Regulation Of Feeding Medium During Adventitious Root Culture Of Oplopanax Elatus And Anticancer Properties Of Extract

Oplopanax elatus Nakai is a kind of deciduous perennial shrub of the Araliaceae,which is a specific medicinal plant in changbai mountain area of China.However,the development and production of O.elatus are restricted because of the shortage of the plant material,and this problem can be alleviated by the application of the plant cell and organ culture technology.The previous studies indicated that O.elatus adventitious roots can be mass produced in bioreactors,and compared with batch culture,the fed-batch culture was more beneficial for biomass and bioactive compound accumulation of adventitious roots.Nevetheless,the fed-batch culture system is not accomplished,in particular,the medium conditions for feeding is not clear yet.Therefore,to select a suitable feeding medium,the present study investigated the effects of sucrose concentration,medium strength,phosphate concentration,and nitrate concentration in feeding medium on biomass and polysaccharide and flavonoid accumulation,which the finding will be used in futher studies for improving the fed-batch culture sytem.At present,the information regarding pharmacological properties of O.elatus adventitious roots is lacking,thereby restricting the commercial utilization.Thus,depending on the O.eluatus adventitious roots containing rich anti-cancer substences（such as polysaccharides and flavonoids）,the present study investigated the effects of extracts from adventitious roots on the cancer cell viability,apoptosis,and expression of apoptosis-related proteins for providing a theoretical basis for the product production in the future.In the experiment of feeding medium selection,the present study found that adventitious root dry weight and total polysaccharide production were favorable in 60 g L^-1 sucrose.However,total flavonoid production did not differ among the sucrose concentrations from 50 g L^-1 to 70 g L^-1,in those concentrations the flavonoid productivities were significantly higher than that in the 40 g L^-1 sucrose treatment;the both flavonoid monomers,the maximum quercetin and kaempferide were produced in 60 g L^-1 sucrose treatment.Among the four feeding medium strengths（0.5×MS,1×MS,1.5×MS,and 2×MS）,adventitious root dry weight was optimal in the 2×MS group,but total polysaccharide production in the 1×MS and 1.5×MS groups,and total flavonoid production in the 1×MS group;quercetin and kaempferide contents were not affected by the feeding medium strength,their productivities reached the maximum in the 2×MS group for quercetin and 1.5×MS and 2×MS for kaempferide.The phosphate concentrations from 0.94 mM to 1.56 mM in the feeding medium（1×MS）did not affected dry weight,but total polysaccharide and flavonoid productivities reached the highest in both concentrations of 0.94 mM and 1.25 mM;the quercetin productivities in 0.63-1.25 mM phosphate did not significantly different,they were all higher than that in 1.56 mM phosphate;the highest kaempferide productivity was determined in 1.25 mM phosphate.Similar to the phosphate effect,nitrate concentrations（30-60 mM）in feeding medium（1×MS）were also not affect adventitious biomass,but total polysaccharide productivities in 30,40,and 50 mM nitrate concentrations were higher than that in 60 mM;total flavonoid and quercetin productivities in 40 mM and 50 mM nitrate were more favorable that those in 30 mM and 60 mM;the highest kaempferide productivity was found in 40 mM nitrate.Therefore,to produce O.elatus polysaccharides and flavonoids via adventitious root culture,the MS medium should be modified for using as the feeding medium,namely,the phosphate and nitrate in basic MS medium should be modified to 1.25 mM and 40 mM,respectively,and 60 g L^-1 sucrose should be added,in which medium 3.6 g L^-1 total polysaccharides,6.9 g L^-1 total flavonoids,4.4 mg L^-1 quercetion,and 1.9 mg L^-1 kaempferide can be produced.In the anti-cancer experiment,the present study determined that the methanol and ethanol extracts from O.elatus adventitious roots possessed anti-cancer activity.Both extracts were found more effective against AGS human gastric cancer and HCT116 human colonic cancer cell lines compared with HepG2 human hepatoma cell line;the anti-cancer activity of the ethanol extract was higher than that of the methanol extract,and the half inhibitory concentration of ethanol extract was 0.32 mg mL^-1 on AGS and 0.1 mg mL^-1 on HCT116.In addition,the ethanol extract can significantly reduce the expression of apoptosis protein inhibitor-Bcl-2,and up-regulate the expression of pro-apoptotic protein-Bax,and subsequently activated the caspase-9 and caspase-3 expression,speculating the adventitious extract-induced apoptosis of cancer cells through mitochondria-dependent pathway...