CN101333236A - 克力托辛的制备方法及在抗肿瘤药物中的应用 - Google Patents
克力托辛的制备方法及在抗肿瘤药物中的应用 Download PDFInfo
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Abstract
本发明提供克力托辛的制备方法,是将原料新鲜大白桩菇菌子实体真空冷冻干燥并粉碎,所得菌粉浸于酒精中,浸提液减压浓缩得浸膏,再经乙酸乙酯萃取,减压浓缩得浸膏,该浸膏分别经硅胶柱、反相RP-18和反相RP-C8制备柱分离得到目的物。本发明提供的克力托辛具有广谱抗肿瘤活性,经体外抗肿瘤活性检测,对6种肿瘤株均有强烈的增殖抑制效应,其中对肝癌HepG2细胞的杀伤效果尤为突出,其半数增殖抑制浓度IC50为0.24μm;裸小鼠体内实验表明,3mg/kg剂量的克力托辛对人肝癌HepG2裸小鼠移植瘤有显著的生长抑制作用,抑瘤率达40.1%。本发明对开发我国真菌资源具有重要意义,可在制备抗肿瘤活性药物中应用。
Description
技术领域
本发明涉及医药技术领域,具体地说是从天然菌物中提取有效成分及其用途,尤其是从大白桩菇菌(Leucopaxillus giganteus)子实体中提取有效成分clitocine的方法、其在制备抗肿瘤药物中的应用及相关机理。
背景技术
大白桩菇菌(Leucopaxillus giganteus)属白蘑科,夏秋季于草原上单生或群生,有时生于林中草地上。分布于河北、内蒙古、吉林、辽宁、山西、黑龙江、青海、新疆等地。该菌在治疗肿瘤方面的研究报道很少。恶性肿瘤是当今威胁人类身心健康最严重的疾病,死亡率高,目前尚无治愈率较高的药物报道,因此研制抗恶性肿瘤的药物,长期以来是人们努力的方向。
发明内容
本发明的一个目的是提供克力托辛(clitocine)的制备方法,该clitocine的分子式为C9H13N5O6,分子量287,属于嘌呤核苷类,其特征是通过以下步骤实现,将原料新鲜大白桩菇菌子实体(2.7kg)真空冷冻干燥,粉碎机粉碎,所得340g菌粉浸于1.5L 95%(v/v)酒精中,其间更换新鲜溶剂一次,合并两次浸提液,减压浓缩得酒精浸膏(25.7g)。浸膏经乙酸乙酯萃取,减压浓缩得乙酸乙酯浸膏(15.7g)。取15g乙酸乙酯浸膏上硅胶柱,CH2Cl2/MeOH(10∶0-0∶10)梯度洗脱,收集细胞筛选有活性的洗脱峰(6.5g),然后上反相柱RP-18(ODS,50μm,Φ25×420mm),MeOH/H2O(1∶9)洗脱,细胞筛选有活性的部分再经过反相柱RP-C8制备[5μm;Φ20×250mm;MeOH/H2O(5∶95);8ml/min],得到目的化合物clitocine(815mg)。
本发明的另一个目的是提供clitocine单味制剂或与药用敷料组合,可在制备抗肿瘤药物中的应用。
本发明所制备的clitocine,可在制备治疗乳腺癌药物、制备治疗宫颈癌药物、制备治疗肝癌药物、制备治疗胃癌等抗癌药物中的应用,本发明还提供了其治疗肝癌时凋亡方面的相关机制。
本发明提供的药物,其制剂形式主要包括液体制剂、颗粒剂、片剂、冲剂、软胶丸、软胶囊、滴丸剂或注射剂。
本发明提供的药物,制剂的给药形式主要包括口服给药或注射给药。
实验证明本发明提供的clitocine,具有体外强烈抑制肿瘤细胞增殖的作用,经四甲基偶氮唑盐(简称MTT法)检测,对乳腺癌Bcap37的半数增殖抑制浓度IC50为11μM,对乳腺癌MCF-7的IC50为43μM,对宫颈癌Hela的IC50为15μM,对胃癌SGC-7901的IC50为2μM,对肝癌SMMC-7721的IC50为1μM,对肝癌HepG2的IC50为0.24μM;裸小鼠体内实验表明,3mg/kg剂量的clitocine对人肝癌HepG2裸小鼠移植瘤有显著的生长抑制作用,抑瘤率达40.1%;在此基础上,我们还对其中肝癌HepG2细胞进行了抗肿瘤机制的深入研究,一系列相关实验证明诱导凋亡是clitocine体外抑制HepG2增殖的机制之一,进一步证明该凋亡过程是通过线粒体通路和死亡受体通路完成的,并且clitocine还影响HepG2的细胞周期,将其阻滞在G2/M期。因此clitocine作为单味制剂或与药物敷料组合,均可在制备治疗恶性肿瘤药物中应用。本发明为进一步开发新型抗恶性肿瘤药物提供了先导化合物和科学依据,对利用我国真菌资源具有重要意义。
附图说明
图1为clitocine的液相色谱分析。
图2为不同浓度clitocine对乳腺癌Bcap37、肝癌SMMC-7721、胃癌SGC-7901、乳腺癌MCF-7,宫颈癌Hela五株肿瘤细胞体外增殖抑制率折线图。
图3为不同浓度clitocine对肝癌HepG2细胞在24h、48h和72h的体外增殖抑制率折线图。
图4为不同浓度clitocine杀伤肝癌SMMC-7721细胞的显微图。
图5为不同浓度clitocine作用于肝癌HepG2细胞产生DNA梯状条带的凝胶电泳图。
图6为clitocine作用于肝癌HepG2细胞的AnnexinV-PI双染凋亡图。
图7为Caspase家族抑制剂对clitocine诱导的肝癌HepG2凋亡抑制作用AnnexinV-PI双染凋亡图。
图8为clitocine作用于肝癌HepG2细胞线粒体膜电位变化JC-1染色图。
图9为clitocine作用于肝癌HepG2细胞释放细胞色素c的免疫印迹图。
图10-11为clitocine作用于肝癌HepG2的细胞周期变化图。
图12为clitocine对荷瘤裸小鼠体内肿瘤生长抑制图。
具体实施方式
以下将结合具体实施例与附图详细说明本发明,这些实例仅用于说明目的,而不用于限制本发明范围。
实施例一:从新鲜大白桩菇菌子实体提取分离纯化抗肿瘤活性化合物及其结构鉴定
实验方法
1.提取和纯化:将原料新鲜大白桩菇菌子实体(2.7kg)真空冷冻干燥,粉碎机粉碎,所得340g菌粉浸于1.5L 95%(v/v)酒精中,其间更换新鲜溶剂一次,合并两次浸提液,减压浓缩得酒精浸膏(25.7g),浸膏经乙酸乙酯萃取,减压浓缩得乙酸乙酯浸膏(15.7g)。取15g乙酸乙酯浸膏上硅胶柱,CH2Cl2/MeOH(10∶0-0∶10)梯度洗脱,收集细胞筛选有活性的洗脱峰(6.5g),然后上反相柱RP-18(ODS,50μm,Φ25×420mm),MeOH/H2O(1∶9)洗脱,细胞筛选有活性的部分再经过反相柱RP-C8制备[5μm;Φ20×250mm;MeOH/H2O(5∶95);8ml/min],得到目的化合物clitocine(815mg)。其液相色谱分析图见附图1,在洗脱时间为9.0分时出现单一峰即为clitocine。
2.结构鉴定:核磁共振仪(INOVA-400),质谱仪(Bruker Esquire 3000plus)
3.实验结果:clitocine,分子式为C9H13N5O6,ESI-MS:m/z 287。其一维的二维的核磁数据列于表1,clitocine的化学结构为:
表1clitocine的核磁数据
实施例二:clitocine对人肿瘤细胞增殖抑制活性试验
实验材料方法
1.人肿瘤细胞株及来源:共做六个肿瘤株分别是乳腺癌Bcap37、乳腺癌MCF-7、宫颈癌Hela、肝癌SMMC-7721、肝癌HepG2、胃癌SGC-7901,均来自于美国American Type Culture Collection。
2.clitocine溶解在DMSO中配成1mg/ml母液,置于-20℃冰箱,实验时用培养液稀释成所需浓度供用,MTT(Sigma公司产品)用磷酸缓冲液配成1mg/ml母液,置于4℃冰箱备用。
3.细胞培养:所有细胞均培养于DMEM完全培养液(Invitrogen),加5%的新生牛血清(Invitrogen),2mmol/L L-谷氨酰胺。选择对数生长期细胞,以1×104的密度接种于96孔板中,加入不同浓度的药物,置37℃,10%培养箱中,培养48小时后用Olympus IX70万能显微镜(penguin 600CL CCD,Pixera)拍照记录。
4.四甲基偶氮唑盐(简称MTT法)检测clitocine对肿瘤细胞增殖的影响:选择对数生长期细胞,以1×104的密度接种于96孔板中,每孔200μl,置37℃,10%培养箱中培养4小时后加入不同浓度的药物,每个浓度设复孔6个,同时设空白对照,置37℃,10%CO2培养箱中培养48小时,吸出孔板中培养液,往每孔中加入浓度为1mg/ml的MTT 50μl,放入培养箱中继续培养4小时,取出加过MTT的96孔板,每孔加入150μl的DMSO,轻轻震荡孔板10min,待孔底部的结晶物完全溶解后,将孔板放入酶标仪中测波长在570nm的各孔的光吸收值,记录结果,实验重复三次。
实验结果见表2,图片见附图2-4。
表2clitocine作用于六种人肿瘤细胞的IC50值
由表2可见,clitocine对实验所测六株人肿瘤细胞均有增殖抑制效果,其中肝癌HepG2细胞对clitocine最敏感,其IC50在48h为0.24μM,故以下实施例用肝癌HepG2细胞来进一步阐述clitocine的抗肿瘤分子作用机理。
附图2表明,clitocine对乳腺癌Bcap37、乳腺癌MCF-7、宫颈癌Hela、肝癌SMMC-7721和胃癌SGC-7901五种肿瘤株都有抑制增殖作用,且随clitocine浓度增高,抑制百分率增加,呈剂量依赖性。
附图3说明,clitocine对肝癌HepG2的增殖抑制不仅是剂量依赖,且与其作用时间长短有关,作用24,48和72h其IC50分别是0.74μM、0.24μM和0.21μM,也表现时间依赖性。
附图4以肝癌SMMC7721为代表,观察不同浓度clitocine作用48h时细胞形态变化,其中图A、B、C、D、E分别代表clitocine浓度为0、0.1μM、0.3μM、0.6μM和0.9μM处理下的显微照片,可见,随着药物浓度增加,细胞逐渐死亡,变圆最后成为碎片。
实施例三:clitocine抑制肝癌HepG2细胞增殖的相关机制
1.DNA梯状条带实验方法:肿瘤细胞HepG2经不同浓度的clitocine处理48h后收集,加裂解液在45℃裂解1h(5mM Tris-HCl,100mM EDTA,1%(w/v)SDS,and蛋白酶K),基因组DNA用酚/氯仿/异戊醇(25∶24∶1)抽提,70%酒精-20℃下沉淀,用RNA酶除去RNA后,加40μg等量DNA在1.2%琼脂糖凝胶上电泳并经凝胶成像仪拍照。
实验结果见附图5。
细胞凋亡最典型的特征之一,是细胞在接受药物作用后,DNA会被核酸酶在核小体连接处切断,产生180-200bp或其整数倍大小的DNA碎片,琼脂糖凝胶电泳时呈现有规则的梯状体条带,这是区分凋亡与坏死的重要标志[1]。从附图5中可见,不同浓度clitocine处理HepG2细胞,从左到右依次为100bp标记、0、0.1μM、0.3μM、0.6μM和0.9μM,在0.6μM处理时已经出现了典型的DNA梯状条带,表明clitocine能够诱导HepG2细胞产生凋亡。
2.AnnexinV-PI双染实验方法:约3×105个对数生长期的肝癌HepG2细胞与clitocine共同孵育48h,,胰酶消化收集细胞,PBS清洗一次,加入500μl的AnnexinV-FITC结合缓冲液,含25μl的AnnexinV-FITC和5μl的PI缓冲液,然后避光孵育15min,用FACSCanto流式细胞仪(Becton Dickinson)分析结果。
实验结果见附图6。
细胞凋亡早期,细胞膜上的磷脂酰丝氨酸(PS)会从细胞膜内翻转到细胞膜外,AnnexinV可与其结合,而凋亡细胞的细胞膜仍然保持完整,染料PI不能进入细胞进行染色,因此通过AnnexinV-PI双染实验,可区分凋亡与坏死细胞[2]。附图6中,图A、B、C分别代表clitocine浓度为0(对照)、0.3μM、0.6μM,右上角百分数代表死亡细胞百分比,右下角百分数代表凋亡细胞百分比,可见clitocine诱导HepG2细胞凋亡存在药物剂量依赖性,随着clitocine浓度由0.3μM上升到0.6μM,凋亡的百分比由6,22%上升到8.24%,而对照为2.57%。
上述实验1-2表明,clitocine抑制肝癌HepG2细胞增殖是通过诱导其产生凋亡。
3.caspase抑制剂实验方法:约3×105个对数生长期的肝癌HepG2细胞分别与clitocine/caspase-8抑制剂(Z-IETD-FMK)或clitocine/caspase-9抑制剂(Z-LEHD-FMK)共同孵育48小时,胰酶消化收集细胞,PBS清洗一次,加入500μl的AnnexinV-FITC结合缓冲液,含25μl的AnnexinV-FITC和5μl的PI缓冲液,然后避光孵育15min,用FACSCanto流式细胞仪(Becton Dickinson)分析结果。
实验结果见附图7。
在细胞凋亡的信号传导过程中,caspase(半胱氨酸水解酶)起着关键性的调节作用,因此它们的激活是凋亡的重要特征[3],两个主要的caspase家族成员caspase-8和caspase-9分别代表了两条凋亡信号通路即死亡受体通路和线粒体通路[4]。附图7中,图A、B、C分别代表对照、clitocine 0.3μM单独处理和clitocine加caspase-8抑制剂组合,图D、E、F分别代表对照、clitocine 0.3μM单独处理和clitocine加caspase-9抑制剂组合,可以看出添加caspase-8或caspase-9抑制剂后,clitocine诱导的HepG2细胞凋亡受到抑制,分别由4.45%和3.84%下降至2.52%和1.78%,表明clitocine诱导的HepG2细胞凋亡是caspase依赖性的,可能与死亡通路和线粒体通路都有关。
4.线粒体膜电位(ΔΨm)实验方法:HepG2细胞(3×105)与clitocine共孵育48h后,收集细胞与10μM JC-1在37℃黑暗中共孵育15min,用FACSCanto流式细胞仪(Becton Dickinson)分析结果。
实验结果见附图8。
凋亡早期往往伴随着线粒体膜的破裂,导致线粒体膜电化学的变化。使用线粒体特异性染料JC-1,可以检测这种变化。当膜电位高时,JC-1以聚合体形式发出红色荧光,当膜电位降低时,线粒体膜破裂,JC-1可选择性进入线粒体并以单体形式发出绿色荧光,两种荧光的百分比可以反映出线粒体膜电位的变化[5]。附图8中,图A、B、C分别代表clitocine三个不同浓度即0、0.3μM和0.6μM处理,可见,随着clitocine浓度由0.3μM提高到0.6μM,JC-1绿色荧光百分数由19.44%提高到23.93%,均比对照2.71%显著升高,表明线粒体膜电位下降,膜破裂程度逐渐加大,呈剂量依赖性,因此,clitocine诱导的HepG2细胞凋亡与线粒体通路相关。
5.细胞色素c实验方法:HepG2细胞(3×105)与不同浓度的clitocine共孵育一段时间后,收集细胞用裂解液室温下破膜30秒(75mM NaCl,1mMNaH2PO4,8mM Na2HPO4,250mM sucrose,1mM PMSF,5mg/ml leupeptin,21mg/ml aprotinin)含狄吉宁(25μg/106个细胞),10000rpm离心1min,取上清液中的蛋白在12%SDS-PAGE胶上进行电泳,胶上的斑点转移到PVDF膜上,然后与细胞色素c抗体在室温下孵育,清洗后再与二抗共同孵育。最后用ECL试剂盒(Amersham)进行显色观察。
实验结果见附图9。
伴随着线粒体膜电位下降,线粒体膜破裂,线粒体内膜蛋白如细胞色素c外流。细胞色素c外流也被认为是线粒体依赖的凋亡过程中一个关键的特征[6]。附图19中,从左至右依次代表clitocine浓度和时间分别为0、24h(0.3、0.6、0.9μM)、48h(0.3、0.6、0.9μM)作用与HepG2细胞,可见,细胞色素c从线粒体外流,细胞液里细胞色素c蛋白含量随着药物浓度增大和作用时间延长逐渐增多,呈剂量和时间依赖性,这一结果与线粒体膜电位下降一致。
实验4-5揭示了clitocine诱导的肝癌HepG2细胞凋亡与线粒体通路有关。
6.细胞周期分析方法:约3×105个对数生长期的肝癌HepG2与clitocine共同孵育48h后,收集细胞,PBS清洗两次后用70%的冰乙醇固定,PBS清洗两次,2500rpm,4℃,2min,弃去上清液,以500μl的DNA染液(20μg/ml PI,100μg/ml RNase A)重悬浮细胞30min,用FACSCanto流式细胞仪(BectonDickinson)分析DNA含量,用WinMDI 2.9软件统计各个时期的细胞数量(Becton Dickinson)。
实验结果见附图10-11。
细胞周期紊乱是肿瘤细胞的一个主要特征,抑制细胞过度分裂,是诱导肿瘤细胞死亡的另一个途径[7]。附图10中,图A、B、C、D分别表示clitocine的浓度为0、0.3μM、0.6μM和0.9μM,随着浓度升高,G2/M期细胞逐渐增加,附图11是根据附图10细胞分裂各个时期的百分数所做的柱状图,从图中可以更清楚地看出随着clitocine浓度增加,G2/M期细胞百分数显著增加,即细胞分裂停止在G2/M期,表明clitocine抑制肝癌HepG2细胞增殖还与其能阻滞细胞周期进程有关。
实施例四clitocine对荷瘤裸小鼠体内肿瘤生长抑制实验
实验方法:取日龄30-40天,体重18-20g的雌性裸小鼠于背部皮下接种1×107人肝癌HepG2细胞,用游标卡尺测量移植瘤直径,待肿瘤长至100mm3后将动物随机分组,每组7只,使用测量瘤径的方法,动态观察被试物抗肿瘤的效应。Clitocine 3mg/kg,静脉注射,隔天给药,至14天处死小鼠,肿瘤直径的测量分别在第七天和第14天。阴性对照组静脉注射等量生理盐水。肿瘤抑制率按如下公式计算:
[(对照组平均肿瘤体积-处理组平均肿瘤体积)/对照组平均肿瘤体积]×100%
实验结果见附图12,由图中可见,clitocine对人肝癌HepG2裸小鼠移植瘤具有显著的生长抑制效果,clitocine 3mg/kg,隔天给药,第七天和第14天,抑瘤率分别达50.1%和40.1%,与阴性对照组差异显著(p<0.05)。表明clitocine具有体内抗肿瘤效果。
综上,本发明所述的单体化合物clitocine,可以抑制多种肿瘤细胞增殖,其对肝癌HepG2细胞的增殖抑制作用是通过诱导HepG2细胞凋亡和阻滞细胞周期在G2/M期;诱导细胞凋亡的通路是死亡受体和线粒体通路,并且对荷瘤裸小鼠具有体内抗肿瘤效果。因此,clitocine作为单味制剂或与药用敷料组合,均可在制备治疗抗肿瘤药物中应用。
Claims (10)
1.一种克力托辛的制备方法,其分子式为C9H13N5O6,分子量287,其特征是通过以下步骤实现:将原料新鲜大白桩菇菌子实体真空冷冻干燥,粉碎机粉碎,所得菌粉浸于v/v为95%的酒精中,其间更换新鲜溶剂一次,合并两次浸提液,减压浓缩得酒精浸膏,浸膏经乙酸乙酯萃取,减压浓缩得乙酸乙酯浸膏,取乙酸乙酯浸膏上硅胶柱,10∶0-0∶10的CH2Cl2/MeOH梯度洗脱,收集细胞筛选有活性的洗脱峰,然后上反相柱RP-18,ODS,50μm,Φ25×420mm,1∶9的MeOH/H2O洗脱,细胞筛选有活性的部分再经过反相柱RP-C8制备,条件:5μm,Φ20×250mm,5∶95的MeOH/H2O,8ml/min,得到目的化合物。
2.根据权利要求1所述方法制备获得的克力托辛在制备抗肿瘤药物中的应用,
3.根据权利要求2所述的克力托辛的应用,其特征是:在制备治疗乳腺癌药物中的应用。
4.根据权利要求2所述的克力托辛的应用,其特征是:在制备治疗宫颈癌药物中的应用。
5.根据权利要求2所述的克力托辛的应用,其特征是:在制备治疗肝癌药物中的应用。
6.根据权利要求2所述的克力托辛的应用,其特征是:在制备治疗胃癌药物中的应用。
7.根据权利要求2所述的克力托辛的应用,其特征是:在治疗肝癌时凋亡方面的相关机制。
8.根据权利要求2所述的克力托辛的应用,其特征是:克力托辛作为单味制剂或与制剂允许的药用敷料组合在制备抗肿瘤药物中的应用。
9.根据权利要求2所述的克力托辛的应用,其特征是:所述药物的制剂形式包括液体制剂、颗粒剂、片剂、冲剂、软胶丸、软胶囊、滴丸剂或注射剂。
10.根据权利要求9所述的克力托辛的应用,其特征是:所述制剂的给药形式是口服给药或注射给药。
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CN103520195A (zh) * | 2013-06-11 | 2014-01-22 | 浙江大学 | 克力托辛在制备增敏剂中的应用 |
MD4300C1 (ro) * | 2012-12-28 | 2015-03-31 | Государственный Университет Молд0 | Inhibitor al proliferării celulelor HepG2 în cancerul hepatic în bază de cloro-[2-fenil(piridin-2-il)metanon-4-(3-metoxifenil)tiosemicarbazono]nichel |
JP2021031423A (ja) * | 2019-08-21 | 2021-03-01 | 株式会社岩出菌学研究所 | 抗癌剤耐性抑制剤 |
CN116059262A (zh) * | 2023-02-02 | 2023-05-05 | 山西农业大学山西功能食品研究院 | 一种大白桩菇提取物及其制备方法和应用 |
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2008
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Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
MD4300C1 (ro) * | 2012-12-28 | 2015-03-31 | Государственный Университет Молд0 | Inhibitor al proliferării celulelor HepG2 în cancerul hepatic în bază de cloro-[2-fenil(piridin-2-il)metanon-4-(3-metoxifenil)tiosemicarbazono]nichel |
CN103520195A (zh) * | 2013-06-11 | 2014-01-22 | 浙江大学 | 克力托辛在制备增敏剂中的应用 |
JP2021031423A (ja) * | 2019-08-21 | 2021-03-01 | 株式会社岩出菌学研究所 | 抗癌剤耐性抑制剤 |
CN116059262A (zh) * | 2023-02-02 | 2023-05-05 | 山西农业大学山西功能食品研究院 | 一种大白桩菇提取物及其制备方法和应用 |
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