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Purity: =98.62%
Hispidol ((Z)-Hispidol) is a potential therapeutic for inflammatory bowel disease; which has an IC50 of 0.50 M and inhibits TNF-induced monocyte adhesion to colon epithelial cells.
| Targets |
The target of Hispidol includes nuclear factor kappa B (NF-κB) signaling pathway proteins (e.g., p65, IκBα) and pro-inflammatory cytokine-related targets (e.g., TNF-α, IL-6); the IC50 values for inhibiting LPS-induced TNF-α and IL-6 release in RAW264.7 cells are 8.5 μM and 7.2 μM, respectively [1]
Hispidol also targets cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), with IC50 values of 9.1 μM (COX-2) and 10.3 μM (iNOS) in cell-free enzyme assays [1] |
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| ln Vitro |
Hispidol shows potent inhibitory effect (>70%) on the TNF-α-induced adhesion of monocytes to colon epithelial cells, which is one of the hallmark events leading to inflammatory bowel disease (IBD). Hispidol is a strong candidate for the development of IBD medications due to its potent inhibitory effects against TNF-α induced monocytic-colonic epithelial cell adhesion as well as LPS-induced TNF-α expression. Hispidol inhibits the transcription of AP-1, a transcription factor also necessary for high levels of TNF-α expression, which corresponds to the additional inhibitory activity against AP-1 transcriptional activity[1].
1. Inhibition of pro-inflammatory cytokine release (Reference [1]): Hispidol (2.5-40 μM) was tested in LPS-stimulated murine macrophage RAW264.7 cells. It dose-dependently inhibited the release of TNF-α and IL-6: (1) At 8.5 μM (IC50 for TNF-α), TNF-α release was reduced by 50% compared to LPS control; (2) At 7.2 μM (IC50 for IL-6), IL-6 release was reduced by 50%; (3) At 20 μM, TNF-α and IL-6 release were inhibited by 68% and 72%, respectively (detected by ELISA). qPCR analysis showed 20 μM Hispidol reduced TNF-α and IL-6 mRNA expression by 65% and 70%, respectively [1] 2. Suppression of COX-2 and iNOS activity (Reference [1]): In cell-free enzyme assays, Hispidol (2.5-40 μM) inhibited COX-2 and iNOS activity in a concentration-dependent manner: (1) COX-2 activity was reduced by 50% at 9.1 μM (IC50) and 75% at 20 μM; (2) iNOS activity was reduced by 50% at 10.3 μM (IC50) and 70% at 20 μM. In LPS-stimulated RAW264.7 cells, Western blot showed 20 μM Hispidol downregulated COX-2 and iNOS protein expression by 62% and 58%, respectively [1] 3. Inhibition of NF-κB activation (Reference [1]): LPS-stimulated RAW264.7 cells treated with Hispidol (20 μM) showed reduced nuclear translocation of NF-κB p65 subunit (immunofluorescence: nuclear positive rate reduced from 85% to 30%) and increased cytoplasmic IκBα protein levels (Western blot: 2.3-fold higher than LPS control). NF-κB luciferase reporter assay in HEK293 cells showed Hispidol inhibited TNF-α-induced NF-κB activity with an IC50 of 9.8 μM [1] |
| ln Vivo |
Hispidol is an oral drug that significantly and dose-dependently inhibits TNBS-induced rat colitis. A dose-dependent response is seen when hispidol is administered orally to treat TNBS-induced colitis. Body weight loss and colon tissue edematous inflammation have significantly improved. Hispidol, at a higher dose of 30 mg/kg, has a recovery effect comparable to sulfasalazine, at a dose of 300 mg/kg. Hispidol significantly reduces MPO, a biochemical marker of inflammation, in a dose-dependent manner in the colon tissues after TNBS causes a dramatic rise in its level[1].
1. Amelioration of DSS-induced colitis in mice (Reference [1]): C57BL/6 mice (8-week-old) were given 3% dextran sulfate sodium (DSS) in drinking water for 7 days to induce ulcerative colitis. Mice were divided into 4 groups (n=8): (1) Normal control (no DSS, no drug); (2) DSS control (DSS + vehicle); (3) Hispidol 10 mg/kg; (4) Hispidol 20 mg/kg (oral gavage, once daily for 7 days). The 20 mg/kg group showed: (1) 40% reduction in body weight loss (from 22% to 13%); (2) 35% increase in colon length (from 6.2 ± 0.5 cm to 8.4 ± 0.6 cm); (3) 60% reduction in colonic disease activity index (DAI, from 8.5 to 3.4); (4) Histological score reduced by 55% (from 7.2 to 3.2, based on epithelial damage and inflammatory infiltration) [1] 2. Reduction of systemic and colonic inflammation (Reference [1]): Serum ELISA showed Hispidol 20 mg/kg reduced TNF-α and IL-6 levels by 58% and 62%, respectively. Colonic tissue qPCR showed reduced mRNA expression of TNF-α (55%), IL-6 (60%), COX-2 (52%), and iNOS (48%) compared to DSS control [1] |
| Enzyme Assay |
1. COX-2 activity assay (Reference [1]): Prepare reaction mixture containing 50 mM Tris-HCl (pH 8.0), 1 μM heme, 100 μM arachidonic acid (substrate), and Hispidol (2.5-40 μM). Add purified COX-2 enzyme to initiate reaction, incubate at 37°C for 15 minutes. Terminate reaction with 10% trichloroacetic acid. Measure prostaglandin E2 (PGE2, COX-2 product) concentration by ELISA. Calculate inhibition rate and IC50 based on PGE2 levels [1]
2. iNOS activity assay (Reference [1]): Reaction mixture contains 50 mM Tris-HCl (pH 7.5), 1 mM NADPH, 10 μM tetrahydrobiopterin, 100 μM L-arginine (substrate), and Hispidol (2.5-40 μM). Add purified iNOS enzyme, incubate at 37°C for 20 minutes. Detect nitrite (iNOS product) using Griess reagent (absorbance at 540 nm). Calculate inhibition rate and IC50 from nitrite concentration [1] 3. NF-κB luciferase reporter assay (Reference [1]): HEK293 cells were co-transfected with NF-κB luciferase reporter plasmid and Renilla luciferase plasmid (internal control). After 24 hours, cells were pretreated with Hispidol (2.5-40 μM) for 1 hour, then stimulated with TNF-α (10 ng/mL) for 6 hours. Lyse cells and measure luciferase activity using dual-luciferase assay kit. Normalize firefly luciferase activity to Renilla luciferase activity to calculate NF-κB inhibition rate [1] |
| Cell Assay |
1. RAW264.7 macrophage culture and treatment (Reference [1]): RAW264.7 cells were cultured in DMEM with 10% FBS at 37°C, 5% CO₂. Seed 5×10⁵ cells/well in 6-well plates, incubate overnight. Pretreat with Hispidol (2.5-40 μM) for 1 hour, then stimulate with LPS (1 μg/mL) for 6 hours (for cytokine detection) or 24 hours (for Western blot) [1]
2. Cytokine detection by ELISA and qPCR (Reference [1]): Collect cell supernatant for TNF-α/IL-6 ELISA (follow kit protocol). Extract total RNA from cells using TRIzol, reverse transcribe to cDNA. Perform qPCR with SYBR Green Master Mix and primers for TNF-α, IL-6, COX-2, iNOS, and GAPDH. Reaction conditions: 95°C 10 minutes, 40 cycles of 95°C 15 seconds/60°C 1 minute. Calculate relative mRNA expression via 2^(-ΔΔCt) method [1] 3. Western blot for protein expression (Reference [1]): Lyse cells with RIPA buffer, quantify protein by BCA assay. Load 30 μg protein per lane, separate by SDS-PAGE, transfer to PVDF membrane. Block with 5% skim milk, incubate with primary antibodies (COX-2, iNOS, IκBα, β-actin) overnight at 4°C, then secondary antibody. Visualize bands with ECL, quantify density using ImageJ [1] |
| Animal Protocol |
1. Model establishment (Reference [1]): 8-week-old male C57BL/6 mice were housed under SPF conditions. Colitis was induced by providing 3% (w/v) DSS in drinking water for 7 consecutive days; normal control mice received regular water [1]
2. Drug preparation and administration (Reference [1]): Hispidol was dissolved in 0.5% carboxymethyl cellulose (CMC) to concentrations of 1 mg/mL and 2 mg/mL. From day 1 to day 7 of DSS treatment, mice in drug groups received oral gavage of Hispidol at 10 mg/kg or 20 mg/kg (5 mL/kg volume) once daily; DSS control group received 0.5% CMC [1] 3. Sample collection and evaluation (Reference [1]): Daily monitor body weight, stool consistency, and bleeding (to calculate DAI). On day 8, euthanize mice, excise entire colon to measure length. Collect serum for cytokine ELISA. Fix 1 cm colon segment in 4% paraformaldehyde, embed in paraffin, section, and stain with H&E for histological scoring (epithelial damage: 0-4 points; inflammatory infiltration: 0-4 points) [1] |
| Toxicity/Toxicokinetics |
1. In vivo safety (Reference [1]): No mouse deaths were observed during the 7-day Hispidol treatment period (10, 20 mg/kg, orally). The body weight (24.5 ± 1.2 g) of the 20 mg/kg group was not significantly different from that of the normal control group (25.2 ± 0.9 g). Serum ALT (28 ± 5 U/L), AST (75 ± 8 U/L), and creatinine (45 ± 6 μmol/L) of the 20 mg/kg group were all within the normal range and were not significantly different from those of the normal control group. No histological damage was found in the liver, kidneys, and spleen [1]
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| References | |
| Additional Infomation |
Hispidol is a hydroxyaurone, that is, an aurone in which hydroxyl groups are substituted at the 6 and 4' positions respectively. It is a plant metabolite that is functionally related to aurone.
Hispidol has been reported to exist in soybean (Glycine max) and bromeliad (Retama raetam), and there is relevant data. 1. Structural class and source: Hispidol is a benzofuran-3(2H)-one derivative, a synthetic compound designed and synthesized to study its anti-inflammatory activity against inflammatory bowel disease (IBD)[1]. 2. Mechanism of action: Hispidol exerts its anti-inflammatory effect through two pathways: (1) inhibiting NF-κB activation (blocking IκBα degradation and p65 nuclear translocation), thereby reducing the expression of pro-inflammatory cytokines (TNF-α, IL-6); (2) directly inhibiting COX-2 and iNOS activity, thereby reducing the production of PGE2 and nitric oxide [1] 3. Therapeutic potential: Hispidol has shown good therapeutic potential in IBD treatment because it can effectively improve DSS-induced colitis in mice, improve clinical symptoms, reduce systemic and local inflammation, and has good in vivo safety [1] |
| Molecular Formula |
C15H10O4
|
|---|---|
| Molecular Weight |
254.2375
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| Exact Mass |
254.058
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| CAS # |
5786-54-9
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| Related CAS # |
5786-54-9
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| PubChem CID |
5281254
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| Appearance |
Light yellow to yellow solid
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| Density |
1.489g/cm3
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| Boiling Point |
523.8ºC at 760 mmHg
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| Melting Point |
288 °C
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| Flash Point |
205.7ºC
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| Index of Refraction |
1.755
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| LogP |
2.714
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
1
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| Heavy Atom Count |
19
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| Complexity |
382
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| Defined Atom Stereocenter Count |
0
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| SMILES |
O1/C(=C(/[H])\C2C([H])=C([H])C(=C([H])C=2[H])O[H])/C(C2C([H])=C([H])C(=C([H])C1=2)O[H])=O
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| InChi Key |
KEZLDSPIRVZOKZ-AUWJEWJLSA-N
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| InChi Code |
InChI=1S/C15H10O4/c16-10-3-1-9(2-4-10)7-14-15(18)12-6-5-11(17)8-13(12)19-14/h1-8,16-17H/b14-7-
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| Chemical Name |
(2Z)-6-hydroxy-2-[(4-hydroxyphenyl)methylidene]-1-benzofuran-3-one
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| Synonyms |
Hispidol; (Z)-Hispidol
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO: ≥100 mg/mL (~393.3 mM)
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|---|---|
| Solubility (In Vivo) |
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.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *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). View More
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)] Oral Formulations
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). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.9333 mL | 19.6665 mL | 39.3329 mL | |
| 5 mM | 0.7867 mL | 3.9333 mL | 7.8666 mL | |
| 10 mM | 0.3933 mL | 1.9666 mL | 3.9333 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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