CN105560269A - 玫瑰花多糖在制备治疗肥胖症的药物中的应用 - Google Patents
玫瑰花多糖在制备治疗肥胖症的药物中的应用 Download PDFInfo
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Abstract
本发明属于生物医药领域,提供一种玫瑰花多糖的应用,具体涉及玫瑰花多糖在制备治疗肥胖症的药物中的应用。所述玫瑰花多糖对秀丽隐杆线虫肥胖症病理模型具有显著的治疗作用,可显著减少秀丽隐杆线虫体内的脂肪储存。提示该玫瑰花多糖具备治疗肥胖症的潜力,可在制备治疗肥胖症的药物中应用,亦可在制备预防和辅助治疗肥胖症的保健品中应用。
Description
技术领域
本发明属于生物医药领域,提供一种玫瑰花多糖的应用,具体涉及玫瑰花多糖在制备治疗肥胖症的药物中的应用。
背景技术
世界卫生组织(WHO)1997年就宣布肥胖是世界首要的健康问题之一(向红丁,2001)。现代医学证明肥胖会影响生长发育和智力发育,并可引发严重的心理问题。肥胖症引发的高血压,糖尿病,高血脂等疾病,已经成为威胁人类生命健康安全的杀手。
对于肥胖症的治疗主要采取限食、体育锻炼等改变生活方式的疗法,肥胖者通常可减少原体重的22%-25%,但三年后仅有一半的受试者能够减少原来体重的5%。外科手术是一种有效的方法,但是对于患者而言不但昂贵而且风险大。FDA仅批准了两个可长期应用的抗肥胖症药物Sibutramine和Orlistat,前者副作用是引发高血压,后者具严重的胃肠道副作用如肛漏。Phentermine是一个短期应用抗肥胖药物,但是需要按时增加剂量才能保持疗效,提示它具潜在的成瘾性。而且,这三个药物减少体重效果仅比安慰剂组高5%(Zhengetal.,2010;2014)。肥胖症是一个慢性病,至今尚无有效的治疗药物。因此,迫切需要筛选与发现高效低毒的抗肥胖症药物。
肥胖症是一种与能量代谢相关的疾病,发病的机理一般是由于机体长期能量摄入大于能量消耗,使得过剩的能量以脂肪的形式储存起来。线虫的脂肪颗粒主要储存于肠道及皮下组织,并且标记方法简单,用苏丹类染料以及尼罗红染色后,利用显微镜就可以直接观察线虫体内贮积的脂肪。秀丽隐杆线虫脂肪酸生化合成通路与人类相似度高,脂代谢各步骤关键酶与其他生物都类似。因此,秀丽隐杆线虫已经成为研究脂肪代谢与分子机制的重要模型(徐蔓玲等,2014)。本发明正是采用秀丽隐杆线虫肥胖症模型发现抗肥胖症药物候选。
玫瑰具有多种药学活性包括催眠、止痛、抗惊厥、止咳和扩张支气管,促进肠蠕动和通便,对心血管系统也有一定的作用。此外,玫瑰的药学活性还包括抗菌、抗HIV、抗糖尿病,抗辐射、抗炎、抗肿瘤等。从玫瑰中提取的活性成分是玫瑰药学活性的基础,这些活性成分多涉及玫瑰精油、甲醇、乙醇、氯仿等提取物,以及从中分离的玫瑰黄酮、多酚,花色素等。在对玫瑰活性成分功效学研究中,大多数集中在对于玫瑰精油药学作用的研究,其次是玫瑰黄酮,而鲜有涉及对于玫瑰花多糖的研究(Boskabadyetal.,2011)。
多糖的结构复杂,多糖的分子量、构型、电荷特性和取代基的位置等均影响着多糖的药学活性(Maetal.,2013),不同来源的多糖决定其结构必然存在着差异,对于不同来源多糖药学活性的研究是这一领域的研究热点。水提醇沉获得的玫瑰粗多糖具有抗肿瘤作用,300mg/Kg/d给药10天,对S180荷瘤小鼠实体瘤的抑制率可达61.27%(白伟芳,2010)。但是,玫瑰花多糖是否具有治疗肥胖症的作用尚未可知。
基于上述研究现状,本发明现提供一种玫瑰花多糖的应用,具体涉及玫瑰花多糖在制备治疗肥胖症的药物中的应用。所述玫瑰花多糖可显著减少秀丽隐杆线虫肥胖症病理模型体内的脂肪储存,提示玫瑰花多糖具备治疗肥胖症的潜力,可在制备治疗肥胖症的药物中应用,亦可在制备预防和辅助治疗肥胖症的保健品中应用。
参考文献
白伟芳.玫瑰花多糖的提取及其功效研究.山东轻工业学院硕士论文,2010.
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徐蔓玲,赵阳,贾熙华,季宇彬.线虫脂肪模型研究.哈尔滨商业大学学报(自然科学版)2014,30:129-132.
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发明内容
本发明的目的是提供一种玫瑰花多糖的应用,具体涉及玫瑰花多糖在制备治疗肥胖症的药物中的应用。所述玫瑰花多糖对秀丽隐杆线虫肥胖症病理模型具有显著的治疗作用,毒性小,成本低廉。
所述玫瑰花多糖作为药效成分的药物剂型为注射剂、散剂、颗粒剂、粉剂、丸剂、口服液、片剂等任一种剂型,这是本领域技术人员可以理解的。
本发明同时提供玫瑰花多糖在制备预防和辅助治疗肥胖症的保健品中的应用。
所述玫瑰花多糖作为作为有效成分的保健品剂型为散剂、颗粒剂、粉剂、丸剂、口服液、片剂等任一种剂型,这是本领域技术人员可以理解的。
上述玫瑰花多糖的制备方法如下:
水提醇沉法制备玫瑰花多糖:称取玫瑰花,按1:20-30质量份数加蒸馏水,浸泡20min,加热煮沸并保持30min,10000rpm离心10min,取上清液,重复三次,合并上清液,加入5倍体积的无水乙醇于室温沉淀12h,10000rpm离心5min,收集沉淀,待溶剂挥干,收集固体,得粗多糖;
粗多糖脱蛋白:将粗多糖加蒸馏水溶解后配置为100mg/ml的水溶液,加入1/5体积5:1v/v的氯仿:正丁醇,剧烈振荡,4000rpm离心20min,弃沉淀,重复多次直至离心后溶剂介面无沉淀;
粗多糖脱色素:取脱蛋白后的粗多糖,加入10倍体积的65%的乙醇,55℃水浴提取5h,4000rpm离心5min,弃上清;
粗多糖脱酚:取脱色素后的粗多糖,加入等体积的丙酮溶液,充分振摇,4000rpm离心20min,收取沉淀,即得所述玫瑰花多糖。其中,所述丙酮为无水丙酮。
采用苯酚-浓硫酸法检测玫瑰花多糖含量,具体方法参见姜琼和谢妤(2013),略改动。简单地说,取所述玫瑰花多糖,用蒸馏水溶解成15mg/ml的玫瑰花多糖水溶液,100倍稀释。之后按照样品、5%苯酚溶液、浓硫酸为2:1:5的比例添加试剂,混匀5min后,沸水浴加热15min,冷却。处理好的待检样品在490nm处测量吸光度值。标准曲线采用葡萄糖为标准品绘制,经检测,所述玫瑰花多糖中多糖含量为85.4%。
本发明的有益效果在于:
本发明以秀丽隐杆线虫肥胖症病理模型进行实验,结果表明:所述玫瑰花多糖对秀丽隐杆线虫肥胖症病理模型具有显著的治疗作用。可以显著减少秀丽隐杆线虫肥胖症病理模型体内的脂肪储存,提示该玫瑰花多糖具有治疗肥胖症的潜力,可在制备治疗肥胖症的药物中应用,亦可在制备预防和辅助治疗肥胖症的保健品中应用。
以下提供具体实施例以实现本发明所述技术方案,但本发明的保护范围不局限于以下所述。
附图说明
图1玫瑰花多糖对秀丽隐杆线虫肥胖症病理模型的治疗作用
Control-空白对照组。
具体实施方式
实施例1本发明所述玫瑰花多糖的制备方法
称取玫瑰花,按1:20-30质量份数加蒸馏水,浸泡20min,加热煮沸并保持30min,10000rpm离心10min,取上清液,重复三次,合并上清液,加入5倍体积的无水乙醇于室温沉淀12h,10000rpm离心5min,收集沉淀,待溶剂挥干,收集固体,得粗多糖;
粗多糖脱蛋白:将粗多糖加蒸馏水溶解后配置为100mg/ml的水溶液,加入1/5体积5:1v/v的氯仿:正丁醇,剧烈振荡,4000rpm离心20min,弃沉淀,重复多次直至离心后溶剂介面无沉淀;
粗多糖脱色素:取脱蛋白后的粗多糖,加入10倍体积的65%的乙醇,55℃水浴提取5h,4000rpm离心5min,弃上清;
粗多糖脱酚:取脱色素后的粗多糖,加入等体积的丙酮溶液,充分振摇,4000rpm离心20min,收取沉淀,即得所述玫瑰花多糖,经检测,所述玫瑰花多糖中多糖含量为85.4%。
实施例2玫瑰花多糖对秀丽隐杆线虫肥胖症病理模型的治疗作用
1、生物材料:
(1)秀丽隐杆线虫野生型株系N2购自CaenorhabditisGeneticsCenter(CGC);
(2)大肠杆菌OP50(尿嘧啶渗漏突变株),购自CaenorhabditisGeneticsCenter(CGC),作为秀丽隐杆线虫的食物。
2、试剂
(1)固体NGM(NematodeGrowthMedium)培养基成分与制作(以1升为例):
成分 | 含量 |
NaCl | 3.00g |
K2HPO4 | 2.34g |
KH2PO4 | 17.23g |
蛋白胨 | 2.50g |
琼脂 | 17.00g |
补充H2O至 | 1000mL |
固体NGM培养基配制好后,121℃下高压恒温灭菌20min,在无菌操作台下加入10mg/mL胆固醇1mL,1MMgSO41mL,1MCaCl21mL摇匀,趁热倒入已灭菌的9cm培养板,约20mL/板。静置等待培养基凝固,备用。
(2)M9液配方
成分 | 含量 |
Na2HPO4 | 6.00g |
KH2PO4 | 3.00g |
NaCl | 5.00g |
1M MgSO4 | 1.00mL |
补充H2O至 | 1000mL |
(3)裂解液的配制:6.4%NaClO溶液和1MNaOH溶液按体积比1:1混合。
3、实验步骤:
(1)秀丽隐杆线虫肥胖症病理模型的建立
将同步化的线虫接在含有10mg/mL胆固醇的高脂NGM培养基上培养,使每板线虫含量为3000条左右,置于20℃生化培养箱中培养,使其储脂增加,成为肥胖模型。
(2)线虫的培养
将线虫接在涂有大肠杆菌OP50的固体NGM板上,然后置于20℃的培养箱中培养,当线虫长到成虫时进行同步化处理。
(3)线虫的同步化
挑选含有大量成虫并且有部分线虫卵已经孵出的NGM培养基,用M9液将线虫从培养基上冲下,转移到离心管中,静置使线虫自由沉降至管底,弃上清。视线虫量多少向离心管中加入裂解液,在漩祸搅拌器上振荡5-7分钟待线虫全部断裂时停止涡旋,并分装于1.5mL离心管中,用M9溶液洗线虫卵三次。孵育48h后备用。
(4)实验组设置
空白对照组:无菌水。
玫瑰花多糖实验组:0.01mg/ml;0.1mg/ml;1.0mg/ml。
配制含有玫瑰花多糖的NGM平板:
将实施例1获得的玫瑰花多糖以无菌水稀释,得到玫瑰花多糖的无菌水溶液,将玫瑰花多糖的无菌水溶液加入NGM培养基中,使玫瑰花多糖在培养基中的终浓度分别达到0.01mg/ml,0.1mg/ml和1.0mg/ml,将NGM倒入各平板,静置等待培养基凝固。在培养基上均匀涂布大肠杆菌OP50作为线虫的食物。空白对照组中,用等量的无菌水替换玫瑰花多糖的无菌水溶液,其他步骤不变。
(5)实验步骤
线虫药物处理:将孵育出的L1期线虫转移到涂布有OP50并混合不同浓度的玫瑰花多糖的NGM平板上,空白对照为涂布有OP50并加有与玫瑰花多糖等体积无菌水的NGM平板上,每个平板上接2000条线虫,20℃培养42h后,用M9将线虫洗下来,转移到离心管中,饥饿处理6h。
线虫的固定与染色:将药物处理后的线虫离心,弃上清,放入-20℃冰箱20min之后取出,加入0.4%甲醛溶液1mL,固定24h,放入4℃冰箱保存备用。
线虫苏丹黑B染色,将固定后的线虫弃去固定液,用蒸馏水洗两次,加70%酒精媒染,之后加苏丹黑B染色液,染色,最后加70%酒精漂洗。用Ti-Ds倒置生物呈像显微镜观察脂滴染色并拍照。
数据处理及统计分析:所拍照片使用image-J软件处理,处理后所得结果用SPSS(17.0)软件分析处理,灰度值高证明线虫体内脂肪储存量高,灰度值低证明线虫体内脂肪储存量低。
(6)实验结果
结果如图1(不同字母表示有显著性差异P<0.05),玫瑰花多糖剂量依赖地降低肥胖秀丽隐杆线虫体内脂滴的含量,1mg/mL的玫瑰花多糖可促使肥胖线虫体内脂滴减少10%。
通过以上实施例证明,本发明所述玫瑰花多糖对秀丽隐杆线虫肥胖症病理模型具有显著的治疗作用。提示该玫瑰花多糖具备治疗肥胖症的潜力,可在制备治疗肥胖症的药物中应用,亦可在制备预防和辅助治疗肥胖症的保健品中应用。有望开发成新一代的抗肥胖症药物或者减肥保健品。
Claims (5)
1.玫瑰花多糖在制备治疗肥胖症的药物中的应用。
2.如权利要求1所述的应用,其特征在于,所述玫瑰花多糖作为作为药效成分的药物剂型为注射剂、散剂、颗粒剂、粉剂、丸剂、口服液、片剂。
3.玫瑰花多糖在制备预防和辅助治疗肥胖症的保健品中的应用。
4.如权利要求3所述的应用,其特征在于,所述玫瑰花多糖作为作为有效成分的保健品剂型为散剂、颗粒剂、粉剂、丸剂、口服液、片剂。
5.如权利要求1或3任一项所述的应用,其特征在于,所述玫瑰花多糖的制备方法如下:
称取玫瑰花,按1:20-30质量份数加蒸馏水,浸泡20min,加热煮沸并保持30min,10000rpm离心10min,取上清液,重复三次,合并上清液,加入5倍体积的无水乙醇于室温沉淀12h,10000rpm离心5min,收集沉淀,待溶剂挥干,收集固体,得粗多糖;
粗多糖脱蛋白:将粗多糖加蒸馏水溶解后配置为100mg/ml的水溶液,加入1/5体积5:1v/v的氯仿:正丁醇,剧烈振荡,4000rpm离心20min,弃沉淀,重复多次直至离心后溶剂介面无沉淀;
粗多糖脱色素:取脱蛋白后的粗多糖,加入10倍体积的65%的乙醇,55℃水浴提取5h,4000rpm离心5min,弃上清;
粗多糖脱酚:取脱色素后的粗多糖,加入等体积的丙酮溶液,充分振摇,4000rpm离心20min,收取沉淀,即得所述玫瑰花多糖。
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