Tanaman Industri dan Produk 42 (2013) 126- 132

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Tanaman Industri dan Produk

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Dalam memengaruhi dari pada pelarut dan antioksidan sifat antimikroba dari kenari ( Juglans regia L.) sekam
hijau ekstrak

A. Fernandez-Agulló itu . E. Pereira b . MS Freire itu . P. Valentão c . PB Andrade c . J. González-Álvarez itu . * .

JA pir b . **
tu Departemen kimia Rekayasa, Sekolah Teknik, Universitas Santiago de Compostela, Rúa Lope Gómez de Marzo s / n, 15.782 Santiago de Compostela, Spanyol
b gunung Pusat Penelitian, Sekolah Pertanian - Polytechnic Institute of Bragança Kampus St itu Apolonia, Apartado 1172, 5301-855 Bragança untuk, Portugal
c REQUIMTE / Laboratorium Farmakognosi, Departemen Kimia, Fakultas Farmasi, Universitas Porto, Rua Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal

articleinfo abstract

Article history: 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
Received 9 November 2011 Received in revised form 31 compounds with antioxidant and antimicrobial properties. At this respect, the effect of the solvent (water, methanol, ethanol and 50% aqueous
March 2012 Accepted 19 May 2012
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
Keywords:
highest extraction yield was achieved with water (44.11%) and high bioactive potential was shown by the samples extracted with water/ethanol
Walnut green husk Solvent extraction
(1:1) (84.46 mg GAE/g extract; EC 50 = 0.95 mg/mL for reducing power and EC 50 = 0.33 mg/mL for DPPH assay). All the antioxidant properties
Total phenols content
analysed showed a concentrationdependent activity. The antimicrobial activity of the aqueous extracts was assessed and showed ability to
Antioxidant activity Antimicrobial activity
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 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
The walnut ( Juglans regia L.) is a tree traditionally cultivated for its valuable wood and fruits. The et al., 2006 ). karya yang berbeda telah Ditandai komposisi fenolik kenari oleh-produk ( Fukuda et al.,
walnut seed is a nut of high economic interest to the food industry and is globally popular and valued 2003; Li dkk., 2006; Stamper et al., 2006; Pereira et al., 2007; Zhang et al., 2009 ). Efek resmi
for its nutritional, health and sensory attributes ( Martínez et al., 2010 ). The Iberian Peninsula yearly manfaat yang berasal dari senyawa fenolik, seperti theire kegiatan anti kanker, antimutagenik dan
produces 13,500 t of walnut kernel, where 75% are produced in Spain and the remaining 25% in Portugal. kardioprotektif telah dikaitkan dengan aktivitas antioksidan theire ( Madhavi et al., 1996; Balasundram
et al., 2006 ). Pada hal ini, Sejumlah penelitian telah difokuskan pada Mendapatkan antioksidan dari
sumber alami. Penelitian telah dipromosikan oleh kebutuhan untuk fi nd pengganti alami untuk
antioksidan sintetik, diduga berpotensi beracun ( Contini et al., 2008 ). Dalam konteks ini, produk
Other by-products derived from the walnut tree have been used in several applications. Thus, green limbah murah dari makanan, hutan atau industri pertanian memiliki Terutama menarik untuk manfaat
walnuts, shells, kernels, bark, and leaves have been used in both cosmetic and pharmaceutical industries lingkungan dan ekonomis Hasil Dari mereka digunakan kembali. Beberapa bahan tanaman telah
dianalisis dengan tujuan ini: tanaman dan agro-industri oleh-produk ( Balasundram et al., 2006 ),
( 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 Kacang-kacangan dan mereka dengan-produk seperti lambung almond ( Pinelo et al., 2004 ), Kernel
et al., 2008 ). Dry walnut leaves are also frequently used as infusions ( Pereira et al., Hazelnut dan shell ( Contini dkk., 2008; Delgado et al., 2010 ) Gevuina hazelnut

∗ Corresponding author. Tel.: +34 881816758; fax: +34 981528050.

∗∗ Corresponding author. Tel.: +351 273303277; fax: +351 273325405.

E-mail addresses: julia.gonzalez@usc.es (J. González-Álvarez),

pereira@ipb.pt (J.A. Pereira). lambung ( Pindahkan et al., 2000 ), Hazel daun ( Oliveira et al., 2007 ) atau

0926-6690 / $ - melihat hal depan © 2012 Elsevier-undang.

http://dx.doi.org/10.1016/j.indcrop.2012.05.021
 A. Fernandez-Agulló et al. / Tanaman Industri dan Produk 42 (2013) 126- 132
kastanye dan buah mereka oleh-produk ( Barrier dkk., 2008; Vázquez et al. 2008, 2009 ). tanaman by-products
2.2. Raw material
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; ˇ
Zivkovi ´ c 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 was calculated from the graph of absorbance at 700 nm against extract concentration in the solution.
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).
127
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 (EC 50)
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
128 A. Fernández-Agulló et al. / Industrial Crops and Products 42 (2013) 126– 132

reduction of the DPPH radical was measured by monitoring the decrease of absorption at 517 nm. respect, the one-way analysis of variance (ANOVA) was used, followed by the Dunnett T3 test. All
DPPH scavenging effect was calculated as the percentage of DPPH discoloration using the following statistical tests were performed at a 5% significance level using SPSS 18.0 software. A regression
equation: % scavenging effect = [( A DPPH − A S)/ A DPPH] × 100, where analysis, using Excel from Microsoft Corporation, was established between the total phenols content
and EC 50 values obtained in the antioxidant assays tested.
A S is the absorbance of the solution when the sample extract has been added at a particular level and A DPPH
s the absorbance of the DPPH solution. The extract concentration providing 50% inhibition (EC 50) was dihitung
dari grafik efek scavenging persentase terhadap ekstrak konsentrasi dalam larutan.

3. Results and discussion

2.5.3. berhubung dgn sendawa oksida aktivitas scavenging Extraction with solvents is frequently used for the isolation of antioxidant compounds, and both
itu berhubung dgn sendawa oksida (NO) aktivitas scavenging dari ekstrak air adalah ditentukan extraction yield and antioxidant activity of the extracts have a strong relationship with the solvent
dengan menggunakan metode yang dijelaskan oleh Sousa et al. (2008b) . itu antiradikal kegiatan itu Ditentukan
employed, mainly due to the different polarity of the compounds obtained ( Moure et al., 2001 ). In
dalam piring Multiskan Pendakian reader. 100 L dari sodium nitroprusside (SNP, 10 mM) diinkubasi dengan particular, for the extraction of phenolic compounds to be used as antioxidants, organic solvents are
100 L ekstrak walnut kulit hijau pada konsentrasi yang berbeda selama 60 menit di ruang temperatur commonly used ( Pokorny and Korczak, 2001 ). The selection of the most appropriate solvent is a
di bawah cahaya. semua solusi emang disusun penyangga fosfat. Setelah inkubasi, 100 L dari Griess reagendeterminant factor on extract properties and due to the diverse structure and composition of the
(1% dan sulphanilamide 0,1% 2% di naphthylethyldiamine asam fosfat) ditambahkan ke setiap sumur. matrix, each matrix-solvent system shows a particular behaviour that cannot be predicted ( Al-Farsi
tu campuran itu diinkubasi pada ruang Suhu selama 10 menit dan absorbansi Selama kromofor and Lee, 2008 ). For this reason, in this work, different solvents were assayed for the extraction of
terbentuk dari diazotisasi dengan nitrit dan sulphanilamide subsequent coupling with walnut green husk (water, methanol, ethanol and 50% aqueous solutions of methanol and ethanol)
naphthylehylendiamine was read at 562 nm. Three assays were performed, each one in triplicate. The and extraction yield, total phenols content and antioxidant and antimicrobial properties of the extracts
NO scavenging effect (%) and the extract concentration providing 50% inhibition (EC 50) were calculated obtained were compared. Table 1 shows the results obtained for extraction yield, total phenols content
as indicated in the DPPH method. and EC 50 values for the reducing power and DPPH assays.

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(water > methanol > ethanol) the extraction yield values decreased in the same order. The high
of University of Minho (Braga, Portugal). The bacterial stocks were maintained at 4 ◦ C on LB agar [tryptone temperature and solid–liquid ratio used in the extraction with water could also explain the high
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 10 6 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
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
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 ).
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

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
 A. Fernández-Agulló et al. / Industrial Crops and Products 42 (2013) 126– 132 129

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 EC 50 ( mg/mL)

GAEs/g extract)

Reducing power DPPH

MeOH 11.26 ± 1.06 ab 65.76 ± 2.29 b 1.65 ± 0.06 b 0.38 ± 0.01 bc

EtOH 3.90 ± 0.86 a 51.87 ± 5.58 ab 1.68 ± 0.18 bc 0.49 ± 0.17 c
MeOH 50% 17.66 ± 2.25 a–c 81.50 ± 2.55 c 1.14 ± 0.14 a 0.34 ± 0.02 ab
EtOH 50% 20.21 ± 1.03 bc 84.46 ± 2.96 c 0.95 ± 0.02 a 0.33 ± 0.02 a
Water 44.11 ± 1.11 d 40.39 ± 1.94 a 2.16 ± 0.06 c 0.72 ± 0.02 d
P- value 0.001 * <0.001 * <0.001 * <0.001 *

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
hydrazine ( Magalhaes et al., 2008 ) and the loss of DPPH colour after reaction with test compounds
( 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 Fe 3+/ ferricyanide complex to the ferrous
ion (Fe 2+) in the presence of antioxidants (reducers). The Fe 2+ concentration can be monitorized by measuring
extracts (WEs) reported the highest EC 50 value (0.72 mg/mL) and, consequently, the lower antioxidant
the absorbance at 700 nm and increased absorbance indicates an increase in reducing power. The
results are expressed as EC 50 values (mg/mL), the concentration required to provide 0.5 of absorbance.
Low values of EC 50 are indicative of high antioxidant activity. As shown in Table 1 , the solvent used
resulted to be a significant factor on reducing power EC 50 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) ( A 700 = 0.12 at 3.6 mg/mL) or -tocopherol ( A 700 = 0.13 at
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
was monitored at 517 nm. The results were expressed as EC 50 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 EC 50 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
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

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

100

1.0
ME EE
75
WME
0.8
WEE
DPPH scavenging effect (%)

WE ME EE

0.6 50
WME
Abs at 700 nm

WEE
WE
0.4
25

0.2

0
0.0
0 0.5 1 1.5 2
0 0.5 1 1.5 2
Concentration (mg/mL)
Concentration (mg/mL)
Fig. 2. Scavenging activity on DPPH radicals (%) of the extracts obtained with different solvents at different extract
Fig. 1. Reducing power values for the extracts obtained with different solvents at different extract concentrations. Each concentrations. Each value is expressed as mean ± standard error (ME – methanol extraction; EE – ethanol extraction;
value is expressed as mean ± standard error (ME – methanol extraction; EE – ethanol extraction; WME – 50% WME – 50% water/methanol extraction; WEE – 50% water/ethanol extraction; WE – water extraction).
water/methanol extraction; WEE – 50% water/ethanol extraction; WE – water extraction).
130 A. Fernández-Agulló et al. / Industrial Crops and Products 42 (2013) 126– 132

75 10

B. cereus

S. aureus
8
B. subtilis

S. epidermis
50
6
NO scavenging effect (%)

Inhibition zone (mm)

4

25

WE 2

0
0
0 0.8 1.6 2.4 3.2 0 25 50 75 100

Concentration (mg/mL)
Concentration (mg/mL)

Fig. 4. Antimicrobial activity against Gram + bacteria of the aqueous extracts from walnut green husk at different extract
Fig. 3. Scavenging of nitric oxide radicals (%) by the water extract (WE). Each value is expressed as mean ± standard
concentrations. Each value is expressed as mean ± standard error.
error of three determinations.

best extract properties were achieved with 50% ethanol. Ethanol resulted to be effective in the extraction
compared to DPPH radical scavenging activity. The maximum nitric oxide radical inhibition (60.45%)
of flavonoids and their glycosides, catecols and tannins from raw plant materials ( Spigno et al., 2007 ). Ethanol,
was achieved for an extract concentration of 3.2 mg/mL.
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,
Comparing the results obtained with those of a previous work conducted with walnut green husk
other factors could influence the extraction process. The most studied were the extraction time,
from five J. regia L. cultivars ( Oliveira et al., 2008 ), similar results were observed for the extraction yield
temperature, solid–liquid ratio and particle size ( Al-Farsi and Lee, 2008; Chew et al., 2011 ). Other important
and total phenols content of the aqueous extracts from the Mellanaise cultivar. However, the reducing
factor is the extraction method. Nowadays, alternative techniques, such as microwaveassisted extraction
power and DPPH EC 50 values were lower. On the other hand, the best extract obtained in this work,
(MAE) or ultrasounds assisted extraction (UAE), are being developed to increase process efficiency.
the 50% ethanol extract from the
These factors could be considered in future studies to improve the extraction process of walnut green
husk.

Mellanaise cultivar, showed higher total phenols content and similar scavenging capacity for the DPPH
radical (84.46 mg GAEs/g and EC 50 = 0.33 mg/mL, respectively) than the aqueous Franquette extracts
(74.08 mg GAEs/g and EC 50 = 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.
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
Significant linear correlations were found between extract total phenols content and antioxidant
that acts as an important biological signalling molecule in a large variety of diverse physiological
activity measured by the reducing power assay (EC 50 ( mg/mL) = − 0.024 × total phenols (mg GAE/g
processes, including neurotransmission, blood pressure regulation, defense mechanisms, smooth
extract) + 3.096; R 2 = 0.903; P < 0.001) and by the DPPH assay (EC 50 ( mg/mL) = − 0.008 × total phenols
muscle relaxation and immune regulation. When the generation of reactive nitrogen species in a
(mg GAE/g extract) + 0.966;
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
R 2 = 0.837; P < 0.001). These results demonstrate the contribution of phenolic compounds to extract
et al., 2007 ). In the performed method NO was generated from sodium nitroprusside (SNP) and was
antioxidant activity. Samples with higher total phenols content showed the higher antioxidant
measured by the Griess reagent. In aqueous solution at physiological pH, SNP spontaneously generates
properties (lower EC 50 values). This kind of relationship has been also reported for other plant
NO, which interacts with oxygen to produce nitrite ions that can be estimated by the use of Griess
materials ( Barreira et al., 2008; Dudonné et al., 2009; Vázquez et al., 2008, 2009; Malheiro et al., 2011,
reagent. Scavengers of NO compete with oxygen leading to reduce the production of NO. As shown in Fig.
2012 ).
3 , aqueous walnut green extract also exhibited concentration dependent activity for NO radical
scavenging capacity. Its EC 50 value (0.96 ± 0.13 mg/mL) was slightly higher

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

B. cereus B. subtilis S. aureus S. epidermis E. coli P. aeruginosa

MIC 20 50 50 50 100 100

(mg/mL) (++) (+++) (++) (+++) ( −) ( −)

nhibition 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 (+++).
 A. Fernández-Agulló et al. / Industrial Crops and Products 42 (2013) 126– 132
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
study of antioxidant properties and total phenolic content of 30 plant extracts of industrial interest using DPPH,
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 extractionand antioxidant activities of heartnut ( Juglans ailanthifolia var. cordiformis) and Persian walnut ( Juglans regia L.).
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 Casal, S., Bento, A., Pereira, J.A., 2011. Cultivar effect on the
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 Espa˜
na-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”.
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