活性分子定点识别技术筛选海洋真菌次级代谢产物的研究
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摘要
本研究设计了一种双模型活性分子定点识别技术,利用HPLC图谱与活性识别谱一一对应即可起到生物活性分子测定定点分离的作用,以此筛选海洋真菌代谢产物中的活性成分。本文得出如下几点主要结论:
(1)将分离自中国东海海域海水样品中的一株海洋真菌编号为ENP701#作为实验研究对象,根据形态学评估初步鉴定其为青霉属真菌(Penicillium sp.),并将其大规模培养分离制备菌体和发酵液提取物浸膏。
(2)采用GC-MS联用仪对不同培养时间的海洋真菌ENP701#发酵液提取样品的挥发性成分进行分析,结果显示主要有芳香族、长链烷烃、生物碱、香豆素和酯类等化合物且不同培养时间下挥发性成分组成和含量不尽相同。
(3)建立海洋真菌ENP701#菌体和发酵液的双模型活性分子定点识别谱图,以此筛选其中的生物活性分子,为后续活性分子的定点分离纯化作参考。在建立抗肿瘤活性分子定点识别谱中,采用H460、PC-12、A549、3T3、HT1080和Hela等6株肿瘤细胞进行筛选,抗氧化活性分子定点识别谱建立在清除自由基DPPH的模型基础上。最终以抗H460肿瘤活性分子定点识别谱为主,抗氧化活性分子定点识别谱为辅建立了菌体和发酵液浸膏的双模型活性分子定点识别谱。
(4)基于已建立的双模型分子定点识别谱,根据确定的最佳HPLC条件,采用中压制备仪直接进行菌体和发酵液浸膏中目标微量分子的定点分离,最终从菌体浸膏中分离到化合物1-6,分别为硫酸酯胆碱(1)、己六醇(2)、丁四醇(3)、喹啉生物碱的二聚物盐(4)、5 ,7 ,22-三烯-3β-羟基-麦角甾(5)、硬脂酸(6)。化合物4为新化合物,其对肿瘤细胞H460显示了明显的抑制活性,其IC_(50)值为19.38±0.15μg·mL~(-1)。此外化合物1-6均显示了清除自由基DPPH的活性,说明化合物1-6均有不同程度的抗氧化活性。
The bioactivity directional screening technology with dual-mode has been designed in this paper, using HPLC correspond with bioactivity profile to identify biological active components, with a view to finding active natural products from marine-derived fungus. Several main conclusions are drawn as follows:
(1) Marine fungus ENP701# which was isolated from seawater sample collected from the East China Sea was analyzed as experimental subjects in this study. It was identified to be Penicillium sp. species based on the morphological evaluation. After large-scale cultivation of marine fungus ENP701#, the mycelium and culture fluid were separated by filtration, and extract were gained, respectively.
(2) The volatile components of EtOAc extract from culture fluid of marine fungus ENP701# were analyzed by GC-MS at different culture time. The results revealed that volatile components from EtOAc extract were mainly complex and aromatic compounds, such as long-chain alkanes, alkaloids, coumarin and ester compounds. Marine-derived fungus can produce different volatile components. The volatile components from marine- derived fungus were different at the different culture time.
(3) The bioactivity directional screening technology with dual-mode of mycelium and culture fluid extract has been established to provide referrence for follow-up seperation and purification of compounds, with a view to screening active natural products from metabolites of marine- derived fungus. During establishment of anti-tumor activity directional screening model, six tumor cell lines including H460, PC-12, A549, 3T3, HT1080 and Hela were selected in order to screen appropriate cell lines. The anti-oxidative activity directional screening model was set up based on DPPH free radical scavenging detection. The bioactivity directional screening with dual-mode of mycelium and culture fluid extract were established mainly on H460 cell inhibition, assisted by DPPH radical scavenging detection profiles eventually.
(4) Based on the bioactivity directional screening spectrum with dual-mode of mycelium and culture fluid extract, six target bioactive compounds were directly isolated from the mycelium extract with middle pressure preparation instrument according to the best conditions of HPLC. They were Choline-O-Sulphate(1), 1,2,3,4,5,6-hexanehexanol(2), 1,2,3,4- erythritol(3), complex quinoline alkaloid(4), 5,7,22- triene-3β-hydroxy- ergosterol(5), stearic acid (6), respectively. Compounds 4 was a new compound, it showed significant inhibitory activity against H460 tumor cells with IC_(50) value of 19.38±0.15μg·mL~(-1). In addition, all the compounds revealed scavenging activity on DPPH free radical so that compounds 1-6 had different degrees of anti-oxidative activity.
(1)将分离自中国东海海域海水样品中的一株海洋真菌编号为ENP701#作为实验研究对象,根据形态学评估初步鉴定其为青霉属真菌(Penicillium sp.),并将其大规模培养分离制备菌体和发酵液提取物浸膏。
(2)采用GC-MS联用仪对不同培养时间的海洋真菌ENP701#发酵液提取样品的挥发性成分进行分析,结果显示主要有芳香族、长链烷烃、生物碱、香豆素和酯类等化合物且不同培养时间下挥发性成分组成和含量不尽相同。
(3)建立海洋真菌ENP701#菌体和发酵液的双模型活性分子定点识别谱图,以此筛选其中的生物活性分子,为后续活性分子的定点分离纯化作参考。在建立抗肿瘤活性分子定点识别谱中,采用H460、PC-12、A549、3T3、HT1080和Hela等6株肿瘤细胞进行筛选,抗氧化活性分子定点识别谱建立在清除自由基DPPH的模型基础上。最终以抗H460肿瘤活性分子定点识别谱为主,抗氧化活性分子定点识别谱为辅建立了菌体和发酵液浸膏的双模型活性分子定点识别谱。
(4)基于已建立的双模型分子定点识别谱,根据确定的最佳HPLC条件,采用中压制备仪直接进行菌体和发酵液浸膏中目标微量分子的定点分离,最终从菌体浸膏中分离到化合物1-6,分别为硫酸酯胆碱(1)、己六醇(2)、丁四醇(3)、喹啉生物碱的二聚物盐(4)、5 ,7 ,22-三烯-3β-羟基-麦角甾(5)、硬脂酸(6)。化合物4为新化合物,其对肿瘤细胞H460显示了明显的抑制活性,其IC_(50)值为19.38±0.15μg·mL~(-1)。此外化合物1-6均显示了清除自由基DPPH的活性,说明化合物1-6均有不同程度的抗氧化活性。
The bioactivity directional screening technology with dual-mode has been designed in this paper, using HPLC correspond with bioactivity profile to identify biological active components, with a view to finding active natural products from marine-derived fungus. Several main conclusions are drawn as follows:
(1) Marine fungus ENP701# which was isolated from seawater sample collected from the East China Sea was analyzed as experimental subjects in this study. It was identified to be Penicillium sp. species based on the morphological evaluation. After large-scale cultivation of marine fungus ENP701#, the mycelium and culture fluid were separated by filtration, and extract were gained, respectively.
(2) The volatile components of EtOAc extract from culture fluid of marine fungus ENP701# were analyzed by GC-MS at different culture time. The results revealed that volatile components from EtOAc extract were mainly complex and aromatic compounds, such as long-chain alkanes, alkaloids, coumarin and ester compounds. Marine-derived fungus can produce different volatile components. The volatile components from marine- derived fungus were different at the different culture time.
(3) The bioactivity directional screening technology with dual-mode of mycelium and culture fluid extract has been established to provide referrence for follow-up seperation and purification of compounds, with a view to screening active natural products from metabolites of marine- derived fungus. During establishment of anti-tumor activity directional screening model, six tumor cell lines including H460, PC-12, A549, 3T3, HT1080 and Hela were selected in order to screen appropriate cell lines. The anti-oxidative activity directional screening model was set up based on DPPH free radical scavenging detection. The bioactivity directional screening with dual-mode of mycelium and culture fluid extract were established mainly on H460 cell inhibition, assisted by DPPH radical scavenging detection profiles eventually.
(4) Based on the bioactivity directional screening spectrum with dual-mode of mycelium and culture fluid extract, six target bioactive compounds were directly isolated from the mycelium extract with middle pressure preparation instrument according to the best conditions of HPLC. They were Choline-O-Sulphate(1), 1,2,3,4,5,6-hexanehexanol(2), 1,2,3,4- erythritol(3), complex quinoline alkaloid(4), 5,7,22- triene-3β-hydroxy- ergosterol(5), stearic acid (6), respectively. Compounds 4 was a new compound, it showed significant inhibitory activity against H460 tumor cells with IC_(50) value of 19.38±0.15μg·mL~(-1). In addition, all the compounds revealed scavenging activity on DPPH free radical so that compounds 1-6 had different degrees of anti-oxidative activity.
引文
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