Design, Synthesis And Neuroprotective Activity Evaluation Of Baicalein-amino Acid Derivative

Background:Huang qin comes from the dry root of the medicinal plant radix scutellariae.This medicine was listed as one of the products in "Sheng Nong’s herbal classic"Radix scutellariae was bitter in taste and cold-natured.It does work in heat-clearing and damp-drying,purging fire for removing toxin,hemostasis and preventing miscarriage.Related pharmacological studies have shown that baicalein is the kind of main active components in radix scutellariae,which has many biological activitys for inhibit inflammatory reaction,anti-microbial,anticoagulation,antioxidant,etc.It suggested that baicalein has good prospect in research in recent years.However,as a natural structure,radix scutellariae still has some shortcomings.For example,there is rarely content in radix scutellariae.And it is difficult to preparat.Baicalein has poor stability and poor solubitity,which make the biological activity weak.The series of problems restrict the further development and clinical application of baicalein.In the past research,we chose a few small molecules and many kinds of Chinese medicine active components to combine according to the methods of extraction and isolation,clinical safe medication,principles of pharmaceutical chemistry.To improve the ingredient solubility,enhance its biological activity.We get a series of compounds with strong activity,low toxicity,high solubility,etc.Therefore,this paper is based on the research approach what is said above.Choosing the baicalein as the basic mother nucleus,combining with the different amine acid molecules.In order to find a compound which has good biological activity and good stability,etc.Research methods:This paper is based on the methods of extraction and isolation,clinical safe medication,principles of pharmaceutical chemistry.Acid hydrolyzation was used to the extract baicalein in baicalin,and the influencing factors of baicalein extraction rate were studied through the single factors and response surface method.At the same time,300g baicalin was prepared.Then choosing the baicalein as the basic mother nucleus,combining with the different amine acid molecules to obtain a series of compounds.Regarding to biological activity,we tested those compounds for neuroprotective effects by MTT assay and cell toxicity to normal cells on various of cells in vitro including SH-SY5Y(liver neurons),and H9C2(human cardiac myocytes).Besides,combined Giemsa,DAPI staining method,this study explored effects of HA-1 on morphology change for SH-SY5Y cells.The quail chorioallantoic membrane experiment is further illustrated the effect of neuroprotective activity after drug administration.Research results:From the single factor test and orthogonal test,the optimum conditions were obtained to extract baicalein from baicalin.We get 300g baicalin.The product had been confirmed by means of 13C-NMR,1H-NMR and HR-MS,which determined the relevant physical parameters.We set up the optimum acid-converted process by marking product yield and content.In the aspect of chemistry,we had designed and syntheses 15 baicalein-amino acid derivatives,and the structure of the compounds had been confirmed by means of 13C-NMR,1H-NMR and HR-MS,which determined the relevant physical parameters.And regarding to biological activity,the experimental results showed that most baicalein-amino acid derivatives of neurons showed good inhibitory activity,and the neuroprotective effect was better than baicalin,among which HA-1 showed that the excellent promising activity of SH-SY5Y and the relatively stronger protective effect on H9C2.In addition,the result of cell staining and quail chorioallantoic membrane experiment had demonstrated that HA-1 displayed the good neuroprotective activity and low cardiac toxicity.HPLC was used to determine the contents of baicalein which has deal with the different extreme conditions.We found that HA-1 was stable in high temperature and high light,but it was unstable with the different pH condition.A specific method developed for the determination of baicalein in biological specimens by using HPLC was adopted in the study of its toxicokinetics in rats.The result reveal baicalein has metabolized and the metabolite is baicalin which was disillusionary.Research conclusion:In this experiment,the preparation of baicalin by acid hydrolysis was optimized,and the production rate was improved as far as possible.The problem of oxidation was avoided.15 compounds were designed and synthesized,and the pharmacological activities of this series of derivatives were evaluated.Among then,HA-1 showed great potential for neuroprotective activity comparable to the positive drug edaravin,It is found that the stability of the product is different due to the different kinds of amino acids.For example,in the thin layer detection,it was found that the impurities in the product of baicalin-bicbz lysine were very difficult to separate.However,the product points of baicalin-Cbz valine are obvious,and the impurity points are less easy to be separated.From the products that were obtained,because of the differences in the activity of the three orthophenol hydroxyl groups in baicalin,it’s 6-OH>7-OH>5-OH.Therefore,in the reaction time,the amino acid preferentially and the 6 phenolic hydroxyl groups have a lot of 8 esterification products.There were 5 products of the esterification of amino acids and 7 phenolic hydroxyl groups.However,due to the difference in space resistance of amino acids,some small molecule amino acids and baicalin reacted with simultaneous esterification reaction on 6 and 7 phenolic hydroxyl groups.In terms of structure-activity relationship,the activity of the derivatives of aliphatic amino acids in vitro was better than aromatic amino acids.In the amino acid derivatives of aliphatic amino acids,the bioactivity of small molecular amino acid derivatives is stronger than that of amino acid derivatives with higher molecular weight.The bioactivity of derivatives with Boc protection group is better than that of Cbz group.The derivatives of 6 or 7 single amino acids were better than 6 or 7 amino acids.In terms of chemotropism,by contrast with baicalin,it was found that the optimal compound ha-1 was unstable in acidic environment.The metabolites in the body were also consistent with baicalin,and the bioavailability was not significantly improved,which was significantly different from the expected results.