CN115531449B - 一种治疗大肠杆菌导致藏猪仔猪腹泻的组合物及制备方法 - Google Patents
一种治疗大肠杆菌导致藏猪仔猪腹泻的组合物及制备方法 Download PDFInfo
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Abstract
本发明公开了一种治疗大肠杆菌导致藏猪仔猪腹泻的组合物及其制备方法，组合物由以下重量配比的原料组成：川西小檗5～15份、华西小石积7～21份，乌樟6～18份。制备方法为将原料按比例混合，加水后提取，提取参数为：固液比1:3～1:10、提取温度55℃～75℃、提取时间70min～90min、提取次数2次～4次。本发明的组合物为纯天然成分，安全、无抗生素残留风险，对致泻大肠杆菌引起的藏猪仔猪腹泻具有非常优异的治疗效果，疗效超过了抗生素组。
Description
技术领域
本发明属于中药组合物领域，具体涉及一种治疗大肠杆菌导致藏猪仔猪腹泻的组合物及其制备方法。
背景技术
藏猪主产于青藏高原，包括云南迪庆藏猪、四川阿坝及甘孜藏猪、甘肃的蕨麻猪以及分布于西藏自治区山南、林芝、昌都等地的藏猪类群。藏猪是世界上少有的高原型猪种，也是我国宝贵的地方品种资源，也是我国国家级重点保护品种中唯一的高原性猪种。藏猪长期生活于无污染、纯天然的高寒山区，具有适应高海拔恶劣气候环境、抗病、耐粗等特点，但繁殖力低。
大肠杆菌(Escherichia coli)，又叫大肠埃希氏菌，Escherich在1885年发现的。大肠杆菌是条件致病菌，在一定条件下可以引起人和多种动物发生胃肠道感染或尿道等多种局部组织器官感染。
由大肠杆菌引发的藏猪腹泻由于其高发病率和高死亡率给养殖户造成巨大的经济损失。虽然通过注射抗生素能治疗，但大量使用抗生素会导致耐药性，而且会导致猪肉抗生素残留。
因此亟待开发出一种能治疗大肠杆菌引发的藏猪仔猪腹泻的安全、有效的药物。
川西小檗(Berberis tischleri Schneid.)是小檗科小檗属落叶或半常绿灌木，高0.5～1.5米左右，最高可达3米。川西小檗的分枝较多，老枝黑灰色，幼嫩枝灰黄色；茎刺三分叉，单叶丛出于刺腋；叶薄纸质，呈长圆状倒卵形或倒卵形，叶缘平展，全缘或每边具细小刺齿，叶柄不显著；松散伞形状总状花序，花序下部花梗无毛，苞片披针形，外萼片狭卵形，内萼片狭长圆状倒卵形，花瓣倒卵形；浆果红色，浆果球形或卵状长圆形，花期5～6月，果期7～9月，果实成熟时红色。川西小檗主要分布于中国四川、西藏，生长于海拔1500～3800米左右的灌木丛林或向阳山坡、路边、林缘处。春秋两季挖根和茎供药用，具有清热解毒，抗菌消炎的作用，在川西北高原牧区常用于目赤肿痛，痢疾腹痛，咽喉肿痛，跌打损伤等疾病的治疗。
华西小石积(Osteomeles schwerinae C.K.Schneid.)别名黑果、地石榴、小黑果、小石积木、沙糖果等，为蔷薇科小石积属落叶或半常绿灌木，高可达1～3米左右。其枝条密集，幼嫩的枝条为近似圆柱形且微弯曲；叶芽小呈扁三角卵形，小叶片对生，为奇数羽状复叶，多为椭圆形、长圆形或倒卵状，叶片上下两面生有疏生柔毛。花序顶生，总花梗和花梗被灰白色柔毛，苞片膜质，线状披针形，萼筒钟状，萼片卵状披针形，花瓣白色，长圆形；果实卵形或近球形，成熟时为蓝黑色；4～5月开花，7月中下旬结果。华西小石积分布于中国四川、云南、贵州、甘肃等省份。生长在海拔1500～3000米的坡灌木丛中或田边路旁向阳干燥地。其药用部位为叶和根，具有清热解毒、收敛止泻、祛风湿的功效，主要用于咽喉炎、腮腺炎、痢疾、肠炎、腹泻、痈疮、无名肿毒等疾病的治疗。夏、秋季节采叶或挖根晒干供药用。
乌樟[Cinnamomum camphora(L.)Presl]又名香樟、油樟、芳樟、樟等，是我国稀有树种之一，成长期较长。为常绿乔木，整树含有丰富的挥发性油脂而具有浓厚的特殊香气，为高品质香水提取源之一。其木质细密、纹理细腻，质地坚韧、轻柔而不易折断，也不易产生裂纹。乌樟仅分布于我国长江以南和西南地区，其中以贵州、四川、湖北、湖南西部及云南东北和东南部最多，多生长于海拔700～1480米左右的路旁、沟边、疏林或灌丛中。乌樟特殊的气味使得其具有广泛的抗菌、祛风、除湿、解毒、杀虫作用，主要用于风湿痹痛、胃痛、疮疡肿毒、慢性下肢溃疡、疥癣、皮肤瘙痒、毒虫咬伤等症的治疗。
发明内容
本发明所要解决的技术问题为：如何提供一种治疗大肠杆菌引起的藏猪仔猪腹泻的安全、有效的药物。
本发明的技术方案为：一种治疗大肠杆菌导致藏猪仔猪腹泻的组合物，由以下重量配比的原料组成：川西小檗5～15份、华西小石积7～21份，乌樟6～18份。
进一步地，由以下重量配比的原料组成：川西小檗15份、华西小石积7份，乌樟12份。
本发明还公开了一种治疗大肠杆菌导致藏猪仔猪腹泻的组合物的制备方法，将上述原料按比例混合，加水后提取，提取参数为：固液比1:3～1:10、提取温度55℃～75℃、提取时间70min～90min、提取次数2次～4次。
进一步地，所述提取参数为：固液比1:10、提取温度75℃、提取时间70min、提取次数2次。
一种组合物提取物，按上述的制备方法将得到的提取溶液冷冻干燥即得。
上述所述的组合物或组合物提取物在制备治疗大肠杆菌导致藏猪仔猪腹泻的药物中的应用
发明人通过归经分析及实践经验，推测川西小檗、华西小石积和乌樟药物组合对相关组织器官系统疾病具有一定的防治作用。通过试验证实，川西小檗、华西小石积和乌樟按一定比例配伍后对标准菌株ATCC25922及致泻大肠杆菌EPEC CVCC1396均具有很好的抑制作用。
通过临床试验证明，该组合物对致泻大肠杆菌引起的藏猪仔猪腹泻具有非常优异的治疗效果，疗效超过了抗生素组。
与现有技术相比，本发明具有以下有益效果：
1、本发明的组合物为纯天然成分，安全、无抗生素残留风险。
2、本发明的组合物对致泻大肠杆菌引起的藏猪仔猪腹泻具有非常优异的治疗效果，疗效超过了抗生素组。
具体实施方式
下述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试验材料，如无特殊说明，均为从商业渠道购买得到的。
菌株来源
标准菌株ATCC25922由西南民族大学兽医药理学与毒理学实验室保存，商品化致泻大肠杆菌EPEC CVCC1396购自中国兽医药品监察所。
1、组合物原料川西小檗、华西小石积和乌樟的归经分析
根据文献资料报道，分别对川西小檗、华西小石积和乌樟三种药物进行性味归经分析，为后续药物组合筛选奠定基础。
川西小檗、华西小石积和乌樟归经分析结果见表1，推测川西小檗、华西小石积和乌樟对相关组织器官系统疾病具有一定的防治作用。
表1川西小檗、华西小石积和乌樟最佳药物组合归经分析
2、川西小檗、华西小石积和乌樟配合比例确定
分别称取一定重量的川西小檗、华西小石积和乌樟，中药粉碎机粉碎，过60目药典筛。以药物组合对标准菌株ATCC25922的抑菌圈大小为评价指标，根据L9(33)正交设计表对川西小檗(A)、华西小石积(B)和乌樟(C)进行不同配合比例的筛选(表2)，确定三者的最佳配合比例。
表2川西小檗、华西小石积和乌樟药物组合的配合比例筛选
根据L9(33)正交设计表，分别将不同配合比例的川西小檗、华西小石积和乌樟溶解于10倍体积的去离子水中浸泡30min后煎煮1h，旋转蒸发仪浓缩后冷冻干燥成粉末状备用。
将不同配合比例的川西小檗、华西小石积和乌樟药物组合冻干粉用去离子水溶解后配制成浓度梯度溶液(2g/mL、1g/mL、0.5g/mL、0.25g/mL)，然后把定性滤纸用打孔器制成直径6mm的纸片，放至预先倍比稀释的药物溶液中过夜，第2天取出纸片后37℃烘干备用。同时以16μg/ml的多西环素作为对照。
将标准菌株ATCC25922过夜培养，菌液调整至0.5麦氏比浊度后稀释1000倍，均匀涂布MH琼脂平板，将预先制备加入梯度稀释的药物组合纸片和多西环素纸片轻轻贴在平板表面，37℃恒温培养12～16h后记录抑菌圈的大小，每个药物组合重复测量3次。
结果如表3所示：每一组试验均产生了明显的抑菌圈，说明组合物的组成为川西小檗5～15份、华西小石积7～21份，乌樟6～18份的情况下，对标准菌株ATCC25922具有明显的抑制作用。
川西小檗、华西小石积和乌樟药物组合的最佳配合为A3B1C2，即川西小檗、华西小石积和乌樟的重量份数分别为15重量份、7重量份和12重量份的条件下，三者最佳药物组合A3B1C2的抗菌活性最强。
表3正交设计筛选川西小檗、华西小石积和乌樟最佳药物组合试验结果
对最佳配合为A3B1C2进行三组平行试验(表4)，重复性试验结果表明筛选的最佳组合抗菌活性稳定，故A3B1C2组合为川西小檗、华西小石积和乌樟最佳药物组合。
表4川西小檗、华西小石积和乌樟最佳药物组合三次平行试验结果
3、川西小檗、华西小石积和乌樟最佳药物组合提取工艺的优化
按照2筛选的川西小檗、华西小石积和乌樟最佳药物组合，分别称取一定重量的川西小檗、华西小石积和乌樟，中药粉碎机粉碎，过60目药典筛。根据L9(34)正交设计表对川西小檗、华西小石积和乌樟药物组合进行最佳提取工艺的影响因素及水平考察(表5)：固液比(1:3，1:5，1:10)、提取温度(55℃、65℃、75℃)和提取次数(2次、3次、4次)，和提取时间(70min、80min、90min)。
表5正交设计试验因素和水平
L9(34)正交设计表将不同提取工艺的川西小檗、华西小石积和乌樟最佳药物组合的水提液进行冷冻干燥后，分别配制成梯度浓度溶液(1g/mL、0.5g/mL、0.25g/mL、0.125g/mL、0.0625g/mL)备用。以多西环素为阳性对照药物、ATCC25922为质控菌株、抑菌活性(抑菌圈直径大小)为评价指标，优化川西小檗、华西小石积和乌樟最佳药物组合的最佳提取工艺。
由表6可知：不同因素对川西小檗、华西小石积和乌樟最佳药物组合提取工艺的影响大小依次为提取时间(D)>固液比(A)>提取次数(C)>提取温度(B)，根据结果可得最佳提取工艺为A3B3C1D1即固液比1:10，提取温度75℃，提取次数2次，提取时间70min。在该提取方法下川西小檗、华西小石积和乌樟最佳药物组合A3B3C1D1的抗菌活性最强。
表6川西小檗、华西小石积和乌樟最佳药物组合正交设计试验结果
按照正交设计所得最优条件对药物组合进行三组平行试验(表7)，重复性试验结果表明优化的最佳提取工艺稳定，故A3B3C1D1组合为最优提取条件。
表7最佳工艺A3B3C1D1平行试验结果
4、川西小檗、华西小石积和乌樟药物组合对致泻大肠杆菌的抗菌活性评价
按照3优化的川西小檗、华西小石积和乌樟最佳药物组合提取工艺，将川西小檗、华西小石积和乌樟最佳药物组合的最优提取条件得到的水提液进行冷冻干燥(命名为：组合物提取物)后，分别配制成梯度浓度溶液(1g/mL、0.5g/mL、0.25g/mL、0.125g/mL、0.0625g/mL)，将定性滤纸用打孔器制成直径6mm的纸片，放至预先倍比稀释好的川西小檗、华西小石积和乌樟最佳药物组合的梯度浓度溶液中浸泡过夜，第2天取出纸片后37℃烘干备用。同时以16μg/ml的多西环素作为对照。
采用K-B纸片法测定筛选的川西小檗、华西小石积和乌樟最佳药物组合的抑菌圈直径，对川西小檗、华西小石积和乌樟组合进行致泻大肠杆菌体外抗菌活性评价：将过夜培养的致泻大肠杆菌EPEC CVCC1396菌液按一定比例稀释后调整至0.5麦氏比浊度，稀释1000倍后均匀涂布MH琼脂平板，将预先制备加入梯度稀释的药物组合纸片和多西环素纸片轻轻贴在平板表面，37℃恒温培养12～16h后测定抑菌圈的大小，每个药物组合重复测量3次。
结果如表8所示，川西小檗、华西小石积和乌樟最佳药物组合A3B3C1D1对致泻大肠杆菌的抗菌活性与16μg/mL的多西环素相当，具有较好的抗菌活性。
表8川西小檗、华西小石积和乌樟最佳药物组合A3B3C1D1对致泻大肠杆菌的抗菌活性
5、川西小檗、华西小石积和乌樟最佳药物组合急性毒性和蓄积毒性评价
取28只体重范围18-25g之间的雌性健康昆明鼠随机分为7组，空白对照组灌胃生理盐水，其余6组分别按0.8倍组距设置药物(组合物提取物)梯度浓度并灌胃，即药物浓度分别为1g/ml、0.8g/ml、0.64g/ml、0.512g/ml、0.4096g/ml、0.33g/ml，灌胃，观察24h。若无死亡及其他异常现象继续进行21天蓄积毒性试验。
急性毒性试验结果表明，川西小檗、华西小石积和乌樟最佳药物组合的LD50为5.04g/kg(>5.0g/kg)，表明其不存在急性毒性；蓄积毒性试验结束时，各剂量组均未出现小鼠死亡，且整个使用过程中，小鼠无其他异常外观变化，表明川西小檗、华西小石积和乌樟最佳药物组合不存在蓄积毒性。
6、组合物提取物对致泻大肠杆菌引起藏猪腹泻治疗效果评价
选取27只健康断奶仔藏猪(空白对照组、多西环素组和组合物提取物组)对致泻大肠杆菌引起的藏猪腹泻治疗效果临床评价。27只仔猪按照109CFU/mL剂量腹腔注射5mL致泻大肠杆菌EPEC CVCC1396，空白对照组不作任何治疗处理，多西环素组和组合物提取物组同时分别按3mg/kg·bw和10g/天/头的剂量灌服7天，期间密切观察各组动物腹泻次数及外观表现反应等，并计算腹泻率和腹泻指数。
腹泻率＝试验期间腹泻仔藏猪头次/(试验仔藏猪头数×试验天数)×100％)
腹泻指数＝腹泻率*腹泻评分
组合物提取物对致泻大肠杆菌引起的藏猪腹泻的临床治疗效果见表9：7天试验中，空白对照组、四环素组和组合物提取物组发生腹泻的仔藏猪的头数分别为7头、3头、2头，腹泻的天数分别3天、2天、2天；空白对照组、四环素组和组合物提取物组仔藏猪腹泻评分分别为4分、2分、2分。与空白对照组相比，四环素组和组合物提取物组腹泻次数、腹泻率及外观表现反应等均有不同程度的改善，其中组合物提取物组的治疗效果与多西环素组差异显著。
表9A3B3C1D1对致泻大肠杆菌所致仔藏猪腹泻的治疗效果
注：*与空白对照组比较差异性显著；#与多西环素组比较差异性显著。
Claims (1)
1.一种治疗大肠杆菌导致藏猪仔猪腹泻的组合物，其特征在于，由以下重量配比的原料制成：川西小檗15份、华西小石积7份，乌樟12份；
将原料按比例混合，加水后提取，提取参数为：固液比1:10、提取温度75 ℃、提取时间70 min、提取次数2次。
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