CN109010420A - 一种川芎和葛根药用组合物在制备改善肠道环境的食品或药品中的用途 - Google Patents
一种川芎和葛根药用组合物在制备改善肠道环境的食品或药品中的用途 Download PDFInfo
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Abstract
本发明涉及一种川芎和葛根药用组合物在制备改善肠道环境的食品或药品中的用途,具体公开了一种川芎和葛根药用组合物在制备改善肠道环境的食品或药品中的用途;所述川芎和葛根药用组合物为葛根和川芎的水提取物,其中葛根和川芎的水提取物中包含葛根素、大豆苷、川芎嗪和大豆苷元;其中葛根素在药用组合物中的含量为0.8mg/g以上,大豆苷在药用组合物中的含量为0.2mg/g以上;川芎嗪在药用组合物中的含量为0.7mg/g以上;大豆苷元含量在0.02mg/g以上。
Description
技术领域
本发明涉及一种中药组合物的用途,具体涉及川芎和葛根药用组合物在制备改善肠道环境的食品或药品中的用途。
背景技术
肠道中栖息着数量庞大、种类繁多的微生物,这些微生物与其所寄居的肠道环境组成了肠道微生态系统。肠道微生物是肠道微生态系统的重要组成部分,人类在长期进化过程中与肠道微生物形成了互利共生的关系。肠道微生物可参与多种疾病如心脑血管系统、消化系统、中枢神经系统、免疫系统以及代谢性疾病的发生与发展[1-7],与健康密切相关。肠道微生物可在肠黏膜表面形成保护性的生物屏障以维持肠上皮结构和功能的完整性,并抑制病原菌的过度增殖和感染,促进并维护免疫系统的正常运行[8-10]。一旦受到宿主自身疾病及外界环境变化的影响,肠道微生物中共生菌、益生菌、致病菌、条件致病菌的相对数量和丰度可能会发生变化,打破肠道微生物本来的平衡状态,进而引发肠道菌群失调,诱发或加速许多疾病的发生与发展[11-13]。如消化系统疾病:IBD、IBS等;神经系统疾病:孤独症、抑郁症等;代谢系统疾病:糖尿病、肥胖等;心脑血管系统疾病:缺血性脑卒中等。肠道菌群失调可直接侵袭损伤肠道上皮细胞,破坏肠道黏膜屏障,增加肠黏膜通透性[14];肠道菌群失调也会导致大量致病菌滋生,使肠道上皮细胞受到各种有毒物质如内毒素、肠毒素等的侵害[15]。因此,维持肠道微生态系统的平衡对宿主的生理健康显得尤为重要。
1、针对上述缺点,说明本发明的目的及能够达到的技术效果。
中药是我国传统医学的重要组成部分,中药用于预防和治疗疾病已经有几千年历史。中药最传统的给药方式为口服,药物口服后在胃肠道内不可避免的与肠道微生物接触,因此,肠道微生物对中药的代谢转化具有非常重要的影响,肠道微生物可将药物转化为代谢产物,起到“增效”或“减毒”的作用。而中药口服后也在很大程度上影响了肠道微生物的结构和组成。例如,中药能够保护胃肠道黏膜屏障功能,促进益生菌的繁殖,抑制有害菌的生长,对维持肠道微生态系统平衡起着关键作用[16]。
中药葛根(Puerariae Lobatae Radix)药食同源,在中国和日本有数千年的食用历史,有“亚洲人参”之美誉,由葛根制的的葛粉被称之为“长寿粉”,在日本被誉为“皇室特供食品”,可见葛根具有极高的食用和药用价值。研究表明,葛根不仅能调控肠道微生物代谢碳水化合物、脂类、蛋白质的能力,还能显著地增加乳杆菌和双歧杆菌等益生菌的数量,提高肠道微生物的抗氧化性,具有极好的改善肠道微生态的作用[17]。中药川芎有活血行气、祛风止痛的功效。研究表明,川芎不仅可以活血化淤,也能够通过其对内毒素的灭活作用来防治肠道微生物失调及内毒素血症[18]。因此,本发明旨在开发一种能够改善肠道微生态的肠内营养药用组合物。本发明根据葛根、川芎等中药的药理活性特点,以中医药配伍理论为依据,结合肠道菌群16S rRNA基因测序和药效学研究,公开一种改善肠道微生态的肠内营养药用组合物。该药物组合物可作为膳食添加剂补充益生元去有效改善正常生理条件下和疾病状态下的肠道微生态,增加益生菌的种类和数量,降低致病菌的种类和数量,维持肠道上皮完整性,阻止肠道微生物及其代谢产物移位,从而有效地发挥肠内营养的作用。有效的预防和治疗某些疾病如消化系统疾病:IBD、IBS等;神经系统疾病:孤独症、抑郁症等;代谢系统疾病:糖尿病、肥胖等;心脑血管系统疾病:缺血性脑卒中等。
发明内容
本发明涉及一种能够改善肠道微生态的肠内营养药用组合物。该药物组合物可作为益生元去有效改善正常生理条件下和疾病状态(如消化系统疾病:IBD、IBS等;神经系统疾病:孤独症、抑郁症等;代谢系统疾病:糖尿病、肥胖等;心脑血管系统疾病:缺血性脑卒中等)下的肠道微生态,增加益生菌的种类和数量,降低致病菌的种类和数量,维持肠道上皮完整性,阻止肠道微生物及其代谢产物移位,从而有效地发挥肠内营养的作用。该组合物主要由以下原料组成:川芎或川芎提取物与葛根或葛根提取物,二者的重量配比范围为川芎或川芎提取物1-500份、葛根或葛根提取物1-1000份。该药用组合物可以单独或与药用辅料组合制成适合于口服的制剂形式。如颗粒剂、胶囊剂、片剂、滴丸剂、液体制剂如口服液、混悬剂、糖浆剂等以及缓释、控释制剂等。通过实验研究表明:本发明提供的药用组合物一方面可明显减少正常生理状态下的肠道微生物群的多样性,改变肠道微生物的组成结构,并能够降低厚壁菌门(Firmicutes)与拟杆菌门(Bacteroidetes)的比例。另一方面,该药用组合物能显著增加疾病模型(缺血性脑卒中)下益生菌的种类和数量,降低致病菌的种类和数量。增加的益生菌主要包括:Alloprevotella,Ruminococcaceae_UCG_005,Ruminococcaceae_NK4A214_group,Ruminococcaceae_UCG_004,Oscillospira,Lachnospiraceae_NK4B4_group,Akkermansia and Megasphaera。降低的致病菌主要包括:Bacteroides,Escherichia_Shigella,Haemophilus,Eubacterium_nodatum_group,Collinsella,Enterococcus,Proteus,Alistipes,Klebsiella,Shuttleworthia和Faecalibacterium。此外,该药物组合物还可以通过增加疾病状态(缺血性脑卒中)下大鼠脑与肠组织中紧密连结蛋白claudin-5和ZO-1的表达,有效地维持了脑与肠组织紧密连接的完整性,从而能够逆转疾病状态(缺血性脑卒中)下血脑屏障和肠道屏障的破坏,因此有效阻止肠道微生物及其代谢产物移位。因此本发明提供的药用组合物对正常生理状态下和心脑血管疾病如(缺血性脑卒中)状态下的肠道微生态均具有很好的调节营养作用。
本发明提供的药用组合物,川芎提取物或川芎以川芎嗪计,其含量不低于10%。
工艺:(水或乙醇提取)
称取质量比例为1:1的葛根和川芎,以每克生药加水10mL,冷水浸泡1h后,煎煮1h,取滤液;药渣再用10倍的水煎煮1h,取滤液,合并两次滤液;浓缩。
本发明经过对正常生理状态大鼠进行药物诱导(使用该药物组合物灌胃处理14天),结合线栓法制备的大鼠右侧大脑中动脉栓塞(Middle Cerebral Artery Occlusion,MCAO)模型及药效学实验验证,结果表明,本发明提供的药用组合物一方面可明显减少正常生理状态下的肠道微生物群的多样性,改变肠道微生物的组成结构,并能够降低厚壁菌门(Firmicutes)与拟杆菌门(Bacteroidetes)的比例。另一方面,该药用组合物能显著增加疾病模型(缺血性脑卒中)下益生菌的种类和数量,降低致病菌的种类和数量。除此之外该药物组合物还可以通过增加Claudin-5和ZO-1的表达,有效地维持了紧密连接的完整性,从而能够逆转缺血性脑卒中疾病状态下血脑屏障和肠道屏障的破坏并能够有效地阻止肠道微生物及其代谢产物移位。因此本发明提供的药用组合物对正常生理状态下和心脑血管疾病如(缺血性脑卒中)状态下的肠道微生态均具有很好的调节作用。
本发明一个方面提供了一种川芎和葛根药用组合物在制备改善肠道环境的食品或药品中的用途;所述川芎和葛根药用组合物为葛根和川芎的水提取物,其中葛根和川芎的水提取物中包含葛根素、大豆苷、川芎嗪和大豆苷元;其中葛根素在药用组合物中的含量为0.8mg/g以上,大豆苷在药用组合物中的含量为0.2mg/g以上;川芎嗪在药用组合物中的含量为0.7mg/g以上;大豆苷元含量在0.02mg/g以上。
在本发明的技术方案中,所述的川芎和葛根药用组合物通过以下方法制备,称取质量比例为1:1的葛根和川芎,以每克生药加水10mL,冷水浸泡1h后,煎煮1h,取滤液;药渣再用10倍的水煎煮1h,取滤液,合并两次滤液;浓缩。
在本发明的技术方案中,改善肠道环境是指提改变肠道微生物的组成结构。
在本发明的技术方案中,改变肠道微生物的组成结构是指增加益生菌的种类和数量,降低致病菌或条件致病菌的种类和数量。
在本发明的技术方案中,所述的益生菌选自Alloprevotella,Ruminococcaceae_UCG_005,Ruminococcaceae_NK4A214_group,Ruminococcaceae_UCG_004,Oscillospira,Lachnospiraceae_NK4B4_group,Akkermansia和Megasphaera,致病菌选自厚壁菌门(Firmicutes),拟杆菌门,Bacteroides,Escherichia_Shigella,Haemophilus,Eubacterium_nodatum_group,Collinsella,Enterococcus,Proteus,Alistipes,Klebsiella,Shuttleworthia,Faecalibacterium。
在本发明的技术方案中,改善肠道环境是指改善了缺血性脑卒中患者的肠道环境。
在本发明的技术方案中,改善肠道环境是指阻止缺血性脑卒中患者肠道微生物移位。
在本发明的技术方案中,阻止缺血性脑卒中患者肠道微生物移位是指维持缺血性脑卒中患者肠屏障功能的完整性。
有益效果
采用川芎与葛根适宜配比的药用组合物无论对正常生理状态下的肠道微生态还是缺血性脑卒中疾病状态下的肠道微生态均有较好的调节作用,可以开发成为膳食添加剂补充益生元达到肠内营养的作用。
附图说明
图1:川芎、葛根药用组合物调节正常生理状态下的肠道微生态,其中a为微生物在门水平上的相对丰度,b为门水平上有差异的微生物,c为正常组与药物组物诱导组的生物标记物
图2:川芎、葛根药用组合物调节缺血性脑卒中疾病状态下的肠道微生态;其中,a为模型对照组,b为空白组,c为尼莫地平组,d为药物组和e为生理盐水组。
a组各菌生物标记丰度从上至下为1Bacteroides,2Escherichia_Shigella,3Allobaculum,4Coprococcus_2,5Ruminococcaceae_UCG_013,6Subdoligranulum,7Turicibacter,8Haemophilus,9Paeniclostridium,10Eubacterium_nodatum_group,11Eubacterium_ruminantiumgroup,12Bacteroides_pectinophilus_group,13Paraprevotella,14Faecalibaculum,15Collinsella,16Ruminiclostridium_1,17Family_Xlll_AD3011_Group,18Eisenbergiella,19Bifidobacterium,20Enterococcus,21Anaerofilum,22Proteus,23Coriobacteriaceae_UCG_002,24Defluviitaleaceae_UCG_011,25uncultured。
b组各菌生物标记丰度从上至下为1Sutterella,2Lachnospiraceae_NK4A136_group,3Parabacteroides,4Intestinimonas,5Eubacterium_coprostanoligenes_group,6Lachnospiraceae_UCG_001,7Eubacterium_xylanophilum_group,8Butyrivibrio,9Roseburia,10Lachnoclostridium,11Fusicatenibacter,12Coprococcus_1,13Oscillibacter,14Tyzzerella,15Acetatifactor,16Mucispirillum,17Anaeroplasma,18Lachnospiraceae_FCS020_group,19Ruminiclostridium,20Peptococcus,21Butyricicoccus,22Lachnospiraceae_ND3007_group,23Eubacterium_ventriosum_group
c组各菌生物标记丰度从上至下为1Helicobacter,2Rikenellaceae_RC9_gut_group,3uncultured_bacterium,4Clostridium_sensu_stricto_1,5Ruminoccaceae_UCG_010,6Christensenellaceae_R_7_group,7Ambiguous_taxa,8uncultured_bacterium,9uncultured,10Butyricimonas,11uncultured_bacterium,12Ambiguous_taxa,13Fusobacterium,14Prevotellaceae_NK3B31_group,15Epulopiscium,16Eubacterium_eligens_group,17uncultured_rumen_bacterium,18Gelria,19Anaerostipes,20Erysipelotrichaceae_UCG_004,21Erysipelotrichaceae_UCG_003,22Trichococcus,23Papillibacter,24Erysipelatoclostridium。
d组各菌生物标记丰度从上至下为1Prevotella_9,2Alloprevotella,3Ruminococcaceae_UCG_005,4Ruminococcaceae_NK4A214_group,5uncultured_Bacteroidales_bacterium,6Ruminococcaceae_UCG_004,7Oscillospira,8Lachnospira,9Lachnospiraceae_NK4B4_group,10Dialister,11Veillonella,12Ruminococcus_gnavus_group,13Odoribacter,14Anaerofustis,15Akkermansia,16Megasphaera,17Ambiguous_taxa。
e组各菌生物标记丰度从上至下为1uncultured_bacterium,2Alistipes,3Prevotellaceae_UCG_001,4Ruminococcaceae_UCG_014,5Ruminococcus_1,6Phascolarctobacterium,7Ruminiclostridium_6,8Klebsiella,9Candidatus_Saccharimonas,10Ruminiclostridium_9,11uncultured_bacterium,12Shuttleworthia,13Ambiguous_taxa,14uncultrred,15Candidatus_Arthromitus,16Megamonas,17Faecalibacterium,18gut_metagenome,19Ruminococcaceae_UCG_002,20Psychrobacter。
图3:Claudin-5在结肠中的表达;
图4:ZO-1在结肠中的表达;
图5:Claudin-5在大脑中的表达;
图6:ZO-1在大脑中的表达;
图7:血清中LPS活性;
图8:血清中二胺氧化酶(DAO)活性;
图9:血清中D-乳酸盐的浓度。
具体实施方式
提供下述实例是为了更好地进一步理解本发明,并不局限于所述实施方式,不对本发明的内容和保护范围构成限制,任何人在本发明的启示下或是将本发明与其他现有技术的特征进行组合而得出的任何与本发明相同或相近似的方法,均落在本发明的保护范围之内。
实施例1川芎、葛根药用组合物的制备及质量控制
称取葛根和川芎,其中葛根和川芎的比例为1:1,以每克生药加水10mL,冷水浸泡1h后,煎煮1h,取滤液。药渣再用10倍的水煎煮1h,取滤液,合并两次滤液。浓缩至含葛根素5mg/mL的葛根川芎合煎液。置4℃冰箱保存备用。给药剂量均按照葛根素50mg/kg。本发明提供的药用组合物主要含的成分有葛根素、川芎嗪、大豆苷、大豆苷元。通过HPLC检测其含量及给药剂量如表1所示。
表1、川芎、葛根药用组合物中主要成分的含量及给药剂量
实施例2川芎、葛根药用组合物调节正常生理状态下的肠道微生态
取至少10只健康雄性SD大鼠(体质量240-260g),灌胃给以实施例1所述给药剂量给予该组合物14天,之后采用腹部按摩法收集新鲜粪便。采用PowerSoil DNA IsolationKit(Mobio,美国)对粪便中的DNA进行提取,通过Illumina Miseq平台对所有样本的16SrRNA基因的V4区进行测序。并对测序结果进行生物信息学分析与挖掘。结果表明,如图1a,1b所示,川芎、葛根药用组合物显著地增强了拟杆菌(Bacteroidetes)和柔膜菌门(Tenericutes)的相对丰度,并显著降低了厚壁菌(Firmicutes)和脱铁杆菌(Deferribacteres)的相对丰度。如图1c所示,川芎、葛根药用组合物显著改变了肠道微生物群的组成结构并降低了厚壁菌与拟杆菌的比例。
实施例3川芎、葛根药用组合物调节缺血性脑卒中疾病状态下的肠道微生态
采用线栓法制备大鼠右侧大脑中动脉栓塞(Middle Cerebral ArteryOcclusion,MCAO)模型用于模拟缺血性脑卒中疾病,采用Longa评分法对其神经功能学进行评分,并结合全脑TTC染色对该模型成功与否进行评价。取至少50只雄性SD大鼠(体质量240-260g),随机分为空白(Control),模型对照组(MCAO),生理盐水组(Saline),实施例1药物组(Decoction)和尼莫地平组(Nimodipine)。其中空白的大鼠未受到任何干预,只是灌胃给以同体积的生理盐水。模型对照组组,大鼠缺血2小时,再灌注24小时。在剩余三组中,分别在模型对照组建模后给大鼠灌胃生理盐水,上述药物组合物(含50mg/kg葛根素)或尼莫地平片剂(用生理盐水溶解,阳性对照组,190mg/kg)并持续14天。同样收集新鲜粪便进行16S rRNA基因测序。如图2所示,结果表明,缺血性脑中风通过富集致病菌和机会致病菌引发肠道微生物紊乱,包括拟杆菌(Bacteroides),大肠志贺氏杆菌(Escherichia_Shigella),嗜血杆菌(Haemophilus),纠缠真杆菌(Eubacterium_nodatum_group),柯林斯氏菌(Collinsella),肠球菌(Enterococcus),变形杆菌(Proteus),另枝菌(Alistipes),克雷伯氏菌(Klebsiella),Shuttleworthia和Faecalibacterium。而本发明提供的川芎、葛根药用组合物可以通过增加益生菌来重塑缺血性脑卒中状态下的肠道微生态,这些益生菌包括拟普雷沃菌(Alloprevotella),瘤胃菌(Ruminococcaceae),颤螺菌(Oscillospira),毛螺旋菌(Lachnospiraceae_NK4B4_group),阿克曼菌(Akkermansia)和巨球型菌属(Megasphaera)。但是口服尼莫地平虽然能够缓解缺血性脑卒中的状态,但是其使肠道中富集了大量的致病菌,加剧了肠道微生物群失调。
实施例4川芎、葛根药用组合物调节缺血性脑卒中疾病状态下的脑屏障和肠道屏障
采用试剂盒(参见表2)对结肠和脑组织中的紧密连接蛋白,包括Claudin-5,ZO-1的蛋白含量进行了测定。如图3-6以及表3所示,我们的结果表明,在缺血性脑卒中疾病模型下,Claudin-5,ZO-1的蛋白含量在结肠和脑组织中显著降低,表明缺血性脑卒中不仅破坏血脑屏障,也破坏肠屏障。阳性药物尼莫地平不会改善这种损伤,然而本发明提供的川芎、葛根药用组合物能够逆转这种损伤,能够很好的维持脑屏障和肠屏障结构和功能的完整性。当肠屏障受到破坏时,肠道微生物及其代谢产物可能会发生移位,通过肠上皮进入血液循环,对机体造成损害。因此我们进一步研究了肠道微生物移位情况。
表2试剂盒的信息表
表3紧密连接蛋白在结肠和脑中的表达
实施例5川芎、葛根药用组合物阻止缺血性脑卒中疾病状态下肠道微生物移位
采用试剂盒测定血清中肠道微生物移位的标记物,主要包括脂多糖(LPS)、二胺氧化酶(DAO)、D-乳酸盐。如图7-9以及表4所示,我们的结果表明,在缺血性脑卒中疾病状态下,DAO,LPS,D-乳酸盐显著增加,表明肠道微生物和其代谢产物发生了移位。阳性药物尼莫地平,加重了对肠屏障的损伤及肠道微生物移位,引起大量内毒素入血。而本发明提供的川芎、葛根药用组合物能够有效的维持肠屏障功能的完整性,抑制肠道微生物和其代谢产物的移位。
表4肠道微生物移位标记物
注;不同字母表示P<0.05,具有统计学差异,相同字母无差异。
Claims (8)
1.一种川芎和葛根药用组合物在制备改善肠道环境的食品或药品中的用途,所述川芎和葛根药用组合物为葛根和川芎的水提取物,其中葛根和川芎的水提取物中包含葛根素、大豆苷、川芎嗪和大豆苷元;其中的葛根素在药用组合物中的含量为0.8mg/g以上,大豆苷在药用组合物中的含量为0.2mg/g以上;川芎嗪在药用组合物中的含量为0.7mg/g以上;大豆苷元含量在0.02mg/g以上。
2.根据权利要求1所述的用途,所述的川芎和葛根药用组合物通过以下方法制备,称取质量比例为1:1的葛根和川芎,以每克生药加水10mL,冷水浸泡1h后,煎煮1h,取滤液;药渣再用10倍的水煎煮1h,取滤液,合并两次滤液;浓缩。
3.根据权利要求1所述的用途,改善肠道环境是指提改变肠道微生物的组成结构。
4.根据权利要求3所述的用途,改变肠道微生物的组成结构是指增加益生菌的种类和数量,降低致病菌的种类和数量。
5.根据权利要求4所述的用途,所述的益生菌选自Alloprevotella,Ruminococcaceae_UCG_005,Ruminococcaceae_NK4A214_group,Ruminococcaceae_UCG_004,Oscillospira,Lachnospiraceae_NK4B4_group,Akkermansia和Megasphaera,致病菌选自厚壁菌门(Firmicutes),拟杆菌门,Bacteroides,Escherichia_Shigella,Haemophilus,Eubacterium_nodatum_group,Collinsella,Enterococcus,Proteus,Alistipes,Klebsiella,Shuttleworthia,Faecalibacterium。
6.根据权利要求1所述的用途,改善肠道环境是指改善了缺血性脑卒中患者的肠道环境。
7.根据权利要求1所述的用途,改善肠道环境是指阻止缺血性脑卒中患者肠道微生物移位。
8.根据权利要求1所述的用途,阻止缺血性脑卒中患者肠道微生物移位是指维持缺血性脑卒中患者肠屏障功能的完整性。
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CN110663950A (zh) * | 2019-09-11 | 2020-01-10 | 南昌大学 | 一种具有增加肠道黏蛋白的提取物及其组合物 |
CN110679799A (zh) * | 2019-09-11 | 2020-01-14 | 南昌大学 | 一种具有增加肠道黏液层毛螺科菌群的提取物及其组合物 |
CN115212225A (zh) * | 2021-11-19 | 2022-10-21 | 南方医科大学 | Rs3型抗性淀粉在防治缺血性脑卒中药物中的应用及其制备 |
CN115212225B (zh) * | 2021-11-19 | 2024-04-12 | 南方医科大学 | Rs3型抗性淀粉在防治缺血性脑卒中药物中的应用及其制备 |
CN114767730A (zh) * | 2022-04-14 | 2022-07-22 | 广东省疾病预防控制中心 | 葛根提取物在防治肠道菌群紊乱中的新应用 |
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