CN116444536A - 一种红树内生真菌中萜类化合物及其制备方法与应用 - Google Patents
一种红树内生真菌中萜类化合物及其制备方法与应用 Download PDFInfo
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Abstract
本发明公开了一种红树内生真菌中萜类化合物及其制备方法与应用,所述萜类化合物通过以下步骤制备:步骤1,配制种子培养基,将Aspergillus sp.菌株接入种子培养基,培养得种子培养液;步骤2,将种子培养液接入发酵培养基中,培养得发酵物;步骤3,将发酵物中的发酵液和菌体分离,发酵液和菌体分别用等体积的乙酸乙酯萃取3~5次,合并萃取液后减压浓缩得到浸膏;步骤4,浸膏经过减压硅胶柱层析,50%乙酸乙酯‑石油醚洗脱部分经Sephadex LH‑20凝胶柱层析,洗脱剂为CHCl3∶MeOH=1∶1,再经高效液相色谱HPLC制备,得到化合物1、化合物2;其中80%乙酸乙酯洗脱部分经Sephadex LH‑20凝胶柱层析,得到上述化合物3、化合物4,四种萜类化合物均具有良好的抗癌活性,可应用于抗癌药物中。
Description
技术领域
本发明涉及微生物技术领域,尤其涉及一种红树内生真菌次级代谢产物,特别是涉及一种红树内生真菌中萜类化合物及其制备方法与应用。
背景技术
海洋药用红树来源的内生真菌是重要的结构新颖活性显著化合物来源之一。近年来,一些结构新颖活性良好的化合物已经从真菌Aspergillus sp.中分离得到,例如:乙酰胆碱酯酶抑制活性化合物millmerranones A-F和terreulactones A-D,具有防污损活性的化合物territrem D,具有抗病毒活性的化合物11a-dehydroxyisoterreulactone A和arisugacin A,具有细胞毒活性的化合物7″-hydroxybutyrolactone III。前期研究发现内生真菌Aspergillus sp.乙酸乙酯粗提物具有一定的抗肿瘤活性,随着恶性肿瘤发病率和死亡率的升高,目前上市的抗肿瘤药物将无法满足病患需求,开发新型抗肿瘤药物已成为药物研发的迫切需求。微生物次级代谢产物能通过发酵工程技术进行工业化生产,原料无后顾之忧,对环境友好等优点,有效解决了从植物中提取抗肿瘤活性物质存在的资源缺乏和环境保护问题。
发明内容
本发明的目的是针对现有技术中存在的技术缺陷,而提供一种红树内生真菌中萜类化合物,所述萜类化合物从红树秋茄内生真菌(Aspergillus sp.)的发酵物中分离得到。
本发明的另一个目的是提供所述萜类化合物的制备方法。
本发明的另一个目的是提供所述萜类化合物的应用。
为实现本发明的目的所采用的技术方案是:
一种红树内生真菌中萜类化合物,所述萜类化合物具有化合物1-4所示的结构:
本发明的另一方面,所述萜类化合物通过以下方法制备:
步骤1,配制种子培养基,将Aspergillus sp.菌株接入种子培养基,培养得种子培养液;
步骤2,将步骤1得到的种子培养液接入发酵培养基中,培养得发酵物;
步骤3,将步骤2得到的发酵物中的发酵液和菌体分离,发酵液和菌体分别用等体积的乙酸乙酯萃取3~5次,合并萃取液后减压浓缩得到浸膏;
步骤4,步骤3得到的浸膏经过减压硅胶柱层析,采用石油醚-乙酸乙酯按100:0至0∶100梯度洗脱,其中50%乙酸乙酯-石油醚洗脱部分经Sephadex LH-20凝胶柱层析,洗脱剂为CHCl3∶MeOH=1∶1,再经高效液相色谱HPLC制备,色谱柱为Agilent C18,9.4×250mm,7μm,流速为2mL/min,流动相为MeOH∶H2O=55∶45最终得到上述化合物1、化合物2;其中80%乙酸乙酯洗脱部分经Sephadex LH-20凝胶柱层析,洗脱剂为CHCl3∶MeOH=1∶3,再经高效液相色谱HPLC制备,色谱柱为Agilent C18,9.4×250mm,7μm,流速为2mL/min,流动相为MeOH∶H2O=35∶65最终得到上述化合物3、化合物4。
在上述技术方案中,所述步骤1中,所述种子培养基中包括葡萄糖1.5wt%–3.0wt%、酵母膏0.1wt%–0.5wt%、蛋白胨0.1wt%–0.5wt%、粗海盐0.11wt%–0.6wt%、余量为水,所述种子培养基配制好后于120~140℃灭25–30分钟。
在上述技术方案中,所述步骤1中,培养温度为26~28℃,培养时间为3~4天。
在上述技术方案中,所述步骤2中,所述发酵培养基中含有葡萄糖1.6wt%–3.5wt%、酵母膏0.1wt%–0.5wt%、蛋白胨0.1wt%–0.5wt%、粗海盐0.11wt%–0.6wt%、余量为水;所述发酵培养基配制好后于120~140℃灭25–30分钟。
在上述技术方案中,所述步骤2中,培养温度为26~28℃,静置培养,培养时间为21~24天。
本发明的另一方面,还包括所述萜类化合物药学上可接受的盐。“药学上可接受的盐”是指非毒性的无机或有机酸和/或碱的加成盐,可参见“Salt selection for basicdrugs”,Int.J.Pharm.(1986),33,201–217。
本发明的另一方面,还包括所述萜类化合物在制备抗肿瘤药物中的应用。
本发明的另一方面,还包括一种抗肿瘤药物,包括所述萜类化合物或者所述萜类化合物药学上可接受的盐以及辅料。
在上述技术方案中,所述辅料为药学上可接受的载体、稀释剂或赋形剂。
在上述技术方案中,所述抗肿瘤药物的剂型为固体制剂、半固体制剂或液体制剂。
与现有技术相比,本发明的有益效果是:
1.本发明的四种萜类化合物均可良好的抑制SW 1990、SW 480、HepG2和MCF-7细胞毒活性,四种萜类化合物均具有良好的抗癌活性,是一种潜在的抗癌药物活性成分。
2.本发明的四种萜类化合物分离提取方法方便快捷,可大范围推广应用,具有良好的市场应用前景。
附图说明
图1是化合物1的1H NMR图
图2是化合物1的13C NMR图
图3是化合物2的1H NMR图
图4是化合物2的13C NMR图
图5是化合物3的1H NMR图
图6是化合物3的13C NMR图
图7是化合物4的1H NMR图
图8是化合物4的13C NMR图
具体实施方式
以下结合具体实施例对本发明作进一步详细说明。应当理解,此处所描述的具体实施例仅仅用以解释本发明,并不用于限定本发明。
实施例1
(1)Aspergillus sp.的菌种培养
配制种子培养基:葡萄糖80g,蛋白胨8g,酵母膏8g,粗海盐12g,水4.0L,平均分装于8个1000mL锥形瓶,120℃灭25–30分钟。
将Genbank登录号MW073438的真菌菌种Aspergillus sp.GXIMD 03004接入配制好的种子培养基中,于26~28℃下,培养3天得种子培养液;
(2)Aspergillus sp.的发酵
配制发酵培养基:葡萄糖1.1kg,蛋白胨100g,酵母膏100g,海盐150g,水50L,平均分装于120个1000mL锥形瓶中,120℃灭25–30分钟。
取适量的步骤(1)中得到的种子培养液接入装有发酵培养基的锥形瓶中,于26~28℃静置培养30天。
(3)化合物1-4的提取分离
取步骤(2)得到的发酵物过滤,分离发酵液和菌体,将滤液浓缩后,分别用等体积的乙酸乙酯萃取浓缩后的滤液和菌体各3次;合并萃取液,浓缩后经过减压硅胶柱层析,采用石油醚-乙酸乙酯按100:0至0:100梯度洗脱,其中50%乙酸乙酯-石油醚洗脱部分经Sephadex LH-20凝胶柱层析,洗脱剂为CHCl3∶MeOH=1∶1,再经高效液相色谱HPLC制备,色谱柱为Agilent C18,9.4×250mm,7μm,流速为2mL/min,流动相为MeOH∶H2O=55∶45最终得到上述化合物1(7.8mg)、化合物2(9.5mg);其中80%乙酸乙酯洗脱部分经Sephadex LH-20凝胶柱层析,洗脱剂为CHCl3∶MeOH=1∶3,再经高效液相色谱HPLC制备,色谱柱为AgilentC18,9.4×250mm,7μm,流速为2mL/min,流动相为MeOH∶H2O=35∶65最终得到上述化合物3(6.8mg)、化合物4(8.5mg)。
化合物1-4的结构式如下所示:
化合物1-4的1H和13C-NMR(400/100MHz,DMSO-d6)数据如下表1。
表1
化合物1结构的确定:根据高分辨质谱数据541.2082[M+H]+,推算出该化合物1的分子式为C29H32O10,不饱和度为14。
1H NMR谱中给出了4个芳香氢信号,包括2个相互耦合的氢信号δH 7.14(d,J=1.6Hz)和7.11(d,J=1.6Hz),2个不耦合的氢信号δH 6.84(s)和5.10(s),提示该化合物存在1个1,3,4,5-四取代苯环系统。除此之外,在连氧区出现1个连氧氢信号δH 6.08(s),2个甲氧基氢信号δH 3.90(s)和3.66(s),3个亚甲基氢信号δH 3.59(m),2.72(m),2.26(m),1.97(m),1.77(m)和1.65(m),以及4个甲基信号δH 1.38(s),1.36(s),1.25(s)和1.14(s)。13C-NMR和DEPT-135谱·共给出了29个碳信号,包括4个甲基,2个甲氧基,4个亚甲基,4个次甲基和15个季碳信号。以上氢谱和碳谱信息,提示该化合物与已知化合物millmerranone D非常相似,不同之处仅在于在化合物1中,多了1个甲氧基信号[δH 3.66(s),δC 55.9]和一个烯氢信号[δH 5.10(s),δC 97.8]。甲氧基和烯氢的连接位置是通过HMBC确定的,在HMBC中,3-OMe与3-C相关,提示甲氧基连接在C-3上;H-2与C-1,C-3,C-4和C-12b相关。化合物1的平面结构进一步通过1H-1H COSY,HMQC和HMBC得以确定,命名为asperlactone A。
化合物2结构的确定:1H NMR和13C-NMR谱与1非常相似,不同之处仅在于化合物1中,多了1个甲氧基信号[δH 3.66(s),δC 55.9]和一个烯氢信号[δH 5.10(s),δC 97.8],在化合物2中多了一个连氧亚甲基信号[δH 3.66(s),δC 55.9],表明化合物2和已知化合物millmerranone D的核磁数据基本一样,因此化合物2的结构鉴定为millmerranone D。
化合物3结构的确定:1H NMR和13C-NMR谱与2非常相似,不同之处仅在于化合物3中,多了一组烯氢信号[δH.04(d,8.0),δC 114.4],在化合物2中多了一个连氧亚甲基信号[δH6.08(s),δC 102.1],此外,化合物3中的甲氧基是连接在C-4'上,化合物3的平面结构进一步通过1H-1H COSY,HMQC和HMBC得以确定,化合物3和已知化合物millmerranone E的核磁数据基本一样,因此化合物3的结构鉴定为millmerranone E。
化合物4结构的确定:对比化合物4和已知化合物terreulactones C的1H NMR和13C-NMR谱,发现化合物4和已知化合物terreulactones C的核磁数据基本一样,因此化合物4的结构鉴定为terreulactones C。
实施例2
(1)Aspergillus sp.的菌种培养
配制种子培养基(5.0L):葡萄糖1.5%(重量百分比,下同)、酵母膏0.5%、蛋白胨0.1%、粗海盐0.11%,其余为水;平均分装于8个1000mL锥形瓶,120~140℃灭25–30分钟。
将Aspergillus sp.菌株接入配制好的种子培养基中,于26~28℃下,培养4天得种子培养液;(2)Aspergillus sp.的发酵
配制发酵培养基(100L):葡萄糖1.6%(重量百分比,下同)、酵母膏0.5%、蛋白胨0.1%、粗海盐0.11%,其余为水;平均分装于150个1000mL锥形瓶,120~140℃灭25–30分钟。
取适量的步骤(1)中得到的种子培养液接入装有发酵培养基的锥形瓶中,于26~28℃静置培养30天。
(3)化合物1-4的提取分离
按照实施例1中类似的分离方法,可得到化合物1-4,结构确证数据与实施例1一致。
实施例3
(1)Aspergillus sp.的菌种培养
配制种子培养基(1.0L):葡萄糖3.0%(重量百分比,下同)、酵母膏0.1%、蛋白胨0.5%、粗海盐0.6%,其余为水;平均分装于3个500mL锥形瓶,120~140℃灭25–30分钟。
将Aspergillus sp.菌株接入配制好的种子培养基中,于26~28℃下,培养4天得种子培养液;(2)Aspergillus sp.的发酵
配制发酵培养基(20L):葡萄糖3.5%(重量百分比,下同)、酵母膏0.1%、蛋白胨0.5%、粗海盐0.6%,其余为水;平均分装于100个1000mL锥形瓶,120~140℃灭25–30分钟。
取适量的步骤(1)中得到的种子培养液接入装有发酵培养基的锥形瓶中,于26~28℃静置培养28天。
(3)化合物1-4的提取分离
按照实施例1中类似的分离方法,可得到化合物1-4,结构确证数据与实施例1一致。
利用相似的培养、发酵以及提取分离方法,Meng Bai,Yue Wang,Ting Liu,Yu-Xing Lian,Qi-Qi Bai,Xiao-Ping Song,Chang-Ri Han,Cai-Juan Zheng,Guang-YingChen.One new piperazinedione isolated from a mangrove-derived fungusAspergillus niger JX-5,Natural Product Research,2022.36:2277-2283,该论文中公开的Aspergillus niger JX-5也可分离出化合物1-4。
实施例4本发明化合物1-4的抗肿瘤活性的测定
试验方法:取化合物1-4对4种肿瘤细胞进行活性测试,分别为SW 1990,SW 480,HepG2,和MCF-7。
具体方法如下:将对数生长期的肿瘤细胞经常规消化、悬浮后,以1×104个/孔接种于96孔板,CO2孵箱中培养24h后,加入不同浓度样品溶液使其终浓度为100,50,25,12.5,6.25μg/mL,每个浓度设4个复孔。另外设空白对照孔3个。分别培养24,48,72h后,每孔加入5mg/mL的MTT,37℃继续孵育培养4h。观察沉淀析出,吸弃上清液,加入DMSO充分振荡溶解结晶物甲瓚。在酶标仪上用波长为570nm,参比波长为630nm测定去除本底光吸收值后的吸光度值(OD),计算细胞增殖抑制率。抑制率=(1–样品OD值/对照组OD值)×100%。实验重复3次求平均值。顺铂为阳性对照药。
实验结果表明,在测试浓度范围内,化合物1-4对上述肿瘤细胞SW 1990,SW 480,HepG2,和MCF-7均显示出一定的细胞毒活性,尤其是在20μg/mL浓度时,化合物1-4对胰腺癌细胞SW 1990的抑制率分别为89.5%、71.6%、67.8%和69.9%(表2)。即化合物1对肿瘤细胞SW 1990的细胞毒活性最强。
表2化合物1-4在20μg/mL对四种肿瘤抑制活性
以上所述仅是本发明的优选实施方式,应当指出的是,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,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 (10)
1.一种红树内生真菌中萜类化合物,其特征在于,所述萜类化合物具有化合物1-4所示的结构:
2.一种红树内生真菌中萜类化合物的制备方法,其特征在于,包括以下步骤:
步骤1,配制种子培养基,将Aspergillus sp.菌株接入种子培养基,培养得种子培养液;
步骤2,将步骤1得到的种子培养液接入发酵培养基中,培养得发酵物;
步骤3,将步骤2得到的发酵物中的发酵液和菌体分离,发酵液和菌体分别用等体积的乙酸乙酯萃取3~5次,合并萃取液后减压浓缩得到浸膏;
步骤4,步骤3得到的浸膏经过减压硅胶柱层析,采用石油醚-乙酸乙酯按100:0至0∶100梯度洗脱,其中50%乙酸乙酯-石油醚洗脱部分经Sephadex LH-20凝胶柱层析,洗脱剂为CHCl3∶MeOH=1∶1,再经高效液相色谱HPLC制备,色谱柱为Agilent C18,9.4×250mm,7μm,流速为2mL/min,流动相为MeOH∶H2O=55∶45最终得到上述化合物1、化合物2;其中80%乙酸乙酯洗脱部分经Sephadex LH-20凝胶柱层析,洗脱剂为CHCl3∶MeOH=1∶3,再经高效液相色谱HPLC制备,色谱柱为Agilent C18,9.4×250mm,7μm,流速为2mL/min,流动相为MeOH∶H2O=35∶65最终得到上述化合物3、化合物4;
化合物1-4所示的结构如下所示:
3.如权利要求2所述的制备方法,其特征在于,所述步骤1中,所述种子培养基中包括葡萄糖1.5wt%–3.0wt%、酵母膏0.1wt%–0.5wt%、蛋白胨0.1wt%–0.5wt%、粗海盐0.11wt%–0.6wt%、余量为水,所述种子培养基配制好后于120~140℃灭25–30分钟。
4.如权利要求2所述的制备方法,其特征在于,所述步骤1中,培养温度为26~28℃,培养时间为3~4天。
5.如权利要求2所述的制备方法,其特征在于,所述步骤2中,所述发酵培养基中含有葡萄糖1.6wt%–3.5wt%、酵母膏0.1wt%–0.5wt%、蛋白胨0.1wt%–0.5wt%、粗海盐0.11wt%–0.6wt%、余量为水;所述发酵培养基配制好后于120~140℃灭25–30分钟。
6.如权利要求2所述的制备方法,其特征在于,所述步骤2中,培养温度为26~28℃,静置培养,培养时间为21~24天。
7.一种基于如权利要求1所述的所述萜类化合物药学上可接受的盐。
8.如权利要求1所述的萜类化合物在制备抗肿瘤药物中的应用。
9.一种抗肿瘤药物,包括如权利要求1所述的萜类化合物或者所述萜类化合物药学上可接受的盐以及辅料。
10.如权利要求9所述抗肿瘤药物,其特征在于,所述辅料为药学上可接受的载体、稀释剂或赋形剂,所述抗肿瘤药物的剂型为固体制剂、半固体制剂或液体制剂。
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