| Polyhydric isoflavone compounds possess good biological properties,and most of them can be abstracted from leguminous plants with little production.6,7,4'-trihydroxyisoflavone(T2)belongs to polyhydric isoflavone compounds and could be brought in the market.However,most of the products cost a lot and lack of high quality of technology in chemical synthesis and purification.Given this,it's necessary to find an efficient way for T2 synthesis to improve the production and quality,and to provide further services for human health.By referring some chemical synthetic ways of T2 and considering the merit and demerit of which,we innovatively worked out a new way for T2 synthesis.This method can be concluded as follows:taking3,4-dihydric phenol and hydroxybenzyl cyanide as substrates,through Hoesch reaction,carbon atoms addition and circle closing,to consecutively achieve the intermediate product deoxybenzoin and T2 precursor.T2 was harvested after the demethylation reaction of T2 precursor.1H NMR,13C-NMR and mass spectrum analysis were utilized to confirm the chemical structure of products,thus to guarantee the accuracy and stability.Experiments of character analysis,residual solvent and content analysis were used to confirm the quality of T2 product.Usually,compounds with better water-solubility showed better biological properties.Therefore,it is a must to improve the water-solubility of T2 for T2 itself shows to be less water-soluble.The sulfonation reaction was used to improve the water-solubility after the purification of T2,and finally the satisfied ramification with better water-solubility was selected out.In the evaluation of biological effects,we mainly determinated and compared the antitumor effect of T2 and its ramification with high water-solubility,including in vitro and in vivo experiments.In vitro experiments were designed to observe the influence of T2 and its ramification on cell proliferation and apoptosis,on mitochondrial membrane potential,on the expression levels of apoptosis-related proteins and cell cycle proteins.In vivo experiments were performed to evaluate the antitumor effect of T2 and its ramification on tumor-bearing mice.At last,we also evaluated the bilogiclal safty or long term toxicity of T2 and its ramification in normal rats,including the peripheral hemogram,liver and kidney functions,and also the pathological changes of liver,kidney and ovary.Through these studies,we successfully achieved the T2 compound with high production and purity.And four T2 ramifications were synthesized,including T2-Na,T2-SO3Na,T2-SO3H.2H2O andT2-SO3(NH4)2,of which T2-SO3(NH4)2 showed highest water-solubility and could be used to assess its antitumor property.Cell assays showed that T2-SO3(NH4)2 and T2 both significantly inhibited Hela cell proliferation and induced obvious apoptosis,decreased mitochondrial membrane potential,promoted the expression of apoptosis-related proteins such as Caspase3/7 and Bcl-2,reduced the expression levels of cell cycle proteins such as Cyclin D1,CDK2/4.What important was that T2-SO3(NH4)2 tended to be more effective than T2.In tumor-bearing mice,we observed that intravenously injection of T2-SO3(NH4)2 or intraperitoneally administration of T2 both suppressed tumor growth and promoted apotosis of In Situ tumor cells,however,T2-SO3(NH4)2 was more effective.In the evaluation of biological safty,T2-SO3(NH4)2 and T2 didn't affect the body weight of rats and also the peripheral hemogram and liver or kidney functions.High doses of T2-SO3(NH4)2 didn't result in any obvious pathological injuries in liver,kidney and ovary,however,T2 casued some injuries in liver and kidney.In conclusion,this kind of synthetic method of T2showed some advantages compared with tranditional method:easier,quicker(8h),cheaper,without side reactions,simpler equipment,high production and purity,et al.Compared with T2,the synthesized ramification T2-SO3(NH4)2 has stronger antitumor property with lower biological toxicity,thus it deserves further application in the tumor chemotherapy. |
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