CN115068488A - 毛蕊花糖苷和异毛蕊花糖苷在制备防治呼吸道合胞体病毒感染的药物或保健品中的应用 - Google Patents
毛蕊花糖苷和异毛蕊花糖苷在制备防治呼吸道合胞体病毒感染的药物或保健品中的应用 Download PDFInfo
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Abstract
本发明公开了毛蕊花糖苷及异毛蕊花糖苷在制备预防和治疗呼吸道合胞体病毒感染的药物或保健品中的应用，本发明在毛蕊花糖苷及异毛蕊花糖苷原有功效的基础上，研发出毛蕊花糖苷及异毛蕊花糖苷在预防和治疗呼吸道合胞体病毒疾病中的新用途，且细胞毒性低，取得了很好的技术效果。
Description
技术领域
本发明涉及中药的新用途，具体涉及毛蕊花糖苷在制备预防和治疗呼吸道合胞体病毒感染的药物或保健品中的应用。
背景技术
人呼吸道合胞体病毒(Human Respiratory Syncytial Virus,HRSV)属于副粘膜病毒科肺炎病毒属。HRSV是引起婴幼儿下呼吸道感染的主要病原体，也是1岁内婴儿细支气管炎和肺炎的最常见病因，2005年HRSV在全球范围内引起340万人住院，6.6-19.9万5岁以下儿童死亡。流行病学调查显示，该病毒有明显的季节性，常于晚秋初冬发病，于冬末春初达到最高峰。目前该疾病的治疗主要依靠吸氧、雾化等支持治疗，临床上批准用于预防和治疗的药物为帕利珠单抗和利巴韦林，其治疗效果存在争议。
毛蕊花糖苷和异毛蕊花糖苷是一种水溶性的苯丙素苷类化合物，存在于多种药用植物中。其药理作用广泛，包具有抗恶性肿瘤、抗阿尔兹海默症、抗炎，抗神经性疼痛、抗氧化等多种药理活性。但未发现毛蕊花糖苷和异毛蕊花糖苷抗病毒的相关报道，也未见毛蕊花糖苷和异毛蕊花糖苷预防和治疗呼吸道合胞体病毒的相关报道。
发明内容
发明目的：本发明的一个目的是通过大量实验筛选，在毛蕊花糖苷和异毛蕊花糖苷原有功效的基础上，研发出毛蕊花糖苷和异毛蕊花糖苷在预防和治疗呼吸道合胞体病毒疾病中的新用途。
技术方案，为了实现以上目的，本发明采取的技术方案为：
毛蕊花糖苷和/或异毛蕊花糖苷在制备防治病毒感染的药物或保健品中的应用。
毛蕊花糖苷和/或异毛蕊花糖苷在制备防治呼吸道合胞体病毒感染的药物或保健品中的应用。
本发明可将毛蕊花糖苷或异毛蕊花糖苷与药学上可接受的载体制备成贴剂、凝胶剂、片剂、颗粒剂、胶囊剂、丸剂或注射剂剂型的药物。
一种组合物在制备防治病毒感染的药物或保健品中的应用，组合物中包含毛蕊花糖苷和/或异毛蕊花糖苷。
有益效果：本发明通过大量实验筛选，实验结果表明，毛蕊花糖苷和/或异毛蕊花糖苷对呼吸道合胞体病毒具有很好的抑制作用，且对正常细胞毒性低，取得了非常好的技术效果。可以用于防治呼吸道合胞体病毒。
具体实施方式
下面结合具体实施例对本发明作进一步阐述，但这些实施例不应解释为限制本发明。
实施例1
1.试验材料：
Hep-2(ATCC CCL-23)细胞、人呼吸道合胞体病毒A型(ATCC VR-26)由中科院上海巴斯德研究所提供。DMEM培养基、胎牛血清、抗生素(青霉素和链霉素)、二甲亚砜(DMSO)、PBS均为Gibco BRL公司产品。96孔细胞培养板及6孔细胞培养板购自Corning公司。XDS-1B型生物倒置显微镜为重庆光学仪器厂产品。酶标仪型号为SpectraMax i3x购于MolecularDevices公司。CCK-8购于Dojindo东仁化学科技(上海)有限公司。受试药物：毛蕊花糖苷自制，纯度为99％。
2.试验方法与结果：
2.1药物对Hep-2细胞的毒性实验
取对数期Hep-2细胞，调整细胞悬液浓度为5×104/ml，向于96孔细胞培养板中每孔接种100μl细胞悬液，置于37℃5％CO2细胞培养箱中培养24小时至细胞长成单层。弃去孔中上清液，用磷酸缓冲液(PBS)洗1遍，将待检测毛蕊花糖苷梯度稀释后，加入到96孔板的细胞中，每个浓度3个复孔，同时设定正常细胞对照及不含细胞的空白对照，将细胞培养板置于37℃5％CO2培养箱继续培养48h。每孔加入10μl的CCK-8，37℃5％CO2培养箱孵育2h，用酶标仪测定在450nm处的吸光度。依照CCK-8细胞毒性实验公式Cellviability/％＝(OD样品-OD空白)/(OD对照-OD空白)×100％计算细胞存活率及药物抑制率，利用改良寇氏法计算CC50(The median cytotoxic concentration，中位浓度即对50％的细胞产生细胞毒性时对应的毛蕊花糖苷浓度)。选择细胞存活率大于50％的毛蕊花糖苷浓度进行后续抗病毒药物筛选实验。
2.2药物抗HRSV-A药效实验
将1×105/ml Hep-2细胞接种于6孔细胞培养板中，置于37℃5％CO2培养箱培养24h。弃掉细胞上清后加入0.1MOI的HRSV-A和以无明显细胞毒性的最高浓度为起点倍比稀释的毛蕊花糖苷溶液，于37℃5％CO2培养箱培养48h，取上清液氮中短时间冷冻后置于-80℃冰箱保存。本实验设不加入抗病毒药物的病毒感染为对照，实验重复两次。
取50ul，-80℃冰箱保存的病毒感染细胞上清，用含2％FBS和100U/ml双抗的DMEM细胞培养基按照10-1-10-8梯度稀释，加入到含有Hep-2细胞的96孔板上，同时设立正常细胞对照，病毒感染细胞对照组，置于37℃5％CO2细胞培养箱中培养48小时后，向每孔加入CCK-8 10μl，37℃培养2h，使用酶标仪测定450nm处的OD值，根据公式Cellviability/％＝(OD样品-OD空白)/(OD对照-OD空白)×100％计算出细胞存活率反映毛蕊花糖苷抗病毒效果，根据Prism software(GraphPadPrism7.04)的非线性回归方法计算(参考Cui R,Wang Y,WangL,et al.Cyclopiazonic acid,an inhibitor of calcium-dependent ATPases withantiviral activity against human respiratory syncytial virus.[J].AntiviralResearch,2016,132:38-45.)半最大效应浓度(concentration for 50％of maximaleffect,EC50)。
3、实验结果
具体检测的实验结果如表1所示。
表1毛蕊花糖苷对HRSV病毒的抑制作用
计算得到毛蕊花糖苷CC50＝0.295mg/ml、EC50＝0.003mg/ml、SI＝98.33,异毛蕊花糖苷CC50＝0.072mg/ml、EC50＝0.002mg/ml、SI＝36.00。具体计算方法如下所示：
“CC50”是指标是说杀死病毒的同时，如果也杀死50％的身体健康细胞，其所需要的药物浓度。根据改良寇式法计算CC50设计要求，设组在4个以上(含4个)；相邻两剂量组间比值相等；最大剂量组死亡率应为100％或相近，最小剂量组应为0％或相近。
根据以上要求，在计算毛蕊花糖苷的CC50时，我们选取浓度0.625mg/ml、0.312mg/ml、0.156mg/ml和0.078mg/ml计算CC50。其中浓度为0.625mg/ml时，细胞死亡率为91.16％，接近100％，浓度为0.312mg/ml时，细胞死亡率为69.15％，在0.156mg/ml和0.078mg/ml浓度时，细胞死亡率均约为0％；根据这个数据结果，计算过程如下：
改良寇式法公式:lg CC50＝Xm-I(P-(3-Pm-Pn)/4)
Xm:lg最大剂量，I:lg(最大剂量/相临剂量)，P:阳性反应率之和，Pm:最大阳性反应率，Pn:最小阳性反应率。
本实验结果为：
Pm＝0.9116；Pn＝0；
P＝0.9116+0.6915+0+0＝1.6031
Xm＝lg0.625＝-0.2041
I＝lg0.625/0.312＝0.3017
lg CC50＝-0.2041-0.3017*(1.6031-(3-0.9116-0)/4)＝-0.53
CC50＝0.295mg/ml。
在计算异毛蕊花糖苷的CC50时，我们选取浓度0.625mg/ml、0.312mg/ml、0.156mg/ml、0.078mg/ml、0.04mg/ml和0.02mg/ml计算CC50。其中浓度为0.625mg/ml、0.312mg/ml和0.156mg/ml时，细胞死亡率分别为96.28％、93.20％和93.54％，接近100％，浓度为0.078mg/ml和0.04mg/ml时，细胞死亡率分别为71.60％和8.15％，浓度为0.02mg/ml时，细胞死亡率约为0％；根据这个数据结果，计算过程如下：
改良寇式法公式:lg CC50＝Xm-I(P-(3-Pm-Pn)/4)
Xm:lg最大剂量，I:lg(最大剂量/相临剂量)，P:阳性反应率之和，Pm:最大阳性反应率，Pn:最小阳性反应率。
本实验结果为：
Pm＝0.9628；Pn＝0；
P＝0.9628+0.9320+0.9354+0.7160+0.0815+0＝3.6277
Xm＝lg0.625＝-0.2041
I＝lg0.625/0.312＝0.3017
lg CC50＝-0.2041-0.3017*(3.6277-(3-0.9628-0)/4)＝-1.1449
CC50＝0.072mg/ml。
本实验计算EC50的方法是基于Prism software(GraphPad Prism 7.04)的非线性回归方法计算(参考Cui R,Wang Y,Wang L,et al.Cyclopiazonic acid,an inhibitor ofcalcium-dependent ATPases with antiviral activity against human respiratorysyncytial virus.[J].Antiviral Research,2016,132:38-45.)。
在计算毛蕊花糖苷的EC50时，选取0.001mg/ml(细胞存活率38.5％)至0.078mg/ml(细胞存活率91.6％)的7个浓度进行计算，得到EC50＝0.003mg/ml。
在计算异毛蕊花糖苷的EC50时，选取0.001mg/ml(细胞存活率45.73％)至0.040mg/ml(细胞存活率107.77％)的6个浓度进行计算，得到EC50＝0.002mg/ml。
SI是一个综合上述有效性和毒副作用的指标，这个指标越大，说明目前试验的药物越可能成功。根据公式SI＝CC50/EC50计算选择指数(selectivity index,SI),毛蕊花糖苷的SI＝0.295/0.003＝98.33,异毛蕊花糖苷的SI＝0.072/0.002＝36.00。
根据SI指数，毛蕊花糖苷的SI优于异毛蕊花糖苷，显示出更加安全有效，说明不同立体构效的化合物其疗效也存在明显的区别。
另外，通过对比可以看出，0.001mg/ml低浓度的毛蕊花糖苷和异毛蕊花糖苷，在其安全性好的同时，反而显示出了非常好的抗HRSV病毒活性。
实施例2
取毛蕊花糖苷，加入崩解剂、粘合剂、润滑剂和填充剂制成片剂。
实施例3
取毛蕊花糖苷，加入粘合剂、润滑剂、填充剂制成贴膏剂。
实施例4
取毛蕊花糖苷，加入溶剂、抛射剂制成气雾剂。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
Claims (4)
1.毛蕊花糖苷和/或异毛蕊花糖苷在制备防治病毒感染的药物或保健品中的应用。
2.毛蕊花糖苷和/或异毛蕊花糖苷在制备防治呼吸道合胞体病毒感染的药物或保健品中的应用。
3.根据权利要求1或2所述的应用，其特征在于，将毛蕊花糖苷提取物与药学上可接受的载体制备成贴剂、凝胶剂、片剂、颗粒剂、胶囊剂、丸剂或注射剂剂型的药物。
4.一种组合物在制备防治病毒感染的药物或保健品中的应用，其特征在于，组合物中包含毛蕊花糖苷或异毛蕊花糖苷。
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