Influence of solvent on the antioxidant and antimicrobial properties of walnut (Juglans regia L.) green husk extracts

Industrial Crops and Products 42 (2013) 126–132

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Influence of solvent on the antioxidant and antimicrobial properties of walnut (Juglans regia L.) green husk extracts A. Fernández-Agulló a , E. Pereira b , M.S. Freire a , P. Valentão c , P.B. Andrade c , J. González-Álvarez a,∗ , J.A. Pereira b,∗∗ a

Department of Chemical Engineering, School of Engineering, University of Santiago de Compostela, Rúa Lope Gómez de Marzoa s/n, 15782 Santiago de Compostela, Spain Mountain Research Centre, School of Agriculture – Polytechnic Institute of Braganc¸a, Campus Sta Apolónia, Apartado 1172, 5301-855 Braganc¸a, Portugal c REQUIMTE/Laboratório de Farmacognosia, Departamento de Química, Faculdade de Farmácia, Universidade do Porto, Rua Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal b

a r t i c l e

i n f o

Article history: Received 9 November 2011 Received in revised form 31 March 2012 Accepted 19 May 2012 Keywords: Walnut green husk Solvent extraction Total phenols content Antioxidant activity Antimicrobial activity

a b s t r a c t Walnut green husk is an agro-forest waste generated in the walnut (Juglans regia L.) harvest that could be valued as a source of natural compounds with antioxidant and antimicrobial properties. At this respect, the effect of the solvent (water, methanol, ethanol and 50% aqueous solutions of methanol and ethanol) on the extraction yields and extracts bioactive properties was analysed. Total phenols content of the extracts was determined by the Folin–Ciocalteau method. Extract antioxidant activity was evaluated using the reducing power assay and by the ability of the extracts to scavenge the DPPH radical. The scavenging effect of the aqueous extracts on the nitric oxide radical was also evaluated. The highest extraction yield was achieved with water (44.11%) and high bioactive potential was shown by the samples extracted with water/ethanol (1:1) (84.46 mg GAE/g extract; EC50 = 0.95 mg/mL for reducing power and EC50 = 0.33 mg/mL for DPPH assay). All the antioxidant properties analysed showed a concentrationdependent activity. The antimicrobial activity of the aqueous extracts was assessed and showed ability to inhibit the growth of Gram positive bacteria. The results obtained demonstrated the potential of the walnut green husk as an economical source of antioxidant and antimicrobial agents. © 2012 Elsevier B.V. All rights reserved.

1. Introduction The walnut (Juglans regia L.) is a tree traditionally cultivated for its valuable wood and fruits. The walnut seed is a nut of high economic interest to the food industry and is globally popular and valued for its nutritional, health and sensory attributes (Martínez et al., 2010). The Iberian Peninsula yearly produces 13,500 t of walnut kernel, where 75% are produced in Spain and the remaining 25% in Portugal. Other by-products derived from the walnut tree have been used in several applications. Thus, green walnuts, shells, kernels, bark, and leaves have been used in both cosmetic and pharmaceutical industries (Stampar et al., 2006). The leaves have been widely used in folk medicine for the treatment of skin inflammations, hyperhidrosis and ulcers and for its antdiarrhoeal, anthelmintic, antiseptic and astringent properties (Almeida et al., 2008). Dry walnut leaves are also frequently used as infusions (Pereira et al.,

∗ Corresponding author. Tel.: +34 881816758; fax: +34 981528050. ∗∗ Corresponding author. Tel.: +351 273303277; fax: +351 273325405. E-mail addresses: [email protected] (J. González-Álvarez), [email protected] (J.A. Pereira). 0926-6690/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.indcrop.2012.05.021

2007). The shell is used as a filtration media to separate crude oil from water (Srinivasan and Viraraghavan, 2008) and the walnut green husk is the basic material for the traditional walnut liqueur (Stampar et al., 2006). Different works have characterised the phenolic composition of walnut by-products (Fukuda et al., 2003; Li et al., 2006; Stampar et al., 2006; Pereira et al., 2007; Zhang et al., 2009). The beneficial effects derived from the phenolic compounds, such as their anticarcinogenic, antimutagenic and cardioprotective activities have been attributed to their antioxidant activity (Madhavi et al., 1996; Balasundram et al., 2006). At this respect, numerous studies have been focused on the obtaining of antioxidants from natural sources. Researches have been promoted by the need to find natural substitutes for the synthetic antioxidants, suspected to be potentially toxic (Contini et al., 2008). In this context, the cheap waste products from the food, forest or agricultural industries are particularly interesting for the environmental and economical benefits resulting from their re-use. Several plant materials have been analysed with this purpose: plants and agro-industrial by-products (Balasundram et al., 2006), nuts and their by-products such as almond hulls (Pinelo et al., 2004), hazelnut shell and kernels (Contini et al., 2008; Delgado et al., 2010), Gevuina avellana hull (Moure et al., 2000), hazel leaves (Oliveira et al., 2007) or

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chestnut fruit and their by-products (Barreira et al., 2008; Vázquez et al., 2008, 2009). Plant by-products could also be used as antimicrobial agents and some studies have demonstrated their antimicrobial activity (Rauha et al., 2000; Pereira et al., 2007; Kavak et al., 2010; Zˇ ivkovic´ et al., 2010). The tendency of the consumers to avoid products prepared with preservatives of chemical origin together with the increased resistance to antibiotics (Rauha et al., 2000; Oliveira et al., 2007) is promoting the interest in using natural antimicrobial compounds, especially extracted from plants (Zhu et al., 2004). The phenolic compound juglone is present in all parts of the walnut and is known for its antimicrobial effect (Stampar et al., 2006). Thus, the walnut by-products could be valorised as sources of natural antioxidants and antimicrobial agents. At this respect, the walnut kernel has been previously evaluated as an antioxidant by Li et al. (2006), Labuckas et al. (2008) and Zhang et al. (2009). Pereira et al. (2008) also evaluated the antimicrobial activity of six different walnut kernels. The antioxidant and antimicrobial capacity of the walnut leaves was demonstrated by the same author (Pereira et al., 2007). The aqueous extracts of walnut green husk were studied by Oliveira et al. (2008) and the methanolic ones by Ghasemi et al. (2011). Carvalho et al. (2010) determined the antioxidant activity of walnut leaf, seed and green husk. The green husk is one of the major waste products from the walnut production that nowadays has a scarce use. The results obtained by Oliveira et al. (2008) and Carvalho et al. (2010) showed the potential of this low cost natural material as source of phenolic compounds with antiradical and antimicrobial activities and demonstrated that the knowledge in green husk should be increased. Then, the aim of this work was to analyse the effect of the solvent on the properties of walnut green husk extracts. Solvents of varying polarity were used: water, methanol, ethanol and their aqueous solutions. These solvents have been frequently used to extract phenolic compounds from natural sources (Moure et al., 2001; Contini et al., 2008; Al-Farsi and Lee, 2008; Vázquez et al., 2008). Extracts were then compared with respect to their total phenols content, reducing power assay and scavenging effect on DPPH (2,2-diphenyl-1-picrylhydrazyl). The scavenging effect on nitric oxide radical and antimicrobial capacity against Gram positive and Gram negative bacteria of the aqueous extracts were also evaluated.

2. Materials and methods 2.1. Reagents and standards Gallic acid, methanol, 2,2-diphenyl-1-picrylhydrazyl, iron (III) chloride, sodium chloride, sulfanilamide and agar-agar were obtained from Sigma–Aldrich (St. Louis, USA). Sodium dihydrogen phosphate dihydrate, potassium hexacyanoferrate (III), N-(1-naphthyl)ethylene-diamine dihydrochloride and phosphoric acid and glucose were purchased from Merck (Darmstadt, Germany). Trichloroacetic acid was obtained from Fluka (Steinheim, Switzerland). Folin–Ciocalteu’s phenol reagent, sodium carbonate anhydrous, hydrochloric acid, ethanol, di-sodium hydrogen phosphate dehydrate, and sodium hydroxide were obtained from Panreac (Barcelona, Spain). Yeast extract, peptone and tryptone were obtained from Himedia (Mumbai, India). Sodium nitroprussiate dihydrate was from Riedelde Haën (St. Louis, MO). The water was treated in a Milli-Q water purification system (Millipore, Bedford, MA, USA).

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2.2. Raw material Samples of walnut green husk from the Cv. Mellanaise variety were collected in Braganc¸a, northeast of Portugal. The orchard has a planting density of 3.5 m × 7 m. The trees were ten years old and no phytosanitary treatments were applied. The fruits were handpicked from the soil and the walnut green husk removed. To preserve antioxidant properties, walnut green husk was stored in plastic bags, immediately frozen at −20 ◦ C, and then freeze dried. 2.3. Extracts preparation Before the extraction process, the walnut green husk was ground in a mill. For the aqueous extraction (WE), 5 g of the powdered sample were extracted with 250 mL of boiling water for 45 min and filtered through Whatman no. 4 paper. In the extractions with absolute methanol (ME), ethanol (EE), methanol–water 50% (v/v, WME) and ethanol–water 50% (v/v, WEE), 1.5 g of sample were extracted with 25 mL of the tested solvent for 45 min at room temperature and filtered through Whatman no. 4 paper. The solvents were evaporated under vacuum in a Büchi R-210 rotavapor and the extracts obtained were redissolved in water to a final concentration of 50 mg/mL and stored in the dark at 4 ◦ C for further use. All the extractions were done in duplicate. 2.4. Total phenols content Total phenols content in the obtained extracts was determined by the method described by Singleton and Rossi (1965) with some modifications. Briefly, 1 mL of an aqueous solution of the extract was mixed with 1 mL of Folin–Ciocalteuˇıs reagent. After 3 min, 1 mL of saturated sodium carbonate solution was added to the mixture and adjusted to 10 mL with distilled water. The reaction was kept in the dark for 90 min, after which the absorbance at 725 nm was measured. The phenols content was calculated as a gallic acid equivalent from the calibration curve of gallic acid standard solutions (0.01–1 mM) and expressed as mg gallic acid equivalents (GAEs)/g of extract. 2.5. Antioxidant activity 2.5.1. Reducing power assay The reducing power was determined according to the procedure of Berker et al. (2007). Several concentrations (0.01–5 mg/mL) of sample extracts (1 mL) were mixed with 2.5 mL of 200 mmol/L sodium phosphate buffer (pH 6.6) end 2.5 mL of 1% potassium ferricyanide. The mixture was incubated at 50 ◦ C for 20 min. After incubation, 2.5 mL of 10% trichloroacetic acid (w/v) were added and then the mixture was centrifuged at 1000 rpm for 8 min (Centorion K24OR-2003 refrigerated centrifuge). The upper layer (2.5 mL) was mixed with 2.5 mL of deionised water and 0.5 mL of 0.1% of ferric chloride. The absorbance was measured spectrophotometrically at 700 nm (higher absorbance readings indicate higher reducing power). Extract concentration providing 0.5 of absorbance (EC50 ) was calculated from the graph of absorbance at 700 nm against extract concentration in the solution. 2.5.2. DPPH scavenging activity The radical scavenging ability of the extracts was monitored using the stable free radical DPPH (2,2-diphenyl-1-picrylhydrazyl) following the method described by Hatano et al. (1988). Aqueous solutions of sample extracts (0.01–2 mg/mL) were prepared. Extract solutions (0.3 mL) were mixed with 2.7 mL of a freshly prepared DPPH solution (6 × 10−5 M in methanol). The mixture was shaken vigorously and left to stand at room temperature for 60 min in the dark (until stable absorbance values were obtained). The

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reduction of the DPPH radical was measured by monitoring the decrease of absorption at 517 nm. DPPH scavenging effect was calculated as the percentage of DPPH discoloration using the following equation: % scavenging effect = [(ADPPH − AS )/ADPPH ] × 100, where AS is the absorbance of the solution when the sample extract has been added at a particular level and ADPPH is the absorbance of the DPPH solution. The extract concentration providing 50% inhibition (EC50 ) was calculated from the graph of scavenging effect percentage against extract concentration in the solution.

respect, the one-way analysis of variance (ANOVA) was used, followed by the Dunnett T3 test. All statistical tests were performed at a 5% significance level using SPSS 18.0 software. A regression analysis, using Excel from Microsoft Corporation, was established between the total phenols content and EC50 values obtained in the antioxidant assays tested.

2.5.3. Nitric oxide scavenging activity The nitric oxide (NO) scavenging activity of the aqueous extract was determined using the method described by Sousa et al. (2008b). The antiradical activity was determined in a Multiskan Ascent plate reader. 100 ␮L of sodium nitroprusside (SNP, 10 mM) was incubated with 100 ␮L of walnut green husk extract at different concentrations for 60 min, at room temperature under light. All solutions were prepared in phosphate buffer. After incubation, 100 ␮L of Griess reagent (1% sulphanilamide and 0.1% naphthylethyldiamine in 2% phosphoric acid) was added to each well. The mixture was incubated at room temperature for 10 min and the absorbance of the chromophore formed during the diazotization of nitrite with sulphanilamide and subsequent coupling with naphthylehylendiamine was read at 562 nm. Three assays were performed, each one in triplicate. The NO scavenging effect (%) and the extract concentration providing 50% inhibition (EC50 ) were calculated as indicated in the DPPH method.

Extraction with solvents is frequently used for the isolation of antioxidant compounds, and both extraction yield and antioxidant activity of the extracts have a strong relationship with the solvent employed, mainly due to the different polarity of the compounds obtained (Moure et al., 2001). In particular, for the extraction of phenolic compounds to be used as antioxidants, organic solvents are commonly used (Pokorny and Korczak, 2001). The selection of the most appropriate solvent is a determinant factor on extract properties and due to the diverse structure and composition of the matrix, each matrix-solvent system shows a particular behaviour that cannot be predicted (Al-Farsi and Lee, 2008). For this reason, in this work, different solvents were assayed for the extraction of walnut green husk (water, methanol, ethanol and 50% aqueous solutions of methanol and ethanol) and extraction yield, total phenols content and antioxidant and antimicrobial properties of the extracts obtained were compared. Table 1 shows the results obtained for extraction yield, total phenols content and EC50 values for the reducing power and DPPH assays. The solvents used for the extraction of walnut green husk showed significantly different extraction capacities (P < 0.05). The values of the extract obtained per 100 g of raw material varied from 3.90% for the ethanolic extraction (EE) to 44.11% for the aqueous one (WE). The extraction yield increased in the following order: ethanol < methanol < 50% methanol < 50% ethanol < water. Extraction yield depended on the polarity of the solvent in such a way that when the polarity of the solvent decreased (water > methanol > ethanol) the extraction yield values decreased in the same order. The high temperature and solid–liquid ratio used in the extraction with water could also explain the high extraction yield obtained. Extraction temperature is an important factor since is related with the solubility and with the diffusion coefficient of the solute. High temperature could also facilitate the disruption of the matrix tissues and more compounds would distribute to the solvent (Al-Farsi and Lee, 2008). In addition, according to mass transfer principles, diffusivity increases with increasing solid–liquid ratio to increase the differences of phenol concentration on the medium (Pinelo et al., 2004).

2.6. Antimicrobial activity The bacterial strains tested with the aqueous extracts of walnut green husks were Bacillus cereus, Bacillus subtilis, Staphyloccocus aureus, Staphyloccocus epidermis (Gram + bacteria), Escherichia coli and Pseudomonas aeruginosa (Gram −). All the microorganisms were obtained from the Biology Department of University of Minho (Braga, Portugal). The bacterial stocks were maintained at 4 ◦ C on LB agar [tryptone 1% (w/v), yeast extract 0.5% (w/v), NaCl 1% (w/v) and agar 2% (w/v)], being sub-cultured periodically at 37 ◦ C. 2.6.1. Preliminary assays for antimicrobial activity The screening of antimicrobial activity against the Grampositive and Gram-negative bacteria as well as the determination of the minimal inhibitory concentration (MIC) values were achieved by an adaptation of the agar streak dilution method based on radial diffusion (Sousa et al., 2006). Suspensions of the microorganisms were prepared and mixed with molten agar (0.8%, w/v) in order to contain approximately 106 cfu/mL. A volume of 8 mL of this mixture was seeded as a lawn onto the surface of plates containing the LB medium. Samples to be tested for antimicrobial potential were placed (85 ␮L) in a hole made in the centre of the solid medium (3 mm depth, 5 mm diameter). The MIC was considered to be the lowest concentration of the tested sample (5–100 mg/mL) able to inhibit the growth of bacteria (after 24 h at 37 ◦ C). The diameters of the inhibition zones were measured using a ruler, with an accuracy of 0.5 mm. Each inhibition zone diameter was measured three times (in three different plates) and the results were expressed as an average of the radius of the inhibition zone in mm. Plates inoculated with each sensitive indicator microorganism were used as controls. 2.7. Statistical analysis All the analyses were done in duplicated and the values averaged. The existence of significant differences among the results for extraction yield, total phenols content and antioxidant properties of the extracts depending on the solvent used was analysed. At this

3. Results and discussion

3.1. Total phenols content and antioxidant activity The Folin–Cioalteau assay, used for the determination of the total phenols content of the walnut green husk extracts, has been employed for many years as a measure of total phenols in natural products. It is simple and widespread method, although it presents some limitations as there are some interfering substances, such as sugars, aromatic amines, sulphur dioxide and ascorbic acid (Prior et al., 2005). The extracts with the highest total phenols content were obtained with 50% ethanol (WEE), followed very closely by 50% methanol and the lowest value was obtained with water. The solvent used resulted to be a significant factor on the total phenols content (P < 0.05). Unlike the behaviour observed for extraction yield, total phenols content did not show dependence on the polarity of the solvent. However, the results obtained for total phenols content were in accordance with previous studies which reported that binary-solvent systems were more favourable in the

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Table 1 Extraction yield, total phenols content and antioxidant capacity of extracts of walnut green husk from Mellanaise cultivar. Solvent

Extraction yield (%)

Total phenols content (mg GAEs/g extract)

MeOH EtOH MeOH 50% EtOH 50% Water P-value

11.26 ± 1.06 3.90 ± 0.86a 17.66 ± 2.25a–c 20.21 ± 1.03bc 44.11 ± 1.11d 0.001*

65.76 ± 2.29 51.87 ± 5.58ab 81.50 ± 2.55c 84.46 ± 2.96c 40.39 ± 1.94a <0.001*

ab

EC50 (mg/mL)

b

Reducing power

DPPH

1.65 ± 0.06 1.68 ± 0.18bc 1.14 ± 0.14a 0.95 ± 0.02a 2.16 ± 0.06c <0.001*

0.38 ± 0.01bc 0.49 ± 0.17c 0.34 ± 0.02ab 0.33 ± 0.02a 0.72 ± 0.02d <0.001*

b

In each column different letters (a–d) mean significant differences P < 0.05. * P-values are those for the effect of the solvent on extraction yield, total phenols content and antioxidant capacity of walnut green husk extracts from one-way Welch ANOVA analysis (SPSS 18.0 software). If there was a significant effect of the solvent and equal variances could not be assumed (P < 0.05 by means of the Levene test), then means were compared by the Dunnett’s T3 test.

extraction of phenolic compounds from plant samples as compared to mono-solvent systems (Spigno et al., 2007; Chew et al., 2011). Antioxidant activity of the extracts is related with those compounds capable of protecting a biological system against the potential harmful effect of oxidative processes. Antioxidants are essential to preserve the biological system from free radicals damage to biological molecules (Mishra et al., 2010). In this study, the antioxidant activity of the walnut green husk extracts was evaluated by the reducing power and scavenging activity on DPPH and NO radical assays. The reducing power assay is based on the reduction of the Fe3+ /ferricyanide complex to the ferrous ion (Fe2+ ) in the presence of antioxidants (reducers). The Fe2+ concentration can be monitorized by measuring the absorbance at 700 nm and increased absorbance indicates an increase in reducing power. The results are expressed as EC50 values (mg/mL), the concentration required to provide 0.5 of absorbance. Low values of EC50 are indicative of high antioxidant activity. As shown in Table 1, the solvent used resulted to be a significant factor on reducing power EC50 values (P < 0.05). In addition, reducing power of the extracts was dependent on the extract concentration (Fig. 1). The highest antioxidant activity was obtained for the 50% ethanol extracts (WEE) and the lowest for the aqueous ones (WE). The results obtained for methanol (ME) and ethanol (EE) extracts and for their 50% aqueous solutions were similar and no significant differences between the results were found. For all the solvents assayed, the antioxidant activity was better than the reference antioxidants such as butylated hydroxytoluene (BHT) (A700 = 0.12 at 3.6 mg/mL) or ␣-tocopherol (A700 = 0.13 at 8.6 mg/mL) (Sousa et al., 2008a). The scavenging activity of the walnut green husk extracts was evaluated by the DPPH assay. The DPPH• radical is one of the few

stable organic nitrogen radicals and the test is simple and rapid which probably explains its widespread use in antioxidant screening (Prior et al., 2005). In this method, the purple chromogen radical DPPH• is reduced by antioxidant/reducing compounds to the corresponding pale yellow hydrazine (Magalhaes et al., 2008) and the loss of DPPH colour after reaction with test compounds was monitored at 517 nm. The results were expressed as EC50 values, that is the amount of antioxidant necessary to decrease by 50% the initial DPPH• concentration. In the same way as for total phenols content and reducing power, the solvent also resulted to be a significant factor (P < 0.05) on DPPH EC50 values. The scavenging effect of the extracts also showed a concentration-dependent activity (Fig. 2), especially for concentrations below 1 mg/mL. The extracts obtained with 50% ethanol (WEE) and 50% methanol (WME) showed the highest antioxidant activity, scavenging 50% of the free DPPH radicals at very low concentrations (0.33 and 0.34 mg/mL, respectively). Once more, water extracts (WEs) reported the highest EC50 value (0.72 mg/mL) and, consequently, the lower antioxidant activity. The influence of the solvent used on extract properties was the same for the total phenols content and antioxidant assays. The extract antioxidant properties increased in the following order: water < ethanol < methanol < methanol 50% < ethanol 50%. As mentioned before for total phenols content, the mixtures of alcohols and water have been more efficient in extracting compounds with antioxidant activity than the corresponding mono-component solvent system. In addition, these results could indicate that the phenolic compounds presented on walnut green husk had a moderately polar characteristic, based on the principle that polar compounds dissolve polar compounds (Chew et al., 2011). The

100

DPPH scavenging effect (%)

Abs at 700 nm

1.0 ME EE WME WEE WE

0.8 0.6 0.4 0.2

75 ME EE WME WEE WE

50

25

0

0.0 0

0.5

1

1.5

2

Concentration (mg/mL) Fig. 1. Reducing power values for the extracts obtained with different solvents at different extract concentrations. Each value is expressed as mean ± standard error (ME – methanol extraction; EE – ethanol extraction; WME – 50% water/methanol extraction; WEE – 50% water/ethanol extraction; WE – water extraction).

0

0.5

1

1.5

2

Concentration (mg/mL) Fig. 2. Scavenging activity on DPPH radicals (%) of the extracts obtained with different solvents at different extract concentrations. Each value is expressed as mean ± standard error (ME – methanol extraction; EE – ethanol extraction; WME – 50% water/methanol extraction; WEE – 50% water/ethanol extraction; WE – water extraction).

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75

10

Inhibition zone (mm)

NO scavenging effect (%)

B. cereus

50

25 WE

0

S. aureus

8

B. subtilis S. epidermis

6

4

2

0

0

0.8

1.6

2.4

3.2

0

25

Concentration (mg/mL) Fig. 3. Scavenging of nitric oxide radicals (%) by the water extract (WE). Each value is expressed as mean ± standard error of three determinations.

best extract properties were achieved with 50% ethanol. Ethanol resulted to be effective in the extraction of flavonoids and their glycosides, catecols and tannins from raw plant materials (Spigno et al., 2007). Ethanol, unlike methanol, is recognised as a GRAS (Generally Recognised as Safe) solvent and therefore can be used safely for applications in the food industry. In addition to the solvent type and concentration, other factors could influence the extraction process. The most studied were the extraction time, temperature, solid–liquid ratio and particle size (Al-Farsi and Lee, 2008; Chew et al., 2011). Other important factor is the extraction method. Nowadays, alternative techniques, such as microwaveassisted extraction (MAE) or ultrasounds assisted extraction (UAE), are being developed to increase process efficiency. These factors could be considered in future studies to improve the extraction process of walnut green husk. The nitric oxide scavenging ability of the aqueous walnut green extracts was also analysed. Research on NO scavenging ability of other walnut wastes is scarce. The study of the effect of extracts against these radical was a novelty point in this work. Only data for NO scavenging effect of methanolic extracts of walnut green husk were available (Ghasemi et al., 2011). Nitric oxide (• NO) is an abundant reactive species that acts as an important biological signalling molecule in a large variety of diverse physiological processes, including neurotransmission, blood pressure regulation, defense mechanisms, smooth muscle relaxation and immune regulation. When the generation of reactive nitrogen species in a system exceeds the system’s ability to neutralise and eliminate them, an overproduction of the radical may occur, reacting and altering the structure of proteins and so inhibiting their normal function (Valko et al., 2007). In the performed method NO was generated from sodium nitroprusside (SNP) and was measured by the Griess reagent. In aqueous solution at physiological pH, SNP spontaneously generates NO, which interacts with oxygen to produce nitrite ions that can be estimated by the use of Griess reagent. Scavengers of NO compete with oxygen leading to reduce the production of NO. As shown in Fig. 3, aqueous walnut green extract also exhibited concentration dependent activity for NO radical scavenging capacity. Its EC50 value (0.96 ± 0.13 mg/mL) was slightly higher

50

100

75

Concentration (mg/mL) Fig. 4. Antimicrobial activity against Gram + bacteria of the aqueous extracts from walnut green husk at different extract concentrations. Each value is expressed as mean ± standard error.

compared to DPPH radical scavenging activity. The maximum nitric oxide radical inhibition (60.45%) was achieved for an extract concentration of 3.2 mg/mL. Comparing the results obtained with those of a previous work conducted with walnut green husk from five J. regia L. cultivars (Oliveira et al., 2008), similar results were observed for the extraction yield and total phenols content of the aqueous extracts from the Mellanaise cultivar. However, the reducing power and DPPH EC50 values were lower. On the other hand, the best extract obtained in this work, the 50% ethanol extract from the Mellanaise cultivar, showed higher total phenols content and similar scavenging capacity for the DPPH radical (84.46 mg GAEs/g and EC50 = 0.33 mg/mL, respectively) than the aqueous Franquette extracts (74.08 mg GAEs/g and EC50 = 0.35 mg/mL), the cultivar that presented the best properties in the study developed by Oliveira et al. (2008). With respect to NO scavenging capacity, the results for water extracts were in the range obtained by Ghasemi et al. (2011) for methanolic extracts. Significant linear correlations were found between extract total phenols content and antioxidant activity measured by the reducing power assay (EC50 (mg/mL) = −0.024 × total phenols (mg GAE/g extract) + 3.096; R2 = 0.903; P < 0.001) and by the DPPH assay (EC50 (mg/mL) = −0.008 × total phenols (mg GAE/g extract) + 0.966; R2 = 0.837; P < 0.001). These results demonstrate the contribution of phenolic compounds to extract antioxidant activity. Samples with higher total phenols content showed the higher antioxidant properties (lower EC50 values). This kind of relationship has been also reported for other plant materials (Barreira et al., 2008; Dudonné et al., 2009; Vázquez et al., 2008, 2009; Malheiro et al., 2011, 2012). 3.2. Antimicrobial activity The aqueous extracts of the walnut green husk were screened for their antimicrobial properties against B. cereus, B. subtilis, S. aureus and S. epidermis (Gram + bacteria) and against E. coli and P. aeruginosa (Gram − bacteria). The inhibition in the growth of the bacteria was evaluated for different extract concentrations and

Table 2 Antimicrobial activity of aqueous extracts from walnut green husk. Bacteria

MIC (mg/mL)

B. cereus

B. subtilis

S. aureus

S. epidermis

E. coli

P. aeruginosa

20 (++)

50 (+++)

50 (++)

50 (+++)

100 (−)

100 (−)

Inhibition zone < 1 mm: no antimicrobial activity (−); inhibition zone 1–3 mm: slight antimicrobial activity (+); inhibition zone 3–5 mm: moderate antimicrobial activity (++); inhibition zone 5–9 mm: high antimicrobial activity (+++).

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the halos of the inhibition zones corresponding to the MICs are presented in Table 2. The response for each microorganism was different. For all Gram positive bacteria tested, the extracts showed antimicrobial activity, while there was no antimicrobial effect against the Gram negative bacteria. The Gram negative bacteria have a lipopolysaccharide outer membrane and the transfer of molecules is achieved through the cell membrane. The antimicrobial effect is related with the ability of the compounds to penetrate the outer membrane and reach its site of action, influenced by their size and shape (Kavak et al., 2010). The compounds present in aqueous walnut green husk extract probably could not pass through the cell membrane. The results obtained for the Gram positive bacteria are shown in Fig. 4. The growth inhibition was dependent on extract concentration and for the lowest concentration tested (5 mg/mL) no inhibition was found. The most sensitive bacteria was B. cereus, the only one that showed inhibition against 20 mg/mL of extract concentration. Comparing the remaining concentrations assayed, the highest inhibition zones were achieved against S. epidermis and the most resistant bacteria was S. aureus. Antimicrobial activity of aqueous walnut green husk extracts of Mellanaise cultivar against B. cereus, S. aureus and B. subtilis was previously tested by Oliveira et al. (2008), who found considerable lower values for the minimal inhibitory concentration (MIC), differences that could be attributed to the effect of the long storage of the walnut green husk sample used in this work which could influence the material properties. 4. Conclusions The influence of the solvent (water, methanol, ethanol and 50% aqueous solutions of methanol and ethanol) on the antioxidant properties of the walnut green husk extracts was demonstrated. The highest extraction yield was obtained with water, although aqueous extracts showed the lowest antioxidant properties. The highest total phenols content and antioxidant activities, measured by the reducing power and DPPH assays, were obtained with 50% aqueous ethanol. Aqueous extracts were effective in the scavenging of NO radical. In addition, aqueous extracts were able to inhibit the growth of Gram positive bacteria, proving the antimicrobial capacity of the extracts. The results obtained demonstrated the potential of the walnut green husk as an economical source of antioxidant and antimicrobial agents for the food or pharmaceutical industries. In order to optimise the extraction process and the quality of the extracts, factors to consider in future studies would be temperature, time and solid–liquid ratio. Acknowledgments Authors are grateful to “Fundación Segundo Gil Dávila” for a research grant awarded to Adela Fernández Agulló, and to the ˜ POCTEP – Programa Cooperac¸ão Transfronteiric¸a Espana-Portugal 2007–2013 for financial support through the project “Mejora de la competitividad del sector agrario de Castilla y León y Norte de Portugal a través de la innovación y el desarrollo de productos diferenciados de alto valor”. References Al-Farsi, M., Lee, C.Y., 2008. Optimization of phenolics and dietary fibre extraction from date seeds. Food Chemistry 108, 977–985. Almeida, I.F., Fernandes, E., Lima, J.L.F.C., Costa, P.C., Bahia, M.F., 2008. Walnut (Juglans regia) leaf extracts are strong scavenger of pro-oxidant reactive species. Food Chemistry 106, 1014–1020. Balasundram, N., Sundram, K., Sundram, S., 2006. Phenolic compounds in plants and agri-industrial by-products: antioxidant activity, ocurrence and potential uses. Food Chemistry 99, 191–203.

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