CN108913606B - 一种海洋抗耐药菌活性物质及制备和用途 - Google Patents
一种海洋抗耐药菌活性物质及制备和用途 Download PDFInfo
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Abstract
本发明提供一种海洋灰黄青霉,分类命名为Penicillium griseofulvum ZZ380,是从海洋动物粗腿厚纹蟹中分离培养而获得。本发明利用海洋灰黄青霉发酵制备获得灰黄青霉碱双醚A,所述灰黄青霉碱双醚A具有抗耐甲氧西林金黄色葡萄球菌的活性,可显著抑制耐药菌MRSA的生长,在治疗耐药菌MRSA感染方面具有应用前景,可在制备抗耐甲氧西林金黄色葡萄球菌药物中应用。灰黄青霉碱双醚A化学结构式为:
Description
技术领域
本发明属医药领域,涉及从海洋灰黄青霉(Penicillium griseofulvumZZ380)的代谢产物中获得的具有抗菌活性化合物灰黄青霉碱双醚A(Penicipyrrodiether A),以及其制备方法和在制备抗耐甲氧西林金黄色葡萄球菌药物中的应用。
背景技术
抗生素滥用引起的致病菌对抗生素的耐药性不断增强,耐药突变菌株的种类和数量不断增多已成为威胁人类健康的一个重大难题。在金黄色葡萄球菌感染的病例中,被称为超级细菌的耐甲氧西林金黄色葡萄球菌(methicillin-resistant Staphylococcusaureus,MRSA)感染所占比例已高达60%,其中30%的病例对万古霉素产生了耐药性并且呈逐年上升的趋势。一直以来万古霉素是治疗MRSA感染的金标药物,然而由于这一金标药物在临床上频繁使用而导致MRSA对其耐药性不断增强。另一方面,微生物出现耐药性变种的速率已经远远超过人类开发新型抗生素的速度,如何对抗耐药菌株尤其是MRSA已成为世界性难题。
海洋特殊的物理化学环境以及生物多样性和复杂性,迫使海洋微生物具有与陆地微生物不同的新陈代谢途径、生存繁殖方式和适应机制,因而产生许多陆地微生物无法产生的化学结构新颖和生物活性独特的代谢产物,是发现新型抗耐药菌株活性物质的重要资源。灰黄青霉碱双醚A(Penicipyrrodiether A)是从海洋灰黄青霉(PenicilliumgriseofulvumZZ380)的次生代谢产物中分离得到的一个结构新颖的生物碱类化合物,对耐甲氧西林金黄色葡萄球菌(MRSA)的生长具有好的抑制作用,因此灰黄青霉碱双醚A在制备抗MRSA药物方面具有应用前景。
发明内容
本发明的目的是提供一种海洋灰黄青霉,分类命名为Penicillium griseofulvumZZ380,已被中国典型培养物保藏中心保藏,保藏编号:CCTCC NO:2018344,保藏日:2018.6.4;保藏地址:中国.武汉.武汉大学。
本发明的第二个目的是提供所述海洋灰黄青霉的提取制备方法,是从海洋动物粗腿厚纹蟹(Pachygrapsus crassipes)中分离海洋真菌ZZ380的方法,通过以下步骤分离培养而获得:
(1)灰黄青霉(Penicillium griseofulvumZZ380)的分离培养
取粗腿厚纹蟹(Pachygrapsus crassipes)在75%的乙醇中浸泡10秒除去表面微生物,然后用无菌水清洗三次后匀浆,离心后取上层液配制成不同浓度的悬浮液。取一定量不同浓度的悬浮液均匀分散到含有固体培养基的培养皿中,在室温条件下培养一定时间后,将不同的菌落分别转移到另一含有固体培养基的培养皿中,在室温条件下继续培养一定时间。最后将生长良好的单一菌落(ZZ380)接种到斜面培养基培养后置4℃冰箱保存备用。
所述的粗腿厚纹蟹(Pachygrapsus crassipes)是从浙江普陀山海滩的岩石缝中获得;所述悬浮液的浓度为1×10-3~1×10-1(取1mL匀浆后离心所得的上层液体,加入9mL无菌水,配成体积浓度为10-1的样品溶液,并逐倍稀释得到体积浓度为10-2和10-3的样品溶液);所述的样品悬浮液的取样量为100~300μL;所述培养皿所含的固体培养基为马铃薯葡萄糖琼脂(PDA,Potato Dextrose Agar,杭州微生物试剂有限公司)培养基或其它固体培养基;所述的斜面培养基为PDA或其它固体斜面培养基;所述的室温培养温度为22~28℃;所述的培养时间为5~15天。
(2)灰黄青霉(Penicillium griseofulvum ZZ380)的菌种鉴定
上述步骤(1)分离培养所获得的菌株ZZ380用目前实验室普遍使用的ITS rDNA序列分析方法鉴定其种类,确定为灰黄青霉,分类命名为Penicillium griseofulvum ZZ380,已被中国典型培养物保藏中心保藏,保藏编号:CCTCC NO:2018344,保藏地址:中国.武汉.武汉大学。
本发明的第三个目的是提供灰黄青霉碱双醚A(Penicipyrrodiether A),其化学结构式为:
本发明的第四个目的是提供灰黄青霉碱双醚A(Penicipyrrodiether A)的制备方法,通过以下步骤实现:
(1)灰黄青霉(Penicillium griseofulvum ZZ380)发酵菌液的制备
将灰黄青霉(Penicillium griseofulvum ZZ380)的菌株接种到含有一定量的液体培养基的大三角烧瓶中,将含有ZZ380菌种的培养液在室温条件下振荡培养一定时间后得到菌种液。最后将菌种液转入含有一定量的液体培养基的大三角烧瓶中,特定温度静置培养一定时间后,得到含有活性物质灰黄青霉碱双醚A的ZZ380培养液。所述的ZZ380菌株为产生抗MRSA活性物质灰黄青霉碱双醚A的海洋灰黄青霉(PenicilliumgriseofulvumZZ380)。
所述的菌种培养基为马铃薯-葡萄糖肉汤(Potato Dextrose Broth,PDB,土豆200g,葡萄糖20g,海盐35g,水1L)液体培养基,所述的用量为250mL;所述的液体发酵培养基为BMPM液体培养基(葡萄糖20克,甘油20克,大豆粉10克,棉粕10克,硫酸铵1克,碳酸钙10克,海盐35g);所述的大三角培养瓶为500mL;所述的培养温度为28℃;所述的培养时间为30天。
(2)灰黄青霉碱双醚A(Penicipyrrodiether A)的提取分离纯化
菌株ZZ380的发酵菌液过滤后分成发酵菌丝体和发酵液两部分。菌丝体用甲醇提取得到甲醇提取物,发酵液用乙酸乙酯萃取得到乙酸乙酯萃取物。将甲醇提取物和乙酸乙酯萃取物合并得到总提取物。总提取物先用十八烷基硅烷键合硅胶(ODS)柱层析分离,用甲醇和水的混合溶剂梯度洗脱,并用薄层层析分析,合并含有相同成分的组分,得到6个组分(Frs.1~6);组分6(Fr.6)再用制备型高效液相色谱(HPLC)仪分离纯化,得到纯化合物灰黄青霉碱双醚A。
所述柱层析的ODS用量与上柱的样品量比例是30~50g:1.0g;所述的甲醇和水的混合溶剂梯度为80:20,90:10,100:0,为体积比;所述的高效液相分离条件是:创新通恒CXTH-3000高效液相色谱仪,富士C18CT-30色谱柱(280×30mm,10μm),甲醇和水为流动相(90/10,体积比),检测波长235nm,流速为15.0mL/min。
(3)灰黄青霉碱双醚A(Penicipyrrodiether A)的结构鉴定
灰黄青霉碱双醚A(Penicipyrrodiether A)的结构是根据它的紫外光谱、红外光谱、一维和二维核磁共振(NMR)光谱、高分辨质谱(HRESIMS)数据、电子圆二色谱(ECD)计算以及单晶X-射线衍射等方法相结合而确定。
本发明的第五个目的是提供灰黄青霉碱双醚A(Penicipyrrodiether A)在制备抗耐甲氧西林金黄色葡萄球菌(MRSA)药物中的应用。所述的灰黄青霉碱双醚A具有抗耐甲氧西林金黄色葡萄球菌(MRSA)的活性,可显著抑制耐药菌MRSA的生长,在治疗耐药菌MRSA感染方面具有应用前景。
本发明的有益之处在于,(1)从海洋动物粗腿厚纹蟹中分离到一株海洋灰黄青霉菌株ZZ380,该菌株在所述的培养条件下可以产生抗耐药菌的活性物质灰黄青霉碱双醚A;(2)灰黄青霉碱双醚A为具有新骨架结构的新化合物,现有抗感染药物没有该结构类型的化合物;(3)灰黄青霉碱双醚A显著抑制耐甲氧西林金黄色葡萄球菌生长,可为耐甲氧西林金黄色葡萄球菌感染的有效治疗提供一种新的药物分子,克服现有抗生素药物对耐甲氧西林金黄色葡萄球菌感染耐药的不足。
附图说明
图1是灰黄青霉(Penicillium griseofulvum ZZ380)的菌落图。
图2是灰黄青霉碱双醚A(Penicipyrrodiether A)的紫外光谱图。
图3是灰黄青霉碱双醚A(Penicipyrrodiether A)的电子圆二色谱(ECD)计算拟合曲线图和实验测得的ECD谱。
图4是灰黄青霉碱双醚A(Penicipyrrodiether A)的红外光谱图。
图5是灰黄青霉碱双醚A(Penicipyrrodiether A)的高分辨质谱。
图6是灰黄青霉碱双醚A(Penicipyrrodiether A)的氢谱。
图7是灰黄青霉碱双醚A(Penicipyrrodiether A)的碳谱。
图8是灰黄青霉碱双醚A(Penicipyrrodiether A)的HSQC谱
图9是灰黄青霉碱双醚A(Penicipyrrodiether A)的1H-1H COSY谱
图10是灰黄青霉碱双醚A(Penicipyrrodiether A)的HMBC谱。
图11-图14是灰黄青霉碱双醚A(Penicipyrrodiether A)的NOESY谱
图15是灰黄青霉碱双醚A(Penicipyrrodiether A)的单晶X-射线衍射结构图。
图16是灰黄青霉碱双醚A(Penicipyrrodiether A)的1H-1H COSY和HMBC相关示意图。
具体实施方式
以下结合附图和实施例对本发明作进一步详细描述。但是,本发明不限于这些实施例。
实施例1
1.灰黄青霉(Penicillium griseofulvum ZZ380)的分离培养
取称重的粗腿厚纹蟹(Pachygrapsus crassipes)(20.1克),在75%的乙醇中浸泡10秒除去表面微生物,然后用无菌水清洗三次后匀浆,离心后取上层液体配制成体积浓度为1×10-1、1×10-2、1×10-3的样品溶液(取1mL匀浆后离心所得的上层液体,加入9mL无菌水,配成体积浓度为10-1的样品溶液,并逐倍稀释得到体积浓度为10-2和10-3的样品溶液)。取各浓度的样品溶液200μL均匀分散到含有马铃薯葡萄糖琼脂(Potato Dextrose Agar,PDA,杭州微生物试剂有限公司)固体培养基的培养皿中,在28℃条件下培养5天后,将不同的菌落分别转移到另一含有PDA固体培养基的培养皿中,在28℃条件下继续培养5天。最后将生长良好的单一菌落(ZZ380)接种到PDA固体斜面培养基培养后,置于4℃冰箱保存备用。
2.灰黄青霉(Penicillium griseofulvum ZZ380)的菌种鉴定
使用ITS rDNA序列分析方法鉴定所获得菌株ZZ380的种类。
2.1实验试剂及仪器
PCR试剂:PrimeSTAR Max DNA Polymerase(TaKaRa),引物(Invitrogen合成),引物序列是:
Marker:DL2000
实验仪器:离心机,电泳仪,PCR仪,ABI 3730XL测序仪。
2.2实验步骤
2.2.1真菌基因组DNA抽提
使用Ezup柱式真菌基因组DNA抽提试剂盒(生工),使用前先对真菌进行液氮研磨。
2.2.2真菌基因组DNA浓度及质量检测
使用Nanodrop超微量分光光度计进行DNA浓度及质量检测。
2.2.3 PCR扩增
a.PCR反应体系
b.PCR反应条件
c.电泳检测
1%琼脂糖凝胶电泳150v,22min
上样量:4μl,Loading buffer 2μl,Marker 4μl
d.测序:切胶纯化测序
e.分析结果:拼接序列。
2.3实验结果
拼接后的序列为:
以上获得的ITS rDNA序列与美国NIH的NCBI GenBank数据库比较,其结果表明:菌株ZZ380的ITS rDNA序列与GenBank库中的Penicilliumgriseofulvum的ITS rDNA序列有99%的相似性(登录号:KF811439.1)。因此,本发明所获得的海洋菌株ZZ380定为灰黄青霉(Penicillium griseofulvumZZ380)(附图1)。灰黄青霉(PenicilliumgriseofulvumZZ380)的菌株已被中国典型培养物保藏中心保藏,保藏编号:CCTCC NO:2018344,保藏日:2018.6.4;保藏地址:中国.武汉.武汉大学。
3.灰黄青霉(Penicillium griseofulvumZZ380)发酵培养物的制备
挑取PDA固体斜面培养基上的灰黄青霉(Penicillium griseofulvumZZ380),接种到含有250mL马铃薯-葡萄糖肉汤(Potato Dextrose Broth,PDB,土豆200g,葡萄糖20g,海盐35g,水1L)液体培养基的500mL三角烧瓶中。将含有ZZ380菌种的培养液在28℃条件下旋转(180rpm)振摇培养3天后得到菌种液。将5mL菌种液转入到含有250mL的BMPM液体培养基(葡萄糖20g,甘油20g,大豆粉10g,棉粕10g,硫酸铵1g,碳酸钙10g,海盐35g,水1L)的500mL三角烧瓶中,在28℃条件下静置培养30天,得到含有活性物质灰黄青霉碱双醚A的ZZ380发酵培养物。
4.灰黄青霉碱双醚A(Penicipyrrodiether A)的提取分离纯化
将实验3获得的发酵培养物(40.0升)离心得到菌丝体和发酵液。菌丝体用甲醇提取三次得到甲醇提取物,发酵液用乙酸乙酯萃取得到乙酸乙酯萃取物,合并得到总提物(10.0g)。总提物用ODS(400g)柱层析分离,用甲醇和水混合溶剂(80/20,90/10,100/0,体积比)梯度洗脱,用薄层层析分析各洗脱液,合并含有相同成分的组分,总共得到6个组分(Frs.1~6)。其中组分6(Fr.6,0.35g)用半制备型高效液相色谱仪分离(仪器:创新通恒CXTH-3000;色谱柱:富士C18CT-30,280×30mm,10μm;流动相:甲醇/水体系,体积比90/10;检测波长:235nm,流速:15.0mL/min),得到化合物灰黄青霉碱双醚A(Penicipyrrodiether A)(9.0mg,保留时间40.5min)。
5.灰黄青霉碱双醚A(Penicipyrrodiether A)的结构鉴定
灰黄青霉碱双醚A(Penicipyrrodiether A):无色晶体;分子式C43H53NO8;紫外光谱(附图2):UV(MeOH)λmax(logε)203(4.46),233(4.24),251(4.15)nm;电子园二色谱(附图3):ECD(0.5mg/mL,MeOH)λmax(Δε)205(+0.67),229(+2.81),251(-1.79),280(+0.41)nm;红外光谱(附图4):IR(CHCl3)vmax 3260,2947,2922,2854,1660,1631,1504,1454,1376,1304,1260,1237,
1221,1091,1040,1004,961,903,808,755,641cm-1;高分辨质谱(附图5)(HRESIMS)为m/z[M+H]+712.3843(计算值C43H54NO8 712.3849)。通过灰黄青霉碱双醚A(Penicipyrrodiether A)的1H谱(附图6)、13C谱(附图7)、HSQC谱(附图8)、1H-1H COSY谱(附图10)、HMBC谱(附图10)、NOESY谱(附图11-14)、X射线单晶衍射(附图15)和电子圆二色谱(ECD,附图3)计算,确定了灰黄青霉碱双醚A(Penicipyrrodiether A)的结构,为一个新化合物,其13C和1H核磁共振信号归属见表一。它的1H-1H COSY和HMBC相关示意图见附图16。
表一、灰黄青霉碱双醚A的13C和1H NMR数据(溶剂:氘代二甲基亚砜DMSO-d6)
6.灰黄青霉碱双醚A(Penicipyrrodiether A)的抗菌活性
采用营养肉汤稀释法,测定灰黄青霉碱双醚A(Penicipyrrodiether A)抑制耐甲氧西林金黄色葡萄球菌(MRSA)生长的作用,具体操作为:
将耐甲氧西林金黄色葡萄球菌(MRSA)接种于营养琼脂(Nutrient Agar,NA,杭州微生物试剂有限公司)平板上,置于37℃恒温培养箱培养24h后,挑取单菌落接种于营养肉汤培养基(Nutrient Broth,NB,杭州微生物试剂有限公司),37℃恒温振荡(180rpm)培养10h获得菌液。以NB培养基作为参比,在550nm波长下检测菌液的OD值,控制在0.08~0.1范围内。
将样品用二甲基亚砜配制成一定浓度(1mg/mL,可根据预设检测浓度适当调整)的母液,经0.22μm无菌有机滤头过滤除菌后加入96孔板内,然后加入一定量的NB培养基和2μL上述菌液,使每个孔内的最终体积为200μL,菌液浓度为106CFU/mL,样品浓度为预设检测浓度。以二甲基亚砜和庆大霉素分别作为阴性和阳性对照药,37℃恒温静置培养12h。
将没有变浑浊并且给药浓度最低的孔内的培养物接种于NA平板,37℃恒温静置培养12h,若无可见菌落生长,则此孔对应的浓度为该样品的最低杀菌浓度(minimumbactericidal concentration,MBC),有可见菌落生长,则此孔对应的给药浓度为该样品的最低抑菌浓度(minimum inhibitory concentration,MIC)。
实验结果显示,灰黄青霉碱双醚A(Penicipyrrodiether A)能够显著抑制耐甲氧西林金黄色葡萄球菌(MRSA)的生长,其MIC值为7.0μM(阳性对照药庆大霉素MIC值为1.47μM,阴性对照二甲基亚砜在1.5M时仍未表现出抑制作用)。实验结果说明灰黄青霉碱双醚A对耐药菌MRSA的生长具有好的抑制作用,在制备抗耐药菌MRSA感染药物方面具有应用前景。
总之,本发明从海洋动物粗腿厚纹蟹(Pachygrapsus crassipes)中分离到一种海洋灰黄青霉(Penicillium griseofulvumZZ380),该菌可以产生抗耐甲氧西林金黄色葡萄球菌(MRSA)的活性化合物灰黄青霉碱双醚A(Penicipyrrodiether A)。本发明提供了灰黄青霉的分离培养方法、活性化合物灰黄青霉碱双醚A(Penicipyrrodiether A)的提取分离纯化方法,以及灰黄青霉碱双醚A(Penicipyrrodiether A)显著抑制耐甲氧西林金黄色葡萄球菌(MRSA)生长的活性。由于灰黄青霉碱双醚A(Penicipyrrodiether A)具有良好的抗耐甲氧西林金黄色葡萄球菌(MRSA)活性,所以灰黄青霉碱双醚A(Penicipyrrodiether A)在制备治疗耐甲氧西林金黄色葡萄球菌(MRSA)感染的药物方面具有应用前景。
序列表
<110> 浙江大学
<120> 一种海洋抗耐药菌活性物质及制备和用途
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 22
<212> DNA
<213> 人工序列(Unknown)
<400> 1
cttggtcatt tagaggaagt aa 22
<210> 2
<211> 20
<212> DNA
<213> 人工序列(Unknown)
<400> 2
gctgcgttct tcatcgatgc 20
<210> 3
<211> 558
<212> DNA
<213> 灰黄青霉(Penicillium griseofulvum ZZ380)
<400> 3
cttccgtagg gggacctgcg gaaggatcat taccgagtgc gggcccctcg gggcccaacc 60
tcccacccgt gttgcccgaa cctatgttgc ctcggcgggc cccgcgcccg ccgacggccc 120
ccctgaacgc tgtctgaagt tgcagtctga gacctataac gaaattagtt aaaactttca 180
acaacggatc tcttggttcc ggcatcgatg aagaacgcag cgaaatgcga taactaatgt 240
gaattgcaga attcagtgaa tcatcgagtc tttgaacgca cattgcgccc tctggtattc 300
cggagggcat gcctgtccga gcgtcattgc tgccctcaag cccggcttgt gtgttgggcc 360
ccgtcccccc cgccgggggg acgggcccga aaggcagcgg cggcaccgcg tccggtcctc 420
gagcgtatgg ggcttcgtca cccgctctag taggcccggc cggcgccagc cgacccccaa 480
cctttaatta tctcaggttg acctcggatc agagtcaggg atacccgctg aacttaagca 540
tatcaataag cggaggaa 558
Claims (1)
1. 一种海洋灰黄青霉(Penicillium griseofulvum),其分类命名为Penicillium griseofulvum ZZ380,已被中国典型培养物保藏中心保藏,保藏编号: CCTCC NO:2018344,保藏日:2018.6.4;保藏地址:中国.武汉.武汉大学。
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CN102268395B (zh) * | 2011-07-22 | 2012-11-21 | 浙江大学 | 一种海洋青霉菌菌株及其应用 |
CN102367421B (zh) * | 2011-12-03 | 2012-09-12 | 云南省农业科学院农业环境资源研究所 | 一株灰黄青霉xj-ep-058菌株及其抗香蕉枯萎病微生物制剂与应用 |
CN103255061B (zh) * | 2012-02-15 | 2014-11-26 | 中国医学科学院医药生物技术研究所 | 灰黄青霉、由其产生的抗菌活性化合物及应用 |
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2018
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