CN104491048A - 一种枇杷叶总倍半萜苷提取物及其制备方法与应用 - Google Patents
一种枇杷叶总倍半萜苷提取物及其制备方法与应用 Download PDFInfo
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Abstract
本发明公开了一种枇杷叶总倍半萜苷提取物及其制备方法与应用。该提取物以枇杷(Eriobotrya japonica)的干燥叶为原料，制备方法包括将干燥枇杷叶经一定浓度乙醇回流提取，过滤，减压浓缩；浓缩液离心，取上清液经大孔树脂吸附，水-乙醇梯度洗脱，收集一定浓度乙醇洗脱部分；收集液减压浓缩，经聚酰胺柱层析，水-甲醇梯度洗脱，收集一定浓度甲醇洗脱部分并回收溶剂，干燥，得到总倍半萜苷提取物。本发明制备的枇杷叶总倍半萜苷提取物能显著促进人肝癌细胞(HepG2)的葡萄糖消耗，提示其具有降血糖活性，可作为治疗糖尿病的药物或保健品进一步研究开发。本发明为枇杷叶治疗糖尿病的应用提供了科学依据，为开发安全高效的新型抗糖尿病药物或保健品提供了新资源。
Description
技术领域
本发明属于中药领域，具体涉及一种从中药枇杷叶中得到的总倍半萜苷提取物及其制备方法和在治疗糖尿病药物或保健品上的应用。
背景技术
枇杷叶是蔷薇科枇杷属植物枇杷(Eriobotryajaponica(Thunb.)Lindl.)的干燥叶，作为一味常用中药，收载于历版中国药典供中医临床使用，具有清肺止咳、降逆止呕等作用，用于治疗肺热咳嗽、气逆喘气、胃热呕逆、烦热口渴等症状，符合中医治疗糖尿病“消渴”的范畴。1988年Noreen等曾报道枇杷叶的乙醇提取物对正常大鼠具有显著但短暂的降低血糖作用。本课题组从2000年起开展“降血糖植物资源调查”工作，在民间走访中发现枇杷叶在我国一些地区作为单验方用于治疗糖尿病，用量少、效果好，曾见有治愈糖尿病的病例，提示枇杷叶可能成为对糖尿病具有良好疗效的新的中药资源。
关于枇杷叶的化学成分，据文献报道的主要有黄酮类、多酚类、三萜和倍半萜苷类以及其它类型化合物。为了验证枇杷叶的降血糖功能，课题组采用不同动物模型开展了药效学评价工作，试验表明枇杷叶乙醇粗提物能显著降低四氧嘧啶糖尿病小鼠的血糖水平(The American Journal of Chinese Medicine，2007，35：705-711)，进一步研究发现其总三萜酸组分和总倍半萜苷组分活性较强(Journal of Ethnopharmacology，2009，122：486-491)。课题组还针对分离得到的三萜酸单体成分蔷薇酸和倍半萜苷单体成分橙花叔醇-3-O-α-L-吡喃鼠李糖基(1→4)-α-L-吡喃鼠李糖基(1→2)-[α-L-吡喃鼠李糖基(1→6)]-β-D-吡喃葡萄糖苷进行了体内降血糖作用评价，结果表明它们分别能显著降低正常小鼠和四氧嘧啶糖尿病小鼠空腹血糖水平(Phytomedicine，2008，15：98-102)。国内已公开的发明专利(200810018820.8，200810024412.3，200410015695.7)分别指出了枇杷叶总三萜酸和总黄酮具有抗糖尿病作用。总倍半萜苷作为枇杷叶的重要活性成分之一，在其降糖活性及其作用机理研究方面，缺乏更广泛而深入的研究。
糖尿病分为1型和2型，2型糖尿病的主要特征是胰岛素抵抗，肝脏、骨骼肌、脂肪等靶组织对葡萄糖的摄取减少，造成血液中葡萄糖水平升高，形成高血糖。因此，提高胰岛素效能，包括促进器官组织对葡萄糖的摄取成为2型糖尿病药物研究重要的研究课题。HepG2细胞源于人肝胚胎瘤细胞，是分化较好的人肝癌细胞，仍有肝细胞的形态和功能。HepG2细胞可以表达胰岛素受体和胰岛素样生长因子的代谢反应，能全面模拟肝细胞对周围环境中葡萄糖的摄入和消耗，因此检测枇杷叶总倍半萜苷干预下HepG2细胞的葡萄糖消耗，探讨其对糖代谢的影响，可以评价枇杷叶总倍半萜苷的降血糖作用，对防治糖尿病及其并发症有积极意义。
发明内容
本发明的目的是为了解决现有技术中存在的缺陷，提供一种具有显著促进人肝癌细胞(HepG2)的葡萄糖消耗活性从而达到抗糖尿病效果的枇杷叶总倍半萜苷提取物及其制备方法以及在降糖药物或保健品制备方面的应用。
为了达到上述目的，本发明提供了一种枇杷叶总倍半萜苷提取物及其制备方法。该提取物以枇杷的干燥叶片为原料，通过以下操作步骤制备得到：
将枇杷叶干燥和粉碎后，用乙醇溶液回流提取，所述乙醇溶液的质量百分比浓度为60-100％，用量为6-10倍药材质量体积，提取次数为1-3次，提取温度为50-80℃，提取时间为1-3小时；提取液过滤，减压浓缩至有沉淀析出，浓缩液分次离心，速率为2000-4000转/分钟；取上清液，流经大孔树脂柱XAD16吸附，先用去离子水冲洗树脂，再用水-乙醇梯度洗脱，收集乙醇浓度为60-80％之间的洗脱液，减压浓缩；浓缩液进一步经聚酰胺柱层析，先用去离子水洗脱，再用水-甲醇梯度洗脱，收集甲醇浓度为0-20％之间的洗脱液，回收溶剂，干燥，得到总倍半萜苷提取物。
以上述方法制备得到的枇杷叶总倍半萜苷提取物，包含以橙花叔醇-3-O-α-L-吡喃鼠李糖基(1→4)-α-L-吡喃鼠李糖基(1→2)-[α-L-吡喃鼠李糖基(1→6)]-β-D-吡喃葡萄糖苷和橙花叔醇-3-O-α-L-吡喃鼠李糖基(1→4)-α-L-吡喃鼠李糖基(1→2)-β-D-吡喃葡萄糖苷两种倍半萜苷为代表的化学成分，该提取物中总倍半萜苷的质量百分含量＞50％。
上述枇杷叶总倍半萜苷提取物在一定浓度下可以显著促进人肝癌细胞(HepG2)的葡萄糖消耗，提示其具有降低血糖的功能，可以应用于制备抗糖尿病药物或保健品。
所述抗糖尿病药物或保健品分别为含有治疗有效剂量或功效成分的枇杷叶总倍半萜苷提取物在药学上可接受的载体和保健食品。
附图说明
图1为本发明实施例2中制备的枇杷叶总倍半萜苷提取物HPLC图。
图2为本发明实施例3中制备的枇杷叶总倍半萜苷提取物对HepG2细胞葡萄糖消耗的影响效果图。
具体实施方式
下面结合实施例对本发明作进一步详细的描述，但本发明的实施方式不限于此。
实施例1：
取干燥枇杷叶1kg，粉碎，用质量百分比浓度为80％的乙醇溶液10L在70℃下回流提取2次，每次2小时，合并提取液，过滤，减压浓缩至有沉淀析出，3000转/分钟离心15分钟，取上清液备用；
称取AmberliteTMXAD16大孔吸附树脂8008，加入适量蒸馏水，在室温下放置过夜，使树脂充分溶胀，湿法装柱，用95％乙醇流动清洗柱子，最后用蒸馏水充分淋洗柱子至流出液无乙醇味，放置过夜；
将上述上清液缓缓流过上述处理和平衡过的大孔树脂柱，先用去离子水冲洗，再用不同浓度的乙醇梯度洗脱，收集乙醇浓度为60-80％之间的洗脱液，流出液经TLC展开，喷洒香草醛-硫酸试剂显淡紫色；减压浓缩收集液，备用。
上述浓缩液进一步流过经处理过的聚酰胺(200mesh，2008)柱，先用去离子水洗脱，再用水-甲醇梯度洗脱，收集甲醇浓度为0-20％之间的洗脱液，回收溶剂，干燥，得到总倍半萜苷提取物。
以上得到的总倍半萜苷提取物用一定体积色谱纯甲醇溶解，45μm微孔滤膜过滤，进行制备型高效液相色谱(岛津LC-6AD)分离，色谱方法如下：
流动相：A水，B甲醇；洗脱方法：等度洗脱，65％B，0-50min；流速6mL/min，室温，检测波长325nm，进样体积每次250μL，运行时间55mins，C18柱(9.4mm ID×250mm，5μm)分离，收集保留时间分别为20.0min和34.0min处馏分。
馏分减压浓缩并经高真空干燥，分别得到2个倍半萜苷类化合物，根据MS和NMR数据与文献比较，鉴定它们的结构分别为：橙花叔醇-3-O-α-L-吡喃鼠李糖基(1→4)-α-L-吡喃鼠李糖基(1→2)-[α-L-吡喃鼠李糖基(1→6)]-β-D-吡喃葡萄糖苷(I)和橙花叔醇-3-O-α-L-吡喃鼠李糖基(1→4)-α-L-吡喃鼠李糖基(1→2)-β-D-吡喃葡萄糖苷(II)。它们的结构如下图所示：
I R1＝Rha-Rha，R2＝Rha
II R1＝Rha-Rha，R2＝H
实施例2：枇杷叶总倍半萜苷提取物的总倍半萜苷含量测定(HPLC法)
分别精密称取化合物I和化合物II各约10mg(自制，HPLC检测其纯度＞95％)为对照品，溶于甲醇，稀释并定容至10mL容量瓶中，分别作为对照品储备液，绘制标准曲线。高效液相色谱条件：C18色谱柱(Dionex-Acclaim 120，4.6×250mm，5μm)；柱温30℃；流速1.0mL/min；进样量20μL；检测波长210nm；流动相为甲醇(A)-水溶液(B)，等度洗脱(0-25min，70％A)。
精确称取枇杷叶总倍半萜苷提取物样品25.0mg，置50mL容量瓶中，甲醇溶解，定容至刻度，过滤得供试品溶液，按上述方法测定。如图1所示，以外标法，按峰面积分别计算化合物I和II的含量和总含量，两个倍半萜苷的总含量＞50％。
实施例3：枇杷叶总倍半萜苷提取物的体外降血糖活性测定-HepG2葡萄糖消耗实验
细胞株及培养：人肝癌细胞株HepG2购于中科院上海细胞所，接种于含10％小牛血清的中糖DMEM培养基中(含葡萄糖11.1mmol/L，补充青霉素、链霉素各100U/L)，置于37℃含5％的细胞培养箱中培养。HepG2细胞呈贴壁生长，0.25％胰酶消化，每3天按1∶3的比例传代1次，取对数生长期的细胞用于实验。试验时将细胞悬液以1.5×105的密度接种于96孔培养板中，置培养箱中培养使之形成单层贴壁细胞。
实验方法：接种24h后细胞已完全贴壁形成单层细胞层，吸掉培养液，用PBS冲洗2次，然后各孔加入终浓度为300μg/mL、200μg/mL和100μg/mL的枇杷叶总倍半萜苷提取物(不同质量的提取物溶解在含11.1mmol/L葡萄糖的DMEM中过滤而成)以及50μg/mL的化合物I和II，同时设立正常对照组和阳性对照组。在培养箱中孵育24h，将培养液移出，用葡萄糖试剂盒测葡萄糖含量。用各组空白孔含糖量的平均值减去其余各孔的含糖量，即是24h各孔细胞的葡萄糖消耗量。板中下层细胞用200g/L KOH裂解，考马斯亮蓝法测定蛋白含量。
测定结果：如图2所示，枇杷叶总倍半萜苷提取物一定浓度下(100～300μg/mL)可显著增加HepG2细胞葡萄糖消耗量，并呈现剂量相关性；化合物I和II在50μg/mL浓度时，也显示显著增加葡萄糖消耗活性，各样品组与不含药的对照组相比有显著性差异(P＜0.01)。该结果提示枇杷叶总倍半萜苷提取物及化合物I和II具有降低血糖的功能。
Claims (4)
1.一种枇杷叶总倍半萜苷提取物及其制备方法与应用，其特征在于：采用植物化学方法，从枇杷叶中分离得到总倍半萜苷提取物，该提取物具有抗糖尿病功能，可以应用于制备抗糖尿病药物或保健品。
2.根据权利要求1所述的枇杷叶总倍半萜苷提取物，其中包含橙花叔醇-3-O-α-L-吡喃鼠李糖基(1→4)-α-L-吡喃鼠李糖基(1→2)-[α-L-吡喃鼠李糖基(1→6)]-β-D-吡喃葡萄糖苷和橙花叔醇-3-O-α-L-吡喃鼠李糖基(1→4)-α-L-吡喃鼠李糖基(1→2)-β-D-吡喃葡萄糖苷两种代表性倍半萜苷化学成分，且该提取物中总倍半萜苷的质量百分含量＞50％。
3.根据权利要求1所述的枇杷叶总倍半萜苷提取物的制备方法为：将枇杷叶干燥和粉碎后，用乙醇溶液回流提取，所述乙醇溶液的质量百分比浓度为60-100％，用量为6-10倍药材质量体积，提取次数为1-3次，提取温度为50-80℃，提取时间为1-3小时；提取液过滤，减压浓缩至有沉淀析出，浓缩液分次离心，速率为2000-4000转/分钟；取上清液，流经大孔树脂柱XAD16吸附，先用去离子水冲洗树脂，再用水-乙醇梯度洗脱，收集乙醇浓度为60-80％之间的洗脱液，减压浓缩；浓缩液进一步经聚酰胺柱层析，先用去离子水洗脱，再用水-甲醇梯度洗脱，收集甲醇浓度为0-20％之间的洗脱液，回收溶剂，干燥，得到总倍半萜苷提取物。
4.根据权利要求1所述的枇杷叶总倍半萜苷提取物的功能和应用在于：该提取物在一定浓度下具有显著促进人肝癌细胞(HepG2)的葡萄糖消耗作用，可以应用于制备抗糖尿病药物或保健品。所述抗糖尿病药物或保健品分别为含有治疗有效剂量或功效成分的枇杷叶总倍半萜苷提取物在药学上可接受的载体和保健食品。
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| CN106511479A (zh) * | 2016-12-19 | 2017-03-22 | 江苏省中国科学院植物研究所 | 一种芡实壳提取物及其制备方法与应用 |
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| CN106511479A (zh) * | 2016-12-19 | 2017-03-22 | 江苏省中国科学院植物研究所 | 一种芡实壳提取物及其制备方法与应用 |
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