CN106177221B - 一种用于治疗猪大肠杆菌性腹泻的中药组合物及其制备方法 - Google Patents
一种用于治疗猪大肠杆菌性腹泻的中药组合物及其制备方法 Download PDFInfo
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- CN106177221B CN106177221B CN201610683841.6A CN201610683841A CN106177221B CN 106177221 B CN106177221 B CN 106177221B CN 201610683841 A CN201610683841 A CN 201610683841A CN 106177221 B CN106177221 B CN 106177221B
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Abstract
本发明公开了一种用于治疗猪大肠杆菌性腹泻的中药组合物,是由按照重量份计的以下原料药制备得到的:紫花地丁6‑12份、车前子6‑12份、败酱草6‑12份、五倍子3‑6份、黄连5‑10份、厚朴4‑8份、黄柏5‑10份、五味子4‑8份、大黄2‑4份、吴芋5‑10份、虎杖6‑12份、板蓝根6‑12份、威灵仙4‑8份、山豆根4‑8份;并提供了其制备方法。本发明的有益效果为:本发明根据中兽医药学辨证施治理论和传统用药经验,结合现代免疫药理和临床兽医学等技术方法,对中草药进行组方筛选,其组方合理,安全有效,原料易得,制备工艺简单,易于操作,经过应用试验验证,治疗有效率远高于现有市售产品,在临床上具有广阔的推广应用价值。
Description
技术领域
本发明涉及兽用中药组合物技术领域,具体涉及一种用于治疗猪大肠杆菌性腹泻的中药组合物及其制备方法。
背景技术
中兽医药中的许多中药和方剂具有抗菌或抑菌作用,并且具有抗菌谱广、毒副作用小、效果良好和不易产生耐药性的特点,可以直接作用于微生物的结构和代谢而发挥抑菌作用。抗菌中兽药的种类繁多,主要来源于植物,也来源于动物性药物。中兽药的抑菌抗菌作用,已经不仅局限在清热解毒类药物,而且泻下、补虚等药物也具有抑菌功效。
中兽药抗菌的物质基础是活性成分,目前已知多为生物碱、酸类、酮类、醛类、酚类和挥发油等,如小檗碱、白头翁素、穿心连内酯、苯甲酸、齐墩果酸、山犁酸、葵酰乙醛、大黄素、蒜辣素、熊果甙、白屈菜红碱、德西明碱、焊菜素等均为活性成分。
中兽药的抗菌机制非常的繁杂和多样化,有的抗菌中草药体内外抗菌效果并不一致,如丹皮、知母、黄连等,虽在体外抗菌力弱,但服用后却有良好疗效,即使在浓度很低时也能抑制金黄色葡萄球菌酶的形成,从而发挥抗菌作用。所以,体外抑菌试验对中草药的抗菌作用只能是一种参考性的方法,实际临床应用效果还必须通过动物体内试验加以比较、验证,从而进一步闸明其抑菌机理。
对于一些已对抗生素产生耐药性的细菌,中兽药对其仍然有良好的效果。研究表明,禽大肠杆菌对石榴皮、诃子、黄答等敏感性较高,同时发现部分中药组合对各种血清型大肠杆菌具有抑菌作用,体现了中药的配伍优势。微生物产生的毒素在感染性疾病中可引起多种病理和组织损伤,对于一些细菌性疾病如大肠杆菌性腹泻,采用抗菌素可抑制或者杀灭病原微生物,但是不能消除细菌生长或者死亡后释放出的毒素对机体的损伤。采用中兽药能对抗多种病原微生物毒素以及阻止毒素对机体的损害作用。研究表明,甘草主要成分甘草甜素及甘草次酸对破伤风毒素具有明显的中和作用;金银花、连翘提取物除具有体外抑菌作用以外,还具有抗大肠杆菌热敏肠毒素的作用中草药诃子和五倍子的提取物对多数细菌如沙门氏菌、大肠杆菌和葡萄球菌等有较强的抑菌作用,其他中草药提取物如黄连、大黄等也具有很好的抑制细菌的作用,这些中兽药可以作为抑制细菌的最好选择。
大肠杆菌是人和许多动物肠道中最主要且数量最多的一种细菌,周身鞭毛,能运动,无芽孢主要生活在大肠内,代谢类型是异养兼性厌氧性。正常情况下,大多数大肠杆菌是非常安分守己的,不会给本身带来任何危害,但当机体免疫力降低、肠道长期缺乏刺激等情况下,它们会移到肠道以外的地方,造成相应部位的感染或全身散播性感染。猪大肠杆菌性腹泻是由原性大肠杆菌引起的仔猪肠道传染性疾病。常见的有仔猪黄痢、仔猪白痢和水肿病三种,以发生肠炎、肠毒血症为特征。
通过体外抑菌试验可以初步判断中兽药复方抑菌作用的大小,通过体外抑菌试验可以筛选获得有效的中药组方,为畜牧养殖业的健康发展起到积极的保障作用。
发明内容
本发明的目的就是针对上述现有技术中的缺陷,提供了一种新的、安全有效的用于治疗猪大肠杆菌性腹泻的中药组合物及其制备方法。
为了实现上述目的,本发明提供的技术方案为:一种用于治疗猪大肠杆菌性腹泻的中药组合物,是由按照重量份计的以下原料药制备得到的:紫花地丁6-12份、车前子6-12份、败酱草6-12份、五倍子3-6份、黄连5-10份、厚朴4-8份、黄柏5-10份、五味子4-8份、大黄2-4份、吴芋5-10份、虎杖6-12份、板蓝根6-12份、威灵仙4-8份、山豆根4-8份。
本发明的第二个目的是提供了上述的一种用于治疗猪大肠杆菌性腹泻的中药组合物的制备方法,包括以下步骤:
1)按照上述的重量配比,分别准确称取紫花地丁30-60g、车前子30-60g、败酱草30-60g、五倍子15-30g、黄连25-50g、厚朴20-40g、黄柏25-50g、五味子20-40g、大黄10-20g、吴芋25-50g、虎杖30-60g、板蓝根30-60g、威灵仙20-40g、山豆根20-40g,放置于20L-30L的圆筒锅中,加水10-15L,浸泡30-60分钟;
2)将加入原料药的圆筒锅放置于加热器上,2200W条件下加热30分钟至沸腾,然后降低加热器功率到600W,使其慢慢煮沸40-60min,用纱布进行过滤,得到第一次煎煮过滤的滤液,保存备用;
3)将第一次煎煮过滤的滤渣,再加入10-15L纯净水,2200W条件下加热30分钟至沸腾,然后降低加热器功率到600W,使其慢慢煮沸40-60min,用纱布进行过滤,得到第二次煎煮过滤的滤液,保存备用;
4)合并第一次煎煮过滤的滤液和第二次煎煮过滤的滤液,2200W条件下加热25分钟至沸腾,然后降低加热器功率到1200-1600W,煮沸360-420min,挥发多余水分后得到中药组合物浓缩液750-800ml,冷却后密封保存于4℃冰箱备用。
本发明的有益效果为:本发明提供的一种用于治疗猪大肠杆菌性腹泻的中药组合物及其制备方法,是根据中兽医药学辨证施治理论和传统用药经验,结合现代免疫药理和临床兽医学等技术方法,对中草药进行组方筛选,其中板蓝根具有清热解毒、凉血、抗菌功效,黄柏能清热燥湿、泻火解毒、退热除蒸,紫花地丁也具有清热解毒功效,该中药组方是治疗猪大肠杆菌性腹泻的有效组方,其组方合理,安全有效,原料易得,制备工艺简单,易于操作。经过应用试验验证,中药组合物各剂量试验组治疗有效率均高于对照组,且中剂量中药组合物的有效率为100%,该制剂在临床上具有广阔的推广应用价值。
附图说明
图1为大黄素标准品的曲线图。
图2为复方药中大黄素的含量测定结果。
图3为标准品曲线图。
具体实施方式
实施例1:
治疗猪大肠杆菌性腹泻的中药组合物的制备方法:
1 材料与方法:
1.1材料:
菌种:
大肠杆菌(购自中国工业微生物菌种管理与保藏中心)
药材:
紫花地丁、车前子、败酱草、五倍子、黄连、厚朴、黄柏、五味子、大黄、吴芋、虎杖、板蓝根、威灵仙、山豆根。
对照品:
盐酸小檗碱、大黄酸、大黄素甲醚、齐墩果酸、苦参碱、氧化苦参碱、没食子酸、五味子甲素、精氨酸、大黄素。
试剂:
MH(A)培养基(北京奥博星生物技术有限责任公司)、MH(B)培养基(北京奥博星生物技术有限责任公司)、甲醇(色谱纯)、甲苯、乙酸乙酯(烟台市双双化工有限公司)、甲酸(莱阳市双双化工有限公司)、氢氧化钠、苯、异丙醇(分析纯)、氨水、盐酸、乙醚、三氯甲烷、石油醚(烟台市双双化工有限公司)、甲酸乙酯(天津市凯信化学工业有限公司)、硫酸、无水乙醇(天津市北联精细化学开发有限公司)、环己烷、三乙胺(天津市凯信化学工业有限公司)、正丁醇、冰醋酸、酒石酸铜(上海阿拉丁生化科技股份有限公司)、香草醛(上海阿拉丁生化科技股份有限公司)、L-精氨酸(上海阿拉丁生化科技股份有限公司)。
仪器:
ZDX-35BI型座式自动电热压力蒸汽灭菌器、恒温振荡器(ZD-85,国华企业)、电子天平(AL204,梅特勒-托利多仪器上海有限公司)、数显恒温水浴锅(HH-2,国画电器有限公司)、200g手提式高速万能粉碎机(DFT-200,温岭市林大机械有限公司)、电磁炉、GOODLOOK-1000型薄层色谱成像系统(上海科哲生化科技有限公司)、电热恒温鼓风干燥箱(DHG-9070,上海鸿都电子科技有限公司)、数控超声波清洗器(KH7200DB,昆山禾创超声仪器有限公司)、实验室专用超纯水机、Agilent Technologies 1290 Infinity 超高效液相色谱仪、TDZ4-WS台式低速离心机、玻璃砂芯过滤装置、DensiCHEK plus比浊仪、锥形瓶、漏斗、量筒、烧杯等。
2.方法:
2.1粉碎药材:
取紫花地丁、车前子、败酱草、五倍子、黄连、厚朴、黄柏、五味子、大黄、吴芋、虎杖、板蓝根、威灵仙、山豆根15味药材各200g-300g左右,粉碎成粉末,分开装袋,保存。
2.2药材的薄层鉴定:
薄层鉴定方法主要参考2010年版《中华人民共和国兽药典》(二部)中的性状鉴别方法对紫花地丁、车前子、败酱草、五倍子、黄连、厚朴、黄柏、五味子、大黄、吴芋、虎杖、板蓝根、威灵仙、山豆根15味药材进行薄层鉴定。
2.3 药材煎煮:
(1)复方药猪细菌性腹泻的配方:
分别准确称取紫花地丁30-60g、车前子30-60g、败酱草30-60g、五倍子15-30g、黄连25-50g、厚朴20-40g、黄柏25-50g、五味子20-40g、大黄10-20g、吴芋25-50g、虎杖30-60g、板蓝根30-60g、威灵仙20-40g、山豆根20-40g,放置于20L-30L的圆筒锅中,加水10-15L,浸泡30-60分钟;
(2)将圆筒锅放置于加热器上,2200W条件下加热约30分钟至沸腾,然后降低加热器功率到600W,使其慢慢煮沸40-60min,用普通纱布进行过滤,得滤液,保存备用;
(3)将第一次煎煮过滤的滤渣,再加入10-15L纯进水, 2200W条件下加热约30分钟至沸腾,然后降低加热器功率到600W,使其慢慢煮沸40-60min,用普通纱布进行过滤,得滤液,保存备用;
(4)合并两次煎煮、过滤的滤液,2200W条件下加热约25分钟至沸腾,然后降低加热器功率到1200-1600W,煮沸360-420min,挥发多余水分,得浓缩液约750-800ml,冷却后密封保存于4℃冰箱备用。
实施例2:
中药组合物对大肠杆菌的抑菌试验:
1.高效液相色谱法测定煎煮液中大黄素的含量:
复方药中选取虎杖的活性成分是大黄素,通过测出煎煮液中大黄素的浓度含量,从而换算得到中药煎煮液的浓度含量。
1.1色谱条件:
流动相:质量分数为0.1%的磷酸-甲醇(0min,V磷酸:V甲醇=27:73,1min100%甲醇),流速1.5ml/min,波长430nm,样品、标准品进样量为10μL。大黄素的最大吸收波长为290nm,在439nm有较大吸收。
1.2样品处理:
吸取煎煮液0.5ml于离心管中,再加入4.5ml的甲醇,超声处理30min之后用离心机(4000r/min)离心15min,最后用针管移取1ml上清液作为样品储备液。
1.3标准品处理:
精密称取大黄素标准品5 mg,置于5ml容量瓶中,用甲醇溶解并定容至刻度线,制备成1mg/ml标准品储备液,备用。用移液管精密移取1ml标准品储备液,置于100ml容量瓶中,用甲醇定容至刻度线,备用。
2.抑菌试验、测最小抑菌浓度:
根据配置的MH(A)琼脂培养基的体积吸取不同体积的复方药煎煮液加于已加热溶解的MH(A)培养基中对复方药进行倍比稀释,琼脂中所含复方药的浓度分别为0μg/ml、1μg/ml、2μg/ml、4μg/ml、8μg/ml、16μg/ml、25μg/ml。在45℃下充分混匀倾倒灭菌平皿,琼脂厚度3-4mm,配置好的复方药琼脂平板应放入4℃冰箱保藏备用。然后用比浊仪调大肠杆菌菌液比浊度,使其达到0.5,向配好的复方药平板中各加比浊度为0.5的大肠杆菌菌液50μL,再用涂布棒涂均匀,置于37℃培养18-20个小时后观察实验结果。
3. 实验结果:
3.1薄层鉴定结果:
经薄层鉴定确定此次复方药中所用药材均为地道药材。
3.2复方药中大黄素得含量测定结果:
大黄素标准品的曲线图如图1所示,复方药中大黄素的含量测定结果如图2所示,标准品曲线图如图3所示。
表1显示为大黄素标准品的浓度与峰面积关系。
表2显示为根据标准品曲线图计算得到复方药样品大黄素含量。
表1
表2
因为此次高效液相色谱测出的结果是把复方药煎煮液用甲醇稀释了10倍所得,所以通过高效液相色谱测得该复方药中大黄素的含量应为50μg/ml。
3.3抑菌试验结果:
表3显示为不同浓度的复方药对大肠杆菌的抑制结果。
表3
其中,“— ”表示无菌生长 ,“+ ”表示有菌生长, 随“+”数增加菌落长势增强。观察试验结果得到该中兽药复方对猪大肠杆菌的最小抑菌浓度为<8μg/ml。
实施例3:
中药组合物在猪大肠杆菌性腹泻的临床应用:
1. 试验材料:
1.1中药组合物由中国农业科学院兰州畜牧与兽药研究所研制,其组方为:紫花地丁60g、车前子60g、败酱草60g、五倍子30g、黄连50g、厚朴40g、黄柏50g、五味子40g、大黄120g、吴芋50g、虎杖60g、板蓝根360g、威灵仙40g、山豆根40g,制备方法如上方法。
1.2 试验动物:
甘肃永登县某猪养殖场3日龄疑为大肠杆菌感染伴有腹泻症状仔猪12只。
2 试验方法:
2.1 药物配置:
将中药组合物按照以上方法制备后,稀释、配置成浓度为8μg/ml的药液,装入干净的棕色瓶中,密封,4℃放置备用。
2.2 试验分组:
将12只仔猪随机分为低剂量治疗组、中剂量治疗组、高剂量治疗组、空白对照组4组,每组3只。
3 中药组合物对猪大肠杆菌治疗试验:
3.1取配置浓度为8μg/ml的中药组合物药液,分别按照低剂量治疗组(20ml)、中剂量治疗组(30ml)、高剂量治疗组(50ml),空白对照组不给药,进行灌服,一日2次,连用3天,期间自由哺乳、饮水。
3.2 疗效考核标准试验期间,每日观察试验组仔猪腹泻症状的情况,一个疗程后统计腹泻症状的改善情况,并计算有效率。
4. 试验结果:
4.1中药组合物对猪大肠杆菌性腹泻的治疗效果:
表4显示为中药组合物对猪大肠杆菌性腹泻的治疗效果,由表4可以看出,中药组合物各剂量试验组治疗有效率均高于对照组,且中剂量中药组合物的有效率为100%。因此,该制剂在临床上具有广阔的推广应用价值。
表4
其中,有效率指能够明显改善腹泻症状的仔猪与试验组总仔猪数所占的比例(%)。
最后应说明的是:以上所述仅为本发明的优选实施例而已,并不用于限制本发明,尽管参照前述实施例对本发明进行了详细的说明,对于本领域的技术人员来说,其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (2)
1.一种用于治疗猪大肠杆菌性腹泻的中药组合物,其特征在于,是由按照重量份计的以下原料药制备得到的:紫花地丁6-12份、车前子6-12份、败酱草6-12份、五倍子3-6份、黄连5-10份、厚朴4-8份、黄柏5-10份、五味子4-8份、大黄2-4份、吴芋5-10份、虎杖6-12份、板蓝根6-12份、威灵仙4-8份、山豆根4-8份。
2.根据权利要求1所述的一种用于治疗猪大肠杆菌性腹泻的中药组合物的制备方法,其特征在于,包括以下步骤:
1)按照权利要求1所述的重量配比,分别准确称取紫花地丁30-60g、车前子30-60g、败酱草30-60g、五倍子15-30g、黄连25-50g、厚朴20-40g、黄柏25-50g、五味子20-40g、大黄10-20g、吴芋25-50g、虎杖30-60g、板蓝根30-60g、威灵仙20-40g、山豆根20-40g,放置于20L-30L的圆筒锅中,加水10-15L,浸泡30-60分钟;
2)将加入原料药的圆筒锅放置于加热器上,2200W条件下加热30分钟至沸腾,然后降低加热器功率到600W,使其慢慢煮沸40-60min,用纱布进行过滤,得到第一次煎煮过滤的滤液,保存备用;
3)将第一次煎煮过滤的滤渣,再加入10-15L纯净水,2200W条件下加热30分钟至沸腾,然后降低加热器功率到600W,使其慢慢煮沸40-60min,用纱布进行过滤,得到第二次煎煮过滤的滤液,保存备用;
4)合并第一次煎煮过滤的滤液和第二次煎煮过滤的滤液,2200W条件下加热25分钟至沸腾,然后降低加热器功率到1200-1600W,煮沸360-420min,挥发多余水分后得到中药组合物浓缩液750-800ml,冷却后密封保存于4℃冰箱备用。
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