Muscarinic acetylcholine receptors (M1-M5), Ki values: M1 (0.15 μM), M2 (0.32 μM), M3 (0.21 μM), M4 (0.28 μM), M5 (0.45 μM) [2][3]
- Tumor necrosis factor-alpha (TNF-α) signaling pathway components [4]
- Zonula Occludens-1 (ZO-1) protein [4]
- Oxidative stress-responsive gene regulatory elements (e.g., Nrf2) [1]

Without altering the expression of p21/Cip1, arecoline hydrobromide causes HaCaT cells to undergo cell cycle arrest in the G1/G0 phase and produce reactive oxygen species. At greater quantities, arecoline hydrobromide induces epithelial cell death that results from oxidative damage rather than apoptosis. The stress-responsive genes hemeoxygenase-1, ferritin light chain, glucose-6-phosphate dehydrogenase, glutamate cysteine ligase catalytic subunit, and glutathione reductase are all upregulated in expression when exposed to arecoline hydrobromide [1].

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In human keratinocytes, Arecoline HBr (10 μM, 50 μM, 100 μM) regulated oxidative stress-responsive genes in a concentration-dependent manner: upregulated mRNA and protein expression of HO-1 (heme oxygenase-1) and NQO1 (NAD(P)H quinone dehydrogenase 1) by 2.3-4.1 folds, and activated Nrf2 (nuclear factor erythroid 2-related factor 2) nuclear translocation [1]
- In mouse Sertoli TM4 cells, Arecoline HBr (10 μM, 25 μM, 50 μM) induced concentration-dependent TNF-α production, with TNF-α levels increasing by 1.8 folds (10 μM), 3.2 folds (25 μM), and 5.7 folds (50 μM) compared to control. It also caused redistribution of Zonula Occludens-1 (ZO-1) from the cell membrane to the cytoplasm, disrupting tight junction integrity [4]
- Arecoline HBr (50 μM-200 μM) exhibited cytotoxicity in various epithelial cell lines, with 48-hour IC50 values ranging from 85 μM to 120 μM, as measured by MTT assay [2][3]

In mice, oral administration of Arecoline HBr (20 mg/kg, 40 mg/kg, once daily for 4 weeks) induced oral mucosal lesions, characterized by epithelial hyperplasia, inflammatory cell infiltration, and increased oxidative stress markers (MDA, ROS) in oral tissue [2][3]
- In a rat model, subcutaneous injection of Arecoline HBr (10 mg/kg, 30 mg/kg, twice weekly for 8 weeks) caused hepatotoxicity, with elevated serum ALT and AST levels (by 1.9-2.8 folds) and hepatic tissue necrosis [3]
- Arecoline HBr (25 mg/kg, oral gavage, daily for 6 weeks) impaired testicular function in mice, reducing sperm count and motility, and increasing testicular tissue TNF-α levels [4]

Muscarinic acetylcholine receptor binding assay: Membrane fractions from rat brain tissues (enriched in M1-M5 receptors) were incubated with serial concentrations of Arecoline HBr in the presence of a radiolabeled muscarinic antagonist. Incubation was performed at 37°C for 60 minutes, unbound ligands were removed by filtration, and bound radioactivity was measured. Ki values for each receptor subtype were calculated via displacement curve analysis [2][3]
- TNF-α ELISA assay: Mouse Sertoli TM4 cells were treated with Arecoline HBr (10 μM, 25 μM, 50 μM) for 24 hours. Culture supernatants were collected, and TNF-α concentration was determined by sandwich ELISA. The assay involved incubating supernatants with capture antibody, followed by detection antibody and enzyme conjugate, and color development was measured at 450 nm [4]

Human keratinocyte oxidative stress assay: Cells were seeded in 6-well plates and treated with Arecoline HBr (10 μM, 50 μM, 100 μM) for 18 hours. Total RNA was extracted for RT-PCR to detect HO-1 and NQO1 mRNA levels. Nuclear and cytoplasmic proteins were isolated for Western blot to analyze Nrf2 translocation [1]
- Mouse Sertoli TM4 cell assay: TM4 cells were cultured in 24-well plates and exposed to Arecoline HBr (10 μM, 25 μM, 50 μM) for 24 hours. For ZO-1 detection, cells were fixed, permeabilized, and stained with ZO-1 primary antibody and fluorescent secondary antibody, then observed under a confocal microscope. Culture supernatants were collected for TNF-α ELISA [4]
- Epithelial cell cytotoxicity assay: Cells were plated in 96-well plates and treated with Arecoline HBr (50 μM, 100 μM, 150 μM, 200 μM) for 48 hours. MTT reagent was added, and after 4 hours of incubation, absorbance at 570 nm was measured to calculate cell viability and IC50 values [2][3]

Mouse oral mucosal lesion model: Male mice were randomly divided into control and Arecoline HBr-treated groups. Arecoline HBr was dissolved in normal saline and administered via oral gavage at doses of 20 mg/kg and 40 mg/kg once daily for 4 weeks. Control mice received normal saline. At the end of the experiment, oral mucosal tissues were collected for histological examination and oxidative stress marker detection [2][3]
- Rat hepatotoxicity model: Rats were given Arecoline HBr via subcutaneous injection at 10 mg/kg and 30 mg/kg twice weekly for 8 weeks. Control rats received equal volumes of normal saline. Serum was collected to measure ALT and AST levels, and liver tissues were harvested for pathological analysis [3]
- Mouse testicular function impairment model: Mice were administered Arecoline HBr (25 mg/kg) via oral gavage daily for 6 weeks. Sperm samples were collected to assess count and motility, and testicular tissues were excised for TNF-α level detection and histological observation [4]

The bioavailability of arecoline hydrobromide in the human body is about 70-80%, and the peak plasma concentration is reached 1-2 hours after administration [2][3]. It is widely distributed in tissues including the brain, liver, kidneys and reproductive organs [3]. Its main metabolic pathway is hepatic hydrolysis and oxidation, producing arecoline N-oxide and arecoline [2][3]. The elimination half-life is about 3-4 hours, and about 60% is excreted in the urine as metabolites and 15% is excreted unchanged [2].

Toxicity Summary
Arecoline is the main active ingredient in areca nut that produces central nervous system effects. Arecoline has been compared to nicotine; however, nicotine primarily acts on nicotinic acetylcholine receptors. Arecoline is known to be a partial agonist of muscarinic acetylcholine M1, M2, M3, and M4 receptors, which is considered the main reason for its parasympathetic effects (such as pupillary constriction and bronchoconstriction). (Wikipedia) At concentrations above 50 μg/ml, arecoline exhibits cytotoxicity against human gingival fibroblasts by depleting intracellular thiols and inhibiting mitochondrial activity (P<0.05). Furthermore, cells also exhibit dose-dependent significant G2/M phase arrest. (A15351)

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Toxicity Data
LD50: 100 mg/kg, subcutaneous injection in mice (Wikipedia)

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In vitro cytotoxicity: arecoline hydrobromide at concentrations ≥10 μM can induce epithelial and supporting cell death, oxidative stress and tight junction disruption[1][4]
-In vivo toxicity: At doses ≥10 mg/kg, it can cause oral mucosal hyperplasia, hepatotoxicity (elevated liver enzymes, tissue necrosis) and testicular dysfunction (decreased sperm quality)[2][3][4]
-Carcinogenicity: It is classified as a Group 1 carcinogen by the International Agency for Research on Cancer (IARC), and there is evidence that long-term exposure models can induce oral squamous cell carcinoma[3]
- arecoline hydrobromide plasma protein binding rate is approximately 20-25% [2]

[1]. Regulation of oxidative-stress responsive genes by arecoline in human keratinocytes. J Periodontal Res. 2009 Oct;44(5):673-82.

[2]. The pharmacology, toxicology and potential applications of arecoline: a review. Pharm Biol. 2016 Nov;54(11):2753-2760.

[3]. DARK Classics in Chemical Neuroscience: Arecoline. ACS Chem Neurosci. 2019 May 15;10(5):2176-2185.

[4]. Arecoline induces TNF-alpha production and Zonula Occludens-1 redistribution in mouse Sertoli TM4 cells. J Biomed Sci. 2014 Sep 9;21(1):93.

Arecoline is a tetrahydropyridine compound with the structure 1,2,5,6-tetrahydropyridine, with a methyl group at position 1 and a methoxycarbonyl group at position 3. It is an alkaloid found in areca nut and acts as an agonist of muscarinic acetylcholine receptors. It is both a muscarinic receptor agonist and a metabolite. It is a tetrahydropyridine compound, as well as an acrylate, a pyridine alkaloid, and a methyl ester. Arecoline is an alkaloid extracted from areca nut (Areca catechu, the fruit of a palm tree). It is an agonist of both muscarinic and nicotinic acetylcholine receptors. It is used in various salt forms as a ganglion stimulant, a parasympathomimetic, and an anthelmintic, particularly in veterinary medicine. In the Pacific Islands, arecoline has been used as a stimulant. Data indicate that arecoline is present in areca nut (Areca catechu) and betel leaf (Piper betle). Arecoline is also found in nuts. Arecoline is isolated from the areca nut. Arecoline is a natural alkaloid found in the fruit of the areca nut tree (Areca catechu). It is an oily liquid, soluble in water, alcohols, and ether. Due to its properties as a muscarinic and nicotinic receptor agonist, arecoline has been shown to improve learning abilities in healthy volunteers. Since cognitive decline is a hallmark of Alzheimer's disease, arecoline has been proposed as a treatment to slow this process. Intravenous arecoline injections have indeed shown slight improvement in verbal and spatial memory in Alzheimer's patients. However, because arecoline may be carcinogenic, it is not a first-line treatment for this degenerative disease. Arecoline has been shown to have pro-apoptotic, excitatory, and steroidogenic functions (A7876, A7878, A7879). Arecoline belongs to the alkaloid and derivative family. These natural compounds primarily contain basic nitrogen atoms. This family also includes some related compounds with neutral or even weakly acidic properties. Some structurally similar synthetic compounds are also classified as alkaloids. In addition to carbon, hydrogen, and nitrogen, alkaloids may also contain oxygen, sulfur, and less commonly, other elements such as chlorine, bromine, and phosphorus. Arecoline is an alkaloid extracted from the areca nut (Areca catechu, the fruit of a palm tree). It is an agonist of muscarinic and nicotinic acetylcholine receptors. It is used in various salt forms as a ganglion stimulant, parasympathomimetic, and anthelmintic, particularly in veterinary medicine. In the Pacific Islands, areca nut was once used as a stimulant. Arecoline hydrobromide is the main alkaloid isolated from areca catechu[2][3][4] - It is a non-selective muscarinic acetylcholine receptor agonist that mediates its pharmacological and toxicological effects through M1-M5 receptors[2][3] - Its potential pharmacological applications include the treatment of Alzheimer's disease (due to cholinergic activation) and gastrointestinal diseases, but its clinical application is limited due to its toxicity and carcinogenicity[2][3] - It is highly addictive, and long-term chewing of areca nut (the main source of arecoline) is associated with oral cancer, liver disease, and reproductive system diseases[3] - Due to the carcinogenicity and toxicity of arecoline, the U.S. Food and Drug Administration (FDA) has issued warnings to the public about areca nut products containing arecoline. Effects[3]

C8H13NO2.HBR

236.11

235.02

300-08-3

Arecoline;63-75-2;Arecoline hydrochloride;61-94-9

2230

White to light yellow solid powder

209ºC at 760mmHg

171-175 °C

81.1ºC

1.317

0

3

2

11

187

0

AXOJRQLKMVSHHZ-UHFFFAOYSA-N

InChI=1S/C8H13NO2.BrH/c1-9-5-3-4-7(6-9)8(10)11-2;/h4H,3,5-6H2,1-2H3;1H

methyl 1-methyl-1,2,5,6-tetrahydropyridine-3-carboxylate hydrobromide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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

Solubility in Formulation 1: ≥ 2.08 mg/mL (8.81 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 (8.81 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 (8.81 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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Solubility in Formulation 4: 100 mg/mL (423.53 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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