Biological activities of essential oils extracted from Thymus capitatus (Lamiaceae)

South African Journal of Botany 128 (2020) 1
9

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Biological activities of essential oils extracted from Thymus capitatus (Lamiaceae) Mohamed Bilal Goudjila,d,*, Souad Zighmib,d, Djamila Hamadaa,d, Zineb Mahcenec, Salah Eddine Bencheikha,e, Segni Ladjela,d a

Applied Sciences Faculty, Process Engineering Laboratory, Ouargla University, Ouargla 30000, Algeria Sciences of the nature and life Faculty, Engineering Laboratory of Water and Environment in Middle Saharian Laboratory, Ouargla University, Ouargla 30000, Algeria c Sciences of the nature and life Faculty, Protection of Ecosystems in Arid and Semi-Arid Zones Laboratory, Ouargla University, Ouargla 30000, Algeria d Applied Sciences Faculty, Process Engineering Department, Ouargla University, Ouargla 30000, Algeria e Science and Technology Faculty, Process Engineering Department, Ghardaia University, Ghardaia, Algeria b

A R T I C L E

I N F O

Article History: Received 23 July 2019 Revised 3 November 2019 Accepted 17 November 2019 Available online xxx Edited by I Vermaak Keywords: Antioxidant activities Antibacterial activity Antifungal activity Chemical composition Essential oil Thymus capitatus GC/MS

A B S T R A C T

The present study aims to investigating, the chemical composition of the essential oil extracted from Thymus capitatus harvested in west, Algeria. It seeks the other objective to the determination of antioxidant, antibacterial and antifungal activities of oil in order to find new metabolite products, which are characterized by a biological activity. The investigations and research was carried out on the essential oil (EO) extracted from the dried leaves of Thymus Capitatus Lamiaceae that was harvested in the region of Tiart (West of Algeria). GC
MS analysis of EO is identified 22 components while thymol, carvacrol and g -terpinene are the major components. The antioxidant activity of EO was evaluated by three methods: the DPPH test (2,2-diphenyl-11-picrirylhydrazil), the FRAP (Ferric reducing antioxidant power) and TAC (Total Antioxidant Capacity) test and compared with ascorbic acid, BHA (Butylated hydroxy anisole) and commercial thymol as standards. The results indicated that Thymus Capitatus oil showed higher antioxidant power in comparison with the standards with values of (0.61, 2.13 and 0.78) mg/mL for the DPPH, FRAP and TAC tests respectively. Also, the antibacterial activity of the oil was tested using the agar disc diffusion method, by determining the inhibition zone and the minimum inhibitory concentration (MIC). In this case, the results have shown a potential of the antibacterial activity than commercial antibiotics (Chloramphenicol, Cefoxitin, Gentamycin) against the tested strains, with MIC values of (29.44, 14.72, 14.72 and 7.36 mg/mL) for Staphylococcus aureus, S. typhi, E. coli and Streptococcus pneumoniae strains respectively. Furthermore, the antifungal study on the fungi of Cladosporium herbarum, Alternaria infectoria, Aspergillus Ochraceus, Trichophyton Sp reported significant results. The effectiveness of the oil showed that the essential oil has a fungicidal effect against the strains Cladosporium herbarum, Alternaria infectoria and a fungistatic effect against strains Aspergillus ochraceus and Trichophyton rubrum. The research outcomes clearly demonstrate that the essential oils of thymus capitatus can present an interesting alternative naturel which can be useful for food preservation and pharmaceutical treatment. © 2019 SAAB. Published by Elsevier B.V. All rights reserved.

1. Introduction The history of medicinal and aromatic plants is linked to the evolution of civilizations. Everywhere in world, the history of peoples shows that these plants always occupied an important place in medicine, in perfumes composition and culinary preparations. The therapeutic utilization of plants is an integral part of the traditions of all cultures. The medicinal value of these practices includes the isolation and identification of new molecules. These compounds

* Corresponding author at: Department of Process Engineering, Faculty of Applied Sciences, University of Ouargla, 30000, Algeria. E-mail address: [email protected] (M.B. Goudjil). https://doi.org/10.1016/j.sajb.2019.11.020 0254-6299/© 2019 SAAB. Published by Elsevier B.V. All rights reserved.

constantly offer new alternatives to modern medicine. In addition, these generally have the advantage of being less toxic than their counterparts of synthetic origin. (Goudjil, 2016) Different aromatic plants are characterized by the biosynthesis of odorant and volatile molecules, which are called essential oils (Bereksi Reguig, 2017). The latter are used as a source of bioactive molecules of natural origin that benefit from biological activities, especially antimicrobial, antioxidant, antiseptic and anti-inflammatory activities (Goudjil, 2016; Jaouadi et al., 2018) Essential oils (EO) are a source of bioactive molecules and are widely studied for their potential use as an alternative to synthetic products in the treatment of infectious diseases and various pathological conditions associated with oxidative stress.

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The lamiaceae family is one of the most widely used families as a global source of spices and extracts with strong antimicrobial and antioxidant properties. In this family, the genus thymus gathers several species widely distributed in the Mediterranean area and used as antibacterial and anti-inflammatory in the traditional pharmacopeia of the region. T. capitatus a dwarf shrub with a strongly aromatic odor of 20
50 cm tall, with upright erect branches, woody, light-colored, young white felted, often only leafy underarm clumps (Bayer et al., 2009). In traditional medicine, T. capitatus is used for its antiseptic, antibacterial, antispasmodic, anticytotoxique benefits (Bayer et al., 2009; D
zamic et al., 2015; El Ajjouri et al. 2008; Kaileh et al., 2007). Because of these ethno-botanical surveys, the most significant side of this study was the ability to determine the chemical composition of leaves essential oils and the evaluation of their biological activities.

This method is utilized to evaluate the scavenger effect of the essential oil of the plant against DPPH radical was according to literature (Braca et al., 2002; Mighri et al., 2010), with some modifications (essential oil diluted in absolute ethanol). 1 ml of EO at different concentrations diluted in ethanol were added to 1 ml of the DPPH solution prepared at 0.4 mM in ethanol. After 30 min of incubation in the dark, the absorbance reading at 517 nm; The mixture of 1 ml of the DPPH solution and 1 ml of ethanol is taken as control product. The reduced level of these molecules by DPPH is expressed in percentage according to the following formula: I% ¼ ðA0
Ae =A0 Þ  100 In which A0 is the absorbance of the control reaction and Ae is the absorbance of the Sample. As an indication, the ascorbic acid, BHA and thymol as the standards known for its anti-radical effect were tested in parallel. As for the inhibitory concentrations (IC50), they are calculated from the curves of linear regression. Tests were carried out in triplicate.

2. Material and methods 2.1. Vegetal material The plant material that is used during the realization of this work consists of T. capitatus leaves species harvested in February 2019 in the region of Tiaret (Western Algeria) coordinates (N 35.362222 °E 1.285555 °) and then dried in the shade at an ambient temperature during 07 days. A specimen is deposited at the university herbarium under number GO2019-2 2.2. Essential oil extraction The Extraction of the EO was achieved by hydrodistillation in a Clevenger apparatus where 100 g of dry leaves immersed in a 1000 ml flask of water for 3 h. The EO obtained is stored in a refrigerator at 4 °C. 2.3. Gas chromatography-mass spectrometry analysis The analysis of T. capitatus EO was performed at the L.G.P (Process Engineering laboratory) in Kasdi Merbah Ouargla University. The gas chromatograph adopted is a Bruker SCION 436 GC, coupled to a mass spectrometer quadrupole ionization voltage of 70 ev. The column that is used is an HP-5MS; 5% Phenyl Methyl Siloxane with a length of 30 m and an internal diameter of 0.25 mm. The wire thickness being 0.25 mm. The operating conditions are: - The temperature of the injector (split mode 1:50): 250 °C - Temperature programming: from 50 °C to 280 °C at a rate of 5 °C/min. - The vector gas used is helium with a flow rate of 1.2 ml/min. The temperatures of the quadrupole source are fixed, respectively, at 250 °C and 280 °C. Linear retention indices (RI) for all compounds were determined using n-alkanes as standards. Identification of individual compounds was performed by matching their mass spectral fragmentation patterns with corresponding data available (Wiley 275 library (6th edition)). 3. Biological activities of thymus capitatus essential oil

3.1.2. Ferric reducing power The iron reducing activity of EO was determined according to the method described by Oyaizu (1986). A volume of 2.5 ml of phosphate buffer (0.2 M, pH 6.6) was added to a 1 ml of various concentrations of plant extract. It is followed by 2.5 ml of potassium ferricyanide (1%). After stirring, the mixture was incubated at 50 °C for 20 min. Then, 2.5 mL of 10% trichloroacetic acid was added to the mixture, which was centrifuged at 3000 rpm for 10 min. After that, 2.5 mL of this mixture was added to 2.5 mL distilled water and 0.5 mL ferric chloride (0.1%) vigorously mixed. Finally, the absorbance was measured at 700 nm. Essential oils were diluted in ethanol. Blank sample is similarly prepared by replacing extracted with ethanol which is used to calibrate the instrument (UV
VIS spectrophotometer). Ascorbic acid, BHA and thymol (in the 10
100 mg mL
1 range) were employed as a positive control where the absorbance was measured in the same conditions as the samples. The EC50 value is the effective concentration at which the absorbance was 0.5 for reducing power and was obtained by interpolation from linear regression analysis (Piaru et al., 2012). Increased absorbance of the reaction mixture indicated an increased reducing power (Singh et al., 2015). Tests were carried out in triplicate. 3.1.3. Total antioxidant capacity (TAC) The total antioxidant capacity (TAC) of EO was evaluated by the phosphomolybdenum method (Chang et al., 1999). It is based on the reduction of molybdates to molybdenum in the presence of the extracts, which giving a green color detectable by UV at a wavelength of 695 nm. 0.2 ml of different concentration of plant extract was mixed with 2 mL of reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). The tubes are screwed and incubated at 95 °C for 90 min. After cooling, the solutions’ absorbance is measured at 695 nm against the blank which contains 2 mL of the reagent solution and 0.2 mL of ethanol and is incubated under the same conditions as the sample. The EC50 value is the effective concentration that reduces molybdenum in an absorbance of 0.5. It is obtained by interpolation from the linear regression analysis. In addition, ascorbic acid, BHA and thymol were used as a positive control where the absorbance was measured in the same conditions as the samples. Those tests were carried out in triplicate. 3.2. Antimicrobial activity

3.1. Antioxidant activity 3.1.1. Free radical scavenging effect The DPPH test (diphenyl picrylhydrazyl) is a widely used method in the analysis of the antioxidant activity.

3.2.1. Antibacterial activity The microbiological material consists of four pathogenic bacterial strains responsible for certain serious infectious diseases. These bacteria are E. coli, S. typhi, Staphylococcus aureus and Streptococcus

M.B. Goudjil et al. / South African Journal of Botany 128 (2020) 1
9 Table 1 Antibiotic used for each bacterium. Species

Positive control

E. coli and S. typhi S. aureus S. pneumoniae

CHLORAMPHENICOL CEFOXITIN GENTAMYCIN

pneumoniae . They come from Microbiology Laboratory in Kasdi Merbah University, Ouargla. 3.2.1.1. Agar diffusion method. The antibacterial effect evaluation of EO is tested by the agar diffusion method according to the NCCLS recommendations (NCCLS 2019). The bacterial strains are prepared in appropriate culture media and adapted to standards. Then disks impregnated in the EO are deposited on the surface of these media and incubated at 37 °C for 24 h. All tests are carried out three times. The results are read by measuring the translucent halo diameter (inhibition zone) around the disc. Antibiotic discs are used as a control reference (Table 1). 3.2.1.2. Determination of the minimum inhibitory concentration (MIC). TaggedPThe MIC of our EO was determined, according to Benabderrahmane et al. (2009), with slight modifications. The EO is diluted in DMSO (dimethyl sulfoxide) to obtain a concentration range of 1, 1/2, 1/4, 1/ 8, 1/16, 1/32, 1/64 and 1/128 mg/mL, then incorporated into 6.0 mm diameter disks with 10 mL of extract. The same volume of DMSO is used as a control. Microbial suspensions are prepared according to the following standards: 0.5 McFarland which is equivalent to (108 CFU/mL)(Jehl et al., 2016); 0.1 mL inoculum was immediately inoculated in the agar using a sterile swab. Disks containing the different concentrations of EO is placed directly on the agar surface. The petri dishes were incubated in an oven at 37 °C. for 24 h. The MIC represents the lowest concentration of EO to completely inhibit the growth of microorganisms tested around the discs. All tests are repeated three times. 3.2.2. Antifungal activity The microbiological material consists of four strains of fungi. They are Cladosporium herbarum, Alternaria infectoria, Trichophyton rubrum and Aspergillus ochraceus, which cause considerable losses of production in several varieties of plants. The species come from Microbiology Laboratory in Kasdi Merbah University, Ouargla. 3.2.2.1. Direct contact method. The antifungal activity evaluation of EO is adopted by the direct contact method in which the five concentrations are obtained by adding 7.5, 15, 75.150 and 225 mL of EO upon 30 mL warm Potatoes Dextrose Agar (PDA) in a vial with adding drops of Tween 20 (concentration of 0.5%). The technique consists of adding oil at different concentrations (0.025%, 0.05%, 0.25%, 0.5% and 0.75%) to the still liquid culture medium followed by 5 min of stirring in order to homogenize the medium PDA with the oil essential. After shaking the vials, the mixture (PDA + HE + Tween 20) is poured into petri dishes. The inoculation is done under the hood by depositing a mycelial disk that is about 0.6 cm of diameter in the center of the box; the controls (fungal PDA Tween 20 strains) are made under the same conditions without essential oil and the measurements are taken after 72 h of incubation. These boxes (controls and tests) are incubated at 25 § 2 °C respectively for 12 days(Goudjil et al., 2016; Mohammedi and Atik 2012). All tests, are restarted three times.

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3.2.2.2. Inhibition rate (TI%). Calculating the percentage of growth inhibition relative to the control makes it possible to evaluate the effect of oil concentrations on fungal growth. The technique involves measuring the diameters of different fungal colonies after incubation time required (Kordali et al., 2003). TI ð%Þ ¼ 100  ðdC
dEÞ=dC TI (%) = inhibition rate expressed as a percentage dC = Colony diameter in "positive control" boxes dE = Diameter of colonies in the boxes containing the plant extract 3.2.2.3. Determination of mycelial growth rate (VC). According to Cahagnier and Richard-Molard (1998), the rate of mycelial growth of each concentration is determined by the formula: VC ¼ ½D1=Te1 þ ½ðD2
D1Þ=Te2 þ ½ðD3
D2Þ=Te3 þ . . . þ ½ðDn
Dn
1 Þ=Ten D: Diameter of the growing zone of each day. Te: incubation time. 4. Results and discussions 4.1. Essential oils analysis EO yield of T. capitatus was determined at a rate of 1.56%. The characterization of essential oils was performed by GC / MS, which allows to simultaneously determine the number of oil components, their respective concentrations and their output orders which provide information on the volatility; that is to say their molecular weights. The peaks of the chromatogram of the plant essential oil are compared with those of the reference compounds present in a spectrum library with a computerized data bank. The GC / MS apparatus gave us the different mass spectra and the indices of retention of substances that may constitute this extract (Goudjil 2016). According to the GC-MS results, T. capitatus EO consists of 22 components (Table 2), whose thymol, carvacrol and g -terpinene respectively are regarded major components (Fig. 1). We found that essential oil is thymol chymotype. Table 2 Chemical composition of the Algerian T. capitatus EO. Compounds

RT

T. Capitatus%

a-thujene a-Pinene

1,716 1,804 1,99 2,465 2,852 3,479 4,047 6,52 7,557 8,038 8,335 8,53 9,372 9,589 9,64 9,703 9961 10,057 10,434 10,461 14,564 16,414

0,38 0,91 0,2 1,49 1,78 10,3 2,29 0,37 51,22 12,59 0,26 2,01 0,34 0,3 0,12 0,24 0,23 0,14 0,87 1,21 1,92 9,04 98,21

Camphene b-Myrcene a-terpinene g -Terpinene Linalool Terpineol Thymol Carvacrol a-gurjunene Caryophyllene (+)-Ledene b-Bisabolene b-copaene delta-Amorphene a-Bisabolene Elemicin (-)-Spathulenol Caryophyllene oxide Pentadecanoic acid trans-13-Octadecenoic acid Total RI : retention indices relative

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M.B. Goudjil et al. / South African Journal of Botany 128 (2020) 1
9 Table 3 Chemical composition of T. capitatus EO in other world regions. Country

year

Ref

Major compositions

rie Alge

2015

(YAHIA and SAMIRA 2015)

g -terpinene (4.3%), p-cymene (12.4%), a-thujone(0.2%)

2018

(Chaima and Ghania 2018)

ne carvacrol (78.78%), p-cyme ne (0.71%) (6.62%), a-pine ne(5%), carvacrol (81%), p-cyme g -terpinene (3.2%), ne carvacrol (57%) g -terpine ne (8.6%) (6.9%), p-cyme carvacrol (81.52%
78.40%), ne ne (4.98%), g -terpine p-cime (3.13%) carvacrol (81.2
14.2%), g -terpinene (34.4
2.6%), ne (22.8
5.0%) p-cyme ne carvacrol (13.4%), p-cyme ne (2.9%) (18.9%), a-pine ne (2.31%), carvacrol p-cyme ne (0.67%) (65.96%), g -terpine ne carvacrol (55.59%), p-cyme ne (0.56%) (11.23%), a-pine ne carvacrol (68.8%), a-pine ne (11.1%). (12.5%), p-cyme ne thymol (89.06%), p-cyme ne (3.19%). (5.04%), g -terpine carvacrol (70%), b-caryophyllene ne (4.3%). (8.5%), g -terpine carvacrol (58.66
81.49%), ne (3.83
13.17%), p-cyme g -terpinene (7.81
3.16%). ne carvacrol (74.31%), g -terpine ne (5.29%). (6.48%), p-cyme carvacrol (68.19%), thymol (12.29%) carvacrol (68.19%), thymol ne (3.09%) (12.29%), g -terpine

2007

(Usai et al., 2010)

Fig. 1. Representation of the structure of the majority compositions.

Italie

2008

(Usai et al., 2010)

Recently, T. capitatus EO has been carefully studied and the diversity in composition from plants have been grown in different countries and even those from different localities in the same country have resulted in many chemotypes (Table 3). The essential oil of T. capitatus from western Algeria is composed mainly of thymol (51.22%), carvacrol (12.59%) and g -terpinene (10.3%) which present 74.11% of the total composition of oil. The composition of EO has shown trends not similar to those published for other geographic areas. According to Miri and Djenane (2018), T. capitatus EO that harvests Mesghana, Tizi Ouzou consists of thymol (25.82%), linalool (23.40%), and geraniol (14.22%) In addition, T. capitatus of Morocco is composed mainly of carvacrol (55.59%), followed by p-cymene (11.23%) and a-pinene (0.56%) (Aissaoui et al., 2018). on the other hand, this chemical composition is substantially similar to that of a native EO Tunisia that are constituted mainly of carvacrol (58,66
81,49%), p-cymene (3,83
13,17%), the g -terpinene (7,81
3,16%) (Hosni et al., 2013). (Giweli et al., 2016) declare that, Thymus capitatus of Zintan-Lybie comprises mainly carvacrol (68.19%), thymol (12.29%), g -terpinene (3.09%). Also the EO of Colombia is formed mainly of carvacrol (59.3%), p-cymene (13.2%), g -terpinene (8.7%) and thymol (6.4%) (Martínez et al., 2018). Generally, it is concluded that the variation in the chemical composition of essential oils could be attributed to the geographical origin of the plant, the extraction technique, the time of harvest and climatic factors (Fidan et al., 2019; Figueiredo et al., 2008).

2013

(Russo et al., 2013)

2015

(Casiglia et al., 2015)

2016

(El Ouadi et al. 2016)

2018

(AINANE et al. 2018)

2018

(Aissaoui et al., 2018)

2010

(Akrout et al., 2010)

2010

(Mkaddem et al., 2010)

2011

(Moujahed et al., 2011)

2013

(Hosni et al., 2013)

2018

(Jayari et al., 2018a)

2015

(D
zamic et al., 2015)

2016

(Giweli et al., 2016)

4.2. Biological activities 4.2.1. Antioxidant activity The antioxidant activity was evaluated by several methods. These methods are based exclusively on the reducing capacity or trapping radicals as an indicator of its antioxidant potential (Javanmardi et al., ~ o et al., 2007). The identification of antioxidant activity in 2003; Villan vitro of the EO of the plant tested was produced by three methods: the scavenger effect against DPPH radical, the reduction of iron and the total antioxidant capacity. Results of the antioxidant activities of T. capitatus EO are summarized in table 4

Maroc

Tunisie

Libya

4.2.1.1. Scavenger effect of the radical DPPH. The antiradical activity was evaluated by using DPPH (2,2-diphenyl-1-picrylhydrazil) which was one of the first free radicals that are used to study the structureantioxidant activity relationship. (Brand-Williams et al., 1995). It is a stable radical, purple solution and having a characteristic absorption maximum at 517 nm. The routine protocol that is applied based on the disappearance of the maximum when DPPH is reduced by an anti-radical property compound, causing discoloration to yellow. The figures below (Fig. 2) show the effectiveness of the EO, BHA, Thymol and A. acid to trap the DPPH radical, this efficiency has resulted in the inhibition rate according to the different concentrations. The IC50 value (otherwise known as the 50% inhibitory concentration) of oil and the standards used is determined and summarized in Table 4. It is defined as the concentration of the sample required to give a 50% decrease in the absorbance of the initial DPPH solution.

Fig. 2. Antiradical activity of T. capitatus EO and the standards.

M.B. Goudjil et al. / South African Journal of Botany 128 (2020) 1
9 Table 4 the antioxidant activities of T. capitatus EO. antioxidant power (mg/ml)

A. acid

Scavenger effect of DPPH 6.42 § 0.36 IC50 Ferric reducing power EC50 66,73 § 0.25 Total antioxidant capacity (TAC) EC50 84.33 § 0.04

BHA

Thymol

T. capitatus

13.58 § 0.06

4.84 § 0.21

0.619 § 0.11

57.47 § 0.19

15.25 § 0.16

2,13 § 0.07

52.62 § 0.03

9.54 § 0.08

0.78 § 0.14

BHA: Butylated hydroxy anisole.

The IC50 are inversely proportional to the scavenger effect whose low values reflect a significant anti-radical effect(Laghouiter et al., 2015). According to the outcomes, it is observed that, the antiradical power has been increased with increasing of oil concentrations. As well, it seems that the EO is the most effective antioxidant as A. acid, BHA and Thymol with an IC50 equal to 0.619, 6.427, 13.58 and 4.84 mg / ml, respectively. Contradictory of our results, it has been reported by several authors that synthetic antioxidants have more ability to scavenge DPPH radical as EO (Kizil et al., 2010; Nanasombat and Wimuttigosol 2011; Tepe et al., 2005). 4.2.1.2. Ferric reducing power. Several previous studies have shown that the reducing power of a compound can serve as a significant indicator of its potential antioxidant activity. Its principle is based on the ability of a compound to donate an electron.(Balasundram et al., 2005; Yi et al., 2008). The reducing power of iron is based on the reduction of the ferric ion (Fe3+) present in the [K3Fe(CN)6], complex to ferrous (Fe2+) ion which causes the transformation of the yellow color of ferricyanide of potassium to a blue color in a reaction medium at 700 nm, the intensity of which depends on the reducing power of the EO. The results of the EO reductive activity, ascorbic acid, BHA and thymol are presented in the curves below. As with the antiradical activity, the concentration of the EO sample has a highly significant effect on the reducing power. From Fig. 3, the reducing power is proportional to the antioxidant concentration. Similar observations have been reported by many authors (Balasundram et al., 2005; Cherrat et al., 2014; Yi et al., 2008). The figure above indicates that the thymus EO has a more effective antioxidant activity than that of A. acid, BHA and Thymol where the EC50 values are 2.13 mg/ml for T. capitatus. Whereas the A. acid, BHA and Thymol, as a control product, gave a value of 66.73, 57.47 and 15.253 mg/ml. 4.2.1.3. Total antioxidant capacity (TAC). This technique is based on the reduction of molybdenum Mo (VI) present in the form of molybdate MoO42
to molybdenum Mo (V) MoO2+ ions in the presence of

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the extract to form a green phosphate / Mo (V) complex at pH acid (Prieto et al., 1999). The EC50 value is the effective concentration that reduces molybdenum in an absorbance of 0.5. It is obtained by interpolation from the linear regression analysis. However, an increase in absorbance corresponds to an increase in the reducing power of the extracts tested. From Fig. 4, it is observed that the HE of T. capitatus has a higher reductive activity than positive controls used (Table 4). According to current results, it is difficult to attribute the antioxidant effect of EO to one or some compounds because from the chemical point of view, an EO is a mixture of several tens of compounds. This complexity often makes it difficult to explain the mode EO activity. However, studies conducted by some researchers have shown that the antioxidant activity of essential oils may be greater than that of the majority of compounds that are tested separately (Bakkali et al., 2008, Safaei-Ghomi et al., 2009) which confirms our results that the reducer and antiradical power of T. capitatus essential oil are famously superior to that of thymol. The studies accomplished by (Marin et al., 2018; Megdiche-Ksouri et al., 2015) concerning the antioxidant activity of EO of T. capitatus from different regions show a very powerful antioxidant power of this oil with an IC50 of the order of 7 and 1.03 mg/ml respectively compared to positive control. (Laghouiter et al., 2015; Laib and Barkat 2011) explain that this domination of the antioxidant power of essences comparing to major component was confirmed by the existence of the synergistic effect that the minor components could bring to the activity of EO. The synergistic interactions between the different constituents of an essential oil are form the origin of a much greater antioxidant power. As well, Amarti et al. (2011) indicate that the synergistic interactions between the different constituents of an essential oil are the origin of its much greater antioxidant power. Indeed, certain compounds other than phenolic compounds such as g -terpinene have also a strong antioxidant activity. In addition, (Ballester-Costa et al., 2017), show that strong radical scavenging potential capacity, measured with DPPH assays, of the EO analyzed could explained by the occurrence of hydroxylated compounds such as terpenoids in their composition. According to (Hinneburg et al., 2006), the reducing power of an extract may be related to the ability of substances to transfer electrons into the reaction medium. For cons, the scavenger activity of an extract can be attributed to the structure of its active compounds that determine their ability to donate a proton (Gulcin et al., 2004) this could be causing the observed results. In several reports, the antioxidant activity of EO can be related to the phenolic content. Phenolic compounds by virtue of their oxidation
reduction properties act as reducing agents, donors of hydrogen and singular oxygen (Amarti et al., 2011). Thus a correlation exists between the essences antioxidant activity and the content of oxygenated monoterpenes (Miladi et al., 2013).

Fig. 3. Reducing power of the essential oil of Thymus capitatus, BHA, thymol and ascorbic acid.

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Fig. 4. The total antioxidant capacity essential oil of Thymus capitatus, BHA, thymol and ascorbic acid.

Indeed, the comparative study on the reduction ability of the DPPH radical by different chemotypes has proved that the phenotypic chemotypes show in vitro antioxidant capacities more expressed and stronger than the non-phenolic chemotypes. (Jukic and Milo
s 2005). The results obtained in the present study confirm the existence of a certain correlation between the content of phenolic compounds and the anti-radical activity. Djeddi et al. (2015), declare that the EO of species belonging to the genus thymus are rich in phenolic monoterpenes such as thymol and carvacrol and the presence of carvacrol at a large percentage in the composition of the essential oil leads to moderate inhibition of the free radical DPPH. 4.2.2. Antimicrobial activities 4.2.2.1. Antibacterial activity. The results of the antibacterial activity of T. capitatus EO against bacteria are summarized in table 5. The inhibitory action results in the appearance of a transparent halo (inhibition zone) around the paper disc impregnated with crude extract studied. It is noticed that, the inhibition zone diameter differs from one bacterium to another as has been reported in literature. We considered that EO has a bacteriostatic or bactericidal action if its inhibition diameter is greater than 8 mm (Moreira et al., 2005). Indeed, the essential oil of T. capitatus showed an important inhibitory effect against the microorganisms studied. It is found that the inhibition zone diameter decreases as a function of the different concentrations, which shows that the antibacterial power is inversely potential for the dilution (the effect decreases with the increase in the dilution of the oil). According to the table (05), we find that the inhibition zones of T. capitatus are important compared to the antibiotics used which shows their antibacterial power, the indicated results are the means of the three measurements. The most sensitive microorganisms to this EO were S. pneumoniae gram-negative and gram-positive E. coli whose growth was arrested at minimum inhibitory concentration (7.36 and 14.72)

Table 5 Antibacterial activity of T. capitatus EO. Values are given as mean § SD (n = 3). Micro-organisms

Gram negative E. coli S. typhi Gram positive Staphylococcus aureus Streptococcus pneumoniae

Inhibition zone (mm) disk diffusion assay

Antibiotic assay

40.00 § 0.32 32.33 § 0.46

24.00 § 0.52 25.13 § 0.22

37.26 § 0.56 57.03 § 0.12

17.33 § 0.76 44.63 § 0.52

mg / mL respectively. Following these results, the EO is considered as a strong activity against strains of S. thypi and S. aureus with inhibition diameter of (32.33, 37.26) mm and minimum inhibitory concentration of (14.72, 29.44) mg/ml respectively. According to present results, it is clear that the EO of studied space and the phenolic monoterpene, the carvacrol and thymol have very high antibacterial activity in comparison with commercial antibiotics namely Chloramphenicol, Cefoxitin and Gentamycin. These results could be related to the percentage of phenolic students compounds such as thymol and carvacrol (Blois 1958; Giordani et al., 2008; Sokovic et al., 2002) . It is also known that bacterial species do not have the same sensitivity towards an antibacterial agent but it is concluded that our EO has a good capacity for destruction and a greater inhibitory power against Gram + and Gram- bacteria. In addition, it is mentioned that the antibacterial activity of oils is related to monoterpene and phenolic compound, as it is demonstrated by previous studies (El Amri et al. 2014; Goudjil et al., 2015; Hay et al., 2018). Concerning the oils’ mechanism of action, most studies have shown that the activity of oils can be attributed to the EO hydrophobicity and their components, which allows them to partition the lipids of the bacterial cell membrane and mitochondria. That is occurred via disrupting cell structures and making them more permeable after distraction of membrane the proton motive force was affected and both pH gradient and the electron flow across the membrane were disrupted. A large leak of bacterial cells or the release of molecules and critical ions will result the cells death (Jouki et al., 2014; Marin et al., 2018; Oulkheir et al., 2017; Sırıken et al., 2018). On the other hand, the previous study which is realized by Jayari et al. (2018b) demonstrated that the strong antibacterial activity of thyme EOs might be due to its major constituents: thymol and carvacrol. Both of them appear to make the cell membrane permeable and are able to disintegrate the outer membrane of Gram-negative bacterial. 4.2.2.2. Antifungal activity. 4.2.2.2.1. Kinetics of mycelial growth. The mycelial growth kinetics was evaluated every 24 h by measuring the mean of three perpendicular diameters through the center of the puck. This reading is always performed in comparison with the control cultures that they started on the same day and under the same conditions. Any slight growth of each fungal will be regarded as negative action that is to say that the EO in question is not inhibitory against fungal growth. The figure below summarizes the results of mycelial growth (cm) of the fungal strains as a function of the incubation time and the concentration of the essential oil of T. capitatus. Fig. 5 represents the kinetics of mycelial growth as a function of time and the concentration of EO studied. It is noticed that there is a highly decrease in mycelial growth with the incubation time for A. ochraceus and T. rubrum with 0.025% and 0.05% concentration, beyond this concentration no growth observed.

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Fig. 5. Mycelial growth kinetics as a function of time and concentration of T. capitatus EO.

Both Cladosporium Herbarum and Alternaria Infectoria are the most sensitive where no growth was recorded for all concentrations of 0.025% to 0.75%. We observed no mycelial growth of any fungal strains for concentrations 0.25%, 0.5% and 0.75%. 4.2.2.2.2. Antifungal index. Fig 6 shows that the concentrations 0.25%, 0.5%, 0.75% of our EO completely prevented the fungal strains growth tested. According to Fig 6, it is observed that the inhibition rate has a fungicidal effect for all the fungal strains. It is increased with the increase of EO concentration for the Aspergillus ochraceus and Trichophyton rubrum strains while the Cladosporium herbarum and Alternaria infectoria keep the maximum peak from low to high concentration. Indeed, the MIC is 0.5, 0.05% for Aspergillus ochraceus and Trichophyton rubrum strains. For Cladosporium herbarum and Alternaria infectoria strains it is estimated that the MIC is 0.0125%. 4.2.2.2.3. Speed mycelial growth. Fig. 7 Shows the mycelial velocity of the four fungal strains according to the concentration of the essential oil: According to Fig. 7, there is a remarkable decrease in the rate of mycelial growth by increasing EO’s concentration whose, decreases

Fig. 6. Strain inhibition rates as a function of essential oil concentration.

up to total inhibition (0 cm/h) in the dose 0.25% for C. herbarum and A. infectoria, and 0.25% for A. ochraceus and T. rubrum. The direct contact technique involves contacting the EO with the micro-organisms and then observing the growth of the latter. T. capitatus’ EO exerted an important inhibitory activity against strains tested. The diameters, the speed and the antifungal index of the growth of mycelium diminish each time that the concentration of EO is increased until non-germination of the disc at the determined MIC and this outcomes are confirmed by the works of Mehani et al. (2014). The complexity of the chemical composition of EO with a dozen compounds makes the component identification process responsible for the antimicrobial activity very difficult. Often, the antimicrobial activity yields from the synergy or antagonism between several components. Even (Daferera et al., 2003) postulated that the antifungal activity of EO is primarily due to their main components, although the possibility of other phenomena, such as synergy or antagonism with minor components(Russo et al., 2013). Much work has highlighted the effectiveness of antifungal terpene phenols and particularly that of thymol and / or carvacrol. These two molecules have a very broad spectrum of antimicrobial activity and are naturally present in the species of most species of thyme and oregano(Rajae and Meryem 2014).

Fig. 7. Mycelial growth rate under the effect of the increase in the concentration of T. capitatus EO.

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The mechanism of phenolic toxicity on fungi is based primarily on the inhibition of fungal enzymes containing the SH moiety in their active site (Arras and Usai 2001). Tabti et al. (2014) reported that the terpene hydrocarbons and phenolic compounds present in the thymus EO were responsible for the effects observed in 14 microorganisms responsible for spoilage and fungal strains such as Aspergillus Niger, A. flavus and Fusarium oxysporum. According to the observations of Soylu et al. (2005), EO can alter the morphology of thalli; they cause the appearance of large vesicles inside the cell wall. In addition, (Carmo et al., 2008; Dai et al., 2013), report that EO damage a series of enzymatic mold systems, affecting the synthesis and energy production structural component which enter into the cell wall composition and disrupting fungal growth. Indeed, a study conducted by Miri and Djenane (2018) clearly shows that the essential oil of Algerian Thymus Capitatus can inhibit the growth of Aspergillus flavus E73. Furthermore, they presented a broad spectrum fungitoxic against some foodborne molds. The tested strains are: Aspergillus carbonarius, Aspergillus fumigatus, Aspergillus niger, Aspergillus ochraceus, Aspergillus tamari, Aspergillus terreus, Fusarium sp., Penicillium sp. and Rhizopus sp. Even, Arras and Usai (2001) found that the essential oil of Thymus capitatus had a strong fungicidal activity against Alternaria citri, Penicillium digitatum, P. italicum and Botrytis cinereal. This significant bioactivity of the essential oil of Thymus Capitatus against the tested pathogenic fungal strains is related to their high content of phenolic compounds (thymol and carvacrol) and the synergistic effect between different minor components of this essential oil. 5. Conclusion The present work has been devoted to study the antibacterial, antifungal and antioxidant activity of the essential oil of Thymus Capitatus specie. It has also been the object of the determination of the chemical composition of the aromatic and medicinal plant’s essential oil in order to contribute to its valorization with the aim of a better exploitation. The qualitative and quantitative analysis of the plant’s essential oil made it possible to identify 22 constituents. The Thymus Capitatus oil is dominated by thymol (51.22%) followed by carvacrol 12.59% and. g -terpinene 10.3%. Bioassays have revealed considerable activity of the studied essential oil compared with antibiotics or standards used. These results are absolutely a rich source of information on chemical properties, antioxidant, antibacterial and antifungal activities of essential oil of endemic species from Algerian flora. The results clearly demonstrate that the essential oil of plant can well present interesting naturel alternative which can be useful for food preservation and pharmaceutical treatment. Declaration of Competing Interest The authors declare no conflict of interest. References Ainane, A., Khammour, F., M’hamad, E., Talbi, M., El Hassan, A., Cherroud, S., Ainane, T.,  anti insecticide des huiles essentielles de 2018. Composition chimique et activite thymus du maroc: thymus capitatus, thymus bleicherianus et thymus satureioides. Proceedings Biosune’ 1, 96–100.  acaricide des huiles Aissaoui, A.B., El Amrani, A., Zantar, S., Toukour, L., 2018. Activite essentielles du mentha pulegium, origanum compactum et thymus capitatus sur L’acarien phytophage tetranychus urticae koch (Acari: tetranychidae). European Scientific Journal, ESJ 14, 118. Akrout, A., El Jani, H., Amouri, S., Neffati, M., 2010. Screening of antiradical and antibacterial activities of essential oils of artemisia campestris L., artemisia herba alba asso, & thymus capitatus hoff. et link. growing wild in the southern of Tunisia. Recent Research In Science And Technology 2, 29–39. Amarti, F., Satrani, B., Ghanmi, M., Aafi, A., Farah, A., Aarab, L., El Ajjouri, M., Guedira, A.,  antioxydante et composition chimique des huiles essenChaouch, A., 2011. Activite ces de thym du maroc. Acta Botanica Gallica 158, 513–523. tielles de quatre espe

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