CN114028415A - 增强防治2019-新冠病毒肺炎功效的桔梗引经药性组分及其组合物和应用 - Google Patents
增强防治2019-新冠病毒肺炎功效的桔梗引经药性组分及其组合物和应用 Download PDFInfo
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- CN114028415A CN114028415A CN202111371148.2A CN202111371148A CN114028415A CN 114028415 A CN114028415 A CN 114028415A CN 202111371148 A CN202111371148 A CN 202111371148A CN 114028415 A CN114028415 A CN 114028415A
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Abstract
本发明公开了增强防治2019‑新冠病毒肺炎功效的桔梗引经药性组分及其组合物和应用。本发明提供了从桔梗中分离得到的能够有效增强防治2019‑新冠病毒肺炎功效的桔梗引经药性组分，该桔梗引经药性组分是由分子量小于3000Da的己糖聚合物组成，其基本糖单元为果糖、半乳糖和葡萄糖。本发明通过进一步的试验发现，将桔梗引经药性组分与桔梗皂苷组分、桔梗单体皂苷、甘草皂苷组分、甘草皂苷单体、连翘咖啡酸苯乙醇苷组分或者连翘咖啡酸苯乙醇苷单体中的任何一种组分进行配伍后，桔梗引经药性组分均能明显增强这些组分的抗2019‑新冠肺炎病毒功效。
Description
技术领域
本发明涉及增强防治病毒功效的引经药性组分及其组合物，尤其涉及增强防治2019-新冠病毒肺炎(COVID-19)功效的桔梗引经药性组分及其组合物，属于2019-新冠病毒肺炎的防治领域。
背景技术
桔梗(Platycodon grandiflorus)，味苦、辛，平，归肺经。具有宣肺，利咽，祛痰，排脓，具有引药上行作用，是典型的引经佐使药物。在中国古代乃至东亚地区常用于治疗呼吸道和肺部疾病的治疗。2019年冠状病毒病(COVID-19)的持续大流行现在是对全球公共卫生的最大威胁。SARS-CoV-2作为呼吸系统疾病，其主要攻击靶器官为肺部，所以桔梗作为传统常用有效的治疗肺部感染药物，具有潜在应用价值。同时中药及其衍生的天然产物在COVID-19的治疗中备受关注，且已经证明中药对流感、HIV、SARS-CoV和MERSCoV10等均显示抗病毒活性。迄今为止，已经提出了许多天然产物来抑制SARS-CoV-2的重要成分之一，包括主蛋白酶(Mpro)、RNA依赖性RNA聚合酶(RdRp)、ACE2和TMPRSS2，并且通过体外及其体内实验也已经证明相关前景。
甘草(Glycyrrhiza uralensis)，味甘，平，归心、肺、脾、胃经。补脾益气，清热解毒，祛痰止咳，缓急止痛，调和诸药。现阶段研究发现甘草及甘草酸类成分有止咳祛痰、平喘及肺保护和抗肺纤维化作用，而肺部是新冠肺炎主要攻击靶器官，所以其具有潜在抗新冠肺炎作用，其可能机制是通过上调过氧化物酶体增殖激活受体-γ(PPARγ)、血管紧张素转化酶2(ACE2)和IκB-α的表达,阻滞ERK/NF-κB信号通路，又能与高迁移率属蛋白B1(HMGB1)结合直接抑制HMGB1的化学趋化和促有丝分裂的活性，抑制炎性细胞因子表达、杯状细胞增生和黏蛋白的过表达，减轻炎症因子风暴发生，并通过上调Smad7的表达，抑制气道重塑，改善肺功能。综上所述甘草及甘草酸类成分有抗冠状病毒潜在作用。并且，甘草与桔梗常以药对出现，属于经典方【桔梗汤】，其主要功效是：宣肺止咳、祛痰排脓，其治疗有显著肺部治疗靶向作用，中医认为新型冠状病毒感染性肺炎为湿热疫,为湿热疫毒之邪，主要从口鼻而入，病位在肺和脾，基本病机特点为"湿、毒、瘀、闭。其治疗主以分消湿热疫毒、宣畅气机，而【桔梗汤】将是理想药对之一。
连翘(Forsythia suspensa)，味苦，微寒。归肺、心、胆经。功效：清热解毒，消痈散结，疏散风热。临床常用于治疗带状疱疹病毒等。其治疗甲型H1N1流感和"非典"(SARS)有效的连花清瘟胶囊，现阶段已经证实具有治疗新冠肺炎作用，以上病毒进入细胞路径多有相似之处，均与ACE2路径有一定关系，作为主要用药成分，起到良好治疗效果，此外“三药三方”中的金花清感颗粒也有连翘成分，其统计数据显示连翘作为高频使用的抗新冠肺炎药物之一，其出现频率为15％，其主要通过Toll样受体信号通路等,作用于PTGS2、TNF-α、NF-κB、IL-6和CASP8等多途径,多靶点的方式发挥抗COVID-19作用。
桔梗皂苷D(PD)是一种在桔梗(PG)中含量丰富的三萜皂苷，属于齐墩果烷型五环三萜皂苷。具有抑制癌细胞和肿瘤细胞增殖,诱导细胞凋亡和保肝等方面的作用，并已被证明具有抗新冠肺炎作用。
甘草酸属于五环三萜皂苷。文献已报道甘草酸可以对抗2型人副流感病毒；对抗甲型H1N1流感病毒相关成果，所以具有潜在抗新冠肺炎活性。同时通过抑制炎症瀑布和氧自由基瀑布，减轻组织器官的损伤，改善患者咳嗽、发热、肺部啰音等体征，同时在改善患者的肝损伤等方面发挥作用，且无激素类药物的不良反应。
连翘脂苷A属于咖啡酸苯乙醇苷类成分，具有抗菌、抗病毒、抗氧化、抗感染和解热等药理作用，现代研究表明可通过调控TLR4抑制NF-κBp65核转移抑制炎症因子表达，从而减轻炎症反应，因此，其具有一定潜在的治疗或辅助治疗新冠肺炎病毒作用。
桔梗作为引经药物，可以体现靶向治疗作用，传统中药化学活性成分(单体)起到药效往往会受到吸收、分布、代谢等多重影响，其生物利用度一般不是很理想，要想提高药物生物利用度，其采用引经药性组分组合是一种较为理想的方法，为此对药物引经组分拆分并获得相关纯化产品，也是相关产品开发提供新的重要途径。
发明内容
本发明的目的之一是提供一种能够有效增强防治2019-新冠病毒肺炎功效的桔梗引经药性组分；
本发明的目的之二是提供一种防治2019-新冠病毒肺炎功效的药物组合物；
本发明的目的之三将将所述的防治2019-新冠病毒肺炎功效的药物组合物制备成相应的临床制剂。
本发明的上述目的是通过以下技术方案来实现的：
本发明的一方面是提供了从中药桔梗中分离得到的能够有效增强防治2019-新冠病毒肺炎功效的桔梗引经药性组分，该桔梗引经药性组分是由分子量小于3000Da的己糖聚合物组成，其基本糖单元为果糖、半乳糖和葡萄糖，其中果糖和半乳糖的质量比例可以是(6-8):(1-3):(0.5-1)，优选为7:2:1。
本发明所述的能够有效增强防治2019-新冠病毒肺炎功效的桔梗引经药性组分可通过以下制备方法制备得到，包括：将桔梗用热水提取方法得到水提取液，将水提取液上大孔吸附树脂柱色谱用水进行洗脱，将水洗脱组分用80％乙醇进行醇沉，收集上清组分，即得；或者是将桔梗用热水提取方法得到水提取液，将水提取液上大孔吸附树脂柱色谱用水进行洗脱，将水洗脱组分进行超滤分离得到分子量小于3000Da组分，即得。
本发明所述的热水提取方法包括不限于煎煮法、回流提取法、超声辅助提取法或微波辅助提取法等中的任何一种提取方法。
本发明中所述的超滤分离包括不局限为中空纤维超滤柱、中空纤维超滤膜、透析袋或切向流膜包等任何一种超滤分离方法。
本发明的另一方面是提供了防治2019-新冠病毒肺炎功效的组合物，该组合物包括以下(1)-(6)所述的组合物中的任何一种：
(1)桔梗引经药性组分与桔梗皂苷组分；优选的，按照质量比计，桔梗引经药性组分与桔梗皂苷组分的比例优选为50:1-100:1。
(2)桔梗引经药性组分与桔梗单体皂苷；优选的，按照质量比计，桔梗引经药性组分与桔梗单体皂苷的比例优选为10:1-200:1。
(3)桔梗引经药性组分与甘草皂苷组分；优选的，按照质量比计，桔梗引经药性组分与甘草皂苷组分的比例优选为60:1-120:1。
(4)桔梗引经药性组分与甘草皂苷单体；优选的，按照质量比计，桔梗引经药性组分与甘草皂苷单体的比例优选为10:1-1000:1。
(5)桔梗引经药性组分与连翘咖啡酸苯乙醇苷组分；优选的，按照质量比计，桔梗引经药性组分与连翘咖啡酸苯乙醇苷组分的比例优选为50:1-100:1。
(6)桔梗引经药性组分与连翘咖啡酸苯乙醇苷单体；优选的，按照质量比计，桔梗引经药性组分与连翘咖啡酸苯乙醇苷单体的比例优选为10:1-100:1。
其中，所述的桔梗皂苷组分可通过以下方法制备得到：将桔梗用水提取得到水提取液，将水提取液上大孔吸附树脂柱色谱吸附，收集50％乙醇洗脱组分，即得。
所述的甘草皂苷组分可通过以下方法制备得到：将甘草用水提取得到水提取液，将水提取液上大孔吸附树脂柱色谱吸附，收集50％乙醇洗脱组分，即得。
所述的连翘咖啡酸苯乙醇苷组分可通过以下方法制备得到：将青连翘用水提取得到水提取液，将水提取液上大孔吸附树脂柱色谱吸附，收集50％乙醇洗脱组分，即得。
本发明中所述的大孔吸附树脂包括不局限为D101、AB-8、HP-20、DM130、HPD300、XAD-1、X-5或NAK-9中的任何一种。
本发明所述的桔梗单体皂苷包括但不限于桔梗皂苷D2、桔梗皂苷D3或桔梗皂苷E中的任何一种；
本发明所述的草皂苷单体包括但不限于甘草酸、次甘草酸、甘草皂苷H2或甘草皂苷C2中的任何一种；
本发明所述的连翘咖啡酸苯乙醇苷单体可以是咖啡酸苯乙醇苷化合物，包括但不限于连翘酯苷A、连翘酯苷B或连翘酯苷I等。
将药学上可接受用量的上述组合物与药学上可接受的载体或稀释剂配合后，按本领域常规的制剂方法将其制备成任意一种适宜的药物制剂。通常该药物制剂适合于口服给药和注射给药，也适合其他的给药方法。该药物制剂包括但不限于颗粒剂、泡腾剂、口服液、喷雾剂、胶囊、软膏、糖浆、片剂等常规的临床制剂形式。
所述的载体是指药学领域常规的载体，例如：稀释剂、赋形剂如水等；粘合剂可以是纤维素衍生物、明胶、聚乙烯吡咯烷酮等；填充剂可以是淀粉、糖粉等物质；崩裂剂可以是淀粉、PEG、纤维素衍生物等；也可以为泡腾剂相关酸相碱相组成；另外，还可以药物组合物加入其它辅助剂如香味剂和甜味剂。
本发明药物制剂的施用量可根据用药途径、患者年龄、体重和疾病严重程度等进行相应的变化，这些都是本领域人员所习知。
本发明药物制剂可通过临床可接受的治疗方式给药，包括口服、吸入、注射或灌注等。
另外，本发明所述的组合物还可制备所形成的系列保健品和食品，不局限为茶叶、泡酒饮、牙膏、口香糖等。
附图说明
图1桔梗引经药性组分UHPLC-MS-MS表征结果。
图2桔梗皂苷D+桔梗引经药性组分组合物细胞毒性试验、抗新冠肺炎感染作用结果。
图3桔梗引经药性组分+连翘酯苷A细胞毒性试验、抗新冠肺炎感染作用结果。
图4桔梗引经药性组分+甘草酸抗新冠肺炎感染作用结果；注：纵坐标是病毒感染率(％)。
具体实施方式
以下结合具体实施例来进一步描述本发明，本发明的优点和特点将会随着描述而更为清楚。但这些实施例仅是范例性的，并不对本发明的范围构成任何限制。本领域技术人员应该理解的是，在不偏离本发明的精神和范围下可以对本发明的细节和形式进行修改或替换，但这些修改和替换均落入本发明的保护范围内。
实施例1桔梗引经药性组分的制备及表征
将桔梗用30-100℃热水进行煎煮提取，将提取液上D101大孔吸附树脂柱色谱用水进行洗脱，将水洗脱组分用80％乙醇进行醇沉，收集上清组分，即得；
桔梗引经药性组分的UPLC-MS-MS的表征，其检测条件如下：流动相：A水(0.1％甲酸)，B乙腈(0.1％甲酸)，柱温35℃，色谱柱：ACQUITY BEH Amide(2.1×100,1.7um)条件：90％水→50％水40min。其表征结果见图1，结果显其为17个单糖组成，分子量小于3000Da，其结构分析见表1。
实施例2桔梗引经药性组分的制备
将桔梗用30-100℃热水进行煎煮提取，将提取液上AB-8大孔吸附树脂柱色谱用水进行洗脱，将水洗脱组分用中空纤维超滤柱进行超滤分离得到分子量小于3000Da组分，即得。
实施例3桔梗皂苷组分的制备
桔梗皂苷组分可通过以下方法制备得到：将桔梗用30-100℃热水进行煎煮提取得到水提取液，将水提取液上DM130大孔吸附树脂柱色谱吸附，收集50％乙醇洗脱组分，即得。
实施例4甘草皂苷组分的制备
所述的甘草皂苷组分可通过以下方法制备得到：将甘草用30-100℃热水进行煎煮提取得到水提取液，将水提取液上HPD300大孔吸附树脂柱色谱吸附，收集50％乙醇洗脱组分，即得。
实施例5连翘咖啡酸苯乙醇苷组分的制备
所述的连翘咖啡酸苯乙醇苷组分可通过以下方法制备得到：将青连翘用30-100℃热水进行煎煮提取得到水提取液，将水提取液上XAD-1大孔吸附树脂柱色谱吸附，收集50％乙醇洗脱组分，即得。
实施例6颗粒剂制备
实施例1制备的桔梗引经药性组分与桔梗皂苷D组成的药物组合物20份(其中，桔梗引经药性组分与桔梗皂苷D的质量比为50：1)、蔗糖50份、糊精5份，淀粉25份，取等体积蔗糖与组合物混合均匀，依次加入淀粉、蔗糖、糊精进一步混合均匀，以10％淀粉浆为粘合剂，采用YK-160摇摆式颗粒机制粒，50℃下烘干，过14-20目筛网整粒，包装即得颗粒剂。
实施例7片剂制备
将实施例6制备的颗粒剂加入1％滑石粉和硬脂酸镁，混合均匀，经过压片可以获得片剂。
实施例8泡腾片制备
将实施例2制备的桔梗引经药性组分与桔梗皂苷D组成的组合物20份(其中，桔梗引经药性组分与桔梗皂苷D的质量比为50：1)、加入柠檬酸10份、碳酸氢钠20份，木糖醇30份、葡萄糖18份、(PEG4000)2份，以上配方采用GFZL-5型干法制粒机干法制粒，过14目筛网整粒，加入适量微粉硅胶混合均匀，压片可以获得泡腾片。
试验例1桔梗引经药性组分与桔梗皂苷D配伍得到药物组合物防治新型冠状病毒的试验
肺A549细胞种板：取对数生长期的A549细胞，细胞融合度到达80％后，胰酶消化，用完全培养基进行重悬，然后用细胞计数器计数，将细胞悬浮液稀释至所需密度，取50μL细胞悬液至96孔板，每孔的细胞数为25000个，37℃、5％CO2培养12h，培养12h后细胞完全贴壁于96孔板上。加药孵育：根据实验设计，在96孔板上每孔加入10μL配制好浓度的待测化合物溶液(桔梗引经药性组分或桔梗皂苷D)，37℃，5％CO2培养24h。24h后更换新鲜培养基，100μL/孔。37℃，5％CO2培养24h。每孔加入10μL CCK-8溶液。继续孵育0.5h，应用酶联免疫监测仪于450nm波长下测定各孔光吸收值，记录结果。
构建新冠假病毒，探究桔梗引经药性组分与桔梗皂苷D配伍后的药物组合物对新冠病毒感染细胞活性的影响：
新冠病毒S蛋白的伪病毒制备及收集：新冠病毒S蛋白的伪病毒制备及收集：伪病毒用三种病毒质粒SARS-CoV-2S蛋白、pAX2和pHB瞬时转染293T细胞，S蛋白作为包膜蛋白，荧光素酶基因被包装在病毒内部。6-8h后更换新鲜培养基，37℃，5％CO2培养48h。从60mm培养皿中收集病毒上清液，病毒上清液用0.45μm滤膜过滤，立即使用病毒或在-80℃保存。
表面表达人ACE2的293T细胞的制备及种板：用ACE2-pc DNA3细胞质粒转染瞬时转染293T细胞，6～8h后更换新鲜培养基，37℃，5％CO2培养48h。48h后，将细胞旋转至生长培养基中，然后用细胞计数器计数，将细胞悬浮液稀释至所需密度，取50μL细胞悬液至96孔板，每孔的细胞数为25000个，37℃、5％CO2培养12h，培养12h后细胞完全贴壁于96孔板上。
加药孵育：根据实验设计，在96孔板上每孔加入10μL配制好浓度的待测化合物溶液(桔梗引经药性组分或桔梗皂苷D)，37℃，5％CO2孵育1h。
假病毒孵育：96孔板上每孔加40μL病毒上清液。37℃，5％CO2培养24h，24h后更换新鲜培养基，100μL/孔，37℃，5％CO2培养24h。
测量：测量前将孔板平衡至室温，每孔加入30μL肾素荧光素酶试剂；在摇床上混合2min，诱导细胞溶解；在室温下孵育5min以稳定发光信号；记录酶标仪上的荧光数据。使用以下公式计算与DMSO处理的对照与加药的孔之间的相对发光(％)：
Relative Luminescence(％)＝RLU compound/RLU control×100％
化合物对于SARS-Co V-2S蛋白假病毒侵染细胞的抑制率为＝100％-RelativeLuminescence(％)。
试验结果表明：桔梗引经药性组分+桔梗皂苷D对细胞毒性较低，药物安全可靠。桔梗引经药性组分+桔梗皂苷D组合配伍后，桔梗引经药性组分明显增强抗新冠肺炎病毒作用，桔梗皂苷D在浓度5μg/mL时，新冠肺炎病毒感染率为17.8％，当加入500μg/mL桔梗上清时其感染率降至11.6％，与桔梗皂苷D比较具有显著差异(P<0.01)。因此，桔梗引经药性组分具有显著提升桔梗皂苷D抗SARS-CoV-2病毒肺炎的作用，其具体结果见图2。
试验例2桔梗引经药性组分与连翘脂苷A配伍得到的药物组合物防治新型冠状病毒试验
具体方法同上述试验例1，所不同的仅是将桔梗皂苷D替换为连翘脂苷A。
试验结果表明：连翘酯苷A浓度10μg/mL时，几乎没有任何新冠肺炎病毒抗感染作用，但是加入100μg/mL桔梗上清，其显示出76％的新冠肺炎病毒感染率，出现抗新冠肺炎病毒作用。说明桔梗引经药性组分具有显著提高连翘脂苷A抗新冠肺炎作用,其具体结果见图3。
试验例3桔梗引经药性组分与甘草酸配伍得到的药物组合物防治新型冠状病毒的试验
本试验的具体方法同上述试验例1，所不同的仅是将桔梗皂苷D替换为甘草酸。其结果表明：甘草酸在浓度10μg/mL时，几乎无抗病毒感染作用，但是加入0.5mg/mL桔梗引经药性组分，其显示出50％左右的新冠肺炎病毒感染率，其新冠肺炎感染率降低了约50％，其与甘草酸组相比有显著差异(P<0.01)，进一步说明桔梗引经药性组分配伍甘草酸，可以显著提升甘草酸抗新冠肺炎活性(图4)。
Claims (10)
1.从桔梗中分离得到的增强防治2019-新冠病毒肺炎功效的桔梗引经药性组分，其特征在于，该桔梗引经药性组分是由分子量小于3000Da的己糖聚合物组成。
2.根据权利要求1所述的桔梗引经药性组分，其特征在于，所述分子量小于3000Da的己糖聚合物的基本糖单元为果糖、半乳糖和葡萄糖，其中，果糖、半乳糖和葡萄糖的质量比例是(6-8)：(1-3)：(0.5-1.0)，优选为7:2:1。
3.根据权利要求1所述的桔梗引经药性组分，其特征在于，所述的桔梗引经药性组分的制备方法，包括：将桔梗用热水提取得到水提取液，将水提取液上大孔吸附树脂柱色谱用水进行洗脱，将水洗脱组分用80％乙醇进行醇沉，收集上清组分，即得；或者是将桔梗用热水提取得到水提取液，将水提取液上大孔吸附树脂柱色谱用水进行洗脱，将水洗脱组分进行超滤分离得到分子量小于3000Da组分，即得。
4.根据权利要求3所述的桔梗引经药性组分，其特征在于，所述的热水提取包括不限于煎煮法、回流提取法、超声辅助提取法或微波辅助提取法等中的任何一种提取方法；所述的大孔吸附树脂包括不限于D101、AB-8、HP-20、DM130、HPD300、XAD-1、X-5或NAK-9中的任何一种；所述的超滤分离包括不限于中空纤维超滤柱、中空纤维超滤膜、透析袋或切向流膜包中的任何一种。
5.一种防治2019-新冠病毒肺炎功效的药物组合物，该药物组合物包括以下(1)-(6)所述的药物组合物中的任何一种：
(1)权利要求1所述的桔梗引经药性组分与桔梗皂苷组分组成的药物组合物；
(2)权利要求1所述的桔梗引经药性组分与桔梗单体皂苷组成的药物组合物；
(3)权利要求1所述的桔梗引经药性组分与甘草皂苷组分组成的药物组合物；
(4)权利要求1所述的桔梗引经药性组分与甘草皂苷单体组成的药物组合物；
(5)权利要求1所述的桔梗引经药性组分与连翘咖啡酸苯乙醇苷组分组成的药物组合物；
(6)权利要求1所述的桔梗引经药性组分与连翘咖啡酸苯乙醇苷单体组成的药物组合物。
6.按照权利要求5所述的药物组合物，其特征在于，按照质量比计，桔梗引经药性组分与桔梗皂苷组分的比例为50:1-100:1；桔梗引经药性组分与桔梗单体皂苷的比例为10:1-200:1；桔梗引经药性组分与甘草皂苷组分的比例为60:1-120:1；桔梗引经药性组分与甘草皂苷单体的10:1-1000:1；桔梗引经药性组分与连翘咖啡酸苯乙醇苷组分的比例为50:1-100:1；桔梗引经药性组分与连翘咖啡酸苯乙醇苷单体的比例为10:1-100:1。
7.按照权利要求5或6所述的药物组合物，其特征在于，所述的桔梗皂苷组分可通过以下方法制备得到：将桔梗用水提取得到水提取液，将水提取液上大孔吸附树脂柱色谱吸附，收集50％乙醇洗脱组分，即得；
所述的甘草皂苷组分可通过以下方法制备得到：将甘草用水提取得到水提取液，将水提取液上大孔吸附树脂柱色谱吸附，收集50％乙醇洗脱组分，即得；
所述的连翘咖啡酸苯乙醇苷组分可通过以下方法制备得到：将青连翘用水提取得到水提取液，将水提取液上大孔吸附树脂柱色谱吸附，收集50％乙醇洗脱组分，即得。
8.按照权利要求5或6所述的药物组合物，其特征在于，所述的桔梗单体皂苷包括但不限于桔梗皂苷D、桔梗皂苷D2、桔梗皂苷D3或桔梗皂苷E中的任何一种；
所述的草皂苷单体包括但不限于甘草酸、次甘草酸、甘草皂苷H2或甘草皂苷C2中的任何一种；
所述的连翘咖啡酸苯乙醇苷单体是咖啡酸苯乙醇苷化合物，包括但不限于连翘酯苷A、连翘酯苷B或连翘酯苷I。
9.权利要求1-4任何一项所述的桔梗引经药性组分在制备防治2019-新冠病毒肺炎的药物或保健品中的用途。
10.权利要求5或6所述的药物组合物在制备防治2019-新冠病毒肺炎的药物或保健品中的用途。
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