CN113480503B - 一种具有降血糖作用的黑桑素b及其制备方法和应用 - Google Patents
一种具有降血糖作用的黑桑素b及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种具有降血糖作用的黑桑素B及其制备方法和应用，该黑桑素B的结构如式I所示，其是从黑桑枝条中提取分离得到的，其具体制备方法包括以下步骤：采用乙醇对粉碎的黑桑枝条进行渗漉提取，将得到的提取液减压浓缩后得到粗浸膏，分离纯化后得到该黑桑素B。本发明提出了一种新型化合物黑桑素B，其对于PTP1B有明显的抑制活性，将其制备为治疗糖尿病、肥胖症及其并发症的药物或食品将具有重要的价值。
Description
技术领域
本发明属于医药技术领域，具体涉及一种具有降血糖作用的黑桑素B及其制备方法和应用。
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 m ellitus，IDDM)与II-型糖尿病(非胰岛素依赖型糖尿病，non-insulin-dependent diabetes mellit us，NIDDM)，其中，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
本发明的目的是解决现有技术的不足，提出了一种具有降血糖作用新型化合物黑桑素B，其结构如式I所示：
上述黑桑素B是发明人从黑桑枝条中提取分离得到的，其具体制备方法包括以下步骤：
(1)采用乙醇对粉碎的黑桑枝条进行渗漉提取，将得到的提取液减压浓缩后得到粗浸膏；
(2)将所述粗浸膏分散于水中得到混悬液，然后依次用石油醚和乙酸乙酯萃取，随后将得到的萃取液浓缩后得到石油醚提取浸膏和乙酸乙酯提取浸膏；
(3)将所述乙酸乙酯提取浸膏进行硅胶柱层析，采用体积比为90:10、85：15、80：20、70：30、60：40、50：50、30:70和0:100的石油醚/丙酮进行梯度洗脱，根据TCL显色合并相似流份得到6个组分A、B、C、D、E、F；采用体积比为1:1的二氯甲烷/甲醇对组分C进行洗脱，根据TCL显色合并相似流份得到8个组分C1、C2、C3、C4、C5、C6、C7、C8；
(4)将组分C4进行硅胶柱层析，采用体积比为80:20的石油醚/丙酮进行洗脱，最后进行HPLC，以体积比为92:8的正己烷/异丙醇进行洗脱，流速为4mL/min，保留时间为25.6min，得到所述黑桑素B；
所述组分C为体积比85:15和80:20的石油醚/丙酮的洗脱部分，所述C4为体积比1:1洗脱体积为100～120mL的二氯甲烷/甲醇的洗脱部分。
优选的，在上述步骤(4)中，所述HPLC的色谱柱填料为正相硅胶。
优选的，在上述步骤(1)中，用于渗漉提取的所述乙醇为95％乙醇。
经过发明人的实验验证，发现上述黑桑素B能够作为PTP1B抑制剂应用在制备治疗糖尿病、肥胖症及其并发症的药物中，或者用于糖尿病患者或肥胖患者的食品中。
上述药物为片剂、胶囊、颗粒剂、口服液、缓释制剂、控释制剂、纳米制剂或注射剂。
本发明的有益效果为：本发明提出了一种新型化合物黑桑素B，其对于PTP1B有明显的抑制活性，其活性强于阳性对照药物齐墩果酸，将其制备为治疗糖尿病、肥胖症及其并发症的药物或食品将具有重要的价值。
附图说明
图1所示为PTP1B抑制活性测试原理图。
具体实施方式
以下将结合实施例和附图对本发明的构思及产生的技术效果进行清楚、完整的描述，以充分地理解本发明的目的、方案和效果。
以下实施例中的黑桑素B的化学结构式如下所示(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：黑桑素B的制备及鉴定
一、黑桑素B的制备：
1.制备黑桑枝条提取物浸膏
(1)制备提取液
将粉碎的中国黑桑(M.nigra)枝条(采自广西金秀县)3.0kg(干重)分别用15L95％乙醇渗漉提取三次，每次渗漉7天，合并提取液；
(2)制备提取物浸膏
将上述提取液在温度≤45℃的条件下减压浓缩回收乙醇，得粗浸膏165g；
2.分离纯化
1)将上述粗浸膏分散于1L水中成混悬液，将混悬液依次用石油醚(1L)和乙酸乙酯(1L)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(48g)和乙酸乙酯提取浸膏(95g)；
2)将乙酸乙酯浸膏进行硅胶柱层析(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)，以体积比90:10、85:15、80:20、70:30、60:40、50:50、30:70和0:100的石油醚/丙酮进行梯度洗脱；根据TLC显色合并相似流份得到6个组分(A、B、C、D、E、F)；
3)组分C(石油醚/丙酮体积比85:15和80:20的洗脱部分)以体积比1:1的二氯甲烷/甲醇洗脱，根据TLC显色合并相似流份得到8个组分(C1-C8)；
4)组份C4(二氯甲烷/甲醇体积比1:1洗脱体积为100～120mL的洗脱部分)经硅胶柱层析，以体积比80:20的石油醚/丙酮洗脱，最后经HPLC(色谱柱的填料为正相硅胶)，以体积比92:8的正己烷/异丙醇洗脱，流速为4mL/min，保留时间为25.6min，得到本实施例的化合物黑桑素B，经鉴定为新化合物。
二、黑桑素B的鉴定：
采用NMR、HRESIMS、UV及IR等多种现代光谱技术，对本实施例制得的黑桑素B进行检测，确定了化合物黑桑素B的化学结构，其理化性质如下：
黄色粉末，分子式为C39H32O9；紫外光谱UV(MeOH)λmax(logε):207(4.48),273(3.32),327(4.02)nm；红外光谱IR(KBr)νmax:3390,2923,1720,1620,1489,1440,1339,1266,1117,1051,969,818cm–1；高分辨质谱(+)-HRESIMS m/z 667.1939[M+Na]+(calcd forC39H32O9Na,667.1944)。
核磁共振氢谱1H NMR(400MHz)及核磁共振碳谱13C NMR(100MHz)数据如表1所示。
表1黑桑素B的1H和13C NMR(ppm in CD3OD)
效果测试试验：
PTP1B抑制活性的测试：
测试原理：如图1所示。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)，pH＝7.6，10mMpNPP，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个稀释浓度，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2.54±0.14μM。
实验结果：本发明化合物黑桑素B对PTP1B酶抑制活性的IC50为1.26±0.34μM。
实验结论：通过分子生物学试验，可以看出化合物黑桑素B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，本发明的黑桑素B可用于制备糖尿病、肥胖症及其并发症的药物中。
以上所述，只是本发明的较佳实施例而已，本发明并不局限于上述实施方式，只要其以相同的手段达到本发明的技术效果，都应属于本发明的保护范围。在本发明的保护范围内其技术方案和/或实施方式可以有各种不同的修改和变化。
Claims (5)
1.一种具有降血糖作用的黑桑素B，其特征在于，其结构如式I所示：
2.一种权利要求1所述黑桑素B的制备方法，其特征在于，包括以下步骤：
（1）采用乙醇对粉碎的黑桑枝条进行渗漉提取，将得到的提取液减压浓缩后得到粗浸膏；
（2）将所述粗浸膏分散于水中得到混悬液，然后依次用石油醚和乙酸乙酯萃取，随后将得到的萃取液浓缩后得到石油醚提取浸膏和乙酸乙酯提取浸膏；
（3）将所述乙酸乙酯提取浸膏进行硅胶柱层析，采用体积比为90:10、85：15、80：20、70：30、60：40、50：50、30:70和0:100的石油醚/丙酮进行梯度洗脱，根据TCL显色合并相似流份得到6个组分A、B、C、D、E、F；采用体积比为1:1的二氯甲烷/甲醇对组分C进行洗脱，根据TCL显色合并相似流份得到8个组分C1、C2、C3、C4、C5、C6、C7、C8；
（4）将组分C4进行硅胶柱层析，采用体积比为80:20的石油醚/丙酮进行洗脱，最后进行HPLC，以体积比为92:8的正己烷/异丙醇进行洗脱，流速为4mL/min，保留时间为25.6min，得到所述黑桑素B；
所述组分C为体积比85:15和80:20的石油醚/丙酮的洗脱部分，所述C4为体积比1:1洗脱体积为100~120mL的二氯甲烷/甲醇的洗脱部分。
3.根据权利要求2所述的制备方法，其特征在于，步骤（4）中，所述HPLC的色谱柱填料为正相硅胶。
4.根据权利要求2所述的制备方法，其特征在于，步骤（1）中，用于渗漉提取的所述乙醇为95%乙醇。
5.权利要求1所述的黑桑素B在制备治疗糖尿病、肥胖症药物中的应用。
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