- Coixol targets three key signaling molecules/pathways: nuclear factor kappa B (NF-κB) pathway, mitogen-activated protein kinase (MAPK) pathway (including p38 MAPK, JNK, ERK), and NLRP3 inflammasome [1]

- Anti-inflammatory activity in LPS-induced RAW 264.7 macrophages: Coixol (10, 20, 40 μM) reduced the secretion of pro-inflammatory cytokines in a concentration-dependent manner. Compared with the LPS-only group, 40 μM Coixol decreased TNF-α secretion by approximately 65%, IL-1β secretion by approximately 70%, and IL-6 secretion by approximately 60% (detected by ELISA). [1]
- Inhibition of NF-κB pathway: Coixol suppressed LPS-induced phosphorylation of NF-κB p65 and degradation of IκBα, thereby reducing the nuclear translocation of p65. Western blot results showed that 40 μM Coixol downregulated the expression of nuclear p-p65 by ~55% and cytoplasmic p-IκBα by ~60% relative to the LPS-only group. [1]
- Inhibition of MAPK pathway: Coixol inhibited LPS-induced phosphorylation of p38 MAPK, JNK, and ERK. At 40 μM, Coixol reduced the expression of p-p38 by ~50%, p-JNK by ~58%, and p-ERK by ~45% compared with the LPS-only group (detected by Western blot). [1]
- Suppression of NLRP3 inflammasome activation: Coixol (10, 20, 40 μM) decreased the expression of NLRP3, ASC, and cleaved caspase-1 (p20) in a concentration-dependent manner. It also reduced the level of cleaved IL-1β (p17) by ~68% at 40 μM compared with the LPS-only group (detected by Western blot and RT-PCR). [1]
- Cytotoxicity evaluation: Coixol (0-40 μM) showed no significant cytotoxicity to RAW 264.7 cells; cell viability remained above 90% even at 40 μM (detected by MTT assay). [1]

- Cell culture and treatment: RAW 264.7 mouse macrophage cells were cultured in DMEM medium supplemented with fetal bovine serum and antibiotics, and maintained at 37°C in a 5% CO₂ incubator. Cells were seeded in 6-well, 24-well, or 96-well plates (based on assay type) and allowed to adhere overnight. Before LPS (1 μg/mL) stimulation, cells were pre-treated with Coixol (10, 20, 40 μM) for 1 hour, then co-incubated with LPS for 12 hours (for protein/mRNA detection) or 24 hours (for cytokine detection and cell viability assay). [1]
- MTT cell viability assay: Cells in 96-well plates were pre-treated with Coixol (0-40 μM) for 1 hour, then stimulated with LPS (1 μg/mL) for 24 hours. MTT solution was added to each well and incubated for 4 hours. The formazan crystals were dissolved with dimethyl sulfoxide, and absorbance was measured at 570 nm. Cell viability was calculated as a percentage relative to the untreated control group. [1]
- ELISA for cytokine detection: After cell treatment, culture supernatants were collected and centrifuged to remove cell debris. TNF-α, IL-1β, and IL-6 levels in supernatants were measured using ELISA kits. Absorbance was read at 450 nm, and cytokine concentrations were determined using standard curves. [1]
- Western blot analysis: Cells were lysed with RIPA buffer containing protease and phosphatase inhibitors. Nuclear and cytoplasmic proteins were separated using a nuclear extraction kit. Protein concentrations were measured by BCA assay. Equal amounts of proteins were separated by SDS-PAGE, transferred to PVDF membranes, and blocked with non-fat milk. Membranes were incubated with primary antibodies against p-p65, p65, p-IκBα, IκBα, p-p38, p38, p-JNK, JNK, p-ERK, ERK, NLRP3, ASC, cleaved caspase-1 (p20), cleaved IL-1β (p17), and β-actin (loading control) overnight at 4°C. After incubation with HRP-conjugated secondary antibodies, protein bands were visualized by ECL, and band intensities were quantified with image analysis software. [1]
- RT-PCR analysis: Total RNA was extracted from cells using an RNA extraction kit and reverse-transcribed into cDNA. PCR amplification was performed with specific primers for TNF-α, IL-1β, IL-6, NLRP3, ASC, and GAPDH (internal control). PCR products were separated by agarose gel electrophoresis, stained with ethidium bromide, and visualized under UV light. Band intensities were quantified, and target gene expression was normalized to GAPDH. [1]

[1]. Coixol Suppresses NF-κB, MAPK Pathways and NLRP3 Inflammasome Activation in Lipopolysaccharide-Induced RAW 264.7 Cells. Molecules

6-Methoxy-2-benzoxazolinone is a 2-benzoxazolinone with a methoxy group substituted at the 6-position. It is a plant metabolite with muscle relaxant, anticonvulsant, antibacterial, and antifungal activities. It is a benzoxazole aromatic ether functionally related to 2-benzoxazolinone. Coixol has been reported in corn, barley, and several other organisms with relevant data. Coixol (chemical name: 4-methylumbelliferone) is a natural coumarin derivative, primarily isolated from Job's tears (Coix lacryma-jobi L., a grass), and has been used in traditional medicine for its anti-inflammatory and analgesic effects. [1] The anti-inflammatory mechanism of coix seed in LPS-induced RAW 264.7 cells is achieved by simultaneously inhibiting the activation of NF-κB, MAPK pathway and NLRP3 inflammasome, suggesting its potential as a treatment for inflammatory diseases associated with overactivation of these pathways, such as sepsis and rheumatoid arthritis. [1]

C8H7NO3

165.15

165.042

532-91-2

10772

Light brown to brown solid powder

1.3±0.1 g/cm3

292.97°C (rough estimate)

151-156ºC(lit.)

1.563

1.07

1

3

1

12

195

0

O1C(N([H])C2C([H])=C([H])C(=C([H])C1=2)OC([H])([H])[H])=O

MKMCJLMBVKHUMS-UHFFFAOYSA-N

InChI=1S/C8H7NO3/c1-11-5-2-3-6-7(4-5)12-8(10)9-6/h2-4H,1H3,(H,9,10)

6-methoxy-3H-1,3-benzoxazol-2-one

6-Methoxy-2-benzoxazolinone; 6-METHOXYBENZO[D]OXAZOL-2(3H)-ONE; MBOA; 6-Methoxybenzoxazolinone; 6-MBOA

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 : ~100 mg/mL (~605.51 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (15.14 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 (15.14 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 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.)