CN116036148B - 一种滇白珠提取物及其制备方法和应用 - Google Patents
一种滇白珠提取物及其制备方法和应用 Download PDFInfo
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- CN116036148B CN116036148B CN202310216226.4A CN202310216226A CN116036148B CN 116036148 B CN116036148 B CN 116036148B CN 202310216226 A CN202310216226 A CN 202310216226A CN 116036148 B CN116036148 B CN 116036148B
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- ethyl acetate
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Abstract
本发明公开了一种滇白珠提取物及其制备方法和应用，其制备方法为：滇白珠采用水提醇沉法制备水提醇沉浸膏，然后使用乙酸乙酯或正丁醇萃取，回收溶剂，得到滇白珠乙酸乙酯萃取部位或正丁醇萃取部位的提取物。本发明制备的滇白珠提取物(乙酸乙酯萃取部位、正丁醇萃取部位)能够抑制ɑ‑葡萄糖苷酶活性，滇白珠提取物给药浓度为25μg/mL时，可明显改善发生IR‑HepG2细胞的葡萄糖消耗，且对细胞活性无明显影响。由此推测滇白珠提取物通过抑制ɑ‑葡萄糖苷酶活性、增加细胞对胰岛素的敏感性等多重起效，实现抗糖尿病作用。
Description
技术领域
本发明属于医药技术领域，具体涉及一种滇白珠提取物及其制备方法和在制备降血糖药物中的应用。
背景技术
2型糖尿病(type 2diabetes，T2DM)是一种以胰岛B细胞功能障碍和外周胰岛素抵抗为特征的进行性代谢性疾病，以持续性高血糖为主要临床特征，可导致糖代谢缺陷和慢性低度炎症。这种慢性病的发展与复杂的遗传、外部环境、膳食以及肠道菌群失调等因素密切相关。据报道，2019年全球有超过4.6亿成年人患有糖尿病，到2045年这一数字将上升到7亿。T2DM是糖尿病最多发的亚型，约占糖尿病患者的90％以上，被称为“非传染性流行病”。迄今为止，T2DM仍不能治愈，其干预措施主要集中在血糖控制以及相关并发症的预防和治疗。糖尿病不仅给患者带来身心痛苦，还加剧医疗经济负担，发病率和死亡率逐年上升，发病人群逐渐趋于年轻化，它已成为一个全球性的健康问题。因此，探索T2DM的发病机制和治疗新方法具有重要意义。现阶段糖尿病的临床药物治疗主要有胰岛素促泌剂、胰岛素增敏剂、减少糖来源类药物、胰岛素及胰岛素类似物以及新型降糖药物。针对新靶点及用药新方式的糖尿病治疗药物研究迅速。尽管当下临床应用的降糖药物种类不断增加，但仍有40％～50％的糖尿病人糖化血红蛋白仍不达标或没有长期达标，胃肠道反应不耐受，随着糖尿病严重程度增加、并发症的进展，治疗方式受到限制，胰岛素不得不成为唯一和最后选择。因此，开发用药方便、针对新靶点、不良反应少、保护胰岛β细胞功能的新型糖尿病药物意义重大。研究发现，中药民族药以其多途径、多靶点、多环节的网络整体调节特点在降糖调脂、改善胰岛素抵抗(insulin resistance，IR)等方面，有着不可替代的竞争优势，其符合现代医学中抗糖尿病联合用药的治疗手段和多靶点药物研发的发展趋势。
滇白珠Gaultheria leucocarpa BI.var.yunnanensis(Franch.)T.Z.Hsu&R.C.Fang)隶属于杜鹃花科(Ericaceae)越桔亚科(Vaccinioideae)白珠树族(Gaultherieae)，白珠树属(Gaultheria L.)。始载于《滇南本草》，《中药大辞典》和《中国民族药志》也有记载。滇白珠是我国重要的民族药用植物，为很多地区(特别是西南地区)的壮族、白族、彝族、侗族、傈僳族、苗族、纳西族、水族、瑶族、布依族等多个少数民族***喘、改善小肠功能及止泻等功效，其全株或根或地上部分，广泛用于治疗风湿性关节炎。滇白珠的主要成分为水杨酸甲酯糖苷类、黄酮类、苯丙素类、有机酸、香豆素类、萜类、甾醇类和挥发油等。
目前国内外对滇白珠的研究大都集中在化学成分研究及其抗风湿、抗炎镇痛、止泻以及抗癌药效方面，但对其运用于降血糖的药效学基础性研究未见报道，滇白珠的药效物质基础不明确，不能指导其临床使用，故有待进一步研究开发，以期为糖尿病临床防治与创新中药民族药的筛选与研发提供新思路。
发明内容
本发明提供了一种滇白珠提取物及其制备方法和在制备降血糖药物中的应用。该提取物作用温和稳定，具有多方位、多靶点的特点，可促进人体的整体调节且降糖作用持久，能顾及病程发展和预后。
为了达到上述目的，本发明采用如下技术方案：
一种滇白珠提取物的制备方法，滇白珠采用水提醇沉法制备水提醇沉浸膏，然后使用乙酸乙酯或正丁醇萃取，回收溶剂，得到滇白珠乙酸乙酯萃取部位或正丁醇萃取部位的提取物。
优选的，取滇白珠药材全株，加水浸泡后水煮多次，合并水煮液，浓缩得水提物；加入乙醇，静置沉淀，离心收集上清液；沉淀物用乙醇洗涤，离心合并上清液，减压旋蒸，回收溶剂，浓缩得到水提醇沉浸膏；得到的水提醇沉浸膏。
优选的，自来水浸泡1～2h后，用8-10倍量水水煮3次，第1次煮2h，第2、3次煮1h，合并水煮液，浓缩至相对密度为1.05-1.10得水提物。
优选的，水提物加体积分数95％乙醇至溶液的乙醇体积分数达70％，静置沉淀，离心收集上清液；沉淀物用体积分数70％乙醇洗涤。
优选的，所述静置沉淀在4℃下沉淀12～24h。
由上述制备方法获得的滇白珠提取物可以应用在制备降血糖药物中。或者由将由滇白珠提取物与医药学上可接受的载体配合应用在制备降血糖药物中。或者将由上述制备方法获得的滇白珠提取物应用在制备抑制ɑ-葡萄糖苷酶的药物中；或者将由上述制备方法获得的滇白珠提取物应用在制备治疗胰岛素抵抗的药物中。
本发明滇白珠提取物配合医药学上可接受的载体，按本领域常规方法制备成片剂、颗粒剂、胶囊剂、滴丸、酊剂等剂型。
本发明的药学上可接受的载体包括但不限于以下：
稀释剂：淀粉、乳糖、糊精、微晶纤维素、无机盐等。
润湿剂与粘合剂：纯化水、乙醇、明胶、聚乙二醇、纤维素衍生物等。
崩解剂：淀粉、羧甲基淀粉钠、纤维素衍生物、交联聚维酮等。
润滑剂：硬脂酸镁、微粉硅胶、滑石粉、聚乙二醇等。
助溶剂：水、乙醇、甘油、丙二醇、液状石蜡、植物油等。
矫味剂：蔗糖、单糖、芳香剂等。
防腐剂：苯甲酸、山梨酸、甲酯、乙酯、丙酯等。
优选的，所述降血糖药物中滇白珠提取物的浓度为25μg/mL。
本发明的有益效果如下：
本发明制备的滇白珠提取物(乙酸乙酯萃取部位、正丁醇萃取部位)具有抑制ɑ-葡萄糖苷酶活性，滇白珠提取物给药浓度为25μg/mL时，可明显改善发生IR-HepG2细胞的葡萄糖消耗，且对细胞活性无明显影响。由此推测滇白珠提取物通过抑制ɑ-葡萄糖苷酶活性、增加细胞对胰岛素的敏感性等多重起效，实现抗糖尿病作用。
附图说明
图1为实施例1中滇白珠不同提取物对HepG2细胞活力的影响；
图2对实施例1中滇白珠不同提取物IR-HepG2细胞葡萄糖消耗的影响，图中：与正常对照组相比，#P<0.05，##P<0.01，###P<0.001；与模型对照组相比，*P<0.05，**P<0.01，***P<0.001。
具体实施方式
下面将对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1
取滇白珠药材1kg(购自广西壮族自治区金秀县，由广西中医药研究院鉴定为杜鹃花科越桔亚科白珠树族白珠树属植物滇白珠Gaultheria leucocarpaBI.var.yunnanensis(Franch.)T.Z.Hsu&R.C.Fang)的全株)，先加水浸泡1.5h，然后水煮3次，每次加10倍量自来水，第1次煮2h，第2、3次分别煮1h，合并3次水煮液，浓缩至水分蒸干相对密度为1.10得水提物，加95％乙醇至溶液的乙醇含量达70％，静置沉淀(4℃冰箱放置18h)，离心收集上清液；沉淀物用70％乙醇洗涤3次，离心合并上清液，减压旋蒸，回收溶剂，浓缩得到水提醇沉浸膏。得到的水提醇沉浸膏依次使用石油醚、二氯甲烷、乙酸乙酯、正丁醇萃取，回收溶剂，分别得到滇白珠石油醚萃取部位、二氯甲烷萃取部位、乙酸乙酯萃取部位、正丁醇萃取部位的提取物。
试验例
(1)药品和药物剂量
①100μg/mL、50μg/mL、25μg/mL、12.5μg/mL、6.25μg/mL系列浓度的滇白珠提取物溶液：分别称取实施例1制备的10.00mg滇白珠水提取物、水提醇沉物、石油醚萃取部位、二氯甲烷萃取部位、乙酸乙酯萃取部位、正丁醇萃取部位，各加入DMSO 100uL，超声使之充分溶解，即得100mg/mL母液；取100mg/mL母液1uL，加入PBS缓冲溶液999uL，混匀即得100μg/mL滇白珠提取物工作液；取100μg/mL滇白珠提取物工作液50uL，加入PBS缓冲溶液50uL，混匀即得50μg/mL滇白珠提取物工作液；按倍半稀释法如上依次配制25μg/mL、12.5μg/mL、6.25μg/mL系列浓度的滇白珠提取物溶液。
②100μg/mL、50μg/mL、25μg/mL、12.5μg/mL、6.25μg/mL阿卡波糖溶液：称取10.00mg阿卡波糖，加入DMSO 100uL超声充分溶解，即得100mg/mL母液；取100mg/mL母液1uL，加入PBS缓冲溶液999uL，混匀即得100μg/mL阿卡波糖工作液；取100μg/mL阿卡波糖50uL，加入PBS缓冲溶液50uL，混匀即得50μg/mL阿卡波糖工作液；按倍半稀释法如上依次配制25μg/mL、12.5μg/mL、6.25μg/mL系列浓度的阿卡波糖溶液。
(2)实验方法
①酶抑制法检测滇白珠提取物对ɑ-葡萄糖苷酶的抑制作用
反应于96孔培养板上进行，分组如下：空白对照组每孔加入50μL PBS(磷酸盐缓冲溶液)；背景对照组加入10μL待测样品和40μL PBS；阴性对照组加入10μL PBS和40μL 1U/mL的ɑ-葡萄糖苷酶溶液；样品组加入不同浓度的10μL待测样品和40μL 1U/mL的ɑ-葡萄糖苷酶溶液，阳性对照组加入不同浓度的10μL阿卡波糖和40μL 1U/mL的ɑ-葡萄糖苷酶溶液，将96孔板置于37℃反应10min后加入50μLɑ-pNPG(对硝基苯-α-D-葡萄糖苷)作为底物，37℃反应20min。反应结束后各组均加入100μL 1mol/L的Na2CO3溶液终止反应，使用酶标仪测定405nm波长处吸光度(OD值)并记录，计算抑制率及半数抑制量(IC50)。
抑制率(％)＝[1-(OD样品-OD背景)/(OD阴性-OD空白)]*100％
②CCK-8法检测滇白珠提取物对HepG2细胞的细胞活性影响
接种细胞：对数生长期的HepG2细胞，用含10％胎牛血清和1％青链霉素混合液的DMEM完全培养基配成单细胞悬液，按5000个/孔接种于96孔板中，每孔100μL，实验设空白组，正常对照组，实验组，每组设置3个复孔，细胞提前12～24h接种培养。
加入提取物溶液：提取物用DMSO溶解，配成100mg/mL母液，以细胞完全培养基稀释配制100μg/mL、50μg/mL、25μg/mL、12.5μg/mL、6.25μg/mL系列浓度的工作液(DMSO占比至少小于千分之一)，每孔终体积100μL，每种处理均设3个复孔。
显色：37℃培养24h后，弃孔内旧培养基，每孔加入10μL CCK-8溶液和100μL新培养基，继续孵育1～2h后，待反应充分进行后测定吸光度值。
比色：选择450nm波长，全波长全自动多功能酶标仪(Multiskan GO)读取各孔吸光度值并记录结果。
数据处理：计算各组细胞存活率：细胞存活率(％)＝(OD实验组－OD空白组)/(OD对照组－OD空白组)×100％。采用SPSS Statistics 26.0软件计算提取物的IC50值。
③葡萄糖氧化酶法检测提取物对IR-HepG2细胞葡萄糖的消耗量的影响HepG2细胞IR模型建立：取对数生长期的HepG2细胞按5000个/孔接种于96孔板，实验设正常对照组，模型对照组，提取物高、中、低浓度组，每组设置3个复孔。培养24h，除正常对照组外，其余各组以75μmol/L的棕榈酸(PA)溶液作用24h诱导HepG2细胞建立IR模型。
加入提取物溶液：根据CCK-8法检测结果，选取细胞活力大于90％的浓度作为给药高剂量组的浓度，用不含酚红的DMEM完全培养基配制提取物高、中、低浓度工作液，每孔终体积100μL，每种处理均设3个复孔，放入培养箱中作用24小时。
检测：取上清液严格按照试剂盒说明书操作以酶标仪检测并计算葡萄糖含量:葡萄糖(mmol/L)＝样本管吸光度(A)/校准管吸光度(A)*校准液浓度。
数据处理：采用SPSS Statistics 26.0软件处理数据，实验数据以均数±标准差表示，经检验数据呈正态分布方差齐，采用单因素方差分析，组间两两比较采用LSD法，以P＜0.05表示差异有统计学意义。
(3)结果
①滇白珠提取物对ɑ-葡萄糖苷酶的抑制作用
分别以100μg/mL、50μg/mL、25μg/mL、12.5μg/mL、6.25μg/mL的滇白珠提取物与酶和底物反应，采用酶标仪检测各孔吸光度值并计算酶抑制率。结果(表1)表明，实验浓度范围6.25～100μg/mL的滇白珠水提物、水提醇沉物、乙酸乙酯萃取部位、正丁醇萃取部位对ɑ-葡萄糖苷酶具有抑制作用，石油醚萃取部位、二氯甲烷萃取部位对ɑ-葡萄糖苷酶不具抑制作用；滇白珠乙酸乙酯萃取部位IC50值为7.50±0.16μg/mL，阿卡波糖IC50值为27.90±2.00μg/mL，相比于阿卡波糖，滇白珠乙酸乙酯萃取部位具有极显著的酶抑制作用(P<0.001)，故对α-葡萄糖苷酶的抑制作用：滇白珠乙酸乙酯萃取部位>阿卡波糖。提示我们滇白珠水提物、水提醇沉物、乙酸乙酯萃取部位、正丁醇萃取部位可作为降糖活性组分候选。
表1滇白珠提取物对ɑ-葡萄糖苷酶的抑制作用
②滇白珠提取物对HepG2细胞增殖的影响
分别以100μg/mL、50μg/mL、25μg/mL、12.5μg/mL、6.25μg/mL的滇白珠提取物(水提物、水提醇沉物、乙酸乙酯萃取部位、正丁醇萃取部位)处理HepG2细胞24h，然后经CCK-8法检测细胞活力，图1为滇白珠不同提取物对HepG2细胞活力的影响。从图1中得出实验结果为：25μg/mL的滇白珠提取物对细胞增殖均无明显影响(细胞存活率大于90％)，即25μg/mL可作为后续实验的药物处理安全剂量参考值。
③滇白珠提取物对胰岛素抵抗的HepG2细胞的影响
以75μmol/LPA作用于HepG2细胞复制IR模型，再以25μg/mL、12.5μg/mL、6.25μg/mL的滇白珠提取物(水提物、水提醇沉物、乙酸乙酯萃取部位、正丁醇萃取部位)作用于IR-HepG2细胞24h后，采用葡萄糖氧化酶法检测细胞上清液中葡萄糖的含量。图2为滇白珠不同提取物对IR-HepG2细胞葡萄糖消耗的影响，从图2中可以得出的结果表明，相比于正常对照组，模型对照组IR-HepG2细胞对葡萄糖的消耗量显著降低(P<0.001)，差异有统计学意义，说明造模成功；相比于模型对照组，滇白珠提取物处理的IR-HepG2细胞，其葡萄糖的消耗量增加且呈剂量依赖性，尤其是高浓度组可显著性IR-HepG2细胞对葡萄糖的消耗量(P<0.001)，说明滇白珠水提物、水提醇沉物、乙酸乙酯萃取部位、正丁醇萃取部位可改善细胞的胰岛素抵抗状态，增加细胞对胰岛素的敏感度。
综上所述，滇白珠提取物(水提物、水提醇沉物、乙酸乙酯萃取部位、正丁醇萃取部位)具有抑制ɑ-葡萄糖苷酶活性，滇白珠提取物给药浓度为25μg/mL时，可明显改善发生IR-HepG2细胞的葡萄糖消耗，且对细胞活性无明显影响。由此推测滇白珠提取物通过抑制ɑ-葡萄糖苷酶活性、增加细胞对胰岛素的敏感性等多重起效，实现抗糖尿病作用。
本说明书中各个实施例采用递进的方式描述，每个实施例重点说明的都是与其他实施例的不同之处，各个实施例之间相同相似部分互相参见即可。
对所公开的实施例的上述说明，使本领域专业技术人员能够实现或使用本发明。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的，本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下，在其它实施例中实现。因此，本发明将不会被限制于本文所示的这些实施例，而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。
Claims (3)
1.一种滇白珠提取物在制备治疗2型糖尿病药物中的应用，其特征在于，所述滇白珠提取物的制备方法为：取滇白珠药材1kg，先加水浸泡1.5h，然后水煮3次，每次加10倍量自来水，第1次煮2h，第2、3次分别煮1h，合并3次水煮液，浓缩至水分蒸干相对密度为1.10得水提物，加95%乙醇至溶液的乙醇含量达70%，4℃冰箱放置18h静置沉淀，离心收集上清液；沉淀物用70%乙醇洗涤3次，离心合并上清液，减压旋蒸，回收溶剂，浓缩得到水提醇沉浸膏；得到的水提醇沉浸膏使用乙酸乙酯萃取，回收溶剂，得到滇白珠乙酸乙酯萃取部位的提取物。
2.根据权利要求1所述的一种滇白珠提取物在制备治疗2型糖尿病药物中的应用，其特征在于，所述的滇白珠提取物与医药学上可接受的载体配合在制备治疗2型糖尿病药物中的应用。
3.一种滇白珠提取物在制备ɑ-葡萄糖苷酶抑制剂中的应用，其特征在于，所述滇白珠提取物的制备方法为：取滇白珠药材1kg，先加水浸泡1.5h，然后水煮3次，每次加10倍量自来水，第1次煮2h，第2、3次分别煮1h，合并3次水煮液，浓缩至水分蒸干相对密度为1.10得水提物，加95%乙醇至溶液的乙醇含量达70%，4℃冰箱放置18h静置沉淀，离心收集上清液；沉淀物用70%乙醇洗涤3次，离心合并上清液，减压旋蒸，回收溶剂，浓缩得到水提醇沉浸膏；得到的水提醇沉浸膏使用乙酸乙酯萃取，回收溶剂，得到滇白珠乙酸乙酯萃取部位的提取物。
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