CN104398572A - 一种玉屏风散加味复方及其制备方法和应用 - Google Patents
一种玉屏风散加味复方及其制备方法和应用 Download PDFInfo
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- CN104398572A CN104398572A CN201410768118.9A CN201410768118A CN104398572A CN 104398572 A CN104398572 A CN 104398572A CN 201410768118 A CN201410768118 A CN 201410768118A CN 104398572 A CN104398572 A CN 104398572A
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Abstract
本发明属于中药组方配伍技术领域,具体涉及一种玉屏风散加味复方及其制备方法和应用。本发明所述玉屏风散加味复方由玉屏风散加入黄芩组成黄芩玉屏风散加味复方或加入荆芥组成荆芥玉屏风散加味复方或加入黄芩和荆芥组成黄芩荆芥玉屏风散加味复方。研究结果表明,玉屏风散加味复方能够显著降低DTH模型小鼠耳廓肿胀程度,能够显著降低脾脏指数,显著升高小鼠血清中IL-4的含量,显著降低小鼠血清中IFN-γ的含量,显著降低脾脏中IL-6表达水平,特别是荆芥YPF和黄芩荆芥YPF对小鼠耳肿胀程度的抑制作用均高于玉屏风散,黄芩YPF和黄芩荆芥YPF的免疫抑制作用高于玉屏风散,荆芥YPF的IFN-γ/IL-4比值明显优于玉屏风散,荆芥YPF降低脾脏中IL-6表达水平的活性也优于玉屏风散。
Description
一、技术领域
本发明属于中药组方配伍技术领域,具体涉及一种玉屏风散加味复方及其制备方法和应用
二、背景技术
正常的免疫应答是机体维持自身平衡、抵御外来侵略的主要机制,过剩的免疫应答则会造成各种自身免疫性疾病、过敏及各种炎症反应等。以抑制过剩免疫应答为目的的现有免疫抑制剂,由于其作用对细胞和组织缺乏选择性,往往造成免疫应答的全面抑制,因而产生严重的毒副作用,如糖皮质激素、环磷酰胺等。环孢素A是第一种专为免疫相关疾病而开发的药物。但是,环孢素A对于不同状态的T细胞(活化和非活化的)以及T细胞介导的所有相关免疫反应的抑制作用并无选择性,因此T细胞所担负的机体正常免疫功能也同时受其抑制。事实上,长期使用环孢素A的病人,常可观察到药物对肾脏和神经系统的毒性,且发生感染性疾病和肿瘤的风险增高。另外,分子靶向类免疫抑制剂是近年来发展起来的以抗体类制剂为主的新型免疫抑制剂,但其对于免疫应答的抑制仍是非选择性的,会造成严重的毒副作用。
寻找能够选择性地抑制在疾病发生发展过程中扮演病理性角色的免疫细胞或分子,而不影响或较少地影响其他正常的免疫细胞的分子,是实现选择性免疫抑制的重要环节。研究发现,一系列清热中药及其有效成分能选择性地作用于免疫应答不同阶段,对迟发型超敏反应(DTH)的不同时相有选择性的抑制作用。
DTH是一种主要由T细胞介导的细胞免疫反应,根据抗原的致敏和攻击,可分为诱导相和效应相两个阶段,分别对应于T细胞的活化过程以及活化T细胞发挥效应的过程。研究发现,方剂四逆散和二妙散、单味中药蒺藜和结血蒿等仅对DTH的诱导相有抑制作用;苦参、地肤子、赤芍、土茯苓和白鲜皮等则主要作用于DTH的效应相;而氢化泼尼松和环孢素A对DTH的诱导相和效应相均有显著的抑制作用。另外,联合使用分别抑制DTH反应诱导相和效应相的中药,可以起到协同的治疗效果。
玉屏风散源于元代朱丹溪的《丹溪心法》,其功效为益气固表止汗,主治表虚不固、恶风自汗、体虚易感风邪者。药物组成为黄芪、防风各一两,白术二两;可研末制丸开水送服,或按相同比例以水煎服饮用。实验研究表明,玉屏风散具有调节人体免疫功能;近年来,被广泛应用于临床,对小儿体虚多汗、预防体弱儿童反复呼吸道感染、荨麻疹、风湿性关节炎、过敏性皮炎、肾炎、美尼尔氏综合征等病症均有良好疗效。
中药是依照七情和合、君臣佐使的原则组成方剂的。研究中药方剂配伍组方的方法有很多,主要是在饮片层次从整体效果及拆方后药物的配伍组合对复方的作用机制进行分析研究。因此,在玉屏风散的基础上,加入一些清热中药,如黄芩和荆芥,形成全新的玉屏风散加味复方,希望获得比玉屏风散疗效更佳的中药复方。继承和发展传统中药,并与现代医药技术相结合,是中药步入国际市场的关键所在。
三、发明内容
本发明需要解决的问题是公开一种玉屏风散加味复方及其制备方法和应用。
本发明的技术方案概括如下:
1.玉屏风散加味复方的组成
本发明所述玉屏风散加味复方由玉屏风散加入黄芩组成黄芩玉屏风散加味复方(黄芩YPF)或加入荆芥组成荆芥玉屏风散加味复方(荆芥YPF)或加入黄芩和荆芥组成黄芩荆芥玉屏风散加味复方(黄芩荆芥YPF)。
2.玉屏风散加味复方的制备方法:
按2:2:1:1的比例分别称取黄芪、白术、防风和黄芩中药饮片60g,加入900ml水,浸泡30分钟,煎煮30-60分钟,过滤,保留滤液,滤液浓缩,获得最终浓度为0.6g生药/ml的黄芩玉屏风散加味复方水煎液。
按2:2:1:1的比例分别称取黄芪、白术、防风和荆芥中药饮片60g,加入900ml水,浸泡30分钟,煎煮30-60分钟,过滤,保留滤液,滤液浓缩,获得终浓度为0.6g生药/ml的荆芥玉屏风散加味复方水煎液。
按2:2:1:1:1的比例分别称取黄芪、白术、防风、黄芩和荆芥中药饮片70g,加入1100ml水,浸泡30分钟,煎煮30-60分钟,过滤,保留滤液,滤液浓缩,获得终浓度为0.7g生药/ml的黄芩荆芥玉屏风散加味复方水煎液。
中药方剂是根据七情和合、君臣佐使的原则组成的。黄芪、白术、防风、黄芩和荆芥出现在“辛芩颗粒”配伍中。“辛芩颗粒”具有益气固表、祛风通窍的功能,主治过敏性鼻炎、鼻窒等症。而且,黄芪、白术和防风配伍构成古方玉屏风散(YPF)。
在玉屏风散(YPF)中,黄芪为君药,益气固表止汗;白术为臣药,补气健脾宁风;防风为佐药,走表散风而祛邪。黄芪、白术,以扶正为主,防风则以祛邪为主,该方通过“标本兼治”,达到提升患者的“正气”以抵御外邪的目的。
黄芩味苦、性寒,有清热燥湿、泻火解毒、止血、安胎等功效,能清肺胃胆及大肠经之湿热,尤善清中上焦湿热,善清肺火及上焦之实热。“黄芩一物汤”来源于张仲景的《伤寒杂病论》,书中介绍用黄芩煮水喝具有清热止血的功效。现代药理学研究发现,黄芩煎剂在体外有较广的抗菌谱,对伤寒杆菌、痢疾杆菌、绿脓杆菌、百日咳杆菌、葡萄球菌、链球菌、肺炎双球菌、脑膜炎双球菌等均有抑制作用,对流感病毒、钩端螺旋体及多种致病真菌亦有抑制作用。将黄芩加入玉屏风散,组成黄芩玉屏风散加味复方(黄芩YPF)。其中黄芪、防风和黄芩与“黄芪防风饮子”(源于《原机启微》卷下)的组成相同。在这里,黄芪,健脾补中,益卫固表,具有增强机体免疫功能、抗菌、降压等作用;防风、白术、黄芩有散风、除湿、清热的作用。
将荆芥加入玉屏风散,组成荆芥玉屏风散加味复方(荆芥YPF)。荆芥,味平、性温,具有镇痰、祛风、凉血的作用。荆芥与防风相配,有达腠理、发汗散邪之效,二者相辅相成,具有疏风凉血、清热解毒的作用。黄芪与荆芥相配,具有疏风益气解表、清热解毒的功效。
将黄芩、荆芥加入玉屏风散,组成黄芩荆芥玉屏风散加味复方(黄芩荆芥YPF)。黄芩、荆芥,源自黄芩荆芥汤(《良朋汇集》卷四),主治小儿感冒发热、痰壅风热、丹毒疼痛等。黄芩、荆芥穗,源自风疹瘙痒茶(《中医良药良方》),具有清热解毒、祛风利湿的作用。黄芩清热燥湿、泄火解毒、抗过敏,其主要成分黄芩甙具有抗炎、抗变态反应、阻止肥大细胞释放组织胺、提高免疫能力的作用,能够降低毛细血管的通透性;荆芥祛风透疹止痒,主治皮肤瘙痒、风疹等;两药相配对以变态反应为主的“痒疹”病有一定疗效。
3.药理实验
使用二硝基氟苯(DNFB)进行外源性刺激,并连续灌胃等量生理盐水5天后,使用较低浓度DNFB进行激发,构建迟发型超敏反应的变应性接触性皮炎小鼠模型。
在此基础上,分别对模型组、地塞米松阳性药物组、玉屏风散(YPF)、黄芩YPF、荆芥YPF和黄芩荆芥YPF给药组的实验小鼠,于激发后6~8小时,分别对实验小鼠的耳厚度和体重变化进行测量;于激发后12小时对小鼠进行摘眼球取血,取离心后得到的上层血清进行ELISA检测,分别检测IL-4和IFN-γ含量并进行统计分析。
小鼠处死后,取其脾脏称重,并计算脾脏指数。
同时,对小鼠脾脏组织加入细胞裂解液和蛋白酶抑制剂,使用匀浆仪进行匀浆;离心,取上清液,用BCA法测定组织蛋白原液蛋白浓度;然后,采用westernblotting方法检测IL-6表达量。
4.实验结果
研究结果表明,玉屏风散加味复方给药后,小鼠体重均明显下降(P<0.05);与地塞米松相比,小鼠体重下降的差异不大;但与模型组小鼠比较,下降幅度明显减小(表1)。由此推断,它们能够抑制DNFB对小鼠的刺激作用,降低药物对小鼠生理活动(包括饮食、饮水)的抑制作用。
玉屏风散加味复方能够显著降低激发后小鼠耳肿胀程度,并表现出显著性差异(P<0.01),而且耳肿胀度抑制率达到50%以上;特别是荆芥YPF和黄芩荆芥YPF对小鼠耳肿胀程度的抑制作用均高于地塞米松和玉屏风散(表1)。
玉屏风散加味复方都能够显著降低脾脏指数(p<0.05),具有一定的免疫抑制作用,而且,黄芩YPF和黄芩荆芥YPF的免疫抑制作用高于地塞米松和玉屏风散(表1)。
玉屏风散加味复方都能够显著升高小鼠血清中IL-4的含量(p<0.01),显著降低小鼠血清中IFN-γ的含量(p<0.05);特别是荆芥YPF的IFN-γ/IL-4比值明显降低,优于地塞米松和玉屏风散的比值。这表明,它们具有调节Th1/Th2失衡的功能(表1)。
表1、玉屏风散复方及其加味复方对DTH小鼠体重、耳肿胀度、脾脏指数、血清中IFN-γ和IL-4水平以及脾脏中IL-6表达水平的影响
注:其中体重变化值=致敏后小鼠体重-致敏前小鼠体重;耳肿胀度为左右耳的厚度差;脾脏指数=脾脏质量/小鼠体重×100;小鼠脾脏IL-6的灰度值除以内参mGAPDH的灰度值代表某样品的目的蛋白相对含量。*P<0.05,**P<0.01,***P<0.001
Western Blotting检测结果表明,与正常对照组相比,模型组小鼠脾脏IL-6表达水平显著升高(p<0.001)。与模型组相比,玉屏风散加味复方能够显著降低IL-6表达水平(p<0.05);特别是荆芥YPF能够极显著降低IL-6表达水平(p<0.001),其活性优于YPF水溶液(表1)。
本发明的特点是:基于临床使用的中药复方进行改造,在玉屏风散复方的基础上进行加味组方,产生全新的玉屏风散加味复方,即黄芩玉屏风散加味复方、荆芥玉屏风散加味复方和黄芩荆芥玉屏风散加味复方。
该加味复方既保留了玉屏风散的原有药物饮片,又增加了黄芩或/和荆芥,确保了中药复方的质量可控性。由于“玉屏风散”、“辛芩颗粒”为临床使用药物,因此,该加味复方又具有可靠的安全性。实验表明,该加味复方的活性优于玉屏风散,具有与玉屏风散类似的有效性。该加味复方具备了创新药物所具有的特征。本发明所述玉屏风散加味复方可在制备治疗自身免疫性疾病或治疗过敏或治疗各种炎症或治疗呼吸道感染或口腔溃疡或支气管哮喘或过敏性鼻炎或面神经麻痹或慢性荨麻疹或皮肤瘙痒症或过敏性皮炎或风湿性关节炎或肾炎或慢性结肠炎或原发性血小板减少性紫癜病症药物中应用。也可在制备提高患恶性肿瘤或慢性肝炎或慢性支气管炎疾病病人的免疫功能药物中应用。以及在制备治疗伤寒杆菌或痢疾杆菌或绿脓杆菌或百日咳杆菌或葡萄球菌或链球菌或肺炎双球菌或脑膜炎双球菌或流感病毒或钩端螺旋体或多种致病性皮肤真菌如堇色毛癣菌、狗小芽胞癣菌、许兰氏黄癣菌、铁锈色小芽胞癣菌、红色表皮癣菌、白色念珠菌所致疾病药中应用。
本发明的有益效果是:本发明是在古方玉屏风散的基础上进行加味组方,产生的玉屏风散加味复方比原方具有更好的活性,而且拓展了原方的治疗领域,展现了良好的应用前景,有利于传统中药复方的推陈出新,有助于实现中药的现代化和国际化。继承和发展传统中药,并与现代医药技术相结合,是中药步入国际市场的关键所在。
四、具体实施方式
1.实验方法
BALB/c小鼠(正常组)
BALB/c纯系小鼠(购自南京大学模式动物中心),清洁级,体重18-22g,雌雄各半。适应性喂养。实验第一天到第五天连续用0.2ml生理盐水(0.9%NaCl)灌胃两次,每次间隔6小时。
2,4-二硝基氯苯致敏小鼠模型的构建(DTH小鼠、病理组/模型组)
BALB/c纯系小鼠(购自南京大学模式动物中心),清洁级,体重18-22g,雌雄各半。适应性喂养数日。实验前一天,剔去小鼠腹部的毛,于第一天早上涂抹1%2,4-二硝基氟苯(DNFB)(丙酮:橄榄油=4:1)20ul致敏。实验第一天到第五天连续用0.2ml生理盐水(0.9%NaCl)灌胃两次,每次间隔6小时。第六天早上,右耳涂抹0.5%二硝基氯苯20ul激发,同时左耳涂抹等量溶剂(橄榄油)进行对照。
地塞米松阳性药物实验(阳性药物组)
取DTH小鼠10只,实验第一天至第四天正常饲养,实验第五天腹腔给药一次。将原浓度5mg/ml的地塞米松醋酸溶液稀释为1mg/ml,每只一次腹腔给药0.2ml,实际给药浓度为10mg/kg。
玉屏风散复方的制备与给药(玉屏风散给药组)
玉屏风散(YPFS)处方来自《医方类聚》卷150《究原方》,药物组成防风1两(30g),黄耆(蜜炙)2两(60g),白术2两(60g)。
按2:2:1的比例分别称取黄芪、白术和防风中药饮片50g,加入500ml水,浸泡30分钟,第一次煎煮,煮沸后保持微沸状态30分钟,过滤,保留滤液;滤渣中加如300ml水继续煎煮,煮沸后保持微沸状态30分钟,过滤,保留滤液;合并两次滤液,滤液浓缩,获得最终浓度为0.5g生药/ml的水煎液。玉屏风散水煎液实际给药剂量为5g生药/kg。
BALB/c纯系小鼠(购自南京大学模式动物中心),清洁级,体重18-22g,雌雄各半。实验前一天,剔去小鼠腹部的毛,于第一天涂抹1%2,4-二硝基氟苯(丙酮:橄榄油=4:1)20ul致敏。从第二天起,连续五天灌胃给药,每只小鼠每天给药一次,每次0.2ml。第六天早上,右耳涂抹0.5%二硝基氟苯20ul激发,同时左耳涂抹等量溶剂进行对照。6~8h后开始进行后续的指标测量及分析。
从实验第一天到第五天,YPFS水煎液组每只小鼠每天给药一次,每次灌胃给药0.2ml即可。
各组小鼠在相同环境下正常饲养,喂食SPF维持型鼠粮和双蒸水。
玉屏风散加味复方的制备与给药(加味玉屏风散给药组)
玉屏风散加味复方的制备与给药方式与玉屏风散的类似。具体制备复方如下:
按2:2:1:1的比例分别称取黄芪、白术、防风和黄芩中药饮片60g,加入600ml水,浸泡30分钟,第一次煎煮,煮沸后保持微沸状态30分钟,过滤,保留滤液;滤渣中加如300ml水继续煎煮,煮沸后保持微沸状态30分钟,过滤,保留滤液;合并两次滤液,滤液浓缩,获得最终浓度为0.6g生药/ml的水煎液。黄芩玉屏风散加味水煎液实际给药剂量为6g生药/kg。
类似地,按2:2:1:1的比例分别称取黄芪、白术、防风和荆芥中药饮片60g,加入适量水、煎煮、浓缩,获得终浓度为0.6g生药/ml的水煎液。荆芥玉屏风散加味水煎液实际给药剂量为6g生药/kg。
按2:2:1:1:1的比例分别称取黄芪、白术、防风、黄芩和荆芥中药饮片70g,加入700ml水,浸泡30分钟,第一次煎煮,煮沸后保持微沸状态30分钟,过滤,保留滤液;滤渣中加如400ml水继续煎煮,煮沸后保持微沸状态30分钟,过滤,保留滤液;合并两次滤液,滤液浓缩,获得终浓度为0.7g生药/ml的水煎液。黄芩荆芥玉屏风散加味水煎液实际给药剂量为7g生药/kg。
从实验第一天到第五天,每只实验小鼠每天给药一次,每次灌胃给药0.2ml即可。各组小鼠在相同环境下正常饲养,喂食SPF维持型鼠粮和双蒸水。
2.实验检测
小鼠的体重变化
分别于实验前一天刺激后和第六天激发后6~8h后,对每一组每只小鼠进行体重记录。计算实验前后小鼠体重变化的绝对值(g)和相对值(百分比)。
小鼠的耳肿胀度
第六天激发后6~8h后,先观察小鼠左右耳形态、颜色上差异。比较红肿、增厚情况。并用测厚仪测量左右耳厚度的差异。
ELISA检测
用乙醚或苯巴比妥钠麻醉小鼠后,摘眼球取血,2500rmp离心10min后取上清液使用ELISA检测盒(Mouse IL-4ELISA Kit 96T,Mouse IFN-gamma ELISAKit,联科生物技术有限公司),测量IL-4和IFN-γ含量。
小鼠的脾脏指数
超净台内无菌取出小鼠脾脏,提前称取空EP管质量,测出EP管和脾脏总重后相减获得脾脏重量再除以小鼠对应体重,获得每100g体重脾脏质量(mg)。
小鼠脾脏蛋白质的提取
分别取小鼠的脾脏组织约50mg,放入2ml离心管中,各加入500ul western及IP细胞裂解液和20ul Roche Protease Inhibitor,匀浆仪匀浆(60Hz、70s)。冰上静置1h,10000g、4℃离心15min。取上清,即为组织蛋白质原液。用BCA法测定组织蛋白原液蛋白浓度,用ddH2O将组织蛋白原液调为等体积等浓度(4.201ug/ul),加入四分之一体积的5×SDS上样缓冲液。沸水浴5min,-20℃保存。
western blotting检测
SDS-PAGE电泳,用半干法转至PVDF膜,5%脱脂奶粉封闭,孵一抗,4℃缓慢摇动孵育过夜,TBST洗膜4次,每次15min。加入二抗,室温孵育1h,TBST洗膜,ECL法检测。用Image J软件分析蛋白灰度值。
统计学处理
利用BM SPSS Statistics 19.0软件进行统计分析。实验数据用x±s表示,两组均数间比较用t检验,以P<0.05为差异有显著性。具体方法为:首先使用“描述统计”中的“探索”对各组数据分布的正态性进行检验,剔除正态分布之外的异常值。对保留的数据利用“分析”下的“比较均值”,以DTH模型组为参照,对各组样本进行独立样本t-test,通过显著性差异大小判断药物的治疗是否有效,若有效根据显著性具体数值判断显著性差异的程度(P<0.001为极显著,标记“***”;P<0.05为很显著,标记“**”;P<0.01为显著,标记“*”)。
Claims (8)
1. 一种玉屏风散加味复方,其特征是在古方玉屏风散的基础上,加入黄芩组成黄芩玉屏风散加味复方或加入荆芥组成荆芥玉屏风散加味复方或加入黄芩和荆芥组成黄芩荆芥玉屏风散加味复方。
2.根据权利要求1所述玉屏风散加味复方的制备方法,其特征是黄芩玉屏风散加味复方的制备由以下步骤构成:
按2:2:1:1的比例分别称取黄芪、白术、防风和黄芩中药饮片60g,加入900ml水,浸泡30分钟,煎煮30-60分钟,过滤,保留滤液,滤液浓缩,获得最终浓度为0.6g生药/ml的黄芩玉屏风散加味复方水煎液。
3.根据权利要求1所述玉屏风散加味复方的制备方法,其特征是荆芥玉屏风散加味复方的制备由以下步骤构成:
按2:2:1:1的比例分别称取黄芪、白术、防风和荆芥中药饮片60g,加入900ml水,浸泡30分钟,煎煮30-60分钟,过滤,保留滤液,滤液浓缩,获得终浓度为0.6g生药/ml的荆芥玉屏风散加味复方水煎液。
4.根据权利要求1所述玉屏风散加味复方的制备方法,其特征是黄芩荆芥玉屏风散加味复方的制备由以下步骤构成:
按2:2:1:1:1的比例分别称取黄芪、白术、防风、黄芩和荆芥中药饮片70g,加入1100ml水,浸泡30分钟,煎煮30-60分钟,过滤,保留滤液,滤液浓缩,获得终浓度为0.7g生药/ml的黄芩荆芥玉屏风散加味复方水煎液。
5.权利要求1所述玉屏风散加味复方在制备治疗自身免疫性疾病或治疗过敏或治疗各种炎症药物中的应用。
6. 权利要求1所述玉屏风散加味复方在制备治疗呼吸道感染或口腔溃疡或支气管哮喘或过敏性鼻炎或面神经麻痹或慢性荨麻疹或皮肤瘙痒症或过敏性皮炎或风湿性关节炎或肾炎或慢性结肠炎或原发性血小板减少性紫癜病症药物中的应用。
7.权利要求1所述玉屏风散加味复方在制备提高患恶性肿瘤或慢性肝炎或慢性支气管炎疾病病人的免疫功能药物中的应用。
8. 权利要求1所述玉屏风散加味复方制备治疗伤寒杆菌或痢疾杆菌或绿脓杆菌或百日咳杆菌或葡萄球菌或链球菌或肺炎双球菌或脑膜炎双球菌或流感病毒或钩端螺旋体或多种致病性皮肤真菌如堇色毛癣菌、狗小芽胞癣菌、许兰氏黄癣菌、铁锈色小芽胞癣菌、红色表皮癣菌、白色念珠菌所致疾病药中的应用。
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CN104800694A (zh) * | 2015-05-11 | 2015-07-29 | 冯洁庆 | 一种防过敏中药 |
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CN113491726A (zh) * | 2020-04-02 | 2021-10-12 | 常州高新技术产业开发区三维工业技术研究所有限公司 | 一种防治肺炎提高机体抗病能力的中药组合物及其应用 |
CN111557979A (zh) * | 2020-05-14 | 2020-08-21 | 云南省中医医院(云南中医药大学第一附属医院) | 一种用于治疗特应性皮炎的中药组合物 |
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