Composition Changes Of Fermented Herb Codonopsis Pilosula Using Fungus Ganoderma Sessile And Its Effect On Proliferation And Apoptosis Of SH-SY5Y Neuroblastoma

Objective:To screen a novel antitumor compound via liquid fermentation technology for the water extract of traditional Chinese medicine Codonopsis pilosula by Ganoderma sessile.Furtherly,the crude ectoenzyme and endoenzyme of G.sessile were extracted to verify the biocatalysis.Subsequently,the proliferation inhibition activity for tumor cell lines was assessed and its possible mechanism was explored.Finally,the acute toxicity of the compound was evaluated.This study provided a certain experimental basis for the research&development of anti-tumor lead compound.Methods:The water extract of C.pilosula was fermented with fungus G.sessile for 7days and the composition changes in the water extract of C.pilosula were detected by ultra performance liquid chromatography?UPLC?.The changed compositions were isolated through preparative ultra performance liquid chromatography,and then the purified compounds were detected and characterized by UPLC,MS,¹H-NMR,and¹³C-NMR,respectively.The supernatant obtained from the fermentation broth as crude ectoenzyme solution and the endoenzyme of G.sessile were extracted by ultrasound combined with homogenate at low-temperature.The compound before fermentation was biocatalyzed by crude ectoenzyme and the endoenzyme,respectively.The optimal catalytic conditions were screened through optimizing the catalytic time,the amount of substrate,and the amount of enzyme.The inhibitory effect of the two compounds on the proliferation of SH-SY5Y,HCT-8,A549 and SGC-7901 cells was detected by cell counting kit-8?CCK-8?or3-?4,5-dimethyl-2-thiazolyl?-2,5-diphenyl-2-H-tetrazolium bromide?MTT?methods.Flow cytometry was applied to analyse changes of cell cycle,apoptosis rate,reactive oxygen species?ROS?level,mitochondrial membrane potential?MMP?and Ca²⁺concentration of sensitive cell.The content of lactate dehydrogenase?LDH?in cell supernatant was measured by a colorimetric kit,the mRNA and protein expressions of CyclinB1,cyclin dependent kinase 1?CDK1?,p21,b-cell lymphoma 2?Bcl-2?and bcl-2 associated X protein?Bax?were determined by Real-time qPCR and Western blot.Finally,the acute toxicity of the two compounds was investigated via SD rats according to the OECD-423 guideline.Results:After fermentation of G.sessile,an original component,5-?hydroxymethyl?furfural?5-HMF?in the water extract of C.pilosula disappeared,meanwhile a new component identified as 2,5-bis?hydroxymethyl?furan?BHMF?was produced.Both endoenzyme and ectoenzyme could convert 5-HMF to BHMF,but the biocatalysis efficiency of ectoenzyme was better than endoenzyme.The conversion efficiency reached 99%when the biocatalysis system consisted of 1%substrate amount,10%enzyme amount and biocatalysis for 72 h.5-HMF exerted a weak inhibitory effect on the proliferation of SH-SY5Y,HCT-8,A549 and SGC-7901 cells with IC₅₀ between6.752⁹.944 mM,while BHMF presented a better inhibitory effect on the proliferation of SH-SY5Y cells with an IC₅₀ of 83?M.Beside,BHMF also showed a certain proliferation inhibitory effect on A549 and SGC-7901 cells with IC₅₀ of 0.697and 0.33 mM,respectively,but it was insensitive to HCT-8 cells.BHMF was able to arrest the cell cycle of SH-SY5Y,and the proportion in the G₂/M phase increased in a concentration-dependent manner?P<0.01?.Low concentration of BHMF could induce early apoptosis of SH-SY5Y cells,which were significantly different from the control?P<0.01?.BHMF contributed to the accumulation of ROS in SH-SY5Y cells,and the level of ROS increased with the increasing of compound concentration,which was significantly different from the control?P<0.01?.BHMF promoted the increase of Ca²⁺concentration in SH-SY5Y cells with the increasing of compound concentration,and the difference was significant compared with the control?P<0.01?.BHMF caused depolarization of MMP in SH-SY5Y cells,MMP decreased with increasing compound concentration,and the difference was significant compared with the control?P<0.01?.BHMF was helpful to the release of LDH in SH-SY5Y cells,the content of LDH in the supernatant increased with the increasing of BHMF concentration?P<0.01?.BHMF could inhibit the CyclinB1,CDK1 and Bcl-2 of SH-SY5Y cells in a concentration-dependent manner,and up-regulated the transcription and translation levels of Bax and p21 at a BHMF concentration of 120?M,the difference reached a significant level compared with the control?P<0.05?.The LD_{50 cut-off} of 5-HMF and BHMF were more than 2000 mg/kg body weight,the body weight and organ index of the rats in the treatment group were no significantly difference?P>0.05?compared with the control,moreover,the pathological section of liver,kidney and spleen showed no pathological changes.Conclusion:The compound 5-HMF in traditional Chinese medicine C.pilosula was biotransformed to BHMF by medicinal fungus G.sessile with high transformation efficiency.Moreover,BHMF exerted less acute toxicity,which could inhibit SH-SY5Y cells proliferation via arrest cell cycle at the G₂/M phase,at the same time,it could induce apoptosis of SH-SY5Y cells.The possible mechanism is that BHMF could inhibit cell proliferation and induces apoptosis by up regulating the transcription and translation levels of Bax and p21,down regulating the transcription and translation levels of cyclinB1,CDK1 and Bcl-2,to induce the accumulation of ROS in SH-SY5Y cells,reduce MMP and increase intracytoplasmic Ca²⁺concentration.Therefore,the possible mechanism of BHMF induced apoptosis in SH-SY5Y neuroblastoma cells lies in the mitochondrial pathway.