Biochemical Effects of the Saharan Myrtle

Plant Material and Essential Oil Isolation

Aerial parts of Myrtus nivellei were collected in July 2016 during the flowering stage. Samples were isolated from plants harvested near Tamanrasset city (Hoggar massif, altitude: 1900 m, latitude: 22°38, longitude: 5°37). The myrtle was identified as Myrtus nivellei at the department of Botany INA-El Harrach (Algeria).samples of aerial parts (leaves and stems) of M. nivellei was submitted to hydrodistillation using a Clevenger type apparatus during 3h. The essential oil obtained was dried over anhydrous sodium sulfate, filtered, and stored in dark glass bottles at +4°C until use.

Fractionation of the Essential Oil

The GC/MS analysis was performed on an HP 5972 mass spectrometer (Agilent technologies, Palo Alto, California, USA) with electron impact ionization (70 eV). An HP-5MS capillary column (30m, 0.25mm coated with 5% phenyl methyl silicone, 95% dimethylpolysiloxane, 0.25µm film thickness) was used. Oven temperature was programmed to rise from 50 to 240°C at a rate of 5°C/min, transfer line temperature was 250°C. The carrier gas was Helium with a flow rate of 1.2 ml/min and a split ratio of 60:1. Scan time and mass range were 1 s and 40–300m/z, respectively. The identification of volatile components was assigned by comparison of their retention indices (RI) with those of literature or with those of authentic compounds available in the authors’ laboratory. Further identification was made by matching their recorded mass spectra with those stored in the Wiley/NBS mass spectral library of the GC/MS data system and other published mass spectra [8].

Preparation of Polar Extracts

A portion (100 g) of dried plant material was extracted with 1l of methanol (MeOH), in a Soxhlet apparatus, the solvent was evaporated using a vacuum evaporator [9]. The ethanolic extract was prepared by macerating 10g of dried plant material with 100ml of ethanol (EtOH) for 72h with intermittent shaking. The solvent was evaporated using a vacuum evaporator. The dry residues were stored until use.

Antimicrobial Activity

Microbial Strains: The essential oil and extracts were individually tested against a panel of microorganisms, including Staphylococcus aureus (ATCC 25923), Staphylococcus epidermidis (ATCC 12228), Streptococcus pneumoniae (ATCC 49619), Moraxella catarrhalis (ATCC 49143), Bacillus cereus (ATCC 11778), Acinetobacter lwoffii (ATCC 19002), Enterobacter aerogenes (ATCC 13043), Escherichia coli (ATCC 25922), Salmonella typhi (ATCC 4404540), Shigella flexineri (ATCC 25936), Klebsiella pneumoniae (ATCC 13883), Proteus mirabilis (ATCC 7002), Pseudomonas aeruginosa (ATCC 27853), Clostridium perfringens, Mycobacterium smegmatis (CMM 2067), Candida albicans (ATCC 10239) and Candida krusei (ATCC 6258). Bacterial strains were cultured overnight at 37°C in Mueller Hinton agar (MHA), with the exception of S. pneumoniae (MHA containing 50 ml citrate blood/l). Yeasts were cultured overnight at 30°C in Sabouraud dextrose agar. Antimicrobial Screening: Two different methods were employed for the determination of antimicrobial activities: agar well-diffusion method for the extracts and disc diffusion method for the essential oil [10]. The MICs of the essential oil and the alcoholic extracts against the test microorganisms were determined by the broth micro-dilution method [11]. All tests were performed in triplicate. Disc Diffusion Method: The agar disc diffusion method was employed for the determination of antimicrobial activities of the essential oil in question [11]. Briefly, a suspension of the tested microorganism (0.1 ml of 108 cells/ml) was spread on the solid media plates. Filter paper discs (6 mm in diameter) were impregnated with 15 μl of the oil and placed on the inoculated plates. These plates were incubated at 37°C for 24 h for bacteria and, at 30°C for 48 h, for yeasts. The diameters of the inhibition zones were measured in millimetres. All tests were performed in triplicate. Determination of Minimum Inhibitory Concentration: A broth micro-dilution assay was used, as recommended by NCCLS, for the determination of the MIC [10]. All tests were performed in Mueller Hinton Broth (MHB; BBL)

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supplemented with Tween 80 detergent (final concentration of 0.5% (v/v), with the exception of the yeasts (Sabouraud dextrose broth-SDB + Tween 80). Bacterial strains were cultured overnight at 37°C in MHB and the yeasts were cultured overnight at 30°C in SDB. Test strains were suspended in MHB to give a final density of 5 × 105 cfu/ml and these were confirmed by viable counts. Geometric dilutions, ranging from 0.036 to 72.0 mg/ml of the essential oil and the alcoholic extracts were prepared in a 96-well microtitre plate, including one growth control (MHB + Tween 80) and one sterility control (MHB + Tween 80 + test oil). Plates were incubated under normal atmospheric conditions, at 37°C for 24 h for bacteria, and at 30°C for 48 h for yeasts. The bacterial growth was indicated by the presence of a white “pellet” on the well bottom.

Assay for Total Phenolics Amount

Total phenolic constituents of the two extracts were determined by the literature methods involving the Folin– Ciocalteu reagent and gallic acid as standard [12]. 100µl of extract solution, containing 1mg extract, was taken in a volumetric flask, 46 ml distilled water and 1 ml Folin- Ciocalteu reagent was added, and the flask was shaken thoroughly. After 3 min, 3 ml of a solution of 2% Na2CO3 were added and the mixture was allowed to stand for 2h with intermittent shaking. Absorbance was measured at 760 nm. The same procedure was repeated for all standard gallic acid solutions.

Antioxidant Activity

DPPH Assay: The hydrogen atom or electron donation abilities of the corresponding extracts and some pure compounds were measured from the bleaching of the purple-coloured methanol solution of 2,2′- diphenylpicrylhydrazyl (DPPH). This spectrophotometric assay uses the stable radical DPPH as a reagent [13]. Fifty microlitre of various concentrations of the extracts in methanol were added to 5 ml of a 0.004% methanol solution of DPPH. After a 30 min incubation period at room temperature, the absorbance was read against a blank at 517 nm. Inhibition of free radical by DPPH in percent (I%) was calculated in following way:

𝐼% = [(𝐴𝑏𝑙𝑎𝑛𝑘−𝐴𝑠𝑎𝑚𝑝𝑙𝑒)/𝐴𝑏𝑙𝑎𝑛𝑘 ]𝑥100 Where A_blank is the absorbance of the control reaction (containing all reagents except the test compound), and _A_sample is the absorbance of the test compound. Extract concentration providing 50% inhibition (IC50) was calculated from the plot of inhibition percentage against extract concentration. Tests were carried out in triplicate. β_-Carotene-linoleic acid assay: In this assay, antioxidant capacity is determined by measuring the inhibition of the volatile organic compounds and the conjugated diene hydroperoxides arising from linoleic acid oxidation [14]. A stock solution of β-carotene-linoleic acid mixture was prepared as follows: 0.5 mg β-carotene was dissolved in 1 ml of chloroform (HPLC grade) and 25 μl linoleic acid and 200 mg Tween 40 were added. Chloroform was completely evaporated using a vacuum evaporator. Then, 100 ml distilled water saturated with oxygen (30 min 100 ml/min) were added with vigorous shaking. 2.5ml of this reaction mixture were dispensed into test tubes and 350 μl portions of the extracts, prepared at 1 g/l concentrations, were added and the emulsion system was incubated for 48 h at room temperature. The same procedure was repeated with synthetic antioxidant, BHT, as positive control, and a blank. After this incubation period, absorbances of the mixtures were measured at 490 nm. Antioxidative capacities of the extracts were compared with those of BHT and blank.

Chemical Composition of the Essential Oil

About 32 compounds, representing 99.821% of the essential oil (EO), were identified. GC/MS analyses revealed that the major constituents of the oil were δ- elemene (15,687%), ρ-menthenol (12.945%), Eucalyptol (12.062%) and β-Citral (7.762%) as listed in Table 1.

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Table1: Components of the essential oil isolated from aerial parts of M. nivellei. * Retention time (as minutes). Various oxygenated monoterpenes were present at appreciable contents: eucalyptol: 12.062%, α-terpineol: 6.455% and bulnesol: 3.049%. The sesquiterpenes are also represented by δ-elemene: 15.687%, azulene: 6.182%, α-patcoulene: 2.873% of the whole composition. The chemical profile of this EO should be classified in the chemo type group of δ-elemene/ ρ-menthenol. However, this chemical composition differs from the EO of M. nivellei harvested in Tassili N’ajjer, reported in previous studies [15, 7]. However, the chemical composition of this EO differs also from the various compositions of EOs isolated from Myrtus communis growing wild all around the Mediterranean basin [15, 3, 16]. It could be due to a number of factors, including the geo-climatic locations and growing conditions, including concentration of nutrients, temperature, humidity, soil type, day length, climate, altitude, amount of available water, ect. The chemical composition also depends on season or vegetative period of plant. According to these factors, plant biosynthetic pathways can change the relative proportion of the primary oil components. These variations in chemical composition led to the notion of chemotypes, which are generally defined as a distinct population within the same species that produces different chemical profiles for a particular class of secondary metabolites [17]. Thus, the same species of plant can produce a similar essential oil, but with different chemical composition and therapeutic activities. Essential oil composition also depends on the plant parts used for oil preparation.

Antimicrobial Activity

As can be seen in Table 2, EO exhibit strong antimicrobial activity, especially against S. epidermidis (43.00±1.13) and S. typhi (24.00±1.30), it didn’t exhibit any antimicrobial activity against four microorganisms (M. catarrhalis, A. iwoffii, E. aerogenes and C. krusei). However this activity was moderate for the other strains. The alcoholic extracts were found to have a good activity against S. epidermidis, S. pneumoniae, E. coli, S. Typhi and S. Flexineri, especially against S.aureus. Five strains (M. catarrhalis, A. iwoffii, K. pneumonia, P. mirabilis and C. perfrengens) were resistant to these extracts.

Table 2: Antimicrobial activity of various extracts of M. Nivellei using agar well diffusion and MIC methods. NA: not active *Diameter on inhibition zone including well diameter of 9mm. MIC: Minimun Inhibitory Concentration, values given as mg/ml for the essential oil and the alcoholic extracts. Results obtained from disc diffusion method, followed by measurements of MIC, indicate that S. epidermidis, is the most sensitive microorganism tested, with the lowest MIC values (0.07 mg/ml) in the presence of the EO of M. nivellei (Table 2). S.aureus, S. Typhi, C. albicans and E. phaecalis were other sensitive ones against the oil with Journal of Natural & Ayurvedic Medicine

MICs values included between 0.28 and 2.25mg/ml. A moderate activity was observed against three gram- negative microorganisms (E. coli, P. mirabilis and P. aeruginosa) known for their resistance to many antibiotics.As far as our literature survey could ascertain, there was only two reports on the antifungal activity of the EO of M. nivellei. As far as this report is concerned, a moderate antifungal activity was observed against some yeasts and dermatophytes including C. albicans and C. krusei [15]. These results support the use of this species in traditional medicine for the treatment of dermatophytosis. Moreover, moderate activities of the alcoholic extracts could also be attributed to the presence of several types of compounds belonging to different classes, such as sterols and their derivatives, flavones and flavonoids in dichloromethane extract and more polar thermolabile and/or thermo-stable phenolics in the hydrophobic subfractions of methanol extract [18, 19].

Amount of Total Phenolics

The determination of the total phenolic amount is based on the absorbance values of the various extracts analysed, reacting with Folin–Ciocalteu reagent and compared with the standard solutions of gallic acid equivalents, as described above, results of the colorimetric analysis of total phenolics are given in Table 3. Total phenolics was highest in the ethanolic extract (784,3 µg eq/mg), followed by the methanolic extract (398,3 µg eq/mg).The lowest amount of total phenolics was recorded in the essential oil (95.4 µg eq/mg). These results agree with those of the DPPH test, the highest amount of total phenolics in the ethanolic extract exibits an important free radical scavenging activity.

aValues are the mean of three replicates bStandard derivation

Antioxidant Activity

The essential oil and the alcoholic extracts were subjected to screening for their possible antioxidative activity. Three positive controls were used (Vitamin C, Quercetione and Vitamin E). Complementary test systems, namely DPPH free radical scavenging and β- carotene/linoleic acid systems were evaluated. Free radical scavenging capacities of the extracts, measured by DPPH assay, are shown in Fig.1. The reaction followed a concentration-dependent pattern. The free radical scavenging activity of ethanolic extract (IC50=0.59mg/ml) was superior to methanolic extract (IC50=0.98mg/ml). Polar extracts exhibited stronger free radical scavenging activity than the EO of M. nivellei (IC50= 12.06mg/ml).

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Figure1: Free radical scavenging of the extracts measured in DPPH essay. The ethanolic extract is more effective than Vitamin E but still less than Vitamin C and Quercetine used as positive controls. Activity should be related to its phenolic content. In the β-carotene/linoleic acid system, oxidation of linoleic acid was effectively inhibited by the ethanolic extract (Figure 2) while the oil was less effective. MW extract shows 93.5% inhibition that is close to the synthetic antioxidant reagent BHT. Antioxidants minimize the oxidation of lipid components in cell membranes or inhibit the volatile organic compounds and the conjugated diene hydroperoxides, arising from linoleic acid oxidation, that are known to be carcinogenic. Alcoholic extracts exhibited stronger activity than essential oil, indicating that polyphenols or flavanones and flavonoids may also play important roles in the activity [3]. .

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