CN111643539A - 甘肃蚤缀的用途 - Google Patents
甘肃蚤缀的用途 Download PDFInfo
- Publication number
- CN111643539A CN111643539A CN202010518923.1A CN202010518923A CN111643539A CN 111643539 A CN111643539 A CN 111643539A CN 202010518923 A CN202010518923 A CN 202010518923A CN 111643539 A CN111643539 A CN 111643539A
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- extract
- gansu
- flea
- ethyl acetate
- methanol
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Abstract
本发明公开了一种抗肝纤维化提取物及其用途，提供了甘肃蚤缀或其提取物在制备预防和/或治疗肝纤维化疾病的产品的用途。本发明通过动物实验说明甘肃蚤缀或其提取物具有确切的抗肝纤维化作用，因此可用来制备预防和/或治疗肝纤维化疾病的产品。
Description
技术领域
本发明涉及肝纤维化的防治技术领域，特别是涉及一种抗肝纤维化提取物及其用途。
背景技术
肝纤维化是一种肝脏内细胞外基质(extracellular matrix,ECM)和***异常积聚和增生从而破坏正常结构和生理功能的疾病，是各种慢性肝病(如慢性病毒性肝炎、酒精性肝病、非酒精性脂肪性肝病以及自身免疫性肝病等)发展至肝硬化和肝癌的必经之路。截止到2019年，关于肝纤维化的临床研究报道很多，但至今没有治愈良策，对于肝纤维化晚期(肝硬化或肝癌)患者，肝脏移植是其最终的救命稻草，但有限的肝脏资源以及移植后的免疫排斥反应仍然是肝脏移植面临的巨大难题。
国际上已有临床报道针对病因的治疗案例，如抗病毒药物替诺福韦酯具有逆转肝纤维化和肝硬化的作用，但是该项研究样本量相对较小、缺乏对照、而且二次肝活检患者较少。国内有一些中成药显示出一定的抗肝纤维化作用，并用于临床治疗，如安络化纤丸、复方鳖甲软肝片、扶正化瘀胶囊等，但存在中药产品质量控制困难、作用机制不清、规范化临床研究较少等问题。多数药物(如多烯磷脂酰胆碱、奥贝胆酸、水飞蓟素、甘草酸类制剂和糖皮质激素等)在动物实验中显示出一定的抗肝纤维化作用，但其具体作用机制不明并伴随有一定的副作用以及耐药性，因此需进一步深入开展研究并寻找替代性治疗药物。
发明内容
为了解决上述技术问题，本发明采取了如下技术方案：
甘肃蚤缀或其提取物在制备预防和/或治疗肝纤维化的产品中的用途。
甘肃蚤缀(Arenaria kansuensis Maxim.)，又名雪灵芝，藏药名为“阿仲嘎保”，藏语义为“采天地之灵气日月之精华的人间仙草”，具有悠久的药用历史，多部藏医学典籍中记载其具有清肺热、止咳、降血压、健胃助消等功效以及治疗“胃肠之溃疡、膨胀、癌症、瘰疬等”。现代药理学研究表明该药具有抑菌、抗氧化、抗炎和免疫调节、抗病毒、保肝等疗效。
本发明中使用的提取物，是从甘肃蚤缀的乙醇提取物中分离而出；更进一步，是从乙酸乙酯提取物中分离而出。
更进一步地，所述乙酸乙酯提取物是以甘肃蚤缀的乙醇提取物为原料，使用乙酸乙酯提取所得的产物。
进一步地，所述提取物是由如下方法制备得到：
取甘肃蚤缀的乙醇提取物，以乙酸乙酯提取后，乙酸乙酯部位再通过反相硅胶色谱，先用50-60％甲醇-水洗脱，再用75～95％甲醇-水洗脱，收集75～95％甲醇-水洗脱产物，即得。
进一步地，取甘肃蚤缀的乙酸乙酯提取物，通过反相硅胶色谱，先用60％甲醇-水洗脱，再用80％甲醇-水洗脱，收集80％甲醇-水洗脱产物，即得。
进一步地，所述提取物是由如下方法制备得到：
取甘肃蚤缀的乙酸乙酯提取物，通过反相硅胶色谱，先用60％甲醇-水洗脱，再用80％甲醇-水洗脱，收集80％甲醇-水洗脱产物，即得。
本发明所述反相硅胶色谱，固定相选自十八烷基键和硅胶或类似材料。
更进一步地，在乙酸乙酯提取前，进行脱脂处理；进一步地，脱脂选用石油醚、***、戊烷或己烷进行。戊烷和己烷是石油醚的主要成分。
进一步地，所述提取物经过HPLC测定，色谱图与图6相似度在90％以上。鉴于天然植物产地、栽培、生长环境等各方面的差异，相同的天然植物中，不同植株也会存在化学成分种类和含量差异，这也势必会导致提取物中的成分差异。因此，在通过HPLC色谱图限定提取物时，与本发明图6相似度在90％以上的提取物色谱图，其色谱图针对的提取物，就可以判定与本发明所述提取物相同。
上述检测的色谱条件如下：
固定相：十八烷基键和硅胶
流动相：流动相A为0.2％的甲酸水溶液，流动相B为乙腈溶液，梯度洗脱程序为：13～46％％B，0-90min。
本发明中，检测波长可以根据实际检测情况进行调整，目前的检测器大部分都可以同时进行全波段检测，在检测结束后可以根据需要任意查看和调取需要的检测波长下的图谱。本发明发现，最佳检测波长可能会在330nm，基于仪器和操作误差等情况，可调取或手动设置的检测波长可以在320～340nm范围。当然，也不排除在其他波长下同样可以得到准确反应提取物所含成分的色谱图。
本发明所述肝纤维化是病毒性肝病、化学性肝损伤、非酒精性脂肪性肝病、脂肪肝或自身免疫性肝病所致。
进一步地，所述肝纤维化是化学性肝损伤所致。
所述产品选自口服、注射或外用剂型。本发明可以将甘肃蚤缀或其提取物与药用辅料可以制备成注射剂、吸入剂、片剂、胶囊、丸剂等。
本发明的有益效果：
本发明在肝纤维化的小鼠模型上验证了甘肃蚤缀乙酸乙酯提取物中富含黄酮木质素具有抗肝纤维化的作用，结果表明，乙酸乙酯提取物可明显减轻对肝脏细胞的损伤程度，对肝组织胶原沉积和肝纤维形成有明显改善作用，可明显抑制肝纤维化相关蛋白TGF-β1、α-SMA的表达，表明乙酸乙酯提取物的有效组分黄酮木质素能够抑制肝纤维化的进程，具有明显的抗肝纤维化作用。
附图说明
图1为甘肃蚤缀乙酸乙酯部位，杂质部位(AS1)和目标部位(AS2)的HPLC分析谱图。图A:乙酸乙酯部位色谱图，图B:杂质部位AS1色谱图，图C:目标部位AS2色谱图。
图2为甘肃蚤缀提取物目标部位AS2对小鼠代表性肝脏图片和苏木精-伊红染色组织切片图片(20×)。
图3为甘肃蚤缀提取物目标部位AS2对小鼠代表性肝组织切片Masson染色图片(20×)。
图4为甘肃蚤缀提取物目标部位AS2对小鼠肝组织中TGF-β1的表达的影响。
图5为甘肃蚤缀提取物目标部位AS2对小鼠肝组织中α-SMA的表达的影响。
图6为甘肃蚤缀目标部位AS2的HPLC分析谱图。
具体实施方式
以下结合具体实施例对本发明甘肃蚤缀黄酮木质素抗肝纤维化的应用做详细说明。应理解，这些实施例用于说明本发明，但不限于本发明的范围。
实施例1
甘肃蚤缀乙酸乙酯提取物目标部位AS2的制备方法如下：
(1)将甘肃蚤缀全草阴干粉碎，过筛后取粉末2kg室温下用95％乙醇进行搅拌提取，料液比为1:10，共提3次，每次8h；
(2)合并提取液、减压浓缩成浸膏，将浸膏溶于2L水，分别用石油醚、乙酸乙酯和正丁醇进行萃取(2L×3)，每种萃取剂萃取3次，将乙酸乙酯萃取液浓缩干燥得到乙酸乙酯提取物部位；
(3)乙酸乙酯提取物部位在C18SPE中进行分段处理，先用60％甲醇-水进行洗脱，去除杂质部位AS1，然后通过80％甲醇-水洗脱，收集80％甲醇-水洗脱产物，即得目标部位AS2。
各个部位可以通过如下方法进行HPLC检测：
甘肃蚤缀乙酸乙酯部位(图1.A)、杂质部位AS1(图1.B)和目标部位AS2(图1.C)分析图在ODS-C18分析柱中进行。流动相A为0.2％的甲酸水溶液，流动相B为乙腈溶液；梯度洗脱步骤为13～46％B洗脱90min，流速为1.0mL/min；注射体积为10μL，色谱图检测波长为330nm。
实施例2
(1)将甘肃蚤缀全草阴干粉碎，过筛后取粉末2kg用乙酸乙酯进行提取，料液比为1:10，共提3次，每次8h；
(2)上述提取物在C18SPE中进行分段处理，先用60％甲醇-水进行洗脱，然后通过80％甲醇-水洗脱，收集80％甲醇-水洗脱产物，即得乙醇提取物中目标部位。
实施例3
甘肃蚤缀全草阴干粉碎，过筛后取粉末2kg室温下用95％乙醇进行搅拌提取，料液比为1:10，共提3次，每次8h，合并提取液，浓缩，即得乙醇提取物。
实施例4
甘肃蚤缀全草阴干粉碎，过筛后取粉末3kg用乙酸乙酯进行提取，料液比为1:12，共提3次，每次12h，合并提取液，浓缩，即得乙酸乙酯提取物。
实施例5
(1)将甘肃蚤缀全草阴干粉碎，过筛后取粉末4kg室温下用85％乙醇进行搅拌提取，料液比为1:8，共提3次，每次6h；
(2)合并提取液、减压浓缩成浸膏，将浸膏溶于2L水，分别用石油醚、乙酸乙酯和正丁醇进行萃取(2L×3)，每种萃取剂萃取3次，将乙酸乙酯萃取液浓缩干燥得到乙酸乙酯提取物部位。
实施例6
(1)将甘肃蚤缀全草阴干粉碎，过筛后取粉末4kg室温下用85％乙醇进行搅拌提取，料液比为1:8，共提3次，每次6h；
(2)合并提取液、减压浓缩成浸膏，将浸膏溶于2L水，乙酸乙酯进行萃取(2L×3)，将乙酸乙酯萃取液浓缩干燥得到乙酸乙酯提取物部位。
实施例7
甘肃蚤缀提取物抗肝纤维化效果研究
1.1实验材料
甘肃蚤缀提取物中的目标部位AS2。
1.2实验方法
(1)实验动物
SPF级健康C57BL/6雄性小鼠，10周龄，体重20±2g。饲养环境适宜，通风良好，安静，保持12h昼夜节律，温度为25±2℃，湿度为45％～60％，每隔2天换一次垫料，自由饮水，每日颗粒饲料喂养，自由饮食。实验动物适应一周后按照体重随机分组进行实验。所有小鼠在试验前饥饿处理12h，实验操作均在同一时间段进行。
(2)动物造模和分组
首次以CCl4 3mL/kg体重皮下注射，以后以50％CCl4-橄榄油3mL/kg体重皮下注射，一周2次，共4周。自造模第2周起，给药组分别灌胃给予AS2高、AS2中剂量下的甘肃蚤缀提取物目标部位AS2(相当于AS2生药量为700mg/kg，350mg/kg)、AS2低剂量组(相当于AS2生药量为170mg/kg)，模型组(造模组，按3mL/kg50％CCl4-橄榄油皮下注射)灌胃给予生理盐水、空白对照组(不造模组，按3mL/kg皮下注射橄榄油)灌胃给予生理盐水，每组10只小鼠，连续给药4周后通过病理组织切片、免疫荧光染色和RT-PCR等方法检测甘肃蚤缀提取物对小鼠肝纤维化的改善作用。
(3)组织切片
小鼠经10％水合氯醛麻醉后，称重，取血后，将小鼠平放于鼠板之上，用75％的酒精轻轻擦拭胸部和腹部的皮肤，用剪刀依次将腹部和颈部剪开，分离出肝组织，用PBS将肝组织表面血迹洗净，将肝放在4％多聚甲醛中固定。大约24h之后取出肝组织进行梯度酒精脱水，脱水完成后，浸入OCT中放入-20℃冰箱中预冻10min，然后放入冷冻切片机冻台上深入冷冻(-40℃)15min，切片(5μm)，捞片，然后按照试剂盒操作规程分别进行HE和Masson染色。镜下观察过程采用从低倍镜到高倍镜，从整体到局部，从宏观到微观的方法。
(4)免疫荧光检测
将以上所得切片滴加pH＝6.0的枸橼酸盐缓冲液至浸没肝组织，加热到98℃，持续5min进行抗原修复。待冷却到室温之后，用PBS反复冲洗三次，每次约5min。加入3％的过氧化氢，用以阻断内源性的过氧化物酶，在室温下阻断15min。用PBS冲洗三次，每次5min。滴加5％BSA进行封闭，室温下孵育30min。弃去封闭液，各组组织切片分别加入一抗(1：50稀释)后，4℃孵育过夜，PBS冲洗三次，每次5min，室温孵育FITC标记的二抗(1：200稀释)3h，DAPI染色3min，PBS冲洗三次，每次5min，然后经过梯度酒精脱水后二甲苯透化，封片后镜检。荧光显微镜拍摄时均采用统一曝光时间。蛋白相对表达量采用Image J软件进行定量分析。检测蛋白包括TGF-β1和α-SMA。
(5)RT-PCR
Collagen-1α序列F:GCTCCTCTTAGGGGCCACT，
R:CCACGTCTCACCATTGGGGGAPDH；
序列F:CGGAGTCAACGGATTTGGTCGTAT，
R:AGCCTTCTCCATGGTGGTGAAGAC。
1.3实验结果
1.3.1甘肃蚤缀提取物对模型小鼠肝脏细胞损伤程度的影响
苏木精-伊红染色表明空白组小鼠的肝脏呈褐色，肝小叶结构正常，肝细胞沿中央静脉呈放射状有序规则排列。模型组小鼠的肝脏呈米黄色，组织切片镜下观察呈现严重的病变坏死状态，肝细胞呈气球样变性，并伴随有严重的脂肪空泡化，加之小叶炎症细胞的浸润，细胞与细胞之间的空隙明显增大。Mallory-Denk小体和大量凋亡的肝细胞也可在小叶中发现。甘肃蚤缀提取物中的目标部位AS2可明显减轻对肝脏细胞的损伤程度，170mg/kg剂量对受损肝组织改善不明显，AS2350mg/kg和AS2700mg/kg给药剂量时，甘肃蚤缀提取物中的目标部位AS2可明显改善CCl4对肝组织的损伤(图2)。
1.3.2甘肃蚤缀提取物对模型小鼠肝组织胶原沉积和肝纤维形成的影响
Masson染色结果和胶原蛋白-ⅠαmRNA转录测试结果表明，与空白组相比，模型组小鼠肝组织呈现大量胶原沉积，表现为大面积蓝染效果。中央桥和肝门桥观察结果表明，与空白组相比，模型组小鼠肝组织的肝小叶中形成明显的中央-中央桥纤维和中央-肝门桥纤维，170mg/kg剂量的甘肃蚤缀提取物中的目标部位AS2对肝组织胶原沉积和桥纤维形成无明显改善作用，350mg/kg和700mg/kg给药剂量时，肝纤维化程度明显好转，并呈剂量依赖性(图3)。
1.3.3甘肃蚤缀提取物对模型小鼠肝组织TGF-β1含量的影响
肝脏中的肝星形细胞(HSCs)激活后分化成的肌成纤维细胞(MFBs)是胞外基质(如胶原蛋白等)的主要来源细胞，HSCs的激活是引发肝纤维化的关键环节。TGF-β1是HSCs的主要激活因子，也是引起肝脏纤维化的重要细胞因子。受损肝脏的枯否细胞、淋巴细胞、窦内皮细胞和HSCs等细胞通过多条信号通路的激活大量产生TGF-β1。TGF-β1通过启动TGF-β1/Smads信号通路激活HSCs并促使其分化成MFBs。首先，TGF-β1与受体作用，激活的膜受体直接作用于Smad2/3使其磷酸化，然后与通用型Smad4结合，转位入核调节靶基因转录，进入细胞核内的Smads复合物可与不同的转录活化因子结合而调节TGF-β1激活HSCs分化成MFBs的生物效应。MFBs大量表达ECM(主要为Ⅰ和Ⅲ型胶原)和α-平滑肌动蛋白(α-SMA)并堆积在受损肝组织病。免疫荧光染色结果表明，与空白组相比，模型组小鼠肝组织TGF-β1表达量明显升高，AS2低剂量甘肃蚤缀提取物对TGF-β1无明显抑制作用，随着给药剂量的增加，AS2350mg/kg和AS2700mg/kg给药剂量下可明显降低肝组织中TGF-β1的表达量(图4)。
1.3.4甘肃蚤缀提取物对模型小鼠肝组织α-SMA水平的影响
α-SMA是MFBs的标志性蛋白，抑制其表达可降低纤维细胞的收缩能力，从而降低纤维化作用，另外，该蛋白的抑制也表明MFBs的数量或者功能受到抑制，从而降低组织纤维化作用。与空白组相比，模型组小鼠肝组织α-SMA表达量明显升高，随着甘肃蚤缀提取物中的目标部位AS2给药剂量的增加，α-SMA表达受到抑制，其抑制作用与给药剂量呈依赖性相关(图5)。
综上所述，实验结果表明甘肃蚤缀提取物可改善肝组织损伤程度及肝组织中胶原蛋白的生成，抑制肝纤维化的作用，甘肃蚤缀提取物具有预防/治疗肝纤维化疾病的新用途。
以上所述仅为本发明的实施例，并非因此限制本发明的专利范围，凡是利用本发明说明书及附图内容所作的等效结构或等效流程变换，或直接或间接运用在其他相关的技术领域，均同理包括在本发明的专利保护范围内。
Claims (10)
1.甘肃蚤缀或其提取物在制备预防和/或治疗肝纤维化的产品中的用途。
2.根据权利要求1所述的用途，其特征在于：所述提取物选自甘肃蚤缀的乙醇提取物或乙酸乙酯提取物；进一步地，所述乙酸乙酯提取物是以甘肃蚤缀的乙醇提取物为原料，使用乙酸乙酯提取所得的产物。
3.根据权利要求1所述的用途，其特征在于：所述提取物是由如下方法制备得到：
取甘肃蚤缀的乙醇提取物，以乙酸乙酯提取后，乙酸乙酯部位再通过反相硅胶色谱，先用60％甲醇-水洗脱，再用80％甲醇-水洗脱，收集80％甲醇-水洗脱产物，即得。
4.根据权利要求1所述的用途，其特征在于：所述提取物是由如下方法制备得到：
取甘肃蚤缀的乙酸乙酯提取物，通过反相硅胶色谱，先用50-60％甲醇-水洗脱，再用75～95％甲醇-水洗脱，收集75～95％甲醇-水洗脱产物，即得；
优选地，取甘肃蚤缀的乙酸乙酯提取物，通过反相硅胶色谱，先用60％甲醇-水洗脱，再用80％甲醇-水洗脱，收集80％甲醇-水洗脱产物，即得。
5.根据权利要求3或4所述的用途，其特征在于：在乙酸乙酯提取前，进行脱脂处理；进一步地，脱脂选用石油醚、***、戊烷或己烷进行。
6.根据权利要求3-5任意一项所述的用途，其特征在于：所述提取物经过HPLC测定，色谱图与图6相似度在90％以上；色谱条件如下：
固定相：十八烷基键和硅胶
流动相：流动相A为0.2％的甲酸水溶液，流动相B为乙腈溶液，梯度洗脱程序为：13～46％％B，0-90min。
7.根据权利要求6所述的用途，其特征在于：HPLC的检测波长为330nm。
8.根据权利要求1所述的用途，其特征在于：所述肝纤维化是病毒性肝病、化学性肝损伤、非酒精性脂肪性肝病、脂肪肝或自身免疫性肝病所致。
9.根据权利要求1所述的用途，其特征在于：所述肝纤维化是化学性肝损伤所致。
10.根据权利要求1所述的用途，其特征在于：所述产品选自口服、注射或外用剂型。
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