Study On The Protective Effect Of Free Phenolics From Lycopus Lucidus Turcz.on Hepatocyte Injury In Vitro

ObjectiveTo clarify the phenolic content and the main phenolic compositions of free phenolic extracts from Lycopus lucidus Turcz.harvested from different sites at different harvest time.The protective effect and protective mechanism of free phenolics from Lycopus lucidus Turcz.on BRL hepatocytes injury induced by carbon tetrachloride was studied through in vitro experiments.And further clarify the functional activity of the free phenolic extracts from Lycopus lucidus Turcz.The results provide a theoretical basis for the development and utilization of Lycopus lucidus Turcz.resources.MethodsUltrasonic-assisted extraction method was used to extract free phenolic from Lycopus lucidus Turcz.,and purified by X-5 macroporous resin.The free phenolic extracts content was determined by Folin-Ciocalteu method.And the monomer composition of free phenolic extracts from Lycopus lucidus Turcz.was analyzed by high performance liquid chromatography?HPLC?.Establishing a model of BRL hepatocytes injury in vitro by carbon tetrachloride,the protective effect of free phenolics from Lycopus lucidus Turcz.on BRL hepatocytes injury was investigated by measuring liver function indexes including alanine transaminase?ALT?,aspertate aminotransferase?AST?and lactate dehydrogenase?LDH?.In addition,in order to investigate the protective mechanism,DAPI staining was used to observe the nuclear morphology,flow cytometry was used to analyze the distribution of cell cycle,and measured the contents of antioxidant indicators including superoxide dismutase?SOD?,malondialdehyde?MDA?,glutathione?GSH?,inducible nitric oxide synthase?iNOS?and nitric oxide?NO?in BRL hepatocytes.ResultsThe phenolic content was in the range of 502.33⁵86.53?g GAE/mg EDW.The results showed that rosmarinic acid was the major phenolic compound in the free phenolic extracts with the content ranging 319.70 to 400.46?g/mg EDW.The results of BRL hepatocytes injury in vitro showed that compared with the model group,the free phenolic extracts from Lycopus lucidus Turcz.had a significant inhibitory effect on the increase of ALT,AST and LDH in BRL hepatocytes injured by CCl₄?p<0.05?.The free phenolic extracts could significantly improve the nuclear morphological's change caused by CCl₄,reduce the increase of apoptosis peak ratio of sub-G₁ cells and promote the cells to enter the mitotic phase.Free phenolic from Lycopus lucidus Turcz.was able to improve the level of SOD and GSH,and reduce the contents of MDA,iNOS and NO in hepatocytes after CCl₄-induced injury.The results indicated that free phenolic extracts from Lycopus lucidus Turcz.had a certain effects on the nuclear morphology and cell cycle distribution.And it had an antioxidant protective effect on BRL hepatocytes injury by CCl₄.ConclusionsThe effects of sampling sites and harvest periods on the phenolic contents and the composition of main phenolic compounds in the free phenolic extracts from Lycopus lucidus Turcz.were significantly different.The main phenolic compound in the free phenolic extracts from Lycopus lucidus Turcz.was rosmarinic acid.The free phenolic extracts had a significant protective effect on BRL hepatocytes injury induced by CCl₄.And there was a good dose-effect relationship between the extracts'concentration and the protective effect in the range of experimental concentration.The protective mechanism of free phenolic extracts on BRL hepatocytes injury induced by CCl₄ was related to the influence of cycle distribution and the antioxidant effect.