Antioxidant and Antibacterial Activities of Essential Oils from Three Moroccan Species (Lavandula mairei Humbert, Lavandula dentata L. and, Lavandula stoechas L.)

This study aimed to evaluate the antioxidant and the antibacterial activities of the essential oils from three Moroccan species of the genus Lavandula (Lavandula dentata L., Lavandula mairei Humbert and, Lavandula stoechas L.). The antibacterial activities were evaluated by the minimal inhibitory concentration (MIC) and the disc diffusion method, against Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Citrobacter koseri) and Gram-positive bacteria (Staphylococcus aureus). The antioxidant potential was evaluated using free radical Original Research Article Ghanimi et al.; JPRI, 33(45B): 64-71, 2021; Article no.JPRI.75106 65 Scavenging against 2,2-diphenyl-1-picrylhydrzyl (DPPH), ferric reducing activity power (FRAP), and total antioxidant capacity (TAC). The antibacterial activity of the essential oils showed higher activity against tested nosocomial bacteria especially against Klebsiella pneumoniae, Citrobacter koseri, and Staphylococcus aureus. The essential oil of Lavandula stoechas L. revealed the higher antioxidant activity as measured by DPPH (IC50 = 3,11 ± 0,13 mg/ml), FRAP (EC50 = 6,88 ± 0,8 mg/ml) and TAC (443,2 ± 38 mg AAE/g EO) assays. The essential oil of Moroccan Lavandula stoechas L. could have potential as antimicrobial compounds for the treatment of many infectious bacterial diseases, including multi-resistant bacterial strains of nosocomial origin.


INTRODUCTION
Nosocomial infections also called healthcareassociated infections (HAI), are infections acquired during the time of receiving healthcare that was not already present at the moment of hospital admission [1]. These infections are commonly caused by bacteria that are resistant to several antibiotics and can lead to delayed recovery or even death [2]. The WHO has launched in May 2015 the Global Antibiotic Research and Development Partnership, to fight antimicrobial resistance through new antibiotics and other therapeutic products [3].
Many studies have shown the potential of aromatic and medicinal plants as sources of compounds with antimicrobial activity [4,5]. One of these is the genus Lavandula that belongs to the Lamiaceae family and comprises more than 40 species [6]. Lavandula species have been used from Antiquity to flavor and preserve food and to cure illnesses, including microbial and viral infections [7]. The different medicinal and therapeutic effects of these plants are attributed to their richness in secondary metabolites [8]. Therefore, the evaluation of essential oil against a microbe is an evaluation of all the existing compounds in this oil at the same time against this microbe (Houël et al. 2015).
The present work aimed to evaluate the antioxidant potential (DPPH, FRAP, and total antioxidant capacity) and the antibacterial activity of the three Lavandula species (Lavandula mairei Humbert, Lavandula dentate L. and Lavandula stoechas L.) against multi-resistant bacterial strains of nosocomial origin.

Plant Materials
Aerial parts of Lavandula mairei Humbert, Lavandula dentata L. and Lavandula stoechas L. were collected in Mai 2019 from the Al-Haouz region (Morocco). The determination was carried out by Professor Ahmed Ouhammou in the regional herbarium "MARK" of the FSSM-UCA (Morocco). Plant material was dried for three weeks in the shade at room temperature. The samples dried were stored in the dark at 5°C until use.

Extraction of Essential Oils
Dried shoot (200 g) was hydro-distilled for 3 h with 700 ml of water using a Clevenger-type apparatus, according to the method recommended by the European Pharmacopoeia (1975). The essential oils (Eos) obtained were dried with anhydrous sodium sulfate and stored in a refrigerator at 5°C until the analysis.

DPPH radical scavenging activity
The ability of the essential oil to scavenge the DPPH radical was measured using the method described by Wu et al. (2003). A volume of 100 µl of various concentrations of the essential oil or standard was added to 1.4 ml of the ethanolic solution containing 0.1 mM of DPPH (2, 2diphenyl-1 picrylhydrazyl). The mixture absorbance was measured at 517 nm by a spectrophotometer after 30 min of incubation in the dark at room temperature. The percentage of inhibition was expressed as I% = ((A0-As)/A0)*100. Where A0 is the Blank absorbance and As is the sample absorbance. Butylated hydroxytoluene (BHT) was used as a positive control and the IC50 values were calculated as the concentration causing 50% inhibition of the DPPH.

Ferric reducing antioxidant power (FRAP) assay
The reducing power activity of the tested oils was measured using the method of Oyaizu 1986. A set of 500 µl of phosphate buffer (0.2 M, pH 6.6) and 500 µl of potassium ferricyanide [K3 Fe (CN)6] 1% were mixed with 200 µl of the essential oils. The mixture was incubated in a bain-marie for 20 min at 50°C. A volume of 500 µl of trichloracetic acid (TCA) 10% was used to acidify the mixture. The obtained solution was then centrifuged for 10 min at 3000 rpm. 500 µl of the solution's upper layer was mixed with 100 µl of FeCl3 (0.1%) and 500 µl of distilled water. The absorbance was measured at 700 nm and Quercetin was used as a standard. EC50 (mg/ml) was calculated by plotting the absorbance against the corresponding concentration (EC50 concentration corresponding to 0.5 of absorbance).

Total antioxidant capacity
The analysis was based on the reduction of Mo (VI) to Mo (V) and the resulting formation of a green phosphate/Mo(V) complex at acidic pH [9]. A volume of 25 µl of essential oil was added to 1 ml of reagent solution (sulfuric acid 0.6 mol/L, sodium phosphate 28 mmol/L, and ammonium molybdate 4mmol/L). The mixtures were incubated in a bain-marie for 90 min at 95° C and then cooled to ambient temperature. The absorbance was measured at 695 nm, and the total antioxidant activity was given as ascorbic acid equivalence number (mg AAE/g EO).

Bacterial strains
The antibacterial activity of Lavandula mairei Humbert, Lavandula dentate L., and Lavandula stoechas L. was tested against; Gram-negative bacteria included Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Citrobacter koseri and Acinetobacter baumannii, and Gram-positive Staphylococcus aureus. The inoculum suspension was prepared by using colonies from 24 hour cultures. The colonies were first suspended in a sterile aqueous solution (0.9%) of NaCl and then shaken for 20s. The density was adjusted at the Standard turbidity of a 0.5 McFarland (1.5 x 10 8 CFU/ml) [10].

Agar disc diffusion assay
The method described by Furtado and Medeiros (1980) was employed for the agar disc diffusion assay in triplicate. The microbial suspensions (1.5 x 10 8 CFU/ml) were inoculated by flooding onto the surface of Mueller Hinton (MH) agar. Sterile 6 mm diameter filter discs were impregnated with 10 µl/disc of the EOs and were delivered onto the inoculated agar MH. The plates were incubated for 18h at 37°C. Antibacterial activity was evaluated by measuring the zones of inhibition against the tested strains. The standard antibiotics for comparison were the discs antibiogram of imipenem (IMP), ampicillin (AMP), cefaclor (CEC), pristinamycin (PR).

Minimum inhibitory concentration (MIC)
The MIC was obtained using a micro-dilution assay in plates of 96-well according to the National Committee for Clinical Laboratory Standards (NCCLS). The different concentrations of the essential oils were prepared in a suspension including 0.2% of agar in sterile distilled water to scatter the components without adding any solvent or detergent [11]. They were performed by successive dilutions 1/2 from 90 to 0.08 mg/ml. The obtained concentrations in the well were from 22.5 to 0.02 mg/ml. The bacterial suspensions were produced in the same way as previously described, diluted in MH broth and placed in 96-well plates at a density of 1.5 x 10 6 CFU/ml. The plates were incubated for 18 h at 37°C, bacterial growth was visualized by adding 20µl of aqueous 2,3,5-triphenyltetrazolium chloride (TTC) solution (1%) to each well, with further incubation for 1 hour. MIC was defined as the lowest concentration that does not produce a red color [12].

Statistical analysis
The mean values and standard deviations were calculated by using SPSS 20. The results were analyzed by one-way ANOVA followed by Tukeytest, using the same software. Differences were considered to be significant at P < 0.05.

Essential oils composition
The phytochemicals contained in essential oils of the three Lavandula species are estimated by more than 23 compounds for L. mairei [4] and 52 for booth L. dentata and L. stoechas [13]. Among these phytochemicals, carvacrol, 1,8-cineole and fenchone were reported as the major components of these species, respectively [4,18,19]. Fig. 1 represents the percentage of inhibition of the essential oils of three studied species according to their concentration (L. stoechas, L. dentata and L. mairei). BHT, known for its antioxidant power [14], was used as a reference to compare the inhibition results of the essential oils. The IC50 value represents the concentration capable of inhibiting 50% of DPPH. The highest antioxidant capacity against DPPH was observed in the essential oil of L. stoechas (3.11 mg/ml), L. dentata (4.75 mg/ml) and L. mairei (6.62 mg/ml), respectively. The difference between the antioxidant capacities of the three essential oils was significant at P <0.05 (Table1).

The free radical-scavenging activity
The antioxidant capacity of the investigated essential oils is due to their ability to act as donors of hydrogen atoms or electrons to transform DPPH • (stable free radical) into its reduced form [15]. The study conducted by El Hamdaoui et al. (2018) on the scavenging of DPPH by the essential oil of L. mairei showed a lower IC50 compared to our results, but remained high compared to the IC50 of the essential oils of L. stoechas, and L. dentata.

Ferric reducing/antioxidant power (FRAP)
Ferric Reducing Antioxidant Power (FRAP) Assay is based on the capacity of the antioxidant to reduce Fe 3+ to Fe 2+ [16]. This colorimetric method was used to compare the effective concentrations (EC50) to reach an absorbance of 0.5 of the different compounds (Fig.2).
The essential oil (EO) of L. stoechas showed the highest antioxidant capacity (6.88 mg/ml), followed by EO of L.dentata (9.23 mg/ml) and EO of L.mairei (13.4 mg/ml), while Quercetin (0.04 mg/ml) was used as reference (Fig.3). The difference in the ferric reducing antioxidant capacities between the essential oils of L. stoechas and L. dentata was not significant, whereas the difference between these two EOs and the essential oil of L. mairei was significant at P <0.05 (Table1).
The antioxidant capacity of L. stoechas and L. dentata has been revealed by several previous studies [18][19][20]. This activity is related to the chemical composition of the essential oils which are rich in phenolic compounds [21]. In addition, the antioxidant capacity of EOs is of high interest since they can preserve foods from the risks of oxidants [22].

The total antioxidant capacity (TAC)
The total antioxidant capacity (TAC) of the three essential oils was expressed as ascorbic acid equivalents ( Table 1). The highest antioxidant activity was observed in the essential oil of L.stoechas (443.2 mg AAE/g EO) followed by L.dentata (262.1 mg AAE/g EO) and L.mairei (125.9 mg AAE/g EO), respectively. The difference in TAC between the three essential oils was significant (P<0.05), whereas the difference between the EO of L. stoechas and the reference compounds (BHT and Quercetin) was non-significant (P<0.05).
The essential oil of L.stoechas has a great antioxidant power which is in correlation with its chemical composition [23][24][25]. However, the phytochemicals that give essential oils their antioxidant activities can be influenced by the environment, agronomic conditions and the harvesting stage [26][27][28]. Table 2 shows the results of antibacterial activity by the agar disc diffusion technique. All essential oils showed significant antibacterial activity. The inhibition diameter of the essential oils ranged from 10±0.1 mm to 18±0.5 mm. The tested essential oils have in the majority of cases a higher antibacterial activity compared to the three antibiotics Ampicillin (AMP), Cefaclor (CEC), Pristinamycin (PR). The imipenem (IMP) antibiotic showed the highest antibacterial activity compared to EOs and the other antibiotics against all tested strains. The obtained results in this paper are in agreement with other studies that revealed the high antibacterial potential of essential oils of the genus Lavanlula [4,23,29].     Table 3 represents the minimum inhibitory concentration of the three essential oils tested. The EO of L.stoechas was the most effective against all strains compared to the other EOs. The minimum inhibitory concentration of L. stoechas was between 0.039 mg/ml (for C. koseri, S. aureus and K. pneumonia) and 0.156 mg/ml for the rest of the tested strains.

Minimal inhibitory concentration (MIC)
The antibacterial capacity of the essential oil of L. mairei, L. dentata and L. stoechas have revealed strong activity against several pathological Gram-negative and Gram-positive bacteria, such as Staphylococcus aureus, Escherichia coli and Klebsiella pneumonia [4,30,31].

CONCLUSION
This study is the first to evaluate the essential oil of L.mairei against some nosocomial bacteria (Escherichia coli, Klebsiella pneumonia, Acinetobacter baumannii and Citrobacter koseri). Our results showed that the essential oil of L.mairei has a significant antimicrobial and antioxidant activity but remains lower than the essential oil of L.stoechas and L.dentata. The highest antioxidant activity was observed in the EOs of L.stoechas as measured by DPPH (IC50 = 3,11 ± 0,13 mg/ml), FRAP (EC50 = 6,88 ± 0,8 mg/ml) and TAC (443,2 ± 38 mg AAE/g EO) assays. The essential oils of the Moroccan Lavandula genus could have potential as antimicrobial compounds for the treatment of many infectious bacterial diseases, including multi-resistant bacterial strains of nosocomial origin.

CONSENT
It's not applicable.

ETHICAL APPROVAL
It's not applicable.

COMPETING INTERESTS
Authors have declared that no competing interests exist.