Preliminary Studies On Formula Screening,Optimized Process And Hepatoprotective Activity Of Zhen-Ju Decoction

As one of the metabolic organs in the digestive system,the liver has many biological functions such as transformation,excretion,and detoxification and is susceptible to injury caused by various infectious and uninfectius factors.Chemical liver injury is most common and can lead to liver fibrosis,or irreversible cirrhosis,which eventually leads to liver cancer.Related studies have shown that the pathogenic mechanism of chemical liver injury is caused by the accumulation of reactive oxygen radicals and results in the inflammatory response.In Chinese traditional medicine,many medicinal plants were used for complementary and alternative therapy of chronic diseases,and many various plant extracts contain a variety of chemical substances with biological activity such as hepatoprotective activity.In this study,we evaluate the hepatoprotective activity of Zhen-Ju Decoction selected from 17 medicinal plants based on antioxidant,antibacterial and anti-inflammatory activities in vitro,the underlying molecular mechanisms in mouse models of carbon tetrachloride(CCl4),paracetamol(APAP)and alcohol-induced liver injury were discussed and evaluated.1.Antioxidant activity evaluation of 17 plant extracts in vitroAntioxidant activities of the total antioxidant capacity,hydroxyl radical scavenging capacity and DPPH radical scavenging capacity of the selected 17 plant extracts were evaluated,and the plant extracts with higher comprehensive scores were screened in vitro.The experimental results showed that Pedicularis longiflora Rudolph.var.tubiformis(Klotz.)Tsoong,Saussurea brunneopilosa H.-M,Lysimachia clethroides Duby,Thalictrum delavayi Franch.,Vicia unijuga A.Br.,Rhododendron aganniphum Balf.f.et K.Ward,Trollius chinensis Bunge and Cardamine tangutorum O.E.Schulz performed the total antioxidant capacity.The scavenging rates of hydroxyl radicals of Pistacia chinensis Bunge,Gentiana waltonii Burk.,Saussurea brunneopilosa H.-M,Primula sikkimensis Hook.f.,Lysimachia clethroides Duby and Chrysanthemum morifolium were all above 60%;Those with DPPH free radical scavenging rates higher than 90% were Pistacia chinensis Bunge,Pedicularis longiflora Rudolph.var.tubiformis(Klotz.)Tsoong,Saussurea brunneopilosa H.-M,Primula sikkimensis Hook.f.,Lysimachia clethroides Duby,Trollius chinensis Bunge and Cardamine tangutorum O.E.Schulz.Lysimachia clethroides Duby,Thalictrum delavayi Franch.and Cardamine tangutorum O.E.Schulz were selected as candidate drugs for subsequent formulation optimization according to the comprehensive scoring criteria.2.Formula optimization and toxicity evaluation of Zhen-Ju DecoctionOrthogonal design was used to optimize the formulations of the 3 plant extracts,and the preliminary toxicity evaluation was carried out.The results of range analysis and visual analysis showed that the ratio of Lysimachia clethroides Duby,Thalictrum delavayi Franch.and Cardamine tangutorum O.E.Schulz(4:4:1)was determined as the best optimized formula based on the highest comprehensive scores of antioxidant,antibacterial and anti-inflammatory activities in vitro.The IC50 of DPPH free radical scavenger and hydroxyl radical scavenger were 2.394 mg/m L and 0.104 g/m L,respectively,and the total antioxidant capacity was 3.73;The anti-inflammatory rate was 75.97 %;The inhibition zone sizes against ATCC25922 and ATCC43300 were 13.67 mm and 12.17 mm,respectively;The toxicity test showed that optimized formula was safe and non-toxic in mice.3.Protective effect of Zhen-Ju Decoction on acute liver injury models in miceAfter continuous gavage of water extract of the optimized formula for 14 days,the pathological models of acute chemical liver injury,drug-induced acute liver injury and alcohol-induced acute liver injury were replicated by intraperitoneal injection of CCl4,APAP and oral administration of alcohol.The results showed that hepatic pathological changes,serum biochemical tests(AST,ALT,TG,T-CHO),hepatic antioxidant indexes(SOD,MDA,T-AOC,GSH-Px)and inflammatory factors(IL-1β,IL-6,TNF-α)and liver-specific expression of mi RNA(mi R-122)were significantly different from those in the blank control group(P<0.05).In the three acute liver injury model groups,pathological changes such as loss of normal hepatic lobules,narrowing of hepatic sinusoids,large areas of hepatic fibrosis around sinusoids,degeneration and necrosis of hepatocytes,interstitial congestion and nuclear lysis were presented.Compared with the model group,serum biochemical indexes,hepatic antioxidant indexes,inflammatory factors and pathological changes were improved to varying degrees,indicating that water extracts of the optimized formula in different dose groups had a good protective effect on the three acute liver injury models.