Search Results (799)

Background: Plant flavonoids are candidate alternatives to ionophores for modulating rumen fermentation, yet the intact glycoside rutin (quercetin-3-O-rutinoside) has received little attention. The objective of this study was to determine whether rutin changes the in vitro ruminal fermentation of total mixed rations (TMR) differing in forage-to-concentrate ratio; monensin was included at a single dose as a reference additive rather than a direct comparator. Methods: In a 2 × 4 factorial batch-culture experiment, a roughage-based (60:40) and a concentrate-based (40:60) TMR were incubated for 24 h without additive (control) or with rutin at 2.5% or 5% of substrate dry matter or monensin at 30 ppm. Results: Rutin did not change the gas production kinetics, ruminal pH, the volatile fatty acid (VFA) profile, or the stoichiometrically estimated methane and carbon dioxide at either dose (p > 0.05), and no dose response was found. Substrate type likewise left the VFA profile and the estimated gases unchanged (p > 0.05). Ammonia nitrogen (NH₃-N) responded to the additive group (p = 0.027) and to the substrate × additive interaction (p = 0.022), but Tukey comparisons separated no individual means, so this response is preliminary. Conclusions: Neither rutin nor monensin markedly altered fermentation of these silage-, alfalfa- and cereal-based rations in the 24 h batch system; the substrate-dependent NH₃-N signal warrants confirmation, and direct methane measurement is required before any anti-methanogenic potential can be claimed. Full article

Background/Objectives: In the search for potent non-sugar α-glucosidase inhibitors with improved safety profiles, a novel flavonoid glycoside was isolated for the first time from the stem bark of Albizia saponaria (Fabaceae). The objective of this study was to elucidate its chemical structure and evaluate its therapeutic potential as an anti-hyperglycemic agent compared to known related flavonoids and a standard clinical drug. Methods: Comprehensive structural elucidation was performed using high-resolution mass spectrometry and multidimensional 1D/2D NMR (¹H, ¹³C, HSQC-DEPT, COSY, and CIGAR). To assess its inhibitory efficacy and pharmacokinetic profiles, an in silico comparative study was conducted against a database of related flavonoids (Quercitrin, Hyperoside, and Isoquercitrin) and the clinical drug Acarbose. This involved molecular docking simulations against human intestinal maltase-glucoamylase (PDB ID: 3TOP) alongside integrated ADMET modeling and toxicological screening. Results: The compound was successfully identified as 4′,7-dihydroxyflavan-3′-O-β-D-glucoside (1). Molecular docking revealed that Compound 1 exhibited a superior predicted binding affinity of −9.5 kcal/mol, outperforming Quercitrin (−9.3 kcal/mol), Hyperoside (−8.3 kcal/mol), Isoquercitrin (−7.9 kcal/mol), and Acarbose (−7.2 kcal/mol). This strong thermodynamic stability is driven by a robust conventional hydrogen-bonding network with key active site residues (Arg1377, Gln1372, and Gly1365), successfully overriding a localized electrostatic strain at Asp1279. Furthermore, ADMET modeling demonstrated a highly desirable local pharmacokinetic framework; its low Caco-2 permeability (−6.432) and low human intestinal absorption (HIA = 0.120) favor targeted luminal retention in the gastrointestinal tract, mirroring Acarbose while minimizing systemic exposure. Crucially, toxicological screening unveiled a significant safety advantage for Compound 1, marked by negligible CYP3A4 interaction (0.004) and a remarkably low risk of Drug-Induced Liver Injury (DILI = 0.213) compared to the high-risk hepatotoxic profile of Acarbose (DILI = 0.882) and the reference flavonoids (DILI > 0.69). Conclusions: These predictive findings establish Compound 1 as a highly promising, low-toxicity natural scaffold for anti-hyperglycemic drug development. Its superior binding affinity and minimized hepatotoxicity risk warrant subsequent in vitro and in vivo functional validation. Full article

Background/Objectives: Berberis species are recognized for their ethnomedicinal importance and phytochemical richness associated with antidiabetic activity. Although B. crataegina is widely distributed in the Turkish flora and has a history of traditional use, its phytochemical properties and biological activities remain less extensively investigated than those of other Berberis species. This study aimed to comparatively evaluate the phytochemical profiles, in vitro antidiabetic potential, and cytoprotective effects of extracts prepared from different parts of B. crataegina in a high-glucose-induced SH-SY5Y cell-based model of diabetic neuropathy. Methods: Crude hydroethanolic extracts (70% ethanol) were prepared from the leaves, flowers, shoots, roots, and fresh fruits of B. crataegina collected from Kızılcahamam, Ankara, Türkiye. The total phenolic and flavonoid contents, antioxidant capacities and α-glucosidase inhibitory activities of leaf, flower, shoot, root, and fruit extracts were determined. The cytoprotective effects of the crude extracts were evaluated in a high-glucose-induced SH-SY5Y cell model. The metabolomic profiles of the extracts were comparatively analyzed using liquid chromatography–quadrupole time-of-flight mass spectrometry (LC-QTOF-MS). The most active fruit extract was fractionated by reversed-phase vacuum liquid chromatography (RP-VLC), and the phytochemical contents and α-glucosidase inhibitory activities of the fractions were evaluated. The major phenolic compounds in the most active fruit extract were quantitatively determined using high-performance liquid chromatography–diode array detection (HPLC-DAD). The relationships between metabolites and biological activities were investigated through correlation analysis. Results: The fruit extract was distinguished by its α-glucosidase inhibitory activity, total phenolic content, antioxidant capacity, and cytoprotective effect in the high-glucose-induced SH-SY5Y cell model. It showed no marked cytotoxicity at concentrations ranging from 25 to 400 µg/mL and, at 25 µg/mL, attenuated the high-glucose-induced reduction in cell viability, restoring viability to a level close to that of the normal control. Untargeted metabolomic analysis identified 191 metabolites detected in at least two crude extracts, while the metabolites detected exclusively in the fruit extract were predominantly flavonoids and flavonoid glycosides. Fractionation showed that α-glucosidase inhibitory activity was mainly concentrated in the medium-polarity fractions; however, none of the fractions reached the activity level of the crude extract. Chlorogenic acid, rutin, caffeic acid, protocatechuic acid, quercetin, and quercetin-3-O-glucoside were detected and quantified in the fruit extract by HPLC-DAD. Correlation analysis indicated that the observed biological activities may be associated with phenolic acids, flavonoids, and other polyphenolic metabolites. Conclusions: The fruit extract of B. crataegina showed promising α-glucosidase inhibitory activity and cytoprotective effects in the high-glucose-induced SH-SY5Y cell model. The findings suggest that these effects may arise from the combined contribution of multiple constituents within a polyphenolic matrix rather than from a single compound; however, synergistic interactions were not experimentally demonstrated. These results support further evaluation of the fruit extract through mechanistic, in vivo, and standardization studies. Full article

Despite known antioxidant activity, the effects of plant-derived compounds on aging-related physiological functions and mechanisms remain unclear, which necessitates investigation at cellular and organismal levels. To examine whether these compounds can mitigate age-related physiological decline, Nelumbo nucifera Gaertn. (sacred lotus) embryo extract–water-soluble fraction (NEW), stilbene glycoside from Pleuropterus multiflorus (Thunb.) Turcz. ex Nakai (PM-SG), and pterostilbene were investigated through chemical profiling, antioxidant activity, HepG2 liver cells, and Caenorhabditis elegans longevity assays, while principal component analysis (PCA) elaborated on the results. Quantitative LC-MS revealed that NEW extract was rich in alkaloids (neferine and liensinine), while PM-SG mainly contained 2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucoside. PM-SG showed the strongest radical-scavenging activity (DPPH, TEAC, and ORAC), followed by pterostilbene. However, pterostilbene exhibited the strongest cellular antioxidant activity among the three test materials (NEW, PM-SG, and pterostilbene), an effect attributed to its superior cellular uptake, although it was less potent than the reference standards EGCG and quercetin. All samples extended the lifespan of C. elegans dose-dependently, with the strongest effect from pterostilbene (40–50%), then PM-SG (25–35%) and NEW (18–22%). Pterostilbene and PM-SG improved health measures, including enhanced resistance to heat and oxidative stress, and maintained feeding function. Discrepancies between in vitro antioxidant rankings and observed cellular effects indicate that radical-scavenging activity alone cannot fully predict in vivo anti-aging efficacy, which depends on cellular uptake, activation of endogenous defense mechanisms, and modulation of longevity pathways. Further clinical studies are needed to determine the potential relevance of these findings to human health. Full article

The Pd(0)-catalysed aryloxylation of 6-O-tert-butyldimethylsilyl-3,4-di-O-isobutyloxycarbonyl-d-glucal (3) with a series of phenolic nucleophiles was investigated as an efficient approach to the synthesis of unsaturated aryl O-glycosides. Under the optimized reaction conditions, the corresponding 2,3- and 3,4-unsaturated O-glycosides were obtained in good to excellent yields and with high β-selectivity. In most cases, the reactions proceeded with a marked preference for the formation of 2,3-unsaturated β-glycosides, whereas chlorophenols exhibited distinct reactivity patterns, resulting in diminished regioselectivity and the additional formation of α-anomeric 2,3-unsaturated glycosides. Comparison with the previously reported reactions of 6-O-tert-butyldiphenylsilyl-3,4-di-O-isobutyloxycarbonyl-d-glucal demonstrated a pronounced influence of the silyl protecting group on the product yields and regioselectivity, with the TBDMS-protected donor generally affording higher yields and enhanced selectivity toward 2,3-unsaturated products. Furthermore, the developed methodology was successfully extended to carbohydrate nucleophiles, enabling the synthesis of model trisaccharides and demonstrating its applicability to oligosaccharide synthesis. Full article

Citrus × aurantium ‘Daidai’ (Citrus aurantium L. var. amara Engl.) is a sour orange cultivar whose dried flower buds are listed in the Chinese national catalogue of substances that serve both as food and as medicine, whereas the dried flower buds of the ordinary species C. × aurantium L. lack this dual recognition. Although the essential oil and hydrosol of these buds have been investigated, the still-bottom water remaining after hydrodistillation is routinely discarded and its chemical composition remains largely unexplored. In this study, we compared the chemical profiles of still-bottom water obtained from the dried flower buds of C. aurantium L. var. amara Engl. (CAVAF) and C. × aurantium L. (CALF) after two pretreatments (simple soaking vs. ultrasonic-microwave synergistic treatment) and evaluated the chemical antioxidant activities of both the hydrosol and the still-bottom water. HPLC-Q-TOF-HRMS identified 215 compounds across the four water samples. The major constituents were neohesperidin (13.18–39.66%), blumeatin (9.78–10.25%), narirutin (13.46–18.01%), and α-D-(6-O-Sinapoyl)-glucopyranosyl(1→22)-β-D-(3-O-sinapoyl)-fructofuranose (7.46–8.57%). Relative to CALF, the CAVAF samples contained a large number of annotated compounds and higher relative amounts of several flavonoids and limonoids (up to nine-fold) in the still-bottom water. Higher relative abundances of the principal flavonoid glycosides were observed after ultrasonic-microwave pretreatment compared with soaking. The hydrosol of both varieties showed only modest radical scavenging activity and weak reducing power. In contrast, the still-bottom water exhibited clear DPPH, ABTS, and hydroxyl radical scavenging, with half-maximal inhibitory concentrations ranging from 0.1901 to 4.873 mg/mL, although reducing power remained limited. These findings suggest that botanical variety and pretreatment method may influence both the metabolite profile and the in vitro radical scavenging behavior of the distillation by-products. The results supply chemical compositional support for the food-medicine homology status of CAVAF and indicate that still-bottom water is a recoverable source of potential polar antioxidants that is currently under-utilized. Full article

Background/Objectives: Co-amorphous solid dispersions (c-ASD) are a promising strategy for enhancing the dissolution of poorly water-soluble drugs. Naringin (NA) and neohesperidin (NE) are dihydroflavonoid components and are poorly soluble. They can form a c-ASD. Concerning the c-ASD formation mechanism, the intermolecular forces between the non-sugar aglycones of NA and NE have been determined; however, the roles of the sugar chains, which account for nearly 50% of the overall molecular weight of both compounds, are unclear. Therefore, the impact of the sugar moiety on the solubility of NA-NE c-ASD needs to be investigated. Methods: The sugar removal products of NA and NE are naringenin-7-O-glucoside, naringenin, hesperetin-7-O-glucoside, and hesperetin. Therefore, in this study, the solubility profiles of NA with hesperetin-7-O-glucoside and hesperetin, and of NE with naringenin-7-O-glucoside and naringenin were assessed by dissolution determination and analyzed by PXRD. Results: These results indicated that the rhamnose and glucose moieties on the NE sugar chain and the glucose moiety on the NA sugar chain are vital to the stability and solubility of NE-NA c-ASD. The rhamnose moiety on the sugar chain of NA is removable, without which a stable soluble aggregator may also be constructed, but the soluble aggregator formation ability is weakened. Conclusions: The results of this study not only inform the rational selection of co-formers for structurally similar flavanone glycosides but can also help chemists modify the c-ASD structure based on the sugar moiety. Full article

Minicitrus (Fortunella hindsii) is a wild kumquat with potential nutritional and medicinal value, but its tissue-specific bioactive metabolites, antioxidant activity, and endophytes remain poorly characterized. Here, we integrated LC-MS profiling, HPLC quantification, chemical and cellular antioxidant assays, 16S rRNA gene sequencing, bacterial isolation, and treatment experiments across five tissues. Among all the metabolites determined by HPLC, 13 were quantified using authentic standards and 24 were semi-quantified as linarin equivalents. Leaves, stems, and fruits accumulated abundant flavone glycosides, with phloretin-3′,5′-di-C-glucoside reaching 1823.19 ± 278.71 μg/g FW in leaves and 847.11 ± 27.28 μg/g FW in fruits, while roots and seeds showed distinct coumarin- and furanocoumarin-rich profiles, respectively. Leaf extracts showed the strongest chemical and cellular antioxidant activities, followed by fruit extracts. Isoorientin 2″-O-rhamnoside and diosmin exhibited strong chemical antioxidant capacity, and several flavone glycosides showed protective effects in cellular assays. Endophytic bacterial communities differed markedly among tissues, with fruit harboring a distinct community dominated by Weissella and Pantoea, which accounted for 65.64% and 19.13% of the relative abundance, respectively. Among two culturable fruit-associated isolates, Weissella sp. SJG-1 treatment was associated with 32.92–104.45% increases in six HPLC-quantified metabolites and higher antioxidant activity of fruit extracts. These findings highlight minicitrus as a source of bioactive metabolites and suggest a potential association between fruit-associated bacteria and fruit functional properties. Full article

Kalanchoe delagoensis is a Crassulacean acid metabolism (CAM) succulent plant native to Madagascar that has spread as an aggressive invader across subtropical and Mediterranean regions. Research on this species has focused almost entirely on its cardiotoxic bufadienolides, leaving its phenolic fraction largely unexplored. Here, we profiled the phenolics of dried K. delagoensis leaves by reversed-phase liquid chromatography coupled with UV-Vis detection and electrospray ionisation high-resolution mass spectrometry (RPLC-ESI-HRMS). Two flavonoid diglycosides produced the most intense signals and were tentatively annotated by MS/MS analyses as quercetin and kaempferol deoxyhexoside-pentosides; the former shares the formula and fragmentation of quercetin 3-O-arabinopyranosyl-rhamnopyranoside (QAR), the chemical marker of the congeneric K. pinnata. The remaining MS/MS supported features form a single combinatorial family of flavonol glycosides on kaempferol, quercetin, and myricetin cores decorated with pentose, deoxyhexose, hexose, and hexuronic-acid units. Minor flavonol glycosides and mono- and digalloyl hexoses were also detected, and a malvidin-hexoside was putatively identified as a candidate contributor to the reddish leaf tint of the more sun-exposed leaves, supported by its absorption maximum at 520 nm and characteristic MS/MS spectrum. Folin-reactive compounds reached 47.0 ± 0.8 mg gallic acid equivalents per gram of dry leaf. These results extend the chemistry of K. delagoensis well beyond its bufadienolides, within the limits of a single clonal genotype and a single harvest. Full article

Indole alkaloids are major bioactive compounds in Strobilanthes cusia. Although methyl jasmonate treatment can promote the accumulation of indole alkaloids in S. cusia, the transcriptional regulation remains unclear. In this study, the jasmonic acid responsive bHLH transcription factor ScMYC2 was cloned and characterized. It encodes a protein containing 477 amino acids, which features typical bHLH-MYC_N and HLH domains. ScMYC2 localized in the nucleus, showed tissue specific expression consistent with indole alkaloid accumulation, and was significantly induced by methyl jasmonate. Furthermore, the coding sequence of ScMYC2 was cloned into an expression vector and overexpressed in Arabidopsis thaliana via Agrobacterium-mediated transformation technology. Alkaloid metabolic analysis was performed on three homozygous A. thaliana lines (ScMYC2-OE1, OE2, OE3) stably transformed using UPLC-MS/MS. The results indicated that, compared with wild-type, 14 differential metabolites were detected in the A. thaliana lines overexpressing ScMYC2. Among them, the content of indole increased to 2.83-fold that of the wild-type, while the indole glycoside component indole-3-cyano-6-O-glucoside increased to 2.35-fold. Yeast two-hybrid screening identified 35 ScMYC2-interacting proteins, including UDP-glycosyltransferase, TOPLESS-related proteins, and E3 ubiquitin-protein ligase. BiFC assays further confirmed the in vivo interaction between ScMYC2 and ScUGT1. These findings suggest that ScMYC2 is associated with methyl jasmonate-responsive regulation of indole alkaloid biosynthesis and provides genetic resources for the cultivation of S. cusia with high medicinal value. Full article

Moss-dominated biological soil crusts are associated with carbon and nitrogen cycling in dryland and alpine sandy ecosystems, but functional differentiation among closely related moss species and their mixed assemblages remains unclear. We used shotgun metagenomic sequencing to compare below-crust soil associated with Didymodon constrictus (Mitt.) K. Saito (D. constrictus) crusts (mossC), ferrugineus (Schimp. ex Besch.) M.O. Hill (D. ferrugineus) crusts (mossF), and visually co-dominated mixed crusts (mossM) in the Gonghe Basin on the northeastern Qinghai–Tibet Plateau. Fifteen spatially separated quadrats per category were pooled into three composite biological replicates (effective n = 3). KEGG, CAZy, and targeted carbon- and nitrogen-cycling annotations showed category-associated differences in relative gene representation. MossC had greater mean representation of glycoside hydrolases and several complex-carbon-processing functions, mossF had greater representation of nitrogen-assimilation and acetate-related functions, and mossM had greater representation of selected carbon-degradation, nitrogen-mineralization, and dissimilatory-nitrate-reduction functions. The full RDA model explained 58.4% of functional variation (adjusted R² = 0.334; exact permutation p = 0.028340), with single-variable associations retained for total carbon and soil water content. Genus-level taxonomic dissimilarity correlated with KEGG, CAZy, carbon-cycling, and nitrogen-cycling dissimilarities after FDR correction. MossM showed both positive and negative descriptive deviations from the approximate unweighted midpoint of mossC and mossF, but no inferential test was applied to these deviations. The small number of composite replicates, visually estimated moss proportions, edaphic confounding, and absence of activity measurements limit causal and confirmatory interpretation. Full article

Rhododendron yedoense var. poukhanense is an important medicinal plant, but still little is known about how its bioactive flavonoids are made in different organs. Comprehensive metabolomic profiling was performed using ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS), coupled with reference-based RNA sequencing (RNA-seq), to analyze root, stem, and leaf tissues from three independent biological replicates. Subsequently, we performed two-way orthogonal partial least squares (O2PLS) regression, canonical correlation analysis (CCA), and Pearson correlation analyses. A total of 2182 metabolites were detected. Among these, 1116, 896, and 1264 metabolites exhibited differential accumulation across the three pairwise comparisons. Concurrently, 9836, 5457, and 8007 genes were differentially expressed across the three pairwise comparisons, yielding a total of 13,019 unique differentially expressed genes (DEGs). The bifunctional flavanone 3-hydroxylase/flavonol synthase (F3H/FLS) enzyme exhibited the highest expression level in roots, consistent with root-preferential biosynthesis of flavonoid skeletons and the subsequent accumulation of flavonol glycosides in this tissue. In contrast, dihydroflavonol 4-reductase (DFR) exhibited predominant activity in roots, consistent with its role in farrerol biosynthesis. The multi-omics integration model demonstrated excellent goodness-of-fit to the experimental data. Canonical correlation analysis (CCA) further revealed a robust positive association between dihydrokaempferol accumulation and kaempferol biosynthesis. These findings collectively support flavanone 3-hydroxylase (F3H) as a candidate regulatory node governing organ-specific flavonoid partitioning. However, functional validation is required to substantiate this inference. Full article

The extracts of the Alcea rosea L. flowers are known to show various kinds of biomedical activities and are widely used as food and cosmetic ingredients. Unfortunately, no comprehensive analysis aimed at the identification of the most abundant flavonoid glycosides present in the extract of Alcea rosea L. has been made as of yet. In this paper we present the LC-MS identification of the most abundant flavonoid glycosides in the methanol and 75% ethanol extracts of Alcea rosea L. obtained from two manufacturers. The identification has been based on the detection of the respective parent and product ions as well as their relative abundances. The differences between identified isomeric and isobaric compounds have also been discussed. In all extracts a number of 3-O-anthocyanidin glycosides (anthocyanins) and 3-O-flavonol glycosides have been identified. Among the anthocyanins, the most abundant were malvidin glycosides, e.g., malvidin 3-O-(6″-[E]-p-coumaroyl)glucoside; among the flavonol glycosides, the most abundant were kaempferol glycosides, e.g., tiliroside. Full article

Alzheimer’s disease (AD) is characterized by the excessive production and aggregation of β-amyloid (Aβ), which serves as key pathological features of disease progression. Although Humulus japonicus Siebold & Zucc. is a traditional medicinal plant rich in flavonoids and phenolic compounds, its specific anti-amyloidogenic constituents remain to be fully elucidated. This study evaluated the effects of the aerial parts of H. japonicus on Aβ production and aggregation, and identified its active constituents through bioassay-guided isolation. The 80% ethanolic extract of H. japonicus aerial parts significantly reduced soluble amyloid precursor protein β (sAPPβ) levels and BACE1 protein expression in Chinese Hamster Ovary cells stably expressing amyloid precursor protein (APP-CHO cells). Furthermore, it inhibited Aβ aggregation and promoted fibril disaggregation in Thioflavin T assays. Among the solvent-partitioned fractions, the ethyl acetate fraction exhibited the most potent anti-amyloidogenic activity and was subjected to chromatographic isolation, leading to the characterization of fourteen compounds (7 flavonoids, 2 phenolic acids, 2 sesquiterpenoids, and 3 phenylpropanoids). Specifically, luteolin 7-O-β-d-glucopyranoside (2), orientin (3), vitexin (5), vomifoliol (8), abscisic acid (9), and p-hydroxyphenylethyl-p-coumarate (12) significantly decreased sAPPβ levels, as well as BACE1 and Presenilin-1 protein expression. Meanwhile, luteolin 7-O-β-d-glucopyranoside (2), orientin (3), vitexin (5), and quercetin 7-O-β-d-glucopyranoside trimer (7) effectively reduced Aβ aggregation and enhanced the disaggregation of pre-formed Aβ aggregates. Collectively, luteolin 7-O-β-d-glucopyranoside (2), orientin (3), and vitexin (5) exhibited the most consistent dual inhibitory effects on both Aβ production and aggregation, supporting their selection as key marker compounds. Among the extracts prepared with varying ethanol concentrations, the 100% ethanol extract yielded the highest levels of these flavonoid glycosides and exhibited the strongest anti-amyloidogenic activity. These findings suggest that the aerial parts of H. japonicus and its constituent flavonoid glycosides represent promising natural resources for modulating amyloidogenic APP processing and Aβ aggregation in AD. Full article

Variegated sweetpotato leaves are of interest for ornamental and functional-food applications, yet the molecular and metabolic basis of their coloration remains poorly understood. A sweetpotato accession with variegated leaves was found incidentally in the field, with rare and vivid Pink-purple coloration in the apical leaves. To investigate the mechanism underlying this interesting phenotype, this material was maintained in the laboratory, and variegated and normal green leaves from this plant were analysed by transcriptome profiling, miRNA sequencing and metabolome analysis. By integrating differential mRNA expression analysis, target-gene prediction, miRNA sequencing and metabolite identification, we screened candidate miRNA-mRNA modules associated with this leaf-colour formation. Using FDR < 0.05 and |log₂FC| ≥ 1 as thresholds, 49 differentially expressed miRNAs and 692 regulatory relationships involving differentially expressed target mRNAs associated with these miRNAs were identified. Functional annotation indicated that the iba_638_x1- G10133|TU16655 and iba_730_x1- G3592|TU5891 modules were associated with anthocyanin/flavonoid biosynthesis and vacuolar transport, respectively. Metabolome analysis showed that the total relative abundance of flavonoids in Pink leaves was 2.98-fold higher than that in CK, and 28 flavonoids accumulated to more than 2-fold higher levels in Pink than in CK. The differentially accumulated metabolites mainly included anthocyanin glycosides, such as petunidin-3-O-glucoside, delphinidin-3-O-glucoside and cyanidin-3,5-O-diglucoside, and flavonol glycosides, such as quercetin-3-O-sophoroside, isorhamnetin-3-O-glucoside and kaempferol-3-O-sophoroside-7-O-glucoside. These results indicate that the variegated phenotype of Pink leaves is mainly associated with differential accumulation of anthocyanin glycosides and flavonol glycosides. Full article