
4.根据权利要求3所述的制备方法,其特征在于,所述步骤(1)中取陈化三年广陈皮青皮,加6倍水浸泡30min。
5.根据权利要求4所述的制备方法,其特征在于,所述步骤(2)中将浸泡后的陈化三年广陈皮青皮煎煮30min并过滤。
6.根据权利要求5所述的制备方法,其特征在于,所述步骤(3)中过滤后的残渣加入8倍水,再次煎煮30min并过滤。
7.根据权利要求6所述的制备方法,其特征在于,所述步骤(4)中将滤液浓缩至浓度为0.5g/ml。
8.使用权利要求3~7任一所述的制备方法制成的陈化三年广陈皮青皮提取物。
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