CN106551962B - 盐肤木果实提取物及其制备方法与应用 - Google Patents
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Abstract
本发明涉及盐肤木果实提取物及其制备方法及应用，属于食品或医药技术领域。本发明以盐肤木的果实盐麸子为原料，用有机溶剂(如乙醇、甲醇、丙酮、乙酸乙酯等)或水与有机溶剂的混合液提取，采用超声波进行辅助提取，得到盐肤木果实提取物，经UPLC‑ESI‑HRMS/MS共鉴定出60多种物质，主要成分为50余种的酚类化合物，这些提取物中酚类物质的含量为200.0‑500.0mg/g提取物干重(约6.0‑50.0mg/g干果重)，体外胰脂肪酶活性测试结果显示，本发明提取物具有优异的胰脂肪酶抑制活性，可用于制备胰脂肪酶抑制剂以及预防或治疗肥胖症及相关脂质代谢性疾病的食品或者药品。
Description
技术领域
本发明涉及盐肤木果实提取物及其制备方法及在用作胰脂肪酶活性抑制剂及用于制备预防或治疗肥胖及相关脂质代谢性疾病的食品或药品中的应用，属于食品或医药技术领域。
背景技术
近十几年来，由于生活物质水平的提高，全世界特别是具有高脂饮食习惯的国家，肥胖病的发病率越来越高。过多的脂质摄入和运动不足导致大量的脂质在身体组织器官中累积，当累积在脂肪细胞中时就会导致肥胖，而累积在脂肪细胞外的组织器官中时，就造成脂质异位沉积，从而引发糖尿病、心血管疾病、脂肪肝和高血脂等脂代谢紊乱相关的疾病。
目前全世界对肥胖症及相关脂代谢疾病的治疗主要是采用药物减少高能量脂质的摄入。食物中的脂肪需经过脂肪酶水解成游离脂肪酸和单酰甘油(或二酰甘油)，经肠道吸收后在体内再次合成甘油三酯，储存在体内，过多的脂肪沉积最终会导致肥胖以及相关的慢性疾病发生(如糖尿病，心血管疾病、脂肪肝和高血脂等)。饮食中的脂肪约70％是由肠道中胰脂肪酶负责水解消化。因此，抑制胰脂肪酶的活性就能够有效地减少脂肪的吸收，从而达到预防和治疗肥胖以及相关疾病的目的。美国FDA通过的用于治疗和预防肥胖的药物有奥利司他和西布曲明，其中奥利司他就是一种胰脂肪酶的抑制剂，但是这种抑制剂具有腹部不适、胃肠胀气和油便等副作用。目前全世界都在积极寻找新型的胰脂肪酶抑制剂以应对日益严峻的肥胖等相关疾病问题。因此，新型胰脂肪酶抑制剂的探寻具有极其重要的意义。
盐肤木(Rhus chinensis Mill.)果实又名盐麸子，是一种传统植物药材，并且也被用作酸味调味料，目前国内外对盐肤木果实提取物的脂肪酶抑制活性以及预防或治疗肥胖相关的研究尚未有专利文献报道。本发明将为开发安全有效纯天然的减肥食品或药品提供一定的科学依据。
发明内容
本发明的目的是提供盐肤木果实的提取物。
本发明的另一个目的是提供盐肤木果实提取物的用途，用于作为胰脂肪酶活性抑制剂，并用于制备预防或治疗肥胖及相关脂质代谢性疾病的食品或药品。
本发明是通过以下技术方案实现的：
将新鲜的或者干燥后的盐肤木(Rhus chinensis Mill.)果实(盐麸子)匀浆或粉碎后，加入有机溶剂(甲醇、乙醇、丙酮、乙酸乙酯等中的任意一种，或是任意几种以任意比例混合而成的混合有机溶剂)或水和有机溶剂的混合溶液，优选乙醇和水(乙醇和水的体积比为4:1)，料液比为1:5-10g/mL，利用超声波在20-50℃下进行辅助提取，超声波功率为50-300W，时间为20-100分钟，提取完成后，过滤得提取液，重复提取2-3次，合并提取液，然后在20-50℃下减压浓缩去除溶剂，干燥得到盐肤木果实提取物。
本发明所得提取物中物质鉴定及酚类物质含量测定：
物质鉴定采用UPLC-ESI-HRMS/MS进行鉴定，UPLC分离条件为：分离柱为shim-packXR-ODS II；流动相A为0.1％甲酸水，B为乙腈；分离梯度为：0-5min，15-30％B；5-8min，30-50％B；8-15min，50-80％B；15-20min，80-85％B；20-25min，85-90％B；25-30min，90-15％B；30-32min，15％B；流速为0.2mL/min；进样量为3μL。ESI-HRMS/MS鉴定条件为：高分辨率质谱为Q-Exactive Orbitrap质谱(Thermo Fisher Scientific，德国)，主要技术参数为：全扫范围为150-2000m/z；鞘气流速为，32L/min；辅助气体流速，8L/min；吹扫气体流速4L/min；正离子模式喷雾电压，3.30kV；负离子模式喷雾电压，3.3kV离子传输管温度，320℃；S-lensRF水平，50％。通过质谱数据与文献及相关数据库中数据比较验证，共鉴定出60多种物质，其中50余种为多酚类物质。
提取物中总酚含量测定采用福林酚法，以没食子酸为标准对照品，计算结果为各样品总酚含量200.0-500.0mg/g提取物干重(约6.0-50.0mg/g干果重)。
本发明中盐麸子各提取物的用途主要采用体外胰脂肪酶活性抑制实验进行考察，结果显示，本发明中各提取物均具有优异的胰脂肪酶抑制活性，可以用作胰脂肪酶活性抑制剂以及用于制备预防或治疗肥胖及相关脂质代谢性疾病的食品或药品。
与现有技术相比，本发明具有如下优点：
1、本发明为盐肤木果实发掘了新的功能食品或医疗用途，开拓了一个新的研究领域；
2、本发明通过实验发现盐肤木果实不同提取物在较低浓度条件下均对胰脂肪酶具有优异的抑制作用，优于阳性对照药品奥利司他，且提取物来自于用作调味料的植物材料，天然安全；
3、盐肤木果实不同提取物中酚类物质含量最高可达约50％，是其主要的活性成分，大量研究表明植物酚类物质具有抗氧化、延缓衰老、预防多种慢性疾病等功效。因此，本发明提取物中大量的酚类物质不仅保证了抑制胰脂肪酶活性，减少脂质吸收，达到预防或治疗肥胖等相关脂质代谢性疾病，还能促进机体健康。
附图说明
图1是盐肤木果实不同溶剂提取物中酚类物质的含量；
图2是盐肤木果实80％乙醇提取物对胰脂肪酶的抑制活性；
图3是盐肤木果实80％甲醇提取物对胰脂肪酶的抑制活性；
图4是盐肤木果实80％丙酮提取物对胰脂肪酶的抑制活性；
图5是盐肤木果实70％甲醇和70％丙酮混合溶剂提取物对胰脂肪酶的抑制活性；
图6是盐肤木果实乙酸乙酯提取物对胰脂肪酶的抑制活性；
图7是阳性对照药品奥利司他对胰脂肪酶的抑制活性。
具体实施方式
下面结合具体实施实例对本发明作进一步说明，但不限制本发明。
实施例1
80％乙醇、80％甲醇、80％丙酮以及乙酸乙酯各溶剂提取物的制备：先将摘取的盐肤木果实盐麸子进行冷冻干燥，然后粉碎作为原料保存于-20℃。称量30g原料，按料液比1:10g/mL，分别加入四种溶剂，在40℃下，进行超声提取，超声波功率为200W，时间为30分钟，然后过滤，将剩余残渣再重复提2次，分别收集各自上清，得到不同溶剂提取液。利用旋转蒸发仪，在40℃下进行浓缩，除去有机试剂，浓缩液保存在-20℃冰箱，然后进行冻干，得到提取物干粉，分别为样品1(80％乙醇提取物)、样品2(80％甲醇提取物)、样品3(80％丙酮)和样品4(乙酸乙酯提取物)。
实施例2
70％甲醇和70％丙酮(1:1)混合溶剂提取物的制备：先将摘取的盐肤木果实盐麸子进行冷冻干燥，然后粉碎作为原料保存于-20℃。称量30g原料，按料液比1:5g/mL，加入70％甲醇和70％丙酮(体积比1:1)混合溶剂，在35℃下，进行超声提取，超声波功率为80W，时间为60分钟，然后过滤，将剩余残渣再重复提2次，收集上清，得到提取液。利用旋转蒸发仪，在30℃下进行浓缩，除去有机试剂，浓缩液保存在-20℃冰箱，然后进行冻干，得到提取物干粉，为样品5。
实施例3
盐肤木果实不同溶剂提取物中多酚类物质的含量测定，采用福林酚法测定各提取物中多酚类物质的总含量，具体操作如下：在室温条件下，将各提取物用甲醇配成一定浓度的溶液，然后每种提取物溶液各取1mL(每种提取物做三次平行)，加入1mL的福林酚试剂，静置1min后分别加入1.5mL的20％Na2CO3和7.5mL的蒸馏水，混匀后于70℃孵育10min，待冷却至室温后，用分光光度计于765nm下测定吸光值，并以没食子酸作标准曲线，通过吸光值计算各提取物中多酚类物质的总含量，表示为mg没食子酸/g干提取物重。结果如图1所示，80％乙醇提取物中多酚类物质的总含量为374.27±12.28mg/g提取物干重，80％甲醇提取物中多酚类物质的总含量为278.09±6.11mg/g提取物干重，80％丙酮提取物中多酚类物质的总含量为359.62±8.27mg/g提取物干重，乙酸乙酯提取物中多酚类物质的总含量为198.79±9.78mg/g提取物干重，70％甲醇和70％丙酮混合溶剂提取物中多酚类物质的总含量为468.70±18.65mg/g提取物干重。
实施例4
采用UPLC-ESI-HRMS/MS对盐肤木果实提取物中化合物组成分析，具体操作如下：UPLC分离条件为：分离柱为shim-pack XR-ODS II；流动相A为0.1％甲酸水，B为乙腈；分离梯度为：0-5min，15-30％B；5-8min，30-50％B；8-15min，50-80％B；15-20min，80-85％B；20-25min，85-90％B；25-30min，90-15％B；30-32min，15％B；流速为0.2mL/min；进样量为3μL。ESI-HRMS/MS鉴定条件为：高分辨率质谱为Q-Exactive Orbitrap质谱(Thermo FisherScientific，德国)，主要技术参数为：全扫范围为150-2000m/z；鞘气流速为，32L/min；辅助气体流速，8L/min；吹扫气体流速4L/min；正离子模式喷雾电压，3.30kV；负离子模式喷雾电压，3.3kV离子传输管温度，320℃；S-lens RF水平，50％。
表1实施例1与2中盐肤木果实五种提取物鉴定出的化合物
实施例5
盐肤木果实各提取物对胰脂肪酶的抑制活性测定。首先将猪胰脂肪酶(购自sigma)溶于蒸馏水中，使其浓度为15mg/mL，在10000rpm转速下离心5分钟，上清备用。将对硝基苯基月桂酸酯(购自sigma)加入到含有0.1％曲通X-100的乙酸钠缓冲液(pH 5.0)中，使得对硝基苯基月桂酸酯的含量为0.1％，作为反应底物。将实施例1和2中所得提取物(或奥利司他)用DMSO溶解，并用pH8.2的Tris缓冲液稀释到合适浓度备用。将400μL的Tris缓冲液，350μL的反应底物，100μL的稀释提取物(或奥利司他)以及150μL酶液混合均匀后，于37℃下孵育2小时。反应结束后，在10000rpm转速下离心1分钟，于400nm下测定吸光值，并以样品溶剂为空白对照，胰脂肪酶抑制率(％)＝(ODcontrol-ODsample)/ODcontrol×100，其中ODcontrol为空白对照组吸光值，ODsample为样品组吸光值。
检测盐肤木果实各提取物对胰脂肪酶的抑制作用结果表明，80％乙醇提取物、80％甲醇提取物、80％丙酮提取物、70％甲醇和70％丙酮混合提取物以及乙酸乙酯提取物对胰脂肪酶的抑制活性IC50值分别为：59.02±3.69μg/mL、62.88±2.38μg/mL、61.42±3.53μg/mL、57.91±2.48μg/mL和81.50±4.57μg/mL。
本结果表明除乙酸乙酯提取物外，其他各提取物对胰脂肪酶的抑制活性均显著强于阳性对照药品奥利司他(40μg/mL浓度条件下)。
Claims (2)
1.一种盐肤木果实提取物，其特征在于，所述盐肤木果实提取物以盐肤木果实为原料，采用甲醇和丙酮水化的混合液提取浓缩而得；具体步骤：
将盐肤木果实匀浆或粉碎后，采用有机溶剂的混合液在20-50℃下，利用超声波辅助提取，料液比为1:5-10g/mL，重复提取2-3次，合并提取液，然后在20-50℃下减压浓缩，干燥后得到盐肤木果实提取物；所述有机溶剂为70%的甲醇和70%的丙酮按体积比1：1的混合溶剂；所述超声波辅助提取的功率为50-300W，时间为20-100分钟。
2.权利要求1所述的盐肤木果实提取物在制备胰脂肪酶抑制剂的应用。
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