CN112336849A - 黄芩三种成分协同增强fgf1促细胞增殖的用途 - Google Patents
黄芩三种成分协同增强fgf1促细胞增殖的用途 Download PDFInfo
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Abstract
本发明公开了黄芩提取物‑FGF1复合物,它是黄芩提取物与FGF1共孵化获得的复合物,所述的黄芩提取物为黄芩苷、千层纸素A‑7‑0‑β‑D‑葡萄糖醛酸苷或汉黄芩苷,获得的复合物为FGF1‑黄芩苷复合物、FGF1‑千层纸素A‑7‑0‑β‑D‑葡萄糖醛酸苷复合物或FGF1‑汉黄芩苷复合物;所述的孵化为:1)用甲醇溶解黄芩提取物,氮气吹干,用水复溶;将FGF1靶蛋白溶解于PBS缓冲液,和黄芩提取物甲醇溶液,混合,在36~38℃下孵化25~35min,真空冷冻干燥;FGF1‑黄芩苷复合物、FGF1‑千层纸素A‑7‑0‑β‑D‑葡萄糖醛酸苷复合物和/或FGF1‑汉黄芩苷复合物在促细胞增殖方面的应用。
Description
技术领域
本发明属于分子生物学技术领域,具体涉及黄芩三种成分协同增强FGF1促NIH3T3细胞增殖的用途。
背景技术
生物活性成分的筛选是中药研究领域的重要内容。传统的化学分离、结构鉴定、活性筛选模式到活性导向化学分离存在目标不明确、操作繁琐、工作量大、周期长、活性成分在分离过程中易丢失等诸多不足。现代药理学研究表明,药物与生物大分子(如酶、受体、DNA、RNA等)的亲和是其发挥作用的第一步。我们开发了一种快速筛选中药中生物活性成分的方法:多室电泳分离系统并联用高效液相色谱串联质谱技术。该技术能在生理状态将溶液状态的蛋白质分离,满足靶分子亲和技术中快速将靶分子-活性分子复合物从孵育溶液中分离出来,减少分离过程中复合物解离的要求。多室电泳的优势在于:1)具有简便快速、作用靶点明确、靶点无需标记、筛选单靶点多成分、筛选多靶点多成分等;2)多室、多维、体外“可视”、动态分析活性成分与靶点的协同作用及变化;3)从单方或复杂组方活性成分中筛选与已知靶点结合的未知活性成分,同时发现促进已知成分与靶点的结合效应的活性成分。该技术与已有筛选技术相比,具有简便快速、作用靶点明确、靶点无需标记、无需固定化、筛选单靶点多成分、筛选多靶点多成分等优势。研究报道:基于多室电泳分离系统在pH为7.4,电压8V,分离时间15min的条件下从孵化后的溶液中分离出酶-活性成分复合物,并联用高效液相色谱串联质谱技术对筛选出的活性成分进行定性分析,并筛选出七种潜在的α–葡萄糖苷酶抑制剂。
黄芩(Scutellaria baicalensis Georgi)是唇形科植物黄芩的根部,主要药用,具有去热燥湿、清火解毒、安胎、调节免疫、防治糖尿病、抗炎抗凋亡等作用。主治湿温,暑湿,胸闷呕恶,湿热痞满,黄疸泻痢,肺热咳嗽,高热烦渴,血热吐衄,胎动不安。相关研究表明,黄芩的抗菌及抗病毒作用较强,其所含的黄芩素及黄芩苷可有效抑制体外多种革兰氏阳性及阴性菌,并能对导致病性皮肤真菌起到有效的抑制作用,此外黄芩苷及黄芩素在抗肿瘤、抗过敏、抗动脉粥样硬化以及对肝脏和中枢神经系统保护中也有一定的作用。黄芩中富含的多种黄酮类化合物,其中汉黄芩苷具有一定的抗肿瘤活性,千层纸素A-7-0-β-D-葡萄糖醛酸苷具有抗氧化作用。因此,其药理应用广泛及开发前景广阔,具有较大的研究应用空间。
FGF1是成纤维细胞生长因子(Fibroblast growth factor,FGF)家族中的一个成员,是由155个氨基酸组成的蛋白质,FGF家族具有促进细胞发育、细胞增殖、转移和分化等功能。FGF1为FGF家族中的一员,因其等电点偏酸性,又被称为酸性成纤维细胞生长因子(acid fibroblast growth factor,aFGF)。FGF1能结合和激活四种FGFRs,FGFRs的激活刺激胞内信号通路,包括PKC/PLCγ、PI3K/Akt 和RAS/MAPK 级联。利用酵母双杂交筛选和质谱分析经串联亲和纯化或与重组FGF1共沉淀后的配合物,在20个新发现的FGF1配合物中有10个与细胞凋亡、细胞周期和增殖有关,提示细胞内FGF1在细胞存活中起着重要作用。FGF1不仅有激活细胞表面特定受体的经典作用方式,也可以独立于受体激活的信号通路来保护细胞免受应激条件的影响,然而越来越多的研究表明FGF1与代谢稳态有关。
发明内容
本发明目的是为解决中药成分之间的协同作用及中药与蛋白药物联合给药的协同作用的问题,而提供
黄芩三种成分协同增强FGF1促NIH3T3细胞增殖的用途。
黄芩提取物-FGF1复合物,它是黄芩提取物与FGF1共孵化获得的复合物,所述的黄芩提取物为黄芩苷、千层纸素A-7-0-β-D-葡萄糖醛酸苷或汉黄芩苷,获得的复合物为FGF1-黄芩苷复合物、FGF1-千层纸素A-7-0-β-D-葡萄糖醛酸苷复合物或FGF1-汉黄芩苷复合物;
所述的孵化为:
1)取黄芩提取物,用其5~20倍重量的甲醇溶解黄芩提取物,得到甲醇溶液,氮气吹干后超纯水回溶,得水溶液;
2)将FGF1靶蛋白溶解于乙酸铵缓冲液,得FGF1乙酸铵溶液;
3)FGF1 乙酸铵溶液和黄芩提取物溶液,混合;
4)在36~38℃下孵化25~35min,得到黄芩提取物-FGF1复合物;
步骤2)所述的乙酸铵缓冲液的浓度为10 mM、pH值为4;
步骤3)所述的FGF1 乙酸铵溶液和黄芩提取物水溶液,按体积比1:1混合;
步骤4)在37℃下孵化30min。
FGF1-黄芩苷复合物、FGF1-千层纸素A-7-0-β-D-葡萄糖醛酸苷复合物和/或FGF1-汉黄芩苷复合物在促细胞增殖方面的应用。
本发明提供了黄芩提取物-FGF1复合物,它是黄芩提取物与FGF1共孵化获得的复合物,所述的黄芩提取物为黄芩苷、千层纸素A-7-0-β-D-葡萄糖醛酸苷或汉黄芩苷,获得的复合物为FGF1-黄芩苷复合物、FGF1-千层纸素A-7-0-β-D-葡萄糖醛酸苷复合物或FGF1-汉黄芩苷复合物;所述的孵化为:1)取黄芩提取物,用其5~20倍重量的甲醇溶解黄芩提取物,得到甲醇溶液,氮气吹干后超纯水回溶,得水溶液2)将FGF1靶蛋白溶解于乙酸铵缓冲液,得FGF1乙酸铵溶液;3)FGF1 乙酸铵溶液和黄芩提取物水溶液混合;4)在36~38℃下孵化30min,得到黄芩提取物-FGF1复合物;FGF1-黄芩苷复合物、FGF1-千层纸素A-7-0-β-D-葡萄糖醛酸苷复合物和/或FGF1-汉黄芩苷复合物在促细胞增殖方面的应用。细胞实验结果表明,黄芩提取物-FGF1复合物对细胞的增值效果比FGF1单独给药效果更佳明显。
附图说明
图1 经多室电泳分离体系黄芩提取液对照组及FGF1+黄芩提取液总离子扫描色谱图FGF1与黄芩提取液孵化谱图(上),黄芩提取液对照组(下);
图2 经多室电泳分离体系黄芩提取液对照组及FGF1+黄芩提取液总离子扫描色谱图(n=3);
图3 经多室电泳分离体系后黄芩提取液+FGF1以及黄芩提取液 MRM分段检测色谱图(n=3);
图4 经多室电泳分离体系后3STD+FGF1以及3STD MRM分段检测色谱图(n=3);
图5 三种成分化合物结构信息;
图6 黄芩活性物质给药24h后CCK-8检测结果结果;BC:黄芩苷,WGS:汉黄芩苷,OXS:千层纸素A-7-0-β-D-葡萄糖醛酸苷,3STD:三种成分混合;
图7 黄芩活性物质给药48h后CCK-8检测结果结果;BC:黄芩苷,WGS:汉黄芩苷,OXS:千层纸素A-7-0-β-D-葡萄糖醛酸苷,3STD:三种成分混合;
图8 FGF1给药浓度筛选结果;
图9 三种成分与FGF1单独给药以及协同给药24h后促细胞增殖结果;
图10 三种成分与FGF1单独给药以及协同给药48h后促细胞增殖结果。
具体实施方式
实施例1 黄芩提取液的制备
药店购买干燥黄芩饮片,除杂质,将干燥的黄芩研磨成均一的粉末,称取2 g粉末加入20 mL甲醇进行超声波萃取,每次20 min,萃取3次,合并萃取液,悬浮液过0.22 μm滤膜,吹氮浓缩至吹干,超纯水回溶,过0.22 μm滤膜在4 ℃下保存直至使用。
实施例2 筛选黄芩提取液中与FGF1结合的有效成分
1、FGF1-黄芩活性成分复合物的制备
将FGF1冻干粉1mg溶解于1ml超纯水中,得FGF1的水溶液1mg/ml,将1mg/ml的FGF1用10mM pH4乙酸铵缓冲液 稀释至0.1 mg/ml,取FGF1 95 ul和20mg/ml 黄芩提取液5 ul,混合,形成FGF1-黄芩活性成分混合液100 ul;37摄氏度下孵育30 min,使黄芩提取液中的活性成分与FGF1结合,形成FGF1-黄芩活性成分复合物。
2、多室电泳分离
孵化后FGF1-黄芩活性成分混合液作为样品溶液,黄芩提取液作为对照溶液,分为实验组和对照组;采用多室电泳技术,装置参考CN104307368A发明专利所述的专用电泳分离装置;具体参数设定如下:电泳装置的样品室两侧选择两个醋酸纤维素膜,其孔径小于0.2um;正负接收室两侧选择10 KD 超滤膜,样品室加入FGF1-黄芩活性成分混合液 100 ul,正负接收室加入等体积乙酸铵缓冲溶液,使其液面高度一直。电压选择为14v,10 mM乙酸铵缓冲溶液 pH 4,电泳时间为15 min,使FGF1-黄芩活性成分复合物由样品室向负接收室迁移。
3、FGF1-黄芩活性成分复合物的解离
用600 ul甲醇解离负接收室中的溶液,12000 rpm离心取上清液;将上清液浓缩到100ul,分别转移到进样瓶中等待检测。黄芩提取液对照组采用相同方法解离。
实施例3 与FGF1结合的黄芩活性成分的定性分析
1. 实施例2解离后的接收室中的溶液,进行高效液相串联质谱分析;
色谱条件:
色谱柱:Tnature C18反向色谱柱(4.6 mm×150mm、5μm)
流动相:流动相A为0.1%甲酸水溶液,流动相B为0.1%甲酸乙腈溶液;
流速:0.5mL/min;
进样量:5μL;
梯度洗脱程序如下:0-10 min,25%-40%B;10-20 min,40%-65%B;20-30 min,65%-80%B;
质谱条件:
质谱条件电喷雾离子源,负离子模式,采用全扫描模式;喷雾气压力:30 psi;干燥气(N2)流速:13.0 L/min,干燥气温度:300℃;毛细管电压:2500 V,毛细管出口电压:135 V;质量数扫描范围:100-600 m/z。
实验组和对照组总离子扫描色谱图如图1、2所示;从图中可以看出黄芩中有三种活性成分与FGF1结合并迁移至负接收室;保留时间见表1;初步确定三种成分依次为为黄芩苷、千层纸素A-7-0-β-D-葡萄糖醛酸苷、汉黄芩苷。
2.标准品建立MRM模式验证三种成分
实验组和对照组分别进行MRM检测,MRM模式分段检测检测色谱图如图3所示,从图中实验组和对照组负接收室扫描色谱图对比,说明黄芩提取液中有三个活性成分与FGF1有结合并对比标准品共定性出3种成分分别为黄芩苷、千层纸素A-7-0-β-D-葡萄糖醛酸苷、汉黄芩苷。三种成分MRM模式检测参数如表2所示。活性成分的结构式信息如图4所示。
实施例4 与FGF2结合的黄芩活性成分的定量分析
1、BCA蛋白浓度检测实验
采用BCA法测定FGF2-黄芩活性成分复合物中蛋白浓度;
具体内容:按照BCA蛋白浓度检测说明书配置标准曲线样品,将标准曲线样品以及经多室电泳后的样品往96孔板加入20 μL,以及加入BCA工作液,60℃孵化30 min,利用酶标仪在吸光值562nm处检查。
BCA标准曲线的回归方程:Y=0.00199x+0.10562,R2=0 .9999;蛋白浓度为0 .1mg/mL;
2、制作标准曲线
HPLC-MS/MS实验平台线性范围和灵敏度的评估 , 分别配制黄芩苷、千层纸素A-7-0-β- D-葡萄糖醛酸苷和汉黄芩苷即制成表中的浓度样本。测的S1~S6系列浓度和相应的峰面积 , 绘制标准浓度-标准浓度面积的曲线 , 并计算曲线方程和相关系数r。
3、三种成分与FGF2结合的黄芩活性成分定量分析及结合比例计算
依据实施例2和实施例3获得与FGF1结合的三种成分,结合实施例4中的标准曲线,进行定量分析;黄芩苷、千层纸素A-7-0-β-D-葡萄糖醛酸苷、汉黄芩苷三种活性成分成分摩尔浓度的具体测定方法为将MRM检测FGF1+3STD负接收室和3STD负接收室结果如图5所示以及制作完的标准曲线使用定量软件计算活性成分摩尔比;结果见表4,可以得出黄芩苷、千层纸素A-7-0-β-D-葡萄糖醛酸苷、汉黄芩苷与FGF1蛋白结合后小分子之间的比例为2:2:1;
实施例5 三种成分与FGF1联合给药促NIH3T3细胞增殖实验
一、给药浓度的筛选
根据实施例3中确定了三种活性物质黄芩苷(BC)、千层纸素A-7-0-β-D葡萄糖醛酸苷(OXS)、汉黄芩苷(WGS),以及三种成分混合(3STD)进行给药浓度筛选;同时筛选FGF1的给药浓度。
具体步骤如下:
1)将NIH3T3细胞用含10%FBS的DMEM调整到浓度为2*104个/mL,接种于96孔板,每孔100uL;
2)在浓度的5%CO2下、37℃恒温培养箱中培养24 h后,化合物组加入无血清DMEM稀释的化合物:化合物单体梯度给药,给药浓度为:0.25、2.5、25、250、2500、25000、50000、100000nM;FGF1组给药浓度为0.625、1.25、2.5、5、10、20、40 nM;
3)设立空白对照组N(加入DMEM培养基),单一成分给药组(加入用DMEM培养基稀释的各种浓度梯度的不同单一成分药物);三种成分混合给药组;蛋白给药FGF1(加入用DMEM培养基稀释的各种浓度梯度的不同FGF1):均设6个复孔;实验重复3次;
4)分别培养24h、48h后,加入CCK-8每孔10μl,5%CO2,37℃恒温培养箱中避光孵育2h后,用酶标仪在450 nm波长下,检测各孔OD值。
结果如图6所示,给药24h后,黄芩苷(BC)、千层纸素A-7-0-β-D葡萄糖醛酸苷(OXS)、汉黄芩苷(WGS)单独给药时对细胞无促增殖作用,在同等浓度下三种成分混合(3STD)给药组促NIH3T3细胞增殖效果显著大于三种成分单独给药组;如图7所示,当三种成分浓度为25000 nM时促增殖效果最好;如图8所示,FGF1单独给药24h、48h后,当给药浓度为2.5 nM 时,促细胞增殖效果最好。并且与空白对照组相比具有显著性差异。
二、联合给药增殖作用效果
基于上述给药浓度筛选结果基础上,三种化合物浓度设置为2.5、250、25000 nM,FGF1给药浓度设置为2.5 nM。给药时间分别为24h和48h;具体步骤如下:
1、将NIH3T3细胞用含10%FBS的DMEM调整到浓度为2*104个/mL,接种于96孔板,每孔100uL;
2、在浓度的5%CO2下、37℃恒温培养箱中培养24 h后,化合物给药浓度设置如下,均设6个复孔:
N:空白对照组,加入无血清培养基;
FGF1:给药浓度固定为2.5 nM;
BC:从左至右依次为黄芩苷2.5 nM、250 nM、2500 nM;
FGF1+BC:从左至右依次为:1)FGF1:2.5 nM BC:2.5 nM;2)FGF1:2.5 nM BC:250 nM;3)FGF1:2.5 nM BC:25000 nM;
WGS:从左至右依次为汉黄芩苷2.5 nM、250 nM、2500 nM
FGF1+WGS:从左至右依次为:1)FGF1:2.5 nM WGS:2.5 nM;2)FGF1:2.5 nM WGS:250nM;3)FGF1:2.5 nM WGS:25000 nM;
OXS:从左至右依次为千层纸素A-7-0-β-D-葡萄糖醛酸苷2.5 nM、250 nM、2500 nM;
FGF1+OXS:从左至右依次为:1)FGF1:2.5 nM OXS:2.5 nM;2)FGF1:2.5 nM OXS:250nM;3)FGF1:2.5 nM OXS:25000 nM;
BC+WGS:黄芩苷与汉黄芩苷浓度和从左至右依次为2.5 nM、250 nM、2500 nM;
FGF1+BC+WGS:从左至右依次是:1)FGF1:2.5 nM BC+WGS(BC与WGS等摩尔质量):2.5nM;2)FGF1:2.5 nM BC+WGS(BC与WGS等摩尔质量):250 nM;3)FGF1:2.5 nM BC+WGS(BC与WGS等摩尔质量):25000 nM;
BC+OXS:从左至右依次是黄芩与千层纸素A-7-0-β-D-葡萄糖醛酸苷分别为2.5 nM、250nM、2500 nM;
FGF1+BC+OXS:从左至右依次是:1)FGF1:2.5 nM BC+OXS(BC与OXS等摩尔质量):2.5nM;2)FGF1:2.5 nM BC+OXS(BC与OXS等摩尔质量):250 nM;3)FGF1:2.5 nM BC+OXS(BC与OXS等摩尔质量):25000 nM;
WGS+OXS:从左至右依次是汉黄芩苷与千层纸素A-7-0-β-D-葡萄糖醛酸苷分别为2.5nM、250 nM、2500 nM;
FGF1+WGS+OXS:从左至右依次是:1)FGF1:2.5 nM WGS+OXS(WGS与OXS等摩尔质量):2.5nM;2)FGF1:2.5 nM WGS+OXS(WGS与OXS等摩尔质量):250 nM;3)FGF1:2.5 nM WGS+OXS:(WGS与OXS等摩尔质量)25000 nM;
3STD:黄芩苷、汉黄芩苷与千层纸素A-7-0-β-D-葡萄糖醛酸苷等摩尔质量,从左至右依次为2.5 nM、250 nM、2500 nM;
FGF1+3STD:从左至右依次是1)FGF1:2.5 nM 3STD:2.5 nM;2)FGF1:2.5 nM 3STD:250nM;3)FGF1:2.5 nM 3STD:25000 nM;
3、在浓度的5%CO2下、37℃恒温培养箱中培养24 h后,加入CCK-8溶液,每孔10 μl,5%CO2,37℃恒温培养箱中避光孵育2h后,用酶标仪在450 nm波长下,检测各孔OD值。
结果如图9、10所示,黄芩三种成分单独给药时对细胞几乎无促进增殖作用,但3种活性成分协同给药时,对细胞是有促增殖作用的,并且三种成分混合后与FGF1结合的促增殖效果显著高于FGF1单独给药、FGF1与单一成分给药和FGF1与两个成分协同给药组。并且FGF1与三种成分协同给药后与FGF1单独给药组相比具有显著性差异。结论:经多室电泳分离体系后筛选到的黄芩中与FGF1结合的三种有效成分,可促进NIH3T3细胞的增殖。
实施例6 FGF1-黄芩苷复合物的制备
取黄芩苷标准品,用其10倍重量的甲醇溶解黄芩苷标准品,得到黄芩苷甲醇溶液,氮气吹干后超纯水回溶,得水溶液;;FGF1靶蛋白溶解于10 mM pH4 乙酸铵缓冲液,得FGF1乙酸铵溶液;按FGF1乙酸铵溶液:黄芩苷溶液(v:v)=1:1配比,混合,形成FGF1-黄芩苷混合液;在37℃下孵化30min,使黄芩苷溶液中的黄芩苷与FGF1结合,形成FGF1-黄芩苷复合物;将孵化后的混合液真空冷冻干燥,即得到FGF1-黄芩苷复合物。
实施例7 FGF1-黄芩苷复合物的制备
取黄芩苷标准品,用其10倍重量的100%二甲基亚砜溶液溶解黄芩苷标准品,得到黄芩苷二甲基亚砜溶液;FGF1靶蛋白溶解于10 mM pH6.8 PBS缓冲液,得FGF1PBS溶液;按FGF1乙酸铵溶液:黄芩苷二甲基亚砜溶液(v:v)=1:1配比,混合,形成FGF1-黄芩苷混合液;在37℃下孵化30min,使黄芩苷二甲基亚砜溶液中的黄芩苷与FGF1结合,形成FGF1-黄芩苷复合物;将孵化后的混合液真空冷冻干燥,即得到FGF1-黄芩苷复合物。
Claims (6)
1.黄芩提取物-FGF1复合物,它是黄芩提取物与FGF1共孵化获得的复合物,所述的黄芩提取物为黄芩苷、千层纸素A-7-0-β-D-葡萄糖醛酸苷或汉黄芩苷,获得的复合物为FGF1-黄芩苷复合物、FGF1-千层纸素A-7-0-β-D-葡萄糖醛酸苷复合物或FGF1-汉黄芩苷复合物。
2.根据权利要求1所述的黄芩提取物-FGF1复合物,其特征在于:所述的孵化为:
1)取黄芩提取物,用其5~20倍重量的甲醇溶解黄芩提取物,得到甲醇溶液,氮气吹干后超纯水回溶,得水溶液;
2)将FGF1靶蛋白溶解于乙酸铵缓冲液,得FGF1乙酸铵溶液;
3)FGF1 乙酸铵溶液和黄芩提取物溶液,混合;
4)在36~38℃下孵化25~35min,得到黄芩提取物-FGF1复合物。
3.根据权利要求2所述的黄芩提取物-FGF1复合物,其特征在于:步骤2)所述的乙酸铵缓冲液的浓度为10 mM、pH值为4。
4.根据权利要求3所述的黄芩提取物-FGF1复合物,其特征在于:步骤3)所述的FGF1 乙酸铵溶液和黄芩提取物水溶液,按体积比1:1混合。
5.根据权利要求4所述的黄芩提取物-FGF1复合物,其特征在于:步骤4)在37℃下孵化30min。
6.FGF1-黄芩苷复合物、FGF1-千层纸素A-7-0-β-D-葡萄糖醛酸苷复合物和/或FGF1-汉黄芩苷复合物在促细胞增殖方面的应用。
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