Efficient and green production of flavone-5-O-glycosides by glycosyltransferases in Escherichia coli.

O-Glycosylflavonoids exhibit diverse biological activities but their low content in plants is difficult to extract and isolate, and chemical synthesis steps are cumbersome, which are harmful to the environment. Therefore, the biosynthesis of O-glycosylflavonoids represents a green and sustainable alternative strategy, with glycosyltransferases playing a crucial role in this process. However, there are few studies on flavone 5-O-glycosyltransferases, which limits the synthesis of rare flavone 5-O glycosides by microorganisms. In this study, we characterized a highly regioselectivity flavone 5-O glycosyltransferase from Panicum hallii. Site-directed mutagenesis at residue P141 switches glucosylation to xylosylation. Using a combinatorial strategy of metabolic engineering, we generated a series of Escherichia coli recombinant strains to biocatalyze glycosylation of the typical flavone apigenin. Ultimately, further optimization of transformation conditions, apigenin-5-O-glucoside and apigenin-5-O-xyloside were biosynthesized for the first time so far, and the yields were 1490 mg/L and 1210 mg/L, respectively. This study provides a biotechnological component for the biosynthesis of flavone-5-O-glycosides, and established a green and sustainable approach for the industrial production of high-value O-glycosylflavones by engineering, which lays a foundation for their further development and application in food and pharmaceutical fields.

Revealing binding mechanism of ß-casein to chrysin, apigenin, and luteolin and locating its binding pockets by molecular docking and molecular dynamics.

Revealing the interaction mechanism of proteins with bioactive molecules and the location of their binding pockets is crucial for predicting the structure-function relationship of proteins in drug discovery and design. Despite some published papers on the interaction of ß-casein with small bioactive molecules, the ambiguity of the location and constituent amino acids of ß-casein binding pockets prompted us to identify them by in silico simulation of its interaction with three polyphenols, chrysin, apigenin, and luteolin. Molecular docking revealed that the primary ß-casein binding pocket for chrysin consists of five nonpolar amino acids (Leu73, Phe77, Pro80, Ile89, and Pro196), three polar neutral amino acids (Ser137, Gln138, and Gln197), and two polar charged amino acids (Glu136, and Arg198). For ß-casein/apigenin and ß-casein/luteolin complexes, Asn83 also contributes to forming the pocket. Molecular dynamics provided more details, such as the relative contribution of determinative amino acids and the role of various forces. For example, we found that Glu210, Glu132, and Glu35 are the most destructive residues in the binding of chrysin, apigenin, and luteolin to ß-casein, respectively. Also, we observed that hydrophobic forces mainly stabilize ß-casein/chrysin and ß-casein/apigenin, and polar solvation (including hydrogen bonds) stabilizes ß-casein/luteolin, all by spontaneous processes.

Biosynthesis of Apigenin Glucosides in Engineered Corynebacterium glutamicum.

Glucosylation is a well-known approach to improve the solubility, pharmacological, and biological properties of flavonoids, making flavonoid glucosides a target for large-scale biosynthesis. However, the low yield of products coupled with the requirement of expensive UDP-sugars limits the application of enzymatic systems for large-scale. C. glutamicum is a Gram-positive and generally regarded as safe (GRAS) bacteria frequently employed for the large-scale production of amino acids and bio-fuels. Due to the versatility of its cell factory system and its non-endotoxin producing properties, it has become an attractive system for the industrial-scale biosynthesis of alternate products. Here, we explored the cell factory of C. glutamicum for efficient glucosylation of flavonoids using apigenin as a model flavonoid, with the heterologous expression of a promiscuous glycosyltransferase, YdhE from Bacillus licheniformis and the endogenous overexpression of C. glutamicum genes galU1 encoding UDP-glucose pyrophosphorylase and pgm encoding phosphoglucomutase involved in the synthesis of UDP-glucose to create a C. glutamicum cell factory system capable of efficiently glucosylation apigenin with a high yield of glucosides production. Consequently, the production of various apigenin glucosides was controlled under different temperatures yielding almost 4.2 mM of APG1(apigenin-4'-O-ß-glucoside) at 25°C, and 0.6 mM of APG2 (apigenin-7-O-ß-glucoside), 1.7 mM of APG3 (apigenin-4',7-O-ß-diglucoside) and 2.1 mM of APG4 (apigenin-4',5-O-ß-diglucoside) after 40 h of incubation with the supplementation of 5 mM of apigenin and 37°C. The cost-effective developed system could be used to modify a wide range of plant secondary metabolites with increased pharmacokinetic activities on a large scale without the use of expensive UDP-sugars.

Apigenin and Rutaecarpine reduce the burden of cellular senescence in bone marrow stromal stem cells.

Introduction: Osteoporosis is a systemic age-related disease characterized by reduced bone mass and microstructure deterioration, leading to increased risk of bone fragility fractures. Osteoporosis is a worldwide major health care problem and there is a need for preventive approaches. Methods and results: Apigenin and Rutaecarpine are plant-derived antioxidants identified through functional screen of a natural product library (143 compounds) as enhancers of osteoblastic differentiation of human bone marrow stromal stem cells (hBMSCs). Global gene expression profiling and Western blot analysis revealed activation of several intra-cellular signaling pathways including focal adhesion kinase (FAK) and TGFß. Pharmacological inhibition of FAK using PF-573228 (5 µM) and TGFß using SB505124 (1µM), diminished Apigenin- and Rutaecarpine-induced osteoblast differentiation. In vitro treatment with Apigenin and Rutaecarpine, of primary hBMSCs obtained from elderly female patients enhanced osteoblast differentiation compared with primary hBMSCs obtained from young female donors. Ex-vivo treatment with Apigenin and Rutaecarpine of organotypic embryonic chick-femur culture significantly increased bone volume and cortical thickness compared to control as estimated by µCT-scanning. Discussion: Our data revealed that Apigenin and Rutaecarpine enhance osteoblastic differentiation, bone formation, and reduce the age-related effects of hBMSCs. Therefore, Apigenin and Rutaecarpine cellular treatment represent a potential strategy for maintaining hBMSCs health during aging and osteoporosis.

Calcium Induces the Cleavage of NopA and Regulates the Expression of Nodulation Genes and Secretion of T3SS Effectors in Sinorhizobium fredii NGR234.

The type III secretion system (T3SS) is a key factor for the symbiosis between rhizobia and legumes. In this study, we investigated the effect of calcium on the expression and secretion of T3SS effectors (T3Es) in Sinorhizobium fredii NGR234, a broad host range rhizobial strain. We performed RNA-Seq analysis of NGR234 grown in the presence of apigenin, calcium, and apigenin plus calcium and compared it with NGR234 grown in the absence of calcium and apigenin. Calcium treatment resulted in a differential expression of 65 genes, most of which are involved in the transport or metabolism of amino acids and carbohydrates. Calcium had a pronounced effect on the transcription of a gene (NGR_b22780) that encodes a putative transmembrane protein, exhibiting a 17-fold change when compared to NGR234 cells grown in the absence of calcium. Calcium upregulated the expression of several sugar transporters, permeases, aminotransferases, and oxidoreductases. Interestingly, calcium downregulated the expression of nodABC, genes that are required for the synthesis of nod factors. A gene encoding a putative outer membrane protein (OmpW) implicated in antibiotic resistance and membrane integrity was also repressed by calcium. We also observed that calcium reduced the production of nodulation outer proteins (T3Es), especially NopA, the main subunit of the T3SS pilus. Additionally, calcium mediated the cleavage of NopA into two smaller isoforms, which might affect the secretion of other T3Es and the symbiotic establishment. Our findings suggest that calcium regulates the T3SS at a post-transcriptional level and provides new insights into the role of calcium in rhizobia-legume interactions.

Leveraging a Y. lipolytica naringenin chassis for biosynthesis of apigenin and associated glucoside.

Flavonoids are a diverse set of natural products with promising bioactivities including anti-inflammatory, anti-cancer, and neuroprotective properties. Previously, the oleaginous host Yarrowia lipolytica has been engineered to produce high titers of the base flavonoid naringenin. Here, we leverage this host along with a set of E. coli bioconversion strains to produce the flavone apigenin and its glycosylated derivative isovitexin, two potential nutraceutical and pharmaceutical candidates. Through downstream strain selection, co-culture optimization, media composition, and mutant isolation, we were able to produce168 mg/L of apigenin, representing a 46% conversion rate of 2-(R/S)-naringenin to apigenin. This apigenin platform was modularly extended to produce isovitexin by addition of a second bioconversion strain. Together, these results demonstrate the promise of microbial production and modular bioconversion to access diversified flavonoids.

Apigenin delays postovulatory oocyte aging by reducing oxidative stress through SIRT1 upregulation.

After ovulation, senescent oocytes inevitably experience reduced quality and defects in embryonic development. Apigenin (API) is a flavonoid with a wide range of pharmacological effects. Therefore, this study examined the protective effects of API on the quality of porcine oocytes during in-vitro ageing and the underlying mechanisms. The results showed that API treatment could reduce the activation rate after aging for 48 h. In addition, API significantly reduced reactive oxygen species, abnormal distribution of mitochondria, early apoptosis in ageing oocytes, increased glutathione, and mitochondrial adenosine triphosphate levels in ageing oocytes. Importantly, API increased the embryonic development rate in aged oocytes. We also examined molecular changes, finding decreased sirtuin 1 expression in in-vitro postovulatory oocytes, but API reversed this effect. Our results suggest that API attenuates the deterioration of oocyte quality during in-vitro ageing, possibly by reducing oxidative stress through the upregulation of sirtuin 1.

Novel Prognostic Model Construction of Tongue Squamous Cell Carcinoma Based on Apigenin-Associated Genes.

BACKGROUND: Clinical indexes are often selected as relevant factors for constructing prognostic models of tongue squamous cell carcinoma (TSCC) patients, while factors related to therapeutic targets are less frequently included. As Apigenin (API) shows anti-tumor properties in many tumors, in this study, we construct a novel prognostic model for TSCC patients based on Apigenin-associated genes through transcriptomic analysis. METHODS: The effect of Apigenin (API) on the cell characteristics of TSCC cells was measured by several phenotype experiments. RNA-seq was executed to ensure differentially expressed genes (DEGs) in squamous cell carcinoma-9 (SCC-9) cells after API treatment. Furthermore, reverse transcription quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry were performed to verify the expression of API-related genes. Then, combined with the gene expression data and relevant individual information of TSCC samples acquired from The Cancer Genome Atlas (TCGA), an API-related model was built through Lasso regression and multivariate Cox regression. A receiver operating characteristic (ROC) curve and a nomogram and calibration curve were created to forecast patient outcomes to improve the clinical suitability of the API-related signature. The relationships between the two risk groups and function enrichment, immune infiltration characteristics, and drug susceptibility were analyzed. RESULTS: We demonstrated that API could inhibit the malignant behavior of TSCC cells. Among API-related genes, TSCC cells treated with API, compared to the control group, have higher levels of transmembrane protein 213 (TMEM213) and G protein-coupled receptor 158 (GPR158), and lower levels of caspase 14 (CASP14) and integrin subunit alpha 5 (ITGA5). An 7 API-associated gene model was built through Lasso regression and multivariate Cox regression that could direct TSCC prognostic status and tumor immune cell infiltration. In addition, we acquired 6 potential therapeutic agents for TSCC based on the prognostic model. CONCLUSIONS: Our research suggested the inhibition effect of API on TSCC cells and provided a novel prognostic model combined with therapeutic factors that can guide the prognosis of TSCC and clinical decision-making in TSCC.

Rational design of mitochondria-targeted fluorescent biosensors for in vivo elucidation of the interaction between breast cancer metastasis and mitochondrial autophagy.

Breast cancer lung metastases (BCLM) are a major cause of high mortality in patients. The shortage of therapeutic targets and rapid drug screening tools for BCLM is a major challenge at present. Mitochondrial autophagy, which involves the degradation of proteins associated with cancer cell aggressiveness, represents a possible therapeutic approach for the treatment of BCLM. Herein, four fluorescent biosensors with different alkyl chains were designed and synthesized to monitor mitochondrial autophagy. Among them, PMV-12 demonstrated the highest sensitivity to viscosity variance, the least impact on polarity, and the longest imaging time. The introduction of the C12-chain made PMV-12 anchored in the mitochondrial membrane without being disturbed by changes of the mitochondrial membrane potential (MMP), thereby achieving the long-term monitor in situ for mitochondrial autophagy. Mitochondria stained with PMV-12 induced swelling and viscosity increase after treating with apigenin, which indicated that apigenin is a potential mitochondrial autophagy inducer. Apigenin was subsequently verified to inhibit cancer cell invasion by 92%. Furthermore, PMV-12 could monitor the process of BCLM in vivo and evaluate the therapeutic effects of apigenin. This work provides a fluorescent tool for elucidating the role of mitochondrial autophagy in the BCLM process and for anti-metastatic drug development.

The Effects of Apigenin in the Treatment of Diabetic Nephropathy: A Systematic Review of Non-clinical Studies.

PURPOSE: Diabetes is one of the important and growing diseases in the world. Among the most common diabetic complications are renal adverse effects. The use of apigenin may prevent the development and progression of diabetes-related injuries. The current study aims to review the effects of apigenin in the treatment of diabetic nephropathy. METHODS: In this review, a systematic search was performed based on PRISMA guidelines for obtaining all relevant studies on "the effects of apigenin against diabetic nephropathy" in various electronic databases up to September 2022. Ninety-one articles were obtained and screened in accordance with the predefined inclusion and exclusion criteria. Seven eligible articles were finally included in this review. RESULTS: The experimental findings revealed that hyperglycemia led to the decreased cell viability of kidney cells and body weight loss and an increased kidney weight of rats; however, apigenin administration had a reverse effect on these evaluated parameters. It was also found that hyperglycemia could induce alterations in the biochemical and renal function-related parameters as well as histopathological injuries in kidney cells or tissue; in contrast, the apigenin administration could ameliorate the hyperglycemia-induced renal adverse effects. CONCLUSION: The results indicated that the use of apigenin could mitigate diabetes-induced renal adverse effects, mainly through its antioxidant, anti-apoptotic, and anti-inflammatory activities. Since the findings of this study are based on experimental studies, suggesting the use of apigenin (as a nephroprotective agent) against diabetic nephropathy requires further clinical studies.

Multiomics Reveals Mechanisms of Alternaria oxytropis Inhibiting Pathogenic Fungi in Oxytropis ochrocephala.

Endophytic fungi can benefit the host plant and increase the plant resistance. Now, there is no in-depth study of how Alternaria oxytropis (A. oxytropis) is enhancing the ability of inhibiting pathogenic fungi in Oxytropis ochrocephala (O. ochrocephala). In this study, the fungal community and metabolites associated with endophyte-infected (EI) and endophyte-free (EF) O. ochrocephala were compared by multiomics. The fungal community indicated that there was more A. oxytropis, less phylum Ascomycota, and less genera Leptosphaeria, Colletotrichum, and Comoclathris in the EI group. As metabolic biomarkers, the levels of swainsonine and apigenin-7-O-glucoside-4-O-rutinoside were significantly increased in the EI group. Through in vitro validation experiments, swainsonine and apigenin-7-O-glucoside-4-O-rutinoside can dramatically suppress the growth of pathogenic fungi Leptosphaeria sclerotioides and Colletotrichum americae-borealis by increasing the level of oxidative stress. This work suggested that O. ochrocephala containing A. oxytropis could increase the resistance to fungal diseases by markedly enhancing the content of metabolites inhibiting pathogenic fungi.

Computational screening of natural MtbDXR inhibitors for novel anti-tuberculosis compound discovery.

DXR (1-deoxy-d-xylulose-5-phosphate reductoisomerase) is an essential enzyme in the Methylerythritol 4-phosphate (MEP) pathway, which is used by M. tuberculosis and a few other pathogens. This essential enzyme in the isoprenoid synthesis pathway has been previously reported as an important target for antibiotic drug design. However, till now, there is no record of any drug-like safe molecule to inhibit MtbDXR. Numerous plant species have been traditionally used for tuberculosis therapies. In this study, we selected six plant species with anti-tubercular properties. The chemoinformatic screening was performed on 352 phytochemicals from those plants against the MtbDXR protein. After molecular docking analysis, we filtered the top five compounds, CID: 5280443 (Apigenin), CID: 3220 (Emodin), CID: 5280863 (Kaempferol), CID: 5280445 (Luteolin), and CID: 6101979 (beta-Hydroxychalcone), based on binding affinity. Molecular dynamics simulations disclosed the stability of the compounds at the active site of the proteins. Finally, in silico ADME and toxicity evaluations confirmed the compounds to be effective and safe for oral administration. Thus, our findings identified three drug-like safe molecules- Apigenin, Kaempferol, and beta-Hydroxychalcone, that showed good stability in the protein's active site. The results of this computational approach may act as an initial instruction for future in vitro and in vivo testing to identify natural drug-like compounds to treat tuberculosis.Communicated by Ramaswamy H. Sarma.

The strong anti-hyaluronidase effect of ellagic acid markedly decreases polyspermy during in vitro fertilization, resulting in sustainment of the developmental potency in porcine oocytes.

The present study investigated the effects of ellagic acid, a type of polyphenol that does not have a glycan and is composed of four hydroxyl groups and two lactone functional groups, on porcine in vitro fertilization (IVF) by focusing on its anti-hyaluronidase activity. A comparative analysis of ellagic acid and apigenin, which is commonly used as a hyaluronidase inhibitor, was performed. It compared the effects of ellagic acid and apigenin on hyaluronidase activity at different concentrations. The results showed that 10, 20, and 40 µM ellagic acid strongly reduced hyaluronidase activity (P < 0.05). The addition of 20 µM ellagic acid, but not apigenin, to porcine IVF medium effectively reduced polyspermy without decreasing sperm penetration or the formation rates of male pronuclei in cumulus-free oocytes. However, neither ellagic acid nor apigenin affected the number of sperm that bound to zona pellucida (ZP) or the induction of zona hardening and protease resistance. The percentage of acrosome-reacting sperm that bound to the ZP was markedly lower in the presence of 20 µM ellagic acid than in the untreated and apigenin-treated groups, even though the antioxidant capacity of ellagic acid was weaker than that of apigenin. Furthermore, a markedly higher percentage of embryos developed to the blastocyst stage in the ellagic acid-treated group, and the apoptotic indexes of expanded blastocysts produced by the ellagic acid treatment during IVF were significantly low. Therefore, the anti-hyaluronidase effect of ellagic acid markedly suppressed the induction of the acrosome reaction in sperm that bound to the ZP, resulting in a marked decrease in polyspermy under conditions that maintained high sperm penetrability during IVF and sustainment of the developmental potency in porcine oocytes.

Intercropping of Narrow-Leafed Lupin (Lupinus angustifolius L.) and Barley (Hordeum vulgare L.) Affects the Flavonoid Composition of Both Crops.

Barley (Hordeum vulgare L.) is a common cereal crop in agricultural production and is often included in legume-cereal intercropping. Flavonoids, a major class of secondary metabolites found in barley, are involved in plant defense and protection. However, the effect of intercropping on barley flavonoids remains unknown. Herein, an intercropping system involving barley and lupin (Lupinus angustifolius L.) was studied. Intercropping increased the level of luteolin in lupin roots. Lupin-barley intercropping considerably increased genistein, rutin, and apigenin in barley shoots. Genistein and apigenin were also detected in intercropped barley roots and rhizosphere soil. The three flavonoids have been reported as defense compounds, suggesting that lupin triggers a defense response in barley to strengthen its survival ability.

Screening of an FDA-approved compound library identifies apigenin for the treatment of myocardial injury.

Apigenin is the active ingredient in Ludangshen. Although previous studies reported the cardioprotective actions of apigenin against doxorubicin (Dox)-induced cardiomyopathy, the underlying mechanisms remain incompletely understood. Since apigenin beneficially regulates various aspects of mitochondrial function and dynamics, we asked whether apigenin improves heart function in mice with Dox-induced cardiomyopathy by regulating the mitochondrial unfolded protein response (UPRmt). Co-administration of apigenin significantly restored heart function, reduced myocardial swelling, inhibited cardiac inflammation, increased cardiac transcription of UPRmt-related genes, and promoted cardiomyocyte survival in Dox-treated mice. In turn, blockade of UPRmt abolished the mito- and cytoprotective effects of apigenin, evidenced by decreased ATP production, suppressed mitochondrial antioxidant capacity, and increased apoptosis, in Dox-treated, cultured HL-1 cardiomyocytes. Furthermore, apigenin treatment prevented Dox-induced downregulation of Sirt1 and Atf5 expression, and the beneficial effects of apigenin were completely nullified in Sirt1 knockout (KO) mice or after siRNA-mediated Sirt1 knockdown in vitro. We thus provide novel evidence for a promotive effect of apigenin on UPRmt via regulation of the Sirt1/Atf5 pathway. Our findings uncover that apigenin seems to be an effective therapeutic agent to alleviate Dox-mediated cardiotoxicity.

Enhancing Glycosylation of Flavonoids by Engineering the Uridine Diphosphate Glucose Supply in Escherichia coli.

Glycosylation can enhance the solubility and stability of flavonoids. The main limitation of the glycosylation process is low intracellular uridine diphosphate glucose (UDPG) availability. This study aimed to create a glycosylation platform strain in Escherichia coli BL21(DE3) by multiple metabolic engineering of the UDPG supply. Glycosyltransferase TcCGT1 was introduced to synthesize vitexin and orientin from apigenin and luteolin, respectively. To further expand this glycosylation platform strain, not only were UDP rhamnose and UDP galactose synthesis pathways constructed, but rhamnosyltransferase (GtfC) and galactosyltransferase (PhUGT) were also introduced, respectively. In a 5 L bioreactor with apigenin, luteolin, kaempferol, and quercetin as glycosyl acceptors, vitexin, orientin, afzelin, quercitrin, hyperoside, and trifolin glycosylation products reached 17.2, 36.5, 5.2, 14.1, 6.4, and 11.4 g/L, respectively, the highest titers reported to date for all. The platform strain has great potential for large-scale production of glycosylated flavonoids.

The flavonoids fisetin, apigenin, kaempferol, naringenin, naringin regulate respiratory activity and membrane potential of rat liver mitochondria and inhibit oxidative processes in erythrocytes.

Flavonoids, secondary plant metabolites, represent the most abundant heterogeneous group of phytochemicals. The aim of this study to compare antioxidant activity and regulatory properties of several representatives of different classes of flavonoids, fisetin, apigenin, kaempferol, naringenin, naringin, using liver mitochondria and erythrocytes as research objects. In the concentration range of 2.5-25 µM fisetin, apigenin, kaempferol, naringenin, and naringin dose-dependently prevented oxidative damage of erythrocytes induced by 700 µM tert-butyl hydroperoxide: accumulation of lipid peroxidation (LPO) products and oxidation of glutathione GSH. The IC50 values corresponding to the flavonoid concentration inhibiting the LPO process in erythrocyte membranes by 50%, were 3.9±0.8 µM in the case of fisetin, 6.5±1.6 µM in the case of kaempferol, 8.1±2.1 µM in the case of apigenin, 37.8±4.4 µM in the case of naringenin, and 64.7±8.6 µM in the case of naringin. The antioxidant effect of flavonoids was significantly higher in the membrane structures compared to the cytoplasm of cells. All flavonoids studied (10-50 µM) effectively inhibited the respiratory activity of isolated rat liver mitochondria and, with the exception of kaempferol, stimulated Ca²âº-induced dissipation of the mitochondrial membrane potential. Cyclosporine A and ruthenium red inhibited flavonoid-stimulated Ca²âº-dependent membrane depolarization, thus indicating that the mitochondrial calcium uniporter and the mitochondrial permeability transition pore opening were involved in the flavonoid effects. Flavonoids, as the redox-active compounds with antioxidant properties, are able to regulate mitochondrial potential and respiratory activity, and prevent mitochondrial oxidative stress. They can be considered as effective pharmacological agents or nutraceuticals.

Oxidation of Naringenin, Apigenin, and Genistein by Human Family 1 Cytochrome P450 Enzymes and Comparison of Interaction of Apigenin with Human P450 1B1.1 and Scutellaria P450 82D.1.

Naringenin, an initial synthesized flavanone in various plant species, is further utilized for production of many biologically active flavonoids, e.g., apigenin, eriodictyol, and genistein, by various plant enzymes including cytochrome P450s (P450s or CYPs). We examined how these flavonoids are oxidized by human P450 family 1 and 2A enzymes. Naringenin was principally oxidized at the 3'-position to form eriodictyol by CYP1 enzymes more efficiently than by CYP2A enzymes, and the resulting eriodictyol was further oxidized to two penta-hydroxylated products. In contrast to plant P450 enzymes, these human P450s did not mediate the desaturation of naringenin and eriodictyol to give apigenin and luteolin, respectively. Apigenin was oxidized at the C3' and C6 positions to form luteolin and scutellarein by these P450s. CYP1B1.1 and 1B1.3 had high activities in apigenin 6-hydroxylation with a homotropic cooperative manner, as has been observed previously in chrysin 6-hydroxylation (Nagayoshi et al., Chem. Res. Toxicol. 2019, 32, 1268-1280). Molecular docking analysis suggested that CYP1B1 had two apigenin binding sites and showed similarities in substrate recognition sites to plant CYP82D.1, one of the enzymes in catalyzing apigenin and chrysin 6-hydroxylations in Scutellaria baicalensis. The present results suggest that human CYP1 enzymes and CYP2A13 in some reactions have important roles in the oxidation of naringenin, eriodictyol, apigenin, and genistein and that human CYP1B1 and Scutellaria CYP82D.1 have similarities in their SRS regions, catalyzing 6-hydroxylation of both apigenin and chrysin.

Apigenin inhibits angiogenesis in retinal microvascular endothelial cells through regulating of the miR-140-5p/HDAC3-mediated PTEN/PI3K/AKT pathway.

BACKGROUND: Diabetic retinopathy (DR) is a common cause of visual impairment. Apigenin has been shown to have antiangiogenic effects in various diseases. Our study aimed to investigate the role of apigenin in DR and elucidate the underlying mechanism. METHODS: Human retinal microvascular endothelial cells (HRMECs) were exposed to high glucose (HG) to establish a DR model. HRMECs were treated with apigenin. Then we knocked down or overexpressed miR-140-5p and HDAC3, and added PI3K/AKT inhibitor LY294002. The expression levels of miR-140-5p, HDAC3, and PTEN were measured using qRT-PCR. Western blot analysis was performed to assess the expression of HDAC3, PTEN, and PI3K/AKT pathway-related proteins. Finally, cell proliferation and migration were evaluated using MTT, wound-healing assay, and transwell assay, while angiogenesis was examined using the tube formation assay. RESULTS: HG treatment resulted in reduced miR-140-5p expression and overexpression of miR-140-5p suppressed proliferation, migration, and angiogenesis of the HG-induced HRMECs. Apigenin treatment significantly restored the decreased level of miR-140-5p caused by HG treatment and inhibited proliferation, migration, and angiogenesis of the HG-induced HRMECs by upregulating miR-140-5p. Moreover, miR-140-5p targeted HDAC3, and overexpression of miR-140-5p reversed the HG-inducted upregulation of HDAC3 expression. HDAC3 was found to bind to the promoter region of PTEN, inhibiting its expression. Knockdown of HDAC3 suppressed the PI3K/AKT pathway by elevating PTEN expression. Furthermore, apigenin inhibited angiogenesis in DR cell models through the regulating of the miR-140-5p/HDAC3-mediated PTEN/PI3K/AKT pathway. CONCLUSIONS: Apigenin effectively suppressed angiogenesis in HG-induced HRMECs by modulating the miR-140-5p/HDAC3-mediated PTEN/PI3K/AKT pathway. Our study may contribute to the development of novel therapeutic approaches and identification of potential targets for the treatment of DR.

Antioxidant and Hepatoprotective Potential of Echinops ritro L. Extracts on Induced Oxidative Stress In Vitro/In Vivo.

Echinops ritro L. (Asteraceae) is traditionally used in the treatment of bacterial/fungal infections and respiratory and heart ailments. The aim of this study was to evaluate the potential of extracts from E. ritro leaves (ERLE) and flowering heads (ERFE) as antioxidant and hepatoprotective agents on diclofenac-induced lipid peroxidation and oxidative stress under in vitro and in vivo conditions. In isolated rat microsomes and hepatocytes, the extracts significantly alleviated oxidative stress by increasing cell viability and GSH levels and reducing LDH efflux and MDA production. During in vivo experiments, the administration of the ERFE alone or in combination with diclofenac resulted in a significant increase in cellular antioxidant protection and a decrease in lipid peroxidation witnessed by key markers and enzymes. A beneficial influence on the activity of the drug-metabolizing enzymes ethylmorphine-N-demetylase and aniline hydroxylase in liver tissue was found. In the acute toxicity test evaluation, the ERFE showed no toxicity. In the ultrahigh-performance liquid chromatography-high-resolution mass spectrometry analysis, 95 secondary metabolites were reported for the first time, including acylquinic acids, flavonoids, and coumarins. Protocatechuic acid O-hexoside, quinic, chlorogenic and 3, 5-dicaffeoylquinic acid, apigenin; apigenin 7-O-glucoside, hyperoside, jaceosidene, and cirsiliol dominated the profiles. The results suggest that both extracts should be designed for functional applications with antioxidant and hepatoprotective capacity.