Antibacterial and Antioxidant Activity of Synthetic Polyoxygenated Flavonoids.

Flavonoids are an abundant class of naturally occurring compounds with broad biological activities, but their limited abundance in nature restricts their use in medicines and food additives. Here we present the synthesis and determination of the antibacterial and antioxidant activities of twenty-two structurally related flavonoids (five of which are new) by scientifically validated methods. Flavanones (FV1-FV11) had low inhibitory activity against the bacterial growth of MRSA 97-7. However, FV2 (C5,7,3',4' = OH) and FV6 (C5,7 = OH; C4' = SCH3) had excellent bacterial growth inhibitory activity against Gram-negative E. coli (MIC = 25 µg/mL for both), while Chloramphenicol (MIC = 25 µg/mL) and FV1 (C5,7,3' = OCH3; 4' = OH) showed inhibitory activity against Gram-positive L. monocytogenes (MIC = 25 µg/mL). From the flavone series (FO1-FO11), FO2 (C5,7,3',4' = OH), FO3 (C5,7,4' = OH; 3' = OCH3), and FO5 (C5,7,4' = OH) showed good inhibitory activity against Gram-positive MRSA 97-7 (MIC = 50, 12, and 50 µg/mL, respectively), with FO3 being more active than the positive control Vancomycin (MIC = 25 µg/mL). FO10 (C5,7= OH; 4' = OCH3) showed high inhibitory activity against E. coli and L. monocytogenes (MIC = 25 and 15 µg/mL, respectively). These data add significantly to our knowledge of the structural requirements to combat these human pathogens. The positions and number of hydroxyl groups were key to the antibacterial and antioxidant activities.

Prenylated Flavanone Isolated from Dalea Species as a Potential Multitarget-Neuroprotector in an In Vitro Alzheimer's Disease Mice Model.

Alzheimer's disease (AD) involves a neurodegenerative process that has not yet been prevented, reversed, or stopped. Continuing with the search for natural pharmacological treatments, flavonoids are a family of compounds with proven neuroprotective effects and multi-targeting behavior. The American genus Dalea L. (Fabaceae) is an important source of bioactive flavonoids. In this opportunity, we tested the neuroprotective potential of three prenylated flavanones isolated from Dalea species in a new in vitro pre-clinical AD model previously developed by us. Our approach consisted in exposing neural cells to conditioned media (3xTg-AD ACM) from neurotoxic astrocytes derived from hippocampi and cortices of old 3xTg-AD mice, mimicking a local neurodegenerative microenvironment. Flavanone 1 and 3 showed a neuroprotective effect against 3xTg-AD ACM, being 1 more active than 3. The structural requirements to afford neuroprotective activity in this model are a 5'-dimethylallyl and 4'-hydroxy at the B ring. In order to search the mechanistic performance of the most active flavanone, we focus on the flavonoid-mediated regulation of GSK-3ß-mediated tau phosphorylation previously reported. Flavanone 1 treatment decreased the rise of hyperphosphorylated tau protein neuronal levels induced after 3xTg-AD ACM exposure and inhibited the activity of GSK-3ß. Finally, direct exposure of these neurotoxic 3xTg-AD astrocytes to flavanone 1 resulted in toxicity to these cells and reduced the neurotoxicity of 3xTg-AD ACM as well. Our results allow us to present compound 1 as a natural prenylated flavanone that could be used as a precursor to development and design of future drug therapies for AD.

Naringin prevents diabetic nephropathy in rats through blockage of oxidative stress and attenuation of the mitochondrial dysfunction.

We have studied the effects of naringin (NAR), a flavonoid from citric fruits, on morphology, ultrastructure and function of the kidney in streptozotocin (STZ)-induced diabetic rats. Two groups of animals were used: (1) control rats and (2) STZ rats (60 mg STZ/kg b.w.). At 3 days after induction, one group of STZ-treated rats received 40 mg NAR/kg b.w. daily. NAR blocked completely alterations in the biochemical renal markers in STZ rats except the increase in serum urea that was partially avoided by the flavonoid. NAR ameliorated the kidney morphological lesions from STZ rats. STZ treatment induced round and smaller mitochondria, which was avoided by NAR. Citrate synthase, isocitrate and malate dehydrogenases, enzyme activities of the Krebs cycle, were decreased in STZ rats. NAR abolished this decrease in the latter proteins. NAR also prevented a decrease in the ATP synthase activity of the mitochondria from renal cortex by about 49% in STZ rats, returning the enzyme activity to control values. The nephroprotection caused by NAR is mediated through counteraction of oxidative stress in mitochondria of proximal tubules. NAR might be a therapeutic strategy to reduce the complication of diabetic nephropathy in type 1 diabetic patients.

Could Naringenin Participate as a Regulator of Obesity and Satiety?

Obesity is a serious health problem worldwide, since it is associated with multiple metabolic disorders and complications such as cardiovascular disease, type 2 diabetes, fatty liver disease and overall metabolic dysfunction. Dysregulation of the hunger-satiety pathway, which includes alterations of central and peripheral signaling, explains some forms of obesity by favoring hyperphagia and weight gain. The present work comprehensively summarizes the mechanisms by which naringenin (NAR), a predominant flavanone in citrus fruits, could modulate the main pathways associated with the development of obesity and some of its comorbidities, such as oxidative stress (OS), inflammation, insulin resistance (IR) and dyslipidemia, as well as the role of NAR in modulating the secretion of enterohormones of the satiety pathway and its possible antiobesogenic effect. The results of multiple in vitro and in vivo studies have shown that NAR has various potentially modulatory biological effects against obesity by countering IR, inflammation, OS, macrophage infiltration, dyslipidemia, hepatic steatosis, and adipose deposition. Likewise, NAR is capable of modulating peptides or peripheral hormones directly associated with the hunger-satiety pathway, such as ghrelin, cholecystokinin, insulin, adiponectin and leptin. The evidence supports the use of NAR as a promising alternative to prevent overweight and obesity.

The Role of Flavanones as Scaffolds for the Development of New Treatments against Malaria and African and American Trypanosomiases.

Parasitic infections are diseases transmitted by parasites usually found in contaminated food, water, or insect bites. Generally classified as neglected tropical diseases, malaria and trypanosomiases are some of the most prominent parasitic diseases that cause significant loss of life annually. In 2020, an estimated 241 million malaria cases were reported, with 627,000 deaths worldwide. An estimated 6 to 7 million people are infected with Trypanosoma cruzi worldwide, whereas an estimated 1000 global cases of African human trypanosomiasis were reported in 2020. Flavanones are a group of compounds that belong to the flavonoid family and are chemically obtained by direct cyclization of chalcones. Recent pharmacological studies have demonstrated the effectiveness of plant flavanones in inhibiting the growth of the parasites responsible for malaria and trypanosomiases. The present work aims to summarize up-to-date and comprehensive literature information on plant flavanones with antimalarial and antitrypanosomal activities. The mechanisms of action of the antiparasitic flavanones are also discussed. A literature search was performed for naturally occurring flavanones and antimalarial and antitrypanosomal activities by referencing textbooks and scientific databases (SciFinder, Wiley, American Chemical Society, Science Direct, National Library of Medicine, Scientific Electronic Library Online, Web of Science, etc.) from their inception until April 2022. Based on in vitro experiments, more than sixty flavanones were reported to exhibit antimalarial, anti-T. cruzi, and anti-T. brucei activities. Previous studies demonstrated that these compounds bind to PGP-like transporters of P. falciparum to reverse the parasite's resistance. Other reports pinpointed the direct effect of these compounds on the mitochondria of the malaria parasite. Moreover, flavanones have shown strong docking to several validated T. cruzi and T. brucei protein targets, including adenosine kinase, pteridine reductase 1, dihydrofolate reductase, and trypanothione reductase, among others. Flavanones, isolated and characterized from diverse plant parts, were reported to exhibit moderate to high activity against P. falciparum, T. cruzi, and T. brucei in in vitro studies. These potentially active flavanones can be used as scaffolds for the development of new antiparasitic agents. However, more studies on the cytotoxicity, pharmacokinetics, and mechanisms of action of potent flavanones should be performed.

Anti-inflammatory effect evaluation of naringenin and its incorporation into a chitosan-based film for transdermal delivery.

Naringenin is a bioflavonoid mainly found in citrus fruits. It presents many pharmacological benefits, including a remarkable anti-inflammatory activity, but its oral bioavailability is poor. To overcome this drawback, this work proposes a transdermal administration of such bioflavonoid, considering its use in the chronic treatment of inflammatory conditions. For this, it aims to develop a chitosan-based film that guarantees a consistent transdermal delivery of the drug. First, naringenin's in vitro anti-inflammatory effect on T-cell proliferation was evaluated, followed by research on the modulation of gene expression for inflammatory factors in peripheral blood mononuclear cells. Chitosan films were then prepared and characterized. Afterward, naringenin release profile from a selected film was determined as well as the drug permeation across porcine skin provided by the film. Naringenin induced the expression of the anti-inflammatory factors IL-10 and TGF-ß1 while inhibiting the expression of the pro-inflammatory cytokine IL-1ß and limiting T-cell proliferation. The chitosan film was successfully developed, and the drug was progressively released to the physiological media following both first order and Korsmeyer-Peppas kinetics. When topically applied, the chitosan film guaranteed a constant and continuous diffusion of naringenin across the skin over 72 h. Indeed, the permeation flux of naringenin was 0.30 ± 0.01 µg/cm2/h, which means a concentration in the receptor solution 14-fold (p < 0.05) higher than that provided by the drug solution. Thus, the chitosan film represents a promising transdermal alternative for the long-term treatment of inflammatory conditions using naringenin.

Therapeutic Effects of Citrus Flavonoids Neohesperidin, Hesperidin and Its Aglycone, Hesperetin on Bone Health.

Flavonoids are natural phytochemicals that have therapeutic effects and act in the prevention of several pathologies. These phytochemicals can be found in seeds, grains, tea, coffee, wine, chocolate, cocoa, vegetables and, mainly, in citrus fruits. Neohesperidin, hesperidin and hesperetin are citrus flavonoids from the flavanones subclass that have anti-inflammatory and antioxidant potential. Neohesperidin, in the form of neohesperidin dihydrochalcone (NHDC), also has dietary properties as a sweetener. In general, these flavanones have been investigated as a strategy to control bone diseases, such as osteoporosis and osteoarthritis. In this literature review, we compiled studies that investigated the effects of neohesperidin, hesperidin and its aglycone, hesperetin, on bone health. In vitro studies showed that these flavanones exerted an antiosteoclastic and anti- inflammatory effects, inhibiting the expression of osteoclastic markers and reducing the levels of reactive oxygen species, proinflammatory cytokines and matrix metalloproteinase levels. Similarly, such studies favored the osteogenic potential of preosteoblastic cells and induced the overexpression of osteogenic markers. In vivo, these flavanones favored the regeneration of bone defects and minimized inflammation in arthritis- and periodontitis-induced models. Additionally, they exerted a significant anticatabolic effect in ovariectomy models, reducing trabecular bone loss and increasing bone mineral density. Although research should advance to the clinical field, these flavanones may have therapeutic potential for controlling the progression of metabolic, autoimmune or inflammatory bone diseases.

Naringin attenuates acute myocardial ischemia-reperfusion injury via miR- 126/GSK-3ß/ß-catenin signaling pathway.

INTRODUCTION: Myocardial ischemia-reperfusion (I/R) injury is one of the mechanisms contributing to the high mortality rate of acute myocardial infarction. PURPOSE: This study intended to study the role of naringin in cardiac I/R injury. METHODS: AC16 cells (human cardiomyocyte cell line) were subjected to oxygen-glucose deprivation/recovery (OGD/R) treatment and/or naringin pretreatment. Then, the apoptosis was examined by flow cytometry and Western blotting. The concentration of IL-6, IL-8 and TNF-α was measured by enzyme-linked immunosorbent assay (ELISA) kits. How naringin influenced microRNA expression was examined by microarrays and quantitative real-time polymerase chain reaction (qRT-PCR). Dual luciferase reporter assay was employed to evaluate the interaction between miR-126 and GSK-3ß. The GSK-3ß/ß-catenin signaling pathway was examined by Western blotting. Finally, rat myocardial I/R model was created to examine the effects of naringin in vivo. RESULTS: Naringin pretreatment significantly decreased the cytokine release and apoptosis of cardiomyocytes exposed to OGD/R. Bioinformatical analysis revealed that naringin upregulated miR-126 expression considerably. Also, it was found that miR-126 can bind GSK-3ß and downregulate its expression, suggesting that naringin could decrease GSK-3ß activity. Next, we discovered that naringin increased ß-catenin activity in cardiomyocytes treated with OGD/R by inhibiting GSK-3ß expression. Our animal experiments showed that naringin pre-treatment or miR-126 agomir alleviated myocardial I/R. CONCLUSIONS: Naringin preconditioning can reduce myocardial I/R injury via regulating miR-126/GSK-3ß/ß-catenin signaling pathway, and this chemical can be used to treat acute myocardial infarction.

Flavanone Glycosides, Triterpenes, Volatile Compounds and Antimicrobial Activity of Miconia minutiflora (Bonpl.) DC. (Melastomataceae).

Chemical composition of the essential oils and extracts and the antimicrobial activity of Miconia minutiflora were investigated. The flavanone glycosides, pinocembroside and pinocembrin-7-O-[4″,6″-HHDP]-ß-D-glucose, were identified, along with other compounds that belong mainly to the triterpene class, besides the phenolics, gallic acid and methyl gallate. Sesquiterpenes and monoterpenes were the major compounds identified from the essential oils. Screening for antimicrobial activity from the methanolic extract of the leaves showed that the MIC and MMC values against the tested microorganisms ranged from 0.625 to 5 mg·mL−1 and that the extract was active against microorganisms, Staphyloccocus aureus, Escherichia coli, and Bacillus cereus.

A New Flavanone from Chromolaena tacotana (Klatt) R. M. King and H. Rob, Promotes Apoptosis in Human Breast Cancer Cells by Downregulating Antiapoptotic Proteins.

Chromolaena tacotana is a source of flavonoids with antiproliferative properties in human breast cancer cells, the most common neoplasm diagnosed in patients worldwide. Until now, the mechanisms of cell death related to the antiproliferative activity of its flavonoids have not been elucidated. In this study, a novel flavanone (3',4'-dihydroxy-5,7-dimethoxy-flavanone) was isolated from the plant leaves and identified by nuclear magnetic resonance (NMR) and mass spectrometry (MS). This molecule selectively inhibited cell proliferation of triple-negative human breast cancer cell lines MDA-MB-231 and MCF-7 whit IC50 values of 25.3 µg/mL and 20.8 µg/mL, respectively, determined by MTT assays with a selectivity index greater than 3. Early and late pro-apoptotic characteristics were observed by annexin-V/7-AAD detection, accompanied by a high percentage of the Bcl-2 anti-apoptotic protein inactivated and the activation of effector Caspase-3 and/or 7 in breast cancer cells. It was verified the decreasing of XIAP more than Bcl-2 anti-apoptotic proteins expression, as well as the XIAP/Caspase-7 and Bcl-2/Bax complexes dissociation after flavanone treatment. Docking and molecular modeling analysis between the flavanone and the antiapoptotic protein XIAP suggests that the natural compound inhibits XIAP by binding to the BIR3 domain of XIAP. In this case, we demonstrate that the new flavanone isolated from leaves of Chomolaena tacotana has a promising and selective anti-breast cancer potential that includes the induction of intrinsic apoptosis by downregulation of the anti-apoptotic proteins XIAP and Bcl-2. New studies should deepen these findings to demonstrate its potential as an anticancer agent.

Insecticidal activity of copper (II) complexes with flavanone derivatives.

In this work, we describe the insecticidal activity against Spodoptera frugiperda of flavanone derivatives (naringin, naringenin and hesperidin) complexed to copper (II). For the assays, the compounds were incorporated into an artificial diet and offered to S. frugiperda during its larval period. The duration of larval and pupal phases, the weight of pupae and the percentage of dead insects at the end of the larval phase were evaluated. Among the tested compounds, the free flavanones 1 and 3 and the complexes 4 and 6 showed a shortening of the larval phase when compared to the control. Complex 9 showed the highest toxicity, with 96.66% larval mortality. This is the first report on the insecticidal activity of the Cu2+ complexes of the flavanones naringin, naringenin and hesperidin.[Formula: see text].

Blood pressure and body fat % reduction is mainly related to flavanone phase II conjugates and minor extension by phenolic acid after long-term intake of orange juice.

Hesperidin and narirutin are the major flavanones present in orange juice, and they are associated with a reduction in risk of cardiometabolic disease. However, there is heterogeneity in their biological responses, which is partly due to the large interindividual variation in these flavonoids' bioavailability. We investigated the relation between interindividual variability in the excretion of phase II conjugates and gut-derived phenolic acids, and cardiometabolic biomarkers response. Seventy-four subjects, both men and women, were included in a single-arm study. Over the 60 days, volunteers consumed 500 mL of orange juice daily. All measurements and blood collections were performed before and after the intervention period. Moreover, 24 h urine collection was performed after first consumption. Individuals were stratified according to the excretion of phase II conjugates and, for the first time, according to phenolic acids in high, medium, and low excretors. Furthermore, for the first time, the ratio between phenolic acids and flavanones-phase II conjugates has shown groups with different metabolization patterns. Groups with a low or intermediate ratio, corresponding to a higher amount of phase II conjugates excreted, showed a significant reduction in body fat % and blood pressure. This finding suggests that these improvements could be associated in a major way to flavanones-phase II conjugates, as well as to phenolic acids and stratification of volunteers according to metabolite excretions could be a good strategy to better understand the effects of orange juice on metabolism and health.

Pinocembrin pretreatment counteracts the chlorpyrifos-induced HO-1 downregulation, mitochondrial dysfunction, and inflammation in the SH-SY5Y cells.

Chlorpyrifos (CPF), an insecticide, induces pro-oxidant, pro-inflammatory, and pro-apoptotic effects in animal cells. Contamination with CPF occurs not only in farms, since CPF is found in the food consumed in homes. Recently, it was demonstrated that CPF affects the mitochondria, inhibiting components of the electron transfer chain (ETC), causing loss of mitochondrial membrane potential (MMP), and reducing the synthesis of adenosine triphosphate (ATP) by the Complex V. Pinocembrin (PB) is found in propolis and exhibits antioxidant, anti-inflammatory, and anti-apoptotic effects in mammalian cells. PB is a potent inducer of the nuclear factor erythroid 2-related factor 2 (Nrf2), which is a major transcription factor controlling the expression of heme oxygease-1 (HO-1), among others. In the present work, we investigated whether PB would be able to prevent the mitochondrial and immune dysfunctions in the human neuroblastoma SH-SY5Y cells exposed to CPF. PB was tested at 1-25 µM for 4 h before the administration of CPF at 100 µM for additional 24 h. We found that PB prevented the CPF-induced inhibition of ETC, loss of MMP, and decline in the ATP synthesis. PB also promoted anti-inflammatory actions in this experimental model. Silencing of Nrf2 or inhibition of HO-1 suppressed the PB-induced effects in the CPF-challenged cells. Thus, PB promoted beneficial effects by a mechanism dependent on the Nrf2/HO-1/CO + BR axis in the CPF-treated cells.

Benzylated Dihydroflavones and Isoquinoline-Derived Alkaloids from the Bark of Diclinanona calycina (Annonaceae) and Their Cytotoxicities.

Diclinanona calycina R. E. Fries popularly known as "envira", is a species of the Annonaceae family endemic to Brazil. In our ongoing search for bioactive compounds from Annonaceae Amazon plants, the bark of D. calycina was investigated by classical chromatography techniques that yielded thirteen compounds (alkaloids and flavonoids) described for the first time in D. calycina as well as in the genus Diclinanona. The structure of these isolated compounds were established by extensive analysis using 1D/2D-NMR spectroscopy in combination with MS. The isolated alkaloids were identified as belonging to the subclasses: simple isoquinoline, thalifoline (1); aporphine, anonaine (2); oxoaporphine, liriodenine (3); benzyltetrahydroisoquinolines, (S)-(+)-reticuline (4); dehydro-oxonorreticuline (3,4-dihydro-7-hydroxy-6-methoxy-1-isoquinolinyl)(3-hydroxy-4-methoxyphenyl)-methanone) (5); (+)-1S,2R-reticuline Nß-oxide (6); and (+)-1S,2S-reticuline Nα-oxide (7); tetrahydroprotoberberine, coreximine (8); and pavine, bisnorargemonine (9). While the flavonoids belong to the benzylated dihydroflavones, isochamanetin (10), dichamanetin (11), and a mixture of uvarinol (12) and isouvarinol (13). Compound 5 is described for the first time in the literature as a natural product. The cytotoxic activity of the main isolated compounds was evaluated against cancer and non-cancerous cell lines. Among the tested compounds, the most promising results were found for the benzylated dihydroflavones dichamanetin (10), and the mixture of uvarinol (12) and isouvarinol (13), which presented moderate cytotoxic activity against the tested cancer cell lines (<20.0 µg·mL-1) and low cytotoxicity against the non-cancerous cell line MRC-5 (>25.0 µg·mL-1). Dichamanetin (11) showed cytotoxic activity against HL-60 and HCT116 with IC50 values of 15.78 µg·mL-1 (33.70 µmol·L-1) and 18.99 µg·mL-1 (40.56 µmol·L-1), respectively while the mixture of uvarinol (12) and isouvarinol (13) demonstrated cytotoxic activity against HL-60, with an IC50 value of 9.74 µg·mL-1, and HCT116, with an IC50 value of 17.31 µg·mL-1. These cytotoxic activities can be attributed to the presence of one or more hydroxybenzyl groups present in these molecules as well as the position in which these groups are linked. The cytotoxic activities of reticuline, anonaine and liriodenine have been previously established, with liriodenine being the most potent compound.

Prenylated Flavonoids with Potential Antimicrobial Activity: Synthesis, Biological Activity, and In Silico Study.

Prenylated flavonoids are an important class of naturally occurring flavonoids with important biological activity, but their low abundance in nature limits their application in medicines. Here, we showed the hemisynthesis and the determination of various biological activities of seven prenylated flavonoids, named 7-13, with an emphasis on antimicrobial ones. Compounds 9, 11, and 12 showed inhibitory activity against human pathogenic fungi. Compounds 11, 12 (flavanones) and 13 (isoflavone) were the most active against clinical isolated Staphylococcus aureus MRSA, showing that structural requirements as prenylation at position C-6 or C-8 and OH at positions C-5, 7, and 4' are key to the antibacterial activity. The combination of 11 or 12 with commercial antibiotics synergistically enhanced the antibacterial activity of vancomycin, ciprofloxacin, and methicillin in a factor of 10 to 100 times against drug-resistant bacteria. Compound 11 combined with ciprofloxacin was able to decrease the levels of ROS generated by ciprofloxacin. According to docking results of S enantiomer of 11 with ATP-binding cassette transporter showed the most favorable binding energy; however, more studies are needed to support this result.

Bioassay-Guided Identification of the Antiproliferative Compounds of Cissus trifoliata and the Transcriptomic Effect of Resveratrol in Prostate Cancer Pc3 Cells.

The bioassay-guided fractionation of a CHCl3-MeOH extract from the stems of Cissus trifoliata identified an active fraction against PC3 prostate cancer cells. The treatment for 24 h showed an 80% reduction in cell viability (p ≤ 0.05) by a WST-1 assay at a concentration of 100 µg/mL. The HPLC-QTOF-MS analysis of the fraction showed the presence of coumaric and isoferulic acids, apigenin, kaempferol, chrysoeriol, naringenin, ursolic and betulinic acids, hexadecadienoic and octadecadienoic fatty acids, and the stilbene resveratrol. The exposure of PC3 cells to resveratrol (IC25 = 23 µg/mL) for 24 h induced significant changes in 847 genes (Z-score ≥ ±2). The functional classification tool of the DAVID v6.8 platform indicates that the underlying molecular mechanisms against the proliferation of PC3 cells were associated (p ≤ 0.05) with the process of differentiation and metabolism. These findings provide experimental evidence suggesting the potential of C. trifoliata as a promising natural source of anticancer compounds.

Naringenin promotes cell autophagy to improve high-fat-diet-induced atherosclerosis in ApoE-/- mice.

Naringenin (NAR) is a major flavanone in citrus fruits that has multiple pharmacological attributes such as anticancer and antiatherogenic. This study aims to investigate the mechanism of NAR in high-fat-diet (HFD)-induced atherosclerosis (AS) in apolipoprotein E-knockout (ApoE-/-) mice. A HFD-induced AS ApoE-/- mouse model was established. The mice were treated with HFD, different doses of NAR and simvastatin (Simv). After drug treatment, the levels of total cholesterol (TC), triglyceride (TG), low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C), glutathione peroxidase (GSH-Px), malondialdehyde (MDA), superoxide dismutase (SOD), and alanine aminotransferase (ALT) were determined. The expression of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) was detected using qRT-PCR and enzyme-linked immunosorbent assay. The plaque area of the aorta of AS mice was determined using oil red O staining. Western blot analysis was applied to measure the levels of autophagy-related proteins [protein 1 light chain 3B (LC3B), beclin 1, and p62]. The TC, TG, LDL-C, TNF-α, ALT, and MDA levels were significantly increased while the HDL-C, SOD, and GSH-Px levels were decreased in the HFD-induced AS ApoE-/- mice. NAR treatment reversed the expression of the above indicators in mice. After they were treated with different doses of NAR, the LC3B and beclin 1 levels were improved while the p62 protein level was decreased. This study suggested that NAR could promote cell autophagy to improve HFD-induced AS in ApoE-/- mice.

Naringenin promotes cell autophagy to improve high-fat-diet-induced atherosclerosis in ApoE-/- mice

Naringenin (NAR) is a major flavanone in citrus fruits that has multiple pharmacological attributes such as anticancer and antiatherogenic. This study aims to investigate the mechanism of NAR in high-fat-diet (HFD)-induced atherosclerosis (AS) in apolipoprotein E-knockout (ApoE-/-) mice. A HFD-induced AS ApoE-/- mouse model was established. The mice were treated with HFD, different doses of NAR and simvastatin (Simv). After drug treatment, the levels of total cholesterol (TC), triglyceride (TG), low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C), glutathione peroxidase (GSH-Px), malondialdehyde (MDA), superoxide dismutase (SOD), and alanine aminotransferase (ALT) were determined. The expression of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) was detected using qRT-PCR and enzyme-linked immunosorbent assay. The plaque area of the aorta of AS mice was determined using oil red O staining. Western blot analysis was applied to measure the levels of autophagy-related proteins [protein 1 light chain 3B (LC3B), beclin 1, and p62]. The TC, TG, LDL-C, TNF-α, ALT, and MDA levels were significantly increased while the HDL-C, SOD, and GSH-Px levels were decreased in the HFD-induced AS ApoE-/- mice. NAR treatment reversed the expression of the above indicators in mice. After they were treated with different doses of NAR, the LC3B and beclin 1 levels were improved while the p62 protein level was decreased. This study suggested that NAR could promote cell autophagy to improve HFD-induced AS in ApoE-/- mice.

Toxicological Screening of Four Bioactive Citroflavonoids: In Vitro, In Vivo, and In Silico Approaches.

Many studies describe different pharmacological effects of flavonoids on experimental animals and humans. Nevertheless, few ones are confirming the safety of these compounds for therapeutic purposes. This study aimed to investigate the preclinical safety of naringenin, naringin, hesperidin, and quercetin by in vivo, in vitro, and in silico approaches. For this, an MTT-based cytotoxicity assay in VERO and MDCK cell lines was performed. In addition, acute toxicity was evaluated on Wistar rats by OECD Guidelines for the Testing of Chemicals (Test No. 423: Acute Oral Toxicity-Class Method). Furthermore, we used the ACD/Tox Suite to predict toxicological parameters such as hERG channel blockade, CYP450 inhibition, and acute toxicity in animals. The results showed that quercetin was slightly more cytotoxic on cell lines (IC50 of 219.44 ± 7.22 mM and 465.41 ± 7.44 mM, respectively) than the other citroflavonoids. All flavonoids exhibited an LD50 value > 2000 mg/kg, which classifies them as low-risk substances as OECD guidelines established. Similarly, predicted LD50 was LD50 > 300 to 2000 mg/kg for all flavonoids as acute toxicity assay estimated. Data suggests that all these flavonoids did not show significant toxicological effects, and they were classified as low-risk, useful substances for drug development.

Effect of enzymatic treatment of citrus by-products on bacterial growth, adhesion and cytokine production by Caco-2 cells.

Citrus by-products are inexpensive sources of polyphenols, important bioactive compounds with wide pharmaceutical and food applications. This study aimed to investigate the effect of enzymatic treatment of citrus by-products on the polyphenolic profile of extracts and assess the influence of extracts on the growth and adhesion of probiotics and foodborne pathogenic bacteria and on the inflammatory response of epithelial cells. Enzyme-assisted extraction altered the polyphenolic profile (as assessed by HPLC-DAD), increasing the content of aglycone flavanones (naringenin and hesperetin). Enzymatic extracts and aglycone flavanones exhibited higher antibacterial and prebiotic activities than non-enzymatic extracts and glycoside flavanones. However, a higher content of aglycones was not associated with higher anti-adhesion activity. Citrus extracts significantly (P ≤ 0.05) decreased the inflammatory response of Caco-2 cells to Salmonella Typhimurium adhesion. These results support the sustainable reuse of citrus agroindustrial wastes and indicate the potential of citrus extracts in preventing infection by foodborne pathogenic bacteria and inducing proliferation of probiotics in foods and the gut environment.