The Main Chemical Constituents From Laba Garlic And Their Biological Activities With Associated Mechanism

Laba garlic is a traditional Chinese processed garlic,which is widely used in the antimicrobial and antiviral treatment and in the prevention of cancer and cardiovascular diseases.At present,the study on Laba garlic is mainly focused on its preparation method and crude pigments,and its chemical composition and biological activities are rarely investigated.In order to clarify the chemical constituents and biological activities of Laba garlic and promote the comprehensive development and utilization of garlic resources,this study elucidated the chemical constituents and evaluated biological activities of Laba garlic and studied the related mechanisms.The results of this study will provide a theoretical basis for the comprehensive utilization of garlic resources and the in-depth study of Laba garlic.The main contents of this paper are as follows:?.Study on the liposoluble chemical constituents and biological activities of Laba garlic.The Laba garlic was extracted by cold ethanol,and the extraction was repeated3 times to obtain the extracts.Then,different polar extracts of Laba garlic were obtained by petroleum ether(PE),ethyl acetate(EA)and n-butanol(n-Bu OH)extraction,and then their antioxidant and anticancer activities in vitro were studied.The results showed that the EA extracts of Laba garlic had significant free radical-scavenging activity on DPPH(P<0.05)and ferric reducing ability(P<0.05),and significantly inhibited the proliferation of human hepatoma Hep G2 cells(P<0.05).By analyzing the chemical composition,the EA extracts showed the highest content of flavonoids.Based on the bioassay-guided method,EA extracts were isolated by silica gel column chromatography,Sephadex LH-20,ODS column chromatography pre-HPLC,and the compounds were identified by thin layer chromatography(TLC),MS and NMR spectrum.There were eight compounds isolated from the EA extracts of Laba garlic as follows:?-sitosterol(1),myricetin(2),kaempferol(3),quercetin(4),apigenin(5),isorhamnetin(6),?-Daucosterol(7)and isoquercitrin(8).Among them,isoquercitrin was first found in garlic.?.The in vitro antioxidant activity of the isolated three compounds of myricetin,isoquercitrin,and isorhamnetin,and the protective effect on H₂O₂-induced oxidative damage in L02 cells were studied.Then,hepatoma Hep G2 cells were used as a tumor model,and the anti-tumor activity of three flavonoids was evaluated by the MTT method.Lactate dehydrogenase(LDH)leakage assay,flow cytometry,and western blotting were used to evaluate the anticancer activity and cytotoxicity in vitro and to elucidate the related anticancer mechanism.The results of antioxidant studies showed that these three compounds showed significant free radical scavenging activity on DPPH(P<0.05)and ferric reducing ability(P<0.05),and had protective effects on H₂O₂-induced oxidative damage in L02 cells,which could repair the damaged mitochondria of cells and maintain the normal morphology of cells,and significantly increased the cell survival rates(P<0.05).The anticancer activity results showed that the three flavonoids could significantly inhibit the proliferation of Hep G2 cells(P<0.05)and showed no obvious toxicity on L02 cells.The half-inhibitory concentrations(IC₅₀)of myricetin,isoquercitrin and isorhamnetin on Hep G2 cells were 44.32±0.213,49.68±0.192 and 54.32±0.176?M,respectively.Flow cytometry analysis results showed that the three compounds were able to induce apoptosis on Hep G2 cells in a dose-dependent manner.The anticancer mechanism studies of the three compounds suggested that all three compounds could promote the apoptosis of Hep G2 cells by up-regulating the expression of Bax and cleaved-Caspase-3 proteins and down-regulating the expression of Bcl-2,Bcl-x L,pro-Caspase-3,and pro-Caspase-9 proteins.By using molecular docking technology,three flavonoid compounds were found to combine with apoptosis-related proteins mainly by hydrogen bonding,Pi-Pi interaction and Pi-cation interaction,and three flavonoid compounds could be the potential inhibitors of Bcl-x L and Bcl-2 and activators of Caspase-3 and Caspase-9.?.The main chemical constituents of the blue pigment of Laba garlic were studied.The biological activities were evaluated by anticancer assay,antioxidant assay,and cytotoxicity in vitro.The role of N-trans-feruloyltyramine(FLA)in promoting apoptosis was examined by LDH assay and flow cytometry analysis and its anticancer mechanism were preliminarily elucidated.FLA was isolated and identified from blue pigment by AB-8 macroporous resin column,Sephadex LH-20 gel column chromatography,pre-HPLC and HR-MS and NMR.The results showed that FLA had significant DPPH free radical scavenging activity and ferric reducing ability(P<0.05),and it showed protective effects on H₂O₂-induced oxidative damage of L02 cells,which could significantly increase cell viability(P<0.05).MTT assay results confirmed that FLA had a significant inhibitory effect on Hep G2 cell growth(P<0.05),and the half-inhibitory concentration was 194±0.894?M.After treatment with FLA(320?M),the apoptosis rate of Hep G2 cells was as high as 83.37±1.02%,and the treatment of FLA had no obvious effects on the survival rate of L02 cells(96.33±0.93%).The experimental results indicated that FLA had selective toxic effects on cells and exerts its toxic effects mainly by inducing apoptosis.?.Identification and antimicrobial activity evaluation of three peptides from Laba garlic and the related mechanism.Pepsin(p H 2.0)and trypsin(p H 8.0)were used to hydrolyze Laba garlic protein to obtain pepsin hydrolysates and trypsin hydrolysates,respectively.The growth inhibition effects of protein,pepsin hydrolysates and trypsin hydrolysates on E.coli,S.aureus,Salmonella enteritidis,and B.subtilis were determined by the Oxford Cup method.The trypsin hydrolysate showed significant antimicrobial activity compared to protein and pepsin hydrolysates(P<0.05).Three antimicrobial peptides were isolated and identified from Laba garlic trypsin hydrolysates,and their antimicrobial mechanisms were studied.Ultrafiltration,Sephadex G25 gel chromatography,and pre-HPLC technology were used to isolate and purify three antimicrobial peptides F3-3-a,F3-3-b,and F3-3-c from Laba garlic.The amino acid sequences of the three peptides were Tyr-Asn-His-Asn-Phe(YNHNF),Trp-Pro-Thr-Ser-Phe-Thr(WPTSFT)and Ala-Val-Asp-Arg-Ala-Val(AVDRAV),respectively.Among them,F3-3-c(AVDRAV)could significantly inhibit the growth of E.coli and S.aureus in vitro(P<0.05),the minimum inhibitory concentration(MIC)was 100?M and exhibited low hemolytic activity.The conformation of the three peptides in PBS,trifluoroethanol and methanol solution was analyzed by circular dichroism.The results showed that the three peptides mainly existed in random coli in PBS.After adding 50%trifluoroethanol,it could induce a random coil to become an?-helix structure,and after adding methanol,it would transform into the?-sheet structure,indicating that the three peptides had amphiphilic properties.Based on the phenomenon of leakage of cell contents,the mechanism of antimicrobial peptides was then investigated by scanning electron microscopy(SEM)and transmission electron microscopy(TEM).The results showed that the antimicrobial peptide of Laba garlic could induce morphological changes of bacteria and destroy the integrity of bacterial cell membrane,leading to extravasation of cell contents and causing bacterial death.In summary,this study clarified the active constituents and structures of the main flavonoids and blue pigments of Laba garlic and elucidated the related mechanism of promoting apoptosis of cancer cells.In addition,three natural antimicrobial peptides were obtained and their amino acid sequence composition and conformational changes were identified.The antimicrobial mechanism was preliminarily studied.The results of this study provided a theoretical reference and experimental basis for the research and development of the health products and medicines of Laba garlic and the comprehensive utilization of garlic resources.