| Gastrodin,a natural active ingredient in Chinese medicinal Rhizoma Gastrodiae,is mainly used for sedation,analgesia,vertigo and cardiovascular disease in clinic.Studies have shown that gastrodin acts on cranial nerve diseases through a variety of mechanisms,and it is a potential therapeutic drug for central nervous system diseases.However,gastrodin is a hydrophilic small molecular substance,which is not easy to penetrate the blood-brain barrier in the drug transmission in the brain.It has the disadvantages of short action time,high cerebral metabolism speed,etc.,which limits its development and application.Acylation modification of natural substances to enhance their fat solubility has been proved to be an effective measure to enhance the blood-brain barrier permeability of drugs and prolong the action time of drugs.In view of the defects of chemical method and biological method at present,a new method for biocatalytic synthesis of gastrodin ester derivatives in solvent-free system is proposed in this paper,and a series of gastrodin ester derivatives with different chain lengths are synthesized.The catalytic activities of commercial immobilized lipase and whole cell catalysts for the synthetic reaction of gastrodin ester in organic solvent and solvent-free system are compared and investigated,and the influence law of some key reaction factors on biocatalytic acylation of gastrodin is discussed and optimized.The biological activities of a series of gastrodin ester derivatives were also evaluated.Firstly,the synthesis of gastrodin esters catalyzed by lipase in organic solvents were studied,and the time course of gastrodin acylation catalyzed by different types of lipase was analyzed.The results showed that the immobilized lipase Novozym 435,lipase IMTL,lipase IMPS and lipase 40086 could all efficiently catalyze the acylation of gastrodin,and the substrate conversion rate could reach 100%.The structure of the product was analyzed by HPLC,mass spectrometry（HRMS）,FT-IR and 13C NMR.It was confirmed that lipase could catalyze the acylation of gastrodin at the 6"-OH position of the sugar ring,the 7-OH position of the benzene ring and the 4"-OH position of the sugar ring.Thus,mono-,di-and tri-esters of gastrodin were isolated and identified.In addition,the effects of the structure and chain length of the acyl donor on the enzymatic synthesis of gastrodin esters were also studied.Sixteen gastrodin ester derivatives with different acylation sites and chain lengths were synthesized by separation,purification and structural analysis of the corresponding reaction products.It was found that the change of the chain length of the acyl donor did not affect the selective acylation site of the enzyme.Compared with the organic solvent system,the solvent-free system has the advantages of more environmental protection,high reaction speed,easy separation and purification of the product.Therefore,lipase-catalyzed acylation of gastrodin in a solvent-free system was further explored in the present study.The results showed that the immobilized lipase Novozym 435,Lipozyme IMTL,Lipozyme IMPS and Lipozyme 40086 had high catalytic efficiency in the solvent-free system.Compared with the organic solvent system,the efficiency of the enzymatic gastrodin esters synthesis reaction in the solvent-free system is higher.Taking the acetylation of gastrodin catalyzed by Novozym 435 as an example,the optimal reaction conditions were determined as follows:enzyme amount 5 mg/m L,reaction temperature 50℃,and substrate concentration 60 mmol/L.After reacting for 24 h under the above reaction conditions,the gastrodin could be completely converted into its tri-esters by lipase;The enzymatic reaction had good batch stability in the absence of solvents.In this study,the effects of acyl donor structure and chain length on the enzymatic reaction in a solvent-free system were further explored,and it was found that the reaction system was suitable for the synthesis of short-and medium-chain gastrodin esters,especially the synthesis of short-chain gastrodin esters.Then,the whole-cell catalytic synthesis of gastrodin esters in organic solvent system and solvent-free system was investigated.The results showed that the products of whole-cell catalytic reaction for the synthesis of gastrodin esters in organic solvent system and solvent-free system were similar to the products of enzymatic reaction.However,the activity of whole-cell catalytic reaction for the synthesis of gastrodin triesters was relatively lower.The reaction conditions for the whole-cell catalyzed synthesis of gastrodin esters in a solvent-free system were further optimized.The optimal conditions were determined as follows:the whole cell amount 20 mg/m L,reaction temperature 50℃,and the substrate concentration 40 mmol/L.After 12 h of reaction,the conversion rate of gastrodin substrate can reach 100%,and the production rate of diester can reach 95.5%.Finally,the blood-brain barrier permeability and activity of a series of gastrodin esters were preliminarily evaluated.First,according to the log P value and relative molecular mass（MW）of gastrodin esters,12 gastrodin esters which met the recommended scope of drugs for brain were screened out from 16 synthetic gastrodin esters.The blood-brain barrier permeability of the selected gastrodin esters was then assessed by determining plasma protein binding and the PAMPA-BBB model.The experiment of plasma protein binding rate showed that with the prolongation of gastrodin ester chain and the increase of the site,the plasma protein binding rate increased gradually with the change of concentration,indicating that the action time of gastrodin in vivo might be prolonged after esterification.PAMPA experiment showed that the permeability coefficients of gastrodin esters were increased to different degrees,and the one with the largest permeability coefficient was benzylbutyl gastrodin monoester,with the Pe value of 8.96±0.18*10–6cm/s,indicating that the blood-brain barrier permeability was further improved after the esterification of gastrodin.Finally,the in vitro inhibitory activity of gastrodin esters on acetylcholinesterase was analyzed.The results showed that compared with gastrodin itself,the inhibitory activity of some gastrodin ester derivatives on acetylcholinesterase was significantly improved.This study had high theoretical and practical significance,and provided a new idea for the development of natural brain drugs represented by gastrodin. |
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