CN112457423B - 一种具有改善胰岛素抵抗作用的天然提取复合多糖及应用 - Google Patents
一种具有改善胰岛素抵抗作用的天然提取复合多糖及应用 Download PDFInfo
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Abstract
本发明属于医药技术开发领域,具体涉及一种具有改善胰岛素抵抗作用的天然提取复合多糖及应用。本发明通过从北柴胡、羊栖菜、温郁金、铁皮石斛中提取复合多糖,发现该复合多糖对高脂膳食诱导的胰岛素抵抗小鼠具有良好的干预治疗作用,能够纠正高脂膳食引起的糖脂代谢紊乱,有效缓解机体的胰岛素抵抗状态,而且相比其中任一种中药材中提取的多糖均效果更优,可以判断北柴胡、羊栖菜、温郁金、铁皮石斛中的部分化合物之间形成协同效应,起到更佳的疗效,作为新型改善胰岛素抵抗综合征药物有着良好的应用前景。
Description
技术领域
本发明属于医药技术开发领域,具体涉及一种具有改善胰岛素抵抗作用的天然提取复合多糖及应用。
背景技术
随着社会生活方式的改变和能量摄入过剩,胰岛素抵抗的发病率日益增高。胰岛素抵抗是指依赖胰岛素的葡萄糖利用器官(如骨骼肌、肝脏、脂肪组织等)对胰岛素的敏感性下降, 细胞糖代谢能力的减低。在胰岛素抵抗初期, 机体通过代偿性胰岛素分泌增多可以维持血糖在正常水平。随着胰腺β细胞功能减退, 当不能再产生足够的胰岛素以增强胰岛素敏感性时, 葡萄糖稳态遭到破坏, 就会出现葡萄糖耐量减低, 最终导致2型糖尿病等代谢综合征的发生。但由于胰岛素抵抗本身并没有给机体带来明显的症状,难以察觉,所以很多胰岛素抵抗的患者并没有得到及时的治疗和干预,而使胰岛素抵抗进一步发展为恶性疾病。
胰岛素抵抗的发生机制十分复杂,目前认为肥胖和高热量饮食是导致胰岛素抵抗的重要原因。有研究表明,肥胖或高脂饮食导致脂质代谢紊乱,游离脂肪酸(FFA)氧化增加,线粒体活性氧簇(ROS)大量产生,进而引起线粒体功能障碍以及体内氧化应激水平升高。氧化应激可致胰岛素信号传导受阻,与脂肪的堆积、血糖升高、胰岛素抵抗均呈显著正相关,并且大量文献证实通过逆转ROS 和抗氧化水平的平衡失调可以改善小鼠和人体的胰岛素抵抗状态。
目前民间有采用中药对人体调养,改善肥胖等问题,但大多数中药材有毒副作用,仅可用作药品,不能当作食品长期食用。多糖是许多中药的活性成分之一,也是食品的重要营养成分,在生物体内作为能量物质,存在于一切细胞膜结构中,参与细胞的各种生理活动。目前对天然植物中的多糖进行开发和应用为目前热门研究的方法。
发明内容
本发明的目的是为了克服现有技术存在的缺点和不足,而提供一种具有改善胰岛素抵抗作用的天然提取复合多糖及应用。
本发明所采取的技术方案如下:一种具有改善胰岛素抵抗作用的天然提取复合多糖,其制备过程如下:
(1)取北柴胡、羊栖菜、温郁金、铁皮石斛,烘干至恒重,粉碎;
(2)向步骤(1)粉碎后的粉末中加入40%~80%的乙醇水溶液,进行浸泡脱脂,取沉淀物;
(3)向步骤(2)得到的沉淀物中加入水,40~65℃下超声处理1~5 h,功率为100~600 W,然后进行沸水煮提1~8 h,过滤除去残渣及不溶物,得到提取液,残渣反复提取2~5次,合并提取液;
(4)对步骤(3)得到的提取液进行减压蒸馏,将提取液浓缩至原体积的1/4~1/10;
(5)将浓缩后的溶液中缓慢加入乙醇,使溶液乙醇浓度为40~95%,4℃静置过夜,2000~12000 rpm离心5~30 min,去上层清液,得到天然提取复合多糖。
优选地,其中各原料的质量份数为:
北柴胡 100~300份;
羊栖菜 300~500份;
温郁金 100~300份;
铁皮石斛 100~200份。
最优选地,其中各原料的质量份数为:
北柴胡 200份;
羊栖菜 500份;
温郁金 100份;
铁皮石斛 200份。
如上述的具有改善胰岛素抵抗作用的天然提取复合多糖在制备改善胰岛素抵抗及糖脂代谢紊乱的保健食品或药物中的应用。
所述胰岛素抵抗及糖脂代谢紊乱为肥胖或高脂饮食引起的。
一种保健食品,其包含如上述的具有改善胰岛素抵抗作用的天然提取复合多糖。其具有改善高脂饮食引起的肥胖的功效。
一种药物制剂,其包含如上述的具有改善胰岛素抵抗作用的天然提取复合多糖和药学上可接受的辅料。
本发明的有益效果如下:本发明通过从北柴胡、羊栖菜、温郁金、铁皮石斛中提取复合多糖,发现该复合多糖对高脂膳食诱导的胰岛素抵抗小鼠具有良好的干预治疗作用,能够纠正高脂膳食引起的糖脂代谢紊乱,有效缓解机体的胰岛素抵抗状态,而且相比其中任一种中药材中提取的多糖均效果更优,可以判断北柴胡、羊栖菜、温郁金、铁皮石斛中的部分化合物之间形成协同效应,起到更佳的疗效,作为新型改善胰岛素抵抗综合征药物有着良好的应用前景。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动性的前提下,根据这些附图获得其他的附图仍属于本发明的范畴。
图1为葡萄糖耐受性实验(A)及曲线下面积AUC(B);
图2 为胰岛素信号通路AKT和IRS-1磷酸化水平变化检测结果;
图3为胰岛素信号通路AKT(A)和IRS-1磷酸化水平变化(B)检测数据对比。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚,下面将结合附图对本发明作进一步地详细描述。
实施例1:
1、复合多糖的提取,具体过程如下:
1)、清洗:取北柴胡200g、羊栖菜500g、温郁金100g、铁皮石斛200g,烘干至恒重;
2)、粉碎:将上述中药混合粉碎至60~100目;
3)、脱脂:向粉碎后的中药粉中加入2~10 L 40%~80%乙醇,在50~80℃回流浸提1~10 h,或者浸泡过夜,其间不断搅拌,静置后弃上清,收集沉淀,反复脱脂2~5次;
4)、将上述沉淀物中加入1~10 L蒸馏水,40~65℃下超声处理1~5 h,功率为100~600 W;
5)、水提:将超声处理后的液料,进行沸水煮提1~8 h,过滤除去残渣及不溶物,得到提取液,残渣反复提取2~5次,合并提取液;
6)、浓缩:将提取液在40~80℃、真空度-0.03~-0.09MPa下减压蒸馏,将提取液浓缩至原体积的1/4~1/10;
7)、将浓缩后的溶液中缓慢加入乙醇,使溶液乙醇浓度为40~95%,4℃静置过夜,2000~12000 rpm离心5~30 min,真空干燥,即得水溶性中药复合多糖(复合多糖1#)。
另外,对步骤1中的原料进行替换,得到不同的多糖,具体如下:
将步骤1中的原料替换为北柴胡300g、羊栖菜300g、温郁金200g、铁皮石斛200g,然后进行步骤2-7,得到复合多糖2#。
将步骤1中的原料替换为北柴胡100g、羊栖菜400g、温郁金300g、铁皮石斛100g,然后进行步骤2-7,得到复合多糖3#。
将步骤1中的原料替换为北柴胡200g、羊栖菜300g、温郁金200g、铁皮石斛100g,然后进行步骤2-7,得到复合多糖4#。
步骤1中,原料仅采用北柴胡1000g,然后进行步骤2-7,得到北柴胡多糖。
步骤1中,原料仅采用羊栖菜1000g,然后进行步骤2-7,得到羊栖菜多糖。
步骤1中,原料仅采用温郁金1000g,然后进行步骤2-7,得到温郁金多糖。
步骤1中,原料仅采用铁皮石斛1000g,然后进行步骤2-7,得到铁皮石斛多糖。
2、胰岛素抵抗模型的建立、分组与实验方法
2.1 实验动物及饲养条件
C57BL/6健康雄性小鼠,体重18~22g,SPF级,由温州医科大学实验动物中心提供。
清洁级动物房,温度为22℃,相对湿度55%,12/12h明暗周期,自由进食饮水。实验过程中对动物的饲养及取材均遵守实验动物管理与保护的有关规定。
2.2 胰岛素抵抗模型的建立与分组给药
经适应性喂养一周后,将70只C57BL/6小鼠随机分成7组,每组10只,分别为:正常对照组、高脂模型组、高脂+多糖治疗组(北柴胡多糖、羊栖菜多糖、温郁金多糖、铁皮石斛多糖分别干预,以及复合多糖干预)。高脂饲料配方为:10%蔗糖、4%胆固醇、0.3%胆酸盐、20%猪油、10%蛋黄粉、1%鱼肝油、55%基础饲料。正常对照组给以基础饲料喂养。高脂喂养8周后,开始灌胃给药,各组每次灌胃剂量均为200 mg//kg/d,每日1次,持续8周。
实验期间小鼠自由饮水进食,每天记录摄食量、饮水量,每3天记录体重,每周记录空腹血糖。连续饲喂8周后,禁食不禁水,心脏取血,常规分离血清,保存于4℃备用。
2.3实验方法
2.3.1 空腹血糖水平的测定
小鼠禁食不禁水过夜,断尾取血,利用血糖试纸测定空腹血糖。
2.3.2 口服葡萄糖耐量的测定
小鼠禁食不禁水过夜,灌胃2 g/kg的葡萄糖溶液,在灌胃0、0.5、1、2 h后,利用血糖试纸测定血糖值,计算葡萄糖曲线下面积(Area Under the Curve,AUC)。
2.3.3 血清胰岛素水平的测定
取血清10 μL,参照ELISA试剂盒说明书测定血清胰岛素水平。
2.3.4 胰岛素抵抗及敏感性指数
按照下述公式分别计算模型评估胰岛素抵抗指数(homeostasis modelassessment of insulin resistance,HOMA-IR)和定量胰岛素敏感性检测指数(quantitative insulin sensitivity check index,QUICKI):
HOMA-IR =空腹血糖水平×血清胰岛素水平/22.5
QUICKI =1/ (lg空腹血糖水平+lg血清胰岛素水平)
2.3.5 Western blot法检测胰岛素信号通路
AKT及IRS-1蛋白表达及磷酸化水平测定。取上述实验中收集的肌肉组织,加入组织裂解液,冰浴中低速匀浆或吹打提取组织总蛋白,4℃下12000 rpm离心15 min,取上清,BCA试剂盒检测其含量。取30 μg总蛋白加入适量2×蛋白上样缓冲液,沸水浴5 min使蛋白变性,-20℃保存备用。
蛋白样品经SDS-PAGE分离、转膜、封闭、抗体孵育、ECL显影,置于化学发光仪中曝光。利用Image J软件分析条带灰度值,计算目的蛋白相对表达量。
2.3.6 统计学分析:
实验结果以平均值±SD表示,采用SPSS 17.0软件进行单因素one-way ANOVA方差分析,同时用Turkeys法进行两两比较,以P<0.05作为具有统计学意义上的差异。
、实验结果
3.1 小鼠一般状况
正常对照组小鼠活泼好动,皮毛光洁整齐,对食物敏感,逃避反应快。高脂模型组小鼠体重明显增加,精神萎靡,反应迟钝,毛竖无光泽,动作迟缓,尾卷缩等现象。北柴胡多糖、羊栖菜多糖、温郁金多糖、铁皮石斛多糖单独给药处理后,模型小鼠均未展现出明显的症状改善作用,与高脂模型组相比,体重增量未见显著差异,各多糖组均未影响动物摄食量(见表1)。而本发明中药复合多糖干预后,与高脂模型组对比,中药复合多糖1#和3#对肥胖小鼠行为学表现展现出一定的改善作用,体重增量显著低于(P<0.05)高脂模型组(见表1),表明复合多糖展现出一定的减肥效果。北柴胡组基本没有起到改善作用,而羊栖菜组和温郁金组可以起到一定的改善作用,但是相比本发明所提供的复合多糖,效果相差较大。
* P<0.05与高脂模型组比较
3.2生化指标与胰岛素抵抗、敏感性分析
由表2可知,高脂模型小鼠空腹血糖水平显著高于正常对照组。北柴胡多糖、羊栖菜多糖、温郁金多糖、铁皮石斛多糖单独给药处理后,展现出不同的作用效果,其中只有羊栖菜多糖展现出较好的降血糖作用,与高脂模型组比较,羊栖菜多糖组小鼠空腹血糖水平显著下降(P<0.05)。中药复合多糖1#组的降糖效果最为明显,与高脂模型组相比,该组小鼠空腹血糖水平极显著下降(P<0.01),而与正常对照比较无统计学差异。此外,复合多糖3#可以显著(P<0.05)降低血清胰岛素水平,但作用效果不及复合多糖1#,具有显著差异(P<0.05)。上述结果表明,中药复合多糖显著降低高脂模型小鼠的空腹血糖水平,具有一定的协同作用。
胰岛素是体内重要的血糖调节激素,对葡萄糖代谢发挥极其重要的生理作用。在胰岛素抵抗状态下,胰岛素促进葡萄糖转运摄取和利用的功能受损,机体为维持血糖的正常水平而代偿性增加胰岛素分泌,导致高胰岛素血症,血清胰岛素含量越高,说明胰岛素抵抗程度越严重。HOMA-IR是用于评价个体胰岛素抵抗程度的指标,QUICKI是用于评价个体胰岛素敏感性的指标。由表2可知,与高脂模型组相比,柴胡多糖、羊栖菜多糖、温郁金多糖、铁皮石斛多糖单独给药时,除羊栖菜多糖外,其余三种多糖在降低胰岛素水平、减轻胰岛素抵抗、增加胰岛素敏感性方面均无显著效果。而中药复合多糖1#则在降低胰岛素水平、减轻胰岛素抵抗、增加胰岛素敏感性方面展现出良好的作用效果,与高脂模型组相比均有显著差异(P<0.01),治疗效果也远优于其它复合多糖组。
3.3葡萄糖耐受性分析
葡萄糖耐受性实验是目前公认的诊断糖尿病的标准之一,葡萄糖耐量是反映短时期内血糖值变化的良好指标,以葡萄糖曲线下面积(AUC)表示,AUC越大,表明个体对葡萄糖的耐受能力越小,发生糖尿病的概率增加。本实验具体采用复合多糖1#进行,图1所示,北柴胡多糖、羊栖菜多糖、温郁金多糖、铁皮石斛多糖分别单独干预时,对葡萄糖耐受性均无显著改善效果(P>0.05)。与高脂模型组相比,经中药复合多糖干预治疗后则可明显缓解葡萄糖耐量,显著降低AUC(P<0.05)。
3.4胰岛素信号转导通路敏感性分析
胰岛素通过激活PI3K/AKT信号转导通路,调控GLUT转位至细胞膜,因此是血浆中葡萄糖转运的主要途径。AKT的Ser473磷酸化是胰岛素信号通路激活的重要标志,而IRS-1的Ser307磷酸化则是胰岛素信号受抑制的标志。本实验检测了中药复合多糖(具体采用复合多糖1#进行)对胰岛素抵抗模型小鼠肌肉中AKT和IRS-1磷酸化水平,从图2和图3可知,与正常对照组比较,高脂模型组、各给药组的AKT总蛋白均无显著变化。而与高脂模型组比较,中药复合多糖组在胰岛素刺激下的AKT蛋白Ser307磷酸化水平显著增加,而IRS-1的Ser307磷酸化水平则显著降低(P<0.05),表明该中药复合多糖可以显著提高胰岛素信号通路敏感性。而北柴胡多糖、温郁金多糖、铁皮石斛多糖并未明显增加AKT磷酸化水平, IRS-1磷酸化水平也未见显著缓解。羊栖菜多糖虽能在一定程度上提高高脂模型小鼠肌肉AKT磷酸化水平,但效果不及复合多糖组显著。上述表明中药复合多糖可以通过增加胰岛素信号敏感性,缓解高脂膳食引起的胰岛素抵抗症状,中药复合多糖的作用效果明显优于北柴胡多糖、温郁金多糖、铁皮石斛多糖和羊栖菜多糖。
综合上述结果,该中药复合多糖能显著降低胰岛素抵抗小鼠的体重增加,降低空腹血糖以及血清胰岛素水平,并改善胰岛素抵抗,增加胰岛素信号通路敏感性,提示四种中药复合提取的多糖较单一中药所提取的多糖具有更佳的药效作用。
此外需要说明的是,以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制,尽管参照实施例对本发明作了详细说明,本领域的普通技术人员应当理解,可以对本发明的技术方案进行修改或者等同替换,而不脱离本发明技术方案的实质和范围。
Claims (6)
1.一种具有改善胰岛素抵抗作用的天然提取复合多糖,其特征在于其制备过程如下:
(1)取北柴胡、羊栖菜、温郁金、铁皮石斛,烘干至恒重,粉碎;
(2)向步骤(1)粉碎后的粉末中加入40%~80%的乙醇水溶液,进行浸泡脱脂,取沉淀物;
(3)向步骤(2)得到的沉淀物中加入水,40~65℃下超声处理1~5 h,功率为100~600W,然后进行沸水煮提1~8 h,过滤除去残渣及不溶物,得到提取液,残渣反复提取2~5次,合并提取液;
(4)对步骤(3)得到的提取液进行减压蒸馏,将提取液浓缩至原体积的1/4~1/10;
(5)将浓缩后的溶液中缓慢加入乙醇,使溶液乙醇浓度为40~95%,4℃静置过夜,2000~12000 rpm离心5~30 min,去上层清液,得到天然提取复合多糖;
各原料的质量份数为:
北柴胡 200份;
羊栖菜 500份;
温郁金 100份;
铁皮石斛 200份。
2.如权利要求1所述的具有改善胰岛素抵抗作用的天然提取复合多糖在制备改善胰岛素抵抗及糖脂代谢紊乱的保健食品或药物中的应用。
3.根据权利要求2所述的应用,其特征在于:所述胰岛素抵抗及糖脂代谢紊乱为肥胖或高脂饮食引起的。
4.一种保健食品,其特征在于,其包含如权利要求1所述的具有改善胰岛素抵抗作用的天然提取复合多糖。
5.如权利要求4所述的保健食品,其特征在于:其具有改善高脂饮食引起的肥胖的功效。
6.一种药物制剂,其特征在于,其包含如权利要求1所述的具有改善胰岛素抵抗作用的天然提取复合多糖和药学上可接受的辅料。
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