Chemical composition, antioxidant/antibacterial activities and ADMET study of the essential oil isolated from the aerial parts of Ziziphora hispanica grown in Morocco

ABSTRACT The present study aimed to characterize the chemical composition and evaluate the antioxidant and antibacterial activities along with the absorption, distribution, metabolism, excretion, toxicity (ADMET) study of the essential oil of Ziziphora hispanica (ZH-EO), collected from the Middle Atlas of Morocco (Boulmane). Results showed that a total of 119 volatile components, characterized by GC-FID and GC-MS analysis, which represents the 88.7% of the total of ZH-EO. ZH-EO displayed a bactericidal effect against Listeria monocytogenes, Pseudomonas aeruginosa and Enterococcus faecalis, and a bacteriostatic effect against Staphylococcus aureus. Furthermore, ZH-EO exhibited strong antioxidant activity against DPPH radical (IC50 = 1.3 mg/mL). The ADMET prediction showed good pharmacokinetic properties of the tested components. The findings obtained from this study suggest that Ziziphora hispanica essential oil could represent a source of bioactive molecules with antioxidant and antibacterial potential in the prevention against diseases related to oxidative stress and pathogenic bacteria.


Introduction
In recent decades, the Lamiaceae family has been the subject of substantial research, primarily focusing on phytochemical and pharmaceutical features. There are more than 245 genera and 7886 species in this family, which are found in temperate and tropical regions such as the Mediterranean, Asia, Europe, United States, and Australia (1). The Lamiaceae family can be found in a variety of traditional remedies, cosmetics, and meals (2). About 25-30 different species of annual and perennial herbs belong to the genus Ziziphora L., which grows in Africa, Asia, and Europe (3). These species are the currently used in the pharmaceutical, cosmetic and food industries for their potential health benefits. Indeed, many studies highlighted various activities for this genus e.g. antioxidant (4,5), antibacterial (5,6), antifungal (7), insecticidal and ovicidal (8), vasodilator activities (9). Ziziphora also showed an inhibitory effect on gastric acid production under basal and vagal stimulation conditions (10), a positive effect on performance and blood biochemical and immune parameters of laying hens (11), and a function as a yogurt starter (12,13). In Morocco, this genus is represented by a single species: Z. hispanica (14), where is called 'Tifliout tbourayt', and besides Morocco it is endemic to Spain, Tunisia and Algeria where it has been employed in traditional medicine for various purposes such as stomach and intestinal ailments, heart disorders, migraine, cough, jaundice as reported by Meratate et al. (15).
The objective of present study was to investigate the composition of the essential oil isolated by hydrodistillation from the aerial parts of Ziziphora hispanica L., growing wild in the Middle Atlas of Morocco (Boulmane) along with their antioxidant and antibacterial activities, and absorption, distribution, metabolism, excretion, toxicity (ADMET) study. So far, the volatile content of Ziziphora hispanica has been investigated only in three works and dealt with either Algerian (16,17) or Spanish (18) species, thus no data are available for the Moroccan one. Other works have focused on different species e.g. Ziziphora clinopodioides from China (19), Turkey (6,20) and Iran (5,21) and Ziziphora taurica from Iran (22). In terms of biological activity, only two have reported antibacterial (16,17) or both antioxidants and antibacterial (15) properties.

Plant material and essential oil extraction
The aerial parts of Ziziphora hispanica were harvested during June 2021, from the Middle Atlas of Morocco (Boulmane) (33.455518° North, − 4.875496° west, altitude 509 m). The plant was identified by the botanist Professor Rahou Abdelilah at the Faculty of Sciences, Moulay Ismail University (Morocco). Voucher specimen was deposited in the Herbarium of the Laboratory of Chemistry-Biology Applied to the Environment, Faculty of Sciences, Moulay Ismail University, of Morocco. The plant was protected from light and dried at room temperature for 5 days. Then the plant was crushed and stored at+4°C until analysis.
An amount of 100g of aerial part powder of Ziziphora hispanica was placed in a flask with 100 mL of distilled water, and then subjected to hydrodistillation for 3 h using a Clevenger type apparatus (23).

GC-MS analysis of ZH-EO
GC-MS analyses were carried out on a GC-QP2020 system (Shimadzu, Kyoto, Japan

GC -FID analysis
GC-FID analyses were carried out on a GC-2010 system (Shimadzu, Kyoto, Japan). The separation of the analytes was effected with the same column, oven temperature program and injection parameters used for MS applications. Helium was used as carrier gas, at a constant linear velocity of 30.0 cm/s. The FID temperature was set at 280°C (sampling rate 200 ms), hydrogen and air flows were 40 mL/min and 400 mL/ min, respectively. Data were collected by LabSolution software ver. 5.92 (Shimadzu, Kyoto, Japan). Quantitative results were determined as peak area percentage without any correction. Samples were analyzed in triplicates.

Antioxidant activity
In order to evaluate the antioxidant activity of Ziziphora hispanica essential oil, the DPPH assay was carried out according to the method described by Bouymajane et al. with some modifications (25). Different concentrations ZH-EO, and ascorbic acid were prepared (0.0625-2 mg/ mL). Then, 0.5 mL of each sample was mixed with 3 mL of methanol DPPH solution (0.1 mM) and stored at room temperature for 20 minutes in the dark. The absorbance was then measured with UV spectrophotometer (Perkin Elmer) at 17 nm. The inhibition percentage was calculated as follows: where IP is the inhibition percentage, A 0 is the absorbance of the control, and A 1 is the absorbance of the ZH-EO. Also, the IC 50 inhibitory concentration and standard deviation were calculated (IC 50 ±SD).

Pathogenic bacterial strains
The

Disc diffusion method
The disc diffusion method was done as a preliminary test to evaluate the antibacterial activity of ZH-EO against bacterial strains according to the protocol previously described by Sadiki et al. (26). Briefly, 1 mL of bacteria suspensions was seeded on Petri dishes containing Mueller Hinton agar. Afterward, 10 µL ZH-EO was dropped on mm diameter sterile paper discs (Biolife, Milan, Italy) placed on the MHA plates. Antibiotics (ampicillin, erythromycin, tetracycline, and imipenem) were used as a reference. After incubation of Petri dishes at 37 ºC for 4 h, the inhibition zone diameter (D) was measured in mm and expressed as means ± (SD). The antibacterial activity was classified into three levels: strong (D ˃ 5 mm), moderate (0 mm ˂ D ˂ 5 mm) and weak (D ˂ 0 mm).

Broth microdilution method
The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of ZH-EO against bacterial strains were determined by the broth microdilution method as described in ref (25).
Then, 100 μL of Z. hispanica essential oil was added to first well plate and mixed, in order to determine a serial dilution. Furthermore, 50 μL of TSYEB + DMSO and 50 μL of bacterium suspensions were added to each well. The well containing bacterial suspension with TSYEB + DMSO and the well containing TSYEB + DMSO and ZH-EO were served as a control and as a black respectively. After all microplate incubation at 37°C for 4 h, 0 μL of TTC (2, 3, 5-triphenyl tetrazolium chloride) was added to each well and re-insulated at 37°C for 0 min. The MIC was determined at the lowest concentration of ZH-EO at which the bacterial growth was not observed. The MBC was determined at the lowest concentration of ZH-EO that did not produce any bacterial colony, by plating 100 μL of samples from the wells in which no growth was observed on the Mueller Hinton agar medium and incubated at 37°C for 4 h. The MBC/MIC ratio is used to determine the bacteriostatic and bactericidal effects of ZH-EO. The ZH-EO effect is bactericidal if the If MBC/MIC≤4, and bacteriostatic if MBC/MIC>4.

ADMET pharmacokinetic properties of major components of ZH-EO
The structure of selected major components of ZH-EO was drawn using Marvin Sketch software for the prediction of pharmacokinetics ADMET study (absorption, distribution, metabolism, excretion and toxicity), then the exported molecules were subjected to minimization using the 6-1 G(d) basis of the B3LYP functional which was a kind of DFT density functional theory method (27). Afterwards, the physicochemical characteristics were assessed using the SwissADME and pKCSM programs (27,28). The term ADMET uses in silico techniques to better forecast and understand how medications affect the body. It can optimize clinical usage, reduce unwanted side effects, direct research toward development, and improve therapeutic options.

Chemical composition of ZH-EO
As illustrated in Figure 1 and summarized in Tables 1 and S1, a total of 119 components were identified representing 88.7% of the total composition of ZH-EO. The main components identified were represented by pulegone (72.7%), followed by 2-Methylcyclopentanone (2.8%), menthofuran (2.6%) and (E)-anethole (2.0%). Ketones were the most abundant class found in ZH-EO (79.0%), followed by monoterpenes hydrocarbons (5.6%) and alcohols (3.4%).. The results achieved are consistent with the same species growing in Algeria. In fact, in Algeria, its EO coming from the province of Tlemcen was mainly dominated by pulegone (79.5%) (16). The work carried out by Negueruela and Rico (29) on three samples of Z. hispanica, one before flowering (ZHNF) and the other two during flowering (ZHF1 and ZHF2) near Arganda, Madrid, in the part central Spain, showed that such samples are mainly composed of pulegone (76.66%, 64.57% and 62.69% respectively), followed by piperitenone (11.65%, 15.64% and 16.67%). According to the same authors, this could be well interpreted by the stereospecific reduction of piperitenone in pulegone which is blocked during flowering (inhibition of the enzyme substrate), thus explaining these differences concerning the percentages of the compounds. Likewise in other works carried out on the essential oil of Z. clinopodioides Lam (19), the content of the pulegone during the growth phase was found to be different with a predominance of pulegone at the flowering stage.
In general, the results of the current research are consistent with other previous studies on other Ziziphora species, which have all been found to be rich in pulegone (6,(20)(21)(22).

Antioxidant activity
The antioxidant potential of ZT-EO was evaluated based on its ability to scavenge stable DPPH radical. ZT-EO exhibited good radical scavenging activity (with IC 50 value of 1.329 ± 0.002), which was found to be higher than that of ascorbic acid (2.560 ± 0.36 mg/mL). These results are lower than those found by Meratate et al. (IC 50 = 0.02 mg/mL) (15). The antioxidant activity of ZT-EO can be attributed to its main components (pulegone (30), menthofuran, piperitenone, (E)-anethole, isopulegone, limonene and caryophyllene oxide), or by synergistic and antagonistic interactions between minor and major components. As reported in the literature, the bioactive components belonging to monoterpenoid class do present the higher great antioxidant properties (15,31,32).

Antibacterial activity
The antibacterial activity of ZH-EO against Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium, Staphylococcus aureus, Enterococcus faecalis, and Listeria monocytogenes was evaluated by disc diffusion and broth microdilution assays. As presented in Table 2, the inhibition zone diameter of ZH-EO ranged from 14.16 ± 0.29 to 23.16 ± 1.6 mm. Except Staphylococcus aureus, all other tested bacteria were sensitive to ZH-   (15,17). The antibacterial activity of the essential oil in Z. hispanica may be associated with the relatively high content of pulegone, piperitenone, limonene and 1-8-cineole. In fact, according to the work of Rabah et al. (17), the presence of pulegone found in the volatile oil as well as limonene and piperitenone are responsible for the antibacterial activity. Similarly, pulegone (the main compound of Z. hispanica) has strong antibacterial properties, especially against S. typhimurium and  S. aureus (32). Linalool and β-caryophyllene (among the constituents of Z. hispanica) also showed good results in the antimicrobial testing (33). The attained results reveal that Z. hispanica essential oil have identical antibacterial activity versus Gram-positive bacteria and Gram-negative bacteria, demonstrating the potent activity of ZH-EO in inhibiting bacterial growth. Nonetheless, other studies have shown that essential oils are much more active towards Gram-positive bacteria than Gram-negative bacteria (34).

ADMET prediction
Additionally, in order to assess the safety of the ZH-EO an ADMET study has been carried out. In this context, the most abundant components detected in the EO (with concentrations above 1%) were selected. Table 3  Limonene, all other compounds tested have a high absorption, which means that the extracted molecules are well digested by the human gastrointestinal system. The second letter, D, indicates the distribution and it is based on the ability of xenobiotics to cross the bloodbrain barrier (35,36): all targeted compounds were able to cross the blood-brain barrier, thus reaching to the bloodstream. The third letter is M, indicates the metabolism and highlights the drug-transforming function in the liver. Some studies have shown that there are many CYP genes from 17 families in humans (35). However, only selected CYPs (1A2, 2C9, 2C19, 2D6, and 3A4) are responsible for the biotransformation of 90% of drugs. In the present case, all the compounds tested are neither substrates nor inhibitors of cytochrome 2D6, 2C19, 2D6 and 3A4; for the rest, Menthofuran (M) and E-Anethole (A) turned out to be inhibitors of cytochrome 1A2, whereas Limonene resulted an inhibitor for cytochrome 2C9. The fourth letter, E, indicates excretion or clearance and represents a value that calculates the relationship between the rate of elimination of the drug and its concentration in the body. All the components of ZH-EO investigated showed high clearance and resulted mutagenic in the Ames test. In terms of toxicity (T), none of the compounds investigated resulted toxic to the human   ------body. The results attained are in agreement with previous findings by Morgan & Wilson (37) which showed that terpenes are not generally toxic.

Conclusions
The present study investigated the chemical composition, antioxidant, antibacterial and ADMET study of Ziziphora hispanica essential oil, collected from the Middle Atlas of Morocco (Boulmane). GC-MS analysis revealed that the chemical composition of ZH-EO are rich in pulegone, menthofuran, piperitenone, (E)-anethole, isopulegone, limonene and caryophyllene oxide. Furthermore ZH-EO displayed the antioxidant capacity and a bactericidal effect against Listeria monocytogenes, Pseudomonas aeruginosa and Enterococcus faecalis, and a bacteriostatic effect against Staphylococcus aureus. The ADMET study has shown that the main compounds present in the ZH-EO have drug-like properties such as low toxicity, and most of the compounds can be easily absorbed through the gastrointestinal tract, with exception of Limonene and can permeate through the blood-brain barrier. Further research may be beneficial for an expanded use of Ziziphora hispanica essential oil as food preservative, flavor enhancer, or natural antioxidants to lessen oxidative stress in humans.