Industrial Crops and Products 62 (2014) 188195

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Industrial Crops and Products

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Phenolic content, antioxidant and allelopathic activities of various

extracts of Thymus numidicus Poir. organs
Imen Ben El Hadj Ali a, , Radhia Bahri b , Maher Chaouachi b , Mohamed Boussad a ,
Fethia Harzallah-Skhiri b
a
Laboratory of Plant Biotechnology, National Institute of Applied Sciences and Technology, Carthage University, B.P. 676, 1080 Tunis, Tunisia
b
Laboratory of Genetic Biodiversity and Valorisation of Bioresources, Higher Institute of Biotechnology of Monastir, University of Monastir, Monastir,
Tunisia

a r t i c l e i n f o a b s t r a c t

Article history: Total phenol content, antioxidant and allelopathic properties of roots, steems and leaves extracts of
Received 12 April 2014 Tunisian endemic T. numidicus were studied. Three solvent systems with varying polarities (petroleum
Received in revised form 12 August 2014 ether, ethyl acetate and methanol) were used. The highest amounts of polyphenols (98.66 3.17 mg
Accepted 17 August 2014
EAG/g DW), avonoids (54.28 1.6 mg RE/g DW), avonols (27.23 1.71 mg RE/g DW) and proan-
thocyanidins (5.12 0.8 g cyanidin chloride/g DW) were shown by the polar subfraction of the leaf
Keywords:
methanolic extracts. The efciency of the solvents used to extract phenols from the three organs
Thymus numidicus
varied considerably. The level of antioxidant activity estimated by DPPH and ABTS test systems was
Organ
Polyphenols
high for leaf (IC50 = 11.06 0.33 g/ml; 235.46 2.14 g TE/mg DW) and stems (IC50 = 15.29 0.9 g/ml;
Antioxidant capacity 233.75 1.2 g TE/mg DW) methanolic extracts. A signicant correlation between radical-scavenging
Phytotoxic activity capacities of extracts with total phenolic compound content was observed. The results also indicate that
the root extracts inhibited the shoot and root growth of Medicago sativa and Triticum stivum seedlings.
Thus, the T. numidicus extracts may be used as a natural herbicide.
2014 Elsevier B.V. All rights reserved.

1. Introduction
Tunisian ora includes more than 350 spontaneous species con-
sidered as medicinal and aromatic plants and used in traditional
phytotherapy (Le Floch, 1983). Among the aromatic plants, the
Thymus genus, belonging to the Lamiaceae family, is noteworthy
for the numerous species and varieties of wild-growing plants.
This genus comprises about 400 species of perennial aromatic
plants with many subspecies, varieties, subvarieties and forms. The
plants are extensively used, fresh and dried, as a culinary herb (Dob
et al., 2006). In recent years, several reports have been published
concerning the composition and the biological properties of the
essential oils and extracts of this genus. In deed, Thymus species are
widely used in pharmaceutical, cosmetic and perfume industry, and
for avoring and preservation of several food products (Hazzit et al.,
2009). Also, they are employed in popular medicinal for its expec-
torant, antitussive, analgesic, antibroncholitic, antispasmodic,
Corresponding author. Tel.: +216 71703829/929.
E-mail address: imenbenelhadjali@yahoo.fr (I. Ben El Hadj Ali).
carminative and diuretic effects (Dob et al., 2006; Hazzit et al.,
2009). Others ndings suggested that Thymus essential oils have
been widely used mainly due to their antibacterial, antifungal,
antioxidant and anti-inammatory properties (Giordani et al.,
2008; Hazzit et al., 2009; Mahmoudi et al., 2008).
Thymus numidicus Poir. is an endemic species of Tunisian
North-West and Algeria (Pottier-Alapetite, 1981). It is a short
lived and outcrossing shrub predominantly bee-pollinated. It
is a hermaphrodite species and possess a capacity for asexual
reproduction by either vegetative propagation (Pottier-Alapetite,
1981). It can reach 1015 cm in height. Leaves are opposite and
linear/lanceolate (415 mm). Flowers are hermaphrodite, large
(15 mm) and grouped in dense terminal heads with an uneven calyx
(3 mm) and a pink corolla (6 mm). Flowering takes place between
April and June (Pottier-Alapetite, 1981). In Tunisia, T. numidicus
populations are distributed from the lower humid to the upper
semi-arid bioclimates at altitudes ranging from 450 to 1100 m
(Nabli, 1995). The species grows on poor fertile calcareous soils
and occurs in scattered small populations.
Previous work on Algerian T. numidicus showed that essen-
tial oils showed strongly antibacterial activity, particularly against

http://dx.doi.org/10.1016/j.indcrop.2014.08.021
0926-6690/ 2014 Elsevier B.V. All rights reserved.
 I. Ben El Hadj Ali et al. / Industrial Crops and Products 62 (2014) 188195
Bacillus subtilus, Staphylococcus aureus and Enterobacter aerogenes
(Kabouche et al., 2005). Moreover, thymol (68.2%), carvacrol
(16.92%) and linalool (11.5%) were the main identied compounds
(Kabouche et al., 2005). The avonoids of T. numidicus was also
investigated (Benkiniouar et al., 2010).
Plant phenols exhibit signicant antioxidant, antitumoral,
antiviral, anti-inammatory and antibiotic properties (Apak et al.,
2007; Zhang et al., 2011). Phenolics are antioxidants with redox
properties, which allow them to act as reducing agents, hydro-
gen donors and singlet oxygen quenchers. In recent years,
there is a wide interest in nding phytochemicals from natural
sources that could replace synthetic antioxidants such as buty-
lated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT)
which are commonly used in food industry because of their toxicity
(Canadanovic-Brunet et al., 2006). Antioxidants such as avonoids,
phenolics, terpenoids, avonols, proanthocyanidins and tannins
are found in various plant products (Jeong et al., 2004). For this
reason, there is a growing interest in separating these plant antiox-
idants and using them as natural antioxidants. However, there are
several methods established for the extraction of polyphenols from
plant materials. Those methods vary in solvents and conditions
used. Therefore, the extraction method is essential for the accu-
rate quantication of antioxidant content and capacity (Alothman
et al., 2009; Santas et al., 2008).
The aims of this study were: (i) to assess the polyphenols,
avonoids, avonols and proanthocyanidins contents of various
extracts from roots, stems and leaves of Tunisian T. numidicus; and
(ii) to investigate their antioxidant and allelopathic activities.
2. Materials and methods
2.1. Plant material
The starting material consists of mature plants, at the vegetative
stage, growing wild in the northwest area of Tunisia, to be precise
from the Jendouba region. From each plant branches with leaves
and roots were collected and transported to the laboratory. The
fresh plants were separated into roots, leaves and stems. Before
analyses, specimens were air-dried at room temperature for 16
days. The moisture content of samples was 3.5%. The studied species
was identied at the Department of Botany, Higher Institute of
Biotechnology of Monastir (ISBM, Tunisia) and voucher specimens
(number: ThN. 1) are kept at the herbarium of the of the Department
of Botany in the cited institute.
2.2. Chemical and reagents
DPPH (1,1-diphenyl-2-picrylhydrazyl), ABTS (2,2 -azinobis-
(3-ethylbenzothiazoline-6-sulphonic acid)), Trolox (6-hydroxy-
2,5,7,8-tetramethylchroman-2-carboxylic acid), gallic acid,
Na2 CO3 , AlCl3 6H2 O, C2 H3 NaO2 , rutin, cyanidin chloride, n-
BuOHHCl, NH4 Fe(SO4 )2 and FolinCiocalteu reagent, were
purchased from SigmaAldrich (St. Louis, MO). All solvents and
reagents used were of the highest purity.
2.3. Preparation of the plant extracts
Plant materials were ground and macerated for extraction.
Eighty grams of each organ were weighed into 1 l Erlenmeyer
asks, and then 400 ml of different solvents of increasing polar-
ity (petroleum ether, ethyl acetate and methanol) were added to
the plant organs. After ltration through lter paper (Whatman
No. 4), the residue was re-extracted twice, and then the combined
extracts of every organ were evaporated at room temperature. For
water extraction, a 20 g of each organ was mixed with 100 ml of
boiling water and ltered through Watman No. 1 paper. Extraction
189
was carried out by shaking at room temperature for 3 days. Each
extract was stored in a brown bottle at 4 C prior to further analysis.
2.4. Phenolic compounds content
2.4.1. Total phenolic content
The total phenolic content of organs was assessed
using the FolinCiocalteu reagent, following Singleton
and Rosis (1965) method, based on the reduction of a
phosphowolframatephosphomolybdate complex by pheno-
lics to blue reaction products and slightly modied by Dewanto
et al. (2002). An aliquot of each diluted sample extract was mixed
with 2.5 ml FolinCiocalteu reagent. 2 ml of sodium carbonate
solution (7.5%) was added to the mixture after 3 min. After incu-
bation (90 min) in dark, the absorbance at 760 nm was read versus
the prepared blank. The standard curve was prepared by solutions
of gallic acid in methanol. The concentration of total phenolic
compounds in the extracts was determined as g of gallic acid
equivalent using an equation obtained from the standard gallic
acid graph, and expressed as mg gallic acid/g dry weight of the
plant material (mg EGA/g DW). The data were presented as the
average of triplicate analyses.
2.4.2. Total avonoid content
The total avonoid contents of plant samples were determined
according to aluminum chloride colorimetric method (Djeridane
et al., 2006). Each extract was dissolved in 1 ml of the appro-
priate solvent. 1 ml of this solution was mixed with 1 ml of 2%
AlCl3 methanolic solution. After incubation at room temperature
for 15 min, the absorbance was measured at 430 nm. Rutin was
chosen as a standard. Using a standard curve, the levels of total
avonoid contents in the sample extracts were determined in trip-
licate, respectively. Total avonoids were expressed as mg rutin
equivalent/g DW (mg RE/g DW). Analyses were done in triplicate.
2.4.3. Total avonol content
The content of avonols was determined according to aluminum
chloride colorimetric method by Miliauskas et al. (2004). Each
extract was dissolved in 1 ml of the appropriate solvent. 1 ml of
this solution was mixed with 1 ml of AlCl3 methanolic and 3 ml
of sodium acetate solutions. The absorption at 440 nm was read
after 2h 30 min at 20 C. Rutin was chosen as a standard. Using a
standard curve, the levels of total avonol contents in the sample
extracts were determined in triplicate, respectively. Total avonols
were expressed as mg rutin equivalent/g DW (mg RE/g DW). All
determinations were carried out in triplicate.
2.4.4. Total proanthocyanidin content
The total amount of proanthocyanidins was measured using
the HCl/butan-1-ol assay described by Bahorun et al. (2003). To
each stoppered tube, 0.5 ml of the extracts was added, owed
by 6 ml of an n-BuOHHCl solution (95:5, v/v) and 0.2 ml of
NH4 Fe(SO4 )2 12H2 O in 2 M HCl. The tubes were thoroughly vor-
texed and incubated for 40 min at 100 C. After cooling in the dark,
the red coloration was read at 550 nm against a blank standard. The
amount of total proanthocyanidins was expressed in g cyanidin
chloride/g of dry weight (DW). All samples were analyzed in three
replications.
2.5. Antioxidant activity evaluation
The antioxidant activity of T. numidicus extracts from all organs
was assessed using free radical-scavenging activity (RSA) with
DPPH (1,1-diphenyl-2-picrylhydrazyl) and ABTS (2,2 -azinobis-(3-
ethylbenzothiazoline-6-sulphonic acid)) radical assay.
190 I. Ben El Hadj Ali et al. / Industrial Crops and Products 62 (2014) 188195
2.5.1. DPPH radical scavenging activity
The evaluation of the free radical-scavenging activity of extracts
was based on the measurement of the reducing ability of
antioxidants toward the DPPH radical. The method described by
Brand-Williams et al. (1995) was used with slight modications.
20 l of different extracts (at different concentrations) was added to
980 l of the methanolic DPPH solution (90 M). The reaction was
allowed to stand at room temperature in the dark for 30 min and the
absorbance was recorded at 517 nm against a blank (methanol solu-
tion) using a UVvis spectrophotometer. The measurements were
performed in triplicate. The radical-scavenging activity was calcu-
lated using the following equation: I% = [(AB AA)/AB] 100, where
I is the DPPH inhibition, %; AB and AA are the absorbance values of
the control and of the test sample, respectively. DPPH scavenging
activity is presented by IC50 value, dened as the concentration of
the antioxidant needed to scavenge 50% of DPPH present in the
test solution. All tests were carried out in triplicate and IC50 values
were reported as means SD of triplicates.
2.5.2. ABTS radical cation decolourisation assay
The determination of ABTS+ radical scavenging was carried out
as reported by Dorman and Hiltunen (2004). This assay assesses
the total radical-scavenging capacity based on the ability of a com-
pound to scavenge the stable ABTS radical (ABTS+ ). The ABTS+
radical cation was produced by reacting ABTS with potassium per-
sulfate (K2 S2 O8 ) (Re et al., 1999). The ABTS+ was produced by the
reaction between 7 mM ABTS in water and 2.45 mM potassium per-
sulfate, stored in the dark at room temperature for 12 h. Before
usage, the ABTS+ solution was diluted to get an absorbance of
0.703 0.025 at 734 nm. 20 l of different extracts was added to
980 l ABTS+ solution and the absorbance at 734 nm was mea-
sured. Sample absorbance was compared to a blank where 20 l
of the solvent were added to 980 l of the ABTS+ solution. The
absorbance was read at ambient temperature at 6 min after addi-
tion of the antioxidant. The standard curve was linear between 0
and 250 g/ml Trolox. Additional dilution was needed if the ABTS
value measured was over the linear range of the standard curve.
All determinations were performed in triplicate. Results were
expressed in inhibition percentage versus samples concentrations
(mg/ml). The percentage decrease of the absorbance at 734 nm was
calculated by formula: %inhibition = [(AB AA)/AB] 100, where I is
the ABTS+ inhibition %; AB and AA are the absorbance values of the
control and of the test sample, respectively. Thereafter, results are
expressed in g of Trolox equivalents (TE) per milligrams of dry
weight (g TE/mg DW).
2.6. Phytotoxic assay
The inhibitory potential of the T. numidicus extracts obtained
from roots, stems and leaves on the seed germination, the hypocotyl
and root lengths and the seedling dry weight of Medicago sativa L.
and Triticum stivum L. seeds was investigated.
Different concentrations (0.2, 0.4, 0.6, 0.8 and 1 mg/ml) of
extracts were dispersed in sterile Petri dishes (9 cm diameter) lined
with double-sterile lter paper (Whatman No. 2). M. sativa and T.
stivum seeds were surface sterilized for 20 min in 1% NaClO before
use. Dishes prepared without solvent were used as a negative con-
trol. Then, 4 ml of distilled water was added to each Petri dishes,
those were sealed with Paralm to prevent water loss and stored
in the dark at 25 C for 7 days. A seed was considered germinated,
when the protrusion of the radical became evident. Seeds that did
not germinate were considered to have a radical length of 0 mm.
All phytotoxic assays were conducted in triplicate, and in total 60
seedlings were measured for each target species.
After 7 days, the germination percentage was determined. Then,
the seedlings of M. sativa and T. stivum seeds were collected,
the hypocotyls and radical lengths were measured, and the fresh
and dry weights per Petri dish were determined to evaluate the
allelopathic activity of the T. numidicus extracts. The inhibitory
or stimulatory effects were calculated using the following equa-
tion, with slight modications from Chung et al. (2001): Inhibition
()/stimulation (+) % = ((EXe Ce)/Ce) 100; where EXe (extract
effect) is the parameter measured in the presence of T. numidi-
cus extracts and Ce (control effect) the parameter measured in the
presence of distilled water.
2.7. Statistical analysis
Each assay was done three times from the same extract in order
to determine their reproducibility. Data were performed using the
software using the SPSS version 13.0 for Windows. Quantitative dif-
ferences were assessed by ANOVA procedure followed by Duncans
multiple range test. Values were expressed as means standard
deviations (SD). Differences were considered signicant at p < 0.05.
Correlations among data obtained were calculated using Spearman
coefcient (r) using SpearmanKendalls rank test (Saez, 1995). The
signicance of the correlation was tested after 1000 permutations.
3. Results and discussion
3.1. Extract yields and total phenolic contents
The yield extracted from the roots, stems and leaves of Tunisian
endemic T. numidicus, collected at the vegetative stage, by three
different solvents with varying polarities (petroleum ether, ethyl
acetate and methanol) is reported in Table 1. It is apparent from
this current work that the highest extract yield was obtained by
methanol extraction of all organs, followed by ethyl acetate and
by petroleum ether. In different organs, the yields of methanolic
extracts were signicantly high: 12.92% for roots, 22.77% for stems
and 28.58% for leaves. However, the petroleum ether efciency as
a solvent was lower than the efciencies of all other solvents. Thus,
the variation in the yields of various extracts can be attributed to the
polarities of the different compounds in the different organs. Such
differences have been reported in the literature (Khli et al., 2011).
There is no other study in the literature which investigated the yield
organs extracted with different solvents in this endemic species.
The total phenolic, avonoid, avonol and proanthocyanidin
contents varied signicantly among the studied parts in the dif-
ferent extracts of the studied species (Table 1). Leaves, determined
in the methanolic extract, had the highest contents of polyphe-
nols, avonoids, avonols and proanthocyanidins: 98.66 3.17 mg
EAG/g DW, 54.28 1.6 mg RE/g DW, 27.23 1.71 mg RE/g DW and
5.12 0.8 g cyanidin chloride/g DW, respectively. However, roots
present lower level of all contents: 23.76 1.29 mg EAG/g DW
of phenols, 13.67 1.1 mg RE/g DW of avonoids, 12.96 1.9 mg
RE/g DW of avonols and 0.35 0.2 g cyanidin chloride/g DW of
proanthocyanidins. This level of total phenols were found to be
lower than the values reported in the literature for other Thymus
species such as T. caramanicus (124.30 2.62 g/mg) reported by
Safaei-Ghomi et al. (2009), T. spathulifolius (141 g/mg of the polar
subfraction of a methanol extract) reported by Sokmen et al. (2004)
and T. serpyllum (113 g/mg of an ethanol extract) reported by Mata
et al. (2007).
The variance analysis performed on averages of these com-
pounds analyzed for roots, stems and leaves showed signicant
organ and solvent effects (Table 1). Results revealed that methanol
was better solvents than the others in extracting polyphenol,
avonoid, avonol and proanthocyanidin compounds due to their
polarity and good solubility for phenolic components from plant
materials. Therefore, the recovery of polyphenols from plant
 I. Ben El Hadj Ali et al. / Industrial Crops and Products 62 (2014) 188195 191

Table 1
Average of total polyphenol, avonoid, avonol and proanthocyanidin contents according to organs and solvent extraction systems.

Assay Yields (%) Polyphenols Flavonoids Flavonols Proanthocyanidins

(mg EAG/g DW) (mg RE/g DW) (mg RE/g DW) (g CC/g DW)

Petroleum ether extracts

Roots 1.4 8.72 0.83a 7.59 2.77a 1.08 0.22a 0.27 0.11a
Stems 2.4 14.29 2.16a 11.87 0.99a 3.9 0.99a 0.44 0.21a
Leaves 4.39 26.31 1.65b 13.48 0.73a 5.36 1.04a 0.83 0.12ab

Ethyl acetate extracts

Roots 6.14 16.73 1.9a 11.52 0.91a 5.91 1.12a 0.64 0.12ab
Stems 8.94 38.04 2.4c 20.16 2.27b 8.08 1.4ab 1.12 0.35b
Leaves 10.5 80.63 1.62d 41.49 2.07c 15.15 1.9b 1.91 0.9b

Methanol extracts
Roots 12.92 23.76 1.29b 13.67 1.1a 12.96 1.9b 0.35 0.2a
Stems 22.77 81.85 2.75d 38.11 0.98c 19.09 1.5c 1.71 0.25b
Leaves 28.58 98.66 3.17d 54.28 1.6d 27.23 1.71c 5.12 0.8c

Means in each column followed by different letters are signicantly different (p < 0.05).

materials is inuenced by the solubility of the phenolic compounds
in the solvent used for the extraction process (Kequan and Liangli,
2006; Roby et al., 2013). Furthermore, solvent polarity will play
a key role in increasing phenolic solubility (Naczk and Shahidi,
2006). So, it could be concluded from the results that polar frac-
tions had more phenolics in them than had non-polar fractions.
Hence, this could be used as an important descriptor to character-
ize the extracts of T. numidicus. These results are almost similar to
those reported by Hernandez-Hernandez et al. (2009). The same
nding is reported for other medicinal plants such as T. vulgaris,
Salvia ofcinalis and Origanum majorana (Roby et al., 2013), Rosmar-
inus ofcinalis and Origanum vulgare (Hernandez-Hernandez et al.,
2009) and Globularia alypum (Khli et al., 2011). On the other hand,
our results revealed also that leaves of T. numidicus at vegetative
stage are characterized by high amounts of phenols, avonoids,
avonols and proanthocyanidins. Thus, the high rate of these com-
pounds should be explained by the beginning of senescence of
organs accompanied by biochemical, physiological and molecular
changes evolving phenol synthesis changes. Then, the differen-
tial accumulation of these compounds between organs should be
related to their specic tissues and cells (i.e. mesophyll, epiderm,
thickness cuticle, chloroplasts, trichomes). Previous reports have
attributed the high presence in leaves of these compounds and their
low content in roots to the close interaction between organs and to
the different processes of biosynthesis and/or degradation, to the
transport involved in the distribution of these polyphenols at the
plant level and to the phenological organ growth (Fico et al., 2000;
Hudaib et al., 2002).
3.2. Antioxidant activity
The different extracts of T. numidicus exhibited remark-
able reduction activities (Table 2). The radical-scavenging
activity, expressed as inhibition concentration (IC50 ), var-
ied according to solvent and organ extracts (Table 2). The
best activity was observed in methanol extracts for leaves
(IC50 = 11.06 0.33 g/ml) and stems (IC50 = 15.29 0.9 g/ml),
and the lowest (IC50 = 66.6 1.22 g/ml) in petroleum ether
extracts for roots. Also, these activities are signicantly higher
than that of the synthetic antioxidant BHT (butylated hydroxy-
toluene) with IC50 = 25 g/ml and the trolox with IC50 = 32.0 g/ml,
as a standard reference product. We can deduce, that the extracts
obtained using high polarity solvents were considerably more
effective radical-scavengers than were those using low polarity
solvents. These extracts exhibited also the highest ABTS value
(235.46 2.14 g TE/mg DW and 233.75 1.2 g TE/mg DW in
methanol extracts for leaves and stems, respectively). This result
conrms the agreement between the solvent polarity and antiox-
idant activity where the methanol extract presented higher total
phenolic quantity. Change in solvent polarity alters its ability to
dissolve a selected group of antioxidant compounds and inu-
ences activity estimation. Our results are comparable to previous
reports which attested an important in vitro antioxidant activity
of various extracts of Thymus genus such T. praecox subsp. skorpilii
var. skorpilii (Ozen et al., 2011) and T. vulgaris (Roby et al., 2013).
While, the Tunisian T. numidicus radical-scavenging activity can
be considered high compared to the results reported on the other
members of Thymus family such as T. caramanicus methanol extract
(IC50 = 43 g/ml) reported by Safaei-Ghomi et al. (2009), highlight-
ing the considerable potential of this plant as an antioxidant food
additive.
In this study, the high free radical-scavenging inhibition activity
evaluated by the DPPH and the ABTS+ radical scavenging test
may be related to the presence of avonoid-type compounds and
other phenolics. So, the high contents of polyphenol compounds
in the studied species contribute to their important antiradical
and antioxidative activities. Others ndings reports the presence
of different phenolic compounds in the Thymus genus such as
rosmarinic acid, caffeic acid, ferulic acid, carnosic acid, quinic
acid, p-coumaric acid, caffeoylquinic acid derivative, quercetin-
7-o-glucoside, cinnamic acid, methyl rosmarenate, nareingenin,
luteolin-7-o-rutinose and ferulic acid derivative, and avonoids
such as 5,7,4 -trihydroxyavone (apigenin); 4 -dihydroxy-6,7,8-
trimethoxyavone (xanthomicrol); 5,7,3 ,4 -tetrahydroxyavone
(luteolin); 5,3 ,4 -trihydroxy-6,7,8-trimethoxyavone (sider-
itoavone) and 5-hydroxy-6,7,3 ,4 -tetramethoxyavone

Table 2
Total antioxidant capacity determined by DPPH and ABTS test systems of Thymus numidicus extracts according to organs obtained from different solvent extraction systems.

Assay DPPH IC50 (g/ml) ABTS (g TE/mg DW)

Roots Stems Leaves Roots Stems Leaves

Petroleum ether extracts 66.6 1.22b 58.1 1.0b 47.31 0.72b 62.12 2.04a 68.26 2.66a 78.97 1.92a
Ethyl acetate extracts 39.1 1.03ab 29.06 0.95a 23.51 1.15a 173.14 2.41b 206.83 1.34b 220.41 1.69b
Methanol extracts 22.51 0.99a 15.29 0.9a 11.06 0.33a 217.32 6.5b 233.75 1.2b 235.46 2.14b

Means in each column followed by different letters are signicantly different (p < 0.05).
192 I. Ben El Hadj Ali et al. / Industrial Crops and Products 62 (2014) 188195

(5-desmethylsinensetin) (Benkiniouar et al., 2010; Jordan et al.,
2009; Loziene et al., 2007; Roby et al., 2013). However, avonoids
interrupt the propagation of the free radical autoxidation chain
by contributing a hydrogen atom from a phenolic hydroxyl group,
with the formation of a relatively stable free radical that does
not initiate or propagate further oxidation processes (Bahramikia
et al., 2009). The presence of these compounds in the subfraction
of T. numidicus extracts from different organs may also be the main
cause of its high radical-scavenging activity and high total phenolic
contents. Furthermore, avonoids are a group of polyphenolic
components with known various properties such as inhibition of
hydrolytic and oxidative enzymes, anti-inammatory action, to
enhance human immunity, reduction blood-lipid and glucose, and
free radical scavenging (Atoui et al., 2005). Those ndings justify
the use of different plant organs of the studied species by ancient
and actual local human populations.
According to Zhang et al. (2011), the antioxidant activity is
generally attributed to phenolic and avonoid compounds in
plant extracts. In deed, a signicant negative correlation between
the polyphenol contents and IC50 antioxidant activity values
were observed indicating that extracts with highest polyphe-
nol contents show lower IC50 values (Khli et al., 2011; Liu
et al., 2007; Sengul et al., 2009; Verzelloni et al., 2007). In the
present study, a negative signicant correlations (0.99 < r < 0.7;
7.68 106 < p < 0.05), estimated by the Spearmans coefcient,
were observed between the total phenol, avonoids, avonols
or proanthocyanidins contents in different organ extracts and
the free radical-scavenging values IC50 (Table 3). Moreover, sig-
nicant correlations were obtained between ABTS and phenols
contents except for proanthocyanidins contents in petroleum
ether extracts of all organs and for phenols contents in ethyl
acetate extracts of roots (Table 3). Thus, the high antioxidant
activity revealed mainly for leaf extracts could be attributed to the
high phenolic content (98.66 mg EAG/g DW) probably due to com-
pounds such as such as 4 -dihydroxy-6,7,8-trimethoxyavone
(xanthomicrol), 5,3 ,4 -trihydroxy-6,7,8-trimethoxyavone
(sideritoavone), 5-hydroxy-6,7,3 ,4 -tetramethoxyavone (5-
desmethylsinensetin), 5,7,4 -trihydroxyavone (apigenin) and
5,7,3 ,4 -tetrahydroxyavone (luteolin) (Benkiniouar et al., 2010).
This is the rst correlation obtained for different extracts of T.
numidicus between polyphenols, avonoids, avonols or proantho-
cyanidins and antioxidant activity, so this correlation can guide the
search for molecules responsible for the antioxidant activity. There-
fore, our results conrm that phenolic compounds contribute to the

Table 3
Spearman correlation coefcient between total polyphenols, avonoids, avonols and proanthocyanidins with antioxidant activity.

Assay DPPH IC50 (g/ml) ABTS (g TE/mg DW)

r p r p

Petroleum ether extracts

Polyphenols 0.91 0.013 0.91 0.016
Flavonoids 0.71 0.046 0.74 0.049
Roots
Flavonols 0.85 0.033 0.84 0.029
Proanthocyanidins 0.49 0.073 0.45 0.076

Polyphenols 0.71 0.046 0.95 0.013

Flavonoids 0.89 0.024 0.98 0.005
Stems
Flavonols 0.90 0.020 0.95 0.012
Proanthocyanidins 0.88 0.028 0.43 0.081

Polyphenols 0.91 0.016 0.71 0.046

Flavonoids 0.98 0.00043 0.48 0.079
Leaves
Flavonols 0.88 0.029 0.85 0.033
Proanthocyanidins 0.34 0.126 0.49 0.064

Ethyl acetate extracts

Polyphenols 0.71 0.046 0.89 0.026
Flavonoids 0.79 0.040 0.39 0.112
Roots
Flavonols 0.82 0.038 0.41 0.110
Proanthocyanidins 0.94 0.011 0.37 0.133

Polyphenols 0.76 0.043 0.72 0.041

Flavonoids 0.75 0.044 0.71 0.046
Stems
Flavonols 0.95 0.014 0.92 0.016
Proanthocyanidins 0.82 0.038 0.84 0.036

Polyphenols 0.89 0.022 0.99 0.003

Flavonoides 0.76 0.043 0.94 0.016
Leaves
Flavonols 0.98 0.002 0.86 0.028
Proanthocyanidins 0.87 0.030 0.98 0.004

Methanol extracts
Polyphenols 0.89 0.024 0.87 0.027
Flavonoids 0.99 7.68 106 0.7 0.048
Roots
Flavonols 0.99 0.001 0.46 0.070
Proanthocyanidins 0.99 5.56 106 0.7 0.049

Polyphenols 0.7 0.049 0.82 0.039

Flavonoids 0.99 0.0002 0.44 0.109
Stems
Flavonols 0.89 0.021 0.48 0.080
Proanthocyanidins 0.74 0.041 0.48 0.083

Polyphenols 0.85 0.034 0.99 0.021

Flavonoids 0.73 0.042 0.7 0.049
Leaves
Flavonols 0.98 0.006 0.98 0.006
Proanthocyanidins 0.85 0.033 0.71 0.046
 I. Ben El Hadj Ali et al. / Industrial Crops and Products 62 (2014) 188195 193

radical scavenging activity of Tunisian T. numidicus extracts. More-

31.4 a
36.3 a
67.2d

46.3b

79.2e
56.1c

53.1c
59.3c
42ab

40ab
47b

46b
over, several studies have focused on the relationship between the

1
antioxidant activity of phenolic compounds as hydrogen donat-
ing free radical-scavengers and their chemical structure. Phenolic

35.1b

30.2b

34.1b
26.6a
42.6c

40.3c

42.3c
23 a
25 a

60d
32b

47c
antioxidants are products of secondary metabolism in plants, and

0.8
the antioxidant activity is mainly due to their redox properties and

29.9ab

29.3ab
chemical structure, which can play an important role in inhibiting

47.1d
34.8b

33.2b

34.1b
23.7a

19.9a

23.2a

39.9c
23a

21a
lipoxygenase and scavenging free radicals (Evans et al., 1996).

0.6

22.8ab

23.1ab
20.2ab

23.1ab
3.3. Phytotoxic potential

26.8b

25.8b
18.8a

17.9a
19.8a

32.8c
Seedling dry weight

15 a
19a
0.4
The phytotoxic effects of the roots, stems and leaves extracts
of T. numidicus by three different solvents with varying polarities

18.1b
11.2a

12.5a

21.2c
13ab

15ab

14ab
9.5 a

10 a

19b
11a

23c
(petroleum ether, ethyl acetate and methanol) and by the water

0.2
are summarized in Tables 4 and 5. The allelopathic inuence on M.

Allelopathic effects of different tissues extracts obtained from different solvent extraction systems of Tunisian T. numidicus at the vegetative stage on Medicago sativa L. seedlings.

74.5d
56.2b

51.1b

57.5b

52.2b

53.7b
48.6a

44.6a
48.8a

49.2a
60.9c

60.5c
sativa L. and T. stivum L. germination and seedling growth varied
signicantly according to the solvent and plant parts.

1
A signicant inhibitory effect on the germination of M. sativa

63.1d
47.6b

44.9b

45.4b
36.5a

33.1a

51.2c
seeds was found (Table 4). In deed, the allelopathic effects were

47b

46b
38a

34a

53c
0.8
signicantly different (p < 0.05) (Table 4). The germination per-
centages varied between 57.5 (of stems petroleum ether extract

31.1ab

32.2ab

31.3ab
52.2d
37.6b
29.7a

27.6a
42.2c
38b

26a
29a

40c
at 0.2 mg/ml) and 22.5% (of leaf methanol extract of at 1 mg/ml) at

0.6
the seventh day of germination. The T. numidicus extracts at differ-
ent concentrations showed very high phytotoxic effects against M.

20.9ab
28.2b

27.1b
15.9a

16.8a

17.6a
24ab

24ab
32 c
sativa, with an inhibition of seed germination of 79.2% at 1 mg/ml.

28b

37c

31c
Hypocotyl length

0.4
Therefore, water, petroleum ether, ethyl acetate and methanol
extracts marked germination and seedling growth inhibition that

12.5ab
13.1ab

13.2ab
17.3b

15.8b

10.2a
20.2c
12ab
7.9 a
9.8 a
was concentration-dependent. Signicant allelopathic effect was

9.3a
10a
0.2
also observed at the lowest concentration tested, 0.2 mg/ml. The
highest allelopathic effects were obtained by methanol extraction

79.5d
56.4b

55.1b

54.7b
55.9b

55.8b
43.4a

40.2a

63.5c

63.1c
of all organs, followed by ethyl acetate then water extract and by

47ab

48ab
petroleum ether. 1
From the results shown in Table 4, it is evident that the recov-

38.2ab

72.8d
48.3b

43.2b

43.4b

41.1b
34.6a

32.5a
34.2a
ery of inhibitory effect was dependent on the solvent used and its

55c

56c
50c
0.8

polarity (for all organs). For roots, methanol extract gave the high-
est inhibition of the seed germination, the hypocotyl and radicle

29.5ab

30.6ab

67.6d
34.2b

33.8b

32.8b
35.9b
27.6a

24.3a

41.3c
30ab

lengths and the seedling dry weight of M. sativa with signicant 32b
0.6

differences between extract concentrations. The inhibition of the

radicle growth varied from 6.8 (stems petroleum ether extract at
21.3b

27.6b

25.6b
14.3a

0.2 mg/ml) to 79.5% (roots methanol extract at 1 mg/ml) and that 31.8c
19ab

19ab

42d
24b

21b
14a

30c
0.4

of the hypocotyl from 7.9 to 74.5%. The biomass production was

Radicle length

Means in each column followed by different letters are signicantly different (p < 0.05).
slightly inhibited in the presence of different extracts at 0.2 mg/ml
12.1ab
13.6ab

11.7ab

10.8ab
11.5ab
16.6b

17.9b

15.6b
21.1c
concentration, and the dry weight of the seedlings treated with
6.8a
8.7a

15b
0.2

1 mg/ml was highly reduced by 79.2% with root methanol extract.

Table 5 showed that the phytotoxic effects of T. numidicus plant 71.2d
55.4b

51.4b

54.1b
43.2a

60.2c

65.4c

parts extracted with different solvents on T. stivum germination

74d
55b

51b
45a
65c
1

and seedling growth was solvent and dose-dependent. In deed, the

allelopathic effects of extracts were signicantly different (p < 0.05).
39.2ab

35.1ab

38.8ab
38.8ab

59.6d

56.7d
41.8b

50.9c
35ab

The best activities were also observed at methanol extracts. The

45b

31a
50c
0.8
Inhibitory effect compared to control (%)

lowest activities were observed for the stems petroleum ether

extracts at different concentration. The root extracts with methanol
26.5ab

48.1d
32.7b

29.6b

33.7b
18.9a

39.4c
27ab

23ab

solvent had the highest allelopathic activity, which the inhibition

35b

30b
20a
0.6

percentage of the germination was 74% (at 1 mg/ml). The inhibi-

tion of the radicle and the hypocotyl growth varied from 6.7 (stems
18.9ab

14.9ab

17.3ab
32.5d

30.8d
20.4b

39.4e
10.8a

27.9c

petroleum ether extract at 0.2 mg/ml) to 79.5% (at 1 mg/ml of roots

30d
10a
25c
Seed germination

0.4

methanol extract), and from 7 (stems water extract at 0.2 mg/ml) to

74.5% (at 1 mg/ml of roots methanol extract), respectively. The dry
13.5cd
14.3d

16.3d
19.2e
11.2c
6.8b

6.8b
2.7a

8.2c
15d

weight of the seedlings treated with roots methanol extract was

5ab
10c
0.2

highly reduced by 73.8% at 1 mg/ml.

Our results demonstrated that the volatile compounds isolated
Petroleum ether extracts
Concentration (mg/ml)

from the Tunisian endemic T. numidicus tissues with various sol-

Parameter controlled

Ethyl acetate extracts

vents signicantly delayed the seed germination and the growth of

Methanol extracts

M. sativa and T. stivum seedlings. This inhibitory activity can be

Water extract

mainly due to toxic compounds present in the roots. In deed, the

Table 4

Leaves

Leaves

Leaves

Leaves
Stems

Stems

Stems

Stems
Roots

Roots

Roots

Roots

reduction in seed germination and shoot length may be attributed

to the reduced rate of cell division and cell elongation due to
194 I. Ben El Hadj Ali et al. / Industrial Crops and Products 62 (2014) 188195

the presence of the allelochemicals (Javaid and Anjum, 2006).

64.8d
49.3b
52.6b

49.1b

73.8e
34.2a
37.5a

59.3c

59.1c
58.3c
42ab

50b
In line with our ndings, few studies reported that some essen-

1
tial oils containing a toxic compounds (e.g. caryophyllene oxide,
limonene, spathulenol, etc.) also have interesting phytotoxic poten-

43.3d 59.6d
36.4b

39.6b

36.1b
38.7b
29.1a
31.8a

32.4a

44.6c

35.8bc 42.4c
28a

22.9 ab 34.5bc 45c

tials (Chung et al., 2001; Mabrouk et al., 2013). The Tunisian T.
0.8

numidicus phytotoxic potential can be considered high compared

32.6b
21.7a
26.9a

20.9 ab 24.8a

19.9ab 25.7a
20.5a

38.5c
to the results reported by Shao et al. (2013). These results conrm

30b

22.3 ab 30b
0.6

that T. numidicus could be used as a potential allelopathic substance

Seedling dry weight

and should be tested as a potential natural herbicide resource.

29.2b

25.8b

28.5b

31.4b

29.9b
16.5a

15.3a

39 c
0.4

4. Conclusion

17.5ab

32.1d
21.2b

21.2b

19.4b
21.6b
14.6a

11.1a
13.6a

15.4a
28.1c
17ab

This study is the rst to investigate the global chemical com-

0.2

position of various extracts from roots, steems and leaves of T.

Allelopathic effects of different tissues extracts obtained from different solvent extraction systems of Tunisian T. numidicus at the vegetative stage on Triticum stivum L. seedlings.

numidicus, endemic species from Tunisia, and their antioxidant

57.5d

52.2d

53.7d
60.5d
32.8b

74.5e
27.7a
28.8a

44.6c
48.8c

49.2c
61d

and allelopathic activities. Our results clearly showed that the

1

leaves seems the organ which give the highest level in polyphe-
53.3d

51.6d
37.7b

34.2b
30.7b

62.9e
21.7a

18.9a

44.7c

45.4c

41.4c nol, avonoid, avonol and proanthocyanidin contents which favor

17a
0.8

them in industrial use for extraction of polyphenol compounds.

Also, we can conclude that leaf methanolic extracts with higher
39.4cd
42.2d

52.2d
27.6b
13.1ab 16.3a
11.6a

37.6c

34.2c

31.3c

antioxidant capacity (IC50 = 11.06 0.33 g/ml by DPPH assay and

11.1a 13 a

26b
29b

235.46 2.14 g TE/mg DW by ABTS assay) could be considered

0.6

a potential natural antioxidant alternative for use as a natural

Hypocotyl length

33.6d
16.3b
11.7a

29.6c

22.2c
28.3c
9.7a

additive in food, cosmetic and pharmaceutical industries for the

40e
29c

22c
0.4

prevention or treatment caused by microorganisms and free radi-

cals. Furthermore, extracts with higher antioxidant capacity also
41.5b 55.1c 11.8ab

12.5ab
13.6ab

14.2ab
10.1a

10.2a
20.2c
16ab
38.5b 47.7b 9.8b

had higher polyphenol contents. Moreover, root extracts showed

30.5ab 40.2ab 7.9a
26.8a 37.8a 7 a
40.6ab 8a
0.2

high allelopathic activity. All those results emphasize the impor-

tance of the chemical composition of this Tunisian endemic plant.
63.5d

63.1d
79.5e
51.7c
55.7c

55.8c
31.3ab 46b
1

Acknowledgments
70.8d
41.5b

42.9b
52.7c

48.7c

52.5c
29a
0.8

This research was supported by a grant of the Ministry of Sci-

entic Research and Technology, the National Institute of Applied
18.3ab 27.7ab

29.5ab

67.6d
33.8b

32.8b
35.9b
30.6b
17.6ab 21.4a

24.3a

41.3c
16.9ab 20 a

Science and Technology (research grant 99/UR/09-10) and the

32b
0.6

Higher Institute of Biotechnology of Monastir (Laboratory of

Genetic Biodiversity and Valorisation of Bioresources).
24.5b

21.5b

21.6b
25.5b
20.2b
12.8a

32.3c

32.9c
30c
Radicle length

0.4

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