蒿红方止咳抗炎的药效研究和机理的初步探讨
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摘要
1、研究目的
国内外研究表明,由于人口密集、吸烟人群增多、环境污染等因素的恶化,使得呼吸系统疾病的发病率在任何年龄组均占多种疾病之首。上呼吸道感染的西医治疗均不外祛痰、镇咳与抗感染等基本方法。化学药品主要是以止咳、化痰等对症治疗为主,作用迅速,但镇咳类药物副作用大且停药后易复发,导致咳嗽缠延难愈；随着抗生素耐药性的不断增多,限制了抗感染药物的应用。中药防治此类疾病具有较大优势,临床疗效确切,既能杀菌、抑菌、抗病毒、抗炎,又能祛痰、止咳、平喘,还可提高机体免疫力,能通过综合作用祛除病邪或者缓解症状,但大多具有起效缓慢,服用不便的特点。因此,汲取中西药的优点,研制一种安全、速效、针对性强、疗效确切的中药制剂,将极为迫切。
针对南方地区的风热感冒、病毒性感冒,开发具有独特疗效的处方,是临床的需求和患者的希望,也是本课题选题的目的。本课题所研究来源于多年临床经验方,具有止咳、抗炎、平喘之功的处方——蒿红方,是由青蒿和满山红经现代工艺提取纯化后组成的中药复方制剂。课题首先通过对小鼠腹腔毛细血管通透性增高的影响试验、枸椽酸喷雾诱咳法对豚鼠的止咳影响试验来阐明处方配伍的合理性,然后选择合理的配伍组成蒿红方,再通过体内抗菌、体内抗病毒、抗内毒素休克死亡、解热化痰止咳、抗炎止痛作用和对免疫功能的影响等实验研究,并以此对蒿红方的止咳抗炎的药效学进行研究,并初步探讨其作用机理。
2、研究方法
2.1处方配伍合理性研究
2.1.1复方药物对小鼠腹腔毛细血管通透性的影响
取SPF级NIH小鼠,雌雄各半,70只,体重为18～22g,于实验环境下常规饲养3天后,按体重随机均分为7组,10只／组,分别为空白对照组、青蒿组、满山红组、复方1组、复方2组、复方3组、阿司匹林组。各给药组按剂量分别灌胃给药,20 ml／kg,1次／天,连续7 d；空白对照组给予同剂量的麻油。末次给药1 h后,小鼠尾静脉注入2%伊文思蓝生理盐水溶液0.1 ml／10g体重,同时于腹腔注射0.8%醋酸生理盐水溶液0.2 ml／只,20 min后脱颈椎处死小鼠,腹腔注射6 ml生理盐水反复冲洗后,收集冲洗液,调整容积至10 ml,1500 rmp离心10 min,取上清液于722型光栅分光光度计中(波长590nm)测定OD值,比较各组的差异。
2.1.2复方药物对枸椽酸喷雾引咳豚鼠咳嗽的影响
取Hartley豚鼠,体重250g左右,雄雌各半,于实验环境下常规饲养3天。于实验前一天将其逐只放入YLS-8A多功能诱咳引喘仪中,由仪器喷雾17.5%枸椽酸溶液,以150 s内出现哮喘(以抽搐、跌倒的时间作为潜伏期)或者五分钟内咳嗽次数多余10次者为合格的敏感动物。
取上述合格豚鼠80只,雄雌各半,随机分为7组,每组10只。分别为空白对照组,可待因组、满山红组、青蒿组、复方1组、复方2组和复方3组。各给药组按剂量灌胃给药,10 ml／kg,每天1次,连续7 d,空白组给予等剂量的麻油。末次给药1 h后,将豚鼠逐只放入YLS-8A多功能诱咳引喘仪中,由仪器喷雾17.5%枸椽酸(每只豚鼠放入前需更换等体积的新鲜枸椽酸溶液),观察并记录豚鼠从接受喷雾开始到发生抽搐跌倒的时间(即引喘潜伏期)及咳嗽次数,引喘潜伏期超过5 min者以5 min计,比较各组差异。
2.2蒿红方体内抗菌、抗病毒的研究
2.2.1蒿红方对感染肺炎双球菌小鼠治疗作用的影响
取SPF级NIH小鼠,200只,雌雄各半,体重为18～22 g,于实验环境下常规饲养3天后,随机分为7组,分别为空白对照组,模型组,清热消炎宁组,头孢组,蒿红方高、中、低剂量组。除空白对照组和模型组外,其余各组按照剂量灌胃,20 ml／kg,每天一次,连续7 d,空白对照组和模型组灌等剂量麻油。末次给药1 h后,腹腔注射肺炎双球菌(菌号31001-20)悬液,每只1.0 ml(6亿菌／ml),空白对照组和给药对照组腹腔注射等容积的注射用蒸馏水。感染后1、6 h再给药一次,观察和记录一周内各组动物死亡数,计算动物死亡率。统计分析各组差异。
2.2.2蒿红方对感染金黄色葡萄球菌小鼠的影响
取SPF级NIH小鼠,200只,雌雄各半,体重为18～22克,适应环境三天后随机分为8组,分别为空白对照组,模型组,清热消炎宁组,头孢组,蒿红方对照组、高、中、低剂量组。除空白对照组和模型组外,其余各组按照剂量灌胃,20 ml／kg,每天一次,连续7 d,空白对照组和模型组灌等剂量麻油。末次给药1 h后,除空白对照组和蒿红方对照组外,其余各组小鼠腹腔注射金黄色葡萄球菌悬液(菌号26112-5),每只1.0 ml(10亿菌／ml)。第一次感染后1、6 h分别重复给药1次,观察和记录一周内各组动物死亡数,计算动物死亡率。统计分析各组差异。
2.2.3蒿红方对感染流感病毒的小鼠死亡率和平均存活天数的影响
(1)病毒接种方法：取9日龄鸡胚尿囊腔接种注入0.1 ml,接种培育3 d取尿囊液置4℃保存,将尿囊液样品作血凝试验,测血凝滴度,取滴度相同的尿囊液合并后分装于无菌小试管,-70℃保存备用,作为试验用毒种。
(2)毒种半数致死量(LD50)测定：在同一批毒种中取出毒种1支,融化后迅速置冰块中,用1640液作10倍稀释,每一稀释度受感染体重位14 g的小鼠10只,乙醚轻度麻醉,将病毒自最大稀释度起依次滴鼻感染小鼠,每只鼠滴入0.05 ml,对照组滴入等剂量1640液。感染后,每天记录小鼠发病死亡数,连续观察14天,按reed和muench法计算出半数致死量为10-1。
(3)感染小鼠：取SPF级NIH小鼠,体重为13～15g,雌雄各半,于实验室适应环境3天后,随机分为8组,分别为空白对照组、模型组、病毒唑组、清热消炎宁组、蒿红方对照组、高、中、低剂量组。除空白对照组和模型组外,其余各组按照剂量灌胃,20ml／kg,每天一次,连续3 d,空白对照组和模型组灌等剂量麻油。末次给药1小时后,在小鼠于乙醚轻度麻醉下,将已稀释好的病毒液(4个LD50／0.05 ml)滴鼻感染小鼠,每鼠0.05 ml。感染后继续按剂量给药,每天1次,连续7天,并每天记录动物发病情况和死亡数,连续观察14天。计算出死亡保护率和延长生命率,比较各组差异。
2.2.4蒿红方对感染流感病毒小鼠肺重量的影响
(1)病毒接种：取9日龄鸡胚尿囊腔接种注入0.1 ml,接种培育3天取尿囊液置4℃保存,将尿囊液样品作血凝试验,测血凝滴度,取滴度相同的尿囊液合并后分装于无菌小试管,-70℃保存备用,作为试验用毒种。
(2)毒种半数致死量(LD50)测定：在同一批毒种中取出毒种1支,融化后迅速置冰块中,用1640液作10倍稀释,每一稀释度受感染体重位14 g的小鼠10只,乙醚轻度麻醉,将病毒自最大稀释度起依次滴鼻感染小鼠,每只鼠滴入0.05 ml,对照组滴入等剂量1640液。感染后,每天记录小鼠发病死亡数,连续观察14天,按reed和muench法计算出半数致死量为10-1
(3)感染小鼠：取SPF级NIH小鼠,130只,体重为13～15g,雌雄各半,于实验室适应环境3天后,随机分为8组,分别为空白对照组、模型组、病毒唑组、清热消炎宁组、蒿红方对照组、高、中、低剂量组。除空白对照组和模型组外,其余各组按照剂量灌胃,20ml／kg,每天一次,连续3 d,空白对照组和模型组灌等剂量麻油。末次给药1小时后,除空白对照组和蒿红方对照组外,其余各组小鼠在乙醚轻度麻醉下,将己稀释好的病毒液(15个LD50／0.05 ml)滴鼻感染小鼠,每鼠0.05 ml。感染后继续按剂量给药,每天1次,连续4天。末次给药前小鼠禁食不禁水8h,称重,末次给药1h后,脱颈椎处死动物,迅速取出全肺,于生理盐水中清洗2次,摘除气管、肺门淋巴结等组织,滤纸吸干表面水份,称重。计算肺指数和抑制率,比较各组差异。
2.2.5蒿红方对内毒素致小鼠休克死亡的影响
取SPF级NIH小鼠,120只,体重为18～22 g,雌雄各半,于实验室适应环境3天后,随机分为6组,分别为空白对照组、模型组、清热消炎宁组、蒿红方对照组、高、中、低剂量组。除空白对照组和模型组外,其余各组按照剂量灌胃,20 ml／kg,每天1次,连续7 d,空白对照组和模型组灌等剂量麻油。末次给药1 h后,除空白对照组小鼠外其余各组小鼠均腹腔注射D-GALN 200 mg／kg和内毒素1.5 mg／kg,空白对照组腹腔注射等剂量的生理盐水,观察72小时,记录动物死亡数。比较各组差异。
2.3蒿红方清热止咳化痰作用的研究
2.3.1蒿红方对干酵母所致大鼠发热的影响
取经帅选合格动物60只,随机分为6组,分别为空白对照组、阿司匹林组、清热消炎宁组、蒿红方高、中、低剂量组。实验前6h禁食不禁水。空白组大鼠颈部以下的背部皮下注射生理盐水10 ml／kg；各组大鼠分别在大鼠颈部以下的背部皮下注射20%酵母悬液10 ml／kg,按摩注射部位以使悬浮液在皮肤下扩散。造模同时各剂量组分别按剂量灌胃给药,空白对照组给予等剂量的麻油。于药后1、2、3、4小时各测体温1次,比较各组动物体温下降值的差异。
2.3.2蒿红方对内毒素所致新西兰兔发热的影响
取经帅选合格的新西兰兔36只,雌雄各半,随机分为6组,分别为空白对照组,阿司匹林组,清热消炎宁组,蒿红方高、中、低剂量组。除空白对照组和模型组外,其余各组按照剂量灌胃,10 ml／kg,每天1次,连续7 d,空白对照组和模型组灌等剂量麻油。末次给药1h后,从兔耳缘静脉注射内毒素10μg／ml·kg体重,于给药后1、2、3、4、6h各测体温1次,比较各组动物体温下降值的差异。
2.3.3蒿红方对小鼠氨水引咳作用的影响
取SPF级NIH小鼠,体重18～22 g,雄雌各半,随机分为7组,每组15只。分别为空白对照组,可待因组,牡荆油组,蒿红方高、中、低剂量组。各给药组按剂量灌胃给药,空白组给予等剂量麻油。20 ml／kg,每天1次,连续7 d。末次给药1 h(可待因组30 min)后,将豚鼠逐只放入恒压氨水喷雾装置内(压力为140mmHg,氨水浓度为28%),由仪器恒压氨水喷雾(每只小鼠放入前需更换等体积的新鲜氨水溶液),氨水喷雾时间按等比级数递增,按序贯法改变氨水喷雾时间,观察并记录小鼠从接受喷雾开始到发生咳嗽的时间,计算小鼠半数引咳时间(EDT50),并按公式计算R值(R值＞130%为有镇咳作用,R值>150%为有明显镇咳作用)。比较各组差异。
2.3.4蒿红方对枸椽酸喷雾引咳法致豚鼠咳嗽的影响
取Hartley豚鼠,体重250～280g,雄雌各半,在实验环境下常规饲养3天。于实验前一天将其逐只放入YLS-8A多功能诱咳引喘仪中,由仪器喷雾17.5%枸椽酸溶液,以150s内出现哮喘(以抽搐、跌倒的时间作为潜伏期)或者五分钟内咳嗽次数多余10次者为合格的敏感动物。
取上述合格豚鼠80只,雄雌各半,随机分为7组,每组10只。分别为空白对照组,可待因组,牡荆油组,蒿红方高、中、低剂量组。各给药组按剂量灌胃给药,空白组给予等剂量麻油,10 ml／kg,每天1次,连续7 d。末次给药1h(可待因组30 min)后,将豚鼠逐只放入YLS-8A多功能诱咳引喘仪中,由仪器喷雾17.5%枸椽酸(每只豚鼠放入前需更换等体积的新鲜枸椽酸溶液),观察并记录豚鼠从接受喷雾开始到发生抽搐跌倒的时间(即引喘潜伏期)及5 min内的咳嗽次数,引喘潜伏期超过5min者以5min计,比较各组差异。
2.3.4蒿红方对小鼠气管酚红排泌量的影响
取SPF级NIH小鼠,60只,体重为18～22 g,雌雄各半。于实验室适应环境3天后,随机分为5组,分别为空白对照组、牡荆油组、蒿红方高、中、低剂量组。各给药组按照剂量灌胃,20 ml／kg,空白对照组灌等剂量麻油。每天1次,连续7 d。末次给药1 h后,各鼠按剂量0.01 ml／g体重腹腔注射5%的酚红溶液,注射后0.5 h,将小鼠颈椎脱臼处死,仰位固定于手术板上剪开颈前皮肤,分离气管,剥离气管周围组织,剪下甲状软骨下缘至气管分叉处之气管,用5%碳酸氢钠溶液0.5 ml冲洗气管,连续4次,洗出液合并,置于紫外可见分光光度计上于546 nm波长处测定OD值,并根据实验前制定的标准曲线计算出气管冲洗液中酚红浓度,计算祛痰率。
标准曲线的制定：用分析天平准确称取一定量酚红以5g／dl碳酸氢钠溶解,使1 ml含酚红1000 ug,依次稀释成每毫升含酚红0.01、0.1、0.5、0.7、1、3、5 ug,测OD值,以酚红剂量为横坐标,OD值为纵坐标,作标准曲线,并计算出每鼠酚红排泌量。比较各组差异。
2.3.5蒿红方对鸽气管纤毛运动的影响
取普通级家鸽,体重400～500 g,雌雄各半,按体重随机均分为5组,分别为空白对照组,牡荆油组,蒿红方高、中、低剂量组。各给药组按剂量分别灌胃给药,10ml／kg,每天一次,连续7 d,空白对照组给予等剂量的麻油。末次给药后1 h,在暗室内将家鸽颈部拉直与水平面平行,然后作颈部切口,剥离气管,使气管尽量地暴露,从靠心脏端,将5号针头插入气管,使针头靠近气管内壁推入0.02 ml墨法,在冷光源下,观察1 min内墨汁向前运动的距离。
2.4蒿红方抗炎止痛作用的研究
2.4.1蒿红方对小鼠腹腔毛细血管通透性的影响
取SPF级NIH小鼠,雄性,60只,体重为18～22g,于实验环境下常规饲养3天后,按体重随机均分为6组,10只／组,分别为空白对照组、阿斯匹林组、清热消炎宁组、蒿红方高、中、低剂量组。除空白对照组外其余分别给药,剂量为20 ml／kg,1次／天,连续7 d；末次给药1 h后,小鼠尾静脉注入2%伊文思蓝生理盐水溶液0.1 ml／10g体重,同时于腹腔注射0.8%醋酸生理盐水溶液0.2 ml／只,20 min后脱颈椎处死小鼠,腹腔注射6 ml生理盐水反复冲洗后,收集冲洗液,调整容积至10 ml,1500 rmp离心10 min,取上清液于722型光栅分光光度计中(波长590nm)测定OD值,比较各组的差异。
2.4.2蒿红方对大鼠蛋清性足肿胀的影响
取SD大鼠60只,体重为130～150 g,随机均分为6组,分别为空白对照组,阿司匹林组,清热消炎宁组,蒿红方高、中、低组,每组10只。各给药组按剂量灌胃给药,空白对照组给予等体积麻油。每天1次,连续灌胃给药7 d。末次给药前,在每只大鼠右后足跖处作一标记,以YLS-7A足趾容积测量仪测量右足后跖体积,作为致炎前右后足基础值。末次给药后1 h,在每只大鼠右后足跖皮腱膜下注射100%新鲜蛋清0.1 mL／只,测定注入致炎剂后1、2、3、4 h右后足跖体积,并减去致炎前大鼠右后足基础值,作为各鼠在不同时间的足肿胀度。比较不同时间段的足肿胀度水平。
2.4.3蒿红方对大鼠甲醛性足肿胀的影响
取SD大鼠60只,体重为140～160g,随机均分为6组,分别为空白对照组、阿司匹林组、清热消炎宁组、蒿红方高、中、低组,每组10只。各给药组按剂量灌胃给药,空白对照组给予等体积麻油。每天1次,连续灌胃给药7 d。在给药前,在每只大鼠右后足跖处作一标记,以YLS-7A足趾容积测量仪测量右足后跖体积,作为致炎前右后足基础值。
第一次给药后1 h,在每只大鼠右后足跖皮腱膜下注射1%甲醛生理盐水溶液0.1mL／只。第2天至第6天继续给药并于给药后1 h测定右足后跖容积,每天1次。以药后1、2、3、4、5天结果计算足肿胀度。比较不同时间段的足肿胀度水平。
2.4.4蒿红方对醋酸所致小鼠疼痛的影响(扭体法)
取SPF级NIH小鼠,72只,雌雄各半,体重为18～22g,按体重随机均分为6组,每组12只,分别为空白对照组、阿司匹林组,清热消炎宁组,蒿红方高、中、低剂量组。各给药组按剂量灌胃给药,空白对照组给予等剂量麻油,20 ml／kg,1次／天,连续7 d。末次给药1 h后,小鼠腹腔注射0.8%醋酸生理盐水溶液0.1mL／10g体重,观察记录20 min内小鼠的扭体次数扭体反应：腹部凹陷、伸展后肢、臀部抬高),比较各组差异。
2.4.5蒿红方对甲醛足跖皮下注射所致小鼠疼痛的影响
取SPF级NIH小鼠,72只,雌雄各半,体重为18～22 g,按体重随机均分为6组,每组12只,分别为空白对照组、阿司匹林组,清热消炎宁组,蒿红方高、中、低剂量组。各给药组按剂量灌胃给药,空白对照组给予等剂量麻油,20 ml／kg,1次／天,连续7 d。末次给药1 h后,各组小鼠左后足足跖皮下注射2.5%甲醛生理盐水溶液0.05 ml／只,立即计时,记录5 min内小鼠舔左后足累积时间,比较各组差异。
2.5蒿红方对免疫功能的研究
2.5.1蒿红方对小鼠免疫器官的影响
取SPF级NIH小鼠,60只,雌雄各半,体重13～15g,适应实验环境三天。按照体重随机均分为5组,分别为空白对照组、绞股蓝组,蒿红方高、中、低剂量组,每组12只。各给药组按剂量灌胃给药,20 ml／kg,每日1次,连续7 d。末次给药后12 h,脱颈椎处死小鼠,迅速剖取胸腺、脾脏,称重,计算脏器指数。脾(胸腺)指数=脾(胸腺)重(mg)／体重(g)。比较各组差异。
2.5.2蒿红方对小鼠体内炭粒廓清功能的影响
取SPF级NIH小鼠,雄性,60只,体重18～22 g,按体重随机均分为5组,分别为空白对照组、绞股蓝组、蒿红方高、中、低剂量组,每组12只。各给药组分别按剂量灌胃给药,20 ml／kg,每日1次,连续7 d,空白对照组给予等容积麻油。末次给药后1h,小鼠尾静脉注入稀释墨汁0.1 ml／10g体重,于注入墨汁后30 s及6 min,从小鼠眼眶后静脉丛取血0.025 ml,立刻吹入2 ml 0.1%碳酸氢钠溶液中；取0.025 ml正常未注射墨汁的血溶于2 ml 0.1%碳酸氢钠溶液校零,675 nm处测定OD值,按下式计算吞噬指数K值。比较各组差异。
2.5.3蒿红方对小鼠血清溶血素抗体生成的影响
取SPF级NIH小鼠,雌雄各半,60只,体重18～22g,按体重随机均分为5组,分别为空白对照组、绞股蓝组、蒿红方高、中、低剂量组,每组12只。各给药组分别按剂量灌胃给药,20 ml／kg,每日1次,连续10 d,空白对照组给予等容积麻油。第6日各组小鼠腹腔注射5%绵羊红细胞0.4 ml／只(相当于4×108SRBC／只)。末次给药后1 h,眼眶取血测定血清溶血素抗体。比较各组差异。
2.5.4蒿红方对小鼠迟发型超敏反应的影响
取SPF级NIH小鼠,雌雄各半,60只,体重18～22g,适应环境三天。按体重随机均分为5组,分别为空白对照组、绞股蓝组、蒿红方高、中、低剂量组,每组12只。各组小鼠腹部皮肤脱毛(1cm2左右),均匀涂布1% DNFB溶液50μl致敏,次日同法强化一次。各给药组分别按剂量灌胃给药,20 ml／kg,每日1次,连续10 d,空白对照组给予等容积麻油。第7天给药后将1%DNFB溶液10μl均匀涂抹于小鼠右耳(两面)进行攻击,攻击后24小时脱颈椎处死小鼠,剪下左右耳壳,用8mm打孔器取同部位的左右两耳片,称重,以左右耳片重量之差为肿胀度(表示DTH的强弱)。
3、结果
3.1配伍合理性研究
3.1.1抑制小鼠腹腔毛细血管通透性与空白对照组相比,复方3组(青蒿200mg／kg+满山红100mg／kg)能降低小鼠腹腔毛细血管通透性并具有显著性统计学差异(P<0.01)；复方1组(青蒿100mg／kg+满山红200mg／kg)、复方2组(青蒿150mg／kg+满山红150mg／kg)也能降低小鼠毛细血管通透性并具有显著性统计学差异(P<0.05)。
3.1.2减轻豚鼠咳嗽与空白组相比,复方2、3组均能延长豚鼠咳嗽的潜伏期、减少5分钟内豚鼠的咳嗽次数,并具有显著性统计学差异(P<0.01)；复方1组能延长咳嗽潜伏期、减少5分钟内豚鼠的咳嗽次数,并具有显著性统计学差异(P<0.05)。
3.2蒿红方体内抗菌、抗病毒的研究
3.2.1降低感染肺炎双球菌病毒小鼠的死亡率与空白对照组相比,模型组小鼠死亡率显著升高且具有统计学差异(P<0.01),蒿红方对照组对未感染肺炎双球菌的小鼠死亡率无影响；与模型组相比较,蒿红方高、中剂量组显著降低小鼠的死亡率且具有统计学差异(P<0.01)。
3.2.2降低感染金黄色葡萄球菌病毒小鼠的死亡率与空白对照组相比,模型组小鼠的死亡率显著升高并具有统计学意义(P<0.01),蒿红方对照组对未感染金黄色葡萄球菌小鼠的死亡率无影响；与模型组相比,蒿红方高剂量组能明显降低金黄色葡萄球菌感染的小鼠的死亡率(P<0.01),蒿红方中、低剂量组亦能降低金黄色葡萄球菌感染的小鼠引起的死亡率但不具有统计学差异(P>0.05)。
3.2.3降低感染流感病毒对小鼠的影响与空白对照组相比较,模型组动物的死亡率显著增高、其平均存活天数显著缩短并且都具有显著性统计学差异(均有P<0.01),蒿红方对照组对未感染小鼠死亡率和平均存活天数无影响；与模型组相比较,蒿红方高、中剂量组显著降低了流感病毒感染的小鼠的死亡率、延长了其平均存活天数并且具有显著性统计学差异(均有P<0.01),蒿红方低剂量组亦显著降低了小鼠的死亡率、延长了其平均存活天数并且具有显著性统计学差异(P<0.05)。
3.2.4抑制感染流感病毒小鼠肺指数与空白对照组相比,模型组肺指数明显增大并具有显著性统计学差异(P<0.01)；与模型组相比较,蒿红方高剂量组能明显降低流感病毒感染小鼠的肺指数并具有显著性统计学差异(P<0.01),蒿红方中剂量组也能明显降低流感病毒感染小鼠的肺指数并具有显著性统计学差异(P<0.05),蒿红方低剂量也能降低小鼠肺指数但不具有统计学意义(P>0.05)。
3.2.5降低内毒素致小鼠的休克死亡与空白对照组相比较,模型组小鼠死亡率增高且具有显著性统计学差异(P<0.01),蒿红方对照组对正常小鼠未见致死；与模型组相比较,蒿红方高剂量组能降低内毒素致休克死亡小鼠的死亡率且具有显著性统计学差异(P<0.01),蒿红方中剂量组、蒿红方低剂量组亦能降低内毒素致休克死亡小鼠的死亡率但不具有显著性统计学差异(P>0.05)。
4、研究结论：
本研究结果表明,蒿红方具有抑菌、抗病毒、抗内毒素、清热化痰止咳、抗炎止痛及增强免疫功能的作用,为蒿红方疏风清热、解毒、止咳化痰的功效提供了药效学依据,从而肯定了蒿红方的有效性。
1、Objective:
Due to population density, increasing smoking crowd, environmental pollution and other factors, research has shown that the incidence of respiratory disease at any age group account for the first of many diseases. The medical treatment of upper respiratory tract infection are the expectorant, antitussive and anti-infection and other basic methods. Chemicals, mainly cough, phlegm and other and other symptomatic treatment based, main effect rapidly, but antitussive drugs have side effects and easy to relapse after drug withdrawal, leading to extended refractory cough bound; with the increasing antibiotic resistance, the application of anti-infectives is restricted. Traditional Chinese medicines have greater advantages, clinical curative effect, which is not only bactericidal, antibacterial, antiviral and anti-inflammatory, but also expectorant, cough, asthma, and improve immunity, eliminate or alleviate the symptoms of pathogenic factors through an integrated role, but many of them have slow onset, taking the characteristics of inconvenience. So, the Development of a safe, quick, targeted, curative effect of Chinese medicine preparations which learn the advantages of Chinese and Western medicine is extremely urgent.
It is the needs of clinical and patients, is also purpose of this topic to develop an unique therapeutic prescription for the southern district of fengreganmao and viral influenza. This topic is from many years of clinical experience. Haohongfang which can suppressing cough、anti-inflammatory、relieving asthma,is a traditional Chinese medicine compound of Modern extraction and purification of Artemisia annua and Folium. Firstly, the topic clarified the rationality of prescription through increased capillary permeability in mice the impact test, CBS spray induced cough cough impact tests on guinea pigs to clarify the prescription to the rationality, secondly selected the rational compatibility of the composition of Haohongfang, thirdly, study the cough and anti-inflammatory pharmacodynamics of Haohongfang and investigate the mechanism of it preliminary.
2、Methods:
2.1 Study of Compatibility Rationality
2.1.1 The impact on the capillary permeability in mice
70 SPF level NIH mice, male and female in half,18-22g, adapted to the environment for three days. And then divided randomly into 7 groups,10 mice/group named control group, Artemisia group, Folium group, compound group 1, compound group 2, compound group 3, aspirin group. Each treatment group were orally administered by 20 ml/kg, once a day,7 days in all; the control group were given equal volume of sesame oil.1 h after the last administration,2% Evans blue saline solution was injected with 0.1 ml/10g weight, and 0.8% acetic acid saline solution intraperitoneal with 0.2 ml each at the same time,20 min later were killed all of the mice,6 ml normal saline flus was injected intraperitoneal, and then the fluid was collected, adjusting the volume to 10 ml,1500 rmp centrifuge for 10 min, tested OD values with 722 grating spectrophotometer(590 nm) and compared the difference between the groups.
2.1.2 Drugs on cough in guinea pigs
Hartley guinea pigs,250g or about, female and male in half, adapted to the environment for three days. The day before the experiment will only add to its by-YLS-8A multi-instrument induced asthma induced cough from 17.5% citric acid solution, spray equipment, to occur within 150 s of asthma (in convulsions, fall time as the incubation period) or cough times within five minutes extra in the 10 who qualified for sensitive animals.
80 qualified guinea pigs, female and male in half, divided into 7 groups of 10 each. Control group, codeine group, Folium group, Artemisia annua group, compound group 1, compound group 2 and compound group 3. Each treatment group were orally administered by 10 ml/kg, once a day,7 days in all; the control group were given equal volume of sesame oil.1 h after the last administration, the guinea pig by only into the YLS-8A multi-functional instrument induced asthma induced cough,17.5% citric acid spray from the apparatus (to be replaced before each guinea pig into the same volume of fresh phosphate histamine solution) were observed and recorded in guinea pigs receiving spray from the beginning to the fall time of the occurrence of seizures (ie, latent period of asthma) and cough frequency, latent period of asthma 5 min to 5 min of were to compare the differences between each group.
2.2 HaoHongfang on antibacterial, antiviral research
2.2.1 HaoHongfang on the treatment in mice infected with Streptococcus pneumoniae
200 SPF level NIH mice, male and female in half,18～22 g, adapted to the environment for three days.And then divided randomly into 7 groups, control group, model group, Qingrexiaoyanning group, cephalosporin group, HaoHongfang high, medium and low dose group. Each treatment group was orally administered by 20 ml/kg, once a day,7 days in all; control group and model group were given equal volume of sesame oil.1 h after the last administration, intraperitoneal injection of Streptococcus pneumoniae (strain No. 31001-20) suspension, each 1.0 ml (0.6 billion bacteria/ml), blank control group and the control group, intraperitoneal administration of the injection volume of distilled water.1,6 h after infection and then administered once a week to observe and record the number of deaths in each group, compute the mortality rate. And compared the difference between the groups.
2.2.2 HaoHongfan on the mortality rate of mice infected with Staphylococcus aureus
200 SPF level NIH mice, male and female in half,18 to 22 g, after three days of adaptation to the environment were randomly divided into 8 groups, control group, model group, Qingrexiaoyanning group, cephalosporin group, HaoHongfang control group, high, medium and low dose group. In addition to the control group and model group, other groups in accordance with the dose administered,20 ml/kg, once a day for 7 days, control group and model group filling the same dose of sesame oil.1 h after the last administration, in addition to the control group and the control group, HaoHongfang side, the remaining mice were injected suspension of Staphylococcus aureus (strain No.26112-5), each 1.0 ml (lbillion bacteria/ml).1,6 h after the first infection were repeated dose 1, observe and record animals in each group died within a week the number of animal mortality calculated. And compared the difference between the groups.
2.2.3 HaoHongfang on the infection of influenza virus in mice
(1) virus inoculation Methods:9 day old chick embryo allantois inoculated into 0.1 ml, inoculation training allantoic fluid obtained 3 d at 4℃, the allantoic fluid samples for hemagglutination test, measuring hemagglutination titer, take the same titer of allantoic fluid after the merger of small-packing in sterile test tubes,-70℃save standby, as a pilot with the virus seed.
(2) toxic species of the median lethal dose (LD50) determination:toxic species in the same group of species of a drug out, home ice melting rapidly, with 1640 as 10-fold dilution, dilution of the infected body weight of each 14-bit 10 g mice, mild ether anesthesia, the maximum dilution of the virus since the beginning followed by intranasal infection of mice, each mouse instilled 0.05 ml, control group instilled such dosage 1640. After infection, the mice daily incidence of mortality, continuous observation for 14 days, according to reed and muench method to calculate the lethal dose was 10-1.
(3) infection Mouse:SPF level obtained NIH mice, weighing 13～15g, male and female in half, to adapt to the environment in the laboratory after 3 days were randomly divided into 8 groups, the control group, model group, ribavirin Group Qingrexiaoyan Ning Group, HaoHongfang control group, high, medium and low dose group. In addition to the control group and model group, other groups in accordance with the dose administered, 20ml/kg, once a day for 3 d, the control group and model group filling the same dose of sesame oil.1 hour after the last administration, the mice in the light ether anesthesia, will have a good virus dilute solution (4 LD50/0.05 ml) intranasally infected mice, each mouse was 0.05 ml. Infection continue to dose,1 time per day for 7 days, and daily records of animal morbidity and mortality, continuous observation for 14 days. Calculate the mortality rate and prolong life protection rate and compared the difference between the groups.
2.2.4 HaoHongfang on Lung-index of mice infected with influenza virus
(1) virus vaccination:from 9 day old chick embryo allantois inoculated into 0.1 ml, inoculation training allantoic fluid obtained 3 days at 4℃, the allantoic fluid samples for hemagglutination, hemagglutination titer test, to take the same titer of allantoic fluid after the merger of small-packing in sterile test tubes,-70℃save standby, as a pilot with the virus seed.
(2) toxic species of the median lethal dose (LD50) determination:toxic species in the same group of species of a drug out, home ice melting rapidly, with 1640 as 10-fold dilution, dilution of the infected body weight of each 14-bit 10 g mice, mild ether anesthesia, the maximum dilution of the virus since the beginning followed by intranasal infection of mice, each mouse instilled 0.05 ml, control group instilled such dosage 1640. After infection, the mice daily incidence of mortality, continuous observation for 14 days, according to reed and muench method to calculate the lethal dose was 10-1.
(3) infection Mouse:SPF level obtained NIH mice,130, weight 13-15g, male and female in half, to adapt to the environment in the laboratory after 3 days, were randomly divided into 8 groups, the control group, model group, ribavirin group, Qingrexiaoyanning group, HaoHongfang control group, high, medium and low dose group. In addition to the control group and model group, other groups in accordance with the dose administered, 20ml/kg, once a day for 3 d, the control group and model group filling the same dose of sesame oil.1 hour after the last administration, in addition to the control group and the control group, HaoHongfang side, the remaining mice in the light ether anesthesia, will have a good virus dilute solution (15 LD50/0.05 ml) intranasally infected with small mice, each mouse was 0.05 ml. Infection continue to dose,1 time per day,4 consecutive days. Fasting before the last administration, mice can not help but water 8h, weighing, 1h after the last administration, animals were sacrificed and dislocation of cervical vertebra, quickly remove the entire lung, in normal saline wash 2, removal of the trachea, hilar lymph nodes and other organizations, the filter paper suction Dry the surface moisture, and weighed. Lung index and the inhibition rate calculated to compare the differences between each group.
2.2.5 HaoHongfang on the death in mice induced by endotoxin
120 SPF level NIH mice,18～22 g, male and female in half, adapted to the environment in the laboratory for 3 days, randomly divided into six groups, control group, model group, Qingrexiaoyanning Group HaoHongfang control group, high, medium and low dose group. In addition to the control group and model group, other groups in accordance with the dose administered,20 ml/kg, once a day,7 days in all, control group and model group were given equal volume of sesame oil.1 h after the last administration, in addition to the blank control group were outside the remaining mice were injected D-GALN 200 mg/kg and LPS 1.5 mg/kg, control group, intraperitoneal dose of saline, observed 72 hours, record the number of animal deaths. There are differences between each group.
2.3 HaoHongfang on clearing heat and relieving cough and phlegm
2.3.1 HaoHongfang on fever induced by dry yeast
60 qualified animals were randomly divided into six groups, control group, aspirin group, Qingrexiaoyanning group, HaoHongfang high, medium and low dose group.6h fasting before the experiment can not help but water. Control back of the neck rats injected with 10 ml/kg; 20% of the yeast suspension were injected 10 ml/kg in the back of rats and normal saline with control group. Modeling the same time each dose group were administered orally by dose and blank control group was given the sesame oil.1,2,3,4 hours after the drug 1 of each measured body temperature, body temperature decreased in all groups compared the value of the difference.
2.3.2 HaoHongfang on endotoxin fever of rabbits
36 qualified New Zealand rabbits were randomly divided into 6 groups, control group, aspirin group, Qingrexiaoyanning group, HaoHongfang high, medium and low dose group. In addition to control group and model group, other groups in accordance with the dose administered,10 ml/kg, once a day,7 days in all, control group and model group were given the same dose of sesame oil. 1h after the last administration,10μg/ml·kg of endotoxin were injection from the rabbit ear vein,1,2,3,4,6 h after the administration temperature were measured one time,and compared the difference between each gtoup.
2.3.3 HaoHongfang on cough in mice with ammonia water
SPF level NIH mice,18～22 g, female and male in half, randomly divided into 7 groups,15 in each group. Control group, respectively, codeine, Vitex oil group, HaoHongfang high, medium and low dose group. Each treatment group by dose oral administration, given control group sesame oil.20 ml/kg, once a day,7 days in all.1 h (codeine 30 min) after the last administration, put constant pressure of guinea pigs by ammonia spray device (pressure of 140mmHg, ammonia concentration of 28%), ammonia by the constant pressure spray equipment fog (to be replaced before each mouse into the same volume of fresh ammonia solution), ammonia spray time, increasing by geometric progression, according to sequential changes in ammonia spray time to observe and record the mice receiving spray from fog began to cough occurred the time to calculate the time mice were half of the cough (EDT50), calculated according to R value (antitussive effects:R value> 130%, significant antitussive effect:R values> 150%). There are differences between each group.
2.3.4 HaoHongfang on the citric acid spray lead cough cough of guinea pigs
Hartley guinea pigs,250～280g, female and male in half, adapted to the environment in the laboratory for 3 days. The day before the experiment will only add to its by-YLS-8A multi-instrument induced asthma induced cough from 17.5% citric acid solution spray equipment to 150s appeared in asthma (in convulsions, fall time as the incubation period) or 5 the number of extra minutes to cough 10 times the sensitivity of animals to qualified persons.
80 qualified guinea pigs, female and male in half, divided into 7 groups of 10 each. Control group, codeine group, Vitex oil group, HaoHongfang high, medium and low dose group. Each treatment group by dose oral administration, control group was given sesame oil,10 ml/kg, once a day,7 days in all. the control group were given equal volume of sesame oil.1 h after the last administration, the guinea pig by only into the YLS-8A multi-functional instrument induced asthma induced cough,17.5% citric acid spray from the apparatus (to be replaced before each guinea pig into the same volume of fresh phosphate histamine solution) were observed and recorded in guinea pigs receiving spray from the beginning to the fall time of the occurrence of seizures (ie, latent period of asthma) and cough frequency, latent period of asthma 5 min to 5 min of were to compare the differences between each group.
2.3.5 HaoHongfang on the mouse trachea phenol red secretion
60 SPF level NIH mice,18～22 g, male and female half and half. adapted to the environment for three days.And then divided randomly into five groups, control group, Vitex oil group, HaoHongfang high, medium and low dose group. The treatment group in accordance with the dose administered,20 ml/kg, such as dosage control group irrigated sesame oil.Once a day,7 days in all.1 h after the last administration,5% phenol red solution were injected with 0.01 ml/g intraperitoneal,0.5 h later, killed the mice by cervical dislocation, Yang-bit fixed-board cut in surgery cervical skin, separation of the trachea, tracheal strip surrounding tissue, cut the lower edge of thyroid cartilage to the tracheal bifurcation of the trachea, with 5% sodium bicarbonate solution,0.5 ml tracheal wash for 4 times, washing the combined solution, placed in UV-visible spectrophotometer on the wavelength of 546 nm measured in OD values, and in accordance with the standards set before the experiment to calculate tracheal fluid curve of phenol red were calculated expectorant rate.
The formulation of the standard curve:analytical balance accurately weighed with a certain amount of phenol red to 5g/dl sodium bicarbonate dissolved in 1 ml phenol red to 1000 ug, followed by diluted per milliliter of phenol red 0.01,0.1,0.5,0.7,1,3,5 ug, test OD values to a dose of phenol red horizontal, OD value of the vertical axis for the standard curve, and calculated for each mouse phenol red secretion. There are differences between each group.
2.3.6 HaoHongfang on the tracheal cilia movement of pigeons
Pigeons,400～500 g, male and female in half, were randomly divided into 5 groups, namely control group, Vitex oil group, HaoHongfang high, medium and low dose group. Each treatment group were administered orally by dose,10 ml/kg, once a day for 7 days, the control group was given the sesame oil.1 h after the last administration, straightened and paralleled levelly to the neck in pigeons in the dark room, and then stripped the trachea with a neck incision so as to expose the trachea, through the heart from the end of the needle NO.5 into the trachea, so that the needle close to the tracheal wall into the 0.02 ml ink, in the cold light source, the observed within 1 min from the forward movement of ink.
2.4 The study of HaoHongfang on anti-inflammatory analgesic
2.4.1 Inhibiting the capillary permeability in mice
60 SPF level NIH mice, male,18-22g, adapted to the environment for three days, were randomly divided into 6 groups,10 each group, named control group, Aspen aspirin group, Qingrexiaoyan Ning group, HaoHongfang high, medium and low dose group. In addition to the rest of control group were administered a dose of 20 ml/kg, once a days,7 days in all; 1 h after the last administration,2% Evans blue saline solution was injected with 0.1 ml/lOg, and 0.8% acetic acid saline solution intraperitoneal with 0.2 ml each at the same time,20 min later were killed all of the mice,6 ml normal saline flus was injected intraperitoneal, and then the fluid was collected, adjusting the volume to 10 ml,1500 rmp centrifuge for 10 min, tested OD values with 722 grating spectrophotometer(590 nm) and compared the difference between the groups.
2.4.2 The influence of HaoHongfang rat paw swelling of egg white
60 SD rats,130～150 g, were randomly divided into six groups, control group, aspirin group, Qingrexiaoyanning group, HaoHongfang high, medium and low groups 10. Each treatment group by dose oral administration, and control group were given equal volume of sesame oil. Once a day,7 days in all. Before the last administration, each rat in the right rear paw Department as a marker to YLS-7A toe right foot volume measurement after measurement plantar volume, as inflammation right foot before the baseline. After the last administration,1 h, each rat in the right foot plantar fascia under the skin injection of fresh egg white 100% 0.1 mL/only measured after injection of inflammatory agent 1,2,3,4 h after the right paw volume and less inflammation in rats before the right foot base value, as the mice at different times of the foot swelling. Comparison of different time levels of foot swelling.
2.4.3 The influence of HaoHongfang rat paw swelling of formaldehyde
SD rats were taken 60,140～160g, were randomly divided into six groups, control group, aspirin group, Qingrexiaoyanning group, HaoHongfang high, medium and low group. Each treatment group by dose oral administration, and control group were given equal volume of sesame oil.1 time per day, continuous intragastric administration 7 d. Prior to administration, in the right rear paw of each rat Office as a marker to YLS-7A toe right foot volume measurement after measurement plantar volume, as inflammation right foot before the baseline.
After the first dose 1 h, each rat in the right foot plantar fascia under the skin of 1% formaldehyde saline solution injection of 0.1 mL/only. Day 2 to 6 days to delivery, and measured at 1 h after administration right foot plantar volume after day 1.1,2,3,4,5 days after drug results to calculate foot swelling. Comparison of different time levels of foot swelling.
2.4.4 The influence of HaoHongfang on the impact of pain in mice induced by acetic acid (writhing)
72 SPF level NIH mice, male and female in half,18-22g, were randomly divided into 6 groups of 12 were named control group, aspirin group, Qingrexiaoyanning group, HaoHongfang high, medium and low dose group. Each treatment group by dose oral administration, the control group was given sesame oil,20 ml/kg, once a day,7 days in all. 1 h after the last administration, mice were injected 0.8% acetic acid saline solution 0.1mL/10g weight, observed within 20 min the number of writhing writhing response: abdominal sag, stretch limbs, buttocks elevation), compared differences between each group.
2.4.5 The influence of HaoHongfang on pain in the foot induced by formalin injection
72 SPF level NIH mice, male and female in half,18～22 g, were randomly divided into 6 groups of 12 rats were named control group, aspirin group Qingrexiaoyan Ning group, HaoHongfang high, medium and low dose group. Each treatment group by dose oral administration, the control group was given sesame oil,20 ml/kg, once a day,7 days in all. 1 h after the last administration, all of the mice left and a full plantar subcutaneous injection of 2.5% formaldehyde saline solution 0.05 ml/only the immediate time, records of mice within 5 min left foot licking accumulated time difference between each group.
2.5 The study of HaoHongfang on the immune function
2.5.1 The influence of HaoHongfang on immune organs
SPF level obtained NIH mice,60, male and female in half, weighing 13～15g, experimental environment to adapt to three days. According to body weight were randomly divided into 5 groups, namely control group, Gynostemma group, HaoHongfang side of high, medium and low dose group,12 in each group. The treatment group according to dose administered orally,20 ml/kg, once a day,7 days in all. After the last administration,12 h, mice were killed dislocation of cervical vertebra, rapidly profile control thymus, spleen, weight, organ index calculation. Spleen (thymus) index= spleen (thymus) weight (mg)/ body weight (g). There are differences between each group.
2.5.2 The influence of HaoHongfang carbon particles clearance function in mice
60 SPF level NIH mice, male,18-22 g, were randomly divided into 5 groups, namely control group, Gynostemma group, HaoHongfang high, medium and low dose group. Each treatment group were administered orally by dose,20 ml/kg, once a day,7 days in all, the control group were given equal volume of sesame oil. After the last administration, 1h, mouse tail vein injection of diluted ink 0.1 ml/lOg weight, at 30 s after injection of ink, and 6 min, from the orbital venous plexus of mouse blood 0.025 ml, immediately cast them into the 2 ml 0.1% sodium bicarbonate solution; taking 0.025 ml normal human blood soluble ink injection 2 ml 0.1% sodium bicarbonate solution, zeroing,675 nm was determined at OD values of phagocytic index calculated by the formula K value. There are differences between each group.
2.5.3 The influence of HaoHongfang on serum hemolysin formation of antibodies
60 SPF level NIH mice, male and female in half,18～22g, according to body weight were randomly divided into 5 groups, namely control group, Gynostemma group, HaoHongfang side of high, medium and low dose groups 12. Each treatment group were administered orally by dose,20 ml/kg, once a day,10 days in all, the control group were given equal volume of sesame oil. Article 6, each mouse was injected with 5% sheep red blood cells 0.4 ml/only (equivalent to 4×108 SRBC/only). After the last administration, 1 h, orbital blood serum hemolytic antibody. There are differences between each group.
2.5.4 The influence of HaoHongfang the impact on delayed-type hypersensitivity in mice
60 SPF level NIH mice, male and female in half,18-22g, adapted to the environment for three days. And then divided randomly into 5 groups, control group, Gynostemma group, HaoHongfang high, medium and low dose group,12 in each. Abdominal skin hair removaled (1 cm2 or so), covered with 50μl 1% DNFB sensitization,and the next day for the same. Each treatment group were orally administered by 20 ml/kg, day once,and for 10 days for all, the control group were given equal volume of sesame oil.7 days after administration of 1% DNFB solution evenly applied 10μl mouse right ear (both sides) to attack,24 hours later killed all of the mice and cut the left and right ears with the 8mm hole puncher, weighed, calculate swelling (DTH).
3% Results:
3.1 Study of Compatibility Rationality
3.1.1 Inhibiting the capillary permeability in mice compared with the control group, Compound groups 3 (Artemisia 200mg/kg+Folium 100mg/kg) can statistically reduce the capillary permeability in mice (P<0.01); Compound group 1 (Artemisia annua 100mg/kg+ Folium 200mg/kg) and Compound group 2 (Artemisia 150mg/kg+Folium 150mg/kg) also can statistically reduce the capillary permeability in mice (P<0.05).
3.1.2 Reduce cough in guinea pigs Compared with the control group, Compound group 2,3 could significant prolong the incubation period of cough and reduce the number of cough in 5 minutes (P<0.01); Compound group 1 could significant prolong the incubation period of cough and reduce the number of cough in 5 minutes (P<0.05).
3.2 The study of HaoHongfang on antibacterial and antiviral
3.2.1 Reduce the mortality rate of mice infected with Streptococcus pneumoniae
Compared with the control group, there were significantly higher mortality in model group (P<0.01),and had no effect on HaoHongfang control group. With model group comparison, HaoHongfang high and middle dose group was significantly lower mortality rate in mice (P＜0.01).
3.2.2 Reduce the mortality rate of mice infected with Staphylococcus aureus
Compared with the control group, model group mice were significantly higher mortality rate(P<0.01), and had no effect on HaoHongfang control group; With model group comparison, HaoHongfang high dose group was significantly lower mortality rate in mice infected by Staphylococcus aureus(P<0.01),and HaoHongfang middle and low group could also reduce mortality rate but there was no statistically different (P>0.05).
3.2.3 Reduce the infection of influenza virus in mice
Compared with the control group, the mortality rate、the average survival times of model group were significantly shorter (both P<0.01), but the were no influence in HaoHongfang control group. HaoHongfang high and middle dose group could significantly reduced the mortality rate in mice infected with influenza virus, prolong the average survival days(both P<0.01),and so did the HaoHongfang low-dose group (P<0.05).
3.2.4 Inhibitory Lung-index of mice infected with influenza virus
Compared with the control group, model group could significantly increased lung-index (P＜0.01); Compared with model group, HaoHongfang high dose group could reduce Lung-index in mice infected with influenza virus (P＜0.01), so did the middle group(P<0.05).
3.2.5 Reduce the death in mice induced by endotoxin
Compared with the control group, model group significantly increased mortality (P<0.01); compared with model group, high HaoHongfang significantly reduced the death in mice (P<0.01), HaoHongfang middle and low dose group could also decrease the death in mice but there was not significant difference (P> 0.05).
4、Conclusion:
The results show that HaoHongfang has antibacterial, antiviral, anti-endotoxin, Clearing heat transforming phlegm and suppressing cough, anti-inflammatory and pain releasement and immune enhancement function; provides pharmacodynamic basis for HaoHongfang in expelling wind-evil and clearing away heat, detoxification, phlegm and suppressing cough.
国内外研究表明,由于人口密集、吸烟人群增多、环境污染等因素的恶化,使得呼吸系统疾病的发病率在任何年龄组均占多种疾病之首。上呼吸道感染的西医治疗均不外祛痰、镇咳与抗感染等基本方法。化学药品主要是以止咳、化痰等对症治疗为主,作用迅速,但镇咳类药物副作用大且停药后易复发,导致咳嗽缠延难愈；随着抗生素耐药性的不断增多,限制了抗感染药物的应用。中药防治此类疾病具有较大优势,临床疗效确切,既能杀菌、抑菌、抗病毒、抗炎,又能祛痰、止咳、平喘,还可提高机体免疫力,能通过综合作用祛除病邪或者缓解症状,但大多具有起效缓慢,服用不便的特点。因此,汲取中西药的优点,研制一种安全、速效、针对性强、疗效确切的中药制剂,将极为迫切。
针对南方地区的风热感冒、病毒性感冒,开发具有独特疗效的处方,是临床的需求和患者的希望,也是本课题选题的目的。本课题所研究来源于多年临床经验方,具有止咳、抗炎、平喘之功的处方——蒿红方,是由青蒿和满山红经现代工艺提取纯化后组成的中药复方制剂。课题首先通过对小鼠腹腔毛细血管通透性增高的影响试验、枸椽酸喷雾诱咳法对豚鼠的止咳影响试验来阐明处方配伍的合理性,然后选择合理的配伍组成蒿红方,再通过体内抗菌、体内抗病毒、抗内毒素休克死亡、解热化痰止咳、抗炎止痛作用和对免疫功能的影响等实验研究,并以此对蒿红方的止咳抗炎的药效学进行研究,并初步探讨其作用机理。
2、研究方法
2.1处方配伍合理性研究
2.1.1复方药物对小鼠腹腔毛细血管通透性的影响
取SPF级NIH小鼠,雌雄各半,70只,体重为18～22g,于实验环境下常规饲养3天后,按体重随机均分为7组,10只／组,分别为空白对照组、青蒿组、满山红组、复方1组、复方2组、复方3组、阿司匹林组。各给药组按剂量分别灌胃给药,20 ml／kg,1次／天,连续7 d；空白对照组给予同剂量的麻油。末次给药1 h后,小鼠尾静脉注入2%伊文思蓝生理盐水溶液0.1 ml／10g体重,同时于腹腔注射0.8%醋酸生理盐水溶液0.2 ml／只,20 min后脱颈椎处死小鼠,腹腔注射6 ml生理盐水反复冲洗后,收集冲洗液,调整容积至10 ml,1500 rmp离心10 min,取上清液于722型光栅分光光度计中(波长590nm)测定OD值,比较各组的差异。
2.1.2复方药物对枸椽酸喷雾引咳豚鼠咳嗽的影响
取Hartley豚鼠,体重250g左右,雄雌各半,于实验环境下常规饲养3天。于实验前一天将其逐只放入YLS-8A多功能诱咳引喘仪中,由仪器喷雾17.5%枸椽酸溶液,以150 s内出现哮喘(以抽搐、跌倒的时间作为潜伏期)或者五分钟内咳嗽次数多余10次者为合格的敏感动物。
取上述合格豚鼠80只,雄雌各半,随机分为7组,每组10只。分别为空白对照组,可待因组、满山红组、青蒿组、复方1组、复方2组和复方3组。各给药组按剂量灌胃给药,10 ml／kg,每天1次,连续7 d,空白组给予等剂量的麻油。末次给药1 h后,将豚鼠逐只放入YLS-8A多功能诱咳引喘仪中,由仪器喷雾17.5%枸椽酸(每只豚鼠放入前需更换等体积的新鲜枸椽酸溶液),观察并记录豚鼠从接受喷雾开始到发生抽搐跌倒的时间(即引喘潜伏期)及咳嗽次数,引喘潜伏期超过5 min者以5 min计,比较各组差异。
2.2蒿红方体内抗菌、抗病毒的研究
2.2.1蒿红方对感染肺炎双球菌小鼠治疗作用的影响
取SPF级NIH小鼠,200只,雌雄各半,体重为18～22 g,于实验环境下常规饲养3天后,随机分为7组,分别为空白对照组,模型组,清热消炎宁组,头孢组,蒿红方高、中、低剂量组。除空白对照组和模型组外,其余各组按照剂量灌胃,20 ml／kg,每天一次,连续7 d,空白对照组和模型组灌等剂量麻油。末次给药1 h后,腹腔注射肺炎双球菌(菌号31001-20)悬液,每只1.0 ml(6亿菌／ml),空白对照组和给药对照组腹腔注射等容积的注射用蒸馏水。感染后1、6 h再给药一次,观察和记录一周内各组动物死亡数,计算动物死亡率。统计分析各组差异。
2.2.2蒿红方对感染金黄色葡萄球菌小鼠的影响
取SPF级NIH小鼠,200只,雌雄各半,体重为18～22克,适应环境三天后随机分为8组,分别为空白对照组,模型组,清热消炎宁组,头孢组,蒿红方对照组、高、中、低剂量组。除空白对照组和模型组外,其余各组按照剂量灌胃,20 ml／kg,每天一次,连续7 d,空白对照组和模型组灌等剂量麻油。末次给药1 h后,除空白对照组和蒿红方对照组外,其余各组小鼠腹腔注射金黄色葡萄球菌悬液(菌号26112-5),每只1.0 ml(10亿菌／ml)。第一次感染后1、6 h分别重复给药1次,观察和记录一周内各组动物死亡数,计算动物死亡率。统计分析各组差异。
2.2.3蒿红方对感染流感病毒的小鼠死亡率和平均存活天数的影响
(1)病毒接种方法：取9日龄鸡胚尿囊腔接种注入0.1 ml,接种培育3 d取尿囊液置4℃保存,将尿囊液样品作血凝试验,测血凝滴度,取滴度相同的尿囊液合并后分装于无菌小试管,-70℃保存备用,作为试验用毒种。
(2)毒种半数致死量(LD50)测定：在同一批毒种中取出毒种1支,融化后迅速置冰块中,用1640液作10倍稀释,每一稀释度受感染体重位14 g的小鼠10只,乙醚轻度麻醉,将病毒自最大稀释度起依次滴鼻感染小鼠,每只鼠滴入0.05 ml,对照组滴入等剂量1640液。感染后,每天记录小鼠发病死亡数,连续观察14天,按reed和muench法计算出半数致死量为10-1。
(3)感染小鼠：取SPF级NIH小鼠,体重为13～15g,雌雄各半,于实验室适应环境3天后,随机分为8组,分别为空白对照组、模型组、病毒唑组、清热消炎宁组、蒿红方对照组、高、中、低剂量组。除空白对照组和模型组外,其余各组按照剂量灌胃,20ml／kg,每天一次,连续3 d,空白对照组和模型组灌等剂量麻油。末次给药1小时后,在小鼠于乙醚轻度麻醉下,将已稀释好的病毒液(4个LD50／0.05 ml)滴鼻感染小鼠,每鼠0.05 ml。感染后继续按剂量给药,每天1次,连续7天,并每天记录动物发病情况和死亡数,连续观察14天。计算出死亡保护率和延长生命率,比较各组差异。
2.2.4蒿红方对感染流感病毒小鼠肺重量的影响
(1)病毒接种：取9日龄鸡胚尿囊腔接种注入0.1 ml,接种培育3天取尿囊液置4℃保存,将尿囊液样品作血凝试验,测血凝滴度,取滴度相同的尿囊液合并后分装于无菌小试管,-70℃保存备用,作为试验用毒种。
(2)毒种半数致死量(LD50)测定：在同一批毒种中取出毒种1支,融化后迅速置冰块中,用1640液作10倍稀释,每一稀释度受感染体重位14 g的小鼠10只,乙醚轻度麻醉,将病毒自最大稀释度起依次滴鼻感染小鼠,每只鼠滴入0.05 ml,对照组滴入等剂量1640液。感染后,每天记录小鼠发病死亡数,连续观察14天,按reed和muench法计算出半数致死量为10-1
(3)感染小鼠：取SPF级NIH小鼠,130只,体重为13～15g,雌雄各半,于实验室适应环境3天后,随机分为8组,分别为空白对照组、模型组、病毒唑组、清热消炎宁组、蒿红方对照组、高、中、低剂量组。除空白对照组和模型组外,其余各组按照剂量灌胃,20ml／kg,每天一次,连续3 d,空白对照组和模型组灌等剂量麻油。末次给药1小时后,除空白对照组和蒿红方对照组外,其余各组小鼠在乙醚轻度麻醉下,将己稀释好的病毒液(15个LD50／0.05 ml)滴鼻感染小鼠,每鼠0.05 ml。感染后继续按剂量给药,每天1次,连续4天。末次给药前小鼠禁食不禁水8h,称重,末次给药1h后,脱颈椎处死动物,迅速取出全肺,于生理盐水中清洗2次,摘除气管、肺门淋巴结等组织,滤纸吸干表面水份,称重。计算肺指数和抑制率,比较各组差异。
2.2.5蒿红方对内毒素致小鼠休克死亡的影响
取SPF级NIH小鼠,120只,体重为18～22 g,雌雄各半,于实验室适应环境3天后,随机分为6组,分别为空白对照组、模型组、清热消炎宁组、蒿红方对照组、高、中、低剂量组。除空白对照组和模型组外,其余各组按照剂量灌胃,20 ml／kg,每天1次,连续7 d,空白对照组和模型组灌等剂量麻油。末次给药1 h后,除空白对照组小鼠外其余各组小鼠均腹腔注射D-GALN 200 mg／kg和内毒素1.5 mg／kg,空白对照组腹腔注射等剂量的生理盐水,观察72小时,记录动物死亡数。比较各组差异。
2.3蒿红方清热止咳化痰作用的研究
2.3.1蒿红方对干酵母所致大鼠发热的影响
取经帅选合格动物60只,随机分为6组,分别为空白对照组、阿司匹林组、清热消炎宁组、蒿红方高、中、低剂量组。实验前6h禁食不禁水。空白组大鼠颈部以下的背部皮下注射生理盐水10 ml／kg；各组大鼠分别在大鼠颈部以下的背部皮下注射20%酵母悬液10 ml／kg,按摩注射部位以使悬浮液在皮肤下扩散。造模同时各剂量组分别按剂量灌胃给药,空白对照组给予等剂量的麻油。于药后1、2、3、4小时各测体温1次,比较各组动物体温下降值的差异。
2.3.2蒿红方对内毒素所致新西兰兔发热的影响
取经帅选合格的新西兰兔36只,雌雄各半,随机分为6组,分别为空白对照组,阿司匹林组,清热消炎宁组,蒿红方高、中、低剂量组。除空白对照组和模型组外,其余各组按照剂量灌胃,10 ml／kg,每天1次,连续7 d,空白对照组和模型组灌等剂量麻油。末次给药1h后,从兔耳缘静脉注射内毒素10μg／ml·kg体重,于给药后1、2、3、4、6h各测体温1次,比较各组动物体温下降值的差异。
2.3.3蒿红方对小鼠氨水引咳作用的影响
取SPF级NIH小鼠,体重18～22 g,雄雌各半,随机分为7组,每组15只。分别为空白对照组,可待因组,牡荆油组,蒿红方高、中、低剂量组。各给药组按剂量灌胃给药,空白组给予等剂量麻油。20 ml／kg,每天1次,连续7 d。末次给药1 h(可待因组30 min)后,将豚鼠逐只放入恒压氨水喷雾装置内(压力为140mmHg,氨水浓度为28%),由仪器恒压氨水喷雾(每只小鼠放入前需更换等体积的新鲜氨水溶液),氨水喷雾时间按等比级数递增,按序贯法改变氨水喷雾时间,观察并记录小鼠从接受喷雾开始到发生咳嗽的时间,计算小鼠半数引咳时间(EDT50),并按公式计算R值(R值＞130%为有镇咳作用,R值>150%为有明显镇咳作用)。比较各组差异。
2.3.4蒿红方对枸椽酸喷雾引咳法致豚鼠咳嗽的影响
取Hartley豚鼠,体重250～280g,雄雌各半,在实验环境下常规饲养3天。于实验前一天将其逐只放入YLS-8A多功能诱咳引喘仪中,由仪器喷雾17.5%枸椽酸溶液,以150s内出现哮喘(以抽搐、跌倒的时间作为潜伏期)或者五分钟内咳嗽次数多余10次者为合格的敏感动物。
取上述合格豚鼠80只,雄雌各半,随机分为7组,每组10只。分别为空白对照组,可待因组,牡荆油组,蒿红方高、中、低剂量组。各给药组按剂量灌胃给药,空白组给予等剂量麻油,10 ml／kg,每天1次,连续7 d。末次给药1h(可待因组30 min)后,将豚鼠逐只放入YLS-8A多功能诱咳引喘仪中,由仪器喷雾17.5%枸椽酸(每只豚鼠放入前需更换等体积的新鲜枸椽酸溶液),观察并记录豚鼠从接受喷雾开始到发生抽搐跌倒的时间(即引喘潜伏期)及5 min内的咳嗽次数,引喘潜伏期超过5min者以5min计,比较各组差异。
2.3.4蒿红方对小鼠气管酚红排泌量的影响
取SPF级NIH小鼠,60只,体重为18～22 g,雌雄各半。于实验室适应环境3天后,随机分为5组,分别为空白对照组、牡荆油组、蒿红方高、中、低剂量组。各给药组按照剂量灌胃,20 ml／kg,空白对照组灌等剂量麻油。每天1次,连续7 d。末次给药1 h后,各鼠按剂量0.01 ml／g体重腹腔注射5%的酚红溶液,注射后0.5 h,将小鼠颈椎脱臼处死,仰位固定于手术板上剪开颈前皮肤,分离气管,剥离气管周围组织,剪下甲状软骨下缘至气管分叉处之气管,用5%碳酸氢钠溶液0.5 ml冲洗气管,连续4次,洗出液合并,置于紫外可见分光光度计上于546 nm波长处测定OD值,并根据实验前制定的标准曲线计算出气管冲洗液中酚红浓度,计算祛痰率。
标准曲线的制定：用分析天平准确称取一定量酚红以5g／dl碳酸氢钠溶解,使1 ml含酚红1000 ug,依次稀释成每毫升含酚红0.01、0.1、0.5、0.7、1、3、5 ug,测OD值,以酚红剂量为横坐标,OD值为纵坐标,作标准曲线,并计算出每鼠酚红排泌量。比较各组差异。
2.3.5蒿红方对鸽气管纤毛运动的影响
取普通级家鸽,体重400～500 g,雌雄各半,按体重随机均分为5组,分别为空白对照组,牡荆油组,蒿红方高、中、低剂量组。各给药组按剂量分别灌胃给药,10ml／kg,每天一次,连续7 d,空白对照组给予等剂量的麻油。末次给药后1 h,在暗室内将家鸽颈部拉直与水平面平行,然后作颈部切口,剥离气管,使气管尽量地暴露,从靠心脏端,将5号针头插入气管,使针头靠近气管内壁推入0.02 ml墨法,在冷光源下,观察1 min内墨汁向前运动的距离。
2.4蒿红方抗炎止痛作用的研究
2.4.1蒿红方对小鼠腹腔毛细血管通透性的影响
取SPF级NIH小鼠,雄性,60只,体重为18～22g,于实验环境下常规饲养3天后,按体重随机均分为6组,10只／组,分别为空白对照组、阿斯匹林组、清热消炎宁组、蒿红方高、中、低剂量组。除空白对照组外其余分别给药,剂量为20 ml／kg,1次／天,连续7 d；末次给药1 h后,小鼠尾静脉注入2%伊文思蓝生理盐水溶液0.1 ml／10g体重,同时于腹腔注射0.8%醋酸生理盐水溶液0.2 ml／只,20 min后脱颈椎处死小鼠,腹腔注射6 ml生理盐水反复冲洗后,收集冲洗液,调整容积至10 ml,1500 rmp离心10 min,取上清液于722型光栅分光光度计中(波长590nm)测定OD值,比较各组的差异。
2.4.2蒿红方对大鼠蛋清性足肿胀的影响
取SD大鼠60只,体重为130～150 g,随机均分为6组,分别为空白对照组,阿司匹林组,清热消炎宁组,蒿红方高、中、低组,每组10只。各给药组按剂量灌胃给药,空白对照组给予等体积麻油。每天1次,连续灌胃给药7 d。末次给药前,在每只大鼠右后足跖处作一标记,以YLS-7A足趾容积测量仪测量右足后跖体积,作为致炎前右后足基础值。末次给药后1 h,在每只大鼠右后足跖皮腱膜下注射100%新鲜蛋清0.1 mL／只,测定注入致炎剂后1、2、3、4 h右后足跖体积,并减去致炎前大鼠右后足基础值,作为各鼠在不同时间的足肿胀度。比较不同时间段的足肿胀度水平。
2.4.3蒿红方对大鼠甲醛性足肿胀的影响
取SD大鼠60只,体重为140～160g,随机均分为6组,分别为空白对照组、阿司匹林组、清热消炎宁组、蒿红方高、中、低组,每组10只。各给药组按剂量灌胃给药,空白对照组给予等体积麻油。每天1次,连续灌胃给药7 d。在给药前,在每只大鼠右后足跖处作一标记,以YLS-7A足趾容积测量仪测量右足后跖体积,作为致炎前右后足基础值。
第一次给药后1 h,在每只大鼠右后足跖皮腱膜下注射1%甲醛生理盐水溶液0.1mL／只。第2天至第6天继续给药并于给药后1 h测定右足后跖容积,每天1次。以药后1、2、3、4、5天结果计算足肿胀度。比较不同时间段的足肿胀度水平。
2.4.4蒿红方对醋酸所致小鼠疼痛的影响(扭体法)
取SPF级NIH小鼠,72只,雌雄各半,体重为18～22g,按体重随机均分为6组,每组12只,分别为空白对照组、阿司匹林组,清热消炎宁组,蒿红方高、中、低剂量组。各给药组按剂量灌胃给药,空白对照组给予等剂量麻油,20 ml／kg,1次／天,连续7 d。末次给药1 h后,小鼠腹腔注射0.8%醋酸生理盐水溶液0.1mL／10g体重,观察记录20 min内小鼠的扭体次数扭体反应：腹部凹陷、伸展后肢、臀部抬高),比较各组差异。
2.4.5蒿红方对甲醛足跖皮下注射所致小鼠疼痛的影响
取SPF级NIH小鼠,72只,雌雄各半,体重为18～22 g,按体重随机均分为6组,每组12只,分别为空白对照组、阿司匹林组,清热消炎宁组,蒿红方高、中、低剂量组。各给药组按剂量灌胃给药,空白对照组给予等剂量麻油,20 ml／kg,1次／天,连续7 d。末次给药1 h后,各组小鼠左后足足跖皮下注射2.5%甲醛生理盐水溶液0.05 ml／只,立即计时,记录5 min内小鼠舔左后足累积时间,比较各组差异。
2.5蒿红方对免疫功能的研究
2.5.1蒿红方对小鼠免疫器官的影响
取SPF级NIH小鼠,60只,雌雄各半,体重13～15g,适应实验环境三天。按照体重随机均分为5组,分别为空白对照组、绞股蓝组,蒿红方高、中、低剂量组,每组12只。各给药组按剂量灌胃给药,20 ml／kg,每日1次,连续7 d。末次给药后12 h,脱颈椎处死小鼠,迅速剖取胸腺、脾脏,称重,计算脏器指数。脾(胸腺)指数=脾(胸腺)重(mg)／体重(g)。比较各组差异。
2.5.2蒿红方对小鼠体内炭粒廓清功能的影响
取SPF级NIH小鼠,雄性,60只,体重18～22 g,按体重随机均分为5组,分别为空白对照组、绞股蓝组、蒿红方高、中、低剂量组,每组12只。各给药组分别按剂量灌胃给药,20 ml／kg,每日1次,连续7 d,空白对照组给予等容积麻油。末次给药后1h,小鼠尾静脉注入稀释墨汁0.1 ml／10g体重,于注入墨汁后30 s及6 min,从小鼠眼眶后静脉丛取血0.025 ml,立刻吹入2 ml 0.1%碳酸氢钠溶液中；取0.025 ml正常未注射墨汁的血溶于2 ml 0.1%碳酸氢钠溶液校零,675 nm处测定OD值,按下式计算吞噬指数K值。比较各组差异。
2.5.3蒿红方对小鼠血清溶血素抗体生成的影响
取SPF级NIH小鼠,雌雄各半,60只,体重18～22g,按体重随机均分为5组,分别为空白对照组、绞股蓝组、蒿红方高、中、低剂量组,每组12只。各给药组分别按剂量灌胃给药,20 ml／kg,每日1次,连续10 d,空白对照组给予等容积麻油。第6日各组小鼠腹腔注射5%绵羊红细胞0.4 ml／只(相当于4×108SRBC／只)。末次给药后1 h,眼眶取血测定血清溶血素抗体。比较各组差异。
2.5.4蒿红方对小鼠迟发型超敏反应的影响
取SPF级NIH小鼠,雌雄各半,60只,体重18～22g,适应环境三天。按体重随机均分为5组,分别为空白对照组、绞股蓝组、蒿红方高、中、低剂量组,每组12只。各组小鼠腹部皮肤脱毛(1cm2左右),均匀涂布1% DNFB溶液50μl致敏,次日同法强化一次。各给药组分别按剂量灌胃给药,20 ml／kg,每日1次,连续10 d,空白对照组给予等容积麻油。第7天给药后将1%DNFB溶液10μl均匀涂抹于小鼠右耳(两面)进行攻击,攻击后24小时脱颈椎处死小鼠,剪下左右耳壳,用8mm打孔器取同部位的左右两耳片,称重,以左右耳片重量之差为肿胀度(表示DTH的强弱)。
3、结果
3.1配伍合理性研究
3.1.1抑制小鼠腹腔毛细血管通透性与空白对照组相比,复方3组(青蒿200mg／kg+满山红100mg／kg)能降低小鼠腹腔毛细血管通透性并具有显著性统计学差异(P<0.01)；复方1组(青蒿100mg／kg+满山红200mg／kg)、复方2组(青蒿150mg／kg+满山红150mg／kg)也能降低小鼠毛细血管通透性并具有显著性统计学差异(P<0.05)。
3.1.2减轻豚鼠咳嗽与空白组相比,复方2、3组均能延长豚鼠咳嗽的潜伏期、减少5分钟内豚鼠的咳嗽次数,并具有显著性统计学差异(P<0.01)；复方1组能延长咳嗽潜伏期、减少5分钟内豚鼠的咳嗽次数,并具有显著性统计学差异(P<0.05)。
3.2蒿红方体内抗菌、抗病毒的研究
3.2.1降低感染肺炎双球菌病毒小鼠的死亡率与空白对照组相比,模型组小鼠死亡率显著升高且具有统计学差异(P<0.01),蒿红方对照组对未感染肺炎双球菌的小鼠死亡率无影响；与模型组相比较,蒿红方高、中剂量组显著降低小鼠的死亡率且具有统计学差异(P<0.01)。
3.2.2降低感染金黄色葡萄球菌病毒小鼠的死亡率与空白对照组相比,模型组小鼠的死亡率显著升高并具有统计学意义(P<0.01),蒿红方对照组对未感染金黄色葡萄球菌小鼠的死亡率无影响；与模型组相比,蒿红方高剂量组能明显降低金黄色葡萄球菌感染的小鼠的死亡率(P<0.01),蒿红方中、低剂量组亦能降低金黄色葡萄球菌感染的小鼠引起的死亡率但不具有统计学差异(P>0.05)。
3.2.3降低感染流感病毒对小鼠的影响与空白对照组相比较,模型组动物的死亡率显著增高、其平均存活天数显著缩短并且都具有显著性统计学差异(均有P<0.01),蒿红方对照组对未感染小鼠死亡率和平均存活天数无影响；与模型组相比较,蒿红方高、中剂量组显著降低了流感病毒感染的小鼠的死亡率、延长了其平均存活天数并且具有显著性统计学差异(均有P<0.01),蒿红方低剂量组亦显著降低了小鼠的死亡率、延长了其平均存活天数并且具有显著性统计学差异(P<0.05)。
3.2.4抑制感染流感病毒小鼠肺指数与空白对照组相比,模型组肺指数明显增大并具有显著性统计学差异(P<0.01)；与模型组相比较,蒿红方高剂量组能明显降低流感病毒感染小鼠的肺指数并具有显著性统计学差异(P<0.01),蒿红方中剂量组也能明显降低流感病毒感染小鼠的肺指数并具有显著性统计学差异(P<0.05),蒿红方低剂量也能降低小鼠肺指数但不具有统计学意义(P>0.05)。
3.2.5降低内毒素致小鼠的休克死亡与空白对照组相比较,模型组小鼠死亡率增高且具有显著性统计学差异(P<0.01),蒿红方对照组对正常小鼠未见致死；与模型组相比较,蒿红方高剂量组能降低内毒素致休克死亡小鼠的死亡率且具有显著性统计学差异(P<0.01),蒿红方中剂量组、蒿红方低剂量组亦能降低内毒素致休克死亡小鼠的死亡率但不具有显著性统计学差异(P>0.05)。
4、研究结论：
本研究结果表明,蒿红方具有抑菌、抗病毒、抗内毒素、清热化痰止咳、抗炎止痛及增强免疫功能的作用,为蒿红方疏风清热、解毒、止咳化痰的功效提供了药效学依据,从而肯定了蒿红方的有效性。
1、Objective:
Due to population density, increasing smoking crowd, environmental pollution and other factors, research has shown that the incidence of respiratory disease at any age group account for the first of many diseases. The medical treatment of upper respiratory tract infection are the expectorant, antitussive and anti-infection and other basic methods. Chemicals, mainly cough, phlegm and other and other symptomatic treatment based, main effect rapidly, but antitussive drugs have side effects and easy to relapse after drug withdrawal, leading to extended refractory cough bound; with the increasing antibiotic resistance, the application of anti-infectives is restricted. Traditional Chinese medicines have greater advantages, clinical curative effect, which is not only bactericidal, antibacterial, antiviral and anti-inflammatory, but also expectorant, cough, asthma, and improve immunity, eliminate or alleviate the symptoms of pathogenic factors through an integrated role, but many of them have slow onset, taking the characteristics of inconvenience. So, the Development of a safe, quick, targeted, curative effect of Chinese medicine preparations which learn the advantages of Chinese and Western medicine is extremely urgent.
It is the needs of clinical and patients, is also purpose of this topic to develop an unique therapeutic prescription for the southern district of fengreganmao and viral influenza. This topic is from many years of clinical experience. Haohongfang which can suppressing cough、anti-inflammatory、relieving asthma,is a traditional Chinese medicine compound of Modern extraction and purification of Artemisia annua and Folium. Firstly, the topic clarified the rationality of prescription through increased capillary permeability in mice the impact test, CBS spray induced cough cough impact tests on guinea pigs to clarify the prescription to the rationality, secondly selected the rational compatibility of the composition of Haohongfang, thirdly, study the cough and anti-inflammatory pharmacodynamics of Haohongfang and investigate the mechanism of it preliminary.
2、Methods:
2.1 Study of Compatibility Rationality
2.1.1 The impact on the capillary permeability in mice
70 SPF level NIH mice, male and female in half,18-22g, adapted to the environment for three days. And then divided randomly into 7 groups,10 mice/group named control group, Artemisia group, Folium group, compound group 1, compound group 2, compound group 3, aspirin group. Each treatment group were orally administered by 20 ml/kg, once a day,7 days in all; the control group were given equal volume of sesame oil.1 h after the last administration,2% Evans blue saline solution was injected with 0.1 ml/10g weight, and 0.8% acetic acid saline solution intraperitoneal with 0.2 ml each at the same time,20 min later were killed all of the mice,6 ml normal saline flus was injected intraperitoneal, and then the fluid was collected, adjusting the volume to 10 ml,1500 rmp centrifuge for 10 min, tested OD values with 722 grating spectrophotometer(590 nm) and compared the difference between the groups.
2.1.2 Drugs on cough in guinea pigs
Hartley guinea pigs,250g or about, female and male in half, adapted to the environment for three days. The day before the experiment will only add to its by-YLS-8A multi-instrument induced asthma induced cough from 17.5% citric acid solution, spray equipment, to occur within 150 s of asthma (in convulsions, fall time as the incubation period) or cough times within five minutes extra in the 10 who qualified for sensitive animals.
80 qualified guinea pigs, female and male in half, divided into 7 groups of 10 each. Control group, codeine group, Folium group, Artemisia annua group, compound group 1, compound group 2 and compound group 3. Each treatment group were orally administered by 10 ml/kg, once a day,7 days in all; the control group were given equal volume of sesame oil.1 h after the last administration, the guinea pig by only into the YLS-8A multi-functional instrument induced asthma induced cough,17.5% citric acid spray from the apparatus (to be replaced before each guinea pig into the same volume of fresh phosphate histamine solution) were observed and recorded in guinea pigs receiving spray from the beginning to the fall time of the occurrence of seizures (ie, latent period of asthma) and cough frequency, latent period of asthma 5 min to 5 min of were to compare the differences between each group.
2.2 HaoHongfang on antibacterial, antiviral research
2.2.1 HaoHongfang on the treatment in mice infected with Streptococcus pneumoniae
200 SPF level NIH mice, male and female in half,18～22 g, adapted to the environment for three days.And then divided randomly into 7 groups, control group, model group, Qingrexiaoyanning group, cephalosporin group, HaoHongfang high, medium and low dose group. Each treatment group was orally administered by 20 ml/kg, once a day,7 days in all; control group and model group were given equal volume of sesame oil.1 h after the last administration, intraperitoneal injection of Streptococcus pneumoniae (strain No. 31001-20) suspension, each 1.0 ml (0.6 billion bacteria/ml), blank control group and the control group, intraperitoneal administration of the injection volume of distilled water.1,6 h after infection and then administered once a week to observe and record the number of deaths in each group, compute the mortality rate. And compared the difference between the groups.
2.2.2 HaoHongfan on the mortality rate of mice infected with Staphylococcus aureus
200 SPF level NIH mice, male and female in half,18 to 22 g, after three days of adaptation to the environment were randomly divided into 8 groups, control group, model group, Qingrexiaoyanning group, cephalosporin group, HaoHongfang control group, high, medium and low dose group. In addition to the control group and model group, other groups in accordance with the dose administered,20 ml/kg, once a day for 7 days, control group and model group filling the same dose of sesame oil.1 h after the last administration, in addition to the control group and the control group, HaoHongfang side, the remaining mice were injected suspension of Staphylococcus aureus (strain No.26112-5), each 1.0 ml (lbillion bacteria/ml).1,6 h after the first infection were repeated dose 1, observe and record animals in each group died within a week the number of animal mortality calculated. And compared the difference between the groups.
2.2.3 HaoHongfang on the infection of influenza virus in mice
(1) virus inoculation Methods:9 day old chick embryo allantois inoculated into 0.1 ml, inoculation training allantoic fluid obtained 3 d at 4℃, the allantoic fluid samples for hemagglutination test, measuring hemagglutination titer, take the same titer of allantoic fluid after the merger of small-packing in sterile test tubes,-70℃save standby, as a pilot with the virus seed.
(2) toxic species of the median lethal dose (LD50) determination:toxic species in the same group of species of a drug out, home ice melting rapidly, with 1640 as 10-fold dilution, dilution of the infected body weight of each 14-bit 10 g mice, mild ether anesthesia, the maximum dilution of the virus since the beginning followed by intranasal infection of mice, each mouse instilled 0.05 ml, control group instilled such dosage 1640. After infection, the mice daily incidence of mortality, continuous observation for 14 days, according to reed and muench method to calculate the lethal dose was 10-1.
(3) infection Mouse:SPF level obtained NIH mice, weighing 13～15g, male and female in half, to adapt to the environment in the laboratory after 3 days were randomly divided into 8 groups, the control group, model group, ribavirin Group Qingrexiaoyan Ning Group, HaoHongfang control group, high, medium and low dose group. In addition to the control group and model group, other groups in accordance with the dose administered, 20ml/kg, once a day for 3 d, the control group and model group filling the same dose of sesame oil.1 hour after the last administration, the mice in the light ether anesthesia, will have a good virus dilute solution (4 LD50/0.05 ml) intranasally infected mice, each mouse was 0.05 ml. Infection continue to dose,1 time per day for 7 days, and daily records of animal morbidity and mortality, continuous observation for 14 days. Calculate the mortality rate and prolong life protection rate and compared the difference between the groups.
2.2.4 HaoHongfang on Lung-index of mice infected with influenza virus
(1) virus vaccination:from 9 day old chick embryo allantois inoculated into 0.1 ml, inoculation training allantoic fluid obtained 3 days at 4℃, the allantoic fluid samples for hemagglutination, hemagglutination titer test, to take the same titer of allantoic fluid after the merger of small-packing in sterile test tubes,-70℃save standby, as a pilot with the virus seed.
(2) toxic species of the median lethal dose (LD50) determination:toxic species in the same group of species of a drug out, home ice melting rapidly, with 1640 as 10-fold dilution, dilution of the infected body weight of each 14-bit 10 g mice, mild ether anesthesia, the maximum dilution of the virus since the beginning followed by intranasal infection of mice, each mouse instilled 0.05 ml, control group instilled such dosage 1640. After infection, the mice daily incidence of mortality, continuous observation for 14 days, according to reed and muench method to calculate the lethal dose was 10-1.
(3) infection Mouse:SPF level obtained NIH mice,130, weight 13-15g, male and female in half, to adapt to the environment in the laboratory after 3 days, were randomly divided into 8 groups, the control group, model group, ribavirin group, Qingrexiaoyanning group, HaoHongfang control group, high, medium and low dose group. In addition to the control group and model group, other groups in accordance with the dose administered, 20ml/kg, once a day for 3 d, the control group and model group filling the same dose of sesame oil.1 hour after the last administration, in addition to the control group and the control group, HaoHongfang side, the remaining mice in the light ether anesthesia, will have a good virus dilute solution (15 LD50/0.05 ml) intranasally infected with small mice, each mouse was 0.05 ml. Infection continue to dose,1 time per day,4 consecutive days. Fasting before the last administration, mice can not help but water 8h, weighing, 1h after the last administration, animals were sacrificed and dislocation of cervical vertebra, quickly remove the entire lung, in normal saline wash 2, removal of the trachea, hilar lymph nodes and other organizations, the filter paper suction Dry the surface moisture, and weighed. Lung index and the inhibition rate calculated to compare the differences between each group.
2.2.5 HaoHongfang on the death in mice induced by endotoxin
120 SPF level NIH mice,18～22 g, male and female in half, adapted to the environment in the laboratory for 3 days, randomly divided into six groups, control group, model group, Qingrexiaoyanning Group HaoHongfang control group, high, medium and low dose group. In addition to the control group and model group, other groups in accordance with the dose administered,20 ml/kg, once a day,7 days in all, control group and model group were given equal volume of sesame oil.1 h after the last administration, in addition to the blank control group were outside the remaining mice were injected D-GALN 200 mg/kg and LPS 1.5 mg/kg, control group, intraperitoneal dose of saline, observed 72 hours, record the number of animal deaths. There are differences between each group.
2.3 HaoHongfang on clearing heat and relieving cough and phlegm
2.3.1 HaoHongfang on fever induced by dry yeast
60 qualified animals were randomly divided into six groups, control group, aspirin group, Qingrexiaoyanning group, HaoHongfang high, medium and low dose group.6h fasting before the experiment can not help but water. Control back of the neck rats injected with 10 ml/kg; 20% of the yeast suspension were injected 10 ml/kg in the back of rats and normal saline with control group. Modeling the same time each dose group were administered orally by dose and blank control group was given the sesame oil.1,2,3,4 hours after the drug 1 of each measured body temperature, body temperature decreased in all groups compared the value of the difference.
2.3.2 HaoHongfang on endotoxin fever of rabbits
36 qualified New Zealand rabbits were randomly divided into 6 groups, control group, aspirin group, Qingrexiaoyanning group, HaoHongfang high, medium and low dose group. In addition to control group and model group, other groups in accordance with the dose administered,10 ml/kg, once a day,7 days in all, control group and model group were given the same dose of sesame oil. 1h after the last administration,10μg/ml·kg of endotoxin were injection from the rabbit ear vein,1,2,3,4,6 h after the administration temperature were measured one time,and compared the difference between each gtoup.
2.3.3 HaoHongfang on cough in mice with ammonia water
SPF level NIH mice,18～22 g, female and male in half, randomly divided into 7 groups,15 in each group. Control group, respectively, codeine, Vitex oil group, HaoHongfang high, medium and low dose group. Each treatment group by dose oral administration, given control group sesame oil.20 ml/kg, once a day,7 days in all.1 h (codeine 30 min) after the last administration, put constant pressure of guinea pigs by ammonia spray device (pressure of 140mmHg, ammonia concentration of 28%), ammonia by the constant pressure spray equipment fog (to be replaced before each mouse into the same volume of fresh ammonia solution), ammonia spray time, increasing by geometric progression, according to sequential changes in ammonia spray time to observe and record the mice receiving spray from fog began to cough occurred the time to calculate the time mice were half of the cough (EDT50), calculated according to R value (antitussive effects:R value> 130%, significant antitussive effect:R values> 150%). There are differences between each group.
2.3.4 HaoHongfang on the citric acid spray lead cough cough of guinea pigs
Hartley guinea pigs,250～280g, female and male in half, adapted to the environment in the laboratory for 3 days. The day before the experiment will only add to its by-YLS-8A multi-instrument induced asthma induced cough from 17.5% citric acid solution spray equipment to 150s appeared in asthma (in convulsions, fall time as the incubation period) or 5 the number of extra minutes to cough 10 times the sensitivity of animals to qualified persons.
80 qualified guinea pigs, female and male in half, divided into 7 groups of 10 each. Control group, codeine group, Vitex oil group, HaoHongfang high, medium and low dose group. Each treatment group by dose oral administration, control group was given sesame oil,10 ml/kg, once a day,7 days in all. the control group were given equal volume of sesame oil.1 h after the last administration, the guinea pig by only into the YLS-8A multi-functional instrument induced asthma induced cough,17.5% citric acid spray from the apparatus (to be replaced before each guinea pig into the same volume of fresh phosphate histamine solution) were observed and recorded in guinea pigs receiving spray from the beginning to the fall time of the occurrence of seizures (ie, latent period of asthma) and cough frequency, latent period of asthma 5 min to 5 min of were to compare the differences between each group.
2.3.5 HaoHongfang on the mouse trachea phenol red secretion
60 SPF level NIH mice,18～22 g, male and female half and half. adapted to the environment for three days.And then divided randomly into five groups, control group, Vitex oil group, HaoHongfang high, medium and low dose group. The treatment group in accordance with the dose administered,20 ml/kg, such as dosage control group irrigated sesame oil.Once a day,7 days in all.1 h after the last administration,5% phenol red solution were injected with 0.01 ml/g intraperitoneal,0.5 h later, killed the mice by cervical dislocation, Yang-bit fixed-board cut in surgery cervical skin, separation of the trachea, tracheal strip surrounding tissue, cut the lower edge of thyroid cartilage to the tracheal bifurcation of the trachea, with 5% sodium bicarbonate solution,0.5 ml tracheal wash for 4 times, washing the combined solution, placed in UV-visible spectrophotometer on the wavelength of 546 nm measured in OD values, and in accordance with the standards set before the experiment to calculate tracheal fluid curve of phenol red were calculated expectorant rate.
The formulation of the standard curve:analytical balance accurately weighed with a certain amount of phenol red to 5g/dl sodium bicarbonate dissolved in 1 ml phenol red to 1000 ug, followed by diluted per milliliter of phenol red 0.01,0.1,0.5,0.7,1,3,5 ug, test OD values to a dose of phenol red horizontal, OD value of the vertical axis for the standard curve, and calculated for each mouse phenol red secretion. There are differences between each group.
2.3.6 HaoHongfang on the tracheal cilia movement of pigeons
Pigeons,400～500 g, male and female in half, were randomly divided into 5 groups, namely control group, Vitex oil group, HaoHongfang high, medium and low dose group. Each treatment group were administered orally by dose,10 ml/kg, once a day for 7 days, the control group was given the sesame oil.1 h after the last administration, straightened and paralleled levelly to the neck in pigeons in the dark room, and then stripped the trachea with a neck incision so as to expose the trachea, through the heart from the end of the needle NO.5 into the trachea, so that the needle close to the tracheal wall into the 0.02 ml ink, in the cold light source, the observed within 1 min from the forward movement of ink.
2.4 The study of HaoHongfang on anti-inflammatory analgesic
2.4.1 Inhibiting the capillary permeability in mice
60 SPF level NIH mice, male,18-22g, adapted to the environment for three days, were randomly divided into 6 groups,10 each group, named control group, Aspen aspirin group, Qingrexiaoyan Ning group, HaoHongfang high, medium and low dose group. In addition to the rest of control group were administered a dose of 20 ml/kg, once a days,7 days in all; 1 h after the last administration,2% Evans blue saline solution was injected with 0.1 ml/lOg, and 0.8% acetic acid saline solution intraperitoneal with 0.2 ml each at the same time,20 min later were killed all of the mice,6 ml normal saline flus was injected intraperitoneal, and then the fluid was collected, adjusting the volume to 10 ml,1500 rmp centrifuge for 10 min, tested OD values with 722 grating spectrophotometer(590 nm) and compared the difference between the groups.
2.4.2 The influence of HaoHongfang rat paw swelling of egg white
60 SD rats,130～150 g, were randomly divided into six groups, control group, aspirin group, Qingrexiaoyanning group, HaoHongfang high, medium and low groups 10. Each treatment group by dose oral administration, and control group were given equal volume of sesame oil. Once a day,7 days in all. Before the last administration, each rat in the right rear paw Department as a marker to YLS-7A toe right foot volume measurement after measurement plantar volume, as inflammation right foot before the baseline. After the last administration,1 h, each rat in the right foot plantar fascia under the skin injection of fresh egg white 100% 0.1 mL/only measured after injection of inflammatory agent 1,2,3,4 h after the right paw volume and less inflammation in rats before the right foot base value, as the mice at different times of the foot swelling. Comparison of different time levels of foot swelling.
2.4.3 The influence of HaoHongfang rat paw swelling of formaldehyde
SD rats were taken 60,140～160g, were randomly divided into six groups, control group, aspirin group, Qingrexiaoyanning group, HaoHongfang high, medium and low group. Each treatment group by dose oral administration, and control group were given equal volume of sesame oil.1 time per day, continuous intragastric administration 7 d. Prior to administration, in the right rear paw of each rat Office as a marker to YLS-7A toe right foot volume measurement after measurement plantar volume, as inflammation right foot before the baseline.
After the first dose 1 h, each rat in the right foot plantar fascia under the skin of 1% formaldehyde saline solution injection of 0.1 mL/only. Day 2 to 6 days to delivery, and measured at 1 h after administration right foot plantar volume after day 1.1,2,3,4,5 days after drug results to calculate foot swelling. Comparison of different time levels of foot swelling.
2.4.4 The influence of HaoHongfang on the impact of pain in mice induced by acetic acid (writhing)
72 SPF level NIH mice, male and female in half,18-22g, were randomly divided into 6 groups of 12 were named control group, aspirin group, Qingrexiaoyanning group, HaoHongfang high, medium and low dose group. Each treatment group by dose oral administration, the control group was given sesame oil,20 ml/kg, once a day,7 days in all. 1 h after the last administration, mice were injected 0.8% acetic acid saline solution 0.1mL/10g weight, observed within 20 min the number of writhing writhing response: abdominal sag, stretch limbs, buttocks elevation), compared differences between each group.
2.4.5 The influence of HaoHongfang on pain in the foot induced by formalin injection
72 SPF level NIH mice, male and female in half,18～22 g, were randomly divided into 6 groups of 12 rats were named control group, aspirin group Qingrexiaoyan Ning group, HaoHongfang high, medium and low dose group. Each treatment group by dose oral administration, the control group was given sesame oil,20 ml/kg, once a day,7 days in all. 1 h after the last administration, all of the mice left and a full plantar subcutaneous injection of 2.5% formaldehyde saline solution 0.05 ml/only the immediate time, records of mice within 5 min left foot licking accumulated time difference between each group.
2.5 The study of HaoHongfang on the immune function
2.5.1 The influence of HaoHongfang on immune organs
SPF level obtained NIH mice,60, male and female in half, weighing 13～15g, experimental environment to adapt to three days. According to body weight were randomly divided into 5 groups, namely control group, Gynostemma group, HaoHongfang side of high, medium and low dose group,12 in each group. The treatment group according to dose administered orally,20 ml/kg, once a day,7 days in all. After the last administration,12 h, mice were killed dislocation of cervical vertebra, rapidly profile control thymus, spleen, weight, organ index calculation. Spleen (thymus) index= spleen (thymus) weight (mg)/ body weight (g). There are differences between each group.
2.5.2 The influence of HaoHongfang carbon particles clearance function in mice
60 SPF level NIH mice, male,18-22 g, were randomly divided into 5 groups, namely control group, Gynostemma group, HaoHongfang high, medium and low dose group. Each treatment group were administered orally by dose,20 ml/kg, once a day,7 days in all, the control group were given equal volume of sesame oil. After the last administration, 1h, mouse tail vein injection of diluted ink 0.1 ml/lOg weight, at 30 s after injection of ink, and 6 min, from the orbital venous plexus of mouse blood 0.025 ml, immediately cast them into the 2 ml 0.1% sodium bicarbonate solution; taking 0.025 ml normal human blood soluble ink injection 2 ml 0.1% sodium bicarbonate solution, zeroing,675 nm was determined at OD values of phagocytic index calculated by the formula K value. There are differences between each group.
2.5.3 The influence of HaoHongfang on serum hemolysin formation of antibodies
60 SPF level NIH mice, male and female in half,18～22g, according to body weight were randomly divided into 5 groups, namely control group, Gynostemma group, HaoHongfang side of high, medium and low dose groups 12. Each treatment group were administered orally by dose,20 ml/kg, once a day,10 days in all, the control group were given equal volume of sesame oil. Article 6, each mouse was injected with 5% sheep red blood cells 0.4 ml/only (equivalent to 4×108 SRBC/only). After the last administration, 1 h, orbital blood serum hemolytic antibody. There are differences between each group.
2.5.4 The influence of HaoHongfang the impact on delayed-type hypersensitivity in mice
60 SPF level NIH mice, male and female in half,18-22g, adapted to the environment for three days. And then divided randomly into 5 groups, control group, Gynostemma group, HaoHongfang high, medium and low dose group,12 in each. Abdominal skin hair removaled (1 cm2 or so), covered with 50μl 1% DNFB sensitization,and the next day for the same. Each treatment group were orally administered by 20 ml/kg, day once,and for 10 days for all, the control group were given equal volume of sesame oil.7 days after administration of 1% DNFB solution evenly applied 10μl mouse right ear (both sides) to attack,24 hours later killed all of the mice and cut the left and right ears with the 8mm hole puncher, weighed, calculate swelling (DTH).
3% Results:
3.1 Study of Compatibility Rationality
3.1.1 Inhibiting the capillary permeability in mice compared with the control group, Compound groups 3 (Artemisia 200mg/kg+Folium 100mg/kg) can statistically reduce the capillary permeability in mice (P<0.01); Compound group 1 (Artemisia annua 100mg/kg+ Folium 200mg/kg) and Compound group 2 (Artemisia 150mg/kg+Folium 150mg/kg) also can statistically reduce the capillary permeability in mice (P<0.05).
3.1.2 Reduce cough in guinea pigs Compared with the control group, Compound group 2,3 could significant prolong the incubation period of cough and reduce the number of cough in 5 minutes (P<0.01); Compound group 1 could significant prolong the incubation period of cough and reduce the number of cough in 5 minutes (P<0.05).
3.2 The study of HaoHongfang on antibacterial and antiviral
3.2.1 Reduce the mortality rate of mice infected with Streptococcus pneumoniae
Compared with the control group, there were significantly higher mortality in model group (P<0.01),and had no effect on HaoHongfang control group. With model group comparison, HaoHongfang high and middle dose group was significantly lower mortality rate in mice (P＜0.01).
3.2.2 Reduce the mortality rate of mice infected with Staphylococcus aureus
Compared with the control group, model group mice were significantly higher mortality rate(P<0.01), and had no effect on HaoHongfang control group; With model group comparison, HaoHongfang high dose group was significantly lower mortality rate in mice infected by Staphylococcus aureus(P<0.01),and HaoHongfang middle and low group could also reduce mortality rate but there was no statistically different (P>0.05).
3.2.3 Reduce the infection of influenza virus in mice
Compared with the control group, the mortality rate、the average survival times of model group were significantly shorter (both P<0.01), but the were no influence in HaoHongfang control group. HaoHongfang high and middle dose group could significantly reduced the mortality rate in mice infected with influenza virus, prolong the average survival days(both P<0.01),and so did the HaoHongfang low-dose group (P<0.05).
3.2.4 Inhibitory Lung-index of mice infected with influenza virus
Compared with the control group, model group could significantly increased lung-index (P＜0.01); Compared with model group, HaoHongfang high dose group could reduce Lung-index in mice infected with influenza virus (P＜0.01), so did the middle group(P<0.05).
3.2.5 Reduce the death in mice induced by endotoxin
Compared with the control group, model group significantly increased mortality (P<0.01); compared with model group, high HaoHongfang significantly reduced the death in mice (P<0.01), HaoHongfang middle and low dose group could also decrease the death in mice but there was not significant difference (P> 0.05).
4、Conclusion:
The results show that HaoHongfang has antibacterial, antiviral, anti-endotoxin, Clearing heat transforming phlegm and suppressing cough, anti-inflammatory and pain releasement and immune enhancement function; provides pharmacodynamic basis for HaoHongfang in expelling wind-evil and clearing away heat, detoxification, phlegm and suppressing cough.
引文
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