Neuroprotective Effect And Mechanism Of Secondary Metabolite CA-1 From Centella Asiatica

Centella asiatica is a medicinal and dietary herb whose main triterpenoids are Asiaticoside（AS）and Aadecassoside（MA）.It has been reported that this ingredient has a positive effect on neuroprotection,and most of the current research on Centella asiatica is based on these two saponins.Therefore,in this study,we will isolate new triterpenoid compounds from Centella asiatica and verify their neuroprotective effects.（1）The dried Centella officinalis was crushed and concentrated under pressure.And then extracted with dichloromethane and n-butanol.Next,Gradient chromatography with methanol-dichloromethane was carried out on silica gel column.Several components were obtained through thin layer chromatography and then several compounds were further prepared.The chemical structure of the compound was analyzed by high resolution mass spectrometry,and two possible structures were speculated,one was a new compound,the other was a known compound.The anti-inflammatory effect of CA-1 was evaluated by measuring the amount of NO released in LPS-induced BV2 cell neuroinflammation model.The results showed that CA-1could reduce the amount of NO released.（2）The Parkinson’s disease model was established with 6-OHDA induction in highly differentiated PC12 cells to study the neuroprotective effect of CA-1 in vitro.Moreover,MTT assay was used to detect the toxicity of CA-1 to PC12 cells and the neuroprotective effects of CA-1,Asiaticoside（AS）and Aadecassoside（MA）.DCFH-DA probe was used to detect intracellular Reactive Oxygen（ROS）levels,semi-quantitative and real-time quantitative polymerase chain reaction（PCR）and Western Blot（WB）to detect the expression of related genes and proteins in PC12 cells.The results showed that CA-1 had stronger neuroprotective effect than AS and MA.Compared with model group,CA-1 increased the survival rate of 6-OHDA-induced PC12 cells in a dose-dependent manner,decreased the intracellular ROS level,and up-regulated the expression of Sod1,Cat and Bcl2 genes.WB showed that CA-1 upregulated the protein expression of P38/ERK/JNK and PI3K/PDK1/Akt pathways,revealing the internal mechanism involved in these two signaling pathways.（3）NLRP3 gene was amplified from c DNA of THP-1 cells,and plasmid NLRP3-PET29A was constructed.NLRP3 protein was induced to express by E.coli BL21,and purified by His tag-nickel column protein purification system.The interaction between NLRP3 protein and compound CA-1 was mediated by Autodock Vina 1.1.2.The results showed that NLRP3 protein could be expressed and purified by His label-nickel column system in E.coli BL21,but it was difficult to obtain a large amount of soluble NLRP3.Molecular docking results showed that the affinity between the compound and the protein was-7.8 kcal mol^-1(the positive compound was-7.9 kcal mol^-1),which could inhibit the active pocket compound.These results indicate that the isolated compound CA-1 has neuroprotective effects and has potential for future development.