CN110772555B - 黄褐毛忍冬不同溶剂提取物在制药中的应用及其制备方法 - Google Patents
黄褐毛忍冬不同溶剂提取物在制药中的应用及其制备方法 Download PDFInfo
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Abstract
本发明公开了黄褐毛忍冬不同溶剂提取物在制药中的应用及其制备方法，本发明发现黄褐毛忍冬水提取物、30％醇提取物、60％醇提取物和95％醇提取物对丙型肝炎病毒NS3/4A蛋白酶具有较强的抑制作用，可用于制备治疗丙型肝炎的药物。
Description
技术领域
本发明涉及中药技术领域，特别是涉及黄褐毛忍冬药材不同溶剂提取物在制备治疗丙型肝炎药物中的应用及不同溶剂提取物的制备方法。
技术背景
人体感染丙型肝炎病毒所致的丙型肝炎，已成为全球最棘手的健康问题,目前全世界已有1.7亿人感染。若无药物的治疗,长期感染丙型肝炎病毒将发展成为慢性肝炎、肝坏死、肝癌等疾病。当前最有效的治疗方法是聚乙二醇干扰素联合利津巴韦，该方法对于广大发展中国家普遍存在的I型丙型肝炎治疗有效率低于50％,长期使用干扰素存在许多副作用，如：白细胞、血小板、嗜中性白血球的减少和抑郁症等；长期使用利津巴韦，也将产生溶血性贫血等副作用，使得患者不能持续治疗，因此发展独特、有效、更安全的丙型肝炎治疗药物尤为紧迫。
丙型肝炎病毒蛋白酶NS3/4A在病毒圆熟过程中起着重要的作用，已被证实是发现丙型肝炎药物的有效靶点之一。目前三种丙型肝炎NS3/4A蛋白酶抑制剂，BILN 2061,VX-950和SCH 503034，已被临床证实疗效显著，但多为肽类抑制剂，合成困难，并有一定的毒副作用。
黄褐毛忍冬系忍冬科植物(Lonicera fulvotomentosa Hsu et S.C.Cheng)的干燥花蕾或带初开的花，具有清热解毒，疏散风热的功效，主治温病发热、热毒血痢、痈肿疔疮、丹毒、热毒血痢、风热感冒、温病发热等多种感染性疾病。该植物主要分布于中国广西西北部、贵州西南部和云南。生长在海拔850-1300米的山坡岩旁灌木林或林中。
发明内容
本发明所要解决的技术问题是克服现有治疗丙型肝炎的药物合成困难、副作用多的缺陷，提供有抑制丙型肝炎蛋白酶作用的黄褐毛忍冬药材不同溶剂提取物的制备方法及该提取物在制药上的新用途。
为了解决上述技术问题，本发明采用如下的技术方案：
本发明提供黄褐毛忍冬不同溶剂提取物在制备治疗丙型肝炎药物中的应用。
其中，前述黄褐毛忍冬为忍冬科植物Lonicera fulvotomentosa Hsu etS.C.Cheng干燥花蕾或带初开的花原药材。
进一步的，前述的不同溶剂包括水、甲醇、乙醇、丙酮、正丁醇、乙酸乙酯、氯仿、二氯甲烷中的一种或两种以上混合溶剂。
本发明还提供了黄褐毛忍冬不同极性溶剂提取物的制备方法，包括如下步骤：黄褐毛忍冬植物花蕾，粉碎，取25克药材，分别加200毫升水、30％甲醇、60％甲醇、95％甲醇，超声2分钟后，加热回流提取3次，第一次回流提取2小时，第二、三次回流1小时，合并提取液，减压浓缩成浸膏，真空干燥，得黄褐毛忍冬水提取、30％醇提物、60％醇提物和95％的醇提物
本发明对黄褐毛忍冬不同极性溶剂提取物的体外抗丙型肝炎活性进行了测试，具体如下；
测试原理：荧光法
检测仪器：Synergy II
试剂盒:
520HCV蛋白酶试剂盒(批号：AS71145-1035，美国圣何塞AnaSpec。)
蛋白酶：HCV NS3/4A蛋白酶(批号：AS-61017-10)，美国圣何塞AnaSpec)
Embelin(Sigma-Aldrich,Lot#:022M4726V)
测试溶液的配制(按试剂盒说明手册操作)：
缓冲溶液：将2X实验缓冲液(Component D)、1M DDT(Component F)和去离子水按1：0.06：1进行混合，冰浴备用。
HCV NS3/4A蛋白酶底物溶解：加0.12ml的DMSO(Component C)到HCV NS3/4A蛋白酶底物[Ac-Asp-Glu-Dap(QXLTM520)-Glu-Glu-Abu-COO-Ala-Ser-Cys(5-FAMsp)-NH2](Component A),作为储备液-20℃冻存。每次实验前取储备液，用缓冲溶液按1：100比例稀释，现配现用。
HCV NS3/4A蛋白酶溶液：用缓冲溶液将蛋白酶溶液稀释20或60倍。测定方法(按试剂盒说明手册操作)：在384孔黑色平板(美国Corning公司)中，先分别加入底物空白溶液(Substrate Control，SC)、溶剂空白溶液(Vehicle Control，VC)、阳性对照(PositiveControl,PC)、抑制剂对照(Inhibitor Control,IC)、测试化合物对照(Test CompoundControl,TCC),再加入HCVNS3/4A蛋白酶和底物。每个浓度均为3个复孔，震荡混匀后，37度孵育30分钟,经Synergy II酶标仪在激发波长485nm和发射波长535nm下测试荧光强度。实验结果见表1。抑制率计算公式：
抑制率％＝100×(FAverageVC-AverageSC-FAveragesample-AverageSC)/FAverageVC-AverageSC
式中的F为对照组和样品组荧光值，抑制率及IC50的计算通过微软Windows Excel软件完成。
表1黄褐毛忍冬不同溶剂提取物对HCV NS3/4A蛋白酶的抑制作用(n＝3)
阳性对照(PC)：Emberlin。
数据为：平均值±相对标准偏差％(三次独立实验值)。
目前离体实验认为，单体化合物的IC50在10μg/ml以下，就具有较强的抗HCV NS3/4A蛋白酶作用。对黄褐毛忍冬不同极性溶剂提取物的HCVNS3/4A蛋白酶测试结果显示：黄褐毛忍冬60％甲醇提取物和95％甲醇提取对HCVNS3/4A蛋白酶抑制率分别达4.0μg/mL和9.0μg/mL，对HCV NS3/4A蛋白酶有较好的抑制作用，可用于研制预防和治疗丙型肝炎药物。
与现有技术相比，本发明发现黄褐毛忍冬水提取物、30％醇提取物60％醇提取物和95％醇提取物对丙型肝炎病毒NS3/4A蛋白酶具有较强的抑制作用，可用于制备治疗丙型肝炎的药物。
具体实施方式
下面结合实施例对本发明作进一步说明，但不作为对本发明的任何限制。
实施例1：黄褐毛忍冬药材于2016年3月采自贵州兴义，由贵阳中医学院陈德媛教授(生药学专家)鉴定为黄褐毛忍冬(Lonicera fulvotomentosa Hsu et S.C.Cheng)，植物标本存放在贵阳中医学院药学系中药化学实验室(标本号LF201607)，采用如下方法提取：
取黄褐毛忍冬干燥花蕾或带初开的花25克，粉碎，用水、30％甲醇、60％甲醇和95％甲醇(料液比：1：8)加热回流提取3次，合并提取液，分别减压浓缩，得水提物浸膏，30％醇提物浸膏，60％醇提物浸膏，95％醇提物浸膏，这四种浸膏即为对丙型肝炎病毒有抑制作用的黄褐毛忍冬提取物。
实施例2：取水提物浸膏，干燥，粉碎，加入辅料，制成中药制剂。例如片剂、颗粒剂等。
实施例3：取30％醇浸膏，干燥，粉碎，加入辅料，制成中药制剂。例如片剂、颗粒剂等。
实施例4：取60％醇浸膏，干燥，粉碎，加入辅料，制成中药制剂。例如片剂、颗粒剂等。
实施例5：取95％醇浸膏，干燥，粉碎，加入辅料，制成中药制剂。例如片剂、颗粒剂等。
实施例6：取各种溶剂浸膏与其他药物组合物的混合物，干燥，粉碎，加入药用辅料，制成药物制剂。
实施例7：取黄褐毛忍冬干燥花蕾或带初开的花，粉碎，装入胶囊，制成胶囊剂。
以上只是本发明的具体应用范例，本发明还有其他的实施方式，凡采用等同替换或等效变换形成的技术方案，均落在本发明所要求的保护范围之内。
Claims (3)
1.黄褐毛忍冬60%甲醇提取物作为唯一活性成分在制备治疗丙型肝炎药物中的应用。
2.根据权利要求1所述的应用，其特征在于：所述黄褐毛忍冬为忍冬科植物Lonicera fulvotomentosa Hsu et S. C. Cheng干燥花蕾或带初开的花原药材。
3.根据权利要求1所述的应用，其特征在于：所述黄褐毛忍冬60%甲醇提取物的制备方法包括如下步骤：黄褐毛忍冬植物花蕾，粉碎，取25克粉碎后药材，加60%甲醇，超声2分钟后，加热回流提取3次，第一次回流提取2小时，第二、三次各回流提取1小时，合并提取液，减压浓缩成浸膏，真空干燥，得黄褐毛忍冬60%甲醇提取物。
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