Redalyc.Total Phenols and Antioxidant Activity of Commercial and Wild

Ciencia y Tecnología Alimentaria ISSN: 1135-8122 [email protected] Sociedad Mexicana de Nutrición y Tecnología de Alimentos México

Alvarez Parrilla, E.; de la Rosa, L. A.; Martínez, N. R.; González Aguilar, G. A. Total phenols and antioxidant activity of commercial and wild mushrooms from Chihuahua, Mexico Ciencia y Tecnología Alimentaria, vol. 5, núm. 5, 2007, pp. 329-334 Sociedad Mexicana de Nutrición y Tecnología de Alimentos Reynosa, México

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TOTAL PHENOLS AND ANTIOXIDANT ACTIVITY OF COMMERCIAL AND WILD MUSHROOMS FROM CHIHUAHUA, MEXICO FENOLES TOTALES Y CAPACIDAD ANTIOXIDANTE DE HONGOS COMERCIALES Y SILVESTRES DE CHIHUAHUA, MÉXICO Alvarez-Parrilla, E.1*; de la Rosa, L. A.1; Martínez, N. R.1; González Aguilar, G. A.2

1Departamento de Ciencias Básicas, Universidad Autónoma de Ciudad Juárez (UACJ), Instituto de Ciencias Biomédicas, Anillo Envolvente del PRONAF y Estocolmo s/n, Ciudad Juárez, Chihuahua, CP 32310, México. 2Centro de Investigación en Alimentación y Desarrollo, A. C. (CIAD, A. C.), Dirección de Tecnología de Alimentos de Origen Vegetal, Carretera a la Victoria Km 0.6, La Victoria, Hermosillo, Sonora, CP 83000, México

Recibido/Received 11-06-2007; aceptado/accepted 07-08-2007 *Autor para la correspondencia/Corresponding author. E-mail: [email protected] Abstract Three wild edible mushrooms (Agaricus sp., Boletus sp., and Macrolepiota sp.) from the North of Mexico (Chihuahua state) and two commercial species (Agaricus bisporus white strain and Portabella: Agaricus bisporus brown strain) were analyzed to determine their proximate composition, total phenols and antioxidant activity. Wild mushrooms presented lower humidity values with respect to commercial species. Other proximate parameters such as total protein, lipids, ashes and carbohydrates were similar to those reported for edible mushrooms. Total phenols and antioxidant activity (Ferric Reducing/ Antioxidant Power assay, FRAP) were determined from 80 % methanol extracts. Wild Mushrooms had higher phenol content and antioxidant capacity than commercial mushrooms. A direct correlation between phenols and antioxidant activity (r2 = 0.986) was observed. Due to these characteristics, wild mushrooms could be considered as a complement in the diet for the health benefits they present.

Resumen Se analizó la composición proximal, fenoles totales y capacidad antioxidante, de tres hongos silvestres (Agaricus sp., Boletus sp., and Macrolepiota sp.) del norte de México (Chihuahua) y dos comerciales (Agaricus bisporus cepa blanca and Portalella: Agaricus bisporus cepa café). Los hongos silvestres presentaron valores menores de humedad que los comerciales. Los demás parámetros proximales (proteínas, lípidos, cenizas y carbohidratos totales) fueron similares a los reportados para hongos silvestres. La concentración de fenoles totales y actividad antioxidante (Ferric Reducing/Antioxidant Power assay, FRAP) se determinaron a partir de extractos con metanol al 80 %. Los hongos silvestres presentaron valores más altos en fenoles totales y actividad antioxidante, frente a los hongos comerciales. Se observó una correlación directa (r2 = 0,986) entre fenoles totales y actividad antioxidante. Estas características de los hogos silvestres hacen que puedan ser considerados, como complemento de la dieta ya que puede presentar buenos beneficios en la salud.

Keywords: Edible mushrooms, proximate composition, polyphenols, antioxidant activity, FRAP Palabras clave: Hongos comestibles, análisis proximal, polifenoles, actividad antioxidante, FRAP INTRODUCTION several diseases (Ferreira et al., 2007). For this reason, during the last decade, an increase in the consumption of Reactive oxygen species (ROS) are formed during antioxidants-rich foods such as fruits and vegetables has the normal cellular metabolism, however, when the been observed. concentration increase, they overcome the physiologic Among the antioxidant compounds, polyphenols antioxidant mechanisms and become toxic. Epidemiological have gained importance due to their large array of studies correlate high ROS values with DNA damage, heart biological actions that include free radical scavenging, diseases, cancer and other chronic and degenerative metal chelation and enzyme modulation activities, diseases (Tesoriere et al., 2004). There are several cellular inhibition of LDL oxidation, among others (Rodrigo and defenses against elevated levels of free radicals, such as Bosco, 2006). The term polyphenol refers to a complex the enzymes superoxide dismutase, catalase or glutathione group of compounds that includes in their structure an peroxidase, which protect the organism against ROS. aromatic ring bearing one or more hydroxyl groups. They Ingestion of exogenous antioxidants through fruit and include simple phenols such as phenolic acids and vegetable rich diets can also help neutralize these free derivatives, as well as complex structures such as flavones, radicals, and consequently decrease the prevalence of flavonoids, anthocyanins, among others.

Mushrooms have been used as traditional foods specimens. We used this classification having in mind that and medicines in different parts of the world, including when people from the region collect wild mushrooms, they Asia, Africa and America. In Mexico, wild edible usually collect different non toxic species of the same mushrooms have been part of the diet, especially among genus, assuming that they are the same mushroom. ethnic groups. They have nutritional relevance due to their Commercial mushrooms (Champignon (champignon c): high fiber, minerals and protein content, as well as low fat Agaricus bisporus white strain and Portabella: Agaricus content (León-Guzmán et al., 1997). Moreover, in the last bisporus brown strain) were purchased at a local few years, an increasing interest in the consumption of supermarket in Ciudad Juarez. Wild and commercial mushrooms has arisen, due to their elevated polyphenol mushrooms were cut, weighted and frozen at -80 °C for 1 concentration, which correlates with an elevated day, lyophilized for 48 h (Labconco Freeze dry/shell freeze antioxidant activity. Several studies analyzing the total system), milled and stored at -80 °C. In order to minimize phenols and antioxidant activity of fresh and cooked wild variability between individuals from the same species, all and commercial mushrooms have been published (Mau et mushrooms from the same specie were homogenized. al., 2001; Hsu et al., 2002; Mau et al., 2002; Yang et al., 2002; Lakshmi et al., 2004; Lo and Cheung, 2005; Choi et Proximate analysis al., 2006; Ferreira et al., 2007). However, as far as we know, Moisture content was determined by lyophilization characterization of species grown in different regions of (48 h) in order to prevent polyphenol and antioxidant México has not been reported. activity losses. Protein, ash and fat were determined The objective of this study was to evaluate the according to the AOAC (2000) procedures. For protein proximate composition, total phenols and antioxidant determination, a conversion factor of 4.38 was used, in activity of wild and commercial edible mushrooms from the accordance with León-Guzmán et al., (1997). Total fat was State of Chihuahua, in the north of Mexico. determined by Soxhlet extraction with hexane and total carbohydrates were calculated by difference. Total energy was calculated according to equation 1 (Manzi et al., 2004): MATERIALS AND METHODS Energy (kcal) = 4(g protein + g carbohydrate) + 9(g fat) (1)

Samples Total phenols determination Wild mushrooms (wild champignon: (Champignon Mushroom extracts were obtained according to the w) Agaricus sp., Boletus sp, and Macrolepiota sp.) were methodology proposed by Kähkönen et al. (1999). Briefly, collected at the end of the rain season at a Holm oak forest, 0.1 g of powdered dried mushrooms was weighted into a near Namiquipa (29°15’ latitude, 107°25’ longitude, and 1828 test tube. 5 mL of 80 % methanolic solution was added, m over sea level), Chihuahua, Mexico, during September stirred and sonicated for 15 min in the dark. Then extract 2004. Five to ten mushrooms of each species (100 g to 1 was centrifuged (3000 g) for 10 min at 4 °C, and the kg, depending of the species) were collected and kept on supernatant was collected. Extraction was repeated and a ice 4-6 h for transportation to the laboratory. Mushrooms total volume of 10 mL was obtained. This extract was used were classified only to genus, using macroscopic for determination of total phenols and antioxidant activity. characteristics. Especial care was taken not to collect toxic Total phenols were determined according to the method

Table 1. Proximate composition (%, fresh weight) of commercial and wild mushrooms. Means with different letters in the same row are significantly different (LSD test, P < 0.05).

Tabla 1. Análisis proximal (% peso fresco) de los hongos comerciales y silvestres. Valores promedio con letras diferentes indican diferencia significativa (prueba DMS, P< 0,05).

Portabella Commercial Macrolepiota Boletus Wild champignon champignon Moisture 91.13 ± 0.20a 90.63 ±2.04a 86.08 ± 0.61b 84.2 ± 2.96b 77.57 ± 0.68c Ash 0.13 ± 0.002a 0.96 ± 0.03b 0.50 ± 0.02c 0.22 ± 0.03d 1.17 ± 0.05e Total fat 0.70 ± 0.09a 0.29 ± 0.04b 0.76 ± 0.11a 0.62 ± 0.04a 1.31 ± 0.06c Protein 2.32 ± 0.16a 3.16 ± 0.10b 1.80 ± 0.20c 2.62 ± 0.15a 4.42 ± 0.16d Total 5.72 4.96 10.86 12.34 15.52 carbohydrates Energy 38.4 35.0 57.5 65.4 91.6 (Kcal/100g FW)

330 SOMENTA ©2007 Alvarez-Parrilla et al.: Total phenols and antioxidant activity of … reported by Georgé et al. (2005), with the Folin-Ciocalteu champignon, which showed clear signs of dehydration, reagent, using caffeic acid in methanol (80 %) as standard. had moisture values in the 85-92 % range. These results Absorbance at 760 nm was determined by using a BioRad are similar to those reported by other authors for fresh Benchmark Plus microplate reader, and results were mushrooms (León-Guzmán et al., 1997; Choi et al., 2006). expressed as mg of caffeic Acid (CAE)/ 100 g fresh weight. As expected, fat levels were low (0.29 to 1.13 % fresh weight, FW; 3.05-7.85 % dried weight, DW), and similar to Antioxidant capacity those reported for several species of commercial and wild Antioxidant capacity was determined by the ferric mushrooms studied elsewhere (León-Guzmán et al., 1997; reducing/antioxidant power assay (FRAP) according to Manzi et al., 2001; Manzi et al., 2004). Commercial and wild Benzie and Strain (1996) and modified by Alvarez-Parrilla champignon presented the lowest and highest fat levels, et al. (2005), with slight modifications. FRAP reagent was respectively. Commercial champignon showed fat content daily prepared and kept at 37 °C, by mixing 0.3 M acetate similar to those reported by Manzi et al. (2001) for raw and buffer, pH 3.6 with 10 mM 2,4,6-tripyridyl-s-triazine (TPTZ, canned champignon. Acrôs Organics, USA) solution in 40 mM HCl plus 20 mM Protein content in the analyzed mushrooms ranged

FeCl3·6H2O, at a 10:1:1 ratio. Assay solutions were prepared from 1.8 to 4.42 % FW (12.95-33.73 % DW). These values by mixing 180 µL of FRAP reagent with 24 µL of 3: 1 water: are higher to those reported by León-Guzman et al. (1997) sample mixture, standard or 80% methanol (blank). for wild mushrooms collected in Queretaro, Mexico (13.2- Methanolic solutions of Fe2+ (80%) in the range of 100- 17.5 % DW), but in the range of the commercial Italian fresh

3000 µM were prepared from a 3000 µM FeSO4·7H2O stock mushrooms reported by Manzi et al. (2001, 2004). solution to obtain the calibration curves. All measurements Portabella (26.2 % DW) and commercial champignon were carried out at 37 °C. Absorbance was measured at (33.73% DW) showed lower protein content than those 595 nm, every 30 s, during 60 min, using a BioRad reported by Dikeman et al. (2005) for the same mushrooms Benchmark Plus microplate reader. Results are reported as (42.4 and 37.5 %, respectively). However, the mushrooms mM Fe2+/100 g fresh mushroom. studied by Dikeman et al. (2005) presented lower moisture content which affects directly protein content. Statistical analysis Total carbohydrate content, calculated by Values are presented as the mean ± SD of four difference, varied from 15.52 to 4.96 % FW (52.9-78.1 % replicates. ANOVA and LSD analyses were performed in DW). These values are in the range of the commercial order to determine differences between mushrooms, using Italian fresh mushrooms reported by Manzi et al. (2001; the commercial software SPSS 13.0 (SPSS Inc. 2004). From the proximate composition, the energy Headquarters Chicago, Illinois, USA). provided by 100 g of fresh samples was calculated and ranged from 38.4 and 91.6 Kcal for Portabella and wild champignon, respectively. These values are similar to those RESULTS AND DISCUSSION reported by Manzi, et al. (2001, 2004) for Italian commercial mushrooms. As already discussed, the higher energy value Proximate composition of the champignon samples is due to the low water content Moisture was determined by lyophilization to of this mushroom. From the low fat content and energy prevent polyphenol degradation and minimize antioxidant value, it can be concluded that wild and commercial activity loss. Table 1 shows the proximate composition of mushrooms can be considered good alternatives for low wild and commercial mushrooms. Moisture values ranged fat/energy diets. from 77.57 to 91.13 %. Commercial samples presented statistically higher moisture values than wild samples. The Antioxidant activity moisture values varied in the order Portabela > commercial It is well known that polyphenols are one of the champignon > Macrolepiota > Boletus > wild champignon. major contributors to the antioxidant activity of fruits, This may be explained by considering that wild mushrooms vegetables and mushrooms (Ferreira et al., 2007). For this were collected at the end of the raining season, when some reason, in this study, total phenols concentration, and dehydration may have occurred. Exposure to different antioxidant activity of commercial and wild mushrooms ambient conditions of wild mushrooms affected were determined, from methanol:water (80:20) extracts. In considerably moisture content. Meanwhile the growth of the literature, total phenolics of different plant tissues have commercial mushrooms is carried out at controlled been reported either as Gallic Acid Equivalent (GAE) or as atmospheres and humidity. The higher moisture of pyrochatecol equivalent. In the present study, caffeic acid Portabella as compared to commercial champignon may be was used as standard, since it has been reported as one of explained considering that the former mushrooms are sold the major phenolic compounds present in some wild and in plastic packages, with moisture controlled atmosphere, commercial mushrooms (Valentão et al., 2005). meanwhile, the later is sold in bulk, and consequently some Figure 1 shows the total phenol concentration of dehydration occurs. All mushrooms, except wild mushrooms, expressed as mg of caffeic acid equivalents

400 3500 Portabella c (A) Champignon (c) 3000 Boletus 300 Macrolepiota 2500 Champignon (w)

d 200 2000

Abs nm 595 1500 mgFW CAE/100 g b 100 a 1000 a

500 0 Portabella Macrolepiota Champignon Champignon Boletus (w) (c) 0 0 1000 2000 3000 Time (s) 5 c Figure 1. Total phenolic content (expressed as caffeic acid (B) equivalents, CAE/ 100 g FW) of 80% methanolic extracts from 4 d wild and commercial mushrooms. Values are mean ± SD from four /100 g /100 g FW 3 determinations. Different letters in the bars indicate statistically 2+ significant differences (LSD test, P < 0.05). 2 b

mmol Fe a,b Figura 1. Concentración fenoles totales (expresado como a equivalentes de ácido caféico, CAE/ 100 g peso fresco) del extracto 1 metanol:agua 80:20 de los hongos comerciales y silvestres. Los 0 valores se reportan como media ± DS de cuatro repeticiones. Letras Portabella Macrolepiota Champignon Champignon Boletus diferentes en las barras indican diferencia significativa (prueba DMS, (w) (c) P < 0,05).

Figure 2. Antioxidant activity, estimated by the FRAP assay (CAE)/ 100 g of fresh weight. Phenolic concentration (expressed as mmol Fe2+/ 100 g FW) of (a) FRAP reaction kinetics of 80 % methanolic extracts from wild and commercial mushrooms, ranged from 45.6 mg CAE/ 100 g FW for commercial measure at 595 nm and (b) Ferric reducing ability of methanolic champignon to 308.3 mg CAE/ 100 g FW for wild extracts determined at 30 min. Values are mean ± SD from four champignon. Figure 1 shows that both commercial species estimations. Different letters in the bars indicate statistically presented statistically the same phenolic concentration, significant differences (LSD test, P < 0.05). and slightly lower than that for Macrolepiota. Boletus Figura 2. Actividad antioxidante, determinada por el método FRAP presented a total phenol concentration of 169.6 ± 26.7 mg (expresado como mmol Fe2+/ 100 g peso fresco) de (a) cinética de CAE/100 g FW. The higher phenolic concentration reacción FRAP de los extractos metanol:agua (80:20) de los hongos observed for wild champignon can be partially explained comerciales y silvestres, determinado a 595 nm y (b) actividad antioxidante de los extractos determinado a los 30 min. Los valores considering its low water content due to dehydration. Wild se reportan como media ± DS de cuatro repeticiones. Letras diferentes mushrooms presented, in general, statistically higher en las barras indican diferencia significativa (prueba DMS, P < 0,05). phenolic concentration than commercial ones. The total phenolic concentration of the samples analyzed in the to Fe2+-TPTZ. In this way, a higher Fe3+-TPTZ reduction present study (4.87 to 13.74 mg CAE/ g DW) were higher means a higher antioxidant activity. than those reported by Mau et al. (2002), and Choi et al. The original FRAP methodology proposed by (2006), but in the range of those reported by Lo and Cheung Benzie and Strain (1996) established a 4 min interval before (2005), Cheung et al. (2003), Yang et al. (2002), and Ferreira the determination of the FRAP value. However, as shown et al. (2007) for different commercial and wild mushrooms in Figure 2a, and in agreement with Pulido et al. (2000) and found world widespread. Alvarez-Parrilla et al. (2005), the reduction of Fe3+ to Fe2+ The antioxidant activity of wild and commercial in the presence of the mushroom extract follows a slow mushrooms has been determined by several methods, such kinetic mechanism, and even after 30 min the reaction was as 2,2-diphenyl-1-pycrilhydracyl radical, DPPH (Mau et al., not totally completed. For this reason, FRAP values were 2001; Mau et al., 2002; Cheung et al., 2003; Lakshmi et al., determined at 30 min, as suggested in the literature (Pulido 2004; Ribeiro et al., 2006; Turkoglu et al., 2007), TEAC et al., 2000; Alvarez-Parrilla et al., 2005). When the FRAP (Lakshmi et al., 2004), conjugated diene method (Mau et values determined at 4 and 30 min were compared, a 32-97 al., 2001; Mau et al., 2002), Ferricyanide reducing power % increment was observed. However, the increment in the (Mau et al., 2001; Mau et al., 2002), and ferric reducing/ FRAP values at 60 min was less than 20 % higher compared antioxidant power assay, FRAP (Lakshmi et al., 2004), to those observed at 30 min. among others. Figure 2b shows the FRAP values of the 80 % In the present work, antioxidant activity was methanolic mushroom extracts determined at 30 min and measured by the FRAP method, which measures the expressed as mmol Fe2+/ 100 g FW. Figure 2b shows that capacity of an antioxidant to reduce a Fe3+-TPTZ complex the antioxidant activity varied in a similar pattern than the

5 found in mushrooms, in agreement with several authors (Lo and Cheung, 2005; Choi et al., 2006; Ferreira et al.,

4 2007). However it is important to evaluate the type of phenol present in mushroom and its individual contribution to the total antioxidant capacity. In this regards, studies 3

/100 g FW /100 g are in progress evaluating other phenols and antioxidants 2+ present in mushrooms. 2 In conclusion, the results reveal that wild Mmol Fe mushrooms could be an important source for low caloric, 1 low fat functional foods, with high level of polyphenols and antioxidant activity, especially for the ethnic groups

0 (Tarahumaras tribes) living in the North of Mexico. From 0 50 100 150 200 250 300 350 the wild mushrooms studied, «Boletus» may be an mg CAE / 100 g FW interesting group due to their high nutritional value, total phenol concentration and antioxidant activity, as well as Figure 3. Linear correlation between total polyphenols (expressed as caffeic acid equivalents, CAE/ 100 g FW) and antioxidant activity its high relative abundance in the region. As far as we (expressed as mmol Fe2+/ 100 g FW) of 80% methanol extract from know, this is the first study of Mexican wild edible wild and commercial mushrooms. Values are mean ± SD from four mushrooms were in vitro antioxidant activity analysis was estimations. Correlation coefficient = 0.9721, significance level P evaluated and compared with commercial species. < 0.002.

Figura 3. Correlación linear entre los fenoles totales (expresado como equivalentes de ácido caféico, CAE/ 100 g peso fresco) y ACKNOWLEDGEMENTS actividad antioxidante (expresado como mmol Fe2+/ 100 g peso fresco) de de los extractos metanol:agua 80:20 de los hongos comerciales y silvestres. Los valores se reportan como media ± DS Financial support from UACJ (internal projects) is de cuatro repeticiones. Coeficiente de correlación = 0,9721, nivel gratefully acknowledged. The authors would like to express de significancia P < 0,002. appreciation to Dr. Marcos Lizarraga for his helpful assistance in collecting and identifying wild mushrooms total phenolic concentration in the order: wild and to Héctor Osvaldo Sosa for technical assistance. champignon> Boletus > Macrolepiota >Portabela H = commercial champignon. FRAP values ranged from 0.94 mmol Fe2+/ 100 g FW (10.01 mmol Fe2+/ 100 g DW) for REFERENCES commercial champignon to 4.49 mmol Fe2+/ 100 g FW (20.03 mmol Fe2+/ 100 g DW) for wild champignon. Both Alvarez-Parrilla, E.; de la Rosa, L. A.; Torres-Rivas, F.; commercial mushrooms presented statistically lower FRAP Rodrigo-García, J.; González-Aguilar, G. A. 2005. values, compared with wild mushrooms. Even though Complexation of apple antioxidants: chlorogenic Lakshmi et al. (2004) determined the antioxidant activity acid, quercetin and rutin by β-cyclodextrin (β-CD). of several Indian mushrooms with the FRAP method, Journal Inclusion Phenomena Macrocyclic observing high FRAP values, however, our results can not Chemistry 53, 121-129. be compared with the former study, since they expressed AOAC. Official Methods of Analysis of the Association the antioxidant activity as TROLOX or ascorbic acid of Official Analytical Chemistry, 17th edition equivalents. When the antioxidant activity values of the Maryland, USA, 2000. wild and commercial mushrooms determined by the FRAP Benzie, I. F. F.; Strain, J. J. 1996. The ferric reducing ability method where compared with other fruits, it was observed of plasma (FRAP) as a measure of «antioxidant that mushrooms presented higher antioxidant activity than power»: The FRAP assay. Analytical Biochemistry those reported for peaches, which ranged from 0.84 to 1.2 239, 70-76. mmol Fe2+/ 100 g FW (Rodrigo-García et al., 2006), but lower Böhm, V.; Kühnert, S.; Rohm, H.; Scholza, G. 2006. than those for strawberries (19.3-24.4 mmol Fe2+/ 100 g DW) Improving the nutritional quality of microwave- (Böhm et al., 2006). vacuum dried strawberries: A preliminary study. It has been reported that polyphenols are the main Food Science and Technology International 12, contributors to the antioxidant activity of fruits and 67-75. vegetables (Mau et al., 2002), therefore a correlation study Cheung, L. M.; Cheung, P. C. K.; Ooi, V. E. C. 2003. between total phenols and antioxidant activity of wild and Antioxidant activity and total phenolics of edible commercial mushrooms was carried out. Figure 3 shows a mushroom extracts. Food Chemistry 81, 249-255. good correlation (R2 = 0.9721, significance level P < 0.002) Choi, Y.; Lee, S. M.; Chun, J.; Lee, H. B.; Lee, J. 2006. between these two parameters. This result indicates that Influence of heat treatment on the antioxidant polyphenols may be the main antioxidant compounds activities and polyphenolic compounds of Shiitake

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