CN109223888B - 抑制产肠毒素大肠杆菌K88菌毛的fae,faeG基因表达的中药药物组合物及应用 - Google Patents
抑制产肠毒素大肠杆菌K88菌毛的fae,faeG基因表达的中药药物组合物及应用 Download PDFInfo
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Abstract
本发明公开了一种抑制产肠毒素大肠杆菌K88菌毛的fae,faeG基因表达的中药药物组合物及应用,它包括黄柏提取物和石榴皮提取物。本发明利用天然植物提取物抑制产肠毒素大肠杆菌菌毛的fae,faeG基因表达,降低ETEC在仔猪肠道上皮细胞的定植和繁殖,而不影响有益微生物在肠道的定植,增加了肠道有益菌的丰度,在降低仔猪发生腹泻风险的同时,保障了肠道菌群的多样性;同时黄柏和石榴皮中的抗氧化物质,保护仔猪断奶应激导致的肠道氧化损伤;与抗生素相比,中药药物组合物不易产生耐药性,且能克服抗生素在杀灭有害菌的同时,也降低了有益菌的丰度的副作用,更有利于生猪健康养殖,可以在临床上作为一种饲料添加剂长期使用。
Description
技术领域
本发明涉及药物组合物,具体涉及一种抑制产肠毒素大肠杆菌K88菌毛的fae,faeG基因表达的中药药物组合物及应用。
背景技术
产肠毒素大肠杆菌(Enterotoxigenic Escherichia coli,ETEC)属于肠杆菌科,埃希菌属,大肠埃希菌种,是致病性大肠杆菌中最常见的一类。ETEC是造成仔猪腹泻的主要致病菌,其致病性决定于在小肠上皮细胞上的定植能力和产生肠毒素的能力。肠毒素是导致腹泻的直接原因,只有大肠杆菌黏附在小肠上皮细胞上,才能避免被肠道蠕动和肠液冲刷所清除,进而在宿主体内定居和繁殖分泌肠毒素。ETEC作用的基础是能够黏附于肠道上皮细胞上,这一功能由细菌表面的菌毛所介导,菌毛黏附素与上皮细胞上的受体结合,两种物质在构型上相互匹配,菌毛黏附素可以接近上皮细胞与受体以共价键结合在一起,引起上皮细胞的蛋白磷酸化,细胞间Ca+升高,随后定植在肠道内大量繁殖,在ETEC生长过程中释放大量的肠毒素引起细胞内水和电解质失衡,从而产生腹泻。菌毛是细菌识别肠上皮细胞受体的重要结构,不同ETEC的菌毛抗原类型不同,其中以K88最为普遍和最为重要。根据血清型不同,K88可分为K88ab、K88ac和K88ad,对于K88的3种血清型,流行病调查表明K88ac是引起仔猪腹泻的主要病原菌,有研究通过检测腹泻仔猪体内44株阳性大肠杆菌发现,96%大肠杆菌有K88ac菌毛基因,仅有4%的大肠杆菌含有K88ab菌毛基因。K88菌毛的化学本质为蛋白质,为直径约2.1nm的丝状、柔毛样结构,K88菌毛蛋白的相对分子量在23.5-26.0之间。菌毛基因表达受温度、pH值和培养基组成的影响调控,37℃时表达良好,18℃~25℃时只产生少量菌毛,18℃以下时不产生菌毛。
编码K88菌毛的基因由faeA至faeJ这10个基因所构成,所编码的蛋白对应为FaeA至FaeJ,见图1。K88菌毛由几百个蛋白亚基构成,其中FaeG是主要结构蛋白,由faeG基因编码,分子量为27.5kDa,FaeG蛋白不仅含有大量的K88菌毛抗原决定簇,而且含有与K88受体结合的位点,除了FaeG亚基蛋白外,一些小分子蛋白如FaeC、FaeF、FaeH也是菌毛的组成部分。FaeC蛋白分子量为16.9kDa,位于菌毛顶端与启动菌毛的合成有关。FaeF(15.2kDa)和FaeH(25.5kDa)是影响菌毛合成的重要因素,参与菌毛的延伸过程,可以形成新生菌毛的支架,常分布于菌毛轴心两侧的菌毛体中,FaeD和FaeE是负责转运菌毛大小亚基蛋白穿过细胞周质和细胞膜的蛋白,K88菌毛中主要亚基和次要亚基在穿过大肠杆菌细胞质膜和外膜时至少需要FaeE和FaeD两种蛋白的协助。FaeE可以与菌毛蛋白FaeG、FaeH和FaeI在细胞周质形成异源蛋白复合物,防止提前聚合或被降解,传递菌毛亚基蛋白给外膜的FaeD转运蛋白,FaeD则转运菌毛亚基蛋白至胞外形成菌毛,同时将菌毛固定于细胞表面。FaeA是阻遏蛋白,FaeB是激活蛋白,两种蛋白主要起调控菌毛表达的作用,不参与K88菌毛的组成。
黄柏为芸香科黄檗属植物。黄柏中含有大量生物活性成分,如生物碱、甾醇、内酯及黏液质等。生物碱是黄柏中最重要的活性物质,目前发现的主要成分为小檗碱,还含有黄柏碱、巴马汀、木兰花碱、药根碱、黄柏酮等。黄柏的化学成分研究始于1926年,日本学者村山义温等从日本产黄柏中得到小檗碱及少量巴马汀。小檗碱是黄柏中最主要的活性成分,因此,小檗碱的功效与黄柏提取物大体一致。小檗碱又名黄连素,是从中国传统中药黄连的根、茎中提炼出的主要成分,是一种异喹啉类生物碱。小檗碱在人医临床中成人每天400mg(成人按照平均60kg计算,相当于6.5mg/kgBW),分4次口服,用于腹泻的治疗。黄柏作为小檗碱的一种来源,因其价格相对黄连具有优势,近年来逐渐受到关注。黄柏的主要成分为小檗碱,黄柏中盐酸小檗碱含量不得低于3.0%(中国药典2015),但是黄柏提取物用于断奶仔猪腹泻的应用研究较少,黄柏提取物对断奶仔猪腹泻的作用机理的研究更是微乎其微。
石榴皮为石榴科植物石榴的干燥果皮。其性酸、温、涩,具有涩肠止泻、止血、驱虫等作用,用于久泻、久痢、便血、脱肛、崩漏、白带、虫积腹痛。现代药理研究证实:石榴皮中的鞣花酸、黄酮类化合物是其活性组分,由于其独特的生理活性如降血脂、抗氧化、抗病毒、抗肿瘤、降糖、抗腹泻及抗菌等,近年来成为科学家研究的热点。抗氧化作用是其生物活性的基础,因此,最近十几年有关石榴皮抗氧化活性方面的药理研究结果表明,石榴皮中的鞣质类化合物是其抗氧化作用的有效组分,石榴皮鞣质对自由基侵害具有保护作用。另外研究发现石榴皮中鞣花酸和黄酮类化合物体外对金黄色葡萄球菌、福氏痢疾杆菌、沙门氏菌、大肠杆菌、绿脓杆菌和白色念珠菌均有不同程度的抑菌作用,且鞣质类化合物呈现广谱抗菌特性,并具有抗耐药菌作用。
K88菌毛fae,faeG基因和蛋白质正常表达后,细菌在动物肠道上皮细胞上定植和繁殖,产生肠毒素导致仔猪肠道损伤和腹泻,危害仔猪健康和生长发育,给养殖生产造成巨大损失。抗生素通过杀灭细菌或者抑制细菌的繁殖,阻止或者减少致病性大肠杆菌在肠道的定植,从而保护仔猪肠道健康和防治腹泻,细菌在临床中的长期频繁使用易造成产肠毒素大肠杆菌产生耐药性,同时可以抑制动物肠道有益菌的增殖,严重影响着养猪产业的可持续发展。
发明内容
本发明的目的在于针对现有技术中存在的问题,提供了一种抑制产肠毒素大肠杆菌K88菌毛的fae,faeG基因表达的中药药物组合物及应用。
为实现上述目的,本发明所设计一种抑制产肠毒素大肠杆菌K88菌毛的fae,faeG基因表达的中药药物组合物,所述中药药物组合物包括黄柏提取物和石榴皮提取物。
进一步地,所述中药药物组合物中,黄柏提取物和石榴皮提取物的重量比1:0.5~2。
再进一步地,所述中药药物组合物中,黄柏提取物和石榴皮提取物的重量比为:1:1或1:2或2:1。
再进一步地,所述黄柏提取物中有效成分为小檗碱,且小檗碱的含量为41~85%,石榴皮提取物中有效成分为鞣花酸,且鞣花酸的含量为2.5~4.5%。
再进一步地,所述小檗碱的含量为84.2%,鞣花酸的含量为4.3%。
再进一步地,所述黄柏提取物和石榴皮提取物的重量比为1:1
再进一步地,所述中药药物组合物中还含有维生素C、山梨醇、甘露醇、木糖醇、果糖、氨基酸、葡甲胺、糊精、硬脂酸镁和蔗糖中任意一种或多种。
再进一步地,所述中药药物组合物为片剂、胶囊剂、颗粒剂和溶液剂中任意一种。
本发明还提供了一种上述中药药物组合物在制备fae,faeG基因表达抑制剂中的应用。
本发明的有益效果:
本发明利用天然植物提取物抑制产肠毒素大肠杆菌菌毛的fae,faeG基因表达,降低ETEC在仔猪肠道上皮细胞的定植和繁殖,而不影响有益微生物在肠道的定植,增加了肠道有益菌的丰度,在降低仔猪发生腹泻风险的同时,保障了肠道菌群的多样性;同时黄柏和石榴皮中的抗氧化物质,保护仔猪断奶应激导致的肠道氧化损伤,抗生素在杀灭有害菌的同时,也降低了有益菌的丰度;与抗生素相比,中药药物组合物不易产生耐药性,有利于生猪健康养殖,可以在临床上作为一种饲料添加剂长期使用。
中药药物组合物极显著降低产肠毒素大肠杆菌fae基因表达水平(P<0.01),显著降低产肠毒素大肠杆菌faeG基因表达水平(P<0.05)。
附图说明
图1为K88菌毛基因组成及其编码蛋白的大小和功能图;
图2为中药药物组合物对fae,faeG基因的表达量的影响的技术路线图。
具体实施方式
下面结合具体实施例对本发明作进一步的详细描述,以便本领域技术人员理解。
1、中药药物组合物中,两种物质的制备:
a.黄柏提取物的制备方法:
1)黄柏经除去杂质,洗净,润透,切片,烘干得净药材,
2)按处方投料量在称量室称量,将烘干后黄柏净药材粉碎为粗粉,粉碎好的黄柏粗粉装入周转容器;
3)酸水提浸泡,按生产指令规定的投入药材量分别加入反应釜,加8倍量0.5%硫酸水溶液浸泡24小时,过滤,再用6倍量硫酸水溶液浸泡24小时,过滤,再用6倍量硫酸水溶液浸泡24小时,过滤备用,合并上述两次提取液,加氯化钠析晶,加盐量使其为饱和溶液,搅匀,静置24小时,过滤得沉淀物;
4)沉淀物加入20~30倍量的沸水搅拌均匀,加入氢氧化钠溶液调pH值至8.5~9,煮沸3~5min.使沉淀物充分溶解。趁热过滤。滤液60℃左右加入盐酸溶液调pH值至2。将上工序的溶液放冷析晶,过滤得结晶,析晶体用蒸馏水充分洗涤,使其洗涤液pH值至5~6,80℃以下低温干燥,粉碎混合,即得到黄柏提取物,测量,黄柏提取物中小檗碱的含量为41~84%。84.2%,鞣花酸的含量为4.3%
b.石榴皮提取物的制备方法:
1)石榴皮经除去杂质,洗净,润透,切片,烘干得净药材;
2)按处方投料量在称量室称量,将烘干后石榴皮净药材粉碎为粗粉,
3)粉碎好的石榴皮粗粉装入提取罐中,加热1mol/L盐酸水溶液,固液比1:10,加热回流提取1h,提取两次,合并两次的提取液,浓缩干燥即得到石榴皮提取物,其中,鞣花酸的含量为2.5~4.5%。
2、产肠毒素大肠杆菌菌液的制备
1)TSB培养基的配制:
按照配方准确称取胰蛋白大豆肉汤培养基于玻璃三角瓶中,加入ddH2O封口,高温高压灭菌处理,待培养基降到不烫手程度时于无菌条件下加入小牛血清,轻摇混匀即可。
2)产肠毒素大肠杆菌菌液的制备:
挑取新鲜的产肠毒素大肠杆菌K88ac单个菌落接种于TSB培养基中,置于37℃生化培养箱培养16-18h取出,采用平皿表面计数法计算含菌量。吸取上述菌液1ml,用生理盐水作10-1梯度稀释,每个梯度取100μl加到预先准备好的TSA固体平板上,将玻璃刮铲在火焰上灼烧灭菌,冷却后将菌液均匀推散在培养基表面,每个浓度涂3个平板,放入37℃生化培养箱中培养18h。
计算原菌液浓度,临用前将菌液用TSB培养基稀释到108CFU/ml。精密称取黄柏提取物和各种药物,用灭菌水配制成药液,药液均在无菌条件下配制,过滤后分装于灭菌的EP管中,置4℃冰箱保存备用。
利用上述黄柏提取物和石榴皮提取物按不同比例制备得到中药药物组合物对fae,faeG基因的表达量的影响
下述实施例中,黄柏提取物和石榴皮提取物的重量比为1:0(单独黄柏提取物)、1:1、1:2、2:1、0:1(单独石榴皮提取物),制备得到黄柏提取物、3种中药药物组合物1~3和石榴皮提取物,且五种物质中,每种物质设置两个浓度梯度。不同比例的黄柏提取物和石榴皮提取物的试验设计如下:
实施例1
黄柏提取物和石榴皮提取物重量比例1:0(单独黄柏提取物),即得到黄柏提取物,分别取黄柏提取物150mg、300mg,精密称定,置于100ml容量瓶中,加PBS 80ml,超声处理40min,放冷,用PBS稀释至刻度,摇匀,滤过备用。
实施例2
黄柏提取物和石榴皮提取物重量比例1:1混合后,即得到中药药物组合物1,分别取中药药物组合物1150mg、300mg,精密称定,置于100ml容量瓶中,加PBS 80ml,超声处理40min,放冷,用PBS稀释至刻度,摇匀,滤过备用。
实施例3
黄柏提取物和石榴皮提取物重量比例1:2混合后,即得到中药药物组合物2,分别取中药药物组合物2150mg、300mg,精密称定,置于100ml容量瓶中,加PBS 80ml,超声处理40min,放冷,用PBS稀释至刻度,摇匀,滤过备用。
实施例4
黄柏提取物和石榴皮提取物重量比例2:1混合后,即得到中药药物组合物3,分别取即得到中药药物组合物3,150mg、300mg,精密称定,置于100ml容量瓶中,加PBS 80ml,超声处理40min,放冷,用PBS稀释至刻度,摇匀,滤过备用。
实施例5
黄柏提取物和石榴皮提取物重量比例0:1混合后,即得到石榴皮提取物,分别石榴皮提取物150mg、300mg,精密称定,置于100ml容量瓶中,加PBS 80ml,超声处理40min,放冷,用PBS稀释至刻度,摇匀,滤过备用。
将5种物质的两个浓度梯度分别处理产肠毒素大肠杆菌:
将制得的产肠毒素大肠杆菌转移到5ml的离心管中,4℃离心机4000r/min离心15min,弃去上清,收集菌体,用1ml PBS清洗,重复一次,沉淀分别用5个中药药物组合物的两个浓度梯度的溶液溶解,37℃作用15min,分装到1.5ml离心管中每管1ml。
不同浓度黄柏提取物石榴皮提取物合剂的大肠杆菌总RNA提取与反转录:将制好的致病菌悬液4℃离心机12000r/min离心2min,收集菌体,利用细菌总RNA提取试剂盒(TIANGEN)进行致病菌总RNA的提取,然后用微量紫外分光光度计测定RNA浓度,琼脂糖凝胶电泳检测RNA质量。利用反转录试剂盒(Thermo)将提取的致病菌总RNA进行反转录。
实时荧光定量PCR:各目的基因和内参基因引物由上海生工合成,各基因的引物如下表
表1 实时荧光定量PCR引物序列
实时荧光定量PCR反应体系(10μl)如下:
PCR反应条件如下:95℃,预变性5min;95℃,10s;退火60℃,10s和延伸30s,共40个循环。每个循环结束时采集一次荧光信号,反应完毕后进行溶解曲线分析。采用ΔΔCt方法,以recA基因的Ct值为量化标准,标准化各目的基因的表达水平得到ΔCt,以对照组ΔCt值为标准,计算各目的基因的ΔΔCt,各目的基因的表达情况用2-ΔΔCt表示。见表2
表2:黄柏提取物及石榴皮提取物单独和中药药物组合物1~3使用的效果
由上表可以看出与对照组相比300μg/ml的黄柏提取物、300μg/ml的中药药物组合物1,300μg/ml的中药药物组合物2,300μg/ml的中药药物组合物3、150μg/ml的中药药物组合物3,可以显著降低fae基因和fae基因的表达量(P<0.05);石榴皮提取物对fae基因和fae基因的表达量无显著性影响。300μg/ml的黄柏提取物、300μg/ml的中药药物组合物1,300μg/ml的中药药物组合物2,300μg/ml的中药药物组合物3对fae基因和fae基因表达量的影响相互之间差异不显著。本试验所使用的同样重量的黄柏提取物的价格显著高于石榴皮提取物的价格,同时黄柏性寒不适宜大剂量和单独使用于断奶仔猪,石榴皮性温,虽然石榴皮提取物单独使用不能显著降低致病性大肠杆菌fae基因和fae基因的表达量,但中药药物组合物1使用效果略优于单独使用相同重量的黄柏提取物,与中药药物组合3的效果相当(但中药药物组合1的价格更低),同时可以缓解单独使用黄柏对仔猪的副作用,所以本实验认为中药药物组合物1是最优的组合。
实施例6
选用36头28d、体重7.5kg左右的杜×长×大三元杂交腹泻断奶仔猪,按体重、性别等相对一致的原则随机分为3组,每组12头腹泻断奶仔猪,公母各半。本试验采用单因子设计,对照组每天注射20mg/kgBW的土霉素,两个试验组每天分别灌服20mg/kgBW的黄柏提取物,20mg/kgBW的中药药物组合物1,连续用药3d,观察黄柏、石榴皮提取物单独使用及中药药物组合物1对断奶仔猪腹泻的治疗效果,试验中黄柏提取物含小檗碱84.2%,石榴皮提取物中鞣花酸的含量为4.3%。结果见表3
表3 黄柏石榴皮提取物对断奶仔猪腹泻的影响
试验处理 | 腹泻率 |
20mg/kg中药药物组合物1 | 45.5%±19.64<sup>a</sup> |
20mg/kg黄柏提取物 | 50.0%±21.32<sup>a</sup> |
20mg/kg土霉素 | 37.5%±17.49<sup>a</sup> |
注:同列相同小写字母表示差异不显著,不同小写字母表示差异显著(P<0.05)。
从上表可以看出中药药物组合物具有与土霉素相似的抗腹泻的效果,土霉素的效果最好,中药药物组合物的效果优于黄柏提取物。
其它未详细说明的部分均为现有技术。尽管上述实施例对本发明做出了详尽的描述,但它仅仅是本发明一部分实施例,而不是全部实施例,人们还可以根据本实施例在不经创造性前提下获得其他实施例,这些实施例都属于本发明保护范围。
Claims (1)
1.一种中药药物组合物在制备fae, faeG基因表达抑制剂中的应用,其特征在于:所述中药药物组合物由黄柏提取物和石榴皮提取物制成;其中,所述中药药物组合物中,黄柏提取物和石榴皮提取物的重量比1:1;所述黄柏提取物中有效成分为小檗碱,且小檗碱的含量为84.2%,石榴皮提取物中有效成分为鞣花酸,且鞣花酸的含量为4.3%;
所述黄柏提取物由以下步骤制备而成:
1)黄柏经除去杂质,洗净,润透,切片,烘干得净药材;
2)按处方投料量在称量室称量,将烘干后黄柏净药材粉碎为粗粉,粉碎好的黄柏粗粉装入周转容器;
3)酸水提浸泡,按生产指令规定的投入药材量分别加入反应釜,加8倍量0.5%硫酸水溶液浸泡24小时,过滤,再用6倍量硫酸水溶液浸泡24小时,过滤,再用6倍量硫酸水溶液浸泡24小时,过滤备用,合并上述三次提取液,加氯化钠析晶,加盐量使其为饱和溶液,搅匀,静置24小时,过滤得沉淀物;
4)沉淀物加入20~30倍量的沸水搅拌均匀,加入氢氧化钠溶液调pH值至8.5~9,煮沸3~5min使沉淀物充分溶解,趁热过滤;滤液在温度为60℃条件下加入盐酸溶液调pH值至2,再将溶液放冷析晶,过滤得结晶,析晶体用蒸馏水充分洗涤,使其洗涤液pH值至5~6,80℃以下低温干燥,粉碎混合,即得到黄柏提取物;
所述石榴皮提取物由以下步骤制备而成:
1)石榴皮经除去杂质,洗净,润透,切片,烘干得净药材;
2)按处方投料量在称量室称量,将烘干后石榴皮净药材粉碎为粗粉;
3)粉碎好的石榴皮粗粉装入提取罐中,加热1mol/L盐酸水溶液,固液比1:10,加热回流提取1h,提取两次,合并两次的提取液,浓缩干燥即得到石榴皮提取物。
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