CN115364144A - 一种余庆小叶苦丁茶提取物及其制备方法与应用 - Google Patents
一种余庆小叶苦丁茶提取物及其制备方法与应用 Download PDFInfo
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- CN115364144A CN115364144A CN202211218981.8A CN202211218981A CN115364144A CN 115364144 A CN115364144 A CN 115364144A CN 202211218981 A CN202211218981 A CN 202211218981A CN 115364144 A CN115364144 A CN 115364144A
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Abstract
本发明公开了一种余庆小叶苦丁茶提取物及其制备方法与应用。该提取物以余庆小叶苦丁茶为原料,采用浸泡、加热、超声提取、浓缩、溶剂萃取、真空冷冻干燥等步骤获得,含有石油醚组分、乙酸乙酯组分、正丁醇组分和水溶性组分四种不同组分,其中提取物中石油醚组分和正丁醇组分能有效降低2型糖尿病小鼠血糖值、改善血清生化指标、上调胰腺组织中INS和Nrf2蛋白表达,还能对小鼠的肝脏、胰腺组织起到保护作用,可应用于制备缓解2型糖尿病症状的药物、辅助药物或健康食品,具有较好的市场前景。该发明对余庆小叶苦丁茶进行了高值化利用,又为治疗2型糖尿病的药物和食品提供了中间体或原料,制备方法简单易行,成本低,适合规模化生产。
Description
技术领域
本发明属于天然产物健康医药领域,具体涉及一种余庆小叶苦丁茶提取物及其制备方法与应用。
背景技术
2型糖尿病是指由遗传和环境因素相互作用而引起的一种代谢异常综合征,以慢性高血糖为主要特征的慢性、非传染性、代谢性疾病,具有起病隐匿、潜伏期长的特点。随着患病时间增加,机体会受到严重损害,改变微量元素、相关酶的浓度与抗氧化防御***,严重影响着人类的生活质量。临床上治疗2型糖尿病有改善胰岛素抵抗和血糖控制两种方式,主要依靠胰岛素或者化学合成降糖药,虽然起效快,但是药物治疗容易产生耐药性,不良反应较多。与临床上常规降糖西药相比,天然产物具有不良反应少、延缓2型糖尿病并发症的优势,且因其毒副作用小、疗效稳定、多通路多靶点的独特优势在2型糖尿病的预防和治疗中具有相当的优势和潜力。
余庆小叶苦丁茶属木樨科粗壮女贞(Ligustrum robustum),生长在贵州境内乌江河流域的喀斯特地貌。在当地主要用于消肿、止痛、抗风湿、降血压等。余庆小叶苦丁茶具有独特的营养成分,相关研究表明,其含有多种营养成分和丰富的药效氨基酸,还含有植物中少见的硒元素、不饱和脂肪酸以及生物活性物质茶多糖等。余庆小叶苦丁茶作为一种宝贵的天然产物资源,其对于2型糖尿病小鼠血糖代谢调控研究未见报道。
发明内容
本发明的目的在于提供一种余庆小叶苦丁茶提取物及其制备方法与应用。该提取物石油醚组分A和正丁醇组分C能有效降低2型糖尿病小鼠血糖值、改善血清生化指标、上调胰腺组织中INS和Nrf2蛋白表达,同时对小鼠的肝脏、胰腺组织起到保护作用,可应用于制备缓解2型糖尿病症状的药物、辅助药物或健康食品,具有较好的市场前景,对余庆小叶苦丁茶进行了高值化利用,同时也为治疗2型糖尿病的药物和食品提供了中间体或原料,制备方法简单易行,成本低,适合规模化生产。
为实现上述目的,所采用的技术方案为:
一种余庆小叶苦丁茶提取物,所述提取物包含有石油醚组分A、乙酸乙酯组分B、正丁醇组分C和水溶性组分D。
所述提取物石油醚组分A包含有三七素、甘露醇、丙酮酸、右旋奎宁酸、5-羟甲基糠醛、达卡巴嗪、7-羟基香豆素、毛蕊花糖苷、金石蚕苷、樟脑、野漆树苷、紫苏烯、乙酰苯、香豆素、白术内酯III、藁本内酯、7-甲氧基-4-甲基香豆素、甘松新酮、芹菜素、松脂酚、莪术烯醇、白术内酯II、梣酮、羌活醇、咖啡酸、白果内酯、8-异戊烯基柚皮素、去甲氧基姜黄素、6-姜烯酚、二氢青蒿酸、诺卡酮、去氢二异丁香酚、α-亚麻酸、刺囊酸、花生四烯酸、油酰、齐墩果酸、油酸酰胺、亚油酸、棕榈酸、亚麻酸乙酯、羽扇烯酮、栎樱酸、花生酸、二十二烷酸其中的任意一种化合物。
所述提取物乙酸乙酯组分B包含有三七素、腺嘌呤、右旋奎宁酸、烟酰胺、柠檬酸、甘露醇、龙胆酸、羟基酪醇、达卡巴嗪、连翘酯苷E、原儿茶醛、对羟基安息香醛、苯甲酸、七叶亭、咖啡酸、5-羟基马鞭草苷马鞭草苷、对羟基肉桂酸、大车前苷、乙酰苯、罗汉果黄素、芦丁、忍冬苷、异槲皮苷、木犀草苷、紫地黄苷C、毛蕊花糖苷、野漆树苷、7-羟基香豆素、金石蚕苷、芹菜素-7-O-β-D-吡喃葡萄糖苷、5-羟基-四氢萘酮、茵芋苷、橄榄苦苷、木犀草素、苍术苷A、香豆素、苔色酸、2-金刚烷酮、异连翘酯苷A其中的任意一种化合物。
所述提取物正丁醇组分C包含有丁香醛、三七素、腺嘌呤、右旋奎宁酸、甘露醇、鸟嘌呤、龙胆酸、羟基酪醇、红景天苷、连翘酯苷E、对羟基苯甲酸、5-羟基马鞭草苷、樟脑、马鞭草苷、维采宁II、大车前苷、乙酰苯、龙血素A、山奈酚、罗汉果黄素、芦丁、忍冬苷、木犀草苷、紫地黄苷C、毛蕊花糖苷、茵芋苷、毛蕊花糖苷、野漆树苷、金石蚕苷、5-羟基-四氢萘酮、橄榄苦苷、5-羟基-四氢萘酮、对羟基肉桂酸、苍术苷A、香豆素其中的任意一种化合物。
所述提取物水溶性组分D包含有丁香醛、右旋奎宁酸、蔗糖、柠檬酸、甘露醇、原儿茶酸、水杨酸、乙酰苯、7-羟基香豆素、毛蕊花糖苷、金石蚕苷、茵芋苷、香豆素其中的任意一种化合物。
所述的余庆小叶苦丁茶提取物的制备方法,包括以下步骤:
步骤一:称取余庆小叶苦丁茶叶,干燥粉碎过50-100目筛;
步骤二:料液比1:10加蒸馏水在温度50-80℃浸提10-50min;
步骤三:设置温度30-60℃,超声频率为100-500W,超声振荡辅助提取,20-40min;
步骤四:重复步骤二、三的操作提取3次,抽滤合并滤液,用0.45μm滤膜过滤后采用旋转蒸发仪减压浓缩;
步骤五:依次用石油醚、乙酸乙酯、正丁醇对步骤四得到浓缩样品进行萃取,各重复3次,分别取上层液,得石油醚组分A、乙酸乙酯组分B、正丁醇组分C,下层即为水溶性组分D;
步骤六:将步骤五得到的四种组分萃取液采用旋转蒸发仪浓缩,-80℃冷冻干燥,即得余庆小叶苦丁茶提取物石油醚组分A、乙酸乙酯组分B、正丁醇组分C、水溶性组分D冻干粉。
步骤七:将步骤六得到的四种组分冻干粉,按照重量比为1:1:1:1混合即为余庆小叶苦丁茶提取物。
上述制备方法中,所述步骤四、六中,旋转蒸发仪设置参数为温度60℃,转速50-60rpm,步骤五中,所述溶剂与浓缩液体积比为2:1。
所述提取物应用于制备缓解2型糖尿病症状的药物、辅助药物或健康食品,所述提取物制备药物、辅助药物或健康食品时,直接使用或者以组合物的形式使用;所述组合物包括0.1–99%的提取物,其余为载体或赋形剂。
前述的应用中,所述提取物石油醚组分A和正丁醇组分C应用于制备缓解2型糖尿病症状的药物、辅助药物或健康食品。
前述的应用中,所述提取物石油醚组分A和正丁醇组分C能有效降低2型糖尿病小鼠血糖值、改善血清生化指标、上调胰腺组织中INS和Nrf2蛋白表达,同时对小鼠的肝脏、胰腺组织起到保护作用。
与现有技术相比,通过动物试验,本发明提供的提取物石油醚组分A和正丁醇组分C能有效降低2型糖尿病小鼠血糖值、改善血清生化指标、上调胰腺组织中INS和Nrf2蛋白表达,同时对小鼠的肝脏、胰腺组织起到保护作用,可应用于制备缓解2型糖尿病症状的药物、辅助药物或健康食品,具有较好的市场前景,对余庆小叶苦丁茶进行了高值化利用,同时也为治疗2型糖尿病的药物和食品提供了中间体或原料,制备方法简单易行,成本低,适合规模化生产。
附图说明
图1为余庆小叶苦丁茶提取物不同组分实物图;
图2为余庆小叶苦丁茶提取物制备流程图;
图3为动物实验2型糖尿病动物模型建立流程图;
图4为余庆小叶苦丁茶提取物不同组分对2型糖尿病小鼠体重毛发对比实物图;
图5为余庆小叶苦丁茶提取物不同组分对2型糖尿病小鼠体重影响的柱形图;
图6为余庆小叶苦丁茶提取物不同组分对2型糖尿病小鼠进食量影响的折线图;
图7为余庆小叶苦丁茶提取物不同组分对2型糖尿病小鼠进水量影响的折线图;
图8为余庆小叶苦丁茶提取物不同组分对2型糖尿病小鼠代谢量影响的折线图;
图9为余庆小叶苦丁茶提取物不同组分对2型糖尿病小鼠血糖值影响的柱形图;
图10为余庆小叶苦丁茶提取物不同组分对2型糖尿病小鼠口服糖耐量影响的折线图;
图11为余庆小叶苦丁茶提取物不同组分对2型糖尿病小鼠脏器指数影响的柱形图;
图12为余庆小叶苦丁茶提取物不同组分对2型糖尿病小鼠血清指标(TG、CHO、HDL-C、GSP、LDL-C)影响的柱形图;
图13为余庆小叶苦丁茶提取物不同组分对2型糖尿病小鼠血清指标(SOD、NEFA、FINS)影响的柱形图;
图14为余庆小叶苦丁茶提取物不同组分对2型糖尿病小鼠肝脏组织形态的影响(×400);
图15为余庆小叶苦丁茶提取物不同组分对2型糖尿病小鼠胰腺组织形态的影响(×400);
图16为余庆小叶苦丁茶提取物不同组分对2型糖尿病小鼠Nrf2蛋白表达的影(×400);
图17为余庆小叶苦丁茶提取物不同组分对2型糖尿病小鼠INS蛋白表达的影响(×400);
图18为余庆小叶苦丁茶提取物不同组分对高血糖小鼠胰腺蛋白INS/Nrf2表达的影响。
具体实施方式
下面结合实施例对本发明作进一步的说明,但并不作为对本发明限制的依据。
实施例1
一种余庆小叶苦丁茶提取物,所述提取物包含有石油醚组分A、乙酸乙酯组分B、正丁醇组分C和水溶性组分D。
所述提取物石油醚组分A包含有三七素、甘露醇、丙酮酸、右旋奎宁酸、5-羟甲基糠醛、达卡巴嗪、7-羟基香豆素、毛蕊花糖苷、金石蚕苷、樟脑、野漆树苷、紫苏烯、乙酰苯、香豆素、白术内酯III、藁本内酯、7-甲氧基-4-甲基香豆素、甘松新酮、芹菜素、松脂酚、莪术烯醇、白术内酯II、梣酮、羌活醇、咖啡酸、白果内酯、8-异戊烯基柚皮素、去甲氧基姜黄素、6-姜烯酚、二氢青蒿酸、诺卡酮、去氢二异丁香酚、α-亚麻酸、刺囊酸、花生四烯酸、油酰、齐墩果酸、油酸酰胺、亚油酸、棕榈酸、亚麻酸乙酯、羽扇烯酮、栎樱酸、花生酸、二十二烷酸其中的任意一种化合物。
所述提取物乙酸乙酯组分B包含有三七素、腺嘌呤、右旋奎宁酸、烟酰胺、柠檬酸、甘露醇、龙胆酸、羟基酪醇、达卡巴嗪、连翘酯苷E、原儿茶醛、对羟基安息香醛、苯甲酸、七叶亭、咖啡酸、5-羟基马鞭草苷马鞭草苷、对羟基肉桂酸、大车前苷、乙酰苯、罗汉果黄素、芦丁、忍冬苷、异槲皮苷、木犀草苷、紫地黄苷C、毛蕊花糖苷、野漆树苷、7-羟基香豆素、金石蚕苷、芹菜素-7-O-β-D-吡喃葡萄糖苷、5-羟基-四氢萘酮、茵芋苷、橄榄苦苷、木犀草素、苍术苷A、香豆素、苔色酸、2-金刚烷酮、异连翘酯苷A其中的任意一种化合物。
所述提取物正丁醇组分C包含有丁香醛、三七素、腺嘌呤、右旋奎宁酸、甘露醇、鸟嘌呤、龙胆酸、羟基酪醇、红景天苷、连翘酯苷E、对羟基苯甲酸、5-羟基马鞭草苷、樟脑、马鞭草苷、维采宁II、大车前苷、乙酰苯、龙血素A、山奈酚、罗汉果黄素、芦丁、忍冬苷、木犀草苷、紫地黄苷C、毛蕊花糖苷、茵芋苷、毛蕊花糖苷、野漆树苷、金石蚕苷、5-羟基-四氢萘酮、橄榄苦苷、5-羟基-四氢萘酮、对羟基肉桂酸、苍术苷A、香豆素其中的任意一种化合物。
所述提取物水溶性组分D包含有丁香醛、右旋奎宁酸、蔗糖、柠檬酸、甘露醇、原儿茶酸、水杨酸、乙酰苯、7-羟基香豆素、毛蕊花糖苷、金石蚕苷、茵芋苷、香豆素其中的任意一种化合物。
所述的余庆小叶苦丁茶提取物的制备方法,包括以下步骤:
步骤一:称取余庆小叶苦丁茶叶,干燥粉碎过60;
步骤二:料液比1:10加蒸馏水在温度60℃浸提30min;
步骤三:设置温度40℃,超声频率为300W,超声振荡辅助提取,30min;
步骤四:重复步骤二、三的操作提取3次,抽滤合并滤液,用0.45μm滤膜过滤后采用旋转蒸发仪减压浓缩;
步骤五:依次用石油醚、乙酸乙酯、正丁醇对步骤四得到浓缩样品进行萃取,各重复3次,分别取上层液,得石油醚组分A、乙酸乙酯组分B、正丁醇组分C,下层即为水溶性组分D;
步骤六:将步骤五得到的四种组分萃取液采用旋转蒸发仪浓缩,-80℃冷冻干燥,即得余庆小叶苦丁茶提取物石油醚组分A、乙酸乙酯组分B、正丁醇组分C、水溶性组分D冻干粉。
步骤七:将步骤六得到的四种组分冻干粉,按照重量比为1:1:1:1混合即为余庆小叶苦丁茶提取物。
上述制备方法中,所述步骤四、六中,旋转蒸发仪设置参数为温度60℃,转速50-60rpm,步骤五中,所述溶剂与浓缩液体积比为2:1。
所述提取物应用于制备缓解2型糖尿病症状的药物、辅助药物或健康食品,所述提取物制备药物、辅助药物或健康食品时,直接使用或者以组合物的形式使用;所述组合物包括0.1–99%的提取物,其余为载体或赋形剂。
前述的应用中,所述提取物石油醚组分A和正丁醇组分C应用于制备缓解2型糖尿病症状的药物、辅助药物或健康食品。
前述的应用中,所述提取物石油醚组分A和正丁醇组分C能有效降低2型糖尿病小鼠血糖值、改善血清生化指标、上调胰腺组织中INS和Nrf2蛋白表达,同时对小鼠的肝脏、胰腺组织起到保护作用。
实施例2
一种余庆小叶苦丁茶提取物,所述提取物包含有石油醚组分A、乙酸乙酯组分B、正丁醇组分C和水溶性组分D。
所述的余庆小叶苦丁茶提取物的制备方法,包括以下步骤:
步骤一:称取余庆小叶苦丁茶叶,干燥粉碎过50目筛;
步骤二:料液比1:10加蒸馏水在温度80℃浸提10min;
步骤三:设置温度60℃,超声频率为100W,超声振荡辅助提取,40min;
步骤四:重复步骤二、三的操作提取3次,抽滤合并滤液,用0.45μm滤膜过滤后采用旋转蒸发仪减压浓缩;
步骤五:依次用石油醚、乙酸乙酯、正丁醇对步骤四得到浓缩样品进行萃取,各重复3次,分别取上层液,得石油醚组分A、乙酸乙酯组分B、正丁醇组分C,下层即为水溶性组分D;
步骤六:将步骤五得到的四种组分萃取液采用旋转蒸发仪浓缩,-80℃冷冻干燥,即得余庆小叶苦丁茶提取物石油醚组分A、乙酸乙酯组分B、正丁醇组分C、水溶性组分D冻干粉。
步骤七:将步骤六得到的四种组分冻干粉,按照重量比为1:1:1:1混合即为余庆小叶苦丁茶提取物。
上述制备方法中,所述步骤四、六中,旋转蒸发仪设置参数为温度60℃,转速50rpm,步骤五中,所述溶剂与浓缩液体积比为2:1。
实施例3
一种余庆小叶苦丁茶提取物,所述提取物包含有石油醚组分A、乙酸乙酯组分B、正丁醇组分C和水溶性组分D。
所述的余庆小叶苦丁茶提取物的制备方法,包括以下步骤:
步骤一:称取余庆小叶苦丁茶叶,干燥粉碎过100目筛;
步骤二:料液比1:10加蒸馏水在温度50℃浸提50min;
步骤三:设置温度30℃,超声频率为500W,超声振荡辅助提取,20min;
步骤四:重复步骤二、三的操作提取3次,抽滤合并滤液,用0.45μm滤膜过滤后采用旋转蒸发仪减压浓缩;
步骤五:依次用石油醚、乙酸乙酯、正丁醇对步骤四得到浓缩样品进行萃取,各重复3次,分别取上层液,得石油醚组分A、乙酸乙酯组分B、正丁醇组分C,下层即为水溶性组分D;
步骤六:将步骤五得到的四种组分萃取液采用旋转蒸发仪浓缩,-80℃冷冻干燥,即得余庆小叶苦丁茶提取物石油醚组分A、乙酸乙酯组分B、正丁醇组分C、水溶性组分D冻干粉。
步骤七:将步骤六得到的四种组分冻干粉,按照重量比为1:1:1:1混合即为余庆小叶苦丁茶提取物。
上述制备方法中,所述步骤四、六中,旋转蒸发仪设置参数为温度60℃,转速50rpm,步骤五中,所述溶剂与浓缩液体积比为2:1。
实施例4
取实施例1制得的提取物,按活性提取物添加量按重量计为0.1%,按常规方法制粒压片,其他辅料包括:淀粉、硬脂酸镁、滑石粉。
以上所述,仅是本发明的较佳实施例而已,并非对本发明作任何形式上的限制,任何未脱离本发明技术方案内容,依据本发明的技术实质对以上实施例所作的任何简单修改、变换材质、等同变化与修饰,均仍属于本发明技术方案的范围内。
为进一步的验证该提取物改善小鼠2型糖尿病症状的效果,采用实施例1制备工艺参数得到的提取物的不同组分(石油醚组分、正丁醇组分)开展实验。
一、实验方法
1.动物模型制备与分组
1.12型糖尿病小鼠模型制备
40只6周龄雄性C57BL/6J小鼠(18~22g),适应性喂养一周;正常组8只常规饲料,其余32只高脂饲料喂养4周;喂养4周之后,禁食不禁水12h,32只左下腹腔注射STZ无菌柠檬酸盐缓冲溶液(链脲佐菌素100mg/kg,均分两次给药,间隔72h),正常对照组8只注射相同体积(0.4ml)的无菌柠檬酸盐缓冲溶液;(1%STZ柠檬酸缓冲溶液,用0.22微米滤膜过滤,配制后30min内注射完毕,并置于冰浴中,现配现用)注射后第3日禁食12h,所有小鼠尾静脉取血,血糖仪测空腹血糖值(FBG),若血糖值≥11.1mmol/L,则认为2型糖尿病小鼠模型制备成功(2型糖尿病动物模型建立流程见图3)。
1.2分组
取32只造模成功且状态好的老鼠,随机分为模型对照组、阳性对照组(二甲双胍50mg/kg·d)、石油醚组分给药组、正丁醇组分给药组、均按照500mg/kg剂量给药。石油醚组分采用玉米油作为溶剂,正丁醇组分采用生理盐水。
1.3给药
每天上午9点灌胃给药,正常对照组和模型对照组每日以相等体积的生理盐水灌胃1mL·kg-1·d-1。每4天记录小鼠进食量,进水量及尿量情况;给药后每周于固定时间给实验组小鼠禁食不禁水12h,尾部取微量血用血糖仪测量空腹血糖值,持续8周。
2.观察指标
2.1.小鼠体征情况
肉眼观察2型糖尿病小鼠在给药灌胃期间饮食情况,毛发是否松散,精神状态是否良好;通过称量观察小鼠体重是否变化等基本情况。
2.2.小鼠进食、饮水、代谢量变化
灌胃给药期间每4天记录小鼠进食量,进水量及代谢量情况。
2.3.血糖值变化
每周于固定时间禁食不禁水12h,小鼠尾静脉取微量血,弃第一滴血,用血糖仪测定空腹血糖值。
2.4.组织标本的采集与处理
于给药8周后,小鼠眼眶静脉丛取血,3000rpm离心10min,分离血清。每只小鼠的血清分别做好标记,测定血清中TG、CHO、HDL-C、LDL-C、GSP、SOD、NEFA的含量水平。摘取小鼠心、肝、脾、肺、肾、胰腺组织并称重记录,切取部分肝脏、胰腺置于4%多聚甲醛溶液中固定48h,用于HE染色和免疫组织化学观察其病理变化和INS、Nrf2蛋白表达情况。
HE染色步骤:
1)取其中一张组织蜡片,放入45℃温水中进行展平、捞片、烘干,再放入90℃烘箱进行除蜡约15min;
2)再依次放入二甲苯Ⅰ与二甲苯Ⅱ各5min→无水乙醇1-2min→95%乙醇1-2min→85%乙醇1-2min→蒸馏水洗2min;
3)滴加Gill改良苏木素染色5min左右→自来水冲洗→75%盐酸酒精分10s→自来水冲洗→温水(50-60℃)返蓝5min→自来水冲洗10min→95%乙醇1min→1%酸化伊红酒***染2-10s→自来水冲洗;
4)95%乙醇l min→90%乙醇l min→100%酒精l min→二甲苯Ⅰ1min→二甲苯Ⅱ1min→中性树胶封片;
HE染色后观察:组织切片经HE染色后在光镜(400×)下观察组织细胞形态,是否有炎症,细胞核是否淡染等情况。
免疫组织化学步骤:
切取部分胰腺组织于4%多聚甲醛溶液中固定,石蜡切片脱蜡至水、柠檬酸抗原修复缓冲液(PH6.0)抗原修复、3%双氧水溶液室温避光孵育25min,阻断内源性过氧化物酶、血清封闭、Nrf2(1:1000)INS(1:2000)一抗,4℃孵育过夜。孵育二抗后加入DAB染色液显色,苏木精复染后脱水封片。于显微镜下采集照片(400×)。
3.数据处理
采用统计软件Excel及SPSS 26.0分析,计量资料用均数±标准差(x±s)表示,多组间数值比较用单因素方差分析,以P<0.05和P<0.01为差异有统计学意义。
二、实验结果
1.小鼠体重变化
2型糖尿病小鼠建立后,模型组小鼠相比于正常组小鼠状态较差,正常组小鼠毛发顺滑,而造模小鼠毛发松散,且“多饮、多食、多尿”等糖尿病典型症状明显,具体见图4,其中(A)表示正常组;(B)表示模型组;(C)表示阳性药组;(D)表示石油醚组;(E)表示正丁醇组。随着灌胃时间增长,石油醚组小鼠体重逐渐增长,显著高于模型组(P<0.01),并趋于正常组水平,余庆小叶苦丁茶提取物不同组分对2型糖尿病小鼠体重影响具体见图5,注:与正常组比较,极显著用“**”表示(P<0.01),显著用“*”表示(P<0.05);与模型组比较,极显著用“##”表示(P<0.01),显著用“#”表示(P<0.05)。
2.小鼠进食、饮水、代谢量变化
与正常组小鼠相比,糖尿病小鼠多饮、多食、多尿症状明显,实验过程中,每隔4日记录各组小鼠的饮水量、进食量。并且通过计算垫料的差值得到其代谢量。模型组小鼠进食量、饮水量和代谢量居高不下,阳性药组和正丁醇组在第6个4日(6x4d)开始减少;石油醚组进食量、饮水量和代谢量在第4个4日(4x4d)开始减少,且逐渐趋于正常组水平,由此可见其改善2型糖尿病症状效果最显著,且优于阳性药。(具体见图6—图8)。
3.血糖值变化
注射链脲佐菌素2~3周后各组小鼠血糖值不断上升到达峰值,而后随着灌胃余庆小叶苦丁茶的不同组分出现不同程度的波动。灌胃期间模型组以及各个给药组的血糖值均与正常组有极显著性差异(P<0.01)。灌胃第6周石油醚组和正丁醇组小鼠血糖下降趋势明显,与模型组具有极显著性差异(P<0.01);第7周石油醚组和正丁醇组与模型组具有显著性差异(P<0.01或P<0.05);第8周阳性药组、石油醚组和正丁醇组血糖值较模型组极显著下降(P<0.01),,具体见图9,注:与正常组比较,极显著用“**”表示(P<0.01),显著用“*”表示(P<0.05);与模型组比较,极显著用“##”表示(P<0.01),显著用“#”表示(P<0.05)。
4.口服糖耐量实验(OGTT)
OGTT能够反应胰岛β细胞的功能和机体对血糖的调节能力。实验灌胃第8周,实验小鼠禁食不禁水,以2g/kg剂量灌胃无水葡萄糖溶液,在灌胃前、灌胃后30min、60min、90min、120min分别采集尾静脉血测定血糖值。给予葡萄糖30min后各组小鼠血糖上升达到峰值,正丁醇组血糖值在30min后下降迅速,与模型组血糖值具有极显著性差异(P<0.01)。石油醚组小鼠血糖值在120min时与模型组具有极显著差异(P<0.01),具体见图10,注:与正常组比较,极显著用“**”表示(P<0.01),显著用“*”表示(P<0.05);与模型组比较,极显著用“##”表示(P<0.01),显著用“#”表示(P<0.05)。
5.小鼠脏器指数的影响
实验结果如图10所示,各组小鼠心脏指数均无显著性差异;患2型糖尿病小鼠肝脏指数与正常组相比具有显著性差异(P<0.01);阳性药组和石油醚组小鼠脾脏指数与模型组相比具有显著性差异(P<0.01或P<0.05);模型组、阳性药组和正丁醇组小鼠肺指数与正常组相比具有极显著性差异(P<0.01);糖尿病患者会出现肾脏水肿现象,从而肾脏加重,患2型糖尿病小鼠肾脏指数显著高于正常组(P<0.01),石油醚组小鼠显著低于模型组(P<0.01);石油醚组和正丁醇组小鼠胰腺指数低于模型组,趋于正常水平,但并未达到显著性水平。结果表明余庆小叶苦丁茶石油醚和正丁醇组分能够改善2型糖尿病小鼠脾、肾和胰腺指数,降低2型糖尿病发病风险,具体见图11,注:与正常组比较,极显著用“**”表示(P<0.01),显著用“*”表示(P<0.05);与模型组比较,极显著用“##”表示(P<0.01),显著用“#”表示(P<0.05)。
6.血清指标
模型组小鼠TG、NEFA含量高于正常组,糖尿病症状明显。石油醚组小鼠HDL-C、GSP含量显著高于模型组(P<0.05),NEFA含量较模型组显著降低(P<0.05)。正丁醇组小鼠CHO、GSP、LDL-C、NEFA含量高于模型组,但并未达到显著性水平。石油醚组和正丁醇组能够增加SOD和FINS合成,并且增加FINS合成效果优于阳性药组(具体见图12-13)。注:与正常组比较,极显著用“**”表示(P<0.01),显著用“*”表示(P<0.05);与模型组比较,极显著用“##”表示(P<0.01),显著用“#”表示(P<0.05)。
7.肝脏、胰腺HE染色
肝脏是葡萄糖代谢的重要器官,肝脏能摄取和利用葡萄糖,降低血糖。若肝功能受损则会影响肝脏对葡萄糖的摄取和利用。胰岛素的分泌取决于胰腺内胰岛细胞的结构和数量。本研究肝脏、胰腺的HE染色结果如图所示。正常组小鼠肝脏、胰腺细胞为圆形、椭圆形细胞团,数量较多,边界清晰,排列紧密,核染色质清晰。而模型组小鼠肝脏和胰岛内分泌的细胞形状不规则,排列稀疏不均,细胞核淡染,胞浆形态不规则。余庆小叶苦丁茶提取物石油醚组和正丁醇组小鼠肝细胞和胰岛细胞形态明显好于模型组,细胞核淡染情况明显改善,胞浆空泡程度减轻,其中石油醚组效果最佳。说明余庆小叶苦丁茶提取物石油醚组分和正丁醇组分对2型糖尿病小鼠胰岛细胞和肝细胞具有一定的保护作用,具体见图14-15,其中(A)表示正常组;(B)表示模型组;(C)表示阳性药组;(D)表示石油醚组;(E)表示正丁醇组。
8.免疫组化结果
免疫组织化学结果显示,模型组小鼠胰腺组织内INS和Nrf2蛋白表达量明显低于正常组(P<0.01),阳性药组、石油醚组和正丁醇组INS和Nrf2蛋白含量较模型组显著提高(P<0.01)。且石油醚组小鼠INS和Nrf2蛋白含量趋于正常水平,与正常组相比无显著性差异。结果表明余庆小叶苦丁茶石油醚和正丁醇组分能够上调2型糖尿病小鼠胰腺组织内INS和Nrf2蛋白的表达,具体见图16-18,其中(A)表示正常组;(B)表示模型组;(C)表示阳性药组;(D)表示石油醚组;(E)表示正丁醇组。
Claims (10)
1.一种余庆小叶苦丁茶提取物,其特征在于:所述提取物包含有石油醚组分A、乙酸乙酯组分B、正丁醇组分C和水溶性组分D。
2.根据权利要求1所述的余庆小叶苦丁茶提取物,其特征在于:所述提取物石油醚组分A包含有三七素、甘露醇、丙酮酸、右旋奎宁酸、5-羟甲基糠醛、达卡巴嗪、7-羟基香豆素、毛蕊花糖苷、金石蚕苷、樟脑、野漆树苷、紫苏烯、乙酰苯、香豆素、白术内酯III、藁本内酯、7-甲氧基-4-甲基香豆素、甘松新酮、芹菜素、松脂酚、莪术烯醇、白术内酯II、梣酮、羌活醇、咖啡酸、白果内酯、8-异戊烯基柚皮素、去甲氧基姜黄素、6-姜烯酚、二氢青蒿酸、诺卡酮、去氢二异丁香酚、α-亚麻酸、刺囊酸、花生四烯酸、油酰、齐墩果酸、油酸酰胺、亚油酸、棕榈酸、亚麻酸乙酯、羽扇烯酮、栎樱酸、花生酸、二十二烷酸其中的任意一种化合物。
3.根据权利要求1所述的余庆小叶苦丁茶提取物,其特征在于:所述提取物乙酸乙酯组分B包含有三七素、腺嘌呤、右旋奎宁酸、烟酰胺、柠檬酸、甘露醇、龙胆酸、羟基酪醇、达卡巴嗪、连翘酯苷E、原儿茶醛、对羟基安息香醛、苯甲酸、七叶亭、咖啡酸、5-羟基马鞭草苷马鞭草苷、对羟基肉桂酸、大车前苷、乙酰苯、罗汉果黄素、芦丁、忍冬苷、异槲皮苷、木犀草苷、紫地黄苷C、毛蕊花糖苷、野漆树苷、7-羟基香豆素、金石蚕苷、芹菜素-7-O-β-D-吡喃葡萄糖苷、5-羟基-四氢萘酮、茵芋苷、橄榄苦苷、木犀草素、苍术苷A、香豆素、苔色酸、2-金刚烷酮、异连翘酯苷A其中的任意一种化合物。
4.根据权利要求1所述的余庆小叶苦丁茶提取物,其特征在于:所述提取物正丁醇组分C包含有丁香醛、三七素、腺嘌呤、右旋奎宁酸、甘露醇、鸟嘌呤、龙胆酸、羟基酪醇、红景天苷、连翘酯苷E、对羟基苯甲酸、5-羟基马鞭草苷、樟脑、马鞭草苷、维采宁II、大车前苷、乙酰苯、龙血素A、山奈酚、罗汉果黄素、芦丁、忍冬苷、木犀草苷、紫地黄苷C、毛蕊花糖苷、茵芋苷、毛蕊花糖苷、野漆树苷、金石蚕苷、5-羟基-四氢萘酮、橄榄苦苷、5-羟基-四氢萘酮、对羟基肉桂酸、苍术苷A、香豆素其中的任意一种化合物。
5.根据权利要求1所述的余庆小叶苦丁茶提取物,其特征在于:所述提取物水溶性组分D包含有丁香醛、右旋奎宁酸、蔗糖、柠檬酸、甘露醇、原儿茶酸、水杨酸、乙酰苯、7-羟基香豆素、毛蕊花糖苷、金石蚕苷、茵芋苷、香豆素其中的任意一种化合物。
6.一种如权利要求1-5任意一项所述的余庆小叶苦丁茶提取物的制备方法,其特征在于:包括以下步骤:
步骤一:称取余庆小叶苦丁茶叶,干燥粉碎过50-100目筛;
步骤二:料液比1:10加蒸馏水在温度50-80℃浸提10-50min;
步骤三:设置温度30-60℃,超声频率为100-500W,超声振荡辅助提取,20-40min;
步骤四:重复步骤二、三的操作提取3次,抽滤合并滤液,用0.45μm滤膜过滤后采用旋转蒸发仪减压浓缩;
步骤五:依次用石油醚、乙酸乙酯、正丁醇对步骤四得到浓缩样品进行萃取,各重复3次,分别取上层液,得石油醚组分A、乙酸乙酯组分B、正丁醇组分C,下层即为水溶性组分D;
步骤六:将步骤五得到的四种组分萃取液采用旋转蒸发仪浓缩,-80℃冷冻干燥,即得余庆小叶苦丁茶提取物石油醚组分A、乙酸乙酯组分B、正丁醇组分C、水溶性组分D冻干粉;
步骤七:将步骤六得到的四种组分冻干粉,按照重量比为1:1:1:1混合即为余庆小叶苦丁茶提取物。
7.根据权利要求6所述的余庆小叶苦丁茶提取物的制备方法,其特征在于:所述步骤四、六中,旋转蒸发仪设置参数为温度60℃,转速50-60rpm,步骤五中,所述溶剂与浓缩液体积比为2:1。
8.一种如权利要求1-5任意一项所述的余庆小叶苦丁茶提取物的应用,其特征在于:所述提取物应用于制备缓解2型糖尿病症状的药物、辅助药物或健康食品,所述提取物制备药物、辅助药物或健康食品时,直接使用或者以组合物的形式使用;所述组合物包括0.1–99%的提取物,其余为载体或赋形剂。
9.根据权利要有8所述的余庆小叶苦丁茶提取物的应用,其特征在于:所述提取物石油醚组分A和正丁醇组分C应用于制备缓解2型糖尿病症状的药物、辅助药物或健康食品。
10.根据权利要有8所述的余庆小叶苦丁茶提取物的应用,其特征在于:所述提取物石油醚组分A和正丁醇组分C能有效降低2型糖尿病小鼠血糖值、改善血清生化指标、上调胰腺组织中INS和Nrf2蛋白表达,同时对小鼠的肝脏、胰腺组织起到保护作用。
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