Cell Viability Protection: In human neuronal SH-SY5Y cells exposed to 20 μM H₂O₂ for 24 hours (oxidative stress model), post-treatment with β-Naphthoflavone (0-20 μM range tested) for 24 hours significantly reversed H₂O₂-inhibited cell viability in a dose-dependent manner. The maximal effect was observed at 10 μM, with higher doses showing a slight decrease in efficacy. At 10 μM, β-Naphthoflavone significantly increased cell viability compared to H₂O₂-only treated cells (p < 0.05). [2]
Apoptosis Inhibition: H₂O₂ treatment increased the apoptosis rate of SH-SY5Y cells to 24.1% (approximately 5-fold increase). Post-treatment with 10 μM β-Naphthoflavone significantly decreased the apoptosis rate compared to H₂O₂-only treated cells (p < 0.05). Co-treatment with α-Naphthoflavone (20 μM) and β-Naphthoflavone (10 μM) reduced apoptosis to 7.2% (p < 0.01 vs. H₂O₂ only). [2]
Total Antioxidant Capacity: H₂O₂ treatment significantly decreased TAC in SH-SY5Y cells (p < 0.01). Post-treatment with 10 μM β-Naphthoflavone significantly reversed this reduction (p < 0.05 vs. H₂O₂ only). Co-treatment with α- and β-Naphthoflavone showed the most marked effect. [2]
Malondialdehyde Levels: H₂O₂ treatment increased MDA levels to approximately 2.5-fold higher than control (13.24 μmol/mg protein). Post-treatment with 10 μM β-Naphthoflavone significantly decreased MDA to 9.01 μmol/mg protein (p < 0.05 vs. H₂O₂ only). Co-treatment reduced MDA to 6.74 μmol/mg protein. [2]
Antioxidant Enzyme Activities: H₂O₂ decreased catalase activity by 82%, SOD by 79%, and GPx by 63% compared to control. β-Naphthoflavone (10 μM) significantly increased CAT and SOD enzyme activities compared to H₂O₂-only treated cells (p < 0.05). GPx activity was not significantly recovered by β-Naphthoflavone alone but was effectively reversed by co-treatment with α- and β-Naphthoflavone. [2]
p38 MAPK Phosphorylation: H₂O₂ increased p38 MAPK phosphorylation at Thr180 and Tyr182 by 2.2-fold (p < 0.05 vs. control). Post-treatment with 10 μM β-Naphthoflavone significantly repressed this phosphorylation (p < 0.05 vs. H₂O₂ only). Phosphorylation at Thr322 was unaffected. [2]
Apoptosis-Related Protein Expression: H₂O₂ significantly increased Bax expression (p < 0.01), cytochrome c release (p < 0.01), and the ratio of cleaved to non-cleaved caspase-3 (p < 0.01). β-Naphthoflavone treatment significantly decreased these apoptosis markers (p < 0.05 vs. H₂O₂ only), with β-Naphthoflavone being more effective than α-Naphthoflavone, particularly in inhibiting cytochrome c expression. [2]
Comparison with α-Naphthoflavone: β-Naphthoflavone was more effective than α-Naphthoflavone against H₂O₂-induced neuron damage in several parameters including cell viability recovery, apoptosis reduction, catalase activity upregulation, and inhibition of cytochrome c release. [2]
Synergistic Effect with α-Naphthoflavone: Co-treatment with α-Naphthoflavone (20 μM) and β-Naphthoflavone (10 μM) produced more marked effects than either compound alone in multiple assays, suggesting a synergistic neuroprotective effect. [2]
Lack of Toxicity: No harmful effects of β-Naphthoflavone were observed in the concentration range used in this study (up to 20 μM). [2]

Protection Against Aristolochic Acid I-Induced Acute Kidney Injury: Male C57BL/6 mice pretreated with BNF (80 mg/kg, i.p., daily for 3 days) before AAI administration (10 mg/kg, i.p.) showed significantly reduced renal damage compared to AAI-only treated mice. BNF pretreatment prevented increases in blood urea nitrogen and serum creatinine at day 7 post-AAI (p < 0.01 vs. AAI group), with levels comparable to control group. [3]
Histopathological Protection: Kidney histology (H&E staining) showed that BNF pretreatment greatly reduced AAI-induced tubular necrosis, tubular dilation, granular and hyaline casts, and interstitial fibrosis at days 3, 7, and 14 post-AAI. [3]
Inhibition of Apoptosis: TUNEL assay revealed that BNF pretreatment significantly inhibited AAI-induced apoptosis in tubular epithelial cells (p < 0.01 vs. AAI group at day 3). [3]
Prevention of Fibrosis: Immunohistochemistry for α-SMA (a marker of myofibroblasts) showed that BNF pretreatment prevented AAI-induced tubulointerstitial fibrosis at day 7 (p < 0.01 vs. AAI group). [3]
Pharmacokinetics of AAI: BNF pretreatment markedly decreased the exposure level of AAI in mice. Pharmacokinetic parameters after a single i.p. dose of 10 mg/kg AAI: Cmax decreased from 8.73 ± 1.57 to 3.08 ± 0.61 μg/mL (p < 0.01); tmax decreased from 16.00 ± 5.48 to 6.25 ± 2.50 min (p < 0.05); AUC decreased from 418.37 ± 126.09 to 149.84 ± 20.30 min·μg/mL (p < 0.05); t1/2 unchanged (27.55 ± 3.37 vs. 31.46 ± 8.80 min, not significant). Serum levels of AAIA (the major metabolite) were higher in BNF-pretreated mice. [3]
Tissue Distribution of AAI and Metabolites: At 30 minutes post-AAI injection, BNF pretreatment resulted in a 3-fold reduction in AAI levels in both liver and kidney (p < 0.01). AAIA levels were not significantly affected by BNF pretreatment. ALI (aristolactam I) levels were decreased in both liver and kidney after BNF pretreatment (p < 0.05 for liver, p < 0.01 for kidney). [3]
CYP1A Induction (mRNA): Real-time RT-PCR showed that after BNF treatment (80 mg/kg daily for 3 days), liver mRNA levels increased: CYP1A1 ~4-fold (p < 0.01), CYP1A2 ~16-fold (p < 0.01), CPR ~3-fold (p < 0.05). In kidney, only CYP1A1 mRNA showed a ~2-fold increase (p < 0.05); CYP1A2 and CPR mRNA were not significantly changed. [3]
CYP1A Induction (Protein): Western blot analysis confirmed that BNF treatment greatly upregulated CYP1A1, CYP1A2, and CPR protein levels in liver microsomes. In kidney microsomes, only CYP1A1 protein was induced; CYP1A2 and CPR protein levels were not significantly changed. [3]

Catalase Activity Assay: Cell homogenates were placed in a cuvette containing 250 mM H₂O₂ for 1-5 minutes. The remaining H₂O₂ was coupled with a chromogenic substrate to generate a red product (N-4-antipyryl-3-chloro-5-sulfonate-p-benzoquinonemonoimine) with maximal absorption at 520 nm. CAT activity was determined by calculating the amount of H₂O₂ that reacted with the enzyme. [2]
Superoxide Dismutase Activity Assay: SOD in samples inhibited the transformation of WST-8 to a water-soluble formazan. This inhibition was evaluated by measuring optical density at 450 nm to determine SOD activity. [2]
Glutathione Peroxidase Activity Assay: GPx activity was determined based on NADPH consumption in the coupled reaction where reduced glutathione is converted to oxidized glutathione (by GPx) and then back to reduced glutathione (by glutathione reductase). Decreased NADPH absorbance at 340 nm indirectly estimated GPx activity. [2]

Cell Culture: Human neuron SH-SY5Y cells were maintained in DMEM containing 10% FBS and 100 U/mL streptomycin/penicillin in a 5% CO₂ incubator at 37°C. [2]
Cell Viability Assay (CCK-8): SH-SY5Y cells were seeded in 96-well plates at 1 × 10⁴ cells/well and cultured in serum-free DMEM at 37°C for 48 hours. Cells were subjected to H₂O₂ (0-320 μM) for 24 hours to establish oxidative stress. In independent experiments, cells were exposed to 20 μM H₂O₂ for 24 hours followed by treatment with β-Naphthoflavone (0-20 μM), α-Naphthoflavone (20 μM), or combination for 24 hours. For p38 MAPK inhibition studies, cells were treated with 0.5 μM SB203580 for 24 hours. CCK-8 solution (10 μL) was added to each well, incubated for 5-8 hours at 37°C, and optical density measured at 450 nm. [2]
Apoptosis Rate Analysis (Flow Cytometry): SH-SY5Y cells (~2 × 10⁵) were stained with 5 μL Annexin V-FITC and 5 μL propidium iodide for 15 minutes at room temperature in the dark. Apoptosis was analyzed using a FACSCalibur flow cytometer and ModFit LT software. [2]
Total Antioxidant Capacity Assay: Cells were lysed with RIPA buffer. TAC was measured using the ABTS method. ABTS was incubated with H₂O₂ and metmyoglobin to produce ABTS•⁺ radical cation. Antioxidants in the sample suppressed color production proportionally. Trolox was used as standard. Results expressed as μmol Trolox equivalent/mg protein. [2]
Malondialdehyde Assay: Cell supernatant (2 mL) was mixed with 2 mL 0.6% thiobarbituric acid, incubated in boiling water for 15 minutes, cooled on ice, and optical density measured at 532 nm. Results expressed as nmol MDA/mg protein. [2]
Western Blot Analysis: Cells were lysed with RIPA buffer containing phosphatase inhibitors. Protein concentration was measured by BCA assay. Proteins (~20 μg) were separated by 10% SDS-PAGE, transferred to PVDF membranes, blocked with 5% non-fat milk, and probed with primary antibodies against p38 MAPK, phospho-p38 (Thr180+Tyr182), phospho-p38 (Thr322), cytochrome c, Bax, caspase-3, and GAPDH. Membranes were incubated with HRP-conjugated secondary antibodies and scanned for optical density quantification. [2]

Animals:** Male C57BL/6 mice (6 weeks old, 18-22 g) were obtained from Shanghai Laboratory Animal Center. All animal experiments were approved by the Shanghai Animal Care and Use Committee [Certificate No. SCXK (Shanghai) 2002-0010]. [3]
* **Treatment Groups:** Mice were divided into three groups (n=15 per group): (1) AAI group: received corn oil (CO) i.p. daily for 3 days, followed by a single i.p. injection of 10 mg/kg AAI in saline 24 h after last CO injection; (2) BNF+AAI group: received 80 mg/kg BNF in CO i.p. daily for 3 days, followed by a single i.p. injection of 10 mg/kg AAI 24 h after last BNF injection; (3) Control group: received CO i.p. daily for 3 days, followed by a single i.p. injection of saline 24 h after last CO injection. [3]
* **Sample Collection:** Blood samples were collected by tail bleeding at various time points after AAI injection (20 μL each) in heparin-coated capillaries. Tissues (liver and kidney) were harvested at 30 min post-AAI for metabolite analysis, or at 24 h after last BNF/CO injection for microsome preparation and RNA extraction. For histopathology and serum biochemistry, mice were sacrificed at 3, 7, and 14 days after AAI/saline injection. [3]
* **Serum Biochemistry:** Blood urea nitrogen and serum creatinine were measured using an automatic HITACHI Clinical Analyzer Model 7080. [3]
* **Histopathology:** Kidneys were fixed in 10% formalin, embedded in paraffin, sectioned at 3 μm, and stained with hematoxylin and eosin. Immunostaining for α-SMA was performed using anti-mouse α-SMA antibody (1:800) and avidin-biotin-peroxidase complex method. TUNEL assay was performed using Roche Diagnostics kit according to supplier's instructions. [3]
* **HPLC Analysis:** AAI and its metabolites (AAIa and ALI) were quantified using an HP1100 HPLC system with UV detection at 250 nm. Separation used a Welchorn XB-C18 column with isocratic mobile phase of methanol:0.1% acetic acid in water (7:3) at 0.8 mL/min. Calibration curves, linear ranges, precision, and recovery were established for serum, liver, and kidney samples. [3]
* **Real-Time RT-PCR:** Total RNA was isolated using UNIQ-10 column & TRIZOL kit. cDNA was synthesized using Cloned AMV Reverse Transcriptase. Real-time PCR used TaKaRa Ex Taq R-PCR kit with gene-specific primers for CYP1A1, CYP1A2, CPR, and β-actin. PCR was monitored for 45 cycles with annealing at 60°C. Relative amounts were normalized to β-actin mRNA. [3]
* **Western Blotting:** Microsomal proteins (30 μg) were separated on 10% SDS-PAGE, transferred to nitrocellulose membranes, and probed with antibodies against CYP1A1 (1:100), CYP1A2 (1:1000), and CPR (1:1000). Signal was detected using ECL system. [3]

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Animals: Male C57BL/6 mice (6 weeks old, 18-22 g) were obtained from Shanghai Laboratory Animal Center. All animal experiments were approved by the Shanghai Animal Care and Use Committee [Certificate No. SCXK (Shanghai) 2002-0010]. [3]
Treatment Groups: Mice were divided into three groups (n=15 per group): (1) AAI group: received corn oil (CO) i.p. daily for 3 days, followed by a single i.p. injection of 10 mg/kg AAI in saline 24 h after last CO injection; (2) BNF+AAI group: received 80 mg/kg BNF in CO i.p. daily for 3 days, followed by a single i.p. injection of 10 mg/kg AAI 24 h after last BNF injection; (3) Control group: received CO i.p. daily for 3 days, followed by a single i.p. injection of saline 24 h after last CO injection. [3]
Sample Collection: Blood samples were collected by tail bleeding at various time points after AAI injection (20 μL each) in heparin-coated capillaries. Tissues (liver and kidney) were harvested at 30 min post-AAI for metabolite analysis, or at 24 h after last BNF/CO injection for microsome preparation and RNA extraction. For histopathology and serum biochemistry, mice were sacrificed at 3, 7, and 14 days after AAI/saline injection. [3]
Serum Biochemistry: Blood urea nitrogen and serum creatinine were measured using an automatic HITACHI Clinical Analyzer Model 7080. [3]
Histopathology: Kidneys were fixed in 10% formalin, embedded in paraffin, sectioned at 3 μm, and stained with hematoxylin and eosin. Immunostaining for α-SMA was performed using anti-mouse α-SMA antibody (1:800) and avidin-biotin-peroxidase complex method. TUNEL assay was performed using Roche Diagnostics kit according to supplier's instructions. [3]
HPLC Analysis: AAI and its metabolites (AAIa and ALI) were quantified using an HP1100 HPLC system with UV detection at 250 nm. Separation used a Welchorn XB-C18 column with isocratic mobile phase of methanol:0.1% acetic acid in water (7:3) at 0.8 mL/min. Calibration curves, linear ranges, precision, and recovery were established for serum, liver, and kidney samples. [3]
Real-Time RT-PCR: Total RNA was isolated using UNIQ-10 column & TRIZOL kit. cDNA was synthesized using Cloned AMV Reverse Transcriptase. Real-time PCR used TaKaRa Ex Taq R-PCR kit with gene-specific primers for CYP1A1, CYP1A2, CPR, and β-actin. PCR was monitored for 45 cycles with annealing at 60°C. Relative amounts were normalized to β-actin mRNA. [3]
Western Blotting: Microsomal proteins (30 μg) were separated on 10% SDS-PAGE, transferred to nitrocellulose membranes, and probed with antibodies against CYP1A1 (1:100), CYP1A2 (1:1000), and CPR (1:1000). Signal was detected using ECL system. [3]

AAI Pharmacokinetics: BNF pretreatment significantly altered the pharmacokinetics of AAI. After a single i.p. dose of 10 mg/kg AAI, BNF-pretreated mice showed lower Cmax (3.08 ± 0.61 vs. 8.73 ± 1.57 μg/mL, p < 0.01), shorter tmax (6.25 ± 2.50 vs. 16.00 ± 5.48 min, p < 0.05), and reduced AUC (149.84 ± 20.30 vs. 418.37 ± 126.09 min·μg/mL, p < 0.05) compared to control mice. Half-life was not significantly changed. [3]
Tissue Distribution: At 30 min post-AAI, BNF pretreatment reduced AAI levels in both liver and kidney by approximately 3-fold (p < 0.01). AAIA levels were not significantly changed. ALI levels were decreased in both liver and kidney after BNF pretreatment. [3]

[1]. Ishida T, Takechi S. β-Naphthoflavone, an exogenous ligand of aryl hydrocarbon receptor, disrupts zinc homeostasis in human hepatoma HepG2 cells. J Toxicol Sci. 2019;44(10):711-720.

[2]. α- and β-Naphthoflavone synergistically attenuate H2O2-induced neuron SH-SY5Y cell damage. Exp Ther Med. 2017 Mar;13(3):1143-1150.

[3]. β-Naphthoflavone protects mice from aristolochic acid-I-induced acute kidney injury in a CYP1A dependent mechanism. Acta Pharmacologica Sinica 30.11 (2009): 1559-1565.

β-naphthoflavone is an extended flavonoid compound formed by the fusion of a benzene ring with the f-side of a flavonoid. It is an aryl hydrocarbon receptor agonist. It is an extended flavonoid compound, an organic heterocyclic tricyclic compound, and a naphtho-γ-pyranone. β-naphthoflavone, also known as 5,6-benzoflavonoid, is a potent agonist of aryl hydrocarbon receptors and can induce cytochrome P450 (CYP) and uridine 5'-bisphosphate glucuronyl transferase (UGT). It may be a chemopreventive agent. β-naphthoflavone is a polycyclic aromatic hydrocarbon consisting of a flavonoid group attached to a benzene ring. β-naphthoflavone is an inducer of hepatic cytochrome P450 CYP1A1 and CYP1A2. A polycyclic aromatic hydrocarbon compound that can induce P4501A1 and P4501A2 cytochromes. (Proc Soc Exp Biol Med 1994 Dec:207(3):302-308)

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Background: β-Naphthoflavone is a synthetic derivative of naturally occurring flavonoids, observed in Passiflora incarnata Linn. It is one of only two structural isomers of Naphthoflavone (along with α-Naphthoflavone). Its potential as a readily available and cheap therapeutic agent for neurological diseases was investigated in this study. [2]
Mechanism of Action (Proposed): β-Naphthoflavone exerts neuroprotective effects against oxidative stress through multiple mechanisms: (1) upregulating antioxidant enzyme activities (CAT, SOD, GPx); (2) inhibiting p38 MAPK phosphorylation at Thr180 and Tyr182; (3) suppressing expression of pro-apoptotic proteins (Bax, cytochrome c release) and caspase-3 activation; (4) increasing total antioxidant capacity and reducing lipid peroxidation (MDA). [2]
Comparison with α-Naphthoflavone: β-Naphthoflavone demonstrated greater efficacy than α-Naphthoflavone in protecting against H₂O₂-induced neuronal damage, particularly in restoring cell viability, reducing apoptosis, upregulating CAT activity, and inhibiting cytochrome c release. [2]
Synergistic Effect: Combined treatment with α- and β-Naphthoflavone produced enhanced neuroprotective effects compared to either compound alone, suggesting potential for combination therapy in neurological diseases. [2]
Clinical Potential: The study suggests that utilizing α- and β-Naphthoflavone together in the clinical setting of neurological disease treatment may be beneficial. [2]
p38 MAPK Signaling: The study confirms that H₂O₂-induced neuronal apoptosis is at least partially dependent on p38 MAPK phosphorylation at specific sites (Thr180/Tyr182), and that β-Naphthoflavone's neuroprotective effects are mediated through inhibition of this pathway. [2]

C19H12O2

272.3

272.083

6051-87-2

2361

White to light yellow solid powder

1.3±0.1 g/cm3

460.9±45.0 °C at 760 mmHg

185-189 °C

215.8±22.3 °C

0.0±1.1 mmHg at 25°C

1.695

4.79

0

2

1

21

433

0

OUGIDAPQYNCXRA-UHFFFAOYSA-N

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

3-phenylbenzo[f]chromen-1-one

beta-Naphthoflavone beta-NF

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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)

Solubility in Formulation 1: 1 mg/mL (3.67 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 10.0 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 1 mg/mL (3.67 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 10.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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