Gingerols From White Ginger:Purification,Anti-Cancerous Potential And The Molecular Mechanism

Ginger is an ancient herb used widely for its many natural medicinal properties and health benefits.Extensively consumed as a spice throughout the world,ginger serves as an excellent source of several bioactive phenolics,including non-volatile pungent compounds such as gingerols,paradols,shogaols and zingerones.Among them,gingerols are the main functional component in fresh ginger and they are a series of homologues collectively with the same phenol acetone functional groups and alkyl side chain with different number of C atoms.They have been extensively studies in the past years for their antioxidant,anti-inflammatory and anti-cancerous attributes.In particular,Tongling White Ginger with unique flavor and composition is a local variety of Tongling city in Anhui province,China.This paper carried out the extraction of gingerols from Tongling White Ginger followed by further purification and evaluating the antioxidants and antitumor activities of 6-gingerol and 10-gingerol.The apoptosis mechanism in cervical cancer Hela cells was studied through supplementation of 6-gingerol and 10-gingerol.The anti-cancerous effects of synergy among 6-gingerol,8-gingerol and 10-gingerol and also with chemotherapeutic drugs?paclitaxel and 5-FU?were also studied.The present research work has the vital significance as it provides a theoretical basis for Tongling White Ginger which can be utilized in special medical purposes.Extraction and separation of Tongling White Ginger and ginger phenol was optmized through ultrasonication with following experimental conditions: extraction time of 53 min,extraction temperature at 47?,liquid-solid ratio of 14:1 and 65% ethanol.The gingerols yield obtained under these conditions was 3.72 mg/g.By using silica gel column chromatography and high speed reflux chromatography,the purification ranges were 90.91%,97.72% and 90.06%.The three components 6-gingerol,8-gingerol and 10-gingerol were identified by the UPLC-TOF-MS/MS and NMR technology.The antioxidant activity of three types of gingerol were compared under in vitro.The six indexes of the antioxidant activity about the three gingerols were tested.With the increasing concentration,higher antioxidant capacity was observed.With the extension of side chain,the reducing power,DPPH radical removal ability,ABTS⁺ ability to remove free radical,hydroxyl free radical removal ability and super oxygen anion removal ability of gingerol were reduced and followed the trend such as: 6-gingerol > 8-gingerol > 10-gingerol.Whereas,the lipid antioxidant ability increases with the extension of the side chain and it was observed as 10-gingerol >8-gingerol > 6-gingerol.Particularly,6-gingerol induced the death of Hela cells in cervical cancer and its mechanism.6-gingerol inhibited the proliferation of Hela cells effectively at the IC₅₀?96.00 ?M?and IC80?133.56 ?M?.The damage to the cells by 6-gingerol is exacerbated by the increase in processing concentration.After the treatment of the 6-gingerol,the morphology of Hela cells was changed with the condensation of the cells on the edges and the presence of certain transparent bubbles in the cells.6-gingerol induced the Hela cell cycle to stagnate at G0/G1,and it was increased with the duration time of the 6-gingerol and processing concentration.The 6-gingerol treatment reduced the levels of mRNA and protein expression in Cyclin A and Cyclin D1 in the Hela cells,and the mRNA and protein expression levels of Cyclin B1 were not significantly reduced.The mRNA of Cyclin E1 was significantly reduced after the 6-gingerol treatment,but the reduction in protein was not significant.6-gingerol is mainly responsible for inducing Hela cell cycle retardation by regulating Cyclin D1 gene.The apoptosis rate of Hela cells treated with the 6-gingerol?100 ?M?for 24 h,48 h and 60 h were 16.71%,33.00% and 38.40%,respectively.After the treatment of Hela cells,the expression of the caspase-3,caspase-8 and caspase-9 protein of the Death pathway were significantly reduced,and the corresponding protein phosphorylation increased significantly.The level of bcl-2 proteins in the mitochondrial pathway was significantly reduced,and the expression of Bax and cytochrome C increased.The p-PI3 K,p-AKT,p-p70S6 K,and p-mTOR protein in PI3K/AKT/AMPK/mTOR pathway were significantly reduced,and the p-AMPK expression increased significantly.Thus,we could summarize that 6-gingerol promoted the apoptosis of Hela cells by regulating the expression of genes in the Death pathway,the mitochondrial signaling pathway,the PI3K/AKT/AMPK/AMPK/mTOR signaling pathway.Simultaneously,10-gingerol also caused cervical cancer Hela cell apoptosis and its mechanism.10-gingerol inhibited the proliferation of Hela cells more than 6-gingerol at IC₅₀?29.19 ?M?and IC80?50.87 ?M?.10-gingerol resulted into certain cytotoxicity to the Hela cells along with changes in cellular morphology.10-ginger phenols effectively induced the Hela cell cycle to stagnate at G0/G1.The G0/G1 phase of the G0/G1 period was increased over time 6 h,12 h and 18 h,respectively,at the ratio of 1.8%,9.8% and 67.48% respectively.10-gingerol treatment effectively reduced the levels of mRNA and protein expression in Cyclin A and Cyclin D1 of Hela cells,which is more than 6-gingerol processing.There was no significant reduction in the level of mRNA and protein expression in Cyclin B1.10-gingerol induced Hela cycle inhibition mainly through regulating the Cyclin D1 gene.As in the case of IC₅₀ concentration,10-gingerol inhibitory effect was stronger than the effect of 6-gingerol.After 10-gingerol processing of Hela,Death Receptor signaling pathways DR3 and DR5 protein expression were significantly increased,and further induced the Caspase 3,Caspase-8 and 9 protein phosphorylation and ultimately activated the Caspase-,p-Caspase-8.The expression volume of the mitochondrial signaling pathway,bcl-2 and the Bad protein were significantly decreased in the treatment of the mitochondrial signaling pathway,and the expression of Bax and cytochrome C was significantly increased.The expression of the PKC epsilon and NF-k? beta of the NF-k? pathway were significantly reduced with 10-gingerol,equivalent to 0.61 and 0.64 times respectively.10-gingerol processing inhibited the Hela PI3K/AKT/AMPK/mTOR pathway of PI3 K protein phosphorylation effectively,and triggered its downstream factor of AKT and P70S6 K protein phosphorylation.10-G induced the phosphorylation of mTOR to reduce the amount of protein expression and pAMPK protein expression eventually.Therefore,10-gingerol could promote the apoptosis of Hela cells by regulating the expression of related genes for Death Receptor signaling pathways,mitochondrial signaling pathways,NF-k? pathways and PI3K/AKT/AMPK/mTOR signaling pathways.The IC₅₀ of the Hela cells inhibition was 45.04 ?M,and the three gingerols inhibited the proliferation of cervical cancer Hela cells.The two combinations among 6-gingerol,8-gingerol and 10-gingerol did not match the suppression of Hela cells.6-phenol combination with 8-gingerol used in low concentration condition showed obvious synergic effect.But under the condition of high concentration,synergic effect was not apparent.The 6-gingerol combination with 10-gingerol acted as an antagonist at lower concentrations,and displayed a stable synergy at high concentrations.Hela cells treated using 8-gingerol combination with 10-gingerol presented an extremely significant synergistic effect.The 6-gingerol combination with 5-FU showed a good synergic effect.6-gingerol combination with paclitaxel,co-processes Hela cells,presenting an extremely significant synergistic effect.The synergistic effect was evident combined use of 10-gingerol and 5-FU and paclitaxel.The combined use of 10-gingerol and 5-FU was observed to be better than that of 6-gingerol and 5-FU under high concentration conditions.The combined effects of 10-gingerol and paclitaxel were similar to the synergistic effects of 6-gingerol and paclitaxel,and showed no significant difference.