白木香内生真菌AS5化学成分与液体发酵工艺研究
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摘要
真菌AS5为分离白瑞香科沉香属植物白木香[Aquilaria sinensis(Lour.)Gilg]茎部的一株内生真菌，经鉴定为光黑壳属(Preussia sp.)真菌，其粗提物具有抗菌活性。本论文应用现代色谱及波谱技术，对真菌AS5的化学成分进行了研究，结合抗菌活性的跟踪，确定了该菌株的抗菌活性成分CH；应用HPLC技术，对活性成分CH的HPLC分析方法进行了初步研究；以真菌AS5的生物量和抗菌成分CH的含量为指标，采用单因子试验和正交试验，对该菌的液体发酵工艺条件进行了系统研究。
(一)从真菌AS5中分离得到了分离得到了13个单体化合物，鉴定了其中的12个，它们分别为：螺光黑壳菌酮A(CH)、螺光黑壳菌酮B(EA2)、螺光黑壳菌素A(EA3)、9-羟基苯嵌萘酮(PE2)、麦角甾醇(PE1)、琥珀酸(EA1)、5-羟甲基糠醛(EA6)、D-甘露醇(B1)、阿洛糖醇(B2)、葡寡糖(B3)、尿嘧啶核苷(B5)、腺嘌呤核苷(B6)，其中化合物CH、EA2、EA3为新化合物，PE2为首次从该属真菌中分离得到的化合物。
(二)化合物CH具有抗菌和抗肿瘤活性，其对金黄色葡萄球菌(Staphylococcus aureus)的最低抑菌浓度(MIC)为8μg／mL；对人肝癌Bel-7402，人卵巢癌A2780细胞的半数抑制浓度(IC_(50))分别为：0.95，0.77μg／mL。
(三)建立了真菌AS5抗菌活性有效成分CH的HPLC定性和定量方法
(四)真菌AS5液体发酵的最佳工艺条件为：培养基组成：葡萄糖20g／L，麦麸15g／L，KH_2PO_4 3g／L，MgSO_4·7H_2O 1.5g／L，初始pH 7；接种量：Φ9mm菌片4片；培养基装量：100-125mL／250mL三角瓶；培养条件：培养温度25-28℃，摇床转速120rpm，发酵周期16天，暗培养。在这一条件下培养，活性成CH的含量可达25.02mg／瓶。
以上工作的开展，为真菌AS5的开发和利用奠定了理论基础。
An endophytic fungus (No. AS5) isolated from Aquilaria sinensis (Lour.) Gilg was identified asPreussia sp. which showed antimicrobial activities. On the basis of classical chemical methods andmodern spectroscopic analysis the chemical, constituents of Preussia sp. were studied guided by theantimicrobial activity screening. Compound CH was found to be the antibiotic component in Preussiasp. It's HPLC analysis method was studied, which established the basis of liquid fermentation optimization.Thirteen compounds were isolated from the fermentation material of Preussia sp. and twelve of themwere determined. They were spiro-preussione A (CH), spiro-preussione B (EA_2), spiro-preussomerin A(EA3), 9-hydroxyphenalenone (PE2), ergosterol (PE1), butanedioic acid (EA1), 5-hydroxymethyle-2-furfurahede (EA6), D-mannitol (B1), D-allitol (B2), B3, uridine (B5), adenosine (B6). Compund Ctt,EA2, EA3 were new compounds and compound PE2 was firstly isolated from Preussia sp.
Compounds CH showed moderately activity aganist Staphylococcus aureus (MIC: 8μg/mL), whichalso suggested cytotoxic activity against Bel-7402 (human liver tumor cell line) and A2780 (humanovarian tumor cell line) with IC_(50) values of 0.95 and 0.77μg/mL, respectively.
The suitabale medium and culture condition for fungus AS5 to accumulate compound CH wereD-glucose 20 g/L, wheat bran 15 g/L, KH2PO4 3 g/L, MgSO4 1.5 g/L, incipient pH 7, inoculation 4-8fungal plate (Φ9 mm), 100-125 mL medium/250 ml triangular flask, temperature 25-28℃, rate ofrotation 120 rpm, culturing for 16 days in dark.
(一)从真菌AS5中分离得到了分离得到了13个单体化合物，鉴定了其中的12个，它们分别为：螺光黑壳菌酮A(CH)、螺光黑壳菌酮B(EA2)、螺光黑壳菌素A(EA3)、9-羟基苯嵌萘酮(PE2)、麦角甾醇(PE1)、琥珀酸(EA1)、5-羟甲基糠醛(EA6)、D-甘露醇(B1)、阿洛糖醇(B2)、葡寡糖(B3)、尿嘧啶核苷(B5)、腺嘌呤核苷(B6)，其中化合物CH、EA2、EA3为新化合物，PE2为首次从该属真菌中分离得到的化合物。
(二)化合物CH具有抗菌和抗肿瘤活性，其对金黄色葡萄球菌(Staphylococcus aureus)的最低抑菌浓度(MIC)为8μg／mL；对人肝癌Bel-7402，人卵巢癌A2780细胞的半数抑制浓度(IC_(50))分别为：0.95，0.77μg／mL。
(三)建立了真菌AS5抗菌活性有效成分CH的HPLC定性和定量方法
(四)真菌AS5液体发酵的最佳工艺条件为：培养基组成：葡萄糖20g／L，麦麸15g／L，KH_2PO_4 3g／L，MgSO_4·7H_2O 1.5g／L，初始pH 7；接种量：Φ9mm菌片4片；培养基装量：100-125mL／250mL三角瓶；培养条件：培养温度25-28℃，摇床转速120rpm，发酵周期16天，暗培养。在这一条件下培养，活性成CH的含量可达25.02mg／瓶。
以上工作的开展，为真菌AS5的开发和利用奠定了理论基础。
An endophytic fungus (No. AS5) isolated from Aquilaria sinensis (Lour.) Gilg was identified asPreussia sp. which showed antimicrobial activities. On the basis of classical chemical methods andmodern spectroscopic analysis the chemical, constituents of Preussia sp. were studied guided by theantimicrobial activity screening. Compound CH was found to be the antibiotic component in Preussiasp. It's HPLC analysis method was studied, which established the basis of liquid fermentation optimization.Thirteen compounds were isolated from the fermentation material of Preussia sp. and twelve of themwere determined. They were spiro-preussione A (CH), spiro-preussione B (EA_2), spiro-preussomerin A(EA3), 9-hydroxyphenalenone (PE2), ergosterol (PE1), butanedioic acid (EA1), 5-hydroxymethyle-2-furfurahede (EA6), D-mannitol (B1), D-allitol (B2), B3, uridine (B5), adenosine (B6). Compund Ctt,EA2, EA3 were new compounds and compound PE2 was firstly isolated from Preussia sp.
Compounds CH showed moderately activity aganist Staphylococcus aureus (MIC: 8μg/mL), whichalso suggested cytotoxic activity against Bel-7402 (human liver tumor cell line) and A2780 (humanovarian tumor cell line) with IC_(50) values of 0.95 and 0.77μg/mL, respectively.
The suitabale medium and culture condition for fungus AS5 to accumulate compound CH wereD-glucose 20 g/L, wheat bran 15 g/L, KH2PO4 3 g/L, MgSO4 1.5 g/L, incipient pH 7, inoculation 4-8fungal plate (Φ9 mm), 100-125 mL medium/250 ml triangular flask, temperature 25-28℃, rate ofrotation 120 rpm, culturing for 16 days in dark.
引文
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[1] 陈树思，唐为萍：白木香Aquilaria sinerisis次生术质部导管分子观察研究。韩山师范学院学报，2004，25(3)：81-84。
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