Deodorizing Effects of Phlorotannins from Edible Brown Alga Eisenia bicyclis on Methyl Mercaptan

In search for new deodorizing compounds, we were identified three phlorotannins; eckol (1), dioxinodehydroeckol (2) and dieckol (3) from edible brown seaweed Eisenia bicyclis and characterized by Fast-atom bombardment mass spectrometry (FAB-MS) and nuclear magnetic resonance (NMR). The deodorizing activity of those compounds was evaluated against methyl mercaptan, which is well known as a major causative material of halitosis and off-flavor. Among them, compound 3 exhibited the highest deodorizing activity against methyl mercaptan at the IC50 values of 26.71 ± 4.16 μg mL, followed by compound 2 (40.57 ± 0.63 μg mL) and compound 1 (43.62 ± 1.52 μg mL). Thus, these results suggest that the phlorotannins derived from E. bicyclis can be an effective deodorizing constituent in the food industry and pharmaceutical industries.


Introduction
Volatile sulfide compounds (VCSs) such as hydrogen sulfide, dimethyl sulfide, and methyl mercaptan have been known as major compounds causing off-flavor from foods and the industrial sites (Tanabe et al., 2012).It is generated through an enzymatic modification of sulfur containing amino acids (cysteine and methionine), which are made available following proteolytic degradation of proteins (Hughes & McNab, 2008;Ali & Nozaki, 2007).The tongue coating consists of desquamated epithelial cells, food debris, bacteria and salivary protein, which provide all the elements for production of volatile sulfide compounds and cause halitosis from the mouth (Gnanasekhar, 2007;Youngnak-Piboonratanakit and Vachirarojpisan, 2010).It has been also known that there is a highly positive correlation between the concentration of methyl mercaptan and the generation of halitosis (Kim et al., 2010;Richter, 1996).Since VSCs are the major causes of the malodor, suppression in production of the VSCs is an effective strategy to prevent the oral malodor.(Du et al., 2012;Sanz et al., 2001) Recently, there has been a growing interest in investigating natural plant extracts as efficacious and safe substances for oral care.More specifically, natural plant extracts such as thyme, rosemary, tea, marine algae, mongolian bark and sage has shown potent qualities as a natural anti-halitosis ingredient (Lourith & Kanlayavattanakul, 2010;Dodds et al., 2009;Lee et al., 1999).Although the function mechanism of the deodorizing effect of phlorotannins has not been unclear, it is assumed that the mechanism of the deodorant action of phenolic compounds in natural extracts is that these compounds are oxidized by oxygen in the atmosphere to form a highly reactive quinone structure (Hiramoto et al., 2001).It is considered that methylmercaptan might relate to the conversion of phenolic hydroxyl group into quinone and the subsequent formation of thio ether with benzene ring due to the nucleophilic addition of methylthio group (Hiramoto et al., 2006).
Among marine algae, edible brown seaweeds have been identified as a potential deodorizer that may be useful in the food industry (Kim et al., 2008).Furthermore, brown algae contain various minerals and dietary fiber, brown algae are widely eaten in East asia.Brown algae such as Eisenia bicyclis, Ecklonia stolonifera, E. cava, and Hizikia fusiformis were reported to contain a high amount of phlorotannins.Phlorotannins, the polymerization of phloroglucinol, are called seaweed polyphenol (Kim et al., 2006;Ragan & Glombitza, 1986).According to recent reports, phlorotannins have known to possess a variety of physiological activities such as antioxidation (Kang et al., 2005;Kang et al., 2007;Zou et al., 2008;Yoon et al., 2011;Eom et al., 2011a), antidiabetic effects (Eom et al., 2012b;Okada et al., 2004), antidementia (Yoon et al., 2008), antimicrobial activity (Eom et al., 2011b), anti-coagulant activity (Jeong et al., 2009), anti-inflammatory activity (Kang et al., 2012), anticonvulsant, and sleep inducer (Cho et al., 2012).However, it has not been investigated on deodorizing activity of isolated compounds from edible brown algae against methyl mercaptan.Therefore, in this study, we isolated its active compounds from E. bicyclis and investigated their effect against methyl mercaptan associated with halitosis.

Materials
E. bicyclis was gathered at Ulleung Island, Korea on September 2010, washed in fresh water to remove undesirable materials.Dried E. bicyclis was ground and then finely powdered with a food mixer (HMF-1000A; Hanil Electronics, Seoul, Korea).The ground powder was stored at 4°C until use.All reagents used in this study were analytical grade.

Preparation of Standard Methyl Mercaptan Solution
Two mL of authentic methyl mercaptan (1 μg μL -1 ) in benzene (Wako Pure Chem., Osaka, Japan) was dissolved in 198 mL of ethanol to be kept at -70°C in a deep-freezer (Samwon Freezing Engineering Co., Busan, Korea).The solution was then diluted to 1 μg mL -1 in distilled water to be used for assay of deodorizing activity.

Determination of Total Phenolic Compounds
The total phenolic contents was determined by the method of Folin-Ciocaleu with slight modifications (Waterman and Mole, 1994).One hundred mL of dilluted sample and 0.5 mL of 1 N Folin-Ciocalteu reagent (Sigma Co., St. Louis, USA) were put in the eppendorf tube and mixed up.The solution was incubated for 3 min at room temperature and then added to 0.4 mL of 7.5% Na 2 CO 3 .the mixture was allowed to stand for 90 min in a darkroom at room temperature and then centrifuged at 1,600 × g for 8 min.After centrifugation process, the supernatant was measured in terms of optical density at 765 nm by using a GENios® microplate reader (Tecan Austria GmbH; Grödig, Austria).The concentration of the total phenolic contents were calculaed using a calibration curve of phloroglucinol (Sigma Co., St. Louis, USA).Total phenolic contents was calculated using the linear equation from a calibration curve and expressed as mg phloroglucinol equivalent (PGE)s g -1 of dry weight.

Deodorizing Activity Assay
Deodorizing activity was evaluated by the method of Tokita et al (1984) with slight modifications.Sample (0.1 mL of different concentrations) and 0.9 mL of 0.2 M potassium phosphate buffer (pH 7.5) were put in the 30 mL of vial, and added 1 mL of standard methyl mercaptan solution (1 μg mL -1 ).The vial was immediately sealed up and agitated with a Vortex mixer (Vortex GENIE 2; Scientific industries, Inc., Bohemia, USA) for 5 sec, and then incubated for 6 min at 37°C.The methyl mercaptan was liberated into the headspace of the vial was extracted with a gas tight syringe (250 μL SYR; Hamilton Co., Reno, USA) and then analyzed with a gas chromatography (GC; HP model 5890, Hewlett-Packard Co., Palo Alto, USA) equipped with a flame photometric detector.Operating conditions of GC are as follows; HP-1 column (5 m×0.53 mm×2.65 μm; Hewlett-Packard Co., Palo Alto, CA), column temperature (35°C), injector temperature (150°C), and detector temperature (200°C).Nitrogen was used as carrier gas at a flow rate of 30 mL min -1 .Deodorizing activity was expressed as the decreased amount of methyl mercaptan and was calculated using the equation: Furthermore, deodorizing activity was expressed as IC 50 (50% inhibitory concentration).IC 50 values were calculated by linear regression analysis of deodorizing activity.All determinations were performed in triplicate.

Extraction and Isolation of Deodorizing Components
One kg of powdered E. bicyclis were putted into 10 L of methanol, and then refluxed for 3 h at 70°C.The process was 3 times repeated to get the extracts and the resulting methanol extract (147.62 g) was obtained by means of the vacuum evaporation.These extracts were dissolved in 1 L of water and transferred into a separating funnel.The solution was treated with n-hexane (1.0 L × 3), and consecutively followed by dichloromethane (DCM) (1.0 L × 3), by ethyl acetate (EtOAc) (1.0 L × 3) and by n-butanol (1.0 L × 3).Finally, the n-hexane  (Kang et al., 2003;Okada et al., 2004).

Instrumental Analysis
Varian VNS600 instrument (Varian, Inc., Walnut Creek, USA) was used for 1 H and 13 C NMR, which was measured at 600 and 150 MHz, respectively.The chemical shifts were expressed in parts-per-million (ppm); and DMSO-d 6 (δ H = 2.49; δ C = 39.7) was used as solution; and tetramethylsilane as standard material.The J CH value was est at 8 Hz in the heteronuclear multiple-bond correlation spectroscopy spectra.Fast-atom bombardment mass spectrometry (FAB-MS) was measured by means of orthogonal acceleration time-of-flight (oaTOF) spectrometer (AutoSpec; Micromass UK, Ltd., Manchester, UK), by using 3-nitrobenzyl alcohol.

Statistical Analysis
All results were indicated as mean ± S.E.M (standard error of the mean) (n=3) and the analysis of variance (ANOVA) was used to make a multiple comparison.SPSS v12.01 (SPSS, Chicago, USA) was used to conduct ANOVA and the difference in statistical significance was estimated by Duncan's Multiple Range tests (P<0.05).

The Deodorizing Activity against Methyl Mercaptan of E. bicyclis Extracts
As shown in Table 1, the deodorizing activities of E. bicyclis methanol extracts and its soluble extract against methyl mercaptan were evaluated.It was revealed that the EtOAc-soluble extract was the highest deodorizing activity exhibiting IC 50 value of 77.49 ± 0.75 µg mL -1 , followed n-butanol (278.98 ± 19.87 µg mL -1 ), DCM (307.90 ± 5.83 µg mL -1 ), n-hexane (733.95 ± 30.77 µg mL -1 ), and water (1,256.16± 54.20 µg mL -1 ) -soluble extracts.The IC 50 values of the EtOAc, n-butanol and DCM -soluble extracts were estimated to possess about 2 times to 8 times more strong deodorizing activity than sodium copper chlorophyllin, which is most widely known as a deodorant for halitosis.Lee et al (1999) reported that deodorizing activity of benxethonium, sodium fluoride, and cetylpyridinium chloride, which are used as the inhibitors of halitosis and antibacterial ingredients for commercial mouthwashes, against methyl mercaptan was less than 50% at concentration of 5 mg mL -1 .Moreover, the total phenolic contents in each extract showed in accordance with the results of deodorizing activity against methyl mercaptan.As shown in Table 2, the EtOAc-soluble extract (767.78 ± 15.46 mg mg PGEs g -1 ), which exhibits the highest deodorizing activity, had the highest total phenolic contents, followed by the DCM (434.17 ± 16.08 mg PGEs g -1 ), n-butanol (343.80 ± 19.18 mg PGEs g -1 ), n-hexane (278.89 ± 7.14 mg PGEs g -1 ), and water -soluble extract (35.19 ± 6.82 mg PGEs g -1 ).Thus, these results strongly suggested that the total phenolic contents of E. bicyclis are closely related with the deodorizing activity.

Instrumental Analysis Data of the Isolated Compounds
In order to elucidate the deodorizing compounds from E. bicyclis, the EtOAc-soluble extract was subjected to open column chromatography on sephadex LH-20 and LiChroprep RP-18 column.Collectively, we isolated three compounds from the EtOAc-soluble extract.The structural analysis using NMR and FAB-MS revealed the three compounds were to be eckol (1), dioxinodehydroeckol (2), and dieckol (3) (Figure 1).

The Identification and Deodorizing Activity of Active Components
Three isolated deodorizing compounds from EtOAc-soluble fraction against methyl mercaptan were shown in Table 3.The results revealed that compound 3 (IC 50 value of 26.71 ± 4.16 µg mL -1 ) shows the highest deodorizing activity, followed by compound 2 (IC 50 value of 40.57± 0.63 µg mL -1 ) and compound 1 (IC 50 value of 43.62 ± 1.52 µg mL -1 ).In addition, as the concentrations of compound 1, 2, and 3 increased, the deodorizing activities increased simultaneously.Thus these phlorotannins showed dose-dependent deodorizing activity (Figure 2).Sodium copper chlorophylline 605.13 ± 17.64 * IC 50 value was defined as the concentration of inhibitor required to inhibit 50% of deodorizing activity.Different letters (a, b) indicate significant differences at the level of P <0.05.

Discussion
VSCs in the mouth consists of hydrogen sulphide, methyl mercaptan and dimethyl sulphide with a unpleasant odor (Sharma et al., 2011).In the above studies, the deodorizing effect was evaluated in the generation of methyl mercaptan, a malodor arising from the oral malodorous breath (Tamaki et al., 2007).
In Table 3, compound 3, which contains the highly polymerized phloroglucinol, showed higher deodorizing activity than compound 1 and 2. The function mechanism of the deodorizing effect has not been cleared, the conversion of hydroxy group into quinone in lignins might be expected for the function mechanism of the deodorizing effect (Hiramoto et al., 2006).Compound 3 was estimated to possess about 22 times more strong deodorizing activity than sodium copper chlorophyllin.For more than 50 years, sodium copper chlorophyllin as positive control in this study has been used in supplements and as an over-the-counter drug used to reduce odor without any serious side effects (Higdon, 2007).However, sodium copper chlorophyll has occasionally been reported to cause the diarrhea related to oral chlorophyllin use (Shiomi et al., 2010).
Moreover, the oral administration of the phlorotannins at a dosage rate of 170-1,500 mg kg -1 bw day -1 for 14 days in mice observed no any cytotoxic effect (Ahn et al., 2004).Since phlorotannins from E. cava has been approved by the U.S. Food and Drug Administration as a new dietary ingredient (FDA-1995-S-0039-0176), phlorotannins from E. bicyclis are also expected to be potential candidates for halitosis control as a usage of toothpaste, chewing gum, and mouthwash without side effect.Therefore, these compounds (1-3) may safe and efficient deodorizing agents for treating malodor.
In conclusion, we obtained active compounds 1, 2, and 3 from E. bicyclis against methyl mercaptan, main cause of bad breath.The results of this study are expected and contributed to develop safe and efficacious substances for the food and pharmaceutical industries.

Table 1 .
Deodorizing activities of Eisenia bicyclis methanol extracts and its soluble extracts IC 50 value was defined as the concentration of inhibitor required to inhibit 50% of deodorizing activity.Different letters(a, b, c, d, e)indicate significant differences at the level of P <0.05.

Table 2 .
Total phenolic contents of Eisenia bicyclis methanol extracts and its soluble extract a PGE means phloroglucinol equivalents.Different letters (a, b, c, d, e) indicate significant differences at the level of P <0.05.† DCM, dichloromethane; EtOAc, ethyl acetate