Natural flavone; anti-inflammatory, anti-tumor, anti-oxidant, neuroprotective, anti-fungal activities
Inhibition of the NF-κB pathway by suppressing phosphorylation of IKKα/β and IκBα, thereby downregulating downstream pro-inflammatory proteins COX-2 and iNOS. [1]
Potential inhibition/modulation of the NorA efflux pump in Staphylococcus aureus. [2]

Cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) are critical pro-inflammatory proteins. The level of protein and mRNA expression of these enzymes induced by lipopolysaccharide (LPS) was dramatically suppressed by treatment with SWE, scutellarein tetramethyl ether/scu, or stigmasterol compounds in a dose-dependent manner. They also reduced PGE2 and NO release. We further analyzed the NF-κB pathway and found that the scu compound suppressed IκB kinase complex alpha/beta (IKKα/β) and Inhibitory-kappa-B-alpha (IκBα), thereby suppressing COX-2 and iNOS expression.
Conclusion: This is the first report of the anti-inflammatory molecular mechanism in SWE and/or its bioactive component scu, indicating alteration NF-κB pathway and further providing potential uses in the treatment of inflammatory-related diseases.[1]

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The purified compound, 4', 5, 6, 7-tetramethoxyflavone, is an active ingredient isolated from Eupatorium odoratum, a Thai indigenous plant that has long been used to stop bleeding. This compound was studied in vitro for the effect on blood clotting factor activities. It was found that the compound enhanced blood coagulation, the observed APTT being shorter than that observed in the control. The result suggested that the compound accelerated clotting time through the intrinsic pathway of the coagulation which may involve the reaction of factor XII, factor XI, factor IX or factor VIII.[3]

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In LPS-stimulated RAW 264.7 murine macrophages, Scutellarein tetramethyl ether at 50 and 100 μM significantly inhibited the protein expression of COX-2 and iNOS in a dose-dependent manner, as determined by Western blot analysis. [1]
The compound at 50 and 100 μM significantly reduced the production of prostaglandin E2 (PGE2) and nitric oxide (NO) in the supernatants of LPS-stimulated RAW 264.7 cells. [1]
At the transcriptional level, Scutellarein tetramethyl ether (50 and 100 μM) suppressed LPS-induced mRNA expression of pro-inflammatory cytokines, including COX-2, iNOS, IL-6, and TNF-α, as shown by RT-PCR analysis. [1]
A luciferase reporter assay demonstrated that Scutellarein tetramethyl ether (10, 50, and 100 μM) significantly inhibited LPS-induced NF-κB promoter activity in a dose-dependent manner in RAW 264.7 cells. [1]
Immunocytochemistry analysis revealed that pretreatment with 50 μM Scutellarein tetramethyl ether inhibited LPS-induced nuclear translocation of the NF-κB p65 subunit in RAW 264.7 cells. [1]
Western blot analysis showed that Scutellarein tetramethyl ether (50 and 100 μM) potently suppressed the phosphorylation of IKKα/β and IκBα in LPS-stimulated RAW 264.7 cells. [1]
A cell proliferation assay (CellTiter 96) indicated that treatment with Scutellarein tetramethyl ether at 50 and 100 μM for 24 hours did not show cytotoxicity in RAW 264.7 cells; instead, a non-significant increase in cell proliferation was observed at 100 μM compared to the vehicle control. [1]
Scutellarein tetramethyl ether, at a concentration up to 256 μg/mL, did not show any direct antibacterial activity against the Staphylococcus aureus SA-1199B strain (which overexpresses the NorA efflux pump). [2]
When used at a sub-inhibitory concentration of 64 μg/mL, Scutellarein tetramethyl ether was able to modulate bacterial drug resistance. It reduced the Minimum Inhibitory Concentration (MIC) of the antibiotic norfloxacin against S. aureus SA-1199B from 128 μg/mL to 8 μg/mL, representing a 16-fold reduction. [2]
Similarly, at 64 μg/mL, it reduced the MIC of ethidium bromide against the same strain from 32 μg/mL to 2 μg/mL, also representing a 16-fold reduction. [2]
The study noted that Scutellarein tetramethyl ether (being the most methoxylated flavone tested) showed the highest efflux pump inhibitory/modulatory activity among the isolated compounds, and this property might be related to its degree of lipophilicity. [2]

Siam weed (Chromolaena odorata (L.) King and Robinson) is a medicinal herb used for wound healing and inflammation-related diseases.
Aim of the study: In this study, we evaluated the molecular mechanism by which Siam weed extract (SWE) and its bioactive components, scutellarein tetramethyl ether (scu), stigmasterol, and isosakuranetin affect anti-inflammatory activity.
Materials and methods: The expression of several inflammatory proteins in RAW 264.7 (murine) macrophages was assessed by Western blot and reverse transcription-polymerase chain reaction (RT-PCR). Biochemical assays including prostaglandin E2 (PGE2) and nitric-oxide (NO) quantification were performed. Luciferase promoter activity and immunocytochemistry of Nuclear factor-κB (NF-κB) were investigated[1].

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Cell Proliferation/Viability Assay: RAW 264.7 cells were seeded at 2.0 x 10^4 cells/well in 96-well plates and grown for 2 days. Cells were then treated with Scutellarein tetramethyl ether at 50 and 100 μM (with 1% serum) for 24 hours. Cell viability was assessed by adding a cell proliferation assay solution, incubating for 1 hour at 37°C, and measuring absorbance at 490 nm. [1]
Western Blot Analysis for Protein Expression: RAW 264.7 cells were grown to 80% confluence and pretreated with Scutellarein tetramethyl ether (50 and 100 μM) for 1 hour in serum-free medium, followed by stimulation with 100 ng/ml LPS for 12 hours. Total cell lysates were prepared, and proteins were separated by SDS-PAGE, transferred to a membrane, and probed with specific primary antibodies against COX-2, iNOS, phospho-IKKα/β, phospho-IκBα, and actin, followed by HRP-conjugated secondary antibodies and chemiluminescence detection. [1]
Measurement of PGE2 and NO: RAW 264.7 cells were pretreated with Scutellarein tetramethyl ether (50 and 100 μM) for 1 hour, followed by LPS (100 ng/ml) stimulation for 12 hours. Cell culture supernatants were collected. PGE2 concentration was measured using a competitive enzyme immunoassay (EIA) kit. NO production was determined by measuring nitrate concentration in the medium using a colorimetric assay kit. [1]
Reverse Transcription-Polymerase Chain Reaction (RT-PCR): Total RNA was extracted from RAW 264.7 cells pretreated with Scutellarein tetramethyl ether (50 and 100 μM) for 1 hour and then stimulated with LPS (100 ng/ml) for 12 hours. RNA was reverse transcribed into cDNA, and PCR amplification was performed using specific primers for mouse COX-2, iNOS, IL-6, TNF-α, CD68, and GAPDH. PCR products were separated on agarose gels and visualized. [1]
Transient Transfection and Luciferase Promoter Assay: RAW 264.7 cells were plated in 12-well plates and transfected with a reporter plasmid containing three NF-κB binding sites linked to a firefly luciferase gene and a control renilla luciferase plasmid. After 24 hours, transfected cells were pretreated with Scutellarein tetramethyl ether (10, 50, and 100 μM) for 1 hour, then stimulated with LPS (100 ng/ml) for 24 hours. Cells were lysed, and luciferase activity was measured and normalized to the control luciferase activity. [1]
Immunocytochemistry for NF-κB Localization: RAW 264.7 cells were seeded on glass-bottom dishes, pretreated with 50 μM Scutellarein tetramethyl ether for 1 hour, and then stimulated with LPS (100 ng/ml) for 12 hours. Cells were fixed, permeabilized, blocked, and incubated with a primary antibody against NF-κB p65, followed by a fluorescent dye-conjugated secondary antibody. Nuclei were stained with DAPI, and cellular localization was observed under a fluorescence microscope. [1]
Bacterial Drug Susceptibility and Modulation Assay: The minimum inhibitory concentrations (MICs) of antibiotics and Scutellarein tetramethyl ether against Staphylococcus aureus strain SA-1199B were determined using a microdilution assay in brain heart infusion (BHI) broth. A bacterial suspension of approximately 10^5 CFU/mL was used. The drug concentration range tested was from 256 to 0.5 μg/mL via twofold serial dilutions. Bacterial growth was detected using a resazurin solution (0.01% w/v), where a color change from blue to pink indicated growth. The MIC was defined as the lowest concentration with no visible growth. To evaluate Scutellarein tetramethyl ether as a modulator of drug resistance, its MIC was first determined. Then, a sub-inhibitory concentration (64 μg/mL, which is 1/4 of its MIC >256 μg/mL) was added to the BHI broth alongside serial dilutions of antibiotics (norfloxacin, pefloxacin) or ethidium bromide. The MICs of these agents were then re-determined in the presence of the flavone to observe any reduction. [2]

Baicalein tetramethyl ether was prepared as a DMSO stock solution. After dilution, the final concentration (4%) of DMSO in the bacterial culture medium did not inhibit bacterial growth, indicating that the compound or solvent did not exhibit acute cytotoxicity under the experimental conditions at the tested concentration. No other toxicity data (e.g., LD50, organ toxicity) were provided. [2]

[1]. Effect of Siam weed extract and its bioactive component scutellarein tetramethyl ether on anti-inflammatory activity through NF-κB pathway. J Ethnopharmacol. 2013 May 20;147(2):434-41.

[2]. Flavonoids from Praxelis clematidea R.M. King and Robinson modulate bacterial drug resistance. Molecules. 2011 Jun 10;16(6):4828-35.

[3]. Effect of Eupatorium odoratum on blood coagulation. J Med Assoc Thai. 1991 May;74(5):283-7.

4',5,6,7-Tetramethoxyflavonoid is a tetramethoxyflavonoid, a tetra-O-methyl derivative of baicalin. It has antimutagenic and plant metabolite effects, and its function is related to that of baicalin.
4',5,6,7-Tetramethoxyflavonoid has been reported to be found in citrus (Citrus reticulata), marjoram (Marrubium peregrinum) and other organisms with relevant data.
See also: orange peel (part); sour orange (Citrus aurantium) peel (part).

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Tetramethoxyflavonoid is one of the major bioactive flavonoids identified in the 70% ethanol extract of Chromolaena odorata leaves. [1]
This study elucidates for the first time the anti-inflammatory molecular mechanism of tetramethoxyflavonoid, confirming that its mechanism of action is through inhibition of the NF-κB signaling pathway. [1]
The compounds used in this experiment are commercially available products. [1]
Baicalein tetramethyl ether (4',5,6,7-tetramethoxyflavonoid) is one of six flavonoid compounds isolated from the aerial parts of Praxelis clematidea RM King & Robinson, a plant in the Asteraceae family. [2]
This study shows that baicalein tetramethyl ether may regulate antibiotic resistance by inhibiting the NorA efflux pump in Staphylococcus aureus, thereby enhancing the activity of antibiotics such as norfloxacin and drugs such as ethidium bromide. [2]
The chemical structure of this compound was confirmed by spectral data analysis (¹H and ¹³C NMR). [2]

C19H18O6

342.34262

342.11

C, 66.66; H, 5.30; O, 28.04

1168-42-9

96118

Off-white to light yellow solid powder

1.243g/cm3

525.7ºC at 760mmHg

142-143ºC

232.4ºC

3.84E-11mmHg at 25°C

1.574

3.494

0

6

5

25

496

0

URSUMOWUGDXZHU-UHFFFAOYSA-N

InChI=1S/C19H18O6/c1-21-12-7-5-11(6-8-12)14-9-13(20)17-15(25-14)10-16(22-2)18(23-3)19(17)24-4/h5-10H,1-4H3

5,6,7-trimethoxy-2-(4-methoxyphenyl)chromen-4-one

Scutellarein tetramethyl ether; 1168-42-9; 4',5,6,7-Tetramethoxyflavone; Tetramethyl-O-scutellarin; Tetra-O-methylscutellarein; Tetramethylscutellarein; Flavone, 4',5,6,7-tetramethoxy-; 5,6,7,4'-Tetramethoxyflavone;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~83.33 mg/mL (~243.41 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (6.08 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 20.8 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: ≥ 2.08 mg/mL (6.08 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 20.8 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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Solubility in Formulation 3: ≥ 2.08 mg/mL (6.08 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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