| In recent years, scientific researches focus on the anti-tumor activity of Ganoderma lucidum. Ganoderma polysaccharides, an water extract, can enhance our immune system, while ethanol extracts have a direct cytotoxic effect on tumor cells. In this paper, the antitumor material basis of alcohol-soluble components is studied. The supercritical CO2 extraction of Ganoderma lucidum (the total component) is divided into the acid component and the neutral component by NaHCO3 solution. The dependablity of components and pharmacodynamic action is investigated in vivo and in vitro. The total component has a higher inhibition rate than the acid component or the neutral component. The acid component and the neutral component simultaneously constitute the anti-tumor material basis of Ganoderma lucidum. And it is shown that the neutral component which is a key active component in the total component exerted more significant anti-hepatoma activities than the acid component. The acid component and the neutral component are analyzed through GC-MS and HPLC-MS. Our studies provide the methods and ideas of the deep development and preparations of Ganoderma lucidum. When the physico-chemical properties of components have been researhed, the solid dispersions of components are prepared, which play a better anti-tumor effect than their suspensions. 1. To study the anti-hepatoma activity of effective components from Ganoderma lucidum in vivo and in vitro (1) The cytotoxicity evaluation on BEL-7402 human hepatoma carcinoma cell line in vitroThe cytotoxicity of Ganoderma lucidum from different areas is compared using MTT assay. The total and neutral component show significant proliferation inhibitory actions, while the acid component has less effects. The acid component from different regions has greater changes in antitumor activities, but it can not be easily removed from the total component because of its relatively weak inhibition rate. The total, neutral and acid components of Ganoderma lucidum from Anji County, Zhejiang Province exert the strongest activity on the proliferation inhibition of BEL-7402 cells. At the dose of 100μg·mL-1, the inhibition rate of the total component, the neutral component and the acid component is 94.06%,99.56% and 60.72%. This is an interesting phenomenon that the acid component from Hainan shows no inhibition on the proliferation of BEL-7402 cell. At the dose of 100μg·mL-1, the inhibition rate of the total component and the neutral component is 66.02% and 85.18%, respectively. We conclude that the neutral component plays an important role on the anti-hepatoma activity of Ganoderma lucidum.According to the results in vitro, Ganoderma lucidum from Zhejiang as raw material is choosen to experimentize studies. The total component (28.08 g) was separated into the neutral component (13.20 g) and the acid component (13.20 g). The content of neutral component and acid component is close to each other, with the ratio of approximate 1:1. Both the total component and neutral component could obviously inhibit the proliferation of BEL-7402 cells, IC50 is 1.04 mg crude drug·mL-1 and 1.48 mg crude drug·mL-1, respectively. IC50 of the acid component is over 5 mg crude drug·mL-1. Under the same amount of crude drug, the total component has better anti-tumor effects than the neutral component alone. By flow cytometry the acid component results in a significant increase percentage at the G1 phase, while the neutral component and the total component induce cell cycle arrest at the G2/M phase. Three components all induce apoptosis in a dose-dependent manner. (2) To investigate the antineoplastic activity in H22-bearing miceThe transplanted tumor is induced by sc injection of H22 hepatoma cells in the right forelimb armpit for each KM mouse. Compared with the control group, the total component group results in 36.14% inhibition of tumor growth compared to the control at the dose of 200 mg·kg-1 per day for 8 consecutive days. While under the same amount of crude drug, the inhibition rate of neutral and acid component groups is 28.02% and 19.45% at the dose of 100 mg·kg-1 per day for 8 consecutive days. At the dose of 200 mg·kg-1, the inhibition rate of the neutral component group is 33.08%, which achieves the total component's effect. The acid and neutral components constitute the anti-tumor material basis of the total component. If we only rely on either component, the anti-tumor effect will diminish.2. To study anti-hepatoma material basis of Ganoderma lucidumGanoderma lucidum from Zhejiang, with a good anti-tumor effect in vivo and in vitro, is selected to investigate the extraction process. The traditional organic solvent extraction is replaced with supercritical fluid extraction, a green pollution-free technology. The solid content of supercritical CO2 extraction,95% ethanol as entrainer, is 28.08±0.02 mg·g-1. The content of ganoderic acid B and ergosterol is 0.363±0.005 mg·g-1 and 0.963±0.007 mg·g-1, respectively. Its extraction time is reduced to 1.5h. Besides, the amount of 95% ethanol is reduced to three times, enormously saving the organic solvent.Ganoderma total component is divided into acid and neutral components, characterized by HPLC. The peak retention time of AC (RT< 80 min) in the chromatogram is shorter than that of NC (RT> 80 min). The appearance of Ganoderma lucidum from different areas has greatly distinction. There are large changes in the content of acid component among different areas, especially Ganoderma hainanense. Its acid component has few peaks, so the proliferation inhibition is undetectable.The HPLC-MS analysis of the acid component shows that it contains a large number of triterpenes. The neutral component, a main anti-tumor component in the total component, contains a large number of fatty acids, triterpenes and sterols through HPLC-MS and GC-MS analysis. The fatty acids and sterols of the neutral component may also contribute to the anti-tumor activity.3. The initial preparation of the solid dispersion of Ganoderma effective componentsThe physico-chemical properties of the different components from Ganoderma lucidum are researched. In weak alkaline environment, the apparent solubility of acid and neutral components is between 100μg·mL-1 and 500μg·mL-1. They belong to very slightly soluble substances. The apparent oil-water partition coefficient (log P) of neutral and acid components in water solution is 2.01 and 1.20, respectively. It is presumed that, with low water solubility and high membrane permeability, their dissolutions are the rate-limiting process of their gastrointestinal absorption.The accumulative dissolution rate of solid dispersions of the total component with different carriers (PVPK30, Poloxamer188, PEG6000) and ratios (1:1,1:4,1:6,1:9) is investigated in vitro. Solid dispersion of the total component with PVPK30 is preparation in the ratio of 1:6. The dissolution of triterpenes at 60 min from the total component, physical mixtures (1:9), solid dispersion (1:9) is 15.74%,31.80% and 97.23%, respectively. The X-ray method is used to verify the formation of solid dispersion. The total component in the solid dispersion existed in the amorphous state. The antitumor activity of solid dispersions in vivo is observed through transplanted hepatoma H22 mice model. There is a statistical difference between the tumor inhibitory rate of the solid dispersions of the three components (the total component,57.27%; the neutral component,55.90%; the acid component,43.80%) and that of their suspensions (36.14%,33.08%,24.04%, respectively) at the dose of 200 mg·kg-1·d-1. Solid dispersions can significantly increase the dissolution of the effect components and enhance their antitumor activity in vivo. |
PDF Full Text Request