Simultaneous determination of five constituents of areca nut extract in rat plasma using UPLC-MS/MS and its application in a pharmacokinetic study.

Areca nuts have been used as a traditional Chinese medicine (TCM) for thousands of years. Recent studies have shown that it exhibits good pharmacological activity and toxicity. In this study, the pharmacokinetics of five major components of areca nut extract in rats were investigated using a highly sensitive ultra-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UPLC-MS/MS) method. Arecoline, arecaidine, guvacoline, guvacine, and catechin were separated and quantified accurately using gradient elution with mobile phases of (A) water containing 0.1 % formic acid-10 mM ammonium formate, and (B) methanol. The constituents were detected under a timing switch between the positive and negative ion modes using multiple reaction monitoring (MRM). Each calibration curve had a high R2 value of >0.99. The method accuracies ranged -7.09-11.05 % and precision values were less than 14.36 %. The recovery, matrix effect, selectivity, stability, and carry-over of the method were in accordance with the relevant requirements. It was successfully applied for the investigation of the pharmacokinetics of these five constituents after oral administration of areca nut extract. Pharmacokinetic results indirectly indicated a metabolic relationship between the four areca nut alkaloids in rats. For further clarification of its pharmacodynamic basis, this study provided a theoretical reference.

Molecular Mechanisms of Malignant Transformation of Oral Submucous Fibrosis by Different Betel Quid Constituents-Does Fibroblast Senescence Play a Role?

Betel quid (BQ) is a package of mixed constituents that is chewed by more than 600 million people worldwide, particularly in Asia. The formulation of BQ depends on a variety of factors but typically includes areca nut, betel leaf, and slaked lime and may or may not contain tobacco. BQ chewing is strongly associated with the development of potentially malignant and malignant diseases of the mouth such as oral submucous fibrosis (OSMF) and oral squamous cell carcinoma (OSCC), respectively. We have shown recently that the constituents of BQ vary geographically and that the capacity to induce disease reflects the distinct chemical composition of the BQ. In this review, we examined the diverse chemical constituents of BQ and their putative role in oral carcinogenesis. Four major areca alkaloids-arecoline, arecaidine, guvacoline and guvacine-together with the polyphenols, were identified as being potentially involved in oral carcinogenesis. Further, we propose that fibroblast senescence, which is induced by certain BQ components, may be a key driver of tumour progression in OSMF and OSCC. Our study emphasizes that the characterization of the detrimental or protective effects of specific BQ ingredients may facilitate the development of targeted BQ formulations to prevent and/or treat potentially malignant oral disorders and oral cancer in BQ users.

Determination of contents of four alkaloids in Pericarpium arecae by quantitative analysis of multi-components by single-marker.

By using a typical component in traditional Chinese medicine Pericarpium Arecae (PA), quantitative analysis of multi-components by single-marker (QAMS) was performed to determine the contents of four alkaloids. With a column packed with strong cation exchange bonded silica particles, the alkaloids were well separated, showing linear relationships within certain ranges. The limit of detection, limit of quantitation, precision, stability, repeatability and recovery all met requirements. By employing arecoline as internal standard, relative correction factors for arecaidine, guvacine and guvacoline at five concentrations were detected with three HPLC systems and three HPLC columns. The peaks of arecaidine, guvacine and guvacoline were positioned, during which the columns with the same packing materials from different manufacturers significantly affected relative retention values and retention time differences of the alkaloids. However, the columns, from different batches, managed to give relative retention values satisfying the requirements of HPLC peak positioning. The Thermo Fisher Scientific column packed with strong cation exchange bonded silica particles was finally selected by considering resolution and peak time. Compared with the external standard method, QAMS detected the alkaloid contents in 12 PA samples more accurately and reliably. The results provide valuable evidence for content determination and quality control of alkaloids in PA.

[Optimization of Preparation of Cutting-processed Arecae Pericarpium with Multi-index Orthogonal Method].

OBJECTIVE: 10 optimize the preparation of cutting-processed Arecae Pericarpium by the orthogonal method. METHODS: With four alkaloids such as arecoline, arecaidine, guvacoline, guvacine and yield of water-soluble decoction as indexes, a L9 (3(4)) orthogonal test was adopted to compare the effect of different factors on cutting-processed Arecae Pericarpium. RESULTS: According to the finalized optimal process, Arecae Pericarpium was washed quickly, moisturized, chopped lengthwise in 1 cm segment and then dried for 2.5 h at 50 °C. CONCLUSION: The optimal process method is reasonable and reliable, it can provide basis for the preparation of cutting-processed Arecae Pericarnium.

Areca nut-induced buccal mucosa fibroblast contraction and its signaling: a potential role in oral submucous fibrosis--a precancer condition.

Betel quid (BQ) chewing is an oral habit that increases the risk of oral cancer and oral submucous fibrosis (OSF), a precancerous condition showing epithelial atrophy and tissue fibrosis. Persistent fibroblast contraction may induce the fibrotic contracture of tissue. In this study, we found that areca nut extract (ANE) (200-1200 µg/ml) stimulated buccal mucosa fibroblast (OMF)-populated collagen gel contraction. Arecoline but not arecaidine-two areca alkaloids, slightly induced the OMF contraction. Exogenous addition of carboxylesterase (2U/ml) prevented the arecoline- but not ANE-induced OMF contraction. OMF expressed inositol triphosphate (IP3) receptors. ANE-induced OMF (800 µg/ml) contraction was inhibited by U73122 [phospholipase C (PLC) inhibitor] and 2-aminoethoxydiphenyl borate (IP3 receptor antagonist), respectively. Ethylene glycol tetraacetic acid and verapamil, two calcium mobilization modulators, also suppressed the ANE-induced OMF contraction. ANE induced calcium/calmodulin kinase II and myosin light chain (MLC) phosphorylation in OMF. Moreover, W7 (a Ca(2+)/calmodulin inhibitor), HA1077 (Rho kinase inhibitor), ML-7 (MLC kinase inhibitor) and cytochalasin B (actin filament polymerization inhibitor) inhibited the ANE-induced OMF contraction. Although ANE elevated reactive oxygen species (ROS) level in OMF, catalase, superoxide dismutase and N-acetyl-L-cysteine showed no obvious effect on ANE-elicited OMF contraction. These results indicate that BQ chewing may affect the wound healing and fibrotic processes in OSF via inducing OMF contraction by ANE and areca alkaloids. AN components-induced OMF contraction was related to PLC/IP3/Ca(2+)/calmodulin and Rho signaling pathway as well as actin filament polymerization, but not solely due to ROS production.

Potential action of betel alkaloids on positive and negative symptoms of schizophrenia: a review.

BACKGROUND: The Areca catechu Linn. is the fourth most used drug in the world after nicotine, ethanol and caffeine. This plant contains nine alkaloids with muscarinic and nicotinic action, which could have an antipsychotic effect. AIM: The aim of this work is reviewing literature data about the potential action of betel alkaloids on positive, negative and cognitive symptoms of schizophrenia. METHOD: We reviewed the clinical literature data since 1980 via a PubMed search for the terms: arecoline, arecaidine, guvacine, guvacoline, betel, Areca catechu, positive and negative symptoms, cognitive symptoms, psychosis and schizophrenia in combination. CONCLUSION: Male high consumption of betel had significantly lower positive symptoms than low consumers or non-betel users.

Activation of TGF-ß pathway by areca nut constituents: a possible cause of oral submucous fibrosis.

Oral submucous fibrosis (OSF) is a chronic inflammatory disease characterized by the accumulation of excess collagen, and areca nut chewing has been proposed as an important etiological factor for disease manifestation. Activation of transforming growth factor-ß signaling has been postulated as the main causative event for increased collagen production in OSF. Oral epithelium plays important roles in OSF, and arecoline has been shown to induce TGF-ß in epithelial cells. In an attempt to understand the role of areca nut constituents in the manifestation of OSF, we studied the global gene expression profile in epithelial cells (HaCaT) following treatment with areca nut water extract or TGF-ß. Interestingly, 64% of the differentially regulated genes by areca nut water extract matches with the TGF-ß induced gene expression profile. Out of these, expression of 57% of genes was compromised in the presence of ALK5 (TßRI) inhibitor and 7% were independently induced by areca nut, highlighting the importance of TGF-ß in areca nut actions. Areca nut water extract treatment induced p-SMAD2 and TGF-ß downstream targets in HaCaT cells but not in human gingival fibroblast cells (hGF), suggesting epithelial cells could be the source of TGF-ß in promoting OSF. Water extract of areca nut consists of polyphenols and alkaloids. Both polyphenol and alkaloid fractions of areca nut were able to induce TGF-ß signaling and its downstream targets. Also, SMAD-2 was phosphorylated following treatment of HaCaT cells by Catechin, Tannin and alkaloids namely Arecoline, Arecaidine and Guvacine. Moreover, both polyphenols and alkaloids induced TGF-ß2 and THBS1 (activator of latent TGF-ß) in HaCaT cells suggesting areca nut mediated activation of p-SMAD2 involves up-regulation and activation of TGF-ß. These data suggest a major causative role for TGF-ß that is induced by areca nut in OSF progression.

Xenobiotic metabolism: a view through the metabolometer.

The combination of advanced ultraperformance liquid chromatography coupled with mass spectrometry, chemometrics, and genetically modified mice provide an attractive raft of technologies with which to examine the metabolism of xenobiotics. Here, a reexamination of the metabolism of the food mutagen PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine), the suspect carcinogen areca alkaloids (arecoline, arecaidine, and arecoline 1-oxide), the hormone supplement melatonin, and the metabolism of the experimental cancer therapeutic agent aminoflavone is presented. In all cases, the metabolic maps of the xenobiotics were considerably enlarged, providing new insights into their toxicology. The inclusion of transgenic mice permitted unequivocal attribution of individual and often novel metabolic pathways to particular enzymes. Last, a future perspective for xenobiotic metabolomics is discussed and its impact on the metabolome is described. The studies reviewed here are not specific to the mouse and can be adapted to study xenobiotic metabolism in any animal species, including humans. The view through the metabolometer is unique and visualizes a metabolic space that contains both established and unknown metabolites of a xenobiotic, thereby enhancing knowledge of their modes of toxic action.

Effect of novel arecoline thiazolidinones as muscarinic receptor 1 agonist in Alzheimer's dementia models.

The discovery of cholinergic deficit in Alzheimer's disease (AD) patient's brain has triggered research efforts, using cholinomimetic approaches for their efficacy in AD therapy. Various therapies may be of potential clinical use in AD. Among these are cholinergic agents, which include muscarinic agonists, acetylcholinesterase inhibitors, and acetylcholine releasing agents. One of the muscarinic agonists tested in AD is arecoline and its bioisosters, which are widely explored as muscarinic receptor 1 agonist (M1 receptor agonist) in AD research. In this regard, five-membered heterocyclic ring system attached arecoline basic nucleus (N-methyl tetrahydropyridines) at third position has been extensively researched on. The present research involved synthesis of arecoline thiazolidinones 5(a-j) by using dipolar addition of 3-aminopyridine and alkyl/aryl carboxaldehydes in presence of gamma ferrite as catalyst. The resulting products were methylated and reduced to get desired products. Subsequently the synthesized arecoline thiazolidinones were subjected to in vitro muscarinic receptor binding studies using male Wistar rat brain (cerebral cortex) membrane homogenate and extended this in vitro study to in vivo pharmacological evaluation of memory and learning in male Wistar rats. Four derivatives (5a-5c and 5e) showed considerable M1 receptor binding affinity (in vitro) and elicited beneficial effects in vivo memory and learning models (Rodent memory evaluation, plus and Y maze studies).

Genetic damage in cultured human keratinocytes stressed by long-term exposure to areca nut extracts.

Chewing betel quid (BQ) is a popular habit worldwide. A causal association between BQ chewing and oral cancer has been well documented. Emerging evidence indicates that sustained exposure to stress induces epigenetic reprogramming of some mammalian cells and increases the mutation rate to accelerate adaptation to stressful environments. In this study, we first confirmed that 24-h treatment with areca nut extracts (ANE; a major component of BQ) at doses over 40 microg/ml induced mutations at the hypoxanthine phosphoribisyltransferase (HPRT) locus in human keratinocytes (HaCaT cells). We then investigated whether the stress of long-term exposure to sublethal doses of ANE (0, 5 and 20 microg/ml for 35 passages) could enhance genetic damage to HaCaT cells. Compared to cells exposed to 0 or 5 microg/ml ANE, cells exposed to 20 microg/ml ANE were slightly but significantly more resistant to a 72-h treatment with ANE and its major ingredients, arecoline and arecaidine, but did not develop cross-resistance to other BQ ingredients or alcohol. The cells that received 20 microg/ml ANE for 35 passages also had a significantly increased mutation frequency at the HPRT locus and an increased frequency in the appearance of micronuclei compared to lower doses. Moreover, increased intracellular levels of reactive oxygen species and 8-hydroxyguanosine in cells exposed to 20 microg/ml ANE suggested that long-term ANE exposure results in the accumulation of oxidative damage. However, cells subjected to long-term treatment of 20 microg/ml ANE contained higher levels of glutathione than unexposed cells. Therefore, after long-term exposure to sublethal doses of ANE, intracellular antioxidative activity may also be enhanced in response to increased oxidative stress. These results suggest that stress caused by long-term ANE exposure enhances oxidative stress and genetic damage in human keratinocytes.

Areca nut extract lowers the permeability of vaginal mucosa to reduced arecoline and arecaidine.

Areca nut chewing has been implicated in the development of oral cancer and oral submucous fibrosis. Arecoline and arecaidine, which are alkaloids present in the areca nut, are thought to play a major role in the development of adverse effects resulting from this chewing habit. Because these alkaloids appear to be associated with the development of the above diseases, we determined their diffusion kinetics through human vaginal mucosa in the presence and absence of a 1% areca nut extract. Seven clinically healthy vaginal mucosa specimens (mean patient age+/-standard deviation, 52+/-13 years; age range, 38-74 years) were obtained during surgery. In vitro flux values of reduced arecoline and arecaidine (r-arecoline and r-arecaidine) were determined through use of a flow-through diffusion apparatus. Analysis of variance, a Duncan multiple range test, and an unpaired t-test were used to determine steady state kinetics and flux differences over time intervals. The flux values across vaginal mucosa of r-arecoline and r-arecaidine were decreased in the presence of 1% areca nut extract. For r-arecoline, these flux values were significantly lower statistically when compared to those obtained in the absence of areca nut extract. These findings concur with results previously obtained for water, where the astringent action of the tannins present in the areca nut extract was thought to alter the barrier properties of the epithelium, resulting in decreased permeability.

Diffusion of reduced arecoline and arecaidine through human vaginal and buccal mucosa.

Because alkaloids from areca nut, arecoline and arecaidine, have been implicated in the development of oral submucous fibrosis, we determined their diffusion kinetics through human buccal and vaginal mucosa. Four clinically healthy vaginal mucosa specimens (mean patient age +/- standard deviation: 47 +/- 15 years; age range: 31-60 years) and 4 buccal mucosa specimens from 2 male patients and 2 female patients (mean patient age +/- standard deviation: 31 +/- 9 years; age range: 17-53 years) were obtained during surgery. In vitro flux rates of reduced arecoline and arecaidine (r-arecoline and r-arecaidine) were determined by use of a flow-through diffusion apparatus. Analysis of variance, a Duncan multiple range test, and an unpaired t-test were used to determine steady state kinetics and flux differences over time intervals. Although statistically significant differences were observed between flux values for both alkaloids and tissues at certain time points, these were not considered to be of biological (clinical) significance. However, the flux rates across both mucosa of r-arecoline were significantly higher statistically than those of rarecaidine. The findings demonstrated the differences in the diffusion kinetics between r-arecoline and r-arecaidine across human buccal and vaginal mucosa, an observation that could be explained in terms of their ionisation characteristics. Additionally, the results obtained further support the hypothesis that human vaginal mucosa can be used as a model for buccal mucosa in studies of permeability to various chemical compounds.

Deficiency in collagen and fibronectin phagocytosis by human buccal mucosa fibroblasts in vitro as a possible mechanism for oral submucous fibrosis.

Oral submucous fibrosis (OSF), a chronic oral mucosal condition commonly found in south Asians, is a disorder characterized by a quantitative as well as a qualitative alteration of collagen deposition within the subepithelial layer of the oral mucosa. Since degradation of collagen by fibroblast phagocytosis is an important pathway for physiological remodelling of soft connective tissues, we have investigated phagocytosis of collagen- and fibronectin-coated latex beads by fibroblast cultures with an in vitro model system. Coated fluorescent latex beads were incubated with human oral mucosa fibroblasts and the fluorescence associated with internalized beads was measured by flow cytometry. Cells from normal tissues that had been incubated with beads for 16 h contained a mean of 75% collagen phagocytic cells and 70% fibronectin phagocytic cells; however, about 15% and 10% of phagocytic cells individually contained more than twice the mean number of beads per cell. In contrast, cells from OSF tissues exhibited a 40% reduction of the proportions of collagen phagocytic cells (mean=35%) and a 48% decrease of the proportions of fibronectin phagocytic cells (mean=22%), none of the cells having a high number of beads as compared to normal fibroblasts. OSF lesions appear to contain fibroblasts with marked deficiencies in collagen and fibronectin phagocytosis. To investigate if inhibition of phagocytosis could be demonstrated in vitro, normal fibroblast cultures were incubated with areca nut alkaloids (arecoline, arecaidine). The cultures had a dose-dependent reduction in the proportions of phagocytic cells. On the other hand, corticosteroid used in the treatment of OSF exhibited a dose-dependent enhancement in the proportion of phagocytic cells. Therefore, our hypothesis for OSF, although oversimplified, is that betel nut alkaloids (arecoline, arecaidine) inhibit fibroblast phagocytosis and this provides a mechanism for the development of OSF. The benefit of a local intralesional injection of corticosteroid is also possibly, at least in part, through an enhancement of fibroblast collagen phagocytosis.

The mutagenicities of alkaloids and N-nitrosoguvacoline from betel quid.

In Taiwan, betel quid is a natural masticatory, which is composed of fresh green areca fruit, Piper betle and slaked lime paste. Areca fruit contains some alkaloids, of which arecoline is the major one. N-Nitrosoguvacoline (NG), one of the N-nitrosation products of arecoline, is the only one N-nitrosamine found in Taiwanese betel quid chewing saliva. The mutagenic studies in Ames Salmonella microsome test showed that crude alkaloid extracts of areca fruit and arecoline were active in Salmonella typhimurium TA100, and NG was weakly active in TA98 and TA100. The activities in both arecoline and NG decreased further in the presence of rat liver S9 mix. Nitrite was significantly consumed during the N-nitrosation of arecoline and sodium nitrite at acidic condition (pH 3), whereas the formation of NG was favored at neutral condition (pH 7). Crude phenolic extracts of leaf and inflorescence of Piper betle inhibited the formation of NG by blocking the nitrite. However, a high amount of crude phenolic extracts of areca fruit enhanced the formation of NG.

Observations on the effect of areca nut extracts on oral fibroblast proliferation.

The effects of aqueous extracts of raw, baked and boiled areca nuts were tested on cultured human buccal mucosa fibroblasts. Cells were exposed to extract concentrations of 0, 50, 100, 150, 300 and 500 micrograms/ml. The arecoline and arecaidine content was determined in the extracts with HPLC and raw nut contained 5.5% m/m, baked nut 6.6% m/m and boiled nut 7.1% m/m. Extract concentrations of 50 to 150 micrograms/ml inhibited cell growth in a concentration-dependent manner but did not lead to total cell death during a 7 day period. However, total cell death did occur with concentrations of 300 and 500 micrograms/ml. It is concluded that areca nut extract is toxic to cultured fibroblasts and inhibits their proliferation in a concentration-dependent manner.

Stimulation of human buccal mucosa fibroblasts in vitro by betel-nut alkaloids.

Oral submucous fibrosis (OSF) is characterized by excessive collagen production by mucosal fibroblasts and is associated with the habitual chewing of betel-nuts (Areca catechu); nut extracts stimulate fibroblast activity in vitro. The metabolism of arecoline, the major alkaloid in the nut, by human buccal mucosa fibroblasts in vitro was investigated; alkaloid metabolites extracted from culture media were analysed by gas chromatography and thin-layer chromatography. [3H]-arecoline was metabolized predominantly to [3H]-arecaidine and this was accompanied by a concentration-dependent stimulation of collagen synthesis and cell proliferation. Arecaidine was a more potent stimulator than arecoline. The rate of hydrolysis of a series of synthetic arecaidine esters (methyl, ethyl, butyl, propyl and pentyl) by fibroblasts was closely correlated with the extent of stimulation of collagen synthesis. Thus fibroblasts are responsive to the major metabolite of arecoline and hydrolysis of the ester group may be necessary for this action. Exposure of buccal mucosa fibroblasts to these alkaloids in vivo may contribute to the accumulation of collagen in OSF.

Induction of in vivo sister chromatid exchanges by arecaidine, a betel nut alkaloid, in mouse bone-marrow cells.

The genotoxicity of arecaidine, an alkaloid of betel nut, was studied on mouse bone marrow cells in vivo by sister chromatid exchange (SCE) method. Arecaidine was administered intraperitoneally to mice at the dose levels of 2.5 mg, 5 mg and 7.5 mg to each mouse weighing 25 +/- 1 g for 5, 10 and 15 days. Significant increase in the number of SCEs was observed in the treated groups, and this increase, although dose-dependent, was not dependent upon the duration of exposure.

Correlation of mutagenicity and tumorigenicity of betel quid and its ingredients.

The mutagenic activity of betel quid and its ingredients was determined using Salmonella typhimurium tester strains TA 100, TA 1535, TA 98, and TA 1538, both in the presence and absence of S9 mixture. Aqueous extracts of betel quid (BQ), betel quid with tobacco (BQT), and betel nut (BN) were mutagenic in strain TA 100. Aqueous extract of betel leaf (BL) was not mutagenic in any of the four strains. Arecoline and arecaidine, which are major alkaloids present in BN, were mutagenic in all four tester strains. Tumorigenicity studies in Swiss mice given the above constituents showed that BN and BQ induced lung tumors (47% and 26%, respectively). However, when BN was fed with BL, tumorigenicity was lowered to 38%. BL alone was not tumorigenic. Thus, the mutagenicity of betel quid and its ingredients is correlated with tumorigenicity.