Naringenin Chalcone Research Articles

Dendrobium Officinale Kimura & Migo Attenuates High Cholesterol Diet-Induced Atherosclerosis

Objectives: This study aimed to investigate the potential of Dendrobium officinale Kimura & Migo (DOKM) in ameliorating high cholesterol-induced atherosclerosis (AS) in zebrafish, focusing on the development of safe and effective drugs with low toxicity for AS treatment. Methods: A zebrafish AS model was established by feeding zebrafish a high cholesterol diet (HCD). Treatment was administered using 1 mg/L and 10 mg/L DOKM water extract. Fluorescence microscopy was utilised to observe the effects of DOKM on HCD-induced cholesterol accumulation, neutrophil accumulation, and macrophage infiltration in the vasculature. The impact of DOKM on HCD-induced disorders of lipid metabolism was assessed using Nile red staining. Total cholesterol (TC), triglycerides (TG), and low-density lipoprotein (LDL-C) levels in zebrafish were measured using kits. The expression of pcsk9, srebf2, and hmgcr genes in zebrafish was determined using RT-PCR. HE staining and Elvitegravir (EVG) staining were employed to assess the effect of DOKM on plaque formation in the blood vessels of AS zebrafish. Results: A total of 145 compounds were collected from Dendrobium officinale, along with 680 corresponding targets. Additionally, 1583 atherosclerosis-related targets were identified, with 202 representing interaction targets between Dendrobium officinale and atherosclerosis-related targets. Key compounds identified included Gigantol, Chrysotobibenzyl, Naringenin Chalcone, Hesperetin, and Tangeretin. The core targets of Dendrobium officinale for atherosclerosis treatment were STAT3, PTPN11, JAK2, epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor alpha (PDGFRA), proto-oncogene tyrosine-protein kinase Src (SRC), STAT1, TP53, ESR1, and AKT1. GO functional enrichment analysis revealed a total of 2090 GO biological functional entries. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis showed 200 AS signalling pathways associated with DOKM treatment for atherosclerosis. DOKM significantly attenuated cholesterol accumulation, neutrophil aggregation, and macrophage infiltration in the blood vessels of AS zebrafish, ultimately leading to reduced plaque formation. AS zebrafish exhibited lipid metabolism disorders, characterised by significant increases in TC, TG, and LDL-C levels, as well as mRNA expression of pcsk9, srebf2, and hmgcr genes. Treatment with DOKM significantly improved these lipid metabolism disorders. Conclusion: DOKM effectively attenuates HCD-induced AS, and this therapeutic effect on AS may be related to its role in improving lipid metabolism dysfunction.

Naringenin chalcone carbon double-bond reductases mediate dihydrochalcone biosynthesis in apple leaves.

Dihydrochalcones (DHCs) are flavonoids produced as a side branch of the phenylpropanoid pathway. DHCs are found at high concentrations in apples (Malus spp.) but not in pears (Pyrus spp.) or other members of the Rosaceae. Biosynthesis of DHCs in apple has been hypothesized to occur via reduction of p-coumaroyl CoA by a Malus × domestica hydroxycinnamoyl CoA double-bond reductase (MdHCDBR) followed by the action chalcone synthase to produce phloretin or via direct reduction of naringenin chalcone to phloretin via an unknown enzyme. In this study, we report that genetic downregulation of MdHCDBR does not reduce DHC concentrations in apple leaves. We used comparative transcriptome analysis to identify candidate naringenin chalcone reductases (NCRs), designated MdNCR1a-c, expressed in apple leaves but not fruit. These MdNCR1 genes form an expanded gene cluster found exclusively in apple. Transient expression of MdNCR1 genes in Nicotiana benthamiana leaves indicated they produced DHCs at high concentrations in planta. Recombinant MdNCR1 utilized naringenin chalcone to produce phloretin at high efficiency. Downregulation of NCR genes in transgenic apple reduced foliar DHC levels by 85-95%. Reducing DHC production redirected flux to the production of flavonol glycosides. In situ localization indicated that NCR proteins were likely found in the vacuolar membrane. Active site analysis of AlphaFold models indicated that MdNCR1a-c share identical substrate binding pockets, but the pockets differ substantially in related weakly active/inactive NCR proteins. Identifying the missing enzyme required for DHC production provides opportunities to manipulate DHC content in apple and other fruits and has other applications, e.g., in biofermentation and biopharming.

FtMYB163 Gene Encodes SG7 R2R3-MYB Transcription Factor from Tartary Buckwheat (Fagopyrum tataricum Gaertn.) to Promote Flavonol Accumulation in Transgenic Arabidopsis thaliana.

Tartary buckwheat (Fagopyrum tataricum Gaertn.) is a coarse grain crop rich in flavonoids that are beneficial to human health because they function as anti-inflammatories and provide protection against cardiovascular disease and diabetes. Flavonoid biosynthesis is a complex process, and relatively little is known about the regulatory pathways involved in Tartary buckwheat. Here, we cloned and characterized the FtMYB163 gene from Tartary buckwheat, which encodes a member of the R2R3-MYB transcription factor family. Amino acid sequence and phylogenetic analysis indicate that FtMYB163 is a member of subgroup 7 (SG7) and closely related to FeMYBF1, which regulates flavonol synthesis in common buckwheat (F. esculentum). We demonstrated that FtMYB163 localizes to the nucleus and has transcriptional activity. Expression levels of FtMYB163 in the roots, stems, leaves, flowers, and seeds of F. tataricum were positively correlated with the total flavonoid contents of these tissues. Overexpression of FtMYB163 in transgenic Arabidopsis enhanced the expression of several genes involved in early flavonoid biosynthesis (AtCHS, AtCHI, AtF3H, and AtFLS) and significantly increased the accumulation of several flavonoids, including naringenin chalcone, naringenin-7-O-glucoside, eriodictyol, and eight flavonol compounds. Our findings demonstrate that FtMYB163 positively regulates flavonol biosynthesis by changing the expression of several key genes in flavonoid biosynthetic pathways.

Flavonoid Synthesis Pathway Response to Low-Temperature Stress in a Desert Medicinal Plant, Agriophyllum Squarrosum (Sandrice).

Background/Objectives:Agriophyllum squarrosum (L.) Moq. (A. squarrosum), also known as sandrice, is an important medicinal plant widely distributed in dunes across all the deserts of China. Common garden trials have shown content variations in flavonoids among the ecotypes of sandrice, which correlated with temperature heterogeneity in situ. However, there have not been any environmental control experiments to further elucidate whether the accumulation of flavonoids was triggered by cold stress; Methods: This study conducted a four-day ambient 4 °C low-temperature treatment on three ecotypes along with an in situ annual mean temperature gradient (Dulan (DL), Aerxiang (AEX), and Dengkou (DK)); Results: Target metabolomics showed that 12 out of 14 flavonoids in sandrice were driven by cold stress. Among them, several flavonoids were significantly up-regulated, such as naringenin and naringenin chalcone in all three ecotypes; isorhamnetin, quercetin, dihydroquercetin, and kaempferol in DL and AEX; and astragalin in DK. They were accompanied by 19 structural genes of flavonoid synthesis and 33 transcription factors were markedly triggered by cold stress in sandrice. The upstream genes, AsqAEX006535-CHS, AsqAEX016074-C4H, and AsqAEX004011-4CL, were highly correlated with the enrichment of naringenin, which could be fine-tuned by AsqAEX015868-bHLH62, AsqAEX001711-MYB12, and AsqAEX002220-MYB1R1; Conclusions: This study sheds light on how desert plants like sandrice adapt to cold stress by relying on a unique flavonoid biosynthesis mechanism that regulating the accumulation of naringenin. It also supports the precise development of sandrice for the medicinal industry. Specifically, quercetin and isorhamnetin should be targeted for development in DL and AEX, while astragalin should be precisely developed in DK.

Step-by-step optimization of a heterologous pathway for de novo naringenin production in Escherichia coli

Naringenin is a plant polyphenol, widely explored due to its interesting biological activities, namely anticancer, antioxidant, and anti-inflammatory. Due to its potential applications and attempt to overcome the industrial demand, there has been an increased interest in its heterologous production. The microbial biosynthetic pathway to produce naringenin is composed of tyrosine ammonia-lyase (TAL), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), and chalcone isomerase (CHI). Herein, we targeted the efficient de novo production of naringenin in Escherichia coli by performing a step-by-step validation and optimization of the pathway. For that purpose, we first started by expressing two TAL genes from different sources in three different E. coli strains. The highest p-coumaric acid production (2.54 g/L) was obtained in the tyrosine-overproducing M-PAR-121 strain carrying TAL from Flavobacterium johnsoniae (FjTAL). Afterwards, this platform strain was used to express different combinations of 4CL and CHS genes from different sources. The highest naringenin chalcone production (560.2 mg/L) was achieved by expressing FjTAL combined with 4CL from Arabidopsis thaliana (At4CL) and CHS from Cucurbita maxima (CmCHS). Finally, different CHIs were tested and validated, and 765.9 mg/L of naringenin was produced by expressing CHI from Medicago sativa (MsCHI) combined with the other previously chosen genes. To our knowledge, this titer corresponds to the highest de novo production of naringenin reported so far in E. coli.Key points• Best enzyme and strain combination were selected for de novo naringenin production.• After genetic and operational optimizations, 765.9 mg/L of naringenin was produced.• This de novo production is the highest reported so far in E. coli.

Structural and Interactional Analysis of the Flavonoid Pathway Proteins: Chalcone Synthase, Chalcone Isomerase and Chalcone Isomerase-like Protein.

Chalcone synthase (CHS) and chalcone isomerase (CHI) catalyze the first two committed steps of the flavonoid pathway that plays a pivotal role in the growth and reproduction of land plants, including UV protection, pigmentation, symbiotic nitrogen fixation, and pathogen resistance. Based on the obtained X-ray crystal structures of CHS, CHI, and chalcone isomerase-like protein (CHIL) from the same monocotyledon, Panicum virgatum, along with the results of the steady-state kinetics, spectroscopic/thermodynamic analyses, intermolecular interactions, and their effect on each catalytic step are proposed. In addition, PvCHI's unique activity for both naringenin chalcone and isoliquiritigenin was analyzed, and the observed hierarchical activity for those type-I and -II substrates was explained with the intrinsic characteristics of the enzyme and two substrates. The structure of PvCHS complexed with naringenin supports uncompetitive inhibition. PvCHS displays intrinsic catalytic promiscuity, evident from the formation of p-coumaroyltriacetic acid lactone (CTAL) in addition to naringenin chalcone. In the presence of PvCHIL, conversion of p-coumaroyl-CoA to naringenin through PvCHS and PvCHI displayed ~400-fold increased Vmax with reduced formation of CTAL by 70%. Supporting this model, molecular docking, ITC (Isothermal Titration Calorimetry), and FRET (Fluorescence Resonance Energy Transfer) indicated that both PvCHI and PvCHIL interact with PvCHS in a non-competitive manner, indicating the plausible allosteric effect of naringenin on CHS. Significantly, the presence of naringenin increased the affinity between PvCHS and PvCHIL, whereas naringenin chalcone decreased the affinity, indicating a plausible feedback mechanism to minimize spontaneous incorrect stereoisomers. These are the first findings from a three-body system from the same species, indicating the importance of the macromolecular assembly of CHS-CHI-CHIL in determining the amount and type of flavonoids produced in plant cells.

Chalcone-Synthase-Encoding RdCHS1 Is Involved in Flavonoid Biosynthesis in Rhododendron delavayi.

Flower color is an important ornamental feature that is often modulated by the contents of flavonoids. Chalcone synthase is the first key enzyme in the biosynthesis of flavonoids, but little is known about the role of R. delavayi CHS in flavonoid biosynthesis. In this paper, three CHS genes (RdCHS1-3) were successfully cloned from R. delavayi flowers. According to multiple sequence alignment and a phylogenetic analysis, only RdCHS1 contained all the highly conserved and important residues, which was classified into the cluster of bona fide CHSs. RdCHS1 was then subjected to further functional analysis. Real-time PCR analysis revealed that the transcripts of RdCHS1 were the highest in the leaves and lowest in the roots; this did not match the anthocyanin accumulation patterns during flower development. Biochemical characterization displayed that RdCHS1 could catalyze p-coumaroyl-CoA and malonyl-CoA molecules to produce naringenin chalcone. The physiological function of RdCHS1 was checked in Arabidopsis mutants and tobacco, and the results showed that RdCHS1 transgenes could recover the color phenotypes of the tt4 mutant and caused the tobacco flower color to change from pink to dark pink through modulating the expressions of endogenous structural and regulatory genes in the tobacco. All these results demonstrate that RdCHS1 fulfills the function of a bona fide CHS and contributes to flavonoid biosynthesis in R. delavayi.

Transcriptomic and targeted metabolomic analyses provide insights into the flavonoids biosynthesis in the flowers of Lonicera macranthoides

BackgroundFlavonoids are one of the bioactive ingredients of Lonicera macranthoides (L. macranthoides), however, their biosynthesis in the flower is still unclear. In this study, combined transcriptomic and targeted metabolomic analyses were performed to clarify the flavonoids biosynthesis during flowering of L. macranthoides.ResultsIn the three sample groups, GB_vs_WB, GB_vs_WF and GB_vs_GF, there were 25, 22 and 18 differentially expressed genes (DEGs) in flavonoids biosynthetic pathway respectively. A total of 339 flavonoids were detected and quantified at four developmental stages of flower in L. macranthoides. In the three sample groups, 113, 155 and 163 differentially accumulated flavonoids (DAFs) were detected respectively. Among the DAFs, most apigenin derivatives in flavones and most kaempferol derivatives in flavonols were up-regulated. Correlation analysis between DEGs and DAFs showed that the down-regulated expressions of the CHS, DFR, C4H, F3’H, CCoAOMT_32 and the up-regulated expressions of the two HCTs resulted in down-regulated levels of dihydroquercetin, epigallocatechin and up-regulated level of kaempferol-3-O-(6’’-O-acetyl)-glucoside, cosmosiin and apigenin-4’-O-glucoside. The down-regulated expressions of F3H and FLS decreased the contents of 7 metabolites, including naringenin chalcone, proanthocyanidin B2, B3, B4, C1, limocitrin-3,7-di-O-glucoside and limocitrin-3-O-sophoroside.ConclusionThe findings are helpful for genetic improvement of varieties in L.macranthoides.

Probing the Deoxyflavonoid Biosynthesis: Naringenin Chalcone Is a Substrate for the Reductase Synthesizing Isoliquiritigenin.

Isoliquiritigenin formation is a key reaction during deoxyflavonoid biosynthesis, which is catalyzed by two enzymes, chalcone synthase (CHS) and reductase (CHR). The substrates for CHS are established. However, the substrate for CHR is unknown. In this study, an in vitro reaction was performed to confirm whether naringenin chalcone can be a substrate. Naringenin chalcone was used as a substrate during the CHR reaction. Analyzing the product revealed that isoliquiritigenin was produced from naringenin chalcone, indicating that naringenin chalcone is a substrate. This study is the first to identify a substrate for CHR, reveals that deoxyflavonoid biosynthesis diverges from naringenin chalcone, endorses the term "chalcone reductase," and answers the long-standing questions about doubly-labeled acetic acid uptake pattern in deoxyflavonoid biosynthesis.

Chromosome-level genome assembly provides insights into the genome evolution and functional importance of the phenylpropanoid–flavonoid pathway in Thymus mongolicus

BackgroundThymus mongolicus (family Lamiaceae) is a Thyme subshrub with strong aroma and remarkable environmental adaptability. Limited genomic information limits the use of this plant.ResultsChromosome-level 605.2 Mb genome of T. mongolicus was generated, with 96.28% anchored to 12 pseudochromosomes. The repetitive sequences were dominant, accounting for 70.98%, and 32,593 protein-coding genes were predicted. Synteny analysis revealed that Lamiaceae species generally underwent two rounds of whole genome duplication; moreover, species-specific genome duplication was identified. A recent LTR retrotransposon burst and tandem duplication might play important roles in the formation of the Thymus genome. Using comparative genomic analysis, phylogenetic tree of seven Lamiaceae species was constructed, which revealed that Thyme plants evolved recently in the family. Under the phylogenetic framework, we performed functional enrichment analysis of the genes on nodes that contained the most gene duplication events (> 50% support) and of relevant significant expanded gene families. These genes were highly associated with environmental adaptation and biosynthesis of secondary metabolites. Combined transcriptome and metabolome analyses revealed that Peroxidases, Hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferases, and 4-coumarate-CoA ligases genes were the essential regulators of the phenylpropanoid–flavonoid pathway. Their catalytic products (e.g., apigenin, naringenin chalcone, and several apigenin-related compounds) might be responsible for the environmental tolerance and aromatic properties of T. mongolicus.ConclusionThis study enhanced the understanding of the genomic evolution of T. mongolicus, enabling further exploration of its unique traits and applications, and contributed to the understanding of Lamiaceae genomics and evolutionary biology.

Metabolism of phenolic compounds catalyzed by Tomato CYP736A61

Cytochrome P450s (CYPs) regulate plant growth and stress responses by producing diverse primary and secondary metabolites. However, the function of many plant CYPs remains unknown because, despite their structural similarity, predicting the enzymatic activity of CYPs is difficult. In this study, one member of the CYP736A subfamily (CYP736A61) from tomatoes was isolated and characterized its enzymatic functions. CYP736A61 was successfully expressed in Escherichia coli through co-expression with molecular chaperones. The purified CYP736A61 showed hydroxylation activity toward 7-ethoxycoumarin, producing 7-hydroxycoumarin or 3-hydroxy 7-ethoxycoumarin. Further substrate screening revealed that dihydrochalcone and stilbene derivates (resveratrol and polydatin) are the substrates of CYP736A61. CYP736A61 also mediated the hydroxylation of resveratrol and polydatin, albeit with low activity. Importantly, CYP736A61 mediated the cleavage of resveratrol and polydatin as well as pinostilbene and pterostilbene. Interestingly, CY736A61 also converted phloretin to naringenin chalcone. These results suggest that CYP736A61 is a novel CYP enzyme with stilbene cleavage activity.

Partial Chemical Characterization of Drought-Related Metabolites in Maize Under Drought Stress Conditions

This study investigates the metabolic response of maize genotypes UASBM13 (drought-tolerant) and UASBM10 (drought-sensitive) to drought stress using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Drought stress significantly impacts crop productivity, with potential consequences for global food security. Secondary metabolites produced by maize under drought conditions, provide insights into molecular response. The identified metabolites include sinapic acid, sinapoyl malate, coumaroyl shikimate, caffeoyl shikimate, syriogenin, sinapaldehyde, hydroxy ferulate, naringenin chalcone, and resveratrol. These compounds play crucial roles in antioxidant defence, lignification, and stress-related pathways. The results suggest that UASBM13 exhibits a more robust metabolic response to drought, with higher levels of key metabolites associated with stress tolerance. The findings contribute to our understanding of plant adaptation to environmental stress and provide valuable information for developing drought-tolerant crop varieties to ensure global food security in the context of a changing climate and growing population.

Comparative Metabolic Profiling of Drought-Tolerant and Drought-Sensitive Maize Inbred Lines under Drought Stress

This study investigates the metabolic responses of plant species under mild drought stress, by comparing drought tolerant UASBM 13 and drought sensitive UASBM10 maize inbred lines. Study emphasized the synthesis and upregulation of crucial metabolites associated with antioxidant activities and cell wall lignification. Metabolites such as syringentin, naringenin chalcone, ferulic acid, sinapic acid, Sinapoyl Malate, and resveratrol were explored for their potential roles in mitigating drought-induced oxidative stress and fortifying cellular structures. Under mild stress, naringenin chalcone exhibited consistent upregulation, indicating its involvement in antioxidant activity and cell wall lignification. The study observed significant upregulation of sinapic acid, Sinapoyl Malate, and resveratrol in tolerant lines, suggesting their role in drought tolerance. Additionally, metabolites related to cell wall lignification were induced in response to drought stress, contributing to the overall drought tolerance in plants. The findings highlight the intricate metabolic mechanisms involved in plant adaptation to drought stress and the potential applications of these metabolites in enhancing drought resilience.

Metabolomics Study of the Effect of Transcription Factor NOR-like1 on Flavonoids in Tomato at Different Stages of Maturity Using UPLC-MS/MS.

Tomato fruits are rich in flavonoids. This study explores the effect of transcription factor SlNOR-like1 on the accumulation of flavonoids in tomato fruits at different ripening stages. We used ultra-pressure liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to analyze wild-type (WT) and NOR-like1 CRISPR/Cas9-edited (NOR-like1) tomato fruits. A total of 50 flavonoid metabolites were accurately identified and determined in tomatoes. The flavonoid metabolic differences were observed among the different tomato sample groups using PCA and OPLS-DA analysis. There were 16 differential flavonoids (13 upregulated and 3 downregulated) identified between WT-GR (WT tomato at the green-ripening stage) and NOR-like1-GR (NOR-like1 tomato at the green-ripening stage), 9 differential flavonoids (six upregulated and three downregulated) identified between WT-BR3 (WT tomato at the color-breaking stage) and NOR-like1-BR3 (NOR-like1 tomato at the color-breaking stage), and 12 differential flavonoids (11 upregulated and 1 downregulated) identified between WT-BR9 (WT tomato at the red-ripening stage) and NOR-like1-BR9 (NOR-like1 tomato at the red-ripening stage). Rutin, nicotiflorin, naringenin chalcone, eriodictyol, and naringenin-7-glucoside were the five flavonoids with the highest content in the ripening stages (BR3 and BR9) in both WT and NOR-like1 tomato fruits. The overall flavonoid contents in WT tomato fruits changed little from GR to BR3 and decreased from BR3 to BR9; meanwhile, in the NOR-like1 tomato fruits, the total amounts of the flavonoids exhibited an increasing trend during all three ripening stages. The accumulation pattern of flavonoid metabolites in NOR-like1 tomato fruits differed from that in WT tomato fruits, especially in the later ripening process of BR9. The transcription factor SlNOR-like1 has an impact on the accumulation of flavonoids in tomato fruits. The results provide a preliminary basis for subsequent research into its regulatory mechanism and will be helpful for attaining future improvements in the nutritional quality and postharvest treatment of tomato fruits.

Quantitative proteomics and metabolomics analysis reveals the response mechanism of alfalfa (Medicago sativa L.) to o-coumaric acid stress

O-coumaric acid (OCA), as a significant phenolic allelochemical found in hairy vetch (Vicia villosa Roth.), that can hinder the growth of alfalfa (Medicago sativa L.), particularly the growth of alfalfa roots. Nonetheless, the mechanism by which OCA inhibits alfalfa root growth remains unclear. In this study, a liquid chromatography tandem mass spectrometry (LC-MS/MS)-based quantitative proteomics analysis was carried out to identify differentially accumulated proteins (DAPs) under OCA treatment. The findings indicated that 680 proteins were DAPs in comparison to the control group. Of those, 333 proteins were up-regulated while 347 proteins were down-regulated. The enrichment analysis unveiled the significance of these DAPs in multiple biological and molecular processes, particularly in ribosome, phenylpropanoid biosynthesis, glutathione metabolism, glycolysis/gluconeogenesis and flavonoid biosynthesis. The majority of DAPs reside in the cytoplasm (36.62%), nucleus (20.59%) and extracellular space (14.12%). In addition, phenylalanine deaminase was identified as a potential chemical-induced regulation target associated with plant lignin formation. DAPs were mainly enriched in flavonoid biosynthesis pathways, which were related to plant root size. Using the UPLC-ESI-MS/MS technique and database, a total of 87 flavonoid metabolites were discovered. The metabolites were predominantly enriched for biosynthesizing naringenin chalcone, which was linked to plant lignin formation, aligning with the enrichment outcomes of DAPs. Consequently, it was deduced that OCA impacted the structure of cell walls by mediating the synthesis of lignin in alfalfa roots, subsequently inducing wilt. Furthermore, a range of proteins have been identified as potential candidates for the breeding of alfalfa strains with enhanced stress tolerance.

Comparative Metabolomics Analysis of Different Perilla Varieties Provides Insights into Variation in Seed Metabolite Profiles and Antioxidant Activities.

Perilla seeds are essential functional foods and key ingredients in traditional medicine. Herein, we investigated the variation in phytochemical profiles and antioxidant activities of twelve different perilla seeds. The seeds showed significant variations in total phenolic and flavonoid contents ranging from 16.92 to 37.23 mg GAE/g (GAE, gallic acid equivalent) and 11.6 to 19.52 mg CAE/g (CAE, catechin equivalent), respectively. LC-QqQ-MS (liquid chromatography triple quadrupole tandem mass spectrometry)-based widely targeted metabolic profiling identified a total of 975 metabolites, including 68-269 differentially accumulated metabolites (DAMs). Multivariate analyses categorized the seeds into four groups based on the seed coat and leaf colors. Most key bioactive DAMs, including flavonoids (quercetin-3'-O-glucoside, prunin, naringenin, naringenin chalcone, butin, genistin, kaempferol-3-O-rutinoside, etc.), amino acids (valine, lysine, histidine, glutamine, threonine, etc.), and vitamins (B1, B3, B6, U, etc.) exhibited the highest relative content in PL3 (brown seed, purple leaf), PL1 (white seed, green-purple leaf), and PL4 (white seed, green leaf) groups, respectively. Meanwhile, key differentially accumulated phenolic acids showed a higher relative content in PL1 and PL4 than in other groups. Both seeds exhibited high antioxidant activities, although those of PL2 (brown seed, green leaf) group seeds were the lowest. Our results may facilitate the comprehensive use of perilla seeds in food and pharmaceutical industries.

Functional characterization and enzymatic properties of flavonoid glycosyltransferase gene CtUGT49 in Carthamus tinctorius

Carthami Flos, as a traditional blood-activating and stasis-resolving drug, possesses anti-tumor, anti-inflammatory, and immunomodulatory pharmacological activities. Flavonoid glycosides are the main bioactive components in Carthamus tinctorius. Glycosyltransferase deserves to be studied in depth as a downstream modification enzyme in the biosynthesis of active glycoside compounds. This study reported a flavonoid glycosyltransferase CtUGT49 from C. tinctorius based on the transcriptome data, followed by bioinformatic analysis and the investigation of enzymatic properties. The open reading frame(ORF) of the gene was 1 416 bp, encoding 471 amino acid residues with the molecular weight of about 52 kDa. Phylogenetic analysis showed that CtUGT49 belonged to the UGT73 family. According to in vitro enzymatic results, CtUGT49 could catalyze naringenin chalcone to the prunin and choerospondin, and catalyze phloretin to phlorizin and trilobatin, exhibiting good substrate versatility. After the recombinant protein CtUGT49 was obtained by hetero-logous expression and purification, the enzymatic properties of CtUGT49 catalyzing the formation of prunin from naringenin chalcone were investigated. The results showed that the optimal pH value for CtUGT49 catalysis was 7.0, the optimal temperature was 37 ℃, and the highest substrate conversion rate was achieved after 8 h of reaction. The results of enzymatic kinetic parameters showed that the K_m value was 209.90 μmol·L~(-1) and k_(cat) was 48.36 s~(-1) calculated with the method of Michaelis-Menten plot. The discovery of the novel glycosyltransferase CtUGT49 is important for enriching the library of glycosylation tool enzymes and provides a basis for analyzing the glycosylation process of flavonoid glycosides in C. tinctorius.

Unveiling targeted spatial metabolome of rice seed at the dough stage using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry imaging

Rice (Oryza sativa) seeds contain a variety of metabolites, which not only provide energy for their own growth and development, but also are an important source of nutrition for humans. It is crucial to study the distribution of metabolites in rice seeds, but the spatial metabolome of rice seeds is rarely investigated. In this study, Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS) imaging was used to reveal the spatial distribution of free soluble sugars (glucose, fructose, sucrose, and maltose), amino acids (9 essential amino acids and 2 amino acids affecting rice eating quality: L-aspartic acid and L-glutamic acid), and 4 metabolites in the flavonoids synthesis pathway (cinnamic acid, naringenin chalcone, naringenin, and dihydrokaempferol) in rice seed at the dough stage. It was found that the 4 free soluble sugars present similar spatial distribution, mainly distributed in the seed cortex and embryo with high abundance. The majority of amino acids are also concentrated in the rice cortex and embryo, while the others are abundant in the whole seed. Besides cinnamic acid distributed in the seed cortex and embryo, the naringenin chalcone, naringenin, and dihydrokaempferol were also found in the endosperm and had lower content. Furthermore, a colocalization phylogenetic tree according to the spatial distribution imaging of each metabolite was constructed. This study revealed the distribution diversity of metabolites in different segmentations of rice seed at the dough stage, providing clues for the nutritional differences between brown rice and white rice, and serving as a reference for people to target a healthy diet.

Phytochemical and Agronomic Characterization of High-Flavonoid Lettuce Lines Grown under Field Conditions.

Flavonoids are antioxidant phytochemicals that confer a beneficial effect on human health. We have previously developed and characterized eight lettuce (Latuca sativa L.) lines that accumulated high levels of diverse flavonoids and their precursors in controlled environment conditions. Three Rutgers Scarlet lettuce (RSL) lines selected in tissue culture for deep-red color (RSL-NAR, RSL-NBR, RSL-NFR) accumulate anthocyanins and quercetin, three lines identified in a chemically mutagenized red lettuce population accumulate kaempferol (KfoA and KfoB) or naringenin chalcone (Nco), and two lines that were spontaneous green mutants derived from the red line RSL-NAR (GSL, GSL-DG) accumulate quercetin. These eight lines were field-grown in the Salinas Valley of California for four years together with seven control accessions of varying colors (light green, dark green, red, and dark red). At market maturity, a substantial variation in plant composition was observed, but the three RSL lines consistently accumulated high levels of cyanidin, GSL and GSL-DG accumulated the highest levels of quercetin, KfoA and KfoB accumulated kaempferol, and Nco amassed naringenin chalcone, confirming that these mutant lines produce high levels of beneficial phytochemicals under field conditions. Mutant lines and control accessions were also assessed for their biomass production (plant weight, height, and width), overall content of pigments (leaf chlorophyll and anthocyanins), resistance to diseases (downy mildew, lettuce drop, and Impatiens necrotic spot virus), postharvest quality of processed tissue (deterioration and enzymatic discoloration), and composition of 23 mineral elements. All but one mutant line had a fresh plant weight at harvest comparable to commercial leaf cultivars; only Nco plants were significantly (p < 0.05) smaller. Therefore, except for Nco, the new, flavonoid hyperaccumulating lines can be considered for field cultivation.

Physicochemical parameters and chemoprofiling of honey of two species of stingless bees in the Peruvian Amazon

The physicochemical and chemical characteristics of stingless bee honey from two commonly grown species in the Peruvian Amazon, Melipona eburnea and Tetragonisca angustula, were evaluated. Key physicochemical findings include humidity levels exceeding Codex-Alimentarius limits (28.13 g/100 g of honey for M. eburnea and 26.33 g/100 g for T. angustula), while sugar content fell below minimum requirements (54.3 g/100 g of honey and 43.5, respectively). Hydroxymethylfurfural levels were 12.5 mg/100 g of honey and 0.4, respectively. Using MALDI-TOF/TOF analysis, biologically and medicinally relevant molecules derived from plants and microbes were tentatively detected in both honey samples. These molecules encompassed properties such as anticancer, antibacterial, antifungal, anti-inflammatory, and antiviral. Notable compounds in M. eburnea honey included naringenin chalcone, caffeine, berberine, and glycosyl trans-zeatin-O-glucoside, previously documented in various honey types. Additionally, quinine, paclitaxel, stigmastonol, and surfactin C, with therapeutic potential, were preliminarily detected for the first time in this honey. T. angustula honey also contained medicinal molecules including lutein, cinnamic acid, fraxin, and hyperoside, along with newly observed compounds such as osthole, culmorin, and uncarine C (cat’s claw). Both honey samples exhibited trace levels of non-natural compounds, including commercial pesticides and herbicides, suggesting environmental contaminants. Further research is needed to verify the identity of these molecules, understand their potential sources, quantify their levels in Amazonian honey, and assess consumption and health implications. This study highlights the necessity for a novel technical standard exclusive to stingless bee honey, encompassing methodologies for measuring the physicochemical parameters and for monitoring the chemical profile to assess the medicinal potential of native honey and define maximum limits for environmental pollutants. This pioneering work underscores the ecological, medicinal, and economic value of stingless bees in the Peruvian Amazon.