Treatment Effects And Mechanisms Of Action Of Triterpenoids Monomer From Ganoderma Lucidum On α-amanitin-induced Liver Injury In Mice

Poisonous mushrooms were the main cause of food poisoning related deaths in our country.Because of its detoxification effect,Ganoderma lucidum decoction has been used to treat the poisoning patients who eat amanita uncarefully.The triterpenoids, as one of the main active ingredients in Ganoderma lucidum, has been knowed that have many pharmacological effects. In this paper,we research the therapeutic effects and mechanisms of action of triterpenoids monomer from G. lucidum on α-amanitin poisoning mice and the results are as following.1.The extraction,separation and purification of Ganoderma lucidum total triterpenoids : The fruiting body powder was extracted with anhydrous ethanol(v:v=1:20), 80 ℃ water bath heating for 4 h, repeated 3 times, then collected and concentrated the crude extraction to obtain extractum. Using macroporous adsorption resin AB-8 to adsord the extractum, then gradient elution(with 70%, 90% ethanol)was be carried out and the eluent was collected,concentrated and lyophilized.After extraction,the extracts was continued to be purified by silica gel column chromatography,and the mobile phase was different proportions(98:2、96:4、94:6、92:8、90:10) of chloroform and methanol. The main product(the purity reachs to 98%) was collected in the first mobile phase(98:2). The result of HPLC analysis showed that it contains more than 18 kinds of triterpenoids monomer compounds, including ganoderic acid A, B, G and ganoderenic acid B.2. The antioxidant activity of G. lucidum triterpenoid monomer compounds and G. lucidum total triterpenoids in vitro: Three methods(the test of DPPH radical scavenging activity, iron ion chelating ability and reducing power) were used to test the oxidation resistance of triterpenoid monomer compounds and total triterpenes from G. lucidum. The results showed that all materials have strong antioxidant capacity, the antioxidant activity of ganoderic acid C2 is strongest, and the weakest one is ganoderic acid A.3. The treatment effect of triterpenoids momomer in α-AMA-injured mice: Compared with normal control mice, α-AMA induced serious hepatocellular injuries, with significant elevation in serum alanine aminotransferase(ALT) and aspartate aminotransferase(AST) activities. Administration of SIL, the total triterpenoids and the five individual triterpenoids to α-AMA treated mice obviously decreased serum ALT and AST levels compared with mice treated with α-AMA alone, indicating that the total triterpenoids and the five individual triterpenoids had the same hepatoprotective effects as SIL.Different dose(5-40mg/kg/day) GAC2 to α-AMA treated mice obviously decreased serum ALT and AST levels, and showed the dose-dependent effects. When the dose reach to 20 mg/kg/day,the treatment effect is the most obvious. Treatment with SIL, total triterpenoids and each of the five individual triterpenoids reduced mortality rates 20–40%.4. The effect of triterpenoid monomer on antioxidant activity in α-AMA-injured mice: Compared with control mice, α-AMA caused a obvious reduction of superoxide dismutase(SOD) and catalase(CAT) activities and a significant increment of malondialdehyde(MDA) content in liver homogenate. Treatment with Ganoderma lucidum triterpenoid monomer compounds 、Ganoderma lucidum total triterpenes or SIL increased significantly SOD and CAT activities and decreased MDA content in liver homogenate. Different dose(5-40mg/kg/day) GAC2 to α-AMA treated mice significantly increased SOD and CAT activities and decreased MDA content, and showed the dose-dependent effects,the treatment effect of the dose of 20 mg/kg/day is most obvious.These results showed that one of the protective mechanisms of the the triterpenoids perhaps because of thier capacity of antioxidant, radical-scavenging and reduction of lipid peroxidation.5. The effect of triterpenoid monomer on apoptosis in α-AMA-injured mice:Analysis of DNA fragmentation by agarose gel electrophoresis showed a characteristic of apoptosis with a distinctive cleavage of liver nuclear DNA in the control α-AMA and Olive oil groups, in the groups treated with the GAC2 at dose of 5, 10, 20 and 40 mg/kg/day respectively, no obvious cleavage of liver nuclear DNA typical for apoptosis were revealed.α-AMA induced an obvious rising in caspase-3,-8 and-9 activities, while treatment with GAC2 at all doses decreased caspase-3,-8 and-9 activities compared with the α-AMA control group. At the dose of 20 mg/kg/day resulted the lowest caspase-3,-8 and-9 activities.The results indicated that α-AMA induced the apoptosis of liver cells in mice,the hepatopective effects of triterpenoids were related to its anti-apoptosis character.In summary, our results demonstrated that triterpenoids exerts hepatoprotective effect on liver injury induced by α-AMA,and the protective mechanisms might be attributed to the co-operation of antioxidative and radical-scavenging and the inhibition of apoptosis.