Identification of endophytic fungus Chaetomium sp. Eef-10 from Eucalyptus exserta and analysis of its active ingredients
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摘要:目的
确定内生真菌Eef-10的分类地位，分离和鉴定该真菌中的次生代谢产物并评价其抗细菌和抗肿瘤细胞活性，以期得到具有抗细菌和抗肿瘤活性的天然活性化合物。
方法内生真菌的鉴定采用形态学和分子生物学相结合的方法；次生代谢产物的分离和纯化主要采用减压硅胶柱层析、葡聚糖凝胶LH-20和半制备高效液相色谱等方法；化合物的鉴定主要依据1H NMR和13C NMR等波谱学数据以及相关的参考文献；采用MTT显色法测定了次生代谢产物对5种不同供试细菌的抑制活性；采用CCK8法测定了次生代谢产物对2种不同癌细胞的抑制活性。
结果从内生真菌Eef-10中分离到的3个化合物，分别鉴定为Atraric acid(化合物Ⅰ)、2, 4−二羟基−3, 6−二甲基苯甲酸乙酯(Ethyl 2, 4-dihydroxy-3, 6-dimethylbenzoate)(化合物Ⅱ)和4−甲基−5, 6−二氢−2H−吡喃−2−酮(4-methyl-5, 6-dihydro-2 H-pyran-2-one)(化合物Ⅲ)。化合物Ⅱ对5种革兰阴性细菌表现出强抑制活性，最大半数抑制质量浓度(IC50)为35.87~55.50 μg/mL，化合物Ⅰ的IC50为67.25~130.55 μg/mL，化合物Ⅲ的IC50均大于200 μg/mL。抗肿瘤细胞活性的测定结果表明，化合物Ⅱ对人肝癌细胞株Hep-G2的IC50为1.50 μg/mL，活性强于阳性对照喜树碱(IC50为3.6 μg/mL)。
结论从内生真菌Chaetomium sp. Eef-10中分离得到化合物Ⅰ~Ⅲ，化合物Ⅱ对桉树青枯病菌和Hep-G2细胞具有较好的抑制作用。
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关键词:
- 内生真菌 /
- Chaetomium sp. Eef-10 /
- 抗细菌活性 /
- 抗肿瘤细胞活性
Abstract:ObjectiveTo determine the classification status of endophytic fungus Eef-10. To isolate and identify secondary metabolites of Eef-10 and evaluate their antibacterial and antitumor activities in order to obtain natural active compounds.
MethodThe endophytic fungus was identified by combining morphology and molecular biology methods. The secondary metabolites were mainly separated and purified by vacuum silica gel column chromatography, sephadex LH-20 column chromatography and semi-preparative HPLC. The compounds were identified mainly based on 1H NMR and 13C NMR spectral data as well as related references. The antibacterial activities against five different test bacteria were determined by the MTT method and the antitumor activities against two cancer cells were determined by the CCK8 method.
ResultThree compounds were isolated from the endophytic fungus Eef-10, namely atraric acid (Compound Ⅰ), ethyl 2, 4-dihydroxy-3, 6-dimethylbenzoate (Compound Ⅱ) and 4-methyl-5, 6-dihydro-2 H-pyran-2-one (Compound Ⅲ). Compound Ⅱ displayed strong inhibitory activities against five test gram-negative bacteria, and the IC50 values were 35.87−55.50 μg/mL. The IC50 values of compound Ⅰ were 67.25−130.55 μg/mL, while compound Ⅲ had IC50 values of more than 200 μg/mL for all five bacteria. The IC50 value of compound Ⅱ for Hep-G2 was 1.50 μg/mL, which was stronger than the positive control camptothecin of 3.6 μg/mL.
ConclusionCompounds Ⅰ−Ⅲ were isolated from the endophytic fungus Chaetomium sp. Eef-10 and compound Ⅱ showed great antibacterial and antitumor activities on R. solanacearum and Hep-G2 tumor cells.
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图 1 内生真菌Eef-10的菌落(a)、菌丝(b)、子囊孢子(c)和子囊果(d)
Figure 1. Colony (a), mycelium (b), ascospore (c) and ascoma (d) of endophytic fungus Eef-10
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图 2 依据内生真菌Eef-10的rDNA-ITS 序列构建的系统发育树
Figure 2. Phylogenetic tree of endophytic fungus Eef-10 based on rDNA-ITS sequence
表 1 内生真菌Eef-10次生代谢产物的抗细菌活性
Table 1 Antibacterial activities of the secondary metabolites isolated from endophytic fungus Eef-10
供试样品
Tested sampleIC50/(μg·mL−1) 大肠埃希菌
Escherichia
coli根癌土壤杆菌
Agrobacterium
tumefaciens黄瓜角斑病菌
Pseudomonas
lachrymans桉树青枯病菌
Ralstonia
solanacearum番茄疮痂病菌
Xanthomonas
vesicatoria化合物Ⅰ Compound Ⅰ 130.55 ± 3.57 105.39 ± 4.52 67.25 ± 1.23 113.11 ± 3.58 93.59 ± 0.43 化合物Ⅱ Compound Ⅱ 48.52 ± 0.33 55.50 ± 1.61 38.91 ± 0.54 35.87 ± 0.18 40.80 ± 0.70 化合物Ⅲ Compound Ⅲ > 200 > 200 > 200 > 200 > 200 硫酸链霉素 Streptomycin sulfate 18.51 ± 0.46 5.10 ± 0.03 30.54 ± 0.89 33.07 ± 2.46 13.81 ± 1.62 
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表 2 内生真菌Eef-10次生代谢产物的抗肿瘤细胞活性
Table 2 Antitumor activities of the secondary metabolites isolated from endophytic fungus Eef-10
供试细胞Tested cell IC50/(μg·mL−1) 化合物Ⅰ
Compound Ⅰ化合物Ⅱ
Compound Ⅱ化合物Ⅲ
Compound Ⅲ喜树碱
CamptothecinHep-G2 > 50 1.50 > 50 3.6 HeLa > 50 > 50 > 50 6.3
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