Chemical Composition And Hepatoprotective Mechanism Of Flavonoids From Ziziphus Jujuba Mill.cv.Jinsixiaozao

Ziziphus jujuba Mill.,as a kind of medicine and food homologous Chinese herb-al medicine,is rich in nutrients and biological active ingredients,and has extremely high nutritional and health care value.Phytochemical studies in recent years have re-vealed that Ziziphus jujuba Mill.contains many functional constituents such as poly-saccharides,triterpenic acid,flavonoids,phenolic acids,Cyclic nucleotides and alka-loids,which have been shown to be responsible for various pharmacological effects including antioxidant,anti-inflammatory,anxiolytic,antiobesity,regulation of im-mune function,antiproliferation of cancer cell,hepatoprotective and gastrointestinal protective activities.China is the country with the richest Ziziphus jujuba Mill.re-sources in the world.Laoling is the hometown of golden jujube in China.Laoling xiaozao is a unique jujube variety in China.It not only has good flavor and rich nutri-tion,but also has high drug value.Flavonoids,as one of the main components of jujube,have a variety of biological activities.Although there have been many reports on flavonoids from jujube in recent years,most of them were concentrated on the extraction,separation and purification of flavonoids from jujube,and the analysis of flavonoids in aqueous ethanol extracts.There are few reports on the analysis of the flavonoids in the purified flavonoids from jujube.In addition,reports on the hepatoprotective effects of jujube were mainly on the hepatoprotective activity of alcohol extracts,aqueous extracts,and polysaccha-rides from jujube.There are few reports available on the hepatoprotective effects of flavonoids from jujube.The purpose of this study was to analyze the chemical composition of flavonoids from Ziziphus jujube Mill,cv.Jinsixiaozao(ZJF)and explored its hepatoprotective mechanism.The main contents of the study were divided into the following three parts:(1)Identification and antioxidant activity evaluation of ZJF;(2)The protective activ-ity and mechanism of ZJF on acetaminophen induced liver injury in vitro;(3)Protec-tive activity and mechanism of ZJF on acetaminophen-induced liver injury in vivo.Firstly,in this study,HPLC and LC-MS/MS methods were used to qualitatively and quantitatively analyze the chemical composition of ZJF.DPPH and ABST radical scavenging assay,ferric ion reducing power(FRAP)were used to evaluate its antiox-idant capacity.In this experiment,we identified two kaempferol derivatives and seven quercetin derivatives,among which quercetin derivatives were the main flavonoids found in ZJF.In vitro,the results of antioxidant experiments showed that ZJF had good scavenging ability for DPPH and ABST free radicals,and had good reducing ability.Secondly,we used acetaminophen(APAP)-induced L-02 cells as a model to study the hepatoprotective activity of ZJF in vitro.The levels of AST,ALT,LDH,NO in culture supernatant and the the antioxidant enzyme(SOD)activity and non-enzymatic antioxidant glutathione(GSH)content in cell lysates were detected by commercial kits;Flow cytometry was used to detect the ROS content in L-02 cells;RT-PCR technique was used to investigate the expression of pro-inflammatory cyto-kines TNF-?,IL-1? and IL-6 mRNA in L-02 cells at the gene level.We also used Western blot to explore the hepatoprotective mechanism of ZJF from the level of pro-tein expression in vitro,not only the expression of COX-2 and iNOS protein related to inflammation were detected,but also the expression of NF-?B p65 and Nrf2 were de-tected in the nucleus and cytoplasm,which are the key regulators of inflammatory re-action and antioxidant response respectively.The experimental results showed that ZJF could inhibit the rise of biochemical markers such as AST,ALT,LDH,NO in-duced by APAP in L-02 cell supernatants,increase the activity of SOD,and reduce the consumption of intracellular GSH.In addition,ZJF could reduce APAP-induced excessive ROS production,thereby inhibiting intracellular oxidative stress.From the gene level,ZJF could reduce the expression of pro-inflammatory cytokines TNF-a,IL-1?,IL-6 mRNA,and then inhibit APAP-induced inflammatory response.Western blot experiments also showed that ZJF can reduce the expression of NF-?B p65 in the nucleus and the important enzymes regulated the inflammatory process such as COX-2 and iNOS,and increase the expression of Nrf2 in the nucleus.These results suggest that ZJF might alleviate APAP-induced cytotoxicity through its strong anti-oxidant and anti-inflammatory effects in vitro.At last,the hepatoprotective activity of ZJF was evaluated in APAP-treated mice We use the commercial kits to detect changes in the levels of the serum AST,ALT,ALP,TB,NO in mice,as well as changes in the antioxidant enzyme(such as SOD,GSH-PX)activity,GSH and lipid peroxidation product malondialdehyde(MDA)lev-els in mice liver.The release of pro-inflammatory cytokines TNF-?,IL-1? and IL-6 in serum was detected by enzyme-linked immunosorbent assay.Histopathological sec-tions were used to observe the extent of liver tissue damage in mice.In addition,Western blot and immunohistochemistry was used to detect the expression of NF-?B p65,Nrf2,and NQO1 protein in liver tissue.The experimental results showed that ZJF could reduce the changes in biochemical parameters associated with liver damage and the MDA level and GSH depletion,reverse the decrease of SOD and GSH-PX ac-tivity in liver tissue induced by APAP.This indicates that ZJF could inhibit APAP-induced oxidative liver injury by enhancing the enzymatic and non-enzymatic antioxidant defense system in the liver of mice.ZJF attenuated APAP-induced in-flammatory mediator production,such as NO,TNF-a,IL-6 and IL-1?.The patholog-ical section of liver tissue showed that ZJF was able to alleviate the severe pathologi-cal changes of liver tissue caused by APAP.The above experimental results show that ZJF might attenuate APAP-induced liver injury in mice through its antioxidant and anti-inflammatory effects.In addition,Western blot and immunohistochemistry re-sults showed that ZJF could inhibit the expression of NF-?B p65 protein and its nu-clear translocation,while enhanced the expression of Nrf2 and NQO1,and promote nuclear translocation of Nrf2.In summary,this is a systematical report on the chemical composition and anti-oxidant capacity assessment of ZJF.Identification of active ingredients showed that ZJF contain mainly kaempferol and quercetin flavonoids.This topic explored the hepatoprotective activity and mechanism of ZJF in vitro and in vivo.From the exper-imental results,we can infer that ZJF exerts their hepatoprotective activity through antioxidant and anti-inflammatory effects.In addition,further signal pathway studies have shown that ZJF may mitigate APAP-induced hepatotoxicity by activating Nrf2 and inhibiting NF-?B signaling pathways.This research will lay a theoretical founda-tion for the further study of the potential value of the hepatoprotective of Ziziphus jujuba cv.Jinsixiaozao,and provide experimental technical guidance for the further development of medicinal products.