Total phenols, antioxidant potential and antimicrobial activity of walnut (Juglans regia L.) green husks.

Abstract

The total phenols content and antioxidant and antimicrobial activities were studied in walnut (Juglans regia L.) green husks aqueous extracts of five different cultivars (Franquette, Mayette, Marbot, Mellanaise and Parisienne). Total phenols content was determined by colorimetric assay and their amount ranged from 32.61 mg/g of GAE (cv. Mellanaise) to 74.08 mg/g of GAE t (cv. Franquette). The antioxidant capacity of aqueous extracts was assessed through reducing power assay, scavenging effects on DPPH (2,2-diphenyl-1-picrylhydrazyl) radicals and beta-carotene linoleate model system. A concentration-dependent antioxidative capacity was verified in reducing power and DPPH assays, with EC50 values lower than 1 mg/mL for all the tested extracts. The antimicrobial capacity was screened against Gram positive and Gram negative bacteria, and fungi. All the extracts inhibited the growth of Gram positive bacteria, being Staphylococcus aureus the most susceptible one with MIC of 0.1 mg/mL for all the extracts. The results obtained indicate that walnut green husks may become important in the obtainment of a noticeable source of compounds with health protective potential and antimicrobial activity.

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Total phenols, antioxidant potential and antimicrobial activity
of walnut (Juglans regia L.) green husks
Ivo Oliveira, Anabela Sousa, Isabel C.F.R. Ferreira, Albino Bento, Letícia Estevinho, José Alberto Pereira*
CIMO/Escola Superior Agrária, Instituto Politécnico de Braganc ?a, Campus Sta Apolónia, Apartado 1 172, 5301-855 Braganc ?a, Portugal
a r t i c l ei n f o
Article history:
Received 4 February 2008
Accepted 11 March 2008
Keywords:
Walnut green husks
Total phenols
Antimicrobial activity
Antioxidant activity
a b s t r a c t
The total phenols content and antioxidant and antimicrobial activities were studied in walnut (Juglans
regia L.) green husks aqueous extracts of five different cultivars (Franquette, Mayette, Marbot, Mellanaise
and Parisienne). Total phenols content was determined by colorimetric assay and their amount ranged
from 32.61 mg/g of GAE (cv. Mellanaise) to 74.08 mg/g of GAE t (cv. Franquette). The antioxidant capacity
of aqueous extracts was assessed through reducing power assay, scavenging effects on DPPH (2,2-diphe-
nyl-1-picrylhydrazyl) radicals and b-carotene linoleate model system. A concentration-dependent anti-
oxidative capacity was verified in reducing power and DPPH assays, with EC50values lower than 1 mg/
mL for all the tested extracts. The antimicrobial capacity was screened against Gram positive and Gram
negative bacteria, and fungi. All the extracts inhibited the growth of Gram positive bacteria, being Staph-
ylococcus aureus the most susceptible one with MIC of 0.1 mg/mL for all the extracts. The results obtained
indicate that walnut green husks may become important in the obtainment of a noticeable source of com-
pounds with health protective potential and antimicrobial activity.
? 2008 Elsevier Ltd. All rights reserved.
1. Introduction
Walnut (Juglans regia L.) is a valuable crop being the nut very
popular and largely consumed. In Portugal, this species is widely
spread throughout the country. Not only dry fruit (nuts) are used
but also green walnuts, shells, kernels, bark, green walnut husks
(epicarp) and leaves have been used in both cosmetic and pharma-
ceutical industry (Stampar et al., 2006).
In recent decades an increasing tendency towards the use of
natural substances instead of the synthetic ones has been ob-
served. As the synthetic materials and products are more complex
in comparison to natural substances, it will take a long time for
them to complete their natural cycles and return to nature; thus
causing a lot of environmental pollution. Also with the increase
in the price of raw materials, the problem of cost benefits for
chemical production is becoming more considerable. Natural anti-
oxidants, such as phenolic compounds, used as natural antioxi-
dants, are gaining importance, due to their benefits for human
health, decreasing the risk of degenerative diseases by reduction
of oxidative stress and inhibition of macromolecular oxidation (Sil-
va et al., 2004; Pulido et al., 2000; Tseng et al., 1997). Their use as
preserving food additives (Zupko et al., 2001) had increasing
interest.
In addition to antioxidant activity, several studies demonstrated
the antimicrobial activity of phenols and/or phenolic extracts
(Fernández et al., 1996; Hoult and Payá, 1996; Pereira et al.,
2006, 2007a,b; Proestos et al., 2005; Puupponen-Pimiä et al.,
2001; Rauha et al., 2000; Sousa et al., 2006; Zhu et al., 2004), mak-
ing them a good alternative to antibiotics and chemical preserva-
tives. There is an extended interest in using natural antimicrobial
compounds, as the consumer’s pressure on the food industry aug-
ments, to avoid chemical preservatives and due to the increasing
resistance to antibiotics (Oliveira et al., 2007; Cowan, 1999).
Walnut’s green husk is a by-product of the walnut production,
having scarce use. Thus, using husk as a source of phytochemicals
will increase the value of the walnut production, as well as offer
utilization for a by-product, which is produced in a large quantity.
Different works demonstrated the potential antioxidant of wal-
nut products, especially fruits (Espín et al., 2000; Li et al., 2006,
2007; Pereira et al., 2008) but also leaves (Pereira et al., 2007b)
andliqueursproducedbygreenfruits (Stamparet al.,2006).Studies
have also demonstrated the antimicrobial activity of walnut prod-
ucts, particularly of bark (Alkhawajah, 1997), leaves (Clark et al.,
1990; Pereira et al., 2007b), fruits (Pereira et al., 2008) and the spe-
cific compound juglone (Clark et al., 1990) but until this time the
information about walnut green husks is almost inexistent.
In the present work, green husk (Fig. 1) from five walnut culti-
vars (cv. Franquette, Marbot, Mayette, Mellanaise and Parisienne)
grown in Portugal, were studied, regarding their total phenols con-
tent, antioxidant and antimicrobial activities. Antioxidant potential
0278-6915/$ - see front matter ? 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fct.2008.03.017
* Corresponding author. Tel.: +351 273 303277; fax: +351 273 325405.
E-mail address: jpereira@ipb.pt (J.A. Pereira).
Food and Chemical Toxicology 46 (2008) 2326–2331
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was accessed by the reducing power assay, the scavenging effect
on DPPH (2,2-diphenyl-1-picrylhydrazyl) radicals and b-carotene
linoleate model system. Antimicrobial capacity was also accessed,
against Gram positive (Bacillus cereus, B. subtilis, Staphylococcus
aureus) and Gram negative bacteria (Pseudomonas aeruginosa, Esch-
erichia coli, Klebsiella pneumoniae) and fungi (Candida albicans,
Cryptococcus neoformans).
2. Materials and methods
2.1. Samples
Walnuts’ green husks were obtained from five J. regia L. cultivars: Franquette,
Marbot, Mayette, Mellanaise and Parisienne that were collected on October 2006
in Braganc ?a, northeast of Portugal (41? 47’ 47.50918” N, 6? 46’ 5.71990” W,
744.341 m). The orchard has a planting density of 3.5 ? 7 m. The trees are ten years
old, being pruned when necessary. No phytosanitary treatments were applied. Per
cultivar, approximately 2 kg of fruits were handpicked from the soil, and the walnut
green husks were removed, put in plastic bags and immediately frozen at ?20?. The
plant material was then freeze dried.
2.2. Samples preparation
Before each kind of analysis (total phenols determination, antioxidant and anti-
microbial activities assays) the walnuts’ green husk were extracted with 250 mL of
boiling water for 45 min, and filtered through Whatman no. 4 paper (for each cul-
tivar, three powdered subsamples, 5 g, 20 mesh). The aqueous extracts were frozen,
lyophilized and redissolved in water at concentrations of 100 mg/mL and 10 mg/mL
for antimicrobial and antioxidant activities assays, respectively.
2.3. Determination of total phenols content
Total phenols content in the obtained extracts were estimated by a colorimetric
assay based on procedures described by Singleton and Rossi (1965) with some mod-
ifications. Briefly, 1 mL of sample was mixed with 1 mL of Folin and Ciocalteu’s phe-
nol 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 was read at 725 nm (Analytik Jena 200–
2004 spectrophotometer). Gallic acid was used for constructing the standard curve
(0.01–0.4 mM). The results are expressed as mg of gallic acid equivalents/g of ex-
tract (GAEs).
2.4. Antioxidant Activity
2.4.1. Reagents
BHA (2-tert-butyl-4-methoxyphenol), TBHQ (tert-butylhydroquinone) and a-
tocopherol were purchased from Sigma (St. Louis, MO, USA). 2,2-Diphenyl-1-pic-
rylhydrazyl (DPPH) was obtained from Alfa Aesar. All other chemicals were
obtained from Sigma Chemical Co. (St. Louis, USA). Methanol was obtained from
Pronalab (Lisboa, Portugal). Water was treated in a Mili-Q water purification system
(TGI Pure Water Systems, USA).
2.4.2. Reducing power assay
The reducing power was determined according to a described procedure (Oyai-
zu, 1986; Ferreira et al., 2007). Various concentrations of sample extracts (2.5 mL)
were mixed with 2.5 mL of 200 mmol/L sodium phosphate buffer (pH 6.6) and
2.5 mL of 1% potassium ferricyanide. The mixture was incubated at 50 ?C for
20 min. After incubation 2.5 mL of 10% tricloroacetic acid (w/v) were added and
then the mixture was centrifuged at 1000 rpm in a refrigerated centrifuge (Cento-
rion K24OR- 2003), for 8 min. The upper layer (5 mL) was mixed with 5 mL of
deionised water and 1 mL of 0.1% of ferric chloride, and the absorbance was mea-
sured spectrophotometrically at 700 nm. The extract concentration providing 0.5
of absorbance (EC50) was calculated from the graph of absorbance registered at
700 nm against the correspondent extract concentration. BHA and a-tocopherol
were used as reference compounds.
2.4.3. Scavenging effect assay
The capacity to scavenge the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical
was monitored according to a method reported before (Hatano et al., 1988). Various
concentrations of sample extracts (0.3 mL) were mixed with 2.7 mL of methanolic
solution containing DPPH radicals (6 ? 10?5mol/L). The mixture was shaken vigor-
ously and left to stand in the dark until stable absorption values were obtained. The
reduction of the DPPH radical was measured by monitoring continuously the de-
crease of absorption at 517 nm. DPPH scavenging effect was calculated as percent-
age of DPPH discolouration using the equation: % scavenging effect = [(ADPPH? AS)/
ADPPH] ? 100, where ASis the absorbance of the solution when the sample extract
has been added at a particular level and ADPPHis the absorbance of the DPPH solu-
tion. The extract concentration providing 25% inhibition (EC25) was calculated from
the graph of scavenging effect percentage against extract concentration. BHA and a-
tocopherol were used as reference compounds.
2.4.4. b-Carotene linoleate model system
The antioxidant activity of walnut leaf extracts was evaluated according to a de-
scribed procedure (Mi-Yae et al., 2003) b-carotene solution was prepared by dis-
solving 2 mg of b-carotene in 10 mL of chloroform. Two millilitres of this solution
were placed in a 100 mL round-bottom flask. After chloroform removal, at 40 ?C un-
der vacuum, 40 mg of linoleic acid, 400 mg of Tween 80 emulsifier, and 100 mL of
distilled water were added to the flask under vigorous shaking. Aliquots (4.8 mL) of
this emulsion were transferred into different test tubes containing 0.2 mL of differ-
ent concentrations of walnut leaf extracts. The tubes were shaken and incubated at
50 ?C in a water bath. As soon as the emulsion was added to each tube, the zero time
absorbance at 470 nm was measured. Absorbance readings were then recorded un-
til the control sample had changed colour. A blank assay, devoid of b-carotene, was
prepared for background subtraction. Antioxidant activity was calculated using the
following equation: Antioxidant activity = (b-carotene content after 2 h of assay/ini-
tial b-carotene content) ? 100. The assays were carried out in triplicate and the re-
sultswere expressed as meanvalues ± standard
concentration providing 50% antioxidant activity (EC50) was calculated from the
graph of antioxidant percentage against extract concentration. TBHQ was used as
reference compound.
deviations. Theextract
2.5. Antimicrobial activity
2.5.1. Reagents
Ampicillin and cycloheximide were of the highest available quality, and pur-
chased from Merck (Darmstadt, Germany). Water was treated in a Mili-Q water
purification system (TGI Pure Water Systems, USA).
2.5.2. Microorganisms and culture conditions
Microorganisms CECT were obtained from the Spanish type culture collection
(CECT) of Valencia University, while microorganisms ESA were clinically isolated
strains identified in Microbiology Laboratory of Escola Superior Agrária de Brag-
anc ?a. Gram + (Bacillus cereus CECT 148, B. subtilis CECT 498 and Staphylococus aureus
ESA 7 isolated from pus) and Gram – (Escherichia coli CECT 101, Pseudomonas aeru-
ginosa CECT 108 and Klebsiella pneumoniae ESA 8 isolated from urine) bacteria, and
fungi (Candida albicans CECT 1394 and Cryptococcus neoformans ESA 3 isolated from
vaginal fluid) were used to screen antimicrobial activity of the three walnut culti-
vars. Microorganisms were cultured aerobically at 37 ?C (Scientific 222 oven model,
2003) in nutrient agar medium for bacteria, and at 30 ?C (Scientific 222 oven model,
2003) in sabouraud dextrose agar medium for fungi.
2.5.3. Test assays for antimicrobial activity
The screening of antibacterial activities against Gram + and Gram - bacteria and
fungi and the determination of the minimal inhibitory concentration (MIC) were
achieved by an adaptation of the agar streak dilution method based on radial diffu-
Fig. 1. Walnut green husks.
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sion (Ferreira et al., 2004; Sousa et al., 2006). Suspensions of the microorganism
were prepared to contain approximately 108cfu/mL, and the plates containing agar
medium were inoculated (100 lL; spread on the surface). Each sample (50 lL) was
placed in a hole (3 mm depth, 4 mm diameter) made in the centre of the agar. Un-
der the same conditions, different solutions of ampicillin (antibacterial) and cyclo-
heximide (antifungal) were used as standards. The MIC was considered to be the
lowest concentration of the tested sample able to inhibit the growth of bacteria
after 24 h and fungi after 48 h. The diameters of the inhibition zones corresponding
to the MICs were measured using a ruler, with an accuracy of 0.5 mm. Each inhibi-
tion zone diameter was measured three times (three different plates) and the aver-
age was considered. A control using only inoculation was also carried out.
3. Results and discussion
3.1. Total phenols content
The total phenols content in walnut green husks aqueous ex-
tracts was different according to the variety. cv. Franquette showed
the high amount of these compounds, with 74.08 mg/g of GAE,
being 1.7–2.3-fold higher than for other varieties. The lowest
amounts were obtained for Mellanaise aqueous extracts with
32.61 mg/g of GAE (Table 1). The extraction yields were very sim-
ilar in the different walnut varieties ranging from 31.63 and
33.69%.
A few studies were developed concerning the phenolic compo-
sition of green walnut husks. Juglone (5-hydroxy-1,4-naphthoqui-
none) is known as a characteristic compound of Juglans spp. being
reported its occurrence in green walnut husks (Mahoney et al.,
2000). These authors report also the existence of other naphtoqui-
nones. On the other hand, Stampar et al. (2006) identified thirteen
phenolic compounds in walnut husks: chlorogenic acid, caffeic
acid, ferulic acid, sinapic acid, gallic acid, ellagic acid, proto-
catechuic acid, syringic acid, vanillic acid, catechin, epicatechin,
myricetin, and juglone. However, in the study developed by Stam-
par et al. (2006) they collected walnuts with green husk just before
the hardening of the endocarp, and probably this fact could inter-
fere in the identified compounds. Recently, Pereira et al. (2007b)
reported, in walnut leaves, the identification and quantification
of ten phenolic compounds, namely 3- and 5-caffeoylquinic acids,
3- and 4-p-coumaroylquinic acids, p-coumaric acid, quercetin
3-galactoside, quercetin 3-pentoside derivative, quercetin 3-arabi-
noside, quercetin 3-xyloside and quercetin 3-rhamnoside. In those
matrices the main constituent was found to be quercetin 3-galac-
toside (Amaral et al., 2004; Pereira et al., 2007b).
3.2. Antioxidant activity
In this study, the antioxidant capacity against ROS species of
walnut green husk samples was accessed by three different assays:
reducing power, scavenging activity on DPPH radicals and lipid
peroxidation inhibition by b-carotene-linoleate system. ROS occurs
in foods and are mainly responsible for the initiation of oxidation
reaction. ROS react with lipids, proteins, sugars and vitamins, pro-
ducing undesirable volatile compounds, destroying essential fatty
acids, amino acids and vitamins and producing carcinogens
(Almeida et al., 2008), and make food products less acceptable or
unacceptable to consumers. In biological systems, oxidative stress,
resulting from an imbalance between the generation of ROS and
the antioxidant defence capacity of the cell affects major cellular
components, including lipids, proteins and DNA. Continuous over-
production of ROS and/or the decrease in antioxidant defences may
contribute to the development of several hearth diseases (Valko
et al., 2007).
Green walnut husks extracts revealed a strong reducing power.
The reducing power of a compound may serve as a significant indi-
cator of its potential antioxidant activity (Meir et al., 1995). The
presence of reducers (i.e. antioxidants) causes the reduction of
the Fe3+/ferricyanide complex to the ferrous form (Fe2+) monitored
at 700 nm (Sousa et al., 2008). In this assay, the yellow colour of
the test solution changes to green depending on the reducing
power of the test specimen. All the aqueous walnut husks extracts
presented a concentration-dependent activity (Fig. 2) increasing
the absorbance at 700 nm with increasing concentration. At a very
low extract concentrations (0.1 mg/mL) the absorbance at 700 nm
varied from 0.09 (cv. Mellanaise) and 0.18 (cv. Franquette). To ob-
tain similar absorbance values with the tested standards we need
to increase 36-fold for BHA (abs at 700 = 0.12 at 3.6 mg/mL) and
86-fold for a-tocopherol (abs at 700 = 0.13 at 8.6 mg/mL). For ex-
tract concentrations of 1 mg/mL the reducing power values varied
from 1.01 (cv. Mellanaise) to 2.00 (cv. Franquette) (Fig. 2). EC50val-
ues obtained for aqueous walnut husks extracts were lower than
1.8 mg/mL. In general, extracts with high total phenols content
presented lower EC50values in reducing power assay and in the
order Franquette < Marbot < Parisinne < Mayette < Mellanaise
(Table 2).
DPPH assay has been widely used to determine the free radical-
scavenging activity of various plants and pure compounds (Pereira
et al., 2006; Ferreira et al., 2007; Sousa et al., 2008). DPPH is a sta-
ble free radical which dissolves in methanol, and its purple colour
shows a characteristic absorption at 517 nm. Antioxidant mole-
cules scavenge the free radical by hydrogen donation, the colour
from the DPPH assay solution becomes light yellow resulting in a
decrease in absorbance at 517 nm. Free radical scavenging is one
of the known mechanisms by which antioxidants inhibit lipid oxi-
dation (Ferreres et al., 2007). In this assay, results are expressed as
the ratio percentage of the absorbance decrease of DPPH radical
solution in the presence of extract at 517 nm to the absorbance
of DPPH radical solution at the same wave length. In the present
work, the scavenging effect on DPPH radicals assay also showed
concentration-dependent activity (Fig. 3). For example to the aque-
ous extract of walnut husks cv. Franquette, at 0.01 mg/mL, pre-
sented a scavenging effect of 3.81% that increase to 91.81% at
1 mg/mL. All the studied extracts exhibited high scavenging prop-
erties against DPPH radicals, varying, at 1 mg/mL extract concen-
tration, from 83.16% (cv. Mellanaise) and 93.92% (cv. Marbot).
The obtained results showed a strong antioxidant potential when
comparing to the ones obtained for the standards (BHA: 96% at
3.6 mg/mL and a-tocopherol: 95% at 8.6 mg/mL). On DPPH assay,
EC50values obtained for aqueous walnut husks extracts varied be-
tween 0.35 mg/mL and 0.59 mg/mL in order of Franquette < Mar-
bot < Parisinne = Mayette < Mellanaise (Table 2). Samples with
higher total phenols showed the strongest free radical-scavenging
effect (lower EC50values), being established a significantly nega-
tive linear correlation between the total phenols content and
EC50values (determination coefficients 0.795; p = 0.042).
In b-carotene linoleate model system free radical arisen from
oxidation of linoleic acid, attacks the highly unsaturated b-caro-
tene molecules, causing decrease of the absorbance at 470 nm.
The presence of different antioxidants can hinder the extent of
b-carotene bleaching by neutralizing the linoleate-free radical
and other free radicals formed in the system (Jayaprakasha et al.,
2001). In the absence of antioxidants the absorbance at 470 nm
Table 1
Extraction yield (in percentage) and total phenols content (mg GAEs/g) aqueous
extracts of walnut green husks from the Franquette, Mayette, Marbot, Mellanaise and
Parisienne cultivars
Cultivar Extraction yield (%) Total phenols contents
Franquette
Marbot
Mayette
Mellanaise
Parisienne
31.63 ± 0.98
33.30 ± 3.33
32.19 ± 1.46
32.41 ± 3.75
33.69 ± 1.24
74.08 ± 0.02
43.77 ± 0.01
41.45 ± 0.01
32.61 ± 0.01
38.76 ± 0.01
2328
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decreases rapidly, whereas in their presence, the colour, and thus
absorbance, is retained for a longer time. The antioxidant activity
of walnut green husks extracts, measured by the inhibition of
b-carotene bleaching is shown in Fig. 4. The results showed a con-
centration-dependent antioxidant capacity and EC25values varied
from 0.1 mg/mL (cv. Mayette) and 1.25 mg/mL (cv. Franquette)
(Table 2).
cv. Franquette proved to possess a much higher content of total
phenols than all of the other cultivars. This occurrence can be re-
lated to the results obtained for the antioxidant assays, as cv. Fran-
quette presents the higher values of total phenols and the better
results for reducing power and scavenging activity on DPPH radi-
cals assays, while cv. Mellanaise presents the worst results and
the lower content of total phenols. In fact, several studies pointed
out the antioxidant potential of phenols (Pereira et al., 2006; Ferre-
ira et al., 2007; Ramadan and Moersel, 2006; Kornsteiner et al.,
2006).
3.3. Antimicrobial activity
The study of antimicrobial capacity of plant phenolics is well-
known (Pereira et al., 2006; Pereira et al., 2007a,b; Proestos et al.,
2005; Rauha et al., 2000). However, crude extracts tested by our
group with other natural products (fruits and leaves) also showed
good antimicrobial activities, such as tables olives (Pereira et al.,
2006; Sousa et al., 2006), hazelnuts (Oliveira et al., 2007), walnuts
(Pereira et al., in press), walnut leaves (Pereira et al., 2007b), olive
leaves (Pereira et al., 2007a), hazelnut leaves (Oliveira et al., 2007)
and different Brassica species flower buds (Sousa et al., in press). In
this work, we propose the use of walnut green husks, a by-product
of walnut production, as antimicrobials source. Borchers et al.
(2004) reported that crude extracts may be more beneficial than
isolated constituents, since a bioactive individual component can
change its properties in the presence of other compounds present
in the extracts.
The walnut green husks aqueous of different cultivars were
screened for their antimicrobial properties against B. cereus, B.
subtilis, S. aureus, E. coli, P. aeruginosa, K. pneumoniae, C. albicans
and C. neoformans. The minimal inhibitory concentration (MIC) val-
ues for the tested bacteria and fungi were determined as an evalu-
ation of the antimicrobial activity of the tested extracts. The halos
of the inhibition zones corresponding to the MICs are also pre-
sented (Table 3). The response for each microorganism tested
was different. All the tested extracts revealed antimicrobial activity
showing different selectivity and MICs for each microorganism.
0.0
0.6
1.2
1.8
2.4
3.0
Concentration (mg/mL)
Abs at 700 nm
Franquette MarbotMayette Mellannaise Parisienne
0.010.05 0.10.250.512
Fig. 2. Aqueous extracts reducing power values of walnut green husks from the Franquette, Mayette, Marbot, Mellanaise and Parisienne cultivars. Each value is expressed as
mean ± standard deviation.
Table 2
EC50values (mg/mL) aqueous extracts of walnut green husks from the Franquette,
Mayette, Marbot, Mellanaise and Parisienne cultivars
CultivarReducing power (EC50)DPPH (EC50)
b-Carotene bleaching (EC25)
Franquette
Marbot
Mayette
Mellanaise
Parisienne
0.50
0.51
0.67
0.70
0.62
0.35
0.42
0.51
0.59
0.51
1.27
0.60
0.10
1.17
0.92
0.0
20.0
40.0
60.0
80.0
100.0
Concentration (mg/mL)
Scavenging effect (%)
FranquetteMarbotMayetteMellannaise Parisienne
0.01 0.050.10.25 0.512
Fig. 3. Aqueous extracts scavenging effect on DPPH of walnut green husks from the Franquette, Mayette, Marbot, Mellanaise and Parisienne cultivars. Each value is expressed
as mean ± standard deviation.
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Gram positive bacteria were the most sensitive being inhibited by
all the extracts. Concerning Gram negative bacteria, only the
extracts of cv. Franquette and cv. Marbot were able to inhibit the
growth of P. aeruginosa at the highest extract concentration tested
(100 mg/mL); fungi were resistant. The pure active compounds
ampiciline and cycloheximide presented lower MICs than the wal-
nut husk extracts.
The extracts presented similar antimicrobial capacity, inhibiting
Gram + bacteria and in the order S. aureus > B. cereus > B. subtilis. S.
aureus was the most susceptible microorganism, presenting MICs
of 0.1 mg/mL. These results are very important considering that
S. aureus can produce several types of enterotoxins that cause gas-
troenteritis, which is a major food-borne disease in most countries
(Halpin-Dohnalek and Marth, 1989). Natural products may be a
particularly rich source of anti-infective agents. For example, flavo-
noids showed antimicrobial activity, and quercetin and other re-
lated compounds acts essentially by enzyme inhibition of DNA
gyrase (Cushnie and Lamb, 2005).
The obtained results are similar, and in some cases better, than
previous results obtained with different aqueous extracts of
walnut leaves (Pereira et al., 2007b). The MICs were very similar
but two of the tested extracts in this work showed inhibition of
P. aeroginosa, which was not observed with leaves extracts. On
the other hand, extracts of walnut fruits (Pereira et al., in press)
were much less effectives.
In conclusion, the results obtained in our work showed that
walnut green husks can be used as an easily accessible source of
natural bioactive compounds. We demonstrate for the first time,
as far we known, that walnut green husks aqueous extracts pre-
sented a strong antioxidant activity and inhibited the growth of
different pathogenic bacteria (Gram +) that can causes health prob-
lems. Further studies will be developed to characterize walnut
green husks extracts and to identify the molecules responsible
for this bioactivity. On other hand, the potential showed by walnut
green husks extracts can lead to the valorization of a by-product,
that nowadays has a scarce use.
Conflict of interest statement
The authors declare that there are no conflicts of interest.
Acknowledgement
The authors are grateful to INTERREG III A Program, Project
PIREFI for financial support of this work.
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0.0
20.0
40.0
60.0
80.0
100.0
Concentration (mg/mL)
Antioxidant activity (%)
Franquette MarbotMayette MellannaiseParisienne
0.10.5125
Fig. 4. Aqueous extracts antioxidant activity (%) by b-carotene bleaching method of walnut green husks from the Franquette, Mayette, Marbot, Mellanaise and Parisienne
cultivars. Each value is expressed as mean ± standard deviation.
Table 3
Antimicrobial activity of aqueous extracts of walnut green husks from different cultivars
Cultivar MIC (mg/mL)
B. cereus B. subtilis S. aureusP. aeruginosa E. coliK. pneumoniae C. albicansC. neoformans
Franquette 0.1
(+ + + +)
1
(+ + + +)
0.1
(+ + + +)
0.1
(+ + + +)
0.1
(+ +)
10
(+ + +)
0.1
(+ + +)
10
(+ +)
0.1
(+ + + +)
0.1
(+ + + +)
0.1
(+ + + +)
0.1
(+ + + +)
0.1
(+ + +)
0.1
(+ + + +)
0.1
(+ + + +)
100
(+ +)
100
(+)
100
(?)
100
(?)
100
(?)
100
(?)
100
(?)
100
(?)
100
(?)
100
(?)
100
(?)
100
(?)
100
(?)
100
(?)
100
(?)
100
(?)
100
(?)
100
(?)
100
(?)
100
(?)
100
(?)
100
(?)
100
(?)
100
(?)
100
(?)
Marbot
Mayette
Mellanaise
Parisienne
No antimicrobial activity (?), inhibition zone < 1 mm. Slight antimicrobial activity (+), inhibition zone 2–3 mm. Moderate antimicrobial activity (+ +), inhibition zone 4–
5 mm. High antimicrobial activity (+ + +), inhibition zone 6–9 mm. Strong antimicrobial activity (+ + + +), inhibition zone >9 mm. Standard deviation ± 0.5 mm.
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