沙漠研究 22-1, 41 -44 (2012 )"ICAL 1 / DT X"

Journal of Arid Land Studies－Refereed Paper－

Utilization of Halophyte Species as New Sources of Bioactive Substances
Riadh KSOURI1), Abderrazak SMAOUI1), Hiroko ISODA2) and Chedly ABDELLY*1)
Abstract: Halophytes grow in a wide variety of saline habitats, from coastal regions, salt marshes and mudflats to inland deserts.
In these saline areas, unfavorable environmental conditions increased production of reactive oxygen species (ROS) in plants, leading tocellular damage, metabolic disorders, and senescence processes. Halophytes are known for their ability to overcome and quench
these toxic ROS, since they are equipped with a powerful antioxidant system. Among them, phenolics have an important effect onoxidative stability and microbial safety. Additionally to their role as antioxidant, these compounds exhibit a wide spectrum of
preventive medicinal properties. In this context, several works studied a large flora of Tunisian halophytic species well known for
their utilization in traditional medicine and investigated their biochemical composition and biological activities in order to reveal theircapacities. For instance, some halophytic species exhibit a higher content of polyphenol between 40 (Suaeda fruticosa) and 200 mg
EAG.g-1MS (Tamarix gallica). Moreover, these halophytes showed a higher activity in each antioxidant system with an IC50 values
more interesting than positive control BHT (scavenging ability against DPPH, ABTS and β-carotene bleaching) and BHA (lipid
peroxidation inhibition). In addition, some halophytic plant extracts showed appreciable antibacterial properties against pathogen
strains and a highest anti-inflammatory activity to inhibit RAW 264.7 macrophages stimulated by (LPS).
Key Words: Antioxidant compounds, Bioactive substances, Biological activities, Halophytes

1. Introduction
In Tunisia, as in several Mediterranean countries, drought
and salinity constitute the two major constraints responsible forthe limitation of crop productivity and the deterioration ofvegetation cover. Several spontaneous species (Extremophile
plants: halophytes, xerophytes and xero-halophytes) have been
facing a strong pressure of selection in these stressing biotopes
and have therefore acquired the necessary features, allowing
them to successfully cope with salinity, water deficit stress,
extreme temperatures and excessive luminosity. It is wellknown that these extreme conditions generate an oxidant stressresulting in the large accumulation of harmful reactive oxygen
species (ROS), which cause lipid membrane peroxidation andprotein and DNA deterioration (Ksouri et al., 2010). Under
these unfavourable conditions, tolerant plants often develop a
powerful antioxidant system implying several components: (i)the increase of osmolyte biosynthesis (or compatible
compounds such as betains, proline, sugars, polyols) involvedin the osmotolerance and in particular in the protection of thecellular structures against the toxic effects of salt, (ii) the
biosynthesis of bioactive substances like polyphenols, vitamins(C and E) and carotenoids, which constitute the principal
category of compounds with high antioxidant capacity, and (iii)
the activation of enzymatic systems and antioxidant substances,involved in the ROS scavenging (glutathione, ascorbate,catalases, peroxidases, superoxide dismutases) (Jaleel et al.,2009). The study of plant response to these constraints (intheir biotope or under laboratory conditions) allows not only
the elucidation of the mechanisms enabling them to survive
under these permanent stressing conditions, but may also haveeconomic implications in the field of the biotechnology applied
to health and agriculture, such as the development of productswith industrial applications. For example, polyphenols have agreat importance considering their multiple virtues andutilizations in the food, cosmetic, pharmaceutical, andmedicinal industries (Macheix et al., 2005). These natural
antioxidants are strongly recommended to replace synthetic
antioxidants (potentially carcinogenic) currently used intherapy and food. Indeed, they present a large spectrum ofphysiologicalactivities(antimicrobial,
anti-allergic,
anti-carcinogens) and preventive action against cardiovasculardiseases (Balasundram et al., 2006). In this context, Tunisianextremophilespecies,
well
known
for
their
ethno-pharmacological uses in traditional medicine andculinary, was making them good candidates for industrial
application.
In order to characterize and valorize local halophytes as
new sources of bioactive substances, the aim of this study was:(i) to evaluate the phenolic content and composition of some
halophytic species, (ii) to investigate antioxidant activities
using different tests, (iii) to estimate antimicrobial capacities
against human pathogen strains, and (iv) anti-inflammatory
activity of lipopolysaccharide (LPS)-stimulated RAW 264.7
macrophages.

* Corresponding Author: chedly.abdelly@cbbc.rnrt.tn; chedly.abdelly@gmail.com
P. O. Box 901, 2050 Hammam-Lif, Tunisia1) Laboratory of Extremophile Plants (LPE), Biotechnology Centre of Borj-Cedria2) Alliance of Research on North Africa, University of Tsukuba, Japan2. Materials and methods
2.1. Plant sampling and preparation for extract
Halophyte species (Tamarix gallica L., Limoniastrum
monopetalum L., Limoniastrum guyonianum L., Suaeda
fruticosa Forssk., and Mesembryanthemum edule L.) were
harvested at full bloom in several Tunisian salt localities (May2007). The harvested shoots were rinsed with distilled water,
left at room temperature for 5 days in the dark, oven-dried for 1
h at 60°C, and grinded to fine powder. Extracts wereobtained by magnetic stirring of 2.5 g dry powder in 25 ml
pure methanol for 30 min. Extracts were kept for 24 h at 4°C,
filtered through a Whatman no. 4 filter paper, and evaporated
under vacuum. Then, they were stored at 4°C until analysis(Falleh et al., 2008).
2.2. Determination of total polyphenol content
Total polyphenols quantification was determined, as
described by Dewanto et al. (2002). An aliquot of 125 µl of
diluted extract were added to 500 µl of distilled water and 125µl of the Folin-Ciocalteu reagent. The mixture was shaken,
before adding 1250 µl Na2CO3 (7%), adjusting with distilledwater to a final volume of 3 ml, and mixed thoroughly. Afterincubation for 90 min at 23°C in the dark, the absorbanceversus prepared blank was read at 760 nm. A standard curveof gallic acid was used. Total phenolic content of organs was
expressed as mg gallic acid equivalents per gram of dry weight(mg GAE/g DW) through the calibration curve with gallic acid,ranging from 0 to 400 µg/ml. All samples were analyzed intriplicate.
2.3. Assessment of antioxidant activities
The most common tests used for the determination ofantioxidant activities of plant extracts such as DPPH test,
Fe-reducing power, and β-carotene linoleate system wereassessed according to Ksouri et al. (2009). All samples wereanalyzed in triplicate.
2.4. Determination of antibacterial activity
The antibacterial activity of shoot extracts was assessed by
the agar disk diffusion assay (Bagamboula et al., 2003) against
five human pathogenic bacteria: Gram-positive cocci including
Staphylococcus epidermidis (Collection Institute Pasteur
106510), Staphylococcus aureus (ATCC 25923), Micrococcus
luteus (NCIMB 8166), and Gram-negative bacteria including
Escherichia coli (ATCC 35218) and Pseudomonas aeruginosa(ATCC 27853). All samples were analyzed in triplicate.

2.5. Measurement of anti-inflammatory activity by nitrite
quantification
Exponentially growing cells were plated in 24-well
microplates (BD Falcon) at a density of 2×105 cells per well in
400 µl of culture medium and were allowed to adhere
overnight. Cells were then treated or not with positive control
N (G)-nitro-l-arginine methyl ester (L-NAME), or increasing
concentrations of extracts dissolved in the appropriate solvent,and incubated at 37°C, 5% CO2 for 24 h. The final
concentration of solvent in the culture medium was maintained
at 0.5% (v/v) to avoid solvent toxicity. Cells were thenstimulated with 100 µg/ml lipopolysaccharide (LPS). After
24 h, cell-free supernatants were collected and stored at −80°C
until NO determination using the Griess reaction (Green et al.,1990) with minor modifications. All samples were analyzed
in triplicate.
2.6. Analysis of individual phenolic compounds
The identification of phenolic compounds was achieved
according to Ksouri et al. (2009).

3. Results and Discussion
Results showed that shoot methanolic extracts of all
halophytes exhibit an important polyphenol contents, ranged
from 38 (Suaeda fruticosa) to 190 mg GAE/g DW (Tamarix
gallica) (Table 1) as compared to glycophyte medicinal plants
such as Mentha pulegium (Bourgou et al., 2008) or Nigellasativa (Karray-Bouraoui et al., 2010). Moreover, these
species exhibited higher antioxidant activities for all
antioxidant tests as compared to the positive control (BHT or
BHA). For example, the antioxidant activity against DPPH
radical was significantly higher in shoot methanolic extracts ofall halophytes except S. fruticosa, IC50 values were ranged
Table 1. Total polyphenol content and antiradical activity against
DPPH radical of studied halophytes. Data concerning
Mentha pulegium, Nigella sativa and BHT are respectively
from Karray-Bouraoui et al. (2010); Bourgou et al. (2008); andKsouri et al. (2009).
Species
Polyphenol content
DPPH test
T. gallica(mgGAE.g/ DW)(IC50 in µg/ml)

190

1

L. monopetalum

40

2

L. guyonianum

99

1

S. fruticosa

38

20

M. edule

71

10

BHT

-

11.5

Mentha pulegium
Nigella sativa

20

44

10

280

Table 2. Assessment of Fe-reducing power and β-carotene bleachingactivity of the studied halophytes, BHT and vitamin C.
SpeciesFe-reducing powerβ-carotene bleaching

Table 3. Antibacterial activity of Tamarix gallica extracts against
pathogen human bacteria.
Bacteria
Tamarix gallica
Control (+)(100mg/ml)
gentamycin(10UI)
S. aureus, ATCC25923
10.00 ± 0.00
22(EC50 in µg/ml)

test (IC50 in µg/ml)

T. gallica

45.2

54.7

S. epidermidis, CIP106510

11.00 ± 0.00

30

L. monopetalum

137

110

Micrococcus luteus, NCIMB 816615.00 ± 0.00

26

L. guyonianum

142

37

E. coli, ATCC 35218

8.00 ± 0.00

27

S. fruticosa

365

30

M. edule

0.2

130

BHT

-

75

Vit C

37

-

P. aeruginosa, ATCC 27853
10.33 ± 0.66
16
No antimicrobial activity (), inhibition zone <1 mm.
Weak
antimicrobial activity (w), inhibition zone = 1 mm. Slight antimicrobialactivity (+), inhibition zone 2–3 mm. Moderate antimicrobial activity(++), inhibition zone 4–5 mm. High antimicrobial activity (+++),
inhibition zone 6–9 mm. Strong antimicrobial activity (++++), inhibitionzone >9 mm. Standard deviation ± 0.5 mm. For all bacteria, theinhibition zone of the control (+) gentamycin (10 UI) was higher than 9
mm (++++). The diameter of disc was 6 mm. SD: standard deviation.

70

Inhibition NO (%)

from 1 to 10 µg/ml, than BHT (IC50=11.5 µg/ml), hence
indicating a notably superior efficiency of halophytes. In
reality, several authors have reported a positive and significant
relationship between the antioxidant components including
phenol acids, flavonoids and tannins, respectively with the
Fe-reducing power and DPPH radical scavenging capacity(Trabelsi et al., 2010). Therefore, the high content of total
phenols in all methanolic extracts might explain the strong
antioxidant properties of these halophytes.
Concerning the other antioxidant tests (Fe-reducing powerand β-carotene bleaching activity), IC50 values wereconsiderably higher in M. edule as compared to vitamin C foriron-reducing activity (Table 2). In addition, T. gallica, L.
guyonianum, and S. fruticosa displayed the highest activity toinhibit β-carotene bleaching as compared to BHT. In fact,
these antioxidant activities depend on phenolic nature, structureand synergistic interactions (Djeridane et al., 2006).
Moreover, T. gallica extracts showed appreciable
antibacterial activities against Human pathogen strains. For
instance, the highest activity was recorded against Micrococcus
luteus and the poorest activity was observed against
Escherichia coli (Table 3). These results suggest thatmethanolic extracts of T. gallica were more efficient to inhibit
bacterial growth, probably in relation to their active molecules.
Several studies attributed the inhibitory effect of plant extracts
against bacterial pathogens to their phenolic composition(Rodriguez Vaquero et al., 2007).
Ksouri et al. (2009) showed after RP-HPLC analysis that T.
gallica phenolic fingerprint was composed of seven phenolicacids (gallic, sinapic, chlorogenic, syringic, vanillic,p-coumaric, and trans-cinnamic acids) and six flavonoids((+)-catechin, isoquercetin, quercetin, apigenin, amentoflavone,and flavone). These data established that the antioxidant andantimicrobial activities of T. gallica shoots could be attributed
to their polyphenol compounds. Recent studies showed that(+)-catechin, and syringic, ferulic, vanillic, and p-coumaric
acids gave high positive correlation with DPPH and ABTS

Methanol extract

60
50
40
30
20
10
0
L-NAME(250 µM)

1,25

5

20

80

160

Concentration (µg/ml)

Fig. 1. Effect of methanol extracts from Suaeda fruticosa on NO
overproduction in LPS-stimulated RAW 264.7 macrophages.

scavenging activities and reducing power. Moreover, gallic
acid and catechin were closely correlated to metal chelating
activity and inhibition of lipid peroxidation, respectively (Tsaiet al., 2007). In addition, catechin, quercetin and gallic acid
were reported for their high antibacterial activity against E. coli,
P. aeruginosa and S. aureus (Rodriguez Vaquero et al., 2007).
On the other hand, the edible medicinal halophyte, Suaeda
fruticosa, showed a higher anti-inflammatory activity,
inhibiting NO release, by 66.4% at 160 µg/ml in
lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages(Fig. 1) as compared to the positive control L-NAME which
inhibit NO release by 58.2%. The elevated anti-inflammatory
activity of methanolic extract could be due, in part, to thepresence of phenolic compounds.
Besides, methanol extract of Saussurea costus was
frequently used in Korean traditional prescriptions for
inflammatory diseases, exhibited more than 50% of inhibition
on tumor necrosis factor (TNF)-alpha production inLPS-stimulated RAW 264.7 cells (Cho et al., 1999).

4. Conclusion
In conclusion, our results showed that the high biologicalcapacity of halophytes were ascribed to the exceptionally highcontent in polyphenols and to the quality of its phenolic acids
and flavonoids identified by RP-HPLC analysis. As a whole,these findings may confirm the interesting potential of these
halophytes as valuable source of natural bioactive molecules.
Acknowledgements
This work was supported by the Tunisian Ministry ofHigher Education and Scientific Research (LR10CBBC02)
and by SATREPES Project: “Valorization of Bio-resources in
Semi-Arid and Arid Land for Regional Development”.
References
Bagamboula M., Uyttendaele J., Debevere M. (2003):
Inhibitory effect of thyme and basil essential oils, carvacrol,
thymol, estragol, linalool and p-cymene towards Shigellasonnei and S. flexneri. Food Microbiol., 21: 33-42.
Balasundram N., Sundram K., Sammar S. (2006): Phenolic
compounds in plants and agri-industrial by-products.
Antioxidant activity, occurrence, and potential uses. Food
Chem., l1: 191-203.
Bourgou S., Ksouri R., Bellila A., Skandrani I., Falleh H.,
Marzouk B. (2008): Phenolic composition and biologicalactivities of Tunisian Nigella sativa L. shoots and roots. C. R.
Biologies, 331 (1): 48-55.
Cho J.Y., Park J., Yoo E.S., Baik K.U., Jung J.H., Lee J., Park
M.H. (1999): Inhibitory effect of sesquiterpene lactones
from Saussurea lappa on tumor necrosis factor-alphaproduction in lipopolysaccharide-stimulated RAW 264.3
cells. Nat. Prod. Sci. , 5: 12-19.
Dewanto V., Wu X., Adom K.K., Liu R.H. (2002): Thermal
processing enhances the nutritional value of tomatoes by
increasing total antioxidant activity. J. Agr. Food Chem., 50:
3010–3014.
Djeridane M., Yousfi B., Nadjemi D., Boutassouna P., Stocker
N. (2006): Antioxidant activity of some Algerian medicinal
plants extracts containing phenolic compounds. Food
Chem., .97: 654-660.

Falleh H., Ksouri R., Chaieb K., Karray-Bouraoui N., TrabelsiN., Boulaaba M., Abdelly C. (2008): Phenolic contents,
antioxidant and antibacterial activities of a Tunisian Cynara
Cardunculus L., C. R. Biologies, 331 (5): 372-379.
Green S.J., Meltzer M.S., Hibbs Jr. J.B., Nacy C.A. (1990):
Activated macrophages destroy intracellular Leishmania
major amastigotes by an l-arginine-dependent killingmechanism. J. Immunol., 144: 278-283.
Jaleel A.C., Ksouri R., Gopi R., Manivannan P., Jallali I.,
Chang-Xing Z. (2009): Antioxidant defense responses:
Physiological plasticity in higher plants under abiotic
constraints. Acta Physiol. Plant, 31 (3): 427-436.
Karray-Bouraoui N., Ksouri R., Falleh H., Rabhi M., Grignon
C., Lachaâl M. (2010): Changes in the antioxidant capacities
of Tunisian Mentha pulegium L. at different developmental
stages as affected by environmental conditions. J. Food
Bioch., 34: 79-89.
Ksouri R., Falleh H., Megdiche W., Trabelsi N., Mhamdi B.,
Chaieb K., Bakrouf A., Magné C., Abdelly C. (2009):
Antioxidant and antimicrobial activities of the edible
medicinal halophyte Tamarix gallica L. and related
polyphenolic constituents. Food Chem Toxicol., 47 (8):2083-2091.
Ksouri R., Megdiche W., Koyro H. W., Abdelly C. (2010):
Responses of halophytes to environmental stresses withspecial emphasis to salinity. Adv. Bot. Res., 53:117-145.
Macheix J.J., Fleuriet A., Jay-Allemand C. (2005): Lescomposés phénoliques des végétaux, un exemple demétabolites secondaires d’importance économique, édition.
Lausanne: Presses Polytechniques et Universitaires
Romandes.
Rodriguez Vaquero M.J., Alberto M.R., Manca de Nadra M.C.
(2007): Antibacterial effect of phenolic compounds fromdifferent wines. Food Control , 18: 93-101.
Trabelsi N., Megdiche W., Ksouri R., Falleh H., Oueslati S.,
Bourgou S., Hajlaoui H., Abdelly C. (2010) : Solvent effects
on phenolic contents and biological activities of the
halophyte Limoniastrum monopetalum leaves. LWT, 43:
632-639.
Tsai M.C., Song T.Y. Shih P.H., Yen G.C. (2007): Antioxidant
properties of watersoluble polysaccharides from Antrodia
cinnamomea in submerged culture. Food Chem., 104,1115-1122.