Zn Superoxide Dismutase from Piper betle Leaf and

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Purification of Cu/Zn Superoxide Dismutase from Piper betle Leaf and Its Characterization in the Oral Cavity Yu-Ching Liu,†,‡ Miau-Rong Lee,§ Chao-Jung Chen,†,∥ Yung-Chang Lin,*,‡ and Heng-Chien Ho*,§ †

Proteomics Core Lab, Department of Medical Research, China Medical University Hospital, No. 2 Yu-De Road, Taichung, 40407, Taiwan R.O.C. ‡ Department of Veterinary Medicine, National Chung Hsing University, 250 Kuo Kuang Road, Taichung 402 Taiwan R.O.C. § Department of Biochemistry, China Medical University, No. 91 Hsueh-Shih Road, Taichung, 40402 Taiwan R.O.C. ∥ Graduate Institute of Integrated Medicine and, China Medical University, No. 91 Hsueh-Shih Road, Taichung, 40402 Taiwan R.O.C. S Supporting Information *

ABSTRACT: The aim of this study was to purify protein(s) from Piper betle leaf for identification and further characterization. A functionally unknown protein was purified to apparent homogeneity with a molecular mass of 15.7 kDa and identified as Cu/Zn superoxide dismutase (SOD). The purified SOD appeared to be monomeric and converted to its dimeric form with increased enzymatic activity in betel nut oral extract. This irreversible conversion was mainly induced by slaked lime, resulting from the increase in pH of the oral cavity. Oral extract from chewing areca nut alone also induced SOD dimerization due to the presence of arginine. The enhanced activity of the SOD dimer was responsible for the continuous production of hydrogen peroxide in the oral cavity. Thus, SOD may contribute to oral carcinogenesis through the continuous formation of hydrogen peroxide in the oral cavity, in spite of its protective role against cancer in vivo. KEYWORDS: superoxide dismutase, Piper betle leaf, dimerization, areca nut, arginine, hydrogen peroxide

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INTRODUCTION Piper betle leaf (PBL) is widely consumed in Asia and valued for its medicinal properties. Its extract has been shown to possess antioxidant activity that highly correlates with its total phenolic content and reducing power.1 Direct analysis using real-time mass spectrometry (DART-MS) has identified chavicol and chavibetol as well as their acetates as the principal components in PBL,2 although its exact constituents have been found to vary among cultivars. Chemical analysis has demonstrated that the most abundant component, estimated to account for nearly 40% in Malaysian PBL, is hydroxychavicol,3 which has multiple pharmacological effects, such as enzyme inhibition4 and inflammatory mediation.5 PBL extract has also been shown to improve halitosis and provide hepatic protection in a rat model.6 It does not possess carcinogenic properties,7 but rather contributes to cancer prevention.8 PBL is rarely consumed by itself in Taiwan. Instead, it is usually coated with slaked lime with calcium hydroxide as one of the major components and then wrapped around an areca nut to produce a betel nut, which is then chewed. There are more than 50 000 betel nut stands in Taiwan, providing easy access to this food. It is estimated that at least two million of the 23 million residents in Taiwan chew betel nuts. A clear correlation between oral cancer and betel nut chewing, smoking, and wine drinking has been established through epidemiological studies and statistical analyses.9 More than 90% of oral cancer patients in Taiwan habitually chew betel nuts. On the basis of the Taiwan Cancer Registry annual report (Ministry of Health and Welfare, Executive Yuan, Taiwan, ROC), there were 6890 cases of oral malignancy in 2011, an increase of 330 cases compared with 2010. More than © XXXX American Chemical Society

90% of the cases are males, consistent with the fact that more males than females chew betel nuts. Oral cancer is the fifth most common cause of mortality in this region. There have been only a few reports demonstrating the characterization of PBL proteins or genes, including invertase,10 β-galactosidase,11 and chlorophyllase.12 In this report, a functionally unknown protein in PBL was purified using DEAE-cellulose chromatography and identified as Cu/Zn SOD. In the presence of slaked-lime aqueous extract and arginine, the monomeric SOD is able to dimerize and exhibits substantially increased enzymatic activity. The dimerization event results in the enhanced production of hydrogen peroxide in the oral cavity, which may damage the tissue.

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MATERIALS AND METHODS

Materials and Reagents. PBL, areca nut, and the combination betel nut were purchased in Taichung City, Taiwan; resin for DEAEcellulose chromatography, from Whatman Inc. (Clifton, NJ); prestained protein marker, from Fermentas (St. Leon-Rot, Germany); trypsin, from Promega (Madison, WI); SOD activity assay kit, from BioVision Inc. (San Francisco, CA); Microcon 3, from Millipore Corp. (Billerica, MA); (+)catechin and L-/D-arginine, from Sigma-Aldrich Corp. (St. Louis, MO); sodium diethyldithiocarbamate trihydrate (SDDC), from Alfa Aesar (Ward Hill, MA); sinapinic acid (SA) and αCyano-4-hydroxycinnamic acid (HCCA), from Bruker Daltonics Inc. (Bremen, Germany). Received: November 27, 2014 Revised: February 3, 2015 Accepted: February 4, 2015

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DOI: 10.1021/jf505753d J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry

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Figure 1. Purification and identification of SOD from Piper betle leaf (PBL). (A) Apparent homogeneity of the purified protein (right lane) detected by SDS-PAGE and Coomassie blue staining. Protein marker (left lane) with molecular mass (kD) is indicated. (B) MALDI-TOF analysis showing the purified protein with a molecular mass of 15.7 kDa. (C) The predicted sequence of Cu/Zn SOD from Zantedeschia aethiopica. The sequences of two adjacent tryptic peptides near the C-terminus are underlined. (D) and (E) Native gel examination of the five collected fractions from DEAEcellulose chromatography by Coomassie staining (D) and zymography (E). described.14 Analysis of the tryptic peptides was performed using an nano-LC-MS/MS system. The peptides were separated using an Ultimate 3000 LC RSLC nanoLC system (Dionex-Thermo Scientific, Chelmsford, MA, U.S.A.), and the mass spectrometry (MS) results were acquired with a quadrupole time-of-flight (Q-TOF) mass spectrometer (maXis impact, Bruker Daltonics Inc. Bremen, Germany). The sample was injected into a C18 trapping column (Acclaim PepMap100:100 μm × 2 cm, Dionex-Thermo Scientific, Chelmsford, MA, U.S.A.), connected with a C18 nano column (Acclaim PepMap RSLC, 75 μm × 15 cm, Dionex-Thermo Scientific, Chelmsford, MA, U.S.A.) to separate peptides. The mass spectrometer was operated by a captive spray source with a spray voltage of 1400 V, and the MS were acquired from 50 to 2000 m/z. The three most intense ions with charge states between 2 and 3 in each survey scan were selected for the MS/MS experiment. The MS/MS data were acquired from 50 to 2000 m/z. The peak list of MS spectra was processed by DataAnalysis v.4.1 software (Bruker Daltonics Inc. Bremen, Germany), and the extracted peak list was searched against the Swiss-Prot database (release 2011727) using MASCOT v.2.2.04 (Matrix Science, London, England). SOD Zymography and Activity Assay. The following protocol for SOD zymography was conducted as described by Shukla et al.15 with a slight modification. Gel electrophoresis is conducted exactly as the above-mentioned SDS-PAGE protocol, except the absence of SDS in the native gel. The native gel was then soaked twice in distilled water with gentle shaking for 30 min per time. The gel was

Purification of the PBL Protein of Interest. Unless otherwise indicated, the following protein purification protocol was performed at room temperature and initiated by homogenizing PBL in 3 volumes of buffer A (20 mM Tris/HCl, pH 7.0). The homogenate was filtrated with 4-layer cheesecloth and centrifuged at 13 000 rpm for 20 min at 4 °C, and the clear supernatant was filtrated again to remove insoluble residue on the surface of the supernatant. The proteins in the clear supernatant were fractionated with 80% ammonium sulfate and centrifuged to collect the precipitate. The precipitate was redissolved in buffer A and dialyzed against buffer A (50X volume) overnight. After repeating the dialysis step, the sample was loaded onto a DEAEcellulose column pre-equilibrated with buffer A. The column was then washed with buffer A until the eluent was colorless, as judged with vision. The proteins in the column were eluted in the linear gradient with 1 M NaCl in buffer A, and the protein eluent was subjected to 15% SDS-PAGE (8 × 10 cm2) at 110 V for 2 h. The fractions containing the protein of interest were pooled and diluted 5-fold with buffer A. The diluted sample was repeatedly subjected to the abovementioned DEAE-cellulose chromatography to purify the protein of interest, except 0.2 M NaCl replaced the 1 M NaCl in the linear gradient. The molecular mass of the purified protein was determined in the linear mode using MALDI-TOF-TOF MS, as previously described.13 Protein Identification by nano-LC-Q-TOF-MS/MS. The protein band on the SDS-PAGE gel was excised, followed by in-gel digestion and peptide extraction according to the protocol previously B



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subsequently incubated in 0.28 mM nitro-blue tetrazolium (NBT) solution in the dark with gentle shaking for 20 min. The gel was then immersed in HEPES buffer (50 mM, pH 7.9) with 28 mM TEMED and 28 μM riboflavin and gentle shaken in the dark for 15 min. A white band on the gel gradually appeared after exposure to light. Activity assay of SOD was conducted exactly as that suggested by the manufacturer. Reduction of WST-1 with superoxide anion, the product of xanthine oxidase-catalyzed reaction, was blocked in the presence of SOD. Absorbance at 450 nm was measured to determine the inhibition rate. In the experiment, 50- and 200-fold dilution for the crude extract and purified SOD, respectively, was used to perform activity assay. One unit/mL of activity was defined as 50% of inhibition rate calculated from the protocol in the kit. Identifying the Mediator of SOD Dimerization by HPLC and LC-MS/MS. Areca nut or PBL alone was chewed in the oral cavity, and the oral extracts were then collected and centrifuged. The smallmolecule components in the clear supernatant were filtrated using Microcon 3 concentrator, followed by HPLC analysis using a LiChroCART column (4 × 250 mm2) (Merck & Co., Inc., Whitehouse Station, NJ). The mobile phases of ddH2O and acetonitrile were used in a linear gradient at a flow rate of 1 mL/ min, and 1.5 mL of eluent was manually collected in each tube. The LC-MS/MS analysis was conducted exactly as previously described.13 Measurement of Hydrogen Peroxide in the Oral Extract. (+)-Catechin (0.5 mM) and 3 μL oral extract, from different preparations were mixed in a 200 μL Tris/HCl (0.1M, pH 7.0) reaction solution. No additional peroxidase was required because peroxidase is present in both areca nuts and PBL. In the presence of hydrogen peroxide, peroxidase catalyzed catechin oxidation to form a product that exhibited absorbance at 475 nm and was measured spectrophotometrically.13 SDDC (a Cu/Zn SOD-specific inhibitor) was employed to evaluate whether SOD was responsible for production of hydrogen peroxide. It was conducted similarly except preincubation of catechin with 5-mM SDDC in Tris/HCl buffer for 5 min. After addition of the oral extract, absorbance at 475 nm was measured spectrophotometrically.

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RESULTS Identification of the Purified PBL Protein of Interest as Cu/Zn SOD. As depicted in Figure 1A, a functionally unknown protein in PBL was purified to apparent homogeneity using repetitive DEAE-cellulose chromatography. It appeared to be monomeric according to the results of SDS-PAGE (Figure 1A) with a 15.7-kDa molecular mass as determined by MALDI-TOF analysis (Figure 1B). The protein was identified as Cu/Zn SOD. Sequence identities were recognized in two adjacent tryptic peptides, both of which were mapped to the Cterminal region of Cu/Zn SOD from Zantedeschia aethiopica, as shown in Figure 1C. Zymography of the native gel clearly established the protein’s SOD activity (Figure 1E). The pattern of Coomassie staining (Figure 1D) indicated that PBL SOD is monomeric at neutral pH. The process represented about 80fold purification, obtained from comparison of specific activity between the crude extract (60 U/mg) and purified SOD (4700 U/mg). SOD Dimerization in the Betel Nut Oral Extract. Zymography was used to examine the status of the SOD in the betel nut oral extract (BPS oral extract). Unexpectedly, the results showed nearly complete conversion of SOD monomer to the dimeric form in all 6 oral extracts (Figure 2A, arrowhead in lanes 1−6). Clearly, SOD was readily dimerized in the alkaline condition caused by slaked lime. Oral extract obtained after chewing PBL combined with areca nut in the absence of slaked lime (BP oral extract) induced SOD dimerization but to a much lesser extent than BPS oral extract (Figure 2A, lanes 7− 9). The conversion was also observed using betel nut

Figure 2. SOD dimerization induced by slaked lime. (A) Zymographic detection of SODs in the oral extract. The oral extract collected after chewing betel nut with slaked lime (BPS oral extract, lanes 1−6) or without slaked lime (BP oral extract, lanes 7−9). Equal volumes of BPS and BP oral extracts were analyzed, and the SOD dimer of interest is indicated by the arrowhead on the left. (B) The presence of SOD dimer in the betel nut homogenate. Betel nut was homogenized with Tris/HCl buffer (20 mM, pH 7.0) instead of oral chewing, and two doses of the homogenate (lanes 1 and 2) were used in a zymographic assay of SOD dimerization (arrowhead on the right). (C) The effect of BPS and BP oral extracts on SOD dimerization. Dimerization was assessed by mixing the purified SOD with a small amount of BPS (lane 3) or BP (lane 5) oral extract, as compared with an equal amount of enzyme alone (lane 1). Lanes 2 and 4 show BPS and BP oral extracts alone. (D) The effect of slaked lime on SOD dimerization. Slaked lime from a betel nut was dissolved in 10 mL ddH2O and centrifuged, and the clear supernatant was used immediately. Increasing amounts of the supernatant were mixed with a given amount of SOD (lanes 2−4) to assess SOD dimerization, as compared with the enzyme control (lane 1). Lanes 5−7 show the C



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addition of a small amount BP oral extract exerted the same effect on SOD dimerization, although to a lesser extent (lane 5). The disappearance of the monomeric SOD in Figure 2A,B is believed to be due to SOD dimerization in the presence of slaked lime. Next, the effect of slaked lime on SOD dimerization was studied. No dimerization was observed with SOD alone (Figure 2D, lane 1), but slaked lime induced SOD dimerization in a dose-dependent manner (Figure 2D, lanes 2−4). Usage of the supernatants, which contained even smaller amounts of slaked lime, resulted in no detectable SOD activity (lanes 5−7). BPS oral extract, which contains slaked lime, is alkaline (pH 8.9), and BP oral extract lacking slaked lime is acidic (pH 5.78). As shown in Figure 2E, the addition of alkaline solution to monomeric SOD induced SOD dimerization (lane 3 of both panels), mirroring the action of slaked lime (lane 4 of both panels), whereas the addition of neutral buffer had no effect (lane 2 of both panels).

Figure 2. continued supernatants alone corresponding to lanes 2−4, respectively. (E) The assessment of SOD dimerization was performed by separately mixing SOD with an equal volume of water (lane 1), 0.1 M Tris/HCl (pH 7, lane 2), 0.1 M Tris/HCl (pH 8.8, lane 3), or the slaked lime supernatant described above (lane 4). The left and right panels show zymography and Coomassie staining, respectively, with the monomer and dimer indicated.

homogenate (Figure 2B, arrowhead), which was derived from homogenizing the betel nut with neutral buffer rather than chewing betel nut in the oral cavity. In the presence of a small amount of BPS oral extract with a lower SOD activity (lane 2 of Figure 2C), the purified SOD remarkably dimerized and possessed at least a 5-fold increase in activity (lane 3) as compared with an equal amount of the monomeric SOD with low activity at neutral pH (lane 1). The

Figure 3. Mediator of SOD dimerization distinct from slaked lime. (A) The component in areca nut that mediates SOD dimerization. The assessment was performed by mixing SOD with saliva (lane 3), areca-nut (AN) oral extract (lane 5), or PBL oral extract (lane 7) and comparing with an equal amount of enzyme control (lane 1). Lanes 2, 4, and 6 show the controls for saliva and AN and PBL oral extracts, respectively. (B) The effect of AN oral extract on SOD dimerization. The effect of increasing amounts of AN oral extract (lanes 2−4) was compared with that of the enzyme control (lane 1). (C) HPLC chromatogram for small-molecule components in AN oral extract. The small-molecule components in AN oral extract were filtrated using Microcon 3, followed by HPLC analysis. The collected fraction for each time interval is shown at the bottom. (D) Each collected fraction, indicated at the top, was speed-vacuum-dried, and mixed with SOD to determine which fraction was able to mediate SOD dimerization. D



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Figure 4. Identification of the mediator in areca nut as arginine. (A) and (B) MS/MS spectrum (A) and extracted ion chromatogram (EIC) (B) of the active component in fraction 2 (upper panel) compared with that of L- and D-arginine (middle and lower panels). (C) The effect of L-arginine on SOD dimerization. SOD dimerization was evaluated in the presence 10 and 20 mM L-arginine (lanes 2 and 3) by zymography (left panel) and Coomassie staining (right panel) and compared with that observed with an equal amount of enzyme control (lane 1). (D) The assessment was extended to D-arginine (lanes 2 and 3) and other basic amino acids, including lysine (lanes 4 and 5), histidine (lanes 6 and 7), and citrulline (lanes 8 and 9). A final concentration of 10 mM (lanes 2, 4, 6, and 8) and 20 mM (lanes 3, 5, 7, and 9) of each amino acid was used, as compared with 20 mM L-arginine (lane 1).

Contribution of Arginine in Areca Nut to SOD Dimerization. An additional component in areca nut distinct from slaked lime contributes to SOD dimerization. When oral extract obtained by chewing areca nut alone was added, dimerization was still observed, although to a less extent than that initiated by slaked lime (Figure 3A, lane 5 vs lane 4). No conversion was detected with the saliva control (lane 3 vs lane 2) or oral extract after chewing PBL alone (lane 7 vs lane 6). However, the oral extract after chewing areca nut alone in an acidic environment stimulated dimerization in a dose-dependent manner (Figure 3B), reminiscent of slaked lime. Furthermore, HPLC analysis revealed a complicated pattern with numerous components in the microcon-3 filtrate of the active oral extract (Figure 3C). The manually collected fractions were examined, and the ability to induce SOD dimerization was stringently restricted to fraction 2 (Figure 3D). The MS/MS spectrum of the active component in fraction 2 with m/z 175 in the positive mode (Figure 4A, upper panel) was equivalent to that of L- and D-arginine (Figure 4A, middle and lower panels). As expected, the analysis of the extracted ion chromatogram (EIC) revealed the same retention time for the active component and both enatiomers of arginine (Figure 4B). As was observed for the enzyme control, 10 mM L-arginine exerted a faint effect that was insufficient to induce dimerization as examined by zymography and Coomassie staining (Figure

4C, lanes 1 and 2 in both panels). In contrast, a nearly complete conversion to the dimeric form occurred in the presence of 20 mM L -arginine (Figure 4C, lane 3 in both panels). Furthermore, D-arginine conferred a similar effect on SOD dimerization (Figure 4D, lanes 2 and 3 compared with Figure 4C, lanes 2 and 3). The absolute specificity of arginine in this process was also reflected in the fact that the other two basic amino acids histidine and lysine as well as arginine-analogous citrulline all lacked the ability to induce SOD dimerization (Figure 4D, lanes 4−9). Production of Hydrogen Peroxide in the Betel Nut Oral Extract. The nearly complete dimerization of SOD in the presence of betel nut oral extract greatly increased its enzymatic activity, potentially resulting in the production of substantial amounts of hydrogen peroxide in the oral cavity. An absorbance increase in response to catechin was observed only with the BPS oral extract (left three bars of Figure 5A); very little change of absorbance occurred with the BP oral extract (middle three bars), clearly indicating the presence of hydrogen peroxide in the BPS oral extract. The increased absorbance was regenerated when hydrogen peroxide was produced in large quantities (right three bars), indicating little or no production of hydrogen peroxide in the BP oral extract. To clarify the source of hydrogen peroxide, additional oral extracts were prepared by chewing PBL or areca nut in the presence of slaked lime and are referred to as PS and BS oral E



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Figure 5. PBL as the source of hydrogen peroxide. (A) BPS and BP oral extracts (left and middle three bars) evaluated for their ability to oxidize catechin. Hydrogen peroxide was used as a control, demonstrating the extremely small amount of hydrogen peroxide in the BP oral extracts (right three bars). (B) PS and BS oral extracts (left and right three bars) were prepared to determine the source of hydrogen peroxide. (C) Continuity for hydrogen peroxide production in the PS oral extract. Formation of hydrogen peroxide immediately ceased in the presence of SDDC, an SOD inhibitor.

was also reported by Klatt et al.18 Furthermore, SOD dimerization in the BP oral extract (Figure 2C, lane 5) was most likely due to the presence of arginine in the areca nut. Previous experiments in animals and cell cultures have demonstrated that arginine plays a role in preventing toxicity such as that induced by free radicals19,20 and may act through the synthesis of nitric oxide. These studies also revealed that arginine treatment alters the activities of antioxidant enzymes. Among them, the increased SOD activity20 might be attributed to the formation of SOD dimers. Based on our results, the increased SOD activity may be independent of nitric oxide formation (Figure 4C). Despite the protective roles of arginine and SOD, the simultaneous presence of both components in the oral cavity resulted in harmful effects, such as the production of hydrogen peroxide as described below. The self-association of arginine molecules in aqueous solution demonstrated its striking ability to stabilize proteins and prevent protein aggregation.21 Arginine monomers have been reported to hydrogen-bond to each other, forming a large cluster.22 The charged amino acids on the surface of SOD may interact with arginine in a cluster and concomitantly link two molecules of SOD together. Plant SODs play a defensive role by detoxifying superoxide radical to oxygen and hydrogen peroxide,23 and numerous studies have demonstrated the protective role of plant Cu/Zn SODs against oxidative stress.24,25 There is increasing evidence establishing hydrogen peroxide as a tumor promoter in carcinogenesis development.26,27 In this study, PBL together

extracts, respectively. As depicted in Figure 5B, a significant change in absorbance was only detected when PS was present, i.e., the SOD in PBL was involved in the production of hydrogen peroxide. SOD may be the primary source for the generation of hydrogen peroxide, as supported by the fact that addition of SDDC immediately blocked the ability of PS oral extract to oxidize catechin (Figure 5C).

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DISCUSSION SOD dimerization and enhancement of enzymatic activity has been previously reported in other studies, using recombinant SOD of sweet potato.16 Unlike other plant SODs, which exist in an equilibrium of monomers and dimers at neutral pH,16,17 purified PBL SOD was entirely monomeric (Figure 1D,E) and existed in the same form within the cell. When chewing betel nut, the presence of slaked lime exerted two significant effects. First, in the presence of slaked lime, significantly more proteins were extracted compared with using oral extract from chewing betel nut alone (Figure 2A). The protein(s) were mainly derived from PBL rather than the areca nut, with trace amounts of proteins that included peroxidase as the major protein constituent.13 Furthermore, the SOD of interest in the experiment was completely converted to the dimeric form in the presence of slaked lime, greatly enhancing its enzymatic activity in the oral cavity. SOD dimerization was further augmented by arginine in the areca nut, i.e., arginine was a distinct mediator of SOD dimerization. The involvement of arginine in SOD dimerization F



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(4) Murata, K.; Nakao, K.; Hirata, N.; Namba, K.; Nomi, T.; Kitamura, Y.; Moriyama, K.; Shintani, T.; Iinuma, M.; Matsuda, H. Hydroxychavicol: a potent xanthine oxidase inhibitor obtained from the leaves of betel, Piper betle. J. Nat. Med. 2009, 63, 355−359. (5) Pandey, A.; Bani, S.; Dutt, P.; Suri, K. A. Modulation of Th1/Th2 cytokines and inflammatory mediators by hydroxychavicol in adjuvant induced arthritic tissues. Cytokine 2010, 49, 114−121. (6) Young, S. C.; Wang, C. J.; Lin, J. J.; Peng, P. L.; Hsu, J. L.; Chou, F. P. Protection effect of piper betel leaf extract against carbon tetrachloride-induced liver fibrosis in rats. Arch. Toxicol. 2007, 81, 45− 55. (7) Bhide, S. V.; Shivapurkar, N. M.; Gothoskar, S. V.; Ranadive, K. J. Carcinogenicity of betel quid ingredients: feeding mice with aqueous extract and the polyphenol fraction of betel nut. Br. J. Cancer 1979, 40, 922−926. (8) Rai, M. P.; Thilakchand, K. R.; Palatty, P. L.; Rao, P.; Rao, S.; Bhat, H. P.; Baliga, M. S. Piper betel Linn (betel vine), the maligned Southeast Asian medicinal plant possesses cancer preventive effects: time to reconsider the wronged opinion. Asian Pac. J. Cancer Prev. 2011, 12, 2149−2156. (9) Ko, Y. C.; Huang, Y. L.; Lee, C. H.; Chen, M. J.; Lin, L. M.; Tsai, C. C. Betel quid chewing, cigarette smoking and alcohol consumption related to oral cancer in Taiwan. J. Oral Pathol. Med. 1995, 24, 450− 453. (10) Hossain, Md. M.; Pervin, F.; Absar, N. Purification, characterization and N-terminal sequence analysis of Betel leaf (Piper betle) invertase. J. Chin. Chem. Soc. 2011, 58, 389−397. (11) Shovon, M. S.; Sharma, S. C. D.; Roy, N. Purification, characterization and effect of physico-chemical agents on the stability of β-galactosidase from betel leaves. J. Biosci. 2010, 18, 108−115. (12) Gupta, S.; Gupta, S. M.; Sane, A. P.; Kumar, N. Chlorophyllase in Piper betle L. has a role in chlorophyll homeostasis and senescence dependent chlorophyll breakdown. Mol. Biol. Rep. 2012, 39, 7133− 7142. (13) Liu, Y. C., Chen, C. J., Lee, M. R., Li, M., Hsieh, W. T., Chung, J. G., Ho, H. C. Peroxidase as the Major Protein Constituent in Areca Nut and Identification of Its Natural Substrates. J. Evidence-Based Complementary Altern. Med. 2013, Article ID 412851. (14) Chang, C. T.; Liao, H. Y.; Chang, C. M.; Chen, C. Y.; Chen, C. H.; Yang, C. Y.; Tsai, F. J.; Chen, C. J. Oxidized ApoC1 on MALDITOF and glycated-ApoA1 band on gradient gel as potential diagnostic tools for atherosclerotic vascular disease. Clin. Chim. Acta 2013, 420, 69−75. (15) Shukla, M. R.; Yadav, R.; Desai, A. Catalase and superoxide dismutase double staining zymogram technique for Deinococcus and Kocuria species exposed to multiple stresses. J. Basic Microbiol. 2009, 49, 593−597. (16) Lin, C. T.; Lin, M. T.; Chen, Y. T.; Shaw, J. F. Subunit interaction enhances enzyme activity and stability of sweet potato cytosolic Cu/Zn-superoxide dismutase purified by a His-tagged recombinant protein method. Plant Mol. Biol. 1995, 28, 303−311. (17) Lin, C. T.; Kuo, T. J.; Shaw, J. F.; Kao, M. C. Characterization of the dimer-monomer equilibrium of the papaya Copper/Zinc superoxide dismutase and its equilibrium shift by a single amino acid mutation. J. Agric. Food Chem. 1999, 47, 2944−2949. (18) Klatt, P.; Schmidt, K.; Lehner, D.; Glatter, O.; Bächinger, H. P.; Mayer, B. Structural analysis of porcine brain nitric oxide synthase reveals a role for tetrahydrobiopterin and L-arginine in the formation of an SDS-resistant dimer. EMBO J. 1995, 14, 3687−3695. (19) Das, B.; Mandal, S.; Chaudhuri, K. Role of arginine, a component of aqueous garlic extract, in remediation of sodium arsenite induced toxicity in A375 cells. Toxicol. Res. 2014, 3, 191−196. (20) Saad, E. A. Kidney response to L-arginine treatment of carbon tetrachloride-induced hepatic injury in mice. Nat. Sci. 2013, 5, 1−6. (21) Shukla, D.; Trout, B. L. Interaction of Arginine with Proteins and the Mechanism by Which It Inhibits Aggregation. J. Phys. Chem. B 2010, 114, 13426−13438.

with the aid of slaked lime facilitated hydrogen peroxide production in the oral cavity (Figure 5A) in response to SOD. The antioxidants catechin and epicatechin in areca nut were in turn oxidized by hydrogen peroxide in the reaction catalyzed by peroxidases of both PBL and areca nut.13 In this case, both antioxidants were completely consumed and undetectable in the BPS oral extract, as shown in the Supporting Information (Figure S1). In contrast, both antioxidants were still present in the BP oral extract due to the low concentration of hydrogen peroxide. The hypothesis that hydrogen peroxide attacks and damages oral tissue is clearly supported by other reports, demonstrating hydrogen peroxide as a mediator of inflammation.28,29 It was noteworthy that the formation of hydrogen peroxide in the oral cavity was immediately blocked by adding the inhibitor SDDC (Figure 5C), i.e., SOD activity was blocked and thus the production of H2O2 was prevented. This clearly indicates that hydrogen peroxide is continuously produced when betel nut is chewed uninterrupted, as is the case with most consumers. Nair et al. reported the effect of slaked lime on the production of reactive oxygen species (ROS) and its involvement in the development of oral cancer.30,31 Taken together with our results, we conclude that slaked lime mediates the production of ROS, which is subsequently converted to hydrogen peroxide via SOD action. Therefore, we conclude that PBL SOD plays a role in oral carcinogenesis through the continuous formation of hydrogen peroxide in the oral cavity.

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ASSOCIATED CONTENT

S Supporting Information *

Figure showing the detection of antioxidants catechin and epicatechin in the BPS and BP oral extracts. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*Tel.: +886 4 22840837 ext 215. Fax:+ 886 4 22862073. Email: [email protected] (Y.-C.L.). *Tel.: +886 4 22053366 ext 2103. Fax: +886 4 22053764. Email: [email protected] (H.-C.H.). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED SOD, superoxide dismutase; DART-MS, direct analysis in real time mass spectrometry; PBL, Piper betle leaf; SA, sinapinic acid; SDDC, sodium diethyldithiocarbamate trihydrate; HCCA, α-cyano-4-hydroxycinnamic acid; DEAE, diethylaminoethyl; BPS, betel nut with slaked lime; BP, betel nut without slaked lime; PS, PBL with slaked lime; BS, areca nut with slaked lime; AN, areca-nut

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REFERENCES

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Zn Superoxide Dismutase from Piper betle Leaf and

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