CN107417559A - 一种倍半萜类化合物及其制备方法和应用 - Google Patents
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Abstract
本发明公开了一种倍半萜类化合物及其制备方法和应用,所述的化合物为式I所示的结构。本发明提供的化合物可用于治疗与急、慢性白血病、淋巴癌、乳腺癌、肺癌、PDGF依赖性肿瘤等癌症,艾滋病,冠心病、糖尿病、老年痴呆症、畸形性骨炎、脑缺血等疾病的药物以及抗白色念珠菌等真菌类药物。本发明抗肿瘤、抗真菌活性的化合物的制备方法,由海洋放线菌大米固体培养基发酵产生,经乙酸乙酯萃取后所得的发酵物,采用凝胶柱色谱和高效液相色谱分离纯化得到,易于操作和实施。
Description
技术领域
本发明涉及海洋放线菌培养制备化合物领域,具体涉及一种具有抗肿瘤、抗真菌活性的倍半萜类化合物及其制备方法和应用。
背景技术
海洋面积约占地球表面积的71%,海洋具有高盐、高压、低温等特殊的环境,促使了海洋微生物形成了独特的代谢途径,决定了海洋微生物种类及其次级代谢产物的化学结构具有极大的复杂性、多样性和高生物活性。海洋放线菌是迄今最经济和最具生物技术价值的原核生物。它们几乎负责了约一半具有生物活性的次级代谢产物的生产,尤其是抗生素、抗肿瘤剂、和酶免疫抑制剂等,主要的代谢产物是生碱类、大环内脂类、肽类、氨基糖苷类、醚类、酮类、萜类、酯类、醌类等,并且具有广泛的生物活性。
海洋放线菌产生的抗生素,不仅结构新颖而且活性代谢产物也是最多的,其中90%以上的海洋放线菌活性产物来自于链霉菌属,仅有少量的来自于放线菌的其他菌属。自1998年以来,从海洋放线菌中发现的新活性化合物数量连年超过了陆地放线菌来源的化合物。其中最为引人注目的加州大学克利普斯海洋研究所Jensen等从海洋盐胞菌属放线菌Salinispora tropica CNB-392中分离得到的β-内酯环-γ-内酰胺类化合物Salinosporamide A(NPI-0052),作为癌症治疗药物已完成非小细胞肺癌、胰腺癌、黑素瘤和淋巴瘤Ⅰ期临床研究,链霉菌N-125来源的吲哚咔唑类生物碱UCN-01已完成淋巴、非小细胞肺癌的Ⅱ期临床研究,并取得良好效果。放线菌素D目前已成药治疗霍奇金病(HD)及神经母细胞瘤且效果显著。
因此,从海洋放线菌中获得具有抗肿瘤活性的化合物具有很大的开发前景,尤其是链霉菌属,并且培养条件简单,易于大规模培养,可作为天然的抗肿瘤活性产物主要来源进行挖掘研究。
发明内容
本发明提供了一种具有抗肿瘤、抗真菌活性的倍半萜类化合物及其制备方法和应用,该式I结构的化合物是微生物代谢产物中结构特征新颖的化合物,本发明采用大米固体培养基培养发酵海洋放线菌,最终由发酵产物分离纯化获得式I结构的化合物。
一种倍半萜类化合物,为式I结构:
所述的倍半萜类化合物的制备方法,由海洋放线菌固体发酵产生,经分离纯化得到,易于操作和实施。
一种倍半萜类化合物的制备方法,包括以下步骤:
1)将海洋放线菌接种于高氏一号液体培养基中,摇床培养,获得种子液;
2)将上述所得的种子液接种于大米固体培养基中,静置培养,萃取获得发酵产物。
3)将上述所得的发酵产物进行分离纯化后,获得式I结构的倍半萜类化合物。
步骤1)中,所述的海洋放线菌,具体可采用市售产品,如采用中国工业微生物菌种保藏管理中心出售的链霉菌Streptomyces sp.CICC 11027,订购网址:http://www.china-cicc.org/。
所述的摇床培养的条件为:在26℃~30℃、130rpm~230rpm的摇床中培养2~4天,进一步优选,在28℃、180rpm的摇床中培养3天。
步骤2)中,所述的大米固体培养基,由大米和海水组成,所述的大米的质量和海水的体积之比为30g~50g:50mL~70mL,进一步优选,所述的大米的质量和海水的体积之比为40g:60mL,即按大米质量40g和海水体积60mL配比后经高压湿热灭菌所得。
所述的静置培养的条件为:在23℃~33℃下静置培养100~140天,进一步优选,在28℃下静置培养120天。
步骤3)中,所述的分离纯化包括:由乙酸乙酯等体积浸泡萃取获得的发酵产物,经凝胶柱色谱、制备液相色谱获得式I结构的倍半萜类化合物。
所述的凝胶柱色谱的条件:采用的填料为羟丙基葡聚糖凝胶(LH-20),采用的洗脱剂为甲醇-水溶液,进一步优选,采用的洗脱剂为甲醇体积百分数20%-100%的甲醇-水溶液。
所述的制备液相色谱的条件:采用的填料为十八烷基硅烷键合硅胶,采用的流动相是甲醇-水的溶液,进一步优选,采用的流动相是甲醇体积百分数40%-100%甲醇-水的溶液,甲醇体积百分数90%甲醇-水的溶液。
本发明采用人前列腺癌细胞株PC3进行活性评价试验,本发明提供的化合物式I能很好抑制PC3细胞的生长,充分说明该类化合物具有癌细胞杀灭作用,因此可制备获得作为抗肿瘤药物。同时采用白色念珠菌进行最小抑菌浓度抗真菌活性评价实验,本发明中式I结构的化合物具有很好的抗真菌活性,因此可制备作为抗真菌类的药物。所述的倍半萜类化合物具有抗肿瘤、抗真菌活性。本发明提供的化合物可用于治疗与急、慢性白血病、淋巴癌、乳腺癌、肺癌、PDGF依赖性肿瘤等癌症,艾滋病,冠心病、糖尿病、老年痴呆症、畸形性骨炎、脑缺血等疾病的药物以及抗白色念珠菌等真菌类药物。所述的式I结构的倍半萜类化合物克用于制备抗肿瘤药物和抗真菌药物,在用于治疗与激酶有关的癌症具有很好的应用前景。
与现有技术相比,本发明具有如下优点:
本发明中具有抗肿瘤、抗真菌活性的倍半萜类化合物,可用于开发治疗非小细胞肺癌、胃癌、前列腺癌、PDGF依赖性肿瘤等癌症药物以及畸形性骨炎、脑缺血等疾病药物和抗白色念珠菌等真菌药物;所述的式I结构的化合物可用于研究抗肿瘤药物的作用靶点筛选,也可作为农药适用研究;本发明实验操作简单,易于扩大生产,具有较好的应用前景。
附图说明
图1为式Ⅰ具有抗肿瘤、抗真菌活性的化合物A68-15R的1H-NMR图谱;
图2为式Ⅰ具有抗肿瘤、抗真菌活性的化合物A68-15R的13C-NMR图谱;
图3为式Ⅰ具有抗肿瘤、抗真菌活性的化合物A68-15R的HSQC图谱;
图4为式Ⅰ具有抗肿瘤、抗真菌活性的化合物A68-15R的HMBC图谱;
图5为式Ⅰ具有抗肿瘤、抗真菌活性的化合物A68-15R的NOESY图谱;
图6为式Ⅰ具有抗肿瘤、抗真菌活性的化合物A68-15R的1H-1H COSY图谱。
具体实施方式
实施例1
一、化合物的发酵
海洋放线菌采用中国普通微生物菌种保藏管理中心出售的链霉菌Streptomycessp.CICC 11027;
1)将海洋放线菌接种于500mL锥形瓶中,每瓶含250mL高氏一号液体培养基,培养条件为28℃、180rpm的摇床中培养3天,获得可用于发酵培养的种子液;
2)将步骤1)所得的种子液接种至大米培养基(大米培养基,由以下组分制成:大米质量40g;海水60mL,置于500ml锥形瓶后经高压湿热灭菌所得),接种体积为12mL,在28℃下静置培养120天,得到含有本发明具有抗肿瘤、抗真菌活性的倍半萜类化合物的固体发酵产物。
二、化合物的制备
将含有本发明具有抗肿瘤、抗真菌活性的倍半萜类化合物的固体发酵产物用乙酸乙酯浸泡萃取3次,溶剂回收浓缩,获得粗体物。将所得粗体物进行凝胶柱色谱(填料为羟丙基葡聚糖凝胶LH-20),洗脱剂为体积百分数20%-100%的甲醇-水体系梯度洗脱,每1/4柱体积为一个馏分,TLC分析合并含有目标化合物的馏分。对目标组分采用中压制备色谱分离(Sepax Amethyst C-18(10μm,30×400mm)色谱柱,检测波长292nm,填料为十八烷基硅烷键合硅胶),流动相为体积百分数40%-100%的甲醇-水溶液梯度洗脱,TLC分析合并含有新化合物的馏分,得到含有新化合物的组分。
所得含有新化合物的组分采用高效制备液相色谱分离(Agilent Pursuit C-18(10μm,21.2×250mm)色谱柱,检测波长292nm,填料为十八烷基硅烷键合硅胶),采用的流动相为甲醇体积百分数90%甲醇/水体系以10mL/min等度洗脱,收集23-26min的色谱峰,回收溶剂,得到化合物A68-15R。如图1至6所示,依据核磁共振数据,其结构如下所示,为式I结构。分子式根据高分辨质谱HR-ESI-MS计算为C22H29NNaO3([M+Na]+378.2135,calculated378.2040),鉴定化合物eudesm-4(15),7-diene-9α-hydroxy-11-anthranilamide,简称为A68-15R,具体结构如下:
化合物的核磁鉴定(1H 500MHz,13C 125.7MHz),其结果如表1所示。
表1
三、抗肿瘤活性实验
采用磺酰罗丹明B(Sulforhodamine B,SRB)比色法检测化合物对人前列腺癌细胞株PC3细胞的增殖抑制作用。取对数生长期的细胞,配置成5×104个/mL,以100μl/孔铺于96孔培养板,CO2培养箱中培养24小时,取出培养板后于每孔中加入不同浓度的待测样品,每个浓度设3个复孔,加药完成后,置于CO2培养箱中继续培养72小时后取出培养板,弃去培养液,每孔加入100μl 4℃冰箱预冷的质量百分数10%的三氯醋酸(TCA)固定,静置5分钟后,再将培养板移至4℃冰箱过夜。倒掉固定液,每孔用去离子水洗涤5遍,甩干,空气干燥。每孔加入70μl SRB溶液,室温25℃放置20分钟,去上清液,用质量百分数1%醋酸洗涤5次,空气干燥。结合的SRB用100μl/孔10mmol/L Tris碱液(pH=10.5)振荡溶解。置于酶标仪中测定各孔光吸收,测定波长为515nm。根据各孔OD值计算药物对细胞增殖抑制率:抑制率=[1-(OD515给药孔/OD515对照孔)]×100%,根据各浓度抑制率计算半数抑制浓度IC50,其结果如表2所示。
表2
化合物 | IC50(μM) |
A68-15R | 10~30 |
四、抗真菌活性实验
采用96孔板倍半稀释法测定式I结构化合物对白色念珠菌的最小抑菌浓度。取白色念珠菌接种于NB营养肉汤培养基中,28℃恒温培养4小时,采用血球计数板计数真菌密度,用空白培养基稀释为约1×108个/ml,菌悬液待用。取空白NB液体培养基加入待测孔和空白对照孔,每孔100ul,将样品用DMSO配制为100μg/ml初始浓度,加入待测孔依次进行半倍稀释,阳性对照为两性霉素B,按照上述方法进行稀释。最后取白色念珠菌悬液,每孔加入5μl并混合均匀,空白孔内加入等量DMSO。将96孔板置于恒温培养箱内培养12小时,取出后和空白对照进行对比,将肉眼观察到有明显抑制作用的测试孔为最小抑菌浓度,其结果如表3所示。
表3
化合物 | MIC(μg/ml) |
A68-15R | 6.25 |
两性霉素B | 0.78 |
结果表明,本发明式I结构的化合物能很好抑制PC3细胞的生长,充分说明该类化合物具有癌细胞杀灭作用,因此可制备获得作为抗肿瘤药物。同时采用白色念珠菌进行最小抑菌浓度抗真菌活性评价实验,本发明中式I结构的化合物具有很好的抗真菌活性,因此可制备作为抗真菌类的药物。
Claims (10)
1.一种倍半萜类化合物,其特征在于,为式I结构:
2.根据权利要求1所述的倍半萜类化合物的制备方法,其特征在于,包括以下步骤:
1)将海洋放线菌接种于高氏一号液体培养基中,摇床培养,获得种子液;
2)将上述所得的种子液接种于大米固体培养基中,静置培养,萃取获得发酵产物;
3)将上述所得的发酵产物进行分离纯化后,获得式I结构的倍半萜类化合物。
3.根据权利要求2所述的倍半萜类化合物的制备方法,其特征在于,步骤1)中,所述的海洋放线菌采用中国工业微生物菌种保藏管理中心出售的链霉菌Streptomyces sp.CICC11027。
4.根据权利要求2所述的倍半萜类化合物的制备方法,其特征在于,步骤1)中,所述的摇床培养的条件为:在26℃~30℃、130rpm~230rpm的摇床中培养2~4天。
5.根据权利要求2所述的倍半萜类化合物的制备方法,其特征在于,步骤2)中,所述的大米固体培养基,由大米和海水组成,所述的大米的质量和海水的体积之比为30g~50g:50mL~70mL。
6.根据权利要求2所述的倍半萜类化合物的制备方法,其特征在于,步骤2)中,所述的静置培养的条件为:在23℃~33℃下静置培养100~140天。
7.根据权利要求2所述的倍半萜类化合物的制备方法,其特征在于,步骤3)中,所述的分离纯化包括:由乙酸乙酯等体积浸泡萃取获得的发酵产物,经凝胶柱色谱、制备液相色谱获得式I结构的倍半萜类化合物。
8.根据权利要求7所述的倍半萜类化合物的制备方法,其特征在于,步骤3)中,所述的凝胶柱色谱的条件:采用的填料为羟丙基葡聚糖凝胶,采用的洗脱剂为甲醇-水溶液;
所述的制备液相色谱的条件:采用的填料为十八烷基硅烷键合硅胶,采用的流动相是甲醇-水的溶液。
9.根据权利要求1所述的倍半萜类化合物在制备抗肿瘤药物中的应用。
10.根据权利要求1所述的倍半萜类化合物在制备抗真菌药物中的应用。
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