Biotransformation Of Coumarins By Cambial Meristematic Cells Of Angelica Sinensis And Camptotheca Acuminate

Objective: The transcriptome de novo sequencing of cambial meristematic cells(CMCs)and dedifferentiated plant cells(DDCs)of Angelica sinensis have been studied in our previously research,and the results showed the gene expression levels of CMCs were significantly different with DDCs.Particularly,the characteristic gene expression levels of enzymes controlled the synthesis of secondary metabolites such as ferulic acid,cinnamic acid,vanillic acid,caffeic acid,p-coumaric acid and p-hydroxybenzoic in CMCs were significantly higher than DDCs.Therefore we evaluated the accumulation of above secondary metabolites in the A.sinensis CMCs,DDCs and the storage root.Now,there are no report about the biotransformation of CMCs suspension cultures.In this study,the A.sinensis CMCs suspension cultures were used to the biotransformation of 6-hydroxycoumarin-3-carboxylicacid ethyl ester and 7,8-dihydroxycoumarin-3-carboxylicacid ethyl ester for the first time.The biotransformation of 4-methylcomarins by CMCs and DDCs of C.acuminate have been studied in our previously research.Among them,6-hydroxy-4,7-methylcoumarin(b)have been transformed to two products,and the one product have been isolated,therefore the other one will be isolated in this study.Methods: The accumulation of cinnamic acid,vanillic acid,caffeic acid,p-coumaric acid and p-hydroxybenzoic in the A.sinensis CMCs,DDCs and the storage root were determined by HPLC.The A.sinensis CMCs suspension cultures were used to the biotransformation of 6-hydroxycoumarin-3-carboxylicacid ethyl ester and 7,8-dihydroxycoumarin-3-carboxylicacid ethyl ester,and the products were isolated by chromatography on ODS column and HPLC column.The biotransformed product in the biotransformation of 6-hydroxy-4,7-methylcoumarin(b)by CMCs and DDCs of C.acuminate were isolated by HPLC column.Results and conclusion: The results showed the concentration of cinnamic acid,caffeic acid,p-coumaric acid and p-hydroxybenzoic in CMCs were higher with 2.05-,2.45-,2.49-and 1.52-fold increase compared with that in DDCs.However,the concentration of vanillic acid in DDCs was higher with 1.67-fold increase compared with that in CMCs.The above results suggested that CMCs can secrete more natural products than DDCs.In addition,the concentration of caffeic acid,p-coumaric acid and p-hydroxybenzoic in CMCs were higher with 1.54-,1.56-and 1.82-fold increase compared with that in A.sinensis root.The above results suggested that CMCs can secrete more natural products than A.sinensis root.In the biotransformation of 6-hydroxycoumarin-3-carboxylicacid ethyl ester,8 compounds were isolated from the EtOAc extract of culture,and were identified by spectral analysis and physic-chemical constants as: p-hydroxyphenylethanol(I-1),4-hydroxy-3-methoxy benzene ethanol(I-2),daucosterol(I-3),Nb-acetyltrytamine(I-4),(3-methoxycar-bonylamino-2-methyl-phenyl)-cabamic acid methy lester(I-5),obtucarbamate A(I-6),1,1?-methanediyl bis(4-methoxybenzene)(I-7),?-sitosterol(I-8).Among them,compounds I-1,I-2,I-4,I-5,I-6 and I-7 were isolated from the culture stimulated by 6-hydroxycoumarin-3-carboxylicacid ethyl ester.Simultanously,we speculated their possible biosynthesis pathways based on the chemical constituents of A.sinensis.However,the compounds I-3 and I-8 were chemical constituents of the A.sinensis according to the previously reports.In order to find products in the biotransformation of 7,8-dihydroxycoumarin-3-carboxylicacid ethyl ester,8 compounds were isolated from the EtOAc extract of culture and were identified as: 1-(4-hydroxyphenyl)-1,2-ethanediol(II-1),5-methyluracil(II-2),p-hydroxyphenylethanol(II-3),daucosterol(II-4),phenazine-1-carboxamide(II-5),2-nonyl-4-hydroxyquinoline N-oxide(II-6),caffeine(II-7),?-sitosterol(II-8).Among them,compounds II-2,II-3,II-5,II-6 and II-7 were isolated from the culture stimulated by 7,8-dihydroxycoumarin-3-carboxylicacid ethyl ester,while compounds II-1,II-4 and II-8 were chemical constituents of the A.sinensis according to the previously reports.Simultanously,we speculated their possible biosynthesis pathways based on the chemical constituents of A.sinensis.Particularly,phenazine-1-carboxamide was isolated firstly from the plant.The above results suggested that the A.sinensis CMCs contained abundant enzymes,and can product a series of compounds after induced by 6-hydroxycoumarin-3-carboxylicacid ethyl ester and 7,8-dihydroxycoumarin-3-carboxylicacid ethyl ester.However,there are no biotransformed products about 6-hydroxycoumarin-3-carboxylicacid ethyl ester and 7,8-dihydroxycoumarin-3-carboxylicacid ethyl ester.We speculated that 6-hydroxycoumarin-3-carboxylicacid ethyl ester and 7,8-dihydroxycoumarin-3-carboxylicacid ethyl ester were allothigenes in the CMCs suspension cultures,and have toxic effects to cultures.Therefore CMCs suspension cultures degraded the allothigenes to product themselves.Four coumarins were obtained by Pechmann condensation in our previously research,named as 7-dihydroxy-4,8-dimethylcoumarin(a),6-hydroxy-4,7-methylcoumarin(b),6,7-dihydroxy-4-methylcoumarin(c)and 5-hydroxy-4,7-methylcoumarin(d),which were transformed by CMCs and DDCs of C.acuminate for the first time.Transformation products were named as 7-O-?-D-glucopyranosyl-4,8-methylcoumarin(a-1),6-O-?-D-glucopyranosyl-4,7-methylcoumarin(b-1),6-hydroxyl-7-methoxy-4-methylcoumarin(c-1)and 5-O-?-D-glucopyranosyl-4,7-methylcoumarin(d-1).In this study,the other product in the biotransformation of 6-hydroxy-4,7-methylcoumarin(b)by CMCs and DDCs of C.acuminate were isolated and identied as 4,7-dimethylcoumarin 6-O-?-D-mannoside(b-2).Furthermore,the monoamine oxidase(MAO)inhibition activity of substrates and biotransformed products was evaluated in vitro assay.The results suggested that substrates synthesized and theire biotransformed products can inhibit MAO activity,and may be used to develop promising drugs for treating neurodegenerative diseases.