茯苓三萜类成分抗肿瘤活性研究与茯苓药材质量分析
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摘要
茯苓Poria cocos (Schw.) Wolf为多孔菌科真菌类药食两用传统中药,收载于历年版《中国药典》,具有渗湿利尿、和胃健脾、宁心安神之功效,是中医临床用于防治肿瘤疾患的常用中药。现代药理研究表明,茯苓具有抗肿瘤、调节免疫功能的良好生物活性,其主要有效成分为多糖和三萜类物质。茯苓多糖抗肿瘤作用研究的文献报道较多,其三萜类物质抗肿瘤活性研究的报道相对较少。本论文在实验室前期研究的基础上,以茯苓三萜提取物(总三萜含量达90%以上)为研究对象,对茯苓三萜抗肿瘤的生物活性进行了较全面的研究,并对不同菌种栽培茯苓药材的质量评价进行较系统的研究。以期进一步诠释茯苓抗肿瘤的药效物质基础,并为提高栽培茯苓药材的品质提供科学依据。
1茯苓三萜抗肿瘤生物活性研究
以多种肿瘤细胞作为受试对象,采用MTT法对茯苓三萜抗肿瘤的生物活性进行了研究。研究结果表明,茯苓三萜对人肝癌(HepG2)株‘、肺癌(A549)株、乳腺癌(MDA-MB-231)株、宫颈癌(Caski)株、神经瘤母细胞(SH-SY5Y)株均有明显的抑制增殖作用。本项研究证明了茯苓三萜对多种肿瘤细胞增殖具有抑制作用,其中人肝癌细胞株HepG2、神经瘤母细胞SH-SY5Y,宫颈癌细胞株caski经药物高浓度(100μg/ml)处理48h、72h,抑制率高达90%以上。
2茯苓三萜抗肿瘤作用机理研究
采用细胞凋亡的形态学观察、琼脂糖凝胶电泳分析凋亡细胞DNA片段化和流式细胞仪检测凋亡率等现代研究方法,对茯苓三萜抗人肝癌细胞HepG2作用机制进行了研究,倒置显微镜和荧光显微镜观察表明,茯苓三萜处理后的HepG2细胞,其形态发生明显的变化,细胞数量减少,可见致密浓染,颜色有些发白的细胞核,说明茯苓三萜具有较强的诱导肝癌细胞凋亡作用；琼脂糖凝胶电泳分析凋亡的细胞DNA片段化研究结果表明,不同浓度茯苓三萜处理HepG2细胞24、48h后,能检测到凋亡中晚期的DNA片段(180-200bp整数倍的寡核苷酸片段)。流式细胞仪检测结果表明,40μg/ml、60μg/ml、80μg/ml、100μg/ml茯苓三萜处理HepG2细胞36h后,其细胞的凋亡率分别为7.2%、12.8%、39.3%、56%；说明茯苓三萜对HepG2细胞具有与药物浓度呈正相关的诱导凋亡作用。
3茯苓三萜化学成分的分离鉴定与药理筛选研究
采用现代色谱技术和波谱技术,对茯苓三萜的化学成分进行分离鉴定,从中分离和鉴定了8个三萜类化合物,分别为茯苓酸(poria acid)、去氢茯苓酸(Dehydropachymic acid)、3β-羟基-16α-乙酰氧基-羊毛甾-7,9(11),24-三烯-21-酸(3β-hydroxy-16a-acetyloxy-lanosta-7,9(11),24-trien-21-oic-acid)、去氢依布里酸(Dehydroeburicoic acid)、3β-羟基-羊毛甾烷-7,9(11),24-三烯-21-酸(3β-hydroxy-lanosta-7,9(11),24-trien-21-oic-acid)3β-羟基-羊毛甾-8,24-二烯-21-酸(Trametenolic acid)、15α-羟基-去氢土莫酸(15α-hydroxydehydrotumulosic acid)、苓酸C(polyporenic acidC)。通过MTT法对8个三萜类化合物进行了抗肿瘤活性评价,结果表明8个三萜类化合物在高浓度时具有不同程度的抗肿瘤活性。因此本项研究进一步诠释了茯苓抗肿瘤的物质基础。
4不同菌种栽培茯苓药材的质量分析研究
茯苓为多孔菌类中药材,其栽培过程的菌种选择是影响药材质量的关键因素。本论文对28种不同菌种栽培茯苓药材进行了薄层色谱、含量分析和指纹图谱比较研究。综合分析表明,湖北麻城茯苓、GZ(贵州)菌种、ACCC50478菌种栽培茯苓药材中的有效物质茯苓酸、总三萜和多糖的含量较高,茯苓药材品质较优。因此,本项研究为我国茯苓药材规范化种植提供了参考依据。
Poria(Poria cocos (Schw.) Wolf), the Polyporaceae fungal is a traditional Chinese medicine (TCM) that is always applied both medicinal and edible. It was documented in the "Chinese Pharmacopoeia" over the years. In the "Chinese Pharmacopoeia", Poria was described to have efficacies such as excreting dampness and diuretic, comforting stomach and tonifying spleen, calming the mind and soothing the nerves, etc., it was also one of the commonly used TCM for the prevention and treatment of cancer. Modern pharmacological studies have shown that Poria has good biological activity of anti-tumor and regulating immune functions. Its main active components are polysaccharides and triterpenoids. Most of the literatures that imported its antitumor activity were about pachymarans, and there are relatively very limited research reports mentioned the antitumor activity of its triterpenoids so far.
My thesis presented here is on the basis of the preliminary studies earried out in our laboratory. For the purpose of in-depth interpretation of the antitumor efficacy material basis of Poria, and to provide scientific evidences of improving the quality of the cultivated Poria, on the one hand, triterpenoids extract of Poria (the content of total triterpenes is above90%) was used for the systematical studies of the anti-tumor activity of Poria triterpenoids, on the other, the medicinal qualities of different strains of cultivated Poria were evaluated.
1. Poria triterpenoids antitumor activity study
Subjects with a variety of tumor cell lines, the anti-tumor activities of Poria triterpenoids were evaluated by using MTT assay. The results showed that Poria triterpenoids could induce noticeable proliferation inhibition effects in HepG2, A549, MDA-MB-231, Caski and SH-SY5Y tumor cell lines. The research results demonstrated that Poria triterpenoids have the anti proliferation activity for several tumor cell lines, in addtion, for HepG2tumor cell line, the anti proliferation activity tend to be very consistant.
2. Poria triterpenoids antitumor mechanisms study
Apoptosis morphology observation, apoptotic cells'DNA fragmentation analysis based on agarose gel electrophoresis and flow cytometry detection of cells apoptosis ratio were performed for understanding the antitumor mechanisms of Poria triterpenoids against human hepatoma cell line HepG2. Observations of the Poria triterpenoids treated HepG2cells under inverted microscope and optical fluorescence microscop showed that the shapes of Poria triterpenoids treated HepG2cells changed significantly, the number of cells decreased, there were visible compact dense stain, and some whitish colored nucleus. Agarose gel electrophoresis analysis of the apoptotic cells' DNA fragmentation results demonstrated that after24,48h treatment with different concentrations of Poria triterpenoids HepG2cells, the typical DNA ladder of late stage apoptotic (180-200bp integer multiple of the oligonucleotides) could be identified. Flow cytometry analysis results illustrated that after40μg/ml,60μg/ml,80米g/ml and100μg/ml Poria triterpenoids36hours treatment, the HepG2cells apoptosis ratios were7.2%12.8%,39.3%and56%respectively, which proved that the Poria triterpenoids have apoptotic induction effects on HepG2cells. All evidences indicated that Poria triterpenoids could induce apoptosis of hepatoma cells efficiently.
3. Poria triterpenoids isolation, identification and pharmacological evaluation
Modern chromatographic and spectroscopic techniques were applied to isolate and identify the chemical components in Poria triterpenoids. Eight terpenoids were isolated and identified, including poria acid, dehydroeburic acid, dehydropachymic acid,3β-hydroxy-lanostane-7,9(11),24-triene-21-acid, 15a-hydroxy-dehydrotumulosic acid,3β-hydroxy-16a-acetyloxy-lanosta-7,9(11),24-trien-21-oic acid, Trametenolic acid and polyporenic acid C. Their antitumor activities were evaluated by using MTT assay, and all the8compounds have shown moderate antitumor activities. The research result further illuminated the antitumor material basis of Poria triterpenoids.
4. Quality analysis of different strains of cultivated Poria
The TCM Poria is a fungus in the Polyporaceae family. As a result, in their cultivation process, the selection of strains is a key factor affecting the medicinal materials quality. In this study,27strains of Poria were analysed by using thin layer chromatography, content analysis and HPLC fingerprint comparison technologies. Comprehensive analysis indicated that the content of active components Poria acid, total triterpenes and polysaccharides in cultivated Porias by using strains from Macheng (Hubei), GZ (Guizhou) and ACCC50478were higher than all other samples. The qualities of the cultivated Porias from these strains were considered to be better than all the others. This study provides a reference for the TCM Poria standardized planting within the GAP project.
1茯苓三萜抗肿瘤生物活性研究
以多种肿瘤细胞作为受试对象,采用MTT法对茯苓三萜抗肿瘤的生物活性进行了研究。研究结果表明,茯苓三萜对人肝癌(HepG2)株‘、肺癌(A549)株、乳腺癌(MDA-MB-231)株、宫颈癌(Caski)株、神经瘤母细胞(SH-SY5Y)株均有明显的抑制增殖作用。本项研究证明了茯苓三萜对多种肿瘤细胞增殖具有抑制作用,其中人肝癌细胞株HepG2、神经瘤母细胞SH-SY5Y,宫颈癌细胞株caski经药物高浓度(100μg/ml)处理48h、72h,抑制率高达90%以上。
2茯苓三萜抗肿瘤作用机理研究
采用细胞凋亡的形态学观察、琼脂糖凝胶电泳分析凋亡细胞DNA片段化和流式细胞仪检测凋亡率等现代研究方法,对茯苓三萜抗人肝癌细胞HepG2作用机制进行了研究,倒置显微镜和荧光显微镜观察表明,茯苓三萜处理后的HepG2细胞,其形态发生明显的变化,细胞数量减少,可见致密浓染,颜色有些发白的细胞核,说明茯苓三萜具有较强的诱导肝癌细胞凋亡作用；琼脂糖凝胶电泳分析凋亡的细胞DNA片段化研究结果表明,不同浓度茯苓三萜处理HepG2细胞24、48h后,能检测到凋亡中晚期的DNA片段(180-200bp整数倍的寡核苷酸片段)。流式细胞仪检测结果表明,40μg/ml、60μg/ml、80μg/ml、100μg/ml茯苓三萜处理HepG2细胞36h后,其细胞的凋亡率分别为7.2%、12.8%、39.3%、56%；说明茯苓三萜对HepG2细胞具有与药物浓度呈正相关的诱导凋亡作用。
3茯苓三萜化学成分的分离鉴定与药理筛选研究
采用现代色谱技术和波谱技术,对茯苓三萜的化学成分进行分离鉴定,从中分离和鉴定了8个三萜类化合物,分别为茯苓酸(poria acid)、去氢茯苓酸(Dehydropachymic acid)、3β-羟基-16α-乙酰氧基-羊毛甾-7,9(11),24-三烯-21-酸(3β-hydroxy-16a-acetyloxy-lanosta-7,9(11),24-trien-21-oic-acid)、去氢依布里酸(Dehydroeburicoic acid)、3β-羟基-羊毛甾烷-7,9(11),24-三烯-21-酸(3β-hydroxy-lanosta-7,9(11),24-trien-21-oic-acid)3β-羟基-羊毛甾-8,24-二烯-21-酸(Trametenolic acid)、15α-羟基-去氢土莫酸(15α-hydroxydehydrotumulosic acid)、苓酸C(polyporenic acidC)。通过MTT法对8个三萜类化合物进行了抗肿瘤活性评价,结果表明8个三萜类化合物在高浓度时具有不同程度的抗肿瘤活性。因此本项研究进一步诠释了茯苓抗肿瘤的物质基础。
4不同菌种栽培茯苓药材的质量分析研究
茯苓为多孔菌类中药材,其栽培过程的菌种选择是影响药材质量的关键因素。本论文对28种不同菌种栽培茯苓药材进行了薄层色谱、含量分析和指纹图谱比较研究。综合分析表明,湖北麻城茯苓、GZ(贵州)菌种、ACCC50478菌种栽培茯苓药材中的有效物质茯苓酸、总三萜和多糖的含量较高,茯苓药材品质较优。因此,本项研究为我国茯苓药材规范化种植提供了参考依据。
Poria(Poria cocos (Schw.) Wolf), the Polyporaceae fungal is a traditional Chinese medicine (TCM) that is always applied both medicinal and edible. It was documented in the "Chinese Pharmacopoeia" over the years. In the "Chinese Pharmacopoeia", Poria was described to have efficacies such as excreting dampness and diuretic, comforting stomach and tonifying spleen, calming the mind and soothing the nerves, etc., it was also one of the commonly used TCM for the prevention and treatment of cancer. Modern pharmacological studies have shown that Poria has good biological activity of anti-tumor and regulating immune functions. Its main active components are polysaccharides and triterpenoids. Most of the literatures that imported its antitumor activity were about pachymarans, and there are relatively very limited research reports mentioned the antitumor activity of its triterpenoids so far.
My thesis presented here is on the basis of the preliminary studies earried out in our laboratory. For the purpose of in-depth interpretation of the antitumor efficacy material basis of Poria, and to provide scientific evidences of improving the quality of the cultivated Poria, on the one hand, triterpenoids extract of Poria (the content of total triterpenes is above90%) was used for the systematical studies of the anti-tumor activity of Poria triterpenoids, on the other, the medicinal qualities of different strains of cultivated Poria were evaluated.
1. Poria triterpenoids antitumor activity study
Subjects with a variety of tumor cell lines, the anti-tumor activities of Poria triterpenoids were evaluated by using MTT assay. The results showed that Poria triterpenoids could induce noticeable proliferation inhibition effects in HepG2, A549, MDA-MB-231, Caski and SH-SY5Y tumor cell lines. The research results demonstrated that Poria triterpenoids have the anti proliferation activity for several tumor cell lines, in addtion, for HepG2tumor cell line, the anti proliferation activity tend to be very consistant.
2. Poria triterpenoids antitumor mechanisms study
Apoptosis morphology observation, apoptotic cells'DNA fragmentation analysis based on agarose gel electrophoresis and flow cytometry detection of cells apoptosis ratio were performed for understanding the antitumor mechanisms of Poria triterpenoids against human hepatoma cell line HepG2. Observations of the Poria triterpenoids treated HepG2cells under inverted microscope and optical fluorescence microscop showed that the shapes of Poria triterpenoids treated HepG2cells changed significantly, the number of cells decreased, there were visible compact dense stain, and some whitish colored nucleus. Agarose gel electrophoresis analysis of the apoptotic cells' DNA fragmentation results demonstrated that after24,48h treatment with different concentrations of Poria triterpenoids HepG2cells, the typical DNA ladder of late stage apoptotic (180-200bp integer multiple of the oligonucleotides) could be identified. Flow cytometry analysis results illustrated that after40μg/ml,60μg/ml,80米g/ml and100μg/ml Poria triterpenoids36hours treatment, the HepG2cells apoptosis ratios were7.2%12.8%,39.3%and56%respectively, which proved that the Poria triterpenoids have apoptotic induction effects on HepG2cells. All evidences indicated that Poria triterpenoids could induce apoptosis of hepatoma cells efficiently.
3. Poria triterpenoids isolation, identification and pharmacological evaluation
Modern chromatographic and spectroscopic techniques were applied to isolate and identify the chemical components in Poria triterpenoids. Eight terpenoids were isolated and identified, including poria acid, dehydroeburic acid, dehydropachymic acid,3β-hydroxy-lanostane-7,9(11),24-triene-21-acid, 15a-hydroxy-dehydrotumulosic acid,3β-hydroxy-16a-acetyloxy-lanosta-7,9(11),24-trien-21-oic acid, Trametenolic acid and polyporenic acid C. Their antitumor activities were evaluated by using MTT assay, and all the8compounds have shown moderate antitumor activities. The research result further illuminated the antitumor material basis of Poria triterpenoids.
4. Quality analysis of different strains of cultivated Poria
The TCM Poria is a fungus in the Polyporaceae family. As a result, in their cultivation process, the selection of strains is a key factor affecting the medicinal materials quality. In this study,27strains of Poria were analysed by using thin layer chromatography, content analysis and HPLC fingerprint comparison technologies. Comprehensive analysis indicated that the content of active components Poria acid, total triterpenes and polysaccharides in cultivated Porias by using strains from Macheng (Hubei), GZ (Guizhou) and ACCC50478were higher than all other samples. The qualities of the cultivated Porias from these strains were considered to be better than all the others. This study provides a reference for the TCM Poria standardized planting within the GAP project.
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