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Journal of Taibah University for Science 8 (2014) 216–224

Total phenolic, ﬂavonoid and tannin contents and antioxidant and

antimicrobial activities of organic extracts of shoots of the plant

Limonium delicatulum

∗

Faten Medini , Hanen Fellah, Riadh Ksouri, Chedly Abdelly

Extremophile Plant Laboratory, BP 9001, Hammam-Lif 2050, Tunisia

Available online 24 January 2014

Abstract

The aim of this study was to assess the antioxidant and antimicrobial activities in vitro of 10 extracts of the halophyte Limonium

delicatulum harvested in two physiological stages, ﬂowering and vegetative, and to determine their phenolic compounds by reverse-

phase high-performance liquid chromatography. The solvent and the physiological development stage signiﬁcantly affected the

quantity of phenols and the biological activity; the best antioxidant and antimicrobial activities and the most phenolic compounds

were found at the ﬂowering stage. Ethanol extracts had the most total antioxidant activity, while the acetone extracts had the greatest

radical scavenging capacity (IC50 = 2 ␮g/mL) and the methanol extract the best inhibition of ␤-carotene bleaching. The ethanol

extract showed the highest total antioxidant activity (177 mg gallic acid equivalents/g dry weight) and antibacterial activity, mainly

against Salmonella (16 mm inhibition diameter). The main phenolic compounds were p-coumaric acid and chlorogenic acid.

Keywords: Limonium delicatulum; Development stage; Extracting solvents; Antioxidant activity; Antibacterial activity

1. Introduction such as salinity, drought, high and low temperatures,

high luminosity and other harsh environmental con-

Saline soil accounts for 9.5 billion hectares of the ditions, which induce oxidative stress in plants, thus

world’s soil [1]. Salty areas are the habitats of a num- generating reactive oxygen species. Higher plants often

ber of shrubby plants and trees, including halophytes, produce such species even under normal conditions dur-

which are naturally salt-tolerant plants that might be ing metabolic processes via the Mehler reaction, in

useful economically to absorb salt from the earth and chloroplasts, electron transport in mitochondria and pho-

serve as new sources of natural antioxidants in food [2]. torespiration in peroxisomes [3]. At low levels, reactive

These habitats are exposed to various abiotic constraints, oxygen species may function as signalling molecules;

however, overexpression of these reactive compounds

can cause protein denaturation, DNA mutation and lipid

∗

Corresponding author. Tel.: +216 22 88 55 79;

peroxidation [4]. To cope with harmful compounds,

fax: +216 79 325 638.

halophytes are equipped with a powerful antioxidant sys-

E-mail address: [email protected] (F. Medini).

tem that has enzymatic and non-enzymatic components

Peer review under responsibility of Taibah University

[5].

Cellular structures are believed to be protected from

oxidative effects under stressful conditions by enhanced

synthesis of secondary metabolites. These include some

F. Medini et al. / Journal of Taibah University for Science 8 (2014) 216–224 217

for normal growth development and defence against L. delicatulum with four complementary test systems:

infection and injury. Structurally, phenols comprise an 2,2-diphenylpicrylhydrazyl (DPPH), total antioxidant

aromatic ring bearing one or more hydroxyl substituents; activity and ferric reducing and ␤-carotene–linoleic acid

they range from simple molecules to highly polyme- tests [17–20].

rized compounds [7]. As antioxidants terminate reactive

oxygen species radical-mediated oxidative reactions 2. Materials and methods

directly, they might be used to prevent ageing-associated

diseases and other health problems, and the search for 2.1. Preparation of plant extracts

antioxidant principles, natural sources and active antiox-

idant molecules has accelerated. Antioxidants have been L. delicatulum was collected from salt ﬂats in Kairoun

detected in many sources, such as agricultural and horti- (semi-arid climate) in August 2012 (ﬂowering stage)

cultural crops and medicinal plants [8]. The antioxidant and in December 2012 (vegetative stage). The harvested

activity of compounds is due mainly to their redox plants were identiﬁed at the Biotechnology Centre at

properties, which allow them to act as reducing agents the Technopark of Borj-Cedria, and a voucher speci-

or hydrogen atom donors. Thus, natural antioxidants men was deposited at the Herbarium of the Laboratory

function as free-radical scavengers and chain breakers, of Extremophile Plants at the Centre. Shoots were air-

◦

complexes of pro-oxidant metal ions and quenchers of dried in an oven at 60 C for 72 h, and plant extracts

singlet-oxygen formation [9]. were obtained by magnetic stirring of 2.5 g of powdered

Previous studies have shown that the amount of dry matter with 25 mL of solvent for 30 min at room

◦

polyphenols in plants and their antioxidant activities temperature (25 C). Extracts were obtained with sol-

depend on both biological factors (genotype, organ and vents of increasing polarity: hexane, acetone/water (8:2,

ontogeny) and edaphic and environmental (tempera- v/v) ethanol/water (9:1, v/v), methanol/water (8:2, v/v)

◦

ture, salinity, water stress and light intensity) conditions and water. The extracts were kept for 24 h at 4 C, ﬁl-

[11]. The solubility of phenolic compounds is governed tered through Whatman No. 4 ﬁlter paper, evaporated

◦

by the type of solvent (polarity) used, the degree of to dryness under vacuum and stored at 4 C until analy-

polymerization of phenols and their interaction [12]. sis.

For instance, absolute methanol was found to be more

effective than water for extracting polyphenols from 2.2. Total phenol content

agricultural wastes [13] and 50% acetone for extracting

total wheat phenols [14]. Total phenols were assayed according to Dewanto

In Tunisia, there is a considerable diversity of halo- et al. [16]. An aliquot of diluted extract was

phytic species, some of which are used therapeutically, added to 0.5 mL of distilled water and 0.125 mL of

such as Limonium spp. (Plumbaginacea family), a genus Folin–Ciocalteu reagent. The mixture was shaken and

of 180 species, which is used in folk medicine [15]. allowed to stand for 6 min, before addition of 1.25 mL

Limonium delicatulum is a rosette plant found in coastal of 7% Na2CO3. The solution was then adjusted with

regions and salt flats. It flowers between April and distilled water to a final volume of 3 mL and mixed

October. This plant can tolerate a wide range of harsh thoroughly. After incubation in the dark, absorbance

environmental conditions and resists abiotic stress such at 760 nm was read versus a prepared blank. The total

as salt, high temperature and water deﬁcits. A litera- phenol content of plant parts was expressed as mil-

ture search revealed no information about the use of this ligrams of gallic acid equivalents per gram of dry

plant as a source of phenolic compounds and biologi- weight (mg GAE/g DW) from a calibration curve with

cal activity. The objectives of this study were therefore gallic acid. All samples were analyzed in three repli-

(i) to investigate its antioxidant activity in different cates.

solvent extracts and physiological stages, (ii) to esti-

mate its antimicrobial activity against human pathogens 2.3. Total ﬂavonoid content

found in food and (iii) to quantify and identify the main

phenolic compounds present in this plant. The total Total ﬂavonoids were measured by a colorimetric

antioxidant activity of vegetables cannot be evaluated assay according to Dewanto et al. [16]. An aliquot of

by a single method, because of the complex nature of diluted sample or standard solution of (+)-catechin was

phytochemicals, and two or more methods are always added to 75 mL of NaNO2 solution (5%) and mixed

used to evaluate their total antioxidative effects [10]. for 6 min before addition of 0.15 mL AlCl3 (10%).

We therefore evaluated the antioxidant capacities of After 5 min, 0.5 mL of NaOH were added. The ﬁnal

218 F. Medini et al. / Journal of Taibah University for Science 8 (2014) 216–224

volume was adjusted to 2.5 mL with distilled water expressed as mg gallic acid equivalent per gram dry

and mixed thoroughly. Absorbance was determined at weight (mg GAE/g DW). The calibration curve range

␮

510 nm against a blank. The total ﬂavonoid content is was 0–500 g/mL. All samples were analyzed in trip-

expressed as milligrams of catechin per gram of dry licate.

weight (mg CE/g DW) against the calibration curve of

(+)-catechin, from 0 to 400 mg/mL. All samples were 2.7. Iron reducing power

analyzed in triplicate.

Another reaction pathway in electron donation is

2.4. Total condensed tannins reduction of an oxidized antioxidant molecule to regen-

erate the “active” reduced antioxidant. The reducing

Condensed tannins (proanthocyanidins) were deter- power of the extracts was determined according to the

mined according to the method of Sun et al. [17]. To method of Oyaizu [20]. Various concentrations of L. del-

␮ ␮

50 ␮L of diluted sample, 3 mL of 4% vanillin solution in icatulum extracts (200 L) were mixed with 500 L of

methanol and 1.5 mL of concentrated HCl were added. 200 mmol/L Na3PO4 buffer (pH 6.6) and 2.5 mL of 1%

The mixture was allowed to stand for 15 min, and absorp- potassium ferricyanide. The mixture was incubated at

◦

tion was measured at 500 nm against methanol as a blank. 50 C for 20 min; then, 2.5 mL of 10% trichloroacetic

The amount of total condensed tannins is expressed as acid (w/v) were added, and the mixture was centrifuged

␮

mg (+)-catechin/g DW. All samples were analyzed in at 650 rpm for 10 min. The upper layer (500 L) was

triplicate. mixed with 500 ␮L deionized water and 100 ␮L of 0.1%

ferric chloride, and absorbance was measured at 700 nm:

2.5. DPPH assay higher absorbance indicates higher reducing power. The

assays were carried out in triplicate, and the results are

The electron donating ability of the obtained extracts expressed as means standard deviations. The extract

was measured by bleaching a purple solution of 1,1- concentration that gave 0.5 absorbance (IC50) was cal-

diphenyl-2-picrylhydrazyl (DPPH) radical according to culated from a graph of absorbance at 700 nm against

the method of Hanato et al. [18]. Extracts (0.1 mL, 5, 10, extract concentration. Ascorbic acid was used as the

50 and 100 ␮g/mL) were added to 0.5 mL of 0.2 mmol/L standard.

DPPH–methanol solution. After incubation for 30 min

at room temperature, the absorbance was determined 2.8. β-Carotene bleaching

against a blank at 517 nm. The percentage inhibition

␤ ␤

of free radical DPPH was calculated from (Ablank − In the -carotene linoleate system, -carotene under-

sample/Ablank) × 100, where Ablank is the absorbance goes rapid discolouration in the absence of antioxidants.

The ␤-carotene bleaching test was performed as

of the control reaction and Asample is the absorbance

described ␤

in the presence of plant extract. The concentration of by Koleva et al. [21]. A solution of 2 mg -

carotene

extract that caused 50% inhibition (IC50) was calculated was prepared in 20 mL chloroform, and 4 mL of

this

from the regression equation for the concentration of solution were pipetted into a 250-mL round-bottom

extract and percentage inhibition. Butylated hydroxy- ﬂask, with 40 mg linoleic acid and 400 mg Tween 40.

toluene was used as a positive control. Samples were Chloroform was removed under vacuum in a rotary evap-

◦

analyzed in triplicate. orator at 40 C, and then the emulsiﬁer was shaken with

100 mL of aerated distilled water. Aliquots of 1500 ␮L

of this emulsion were transferred into a series of test

2.6. Total antioxidant capacity

tubes containing 100 ␮L of the extracts or methanol

◦

The assay is based on the reduction of Mo(VI) to (control), then incubated at 50 C for 120 min. Butylated

Mo(V) by the extract and subsequent formation of hydroxytoluene was used as the reference antioxidant.

a green phosphate–Mo(V) complex at acid pH [19]. Absorbance was measured at 470 nm immediately after

An aliquot (0.1 mL) of plant extract was added to addition of the emulsion to each tube and then at 120 min.

1 mL of reagent solution (0.6 mol/L H2SO4, 28 mmol/L The capacity of the extracts to protect against oxida-

␤

× A −

Na3PO4 and 4 mmol/L ammonium molybdate). The tion of -carotene was determined from 100 ( 0

◦

A /A − A A

tubes were incubated in a thermal block at 95 C for 1 1 2), where 0 is the absorbance of the sample

90 min. Once the mixture had cooled to room temper- at 120 min, 1 is the absorbance of the control at 0 min,

ature, the absorbance of each solution was measured and 2 is the absorbance of the control at 120 min. The

␮

at 695 nm against a blank. Antioxidant capacity was results are expressed as IC50 ( g/mL).

F. Medini et al. / Journal of Taibah University for Science 8 (2014) 216–224 219

2.9. Antimicrobial activity by comparing their retention times with those of the standards.

2.9.1. Microorganisms

We screened antimicrobial activity against seven 2.11. Statistical analysis

human pathogenic Gram-positive (Staphylococcus

aureus ATCC 25923, Micrococcus luteus NCIMB Data were analyzed with statistical software. Analy-

8166, Enterococcus faecalis ATCC 29212 and Lis- sis of variance (ANOVA) and Duncan’s multiple range

teria monocytogenes ATCC 19115) and Gram-negative method were used to compare any signiﬁcant differences

bacteria (Escherichia coli ATCC 35218, Pseudomonas between solvents and samples. Values were expressed as

aeruginosa ATCC 27853 and Salmonella typhi LT2). means ± standard deviations. Differences were consid-

ered signiﬁcant at p < 0.05.

2.9.2. Preparation of test discs

The extracts were dissolved in dimethylsulfoxide to 3. Results

ﬁnal concentrations of 50 and 12.5 mg/mL. Sterile discs

␮

(6 mm in diameter) were impregnated with 20 L of the 3.1. Total phenolic, ﬂavonoid and tannin contents

extracts (50 mg/mL and 25 mg/mL, respectively). Neg-

ative controls were prepared on discs impregnated with The total phenol contents of the extracts are shown

dimethylsulfoxide (solvent control). Gentamicin (10 UI) in Table 1. The total polyphenol content of L. deli-

was used as the positive reference for all bacterial strains. catulum shoots was higher in plants at the ﬂowering

than at the vegetative stage, ranging from 0.19 to

2.9.3. Disc diffusion assay 92.9 mg GAE/g DW during ﬂowering and from 0.13 to

Inocula of bacterial strains were prepared from 18-h 44.13 mg GAE/g DW at the vegetative stage. In both

cultures, and suspensions were adjusted to 0.5 at 570 nm stages, the maximum content was recorded in acetone

with a spectrophotometer. Petri dishes were prepared extracts and the minimum in hexane extracts; a simi-

with 20 mL of Mueller Hinton agar; the inocula were lar tendency was observed for ﬂavonoid and condensed

spread on top of the solidiﬁed medium and allowed to tannin contents, 80% acetone being the best solvent

dry for 60 min. The discs with extract were then applied, for extracting ﬂavonoids at the ﬂowering stage, while

and the plates were left for 30 min at room temperature to methanol extracts contained more condensed tannins.

allow diffusion of the extract, before incubation for 24 h

◦

at 37 C. The diameter of the inhibition halo was evalu- 3.2. DPPH radical-scavenging activity

ated in millimetres. Each assay was repeated in triplicate

[22]. Extracts of L. delicatulum collected in winter

(December) were signiﬁcantly less potent against DPPH

2.10. Analysis of phenols by reverse-phase synthetic radical than those collected in summer

high-performance liquid chromatography (August) (Table 2). Hexane extracts had the lowest anti-

radical activity, while the most polar solvent (water) had

High-performance liquid chromatography (HPLC) moderate activity and 80% acetone or methanol and 95%

was performed on an AGILANT apparatus equipped ethanol were the most potent. Thus, phenolic compounds

with an autosampler model 1100, a Prostar Pump from acetone, ethanol and methanol extracts of L. deli-

model 1100, a Prostar diode array detector model 1100 catulum were more efﬁcient antioxidants than butylated

and an RPC18 column (Prontosil, 250 mm × 4.0 mm, hydroxytoluene.

5 ␮m, Bischoff). The mobile phase was composed

of two solvents: 0.025% triﬂuoroacetic acid in H2O 3.3. Total antioxidant activity

(A) and acetonitrile (B). The sample was dissolved

in methanol/water (1/1, v/v) and ﬁltered with What- The total antioxidant capacity of L. delicatulum was

␮

man paper and through a 0.45- m Millipore ﬁlter. The much higher in plants collected at the ﬂowering than

total run time was 60 min. The elution programme at the vegetative stage (Table 3) and varied according

at 1 mL/min was: 90 A/10 B (0–40 min), 50 A/50 to the solvent. For example, an ethanol extract of plants

B (40–41 min), 100% B (41–50 min) and 90 A/10 B collected in the summer had the highest total antioxidant

(50–59 min). Each sample was injected directly, and activity, followed by the methanol extract, the acetone

chromatograms were monitored at 280 nm. The sample extract, the hexane extract and the extract in water. For

␮

injection volume was 20 L. Compounds were identiﬁed plants collected in winter, the methanol extract had the

220 F. Medini et al. / Journal of Taibah University for Science 8 (2014) 216–224

Table 1

Contents of total polyphenol (expressed as mg gallic acid equivalents/g dry weight), ﬂavonoids and condensed tannin (expressed as mg catechin/g

dry weight) in Limonium delicatulum shoot extracts.

Solvent Polyphenols Tannins Flavonoids

Flowering Vegetative Flowering Vegetative Flowering Vegetative

± ± ± ±

Hexane 0.19 0.03g 0.13 0.05g 2.35 0.47g 0.2 0.13g 0.07 0.01h 0.83 ± 0.31g

± ±

Acetone 92.9 1.45a 44.13 3.43c 38.35 ± 5.02b 8.93 ± 2.80e 15.09 ± 1.03a 6.19 ± 0.49c

Methanol 46.5 ± 1.81b 35.88 ± 1.31d 48.38 ± 0.75a 17.6 ± 7.67d 5.55 ± 2.95c 9.23 ± 0.60b

Ethanol 29.58 ± 7.21e 14.25 ± 5.62f 23.2 ± 4.47c 4.87 ± 1.69f 2.62 ± 0.57de 3.14 ± 0.14d

Water 12.3 ± 0.52f 11.83 ± 0.08f 8.14 ± 0.44e 0.58 ± 0.30g 2.15 ± 0.16ef 1.47 ± 0.22fg

Values (means of three replicates) followed by different letters are signiﬁcantly different at p < 0.05.

Table 2

Scavenging activity, expressed as median inhibitory concentration (␮g/mL), in the DPPH test with Limonium delicatulum extracts and butylated

hydroxytoluene.

Stage Hexane Acetone Methanol Ethanol Water Butylated hydroxytoluene

± ±

Flowering >1000a 2 0.68e 5.25 0.62e 4.3 ± 0.14e 29 ± 5.73c 11.5 ± 0.2cd

Vegetative >1000a 9.3 2.52cd 5.5 0.94e 19.2 ± 4.18cd 410 ± 37.93b

Values (means of three replicates) followed by different letters are signiﬁcantly different at p < 0.05.

most activity, followed by ethanol and acetone and then tive stage showed the best capacity to inhibit ␤-carotene

water and hexane extracts. bleaching (Table 4).

3.4. Iron reducing power

3.6. Antimicrobial activity

As shown in Table 4, the potential of Limonium to

reduce the ferrous ion also depended on the physiological As the initial results showed that L. delicatulum at the

stage and solvent. Plants in the ﬂowering stage produced ﬂowering stage had a higher phenol content and greater

more Fe than plants collected in winter. The greatest antioxidant activity than plants in the vegetative stage,

activity was observed with an ethanol extract. the antimicrobial activity of this plant was evaluated only

at ﬂowering. Table 5 shows the antibacterial activity of

3.5. Antioxidant assay in the -carotene linoleate extracts of L. delicatulum shoots. Hexane and aqueous

system extracts had no antimicrobial activity, methanol and ace-

tone extracts had moderate activity against S. aureus,

␤

Addition of Limonium extracts to the -carotene P. aeruginosa, L. monocytogenes and M. luteus, while

linoleate system prevented bleaching. Methanol extracts ethanol extracts had good inhibitory activity against

of the ﬂowering stage and acetone extracts of the vegeta- Salmonella, E. coli, S. aureus, and L. monocytogenes and

moderate activity against Enterococcus faecium, Pseu-

Table 3

domonas and M. luteus. The antibacterial potential was

Total antioxidant capacity (expressed as mg gallic acid equivalents/g

dose-dependent against Salmonella, Pseudomonas and

dry weight) in Limonium delicatulum shoots at ﬂowering and vegeta-

tive stages. Micrococcus.

Solvent Flowering stage Vegetative stage

Hexane 71.96c 10.41b 3.7. Phenols identiﬁed in L. delicatulum

Acetone 92.9b 43.35b

Methanol 98.07b 46.24b

Six phenolic compounds, mostly phenolic acids and

Ethanol 177.41a 44.56b

one ﬂavonoid (Fig. 1), were identiﬁed by reverse-phase

Water 35.26d 10.31a

HPLC in L. delicatulum shoot extracts: p-coumaric acid,

Values (means of three replicates) followed by different letters are

chlorogenic acid, 1,2-p-hydroxybenzoic acid, 4,3,5-

signiﬁcantly different at p < 0.05.

dimethoxyhydrobenzoic acid, gallic acid and rutin.

F. Medini et al. / Journal of Taibah University for Science 8 (2014) 216–224 221

Table 4

Reducing power and ␤-carotene bleaching activity of aerial parts of Limonium delicatulum.

Solvent Reducing power ␤-Carotene bleaching

Flowering Vegetative Flowering Vegetative

Hexane >1000a >1000a >1000a >1000a

± ± ±

Acetone 105 4.30f 330 21.50c 530 24.92d 380 ± 37.98e

Methanol 130 ± 2.87e 200 ± 13.13d 290 ± 24.82f 690 ± 28.26c

Ethanol 99 ± 6.25f 880 ± 49.64b 620 ± 79.79cd 760 ± 100.31b

Water >1000a >1000a >1000a >1000a

Ascorbic acid 40.5 ± 0.5g

Butylated hydroxytoluene 75 ± 0.2g

Values (means of three replicates) followed by different letters are signiﬁcantly different at p < 0.05 according to the Newman–Keuls post hoc test.

Table 5

Antibacterial activity of Limonium delicatulum extracts against human pathogenic bacteria.

Solvent Concentration (␮g/disc) E. coli E. faecum S. typhi S. aureus P. aerigunosa L. mono M. luteus

Hexane 1000 – – – – – – –

500 – – – – – –

Acetone 1000 – 10 8 9bc 9b

500 – – – 11 8 8c 9b

Methanol 1000 – – – 8 – 10b 9b

500 – – – 8 – 9bc 12b

Ethanol 1000 14b 12b 15b 14b 12b 14a 12b

500 14b 12b 11b 14b 13b 14a –

Water 1000 – – – – – – –

500 – – – – – – –

Gentamicin 10 UI 25a 19a 22a 20a 16a 0 26a

–, no antimicrobial activity; E. coli, Escherichia coli; E. faecium, Enterococcus faecium; S. typhi, Salmonella typhi; S. aureus, Staphylococcus

aureus; L. mono, Listeria monocytogenes; M. luteus, Micrococcus luteus. The inhibition zone of the control, gentamycin (10 UI), was >15 mm for

all bacteria; the diameter of disc was 6 mm. Each experiment was done in triplicate.

Fig. 1. Reverse-phase high-performance liquid chromatography proﬁles of L. delicatulum extract monitored at 280 nm. The peak numbers correspond

to: 1: 1,2-p-hydroxybenzoic acid; 4: 3,5-dimethoxy-4-hydroxybenzoic acid; 9: chlorogenic acid; 10: gallic acid; 14: p-coumaric acid; 18: rutin. NI,

not identiﬁed.

222 F. Medini et al. / Journal of Taibah University for Science 8 (2014) 216–224

p-Coumaric acid was the major compound, followed by radicals, by inhibiting lipid peroxidation or chelating

chlorogenic acid. metal. In our study, the different extracts of L. delicatu-

lum showed high total antioxidant and DPPH activity and

␤

4. Discussion moderate ferrous iron reducing and -carotene bleaching

inhibition capacities. Antioxidant capacity may be asso-

We have determined the phenolic composition and ciated with a high phenol content, as Mansouri et al. [29]

antioxidant and antibacterial activities of L. delicatu- reported that most of the antioxidant activity of plants is

lum, a halophyte found in a semi-arid region of Tunisia. derived from phenols. Structurally, phenols comprise an

The phenol content depended on the solvent used and its aromatic ring bearing one or more hydroxyl substituents.

polarity. Acetone extracts gave the highest yield of total The antioxidant activity of this type of molecule is due to

polyphenols, while methanol gave the highest yield of their ability to scavenge free radicals, donate hydrogen

tannins. The highest yields of ﬂavonoids were obtained atoms or electrons or chelate metal cations [30].

with acetone from plants at the ﬂowering stage and with In our study, hexane and aqueous extracts had

methanol from plants in the vegetative stage. In previ- no antimicrobial activity, while methanol and acetone

ous studies, methanol, ethanol, acetone, propanol, ethyl extracts had moderate activity against some organisms.

acetate and dimethylformamide have been used to extract Ethanol extracts were found to be the most effective,

phenols from fresh produce in water [23]. The recovery with a broad antimicrobial spectrum against both Gram-

of polyphenols from plant materials is inﬂuenced by their positive and Gram-negative bacteria, the most suscepti-

solubility in the extraction solvent, the type of solvent, ble bacteria being Salmonella and Pseudomonas. This

the degree of polymerization of phenols, the interaction ﬁnding is important, because these bacteria are resis-

of phenols with other plant constituents and the for- tant to a number of antibiotics and produce toxins that

mation of insoluble complexes [24]. Differences in the cause many types of enteritis and septicaemia. Phenols

polarity (and thus the extractability) of antioxidants may have previously been reported to have a wide spectrum

explain differences in extraction yield and antioxidant of biological activity, including anti-thrombotic, cardio-

activity. Furthermore, solvent polarity plays a key role protective, vasodilator and antimicrobial activities [31].

in increasing phenolic solubility [25]. It is therefore dif- L. delicatulum at the ﬂowering stage had a greater

ﬁcult to deﬁne a standard procedure for the extraction of phenol content and biological capacity than in the veg-

plant phenols. The least polar solvents are generally con- etative stage and might be considered a potential source

sidered to be suitable for extracting lipophilic phenols, of antioxidants. Reverse-phase HPLC analysis identi-

unless very high pressure is used, and polar solvents are ﬁed many phenolic acids, with p-coumaric acid as the

used for hydrophilic phenols [26]. major type. p-Coumaric acid has known applications in

Physiological stage can also affect the composition the food, health, cosmetic and pharmaceutical industries.

and content of polyphenols and biological activity. We The other phenols identiﬁed also have important protec-

found that Limonium at the ﬂowering stage had a higher tive effects. Bouayed et al. [32] found that chlorogenic

level of phenol compounds than the vegetative stage. acid has anxiolytic and antioxidant activity. In addition,

Ichiho et al. [27] reported that the antioxidant capac- gallic acid and rutin have been reported to be protective

ity and phenol content of six crops cultivated in Japan against the deleterious effects of H2O2 [33].

was strongly affected by the growing season, and Bano

et al. [11] reported that the distribution of secondary 5. Conclusion

metabolites may change during plant development, per-

L. delicatulum

haps related to the harsh climatic conditions of the Extracts of in acetone, methanol and

ethanol

plant’s usual habitat (hot temperature, high solar expo- were more effective than those in hexane or

water.

sure, drought, salinity), which stimulate the biosynthesis In addition, the plant at the ﬂowering stage had

of secondary metabolites such as polyphenols. greater activity than at the vegetative stage. -Coumaric

The phenol content of a plant depends on a num- acid and chlorogenic acid, identiﬁed as the dominant

ber of intrinsic (genetic, extracting solvent) and extrinsic phenol compounds in the extracts, may contribute to the

(environmental, handling and development stage) factors high antioxidant activities of this species. These results

[28]. The plants contain a wide variety of antioxidants, indicate that selective extraction of bioactive molecules

and it is difﬁcult to measure the antioxidant capacity from natural sources such as halophyte species, with

of each compound separately. Most methods developed appropriate solvents, can provide fractions with high bio-

logical

to estimate the antioxidant capacity of different plant activity that could be used as preservatives in food

materials [10] measure the ability to scavenge speciﬁc or pharmaceuticals.

F. Medini et al. / Journal of Taibah University for Science 8 (2014) 216–224 223

Acknowledgements horseradish roots (Armoracia rusticana), World Acad. Sci. Eng.

Technol. 64 (2012) 903–908.

[14] N. Melissa, M. Maria Valentina, L.C. Francesco, D.M. Giovanni,

This work was supported by the Tunisian Min-

P.A. Rita, V. Maria Grazia, Extraction and characterization of

istry of Higher Education and Scientiﬁc Research

biomolecules from agricultural wastes, Chem. Eng. Technol. 12

(LR10CBBC02), by the Tunisian-French “Comité Mixte (2012) 1164–1169.

de Coopération Universitaire” (CMCU) network # [15] R. Ksouri, W.M. Ksouri, I. Jallali, A. Debez, C. Magné, I.

Hiroko, C. Abdelly, Medicinal halophytes: potent source of health

08G0917 and by the SATREPES project “Valorization of

promoting biomolecules with medical, nutraceutical and food

Bio-resources in Semi-arid and Arid Land for Regional

applications, Crit. Rev. Biotechnol. 32 (2011) 289–326.

Development.

[16] X. Dewanto, K. Wu, K. Adom, R.H. Liu, Thermal processing

enhances the nutritional value of tomatoes by increasing

total antioxidant activity, J. Agric. Food Chem. 50 (2002)

References 3010–3014.

[17] B. Sun, J.M. Richardo-Da-Silvia, I. Spranger, Critical factors of

vanillin assay for catechins and proanthocyanidins, J. Agric. Food

[1] A. Jones, P. Panagos, S. Barcelo, F. Bouraoui, C. Bosco, O.

Chem. 46 (1998) 4267–4274.

Dewitte, C. Gardi, M. Erhard, J. Hervas, R. Heiderer, S. Jef-

[18] T. Hatano, H. Kagawa, T. Yasuhara, T. Okuda, Two new ﬂavonoids

fery, A. Lukewille, L. Marmo, L. Montanarella, C. Olazabal, J.E.

and other constituents in licorice root their relative astringency

Petersen, V. Penizek, T. Strassburger, G. Toth, M. Van Den Eeck-

and radical scavenging effect, Chem. Pharm. Bull. 36 (1988)

aut, M. Van Liedekerke, F. Verheigen, E. Viestova, Y. Yigini,

2090–2097.

The State of Soil in Europe, Copenhagen, European Environ-

[19] P. Prieto, M. Pineda, M. Aguilar, Spectrophotometric quantitation

ment Agency, 2012 http://ec.europa.eu/dgs/jrc/downloads/jrc

of antioxidant capacity through the formation of a phospho-

reference report 2012 02 soil.pdf

molybdenum complex: speciﬁc application to the determination

[2] L. Meot-Duros, G. Le Floch, C. Magne, Radical scavenging,

of vitamin E, Anal. Biochem. 269 (1999) 337–341.

antioxidant and antimicrobial activities of halophytic species, J.

[20] M. Oyaizu, Studies on products of the Browning reaction prepared

Ethnopharmacol. 116 (2008) 258–262.

from glucose amine, Jpn. J. Nutr. 44 (1986) 307–315.

[3] S. Neill, R. Desikan, J. Hancock, Hydrogen peroxide signaling,

[21] I.I. Koleva, T.A. van Beek, J.P.H. Linssen, A. de Groot, L.N.

Curr. Opin. Plant Biol. 5 (2002) 388–395.

Evstatieva, Screening of plant extracts for antioxidant activity: a

[4] G.J.M. Beckers, S.H. Spoel, Fine-tuning plant defence signaling:

comparative study on three testing methods, Phytochem. Anal.

salicylate versus jasmonate, Plant Biol. 8 (2006) 1–10.

13 (2002) 8–17.

[5] R. Ksouri, W. Megdiche, H. Falleh, N. Trabelsi, M. Boulaaba, A.

[22] J. Freney, F. Renaud, W. Hansen, C. Bollet, Précis de Bactériolo-

Smaoui, C. Abdelly, Inﬂuence of biological, environmental and

gie Clinique, Editions ESKA, Paris, 2002, pp. 565.

technical factors on phenolic content and antioxidant activities of

[23] E.S. Tan, A. Aminah, H.M. Khalid, M.Y. Maskat, M.A. Ghani,

Tunisian halophytes, C. R. Biol. 331 (2008) 865–873.

Antioxidant properties of three banana cultivars (Musa acuminata

[6] M. Mazid, T.A. Khan, F. Mohammad, Role of secondary metabo-

‘Berangan’, ‘Mas’ and ‘Raja’) extracts, Sains Malays. 41 (2012)

lites in defense mechanisms of plants, Biol. Med. 3 (2011)

232–249. 319–324.

[24] C.J. Galvez, P. Martin-Cordero, A.M. Houghton, Antioxidant

[7] L. Bravo, Polyphenols: chemistry, dietary sources, metabolism,

activity of methanol extracts obtained from Plantago species, J.

and nutritional signiﬁcance, Nutr. Rev. 56 (1998) 317–333.

Agric. Food Chem. 53 (2005) 1927–1933.

[8] R. Ksouri, H. Falleh, W. Megdiche, N. Trabelsi, B. Mhamdi,

[25] M. Naczk, F. Shahidi, Phenolics in cereals, fruits and vegetables:

K. Chaieb, A. Bakrouf, C. Magné, C. Abdelly, Antioxidant and

occurrence, extraction and analysis, J. Pharm. Biomed. Anal. 41

antimicrobial activities of the edible medicinal halophyte Tamarix

(2006) 1523–1542.

gallica L. and related polyphenolic constituents, Food Chem.

[26] M. Alothman, A. Rajeev, A. Karim, Antioxidant capacity and

Toxicol. 47 (2009) 2083–2091.

phenolic content of selected tropical fruits from Malaysia,

[9] M. Tosun, F. Celik, S.O. Ercisli, S. Yilmaz, Bioactive contents

extracted with different solvents, Food Chem. 115 (2009)

of commercial cultivars and local genotypes of walnut (Juglans

785–788.

regia L.), in: International Conference on Environmental and

[27] M.K. Ichiho, M. Shiro, N. Keijiro, T. Yumico, Y. Minako, S.

Agriculture Engineering (IPCBEE), Vol. 15, 2011.

Tetsuo, W. Jun, H. Akihiro, Antioxidant capacity and polyphenols

[10] N. Swapana, N. Lokendrajit, S. Warjeet, C. Laitonjam, B. Singh,

contents of extracts from crops cultivated in Japan and the effect

Antioxidant activities of the rhizomes of different Zingiberaceae

of the cultivation, Environ. Food Sci. Technol. Res. 19 (2013)

plants of north-east India, Asian J. Biol. Life Sci. 2 (2013) 19–22.

69–79.

[11] M.J. Bano, J. Lorente, J. Casstillo, G.O. Benavente, J.A. Rio, A.

[28] F. Fratianni, M. Tucci, M. De-Palma, R. Pepe, F. Nazzaro,

Ortuno, K.W. Quirin, D. Gerard, Phenolic diterpenes, ﬂavones,

Polyphenolic composition in different parts of some cultivars of

and rosmarinic acid distribution during the development of leaves,

globe artichoke (Cynara cardunculus L. var. scolymus (L.) Fiori),

ﬂowers, stems and roots of Rosmarinus ofﬁcinalis antioxidant

Food Chem. 104 (2007) 1282–1286.

activity, J. Agric. Food Chem. 51 (2003) 4247–4253.

[29] A. Mansouri, G. Embared, E. Kokkalou, P. Kefalas, Phe-

[12] A. Djeridane, M. Yousﬁ, B. Nadjemi, D. Boutassouna, P. Stocker,

nolic proﬁle and antioxidant activity of the Algerian ripe

N. Vidal, Antioxidant activity of some Algerian medicinal plants

date palm fruit (Phoenix dactylifera), Food Chem. 89 (2005)

extracts containing phenolic compounds, Food Chem. 97 (2006)

654–660. 411–420.

[30] R. Amarowicz, R.B. Pegg, M. Rahimi, B. Barl, J.A. Weil, Free-

[13] T. Lolita, K. Zanda, G. Ruta, Comparison of different solvents

radical scavenging capacity and antioxidant activity of selected

and extraction methods for isolation of phenolic compounds from

224 F. Medini et al. / Journal of Taibah University for Science 8 (2014) 216–224

plant species from the Canadian prairies, Food Chem. 84 (2004) [32] J. Bouayed, H. Rammal, A. Dicko, C. Yonos, R. Soloumani,

551–562. Chlorogenic acid, a polyphenol from Prunus domestica

[31] A. García-Lafuente, E. Guillamón, A. Villares, M.A. Rostagno, (Mirabelle), with coupled anxiolytic and antioxidant effects, J,

J.A. Martínez, Flavonoids as anti-inﬂammatory agents: impli- Neurol. Sci. 262 (2007) 77–84.

cations in cancer and cardiovascular disease, Inﬂamm. Res. 58 [33] E. Cemeli, B. Adolf, A. Diana, Antioxidant and the comet assay,

(2009) 537–552. Mutat. Res. 681 (2008) 51–67.