| Objective To optimize the extraction process of dihydroquercetin, study on the extraction yieled of the effect on crushing particle size, and improve the bi oactivity of Dihydroquercetin through the structure modification of amino acid.Methods(1) Hot refluxing, ethanol refluxing, microwave, ethanol ultrasonic ation, and flash extraction were used to extracted dihydroquercetion, which usin g larch as the raw material. The Box-Behnken response surface method was used to optimizate extraction method of dihydroquercetion. Dihydroquercetion w as extracted by dihydroquercetion, dihydroquercetion was purificated by organic reagent and hot water recrystallization method.(2) Dihydroquercetion and amin oacid(glutamic acid cysteine alanine phenylalanine glycine lysine) were used a s raw materials, the dihydro quercetin-amino acid schiff base compounds were synthsized by conventional method, the structure of the product were determina ted through carbon nuclear magnetic resonance spectrum mass spectrum and inf rared spectrum.(3) The biological activity of dihydro quercetin-amino acid schi ff base were studied by antitumor experiment and antioxidant experiment in vit ro.Results(1) Five kinds of extraction methods of extraction rate were 0.17%, 0.20%, 0.21%, 0.22%, 0.21%, the extraction yield of hydrogen quercetin by flash type extraction method has the remarkable difference compared with other fo ur kinds of methods.Response surface optimization of flash type extraction method t o determinet-heoptimal conditions for: xtraction time 79 s, the ratio of 81% concentrati on of ethanol with solid-liquid as 1:10. On this condition dihydroquercetin extraction rate can reach 0.47%. After the raw material crushed and sieved(1-9 pharmacopoei a sieve), dihydro-quercetin extraction rate was 0.20%, 0.21%, 0.20%, 0.43%, 0.53%, 0.36%, 0.35%, 0.33%, 0.32 %. One of the 5 sieve dihydrogen extraction yield o f quercetin was obviously higher than that of eight other screen size.The purity of dih ydro quercetin crude product was 98% by organic reagent extraction and recrystallizat ion by hot water.(2) Synthesized and characterized six dihydro quercetin-amino aci d Schiff base compounds were dihydro quercetin-cysteine(compound I), dihydro-que rcetin-alanine(Compound II), dihydro quercetin-phenylalanine(compound III),dihydr o-quercetin-glutamic acid(compound IV), dihydro-quercetin-glycine(compoun d V), dihydro-quercetin-lysine(compound VI).The water-soluble of parent compou nd was improved, The compounds were found that the thermal stability of co mpounds II and III were better than two hydrogen quercetin, thermal stability of compound I, V and VI had barely changed, thermal stability of compound I V had reduced by melting point determination. The compounds I~VI had good stability within 3~8 by acid and alkali stability test.(3) Oxidation experiment s show that the dihydroquercetin and target compounds I~VI on the free radical scave nging effect increased with the concentration increased, were positively correlated.Co mpound I~VI of DPPH free radical scavenging effect results is 79.54%, 64.63%, 64.68%, 64.63%, 76.73% and 65.15%, the dihydroquercetin clearance rate of 62.83%.Compound I~VI the result of the ultra oxygen anion scavenging effe ct was 59.54%, 48.72%, 50.37%, 48.72%, 50.53% and 54.87%.Dihydroquercetin clearance rate of 60.72%.Compound I~VI with the result of clearing hydroxyl free radicals was 48.25%, 49.42%, 46.65%, 49.42%, 47.53% and 45.46%, resp ectively. Dihydroquercetin clearance rate of 53.77%. In vitro experiments showe d anti-tumor target compounds I~VI and dihydro quercetin on human breast cancer ce lls MCF-7 and human cervical cancer cells Hela, has improved to some extent. Dihy droquercetin-amino acid schiff base compounds I~VI to act on human breast ca ncer cells MFC-7, 24 h after the results of the inhibition rate was 71.2%, 46.9%, 48.7%, 59.4%, 68.3%, 54.6%;Dihydro quercetin in human breast cancer ce lls MFC-7 inhibition rate of 38.8%.Compound I~VI role in cervical cancer Hel a 24 h after the results of the inhibition rate was 66.9%, 41.4%, 45.1%, 58.2%, 61.7%, 51.9%; Dihydroquercetin act on human cervical cancer Hela inhibiti on rate of 39.6%.Conclusions The optimal extraction process of dihydroquercetin was deter mined by five extraced methods, and the effect of grinding particle size on the extraction yield was investigated to maximize the extraction of dihydroquerceti n. six dihydroquercetin-amino acid schiff base compounds were synthesized and characterized, the water-soluble of parent compound was improved.The compo unds were found that the thermal stability of compounds II and III were better than two hydrogen quercetin, thermal stability of compound I, V and VI had barely changed, thermal stability of compound IV had reduced by melting poin t determination. The compounds I~VI had good stability within 3~8 by acid an d alkali stability test. The new synthesis of flavonoids-amino acid schiff base c ompounds, and the corresponding matrix of flavonoids were studied by antitum or activity in vitro. The inhibition rate of target compounds I~VI on MCF-7 a nd Hela had a certain degree of improvement compared with dihydroquercetin. |
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