5-Demethylnobiletin inhibits 5-lipoxygenase (5-LOX) in rat peritoneal neutrophils (IC50 = 0.35 μM for LTB4 production from endogenous arachidonic acid). [1]
In PC12 cells, 5-Demethylnobiletin activates the MAPK/ERK, PKA, and PKC signaling pathways, leading to the phosphorylation of cAMP response element-binding protein (CREB) at Ser-133. This activation is independent of the TrkA receptor. [2]

5-O-Demethylnobiletin (5-demethylnobiletin) stimulates signaling pathways that are dependent on PKC, PKA, and MAPK/ERK, hence promoting neuritogenesis [2].

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In rat peritoneal neutrophils, 5-Demethylnobiletin inhibited leukotriene B4 (LTB4) production with an IC50 of 0.35 μM. In lysed cell preparations, it inhibited LTB4 synthesis by 57% at 0.25 μM. [1]
5-Demethylnobiletin inhibited elastase release from human neutrophils by 48% at a concentration of 10 μM but did not affect the activity of the elastase enzyme itself. [1]
In LPS-stimulated RAW 264.7 macrophages, 5-Demethylnobiletin showed little effect on cyclooxygenase-2 (COX-2) expression, with an inhibition range of only ≤20% at concentrations of 10, 5, and 2.5 μM. [1]
In PC12 cells, 5-Demethylnobiletin (10-20 μM) potently induced neurite outgrowth, with the percentage of neurite-bearing cells reaching 49.1±2.2% at 10 μM and 53.5±2.5% at 20 μM after 48 hours. This was accompanied by increased expression of the neuronal differentiation and synapse formation marker proteins, growth-associated protein-43 (GAP-43) and synaptophysin. [2]
The neurite outgrowth induced by 5-Demethylnobiletin in PC12 cells was not affected by the TrkA antagonist K252a (100 nM), indicating a TrkA-independent mechanism. [2]
5-Demethylnobiletin (10 μM) markedly induced the phosphorylation of CREB in PC12 cells, with a 1.7-fold induction observed after 120 minutes. It also increased cAMP response element (CRE)-mediated transcription by approximately 10-fold. [2]
The CREB antagonist KG-501 (10 μM) or transfection with CREB siRNA significantly attenuated the promotion of neurite outgrowth by 5-Demethylnobiletin. [2]
5-Demethylnobiletin (10 μM) activated ERK1/2 phosphorylation in PC12 cells, with phosphorylation significantly increased at 15 min, peaking at 60 min and remaining elevated for 120 min. It also increased PKC and PKA activity, peaking at 30 min and remaining elevated for 120 min. [2]
MEK1/2 inhibitors (U0126, PD98059), a PKC inhibitor (BIM), and PKA inhibitors (H-89, Rp-cAMPS) blocked the CRE transcription activity and neurite outgrowth induced by 5-Demethylnobiletin in PC12 cells. CaMK II inhibitor (KN-62) and PI3-K inhibitor (LY294002) did not affect the neurite outgrowth. [2]

In the mouse paw oedema induced by carrageenan, oral administration of 5-Demethylnobiletin at 100 mg/kg significantly reduced the oedema by 47% at 3 hours after challenge. The effect was dose-dependent (4% at 25 mg/kg, 20% at 50 mg/kg). [1]
In the mouse paw oedema induced by phospholipase A2 (PLA2), oral administration of 5-Demethylnobiletin at 100 mg/kg significantly reduced the oedema by 65% at 1 hour after challenge. The effect was dose-dependent (30% at 25 mg/kg, 40% at 50 mg/kg), with an inhibitory dose-50 (ID50) of 69 mg/kg. [1]
In a mouse ear inflammation model induced by repeated application of TPA, topical application of 5-Demethylnobiletin at 0.25 mg/ear significantly reduced the subchronic inflammation by 41%, as measured by ear weight increase. It also inhibited neutrophil infiltration, measured indirectly as myeloperoxidase (MPO) activity in the tissue, by 78%. [1]
Histological analysis of TPA-treated mouse ears showed that 5-Demethylnobiletin treatment reduced oedema, led to a reduction of inflammatory cell infiltration, and attenuated epithelium thickening, papillomatosis, acanthosis, hyperkeratosis, and spongiosis. [1]

To assess 5-LOX activity, rat peritoneal neutrophils were lysed by sonication and subjected to sequential centrifugation. The supernatant (containing the enzyme) was incubated with 5-Demethylnobiletin for 5 minutes. Arachidonic acid was then added to a final concentration of 20 μM to initiate the reaction. After 5 minutes, the 5-LOX products, specifically LTB4, were quantified using a specific enzyme immunoassay kit. [1]
For the elastase activity assay, human neutrophils were stimulated with cytochalasin and fMLP. The supernatant was then mixed with a BOC substrate and incubated at 37°C for 30 minutes. The absorbance, measured at 414 nm, was used to determine elastase activity, and the effect of 5-Demethylnobiletin on this activity was assessed. [1]

Cytotoxicity was measured by the MTT assay. Neutrophils or RAW 264.7 macrophages were exposed to 5-Demethylnobiletin (up to 10 μM) for 30 minutes. MTT solution was then added and incubated until blue deposits formed. The colored metabolite was dissolved in DMSO, and absorbance was measured at 490 nm to assess cell viability. [1]
For the LTB4 production assay in intact cells, rat peritoneal neutrophils were preincubated with 5-Demethylnobiletin (0.01 to 10 μM). LTB4 production was stimulated, and the amount of LTB4 was analyzed using a specific enzyme immunoassay kit. [1]
For Western blot analysis of COX-2, RAW 264.7 macrophages were pretreated with 5-Demethylnobiletin (2.5, 5, 10 μM) or vehicle for 1 hour and then incubated with LPS for 18 hours. Membranes were incubated with anti-COX-2 polyclonal antiserum and anti-β-actin antibody. Blots were then incubated with a peroxidase-conjugated secondary antibody, and the immunoreactive bands were visualized using an enhanced chemiluminescence system. [1]
For the elastase release assay, human neutrophils were preincubated with 5-Demethylnobiletin (10 μM) for 5 minutes. Elastase release was stimulated by the addition of cytochalasin and then fMLP. After centrifugation, the supernatant was mixed with a BOC substrate, and the absorbance was measured at 414 nm to quantify elastase release. [1]
For PC12 cell neurite outgrowth, cells were seeded on poly-L-lysine-coated plates and after 24 hours, the medium was changed to low serum medium. 5-Demethylnobiletin (2-20 μM) was added for 48 hours. Cells with at least one neurite with a length equal to the cell body diameter were counted as neurite-positive. [2]
For RT-Q-PCR analysis of GAP-43, PC12 cells were treated with 5-Demethylnobiletin (10 μM) for 48 hours. Total RNA was isolated, reverse transcribed, and quantitative real-time PCR was performed with specific primers for GAP-43 and β-actin. [2]
For Western blot analysis of GAP-43, synaptophysin, CREB, and ERK1/2, PC12 cells were treated with 5-Demethylnobiletin (10 μM) for various time points. Cell lysates were separated on SDS-PAGE, transferred to a PVDF membrane, and probed with specific primary antibodies and HRP-conjugated secondary antibodies. The signal was detected using a chemiluminescence reagent. [2]
For immunofluorescence staining of GAP-43, PC12 cells were treated with 5-Demethylnobiletin for 48 hours, fixed with formaldehyde, permeabilized, and incubated with anti-GAP-43 antibody. This was followed by incubation with an Alexa Fluor 488-conjugated secondary antibody and DAPI for nuclear staining. [2]
For the CRE-mediated transcription activity assay, PC12 cells were co-transfected with a pCRE-Luc reporter plasmid and a Renilla luciferase vector. After 24 hours, cells were treated with 5-Demethylnobiletin (10 μM) for 8 hours. Luciferase activities were measured and normalized to Renilla luciferase activity. [2]
For siRNA transfection, PC12 cells were transfected with CREB-specific siRNA or a negative control for 24 hours before treatment with 5-Demethylnobiletin. [2]
For PKC and PKA activity assays, PC12 cells were treated with 5-Demethylnobiletin (10 μM). Cell lysates were added to PKC or PKA substrate microtiter plates, and the reaction was initiated by adding ATP. After incubation, a phosphospecific substrate antibody and HRP-conjugated secondary antibody were added. The TMB substrate solution was then added, and the plate was read at 450 nm. [2]

Carrageenan-induced hind-paw mouse oedema: Oedema was induced by subplantar injection of carrageenan (3% w/v in saline, 25 μL) into the right hind paw of female Swiss mice. 5-Demethylnobiletin (25, 50, and 100 mg/kg) was suspended in saline buffer (0.9% NaCl, 0.1% NaHCO3, pH 7.4) and administered orally 1 hour before carrageenan injection. Paw volume was measured plethysmographically 1, 3, and 5 hours after the challenge. [1]
PLA2-induced hind-paw mouse oedema: PLA2 from Naja mosambica (1.18 units in 25 μL saline) was injected subcutaneously into the right hind paw of mice. 5-Demethylnobiletin (25, 50, and 100 mg/kg) was administered orally 1 hour before PLA2 injection. Paw volume was measured 30, 60, and 90 minutes after the challenge. [1]
Mouse ear inflammation induced by multiple applications of TPA: A subchronic inflammatory process was induced in mouse ears by repeated topical application of TPA (2 μg in 20 μL acetone) on alternate days (days 1, 3, 5, 8, and 10). 5-Demethylnobiletin (0.25 mg/ear in 20 μL acetone) was applied topically twice daily for 4 days (days 8-11) in the morning simultaneously with TPA and 6 hours later without TPA. On the last day (day 11), it was applied only in the morning. Mice were killed 6 hours after the last application. Ear swelling was assessed by weight, and MPO activity was measured as an index of neutrophil infiltration. [1]

5-Demethylnobiletin displayed no toxicity against rat and human neutrophils and murine RAW 264.7 macrophages at the assayed doses (10 μM and lower), as measured by the MTT assay. [1]
In PC12 cells, 5-Demethylnobiletin did not exert detectable cytotoxicity after 48 hours of incubation in low serum medium at concentrations up to 20 μM, as measured by the MTT assay. [2]

[1]. Anti-inflammatory activity of 5-O-demethylnobiletin, a polymethoxyflavone isolated from Sideritis tragoriganum. Planta Med. 2006 Feb;72(2):136-42.

[2]. Neurotrophic action of 5-hydroxylated polymethoxyflavones: 5-demethylnobiletin and gardenin A stimulate neuritogenesis in PC12 cells. J Agric Food Chem. 2013 Oct 2;61(39):9453-63.

Desmethylnorbiglitin is an ether compound belonging to the flavonoid family. It has been reported to be found in citrus (Citrus reticulata), thyme-leaved hawthorn (Clinopodium thymifolium), and other organisms with relevant data. See also: orange peel (partial); sour orange (Citrus aurantium) peel (partial).

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5-Demethylnobiletin (5-hydroxy-6,7,8,3',4'-pentamethoxylflavone) is a common flavonoid present in species of Citrus, and was also isolated from Sideritis mugronensis. [1]
The anti-inflammatory activity of 5-Demethylnobiletin is hypothesized to involve a direct inhibition of 5-LOX, without affecting the expression of COX-2. Its effect on reducing leukocyte infiltration is attributed to its capacity to inhibit LTB4 formation directly, rather than by an antioxidant mechanism. [1]
The neurotrophic action of 5-Demethylnobiletin in PC12 cells is mediated through MAPK/ERK-, PKC-, and PKA-dependent, but TrkA-independent, CREB signaling pathways. The substitution of a methoxy group with a hydroxyl at the 5-position of PMFs might increase neurotrophic activities. [2]

C20H20O8

388.37

388.115

2174-59-6

358832

Light yellow to green yellow solid powder

1.304±0.06 g/cm3

601.4±55.0 °C at 760 mmHg

145-146 ºC

213.9±25.0 °C

0.0±1.8 mmHg at 25°C

1.583

2.1

1

8

6

28

579

0

DOFJNFPSMUCECH-UHFFFAOYSA-N

InChI=1S/C20H20O8/c1-23-12-7-6-10(8-14(12)24-2)13-9-11(21)15-16(22)18(25-3)20(27-5)19(26-4)17(15)28-13/h6-9,22H,1-5H3

2-(3,4-Dimethoxyphenyl)-5-hydroxy-6,7,8-trimethoxychromen-4-one

5 Demethylnobiletin 5-Demethylnobiletin

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 : ~25 mg/mL (~64.37 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.44 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 (6.44 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.)