Plant Biosystems - An International Journal Dealing with

all Aspects of Plant Biology

Official Journal of the Societa Botanica Italiana

ISSN: 1126-3504 (Print) 1724-5575 (Online) Journal homepage: http://www.tandfonline.com/loi/tplb20

Phytochemical analysis and antioxidant activity

of Tamarix africana, Arthrocnemum macrostachyum
and Suaeda fruticosa, three halophyte species from
Algeria

N. Chekroun-Bechlaghem, N. Belyagoubi-Benhammou, L. Belyagoubi, A.

Gismondi, V. Nanni, G. Di Marco, L. Canuti, A. Canini, I. A. El Haci & F. Atik
Bekkara

To cite this article: N. Chekroun-Bechlaghem, N. Belyagoubi-Benhammou, L. Belyagoubi, A.

Gismondi, V. Nanni, G. Di Marco, L. Canuti, A. Canini, I. A. El Haci & F. Atik Bekkara (2019):
Phytochemical analysis and antioxidant activity of Tamarix�africana,�Arthrocnemum�macrostachyum
and Suaeda�fruticosa, three halophyte species from Algeria, Plant Biosystems - An International
Journal Dealing with all Aspects of Plant Biology, DOI: 10.1080/11263504.2018.1555191

To link to this article: https://doi.org/10.1080/11263504.2018.1555191

Published online: 17 Jan 2019.

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PLANT BIOSYSTEMS - AN INTERNATIONAL JOURNAL DEALING WITH ALL ASPECTS OF PLANT BIOLOGY
https://doi.org/10.1080/11263504.2018.1555191

Phytochemical analysis and antioxidant activity of Tamarix africana,

Arthrocnemum macrostachyum and Suaeda fruticosa, three halophyte species
from Algeria
N. Chekroun-Bechlaghema, N. Belyagoubi-Benhammoua , L. Belyagoubia, A. Gismondib , V. Nannib, G. Di
Marcob, L. Canutib, A. Caninib , I. A. El Hacic and F. Atik Bekkaraa
a
Laboratoire des Produits Naturels (LAPRONA), D
epartement de Biologie, Facult
e des Science de la Nature et de la Vie, des Sciences de la
Terre et de l’Univers, Universit
e Abou Bekr Belkaid, Imama, Tlemcen, Alg
erie; bDepartment of Biology, University of Rome Tor Vergata, Via
della Ricerca Scientifica 1, Rome, Italy; cCentre de Recherche scientifique et technique en Analyses Physico-Chimiques (CRAPC), Zone
industrielle Bou-Ismail, RP, Tipaza, Alg
erie

ABSTRACT ARTICLE HISTORY

Extracts and fractions using six solvents of increasing polarities from Northwest Algeria (Tamarix afri- Received 15 February 2018
cana, Arthrocnemum macrostachyum and Suaeda fruticosa) were studied for phytochemical analysis Accepted 28 November 2018
and in vitro antioxidant properties. Methanol and water fractions were found to be the more suitable
KEYWORDS
solvents used for extraction of polyphenolic compounds. Aqueous leaf fraction of T. africana showed
Halophytes; bioactive
the highest content of phenolics (61.06 ± 0.40 mg GAE/g DW) and condensed tannins substances; solvent affinity;
(118.43 ± 11.79 mg CE/g DW). Dichloromethane stem fraction of T. africana had the highest 2,2- antioxidant activity; high-
diphenyl-1-picrylhydrazil radical scavenging ability (0.34 ± 0.00 mg/ml). Methanol leaf fraction of the performance liquid
same plant exhibited the highest antioxidant power against the inhibition of b-carotene bleaching, chromatography with a
while the maximum total antioxidant capacity was recorded in the leaf extract of S. fruticosa. Phenolic diode-array detector (HPLC-
content was not influenced by the species but very affected by the extraction solvent, while antioxi- DAD) analysis
dant activities were not influenced by these two parameters. High-performance liquid chromatography
with a diode-array detector analysis of methanol and aqueous leaf fractions of T. africana revealed the
presence of six phenolic acids; chlorogenic and gallic acids were predominant and 10 flavonoid com-
pounds among which rutin and quercetin-3-O-arabonoside were the major constituents. These find-
ings suggest that these species may be considered as an interesting source of antioxidants.

GRAPHICAL ABSTRACT

Introduction
In Africa, several medicinal plants and vegetables are widely powerful machinery, are also able to withstand and quench
used for therapeutic purposes, as food and in folk medicine. toxic reactive oxygen species (Ksouri et al. 2007). Plant
More than 20% of cultivated land is affected by salt stress metabolites resulting from oxidative stress have been deter-
(Gupta and Huang 2014). Extremophile plants including halo- mined but it depends on multiple simultaneous analytical
phytes have the particularity to support and resist severe methods used (Sanchez et al. 2016), for example, the pres-
weather conditions like salinity and high temperatures (Le ence of cuticular wax on leaf surface is an interference prob-
Hou
erou 2001). Furthermore, halophytes, due to their lem in need of resolution (Bjarnholt et al. 2014).

CONTACT N. Belyagoubi-Benhammou nabila.benhammou79@yahoo.fr Department of Biology, Faculty of Natural and Life Sciences, Earth and Universe
Sciences, Abou Bekr Belkaid University, Imama, Tlemcen, BP 119, Algeria
ß 2019 Societ
a Botanica Italiana

Published online 17 Jan 2019

2 N. CHEKROUN-BECHLAGHEM ET AL.

Polyphenols represent the main powerful non-enzymatic sodium carbonate, sodium acetate, sodium phosphate, gallic
antioxidant plant compounds, being characterized by a acid, ascorbic acid, catechin, quercetin, vanillin, NaNO2, AlCl3,
strong biological activity. Therefore, they should replace syn- NaOH, HCl, sulfuric acid, ammonium molybdate, 2,2-
thetic antioxidants in pharmacology and medicine. In this diphenyl-1-picrylhydrazil (DPPH), b-carotene, Tween 40, oxy-
framework, our researchers try to find new Algerian extremo- genated distilled water and butylhydroxyanisole (BHA).
phile species, which are used for their ethno-pharmacological
uses, as good candidates for industrial application.
Salty areas like Sabkha, localized in arid and semi-arid Plant collection
regions, are convenient habitats for halophyte plants (salt- A. macrostachyum and S. fruticosa plants were collected in
tolerant plants) which may be potentially useful new sources October 2013 from Sebkha to 50 km north of Oran city
of natural antioxidants in dietary food (Meot-Duros (Northwest Algeria) (0
410 west longitude, 35
370 north lati-
et al. 2008). tude) and T. africana plants were collected from Sebkha
The genus Tamarix from Tamaricaceae family includes sev- about 45 km of Tlemcen city (1
260 west longitude, 35
160
eral species, which can grow in a wide range of environmen- north latitude), in Algeria. All plants were identified by Dr.
tal conditions and different abiotic stresses (high salt Hassani F, Department of Biology and Environment, Tlemcen
concentration, drought and high temperature) (Mahfoudhi University, Algeria. Voucher specimens numbers (T. africana
et al. 2014). In recent years, these salt-tolerant plants may be Poir.: L-2038; A. macrostachyum (Moric.: Moq-745); S. fruticosa
potentially useful for economical applications as new sources Forssk.: Forssk-767) were deposited in the laboratory of
of natural antioxidants in dietary food, encouraging the inter- Natural Products, Tlemcen. After collection, leaves and stems
est of many researchers (Meot-Duros et al. 2008; Ksouri et al. were manually separated.
2009). Tamarix africana Poir is rich in polyphenols (phenolic
acids, tannins and flavonoids) and is widely used in Algerian
traditional medicine for the treatment of gastroduodenal dis- Preparation of plant extracts
eases (Benabdallah et al. 2014). In particular, the leaves are
Dried powdered samples (leaves and stems) (1 g) were
traditionally used in decoction and infusion for the treatment
extracted with 20 ml of methanol/water mixture (70:30; V/V)
of disorders of the digestive tract (Khennouf et al.
at room temperature, for 24 h. The extract was then filtered
2003). Arthrocnemum macrostachyum (Moric.) K. Koch
through filter paper. The filtrate was concentrated using vac-
(Amaranthaceae family) has been reported to be a medicinal
uum rotary evaporator at 60
C to yield the crude extract.
plant (El-Wahab et al. 2008). Secondary metabolites present
For extracting the fractions, 2 g of each part of different
in this plant include phenolic compounds, alkaloids, flavo-
plants was partitioned successively with 40 ml of n-hexane,
noids and tannins. A. macrostachyum has antioxidant and
reductive activities which are attributable to the high phen- dichloromethane, acetone, ethyl acetate, methanol and
olic content (55%) (Custo
dio et al. 2012). Also, a high radical water. Each fraction was then filtered and concentrated using
scavenging activity indicates that A. macrostachyum can be a vacuum rotary evaporator at 60
C.
potential source of antioxidant compounds (Custo
dio et al.
2012). Finally, Suaeda fruticosa Forssk. (Amaranthaceae fam- Phytochemical analysis
ily) is a succulent perennial halophytic shrub, which can tol-
erate high salt conditions; it is widely distributed in land and Total phenolic content
coastal salt marshes (Ksouri et al. 2012a). Suaeda species are The total phenolic content was determined according to the
well known for their use in folkloric medical treatments. In method of Singleton and Rossi (1965) using Folin-Ciocalteu
this context, the edible S. fruticosa has hypoglycaemic and reagent. At a volume of 0.2 ml of the extract, 1 ml of Folin-
hypolipidaemic activities (Ksouri et al. 2012a). Ciocalteu reagent diluted 10 times with water and 0.8 ml of
To the best of our knowledge, these results represent the 7.5% sodium carbonate solution were added in a test tube.
first report on secondary metabolites content and biological After stirring, 30 min later, the absorbance was measured by a
activity for halophytes of Algerian saline habitats. In this art- spectrophotometer at 765 nm. Gallic acid was used as a stand-
icle, we quantified total phenolic, flavonoid, flavonol and ard for the calibration curve (y ¼ 2.916 x; R2 ¼ 0.997). The total
condensed tannin contents and evaluated in vitro antioxidant phenolic content was expressed as milligrams of gallic acid
activities of various crude extracts and fractions obtained equivalents per gram of dry weight (mg GAE/g DW).
from leaves and stems of the aerial part of T. africana, A.
macrostachyum and S. fruticosa. Our study was conducted to
find new sources for natural antioxidants. Total flavonoid content
Total flavonoid content was determined by a colorimetric
assay using a method described by Zhishen et al. (1999).
Materials and methods Briefly, 0.5 ml of different concentrations of each extract was
mixed with 1.5 ml of distilled water in a test tube. At time
Chemical products
zero, 0.15 ml of a 5% (w/v) NaNO2 solution was added to the
All solvents and chemical agents were obtained from Sigma- mixture. After 5 min, 0.15 ml of 10% AlCl3 (m/v) was added
Aldrich (Taufkirchen, Germany): n-hexane, dichloromethane, and incubated for 6 min, at room temperature. Then, 0.5 ml
chloroform, acetone, ethyl acetate, methanol, Folin-Ciocalteu, of NaOH (1 M) was added. After agitation, the absorbance of
 PLANT BIOSYSTEMS - AN INTERNATIONAL JOURNAL DEALING WITH ALL ASPECTS OF PLANT BIOLOGY 3

Table 1. Yield of plant extracts percentage.

Yield % TL TS AL AS SL SS
HF 0.07 ± 0.01 0.06 ± 0.04 0.09 ± 0.06 0.09 ± 0.04 0.07 ± 0.00 0.12 ± 0.09
DFM 0.43 ± 0.09 1.01 ± 0.13 0.55 ± 0.39 0.51 ± 0.26 0.51 ± 0.10 0.34 ± 0.16
DFC 0.32 ± 0.06 – 0.38 ± 0.00 – 0.60 ± 0.24 –
ACF 0.26 ± 0.17 0.41 ± 0.03 0.56 ± 0.17 0.49 ± 0.26 0.90 ± 0.65 1.37 ± 1.51
EAF 0.30 ± 0.23 0.34 ± 0.32 0.33 ± 0.21 0.27 ± 0.20 0.48 ± 0.27 0.34 ± 0.30
MF 3.90 ± 0.98 3.27 ± 0.61 8.62 ± 1.32 5.51 ± 0.06 11.80 ± 0.25 7.27 ± 1.90
MFW – – 5.99 ± 0.01 – – –
AF 14.60 ± 0.31 7.96 ± 0.54 26.00 ± 0.86 10.95 ± 1.35 15.22 ± 0.71 7.77 ± 0.03
MWE 7.45 4.68 8.78 6.83 9.72 7.96
Values were the means of three replicates ± standard deviation (p < 0.05, stem sample versus leaf sample for
each species).
TL: T. africana leaf; TS: T. africana stem; AL: A. macrostachyum leaf; AS: A. macrostachyum stem; SL: S. fruticosa leaf;
SS: S. fruticosa stem; HF: n-hexane fraction; DFM: dichloromethane fraction recovered in methanol; DFC: dichlorome-
thane fraction recovered in chloroform; ACF: acetone fraction; EAF: ethyl acetate fraction; MF: methanol fraction
recovered in methanol; MFW: methanol fraction recovered in water; AF: aqueous fraction; MWE: methanol/
water extract.

the solution was measured at 510 nm against a blank. ascorbic acid equivalents per gram of dry weight (mg AAE/
Catechin was used as a standard for the calibration curve g DW).
(y ¼ 5.140 x; R2 ¼ 0.991). The total flavonoid content was
expressed as milligrams of catechin equivalents per gram of
DPPH radical scavenging activity
dry weight (mg CE/g DW).
DPPH scavenging activity was determined using the method
of Sanchez-Moreno et al. (1998) based on the scavenge of
Total flavonol content DPPH free radical hydrogen atom by donation ability of phen-
The content of flavonols was performed as described by olic compounds. A solution of a final volume of 2 ml composed
Kumaran and Karunakaran (2007). Aliquots (0.25 ml) of of 50 ml of various concentrations of the extracts in methanol
extracts were mixed with 0.25 ml AlCl3 (2 mg/ml) and 1.5 ml (0.062; 0.125; 0.25; 0.5; 1; 2; 4 mg/ml) and 1.95 ml of a 0.025 g/l
sodium acetate (50 mg/ml). The absorbance at 440 nm was methanol solution DPPH. After 30 min of incubation at room
recorded after 2.5 h of incubation. Quercetin was used as a temperature, the absorbance was read against a blank at
standard for the calibration curve (y ¼ 7.601 x; R2 ¼ 0.991). 515 nm. DPPH free radical scavenging activity was calculated
The content of flavonols was expressed as milligrams of as percentage (%) using the following formula:
quercetin equivalents per gram of dry weight (mg QE/g DW). As reported in Di Marco et al. (2018), DPPH scavenging
activity (%) ¼ (Ablank – Asample/Ablank)  100, where Ablank is
the absorbance of the control reaction (containing all reagents
Total condensed tannins except the test compound); Asample is the absorbance of the
Proanthocyanidins were evaluated using method described by test compound. Extract concentration providing 50% inhibi-
Julkunen-Titto (1985) by vanillin assay. At a volume of 50 ml of tory concentration (IC50) was calculated from the plotted
each extract, 1500 ml of vanillin/methanol solution (4%, w/v) graph representing the inhibition percentage against extract
was added. After the addition of 750 ml of concentrated HCl, concentrations. Ascorbic acid was used as positive control.
the samples were incubated at room temperature for 20 min.
The absorbance at 550 nm was measured against a blank.
b-Carotene bleaching assay
Catechin was used as a standard for the calibration curve
The antioxidant activity of methanolic extracts was measured
(y ¼ 0.116 x; R2 ¼ 0.996). The amount of total condensed tan-
using b-carotene-linoleate model system, as descrided by
nins was expressed as milligrams of catechin equivalents per
Moure et al. (2000). Two milligrams of b-carotene were dis-
gram of dry weight (mg CE/g DW) from the calibration curve.
solved in 10 ml chloroform and 1 ml b-carotene solution.
Then they were mixed with 0.02 ml of purified linoleic acid
Antioxidant activity and 200 mg of Tween 40 emulsifier. After evaporation of
chloroform under vacuum, oxygenated distilled water
Phosphomolybdate assay (100 ml) was added by vigorous shaking. To an aliquot of
The total antioxidant capacity (TAC) of the plant extracts was 4 ml of this emulsion, 0.2 ml of extracts or the BHA were
evaluated by the phosphomolybdenum method of Prieto added and well mixed. The absorbance at 470 nm, which
et al. (1999). A 0.3 ml aliquot of the plant extract was mixed was regarded t ¼ 0 min, was measured immediately, against
with 3 ml of the reagent solution (0.6 M sulfuric acid, 28 mM a blank consisting of the emulsion without b-carotene. The
sodium phosphate and 4 mM ammonium molybdate). The capped tubes were placed in a water bath at 50
C for 2 h.
tubes were capped and incubated at 95
C for 90 min. After Thereafter, the absorbance of each sample was measured at
the samples were cooled to room temperature and the 470 nm. For the positive control sample was replaced with
absorbance of the mixture was measured at 695 nm against the BHA. A negative control consisted of 0.2 ml of methanol
a blank. The values of TAC were expressed as milligrams of instead of extract or BHA. The antioxidant activity (AA) was
4 N. CHEKROUN-BECHLAGHEM ET AL.

Table 2. Results of quantitative estimation of total phenolic, flavonoid, flavonol and condensed tannin contents.
Total phenolics Total flavonoids Condensed tannins Total flavonols
Plant extracts (mg GAE/g DW) (mg CE/g DW) (mg CE/g DW) (mg QE/g DW)
T. africana leaf TLHF 1.15 ± 0.12 0.13 ± 0.00 0.47 ± 0.04 0.19 ± 0.01
TLDFM 0.86 ± 0.00 0.69 ± 0.00 3.59 ± 0.03 0.80 ± 0.01
TLDFC 1.44 ± 0.28 0.36 ± 0.01 0.51 ± 0.00 0.60 ± 0.04
TLACF 0.87 ± 0.02 0.45 ± 0.00 2.08 ± 0.00 0.65 ± 0.01
TLEAF 0.30 ± 0.02 0.46 ± 0.00 0.34 ± 0.02 0.60 ± 0.13
TLMF 19.75 ± 0.06 1.05 ± 0.00 61.64 ± 2.54 1.60 ± 0.20
TLAF 61.06 ± 0.40 2.21 ± 0.01 118.43 ± 11.79 1.92 ± 0.06
TLMWE 24.53 ± 1.03 1.37 ± 0.03 61.34 ± 0.46 1.91 ± 0.01
T. africana stem TSHF 0.10 ± 0.00 0.05 ± 0.00 0.06 ± 0.04 0.14 ± 0.01
TSDFC 2.39 ± 0.00 1.17 ± 0.00 1.28 ± 0.02 0.78 ± 0.03
TSACF 1.66 ± 0.02 0.37 ± 0.00 1.98 ± 0.03 0.25 ± 0.03
TSEAF 0.35 ± 0.04 0.21 ± 0.02 0.01 ± 0.00 0.34 ± 0.03
TSMF 17.74 ± 0.17 0.49 ± 0.00 19.33 ± 0.32 0.24 ± 0.02
TSAF 33.99 ± 0.07 0.90 ± 0.02 42.80 ± 4.68 0.57 ± 0.02
TSMWE 18.24 ± 0.33 0.67 ± 0.10 34.52 ± 1.22 0.61 ± 0.01
A. macrostachyum leaf ALHF 0.09 ± 0.00 0.06 ± 0.00 0.05 ± 0.01 0.18 ± 0.00
ALDFM 0.61 ± 0.01 0.44 ± 0.00 2.10 ± 0.06 0.56 ± 0.03
ALDFC 3.58 ± 0.02 0.35 ± 0.09 1.12 ± 0.00 0.56 ± 0.02
ALACF 0.44 ± 0.01 0.44 ± 0.00 0.71 ± 0.10 0.45 ± 0.03
ALEAF 0.51 ± 0.00 0.27 ± 0.02 0.12 ± 0.01 0.34 ± 0.07
ALMF 3.01 ± 0.02 0.29 ± 0.02 1.74 ± 0.04 0.71 ± 0.03
ALMFW 0.40 ± 0.02 0.09 ± 0.00 0.22 ± 0.05 0.29 ± 0.04
ALAF 10.24 ± 0.01 1.17 ± 0.04 7.50 ± 0.80 1.75 ± 0.06
ALMWE 2.96 ± 0.26 0.62 ± 0.04 0.87 ± 0.15 1.77 ± 0.23
A. macrostachyum stem ASHF 0.07 ± 0.00 0.04 ± 0.00 0.04 ± 0.01 0.18 ± 0.02
ASDFM 0.95 ± 0.00 0.33 ± 0.00 2.30 ± 0.08 0.38 ± 0.03
ASACF 0.61 ± 0.00 0.37 ± 0.00 0.50 ± 0.01 0.34 ± 0.01
ASEAF 0.40 ± 0.01 0.29 ± 0.02 0.04 ± 0.01 0.29 ± 0.01
ASMFW 3.54 ± 0.08 0.36 ± 0.01 0.81 ± 0.06 0.54 ± 0.06
ASAF 5.76 ± 0.16 0.63 ± 0.00 1.53 ± 0.26 0.46 ± 0.01
ASMWE 1.81 ± 0.88 0.39 ± 0.00 2.16 ± 0.18 0.53 ± 0.01
S. fruticosa leaf SLHF 0.06 ± 0.00 0.05 ± 0.00 0.05 ± 0.04 0.19 ± 0.01
SLDFM 1.31 ± 0.01 1.04 ± 0.00 4.11 ± 0.07 0.82 ± 0.04
SLDFC 1.73 ± 0.19 0.41 ± 0.07 2.18 ± 0.03 1.07 ± 0.12
SLACF 3.38 ± 0.01 2.38 ± 0.01 3.07 ± 0.02 0.77 ± 0.02
SLEAF 0.64 ± 0.00 0.85 ± 0.01 0.67 ± 0.05 0.39 ± 0.07
SLMF 29.36 ± 0.01 12.14 ± 0.19 9.54 ± 0.82 2.36 ± 0.11
SLAF 16.06 ± 0.00 4.26 ± 0.02 0.65 ± 0.10 1.16 ± 0.05
SLMWE 47.73 ± 1.17 4.27 ± 0.12 7.76 ± 0.28 1.75 ± 0.13
S. fruticosa stem SSHF 0.05 ± 0.00 0.04 ± 0.00 0.03 ± 0.00 0.16 ± 0.01
SSDFC 0.98 ± 0.00 0.62 ± 0.08 0.90 ± 0.03 0.34 ± 0.01
SSACF 1.56 ± 0.00 1.10 ± 0.09 2.42 ± 0.00 1.07 ± 0.07
SSEAF 0.37 ± 0.02 1.26 ± 0.01 0.32 ± 0.00 0.42 ± 0.07
SSMF 9.56 ± 0.02 3.82 ± 0.07 2.18 ± 0.02 0.89 ± 0.10
SSAF 4.49 ± 0.01 1.18 ± 0.01 0.39 ± 0.04 0.20 ± 0.00
SSMWE 10.85 ± 0.49 1.05 ± 0.01 2.29 ± 0.11 0.71 ± 0.04
 
Values were the means of three replicates ± standard deviation ( p < 0.05; p < 0.01;  p < 0.001, stem sample versus leaf sample for each species in
each fraction).
TL: T. africana leaf; TS: T. africana stem; AL: A. macrostachyum leaf; AS: A. macrostachyum stem; SL: S. fruticosa leaf; SS: S. fruticosa stem; HF: n-hexane fraction;
DFM: dichloromethane fraction recovered in methanol; DFC: dichloromethane fraction recovered in chloroform; ACF: acetone fraction; EAF: ethyl acetate fraction;
MF: methanol fraction recovered in methanol; MFW: methanol fraction recovered in water; AF: aqueous fraction; MWE: methanol/water extract.

calculated according to the following equation: AA¼ [(AA(120) chromatographic peak area. The analysis was carried out using
– AC(120))/(AC(0) – AC(120))]  100, where AA(120) is the a Phenomenex Luna 3 u C18(2) (3 mm  4.6 mm  150 mm)
absorbance of the sample at t ¼ 120 min; AC(0) is the absorb- column, formic acid 1% (phase A) and methanol (phase B) as
ance of the control at t ¼ 0 min. solvents and an elution gradient set as reported: t0 min (A
85%, B 15%); t20 min (A 65%, B 35%); t55 min (A 10%, B 90%);
t68 min (A 85%, B 15%); t70 min (end run). The injection volume
HPLC-DAD analysis
was 100 ml and the column temperature kept at 30
C.
Methanol and aqueous fractions of T. africana leaves (TLMF;
Each metabolite concentration was expressed as milli-
TLAF)) were subjected to chromatographic analysis by high-
grams of metabolite equivalent per gram of dry weight (mg
performance liquid chromatography (HPLC) system equipped
ME/g DW).
with SPD-M20A diode array detector (DAD, Shimadzu, Tokyo,
Japan). Sixteen different plant compounds were detected,
identified and quantified. Quantitation of each molecule was
Statistical analysis
performed by direct comparison with different concentration
of relative pure standard (Sigma-Aldrich), on the basis of reten- All analyses were carried out in triplicates. Data were pre-
tion time, absorbance spectrum (at 280 or 340 nm) and sented as mean ± standard deviation. Microcal Origin 6 and
 PLANT BIOSYSTEMS - AN INTERNATIONAL JOURNAL DEALING WITH ALL ASPECTS OF PLANT BIOLOGY 5

Table 3. Total antioxidant capacity (TAC), DPPH radical scavenging activity and b-carotene bleaching assay of MWE and fractions.
Plant extracts TAC (mg AAE/g DW) DPPH IC50 (mg/ml) b-carotene assay IC50 (mg/ml)
T. africana leaf TLHF 0.23 ± 0.28 ND 0.66 ± 0.03
TLDFM 2.12 ± 0.39 4.83 ± 0.17 0.43 ± 0.05
TLDFC 1.42 ± 0.13 2.02 ± 0.02 0.22 ± 0.00
TLACF 1.53 ± 0.06 0.81 ± 0.00 1.00 ± 0.02
TLEAF 0.83 ± 0.25 ND 0.27 ± 0.03
TLMF 8.61 ± 1.11 0.74 ± 0.00 0.01 ± 0.00
TLAF 15.60 ± 1.70 1.73 ± 0.01 0.64 ± 0.11
TLMWE 10.30 ± 0.10 2.28 ± 0.02 2.03 ± 0.23
T. africana stem TSHF 1.08 ± 0.02 ND 0.66 ± 0.09
TSDFC 1.50 ± 0.20 0.34 ± 0.00 0.23 ± 0.03
TSACF 1.20 ± 0.17 0.65 ± 0.00 0.24 ± 0.09
TSEAF 0.89 ± 0.05 ND 0.56 ± 0.04
TSMF 4.58 ± 0.12 1.12 ± 0.01 0.18 ± 0.03
TSAF 8.12 ± 0.08 6.60 ± 0.44 2.14 ± 0.13
TSMWE 6.88 ± 0.15 1.93 ± 0.01 1.90 ± 0.55
A. macrostachyum leaf ALHF 0.60 ± 0.00 ND 0.61 ± 0.15
ALDFM 1.08 ± 0.00 7.09 ± 0.02 0.59 ± 0.11
ALDFC 1.41 ± 0.06 ND 0.15 ± 0.04
ALACF 1.28 ± 0.07 ND 0.44 ± 0.05
ALEAF 0.63 ± 0.06 ND 3.52 ± 1.19
ALMF 2.93 ± 0.05 ND /
ALMFW 0.16 ± 0.05 ND 3.95 ± 0.21
ALAF 7.80 ± 0.06 ND 0.23 ± 0.03
ALMWE 4.87 ± 0.23 2.54 ± 0.03 2.20 ± 0.48
A. macrostachyum stem ASHF 0.69 ± 0.00 ND 0.55 ± 0.19
ASDFM 1.36 ± 0.08 2.98 ± 0.00 0.19 ± 0.03
ASACF 0.62 ± 0.05 5.70 ± 0.02 1.15 ± 0.27
ASEAF 0.85 ± 0.22 ND 0.34 ± 0.01
ASMF 2.98 ± 0.07 ND /
ASAF 7.47 ± 0.06 ND 0.82 ± 0.22
ASMWE 6.26 ± 0.23 ND /
S . fruticosa leaf SLHF 0.46 ± 0.06 ND 0.73 ± 0.54
SLDFM 2.52 ± 0.00 3.27 ± 0.04 5.14 ± 0.28
SLDFC 8.15 ± 0.08 3.05 ± 0.07 0.12 ± 0.04
SLACF 4.17 ± 0.15 0.83 ± 0.00 3.47 ± 0.95
SLEAF 1.02 ± 0.01 ND /
SLMF 12.89 ± 0.12 0.79 ± 0.00 4.04 ± 1.27
SLAF 10.85 ± 0.06 2.29 ± 0.02 3.68 ± 0.00
SLMWE 66.78 ± 3.85 0.44 ± 0.00 2.37 ± 0.26
S. fruticosa stem SSHF 0.48 ± 0.14 ND /
SSDFC 0.97 ± 0.04 ND 4.18 ± 1.10
SSACF 2.33 ± 0.12 12.23 ± 2.64 3.43 ± 1.21
SSEAF 0.95 ± 0.07 ND 1.48 ± 0.30
SSMF 6.62 ± 0.08 2.03 ± 0.03 3.81 ± 0.55
SSAF 4.18 ± 0.00 6.91 ± 0.06 7.95 ± 0.18
SSMWE 19.24 ± 2.74 1.37 ± 0.04 1.74 ± 0.41
Ascorbic acid 0.09 ± 0.00 0.03 ± 0.00
BHA
Values were the means of three replicates ± standard deviation (p < 0.05; p < 0.01; p < 0.001, stem sample versus leaf sample for
each species in each fraction).
TL: T. africana leaf; TS: T. africana stem; AL: A. macrostachyum leaf; AS: A. macrostachyum stem; SL: S. fruticosa leaf; SS: S. fruticosa stem; HF: n-
hexane fraction; DFM: dichloromethane fraction recovered in methanol; DFC: dichloromethane fraction recovered in chloroform; ACF: acetone
fraction; EAF: ethyl acetate fraction; MF: methanol fraction recovered in methanol; MFW: methanol fraction recovered in water; AF ¼ aqueous
fraction; MWE: methanol/water extract; ND: not determined under these operating conditions.

Microsoft Excel 2003 were used for statistical and graphical 26.00 ± 0.86%. The hexane fractions of all stem and leaf sam-
evaluations. One way analysis of variance (ANOVA) test was ples showed the lowest yields (from 0.06 ± 0.04% to
performed to evaluate the statistical significance of data. p < 0.12 ± 0.09%). The leaf aqueous fraction of A. macrostachyum
0.05 was considered statistically significant (p < 0.05; p < (ALAF) had the highest yield (26.00 ± 0.86%). For each spe-
0.01; p < 0.001). Moreover, two-way ANOVA was also car- cies, the yields of stem aqueous fractions were significantly
ried out to test any significant differences between species less than that of the leaf aqueous fractions (p < 0.05).
and extraction solvents at p < 0.05.

Phytochemical analysis
Results and discussion The results of total phenolic, flavonoid, flavonol and con-
densed tannin contents are reported in Table 2.
Yields
Total phenolic content of the different fractions and
Result reported in Table 1 showed that the yields obtained extracts of T. africana, A. macrostachyum and S. fruticosa was
of crude extracts and fractions varied from 0.06 ± 0.04% to solvent dependent. Table 2 shows that total phenolic
6 N. CHEKROUN-BECHLAGHEM ET AL.

Figure 1. HPLC-DAD chromatograms of methanol and aqueous fractions of T. africana leaves. Signals were recorded at 280 nm (A) and 340 nm (B). Black: methanol
fraction; Red: WT 100% (water or aqueous fraction).

compounds in fractions varied between 0.10 ± 0.00 and way ANOVA analysis (at a ¼ 0.05). The result showed no sig-
61.06 ± 0.40 mg GAE/g DW for T. africana, 0.07 ± 0.00 and nificant difference between species (F ¼ 2.45, p ¼ 0.05) but a
10.24 ± 0.01 mg GAE/g DW for A. macrostachyum significant difference between extraction solvents (F ¼ 5.06, p
and 0.05 ± 0.00 and 47.73 ± 1.17 mg GAE/g DW for S. fruti- ¼ 0.001).
cosa. Aqueous leaf fraction of T. africana (TLAF) exhibited the The content of flavonoids, total flavonols and condensed
highest total phenolic content (61.06 ± 0.40 mg GAE/g DW). tannins varied from 0.04 ± 0.00 to 12.14 ± 0.19 mg CE/g DW,
This difference between the phenolic content may be due to 0.16 ± 0.01 to 2.36 ± 0.11 mg QE/g DW and 0.01 ± 0.00 to
the nature of the molecules present in plants and their solu- 118.43 ± 11.79 mg CE/g DW, respectively. Methanol leaf frac-
bility in each solvent. This suggestion was confirmed by two- tion of S. fruticosa (SLMF) exhibited the highest values of
 PLANT BIOSYSTEMS - AN INTERNATIONAL JOURNAL DEALING WITH ALL ASPECTS OF PLANT BIOLOGY 7

0.16
Methanolic fraction Aqueous fraction
0.14 ***

0.12 **
***

mg ME/g DM
0.1

0.08 **

0.06
*
* **
0.04
*
0.02

Figure 2. Detection and quantification of 16 different plant metabolites in methanolic and water fractions of T. africana leaves (p < 0.05; p < 0.01; p <
0.001, water versus methanolic fraction).

flavonoids (12.14 ± 0.19 mg CE/g DW) and flavonols analysis (Kumaran and Karunakaran 2007). Methanol/water
(2.36 ± 0.11 mg QE/g DW). TLAF revealed a high content in leaf extract of S. fruticosa (SLMWE) exhibited the highest TAC
condensed tannins (118.43 ± 11.79 mg CE/g DW). Results (66.785 ± 3.859 mg EAA/g MS), followed by methanol/water
showed that the species influenced significantly the amounts stem extract of S. fruticosa (SSMWE) (19.24 ± 2.74 mg/g) and
of these classes of polyphenolic compounds (p < 0.05) but TLAF (15.60 ± 1.70 mg/g). The SSMWE extract was significantly
no significant difference was observed on flavonoid and con- less than SLMWE extract (p < 0.01). Results of Oueslati et al.
densed tannins contents (p > 0.05). (2012b) revealed that the highest TAC of S. fruticosa
Results of Oueslati et al. (2012a) founded that the acetone (53.62 mg GAE/g DW) was observed in acetone extract. In
extract had a value of 31.7 ± 0.51 mg GAE/g DW for phenolic addition, S. fruticosa extract was fourfold higher when com-
contents, 26.2 ± 0.44 and 1.50 ± 0.13 mg CE/g DW for flavon- pared with the leaves (14.66 ± 1.25 mg GAE/g DW) and flow-
oid and condensed tannin contents in shoot extracts of S. ers (33.73 ± 3.65 mg GAE/g DW) of T. gallica (Ksouri et al.
fruticosa. Another study of Ksouri et al. (2012a, 2012b) 2009). Our result indicated that there was no significant dif-
showed that the methanolic extracts of this species reveled ference between the species (F ¼ 1.63, p ¼ 0.18; at a ¼ 0.05)
high polyphenol content of about 38 mg GAE/g DW. This but a significant difference between the solvents (F ¼ 3.41, p
variation in the contents between our results and previous ¼ 0.01; at a ¼ 0.05). This effect could be due to the solubil-
findings can be explained by the influence of species, extrac- ity of chemical substances with potent antioxidant activity
tion solvent and others intrinsic and extrinsic factors such as present in these extracts and their nature.
the nature of the bioactive molecules present, origin and
vegetative stage of the plant.
DPPH radical scavenging activity
All extracts had dose-dependent activity, i.e. DPPH scaveng-
In vitro antioxidant capacity
ing activity proportionally increased according to the
Results of in vitro antioxidant capacity of plant extracts and increase in concentration of the extracts. The scavenging
fractions are reported in Table 3. The evaluation of the anti- effect of phenolic extracts on the DPPH radical, expressed as
oxidant capacity of plant extracts and fractions was per- IC50 value, varied widely from 0.34 ± 0.00 to 12.23 ± 2.64 mg/
formed using three different assays: total antioxidant activity, ml (Table 3). In particular, the antioxidant activity against
DPPH radical scavenging activity and b-carotene assay. DPPH radical was significantly higher in dichloromethane
stem fraction of T. africana (TSDFC) (0.34 ± 0.00 mg/ml), fol-
lowed by SLMWE (0.44 ± 0.00 mg/ml). Though the antioxidant
Total antioxidant activity
potential of extracts and fractions was found to be low than
Total antioxidant capacity of the extracts is shown in Table 3. that of ascorbic acid (0.09 ± 0.00 mg/ml). For the DPPH free
The phosphomolybdate method is based on the reduction of radical scavenging assay, no significant difference was
Mo (VI) to Mo (V) by the antioxidant compounds and the for- recorded between species (two-way ANOVA, F ¼ 2.13, p ¼
mation of a green phosphate/Mo (V) complex with a max- 0.16, at a ¼ 0.05) and solvents (two-way ANOVA, F ¼ 1.23, p
imal absorption at 695 nm. This method is a quantitative ¼ 0.35, at a ¼ 0.05). In reality, several authors have reported
8 N. CHEKROUN-BECHLAGHEM ET AL.

O O OH
OH HO
O OH
OH
HO OH H H H
H
O
H
HO O OH
H HO
HO
O OH HO OH OH

Gallic acid Chlorogenic acid Caffeic acid ρ-Coumaric acid

OH O
OH O
OH
OH O OH

OH
HO O HO O
HO O
OH OH
HO HO

Chrysin Myricetin Quercetin

OH O
OH O
OH
OH
OH O

HO O HO O

HO O OH OH

Kaempferol Apigenin
Genistein
OH OH
OH

O
HO O OH O OH OH O
O
O OH
HO O O
OH HO O
HO O
HO OH OH
OH OH

Kaempferol-3-O-glucoside Quercetin-3-O-arabonoside Galangin

OH
OH

HO O

OH O OH O O
O
H O CH3
OH H
H O OH
O HO
O CH3

HO OH HO
OH OH HO

Rutin Caffeic acid phenethyl ester Caffeic acid 1,1 dimethyl allyl ester
Figure 3. Chemical structures of identified phenolic acids and flavonoids of methanol and aqueous fractions of T. africana leaves.

a positive and significant relationship between the antioxi- hypothesis. According to another study of Khabtane et al.
dant components including phenol acids, flavonoids and tan- (2017), the results showed that the ethyl acetate extracts of
nins, respectively with the DPPH radical scavenging capacity T. africana had an excellent power to neutralize the
(Trabelsi et al. 2010) and our data can also support this DPPH radical.
 PLANT BIOSYSTEMS - AN INTERNATIONAL JOURNAL DEALING WITH ALL ASPECTS OF PLANT BIOLOGY 9

b-Carotene bleaching activity of their peaks against those of standards as well as by spik-
ing the sample with standards. Chromatogram analysis
This method is based on the discoloration of b-carotene by
showed important differences between methanol and aque-
the peroxides generated during the oxidation of linoleic acid
ous fractions phenolic composition. Indeed, phenolic acids
at elevated temperature. In this model, b-carotene undergoes
and flavonoids were more largely represented in aqueous
rapid discoloration in the absence of an antioxidant. The per-
fraction than in methanol fraction of T. africana (Figure 2).
centage inhibition of the antioxidant activity by the b-caro-
Chemical structures of these components are indicated in
tene/acid linoleic system is proportional to the concentration.
Figure 3.
All plant extracts inhibited the b-carotene bleaching effect at
These data established that the antioxidant activities of T.
different levels, thanks to the scavenging free radicals. Table 3
africana leaves could be attributed to their polyphenol com-
reports the inhibition of b-carotene bleaching by the crude
pounds. In this way, the superiority of antioxidant activity of
extracts and fractions. IC50 value was considerably higher in
methanol leaf fraction of T. africana (TLMF) (0.01 ± 0.00 mg/ml) the fractions compared to that of the crude extracts may be
as compared to BHA (0.03 ± 0.00 mg/ml). In addition, dichloro- explained by amount and nature of individual phenolic com-
methane fractions recovered in chloroform gave a good anti- pounds. Previous studies indicated that benzoates are potent
oxidant activity: 0.22 ± 0.00 mg/ml for leaf fraction of T. antioxidant agents, quenching radicals, singlet oxygen and
africana (TLDFC), 0.15 ± 0.04 mg/ml for leaf fraction of A. mac- hydrogen peroxide (Bourgou et al. 2008). Moreover, phenolic
rostachyum (ALDFC) and 0.12 ± 0.04 mg/ml for leaf fraction of acids, such as gallic and vanillic acids, account for almost
S. fruticosa (SLDFC). Two-way ANOVA analysis (at a ¼ 0.05) one-third of dietary phenols and are associated with the
applied on the b-carotene showed no significant difference most organoleptic, nutritional and antioxidant properties of
between neither species (F ¼ 0.95, p ¼ 0.45) nor between sol- plant foods (Rodr
ıguez et al. 2008). Concerning leaves of T.
vents (F ¼ 1.45, p ¼ 0.26). These data indicated that the b-caro- gallica, literature data report the identification of 12 phenolic
tene bleaching assay of extracts is strongly dependent on oil- compounds, including phenolic acids and flavonoids, and
in-water emulsion system, which influence the reactivity of among them syringic acid, isoquercitin and catechin were
chemical compounds. Compared with the literature, several the major compounds (Ksouri et al. 2009). Finally, Mahfoudhi
authors (Djeridane et al. 2006) confirm this suggestion. et al. (2014) revealed 16 phenolic compounds from leaves
Moreover, Ksouri et al. (2012a, 2012b) reported the highest and stems of T. aphylla (L.) using HPLC-UV/DAD, HPLC-ESI-MS
antioxidant effect of T. gallica, and S. fruticosa to inhibit the and MS2, and an ion trap mass analyzer, thus confirming our
bleaching of the b-carotene by linoleic acid as compared to results on the presence of such molecules in halo-
others halophyte plants tested (T. gallica, Limoniastrum phyte plants.
monopetalum, L. guyonianum, and Mesembryanthemum edule).
The same activity was observed for S. pruinosa and S. mollis
(IC50 ¼ 540 mg/ml), while S. maritima was less active with an Conclusion
IC50 over 1000 mg/ml (Oueslati et al. 2012b). Other research This study provides valuable information regarding the
showed that S. fruticosa has higher antioxidant activity than potential role of halophytes as natural sources of antioxi-
the halophyte T. gallica, which exhibited an IC50 equal to 54.7 dants. Until now, the halophyte A. macrostachyum has never
mg/ml (Ksouri et al. 2009) and M. nodiflorum (IC50 ¼ 205 mg/ml) been subjected to antioxidant studies and this aspect consti-
(Falleh et al. 2009). These results suggest that S. fruticosa con- tutes the originality of our work. Indeed, our results suggest
tains antioxidant substances that are capable to scavenge free that halophytes possess optimal biological activities due to
radicals and block the spread of radical chains. their exceptionally high content and quality in phenolic acids
and flavonoids. As a whole, these findings may confirm the
HPLC-DAD analysis interesting potential of these halophytes as valuable source
of natural bioactive molecules. Moreover, choose of species
Figure 1 shows HPLC-DAD chromatograms of methanol and and extraction solvent was very important to extract phen-
aqueous fractions of T. africana leaves. Signals were recorded olic compounds with strong antioxidant activity. As phenolic
at 280 nm and 340 nm. The retention time of authentic compounds have several activities, beyond the antioxidant
standards and milligrams of metabolite equivalent per gram effect, they can replace synthesis antioxidants, such as ascor-
of dry weight (mg ME/g DW) are indicated. Concerning leaf bic acid. Considering that the composition of each extract is
methanolic fraction, 16 phenolic compounds were success- complex, it is necessary to isolate and assess the major active
fully identified. They include six phenolic acids (Gallic acid,
principles of these plants to test their effect against sev-
Chlorogenic acid, Caffeic acid, p-Coumaric acid, Caffeic acid
eral diseases.
1,1 dimethyl allyl ester and Caffeic acid phenethyl ester) and
10 flavonoids (Chrysin, Rutin, Myricetin, Quercetin, Genistein,
Kaempferol, Apigenin, Kaempferol-3-O-glucoside, Quercetin- Acknowledgments
3-O-arabonoside and Galangin). Moreover, the same 16
Nabila Belyagoubi-Benhammou sincerely thanks Dr. Angelo Gismondi
phenolic compounds were successfully identified in leaf and Prof. Antonella Canini, Department of Biology, University of Rome
aqueous fraction. All these compounds have been identified Tor Vergata, Italy, for their help in the HPLC-DAD analysis and for their
according to their retention times and spectral characteristics linguistic revision and correction of this manuscript.
10 N. CHEKROUN-BECHLAGHEM ET AL.

Disclosure statement leaves of the halophyte Cakile maritime. Plant Physio Biochem. 45(3-
4):244–249.
No potential conflict of interest was reported by the authors. Ksouri R, Falleh H, Megdiche W, Trabelsi N, Mhamdi B, Chaieb K, Bakrouf
A, Magn
e C, Abdelly C. 2009. Antioxidant and antimicrobial activities
of the edible medicinal halophyte Tamarix gallica L. and related poly-
ORCID phenolic constituents. Food Chem Toxicol. 47(8):2083–2091.
Ksouri R, Ksouri WM, Jallali I, Debez A, Magn
e C, Hiroko I, Abdelly C.
N. Belyagoubi-Benhammou https://orcid.org/0000-0001-5624-4627 2012a. Medicinal halophytes: potent source of health promoting bio-
A. Gismondi http://orcid.org/0000-0002-9257-9667 molecules with medical, nutraceutical and food applications. Critl Revi
A. Canini http://orcid.org/0000-0003-1132-8899
Biotechnol. 32(4):289–326.
I. A. El Haci http://orcid.org/0000-0001-5364-377X
Ksouri R, Smaoui A, Isoda H, Abdelly C. 2012b. Utilization of halophyte
species as new sources of bioactive substances. J Arid Land Stud. 22:
41–44.
Kumaran A, Karunakaran RJ. 2007. In vitro antioxidant activities of
References methanol extracts of five Phyllanthus species from India. LWT-Food
Sci Technol. 40(2):344–352.
Benabdallah H, Gharzouli K, Khennouf S, Amira S, Soufane S. 2014.
Le Hou
erou HN. 2001. Biogeography of the arid steppeland north of the
Phytochemical analysis and anti-lipid peroxidation activity of Tamarix
Sahara. J Arid Environ. 48(2):103–128.
africana L. extracts. Global J Res Med Plants Indigen Med. 3:278–285.
Mahfoudhi A, Pio Prencipe F, Mighri ZF, Pellat F. 2014. Metabolite profil-
Bjarnholt N, Li B, D’Alvise J, Janfelt C. 2014. Mass spectrometry imaging
ing of polyphenols in the Tunisian plant Tamarix aphylla (L.) Karst. J
of plant metabolites-principles and possibilities. Nat Prod Rep. 31(6):
Pharm Biomed Anal. 99:97–105.
818–837.
Meot-Duros L, Le Floch G, Magne C. 2008. Radical scavenging, antioxi-
Bourgou S, Ksouri R, Bellila A, Skandrani I, Falleh H, Marzouk B. 2008.
dant and antimicrobial activities of halophytic species. J
Phenolic composition and biological activities of Tunisian Nigella sat-
Ethnopharmacol. 116(2):258–262.
iva L. shoots and roots. CR Biol. 331(1):48–55.

dio L, Ferreira AC, Pereira H, Silvestre L, Vizetto-Duarten C, Barreira Moure A, Franco D, Sineiro J, Dominguez H, Nunez MJ, Lema GM. 2000.
Custo
Evaluation of extracts from Gevuina avellana hulls as antioxidants. J
L, Rauter AP, Albericio F, Varela J. 2012. The marine halophytes
Agric Food Chem. 48(9):3890–3897.
Carpobrotus edulis L. and Arthrocnemum macrostachyum L. are
Oueslati S, Ksouri R, Falleh H, Pichette A, Abdelly C, Legault J. 2012a.
potential sources of nutritionally important PUFAs and metabolites
with antioxidant, metal chelating and anticholinesterase inhibitory Phenolic content, antioxidant, anti-inflammatory and anticancer activ-
activities. Botanica Marina. 55:281–288. ities of the edible halophyte Suaeda fruticosa Forssk. Food Chem.
Di Marco G, Gismondi A, Panzanella L, Canuti L, Impei S, Leonardi D, 132(2):943–947.
Canini A. 2018. Botanical influence on phenolic profile and antioxi- Oueslati S, Trabelsi N, Boulaaba M, Legault J, Abdelly C, Ksouri R. 2012b.
dant level of Italian honeys. Int J Food Sci Technol. 55(10):4042–4050. Evaluation of antioxidant activities of the edible and medicinal
Djeridane A, Yousfi M, Nadjemi B, Boutassouna D, Stocker P, Vidal N. Suaeda species and related phenolic compounds. Ind Crops Prod.
2006. Antioxidant activity of some Algerian medicinal plants extracts 36(1):513–518.
containing phenolic compounds. Food Chem. 97(4):654–660. Prieto P, Pineda M, Aguilar M. 1999. Spectrophotometric quantitation of
El-Wahab RHA, Zaghloul MS, Wafaa MW, Kamel WM, Moustafa RAA. antioxidant capacity through the formation of a phosphomolybde-
2008. Diversity and distribution of medicinal plants in North Sinai, num complex: specific application to the determination of vitamin E.
Egypt. Afr J Environ Sci Technol. 2:157–171. Anal Biochem. 269(2):337–341.
Falleh H, Ksouri R, Oueslati S, Guyot S, Magn
e C, Abdelly C. 2009. Rodr
ıguez H, Landete JM, De las Rivas B, Muno ~z R. 2008. Metabolism of
Interspecific variability of antioxidant activities and phenolic compos- food phenolic acids by Lactobacillus plantarum CECT 748T. Food
ition in Mesembryanthemum genus. Food Chem Toxicol. 47: Chem. 107(4):1393–1398.
2308–2313. Sanchez MIG, Cullagh JM, Guy RH, Compton RG. 2016. Reverse iontopho-
Gupta B, Huang B. 2014. Mechanism of salinity tolerance in plants: retic extraction of metabolites from living plants and their identifica-
physiological, biochemical, and molecular characterization. Int J tion by ion-chromatography couple to high resolution mass
Genomics. 2014:701596. spectrometry. Phytochem Anal. 28:195–201.
Julkunen-Titto R. 1985. Phenolic constituents in the leaves of northern Sanchez-Moreno C, Larrauri JA, Saura-Calixto F. 1998. A procedure to
Willows methods for the analysis of certain phenolics. J Agric Food measure the antiradical efficiency of polyphenols. J Sci Food Agric.
Chem. 33:213–217. 76:270–276.
Khabtane A, Zeraib A, Aouidane L, Kara Ali W, Belguidoum FZ, Singleton VL, Rossi JR. 1965. Colorimetry of total phenolics with phos-
Rahmoune C. 2017. In vitro evaluation of the antimicrobial activity phomolybdic–phosphotungstic acid reagents. Am J Enol Vitic. 16:
and the antioxidant activity of the flavonoids extracted from the flow- 144–158.
ers of the Tamarix africana Poir. Int J Biosci. 11:417–426. Trabelsi N, Megdiche W, Ksouri R, Falleh H, Oueslati S, Bourgou S,
Khennouf S, Benabdallah H, Gharzouli K, Amira S, Ito H, Kim TH, Yoshida Hajlaoui H, Abdelly C. 2010. Solvent effects on phenolic contents and
T, Gharzouli A. 2003. Effect of tannins from Quercus suber and biological activities of the halophyte Limoniastrum monopetalum
Quercus coccifera leaves on ethanol-induced gastric lesions in mice. J leaves. LWT- Food Sci Technol. 43(4):632–639.
Agric Food Chem. 51(5):1469–1473. Zhishen J, Mengcheng T, Jianming W. 1999. The determination of flavon-
Ksouri R, Megdiche W, Debez A, Falleh H, Grignon C, Abdelly C. 2007. oid contents in mulberry and their scavenging effects on superoxide
Salinity effects on polyphenol content and antioxidant activities in radicals. Food Chem. 64(4):555–559.