Pharmaceutical Biology

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Antioxidant and antimicrobial activities of ethanol

and aqueous extracts from Urtica urens

Massara Mzid, Sameh Ben Khedir, Maryem Ben Salem, Wafa Regaieg & Tarek
Rebai

To cite this article: Massara Mzid, Sameh Ben Khedir, Maryem Ben Salem, Wafa Regaieg &
Tarek Rebai (2017) Antioxidant and antimicrobial activities of ethanol and aqueous extracts from
Urtica�urens, Pharmaceutical Biology, 55:1, 775-781, DOI: 10.1080/13880209.2016.1275025

To link to this article: https://doi.org/10.1080/13880209.2016.1275025

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PHARMACEUTICAL BIOLOGY, 2017
VOL. 55, NO. 1, 775–781
http://dx.doi.org/10.1080/13880209.2016.1275025

RESEARCH ARTICLE

Antioxidant and antimicrobial activities of ethanol and aqueous extracts from

Urtica urens
Massara Mzida, Sameh Ben Khedira, Maryem Ben Salemb, Wafa Regaiegb and Tarek Rebaia
a
Laboratory of Histology Embryology and Reproductive Biology, Faculty of Medicine of Sfax, University of Sfax, Sfax, Tunisia; bLaboratory of
Pharmacology, Faculty of Medicine of Sfax, University of Sfax, Sfax, Tunisia

ABSTRACT ARTICLE HISTORY

Context: Urtica urens L. (Urticaceae) is an important and commonly used plant for its medicinal and Received 15 March 2016
pharmacological properties. Revised 1 November 2016
Objective: We analyzed the antioxidant and antimicrobial activities of the leaves of Urtica urens in ethanol Accepted 17 December 2016
(EtOH) and water (WA) solvents, employing standard analytical methods.
KEYWORDS
Materials and methods: Polyphenol, flavonoid and tannin content of Urtica urens leaves were deter- Flavonoids; MIC;
mined, after their extraction, using EtOH (70%) and WA extracts as well as the antioxidant (DPPH, ABTS, Polyphenols
b-carotene and FRAP) and the antibacterial (via the method of dilution tests) activities of EtOH and WA
extracts.
Results: The 70% EtOH of Urtica urens showed the highest values of total phenolic (31.41 mg GAE/g DW),
flavonoids (6.81 mg quercetin/g DW), tannin (8.29 mg GAE/g DW) and TEAC (560 mmol Trolox/g DW), com-
pared to the WA. The results of DPPH for EtOH (95.56%) were higher than that of WA (64.56%) at a con-
centration of 40 mg/L. The extracts displayed a FRAP 106.23 for EtOH and 30.55 lmol Fe(II)/g DW for WA.
The results clearly indicated that EtOH was the strongest radical scavenger (IC50 ¼ 245.65 ± 10.2 lg/mL).
Ethanol was the most effective with minimum inhibitory concentration (MIC) < 250 lg/mL. WA has no
antibacterial activity.
Discussion and conclusion: The results indicate that leaves of Urtica urens could be used as natural anti-
oxidant and antimicrobial agents.

Introduction Urtica urens is widely distributed in Tunisia and traditionally

used as an herbal medicine for a wide variety of purposes. Other
The world is presently over-dependent on a few plant species species of Urtica has been studied the microbial and antioxidant
(Correa et al. 2013). Diversification of production and consump- activities (Gulcin et al. 2004; Kukric et al. 2012; Sidaoui et al.
tion habits to include a broader range of plant species, particu- 2015). But, in our knowledge, the antimicrobial and antioxidant
larly those currently identified as under-utilized, could activities of the species of Urtica urens from Tunisia have not yet
significantly contribute to improve health and nutrition, liveli- studied.
hoods and ecological sustainability. Wild plants have played a The use of such antioxidant and antimicrobial compounds
significant role in supplementing staple foods by supplying trace from natural sources has been considerable interest not only for
elements, vitamins, and minerals in order to obtain a balanced the preservation of foods and improving the shelf life of food
diet, and they may do so again in the future. Their interest as a products, but also for increasing the stability of fats and oils and
source of nutraceuticals has been highlighted in recent studies to control the human and plant diseases of microbial origin
(Leonti 2012). Several epidemiological studies suggest that a high (Benkeblia 2004).
intake of foods rich in natural antioxidants reduces the risk of This study evaluates the total phenolic compounds, total flavon-
some cancers, heart, and degenerative diseases. Urtica urens L. oid compounds, total tannin contents, vitamins (D, E and C), the
(Urticaceae) leaves, have a relatively high level of protein (66%), antioxidant properties (DPPH, ABTS, b-carotene and FRAP), and
which is of better quality if compared with the proteins of other the in vitro antimicrobial activities of ethanol and aqueous extracts
leafy vegetables (Hughes et al. 1980). The leaves of nettle are of leaves of Urtica urens against strains of bacteria which known
good sources of different significant minerals and vitamins as multi-resistant organisms or involved in diverse pathology.
(Adamski & Bieganska 1980; Kukric et al. 2012). Nettles contain
flavonoids, fatty acids, terpenes, protein, vitamins, and minerals.
Stinging nettle leaves are rich in vitamin C, B groups vitamins, Materials and methods
vitamin K, and some minerals mainly calcium, iron, magnesium, Chemicals
phosphorus, potassium, and sodium (Upton 2013). Nettle leaves
contain nine carotenoids: Lutein, lutein isomers and b-carotene 1,1-Diphenyl-2-picrylhydrazyl (DPPH), butylated hydroxytoluene
are the basic carotenoids (Guil-Guerrero et al. 2003). (BHT), a-tocopherol, b-carotene, and linoleic acid were

CONTACT Massara Mzid mzid.masarra@gmail.com Faculty of Medicine of Sfax, University of Sfax, Tunisia, Avenue Majida Boulila, 3029 Sfax, Tunisia
ß 2017 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distri-
bution, and reproduction in any medium, provided the original work is properly cited.
776 M. MZID ET AL.

purchased from Sigma Chemical Co. (St. Louis, MO). All other Determination of hydrolyzable tannin
chemicals, namely L-ascorbic acid, Folin-Ciocalteu reagent (mix-
ture of phosphomolybdate and phosphotungstate, Sigma) and The hydrolyzable tannin content was determined from AECS
other solvents were of analytical grade. All solutions were freshly using the potassium iodide test (Saad et al. 2012). The analysis
prepared in distilled water. was performed in triplicate and the results were expressed in mg
equivalent of gallic acid per gram of dried extract (mg GAE/g
DW) using the calibration curve of gallic acid
Plant material (y ¼ 0.121x þ 0.011, r2 ¼ 0.9819).

Aerial parts of Urtica urens were collected from Sfax, Tunisia,

during December 2014 and identified by Professor Mohamed Antioxidant activity
Chaieb from the Faculty of Sciences of Sfax (Laboratory of DPPH assay
Biology and Vegetable Ecophysiology, Faculty of Science, Sfax,
Tunisia). The voucher sample was deposited at The National The DPPH radical-scavenging activity of Urtica urens extracts
Botanical Research Institute Tunisia (INRAT). It was washed was determined using the method described by Kirby and
with distilled water and then dried at room temperature for two Schmidt (1997) with some modifications. The DPPH scavenging
days, afterwards, crushed, milled in a knife mill to obtain 100 g percentage was calculated as follows:
of Urtica urens powder and subsequently stored in glass bottles
% DPPH scavenging ¼
at room temperature.
½ðabsorbance of negative control  absorbance of sampleÞ=
absorbance of negative control  100:
Preparation of plant extract
Tests were carried out in triplicate. The free radical scaveng-
The dried powder (50 g) was extracted (by a maceration method) ing activity of each extract was expressed in terms of Trolox
by adding ethanol (70%) at 37  C for 72 h. The extract was fil- equivalents (TE) (mg TE/g extract) and at the extract concentra-
tered through Whatman No.1 filter paper in a Buchner funnel. tion where 50% of DPPH was attenuated (IC50).
The filtrate was evaporated to dryness under reduced pressure in
a rotatory vacuum evaporator (EYELA N1000, Tokyo, Japan)
(Nassiri-Asl et al. 2009). Antioxidant capacity by ABTSþ method
The other dried powder (50 g) was subjected to an extraction The 2,2-azinobis-3-ethylbenzothiazoline-6-sulfonic acid free rad-
with water for 20 min by infusion (1/10, plant/solvent). After the ical (ABTSþ) neutralization was determined using a spectro-
treatment, the aqueous filtrate was concentrated by rotatory photometric, 96-well microplate method described by Re et al.
evaporator in order to obtain the aqueous extract of Urtica urens (1999) with minor modifications. The antioxidant capacity of
in powder form. each extract was expressed quantitatively in terms of trolox
The dried sample of each extract was weighed and the equivalents (TE) (mg TE/g extract) and at the extract concentra-
yield of soluble constituents was determined. The dried tion where 50% of ABTSþ was neutralized (IC50).
extracts were kept in dark at þ4  C until further analyses. The
yield of the extract under study was calculated by the follow-
ing equation: b-Carotene bleaching by linoleic acid assay
Yield ð%Þ ¼ ðA1  100Þ=A2 The ability of Urtica urens extract to prevent bleaching of b-caro-
tene was assessed as described by Koleva et al. (2002).
Where A1 corresponds to the Urtica urens concentrated by
evaporator and A2 corresponds to the powdered dried plant
material used for extraction with ethanol or water. Ferric reducing antioxidant power (FRAP assay)
A modified method of Benzie and Strain (1999) was adopted for the
FRAP assay. Results are expressed in lM Fe (II)/g dry mass (DW).
Total phenolic compound
The total phenolic compound (TPC) of extracts was determined
by the Folin-Ciocalteu method (Singleton & Rossi 1965). TPC Estimation of vitamin D
was expressed as mg gallic acid equivalents (GAE) per gram Vitamin D levels was measured by colorimetric methods using
extract. Values presented are the average of three measurements. commercial reagent kits (Ref: 20151 and 20091), respectively,


y ¼ 0:121 þ 0:011; r2 ¼ 0:9819 : purchased from Biomaghreb (Ariana, Tunis, Tunisia).

Estimation of vitamin E
Total flavonoids compound
The extraction of vitamin E from the method has been described
The total flavonoid content of Urtica urens extracts was deter- by both Katsanidis and Addis (1999).
mined by the method described by Zhishen et al. (1999).
Flavonoid content was expressed as mg quercetin equivalent
(QE)/g dried extract. Values presented are the average of three Estimation of vitamin C
measurements.

Ascorbic acid was determined as described by Jacques-Silva et al.
y ¼ 0:0182x  0:004; r2 ¼ 0:9949 : (2001).
 PHARMACEUTICAL BIOLOGY 777

Antimicrobial activity phenolic compound, 29.56 ± 1.56 mg R/g E to flavonoids and

4.05 ± 0.52 mg GAE/g E to hydrolyzable tannin (Table 1).
Microbial strains
Antimicrobial activities of Urtica urens extracts were tested
against eight strains of bacteria: Staphylococcus aureus (ATCC Antioxidant capacity by DPPH method
6538), Pseudomonas aeruginosa (ATCC 27893), Bacillus subtilis
The DPPH method has been widely applied for estimating anti-
(JN 934392), Salmonella enteritidis, Escherichia coli (ATCC
oxidant activity in recent years. DPPH, a stable free radical with
25922), Staphylococcus epidermidis (MTCC 3615), Micrococcus
a purple colour, changes into a stable yellow compound upon
luteus (ATCC 4698) and Enterococcus faecalis (ATCC 29212).
reacting with an antioxidant. In brief, the reduction capacity of
These strains were kindly provided by the Centre of
DPPH was determined by the decrease in its absorbance at
Biotechnology of Sfax, Tunisia.
517 nm, which is reduced by the antioxidant (Duh 1998). As can
be seen in Figure 1, the results clearly indicated that ethanol
Agar diffusion method extract, which contained the highest amount of phenolics and
flavonoids compounds, was the strongest radical scavenger
Antimicrobial activity was performed according to the method (IC50 ¼ 245.65 ± 10.2 lg/mL). The WA extract displayed free rad-
described by Vanden Berghe and Vlietinck (1991). ical scavenging activity (IC50 ¼ 142.94 ± 10.54 lg/mL) (Table 2).
The highest inhibition effect of WA and EtOH extracts of
Determination of the minimal inhibitory concentration (MIC) Urtica urens were 64.56 and 93.56%, respectively, compared with
ascorbic acid with 99.83% and BHT with 95.56% at concentra-
MIC values, which represent the lowest plant extracts concentra- tion of 40 mg/L. However, in our results further increase in the
tion that completely inhibits the growth of microorganisms, were concentration of the Urtica urens extracts indicated increasing
determined based on a micro-well dilution method (1998). inhibition of DPPH radical (Figure 1).

Determination of minimum bactericidal concentration (MBC)

Antioxidant capacity by the method of ABTS1
To determine the minimum bactericidal concentration (MBC), a
The free radical scavenging ability of Urtica urens phenolics was
loop full from those tubes, which did not show any visible
determined using ABTS radical cation, too. ABTS radical cation
growth in MIC assay, was cultured on MHA and incubated at
has often been used in the evaluation of antioxidant activity of
37  C for 18 to 24 h. The highest dilution that did not yield col-
single compounds and complex mixtures of various origins
ony formation on MHA was considered as MBC (Testai et al.
(body fluids, foods, beverages, plant extracts). In this assay,
2002).
ABTS radical cation was directly generated in stable form using
potassium persulfate. Generation of radicals before the antioxi-
Statistical analysis dants are added to prevent the interference of compounds, which
affect radical formation. This modification makes the assay less
Values were expressed as mean ± standard error of the mean susceptible to artifacts and inhibits overestimation of antioxidant
(SEM) of many parallel measurements. Differences at p  0.05 capacity. EtOH extract had an activity of 560.33 ± 29.45 mol
were considered statistically significant. Database management
and statistical analysis were performed using SPSS (Microsoft
Corporation) statistical software package (SPSS Inc, Chicago, IL).

Results
Extraction yield and polyphenol, flavonoid and hydrolyzable
tannin contents
The yield of extractable components relative to the weight of
dried plant material ranged from 4.768% for EtOH extract and
0.87% for WA extract. The results of the phytochemical analysis
Figure 1. Antioxidant capacity of ethanol and aqueous extracts of Urtica urens
evinced the quantitative values of EtOH extract: 31.41 ± 0.31 mg by the DPPH· method at different concentrations. BHT and ascorbic acid (Vitamin
GAE/g E to phenolic compound, 6.81 ± 1.72 mg R/g E to flavo- C) were used as positive control. Values are means ± SEM (n ¼ 3).
noids and 8.29 ± 0.3 mg GAE/g E to hydrolyzable tannin
(Table 1). For WA extract; 5.34 ± 0.21 mg GAE/g E to total

Table 1. Extract yield and total flavonoids, phenolic and tannin contents from Table 2. Antioxidant activity of Urtica urens extracts (ABTSþ, DPPH).
Urtica urens extracts. DPPH ABTSþ DPPH ABTSþ
Flavonoids Phenolics Tannins scavenging scavenging scavenging scavenging
Sample Yield (%) (mg quercetin/g DW) (mg GAE/g DW) (mg GAE/g DW) Sample (IC50, lg/mL) (IC50, lg/mL) (mg TE/g extract) (mg TE/g extract)
Ethanol 4.768 6.81 ± 1.72 31.41 ± 0.31 8.29 ± 0.3 Ethanol 245.65 ± 10.2 30.88 ± 3.03 65.33 ± 10.72 560.33 ± 29.45
Aqueous 0.87 5.34 ± 0.21 29.56 ± 1.56 4.05 ± 0.52 Aqueous 142.94 ± 10.54 14.65 ± 1.09 45.67 ± 10.21 350.33 ± 18.73
Values are expressed as mean ± SEM of three independent determinations. Values are expressed as mean ± SEM of three independent determinations.
p < 0.05. p < 0.001.
DW: dry weight. TE: Trolox equivalents; IC: Inhibition Concentration.
778 M. MZID ET AL.

Figure 2. ABTS (TEAC) activities of Urtica urens extracts. Values are means ± SEM Figure 4. FRAP activities in ethanol and water extracts of Urtica urens. Values
(n ¼ 3). p < 0.001. represent means ± SEM (n ¼ 3). p < 0.001. a: compared to BHT; b: compared
to Ethanol.

Table 3. Rapport MBC/MIC.

Ethanol
Aqueous
Stains MBC/MIC MBC/MIC
Gramþ Bacillus subtilis 2 bactericidal –
Staphylococcus aureus 2 bactericidal –
Micrococcus luteus 2.01 bactericidal –
Staphylococcus epidermidis 2 bactericidal –
Enterococcus faecalis – – –
Gram Escherichia coli – – –
Salmonella enteritidis 1.93 bactericidal –
Pseudomonas aeruginosa 2 bactericidal –
Rapport MBC/MIC MBC/MIC 4 bacteriostatic
Canillac & Mourey 2001
MBC/MIC <4 bactericidal
Figure 3. b-Carotene bleaching percentage of Urtica urens extracts. Values are
means ± SEM (n ¼ 3). p < 0.001. a: compared to BHT (control); b: compared
to Ascorbic Acid; £: compared to Ethanol.

Trolox/g DW versus WA extract had much less activity com- S. aureus, M. luteus and E. faecalis) and Gram-negative (E. coli,
pared to EtOH, 350.33 ± 18.73 mol Trolox/g DW (Figure 2). P. aeruginosa and S. enteritidis) bacteria. The evaluation of the
antibacterial activity was realized by the determination of MIC
and MBC values. Table 3 reveal that all extracts vary the degrees
b-carotene bleaching by linoleic acid assay of antibacterial activity against most of the tested Gram-positive
and Gram-negative bacteria, and the EtOH extract was found to
The b-carotene scavenging activity of the two extracts at different be the most effective. In fact, it displayed a large antimicrobial
concentrations was compared with that of butylated hydroxyto- spectrum and exerted a major antibacterial effect against both
luene (BHT), as shown in Figure 3. In this study, the addition of tested Gram-positive and Gram-negative bacteria. While the
Urtica urens extracts and BHT prevented bleaching of the most vulnerable bacteria for the EtOH extract were for Gram-
b-carotene at different degrees. The WA and EtOH extracts of positive (Bacillus subtilis, Staphylococcus aureus, Micrococcus
Urtica urens demonstrated high ability to prevent bleaching luteus and Staphylococcus epidermidis), and two Gram-negative
of the b-carotene. Interestingly, they exhibited a strong inhibition bacteria (Salmonella enteritidis and Pseudomonas aeruginosa) at
of the b-carotene bleaching up to 71.12% and 60.52%, respect- concentration of 150 lg/mL. However, the WA extract did not
ively, at concentrations up to 500 lg/mL. show any effect on almost all the tested bacteria.

Ferric reducing antioxidant power ability Vitamins D, E and C

In this study, the ferric reducing power of plant extracts is dose- In our results (Table 4), we noted that the concentrations of vita-
dependent as illustrated in Figure 4. The strongest activity of the min D, vitamin C and E were 1.45 ± 0.14 mg/100 g;
reducing power is exhibited by the EtOH extract 238 ± 2.95 mg/100 g; 356 ± 0.15 mg/100 g, respectively, in EtOH
(106.23 ± 3.45 lmol Fe (II)/g DW). This is followed by WA extract of Urtica urens and of water extract 0.23 ± 0.04;
extract (30.55 ± 2.67 lmol Fe(II)/g DW). This activity of the 160.55 ± 3.09; 2.3 ± 0.01 mg/100 g, respectively.
aqueous extracts is significantly lower than the EtOH and BHT
(75.56 ± 3.57 lmol Fe (II)/g DW).
Discussion

Antibacterial activity The total polyphenol, flavonoid and tannin content in the EtOH
and WA extracts were determined. It is well known that phenolic
The antibacterial activity of the various Urtica urens extracts and flavonoid compounds contribute directly to the biological
was assessed against Gram-positive (B. subtilis, S. epidermidis, activity of plant materials (Rice-Evans et al. 1996).
 PHARMACEUTICAL BIOLOGY 779

Table 4. Levels of vitamins D, C and E in Urtica urens leaves. could be attributed to the fact that the ABTSþ assay is aqueous
Sample Vitamin D (mg/100 g) Vitamin C (mg/100 g) Vitamin E (mg/100 g) based, favouring hydrophilic compounds; whereas the DPPH
Ethanol 1.45 ± 0.14 238 ± 2.95  356 ± 0.15  assay is an organic based assay, favouring hydrophobic phytocon-
Aqueous 0.23 ± 0.04 160.55 ± 3.09a 2.3 ± 0.01a stituents (Schlesier et al. 2002).
Values are expressed as mean ± SEM of three independent determinations. On the other hand, EtOH extract was ineffective regarding
p < 0.001.
cellular-ROS neutralization. Instead, it caused a significant over-
a: compared to Ethanol.
production of intracellular ROS. It has been shown that Urtica
urens can have an antioxidant or pro-oxidant effect depending
With 4.768% of yield in the EtOH extract, the results of the on their concentration and/or structural properties.
phytochemical analysis exhibited the quantitative values of Urtica The antioxidant activity increased with increasing extract con-
urens 31.41 ± 0.31 mg GAE/g E to phenolic compound, centration. The EtOH extract of Urtica urens displayed a signifi-
6.81 ± 1.72 mg R/g E to flavonoids and 8.29 ± 0.3 mg GAE/g E to cant (p < 0.001) cell-free (IC50 ¼ 30.88 ± 3.03 lg/mL of ABTSþ)
hydrolyzable tannin. These results demonstrate that the Urtica as comparison with WA extract (Table 2). This could be ascribed
urens has high quantities of phenolic compounds. Other studies to patuletin, an abundant and potent aglycon flavonol extracted
performing quantification of the phenolic compounds and flavo- from Urtica urens (Ataa et al. 1995). This phytochemical is lipo-
noids in EtOH extract of leaves showed their presence in large philic and readily crosses the cell membrane, making it easily
quantity as compared with our results for the phenolic com- available in the cytosol to exert its protective antioxidant action
pounds and flavonoids (Manu Kumar et al. 2013). (Abdel-Wahhab et al. 2005).
The content of phenolic compounds found in plants may vary The b-carotene bleaching method is based on the loss of the
during processing steps such as growing, harvesting, storage and orange colour of b-carotene due to its reaction with radicals
technological procedures used (Lombardo et al. 2010). The phen- formed by linoleic acid oxidation in an emulsion. The rate of
olic compounds present in plants have received great attention b-carotene bleaching can be slowed down in the presence of anti-
because of their antioxidant properties and they can potentially oxidants (Kulisic et al. 2004). Interestingly, the WA and EtOH
interact with biological systems and play an important role in extracts exhibited strong inhibition of b-carotene bleaching com-
anticancer, anti-inflammatory, and antimicrobial activity (Wang parable to BHT and ascorbic acid. The control, without addition
et al. 2003; Abu-Reidah et al. 2013). The antioxidant properties
of sample, oxidized rapidly and the absorbance at 470 nm tends
are attributed to flavonoids due to their hydroxyl groups that
to zero. Therefore, the higher antioxidant activity of compounds
can act as free radical scavengers, reducing agents and metal
from the Urtica urens extracts in this assay suggests a possible
chelation (Agati et al. 2012). It has been reported that free
biological functionality in preventing the oxidative degradation of
radical-scavenging activity is greatly influenced by the phenolic
membrane lipids.
composition of the sample (Cheung et al. 2003). The antioxidant
In this study, we used FRAP assay because it is quick and
activity of the WA and EtOH extracts may be attributed to their
simple to perform, and its reaction is reproducible and linearly
phenolic and flavonoid contents.
related to the molar concentration of the antioxidant(s) present.
The antiradical activity assay is based on the reduction of 1,1-
This method was initially developed to assay plasma antioxidant
diphenyl-2-picrylhydrazyl (DPPH). The ability of the extracts and
capacity, but could be used to measure the antioxidant capacity
standard ascorbic acid to scavenge free radicals and pair off the
odd electron was shown in this assay. It was observed that WA from a wide range of biological samples and pure compounds to
and EtOH extracts of Urtica urens are a similar scavenger of fruits, wines, and animal tissues. In our study, the EtOH extract
DPPH radical compared with the standard BHT. The highest had higher ferrous reducing antioxidant power compared to
inhibition effects of WA and EtOH extracts of Urtica urens were standard BHT which had 75.56 ± 3.57 lmol Fe (II)/g DW (Figure 4).
64.56% and 93.56%, respectively, compared with ascorbic acid FRAP assay demonstrated that EtOH extract had higher activity
with 99.83% and BHT with 95.56% at concentration of 40 mg/L. (106.23 ± 3.45 lmol Fe (II)/g DW) than that of BHT. Based on
However, in our results, further increase in the concentration of this background, this study concludes that Urtica urens is consid-
the Urtica urens extracts displayed increasing inhibition of DPPH ered as a potential source of natural antioxidants. The presence
radical. of general phytochemicals and specific active compounds might
According to Manu Kumar et al. (2013), using DPPH to be responsible for their therapeutic effects. Their reducing power
measure the results of radical scavenging activities, Urtica urens is probably attributed to the presence of polyphenols that may
leaf extract by methanol showed higher activity (78% at 0.5 mg/mL) act in a similar way as reductones by donating the electrons and
among the other solvents and ethyl acetate displayed lower activity reacting with free radicals to convert them into more stable
compared to ethanol, water and acetone. products and terminate the free radical chain reaction
As is well known, the antioxidant activity of plant extracts (Siddhuraju & Becker 2007). Reductones also react with certain
containing polyphenol components is assigned to their capacity precursors of peroxide, thus preventing the formation of the lat-
to be donors of hydrogen atoms or electrons and to capture the ter (Matsushige et al. 1996). The antioxidant activity has con-
free radicals (Shon et al. 2003). The DPPH radical-scavenging firmed the medicinal importance of plants as naturally-occurring
assay is a widely used method to evaluate the ability of plant antioxidants (Venkatachalam & Muthukrishnan 2012).
extracts to scavenge free radicals generated from DPPH reagent According to Manu Kumar et al. (2013), FRAP assay exhib-
(Chung et al. 2006). ited ethanol fraction activity of 80 lmol Fe(II)/g DW. The anti-
Like DPPH, the method of ABTSþ or TEAC (Trolox bacterial activity of Urtica urens extracts was evaluated against
Equivalent Antioxidant Capacity) is widely used to evaluate the Gram-positive (B. subtilis, S. epidermidis, S. aureus, M. luteus and
antioxidant capacity of a variety of substances including plant E. faecalis) and Gram-negative (E. coli, P. aeruginosa and S.
extracts. enteritidis) bacteria. These strains are available in the laboratory.
The TEAC value takes into account the capacity of a sub- The antibacterial activity was assessed by the determination of
stance to react with ABTSþ radical (Arts et al. 2004). The water MIC and the minimal bactericidal concentration (MBC) (the
extract caused a greater attenuation of ABTSþ than DPPH. This minimum concentration that inhibits all visible growth of
780 M. MZID ET AL.

microorganisms for 48 h at 37  C) (Diao et al. 2014). As can be Urtica urens extracts are a substantial source of vitamins C, E
seen in Table 3, Urtica urens showed varying degrees of antibac- and D.
terial activity against most of the Gram-positive and Gram-nega-
tive bacteria tested. The aqueous extract was found to be not
active against all tested bacteria. Ethanol extract exhibited anti- Conclusion
bacterial activity (bactericidal) against Gram-positive (Bacillus
The results of this study concluded that the leaves of Urtica urens
subtilis, Staphylococcus aureus, Micrococcus luteus and
contain appreciable number of flavonoids, polyphenols and tan-
Staphylococcus epidermidis), and two Gram-negative bacteria
nins. The extracts seem to present a real interest and potential
(Salmonella enteritidis and Pseudomonas aeruginosa) at concen-
through their antioxidant activities that have been made by the
tration of 150 lg/mL.
different tests (DPPH, ABTS, b-carotene and FRAP).
The reason for the difference in sensitivity between
Their antioxidant activities further lend credence to the bio-
Gram-positive and Gram-negative bacteria might be ascribed to
logical value of this plant. The EtOH extract shows activity
the differences in morphological constitutions between these
against bacteria. This may be due to high phenolic content and
microorganisms; Gram-negative bacteria having an outer phos-
the presence of active compounds such as tannins.
pholipidic membrane carrying the structural lipopolysaccharide
components. This makes the cell wall impermeable to antimicro-
bial chemical substances. On the other hand, Gram-positive bac-
Acknowledgements
teria are more susceptible, having only an outer peptidoglycan
layer which is not an effective permeability barrier. Therefore, This research was supported by the Tunisian Ministry of Higher
the cell walls of Gram-negative organisms are more complex in Education and Scientific Research.
lay out than Gram-positive ones acting as a diffusional barrier
and making them less susceptible to the antimicrobial agents
than Gram-positive bacteria (Kaushik et al. 2015). Disclosure statement
The most susceptible bacteria for the EtOH extract were S.
The authors declare no conflict of interest.
aureus, M. luteus, S. enteritidis, S. epidermidis, P. aeruginosa and
B. subtilis with a rapport MBC/MIC values of 2, 2.01, 1.93, 2 and
2, respectively. Funding
These results are of great importance, particularly in the case
of S. aureus which is well-known for being resistant to a number This research was supported by the Tunisian Ministry of Higher
of antibiotics and for the production of several types of entero- Education and Scientific Research via Sfax University.
toxins that cause many enteritis types and septicemia (Kaushik
et al. 2015). P. aeruginosa is a naturally resistant Gram-negative
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