Study On Separation,Purification And In Vitro Anti-inflammatory Effect Of Ganoderma Lucidum Sterols

Objective The purpose of this study was to establish a single-step method to separate sterols from Ganoderma lucidum by aqueous two-phase system（ATPS）and to study the in vitro inflammatory inhibition effect and molecular mechanism of sterols purified from Ganoderma Lucidum.It will provide technical supports for the application and development of Ganoderma lucidum sterols.Methods（1）Aqueous two-phase system formed by hydrophilic solvents and inorganic salts was used to extract sterols from Ganoderma lucidum by a single-step.In oder to assess the efficiency of the ATPS,the recovery efficiency of ergosterol was explored.Furthermore,the optimal ATPS was investigated in detail including the type of hydrophilic alcohol,the type and concentration of inorganic salt,the solid-liquid ratio,the ultrasonic temperature and time.On this basis,the samples were purified by column chromatography to isolated monomeric compounds and their chemical structures were also elucidated.（2）The mice macrophage RAW264.7 inflammatory status which induced by LPS was used to investigate the anti-inflammatory effects and molecular pathways.MTT was used to assay the drug dosage and time effect on the cell viability.Cell morphology was used to observed using microscope,the contents of cytokines NO,IL-6,TNF-αand IL-1βin the culture supernatant was deteced,qPCR were used to test the relative expression of the proinflammatory cytokines,and Western-Blot were used to detected protein expression in NF-κB pathway.Results（1）An aqueous two-phase system was formed,using n-propyl alcohol and K₂CO₃ to extract sterols from Ganoderma lucidum by a single-step.The optimal condition was 27.5%n-propyl alcohol and 8.65%K2CO3 with a solid-liquid ratio of1:100 and treated with ultrasonics for 30min.Under this condition the extraction efficiency of Ganoderma lucidum sterols could reach 2.37mg/g,compared to the traditional acid-base conversion method the extraction efficiency was improved 38.8%.（2）The total sterols extracted from the ATPS was subjected to gel columns and ODS columns chromatography to give two monomeric compounds ergosterol and ergosterol peroxide（EP）,they were identified through comparison of NMR and mass spectral data with those reported previously.（3）The studies on cell morphology and cell viability showed that LPS can change the morphology of RAW264.7（enlarged cell size,irregular morphology,pseudopodia outstretched）and the EP intervention can inhibit the cell morphology change.Cell viability in experimental groups at the concentration range of 0.5～5μg/mL EP or co-culture with 1 mg/mL LPS show no cytotoxic effect on RAW264.7 cells compared with the control group.Ergosterol showed a strong cytotoxic effect at the concentration of0.01～10μg/mL,therefore it was excluded from anti-inflammatory study.（4）Compared with the LPS group,EP（does of 0.5,2.5,5μg/mL）intervention can significantly inhibit the secretion of inflammatory cytokines NO,TNF-α,IL-6,IL-1βwhich induced by LPS and the inhibit effect was in dose-dependent.In addition,EP significantly reduced the mRNA expression in IL-6,TNF-α,IL-1β.The mRNA expression levels of the proinflammatory cytokines in the EP groups were significantly lower than that in the LPS groups.（5）The Western-blot results showed that in this process compared with the LPS group EP（does of 0.5,2.5,5μg/mL）intervention can significantly inhibit the expression of phosphorylated nuclear transcription factor protein p65 and IκBα.The molecular mechanism of anti-inflammatory effects of EP were associated with NF-κB signal pathway.Conclusion（1）In this study,a new single-step ATPS method has been used to extract sterols from Ganoderma lucidum.The system formed by n-propyl alcohol/K₂CO₃ is simple and efficient,and it can take the place of the traditional method which need acid-base conversion and multi-step extraction.The method is characterized by high efficiency,accuracy,reliability,and strong operability.（2）The anti-inflammatory effects of EP were founded on RAW264.7macrophages induced by LPS.EP was found to significantly suppress nitric oxide（NO）production,decrease the levels of pro-inflammatory cytokines TNF-α,IL-6,IL-1βand down regulation the transcription of genes involved in TNF-α,IL-6,IL-1β.（3）The molecular mechanism of anti-inflammatory effects of EP founded on RAW264.7 macrophages which induced by LPS were associated with NF-κB signaling pathway,EP through inhibit the activation of NF-κB signaling achieve anti-inflammatory effects.