In Vitro Anti-inflammatory Activity of Tyrosol and Tryptophol: Metabolites of Yeast via the Ehrlich Pathway

Abstract

Soy isoflavonoids were applied to commercially available baker’s yeast in vitro to find metabolites. Tyrosol, an ingredient in olive oil and wine, and tryptophol were found in the culture media. To test whether tyrosol is a metabolite of soy isoflavonoids, we prepared 2,4-dideuterated equol and applied it to yeast. According to LC-MS analysis of the culture media, deuterated tyrosol was not produced. Therefore, tyrosol is assumed to be a tyrosine metabolite of yeast known as the Ehrlich pathway. We then evaluated the in vitro activities of the 2 amino acid-derived alcohols. Both tyrosol and tryptophol similarly showed anti-inflammatory activity, as evaluated by monocyte chemoattractant protein-1 in 3T3-L1 murine adipocytes in vitro. Our results suggested that the amino acid-derived alcohols may contribute to the anti-inflammatory activity of fermented foods.

INTRODUCTION

Polyphenols have recently attracted great interest for their contribution to human health. Curcumin is a well-known natural compound because of its wide scope of activity, even though its characteristic yellowish color limits its usage as a food additive. We recently reported the metabolism of tetrahydrocurcumin, a well-known colorless metabolite of curcumin, by baker’s yeast.¹⁾ This finding suggests that some other polyphenols could be metabolized via yeast fermentation.

Because we have studied soy isoflavonoids including their bacterial metabolites,^2,3) we thought that yeast might react with them, likewise curcumin did. Soy contains unique isoflavones, such as daidzein and genistein, that predominantly present as the conjugates, daidzin and genistin.^4,5) Recent studies suggested that they are metabolized by bacteria in the animal and human intestines.⁶⁾ Among the metabolites of soy isoflavones, equol is interesting because it exhibits more potent activity than any other isoflavone.⁷⁾ Therefore, we tested whether soy isoflavonoids were metabolized by baker’s yeast in vitro using daidzein-, genistein-, and equol-containing media. In addition, we evaluated in vitro activity of the isolated compounds from the culture media.

MATERIALS AND METHODS

Chemicals and Reagents

Racemic equol, genistein, and daidzein were obtained from LC Laboratories (Woburn, MA, U.S.A.). Tyrosol and tryptophol were obtained from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Mushroom tyrosinase and NaBD₄ were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). d-Glucose, l-tyrosine, and 5% Pd/C were sourced from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan).

Isolation of the Compounds in a Culture Medium of Yeast and Equol

Racemic equol in ethanol (EtOH) (10 mM, 20 mL) was added to sterilized 10% glucose (2.0 L). Commercially available baker’s yeast (6 g) was then added, and the reaction mixture was incubated under static aerobic conditions at 30°C. After 3 d, the medium was extracted with ethyl acetate (EtOAc) (600 mL × 2), and the extract was washed with water and saturated brine. After the extract was dried over anhydrous Na₂SO₄, it was concentrated in vacuo. The concentrate was then separated by elution, using a 1-mm-thick Merck silica gel plate, with a solvent mixture of hexane/EtOAc (1 : 1). The metabolite was finally purified using preparative HPLC on a 250 × 20 mm i.d. Wakosil-II 5C18HG column (Wako Pure Chemical Corporation, Osaka, Japan) and a UV detector (254 nm). A solvent mixture containing 45% methanol (MeOH) was used to elute 2 fractions, 5.6 and 0.6 mg, at a flow rate of 6.0 mL/min at ambient temperature. The 5.6 mg sample was then purified by the same column eluting with 25% MeOH. Finally, 5.0 mg of the product was obtained.

Preparation of 2,4-Dideuterated Equol

Deuterated equol was prepared via the dehydroequol.⁸⁾ Briefly, deuterated 4-hydroxyequol was obtained using NaBD₄ and treated by acid-catalyzed dehydration. Then, the double bond of dehydroequol was catalytically reduced using 5% Pd/C under an H₂ atmosphere to produce the equol. These reactions were followed by comparison with intermediates²⁾ and equol using TLC and HPLC. The product was finally purified using an i.d. 20 mm Wakosil-II 5C18HG column eluted with 50% MeOH.

In Vitro Activities

Evaluation of in vitro monocyte chemoattractant protein-1 (MCP-1) attenuation and mushroom tyrosinase inhibition were performed as we described previously.^3,9) Each analysis was performed in triplicate, and the results were reported as mean ± standard deviation. The p-values were calculated using Dunnett’s test, which was carried out with EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan) and was compared with the vehicle control (EtOH).

RESULTS AND DISCUSSION

Isolation and Identification of Tyrosol and Tryptophol

Daidzein, genistein, and equol were applied to a commercially available baker’s yeast in vitro. In the EtOAc extracts of their culture media, each of the added isoflavonoids was detected by TLC and HPLC as described below, and novel products other than the initial materials were observed in all 3 media. Therefore, the products were isolated for structural elucidation. From 2.0 L of medium incubated with 100 µM equol and yeast, we isolated 2 compounds weighted at 5.0 and 0.6 mg. The ¹H-NMR spectrum (400 MHz, acetone-d₆) suggested that the former compounds had one para-disubstituted benzene ring and an ethyl moiety. The substrate equol has a para-disubstituted benzene ring at the B-ring, and an ethyl moiety connected to the phenol. The isolated compound was then deduced to be tyrosol (Fig. 1a, 1), which was supported by comparison with the reported ¹H-NMR spectrum.¹⁰⁾ In addition, commercially available tyrosol showed the same profiles as those of the isolated compound on TLC and HPLC.

Fig. 1. Structures of Tyrosol, Tryptophol, and 2,4-Dideuterated Equol

(a) Structures of tyrosol (1) and tryptophol (2). (b) The structure of 2,4-dideuterated equol (3) and its mass spectrum (ESI–) compared to that of equol. The separation was performed using a Hypersil Gold column (2.1 × 100 mm, Thermo Fisher Scientific) and was carried out using gradient elution with 0.1% formic acid in water (solvent A) and acetonitrile (solvent B) at a flow rate of 0.4 mL/min at ambient temperature. The linear gradient program was as follows: 0 min (10% B), 4.0 min (50% B), 5.0 min (50% B), and 10 min (10% B). Mass spectra for the peaks eluted at 4.1 min, monitored at UV 275 nm, were analyzed using Sciex X500R quadrupole time-of-flight mass spectrometer.

The ¹H-NMR spectrum of another compound revealed an ethanol moiety similar to that of tyrosol; however, it may feature a para-disubstituted benzene ring. LC-MS (X500R, Sciex; Framingham, MA, U.S.A.) in positive-ion mode electrospray ionization (ESI+) indicated the presence of nitrogen, leading us to consider it could be an indole compound. Thus, this structure was proposed to be an ethanol-substituted indole. The ¹H-NMR analysis suggested a para-disubstituted benzene ring with two D₂O-exchangable peaks (δ_H 10.0, 3.63). These results suggest that the isolated compound was 2- or 3-(2-hydroxyethyl)indole, and the profiles of commercially available 3-(2-hydroxyethyl)indole, known as tryptophol (Fig. 1a, 2), closely matched those of the isolated compound in TLC and HPLC analysis. The ¹H-NMR spectra of tryptophol reported in the literature used CDCl₃ as a solvent,^11,12) with data provided by the supplier (Tokyo Chemical Industry). However, slight variations in chemical shifts and coupling constants were observed, likely owing to long-range coupling. Nevertheless, commercially available tryptophol exhibited profiles identical to those of the isolated compound, including ¹H-NMR and LC-tandem mass spectrometry results. Therefore, we present a ¹H-NMR spectrum of the reagent, obtained by zero filling without line broadening, for clearer analysis.

Tryptophol (2): ¹H-NMR (400 MHz, acetone-d₆) δ_H 10.0 (1H, brs), 7.57 (1H, dddd, J = 0.8, 0.8, 1.2, 8.0 Hz), 7.36 (1H, ddd, J = 0.8, 1.0, 8.1 Hz), 7.17 (1H, m), 7.08 (1H, dddd, J = 0.4, 1.0, 7.0, 8.1 Hz), 7.00 (1H, ddd, J = 1.0, 7.0, 7.0 Hz), 3.81 (2H, dd, J = 5.4, 7.2 Hz), 3.63 (1H, t, J = 5.6 Hz), 2.96 (2H, dd, J = 0.9, 7.2 Hz). The magnified spectra for the indole moiety are illustrated in Supplementary Figs. 1A and 1B, along with the parameters. LC-MS (ESI+) m/z: 162.0913 [M + H]⁺ (Calcd. for C₁₀H₁₂ON: 162.0920).

However, tryptophol is not expected to be a metabolite of equol because it contains an indole ring. Tryptophol is produced from tryptophan by yeast and tyrosol is produced from tyrosine, known as the Ehrlich pathway.^13,14) However, soy isoflavonoids contain a skeleton for tyrosol in their B- and C-rings. Therefore, it was still unclear whether any tyrosol was produced from isoflavonoids or not.

Origin of Tyrosol

For this purpose, equol was marked with deuterium via the catalytic hydrogenation of the corresponding dehydroequol, which was prepared according to the scheme described by Cho et al.,⁸⁾ using NaBD₄. The product may contain stereoisomers because of the presence of three asymmetric carbons. However, it is difficult to separate isotopes; therefore, we did not record the ¹H-NMR spectrum because this could be the sum of the isomers. The product gave a clear spot on TLC at the same position as that of equol, even though it could be a mixture of stereoisomers. LC-MS analysis (ESI–) revealed peaks at m/z = 243.0994 (Calcd. for C₁₅H₁₁D₂O₃: 243.0990) as [M–H]^– and m/z = 289.1050 (Calcd. for C₁₆H₁₃D₂O₅: 289.1045) as [M + HCOO]^– (Fig. 1b). Therefore, we confirmed the successful preparation of 2,4-dideuterated equol (Fig. 1b, 3). At 100 µM, the marked equol was applied to the yeast in 6.0 mL of 10% glucose in a 6-well plate and incubated for 23 h. Then, the EtOAc extract was subjected to LC-MS with ESI–, and the produced tyrosol showed the same ratio for m/z = 138 and 137 as the reagent. This result suggested that equol was not metabolized to tyrosol by baker’s yeast in vitro and that both tyrosol and tryptophol are bacterial products obtained from the corresponding amino acids via the Ehrlich pathway.^13,14) Consequently, the 3 isoflavonoids may remain stable even after yeast fermentation. To verify this, they were incubated with or without yeast for approximately 2 d. Each isoflavonoid was found to maintain over 80% of its original concentration compared to samples without yeast (Supplementary Fig. 2: left y-axis). This finding further supports the idea that equol is not converted into tyrosol. Additionally, the experiment indicated that soy isoflavonoids did not change the levels of tyrosol and tryptophol produced by yeast (Supplementary Fig. 2: right y-axis). This result was corroborated by a test that compared 100 µM equol with the vehicle (EtOH) over the same incubation time (23 h), which showed no statistically significant difference in their tyrosol concentrations (data not shown). These results follow that tyrosol is present in soy-fermented foods.¹⁵⁾

Anti-inflammatory Activity

Next, we investigated the functionality of the amino acid-derived fermentation products, even though there have been many reports of tyrosol in relation to the health benefits of olive oil and wine. Some animal studies have claimed that tyrosol attenuates the secretion of inflammatory adipocytokines. However, the direct activity in cells is limited,^16–18) and tryptophol was rarely tested.¹⁹⁾ We applied the amino acid derivatives to murine adipocytes in vitro as we had done previously.⁹⁾ First, we checked the cytotoxicity of the test samples using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) method, and we found no cytotoxicity under 100 µM (Fig. 2a). As shown in Fig. 2b, both tyrosol and tryptophol reduced MCP-1 production in a dose-dependent manner. Previous studies have reported MCP-1 reduction by drinking alcohol.^20,21) According to a report, white wine reduces MCP-1 levels, similar to red wine, which typically has a higher polyphenol content, including resveratrol. Further study is required but we expect that yeast products play a role in the anti-inflammatory action of alcoholic drinks, at least in part.

Fig. 2. Inhibitory Effects of Tyrosol and Tryptophol in Vitro

As described previously, 3T3-L1 cells were differentiated into adipocytes in a 24-well plate.⁹⁾ Fresh media containing samples were applied to the cells, and the cells were incubated for 24 h. (a) Cell viability was evaluated using the MTT assay. (b) Monocyte chemoattractant protein-1 (MCP-1) secretion was evaluated using a commercially available ELISA kit. Data are presented as the mean ± standard deviation (S.D.) of 3 experiments. * p < 0.05 vs. Vehicle (EtOH), ** p < 0.01 vs. Vehicle (EtOH). (c) Dose-dependent experiment against mushroom tyrosinase in vitro was performed using a 96-well plate.³⁾ The amount of dopaquinone produced was determined by measuring the absorbance at 490 nm. Data are presented as the mean ± S.D. of three experiments. *p <0.01 vs. Vehicle (EtOH).

We were also interested in mushroom tyrosinase owing to the strong in vitro inhibition observed with equol,³⁾ which forms a complex with tyrosinase. As noted in the previous section on tyrosol isolation, equol has a structure similar to that of tyrosol concerning its B- and C-rings. Therefore, we expected that tyrosol would inhibit mushroom tyrosinase in a manner to equol. However, tyrosol exhibited no inhibitory activity, nor did tryptophol under our in vitro conditions,³⁾ as illustrated in Fig. 2c.

Acknowledgments

Part of this study was supported by The Public Foundation of Elizabeth Arnold-Fuji. We also thank Mr. Yuzo Ishigaki (Nagoya Municipal Industrial Research Institute) for his professional technique and useful advice on NMR.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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