CN114869985B - 一种中药组合物在制备治疗冠状病毒的药物上的应用 - Google Patents
一种中药组合物在制备治疗冠状病毒的药物上的应用 Download PDFInfo
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Abstract
本发明涉及医药领域,具体公开了一种中药组合物在制备治疗冠状病毒的药物上的应用,中药组合物由以下组分制成:芦根、冬瓜子、滑石、薏苡仁、黄芩、青蒿、射干、枇杷叶、法半夏、淡豆豉、苦杏仁、葶苈子、郁金;中药组合物的应用是用于制备治疗冠状病毒(HCoV‑229E)的药物。本发明具有更好的治疗冠状病毒(HCoV‑229E)感染引发的病症的优点。
Description
技术领域
本发明涉及医药领域,尤其是涉及一种中药组合物在制备治疗冠状病毒的药物上的应用。
背景技术
冠状病毒是目前已知的RNA病毒中基因组最大的病毒,其仅感染脊椎动物,如人、鼠、猪、猫、犬、狼、鸡、牛等。
冠状病毒(HCoV-229E)是已知的可以感染人的七种冠状病毒之一。人体感染冠状病毒(HCoV-229E)后会引发轻中度上呼吸道疾病,会出现流鼻涕、头痛、咳嗽、咽喉痛、发热等症状,严重时还会诱发肺炎或支气管炎等下呼吸道疾病。
冠状病毒最早发现于1937年,冠状病毒(HCoV-229E)可通过空气飞沫传播,具有较强的传染能力,但截止目前仍然没有针对性的特效药,也没有可用的疫苗,导致人类长期受到冠状病毒的困扰,因此还有改善空间。
发明内容
为了更好的治疗冠状病毒(HCoV-229E)感染引发的病症,本申请提供一种中药组合物在制备治疗冠状病毒的药物上的应用。
本申请提供的一种中药组合物在制备治疗冠状病毒的药物上的应用采用如下的技术方案:
一种中药组合物在制备治疗冠状病毒的药物上的应用,所述中药组合物由以下组分制成:
芦根、冬瓜子、滑石、薏苡仁、黄芩、青蒿、射干、枇杷叶、法半夏、淡豆豉、苦杏仁、葶苈子、郁金;
所述中药组合物的应用是用于制备治疗冠状病毒(HCoV-229E)的药物。
中药组合物即将多种以中国传统医药理论指导采集、炮制、制剂的药物根据病情的不同需要和药物的不同特点配合在一起使用的混合药材。
申请人于2010年申请了治疗支气管炎的中药组合物的专利,专利申请名称:一种治疗小儿急性支气管炎的中药组合物及制备方法,申请号为201010125894.9。其主要公开了该中药组合物具有清热利湿、化痰止咳之功效,用于治疗儿童急性支气管炎;对于儿童急性支气管炎湿热蕴肺证尤为有效,但该中药组合物在人体感染冠状病毒(HCoV-229E)的治疗中的应用还处于空白状态。
通过采用上述技术方案,添加了由芦根、冬瓜子、滑石、薏苡仁、黄芩、青蒿、射干、枇杷叶、法半夏、淡豆豉、苦杏仁、葶苈子、郁金制成的中药组合物的治疗冠状病毒(HCoV-229E)的药物对冠状病毒(HCoV-229E)的选择指数SI为6.80,对冠状病毒(HCoV-229E)感染导致的细胞病变具有显著的抑制作用,可见该中药组合物制成的治疗冠状病毒的药物对感染冠状病毒(HCoV-229E)的患者有明显的治疗作用,能使得感染冠状病毒(HCoV-229E)的病症能被更为快速的治愈,减少病症对患者的折磨,填补了治疗感染冠状病毒(HCoV-229E)领域的空白。
优选的,所述中药组合物由以下质量份数的组分制成:
芦根500-600份;
冬瓜子380-460份;
滑石380-460份;
薏苡仁300-380份;
黄芩250-320份;
青蒿250-320份;
射干250-320份;
枇杷叶250-320份;
法半夏180-250份;
淡豆豉180-250份;
苦杏仁180-250份;
葶苈子180-250份;
郁金180-250份。
通过采用上述技术方案,通过芦根、冬瓜子、滑石、薏苡仁、黄芩、青蒿、射干、枇杷叶、法半夏、淡豆豉、苦杏仁、葶苈子、郁金以特定比例范围配合时,制得的治疗冠状病毒(HCoV-229E)的药物具有更好且更快的治疗效果,有效抑制细胞病变,治疗效果较佳。
优选的,所述中药组合物由以下质量份数的组分制成:
芦根563份;
冬瓜子422份;
滑石422份;
薏苡仁340份;
黄芩282份;
青蒿282份;
射干282份;
枇杷叶282份;
法半夏225份;
淡豆豉225份;
苦杏仁225份;
葶苈子225份;
郁金225份。
通过采用上述技术方案,通过芦根、冬瓜子、滑石、薏苡仁、黄芩、青蒿、射干、枇杷叶、法半夏、淡豆豉、苦杏仁、葶苈子、郁金以特定比例配合时,制得的治疗冠状病毒(HCoV-229E)的药物更为有效抑制细胞病变,治疗效果更好,见效更快。
优选的,所述冬瓜子为炒冬瓜子、黄芩为酒制黄芩、枇杷叶为蜜炙枇杷叶、葶苈子为炒葶苈子。
通过采用上述技术方案,通过具体选择炒冬瓜子、酒制黄芩、蜜炙枇杷叶、炒葶苈子,使得治疗冠状病毒(HCoV-229E)的药物疗效更佳,治疗冠状病毒(HCoV-229E)诱发的疾病的效果更好。
优选的,所述中药组合物制备的治疗冠状病毒的药物可以为片剂、胶囊剂、丸剂、颗粒剂、口服液。
通过采用上述技术方案,通过制成不同的剂型,使得治疗冠状病毒(HCoV-229E)的药物可满足不同使用者的不同使用需求,应用较为广泛。
综上所述,本申请具有以下有益效果:
1、由于本申请采用添加了由芦根、冬瓜子、滑石、薏苡仁、黄芩、青蒿、射干、枇杷叶、法半夏、淡豆豉、苦杏仁、葶苈子、郁金制成的中药组合物的治疗冠状病毒(HCoV-229E)的药物对冠状病毒(HCoV-229E)的选择指数SI为6.80,对冠状病毒(HCoV-229E)感染导致的细胞病变具有显著的抑制作用,可见该重要组合物制成的治疗冠状病毒的药物对感染冠状病毒(HCoV-229E)的患者有明显的治疗作用,能使得感染冠状病毒(HCoV-229E)的病症能被更为快速地治愈,减少病症对患者的折磨,填补了治疗感染冠状病毒(HCoV-229E)领域的空白。
2、本申请中优选通过芦根、冬瓜子、滑石、薏苡仁、黄芩、青蒿、射干、枇杷叶、法半夏、淡豆豉、苦杏仁、葶苈子、郁金以特定比例配合时,制得的治疗冠状病毒(HCoV-229E)的药物更为有效抑制细胞病变,治疗效果更好,见效更快。
附图说明
图1为实验例1的治疗冠状病毒(HCoV-229E)的口服液对Huh-7细胞毒性结果图;
图2为实验例1的治疗冠状病毒(HCoV-229E)的口服液对冠状病毒HCoV-229E药效检测结果图;
图3为实验例1的治疗冠状病毒(HCoV-229E)的口服液对冠状病毒HCoV-229E炎症药效图。
具体实施方式
以下结合实施例对本申请作进一步详细说明。
实施例1
一种治疗冠状病毒(HCoV-229E)的片剂的制备方法,包括以下步骤:
步骤1),将2.5kg青蒿和1.8kg郁金与25.8kg水混合,水蒸气蒸馏,收集挥发油至无油滴产生,将挥发油用β环糊精包结,保存备用。
步骤2),向步骤1)中蒸馏后剩下的水液及药渣混合物中加入5kg芦根、3.8kg冬瓜子、3.8kg滑石、3kg薏苡仁、2.5kg黄芩、2.5kg射干、2.5kg枇杷叶、1.8kg法半夏、1.8kg淡豆豉、1.8kg苦杏仁、1.8kg葶苈子,然后加水煎煮2次,每次加276.8kg水,煮沸1小时,分别过滤,合并2次滤液,浓缩至相对密度为1.15(50℃)的清膏,加入乙醇使含醇量达50%,静置12h,过滤,然后回收滤液中的乙醇至无醇味,然后浓缩至相对密度为1.3(60℃)的稠膏,减压干燥,粉碎成细粉,得到药粉。
步骤3),将步骤1)得到的挥发油-β环糊精包结物和羟甲基淀粉钠(羟甲基淀粉钠的投入质量为稠膏质量的4%)加入稠膏中,混匀,压制成片剂,得治疗冠状病毒(HCoV-229E)的片剂。
实施例2
一种治疗冠状病毒(HCoV-229E)的颗粒剂的制备方法,包括以下步骤:
步骤1),将3.2kg青蒿和2.5kg郁金与51.3kg水混合,水蒸气蒸馏,收集挥发油至无油滴产生,用57kg乙醇将挥发油溶解后备用。
步骤2),向步骤1)中蒸馏后剩下的水液及药渣混合物中加入6kg芦根、4.6kg冬瓜子、4.6kg滑石、3.8kg薏苡仁、3.2kg黄芩、3.2kg射干、3.2kg枇杷叶、2.5kg法半夏、2.5kg淡豆豉、2.5kg苦杏仁、2.5kg葶苈子,然后加水煎煮3次,第1次加531.6kg水煮沸1.5小时,第2、3次加354.4kg水煮沸1小时,分别过滤,合并滤液,浓缩至相对密度为1.15(50℃)的清膏,加入乙醇使含醇量达70%,静置12h,过滤,然后回收滤液中的乙醇至无醇味,然后浓缩至相对密度为1.25(60℃)的稠膏,喷雾干燥成细粉,得到药粉。
步骤3),加入稠膏质量0.2%的蛋白糖和稠膏质量0.01%的微晶纤维素,以70%乙醇制粒,加入步骤1)的挥发油乙醇溶液,烘干,分装成颗粒剂,得治疗冠状病毒(HCoV-229E)的颗粒剂。
实施例3
一种治疗冠状病毒(HCoV-229E)的胶囊剂的制备方法,包括以下步骤:
步骤1),将2.5kg青蒿和2.5kg郁金与60kg水混合,水蒸气蒸馏4小时,收集挥发油,将挥发油用β环糊精包结,保存备用。
步骤2),向步骤1)中蒸馏后剩下的水液及药渣混合物中加入5kg芦根、3.8kg冬瓜子、3.8kg滑石、3kg薏苡仁、2.5kg黄芩、2.5kg射干、2.5kg枇杷叶、2.5kg法半夏、2.5kg淡豆豉、2.5kg苦杏仁、2.5kg葶苈子,然后加水煎煮2次,第1次加381kg水煮沸1小时,第2次加304.8kg水煮沸0.5小时,分别过滤,合并滤液,浓缩至相对密度为1.15(50℃)的清膏,加入乙醇使含醇量达60%,静置12h,过滤,用浓度为60%的乙醇洗涤滤渣,合并洗液与滤液后回收乙醇至无醇味,然后浓缩至相对密度为1.20(60℃)的稠膏,减压干燥,粉碎成细粉,得到药粉。
步骤3),向药粉中加入步骤1)的挥发油的β环糊精包结物,以70%乙醇制粒,加入稠膏质量0.01%的微粉硅胶,混匀,制得胶囊剂,得治疗冠状病毒(HCoV-229E)的胶囊剂。
实施例4
一种治疗冠状病毒(HCoV-229E)的口服液的制备方法,包括以下步骤:
步骤1),将2.82kg青蒿、2.25kg苦杏仁和kg2.25郁金与65.88kg水混合,收集7500ml蒸馏液,将蒸馏液进行重蒸馏,收集重蒸馏液3500ml,加入15ml聚山梨酯80,混匀备用。
步骤2),向步骤1)中蒸馏后剩下的水液及药渣混合物中加入5.63kg芦根、4.22kg冬瓜子(炒)、4.22kg滑石、3.40kg薏苡仁、2.82kg黄芩(酒制)、2.82kg射干、2.82kg枇杷叶(蜜炙)、2.25kg法半夏、2.25kg淡豆豉、2.25kg葶苈子(炒),然后加水煎煮2次,每次加48kg水煮沸1小时,分别过滤,合并滤液,减压浓缩至相对密度为1.25(40℃),放冷,加入乙醇使含醇量达60%,静置12h,过滤,用浓度为60%的乙醇洗涤沉淀,合并洗液与滤液后回收乙醇至无醇味,加入5g羟苯乙酯和25g苯甲酸,混匀,放冷,加入步骤1)的重蒸馏液,混匀,静置,过滤,取滤液备用。
步骤3),取200g蜂蜜、2000g蔗糖、适量甜蜜素和安赛蜜,加水煮沸至溶解,过滤,放冷,加入步骤2)的滤液中,混匀,分装成口服液,得治疗冠状病毒(HCoV-229E)的口服液。
实验例1
一种治疗冠状病毒(HCoV-229E)的口服液的制备方法,包括以下步骤:
步骤1),将2.82kg青蒿、2.25kg苦杏仁和kg2.25郁金与65.88kg水混合,收集7500ml蒸馏液,将蒸馏液进行重蒸馏,收集重蒸馏液3500ml,加入15ml聚山梨酯80,混匀备用。
步骤2),向步骤1)中蒸馏后剩下的水液及药渣混合物中加入5.63kg芦根、4.22kg冬瓜子(炒)、4.22kg滑石、3.40kg薏苡仁、2.82kg黄芩(酒制)、2.82kg射干、2.82kg枇杷叶(蜜炙)、2.25kg法半夏、2.25kg淡豆豉、2.25kg葶苈子(炒),然后加水煎煮2次,每次加48kg水煮沸1小时,分别过滤,合并滤液,减压浓缩至相对密度为1.25(40℃),放冷,加入乙醇使含醇量达60%,静置12h,过滤,用浓度为60%的乙醇洗涤沉淀,合并洗液与滤液后回收乙醇至无醇味,加入5g羟苯乙酯和25g苯甲酸,混匀,放冷,加入步骤1)的重蒸馏液,混匀,静置,过滤,取滤液备用。
步骤3),将滤液加水稀释,分装,分别获得浓度为100mg生药/ml、50mg生药/ml、25mg生药/ml、12.5mg生药/ml、6.25mg生药/ml、3.125mg生药/ml、1.56mg生药/ml、5mg生药/ml、2.5mg生药/ml、1.25mg生药/ml、0.6125mg生药/ml的治疗冠状病毒(HCoV-229E)的口服液。
检测实验
一、实验材料
1.受试药物:实验例1制得的治疗冠状病毒(HCoV-229E)的口服液试样。
阳性对照药物瑞德西韦(GS-5734)购于上海吉至生化科技有限公司。
2.细胞:Huh-7细胞,由广州呼吸健康研究院呼吸疾病国家重点实验室病毒室保存。
3.病毒:冠状病毒HCoV-229E,滴度为TCID50=10-5/100μL,由广州呼吸健康研究院呼吸疾病国家重点实验室病毒室-80℃保存;使用病毒滴度为100TCID50。
4.试剂及耗材,详见表1。
表1:常用试剂及耗材列表
5.主要仪器,详见表2。
表2:主要仪器列表
二、具体实验
实验1
细胞毒性试验(MTT法),具体如下:
(1)受试药物,详见表3。
表3:药物名称、实验浓度和分组
(2)无菌96孔培养板,每孔加入100μL浓度为2×105cells/mL Huh-7细胞,37℃5%CO2培养24小时。
(3)96孔板单层细胞用PBS洗涤1次,每孔加入100μL2倍梯度稀释的药物。空白对照组和细胞对照组每孔加入等体积培养液,37℃培养4日。
(4)每孔加入浓度为5mɡ/mL的MTT溶液20μL,继续孵育4小时。弃上清,每孔加入100μL DMSO,低速振荡5分钟,使结晶物充分溶解。
(5)选择490nm波长,在酶联免疫仪上测定吸光值,计算抑制率。抑制率=(细胞对照组平均OD值-实验组平均OD值)÷(细胞对照组平均OD值-空白对照组平均OD值)×100%。Reed-Meunch法计算药物半数毒性浓度(TC50)。
(6)实验条件:以上实验操作均在BSL-2实验室内完成。
实验2
受试药物抗病毒实验,具体如下:
(1)受试药物:
表4:药物名称、实验浓度和分组
(2)无菌96孔培养板,每孔加入100μL浓度为2×105cells/mL Huh-7细胞,37℃5%CO2培养24小时。
(3)培养板实验组和病毒对照组加入2%血清培养基稀释的100TCID50病毒液100μL/孔,细胞对照组加入等体积2%血清培养基,37℃5%CO2培养箱吸附2h。
(4)2h后,弃去96孔培养板中细胞培养液;受试药物使用2%血清培养基稀释成表4中的各个浓度,每个浓度3个复孔,100μL/孔加入上述药液。
(5)细胞对照组和病毒对照组加入等体积2%血清培养基。
(6)细胞37℃5%CO2孵箱孵育3-4天。
(7)每孔加入浓度为5mɡ/mL的MTT溶液20μL,继续孵育4小时。弃上清,每孔加入100μL DMSO,低速振荡5分钟,使结晶物充分融解。
(8)选择490nm波长,在酶联免疫仪上测定吸光值,计算抑制率。抑制率=(实验组平均OD值-病毒对照组平均OD值)÷(细胞对照组平均OD值-病毒对照组平均OD值)×100%。能够抑制50%及以上病毒引起的细胞病变(CPE)的药物浓度视为有效浓度,采用Reed-Muench法或GraphPad Prism8.0计算半数抑制浓度(IC50)。
(9)实验条件:以上实验操作均在BSL-2实验室内完成。
实验3
RT-qPCR检测炎症因子,具体如下:
(1)受试药物:
表5:药物名称、实验浓度和分组
(2)Huh-7细胞接种至6孔板中,分为细胞对照组、病毒对照组、阳性对照组和实验组。细胞长至单层后,弃去培养液,用PBS洗一次,病毒对照组、阳性对照组和实验组使用含2%血清培养基稀释并接种100TCID50的冠状病毒(HCoV-229E),正常组加入含2%血清培养基,37℃吸附2小时。
(3)弃去病毒液,实验组加入含不同浓度药物的2%血清培养基2mL,阳性对照组加入含5μM瑞德西韦的2%血清培养基2mL,正常组和病毒组加入等体积含2%血清培养基。37℃作用48小时后,弃上清,PBS洗细胞一次,加入TRIzol试剂裂解细胞并转移至1.5mL EP管。
(4)RNA的提取
向细胞中加入氯仿200μL,震荡15s,在室温孵育5min,4℃、10000rpm离心10min;离心后,取上清至新的1.5mL EP管中,加入异丙醇500μL,室温孵育10min,4℃、10000rpm离心10min;离心弃上清后加入75%乙醇1000μL混匀,4℃、10000rpm离心10min;弃上清,室温中放置5min,加入适量无RNA酶的ddH2O溶解RNA,检测RNA浓度并进行逆转录cDNA。
(5)样本RNA的逆转录
mRNA逆转录成cDNA;配置逆转录反应体系如下:
逆转录反应条件如下:
(6)实时荧光定量PCR检测
反应体系如下表:
PCR反应条件如下表:
(7)引物的设计和合成
利用引物设计软件Primer Premier 5.0进行引物设计,并进行合成,引物序列如下:
实验条件:以上实验操作均在BSL-2实验室内完成。
抗病毒药效结果判定标准:能够抑制50%及以上病毒引起的细胞病变(CPE)的药物浓度视为有效浓度,,以选择指数判定药效标准,SI>1表示有效,选择指数越大,抗病毒效果越好。
实验结果
实验1,在Huh-7细胞中,治疗冠状病毒(HCoV-229E)的口服液半数毒性浓度(TC50)为16.99mg生药/ml,详见图1。
实验2,治疗冠状病毒(HCoV-229E)的口服液在此次实验的治疗模式中IC50为2.5mg生药/ml,低于无毒浓度,SI为6.80,详见图2。
实验3,通过RT-qPCR检测细胞因子mRNA的表达,采用GraphPad Prism 8.0分析计算结果详见图3。
结论
上述实验结果表明治疗冠状病毒(HCoV-229E)的口服液在体外对冠状病毒(HCoV-229E)感染Huh-7细胞致细胞病变具有较强的抑制作用(选择指数SI为6.80);体外能抑制冠状病毒(HCoV-229E)诱导的炎症因子IL-6、IL-8、MCP-1和TNF-α的mRNA过度表达,具剂量依赖关系。
证明,采用中药组合物制成的治疗冠状病毒(HCoV-229E)的口服液能对冠状病毒(HCoV-229E)感染导致的细胞病变具有较强的抑制作用,还能缓解因冠状病毒(HCoV-229E)感染而引发的炎症,缓解病患痛楚,使病患能在较为舒适的过程中快速治愈病症,以较快达到痊愈的效果,同时也减少了因病症严重引发并发症或其他感染的情况,治愈效果较佳。
本具体实施例仅仅是对本申请的解释,其并不是对本申请的限制,本领域技术人员在阅读完本说明书后可以根据需要对本实施例做出没有创造性贡献的修改,但只要在本申请的权利要求范围内都受到专利法的保护。
Claims (4)
1.一种中药组合物在制备治疗冠状病毒的药物上的应用,其特征在于:所述中药组合物由以下质量份数的组分制成:
芦根500-600份;
冬瓜子380-460份;
滑石380-460份;
薏苡仁300-380份;
黄芩250-320份;
青蒿250-320份;
射干250-320份;
枇杷叶250-320份;
法半夏180-250份;
淡豆豉180-250份;
苦杏仁180-250份;
葶苈子180-250份;
郁金180-250份;
所述冠状病毒为冠状病毒HCoV-229E。
2.根据权利要求1所述的一种中药组合物在制备治疗冠状病毒的药物上的应用,其特征在于:所述中药组合物由以下质量份数的组分制成:
芦根563份;
冬瓜子422份;
滑石422份;
薏苡仁340份;
黄芩282份;
青蒿282份;
射干282份;
枇杷叶282份;
法半夏225份;
淡豆豉225份;
苦杏仁225份;
葶苈子225份;
郁金225份。
3.根据权利要求1或2所述的一种中药组合物在制备治疗冠状病毒的药物上的应用,其特征在于:所述冬瓜子为炒冬瓜子、黄芩为酒制黄芩、枇杷叶为蜜炙枇杷叶、葶苈子为炒葶苈子。
4.根据权利要求3所述的一种中药组合物在制备治疗冠状病毒的药物上的应用,其特征在于:所述中药组合物制备的治疗冠状病毒的药物为片剂、胶囊剂、丸剂、颗粒剂、口服液。
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