Formation of reactive oxygen species and of 8-hydroxydeoxyguanosine in DNA in vitro with betel quid ingredients

The formation of reactive oxygen species (ROS) from betel quid ingredients, namely areca nut, catechu and tobacco, was studied using a chemiluminescence (CL) technique. Aqueous extracts of areca nut and catechu were capable of generating superoxide anion and hydrogen peroxide at pH greater than 9.5. The formation of O2 was enhanced by Fe2+, Fe3+ and Cu2+ but inhibited by Mn2+. Tobacco extract failed to generate ROS under similar conditions. Saliva was found to inhibit both O2 and H2O2 formation from betel quid ingredients. Upon incubation of DNA at alkaline pH with areca nut extract and Fe3+ or catechu, 8-hydroxydeoxyguanosine was formed as quantified by high performance liquid chromatography (HPLC)/electrochemical detection. The data suggest a possible role of reactive oxygen species in the etiology of oral cancer in betel quid chewers.     

Study of unscheduled DNA synthesis following exposure of human cells to arecoline and extracts of betel nut in vitro.

Aqueous, acetic acid, hydrochloric acid and ethanol extracts of betel nut (Areca catechu L.) have been found to induce unscheduled DNA synthesis in Hep 2 cells obtained from human larynx carcinoma, in vitro. Different concentrations of extracts of betel nut induced dose-dependent unscheduled DNA synthesis in Hep 2 cells. Together with the viability of the Hep 2 cells, our results indicate that the aqueous and acetic acid extracts of betel nut induce relatively more unscheduled DNA synthesis than the hydrochloric acid and ethanol extracts and arecoline. The carcinogenic potency of raw and unprocessed betel nut of North-East India used in this study is discussed.     

Betel nut extract and arecoline block insulin signaling and lipid storage in 3T3-L1 adipocytes

According to several population-based studies, betel nut chewing is associated with metabolic syndrome and diabetes in British South Asians and Taiwanese. However, the underlying molecular mechanism is not yet clear. Arecoline is an alkaloid-type natural product found in betel nuts. Our aim was to clarify the influence of betel nut extract and arecoline on lipid accumulation and insulin signaling in adipocytes. We found that betel nut extract and arecoline blocked lipid storage in 3T3-L1 adipocytes. The possible mechanism may function by inhibiting the expression of the insulin receptor, glucose transporter-4, fatty acid synthase, and the lipid droplet proteins perilipin and adipophilin. In addition, betel nut extract and arecoline increased the basal level of IRS-1 serine³⁰⁷ phosphorylation and decreased insulin-stimulated IRS-1 tyrosine, Akt, and PI3 kinase phosphorylation. In conclusion, betel nut extract and arecoline have diabetogenic potential on adipocytes that may result in insulin resistance and diabetes at least in part via the obstruction of insulin signaling and the blockage of lipid storage.     

Effect of chewing betel nut on measurements of salivary progesterone and estradiol

The measurement of steroids in saliva is both simple and non-invasive and has been widely used in field and clinical-based research. The observance of particular cultural practices by some populations, however, may hamper accurate hormonal analyses. The present study evaluated the effects of one such practice-the chewing of betel nut-on the accurate measurement of salivary progesterone and estradiol. A time series experiment was conducted among Bangladeshi women who are regular users of betel nut. Salivary steroids were analyzed by radioimmunoassay in samples collected prior to and then 30, 60, 120, and 240 min following betel quid use. Results show no significant difference between basal steroid levels and those obtained 60, 120, and 240 min after chewing betel nut. We conclude that with specific collection protocols that take into account time since chewing, salivary steroid analyses can be undertaken in populations among whom the practice of chewing betel nut is endemic.     

Salivary Proteomic Analysis of Betel Nut (Areca catechu) Consumers by Mass Spectrometry Revealed Primary Indication of Oral Malignancies

Areca nut is the fourth most widely used addictive and psychoactive substance consumed by approximately 10% of the world’s population. The use of areca nut is estimated to account for up to 50% of oral cancer in the low-income, and middle-income countries. In the present study, the effect of betel nut chewing on saliva proteomics was investigated by using mass spectrometry. Matrix-assisted laser desorption ionization mass spectrometry was used to generate a profile of the peptides in betel nut consumers and control group. We found 13 peptide peaks which were significantly altered (p?<?0.05) in the betel nut addicts when compared with the control group. These significant peptides signals were corresponding to protein cystatin SN (CST1), cystatin S (CST4), alpha 2 macroglobulin (A2M), complement C3 (C3), apolipoprotein E (APOE), serum albumin (ALB), matrix metalloproteinase-9 (MMP-9), deleted in malignant brain tumor protein 1 (DMBT1), zinc-alpha-2-glycoprotein (ZAG), and protein S100A8. The correlation analysis of significant peptides intensities with the history of betel nut chewing was also performed. The peptides of CST1 and CST4 showed negative correlation, whereas the peptides of the MMP-9, DMBT1, APOE, and C3 showed positive correlation with significant differences. STRING analysis of these proteins revealed that most of these proteins are interacting with each other. The present study identifies a number of proteins in a significantly different abundance in the betel nut consumers group. Some of these proteins are the reported biomarkers of several oral malignancies, which implies that the usage of betel nut could lead to inflammation, and development of oral cancer.

     

Betel Nut Chewing Is Strongly Associated With General and Central Obesity in Chinese Male Middle‐aged Adults

Betel nut chewing has been reported to increase the risk of cardiovascular disease and all‐cause mortality. The reason is unclear. In this study, we investigated the association between betel nut chewing and general obesity (BMI ≥25 kg/m²) and central obesity (waist circumference (WC) ≥90 cm). A total of 1,049 male subjects, aged ≥40 years, were recruited from Taichung city in Taiwan in 2004. The relationships between betel nut chewing and general and central obesity were studied by multiple linear and logistic regression analyses. The prevalence of current and former betel nut chewing was 7.0 and 10.5% in our male Taiwanese cohort. Current/former betel nut chewers had a higher prevalence of general and central obesity when compared with individuals who had never chewed betel nut. Adjusted for age, diabetes, hypertension, lipids, smoking, alcohol drinking, physical activity, income, and education level, the odds ratios (ORs; 95% confidence intervals) of general and central obesity among the lower consumption of betel nut chewers were 1.78 (1.07, 2.96) and 1.19 (0.70, 2.02), respectively, compared to 2.01 (1.18, 3.41) and 1.89 (1.10, 3.23), respectively, among higher consumption chewers compared to individuals who had never chewed betel nut. The increasing ORs of general and central obesity with higher betel nut consumption revealed dose–response effects. Using multiple linear regression analyses, after adjusting for potential confounders, betel nut consumption was statistically significantly associated with BMI and WC. In conclusion, betel nut chewing was independently associated with general and central obesity in Taiwanese men. Dose–response effects of the association between betel nut consumption and general obesity as well as central obesity were found.     

Epidemiology of areca (betel) nut use in the mariana islands: Findings from the University of Guam/University of Hawai`i cancer center partnership program

BackgroundAreca (betel) nut is considered a Group 1 human carcinogen shown to be associated with other chronic diseases in addition to cancer. This paper describes the areca (betel) nut chewing trend in Guam, and health behaviors of chewers in Guam and Saipan.MethodsThe areca (betel) nut module in the Guam Behavioral Risk Factor Surveillance Survey was used to calculate the 5-year (2011–2015) chewing trend. To assess the association between areca (betel) nut chewing and health risks in the Mariana Islands, a cross-section of 300 chewers, ≥18 years old, were recruited from households in Guam and Saipan. Self-reported socio-demographics, oral health behaviors, chronic disease status, diet, and physical activity were collected. Anthropometry was measured. Only areca (betel) nut-specific and demographic information were collected from youth chewers in the household.ResultsThe 5-year areca (betel) nut chewing prevalence in Guam was 11% and increased among Non-Chamorros, primarily other Micronesians, from 2011 (7%) to 2015 (13%). In the household survey, most adult chewers (46%) preferred areca nut with betel leaf, slaked lime, and tobacco. Most youth chewers (48%) preferred areca nut only. Common adult chronic conditions included diabetes (14%), hypertension (26%), and obesity (58%).ConclusionThe 5-year areca (betel) nut chewing prevalence in Guam is comparable to the world estimate (10–20%), though rising among Non-Chamorros. Adult and youth chewers may be at an increased risk for oral cancer. Adult chewers have an increased risk of other chronic health conditions. Cancer prevention and intervention strategies should incorporate all aspects of health.     

Areca (betel) nut chewing practices of adults and health behaviors of their children in the Freely Associated States,Micronesia: Findings from the Children’s Healthy Living (CHL) Program

BackgroundChewing areca (betel) nut has been deemed carcinogenic. The practice has become a public health concern in Micronesia. The Children's Healthy Living (CHL) Program included an areca (betel) nut questionnaire in a survey of household characteristics in the Freely Associated States (FAS). This paper describes areca (betel) nut chewing practices of adults and the health behaviors of their children.MethodsA cross-section of 1200 children (2–8 year-olds) and their caregivers in Chuuk, Kosrae, Pohnpei, Republic of Palau, Republic of the Marshall Islands (RMI), and Yap were recruited. Socio-demographics, adult areca (betel) nut chewing practices, and other health behaviors of children and adults were assessed. Child anthropometric measurements were collected to estimate weight status.ResultsThe FAS areca (betel) nut chewing prevalence was 42%, ranging from 3% (RMI) to 94% (Yap). Among chewers, 84% added tobacco, 97% added slaked lime, 85% added betel leaf, and 24% mixed the components with alcohol. Among FAS children, 95% practiced daily teeth-brushing and 53% visited the dentist annually. Compared to non-chewing households, areca (betel) nut chewing households were more likely to have very young children enrolled, more highly educated adults, and members that used tobacco and alcohol.ConclusionThe FAS areca (betel) nut chewing prevalence (42%) is above the world prevalence of 10–20%, with wide variability across the islands. The oral health findings in this study may inform future oral cancer prevention programs or policies. Regular monitoring of areca (betel) nut use is needed to measure the impact of such programs or policies.     

Mutagenic activation of betel quid-specific N-nitrosamines catalyzed by human cytochrome P450 coexpressed with NADPH-cytochrome P450 reductase in Salmonella typhimurium YG7108

Betel quid chewing is known to cause cheek cancer in a wide area covering Africa to Asia. Areca nut contained in the betel quid is believed to give rise to carcinogenic N-nitrosamines. In the present study, the roles of human cytochromes P450 (P450 or CYP) in the mutagenic activation of betel quid-specific N-nitrosamines such as 3-(N-nitrosomethylamino)propionitrile (NMPN), 3-(N-nitrosomethylamino)propionaldehyde (NMPA) and N-nitrosoguvacoline (NG) were examined by using genetically engineered Salmonella typhimurium YG7108 expressing each form of human P450 together with NADPH-P450 reductase, which had been established in our laboratory. Among typical P450s (CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2A13, CYP2D6 or CYP3A4) examined, CYP2A6 was the most efficient activator of NMPN, followed by CYP1A1 and CYP1B1. The mutagenic activation of NMPN by CYP2A6 was seen at the substrate concentrations of microM levels (approximately 100 microM). The activation of NMPA was catalyzed predominantly by CYP2A13 and to lesser extents by CYP2A6, CYP1A1, CYP1A2 and CYP1B1. The activation of NMPA by CYP2A13 was detectable at the substrate concentrations of microM levels (approximately 1 microM). NG was activated by CYP2A13 and CYP2A6, the genotoxicity of NG being much lower than that of NMPA or NMPN. Based on these data, we conclude that human CYP2A subfamily members play important roles in the mutagenic activation of essentially all betel quid-related N-nitrosamines tested in the present study.     

Presence of cholinomimetic and acetylcholinesterase inhibitory constituents in betel nut

In this investigation, we report the presence of cholinomimetic and acetylcholinesterase (AChE) inhibitory constituents in betel nut, the most commonly used drug in the world after tobacco, ethanol and caffeine. The crude extract of betel nuts or Areca catechu (Ac.Cr) caused a dose-dependent (0.3-300 microg/mL) spasmogenic effect in the isolated rabbit jejunum. The spasmogenic effect was blocked by atropine, similar to that of acetylcholine (ACh), suggestive of muscarinic receptor mediated effect. Both the extract (0.3-10 microg/mL) and physostigmine (0.1-3.0 microM) potentiated the effect of a fixed dose of ACh (10 microM) in a dose-dependent fashion, suggesting acetylcholinesterase (AChE) inhibitory effect. This effect was confirmed in the in vitro assay where both the crude extract (1-100 microg/mL) and physostigmine inhibited the enzyme. In the in vivo model of gastrointestinal transit, Ac.Cr (10-30 mg/kg) enhanced the travel of charcoal meal and also exhibited a laxative effect in mice. The plant extract was subjected to activity-directed fractionation and all resultant fractions showed atropine-sensitive spasmogenicity in rabbit jejunum and also AChE inhibitory effect at doses similar to that for the parent crude extract, the ethyl acetate fraction being slightly less potent. Some of the known constituents of betel nut, including arecoline, were tested for the possible inhibitory effect on AChE, none were found active. The study provides first evidence for the presence of AChE inhibitory constituents in betel nut, though additional direct muscarinic stimulatory effect cannot be ruled out and this study provides sound scientific basis for some of the folkloric uses associated with betel nut chewing.     

Soil chemistry and plant distributions in rock habitats of southern Sweden

The vascular plant flora of open land on superficial bedrock in southem Sweden (northwards to 59°N) is described and related to soil chemical properties. including soil acidity (pH), exchangeable Al, Ca, Fe, Mn, Mg, and phosphate, as well as to soil solution pH and concentrations of Al, Ca and Mg, and to the contents of soil organic matter, soil depth and bedrock types (sandstone, gneiss, granites, various dark igneous rocks and limestones). About 120 localities with totally 652 sites (4 m²) have been examined. Experimental evidence for the toxicity of acid soils and mineral nutrient deficiency of neutral and alkaline soils is related to field data. Hydrogen and Al ion toxicity in acid soils and low phosphate solubility in neutral - alkaline soils are identified as major factors limiting the field distributions of rock habitat plants. Some species (e.g., Rumex acetosella and Sedum telephium ) were limited by phosphate also in acid soils. The relative importance of H and Al ion concentrations to plant performance under variously acid soil conditions is discussed, and strong evidence is given for a decisive influence of Al ion toxicity on species diversity at pH-KCI > 4.5. The importance of grazing and former land use is considered briefly and the floristic differences between the western and the eastern half of the study area are discussed originating from differences in general distribution patterns of species and soil chemical properties.     

Effects of aluminum on plasma membrane as revealed by analysis of alkaline band formation in internodal cells of Chara corallina.

To study the mechanism of aluminum toxicity in plant cells, the effects of aluminum on alkaline band formation were analyzed in the internodal cells of Chara. After cells were treated with AlCl3, they were examined for their capacity to develop alkaline bands. Treating cells with AlCl3 medium at pH 4.5 completely inhibited alkaline band formation. When either CaCl2 or malic acid was added to the AlCl3 medium (pH 4.5), it did not produce an ameliorative effect, whereas addition of both CaCl2 and malic acid induced a significant ameliorative effect. It was found that treatment at pH 4.5 in the absence of AlCl3 strongly inhibited alkaline band formation. This inhibition by the low pH (4.5) treatment was effectively ameliorated by CaCl2. At higher pH (5.0), malic acid alone produced a significant ameliorative effect on aluminum inhibition of alkaline band formation, but CaCl2 did not. Recovery from aluminum inhibition was also studied. When cells treated with AlCl3 at pH 4.5 were incubated in artificial pond water, they could not recover the capacity to develop alkaline band. When either malic acid or CaCl2 was added to artificial pond water, cells recovered their alkaline band formation. It was concluded that one of the primary targets of aluminum is the plasma membrane and that aluminum affects the plasma membrane from the cell exterior at the beginning of the treatment (within 24 h). It was also suggested that the aluminum treatment impairs the HCO3- influx mechanism but not the OH- efflux mechanism.     

Effects of betel chewing on the central and autonomic nervous systems

Betel chewing has been claimed to produce a sense of well-being, euphoria, heightened alertness, sweating, salivation, a hot sensation in the body and increased capacity to work. Betel chewing also leads to habituation, addiction and withdrawal. However, the mechanisms underlying these effects remain poorly understood. Arecoline, the major alkaloid of Areca nut, has been extensively studied, and several effects of betel chewing are thought to be related to the actions of this parasympathomimetic constituent. However, betel chewing may produce complex reactions and interactions. In the presence of lime, arecoline and guvacoline in Areca nut are hydrolyzed into arecaidine and guvacine, respectively, which are strong inhibitors of GABA uptake. Piper betle flower or leaf contains aromatic phenolic compounds which have been found to stimulate the release of catecholamines in vitro. Thus, betel chewing may affect parasympathetic, GABAnergic and sympathetic functions. Betel chewing produces an increase in heart rate, blood pressure, sweating and body temperature. In addition, EEG shows widespread cortical desynchronization indicating a state of arousal. In autonomic function tests, both the sympathetic skin response and RR interval variation are affected. Betel chewing also increases plasma concentrations of norepinephrine and epinephrine. These results suggest that betel chewing mainly affects the central and autonomic nervous systems. Future studies should investigate both the acute and chronic effects of betel chewing. Such studies may further elucidate the psychoactive mechanisms responsible for the undiminished popularity of betel chewing since antiquity.     

HPLC法测定槟榔中的多酚类物质

采用高效液相色谱法对槟榔中的多酚类物质进行分析.用甲醇提取槟榔中的多酚类物质,并依次用石油醚、乙酸乙酯、正丁醇萃取,萃取物经抽真空浓缩,流动相定容,高效液相色谱法测定.分析结果表明:槟榔幼果较槟榔成熟果中所含的多酚种类和数量少,槟榔成熟果中果仁的多酚种类和数量都远较皮中多;槟榔的甲醇提取物乙酸乙酯萃取部分中所含的多酚种...     

不同pH的酚酸溶液对杉木及萝卜幼苗生长的影响

通过四因素五水平的二次回归正交旋转试验设计 ,研究 pH值、肉桂酸、对羟基苯甲酸、香草醛四个因素对杉木和萝卜幼苗生长的影响。结果表明 ,杉木幼苗的鲜重生长随酚酸溶液的pH值的增大而增加 ,而随肉桂酸、对羟基苯甲酸和香草醛浓度的增大而减小。比较 3种不同酚类物质对杉木幼苗生长的影响 ,肉桂酸对杉木幼苗生长的影响 >对羟基苯甲酸 >香草醛 ,且 pH值与肉桂酸之间 ;肉桂酸与香草醛之间 ;对羟基苯甲酸与香草醛之间的交互作用都达到显著水平。萝卜幼苗生长对不同pH的酚酸溶液的反应与杉木幼苗相类似 ,随酚酸溶液的pH值的增大而增加 ,而随肉桂酸、对羟基苯甲酸和香草醛浓度的增大而减小。但对羟基苯甲酸对萝卜幼苗生长的影响 >肉桂酸 >香草醛 ,酚酸溶液的 pH值与所有 3种酚酸之间的交互作用都达到显著水平 ;肉桂酸与香草醛的交互作用对萝卜幼苗生长的影响也达到F0 0 1水平显著。     

Transplacental micronucleus inducing ability of arecoline, a betel nut alkaloid, in mice

Arecoline, a major betel nut alkaloid, was tested for its effectiveness in inducing micronuclei in fetal mouse blood after transplacental exposure late in the gestation period. Positive results were obtained and a linear dose-response relationship was expressed when pregnant mice were treated with arecoline at dose levels of 20, 40 and 80 mg/kg and micronucleated polychromatic erythrocytes from fetal blood were subsequently analysed.     

Nicotinic Activity of Arecoline,the Psychoactive Element of "Betel Nuts", Suggests a Basis for Habitual Use and Anti-Inflammatory Activity

Habitual chewing of "betel nut" preparations constitutes the fourth most common human self-administration of a psychoactive substance after alcohol, caffeine, and nicotine. The primary active ingredient in these preparations is arecoline, which comes from the areca nut, the key component of all such preparations. Arecoline is known to be a relatively non-selective muscarinic partial agonist, accounting for many of the overt peripheral and central nervous system effects, but not likely to account for the addictive properties of the drug. We report that arecoline has activity on select nicotinic acetylcholine receptor (nAChR) subtypes, including the two classes of nAChR most related to the addictive properties of nicotine: receptors containing α4 and β2 subunits and those which also contain α6 and β3 subunits. Arecoline is a partial agonist with about 6–10% efficacy for the α4* and α6* receptors expressed in Xenopus oocytes. Additionally, arecoline is a silent agonist of α7 nAChR; while it does not activate α7 receptors when applied alone, it produces substantial activation when co-applied with the positive allosteric modulator PNU-120696. Some α7 silent agonists are effective inhibitors of inflammation, which might account for anti-inflammatory effects of arecoline. Arecoline''s activity on nAChR associated with addiction may account for the habitual use of areca nut preparations in spite of the well-documented risk to personal health associated with oral diseases and cancer. The common link between betel and tobacco suggests that partial agonist therapies with cytisine or the related compound varenicline may also be used to aid betel cessation attempts.     

Phenolics removal from transgenic Lemna minor extracts expressing mAb and impact on mAb production cost

Transgenic Lemna minor has been used successfully to produce several biotherapeutic proteins. For plant-produced mAbs specifically, the cost of protein A capture step is critical as the economic benefits of plant production systems could be erased if the downstream processing ends up being expensive. To avoid potential modification of mAb or fouling of expensive protein A resins, a rapid and efficient removal of phenolics from plant extracts is desirable. We identified major phenolics in Lemna extracts and evaluated their removal by adsorption to PVPP, XAD-4, IRA-402, and Q-Sepharose. Forms of apigenin, ferulic acid, and vitexin comprised ～ 75% of the total phenolics. Screening of the resins with pure ferulic acid and vitexin indicated that PVPP would not be efficient for phenolics removal. Analysis of the breakthrough fractions of phenolics adsorption to XAD-4, IRA-402, and Q-Sepharose showed differences in adsorption with pH and in the type of phenolics adsorbed. Superior dynamic binding capacities (DBC) were observed at pH 4.5 than at 7.5. To evaluate the cost impact of a phenolics removal step before protein A chromatography, a mAb purification process was simulated using SuperPro Designer 7.0. The economic analysis indicated that addition of a phenolics adsorption step would increase mAb production cost only 20% by using IRA-402 compared to 35% for XAD-4 resin. The cost of the adsorption step is offset by increasing the lifespan of protein A resin and a reduction of overall mAb production cost could be achieved by using a phenolics removal step.     

Isolation and characterization of polymorphic microsatellite loci from Areca catechu (Arecaceae) using PCR-based isolation of microsatellite arrays

Betel nut (Areca nut, Areca catechu L.) is a conspicuous and important cultivated plant of tropical and subtropical habitats throughout Southeast Asia and Oceania. As a significant cultural and social offering, the migration of betel nut associated with human dispersal is an important issue in ethnobotany and anthropology. In this study, we described the development of nine microsatellite loci from A. catechu. The number of alleles per locus ranged from 5 to 15. The expected and observed heterozygosities ranged from 0.71to 0.94 and from 0 to 0.88, respectively. All microsatellite loci, except for AC30, significantly deviated from Hardy-Weinberg equilibrium possibly due to artificially selected cultivation or the existence of excessive null alleles. No linkage disequilibrium was observed from pairwise comparisons of loci, except for AC06 and AC08.     

Arecoline induced disruption of expression and localization of the tight junctional protein ZO-1 is dependent on the HER 2 expression in human endometrial Ishikawa cells

Background  

Approximately 600 million people chew Betel nut, making this practice the fourth most popular oral habit in the world. Arecoline, the major alkaloid present in betel nut is one of the causative agents for precancerous lesions and several cancers of mouth among those who chew betel nut. Arecoline can be detected in the human embryonic tissue and is correlated to low birth weight of newborns whose mothers chew betel nut during pregnancy, suggesting that arecoline can induce many systemic effects. However, few reports exist as to the effects of arecoline in human tissues other than oral cancer cell lines. Furthermore, in any system, virtually nothing is known about the cellular effects of arecoline treatment on membrane associated signaling components of human cancer cells.