CN115040633A - 芍药甘草汤蛋白提取物的新用途 - Google Patents
芍药甘草汤蛋白提取物的新用途 Download PDFInfo
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- CN115040633A CN115040633A CN202210879400.9A CN202210879400A CN115040633A CN 115040633 A CN115040633 A CN 115040633A CN 202210879400 A CN202210879400 A CN 202210879400A CN 115040633 A CN115040633 A CN 115040633A
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- protein
- peony
- licorice
- decoction
- drying
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Abstract
本发明公开芍药甘草汤蛋白提取物在制备用于治疗、预防、减轻和/或缓解与疼痛相关疾病和/或炎症相关疾病的药物中的用途。本发明还公开了一种药物组合物,包括有效量的芍药甘草汤蛋白提取物和药学上可接受的辅料。本发明能够拓宽芍药甘草汤的应用范围,提示在方剂使用、开发时重视蛋白类成分的作用,避免药效的损失。
Description
技术领域
本发明属于中医药技术领域,具体地,本发明涉及一种芍药甘草汤蛋白提取物的新用途。
背景技术
疼痛是与实际存在或潜在的组织损伤相关联的不愉快的感受和情绪体验,是人体的一种主观感觉。疼痛的性质是复杂的,根据疼痛产生的原因可以分为以下六大类:炎性疼痛、神经病理性疼痛、癌性疼痛、痉挛性疼痛、心因性疼痛等。
疼痛严重影响心理健康,导致抑郁、焦虑、快感缺失并影响总体生活质量。目前西医对炎症性疼痛药物的开发主要集中在对非阿片类药物的研究上,但这类药物会增加肾脏和心血管不良事件的风险。中药的药性温和、效果明显并且对人体的副作用小,因此,越来越多的研究人员已经开始从中药中寻找适合炎症性疼痛药物。
芍药甘草汤出自《伤寒论》29条“伤寒,脉浮,自汗出,小便数,心烦,微恶寒,脚挛急……若厥愈足温者,更作芍药甘草汤与之,其脚即伸。芍药(四两),甘草(四两,炙),上二味,以水三升,煮取一升五合,去滓,分温再服”。芍药甘草汤作为解痉镇痛的经典名方,因其药味少,作用显著,被历代医家推崇并沿用至今。
现代药理研究发现芍药甘草汤主要含有黄酮类、三萜皂苷类、单萜糖苷类、等化学成分,具有解痉、抗炎、镇痛、止咳平喘、保肝作用、保护胃黏膜、免疫调节等作用。芍药苷、芍药内酯苷、没食子酸、甘草苷、甘草酸等为其主要药效成分。
不难看出,当前针对芍药甘草汤药效成分的研究主要集中在小分子物质上。因此,目前亟需对芍药甘草汤进行更加深入的研究以便开发出更多效果更好的抗炎镇痛类药物。
发明内容
本发明的发明目的在于针对现有技术中存在的缺陷,提供芍药甘草汤蛋白提取物的新用途。
作为本发明的一个方面,本发明提供了芍药甘草汤蛋白提取物在制备用于治疗、预防、减轻和/或缓解与疼痛相关疾病和/或炎症相关疾病的药物中的用途。
优选地,所述与疼痛相关疾病是腓肠肌痉挛、肋间神经痛、胃痉挛、胃痛、腹痛、坐骨神经痛、妇科炎性腹痛或痛经。
优选地,所述炎症相关疾病是十二指肠溃疡、萎缩性胃炎、胃肠神经官能症或急性乳腺炎。
优选地,所述芍药甘草汤蛋白提取物通过如下方法获得:
制备芍药甘草汤;
从所述芍药甘草汤中提取蛋白初提液;
对所述蛋白初提液进行纯化,得到纯化蛋白;
对所述纯化蛋白进行透析和干燥。
进一步优选地,所述芍药甘草汤蛋白提取物通过如下方法获得:
重量比是(1~3):1的芍药和甘草饮片用水浸泡、煎煮、过滤、浓缩,得到芍药甘草汤;
所述芍药甘草汤使用乙醇进行分离提纯,随后离心分离,得到的沉淀使用PBS缓冲液溶解,得到蛋白初提液;
采用离子交换色谱分离法对所述蛋白初提液进行纯化,得到纯化蛋白;
采用截留分子量是8000~14000Da的透析袋对所述纯化蛋白进行透析,得到的透析液进行干燥;优选地,所述干燥是减压干燥、冷冻干燥或鼓风干燥。
作为本发明的另一个方面,本发明还提供了一种能够用于治疗、预防、减轻和/或缓解与疼痛相关疾病和/或炎症相关疾病的药物组合物,包括芍药甘草汤蛋白提取物和药学上可接受的辅料。
优选地,所述与疼痛相关疾病是腓肠肌痉挛、肋间神经痛、胃痉挛、胃痛、腹痛、坐骨神经痛、妇科炎性腹痛或痛经。
优选地,所述炎症相关疾病是十二指肠溃疡、萎缩性胃炎、胃肠神经官能症或急性乳腺炎。
优选地,所述芍药甘草汤蛋白提取物通过如下方法获得:
制备芍药甘草汤;
从所述芍药甘草汤中提取蛋白初提液;
对所述蛋白初提液进行纯化,得到纯化蛋白;
对所述纯化蛋白进行透析和干燥。
进一步优选地,所述芍药甘草汤蛋白提取物通过如下方法获得:
重量比是(1~3):1的芍药和甘草饮片用水浸泡、煎煮、过滤、浓缩,得到芍药甘草汤;
所述芍药甘草汤使用乙醇进行分离提纯,随后离心分离,得到的沉淀使用PBS缓冲液溶解,得到蛋白初提液;
采用离子交换色谱分离法对所述蛋白初提液进行纯化,得到纯化蛋白;
采用截留分子量是8000~14000Da的透析袋对所述纯化蛋白进行透析,得到的透析液进行干燥;优选地,所述干燥是减压干燥、冷冻干燥或鼓风干燥。
作为一个特别优选的实施例,所述芍药甘草汤蛋白提取物通过如下方法获得:
(1)芍药甘草汤的制备
按照重量比1:1称取芍药和甘草饮片,加入芍药和甘草饮片重量之和的8倍量水浸泡30min,煎煮40min后,无纺纱布过滤,药渣中加6倍量水煎煮30min,无纺纱布过滤,合并两次滤液,旋转蒸发仪回收浓缩至1g生药/mL,得芍药甘草汤;
(2)芍药甘草汤中蛋白的粗提
向芍药甘草汤中缓慢加入95%乙醇并不断搅拌,直至乙醇饱和度为80%,4℃静置24h,4000rpm离心20min,分离上清和沉淀,沉淀用PBS缓冲液(0.05mol/L、PH 7.4)充分溶解,即得芍药甘草汤蛋白初提液;
(3)离子交换色谱分离纯化蛋白
取芍药甘草汤蛋白初提取液100mL上样到层析柱上。流动相A:PBS缓冲液(0.05mol/L、PH 7.4);流动相B:含0.5mol/L NaCl的PBS缓冲液(0.05mol/L、PH 7.4);流速1mL/min,分别采用100%的流动相A和100%的流动相B对蛋白进行洗脱,收集洗脱组分;
(4)透析
上述流出组分混合后装入预处理的透析袋(截留分子量为8000-14000Da),置于PBS缓冲液(0.05mol/L、PH 7.4)中12h,此期间至少更换五次缓冲液。透析液即为芍药甘草汤蛋白提取液,进行蛋白浓度测定后,冻干,得到的冻干粉即为芍药甘草汤蛋白提取物。
优选地,药学上可接受的辅料是填充剂、崩解剂、润滑剂、助悬剂、粘合剂、甜味剂、矫味剂、防腐剂和基质中的任意一种或多种。
优选地,所述药物组合物包括但不限于片剂、硬胶囊剂、软胶囊剂、颗粒剂、丸剂、散剂、口服液、吸入剂或注射剂。所述药学可接受的辅料包括:填充剂、崩解剂、润滑剂、助悬剂、粘合剂、甜味剂、矫味剂、防腐剂、基质等。填充剂包括:淀粉、预胶化淀粉、乳糖、甘露醇、甲壳素、微晶纤维素、蔗糖等;崩解剂包括:淀粉、预胶化淀粉、微晶纤维素、羧甲基淀粉钠、交联聚乙烯吡咯烷酮、低取代羟丙纤维素、交联羧甲基纤维素钠等;润滑剂包括:硬脂酸镁、十二烷基硫酸钠、滑石粉、二氧化硅等;助悬剂包括:聚乙烯吡咯烷酮、微晶纤维素、蔗糖、琼脂、羟丙基甲基纤维素等;粘合剂包括,淀粉浆、聚乙烯吡咯烷酮、羟丙基甲基纤维素等;甜味剂包括:糖精钠、阿斯帕坦、蔗糖、甜蜜素、甘草次酸等;矫味剂包括:甜味剂及各种香精;防腐剂包括:尼泊金类、苯甲酸、苯甲酸钠、山梨酸及其盐类、苯扎溴铵、醋酸氯乙定、桉叶油等;基质包括:PEG6000,PEG4000,虫蜡等。
本发明所述的“有效量”或“治疗有效量”是指无毒性,但足够量的提供所需的作用的药物或药剂。在本发明的药物组合物中,一种成分或制剂单元的“有效量”是指该成分在和其他成分联合应用时有效提供所需效应的量。“有效量”会因受试者的不同而不同,依据年龄和个体的一般情况,特定的活性药物等等。因此,不可能总是指精确的“有效量”,然而,任何个体病例中合适的“有效量”可以由本领域普通技术人员应用常规的实验方法来确定。例如,对于正常成年人来说,可以按照0.55~0.70mg/kg的有效量进行给药,当然,这只是示例性的。
本发明所述的芍药和甘草饮片(或甘草)均为《中国药典》(2015版)所收载,符合药典项下的各项规定。
芍药甘草汤作为具有镇痛作用的经典方剂,主要的研究对象是以芍药苷、芍药内酯苷为代表的小分子化合物,但是目前的研究没有发现大分子如蛋白类成分的药效作用。本发明的发明人通过研究发现,芍药甘草汤的蛋白提取物具有较好的镇痛及抗炎作用,其镇痛效果与化学药对乙酰氨基酚相似,但其抗炎能力优于对乙酰氨基酚,且其来源于中药,安全性较好。本发明能够拓宽芍药甘草汤的应用范围,提示在方剂使用、开发时重视蛋白类成分的作用,避免药效的损失。
附图说明
图1是本发明实施例1中BSA的标准曲线图;
图2显示了本发明实施例2中扭体实验结果;
图3显示了本发明实施例3中小鼠血清PGE2水平(**代表和模型组有极显著性差异P<0.01);
图4显示了本发明实施例3中小鼠血清NO水平(**代表和模型组有极显著性差异P<0.01);
图5显示了本发明实施例3中小鼠血清IL-10水平(**代表和模型组有极显著性差异P<0.01)。
具体实施方式
实施例1:芍药甘草汤蛋白提取物的制备
(1)芍药甘草汤的制备
称取芍药和甘草饮片各62g,加八倍量水浸泡30min,煎煮40min后,无纺纱布过滤,药渣中加6倍量水煎煮30min,无纺纱布过滤,合并两次滤液,旋转蒸发仪回收浓缩至1g生药/mL,得芍药甘草汤。
(2)芍药甘草汤中蛋白的粗提
向芍药甘草汤中缓慢加入95%乙醇并不断搅拌,直至乙醇饱和度为80%,4℃静置24h,4000rpm离心20min,分离上清和沉淀,沉淀用PBS缓冲液(0.05mol/L、PH 7.4)充分溶解,即得芍药甘草汤蛋白初提液。
(3)离子交换色谱分离纯化蛋白
a将50mL填料倒入500mL烧杯中,向烧杯中添加200mL的蒸馏水(约为填料体积的4倍),搅拌后静置45min,小心倒出表面的悬浮液(含小颗粒),重复浸泡洗涤3-4次。
b将色谱空柱竖直固定,柱子下端预先注入0.5mL蒸馏水润湿底部,将填料上层水弃去,玻璃棒搅拌至填料为匀浆状态,用玻璃棒引流将其缓慢匀速地倒入色谱柱中,保证柱中无气泡和裂缝。
c以蒸馏水为流动相,设置流速为10mL/min,压力在0.3MPa以下,冲洗10min后,装柱完成。
流动相A:PBS缓冲液(0.05mol/L、PH 7.4);流动相B:含0.5mol/L NaCl的PBS缓冲液(0.05mol/L、PH 7.4);流速1mL/min,分别采用100%的流动相A和100%的流动相B对蛋白进行洗脱,收集洗脱组分,进行进一步的透析纯化。
(4)透析
透析过程中所用透析袋的截留分子量为8000-14000Da,洗脱组分装入预处理的透析袋,置于PBS缓冲液(0.05mol/L、PH 7.4)中12h,此期间至少更换五次缓冲液,透析后冻干即得芍药甘草汤蛋白冻干粉。
(5)汤剂中蛋白类成分含量的测定
使用BCA试剂盒(碧云天生物技术公司)测定汤剂中蛋白类成分的含量。以牛血清白蛋白(bovine albumin,BSA)为标准品,用PBS缓冲液将其配制成0.5mg/mL的标准品母液。精密吸取0,1,2,4,8,12,16,20μL的0.5mg/mLBSA标准品溶液至96孔板中,再依次加PBS缓冲液补足至20μL,相当于标准品浓度分别为0,0.025,0.05,0.1,0.2,0.3,0.4,0.5mg/mL。将汤剂样品用PBS缓冲液稀释20倍,精密吸取20μL至96孔板。以上标准品和样品均设置三个复孔。根据标准品孔和样品孔数量,将BCA试剂A和BCA试剂B按体积比为50:1配制适当体积的工作液,充分混匀,精密吸取200μL至各标准品孔和样品孔中,37℃孵育0.5h后,570nm波长下测定吸光度(OD值),以OD值为纵坐标,以BSA的浓度为横坐标绘制标准曲线,见图1,可得线性回归方程为Y=1.0262X+0.1168(R2=0.9977),说明BSA在0-0.5mg/mL浓度范围内具有良好的线性关系,可用于测定样品中蛋白含量。根据BSA的线性回归方程计算汤剂中的蛋白含量。
本实施例的研究表明,将芍药甘草汤制成冻干粉,该冻干粉中的蛋白含量为207mg/g。芍药甘草汤经提取、分离、纯化、透析、冻干处理后得到的芍药甘草汤蛋白提取物冻干粉,该冻干粉中的蛋白含量为783mg/g。这说明本发明的方法能够有效的富集提纯芍药甘草汤蛋白。
实施例2:芍药甘草汤蛋白提取物对急性疼痛的影响
(1)实验方法:
扭体法实验参照《实验药理方法学》(药理实验方法学(第四版)主编:魏伟,吴希美,李元建出版社:人民卫生出版社出版日期:2010年)中的醋酸扭体实验。取昆明种小鼠24只,随机分为3组,每组8只,分别为模型组、芍药甘草汤蛋白组、阳性药组。按照0.1ml/10g的剂量向模型组的小鼠腹腔注射生理盐水。用生理盐水溶解实施例1制备的芍药甘草汤蛋白冻干粉,配置成浓度是11.8mg/ml的溶液,按照0.1ml/10g的剂量向芍药甘草汤蛋白组的小鼠腹腔注射药物。用生理盐水稀释对乙酰氨基酚注射液(乐夫瑞),配置成浓度是0.5mg/ml的溶液,按照0.1ml/10g的剂量向阳性药组的小鼠腹腔注射药物。给药一次。
芍药甘草汤蛋白组和阳性药组小鼠给药60min后,建立急性疼痛模型,方法是以0.1ml/10g的剂量给模型组、芍药甘草汤蛋白组和阳性药组的每只小鼠腹腔注射0.6%的醋酸溶液,记录注射醋酸后15min内小鼠的首次扭体(以小鼠出现腹部内凹、躯干与后肢伸张,臀部高起为扭体反应)时间与扭体反应次数,计算扭体抑制率。
扭体抑制率(%)=(模型组平均扭体次数-给药组平均扭体次数)/模型组平均扭体次数×100%。
(2)扭体实验结果:
扭体实验结果见图2和表1,与模型组相比,芍药甘草汤蛋白组小鼠的扭体次数明显减少(p<0.01)。
表1:芍药甘草汤蛋白对醋酸扭体实验扭体次数的影响(xˉ±s)
组别 | 例数 | 扭体次数 | 扭体抑制率(%) |
模型组 | 8 | 38.4±16.99 | |
阳性药组 | 8 | 12.5±6.99** | 67.44 |
芍药甘草汤蛋白组 | 8 | 13.13±5.817** | 65.82 |
(**代表和模型组有极显著性差异P<0.01)
从图2和表1可以看出,芍药甘草汤蛋白可以有效的减少扭体次数,缓解急性疼痛,芍药甘草汤蛋白组的扭体抑制率与阳性药接近,说明芍药甘草汤蛋白具有和对乙酰氨基酚相似的镇痛作用,但对乙酰氨基酚作为化学药物具有一定的副作用,芍药甘草汤蛋白提取自中药汤剂,生物相容性较好。
实施例3:芍药甘草汤蛋白提取物对急性疼痛模型的炎症因子水平的影响
(1)实验方法
取昆明种小鼠32只,随机分为4组,每组8只,分别为正常组、模型组、芍药甘草汤蛋白组、阳性药组。实验前小鼠禁食12h,不禁水。
按照0.1ml/10g的剂量向正常组和模型组的小鼠腹腔注射生理盐水。用生理盐水溶解实施例1制备的芍药甘草汤蛋白冻干粉,配置成浓度是11.8mg/ml的溶液,按照0.1ml/10g的剂量向芍药甘草汤蛋白组的小鼠腹腔注射药物。用生理盐水稀释对乙酰氨基酚注射液(乐夫瑞),配置成浓度是0.5mg/ml的溶液,按照0.1ml/10g的剂量向阳性药组的小鼠腹腔注射药物。给药一次。
芍药甘草汤蛋白组和阳性药组小鼠给药60min后,建立急性疼痛模型,方法是以0.1ml/10g的剂量给模型组、芍药甘草汤蛋白组和阳性药组的每只小鼠腹腔注射0.6%的醋酸溶液,正常组小鼠腹腔注射生理盐水。
建立疼痛模型60min后,使用10%水合氯醛0.04ml/10g腹腔注射麻醉小鼠,眼球取血,血样于室温下静置30min,4℃下3000r/min离心15min,取上清于EP管中,将得到的血清样品在-80℃生物样品库保存。
使用前列腺素E2(PGE2)测试盒(南京建成生物工程研究所)、一氧化氮(NO)测定试剂盒(微板法)(南京建成生物工程研究所)和白介素-10(IL-10)测试盒(南京建成生物工程研究所)分别检测血清里炎症因子PGE2、NO、IL-10的水平。结果如图3、图4和图5所示。
由图3、图4和图5可以看出,给予小鼠芍药甘草汤蛋白后能够有效降低血清中促炎因子PGE2及NO的水平(p<0.01),也能够提高抗炎因子IL-10的水平。其中,芍药甘草汤蛋白降低促炎因子水平的能力比阳性药对乙酰氨基酚更好。
Claims (10)
1.芍药甘草汤蛋白提取物在制备用于治疗、预防、减轻和/或缓解与疼痛相关疾病和/或炎症相关疾病的药物中的用途。
2.根据权利要求1所述的用途,其特征在于,所述与疼痛相关疾病是腓肠肌痉挛、肋间神经痛、胃痉挛、胃痛、腹痛、坐骨神经痛、妇科炎性腹痛或痛经。
3.根据权利要求1所述的用途,其特征在于,所述炎症相关疾病是十二指肠溃疡、萎缩性胃炎、胃肠神经官能症或急性乳腺炎。
4.根据权利要求1所述的用途,其特征在于,所述芍药甘草汤蛋白提取物通过如下方法获得:
制备芍药甘草汤;
从所述芍药甘草汤中提取蛋白初提液;
对所述蛋白初提液进行纯化,得到纯化蛋白;
对所述纯化蛋白进行透析和干燥。
5.根据权利要求4所述的用途,其特征在于,所述芍药甘草汤蛋白提取物通过如下方法获得:
重量比是(1~3):1的芍药和甘草饮片用水浸泡、煎煮、过滤、浓缩,得到芍药甘草汤;
所述芍药甘草汤使用乙醇进行分离提纯,随后离心分离,得到的沉淀使用PBS缓冲液溶解,得到蛋白初提液;
采用离子交换色谱分离法对所述蛋白初提液进行纯化,得到纯化蛋白;
采用截留分子量是8000~14000Da的透析袋对所述纯化蛋白进行透析,得到的透析液进行干燥;优选地,所述干燥是减压干燥、冷冻干燥或鼓风干燥。
6.一种药物组合物,其特征在于,包括有效量的芍药甘草汤蛋白提取物和药学上可接受的辅料。
7.根据权利要求6所述的药物组合物,其特征在于,所述芍药甘草汤蛋白提取物通过如下方法获得:
制备芍药甘草汤;
从所述芍药甘草汤中提取蛋白初提液;
对所述蛋白初提液进行纯化,得到纯化蛋白;
对所述纯化蛋白进行透析和干燥。
8.根据权利要求7所述的药物组合物,其特征在于,所述芍药甘草汤蛋白提取物通过如下方法获得:
重量比是(1~3):1的芍药和甘草饮片用水浸泡、煎煮、过滤、浓缩,得到芍药甘草汤;
所述芍药甘草汤使用乙醇进行分离提纯,随后离心分离,得到的沉淀使用PBS缓冲液溶解,得到蛋白初提液;
采用离子交换色谱分离法对所述蛋白初提液进行纯化,得到纯化蛋白;
采用截留分子量是8000~14000Da的透析袋对所述纯化蛋白进行透析,得到的透析液进行干燥;优选地,所述干燥是减压干燥、冷冻干燥或鼓风干燥。
9.根据权利要求6所述的药物组合物,其特征在于,所述药学上可接受的辅料是填充剂、崩解剂、润滑剂、助悬剂、粘合剂、甜味剂、矫味剂、防腐剂和基质中的任意一种或多种。
10.根据权利要求6所述的药物组合物,其特征在于,所述药物组合物是片剂、硬胶囊剂、软胶囊剂、颗粒剂、丸剂、散剂、口服液、吸入剂或注射剂。
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