CN115364169A - 抗幽门螺杆菌的中药组合物及其制备方法与应用 - Google Patents
抗幽门螺杆菌的中药组合物及其制备方法与应用 Download PDFInfo
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Abstract
一种抗幽门螺杆菌的中药组合物及其制备方法与应用,涉及中药领域。本发明的地黄、黄精和蒲公英中药组合制备的提取物,可作为活性成分用于制备治疗幽门螺旋杆菌病的药物。本发明为传统中药材的应用开拓了新领域,为高效低毒抗幽门螺杆菌药物研发或保健食品开发提供了新方向。
Description
技术领域
本发明属于中药技术领域,涉及一种抗幽门螺杆菌的中药组合物及其制备方法与应用。
背景技术
幽门螺杆菌(Helicobacter pylori,HP)是一种定植于人胃黏膜的微需氧、螺旋状弯曲的革兰阴性杆菌,可以导致慢性胃炎、消化性溃疡,并与胃黏膜相关的淋巴样组织淋巴瘤及胃癌的发生密切相关。美国卫生及公共服务部发布了第15版致癌物名单,其中新增8种致癌物,HP被列为明确致癌物。随着HP耐药率升高,其根除治疗面临巨大挑战,其中以克拉霉素、左氧氟沙星及甲硝唑的耐药情况最为严重,导致HP根除难度逐渐增大,根除率下降,严重威胁人民健康。抗生素耐药性及不良反应多是西药治疗过程遇到的主要问题,且HP的耐药机制仍未阐明,因此需要寻找安全有效、毒副作用少且无耐药性的药物用于HP感染预防和治疗。目前很多中药对 HP感染相关的慢性胃炎和消化性溃疡等消化道疾病具有较好的疗效,某些药物的疗效可能与药物中含有特殊抗菌成分有关。
地黄为玄参科植物地黄(Rehmannia glutinosa Libosch)的新鲜或干燥块根,始载于《神农本草经》,地黄化学成分主要为环烯醚萜苷类、苯乙醇类、紫罗兰酮类、糖类、氨基酸等,具有降血糖、免疫抑制、抗肿瘤等方面作用,地黄中内生菌对革兰氏阴性和阳性细菌以及霉菌都有抗性,显示出抗菌谱广、抗菌活性强的特性。黄精(Polygonatum sibiricum)为百合科 (Liliaceae)黄精属(Pilygonatum)多年生草本植物根茎,多糖和皂苷类化合物是其主要活性成分,具有抗氧化、抗疲劳、抗肿瘤、增强免疫力、提高记忆力、降血脂、降血糖、抗炎、抗病毒、抗菌等生理功能。蒲公英是菊科植物蒲公英(Taraxacum mongolicumHand.-Mazz.)、碱地蒲公英(Taraxacum borealisinense Kitam.)或同属数种植物的干燥全草,含有挥发油、多糖、有机酸、黄酮、生物碱等活性成分,具有降糖、抗菌、抗炎、抗氧化、抗肿瘤、调节肠道微生态等多种生物学活性。目前应用地黄、黄精和蒲公英中药组合作为药效成分预防和/或治疗幽门螺杆菌相关疾病还末见报道,有望开发出安全有效的药剂。
发明内容
本发明的目的在于提供一种抗幽门螺杆菌的中药组合物及其制备方法与应用。
为实现上述目的及其他相关目的,本发明提供的技术方案是:一种抗幽门螺杆菌的中药组合物,原料配方包括以下重量份的原料:地黄15-25重量份、黄精32-42重量份、蒲公英 30-40重量份。
优选的技术方案为:原料配方由以下重量份的原料组成:地黄18重量份、黄精36重量份、蒲公英32重量份。
为实现上述目的及其他相关目的,本发明提供的技术方案是:一种采用上述原料配方制备中药组合物的方法,按所述原料配方取原料,经溶剂提取、过滤、合并滤液后浓缩得到浓缩浸膏。
优选的技术方案为:所述溶剂为水和体积分数为25-90%的乙醇水溶液中的至少一种。
为实现上述目的及其他相关目的,本发明提供的技术方案是:一种抗幽门螺杆菌的中药的制备方法,将步骤3得到的浓缩浸膏,加入或不加入药用辅料,制备成粉剂、颗粒剂、片剂、胶囊剂、丸剂、袋泡剂或者口服液。
为实现上述目的及其他相关目的,本发明提供的技术方案是:一种抗幽门螺杆菌的保健品,取步骤3得到的浓缩浸膏,加入或不加入辅料,制备保健饮品或保健酒。
由于上述技术方案运用,本发明与现有技术相比具有的优点是:
本发明的中药组合物药材选取合理,具有协同增效作用,大大增强中药组合物的抗炎抑菌的效果,对HP具有优异的抑制增殖和杀灭作用,安全性高,且能在短期内获得抑菌除菌效果。因此,本发明对抗HP和HP相关疾病的治疗、预防或改善具有积极作用,也为传统中药材的应用开拓了新领域,为高效低毒抗HP药物研发或保健食品开发提供了新方向。
附图说明
图1为HP镜检图片(1000×)。
图2为不同浓度中药组合物对菌液脲酶活性的抑制作用。
图3为80mg/L中药组合物对脲酶活性的抑制作用。
图4为80mg/L中药组合物不同孵育时间对脲酶活性的抑制作用。
具体实施方式
以下由特定的具体实施例说明本发明的实施方式,熟悉此技术的人士可由本实施例所揭露的内容轻易地了解本发明的其他优点及功效。
请参阅图1-4。须知,本说明书所附图式所绘示的结构、比例、大小等,均仅用以配合说明书所揭示的内容,以供熟悉此技术的人士了解与阅读,并非用以限定本发明可实施的限定条件,故不具技术上的实质意义,任何结构的修饰、比例关系的改变或大小的调整。提供以下实施例以便更好地理解本发明,而非限制本发明。以下实施例中的实验方法如无特殊说明,均为常规方法。下述实施例中所用的实验材料如无特殊说明,均为常规生化试剂商店购买所得。
实施例1:抗幽门螺杆菌的中药组合物及其制备方法与应用
1、主要材料:HP标准菌株H.pylori NCTC11637来源于安徽医科大学第一附属医院消化内科实验室。熟地黄、制黄精及蒲公英购自河南中科怀药研究院有限公司。
2、菌体培养:供试菌种接种于HP液体培养基试管内,在放有微需氧产气袋的密闭培养罐中37℃震荡培养36h后,将各菌转移至HP固体培养基进一步培养24-72h,处于对数生长期的菌落用刮铲刮下菌苔,溶于5mL生理盐水中,调整浓度至0.5麦氏浊度(1×l08 CFU/mL)。
3、中药组合物的最小抑菌浓度(MIC)测定
向33支无菌试管中分别加无菌HP液体培养基1mL,分成11组,每组设3个平行,用倍比稀释法将中药组合物依次稀释为最终浓度分别为512、256、128、64、32,16、8、4、2 mg/L。向每支1mL含不同药液浓度试管中加入108CFU/mL的HP菌液100mL保证每支试管含HP数为107CFU/mL,同时设不加中药组合物只加菌液为阳性对照,不加菌液不加中药组合物作为阴性对照。混匀后置密封培养罐中,并保持试管直立状态,放入产气袋、湿棉球,37℃震荡(100rpm/min)培养72h观察结果。将上述的各管培养物取出0.5mL溶液进行涂片染色鉴定,另取5uL转至新鲜哥伦比亚非选择性培养基培养72h无细菌生长时认为有抑菌作用,能够抑制HP生长的最低浓度为中药组合物的MIC值。
4、实验结果:涂片染色如图1所示,中药组合物对HP的最小抑菌浓度为8mg/L,说明中药组合物具有较好的抑菌效果。
实施例2:抗幽门螺杆菌的中药组合物及其制备方法与应用
脲酶是HP重要的定植因子与毒力因子,可分解尿素产生氨以中和胃酸,使HP可以在胃部生长繁殖,因此,抑制脲酶活性药物对抗HP活性的重要指标。
1、主要材料:HP和中药材来源同实施例1,脲酶提取和检测试剂盒来源于南京建成生物过程研究所。
2、菌体培养同实施例1。
3、菌液脲酶活性抑制实验:中药组合物与HP菌液混合,设置各组中药组合物终浓度分别为0、10、20、40、80、160、320、640mg/L,37℃震荡(100rpm/min)微氧培养6h,评价其脲酶活性差异。
脲酶活性抑制实验:常规方法提取HP脲酶,取中药组合物与脲酶于37℃孵育20min后测定其酶活性,中药组合物浓度取上述菌液脲酶活性抑制实验中最佳中药组合物浓度,设置 pH 3和4的柠檬酸溶液作为空白对照,试验重复3次。
4、实验结果:
(1)不同浓度中药组合物对菌液脉酶活性影响试验结果如图1所示。随着中药组合物浓度的增加,反应体系中脲酶活性逐渐减小,当中药组合物浓度达80mg/L时,残留脲酶活性显著降低,继续增加中药组合物浓度,各组间脲酶残留活性无显著差异。表明低浓度时中药组合物对菌液脲酶活性抑制作用与中药组合物浓度呈正相关性(如图2)。
(2)80mg/L中药组合物对脲酶活性具有明显抑制作用(P<0.01如图3),作用20min后,脲酶活性降至17%。随着孵育时间延长,脲酶活性更降至9%(如图4)。表明适当延长作用时间,有助于中药组合物发挥抑菌作用。
实施例3:抗幽门螺杆菌的中药组合物及其制备方法与应用
1、主要材料:SPF级小鼠由安徽医科大学动物实验中心提供,标准四联药物:艾司奥美拉唑肠溶胶囊、阿莫西林克拉维酸钾片、克拉霉素片、胶体果胶铋干混悬剂购于安徽百姓缘大药房,其他材料同实施例1。
2、给药方式:参考成人(60kg)每次用药剂量:艾司奥美拉唑20mg、阿莫西林1000mg、克拉霉素500mg、胶体果胶铋300mg,小鼠给药剂量均按60kg成人体表面积换算,小鼠每日等效剂量为262.5mg/kg。先将各药物按比例研碎混合,灌胃前溶于蒸馏水中。
3、实验方法:
小鼠适应性饲养1周后,按随机数字表法将小鼠随机分为正常组与造模组。造模组第1 天开始感染HP菌液(1×109CFU/ml,0.2ml/只),隔日感染1次,共5次,感染后定植2周。
造模组按随机数字表分为5组,每组8只,即模型组,标准四联组,中药高、中、低剂量组。正常组和模型组小鼠每日予以生理盐水灌胃。中药组给予不同浓度中药组合物(400,200,100mg/kg)灌胃,标准四联组予以四联药物混悬液灌胃。每日一次,共给药2周。
末次给药1h后脱颈处死小鼠,剖腹取全胃,沿胃大弯剪开,生理盐水冲洗胃内容物后,暴露胃黏膜,肉眼观察胃窦及胃体黏膜有无糜烂、出血和溃疡等。分别在胃窦、胃体、胃底处各取胃黏膜组织进行快速尿素酶试验。
4、实验结果:
(1)正常组小鼠胃黏膜组织完整,未见明显充血;模型组小鼠黏膜局部脱失,充血明显。中药组和标准四联组见少许出血现象。
(2)标准四联组及中药组中、高剂量给药两周后,快速尿素酶实验显示HP根除率均达到75%以上(表1)。
表1小鼠胃黏膜组织快速尿素酶实验结果
组别 | 数量 | HP(-) | HP(+) | HP清除率 |
正常组 | 8 | 8 | 0 | - |
模型组 | 8 | 0 | 8 | - |
标准四联组 | 8 | 7 | 1 | 100% |
中药组(400mg/kg) | 8 | 7 | 4 | 87.5% |
中药组(200mg/kg) | 8 | 6 | 75% | |
中药组(100mg/kg) | 8 | 4 | 50% |
实施例1:抗幽门螺杆菌的中药组合物及其制备方法与应用
中药组合在制备抗HP药物中的应用,或由于HP导致的慢性胃炎、消化性溃疡或胃黏膜相关疾病的药物或保健品中的应用。
优选的技术方案为:中药组合中,以重量份计,含有地黄15-25份、黄精32-42份、蒲公英30-40份。
优选地,以重量份计,所述中药组合的活性成分由包括如下组分的原料制备而成:地黄 18份、黄精36份、蒲公英32份。
本发明所述中药组合物精选药材原料的药物性味与归经如下:
熟地黄:甘,微温。归肝、肾经。补血滋阴,益精填髓。
制黄精:甘,平。归脾、肺、肾经。补气养阴,健脾,润肺。
蒲公英:味苦甘,性寒。归肝、胃经。清热解毒,消肿散结。
上述各味药材协同配合,使所述中药组合物具有杀菌抗炎作用,能用于HP引发胃黏膜相关疾病的预防或治疗。
本发明提供的中药组合物制备方法的技术方案包括:溶剂提取,过滤,合并滤液,浓缩,合并浓缩液等。具体步骤为:
1.称取原料药熟地黄、制黄精和蒲公英,分别粉碎备用;
2.将熟地黄和制黄精的粉碎物料混合制成混合物料,加水浸泡后,加热保温搅拌提取,过滤收集提取液;
3.将蒲公英粉碎物料,加水浸泡后,加热保温搅拌提取,过滤收集提取液;
4.对步骤2和3提取液分别浓缩处理,获得熟地黄、制黄精浓缩浸膏和蒲公英浓缩浸膏;
5.将步骤4浓缩浸膏混合搅拌均匀即可。
其中,步骤1所述熟地黄和制黄精粉碎为0.1-0.5cm3块状,蒲公英粉碎粒度为60-180目。
其中,步骤2和3所述加热提取包括步骤:
(1)将物料加水浸泡后,开始第一次加热提取,水与混合物料的总重量比为7-10:1,水温维持在50-90℃,50-200rpm/min超声搅拌提取1-3h后,过滤收集滤液;
(2)向滤渣中加25-90%的乙醇水溶液,开始第二次加热提取,乙醇水溶液与混合物料的重量比为7-10:1,加热温度为45-85℃,50-200rpm/min搅拌提取1-3h后,过滤收集滤液;
(3)向滤渣中加水,开始第三次加热提取,水与混合物料的重量比为3-6:1,水温维持在80-100℃,50-200rpm/min超声搅拌提取1-2h后,过滤收集滤液;
(4)将三次过滤的滤液合并。
优选的,步骤(1)中所述浸泡时间为30-90min,优选为45-60min;水与混合物料的重量比优选为8-9:1;温度优选为60-80℃,转速优选为100-150rpm/min,加热提取时间优选为 2-3h。
优选的,步骤(2)中所述为40-80%的乙醇水溶液;乙醇水溶液与混合物料的重量比优选为8-9:1,温度优选为50-80℃,转速优选为100-150rpm/min,加热提取时间优选为2-3h。
优选的,步骤(3)中水与混合物料的重量比优选为4-5:1;温度优选为90-100℃,转速优选为150-200rpm/min,加热提取时间优选为1-1.5h。
其中,步骤4所述浓缩温度为50-90℃,压力为-0.01~-0.1Mpa。
其中,步骤5所述浓缩浸膏的相对密度为1.2-1.6,优选为1.3-1.5,进一步优选为1.4-1.5。
为实现上述目的及其他相关目的,本发明提供的技术方案是:一种抗幽门螺杆菌的药物,取浓缩浸膏,加入或不加入药用辅料,按常规方法制备成粉剂、颗粒剂、片剂、胶囊剂、丸剂、袋泡剂或者口服液。
为实现上述目的及其他相关目的,本发明提供的技术方案是:一种抗幽门螺杆菌的保健品,取浓缩浸膏,加入或不加入辅料,按常规方法制备保健饮品或保健酒。
以上所述者仅为用以解释本发明之较佳实施例,并非企图具以对本发明做任何形式上之限制,是以,凡有在相同之发明精神下所作有关本发明之任何修饰或变更,皆仍应包括在本发明意图保护之范畴。
Claims (6)
1.一种抗幽门螺杆菌的中药组合物,其特征在于:原料配方包括以下重量份的原料:地黄15-25重量份、黄精32-42重量份、蒲公英30-40重量份。
2.根据权利要求1所述的抗幽门螺杆菌的中药组合物,其特征在于:原料配方由以下重量份的原料组成:地黄18重量份、黄精36重量份、蒲公英32重量份。
3.一种采用权利要求1或2所述的原料配方制备中药组合物的方法,其特征在于:按所述原料配方取原料,经溶剂提取、过滤、合并滤液后浓缩得到浓缩浸膏。
4.根据权利要求1-2所述中药组合物的制备方法,其特征在于:所述溶剂为水和体积分数为25-90%的乙醇水溶液中的至少一种。
5.一种抗幽门螺杆菌的中药的制备方法,其特征在于:将步骤3得到的浓缩浸膏,加入或不加入药用辅料,制备成粉剂、颗粒剂、片剂、胶囊剂、丸剂、袋泡剂或者口服液。
6.一种抗幽门螺杆菌的保健品,其特征在于:取步骤3得到的浓缩浸膏,加入或不加入辅料,制备保健饮品或保健酒。
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