Extraction And Biological Activity Evaluation Of Flavonoids From Different Years Of Citrus Reticulata ’Chachi’

XinhuiChenPi（Citrus reticulata’Chachi’）is the dried and mature peel of Chachiensis produced in Xinhui area,Jiangmen City,Xinhuidong Province.As a traditional Chinese medicinal material,it has both medicinal value and edible value.The XinhuiChenPi is rich in flavonoids,especially Polymethoxylated flavones（PMFs）,which have anti-inflammatory,antioxidant,anti-cancer and other biological activities.Previous studies suggested that the flavonoids of XinhuiChenPi will change during aging,but whether this change will lead to the change of its biological activity remains unclear.Therefore,the study on the changes of flavonoids and their biological activities during the aging process of XinhuiChenPi is related to the aging time,providing guidance for revealing the effects of flavonoids on biological activities during the aging process of XinhuiChenPi,and providing an important theoretical basis for targeted development of XinhuiChenPi.In this study,the XinhuiChenPi from 2011 to 2020 in Xinhui area of Xinhuidong Province was used as the research object.The optimal extraction conditions of ultrasonic assisted solvent were obtained by response surface method.The content of flavonoids in XinhuiChenPi was compared with that of HPLC.Saccharomyces cerevisiae model was used to evaluate the antioxidant activity of XinhuiChenPi bark of different years,and human monocyte macrophage THP-1 was used to evaluate the anti-inflammatory activity of XinhuiChenPi bark of different years.The main results can be summarized as follows:（1）Combined with the single factor test results,response surface methodology（RSM）was applied to optimize the extraction conditions.And the content of total flavonoids as the response value.The results showed that under the conditions of 64%ethanol as extraction solvent,solid-liquid ratio of 1:40 g/m L and extraction time of69min,the total flavonoid content was 6.013 mg/g,the actual extraction content was5.955 mg/g,and the relative error was 0.92%.The optimum technological conditions can be applied to the extraction of total flavonoids from XinhuiChenPi.（2）After methodological investigation,it was confirmed that the established HPLC detection conditions met the experimental requirements.Rutin decreased with the aging time during the 10-year aging process,and the contents of naringin and 5-demethylcitrine did not change significantly during the aging process,while the contents of nobiletin,3,5,6,7,8,3’,4’-heptamethoxyl flavonoid,tangeretin and 5-demethylnobiletin tended to increase.（3）The antioxidant activities of 2011,2016 and 2020 XinhuiChenPi were compared using Saccharomyces cerevisiae.The results showed that XinhuiChenPi could significantly improve the survival rate of yeast cells under H₂O₂ and CCl₄ stress,reduce the level of lipid peroxidation and intracellular peroxidation,and the antioxidant capacity increased with aging time.（4）The inflammatory model was established on human mononuclear macrophages（THP-1）by lipopolysaccharide（LPS）,and the anti-inflammatory activity of XinhuiChenPi from 2011,2016 and 2020 was compared.The results showed that the expression levels of inflammatory factors TNF-α,IL-6 and IL-1βwere significantly down-regulated by XinhuiChenPi,and the down-regulation ability increased with aging time.XinhuiChenPi at the early stage of aging seemed to have a better ability to inhibit the phosphorylation of IκBαand P65 protein in NF-kB pathway.In contrast,ERK1/2and P38 phosphorylation in MAPKs pathway were more inhibited by XinhuiChenPi at later aging stage.