CN105963567A - 治疗糖尿病的药物及制备方法 - Google Patents
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Abstract
本发明一种原料来源广泛、成本低廉、可有效提高降糖效果且无副作用的治疗糖尿病的药物及制备方法,是以牡蛎多糖、玉米须多糖及苦瓜多糖按质量比为5~6:2~3:2~3组合而成。降血糖的效果及综合药效显著好于牡蛎多糖、玉米须多糖或者苦瓜多糖单独作用效果,同时本发明还可对糖尿病患者的肾功能起到了明显地改善作用。
Description
技术领域
本发明涉及一种治疗糖尿病的药物及制备方法,尤其是一种原料来源广泛、成本低廉、可有效提高降糖效果且无副作用的治疗糖尿病的药物及制备方法。
背景技术
糖尿病是以高血糖为特征的一系列代谢紊乱的疾病,长期存在的高血糖可导致各种组织(眼、肾、心脏、血管、神经等)的慢性损害、功能障碍,目前对于糖尿病的治疗大多采用磺脲类、双胍类等降糖药物。磺脲类、双胍类等药物在降糖的同时会对人体产生副作用,最常见表现为恶心、呕吐、食欲下降、腹痛、腹泻;头痛、头晕、金属味;乳酸酸中毒,伴有肝、肾功能减退等,因此药物应用禁忌症较多,如严重肝、肾、心、肺疾病,消耗性疾病,营养不良,缺氧性疾病,妊娠期等均禁止使用。
牡蛎是我国重要的养殖贝类,也是全世界分布最广的贝类。人们对于牡蛎的药用价值早有认识。《本草纲目》中记载牡蛎肉具有治疗虚损、调中、解丹毒、妇人血气的作用。以姜、醋生食,治丹毒,酒后烦热,止渴。牡蛎多糖是牡蛎中重要的活性物质,对于它的研究已有很多报道。Shi等以太平洋牡蛎为原料,采用热水萃取方法得到水溶性牡蛎多糖,研究了牡蛎多糖对四氯化碳诱导的急性肝损伤以及酒精诱导的慢性肝损伤的保护作用,结果表明灌胃牡蛎多糖的小鼠血清中谷草转氨酶(AST)、谷丙转氨酶(ALT)活性以及丙二醛(MDA)含量降低,超氧化物歧化酶(SOD)活性升高,说明牡蛎多糖对肝脏具有较强的保护作用(Shi等发表于2015年第78卷第142-148页的《International Journal of BiologicalMacromolecules》上)。贾淑杰等研究了玉米须乙醇及水提取物对小鼠的降血糖作用,发现两种提取物对高血糖和肾上腺素所致的小鼠高血糖有降低作用(贾淑杰等发表于2012年第8期第40卷第809-811页的《天津医药》上)。苦瓜果实含有丰富的皂苷、黄酮、多肽、多糖等多种生物活性物质,具有降血糖、抗癌、抗病毒等作用。但是,迄今为止,还没有将牡蛎多糖、玉米须多糖及苦瓜多糖进行组合进行降糖的研究报道。
发明内容
本发明是为了解决现有技术所存在的上述技术问题,提供一种原料来源广泛、成本低廉、可有效提高降糖效果且无副作用的治疗糖尿病的药物及制备方法。
本发明的技术解决方案是:一种治疗糖尿病的药物,其特征在于是以牡蛎多糖、玉米须多糖及苦瓜多糖按质量比为5~6:2~3:2~3组合而成。
一种上述治疗糖尿病的药物的制备方法,所述的牡蛎多糖按如下方法制备:将牡蛎去壳及去内脏之后进行匀浆处理,用体积/质量为15倍的沸水浸提10min,过滤得滤液;对滤渣用体积/质量为15倍的沸水浸提10min,过滤得滤液;合并滤渣再用体积/质量为15倍的沸水浸提10min,过滤得滤液;将3次滤液合并调节pH为5~6,过夜放置,6000rpm离心20min,收集上清;上清液调节pH为1.5~2.5,向上清液加入胃蛋白酶,在温度为37℃条件下进行保温酶解4h,再次6000rpm离心20min,胃蛋白酶的加入量为上清液质量1%×6000活性单位/克;获得的酶解上清液经过截留分子量10kDa的超滤膜过滤,再将过膜截留的滤液进行喷雾干燥,得到牡蛎多糖。
所述玉米须多糖按如下方法制备:将玉米须粉碎,用体积/质量为15倍的沸水浸提10min,过滤得滤液;对滤渣用体积/质量为15倍的沸水浸提10min,过滤得滤液;合并滤渣再用体积/质量为15倍的沸水浸提10min,过滤得滤液;将3次滤液合并调节pH为4~5,过夜放置,6000rpm离心20min,收集上清;上清液调节pH为1.5~2.5,向上清液加入胃蛋白酶,在温度为37℃条件下进行保温酶解4h,再次6000rpm离心20min,胃蛋白酶的加入量为上清液质量1%×6000活性单位/克;获得的酶解上清液经过截留分子量10kDa的超滤膜过滤,再将过膜截留的滤液进行喷雾干燥,得到玉米须多糖。
所述苦瓜多糖按如下方法制备:新鲜苦瓜去籽去瓤,进行匀浆处理,用体积/质量为10倍的沸水浸提10min,过滤得滤液;对滤渣用体积/质量为10倍的沸水浸提10min,过滤得滤液;合并滤渣再用体积/质量为10倍的沸水浸提10min,过滤得滤液;将3次滤液合并调节pH为5~6,过夜放置,6000rpm离心20 min,收集上清;上清液调节pH为6.5~7.5,向上清液加入中性蛋白酶,在温度为37℃条件下进行保温酶解4h,再次6000rpm离心20min,中性蛋白酶的加入量为上清液质量1%×6000活性单位/克;获得的酶解上清液经过截留分子量10kDa的超滤膜过滤,再将过膜截留的滤液进行喷雾干燥,得到苦瓜多糖。
本发明原料来源广泛、成本低廉、可有效提高降糖效果且无副作用,降血糖的效果及综合药效显著好于牡蛎多糖、玉米须多糖或者苦瓜多糖单独作用效果,同时本发明还可对糖尿病患者的肾功能起到了明显地改善作用。
具体实施方式
实施例1:
以牡蛎多糖、玉米须多糖及苦瓜多糖按质量比为6:2:2混合而成,可制成片剂、散剂、颗粒剂、口服液或注射液等各种剂型。
所述的牡蛎多糖按如下方法制备:将牡蛎去壳及去内脏之后进行匀浆处理,用体积/质量为15倍的沸水浸提10min,过滤得滤液;对滤渣用体积/质量为15倍的沸水浸提10min,过滤得滤液;合并滤渣再用体积/质量为15倍的沸水浸提10min,过滤得滤液;每次浸提过程中均需间歇搅拌,将3次滤液合并调节pH为6,过夜放置,6000rpm离心20min,收集上清;上清液调节pH为2,向上清液加入胃蛋白酶,在温度为37℃条件下进行保温酶解4h,再次6000rpm离心20min,胃蛋白酶的加入量为上清液质量1%×6000活性单位/克,即若上清液为1000克,则加入60000活性单位的胃蛋白酶;若上清液为800克,则加入48000活性单位的胃蛋白酶。在温度为37℃条件下进行保温酶解4h,再次6000rpm离心20min;获得的酶解上清液经过截留分子量10kDa的超滤膜过滤,再将过膜截留的滤液进行喷雾干燥,得到牡蛎多糖,牡蛎多糖纯度≥90%。
所述玉米须多糖按如下方法制备:将玉米须粉碎,用体积/质量为15倍的沸水浸提10min,过滤得滤液;对滤渣用体积/质量为15倍的沸水浸提10min,过滤得滤液;合并滤渣再用体积/质量为15倍的沸水浸提10min,过滤得滤液;每次浸提过程中均需间歇搅拌,将3次滤液合并调节pH为4,过夜放置,6000rpm离心20min,收集上清;上清液调节pH为2,向上清液加入胃蛋白酶,在温度为37℃条件下进行保温酶解4h,再次6000rpm离心20min,胃蛋白酶的加入量为上清液质量1%×6000活性单位/克;获得的酶解上清液经过截留分子量10kDa的超滤膜过滤,再将过膜截留的滤液进行喷雾干燥,得到玉米须多糖,玉米须多糖纯度≥90%。
所述苦瓜多糖按如下方法制备:新鲜苦瓜去籽去瓤,进行匀浆处理,用体积/质量为10倍的沸水浸提10min,过滤得滤液;对滤渣用体积/质量为10倍的沸水浸提10min,过滤得滤液;合并滤渣再用体积/质量为10倍的沸水浸提10min,过滤得滤液;每次浸提过程中均需间歇搅拌,将3次滤液合并调节pH为5,过夜放置,6000rpm离心20 min,收集上清;上清液调节pH为7,向上清液加入中性蛋白酶,在温度为37℃条件下进行保温酶解4h,再次6000rpm离心20min,中性蛋白酶的加入量为上清液质量1%×6000活性单位/克;获得的酶解上清液经过截留分子量10kDa的超滤膜过滤,再将过膜截留的滤液进行喷雾干燥,得到苦瓜多糖,苦瓜多糖纯度≥90%。
实施例2:
以牡蛎多糖、玉米须多糖及苦瓜多糖按质量比为5:3:2混合而成,可制成片剂、散剂、颗粒剂、口服液或注射液等各种剂型。
制备方法同实施例1。
实施例3:
以牡蛎多糖、玉米须多糖及苦瓜多糖按质量比为6:2:3混合而成,可制成片剂、散剂、颗粒剂、口服液或注射液等各种剂型。
制备方法同实施例1。
实验:
实验材料:
四氧嘧啶、乙酸铵购于Sigma公司。盐酸二甲双胍购于北京京丰制药集团。尿肌酐测定试剂盒、尿素氮测定试剂盒、糖化血清蛋白测定试剂盒、胆固醇测定试剂盒、甘油三酯测定试剂盒购于南京建成生物工程研究所。
实验仪器:
三诺安稳血糖仪(三诺生物传感股份有限公司);HH-2型数显恒温水浴锅(国华电器有限公司);UV-1750紫外分光光度计(苏州岛津仪器有限公司);IEC MicroCL 17R微量台式小型高速离心机(德国Thermo公司);电子天平(北京赛多利斯仪器系统有限公司)。
实验分组及用药:
SPF级ICR雄性小鼠,体质量18~22 g,购自大连医科大学实验动物中心。取SPF级ICR雄性小鼠180只,适应性饲养3 d,随机挑选10只作为正常对照组,其余动物禁食15 h(不禁水),由尾静脉快速注射新鲜配制的四氧嘧啶生理盐水溶液(80mg/kg),建立糖尿病动物模型(血糖值≥11.1 mmol/L的小鼠,且连续出现多饮、多食、多尿、体质量减轻症状者为糖尿病小鼠)。正常对照组注射等体积的生理盐水,96 h后(禁食12 h)尾静脉取血测定小鼠空腹血糖水平。
将成模的动物按体质量和血糖水平随机分高血糖组、盐酸二甲双胍组和给药组(200、400、800 mg/kg/day)12个剂量组。盐酸二甲双胍组给予200mg/kg/day盐酸二甲双胍,给药组(本发明实施例1、牡蛎多糖组、玉米须多糖组以及苦瓜多糖组)按不同剂量灌胃给药。给药组每天给药2次,正常对照组、高血糖组每天灌胃蒸馏水2次,间隔6 h,连续21d。第7、14、21d测小鼠空腹血糖,测试前禁食12 h。第7d测小鼠体重、饮水量、饮食量。
生化指标检测:
第21d收集尿液,用检测试剂盒测定尿肌酐、尿素氮。
第21d测定空腹血糖后,摘眼球取血,分离血清,果糖胺法测定其中糖化血清蛋白含量(GSP),酶偶联比色法测定甘油三酯(TG)、总胆固醇(TC)含量。
实验结果:
一.对四氧嘧啶致ICR小鼠糖尿病模型降血糖的影响如表1。
表1 牡蛎多糖对四氧嘧啶致ICR小鼠糖尿病模型降血糖的影响
注:(1)同一行数据中上标字母不同代表存在显著差异(P <0.05);
(2)与高血糖组相比,*代表差异显著(P <0.05);**代表差异极显著(P <0.01)
表1结果表明:给药7 d与给药0 d相比,盐酸二甲双胍组(200 mg/kg/day)、本发明实施例1组(200、400、800 mg/kg/day)、牡蛎多糖组(400、800 mg/kg/day)、玉米须多糖组(200、400、800 mg/kg/day)和苦瓜多糖组(400、800 mg/kg/day)的空腹血糖值下降显著(P <0.05),牡蛎多糖组(200 mg/kg/day)、苦瓜多糖组(200 mg/kg/day)的空腹血糖值下降不显著(P >0.05)。与高血糖组相比,盐酸二甲双胍组(200 mg/kg/day)、本发明实施例1组(200、400 mg/kg/day)、牡蛎多糖组(400、800 mg/kg/day)、玉米须多糖组(200、400、800 mg/kg/day)和苦瓜多糖组(800 mg/kg/day)的空腹血糖值下降极显著(P <0.01),本发明实施例1组(800 mg/kg/day)和牡蛎多糖组(200 mg/kg/day)的空腹血糖值下降显著(P <0.05)。与给药7d相比,给药14d、21d的盐酸二甲双胍组(200 mg/kg/day)、本发明实施例1组(200、400、800 mg/kg/day)、牡蛎多糖组(200、400、800 mg/kg/day)、玉米须多糖组(200、400、800mg/kg/day)和苦瓜多糖组(200、400、800 mg/kg/day)的空腹血糖值变化不显著(P >0.05),表明给药后血糖值维持稳定。
二.对四氧嘧啶致ICR小鼠糖尿病TG、TC、GSP的影响见表2。
表2 对四氧嘧啶致ICR小鼠糖尿病TG、TC、GSP的影响
注:(1)与正常组相比,*代表差异显著(P <0.05);**代表差异极显著(P <0.01)
(2)与高血糖组相比,#代表差异显著(P <0.05);##代表差异极显著(P <0.01)
表2结果表明,与高血糖组相比,盐酸二甲双胍组(200 mg/kg/day)、本发明实施例1组(200、400、800 mg/kg/day)、牡蛎多糖组(200、400 mg/kg/day)和玉米须多糖组(200、400mg/kg/day)的TG含量均显著下降(P <0.05);盐酸二甲双胍组(200 mg/kg/day)、发明实施例1组(200 mg/kg/day)、牡蛎多糖组(200 mg/kg/day)、玉米须多糖组(400 mg/kg/day)和苦瓜多糖组(200 mg/kg/day) TC含量显著下降(P <0.05),发明实施例1组(400 mg/kg/day)、牡蛎多糖组(400 mg/kg/day)和玉米须多糖组(200 mg/kg/day) TC含量下降极显著(P <0.01);盐酸二甲双胍组(200 mg/kg/day)、发明实施例1组(200、400、800 mg/kg/day)、牡蛎多糖组(200、400、800 mg/kg/day)和玉米须多糖组(200、400、800 mg/kg/day)、苦瓜多糖组(200 mg/kg/day)的GSP含量下降极显著(P <0.01),苦瓜多糖组(400、800 mg/kg/day)的GSP含量下降显著(P <0.05)。
三.对四氧嘧啶致ICR小鼠糖尿病的综合药效评价
按照综合药效评价公式进行评价:
综合药效评价(%)=体质量增加率(%)×0.2+饮水降低率(%)×0.15+摄食降低率(%)×0.15+血糖降低率(%)×0.5
实验结果如表3:
表3 对糖尿病小鼠的综合药效评价
表3结果表明:给药后小鼠表现出体重增加,饮水量和摄食量下降。本发明实施例1组(200、400、800 mg/kg/day)的综合药效评价高于牡蛎多糖组(200、400、800 mg/kg/day)、玉米须多糖组(200、400、800 mg/kg/day)、苦瓜多糖组(200、400、800 mg/kg/day),并且本发明实施例1(800 mg/kg/day)的综合药效评价值最高。
四.各组小鼠尿肌酐和尿素氮水平
表4 各组小鼠尿肌酐和尿素氮水平
注:(1)与正常组相比,*代表差异显著(P <0.05);**代表差异极显著(P <0.01)
(2)与模型组相比,#代表差异显著(P <0.05);##代表差异极显著(P <0.01)
表4结果表明:与高血糖组相比,本发明实施例1组(400、800 mg/kg/day)及牡蛎多糖组(400、800 mg/kg/day)的尿素氮和尿肌酐下降极显著(P <0.01),本发明实施例1组(200mg/kg/day)和牡蛎多糖组(200 mg/kg/day)组的尿素氮和尿肌酐下降显著(P <0.05)。本发明实施例1组肾功能指标参数显著降低。
结论:将本发明的药物对四氧嘧啶导致的糖尿病ICR小鼠的降血糖作用实验,结果显示本发明的药物对四氧嘧啶导致的糖尿病ICR小鼠的降血糖及综合药效显著好于牡蛎多糖、玉米须多糖或者苦瓜多糖单独作用,表现出显著的增效作用,同时本发明的药物明显改善ICR小鼠的肾功能。
Claims (4)
1.一种治疗糖尿病的药物,其特征在于是以牡蛎多糖、玉米须多糖及苦瓜多糖按质量比为5~6:2~3:2~3组合而成。
2.一种如权利要求1所述治疗糖尿病的药物的制备方法,其特征在于所述的牡蛎多糖按如下方法制备:将牡蛎去壳及去内脏之后进行匀浆处理,用体积/质量为15倍的沸水浸提10min,过滤得滤液;对滤渣用体积/质量为15倍的沸水浸提10min,过滤得滤液;合并滤渣再用体积/质量为15倍的沸水浸提10min,过滤得滤液;将3次滤液合并调节pH为5~6,过夜放置,6000rpm离心20min,收集上清;上清液调节pH为1.5~2.5,向上清液加入胃蛋白酶,在温度为37℃条件下进行保温酶解4h,再次6000rpm离心20min,胃蛋白酶的加入量为上清液质量1%×6000活性单位/克;获得的酶解上清液经过截留分子量10kDa的超滤膜过滤,再将过膜截留的滤液进行喷雾干燥,得到牡蛎多糖。
3.根据权利要求2所述治疗糖尿病的药物的制备方法,其特征在于所述玉米须多糖按如下方法制备:将玉米须粉碎,用体积/质量为15倍的沸水浸提10min,过滤得滤液;对滤渣用体积/质量为15倍的沸水浸提10min,过滤得滤液;合并滤渣再用体积/质量为15倍的沸水浸提10min,过滤得滤液;将3次滤液合并调节pH为4~5,过夜放置,6000rpm离心20min,收集上清;上清液调节pH为1.5~2.5,向上清液加入胃蛋白酶,在温度为37℃条件下进行保温酶解4h,再次6000rpm离心20min,胃蛋白酶的加入量为上清液质量1%×6000活性单位/克;获得的酶解上清液经过截留分子量10kDa的超滤膜过滤,再将过膜截留的滤液进行喷雾干燥,得到玉米须多糖。
4.根据权利要求3所述治疗糖尿病的药物的制备方法,其特征在于所述苦瓜多糖按如下方法制备:新鲜苦瓜去籽去瓤,进行匀浆处理,用体积/质量为10倍的沸水浸提10min,过滤得滤液;对滤渣用体积/质量为10倍的沸水浸提10min,过滤得滤液;合并滤渣再用体积/质量为10倍的沸水浸提10min,过滤得滤液;将3次滤液合并调节pH为5~6,过夜放置,6000rpm离心20min,收集上清;上清液调节pH为6.5~7.5,向上清液加入中性蛋白酶,在温度为37℃条件下进行保温酶解4h,再次6000rpm离心20min,中性蛋白酶的加入量为上清液质量1%×6000活性单位/克;获得的酶解上清液经过截留分子量10kDa的超滤膜过滤,再将过膜截留的滤液进行喷雾干燥,得到苦瓜多糖。
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CN102702582A (zh) * | 2011-11-07 | 2012-10-03 | 赛珂睿德生物医药科技(上海)有限公司 | 玉米须多糖及其制备方法和用途 |
CN104861083A (zh) * | 2015-06-14 | 2015-08-26 | 威海紫光金奥力生物技术有限公司 | 一种海洋牡蛎生物多糖提取制备方法 |
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CN1872134A (zh) * | 2006-04-21 | 2006-12-06 | 江苏大学 | 一种苦瓜多糖降血糖组合物及其制备方法 |
CN102702582A (zh) * | 2011-11-07 | 2012-10-03 | 赛珂睿德生物医药科技(上海)有限公司 | 玉米须多糖及其制备方法和用途 |
CN104861083A (zh) * | 2015-06-14 | 2015-08-26 | 威海紫光金奥力生物技术有限公司 | 一种海洋牡蛎生物多糖提取制备方法 |
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CN111450136A (zh) * | 2020-06-12 | 2020-07-28 | 广西医科大学 | 一种牡蛎葛根复合颗粒及其制备方法 |
CN111450136B (zh) * | 2020-06-12 | 2021-12-14 | 广西医科大学 | 一种牡蛎葛根复合颗粒及其制备方法 |
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