CN117448174A - 一株红树林沉积物来源真菌及其应用 - Google Patents
一株红树林沉积物来源真菌及其应用 Download PDFInfo
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Abstract
本发明提供了一株红树林沉积物来源真菌及其应用。该Penicillium sp.UJNMF0740菌株的保藏编号为CGMCC No.40521;上述青霉属真菌及其发酵产物可用于生产抗菌药物。本发明还提供了上述青霉属真菌产生的具有抗菌作用的吲哚二萜类化合物:
Description
技术领域
本发明属于海洋生物领域,具体涉及一株青霉属真菌及其在抗菌方面的应用。
背景技术
金黄色葡萄球菌(Staphylococcu saureus),又称金葡菌,是一种常见的革兰阳性菌,是一种***共患病原菌,是全世界公认的食源性病原菌,在水产品(鱼、虾等)、肉食品(猪肉、家禽肉、牛羊肉等)以及乳制品中均广泛分布,可导致人和动物的多种疾病,如膜性肠炎、肺炎、心包炎等局部化脓感染,甚至全身感染性疾病如脓毒症、败血症等,严重威胁人类和动物安全。随着耐甲氧西林金黄色葡萄球菌等耐药菌株的出现,金黄色葡萄球菌已成为世界性的卫生问题。
红树林指生长在热带、亚热带海岸潮间带上部,以红树林植物为主体的常绿灌木或乔木组成的潮滩湿地木本生物群落,是陆地向海洋过渡的特殊生态***。红树林生态***周期性遭受海水浸淹,其动态盐度水平高,有机物储量大,养分循环率高,兼有海洋和陆地的性质却又有其独特的特点,孕育着丰富的微生物资源。真菌是红树林微生物中的一个主要类群,具有产生各种新型化合物的生物合成潜力,能够产生种类丰富的生理活性物质,是抗菌、抗肿瘤等药用价值活性代谢物的重要来源,因此筛选具有抗菌作用的红树林沉积物真菌,对新型抗菌药物的研发具有重要意义。
发明内容
针对目前抗菌药物的迫切需求,本发明提供一种青霉属真菌(Penicilliumsp.UJNMF0740),其发酵产物具有抗菌作用。
本发明的另一目的是提供一种青霉属真菌发酵物中分离的具有抗菌活性的吲哚二萜类化合物及其制备方法。
为实现上述目的,本发明采用如下技术方案。
一株青霉属真菌Penicilliumsp. UJNMF0740,保藏于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC),保藏编号为CGMCC No. 40521。
一种青霉属真菌Penicilliumsp. UJNMF0740及其发酵产物在制备抗菌药物中的应用。所述青霉属真菌Penicilliumsp. UJNMF0740培养后可产生发酵产物,所述发酵产物具有抗菌活性,可用于生产具有抗菌活性的药物或添加剂。
青霉属真菌Penicilliumsp. UJNMF0740产生的吲哚二萜类化合物,其结构如式(I)所示:
式(I)
上述吲哚二萜类化合物的制备方法,包括以下步骤:
(1)青霉属真菌Penicilliumsp. UJNMF 0740的发酵获得发酵物;
(2)从步骤(1)所得发酵物中经乙醇提取、乙酸乙酯萃取得到发酵粗提物;
(3)步骤(2)中的发酵粗提物按照以下方式进行分离:
(3-1)发酵粗提物以二氯甲烷:甲醇按照体积比1:0-0:1为洗脱液通过100-200目正相硅胶柱梯度洗脱,获得馏分Fr.1-Fr.9;
(3-2)馏分Fr.2以石油醚:丙酮按照体积比1:0-0:1为洗脱液通过200-300目正相硅胶柱梯度洗脱,获得馏分Fr.2.1-Fr.2.7;Fr.2.3以体积比为1:1的二氯甲烷:甲醇为洗脱液经过Sephadex LH-20,获得馏分Fr.2.3.1-Fr.2.3.5,Fr.2.3.2以体积比为80:20的甲醇:水为流动相通过C18柱半制备高效液相色谱,获得式(I)所示化合物。
本发明提供一种步骤(2)中的发酵粗提物,或式(I)所示化合物在制备抗菌药物、药物中间体以及抗菌添加剂的用途。
本发明提供一种含有步骤(2)中的发酵粗提物,或式(I)所示化合物制备的抗菌药物或抗菌添加剂。所述抗菌药物还包括医学上可接受的辅料或/和其他有效成分,以扩大杀菌谱或增强抗菌效果。
本发明具有以下优点:
本发明的吲哚二萜类化合物可以通过Penicilliumsp.UJNMF0740的发酵提取分离获得,能够抑制金葡萄球菌的生长,在制备抗菌药物或抗菌添加剂方面具有应用潜力。
生物保藏信息
Penicilliumsp. UJNMF 0740,于2023年03月09日保藏于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC),保藏地址为中国北京,北京市朝阳区北辰西路1号院3号中国科学院微生物研究所,保藏编号为CGMCC No.40521。
附图说明
图1为真菌Penicilliumsp.UJNMF0740平板培养图片;
图2为式(I)所示化合物主要的1H-1H COSY、HMBC信息;
图3为式(I)所示化合物主要的NOESY信息;
图4为式(I)所示化合物的ECD谱图;
图5为Penicilliumsp. UJNMF0740发酵提取物及式(I)所示化合物的细胞毒活性。
具体实施方式
下面结合实施例和附图对本发明做进一步说明,但本发明不受下述实施例的限制。
实施例1 UJNMF 0740的分离纯化与鉴定
将采集的自海南省东寨港红树林秋茄根际土壤,按照梯度稀释法获得土壤悬液,在超净工作台中,将上述土壤悬液用移液枪吸取100 μL加到分离培养基中,并用涂布棒涂布均匀,将培养皿倒置放入28℃恒温培养室中,静置培养1-2周,观察是否有菌落长出,然后将菌落的菌丝用无菌竹签通过平板划线法接种到PDA培养基中纯化。经分离纯化获得菌种Penicilliumsp.UJNMF0740,其典型培养物图片如图1所示:菌落呈圆形,外周白色,菌丝绿色。经ITS rDNA检测,与Gene Bank中Penicillium svalbardense(NR_111508.1)同源性为99.08%,鉴定为青霉属真菌。
实施例2 UJNMF 0740的发酵和粗提物制备
种子培养基的配制方法:土豆浸出液200 mL,葡萄糖20 g,海盐30 g,用水定容到1L。将培养基装入20个500 mL的三角烧瓶中,每瓶约150 mL,在121℃高压蒸汽灭菌25分钟,备用。
大米发酵培养基配置方法:大米80g,酵母膏0.4g,葡萄糖0.4g,水120 ml,海盐3%。置于1 L三角瓶中,共150瓶。121℃高压蒸汽灭菌25分钟,备用。
用无菌竹签挑取适量的真菌Penicilliumsp. UJNMF 0740菌种接种入种子培养基中,28℃摇床(200 rpm)培养3天得到种子液,然后用移液枪接种10 mL种子液到1 L的装有大米发酵培养基的三角烧瓶中,于28℃静置培养30天后,收取发酵物。
将发酵30天之后的大米发酵培养基转移出来进行粉碎,使用95%乙醇进行浸泡,浸泡4次(单次浸泡时间为6-7天),将每次浸泡完成之后的95%乙醇收集起来,用大型旋转蒸发仪浓缩得到溶液,而后使用等量的乙酸乙酯进行萃取,共萃取五次,合并萃取液旋干溶剂得到发酵粗提物约35g。
实施例3 UJNMF 0740发酵粗提物的抗菌活性
(1)抑菌圈
以纸片扩散法测定UJNMF 0740提取物对Staphylococcus aureusATCC25923的抗菌活性:分别将上述菌种接种到LB液体培养基中37℃摇床(180 rpm)培养24小时,然后通过麦氏比浊法,用LB液体培养基将培养液的菌种浓度稀释到约108cfu/mL备用;在50-60℃时,每200 mL无菌LB固体培养基中加入200 μL上述菌种稀释液后迅速倒入直径为9cm的灭菌培养皿中,待平板冷却后,将6 mm的无菌纸片(含100 μg待测样品)置于上述平板中37℃培养18小时,观察抑菌圈大小。
实验结果显示,Penicilliumsp.UJNMF0740的发酵粗提物在100 μg/纸片的浓度下能够抑制Staphylococcus aureus的生长,抑菌圈为7.5 mm。
(2)IC50测定
以Staphylococcus aureusATCC25923作为测试菌株,首先将菌株接种到LB固体培养基上进行复苏,然后挑取适量菌落接种到液体培养基37℃、160rpm摇床震荡培养,使用空白培养基将培养好的菌株稀释到108cfu/mL。使用移液枪量取190 μL加入至96孔板中,再加入10 μL含粗提物的DMSO溶液,37℃过夜培养,使用酶标仪在600 nm下测定吸光度(OD值)并计算抑制率,使用空白培养基作为对照。
实验结果表明,Penicilliumsp.UJNMF0740发酵提取物对S. aureus有抗菌活性,其IC50为73.2 μM,具有抑制S. aureus生长的活性。
实施例4 吲哚二萜类化合物的制备
按照实施例2的方法获得发酵粗提物,将其经过硅胶柱层析(100-200目),以二氯甲烷:甲醇(1:0-0:1v/v)进行梯度洗脱,洗脱期间使用薄层层析硅胶板进行检测并合并化合物相似馏分,共得到9个馏分(Fr.1-Fr.9)。
Fr.2通过硅胶柱层析(200-300目),以石油醚:丙酮(1:0-0:1v/v)进行梯度洗脱,结合薄层色谱硅胶板进行检测,合并得到7个馏分(Fr.2.1-Fr.2.7)。Fr.2.3经过SephadexLH-20(二氯甲烷:甲醇= 1:1),得到了5个馏分(Fr.2.3.1-Fr.2.3.5),Fr.2.3.2通过半制备高效液相色谱(甲醇/水,80:20v/v,3 mL/min),得到式(I)所示化合物(tR= 12 min、1.5mg)。
式(I)所示化合物为无定型白色粉末,旋光度值为 [α]25 D15.7(c0.23,MeOH)。
对分离获得的化合物进行高分辨质谱(HR-ESIMS)、1H NMR、13C NMR、2D1H-1H COSY、HSQC、HMBC分析,确定了平面结构,再结合NOESY和ECD谱图确定了化合物的构型。化合物的1H和13C NMR数据见表1,主要的1H-1H COSY和HMBC相关信息见图2。
表1 式(I)所示化合物在CDCl3中的1H(600 MHz)和13C(150 MHz)NMR数据
式(I)所示化合物的高分辨质谱(HR-ESIMS)在m/z处给出准分子离子峰598.3166([M + H]+, 计算598.3163),结合NMR波谱数据,推测化合物的分子式为C37H43NO6,不饱和度为17。分析该化合物的NMR数据发现,该化合物与文献报道的化合物shearinine D核磁数据非常相似(Xu M., 2007),表明化合物具有相似的结构片段。不同的是化合物shearinine D在δC/δH76.7/4.86(CH-22)的连羟基次甲基碳信号在式(I)所示化合物中消失,同时δC201.7处出现羰基信号,推测式(I)所示化合物中C-22位变成了羰基。HMBC谱中从H-23到C-22/C-24、以及从H-20到C-22的相关信号证实了这一点(图2)。
NOE谱图中(图3),H-16与H-14β,H-16与H3-33,H3-32与H-17α,H3-32与H-5α,H3-32与H-15α以及 H-14α与H-11有相关可以推测该化合物的相对构型。然后,通过计算(3S,4R,7S,9R,13S,16S,23R)-1和(3S,4R,7S,9R,13S,16S,23S)-2的理论ECD谱,式(I)所示化合物实验ECD曲线与2一致(图4),确定其绝对构型为3S,4R,7S,9R,13S,16S,23S:
实施例5 式(I)化合物的抗菌活性
以Staphylococcus aureusATCC25923作为测试菌株,首先将菌株接种到LB固体培养基上进行复苏,然后挑取适量菌落接种到液体培养基37℃、160rpm摇床震荡培养,使用空白培养基将培养好的菌株稀释到108cfu/mL。使用移液枪量取190 μL加入至96孔板中,再加入10 μL含一定浓度化合物的DMSO溶液,37℃过夜培养,使用酶标仪在600 nm下测定吸光度(OD值)并计算抑制率,实验使用空白培养基作为对照。
实验结果表明,式(I)所示化合物对S. aureus有抗菌活性,其IC50为51.0 μM,具有抑制S. aureus生长的活性。
实施例6 Penicilliumsp.UJNMF0740发酵提取物及式(I)所示化合物的细胞毒性
将PC12细胞按照1.5×105个/mL (100 μL/孔)的密度接种到96孔板中,37C 5% CO2的恒温培养箱中培养细胞24小时。设置对照组、实验组,实验组用100 μM待测物孵育细胞12小时,对照组加等体积DMSO溶液。随后,每孔加入MTT溶液10μL,37℃孵育4小时,除去培养基,加入150 μL DMSO溶解甲醛晶体,使用酶标仪490 nm处测量光密度(OD)。
实验结果表明(图5),Penicilliumsp.UJNMF0740发酵粗取物和式(I)所示化合物在100 μM对PC12细胞没有明显的细胞毒活性,且式(I)所示化合物对PC12细胞具有一定的促进增殖的活性。
Claims (7)
1.一株青霉属真菌Penicillium sp. UJNMF0740,其特征在于,保藏编号为CGMCC No.40521。
2.一种青霉属真菌Penicillium sp. UJNMF0740及其发酵产物在制备抗菌药物或添加剂中的应用,其特征在于,青霉属真菌Penicillium sp. UJNMF0740的保藏编号为CGMCCNo. 40521。
3.一种吲哚二萜类化合物,其结构如式(I)所示:
式(I)。
4.一种如权利要求3所述的吲哚二萜类化合物的制备方法,其特征在于,包括以下步骤:
(1)青霉属真菌Penicillium sp. UJNMF 0740的发酵获得发酵物;
(2)从步骤(1)所得发酵物中经乙醇提取、乙酸乙酯萃取得到发酵粗提物;
(3)步骤(2)中的发酵粗提物按照以下方式进行分离:
(3-1)发酵粗提物以二氯甲烷:甲醇按照体积比1:0-0:1为洗脱液通过100-200目正相硅胶柱梯度洗脱,获得馏分Fr.1-Fr.9;
(3-2)馏分Fr.2以石油醚:丙酮按照体积比1:0-0:1为洗脱液通过200-300目正相硅胶柱梯度洗脱,获得馏分Fr.2.1-Fr.2.7;Fr.2.3以体积比为1:1的二氯甲烷:甲醇为洗脱液经过Sephadex LH-20,获得馏分Fr.2.3.1-Fr.2.3.5,Fr.2.3.2以体积比为80:20的甲醇:水为流动相通过C18柱半制备高效液相色谱,获得式(I)所示化合物。
5.一种具有抗菌作用的药物,其特征在于,包含有效量的作为活性成分的利要求4步骤(2)所述的青霉属真菌Penicillium sp.UJNMF0740发酵粗提物,或如权利要求3所述的吲哚二萜类化合物。
6.根据权利要求5所述的药物,其特征在于,还包括医学上可接受的辅料或/和其他有效成分。
7.一种权利要求4步骤(2)所述的青霉属真菌Penicillium sp.UJNMF0740发酵粗提物,或权利要求3所述的吲哚二萜类化合物在制备具有抗菌作用的药物、药物中间体中或抗菌添加剂中的应用。
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