CN113861208B - 一种细胞松弛素类化合物及其制备方法和应用 - Google Patents
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Abstract
本发明涉及微生物代谢产物及其在药物研究领域的应用技术,具体是微生物代谢产生细胞松弛素类化合物的制备方法与应用。所述细胞松弛素类化合物的化学结构如式I所示,由糙壁弯孢菌Curvularia verruculosa经发酵培养制备获得。经实验表明此类化合物具有抗肿瘤活性,可作为细胞增殖抑制剂或抗肿瘤制剂。本发明制备所得的细胞松弛素类化合物是利用微生物进行发酵培养生产的,具有操作简单、生产周期短、成本低等特点。
Description
技术领域
本发明涉及微生物代谢产物及其在药物研究领域的应用技术,具体是一种从糙壁弯孢菌(Curvularia verruculosa)的发酵产物中获得细胞松弛素类化合物及其制备方法与应用。
背景技术
恶性肿瘤是严重威胁人类健康的重要疾病。中国是恶性肿瘤的发病大国,据国家癌症中心的《2017中国肿瘤登记年报》报道,中国恶性肿瘤病人占全球癌症病人总量的将近40%。每天约1万人确诊癌症,平均每分钟就有7人确诊。肝癌、结肠癌、女性乳腺癌等依然是我国主要的恶性肿瘤,而乳腺癌为女性发病首位。
虽然研究人员一直致力于恶性肿瘤的研究,但目前来看我国恶性肿瘤发病、死亡数持续上升,医疗支出巨大,社会负担沉重,急需开发新型药物来防控日益严峻的恶性肿瘤发展趋势。
目前市面上用于治疗恶性肿瘤的药物多为有机合成产物或微生物的次级代谢产物。
糙壁弯孢菌(Curvularia verruculosa)可从多种植物中提取获得,对于该菌株形态学特征及其生物学作用已经被本领域所熟知,并且有文献对其进行报道,可从狗牙根(习平根等,华南农业大学学报,2005,26(2),31-34)中分离得到,也可从长春花中分离得到(R.Parthasarathy et al,Analytical Biochemistry,2020,593,113530)。据报道,该菌经发酵可产生长春花碱,对海拉细胞具有细胞毒活性,可用于治疗恶性淋巴瘤、绒毛膜癌等(R.Parthasarathy et al,Analytical Biochemistry,2020,593,113530)。但目前为止并未有对糙壁弯孢菌(Curvularia verruculosa)次级代谢产物进行系统研究的报道,本发明首次从该菌中分离得到具有抗肿瘤活性的细胞松弛素类化合物。
发明内容
本发明的目的在于提供一种从糙壁弯孢菌(Curvularia verruculosa)的发酵产物中获得细胞松弛素类化合物及其制备方法与肿瘤防治方面的应用。
为实现上述目的,本发明采用的技术方案为:
一种细胞松弛素类化合物,细胞松弛素类化合物结构如式I中所示:
一种细胞松弛素类化合物的制备方法,
1)将糙壁弯孢菌Curvularia verruculosa经发酵培养,而后将发酵液用乙酸乙酯萃取3-4次,合并提取液进行浓缩,获得发酵液粗提物;
所述糙壁弯孢菌(Curvularia verruculosa)孢子通常呈弯曲状,壁粗糙;该菌株在多篇文献中报道,并且可在多个保藏中心通过流通方式获得;
比如中国典型培养物保藏中心(China Center for Type Culture Collection,CCTCC,保藏号CCTCC HF2008198)、美国典型培养物保藏中心(American Type CultureCollection,ATCC,保藏号ATCC 60943-60948)、德国微生物菌种保藏中心(DeutscheSammlung von Mikroorganismen und Zellkulturen,DSMZ,保藏号DSM 1157)、荷兰真菌菌种保藏中心(Central Bureau voor Schimmel-Cultures,CBS,保藏号CBS 147.63和CBS444.70)等。另外,本领域的技术人员可根据文献公开报道的方法方便地从狗牙根(习平根等,华南农业大学学报,2005,26(2),31-34)和长春花(R.Parthasarathy et al,Analytical Biochemistry,2020,593,113530)等植物中分离获得;并可方便地获得菌株ITS在基因库中的登记信息,例如,NCBI(MK995628)、NCBI(KJ748697.1)等等。
2)将步骤1)中的发酵液粗提物进行减压硅胶柱层析,依次采用梯度为50:1至1:1(v/v)的石油醚-乙酸乙酯和梯度为20:1至1:1(v/v)的二氯甲烷-甲醇进行洗脱;
3)将步骤2)中以二氯甲烷-甲醇10:1(v/v)梯度洗脱下的组分以甲醇-水(甲醇-水的比例10:90至100:0)作为洗脱液进行反相柱层析,收集甲醇-水60:40(v/v)洗脱得到的组分,再用薄层层析(TLC)纯化,所采用的展开体系为20:1(v/v)的二氯甲烷-甲醇,后经过凝胶柱层析制得目标化合物。
具体为:
1)将所述糙壁弯孢菌(Curvularia verruculosa)在PDA(马铃薯蔗糖琼脂)培养基上于28℃下培养7天,取其菌丝体接种于固体培养基中,室温下静置发酵30天,发酵产物经石油醚萃取脱除小极性产物后,采用乙酸乙酯充分浸泡反复萃取,合并萃取液进行浓缩,获得粗提物;
2)将上述粗提物进行减压硅胶柱层析,依次使用梯度分别为50:1至1:1(v/v,下同)的石油醚-乙酸乙酯和20:1至1:1的二氯甲烷-甲醇洗脱体系进行梯度洗脱;
3)收集上述步骤2)中的二氯甲烷-甲醇10:1洗脱的组分浓缩,进行反相硅胶柱层析,使用梯度为10:90至100:0的甲醇-水洗脱体系进行梯度洗脱;
4)收集上述步骤3)中的甲醇-水60:40的组分,进行薄层层析(TLC)纯化,后采用Sephadex LH-20甲醇凝胶柱层析进行纯化(以MeOH作为洗脱剂),得到式I所示细胞松弛素类化合物。
一种细胞松弛素类化合物的应用,所述式I中的细胞松弛素类化合物在作为细胞增殖抑制剂或抗肿瘤制剂或其先导化合物中的应用。
所述式I中的细胞松弛素类化合物在作为抗肝癌、结肠癌和乳腺癌药物或先导化合物中的应用。
所述肝癌为HepG-2肝癌细胞;所述结肠癌为HCT-16结肠癌细胞;所述乳腺癌为MCF-7乳腺癌细胞。
本发明所具有的优点:
1.本发明制备所得的细胞松弛素类化合物来源于糙壁弯孢菌(Curvulariaverruculosa)的发酵产物,采用微生物发酵的方法制备该化合物,具有高效、环保的特点;
2.本发明制备所得的细胞松弛素类化合物具有显著抗HepG-2肝癌细胞,HCT-16结肠癌细胞,MCF-7乳腺癌细胞的活性,且化合物为尚未被报道的新化合物,可进一步探索其作用机制,以期开发为新型抗肝癌药物或其先导化合物。经实验证明该化合物对肝癌细胞株(HepG-2)、结肠癌细胞株(HCT-116)、乳腺癌细胞株(MCF-7)具有较强的抑制活性,其半数抑制浓度IC50分别为6.40、9.51和6.04μM。而阳性对照顺铂的IC50分别为15.6、10.7和11.0μM。
具体实施方式
下面结合一些非限定性的具体实施实例对本发明做进一步阐述。
实施例1.化合物结构式
从糙壁弯孢菌(Curvularia verruculosa)发酵产物中分离获得的细胞松弛素类化合物结构如式(I)所示(式中的阿拉伯数字代表化学结构中的碳原子标位):
实施例2.式I所示化合物的制备方法
1)发酵培养
所述糙壁弯孢菌(Curvularia verruculosa)取适量菌种接种在PDA平板上,在PDA(马铃薯蔗糖琼脂)培养基上菌丝体呈白灰色,后期产生白色孢子,于28℃下培养7天后待用。
将上述PDA平板的菌丝体接种于无菌的固体培养基中,室温下静置培养30天,发酵产物通过石油醚萃取去除小极性产物后,使用乙酸乙酯浸泡萃取4次,合并浓缩后获得发酵粗提物;
所述固体培养基配方为:每100毫升海水中含大米70克,玉米浆0.1克,蛋白胨0.3克。
2)粗提物分离纯化
将上述粗提物通过减压硅胶(100-200目)柱层析分段,并使用梯度为50:1至1:1(50:1、20:1、10:1、5:1、2:1、1:1,v/v,下同)的石油醚-乙酸乙酯和20:1至1:1(20:1、10:1、5:1、1:1)的二氯甲烷-甲醇作为洗脱溶剂,依次进行梯度洗脱;收集二氯甲烷-甲醇10:1洗脱得到的组分;
将上述收集到的组分进行反相硅胶柱层析,以10:90至100:0的甲醇-水依次进行梯度洗脱;
收集上述步骤中的甲醇-水60:40的组分,进行薄层层析(TLC)纯化,所采用的展开体系为20:1(v/v)的二氯甲烷-甲醇,而后采用Sephadex LH-20甲醇凝胶柱层析进行纯化,以MeOH作为洗脱剂,收集洗脱组分,干燥得到式I所示细胞松弛素类化合物。
该化合物具有以下理化和波谱特性:
淡黄色油状固体,化学式C29H37NO6,比旋光度(c 0.14,MeOH);ECD(0.71mM,MeOH)λmax(Δε))212(–8.09),248(+1.07)nm;ESI-MSm/z 496[M+H]+;HR-ESI-MSm/z 496.2680[M+H]+(calcd for C29H38NO6,496.2694)。1H-NMR和13C-NMR见表1。
表1.核磁共振氢谱(500MHz,in DMSO)碳谱(500MHz,in DMSO)数据
实施例3.抗肿瘤活性实验
选择以下3株供试细胞株:肝癌细胞株(HepG-2)、结肠癌细胞株(HCT-116)、乳腺癌细胞株(MCF-7)进行抗肿瘤活性测试。
1)供试细胞株及其培养
本发明采用的供试细胞株为肝癌细胞株(HepG-2)、结肠癌细胞株(HCT-116)、乳腺癌细胞株(MCF-7),将细胞在含10%胎牛血清、100U/ml青霉素和100mg/ml链霉素的RPMI-1640培养基中培养。所有的实验都是采用第2代和第5代之间的同一批细胞系。
2)待测样品的配制
测试样品为上述实施例中获得的纯化合物准确称取适量样品,用二甲基亚砜(DMSO)配制成所需浓度的溶液,供活性测试。阳性对照顺铂配制方法同上。
3)肿瘤细胞生长抑制活性测试(MTT法):
本发明采用四氮唑盐染色法(MTT法)进行肿瘤细胞的生长抑制活性测试,以评价化合物的作用效果。
该测试方法的原理为:四氮唑盐MTT是一种能接受H+的黄色染料。在活细胞的线粒体中,外源性MTT能在琥珀酸脱氢酶和细胞色素C的作用下还原为甲臜(Formazan),一种不溶于水的蓝紫色结晶,其结晶后会沉积在细胞中,而在死细胞中是没有这种琥珀酸脱氢酶的,因此不能形成甲臜结晶。而且,在一定细胞数量范围内,甲臜结晶的生成量与活细胞的数目成正比,二甲亚砜(DMSO)能够溶解这些沉积在细胞中的甲臜,且在570纳米处有最大吸收峰,因此,使用酶标仪在570纳米波长处检测其吸光值,就可间接反映活细胞的数目,从而来评价药物对细胞增殖的影响。
测试流程:取对数生长期的肿瘤细胞,将细胞密度调至2×105个细胞/毫升,按每孔200微升接种于96孔细胞培养板中,于37℃通入5%二氧化碳的培养箱中培养4小时。样品分别设定5个浓度梯度10-8,10-7,10-6,10-5和10-4mol/L,每个浓度设三个平行样,每孔加样品液或空白液各2微升,培养48小时,然后每孔加浓度为5毫克/毫升的MTT液10微升,继续培养4小时,除去上清液。每孔加入DMSO各100微升,在微量震荡器上震荡10分钟,至结晶完全溶解后,酶标仪测定每孔570纳米处的吸光值(OD值)。
抑制率(IR%)计算:OD值取三孔平均,公式:IR%=(OD空白对照-OD样品)/OD空白对照×100%,计算所得值即为样品对细胞增殖的抑制率(IR%)。
IC50值:抑制率(IR%)为50%时对应的样品溶液浓度。
实验结果见表2。
表2.实施例获得化合物的抗肿瘤活性(IC50,μM)
实验结果表明该化合物对肝癌细胞株(HepG-2)、结肠癌细胞株(HCT-116)、乳腺癌细胞株(MCF-7)具有较强的抑制活性,IC50值均小于10μM,优于阳性对照顺铂的活性。
上述实验结果证明本发明所涉及的化合物对所试肝癌细胞株(HepG-2)、结肠癌细胞株(HCT-116)、乳腺癌细胞株(MCF-7)具有很强的生长抑制作用,其效果优于阳性对照顺铂的活性,可制备为抗肿瘤制剂或先导化合物,且有望以任何药学上可接受的盐或药学上可接受的辅料包括载体如赋形剂、稀释剂等的形式在相关药物的开发中应用。
Claims (2)
1.一种细胞松弛素类化合物的制备方法,其特征在于,包括如下步骤:
1)将糙壁弯孢菌Curvularia verruculosa经发酵培养,然后将发酵液用乙酸乙酯萃取3-4次,合并提取液进行浓缩,获得发酵液粗提物;
2)将步骤1)中的发酵液粗提物进行减压硅胶柱层析,依次采用梯度体积比为50:1至1:1的石油醚-乙酸乙酯和梯度体积比为20:1至1:1的二氯甲烷-甲醇进行洗脱;
3)将步骤2)中以二氯甲烷-甲醇体积为10:1梯度洗脱下的组分以体积为10:90至100:0的甲醇-水作为洗脱液进行反相柱层析,收集体积比60:40的甲醇-水洗脱得到的组分,再用制备型薄层层析纯化,所采用的展开体系为体积比20:1的二氯甲烷-甲醇,然后经过凝胶柱层析,制得式I所示细胞松弛素类化合物;
式I。
2.按权利要求1所述的细胞松弛素类化合物的制备方法,其特征在于:
1)将所述糙壁弯孢菌Curvularia verruculosa在PDA培养基上于28℃下培养7天,取其菌丝体接种于固体培养基中,室温下静置发酵30天,发酵产物经石油醚萃取脱除小极性产物后,采用乙酸乙酯充分浸泡反复萃取,合并萃取液进行浓缩,获得粗提物;
2)将上述粗提物进行减压硅胶柱层析,依次使用梯度分别为体积比50:1至1:1的石油醚-乙酸乙酯和体积比20:1至1:1的二氯甲烷-甲醇洗脱体系进行梯度洗脱;
3)收集上述步骤2)中的体积比10:1的二氯甲烷-甲醇洗脱的组分浓缩,进行反相硅胶柱层析,使用梯度体积比为10:90至100:0的甲醇-水洗脱体系进行梯度洗脱;
4)收集上述步骤3)中的体积比60:40甲醇-水的组分,进行制备型薄层层析纯化,然后采用Sephadex LH-20甲醇凝胶柱层析进行纯化,得到式I所示细胞松弛素类化合物。
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