The Active Component Identification Of Polyphenols From Ziziphi Spinosae Semen Against Colon Cancer And Its Molecular Mechanism

Colorectal cancer(CRC)is a common malignant disease of digestive tract.Incidence rate and mortality rate of CRC are increasing year by year,which seriously threatens human health.Therefore,it has been a research hotspot for scientists to screen functional molecules and effective drugs for the prevention and theatment of CRC.Ziziphi Spinosae Semen(ZSS)is dried and mature seed of Ziziphi Spinosae,which is a traditional Chinese medicine with a long history of medicine and food.ZSS has significant sedative,hypnotic and antidepressant pharmacological activities,such as saponins,flavonoids and triterpenoids.In present,the research on polyphenols from ZSS is mainly focused on sedation and hypnosis,but there are few reports about its antitumor activity.Plant polyphenols,which are rich in active groups,have been widely reported for their antitumor activities,and have the potential to be developed into natural antitumor drugs.The water-soluble polyphenols were extracted from ZSS by acid digestion,named ZSSP.The anti-CRC effects of ZSSP were evaluated in vitro and in vivo.Further,the main component of ZSSP with anti-CRC activity was isolated and identified by various chromatographic techniques,and its molecular mechanism of anti-CRC was preliminarily discussed.The results were as follows:ZSSP displays significantly anti-CRC activity in vitro.MTT assay showed that the IC50 values of ZSSP to CRC cell lines HCT-116,HCT-8 and HCT-8FU were128.911±14.88,208.509±23.84 and 266.931±31.89 ?g/m L,respectively.Flow cytometry showed that ZSSP treatment significantly induced the apoptosis of HCT-116 and HCT-8 cells and promoted cell cycle in S phase arrest.Endocytosis assay showed that ZSSP could reverse the resistance of HCT-8FU cell to 5-FU through increasing the accumulation of rhodamine-123 in cells.Inhibitory effect of ZSSP on CRC induced by AOM/DSS in mice.Mice CRC model was induced by AOM/DSS.The results showed that compared with CRC model group induced by AOM/DSS,the weight,intestinal structure and length of mice in ZSSP treatment groups were recovered,and Kaplan Meier survival curveshowed ZSSP intervention improved the survival rate of the CRC mice in a certain extent.ZSSP treatment significantly reduced the number of colonic polyps and tumor volume of mice induced by AOM/DSS.Immunohistochemistry results showed that the expressions of early marker of CRC,such as COX-II,EMR1 and Ki67,were effectively inhibited in ZSSP treatment groups;HE-staining showed that ZSSP had no significant effect on the growth and main organs of CRC mice.The active component of ZSSP against CRC,spinosin-Na,was isolated by various chromatography techniques.Firstly,according to the adsorption and resolution of different macroporous resins to ZSSP,we determined that SP207 was an ideal resin for the separation of ZSSP.Next,ZSSP adsorbed by SP207 were respectively eluted with acetone solution of increasing concentrations,and the anti-CRC activity of each elution component was detected by MTT assay.The results showed that the elution fraction of 50 % acetone exhibited significant anti-CRC activity.RP-HPLC was used for refined separation of the elution fraction of 50 %acetone,the fraction with peak time of 22.444 minutes(peak 5)was the main active component of ZSSP against CRC.Then,it was further identified that the peak 5 was spinosin-Na with through RP-HPLC-MS/MS and standard comparison,and its purity reached 98.25±8.23%.MTT results showed that spinosin-Na significantly inhibited the proliferation of CRC cells,but had no effect on normal cell FHC.Spinosin-Na suppresses the proliferation of CRC cells by remodeling serine biosynthesis pathway(SSP)and its the downstream of redox homeostasis and cell cycle.The abnormal activation of SSP plays an important role in the occurrence and development of malignant tumors.Transcriptome sequencing results showed that spinosin-Na intervention significantly inhibited the expression levels of SSP-related metabolic enzymes of CRC cells,such as PHGDH,PSAT1,PSPH,SDSL and SHMT2.Meanwhile,the enrichment of KEGG signaling pathway showed that serine and glycine metabolism pathways were significantly enriched by spinosin-Na treatment.Next,q PCR and western blot assays found that spinosin-Na intervention increased the level of intracellular ROS and decreased GSH content and GSH/GSSG ratio in HCT-116 cells,which further was verified in HCT-116 cells with knock-down PSPH.Meantime,cell cycle was arrested in S phase of HCT116 and HCT-8 cells.In conclusion,water-soluble polyphenol ZSSP was extracted from ZSS by acid digestion method.It was found that ZSSP had significant anti-CRC activity in vitro and in AOM/DSS induced CRC mice.Further,the results showed that spinosin-Na was the active component of ZSSP to exert anti-CRC effect,which mainly attributed to the blocken of SSP pathway in CRC cells.This study reveals the new pharmacological function of spinosin-Na in anti-CRC activity,and elucidates the molecular mechanism of its anti-CRC activity from the perspective of regulation of serine metabolism.These new findings will provide new ideas for the development of anti-CRC drug and diet intervention of ZSS polyphenol,as well as new utilization value.