Natural biflavonoid; antioxidative; anti-inflammatory; anti-viral; anti-tumor
Nuclear Factor-κB (NF-κB) - Amentoflavone inhibits the activation and nuclear translocation of the p65 subunit of NF-κB, thereby suppressing downstream inflammatory mediators [1]
.
- Inflammatory Mediators - Amentoflavone inhibits the production of nitric oxide (NO), prostaglandin E2 (PGE2), interleukin-1β (IL-1β), and interleukin-6 (IL-6) [1]
.
- Inducible Nitric Oxide Synthase (iNOS) - Amentoflavone inhibits NF-κB-mediated iNOS expression, reducing NO generation [1]
.

In RAW 264.7 cells, amenoflavone (1-60 μM) suppresses the generation of nitric oxide in a concentration-dependent way [2]. U-87 MG cell viability is inhibited by amentoflavone (50-200 μM) with an IC50 value of 100 μM after 48 hours [3]. In the sub-G1 phase, amentoflavone (0, 50, and 100 μM; 48 hours) causes cell cycle arrest and apoptosis [3]. In U-87 MG cells, amentoflavone (0, 50, 100 μM; 48 h) suppresses NF-κB activation and lowers MCL1 and C-FLIP protein expression [3]. With minimum inhibitory concentrations (MICs) of 8, 4, 32, 8, 16, and 8 μg/ml, amentoflavone (0-32 μg/ml) exhibits antibacterial action against Enterococcus faecium ATCC 19434, Staphylococcus aureus ATCC 25923, Streptococcus mutans ATCC 3065, and Escherichia coli. ml. They're E. O. 157 ATCC 25922, E. coli. Pseudomonas aeruginosa ATCC 27853 and Escherichia coli ATCC 43895 [4].

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Cytotoxicity in Glioblastoma Cells: Amentoflavone significantly inhibited the viability of U-87 MG glioblastoma cells in a dose-dependent manner. At concentrations of 50-200 μM, cell viability was reduced by 23-71% after 48 hours treatment compared to controls. The IC50 was determined to be 100 μM [3]
.
- Induction of Apoptosis - Sub-G1 Accumulation: Flow cytometry analysis showed that Amentoflavone treatment (50 and 100 μM for 48 hours) significantly induced accumulation of cells in the sub-G1 phase (a marker of late apoptosis) by 14-52% compared to controls [3]
.
- Activation of Caspase-3: Amentoflavone significantly increased the expression of active caspase-3 by 24-42% at 48 hours compared to controls, as measured by flow cytometry using FITC-DEVD-FMK staining [3]
.
- Activation of Caspase-8: Amentoflavone significantly increased the expression of active caspase-8 by 25-50% at 48 hours compared to controls, as measured by flow cytometry using Red-IETD-FMK staining [3]
.
- Loss of Mitochondrial Membrane Potential (ΔΨm): Amentoflavone treatment significantly induced loss of mitochondrial membrane potential by 23-53% at 48 hours compared to controls, as measured by DiOC6(3) staining and flow cytometry [3]
.
- Inhibition of NF-κB Activation: Using NF-κB luciferase reporter gene assay, Amentoflavone significantly suppressed NF-κB activation in a dose-dependent manner by 23-71% at 48 hours. At 50-200 μM concentrations, NF-κB activity was reduced by 25-87% [3]
.
- Reduction of Anti-apoptotic Proteins: Western blot analysis showed that Amentoflavone (50 and 100 μM for 48 hours) significantly reduced protein expression of MCL1 by 50-80% and C-FLIP by 38-57% compared to controls. This effect was similar to that observed with the NF-κB inhibitor QNZ, which reduced MCL1 by 87% and C-FLIP by 79% [3]
.
- Comparison with NF-κB Inhibitor QNZ: The NF-κB inhibitor QNZ (0.3 μM) significantly reduced NF-κB activation and decreased MCL1 and C-FLIP protein levels, confirming that Amentoflavone's effects on these anti-apoptotic proteins are mediated through NF-κB inhibition [3]
.

Through its anti-inflammatory properties in mice, amentoflavone (25 mg/kg; oral; once daily for 3 days) has been demonstrated to be neuroprotective in epilepsy [1].

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Animal Model: Male Kunming mice (28-32 g, 5-6 weeks old) were used to establish a pilocarpine-induced epilepsy model (kindling model). Mice were assigned to five groups: blank control, non-treated epilepsy, valproate-treated (20 mg/kg, positive control), amentoflavone-treated (25 mg/kg given 1 hour after status epilepticus), and amentoflavone pre-treated (25 mg/kg intragastrically for 3 consecutive days before pilocarpine injection). Seizures were induced by subcutaneous injection of N-methylscopolamine bromide (1 mg/kg) followed by intraperitoneal injection of pilocarpine (300 mg/kg) 30 minutes later [1]
.
- EEG Recording: Electrodes were implanted into the bilateral hippocampus (2.3 mm posterior to bregma, 2.1 mm lateral, 2.0 mm subdural). EEG was recorded using a 4-channel signal acquisition system. Amentoflavone pre-treatment significantly stabilized EEG signals, reduced epileptiform discharges, and shortened seizure duration compared to the non-treated epilepsy group [1]
.
- NF-κB p65 Expression: Immunohistochemistry and western blot showed that amentoflavone pre-treatment significantly decreased NF-κB p65 expression and nuclear translocation in hippocampal CA1 neurons compared to the non-treated epilepsy group (P < 0.05 to P < 0.01). Valproate treatment also reduced NF-κB p65 expression but to a lesser extent [1]
.
- Neuronal Protection (Nissl Staining): Nissl staining of hippocampal CA1 region at 72 hours post-status epilepticus showed that amentoflavone pre-treatment preserved neuronal integrity, with neatly arranged neurons and visible Nissl bodies. The non-treated epilepsy group showed severe neuronal degeneration, loose arrangement, and absent or indistinct nucleoli. Neuronal counts were significantly higher in the amentoflavone pre-treated group compared to the non-treated epilepsy group (P < 0.05 to P < 0.01) [1]
.
- Apoptosis (TUNEL Staining): TUNEL staining revealed significantly fewer apoptotic cells in the hippocampal CA1 region of amentoflavone pre-treated mice compared to the non-treated epilepsy group (P < 0.05 to P < 0.01). No significant difference was observed between the blank control and amentoflavone pre-treated groups [1]
.
- Inflammatory Mediators: Amentoflavone pre-treatment significantly reduced NO and PGE2 levels in hippocampal tissue compared to the non-treated epilepsy group (P < 0.01). It also significantly decreased IL-1β and IL-6 production (P < 0.01) [1]
.
- Seizure Behavior: In the amentoflavone pre-treated group, no grand mal seizures occurred after model establishment; only occasional grade I-II seizures were observed, and attack time was significantly shortened compared to the non-treated epilepsy group which exhibited grade III-V seizures [1]
.

NO Measurement: Nitric oxide production in hippocampal tissue supernatants was assayed using the Griess reagent system. Samples were mixed with equal volume of Griess reagent, incubated at room temperature for 15 minutes, and absorbance measured at 540 nm. Nitrite concentration was determined using a sodium nitrite standard curve [1]
.
- PGE2, IL-1β, IL-6 ELISA: Prostaglandin E2, IL-1β, and IL-6 concentrations in hippocampal tissue supernatants were measured using commercial ELISA kits according to manufacturer's instructions [1]
.

Cell viability assay [3]
Cell Types: U-87 MG Cell
Tested Concentrations: 0, 50, 75, 100, 200 µM
Incubation Duration: 48 hrs (hours)
Experimental Results: Dramatically inhibited the viability of U-87 MG cells by 23-71% at 48 hrs (hours) The IC50 value is 100 µM.

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Apoptosis analysis [3]
Cell Types: U-87 MG Cell
Tested Concentrations: 0, 50, 100 µM
Incubation Duration: 48 hrs (hours)
Experimental Results: Dramatically induced the accumulation of cells in the sub-G1 population and increased the levels of active caspase-3 respectively 14-52% and 24-42%, and Dramatically induced Ψm loss and active caspase-8 expression by 23-53% and 25-50%, respectively.

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Western Blot Analysis[3]
Cell Types: U-87 MG Cell
Tested Concentrations: 0, 50, 100 µM
Incubation Duration: 48 hrs (hours)
Experimental Results: Dramatically diminished NF-ĸB activation by 25-87% in a dose-dependent manner and diminished protein expression MCL1 and C-FLIP increased by 50-80% and 38-57% respectively.

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Cell Culture: Human U-87 MG glioblastoma cells were cultured in Minimum Essential Medium Eagle supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin, and 1% sodium pyruvate at 37°C with 5% CO₂ [3]
.
- MTT Cell Viability Assay: Cells were seeded in 96-well plates (2×10⁴ cells/well) overnight and treated with different concentrations of Amentoflavone (0-200 μM) for 48 hours. MTT reagent was added and absorbance measured at 570 nm using a microplate spectrophotometer. IC50 was calculated [3]
.
- Sub-G1 Analysis by Flow Cytometry: Cells (5×10⁵) were seeded in 6-well plates and treated with 50 or 100 μM Amentoflavone for 48 hours. Cells were harvested, fixed with 70% ethanol overnight, and stained with PI/RNase solution (40 μg/mL PI, 100 μg/mL RNase, 1% Triton X-100 in PBS) for 30 minutes. At least 10,000 cells per group were analyzed by flow cytometry. The percentage of sub-G1 phase cells was measured using FlowJo 7.6.1 software [3]
.
- Active Caspase-3 Detection: Cells were treated with 0, 50, or 100 μM Amentoflavone for 48 hours, harvested, washed with PBS, and stained with FITC-DEVD-FMK working solution (1 μL in 300 μL PBS) according to manufacturer's instructions (CaspGLOW Fluorescein Active Caspase-3 assay kit). Expression was evaluated by flow cytometry [3]
.
- Active Caspase-8 Detection: Cells were treated with 0, 50, or 100 μM Amentoflavone for 48 hours, harvested, washed with PBS, and stained with Red-IETD-FMK working solution (1 μL in 300 μL PBS) according to manufacturer's instructions (CaspGLOW Fluorescein Active Caspase-8 assay kit). Expression was evaluated by flow cytometry [3]
.
- Mitochondrial Membrane Potential Assay: Cells were treated with 0, 50, or 100 μM Amentoflavone for 48 hours, harvested, washed with PBS, and stained with DiOC6(3) working solution (4 μM in 500 μL PBS) for 30 minutes in the dark. ΔΨm was analyzed by flow cytometry [3]
.
- Western Blotting: Cells (3×10⁶) were seeded in 10 cm dishes, treated with 0, 50, 100 μM Amentoflavone or 0.3 μM QNZ for 48 hours. Total protein was extracted using lysis buffer. Protein expression of MCL1 and C-FLIP was determined by western blotting. Protein bands were quantified using ImageJ software. β-actin was used as loading control [3]
.

Animal/Disease Models: 5-6 weeks, 28-32 g, Kunming mice [1]
Doses: 25 mg/kg
Route of Administration: Po; one time/day for 3 days
Experimental Results: Inhibition of the activation and nuclear activation of NF-κB subunit p65 translocation, reducing the production of IL-6 and IL-1β, and Dramatically reducing the production of NO and prostaglandin E2.

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Animals and Grouping:** Male Kunming mice (28-32 g, 5-6 weeks old) were randomly divided into five groups (n=30 per group): blank control, non-treated epilepsy, valproate-treated (20 mg/kg), amentoflavone-treated (25 mg/kg given 1 hour after status epilepticus), and amentoflavone pre-treated (25 mg/kg intragastrically once daily for 3 consecutive days before pilocarpine injection) [1]
.
- **Drug Administration:** Amentoflavone was dissolved and suspended in 1% gum acacia solution for intragastric administration. Valproate was administered at 20 mg/kg. Control groups received equal volumes of 0.9% saline [1]
.
- **Epilepsy Model Induction:** Mice received subcutaneous N-methylscopolamine bromide (1 mg/kg) to reduce peripheral cholinergic effects, followed 30 minutes later by intraperitoneal pilocarpine (300 mg/kg). Seizures were terminated with chloral hydrate 2 hours after status epilepticus onset. Seizure severity was scored according to Racine's scale (stages 1-2: partial seizures; stages 3-5: generalized seizures) [1]
.
- **EEG Electrode Implantation:** Under sodium pentobarbital anesthesia (80 mg/kg, i.p.), mice were fixed in a stereotaxic instrument. Bipolar insulated electrodes (0.15 mm diameter) were implanted into the bilateral hippocampus at coordinates: 2.3 mm posterior to bregma, 2.1 mm lateral to sagittal suture, and 2.0 mm subdural. Electrodes were fixed with dental cement. EEG recording was performed 1 week after surgery using a 4-channel signal acquisition system [1]
.
- **Tissue Collection:** At 72 hours after pilocarpine injection, mice were anesthetized and perfused intracardially with heparinized saline followed by 4% paraformaldehyde for histology. For biochemical assays, hippocampal tissues were collected on ice and stored at -80°C [1]
.

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Animals and Grouping: Male Kunming mice (28-32 g, 5-6 weeks old) were randomly divided into five groups (n=30 per group): blank control, non-treated epilepsy, valproate-treated (20 mg/kg), amentoflavone-treated (25 mg/kg given 1 hour after status epilepticus), and amentoflavone pre-treated (25 mg/kg intragastrically once daily for 3 consecutive days before pilocarpine injection) [1]
.
- Drug Administration: Amentoflavone was dissolved and suspended in 1% gum acacia solution for intragastric administration. Valproate was administered at 20 mg/kg. Control groups received equal volumes of 0.9% saline [1]
.
- Epilepsy Model Induction: Mice received subcutaneous N-methylscopolamine bromide (1 mg/kg) to reduce peripheral cholinergic effects, followed 30 minutes later by intraperitoneal pilocarpine (300 mg/kg). Seizures were terminated with chloral hydrate 2 hours after status epilepticus onset. Seizure severity was scored according to Racine's scale (stages 1-2: partial seizures; stages 3-5: generalized seizures) [1]
.
- EEG Electrode Implantation: Under sodium pentobarbital anesthesia (80 mg/kg, i.p.), mice were fixed in a stereotaxic instrument. Bipolar insulated electrodes (0.15 mm diameter) were implanted into the bilateral hippocampus at coordinates: 2.3 mm posterior to bregma, 2.1 mm lateral to sagittal suture, and 2.0 mm subdural. Electrodes were fixed with dental cement. EEG recording was performed 1 week after surgery using a 4-channel signal acquisition system [1]
.
- Tissue Collection: At 72 hours after pilocarpine injection, mice were anesthetized and perfused intracardially with heparinized saline followed by 4% paraformaldehyde for histology. For biochemical assays, hippocampal tissues were collected on ice and stored at -80°C [1]
.

The introduction mentions that Amentoflavone is able to cross the blood-brain barrier and afford neuroprotection against neonatal hypoxic-ischemic brain injury, citing previous literature. However, no specific ADME/PK data from this study are provided [3]

[1]. Amentoflavone protects hippocampal neurons: anti-inflammatory, antioxidative, and antiapoptotic effects. Neural Regen Res. 2015 Jul;10(7):1125-33.

[2]. Amentoflavone inhibits the induction of nitric oxide synthase by inhibiting NF-kappaB activation in macrophages. Pharmacol Res. 2005 Jun;51(6):539-46.

[3]. Amentoflavone Induces Apoptosis and Inhibits NF-ĸB-modulated Anti-apoptotic Signaling in Glioblastoma Cells. In Vivo. 2018 Mar-Apr;32(2):279-285.

[4]. Antibacterial effect of amentoflavone and its synergistic effect with antibiotics. J Microbiol Biotechnol. 2013;23(7):953-8.

[5]. Semisynthetic preparation of amentoflavone: A negative modulator at GABA(A) receptors. Bioorg Med Chem Lett. 2003 Jul 21;13(14):2281-4.

Amentoflavone are biflavonoids formed by the oxidative coupling of two apigenin molecules, with a chemical bond between the C-3 position of the hydroxybenzene ring and the C-8 position of the chromene ring. It is a natural product, primarily found in Ginkgo biloba and St. John's wort. Amentoflavone possess various activities, including being a cathepsin B inhibitor, antiviral agent, angiogenesis inhibitor, P450 inhibitor, and plant metabolite. It is a biflavonoid, hydroxyflavonoid, and cyclic compound. Amentoflavone have been reported in cycads, cruciferous microbiota, and other organisms with relevant data. See also: ... (See more...)

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Background and Source: Amentoflavone is a natural biflavone compound derived from extracts of Selaginella tamariscina and other plants. It possesses multiple biological properties including anti-inflammatory, antioxidative, anti-viral, anti-tumor, anti-radiation, and neuroprotective effects [1]
.
- Mechanism of Action in Epilepsy: Amentoflavone exerts neuroprotective effects in pilocarpine-induced epilepsy through multiple mechanisms: (1) inhibition of NF-κB activation and nuclear translocation, (2) suppression of inflammatory mediators (NO, PGE2, IL-1β, IL-6), (3) reduction of neuronal apoptosis, and (4) protection of hippocampal neuronal integrity. These combined effects result in reduced seizure severity and frequency [1]
.
- Anti-inflammatory Mechanism: Amentoflavone blocks the nuclear translocation of NF-κB p65 and inhibits I-κBα degradation, which is mediated by I-κBα kinase. This prevents the transcription of pro-inflammatory genes including iNOS, COX-2, and cytokines [1]
.
- Comparison with Valproate: Amentoflavone pre-treatment showed comparable or superior effects to valproate (20 mg/kg) in reducing NF-κB expression, protecting neurons, and decreasing inflammatory mediators. Both treatments effectively prevented severe seizures, but amentoflavone appeared more effective in preserving neuronal integrity [1]
.
- Therapeutic Potential: The study suggests that amentoflavone could be developed as a novel therapeutic agent for epilepsy, particularly for preventing epileptogenesis rather than merely treating symptoms. Its anti-inflammatory and neuroprotective properties make it a promising candidate for disease-modifying therapy [1]
.
- First Study in Epilepsy: This is the first study to report the effects of amentoflavone on epilepsy, demonstrating its ability to prevent seizures and protect hippocampal neurons through anti-inflammatory, antioxidative, and anti-apoptotic mechanisms [1]
.
- Source: Amentoflavone used in this study was purchased from Sigma-Aldrich [1]

C30H18O10

538.46

538.09

C, 66.92; H, 3.37; O, 29.71

1617-53-4

1617-53-4 38Biapigenin

5281600

Light yellow to yellow solid powder

1.7±0.1 g/cm3

910.5±65.0 °C at 760 mmHg

>300ºC

308.5±27.8 °C

0.0±0.3 mmHg at 25°C

1.793

3.11

6

10

3

40

1050

0

YUSWMAULDXZHPY-UHFFFAOYSA-N

InChI=1S/C30H18O10/c31-15-4-1-13(2-5-15)24-12-23(38)29-21(36)10-20(35)27(30(29)40-24)17-7-14(3-6-18(17)33)25-11-22(37)28-19(34)8-16(32)9-26(28)39-25/h1-12,31-36H

8-[5-(5,7-dihydroxy-4-oxochromen-2-yl)-2-hydroxyphenyl]-5,7-dihydroxy-2-(4-hydroxyphenyl)chromen-4-one

Didemethylginkgetin; Amentoflavone; 1617-53-4; Didemethyl-ginkgetin; 3',8''-Biapigenin; Amenthoflavone; I3,II8-biapigenin; Tridemethylsciadopitysin; MFCD00017470;

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 : ~125 mg/mL (~232.14 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.64 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (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 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL 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: 2.5 mg/mL (4.64 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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