CN112795617B - 海洋真菌次级代谢物及其制备与应用 - Google Patents

海洋真菌次级代谢物及其制备与应用 Download PDF

Info

Publication number
CN112795617B
CN112795617B CN202110035660.3A CN202110035660A CN112795617B CN 112795617 B CN112795617 B CN 112795617B CN 202110035660 A CN202110035660 A CN 202110035660A CN 112795617 B CN112795617 B CN 112795617B
Authority
CN
China
Prior art keywords
fraction
compound
volume ratio
preparation
secondary metabolite
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202110035660.3A
Other languages
English (en)
Other versions
CN112795617A (zh
Inventor
董存柱
侯宗敏
陶敏
夏玉莲
伍显锋
曹云
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Hainan University
Original Assignee
Hainan University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Hainan University filed Critical Hainan University
Priority to CN202110035660.3A priority Critical patent/CN112795617B/zh
Publication of CN112795617A publication Critical patent/CN112795617A/zh
Application granted granted Critical
Publication of CN112795617B publication Critical patent/CN112795617B/zh
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P39/00—Processes involving microorganisms of different genera in the same process, simultaneously
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N35/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having two bonds to hetero atoms with at the most one bond to halogen, e.g. aldehyde radical
    • A01N35/04—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having two bonds to hetero atoms with at the most one bond to halogen, e.g. aldehyde radical containing aldehyde or keto groups, or thio analogues thereof, directly attached to an aromatic ring system, e.g. acetophenone; Derivatives thereof, e.g. acetals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
    • A01N37/08—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing carboxylic groups or thio analogues thereof, directly attached by the carbon atom to a cycloaliphatic ring; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
    • A01N43/02—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms
    • A01N43/04—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom
    • A01N43/14—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings
    • A01N43/16—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings with oxygen as the ring hetero atom
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
    • A01N43/02—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms
    • A01N43/04—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom
    • A01N43/22—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom rings with more than six members
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
    • A01N43/90—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having two or more relevant hetero rings, condensed among themselves or with a common carbocyclic ring system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C45/00—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds
    • C07C45/78—Separation; Purification; Stabilisation; Use of additives
    • C07C45/79—Separation; Purification; Stabilisation; Use of additives by solid-liquid treatment; by chemisorption
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C47/00—Compounds having —CHO groups
    • C07C47/52—Compounds having —CHO groups bound to carbon atoms of six—membered aromatic rings
    • C07C47/56—Compounds having —CHO groups bound to carbon atoms of six—membered aromatic rings containing hydroxy groups
    • C07C47/565—Compounds having —CHO groups bound to carbon atoms of six—membered aromatic rings containing hydroxy groups all hydroxy groups bound to the ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
    • C07C51/42—Separation; Purification; Stabilisation; Use of additives
    • C07C51/47—Separation; Purification; Stabilisation; Use of additives by solid-liquid treatment; by chemisorption
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C61/00—Compounds having carboxyl groups bound to carbon atoms of rings other than six-membered aromatic rings
    • C07C61/16—Unsaturated compounds
    • C07C61/35—Unsaturated compounds having unsaturation outside the rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D309/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings
    • C07D309/16—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member
    • C07D309/28—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D309/30—Oxygen atoms, e.g. delta-lactones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D309/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings
    • C07D309/34—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
    • C07D309/36—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with oxygen atoms directly attached to ring carbon atoms
    • C07D309/38—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with oxygen atoms directly attached to ring carbon atoms one oxygen atom in position 2 or 4, e.g. pyrones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D313/00—Heterocyclic compounds containing rings of more than six members having one oxygen atom as the only ring hetero atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
    • C07D493/02—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains two hetero rings
    • C07D493/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/02—Oxygen as only ring hetero atoms
    • C12P17/06—Oxygen as only ring hetero atoms containing a six-membered hetero ring, e.g. fluorescein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/02—Oxygen as only ring hetero atoms
    • C12P17/08—Oxygen as only ring hetero atoms containing a hetero ring of at least seven ring members, e.g. zearalenone, macrolide aglycons
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
    • C12P17/181—Heterocyclic compounds containing oxygen atoms as the only ring heteroatoms in the condensed system, e.g. Salinomycin, Septamycin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl group
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2602/00—Systems containing two condensed rings
    • C07C2602/02—Systems containing two condensed rings the rings having only two atoms in common
    • C07C2602/14—All rings being cycloaliphatic
    • C07C2602/26—All rings being cycloaliphatic the ring system containing ten carbon atoms
    • C07C2602/28—Hydrogenated naphthalenes

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Environmental Sciences (AREA)
  • Dentistry (AREA)
  • Plant Pathology (AREA)
  • Agronomy & Crop Science (AREA)
  • Pest Control & Pesticides (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Botany (AREA)
  • Mycology (AREA)
  • Medicinal Chemistry (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Biomedical Technology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)

Abstract

本发明公开了海洋真菌次级代谢物及其制备与应用。其中所述制备方法包括：将哈茨木霉菌与短柄青霉菌进行混合发酵，提取发酵物，得到所述次级代谢物。本发明的制备方法通过一般环境条件获得了海洋真菌哈茨木霉菌与短柄青霉菌的混合发酵物，并从中成功获取到天然活性物，创造性地从海洋真菌中制得了生物杀虫剂先导化合物。

Description

海洋真菌次级代谢物及其制备与应用
技术领域
本发明涉及海洋真菌的技术领域。
背景技术
海洋真菌在特殊的生存环境下，生命力强，具有奇特的代谢途径和防御体制， 能够产生大量结构新颖、功能丰富的生物活性代谢产物，引起了国内外科学家的 密切关注，成为当前最具有开发前景的海洋天然产物新药源，同时也是世界药物 研发新型先导化合物的热点之一。到目前为止，研究者们从海洋真菌的次生代谢产物中分离鉴定出大量的结构新颖的化合物，这些化合物表现出良好的抗肿瘤、 抗菌或抗病毒等药理学活性，已从海洋真菌的发酵产物中发现了1500余种新的 次生代谢产物，这些化合物的性质非常典型地代表了海洋真菌的化学防御机制。 近些年，不断有结构新颖的生物活性代谢产物从海洋真菌中分离得到。
虽然对海洋真菌的研究已经被证明可以发现大量结构新颖的活性化合物，但 是很多海洋真菌只是当存在特定养分，或为了竞争营养物质、抵御天敌、维持物 种间信息传递等情况下，才会发展出独特的代谢方式而产生特殊的化学成分，而 在一般生境下并不产生特殊的代谢产物，使其研究和应用都受到了极大的限制。
发明内容
本发明的目的在于提出一种将海洋真菌哈茨木霉菌Trichoderma harzianumABC19819与短柄青霉菌Penicillium brefeldianum ABC190807在一般环境条件下 进行混合发酵的次级代谢物制备方法。
本发明的目的还在于提出通过上述制备方法获得的次级代谢物。
本发明的目的还在于提出上述次级代谢物的一些应用方法。
本发明首先提供了以下技术方案：
海洋真菌次级代谢物的制备方法，其包括：将哈茨木霉菌与短柄青霉菌进行 混合发酵，提取发酵物，得到所述次级代谢物。
根据本发明的一些优选实施方式，所述混合发酵包括：将哈茨木霉菌种子液 与短柄青霉菌种子液共同接种至发酵培养基中，于23-28℃下静置发酵30-50天。
根据本发明的一些优选实施方式，所述发酵培养基选自大米培养基，所述大 米培养基包括大米和海水。
优选的，所述大米培养基的固液比为70-90g/100mL
根据本发明的一些优选实施方式，所述种子液的获得包括：将纯化的哈茨木 霉菌与短柄青霉菌各自接入于种子液培养基中，于23-28℃、150-200r·min-1的 条件下培养2-4天得到所述种子液。
根据本发明的一些优选实施方式，所述种子液培养基选自PDB培养基；其中， 所述PDB培养基包括马铃薯、葡萄糖和海水。
优选的，所述PDB培养基中马铃薯的含量为180-230g/1000mL，和/或葡萄 糖的含量为18-23g/1000mL。
根据本发明的一些优选实施方式，所述提取包括：
将所述发酵物进行浓缩，获得发酵物浸膏；
向所述发酵物浸膏中加入60～80目硅胶进行拌样，并将拌样后的混合样品加 入装填有200～300目硅胶的硅胶色谱柱中进行溶剂洗脱，得到基础馏分；
通过薄层层析法分析所述基础馏分，合并其中的相似馏分，得到合并馏分；
对不同合并馏分通过凝胶色谱柱或硅胶色谱柱进行分别洗脱，得到所述次级 代谢物。
其中，所述相似馏分为：通过薄层层析法计算各馏分中主要成分的比移值(Rf值)，主要成分的比移值相同的馏分为相似馏分。
根据本发明的一些优选实施方式，所述溶剂洗脱包括通过体积比为100:0, 95:5,90:10,85:15,80:20,75:25,70:30,65:35,60:40,55:45,50:50,45:55,40:60,35:65,20:80,0:100的石油醚和乙酸乙酯依次对所述硅胶色谱柱进行的洗脱，及通 过体积比为90:10,65:35,30:70,0:100的乙酸乙酯和甲醇依次对所述硅胶色谱柱进 行的洗脱。
根据本发明的一些优选实施方式，所述分别洗脱包括：
将合并后的第三馏分经体积比为1:1的甲醇和氯仿进行凝胶柱色谱洗脱得到 第一化合物；
将合并后的第四馏分经体积比为90:10的石油醚和乙酸乙酯进行硅胶柱色谱 洗脱、再经体积比为1:1的甲醇和氯仿进行凝胶柱色谱洗脱得到第二化合物；
将合并后的第四馏分经体积比为85:15的石油醚和乙酸乙酯进行硅胶柱色谱 洗脱、再经体积比为1:1的甲醇和氯仿进行凝胶柱色谱洗脱得到第三化合物；
将合并后的第六馏分经体积比为65:35的石油醚和乙酸乙酯进行硅胶柱色谱 洗脱、再经甲醇进行凝胶柱色谱洗脱得到第四化合物；
将合并后的第七馏分经体积比为70:30的石油醚和乙酸乙酯进行硅胶柱色谱 洗脱、再经体积比为1:1的甲醇和氯仿进行凝胶柱色谱洗脱得到第五化合物；
将合并后的第八馏分经甲醇进行凝胶柱色谱洗脱得到第六化合物；
其中，所述第三馏分为由体积比85:15的石油醚和乙酸乙酯进行洗脱对应得 到的馏分及其相似馏分，所述第四馏分为由体积比80:20的石油醚和乙酸乙酯进 行洗脱对应得到的馏分及其相似馏分，所述第六馏分为由体积比70:30的石油醚 和乙酸乙酯进行洗脱对应得到的馏分及其相似馏分，所述第七馏分为由体积比65:35的石油醚和乙酸乙酯进行洗脱对应得到的馏分及其相似馏分，所述第八馏 分为由体积比60:40的石油醚和乙酸乙酯进行洗脱对应得到的馏分及其相似馏分。
本发明还提供了上述任一制备方法制备得到的次级代谢物。
根据本发明的一些具体实施方式，所述的次级代谢物包括如下的化合物1～6：
Figure SMS_1
Figure SMS_2
本发明还提供了其中化合物1和/或化合物3的一种应用方法，为将其作为杀 虫活性物进行应用。
本发明通过一般环境条件获得了海洋真菌哈茨木霉菌Trichoderma harzianumABC19819与短柄青霉菌Penicillium brefeldianum ABC190807的混合发酵物，并 从中成功获取到天然活性物，且所得天然活性物具有良好的杀虫活性，创造性地 从海洋真菌中制得了生物杀虫剂先导化合物。
具体实施方式
以下结合实施例对本发明进行详细描述，但需要理解的是，所述实施例仅用 于对本发明进行示例性的描述，而并不能对本发明的保护范围构成任何限制。所有包含在本发明的发明宗旨范围内的合理的变换和组合均落入本发明的保护范 围。
以下实施例中：
所用菌株ABC19819和ABC190807分离于海南东寨港红树林海泥，经ITS 序列比对及形态学分析，确定种属为Trichoderma harzianum和Penicillium brefeldianum。菌种保藏于海南大学植物保护学院天然产物研究实验室。
供试埃及伊蚊(Aedes aegypti)3龄幼虫、家蝇成虫(Musca demestica)和斜 纹夜蛾(Spodoptera litura)3龄幼虫由海南大学植物保护学院化保实验室提供。
分离及色谱分析通过Sephadex LH-20凝胶柱色谱、含有60～80目硅胶的硅胶 薄层色谱板GF254、含有200～300目硅胶的柱色谱实现。
所用核磁共振仪为Bruker DRX-600型核磁共振波谱仪。
所得ESI-MS谱通过Finnigan LCQ Advantage MAX型LC-MS质谱仪得到。
死亡率和校正死亡率计算方法如下：
死亡率＝死虫数/总虫数×100％
校正死亡率＝(处理死亡率-对照组死亡率)/(1-对照组死亡率)×100％
通过DPS v9.50软件统计分析不同处理间的差异显著性(Duncan’s新复极差 测定法)，毒力测定采用毒力回归方法。
实施例1
将哈茨木霉菌Trichoderma harzianum ABC19819与短柄青霉菌Penicilliumbrefeldianum ABC190807进行混合发酵，发酵过程为：
(1)将马铃薯200g、葡萄糖20g，经海水混合、定容至1000mL，得到种 子液培养基；
(2)将大米80g、海水100mL混合得到发酵培养基；
(3)将保藏的两个菌种活化备用；
(4)将纯化的2株真菌各自接入5瓶种子液培养基中，于26℃、180r·min-1的条件下培养3d作为种子液；
(5)将2株真菌的种子液各接种3mL至发酵培养基中，通过135瓶发酵培 养基于26℃静置混合发酵培养45d，得到发酵物。
实施例2
对实施例1得到的发酵物中的次级产物进行分离提取，其过程为：
(1)发酵结束后，通过乙酸乙酯对每个发酵瓶进行杀菌浸泡3d，其后过滤， 经抽滤减压浓缩得到发酵物浸膏，重复该过程3次，将全部发酵物浸膏合并，共 394.14g；
(2)向发酵物浸膏中加入与其质量比为1:1的60～80目的硅胶进行拌样；
(3)将拌样后的混合样品加入装填有200～300目硅胶的硅胶柱中：
(4)通过石油醚-乙酸乙酯系统按体积比100:0,95:5,90:10,85:15,80:20, 75:25,70:30,65:35,60:40,55:45,50:50,45:55,40:60,35:65,20:80,0:100依次对硅 胶柱色谱进行常压溶剂洗脱，每个体积比下，每收集洗脱液500mL，进行一次减 压浓缩，得到一个小馏分，多次收集后合并该体积比下的全部小馏分，并进行下 一个体积比的溶剂洗脱，至完成全部体积比的溶剂洗脱；
(5)通过乙酸乙酯-甲醇系统按体积比90:10,65:35,30:70,0:100依次继续进 行对该硅胶柱色谱的常压溶剂洗脱，并采用与步骤(4)相同的方法收集各体积 比下浓缩得到的小馏分；
(6)通过薄层层析法(TLC)对步骤(4)和(5)洗脱所得的各小馏分进行 分析，计算各小馏分中主要成分的比移值(Rf值)，将主要成分的比移值相同的小馏分进行合并，得到18个馏分，即馏分Fr.1～18；
其中馏分Fr.3为由石油醚-乙酸乙酯体积比85:15洗脱得到，馏分Fr.4由石油 醚-乙酸乙酯体积比80:20洗脱得到，馏分Fr.6由石油醚-乙酸乙酯体积比70:30洗脱得到，馏分Fr.7由石油醚-乙酸乙酯体积比65:35洗脱得到，馏分Fr.8由石油 醚-乙酸乙酯体积比60:40洗脱得到；
(7)通过凝胶色谱柱或硅胶柱将所得18个馏分进行洗脱，获得次级代谢化 合物，如下：
Fr.3经体积比为1:1的甲醇-氯仿凝胶柱色谱洗脱得到63.7mg的次级代谢化 合物1。
Fr.4经体积比为90:10的石油醚-乙酸乙酯硅胶柱色谱洗脱得到Fr.4-1、经体 积比为85:15的石油醚-乙酸乙酯硅胶柱色谱洗脱得到Fr.4-2，
其中，Fr.4-1经体积比为1:1的甲醇-氯仿凝胶柱色谱洗脱得158.8mg的化合 物2。
其中，Fr.4-2经体积比为1:1的甲醇-氯仿凝胶柱色谱洗脱得78.9mg的化合 物3。
Fr.6经体积比为65:35的石油醚-乙酸乙酯硅胶柱色谱洗脱得到Fr.6-1，
其中，Fr.6-1经甲醇凝胶柱色谱洗脱得58.2mg的化合物4。
Fr.7经体积比为70:30的石油醚-乙酸乙酯硅胶柱色谱洗脱得到Fr.7-1，
其中，Fr.7-1经体积比为1:1的甲醇-氯仿凝胶柱色谱洗脱得84.6mg的化合 物5。
Fr.8分别经甲醇凝胶柱色谱洗脱得103.5mg的化合物6。
对上述化合物进行分析，其结果为：
化合物1：
为白色针状晶体(甲醇)；
其核磁共振氢谱数据为：
1H-NMR(600MHz,CD3OD)δ:5.60(s,H-C(14),1H),4.88(s,H-C(17),1H), 4.53(s,H-C(17),1H),2.41(m,H-C(7),1H),2.29(m,H-C(12),1H),2.13(m,H-C(2), 1H),2.12(d,J＝1.3Hz,3H),2.00(m,H-C(6,12),2H),1.89(m,H-C(1,3,6,7),4H),1.72(m,H-C(11),1H),1.63(m,H-C(9),1H),1.52(m,H-C(2,11),2H),1.19(s, H-C(18),3H),1.08(m,H-C(1,3,5),3H),0.64(s,H-C(20),3H)；
其核磁共振碳谱数据为：
13C-NMR(151MHz,CD3OD)δ:181.2(C＝O,C-19),170.3(C＝O,C-15),161.9 (C＝C,C-13)，149.5(C＝C,C-8)，116.8(C＝C,C-14)，106.9(C＝C,C-17),57.5(CH, C-5),56.7(CH,C-9),45.2(C,C-4),41.5(C,C-10),40.8(CH2,C-1),40.4(CH2,C-7), 39.9(CH2,C-3),39.3(CH2,C-12),29.6(CH3,C-18),27.6(CH2,C-6),22.9(CH2, C-11),21.2(CH2,C-2),18.9(CH3,C-16),13.4(CH3,C-20)。
其ESI-MS(m/z)为:333.2062[M-H]-给出分子离子峰，结合1H-NMR和13C-NMR谱推导其分子式为C20H30O4，根据具体的NMR数据，可鉴定出该化合 物为玛瑙酸(Agathic acid)，具有如下的结构式：
Figure SMS_3
化合物2：
为白色针状晶体(甲醇)；
其核磁共振氢谱数据为：
1H-NMR(600MHz,CD3OD)δ:6.91(d,J＝8.6Hz,H-C(2，6),2H),7.77(d, J＝8.6Hz,H-C(3,5),2H)；9.75(s,H-C(7),1H)；
其核磁共振碳谱数据为：
13C-NMR(151MHz,CD3OD)δ:193.0(C＝O,C-7),165.2(C＝C,C-4), 135.5(C＝C,C-2,6),130.3(C＝C,C-1),116.9(C＝C,C-3,5)；
其ESI-MS(m/z)为:121.0366[M-H]-给出分子离子峰，结合1H-NMR和13C-NMR谱推导其分子式为C7H6O2，根据具体的NMR数据，可鉴定出该化 合物为4-对羟基苯甲醛，结构式如下：
Figure SMS_4
化合物3：
白色针状晶体(甲醇)；
其核磁共振氢谱数据为：
1H-NMR(600MHz,CDCl3)δ:6.36(dd,J＝15.1,10.4Hz,H-C(7),1H), 6.16(ddd,J＝15.1,10.8,1.0Hz,H-C(14),1H),6.00(dd,J＝15.3,10.4Hz, H-C(8),1H),5.78(m,H-C(9,13),2H),5.48(dd,J＝13.6,6.4Hz,H-C(6),1H),5.45(m,H-C(15),1H),4.91(d,J＝7.9Hz,H-C(5),1H),4.57(s,H-C(2),1H), 3.51(m,H-C(3),1H),2.42(m,H-C(10),1H),2.07(m,H-C(11,16),2H),1.96(m,H-C(11),1H),1.29(m,H-C(17),2H),1.69(s,H-C(21),3H),1.45(s,H-C(19), 3H),0.98(d,J＝0.9Hz,H-C(22),3H),0.97(d,J＝0.9Hz,H-C(20),3H),0.84(d,J＝7.4Hz,H-C(18),3H)；
其核磁共振碳谱数据为：
13C-NMR(151MHz,CDCl3)δ:170.5(C＝O,C-1),145.1(C＝C,C-9),138.8 (C＝C,C-15),137.9(C＝C,C-7),133.6(C＝C,C-12),127.0(C＝C,C-13),126.1 (C＝C,C-8),124.6(C＝C,C-14),122.1(C＝C,C-6),80.2(C-O,C-5),68.0(C-OH, C-2),58.7(C-O,C-3),58.2(C-O,C-4),47.2(CH2,C-11),38.5(CH,C-16),34.8 (CH,C-10),29.8(CH2,C-17),20.1(CH3,C-22),19.4(CH3,C-20),17.7(CH3, C-19),16.5(CH3,C-20),11.7(CH3,C-18)；
其ESI-MS(m/z)为:361.2440[M+H]+给出分子离子峰，结合1H-NMR和13C-NMR谱推导其分子式为C22H32O4，根据具体的NMR数据，可鉴定出该 化合物为萘呋定(Nafuredin)，结构式如下：
Figure SMS_5
化合物4：
白色针状晶体(甲醇)；
其核磁共振氢谱数据为：
1H-NMR(600MHz,DMSO-D6)δ:5.98(s,H-C(5),1H),2.13(s,H-C(8), 3H),1.73(s,H-C(7),3H)；
其核磁共振碳谱数据为：
13C-NMR(151MHz,DMSO-D6)δ:165.0(C＝O,C-2),164.9(C＝C,C-4), 159.2(C＝C,C-6),99.7(C＝C,C-5),96.2(C＝C,C-3),19.1(CH3,C-8),8.2(CH3, C-7)。
其ESI-MS(m/z)为:139.0488[M-H]-给出分子离子峰，结合1H-NMR和13C-NMR谱推导其分子式为C7H8O3，根据具体的NMR数据，可鉴定出该化 合物为4-羟基-3,6-二甲基-2H-吡喃-2-酮，结构式如下：
Figure SMS_6
化合物5：
白色针状晶体(甲醇)；
其核磁共振氢谱数据为：
1H-NMR(600MHz,CDCl3)δ:4.77(s,H-C(4),1H),4.35(m,H-C(6),1H), 4.61(m,H-C(6),1H),2.67(ddd,J＝17.3,2.0,0.6Hz,H-C(4),2.53(d,J＝17.4 Hz,H-C(4),1H),1H),1.92(m,H-C(5),2H),1.40(s,H-C(7),3H)；
其核磁共振碳谱数据为：
13C-NMR(151MHz,CDCl3)δ:170.6(C＝O,C-2),68.2(C-O,C-4),66.0 (C-O,C-6),44.7(CH2,C-3),35.9(CH2,C-5),29.7(CH3,C-7)。
其ESI-MS(m/z)为:131.0932[M+H]+给出分子离子峰，结合1H-NMR和13C-NMR谱推导其分子式为C6H10O3，根据具体的NMR数据，可鉴定出该 化合物为甲瓦龙酸内酯，结构式如下：
Figure SMS_7
化合物6：
白色针状晶体(甲醇)；
其核磁共振氢谱数据为：
1H-NMR(600MHz,CD3OD)δ:7.47(dd,J＝15.6,3.0Hz,H-C(3),1H), 5.84(dd,J＝15.6,1.9Hz,H-C(10),1H),5.79-5.75(m,H-C(11),1H),5.29(dd,J ＝15.2,9.6Hz,H-C(2),1H),4.85-4.77(m,H-C(7),1H),4.26-4.20(m,H-C(4), 1H),4.05(ddd,J＝9.7,3.1,2.0Hz,H-C(15),1H),2.40(p,J＝8.4Hz,H-C(9), 1H),2.14(ddd,J＝13.6,8.7,5.4Hz,H-C(14),1H),2.07-1.98(m,H-C(12),2H),1.94-1.74(m,H-C(6,8,13,14),5H),1.64-1.56(m,H-C(8),1H),1.46(ddd,J＝ 13.3,7.5,3.1Hz,H-C(14),1H),1.26(d,J＝6.3Hz,H-C(16),3H),0.96-0.87(m, H-C(13),1H)；
其核磁共振碳谱数据为：
13C-NMR(151MHz,CD3OD)δ:167.0(C＝O,C-1),153.7(CH,C-3),136.7 (CH,C-10),130.0(CH,C-11),116.4(CH,C-2),75.2(CH,C-7),71.8(CH,C-4), 71.6(CH,C-15),51.8(CH,C-9),44.1(CH,C-5),42.7(CH2,C-14),40.5(CH2, C-12),33.6(CH2,C-8),31.6(CH2,C-6),26.6(CH2,C-13),19.6(CH3,C-16)；
其ESI-MS(m/z)为:279.1610[M-H]-给出分子离子峰，结合1H-NMR和13C-NMR谱推导其分子式为C16H24O4，根据具体的NMR数据，可鉴定出该 化合物为布雷菲尔德菌素A(Brefeldin A)，结构式如下：
Figure SMS_8
实施例3
对分离得到的代谢化合物进行杀虫活性筛选，如下：
将所得6种化合物配制为500μg·mL-1的溶液，通过浸渍法对其进行杀埃及伊 蚊3龄幼虫活性筛选。并以相同方法配制同等浓度鱼藤酮为对照组，以丙酮为空 白对照。
分别向一次性杯子中移入10mL配好的化合物药液或鱼藤酮溶液或丙酮溶液， 每个杯子里加入20只3龄埃及伊蚊幼虫，每个处理重复三次，观察记录12、24、 36、72h埃及伊蚊幼虫死亡数，统计死亡率，结果如下：
表1化合物1～6对埃及伊蚊3龄幼虫的致死率(
Figure SMS_9
n＝3)
Figure SMS_10
1)同列数据后不同小写字母表示差异显著(P<0.05，DMRT法)
2)-表示无毒杀作用。
可以看出，化合物Agathic acid(1)和Nafuredin(3)表现出不同程度的毒杀活性。随处理时间增加，不同化合物杀埃及伊蚊3龄幼虫死亡率均增加，处理72h 后，化合物Agathic acid(1)对埃及伊蚊3龄幼虫的致死率达到100％，化合物 Nafuredin(3)对埃及伊蚊3龄幼虫致死率达到80％以上。
实施例4
根据活性筛选结果，对化合物1(Agathic acid)和化合物3(Nafuredin)进 行毒力测定，包括：
(1)对埃及伊蚊3龄的LC50测试
准确称取该2种化合物各40mg，加1mL丙酮助溶，以脱氯水定容至10mL 为母液，其母液质量浓度为4mg·mL-1，取1mL母液加9mL脱氯水定容10mL， 得到400μg·mL-1的质量浓度，最终配制成质量浓度分别为400、300、200、100、 50μg·mL-1的药液，采用与所述活性筛选相同的处理方法，于72h观察记录死亡 虫数、测定LC50值，结果如下：
表2化合物1和3对埃及伊蚊3龄幼虫的LC50值
Figure SMS_11
可以看出，化合物Agathic acid(1)和Nafuredin(3)对埃及伊蚊3龄幼虫的毒 力在72h后均呈现清晰的剂量-效应关系，相关性良好。化合物Agathic acid(1) 和Nafuredin(3)对埃及伊蚊的LC50值分别为163.16μg·mL-1和243.55μg·mL-1。
(2)对家蝇成虫的LC50测试
称取1.00g蔗糖置于透明一次性杯子中备用，准确称取该2种化合物各40mg， 以丙酮定容至10mL为母液，其母液质量浓度为4mg·mL-1，取母液进行等差梯 度稀释，最终配制成质量浓度分别为2、1.8、1.6、1.4、1.2mg·mL-1的药液，通 过移液枪移取1mL配制好的药液于杯中，每个浓度设3个重复，通过相同的过程以同等浓度鱼藤酮为药效对照组，空白对照组用丙酮处理后置于风扇下吹12h， 使丙酮挥发完全。其后通过三角瓶抓取家蝇，纱布蒙口，将乙醚湿润的棉花置于 瓶口，麻醉后迅速计数20只家蝇放入杯中，纱布封口，纱布外放一润湿棉花球 供水，于72h观察记录死亡虫数、测定LC50值，结果如下：
表3化合物1和3对家蝇成虫的LC50值
Figure SMS_12
Figure SMS_13
可以看出，化合物Agathic acid(1)和Nafuredin(3)对家蝇成虫的毒力在72h 后均呈现清晰的剂量-效应关系，相关性良好。化合物Agathic acid(1)和Nafuredin (3)对家蝇成虫的LC50值分别为2.443mg·mL-1和1.260mg·mL-1。
(3)对斜纹夜蛾3龄幼虫的LC50测试
准确称取该2种化合物各40mg，以丙酮定容至10mL为母液，其母液质量 浓度为4mg·mL-1，取母液进行等比梯度稀释，最终配制成质量浓度分别为2、1、 0.5、0.25、0.125mg·mL-1的药液。采用饲料浸渍饲喂法，将质量一致的饲料在不 同质量浓度的药液中浸渍15s，晾干后放入培养皿(直径9cm)中，每皿移入30 头浸过药的斜纹夜蛾幼虫；通过相同的过程以同等浓度的鱼藤酮为药效对照组，以丙酮为空白对照，每个处理重复3次，皿上加盖；将培养皿置于避光处饲养， 于72h观察记录死亡虫数、测定LC50值，结果如下：
表4化合物1和3对斜纹夜蛾3龄幼虫的LC50值
Figure SMS_14
可以看出，化合物Agathic acid(1)和Nafuredin(3)对斜纹夜蛾3龄幼虫的毒 力在72h后均呈现清晰的剂量-效应关系，相关性良好。化合物Agathic acid(1) 和Nafuredin(3)对斜纹夜蛾3龄幼虫的LC50值分别为1.422mg·mL-1和1.735 mg·mL-1。
综上，次级化合物Agathic acid和Nafuredin对埃及伊蚊(Aedes aegypti)3 龄幼虫、家蝇成虫(Musca demestica)和斜纹夜蛾(Spodoptera litura)3龄幼虫 均表现出明显的毒杀活性。
以上实施例仅是本发明的优选实施方式，本发明的保护范围并不仅局限于上 述实施例。凡属于本发明思路下的技术方案均属于本发明的保护范围。应该指出， 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的改进和润 饰，这些改进和润饰也应视为本发明的保护范围。

Claims (1)

1.一种海洋真菌次级代谢物作为杀虫活性物的应用，其中，所述虫选自埃及伊蚊、家蝇、斜纹夜蛾中的一种或多种；所述海洋真菌次级代谢物具有如下式1的结构式：
Figure FDA0004256232470000011
CN202110035660.3A 2021-01-12 2021-01-12 海洋真菌次级代谢物及其制备与应用 Active CN112795617B (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202110035660.3A CN112795617B (zh) 2021-01-12 2021-01-12 海洋真菌次级代谢物及其制备与应用

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202110035660.3A CN112795617B (zh) 2021-01-12 2021-01-12 海洋真菌次级代谢物及其制备与应用

Publications (2)

Publication Number Publication Date
CN112795617A CN112795617A (zh) 2021-05-14
CN112795617B true CN112795617B (zh) 2023-07-07

Family

ID=75810050

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202110035660.3A Active CN112795617B (zh) 2021-01-12 2021-01-12 海洋真菌次级代谢物及其制备与应用

Country Status (1)

Country Link
CN (1) CN112795617B (zh)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113583881B (zh) * 2021-09-13 2023-03-21 福建省微生物研究所 一种发酵产布雷费尔德菌素a的正青霉菌株及其应用

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105602994B (zh) * 2016-04-08 2019-03-12 广东海洋大学 一种海洋真菌哈茨木霉dlen2008005的发酵提取物及其制备方法和应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Divergent synthesis of complex diterpenes through a hybrid oxidative approach;Zhang X等;Science;第369卷(第6505期);199-806 *

Also Published As

Publication number Publication date
CN112795617A (zh) 2021-05-14

Similar Documents

Publication Publication Date Title
Venkateswarulu et al. Isolation and characterization of plumbagin (5-hydroxyl-2-methylnaptalene-1, 4-dione) producing endophytic fungi Cladosporium delicatulum from endemic medicinal plants
CN107353274B (zh) 源于草酸青霉的黑麦酮酸i及制备抗人食管癌药物的应用
CN107298672B (zh) 源于草酸青霉的黑麦酮酸i在制备抗人结肠癌药物的应用
CN109970538A (zh) 一类海洋真菌来源的二倍半萜类化合物及其制备方法和在制备抗炎药物中的应用
CN110776518B (zh) Azaphilone类螺环化合物及其制备方法与应用
CN103183666B (zh) 一种环匹阿尼酸类化合物及其制备和应用
CN112795617B (zh) 海洋真菌次级代谢物及其制备与应用
CN108315264A (zh) 一种海漆内生真菌来源的聚酮化合物及其在制备抗炎药物中的应用
CN104098585B (zh) 米尔贝霉素类似物、其制备方法和应用
CN109295122A (zh) 一种窿缘桉内生真菌Chaetomium sp次生代谢产物的制备方法及用途
CN107603922B (zh) 海绵共生链霉菌及其发酵生产星形孢菌素的方法和应用
CN107417559B (zh) 一种倍半萜类化合物及其制备方法和应用
CN110330544A (zh) 一种4,4,1-双环甾类化合物及其制备方法和用途
CN109400444B (zh) 抑制植物病源真菌的倍半萜类化合物及其制备方法
CN113527247B (zh) 一种嗜氮酮类多聚体化合物及其制备方法和应用
CN111602662B (zh) 一种杀锥曲菌素的制备方法和应用
CN106831696A (zh) 大环内酯类衍生物及其制备方法和应用
CN103361276B (zh) 刺糖多胞菌hberc‑25376及其培养、活性物质分离方法与应用
CN114149445A (zh) 一种氧杂蒽酮类化合物的制备方法及其在抗耐药细菌方面的应用
CN114409624B (zh) 一种从哈茨木霉菌中分离得到的色酮类化合物及其制备方法和用途
CN110746338A (zh) 一种百部内生链霉菌中吡咯-2-羧酸酯类化合物的制备方法及其应用
CN115287236B (zh) 一类昆虫共生放线菌来源的生物碱化合物的制备方法和用途
CN104450528A (zh) 栀子内生真菌的分离筛选方法
CN117510451B (zh) 一种苯骈吡喃二聚体轮层炭菌素及其制备方法和应用
CN106967622A (zh) 一株产紫杉醇的黄曲霉bp6t2及其应用

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant