| Dendrobium catenatum is a perennial herb of the genus Dendrobium orchidaceae.It has been known as “Golden Grass,Soft Gold” since ancient times with a sweet and slightly cold nature,and belongs to the stomach meridian and kidney meridian.It has the effects of strengthening the body,benefiting the stomach,generating body fluid,nourishing Yin and clearing internal heat.Modern medical research has found that D.catenatum has the functions of lowering blood pressure,anti-oxidation,anti-aging,antipyretic and analgesic,and regulating immunity.The whole herb of D.catenatum can be used as an edible herb.According to the metabolome analysis of the research group,polysaccharides were mostly concentrated in the stem,and flavonoids and their derivatives were gathered in flowers.The composition analysis of flower proved that the content of flavone O-glycosides and C-glycosides were in the first place.Under different treatments,the content of flavonoid glycosides changed significantly.At the same time,flavonoids as secondary plant metabolites with pharmacological activity,the pharmacological activity of flavonoids is higher than that of flavonoids and has important research value.Studies have shown that D.catenatum has antioxidation,improve Yin deficiency body and immune activation.In order to fully develop the research potential of flavonoid glycosides,some key questions need to be resolved.1.Does the floral tissue have the same role in the dynamic regulation of the body’s immunity as the stem? Whether flavonoid glycosides as major components are key factors in the pharmacological activity of D.catenatum flowers?2.The mining and functional studies of key catalytic enzymes as the biosynthetic pathway of flavonoid glycosides,the main component of D.catenatum flowers,need to be further explored.3.The exploration of the catalytic mechanism involving flavonoid glycosyltransferases still faces various challenges.Based on the present analysis of the composition of D.catenatum flowers and the evaluation of clinical efficacy,this study explored the following aspects of D.catenatum flowers: 1.The infusions of D.catenatum flowers after treatment in different ways,were analyzed qualitatively for composition of flavonoid glycosides by HPLC.The ability of each extract to dynamically modulate immune activity was evaluated by pharmacological experiments at the cellular and molecular biological levels,mechanism exploration was performed via Western Blot.2.Medicinal prospects based on flavonoid glycoside components,the genomic data analysis and evolutionary tree construction were used to screen for glycosyltransferases that catalyzed the generation of glycosidic components from flavonoid glycosides.After the selection of candidate UGT in D.catenatum,bioinformatics analysis and subcellular localization were performed.The catalytic products of DcUGT were detected by UPLC-Q-TOF/MS and analyzed for their functional and catalytic properties."Spatiotemporal" and stress expression patterns of DcUGT analyzed by q RT-PCR experiments and transcriptome data;3.Based on the difference in catalytic activity of DcUGT,the key amino acid sites affecting the catalytic properties and catalytic efficiency of the enzyme were analyzed by structural biology and molecular docking.Structurefunction relationships were explained by comparing the products catalyzed by the fixedpoint mutant protein and the wild-type protein.In order to determine the pharmacological activity and mechanism of D.catenatum flowers and speculate the active ingredients,HPLC analysis of D.catenatum flower extracts after different treatments revealed that most of the flavonoid glycoside components in the flowers were the same,but the contents were different,The RAW264.7 cell assay showed that all three groups of D.catenatum extracts promoted phagocytosis of macrophages and increased the release of inflammatory factors(NO,IL-1β,IL-6,TNF-α and IFN-γ)to achieve a good immune activation effect.In the LPS-stimulated RAW264.7 cellular immune stress model,all three extracts dynamically reduced NO secretion and the best inhibition efficiency was observed in treatment group 2(fresh D.catenatum flowers).Meanwhile,treatment group 2 reduced the expression of IL-6,IL-1β,TNF-α and IFN-β at the mRNA level,achieving the effect of inhibiting immune hyperactivation under stressful conditions.By exploring the c GAS-STING signaling pathway,the second group reduced IRF3 expression at the mRNA and protein levels,leading to the hypothesis that inhibition of hyperimmunity by suppressing IRF3 secretion,a key target of the pathway,in turn affects the mutual recruitment of STING,TBK1 and IRF3,reduces the entry of IRF3 into the nucleus and decreases the release of interferon to achieve the effect of hyperimmunity suppression.In summary,we hypothesized that flavonoid glycosides were key factors in the dynamic immunomodulatory activity of D.catenatum flowers,differences in the content of flavonoid glycosides affected the pharmacological activity strength.Based on the pharmacological prospects of flavonoid glycoside components in D.catenatum flowers,the glycosyltransferases in flowers with the ability to catalyze the formation of flavonoid glycosides were explored.DcUGT(UGT84P1,UGT84P2,UGT71C4,UGT708S3,UGT708S4,UGT708S5)were screened by homology comparison and evolutionary tree analysis.They were identified by bioinformatic analysis as welldefined,hydrophilic,non-secretory soluble proteins in the family.Tobacco transient transformation determined that all DcUGT were expressed in the cytoplasm and that UGT708S3,UGT708S5 and UGT84P1 could also localize in the nucleus,presumably involved in transcriptional regulation of the organism.Analysis of transcriptome data and q RT-PCR experiments showed that UGT84P1 was most highly expressed in leaves,UGT708S3 in stems,UGT84P2 in calyx labellum,UGT71C4 and UGT708S5 in petals,and UGT708S4 in pollen mass,which were consistent with the enrichment location of flavonoid and flavonoid glycoside components.UGT84P1 and UGT84P2 had the highest transcript levels at 1 month of age and might be involved in the growth and development of the organism,whereas UGT71C4,UGT708S3,UGT708S4 and UGT708S5 had the highest transcript levels at 9 months of age and might be involved in nutrient accumulation and reproductive activities of the plant.UGT84P1,UGT708S4 and UGT708S5 responded positively to cold stress,possibly by synergistically regulating changes in plant physiology and metabolism to improve plant cold tolerance.Expression of UGT71C4,UGT84P1 and UGT84P2 increased dramatically after stimulation by P1 fungal infection,possibly in response to White Silk prevention.And UGT84P1 expression increased in response to Me JA induction,possibly in response to biotic and abiotic stress.Purification by metal affinity chromatography,ion exchange chromatography and gel filtration chromatography to obtain purer target proteins,characterization of the proteins,and examination to determine the optimal reaction conditions(p H=7-8,reaction temperature around 35°C,reaction time 12 hours,not exactly the same metal ion effect for each protein)to lay the foundation for subsequent identification of the enzyme activity.DcUGT catalyzed different flavonoid sugar acceptors and donors and determined their catalytic properties.It was found that DcUGT exhibited substrate heterogeneity and product diversity,with sugar donors preferring glucose,while UGT708S3,UGT708S4 and UGT708S5 could exhibit simultaneous catalytic production of O-glycosides and C-glycosides when using Phloretin as substrates,and UGT708S4 had higher catalytic efficiency and better product diversity compared to other DcUGT,followed by UGT84P2.Based on the enzymatic activity of DcUGT,in order to clarify the relationship between the structure and function of DcUGT,the catalytic mechanism was explained by the resolution of UGT708S4 protein crystals in this study,but due to the inability to collect valid data,the results of molecular docking between peroxidase and substrate were analyzed using Discovery studio,and the catalytic mechanism was analyzed using Swiss-Model to infer the key sites that determined the catalytic properties and catalytic efficiency of the enzyme.In contrast to the catalytic product of the wild-type protein,the mutant protein was obtained by narrowing/increasing the spatial site resistance or changing the polarity of the key site,catalyzed by Phloretin as substrate and UDP-Glc as sugar donor.Leucine at position 99 was found to be a key catalytic site for UGT84P1 to regulate catalytic C-glycosylation and Oglycosylation.Other key sites identified in the DcUGT site exploration that can control the conversion of glycosylation products,and their presence or absence,provided a theoretical basis for exploring the catalytic mechanism of glycosyltransferases.In summary,this study identified that the flavonoid glycosides in D.catenatum flowers had the effect of dynamically regulating immune activity,and by reducing the accumulation of IRF3 protein,a key site on the cGAS-STING signaling pathway,the effect of suppressing immune response could be achieved,which provided a new idea and reference for the development of immune-related clinical Chinese medicine.The expression characteristics and catalytic properties of DcUGT were comprehensively studied and verified,and it was demonstrated that all DcUGT had the activity of catalytic formation of O-glycosides,among which UGT708S3,UGT708S4 and UGT708S5 also had the activity of catalytic formation of C-glycosides,which laid the foundation for the biosynthetic pathway of D.catenatum flavonoids.The key amino acid residues affecting the nature of enzyme activity were identified by targeted mutagenesis,which provided a referenceable basis for the mechanistic study of plant glycosyltransferases.It was proved that D.catenatum flowers had great medicinal value and prospects for further development. |
PDF Full Text Request