Extraction And Purification Of Polyphenols From Artemisia Integrifolia L. And It's Anti-inflammatory And Hepatoprotective Effects

The Artemisia integrifolia L.Polyphenols?AILP?was extracted and purified from the bud of Artemisia integrifolia L.,and its anti-inflammatory and hepatoprotective effects on mice with acute liver injury induced by carbon tetrachloride were explored.In this study,Ultrasound-assisted,cellulose-breaking method was used to extract AILP.Eight factors that will affect the yield of AILP,including enzymatic hydrolysis,ultrasonic time,ethanol concentration,liquid-to-material ratio,enzymolysis temperature,enzymatic hydrolysis time,ultrasonic power and enzymolysis pH were used for single factor experiments.Plackett-Burman test and Box-Behnken test to determine the optimal extraction scheme;The crude polyphenols were purified by D101 macroporous adsorption resin.The factors such as sample solution concentration,sample solution pH,sample loading rate,eluent volume fraction,elution rate,and eluent usage amount were selected for factor selection,the orthogonal experiment analysis to get the best purification process conditions;After purification by D101 macroporous adsorption resin,the AILP were identified by high performance liquid chromatography to determine the polyphenols species.Acute liver injury model was established in mice induced by carbon tetrachloride?CCl4?and intragastric administration of AILP.Detection of serum alanine aminotransferase?ALT?,aspartate aminotransferase?AST?activity,superoxide dismutase?SOD?activity,reduced glutathione?GSH?and malondialdehyde?MDA?content in liver homogenate and calculation Liver index,determination of serum inflammatory cytokines:tumor necrosis factor-alpha?TNF-??,interleukin-6?IL-6?,and nitric oxide?NO?levels.and discuss the mechanisms associated with the anti-inflammatory and hepatoprotective effects of AILP.Test results are as follows:1.According to the Plackett-Burman analysis in Design-Expert 8.06 software,the factors influencing the yield of AILP were ranked according to their importance:enzymolysis temperature?E?,enzymolysis pH?H?,ethanol concentration?C?,enzymolysis time?F?,enzyme addition amount?A?,liquid to material ratio?D?,ultrasonic power?G?and ultrasonic time?B?.The three factors of E,H,and C have the greatest influence on the extraction of AILP.2.By Box-Behnken analysis in Design-Expert8.06 software,the order of ethanol concentration?A?,enzymolysis temperature?B?and enzymolysis pH?C?on the AILP yield was B>A>C.The fixed extraction process of AILP was determined as follows:ethanol concentration 70%,enzymolysis temperature 60°C,enzymatic hydrolysis pH5.5,enzyme dosage 50 mg/g,enzymolysis time 2 h,solid-liquid ratio 1:30?g:mL?,ultrasonic time 30min,ultrasonic power 400W,extracted twice under conditions,the yield was 9.41%,3.The purification test results showed that,the adsorption capacity of D101macroporous resin to ALPD was 18.9?mg/g?,the adsorption rate was 94.3%,the desorption rate was 97.3%,and It is the best resin in these five macroporous resin of D101,D4020,HPD100C,HPD600,NKA-9.Orthogonal test results showed that the influence of the three factors of sample concentration?A?,sample solution pH?B?and sample rate?C?on the adsorption rate was C>A>B,and the effect was significant.Combined with the results of dynamic desorption test,the optimum conditions for the purification of AILP with D101 macroporous adsorption resin were as follows:sample solution concentration1.0 mg/mL,sample solution pH5,loading rate 1.5mL/min,eluent volume fraction 75%,the elution rate was 1.0 mL/min,and the eluent amount was 200 mL,the AILP with a purity of 76.2%.4.HPLC results showed that the main components of AILP were chlorogenic acid,ferulic acid,gallic acid,catechin,and quercetin.Among them,the highest chlorogenic acid content was 43.04.%,the content of gallic acid and quercetin was the second highest,accounting for 11.67%and 11.31%of the total,respectively.The content of catechin and ferulic acid was the lowest,accounting for 4.34%and 7.39%of the total,respectively.These five phenolic substances a total of 77.75%of the total,can be basically confirmed that the purified AILP contains chlorogenic acid,ferulic acid,gallic acid,catechin and quercetin five phenolic substances.5.The results of animal experiments showed that AILPH?400 mg/kg?,AILPM?200 mg/kg?,and AILPL?100 mg/kg?all inhibited ALT,AST,and TNF-?in the serum caused by CCl₄-induced acute liver injury in mice.The levels of IL-6 and NO increased?P<0.01?,decreased MDA content and liver coefficient?P<0.01?,increased SOD and GSH vitality?P<0.01?,and improved liver tissue morphological lesions.These results indicate that AILP has anti-inflammatory and hepatoprotective effects on acute liver injury induced by CCl₄ in mice.The mechanism may be related to the scavenging of free radicals,anti-lipid peroxidation and inhibition of inflammatory cytokine activation,and protection of hepatocyte metabolism balance.