Study On The Inhibitory Mechanism And Effects Of Lonicera Caerulea Berry Anthocyanin Extracts On Hepatitis Induced By Lipopolysaccharide

Lonicera caerulea L.is belonging to Caprifoliaceae and honeysuckle.L.caerulea berry,with the "third generation fruit" reputation,is rich in anthocyanins and has higher nutritional,healthy and medical values.Researches have shown that anthocyanins have a wide range of biological activities,such as antioxidant and anti-aging.Long-term intakes of anthocyanins have been suggested to reduce the incidence of chronic diseases.Lipopolysaccharide(LPS)is a kind of endotoxin produced during the body's metabolism.LPS can stimulate various pro-inflammatory cytokine expressions and induce liver Immune response,thereby promoting liver inflammation,which is the etiology of some chronic liver diseases such as liver cirrhosis and cancer.Therefore,the study on the inhibitory mechanism of L.caerulea berry anthocyanins in LPS-induced liver inflammation,which provides theoretical references for seeking a potential drug or health care product for preventing chronic hepatitis,is of great significance.In this study,based on the qualitative and quantitative analysis of individual anthocyanins from various cultivars of L.caerulea berries,in vitro and in vivo experiments were used to investigate the inhibitory mechanism and effects of L.caerulea berry anthocyanin extracts on LPS-induced liver inflammation from oxidative stress,energy metabolism,liver function,antioxidant capacity,inflammatory factors and apoptosis related proteins expression,as well as mitogen-activated protein kinase(APK)signaling pathway.The main results are as follows:HCl(0.10%)acidized methanol was used to extract L.caerulea anthocyanins assisted with ultrasonic.According to the results of single factor experiment,response surface analysis was used to optimize the extraction process conditions.After slightly adjustment,the optimal extraction process:solid-liquid ratio was 1:10,extraction time was 90 min,extraction temperature was 40?;the optimal conditions for the static adsorption and desorption of D101 macroporous resin:adsorption equilibrium time was 3 h,desorption equilibrium time was 2 h,sample concentration was 2.52 mg/mL,methanol concentration was 100%;the optimal conditions for the dynamic adsorption and desorption:sample velocity was 2 BV/h,sample volume was 490 mL,elution velocity was 2 BV/h and distilled water consumption was 2 BV.The purified L.caerulea anthocyanins extract under these conditions was purple black powder with the color value of 47.8,which exhibited 11-fold enhancement compared with that of unpurified anthocyanins,and the anthocyanins content was 484.2 mg CYD-3-G/L.The individual anthocyanins composition and proportion are different from various cultivars of L.caerulea berries.In the present study,14?11?11 and 12 individual anthocyanins were identified in wild,'Beilei','NO.1' and 'NO.2' L.caerulea berries,respectively.The major anthocyanin of the above four cultivar L.caerulea berries was cyanidin-3-glucoside,which respectively accounted for 89.8%?90.7%?89.6%and 91.3%of total anthocyanins.Cyanidin-3-acetylhexoside(found in 'Beilei','No.1' and 'No.2' cultivars)and peonidin-3-acetylhexoside(found in 'No.2' cultivar),were found for the first time in L caerulea berry extracts.No significant differences in total anthocyanins content were found in the four cultivar L.caerulea berries.Relatively high antioxidant capacity and total polyphenols content were measured in wild and 'Beilei' L.caerulea berries.Total soluble solid of 'NO.2' L.caerulea berry was the highest.After comparing the anthocyanins composition and antioxidant activity of various L.caerulea berries,'Beilei' L.caerulea berry,which is suitable for eating and with relatively high antioxidant activity,was selected for the subsequent experiments.The results of anthocyanins content and antioxidant activity before and after digestion suggested that in vitro digestion decreased anthocyanins content and antioxidant activity:anthocyanins content decreased by 67.6%,oxygen radical absorbance capacity(ORAC)and total antioxidant capacity(T-AOC)decreased by 80%and 55.6%,respectively.Additionally,compared to the undigested extracts,the number of individual anthocyanins and phenolic acid of extracts after in vitro digestion reduced 3 and 1,respectively.The decreases in anthocyanins content and antioxidant activity indicated that the biological activity of the anthocyanin extracts subjected to in vitro digestion might decline.L.caerulea berry anthocyanin extracts before and after in vitro digestion could effectively suppress LPS-induced reactive oxygen species(ROS),8-hydroxydeoxyguanosine(8-OHdG),protein hydroxyl,lipid peroxidation products generation and glutathione(GSH)depletion;inhibit the decreases in ATP synthase,cytochrome oxidase(COX)and succinate dehydrogenase(SDH)activities and the increases in pro-inflammatory cytokines expression(interleukin(IL)-1? IL-6);modulate aspartate aminotransferase(AST),alanine aminotransferase(ALT)and cholinesterase activities,as well as maintain cellular structure.Therefore,L.caerulea berry anthocyanin extracts could prevent against the development of LPS-induced BRL-3A cells inflammation by inhibiting oxidative stress,maintaining energy metabolism and modulating liver function.Moreover,the effects of undigested anthocyanin extracts were superior to digested anthocyanin extracts.L.caerulea berry anthocyanin extracts before and after in vitro digestion could attenuate the LPS-caused decreases in catalase(CAT)and superoxide dismutase(SOD)activities,thereby maintaining the total antioxidant activity(T-AOC),which was identified by the higher T-AOC value;inhibit the up-regulation of nuclear factor-kappa B(NF-?B)and tumor necrosis factor-?(TNF-?)mRNA expressions and down-regulation of IL-10 mRNA expression,which suppressed inflammation development;attenuate the increases in Caspase-3,cleaved Caspase-3,Bax and cleaved PARP protein expressions and decreases in poly(ADP-ribose)polymerase(PARP)and B-cell lymphoma(Bcl-2)protein expressions,which attenuated cell apoptosis.L.caerulea berry anthocyanin extracts attenuated LPS-induced liver toxicity via antioxidant,anti-inflammatory,and anti-apoptotic pathways,and exhibited protective effects on liver cells.Additionally,L.caerulea berry anthocyanin extracts could inhibit the development of hepatitis by suppressing oxidative stress(ROS,GSH),liver dysfunction(AST,ALT),abnormal cell cycle,organization structure damage,inflammatory factors(C-reactive protein(CRP),IL-6)expressions.as well as toll-like receptor and oxidative stress-associated MAPK signaling transduction.Moreover.L.caerulea berry anthocyanin extracts protected liver in a dose-dependent manner in the range of 50-200 mg/kg bw.The present results supplement the insight into underlying anti-inflammatory mechanisms of L.caerulea berry anthocyanins,which provide a basis for its use as functional foods or alternative drugs to prevent hepatitis.