Antioxidant and antimicrobial activity of Cotula coronopifolia

65
Tunisian Journal of Medicinal Plants and Natural Products
TJMPNP 2, (2009), 65-73
Antioxidant and antimicrobial activity of Cotula coronopifolia
(Asteraceae) growing in Tunisia
Kaouthar Liouane2, Houda Ben Abdelkader1, Karima Bel Hadj Saleh3, Awatef
Debbabi1 , Mohamed Ali Mahjoub1, Khaled Said2, Zine Mighri1 .
1
Laboratory of Natural Substances Chemistry and Organic Synthesis, Faculty of Sciences, 5000 Monastir,
Tunisia.
2
Laboratory of Genetic, Biodiversity and Bio-resources valorisation, Biotechnology Institute of Monastir,
5000 Tunisia.
3
Laboratory of Transmissible Diseases and Biologically Active Substances, Faculty of Pharmacy, 5000
Monastir, Tunisia.
Received 16 October 2009; received in revised form 24 November 2009; Accepted 22 December 2009
Abstract
The in vitro antimicrobial and antioxidant activities of the Petroleum extract, and two fatty acids
(hexadecanol and pentanol) isolated from the plant Cotula coronopifolia were investigated.
Antibacterial and antifungal activities were tested against bacteria and fungi using Disc diffusion
method and Minimum Inhibitory Concentrations (MIC). The fatty acids were confirmed using 1D
NMR experiments (1H and 13C). Total antioxidant capacities were assessed by ABTS (2,2’Azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)) and DPPH(1,1-diphenyl-2-picrylhydrazyl).Total
phenolic contents were measured by Folin-Ciocalteu assay.
Petroleum extract showed promising antibacterial and antifungal activity and isolated fatty acids
showed moderate activity against bacteria and fungi and reasonable antioxidant properties, and they
can therefore be used as a natural additive in food, cosmetic and pharmaceutical industries.
Key words: Cotula coronopifolia, hexadecanol, pentanol, antibacterial, antifungal antioxidant,DPPH, ABTS,
Phenolic content.
1. Introduction
Because of the resistance that pathogenic build against antibiotics, there is a great interest in
the search for new antimicrobial drugs also from nature. Natural crude drug extracts and
biologically active compounds isolated from plant species used in traditional medicine can
be prolific resources for such new drugs. Some of the antimicrobial compounds produced
by plants are active against plant and human pathogenic microorganisms [1].
Therefore in the present study, we initiated an investigation for the evaluation of
antimicrobial and antioxidant properties of the aquatic plant Cotula coronopifolia
(Asteraceae) from the Tunisian littoral.The genus Cotula comprises about 55 species, most
of them endemic to South Africa, two occur in Australia, two in Asia, two on the Tristan da
Cunha Islands in the South Atlantic Ocean, and one each in North Africa. [2].
The Asteraceae family includes a large number of plants that are well known for their
antioxidant and antioxidant properties. In particular, the essential oil and methanol extract
of Achillea millefolium [3]. Moreover Kokoska et al. (2002) [4], reported antimicrobial
activity of Tussilago farfara, Arctium lappa, Cichorium intybus and Rhaponticum
carthamoides. Concerning the genus Cotula, Markouk et al. (1999) [5] announced
antibacterial activity of Cotula cinerea extracts.
The aim of this study was to evaluate the in vitro antioxidant the antimicrobial properties
Cotula Coronopifolia (Asteraceae) growing in Tunisia.
* Corresponding author: Kaouthar Liouane ; E- mail: Kaouthar_Liouane @yahoo.fr.
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Z. MIGHRI et al. / TJMPNP 2 (2009) 65-73
2. Materials and methods
2.1. Plant material
Aerial parts (leaves, stems and flowers) of the aquatic plant Cotula coronopifolia were
collected at the beginning of mars 2007 in Monastir (Tunisian littoral). A voucher specimen
was deposited at the herbarium in the Faculty of Sciences, University of Monastir, Tunisia.
2.2. Preparation of extract
Dried and finely powdered Cotula coronopifolia aerial part (leaves, stems and flowers)
(2500 g) was immersed in Methanol (MeOH) at room temperature for a week and was
extracted three times (3x3L). The extract obtained was filtered and evaporated to dryness.
The crude methanolic extract was extracted with equal volume of organic solvent the
Petroleum ether, the Petroleum extract was taken to dryness under vacuim and stored at 4°C.
2.3. Isolation and identification of active compound
Since the Petroleum extract showed promising antimicrobial and antifungul activity,
it(57,2g) was fractioned by column chromatography over silica gel (850 g) using as eluent
Petroleum ether/ethyl acetate.We collected 170 fractions (each 200ml) by increasing the
polarity of solvant. Based on thin layer chromatography profile the fractions were pooled
together to get 24 fractions. The pentanol was extracted from the fraction F6 and the
hexadecanol from the fraction F10 .The total structural elucidation of these compounds was
performed by 1D NMR experiments (1H and 13C) and GC-MS spectrometry.
2.4. Antibacterial activity
The antibacterial activity was evaluated against two Gram negative bacteria such as
Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853) and Gram
positive strain Staphylococcus aureus (ATCC 25923) and Enterococcus fecalis (ATCC
29212) and the two clinical bacteria Citrobacter freundei and proteus mirabilis.
The microorganisms were obtained from the Laboratory of Transmissible Diseases and
Biologically Active Substances, Faculty of Pharmacy, Monastir, Tunisia. Cultures of these
bacteria were done on Nutrient Muller-Hinton medium and were incubated at 37°C for 24h.
Gentamycin was used as positive control. Three replicate runs were carried out for each
concentration and for each micro-organism. The antimicrobial activity was evaluated by
paper disc diffusion [6] and dilution methods [7].
2.5. Antifungal activity
Samples were tested against seven strains of fungi, comprising two opportunist pathogenic
yeasts (Candida albicans and Cryptococcus neoformans); three dermatophytes
(Trichophyton rubrum, Trichophyton soudanense and Microsporum canis) and two
hyphamycet (Aspergillus fumigatus and Scopulariopsis brevicaulis.)
Antifungal activity was assayed by the method of agar incorporation (dilution in a solid
medium) including a negative control, as described previously [8-10]. Briefly, the test was
performed in sterile Petri dishes (33mm Ø) containing sabouraud Glucose Agar (SGA).
Samples were mixed aseptically with SGA (100 mL) to give stocks with concentrations of
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Z. MIGHRI et al. / TJMPNP 2 (2009) 65-73
1000, 500 and 250 µl/mL. Stocks were dissolved in 99% EtOH, and this solvent was used as
the negative control. After cooling and solidification, the medium was inoculated with a
small amount (5mm Ø) of a 7-day-old mycelium culture (for dermatophytes and
Scytalidium), a 3-day-culture suspension adjusted to 105 conidies/mL (Aspergillus and
Scopulariopsis) or a 3-day culture suspended in sterile distilled water and adjusted to 105
spores mL-1 (yeasts).The Petri dishes were then incubated for 7 days at 24°C for
dermatophytes and Scopulariopsis, 24 h at 37°C for Candida and Aspergillus and 48 h at
37°C for Cryptococcus. The antifungal agent, Fluconazol was included in the essays as
positive control. Three replicate runs were carried out for each concentration and for each
micro-organism. The antifungal activity of the samples was evaluated using two methods:
(a) by calculating the percentage inhibition (%I) from the diameters of the colonies in the
control plate (dC) and the colonies in the treated plate (dE); %I=(dC-dE)/dC, according to
the method of Singh et al [11].
(b) by determining the minimal inhibitory concentration (MIC), defined as the lowest
concentration which inhibits the visible growth of fungi during the defined incubation period
for each species.
The micro-organisms were obtained from the Laboratory of Transmissible Diseases and
Biologically Active Substances, Faculty of Pharmacy, Monastir, Tunisia.
2.6. Antioxidant activity
2.6.1. Radical cation ABTS+• scavenging activity
The standard method described by Miller et al. (1993)[12] has been adopted with minor
modifications. This assay assesses the total radical scavenging capacity based on the ability
of a compound to scavenge the stable ABTS radical (ABTS+•). The blue green ABTS
radical form was produced through the reaction between ABTS and potassium persulfate in
water. A concentrated ABTS+• stock solution was diluted with phosphate buffered saline
(PBS), pH 7.4 to a final absorbance of 0.70±0.02 with a wavelength of 734 nm and at a
temperature of
25°C. Solutions with different diluted concentrations of our samples
(Extracts and natural products) were prepared in ethanol. Ten microliters of an antioxidant
containing studied solution were added to 990 ml ABTS+• solution and the absorbance at
734 nm was measured. Sample absorbance was compared to a blank where 10 µl of the
solvent were added to 990 ml of the ABTS+• solution. Absorbance was measured at 5, 10,
15 and 20 min after addition of the antioxidant. All measurements were performed in
triplicate. Results were expressed in inhibition percentage versus samples concentrations
(mg/ml) at different times.
2.6.2. DPPH• scavenging capacity
The DPPH• radical scavenging capacity of studied samples (Extracts and natural products)
was measured from the bleaching of purple colored ethanol solution of DPPH•. Method
described by Hatano et al. (1988) [13] has been used. 0.5 ml of each sample concentration
was mixed with a same volume of DPPH• ethanolic solution. After incubation of 30 min in
darkness and at a temperature of 25°C, absorbance was read at 520 nm wavelength. A
mixture of 0.5 ml of DPPH• solution and 0.5 ml of ethanol was taken as a blank. Decrease in
absorption induced by the tested samples was compared to that of the positive control
Trolox. EC50 values calculated denote the concentration required to scavenge 50% of
DPPH• radicals. All measurements were performed in triplicate. Results were expressed in
inhibition percentage versus samples concentrations (mg/ml) at 30 min.
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Z. MIGHRI et al. / TJMPNP 2 (2009) 65-73
2.6.3. TEAC assay
TEAC assay was performed with minor modifications according to the original method
proposed by van den Berg et al. (1999) [14]. Solutions were prepared as the ABTS+•
scavenging activity section. Measurements were performed in triplicate at four
concentrations. The TEAC values of the antioxidant (expressed in mmol of trolox per
milligrams of samples) were calculated at 5, 10, 15 and 20 minutes after the mixing of the
reactants and by relating this decrease in absorbance to that of a trolox solution on a molar
basis.
2.7. Determination of total phenolic content
The amount of total phenolic was determined according to the method of Velioglu et al.
(1998)[15], which used Folin-Ciocalteu reagent. Tested extracts were prepared at a
concentration of 1 mg/ml. 100 µl of extract was transferred into a test tube and 0.75 ml of
Folin-Ciocalteu reagent (previously diluted 10-fold with deionised water) were added and
mixed. The mixture was allowed to stand at a temperature of 25°C for 5 min. 0.75 ml of
saturated sodium carbonate solution was added to the mixture and then mixed gently. After
standing at 25°C for 90 min, the absorbance was read at 725 nm using an UV–Vis
spectrophotometer. The standard calibration (0.01–0.05 mg/ml) curve was plotted using
catechin. The total phenolic content was expressed as catechin equivalents in milligrams per
1 g vegetable extract.
2.8. Statistical analysis
All analyses were carried out in triplicate and the data were expressed as means ±standard
deviations (SD).Correlations were obtained by Pearson’s correlation coefficient (r) in
bivariate linear correlations.
3. Results and discussion
3.1. Antibacterial activity
The in vitro antibacterial activity of the petroleum extract, the fractions and the naturel
product and fractions were qualitatively and quantitatively tested by using disc diffusion and
liquid dilution method with the microorganisms as seen in table1.
The results of the bioassays showed that extracts exhibited moderate to appreciable
antibacterial activities against all bacteria. However, this activity varies with the kind of
bacteria. The petroleum extract present an important activity against Escherichia coli
(MIC=250µg/ml) and Enterococcus fecalis (MIC=125µg/ml).
The samples showed no antimicrobial profile against the proteus mirabilis in exception the
two fatty acids (MIC=166 µg/ml) who are active against all tested bacteria.The fraction F6
possessed stronger antimicrobial activity against Escherichia coli, Staphylococcus aureus,
Enterococcus fecalis and Citrobacter freundei, and no activity against proteus mirabilis and
Citrobacter freundei. Concerning the fraction F10 was strongly inhibit the three bacteria
Staphylococcus aureus, Enterococcus fecalis and Citrobacter freundei but are resistant
Escherichia coli, Pseudomonas aeruginosa and proteus mirabilis. Cotula coronopifolia
samples appeared slightly more active against Gram-positive than Gram-negative bacteria.
This higher resistance among Gram-negative bacteria could be due to the differences in the
cell membrane of these bacterial groups.
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Table1. Antibacterial activity of Cotula coronopifolia
Microorganisms a
*
Petroleum ether extract
Fraction F6
Pentanol
Fraction F10
Hexadecanol
IZ
MIC**
IZ*
MIC**
IZ*
MIC**
IZ*
MIC**
IZ*
MIC**
EC
8
250
11.5
166
7
nt
8
1000
9.5
166
PA
9.5
500
7
nt
10
250
8
1000
10
250
SA
9.5
500
17
142
17
142
17
142
13.5
166
EF
15
125
12.5
166
12,5
142
12,5
166
15.5
166
CF
7
nt
12
166
12
166
12
166
10
333
PM
9
1000
7
nt
14
166
8
>1000
14
166
*IZ: inhibition zone (mm) including disk diameter of 6 mm.
**MIC: minimum inhibitory concentration (µg/ml).
nt : not tested.
a
Microorganisms : EC:Escherichia coli (ATCC 25922), PA:Pseudomonas aeruginosa (ATCC 27853), SA: Staphylococcus aureus (ATCC
25923), EF: Enterococcus fecalis (ATCC 29212) CF: Citrobacter freundei and PM: proteus mirabilis
Indeed, the external membrane of Gram-negative bacteria renders their surfaces highly
hydrophilic [16],whereas the lipophilic ends of the lipoteichoic acids of the cell membrane
of Gram positive bacteria may facilitate penetration by hydrophobic compounds [17].
3.2. Antifungal activity
The antifungal activity of cotula coronopifolia samples are presented in table 2. The
inhibition varied widely (0–100%) in the presence of 250µg/ml. We notice no inhibition of
the two pathogenic yeasts C. albicans and C. neoformans except the petroleum extract with
strong inhibition (MIC=500µg/ml) and the fraction F6 with weak inhibition
(MIC=750µg/ml).
Table2. Antifungal activity of Cotula coronopifolia.
Microorganismsa
I%*
Petroleum
MIC**
ether extract
I%*
Fraction F6
MIC**
I%*
Pentanol
MIC**
I%*
Fraction F10
MIC**
I%*
Hexadecanol
MIC**
TR
61
500
88
125
92
125
100
125
100
125
TS
75
250
95
125
50
500
63
500
61
500
MC
51
500
100
125
100
125
100
125
100
125
SB
59
500
100
125
93
125
73
250
73
250
AF
100
125
100
125
93
125
100
125
100
125
CA
nt
500
nt
>1000
nt
>1000
nt
>1000
nt
>1000
CN
nt
500
nt
750
nt
>1000
nt
>1000
nt
>1000
*I%: Percentage inhibition of microorganisms in the presence of 125 µg/ml of samples (0-25%,no or little inhibition; 25-50%, average
inhibition; 50-75%, strong inhibition; 75-100%, very strong inhibition)
**MIC: minimum inhibitory concentration (µg/mL)
a
Microorganisms : TR,Trichophyton rubrum; TS,Trichophyton soudanense; MC, Microsporum canis ; SB, Scopulariopsis brevicaulis;
AF ,Aspergillus fumigatus ;CA,Candida albicans; CN,Cryptococcus neoformans.
nt, not tested.
The results of the bioassays showed that extracts and the naturel product exhibited an
interesting antifungal activity against all fungi except the pathogenic yeasts C. albicans and
C. neoformans.. Indeed samples schowed strong to very strong inhibition ( 50-100%). The
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results of MIC confirm those obtained using the percentage inhibition method.
In this study, the dermatophytes was clearly the most sensitive fungal specie and the yeasts
was resistant.
3.3. Radical cation ABTS֥ scavenging activity
Results of our investigation on ABTS֥ cation radicals scavenging activity of extract,
fractions and pure natural products from Cotula coronopifolia are represented in Table 3.
Table 3. ABTS֥ and DPPH. inhibition percentage in the presence of different concentrations of Cotula
coronopifolia..
ABTS percentage inhibition
Concentration
Time (min)
(mg.mL-1)
5
10
15
20
79.9±0.4 78.5±0.3 79.8±0.6 84.1±1
1.00
49.7±0.3 62.3±0.3 67.4±0.3 66.7±06
0.50
37.4±1.4 50.1±0.8 54.8±1.2 58.8±1.4
0.25
Petroleum
25.3±0.4 39.2±1.7 53.9±0.4 57.3±0.4
0.12
ether extract
19±1.2
32.2±0.8 50.5±0.8 52.5±0.7
0.06
6.2±0.6 21.6±1.2 32±0.9
35.7±0.4
0.03
DPPH
30
79.4±0.1
77.9±0.6
73.8±1.2
53.8±1
48.2±0.9
33.2±0.2
F6
1.00
0.50
0.25
0.12
0.06
0.03
70.7±1.2
56±0.1
51.1±1.2
43.9±0.1
20.5±0.3
10.1±0.6
79.6±0.8
71.5±0.3
68.5±0.9
57.8±0.2
40.4±0.4
20±1.8
81.3±0.3
69.9±0.2
68.8±1.4
56.7±0.1
41±0.6
24.3±0.3
83±0.21
71.6±0.2
70.3±0.4
48.2±0.4
40.7±0.1
16.3±0.3
97.7±0.5
95.8±0.2
92.6±1.2
91.7±0.5
89.3±0.8
87.5±1.6
Pentanol
1.00
0.50
0.25
0.12
0.06
0.03
72.2±1.5
70±0.5
52.7±0.2
32.2±0.4
21.7±0.3
2.2±0.3
80.2±0.4
67±0.5
48.1±0.3
34.9±0.3
23.2±0.4
2.5±0.3
77.5±0.9
70.1±0.5
52.6±0.8
33.1±1.2
19.8±0.1
1.46±0.3
78.6±0.4
62.8±0.2
53.6±0.4
48.2±0.2
30.5±0.4
17.7±0.1
83.7±0.8
75.8±0.4
50.6±0.1
33.1±5.8
8.2±0.7
3.8±0.1
F10
1.00
0.50
0.25
0.12
0.06
0.03
77.7±0.5
66.5±0.2
50.1±0.8
35.1±0.7
20.3±1.4
2.6±2.1
83.5±0.5
68.8±0.3
54.4±0.6
39.15±0.5
23.6±0.5
11.5±0.2
82.3±0.3
70.2±0.2
67.9±1.1
52.2±0.3
35.1±0.4
6.3±0.3
83.8±0.4
69.1±0.4
55.1±0.2
39.1±0.3
26.1±0.4
12.03±0.2
69.1±0.8
66.7±0.7
65.3±0.9
64.5±2.7
65.1±0.3
62.5±0.3
Hexadecanol
1.00
0.50
0.25
0.12
0.06
0.03
79±0.2
68.5±0.1
56.1±0.5
41.3±0.6
17.5±1.1
2.93±0.3
79.3±0.3
64.5±0.3
51.6±0.5
34.4±0.6
16.6±0.3
1.2±0.2
80.3±0.4
67.7±0.3
53.2±0.5
40.3±0.3
20±0.4
4±0.3
83.6±0.4
68.5±1.2
51.9±0.3
37.5±0.3
22.7±0.4
12.7±1.2
67.4±0.3
63.3±0.6
57.1±0.3
44.4±0.6
33.8±0.39
22.3±1.7
We note that inhibition of samples was function of concentration and was dependent of time.
The Petroleum extract (E1) shows the heights inhibition, with values rising from 35.7–
84.1% at different concentrations and after 20 minutes of reaction.
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The antioxidant activity of Cotula coronopifolia at 1g/ml reached its maximum at 20min,
fraction F6 (83%), fraction F10 (83,8%), pentanol (78,6%), hexadecanol (83,6%).
At feeble concentration (0.03 mgmL-1) and immediately after 5 min of incubation the new
petroleum extract display a value of 6.2% of ABTS֥ inhibition. Compared to Trolox
(97.9%inhibition at 1mgmL-1and after 5 min of incubation), the petroleum extract is a
powerful antioxidant substance against ABTS֥ cation radicals at a concentration of
1mgmL-1.
3.4. DPPH• scavenging capacity
Results of the antioxidant activity of Cotula coronopifolia extract, the two fractions and the
two fatty acids are represented by Table 4. Compared with trolox as a standard reference
product (99.3% inhibition at 1mgmL-1 and after 30 min of incubation), We note that
inhibition of samples was function of concentration. All tested samples were active against
DPPH• radicals. With weak concentrations the percentage of inhibition decreases
considerably and those for the pentanol (3,8%) and the Hexadecanol (22,3%). We note that
the fraction F6 was the most active one even at feeble concentration 0.06 mgmL-1, with
inhibition reactivity similar to that of trolox and with a maximal inhibition percentage of
97,7% at a concentration of 1mg.ml-1.
The in vitro test of samples against toxic radical entities ABTS֥ and DPPH confirmed that
this plant cotula coronopifolia is a good source of compounds that could help to increase the
overall antioxidant capacity of an organism.
The results of the DPPH• scavenging capacity of Cotula coronopifolia samples were in
agreement with that recorded in the ABTS֥ cation radicals test. Differences in inhibition
percentage values between the two methods may due to the difference in the reactivity of the
two radical forms towards Cotula coronopifolia samples.
Values of IC50 calculated for Cotula coronopifolia samples (Table 4) confirm the reactivity
of these samples against DPPH• free radicals. IC50 values of 0.15, 0.002, 0.002, 0.22 and
0.17, mgml-1 respectively for petroleum extract, fraction F6,fraction F10, pentanol and
hexadecanol, indicated a good inhibition of these samples versus DPPH• free radicals.
3.5. TEAC assay
Cotula coronopifolia samples values of the Trolox Equivalent Antioxidant Capacity (TEAC)
at 5, 10, 15, and 20 minutes were represented in Table 4. The values of the TEAC analysis at
20 minutes show that fraction F10 and hexadecanol possessed the highest antioxidant
capacity (2.01 mM trolox/mg sample), followed by the petroleum extract and fraction F6
(1.99 mM trolox/mg sample), the pentanol (1.89 mM trolox/mg sample).
Table 4. TEAC, total phenolic content, IC50 of DPPH• scavenging capacity of Cotula coronopifolia samples
Petroleum
extract
F6
Pentanol
F10
Hexadecanol
TEAC (mM equivalent Trolox
per mg of sample)
Time (min)
5
10
15
20
1.44
1.88
1.92
1.99
1.7
1.73
1.87
1.9
1.91
1.92
2.01
1.91
1.95
1.86
1.98
1.93
1.99
1.89
2.01
2.01
Total phenolic content
(mg catechin/g sample)
DPPH
IC50
(mg/ml)
308.65
0.15
713.06
356.57
306.23
127.9
0.002
0.22
0.002
0.17
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3.6. Determination of total phenolic content
The contents of total phenols content of the different extracts issued from Cotula
coronopifolia was evaluated, using the Folin Ciocalteu method (Table 4).The amount of
total phenolic compounds ranged from127.9 to 713.06 mg Catechin/g sample. The Fraction
F6 contained the highest phenolic content (713.06mg Catechin/g extract). A positive linear
correlation between the total antioxidant activity, determined by the TEAC method, and
phenolic content of Cotula coronopifolia samples was observed (Table 4; Fig. 1).
To evaluate the suitability and reliability of antioxidant activity and total phenolic content,
the coefficient of determination (R2) was calculated and represented by figure 1.
Fig. 1 Correlation between the antioxidant capacity and total phenolic content of Cotula coronopifolia
We note that correlation coefficient were feeble (R2 = 0,0147). Although of the important
antioxidant capacity, we note that the majority of tested samples present a moyen total
phenolic content. This antioxidant activity may be caused by other metabolites. These
metabolites may be vitamins as vitamin E and C or carotenoids or metals ions as Se, Zn, Cu,
Mn and Fe or enzymes implicated in antioxidant mechanisms.
4. Conclusion
In conclusion our observations confirm that petroleum extract, fractions and the two fatty
acids possess strong antimicrobial and antioxidant activities.
Future studies are required to determine the types of other bioactive compounds in the plant
Cotula coronopifolia, as well as the synergistic effects responsible for the antimicrobial and
antioxidant activity of the plant Cotula coronopifolia.
Due to the high antioxidant, antimicrobial and antifungal activities the Cotula coronopifolia
samples have promising potential as natural antioxidants in the food industries, in the
preservation of foodstuffs against a range of food-related bacterial and fungal species or in
the pharmaceutical and cosmetic industries.
Acknowledgements
The authors are grateful to Pr. Aouni Mahjoub (Laboratory of Transmissible Diseases and
Biologically Active Substances, Faculty of Pharmacy, Monastir, Tunisia.) for assistance in
antibacterial assays.
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