Distinct phenolic, alkaloid and antioxidant profile in betel quids from four regions of Indonesia

Betel quid (BQ) is a chewing mixed package that mainly contains areca nut (AN), betel leaf (Leaf) or betel stem inflorescence (SI), and slaked lime, and is consumed with or without tobacco BQ chewing is common in South East Asia and has been strongly associated with malignant and potentially malignant diseases of the oral cavity. Alkaloids such as arecoline are often accounted for the carcinogenic potential of BQ, however the chemical composition of BQ has not been studied in detail. In the current study, we investigated the total phenolic content (TPC), antioxidant activity (by mean of ferric reducing antioxidant power, FRAP), radical scavenging activity (DPPH test), polyphenolic profile and arecoline content in different components of BQ, namely AN, Leaf or SI, Husk, and blended BQ (BQ mix, containing AN, Leaf or SI and slaked lime). Samples were imported from 4 major regions of Indonesia, namely: Banda Aceh (BA), North Sumatra (NS), West Kalimantan (WK) and West Papua (WP). The highest TPC, FRAP, and DPPH values were detected in AN samples compared to other BQ components, while samples from WP region were of higher values compared to the other regions. High performance liquid chromatography—Mass Spectrometry (LC–MS) analysis showed that Husk contains the widest range of polyphenols, including hydroxybenzoic acids, hydroxycinnamic acids, flavanols, flavonols and stilbenes. Catechin and epicatechin were the main polyphenols detected in BQ, and they were present at the highest concentrations in WP–AN sample. Arecoline was detected in all AN and BQ mix samples and was significantly correlated with catechin and epicatechin, and significantly negatively correlated with p-hydroxybenzoic acid. Notably, arecoline concentration changed significantly when AN was blended in BQ mixtures. The current study is the first to extensively characterise the chemical composition of BQ and provides insight for a better understanding of the interactions of BQ alkaloids and phenolics in the development of oral submucous fibrosis and oral cancer.

Methods and experimental design chemicals and reagents. The reagents and solvents used for sample preparation and chemical assay were analytical reagent (AR) grades. AR grade methanol, sodium carbonate anhydrous, sodium hydroxide pellets and iron (II) sulfate heptahydrate were supplied by Chem-Supply Pty. Ltd. (Gillman SA, Australia). Hydrochloric acid (HCl) 36% grade AR, and iron (III) chloride anhydrous were purchased from Thermo Fisher Scientific (Scoresby VIC, Australia). Gallic acid, 2.0 N Folin-Ciocalteu's phenol reagent (FCR), 2,4,6,-Tris(2-pyridyl)-s-triazine (TPTZ), 2,2-Diphenyl-1-picrylhydrazyl (DPPH), and (±)-6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid 97% (Trolox) were obtained from Sigma-Aldrich (Castle Hill NSW, Australia). Acetic acid glacial was supplied by VWR International Pty. Ltd. (Tingalpa QLD, Australia). The chemicals and standard compounds applied in HPLC analysis were in HPLC grades. Acetic acid 50% solution and standard compounds [gallic acid, 5-(hydroxymethyl)furfural, protocatechuic acid, caftaric acid, p-hydroxybenzoic acid, caffeic acid, syringic acid, p-coumaric acid, trans-sinapic acid, ferulic acid, procyanidin B1, procyanidin B2, catechin, epicatechin, epicatechin gallate, quercetin, quercetin 3-O-galactoside, quercetin 3-O-glucuronide, quercetin 3-O-rhamnoside, kaempferol, kaempferol 3-O-glucoside, resveratrol, polydatin were purchased from Sigma-Aldrich (Castle Hill NSW, Australia). Acetonitrile was obtained from Merck Pty. Ltd. (Bayswater, VIC, Australia). All water used was prepared by Milli-Q Gradient water purification system (Millipore Australia Pty. Ltd., North Ryde, NSW, Australia). The alkaloids analytical standard were arecoline hydrobromide 250 mg and guvacine hydrocloride 250 mg from Merck Pty.Ltd. (Castle Hill, NSW, Australia). Guvacoline hydrobromide 10 mg and arecidine hydrobromide 10 mg from Sapphire Bioscience Pty. Ltd (Redfern NSW 2016, Australia). experimental design and sample preparation. Raw BQ components were collected from four regions of Indonesia; Banda Aceh (BA), North Sumatra (NS), West Kalimantan (WK), and West Papua (WP). The four regions selected represented the west, middle, and east part of Indonesia. BQs were bought from local traditional market from each region. For BA, NS and WK regions, dried AN, Leaf, Husk, and BQ mix samples were analyzed, and the BQ mix contains dried AN, Leaf and lime at the ratio of 80.5:12.5:7 by weight 28 (Fig. 1). For WP region, dried AN, SI, Husk, and BQ mix samples were analyzed, and different from other regions, the BQ from WP region contains, betel inflorescence stem (SI) that commonly named as flower, instead of betel leaf 26 . The outer cap colour differences of AN is a manifestation of the ripeness of AN, where the unripe AN looks green Scientific RepoRtS | (2020) 10:16254 | https://doi.org/10.1038/s41598-020-73337-0 www.nature.com/scientificreports/ (shown by An from WP and WK regions), while the ripe AN looks yellow-orange colour 26 shown by AN from BA and NS (Fig. 1). Extraction of polyphenols from BQ samples were performed following published method with modifications 29 . Seeds and husks of AN were manually separated. All samples were freeze-dried for 72 h using FD3 Freeze Drier (Dynavac Engineering, Australia), and finely grinded using electronic grinder (Multigrinder II, model EM0405, Sunbeam, Auckland, New Zealand). 5 g of each sample powder was weighed into a 50 mL centrifuge tube prefilled with nitrogen gas (99.99% Grade 4.0, Coregas, Yennora NSW, Australia), added with 50 mL methanol as extraction solution. Extractions were carried out at 20 °C for 48 h under shaking condition at 150 rpm (Incubator shaker ZWYR-240, LABWIT scientific, Australia). After the extraction, the samples were centrifuged at 6500 rpm for 10 min at 20 °C. The supernatants were passed to a clean tube and evaporated at 40 °C to remove the solvent entirely using Hei-VAO Value rotary evaporator (Heidolp Instruments GmbH & Co.KG, Schwabach, Germany), then redissolved in 10 mL of LC grade methanol. The final extract was flushed with nitrogen gas and sealed with parafilm to avoid oxidation and storage at 4 °C in darkness until analysis. The extract stock solution was filtered using 30 mm × 0.45 µm nylon syringe filter (Thermo Fisher Scientific, VIC, Australia) to gain a clear solution before further analyses.
Determination of total phenolic content (tpc). The sample extract total phenolic content was examined using a modified Folin-Ciocalteu reagent (FCR) based on Singleton and Rossi (1965) method 30 . TPC measurement based on the spectrophotometric detection of chromogens established by the reaction between the phenols from the functional hydroxyl groups with the phosphomolybdic/phosphotungstic acid complexes reagents. A standard calibration curve was constructed using gallic acid at concentrations from 50 mg/L to 500 mg/L, with 50 mg/L increment. The total phenolic content of sample was expressed as gallic acid equivalents (GAE) [mg GAE/g sample dry mass (DM)]. The assay was conducted by adding 100 µL of diluted samples into clean tube containing 2.5 mL 0.2 N FCR (tenfold diluted 2.0 N FCR with Milli-Q water). Add 2 mL of saturated sodium carbonate solution (75 g/L) after 5 min. The blend solutions were vortexed and allowed to react in the dark room for 1 h. The absorbance was further quantified using Multiskan GO Microplate Spectrophotometer (Thermo Fisher Scientific Oy, Vantaa, Finland) at 765 nm. Technical duplicates were performed for each sample replicate.

Determination of ferric reducing antioxidant power (fRAp).
The FRAP test of the sample extract was performed following the Benzie and Strain (1996) method 31 . FRAP assay uses antioxidants iron (III) as reductants in a redox-linked colorimetric method, employing an easily reduced oxidant system presenting stoichiometric excess. Furthermore, Iron (II) sulfate heptahydrate was used as the standard composite to produce  Lc-DAD-eSiMS analysis. LC-MS analysis of polyphenols was performed with Agilent 1260 Infinity II LC system (Agilent Technology, Santa Clara, United States), coupled to Agilent ESI MSD (G6125B, Agilent Technology). Data acquisition and machine control were achieved using the Openlab Chemstation software. Negative ion mass spectra of the column elute were analysed in simultaneous sim/scan mode for polyphenols based on a published method 29 . The scan covered the range m/z 90-600, while the target sim ions used for individual compounds are listed in Table S1. Nitrogen was used both as drying gas at the flow of 11.0 L/min and as nebulizing gas at pressure of 55 psi. The nebulizer temperature was set at 350 °C. The LC elusion program used was the same as the System I, with DAD synchronous detection wavelength set at 280 nm, 320 nm and 370 nm.

Statistical analysis. Statistical analysis was conducted using MS-Excel, Minitab 17 2016, MATLAB by
Mathworks, XLStat, and R software (v3.5.2 The R foundation). One-way ANOVA with Odd Ratio test were executed at p ˂ 0.05. Group indicated with different letters showed statistically significant difference. The results were exhibited as the means ± standard deviation (SD) from four observations of individualistic replicates.

Results
Total phenolic content (TPC) and antioxidant activities in betel quid samples. The  www.nature.com/scientificreports/ twofold that of nut from other regions, indicating that WP-AN contains the most abundant phenolics among nut group (Table 1). There was a considerable reduction of TPC and antioxidant activities (FRAP and DPPH scavenging activities) when AN was in a BQ mixture with slaked lime (AN: betel leaf/stem: slaked lime = 80.5:12.5:7 by weight). In the betel leaf/stem group alone, WK-Leaf had the highest phenolics and antioxidant activities, while the least phenolics content was found in NS-Leaf.

Polyphenolic content in betel quid components.
Our study shows that phenolic acids were mostly detected in husk, leaf and SI samples, but traced in nuts and BQ mixtures. The most abundant phenolic acids detected were protocatechuic acid, p-hydroxybenzoic acid and syringic acid, while gallic acid, caffeic acid, p-coumaric acid, ferulic acid and sinapic acid were detected at low concentrations. The highest protocatechuic acid content was observed in NS-Husk followed by WK-Husk and BA-Husk respectively. The highest p-hydroxybenzoic acid was observed in WP-SI, followed by NS-Leaf, BA-Leaf, and WK-Leaf respectively. NS-Leaf has the highest concentrations of syringic acid (Table 2 and Fig S2).
Flavanols, mainly catechin and epicatechin, were the most abundant polyphenols detected in nearly all samples except WK-leaf. Higher flavanols concentrations were observed in nuts compared to leaf, husk and SI. Epicatechin gallate was also detected in BA-Leaf at high concentrations, but not in other leaf samples. Flavonols, including quercetin-3-galactoside, quercetin-3-glucuronide, quercetin-3-rhamnoside, kaempferol-3-glucoside and quercetin were also detected in the leaf and husk samples mainly from BA, but low or absent in samples from NS, WK and WP. Resveratrol was the only stilbene detected in BQ mainly in leaf and husk, but absent or at low concentrations in nut samples.
Alkaloid content in betel quid components. Only arecoline analytical standard showed a stable peak using the current method, while arecaidine, guvacine and guvacoline showed unstable peaks for standard curve. Therefore, arecaidine, guvacine, and guvacoline could not be identified and quantified with this method.
The highest arecoline concentration in the current study was observed in WP, followed by BA, WK, and NS respectively in a nut group alone (Table 3). Interestingly, in the mixture group, the concentration of arecoline in BQ from WP and WK dropped only 0.38 and 1.3 mg/g DM, respectively, compared to arecoline concentration in their AN. However, BQ mixtures from the other 2 regions showed a considerable reduction of arecoline concentration to more than 50% from their nut. No arecoline was detected in betel leaf and stem inflorescence samples. Low concentrations of arecoline was detected in husk from most regions, where husk from BA was the only husk which does not contain arecoline. Table 1. Total phenolics content and antioxidant activities in AN, betel leaf/ betel stem inflorescence, BQ mixture and husk. Data are expressed as mean ± standard deviation of triplicate experiments (n = 3) with confident interval significantly (P < 0.05). Mean values in the same column followed by the same letter do not differ significantly. An a letter besides mean values refer to the highest value, and its value decreases following the descending letter in the same column. BA = Banda Aceh, NS = North Sumatra, WK = West Kalimantan, WP = West Papua, AN = Areca Nut, SI = stem inflorescence BQ Mix = Areca nut + betel leaf/betel stem inflorescence stem + slaked lime, TPC = total phenolic content, FRAP = ferric reducing antioxidant power, DPPH = 1,1-diphenil-2-picrylhydrazyl. The bold value is significantly higher compared to another mean in the same column. The unbold value is significantly lower mean values in the same column.  (Fig. 2). Agglomerative hierarchical clustering (AHC) was further used to classify samples into different groups (Fig S3). A total of 42.45% of variation was explained by PC 1 (24.11%) and PC 2(18.34%). Arecoline, catechin, quercetin, kaempferol and epicatechin mainly contributed to positive aspect of PC 1, while sinapic acid, quercetin-3-galactoside, quercetin-3-rhamnoside, quercetin-3-glucuronide, sinapic acid, p-coumaric acid and ferulic acid contributed to negative aspect of PC 1 and positive aspect of PC 2. Protocatechuic acid, caffeic acid, syringic acid, p-hydroxybenzoic, gallic acid, epicatechin gallate and resveratrol contributed to the negative aspects of PC 1 and PC 2. Catechin, epicatechin on PC 1 were significantly positively correlated with arecoline, while p-hydroxybenzoic on the negative aspect of PC 2 was significantly negatively correlated with arecoline (Table 4). Samples were well separated long PC 1 vectors (mainly arecoline, catechin and epicatechin) into two groups, while samples on the negative side of PC 1 would further be divided into three groups based on vectors of PC 2 (Fig. 2)

Discussion
In this study we performed a systematic, thorough chemical characterisation of the individual constituents of BQ. Our data show, for the first time, that BQ has distinct chemical features that are mixture and region specific. Interestingly, the alkaloid and polyphenolic content as well as antioxidant activity of individual ingredients change significantly when these are combined into a BQ mixture. Different altitude and climate in each region may also account for the variation in polyphenols and arecoline content in the various BQ.
The results of the present study indicate that BQ from WP is of particular interest, and this is consistent with our previous epidemiology research 6 . Specifically, all forms of BQ from WP contain the highest concentrations of arecoline, phenolic acid, anthoxanthins, and stilbenes which may help explain the high prevalence of OSMF and oral cancer in this region. Further research is required to better elucidate the association between the identified arecoline and phenolics from four regions of Indonesia with the development of OSMF.
The highest arecoline concentration in the current study was observed in AN from WP, followed by AN from BA, WK, and NS respectively. The amount of arecoline changed significantly when AN was used in the BQ mixture but did so to a different extent. For example, the concentration of arecoline in WP-BQ mix and WK-BQ mix dropped only 1.35 and 1.3 mg/g DM respectively (WP-AN: 6.92 ± 1.64a, WP-mix: 5.57 ± 0.66b, WK-AN: 4.4 ± 0.27ac, WK-mix: 3.11 ± 0.15d). However, BQ mixtures from other 2 regions showed a considerable reduction of arecoline concentration to more than 50% from their nut with addition of slaked lime and leaf/SI (BA-AN: 6.10 ± 0.99ab, BA-mix: 2.58 ± 0.39d, NS-AN: 4.11 ± 0.22c, NS-mix: 1.54 ± 0.23e) ( Table 3). These findings show that adding slaked lime not only decreases the overall concentration of polyphenols but, also, of arecoline. This might be due to extreme increases of pH up to 10 by slaked lime as most alkaloids are only stable in the range of pH 4-7 34 . This extreme pH has been shown to damage the alkaloid 35,36 , which might lead to decreased arecoline concentration. However, immature ANs from WK and WP were interestingly less affected by the addition of slaked lime, potentially due to different chemical and biological processes occurring in the mixture 37 . It could indicate that polyphenols and arecoline have a synergic protection from pH change that preserve the inclination number of arecoline i.e. the more phenolics in AN, the less arecoline damage by slaked lime in BQ mixture. However, further research is needed to clarify this hypothesis. One study from India reported that Mangalore variety of AN, extracted with various solvents, showed arecoline quantification of 2.3-12.79 mg/g DM 22,38 , whereas our current study using methanol extraction solvent showed a range of 0.37-6.9 mg/g DM (Table 3). This difference could be due to the origin of BQ products, different process or method of extraction, and its degree of maturity 39 . No arecoline was detected in betel leaf and SI samples. This finding was consistent with other studies, which reported high polyphenolic contents and absence of alkaloids in betel leaf and SI 26,40,41 . Low Table 4. Correlation (Pearson) between arecoline and phenolic compounds. The bold phenolic acid compound is significantly correlated with arecoline. The unbold phenolic acid compound is insignificantly correlated with arecoline. www.nature.com/scientificreports/ concentrations of arecoline was detected in husk from most regions, except BA-Husk. No previous study has investigated alkaloid and phenolics composition of husk, thus the present study is the first to assess the chemical composition of areca husk. It has been suggested that regular and frequent polyphenols intake may be helpful to protect against oral cancer 19 . However, polyphenols can also act as pro-oxidant at high concentrations under certain conditions 14,15 . In the present study, we found polyphenols (mainly catechin and epicatechin) in high amount (Table 2), especially in WP-AN sample. Polyphenols induce cell apoptosis by triggering reactive oxygen species (ROS) through two mechanisms 13 , direct formation of labile aroxil radical or a labile radox complex with a metal cation 42 and indirect activation of intracellular production of ROS by NADPH oxidases 43 . Cell culture study showed catechin-induced DNA damage when treating human leukemia HL-60 cells with epigallocatechin-3-gallate (EGCG) at 50 µM 16 . Further, in vivo studies show the dose-dependent hepatotoxicity of EGCG at single dose from 750-1500 mg/kg, where 1500 mg/kg treatment reduce survival rate by 85% 18 . Consistently, increasing numbers of hepatoxicity cases in humans have been associated with intake of green tea polyphenols, mainly EGCG 44 and in some cases, associated with liver inflammation and necrosis 45 . Despite the results of a recent study that EGCG transiently inhibits both cell proliferation and migration of oral cavity cancer cells 46 , there is limited evidence showing the pro-oxidant effect of catechins and cytotoxicity to oral cells, which warrants further investigation.
Catechin as the main flavanol present in BQ can also act as antioxidant, that has been shown to prevent cell mutation by inhibiting the production of metalloproteases, leading to potentially reducing invasion and migration, inducing apoptosis and growth arrest in both oral cancer and oral leukoplakia cell lines 19 . The lower concentrations of catechin and other flavanols in mixture compared to nut observed in the current study may likely be due to the dilution effects of other BQ components. Furthermore, addition of lime can increase pH to 10 and damage the polyphenols 34 . Further, the polyphenols in AN have been proven to possess many pharmacological activities 47,48 . These include antiparasitic effects, anti-depressive effects, anti-fatigue effects, antioxidant effects, antibacterial and antifungal effects, antihypertensive effects, anti-inflammatory and analgesic effects, anti-allergic effects, the promotion of digestive functions, suppression of platelet aggregation, regulatory effects on blood glucose and lipids, etc. 20,47,48 . The large variations in BQ polyphenol contents of different Indonesian regions may be due to various reasons, such as environment conditions, germination, variety of AN, degree of ripeness 26,47 . It is to be noted that variations in the concentration of the various constituents may occur in nuts from different geographical locations and according to the degree of maturity of the nut. Similarly, the different altitude and climate in each region may play a role in the content of polyphenols and arecoline detected in our study, which makes it difficult to compare our results with those available in the literature.
In the current study, alkaloid and flavonoid (catechin and tannin) components were simultaneously identified in AN. The alkaloids are considered to be the most potent inducers of OSMF, while flavonoids have a synergistic role 7 . Flavonoids could decrease collagenase, stabilize the collagen fibrils to degradation by collagenase 8 .
Other polyphenols identified were ferulic acid and resveratrol that have been previously reported in AN 48,49 . However, in the present study, ferulic acid was not detected in any nuts samples and resveratrol was only detected in nuts from WP. Further polyphenols such as quercetin and kaempferol were also reported in AN 48,49 , and these compounds were detected in the current study and interestingly observed in leaf and husk samples as well. Previous studies on BQ polyphenols mainly reported phenolic compounds in the nuts 21,48-52 , but few study reported these compounds in husk, leaf and SI. Therefore, the polyphenols that were identified in husk, leaf and SI reported in the current study might explain the antioxidant capacity of these samples and provided insights for future research investigating antioxidants in BQ. Sarode et al. 11 hypothesized that only AN alone cannot trigger OSMF, without including other necessary required factors such as slaked lime and inflammation. These factors possibly result in conversion to phenotypically altered fibroblasts, which leads to increased fibrosis in the oral mucosa causing OSMF. Previous research has shown that powdered slaked lime applied to the chewed AN and betel inflorescence at the corner of the mouth in Papua New Guinea, causes the pH change to 10, at which reactive oxygen species (ROS) are produced from BQ in vitro 34 . This high pH change and excessive polyphenols can alter the polyphenols to act as pro-oxidant 13 . Thus, we hypothesise that high concentration of polyphenols in AN, especially catechin and epicatechin, together with high pH altered by slaked lime, are the main factors triggering OSMF. Further, our study showed that adding slaked lime into BQ mixture decreased by more than 70% all the antioxidant potency (TPC, FRAP and DPPH scavenging activities), compared to AN without slaked lime (Table 1). However, only WP-BQ mixture showed a slight inclination of polyphenols and arecoline concentration. In this case, consuming WP-BQ mixture results in having BQ with high concentration of polyphenols, arecoline and high pH due to added slaked lime. This could explain why people living in WP, who consume WP-BQ mixture, are more likely to develop OSMF as shown in our previous study 6 . Further in vitro and in vivo research needs to be conducted to elucidate the association between Indonesian BQ and pathogenesis of OSMF.
The observed high TPC value of WP-AN compared to AN from other regions could reflect the different amount of immature nuts from this region, as unripe ANs commonly contain more polyphenols compared to ripe AN 26 . This theory is consistent with our current study that the unripe AN from WP and WK showed the highest total phenolics content. Further, we also found that FRAP and DPPH antioxidant activities are higher in unripe nut rather than in the ripe AN from BA and NS (Table 1). The TPC value of nut may also depend on the region where areca catechu is grown, and its processing method 39 . In present study, we could recognize unripe AN by its green in colour, while the ripe AN looks yellow (Fig. 1) 26 . In some countries, such as Taiwan, Guam, and China, the unripe AN is mostly consumed 26 , similar to WP and WK. Whereas in BA and NS, the ripe AN is favoured 6 .
An interesting finding was that the phenolic content and antioxidant activities dropped in the BQ mixture featuring slaked lime (AN: betel leaf/SI: slaked lime = 80.5:12.5:7 by weight). A previous study also reported that BQ without slaked lime had higher TPC, antioxidant and cytoprotective activities compared to BQ with slaked lime 22 . It may be that slaked lime raises the pH up to 10 leading to phenolics damage 34  www.nature.com/scientificreports/ BA, NS, and WK dropped by 94.5%, 90.5%, and 74.5% compared to TPC of AN alone, respectively. These results were consistent with FRAP and DPPH tests as well (Table 1). Interestingly, total phenolics content in WP-BQ mixture only dropped by 28.4% from the amount in AN alone. This might be due to the degree of immaturity of WP-AN and the form of slaked lime. The excessive amount of phenolics could lead to cytotoxicity instead of exerting protective action 19 . The results of our recent survey supports the theory that consuming excessive polyphenols in BQ could be detrimental to, rather than protective for oral health 19 . Our previous study showed that 60 out of 139 participants (43.17%) who had OPMD were living in WP region and mostly BQ consumers 6 . This correlates in this current study with BQ containing the most abundant phenolics levels. Thus, we suggest consuming BQ containing ripe AN is better compared to consuming BQ containing unripe AN, as ripe AN contains less arecoline, known to trigger OSMF development 8 , and contains moderate number of polyphenols that can act to protect oral cavity 19 . WK-leaf had the highest antioxidant activities and the widest range of phenolic acids among betel leaf/ stem group alone. WP-SI was observed to have the highest concentration of phenolic compounds, such as p-hydroxybenzoic acid. Previous study reported that betel SI may contain polyphenols such as hydroxychavicol, eugenol, isoeugenol, eugenol methyl ester and safrole 40 . Safrole as the major polyphenols in betel inflorescence is also considered to be a carcinogen 41 . However, safrole was not profiled in the present study. The values of FRAP and DPPH were consistent with TPC results in most samples, indicating that FRAP and DPPH attributes of the samples were due to the presence of polyphenol compounds.
Some people living in Taiwan, China, Guam, and Papua New Guinea include the husk of outer AN pericarp as part of the BQ mixture 26 . This might increase the risk of developing OSMF, as our study revealed that husk from WK, NS, and WP contains 0.16, 0.37, 1.1 mg/g DM of arecoline respectively (Table 3). Further, an improper management of waste of husk could also possess potential hazardous to environment as it contains carcinogenic properties. The recent study suggested the use of areca husk as self-assembly of oleylamine modified nanofibrillated cellulose from areca husk fibers into giant vesicles formed that can have applications in storage and delivery of drugs in topical applications 53 . Thus, our findings describing the chemical composition of areca husk is important to avoid or manage future risks. Further study is required to clarify how husk can hazardous the environment. There betel husk should be carefully managed to avoid entering human food chain. conclusion BQ compounds have distinct chemical features that are mixture and region specific. Interestingly, the alkaloid and phenolic content as well as antioxidant activity of individual ingredients change significantly when these are combined into a BQ mixture. We observed that BQ compounds from WP was distinctive from other regions, and that AN and BQ mixture from this region contained the highest TPC, antioxidant activities, polyphenols and arecoline content.
Catechin and epicatechin are the main polyphenols detected in BQ, while WP had the highest concentrations in nut sample. The high amount of catechin and epicatechin could change the effect of antioxidant into pro-oxidant leading to raise the potential of developing OSMF.
Arecoline was detected in all ANs and mixture samples and was significantly correlated with catechin and epicatechin, and significantly negatively correlated with p-hydroxybenzoic acid. Phenolic acids and flavanols content in betel quid mixture were low, likely due to the addition of lime, which also led to lower antioxidant activity and radical scavenging capacity in the mixture.
The ripeness degree of AN is directly related to the amount of both polyphenols and arecoline. The unripe AN contained a higher concentration of polyphenols and arecoline compared to ripe AN. Suggesting that consume the ripe AN could lower the potential in developing OSMF.
In summary, our results shed light on the chemical composition of different Indonesian BQs and may inform the development of chemo-preventive strategies to contrast the development of OSMF. For example, consuming mature type AN, avoiding the husk because of its high arecoline content, as well as not adding slaked lime, could decrease the potential of developing OSMF in betel chewers".