平盖灵芝生物活性物质的分离纯化及萃取动力学研究
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摘要
灵芝俗称灵芝草，古称瑞草，在中国的临床应用历史有两千多年，是一种传统中药。本论文对平盖灵芝(G.applanatum (pers) pat)生物活性组分进行分离和纯化，并分析超声和磁化外场存在条件下的萃取动力学。主要研究包括四个方面的内容：
1．声磁辅助萃取平盖灵芝子实体活性组分及声磁萃取动力学研究。分析超声功率、磁场强度、萃取时间和固液比对萃取平盖灵芝活性组分过程的影响，通过四因素二次回归正交旋转组合实验设计和响应面分析，对声磁萃取平盖灵芝子实体工艺条件进行探讨和优化。结果表明：声磁辅助萃取可以快速有效地获取平盖灵芝子实体的生物活性组分，不仅增加了生物碱的溶出率，同时一些小分子低极性的易挥发化合物也损失很少；优化的萃取工艺参数为：超声功率0.40w·cm-2、磁场强度0.196T、萃取时间68.59min和物料比为18.89g/mL，此时平盖灵芝的子实体萃取率为13.89%；声磁条件下的萃取过程受到了涡流扩散效应的影响，研究得到的声磁萃取动力学方程既适合于平盖灵芝子实体总灵芝酸的萃取，也适用于以总萃取率为研究目标的中药声磁萃取过程。
2．平盖灵芝富萜菌丝体培养及灵芝酸含量监测。采用先摇床后长时间静置相结合的培养方法，分析培养过程中培养时间，培养基pH值，培养温度和摇床转速对平盖灵芝菌丝体灵芝酸含量的影响。运用RT-PCR技术监测菌丝体细胞内灵芝酸合成过程，通过检测灵芝酸合成相关调控基因(Gl-hmt, Gl-hmgr, Gl-mvd, Gl-fps, Gl-sqs, Gl-osc, Gl-gpd)的表达水平进而获得菌丝体细胞内灵芝酸含量的变化。结果表明：培养时间和摇床转速是影响菌丝体生物量和灵芝酸含量的主要因素，偏酸的培养条件更适合菌丝体的生长和灵芝酸的生成；RT-PCR分子生物学方法对富萜菌丝体培养过程中灵芝酸含量变化的监测准确有效。
3．平盖灵芝萃取组分的分离和纯化。研究平盖灵芝子实体和菌丝体萃取组分的硅胶柱和HPLC的分离和纯化，依次用石油醚、氯仿、乙酸乙酯、甲醇等对浸膏进行分级淋洗过硅胶柱，得到的组分经过筛选后进行HPLC分离和纯化；运用NMR、HPLC/MS等手段，对实验中得到的单体化合物进行结构分析和确定。本论文从平盖灵芝子实体浸膏纯化得到7种化合物单体，其中1种是首次从灵芝中得到的倍半萜化合物；通过半制备HPLC从平盖灵芝的子实体中分离到4种灵芝酸化合物，菌丝体中分离到4种灵芝酸化合物。
4．平盖灵芝萃取组分的生物活性研究。对平盖灵芝子实体和菌丝体的分离组分进行癌细胞致死率、修复药物和环境毒物导致的肝脏损伤修复及抗炎症的生物活性研究，并对具有生物活性的组分进行分析。结果共检测到具有较高生物活性的单体化合物11种及活性组分19种；平盖灵芝萃取组分和化合物对癌细胞具有明显的致死作用，且呈剂量效应，其机理主要是通过p53-JNK线粒体途径促使癌细胞凋亡；对抗癌药物及环境毒物导致的肝损伤及炎症具有很好的修复作用。
G.applanatum(pers) pat also called Lingzhi, In this paper, G. applanatum was extracted byseveral solvents. Biological active substances were isolated from the extracts of G. applanatum.The ultrasound magnetic extraction kinetics of G. applanatum were analyzed. Content of thepaper consists of four aspects:
1. The fruiting bodies of G. applanatum was extracted by using the ultrasound magneticgenerator and the kinetics was studied. The parameters(extraction time, magnetic intensity,ultrasound frequency, material ratio), which impact on the compounds in extracts of G.applanatum were evaluated. The extraction kinetics of G. applanatum were analyzed. Theeffects using the ultrasound magnetic generator, Soxhlet extraction were compared. The resultsshowed that bioactive substances can be effectively extracted from G. applanatum fruitingbodies using the ultrasound magnetic generator. The extraction process can be described by theextraction kinetics.
2. The culture process of terpene-rich G. applanatum mycelium was improved. Effect ofmetal trace elements culture time, pH, table speed and temperature on the G. applanatumterpenoid content was analyzed.The produce terpenoid related gene expression levels inmycelium cells were detected using semi-quantitative RT-PCR technique. The results showedthat of mycelium significantly effect on content of terpenoids in mycelium. The emulsionobtained as described above was homogeneous and the result to detect terpenoid content by themolecular biology methods was correspondent to the HPLC area normalization method methods.
3. The extracts of G. applanatum were purified by applying to a silica gel column andHPLC. The extracts were eluted using a silica gel column with petroleum ether, acetone, ethylacetate, methanol. The soluble fractions are analyzed by HPLC. The fifteen pure compoundswere isolated from the extracts of G. applanatum fruiting body and mycelium. The one purecompound was for the first isolated from G. applanatum.
4. The biological active of the extracts of G. applanatum was detected in cells and animaltissue. The inhibition effect of G. applanatum extract on human cancer cell lines and apoptosismechanism was investigated. The data showed that the extracts of G. applanatum induced cancercell apoptosis process by p53/JNU/mitochondria pathway. In this study, we also evaluated theprotective effect of G. applanatum extract against cisplatinum or BaP induced oxidative stressand inflammation in mouse liver, and explored the potential mechanism of its action. The resultssuggested that G. applanatum extract could protect the mouse liver against cisplatinum andbenzo(a) pyrene induced injury by improving hepatic function, attenuating histopathologicchanges, decreasing levels of ROS and MDA, renewing the activities of antioxidant enzymes andsuppressing inflammatory response.
1．声磁辅助萃取平盖灵芝子实体活性组分及声磁萃取动力学研究。分析超声功率、磁场强度、萃取时间和固液比对萃取平盖灵芝活性组分过程的影响，通过四因素二次回归正交旋转组合实验设计和响应面分析，对声磁萃取平盖灵芝子实体工艺条件进行探讨和优化。结果表明：声磁辅助萃取可以快速有效地获取平盖灵芝子实体的生物活性组分，不仅增加了生物碱的溶出率，同时一些小分子低极性的易挥发化合物也损失很少；优化的萃取工艺参数为：超声功率0.40w·cm-2、磁场强度0.196T、萃取时间68.59min和物料比为18.89g/mL，此时平盖灵芝的子实体萃取率为13.89%；声磁条件下的萃取过程受到了涡流扩散效应的影响，研究得到的声磁萃取动力学方程既适合于平盖灵芝子实体总灵芝酸的萃取，也适用于以总萃取率为研究目标的中药声磁萃取过程。
2．平盖灵芝富萜菌丝体培养及灵芝酸含量监测。采用先摇床后长时间静置相结合的培养方法，分析培养过程中培养时间，培养基pH值，培养温度和摇床转速对平盖灵芝菌丝体灵芝酸含量的影响。运用RT-PCR技术监测菌丝体细胞内灵芝酸合成过程，通过检测灵芝酸合成相关调控基因(Gl-hmt, Gl-hmgr, Gl-mvd, Gl-fps, Gl-sqs, Gl-osc, Gl-gpd)的表达水平进而获得菌丝体细胞内灵芝酸含量的变化。结果表明：培养时间和摇床转速是影响菌丝体生物量和灵芝酸含量的主要因素，偏酸的培养条件更适合菌丝体的生长和灵芝酸的生成；RT-PCR分子生物学方法对富萜菌丝体培养过程中灵芝酸含量变化的监测准确有效。
3．平盖灵芝萃取组分的分离和纯化。研究平盖灵芝子实体和菌丝体萃取组分的硅胶柱和HPLC的分离和纯化，依次用石油醚、氯仿、乙酸乙酯、甲醇等对浸膏进行分级淋洗过硅胶柱，得到的组分经过筛选后进行HPLC分离和纯化；运用NMR、HPLC/MS等手段，对实验中得到的单体化合物进行结构分析和确定。本论文从平盖灵芝子实体浸膏纯化得到7种化合物单体，其中1种是首次从灵芝中得到的倍半萜化合物；通过半制备HPLC从平盖灵芝的子实体中分离到4种灵芝酸化合物，菌丝体中分离到4种灵芝酸化合物。
4．平盖灵芝萃取组分的生物活性研究。对平盖灵芝子实体和菌丝体的分离组分进行癌细胞致死率、修复药物和环境毒物导致的肝脏损伤修复及抗炎症的生物活性研究，并对具有生物活性的组分进行分析。结果共检测到具有较高生物活性的单体化合物11种及活性组分19种；平盖灵芝萃取组分和化合物对癌细胞具有明显的致死作用，且呈剂量效应，其机理主要是通过p53-JNK线粒体途径促使癌细胞凋亡；对抗癌药物及环境毒物导致的肝损伤及炎症具有很好的修复作用。
G.applanatum(pers) pat also called Lingzhi, In this paper, G. applanatum was extracted byseveral solvents. Biological active substances were isolated from the extracts of G. applanatum.The ultrasound magnetic extraction kinetics of G. applanatum were analyzed. Content of thepaper consists of four aspects:
1. The fruiting bodies of G. applanatum was extracted by using the ultrasound magneticgenerator and the kinetics was studied. The parameters(extraction time, magnetic intensity,ultrasound frequency, material ratio), which impact on the compounds in extracts of G.applanatum were evaluated. The extraction kinetics of G. applanatum were analyzed. Theeffects using the ultrasound magnetic generator, Soxhlet extraction were compared. The resultsshowed that bioactive substances can be effectively extracted from G. applanatum fruitingbodies using the ultrasound magnetic generator. The extraction process can be described by theextraction kinetics.
2. The culture process of terpene-rich G. applanatum mycelium was improved. Effect ofmetal trace elements culture time, pH, table speed and temperature on the G. applanatumterpenoid content was analyzed.The produce terpenoid related gene expression levels inmycelium cells were detected using semi-quantitative RT-PCR technique. The results showedthat of mycelium significantly effect on content of terpenoids in mycelium. The emulsionobtained as described above was homogeneous and the result to detect terpenoid content by themolecular biology methods was correspondent to the HPLC area normalization method methods.
3. The extracts of G. applanatum were purified by applying to a silica gel column andHPLC. The extracts were eluted using a silica gel column with petroleum ether, acetone, ethylacetate, methanol. The soluble fractions are analyzed by HPLC. The fifteen pure compoundswere isolated from the extracts of G. applanatum fruiting body and mycelium. The one purecompound was for the first isolated from G. applanatum.
4. The biological active of the extracts of G. applanatum was detected in cells and animaltissue. The inhibition effect of G. applanatum extract on human cancer cell lines and apoptosismechanism was investigated. The data showed that the extracts of G. applanatum induced cancercell apoptosis process by p53/JNU/mitochondria pathway. In this study, we also evaluated theprotective effect of G. applanatum extract against cisplatinum or BaP induced oxidative stressand inflammation in mouse liver, and explored the potential mechanism of its action. The resultssuggested that G. applanatum extract could protect the mouse liver against cisplatinum andbenzo(a) pyrene induced injury by improving hepatic function, attenuating histopathologicchanges, decreasing levels of ROS and MDA, renewing the activities of antioxidant enzymes andsuppressing inflammatory response.
引文
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