Aromatase; Synthetic and natural flavonoid
Alpha-Naphthoflavone targets multiple enzymes and receptors. It antagonizes the aryl hydrocarbon receptor (AhR), blocking the expression of phase I and II genes at nanomolar concentrations, although it can agonize AhR at higher concentrations (10 μM). The compound is an inhibitor of CYP1A1 gene expression. It is a potent and competitive aromatase inhibitor with IC50 and Ki values of 0.5 and 0.2 μM respectively. Alpha-Naphthoflavone is a CYP1A2 inhibitor.

The effect of α-naphthoflavone (α-NF) on vascular function was studied in isolated ring segments of the rat thoracic aorta and in primary cultures of human umbilical vein endothelial cells (HUVECs). α-NF induced concentration-dependent relaxation of the phenylephrine-precontracted aorta endothelium-dependently and -independently at lower and higher concentrations, respectively. The cGMP, but not cAMP, content was increased significantly in α-NF-treated aorta. Pretreatment with N ω-nitro-L-arginine methyl ester (L-NAME) or methylene blue attenuated both α-NF induced vasorelaxation and the increase of cGMP content significantly. The increase of cGMP content induced by α-NF was also inhibited by chelating extracellular Ca2+ with EGTA. These results suggest that the endothelium-dependent vasorelaxation induced by α-NF is mediated most probably through Ca2+-dependent activation of NO synthase and guanylyl cyclase. In HUVECs, α-NF induced concentration-dependent formation of NO and Ca2+ influx. α-NF-induced NO formation was abolished by removal of extracellular Ca2+ and by pretreatment with the Ca2+ channel blockers SKF 96365 and Ni2+, but not by the L-type Ca2+ channel blocker verapamil. The Ca2+ influx, as measured by 45Ca2+ uptake, induced by α-NF was also inhibited by SKF 96365 and Ni2+. Our data imply that α-NF, at lower concentrations, induces endothelium-dependent vasorelaxation by promoting extracellular Ca2+ influx in endothelium and the activation of the NO-cGMP pathway[2].
In vitro, Alpha-Naphthoflavone is a potent and competitive aromatase inhibitor with IC50 and Ki values of 0.5 and 0.2 μM respectively. It antagonizes AhR, blocking the expression of phase I and II genes at nanomolar concentrations. The compound inhibits CYP1A1 gene expression. It shows selective binding to P4503A4. Alpha-Naphthoflavone is a synthetic flavonoid found naturally in Passiflora.

Non-alcoholic fatty liver disease (NAFLD) is a chronic liver disease. The literature suggests that the aryl hydrocarbon receptor (AHR) may be a key player in the pathogenesis of NAFLD, and it can modulate the synthesis of cytochrome P450 1A1 (CYP1A1) and tumor necrosis factor-α (TNF-α). Previous studies have shown that CYP1A1 is a key enzyme of oxidative stress, TNF-α is involved in the formation of insulin resistance (IR), oxidative stress and insulin resistance are the key factors for the formation of NAFLD. Therefore, it can be said that AHR may participate in contributing to NAFLD by regulating CYP1A1 and TNF-α. Alpha-naphthoflavone (ANF) is an effective AHR inhibitor. The present study was designed to explore the hepatoprotective effect of ANF in high fat diet (HFD)-induced NAFLD mice and oleic acid (OA)-treated HepG2 hepatocytes. Mice were fed HFD to induce NAFLD, HepG2 cells were exposed to OA to induce hepatocyte injury, and ANF significantly reduced mouse and cellular liver damage compared to the HFD-induced NAFLD and OA-treated HepG2 hepatocytes. ANF treatment reduces liver damage by reducing ROS and IR, the data show that ANF inhibits the expression of AHR, CYP1A1 and TNF-α in NAFLD. Taken together, these findings show that ANF alleviate NAFLD via regulation of AHR/CYP1A1 and AHR/TNF-α pathways, which may have potential for further development as novel therapeutic agents for NAFLD[4].
In vivo, Alpha-Naphthoflavone has been studied for its effects on xenobiotic metabolism through AhR modulation and enzyme inhibition. As an aromatase inhibitor, it may have potential for hormone-dependent cancer research. The compound's AhR antagonism and CYP inhibition suggest applications in toxicology and drug metabolism research. Further in vivo studies are needed to fully characterize its therapeutic potential.

Biochemical analysis[4]
According to the manufacturer's instructions. Detection of aspartate aminotransferase (AST), alanine aminotransferase (ALT), triglyceride (TG) and total cholesterol (TC), catalase (CAT), glutathione (GSH), malondialdehyde (MDA) and superoxide dismutase (SOD) enzyme activity using a commercial assay kit.

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Eleven flavonoid compounds were compared with aminoglutethimide (AG), a pharmaceutical aromatase inhibitor, for their abilities to inhibit aromatase enzyme activity in a human preadipocyte cell culture system. Flavonoids exerting no effect on aromatase activity were catechin, daidzein, equol, genistein, beta-naphthoflavone (BNF), quercetin and rutin. The synthetic flavonoid, alpha-naphthoflavone (ANF), was the most potent aromatase inhibitor, with an I50 value of 0.5 microM. Three naturally-occurring flavonoids, chrysin, flavone, and genistein 4'-methyl ether (Biochanin A) showed I50 values of 4.6, 68, and 113 microM, respectively, while AG showed an I50 value of 7.4 microM. Kinetic analyses showed that both AG and the flavonoids acted as competitive inhibitors of aromatase. The Ki values, indicating the effectiveness of inhibition, were 0.2, 2.4, 2.4, 22, and 49 microM, for ANF, AG, chrysin, flavone, and Biochanin A, respectively. Chrysin, the most potent of the naturally-occurring flavonoids, was similar in potency and effectiveness to AG, a pharmaceutical aromatase inhibitor used clinically in cases of estrogen-dependent carcinoma. These data suggest that flavonoid inhibition of peripheral aromatase activity may contribute to the observed cancer-preventive hormonal effects of plant-based diets[1].
In vitro enzyme assays for Alpha-Naphthoflavone involve measuring aromatase inhibition. Aromatase activity is assessed by measuring the conversion of androstenedione to estrone using radiometric or fluorometric methods. The compound's IC50 of 0.5 μM and Ki of 0.2 μM are determined through dose-response curves. AhR binding is assessed using reporter gene assays or DNA binding assays. CYP1A1 and CYP1A2 inhibition is measured using specific substrates. Assays are performed in appropriate buffer systems with positive controls such as known aromatase inhibitors.

Cell culture and cell treatment[4]
Human hepatocellular carcinoma cell line HepG2 (ATCC, US) was cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution. The culture was maintained at 37 °C in a humidified 5% CO2 incubator. To induce excessive lipid accumulation in an in vitro hepatic steatosis model, The cells were divided into 6 groups: (1) control group (C), (2) model control group (M), (3) model control group containing α-Naphthoflavone/ANF (5 μM) (ANF5) (5), (4) model control group ANF (10 μM) (ANF10), (5) model control group containing ANF (20 μM), (6) model control group containing ANF (40 μM) (ANF40), except for the control group, the remaining groups of cells were exposed in medium, and the medium was supplemented with 0.1 mM oleic acid for 48 h. ANF was dissolved in dimethyl sulfoxide (DMSO) and cells were incubated with different concentrations of ANF (0/5/10/20/40 μM) during the last 24 h of the 48 h treatment.

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MTT assay[4]
HepG2 cells (1 × 104cells/mL) were seeded in 96-well plates, and inhibition experiments were carried out in the presence of 5 μM, 10 μM, 20 μM, 40 μM α-Naphthoflavone/ANF and 0.1 mM oleic acid. Cells were incubated in the incubator for 48 h before detection.

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Human papilloma viruses 16 and 18 express E6 and E7 oncoproteins. E6 activates and redirects E6-associated protein (E6AP), an E3 ubiquitin ligase. E6AP interacts with Ube2l3, an E2 ubiquitin conjugating enzyme protein (also known as UbcH7), to promote p53 ubiquitination and degradation by the 26S proteasome. Therefore, blocking E6-mediated p53 degradation might be an alternative treatment for cervical cancer. In addition, activation of the aryl hydrocarbon receptor (AHR) induces Ube2l3 expression, resulting in p53 ubiquitination and degradation. The aim of the present study was to determine whether inhibition of AHR in HeLa cells resulted in an increase in p53 and apoptosis along with a decrease in cell proliferation. The results demonstrate that two AHR antagonists, α-naphthoflavone (α-NF) and resveratrol, decreased cell proliferation, arrested cells in the gap 1/synthesis (G1/S) phases, and increased p53 levels and apoptosis. However, knocking out the Ahr gene did not abrogate the effects of α-NF and resveratrol. Moreover, Ahr-null cells presented similar cell proliferation rates and apoptosis levels when compared to control HeLa cells. Taken together, the results indicate that α-NF's and resveratrol's cytostatic and cytotoxic actions, respectively, occur through an AHR-independent mechanism, and that AHR is not required for HeLa cell proliferation[3].
In vitro cell-based assays for Alpha-Naphthoflavone are conducted in various cell lines. Cells are cultured in appropriate media at 37°C with 5% CO2 and treated with the compound at varying concentrations. AhR activity is assessed by measuring CYP1A1 gene expression. Aromatase inhibition is assessed by measuring estrogen production. Cell viability is assessed by standard assays. Experiments are performed in triplicate with appropriate positive and negative controls.

Male C57 mice (18–20 g, 6–8 weeks) were housed in plastic cages, casual food and water, and maintained at room temperature (25 ± 2 °C) under a 12 h light/dark cycle. After one week of adaptation, animals were randomized into 4 groups (6 in each group): (1) normal control group (C), (2) high-fat diet control group (M), (3) high-fat diet containing 80 mg/kg/day α-Naphthoflavone/ANF (ANF80), (4) high-fat diet containing 160 mg/kg/day ANF (ANF160). Control mice were fed a normal diet (fat 10% calories, protein 20% calories, carbohydrate 70% calories; 3.5 Kcal/g diet), and the other 3 groups of mice fed a high-fat diet (fat 42% calories), protein 15% calories, carbohydrate calories 43%; diet 4.5 Kcal/g). The total feeding duration is 12 weeks. The normal control group was fed a normal diet and the remaining 3 groups were fed a high-fat diet. Three groups of mice fed high-fat diets were given different ANF doses (0, 80, 160 mg/kg/day) by gavage in the last four weeks of feeding. After 12 weeks, the mice were euthanized and tissue samples and blood were collected for further analysis.[4]
Alpha-Naphthoflavone in vivo studies are conducted in animal models of hormone-dependent cancers and toxicology. Animals are treated with Alpha-Naphthoflavone via oral administration or injection. For cancer studies, tumor growth is monitored. For toxicology studies, xenobiotic metabolism and enzyme activities are assessed. Animals are monitored for clinical signs. Tissues and blood samples are collected for histopathological and biomarker analysis at study endpoints. Studies are conducted in accordance with institutional animal care guidelines.

Alpha-Naphthoflavone (MW 272.30 g/mol, C19H12O2) has a melting point of 154.0 to 158.0°C. It appears as a white to yellow to orange powder to crystal. Its purity is min. 98.0 area% by HPLC. The compound is soluble in hot ethanol. Alpha-Naphthoflavone is a synthetic flavonoid and AhR antagonist. Pharmacokinetic parameters such as half-life, bioavailability, and tissue distribution would be determined in species-specific studies.

Alpha-Naphthoflavone is generally well-tolerated in preclinical studies. The compound is a synthetic flavonoid with established safety profiles. Its AhR antagonism and enzyme inhibition activities have been demonstrated with acceptable safety profiles. No significant adverse effects have been reported in the available literature at research-use concentrations. The compound is intended for research use only. Standard safety precautions should be followed when handling. Comprehensive toxicological evaluation would be required for therapeutic development.

[1]. Flavonoid inhibition of aromatase enzyme activity in human preadipocytes. J Steroid Biochem Mol Biol. 1993 Sep;46(3):381-8.

[2]. Alpha-naphthoflavone induces vasorelaxation through the induction of extracellular calcium influx and NO formation in endothelium. Naunyn Schmiedebergs Arch Pharmacol. 2003 Nov;368(5):377-85.

[3]. Apoptosis induction and inhibition of HeLa cell proliferation by alpha-naphthoflavone and resveratrol are aryl hydrocarbon receptor-independent. Chem Biol Interact. 2018 Feb 1;281:98-105.

[4]. Alpha-naphthoflavone attenuates non-alcoholic fatty liver disease in oleic acid-treated HepG2 hepatocytes and in high fat diet-fed mice. Biomed Pharmacother. 2019 Oct;118:109287.

α-Naphthylflavonoid is an extended flavonoid compound formed by the fusion of a benzene ring with the h-side of a flavonoid. It is a synthetic compound and an inhibitor of aromatase (EC 1.14.14.14). It possesses multiple functions, including as an aromatase inhibitor, aryl hydrocarbon receptor antagonist, and aryl hydrocarbon receptor agonist. It is an organic heterocyclic tricyclic compound belonging to the extended flavonoid and naphtho-γ-pyranone classes. α-Naphthylflavonoid has been reported to exist in Rhaponticum repens, and relevant data are available.
Alpha-Naphthoflavone (7,8-benzoflavone) is a synthetic flavonoid found in Passiflora that modulates xenobiotic metabolism. It antagonizes AhR (blocks phase I/II gene expression) and is a potent aromatase inhibitor (IC50 = 0.5 μM, Ki = 0.2 μM). The compound inhibits CYP1A1 and CYP1A2. Its molecular formula is C19H12O2 with a molecular weight of 272.30 g/mol. All applications are limited to non-human research use.

C19H12O2

272.2974

272.083

604-59-1

11790

White to light yellow solid powder

1.3±0.1 g/cm3

460.9±45.0 °C at 760 mmHg

153-157 °C(lit.)

215.8±22.3 °C

0.0±1.1 mmHg at 25°C

1.695

4.79

0

2

1

21

433

0

VFMMPHCGEFXGIP-UHFFFAOYSA-N

InChI=1S/C19H12O2/c20-17-12-18(14-7-2-1-3-8-14)21-19-15-9-5-4-6-13(15)10-11-16(17)19/h1-12H

2-phenylbenzo[h]chromen-4-one

alpha-Naphthoflavone; 7,8-Benzoflavone; 604-59-1; 2-Phenyl-4H-benzo[h]chromen-4-one; alpha-Naphthylflavone; 2-phenylbenzo[h]chromen-4-one; Benzo(h)flavone; 4H-Naphtho[1,2-b]pyran-4-one, 2-phenyl-;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~25 mg/mL (~91.81 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)