CN104306826A - 一种黄萱益肝片的制备方法及其在制备抑制白血病细胞l6565细胞增殖药物中的应用 - Google Patents
一种黄萱益肝片的制备方法及其在制备抑制白血病细胞l6565细胞增殖药物中的应用 Download PDFInfo
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Abstract
本发明属于中药技术领域,具体涉及一种黄萱益肝片的制备方法及应用。本发明的黄萱益肝片的制备方法,由土大黄291g、萱草103g、千里光92g、猕猴桃92g、红土茯苓92g、野蔷薇62g、獐芽菜62g、骚羊古62g、南五味子62g作为原料药制成,采用超临界萃取、微波萃取和大孔树脂吸附制备而成,使得大黄素含量有很大提高。本发明还提供了黄萱益肝片在制备抑制小鼠白血病细胞L6565细胞增殖药物中的应用。
Description
技术领域
本发明属于中药技术领域,具体涉及一种黄萱益肝片的制备方法及其在制备抑制小鼠白血病细胞L6565细胞增殖药物中的应用。
背景技术
黄萱益肝散标准号WS-10465(ZD-0465)-2002,记载于国家中成药标准汇编内科肝胆分册。由土大黄291g、萱草103g、千里光92g、猕猴桃92g、红土茯苓92g、野蔷薇62g、獐芽菜62g、骚羊古62g、南五味子62g作为原料药制成,具有清热解毒,疏肝利胆的功效。用于肝胆湿热所致的慢性乙型肝炎。
现有技术中,尚未有黄萱益肝散在提取制备方面采用CO2超临界萃取、微波技术和大孔树脂吸附技术提取的报道,而中药直接打粉取的方法,工艺粗糙、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
为了解决上述的技术问题,本发明提供了一种黄萱益肝片的制备方法。
本发明的另一个目的在于提供一种黄萱益肝片在制备抑制小鼠白血病细胞L6565 细胞增殖药物中的应用。
本发明的目的是通过如下的方案实现的:
一种黄萱益肝片的制备方法,由土大黄291g、萱草103g、千里光92g、猕猴桃92g、红土茯苓92g、野蔷薇62g、獐芽菜62g、骚羊古62g、南五味子62g作为原料药制成,所述的制备方法由下列步骤组成:取野蔷薇、骚羊古、南五味子,粉碎成细粉,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20-30MPa,温度30-50℃,CO2流量l-3ml/g生药·min,萃取时间130-170min,得超临界萃取物,备用;萃取后的野蔷薇、骚羊古、南五味子残渣与其他六味药材,粉碎,加入5L的60%乙醇,投入微波萃取装置中进行微波萃取,萃取功率600-800W,萃取2次,每次5-10分钟,合并萃取液,浓缩,加到D101大孔吸附树脂柱上,60%乙醇洗脱,收集5倍量柱体积洗脱液,减压回收乙醇,浓缩并干燥,得微波提取物,将超临界萃取物、微波提取物混合加入淀粉,60%乙醇制颗粒,干燥,压片,制成200片,每片重0.5g。
上述的黄萱益肝片的制备方法中,所述CO2超临界萃取中夹带剂占总萃取溶剂的体积百分比为5%。
上述的黄萱益肝片的制备方法中,所述CO2超临界萃取中萃取压力为25 MPa。
上述的黄萱益肝片的制备方法中,所述CO2超临界萃取中萃取温度为60℃。
上述的黄萱益肝片的制备方法中,所述CO2超临界萃取中CO2流量2ml/g生药·min。
上述的黄萱益肝片的制备方法中,所述CO2超临界萃取中萃取时间为150min。
上述的黄萱益肝片的制备方法中,所述微波萃取功率为700W。
上述的黄萱益肝片的制备方法中,所述微波萃取每次萃取时间为8分钟。
上述的黄萱益肝片在制备抑制小鼠白血病细胞L6565 细胞增殖药物中的应用。
现有技术中,黄萱益肝散口服,成人一次9g,一日3次。黄萱益肝散服药量大。采用本发明方法制备成的黄萱益肝片每片重0.5g,每次仅需2片,一日服用3次。在具有更多活性成分的条件下大大减少了服用量。此结论可以通过下述试验证明。
试验一、不同方法制备的黄萱益肝片中大黄素含量的比较
l、仪器及试药本发明黄萱益肝片:按实施例1方法制备,使用918g原料药,经提取制成200片,每片重0.5g。原黄萱益肝散,按照WS-10465(ZD-0465)-2002标准方法制备,作为对照。Agilent1200高效液相色谱仪; AE240电子分析天平;大黄素对照品(中国药品生物制品检定所)。
2、方法
照高效液相色谱法(中国药典2010年版一部附录Ⅵ D)测定。
色谱条件与系统适用性试验 用十八烷基硅烷键合硅胶为填充剂;甲醇-0.25%磷酸溶液(80∶20)为流动相;检测波长为436nm。理论板数按大黄素峰计算应不低于3000。
对照品溶液的制备 取经五氧化二磷干燥至恒重的大黄素对照品适量,精密称定,加甲醇制成每1ml含0.05mg的溶液,即得。
本发明产品供试品溶液的制备 取本发明的黄萱益肝片,研细,取0.4g,精密称定,置索氏提取器中,加乙醇适量,加热回流提取至无色,提取液浓缩至约5ml,放冷,加入稀盐酸5ml,氯仿30ml,加热回流30分钟,放冷,分取氯仿液,水液用氯仿振摇提取2次(20ml、15ml),合并氯仿液,蒸干,残渣加甲醇使溶解并移至10ml量瓶中,加甲醇稀释至刻度,摇匀,离心,取上清液,即得。
对照品供试品溶液的制备 取本对照的黄萱益肝散,研细,混匀,取2g,精密称定,置索氏提取器中,加乙醇适量,加热回流提取至无色,提取液浓缩至约5ml,放冷,加入稀盐酸5ml,氯仿30ml,加热回流30分钟,放冷,分取氯仿液,水液用氯仿振摇提取2次(20ml、15ml),合并氯仿液,蒸干,残渣加甲醇使溶解并移至10ml量瓶中,加甲醇稀释至刻度,摇匀,离心,取上清液,即得。
测定法 分别精密吸取对照品溶液、本发明产品供试品溶液与对照品供试品溶液各10μl,注入液相色谱仪,测定,即得。
3、结果
结果表明,本发明黄萱益肝片中大黄素的含量为2-4mg/片;而原黄萱益肝散中大黄素的含量为0.21mg/粒,每片大黄素含量相当于散剂每g散剂含量的10-20倍,在服用量减少的情况下,大黄素含量有很大提高。
上述研究表明,采用本发明制备方法制备的黄萱益肝片,有效成分含量远远高于WS-10465(ZD-0465)-2002标准记载的方法制备的黄萱益肝散。
具体实施方式
下面结合具体实施例对本发明作更进一步的说明,以便本领域的技术人员更了解本发明,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
取土大黄291g、萱草103g、千里光92g、猕猴桃92g、红土茯苓92g、野蔷薇62g、獐芽菜62g、骚羊古62g、南五味子62g:取野蔷薇、骚羊古、南五味子,粉碎成细粉,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25MPa,温度40℃,CO2流量2ml/g生药·min,萃取时间150min,得超临界萃取物,备用;萃取后的野蔷薇、骚羊古、南五味子残渣与其他六味药材,粉碎,加入5L的60%乙醇,投入微波萃取装置中进行微波萃取,萃取功率700W,萃取2次,每次8分钟,合并萃取液,浓缩,加到D101大孔吸附树脂柱上,60%乙醇洗脱,收集5倍量柱体积洗脱液,减压回收乙醇,浓缩并干燥,得微波提取物,将超临界萃取物、微波提取物混合加入淀粉,60%乙醇制颗粒,干燥,压片,制成200片,每片重0.5g。
经检测,成品中大黄素的含量为4.02mg/片。
实施例2
取土大黄291g、萱草103g、千里光92g、猕猴桃92g、红土茯苓92g、野蔷薇62g、獐芽菜62g、骚羊古62g、南五味子62g:取野蔷薇、骚羊古、南五味子,粉碎成细粉,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50℃,CO2流量3ml/g生药·min,萃取时间130min,得超临界萃取物,备用;萃取后的野蔷薇、骚羊古、南五味子残渣与其他六味药材,粉碎,加入5L的60%乙醇,投入微波萃取装置中进行微波萃取,萃取功率600W,萃取2次,每次8分钟,合并萃取液,浓缩,加到D101大孔吸附树脂柱上,60%乙醇洗脱,收集5倍量柱体积洗脱液,减压回收乙醇,浓缩并干燥,得微波提取物,将超临界萃取物、微波提取物混合加入淀粉,60%乙醇制颗粒,干燥,压片,制成200片,每片重0.5g。
经检测,成品中大黄素的含量为2.03mg/片。
实施例3
取土大黄291g、萱草103g、千里光92g、猕猴桃92g、红土茯苓92g、野蔷薇62g、獐芽菜62g、骚羊古62g、南五味子62g:取野蔷薇、骚羊古、南五味子,粉碎成细粉,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20MPa,温度30℃,CO2流量1ml/g生药·min,萃取时间170min,得超临界萃取物,备用;萃取后的野蔷薇、骚羊古、南五味子残渣与其他六味药材,粉碎,加入5L的60%乙醇,投入微波萃取装置中进行微波萃取,萃取功率800W,萃取2次,每次10分钟,合并萃取液,浓缩,加到D101大孔吸附树脂柱上,60%乙醇洗脱,收集5倍量柱体积洗脱液,减压回收乙醇,浓缩并干燥,得微波提取物,将超临界萃取物、微波提取物混合加入淀粉,60%乙醇制颗粒,干燥,压片,制成200片,每片重0.5g。
经检测,成品中大黄素的含量为3.17mg/片。
实施例4:黄萱益肝片抑制小鼠白血病细胞L6565 细胞增殖的实验研究资料
1.实验材料
1.1实验用细胞株
小鼠白血病细胞L6565 细胞,山东大学实验室细胞库,DMEM+10%FBS常规培养。
1.2实验药物
研究药物:本发明黄萱益肝片:按实施例1方法制备。
药液储液:称取100mg黄萱益肝片,溶于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.12100-061 Lot.No.758137);胎牛血清(浙江天杭生物科技有限公司Lot.No.100419);NaHC03(上海久亿化学试剂有限公司Cat.No.11810-033 Lot.No. 1088387);Trypsin(AMRESCO公司);EDTA(AMRESCO公司);Penicillin G Sodium Salt(AMRESCO公司1);Streptomycin Sulfate(AMRESCO);无水乙醇(淄博亚杜兰经贸有限公司);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:DMIL);可见-紫外光微孔板检测仪(美国MD公司型号:SPECTRAMAX 190);C02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Sprlng公司型号:S/N 020579);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);1cm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)L6565 细胞用DMEM+10%FBS于37℃、5%C02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3ml 0.25%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%C02)常规培养。
3)根据细胞生长情况,一般长至50%-70%,加入黄萱益肝片溶液,继续培养24h。
4)24h后加入20μl MTT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,每孔如入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.统计处理
采用Microsoft Excel 2007软件中的相关分析和Student t检验,数据以mean±S.D.表示。
4.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L6565 细胞增殖抑制有差异(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黄萱益肝片对L6565 细胞增殖抑制影响研究 (X±SD)
组别 | 药物浓度(mg/ml) | 抑制率(%) |
对照组 | 0 | 0 |
1 | 5 | 15.17±9.21 |
2 | 10 | 29.05±12.54* |
3 | 15 | 37.02±14.36** |
4 | 20 | 45.06±17.08** |
注:与对照组比较,*P<O.01;**P<0.001。
5.实验结论
黄萱益肝片可以抑制L6565 细胞增殖,减少L6565 细胞的细胞生长数目,该作用呈剂量依赖性。
Claims (9)
1.一种黄萱益肝片的制备方法,由土大黄291g、萱草103g、千里光92g、猕猴桃92g、红土茯苓92g、野蔷薇62g、獐芽菜62g、骚羊古62g、南五味子62g作为原料药制成,其特征在于,所述的制备方法由下列步骤组成:取野蔷薇、骚羊古、南五味子,粉碎成细粉,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20-30MPa,温度30-50℃,CO2流量l-3ml/g生药·min,萃取时间130-170min,得超临界萃取物,备用;萃取后的野蔷薇、骚羊古、南五味子残渣与其他六味药材,粉碎,加入5L的60%乙醇,投入微波萃取装置中进行微波萃取,萃取功率600-800W,萃取2次,每次5-10分钟,合并萃取液,浓缩,加到D101大孔吸附树脂柱上,60%乙醇洗脱,收集5倍量柱体积洗脱液,减压回收乙醇,浓缩并干燥,得微波提取物,将超临界萃取物、微波提取物混合加入淀粉,60%乙醇制颗粒,干燥,压片,制成200片,每片重0.5g。
2.根据权利要求l所述的黄萱益肝片的制备方法,其特征在于,所述CO2超临界萃取中夹带剂占总萃取溶剂的体积百分比为5%。
3.根据权利要求l所述的黄萱益肝片的制备方法,其特征在于,所述CO2超临界萃取中萃取压力为25MPa。
4.根据权利要求l所述的黄萱益肝片的制备方法,其特征在于,所述CO2超临界萃取中萃取温度为60℃。
5.根据权利要求l所述的黄萱益肝片的制备方法,其特征在于,所述CO2超临界萃取中CO2流量2ml/g生药·min。
6.据权利要求l所述的黄萱益肝片的制备方法,其特征在于,所述CO2超临界萃取中萃取时间为150min。
7.根据权利要求l所述的黄萱益肝片的制备方法,其特征在于,所述微波萃取功率为700W。
8.根据权利要求l所述的黄萱益肝片的制备方法,其特征在于,所述微波萃取每次萃取时间为8分钟。
9.权利要求l所述的黄萱益肝片在制备抑制小鼠白血病细胞L6565 细胞增殖药物中的应用。
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