The Extraction And Derivatization Of Esculetin In Cortex Fraxini And Anti-tumor Activity Of Esculetin Derivatives

Cortex Fraxini, dried barks of Fraxinus chinensis Roxb of Oleaceae, is a commonly used Chinese medicine, mainly distributed in Northeast China, North China, West China and other domestic regions. Esculetin is the main active ingredient of Cortex Fraxini, endowed with chemical name 6,7-dihydroxycoumarin. Esculetin has many pharmacological activities such as anti-tumor, anti-inflammatory, antioxidant, protecting the central nervous system and other bio-activities. According to these pharmacological activities, esculetin has broad prospects in the development of medicinal utilization. In medicinal chemistry, the biological activities of natural products and their derivatives are very important research topics as they often have specific target function. Besides, natural products derivatives can obtain higher pharmacological activity and enrich the structural diversity of the drug molecule library by molecular designing and drug synthesis.6,7-dihydroxy in esculetin is the significant reaction site, which can interact with the substituent group or the functional group to modify its chemical structure to gain higher activity esculetin derivatives.Based on the established researches and drug development theories, esculetin was selected as the research object in the present research. The extraction and separation, derivatization of esculetin and preliminary anti-tumor activities of esculetin derivatives were studied. Raw esculetin was extracted from Cortex Fraxin, additionally, high purity esculetin was obtained by liquid-liquid extraction separation followed by recrystallization. Based on medicinal hybrid design theory, esculetin was hybridized with cinnamic acid, salicylic acid and other organic acid compounds to afford new esculetin derivatives, which were purified by silica gel column chromatography. Last but not least, the anti-tumor activities of new synthesized esculetin derivatives were tested by HepG2 and HCT-116 cell-lines based on MTT methods. All the experiment results can be concluded as follows:1.The extraction and separation processes of esculetin were studied. Ethanol refluxing extract method was used to extract the esculetin from Cortex Fraxini. The esculetin content was taken as a key extract experimental index, the extraction method of esculetin was optimized by the single factor and orthogonal experiments. The optimum conditions were confirmed as follows: ethanol concentration 75%, reagent ratio 1:7, extracted 3 times,2 h. Under this optimum conditions, the esculetin crude content could achieve 1.83 mg/g finally. After extraction under the optimum conditions, esculetin was isolated and purified by liquid-liquid extraction and recrystallization. Esculetin crude was obtained by liquid-liquid extraction and recrystallization procedures. Esculetin purity and yield were taken as comprehensive efficiency index, the recrystallization of esculetin was optimized by single factor experiment. The optimum recrystallization condition was confirmed as follows:the ethanol-water ratio at 1:3. Under this condition, the esculetin purity was 98.38%, the yield was 39.7%.2. In terms of esterification modification of 7-hydroxy of esculetin scaffold,10 kinds of esculetin derivatives were synthesized by esculetin and organic acids including cinnamate, salicylate etc. All the esculetin derivatives were purified by silica gel column chromatography as well as the structures of esculetin derivatives were identified by 1H NMR and 13C NMR.3. The inhibition of 10 different kinds of esculetin derivatives on tumor cell proliferation were investigated with esculetin as control. The results showed that 6 kinds esculetin derivatives had better effects than esculetin on inhibiting the proliferation of human hepatoma cells HepG2,5 kinds esculetin derivatives had better effects than esculetin on inhibiting the proliferation of human colon cancer cells HCT-116. Among which, esculetin derivatives hybridized with cinnamic acids had better anti-tumor activities than the others, and the anti-tumor activities showed good dose dependence at the range of 1?100 ?mol/L. (45) (E)-6-hydroxy-2-oxo-7-chromen-3-(3,4-dimethoxyphenyl) acrylate had the most significantly inhibition on the proliferation of human hepatoma HepG2 and human colon cancer cells HCT-116 with IC50 28.624 ?mol/L and 45.908?mol/L.