Antimutagenic and antioxidant activities of quebracho phenolics (Schinopsis balansae) recovered from tannery wastewaters

Bioresource Technology 100 (2009) 434–439

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Antimutagenic and antioxidant activities of quebracho phenolics (Schinopsis balansae) recovered from tannery wastewaters Raúl Marín-Martinez b, Rafael Veloz-García b, Rafael Veloz-Rodríguez b, Salvador H. Guzmán-Maldonado c, Guadalupe Loarca-Pina d, Anabertha Cardador-Martinez d,e, Lorenzo Guevara-Olvera b, Rita Miranda-López b, Irineo Torres-Pacheco a, Cristina Pérez Pérez b, Guadalupe Herrera-Hernández b,c, Francisco Villaseñor-Ortega b, Mario González-Chavira c, Ramón G. Guevara-Gonzalez a,* a

Facultad de Ingeniería, Universidad Autónoma de Querétaro, Centro Universitario Cerro de las Campanas, s/n, Querétaro, Qro 76010, Mexico Departamento de Ingeniería Bioquímica, Instituto Tecnológico de Celaya. Av. Tecnológico s/n, Celaya, Gto., Mexico Unidadde Biotecnología, Campo Experimental Bajío, INIFAP. Km 6.5 Carretera Celaya-SanMiguel de Allende, Celaya, Gto, 38010, México d Programa de Posgrado en alimentos del centro dela república (PROPAC), Facultad de Química, Universidad Autónoma de Querétaro, Centro Universitario Cerro de las Campanas, s/n, Querétaro, Qro, 76010, México e Unidad Académica de Agricultura y Tecnología de Alimentos. ITESH, Campus Queretaro. Av. Epigmenio Gonzalez No. 500. Fracc. San Pablo. Querétaro, Qro, 76130, México b c

a r t i c l e

i n f o

Article history: Received 22 June 2006 Received in revised form 25 April 2008 Accepted 1 May 2008 Available online 9 July 2008 Keywords: Schinopsis balansae Phenolics Antimutagenic Antioxidant

a b s t r a c t Quebracho extracts are used in tannery due to their high concentration of phenolics. The Mexican tannery industry uses around 450 kg/m3 of which, 150 kg/m3 remains in wastewaters and are discharged in drain pipe systems or rivers. The quebracho phenolics recovered from tannery wastewater (QPTW) was characterized by HPLC. The antimutagenic and antioxidant activities as well as the microbiological quality were evaluated. Total phenolic content of QPTW was 621 mg catechin equivalent/g sample. Gallic and protocatechuic acids were the major components characterized by HPLC. QPTW showed an inhibition range on aflatoxin B1 mutagenicity from 16 to 60% and was dose-dependent. Antioxidant activity (defined as b-carotene bleaching) of QPTW (64.4%) at a dose of 12.3 mg/mL was similar to that of BHT (68.7%) at a dose of 0.33 mg/mL, but lower than Trolox (90.8% at a dose of 2.5 mg/mL); meanwhile antiradical activity (measured as reduction of DPPH) (60.8%) was higher than that of BHT (50.8%) and Trolox (34.2%). Quebracho residues were demonstrated to be an outstanding source of phenolic acids and for research and industrial uses. Ó 2008 Elsevier Ltd. All rights reserved.

1. Introduction Two species of quebracho tree, Schinopsis balansae and Schinopsis lorentzii, are grown in South America, particularly in north of Argentina and eastern Paraguay. These species are of interest given the high concentration of phenolics that accumulate. The extraction of quebracho phenolics is relatively easy because of their solubility in hot water (Acosta, 1999). Extracts of quebracho phenolics have been tested to control gastrointestinal parasites in ruminants (Paolini et al., 2003a, b; Athanasiadou et al., 2001a, b, 2000), and to evaluate their effect on protein consumption and hepatic concentration of vitamin A (Suschetet and de Larturiere, 1978). Quebracho phenolics have been classified as condensed tannins (Suschetet and de Larturiere, 1978; Athanasiadou et al., 2001a; Lopez-Fluza et al., 2003) and determined as gallic acid (Kratzer et al., 1975) or catechin (Acosta, 1999) equivalents; however, the separa-

* Corresponding author. Tel.: +52 01 442 192 12 64; fax: +52 01 442 192 12 00. E-mail address: [email protected] (R.G. Guevara-Gonzalez). 0960-8524/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2008.05.029

tion and quantification of quebracho phenolics have not been carried out. The most common use of quebracho phenolics is in tannery processes. In Mexico, the tannery industry uses around 40,000 ton of quebracho extracts/year; which represent 450 kg/m3 of quebracho extracts for tanning. After tanning treatment, the wastewaters discharged into drain pipe systems and likewise in rivers were still found to contain about 33% or 150 kg/m3 of quebracho extract. Given the high concentration of phenolics and collagen, this practice is a problem of contamination for agriculture fields and underground waters. Unfortunately, efforts for recycling and use of quebracho phenolic residues recovered from tannery have been scarce. Acosta (1999) demonstrated that it is possible to recover 62% of quebracho phenolics from wastewaters by treatments with sulfuric acid in order to separate them from collagen. Flavonoids are the most important group of the family of phenolics and represent two thirds of dietary phenolics; the rest are mostly represented by the phenolic acids (Scalbert and Williamson, 2000). There is increasing awareness and interest in the antioxidant behavior and potential health benefits associated with

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phenolics because these compounds have been related to the prevention of chronic diseases, such as cancer, cardiovascular problems and diabetes (Robbins, 2003). Dietary sources of phenolics are fruits, grains, teas and spices (Parr and Bowell, 2000), their content, expressed as catechin equivalents, vary from traces to 0.39 g/ kg (Santos-Buelga and Scalbert, 2000; Clifford, 2000). Phenolics are linked to a negative association with stomach cancer incidences (Powles and Ness, 1996) and tumor growth (Block et al., 1992). They also exhibit antimutagenic effects against aflatoxin B1 (Loarca-Piña et al., 1996), benzo(a)pyrene and 1-nitropyrene (Gonzalez de Mejia et al., 1999). Phenolics also exhibited an antioxidant effect, which has been linked to inhibition of oxidative damage diseases such as coronary heart diseases, stroke, and cancers (Block et al., 1992; Powles and Ness, 1996; Singh et al., 2002). Antioxidant activity of phenolics is of interest for the food industry given the demand for natural antioxidants (Ibáñez et al., 2003). Additionally, the food industry has investigated the effect of phenolics on fruit maturation, prevention of enzymatic browning, and their role as food preservative (Robbins, 2003). Phenolics have been also used as antimicrobial agent against phytopathogens such as fungi, bacteria and yeast (Field and Letinga, 1992). Thus, it will be important the search of novel sources of phenolics from unexploited resources (e.g. quebracho phenolics from tannery wastewaters). The aim of the present work was to use HPLC to characterize quebracho phenolics recovered from tannery wastewaters. Also evaluated were the antimutagenic effect against aflatoxin B1 produced by Aspergillus flavus and Aspergillus parasiticus, which is frequently found in foodstuffs such as corn and peanuts (Hsieh, 1989; Liu and Mases, 1992), and the antioxidant activity using the b-carotene-linoleate and the 1,1-diphenyl-2-picryl hydrazyl (DPPH) model systems. To complete the characterization of the recovered quebracho phenolics, a microbiological analysis was performed. 2. Methods 2.1. Quebracho extract Wastewaters of quebracho (S. balansae) were collected from a local tannery (Leon, Guanajuato, Mexico). The aqueous solution was placed in a tank at room temperature for decantation of solid materials. After the solid material was removed, the solution was centrifuged (5000g) to recover the supernatant, which was treated with HCl 0.1 N and sodium bisulfite 3% (w/v) keeping at pH of 4.5 ± 0.2 in order to prevent precipitation (pH < 4.0) or oxidation (pH > 5.0). After chemical addition, sample was placed at room temperature for 12 h with shaking. Immediately after shaking, the sample was centrifuged to recover the phenolic-rich aqueous solution and the collagen fraction was discarded. The phenolic-rich solution was evaporated at 80 °C and spray-dried at 4 kg/cm2 and 220 °C. With comparative purposes, commercial quebracho ‘‘tannins” were purchased from a local industry. 2.2. Chemicals Aflatoxin B1 (AFB1), vainillin, (+)-catechin, dimethyl sulphoxide (DMSO), butylated hydroxytoluene (BHT), 1,1-diphenyl-2-picrylhydrazyl (DPPH) and b-carotene and gallic, protocatechuic, chlorogenic, caffeic, 4-hydroxy-3-metoxybenzoic, syringic, benzoic, and salicylic acids were obtained from Sigma Chemical Co. (St. Louis, MO, USA). All the other chemicals and high-performance liquid chromatography solvents were of HPLC grade. 2.3. Total phenolics The spray-dried powder of quebracho phenolics recovered from tannery wastewaters (QPTW) was dissolved in distilled water

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(200 g/L) before total phenolic determination. Phenolics were assayed using the method described by Price and Butler (1977) and reported as mg of (+) catechin equivalents/g of sample (mg CE/g). 2.4. HPLC analysis Individual phenolic acids in QPTW were separated and quantified by high-performance liquid chromatography equipped with an Agilent 1100 controlled, 1100 quaternary gradient pump, inline degasser autosample, dual wavelength UV/VIS detector and acquisition system (Agilent Sofware 1100) (Agilent 1100 Model). A 15  4.6 mm i.d. reversed-phase Zorbax octadecilsilane (ODSC18) (Agilent) was used and operated at room temperature. The phenolic acids were eluted at 1 mL/min using a gradient systems consisting of two solvents: (A) acetic acid–water 2:98 v/v and (B) acetic acid–acetonitrile–water (2:30:68 v/v). The gradient was programmed at 10:90 (A:B v/v) in 30 min. The separated compounds were identified at 280 nm, and quantified using a diode array detector on the basis of chromatographic retention times and coelution with added standards. Eight pure phenolic acids (gallic, protocatechoic, chlorogenic, caffeic, 4-hydroxi-3-metoxibenzoic, syringic, benzoic, and salicylic) and vanillin were used for calibration and quantification. The standard phenolic acids exhibited a linear relationship with a HPLC peak area in a concentration of 0.0–20 g. The HPLC chromatogram for phenolic acids standards is presented in Fig. 1A. The linear standard calibration curves (0.9909 R2) were generated by injecting 1.5– 20 lg/mL of a specific phenolic acid in 50 lL of 70% H2O–30% acetonitrile. 2.5. Mutagenic activity Tester strain TA100 of Salmonella typhymurium, kindly provided by Dr. Bruce Ames (Berkeley, CA), was used in order to evaluate a potential mutagenic effect of QPTW (Ames et al., 1975). Bacteria grown overnight in Oxoid nutrient broth No. 2 (Oxoid Ltd., Hants, England) up to approximately 1  109 to 2  109 cells/mL and harvested by centrifugation (4500 g, 4 °C, 10 min). The bacteria were suspended in ice cold phosphate-buffered saline (PBS) (0.15 M, pH 7.4) to a concentration of 1010 cells/mL. The rat liver microsome S9 from Aroclor 1254 pretreated Sprague-Dawley male rats was obtained from Molecular Toxicology (Annapolis, MD) which contained 40 mg protein/mL as determined by a standard method (Lowry et al., 1954). The concentration of the S9 in the mix (enzyme + cofactors, 50:50) was 300 lg/mL for all experiments. The mutagenic activity of QPTW was evaluated using the microsuspension assay (Kado et al., 1986, 1983). The following ingredients were added to different tubes: 0.1 mL S9 mix, 0.1 mL concentrated bacteria (1010 cells/mL PBS) and 0.01 mL of QPTW at the following concentrations: 160, 320, 640 and 1280 lg/mL to give a final concentration of quebracho extract of 1.6, 3.2, 6.4 and 12.8 lg/tube, respectively. A 12  75 mm sterile glass culture tubes were used in all experiments. For comparative purposes, an experiment without S9 was carried out under the same conditions. The tubes were incubated in the dark at 37 °C with rapid shaking for 90 min. After incubation the tubes were placed in an iced bath, and 2 mL molten top agar containing 45 nmol/mL histidine and biotin was added (Ames et al., 1975). The combined solutions were vortexed and poured into minimal glucose plates. Plates were incubated at 37 °C in the dark for 48 h after which the numbers of revertant colonies were counted using a colony counter (AccuCount 1000, BioLogics Inc.). Strain markers and bacterial survival were routinely monitored for each experiment. Extracts were tested in triplicate in each independent experiment performed.

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A

mAU

1 70 60

6 50 40 30

7

4 2

20

5

8

10

3

9

0 0

5

10

15

20

25

B

mAU 1 350

2.7. Antioxidant activity

300 250

b

200 150

a

100

2

50 0 0

5

10

15

20

25

C mAU 1 100

activity of QPTW. Bacteria were grown as reported elsewhere (Ames et al., 1975). A dose–response of AFB1 mutagenicity was evaluated in order to define the dose at which AFB1 was not toxic. The following ingredients were added to different tubes: 0.1 mL S9 mix, 0.1 mL concentrated S. typhimurium (1010 cells/mL PBS) and 0.01 mL AFB1 at the following concentrations: 12.5, 25, 50 and 100 lg/mL to give a final concentration of 0.125, 0.25, 0.50 and 1 lg/tube, respectively. Incubation conditions for bacteria and counting of revertant colonies were carried out as mentioned before (Ames et al., 1975). To evaluate the antimutagenic effect of QPTW, the following ingredients were added to different tubes: 0.1 mL S9 mix, 0.1 mL concentrated bacteria (1010 cells/mL PBS) and 0.005 mL of AFB1 at concentration of 100 lg/mL. Finally, 0.005 mL of QPTW was added to each tube at different concentrations to give a final concentration of 1.6, 3.2, 6.4 and 12.8 lg/tube. Incubation conditions for S. typhimurium bacteria and counting of revertant colonies were carried out as mentioned in Ames et al. (1975). Extracts were tested in triplicate for each independent experiment performed.

b

2.7.1. b-carotene bleaching method The method reported by Fukumoto and Mazza (2000) was used to evaluate the antioxidant activity of QPTW. The procedure was as follows: Aliquots of QPTW, 100 lg of BHT or Trolox and 200 lL of the b-carotene solution were added to a well in a 96-well flat-bottom microtitration plate (ICN Biomedical Inc., Aurora, OH). The sample mixture was diluted by transferring 30 lL to another plate containing air-sparged distilled water (210 lL) to give a final concentrations of 0.15, 0.3, 0.6 and 1.2 mg of QPTW in each well. Dilutions were done in triplicate since the b-carotene bleaching reaction was subject to noticeable variations. ADIBA (a-a0 azodiisobutyramidine dihydrochloride, 20 lL, 0.3 M), which generates peroxyl radicals from linoleic acid, was added to each well to initiate the reaction. Absorbance readings were recorded in a plate reader (Multiskan Multisoft, Labsystems) using a 450-nm filter at 0 min and at intervals of 10 min until 90 min. Plates were kept in the dark at room temperature between readings. Antioxidant activity (AA) was calculated as percentage of inhibition relative to the control using the following relationship (Fukumoto and Mazza, 2000):

80

AA ¼ ½ðRcontrol —Rsample Þ  100=Rcontrol

60

where Rcontrol and Rsample are the degradation of b-carotene in reactant mixture without and with quebracho extract, respectively. The AA for different times was averaged to give one AA value for each triplicate sample.

a 40

4 20

2

0

0

5

10

15

20

25

Minutes Fig. 1. HPLC profiles obtained from (A) pure standards 1, gallic acid; 2, protocatechoic acid; 3, chlorogenic acid; 4, caffeic acid; 5, 4-hydroxy-3-metoxibenzoic acid; 6, syringic acid; 7, vanillin; 8, benzoic acid; 9, salicylic acid (B) QPTW and (C) quebracho phenolics from a commercial product. Peaks a and b on panels B and C, corresponds to compounds that have not yet been identified.

2.6. Antimutagenic activity Tester strain TA100 of S. typhymurium, was also used to evaluate the dose–response of aflatoxin B1 (AFB1) and the antimutagenic

2.7.2. DPPH method The 1,1-diphenyl-2-picrylhydrazyl (DPPH) method of BrandWilliams et al. (1995) modified by Fukumoto and Mazza (2000), which measures the ability of test sample to scavenge peroxyl radicals, was also used to evaluate the antioxidant activity of the QPTW. Reduction of DPPH by an antioxidant results in a loss of absorbance at 515 nm. The degree of discoloration of the solution indicates the scavenging efficiency of the added substance. The method for evaluating reduction of DPPH was as follows: A solution of DPPH (150 lM) was prepared in 80% methanol. Using 80% methanol had the advantage of a faster reaction rate for some compounds such as butylated hydroxitoluene (BHT) and lower evaporative losses. Aliquots of QPTW, to give a final concentration of 0.15, 0.3, 0.6 and 1.2 mg in each well and standards (BHT and Trolox) were added in a 96-well flat-bottom visible light plate containing 200 ll of DPPH solution. QPTW was prepared in triplicate for each concentration used. The plate was read from 0–120 min

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each 10 min in a plate reader at 520 nm (Multiskan Multisoft, Labsystems). The plate was covered and left in the dark at room temperature between readings. A plot of the absorbance at 520 nm versus concentration was made in each interval time. Antiradical activity (ARA) was calculated by the following equation from Burda and Oleszek (2001):

ARA ¼ 100  ð1  absorbance of sample=absorbance of controlÞ:

2.8. Microbiological analysis For the analysis of fungi and yeast in QPTW 20 mL aliquots of agar Sabouraud, and nutritive agar for bacteria (Rasool et al., 2005) were placed (45 °C) on Petri dishes and allowed to cool at room temperature under sterile conditions. Aliquots (1 mL) of diluted QPTW in sterile phosphate buffer (1:1000 v/v) were used to inoculate the agar media. The Petri dishes were incubated at 37 °C for 48 h. For the analysis of coliforms, 20 mL aliquots of agar red bile agar (Musgrove et al., 2008) were placed on Petri dishes and allowed to cool at room temperature. To inoculate the agar media, aliquots (1 mL) of diluted QPTW (1:1000) were used. The Petri dishes were incubated at 35 °C for 24 h. Colonies on agar plates were counted and a proportional subsampling procedure was used to select colonies for identification. Colonies are reported as colony-forming units (CFU/Kg). All microbiological analyses were carried out in duplicate. 2.9. Statistical analysis Data were subjected to analysis of variance by the general linear models (GLM) procedure, means comparison by Tukey´s test and comparison to a control by Dunnett according to SAS methods (1990). 3. Results and discussion 3.1. Yield and total phenolics The yield of dry QPTW was 92 ± 4 kg/m3, was in agreement with the report of Acosta (1999). This result could be of interest for industrial production of phenolics from tannery wastewaters and could contribute to reduce the contamination problems of rivers, underground waters and agriculture fields. Total phenolic contents of QPTW was 621.5 ± 2.9 mg Catechin Equivalent (CE)/g sample (equivalent in wet weight to 14 g CE/L of wastewater), similar to that of commercial product of quebracho phenolics (643.3 ± 1.9 mg CE/g sample) although they were statistically different (p < 0.05). Santos-Buelga and Scalbert (2000) reported levels of phenolics in a wet weight base from 0.01 to 3.99 g CE/g, among cereals, fruits and berries; and from 0.004 to 3.71 g CE/L among juices and drinks. Therefore, the phenolic concentration in QPTW was far higher than that of those presented in cereals, fruits and berries as well as juices and drinks. With regard to the microbiological quality of the QPTW, fungi, yeast, bacteria and coliforms were not detected at any of the repetitions carried out (data not shown). The presence of such microorganisms is a well-known indicator of contamination during the process or from polluted water. 3.2. HPLC analysis The HPLC chromatographic analysis shows that QPTW contain gallic acid (4.25 mg/g) and protocatechuic acids (3,4, dihydroxybenzoic acid) (2.17 mg/g). These results were obtained based on retention time comparisons with phenolic acids and other poly-

phenols standards (Fig. 1A and B). Interestingly, quebracho phenolics from a commercial product show the same HPLC profile with exception of caffeic acid (8.1 mg/g) (Fig. 1C). This product is the same that industry used for tannery purposes with caffeic acid as the primary tanning agent, which it could be a possible explanation of the absence of this acid in QPTW, because QPTW are recovered after the tannery process. It can be concluded that gallic acid is the major component in QPTW followed by protocatechuic acid. It is well-known that gallic acid is one of the major phenolics present in plants (Parr and Bowell, 2000). The high level of gallic and protocatechuic acids, makes QPTW an outstanding source of these phenolic acids for possible industrial production. 3.3. Mutagenic activity The concentrations of phenolic acids from QPTW used to evaluate the mutagenic activity were neither toxic or mutagenic, since the number of revertant colonies were similar (p < 0.05) to that of spontaneous revertants (102) for tester strain TA100 with or without S9 (data not shown). For all treatments tested with QPTW, the range of revertant colonies was from 106 to 115 when S9 was present and from 100 to 110 in the treatments without S9. The range on revertant colonies was similar to those reported previously by several authors (Maron and Ames, 1983; Gonzalez de Mejia et al., 1999; Cardador-Martínez et al., 2002). It has been reported that several phenolic compounds tested, including gallic and protocatecuic acids, had non-mutagen or toxic activity (Cardador-Martínez et al., 2002; Ferguson, 2001 Cardador-Martínez et al., 2006; Santos-Cervantes et al., 2007; Aparicio-Fernández et al., 2005). 3.4. Antimutagenic activity Dose–response curves of AFB1 mutagenicity in tester strain TA100 showed that at 5 lg/tube QPTW was not toxic to the bacteria (data not shown). In accordance, these doses were chosen for all subsequent antimutagenicity assays. The antimutagenic evaluation of QPTW showed an inhibition range on AFB1 mutagenicity from 16 to 60% (Table 1). These results suggest that QPTW possess dose-dependent antimutagenic properties against AFB1 between 0.0 and 12.8 lg/mL. It will be necessary to evaluate if doses above 12.8 lg/mL exhibit any further increased inhibition. However, the antimutagenic activity of QPTW could be defined as ‘‘strong” since it presented more than 50% inhibition of mutagenic activity of AFB1 (Tzyh-Chyuan et al., 1999); these results agree with early reports which indicate that gallic and protocatechuic acids have been associated with antimutagenic and anticarcinogenic activity at least in vitro or in animal systems (Ferguson, 2001).

Table 1 Antimutagenic activity of QPTW against Aflatoxin B1 (AFB1) Mutagen AFB1 (lg tube1)a

QPTW (lg tube1)

Revertant colonies plate1b

Inhibition (%)

0.5 0.5 0.5 0.5 0.5

0.0 1.6 3.2 6.4 12.8

2186 ± 175a 1844 ± 19b 1555 ± 26c 1102 ± 36d 875 ± 16e

0 16 29 50 60

Averages in the same column with different letters are statistically different (a < 0.05). QPTW, quebracho phenolics recovered from tannery wastewaters. a Total volume in tube was 10 mL. b Results are the average of three independent experiments ± SD. Triplicate plates were tested per dose per experiment. Spontaneous revertant colonies were from 106 to 115/plate.

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Table 2 Antioxidant and antiradical activities of QPTW evaluated by b-carotene-linoleate and DPPH methods Sample

Antioxidant activitya (%)

Antiradical activityb (%)

DMSO BHT (0.33 mg/mL) Trolox (2.5 gm/mL)

0.0 68.7 ± 2.25b 90.8 ± 1.84a

0.0 50.8 ± 2.14b 34.2 ± 2.06c

QPTM (mg/mL) 12.3 9.2 6.1

64.4 ± 1.96b 35.5 ± 1.57c 32.0 ± 1.85c

60.8 ± 3.08a 30.0 ± 2.42c 15.5 ± 2.79d

BHT, butyrated hydroxytoluene; QPTW, quebracho phenolics recovered from tannery wastewaters. Averages in the same column with different letters are statistically different (a < 0.05). Data were obtained from triplicate assays. a b-carotene-linoleate method. b DPPH method (1,1-diphenyl-2picryl hydrazyl).

Due to the level of inhibition of mutagenic activity of AFB1, it will be of interest to evaluate the capacity of QPTW to inhibit AFB1 found in food stuffs, like maize and their sub products (Hsieh, 1989; Guzman de Peña et al., 1995); and additionally studying their toxicity to mammals in vivo due to potential impurities, to even consider these extracts as a possible means of protection against aflatoxin B1. 3.5. Antioxidant activity QPTW exhibited antioxidant activity at all concentrations tested (6.1, 9.2 and 12.3 mg/mL). However, the antioxidant activity was lower than that of Trolox at dose tested (2.5 mg/mL) (p < 0.05) (Table 2). The antioxidant activity of QPTW (64.4%) at dose of 12.3 mg/ mL was similar (p < 0.05) to that of BHT (68.7%) at dose of 0.33 mg/ mL. Clearly the antioxidant activity of QPTW was significantly lower compared to that of BHT at the concentrations sampled. The antioxidant activity of QPTW was dose-dependent (Table 2). QPTW showed antiradical activity at all concentrations assayed. Interestingly, QPTW at dose of 12.3 mg/mL showed an antiradical activity of 60.8%, higher (p < 0.05) than that of BHT (50.8%) and Trolox (34.2%) (Table 2). Even at dose of 9.2 mg/mL QPTW antiradical activity (p < 0.05) was similar to that of Trolox. Furthermore, reports indicate that protection against oxidative damage is one of the most widely described attributes of phenolics in general, including gallic and protecatecuic acids (Robbins, 2003; Ferguson, 2001). Additionally, Tamural et al. (2004) reported that protocatechuic acid reduced serum cholesterol by reducing serum and hepatic high- and low-density lipoproteins in rats, defining the antioxidant properties of this phenolic acid. Quebracho tree extracts have been used to evaluate toxicity, as well as protein and food conversion in ruminants (Paolini et al., 2003a, b; Athanasiadou et al., 2000, 2001a, b); doses of 0.5–1.5 g/ kg live-weight were not toxic to ruminants (Hervas et al., 2003); meanwhile doses of 30 and 60 g/kg food (fresh weight) showed no effect on live-weight gain and food conversion efficiency of sheep in a long-term evaluation (Athanasiadou et al., 2000). Doses used to evaluate toxic, mutagenic, antimutagenic and antioxidant activities of QPTW in the present work, are far lower than those used by Hervas et al. (2003) and Athanasiadou et al. (2000); however, it will be of interest to evaluate the toxicity of QPTW in ruminants, rats or chickens. 4. Conclusions It was demonstrated that QPTW could be an outstanding source of phenolics such as gallic or protocatechuic acids. We also demonstrated that QPTW did not display either toxic or mutagenic activ-

ity in bacteria and in turn, may be not surprisingly, they showed antimutagenic and antioxidant activities at evaluated doses. Currently we are carrying out studies to evaluate the potential of QPTW as antimicrobial agent against pathogenic fungi. The discrepancies found between the two methods used in this work to assess antioxidant activity of QPTW could be due to the fact that individual antioxidants may in some cases act by multiple mechanisms in a single system depending on the reaction system. Furthermore, antioxidants may respond in a different manner to different radical or oxidant sources (Huang et al., 2005; Prior et al., 2005). QPTW could be an interesting alternative to evaluate from different aspects for industrial exploitation, because of the high yield and concentration of phenolics obtained from the aqueous solution. 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