Biotransformation Of Bioactive Compounds In Pannax Notoginseng Fermented By Eurotium Cristatum And Its Mechanism

Eurotium cristatum is the dominant probiotics in the fermentation process of Fuzhuan brick tea,which is the decisive strain to form the special flavor of its,also known as"Jinhua".Japanese researchers have processed"Jinhua"into health products,and its price even more expensive than gold.As a result,people are paying more and more attention to its health functions.However,in our country,the research on E.cristatum is mostly based on its influence on tea and we all ignored its medical value as a important medicinal fungus with good development prospect.The metabolites of E.cristatum are rich,including various exopolysaccharides,amino acids,tea polyphenols and so on,which have high medicinal value.Meanwhile,its rich and powerful enzyme system has great potential for the transformation and modification of the active ingredients of medicinal plants.Therefore,we believe that E.cristatum is one of the best strains to construct the combination of medicinal fungi and traditional Chinese medicine fermentation.Panax notoginseng is a common kind of TCM medicine,which can be applied differently by raw and cooked in TCM.Due to the different effects of raw and cooked Sanqi,“Sheng Xiao Shu Bu”can be found in it.As a common and valuable Chinese herbal medicine in China,it has been widely used in treating various traumas and replenishing qi and blood and so on since ancient times.In recent years,more attention has been paid to the research on Panax notoginseng,mainly focusing on the chemical constituent,pharmacological action,clinical application and processing method of Panax notoginseng,with the acceleration of the modernization of traditional Chinese medicine and the urgent needs of people.Although the research on processed Panax notoginseng has been increasing in these years,the mechanism of processing and the changes of bioactive constituents before and after processing are still unclear.At the same time,with the development of modern biotechnology and the progress of various chemical analysis technologies,more and more researchers have begun to apply microbial fermentation technology to the processing of traditional Chinese medicine,and explore the research and development of new drugs in traditional Chinese medicine.The present study aim to illuminate the changes of bioactive components before and after processing and the mechanism of processing of Panax notoginseng fermented by Eurotium cristatum.There were three groups of samples:fermented Panax notoginseng,notes FS?medium fermentation stage Panax notoginseng,notes FS1;fermentation stage Panax notoginseng,notes FS2?;unfermented Panax notoginseng,notes WS;culture Eurotium cristatum in rice mediumrice,notes DM.It is proposed that modern technologies should be put forward to study the correlation between the chemical constituent transformation and microbial fermentation,in order to explain the processing mechanism of Panax notoginseng,improve the quality evaluation system of this traditional Chinese medicine,and provide guarantee for the safety and effectiveness of its clinical application.This paper also provided new ideas for the deep exploitation of Panax notoginseng as well as promoting the modernization of traditional Chinese medicine.The main results were as follows:1.DPPH radical,ABTS radical and hydroxy radical scavenging capacities of fermentation products were evaluated.The results show that each sample has certain scavenging ability to DPPH,ABTS and hydroxyl radicals at a certain concentration range.The IC₅₀ of DPPH free radical scavenging rate of fermented product,unfermented Panax notoginseng and Vc and DM were 2.6047 mg/m L,6.3113 mg/m L,0.0018 mg/m L,0.8374mg/m L,respectively.The IC₅₀ of ABTS radical scavenging rate were13.4067 mg/m L,14.4333 mg/m L,0.06 mg/m L,34.9033 mg/m L,respectively.The IC₅₀ of hydroxy radical scavenging rate were as flows:FS is 6.4213±0.6318 mg/m L,WS is 11.8233±0.5050 mg/m L,Vc is11.4033±0.1960 mg/m L,DM is 15.9500±2.3797 mg/m L?There was significant difference between them,which indicating that the antioxidant activity of fermented Panax notoginseng was superior to the unfermented Panax notoginseng.2.The inhibitory activity of each sample on?-glucosidase and?-amylase was determined by PNPG method and iodine-starch coloration method,respectively.The results were as follows:each group has certain inhibition ability to?-glucosidase but DM group in the concentration range of 0.0625?2 mg/m L.At the same time,The inhibitory effect of acarbose,FS group and WS group on?-glucosidase were flows:acarbose>FS>WS.However,the inhibitory activity of each sample on?-amylase were:FS>WS>DM>acarbose.3.High pressure liquid chromatograph?HPLC?analysis was applied to identify dynamic changes of some specific bioactive components in Panax notoginseng,including Notoginsenoside R1,Ginsenoside Re,Ginsenoside Rb1 and Ginsenoside Rd.The results showed that the average content of the four standard substance in FS and WS groups?values in brackets?were Notoginsenoside R1 5.6915 mg/g?3.1814 mg/g?,Ginsenoside Re 11.1207mg/g?5.0011 mg/g?,Ginsenoside Rb1 19.2661 mg/g?9.1570 mg/g?,Ginsenoside Rd 5.5865 mg/g?2.3841 mg/g?.4.Moreover,the fingerprints of panax notoginseng before and after fermentation were compared.The results showed that more substance absorption peaks were detected in FS group and WS group compared with DM group.There were 11 special absorption peaks?peaks number:1-11?were labeled,of which 6 absorption peaks?peaks number:1,2,3,6,8,10?were unique to FS group,and 5 absorption peaks?peaks number:4,5,7,9,11?which were unique to FS group and DM group.5.The original data obtained after UHPLC–Triple TOF/MS analysis were annotated to 756 metabolites,including 215 metabolites annotated to KEGG database and 673 metabolites annotated to HMDB and Lipidmaps database.There were 622 metabolites annotated to the HMDB database.They were classified into 14 HMDB super classes,of which 266 were"lipids and lipids"and 115 metabolites were"organic acids and their derivatives".Both accounted for about 61%of all HMDB annotated metabolites.6.The identification and quantification results of metabolites showed that a total of 764,866 and 850 metabolites had been obtained in DWS,DFS1and DFS2 groups respectively,of which 12 metabolites were unique to DWS and DFS1,and 28 metabolites unique to DFS2.In comparison of DWS and DFS1 and DFS2,385 metabolites were identified with significant difference.In comparison of DFS2 vs.DFS1,263 metabolites were significant difference,of which 96 were up-regulated and 167 were down-regulated.There were 261 were significant difference in DFS2 vs.DWS,of which 114 were down-regulated and 117 were up-regulated.By cluster analysis of differential metabolites before and after fermentation and KEGG pathway enrichment analysis,it was found that there were significant differences in metabolite expression before and after fermentation.The related metabolic pathways of metabolites with significant difference in DFS2 vs.DWS groups included phenylalanine metabolism,choline metabolism in cancer,bile secretion,glycerophospholipid metabolism and phenylpropane biosynthesis.Conclusion:1.The results showed that the fermentation process of Panax notoginseng by Eurotium cristatum was feasibleand and laid the foundation for the next experiment.2.The results,which the mensuration of in vitro biological activity of Panax notoginseng samples before and after fermentation,including hypoglycemic effect and antioxidant activity,indicate that the biological activity of fermented Panax notoginseng was greatly improved.It further confirmed the great potential of E.cristatum in the processing of traditional Chinese medicine and the possibility of Panax notoginseng fermentation.3.The contents of several main saponins in Panax notoginseng were determined by high performance liquid chromatography,including Panax notoginseng saponins R1,ginsenoside Re,ginsenoside Rb1 and ginsenoside Rd.The results showed that the contents of the four substances were improved after fermentation.4.Through LC-MS analysis,the changes of substances before and after fermentation were further clarified:a total of 385 differential metabolites were screened,of which 263 were DFS2 and DFS1,261 were DFS2 and DWS,and 12 were unique to DWS and DFS1,and there are 28differential metabolites were unique in DFS2.