Antioxidant activity and phenolic compounds in conventional and organic red grapes (var. Monastrell)
Actividad antioxidante y compuestos fenólicos en uva tinta (var. Monastrell) obtenida por cultivo tradicional y ecológico

Abstract

Growing conditions appear to influence the nutritional composition of different types of fruit. Grape contains a large amount of phenolic compounds in the skin, pulp, and seeds. In this research work, a comparison of the phenolic composition and antioxidant activity between red grapes (Vitis vinifera) var “Monastrell” obtained from organic and conventional cultures has been studied. The identification and quantification of phenolic compounds (anthocyanins, flavonols, and hidroxycinnamic acids) have been realized by high pressure liquid chromatography (HPLC) and analysis of antioxidant activity using the DPPH^· radical. No differences were found in the total phenolic composition as well as in the antioxidant activity in both types of grapes (organic and conventional).

Las condiciones de cultivo parecen influir en la composición nutricional de diferentes tipos de fruta. La uva contiene una gran cantidad de compuestos fenólicos en la piel, la pulpa y las semillas. En el presente trabajo se ha realizado la identificación y cuantificación de compuestos fenólicos (antocianos, derivados hidroxicinámicos y flavonoles) por cromatografía de alta presión (HPLC) y el análisis de la actividad antioxidante utilizando el radical DPPH^· en uva tinta de la variedad Monastell obtenida por técnicas de cultivo ecológico y tradicional. No se han encontrado diferencias en la composición fenólica total en ambos tipos de uva (ecológica y tradicional), así como tampoco en la actividad antioxidante de las mismas.

Palabras clave:

• uva ecológica
• compuestos fenólicos
• actividad antioxidante

Keywords:

• organic grape
• phenolic compounds
• antioxidant activity

Introduction

Organic agriculture is an organic production management system that promotes and enhances biodiversity, biological cycles, and soil biological activity. It is based on minimal use of off farm inputs and on management practices that restore, maintain, and enhance organic harmony. Organic is a labeling term that denotes products produced under the authority of the Organic Foods Production Act (CEE, 1991; Le Guillou & Scharpé, 2001). The primary goal of organic agriculture is to optimize the health and productivity of interdependent communities of soil life, plants, animals, and humans.

Grapes contain a large amount of different phenolic compounds in skin, pulp, and seeds and these are the main compounds responsible for color, taste, mouth feel, oxidation and other chemical reactions in wine. Vineyard location and specific site are very important. In dry climates irrigation generally increases grapevine vigor, berry size, and yield (Roggero, Coen, & Ragonnet, 1986). Within the same fruit type, the growing season, variety, environmental and climatic conditions, plant disease, soil type, geographic locations, and maturity seem to influence the concentration of phenolic compounds (Subramani, Casimir, & Krewer, 2002). These compounds are an integral part of the human diet and considered as biologically active non-nutrient compounds (Subramani et al., 2002). Among these phenolic substances, flavonoids, and in particular anthocyanins, are of interest because of their high occurrence in foods.

The presence of hydroxycinnamic acids and flavonols in grapes is well documented (Wulf & Nagel, 1980). The flavonols are localized in the solid parts of the cluster. In red grapes, flavonols are present in much smaller quantities than anthocyanins and in most cases they have been considered to be negligible.

As polyphenolic compounds, flavonoids share the ability to act as antioxidants by a free radical scavenging mechanism and metal ion chelation (Lodovici et al., 2001). Growing evidence of the role of radicals and antioxidants in the health and ageing has promoted a great interest on these compounds. A wide range of studies has shown that the antioxidative properties of these compounds may protect against arteriosclerosis and coronary heart disease (Hooper et al., 2008; Struch, 2000; Sun, Simonyi, & Sun, 2002; Terao, Kawai, & Murota, 2008).

Materials and methods

Reagents

Formic acid and methanol (MeOH) of analytical grade were supplied by Merck (Darmstad, Germany). Mili-Q system (Millipore Corp., Bedford, MA) ultrapure was used throughout this research. Cyanidin 3-rutinoside was purchased from Polyphenols A.S (Sandnes, Norway). Rutin was purchased from Merck (Darmstadt, Germany). Chlorogenic acid, Trolox (6-hidroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), and 2,2- diphenylpicrylhydrazyl (DPPH) were purchased from Sigma (Madrid, Spain).

Grape samples

The study was conducted during 2 consecutive years (2002 and 2003) on conventional and organic red grapes (Vitis vinifera) var. “Monastrell”, which is the variety most frequently used in Origin Denomination Jumilla wines (Murcia, Spain). The vineyard was located in Jumilla (two conventional and two organics). The vineyards were located on the same characteristic terroir of the Hoya Torres. This distinctive soil is characterized by sandy sediment. The selection of neighboring parcels allowed us to compare organic and conventional vineyards in the same climate and soil conditions.

Samples of 100 berries were carefully taken from different parts of various clusters in every vineyard during the first morning hours, transported to the laboratory, and processed on the same day. The samples were obtained in September at the technological maturity of grapes.

Conventional grapes were treated with plaguicides: Benomyl (Methyl N-[1-(butylcarbamoyl) benzimidazol-2-yl]carbamate), fenarimol (alpha-(2-chlorophenyl)-alpha-(4-chlorophenyl) -5-pyrimidinemethanol), trichlorfon (2,2,2-trichloro-1-dimethoxyphosphorylethanol), whereas organic grapes were treated with natural pesticide such as sulfur and treatment with feromones, allowed by organic agriculture. These chemicals are subject to rigorous testing and authorization procedures before they can be used and winegrowers have to respect precise guidelines and restrictions to use them. Organic grapes were treated with natural pesticides such as dry flowable sulfur, copper salts (reduction of application compared to official doses), and oligoelements.

Physicochemical analysis of grapes

Baume degree, pH, total acidity, were determined following the procedures of OIV (Office International de la Vigne et du vin, 1990).

Extraction of phenolic compounds

The grapes were peeled with a sharp knife. The skin represented ∼13% of the total fresh weight of the grape berry. The samples were homogenized in Ultraturrax T-25 equipment (Janke and Kunkel, Ika-Labortechnick) at 24,000 rpm for 1 min after addition of 4 mL of a solution of MeOH/formic acid (97:3) per gram of skin. The extracts were centrifuged at 5000g for 5 min in a Centromix centrifuge (Selecta, Barcelona), filtered through a 0.45 nm membrane filter Millex-HV13 (Millipore), and analyzed by high pressure liquid chromatography (HPLC). Three extracted were made for each sample collected and sampling was done by triplicate.

HPLC-DAD analysis of phenolics

Twenty microliters of every sample were injected for HPLC analysis using a Merck-Hitachi pump L-7100 (Merck-Hitachi, Darmstadt, Germany) and a diode array detector Merck-Hitachi 7455, employing a reversed-phase column Lichrochart 100 RP-18 column (Merck, Darmstadt, Germany) (25 × 0.4 cm², 5 mm particle size), and water plus 5% formic acid (solvent A) or HPLC grade methanol (solvent B) as solvents at a flow rate of 1 mL min-1. The elution was performed with a gradient starting with 2% B to reach 32% B at 30 min, 40% B at 40 min, and 95% B at 50 min, becoming then isocratic for 5 min (Cantos, Garcia-Viguera, De Pascual-Teresa, & Tomas Barberan, 2000). The HPLC experiments were repeated three times. The phenolic content was expressed as mg/kg of fresh weight taking into account the percentage represented by the skin out of the total berry weight. The chromatograms were recorded at 510, 320, and 360 nm.

Phenolic compounds identification and quantification

The phenolic compounds in grapes were identified by their UV-VIS spectra, recorded with a diode-array-detector and, when possible, by chromatographic comparison with authentic markers. Individual anthocyanins were quantified by comparisons with an external standard of cyanidin 3-rutinoside at 510 nm. Flavonols were quantified as rutin at 360 nm and the hydroxycinnamic acid derivatives at 320 nm as chlorogenic acid. All analyses were repeated three times and the results were expressed as mean values in mg/kg of grapes ± SD. The reproducibility of the HPLC analyses was within 5% error. The total phenolic compounds were calculated by adding the amounts of anthocyanins, flavonols, and hidroxycinamic acids detected in each chromatogram, as previously reported (Cantos et al., 2000).

Statistical data treatment

To assess the differences between the compounds quantified and the antioxidant properties, a statistical analysis was performed using SPSS 12.0. The data are presented as mean ± SE. (standard error). Significance of differences was determined by analysis of variance (ANOVA). A p value of 0.05 was considered statistically significant.

Antioxidant activity

The samples were analyzed according to the technique reported by Brand-Williams et al. (1995). Briefly, a volume of 5 μL methanolic extract (1:1, v/v with methanol) was added to a volume of 2,2-diphenyl-1-picrylhydrazyl (DPPH •) (Sigma, Steinheim, Germany) 0.094 mM in methanol up to completing 1 mL. The free radical scavenging activity using the free radical DPPH reaction was evaluated by measuring the absorbance at 515 nm after 60 min of reaction at 20°C in a spectrophotometer (Varian Cary 50-Bio, Victoria, Australia). The results were expressed as mmol/L Trolox equivalents, a vitamin E analogue (Yamaguchi, Takamura, Matob, & Terao, 1998).

Results and discussion

Physicochemical analysis of grapes

No significant differences in the physicochemical parameters were observed in both types of grapes throughout any of the years studied (Table 1).

Table 1. Enological parameters of both conventional and organic grapes.
Tabla 1. Parámetros enológicos de uva ecológica y convencional.

	Year	Weight 100 berry (g)	°Be	TAc g/L tartaric acid	pH
P1	2002	151.30	12.5	5.10	3.71
	2003	148.32	11.8	5.25	3.73
P2	2002	143.75	11.5	5.71	3.68
	2003	159.51	12.5	6.15	3.61
P3	2002	148.32	11.8	6.00	3.65
	2003	160.78	11.5	7.20	3.40
P4	2002	130.43	12.7	7.16	3.63
	2003	118.24	12.9	6.45	3.66
Notes: TAc, total acidity; °Be, Baume degree; P1, conventional vineyard; P2, conventional vineyard; P3, organic vineyard; P4, organic vineyard. Notas: TAc, acidez total; °Be, grados Baume; P1, viña convencional; P2, viña convencional; P3, viña ecológica; P4, viña ecológica.

Antioxidant activity

No significant differences were observed in antioxidant activity in organic and conventional grapes (4.70 ± 0.11 mM Trolox/g and 4.80 ± 0.25 mM Trolox/g, respectively) in 2002 vintage. In 2003, the average antioxidant activity of organic grapes 4.61 ± 0.02 mM Trolox/g), was higher than observed in conventional grapes (3.99 ± 0.10 mM Trolox/g) although there was no significant difference (Figure 1).

Figure 1. Antioxidant activity in conventional and organic grapes. (-□-) Antioxidant activity organic grape. (-▪-) Antioxidant activity conventional grape.

Figura 1. Actividad antioxidante en uva ecológica y convencional. (-□-) Actividad antioxidante de uva ecológica. (-▪-) Actividad antioxidante de uva convencional.

These values were higher to those found in the Chardonnay and Merlot varieties by Yilmaz and Toledo (2004), and in Norton, Concord and Marechal varieties by Muñoz-Espada, Wood, Bordelon, and Watkins (2004), and lower than the values described in the Muscadina variety by Pastrana-Bonilla, Ago, Sellappan, and Krewer (2003) and in Norton and Cabernet Franc by Hogan, Zhang, Li, Zoecklein, and Zhou (in press).

The variations of the antioxidant activity of the grapes have been previously reported by Lee and Talcott, (2004) in Muscadina red grapes. They found a different behavior for the antioxidant activity of the grapes based on the geographic location of the cultures, and they attributed this different pattern to the ripening, the physiology and the processing of the grapes.

Anthocyanins

Ten anthocyanin-glucosides (five of them acylated) were identified in organic and conventional red grapes skins: delphinidin 3-glucoside, cyanidin 3-glucoside, petunidin 3-glucoside, peonidin 3-glucoside, malvidin 3-glucoside, cyanidin 3-p-coumaroilglucoside, petunidin 3-p-coumaroilglucoside, peonidin 3-p-coumaroilglucoside, petunidin 3-p coumaroilglucoside, and malvidin 3-p-coumaroilglucoside, as described by Fernandez-Lopez, Hidalgo, Almela, and Lopez-Roca (1992) and Cantos, Espin, Fernandez, Oliva, and Tomas-Barberan (2003). The acylated derivatives were globally quantified because of the small individual concentrations observed.

Malvidin3-glucoside was the major anthocyanin in both types of grapes, with mean contributions of about 35.7% and 29.0% of the total anthocyanins in conventional and organic grapes, respectively, in 2002 vintage, and about 42.0% and 32.0% of the total anthocyanins also in conventional and organic grapes, in 2003 vintage (Table 2). These results agree with those presented previously by Fernandez-Lopez et al., (1992) and Cantos et al., (2003).

Table 2. Anthocyanins. Composition of conventional and organic grapes.
Tabla 2. Antocianos. Composición de uva ecológica y convencional.

	Year	Dp-3-glc	Cy-3-glc	Pt-3-glc	Pn-3-glc	Mv-3-glc	Acyl derv
P1	2002	78.50 ± 2.70	62.65 ± 1.57	96.55 ± 1.80	87.60 ± 3.84	270.9 ± 10.50	110.35 ± 17.70
	2003	60.04 ± 2.30	55.36 ± 3.50	70.01 ± 1.09	75.02 ± 2.57	250.04 ± 2.50	112.75 ± 2.00
P2	2002	66.85 ± 3.22	93.35 ± 5.44	68.55 ± 5.29	130.30 ± 8.49	234.02 ± 5.85	115.75 ± 5.27
	2003	83.20 ± 2.08	63.72 ± 3.60	88.30 ± 2.51	75.26 ± 1.50	330.48 ± 2.50	100.23 ± 3.40
P3	2002	91.20 ± 3.02	114.50 ± 5.61	95.15 ± 4.16	143.50 ± 8.15	215.35 ± 10.40	90.05 ± 2.64
	2003	72.25 ± 3.06	62.50 ± 3.72	92.90 ± 3.65	95.66 ± 5.43	300.00 ± 9.50	131.75 ± 4.90
P4	2002	102.50 ± 7.63	100.01 ± 8.74	58.60 ± 3.93	161.20 ± 8.94	211.70 ± 10.16	100.80 ± 4.26
	2003	130.44 ± 1.26	110.30 ± 3.63	140.81 ± 5.10	100.10 ± 10.94	240.23 ± 6.35	165.90 ± 4.71
Notes: Values are expressed as mg/kg fresh weight of total grape berry. Mean values are shown (n = 3) ± standard error. P1, conventional vineyard, P2, conventional vineyard, P3, organic vineyard, P4, organic vineyard; Dp-3–glc, delphinidin 3-glucoside; Cy-3-glc, cyanidin 3-glucoside; Pt-3-glc, petunidin 3-glucoside; Pn-3-glc, peonidin 3-glucoside; Mv-3-glc, malvidin 3-glucoside; Acyl derv, acylated derivatives. Notas: Los valores se expresan por mg/kg de peso fresco de la uva. Los valores se expresan como medias (n = 3) ± error estándar. P1, viña convencional; P2, viña convencional; P3, viña ecológica; P4, viña ecológica; Dp-3–glc, delfinidina-3-glucósido; Cy-3-glc, cianidina-3-glucósido; Pt-3-glc, petunidina-3-glucosido; Pn-3-glc, peonidina-3-glucósido; Mv-3-glc, malvidina-3-glucósido; Acyl derv, derivados acilados.

The anthocyanins concentration was slightly higher in organic grapes (741.93 ± 38.82 mg/kg) than in conventional ones (706.50 ± 35.82 mg/kg) in 2002 vintage (Table 2).

In the year 2003, significant differences were observed between both types of grapes (p = 0.035) at confidence level of 95%, in which the average concentration of anthocyanins reached up to 682.27 ± 14.82 mg/kg in conventional grapes and 821.37 ± 31.14 mg/kg in organic grapes (Table 2). The content in anthocyanins found in 2002 and 2003 was in accordance with the values obtained in Monastrell grapes by Fernández et al., (2003) and in different varieties of red wine grapes by Cho, Howard, Prior, and Clarck (2003). These concentrations were also similar to those obtained by Valls, Lampreave, Nadal, and Arola (2000) in Tempranillo and Cabernet Sauvignon varieties and lower than those reported in Monastrell grapes by Cantos et al., (2003).

The culturing practices and the climatic conditions, in addition to ripening, can affect the composition of grapes (Kennedy, Matthews, & Waterhouse 2002; Scienza, Fregoni, & Boselli, 1981). Those three factors could explain the different concentration of anthocyanins found in both types of grapes during 2002 and 2003.

Hydroxycinnamic acids

Three hydroxycinnamic derivatives were quantified: trans-caffeoyltartaric acid, trans-p-coumaroiltartaric acid and a third derivative quantified, but not identified, which has been named compound 2 (Table 3).

Table 3. Hydroxycinnamic acid derivatives. Composition in conventional and organic grapes.
Tabla 3. Derivados hidroxicinámicos. Composición de uva ecológica y convencional.

	Year	Trans-caffeoyltartaric	Compound 2	Trans-p-coumaroyltartaric
P1	2002	2.80 ± 0.05	2.47 ± 0.35	9.80 ± 0.05
	2003	4.23 ± 0.43	3.55 ± 0.17	6.04 ± 0.02
P2	2002	4.35 ± 0.45	3.09 ± 0.35	10.75 ± 0.15
	2003	8.09 ± 0.70	6.26 ± 0.70	14.04 ± 0.25
P3	2002	3.90 ± 0.50	4.54 ± 0.06	10.07 ± 0.15
	2003	14.01 ± 0.50	4.53 ± 0.50	11.47 ± 0.50
P4	2002	5.48 ± 0.50	4.65 ± 0.06	13.09 ± 0.15
	2003	7.50 ± 0.80	5.70 ± 0.20	13.53 ± 0.28
Notes: Values are expressed as mg/kg fresh weight of total grape berry. Mean values are shown (n = 3) ± standard error; P1, conventional vineyard; P2, conventional vineyard; P3, organic vineyard; P4, organic vineyard. Notas: Los valores se expresan por mg/kg de peso fresco de la uva. Los valores se expresan como medias (n = 3) ± error estándar. P1, viña convencional; P2, viña convencional; P3, viña ecológica; P4, viña ecológica.

In both types of grapes, conventional and organic, the major hydroxycinnamic acids derivatives, were trans-p-coumaroyltartaric and trans-caffeoyltartaric acid throughout 2002 and 2003 (Table 3).

The average concentration of hydroxycinnamic acids derivatives in 2002 vintage was higher in organic grapes (20.86 ± 0.71mg/kg) than in conventional ones (16.63 ± 0.70 mg/kg), though these differences were not significant. Just as in the previous year, the average concentration of hydroxycinnamic acids derivatives in 2003 vintage was higher in organic grapes (28.37 ± 1.39 mg/kg) than in conventional grapes (21.10 ± 1.13 mg/kg), and these differences were not significant. These values were lower than those obtained by Kammerer, Claus, Carle, and Schieber (2004) in different varieties of red grapes (Table 3).

Flavonols

Six flavonols were identified in organic and conventional red grapes skins: myricetin 3-glucoside, quercetin 3-glucoside, quercetin 3-rutinoside, kaempferol 3-glucoside, myricetin and quercetin. In both types of grapes, conventional and organic, myricetin 3-glucoside, quercetin 3-glucoside, and quercetin 3-rutinoside were the most abundant flavonols during 2002 and 2003, followed by kaempferol 3-glucoside, myricetin and quercetin (Table 4).

Table 4. Flavonols. Composition in conventional and organic grapes.
Tabla 4. Flavonoles. Composición de uva ecológica y convencional.

	Year	Myr-3-glc	Q-3-glc	Q-3-Rut	Kp-3-glc	Myricetin	Quercetin
P1	2002	96.40 ± 6.13	46.65 ± 2.59	143.70 ± 10.10	13.90 ± 2.95	21.50 ± 4.15	4.30 ± 0.55
	2003	54.85 ± 2.00	42.40 ± 1.55	55.58 ± 2.58	10.35 ± 0.30	8.45 ± 0.75	10.40 ± 0.20
P2	2002	38.80 ± 3.10	23.30 ± 1.80	92.35 ± 4.90	4.85 ± 1.01	10.75 ± 3.80	2.85 ± 0.15
	2003	77.75 ± 4.10	79.05 ± 4.30	82.05 ± 5.75	7.50 ± 0.10	7.85 ± 0.30	16.85 ± 1.55
P3	2002	38.30 ± 4.10	28.05 ± 5.94	93.85 ± 6.80	4.05 ± 0.75	10.60 ± 0.51	2.75 ± 0.50
	2003	84.05 ± 3.50	80.05 ± 2.35	79.05 ± 3.30	12.25 ± 0.40	12.55 ± 0.50	20.80 ± 0.70
P4	2002	51.00 ± 5.10	50.02 ± 7.80	127.65 ± 5.18	5.50 ± 0.90	19.10 ± 1.80	3.75 ± 0.45
	2003	111.25 ± 8.30	78.35 ± 8.20	84.65 ± 3.77	13.25 ± 0.40	13.50 ± 0.30	16.90 ± 0.70
Notes: Values are expressed as mg/kg fresh weight of total grape berry. Mean values are shown (n = 3) ± standard error.
P1, conventional vineyard; P2, conventional vineyard; P3, organic vineyard; P4, organic vineyard; Myr 3-glc, myricetin 3- glucoside; Q-3-glc, quercetin 3-glucoside; Q-3-rut, quercetin 3-rutinoside; Kp-3-glc, kaempferol 3-glucoside. Notas: Los valores se expresan por mg/kg de peso fresco de la uva. Los valores se expresan como medias (n = 3) ± error estándar.
P1, viña convencional; P2, viña convencional; P3, viña ecológica; P4, viña ecológica; Myr 3-glc, miricetina-3-glucósido; Q-3-glc, quercetina-3-glucósido; Q-3-Rut, quecetina-3-rutinósido; Kp-3-glc, kaempferol-3-glucósido.

In 2002 and 2003 vintage, the average concentration of flavonols was similar 199 in organic grapes than in conventional ones (217.30 ± 19.91 and 249.60 ± 20.61 mg/kg in organic and conventional grapes, respectively) in 2002 vintage, and (303.35 ± 17.86 and 317.55 ± 7.24 mg/kg in organic and conventional grapes, respectively) in 2003 vintage (Table 4).

The concentration of flavonols observed of grapes in 2002 and 2003 was slightly higher to that found in Monastrell grapes by Cantos et al., (2003). Nevertheless, they are within the concentrations described by Cho, Howard, Prior, and Clarck (2003) in different varieties of red grapes and by Lee and Talcott, (2004) in Muscadina grapes.

Total phenolic compounds

In 2002 vintage, the average concentration of the total phenolic compounds was similar in both types of grapes; 979.93 ± 59.22 mg/kg in organically cultured grapes and 991.68 ± 42.26 mg/kg in conventionally cultured ones (Figure 2). These values are lower than those obtained by Valls, Lampreave, Nadal, and Arola (2000) in the Tempranillo and Cabernet sauvignon varieties.

Figure 2. Total phenols in conventional and organic grapes. (-□-) Total phenols in conventional grape. (-▪-) Total phenols in organic grape.

Figura 2. Compuestos fenólicos totales en uva ecológica y convencional. (-□-) Compuestos fenólicos totales en uva ecológica. (-▪-) Compuestos fenólicos totales en uva convencional.

In 2003 vintage, the average concentrations of phenolic compounds was slightly higher in organic grapes (1153.11 ± 49.82 mg/kg) than in conventional grapes (968.72 ± 48.63 mg/kg) although, these differences in the concentration of total phenolic compounds between both types of grapes were found to be not significant.

Conclusion

The antioxidant activity of organic and conventional grape var. Monastrell was similar in both years of study 2002 and 2003. Although in year 2003 it appreciates an antioxidant activity slightly major in organic grape that in the traditional one, significant differences do not exist between both types of culture in any of two vintages analyzed.

As for the analysis of the phenolic compounds only significant differences have been observed in the concentration of anthocyanins between the organic and conventional grape of the vintage 2003. In spite of this, we can conclude that we have not found significant differences as for phenolic compounds and antioxidant activity between grapes obtained by conventional and organic cultures.

Nevertheless and due to the multiple factors that can concern the synthesis of the phenolic compounds in grapes, it would be interesting to continue during successive years with the study of the same ones to be able to conclude on the effect of the organic culture in the synthesis of phenolic compounds of grapes.

Acknowledgements

The authors are grateful to Bodegas San Isidro of Jumilla (Murcia) for supplying wine and grapes. The authors acknowledge the financial support of Consejeria de Ciencia, Tecnologia, Industria y Comercio de la Region de Murcia and Catholic University San Antonio de Murcia.