Khadidja Houda Benabed

Nadhir Gourine

Mohamed Ouinten

Isabelle Bombarda

Mohamed Yousfi

Chemical Composition, Antioxidant and Antimicrobial Activities of the Essential Oils of Three Algerian Lamiaceae Species

Keywords: Thymus vulgaris L., Thymus algeriensis Boiss. & Reut., Mentha pulegium L., DPPH assay, phosphomolybdenum assay, antimicrobial activity, essential oil

The present work investigates the chemical composition, the antioxidant and the antimicrobial activities of the Essential Oils (EOs) of three species of the Lamiaceae family gowning in Algeria: Thymus vulgaris L., Thymus algeriensis Boiss. & Reut. and Mentha pulegium L.

Methods: Essential Oils (EOs) obtained by hydrodistillation of the aerial parts of the studied plants were analyzed by GC and GC-MS. The antioxidant activity of the EOs was determined using two different assays: free radical scavenging activity of DPPH • (2,2-diphenyl-1-picrylhydrazyl) and Phosphomolybdenum reducing power. The EOs were also tested for their antibacterial and antifungal activities against eight pathogenic bacteria (Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella typhi, Escherichia coli, Bacillus cereus, Staphylococcus aureus, Methicillin resistant strain of S. aureus "MRSA" and Enterococcus faecalis); a yeast (Candida albicans); and a fungi (Fusarium oxysporum).

Results: For DPPH assay, T. vulgaris presented very interesting activity. At the opposite, T. algeriensis (Aflou) and M. pulegium, were the most active EOs in term of Phosphomolybdenum assay. The antimicrobial activity of T. vulgaris was found to be the most active EO and exhibited important resistance against most of studied bacteria. For disc diffusion test, the most active EO plant was T. algeriensis. Alternatively, and for antifungal activity, T. vulgaris presented the highest value of MFC.

Conclusion:

The antioxidant activity test's results showed that the EOs exhibited important reducing powers but weak scavenging activities. On the other hand, it was found that some EO samples have shown very interesting antimicrobial activities. Actually, among the investigated EOs, T. vulgaris presented the strongest antibacterial and antifungal activities.

INTRODUCTION

To cure its ills, man had always relied on animals and plants. These have always been the basis of the traditional systems of medicine for thousands of years and continue to date to provide new remedies [START_REF] Gurib-Fakim | Medicinal plants: traditions of yesterday and drugs of tomorrow[END_REF]. Recently, the introduction of traditional medicine, as an alternative form, as well as the development of microbial resistance to antibiotics has led researchers to study the antimicrobial activity of medicinal plants [START_REF] Nostro | Extraction methods and bioautography for evaluation of medicinal plant antimicrobial activity[END_REF]. On the other hand, the different synthetic antioxidants used in food, were suspected to have negative health effects. It is for this reason that the interest for the study of *Address correspondence to this author at the Laboratoire des Sciences Fondamentales LSF, Université Amar Télidji, Laghouat, BP. 37G, Laghouat, Algérie; E-mails: gourine.nadir@gmail.com or n.gourine@mail.lagh-univ.dz natural products such as antioxidants is increasingly important. Thus, various sources of antioxidants of plant origin have been studied in recent years [START_REF] Kulisic | Use of different methods for testing antioxidative activity of oregano essential oil[END_REF].

The use of essential oils as functional ingredients in foods, drinks, toiletries and cosmetics is gaining momentum [START_REF] Chouliara | Combined effect of oregano essential oil and modified atmosphere packaging on shelflife extension of fresh chicken breast meat, stored at 4 °C[END_REF]. Additionally, the use of essential oils is becoming popular to increase the shelf-life of food products, since consumers are more conscious about the health problems caused by several synthetic preservatives [START_REF] Gómez-Estaca | Biodegradable gelatin-chitosan films incorporated with essential oils as antimicrobial agents for fish preservation[END_REF][START_REF] Holley | Improvement in shelf-life and safety of perishable foods by plant essential oils and smoke antimicrobials[END_REF].

The purpose of this research was to investigate, firstly the chemical composition and the antioxidant properties of three EOs (T. vulgaris L., T. algeriensis Boiss. & Reut. and M. pulegium L.) collected from two regions in the center of Algeria; and secondly, to examine their antimicrobial activities against eight pathogenic bacteria (Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella typhi, Escherichia coli, Bacillus cereus, Staphylococcus aureus, Methicillin resistant strain of S. aureus "MRSA" and Enterococcus faecalis); a yeast (Candida albicans); and a fungi (Fusarium oxysporum).

Only very few reports on the determination of the antioxidant activity of the essential oil using Phosphomolybdenum assay and applied for of T. vulgaris were found in literature [START_REF] Silva | Chemical characterization, antibacterial and antioxidant activities of essential oils of Mentha viridis L. and Mentha pulegium L. (L)[END_REF][START_REF] Grigore | Chemical composition and antioxidant activity of Thymus vulgaris L. volatile oil obtained by two different methods[END_REF][START_REF] Ramadan | Comparative evaluation of three essential oils as functional antioxidants and natural flavoring agents in ice cream[END_REF]. For these reports, the antioxidant values are difficult to use for comparison purpose since different methods of expressions or different antioxidant of reference were engaged. In the same context, and as best of our knowledge, there are no reports dealing with the Phosphomolybdenum assay of the EO for T. algeriensis Boiss. & Reut. and M. pulegium L. As far as we know, this is the first report that investigates the antioxidant antimicrobial activities of the essential oils of the plants from these regions of collection (Djelfa and Aflou "Laghouat") from Algeria. Furthermore, we have used for the first time a special form of Fusarium oxysporum which was isolated from a fragment of date palm from Ghardaia region to investigate the antifungal activity. In addition, the antioxidant activity of the EOs determined by DPPH and Phosphomolybdenum assays were examined for the first time for these regions. Finally, correlations between chemical compositions of the EOs and their antioxidant and antimicrobial activities were investigated using statistical analysis; this was done in order to determine the nature of the chemical component(s) responsible(s) for the different activities of the EOs.

MATERIALS AND METHODS

Plants Descriptions

Thymus vulgaris L. is a perennial herb indigenous in central and southern Europe, Africa and Asia. It is widely used in folk medicine in the treatments of variety of diseases such as gastroenteric and bronchopulmonary disorders, as well as due to its anthelmintic, carminative, sedative and diaphoretic properties [START_REF] Rustaiyan | Volatile constituents of three Thymus species grown wild in Iran[END_REF]. It has been reported that its EO possesses numerous biological activities including antiworm, antiseptic, antispasmodic, antimicrobial [START_REF] Marino | Antimicrobial activity of the essential oils of Thymus vulgaris L. measured using a bioimpedometric method[END_REF] and antioxidant [START_REF] Vaz | Antifungal activity of Thymus oils and their major compounds[END_REF]. In addition, it is a well-known species of the genus Thymus, extensively studied for its chemical and biological activities [START_REF] Simandi | Antioxidant activity of pilot plant alcoholic and supercritical carbon dioxide extracts of thyme[END_REF][START_REF] Sokovi | Antifungal activity of the essential oil of Thymus vulgaris L. and thymol on experimentally induced dermatomycoses[END_REF][START_REF] Sokovi | Chemical composition of essential oils of Thymus and Mentha species and their antifungal activities[END_REF].

In Algeria, T. vulgaris is one of the most useful popular remedies in the treatment of respiratory affections (colds, flu, angina) and gastric disorders (dyspepsia, cramps) [START_REF] Baba-Aissa | Encyclopédie des plantes utiles: Flore méditerranéenne (Maghreb,Europe méridionale) Substances végétales d'Afrique , d'Orient dt d'Occident[END_REF].

Thymus algeriensis

Boiss. & Reut. is the most widespread North African species, endemic to Libya, Tunisia, Algeria and Marocco. Fresh or dried, it is largely used only as a culinary herb. T. algeriensis is also used in traditional medicine, as a fresh or dry seasoning, in respiratory and digestive tube disorders and against abortion [START_REF] Nikoli | Chemical composition, antimicrobial, antioxidant and antitumor activity of Thymus serpyllum L., Thymus algeriensis Boiss. and Reut and Thymus vulgaris L. essential oils[END_REF].

In Algeria, T. algeriensis is used as a treatment for stomachic, diaphoretic, antispasmodic and stimulating. The summits and the young flowering twigs are used. Infusion is useful against all infectious diseases, such as influenza, pneumonia and respiratory tract diseases. It is used in friction in cases of neuralgia and sciatica and as odontalgic on decayed toothache [START_REF] Beloued | Plantes médicinales d'Algérie: Alger[END_REF].

Mentha pulegium L. is one of the Mentha species commonly known as pennyroyal. It is native species of Europe, North Africa and in Asia Minor and near East. The flowering aerial parts of M. pulegium L. has been traditionally used for its antiseptic for treatment of cold, sinusitis, cholera, food poisoning, bronchitis and tuberculosis, and also as antiflatulent, carminative, expectorant, diuretic, antitussive, menstruate. Some pharmacological effect of M. pulegium L. essential oil such as abortifacient effect in rat myometrium, cytotoxic activity against different human cell lines and its antioxidant effect were confirmed [START_REF] Boukhebti | Chemical composition and antibacterial activity of Mentha pulegium L. and Mentha spicata L. essential oils[END_REF].

In Algeria it is much appreciated, so much so that it is used to prepare a traditional dish: the potato stew with the pennyroyal "Batata Fliou". Its pleasant odor seems to displease some kind of parasites, and its insecticidal power is well established. Formerly, it was burned in fleece infested premises. It was also used as a lotion on the coat of domestic animals to rid them of their parasitic pests [START_REF] Baba-Aissa | Encyclopédie des plantes utiles: Flore méditerranéenne (Maghreb,Europe méridionale) Substances végétales d'Afrique , d'Orient dt d'Occident[END_REF].

Plants Materials Collections

The study was carried out using four EO samples obtained from three species belonging to the Lamiaceae family: M. pulegium L. (Djelfa), T. vulgaris L. (Djelfa) and T. algeriensis Boiss. & Reut. (from two different stations: Djelfa and Aflou-Laghouat). Since T. algeriensis, wide spreads over a larger region, we have picked up two samples from different locations in order to compare theirs EOs composition differences as well as theirs antioxidant and antibacterial activities behaviors. The different plant samples were collected in 2010 during the flowering stage; (end of May for M. pulegium and T. algeriensis, and the beginning June for T. vulgaris). Voucher specimens (TV/06/10, TA-AFL/06/10, TA-DJF/06/10, MP/06/10) were deposited in the herbarium of the Fundamental Sciences Research Laboratory at Laghouat University.

Essential Oil Extraction

The different samples of EOs were obtained by hydrodistillation using a Clevenger type apparatus; subsequently the obtained EOs were dried over anhydrous sodium sulfate, and stored in a dark at 4 °C.

Essential Oil Analysis

The GC analysis was performed using a gas chromatograph type Chrompack CP 9002, equipped with a fused silica capillary column DB-5 (30m 0.32mm, 0.25 m film thickness) and Flame Ionization Detector (FID). The carrier gas was nitrogen at a flow rate of 1mL/min. The column temperature was programmed from 50 °C (3 minutes) to 250 °C at 2 °C/min, and then maintained at 250 °C for 10 min. The temperatures of the injector and detector were set at 250 °C. Volumes of 1 L of diluted samples (1:100 v/v, in ethanol) were injected manually using split less mode. Linear retention indices of the components were calculated using a series of n-alkane (C 9 -C 25 ) analyzed under the same operating conditions as those of the EOs samples.

The GC/MS analysis was performed on an AGILENT 6890 GC/CMSD 5973 equipped with a capillary column UB-Wax (30m 0.25mm, 0.25 m film thickness) and a 70 eV EI Quadruapole detector. Helium was the carrier gas, at a flow rate of 1mL/min. Injector and MS transfer line temperatures were set at 250 °C and 220 °C, respectively. Column temperature was programmed same as gas chromatography. Diluted samples (1:100 v/v, in ethanol) of 1 L were also injected manually using split less mode.

The EOs constituents were identified by comparison of their linear retention indices and mass spectra with those in the computer library (NIST MS Library) and with literature data [START_REF] Adams | Identification of essential oil components by gas chromatography/ mass spectrometry[END_REF].

DPPH Assay (Free Radical Scavenging Activity)

For this test, the ability of hydrogen donating or radical scavenging was measured using the stable radical DPPH (2,2-diphenyl-1-picrylhydrazyl) [START_REF] Molyneux | The use of the stable free radical diphenylpicrylhydrazyl (DPPH) for estimating antioxidant activity[END_REF]. The EOs dilutions were prepared in absolute ethanol, and then 1mL of each dilution was added to 1mL of a 200 M DPPH solution. The mixtures were incubated in dark for 30 min; afterwards the absorbance was measured at 517 nm against a blank. The inhibition percentage was calculated using the following formula:

100 (%) 0 0 = A A A I
Where:

A 0 is the absorbance of the control reaction (containing all reagents except the test compound) and A is the absorbance of the test compound.

Phosphomolybdenum Assay

This assay is based on the reduction of Molybdate (VI) to Molybdate (V) by the antioxidant compounds and the formation of a green phosphate/Molybdate (V) complex at acidic pH with a maximum absorbance at 695 nm [START_REF] Prietto | Spectrophotometric quantification of antioxidant capacity through the formation of phosphomolybdenum complex: Specification application to the determination of vitamin E[END_REF]. The EOs samples were diluted in absolute ethanol. An amount of 200 L of each dilution was mixed with 2 mL of reagent (0.6 M sulfuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate). The mixture was then incubated in a water bath set at 70 °C for 90 min [START_REF] Prietto | Spectrophotometric quantification of antioxidant capacity through the formation of phosphomolybdenum complex: Specification application to the determination of vitamin E[END_REF]. The absorbance was determined at 695 nm. The results were expressed in terms of ascorbic acid equivalent AEAC.

Microbial Strains

The different samples of EOs were tested against eight bacteria: Klebsiella pneumoniae ATCC 700603, Pseudomonas aeruginosa ATCC 27853, Salmonella typhi (isolate), Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, Methicillin resistant strain of S. aureus ATCC 43300, Bacillus cereus ATCC 11779, Enterococcus faecalis ATCC 29212 ; a yeast: Candida albicans (isolate) and a filamentous fungi Fusarium oxysporum f. sp. albedinis. The fungal species Fusarium oxysporum special form albedinis was isolated from a fragment of date palm provided by the Regional Station of Plant Protection of Ghardaia (Algeria). The culture was then purified by performing a monospore culture.

Antimicrobial Tests

The antimicrobial activity of the different EO samples was screened by the disk diffusion test [START_REF] Billerbeck | Huiles essentielles et bactéries résistantes aux antibiotiques[END_REF], and then the minimal inhibitory and bactericidal concentrations were determined using a dilution test [START_REF] Cao | Essential oil composition, antimicrobial and antioxidant properties of Mosla chinensis Maxim[END_REF]. The antifungal activity was tested using a direct contact test as described by El Ajjouri et al. [START_REF] El Ajjouri | Activité antifongique des huiles essentielles de Thymus bleicherianus Pomel et Thymus capitatus (L.) Hoffm. & Link contre les champignons de pourriture du bois d'oeuvre[END_REF].

RESULTS

Essential Oil Yield and Chemical Composition

The yields of the studied EOs were varying from moderate to low values. They were somehow important for M. pulegium and T. vulgaris. In the other hand, they were ranging from low to poor values for T. algeriensis (Table 1).

The chemical compositions of the different EO samples are presented in Table 1. For the EO of T. vulgaris, thirty two different compounds representing 96.47% of the total oil were identified, with proportions of monoterpene hydrocarbons and oxygenated monoterpenes equal to 52.98% and 39.14%, respectively. The major component of this oil was the -terpinene (25.70%) followed by thymol (20.83%), then p-cymene (20.04%). The oxygenated compounds were also present in lower amounts than the hydrocarbons: thymol methyl ether (7.82%), linalool (4.76%) and borneol (2.54%). The -caryophyllene (2.28%) was the main sesquiterpene compound for this EO.

For the EO of pennyroyal (M. pulegium), twenty eight compounds were identified representing 76.38% of the total EO. The oil was mainly composed of pulegone (54.92%), which accounts for almost the majority of oxygenated monoterpenes. Monoterpene hydrocarbons represented only a small fraction of the EO content (2.67%).

For the case of T. algeriensis, forty four components were identified for the sample of Djelfa and thirty three components of that of Aflou (Laghouat), both accounting for 83.30 and 81.06% of the total EOs, respectively. Camphor was the predominant compound in the sample of Aflou (Laghouat) with a percentage of 17.68%, this content agrees with the results of Amarti et al. [START_REF] Amarti | Composition chimique et activité antimicrobienne des huiles essentielles de Thymus algeriensis Boiss. & Reut. et Thymus ciliatus (Desf.) Benth. du Maroc[END_REF] and Zouari et al. [START_REF] Zouari | Chemical composition, angiotensin I-converting enzyme inhibitory, antioxidant and antimicrobial activities of essential oil of Tunisian Thymus algeriensis Boiss. et Reut.(Lamiaceae)[END_REF] who have detected camphor as major compound for T. algeriensis from Morocco and Tunisia, respectively. The sample of Djelfa was predominantly composed of -terpinenyle acetate (27.32%), a compound completely absent in the sample of Aflou. Camphor was also present, but with a lower content (10.77%) than recorded for the sample of Aflou (Laghouat). The eucalyptol was one of the major compounds in the EO sample of Aflou (10.04%), whereas it represented only 2.47% for the sample of Djelfa. The EO of Aflou sample showed a high content of monoterpene hydrocarbons (41.29%) compared to that of Djelfa (13.71%), with a predominance of camphene (8.73%), -pinene (7.84%) and myrcene (6.95%). On the other hand, the two samples showed almost equal percentages of borneol (5.58 and 5.68%). Sesquiterpenes were minor compounds in the EOs of the two regions with a total percentage of 5.46 and 3.04% for Djelfa and Aflou respectively.

Antioxidant Activity

DPPH Assay

Antioxidant activities of the different EO samples were evaluated using two complementary methods: the DPPH assay, and the phosphomolybdenum assay. The DPPH test measures the ability of the sample to provide protons, whereas the phosphomolybdenum assay measures the capacity of the sample to donate electrons.

The results of the tests of DPPH radical scavenging activities of the different samples on the EOs are shown in Table 2. These results are expressed as EC 50 values, that are defined as the concentration of substrate that causes 50% loss of the concentration of the free radicals DPPH initially introduced; the higher the antioxidant activity, the lower is the value of EC 50 [START_REF] Molyneux | The use of the stable free radical diphenylpicrylhydrazyl (DPPH) for estimating antioxidant activity[END_REF]. In comparison with antioxidants of reference, the EO samples showed weak DPPH-radicalscavenging activities. Their activities can be classified as follows: T. vulgaris>T. algeriensis (Djelfa)>M. pulegium>T. algeriensis (Aflou-Laghouat). More effectively, T. vulgaris presented a very interesting EC 50 value, and it could be considered active in comparison with the majority of common EOs.

Phosphomolybdenum Assay

The second test named Phosphomolybdenum test, measures the reducing power capacity in aqueous medium. Antioxidant reducing power is expressed as AEAC: Ascorbic acid Equivalent Antioxidant Power. By definition, AEAC is defined as the concentration of an antioxidant of reference (ascorbic acid in this case) in mg/mL, which gives the equivalent antioxidant power for a concentration of 1 mg/mL of the EO. High value of AEAC indicates high antioxidant capacity.

In comparison with vitamin E, the EOs showed moderate antioxidant capacities (Table 3). The highest antioxidant capacity was observed for the EO of T. algeriensis (Djelfa); while the EO with the lowest activity was recorded for T. vulgaris. As a matter of fact, the EOs samples presented practically close values of antioxidant activities, and they were approximately 2 to 5 times lower than vitamin E; whereas, vitamin E presented almost two times lower antioxidant power than vitamin C.

Antimicrobial Activity

Disc Diffusion Test

The antibacterial activities of the EOs were also investigated. Inhibition zone diameters are includes in Table 4 for the four EO samples (values are average of three repetitions). The results showed a sensitivity of the microorganisms towards all the EOs, with a remarquable resistance for Pseudomonas aeruginosa. This resistance was observed by Cosentino et al. [START_REF] Cosentino | In vitro antimicrobial activity and chemical composition of Sardinian thymus essential oils[END_REF] for the EOs of T. capitatus and T. herba barona, Bouhdid et al. [START_REF] Bouhdid | Thymus essential oils: Chemical composition and in vitro antioxidant and antibacterial activities[END_REF] for three species of T. genus and Hussain et al. [START_REF] Hussain | Antibacterial activity of some Lamiaceae essential oils using resazurin as an indicator of cell growth[END_REF] for six plants within the Lamiaceae family. Let's mention that comparisons of results of the founded antimicrobial activities with those of literature "Tables 4(c) and 5(b)" are not evident since the evaluations conditions are somehow different.

T. vulgaris EO sample had the strongest activity with the highest inhibition zone diameters, while the EOs of T. algeriensis and M. pulegium showed moderate activities.

In a diffusion test, the inhibition zone diameter for a component is determined by its antimicrobial activity, its solubility and capacity of diffusion in the media and by the characteristics of the microorganism itself [START_REF] Carson | Antimicrobial activity of the major components of the essential oil of Melaleuca alternifolia[END_REF]. Diffusion methods are likely appreciated because their simplicity and low cost, but these methods are not always reliable for the evaluation of antimicrobial activities of plant extracts, because the absence of inhibition zone doesn't mean that the tested compound is inactive, especially for compounds with low polarity that diffuse slowly in the culture media [START_REF] Klan Nik | Evaluation of diffusion and dilution methods to determine the antibacterial activity of plant extracts[END_REF].

Minimal Inhibitory Concentration (MIC) and Bactericidal Concentrations (MBC) Tests

The determined MIC values were ranged from 0.127 mg/mL (T. vulgaris EO for B. cereus and E. faecalis) to higher than 4.5 mg/mL (for Candida albicans for the two samples of T. algeriensis). The same resistance was observed for P. aeruginosa towards all the EO samples (Table 5).

T. vulgaris EO showed MICs values between 0.12 mg/mL (for the strains B. cereus and E. faecalis) and 1.01 mg/mL (for K. pneumoniae and S. aureus). The growth of MRSA, S. typhi and E. coli was inhibited at a concentration equal to 0.5 mg/mL. The resistance of P. aeruginosa was observed even with a concentration of 2.03 mg/mL. Imelouane et al. [START_REF] Imelouane | Chemical composition and antimicrobial activity of essential oil of thyme (Thymus vulgaris) from Eastern Morocco[END_REF] found for this same species EO MIC values equal to 1.33 mg/mL for the strains E. coli, S. aureus and S. epidermidis. Kaloustian et al. [START_REF] Kaloustian | Étude de six huiles essentielles: Composition chimique et activité antibactérienne[END_REF] found MICs equal to 1 and 2 mg/mL for the strains E. coli and S. aureus respectively, while testing the EOs of T. vulgaris and T. zygis.

For the MBCs, B. cereus was the most sensitive strain, with MBC equal to 0.5 mg/mL. MRSA, S. typhi, E. coli, S. aureus and E. faecalis were inhibited at an oil concentration of 1.01 mg/mL. Many previous studies confirm the antimicrobial properties of EOs from Thymus genus [START_REF] Govaris | Antibacterial activity of oregano and thyme essential oils against Listeria monocytogenes and Escherichia coli O157: H7 in feta cheese packaged under modified atmosphere[END_REF][START_REF] Kabouche | Comparative antibacterial activity of five Lamiaceae essential oils from Algeria[END_REF][START_REF] Naeini | Anti-Candida albicans activity of some Iranian plants used in traditional medicine[END_REF][START_REF] Rota | Antimicrobial activity and chemical composition of Thymus vulgaris, Thymus zygis and Thymus hyemalis essential oils[END_REF][START_REF] Saad | Anticandidal activity of the essential oils of Thymus maroccanus and Thymus broussonetii and their synergism with amphotericin B and fluconazol[END_REF][START_REF] Smith-Palmer | Antimicrobial properties of plant essential oils and essences against five important food-borne pathogens[END_REF][START_REF] Tohidpour | Antibacterial effect of essential oils from two medicinal plants against Methicillinresistant Staphylococcus aureus (MRSA)[END_REF], that is in correlation with our results. T. algeriensis EO showed a moderate to weak activity with more effectiveness on Gram positive bacteria (Table 6), this weak activity has been also observed by Hazzit et al. [START_REF] Hazzit | Chemical composition and biological activities of Algerian Thymus oils[END_REF] for two samples of T. algeriensis.

Antifungal Activity

Inhibition growth percentages observed for the different EO samples against the filamentous fungi Fusarium oxysporum sp.f. albedinis are shown in Table 6. All the EO tested exhibited inhibition effects against the tested fungi. The EO of T. vulgaris showed the strongest activity with a fungistatic and fungicide concentration equal to 1.753 mg/mL, and 3.438 mg/mL, respectively. While the EOs of T. algeriensis (Djelfa) and M. pulegium showed a moderate activity with MICs values equal to 8.963 mg/mL and 8.781 mg/mL, respectively. In another hand, these latest EOs did not exhibit any measured MFCs activity in the range of the concentration studied. These observed differences are proba-

Table 4c. Antimicrobial activity of essential oil samples of Thymus vulgaris, Thymus algeriensis and Mentha pulegium by discdiffusion diameter inhibition zones (values from literature).

Essential Oil Plant

Thymus vulgaris Thymus algeriensis Mentha pulegium

Country Morocco [START_REF] Imelouane | Chemical composition and antimicrobial activity of essential oil of thyme (Thymus vulgaris) from Eastern Morocco[END_REF] Morocco [START_REF] Zantar | Effect of gamma irradiation on chemical composition, antimicrobial and antioxidant activities of Thymus vulgaris and Mentha pulegium essential oils[END_REF] Libya [START_REF] Nikoli | Chemical composition, antimicrobial, antioxidant and antitumor activity of Thymus serpyllum L., Thymus algeriensis Boiss. and Reut and Thymus vulgaris L. essential oils[END_REF] Spain [START_REF] Rota | Antimicrobial activity and chemical composition of Thymus vulgaris, Thymus zygis and Thymus hyemalis essential oils[END_REF] Algeria [START_REF] Hazzit | Chemical composition and biological activities of Algerian Thymus oils[END_REF] Tunisia [START_REF] Zouari | Chemical composition, angiotensin I-converting enzyme inhibitory, antioxidant and antimicrobial activities of essential oil of Tunisian Thymus algeriensis Boiss. et Reut. (Lamiaceae)[END_REF] Libya [START_REF] Nikoli | Chemical composition, antimicrobial, antioxidant and antitumor activity of Thymus serpyllum L., Thymus algeriensis Boiss. and Reut and Thymus vulgaris L. essential oils[END_REF] Iran [START_REF] Mahboubi | Antimicrobial activity and chemical composition of Mentha pulegium L. essential oil[END_REF] Morocco [START_REF] Cherrat | Chemical composition, antioxidant and antimicrobial properties of Mentha pulegium, Lavandula stoechas and Satureja calamintha Scheele essential oils and an evaluation of their bactericidal effect in combined processes[END_REF] Morocco [START_REF] Ait-Ouazzou | Evaluation of the chemical composition and antimicrobial activity of Mentha pulegium, Juniperus phoenicea, and Cyperus longus essential oils from Morocco[END_REF] Portugal [START_REF] Teixeira | European pennyroyal (Mentha pulegium) from Portugal: Chemical composition of essential oil and antioxidant and antimicrobial properties of extracts and essential oil[END_REF] Inhibition Zone Diameters (mm) a

Plant

Thymus vulgaris Thymus algeriensis Mentha pulegium

Country Libya Soliman & Badiaa [START_REF] Soliman | Effect of oil extracted from some medicinal plants on different mycotoxigenic fungi[END_REF] tested the antifungal effect of EOs of three species of Lamiaceae: T. vulgaris, M. viridis and Ocimum basilicum. They revealed that the three oils have inhibitory activities against the tested fungal species (Aspergillus flavus, A. parasiticus, A. ochraceus and Fusarium moniliforme). For the same family Dambolena et al. [START_REF] Dambolena | Essential oils composition of Ocimum basilicum L. and Ocimum gratissimum L. from Kenya and their inhibitory effects on growth and fumonisin production by Fusarium verticillioides[END_REF] found that several samples of Ocimum basilicum and Ocimum gratissimum have an antifungal activity against Fusarium verticillioides. Ouraïni et al. [START_REF] Ouraïni | Activité antifongique de l'acide oléique et des huiles essentielles de Thymus saturejoides L. et de Mentha pulegium L., comparée aux antifongiques dans les dermatoses mycosiques[END_REF] obtained significant antifungal activity for EOs of T. saturejoides and M. pulegium. The antifungal potency of the EO of thyme has also been shown by Yang & Clausen [START_REF] Yang | Antifungal effect of essential oils on southern yellow pine[END_REF].

The results show that the extracts with high phenol concentration (T. vulgaris) have strong antifungal activities. Several in vitro and in vivo studies conducted by Figueiredo et al. [START_REF] Figueiredo | Portuguese Thymbra and Thymus species volatiles: chemical composition and biological activities[END_REF] showed that the EOs containing phenolic structures are very active against a wide spectrum of pathogenic fungi. The mechanism of toxicity of the phenols towards fungi is based on the inactivation of fungal enzymes. Phenolic terpene also act by binding to the amino groups of the microbial membrane proteins causing the alteration of the permeability and leakage of intracellular components [START_REF] El Ajjouri | Activité antifongique des huiles essentielles de Thymus bleicherianus Pomel et Thymus capitatus (L.) Hoffm. & Link contre les champignons de pourriture du bois d'oeuvre[END_REF]. This effect was also mentioned by Pinto et al. [START_REF] Pinto | Antifungal activity of the essential oil of Thymus pulegioides on Candida, Aspergillus and dermatophyte species[END_REF] by studying the EO of T. pulegioides effect on the membrane of Candida and Aspergillus.

DISCUSSION

Essential Oil Yield and Chemical Composition

According to previous reports [START_REF] Ring | Beyond six scents: Defining a seventh Thymus vulgaris chemotype new to southern France by ethanol extraction[END_REF], several chemotypes of T. vulgaris were determined: geraniol, -terpineol, sabinene hydrate (thuyanol), linalool, carvacrol, thymol and 1,8-cineole. Several species belonging to Thymus genera have similar compositions with a high content of phenolic terpenes (thymol or carvacrol) followed by a high percentage of -terpinene and p-cymene: T. hyemalys [START_REF] Jordán | Seasonal variation of Thymus hyemalis Lange and Spanish Thymus vulgaris L. essential oils composition[END_REF] T. pulegioides [START_REF] Pinto | Antifungal activity of the essential oil of Thymus pulegioides on Candida, Aspergillus and dermatophyte species[END_REF], T. capitatus and T. herba-barona [START_REF] Cosentino | In vitro antimicrobial activity and chemical composition of Sardinian thymus essential oils[END_REF].

Referring to literature, several major compounds were identified for the EO of this plant; previous studies confirm the predominance of pulegone as major compound: 73.40% on populations of Uruguay [START_REF] Lorenzo | Essential oils of Mentha pulegium and Mentha rotundifolia from Uruguay[END_REF]; 85.40% on populations of Morocco [START_REF] Bouchra | Chemical composition and antifungal activity of essential oils of seven Moroccan Labiatae against Botrytis cinerea Pers: Fr[END_REF]; while others have identified piperitone and piperitenone as the two dominant compounds [START_REF] Derwich | Comparative essential oil composition of leaves of Mentha rotundifolia and Mentha pulegium a traditional herbal medicine in Morocco[END_REF][START_REF] Mahboubi | Antimicrobial activity and chemical composition of Mentha pulegium L. essential oil[END_REF]. According to literature, three chemotypes have been established, pulegone type, piperitenone/piperitone type, and isomenthone / neoisomenthol type [START_REF] Stoyanova | Chemical composition of the essential oil of Mentha pulegium L. from Bulgaria[END_REF]. Beghidja et al. [START_REF] Beghidja | Composition of the oils from Mentha pulegium grown in different areas of the East of Algeria[END_REF] studied the composition of several samples of pennyroyal from eastern Algeria, and claimed that these oils can be classified into two chemotypes: one of pulegone (pulegone with a percentage between 52 and 87%), and a new chemotype poor in pulegone and rich of nonoxygenated terpenic fractions ( -pinene, -thujene, -pinene, camphene, sabinene,phellandrene) and relatively high level of 1,8-cineol.

According to previous works which dealt with this plant from Algeria, it seems that there were some differences in the composition of the main compounds and which were related to the regions of collection [START_REF] Hazzit | Chemical composition and biological activities of Algerian Thymus oils[END_REF][START_REF] Dob | Studies on the essential oil composition and antimicrobial activity of Thymus algeriensis Boiss. et Reut[END_REF]. Indeed, Dob et al. identified for a sample of T. algeriensis (Medea -Algeria) the following major compounds: linalool (47.30%), thymol (29.20%) and p-cymene (6.80%) [START_REF] Dob | Studies on the essential oil composition and antimicrobial activity of Thymus algeriensis Boiss. et Reut[END_REF]; While Hazzit et al. have suggested the existence of two chemotypes (samples were collected from the Chrea National Park, and the Chlef regions); the first was characterized by terpinyl acetate (18.00%), trans-nerolidol (12.60%), -pinene (11.10%), borneol (9.00%) and bornyl acetate (7.70%), while the second has presented the following major compounds: terpinen-4-ol (10.60%), camphor (10.10%), p-cymene (9.90%),pinene (6.50%) and 1,8-cineole (6.50%) [START_REF] Hazzit | Chemical composition and biological activities of Algerian Thymus oils[END_REF]. Comparing the founding of these In a matter of fact, the difference in composition of the EOs of T. algeriensis might be due to several factors, such as climate, season of harvest, stage of development and even genetic profiles of the species [START_REF] Amarti | Composition chimique et activité antimicrobienne des huiles essentielles de Thymus algeriensis Boiss. & Reut. et Thymus ciliatus (Desf.) Benth. du Maroc[END_REF][START_REF] Jordán | Seasonal variation of Thymus hyemalis Lange and Spanish Thymus vulgaris L. essential oils composition[END_REF][START_REF] Nan | Effect of harvest time on essential oil composition of Thymbra spicata L. growing in flora of Adıyaman[END_REF][START_REF] Sellami | Effect of growth stage on the content and composition of the essential oil and phenolic fraction of sweet marjoram (Origanum majorana L.)[END_REF][START_REF] Telci | Environmental variation on aroma components of pulegone/piperitone rich spearmint (Mentha spicata L.)[END_REF].

Antioxidant Activity

The obtained results suggested that when the EOs of the studied plants exhibited high antioxidant activity in term of DPPH assay, they simultaneously exhibited low activity in term of phosphomolebdenum assay; this finding could be explained by the different reactions mechanisms involved in each assay and related to different active chemicals in each case.

DPPH Assay

The antioxidant activity of the EO of T. vulgaris is found to be four times more active than the EO of T. algeriensis (Djelfa), and twelve times more active than the EO of M. pulegium.

Since the results of the investigated antioxidant activity showed a large variation in the values of EC 50 and which were depending on the nature of the studied plant, it was mandatory to try to find a correlation between the chemical composition of the EOs and their antioxidant activity power AAP (=1/EC 50 ) values. This was carried out by applying statistical methods involving Principal Component Analysis PCA (Fig. 1). It was found that highest value of AAP (as it was the case of T. vulgaris) was correlated with the previously major identified components: p-cymene, thymol, linalool, thymol-ethyl-ether and -terpinene. At the opposite, T. algeriensis and M. pulegium presented the lowest activities, and where each correlated with their main EOs components, i.e. -terpinenyl acetate and pulegone, respectively. Although, terpinen-4-ol and -terpineol were minor compo- nents for T. algeriensis and M. pulegium, their relatively high percentages was also correlated with low antioxidant activity. Finally, T. algeriensis from Aflou, had presented practically no activity, and its EO was correlated with the presence of simultaneously higher percentages of -pinene, camphene, myrcene, eucalyptol, camphor and borneol.

Phosphomolybdenum Assay

First, let's mention that vitamin E (antioxidant of reference) which was used for comparison is almost 2 times less active than ascorbic acid (Table 3). Second, the values of the antioxidant power for this assay were completely different to those found using DPPH assay. This time, T. algeriensis (Aflou) and M. pulegium presented almost similar activities and were the most active EOs (6 to 7 times less active than ascorbic acid). At the opposite, T. vulgaris EO exhibited the lowest antioxidant power (almost 10 times less active than ascorbic acid).

It was found that highest reduction power of the EOs of T. algeriensis (Djelfa) is highly correlated with -terpinenyl acetate, terpinen-4-ol and -terpineol. In another hand both T. algeriensis (Aflou) and M. pulegium with practically close reduction powers were correlated positively with different components: -pinene, camphene, myrcene, eucalyptol, camphor and borneol for T. algeriensis (Aflou); and only pulegone for M. pulegium. At the contrary, T. vulgaris with the lowest reduction power was related to higher percentages of p-cymene, thymol, linalool, thymol-ethyl-ether andterpinene.

Antimicrobial Activity

According to Fig. 2, the T. vulgaris was found to be the most active EO and exhibited important resistance against most of studied bacteria: Staphylococcus aureus, Escherichia coli, Salmonella typhi, Klebsiella pneumoniae and Candida albicans. According to PCA analysis, this activity is due mainly to presence of thymol and thymol-methyl-ether. Alternatively, the activity against Bacillus cereus was correlated with high percentages of camphor, borneol,terpinenyl acetate, terpinen-4-ol and -terpineol. Finally, the relatively slight improvement of activity of M. pulegium EO against Pseudomonas aeruginosa is probably due to the presence of the high percentage of pulegone. As a result, and based on the PCA, it seems that pulegone does not contribute to any practical antimicrobial activity, since high percentage of pulegone imply slight improvement of this activity.

Disc Diffusion Test

The analysis of the simultaneous values of MIC and MIB with the chemical composition of the EOs against different bacteria using PCA revealed that the most active EOs plants were those of T. algeriensis (Djelfa & Aflou) (Fig. 3). This strong activity was in strong correlation with several chemicals: eucalyptol, camphor, myrcene, -pinene, camphene,terpineol, -terpinenyl acetate and terpinen-4-ol. For M. pulegium where its EO presented relatively moderate activity (MBC) against Bacillus cereus, Staphylococcus aureus and Enterococcus faecalis; this moderate activity is believed to be correlated to high content of pulegone.

Antifungal Activity

It was found that the EOs were exhibiting high MIC and at the same time presenting low or undetected activities in term of MIB values.

The results of PCA showed (Fig. 4), that the highest MIC values were correlated for different sets of EOs components for both T. algeriensis (Djelfa) and M. pulegium. Moreover, for almost the same components observed previously with disc diffusion test, the high MIC value for M. pulegium was attributed to the high composition of pulegone, but for T. algeriensis (Djelfa) it was due to the presence of eucalyptol, -terpinenyl acetate, terpinen-4-ol, -terpineol, -pinene, borneol and camphene. The EO of T. vulgaris with the lowest value of MIC was correlated with the following components: thymol-ethyl-ether, -terpinene, thymol, p-cymene and linalool. At the opposite, the EO of this later plant presented a good MFC value in comparison with the rest of the studied plants, in which they did not offer any measurable activity in the range of the studied concentrations. This high activity is due to the presence of -terpinene, thymol, p-cymene and linalool.

CONCLUSION

As a conclusion, the EOs tested in this study showed moderate to strong antioxidant and antimicrobial activities. Almost all EOs exhibited a radical scavenging activity (DPPH assay), with different values of EC 50 . T. vulgaris EO had the lowest value of EC 50 which makes it the strongest EO among the tested samples. For antimicrobial activity, all the samples had inhibited the growth of the tested microbial strains, with the strongest antibacterial and antifungal activity attributed to T. vulgaris EO. The study of the relation between the chemical composition of the EOs and the antioxidant or the antimicrobial activity revealed the presence of different strong correlations with some major identified compounds in each case of study. It could be concluded that, thyme, T. vulgaris EO were effective as antioxidant activity due, in part, to the presence of several compounds, like thymol, thymol-methyl-ether, linalool and carverol, in their chemical compositions. They could be used as flavoring agents and good sources of antioxidants in making food with healthy benefits and good sensory acceptability. Further studies are required to collect more data about the toxicity of these EOs.

  the difference in the compositions between the EOs.

Fig. ( 1

 1 Fig. (1).Two dimensional plot on axes F1 and F2 of the essential oil samples end their antioxidant activities using Principal Component Analysis.

Fig. ( 2

 2 Fig. (2).Two dimensional plot on axes F1 and F2 of the essential oil samples end their antimicrobial activity (disk diffusion test) using Principal Component Analysis.

Fig. ( 3

 3 Fig. (3).Two dimensional plot on axes F1 and F2 of the essential oil samples end their antimicrobial activity (minimal inhibitory and bactericidal concentrations) using Principal Component Analysis.

Fig. ( 4 ).

 4 Fig. (4).Two dimensional plot on axes F1 and F2 of the essential oil samples end their antifungal activity using Principal Component Analysis.

Table 1 . Chemical composition of the essential oil samples: Thymus vulgaris, Thymus algeriensis and Mentha pulegium.

 1

	Thymus algeriensis

Table 1 . contd…

 1

	Thymus algeriensis

a LRI: Linear Retention Indices determined experimentally on apolar column DB-5. b LRI: Linear Retention Indices obtained from literature

[START_REF] Adams | Identification of essential oil components by gas chromatography/ mass spectrometry[END_REF]

. c LRI: Linear Retention Indices obtained from literature

[START_REF] Senatore | Essential oil of Eremocharis triradiata (Wolff.) Johnston (Apiaceae) growing wild in Peru[END_REF]

.

Table 2 . Antioxidant acitivity of the essential oil samples of Thymus vulgaris, Thymus algeriensis and Mentha pulegium using DPPH free radical scavenging.

 2

	Essential Oil Sample	EC50 (mg/mL)
	Thymus vulgaris	2.3 ± 0.1
	Thymus algeriensis (Djelfa)	10.2 ± 0.9
	Thymus algeriensis (Aflou)	> 45.0
	Mentha pulegium	27.7± 2.4
	Vitamin E	0.0207 ± 0.0007
	Ascorbic acid	0.0078 ± 0.0001
	Butylhydroxyanisole (BHA)	0.0011 ± 0.0003

Table 3 . Antioxidant acitivity of the essential oil samples of Thymus vulgaris, Thymus algeriensis and Mentha pulegium using Phosphomolybdenum assay.

 3

	Essential Oil	AEAC a (mg/mL)
	Thymus vulgaris (Djelfa)	0.107 ± 0.007
	Thymus algeriensis (Djelfa)	0.220 ± 0.022
	Thymus algeriensis (Aflou)	0.148 ± 0.003
	Mentha pulegium (Djelfa)	0.159 ± 0.002
	Vitamin E	0.572 ± 0.023
	a Antioxidant reducing power is expressed as AEAC (Ascorbic acid Equivalent Antioxi-
	dant Power).

Table 4a . Antimicrobial activity of essential oil samples of Thymus vulgaris, Thymus algeriensis and Mentha pulegium by disc- diffusion diameter inhibition zones. Thymus algeriensis Essential Oil Plant Thymus vulgaris Djelfa Aflou Mentha pulegium

 4a

	Essential oil concentration (mg/disc)	2.23	2.32	2.23	2.27
			Inhibition Zone Diameters (mm) a
	Klebsiella pneumoniae	24,50 ± 2,12	16.00 ± 1.41	11.78 ± 2.27	10.33 ± 2.31
	Pseudomonas aeruginosa	9.00 ± 0.00	7.83 ± 0.76	7.50 ± 0.71	9.17 ± 0.29
	Salmonella typhi	24.00 ± 1.41	12.22 ± 0.70	16.67 ± 1.15	10.50 ± 0.71
	Escherichia coli	31.17 ± 2.84	13.88 1.24	14.33 ± 0.47	10.75 ± 0.35
	Bacillus cereus	19.33 ± 2.52	17.54 ± 0.30	17.00 ± 0.00	11.33 ± 1.53
	Staphylococcus aureus	22.33 ± 1.15	8.33 ± 0.58	9.00 ± 0.00	9.22 ± 1.07
	MRSA	29.00 ± 1.00	14.17 ± 1.18	9.83 ± 1.44	9.33 ± 1.15
	Enterococcus faecalis	15.33 ± 1.04	8.33 ± 0.58	9.00 ± 1.41	9.00 ± 1.41
	Candida albicans	19.67 ± 1.53	10.33 ± 2.31	10.33 ± 1.53	7.50 ± 0.71

a three repetitions average ± SD

Table 4b . Antimicrobial activity of the controls used by disc-diffusion diameter inhibition zones.

 4b

		GM (10UI)	AMC (10 g)	OX1 (1 g)	E15 (15 g)
			Inhibition Zone Diameters (mm)
	Klebsiella pneumoniae	9	9	0	10
	Pseudomonas aeruginosa	21	0	0	8
	Salmonella typhi	21	30	0	16
	Escherichia coli	31	24	0	15
	Bacillus cereus	31	28	18	33
	Staphylococcus aureus	22	32	24	29
	MRSA	12	22	16	0
	Enterococcus faecalis	23	25	0	29

GM: Gentamicine ; UI: unité internationale ; AMC: Amoxicilline -acide clavulanique ; OX1: Oxacilline ;E15: Erythromycine.

Table 5a . MIC and MBC for the essential oil samples of Thymus vulgaris, Thymus algeriensis and Mentha pulegium.

 5a

		Thymus vulgaris		Thymus algeriensis		Mentha pulegium
	Microbial Strains	MIC	MBC		(Djelfa)	(Aflou-Laghouat)	MIC	MBC
		(mg/mL)	(mg/mL)	MIC	MBC	MIC	MBC	(mg/mL)	(mg/mL)
				(mg/mL)	(mg/mL)	(mg/mL)	(mg/mL)
	Klebsiella pneumoniae	1.016	2.032	2.114	4.227	2.030	> 4.059	2.070	2.070
	Pseudomonas aeruginosa	> 2.032	> 2.032	> 4.227	> 4.227	> 4.059	> 4.059	> 2.485	> 2.485
	Salmonella typhi	0.508	1.016	2.114	3.044	3.004	4.059	1.035	2.070
	Escherichia coli	0.508	1.016	3.044	4.227	3.004	> 4.059	1.035	1.035
	Bacillus cereus	0.127	0.508	0.264	0.528	1.015	1.015	1.035	2.485
	Staphylococcus aureus	1.016	1.016	1.057	1.057	1.015	1.015	1.035	2.070
	MRSA	0.508	1.016	0.528	3.044	1.015	2.030	1.035	2.070
	Enterococcus faecalis	0.127	1.016	0.528	1.057	0.507	1.015	1.035	2.070
	Candida albicans	0.564	1.129	4.697	> 4.697	4.510	4.510	1.150	2.301

MIC: Minimum Inhibitory Concentration; MBC:Minimum Bactericide Concentration.

Table 5b . MIC (mg/mL) for the essential oil samples of Thymus vulgaris, Thymus algeriensis and Mentha pulegium (values from literature).

 5b

Current Nutrition & Food Science, 2017, Vol. 13, No. 0 Benabed et al.

Current Nutrition & Food Science, 2017, Vol. 13, No. 0 Benabed et al.

ACKNOWLEDGEMENTS

The authors wish to thank the Algerian Ministry of High Education and Scientific Research for funding the Research Laboratory of Fundamental Sciences-Laghouat, Algeria.

CONFLICT OF INTEREST

The authors confirm that this article content has no conflict of interest.


Rekia Cherbi

Chahrazed Hamia

Nadhir Gourine

Isabelle Bombarda

Mokhtar Saïdi

Mohamed Yousfi
email: yousfim8@gmail.com

Lipid Classes, Fatty Acids, Tocopherols Compositions and Antioxidant Activity of Lawsonia alba Seed Oils Growing in Algeria

Keywords: Lawsonia alba, seeds, lipids, fatty acids, tocopherols, DPPH assay, Phosphomolybdenum assay, Hierarchy Ascendant Classification (HAC)

Background: Lawsonia alba seed oils are not widely used commercially even though they have characteristics that well suit for industrial applications and can contribute to healthy human diets. The most important aim of the present work is the evaluation of the tocopherols composition of these oils. Moreover, lipid classes, fatty acids compositions and antioxidant activity of the seed oils were also investigated.

Methods: Lawsonia alba seed oils were extracted with Soxhlet device using n-hexane and chloroform/methanol. Tocopherols were analyzed by HPLC The antioxidant activity were determined using two different assays: DPPH (1,1-diphenyl-2-picrylhydrazyl), and Phosphomolybdenum.

Results:

The content of the oils ranged from 7.52 to 10.45%. Polyunsaturated fatty acids dominated all the studied samples. Linoleic, followed by palmitic and oleic, were the major fatty acids found in the crude seed oils and their lipid classes. Results of tocopherols identification showed that the seed oils were rich in tocopherols, especially -and -tocopherols. For DPPH assay, the antioxidant powers varied from medium to low values (IC 50 = 0.11 to 16.54 mg/mL). The results of the Phosphomolybdenum assay showed very important activities of the oils that were decreasing in the following order: polar lipids > neutral lipids (Chloroform/Methanol) > total lipids > neutral lipids (n-hexane).

Conclusion:

this current report reveals the richness of the Lawsonia alba seeds in tocopherols, which in fact is studied for the first time. Moreover, cluster analysis revealed that fatty acid composition of the seed oils of Lawsonia alba were very close to those of Walnut.

INTRODUCTION

Lawsonia alba (syn. Lawsonia inermis Linn.) locally known as El-Henna is (Henna) is a plant from the Lythraceae. It is native to North Africa and South East Asia, and is often cultivated as an ornamental plant throughout India, Persia, and along the African coast of the Mediterranean Sea [START_REF] Bakkali | Lawsone accumulation in normal and transformed cultures of henna, Lawsonia inermis[END_REF][START_REF] Chaudhary | Lawsonia inermis Linnaeus: A phytopharmacological review[END_REF][START_REF] Goswami | Antiulcer potential of Lawsonia inermis L. Leaves against gastric ulcers in rats[END_REF][START_REF] Makhija | Lawsonia inermis-From traditional use to scientific assessment[END_REF][START_REF] Uddin | Bioactive flavonoids from the leaves of Lawsonia alba (Henna)[END_REF]. It is a unique source of various pharmacologically important compounds such as naphthoquinone (Lawsone) derivatives, aliphatic components, triterpenes, sterols, essential oils, phenolic derivatives, fatty acids, amino acids, coumarins, xanthones, flavonoids and other constituents [START_REF] Ghosh | Antibacterial activity of some medicinal plant extracts[END_REF][START_REF] Chaudhury | Efficient method for micropropagation of Lawsonia inermis[END_REF]. Lawsonia alba is known to contain antibacterial [START_REF] Ghosh | Antibacterial activity of some medicinal plant extracts[END_REF], antifungal [START_REF] Singh | Fungitoxic studies on bark extract of Lawsonia inermis against ringworm fungi[END_REF], antiparasitic [START_REF] Okpekon | Antiparasitic activities of medicinal plants used in Ivory Coast[END_REF], antiviral [START_REF] Khan | Occurrence of some antiviral sterols in Artemisia annua[END_REF], anticancer [START_REF] Endrini | Comparing of the cytotoxicity properties and mechanism of Lawsonia inermis and Strobilanthes crispus extract against several eancer cell lines[END_REF], antidiabetic [START_REF] Abdillah | Hypoglycaemic and antihyperlipidemic effects of henna leaves extract (Lawsonia inermis Linn) on alloxan induced diabetic mice[END_REF][START_REF] Syamsudin | The effect of Inai (Lawsonia inermis Linn) leaves extract on blood sugar level: An experimental study[END_REF], antioxidant [START_REF] Hosein | Phenolic compounds and antioxidant activity of henna leaves extracts (Lawsonia inermis)[END_REF][START_REF] Rahmat | Anticarcinogenic properties and antioxidant activity of henna (Lawsonia inermis)[END_REF], tuberculostatic [START_REF] Sharma | Tuberculostatic activity of henna (Lawsonia inermis Linn.)[END_REF], anti-inflammatory [START_REF] Gupta | Evaluation of antiinflammatory activity of some constituents of Lawsonia inermis[END_REF], antifertility [START_REF] Munshi | Antifertility activity of three indigenous plant preparations[END_REF] and wound healing properties [START_REF] Nayak | The evidence based wound healing activity of Lawsonia inermis Linn[END_REF]. Only a few works on the chemical composition of the fatty acids of the seed oils of Lawsonia alba have been reported [START_REF] Aggarwal | Chemical examination of the seed oil of Lawsonia alba[END_REF][START_REF] Ansari | Component acids of minor seed oils[END_REF][START_REF] Raie | Physico-chemical characterization of Lawsonia alba oil of the family myrtaceae[END_REF][START_REF] Ahmad | Physio-chemical analysis of seven seed oils[END_REF][START_REF] Datta | Determination of some characteristics and fatty acid composition of the seed oil of Lawsonia alba[END_REF]. Lawsonia alba seed oils are not widely used commercially even though they have characteristics that well suit for industrial applications and can contribute to healthy human diets.

To our knowledge, no report has been found on the evaluation of the composition of the tocopherols of Lawsonia alba seeds grown in Algeria, nor on their antioxidant activities.

MATERIALS AND METHODS

Chemicals

Methanol, chloroform, 2-propanol, absolute ethanol, sulfuric acid, anhydrous sodium sulphate, sodium chloride, silica gel (60 G), sodium, ascorbic acid, sodium thiosulfate, potassium hydroxide, ammonium molybdate, sodium phosphate were purchased from Merck, Germany. n-hexane, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), -tocopherol (Vitamin E), DPPH • (1,1-diphenyl,2picrylhydrazyl) were obtained from Sigma-Aldrich Chemical Co. USA. All reagents and chemicals used were of analytical grade.

Collection of Plant Material

The seeds of Lawsonia alba samples used in this study were collected during the maturation stage in October 2011, from three different locations (Adrar, Ouargla and Biskra), located in the south of Algeria. The sizes of the seeds were 7.0±0.3 mm, 5.0±0.3 mm and 4.0±0.3 mm for Adrar, Ouargla and Biskra regions, respectively (Fig. 1).

Oil Extraction

The oil was extracted from seeds using n-hexane solvent. The dried grounded seeds of L. alba (100 g) were extracted with 300 mL n-hexane using a soxhlet extraction apparatus for 24 h. The n-hexane was evaporated in a rotary vacuum evaporator at 40 °C.

Extraction of Total Lipids from L. alba

The total lipids were extracted by the method of Folch et al [START_REF] Folch | A simple method for the isolation and purification of total lipids from animal tissues[END_REF]. The seeds used in this study were dried under the shade, ground in a coffee grinder to fine powder and weighed accurately; about 100 g of the powder was extracted in 400 mL of chloroform/methanol (2:1, v/v) for 24 hours using Soxhlet apparatus (extractor). The solvent was evaporated in a rotary vacuum apparatus at 40 °C. The residue was dissolved in 200 mL of aqueous sodium chloride (9%), then 200 mL of chloroform was added and the phases were mixed vigorously. After phase separation, the chloroform layer was withdrawn, dried over anhydrous sodium sulphate Na 2 SO 4 and filtered. The filtrate was then concentrated under vacuum. The extracted lipids were weighed to determine the total lipids content, and then stored at +4 °C for further analysis.

Isolation of Neutral Lipids, Polar Lipids by Column Chromatography

The extracted lipids (2 g) were separated into Neutral Lipids (NLs) and polar lipids Glycolipids (GLs) and Phospholipids (PLs) by column chromatography with 10 g of activated silica gel (60G). Neutral lipids and polar lipids were eluted successively using chloroform and methanol, respectively. The individual fractions were evaporated under vacuum to remove solvent and the weight of each lipid class was expressed as a percentage of the total lipid dry weight.

Fatty Acid (FA) Composition

The FA composition of the oils was determined after having converted the FA into FA Methyl Esters (FAMEs) then analysed by Gas Chromatography (GC). The methyl esters were prepared by the following procedure: Oils (50 mg) were refluxed for 30 min in 20 mL of 1% sodium methylate (NaOMe), and then 20 mL of water was added. The Fatty Acid Methyl Esters (FAMEs) were extracted by nhexane and washed with distilled water. The combined extracts were dried over anhydrous sodium sulphate Na 2 SO 4 and evaporated under vacuum, then analysed by capillary gas chromatography.

Chromatographic Analysis of FAME(s)

A gas chromatographic GC (model Chrompack CP 9002) was equipped with a FID detector and DB-23 capillary column (30 m 0.32 mm internal diameter, 0.25 μm film thickness). Two μL of the sample was injected in the Split injection mode. The oven analysis temperature started at 200 °C for 10 min and was raised at a rate of 4 °C/min to 250 °C. The injector temperature was 250 °C and the detector temperature was 280 °C, the flow rate of nitrogen (carrier gas) was 1 mL/min. Identification and quantification of FAMEs were accomplished by comparing the retention times of peaks with those of pure standards purchased from Sigma and analysed under the same conditions. The results were expressed as total area percentage of identified individual fatty acids in the lipid fraction.

Tocopherol Analysis

Prior to HPLC analysis, 35 mg of seed oil extracted with n-hexane was diluted with 1 mL of n-hexane, and then 20 μL of the solution was injected. Tocopherols were analyzed by HPLC using an Agilent 1290 Infinity apparatus equipped with a Quaternary Pump Model 1260, an automatic liquid sample and a Fluorimetric Detector (FLD 1260). The detection was set at 295 nm for excitation wavelength and at 330 nm for emission wavelength. The separation column was a SI60 (250 mm 4.6 mm i.d., 5 μm, Lichrosorb, Merck KGaA Darmstadt, Germany). The mobile phase was nhexane/isopropanol, 99/1, v/v, in isocratic mode, with a flow rate set at 1 mL/min, during 20 min. Column compartment was set at 25 ºC. Commercial standard ( -Toc) and a mixture ( -Toc, -Toc, -Toc and -Toc) were co-injected with samples for the identification of tocopherol isomers.

Chemical Analysis

Refractive Index (RI), Acid Value (AV), Iodine Value (IV), Saponification Value (SV) and ester Value (EV) of the extracted Lawsonia alba seed oils were determined according to the standard methods of the French Association of Normalisation ''Association Française de Normalisation'' AFNOR NF T 60-206, 1984) [START_REF]Corps gras, graines oléagineuses et produits dérivés[END_REF]. All experiments were performed in triplicate.

Radical-Scavenging Activity (DPPH Assay)

The antioxidant activity was determined by DPPH method, which is based on the evaluation of the free-radical scavenging capacity. In this method, the 1,1 diphenyl-2picrylhydrazyl (DPPH • ) free stable radical was used to measure the antioxidant activity. Briefly, 1 mL of the sample diluted in ethanol or 2-propanol was mixed with 1 mL of 0.25 mM DPPH • in ethanol. The reaction mixture was well shaken and incubated in the dark at room temperature for a time period of 30 min. The absorbance of the resulting solution was read at 517 nm against a blank [START_REF] Molyneux | The use of the stable free radical diphenylpicrylhydrazyl (DPPH) for estimating antioxidant activity[END_REF]. The antiradical activity was expressed as IC 50 (mg/mL): the concentration required to cause 50% initial DPPH • inhibition. The inhibitory percentage of DPPH • was calculated according to the following equation:

IC "Scavenging activity %" = [1-(A/A 0 )] 100.
Where A: Absorbance of the sample; A 0 : Absorbance of the control (Initial value) All experiments were performed in triplicate.

Total Antioxidant Activity (Phosphomolybdenum Assay)

The total antioxidant activity of the samples was determined by the phosphomolybdenum method PPM of Prieto et al. [START_REF] Prieto | Spectrophotometric quantitation of antioxidant capacity through the formation of a phosphomolybdenum complex: specific application to the determination of vitamin E[END_REF]. Briefly, 0.3 mL of oil extract dissolved in ethanol or 2-propanol was mixed with 3 mL of the reagent solution (0.6 mol/L sulphuric acid, 28 mmol/L sodium phosphate and 4 mmol/L ammonium molybdate). The tubes were capped and incubated in a water bath at 95 °C for 90 min. The tubes were cooled to room temperature and the absorbance of the green phosphomolybdenum complex was measured at 695 nm. In the case of the blank, 0.3 mL of solvent was used in place of the sample (oil). Ascorbic acid was used as a standard. Total antioxidant capacity was expressed as equivalents of vitamin E: VEAEC= "Vitamin E Equivalent Antioxidant Capacity"). All experiments were performed in triplicate.

Cluster Analysis

Cluster analysis was performed using hierarchical clustering (Ward's technique) with Euclidean distance measure. The calculus was performed using a set of data composed of 9 different plants (representing 18 individuals) and 14 variables (fatty acids: C 10:0 , C 12:0 , C 14:0 , C 16:0 , C 16:1 , C 17:0 , C 17:1 , C 18:0 , C 18:1 , C 18:2 , C 18:3 , C 20:0 , C 20:1 , C 20:5 ).

RESULTS AND DISCUSSION

Physico-Chemical Properties

The oils of Lawsonia alba seeds from the three different regions in Algeria (Adrar, Biskra, and Ouargla) were Soxhlet extracted with chloroform/methanol 2:1, v/v) and with nhexane. The oils (total lipids) content is summarized in Tables 1 and2. The results showed that the yields of extraction obtained with chloroform/methanol solvent were slightly higher than those obtained with n-hexane. Moreover, the values of the yields were practically the same for the three studied regions. In fact, the yield values were in the range of 7.52-9.56% w/w for the n-hexane solvent extraction; and were in the range of 9.88-10.45% w/w when the chloroform/methanol solvent was used. As a result, the nature of solvent did not strongly affect the extraction yield. Furthermore, the crude lipid contents obtained by the two solvents revealed that these seeds cannot be considered as oil-bearing: since their oil contents were low and were in the range of some other vegetable materials such as wheat germ, acorns (Quercus sp), Pistacia lentiscus, Sorghom, Hippophae rhamnoides, Myrtus communis and Rhanterium adpressum flowers [START_REF] Charef | Determination of the fatty acid composition of acorn (Quercus), Pistacia lentiscus seeds growing in Algeria[END_REF][START_REF] León-Camacho | Acorn (Quercus spp.) fruit lipids: Saponifiable and unsaponifiable fractions: A detailed study[END_REF][START_REF] Hadbaoui | Fatty acid, tocopherol composition and the antioxidant activity of the lipid extract from the sorghum grains growing in Algeria[END_REF][START_REF] Hamia | Chemical composition and antioxidant activity of the essential oil and fatty acids of the flowers of Rhanterium adpressum[END_REF][START_REF] Sonntag | Composition and characteristics of individual fats and oils[END_REF]. The yields found here in this investigation are in very good agreement with those of literature (India and USA): 7.5% "hexane extract" [START_REF] Atal | Studies on Indian seed oils. Part I. Preliminary screening of linoleic acid rich oils[END_REF], and 9.6% "pentane extract" [START_REF] Graham | Seed lipids of the Lythraceae[END_REF]. On the other hand, these seed oil contents are important compared to other seed oils such as corn (3.0-6.5%) [START_REF] Nehdi | Characteristics, composition and thermal stability of Acacia senegal (L.) Willd. seed oil[END_REF]. In addition, the slight difference in the yields may be due to the different origins of the seeds as well as the different environmental conditions that affect lipid development [START_REF] Cerchiara | Characterization and utilization of Spanish Broom (Spartium junceum L.) seed oil[END_REF].

The results of the physico-chemical characteristics of the oils are presented in Tables 1 and2. All the oils had a pleasant odour and a dark green colour, while the colour of the oil extracted by chloroform/methanol (Ouargla) was brown. These colours indicate the presence of chlorophylls and various carotenoids pigments [START_REF] Sam | Extraction and classification of lipids from seeds of persea Americana miller and Chrysophyllum albidum g[END_REF][START_REF] Therios | CAB International[END_REF]. The seed oil states at room temperature (25.0±1 °C) were generally in the form of semi liquids. However, the oils from Adrar were liquid for both solvents.

The refractive indexes of the six investigated neutral lipid extracts were found to be pretty similar. The results also indicated that these oils presented high unsaturation bonds. The greater the degree of unsaturation, the higher refractive index will be [START_REF] Cerchiara | Characterization and utilization of Spanish Broom (Spartium junceum L.) seed oil[END_REF]; this relationship was confirmed by the values of fatty acid composition of the oils presented in Tables 1 and2.

The acid value is a measure of the total acidity of the lipid [START_REF] Onyeike | Chemical composition of selected Nigerian oil seeds and physicochemical properties of the oil extracts[END_REF]. The acid values of L. alba seed oils from Biskra, extracted by n-hexane and CHCl 3 /MeOH were very high: 169.47 and 146.59 "mg KOH/g oil", respectively, while, the Acid Values (AVs) of L. alba seed oils from the rest of the regions (Adrar and Ouargla) varied form weak to low values 4.39-43.36 "mg KOH/g oil". The weakest value of AV was recorded for the region of Adrar when n-hexane solvent was used. These results can be attributed to the action of lipolytic enzymes [START_REF] Cerchiara | Characterization and utilization of Spanish Broom (Spartium junceum L.) seed oil[END_REF], the bad conservation of the seeds before extraction [START_REF] Charef | Determination of the fatty acid composition of acorn (Quercus), Pistacia lentiscus seeds growing in Algeria[END_REF], or the incomplete ripeness of the seeds.

The saponification value (SV) is a measure of the fatty acids molecular weight, and defined as the amount of alkali required to saponify fatty acids in a given weight of oil [START_REF] Boran | Changes in the quality of fish oils due to storage temperature and time[END_REF]. The saponification values of the oils prepared by Ch/MeOH were higher than those of the oils extracted by n-hexane. This indicates that the oils achieved by Ch/MeOH contain high amounts of low molecular weight of fatty acids [START_REF] Cerchiara | Characterization and utilization of Spanish Broom (Spartium junceum L.) seed oil[END_REF], because a high saponification value indicates very high content of low molecular weight triacylglycerols [START_REF] Nehdi | Characteristics and chemical composition of date palm (Phoenix canariensis) seeds and seed oil[END_REF]. This value makes them useful in the production of liquid soaps and shampoos [START_REF] Nehdi | Characteristics, chemical composition and utilisation of Albizia julibrissin seed oil[END_REF][START_REF] Ku | Characterization of seed oils from fresh Bokbunja (Rubus coreanus Miq.) and wine processing waste[END_REF].

The iodine value is used to assess the unsaturation of the oils and in assessing the stability of oils in industrial applications [START_REF] Wu | Essential oil extracted from peach (Prunus persica) kernel and its physicochemical and antioxidant properties[END_REF]. The range of iodine numbers were 68.0-113.3 "mg/100g oil", thus, the L. alba seed oil could be classified as a non-drying oil. The high iodine value is due to the high content of unsaturated fatty acids of a sample. For both solvents, the highest IVs were recorded for Adrar oils, and the lowest ones were observed for the Biskra region.

Lipid Classes and Fatty Acid Composition of L. alba Seed Oil and Its Lipid Classes

After quantification of the lipid classes, it was noted that neutral and polar lipids were absent (not detected) in the total lipids obtained by n-hexane solvent. In the opposite side, when chloroform/methanol solvent was used, the content of neutral lipids was very significant (85.82-93.08%), and the Moreover, the difference in interaction between solvent and solutes, caused by both solvent polarities and their boiling temperature, may contribute to the extraction efficiency and yield, as well as the composition of the extracted oils [START_REF] Wu | Essential oil extracted from peach (Prunus persica) kernel and its physicochemical and antioxidant properties[END_REF]. The total Fatty Acid (FA) composition of the oils obtained in this study was determined by GC, and is shown in Tables 1 and2. The first table presents the FA compositions of lipid fractions obtained by n-hexane solvent; while the second table gathers the lipids obtained by chloroform/methanol solvent (total lipids fraction and both neutral and polar sub-extractions or fractionations).

The FA profile is a main determinant of the oil quality. Fatty acid analysis has shown that the seed oils studied contained twelve fatty acids: Five were saturated FAs (SFAs), four were monounsaturated FAs (MUFAs) and three were polyunsaturated FAs (PUFAs) with 14 to 20 carbons.

The major fatty acids identified in all tested samples were the linoleic acid (18:2) (45.10-74.39%), palmitic acid (16:0) (8.84-21.52%), oleic acid (18:1) (8.48-17.19%), stearic acid (C18:0) (3.29-8.01%), arachidic acid (C20:0) (1.39-2.48%) and eicosapentaenoic acid (C20:5) (0.47-2.43%) (Tables 1 and2). The linoleic, palmitic and oleic acids accounted for more than 83% of the total fatty acid content. Moreover, FA (14:0, 16:1, 17:0, 17:1, 18:3, 20:1) were absent or detected at low percentages (0.03-1.22%) in these lipids. The results of fatty acids profile of the oils are similar to those reported previously for Lawsonia alba seed oils [START_REF] Aggarwal | Chemical examination of the seed oil of Lawsonia alba[END_REF][START_REF] Ansari | Component acids of minor seed oils[END_REF][START_REF] Raie | Physico-chemical characterization of Lawsonia alba oil of the family myrtaceae[END_REF][START_REF] Ahmad | Physio-chemical analysis of seven seed oils[END_REF][START_REF] Datta | Determination of some characteristics and fatty acid composition of the seed oil of Lawsonia alba[END_REF], and Lawsonia inermis seed oils [START_REF] Graham | Seed lipids of the Lythraceae[END_REF]. Furthermore, the three main fatty acid components were the same as those of Pistacia atlantica fruits (whose oils were considered as dietary nutriment) [START_REF] Guenane | Effect of maturation degree on composition of fatty acids and tocopherols of fruit oil from Pistacia atlantica growing wild in Algeria[END_REF].

All oils had high amounts of the Polyunsaturated Fatty Acid (PUFA) that primarily was linoleic acid. The polyunsaturation was found to be 48.22-75.06% as shown in Tables 1 and2. The ratio of unsaturated fatty acids to saturated fatty acids was mainly higher in the oils than in the polar lipid fractions.

The results suggested that L. alba oil is a good source of unsaturated fatty acids; it means that it should exhibit high antioxidant activity. Let's remind that unsaturated fatty acids are very important compounds for the stability of oils, because of the chemical reactions occurring at the double bonds [START_REF] Wu | Essential oil extracted from peach (Prunus persica) kernel and its physicochemical and antioxidant properties[END_REF]. The high unsaturation degree of the oils allows them to be oxidized easily when used at high temperatures. For example, linoleic acid oxidizes approximately 50 times faster than oleic acid [START_REF] Luzia | Bioactive substance contents and antioxidant capacity of the lipid fraction of Annona crassiflora Mart. seeds[END_REF].

Tocopherol Contents and Compositions of L. alba Seed Oils

In spite of the nutritional importance of tocopherols [START_REF] Kamal-Eldin | The chemistry and antioxidant properties of tocopherols and tocotrienols[END_REF][START_REF] Hussain | Biosynthesis, structural, and functional attributes of tocopherols in planta; past, present, and future perspectives[END_REF], their content and composition in L. alba seed oils remain unknown. The quantification of tocopherols by liquid chromatography was based on an external standard method using -tocopherol. The results are expressed as mg oftocopherol equivalents per 100 g of oil. Results are presented in Table 3, which shows clearly that the seed oils are rich in tocopherols, and especially for -and -tocopherols. For the region of Biskra no presence of tocopherols was detected. However, the total tocopherol content of the Ouargla region (752.5 mg/100 g) was found to almost twice as big as the one of the Adrar region (401.09 mg/100 g). In addition, the contents of both -and -tocopherols were twice as high as the one of the Adrar region; although the -tocopherol contents in both regions were similar (17.60% and 15.59%). Furthermore, the compositions of -and -tocopherols for these two last regions were very close to each other: 38.9% and 37.25%, respectively. The percentage of -tocopherol in the Ouargla region (58.71%) was slightly higher than the Adrar region (51.77%). Conversely, and unlike the Adrar region the -tocopherol was absent in the Ouargla region. It was noted that the sum of -and -tocopherols percentages were practically the same for the two studied regions (Adrar: 58.71%, Ouargla: 58.87%).

Antioxidant Activities

Free Radical Scavenging Activity

The antioxidant activities of L. alba seed oils were determined using the DPPH free radical assay. The DPPH is a stable free radical which has been widely used to evaluate the radical scavenging ability of antioxidants [START_REF] Murugan | Comparative evaluation of different extraction methods for antioxidant and anti-inflammatory properties from Osbeckia parvifolia Arn. An in vitro approach[END_REF]. DPPH radical scavenging assays are based on the transfer of electrons from a donor molecule to the corresponding radical. The reduction capacity of DPPH radicals was determined by the decrease of absorbance at 517 nm. This activity can be evaluated by the determination of the IC 50 values. Low IC 50 value indicates strong antioxidant activity in a tested sample [START_REF] Loo | Antioxidant activity of compounds isolated from the pyroligneous acid, Rhizophora apiculata[END_REF]. The IC 50 value can be defined as the amount of antioxidant necessary to decrease the initial DPPH concentration by 50% [START_REF] Antolovich | Methods for testing antioxidant activity[END_REF]. Vitamin C, BHA, BHT and vitamin E were used as standards.

According to Table 4, IC 50 values of the DPPH • radical scavenging activity ranged from 0.11 to 31.96 mg/mL for the twelve oils extracts. PLB (Biskra), PLO (Ouargla), TLO (Ouargla), PLA (Adrar) and TLB (Biskra) oils extracts gave good values of the IC 50 : 0.11, 0.12, 0.38, 0.51 and 0.56 mg/mL respectively, while the IC 50 values of the standards BHA, ascorbic acid, Vitamin E and BHT, which showed an excellent scavenging activity, were 0.0086, 0.0096, 0.0188 and 0.1 mg/mL respectively. NLB (n-hexane extract) did not show any antioxidant activity (very weak value).

Referring to the results of DPPH assay (Table 4), the lowest antioxidant activities were detected for NL (n-hexane extract) fractions, whereas the highest antioxidant activities were detected for Polar Lipids (PL) fractions in all the samples. Also Total Lipids (TL) fractions appeared to be more effective scavengers against DPPH radical than NL (Chloroform/MeOH extract) fractions. Finally it seemed that the region of collection did not affect strongly the antioxidant activities of the tested fractions.

In the literature, the inhibition of DPPH radical scavenging by plant oils can be attributed to the unsaturated fatty acids (PUFA), phospholipids, total phytosterols, total tocopherols, carotenoids and phenolics [START_REF] Luzia | Bioactive substance contents and antioxidant capacity of the lipid fraction of Annona crassiflora Mart. seeds[END_REF][START_REF] Gai | Microwave-assisted aqueous enzymatic extraction of oil from Isatis indigotica seeds and its evaluation of physicochemical properties, fatty acid compositions and antioxidant activities[END_REF][START_REF] Jiao | Microwave-assisted aqueous enzymatic extraction of oil from pumpkin seeds and evaluation of its physicochemical properties, fatty acid compositions and antioxidant activities[END_REF]. Therefore, the antioxidant activities of the Lawsonia alba oils can be attributed to the unsaturated fatty acids, tocopherols and polar lipid compound present in the samples.

Antioxidant Capacity by Phosphomolybdenum Method "Total Antioxidant Activity"

The phosphomolybdenum PPM is a method based on the reduction of Mo (VI) to Mo (V) by the antioxidant compound; this reaction is accompanied by the appearance of a green colour phosphate/Mo (V) (phosphomolybdenum) complex with a maximal absorption at 695nm and acidic pH [START_REF] Prieto | Spectrophotometric quantitation of antioxidant capacity through the formation of a phosphomolybdenum complex: specific application to the determination of vitamin E[END_REF]. A higher absorbance indicates a higher total antioxidant capacity [START_REF] Loo | Antioxidant activity of compounds isolated from the pyroligneous acid, Rhizophora apiculata[END_REF].

Total antioxidant activities of the lipid extracts, expressed as VEEAC, are shown in Table 4. Ascorbic acid "Vitamin C" (VC), which was used as a positive control, presented the highest antioxidant capacity from all tested samples (VEEAC = 0.95 mg/mL). In lipids extracts, total antioxidant activities were found to be high in PLB (VEEAC = 0.57 mg/mL) followed by PLA (0.52 mg/mL), and PLO (0.41 mg/mL). For these PLB and PLA fractions, the antioxidant power was almost 2 times less active than vitamin C while PLO was more than 2 times less active than vitamin C. On the other hand, the rest of the fractions exhibited lower antioxidant activities with very close values of VEEAC and ranging from 0.16 to 0.27 mg/mL. This range can be roughly approximated to a mean value of 0.2 mg/mL, which means that these extracts exhibited 5 times lower activities than vitamin E. Moreover, and no matter the region of collection, it was found that strong activities of the oils were mostly decreasing in the following order: PL>NL (chloroform/methanol) > TL > NL (n-hexane). These results were expected, since most known strong antioxidant chemicals include polar functions responsible for their activities.

Correlation between total tocopherol contents and antioxidant activities using DPPH and phosphomolybdenum assays were investigated. After a close examination of the results of Table 4, it was noticed that the antioxidant activity powers of the seed oils (NL obtained by n-hexane extracts) from the three investigated regions were decreasing in the following order Ouargla > Adrar > Biskra i.e. (DPPH: 5.31 > 16.54 > 31.96 mg/mL; Phosphomolybdenum: 0.14 > 0.16 > 0.21 mg/mL). This decline in activities may be attributed to the total contents of tocopherols which were also decreasing in the same order for the same regions. In other words, a direct correlation between total tocopherol contents and antioxidant powers was observed.

Statistical Analysis Using Hierarchy Ascendant Classification Method (HAC)

In order to highlight the difference in the contents of the main fatty acids FAs (n-hexane extracts) of the seeds of Lawsonia alba (collected from different regions: Adrar, Biskra, Ouargla) with some chosen common seed plants (data provided from the literature [START_REF] Hadbaoui | Fatty acid, tocopherol composition and the antioxidant activity of the lipid extract from the sorghum grains growing in Algeria[END_REF][START_REF] Boukouada | chemical composition and antioxidant activity of seed oil of two Algerian date palm cultivars (Phoenix dactylifera)[END_REF][START_REF] Sabudak | Fatty acid composition of seed and leaf oils of Pumpkin, Walnut, Almond, Maize, Sunflower and Melon[END_REF][START_REF] Besbes | Date seeds: chemical composition and characteristic profiles of the lipid fraction[END_REF][START_REF] Benalia | Fatty acid profile, tocopherols content and antioxidant activity of algerian pumpkin seeds oil (Cucurbita pepo L)[END_REF]), we have ). Let's remind that the first cluster could be separated into two close sub groups SG-1 and SG-2. The SG-1 showed that the fatty acids profiles of Lawsonia alba samples are pretty similar to Walnut; but the tiny difference between them is due to a significant content of C 18:3 (11.37%) for Walnut when compared to Lawsonia alba samples where it was insignificant (0.11-0.37%). As a general conclusion, the HAC analysis revealed that the studied seeds have very similar fatty acids profiles to most common considered edible plants.

CONCLUSION

In conclusion, it was found that the seed oils coming from three different regions in Algeria (Adrar, Ouargla, and Biskra) presented practically the same chemical compositions of fatty acids compound; and despite the fact that there was no significant impact of the regions of collection on the values of the antioxidant activities in terms of both DPPH and Phosphomolebdenum assays this current report reveals the richness of the Lawsonia alba seeds in tocopherols, which in fact is studied for the first time.

The physico-chemical characterization and the fatty acid profile suggest the possible application of the oils in the industry of liquid soaps and hair shampoo. The results suggested that Lawsonia alba oils are a good source of unsaturated fatty acids with good antioxidant activity in terms of Phosphomolybdenum assay (total antioxidant power).

On the other hand, cluster analysis revealed that fatty acid composition of the seed oils of Lawsonia alba was very close to those of Walnut.

CONFLICT OF INTEREST

The author(s) confirm that this article content has no conflict of interest.

ACKNOWLEDGEMENTS

Declared none.

Fig. ( 1 ).

 1 Fig. (1). Seeds of Lawsonia. alba from different regions in Algeria.

  adopted the method of cluster analysis, using Ward's technique; 14 fatty acids representing the main identified compounds in these plants(18 individuals) were employed. The results show the existence of two principal clusters (I and II) within the fatty acids of the individuals of the investigated plants (Fig. 2). First cluster (I) includes the following seed plants: Lawsonia alba, Walnut, Pumpkin, Sorghum, Maize, Sunflower, Melon while the second cluster (II) is composed of only Date palm varieties and Almond. The last cluster shows that the fatty acids contents of the Date palm seed varieties have a similar pattern in comparison with Almond; indeed this group is characterized by roughly close contents of C 16:1 , C 17:0 , C 17:1 , C 18:0 , C 18:1 , C 18:3 , C 20:0 , C 20:1 , C 20:5 . The major difference between them lies in the moderate/high content of C 12:0 and C 14:0 (9.84-11.6% and 17.8-28.4%, respectively) for the Date palm varieties versus their negligible contents for the Almond (C 12:0 : 0%, C 14:0 : 0.03%). The data examination of the cluster (I) revealed that this group is characterized by high contents of C 16:0 , C 18:1 and C 18:2 (mean±sd: 10.47±6.06%, 25.67±16.94% and 57.23±18.72%, respectively) and low contents of C 10:0 , C 12:0 , C 14:0 , C 16:1 , C 17:0 , C 17:1 , C 18:0 , C 18:3 , C 20:0 , C 20:1 , C 20:5 (<5.3%

Fig. ( 2 ).

 2 Fig. (2). Dendrogram obtained from a cluster analysis of the fatty acids obtained from different seed oils of Algerian cultivars of Lawsonia alba and a selection of seed oils of different edible plants. Samples are clustered using Ward's technique with a Euclidean distance measure.

Table 1 . Physicochemical proprieties and fatty acid compositions of Lawsonia alba seed oils (n-hexane solvent).

 1

	Composition (%)

tr: traces content of polar lipids ranged from weak to moderate values (3.9-13.85%); the lowest value was recorded for the region of Adrar. As a result, L. alba seed oils exhibited high amounts of neutral lipids (85.82-93.08%) when chloroform/methanol solvent was employed.

Table 2 . Physicochemical proprieties and fatty acid compositions of Lawsonia alba seed oils (Chloroform/Methanol solvent extrac- tion and fractionations).

 2

	Composition (%)

Table 3 . Individual tocopherols content and their relative composition in Lawsonia alba seed oils (n-hexane).

 3

		Biskra		Ouargla		Adrar
	Tocopherols	Content	Composition	Content	Composition	Content	Composition
		(mg/100 g)	(%)	(mg/100 g)	(%)	(mg/100 g)	(%)
	-tocopherol	nd	nd	0.00	0.00	28.46±1.34	7.10±0.09
	-tocopherol	nd	nd	17.60 ±0.15	2.34±0.07	15.59±0.13	3.89±0.02
	-tocopherol	nd	nd	441.77±1.81	58.71±0.92	207.64±1.11	51.77±0.89
	-tocopherol	nd	nd	293.13±1.03	38.95±0.06	149.40±0.89	37.25±0.07
	Total	nd	nd	752.5	100.00	401.09	100.00
	nd: not determined.

Table 4 . Antioxidant activities of the lipids extracts of L. alba seeds.

 4

	Region			Biskra	Ouargla		Adrar
	Antioxidant assay	DPPH IC50 (mg/mL)	PPM VEEAC (mg/mL)	DPPH IC50 (mg/mL)	PPM VEEAC (mg/mL)	DPPH IC50 (mg/mL)	PPM VEEAC (mg/mL)	DPPH IC50 (mg/mL)	PPM VEEAC (mg/mL)
	n-hexane extracts	> 31.96	0.21 ± 0.01	5.31 ± 0.31	0.14 ± 0.01	16.54 ± 0.20	0.16 ± 0.01	-	-
		Total lipids	0.56 ± 0.01	0.27 ± 0.01	0.38 ± 0.02	0.20 ± 0.01	2.21 ± 0.12	0.23 ± 0.01	-	-
	CHCl3/MeOH extracts	Neutral lipids	1.94 ± 0.04	0.23 ± 0.01	1.91 ± 0.05	0.20 ± 0.01	5.3 ± 0.30	0.27 ± 0.01	-	-
		Polar lipids	0.11 ± 0.01	0.57 ± 0.01	0.12 ± 0.01	0.41 ± 0.03	0.51 ± 0.05	0.52 ± 0.02	-	-
		Vit. E	-	-	-	-	-	-	0.02 ± 0.01	-
	Standards	Vit. C	-	-	-	-	-	-	0.0096± 0.0005	0.95 ± 0.03
		BHA	-	-	-	-	-	-	0.0086 ± 0.0006	-