CN110051682A - 圆柏属植物多糖在制备防治病毒性急性肺损伤的药物中的用途 - Google Patents
圆柏属植物多糖在制备防治病毒性急性肺损伤的药物中的用途 Download PDFInfo
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Abstract
本发明属于中药领域，具体涉及圆柏属植物多糖在制备防治病毒性急性肺损伤药物中的用途。本发明从圆柏植物香柏和滇藏方枝柏中分离提取得到总多糖，糖含量均超过60％。经整体实验证实，所述多糖对甲型流感病毒H1N1诱导的小鼠急性肺损伤均有显著的治疗作用，能抑制流感病毒在鼠肺内的复制、降低肺部的毛细血管通透性、减轻肺部出血及肺水肿、抑制全身和肺部局部炎症反应，显著提高重症流感病毒感染小鼠的生存率；所述的圆柏属植物多糖可进一步用于制备防治病毒性急性肺损伤的药物。
Description
技术领域
本发明属中药技术领域，涉及柏科圆柏属植物多糖新的药物用途。具体涉及圆柏属植物多糖在制备防治病毒性急性肺损伤的药物中的用途。
背景技术
研究报道，病毒性急性肺损伤是由多种病毒感染而引起的肺部损伤，通常是由上呼吸道病毒感染向下蔓延所致。研究显示，引起急性肺损伤的病毒以流感病毒为常见，还包括副流感病毒、巨细胞病毒、腺病毒、鼻病毒、冠状病毒等。有研究报道，甲型流感病毒是引起人和禽类呼吸道疾病的重要病原体，病毒侵入细支气管上皮引起细支气管炎，感染波及肺间质与肺泡而致肺炎；研究显示，病毒等一方面在呼吸道上皮细胞和血管内皮细胞内复制，损伤肺组织，破坏气体交换屏障；另一方面，通过巨噬细胞、分叶核中性粒细胞、淋巴细胞以及内皮细胞等释放大量炎症因子，宿主免疫机制被过度激活并失控，导致急性肺损伤。
研究显示了急性肺损伤主要病理改变是肺泡水平的气体交换障碍，感染后所致的急性肺损伤(acute lung injury，ALI)可引发患者出现呼吸窘迫和严重的低血氧症。研究进一步表明单核细胞浸润、肺泡水肿是形成肺毛细血管通透性增高及肺水肿的病理基础，细菌，病毒感染诱发的肺损伤，临床表现为呼吸困难，严重的可以导致急性呼吸窘迫综合征，甚至死亡。
柏科圆柏属植物香柏(S.pingii var.wilsonii)、滇藏方枝柏(S.wallichiana)分布于我国主产西北、西部和西南高山地区，枝叶作为藏药徐巴入药，功效清热、祛湿、解毒，能利肺、肝胆、肾热，传统上用于治疗风湿性关节炎、类风湿性关节炎、胆囊炎、肾炎、炭疽病、痈疖肿毒、黄水病等疾病，迄今，其具体药效物质基础尚不明确。纵观国内外的研究，尚未见有关圆柏属植物多糖治疗病毒性急性肺损伤的研究报道。
基于现有技术的现状，本申请的发明人拟提供圆柏属植物多糖新的药物用途，尤其是圆柏属植物多糖在制备防治病毒性急性肺损伤的药物中的用途。
发明内容
本发明的目的是提供圆柏属植物多糖新的药物用途，具体涉及圆柏属植物多糖在制备防治病毒性急性肺损伤的药物中的用途。
本发明从圆柏属植物香柏和滇藏方枝柏中分离提取得到多糖提取物，所述多糖提取物经整体动物模型试验证实，具有显著治疗流感病毒诱导的急性肺损伤的作用。
本发明的圆柏属植物多糖通过下述方法制备：
取圆柏属植物枝叶，粉碎，以95％乙醇冷浸提取3次，药渣于室温下置通风处晾干，然后用热水提取3次，过滤，合并提取液，浓缩，加乙醇至含醇量80％，沉淀复溶，以三氯乙酸去游离蛋白，离心，上清液用水透析3天，透析液浓缩，冷冻干燥即得总多糖；采用硫酸-苯酚法测得圆柏属植物多糖的糖含量超过60％；采用间羟基联苯法测得圆柏属植物多糖的糖醛酸含量超过4％；采用考马斯亮蓝法测得圆柏属植物多糖的蛋白质含量不超过2％。
本发明所述的圆柏属植物是香柏(Sabina pingii var.wilsonii)或滇藏方枝柏(S.wallichiana)。
本发明的圆柏属植物多糖进行了如下整体动物试验，尤其是针对H1N1甲型流感病毒毒株的试验：
1、圆柏属植物多糖用于治疗H1N1甲型流感病毒诱导急性肺损伤试验
选用BALB/C小鼠，以H1N1甲型流感病毒滴鼻感染，建立流感病毒诱导的急性肺损伤模型。圆柏属植物多糖灌胃给药，96h后观察动物肺损伤程度及干扰素、炎性因子等指标。结果证实口服圆柏属植物多糖后，对H1N1流感病毒诱导急性小鼠肺损伤有显著的保护作用。
2、圆柏属植物多糖对病毒诱导鼠肺急性损伤的抗炎相关研究指标检测方法
1)肺指数
病毒感染第四天，动物称重，充分取血，解剖完整去下肺脏，将整个肺叶置于滤纸，待滤纸吸干表面水分后称重，以此为肺湿重；计算肺指数，肺指数＝肺湿重(mg)/体重(g)。
2)肺总蛋白测定
整个全肺留取用于病理切片的肺叶后，其余部分的肺叶生理盐水漂洗，用适量冰浴的PBS匀浆，肺匀浆液分装，100ul肺匀浆液使用RIPA裂解液提取总蛋白，裂解液低温高速离心，收获上清分装，应用BCA法检测蛋白含量。
3)肺匀浆神经氨酸酶活性测定
神经氨酸酶为流感病毒表面主要的糖蛋白，其含量能反映流感病毒的浓度。应用神经氨酸检测试剂盒检测病毒浓度。
4)肺匀浆TNF-α，IL-6，IFN-γ，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TNF-α，IL-6，IFN-γ，IL-10试剂盒说明书测定。
3、圆柏属植物多糖对重症H1N1甲型流感病毒感染鼠的生存保护试验
选用BALB/C小鼠，以H1N1甲型流感病毒滴鼻感染，建立流感病毒诱导重症肺损伤模型，给药7天，停药观察7天，观察本发明药物对重症流感感染鼠的14天观察期的生命保护作用。
本发明实验结果显示，所述的圆柏属植物多糖对流感病毒H1N1诱导的急性肺损伤有显著的治疗作用；圆柏属植物多糖可显著提高重症流感病毒感染小鼠的生存率，降低肺指数(肺组织湿重/体重比值)和病毒神经氨酸酶活性，减少肺内总蛋白含量，抑制血清及肺内TNF-α，IL-6的释放，促进IFN-γ，IL-10的分泌。
本发明所述的圆柏属植物多糖能抑制流感病毒在鼠肺内的复制，从而减轻病毒感染造成的炎性免疫损伤；并通过抗炎作用来降低肺部毛细血管通透性，减少淋巴细胞浸润及炎性蛋白的募集，抑制全身和肺部局部炎症反应。
本发明所述的圆柏属植物多糖可进一步制备防治病毒性急性肺损伤的药物。
附图说明
图1香柏多糖对H1N1病毒感染小鼠肺指数的影响。
图2香柏多糖对H1N1病毒感染小鼠肺总蛋白的影响。
图3香柏多糖对H1N1病毒感染小鼠神经氨酸酶活性的影响。
图4香柏多糖对H1N1病毒感染小鼠肺匀浆细胞因子的作用。
图5香柏多糖对H1N1病毒感染小鼠肺切片HE染色(×200)。
图6香柏多糖对H1N1病毒感染小鼠的生存保护作用。
图7滇藏方枝柏多糖对H1N1病毒感染小鼠肺指数的影响。
图8滇藏方枝柏多糖对H1N1病毒感染小鼠肺切片HE染色(×200)。
具体实施方式
实施例1
取香柏药材100g，粉碎，以95％乙醇冷浸提取3次，药渣于室温下置通风处晾干，然后用热水提取3次，过滤，合并提取液，浓缩，加乙醇至含醇量80％，沉淀复溶，以三氯乙酸去游离蛋白，离心，上清液用水透析3天，透析液浓缩，冷冻干燥即得总多糖。采用硫酸-苯酚法测得香柏多糖的糖含量为81.2％；采用间羟基联苯法测得香柏多糖的糖醛酸含量11.1％；采用考马斯亮蓝法测得香柏多糖的蛋白质含量1.1％。
实施例2
BALB/C小鼠36只(14-16g)，按体重随机分成6组(N、M、A、B、C、P)：N组为正常组，M组为H1N1病毒模型组，A组为香柏多糖7.5mg/kg，B组为香柏多糖15mg/kg，C组为香柏多糖30mg/kg，P组为阳性药利巴韦林100mg/kg，每组6只，所有动物经丙泊酚尾静脉注射麻醉，N组滴鼻1640培养液30μL作为对照；其他组滴鼻感染5LD50H1N1病毒液30μL，感染H1N1后两小时灌胃给药，N组与M组给予0.5％CMC灌胃给药，作为正常和病毒对照，一天给药一次，连续给药四天，H1N1病毒攻击96h后，称重体重，摘眼球取血，小心剪下全肺，滤纸沾干血迹，称重记录；小心剪下右肺上叶置于10％福尔马林中；右肺中叶、下叶及左肺用生理盐水漂洗干净，置于-80℃保存。冻存的肺组织之后使用预冷的PBS，用匀浆器在冰浴中匀浆，收货匀浆液，离心，收集上清，分装，-80℃保存，用于检测肺指数、神经氨酸酶活性、肺总蛋白、肺炎性细胞因子、干扰素等指标；
肺指数是小鼠的肺重与体重的比值，其值越大说明肺病变程度越严重，与正常组相比，模型组小鼠肺指数明显升高；给予药物后，实验结果显示，与模型组相比，香柏多糖15mg/kg和30mg/kg可显著抑制小鼠肺指数的升高(P<0.001，P<0.001)(如图1所示)；
肺部蛋白含量是衡量小鼠肺毛细血管通透性增高的重要指标，是肺部炎症细胞募集和肺水肿的重要标志，结果显示，与模型相比，7.5mg/kg、15mg/kg和30mg/kg香柏多糖可显著抑制小鼠肺总蛋白水平的升高(P<0.01，P<0.001，P<0.001)，抑制病毒诱导后肺部对炎性蛋白的募集(如图2所示)；
神经氨酸酶(NA)在感染宿主细胞的过程中扮演了重要角色，实验结果显示，与模型组相比，15mg/kg和30mg/kg香柏多糖可显著抑制病毒感染小鼠肺匀浆神经氨酸酶活性升高(P<0.05，P<0.001)，即能有效抑制病毒在鼠肺中的复制(如图3所示)；
对肺匀浆中炎症因子的测定结果显示，与正常组相比，模型组小鼠肺匀浆的TNF-α、IL-6水平显著升高，给药组与模型组相比，7.5mg/kg、15mg/kg和30mg/kg香柏多糖可显著抑制小鼠肺匀浆TNF-α(P<0.05，P<0.05，P<0.01)的释放，15mg/kg和30mg/kg香柏多糖可显著抑制小鼠肺匀浆IL-6(P<0.05，P<0.01)炎性细胞因子的释放，与正常组相比，模型组小鼠肺匀浆IFN-γ，IL-10水平显著下降，给药组与模型组相比，15mg/kg和30mg/kg香柏多糖可明显促进IFN-γ(P<0.05，P<0.01)，IL-10(P<0.05，P<0.01)的分泌(如图4所示)；
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明显增厚，肺泡萎缩变形，大量白细胞聚集，炎症严重，香柏多糖30mg/kg给药组肺泡轮廓清晰，结构较完整，无严重出血现象，炎症症状显著减轻(如图5所示)。
实施例3
BALB/C小鼠60只(14-16g)，按体重随机分成6组(N、M、A、B、C、P)：N组为正常对照组，M组为H1N1病毒模型组，A组为香柏多糖组7.5mg/kg，B组为香柏多糖组15mg/kg，C组为香柏多糖组30mg/kg，P组为利巴韦林100mg/kg，每组10只；所有组动物经丙泊酚麻醉，N组滴鼻1640培养基30μL作为对照；滴鼻感染10LD50H1N1病毒30μL，A、P组感染H1N1后2小时灌胃给药，同时N组与M组给予0.5％CMC灌胃给药，作为正常和病毒对照，一天给药一次，连续给药七天，停药观察到14天，每天记录动物的存活数量及死亡数，计算药物对重症流感感染肺炎小鼠的生命保护率；
结果显示，口服香柏多糖30mg/kg后，能提高重症流感病毒感染肺炎小鼠的生存率(P<0.01)，具有显著的生命保护作用(如图6所示)。
实施例4
取滇藏方枝柏药材100g，粉碎，以95％乙醇冷浸提取3次，药渣于室温下置通风处晾干，然后用热水提取3次，过滤，合并提取液，浓缩，加乙醇至含醇量80％，沉淀复溶，以三氯乙酸去游离蛋白，离心，上清液用水透析3天，透析液浓缩，冷冻干燥即得总多糖；采用硫酸-苯酚法测得滇藏方枝柏多糖的糖含量65％；采用间羟基联苯法测得滇藏方枝柏多糖的糖醛酸含量4.51％；采用考马斯亮蓝法测得滇藏方枝柏多糖的蛋白质含量1.9％。
实施例5
BALB/C小鼠24只(14-16g)，按体重随机分成4组(N、M、A、P)：N组为正常组，M组为H1N1病毒模型组，A组为滇藏方枝柏多糖30mg/kg，P组为阳性药利巴韦林100mg/kg，每组6只。所有动物经丙泊酚尾静脉注射麻醉，N组滴鼻1640培养液30μL作为对照；其他组滴鼻感染5LD50H1N1病毒液30μL，感染H1N1后两小时灌胃给药，N组与M组给予0.5％CMC灌胃给药，作为正常和病毒对照。一天给药一次，连续给药四天。H1N1病毒攻击96h后，称重体重，摘眼球取血。小心剪下全肺，滤纸沾干血迹，称重记录；小心剪下右肺置于10％福尔马林中；
模型组小鼠肺指数明显升高；给予药物后，实验结果显示，与模型组相比，滇藏方枝柏多糖30mg/kg可显著抑制小鼠肺指数的升高(P<0.05)(如图7所示)；
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明显增厚，肺泡萎缩变形，大量白细胞聚集，炎症严重，滇藏方枝柏多糖30mg/kg给药组肺泡轮廓清晰，结构较完整，无严重出血现象，炎症症状显著减轻(如图8所示)。
本发明实验证实，圆柏属植物多糖对流感病毒H1N1诱导的急性肺损伤有显著的治疗作用，可进一步制备防治流感和病毒性肺损伤的药物。
Claims (6)
1.圆柏属植物多糖在制备防治病毒性急性肺损伤药物中的用途。
2.按权利要求1所述的用途，其特征在于，所述的圆柏属植物是香柏(Sabina pingiivar.wilsonii)或滇藏方枝柏(S.wallichiana)。
3.按权利要求1或2所述的用途，其特征在于，所述的圆柏属植物多糖通过下述方法制备：
取圆柏属植物枝叶，粉碎，以95％乙醇冷浸提取3次，药渣于室温下置通风处晾干，然后用热水提取3次，过滤，合并提取液，浓缩，加乙醇至含醇量80％，沉淀复溶，以三氯乙酸去游离蛋白，离心，上清液用水透析3天，透析液浓缩，冷冻干燥得总多糖；采用硫酸-苯酚法测得圆柏属植物多糖的糖含量超过60％；采用间羟基联苯法测得圆柏属植物多糖的糖醛酸含量超过4％；采用考马斯亮蓝法测得圆柏属植物多糖的蛋白质含量不超过2％。
4.按权利要求1所述的用途，其特征在于，所述的病毒性急性肺损伤是流感病毒H1N1诱导的急性肺损伤。
5.按权利要求1或4所述的用途，其特征在于，所述的圆柏属植物多糖降低重症流感病毒感染小鼠的肺指数和病毒神经氨酸酶活性，减少肺内总蛋白含量，抑制血清及肺内TNF-α，IL-6的释放，促进IFN-γ，IL-10的分泌。
6.按权利要求1或4所述的用途，其特征在于，所述的圆柏属植物多糖抑制流感病毒在鼠肺内的复制，减轻病毒感染造成的炎性免疫损伤；降低肺部毛细血管通透性，减少淋巴细胞浸润及炎性蛋白的募集，抑制全身和肺部局部炎症反应。
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| CN111773251A (zh) * | 2020-06-15 | 2020-10-16 | 江苏安泰生物技术有限公司 | 一种肺泡吸入组合物及其制备方法和其应用 |
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