Extraction Of Total Flavonoids From Equisetum Hyemale L. And The Study On The Protective Of APAP-induced Liver Injury In Mice

This product was taken from the dried aerial parts of Equisetum hiemaleL..which was included in the 2015 edition of the Chinese Pharmacopoeia.Its nature is sweet,bitter,and gentle in taste,and belongs liver,lung channel.Equisetum hiemale L.can dispel wind,heat and retire eyesight.It is mainly used to treat dizzy,tears,blood under the intestinal wind,trauma bleeding.Equisetum hiemale L.is mainly produced in the Northeast,North China,and Inner Mongolia,which is a traditional Chinese medicine with a long history.The main active ingredient of Equisetum hiemale L.is flavonoids.This paper studied the extraction process of total flavonoid content of Equisetum hiemale L.and explored its protective effect of liver damage preliminarily,which is of great significance to deeply develop the clinical application value of Equisetum hiemale L..The root of Equisetum hiemale L should be removed.The rest part shoud be dried,smashed,sifted and reserced.A suitable amount of fine powder was used to conduct a single-factor investigation of ethanol concentration,extraction time,and feed-liquid ratio under certain conditions.The optimum extraction process of total flavonoids from Equisetum hiemale L.was screened by Box-Behnken Design response surface optimization method,and the antioxidant activity was studied.The results showed that when the ethanol concentration was 70%,the extraction time was 50 min,and the solid-liquid ratio was 1:25?g/ml?,the yield was 10.06%and the total flavonoid content of Equisetum hiemale L.was up to 13.57 mg/g,which was in good agreement with the predicted value of up to 98%.The effect of factors on total flavonoid content was:ethanol concentration>extraction time>solid-liquid ratio.The paper also explored the antioxidant activity of total flavonoids obtained by the optimal process.The experiment showed that the total flavonoids of Equisetum hiemale L.oxysporum had a certain reducing ability,and the scavenging effect of hydroxyl radicals?�OH?,ABTS+�,DPPH�increased with the increase of flavonoid concentrations.The corresponding IC50 values were 1.02�0.01,0.75�0.00,and0.35�0.00 mg/ml,respectively.This study laid the foundation for the extraction of total flavonoids from Equisetum hiemale L.,the development of natural antioxidants and follow-up experiments.On the basis of previous experiments,10 kg of ground fine powder was taken and the total flavonoids of Equisetum hiemale L.were extracted according to the best technology.After petroleum ether degrease?181 g?,826 g of dry powder was obtained,and appropriate amount of distilledwater was added to dissolve the sample to the maximum extent.The D101 macroporous resin column was used to separate and purify the sample.Gradient elution was performed with 5column volumes of distilled water,30%,50%,and 70%ethanol.Collected the eluate of 30%,50%,70%ethanol macroporousresin,The ethanol was recovered by reduced pressure,then the30%macroporous resin elution site?EHLA?,50%macroporous resin elution site?EHLB?,70%macroporous resin elution site?EHLC?were obtained by freeze-dry,the flavonoid content of Equisetum hiemale L.was assayed and its antioxidant effect was explored.The results showed that the total flavonoid content of Equisetum hiemale L.rose from 16.43%of total extract to70.95,83.61,and 68.37%of EHLA,EHLB,and EHLC after the purification of D101macroporous resins,the contents from high to low were:EHLB>EHLA>EHLC.EHLA showed a strong reduction ability.The IC₅₀ values of EHLA,EHLB,and EHLC on ABTS+�radical scavenging capacity were:0.36,0.20,and0.30 mg/ml,the scavenging capacity is:EHLB>EHLC>EHLA;The IC₅₀ values for DPPH�scavenging capacity were:0.06,0.02,0.14mg/ml,The scavenging capacity is:EHLB>EHLA>EHLC.The comprehensive yield EHLB had the strongest antioxidant capacity,followed by EHLA.The liver is an important metabolic organ of the body and plays an irreplaceable role.Studies found that the widely used antipyretic analgesic,acetaminophen?abbreviated as APAP?,had a certain degree of damage effect on the liver.APAP at a safe dose will be excreted by the kidneys after exerted the therapeutic effect,while excessive APAP can cause severe liver damage and even cause liver failure.This study,based on the antioxidation of total flavonoid content of Equisetum hiemale L.and the anti-inflammatory effects of flavonoids,established a model of APAP-induced acute liver injury to observe the hepatoprotective effects of EHLA and EHLB.The results showed that compared with the model group,the growth status of the EHLA,EHLB-administered mice was significantly improved,both drugs can significantly reduce the serum ALT,AST levels in mice,increased the MDA content in the liver tissue,decreased the levels of SOD,GSH and GSH-Px in liver tissue to some extent,and inhibited the increase of TNF-?and IL-8 content.H&E staining and Western blot results showed that the drug groups can relieve APAP-induced liver cell apoptosis.The overall results indicated that EHLB-H had a protective effect on APAP-induced acute liver injury,and its mechanism may be related to the inhibition of lipid peroxidation and oxidative stress,inflammatory response and anti-apoptosis.