| Background and ObjectivesGanoderma lucidum has been used in our country for more than 2000 years. It can bring about nourishing and strengthening, strengthening the body. It is well known that there are about more than 100 species of Ganoderma lucidum. The most widely distributed specie is Ganoderma lucidum, followed by Ganoderma japonicum (G. japonicum, Ganoderma applanatum, Ganoderma tsugae and Bu Shuzhi. It consists amino acids, peptides, proteins, fungal lysozyme (fungal lysozyme), and sugar (reducing sugars and polysaccharides), ergosterol, triterpene, coumarin glycosides, essential oil, stearic acid, benzoic acid, alkaloid, vitamin B2 and C. Spores also contain mannitol, trehalose (trehalose). A large number of clinical studies show that Ganoderma lucidum have varying degrees of efficacy for neurasthenia, high fat, hyperlipidemia, coronary heart disease angina pectoris, arrhythmia, Keshan disease and plateau of discomfort mononucleosis, hepatitis, hemorrhagic fever, indigestion, tracheitis.Statistics show that there are 98 kinds of Ganoderma lucidum fungi in China, belonging to 4 genera. More than ten species, mostly Ganoderma lucidum, have been developed and utilized. However, the current research of Ganoderma lucidum are more common in Ganoderma lucidum Ganoderma species, a large number of species of the genus Ganoderma has not yet been developed. Ganoderma lucidum generally grow solitary or clustered in tree stem or stakes. The decomposition of wood lignin is a nutrient and growth. Ganoderma lucidum fungi of the genus grow mostly in a sparse scattering sunlight, the trees, local or open ground. Ganoderma lucidum is widely distributed in all continents of the world, there are more than 250 species are described, most of them grow in tropical, subtropical, temperate zone. Ganoderma in China are mainly distributed in Hebei, Shanxi, Liaoning, Jilin, Fujian, Jiangsu, Henan, Guangdong, Guangxi, Sichuan, Hainan and other places in the mountains.Ganoderma lucidum contains mainly triterpenoid compounds and polysaccharides compounds and sterols, and other chemical composition such as nucleosides, alkaloids, furan derivatives, amino acid polypeptide, inorganic elements and fatty acids. The ganoderma lucidum triterpenes and ganoderma lucidum polysaccharides are the main active substances in Ganoderma lucidum. Ganoderma lucidum has the effect of nourishing the body, supporting the healthy energy and alleviating pain. They have been used for a malignant tumor of digestive tract and postoperative chemotherapy induced weight loss, insomnia, pain, anorexia, neutropenia, fatigue weakness in rehabilitation and health care, to get a good effect. Previous studies showed that Ganoderma lucidum spore powder can increase the immune function of mice and had some inhibitory effect on tumor growth in mice, and can enhance the mitomycin antitumor effects. Some studies showed that Ganoderma oral liquid has obvious inhibitory effect on human nasopharyngeal carcinoma cells in nude mice tumor. Ganoderma lucidum polysaccharide can induce cultured mouse spleen cell nuclear DNA, RNA content increased significantly and intracellular ultrastructural changes, in both the cytoplasm and nucleus of the average cross-sectional area increased significantly, nucleo cytoplasmic ratio was decreased trend, indicating that Ganoderma lucidum polysaccharides in promoting spleen cells and protein synthesis at the same time, promote cell proliferation.Since 1982, Ganoderma lucidum such as Ganoderma lucidum acid A and Ganoderma lucidum acid B were firstly isolated by Kubota. Then, the research on the three kinds of Ganoderma lucidum were carried out. At present, more than 140 kinds of triterpenes compounds have been isolated from the fruiting body, mycelium and spore powder of Ganoderma lucidum. The study found that the traditional Chinese medicine Ganoderma lucidum contains more than 122 components of triterpenes compounds Ganoderma lucidum, which can inhibit the release of histamine, blood sugar, anti hypertension, anti-tumor and many other pharmacological activities. Ganoderma triterpenoids decreased significantly rats with hyperlipidemia fatty liver lipid content, and possibly by reducing lipid peroxidation, maintaining the in vivo free radical homeostasis and balance, improve the injury of liver tissue, to repression of hyperlipidemic fatty liver and the role of development. It is found that the Ganoderma lucidum has the antimicrobial effect to the mycobacterium tuberculosis, and the effective ingredient is the Ganoderma triterpenoids. When the total concentration of Ganoderma triterpenoids was 500 ug/ml, the growth of the standard strains and drug resistant strains of mycobacterium tuberculosis were inhibited. Su et al. reported that Ganodermic acids on TXA2 quasi U46619 induced source of silica gel filtration treatment of platelet aggregation significantly inhibited, platelet shape change of the time delay, and reduce the initial platelet aggregation, reduce the aggregate percentage and the inhibition effect is dose dependent.In addition, It has also been reported that Ganoderma lucidum can inhibit cholesterol synthesis. Komoda et al. found that Ganoderma lucidum mycelium was added to rat high cholesterol diet can significantly reduce the content of triglyceride and cholesterol in serum and liver, and it is pointed out that the active ingredient is triterpenoid compounds.. this substances active ingredients of Ganoderma lucidum alcohol A, Ganoderma lucidum alcohol B, Ganoderma lucidum aldehyde A, ganoderic acid Y in Ganoderma lucidumbiological have the effect of inhibiting human liver cell lines synthesize cholesterol and sterol in Ganoderma lucidum inhibit cholesterol biosynthesis by changing the precursors of cholesterol acetate or methyl hydroxy acid. Therefore, Ganoderma lucidum also has the effect of decreasing blood lipids. Some compounds in ganoderma triterpenes have inhibitory effects on the inflammatory mediators released by neutrophils. In the mouse model of asthma, the extract of Ganoderma lucidum triterpenes significantly attenuated the airway high reactivity, reducing the overall granulocyte and eosinophilic infiltration and the level of inflammatory mediators.Ganoderma lucidum triterpenes can inhibit the growth and metastasis of a variety of tumor cells. A lot of experiments and clinical studies have indicated that Ganoderma lucidum has a good therapeutic effect on many diseases, especially for the prevention and treatment of cancer. Many studies have reported that Ganoderma lucidum triterpenes has a certain anti-tumor effect, such as breast cancer and oral cancer. The main mechanism is that Ganoderma lucidum triterpenes can activate the immune system of the body. Glossy ganoderma alcohol F, Ganoderma lucidum ketone, glycol, ganodermanontriol can effectively inhibit complement activation of the classical pathway. Ganoderma lucidum acid can increased the content of IL-2 in pigs of lung cancer, and activate the immune activity of NK cells, with enhanced immune function. Ganoderic Acid B can block the cell cycle of human hepatoma cell line BEL7402 from phase G1 to phase S transition, and inhibit the growth of cancer cells.Nowadays, the use of traditional Chinese medicine extract in the treatment of prostate cancer has been extensively investigated, the research about triterpenoids of Ganoderma lucidum is gradually increasing. However, the studies on benign prostatic hyperplasia and prostate cancer have rarely been reported. Ganoderma lucidum triterpenoids can inhibit the growth of a variety of tumor cells in vitro and in vivo, and induce the apoptosis of tumor cells. At present, the country vigorously promotes the development of traditional Chinese herbal medicine. By means of molecular biology gives a new explanation is committed to research and development of new drugs to vigorously carry forward the traditional Chinese medicine for its pharmacological effects, has become the development trend of Chinese medicine research. It is undoubtedly a promising and valuable topic to study the molecular mechanism of the anti-tumor effect of Ganoderma lucidum triterpenoids.This study aimed to study on the extraction and purification of triterpenoids to establish a more rapid, stable and convenient industrialized extraction and purification process and provide technical support For the subsequent product development. To clarify whether Ganoderma lucidum triterpenoids has the effect of inhabiting prostate cancer cells and to explore its mechanism. First, the effect of Ganoderma lucidum triterpenoids on the growth and proliferation of prostate cancer cells were observed, and the effect of Ganoderma lucidum three on the apoptosis of prostate cancer cells were analyzed. Second, the effect of Ganoderma lucidum triterpenoids on the cell cycle of prostate cancer cell cycle and the regulation of the cell cycle were observed, then, the influence of the most Caspase-3 and Caspase-9 levels were detected. In order to find out the related signal transduction pathway or target of the apoptosis of prostate cancer cell induced by Ganoderma lucidum triterpenoids, to elucidate the possible mechanism of prostate cancer, and to provide scientific basis for its clinical application and product development.Methods1. The study of extraction and purification technics of Ganoderma lucidum triterpenoidsThe fruiting body of Ganoderma lucidum was identified according to the pharmacopoeia standards. The original medicinal herbs which meet the standards were crushed. The edible fungus coarse powder were put in some size suitable chromatography column of leakage device. The column ratio was between the 10:1-15:1.8-12 times amount of petroleum ether were put in and soak for 24 h, and then, collect fluid leakage by adding 8 to 12 times the amount of ethanol and water to collect fluid leakage. The petroleum ether and ethanol part leakage liquid were mixed, added water to 20%, then shaking and layering, organic layer and water layer were observed. The liposoluble constituent were extracted from Organic layer recovery solvent (extract 1). The alcohol extracts were extracted from water recovery solvent (extract 2). The ethanol is added into leakage of liquid water to 80%. They were shaked and cold storage at 4℃ for 24 h. The crude polysaccharide were filtered and obtained (extract 3). The main components of the extract were determined to be the Ganoderma lucidum triterpenes by using HPLC chromatography and the relevant literature.2. The study of extraction and purification technics of triterpenoids from Ganoderma lucidumC18 were packed into the column, extract 2 were added into the columns, and eluted with different concentrations of methanol. the eluent were collected. HPLC was used to detect and track to determine the best elution purification process. Macroporous resin were filled into the column, extract 2 were added into the column, and eluted with different concentrations of ethanol. Each part of the elution liquid were collected. HPLC was used to detect and track to determine the best elution purification process.3. Effect of Ganoderma lucidum triterpenes on the growth of prostate cancer cellsThe different concentrations of triterpenoids from Ganoderma lucidum (50,100 UG/ml) were used to treat normal prostate epithelial cell line (BPH) and four human prostate cancer (LNCaP and 22Rvl, PC-3, DU-145) cells for 48 hours. The cells were harvested and the effect of growth inhibitory of prostate cancer cells were assessed with cell counting kit-8 proliferation assay. Death Detection ELISA kit Cell was used to detect the effect of Ganoderma lucidum triterpenoids on the apoptosis of prostate cancer cells.4. The study on the apoptosis of prostate cancer cells induced by Ganoderma lucidum triterpenesThe different concentrations of triterpenoids from Ganoderma lucidum (50,100 UG/ml) were used to treat normal prostate epithelial cell line (BPH) and four human prostate cancer (LNCaP and 22Rvl, PC-3, DU-145) cells for 48 hours. The flow cytometry was used to detect the effect of Ganoderma lucidum triterpenoids on the cell cycle of prostate cancer cells.5. Effect of Ganoderma lucidum triterpenoids on apoptosis related genes in prostate cancer cell lineThe different concentrations of triterpenoids from Ganoderma lucidum (50,100 UG/ml) were used to treat normal prostate epithelial cell line (BPH) and four human prostate cancer (LNCaP and 22Rv1, PC-3, DU-145) cells for 0,12,24,48 hours. Cells were collected, and the total RNA of LNCaP,22Rv1, DU-145 and PC-3 prostate cancer cells were extracted respectively. Apoptosis related proteins p21, CDK4, E2F1 and D1 Cyclin primers were designed by primer 5 software. Western, qPCR and Blot RT-PCR were used to detect the expression levels of these genes and proteins in LNCaP,22Rvl, PC3 and DU-145 prostate cancer cells.6. Effect of Ganoderma lucidum triterpenoids on the expression of apoptosis factor Caspase 3 and Caspase 9 protein in prostate cancer cell lineThe activity of Caspase-3 and Caspase-9 in prostate cancer cells was detected by ELISA, and the possible molecular mechanism of the effect of Ganoderma lucidum triterpenoids were further explored.Results1. The extraction and purification technology of Ganoderma lucidum triterpenoidsThe protocol was carried out with the percolation method and petroleum ether and ethanol water solvent system. All ingredients of Ganoderma lucidum were extracted by "one step" extraction. The crude extract of Ganoderma lucidum triterpenes were recycled and extracted from the ethanol extract.The crude extract of Ganoderma lucidum triterpenes were obtained, adsorbed and purified by C18 and macroporous resin, and the three components of Ganoderma lucidum were divided into. The ingredients were not significant among the different groups. The middle part of the extract was taken as the pure extract of Ganoderma lucidum triterpenes.2. Effect of Ganoderma lucidum triterpenes on the growth of prostate cancer cellsThe different concentrations of Ganoderma lucidum triterpenes extract (10,50,100 g/ml) were treated cells. Cell viability of prostate cancer cells was detected by cell counting kit-8. The results showed that GLT inhibited cell growth in all 5 cell lines in a dose-dependent manner and inhibited growth. Compare with the normal prostate epithelial cell line (BPH), the other four prostate cancer cell line (LNCaP,22Rv1, PC-3, DU-145) were more sensitive for of GLT (p<0.05 for all). When the concentration reached 50 ug/ml (p<0.05 for all), the number of cells in the four cell lines decrease by 55% to 45%, However, the BPH only decreased by 12%(p>0.05 for all).3. Effect of Ganoderma lucidum triterpenes on the growth of prostate cancer cellsThe Morphologic changes of apoptosis nuclei and the expression of apoptosis protein PARP were observed. The results showed that compared with the control group, the 50 ug/ml GLT treatment group significantly increased the number of nuclear bodies (p<0.05), the PARP expression was also significantly increased in a does dependent manner (p<0.05).The percentage of phase G1 (G0), S, and G2 (M) cell were analyzed by flow cytometry. Results showed that 10g/ml GLT can significantly increase the percentage of LNCaP,22Rvl cells in the phase G1/G0, but significantly reduced the percentage of the S phase cells. For PC-3 and DU-145 cells,100 g/ml GLT treatment group significantly increased the percentage of cells in the G1/G0 phase, while decrease in the percentage of phase S cells.4. The study on the apoptosis of prostate cancer cells induced by Ganoderma lucidum triterpenesThe expression of modulators of cell cycle regulation (p21, CDK4, E2F1 and cyclin Dl) was detected by Western blot and PCR. The results showed that after 48 h treatment with 50 ug/ml GLT, the expression of p21 was significantly increased in four prostate cancer cells (p<0.05 for all), while the levels of CDK4, E2F1 and D1 Cyclin were significantly decreased (p<0.05 for all).5. Effect of Ganoderma lucidum triterpenoids on the expression of apoptosis factor Caspase 3 and Caspase 9 protein in prostate cancer cell lineThe activity of Caspase-3 and Caspase-9 were detected by ELISA kit. The results showed that after 48h, treatment with 50 and 100 ug/ml GLT, the expression of Caspase-3 in all four prostate cancer cell line were significantly increased (p< 0.05) and the activity of caspase-9 level also showed the same tendency.ConclusionThe method of one-step solvent-penetration was used in this experiment to extract all components from Ganoderma lucidum. Solvent extraction followed by column chromatographic purification was processed to obtain the GLT. Thus, we established methods of rapid and efficient extraction from Ganoderma lucidum and the quality control method of GLT. GLT can significantly inhibit the growth of prostate cancer cells. The mechanism may involve the regulation of cell cycle, which can affect cell proliferation and apoptosis. However, the precise mechanism needs to be further verified by using in-vitro and in-vivo experiments. |
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