CN112961170B - 一株海绵来源放线菌及所产含硫生物碱的制备方法和应用 - Google Patents
一株海绵来源放线菌及所产含硫生物碱的制备方法和应用 Download PDFInfo
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- C07D513/12—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for in groups C07D463/00, C07D477/00 or C07D499/00 - C07D507/00 in which the condensed system contains three hetero rings
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Abstract
Description
技术领域
本发明属于微生物药物技术领域,具体涉及一株海绵来源的拟诺卡氏放线菌及所产的两个含硫生物碱dassonmycins A(1)和B(2)的制备方法和应用。
背景技术
海绵(Marine sponge)是地球上最原始的多细胞动物,海绵细胞的主要成分是碳酸钙或碳酸硅以及大量的胶原质。海绵虽然是动物,但是并不能自己行走,只能附着固定在海底的礁石上,从流过身边的海水中获取食物。布满海绵全身的小孔内长着许多鞭毛和一个筛子状的环状物,可以利用海流和鞭毛的摆动吸纳海水,带进氧气、细菌、微小藻类和其它有机碎屑,再经环状物过滤,最后变为海绵的养料。海绵具有独特的空腔结构和滤食性生活方式,使其拥有“微生物资源库”的美誉。近年来,越来越多的研究表明从海绵共附生微生物发酵产物中分离得到多种生物活性物质,可以用来制药,***、心血管和呼吸***等疾病。
革兰氏阳性菌泛指革兰氏染色反应呈紫色的细菌,大多数化脓性球菌都属于革兰氏阳性菌,能产生外毒素使人致病,以金黄色葡萄球菌为代表。临床常见的革兰氏阳性病原菌为金黄色葡萄球菌(Staphylococcus aureus)、耐甲氧西林金黄色葡萄球菌(methicillin-resistant Staphylococcus aureus)。粪肠球菌(Enterococcus faecalis)是一种肠道共生菌,而部分粪肠球菌可引起广泛的感染。由于抗生素的长期和大量不规范使用,粪肠球菌获得性耐药性不断上升,治疗粪肠球菌感染日益困难。藤黄微球菌(Micrococcus luteus)为条件致病菌,引起伤口等局部组织感染,也能引起严重感染,如心内膜炎等疾病。在水产养殖方面,革兰氏阴性菌溶藻弧菌(Vibrio alginolyticus)是海水养殖动物的常见致病菌,养殖的贝类、虾类等有壳动物比养殖鱼类更易感染该菌。溶藻弧菌是引起对虾黑鳃、褐斑综合症的病原菌。该菌同时还是文蛤、合浦珠母贝体内的的病原菌,还可引起尖吻鲈、赤点石斑鱼、真鲷等鲷科鱼类的死亡。
发明内容
本发明的第一个目的是提供两个具有抗菌和抗肿瘤活性的含硫生物碱dassonmycins A(1)和B(2)。
本发明的含硫生物碱dassonmycins A(1)和B(2),其结构式如下所示:
两个含硫生物碱是指dassonmycins A(1)和B(2),Dassonmycin A的分子式为C17H16N2O4S(分子量为345.0904),dassonmycin B的分子式为C17H14N2O4S(分子量为343.0747)。化合物1和2是从自然界中首次发现的具有萘醌[2,3-e]哌嗪[1,2-c]硫代吗啉(naphthoquinone[2,3-e]piperazine[1,2-c]thiomorpholine)骨架化合物。
本发明的第二个目的是提供一种生产上述含硫生物碱的拟诺卡氏放线菌Nocardiopsis dassonvillei SCSIO 40065,其保藏编号为GDMCC No.61460。
本发明的第三个目的是提供一种利用上述放线菌制备含硫生物碱dassonmycinsA(1)和B(2)的方法。
优选,具体步骤如下:
制备Nocardiopsis dassonvillei SCSIO 40065的发酵培养物,分离出上清液和菌丝体,上清液用树脂吸附,用丙酮洗脱浓缩后得到上清液提取液;菌丝体经甲醇浸提,浸提液经浓缩去除甲醇后得到菌丝体提取液,合并上清液提取液和菌丝体提取液用丁酮再萃取,萃取物浓缩后得到浸膏;
浸膏经中压反相柱层析梯度洗脱,从体积比水:乙睛=100:0~0:100梯度洗脱,收集馏份F14和F15;流份F14经纯化得到化合物1;流份F15经纯化得到化合物2。
优选,所述的浸膏经中压反相柱层析梯度洗脱,从体积比水:乙睛=100:0~0:100梯度洗脱,收集馏份F14和F15是经中压反相柱层析梯度洗脱,从体积比水:乙睛=100:0~0:100梯度洗脱,收集体积分数60%乙睛水洗脱而得的流份F14以及体积分数65%乙睛-水洗脱而得的流份F15。
优选,所述的流份F14经纯化得到化合物1、流份F15经纯化得到化合物2是利用高效液相色谱仪进行纯化。
优选,所述的制备Nocardiopsis dassonvillei SCSIO 40065的发酵培养物是以N4液体培养基为发酵培养基进行发酵培养获得发酵培养物。
本发明的第四个目的是提供含硫生物碱dassonmycins A(1)和B(2)在制备抗菌或抗肿瘤药物方面的应用。
所述的抗菌药物是指具有抑制藤黄微球菌、枯草芽孢菌、金黄色葡萄球菌、耐甲氧西林金黄色葡萄球菌、粪场球菌或溶藻弧菌的药物。
所述的抗肿瘤药物是抗人神经癌、人乳腺癌、人肝癌或人非小细胞肺癌的药物。
本发明从Nocardiopsis dassonvillei SCSIO 40065分离出含硫生物碱类化合物1和化合物2,生物活性筛选发现两个化合物都具有抗菌、抗肿瘤细胞株活性,因此可以将它们用于抗菌、抗肿瘤药物中的应用。
Nocardiopsis dassonvillei SCSIO 40065于2021年1月21日保藏于广东省微生物菌种保藏中心(GDMCC),地址:广州市先烈中路100号大院59号楼5楼,邮编:510070,保藏编号:GDMCC No:61460。
附图说明:
图1是Nocardiopsis dassonvillei SCSIO 40065的菌株形态图;
图2是Nocardiopsis dassonvillei SCSIO 40065***发育树;
图3是化合物1(Dassonmycin A)核磁共振氢谱图(氘代DMSO,700兆赫兹);
图4是化合物1(Dassonmycin A)核磁共振碳谱(氘代DMSO,175兆赫兹);
图5是化合物1(Dassonmycin A)HR-ESI-MS分析图(正离子模式,[M+H]+);
图6是化合物1(Dassonmycin A)X-射线单晶衍射图;
图7是化合物2(Dassonmycin B)核磁共振氢谱图(氘代DMSO,700兆赫兹);
图8是化合物2(Dassonmycin B)核磁共振碳谱(氘代DMSO,175兆赫兹);
图9是化合物2(Dassonmycin B)HR-ESI-MS分析图(正离子模式,[M+H]+);
图10是化合物2(Dassonmycin B)X-射线单晶衍射图。
具体实施方式:
以下实施例是对本发明的进一步说明,而不是对本发明的限制。
实施例1:
1、发明人从南海永兴岛附近浅海底(-20米)采集到Petrosia sp.海绵样品,经乙醇表面消毒后使用匀浆机研碎,取部分研碎后的样品采用印迹法接种于无菌的ISP2琼脂平板(每1升培养基中含有酵母提取粉4.0g,葡萄糖4.0g,麦芽提取粉5.0g,复合维生素500微升,海盐30.0g,琼脂粉20.0g,pH 7.0,115℃灭菌30分钟);另取部分研碎后的样品用无菌水洗涤,取上清液在无菌的ISP2琼脂平板上进行稀释涂布。之后将ISP2琼脂平板置于28摄氏度培养箱进行培养。从平板上长出的菌落中挑取具有放线菌形态的菌株作进一步的分离纯化,从而获得了拟诺卡氏放线菌Nocardiopsis dassonvillei SCSIO 40065(图1)。提取该菌株的基因组DNA,通过聚合酶链反应(PCR)扩增获得16S rDNA序列,经序列比对并建立***发育树。结果显示:该菌与Nocardiopsis dassonvillei HZNU N1的相似度达到100%(图2),说明菌株SCSIO 40065为放线菌拟诺卡氏菌属,命名为拟诺卡氏菌Nocardiopsisdassonvillei SCSIO 40065,其于2021年1月21日保藏于广东省微生物菌种保藏中心(GDMCC),地址:广州市先烈中路100号大院59号楼5楼,邮编:510070,保藏编号:GDMCC No:61460。
2、将拟诺卡氏菌Nocardiopsis dassonvillei SCSIO 40065从保藏管接种于ISP2琼脂平板活化,之后挑取孢子接种到250毫升锥形瓶中,每瓶含有50毫升N4液体培养基(每1升培养基中含有可溶性淀粉15克,鱼蛋白胨8克,细菌学蛋白胨5克,甘油7.5克,碳酸钙2g,溴化钾0.2g,海盐30g,余量为水,将各成分混合均匀,调pH 7.0,115℃灭菌30分钟),共30瓶(1.5升)。置于28摄氏度摇床上,以200转/分钟振荡培养2天后获得发酵种子。将25毫升发酵种子液倒入1升锥形瓶中,每瓶含有250毫升N4液体培养基(每1升培养基中含有可溶性淀粉15克,鱼蛋白胨8克,细菌学蛋白胨5克,甘油7.5克,碳酸钙2g,溴化钾0.2g,海盐30g,余量为水,将各成分混合均匀,调pH 7.0,115℃灭菌30分钟),共64瓶(16升)。置于28摄氏度摇床上,以200转/分钟振荡培养7天后获得发酵培养物。以4000转/分钟的转速离心分离发酵培养物中的上清液和菌丝体。上清液用大孔树脂XAD-16吸附,丙酮冲洗得到萃取液,减压浓缩回收有机溶剂得到上清液提取液;菌丝体部分用甲醇浸提四次,减压浓缩回收有机溶剂得到菌丝体提取液。合并上清液提提液+菌丝体提提液,再用等体积的丁酮萃取四次,萃取物减压浓缩后得到提取物12.69克。
Nocardiopsis dassonvillei SCSIO 40065粗提物(12.69g)经中压反相柱层析梯度洗脱(从体积比水:乙睛=100:0~0:100梯度洗脱),收集体积分数60%乙睛水洗脱而得的流份F14以及体积分数65%乙睛-水洗脱而得的流份F15。
流份F14进一步通过半制备HPLC法(乙腈B相-水A相;梯度洗脱(洗脱程序为:0-26min,A:95%-0%,B:5%-100%;26-30min,A:0%;B:100%;30-32min,A:95%;B:5%,流速为2.5ml/min),Kinetex C-18柱,250×10mm,流速2.5ml/mim)获得了化合物1(20毫克,保留时间为16分钟)。此外,化合物1在室温下放置后于甲醇溶液中析出红色晶体。
流份F15进一步通过半制备HPLC法(乙腈B相-水A相;等度洗脱(洗脱程序为:A:65%,B:35%,流速为2.5ml/min),Kinetex C-18柱,250×10mm,流速2.5ml/mim)获得了化合物2(13.8mg毫克,保留时间为6分钟)。此外,化合物2在室温下放置后于甲醇溶液中析出红色晶体。
化合物1的核磁共振氢谱图(氘代DMSO,700兆赫兹)如图3所示,核磁共振碳谱(氘代DMSO,175兆赫兹)如图4所示,HR-ESI-MS分析图(正离子模式,[M+H]+)如图5所示。化合物1(Dassonmycin A的X-射线单晶衍射图如图6所示。化合物1:红色晶体,1H-NMR、13C-NMR数据和晶体结构比对一致,高分辨率质谱(LR-ESI-MS)给出准分子离子峰m/z 345.0904([M+H]+),该数据与Dassonmycin A的分子式C17H16N2O4S相一致。因此确定为Dassonmycin A。
化合物2的核磁共振氢谱图(氘代DMSO,700兆赫兹)如图7所示,核磁共振碳谱(氘代DMSO,175兆赫兹)如图8所示,HR-ESI-MS分析图(正离子模式,[M+H]+)如图9所示。化合物2(Dassonmycin B的X-射线单晶衍射图如图10所示。化合物2:红色晶体,1H-NMR、13C-NMR数据和晶体结构比对一致,LR-ESIMS给出准分子离子峰m/z 343.0747([M+H]+),该数据与Dassonmycin B的分子式C17H14N2O4S相一致。因此确定为Dassonmycin B。
表1.化合物1和2的NMR数据
鉴定化合物1为Dassonmycin A,其结构式如式I中的1所示;
鉴定化合物2为Dassonmycin B,其结构式如式I中的2所示;
实施例2:化合物1和2的抗菌活性的测定
采用微量培养基稀释法测定了化合物1和2对6种指示菌藤黄微球菌(Micrococcusluteus SCSIO ML01)、枯草芽孢杆菌(Bacillus subtilis 1064)、金黄色葡萄球菌(Staphylococcus aureus ATCC 29213)、耐甲氧西林金黄色葡萄球菌(methicillin-resistant Staphylococcus aureus shhsA1)、粪肠球菌(Enterococcus faecalis ATCC29212)、溶藻弧菌(Vibrio alginolyticus)的抑制活性。6种指示菌经摇床37℃,200rmp培养16h,用无菌培养基稀释到OD值(600nm)为0.04-0.06,再稀释10倍加入到96孔板中;加入样品后,等倍稀释到终浓度64-0.125μg mL-1,每个浓度3个平行;37℃培养18h,用酶标仪测定各孔的吸收值,确定各化合物的最小抑菌浓度(MIC)。以三甲氧基苄啶(TMP)为阳性对照,其结果见表2。
结果如表2所示:
表2.化合物1和2对六种指示菌的最小抑菌浓度测试结果(单位:μg/ml)
从表2可以发现化合物1和化合物2对以上六种指示菌均有抑制作用,其中化合物1对枯草芽孢杆菌的抑制活性比较强(8μg mL-1)。
实施例3:化合物1和2抗肿瘤活性的测定
采用SRB法测定了化合物1和2对4种肿瘤细胞株SF-268,HepG2,MCF-7和A549的抑制活性。4种肿瘤细胞株采用RPMI培养基培养,将180μL培养物(浓度为3×104个细胞每mL)加入到96孔板中,37℃,5%CO2培养18h;将20μL的待测样品(终浓度为1、10和100μM,溶剂为DMSO)加入到96孔板相应的孔中,用DMSO作为阴性对照,每个浓度做3个平行,继续培养72小时;加入50%的三氯乙酸50μL混合,再加入0.4%的SRB(溶解在1%的乙酸中)放置30分钟;去除上清,将与染料结合的蛋白溶解200μL10 mM的Tris缓冲液中,用酶标仪测定各孔的OD值(570nm),采用SigmaPlot 14.0软件中非线性曲线拟合(non-linear curve-fitting)的方法计算相应的IC50;以阿霉素(Adriamycin)作为阳性对照。其结果见表3,结果表明,Dassonmycin A(1)及Dassonmycin B(2)对4种指示细胞株的IC50范围为11.94到34.39μM。
表3.化合物1和2对四种肿瘤细胞株的IC50抑制浓度测试结果(单位:μM)
从表3可以发现化合物1和化合物2对以上四种肿瘤细胞株均有抑制作用,其中化合物2对人神经癌细胞株SF-268具有较强的肿瘤细胞株抑制活性(IC50=11.94μM)。
以上仅是本发明的优选实施方式,应当指出的是,上述优选实施方式不应视为对本发明的限制,本发明的保护范围应当以权利要求所限定的范围为准。对于本技术领域的普通技术人员来说,在不脱离本发明的精神和范围内,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (11)
2.权利要求1所述的含硫生物碱或其药用盐在制备抗菌或抗肿瘤药物中的应用。
3.根据权利要求2所述的应用,其特征在于,所述的抗菌药物是指具有抑制藤黄微球菌、枯草芽孢菌、金黄色葡萄球菌、粪肠球菌或溶藻弧菌的药物。
4.根据权利要求3所述的应用,其特征在于,所述的金黄色葡萄球菌是耐甲氧西林金黄色葡萄球菌。
5.根据权利要求2所述的应用,其特征在于,所述的抗肿瘤药物是抗人神经癌、人乳腺癌、人肝癌或人非小细胞肺癌的药物。
6.一种权利要求1中所述的含硫生物碱的制备方法,其特征在于,是从菌株Nocardiopsis dassonvillei SCSIO 40065的发酵培养物中制备分离得到的。
7.根据权利要求6所述的制备方法,其特征在于,具体步骤如下:
制备Nocardiopsis dassonvillei SCSIO 40065的发酵培养物,分离出上清液和菌丝体,上清液用树脂吸附,用丙酮洗脱后经浓缩得到上清液提取液;菌丝体经甲醇浸提,浸提液经浓缩去除甲醇后得到菌丝体提取液,合并上清液提取液和菌丝体提取液,再用丁酮萃取,萃取物浓缩后得到浸膏;
浸膏经中压反相柱层析梯度洗脱,从体积比水:乙睛=100:0~0:100梯度洗脱,收集流份F14和F15;流份F14经纯化得到化合物1;流份F15经纯化得到化合物2。
8.根据权利要求7所述的制备方法,其特征在于,所述的浸膏经中压反相柱层析梯度洗脱,从体积比水:乙睛=100:0~0:100梯度洗脱,收集流 份F14和F15是经中压反相柱层析梯度洗脱,从体积比水:乙睛=100:0~0:100梯度洗脱,收集体积分数60%乙睛水洗脱而得的流份F14以及体积分数65%乙睛-水洗脱而得的流份F15。
9.根据权利要求7所述的制备方法,其特征在于,所述的流份F14经纯化得到化合物1、流份F15经纯化得到化合物2是利用高效液相色谱仪进行纯化。
10.根据权利要求7所述的制备方法,其特征在于,所述的制备Nocardiopsisdassonvillei SCSIO 40065的发酵培养物是以N4液体培养基为发酵培养基进行发酵培养获得发酵培养物。
11.Nocardiopsis dassonvillei SCSIO 40065,其保藏编号为GDMCC No.61460。
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