CN112426442A - 一种沙棘总黄酮提取物及其应用 - Google Patents
一种沙棘总黄酮提取物及其应用 Download PDFInfo
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Abstract
一种沙棘总黄酮提取物，所述的沙棘总黄酮提取物按如下方法制备：取脱脂后的沙棘干粉，加入碳酸钠作为助剂，100～400rpm球磨5～60min，取所得混合物加去离子水A，室温下搅拌提取10～60min，所得混合液离心去除沉淀，用去离子水B洗涤沉淀，合并上清，浓缩，得粗提物，所述粗提物经大孔树脂分离，去离子水C冲洗至流出液澄清后，以体积分数80％乙醇水溶液为洗脱液洗脱，所得洗脱液经后处理即得所述沙棘总黄酮提取物；本发明提取出的沙棘总黄酮呈无定形态，对其溶解度的增加、生物利用度的提高均具有有益作用。
Description
技术领域
本发明涉及天然产物领域，特别涉及一种沙棘总黄酮提取物及其在制备保肝护肝药物上应用。
背景技术
沙棘(拉丁学名：Hippophae rhamnoides Linn.)是一种胡颓子科、沙棘属落叶性多年生的落叶灌木，全株富含各类有效成分，其中以黄酮类含量最高。沙棘果实中含有的大量且种类繁多的生物活性成分，已有接近200种活性成分被报道。其中包括黄酮类的山奈酚、鼠李素、异鼠李素、槲皮素等，酚类化合物，脂肪酸类，维生素，游离氨基酸等。黄酮类化合物作为重要的食品成分在人类饮食中起着核心作用，他们是一类具有两个酚羟基的苯环通过中央三碳原子相连构成的一系列化合物。当日常饮食中摄入适量的黄酮时，可以降低人们患慢性疾病的风险。
黄酮类化合物具有保肝护肝的作用，药理作用如下。肝纤维化易发展为肝硬化或者肝癌，是影响现代人们健康的一大杀手。肝纤维化是因为细胞外基质过量聚集，破坏了肝脏的正常结构，细胞外基质有激活肝星状细胞的能力，健康肝脏中肝星状细胞是处于停滞增长状态的，一旦被激活的肝星状细胞可以持续增殖，具有收缩性、呈纤维性和炎症特性，如果没有采取适当的干预措施这些细胞将从受损的DNA信号通路诱导的死亡细胞中逃逸，肝星状细胞的持续增值导致细胞外基质过度聚集加重肝纤维化。有研究表明，沙棘中的活性物质可以上调DNA损伤信号的传导，使下游细胞能够诱导肝星状细胞凋亡，从而减轻肝纤维化。黄酮类物质在体外可以抑制肝星状细胞活化，在体内可以通过抑制阻碍转化生长因子-β/Smad信号通路，同时减轻氧化应激反应，阻碍TGF-β诱导的成纤维基因(包括，α-平滑肌肌动蛋白，纤溶酶原激活物抑制剂-1及COL1-A1)的表达。因此，一种有效的沙棘植物的总黄酮提取物对新的保肝护肝药物的制备具有重要意义。
发明内容
为了解决上述问题，本发明提供了一种沙棘总黄酮提取物，所述的沙棘总黄酮提取物按如下方法制备：取脱脂后的沙棘干粉，加入碳酸钠作为助剂，100～400rpm球磨5～60min，取所得混合物加去离子水A，室温下搅拌提取10～60min，所得混合液离心去除沉淀，用去离子水B洗涤沉淀，合并上清，浓缩，得粗提物，所述粗提物经大孔树脂分离，去离子水C冲洗至流出液澄清后，以体积分数80％乙醇水溶液为洗脱液洗脱，所得洗脱液经后处理即得所述沙棘总黄酮提取物；所述脱脂后的沙棘干粉与碳酸钠的质量比为1：1～1：100，所述去离子水A的体积以所述混合物的质量计为5～20mL/g，所述去离子水B的体积为2～20个大孔树脂的柱体积，所述80％乙醇水溶液的体积为2-10个大孔树脂的柱体积。去离子水A、B和C都是去离子水，此处只是为了方面描述，因此用A、B、C将它们区分开来。
优选地，球磨条件为400rpm，30min。所述球磨采用行星式球磨机。
优选地，所述搅拌提取的条件为室温下搅拌提取30min
优选地，所述大孔树脂为D101大孔树脂。
优选地，所述脱脂后的沙棘干粉与碳酸钠的质量比为10:1。
优选地，所述去离子水B的体积为5个大孔树脂的柱体积；所述80％乙醇水溶液的体积为3.3个大孔树脂的柱体积。
优选地，所述80％乙醇水溶液的体积以大孔树脂的体积比计为3.3BV。
进一步，所述脱脂的过程为：将沙棘果实烘干至恒重，粉碎至20目后用石油醚脱酯，即得脱脂后的沙棘干粉。
进一步，所述后处理的方法为：将所述洗脱液浓缩除去乙醇后，冷冻干燥，即得所述沙棘总黄酮提取物。
本发明还提供一种上述的沙棘总黄酮提取物在制备预防或治疗非酒精性脂肪肝的药物中的应用。
与现有技术相比，本发明的有益效果体现在：机械化学辅助提取中国传统药用植物依据的基本原理是植物细胞壁的破裂和目标有效成分的提取。机械化学辅助提取的沙棘总黄酮保肝护肝的药理活性未见报道，该法提取出的沙棘总黄酮呈无定形态，对其溶解度的增加、生物利用度的提高均具有有益作用。
附图说明
图1沙棘研磨物的扫描电镜图:a)500X下研磨前沙棘粉末；b)10.00KX下研磨前沙棘粉末；c)500X下研磨后沙棘粉末；d)10.00KX下研磨后沙棘粉末；e)500X沙棘总黄酮提取物。
图2为各组小鼠肝脏HE染色图1)正常组；2)NAFLD组；3)高剂量组；4)低剂量组；5)药物对照组。
具体实施方式
下面结合具体实施例及附图对本发明进行进一步说明。
实施例1：沙棘总黄酮的制备与表征
1.沙棘总黄酮的制备与分离
本实验所用沙棘果实，采自于青海，经青海民族大学鉴定为沙棘Hippophaerhamnoides Linn.，随后将沙棘原料在烘箱中以40-60℃温度烘干至恒重，并用中药粉碎机粉碎过筛至颗粒细度20目以下，称取粉碎后的沙棘1000g,加入10升石油醚，升温至60℃回流提取30min,过滤收集滤渣，在烘箱中以40-60℃温度烘干至恒重，收集脱脂后的沙棘干粉备用。
本实验所用的沙棘总黄酮提取方法为机械化学辅助提取，准确称取脱脂后的沙棘干粉50g，加入100mL不锈钢球磨罐中，加入20颗直径2cm不锈钢球磨珠，加入无水碳酸钠0.5g，放入行星式球磨机中，400rpm球磨30分钟，结束后获得的粉末除去球磨珠后放置于500ml烧杯中，加入250ml去离子水，室温(25℃)搅拌提取30分钟，提取完毕后离心除去沉淀，并用100ml水洗涤沉淀2次，合并提取液浓缩至200ml进行后续大孔树脂分离。量取预处理后的D101大孔树脂30mL放入直径5ml色谱柱中,加入提取液并以1ml/min的速率上样，收集上样后流出液。上样结束后以去离子水2ml/min的速率冲洗色谱柱至流出液澄清无浑浊，需使用150ml去离子水。随后，以80％乙醇水溶液作为洗脱剂以1ml/min的速率洗脱沙棘黄酮，共计消耗100ml 80％乙醇溶液。收集80％乙醇溶液浓缩除去乙醇后，采用冷冻干燥获得沙棘总黄酮。
2.沙棘黄酮中总黄酮含量测定与结构表征
采用黄酮标准测定方法铝盐法对获得的沙棘总黄酮含量进行测定，具体操作为准确称取沙棘总黄酮记录质量m置于25ml容量瓶中，加入去离子水充分溶解后定容，随后精确量取0.2mL溶液至25mL容量瓶中，依此加入0.2mL 5％亚硝酸钠溶液，0.2mL 10％硝酸铝溶液，0.5mL 5％氢氧化钠溶液，用去离子水定容，静置15min后用紫外分光光度计于510nm处测得吸光度为A，代入标准曲线y＝10.73x-0.01027(吸光度为纵坐标，浓度为横坐标，单位为mg/mL，R2值为0.9996)得浓度x mg/mL，所以称取样品中黄酮含量(以芦丁计)＝(x×25×25÷0.2/1000)/m。
实施例2：沙棘黄酮制备工艺优化
针对机械化学主要参数球磨转数、球磨时间以及碳酸钠质量进行优化，并按照实施例1所述的提取与后处理方法获得沙棘黄酮，测定其中沙棘黄酮的含量，确定获得最佳的提取球磨条件，实验结果见表1。
表1：沙棘黄酮球磨处理条件优化
实验结果表明，最佳机械化学处理条件下：无水碳酸钠的比例10％g/g，球磨转数400rpm，球磨30分钟后，按照实施例1精制后获得的沙棘黄酮的纯度最佳，铝盐法测得溶液吸光度为0.515(粗黄酮质量0.494g)，通过实施例1中所述公式计算黄酮含量为30.99％(以芦丁计)。
用扫描电子显微镜观察机械化学研磨后的沙棘颗粒表面形貌，粉末需置于载物台上进行喷金处理，再放入透射电镜中进行观察，沙棘黄酮电镜见图1，沙棘颗粒研磨后呈无定形态，细胞壁破裂且无明显细胞结构残留，说明机械力粉碎与破壁作用提高沙棘的提取率。
实施例3：非酒精性脂肪肝小鼠的实验方法
1、沙棘总黄酮治疗小鼠实验性非酒精性脂肪肝的药理研究实验
模型建立方法为：将40只健康雄性ICR小鼠在动物房饲养7天以适应新环境，然后随机分为5组，每组各8只：正常对照组，给予小鼠普通饲料喂养，连续5天每天根据体重进行10mg/kg生理盐水腹腔注射作为空白；非酒精性脂肪肝模型组、高剂量组(30mg/100g)、低剂量组(10mg/100g)，给予小鼠高脂饲料喂养，连续5天每天按照15mg/100g剂量进行四环素生理盐水溶液腹腔注射；造模结束后每组随机抽取一只小鼠断颈处死后解剖摘取肝脏，观察肝脏外观并称量肝脏湿重进行病理检测。
给药方式为：小鼠每天正常灌胃0.5～1mL，按照正常对照组、NAFLD模型组、低剂量组、高剂量组、药物对照组的顺序。高剂量组按照30mg/100g的剂量，将按照实施例1-1中所述方案制备的沙棘总黄酮溶于纯净水中配置不同剂量的药液；低剂量组按照10mg/100g的剂量，将沙棘总黄酮提取物溶于纯净水中给药；药物对照组为20mg/100g的剂量，将姜黄素溶于纯净水中给药；正常对照组和NAFLD模型组按照体重给予纯净水灌胃；连续给药15天。
2、沙棘总黄酮治疗非酒精性脂肪肝药效评价
生物样本采集：给药结束后，前一天晚断粮不断水12h眼眶取血后断颈处死，血液样本于4℃下，3000r/min离心10min，收集血清。给药结束后，前一天晚21点断粮不断水12h，第二天称量各组小鼠体重，在解剖摘取肝脏后称取湿重并记录，按照下面公式计算肝指数，结果见表2，每组取一块肝脏于多聚甲醇中固定，用于蜡块包埋组织切片。
肝指数(％)＝肝脏湿重(mg)/体重(g)×100％
血清、肝脏生化指标检测：血清生化指标进行TC、TG、HDL-C、LDL-C、ALT、AST的检测，肝脏生化指标进行TC、TG、HDL-C、LDL-C的检测。检测方法按血清生化试剂盒说明操作，检测结果见表2与表3。
表2小鼠血清指数实验结果
血脂是血浆中的中性脂肪甘油三酯和总胆固醇及类脂(磷脂、糖脂、类固醇等)的总称，TG和TC含量指数在临床上用来反映血脂水平。高密度脂蛋白和低密度脂蛋白与TC的代谢相关，HDL负责将TC转运至肝脏，LDL负责将TC转运出细胞外，血清中的HDL-C水平的提高和LDL-C水平的降低可显示出缓解NAFLD的疗效。
由表2可以看出，较正常对照组，NAFLD模型组小鼠血清TG有所升高(P＜0.01)；较NAFLD模型组，高剂量组、低剂量组和药物对照组血清TG有所降低(P＜0.05，P＜0.05，P＜0.05)。较正常对照组，NAFLD模型组小鼠血清TC显著升高(*P＜0.01)；较NAFLD模型组，高剂量组、低剂量组和药物对照组血清TC较明显降低(P＜0.01，P＜0.01P＜0.01)。较正常对照组，NAFLD模型组小鼠血清HDL-C显著升高(P＜0.01)；较NAFLD模型组，高剂量组、低剂量组和药物对照组血清HDL-C较明显降低(P＜0.01，P＜0.01，P＜0.01)。较正常对照组，NAFLD模型组小鼠血清LDL-C显著升高(P＜0.01)；较NAFLD模型组，高剂量组、低剂量组和药物对照组血清LDL-C有所降低(P＜0.05，P＜0.05，P＜0.01)。
表3小鼠肝脏指数实验结果
由表2可以看出，较正常对照组，NAFLD模型组小鼠肝脏TG有所升高(*P＜0.01)；较NAFLD模型组，高剂量组、低剂量组和药物对照组肝脏TG有所降低。较正常对照组，NAFLD模型组小鼠肝脏TC显著升高(*P＜0.01)；较NAFLD模型组，高剂量组、低剂量组和药物对照组肝脏TC较明显降低。较正常对照组，NAFLD模型组小鼠肝脏HDL-C显著降低(*P＜0.01)；较NAFLD模型组，高剂量组、低剂量组和药物对照组肝脏HDL-C较明显升高(#P＜0.01，&P＜0.01，+P＜0.01)。较正常对照组，NAFLD模型组小鼠肝脏LDL-C显著升高(*P＜0.01)；较NAFLD模型组，高剂量组、低剂量组和药物对照组血清LDL-C有所降低(#P＜0.05，&P＜0.05，+P＜0.01)。
造模及给药至药理实验结束所有组的小鼠体重均呈增长态势，除第十四天晚断食不断水导致第十五天体重略微下降，解剖后肝指数反映出小鼠肝脏脂质积累的情况，NAFLD模型组小鼠的肝指数较正常对照组增加了35.40％(P＜0.05)，高剂量组、低剂量组、药物对照组较NAFLD模型组分别降低了14.81％(无统计学意义)、32.51％(P＜0.05)、39.09％(P＜0.05)，得出结论为机械化学提取获得的沙棘黄酮类化合物有降低肝指数，减少肝细胞内脂肪的堆积，保肝护肝的作用，可以作为保肝护肝的产品。
实施例4：沙棘总黄酮对肝脏组织形态学的作用
切片制作：
固定：将新鲜肝脏组织固定在4％多聚甲醛中，固定24h以上。将组织从固定液取出修整目的部位，修好的组织放于脱水盒内。
脱水：将脱水盒放进吊篮中于脱水机内进行梯度脱水，顺序为75％乙醇4h，85％乙醇2h，90％乙醇2h，95％乙醇1h，无水乙醇1h，醇苯5min，二甲苯5min，重复一次，浸蜡1h，重复两次。
包埋：将融化的蜡放入包埋框，蜡凝固前将组织从脱水盒内取出并放入包埋框，在-20℃冻台冷却，凝固后从包埋框中取出并修整蜡块。
切片：将修整好的蜡块置于石蜡切片机上切片，每片厚4μm。切片放于摊片机40℃温水中漂浮展平，用载玻片捞起组织，再放入60℃烘箱中烤片，水分蒸发完全后取出常温保存备用。
HE染色：
切片脱蜡至水：将切片按以下顺序放入二甲苯20min，重复1次，无水乙醇10min，重复1次，95％乙醇5min，90％乙醇5min，80％乙醇5min，70％乙醇5min，蒸馏水。
苏木素染细胞核：将切片放入Harris苏木素溶液染1-3min，取出后用蒸馏水清洗，再用1％的HCl乙醇溶液分化数秒，再用蒸馏水清洗，之后用0.6％的氨水返蓝，再用蒸馏水冲洗。
伊红染细胞质：染核后的切片放入伊红染液中染色1-3min。正置光学显微镜200倍视野下随机选择5个视野拍照，照片见图2.
正常对照组：肝小叶结构清晰，肝索排列整齐，肝细胞胞质丰富、形态结构正常，肝窦未见明显扩张或挤压，无明显炎症可见。NAFLD模型组：广泛可见肝细胞胞质疏松(黑色箭头)，少量肝细胞脂肪变性，胞质中可见大小不一的圆形空泡(黄色箭头)；多见中央静脉周围有肝细胞坏死溶解，被增生的结缔组织取代(红色箭头)，伴有较多淋巴细胞浸润(蓝色箭头)。高剂量组：肝小叶结构清晰，肝索排列整齐，肝窦未见明显扩张或挤压，未见明显炎症；广泛可见大量肝细胞胞质疏松或空泡化(黑色箭头)。低剂量组：肝小叶结构清晰，肝索排列整齐，肝窦未见明显扩张或挤压；组织中可见较多肝细胞胞质轻度疏松(黑色箭头)；少见淋巴细胞与中性粒细胞浸润(黄色箭头)。药物对照组：组织中可见中央静脉周围有大量肝细胞脂肪变性，胞质中可见大小不一的圆形空泡(黑色箭头)；局部汇管区胆管周围可见少量淋巴细胞浸润(黄色箭头)。
Claims (10)
1.一种沙棘总黄酮提取物，其特征在于所述的沙棘总黄酮提取物按如下方法制备：取脱脂后的沙棘干粉，加入碳酸钠作为助剂，100～400rpm球磨5～60min，取所得混合物加去离子水A，室温下搅拌提取10～60min，所得混合液离心去除沉淀，用去离子水B洗涤沉淀，合并上清，浓缩，得粗提物，所述粗提物经大孔树脂分离，去离子水C冲洗至流出液澄清后，以体积分数80％乙醇水溶液为洗脱液洗脱，所得洗脱液经后处理即得所述沙棘总黄酮提取物；所述脱脂后的沙棘干粉与碳酸钠的质量比为1：1～1：100，所述去离子水A的体积以所述混合物的质量计为5～20mL/g，所述去离子水B的体积为2～20个大孔树脂的柱体积，所述80％乙醇水溶液的体积为2-10个大孔树脂的柱体积。
2.如权利要求1所述的沙棘总黄酮提取物，其特征在于：球磨条件为400rpm，30min,所述球磨采用行星式球磨机。
3.如权利要求1所述的沙棘总黄酮提取物，其特征在于：所述搅拌提取为室温下搅拌提取30min。
4.如权利要求1所述的沙棘总黄酮提取物，其特征在于所述大孔树脂为D101大孔树脂。
5.如权利要求1所述的沙棘总黄酮提取物，其特征在于：所述脱脂后的沙棘干粉与碳酸钠的质量比为10:1。
6.如权利要求1所述的沙棘总黄酮提取物，其特征在于：所述去离子水A的体积以所述混合物的质量计为5mL/g。
7.如权利要求1所述的沙棘总黄酮提取物，其特征在于：所述去离子水B的体积为5个大孔树脂的柱体积；所述80％乙醇水溶液的体积为3.3个大孔树脂的柱体积。
8.如权利要求1所述的沙棘总黄酮提取物，其特征在于所述脱脂的过程为：将沙棘果实烘干至恒重，粉碎至20目后用石油醚脱酯，即得脱脂后的沙棘干粉。
9.如权利要求1所述的沙棘总黄酮提取物，其特征在于所述后处理的方法为：将所述洗脱液浓缩除去乙醇后，冷冻干燥，即得所述沙棘总黄酮提取物。
10.如权利要求1所述的沙棘总黄酮提取物在制备预防或治疗非酒精性脂肪肝的药物中的应用。
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