CN111346117A - 一种具有抑制血管生成作用的榆黄菇提取物pce及其制备方法和用途 - Google Patents
一种具有抑制血管生成作用的榆黄菇提取物pce及其制备方法和用途 Download PDFInfo
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Abstract
本发明公开了一种具有抑制血管生成作用的榆黄菇提取物PCE及其制备方法和用途,其中榆黄菇提取物PCE是从榆黄菇子实体中提取获得,主要成分包括多糖、蛋白质、多酚、三萜和甾醇。体内外实验均证明这种提取物具有抑制血管生成的作用,可用于治疗或预防与血管生成相关的疾病,在医药和食品等领域具有巨大的应用前景。
Description
技术领域
本发明涉及一种具有抑制血管生成作用的榆黄菇提取物PCE及其制备方法和用途,属于食用菌应用技术领域。
背景技术
血管是供应营养物质、氧气和代谢产物运送的至关重要的途径,但是如果不受机体调控而以不正常的速度增殖,打破原有的平衡系统,那就是病态的血管生成。血管生成参与肿瘤、视网膜病变等各种疾病的形成过程,因此血管生成抑制剂被开发应用于治疗和预防血管性疾病。然而,已知目前正在开发的血管生成抑制剂具有诸如咯血、血栓形成和增加高血压发病率等副作用。由于血管生成抑制剂应用范围广,而且肿瘤等疾病治疗需长期服用血管生成抑制剂,对安全的血管生成抑制剂的需求量增加。因此,寻找低副作用的天然活性物质作为新生血管抑制剂,具有重要意义。
目前已有研究发现,植物蜜蜂花叶水溶剂提取物,具有抑制血管生成作用(CN100336521 C),多种合欢属植物的根茎叶等多个部位进行水或有机溶剂萃取,其萃取物具有抑制血管生成等作用(CN101167787 A),对香豆酸甲酯具有抑制HUVEG细胞迁移及生长的作用和抑制斑马鱼血管生成等作用(Phytomedicine,2018,48,10–20)。榆黄菇是一种食药两用菌,营养价值高,功能成分丰富。其中的酚类、三萜等多种物质具有抗高血脂、保护肝脏免于酒精的损伤和抑制癌症等作用(JAgric.Food Chem,2018,66,13183-13190;2006,54,2103–2110;2010,58, 12117–12122),而榆黄菇纯水提取物含有多糖和麦角硫因等活性物质具有抗氧化和抑制酪氨酸酶活性(CN109293572 A)。然而对于榆黄菇提取物是否具有抑制血管生成作用目前未见相关报道。
发明内容
本发明目的在于提供一种具有抑制血管生成作用的榆黄菇提取物PCE及其制备方法和用途。本发明从榆黄菇中获得的提取物组分PCE,具有良好的抑制血管生成的作用,可用于治疗或预防与血管生成相关的疾病,在医药和食品等领域具有巨大的应用潜力。
本发明榆黄菇提取物PCE,是从榆黄菇子实体中提取获得,通过苯酚硫酸法、考马斯亮蓝法、福林酚法、香草醛-冰醋酸-高氯酸法和Liebermann–Burchard法检测得知其主要成分为多糖、蛋白质、多酚、三萜和甾醇,含量分别为220.12-303.81mg/g、8.56-23.41mg/g、80.24-124.05mg/g、140.26-171.43mg/g、5.62-10.57mg/g。
本发明榆黄菇提取物PCE的制备方法,包括如下步骤:
步骤1:将榆黄菇子实体粉碎,过80目筛,得粉料;
步骤2:称取所得粉料20g,按照料液比20mL/g的比例加入水,35℃下超声提取2h,超声功率为400W-800W,每提取15min,间歇5min,重复三次,离心合并提取液;
步骤3:将步骤2所得提取液离心处理,以去除浸出液中的不溶物,得到榆黄菇的水萃取物;
步骤4:固液分离后用0.22um滤膜进行抽滤,进一步去除不溶性物质;
步骤5:将步骤4所得滤液减压浓缩(35℃-40℃,减压浓缩的压力为–1MPa至–0.5MPa),真空冷冻干燥(温度为–56℃,气压0.10Mbar,时间4天),即得榆黄菇提取物PCE。
本发明榆黄菇提取物PCE的用途,是在制备新生血管抑制剂中的应用。本发明榆黄菇提取物PCE具有良好的抑制血管生成的作用,可用于治疗或预防与血管生成相关的疾病,在医药和食品等领域具有巨大的应用潜力。
本发明榆黄菇提取物PCE的活性检测,包括如下内容:
1、通过MTT实验测定EC.h926细胞的存活性,直接观察PCE抑制血管作用。
2、通过细胞迁移实验,测定单层铺满的细胞在划痕处理后,细胞迁移至无细胞处的数量及距离。
3、通过荧光斑马作为模型生物,于显微镜下直接观察并拍摄图片,并进行血管长度、数量及面积分析,定量分析PCE作用效果。
与现有技术相比,本发明的有益效果体现在:
1、本发明提供了一种用于抑制血管生成的榆黄菇提取物,目前查阅资料中,尚未发现榆黄菇提取物的相关活性报道,拓展了榆黄菇提取物的应用范围。
2、寻找低副作用的天然活性物质作为新生血管抑制剂具有重要意义,本发明提供了一种新型新生血管抑制剂,体内外实验均证实其具有良好的血管生成抑制能力。
3、本发明以超声辅助方法进行提取,步骤简单,效率高,与常规水浸提相比,提取物中活性功能成分含量更高,血管生成抑制活性更佳。
附图说明
图1是显示在实施例5的不同条件下进行培养的结果中,对细胞存活力检测的MTT结果图。测量结果表明血管生成抑制剂对EC.hy926呈现浓度依赖性抑制关系。血管生成抑制剂的添加量越大,EC.hy926的生长受到抑制越多,与仅添加VEGF的情况(对照)相比,与阳性对照效果类似,发现显著差异(图仅列出未进行超声处理的实验组:对比例1,超声处理实验组效果类似)。
图2是实施例3结果分析,分别为对照组、VEGF、VEGF+0.3mg/mL PCE、VEGF+0.6mg/mL PCE和VEGF+0.9mg/mL PCE进行培养24h后形态观察图。迁移状态的测量结果表明PCE 对EC.hy926迁移作用抑制呈现浓度依赖性抑制关系。PCE的添加量越大,EC.hy926的迁移被抑制越多。与仅添加VEGF的情况(对照)相比,发现显著差异(图仅列出未进行超声处理的实验组:对比例1,超声处理实验组效果类似)。
图3是显示实施例3的结果,分别为对照组、VEGF、VEGF+PCE(0.3、0.6和0.9mg/mL)细胞数量及形态观察图。图3结果显示,PCE添加量越大,EC.h926的细胞外伸伪足减少,细胞数量明显减少,与对照组相比发现显著差异(图仅列出未进行超声处理的实验组:对比例1,超声处理实验组效果类似)。
图4是显示实施例4中以斑马鱼模型的结果:(A)对照组和PCE(0.6、1.2和1.8mg/mL) 的结果图片;(B)血管长度定量分析图;(C)血管篮面积定量分析图。如图4所示,血管生成结果图表明PCE对斑马鱼血管生成的抑制作用呈浓度依赖关系,随着浓度增加,与对照组相比,肠下血管条数明显减少,整个血管篮面积也明显减小。PCE的添加量越大,血管的形态、长度和血管篮面积等被抑制效果越明显(图仅列出未进行超声处理的实验组:对比例1,超声处理实验组效果类似)。
具体实施方式
下面通过具体的实施例对本发明进行详细说明上述本实施方案的血管生成抑制剂的效果。应当理解的是,这些具体实施方式仅用来帮助理解本发明,并非对本发明的实际保护范围构成任何形式的任何限定。
实施例1:榆黄菇提取物PCE的制备
(1)将榆黄菇子实体粉碎,过80目筛,得粉料;
(2)将所得粉料20g,按照料液比20mL/g加入一定体积的水,温度35℃超声2h,超声功率为400W,每提取15min,间歇5min,重复三次,离心合并提取液;
(3)将步骤2所得提取液,离心去除浸出液中的不溶物,得到榆黄菇的水萃取物;
(4)固液分离后用0.22um滤膜进行抽滤,进一步去除不溶性物质;
(5)将步骤4所得滤液减压浓缩(35℃-40℃,减压浓缩的压力为–1MPa至–0.5MPa),真空冷冻干燥(温度为–56℃,气压0.10Mbar,时间4天),即得血管抑制剂PCE400。
PCE400的提取率为34.33%,提取物中蛋白含量为8.52mg/g,多糖含量为246.86mg/g,总酚含量为85.03mg/g,三萜类物质含量为153.07mg/g,甾醇类物质含量为6.15mg/g。
实施例2:榆黄菇提取物PCE的制备
(1)将榆黄菇子实体粉碎,过80目筛,得粉料;
(2)将所得粉料20g,按照料液比20mL/g加入一定体积的水,温度35℃超声2h,超声功率为600W,每提取15min,间歇5min,重复三次,离心合并提取液;
(3)将步骤2所得提取液,离心去除浸出液中的不溶物,得到榆黄菇的水萃取物;
(4)固液分离后用0.22um滤膜进行抽滤,进一步去除不溶性物质;
(5)将步骤4所得滤液减压浓缩(35℃-40℃,减压浓缩的压力为–1MPa至–0.5MPa),真空冷冻干燥(温度为–56℃,气压0.10Mbar,时间4天),即得血管抑制剂PCE600。
PCE600的提取率为35.12%,提取物中蛋白含量为23.41mg/g,多糖含量为303.8mg/g,总酚含量为110.48mg/g,三萜类物质含量为162.87mg/g,甾醇类物质含量为10.57mg/g。
实施例3:榆黄菇提取物PCE的制备
(1)将榆黄菇子实体粉碎,过80目筛,得粉料;
(2)将所得粉料20g,按照料液比20mL/g加入一定体积的水,温度35℃超声2h,超声功率为800W,每提取15min,间歇5min,重复三次,离心合并提取液;
(3)将步骤2所得提取液,离心去除浸出液中的不溶物,得到榆黄菇的水萃取物;
(4)固液分离后用0.22um滤膜进行抽滤,进一步去除不溶性物质;
(5)将步骤4所得滤液减压浓缩(35℃-40℃,减压浓缩的压力为–1MPa至–0.5MPa),真空冷冻干燥(温度为–56℃,气压0.10Mbar,时间4天),即得血管抑制剂PCE800。
PCE800的提取率为38.36%,提取物中蛋白含量为22.65mg/g,多糖含量为291.12mg/g,总酚含量为124.05mg/g,三萜类物质含量为171.43mg/g,甾醇类物质含量为8.43mg/g。
对比例1:
在对比例1中,常规榆黄菇水提物的制备方法如下:
(1)将榆黄菇子实体粉碎,过80目筛,得粉料;
(2)将所得粉料20g,按照料液比20mL/g加入一定体积的水,35℃纯水提取2h,重复三次,离心合并提取液;
(3)将步骤2所得提取液,离心去除浸出液中的不溶物,得到榆黄菇的水萃取物;
(4)固液分离后用0.22um滤膜进行抽滤,进一步去除不溶性物质;
(5)将步骤4所得滤液减压浓缩(35℃-40℃,减压浓缩的压力为–1MPa至–0.5MPa),真空冷冻干燥(温度为–56℃,气压0.10Mbar,时间4天),即得血管抑制剂PCEw。
PCEw的提取率为18.21%,提取物中蛋白含量为14.32mg/g,多糖含量为220.121mg/g,总酚含量为80.24mg/g,三萜类物质含量为140.26mg/g,甾醇类物质含量为5.62mg/g。
实施例4:
使用人脐静脉内皮细胞(EC.hy926)作为血管生成抑制模型的靶标。通过空白组、VEGF组、血管生成抑制剂PCE组(0.3、0.6、0.9mg/mL),与EC.h926并培养。EC.hy926培养24小时后,通过分别不加VEGF,SU5416作阳性对照,加入适量的VEGF,加入PCE(0.3、0.6和0.9mg/mL)。另外,向每个培养的样品中加入100μL MTT,经过4小时后用酶标仪于570nm 吸光值处测量得到结果。
图1,仅显示为不超声提取的PCE作用后,其IC50=1.17mg/mL,下面依次为超声功率400W、600W和800W提取物测定结果:IC50,400=0.67mg/mL,IC50,600=0.50mg/mL, IC50,800=0.35mg/mL。
实施例5:
为了测量细胞的迁移,培养24小时,在六孔板上成长成铺开的单层EC.hy926。以片状生长的EC.hy926被无菌划成一定长度划痕,从而形成没有细胞的部分。除去由于划痕浮起的细胞,通过不加VEGF,加入适量的VEGF,加入PCE(0.3、0.6和0.9mg/mL)培养24小时。用光学显微镜观察每个培养样品的划痕部分。图2显示样品的光学显微照片分别是:空白组、SU5416组、VEGF组、和VEGF+PCE(0.3、0.6和0.9mg/mL)进行的培养结果的照片,图2仅示为纯水提物的细胞迁移实验图,迁移定量分析发现IC50=0.91mg/mL,下面依次为超声功率400W、600W和800W提取物测定结果:IC50,400=0.65mg/mL,IC50,600=0.57mg/mL, IC50,800=0.43mg/mL,图3仅显示为不超声提取的PCE作用后细胞形态和数量图,细胞覆盖面积定量分析发现IC50=0.65mg/mL,下面依次为超声功率400W、600W和800W提取物测定结果:IC50,400=0.43mg/mL,IC50,600=0.40mg/mL,IC50,800=0.23mg/mL。
实施例6:
为了进一步测量新生血管抑制作用,采用6hpf fli1a:EGFP斑马鱼胚胎进行血管生成定量和定性分析。通过空白组和PCE(0.6、1.2和1.8mg/mL)培养96hdf。用荧光显微镜观察每组斑马鱼的血管生成情况。图3显示样品的照片分别是:空白组和PCE(0.6、1.2和1.8mg/mL) 进行的培养结果的照片。此外,图4仅示出纯水提物的结果示意图,通过光学显微镜拍照后(图 4A),经过图片处理软件定量测定的血管形成结果(面积和长度)。该定量测定是通过处理上述图4中的一系列图片数据而进行的。图4B仅显示为不超声提取的PEW作用后血管长度定量测定结果图,IC50,longth=0.92mg/mL,下面依次为超声功率400W、600W和800W提取物测定结果:IC50,400=0.67mg/mL,IC50,600=0.50mg/mL,IC50,800=0.43mg/mL。
Claims (6)
1.一种具有抑制血管生成作用的榆黄菇提取物PCE,其特征在于:
所述榆黄菇提取物PCE是从榆黄菇子实体中提取获得,主要成分包括多糖、蛋白质、多酚、三萜和甾醇。
2.根据权利要求1所述的榆黄菇提取物PCE,其特征在于:
所述榆黄菇提取物PCE中多糖、蛋白质、多酚、三萜和甾醇的含量分别220.12-303.81mg/g、8.56-23.41mg/g、80.24-124.05mg/g、140.26-171.43mg/g和5.62-10.57mg/g。
3.一种权利要求1或2所述的榆黄菇提取物PCE的制备方法,其特征在于包括如下步骤:
步骤1:将榆黄菇子实体粉碎,过80目筛,得粉料;
步骤2:称取所得粉料20g,按照料液比20mL/g的比例加入水,35℃下超声提取2h,每提取15min,间歇5min,重复三次,离心合并提取液;
步骤3:将步骤2所得提取液离心处理,以去除浸出液中的不溶物,得到榆黄菇的水萃取物;
步骤4:固液分离后用0.22um滤膜进行抽滤,进一步去除不溶性物质;
步骤5:将步骤4所得滤液减压浓缩,真空冷冻干燥,即得榆黄菇提取物PCE。
4.根据权利要求3所述的制备方法,其特征在于:
步骤2中,超声功率为400W-800W。
5.根据权利要求3所述的制备方法,其特征在于:
步骤5中,减压浓缩的温度为35-40℃,压力为–1MPa至–0.5MPa;真空冷冻干燥的温度为–56℃,气压0.10Mbar,时间4天。
6.一种权利要求1或2所述的榆黄菇提取物PCE的用途,其特征在于:
所述榆黄菇提取物PCE在制备新生血管抑制剂中的应用。
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