CN107158005A - 救必应酸在制备抗流感病毒药物中的应用 - Google Patents
救必应酸在制备抗流感病毒药物中的应用 Download PDFInfo
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Abstract
本发明首次公开救必应酸在制备抗流感病毒药物中的应用。并进一步公开救必应酸在制备治疗由流感病毒引起的肺炎药物中的应用。所述流感病毒优选甲型H1N1流感病毒。经体外细胞实验，证明救必应酸抗H1N1流感病毒的IC50值为5.8μg/mL，与阳性药利巴韦林作用相当。小鼠流感模型药理实验证实，救必应酸可显著降低流感感染小鼠的肺指数，并且对H1N1呈现一定的量效特征。
Description
技术领域
本发明公开了救必应酸的新用途，具体为救必应酸在制备抗流感病毒药物中的应用。
背景技术
流行性感冒病毒(简称流感病毒)可引起急性呼吸道感染，会对人类的健康造成威胁。其中，甲型流感病毒由于抗原性易发生变异，由其引发的甲型H1N1流感具有流行面广、传染性强、发病率高等特点。目前，应对流感病毒主要是通过疫苗和药物治疗。其中，M2离子通道抑制剂金刚烷胺类以及神经氨酸酶抑制剂是许多国家在临床上常用的抗流感病毒药物，但是这两类抗病毒药物现在均出现不同程度的耐药性，且有一定的毒副作用。因此，寻找新的、高效低毒的抗流感病毒药物显得十分重要和紧迫。
救必应酸(分子式为C30H48O5)，可提取自冬青科冬青属植物铁冬青Ilex rotundaThunb.的干燥树皮，其原料中药救必应来源丰富，低廉，显示出救必应酸良好的药物应用前景。
救必应酸属五环三萜化合物，具有五环三萜母核。五环三萜类化合物的结构通式为：
式中，R为COOH、COO―β―D―葡萄糖基或其他药学上可接受的基团。当式中的R为COOH时，化合物名为救必应酸(Rotundic acid)。
目前，专利文献CN101856357A公开了救必应酸具有防治心脑血管疾病，包括心绞痛、心肌梗死、脑梗死的作用。专利文献CN102391352A公开了救必应酸的氨基酸衍生物具有体外抗肿瘤活性。但尚未见关于救必应酸抗流感病毒作用的研究报道。
发明内容
本发明目的在于公开救必应酸的新药用用途，具体涉及救必应酸在制备抗流感病毒药物中的应用。包括H1N1流感病毒。
并进一步公开，救必应酸在制备治疗由流感病毒引起的肺炎药物中的应用。
与现有技术相比，本发明首次公开救必应酸在制备抗流感病毒药物中的新用途。经体外细胞实验，证明救必应酸抗H1N1流感病毒的IC50值为5.8μg/mL，与阳性药利巴韦林作用相当。小鼠流感模型药理实验证实，救必应酸可显著降低流感感染小鼠的肺指数，并且对H1N1呈现一定的量效特征。
具体实施方式
为更好理解本发明内容，下面结合具体实施例对本发明的内容作进一步说明，但本发明的保护内容不局限于以下实施例。
实施例1救必应药材提取物的制备
取救必应药材适量，粉碎，过1号筛，药材加8倍量70％(体积分数)乙醇，加热回流提取2次，每次1h，过滤，合并提取液，置旋转蒸发器中，在80℃下减压浓缩至稠膏状。将浓缩液转移置减压干燥箱中，在80℃下继续进行干燥、粉碎，即得救必应药材提取物。
实施例2体外流感病毒抑制实验
1.实验材料
1.1受试药物：救必应酸(从救必应中提取得到，纯度＞98％)。
1.2对照药物：利巴韦林，齐墩果酸，救必应药材提取物(见实施例1)
1.3病毒株：甲型H1N1流感病毒鼠肺适应株(FM/1/47株)。鸡胚传代，BSL-3(生物安全实验室三级)实验室内检测，分装，-80℃保存。
1.4细胞模型：狗肾细胞MDCK细胞
上述药物均经二甲基亚砜(DMSO)充分溶解后-20℃保存备用。
2.实验方法
2.1病毒毒力的测定
将MDCK细胞按5×104/mL浓度接种96孔培养板，每孔100μl，37℃、5％CO2培养，形成细胞单层铺满状态。将100μl10倍系列稀释得到的6个浓度的病毒液，依次接种到长满单层MDCK细胞层的96孔板中，同时设细胞对照。37℃、5％CO2病毒培养箱中培养，逐日用倒置显微镜观察细胞病变(CPE)，连续观察3天，用MTT法进行检测,在酶标仪570nm波长测定吸光度OD值，用Reed-Muench法计算病毒半数感染剂量(TCID50)，每浓度设8个复孔，整个实验重复3次，取平均值。
测定结果：MDCK细胞受到流感病毒攻击后，细胞会发生凋亡，在细胞形态学上主要表现为细胞圆缩，核固缩，碎裂等，在细胞周围形成典型的圆形凋小体。用Reed-Muench法计算结果得：病毒毒力TCID50为10-4.5。
2.2救必应酸细胞毒性测定
取MDCK细胞已长成单层的96孔细胞培养板，倾去培养液，用PBS洗涤2次，加入不含血清的DMEM培养基连续2倍系列稀释(488μg/mL-3.81μg/mL)的救必应酸药液，100μl/孔，每个浓度设4个复孔，设正常细胞对照4孔。37℃、5％CO2病毒培养箱中培养72h，用MTT法进行检测，在570nm波长下测出各实验组与细胞对照组的吸收度。以对照孔为参照，计算各实验孔细胞的活力比值。根据Reed-Muench法计算细胞毒性浓度CC50，整个实验重复3次，取平均值。
测定结果：救必应酸的细胞半数毒性浓度CC50>488μg/mL。
2.3救必应酸抗H1N1活性测试
取MDCK细胞已长成单层的96孔细胞培养板，倾去培养液，用PBS洗涤2次，加入100TCID50病毒液，100μl/孔，37℃、5％CO2吸附2h。加入不含血清的DMEM培养基连续2倍系列稀释(244μg/mL-3.81μg/mL)的救必应酸药液,同时设病毒对照组、细胞对照组、阳性药组、救必应药材提取物组和齐墩果酸组。在37℃、5％CO2培养箱中培养72h后，用MTT法进行检测，用酶标仪测570nm波长处的吸光度OD值。按下述公式计算药物对病毒的抑制率：病毒抑制率(％)＝(药物处理组OD均值-病毒对照组OD均值)/(细胞对照组OD均值-病毒对照组OD均值)×100％，根据Reed-Muench法计算药物对流感病毒致CPE产生50％抑制的浓度，即半数抑制浓度IC50，整个实验重复3次，取平均值。
实验结果：根据MTT结果，采用Reed-Muench法计算救必应酸的半数抑制浓度IC50为5.8μg/mL，齐墩果酸的半数抑制浓度IC50为37.5μg/mL，救必应药材提取物的半数抑制浓度IC50>244μg/mL。阳性药物利巴韦林的半数抑制浓度IC50为6.8μg/mL。
结论：救必应酸在对H1N1流感病毒具有较强的抑制作用，其IC50值与利巴韦林IC50值大小接近，且比齐墩果酸以及救必应药材提取物IC50值都要小，证明救必应酸对H1N1流感病毒的抑制作用与阳性对照药物利巴韦林相当，且强于齐墩果酸和救必应药材提取物。
实施例3体内流感抑制实验
1.实验材料
1.1受试药物：救必应酸(从救必应中提取得到，纯度＞98％)。
1.2对照药物：达菲，齐墩果酸，救必应药材提取物(见实施例1)
1.3病毒：甲型H1N1流感病毒鼠肺适应株(FM/1/47株)。鸡胚传代，BSL-3(生物安全实验室三级)实验室内检测，分装，-80℃保存。
2.实验动物
BALB/c小鼠体重18g-22g，80只，购自广东省实验动物中心。
3.实验方法
将BALB/c小鼠随机分为8组，分别为空白对照组、模型对照组、达菲对照组、齐墩果酸组、救必应药材提取物组、救必应酸高、中、低3个剂量组，每组10只。除空白对照组外，各实验组动物用乙醚轻度麻醉，以15个LD50的FM1流感病毒液滴鼻感染，每只35μL。感染前一天开始灌胃给药，每次按0.2ml/10g体重灌胃，每天1次，连续给药5天。空白组和模型对照组在同等条件下灌胃给予等体积蒸馏水。第6天称重后解剖，取肺，称重量，计算肺指数与肺指数抑制率。
计算公式：肺指数＝肺质量(g)/体重(g)×100％；肺指数抑制率＝(模型对照组平均肺指数－实验组平均肺指数)/模型对照组平均肺指数×100％数据处理与统计方法：肺指数用平均值±SD值表示，组间比较采用单因素方差分析；采用SPSS19.0软件处理数据。
4.实验结论
肺指数值越大，表示肺炎越严重。由表1的实验结果显示，本发明的救必应酸能够显著降低流感感染小鼠的肺指数，且对H1N1呈现一定的量效特征，表明救必应酸对流感病毒具有防治作用。此外，救必应酸对于流感病毒的防治效果明显强于齐墩果酸与救必应药材提取物。本发明所述的救必应酸可作为活性成分制备用于抗流感病毒的药物。
表1药物对流感病毒FM/1/47株感染小鼠肺炎模型肺指数的影响(n＝10)
注：##与空白对照组比较，P<0.05，**与模型对照组比较，P<0.01。
Claims (4)
1.救必应酸在制备抗流感病毒药物中的应用。
2.根据权利要求1所述的应用，其特征在于，所述流感病毒为甲型H1N1流感病毒。
3.救必应酸在制备治疗由流感病毒引起的肺炎药物中的应用。
4.根据权利要求3所述的应用，其特征在于，所述流感病毒为甲型H1N1流感病毒。
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