CN116407533A - 降倍半萜类化合物loliolide在制备治疗病毒性肺炎药物中的用途 - Google Patents
降倍半萜类化合物loliolide在制备治疗病毒性肺炎药物中的用途 Download PDFInfo
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Abstract
本发明属于中药制药技术领域，具体涉及一种降降倍半萜类化合物loliolide在治疗病毒性肺炎药物中的用途。本发明从青黛马勃方中分离提取到降倍半萜类化合物loliolide。所述化合物经整体动物实验结果证实，对甲型流感病毒H1N1诱导的小鼠病毒性肺炎有良好的治疗作用，对病毒感染小鼠体重、肺指数、肺部炎症、肺部病毒滴度和病理变化均有明显改善。所述的loliolide可以进一步用于制备治疗病毒性肺炎的新药。
Description
技术领域
本发明属于中药技术领域，涉及降倍半萜类化合物loliolide新的制药用途， 具体涉及降倍半萜类化学物loliolide在制备治疗病毒性肺炎药物中的用途。
背景技术
现有技术公开了病毒性肺炎是由上呼吸道病毒感染、向下蔓延引发肺部炎 症，严重时导致肺换气功能障碍。流感病毒首先入侵上呼吸道粘膜,复制并释放 病毒颗粒和毒素引发感染。在疾病发生发展的过程中伴随免疫细胞的聚集、激活 及肺泡上皮细胞和血管内皮细胞的凋亡，释放大量炎症介质，如肿瘤坏死因子 (TNF-α)、白三烯、IL-1、IL-6、IL-8等(Burk M,El-Kersh K,Saad M,et al.Viral infection in community-acquiredpneumonia:A systematic review and meta-analysis[J].European RespiratoryReview,2016,25(140):178-188.)。由于病毒 的大量快速复制，机体的免疫系统被过度激活，进一步导致免疫调节失控，严重 时引发细胞因子风暴。而过度激活的免疫系统会诱导肺泡上皮细胞死亡，加剧上 皮细胞通透性，造成血管渗漏和肺部水肿，临床表现为非心源性肺水肿和顽固性 低氧血症，甚至出现ARDS，(Channappanavar R,Perlman S.Pathogenichuman coronavirus infections:causes and consequences of cytokine storm andimmunopathology[J].Seminars in Immunopathology,2017,39(5):529-539)。
青黛马勃方源于近代中医名家施今墨所著《施今墨对药》。施今墨在临床处 方时，用马勃、青黛两味中药配伍，青黛是君药，马勃为臣药。马勃清热解毒， 宣肺气，利咽喉；青黛凉血止血。马勃性平，宣散之力尤著，青黛性味苦寒，清 热之效力胜。二者配伍使用，并走上焦，用以消肿止痛，清热利咽。急性咽喉发 炎、慢性咽喉炎症以及扁桃体炎症均可使用(施今墨对药，[M]北京：人民卫生 出版社，2002)。现代药理学研究表明，青黛马勃方水提物体外具有抗补体、抗 氧化、抗病毒活性，对病毒性肺炎小鼠的肺部损伤也有显著改善(涂鹏.马勃青 黛方的清热解毒物质基础研究对病毒性急性肺损伤小鼠的保护作用：[D]上海：复旦大学，2018)。但青黛马勃方治疗病毒性肺炎的药效物质基础未见报道。
基于现有技术的现状，本申请的发明人拟提供降倍半萜类化合物loliolide新 的制药用途，具体涉及降倍半萜类化学物loliolide在制备治疗病毒性肺炎药物中 的用途。
发明内容
本发明的目的在于基于现有技术的基础与现状，提供降倍半萜类化合物loliolide新的制药用途，具体涉及降倍半萜类化学物loliolide在制备治疗病毒性 肺炎药物中的用途。
本发明提出的一种降倍半萜类化合物loliolide在治疗病毒性肺炎药物中的用途，所述降倍半萜类化合物loliolide的结构式如下：
本发明中，所述降倍半萜类化合物loliolide的制备方法如下：取青黛、马勃 两种药材按照8:3的质量比，将马勃剪碎，以10倍体积的纯水于78℃煎煮提取， 水提液浓缩；然后用乙酸乙酯萃取得到乙酸乙酯浸膏(EA)，所得乙酸乙酯浸膏 经硅胶柱、ODS反相柱和凝胶柱富集和分离，结合UPLC-MS/MS追踪，最终通 过半制备HPLC分离得到化合物loliolide。
本发明经实验证实降倍半萜类化合物loliolide为青黛马勃方治疗病毒性肺炎的药效物质。本发明从青黛、马勃中分离制备得到loliolide，经整体动物模型试 验证实，其对病毒性肺炎具有显著的治疗作用，可用于病毒性肺炎药物的开发。
本发明中，loliolide用于治疗流感病毒H1N1诱导小鼠病毒性肺炎的体内实 验：
(1)肺指数
病毒感染第四天，动物称重，充分取血，解剖完整取下肺脏，将整个肺叶置 于滤纸，待滤纸吸干表面水后称重，以此为肺脏湿重；计算肺指数，肺指数＝肺 湿重(mg)/体重(g)；
(2)肺总蛋白测定
留取整个全肺的部分用于病理切片的肺叶后，剩余部分的肺叶以生理盐水漂 洗，再用适量冰浴的PBS匀浆，肺匀浆液分装，100μL肺匀浆液加入适量的PBS， 此后低温高速离心，收获上清分装。应用BCA法检测蛋白含量。以牛血清白蛋 白作为标准蛋白溶液，制作标准曲线。BCA工作液加入待测样品后，于多功能 自动酶标仪(Thermo,Bio-Tek)A562 nm处读取吸收度(A)值；
(3)小鼠肺匀浆IL-6,TNF-α,IL-10测定
小鼠肺匀浆液，高速离心收获上清，分装，冻存。应用ELISA法，将匀浆 上清按照IL-6,TNF-α,IL-10试剂盒说明书测定；
(4)神经氨酸酶活性测试
96孔荧光酶标板加入神经氨酸酶检测缓冲液，70μL/孔，每孔加入10μL神 经氨酸酶样品；每孔再加入10μL Milli-Q水使每孔中的体积为90μL。检测时每 孔加入10μL神经氨酸酶荧光底物后在混匀1分钟。37摄氏度孵育半小时进行 荧光检测，波长设置：322nm为激发波长，450nm发射波长；
(5)肺脏和核蛋白表达水平检测
肺部组织白片依次经脱蜡、抗原修复、抗体结合(一抗：Anti-virusnucleoprotein)、复染等步骤，最终获得免疫组化样本玻片，通过显微镜观察各组 肺部病毒的复制情况；
(6)肺部病理变化检测
采用HE染色法观察病毒性肺炎小鼠肺部的病理变化，并拍照。
经实验证实，所述的loliolide对流感病毒H1N1诱导的急性肺损伤有显著的 治疗作用；loliolide可显著降低小鼠肺指数，改善肺部病理损伤，抑制肺部炎症， 降低肺部病毒滴度。
本发明所述的loliolide可制备防治病毒性肺炎的药物。
本发明的有益效果在于：从青黛、马勃中分离制备得到loliolide，经整体动 物模型试验证实，其对病毒性肺炎具有显著的治疗作用，可用于制备病毒性肺炎 的药物。
附图说明
图1为loliolide对H1N1病毒感染小鼠体重的影响。
图2为loliolide对H1N1病毒感染小鼠肺指数的影响，**P<0.01,***P<0.001, vsModel Control。
图3为loliolide对H1N1病毒感染小鼠肺部IL-6、TNF-α和IL-10的影响， 其中：A为IL-6、B为TNF-α、C为IL-10，*P<0.05,**P<0.01,***P<0.001,vs Model Control。
图4为loliolide对H1N1病毒感染小鼠肺部神经氨酸酶的影响，***P<0.001,vsModel Control。
图5为loliolide对H1N1病毒感染小鼠肺部病毒核蛋白表达的影响。
图6为loliolide对H1N1病毒感染小鼠肺部病理改变的影响。
具体实施方式
下面通过实施例进一步说明本发明。
实施例1：loliolide的分离与制备
分别取青黛马勃药材29kg和11kg,按照8:3配比，脱皮马勃剪碎，以10倍 体积的纯水于78℃煎煮提取。然后用乙酸乙酯萃取，合并萃取液得到乙酸乙酯 浸膏(EA)。所得浸膏经硅胶柱、ODS反相柱、凝胶柱富集和分离，结合 UPLC-MS/MS追踪，最终通过半制备HPLC制备得到化合物loliolide 30mg。
实施例2：loliolide对流感病毒病毒性肺炎小鼠的保护作用
BALB/c雄性小鼠30只(14-15g)，按体重随机分为5组(A,B,C,D,E)： A组为正常对照组，B组为H1N1病毒模型组，C组为EA组(5mg/kg)，D组 为loliolide(5mg/kg)，E组为药性药奥司他韦(20mg/kg)，每组6只。所有动 物经异氟烷气体麻醉，滴鼻感染3LD50 H1N1病毒液30μL，其中A组滴鼻感染 DMEM培养基30μL，B,C,D,E组感染H1N1稀释液。C,D感染2小时后分别 灌胃给药EA和loliolide，剂量为5mg/kg；E组给予阳性药奥司他韦，剂量为20 mg/kg。一天给药一次，连续给药四天并记录每日小鼠体重。小鼠感染病毒96h 后，称量体重，摘眼球取血。小心剪下全肺，滤纸沾干血迹，称重记录；小心剪 下左肺置于10％福尔马林中，用于病理评估；右肺用生理盐水漂洗干净；置于 -80℃冰箱保存，用于炎症因子、神经氨酸酶活性等指标。
(1)loliolide对H1N1病毒感染小鼠体重的影响
体重变化是评估药物对病毒性肺炎小鼠保护作用的一个宏观指标。记录小鼠 每日体重，绘制体重变化情况。结果如图1所示：与正常组相比，模型组的小鼠 体重显著下降；与模型组相比，经loliolide治疗后小鼠体重略微上升。
(2)loliolide对H1N1病毒感染小鼠肺指数的影响
肺指数可以反映感染病毒小鼠肺部的水肿情况，其值越大说明肺脏病变程度 越严重。结果如图2所示，与正常组比较，模型组小鼠肺指数显著升高(P<0.001)； loliolide(5mg/kg)给药后，小鼠肺指数显著下降(P<0.001)。
(3)loliolide对H1N1病毒感染小鼠肺部炎症的影响
对肺部炎症因子检测结果表明，与正常组比较，模型组小鼠肺匀浆液中的 IL-6,TNF-α释放水平显著升高(均P<0.001)，IL-10释放水平明显下降(P<0.001)； 给药后，loliolide组肺匀浆中IL-6和TNF-α水平显著下降(IL-6:P<0.01；TNF-α： P<0.05),IL-10释放水平明显上升(P<0.05)(如图3所示)。
(4)loliolide对H1N1病毒感染小鼠肺部病毒滴度的影响对病毒感染小鼠 肺部神经氨酸酶活性的测试结果表明(如图4所示)，与正常组相比，小鼠经流 感病毒感染后，肺部神经氨酸酶活性显著上升(P<0.001)；给药后，loliolide 组神经氨酸酶活性显著下降(P<0.001)。同样的，H1N1病毒核蛋白的免疫组化 结果也表明，小鼠在感染流感病毒后，病毒核蛋白的表达增强，表明病毒在肺部 的肤质增加；与模型组相比，loliolide给药后，病毒核蛋白表达水平下降(如 图5所示)。
(5)loliolide对H1N1病毒感染小鼠肺部病理变化的影响
病理检查结果表明，正常组肺泡轮廓清晰，结构完整，无渗血现象，基本无 炎症；模型组病理切片显示肺泡壁显著增厚，肺泡萎缩变形，免疫细胞大量聚集， 炎症严重。loliolide组能显著改善肺脏病理损伤，肺泡轮廓较为清晰，结构相 对较为完整，出血现象得到缓解，炎症症状明显减轻，如图6所示。
综上所述，loliolide对H1N1流感病毒诱导的小鼠病毒性肺炎有显著治疗作 用；缓解小鼠体重下降、显著降低肺指数、肺脏神经氨酸酶活性、病毒核蛋白 NP蛋白表达水平、抑制肺部炎症反应，减少淋巴细胞浸润、缓解肺损伤。
本发明所述的loliolide可进一步制备治疗病毒性肺炎的药物。
Claims (5)
1.降倍半萜类化合物loliolide在制备治疗病毒性肺炎药物中的用途，所述降倍半萜类化合物loliolide的结构式如下：
2.根据权利要求1所述的用途，其特征在于，所述降倍半萜类化合物loliolide通过下述方法制备：
取青黛、马勃两种药材按照8:3的质量比，将马勃剪碎，以10倍体积的纯水于78℃煎煮提取，水提液浓缩；然后用乙酸乙酯萃取得到乙酸乙酯浸膏(EA)，所得乙酸乙酯浸膏经硅胶柱、ODS反相柱和凝胶柱富集和分离，结合UPLC-MS/MS追踪，最终通过半制备HPLC分离得到化合物loliolide。
3.根据权利要求1所述的用途，其特征在于，所述降倍半萜类化合物loliolide显著降低H1N1病毒感染小鼠肺指数。
4.根据权利要求1所述的用途，其特征在于，所述降倍半萜类化合物loliolide改善H1N1病毒感染小鼠肺部病理损伤。
5.根据权利要求1所述的用途，其特征在于，所述降倍半萜类化合物loliolide抑制H1N1病毒感染小鼠肺部炎症，降H1N1病毒感染低小鼠肺部病毒滴度。
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