CN113967230B - 治疗马腺疫的中药组合物 - Google Patents
治疗马腺疫的中药组合物 Download PDFInfo
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Abstract
本发明提供一种治疗马腺疫的中药组合物,所述中药组合物由如下原料复配而成:赤芍提取液0.5g/mL‑2g/mL、甘草提取液0.25g/mL‑1g/mL、大黄提取液0.25g/mL‑1g/mL、雪白睡莲提取液0.5g/mL‑2g/mL、山豆根提取液0.5g/mL‑2g/mL、苦参提取液0.125g/mL‑0.5g/mL以及黄芪和当归质量比为1:1配伍提取液0.25g/mL‑1g/mL。本发明采用纯中草药材制成,抗马腺疫链球菌效果显著,患马治愈率高,转归良好。
Description
技术领域
本发明涉及医药领域,具体地说,涉及一种治疗马腺疫的中药组合物。
背景技术
马腺疫是由马链球菌马亚种(又称马腺疫链球菌)引起的一种马属动物急性接触性传染病,主要侵害半岁至四岁的幼驹,以鼻、咽粘膜及颌下淋巴结化脓溃烂,体温升高等全身症状为主要特征。患马腺疫病的马骡在咳嗽、喷嚏时,可通过飞沫经呼吸道传播。目前,因抗生素见效快,价格低廉,所以以其治疗为主,但易产生耐药性、药物残留等问题,且毒副作用较大,整体治疗效果不理想。中药作为我国国粹,拥有天然无残留,不易产生耐药性且价格低廉等优点,因此可以作为开发新药的重要原材料。
发明内容
本发明的目的是提供一种治疗马腺疫的中药组合物。
为了实现本发明目的,第一方面,本发明提供一种治疗马腺疫的中药组合物,所述中药组合物由如下原料复配而成:赤芍提取液0.5g/mL-2g/mL、甘草提取液0.25g/mL-1g/mL、大黄提取液0.25g/mL-1g/mL、雪白睡莲提取液0.5g/mL-2g/mL、山豆根提取液0.5g/mL-2g/mL、苦参提取液0.125g/mL-0.5g/mL以及黄芪和当归质量比为1:1配伍提取液0.25g/mL-1g/mL。
优选地,所述中药组合物由如下原料复配而成:赤芍提取液1g/mL、甘草提取液0.5g/mL、大黄提取液0.5g/mL、雪白睡莲提取液1g/mL、山豆根提取液1g/mL、苦参提取液0.25g/mL以及黄芪和当归质量比为1:1配伍提取液0.5g/mL。
各中药提取液是将药材通过水提法制备得到的,浓缩至4g/mL。
进一步地,所述中药提取液的制备方法包括:称取40g药材,粉碎后按1:15料液比加入蒸馏水,浸泡2h,然后95℃回流提取2h,倒出提取液,再按1g:10mL料液比加入蒸馏水95℃回流提取2h;合并2次提取液,离心,滤纸抽滤,旋转蒸发浓缩药液至4g/mL。
第二方面,本发明提供所述中药组合物在抗马腺疫链球菌以及治疗由其感染所致疾病方面的应用。
第三方面,本发明提供一种治疗马腺疫的方法,所述方法包括:给患马腺疫病的马属动物饲喂所述中药组合物。
各味中药的特性如下:
赤芍:苦,微寒。归肝经。具有清热凉血,活血祛瘀的功效。
甘草:味甘,性平。补脾益气,清热解毒,祛痰止咳,缓急止痛,调和诸药。
大黄:性寒;味苦;归脾、胃、大肠、心包、肝经。功效泻热通肠,凉血解毒,逐瘀通经。
雪白睡莲:味甘,性平,降热止咳、益心护脑、安神止痛等。
山豆根:苦,寒;有毒。有清热解毒,消肿利咽之功效。
苦参:味苦,性寒。清热燥湿,利尿。
黄芪当归:益气养血,可以用于气血亏虚之头晕、神疲乏力。
中药组合物的配伍原则是:组方的配伍原则是“增效减毒”。本病为三喉症之一,以理气泻心肺,清咽喉为治则。赤芍、大黄、山豆根清热凉血,解毒祛瘀,甘草、雪白睡莲平和药性,益心护脑,黄芪当归益气养血,共同达到理气清热袪脓的功效。
借由上述技术方案,本发明至少具有下列优点及有益效果:
本发明以理气清热袪脓为治则,组方合理,制备工艺简单,在参考传统复方的基础上,进行药物筛选试验,选取最佳治疗浓度,具有夯实的科学依据,可为临床提供参考。本组方采用天然中草药材制成,抗马腺疫链球菌效果显著,马腺疫病治愈率高,可有效缩短恢复期。
附图说明
图1为本发明较佳实施例中治疗马腺疫链球菌中药组方的筛选流程示意图。
图2为本发明较佳实施例中马链球菌马亚种致病菌在培养基上的复壮情况。
图3为本发明较佳实施例中马链球菌的细菌形态学观察。
图4为本发明较佳实施例中马链球菌马亚种的16S rRNA特异引物扩增结果。
图5为本发明较佳实施例中心脏、肝脏、脾脏、肺脏、肾脏、脑样品中致病菌的扩增结果。
图6为本发明较佳实施例中中药组合物体外杀菌曲线测定结果。
图7为本发明较佳实施例中小鼠对中药组合物的最大耐受量。其中,A:肺脏,B:心脏,C:肝脏,D:肾脏,E:脾。
图8为本发明较佳实施例中肺脏病理切片。其中,A:模型组小鼠,B:空白组小鼠,C:高浓度中药组合物,D:中浓度中药组合物,E:低浓度中药组合物,F:基础组方,G:西药组。
图9为本发明较佳实施例中心脏的病理切片。其中,A:模型组小鼠,B:空白组小鼠,C:高浓度中药组合物,D:中浓度中药组合物,E:低浓度中药组合物,F:基础组方,G:西药组。
图10为本发明较佳实施例中肝脏病理切片。其中,A:模型组小鼠,B:空白组小鼠,C:高浓度中药组合物,D:中浓度中药组合物,E:低浓度中药组合物,F:基础组方,G:西药组。
图11为本发明较佳实施例中肾脏的病理切片。其中,A:模型组小鼠,B:空白组小鼠,C:高浓度中药组合物,D:中浓度中药组合物,E:低浓度中药组合物,F:基础组方,G:西药组。
图12为本发明较佳实施例中脾脏的病理切片。其中,A:模型组小鼠,B:空白组小鼠,C:高浓度中药组合物,D:中浓度中药组合物,E:低浓度中药组合物,F:基础组方,G:西药组。
具体实施方式
以下实施例用于说明本发明,但不用来限制本发明的范围。若未特别指明,实施例中所用的技术手段为本领域技术人员所熟知的常规手段,所用原料均为市售商品。
实施例1治疗马腺疫链球菌中药组方的筛选
治疗马腺疫链球菌中药组方的筛选流程见图1。
1实验方法
1.1马链球菌马亚种致病菌复壮
取-20℃中保存的马链球菌马亚种,在超净工作台中吸取400μL的冻存菌液加入4mL THB液体培养基中,置于恒温摇床中37℃,180rpm培养18-24h,至培养液浑浊即可。
配置THB绵羊血琼脂培养基,蘸取一环已经复苏的菌液,划线接种。将接种后的血平板放入恒温培养箱中37℃,过夜培养。选取针尖大小且具有溶血环的目标菌落,挑菌,再次置于4mL液体THB培养基中,于恒温摇床中37℃过夜培养,重复三代,直至菌落稳定生长。
1.2鉴定
1.2.1菌株形态观察
观察并记录菌落的大小,形状,颜色,是否凸起以及有无溶血环等。挑取纯化的单菌落,对其进行革兰氏染色,于油镜下观察并记录细菌形态。
1.2.2 PCR鉴定
1.2.2.1引物合成
根据NCBI收录的马链球菌马亚种4047全基因组(FM204883.1)中的fneB基因序列,利用primer5.0设计引物,送由生工生物工程(上海)股份有限公司合成。PCR扩增引物序列和反应运行参数如下:
1.2.2.2 fneB基因扩增
挑取一个在5%绵羊鲜血培养基上纯化完成的单菌落于20μL TE buffer中,吹打混匀,PCR仪95℃5min,得到菌体DNA作为扩增模板。PCR反应体系制备:2×Taq PCR GreenMix 12.5μL,上、下游引物各1μL,DNA模板1μL,dd H2O补至25μL。PCR完成后,用凝胶成像分析系统观察结果。如果出现目标片段,将PCR反应体系送至生工生物工程(上海)股份有限公司测序。
1.3抗菌中药复方的筛选
1.3.1中药煎剂的制备
称取40g药材,粉碎后按1:15料液比加入蒸馏水,室温浸泡2h,95℃回流提取2h,倒出提取液,再按1:10料液比加入蒸馏水95℃回流提取2h;合并2次提取液,离心,滤纸抽滤,旋转蒸发浓缩药液至4g/mL,冷却后置于4℃冰箱中备用。各味药材处理均采用上述方法。
1.3.2试验菌悬液的制备
将18-24h活化好的马链球菌马亚种菌液,加入透明试管中,用生理盐水对比麦氏比浊管,将菌液浓度校正到1×106CFU/mL。校正后的菌液需在15min内接种。
1.3.3培养基的制备
在超净工作台中,将高压好的THB固体培养基倒入平皿中,待培养基凝固,将校对好的菌液,吸取100μL加入培养基表面,用玻璃涂布棒均匀涂布,定点放置牛津杯6个,盖上培养皿盖备用。
1.3.4单味中药对马链球菌马亚种体外抑菌实验
采用牛津杯法,将200μL提取好的单味药物加入牛津杯中,每种单味药物做三个重复,37℃培养16-18h,用游标卡尺测量抑菌圈直径,选取对马链敏感的药物,采用2倍稀释法测其MIC及MBC和部分药物间的FIC。FIC指数=MIC甲药联合/MIC甲药单用+MIC乙药联合/乙药单用。
1.3.5结果判定标准
抑菌圈直径大于15mm为极度敏感;大于10mm为高度极敏;大于8mm,小于10mm为敏感;小于8mm为不敏感。
1.4中药抗菌组方的筛选
1.4.1初步筛选
利用正交试验法。按照1.3.3方法制备含菌培养基。药物按照L8(26)正交表(表1)配制成相应的8种溶液,药物剂量水平设置见表2。采用牛津杯法,分别将8种配制好药液取100μL,加入制备好的含含菌培养基上相应的牛津杯内,做好标记,37℃培养24h,观察其结果并用游标卡尺测量抑菌直径。每种药重复3次,取平均值,同时设空白对照和抗生素对照。结果采用极差分析法进行统计。
表1 L8(26)正交表
表2各单味药物剂量水平设置
注:“/”表示不加药
1.4.2药物剂量优化
根据药物效用的初筛试验结果,采用L18(63)正交表,进一步作剂量优化,药物的剂量水平设置见表4。各药按照正交表(表3)配制成相应的18种溶液,分别测定抑菌圈直径,平行测定3次,取平均值,方法同1.4.1。剂量优化后的最优组合即为中药抗菌基础组方。
表3 L18(63)
表4各单味药水平设置
1.5抗菌组方的加味实验
1.5.1致病菌MLD测定
将活化好的马链球菌马亚种接种于4mL THB肉汤中,37℃培养18h,用生理盐水校准至4.0麦氏比浊度,然后再用生理盐水进行不同Lg的稀释109,108,107,106,105;从中选出3种菌液浓度,每一浓度腹腔注射感染10只雌雄对半,体重20±2g昆明小鼠,每只注射量为0.5mL,观察动物2d内的死亡状况。当出现高浓度组动物全部感染死亡,中浓度组动物部分感染死亡,低浓度组动物全部存活时,将高浓度与中浓度菌液之间用生理盐水稀释出5个浓度梯度,分别对小鼠进行腹腔注射,以确定菌液的最小致死浓度(MLD),取80%~90%MLD浓度作为后续试验的攻毒菌量。
1.5.2抗菌中药组方的加味试验
根据中兽医理论,将已筛选得到的抗菌基础组方内加入不同配比及相同配比不同浓度的补益药,即黄芪当归质量比为1:1,浓度为1g/mL、0.5g/mL,黄芪当归质量比为5:1,浓度为1g/mL、0.5g/mL,从而开展小鼠的体内抗菌试验,观察加味后各组方对感染小鼠的保护率。
保护率=(感染对照组死亡数-试验组死亡数)/感染对照组死亡数×100%
1.6 LD50测定及最大耐受量测定
根据剂量优化和加味实验设定组方各药的初始浓度,在此浓度基础上,向上增加2倍、4倍药物浓度,向下降低2倍、4倍药物浓度,共分为5个用药组(表5),一个空白对照组(生理盐水),第1d按0.04mL/g·bw灌胃小鼠,连续观察七天,第7d采取肝肾组织固定,判定药物毒性。因预实验中无动物死亡,选取最高药物浓度组开展最大耐受量实验,于第1d灌胃3次,每次0.8mL,连续观察14d。小鼠脱颈处死,采心肝脾肺肾,保存在4%的福尔马林中,HE染色制作病理切片。
表5中药组合物最大耐受量各浓度配比
2中药组合物治疗效果测定
2.1马腺疫造模
造模方法同上。
2.2中药组合物治疗
共分为六组,每组10只雌雄各半,体重为20±2kg的昆明小鼠,按照0.01mL/g·bw的药量进行灌胃,第五天灌胃后1h,给予攻毒,再继续灌胃2d,计算保护率。每组存活小鼠脱颈处死,采心肝脾肺肾保存于4%福尔马林中,HE染色制作病理切片。
3结果
3.1马链球菌马亚种致病菌复壮
将-20℃保存的菌种活化后。接种于5%绵羊血培养基上,呈现针尖样大小灰色菌落,并伴随β溶血(图2)。
3.2鉴定
3.2.1细菌形态学观察
挑取单菌落固定于载玻片上,对其进行革兰氏染色,油镜下可见深紫色,排列成短链状的球菌(图3)。
3.2.2 fneB序列的生物信息学分析
3.2.2.1 fneB基因扩增
按照所设计的fneB基因引物,所得菌株能在435bp处扩增出目的条带(图4)。
3.2.2.2fneB基因的同源性比较
将测序结果与GenBank登录的fneB基因序列进行同源性比较,测序结果99%同源。
3.3抗菌组方筛选
3.3.1单味药体外抑菌效果,结果见表6。
表6各中药水煎剂对两种病原菌的抑菌圈直径(mm)
3.3.2高敏药物的MIC及MBC,结果见表7。
表7高敏药物MIC及MBC(g/mL)
3.3.3高敏药物之间的FIC
根据表8结果显示,浙贝母和雪白睡莲FIC指数为4,二者有一定的拮抗作用,综合考虑删减浙贝母。
表8部分敏感中草药间的FIC指数
注:FIC指数<0.5,协同作用;0.5≤FIC<4,相加、无关;4≥FIC指数,拮抗作用。
3.3.4组方药物主次作用分析
按照方案各药剂量水平设置,正交试验结果表明,药物的主次顺序为苦参、大黄、雪白睡莲、甘草、山豆根、赤芍,各单药的极差均未达到负值,6种单味药物全部保留,作为组方基础药物(表9)。
表9组方药物主次作用分析(mm)
3.3.5基础组方剂量优化
由表10可知,赤芍、大黄、雪白睡莲、山豆根选择剂量水平为2MIC时最有效,甘草、苦参在选择MIC水平的时候药效最佳。
表10筛选单味药物剂量优化(mm)
3.4加味实验
3.4.1致病菌MLD的测试结果
由表11可知,马链球菌马亚种的最适攻毒剂量为1×109CFU/mL,0.5mL/只。选取两只死亡的小鼠解剖,进行脏器S.equi分离,在哥伦比亚血平板上划线接种脏器组织液,结果显示心脏、肝脏、脾脏、肺脏、肾脏、脑样品均出现β溶血菌落,分离菌经提取DNA,通过16SrRNA特异引物扩增,显示为马链球菌马亚种(图5)。
表11最小致死量筛选(CFU/mL)
3.4.2中药抗菌基础组方的加味试验结果
加味后各组合对感染小鼠的保护率见表12。由结果可知,黄芪当归按质量比1:1,浓度为0.5g/mL加味的疗效最佳,保护率可达87.5%,因此选用基础方加味1:1,0.5g/mL黄芪当归,将最终组方命名为中药组合物。
表12加味组方保护率
3.5最大耐受量及急性毒性试验结果
按照赤芍提取液1g/mL、甘草提取液0.5g/mL、大黄提取液0.5g/mL、雪白睡莲提取液1g/mL、山豆根提取液1g/mL、苦参提取液0.25g/mL以及质量比为1:1的黄芪当归提取液0.5g/mL,各药物按体积1:1配伍下,配置中药组合物。在急性毒性试验和最大耐受量试验条件下,均无动物死亡,且相病理切片证实没有产生眼观可见的病理变化(图12)。证明该组方的毒性低,在正常给药浓度下,不会对动物机体造成伤害。
3.6体内治疗效果
中药组合物治疗效果见表13,结果显示两次治疗效果均仅次于西药,证明该组方有效。与造模组小鼠比较,治疗效果显著。图8肺脏病理切片可见模型组小鼠的肺脏充血且有炎性细胞浸润,有少量的浆液性渗出,经过不同浓度的中药组合物治疗后,效果显著,肺脏回归正常状态。图9心脏的病理切片可见模型组心肌细胞变性坏死,心肌断裂,经药物治疗后,心肌基本恢复正常,但基础组方对心脏治疗效果极差,这可能也是其治疗效果差于黄连清瘟汤的原因。图10肝脏病理切片可见模型组肝细胞死亡溶解,细胞核固缩,再经过药物治疗后,基本恢复正常。图11肾脏的病理切片可见模型组肾小球毛细血管壁坏死溶解,其周围肾小管上皮细胞有局部溶解坏死,经药物治疗后效果显著,肾脏未出现眼观可见的病理变化。图12脾脏的病理切片可见模型组脾脏结构被破坏,有少量的中性粒细胞浸润,经药物治疗后,仍然存在结构不完整,药物对脾脏的保护效果不显著。
表13中药组合物治疗效果
3.7中药组合物体外杀菌作用
3.7.1体外MIC及MBC的测定,结果见表14。
表14中药组合物MIC及MBC(g/mL)
3.7.2体外杀菌曲线测定
由图6可以看出抑菌效果与药物浓度成正比。小鼠对中药组合物的最大耐受量见图7。
实施例2利用本发明的中药组合物治疗患马腺疫病的马属动物
实例1:昭苏某马场,一匹2岁公马,食欲减退,体温升高至40.1℃,流脓性鼻液,颈部淋巴结脓肿溃烂,经实验室诊断确诊为马腺疫链球菌感染所致。使用实施例1中的中药组合物(赤芍提取液1g/mL、甘草提取液0.5g/mL、大黄提取液0.5g/mL、雪白睡莲提取液1g/mL、山豆根提取液1g/mL、苦参提取液0.25g/mL以及质量比为1:1的黄芪当归提取液0.5g/mL),每日灌服一次,连续5天,肿胀消失愈合,体温正常。
实例2:喀什地区某驴场,多头驴出现流脓性鼻液,淋巴结破溃,食欲降低,背毛杂乱,低热,经实验室诊断确诊为马腺疫链球菌感染所致。按照实例1的方法灌服药剂,7天后,恢复正常。
本发明在参考古籍记载,以及配伍禁忌的前提下,通过科学方法从27种单味药材中筛选出7种体外抑菌效果较强的单味药物,再通过两次正交试验,科学地配制出中药组合物,并通过急性毒性试验以及最大耐受量验证了药物毒性。以上实验结果表明,本发明提供的中药组合物抗马腺疫链球菌效果显著,马腺疫病治愈率高,可有效缩短恢复期。
虽然,上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述,但在本发明基础上,可以对之做一些修改或改进,这对本领域技术人员而言是显而易见的。因此,在不偏离本发明精神的基础上所做的这些修改或改进,均属于本发明要求保护的范围。
Claims (2)
1.治疗马腺疫的中药组合物,其特征在于,所述中药组合物由如下原料复配而成:赤芍提取液1g/mL、甘草提取液0.5g/mL、大黄提取液0.5g/mL、雪白睡莲提取液1g/mL、山豆根提取液1g/mL、苦参提取液0.25g/mL以及黄芪和当归质量比为1:1配伍提取液0.5g/mL;
上述各提取液按体积比1:1配伍配制中药组合物;
各中药提取液是将药材通过水提法制备得到的。
2.根据权利要求1所述的中药组合物,其特征在于,所述中药提取液的制备方法包括:称取40g药材,粉碎后按1:15料液比加入蒸馏水,浸泡2h,然后95℃回流提取2h,倒出提取液,再按1:10料液比加入蒸馏水95℃回流提取2h;合并2次提取液,离心,滤纸抽滤,旋转蒸发浓缩药液至4g/mL。
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