CN113813300B - 一种光果甘草提取物的制备方法及用途 - Google Patents
一种光果甘草提取物的制备方法及用途 Download PDFInfo
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- CN113813300B CN113813300B CN202111287796.XA CN202111287796A CN113813300B CN 113813300 B CN113813300 B CN 113813300B CN 202111287796 A CN202111287796 A CN 202111287796A CN 113813300 B CN113813300 B CN 113813300B
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- extract
- glycyrrhiza glabra
- ethanol
- ethyl acetate
- glabridin
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Abstract
本发明涉及一种光果甘草提取物的制备方法及其用途，通过有机溶剂乙醇回流提取，大孔树脂分离纯化，石油醚、乙酸乙酯沉降处理从光果甘草中获得降糖有效组分，其光甘草定含量在40‑70%，经初步的活性筛选试验证明：本发明所述的方法获得的有效部位具有蛋白酪氨酸磷酸酶1B（PTP‑1B）和α‑葡萄糖苷酶抑制作用，可作为原料或辅助剂用于防治糖尿病的药物或保健品。本发明所述的方法工艺简单，生产成本低，有效成分质量稳定，易于控制。
Description
技术领域
本发明涉及一种光果甘草提取物的制备方法，以及该提取物在降血糖的药物、保健品或其他功能性食品中的用途。
背景技术
甘草是重要的大宗中药材，具有补脾益气，清热解毒，祛痰止咳，缓急止痛，调和诸药的功效，在中药方剂中应用十分广泛。有统计表明，甘草的需求量居大宗药材之首，且逐年递增，但甘草产业目前仍以药材饮片和提取物等粗加工产业为主，甘草产品的深加工技术落后，产业规模小，严重限制了甘草产业链的延伸和甘草产业的发展。
光果甘草Glycyrrhiza glabra L.是甘草属(Glycyrrhiza)的一种植物，具有甘草酸、光甘草定等多种功能成分。而光甘草定具有出色的抗炎、抗氧化和抗黑色素形成的作用，在世界化妆品界，被誉为美白黄金，是目前国际上高档美白化妆品的主要功效成分，如雅诗兰黛、兰蔻、谷雨、赫莲娜这些品牌的美白产品。市面常规销售光甘草定40％，但由于含量珍稀、提取难度大，导致光甘草定做护肤品的成本非常高。
光甘草定的提取纯化方法包括重结晶、硅胶柱层析、树脂法等。专利CN107510710A采用大孔树脂辅以聚酰胺、MCI的一种或两种混合制备甘草总黄酮，主要制备的是甘草查尔酮类化合物，其中查尔酮A占提取物含量的10-20％，由于聚酰胺、MCI粒径较小，柱层析容易发生堵塞问题，在实际生产中使用不多。专利CN112778323A采用乙醇提取、萃取、大孔树脂纯化、反相硅胶柱层析得到高纯度的光甘草定，但工艺周期长、有机溶剂耗费多。专利CN105777771A采用硅胶柱层析、重结晶得到光甘草定，也存在硅胶和有机溶剂耗费多，不环保等问题。
随着社会的进步以及人们生活水平的提高，糖尿病患者，尤其是Ⅱ型糖尿病(非胰岛素依赖型糖尿病)人数成倍增长。蛋白质酪氨酸磷酸酶(PTP-1B)属于蛋白质酪氨酸磷酸酶家族，与蛋白酪氨酸激酶(Protein Tyrosine Kinases，PTK)共同维持着酪氨酸蛋白磷酸化的平衡，参与细胞的信号转导，调节细胞的生长、分化、代谢、基因转录和免疫应答等。PTP-1B通过对胰岛素受体激酶(insulin receptor kinase，IRK)或IRK活性片段的磷酸化酪氨酸残基去磷酸化作用对胰岛素信号传导进行负调节，通过抑制PTP1B可有效治疗2型糖尿病。
α-葡萄糖苷酶抑制剂是一类具有糖结构的新型抗糖尿病药物，目前临床上应用的有阿卡波糖、伏格列波糖和米格列醇。现已作为治疗2型糖尿病的首选药和1型糖尿病的辅助药物普遍应用于临床。α-葡萄糖苷酶位于小肠刷状缘膜上皮细胞，它能将双糖，如蔗糖、麦芽糖等水解成可被小肠吸收的单糖，是食物中碳水化合物水解的关键酶。因此，抑制该酶能够延缓碳水化合物的消化，减少葡萄糖吸收入血，进而抑制餐后高血糖，改善对血糖的控制，缓解高胰岛素血症，同时可以提高糖耐量，从而可达到防治糖尿病及其并发症发生的目的。
本发明直接针对蛋白酪氨酸磷酸酶(PTP-1B)和α-葡萄糖苷酶为靶点进行筛选。至今为止，尚未见文献记载或研究报道光甘草定为主要活性成分的甘草提取物在治疗糖尿病方面的应用。光果甘草在民间用药历史悠久，有着长期稳定的临床应用，因此其开发应用前景广阔。
发明内容
本发明目的在于，提供一种光果甘草提取物的制备方法及用途，该方法将光果甘草的根或提取甘草酸后的光果甘草药渣通过有机溶剂乙醇回流提取，大孔树脂分离纯化，石油醚、乙酸乙酯沉降处理从光果甘草中获得降糖有效组分，并阐明其医学用途，经初步的活性筛选试验证明：通过本发明所述的方法获得的光果甘草提取物具有蛋白酪氨酸磷酸酶1B(PTP-1B)和α-葡萄糖苷酶抑制抑制作用，可作为原料或辅助剂用于防治糖尿病的药物或保健品。
本发明所述的一种光果甘草提取物的制备方法，按下列步骤进行：
a.将光果甘草的根或提取甘草酸后的光果甘草药渣粉粹后，用浓度为80-95％乙醇水溶液，在温度50-80℃的条件下加热提取1-3次，每次1-3小时，过滤，合并滤液，减压浓缩，调至含醇量30-50％，过滤，得到醇提物；
b.将步骤a得到的醇提物加入预先准备好的型号为HPD-300、HPD-400、HPD-600、AB-8或D101大孔树脂柱，先用30-50％乙醇洗脱，再用3-8倍柱体积的70-85％乙醇洗脱，收集70-85％乙醇洗脱液，浓缩，得到树脂纯化物；
c.将步骤b得到的树脂纯化物用乙酸乙酯萃取1-3次，得到乙酸乙酯萃取物，加入乙酸乙酯3-5倍体积量石油醚沉降处理1-3次，收集上清液，浓缩，干燥，即可得到光甘草定含量40-70％的光果甘草提取物。
所述方法获得的光果甘草提取物在抑制糖尿病药物筛选靶点-蛋白质酪氨酸磷酸酶1B中的作用。
所述方法获得的光果甘草提取物在抑制糖尿病药物筛选靶点α-葡萄糖苷酶中的作用。
所述方法获得的光果甘草提取物在制备治疗降血糖的药物中的用途。
所述方法获得的光果甘草提取物在制备保健品或其他功能性食品中的用途。
本发明所述的光果甘草提取物的制备方法相对于现有技术具有如下的优点及效果：
(1)采用本发明所述方法获得的光果甘草提取物，光甘草定的含量为40-70％，该生产方法简单易行，适合工业化生产。
(2)初步的活性筛选试验证明，该提取物具有显著的蛋白酪氨酸磷酸酶1B(PTP-1B)和α-葡萄糖苷酶抑制作用，其中对α-葡萄糖苷酶抑制活性是阳性药阿卡波糖的200倍，同时该提取物活性优于含量90％的光甘草定。
附图说明
图1为本发明光甘草定对照品高效液相图；
图2为本发明光果甘草提取物高效液相图。
具体实施方式
实施例1
a、取光果甘草的根10kg粉粹后，加入8倍体积浓度为80％乙醇加热回流提取3次，温度50℃，时间1小时，过滤，合并提取液，减压浓缩，加醇至含醇量为50％，过滤，得到醇提物；
b、将步骤a得到的醇提物加入预先准备好的AB-8大孔树脂柱，先用6倍柱体积浓度为50％乙醇洗脱，再用5倍柱体积浓度为80％乙醇洗脱，收集乙醇洗脱液，减压浓缩，得到树脂纯化物；
c、将步骤b得到的树脂纯化物用0.3L乙酸乙酯萃取2次，得到乙酸乙酯萃取物，加入1.8L石油醚沉降处理2次，收集上清液，浓缩，干燥，即得到光果甘草提取物30g，光甘草定含量63％。
实施例2
a、取光果甘草的根10kg粉粹后，加入8倍体积浓度为85％乙醇加热回流提取3次，温度60℃，时间1小时，过滤，合并提取液，减压浓缩，加醇至含醇量为40％，过滤，得到醇提物；
b、将步骤a得到的醇提物加入预先准备好的HPD-300大孔树脂柱，先用4倍柱体积浓度为40％乙醇洗脱，再用4倍柱体积浓度为70％乙醇洗脱，收集乙醇洗脱液，减压浓缩，得到树脂纯化物；
c、将步骤b得到的树脂纯化物用0.4L乙酸乙酯萃取3次，得到乙酸乙酯萃取物，加入3.6L石油醚沉降处理1次，收集上清液，浓缩，干燥，即得到光果甘草提取物25g，光甘草定含量51％。
实施例3
a、提取甘草酸后的光果甘草药渣10kg粉粹后，加入6倍体积浓度为95％乙醇加热回流提取1次，温度50℃，时间3小时，过滤，减压浓缩，加水至含醇量为30％，过滤，得到醇提物；
b、将步骤a得到的醇提物加入预先准备好的HPD-400大孔树脂柱，先用3倍柱体积浓度为30％乙醇洗脱，再用3倍柱体积浓度为85％乙醇洗脱，收集乙醇洗脱液，减压浓缩，得到树脂纯化物；
c、将步骤b得到的树脂纯化物用0.2L乙酸乙酯萃取3次，得到乙酸乙酯萃取物，加入2.4L石油醚沉降处理1次，收集上清液，浓缩，干燥，即得到光果甘草提取物35g，光甘草定含量68％。
实施例4
a、取光果甘草的根10kg粉粹后，加入8倍体积浓度为95％乙醇加热回流提取2次，温度80℃，时间2小时，过滤，减压浓缩，加水至含醇量为40％，过滤，得到醇提物；
b、将步骤a得到的醇提物加入预先准备好的HPD-600大孔树脂柱，先用5倍柱体积浓度为50％乙醇洗脱，再用3倍柱体积浓度为85％乙醇洗脱，收集乙醇洗脱液，减压浓缩，得到树脂纯化物；
c、将步骤b得到的树脂纯化物用0.1L乙酸乙酯萃取2次，得到乙酸乙酯萃取物，加入1L石油醚沉降处理3次，收集上清液，浓缩，干燥，即可得到光果甘草提取物32g，光甘草定含量43％。
实施例5
a、取光果甘草药渣10kg粉粹后，加入10倍体积浓度为85％乙醇加热回流提取2次，温度50℃，时间2小时，过滤，减压浓缩，加水至含醇量为45％，过滤，得到醇提物；
b、将步骤a得到的醇提物加入预先准备好的D101大孔树脂柱，先用4倍柱体积浓度为45％乙醇洗脱，再用6倍柱体积浓度为80％乙醇洗脱，收集乙醇洗脱液，减压浓缩，得到树脂纯化物；
c、将步骤b得到的树脂纯化物用0.2L乙酸乙酯萃取2次，得到乙酸乙酯萃取物，加入1.2L石油醚沉降处理2次，收集上清液，浓缩，干燥，即可得到光果甘草提取物28g，光甘草定含量61％。
实施列6
光果甘草提取物中光甘草定含量的测定方法：
将实施例1-5获得的任意一种光果甘草提取物测定甘草定含量，色谱条件：以十八烷基硅烷键合硅胶为填充剂；以乙腈-水溶液(46:54)为流动相；检测波长为282nm；
对照品溶液的制备：精密称取光甘草定对照品，精密称定，加无水乙醇制成每1ml含有40μg的溶液，即得；
供试品溶液的制备：光果甘草提取物样品30mg，置25mL容量瓶中，无水乙醇溶解并稀释至刻度，精密量取2ml溶液置于25mL容量瓶中，加无水乙醇稀释至刻度，摇匀，即得；含量测定：分别精密吸取对照品溶液及供试品溶液各10μL，注入液相色谱仪，测定，计算光果甘草提取物中光甘草定含量，应在40％至70％之间。
实施例7
将实施例1-5获得的光果甘草提取物作为蛋白酪氨酸磷酸酶抑制剂和α-葡萄糖苷酶抑制剂的药物或保健品的用途的生物活性筛选：
蛋白质酪氨酸磷酸酶1B(PTP-1B)筛选方法：用对-硝基苯基磷酸二钠(pNPP)作为底物，通过检测产物对PNPP的变化，确定蛋白酪氨酸磷酸酯酶1B的活性。反应总体积为200μL：179μL含PTP1B蛋白溶液的磷酸盐缓冲溶液，1μL待测样品,20μL 35mmol/L PNPP，振荡混匀，室温反应30min后，405nm处测定吸收值，以不含酶溶液体系为空白，3-(3,5-Dibromo-4-hydroxy-benz-oyl)-2-ethyl-benzofuran-6-sulfonicacid-(4-(thiazol-2-ylsulfamyl)-phenyl)-amide为阳性对照。
抑制率(I％)＝[酶活组-酶活对照组-(药品组-药品对照组)]/[(酶活组-酶活对照组)]×100％，IC50用软件计算。
α-葡萄糖苷酶筛选方法：实验以PNPG为底物，通过检测产物对硝基苯酚(p-nitrophenol，PNPG)的变化，确定α-葡萄糖苷酶的活性。反应总体积为100μL：2μL待测样品,71.5μL pH 6.8的磷酸盐缓冲溶液，1.5μLα-葡萄糖苷酶，室温孵育10min后加入25μL5mmol/L PNPG，振荡混匀，37℃反应30min后，405nm处测定吸收值，以不含酶溶液体系为空白，以阿卡波糖为阳性对照。
抑制率(I％)公式＝[酶活组-(药品组-药品对照组)/(酶活组-酶活对照组)]×100％
表光果甘草提取物对PTP1B抑制剂和α-葡萄糖苷酶抑制剂的IC50浓度
结论：
从实验结果表明，通过本发明所述方法获得的光果甘草提取物具有蛋白酪氨酸磷酸酶1B抑制剂和α-葡萄糖苷酶的抑制功效。
Claims (2)
1.一种光果甘草提取物的制备方法，其特征在于按下列步骤进行：
a. 将光果甘草的根或提取甘草酸后的光果甘草药渣粉粹后，用浓度为80-95%乙醇水溶液，在温度50-80℃的条件下加热提取1-3次，每次1-3小时，过滤，合并滤液，减压浓缩，调至含醇量30-50%，过滤，得到醇提物；
b. 将步骤a得到的醇提物加入预先准备好的型号为HPD-300、HPD-400、HPD-600、AB-8或D101大孔树脂柱，先用30-50%乙醇洗脱，再用3-8倍柱体积的70-85%乙醇洗脱，收集70-85%乙醇洗脱液，浓缩，得到树脂纯化物；
c. 将步骤b得到的树脂纯化物用乙酸乙酯萃取1-3次，得到乙酸乙酯萃取物，加入乙酸乙酯3-5倍体积量石油醚沉降处理1-3次，收集上清液，浓缩，干燥，即可得到光甘草定含量40-70%的光果甘草提取物。
2.根据权利要求1 所述的方法获得的光果甘草提取物在制备治疗降血糖的药物中的用途。
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