NADPH-oxidase (IC50: 10 μM in activated human neutrophils) [1]

After promoting osteogenesis, apocynin (100 μM, 1-7, 14 days) dramatically raises the expression levels of osteogenic markers in aging BMSC cells [3]. Apocynin (1, 10, 100 μM; 48 hours) specifically prevents fibronectin staining and vH-ras-transfected 3Y1 cell proliferation [4]. Apocynin (1, 10, 100 μM; 3, 6, 12 hours) inhibits 3Y1 and HR-3Y1-2 cell growth.

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Acetovanillone inhibits NADPH-oxidase activity and concomitant ROS production with an IC50 value of 10 μM in activated human neutrophils [1].
Apocynin prevents the translocation of p47phox to Nox2 in leukocytes, monocytes, and endothelial cells. The inhibitory action occurs after a lag time and requires activation by myeloperoxidase (MPO) because apocynin does not inhibit the oxidase in cells devoid or deficient of MPO [1].
Apocynin is activated by H2O2 and MPO to form an apocynin radical, which then oxidizes thiols critical for the function of p47phox. Supplementation of thiol (glutathione or cysteine) prevents the inhibitory effect. Alternatively, an apocynin dimer is formed and may be the active inhibitory compound [1].
In vitro anti-inflammatory effects include: reduction of neutrophil oxidative burst and neutrophil-mediated oxidative damage; decreased adhesion of monocytic U937 cells to TNF-treated endothelial cells; reduction of polymorphonuclear granulocyte chemotaxis; inhibition of peroxynitrite formation; inhibition of inflammation-mediated cartilage destruction [1].
In endothelial cells incubated with 600 μM apocynin and stimulated with thrombin, NADPH-oxidase inhibition resulted in significantly impaired ROS production. Apocynin also markedly diminished high LDL-induced increases in cellular H2O2 concentrations [1].
Apocynin inhibited NADH- and NADPH-oxidase-mediated O2- generation in human arteries and veins, improved endothelium-dependent vasodilation, and enhanced NO production from human endothelial cells [1].
Apocynin prevented COX-2 expression in stimulated human monocytes via inhibition of NADPH-oxidase-dependent superoxide production, reduction of intracellular GSH/GSSG ratio, and prevention of NF-κB activation [1].
In breast cancer cell line MDA-MB-435, apocynin derivatives (but not apocynin itself) inhibited migration at subtoxic concentrations, caused actin cytoskeleton rearrangement, cell rounding, and decreased levels of active Rac1 and Cdc42. Nonmalignant MCF10A cells were unaffected [1].
Apocynin per se can induce oxidative stress in resting cells: it increases H2O2 concentration, decreases intracellular glutathione/glutathione disulfide ratio, increases efflux of glutathione and glutathione disulfide, induces activation of pentose phosphate pathway and tricarboxylic acid cycle, increases malonildialdehyde generation and lactate dehydrogenase leakage. In a cell-free system, apocynin evokes faster oxidation of thiols (glutathione, dithiothreitol) and elicits generation of ROS, mainly superoxide anions [1].

In SAMP6 samples, apocynin (0.1 mg/kg/day, intraperitoneally, three times a week for three months) enhanced bone mass and density [3]. Apocynin injection intraperitoneally (5 mg/kg)
In animal models of ischemia-reperfusion lung injury, Acetovanillone prevented increased vascular permeability caused by ischemia and reperfusion in isolated sheep lungs in a dose-dependent manner [1].
In mild asthmatics, apocynin significantly reduced ozone-induced bronchial hyperresponsiveness to methacholine measured 16 hours after ozone exposure, but did not prevent the decline in FEV1 directly after ozone exposure [1].
In a collagenase-induced rat model of intracerebral hemorrhage (ICH), apocynin reduced cerebral and vascular injury at doses similar to those used in stroke models [1]. However, another study reported that apocynin had no effects on enhanced NADPH-oxidase activity, lipid peroxidation, brain water content, or neurological dysfunction after ICH [1].
In a mouse model of transient middle cerebral artery occlusion (2-hour occlusion followed by 22-hour reperfusion), apocynin given at 2.5 mg/kg 30 minutes before reperfusion improved neurological function, reduced infarct volume, and reduced the incidence of cerebral hemorrhage. Higher doses (3.75 and 5 mg/kg) increased brain hemorrhage. Apocynin tended to reduce mortality at the lower dose but not at higher doses [1].
In rabbit models, apocynin suppressed atherogenesis in spite of highly elevated serum LDL levels. Local adventitial application of apocynin to collared arteries reduced superoxide production and prevented vascular injury associated with remodelling, without affecting Nox2 mRNA expression [1].
In deoxycorticosterone acetate salt-induced hypertensive rats, extended oral treatment with apocynin reduced superoxide generation in arteries and reduced blood pressure. Apocynin prevented and reversed dexamethasone-induced increases in systolic blood pressure [1].
In Dahl salt-sensitive rats, apocynin inhibited superoxide production in the renal medulla and decreased hypertension [1].
In mice, oral administration of apocynin partially reversed inflammation-induced inhibition of cartilage proteoglycan synthesis and reduced joint swelling in collagen-induced arthritis [1].
Apocynin prevented airway hyperresponsiveness during allergic reactions in mice and reduced airway hyperreactivity to methacholine when inhaled by humans with mild atopic asthma [1].

NADPH-oxidase activity assay: Apocynin's ability to inhibit superoxide production was assessed in activated human neutrophils. The IC50 value was determined to be 10 μM [1].
Myeloperoxidase (MPO)-dependent activation assay: Apocynin requires activation by MPO and H2O2. In cells devoid or deficient of MPO, apocynin does not inhibit NADPH-oxidase. Agents that promote MPO release (e.g., zymosan) enhance apocynin efficacy. The oxidation of apocynin catalyzed by MPO produces an apocynin radical that oxidizes thiols [1].
Thiol oxidation assay: In a cell-free system, apocynin evoked faster oxidation of thiols (glutathione and dithiothreitol) and elicited generation of reactive oxygen species, mainly superoxide anions. Neither apocynin nor its dimer/trimer derivatives were able to conjugate with GSH, but GSH reacted with apocynin radical and/or its dimer radical formed during MPO-catalyzed oxidation [1].
Scavenger property assay: Apocynin did not scavenge superoxide anion generated by xanthine oxidase, but it scavenged hydrogen peroxide in a dose-response manner as shown by inhibition of luminol-enhanced chemiluminescence generated by H2O2 [1].
Cytochrome P450 inhibition: Apocynin inhibited cytochrome P450 activity in endothelial cells [1].
Thromboxane synthase inhibition: Apocynin inhibited thromboxane synthase [1].

Western Blot analysis [3]
Cell Types: Bone marrow stromal cells (BMSCs)
Tested Concentrations: 100 μM
Incubation Duration: 1-7, 14 days
Experimental Results: Increased intracellular reactive oxygen species (ROS) levels [4]. "Dry Markers" Nanog and Oct-4. Reduces the expression levels of p53, p21 and p16 at both the mRNA and protein levels. Compared with the negative control group, the expressions of sox-2 and klf-4 increased by 82.4% and 38.7%, respectively. Compared with the negative control, the expression of NADPH oxidase was diminished by 66.5%. There were no changes in cell cycle or proliferation. The percentage of DSA-β-gal positive (green staining) cells was diminished by 42.5%. Increased expression levels of four key osteogenic markers (Runx2, OSX, Ocn, and Col1). Cell proliferation experiment [4]
Cell Types: HR-3Y1-2, 3Y1 Cell
Tested Concentrations: 0, 1, 10 or 100 μM
Incubation Duration: 48 hrs (hours)
Experimental Results: 10 μM inhibited HR-3Y1-2 cell proliferation, but not 3Y1 cells Proliferate 100 μM.

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RT-PCR[3]
Cell Types: HR-3Y1-2, 3Y1 cells
Tested Concentrations: 0, 1, 10, or 100 μM
Incubation Duration: 24, 36, 48 h
Experimental Results: Selectively down-regulated 1-integrin cell surface expression on the HR-3Y1-2 cells. diminished adhesion of HR-3Y1-2 cells to fibronectin-coated plates.

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Cell culture studies: Human neutrophils, monocytes, endothelial cells, U937 cells, glial cells (N11), alveolar epithelial cells, chondrocytes, and breast cancer cell lines (MDA-MB-435, MCF10A) were used. Apocynin was dissolved in DMSO and added to cell cultures at various concentrations (e.g., 600 μM for endothelial cells, 10 μM for neutrophil IC50 determination). Cells were stimulated with agents such as thrombin, LDL, or zymosan [1].
Cell viability and cytotoxicity assays: MTT or LDH leakage assays were used. Apocynin induced a dose-dependent increase in oxidative stress markers, increased H2O2 concentration, decreased intracellular GSH/GSSG ratio, increased efflux of glutathione and glutathione disulfide, and induced activation of pentose phosphate pathway and tricarboxylic acid cycle. Co-incubation with glutathione prevented apocynin-induced increase of malonildialdehyde generation and lactate dehydrogenase leakage [1].
COX-2 expression assay: Human monocytes were stimulated, and apocynin prevented COX-2 expression. The mechanism involved inhibition of NADPH-oxidase-dependent superoxide production, reduction of intracellular GSH/GSSG ratio, and prevention of NF-κB activation [1].
Cell migration assay: An in vitro screening assay was used to identify apocynin-derived inhibitors of Rac1-based tumor cell migration. Apocynin itself was not effective, but its derivatives inhibited migration of MDA-MB-435 cells at subtoxic concentrations without affecting nonmalignant MCF10A cells. Actin cytoskeleton rearrangement and decreased active Rac1/Cdc42 levels were observed [1].
Chemotaxis assay: Apocynin reduced polymorphonuclear granulocyte chemotaxis, right angle light scatter, and actin polymerization [1].
Adhesion assay: Apocynin decreased adhesion of monocytic U937 cells to TNF-treated human umbilical vein endothelial cells [1].
Peroxynitrite formation assay: Apocynin inhibited peroxynitrite formation by murine macrophages [1].

Animal/Disease Models: SAMP6 mouse model (pharmacokinetic/PK/PK determination) [3]
Doses: 0.1 mg/kg/day
Route of Administration: intraperitoneal (ip) injection, weekly 3 times for 3 months.
Experimental Results: Compared with the control group, the bone value was higher and the percentage of SA-β-gal positive cells was lower. The expression of Ki67 and Oct-4 mRNA was increased. Changes the osteoblast-osteoclast balance in bone and promotes osteoblast activity.

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Animal/Disease Models: Carrageenan induced pleurisy in male adult CD1 mice [5]
Doses: 5 mg/kg
Route of Administration: intraperitoneal (ip) injection
Experimental Results: Blocked NADPH oxidase activation and attenuated neutrophils in lung tissue Cellular infiltration and lipid peroxidation. Reduce PARP activation and IL-1b expression. Prevents carrageenan-induced IkB-a degradation and reduces NF-kB p65 levels. Attenuate iNOS expression and reduce the degree of positive staining of Fas ligand in lung tissue. Inhibition of apoptosis in carrageenan-treated mice. Blocks Bax expression and r

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Rat models: Apocynin administered intraperitoneally (IP) at doses of 2.5, 3.75, 5 mg/kg in stroke model; 120 mg/kg IP for pharmacokinetics; various doses in hypertension models (e.g., DOCA-salt rats). Oral administration in drinking water or by gavage in some studies. In ischemia-reperfusion lung injury, apocynin was given before ischemia [1].
Mouse models: Collagen-induced arthritis, allergic airway hyperresponsiveness, intracerebral hemorrhage. Apocynin given orally or IP. Doses: e.g., 2.5 mg/kg IP in stroke model [1].
Rabbit models: High LDL diet-induced atherosclerosis, carotid artery collar model. Apocynin administered orally or locally via adventitial application. Oral treatment extended over weeks [1].
Sheep lung model: Isolated perfused sheep lungs, apocynin administered to prevent ischemia-reperfusion-induced vascular permeability [1].
Human study: Mild asthmatics inhaled apocynin or received via inhalation? The study by Peters et al. (2001) described inhalation of apocynin to assess ozone-induced bronchial hyperresponsiveness [1].
Formulation: Apocynin was dissolved in appropriate vehicles (e.g., saline, DMSO, or drinking water). For IP injection, typical doses as above. For oral administration, apocynin was given in drinking water or by gavage [1].

After intraperitoneal administration of 120 mg/kg apocynin to rats, 80% of the apocynin was recovered unchanged in the urine after 20 hours [1].
Apocynin is metabolically activated by myeloperoxidase and H2O2 to form an apocynin radical and subsequently a dimer, which is more efficient than apocynin itself as an NADPH-oxidase inhibitor [1].
Apocynin undergoes O-methylation and demethylation as part of its metabolism (based on Daly et al., 1960) [1].

Oral LD50 in mice: 9 g/kg [1].
Apocynin has very low toxicity and no known side effects in general use [1].
Apocynin may increase oxidative stress under some conditions: it decreases glutathione expression in alveolar epithelial cells, produces an apocynin free radical that oxidizes glutathione, and increases H2O2 concentration in resting cells. In glial cells, apocynin shows a dose-dependent increase in oxidized glutathione [1].
Apocynin can induce oxidative damage and cytotoxic effect in resting cells (e.g., N11 glial cell line) after longer exposure, suggesting that in the absence of NADPH-oxidase stimulation, the oxidative effect of apocynin itself may predominate [1].
Apocynin may cause a defect in bactericidal phagocytosis that can mimic chronic granulomatous disease by indirect inhibition of respiratory burst. However, data are ambiguous and this requires further investigation [1].
Apocynin inhibits cytochrome P450 activity in endothelial cells and interferes with actin polymerization and cytoskeletal rearrangement [1].
Apocynin can increase H2O2 concentration and decrease intracellular GSH/GSSG ratio in various cell types [1].

[1]. Mediators Inflamm, 2008, 106507.

[2]. Am J Respir Cell Mol Biol, 1994, 11(1), 95-102.

[3]. Sun J, et.al. Apocynin suppression of NADPH oxidase reverses the aging process in mesenchymal stem cells to promote osteogenesis and increase bone mass. Sci Rep. 2015 Dec 21;5:18572.

[4]. Yamasaki M, et.al. Selective inhibition by apocynin of the proliferation and adhesion to fibronectin of v-H-ras-transformed 3Y1 cells. Biosci Biotechnol Biochem. 2012;76(6):1177-81.

[5]. Effect of apocynin, a NADPH oxidase inhibitor, on acute lung inflammation. Biochem Pharmacol. 2011 Mar 1;81(5):636-48.

Apoxinin is an aromatic ketone, a 1-acetophenone with a hydroxyl group at the 4-position and a methoxy group at the 3-position. It can be used as a non-narcotic analgesic, nonsteroidal anti-inflammatory drug, antirheumatic drug, peripheral nervous system drug, EC 1.6.3.1. [NAD(P)H oxidase (H2O2 generation)] inhibitor, and plant metabolite. It belongs to the acetophenone class of compounds and is a methyl and aromatic ketone. Acetylvanillin has been used in research trials for the treatment of bronchial asthma and chronic obstructive pulmonary disease. Acetylvanillin has been reported in iris (Iris tectorum), Lagochilus leiacanthus, and other organisms with relevant data. Acetylvanillin is a metabolite of Saccharomyces cerevisiae.

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Acetovanillone (apocynin) is a naturally occurring methoxy-substituted catechol first described by Schmiedeberg in 1883, isolated from Apocynum cannabinum and Picrorhiza kurroa. It has been used in traditional Indian medicine (Ayurveda) for treatment of asthma and other ailments [1].
Apocynin is a prodrug that requires peroxidase-mediated oxidation to form a dimer, which is more efficient as an NADPH-oxidase inhibitor. The mechanism involves impairment of translocation of the cytosolic component p47phox to the membrane [1].
Apocynin inhibits peroxynitrite (ONOO-) formation by reacting with superoxide and NO, thus reducing epithelial damage and hyperresponsiveness in airways [1].
Apocynin has shown promising application in animal studies of hypertension, atherosclerosis, stroke, arthritis, and asthma. However, its utility in human inflammatory diseases remains to be determined. Some studies report that apocynin acts predominantly as an antioxidant (radical scavenger) rather than as an NADPH-oxidase inhibitor in nonphagocytic cells [1].
Apocynin may have a narrow therapeutic window in stroke (protective at 2.5 mg/kg but harmful at higher doses) [1].
Apocynin is a reversible inhibitor of NAD(P)H oxidase and has been proposed as a potential therapeutic agent for atherosclerotic disease [1].
Apocynin inhibits thromboxane synthase and cytochrome P450, which may contribute to its protective effects in ischemia-reperfusion injury [1].
Apocynin derivatives (but not apocynin itself) inhibit migration of breast cancer cells via Rac1 inhibition [1].

C9H10O3

166.18

166.062

498-02-2

Apocynin-d3;80404-23-5

2214

Off-white to gray solid powder

1.2±0.1 g/cm3

297.5±0.0 °C at 760 mmHg

112-115 °C(lit.)

125.5±15.8 °C

0.0±0.6 mmHg at 25°C

1.538

1.39

1

3

2

12

167

0

DFYRUELUNQRZTB-UHFFFAOYSA-N

InChI=1S/C9H10O3/c1-6(10)7-3-4-8(11)9(5-7)12-2/h3-5,11H,1-2H3

1-(4-hydroxy-3-methoxyphenyl)ethanone

Acetovanillone NSC-209524 NSC 209524

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 (~601.79 mM)
H2O : ~3.33 mg/mL (~20.04 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (15.04 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 (15.04 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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Solubility in Formulation 3: ≥ 2.5 mg/mL (15.04 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 25.0 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.)