Comprehensive Insights into Arecoline Hydrobromide: Pharmacology, Toxicity, and Pharmacokinetics

Abstract

Arecoline hydrobromide (AH) is an active alkaloid found in betel nut. AH was first extensively employed for treatment of tapeworm infection in dogs in Australia. In the last 2 decades, AH has gained increasing attention due to its multiple and notable pharmacological activities in various diseases, including: acaricidal activity against cattle ticks; anticataract activity; therapeutic and alleviating effects against diabetes complications, including male reproductive damage and cataract; treatment of rheumatoid arthritis (RA); and protection against gastric ulcer. In addition, AH may have potassium channel inhibitory activity, regulate of CYP2B (Cytochrome P450 2B) expression, and regulate mRNA expression of hepatorenal transporters. In terms of toxicity, the data showed that AH may have sub-chronic, long-term and acute toxicities, and teratogenic effects. Additionally, in pharmacokinetic studies, rapid LC-MS/MS methods have been applied to determine AH or arecoline quantitatively. In summary, the above studies suggested that AH may have considerable application prospects and great potential value in clinical practice in the future, but limitations of current studies and new challenges in AH were also be discussed, and future development directions were suggested in toxicities and pharmacokinetics. This article aims to review the pharmacological activity, pharmacokinetics, and toxicity of the natural alkaloid, arecoline hydrobromide.

Introduction

Arecoline hydrobromide (AH) is an alkaloid originally found in Areca catechu (A. catechu) [1], an ornamental plant belonging to the Arecaceae family, thriving abundantly in Southern and Southeast Asia [2,3]. Areca nut (also called betel nut) is the fruit of A. catechu, which is used as a masticatory agent with betel leaf and lime and chewed by at least 10% of the world’s population daily [4,5]. A. catechu is a popular traditional medicine for promoting digestion and killing parasites, widely used in traditional Chinese medicine prescriptions, such as Binglang Sixiao Wan, Muxiang Binglang Wan, and Yueju Baohe Wan [6]. Multiple types of compounds have been extracted and isolated from betel nut, including tannins, fatty acids, alkaloids, and flavonoids, of which the most active constituent is arecoline [7,8]. Numerous researchers have suggested that arecoline displays extensive pharmacological activities, including regulatory effects on the digestive, cardiovascular, nervous, and endocrine systems, as well as anti-parasitic effects, among others [9,10].

However, large-scale application of arecoline is constrained by its unstable properties and the inefficiency of extraction methods [11]. AH is the arecoline salt of hydrobromic acid [12]. In industry, AH was crystallized by adding hydrobromic acid to arecoline. In the last century, AH has been applied to diagnose and treat tapeworm infections in dogs in New Zealand, Tasmania, New South Wales, Victoria, and Western Australia [13]. At present, many studies have demonstrated that AH has numerous biological activities, but its pharmacological and toxicological mechanism is not clear, so it is difficult to carry out large-scale clinical trials. This article aims to review the pharmacological activity, pharmacokinetics, and toxicity of the natural alkaloid, arecoline hydrobromide.

Physicochemical Properties

The IUPAC name of AH is methyl 1-methyl-3,6-dihydro-2H-pyridine-5-carboxylate; hydrobromide, its molecular formula is C₈H₁₄BrNO₂ and its molecular weight is 236.11 g/mol. Its chemical structure is shown in Figure 1. Its mean solubility at pH 7.4 is more than 35.4 μg/mL and the melting point is 171–175°C. These data were retrieved from Chemical Book (http://www.chemicalbook.com/).

Figure 1.

Chemical structure of arecoline hydrobromide. Created using KingDraw Software, version 3.0 for Windows, Qingyuan Precision Agriculture Technology Co., Ltd., Qingdao, China.

Pharmacological Activities of AH

AH has various pharmacological activities, and the comprehensive pharmacological activity information of AH is listed in Table 1 and Figure 2.

Table 1.

Summary of biological activities of arecoline hydrobromide.

Pharmacological effects	Types	Cell lines/model	Doses	Effects/mechanisms	Reference
Acaricidal activity	In vitro	Cattle tick Rhipicephalus (Boophilus) microplus	0.1, 0.5, 1.5, 2.5, 5, 7.5, 10 and 12.5 mg/mL	Larvae numbers (↓); Tick number, egg weight, and hatching capability (↓); SOD (↓), GST (↓), NOS (↑), MAO (↓), AChE (↓)	[17]
	In vivo	Cattle tick Rhipicephalus (Boophilus) microplus-infected calves	12.5 mg/mL	Tick number (↓); Total erythrocyte count (↑), neutrophil count (↑), monocyte count (↑)	[18]
Treatment of rheumatoid arthritis	In vitro	Rheumatoid arthritis fibroblast-like synoviocytes (RA-FLSs)	0, 12.5, 25, 50, 75, and 100 μg/ml	Cell viability (↓); DNA replication (↓); CDK1, cyclin A2, and cyclin B1 (↓); Cell apoptosis (↑); Bcl-2 (↓); Cell migration and invasion abilities (↓); PI3K/AKT pathway	[1]
	In vivo	Collagen-induced arthritis (CIA) mice	60 mg/kg, i.p.	Joint swelling, arthritis inflammation, cartilage degradation, bone erosion (↓)	[1]
Potassium channel inhibitory activity	In vitro	Human embryonic kidney (HEK) 293 cells	1.0, 10, and 100 μmol/L	hERG K+ current (↓)	[24]
	In vivo	Jejunum muscle cells in W/Wv	10−8, 10−7, 10−6, and 10−5 M	Jejunum smooth muscle contraction frequency (↑)	[25]
Reproductive protection	In vivo	Experimentally induced type 1 diabetic rats	10 mg/kg, i.p.	Serum insulin (↑), blood glucose (↓); Liver glycogen content (↑); Testosterone (↑); Serum FSH and LH (↑); Recovering Leydig cell ultrastructure; Fructose and sialic acid (↑); Expression of PDX-1 and GLUT-2 (↑)	[29]
Protective effect of gastric ulcer	In vivo	Alcohol-induced gastric ulcer rat model, acetic acid-induced gastric ulcer rat model	4.41, 8.82 and 17.64 mg/kg, i.g.	Free acid, total acid, and activity of pepsin in stomach (↓); Ulcer index (↓)	[35]
	In vivo	Stress gastric ulcer mouse model	9, 18 and 36 mg/kg, i.g.	Ulcer index (↓)	[35]
Regulation of CYP2B expression	In vivo	Wistar rats	4, 20 and 100 mg/kg, i.g.	mRNA expression of hepatic CYP2B1 (↑), activity of hepatic CYP2B1/2 (↑); Protein content of hepatic endonuclear CAR (↑)	[39]
Regulation of mRNA expression of hepatorenal transporters	In vivo	Wistar rats	0.8, 4 and 20 mg/kg, i.g.	mRNA expression of hepatic OCT2, OAT2, OCTN2, OATP1A1, OATP1A4, OATP2B1, MRP2, and MDR1A, and renal MRP2, BCRP, and MDR1A (↓); mRNA expression of renal OCTN2, OATP1A1, OATP1A4, and MRP5 (↑)	[41]

↑ – improve or promote; ↓ – inhibit or reduce.

Figure 2.

Pharmacological activities of arecoline hydrobromide. Created using MedPeer Scientific Research Platform, version 2.1.240807, Beijing MedPeer Information and Technology Co., Ltd., Beijing, China.

Acaricidal Activity

Rhipicephalus microplus is an ectoparasites that mainly parasitize cattle in tropical and subtropical regions worldwide [14]. Apart from causing milk production loss, weight loss, and physical decline of parasitic cattle, they are also vectors for transmission of diseases such as anaplasmosis and babesiosis, indirectly causing significant economic losses to cattle producers [15,16]. AH was found to be efficient in eliminating ticks and their larvae, as well as reducing egg weight in the larval grouping experiment and adult soaking test conducted by Jain et al. In biochemical estimation, AH decreased the levels of glutathione S-transferase (GST) and superoxide dismutase (SOD), and decreased the activities of acetylcholinesterase (AChE) and monoamine oxidase (MAO) enzymes, indicating that it has oxidative stress-inducing activity [17]. Subsequently, Jain et al did an experiment on the acaricidal activity of AH in cattle. In comparison to the control group, which exhibited a 6.21% reduction, the results revealed an 82.26% decrease in the number of ticks after 144 hours of treatment with AH, and AH had no toxicity when applied to animal skin. Consequently, these findings suggested that AH has potential as an herbal acaricide to replace synthetic acaricidal drugs that are harmful to organisms and the environment [18].

Treatment of Rheumatoid Arthritis (RA)

RA is a common chronic inflammatory systemic disease primarily characterized by persistent synovitis and inflamed hyperplastic synovial tissue, and may eventually cause joint deformities [19,20]. He et al investigated the effects of AH on the functions of collagen-induced arthritis (CIA) mice and rheumatoid arthritis fibroblast-like synoviocytes (RA-FLSs). AH exhibited inhibitory effects on the proliferation and DNA replication of RA-FLSs, with an IC₅₀ of 71.55–105.16 μg/ml, promoted cell cycle arrest and cell apoptosis by inhibiting B-cell lymphoma-2 (Bcl-2), cyclin A2, cyclin B1, and cyclin-dependent kinase 1 (CDK1) expression, and attenuated migratory and invasive abilities by suppressing the expression of vimentin in RA-FLSs. Mechanistically, RNA sequencing analyses suggested that AH inactivated PI3K/AKT signaling pathway. In addition, AH can alleviate the arthritis symptoms of CIA mice in vivo. These findings clearly indicate that AH has high therapeutic potential for RA management [1].

Potassium Channel Inhibitory Activity

The potassium channel is the ion channel with the strongest heterogeneity and the most widely distributed ion channel, and is recognized as a potentially significant therapeutic target in the management of numerous diseases [21,22]. Potassium channel blockers inhibit the passage of K⁺ through the membrane channel to improve the pathological state [23]. Zhao et al recorded the human ether-a-go-go-related gene (hERG) K⁺ current (I_hERG) before and after exposure to AH using a standard whole-cell patch-clamp technique and found that AH exhibited a potent ability to block I_hERG in both frequency- and state-dependent manners [24]. Chen et al found that resting membrane potential of jejunum smooth muscle cells (SMCs) in W/W^v was depolarized by AH through voltage-gated potassium channels, which increased the contraction activity of jejunum smooth muscle in mice. The contraction frequency was dramatically changed from 129.6±22.9 mg in the control group to 157.5±24.4 mg after treatment with 10⁻⁶ M AH in W/W^v. The K⁺ channel blocker tetraethylammonium (TEA) could not block the enhancement of AH on jejunal smooth muscle of wild type mice, but was able to block the excitatory action in W/W^v. Intracellular results showed that AH significantly depolarized resting membrane potential of jejunal SMCs, and AH inhibited the voltage-gated potassium currents of acutely isolated mouse jejunal SMCs by whole-cell patch-clamp technique [25]which were lacking of interstitial cells of Cajal (ICC.

Reproductive Protection

Worldwide, the prevalence of diabetes is growing and it is becoming a common disease, including in children and adolescents and in reproductive-age men [26]. Male reproductive system disorders are common complications of diabetes [27,28]. Saha et al intraperitoneally injected AH (10 mg/kg body weight) into experimentally-induced type 1 diabetic rats for 10 consecutive days, showing that the levels of serum gonadotropins and insulin in diabetic rats were notably improved after AH treatment. As a result, testicular and sex accessory dysfunctions were restored, and β-cell regeneration was obviously promoted. At the protein level, AH activated key genes related to β-cell regeneration, including pancreatic and duodenal homeobox 1 (PDX-1) and glucose transporter 2 (GLUT-2). Therefore, it may be concluded that AH can effectively improve the adverse effects of insulin deficiency on gonads and male sexual organs of type 1 diabetic rats [29].

Anticataract Activity

Cataract is a visual impairment characterized by lens opacities [30]. Excessive accumulation of polyols and osmotic stress caused by diabetes are important contributors to cataract [31]. Anusha et al investigated aldose reductase (AR) inhibitory activity of AH on rat lens and kidneys in vitro and found that the inhibition of AH on AR was concentration-dependent, achieving the highest activity at the highest concentration (10 μg/ml). Additionally, they utilized a sugar-induced lens opacity model to determine anticataract activity of AH. The results clearly demonstrate that the transparency of the lens increased with AH treatment, indicating that AH could slow the development of lens opacification. Furthermore, the ability of AH to reduce osmotic stress induced by elevated polyol levels also confirmed its inhibitory effect on the formation of cataracts [32].

Protective Effect of Gastric Ulcer

Gastric ulcer (GU) is a digestive system disease attributed to various endogenous or exogenous factors, such as free oxygen radicals, stress, alcohol, Helicobacter pylori infection, and excessive secretion of pepsin and gastric acids [33,34]. Tang et al used 3 different mouse GU models to investigate the therapeutic effect of AH on GU. The experimental results indicated that AH significantly inhibits the formation of stress GU in mice, acetic acid GU and ethanol injury GU in rats, and had good preventive and protective effects on GU, and they also found that AH had a significant inhibitory effect on pepsin and gastric acid activity in pylorus-ligated rats. These results suggest that the mechanism of AH in treatment of GU is related to reduction of the erosion caused by gastric acid and pepsin and inhibition of gastric juice secretion [35].

Regulation of CYP2B Expression

CYP (cytochrome P450) is a superfamily of heme proteins that can metabolize many exogenous and endogenous compounds with different structures [36]. Belonging to the important CYP subfamilies, cytochrome P450 2B (CYP2B) are mainly involved in the metabolism of xenobiotics [37,38]. Huang et al assessed the hepatic CYP2B activity of AH-treated rats by use of LC-Ms/MS and found low doses of AH had more effect than high doses on hepatic CYP2B activity. They also used real-time PCR to determine hepatic CYP2Bl mRNA levels and used Western blotting to determine the protein levels of hepatic CAR and CYP2B. They found that the protein level of CYP2B in rat livers was not markedly altered after AH treatment, while the mRNA level of CYP2Bl showed a dose-dependent increase. Furthermore, AH caused obvious upregulation of CAR protein expression in the nucleus, but not of total CAR [39].

Regulation of mRNA Expression of Hepatorenal Transporters

Changes in the expression or activity of hepatorenal transporters affect the pharmacokinetics, pharmacodynamics, and toxicity of clinical drugs in vivo, subsequently causing drug interactions [40]. Thus, Zhai et al explored the effect of AH on mRNA expression of hepatorenal transporter in rats to provide a useful reference for clinically safe consumption of betel nut. The PCR results indicated that the mRNA expression of hepatic efflux transporter MRP2 and MDR1A were inhibited in the low-dose group, but the mRNA expression of hepatic efflux transporters OCT1, OCT2, OAT2, OCTN2, OATP2B1, OATP1A1, and OATP1A4, as well as MRP2, MRP5, and MDR1A, were significantly inhibited in the high-dose group. On the other hand, high-dose AH treatment inhibited the mRNA expression of renal efflux transporters BCRP, MRP2, and MDR1A, and significantly upregulated the mRNA expression of renal uptake transporters OCTN2, OATP1A1, and OATP1A4 and efflux transporter MRP5, while low-dose AH treatment only increased the renal MRP5 mRNA level. These findings suggest the need to pay attention to the safety of betel nut users in clinical practice [41].

Toxicology of AH

Although AH has many pharmacological activities, its toxicity also requires research. Wei et al conducted a 14-day toxicity study to evaluate sub-chronic toxicity of AH at doses of 100, 200, and 1000 mg/kg bw. Following 14 days of AH administration, the food intake and body weight of rats in all dosing groups had decreased. The hemoglobin, red blood cells, platelets, and leukocyte counts in the high-dose group were much lower than in the control group, and blood biochemical parameters (alanine aminotransferase, total protein and blood urea nitrogen) were also inhibited. In addition, organ coefficients (liver and kidney) of rats in the high-dose group showed a significant increasing trend. It is critical to ensure the safety of patients receiving AH, accordingly, high doses and prolonged use of AH should be avoided [42]. Forbes et al conducted a study to assess the effect of AH on oral, esophageal, gastric, and intestinal absorption in dogs. It was found that oral administration of AH locally acts on the intestinal wall, resulting in vomiting. After immersion administration, AH is absorbed through the mouth, pharynx and lungs, resulting in acute toxic reactions [43].

A study suggested that AH could induce teratogenic effects in chicken embryos. Paul et al injected AH aseptically dissolved in distilled water into the air-sac of the fertilized egg, and then observed and recorded the abnormal development of these chick embryos. Edema, exencephaly, and fetotoxicity were common in the AH group, among which arthrogryposis or clubfoot was one of the most consistent deformities [44]. Although the toxicological mechanisms behind teratogenic effects of AH were not explored in this study, they inferred arecoline may disrupt multiple biochemical pathways, leading to teratogenesis. More detailed toxicological studies on AH are needed before it can be clinically applied.

Pharmacokinetics of AH

There have been few studies on the pharmacokinetics of AH. Li et al established and validated a rapid and sensitive high-performance LC-MS/MS method and used it to determine the pharmacokinetic parameters of arecoline. AH is decomposed into arecoline when it enters the body, and then is further decomposed into arecaidine and arecoline N-oxide [45,46]. Following a single oral dose (3 mg/kg) of AH tablets, plasma samples were obtained from beagle dogs at 0.5, 0.67, 0.83, 1, 1.25, 1.5, 2, 3, 4, 5, 6, 8, 12, and 24 hours. C_max (peak plasma concentration) and T_max (peak time) of arecoline were 60.61 ng/mL and 120.07 min, respectively, the t_1/2 (half-life) was 69.32 min. the C_L/F (plasma clearance) was 0.19 L/min/kg, and AUC_0–t and AUC_0–∞ (area under the concentration–time curve values) were 15116.86 and 15771.37 min*ng/mL, respectively. The data of this investigation suggested that arecoline was rapidly absorbed and eliminated in dogs [47]. More research is needed on the pharmacokinetics of AH to provide more reliable data for its clinical use.

Future Directions

In nearly 3 decades of research, AH has been found to have a variety of pharmacological activities, including acaricidal activity, treatment of RA, potassium channel inhibitory activity, reproductive protection, protecting against gastric ulcers, regulation of CYP2B expression, and regulation of mRNA expression of hepatorenal transporters. AH has drawn increasing attention in the past decade, and the number of related patents has grown (Figure 3). However, most studies of AH did not involve its specific action target and mechanism, which hinders its application in many aspects, so further research is needed.

Figure 3.

Number of patents related to arecoline hydrobromide applied for and published per year through 2024 worldwide of the final data set. Generated using the Lens.org platform, version 9.2.2, Cambia, an international non-profit research organization in Australia.

In the last century, AH has been widely used in the treatment and diagnosis of tapeworm infection, but unfortunately its therapeutic effect is highly erratic [48]. Gregory et al assessed the effectiveness of AH during the initial 11-year phase of the hydatid control program in Tasmania. Between 1973 and 1975, 2563 dogs were infected with Taenia spp, of which 203 (7.9%) were not cured after treatment with AH [13]. Trejos et al used AH to treat 40 dogs infected with Echinococcus granulosus (E. granulosus) at 4 mg/kg, and necropsies were done 9 weeks later. Although AH demonstrated a certain deworming efficacy on E. granulosus of no more than 5 weeks of age, almost half of the dogs still had E. granulosus at necropsy [49]. AH did not have a good deworming effect at a safe clinical dosage, and doses beyond the safe clinical dosage lead to adverse reactions such as vomiting and convulsions in animals infected with tapeworm; thus, AH is basically no longer used as an anthelmintic [13]. The toxicity of AH limits its clinical application. However, given the scarcity of studies on the toxicological and pharmacokinetic properties of AH, this review can only provide a brief summary of the available information. Conducting more extensive toxicological and pharmacokinetic studies on AH in different administration modes or for different experimental animals is urgently needed to ensure the safe use of AH.

There are still multiple opportunities and challenges in the field of clinical application of AH. Changing the mode of administration and developing new drug preparations may expand the application of AH and inhibit its toxicity. Jain et al studied the acute toxicity of AH on animal skin and found that there was no toxic reaction [18], suggesting that topical dosage forms such as ointment and solution may be good directions for future development. In addition, chemical structural modification is also a promising research subject.

Conclusions

In summary, AH has been proved to be a valuable compound with a variety of pharmacological activities. However, the pharmacological mechanism and metabolic pathway of AH remain unclear, and the toxicity is also controversial. Therefore, further research on the safety, efficacy, and pharmacokinetics of AH should be carried out.