Research On The Key Anti-tumor Ingredients Of Oleophenols Based On The Spectrum-effect Relationship

As an endemic Chinese species,the Chinese dwarf cherry（Cerasus humilis（Bge.）Sok.）belongs to the Rosaceae family of Cerasus.It is a perennial deciduous shrub that is particularly valued for its possession of both edible and medicinal characteristics.As one of the third-generation fruits,Chinese dwarf cherry has a unique and rich aroma with abundant nutrition,thereby indicating promise as novel nutraceuticals and dietary supplements.In this experiment,phenolic compounds were extracted and purified from the fruits of 16 different Chinese dwarf cherry germplasms.Then,the proper concentrations of purified extracts were selected to act on three different cancer cells（HepG2,HCT116 and BGC823）.The inhibition rate of cancer cells was observed 24,48 and 72 hours after administration.The chemical profiles of different germplasms were analyzed by UPLC and UPLC-MS technology.According to the spectral relationship between the chemical profiles and the in vitro anti-cancer activity,the key components affecting the anti-tumor activity were elucidated.The main results are as follows:（1）The crude extracts in the fruits of 16 Chinese dwarf cherry germplasms were obtained by organic solvent extraction with 40%ethanol as solvent.Concentrated by rotary evaporation,it was purified by AB-8 macroporous resin with 80%ethanol as eluent,and freeze-dried.Fruit phenolic compounds contents were determined before and after purification by UPLC.Cluster analysis was carried out on the contents of purified products of different germplasms.The results showed that the content of phenolic compounds in different germplasms varied greatly,in addition,the content of phenolic compounds in light-colored fruits was distributed in both high and low content categories.The average recovery rate of purified phenolic compounds was 84.24%.It was found that the purified phenolic compounds extracts have a stronger response value and their components are more diversity than the unpurified samples,indicating AB-8 macroporous resin could effectively gather and purify the crude fruit extract.The purified phenolic compounds also have better dissolution in cell culture medium.（2）Using Waters Acquity ultra-high performance liquid chromatography（UPLC）system.UPLC-MS/MS characteristic maps of 16 germplasms of Chinese dwarf cherry phenolic compounds were obtained.Thirty-one phenolic compounds were identified,they are procyanidin trimer T2,vanillic acid,sesamolin,cinnamtannin A2,procyanidin dimer B7,p-coumarin-dihexyl glycoside,pelargonidin-3-glucoside,4-p-coumaroylquinic acid,（-）-catechin,cyanidin-3-glucoside,（-）-L-epicatechin,eriodictyol-7-O-glucoside,verbascoside,demethyloleuropein,apigenin-7-O-glucoside,dihydromyricetin-3-O-rhamnoside,kaempferol,（-）-epicatechin-3-（3-O-methyl）-gallate,delphinidin-3-O-glucoside,（-）-epicatechin gallate,peonidin-3-O-rutinoside,（-）-catechin gallate,peonidin-3-O-galactoside chloride,kaempferol-3-O-xylosyl-glucoside,isorhoifolin,pelargonodin-3-glucoside,chrysin-6-O-β-D-glucoside,pelargonodin-3-glucoside,luteolin-7-O-glucoside,quercetin,isorhamnetin-3-glucoside,matairesinol.（3）Phenolic compounds extracts from 16 different germplasms were purified by macroporous resin,and to test on three different cancer cells（HepG2,HCT116 and BGC823）.MTT（tetrazolium salt）colorimetry was used to evaluate their inhibitory effects on the proliferation of three cancer cells after 24,48 and 72 hours of administration,and cluster analysis was applied.The results showed that Chinese dwarf cherry phenolic compounds had inhibitory effect on the three cancer cells.After 72 hours of action,their inhibitory rate almost reached 70%.According to cluster analysis,yellow and orange germplasms had better anti-cancer effects.Among them,the germplasms of No.6,No.8 and No.16 all had significant proliferation inhibition effect on three cancer cells.（4）Based on the purified phenolic compounds UPLC peak area of 16 different germplasms and their inhibition effects on human hepatocellular carcinoma cell HepG2,human colon cancer cell HCT116 and human gastric cancer cell BGC823,the"effective components"of the extracts were determined by the Spectrum-Effect analysis,which based on partial least squares regression method.Above all,it is inferred that peaks X₃,X₁₆,X₁₇,X₁₈,X₂₀,X₂₂,X₂₇,X₃₀,X₃₁,X₃₂ and X₃₃ are the"effective components"of inhibiting human hepatocellular carcinoma cell line HepG2,peaks X₁,X₂,X₃,X₁₁,X₁₇,X₂₆,X₂₇ and X₃₁ are the"effective component group"of inhibiting human colon cancer cell line HCT116,and peaks X₁,X₂,X₄,X₅,X₆,X₁₆,X₁₇,X₂₁,X₂₂ and X₂₉ are the"effective component"group of inhibiting human gastric cancer cell line BGC823.（1）According to the identification results of UPLC-MS/MS,the peaks X₃,X₁₆,X₁₇,X₁₈,X₂₀,X₂₂,X₂₇,X₃₀,X₃₁,X₃₂ and X₃₃ which inhibit the growth of HepG2 were sesamolin,apigenin-7-O-glucoside,dihydromyricetin-3-O-rhamnoside,kaempferol,delphinidin-3-O-glucoside,peonidin-3-O-rutinoside,pelargonodin-3-glucoside,luteolin-7-O-glucoside,quercetin,isorhamnetin-3-glucoside,matairesinol.（2）According to the identification results of UPLC-MS/MS,the peaks X₁,X₂,X₃,X₁₁,X₁₇,X₂₆,X₂₇ and X₃₁ which inhibit the growth of HCT116 were procyanidin trimer T2,vanillic acid,sesamin,（-）-L-epicatechin,dihydromyricetin-3-O-rhamnoside,isorhoifolin,pelargonodin-3-glucoside and quercitrin.（3）According to the identification results of UPLC-MS/MS,the peaks X₁,X₂,X₄,X₅,X₆,X₁₆,X₁₇,X₂₁,X₂₂ and X₂₉ which inhibited the growth of BGC823 were procyanidin trimer T2,vanillic acid,cinnamtannin A2,procyanidin dimer B7,p-coumarin-dihexyl glycoside,apigenin-7-O-glucoside,dihydromyricetin-3-O-rhamnoside,（-）-epicatechin gallate,peonidin-3-O-rutinoside,pelargonodin-3-glucoside.