Altitude impact on the chemical profile and biological activities of Satureja thymbra L. essential oil

Background Several agricultural or environmental factors affect plants’ chemical and pharmacological properties. Methods In this study, the essential oil of Libyan Satureja thymbra was isolated from plants collected during two successive years at two different altitudes; Wasita (WEO) and Safsaf (SEO), 156 and 661 m above sea level, respectively. Results GC/MS allowed the identification of 21 and 23 compounds, respectively. Thymol prevailed in WEO (26.69%), while carvacrol prevailed in SEO (14.30%). Antimicrobial activity was tested by agar-well diffusion method, and MIC/MLC values were determined by broth dilution method. Values of MIC/MLC were 0.125/0.25 μg/ml for SEO against S. aureus, P. mirabilis and K. pneumonia and for WEO against B. subtilus. It was observed that plants growing at lower altitude in Wasita locality had better antifungal activity, while those growing at higher altitude at Safsaf locality had better antibacterial activity. Both essential oils had a better anthelmintic activity than the standard piperazine citrate against a tested earthworm. However, SEO oil had a significantly higher anthelmintic activity than WEO. Cytotoxicity of the oils tested using SRB assay on human breast cancer (MCF-7) and colon cancer cell lines (HCT-116) showed better activity for SEO, especially against HCT-116 with IC50 2.45 ± 0.21 μg/ml. Conclusions Thus, altitude is an important factor that should be considered as it affected the yield, composition and biology of the plant extracts.


Background
The Libyan flora is spread over a diversity of geomorphologic regions which in return gives rise to a variety of climatic conditions suitable for the growth of different types of plants [1]. One of the widespread plants in Al Jabal Al Akhdar region in Libya are members of family Lamiaceae like Satureja genus [2]. Satureja comprises more than 200 species of popular herbs or shrubs that are often aromatic [3]. In Al Jabal Al Akhdar, two main species of Satureja dominate, S. thymbra and S. fortii [4]. Satureja thymbra L., known as pink savory, is an aromatic plant with oregano-like smell that is endemic to the Mediterranean region. The main chemical classes identified in the essential oil include monoterpene hydrocarbons such as p-cymene and γ-terpinene, oxygenated monoterpenes, especially phenolic constituents as thymol and carvacrol. Also, sesquiterpene hydrocarbons were detected as β-caryophyllene and α-humulene as well as oxygenated sesquiterpenes such as caryophyllene oxide [5][6][7][8][9]. The plant is also rich in other phenolic constituents like flavonoids and flavonoid glycosides as well as tannins, acids and exudates [10]. Due to the presence of these compounds, S. thymbra L. has been evaluated as antibacterial, antifungal and antiviral agent [11]. The essential oil proved an antimicrobial activity against several bacteria and fungi like Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium, Shigella sonnei, Staphylococcus aureus and some resistant strains such as Stenotrophomonas maltophilia, S. maltophilia, and Chryseomonas luteolaas as well as the yeast Candida albicans [8,[12][13][14][15]. The essential oil of S. thymbra (1%) has also been reported to possess a strong bactericidal activity against bacterial biofilms formed on stainless steel by some pathogenic bacteria [16]. The oil has also shown an in-vitro inhibitory activity against SARS-CoV and HSV-1 replication by visually scoring of the virus-induced cytopathogenic effects post-infection with a high selectivity index (SI) against HSV-1 [17]. The oil also had a high SI when tested for its cytotoxicity in Vero cells using MTT assay [18]. Essential oil of S. thymbra has also been reported to possess an insecticidal activity against the mosquito Culex pipiens biotype molestus, acaricidal effect against Hyalomma marginatum, genotoxic activity against Drosophila as well as insecticidal activity against three stored-product insects [19][20][21][22]. Other reported activities for the oil included improving circulation and thus being used in the treatment of arthritis, rheumatism, as a wound healing promoter and in painful joints and muscles [23]. Being evaluated for cholinesterase inhibitory and antioxidative effects, the essential oil is suggested to treat amnesia and Alzeheimer's diseases [24]. Traditionally, it has been used in food flavoring, perfumery and in local home remedies as antiseptic and diuretic [25].
Plants' essential oil content and composition, and inturn, its biological activity, has proved to be related to several factors including altitude, genetic, climate, topography, genotype, growing conditions and harvest time. Also, different chemotypes have been described for a number of medicinal plants [26][27][28][29]. Moreover, studies have shown that the different characteristics of medicinal plant may be affected by a variety of ecological factors such soil composition, temperature, humidity and other climatic conditions [30][31][32][33]. Consequently, these changes in content and composition would definitely affect the pharmacological activity of the essential oil. Specifically, several studies show that chemical metabolic profile of Lamiaceae plants is strongly affected by environmental conditions such as geographical cultivation area, cultivation and harvesting period as well as local ecological conditions [34]. For example, altitude is one of the factors that affect plant metabolism [35]. Previous reports revealed that phenol-rich Lamiaceae species have shown that the great diversity in the content of thymol and carvacrol in the essential oils is associated with climatic factors [36,37]. Altitude affected the quantity and quality of the essential oil of several studied plants like Thymus kotschyanus (Lamiaceae), Teucrium hyrcanicum (Lamiaceae), Cymbopogon olivieri (Poaceae), Mentha piperita (Lamiaceae), Tanacetum polycephalum (Asteraceae), Ziziphora clinopodioides (Lamiaceae) and Lavandula angustifolia (Lamiaceae) [33,34,38]. To further investigate the effect of altitude on the Libyan-growing medicinal plant; Satureja thymbra, the present work aimed at studying the impact of two different altitudes (Wasita and Safsaf in Al Jabal Al Akhdar region in Libya), on the chemical profile, antimicrobial, anthelmintic and cytotoxic activity of the essential oils of S. thymbra aerial parts collected during fall 2017 and 2018.

Plant material
Samples of aerial parts of S. thymbra were obtained from two different localities (50 plants from random locations in each locality), during fall 2017 and fall 2018 from plants in Al-Jabal Al-Akhdar in Libya, from Wasita locality (32°52′30.68″N, 13°11′14.86″E) at 150 m above sea level and from Safsaf locality (32°78′19.51″N, 21°95′ 23.92″E) at 661 m above sea level. The plant is not endangered, so no permission was needed for its collection. Plants identity was authenticated by staff members of the Plant Taxonomy Department of the Faculty of Science at Omar Al Mokhtar University, Al Bydaa, Lybia. Voucher specimens were deposited at the Faculty of Pharmacy, Omar Al-Moukhtar University (STTB-8 and STTB-9). Plants were dried in shade, powdered and refrigerated in closed containers until use.

Isolation of essential oils
Samples (100 g of dried plant in 500 ml distilled water), were separately hydro-distilled with a Clevenger apparatus for 4 h. Percentage oil yield was calculated/dry weight using the eq. (W1/W2 × 100, where W1 is the weight if the oil in grams and W2 is the total weight of dried plant used). The obtained essential oil was dried over anhydrous sodium sulfate and kept refrigerated in sealed amber vials till analysis. Specific gravity of the obtained essential oils was determined according to the Egyptian Pharmacopoeia (2005).

GC/MS analysis of the essential oil samples
An Agilent 7890A gas chromatograph (Agilent Technologies, Palo Alto, CA, USA) with a capillary column RTX-5MS (30 m × 0.32 mm, film thickness 0.25 μm) was used for the GC/MS analysis of the essential oils. This was coupled to an Agilent 5975C mass selective detector. The initial oven temperature was 40°C for 2 min, then it was raised at the rate of 5°C/min until it reached 210°. The injector and detector temperatures were 290 and 300°C, respectively. Helium carrier gas was used at a flow rate of 2 ml/min. Manual split mode injection was applied (0.1 μl, each). EI mode was used for recording the mass spectra. The range for m/z was 35-500. Ionization voltage was 70 eV and ion source temperature was set at 230°C. The above conditions were applied for the analysis of a homologous series of n-alkanes to calculate retention index (RI). Identification was based on comparison of KI with literature [39], in addition to obtained data from Wiley's MS libraries. Authentic compounds (Sigma-Aldrich, Germany) were also used for identification of some compounds ( Table 2).

Gas chromatography/flame ionization detection (GC-FID)
The GC analyses were carried out on a Focus GC® (Thermo fisher scientific®, Milan, Italy) equipped with TR5-MS fused bonded column (30 m × 0.25 mm × 0.25 μm) (Thermo fisher scientific®, Florida, USA) and FID detector; carrier gas was nitrogen (1.5 ml/ min); the operating conditions were: initial temperature 40°C, 1 min. Isothermal followed by linear temperature increase till 230°C at a rate of 4°C / min. 230°C, then 5 min. Isothermal. Detector and injector temperatures were 300 and 220°C, respectively. The split ratio was 1: 20. Chrom-card® chromatography data system ver. 2.3.3 (Thermo Electron Corp.®, Florida, USA) was used for recording and integrating of the chromatograms. Average areas under the peaks of three independent chromatographic runs were used for calculation the % composition of each component.

Source of microbial cultures
Standard reference strains (American Type Culture Collection "ATCC" for bacteria and fungi) were used for assessing the antimicrobial activity of the essential oils.

Agar-well diffusion method
Satureja thymbra essential oils were prepared as an emulsion (10% w/w) using Tween 80. Sterilization of the obtained emulsion was achieved using a 0.45 μm membrane filter. Agar-well diffusion method was performed according to the Clinical and Laboratory Standards Institute [40]. The resultant inhibition zones were measured in mm, and the average values were taken. Ampicillin, Ciprofloxacin and Amphotericin B were used as standard antimicrobial agents. A blank prepared from the same concentration of Tween 80 was included.

Determination of minimum inhibitory and lethal concentrations (MIC/MLC) by broth dilution method
Broth dilution method was used for determination of minimum inhibitory and minimum lethal concentrations of the oil (MIC/MLC) against the previously mentioned standard microorganisms [40]. Determination was achieved on 96 well culture plates using a microorganism suspension at a density of 10 5 CFU/ml with Casein Soy Broth (CSB) incubated for 24 h at 37°C for bacteria, and Sabouraud Dextrose Broth (SDB) incubated for 48 h at 25°C for yeasts. Serial dilutions of essential oils (0.125-5 μl/ml) were prepared with Brain Heart infusion (BHI) broth medium in test tube and mixed with bacterial suspensions to give a volume of 4 ml and a final concentration of bacteria of approximately 5 × 10 4 CFU/ml. Final solutions were incubated at the temperatures mentioned earlier. The MIC is the lowest concentration which gave no visible growth, while the MLC was determined by sub-culturing 100 μl from each negative test tube onto plate count agar (PCA) plates. MLC was defined as the lowest concentration resulting in a negative subculture or giving presence of only one colony after incubation. Negative controls lacking the oil were assayed simultaneously. The experiments were carried out in three replicates.
Anthelmintic activity of the essential oils Allolobophora caliginosa; an adult earthworm, was used to evaluate the anthelmintic activity of the essential oils at different concentrations. To obtain different concentrations of the essential oil (1, 2 and 3% v/v), 3% essential oil was diluted in 1% aqueous tween 80. Anthelmintic assay was carried out by dividing the earth worms into three groups each containing 6 worms (each worm ≥10 cm long). A control containing 3% aqueous tween 80 was used. The reference anthelmintic drug piperazine citrate (Sigma-Aldrich, USA) was also prepared in 0.1% solution using tween 80. Time of paralysis was recorded when no movement was observed, while time of death when the worms didn't move after being dipped in warm water (50°C) [41].

In-vitro screening of cytotoxic activity Human tumor cell lines
The HCT-116 (human colon carcinoma) cell lines and MCF-7 (human breast adenocarcinoma) cells, maintained in the laboratory of Cancer Biology Department of National Cancer Institute, Cairo, Egypt (obtained originally from Sigma-Aldrich, Germany) were used for cytotoxicity assay. Normal hamster lung fibroblasts (V79 cells) were included as control.

Cytotoxicity assay
The essential oils at different concentrations (0-10 μg/ml in 2% DMSO) were tested for cytotoxicity against the fore mentioned human tumor cell lines adopting sulforhodamine B stain (SRB) assay [42]. The relation between survivals and the oil concentration was plotted to get the survival curve of each tumor cell line after the application of specific concentration. The results were compared to those of the standard cytotoxic drug; doxorubicin (10 mg adriamycin hydrochloride, in 5 ml IV injection, Pharmacia, Italy) at the same concentration was used as standard anti-tumor. The dose of the test solutions which reduces survivals to 50% (IC 50 ) was calculated as well as selectivity index. A blank prepared from the same concentration of Tween 80 was included.

Data analysis
Tests were conducted in triplicate and values recorded as mean ± SEM. Results were analyzed by GraphPad Prism® v.5 software. Significant differences among means of different samples were analyzed using paired-t-test at p ≤ 0.05.

Results
Effect of altitude on essential oil physical properties, yield and composition Both Wasita essential oil (WEO) and Safsaf essential oil (SEO) were obtained in yellow color with a strong aromatic odor. During fall 2017, WEO had a significantly higher yield and specific gravity (1.6 ± 0.03%v/w, 0.9 ± 0.04, respectively, p < 0.01, paired-t-test) than SEO (1.4 ± 0.02%v/w and 0.6 ± 0.02, respectively). No significant difference in the results was observed during fall 2018 than those obtained during fall 2017 (Table 1).
GC/MS analysis of WEO and SEO allowed the identification of 21 and 23 compounds, representing 88.17 and 93.84% in both oils, respectively during fall 2017 ( Table 2). Monoterpene hydrocarbons had almost the same percentage in both oils (16.6 and 16.1% in WEO and SEO, respectively) represented mainly by α-thujene and p-cymene which had higher percentage in SEO (8.7 and 2.3% in SEO and 3.8 and 7.5% in WEO, respectively). On the other hand, two monoterpene hydrocarbons; α-terpinolene and allo-ocimene were detected in WEO only. Oxygenated monoterpenes was the most prevailing class in both oils (35.6 and 32.1% in WEO and SEO, respectively) represented mainly by the phenolic constituents; thymol and carvacrol. Thymol was the major identified compound in WEO (29.6%), while carvacrol was the major identified compound in SEO (14.3%). Sesquiterpene hydrocarbons were represented mainly by viridiflorine in WEO (3.4%) and δcadinene in SEO (6.4%). Oxygenated sesquiterpenes had a much higher percentage in WEO than SEO (27.2 and 2.1%, respectively). Iso-spathulenol was the only oxygenated sesquiterpene in SEO (2.1%) which was also the major oxygenated sesquiterpene detected in WEO (13.4%) among other oxygenated sesquiterpenes present. Other classes of compounds were 27.2% in SEO and only 1.1% in WEO, represented by 2-tetra-butyl-4-methyl-phenol, which was completely absent in SEO. Generally, no significant difference in the results was observed during fall 2018 than those obtained during fall 2017.

Effect of altitude on antimicrobial activity of the essential oils
Testing of antimicrobial activity of the oils by agar-well diffusion method revealed that highest inhibition zone was observed for SEO collected during fall 2017 (55.7 ± 0.7 mm) against the gram-negative K. pneumonia, while the weakest activity was observed for WEO collected during the same season, against P. aeruginosa with inhibition zone 11.4 ± 0.2 mm (Table 3). Both oils had significantly (p < 0.05, paired-t-test) higher activity than ampicillin against the tested gram-positive bacteria. SEO had significantly higher activity than WEO against B. subtilus with inhibition zones 49.2 ± 1.1 mm and 39.2 ± 1.5 mm, respectively. However, the difference in activity between both oils against S. aureus was not significant. Also, activity of both oils was significantly higher than ciprofloxacin against all tested gram-negative bacteria, except for SEO against P. aeruginosa, which had the same activity as ciprofloxacin, while WEO had  significantly lower activity than ciprofloxacin. Antibacterial activity of WEO was significantly higher than SEO against E. coli and P. mirabilis. WEO had significantly higher antifungal activity than SEO; however, both oils' antifungal activity was significantly higher than the standard amphotericin B against C. albicans, but significantly lower than the standard amphotericin B against A. niger. Determination of MIL/MLC for the essential oils was performed using broth dilution method ( Table 4). The results showed variable effects of the oils against the tested microorganisms. Highest bacteriostatic and bactericidal effects were observed for SEO against S. aureus, P. mirabilis and K. pneumonia with MIC/MLC 0.125/ 0.25 μg/ml, while lowest activity was for both oils against P. aeruginosa and the fungi A. niger with MIC/MLC ranging 1-> 1.5 μg/ml. SEO collected during fall 2018 had also better effect against B. subtilus with MIC/MLC 0.125/0.25 μg/ml. WEO had better bacteriostatic and bactericidal activity than SEO against E. coli, while no significant effect was observed between the activity of both tested samples against C. albicans.

Effect of altitude on anthelmintic activity of the essential oils
The essential oils collected from cuttings of two successive years from two different altitudes had a strong anthelmintic activity against the tested earthworm which  was significantly higher than the standard piperazine citrate used. SEO had a slightly higher anthelmintic activity with shorter time recorded for the paralysis and death of the tested worms ( Table 5).

Effect of altitude on cytotoxic activity of the essential oils
Both WEO and SEO had a strong cytotoxic activity (IC 50 < 10 μg/ml) on both breast and colon cancer cell lines, with significantly higher activity and better selectivity index observed for SEO (Table 6, Fig. 1).

Discussion
At higher altitudes, average temperatures are lower than those at the base of the mountains. Elevation plays a great role in the health and growth of plants. The type and amount of sunlight, amount of water that plants can absorb and the nutrients that plants receive differ at different altitudes. The main difference observed in the composition of the essential oils was in the major identified compound which was thymol in WEO and carvacrol in SEO. It was also observed that the percentage of total phenolic constituents of WEO is 36.77% while that of SEO is 32.18%. Present results are in agreement with the previously identified compounds in S. thymbra essential oil (thymol and carvacrol as well as their precursors; pcymene and γ-terpenene) [8,43]. Several studies showed that a variation in the essential oil composition occurred in S. thymbra plants due to the effect of different factors such as different ecological conditions and even harvesting time [44,45]. Other studies showed that altitude affects the chemical composition of the essential oils of several plants [31,46]. Essential oil of S. hortensis gave a higher yield of carvacrol at higher altitude [47], which is the case in this present work. Qualitative and quantitative variation in secondary metabolites may be due to several other different environmental, agricultural and  genetic factors such as temperature, humidity, amount of rainfall, amount of sunlight, variety of the plant, collection season and even collection timing through the day as well as method of drying and extraction of the plant [13,34,[48][49][50]. It is thus interesting to study the composition and biological activity at different geographical areas in order to choose the optimum source of possible drug. The tested essential oils gave a promising antimicrobial activity against bacteria and fungi. This was evidenced by larger inhibition zones given by the oils compared to the standard antimicrobial agents used, especially against S. aureus, B. subtilus, E. coli, P. mirabilis, K. pneumonia as well as C. albicans. Values of MIC/ MLC were 0.125/0.25 μg/ml for SEO against S. aureus, P. mirabilis and K. pneumonia and for WEO against B. subtilus. It was observed that plants growing at lower altitude in Wasita locality had better antifungal activity, while those growing at higher altitude at Safsaf locality had better activity antibacterial activity. Compared with other studies, S. thymbra essential oil proved to be an available and natural antimicrobial agent that could be used in food industry to prevent the growth of food borne bacteria or to increase the shelf life of different processed foods [16]. A study showed that the oil of S. thymbra showed bacteriostatic activity at 0.001-0.1 mg/ ml and was bactericidal at 0.002-0.2 mg/ml and fungistatic effects at 0.001-0.025 mg/ml and fungicidal effects at 0.001-0.1 mg/ml, while the oil main constituents thymol and carvacrol e also showed strong antimicrobial activity and antifungal activity that was even higher than the commercial fungicide bifonazole [8]. The oil is active against certain microbial strains like Salmonella typhimurium, E. coli, S. aureus, P. aeruginosa, C. albicans and Moniliophthora perniciosa [11]. This strong antimicrobial activity may be attributed to the phenolic constituents: thymol and carvacrol which proved to interfere with the cell wall enzymes of bacteria and fungi [51]. Studies also revealed that thymol's antimicrobial effect, especially against S. aureus and E. coli could be due to the perturbation of the lipid fraction of the bacterial plasma membrane resulting in the leakage of  intracellular materials [52]. Also data show that both carvacrol and thymol had desired antimicrobial effect on E. coli, possibly due to their ability to permeabilize and depolarize the cytoplasmic membrane [53]. A study showed that at higher altitudes, plants are exposed to extreme environmental conditions, which stimulate the accumulation of secondary metabolites that causes a further enhancement in the medicinal activity including the antimicrobial effect [54]. Results of the anthelmintic studies augmented that of the antimicrobial results. In literature, the essential oil of S. thymbra is well recorded for its anthelmintic, larvicidal, acaricidal and genotoxic activity [3].
Previously, it has been reported that the essential oil of S. thymbra possesses a moderate anti proliferative and concentration dependant inhibition of viability of C32 (human amelanotic melanoma cell line) and ACHN (renal cell adenocarcinoma) [6]. Cytotoxicity of the essential oils of other Satureja species were evaluated on several cell lines such as J774 macrophage, 5637, KYSE, Fem-X Human Malignant Melanoma, Vero, SW480, MCF7, JET3, A549, THP-1 and HT29/219 [3].

Conclusion
In conclusion, altitude may influence the chemical composition and biological activity due to a collection of conditions like temperature and climate changes. In this study, S. thymbra plants collected during two successive years, growing at a lower altitude gave a better essential oil yield, higher yield of thymol as well as better antifungal activity. However, plants growing at higher altitudes gave better yield of carvacrol, improved antibacterial, anthelmintic activity and better cytotoxic activity against colon and breast cancer cell lines. Therefore, altitude at which the plant grows should be considered according to its intended use. Moreover, further studies on samples collected from more altitudes, as well studying the effect of other different environmental factors at these altitudes, such as temperature, humidity and amount of sunlight, should be employed in order to augment the results of this study.