- Aryl hydrocarbon receptor (AHR): Visnagin activates AHR signaling, as shown by XRE-driven luciferase reporter gene activity (maximal 24-fold induction at 20 μM) and induction of CYP1A1 transcription, which is abolished by the specific AHR antagonist MNF (3'-methoxy-4'-nitroflavone) [1].
- CYP1A enzymes (CYP1A1/1A2): Visnagin inhibits CYP1A catalytic activity in a dose-dependent manner. In TCDD-induced HepG2 cells, Visnagin decreased EROD activity with a reverse dose-response; at 20 μM, it significantly inhibited activity [1].
- Nrf2 (Nuclear factor erythroid 2-related factor 2): Visnagin upregulates Nrf2 expression in pancreatic acinar cells [2].
- NFκB (p65): Visnagin reduces nuclear translocation of p65-NFκB in pancreatic tissue [2].
- Inflammatory cytokines: Visnagin decreases IL-1β, IL-6, IL-17, and TNF-α levels [2].
- Nitrotyrosine: Visnagin reduces nitrotyrosine expression in pancreatic acinar cells [2].

HepG2 cells exposed to visnagin (10 µM; 4, 8, 16, 24 hours) exhibit CYP1A1 transcription induction [1]. MNF (3'-methoxy-4'-nitroflavone; 20 µM; pretreatment 1 hour) effectively counteracts this induction, while visnagin (10 µM; for 16 hours) increases CYP1B1 gene expression in an aryl hydrocarbon receptor (AHR)-dependent manner. In an AHR-dependent way, visnagin also increases PAI-2 transcription [1].

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- In stably transfected AZ-AHR reporter cells (derived from HepG2), treatment with Visnagin (0.001 μM to 20 μM) for 24 hours resulted in a dose-dependent increase of XRE-driven luciferase reporter gene activity, with a maximum 24-fold induction at 20 μM. A statistically significant increase was observed at 10 μM visnagin [1].
- In HepG2 cells, Visnagin (10 μM) induced CYP1A1 mRNA expression in a time-dependent manner. A slight induction was observed after 4 hours, with peak expression at 16 hours (approximately 160-fold induction). The induction was abolished by pre-treatment with the AHR antagonist MNF (20 μM for 1 hour) [1].
- Western blot analysis in HepG2 cells showed that Visnagin (1 μM to 20 μM) for 48 hours induced CYP1A1 protein expression, with levels exceeding DMSO- and 3MC-treated cells. Visnagin (20 μM) also induced CYP1B1 protein expression after 16 hours [1].
- CYP1A enzyme activity (EROD assay) in HepG2 cells: Treatment with Visnagin (1-20 μM) for 16 hours did not significantly induce activity; after 48 hours, a slight but significant enhancement of EROD activity was observed, with a reverse dose-response suggesting inhibition. In TCDD-pretreated cells (5 nM for 48 hours), Visnagin (1 nM to 20 μM) decreased TCDD-induced catalytic activity in a dose-dependent manner [1].
- In HepG2 cells, Visnagin (10 μM for 16 hours) increased expression of AHR target genes: CYP1B1, AHRR, PAI-2, and VEGF. VEGF induction was significant only for visnagin (not for khellin). These inductions were attenuated by MNF pre-treatment [1].
- In primary human hepatocytes from four donors (LH40, LH42, HEP220586, HEP220624), Visnagin (1, 10, 20 μM for 24 hours) induced CYP1A1 mRNA expression, but with high interindividual variability (e.g., at 10 μM visnagin: 63.3, 12.0, 20.8, 28.9 fold induction). At protein level, Visnagin (10 and 20 μM for 48 hours) increased CYP1A1 protein expression approximately 3- to 6-fold, comparable to 1 μM 3MC [1].
- In primary human hepatocytes, EROD assay showed only a very weak increase in CYP1A enzyme activity upon exposure to Visnagin (1, 10, 20 μM for 48 hours), despite significant protein induction, suggesting inhibitory properties [1].
- In BV-2 microglial cells (from literature cited, but not detailed in the provided text), Visnagin inhibited nitric oxide (NO) production and reduced gene expression and production of IL-1β, IL-6, and TNF-α [2, reference 17 within the paper].

Visnagin (10, 30, 60 mg/kg; intraperitoneally; for 7 days) efficiently lowers plasma levels of lipase and amylase, and lowers Cerulein (50 μg/kg, i.p., six times hourly).Swiss albino male induced Mice under oxidative stress (age: 6–8 weeks, body weight: 20–25 g) [1]. The expression of TNF-α, IL-17, IL-6, and IL-1β is dose-dependently decreased by visnagin. Nuclear p65-NFκB levels are attenuated by it. By enhancing Nrf2 expression, visnagin enhances antioxidant defense and suppresses pancreatic inflammation by preventing NFκB and nitrotyrosine expression in acinar cells [1].

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- In male Swiss albino mice (6-8 weeks, 20-25 g), cerulein-induced acute pancreatitis (AP) was established by six hourly intraperitoneal injections of cerulein (50 μg/kg). Pre-treatment with Visnagin (10, 30, 60 mg/kg, i.p. for 7 days) dose-dependently reduced pancreatic edema (pancreas weight and pancreas/body weight ratio), plasma amylase and lipase levels compared to cerulein control (p < 0.05 to p < 0.001) [2].
- Visnagin (10, 30, 60 mg/kg) pre-treatment decreased the elevated levels of pro-inflammatory cytokines IL-1β, IL-6, IL-17, and TNF-α in plasma or tissue (p < 0.05 to p < 0.001). It also reduced cytosolic and nuclear p65-NFκB levels (p < 0.05 to p < 0.001) [2].
- Histological analysis (H&E staining) of pancreas showed that Visnagin preserved acinar cell architecture, reduced leukocyte infiltration, and prevented distortion compared to cerulein control. In lungs and intestines (MODS assessment), Visnagin prevented alveolar wall thickening, interstitial edema, neutrophil infiltration, and maintained intestinal villi and mucosal barrier integrity [2].
- Visnagin (10, 30, 60 mg/kg) reduced inflammatory cytokine levels (IL-1β, IL-6, TNF-α) in lungs and intestines, and decreased serum markers of MODS (SGOT, SGPT, ALP, LDH) in a dose-dependent manner (p < 0.05 to p < 0.001) [2].
- Visnagin (30, 60 mg/kg) significantly reduced pancreatic lipid peroxidation (MDA levels, p < 0.01 and p < 0.001), restored glutathione (GSH) levels (only at 60 mg/kg, p < 0.05), decreased nitrite levels (at 60 mg/kg, p < 0.01), and reduced myeloperoxidase (MPO) levels in pancreas and lungs (p < 0.01 to p < 0.001) [2].
- Immunohistochemistry of pancreatic tissue showed that Visnagin (30, 60 mg/kg) reduced nitrotyrosine expression (p < 0.001), increased Nrf2 expression (p < 0.01 to p < 0.001), decreased NFκB expression (p < 0.001), and reduced IL-6 and TNF-α immunoreactivity (p < 0.05 to p < 0.001) compared to cerulein control [2].

- CYP1A enzyme activity (EROD) assay in intact HepG2 cells or primary human hepatocytes: Cells were washed twice with PBS and then incubated with 100 μl of PBS containing 8 μM 7-ethoxyresorufin and 10 μM dicumarol to prevent further metabolism of resorufin. After 30 minutes of incubation at 37°C, 75 μl of the supernatant was transferred to a black 96-well plate together with 125 μl of methanol. Fluorescence of resorufin was measured at 530 nm excitation and 590 nm emission wavelengths. Results were normalized to cell viability determined by MTT assay to exclude cytotoxicity [1].
- For inhibition studies, HepG2 cells were pretreated with 5 nM TCDD for 48 hours, then the substrate mixture (7-ethoxy-O-resorufin) was supplemented with increasing doses of Visnagin (1 nM to 20 μM) and EROD activity was measured as above. Data were expressed as percentage of TCDD-mediated induction [1].
- Estimation of malondialdehyde (MDA) in pancreatic tissue: Tissue was homogenized and MDA was measured using the thiobarbituric acid (TBA) reaction method, with absorbance at 532 nm. Results expressed in μM/mg protein [2].
- Estimation of non-protein sulfhydryl group (reduced glutathione, GSH): Pancreatic tissue (10 mg) was homogenized in Triton-X100 based buffer, centrifuged, and supernatant was mixed with Ellman's reagent (DTNB). Absorbance measured at 412 nm. GSH levels calculated using a standard curve [2].
- Estimation of tissue nitrite (nitrosative stress): Nitrite levels were measured using Griess reagent (0.1% naphthylethylenediamine dihydrochloride + 1% sulfanilamide in 5% phosphoric acid). Absorbance at 540 nm [2].
- Myeloperoxidase (MPO) assay: Tissue MPO activity was measured as a marker of neutrophil sequestration [2].

- AZ-AHR reporter gene assay: Stably transfected HepG2 cells containing XRE-driven luciferase reporter gene were plated, stabilized for 16 hours, then treated for 24 hours with Visnagin (0.001-20 μM), 5 μM 3MC, or vehicle (DMSO 0.1% v/v). After treatment, cells were lysed and luciferase activity was measured using a luminometer [1].
- qRT-PCR in HepG2 cells: Total RNA was isolated using peqGOLD total RNA kit. 0.5 μg of total RNA was reverse transcribed using MMLV reverse transcriptase. 3 μl of 1:3 diluted cDNA were used for qRT-PCR with QuantiFast SYBR Green. Gene expression was normalized to β-actin. Primers for CYP1A1, CYP1B1, AHRR, PAI-2, and VEGF were used as described previously [1].
- qRT-PCR in primary human hepatocytes: Total RNA was isolated using TRI Reagent. cDNA was synthesized from 1000 ng RNA using M-MLV Reverse Transcriptase at 42°C for 60 min in presence of random hexamers. qRT-PCR was performed using LightCycler FastStart DNA Master SYBR Green I on a Light Cycler 480 II. CYP1A1 and GAPDH mRNA expression was determined. Measurements in triplicates, normalized to GAPDH [1].
- Western blot analysis in HepG2 and primary hepatocytes: Total protein extracts were prepared by lysing cells in ice-cold lysis buffer (150 mM NaCl, 10 mM Tris pH 7.2, 0.1% SDS, 1% Triton X-100, 1% sodium deoxycholate, 5 mM EDTA, protease and phosphatase inhibitors). Protein concentration determined by Bradford reagent. 8% SDS-PAGE gels were run, proteins transferred to PVDF membrane, saturated with 5% non-fat dry milk, probed with primary antibodies against CYP1A1, CYP1B1, actin, or GAPDH overnight at 4°C, followed by HRP-conjugated secondary antibodies and chemiluminescence detection. Densitometric analysis was performed [1].
- Immunohistochemistry in pancreatic, lung, and intestinal tissues: Tissues were fixed in neutral buffered formalin, embedded in paraffin, sectioned, and stained with H&E for histopathology. For immunohistochemistry, primary antibodies against nitrotyrosine, Nrf2, NFκB, IL-6, and TNF-α were used, followed by detection using an immunohistochemistry kit. Images were captured and quantified [2].
- ELISA for cytokines and p65-NFκB: Plasma or tissue homogenates were used to measure IL-1β, IL-6, IL-17, TNF-α, and p65-NFκB levels using commercial ELISA kits according to manufacturer's instructions [2].
- MTT assay for cell viability: Used to normalize EROD activity results in HepG2 cells and primary hepatocytes to exclude cytotoxicity [1].

- Animal model of acute pancreatitis (AP): Male Swiss albino mice (6-8 weeks, 20-25 g) were randomly divided into six groups (n=6 per group). Visnagin was dissolved in a mixture of 3% ethanol + 97% olive oil (vehicle). Groups: normal control (vehicle only), cerulein control (vehicle daily + cerulein induction), Visnagin low dose (10 mg/kg, i.p. for 7 days), Visnagin mid dose (30 mg/kg, i.p. for 7 days), Visnagin high dose (60 mg/kg, i.p. for 7 days), and Visnagin control (60 mg/kg alone). On day 7, AP was induced by six hourly intraperitoneal injections of cerulein (50 μg/kg). Animals were sacrificed 6 hours after the last cerulein injection. Pancreas, lungs, intestines, and blood were collected for analysis [2].
- For primary human hepatocyte experiments: Human hepatocytes were isolated from liver tissue obtained from multiorgan donors (LH40, LH42) or purchased as long-term cultures (HEP220586, HEP220624). Cells were cultured in serum-free medium and treated for 24 or 48 hours with Visnagin (1, 10, 20 μM), 1 μM 3MC, 5 nM TCDD, or vehicle (DMSO 0.1% v/v). Cultures were maintained at 37°C, 5% CO2 in a humidified incubator [1].
- For HepG2 cell experiments: HepG2 cells were cultured in DMEM supplemented with 10% fetal calf serum, 100 U/mL streptomycin, 100 μg/mL penicillin, 4 mM L-glutamine, 1% non-essential amino acids, and 1 mM sodium pyruvate. Cells were maintained at 37°C, 5% CO2. Treatments were performed as described for various assays [1].

- Serum levels in humans: After ingestion of a single dose of 100 mg khellin (structurally related, not visnagin itself) during KUVA therapy, peak serum levels of 4.9 μM to 8.4 μM were reached in vitiligo patients 2 to 5 hours post-ingestion. It is noted that Visnagin is rapidly bioavailable, and the liver may be exposed to even higher concentrations [1].

- Visnagin at 60 mg/kg (i.p. for 7 days) in male Swiss albino mice showed no adverse effects as evidenced by normal pancreatic weight, organ index, plasma amylase, lipase, cytokine levels, and histology compared to normal control, indicating safety at this dose [2].
- Visnagin inhibits CYP1A enzyme activity, which may lead to drug-drug interactions by altering the pharmacokinetics of co-administered drugs that are CYP1A substrates (e.g., verukast, clozapine, verapamil). This could result in altered metabolic fate of pro-carcinogens, drugs, and steroid hormones [1].

[1]. Khellin and visnagin differentially modulate AHR signaling and downstream CYP1A activity in human liver cells. PLoS One. 2013 Sep 19;8(9):e74917.

[2]. Visnagin attenuates acute pancreatitis via Nrf2/NFκB pathway and abrogates associated multiple organ dysfunction. Biomed Pharmacother. 2019 Apr;112:108629.

Visnagin is a furanochrome ketone compound with the structure furano[3,2-g]chromen-5-one, substituted at positions 4 and 7 with methoxy and methyl groups, respectively. It is found in Ammi visnaga. Visnagin possesses various pharmacological activities, including phytotoxicity, EC 1.1.1.37 (malate dehydrogenase) inhibitor, vasodilator, antihypertensive agent, anti-inflammatory agent, and plant metabolite. It is a furanochrome ketone compound belonging to the aromatic ether and polyketide class. Its function is similar to that of 5H-furano[3,2-g]chromen-5-one. Visnagin has been reported to exist in Actaea dahurica, Musineon divaricatum, and Ammi visnaga, and relevant data are available for reference.

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- Visnagin is a furanochromone found in Ammi visnaga, traditionally used as an herbal remedy in Asia and the Middle East. It is structurally related to khellin and has been used in photochemotherapy (KUVA) for vitiligo, though khellin is more commonly used [1].
- Visnagin acts as both an activator of AHR-dependent signaling (inducing CYP1A1, CYP1B1, AHRR, PAI-2, VEGF) and an inhibitor of CYP1A catalytic activity. This dual action may have implications for therapeutic use, especially regarding drug-drug interactions and potential side effects [1].
- In acute pancreatitis, Visnagin attenuates disease severity by reducing oxidative stress, nitrosative stress, and inflammation via upregulation of Nrf2 and downregulation of NFκB and nitrotyrosine. It also prevents multi-organ dysfunction syndrome (MODS) involving lungs and intestines [2].
- Visnagin has been shown in other studies (referenced within) to protect against doxorubicin-induced cardiotoxicity by inhibiting mitochondrial malate dehydrogenase 2 (MDH2), and to have vasodilator effects via calcium channel blockade [2].

C13H10O4

230.2

230.057

C, 67.82; H, 4.38; O, 27.80

82-57-5

6716

White to off-white solid powder

1.3±0.1 g/cm3

378.2±42.0 °C at 760 mmHg

139-142 °C

182.5±27.9 °C

0.0±0.9 mmHg at 25°C

1.613

2.26

0

4

1

17

362

0

O=C1C2C(=CC3=C(C=2OC)C=CO3)OC(C)=C1

NZVQLVGOZRELTG-UHFFFAOYSA-N

InChI=1S/C13H10O4/c1-7-5-9(14)12-11(17-7)6-10-8(3-4-16-10)13(12)15-2/h3-6H,1-2H3

4-methoxy-7-methyl-5H-furo[3,2-g]chromen-5-one

NSC100593; Visnagin; NSC-100593; NSC 100593

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 (~217.18 mM)
Ethanol :< 1 mg/mL DMF :< 1 mg/mL

Solubility in Formulation 1: ≥ 2.5 mg/mL (10.86 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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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 (Please use freshly prepared in vivo formulations for optimal results.)