Synergistic actions of three MYB transcription factors underpins the high accumulation of myricetin in Morella rubra.

Flavonols are health-promoting bioactive compounds important for human nutrition, health, and plant defense. The transcriptional regulation of kaempferol and quercetin biosynthesis has been studied extensively, while little is known about the regulatory mechanisms underlying myricetin biosynthesis, which has strong antioxidant, anticancer, antidiabetic, and anti-inflammatory activities. In this study, the flavonol-specific MrMYB12 in Morella rubra preferred activating the promoter of flavonol synthase 2 (MrFLS2) (6.4-fold) rather than MrFLS1 (1.4-fold) and upregulated quercetin biosynthesis. Furthermore, two SG44 R2R3-MYB members, MrMYB5 and MrMYB5L, were identified by yeast one-hybrid library screening using the promoter of flavonoid 3',5'-hydroxylase (MrF3'5'H), and transcript levels of these R2R3-MYBs were correlated with accumulation of myricetin derivatives during leaf development. Dual-luciferase and electrophoretic mobility shift assays demonstrated that both MrMYB5 and MrMYB5L could bind directly to MYB recognition sequence elements in promoters of MrF3'5'H or MrFLS1 and activate their expression. Protein-protein interactions of MrMYB5 or MrMYB5L with MrbHLH2 were confirmed by yeast two-hybrid and bimolecular fluorescence complementation assays. MrMYB5L-MrbHLH2 showed much higher synergistic activation of MrF3'5'H or MrFLS1 promoters than MrMYB5-MrbHLH2. Studies with Arabidopsis thaliana homologs AtMYB5 and AtTT8 indicated that similar synergistic regulatory effects occur with promoters of MrF3'5'H or MrFLS1. Transient overexpression of MrMYB5L-MrbHLH2 in Nicotiana benthamiana induced a higher accumulation of myricetin derivatives (57.70 µg g-1 FW) than MrMYB5-MrbHLH2 (7.43 µg g-1 FW) when MrMYB12 was coexpressed with them. This study reveals a novel transcriptional mechanism regulating myricetin biosynthesis with the potential use for future metabolic engineering of health-promoting flavonols.

A multifunctional true caffeoyl coenzyme A O-methyltransferase enzyme participates in the biosynthesis of polymethoxylated flavones in citrus.

Polymethoxylated flavones (PMFs) have received extensive attention due to their abundant bioactivities. Citrus peels specifically accumulate abundant PMFs, and methylation modification is a key step in PMF biosynthesis; however, the function of reported O-methyltransferase (OMT) in citrus is insufficient to elucidate the complete methylation process of PMFs. In this study, we analyzed the accumulation pattern of PMFs in the flavedo of the sweet orange (Citrus sinensis) cultivar "Bingtangcheng" at different developmental stages. We found that accumulation of PMFs was completed at the early stage of fruit development (60-d after flowering). Furthermore, we characterized a true caffeoyl-CoA O-methyltransferase (named CsCCoAOMT1) from C. sinensis. Functional analysis in vitro showed that CsCCoAOMT1 preferred flavonoids to caffeoyl-CoA and esculetin. This enzyme efficiently methylated the 6-, 7- 8-, and 3'-OH of a wide array of flavonoids with vicinal hydroxyl groups with a strong preference for quercetin (flavonol) and flavones. The transient overexpression and virus-induced gene silencing experiments verified that CsCCoAOMT1 could promote the accumulation of PMFs in citrus. These results reveal the function of true CCoAOMTs and indicate that CsCCoAOMT1 is a highly efficient multifunctional O-methyltransferase involved in the biosynthesis of PMFs in citrus.

Integrated Metabolomic-Transcriptomic Analyses of Flavonoid Accumulation in Citrus Fruit under Exogenous Melatonin Treatment.

The flavonoids in citrus fruits are crucial physiological regulators and natural bioactive products of high pharmaceutical value. Melatonin is a pleiotropic hormone that can regulate plant morphogenesis and stress resistance and alter the accumulation of flavonoids in these processes. However, the direct effect of melatonin on citrus flavonoids remains unclear. In this study, nontargeted metabolomics and transcriptomics were utilized to reveal how exogenous melatonin affects flavonoid biosynthesis in "Bingtangcheng" citrus fruits. The melatonin treatment at 0.1 mmol L-1 significantly increased the contents of seven polymethoxylated flavones (PMFs) and up-regulated a series of flavonoid pathway genes, including 4CL (4-coumaroyl CoA ligase), FNS (flavone synthase), and FHs (flavonoid hydroxylases). Meanwhile, CHS (chalcone synthase) was down-regulated, causing a decrease in the content of most flavonoid glycosides. Pearson correlation analysis obtained 21 transcription factors co-expressed with differentially accumulated flavonoids, among which the AP2/EREBP members were the most numerous. Additionally, circadian rhythm and photosynthesis pathways were enriched in the DEG (differentially expressed gene) analysis, suggesting that melatonin might also mediate changes in the flavonoid biosynthesis pathway by affecting the fruit's circadian rhythm. These results provide valuable information for further exploration of the molecular mechanisms through which melatonin regulates citrus fruit metabolism.

Nobiletin as a chemopreventive natural product against cancer, a comprehensive review.

As a leading cause of death, second only to heart disease, cancer has always been one of the burning topics in medical research. When targeting multiple signal pathways in tumorigenesis chemoprevention, using natural or synthetic anti-cancer drugs is a vital strategy to reduce cancer damage. However, toxic effects, multidrug resistance (MDR) as well as cancer stem cells (CSCs) all prominently limited the clinical application of conventional anticancer drugs. With low side effects, strong biological activity, unique mechanism, and wide range of targets, natural products derived from plants are considered significant sources for new drug development. Nobiletin is one of the most attractive compounds, a unique flavonoid primarily isolated from the peel of citrus fruits. Numerous studies in vitro and in vivo have suggested that nobiletin and its derivatives possess the eminent potential to become effective cancer chemoprevention agents through various cellular and molecular levels. This article aims to comprehensively review the anticancer efficacy and specific mechanisms of nobiletin, enhancing our understanding of its chemoprevention properties and providing the latest research findings. At the end of this review, we also give some discussion and future perspectives regarding the challenges and opportunities in nobiletin efficient exploitation.

Genome-wide identification and expression analysis of MATE gene family in citrus fruit (Citrus clementina).

Multidrug and toxic compound extrusion (MATE) proteins are a class of secondary active multidrug transporters. In plants, this family has significantly expanded and is involved in numerous plant physiological processes. Although MATE proteins have been identified in an increasing number of species, the understanding about this family in citrus remains unclear. In this study, a total of 69 MATE transporters were identified in the citrus genome (Citrus clementina) and classified into four groups by phylogenetic analysis. Tandem and segmental duplication events were the main causes of the citrus MATE family expansion. RNA-seq and qRT-PCR analyses were performed during citrus fruit development. The results indicated that CitMATE genes showed specific expression profiles in citrus peels and flesh at different developmental stages. Combined with the variations of flavonoids and citrate levels in citrus fruit, we suggested that CitMATE43 and CitMATE66 may be involved in the transport process of flavonoids and citrate in citrus fruit, respectively. In addition, two flavonoids positive regulators, CitERF32 and CitERF33, both directly bind to and activated the CitMATE43 promoter. Our results provide comprehensive information on citrus MATE genes and valuable understanding for the flavonoids and citrate metabolism in citrus fruit.

Preparation of Neohesperidin-Taro Starch Complex as a Novel Approach to Modulate the Physicochemical Properties, Structure and In Vitro Digestibility.

Neohesperidin (NH), a natural flavonoid, exerts multiple actions, such as antioxidant, antiviral, antiallergic, vasoprotective, anticarcinogenic and anti-inflammatory effects, as well as inhibition of tumor progression. In this study, the NH-taro starch complex is prepared, and the effects of NH complexation on the physicochemical properties, structure and in vitro digestibility of taro starch (TS) are investigated. Results showed that NH complexation significantly affected starch gelatinization temperatures and reduced its enthalpy value (ΔH). The addition of NH increased the viscosity and thickening of taro starch, facilitating shearing and thinning. NH binds to TS via hydrogen bonds and promotes the formation of certain crystalline regions in taro starch. SEM images revealed that the surface of NH-TS complexes became looser with the increasing addition of NH. The digestibility results demonstrated that the increase in NH (from 0.1% to 1.1%, weight based on starch) could raise RS (resistant starch) from 21.66% to 27.75% and reduce RDS (rapidly digestible starch) from 33.51% to 26.76% in taro starch. Our work provided a theoretical reference for the NH-taro starch complex's modification of physicochemical properties and in vitro digestibility with potential in food and non-food applications.

Elucidation of myricetin biosynthesis in Morella rubra of the Myricaceae.

Flavonols are health-promoting bioactive compounds important for plant defense and human nutrition. Quercetin (Q) and kaempferol (K) biosynthesis have been studied extensively while little is known about myricetin (M) biosynthesis. The roles of flavonol synthases (FLSs) and flavonoid 3',5'-hydroxylase (F3'5'H) in M biosynthesis in Morella rubra, a member of the Myricaceae rich in M-based flavonols, were investigated. The level of MrFLS transcripts alone did not correlate well with the accumulation of M-based flavonols. However, combined transcript data for MrFLS1 and MrF3'5'H showed a good correlation with the accumulation of M-based flavonols in different tissues of M. rubra. Recombinant MrFLS1 and MrFLS2 proteins showed strong activity with dihydroquercetin (DHQ), dihydrokaempferol (DHK), and dihydromyricetin (DHM) as substrates, while recombinant MrF3'5'H protein preferred converting K to M, amongst a range of substrates. Tobacco (Nicotiana tabacum) overexpressing 35S::MrFLSs produced elevated levels of K-based and Q-based flavonols without affecting M-based flavonol levels, while tobacco overexpressing 35S::MrF3'5'H accumulated significantly higher levels of M-based flavonols. We conclude that M accumulation in M. rubra is affected by gene expression and enzyme specificity of FLS and F3'5'H as well as substrate availability. In the metabolic grid of flavonol biosynthesis, the strong activity of MrF3'5'H with K as substrate additionally promotes metabolic flux towards M in M. rubra.

Unravelling the consecutive glycosylation and methylation of flavonols in peach in response to UV-B irradiation.

Flavonol glycosides are bioactive compounds important for plant defence and human nutrition. Glycosylation and methylation play an important role in enriching the diversity of flavonols in response to the environment. Peach flowers and fruit are rich in flavonol diglycosides such as isorhamnetin 3-O-rutinoside (I3Rut), kaempferol 3-O-rutinoside and quercetin 3-O-rutinoside, and flavonol monoglycosides such as I 3-O-glucoside and Q 3-O-galactoside. UV-B irradiation of fruit significantly induced accumulation of all these flavonol glycosides. Candidate biosynthetic genes induced by UV-B were identified by genome homology searches and the in vitro catalytic activities of purified recombinant proteins determined. PpUGT78T3 and PpUGT78A2 were identified as flavonol 3-O-glucosyltransferase and 3-O-galactosyltransferase, respectively. PpUGT91AK6 was identified as flavonol 1,6-rhamnosyl trasferase catalysing the formation of flavonol rutinosides and PpFOMT1 was identified as a flavonol O-methyltransferase that methylated Q at the 3'-OH-OH to form isorhamnetin derivatives. Transient expression in Nicotiana benthamiana confirmed the specificity of PpUGT78T3 as a flavonol 3-O-glucosyltransferase, PpUGT78A2 as a 3-O-galactosyltransferase, PpUGT91AK6 as a 1,6-rhamnosyltrasferase and PpFOMT1 as an O-methyltransferase. This study provides new insights into the mechanisms of glycosylation and methylation of flavonols, especially the formation of flavonol diglycosides such as I3Rut, and will also be useful for future potential metabolic engineering of complex flavonols.

Citrus flavonoids and their antioxidant evaluation.

The antioxidant ability is the link and bridge connecting a variety of biological activities. Citrus flavonoids play an essential role in regulating oxidative stress and are an important source of daily intake of antioxidant supplements. Many studies have shown that citrus flavonoids promote health through antioxidation. In this review, the biosynthesis, composition and distribution of citrus flavonoids were concluded. The detection methods of antioxidant capacity of citrus flavonoids were divided into four categories: chemical, cellular, animal and clinical antioxidant capacity evaluation systems. The modeling methods, applicable scenarios, and their relative merits were compared based on these four systems. The antioxidant functions of citrus flavonoids under different evaluation systems were also discussed, especially the regulation of the Nrf2-antioxidases pathway. Some shortcomings in the current research were pointed out, and some suggestions for progress were put forward.

Three AP2/ERF family members modulate flavonoid synthesis by regulating type IV chalcone isomerase in citrus.

Flavanones and flavones are excellent source of bioactive compounds but the molecular basis of their highly efficient production remains elusive. Chalcone isomerase (CHI) family proteins play essential roles in flavonoid biosynthesis but little are known about the transcription factors controlling their gene expression. Here, we identified a type IV CHI (designated as CitCHIL1) from citrus which enhances the accumulation of citrus flavanones and flavones (CFLs). CitCHIL1 participates in a CFL biosynthetic metabolon and assists the cyclization of naringenin chalcone to (2S)-naringenin, which leads to the efficient influx of substrates to chalcone synthase (CHS) and improves the catalytic efficiency of CHS. Overexpressing CitCHIL1 in Citrus and Arabidopsis significantly increased flavonoid content and RNA interference-induced silencing of CitCHIL1 in citrus led to a 43% reduction in CFL content. Three AP2/ERF transcription factors were identified as positive regulators of the CitCHIL1 expression. Of these, two dehydration-responsive element binding (DREB) proteins, CitERF32 and CitERF33, activated the transcription by directly binding to the CGCCGC motif in the promoter, while CitRAV1 (RAV: related to ABI3/VP1) formed a transcription complex with CitERF33 that strongly enhanced the activation efficiency and flavonoid accumulation. These results not only illustrate the specific function that CitCHIL1 executes in CFL biosynthesis but also reveal a new DREB-RAV transcriptional complex regulating flavonoid production.

Characterization of a caffeoyl-CoA O-methyltransferase-like enzyme involved in biosynthesis of polymethoxylated flavones in Citrus reticulata.

Polymethoxylated flavones (PMFs), which accumulate exclusively in fruit peel of citrus, play important physiological and pharmacological roles but the genetic basis for the methylation of flavonoids has not been fully elucidated in citrus. Here we characterize a caffeoyl-CoA O-methyltransferase-like enzyme, designated CrOMT1. The expression pattern of CrOMT1 was highly correlated with the concentration of the three major PMFs in two different citrus fruit tissues during fruit maturation. Exposure of fruit to UV-B radiation sharply increased the level of CrOMT1 transcripts and also led to the accumulation of three PMFs. The potential role of CrOMT1 was studied by testing the catalytic activity of recombinant CrOMT1 with numerous possible substrates in vitro. The enzyme could most efficiently methylate flavones with neighboring hydroxy moieties, with high catalytic efficiencies found with 6-OH- and 8-OH-containing compounds, preferences that correspond precisely with the essential methylation sites involved in the synthesis of the three naturally occurring PMFs in Citrus reticulata. This indicates that CrOMT1 is capable of in vitro methylation reactions required to synthesize PMFs in vivo. Furthermore, transient overexpression of CrOMT1 increased levels of the three major PMFs in fruit, indicating that CrOMT1 is likely to play an essential role in the biosynthesis of PMFs in citrus.

Characterization of a Flavonoid 3'/5'/7-O-Methyltransferase from Citrus reticulata and Evaluation of the In Vitro Cytotoxicity of Its Methylated Products.

O-methylation of flavonoids is an important modification reaction that occurs in plants. O-methylation contributes to the structural diversity of flavonoids, which have several biological and pharmacological functions. In this study, an O-methyltransferase gene (CrOMT2) was isolated from the fruit peel of Citrus reticulata, which encoding a multifunctional O-methyltransferase and could effectively catalyze the methylation of 3'-, 5'-, and 7-OH of flavonoids with vicinal hydroxyl substitutions. Substrate preference assays indicated that this recombinant enzyme favored polymethoxylated flavones (PMF)-type substrates in vitro, thereby providing biochemical evidence for the potential role of the enzyme in plants. Additionally, the cytotoxicity of the methylated products from the enzymatic catalytic reaction was evaluated in vitro using human gastric cell lines SGC-7901 and BGC-823. The results showed that the in vitro cytotoxicity of the flavonoids with the unsaturated C2-C3 bond was increased after being methylated at position 3'. These combined results provide biochemical insight regarding CrOMT2 in vitro and indicate the in vitro cytotoxicity of the products methylated by its catalytic reaction.

Rapid Classification and Quantification of Camellia (Camellia oleifera Abel.) Oil Blended with Rapeseed Oil Using FTIR-ATR Spectroscopy.

Currently, the authentication of camellia oil (CAO) has become very important due to the possible adulteration of CAO with cheaper vegetable oils such as rapeseed oil (RSO). Therefore, we report a Fourier transform infrared (FTIR) spectroscopic method for detecting the authenticity of CAO and quantifying the blended levels of RSO. In this study, two characteristic spectral bands (1119 cm-1 and 1096 cm-1) were selected and used for monitoring the purity of CAO. In combination with principal component analysis (PCA), linear discriminant analysis (LDA), and partial least squares regression (PLSR) analysis, qualitative and quantitative methods for the detection of camellia oil adulteration were proposed. The results showed that the calculated I1119/I1096 intensity ratio facilitated an initial check for pure CAO and six other edible oils. PCA was used on the optimized spectral region of 1800-650 cm-1. We observed the classification of CAO and RSO as well as discrimination of CAO with RSO adulterants. LDA was utilized to classify CAO from RSO. We could differentiate and classify RSO adulterants up to 1% v/v. In the quantitative PLSR models, the plots of actual values versus predicted values exhibited high linearity. Root mean square error of calibration (RMSEC) and root mean square error of cross validation (RMSECV) values of the PLSR models were 1.4518%-3.3164% v/v and 1.7196%-3.8136% v/v, respectively. This method was successfully applied in the classification and quantification of CAO adulteration with RSO.

Transcriptomic Analyses of Root Restriction Effects on Phytohormone Content and Signal Transduction during Grape Berry Development and Ripening.

Phytohormones strongly influence growth, development and nutritional quality of agricultural products by modulating molecular and biochemical changes. The purpose of the present study was to investigate the influence of root restriction (RR) treatment on the dynamic changes of main phytohormones during the berry development and ripening of "Summer Black" early ripening seedless grape (Vitis vinifera × V. labrusca), and to analyze the changes in the biosynthesis and signal transduction pathways of phytohormones by transcriptomics. Enzyme-linked immunosorbent assay (ELISA) and Ultra Performance Liquid Chromatography-High Resolution Mass Spectrometry (UPLC-HRMS) were used to quantify the phytohormone levels, and RNA-Seq was used to analyze the transcript abundance. The results showed that 23 transcripts involved in the phytohormone biosynthesis and 34 transcripts involved in the signal transduction pathways were significantly changed by RR treatment. RR also increased abscisic acid, brassinosteroid, ethylene, jasmonic acid and salicylic acid levels, while decreasing auxin, cytokinin, and gibberellin contents. The results of the present study suggest that RR treatment can accelerate the grape ripening process, and specific candidate genes were identified for further functional analysis.

Neohesperidin Exerts Lipid-Regulating Effects in vitro and in vivo via Fibroblast Growth Factor 21 and AMP-Activated Protein Kinase/Sirtuin Type 1/Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1α Signaling Axis.

The purpose of this study is to prove the lipid-regulating effects of neohesperidin (NHP) and explore the potential mechanisms related to fibroblast growth factor 21 (FGF21) and AMP-activated protein kinase (AMPK). Free fatty acids (FFAs)-induced lipid-accumulated HepG2 cells, acutely egg yolk-induced dyslipidemia and chronically diet-induced obese (DIO) model mice were treated with NHP. Biochemical analyses were carried out to determine the lipid profiles. Western blotting and real-time PCR were employed to analyze FGF21, AMPK and the related proteins or mRNA expressions. Body weight and food intake were measured in DIO mice. siRNA or inhibitors of FGF21 or AMPK were utilized in further study. NHP showed potent hypolipidemic effect in HepG2 cells loaded with FFAs and reversed the pathological changes of lipid in the acute or chronic dyslipidemia mouse model. It obviously improved the lipid profiles in plasma, liver and gastrocnemius muscles in DIO mice, and led to a significant body weight loss. Simultaneously, FGF21 protein expression or secretion, and AMPK/sirtuin type 1 (SIRT1)/peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α) axis or related molecules, was improved by NHP in HepG2 cells and/or DIO mice. Furthermore, the siRNA or inhibitor targeting FGF21 or AMPK rejected the triglyceride-lowering effect of NHP. In conclusion, NHP regulates lipid metabolism in vivo and in vitro via FGF21 and AMPK/SIRT1/PGC-1α signaling axis.

Characterization and Purification of Bergamottin from Citrus grandis (L.) Osbeck cv. Yongjiazaoxiangyou and Its Antiproliferative Activity and Effect on Glucose Consumption in HepG2 cells.

Bergamottin is a natural furanocoumarin compound with weak polarity. Characterization and quantification of bergamottin were carried out in different fruit tissues of various citrus cultivars. Among the four citrus tissues tested, i.e., flavedo, albedo, segment membrane (SM), and juice sacs (JS) in eight citrus cultivars, the highest bergamottin content was found in the flavedo of Citrus grandis (L.) Osbeck cv. Yongjiazaoxiangyou (YJZXY, 666.54 µg·g-1 DW). A combination of silica gel column chromatography and high-speed counter-current chromatography (HSCCC) was established to efficiently purify bergamottin from the flavedo of YJZXY. Bergamottin showed significant antiproliferative activity on three cancer cell lines, i.e., human liver cancer HepG2, promyelocytic leukemia HL-60, and gastric cancer BGC-823 cells, which showed a marked inhibition effect on these cell lines in a dose-dependent manner. In addition, bergamottin significantly increased glucose consumption in HepG2 cells also in a dose-dependent manner, which is the first report of its potential in anti-diabetes applications.

Antioxidant Capacity, Anticancer Ability and Flavonoids Composition of 35 Citrus (Citrus reticulata Blanco) Varieties.

Citrus (Citrus reticulate Blanco) is one of the most commonly consumed and widely distributed fruit in the world, which is possessing extensive bioactivities. Present study aimed to fully understand the flavonoids compositions, antioxidant capacities and in vitro anticancer abilities of different citrus resources. Citrus fruits of 35 varieties belonging to 5 types (pummelos, oranges, tangerines, mandarins and hybrids) were collected. Combining li quid chromatography combined with electrospray ionization mass spectrometry (LC-ESI-MS/MS) and ultra-performance liquid chromatography combined with diode array detector (UPLC-DAD), a total of 39 flavonoid compounds were identified, including 4 flavones, 9 flavanones and 26 polymethoxylated flavonoids (PMFs). Each citrus fruit was examined and compared by 4 parts, flavedo, albedo, segment membrane and juice sacs. The juice sacs had the lowest total phenolics, following by the segment membrane. Four antioxidant traits including 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, ferric reducing antioxidant power (FRAP), oxygen radical absorbance capacity (ORAC) and cupric reducing antioxidant capacity (CUPRAC) were applied for the antioxidant capacities evaluation. Three gastric cancer cell lines, SGC-7901, BGC-823 and AGS were applied for the cytotoxicity evaluation. According to the results of correlation analysis, phenolics compounds might be the main contributor to the antioxidant activity of citrus extracts, while PMFs existing only in the flavedo might be closely related to the gastric cancer cell line cytotoxicity of citrus extracts. The results of present study might provide a theoretical guidance for the utilization of citrus resources.

Comparative Transcriptomic Analysis of Grape Berry in Response to Root Restriction during Developmental Stages.

Root restriction improved berry quality by being involved in diverse aspects of grapevine life. However, the molecular mechanism driving this process is not understood very well. In this study, the 'Summer Black' grape berry (Vitis vinifera × V. labrusca) under root restriction was investigated, which showed an increase of total soluble solids (TSS), color index of red grapes (CIRG) value, anthocyanins accumulation, total phenolics and total procyanidins contents during berry development compared with those in control berries. The transcriptomic changes induced by root restriction in 'Summer Black' grape over the course of berry development were analyzed by RNA-Seq method. A total of 29,971 genes were generated in 'Summer Black' grape berry during development, among which, 1606 genes were significantly responded to root restriction. Furthermore, 1264, 313, 141, 246 and 19 sequences were significantly changed at S1, S2, S3, S4 and S5 sample points, respectively. The gene (VIT_04s0023g02290) predicted as a salicylate O-methyltransferase was differentially expressed in all developmental stages. Gene Ontology (GO) enrichment showed that response to organic nitrogen, response to endogenous stimulus, flavonoid metabolic process, phenylpropanoid biosynthetic process and cell wall macromolecule metabolic process were the main significant differential categories. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment revealed plant-pathogen interaction, plant hormone signal transduction, flavone and flavonol biosynthesis, flavonoid biosynthesis and glucosinolate biosynthesis were the main significant differential pathways. The results of the present study provided a genetic base for the understanding of grape berry fruit quality improvement under root restriction.

Anti-Obesity and Hypoglycemic Effects of Poncirus trifoliata L. Extracts in High-Fat Diet C57BL/6 Mice.

The present study investigated the possible anti-obesity and hypoglycemic effects of Poncirus trifoliata L. extracts. Mature fruit were divided into flavedo (PF) and juice sacs (PJ), and extracts from them were tested on C57BL/6 mice fed a high-fat diet (HFD) for thirteen weeks. Both fruit extracts (40 mg/kg body weight, respectively) showed anti-obesity and hypoglycemic effects. Consumption of PF and PJ extracts reduced body weight by 9.21% and 20.27%, respectively. Liver and adipose weights, fasting glucose, serum triglyceride (TG), and low density lipoprotein cholesterol (LDL-c) levels decreased significantly, while serum high density lipoprotein cholesterol (HDL-c) and oral glucose tolerance levels increased significantly in response to two fruit extracts. These effects were due in part to the modulation of serum insulin, leptin, and adiponectin. Furthermore, transcript levels of fatty acid synthase (FAS) and stearoyl-CoA desaturase 1 (SCD1) were reduced while those of carnitine palmitoyltransferase 1α (CPT1α) and insulin receptor substrate 2 (IRS2) were increased in the liver of C57BL/6 mice, which might be an important mechanism affecting lipid and glucose metabolism. Taken together, P. trifoliata fruit can be potentially used to prevent or treat obesity and associated metabolic disorders.

Purification of Flavonoids from Chinese Bayberry (Morella rubra Sieb. et Zucc.) Fruit Extracts and α-Glucosidase Inhibitory Activities of Different Fractionations.

Chinese bayberry (Morella rubra Sieb. et Zucc.) fruit have a diverse flavonoid composition responsible for the various medicinal activities, including anti-diabetes. In the present study, efficient simultaneous purification of four flavonoid glycosides, i.e., cyanidin-3-O-glucoside (1), myricetin-3-O-rhamnoside (2), quercetin-3-O-galactoside (3), quercetin-3-O-rhamnoside (4), from Chinese bayberry pulp was established by the combination of solid phase extract (SPE) by C18 Sep-Pak(®) cartridge column chromatography and semi-preparative HPLC (Prep-HPLC), which was followed by HPLC and LC-MS identification. The purified flavonoid glycosides, as well as different fractions of fruit extracts of six bayberry cultivars, were investigated for α-glucosidase inhibitory activities. The flavonol extracts (50% methanol elution fraction) of six cultivars showed strong α-glucosidase inhibitory activities (IC50 = 15.4-69.5 µg/mL), which were higher than that of positive control acarbose (IC50 = 383.2 µg/mL). Four purified compounds 1-4 exerted α-glucosidase inhibitory activities, with IC50 values of 1444.3 µg/mL, 418.8 µg/mL, 556.4 µg/mL, and 491.8 µg/mL, respectively. Such results may provide important evidence for the potential anti-diabetic activity of different cultivars of Chinese bayberry fruit and the possible bioactive compounds involved.