| Apocyni Veneti Folium is a commonly used Chinese medicinal material.It is the dried leaf of Apocynum venetum L.(Apocynaceae),which has the traditional functions of calming livers and nerves,clearing heat and water,and pharmacological effects of antihypertensive,antihyperlipidemic,antidepressant,liver protection,strengthening immunity,anti-aging,anti-diabetic vascular lesions.It has a long history of medicinal use and tea drinking.Apocyni Veneti Folium is clinically used for liver-yang dizziness,palpitations,insomnia,edema and oliguria,hypertension,neurasthenia,and nephritis edema embolism.Apocynum venetum L.is a widely available salt-tolerant Chinese medicinal resource.The salt stress environment is closely related to the quality of Apocyni Veneti Folium.Moderate level of salt stress can promote the synthesis and accumulation of the active ingredients of Apocyni Veneti Folium and improve its quality.However,in the process of quality formation,how salt stress affects the synthesis and accumulation of secondary metabolites in Apocyni Veneti Folium and their response to salt stress mechanisms are still unclear.Therefore,how to elucidate the quality formation mechanism has become a key issue in the quality improvement project of Apocyni Veneti Folium.Under the preamble of the research on the quality evaluation of Apocyni Veneti Folium,this subject is based on the law that the differential expression of transcriptome and proteome induced by salt stress affects the synthesis and accumulation of secondary metabolites.With the advantages and characteristics of multi-omics integrated analysis,transcriptomics and proteomics techniques were used to screen and analyze differentially expressed genes(DEGs)and proteins(DEPs)in salt-treated Apocyni Veneti Folium;plant metabolomics techniques were used to screen and identify characteristic metabolic markers of Apocyni Veneti Folium after salt stress;through multi-omics correlation analysis,the causes of the differences in the secondary metabolites of Apocyni Veneti Folium under salt stress and the internal quality formation mechanism of the medicinal material were explored.The main research contents and innovative results are as follows:1.Literature research Related literatures of Apocyni Veneti Folium and multi-omics analysis under salt stress were reviewed and summarized.2.Analysis of multiple bioactive constituents in Apocyni Veneti Folium An optimized method based on UFLC-QTRAP-MS/MS technology to simultaneously determine the content of 43 bioactive ingredients in flavonoids,organic acids,amino acids and nucleosides of Apocyni Veneti Folium was established.Combined with principal component analysis(PCA)and gray relational analysis(GRA)to classify and evaluate the samples,total of 41 batches of Luobumaye samples were compared and analyzed.The results showed that the established method is accurate and reliable;Apocyni Veneti Folium is clearly distinguished from Pocynum hendersonii(Hook.f.)Woodson leaf,and the quality of Apocyni Veneti Folium is good.This article provides a new method reference for comprehensive evaluation and internal quality control of Apocyni Veneti Folium.3.Site-specific accumulation and dynamic change of flavonoids in Apocyni Veneti Folium Site-specific accumulation of flavonols in Apocyni Veneti Folium was determined by laser scanning confocal microscope(LSCM)and the localization of flavanols also was observed via vanillin-HCl staining.LSCM observation showed that flavonols were accumulated in cuticle of epidermal cells and vessel walls,especially in protoplasts and nucleolus of the collenchyrma cells and the epidermal cells.Flavanols were localized in the palisade parenchyma cells and vessel walls,particularly in the laticifers of the phloem.On the basis of the difference of the maximal emission wavelength between quercetin and kaempferol derivatives which had fluorescence behavior by Naturstoffreagenz A treatment,kaempferol and its derivatives were localized exclusively in the cuticle.The contents of five flavonoids in Apocyni Veneti Folium from different harvest times and growth parts were measured using HPLC method.Results showed that the content of astragalin in Apocyni Veneti Folium from different parts revealed the decreasing trend,while hyperoside and isoquercitrin were higher in June and July.In summary,the site-specific accumulation of flavonoids in Apocyni Veneti Folium can be determined by LSCM and vanillin-HCl staining.The contents of flavonoids in Apocyni Veneti Folium were correlated with harvest times and growth parts.4.Quality analysis of Apocyni Veneti Folium under salt stress(1)Effects of salt stress on the physiological indicators and multiple bioactive constituents of salt-treated Apocyni Veneti Folium To systematically evaluate the quality of Apocyni Veneti Folium from the perspective of physiological and the wide variety of bioactive components response to various concentrations of salt stress,this experiment was arranged on the basis of UFLC-QTRAP-MS/MS technology and multivariate statistical analysis.Physiological characteristics of photosynthetic pigments,osmotic homeostasis,lipid peroxidation product,and antioxidative enzymes were introduced to investigate the salt tolerance mechanism of Apocyni Veneti Folium under salinity treatments of four concentrations(0,100,200,and 300 mM NaCl,respectively).Furthermore,a total of 43 bioactive constituents,including 14 amino acids,nine nucleosides,six organic acids,and 14 flavonoids were quantified in Apocyni Veneti Folium under salt stress.In addition,multivariate statistical analysis,including hierarchical clustering analysis(HCA),PCA,and gray relational analysis(GRA)was employed to systematically cluster,distinguish,and evaluate the samples,respectively.Compared with the control,the results demonstrated that low and moderate levels of salt stress contributed to maintain high quality of photosynthesis,osmotic balance,antioxidant enzyme activity,and the accumulation of metabolites,except for total organic acids,and the quality of Apocyni Veneti Folium obtained by these two groups was better than others;however,under severe stress,the accumulation of the oxidative damage and the reduction of metabolite caused by inefficiently scavenging reactive oxygen species(ROS)lead to lower quality.In summary,the proposed method may be helpful for the quality evaluation of Apocyni Veneti Folium and other salt-tolerant Chinese medicines.(2)Quality analysis of salt-treated Apocyni Veneti Folium by fingerprint-activity relationship A fingerprint-activity relationship between chemical fingerprints and hepatoprotective activity was established to evaluate the quality of salt-treated Apocyni Veneti Folium.Characteristic fingerprints of Apocyni Veneti Folium samples exposed to different concentrations of salt were generated by UFLC-Triple TOF-MS/MS,and a similarity analysis was performed based on common characteristic peaks by cluster analysis.The hepatoprotective activity of Apocyni Veneti Folium against CCl4-induced acute liver damage in mice was investigated by assessing biochemical markers and histopathology,which showed that a high dose of Apocyni Veneti Folium exposed to low levels of salt stress produced a marked amelioration of hepatic damage in mice compared with the other salt-treated Apocyni Veneti Folium.Fingerprint-activity relationship was investigated by the chemical fingerprints and the hepatoprotective activities utilizing multivariate statistical analysis,gray correlation analysis and bivariate correlation analysis.The results showed that the accumulation of polyphenols,such as flavonoids and phenolic acids,in Apocyni Veneti Folium subjected to low levels of salt stress could result in the effective scavenging of free radicals.Therefore,the present study may provide a powerful strategy to holistically evaluate the quality of salt-treated Apocyni Veneti Folium in combination with chemical fingerprint and bioactivity evaluation.5.Multi-omics analysis of Apocyni Veneti Folium under salt stress(1)Transcriptomics analysis of Apocyni Veneti Folium under salt stress based on RNA-seq technology Transcriptomics analysis of Apocyni Veneti Folium under four different levels of salt stress was performed using De novo assembly RNA-seq technology.BLAST was used to compare all genes to NT,NR,Swiss-prot,COG,KEGG,GO,and Interpro databases for functional annotation.DEGs were selected based on fold changes and false discovery rate;Apocyni Veneti Folium salt tolerance mechanism was analyzed based on KEGG pathways of DEGs.Results showed that more than 46892898 raw reads were obtained by double-end sequencing,and 54276 trinity were assembled,with a total length of 56485295 bp and an average length of 1040 bp.Compared with the control group,there were 807,1512,and 1258 DEGs of under low,moderate,and high levels of salt-stressed Apocyni Veneti Folium,respectively.These DEGs were involved in 164 KEGG pathways,such as phenylpropanoid biosynthesis,flavonoid and terpenoid biosynthesis and biosynthesis of amino acid.It was found that salt stress significantly up-regulated gene expression of starch and sucrose metabolism,glycolysis,terpene synthesis,plant hormone signal transduction,plant-pathogen interaction,glutathione metabolism,and ribosome.The transcriptomics analysis has certain significance for revealing the quality formation mechanism of the salt-tolerant medicinal Apocyni Veneti Folium.(2)iTRAQ-based proteomics analysis of Apocyni Veneti Folium under salt stress iTRAQ-based proteomic profiles of Apocyni Veneti Folium exposed to four levels of salt treatments were performed to investigate protein changes.A total of 143,162 and 167 DEPs were found between salt-treated Apocyni Veneti Folium compared with control,respectively.They were mainly involved in carbohydrate and energy metabolism,biosynthesis of metabolites and signal transduction.Furthermore,results showed that carbon and nitrogen metabolisms were altered under salt stress;low and moderate levels of salt stress enhanced photosynthetic functions and ramped up carbohydrate metabolism.However,severe salt stress depressed biosynthesis of secondary metabolites.(3)Metabolomics analysis of Apocyni Veneti Folium in response to salt stress based on UFLC-Triple TOF-MS/MS To understand the salt-tolerant mechanisms of the halophyte,metabolomics platform based on UFLC-Triple TOF-MS/MS was applied in this study.The results showed that metabolic profiles were separated and differentiated among groups based on multivariate statistical analysis;different metabolites belonged to various chemical classes.Besides,phenylpropanoid pathway and terpenoid biosynthesis were disturbed in all salt-stressed Apocyni Veneti Folium and low level of salt-treated group was better than other samples in terms of relative contents of the wide variety of bioactive components and physiological variations of photosynthetic pigments,osmotic homeostasis,lipid peroxidation product and antioxidative enzymes.This study may provide additional insight into the salt-tolerant mechanisms and the quality assessment of Apocyni Veneti Folium in a holistic level based on the plant metabolites.(4)Correlation analysis of omics on salt-treated Apocyni Veneti Folium Through the correlation analysis among multiple bioactive constituent content,spectrum-effect relationship,and metabolomics,seven markers of quality difference,including proanthocyanidin B2,glaucolide B,allamandin,quercetin,kaempferol,isoquercitrin and acetylated isoquercitrin.Through the integrated analysis of transcriptomics,proteomics and metabolomics,the target genes/proteins related to the quality formation of Apocyni Veneti Folium under salt stress were identified,and 8 key enzyme genes encoding dehydrin 1,annexin,pathogenesis-related protein,prolyl oligopeptidase,peroxidase,cinnamyl alcohol dehydrogenase,4-hydroxycinnamoyl-CoA ligase 3,cytochrome P450 enzyme were screened.These quality markers and key enzymes/genes provide a reference for quality improvement and functional gene verification of Apocyni Veneti Folium. |
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