Isolation And Identification Of Quinone Reductase Inducers From Ginger And Their Inducing Effects

Exogenous carcinogenic chemicals have been increasingly recognized asthe important factors to cause cancer and other malignancies in human body.In vivo detoxification of these chemicals is commonly consisted of phase Iand phase II reactions.Phase I reaction might activate some exogenous pro-carcinogens in some cases,while phase IIreaction can effectively eliminate free radicals and electrophilic reagents,and thus lead to detoxification of the carcinogen chemicals.Therefore,to selectively induce phase II enzymes is helpful for detoxification of exogenous carcinogens in the body,which greatly provoked the interest of the researchers in screening the phase II enzymes inducers.With the deepening of the research,it is found that synthetic inducers have strong toxic effects,thus more and more attention are paid to those from natural origins,which might be promising sources for phase II enzymes inducers with higher efficiency and lower toxicity.Ginger is a kind of medicinal and edible plant with a long history,since it is rich invarious nutrients and bioactive ingredients.Ginger is also widely utilized as spice and medical herb in China and southeast countries.Results from pharmacological studies show that ginger and its extracts possess many pharmacological effects,including anti-inflammation,anti-sickness,antitumor and antibacterial activity.However,to the best of our knowledge,little informationis available regarding the phase II enzymes inducing effects of ginger compounds and their inducing mechanism.The objective of this study is to isolate the potential phase II enzymes inducing compounds from ginger guided by quinone reductase(QR)inducing assay,and further investigate theirinducingmechanism.The main work carried out and the results obtained were summarized as follows:1.Thirty-two kinds of ginger were collected,and their ethanolic extracts were prepared by Sohlext extraction.The DPPH??ABTS?+ and OH radical scavenging activity of 32 kinds of ginger extract were analyzed.Results showed that YKHJ and YNHJ exhibited higher(P< 0.05)potency in scavenging DPPH? radicals than other samples,while ZYDJ had the weakest potency.YKHJ,YNHJ and HNDJ presented the strongest potency in scavenging ABTS?+ radicals,while the weakest activity was observed for HWHJ.As for OH-scavenging ability,GSHJ,ZJHJ,GXHJ,YNHJ and FJXJ presented the strongest activity,while LPHJ had the least ability.2.The total phenol and flavonoid contents of 32 kinds of ginger extracts were investigated.Results showed that YKHJ had the highest total phenol content,followed by HNXJ and HNDJ,while the lowest total phenolic content was observed for GMBJ.YJHJ had the highest total flavonoid content,whileYKHJ had the lowest content of total flavonoid.The correlation analysis between total phenolic and flavonoid content and antioxidant capacity were carried out,and results showed that both DPPH? and ABTS radicals scavenging capacity of these samples were significantly(P< 0.05)correlated with their total phenol content.There was no significant(P> 0.05)correlation between the total flavonoid content and their free radicals-scavenging capacities.3.Their cytotoxic effects were firstly investigated,and then their inducing potency towards QR was compared.Results showed that XMTJ presented the strongest effect in inducing QR activity.Therefore,it was subjected to thin layer chromatography,silica gel chromatography and HPLC for further isolation,and the potential compounds were identified based on LTQ-Orbitrap-MSn data,namely 6-shogaol,6-gingerol,demethoxycurcumin curcumin and tetrahydrocurcumin.4.In order to further explore the possible mechanism by which ginger agents induce the QR,the effects of these compounds on Nrf2-Keap1 signaling pathway were determined by Western Blot technique.The results showed that ginger inducers were able tostimulate the translocation of Nrf2 from cytoplasm to nucleus after incubation for 24 h,meanwhile inhibit the expression of Keap1,with demethoxycurcuminbeing the strongest inducer among the samples.These findings suggest that the inducing potency of these ginger compounds is related to Nrf2-Keap1 pathway.RNA-seq analysis was also performed to explore the other pathways that the ginger compounds exert their inducing actions.It was found that curcumin,6-shogaol and demethoxycurcuminwere related to oxytocin,chemokine and cGMP-PKGsignaling pathway.