CN105770200A - 一种黄连上清片的制备方法及应用 - Google Patents

一种黄连上清片的制备方法及应用 Download PDF

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CN105770200A
CN105770200A CN201610116335.9A CN201610116335A CN105770200A CN 105770200 A CN105770200 A CN 105770200A CN 201610116335 A CN201610116335 A CN 201610116335A CN 105770200 A CN105770200 A CN 105770200A
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preparation
supercritical extract
huanglianshangqing
root
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赵明亮
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Nanjing Zhengliang Pharmaceutical Technology Co Ltd
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Abstract

本发明属于中药制剂领域，具体提供一种黄连上清片的制备方法，由黄连5g、连翘40g、防风20g、白芷40g、菊花80g、大黄160g、桔梗40g、石膏20g、甘草20g、栀子40g、炒蔓荆子40g、荆芥穗40g、黄芩40g、薄荷20g、黄柏20g、川芎20g、旋覆花10g作为原料药制成，采用超临界萃取制备而成，使得含量有很大提高，用量减少，本发明还提供了黄连上清片在制备抑制小鼠巨噬细胞瘤RAW‑26407增殖药物中的应用。

Description

一种黄连上清片的制备方法及应用
技术领域
本发明涉及中药制剂技术领域，具体涉及一种黄连上清片的制备方法及应用。
背景技术
黄连上清片记载于药典标准，处方为黄连5g、连翘40g、防风20g、白芷40g、菊花80g、大黄160g、桔梗40g、石膏20g、甘草20g、栀子40g、炒蔓荆子40g、荆芥穗40g、黄芩40g、薄荷20g、黄柏20g、川芎20g、旋覆花10g，以上十七味，大黄、白芷、黄连、石膏粉碎成粉；连翘、荆芥穗、薄荷提取挥发油，药渣加水煎煮二次，每次小时，滤过，合并滤液并浓缩成清膏；其余旋覆花等十味用70％乙醇加热回流2小时，滤过，滤液回收乙醇并浓缩成清膏，药渣再加水煎煮二次，每次1小时，滤过，合并滤液并浓缩成清膏；合并三种清膏，浓缩至适量，与大黄等粉末混匀；或清膏喷雾干燥后，与大黄等粉末混匀；加入适量辅料，制成颗粒，干燥，喷入连翘等挥发油，混匀，压制成1000片，包糖衣或薄膜衣，即得。散风清热，泻火止痛。用于风热上攻、肺胃热盛所致的头晕目眩、暴发火眼、牙齿疼痛、口舌生疮、咽喉肿痛、耳痛耳鸣、大便秘结、小便短赤。口服，一次6片，一日3次，每粒装0.3g。
现有技术中，尚未有黄连上清片在提取制备方面采用超临界技术的报道，而采用打粉等方法，工艺粗糙、复杂、落后，杂质多，导致患者用量过大，不方便服用，严重影响了本品在临床上应用。
发明内容
发明目的：为了解决上述问题，本发明的目的在于提供一种黄连上清片的制备方法。
本发明的另一个目的在于提供一种黄连上清片在制备抑制小鼠巨噬细胞瘤RAW-26407增殖药物中的应用。
技术方案：本发明的目的是通过如下的方案实现的：
一种黄连上清片的制备方法，由黄连5g、连翘40g、防风20g、白芷40g、菊花80g、大黄160g、桔梗40g、石膏20g、甘草20g、栀子40g、炒蔓荆子40g、荆芥穗40g、黄芩40g、薄荷20g、黄柏20g、川芎20g、旋覆花10g作为原料药制成，所述的方法由下列步骤组成：取上述药材加入到CO2超临界萃取器中，乙醇作为夹带剂，夹带剂占总萃取溶剂的体积百分比为4-6％，萃取压力15-30MPa，温度30-50℃，CO2流量1-3m1/g生药·min，萃取时间150-180min，得超临界萃取物，制粒，压片，每片重0.3g。
所述黄连上清片的制备方法，所述CO2超临界萃取夹带剂占总萃取溶剂的体积百分比为5％。
所述黄连上清片的制备方法，所述CO2超临界萃取的萃取压力20MPa，温度40℃，CO2流量2ml/g生药·min，萃取时间160min。
所述黄连上清片在制备抑制小鼠巨噬细胞瘤RAW-26407增殖药物中的应用，黄连上清片由黄连5g、连翘40g、防风20g、白芷40g、菊花80g、大黄160g、桔梗40g、石膏20g、甘草20g、栀子40g、炒蔓荆子40g、荆芥穗40g、黄芩40g、薄荷20g、黄柏20g、川芎20g、旋覆花10g作为原料药制成，制备方法由下列步骤组成：取上述药材加入到CO2超临界萃取器中，乙醇作为夹带剂，夹带剂占总萃取溶剂的体积百分比为4-6％，萃取压力15-30MPa，温度30-50℃，CO2流量1-3m1/g生药·min，萃取时间150-180min，得超临界萃取物，粉碎成细粉，制粒，压片，每片重0.3g。
所述黄连上清片在制备抑制小鼠巨噬细胞瘤RAW-26407增殖药物中的应用，其特征在于黄连上清片的制备方法中所述CO2超临界萃取夹带剂占总萃取溶剂的体积百分比为5％。
所述黄连上清片在制备抑制小鼠巨噬细胞瘤RAW-26407增殖药物中的应用，其特征在于黄连上清片的制备方法中所述CO2超临界萃取的萃取压力20MPa，温度40℃，CO2流量2ml/g生药·min，萃取时间160min。
现有技术中，黄连上清片一次3片，一日3次，每片0.3g，采用本发明制备成的黄连上清片每片0.3g，但含有的药材量比原来多，在同样服用量的情况下具有更多活性成分。
具体实施方式
以下通过实施例形式，对本发明的上述内容再作进一步的详细说明，但不应将此理解为本发明上述主题的范围仅限于以下的实例，凡基于本发明上述内容所实现的技术均属于本发明的范围。
实施例1：取黄连5g、连翘40g、防风20g、白芷40g、菊花80g、大黄160g、桔梗40g、石膏20g、甘草20g、栀子40g、炒蔓荆子40g、荆芥穗40g、黄芩40g、薄荷20g、黄柏20g、川芎20g、旋覆花10g，加入到CO2超临界萃取器中，乙醇作为夹带剂，夹带剂占总萃取溶剂的体积百分比为4％，萃取压力30MPa，温度30℃，CO2流量1m1/g生药·min，萃取时间180min，得超临界萃取物，粉碎成细粉，制粒，压片，每片重0.3g。
实施例2：取黄连5g、连翘40g、防风20g、白芷40g、菊花80g、大黄160g、桔梗40g、石膏20g、甘草20g、栀子40g、炒蔓荆子40g、荆芥穗40g、黄芩40g、薄荷20g、黄柏20g、川芎20g、旋覆花10g，加入到CO2超临界萃取器中，乙醇作为夹带剂，夹带剂占总萃取溶剂的体积百分比为6％，萃取压力15MPa，温度50℃，CO2流量1m1/g生药·min，萃取时间180min，得超临界萃取物，粉碎成细粉，制粒，压片，每片重0.3g。
实施例3：取黄连5g、连翘40g、防风20g、白芷40g、菊花80g、大黄160g、桔梗40g、石膏20g、甘草20g、栀子40g、炒蔓荆子40g、荆芥穗40g、黄芩40g、薄荷20g、黄柏20g、川芎20g、旋覆花10g，加入到CO2超临界萃取器中，乙醇作为夹带剂，夹带剂占总萃取溶剂的体积百分比为5％，萃取压力20MPa，温度40℃，CO2流量2m1/g生药·min，萃取时间160min，得超临界萃取物，粉碎成细粉，制粒，压片，每片重0.3g。
实施例4：黄连上清片抑制小鼠巨噬细胞瘤RAW-26407增殖的实验研究资料
1实验材料
1.1实验用细胞株：小鼠巨噬细胞瘤RAW-26407，南京医科大学实验室细胞库，DMEM+10％FBS常规培养。
1.2实验药物：研究药物：本发明黄连上清片：按实施例3方法制备。药液储液：称取100mg黄连上清片内容物，混悬于5ml无水乙醇中，0.2μm滤器过滤，500μldoff管分装，-20℃存储，同时0.2μm滤器过滤无水乙醇以备对照组之用。
1.3实验试剂：DMEM(GIBCO公司Cat.No.12100-061Lot.No.758137)；胎牛血清(浙江天杭生物科技有限公司Lot.No.100419)；NaHCO3(上海久亿化学试剂有限公司Cat.No.11810-033Lot.No.1088387)；Trypsin(AMRESCO公司批号：2010/04)；EDTA(AMRESCO公司批号：2009/10)；PenicillinGSodiumSalt(AMRESCO公司批号：2010242)；StreptomycinSulfate(AMRESCO公司批号：2010382)；无水乙醇(南京化学试剂有限公司批号：080310182)；MTT(Biosharp批号：0793)；PBS(实验室自配)；
1.4实验器材：莱卡倒置显微镜(德国Leica型号：DM1L)；可见-紫外光微孔板检测仪(美国MD公司型号：SPECTRAMAX190)；CO2培养箱(FORMA型号：3111)；超净台(苏净集团安泰公司制造型号：SW-CJ-ZFD)；纯水仪(美国Spring公司型号：S/N020579)；精密移液器(法国吉尔森公司型号：P2)；电子天平(德国赛多利斯有限公司型号：BT323S)；全自动高压灭菌锅(日本SANYO公司型号：MLS-3020)；台式电热鼓风干燥箱(上海精密实验设备公司型号：DHG9123A)；冰箱(西门子公司型号：KG18V21TI)；液氮罐(CBS型号：2001)；低速离心机(上海安亭科学仪器厂型号：KA-1000)；0.2μm滤器(MILLIPORE型号：SLGP033RB)；10cm培养皿(NEST公司)、96孔培养板(NEST公司)；细胞计数板；离心管、移液管、Tips若干。
2实验方法：1)小鼠巨噬细胞瘤RAW-26407用DMEM+10％FBS于37℃、5％CO2进行常规培养(10cm培养皿)，当细胞生长至对数期时，收集细胞，弃去培养液，PBS轻洗3遍，加入3ml0.25％胰蛋白酶-0.04％EDTA，37℃消化2min后，向其中加入5ml完全培养基中和反应，吹打细胞后将其转入离心管中，1000rpm离心5min，调整细胞悬液浓度3×104个/ml。2)将细胞种入96孔培养板中，每孔加入细胞悬液180μl，培养板放入细胞培养箱中(37℃，5％CO2)常规培养。3)根据细胞生长情况，一般长至50％-70％，加入黄连上清片溶液，继续培养24h。4)24h后加入20μlMTT溶液(5mg/ml，即0.5％MTT)，继续培养4h。5)4h后扣板法倒去上清，用吸水纸轻轻拍干，每孔加入200μl二甲基亚砜，置摇床上低速振荡10min，使结晶物充分溶解。在酶联免疫检测仪490nm处测量各孔的吸光值。6)同时设置本底(不加细胞，只加培养液)，对照孔(细胞、相同浓度的药物溶解介质、培养液、MTT、二甲基亚砜)，每组设定6个复孔。7)结果以药物对细胞的抑制率表示：细胞增值抑制率(％)＝(对照孔OD值-给药孔OD值)/对照孔OD值×100％。实验重复3次。
3实验结果：MTT法实验后统计结果显示，与对照组比较，当剂量达到5mg/ml时，对小鼠巨噬细胞瘤RAW-26407增殖抑制有差异(P<0.05)，剂量在10mg/ml时该差异具有显著性(P<0.01)，当剂量达到15-20mg/ml时有极显著性差异(P<0.001)。
表1黄连上清片对小鼠巨噬细胞瘤RAW-26407增殖抑制影响研究
注：与对照组比较，*P<0.01；**P<0.001
4实验结论：黄连上清片可以抑制小鼠巨噬细胞瘤RAW-26407增殖，减少小鼠巨噬细胞瘤RAW-26407的细胞生长数目，该作用呈剂量依赖性。

Claims (6)

1.一种黄连上清片的制备方法，由黄连5g、连翘40g、防风20g、白芷40g、菊花80g、大黄160g、桔梗40g、石膏20g、甘草20g、栀子40g、炒蔓荆子40g、荆芥穗40g、黄芩40g、薄荷20g、黄柏20g、川芎20g、旋覆花10g作为原料药制成，其特征在于所述的方法由下列步骤组成：取上述药材加入到CO2超临界萃取器中，乙醇作为夹带剂，夹带剂占总萃取溶剂的体积百分比为4-6％，萃取压力15-30MPa，温度30-50℃，CO2流量1-3m1/g生药·min，萃取时间150-180min，得超临界萃取物，粉碎成细粉，制粒，压片，每片重0.3g。
2.根据权利要求1所述黄连上清片的制备方法，其特征在于所述CO2超临界萃取夹带剂占总萃取溶剂的体积百分比为5％。
3.根据权利要求1所述黄连上清片的制备方法，其特征在于所述CO2超临界萃取的萃取压力20MPa，温度40℃，CO2流量2ml/g生药·min，萃取时间160min。
4.根据权利要求1所述黄连上清片在制备抑制小鼠巨噬细胞瘤RAW-26407增殖药物中的应用，其特征在于黄连上清片由黄连5g、连翘40g、防风20g、白芷40g、菊花80g、大黄160g、桔梗40g、石膏20g、甘草20g、栀子40g、炒蔓荆子40g、荆芥穗40g、黄芩40g、薄荷20g、黄柏20g、川芎20g、旋覆花10g作为原料药制成，制备方法由下列步骤组成：取上述药材加入到CO2超临界萃取器中，乙醇作为夹带剂，夹带剂占总萃取溶剂的体积百分比为4-6％，萃取压力15-30MPa，温度30-50℃，CO2流量1-3m1/g生药·min，萃取时间150-180min，得超临界萃取物，粉碎成细粉，制粒，压片，每片重0.3g。
5.根据权利要求4所述黄连上清片在制备抑制小鼠巨噬细胞瘤RAW-26407增殖药物中的应用，其特征在于黄连上清片的制备方法中所述CO2超临界萃取夹带剂占总萃取溶剂的体积百分比为5％。
6.根据权利要求4所述黄连上清片在制备抑制小鼠巨噬细胞瘤RAW-26407增殖药物中的应用，其特征在于黄连上清片的制备方法中所述CO2超临界萃取的萃取压力20MPa，温度40℃，CO2流量2ml/g生药·min，萃取时间160min。
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