IC50: EV71; bacterial
For anti-EV71 activity: the compound inhibits EV71 replication, specifically viral VP2 protein expression, thereby inhibiting viral capsid protein synthesis; it affects the initial stage of virus infection but does not block viral attachment and entry (IC50 = 37.72 ± 0.78 μg/mL for EV71-induced cytopathic effect). [1][3]
For anti-Pseudomonas aeruginosa activity: the compound targets quorum sensing (QS) regulatory proteins LasR and RhlR by competitively inhibiting the binding of natural autoinducers, as evidenced from molecular dynamics simulation studies. [2]

Mosloflavone inhibits TNF-α, IL-1β, and iNOS levels in the supernatant of mouse macrophage cell line J774A in a dose-dependent manner. It can also be used as a starting point to find lead structures for the treatment of inflammatory and immunomodulatory diseases[2]. Mosloflavone exhibits promising anti-inflammatory activity via inhibition of TNF-α and IL-1β with IC50 values of 16.4 uM and 6.4 uM, respectively.

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5-Hydroxy-6,7-dimethoxylflavone exhibited strong anti-EV71 activity against EV71 in Vero cells in a dose-dependent manner; at a concentration of 50 μg/mL, it strongly decreased the formation of visible cytopathic effects (CPE) and enhanced cell viability (EC50 = 37.72 ± 0.78 μg/mL, CC50 > 50 μg/mL). [1][3]
In time-of-addition assays, the compound suppressed EV71 infection when added just after virus inoculation (0 h) and during early stages after inoculation (1, 2, and 4 h), with inhibitory levels higher than 80%; however, when added prior to infection (-1 h), the inhibitory level declined to ≤20%, indicating it affects the initial stage of EV71 infection but does not block viral attachment and entry. [1][3]
Real-time PCR analysis showed that at 48 h after EV71 infection, the compound had a marginal effect on expression of the EV71 NCR gene (at 50 μg/mL). [1][3]
Western blot analysis showed that viral VP2 protein expression (34 kDa) was decreased dramatically by 5-Hydroxy-6,7-dimethoxylflavone at 50 μg/mL at 48 h after infection with EV71. [1][3]
Against Pseudomonas aeruginosa PAO1, 5-Hydroxy-6,7-dimethoxylflavone exhibited anti-quorum sensing activity with a 16 mm zone of inhibition at sub-MIC dose. It inhibited pyocyanin production by 59.52 ± 2.74%, LasB elastase activity by 35.90 ± 4.34%, and chitinase activity by 61.18 ± 5.52% at sub-MIC concentration. [2]
The compound also reduced biofilm formation, exopolysaccharide (EPS) production, alginate production, and rhamnolipid production. It significantly reduced swimming and swarming motilities. Light microscopy, confocal laser scanning microscopy (CLSM), and scanning electron microscopy (SEM) analyses revealed that treatment with 5-Hydroxy-6,7-dimethoxylflavone resulted in thinner biofilm architecture compared to thick aggregated biofilm in control. [2]
RT-PCR gene expression studies demonstrated that the compound down-regulated the expression levels of QS-regulated virulence genes: lasI (60.64%), lasR (91.70%), rhl (57.30%), chiC (90.20%), rhlA (47.87%), rhlR (21.55%), lasB (37.80%), phzM (42.40%), toxA (61.00%), aprA (58.4%), exoS (78.01%), algD (46.60%), and pelA (50.45%). [2]
Molecular dynamics simulation studies (using GROMACS 5.1.2 with GROMOS force field) validated the stability of LasR-5-Hydroxy-6,7-dimethoxylflavone and RhlR-5-Hydroxy-6,7-dimethoxylflavone complexes. Post-simulation binding energy calculation using MM/PBSA was performed to measure protein-ligand interaction. [2]

In a Caenorhabditis elegans liquid killing assay, 5-Hydroxy-6,7-dimethoxylflavone significantly enhanced the survival rate of C. elegans infected with P. aeruginosa PAO1, indicating attenuation of bacterial virulence factors. [2]
In an in vivo colonization assay using GFP-tagged P. aeruginosa PAO1 and C. elegans (L4 stage) on nematode growth medium, treatment with 5-Hydroxy-6,7-dimethoxylflavone reduced bacterial colonization inside the nematodes as observed under a fluorescence microscope. [2]

For anti-EV71 activity: Vero cells (kidney epithelial cell line from African green monkey) were seeded onto 96-well culture plates at 2×10⁴ cells per well. The next day, medium was removed and cells were washed with PBS. Then 0.09 mL of diluted virus suspension of EV71 (containing TCID₅₀ to produce cytopathic effects within 2 days) and 0.01 mL of 5-Hydroxy-6,7-dimethoxylflavone (at concentrations 0.4, 2, 10, and 50 μg/mL) were added. After incubation at 37°C in 5% CO₂ for 2 days, cell morphology was observed under a microscope at 32×10 magnifications, and cell viability was evaluated using the sulforhodamine B (SRB) assay for cytopathic effect reduction. [1][3]
For time-of-addition assays: Vero cells were seeded onto 96-well plates at 2×10⁴ cells per well and incubated for 1 day. After washing with PBS, 50 μg/mL of 5-Hydroxy-6,7-dimethoxylflavone was added either before (-1 h), during (0 h), or after (1, 2, 4, 6, and 8 h) EV71 infection. After 2 days, antiviral activity was tested using the SRB assay. [1][3]
For real-time PCR analysis: Vero cells were seeded onto 96-well plates at 2×10⁴ cells per well. The next day, medium was removed and cells were washed with PBS. Then 90 μL of diluted virus suspension and 10 μL of medium containing 50 μg/mL 5-Hydroxy-6,7-dimethoxylflavone were added. After incubation at 37°C in 5% CO₂ for 48 h, total RNA was extracted, reverse transcription was performed, and real-time PCR was carried out using primers for β-actin and EV-NCR. [1][3]
For Western blot analysis: Vero cells were plated onto 6-well plates at 5×10⁵ cells/well 24 h before infection with EV71. Infected cells were treated with 5-Hydroxy-6,7-dimethoxylflavone at 50 μg/mL for 48 h for detection of viral VP2 protein. Mock-infected cells treated with 0.1% DMSO and EV71-infected cells treated with 0.1% DMSO were used as controls. Cells were lysed in ice-cold RIPA lysis buffer, and protein samples (30 mg) were boiled and separated in 12% acrylamide gels. Proteins were transferred to nitrocellulose membranes, blocked with 5% skim milk, and incubated with primary mouse anti-EV71 monoclonal antibody (1:1,000) or α-tubulin mouse monoclonal IgG1 (1:1,000), followed by secondary polyclonal goat anti-mouse IgG-HRP (1:5,000). Membranes were developed by enhanced chemiluminescence. [1][3]
For anti-P. aeruginosa assays: Various cell-based assays were performed using P. aeruginosa PAO1. Pyocyanin inhibition was measured by mixing supernatant with chloroform (3:2) and extracting with 0.2N HCl, recording OD at 520 nm. LasB elastolytic activity was determined using Elastin Congo Red (ECR) buffer incubated with supernatant for 3 h at 37°C, then mixed with sodium phosphate buffer, and OD measured at 495 nm. Chitinase activity was assayed using chitin azure as substrate in sodium citrate buffer, agitated for 7 days, centrifuged, and OD recorded at 570 nm. Biofilm inhibition was performed using microtiter plate assay with crystal violet staining (0.4%) and OD at 590 nm. Exopolysaccharide quantification was done by total carbohydrate assay (phenol-sulphuric acid method) with OD at 490 nm. Alginate inhibition was quantified by carbazole method with OD at 530 nm. Cell surface hydrophobicity was evaluated using toluene and OD at 600 nm. Rhamnolipid production was determined by Orcinol assay with OD at 421 nm. Swimming and swarming motility were assessed on agar media (0.3% and 0.5% agar respectively) after 24 h incubation at 37°C. Light microscopy, CLSM (with acridine orange staining), and SEM (with glutaraldehyde fixation) were used to visualize biofilm architecture. [2]
For RT-PCR gene expression: Total RNA from P. aeruginosa PAO1 treated with 5-Hydroxy-6,7-dimethoxylflavone was extracted using Trizol. cDNA was constructed using a cDNA kit. Genes were amplified using SYBR Green Master Mix in a real-time PCR system with primers listed in Table 1 (including lasI, lasR, rhl, rhlR, phzM, lasB, rhlA, toxA, exoS, aprA, algD, chiC, pelA, proC). Cycling conditions: initial denaturation at 95°C for 10 min, then 45 cycles of 95°C for 30 s, annealing at specific temperatures for 15 s, extension at 72°C for 15 min, and final extension at 72°C for 5 min. [2]

For C. elegans survival assay (liquid killing assay): C. elegans (L4 stage) infected with P. aeruginosa PAO1 were incubated for 12 h at 25°C in a 24-well microtiter plate. Fifteen worms were relocated to wells containing M9 buffer with 10% LB broth and incubated at 25°C with or without 5-Hydroxy-6,7-dimethoxylflavone at sub-MIC concentration. C. elegans fed with E. coli OP50 was used as control. Dead and live worms were counted every 12 h for 4 days and a survival curve was generated. [2]
For in vivo colonization assay: C. elegans (L4 stage) were grown on nematode growth medium with GFP-tagged P. aeruginosa PAO1 with and without treatment of 5-Hydroxy-6,7-dimethoxylflavone. After 24 h incubation, nematodes were washed with M9 buffer and analyzed under a fluorescence microscope. [2]

In Vero cells, 5-Hydroxy-6,7-dimethoxylflavone showed no signs of cytotoxicity at concentrations up to 50 μg/mL (CC50 > 50 μg/mL). [1][3]
In P. aeruginosa PAO1, the minimum inhibitory concentration (MIC) was determined to be 250 μg/mL, and sub-MIC concentrations were used for all biological activities without observed growth inhibition. [2]

[1]. Inhibitory Effects of Norwogonin, Oroxylin A, and Mosloflavone on Enterovirus 71.Biomol Ther (Seoul). 2016 Sep 1;24(5):552-8.

[2]. Mosloflavone attenuates the quorum sensing controlled virulence phenotypes and biofilm formation in Pseudomonas aeruginosa PAO1: In vitro, in vivo and in silico approach.Microb Pathog. 2019 Jun;131:128-134.

[3]. Anti-inflammatory and immunomodulatory flavones from Actinocarya tibetica Benth.Nat Prod Res. 2013;27(23):2227-30.

Mosloflavone belong to the flavonoid class of compounds and are a type of ether compound. They have been reported to be found in Candida dulcis, Candida suans, and other organisms with relevant data.

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5-Hydroxy-6,7-dimethoxylflavone is a flavonoid isolated from the aerial parts of Scutellaria baicalensis Georgi (using methanol extraction, partitioning into chloroform, ethyl acetate, n-butanol, and water fractions, followed by C18 column chromatography and Sephadex LH20 column chromatography; chemical structures identified using electrospray ionization mass spectrometry, ¹H-NMR, and ¹³C-NMR). [1][3]
It was also derived from the herbs of Mosla soochouensis Matsuda. [2]
The compound exhibits anti-EV71 activity by inhibiting viral VP2 protein expression and acts as an early-stage inhibitor of EV71 replication or translation without blocking viral attachment and entry. [1][3]
It attenuates quorum sensing controlled virulence phenotypes and biofilm formation in Pseudomonas aeruginosa PAO1 by down-regulating QS-regulated virulence genes and competitively inhibiting autoinducer binding to LasR and RhlR. [2]
The compound showed promising potential in controlling bacterial infection in the Caenorhabditis elegans model system in vivo. [2]

C17H14O5

298.2901

298.084

740-33-0

471722

Light yellow to yellow solid powder

1.3±0.1 g/cm3

508.5±50.0 °C at 760 mmHg

163-164ºC (chloroform , acetone )

190.5±23.6 °C

0.0±1.4 mmHg at 25°C

1.622

2.74

1

5

3

22

440

0

SIVAITYPYQQYAP-UHFFFAOYSA-N

InChI=1S/C17H14O5/c1-20-14-9-13-15(16(19)17(14)21-2)11(18)8-12(22-13)10-6-4-3-5-7-10/h3-9,19H,1-2H3

5-hydroxy-6,7-dimethoxy-2-phenylchromen-4-one

5-Hydroxy-6,7-dimethoxylflavone;F4DL1FN60Q;

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 : ~50 mg/mL (~167.62 mM)

Solubility in Formulation 1: 2.5 mg/mL (8.38 mM) in 10% DMSO + 90% (20% SBE-β-CD in 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 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.)