Radical scavenging effects of 1-naphthol, 2-naphthol, and their sulfate-conjugates

Abstract

1-Naphthol (1-Nap) and 2-naphthol (2-Nap) are phenolic isomers that may be subjected to sulfate conjugation in vivo. Phase-II sulfate conjugation of phenolic compounds is generally thought to result in their inactivation. This study aimed to investigate the antioxidative effects of 1-NapS and 2-NapS, in comparison with their unsulfated counterparts, using established free radical scavenging assays. Based on the calculated EC₅₀ values, 1-NapS resulted in 5.60 to 7.35-times lower antioxidative activity than 1-Nap. In contrast, 2-NapS showed comparable activities as did the unsulfated 2-Nap. Collectively, the results obtained indicated that sulfate conjugation of the Nap isomers did not always result in the decrease of their antioxidant activity, and the antioxidant activity that remained appeared to depend on the position of sulfation.

INTRODUCTION

In vertebrates, sulfate conjugation constitutes a major pathway in the biotransformation and excretion of xenobiotics as well as the homeostasis of key endogenous compounds such as catecholamines and steroid/thyroid hormones (Mulder and Jakoby, 1990; Falany and Roth, 1993; Weinshilboum and Otterness, 1994). The responsible enzymes, the so-called “cytosolic sulfotransferases (SULTs)”, catalyze the transfer of a sulfonate group from the active sulfate, 3'-phosphoadenosine 5'-phosphosulfate, to an acceptor substrate compound containing a hydroxyl or an amino group (Lipmann, 1958). Sulfate conjugation generally leads to the inactivation of the substrate compounds and/or the increase in their water-solubility, thereby facilitating their removal from the body (Mulder and Jakoby, 1990; Falany and Roth, 1993; Weinshilboum and Otterness, 1994). However, for certain compounds such as hydroxyarylamines, sulfate conjugation has been shown to result in their activation to become chemically reactive carcinogens (DeBaun et al., 1970). In regard to the metabolism of xenobiotics, sulfate conjugation may therefore act as a double-edged sword, being beneficial in the detoxification of some and deleterious in the activation of others. It is therefore an important toxicological issue whether upon sulfate conjugation, a xenobiotic compound may become inactivated or, instead, activated.

Naphthalene is an industrial chemical commonly used as a fumigant or as a pesticide (Sudakin et al., 2011). In the human body, naphthalene has been reported to undergo hepatic metabolism under the action of cytochrome P450 enzymes, generating 1-naphthol (1-Nap) and 2-naphthol (2-Nap) that carry a hydroxyl group at, respectively, the 1- and 2-position of the naphthalene ring (Fukami et al., 2008) (see Fig. 1 for chemical structures). Notably, 1-Nap and 2-Nap may further be metabolized, particularly through sulfate conjugation in vitro and in vivo (Isozaki and Tamura, 2001; Ayala et al., 2015). Incidentally, in drug metabolism research, 1-Nap and 2-Nap are often used as prototype substrates for certain human SULTs, e.g., SULT1A1, SULT1A3, and SULT1E1 (Allali-Hassani et al., 2007). Glucuronide and sulfate conjugates of Nap have been shown to be metabolites generated upon exposure to Nap and/or naphthalene (Déchelotte et al., 1993; Ayala et al., 2015). A recent study demonstrated the presence of additional naphthalene metabolites including mercapturic acid and N-acetyl glutathione conjugate in urine (Ayala et al., 2015). In industry, 1-Nap and 2-Nap per se have been used in the preparation of dyes, perfumes, insecticides, and pharmaceutical compounds such as antiseptic skin medications, as well as in the production of rubber for antioxidant purposes (Dorland, 2011; Shindy, 2016). In view of their widespread occurrence and thus the risk for human exposure, it is an interesting question whether sulfate conjugation, generating 1-NapS and 2-NapS, may play a role in influencing the biological activities/effects of 1-Nap or 2-Nap, particularly in regard to their antioxidant capacity. With regard to potential biochemical effects of Nap, 1-Nap has been reported to be a phenolic reactive reagent against a peroxyl radical (Lissi et al., 2000). Since there is currently limited information available concerning the biochemical effects of xenobiotics upon sulfate conjugation, we were interested in using Nap isomers as model compounds for determining the effects of sulfate conjugation on their antioxidant activity.

Fig. 1

Chemical structures of Nap isomers and their derivatives used in this study.

In this study, we quantitatively evaluated the antioxidative effects of 1-NapS and 2-NapS using three different free radical scavenging assays. Representative synthetic free radicals, including 1,1-diphenyl-2-picryl hydrazyl (DPPH) radical (Blois, 1958), 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) cation (ABTS⁺) radical (Thaipong et al., 2006), and chlorpromazine cation (CPZ⁺) radical (Nagaraja et al., 2014), were used as free radicals for the determination of antioxidative capacity of test samples. These free radical assays were chosen they have been shown to be particularly useful for examining the antioxidant effects of agricultural products as well as industrial phenolic compounds (Sugahara et al., 2015). The antioxidative capacity of 1-NapS and 2-NapS was analyzed in comparison with unsulfated 1-Nap and 2-Nap, respectively.

MATERIALS AND METHODS

Materials

Naphthalene, 1-Nap, ABTS, DPPH, potassium peroxodisulfate, phosphoric acid were products of Nacalai Tesque Inc. (Kyoto, Japan). 1-NapS was from Research Organics Inc. (Cleveland, OH, USA). 1-Methoxynaphthalene (1-NapM), 2-Nap, and potassium dichromate were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). 2-NapS was a product of Alfa Aesar (Ward Hill, MA, USA). 2-Methoxynaphtalene (2-NapM) and CPZ were products of Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). (±)-6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox) was from Sigma-Aldrich, Co. (St Louis, MO, USA). All other chemicals were of the highest grade commercially available. Figure 1 shows the chemical structures of Nap isomers and their derivatives used in this study.

DPPH radical scavenging assay

The DPPH radical scavenging activity was measured based on the method of Blois (Blois, 1958). Briefly, the reaction was started by the addition of 0.5 mM DPPH dissolved in ethanol (50 µL) to the pre-assay mixture (200 µL) containing varying concentrations of a test sample (10 µL), 70% ethanol (90 µL), and 0.1 M sodium acetate buffer (pH 5.5, 100 µL). The reaction, in a final volume of 250 µL, was allowed to proceed for 30 min at room temperature. The absorbance of the resulting solution was measured at 517 nm. Trolox was used as a control standard.

DPPH radical scavenging activity (%) = [1-{(A_Sample-A_Blank)/(A_Control-A_Blank)}]x100

where A_Sample is the absorbance measured in the presence of the test sample and A_Blank is the absorbance measured in the presence of neither the tested sample nor the synthetic radical solution. A_Control is the absorbance measured in the absence of the sample.

ABTS⁺ radical scavenging assay

The ABTS⁺ radical scavenging activity was measured based on the method of Thaipong et al. (2006). Briefly, ABTS-mixture solution was first prepared by mixing equal volumes of 7.4 mM ABTS and 2.6 mM potassium peroxodisulfate solutions by rotation for 15-hr in the dark at room temperature. Thereafter, ABTS-working solution was prepared by diluting the ABTS-mixture solution (150 µL) in methanol (2.9 mL). The reaction was started by the addition of the ABTS-working solution (190 µL) to varying concentrations of a test sample (10 µL). The reaction, in a final volume of 250 µL, was allowed to proceed at room temperature for 2 hr in the dark. The absorbance of the resulting solution was measured at 734 nm. Trolox was used as a control standard.

ABTS⁺ radical scavenging activity (%) = [1-{(A_Sample-A_Blank)/(A_Control-A_Blank)}]x100

where A_Sample is the absorbance measured in the presence of the test sample and A_Blank the absorbance measured in the presence of neither the tested sample nor the synthetic radical solution. A_Control is the absorbance measured in the absence of the sample.

CPZ⁺ radical scavenging assay

The CPZ⁺ radical scavenging activity was measured based on the method of Nagaraja et al. (2014). Briefly, the reaction was started by the addition of 0.5 mM chromium(VI) solution (10 µL) to the pre-assay mixture (240 µL) containing varying concentrations of a test sample (12.5 µL), 10 mM CPZ dissolved in ethanol (20 µL), 1:1 phosphoric acid plus ethanol mixture (200 µL), and ethanol (7.5 µL). The reaction, in a final volume of 250 µL, was allowed to proceed for 10 min at room temperature. The absorbance of the resulting solution was measured at 530 nm. Trolox was used as a control standard.

CPZ⁺ radical scavenging activity (%) = [1-{(A_Sample-A_Blank)/(A_Control-A_Blank)}]x100

where A_Sample is the absorbance measured in the presence of the test sample and A_Blank the absorbance measured in the presence of neither the tested sample nor the synthetic radical solution. A_Control is the absorbance measured in the absence of the sample.

Preparation of liver microsomal fraction and lipid-peroxidation-suppressing assay

To prepare the liver microsomal fraction, 10 g of commercial chicken liver was homogenized at 4°C in 10 mL of 0.25 M sucrose solution and subjected to centrifugation at 12,000 x g for 15 min. The supernatant collected was further centrifuged at 105,000 x g for 60 min. The microsomal pellet was suspended in 50 mM Tris-HCl buffer (pH 7.4). Protein concentration of the microsomal fraction thus prepared was determined based on the method of Bradford with BSA as the standard. (Bradford, 1976).

Lipid-peroxidation-suppressing activity of the liver microsomal fraction was measured based on an established procedure (Miyazawa and Nakagawa, 1998). Briefly, the reaction was started by the addition of 1.0 mM NADH (13.5 µL) to the pre-assay mixture (136.5 µL) containing varying concentrations of a test sample (7.5 µL), 0.691 mg/mL microsomal fraction (82 µL), ADP-FeCl₃ (12.3 µL; 22 mM ADP and 1.3 mM FeCl₃ in 50 mM Tris-HCl buffer (pH 7.4)), and 50 mM Tris-HCl buffer (pH 7.4) (34.7 µL). The reaction, in a final volume 150 µL, was allowed to proceed for 20 min at 37°C, and terminated by the addition of TCA/TBA/HCl solution (300 µL; trichloroacetic acid, 1,000 mg and 2-thiobarbituric acid, 37.5 mg in 10 mL of 0.25N hydrochloric acid), followed by heating at 100°C for 15 min and centrifugation at 13,000 x g. The absorbance of the supernatant collected (200 µL) was measured at 532 nm.

Statistical analysis

For statistical analysis, the values were expressed as mean ± standard deviation as calculated from the data derived from four experiments. Data in part were analyzed using statistical add-on software program (Statcel, OMS Co., Saitama, Japan) for Excel 2004 (Microsoft Co., Redmond, WA, USA). With a one-factor analysis of variance (ANOVA), a post-hoc Bonferroni-Dunn test was conducted for the multiple comparison and differences at P < 0.05 were considered significant.

RESULTS AND DISCUSSION

The current study was designed to determine the antioxidative effects of O-sulfated 1-Nap and 2-Nap and compare them with their unsulfated counterparts. Three different free radical scavenging assays were employed in this study.

Determination of free radical scavenging activities of sulfated vs. unsulfated 1-Nap and 2-Nap

In a previous report, 1-Nap was used as a reactive reagent for a peroxyl radical assay model (Lissi et al., 2000). Because the biochemical effects of Nap isomers and their sulfated derivatives have not been systematically investigated, we first investigated the potential antioxidative capacity of these compounds in several free radical scavenging assays. To evaluate their antioxidative effects, 1-NapS vs. 1-Nap and 2-NapS vs. 2-Nap were tested at different concentrations in three different free radical assays. DPPH is a stable free radical with a deep violet color, which becomes colorless upon reduction by proton-donating antioxidants (Blois, 1958; Molyneux, 2004). As shown in Fig. 2A, 1-NapS showed a lower DPPH radical scavenging activity (EC₅₀; 172 µM) than unsulfated 1-Nap (EC₅₀; 23.4 µM). In contrast, 2-NapS displayed a DPPH radical scavenging activity (EC₅₀; 29.8 µM) comparable to that of 2-Nap (EC₅₀; 22.6 µM) (Fig. 2B).

Fig. 2

Antioxidative effects of Nap isomers and their sulfate-conjugate in DPPH (A and B), ABTS⁺ (C and D), and CPZ⁺ radical scavenging assays (E and F). 1-Nap (gray circle) and 1-NapS (black circle) were assayed in panels A, C, and E, while 2-Nap (gray circle) and 2-NapS (black circle) were used in panels B, D, and F. Data shown represent mean ± S.D. from four experiments. Trolox (white triangle) was used as a control standard. Naphthol; Nap, naphthyl sulfate; NapS.

ABTS, a stable cation radical, has been widely used to screen the free radical scavenging capacity of both lipophilic and hydrophobic antioxidant that display electron and hydrogen-donating properties (Pellegrini et al., 1999; Re et al., 1999; Thaipong et al., 2006). A similar trend was observed for the two sets of test compounds in ABTS⁺ radical scavenging assays performed. As shown in Fig. 2C, 1-NapS demonstrated lower ABTS⁺ radical scavenging activity (EC₅₀; 75.4 µM) than that (EC₅₀; 11.2 µM) of 1-Nap, whereas 2-NapS and 2-Nap showed comparable strong activities (with EC₅₀ at 10.4 and 11.2 µM, respectively) (Fig. 2D).

CPZ, a neuroleptic drug used for treating schizophrenia, has been shown to form a stable cation radical under acidic conditions upon oxidization by chromium(IV) (Nagaraja et al, 2014) or Fe(III) (Miftode et al., 2010), and thus has been used for analyzing antioxidation capacity of electron transferring compounds. As shown in Fig. 2E, 1-NapS demonstrated a lower CPZ⁺ radical scavenging activity (EC₅₀; 117 µM) than that (EC₅₀; 20.9 µM) of 1-Nap in the CPZ⁺ radical scavenging assay. While 2-NapS and 2-Nap failed to achieve EC₅₀ for all concentrations tested (Fig. 2F), 2-NapS and 2-Nap displayed comparable but lower activities (43.2 ± 4.8% and 43.2 ± 2.7%, respectively) at a maximum concentration of 1,000 µM.

Table 1 shows the EC₅₀ values of Nap and NapS isomers and trolox determined using the three radical scavenging assays. Interestingly, 1-Nap and 2-Nap showed comparable activity in DPPH radical scavenging assay (EC₅₀; 23.4 µM vs. 22.6 µM, respectively). A similar trend was observed in ABTS⁺ radical scavenging assay (EC₅₀; 11.2 µM vs. 11.2 µM, respectively). On the other hand, the activity of 2-Nap toward CPZ⁺ radical (EC₅₀; > 1,000 µM) was lower than that of 1-Nap (EC₅₀; 20.9 µM). These data implied that the antioxidant capability of 2-Nap might be lowered under acidic environment because the CPZ⁺ radical scavenging assay was carried out in the presence of phosphoric acid. The ratio of EC₅₀ values (or called the ‘1/Ratio of EC₅₀ values’) from two test compounds reflects the quantitative difference between their activities. The calculated ratio of EC₅₀ values (Table 1) indicated that the DPPH, ABTS⁺, and CPZ⁺ radical scavenging activities of 1-NapS were 5.60 to 7.35-times lower than that of 1-Nap. These results are in line with the general perception that sulfation represents a deactivation pathway. It is noted that the radical scavenging capability of 1-NapS amounted to 0.136 to 0.179-fold level of that of 1-Nap. In contrast, 2-NapS showed comparable effects as 2-Nap in DPPH and ABTS⁺ radical scavenging assays. The calculated ‘1/Ratio of EC₅₀ values’ indicated that the radical scavenging capacity of 2-NapS ranged 0.758 to 1.08-fold of that of 2-Nap in these assays. Although the EC₅₀ values of 2-NapS and 2-Nap could not be determined in the CPZ⁺ radical scavenging assay, comparable effects of these two compounds were found.

Table 1. EC₅₀ values and ratio of EC₅₀ values of Nap isomers and their sulfate conjugate as determined in three different free radical scavenging assays.*

	DPPH Radical			ABTS+ Radical			CPZ+ Radical
	EC50 (µM)**
1-Nap	23.4	±	0.7a	11.2	±	0.3a	20.9	±	0.4a
1-NapS	172	±	2b	75.4	±	1.1b	117	±	18b
2-Nap	22.6	±	0.5a	11.2	±	0.5a	>1,000
2-NapS	29.8	±	0.4c	10.4	±	0.7a	>1,000
Trolox	31.1	±	0.8c	28.4	±	0.4c	17.2	±	0.2a

	Ratio of EC50 values***
1-NapS/1-Nap	7.35 (0.136)	6.73 (0.149)	5.60 (0.179)
2-NapS/2-Nap	1.32 (0.758)	0.929 (1.08)	-
1-Nap/2-Nap	1.04 (0.962)	1.00 (1.00)	-
1-NapS/2-NapS	5.77 (0.173)	7.25 (0.138)	-

*Data shown represent mean ± S.D. from four experiments and values not sharing a common superscript letter are considered significantly different at P < 0.05. Trolox was used as a control standard.

**EC₅₀ values indicate effective concentration at which the activity was 50% from the data obtained in Fig. 2.

***Data shown in parentheses indicate calculated ‘1/Ratio of EC₅₀ values’.

Naphthol; Nap, naphthyl sulfate; NapS.

How 1-NapS could scavenge radicals and donate protons to the synthetic radicals still remains unclear. One possibility is that the chemicals used in this study may be substances possessing no radical scavenging activities and/or that the solvent used may interfere with the reactions. DPPH is a stable synthetic radical that occurs spontaneously in ethanolic solvent prior to starting the scavenging assay. ABTS⁺ radical was thoroughly prepared overnight in the presence of peroxodisulfate prior to starting the assay. In contrast, the CPZ⁺ radical scavenging assay was started by the addition of chromium(VI) in the assay mixture, concomitantly with the generation of CPZ⁺ radical. A pilot experiment on 2-NapS using a pre-mixed CPZ in the presence of chromium(VI) demonstrated similar results. Therefore, it is conceivable that 2-NapS may not directly interfere with the generation of DPPH, ABTS⁺, and also CPZ⁺ radicals in these assays. In this study, 1-Nap, 2-Nap, 1-NapS and 2-NapS tested in the radical scavenging assays were dissolved in DMSO. We observed that there were: a) no precipitates or crystals of these four chemicals were observed as dissolved in DMSO or in each of the three different reaction mixtures, b) no clear change of pH in the presence of these four chemicals in reaction mixture, and c) negative control with DMSO only in these three radical scavenging assays showed results comparable to those found with MilliQ water. Thus, it appeared that the solvent may not directly interfere with the determination of radical scavenging activities of Nap and NapS isomers in these assays.

It is at present unclear why 1-NapS vs. 1-Nap and 2-NapS vs. 2-Nap showed different results in radical scavenging assays. The sulfate group of NapS is attached to a sp² carbon of the benzene ring in a double bond situation. In some cases, the sulfate anions of sulfated compounds can act as good leaving groups to form resonance-stabilized intermediates, except for sulfated phenol (Yi et al., 2006). Whether the sulfate group at specific positions of the Nap molecule may form resonance-stabilized intermediates or act as proton/electron/hydrogen donor to stabilize targeting free radical(s) will be an issue for further investigation. Further investigation on the O-H bond dissociation energy/enthalpies as well as stability of the target group at different position may provide useful clues (Pino et al., 2006; Nantasenamat et al., 2008). To gain insight into the stoichiometric relationship, we proceeded to determine the stoichiometric values of test samples as previously reported (Brand-Williams et al., 1995). Based on that the anti-radical power being defined as 1/EC₅₀ in the number of moles of test sample/mol of DPPH radical, the stoichiometric values of 1-Nap, 1-NapS, 2-Nap, 2-NapS and trolox were calculated to be 0.468, 3.45, 0.451, 0.596, and 0.621, respectively. Among the five compounds, 1-NapS showed a relatively higher value than the others. These results indicated that the number of DPPH radicals reduced by 1-NapS was much lower than those reduced by others. It is noted that a variety of phenolic sulfates were reported to be present in human plasma upon ingestion of a mixed berry fruit puree (Pimpão et al., 2015). While sulfation may play a role in diminishing the biological activities of these phenolic compounds, the degree of the actions of individual sulfated metabolites may be worth further investigation.

Structure specificity of Nap derivatives on the free radical assays

To gain insight into the structural determinants of the free radical scavenging activity of Nap and NapS, Nap derivatives including 1- and 2-methoxynaphthalene (1-NapM and 2-NapM) and naphthalene were tested at 100 and 1,000 µM concentrations in the three free radical scavenging assays described above. As shown in Fig. 3A, 1-NapM, 2-NapM and naphthalene showed lower DPPH radical scavenging activities compared with 1-NapS and 2-NapS. These results implied that the free radical scavenging activity of 1-Nap depends on the -OH group at 1-position of the naphthalene ring, and the activity may be diminished by O-sulfation or methoxylation. Interestingly, the free radical scavenging activity of 2-Nap, which seemed to depend on the -OH group at 2-position of the naphthalene ring, was diminished by methoxylation, but not by O-sulfation. The DPPH radical scavenging activities of 2-NapS and 1-NapS were relatively higher than those of 1-NapM, 2-NapM, and naphthalene. It therefore appears that certain O-sulfated form, but not the methoxylated form, of phenolic compounds may retain antioxidant capacity. A similar trend was observed when these compounds were tested in ABTS⁺ radical scavenging assay (Fig. 3B). It is noted that in Figs. 3B and 3C, 1-NapM, 2-NapM, and naphthalene showed the lowest activity. Intriguingly, the antioxidant activities of 1-NapS and 2-NapS were significantly higher than those of 1-NapM, 2-NapM, and naphthalene in these free radical assays.

Fig. 3

Antioxidative effects of Nap isomers and their derivatives in DPPH (A), ABTS⁺ (B), and CPZ⁺ radical scavenging assays (C). Activities were determined with each compound at 100 µM (gray) and 1,000 µM (black). Data shown represent mean ± S.D. from four experiments and values not sharing a common superscript letter (small at 100 µM, or capital at 1,000 µM) are considered significantly different at P < 0.05. Trolox was used as a control standard. Naphthol; Nap, naphthyl sulfate; NapS, methoxynaphthalene; NapM.

An important issue is with regard to determining the biological relevance of sulfate-conjugated metabolites. 1-Nap and 2-Nap and their sulfated form, 1-NapS and 2-NapS, were selected as model compounds in this study. As noted earlier, 1-Nap and 2-Nap are often used as prototype substrates for some SULTs in Phase-II drug metabolism research (Allali-Hassani et al., 2007; Arand et al., 1987). In the human body, urinary Nap has been proposed as a biomarker for the exposure to carbaryl (carbamate insecticide; 1-naphthyl methylcarbamate) applied in a variety of crops (Sams, 2017), and for workers occupationally exposed to naphthalene (Preuss et al., 2003, 2004; Sams, 2017). Sulfate conjugation of xenobiotics by SULTs may alter the physico-chemical properties of those compounds that are bioactive, and is widely assumed to result in the inactivation of their biological activity (Mulder and Jakoby, 1990; Falany and Roth, 1993; Weinshilboum and Otterness, 1994). In a previous paper, possible effects of metabolic conjugation reactions on the biological activity of phenolic flavonoids were discussed (Beekman et al., 2012). Although only a limited amount of information is currently available, Phase II conjugation reactions including glucuronidation, methoxylation, and sulfation seem not always result in the complete inactivation of the actions of flavonoids. Nevertheless, the radical scavenging assays we tested in this study were all simple chemical reaction models in vitro. It will be helpful in the future to determine the antioxidative activity of the test samples using more biologically relevant assays. To investigate lipid-peroxidation-suppressing capability of 2-Nap and 2-NapS, we performed an additional experiment using the microsomal fraction prepared from the chicken liver. Varying concentrations of 2-Nap and 2-NapS incubated in the presence of the chicken liver microsomal fraction resulted in comparable suppressing activities with EC₅₀ value of 254 ± 26 µM and 245 ± 6 µM, respectively (figure not shown). The comparable antioxidative activity of 2-Nap and 2-NapS was thus demonstrated in this lipid-peroxidation assay as well. The results presented in this study provided another example that sulfate conjugation of Nap does not always lead to the decreased biological activity of sulfated products. For Nap isomers, the remaining antioxidant activity may depend on the position of sulfate conjugation.

In conclusion, we reported in this communication the antioxidative effects of 1-NapS and 2-NapS determined using three different free radical scavenging assays. Based on the calculated EC₅₀ values, 1-NapS resulted in 5.60 to 7.35-times lower antioxidative activity than unsulfated 1-Nap. In contrast, 2-NapS showed antioxidative activities comparable to the unsulfated 2-Nap. These findings provided additional evidence that sulfate conjugation does not always lead to decreased biological/biochemical activity and the remaining activity may depend on the position of sulfation. The current study using sulfated Nap isomers as model O-sulfated phenolic compounds demonstrated clearly this interesting and important issue.

ACKNOWLEDGMENTS

This work was supported in part by Grant-in-Aid for Young Scientists (B) (JSPS KAKENHI Grant Numbers 23780143 and 15K18700), Research and Study Program/Project of Tokai University Educational System General Research Organization (Kanagawa, Japan).

Conflict of interest

The authors declare that there is no conflict of interest.

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