Inhibition of Thyroid Peroxidase by Myrcia uniflora Flavonoids

Chem. Res. Toxicol. 2006, 19, 351-355

351

Inhibition of Thyroid Peroxidase by Myrcia uniflora Flavonoids Andrea C. F. Ferreira,† Jair C. Neto,‡ Alba C. M. da Silva,† Ricardo M. Kuster,‡ and Denise P. Carvalho*,† Laborato´ rio de Fisiologia Endo´ crina, Instituto de Biofı´sica Carlos Chagas Filho, and Laborato´ rio de Fitoquı´mica, Nu´ cleo de Pesquisas de Produtos Naturais, UniVersidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil ReceiVed June 21, 2005

Thyroid peroxidase (TPO), the key enzyme in thyroid hormone biosynthesis, is inhibited by dietary flavonoids; thus, a high consumption of plants containing inhibitory flavonoids may affect thyroid function and lead to hypothyroidism. In this work, TPO inhibition by the aqueous partition of Myrcia uniflora and its isolated compounds has been evaluated. The aqueous partition of the methanolic extract of M. uniflora is able to inhibit TPO activity in vitro. Two known flavonoids were isolated and characterized by mass spectrometry and 1H NMR from plant extracts: mearnsitrin and myricitrin. The degree of TPO inhibition produced by the aqueous solution of the flavonoids was very high, with a 50% inhibition of the original TPO activity (IC50) obtained at 1.97 µM mearnsitrin and at 2.88 µM myricitrin. These results suggest that the indiscriminated consumption of M. uniflora pharmaceutical products allied to the nutritional deficiency of iodine might contribute to the development of hypothyroidism and goiter. Introduction Environmental factors, such as pollution and diet, can affect thyroid function (1). There are reports in the literature about the effects of goitrogenic substances (2), and it is known that goiter prevalence is still higher if nutritional iodine deficiency is associated with the presence of thyroid function inhibitors (3). People around the world employ a large variety of plant extracts for the treatment of many diseases; consequently, plants with unknown chemical composition and biological activity are indiscriminately consumed. Flavonoids are a group of natural compounds widely found in the plant kingdom, and diversified biological and pharmacological activities of these compounds have been reported during the last years. Flavonoids may inhibit many enzymes, including thyroid peroxidase (TPO) and 5′-deiodinase, key enzymes for thyroid hormone synthesis and metabolism (37). Myrcia uniflora, a plant popularly called “pedra hume kaa´” in Brazil, is sold as a dry extract in capsules or as tinctures for the treatment of diabetes mellitus (8, 9). In the present study, we investigated the action of this plant extract on TPO activity. The phytochemical analysis of the alcoholic extract obtained from the dry and triturated plant furnished by the Flora Medicinal industry and its purified fractions and respective isolated compounds allowed us to determine the inhibitory compounds present in this plant.

Experimental Procedures TPO Preparation. TPO was extracted from human toxic diffuse goiter tissue samples obtained during thyroidectomies (informed consent given by patients) as described by Moura et al. (10) and Carvalho et al. (11). These glands usually have a high TPO activity. * To whom correspondence should be addressed. Tel: 55 21 2590 7147. Fax: 55 21 2280 8193. E-mail: [email protected]. † Laborato ´ rio de Fisiologia Endo´crina, Instituto de Biofı´sica Carlos Chagas Filho. ‡ Laborato ´ rio de Fitoquı´mica, Nu´cleo de Pesquisas de Produtos Naturais.

After the tissue was cleaned on an ice-cooled glass plate, the thyroid samples (1 g) were minced and homogenized in 3 mL of 50 mM Tris-HCl buffer, pH 7.2, containing 1 mM KI, using an Ultra-Turrax homogenizer. The homogenate was centrifuged at 100000g, 4 °C, for 1 h. The pellet was suspended in 2 mL of digitonin (1%, w/v) and incubated at 4 °C for 24 h, and the supernatant fluid containing the solubilized TPO was used for the assays. Plant Extract, Fractions, and Flavonoids. The dry and triturated plant was extracted with methanol and concentrated under reduced pressure. After the addition of water, a mixture of hexane, dichloromethane, and ethyl acetate was added in order to obtain the different fractions. The HPLC (high-performance liquid chromatography) screening analysis of the ethyl acetate fraction showed peaks with UV spectroscopic characteristics of flavonoids, and then, the ethyl acetate fraction was submitted to a MPLC (middle-pressure liquid chromatography) with Toyopearl HW-40C as the stationary phase to a preliminary purification, followed by an open column chromatography with Sephadex LH-20 gel as the stationary phase. Applying this methodology, two known flavonoids, mearnsitrin and myricitrin, were isolated and characterized by mass spectrometry and 1H and 13C NMR spectroscopy. TPO iodide-oxidation inhibitory tests of the aqueous and ethyl acetate fractions and of the isolated flavonoids were performed. In the ethyl acetate fraction, a more potent TPO inhibition was detected, and this fraction was thus used to isolate the flavonoids. TPO Iodide-Oxidation Activity Inhibition. The TPO iodide oxidation was measured as previously described (6, 10, 11). The control assay mixture contained 1.0 mL of freshly prepared 50 mM sodium phosphate buffer, pH 7.4, containing 24 mM KI and 11 mM glucose, and the amount of solubilized TPO producing an iodide-oxidation activity of 0.1 ∆A353nm/min. The final volume was adjusted to 2.0 mL with 50 mM sodium phosphate buffer, pH 7.4, and the reaction was started by the addition of 10 µL of 0.1% glucose oxidase (Boehringer Grade I). The increase in absorbance at 353 nm (tri-iodide production) was registered for 4 min on a Hitachi spectrophotometer (U-3300). Taking into account the kinetical parameters of TPO iodide-oxidation reaction and assuming that V/Vmax ) [S]/[S] + Km, we calculated that the rate of oxidation of I- was 11.3 mM when the absorbance change at 353 nm was 0.1 per min. The rate of H2O2 production by glucose-glucose oxidase is about 0.59 mmol H2O2/h/mL.

10.1021/tx0501684 CCC: $33.50 © 2006 American Chemical Society Published on Web 02/22/2006

352 Chem. Res. Toxicol., Vol. 19, No. 3, 2006 To test the inhibitory effects, the desired amount of M. uniflora or isolated flavonoids in 50 mM sodium phosphate buffer, pH 7.4, was added to the assay mixture before adjusting the final volume to 2 mL. The ∆A353nm/min in the presence or absence of inhibitors was determined from the linear portion of the reaction curve. As a positive control, we compared the M. uniflora TPO inhibition curve with the inhibition produced by PTU (6-n-propylthiouracyl), a wellknown TPO inhibitor used as an antithyroid drug (13). The inhibitory activity was expressed as the concentration necessary to produce a 50% inhibition of the original TPO activity (IC50). Each component was tested in at least three series of experiments, in which 8-12 different concentrations were assayed. Iodide Oxidation Inhibitory Kinetics. To evaluate the kinetic parameters of TPO-catalyzed iodide-oxidation inhibition, a given TPO activity (0.1∆abs/min) was assayed as described above, with or without M.uniflora flavonoids (IC50) and variable iodide concentrations. Each iodide concentration was tested twice in the presence and absence of M.uniflora flavonoids, and the ∆abs/min values obtained were plotted against KI concentrations. We also compared the kinetic parameters of TPO-catalyzed iodide-oxidation inhibition produced by PTU and M. uniflora flavonoids. Horseradish Peroxidase (HRP) Activity Inhibition by M. uniflora Flavonoids. To evaluate if M. uniflora flavonoids could inhibit HRP (Boheringer), HRP iodide-oxidation activity was measured as described above for TPO iodide-oxidation activity. An amount of HRP producing an iodide-oxidation activity of 0.1∆A353nm/min) was added to a reaction mixture, in the presence of the desired amount of isolated flavonoids in 50 mM sodium phosphate buffer, pH 7.4. The inhibitory activity was expressed as the concentration necessary to produce a 50% inhibition of the original HRP activity (IC50). H2O2 Trapping Effect. To study if M. uniflora flavonoids were able to scavenge H2O2, 4.0 µM H2O2 (Merck) was incubated for 20 min in the presence or absence of 2.88 µM myricitrin and 1.97 µM mearnsitrin (the respective IC50) and 28.8 µM myricitrin and 19.7 µM mearnsitrin (the respective IC100). Aliquots of 100 µL were then added to 1 mL of 0.2 M sodium phosphate buffer, pH 7.8, containing scopoletin (5.0 µM) and HRP (5 µg/mL), and the fluorescence was immediately measured in a Hitachi (F4000) spectrofluorometer (excitation, 360 nm; emission, 460 nm), as previously described (12, 13). The fluorescence measurements were plotted against H2O2 concentrations. To evaluate whether these flavonoids could directly interfere with the decreased fluorescence of oxidized scopoletin, we added the same amounts of flavonoids at the end of scopoletin incubation with 4.0 µM H2O2 and HRP. We detected no changes in the previous fluorescence measurement, demonstrating that flavonoids do not interfere with the decrease in fluorescence detected when scopoletin is in the presence of H2O2 (data not shown). The assay described above was based on the measurement of H2O2 concentrations using the HRP-catalyzed scopoletin oxidation method. To exclude the possibility of a direct HRP inhibition by M. uniflora flavonoids, we also used a greater amount of HRP in the assay (10-3% instead of 5 × 10-4%, w/v).

Results TPO iodide-oxidation activity was significantly inhibited by M. uniflora extract, with a 50% inhibition of the original TPO activity (IC50) obtained at a 0.0017% (w/v) M. uniflora aqueous extract concentration (Figure 1A). PTU produced 50% inhibition of the initial TPO iodide-oxidation activity at a concentration of 10 µM (Figure 1B) (13). We have isolated two known flavonoids: mearnsitrin and myricitrin (Figure 2) (14-17), which significantly inhibited TPO iodide-oxidation activity with a 50% inhibition of the original TPO activity (IC50) obtained at 2.9 µM myricitrin and 2.0 µM mearnsitrin (Figure 3). The possibility of a TPO inhibition caused by competition with the substrate (iodide) was evaluated. Kinetic iodideoxidation studies show that in the presence of mearnsitrin, but

Ferreira et al.

Figure 1. TPO iodide-oxidation activity inhibition by M. uniflora aqueous extract (A) and PTU (B). The TPO iodide-oxidation activity was measured in the presence of increasing amounts of M. uniflora aqueous extract (A) and PTU (B), as previously described (6, 10, 11).

Figure 2. Mearnsitrin and myricitrin structures.

not myricitrin, TPO K0.5 was significantly increased (without flavonoid, 13.41 ( 5.514 mM; with mearnsitrin, 260.8 ( 80.32 mM; and with myricitrin, 8.310 ( 1.651 mM) (Figure 4). Vmax was decreased in the presence of myricitrin but not mearnsitrin (without flavonoid, 0.2194 ( 0.0231 ∆abs353 nm/min; with mearnsitrin, 0.2134 ( 0.0560 ∆abs353 nm/min; and with myricitrin, 0.1331 ( 0.0178 ∆abs353 nm/min) (Figure 4). These results indicate that the mechanism of TPO inhibition differs between these two flavonoids. Mearnsitrin is a competitive inhibitor, whereas myricitrin is a noncompetitive inhibitor of TPO iodide-oxidation reaction. As previously described (13), we found that PTU (Figure 4, insert) is a competitive inhibitor of TPO iodide oxidation reaction, since Vmax was not decreased in the presence of PTU but TPO K0.5 was significantly increased (without PTU, K0.5 ) 17.04 ( 2.82 mM; Vmax ) 0.2505 ( 0.0215 ∆abs353 nm/min; in the presence of PTU, K0.5 ) 32.24 ( 6.31 mM; Vmax ) 0.2848 ( 0.0450 ∆abs353 nm/min). M. uniflora flavonoids also inhibited HRP iodide-oxidation activity, with an IC50 value of 5.7 µM for myricitrin and 4.8 µM for mearnsitrin (Figure 5). PTU produced 50% inhibition of the initial HRP iodide-oxidizing activity at a concentration of 25 µM (Figure 5, insert).

TPO Inhibition by Myrcia uniflora FlaVonoids

Chem. Res. Toxicol., Vol. 19, No. 3, 2006 353

Figure 3. TPO iodide-oxidation activity inhibition by myricitrin (A) and mearnsitrin (B). The TPO iodide-oxidation activity was measured in the presence of increasing amounts of M. uniflora flavonoids, as previously described (6, 10, 11).

To further evaluate the possible mechanism of peroxidase inhibition by M. uniflora flavonoids, we tested whether they interfere with H2O2. Our results show that mearnsitrin and myricitrin significantly destroy H2O2 added to the incubation mixture in both the IC50 and the IC100 concentrations (Figure 6), while PTU did not decrease H2O2 in the incubation mixture (Figure 6, insert) (13). The flavonoid concentrations needed to inhibit HRP iodideoxidation activity were higher than those used to test H2O2 trapping effects. H2O2 was incubated in the presence of TPO IC50 flavonoids concentrations, but after the addition of the solution containing scopoletin and HRP, the final concentration of flavonoids was at least 10 times lower (0.26 µM for myricitrin and 0.18 µM for mearnsitrin). Thus, the interference of flavonoids in the scopoletin oxidation by HRP does not occur in the final assay mixture. To confirm that H2O2 trapping effects of flavonoids were not due to their effect on HRP, the H2O2 trapping assay was performed using two diferent HRP concentrations. There were no differences in H2O2 trapping effects detected in either assay, indicating again that flavonoids were in fact able to directly interact with H2O2 present in the assay mixture (Figure 6). Also, we have ruled out the possibility of flavonoids directly interfering with the fluorescence by the addition of flavonoids after scopoletin has been incubated with H2O2 and HRP. Under this circumstance, the flavonoids were not able to modify the previous decrease in fluorescence (data not shown).

Discussion M. uniflora extract significantly inhibited TPO iodideoxidation activity. We have previously shown that Kalanchoe brasiliensis aqueous extract, another Brazilian medical herb, is a potent TPO inhibitor. In the present study, we demonstrate

Figure 4. Iodide concentration dependence of the TPO-catalyzed iodide-oxidation inhibitory activity produced by mearnsitrin (A) and myricitrin (B). The amount of solubilized TPO producing a fixed iodide oxidation activity (∆abs353 nm/min ) 0.1) was assayed in the presence (2) or absence (b) of IC50 flavonoids (2.9 µM myricitrin and 2.0 µM mearnsitrin). Different concentrations of KI were added, and the final volume was adjusted to 2.0 mL. The reaction was started by the addition of 10 µL of 0.1% glucoseoxidase. The increase in absorbance at 353 nm (A353nm) was followed for 4 min on a computerized Hitachi spectrophotometer (U-3300). The iodide-oxidation activity (∆A353nm/ min) was determined from the linear portion of each reaction curve and plotted against different iodide concentrations. Each iodide concentration was tested twice in the presence or absence of M. uniflora flavonoids, and the ∆A353nm/min values obtained were plotted against KI concentrations. For comparison, iodide concentration dependence of the TPO-catalyzed iodide-oxidation inhibitory activity produced by PTU was done (insert).

that M. uniflora aqueous extract is more potent than K. brasiliensis aqueous extract (K. brasiliensis IC50 ) 0.07%; M. uniflora IC50 ) 0.0017%) (6). We have isolated from M. uniflora extract two previously known flavonoids: mearnsitrin and myricitrin. Both flavonoids significantly inhibited TPO and HRP iodide-oxidation activity, although potencies and kinetics seem to be different. Mearnsitrin inhibits TPO iodide-oxidation activity competitively, since the TPO K0.5 for iodide was significantly increased in the presence of mearnsitrin but Vmax was not altered, while myricitrin inhibits TPO noncompetitively, since Vmax was significantly decreased but K0.5 was not altered. Divi and Doerge (3) studied the inhibition of TPO tyrosine-iodination activity by myricetin, the aglycone form of myricitrin, which showed an IC50 of 0.6 µM, suggesting that the aglycone compound is even more potent than the glycone form presented herein, although the method of measuring TPO activity was different. It is important to notice that M. uniflora flavonoids are more potent than PTU and could thus promote sufficient TPO inhibition as to impair thyroid

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Figure 6. Effect of different HRP concentrations on H2O2 trapping effect of M. uniflora flavonoids. A 4.0 µM concentration of H2O2 (Merck) was incubated in the presence or absence of 2.88 µM myricitrin and 1.97 µM mearnsitrin (the respective TPO IC50) and 28.8 µM myricitrin and 19.7 µM mearnsitrin (the respective TPO IC100). In the insert, PTU was used (10 and 100 µM, the respective TPO IC50 and IC100). Two different HRP concentrations were used as follows: 5 × 10-4 and 10-3% (w/v). The H2O2 concentration was measured as previously described (12, 13). Figure 5. HRP iodide-oxidation activity inhibition by M. uniflora flavonoids: mearnsitrin (A) and myricitrin (B). The HRP iodideoxidation activity was measured in the presence of increasing amounts of M. uniflora flavonoids, as previously described (6, 10, 11). A representative curve is shown. In the insert, PTU inhibition of HRP is shown.

hormone biosynthesis. Mearnsitrin and myricitrin significantly trapped H2O2 added to the incubation mixture. Mearnsitrin seems to destroy H2O2 in proportion mol:mol, while myricitrin seems to destroy H2O2 in a higher proportion than mearnsitrin. However, apart from trapping H2O2, these flavonoids certainly directly inhibit TPO since the concentrations of H2O2 present in the assay conditions of the iodide oxidation reaction are in the millimolar range and M. uniflora flavonoids scavenged H2O2 in the micromolar range. The H2O2 trapping effect described herein is probably not due to HRP inhibition. In fact, M. uniflora flavonoids were able to inhibit HRP iodide-oxidation activity. However, the concentration of flavonoids needed to inhibit HRP iodide-oxidation activity is much higher than that used in the H2O2 trapping assay. These results support a direct effect of M. uniflora flavonoids on H2O2. Moreover, using a higher HRP concentration, H2O2 destruction by flavonoids was similar, which reinforces the idea that M. uniflora flavonoids do not diminish the oxidation of scopoletin by inhibiting HRP activity. PTU, which is a known peroxidase inhibitor and significantly inhibited HRP activity, did not significantly scavenge H2O2, which suggests that in this condition HRP inhibition is not the explanation for the decreased scopoletin oxidation detected. These data suggest that M. uniflora flavonoids have antioxidant properties, as previously reported for other flavonoids (18-20) and for mearnsitrin and myricitrin (21-24). We conclude that M. uniflora extract contains flavonoids that are potent TPO inhibitors. In fact, their inhibitory potencies in vitro are greater than that of PTU, a well-known antithyroid drug. The pharmacokinetic behavior of naturally occurring

isoflavones in humans shows an extensive distribution of these substances after absorption (25). Furthermore, some previous reports have shown that orally administered flavonoids are distributed and accumulated in various endocrine organs, including the thyroid (26). It has already been reported that the concentrations of genistein (the principal soy isoflavone) achieved in plasma from humans consuming soy products can reach approximately 1 µM (27), a concentration similar to the IC50 for TPO inhibition in vitro. Considering our knowledge at this point, we believe that intermittent or low-dose exposure to M. uniflora extract would have no significant impact on thyroid hormone status, but it is possible that chronic exposure could elicit prolonged blockage of thyroid hormone synthesis. Because a chronic inhibition of thyroid hormone synthesis induces increased secretion of thyroid stimulating hormone, which can lead to thyroid growth, our results suggest that chronic consumption of M. uniflora has the potential to induce hypothyroidism and goiter, especially in areas with a low iodine intake and in patients with a subclinical thyroid dysfunction. Acknowledgment. This work was supported by Grants from Fundac¸ a˜o Carlos Chagas Filho de Amparo a` Pesquisa do Estado do Rio de Janeiro (FAPERJ) and Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq). We are grateful for the technical assistance of Norma Lima de Arau´jo Faria, Advaldo Nunes Bezerra, and Wagner Nunes Bezerra.

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