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PHYTOCHEMICAL SCREENING AND ASSESSMENT OF ANTIOXIDANT,

ANTIBACTERIAL AND CYTOTOXICITY ACTIVITIES OF FIVE TUNISIAN MEDICINAL

PLANTS

EL ABED N 1, GUESMI F2, MEJRI M3, MARZOUKI MN1, BEN HADJ AHMED S1

1. Laboratory of Protein and Bioactive Molecules Engineering (LIP-MB), National Institute of Applied Sciences and

Technology (INSAT), Centre Urbain Nord, University of Carthage, 1080, Tunis cedex, Tunisia

2. Department of Biochemistry, Faculty of Sciences Bizerte.

3. Higher Institute of Biotechnology of Béja, Béja, Tunisia.

Accepted Date: 07/08/2014; Published Date: 27/08/2014

Abstract: The present study was carried out to investigate the phytochemical compositions, antioxidant and antibacterial activities, and the cytotoxicity of aqueous and methanolic crude extracts of five wild medicinal plants from the south of Tunisia. The phytochemical screening was quantified by calorimetric techniques in terms of the total contents of phenolic compounds and total condensed tannin content. The results showed that the methanolic extracts of Thymelaea hirsuta L (44. 23 ± 1.96 mg GAE/g DW) possessed the highest total phenolic content and the total condensed tannins. The antioxidant activity was assessed in vitro by using both the scavenging activity of DPPH radical and phosphomolybdenum total antioxidant assay. The findings showed that the methanolic extract of Thymelaea hirsuta L, and the aqueous extract of Artemesia campestris L showed the strongest radical scavenging activity by the DPPH assay (IC50 values = 1.74 ± 0.12 µg/mL and 0.77 ± 0.18 µg/mL, respectively) compared to the standard, ascorbic acid and BHT. The height antioxidant capacity by the formation of phosphomolybdenum complex was obtained for the methanolic extracts of T. hirsute and A. campestris (57.54± 0.46 mg AAE /g and 55.75± 3.76 mg AAE /g). Furthermore, the extracts were evaluated for its antibacterial activity using the disc agar diffusion. The results have demonstrated that the methanolic extracts of Thymelaea hirsuta L and Artemesia campestris L were most effective against the selected bacteria. The cytotoxicity was analyzed by examining haemolytic activity against chicken erythrocytes, the results clarified that only the methanolic extract of Cleome arabica L was able to generate haemolytic activity with 30.45 ± 0.63 %.

Keywords: Plant extracts, phenolic compounds, Antioxidant activity, Antibacterial activity, Cytotoxicity,

haemolytic activity

INTERNATIONAL JOURNAL OF

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Corresponding Author: MS. NARIMAN EL ABED

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El Abed N, Guesmi F, Mejri M, Marzouki MN, Ben Hadj Ahmed S;

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INTRODUCTION

Nowadays, in spite of the great improvements in food production techniques, preservation of

food products from degradation and poisoning, caused by spoilage microorganisms or by

oxidation processes, remains an important issue, during production and storage in the food

industry (1, 2) . In order to prolong the storage stability of foods and protect human beings

against oxidative damage, several synthetic antioxidants, such as butylated hydroxyanisole

(BHA), butylated hydroxytoluene (BHT), tert-butylhydroquinone (TBHQ) and propylgallate (PG)

have been extensively used as food preservatives for industrial processing. Nevertheless,

restrictions on the use of these compounds are being imposed because of their possible

injurious properties to human health (3) . Indeed, the metabolites produced by additives or

preservatives in foods may themselves trigger hormonal or chemical (s) processes inside the

body that can generate negatives physiological responses (4). Consequently, some of the

commercial antioxidants are known to be carcinogenic or toxic (5). Furthermore, the recent

emergence of food-borne pathogens such as the enterohemorrhagic Escherichia coli O157:H7,

Listeria monocytogenes, and Enterococcus faecium have made food safety a high priority (6).

In addition, the uncontrolled use and the overuse of conventional antibiotics and synthetic

antimicrobial drugs are the main factor responsible for the emergence of the phenomenon of

antibiotic resistant microbes (ARM), such as methicillin-resistant Staphylococcus aureus

(MRSA), and multidrug-resistant strains of Pseudomonas aeruginosa and Klebsiella pneumonia

(7).

Therefore, there is a growing interest in discovering natural antioxidant and antimicrobial

compounds that might be used as alternatives to the chemical preservatives used in food

industry, and to antibiotics, thus decreasing their use, and reducing the probability of the

occurrence of human resistance to these chemicals (8, 9). Accordingly, plant extracts and their

derived secondary metabolites offer the opportunity in this regard (10) and they have been

known to possess very interesting biological properties as anti-inflammatory, anticancer, and

anti-allergic activities and some of them are classified as Generally Recognized as Safe

Substances (GRAS) (3). Nevertheless, several plants have been used for food and medicinal

purposes for thousands of years in unmodified form, and then employed as concentrated

extract to improve their intensity and uniformity of action. Subsequently, pure chemical

compounds responsible for the therapeutic effects were isolated and served as prototypes for

the development of synthetic chemical entities that possessed even greater activity.

Therefore, some plants revealed a very strong and even toxic activity in humans, which

especially refers to their extracts or their pure compounds. For this reason, it seems very

important to conduct screening tests to assess the toxic and beneficial effects of plant materials (11). In fact, the haemolytic activity represents a useful starting point in this regard; it provides

the primary information about the interaction between molecules and biological entities at

cellular levels. Moreover, study of Haemolytic activity of any compounds could give us an idea

of cytotoxicity profile (12) .

The purposes of the current work are to quantify phenolic compounds of methanolic and

aqueous extracts obtained from five species and to evaluate their antioxidant capacity as well

as their antibacterial activity and cytotoxicity.

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2. Material and methods

2.1. Chemicals

The reagents used in this study, such as Folin-Ciocalteu’s phenol reagent, Sodium carbonate

(Na2CO3) carbonate were from Merck Chemical Supplies (Damstadt, Germany), 1, 1’-diphenyl-

1picrylhydrazyl (DPPH.), Vanillin reagent, ascorbic acid, butylated hydroxytoluene (BHT), Gallic

acid, catechin, were purchased from Sigma Chemical Co. (St. Louis, MO, USA), Hydrochloric acid

(HCl), Sodium hydroxide (NaOH), Sulfuric acid (H2SO4), Ammonium molybdate ((NH4) 6

Mo7O24.4H2O), Sodium phosphate (Na2 HPO4), Phosphate buffered (PBS). All the other

chemicals used, including the solvents, were of analytical grade.

2.2. Plant materials

Five plant species were assessed in the present study. The plant materials were collected during

July-September 2009 from the south area of Tunisia, precisely from the Gafsa region.

Specimens were identified by a specialist in botany. Collected materials were dried in the dark

at room conditions, until weight stabilization. Then, each dried sample was ground by grinding

machine into a powdered form; filtered and fine powder was stored in a non toxic polyethylene

bag at room temperature before use. The used parts of the plants and the location from which

they are gathered are illustrated in Table 1.

Table 1: Names, used parts and location of the different plant species studied.

Scientific name Botanical Family

Plant part

Location

Artemisia campestris L Asteraceae

Leaves Gafsa

Peganum harmala L Zygophyllaceae Arial parts: Leaves, seeds, stems

Gafsa

Thymelaea hirsuta L

Thymelaeaceae

Leaves + Fruits

Gafsa

Cleome arabica L

Capparaceae

Arial parts: Leaves, Flowers, Stems

Gafsa

Haplophyllum tuberculatum

Rutaceae Arial parts: Leaves, flowers, Fruits, Stems

Gafsa

2.3. Preparation of extracts

The air-dried and powdered plant materials (9 g) were extracted in a soxhlet apparatus using

separately two solvents of croissant polarity: methanol (MeOH) and distilled water for 6 hours

at a temperature not exceeding the boiling point of the solvent (13). The whole extracts were

filtered and concentrated by rotary evaporation under pressure at 40°C to get crude extracts.

The extracts were stored in the refrigerator at 4°C for subsequent analysis.

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2.4. Determination of plant extract yield

The dried extract was weighed, and then the extraction yield was calculated and expressed as

the percentage of the weight of the crude extracts to the raw material according to the method

of Zhang (14). To determine the percentage (%) yield, the following equation will be used:

Percentage yield (%) = (W1 × 100) / W2

Where W1 was the weight of the extract after evaporation of solvent and W2 was the weight of

the dry.

2.5. Quantification of the phenolic constituents of the extracts

2.5.1. Determination of total phenolic contents

The total phenolic contents of the plant extracts were determined by the Folin-Ciolcalteu’s

colorimetric assay, which was based on the method described by Adedapo (15), with slight

modification. Briefly, one hundred microliteres of each diluted extract was mixed with one

hundred microliteres of distilled water, and then 0.5 mL of 10 % (v/v) Folin-Ciocalteu reagent

was added to each sample followed by thoroughly mixing with a vortex. The mixture was

allowed to stand in the dark for 10 minutes at room temperature. Subsequently, 1.0 mL of 7.5%

(w/v) sodium carbonate solution was added, and the mixture was incubated at room

temperature in the dark for 60 minutes at room temperature. The absorbance was measured at

725 nm using a UV–vis spectrophotometer. Quantification was obtained by reporting the

absorbance in the calibration curve of gallic acid used as standard phenol. The results were

expressed as milligram gallic acid equivalents per gram of dry weight of plant material (mg

GAE/g DW). The assay was performed in triplicate for each extract.

2.5.2. Determination of condensed tannins (proanthocyanidins)

Quantitative estimation of condensed tannins was carried out according to a modified vanillin-

HCl method described by Sefi et al. (2010) as adopted from Julkunen-Titto(16. Briefly, 50 µL of

each properly diluted sample was reacted with 1.5 mL of 4% vanillin solution in methanol, and

then 750 µL of concentrated hydrochloric acid was added. The mixture was allowed to stand for

15 min at ambient temperature, and the absorbance was measured at 500 nm against the blank

where the sample was omitted. Catechin was used as a reference standard, and the results

were expressed as milligram catechin equivalents (mg CE/g DW). All samples were analyzed in

triplicate.

2.6. Determination of antioxidant activity

The antioxidant activity of different extracts was carried out using two antioxidants assays in

vitro.

2.6.1. DPPH radical-scavenging activity

The antioxidant activity of the extracts was assessed on the basis of scavenging ability of the

stable 1, 1-diphenyl-2-picrylhydrazyl (DPPH) free radical by the method described by Kyung (17).

Therefore, in the presence of an antioxidant which can donate an electron to DPPH, the purple

color which is typical to free DPPH radical decays and the absorbance was measured

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colorimetrically at 517 nm. Briefly, 0.2 mL of DPPH solution (0.2 mM, in methanol) was added

to 1 mL of extract solutions in methanol at different concentrations (1, 10, 100 and 200 µg/mL).

Then, these solutions were vortexed thoroughly and incubated in the dark at room

temperature for 30 min. The absorbance of the mixture was measured spectrophotometrically

at 517 nm. Ascorbic acid and BHT were used as references. The ability to scavenge DPPH radical

was calculated by the following equation:

DPPH radical scavenging activity (%) = [(Abs Control –Abs Sample) / (Abs Control)] * 100

Where Abs Control is the absorbance of DPPH radical + methanol; Abs Sample is the absorbance of

DPPH radical + sample extract / standard.

The antioxidant activity was also expressed as IC50, which was defined as the concentration of

the sample that could scavenge 50% of the DPPH radicals. Each essay was carried out in

triplicate, and the average result and standard deviation were reported.

2.6.2. Determination of total antioxidant capacity by phosphomolybdenum method

The total antioxidant capacities of the extracts were evaluated by the phosphomolybdenum

method as described by Prieto (18). The assay is based on the reduction of Mo (VI) to Mo (V) by

the extract and subsequent formation of a green phosphate/Mo (V) complex at acid pH. An

aliquot of 0.4 mL of each sample solution and ascorbic acid was combined with 3.6 mL of

reagent (600 mM sulphuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate).

In case of blank, 0.1 ml of methanol was used in place of the sample. The tubes containing the

reaction solution were capped and incubated in a water bath at 95 °C for 90 min. After the

samples had been cooled to room temperature, and then the absorbance of the solution of

each sample was measured at 695 nm against the blank using a UV–Vis spectrophotometer.

The total antioxidant activity was expressed as ascorbic acid equivalents (AAE) in milligrams per

gram of dry weight of plant material.

2.7. Determination of antibacterial activity

In recent years due to an upsurge in antibiotic-resistant infection, the search for new prototype

drugs to combat infections is an absolute necessity and in this regard natural products may

offer great potential and hope. As is well known that the bioactive plant extracts are a

promising source of antimicrobial compounds and hence antibiotic in nature (19).

2.7.1. Bacterial strains and Culture Conditions

The plant extracts were individually tested against a panel of bacterial pathogens, including

food spoilage bacteria, such as Bacillus subtilis, Bacillus cereus and Pseudomonas aeruginosa

and food-borne pathogens, namely Salmonella enteridis ATCC 502, Salmonella typhimurium,

Staphylococcus aureus ATCC 25923 and Escherichia coli GM 109 were obtained from the culture

collection of Pasteur Institute of Tunis. The bacterial strains were cultivated in the Luria-Bertani

(LB) agar at 37°C, except Bacillus species, which were grown at 30°C. The cultures of each

bacterial strain were maintained in their appropriate agar slants at 4°C.

2.7.2. Agar disc diffusion method

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The agar disk diffusion method was employed for the determination of antibacterial activity of

plant extracts according to Mackeen(20) with some modifications. Briefly, a fresh inoculum of

the tested bacteria was developed by suspending activated colonies in physiological saline

solution (0.9 %). After, each suspension was adjusted to a concentration (107–108 CFU/mL). The

prepared inoculum of each microorganism was uniformly spread on the LB agar plates. Sterile

filter paper discs, 9mm in diameter were individually impregnated with 30 µL of plant extracts

(300µg/ disc) and then placed on the inoculated plates; these plates were placed at 4°C for 2

hours and were then incubated at 37°C for 24 hours. Antibacterial activity was evaluated by

measuring the diameter of zones of inhibition against the tested bacteria. All tests were

performed in triplicate, Streptomycin B (15μg/mL) and Chloramphenicol (30 μg/mL) were used

as positive controls.

2.8. Cytotoxicity activity

2.8.1. In Vitro Haemolysis Assay

Haemolytic activity of different extracts was performed as described by Malagoli (21) with slight

modification. In brief, freshly collected chicken blood samples were immediately mixed with

anticoagulant, ethylenediaminetetraacetic (EDTA) solution to prevent blood coagulation.

Erythrocytes were isolated from the blood samples by centrifugation at 1500 RPM for 10 min at

4°C and were washed three times with phosphate buffered saline (PBS, pH 7.4). The

supernatant was then discarded by gentle aspiration, and the above process was repeated two

more times. Erythrocytes were finally resuspended in PBS to obtain 1% solution and stored at

4°C until use.

The hemolytic activity was tested under in vitro conditions in 96-well microplates. For this,

various concentrations of different extracts (125 – 8000 µg/mL) prepared in PBS buffer were

added to the erythrocyte suspension. The mixtures were incubated at 37 °C for 3 h in a shaking

water bath and then centrifuged at 1500 rpm for 10 min at 4°C. The supernatants were

transferred to 96-well Microplates and the absorbance were measured at 540 nm by a

microplate reader to determine the extent of erythrocyte lysis. Pure PBS was used as a negative

control (0% hemolysis) and triton X-100 (1%, v/v) was used as a positive control. The haemolytic

activity was expressed as the concentration producing 50 % of the maximum haemolysis (HD50) (22) .

2.9. Statistical Analysis

All data were reported as mean ± standard deviation of triplicate measurements. Significant

differences were examined by contrast of means using one-way variance analysis (One Way

ANOVA) followed by Tukey’s test, and statistical analyses were carried out with the software

IBM SPSS Statistics 20 for windows. Correlations between the antioxidant activity and total

phenolic content were examined using Pearson’s correlation. In addition, the inhibitory

concentration 50 % (IC50 values) for antioxidant activities was calculated by nonlinear regression

analysis using the Graphpad Prism version 5.0. The dose–response curve was obtained by

plotting the percentage of inhibition versus the concentrations. On the other hand, the

inhibitory concentration required for 50% cytotoxicity (IC50) value was analyzed using Sigmaplot

12.0 software.

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3. Results and Discussion

3.1. Extraction yield

Different plant extracts were prepared from five selected species using two polar solvent. The

extraction yield of these plants varied from 5.51 ± 2.26 % to 28.08 ± 0.35 % (Fig. 1). It can be

noted from these results that the amount of residue extracted with water, in the most cases, is

higher than methanol. In general, the variation in extraction yield among the different plants

studied in the present analysis might be ascribed to the different availability of extractable

components, resulting from the various chemical compositions of plants (23). Furthermore, it

should be noted that the characteristics of the solvent influenced the global extraction yield. In

fact, it has been reported in the literature data that the yield of extractable compounds

increased with the polarity of the solvent, so the extraction with water had the highest yield (24).

Figure 1: Extraction yield (%) of several methanolic and aqueous extracts. Data are given as

mean ± standard deviation (n=3) from three replicates in each extract.

3.2. Quantification of the phenolic constituents of the extracts

Generally, the phenolic compounds are one of the most diverse groups of secondary

metabolites found in edible plants. Chemically, they can be defined as substances possessing an

aromatic ring bearing one or more hydroxyl groups, including their functional derivatives25. In

fact, there are different classes of polyphenols such as, phenolic acids, tannins, flavonoids.

Tannins may be subdivided into hydrolysble and condensed tannins, which are also known as

proanthocyanindins. In the present study, concentrations of total phenolics and

proanthocyanindins in the different plant extracts were determined.

3.2.1. Determination of total phenolics content and tannins content

The content of total phenolic was carried out based on the absorbance values of the various

extract solutions, reacted with Folin-Ciocalteu reagent and compared with standard solutions of

gallic acid equivalents, as described above, and the results of the colorimetric analysis of total

phenolics are presented in Fig.2 (A). The amount of total phenolic compounds varied widely in

herbal materials and ranged from 3.64 ± 0.87 to 44. 23 ± 1.96 (mg GAE/g DW) for methanolic

extracts, and from 1.89 ± 0.39 to 13.28 ± 0.08 (mg GAE/g DW) for aqueous extracts. Indeed, the

results clearly demonstrate that the total phenolics content were higher in methanolic extracts

compared to aqueous for different species. The phenolic content of the methanolic extracts of

0

5

10

15

20

25

30

Artemisia

campestris L

Thymelaea

hirsuta L

Haplophyllum

tuberculatum

Peganum

harmala L

Cleome arabica L

Yie

ld (

%)

Methanolic extracts

Aqueous extracts

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Thymelaea hirsuta L and Artemisia campestris L (44. 23 ± 1.96 mg GAE/g DW and 36.05± 2.06

mg GAE/g DW, respectively) were significantly higher than those of other extracts. Whereas,

the phenolic content of the aqueous extracts of A. campestris and T. hirsuta showed the highest

amounts (13.28 ± 0.08 mg GAE/g DW and 10.10± 0.04 mg GAE/g DW, respectively).

Therefore, the results obtained indicated that the total phenolic content of the aqueous extract

of T. hirsuta was lower than that obtained by Trigui (3), who reported a value of 31.5 ± 1.5 mg

GAE/g DW. Quantitatively, total phenolic content obtained with the methanolic extract of A.

campestris in our study was significantly higher than that reported by Tlili (26).

On the other hand, the results of total condensed tannins of different extracts are shown in

Fig.2 (B). These contents were estimated by vanillin assay and compared with standard

solutions of catechin equivalents. The amount of condensed tannins ranged from to 9.45± 1.22

to 0.09 ± 0.02 mg EC/gDW among the different plant extracts. Methanol extracts have been

found to be rich in tannins compared with aqueous extracts. From the analysis of Fig.2 (B), we

can conclude that the methanolic extract of Thymelaea hirsuta L presented a significantly

higher level of condensed tannins, while the lowest was found in aqueous extracts of

Haplophyllum tuberculatum.

Moreover, it has been reported by Sefi (27) that amount of total condensed tannin of the

aqueous extract of A. campestris was higher than the finding obtained in this study. The level

found for P. harmala was lower than that reported by Khlifi (28) , who reported a total

condensed tannin of 2.03 ± 0.06 mg EC/g DW.

(A)

(B)

0

10

20

30

40

50

60

Artemisia

campestris L

Thymelaea

hirsuta L

Haplophyllum

tuberculatum

Cleome

arabica L

Peganum

harmala L

To

tal

ph

eno

lic

com

po

un

ds

(mg

GA

E/g

DW

)

Methanolic extracts Aqueous extracts

0.01 0.02 0.04 0.08 0.16 0.32 0.64 1.28 2.56 5.12

10.24

Artemisia

campestris L

Thymelaea

hirsuta L

Haplophyllum

tuberculatum

Cleome

arabica L

Peganum

harmala L

To

tal

con

den

sed

ta

nn

ins

(mg

CE

/gD

W)

Methaonolic extracts Aqueous extracts

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Figure 2: Quantification of the phenolic constituents of the different extracts. (A) Total phenolic

content (mg GAE /g dry weight of plant material) as affected by extracting solvents. (B) Total

condensed tannins (mg CE/g dry weight of plant material) as affected by extracting solvents.

Values are expressed as means ± standard deviation (n = 3).

On the other hand, these variations in the phenolic compound levels may be due to phenolic

metabolism, the environmental conditions and geographical distribution, which can modify the

constituents of the plant. Under field conditions, the phenolic composition of plant tissues

varied considerably with seasonal, genetic and agronomic factors (28). In addition, a large

variability at various stages of maturation and for different growing conditions, such as

temperature and rainfall, is known to affect the contents of phenolic compounds (29).

Moreover, it has been suggested that the phenolic content of plant materials is correlated with

their antioxidant and antibacterial activity. It is considered that the antioxidant activity of

phenolic compounds is due to their high redox potentials, which allow them to act as reducing

agents, hydrogen donors and singlet oxygen quenchers (30) .

3.3. Antioxidant activity

It is important to select and employ a stable and rapid method to assay antioxidant activity,

because the determination of many samples is time-consuming. Owing to the complex reactive

facets of phytochemicals, the antioxidant activities of plant extracts cannot be evaluated by

only a single method, but least two test systems have been recommended for the

determination of antioxidant activity to establish authenticity (31,32). Therefore, in the present

study, this activity was evaluated in vitro by two complementary colorimetric methods, namely

DPPH assay and phosphomolybdenum methods.

3.3.1. DPPH radical scavenging activity

The DPPH method is one of the most extensively used and stable chromogen compounds to

measure the antioxidant activity of biological material (33). DPPH is a free radical that accepts on

electron or hydrogen radical to become a stable diamagnetic molecule(34). In the DPPH assay,

the antioxidants were able to reduce the stable radical DPPH and the color changes from purple

to yellow after reduction. The method is based on the reduction of DPPH in alcoholic solution in

the presence of a hydrogen-donating antioxidant due to the formation of the non-radical form

DPPH-H in the reaction(35). In this assay, the results were compared to the reference standards

butylated hydroxyl toluene (BHT) and ascorbic acid (AA).

The results showed that all tested plant extracts exhibited interesting antiradical activity (Fig.3

(A) and Fig.3 (B)). From the graphs previously mentioned, we can see that these extracts

showed a concentration-dependent manner. Moreover, the findings obtained in this study

indicated that methanolic extracts were the most effective DPPH radical scavengers compared

to aqueous extracts for different species.

It can be observed from the data in Fig.3 (A) that the methanolic extract of Thymelaea hirsuta L

showed the strongest (p <0.05) radical scavenging effect (91.89 ± 1.82 %), while the methanolic

extract of Peganum harmala (62.33 ± 0.69) showed the lowest radical scavenging activity (p

<0.05) at the same concentration of 200µg/mL. In order to quantify the antioxidant activity

further, the IC50 for each methanolic extract was calculated and defined as the concentration of

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extract causing 50 % inhibition of absorbance, thus, a lower IC50 value would reflect greater

antioxidant activity of the sample (Table 2). The results were demonstrated that the most active

radical scavenging activities of the methanolics extracts were T. hirsuta (IC50 =1.74 ± 0.12

µg/mL), followed by Artemisia campestris L (IC50 = 2.23 ± 0.07 µg/mL), and Haplophyllum

tuberculatum (IC50= 25. 3 ± 0.4 µg/mL), which were lower than that observed for the positive

control ascorbic acid (IC50 = 0.27± 0.71 µg/mL), but higher than that of positive control BHT

(IC50= 29.62 ± 1.66 µg/mL).

On the other hand, the investigated aqueous extracts of T. hirsuta and A. campestris exhibited

the highest (p <0.05) DPPH radical-scavenging activity, and their maxima of inhibition reached

to 85.8 ± 0.7 % and 85.48± 1.13 %, respectively at a concentration of 200µg/mL, whereas the

extracts of Cleome arabica (61.16 ± 3.05 %) and H. tuberculatum (59.38 ± 3.69 %) showed a

moderate radical scavenging activity (p <0.05) in the same conditions (Fig.3 (B)). With this

assay, the extract of P. harmala presented the lower amount of radical scavenging compounds,

and its percentage inhibition reached to 41.4 ± 0.72 %. The calculated IC50 values are presented

in the Table 2. It can be concluded from these findings that the highest radical scavenging

activities were presented by A. campestris (IC50 = 0.77 ± 0.18 µg/mL), T. hirsuta (IC50 = 2.65 ±

0.49 µg/mL), C. arabica (IC50 = 20.3 ± 0.65 µg/mL), and Haplophyllum tuberculatum (IC50=

21.82± 0.77 µg/mL), which were lower than that of positive control ascorbic acid, whilst higher

than that of positive control BHT.

(A)

(B)

Figure 3: DPPH free radical scavenging activity at different concentrations (1–200 μg/mL) of

methanolic extracts (A), aqueous extracts (B) and reference antioxidants; BHT (butylated

0

20

40

60

80

100

1 10 100 200

DP

PH

(%

) in

hb

itio

n

Concentration (µg/mL)

Artemisia campestris L

Thymelaea hirsuta L

Haplophyllum tuberculatum

Cleome arabica L

Peganum harmala L

BHT

Ascorbic acid

0

20

40

60

80

100

1 10 100 200

DP

PH

(%

) in

hib

itio

n

Concentration (µg/mL)

Artemisia campestris L

Thymelaea hirsuta L

Haplophyllum tuberculatum

Cleome arabica L

Peganum harmala L

BHT

Ascorbic acid

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hydroxytoluene) and ascorbic acid. The absorbance values were converted to scavenging

effects (%) and data plotted as the means of replicate scavenging effect (%) values. (Results are

expressed as means ± standard deviation of three measurements).

As regards to the antioxidant activity of A. campestris by DPPH assay, previous studies have

reported that this species had significantly lower antioxidant activity of ours (36,37). Moreover, it

has been previously reported that the antioxidant activity of T. hirsuta was lower than that

obtained in this study(3). The data obtained in earlier studies demonstrated that the antioxidant

activity of P. harmala was higher than that obtained in this investigation study(28). The free

radical-scavenging activity funded by DPPH assay concerning C. arabica was well in accordance

with the recent reports in the literature (38) .

Table 2: The DPPH free radical scavenging activity (IC50 values) of different extracts

IC50 (µg/mL) a Species and standard

Aqueous extracts Methanolic extracts

0.7788 ± 0.18* 22.233 ± 0.07 * Artemisia campestris L

2.650 ± 0.49* 1.749 ± 0.12*

Thymelaea hirsuta L

21.82 ± 0.77** 25.3 ±0.4**

Haplophyllum tuberculatum

20.3 ±0.65* 39.43 ± 2.01*

Cleome arabica L

NDb 142.5 ± 1.65** Peganum harmala L

29.62 ± 0.71* BHT 0.2704 ± 1.66*

Ascorbic acid (a) IC50 (50% concentration of inhibition for radical scavenging activity) value expresses the

concentration in µg/ml for each sample. Values are mean ± SD of triplicates.

* / ** The values with asterisks in columns are significantly different (p<0.05).

(b)ND: not determined.

3.3.2. Total antioxidant capacity by phosphomolybdenum method

Total antioxidant capacities of different plant extracts were quantitatively determined by the

formation of phosphomolybdenum complex and, the results expressed as ascorbic acid

equivalents (mg AAE/g DW) are illustrated in Fig. 4. Thus, the plant extracts demonstrated

electron-donating capacity showing their ability to act as chain terminators, transforming

relative free radical species into more stable non-reactive products (39). It can be seen from this

Figure that the methanolic extracts of five different species exhibited an excellent potent total

antioxidant capacity compared to aqueous extracts, and these data were in agreement with

those obtained by the DPPH assay. In fact, the total antioxidant activity of the various extracts

ranged between 5.72 ±0.01 mg AAE/g DW and 57.54 ± 0.46 mg AAE/g DW. Among all the plant

extracts, the methanolic extracts of T. hirsuta and A. campestris were found to be the most

actives (57.54±0.46 and 55.75±3.76 mg AAE/g DW, respectively), while the lowest values are

detected in the methanolic and aqueous extracts of P. harmala (8.98±0.73 and 5.72 ±0.01 mg

AAE/g DW, respectively).

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Figure 4: total antioxidant capacity by phosphomolybdenum method of different methanolic

and aqueous extracts (mg AAE /g dry weight of plant material). Data are expressed as means ±

standard deviation of three measurements.

Indeed, this investigation suggests that phytochemicals necessary for antioxidant activity are

present in abundance in methanol extracts. This could be the reason why the methanol extracts

have a high content of phenolic compounds compared to the aqueous extracts.

On the other hand, the results showed that the antioxidant activities of different species varied

markedly, and this may be attributed to the differences in their chemical composition and

primarily related to their hydroxylation and methylation patterns (40) . Moreover, recent studies

showed that many phenolic compounds, such as flavonoids and related polyphenols contribute

significantly to the total antioxidant activity of many medicinal plants (41). However, plants may

contain other antioxidants such as proteins, ascorbate, β carotene, α-tocopherol and lycopene,

which could play some role in the increase of the antioxidant activity (40).

3.3.3. Relationship between antioxidant activity and phenolic compounds

There was a direct relationship between the total polyphenol content and antioxidant activity

of the various extracts of medicinal plants, and their correlation coefficients were calculated.

The number of tested samples and range of parameter values normally influence the

calculation of correlation coefficients (R2). The overall relationship between antioxidant activity

and total phenolic content for all tested plants was a positive and significant linear correlation.

The results obtained in our study show that total phenolic compounds of methanolic extracts

are correlated with DPPH scavenging activity assay (R2 = 0.876). On the other hand, there is a

high correlation between this assay and the total phenolic content of aqueous extracts (R2 =

0.9543). Statistically significant relationships were also observed between total phenolics and

total antioxidant capacity by the phosphomolybdenum method of methanolic and aqueous

plant extracts (R2 = 0.9229 and R2 = 0.8045, respectively). The results suggested that the

phenolic compounds contributed significantly to the antioxidant capacity of the medicinal

herbs.

3.4. Antibacterial activity

In the present study, the in vitro antibacterial activity of different plant extracts against the

studied microorganisms and their activity potentials was quantitatively assessed by the

0

10

20

30

40

50

60

70

Artemisia

campestris L

Thymelaea

hirsuta L

Haplophyllum

tuberculatum

Cleome

arabica L

Peganum

harmala L

To

tal

an

tio

xid

an

t ca

pa

city

(m

g

AA

/g D

W)

Methanolic extracts

Aqueous extracts

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presence or absence of inhibition zones using the agar disk diffusion method as shown in

Tables 3and 4. The results showed great variations in the potency of the antibacterial activity of

selected species. These data were compared to antibiotics (Chloramphenicol and Streptomycin

B), used as positive controls tested.

In fact, it can be noted from the data in Table 3 that the methanolic extracts of Artemisia

campestris L and Thymelaea hirsuta L were found to be the most actives against all the tested

microorganisms studied, except for Pseudomonas aeruginosa and the size of their inhibition

zones varied between 11 to 25 mm. Therefore, the strong antibacterial activity of these species

may be attributed to the presence of bioactive metabolites of various chemical types, such as

phenolic compounds. Thus, Shoko(42) confirmed that phenolic compounds were the most

important substances active against bacteria. However, the methanolic extract of Peganum

harmala revealed a moderate activity against E. coli with inhibition zone of 12 mm. Thus, it can

be hypothesized that the antibacterial activity detected in P. harmala was due to the β-

carbolines alkaloids (43). Further, our results showed that Haplophyllum tuberculatum was not

active against any tested strains. To our knowledge, there are no data in the literature on the

antibacterial activity of the extracts of H. tuberculatum.

Table 3: Antibacterial activity of the methanolic extracts, using paper disc-diffusion method

Methanolic extracts and standards

Disc diffusion method (DD) (a)

Bacterial strains E. coli

P. aeruginosa

S. typhi

S. enteridis

S. aureus

B. subtilis

B. cereus

Methanolic extracts Artemisia campestris L

17 NA 13 13 20 21 25

Thymelaea hirsuta L 15 NA 13 11 14 15 11 Cleome arabica L NA NA NA NA NA NA NA Peganum harmala L 12 NA NA NA NA NA NA Haplophyllum tuberculatum

NA NA NA NA NA NA NA

Antibiotics

Chloramphenicol (b) NA 28 27 27 20 33 NA

Streptomycin B (c) 12 17 15 13 22 15 NA

(a )(DD) Disc-diffusion method. Inhibition zone in diameter around the discs impregnated with

30 µL of plant extracts (300µg/ disc). The diameter (9 mm) of the disc is included.

(b): Chloramphenicol (30 µg/µL)

(c): Streptomycin B (10 µg/µL)

NA: Not Active

Nevertheless, it can be observed from the data in Table 4 that the aqueous extracts of the five

selected species were not active with the exception of the extract derived from Cleome arabica

L, which was moderately active against only E. coli strain with inhibition zone of 12 mm. On the

other hand, the inhibition zones of the Chloramphenicol and Streptomycin B ranged from 20-

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33 mm and 12-22 mm, respectively, while solvents when used alone (negative control) did not

show any zone of inhibition.

However, negative results do not mean absence of bioactive constituents, nor that plant is

inactive. The active compound (s) could be present in insufficient quantities in the crude

extracts to show activity with the dose applied (44) .

Our previous data indicate that Gram-positive bacteria were found to be more susceptible to

growth inhibition by plant extracts than Gram-negative bacteria. The best activity was exhibited

by the methanolic extract derived from A. campestris against B. cereus, one of the most

common Gram-positive, causing food poisoning, followed by S. aureus and B. subtilus strains.

Indeed, the greater susceptibility of Gram-positive bacteria towards various plant extracts was

also previously reported (45). In fact, the pathogenic capability of Gram-negative bacteria is

usually associated with the presence of lipopolysaccharide layer, which might be involved in

reducing the sensitivity of these bacteria (5) .

Table 4: Antibacterial activity of the aqueous extracts, using paper disc-diffusion method

Aqueous extracts and standards

Disc diffusion method (DD) (a)

Bacterial strains E. coli

P. aeruginosa

S. typhi

S. enteridis

S. aureus

B. subtilis

B. cereus

Aqueous extracts Artemisia campestris L

NA NA NA NA NA NA NA

Thymelaea hirsuta L NA NA NA NA NA NA NA Cleome arabica L 12 NA NA NA NA NA NA Peganum harmala L NA NA NA NA NA NA NA Haplophyllum tuberculatum

NA NA NA NA NA NA NA

Antibiotics Chloramphenicol (b) NA 28 27 27 20 33 NA

Streptomycin B (c) 12 17 15 13 22 15 NA

(a) (DD) Disc-diffusion method. Inhibition zone in diameter around the discs impregnated with

30 µL of plant extracts (300µg/ disc). The diameter (9 mm) of the disc is included.

(b): Chloramphenicol (30 µg/µL)

(c): Streptomycin B (10 µg/µL)

NA: Not Active

On the other hand, previous literatures have reported that the aqueous and methanolic

extracts of A. campestris L have lower or any inhibitory activity against B. subtili S. aureus, E.

coli and P. aeruginosa (46, 36) . However, to our knowledge, there are few data in the literature

on antibacterial activity in vitro of T. hirsuta. Trigui (3) reported that acetone and ethyl acetate

extracts of T. hirsuta have a strong antibacterial activity against S. aureus and B. cereus, while

the aqueous extract of this specie was found to be inactive. In addition, there are no published

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studies that have evaluated the antibacterial activity of C. arabica, but this activity of other

Cleome species was studied, such as Cleome gynandra, Cleome viscosa and Cleome droserifolia 47,48,49. Furthermore, these differences of results in the antibacterial activity of different plants

could be attributed to phytochemical properties and differences among species; also they are

certainly related to the different bacteria tested.

3.5. Cytotoxicity activity

Bioactive compounds in plants have been promising potential usefulness in safety and human

health concerns. However, there has been an increasing demand for the development of drugs

and effective compounds from natural sources with lower toxicity for eukaryotic systems.

Indeed, the haemolytic activity represents of any compounds is a good indicator for the

screening of cytotoxicity towards normal healthy cells.

3.5.1. In Vitro Haemolysis Assay

In the present study, the different plant extracts were assessed for their haemolytic activity

using the blood samples of chicken. The results were constructed by the software of Sigmaplot

and presented in Fig. 5. From the graph, we can see that among all tested extracts, only the

methanolic extract of C. arabica (Cleome amblyocarpa) exhibited haemolytic activity, and its

percentage inhibition reached to 30.45 ± 0.63 % at a concentration of 8000 µg/mL. Whereas,

this finding indicated that this plant had a little hemolytic effect, and its half hemolytic dosage

(HD50) was 11.301 mg/mL (Pearson’s correlations r = 0.9533 with p <0.05). However, the

standard Triton-x- presented very high haemolytic activity with HD50 was 0.250 mg/mL

(Pearson’s correlations r= 0.7751 with p <0.05). It can be seen also from the data in Fig. 6 that

the methanolic extract of C. arabica showed concentration-dependent haemolytic activity.

Figure 5: Haemolytic activity of extract and positive control (Tritonx-100) at various

concentrations (125 – 8000 µg/mL).

On the other hand, most previous studies have reported that some secondary metabolites in

several plant species could show cytotoxic and haemolytic activities, such as saponins, alkaloids,

dammarane triterpene and quercetin. In fact, Gleason and Raymond (50), have reported that the

alkaloids disrupt cell membranes were found to be responsible for haemolytic activity. In

Concentration (µg/mL)

0 2000 4000 6000 8000 10000

Hem

olyt

ic D

egre

e (%

)

0

20

40

60

80

100

120

Triton X-100

Cleome arabica L

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addition, quercetin showed a weak haemolytic activity on human erythrocytes (51). Thus, the

presence of a number of bioactive compounds, including alkaloids, dammarane triterpene and

quercetin in C. arabica has already been reported (52), which may explain the haemolytic effect.

Moreover, Hassan (53) have reported that the haemolytic activity may be due to effects on cell

membrane permeability by forming pores in membranes, altering the sodium-potassium and

calcium-magnesium ATPase activities.

Elsewhere, no research has evaluated the haemolytic activity of C. arabica. Thus, the study of

Edziri (54) has demonstrated that C. amblyocarpa should be considered as a potent in vitro toxic

compound. Besides, Hidekazu55 have reported that the dammarane-triterpenes isolated from

C. africana have significant cytotoxic activity against P388 leukemia cells.

4. Conclusion

According to the results of this study, it is concluded that the methanolic extracts of the five

species possess a high amount of phenolic compound and condensed tannin. In addition, it can

also be inferred that the methanolic extracts and especially those of T. hirsuta and A. campestis

have good antioxidative and antibacterial activities and could be considered as natural sources

for the use as a preservative in food systems and pharmaceutical industries. Moreover, the

results obtained in the present study illustrated that correlations exist between biological

activities and the amount of phenolic of plant extracts. To understand their mechanism of

action as bioactive components, further isolation of phenolic compounds, and determination of

their biological activities are necessary.

Conflict of Interests

The authors declare that there is no conflict of interests regarding the publication of this paper.

ACKNOWLEDGMENT

This work was supported by a grant from the Ministère de l’Enseignement Supérieur, de la

Recherche Scientifique et de la Technologie (Tunisia). We are very grateful Dr Ghrabi-Gammar

Zeineb (INAT) for identification of the harvested plants used in this study. The authors equally

thank Dr. Nazek Gallas (Institut Pasteur de Tunis, Tunisia) for providing us with bacteria.

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