Preparation Of Ursolic Acid And Its Derivatives And Reaserch On Anti-cancer Efficacy

Cancer is a horrible threat to human health. Currently, chemotherapy remains theprimary means to cure cancer.Recently, research on natural anti-cancer drugs withspecial effects and low toxicity develops as a hotspot. Prominent anti-tumor activityof ursolic acid was reported widely. The chemical modification to ursolic acid in orderto screen better anti-cancer drugs has been develped as the frontier of research onursolic acid.Presently, effective derivatives and its mechanism on anti-tumor are both notreported. Therefore, this article aimed to synthesize and screen derivatives with highactivity, low toxicity,in addition, study on the mechanisms, in order to providevaluable experimental evidence for research on ursolic acid and its derivatives.Separated by column and purified by crystallization, ursolic acid was prepared fromthe loquat leaf extract and finally analyzed with HPLC.After different chemicalstructure modification including acylation and esterification on the hydroxyl andcarboxyl, a series of ursolic acid derivatives were synthesized and identified byinfrared and NMR. Anti-proliferation effects of ursolic acid and its derivatives onhuman hepatoma cells HepG2, human prostate cancer cells PC-3, human colon cancercells Caco-2, human gastric cancer cells AGS were assessed by MTT assay. Growthinhibition effects of ursolic acid and its potential derivatives on human gastric cancercells MKN45, BGC-823and human gastric mucosa epithelial cells GES-1wereassessed by further MTT test.Effects on morphology of different cells caused byursolic acid and its derivatives were observed by ordinary optical microscope.Fluorescence microscopy morphology of cells binded by AO/EB was observed byfluorescence microscope. DNA Ladder was detected by agarose gel electrophoresis.Stained with both Annexin V and PI, Apoptotic cells were examined by flowcytometry. Flow cytometry also was used to detect cell cycle distribution.Mitochondrial membrane potential was tested by staining with rhodamine123. Theexpression of relative apoptotic protein was determined by Western Blot Detection.HPLC analysis showed that the purity of the prepared ursolic acid was more than95%. IR and NMR characterization indicated that derivatives of UA-1~UA-6with different chemical modification were synthesized ultimately. MTT assay showed thatUA-1displayed stronger anti-cancer activity on different human cancer cells. FurtherMTT tests showed that UA-1also exhibited stronger anti-tumor activity on differentgastric cancer cells. The apoptotic bodies and the nucleosome fragmentation wereobserved by Inverted microscope and fluorescence microscope respectively inMKN45cells treated with UA-1of30~50μmol/L for24h. Under the same conditions,typical DNA Ladder was observed by agarose gel electrophoresis.AnnexinV/PIstaining results showed that the UA-1could obviously induce apoptosis in MKN45cells. Flow cytometry results showed that UA-1mainly blocked MKN45cells inG0/G1phase and prominently induced apoptosis. Western Blot detection showed thatthe expression of bax was upregulated obviously,on the contrary, the expression ofsurvivin was downregulated gradually.Consequently, UA-1has been demonstrated of strong anti-proliferation activityagainst several malignant tumors. UA-1mainly blocked MKN45cells in G0/G1phaseand prominently induced apoptosis, which may be associated with upregulation ofexpression of Bax and downregulation of expression of Survivin. UA-1has apromising prospect to become a new effective and low-toxic anti-cancer drug forclinical cancer therapy.