CYP3A4 enzyme. It inhibits the debenzylation activity of CYP3A4 with an IC50 value of 24.92 μM [1].

According to ABTS and DPPH tests, bergaptol (0-800 ppm) exhibits definite activity [1]. In RAW264.7 cells, LPS-induced NO, IL-6, and TNF are inhibited by 50 μg/mL of Bergaptol for 24 hours. Additionally, LPS-induced MAPK phosphorylation and NF-κB activation are inhibited in RAW264.7 cells [3].

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Bergaptol demonstrated potent inhibitory activity against the CYP3A4 enzyme. At a concentration of 0.1 mM, it showed 98.9 ± 0.75% inhibition of the enzyme's debenzylation activity [1].
Bergaptol exhibited radical scavenging activity in a concentration-dependent manner. In the ABTS assay, it showed 29.7%, 60.9%, and 91.1% radical scavenging activity at concentrations of 200, 400, and 800 ppm, respectively. In the DPPH assay, it showed 42.5%, 65.2%, and 82.9% radical scavenging activity at the same respective concentrations [1].

LPS-treated mice's cognitive function is improved when bergamot alcohol (10–40 mg/kg) is administered intraperitoneally once a day, during sleep [Disorder 4].

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In a mouse model of neuroinflammation induced by intra-hippocampal injection of LPS (40 μg/kg), intraperitoneal administration of Bergaptol (20 and 40 mg/kg, once daily for two weeks) significantly improved LPS-induced cognitive impairment, as evidenced by reduced escape latency and increased time spent in the target quadrant in the Morris water maze test. The 10 mg/kg dose showed no significant effect [4].
Bergaptol treatment (20 and 40 mg/kg) significantly reduced LPS-induced neuronal damage in the hippocampal CA1 region, as shown by H&E and Nissl staining. It also alleviated the reduction in dendritic spine density in hippocampal pyramidal neurons (Golgi staining) and restored the expression of synaptic proteins PSD-95 and synapsin-1 (immunofluorescence) [4].
Bergaptol treatment (20 and 40 mg/kg) significantly reduced the mRNA levels and protein release of TNF-α, IL-6, and IL-1β in the hippocampus of LPS-treated mice, as measured by RT-PCR and ELISA [4].
Immunofluorescence analysis showed that Bergaptol treatment significantly reduced the density of Iba-1 positive microglia in the hippocampus of LPS-treated mice, indicating inhibition of microglial activation [4].

The CYP3A4 enzyme inhibition assay was performed to determine the effect of Bergaptol on CYP3A4 activity. To a 96-well microtiter plate, 12.5 μL of luciferin-free water (positive control), ketoconazole (positive inhibition control), and test compound were added at 4× concentrations. Then, 12.5 μL of control reaction mixture and membrane preparations containing CYP3A4 were added at 4× concentration. The plate was mixed and pre-incubated at 37°C for 10 minutes. The CYP3A4 assay reaction was initiated by adding 25 μL of 2× NADPH regeneration system to all wells. After mixing, the plate was incubated at 37°C for 30 minutes. Following incubation, 50 μL of reconstituted luciferin detection reagent was added to all wells. The plate was mixed briefly and incubated at 37°C for 20 minutes. Luminescence was recorded using a luminometer in terms of relative light units (RLU). Inhibition was calculated based on the relative activity of the positive control (set as 100%). The percent inhibition was calculated as 100 - [(relative CYP activity/relative activity of positive control) × 100]. IC50 values were calculated using GraphPad Prism software [1].

Western Blot Analysis[3]
Cell Types: Production of LPS--α[3]. Induction of RAW264.7 cells
Tested Concentrations: 50 μg/mL
Incubation Duration: 24 hrs (hours)
Experimental Results: Inhibition of JNK phosphorylation and NF-κB activation. Inhibits the translocation of NF-κB P65 from the cytoplasm to the nucleus.

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RAW264.7 murine macrophages were used for all experiments. Cells were cultured in DMEM with 10% FBS at 37°C in 5% CO₂. Bergaptol was dissolved in DMSO and diluted in DMEM to final concentrations of 3.13-50 μg/mL (final DMSO <0.1%) [3].
For cytokine production, cells were seeded in 24-well plates, treated with LPS (1 μg/mL) with or without BER for 24 h. Supernatants were collected, and NO was measured using Griess reagents. IL-6, TNF-α, and IL-1β were quantified by ELISA kits [3].
For gene expression, total RNA was extracted with Trizol, reverse transcribed, and analyzed by qPCR with SYBR Green. Primer sequences are provided in the supplementary information. GAPDH was used as the housekeeping gene [3].
For western blotting, cells were lysed in RIPA buffer on ice. Proteins (40 μg) were separated by SDS-PAGE, transferred to PVDF membranes, blocked with 5% BSA, and incubated with primary antibodies against ERK, p-ERK, JNK, p-JNK, P38, p-P38, P65, p-P65, p-IκBα, p-IκBα/β, COX-2, ABCA1, ABCG1, SRB1, SRA1, CD36, and GAPDH overnight at 4°C. Membranes were then incubated with HRP-conjugated secondary antibodies and detected with ECL reagents. Band densities were analyzed with ImageJ software [3].
For Oil Red O staining, cells were fixed with 4% paraformaldehyde for 30 min, stained with 0.5% Oil Red O (in 60% isopropanol) for 30 min, and examined by microscopy [3].
For Dil-ox-LDL uptake, cells were incubated with Dil-ox-LDL (10 μg/mL) with or without BER for 4 h, washed with PBS, and visualized by confocal microscopy. Uptake was quantified by flow cytometry [3].
For BODIPY staining, cells were fixed with 4% paraformaldehyde, stained with BODIPY (10 μg/mL) at 37°C for 30 min, counterstained with DAPI (100 ng/mL) for 15 min, and examined by confocal microscopy [3].
For 25-NBD cholesterol uptake, cells were incubated with ox-LDL and BER for 24 h, then labeled with 25-NBD cholesterol (5 μg/mL) for 4 h. Cells were lysed with 0.1% Triton X-100 and sonicated. Fluorescence intensity was measured at Ex/Em 469/537 nm [3].
For cholesterol efflux, cells were treated with ox-LDL and BER for 24 h, labeled with 25-NBD cholesterol for 4 h, then stimulated with HDL (50 μg/mL) for 4 h. Fluorescence intensity in the culture medium and cell lysates was measured, and efflux efficiency was calculated [3].

Animal/Disease Models: LPS (40 μg/kg, icv) treated mice [4] ]
Doses: 10-40 mg/kg
Route of Administration: intraperitoneal (ip) injection, one time/day for two weeks
Experimental Results: diminished LPS-induced hippocampal CA1 area nerves Fixation and lysis of metanuclei (H&E staining). Increased dendritic spine density in mice. Inhibits LPS-induced neuroinflammation.

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Male C57BL/6 mice (6 weeks old) were randomly divided into 5 groups: Sham, LPS, LPS + Bergaptol (10 mg/kg), LPS + Bergaptol (20 mg/kg), and LPS + Bergaptol (40 mg/kg). Mice were anesthetized and fixed in a stereotaxic apparatus. LPS (40 μg/kg) or an equivalent volume of normal saline was injected into the hippocampal CA1 region (coordinates: 0.8 mm posterior to bregma, ±1.4 mm lateral, 4 mm depth) using a microsyringe. Bergaptol was dissolved in normal saline to 4 mg/mL and administered intraperitoneally once daily for two weeks. Sham and LPS groups received the same volume of normal saline [4].
After treatment, the Morris water maze test was performed to assess learning and memory. Mice were trained for 6 consecutive days to find a hidden platform, followed by a spatial probe test on day 7 where the platform was removed, and swimming tracks were recorded for 60 seconds [4].
Brain tissues were collected for histological analysis. For Golgi-Cox staining, brain tissue was immersed in impregnation solutions for two weeks, sectioned (100 μm), and stained. For H&E and Nissl staining, paraffin sections were prepared and stained with hematoxylin-eosin or 1% toluidine blue. For immunofluorescence, sections were incubated with primary antibodies against Iba-1, PSD-95, or synapsin-1, followed by fluorescent secondary antibodies, and observed by confocal microscopy [4].
Hippocampal tissue was homogenized for RNA extraction (RT-PCR) and protein extraction (ELISA) to measure TNF-α, IL-6, and IL-1β levels [4].

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Male C57BL/6 mice (6 weeks old) were randomly divided into 5 groups: Sham, LPS, LPS + Bergaptol (10 mg/kg), LPS + Bergaptol (20 mg/kg), and LPS + Bergaptol (40 mg/kg). Mice were anesthetized and fixed in a stereotaxic apparatus. LPS (40 μg/kg) or an equivalent volume of normal saline was injected into the hippocampal CA1 region (coordinates: 0.8 mm posterior to bregma, ±1.4 mm lateral, 4 mm depth) using a microsyringe. Bergaptol was dissolved in normal saline to 4 mg/mL and administered intraperitoneally once daily for two weeks. Sham and LPS groups received the same volume of normal saline [4].
After treatment, the Morris water maze test was performed to assess learning and memory. Mice were trained for 6 consecutive days to find a hidden platform, followed by a spatial probe test on day 7 where the platform was removed, and swimming tracks were recorded for 60 seconds [4].
Brain tissues were collected for histological analysis. For Golgi-Cox staining, brain tissue was immersed in impregnation solutions for two weeks, sectioned (100 μm), and stained. For H&E and Nissl staining, paraffin sections were prepared and stained with hematoxylin-eosin or 1% toluidine blue. For immunofluorescence, sections were incubated with primary antibodies against Iba-1, PSD-95, or synapsin-1, followed by fluorescent secondary antibodies, and observed by confocal microscopy [4].
Hippocampal tissue was homogenized for RNA extraction (RT-PCR) and protein extraction (ELISA) to measure TNF-α, IL-6, and IL-1β levels [4].

The CCK-8 assay on BV-2 cells showed that Bergaptol at concentrations up to 20 μg/mL had no significant cytotoxic effect. In vivo, no obvious adverse effects were reported in mice treated with Bergaptol at doses up to 40 mg/kg for two weeks [4].

[1]. Radical scavenging and cytochrome P450 3A4 inhibitory activity of bergaptol and geranylcoumarin from grapefruit. Bioorganic & Medicinal Chemistry.June 2007, Pages 3684-3691.

[2]. Cloning, Functional Characterization, and Catalytic Mechanism of a Bergaptol O-Methyltransferase from Peucedanum praeruptorum Dunn. Front Plant Sci, 2016, 7: 722.

[3]. Bergaptol from blossoms of Citrus aurantium L. var. amara Engl inhibits LPS-induced inflammatory responses and ox-LDL-induced lipid deposition. Food Funct. 2020 Jun 24;11(6):4915-4926.

[4]. Bergaptol Alleviates LPS-Induced Neuroinflammation, Neurological Damage and Cognitive Impairment via Regulating the JAK2/STAT3/p65 Pathway. J Inflamm Res. 2022 Nov 9;15:6199-6211.

Bergamot lactone belongs to the psoralen class of compounds and is a 5-hydroxyfuranocoumarin. It is the conjugated acid of bergamot lactone (1-). Bergamot lactone has been reported to exist in Angelica dahurica, plants of the genus Dorstenia, and other organisms with relevant data.

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Bergaptol is a furocoumarin compound isolated from grapefruit juice and grapefruit peel oil. Its structure was elucidated through extensive NMR studies (including 1D and 2D experiments such as DQF-COSY, edited HSQC, HMBC, and 1,1-ADEQUATE) and mass spectrometry. It has a molecular mass of 202 and UV absorption maxima at 268, 248, and 313 nm [1].
Furocoumarins like Bergaptol are responsible for the grapefruit juice effect, which markedly increases the oral bioavailability of many drugs by inhibiting CYP3A4-mediated first-pass metabolism in the gut. The potent CYP3A4 inhibitory activity of Bergaptol is attributed to the presence of the furan moiety in its structure, which is involved in bioactivation and covalent interaction with the enzyme [1].
This is reported as the first study on the isolation, characterization, and evaluation of radical scavenging activity and CYP3A4 inhibition properties of Bergaptol from grapefruit [1].

C11H6O4

202.165

202.026

486-60-2

5280371

Light yellow to yellow solid powder

1.5±0.1 g/cm3

311.9±11.0 °C at 760 mmHg

287-290ºC

142.4±19.3 °C

0.0±0.7 mmHg at 25°C

1.711

0.93

1

4

0

15

312

0

GIJHDGJRTUSBJR-UHFFFAOYSA-N

InChI=1S/C11H6O4/c12-10-2-1-6-9(15-10)5-8-7(11(6)13)3-4-14-8/h1-5,13H

4-hydroxyfuro[3,2-g]chromen-7-one

Psoralin, 5-hydroxy- 5-Hydroxypsoralen

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 : ≥ 50 mg/mL (~247.33 mM)
H2O : ~0.67 mg/mL (~3.31 mM)

Solubility in Formulation 1: 2.5 mg/mL (12.37 mM) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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 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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 (Please use freshly prepared in vivo formulations for optimal results.)