The study shows that arecoline activates ERK1/2 and JNK MAPKs signaling pathways, leading to downstream effects. [4]

Without altering the expression of p21/Cip1, arecoline causes the generation of reactive oxygen species and cell cycle arrest in the G1/G0 phase in HaCaT cells. Higher arecoline concentrations promote epithelial cell death, however oxidative damage rather than apoptosis is the reason. 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 [1].

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In confluent mouse Sertoli TM4 cells, treatment with Arecoline (400 μM for 6 hours) induced a redistribution of the tight junction protein ZO-1. This was evidenced by a decrease in ZO-1 protein in the Triton-insoluble fraction (membrane-associated) and a corresponding increase in the soluble fraction. Immunofluorescence staining showed reduced punctate ZO-1 signal at the plasma membrane. This effect was dose-dependent, with insoluble ZO-1 reduced to 74.7% at 200 μM and 42.5% at 400 μM. [4]
Arecoline (400 μM) induced the phosphorylation of multiple signaling proteins in TM4 cells, including ERK1/2, JNK, p38 MAPK, and IkappaB-alpha, as determined by Micro-Western Array and confirmed by Western blot. It also decreased phosphorylation of PP2A and STAT1. [4]
Arecoline (100-400 μM for 6 hours) dose-dependently increased TNF-alpha mRNA expression and soluble protein secretion in TM4 cells. Significant induction was observed at 300 μM for mRNA and 100 μM for protein. [4]
In human monocytic THP1 cells, Arecoline (100-400 μM for 6 hours) also dose-dependently increased TNF-alpha mRNA and soluble protein expression. [4]
Pretreatment with the ERK1/2 inhibitor PD98059 (10 μM for 1 hour) significantly reversed the Arecoline-induced increase in TNF-alpha (mRNA and protein) and the redistribution of ZO-1 (restoring insoluble ZO-1 levels) in TM4 cells. It also inhibited Arecoline-induced TNF-alpha production in THP1 cells. [4]
Arecoline treatment (400 μM for 6 hours) caused a slight but significant increase in LDH release (122.6% of control) but did not significantly reduce cell viability in MTS assays over 6, 12, or 24 hours, suggesting minimal acute cytotoxicity. [4]

Cell Culture and Viability: TM4 (mouse Sertoli) and THP1 (human monocytic) cells were cultured. Cell viability was assessed using the MTS assay, and cytotoxicity was evaluated by measuring lactate dehydrogenase (LDH) release, both according to the manufacturer's protocols. [4]
Micro-Western Array: Confluent TM4 cells were treated with 400 μM Arecoline for 10 or 60 minutes. Cell lysates were analyzed by Micro-Western Array using 48 antibodies to detect changes in protein phosphorylation and expression. [4]
Western Blotting: TM4 cells were treated with Arecoline. For phosphorylated protein analysis, cells were lysed in buffer A. For ZO-1 fractionation, cells were lysed in buffer B containing 1.0% Triton X-100. The lysate was centrifuged to separate Triton-insoluble (pellet) and soluble (supernatant) fractions. Proteins were separated by SDS-PAGE, transferred to membranes, and probed with specific antibodies. [4]
RT-qPCR: TM4 or THP1 cells were treated with Arecoline (100-400 μM) for 6 hours. Total RNA was extracted, reverse-transcribed, and subjected to quantitative real-time PCR with specific primers for TNF-alpha and GAPDH. [4]
ELISA for TNF-alpha: TM4 or THP1 cells were treated with Arecoline (100-400 μM) for 6 hours. The culture medium was collected, and levels of mouse or human TNF-alpha were measured using commercial ELISA kits. [4]
Immunofluorescence: TM4 cells treated with Arecoline (400 μM, 6 hours) were fixed, permeabilized, and incubated with anti-ZO-1 antibody followed by a fluorescein-conjugated secondary antibody. ZO-1 localization was visualized by fluorescence microscopy. [4]
Inhibitor Studies: TM4 or THP1 cells were pretreated with the ERK1/2 inhibitor PD98059 (10 μM) for 1 hour before co-treatment with Arecoline (400 μM) for the indicated times. The effects on ERK phosphorylation, TNF-alpha expression, and ZO-1 distribution were then assessed. [4]

During betel quid chewing, Arecoline can diffuse into the blood through the buccal mucosa or be absorbed by the gastrointestinal tract. The concentration of Arecoline in saliva can reach approximately 140 μg/mL (about 902.12 μM). Arecoline has been detected in blood, placenta, urine, and breast milk. [4]
Arecoline has been reported to cross the blood-brain barrier. [4]

Arecoline is known to be cytotoxic to various cell types, including normal human gingival fibroblasts, by inducing ROS production, cell cycle arrest, and DNA damage. [4]
In this study, short-term (6-24 h) exposure of TM4 cells to Arecoline (up to 400 μM) did not cause significant cell death, suggesting that the observed effects on ZO-1 and TNF-alpha are early events that may precede cytotoxicity. A slight but significant increase in LDH release was observed only at the highest dose (400 μM) after 6 hours. [4]

[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. Arecoline has been reported to be found in areca nut (Areca catechu) and betel leaf (Piper betle), and there is available data. 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 can indeed slightly improve verbal and spatial memory in Alzheimer's patients, but because of its potential carcinogenicity, it is not a first-line treatment for this degenerative disease. Arecoline has been shown to have pro-apoptotic, excitatory, and steroid-producing 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. It was once used as a stimulant on Pacific islands.

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Arecoline is the major alkaloid found in the Areca nut (betel nut). Betel quid chewing is a common addiction in many Asian populations, and the Areca nut has been classified as a Group I carcinogen by the IARC. [4]
Epidemiological and experimental studies have shown that betel quid chewing or exposure to Arecoline has harmful effects on male reproductive function, including reduced sperm count and motility, and abnormal sperm morphology. [4]
This study provides a molecular mechanism for these observations. It demonstrates that Arecoline activates ERK1/2 signaling in Sertoli cells, leading to the production of the pro-inflammatory cytokine TNF-alpha and the redistribution of the tight junction protein ZO-1. These effects compromise the integrity of the blood-testis barrier (BTB), which is essential for normal spermatogenesis, and may represent an early step in Arecoline-induced male reproductive dysfunction. [4]

155.095

63-75-2

Arecoline hydrobromide;300-08-3;Arecoline hydrochloride;61-94-9

2230

Light yellow to brown liquid

1.059g/cm3

209ºC at 760mmHg

< 25 °C

81.1ºC

1.483

0.359

0

3

2

11

187

0

HJJPJSXJAXAIPN-UHFFFAOYSA-N

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

methyl 1-methyl-3,6-dihydro-2H-pyridine-5-carboxylate

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)

H2O : ~250 mg/mL (~1610.93 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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