Extraction And Purification Of Gingerols In Rucheng Xiaohuang Ginger And Their Intervention On Non-alcoholic Fatty Liver Disease

Xiaohuang Ginger,a variety of Zingiber officinale Roscoe grown in Rucheng County,Chenzhou City,Hunan Province,China,possesses various bioactivities such as antioxidant,anti-tumor,anti-inflammatory,and hypoglycemic effects.Non-alcoholic fatty liver disease(NAFLD),closely associated with obesity,includes simple steatosis,non-alcoholic steatohepatitis,and liver cirrhosis.The global prevalence of NAFLD is approximately 25%,with a proportion of nearly 30% in China.Therefore,investigating effective interventions for preventing and treating NAFLD is of great significance.Previous studies have indicated that Xiaohuang Ginger extract exhibits lipid-lowering activity,but its underlying substance and mechanisms of action in preventing NAFLD remain unclear.In this study,we optimized the extraction and purification process of 6-gingerol from Xiaohuang Ginger,predicted its pharmacological targets using network pharmacology,and established NAFLD cell models to verify the protective mechanisms of the main components of 6-gingerol.The main results are as follows:(1)Optimization of Xiaohuang Ginger extraction process: Through single-factor and response surface experimental designs,the optimal extraction process was determined as follows: extraction time of 83 min,temperature of 39 ℃,and solid-liquid ratio of 15(m L/g).The total phenolic content of the extract was 11.52 mg/g.HPLC analysis revealed that the content of 6-gingerol was 203 mg/g,8-gingerol was 16 mg/g,and 10-gingerol was 16 mg/g in the crude extract.(2)Optimization of 6-gingerol purification process: AB-8 macroporous resin was determined as the optimal filler through static adsorption and desorption experiments.The effects of p H,ethanol concentration,and elution time on the desorption process of6-gingerol were investigated.The highest desorption rate was achieved at p H 7,ethanol concentration of 80%,and elution time of 6 h.Dynamic adsorption and desorption experiments showed that the content of 6-gingerol in the fraction eluted with 80%ethanol was 729 mg/g,which was 2.59 times higher in purity compared to the crude extract.Antioxidant activity experiments indicated that 6-gingerol was the major active substance,with DPPH and ABTS radical scavenging rates reaching 60.3% and 20.2%for the fraction eluted with 80% ethanol,and 80.9% and 50.9% for pure 6-gingerol,respectively,while the fractions with lower 6-gingerol content exhibited lower scavenging rates.(3)Effects of 6-gingerol on NAFLD intervention: 6-gingerol could partially restore the damage caused by free fatty acids and reduce lipid accumulation in a dosedependent manner.Treatment with 10 μmol/L 6-gingerol reduced the triglyceride content by 42.2% in Hep G2 cells and 48.7% in LO-2 cells.Through network pharmacology analysis,potential targets related to the interaction between 6-gingerol and NAFLD were explored,and FABP1,ACOX1,CD36,PPARα,and PTGS2 were identified as key proteins.RT-PCR and Western blot experiments,with GAPDH as the reference protein,were performed on Hep G2 and LO-2 NAFLD cell models.In Hep G2 cells,low concentrations of 6-gingerol decreased the expression of FABP1,ACOX1,CD36,and PPARα proteins(p<0.05)but had no significant effect on PTGS2 expression.High concentrations of 6-gingerol significantly reduced the expression of all five proteins in the model groups(p<0.05).In LO-2 cells,low concentrations of 6-gingerol increased the expression of ACOX1 and PPARα(p<0.05)while decreasing the expression of the other three proteins(p<0.05).High concentrations of 6-gingerol reduced the expression of PPARα(p<0.05)and exhibited enhanced effects on the other four proteins compared to low concentrations(p<0.05).These results suggest that 6-gingerol may mainly reduce lipid accumulation by upregulating ACOX1 expression,but it exhibits different mechanisms and effects in different cell types.