Research On Antitumor Activity Of Suspension Cells And Endophytic Fungi From Bupleurum Scorzonerifouum Willd

Bupleurum scorzonerifolium Willd. is a kind of Bupleurum L. According to2010"Chinese Pharmacopoeia", its driedroots are regarded as original medicinal materials, and the main active ingredients are saikosaponins. Modern pharmacological studies have shown thatB. scorzonerifoliumhaverole of antipyretic, anti-pathogenic microbial, anti-inflammatory, anti-tumor and promoting immune function.According to LC-MS analysis for metabolites of endophytic fungi CHS2, CHS3, CHS4, CHS5, CHL2and CHR4, the results showed that these fungi could produce same compounds to host plant, CHS2and CHS3had the capability to synthesize saikosaponin d. The effects of thesestrains on anti-breast cancer MCF-7cells, anti-stomach cancer SGC-7901cells and anti-liver HepG2cells were tested in vitro. The results showed that the crude ethanol extracts of same strain metabolites had different antitumor effects on different tumor cells and the crude ethanol extracts of different strains metabolites had different antitumor effects on same tumor cells.Among them, the50%inhibitory concentration (IC50) of CHS4, CHR4and CHS5was75.11ug/mL,87.17ug/mL and88.38ug/mL to HepG2, MCF-7and SGC-7901respectively.Those endophytic fungi which could produce the same compounds as B.scorzonerifoliumwere identified by ITS molecular identification, and the results showed that these strains were classified as Fusarium oxysporum, Fusarium acuminatum, Alternaria alternate, Aspergillus versicolor,Penicillium sanguifluumandPenicillium cordubense respectively.Effective parts of CHS4and CHS5were screened for their strong inhibitory activity on HepG2cell in vitro. And the results showed that crude extracts of different effective parts of the same strain metabolites had different inhibitory activity on HepG2cells. Among them, the n-butanol crude extractsof CHS5metabolites showed high activity on HepG2cells with an IC50value of14.53ug/mL and the petroleum ether crude extractsof CHS4metabolites showed high activity on HepG2cells with an IC50value of20.06ug/mL.In this paper, fermentation conditions of B. scorzonerifoliumsuspension cells were optimized by observing effects about initial inoculation of callus, ratio of inoculation and fermentation volume and dose of exogenous substances (casein hydrolyzate, salicylic acid and methyl jasmonate).The optimum fermentation conditions ofB. scorzonerifolium suspension cells were determined as2.0g initial inoculation of callus,100mL culture volume (250mL Erlenmeyer flask) and MS liquid medium supplemented with6-BA2.5mg/L,2,4-D0.1mg/L and KT0.9mg/L and optimum additive concentration of hydrolyzed casein, salicylic acid and methyl jasmonate were250mg/L,16mg/L and22.5mg/L respectively. According to LC-MS analysis for metabolites of callus, suspension cells and suspension cell culture medium, the results showed that saikosaponin d could be found in callus, suspension cells and suspension cell culture medium.In this paper, the different effective parts of callus and suspension cells metabolites were screened for anti-breast cancer MCF-7cells, anti-liver cancer HepG2cells and anti-stomach cancer SGC-7901in vitro. The results showed that ethyl acetate effective part of callus and suspension cells metabolites had-the strongest inhibitory effect on MCF-7cells and SGC-7901cells. And the50%inhibitory concentration (IC50) of calluswas26.06ug/mLand31.45ug/mL toMCF-7and SGC-7901respectively; the50%inhibitory concentration (IC50) of suspension cells was18.21ug/mL and32.43ug/mL toMCF-7and SGC-7901respectively. N-butanol effective part of callus and suspension cells metabolites had the strongest inhibitory effect on HepG2cells with an IC50value of17.77ug/mL and30.06ug/mL.