Dietary Flavonoid Glycosides Research Articles

Antiviral Significance of Isoquercetin (Quercetin-3-O-Glucoside) With Special Reference to its Anti-Coronaviral Potential

Isoquercetin, a naturally occurring dietary flavonoid glycoside, has shown many promising biological activities, such as antioxidant, anti-inflammatory, and anticancer. It also exhibits broad-spectrum antiviral activity, significantly reducing cell infection by influenza, Zika, Ebola, and dengue viruses, among others. Its beneficial effect for mitigating the disease of COVID-19 can be explained due to its inhibitory effects on several stages of the viral life cycle, from entry to replication and virus infectivity, and thus isoquercetin can be a promising anticoronaviral flavonoid to manage severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection and warrants further investigation into its potential use in clinical settings.

Didymin alleviates metabolic dysfunction-associated fatty liver disease (MAFLD) via the stimulation of Sirt1-mediated lipophagy and mitochondrial biogenesis

BackgroundMetabolic dysfunction-associated fatty liver disease (MAFLD) is one of the most prevalent metabolic syndromes worldwide. However, no approved pharmacological treatments are available for MAFLD. Chenpi, one kind of dried peel of citrus fruits, has traditionally been utilized as a medicinal herb for liver diseases. Didymin is a newly identified oral bioactive dietary flavonoid glycoside derived from Chenpi. In this study, we investigated the therapeutic potential of Didymin as an anti-MAFLD drug and elucidated its underlying mechanisms.MethodsHigh-fat diet (HFD)-induced MAFLD mice and alpha mouse liver 12 (AML12) cells were utilized to evaluate the effects and mechanisms of Didymin in the treatment of MAFLD. Liver weight, serum biochemical parameters, and liver morphology were examined to demonstrate the therapeutic efficacy of Didymin in MAFLD treatment. RNA-seq analysis was performed to identify potential pathways that could be affected by Didymin. The impact of Didymin on Sirt1 was corroborated through western blot, molecular docking analysis, microscale thermophoresis (MST), and deacetylase activity assay. Then, a Sirt1 inhibitor (EX-527) was utilized to confirm that Didymin alleviates MAFLD via Sirt1. Western blot and additional assays were used to investigate the underlying mechanisms.ResultsOur results suggested that Didymin may possess therapeutic potential against MAFLD in vitro and in vivo. By promoting Sirt1 expression as well as directly binding to and activating Sirt1, Didymin triggers downstream pathways that enhance mitochondrial biogenesis and function while reducing apoptosis and enhancing lipophagy.ConclusionsThese suggest that Didymin could be a promising medication for MAFLD treatment. Furthermore, its therapeutic effects are mediated by Sirt1.

Multiple bioactivities of choerospondin obtained from blossom of Citrus aurantium L. Var. Amara engl. And its anti-inflammation and anti-atherosclerosis action pathways

Nutritional intervention has been emerged as a popular strategy to improve quality of human life. Here we extracted and identified choerospondin (CHO), a dietary flavonoid glycoside from blossom of Citrus aurantium L. var. amara Engl. Different models were employed to screen the antioxidant, anti-tumor, anti-obesity, anti-inflammation and anti-atherosclerosis effects of CHO. The data showed that CHO displayed enhanced resistance to oxidative stress. Anti-proliferative effects against MCF-7, HepG2 and H1299 cells were also observed by CHO administration. CHO intervention potently inhibited 3 T3-L1 preadipocytes differentiation by suppressing transcription of some key adipogenic factors. Especially, CHO supplementation exhibited marked decreases of nitric oxide production in LPS-induced macrophages, significant reductions of cholesterol uptake and increases of cholesterol efflux in ox-LDL-loaded macrophages. These facts indicated that CHO showed greater anti-inflammation and anti-atherosclerosis potential. Futher, CHO markedly inhibited inflammatory responses through blunting mitogen-activated protein kinase phosphorylation and nuclear factor-kappa B activation. CHO application also reversed foam cell formation by regulating cluster of differentiation 36, phospholipid ATP-binding cassette transporter A1, phospholipid ATP-binding cassette transporter G1 and scavenger receptor class B type 1 expression. In conclusion, these data provided strong evidences that CHO possessed various pharmacological effects, including antioxidant, anti-tumor, anti-obesity and especially anti-inflammation and anti-atherosclerosis properties.

Microbial Metabolism of Naringin and the Impact on Antioxidant Capacity.

Naringin is a dietary flavonoid glycoside with broad bioactivities, and it has been found to undergo extensive microbial metabolism in human gut. Microbial metabolites are believed to play an important role in the overall bioactivity of naringin. However, knowledge is scarce about its microbial metabolism in laboratory rats, which are the most commonly used animal model for naringin-related biomedical studies. Herein, we profiled the microbial metabolism of naringin in rat by an in vitro anaerobic fermentation combined with LC-MS/MS methods. A total of 35 microbial metabolites were identified, and corresponding metabolic pathways were proposed. Naringin and its metabolites were further quantified in fermentation samples. Rhoifolin, neoeriocitrin, neohesperidin, naringenin, methylated naringin, and hydroxylated naringin were detected as the primary microbial metabolites. Moreover, antioxidant capacity assays suggested that fermentation-associated microbial metabolites exhibited higher antioxidant activity than original naringin. Obtained results contribute to a more comprehensive understanding of the microbial metabolism and antioxidant capacity of naringin.

A Rapid LC-MS/MS Method for Simultaneous Determination of Ten Flavonoid Metabolites of Naringin in Rat Urine and Its Application to an Excretion Study.

Naringin is a dietary flavonoid glycoside with multiple bioactivities. It has been involved in numerous metabolism and excretion studies, and its metabolic properties are clear. However, information concerning the excretion profile of its original metabolites are still scarce, and few methods for simultaneous determination of multiple original metabolites of naringin in biological samples have been reported so far. In this study, a rapid and sensitive method for simultaneous determination of ten flavonoid metabolites of naringin in rat urine was developed with an UHPLC-Q-Trap-MS/MS system. One-step protein precipitation method with acetonitrile was used to extract analytes. A rapid chromatographic separation within 11 min was performed on an ACQUITY UPLC® BEH C18 column (2.1 mm × 50 mm, 1.7 μm) using gradient elution with a mobile phase of water and methanol, both with 0.1% formic acid (v/v). MS/MS detection was conducted in negative ion mode and multiple reactions monitoring scanning mode. The analytical method was fully validated and successfully applied to monitor the excretion profiles of naringin in rat urine. Quantitative results revealed the visible individual difference and low urinary recovery of flavonoid metabolites in the excretion of naringin, which may be helpful for further study to understand the in vivo behavior and action mechanism of naringin.

Didymin by suppressing NF-κB activation prevents VEGF-induced angiogenesis in vitro and in vivo

Although didymin, a dietary flavonoid glycoside from citrus fruits, known to be a potent antioxidant with anti-cancer activities, its role in angiogenesis is not known. In this study, we examined the effect of didymin on VEGF-induced angiogenesis in vitro and in vivo models. Our results suggest that treatment of human umbilical vein endothelial cell (HUVECs) with didymin significantly prevented the VEGF-induced cell proliferation, migration, and invasion. Further, didymin significantly prevented the VEGF-induced endothelial tube formation in culture. Didymin also attenuated the VEGF-induced generation of ROS, activation of NF-κB and the expression of adhesion molecules such as VCAM-1, ICAM-1, and E-selectin in HUVECs. Further, didymin also prevented the VEGF-induced microvessel sprouting in ex vivo mouse aortic rings. Most importantly, didymin significantly prevented the invasion of endothelial cells and formation of blood capillary-like structures in Matrigel plug model of angiogenesis in mice. Thus, our results suggest a novel antiangiogenic efficacy of didymin in addition to its reported anti-cancer properties, which warrant further development of this agent for cancer therapy.

Recent Trends in Potential Therapeutic Applications of the Dietary Flavonoid Didymin

Didymin (isosakuranetin 7-O-rutinoside) is an orally bioactive dietary flavonoid glycoside first found in citrus fruits. Traditionally, this flavonoid has long been used in Asian countries as a dietary antioxidant. Recent studies have provided newer insights into this pleiotropic compound, which could regulate multiple biological activities of many important signaling molecules in health and disease. Emerging data also presented the potential therapeutic application of dietary flavonoid glycoside didymin against cancer, neurological diseases, liver diseases, cardiovascular diseases, and other diseases. In this review, we briefly introduce the source and extraction methods of didymin, and summarize its potential therapeutic application in the treatment of various diseases, with an emphasis on molecular targets and mechanism that contributes to the observed therapeutic effects. The dietary flavonoid didymin can be used to affect health and disease with multiple therapeutic targets, and it is anticipated that this review will stimulate the future development of this potential dietary medicine.

3,4-Dihydroxyphenylacetic acid is a predominant biologically-active catabolite of quercetin glycosides

Since dietary flavonoid glycosides, including quercetin 4′-glucoside from onion, are poorly absorbed from the gastrointestinal tract, they are converted into smaller phenolic acids, which can be absorbed into the circulation. The purpose of this study was to compare the effects of the major phenolic acid catabolites of quercetin 4′-glucoside, including 3,4-dihydroxyphenylacetic acid (DOPAC), 3-hydroxyphenylacetic acid, 3,4-dihydroxybenzoic acid (protocatechuic acid) and hippuric acid, on the antioxidant activity and phase II cytoprotective enzyme induction in vitro. Both DOPAC and protocatechuic acid, having a catechol moiety, exhibited both DPPH radical scavenging and superoxide dismutase-like activities, whereas 3-hydroxyphenyl acetic acid and hippuric acid did not. DOPAC also more potently enhanced the gene expression of several phase II drug-metabolizing enzymes than the other phenolic acid catabolites. DOPAC significantly inhibited the hydrogen peroxide-induced cytotoxicity in hepatocytes with the enhancement of the total glutathione S-transferase activity. In conclusion, DOPAC may play a key role in the antioxidative potential of the colonic lumen after the ingestion of the quercetin glycoside-rich onion.

Didymin reverses phthalate ester-associated breast cancer aggravation in the breast cancer tumor microenvironment.

The present study demonstrated two novel findings. To the best of our knowledge, it is the first study to demonstrate that regulated upon activation, normal T-cell expressed and secreted (RANTES), produced by breast tumor-associated monocyte-derived dendritic cells (TADCs) following breast cancer cell exposure to phthalate esters, may contribute to the progression of cancer via enhancement of cancer cell proliferation, migration and invasion. Furthermore, the present study revealed that didymin, a dietary flavonoid glycoside present in citrus fruits, was able to reverse phthalate ester-mediated breast cancer aggravation. MDA-MB-231 cells were treated with butyl benzyl phthalate (BBP), di-n-butyl phthalate (DBP) or di-2-ethylhexyl phthalate (DEHP). Subsequently, the conditioned medium (CM) was harvested and cultured with monocyte-derived dendritic cells (mdDCs). Cultures of MDA-MB-231 cells with the conditioned medium of BBP-, DBP- or DEHP-MDA-MB-231 tumor-associated mdDCs (BBP-, DBP- or DEHP-MDA-TADC-CM) demonstrated enhanced proliferation, migration and invasion. Exposure of the MDA-MB-231 cells to DBP induced the MDA-TADCs to produce the inflammatory cytokine RANTES, which subsequently induced MDA-MB-231 cell proliferation, migration and invasion. Depleting RANTES reversed the effects of DBP-MDA-TADC-mediated MDA-MB-231 cell proliferation, migration and invasion. In addition, didymin was observed to suppress phthalate-mediated breast cancer cell proliferation, migration and invasion. The present study suggested that didymin was capable of preventing phthalate ester-associated cancer aggravation.

Diosmin induces genotoxicity and apoptosis in DU145 prostate cancer cell line

Plant-derived dietary polyphenolic compounds, such as flavonoids, with cancer cell-specific pro-apoptotic activity and chemopreventive potential are thought to be promising anticancer agents. In the present study, we were interested in determining if flavonoid-induced genotoxicity may also provoke cancer cell death. Cyto- and genotoxicity of three selected flavonoid glycosides (naringin, diosmin and hesperidin) in DU145 prostate cancer cell line were investigated. Flavonoid glycosides decreased cancer cell number and proliferative activity in a different manner. Flavonoid glycosides induced oxidative stress: intracellular total ROS and superoxide production were augmented after flavonoid treatment. Flavonoid glycosides stimulated DNA double strand breaks (DSBs) and micronuclei production. Diosmin was found the most potent genotoxic agent in DU145 cells, which, in turn, resulted in its pro-apoptotic activity. The more robust recruitment of 53BP1 was correlated with lower DNA and chromosomal damage after naringin and hesperidin treatment compared with diosmin treatment. Flavonoid glycosides were also found to be DNA hypomethylating agents with an ability to modulate cancer cell epigenome leading to changes in the gene expression patterns. Taken together, diosmin, a dietary flavonoid glycoside, was found active against DU145 cells by promoting genotoxic events and a concomitant apoptotic cell death. Thus, a comprehensive analysis of biological activity of diosmin against cancer cells both in vitro and in vivo deserves further investigation.

Flavonoid glycosides inhibit oral cancer cell proliferation — role of cellular uptake and hydrolysis to the aglycones

Epidemiologic evidence supports the view that dietary flavonoids exert protective effects in oral diseases, including cancer. However, the dietary forms of flavonoids, the flavonoid glycosides, are thought to be inactive, thus they must first be hydrolysed to their active aglycones. This may occur in the saliva in the oral cavity. We have examined if the flavonoid glycosides directly could affect cell proliferation, using the human oral squamous carcinoma SCC-9 cells. The cellular uptake and hydrolysis of the glycosides were assessed also. The four flavonoid glycosides tested each behaved differently. Genistin, the 7-glucoside of genistein, showed clear and consistent inhibition of cell proliferation, which appeared to be the result of rapid cellular uptake of the glucoside and hydrolysis to genistein. Spiraeoside, the 4'-glucoside of quercetin, showed a similar inhibition of cell proliferation, which also appeared to be associated with its hydrolysis to quercetin. Diosmin, the 7-rutinoside of diosmetin, surprisingly, was more potent and effective than diosmetin. In contrast, quercitrin, the 3-rhamnoside of quercetin, showed no effect and only minimal cellular uptake and no hydrolysis. In summary, dietary flavonoid glycosides may exert cellular effects in the oral cavity, but this varies greatly with the nature of the glycoside.

Didymin, a dietary flavonoid glycoside from citrus fruits, induces Fas-mediated apoptotic pathway in human non-small-cell lung cancer cells in vitro and in vivo

Epidemiological studies provided evidence that the high dietary intake of flavonoids with fruits and vegetables could be associated with lower cancer prevalence in humans. Didymin, a dietary flavonoid glycoside from citrus fruits, possesses antioxidant properties. This study first investigates the anticancer effect of didymin in human non-small-cell lung cancer A549 and H460 cells. To identity the anticancer mechanism of didymin, we assayed its effect on apoptosis, cell cycle distribution, and levels of p53, p21/WAF1, Fas/APO-1 receptor, and Fas ligand. The results showed that didymin-induced apoptosis of A549 and H460 cells without mediation of p53 and p21/WAF1. We suggest that Fas/Fas ligand apoptotic system is the main pathway of didymin-mediated apoptosis of A549 and H460 cells. Importantly, a novel chemotherapeutic agent for the treatment of non-small-cell lung cancer, and is supported by animal studies which have shown didymin delay the tumor growth in nude mice. Our study reports here for the first time that the activity of the Fas/Fas ligand apoptotic system may participate in the antiproliferative activity of didymin in A549 and H460 cells.

Deglycosylation by small intestinal epithelial cell beta-glucosidases is a critical step in the absorption and metabolism of dietary flavonoid glycosides in humans.

Pharmacokinetic studies have shown that the small intestine is the major site of absorption for many flavonoid glucosides. Flavonoids are generally present as glycosylated forms in plants and foods, but there is increasing evidence that the forms reaching the systemic circulation are glucuronidated, sulphated and methylated derivatives. Hence, first-pass metabolism (small intestine-liver) appears to involve a critical deglycosylation step for which the mechanisms are not known. To explore the hypothesis that deglycosylation is a prerequisite to absorption and metabolism of dietary flavonoid glycosides, to identify the enzymes responsible, and relate their specificities with absorption kinetics. Flavonoid glycoside hydrolysing enzymes were isolated from samples of human small intestine and liver using chromatographic techniques. The proteins were characterised with respect to the cellular fraction with which they were associated, molecular weight, specificity for various substrates, and cross-reactions with antibodies. Cellular models were used to mimic the small intestine. Protein extracts from human jejunal mucosa were highly efficient in hydrolysing flavonoid glycosides, consistent with an enterocyte-mediated deglycosylation process. Considerable inter-individual variation was observed [e. g. range, mean and standard deviation for rate of hydrolysis of quercetin-3-glucoside (n = 10) were 6.7-456, 96, and 134 nmol min(-1) (mg protein)(-1), respectively]. Two beta-glucosidases with activity towards flavonoid glycosides were isolated from human small intestine mucosa: lactase-phlorizin hydrolase (LPH; localised to the apical membrane of small intestinal epithelial cells) and cytosolic beta-glucosidase (CBG), indicating a role of human LPH and CBG from small intestine in flavonoid absorption and metabolism. Hydrolysis of flavonoid glycosides was only detected in cultured cells exhibiting beta-glucosidase activity. The absorption of dietary flavonoid glycosides in humans involves a critical deglycosylation step that is mediated by epithelial beta-glucosidases (LPH and CBG). The significant variation in beta-glucosidase activity between individuals may be a factor determining variation in flavonoid bioavailability.

Identification and Quantification of Flavonoids in Human Urine Samples by Column-Switching Liquid Chromatography Coupled to Atmospheric Pressure Chemical Ionization Mass Spectrometry

A rapid and sensitive high-performance liquid chromatographic mass spectrometric (HPLC-MS) method is described for the determination and quantification of 12 dietary flavonoid glycosides and aglycons in human urine samples. Chromatographic separation of the analytes of interest was achieved by column-switching, using the first column (a Zorbax 300SB C-3 column) for sample cleanup and eluting the heart-cut flavonoid fraction onto the second column (a Zorbax SB C-18 column) for separation and detection by ultraviolet and atmospheric pressure chemical ionization MS using single ion monitoring in negative mode. The fragmentor voltage was optimized with regard to maximum abundance of the molecular ion and qualifier ions of the analytes. Calibration graphs were prepared for urine, and good linearity was achieved over a dynamic range of 2.5-1000 ng/mL. The inter- and intraassay coefficients of variation for the analysis of the 12 different flavonoids in quality control urine samples were 12.3% on average (range 11.0-13.7%, n = 24, reproducibility) and the repeatability of the assay were 5.0% (mean, range 0.1-14.8%, n = 12). A subset of 10 urine samples from a human dietary intervention study with high and low flavonoid content was analyzed, and the results are reported.

Purification of cytosolic β-glucosidase from pig liver and its reactivity towards flavonoid glycosides

Flavonoid glycosides are common dietary components which may have health-promoting activities. The metabolism of these compounds is thought to influence their bioactivity and uptake from the small intestine. It has been suggested that the enzyme cytosolic β-glucosidase could deglycosylate certain flavonoid glycosides. To test this hypothesis, the enzyme was purified to homogeneity from pig liver for the first time. It was found to have a molecular weight (55 kDa) and specific activity (with p-nitrophenol glucoside) consistent with other mammalian cytosolic β-glucosidases. The pure enzyme was indeed found to deglycosylate various flavonoid glycosides. Genistein 7-glucoside, daidzein 7-glucoside, apigenin 7-glucoside and naringenin 7-glucoside all acted as substrates, but we were unable to detect activity with naringenin 7-rhamnoglucoside. Quercetin 4′-glucoside was a substrate, but neither quercetin 3,4′-diglucoside, quercetin 3-glucoside nor quercetin 3-rhamnoglucoside were deglycosylated. Estimates of K m ranged from 25 to 90 μM while those for V max were about 10% of that found with the standard artificial substrate p-nitrophenol glucoside. The non-substrate quercetin 3-glucoside was found to partially inhibit deglycosylation of quercetin 4′-glucoside, but it had no effect upon activity with p-nitrophenol glucoside. This study confirms that mammalian cytosolic β-glucosidase can deglycosylate some, but not all, common dietary flavonoid glycosides. This enzyme may, therefore, be important in the metabolism of these compounds.

The sugar moiety is a major determinant of the absorption of dietary flavonoid glycosides in man

Flavonoids are antioxidants present in plant foods. They occur mainly as glycosides, i.e. linked with various sugars. It is uncertain to what extent dietary flavonoid glycosides are absorbed from the gut. We investigated how the nature of the sugar group affected absorption of one major flavonoid, quercetin. Quercetin linked with glucose, i.e. quercetin glucoside and quercetin linked with rutinose, i.e. quercetin rutinoside, both occur widely in foods. When we fed these compounds to nine volunteers, the peak concentration of quercetin (Cmax) in plasma was 20 times higher and was reached (Tmax) more than ten times faster after intake of the glucoside (Cmax = 3.5 ± 0.6 μM (mean ± SE); Tmax < 0.5 h) than after the rutinoside (Cmax = 0.18 ± 0.04 μM; Tmax = 6.0 ± 1.2 h). The bioavailability of the rutinoside was only 20% of that of the glucoside. We suggest that quercetin glucoside is actively absorbed from the small intestine, whereas quercetin rutinoside is absorbed from the colon after deglycosylation. Absorption of other food components might also be enhanced by attachment of a glucose group.

Hydrolysis of dietary flavonoid glycosides by strains of intestinal Bacteroides from humans.

Rutin and quercitrin are hydrolysed to quercetin, and robinin is hydrolysed to kaempferol, by faecal flora from healthy subjects. The enzymes required for these hydrolyses, namely alpha-rhamnosidase and beta-galactosidase, were produced by some strains of Bacteroides distasonis; other strains, however, synthesized beta-glucosidase. The last-named enzyme was also elaborated by Bacteroides uniformis and Bacteroides ovatus. All the enzymes were produced constitutively. A cell-free extract of B. distasonis containing beta-glucosidase displayed an enzymic activity of 1 mumol/10 min per 10 mg of protein.