CN110179814B - 一种蝉花虫草多糖、制备方法及在制备预防肾间质纤维化药物中应用 - Google Patents
一种蝉花虫草多糖、制备方法及在制备预防肾间质纤维化药物中应用 Download PDFInfo
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Abstract
本发明公开了一种蝉花虫草多糖、制备方法及在制备预防肾间质纤维化药物中应用,将蝉花虫草子实体粉碎,过筛得到蝉花虫草子实体粉料;蒸馏水超声提取子实体粉料后进行超高压处理;处理后制备蝉花虫草子实体粗多糖;然后使用DEAE‑纤维素阴离子柱色谱分离,冷冻干燥后得到纯化多糖。本发明成功制备超高压处理修饰的蝉花虫草多糖,步骤简单,效率较高。通过对大鼠肾小球上皮细胞模型的干预发现,多糖可以有效抑制大鼠肾小球上皮细胞(HMC)的增殖,其在预防肾间质纤维化方面具有广阔的应用前景,蝉花虫草多糖抗氧化性得到明显提高,诱导了对高糖大鼠肾小球系膜细胞凋亡,而且对正常细胞无毒性,具有较好的研究价值和商业价值。
Description
技术领域
本发明涉及一种虫生真菌的制备方法及应用,尤其涉及的是一种蝉花虫草多糖、制备方法及在制备预防肾间质纤维化药物中应用。
背景技术
蝉花虫草(Cordyceps cicadae)又称金蝉花,是由麦角菌科虫草属真菌蝉拟青霉寄生于蝉类若虫的干燥复合体,属于农产品中的虫生真菌,是我国传统的名贵中药材之一。蝉花虫草的记载历史比冬虫夏草早800年,某些活性成分比冬虫夏草还高。现代医学研究表明,蝉花虫草含有大量的虫草酸、虫草多糖、腺苷、多种生物碱及麦角甾醇等功能成分,具有改善肾功能、缓解慢性肾衰竭、调节脂代谢、解热镇痛、镇静催眠,增强机体免疫力,抗辐射和明目等作用,对慢性代谢疾病有一定的预防和辅助治疗效果。
糖尿病肾病(diabetic nephropathy,DN)为糖尿病常见并发症,是导致终末期肾脏疾病主要原因之一。研究结果表明,DN是典型衰老相关性疾病,肾脏多种固有细胞衰老和功能减退(对抗损伤的应激能力减弱)是DN各种病理损害加速进展的重要原因。糖尿病肾病的基本病理改变为细胞外基质聚集,基膜增厚,晚期出现弥漫性肾小球硬化,最终导致肾衰竭。在肾小球疾病中,系膜细胞是重要的靶细胞及效应细胞,是糖尿病肾病重要的病理改变。糖尿病肾病的一个主要特征是肾小球系膜细胞的早期增殖以及细胞外基质的增多,而肾小球系膜细胞增殖抑制剂可能具有潜在的治疗肾病的作用。而能够诱导高糖大鼠肾小球系膜细胞凋亡的药物,对肾脏具有一定的保护作用,可以在一定程度上缓解糖尿病肾病的症状。
真菌多糖是由10个分子以上的醛糖和酮糖通过糖苷键缩合而成,有的还结合蛋白质或多肽,广泛存在于真菌的子实体、菌核或菌丝体中。国内外大量研究表明,天然真菌多糖具有多种生物药理活性,如抗氧化、调节免疫、护肝保肾等活性,在国际上被称为“生物反应调节剂”,对疾病有着特殊的治疗效果。以天然真菌多糖为基础的多糖药物的研究,已成为生物化学与生物医学领域的一个研究热点,可以用于慢性代谢疾病的防治。但是国内外对蝉花虫草研究得并不深入,人工栽培、选育的品种较少,而且往往集中在蝉花菌丝活性的研究。
发明内容
本发明所要解决的技术问题在于:如何发掘新的调节肾功能、改善肾损伤的多糖类药物,提供了一种蝉花虫草多糖、制备方法及在制备预防肾间质纤维化药物中应用。
本发明是通过以下技术方案解决上述技术问题的,本发明的发明目的之一是公开了一种蝉花虫草多糖在制备预防肾间质纤维化药物中的应用。
一种蝉花虫草多糖的制备方法,包括以下步骤:
(1)将蝉花虫草子实体粉碎,过筛得到蝉花虫草子实体粉料;
(2)按照1:6~10的料液比加入蒸馏水超声提取,然后80~90℃水浴1.5~2.5h,多次过滤收集并合并上清液;
(3)将上清液进行超高压处理,压力为400~450MPa、超高压处理时间为10~15min,保持温度为20℃;
(4)将处理后的上清液浓缩后加入乙醇,离心收集沉淀,加水溶解沉淀后得到粗多糖水溶液;
(5)在粗多糖水溶液中加入同体积的Sevag试剂,磁力搅拌后离心收集上清液;重复至蛋白质完全去除,然后将多糖溶液浓缩,去除小分子,使用透析袋流水透析,旋蒸浓缩至浓稠状液体,真空冷冻干燥,即得蝉花虫草子实体粗多糖;
(6)然后使用DEAE-纤维素阴离子柱色谱分离,冷冻干燥后得到纯化多糖。
作为本发明的优选方式之一,所述步骤(1)中,将蝉花虫草子实体粉碎后过60目筛。
作为本发明的优选方式之一,所述步骤(5)中,多粗糖水溶液与Sevag试剂多糖水溶液中的三氯甲烷和正丁醇的体积比为5:4:1。
作为本发明的优选方式之一,所述步骤(5)中,透析袋的截留分子量为3500Da。
作为本发明的优选方式之一,所述步骤(6)中,冷冻干燥的参数为-50℃,气压0.1mbar,时间为72h。
一种如所述方法制备得到的蝉花虫草多糖。
所述蝉花虫草多糖的分子量为1.5×106Da,多糖提取率为5.8%。
本发明相比现有技术具有以下优点:本发明成功制备超高压处理修饰的蝉花虫草多糖,步骤简单,效率较高。通过对大鼠肾小球上皮细胞模型的干预发现,多糖可以有效抑制大鼠肾小球上皮细胞(HMC)的增殖,其在预防肾间质纤维化方面具有广阔的应用前景,蝉花虫草多糖抗氧化性得到明显提高,诱导了对高糖大鼠肾小球系膜细胞凋亡,而且对正常细胞无毒性,具有较好的研究价值和商业价值。
附图说明
图1是不同葡萄糖浓度对HMC细胞生长率的影响;
图2是不同浓度的蝉花虫草多糖对MRC-5细胞抑制率的影响;
图3是不同浓度的蝉花虫草多糖对HMC细胞生长抑制率的影响;
图4是不同浓度的蝉花虫草多糖对HMC细胞形态的影响图,正常组、模型组、蝉花虫草多糖组(100μg/ml、200μg/ml、400μg/ml、600μg/ml、800μg/ml);
图5是不同浓度的蝉花虫草多糖对HMC细胞凋亡率的影响,a是DMEM低糖正常培养组,b是FITC单染组,c是PI单染组,d是DMEM高糖正常培养组,e是200μg/ml多糖处理组,f是400μg/ml多糖处理组,g是600μg/ml多糖处理组,h是800μg/ml多糖处理组。
具体实施方式
下面对本发明的实施例作详细说明,本实施例在以本发明技术方案为前提下进行实施,给出了详细的实施方式和具体的操作过程,但本发明的保护范围不限于下述的实施例。
实施例1
一、本实施例首先制备蝉花虫草多糖,具体过程如下:
(1)将蝉花虫草子实体粉碎,过60目筛得到蝉花虫草子实体粉料;
(2)按1:10比例加入蒸馏水超声提取20min,然后于80℃水浴2h,四层纱布过滤,收集上清液,重复3次,合并上清液;
(3)将上清液进行超高压处理,压力为400MPa、超高压处理时间为12min,保持温度为20℃;
(4)将处理后的上清液旋蒸浓缩至150ml,然后加入600ml无水乙醇,于4℃下静置12h,5000rpm/min离心收集沉淀,加水溶解沉淀即得粗多糖水溶液;
(5)加入与步骤(4)中大概相同体积的Sevag试剂,多粗糖水溶液与Sevag试剂多糖水溶液中的三氯甲烷和正丁醇的体积比为5:4:1,磁力搅拌2h,离心收集上清液;重复至蛋白质完全去除,然后将多糖溶液浓缩,去除小分子,使用透析袋流水透析,旋蒸浓缩至浓稠状液体,真空冷冻干燥,即得蝉花虫草子实体粗多糖;
(6)通过DEAE-纤维素阴离子柱色谱分离,-50℃,气压0.1mbar,冷冻干燥72h后得到纯化多糖。
二、高糖诱导的大鼠肾小球系膜细胞模型的建立
取冻存于-80℃冰箱中肾小球系膜细胞(HMC细胞)细胞株,水浴后离心,1000r/min离心3min,加入含体积分数10%胎牛血清的DMEM完全培养基移入细胞培养瓶于37℃培养箱中静置培养。待细胞融合达80%以上后,加入2mL0.25%胰蛋白酶消化液室温消化1min,弃去胰蛋白酶,加2mLDMEM完全培养基吹打使细胞完全脱壁,制成细胞悬液,进行传代培养。取对数生长期的肾小球系膜细胞接种于6孔板,培养24h后,换含有0.5%血清的培养基同步化24h。加入不同浓度的葡萄糖溶液建立高糖刺激的大鼠肾小球系膜细胞模型。
如图1所示,通过测定HMC细胞的生长率,判断建立模型的最佳葡萄糖浓度为25mmol/L,此时细胞达到最大吸光值。
三、蝉花虫草多糖对肾小球系膜细胞的凋亡诱导作用
1、蝉花虫草多糖对正常细胞毒性检测
在96孔板中接入生长状态良好的MRC-5细胞,每孔加入3×103个细胞。在5%CO2培养液箱中适应培养24h后,加入0-1mg/mL的蝉花虫草多糖处理,每个浓度设置8个复孔。然后把10μL 5mg/mL的MTT溶液加入96孔板中,将细胞板放进培养箱内孵育4h,吸尽培养板中的上清,各孔中加入150μL二甲亚飒(DMSO),锡箔纸将细胞板包裹,然后放入摇床内100rpm震荡15min,让生成的甲瓒结晶物充分溶解,在570nm处应用酶联免疫检测仪检测各个孔细胞的吸光值,计算细胞的存活率。分析复合物对MRC-5细胞是否具有毒性。如图2所示,说明蝉花虫草多糖对正常细胞MRC-5细胞不具有任何毒副作用。
2、蝉花虫草多糖对肾小球系膜细胞(HMC细胞)的抑制率
HMC细胞复苏传代,将盖玻片放入6孔板。调整细胞浓度约100000个/ml,接种于含盖玻片的6孔板中。用DMDE低糖培养基(不含血清和双抗)2000μL同步化培养24h。用DMEM高糖培养基(含血清和双抗、葡萄糖浓度25mmol/L)2000μL含不同浓度蝉花虫草多糖(100、200、400、600、800ug/ml)培养48h。去除培养基,加入DMSO 100ul震荡10min(避光),570nm检测吸光值。测定蝉花虫草多糖对HMC细胞的抑制率。如图3所示,蝉花虫草多糖对HMC细胞的增殖有显著抑制作用,而且随着蝉花虫草多糖浓度的增加,抑制率升高。
3、采用形态学方法观察蝉花虫草多糖对肾小球系膜细胞(HMC细胞)凋亡的影响
HMC细胞接种于含盖玻片的6孔板中。用DMDE低糖培养基(不含血清和双抗)2000μL同步化培养24h。用DMEM高糖培养基(含血清和双抗、葡萄糖浓度25mmol/L)2000μL含不同浓度蝉花虫草多糖(100、200、400、600、800ug/ml)培养48h。取出盖玻片PBS洗三次(1min/次)。放入4%多聚甲醛中,固定15-20min。PBS洗三次(1min/次),放入苏木精染液中,染色15min。自来水冲洗,浸入1%盐酸乙醇溶液中分色2-3s。自来水冲洗,浸入伊红染液染色5min。依次过70%、80%、90%、90%、100%、100%、100%乙醇。自然晾干,放入二甲苯溶液3次,固定盖玻片于载玻片,在倒置显微镜下观察细胞形态并拍照。如图4所示,蝉花虫草多糖对HMC细胞凋亡形态有明显影响,正常组为正常HMC细胞形态,模型组中HMC增殖明显,经蝉花虫草多糖处理后,随着蝉花虫草多糖的浓度增大,HMC细胞形态发生明显变化,细胞膜和细胞核发生固缩,细胞凋亡量增大。说明蝉花虫草多糖可以抑制细胞增殖,诱导细胞凋亡,而且HMC细胞的凋亡具有浓度依赖性。
4、流式细胞分析仪检测肾小球系膜细胞(HMC细胞)的凋亡比率
调整细胞浓度约100000个/ml,接种于6孔板中。用DMDE低糖培养基(不含血清和双抗)2000μL同步化培养24h。用DMEM高糖培养基(含血清和双抗、葡萄糖浓度25mmol/L)2000Ul含不同浓度蝉花虫草多糖(200、400、600、800、ug/ml)培养48h。用不含EDTA的胰酶消化后,利用离心机1000r/min,离心5min收集细胞。胰酶消化时间不宜过长,以防引起假阳性。用预冷的PBS洗涤细胞2次,每次均需1000r/mim,4℃离心5min。收集细胞。吸弃PBS,加500μL 1×Binding Buffer重悬细胞。加入5μL Annexin V-FITC避光孵育10min。然后,10μLPI Staining Solution避光孵育5min。然后应用流式细胞仪进行检测,SPSS16.0软件分析细胞凋亡率。如图4所示,蝉花虫草多糖对HMC细胞凋亡形态有明显影响,正常组为正常HMC细胞形态,模型组中HMC增殖明显,经蝉花虫草多糖处理后,随着蝉花虫草多糖的浓度增大,HMC细胞形态发生明显变化,细胞膜和细胞核发生固缩,细胞凋亡量增大。说明蝉花虫草多糖可以抑制细胞增殖,诱导细胞凋亡。如图5所示,与DMEM低糖培养基组(正常组)相比,蝉花虫草多糖促进了HMC细胞的凋亡,而且随着浓度的增大,凋亡细胞增多,而且HMC细胞的凋亡具有浓度依赖性。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等,均应包含在本发明的保护范围之内。
Claims (4)
1.一种蝉花虫草多糖在制备预防肾间质纤维化药物中的应用,其特征在于,蝉花虫草多糖通过抑制大鼠肾小球上皮细胞的增殖预防肾间质纤维化,所述蝉花虫草多糖的分子量为1.5×106Da;
所述蝉花虫草多糖的制备方法包括以下步骤:
(1)将蝉花虫草子实体粉碎,过筛得到蝉花虫草子实体粉料;
(2)按照1:6~10的料液比加入蒸馏水超声提取,然后80~90℃水浴1.5~2.5h,多次过滤收集并合并上清液;
(3)将上清液进行超高压处理,压力为400~450MPa、超高压处理时间为10~15min,保持温度为20℃;
(4)将处理后的上清液浓缩后加入乙醇,离心收集沉淀,加水溶解沉淀后得到粗多糖水溶液;
(5)在粗多糖水溶液中加入同体积的Sevag试剂,磁力搅拌后离心收集上清液;重复至蛋白质完全去除,然后将多糖溶液浓缩,去除小分子,使用透析袋流水透析,旋蒸浓缩至浓稠状液体,真空冷冻干燥,即得蝉花虫草子实体粗多糖,粗多糖水溶液与Sevag试剂多糖水溶液中的三氯甲烷和正丁醇的体积比为5:4:1;
(6)然后使用DEAE-纤维素阴离子柱色谱分离,冷冻干燥后得到纯化多糖。
2.根据权利要求1所述的一种蝉花虫草多糖在制备预防肾间质纤维化药物中的应用,其特征在于,所述步骤(1)中,将蝉花虫草子实体粉碎后过60目筛。
3.根据权利要求1所述的一种蝉花虫草多糖在制备预防肾间质纤维化药物中的应用,其特征在于,所述步骤(5)中,透析袋的截留分子量为3500Da。
4.根据权利要求1所述的一种蝉花虫草多糖在制备预防肾间质纤维化药物中的应用,其特征在于,所述步骤(6)中,冷冻干燥的参数为-50℃,气压0.1mbar,时间为72h。
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