CN106748666B - 一种降血糖天然药物及其制备糖尿病或肥胖症药物用途 - Google Patents
一种降血糖天然药物及其制备糖尿病或肥胖症药物用途 Download PDFInfo
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Abstract
本发明涉及医药技术领域，涉及从中国碧绿米仔兰Aglaia abbreviata C.Y.Wu叶片中提取分离得到的、具有降血糖功能的新天然药物，该药物为联苄类化合物联苄米仔兰素D(aglaiabbrevin D)。体外PTP1B抑制试验表明，该化合物对蛋白络氨酸酯酶1B(PTP1B)具有显著抑制活性，其活性强于阳性对照药物齐墩果酸，因此可制成药物制剂作为PTP1B抑制剂，用于治疗糖尿病、肥胖症及其并发症，也可以制造有益于糖尿病患者或肥胖患者的保健食品。
Description
技术领域
本发明涉及医药技术领域，涉及从中国碧绿米仔兰Aglaia abbreviata C.Y.Wu叶片中提取分离得到的、具有降血糖功能的新天然药物，该药物为联苄类化合物联苄米仔兰素D(aglaiabbrevin D)。可制成药物制剂作为PTP1B抑制剂，用于治疗糖尿病、肥胖症及其并发症，也可以制造有益于糖尿病患者或肥胖患者的保健食品。
背景技术
糖尿病(diabetes mellitus)是一组由遗传和环境因素相互作用而引起的临床综合症。目前，一般将糖尿病分为两类，I-型糖尿病(胰岛素依赖型糖尿病，insulin-dependent diabetes mellitus，IDDM)与II-型糖尿病(非胰岛素依赖型糖尿病，non-insulin-dependent diabetes mellitus，NIDDM)。糖尿病中90％以上是II-型糖尿病。
II-型糖尿病的特点是胰岛素敏感组织如骨骼肌、肝、脂肪组织对胰岛素作用的抵抗。蛋白酪氨酸磷酸酯酶(PTPases)在平衡细胞内胰岛素作用通路中相关蛋白酪氨酸磷酸化水平中的作用越来越受到重视，成为治疗II-型糖尿病的新途径。PTPase包括一大族跨膜(受体型)和胞内(非受体型)酶，参与调控一系列重要生命过程。目前，对PTPase在胰岛素通路中受体或受体后环节影响正常胰岛素作用的研究，主要集中在LAR-PTPase、SHPTP-2、PTP1B。
PTP1B是第一个被鉴定的蛋白酪氨酸磷酸酯酶(protein tyrosinephosphatase),通过PTP1B剔除的老鼠实验表明,PTP1B通过对胰岛素受体的脱磷酰化，进而在调节胰岛素敏感性和脂肪代谢过程中起着非常重要的作用。因而，选择性的、高活性的PTP1B抑制剂在II-型糖尿病、肥胖症及其并发症的治疗中有重要的价值。
发明内容
本发明是从中国碧绿米仔兰(A.abbreviata)叶片中提取分离得到的、具有降血糖作用的新联苄类化合物联苄米仔兰素D(aglaiabbrevin D)。经药理试验研究表明，该化合物对蛋白络氨酸磷酸酯酶1B(PTP1B)具有显著抑制活性，其活性强于阳性对照药齐墩果酸。
因此，本发明的一个目的是提供新的联苄类化合物联苄米仔兰素D。
本发明的另一个目的是提供所述联苄米仔兰素D的制备方法。
本发明的进一步目的是提供所述联苄米仔兰素D的用途。具体地说，所述联苄米仔兰素D在制备蛋白酪氨酸磷酸酯酶1B(PTP1B)抑制剂的药物中的应用，进一步在制备治疗糖尿病、肥胖症及其并发症的药物或保健食品中的应用。
根据本发明的第一个目的，本发明首次从碧绿米仔兰叶片中发现了联苄米仔兰素D，其化学结构如下所示：
根据本发明的第二个目的，本发明提供所述联苄米仔兰素D的制备方法，它是从碧绿米仔兰叶片中分离得到的，具体步骤如下：
1)制备提取物浸膏
将粉碎的碧绿米仔兰(A.abbreviata)叶片用乙醇按常规渗漉提取，得提取液，将提取液减压浓缩回收乙醇，得粗浸膏；
2)分离纯化
(1)将上述粗浸膏分散于水中成混悬液，将混悬液依次用石油醚、乙酸乙酯和正丁醇萃取，所得萃取液浓缩分别得到石油醚提取浸膏、乙酸乙酯提取浸膏和正丁醇提取浸膏；
(2)将乙酸乙酯浸膏进行硅胶柱层析，以石油醚/丙酮梯度洗脱，根据TLC显色合并相似流份得到4个组分A、B、C、D；其中组分B即石油醚/丙酮体积比8:2和7:3洗脱部分经Sephadex LH-20凝胶柱层析【层析柱规格：4.0(直径)×120(长度)cm；Sephadex LH-20凝胶干重：150g】，以二氯甲烷/甲醇体积比1:1洗脱，根据TLC显色合并相似流份得到6个组分(B1-B6)；组份B3即二氯甲烷/甲醇体积比1:1洗脱体积为90～120mL洗脱部分，再经硅胶柱层析，以石油醚/丙酮体积比75:25洗脱，最后经制备型HPLC，以甲醇/水80:20体积比洗脱，得到本发明化合物联苄米仔兰素D。
在上述制备方法中，在制备提取物浸膏步骤中，所述提取采用的乙醇为95％乙醇。
在上述制备方法中，在分离步骤中，石油醚/丙酮梯度洗脱的浓度依次为体积比100:0、90:10、80:20、70:30、50:50、30:70和0:100。
在上述制备方法中，在分离步骤中，所述制备型HPLC的色谱柱的填料为RP-18。
根据本发明的第三个目的，本发明提供了所述联苄米仔兰素D在制备PTP1B抑制剂、糖尿病药物、肥胖症药物的用途。以及制备用于糖尿病患者或肥胖患者保健食品的用途。
为了达到应用目的，可以制成片剂、胶囊、颗粒剂、口服液、缓释制剂、控释制剂、纳米制剂或注射剂等形式。
本发明对所得联苄米仔兰素D进行了体外PTP1B抑制试验，结果表明该化合物对PTP1B有明显的抑制活性。因此，可用来制备PTP1B抑制剂，用于治疗糖尿病、肥胖症及其并发症。
附图说明
图1PTP1B抑制活性测试原理
具体实施方式
在如下的实施例中所指的联苄米仔兰素D的化学结构式(结构式中的阿拉伯数字是化学结构中碳原子的标位)：
实施例1所述联苄米仔兰素D的制备
1.制备碧绿米仔兰叶片提取物浸膏
(1)制备提取液
将粉碎的中国碧绿米仔兰(A.abbreviata)叶片(采自云南西双版纳)7.8kg(干重)分别用40L 95％乙醇渗漉提取三次，每次渗漉2天，合并提取液；
(2)制备提取物浸膏
将上述提取液在温度≤45℃减压浓缩回收乙醇，得粗浸膏530g；
2.分离纯化
1)将上述粗浸膏分散于6L水中成混悬液，将混悬液依次用石油醚(4L)、乙酸乙酯(4L)和正丁醇(2L)分别萃取三次，所得萃取液减压浓缩分别得到石油醚提取浸膏(58g)、乙酸乙酯提取浸膏(186g)和正丁醇提取浸膏(152g)；
2)将乙酸乙酯浸膏进行硅胶柱层析，以石油醚/丙酮梯度洗脱；梯度洗脱的浓度依次为体积比100:0、90:10、80:20、70:30、50:50、30:70和0:100，根据TLC显色合并相似流份得到4个组分(A、B、C、D)；
3)组分B即石油醚/丙酮体积比8:2和7:3洗脱部分经Sephadex LH-20凝胶柱层析【层析柱规格：4.0(直径)×120(长度)cm；Sephadex LH-20凝胶干重：150g】，以二氯甲烷/甲醇体积比1:1洗脱，根据TLC显色合并相似流份得到6个组分(B1-B6)；
4)组份B3，即二氯甲烷/甲醇体积比1:1洗脱体积为90～120mL洗脱部分，再经硅胶柱层析，以石油醚/丙酮体积比75:25洗脱，最后经制备型HPLC(色谱柱的填料为RP-18)，以甲醇/水体积比80:20洗脱，流速为3.5mL/min，保留时间为11.5min，得到本发明化合物联苄米仔兰素D，经鉴定为新化合物。
3.结构鉴定
按常规经NMR、HRESIMS、UV及IR等多种现代光谱技术，确定了化合物联苄米仔兰素D的化学结构，其理化性质如下：
黄色粉末,分子式为C24H30O3；
紫外光谱UV(MeOH)λmax(logε):216(4.33),232(3.98),285(3.72)nm；
红外光谱IR(KBr)νmax:3320,1612,1515,1420,1203cm–1；
高分辨质谱HR-ESI-MS m/z 365.2114[M–H]–(calcd for C24H29O3 –,365.2117)；
核磁共振氢谱1H NMR(600MHz)及核磁共振碳谱13C NMR(150MHz)数据见表一
表一 所述联苄米仔兰素D的1H和13C NMR(ppm in CDCl3)
实施例2 PTP1B抑制活性的测试：
测试原理：见图1。利用分子生物学手段在大肠杆菌系统表达人源蛋白络氨酸磷酸酯酶1B(hPTP1B)催化结构域，经纯化后的hPTP1B重组蛋白能水解底物对硝基苯磷酸(p-Nitrophenyl phosphate，pNPP)的磷脂键，得到黄色可溶产物对硝基苯酚(p-Nitrophenol)，该产物在410nm处有很强的光吸收，因此可以直接检测410nm处光吸收的变化及观察酶的活性变化以及化合物对酶活性的抑制情况。
标准的测活体系：10mM Tris.Cl三(羟甲基)氨基甲烷盐酸盐)，pH7.6，10mM pNPP，2％DMSO，100nM hPTP1B。
观察指标：动态测定波长为410nm处的光吸收，时间为3分钟，其动力学曲线一级反应的斜率作为酶的活性指标。
试验方法：用于筛选的蛋白酪氨酸磷酸酯酶PTP1B是从大肠杆菌中表达并纯化的GST融合蛋白。采用紫外适用底物对硝基苯磷酸(pNPP)，观察不同浓度对重组酶的活性的抑制作用，以初步评价化合物的药用效果。临用前将样品溶于DMSO配成适当浓度，3倍稀释，7个稀释度，设置三复孔，取2μL样品溶液加入96孔板，然后加入88μL assay mix(assaybuffer，pNPP，H2O)，再加入10μL PTP1B。将96孔板置于VERSAmax上动态检测波长为410nm处检测光吸收值，时间为3分钟。
实验结果的评判与解释：
筛选结果是化合物浓度为20μg/ml时对酶活性的百分抑制率，抑制率高于50％时，按常规筛选(将抑制率高于50％的被检测化合物稀释成不同的浓度，依上述测试方法进行反应，所有试验均设置复孔)得出IC50，阳性对照齐墩果酸的IC50为2.74±0.20μM。
实验结果：本发明化合物联苄米仔兰素D对PTP1B酶抑制活性的IC50为2.44±0.35μM。
实验结论：通过分子生物学试验，可以看出化合物联苄米仔兰素D对蛋白络氨酸酯酶1B(PTP1B)显著的抑制活性，其活性强于阳性对照药物齐墩果酸。因此，本发明的联苄米仔兰素D可用于制备糖尿病、肥胖症及其并发症的药物中。
Claims (1)
1.一种具有降血糖作用的联苄米仔兰素D的制备方法，其特征在于，制备步骤如下：
1)制备提取物浸膏
将粉碎的碧绿米仔兰A.abbreviata叶片用乙醇按常规渗漉提取，得提取液，将提取液减压浓缩回收乙醇，得粗浸膏；
2)分离纯化
2.1)将上述粗浸膏分散于水中成混悬液，将混悬液依次用石油醚、乙酸乙酯和正丁醇萃取，所得萃取液浓缩分别得到石油醚提取浸膏、乙酸乙酯提取浸膏和正丁醇提取浸膏；
2.2)将乙酸乙酯浸膏进行硅胶柱层析，以石油醚/丙酮梯度洗脱，根据TLC显色合并相似流份得到4个组分A、B、C、D；其中组分B即石油醚/丙酮体积比8:2和7:3洗脱部分经SephadexLH-20凝胶柱层析；层析柱的规格为直径4.0cm，长度120cm，Sephadex LH-20凝胶干重：150g；以二氯甲烷/甲醇体积比1:1洗脱，根据TLC显色合并相似流份得到6个组分，即B1-B6；组份B3即二氯甲烷/甲醇体积比1:1洗脱体积为90～120mL洗脱部分，再经硅胶柱层析，以石油醚/丙酮体积比75:25洗脱，最后经制备型HPLC，以甲醇/水80:20体积比洗脱，得到所述联苄米仔兰素D；
在制备提取物浸膏步骤中，所述提取采用的乙醇为95％乙醇；
在分离步骤中，石油醚/丙酮梯度洗脱的浓度依次为体积比100:0、90:10、80:20、70:30、50:50、30:70和0:100；
在分离步骤中，所述制备型HPLC的色谱柱的填料为RP-18；
所述具有降血糖作用的联苄米仔兰素D，具有如下化学结构：
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