Study On Enzymatic Synthesis,Biological Activity,Digestion And Absorption Characteristics Of Baicalin Ester Derivatives

Baicalin is a kind of flavonoids from Scutellaria baicalensis with various biological activities including antioxidant,antimicrobial and lipid-lowering activity.However,the low lipophilicity of baicalin makes it difficult to penetrate the intestinal epithelial cells,resulting in poor oral bioavailability,which further limits its application in the health field.The introduction of a hydrophobic fatty chain into the compound can increase its lipophilicity and thus improve its oral bioavailability.The current research mainly uses chemical method to modify the structure of baicalin to improve its lipophilicity.The chemical method has some problems such as complicated operation,poor regioselectivity and loss of biological activity.Enzymatic catalysis has the advantages of simple operation,mild reaction conditions,green biocatalysts and high regioselectivity.However,the enzymatic catalysis has not been used to improve the lipophilicity of baicalin.In this study,the immobilized lipase was used to modify the structure of baicalin through esterification,and a series of baicalin ester derivatives with different chain lengths were synthesized.Cell models and animal experiments were used to study their biological activities and gastrointestinal digestion and absorption characteristics of these baicalin derivatives were investigated.The results are as follows:(1)In this study,Novozym 435(a lipase from C.antarctica)was screened from seven lipases with different microorganisms as the enzyme catalyst for the esterification of baicalin,and the optimal reaction conditions were explored.The results showed that the most suitable solvents,molar ratio of fatty alcohol to baicalin,enzyme dosage and temperature were the binary organic solvent consisting of cyclohexane and pyridine(1:1),40:1,40 mg/mL and50?.Under the optimal reaction conditions,all conversions were above 90%.HPLC,LC-MS,¹H-NMR and ¹³C-NMR were used to determine the structure of synthesized baicalin ester derivatives,including ethyl ester,propyl ester,butyl ester,hexyl ester,octyl ester and decyl ester.And it was found that the esterification site was on the carboxyl group of the glycosyl group of baicalin.The oil-water partition coefficients of these ester derivatives are1.59,1.94,2.18,2.97,4.01 and 5.37,respectively.(2)In this study,the lipid-lowering activities and mechanism of baicalin and its ester derivatives were investigated.Baicalin,ethyl ester,propyl ester,butyl ester,hexyl ester,octyl ester and decyl ester could significantly reduce the lipid accumulation in hepatocytes(LO2)induced by oleic acid and palmitic acid,by 25.1%,34.8%,41.6%,49.7%,26.4%,26.8%and19.6%,respectively.Baicalin ethyl ester,propyl ester and butyl ester have better lipid-lowering activity than baicalin.There are two main mechanisms for the better lipid-lowering activity of ethyl ester,propyl ester and butyl ester.One is that the accumulation of ethyl ester,propyl ester and butyl ester in LO2 cell is 24-110 times that of baicalin at the same concentration.On the other hand,the activation of ethyl ester,propyl ester and butyl ester on carnitine palmitoyltransferase 1A(CPT1A)is 1.8-2.2 times that of baicalin.In addition,in mouse 3T3-L1 adipocytes and oxidized low-density lipoprotein-induced foamed macrophages,baicalin ethyl ester,propyl ester and butyl ester reduced 30-40%lipids at 5?mol/L,while baicalin only reduced 6%lipids,and even showed no lipid-lowering activity on foam cells.(3)The antioxidant and antibacterial activities of baicalin and its ester derivatives were also investigated.The results showed that the antioxidant activities of baicalin and its ester derivatives were different in different antioxidant evaluation systems.In ORAC,DPPH and ABTS free radical scavenging experiments,the antioxidant activity of baicalin was 1.1-1.2times than that of its ester derivatives,but in AAPH-induced erythrocyte hemolysis and CAA experiments,the antioxidant activity of ester derivatives was 1.2-1.4 times than that of baicalin.The reason may be related to the distribution of baicalin derivatives in different systems.The results of antibacterial experiment showed that the inhibitory effect of ethyl,propyl and butyl esters with shorter fatty chains and lauryl esters with longer fatty chains on Staphylococcus aureus,Escherichia coli and Candida albicans was close to that of baicalin,with MICs of 6.4-12.8 mmol/L,but the inhibitory activity of hexyl,octyl,geranyl,citronellyl,undecyl and 10-undecenyl ester with medium-length fatty chain on these three target strains was better than that of baicalin,with MICs of 0.4-6.4 mmol/L.The reason may be that baicalin esters with medium-length fatty chain can exert the nonspecific antibacterial activity by destroying microbial cell membrane.(4)In this study,the absorption characteristics of baicalin and its ester derivatives in the Caco-2 cell monolayer,as well as the stability and bioaccessibility in gastrointestinal digestion were also investigated.The results showed that the P_app values of ethyl,propyl and butyl ester in Caco-2 monolayer were about 2-10 times higher than that of baicalin,while the absorption of hexyl,octyl and decyl ester were not evaluated due to their low cytotoxicity and solubility.It was also found that carboxylesterase(CES)in Caco-2 cells could affect the absorption of ester derivatives.After inhibition by CES inhibitor BNPP,the P_app values of these ester derivatives were about 1.5 times higher than those without BNPP treatment.The results of in vitro gastrointestinal digestion showed that the digestion stability of baicalin ethyl ester,propyl ester,butyl ester,hexyl ester,octyl ester and decyl ester were 27.9%,34.4%,58.6%,52.7%,50.8%and 43.6%,respectively.And their bioaccessibility were 23.9%,29.4%,51.4%,47.3%,40.3%and 32.9%,respectively,which were significantly lower than of baicalin(80.0%for digestion stability and 75.9%for bioaccessibility).The reason is that baicalin ester derivatives in the gastrointestinal digestive juice were hydrolyzed,and some ester derivatives were precipitated because of low solubility in water.(5)In this study,the digestion and absorption characteristics and pharmacokinetic properties of baicalin ester derivatives-loaded liposomes were investigated.The results showed that the embedding of baicalin butyl ester and octyl ester by liposomes could reduce the cytotoxicity by more than 4 times,increased their solubility in aqueous solution by nearly38 and 136.8 times,and inhibit the hydrolysis.In particular,the embedding of hexyl,octyl and decyl ester by liposomes can reduce the toxicity to Caco-2 monolayer,and the P_app value of baicalin esters-loaded liposomes is 13.5-24.4 times higher than that of baicalin-loaded liposomes.The pharmacokinetic study showed that,compare with free baicalin butyl ester and octyl ester,the C_max value of butyl ester liposomes in rats increased from 1.46±0.15 to2.06±0.20?mol/L,and the AUC_0-?value increased from 13.81±1.72 to 26.13±1.76?mol/L·h,which increased by 1.41 and 1.89 times,respectively;the C_max value of octyl ester liposome in rats did not increase significantly,which were 1.40±0.09 and 1.32±0.33?mol/L,respectively,while the AUC_0-?value increased from 6.92±0.91 to 22.24±2.71?mol/L·h,which increased by 3.21 times.These results indicated that the encapsulation of butyl ester and octyl ester by liposomes increased their oral ansorption.In conclusion,this study established a new method for the synthesis of baicalin ester derivatives catalyzed by Novozym 435,which effectively improve the lipophilicity of baicalin.Some of the baicalin ester derivatives showed significantly enhanced lipid-lowering,antioxidant and antimicrobial activity.Aiming at the problem that the introduction of aliphatic chains reduces the bioaccessibility of baicalin,the liposome embedding method of baicalin ester derivatives and its pharmacokinetic properties have been explored.The combined use of enzymatic modification and liposome embedding is expected to further solve the problem of low bioavailability of baicalin.