GC-MS Analysis of the Compositions and Antimicrobial Activities of Essential Oils From Five Species of Lamiaceae in Iran

Essential oils (EOs) separated from Lamiaceae species attract more attention due to their abundant use in the preservation of natural foods and pharmaceutics and have gained considerable interester in research and industrial. The aim of this study was to evaluate composition and antimicrobial activity of EOs obtained from ve species Lamiaceae in Iran. After extraction of EOs by Clevenger, their composition was evaluated by gas chromatography-mass spectrometry (GC–MS). Antimicrobial properties were assayed by measuring inhibition zones, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). The results showed that the effects of species, on yield and predominant compounds amount of EOs of ve species were signicant with a probability of error of 1 %. The yield of oils were varied from ~ 0.02 to ~ 1.88%. About 66 components were identied by gas chromatography-mass spectrometry (GC-MS), and the dominant compounds were included thymol (67.71%), oleic acid (0.49–62.09%), (-)-caryophyllene oxide (0.41–24.81%), α-pinene (1.09– 19.41%), 1,8-cineole (0.22–15.40%), palmitic acid (0.32–13.28%), (+)spathulenol (11.16%), and germacrene D (0.30-10.26%) in different species. The results of analysis of variance showed that there was a signicant difference between the mean of the inhibition zone obtained treating the different microorganisms with the essential oil of ve species (P ≤ 0.01). The highest inhibition zone belonged to TDEO (39.33 ± 0.58 and 25.00 ± 0.00 mm) against Gram-positive S. aureus and A. brasiliensis. The Gram-negative P. aeruginosa showed the lowest inhibitory resistance to HIEO, SIEO, and ROEO (with a MIC value of 31.25 µg/mL), which was very signicant compared to rifampin. Therefore, EOs of ve species have potential applications in the control of various bacteria and fungi and can be a natural alternative to some antibiotics. 2005) and EO (Bajalan et al., 2017; Messaoudi Moussi et al., 2019 and Risaliti et al., 2019), recorded. Rosemary EO is used in the treatment of indigestion and milder forms of gastrointestinal disorders, circulatory disorders, as a supplement in the treatment of musculoskeletal pain and inammation et Gram-positive bacteria B. subtilis (inhibition zone diameter 9.00±0.00 mm) and S. aureus (inhibition zone diameter 8.50 ± 0.50 mm). The present results are remarkable when compared to the report of Ebrahimabadi et al., (2009), for the lack of SIEO inhibition zone diameter against these micro-organisms. Inhibition zone diameter of ROEO against three gram-positive bacteria B. subtilis, S. aureus (9.00 ± 0.00 mm) and S. epidermidis (9.33 ± 0.58 mm), which had little activity compared to control antibiotics rifampin and gentamicin.

Lamiaceae EOs because of their interesting physicochemical characteristics of substantial value, have garnered research and industrial interests for use as natural products. These EOs mainly contain various phenolic compounds, such as monoterpene perillaldehyde, polyphenols, coumarins, tannins, iridoids, diterpenoids, triterpenoids, quinones, saponins, and pyridine and pyrrolidine alkaloids (El Asbahani et al., 2015). Studies have shown that many mint family plants such as thyme, peppermint, lavender, rosemary, peppermint, savory, marjoram have antimicrobial effects (Zargari, 2012 andSekman et al., 2004).
Thymus daenensis Celak. is an Iranian herbaceous, that is distributed in most parts of Iran, especially throughout the Zagros and some parts of the Alborz Mountains (Jamzad, 2012;Zarshenas and Kern, 2015). Thymol, carvacrol, and p-cymene have been reported as the most important constituents in the EO of this plant (Sajjadi and Khatamsaz, 2003  Hymenocrater incanus Bunge is one of the exclusive species of this genus in Iran. The herbaceous plant is perennial with a wooden base 30 to 50 cm high (Jamzad, 2012). The major constituents of the EO of this plant have been reported as β-caryophyllene and 1,8-cineole, αpinene, β-pinene, trans-β -ocimene, germacrene-D, and caryophyllene oxide ( Rosmarinus o cinalis L. The plant is in the form of durable small shrubs with aromatic leaves and small blue owers (Inouye et al., 2001). Rosemary EO is one of the compounds that have antimicrobial and antioxidant properties in many cases and antimicrobial compounds such as phenolic compounds are found in abundance in it. Studies on these species indicate the importance of these plants in terms of their essential oil composition and antimicrobial properties.
However, no comprehensive study of these species has been performed simultaneously using Twelve strains of microorganisms. Since these plants have not been studied in the habitats of Isfahan province in Iran, this study is of particular importance. In particulary, most of the species studied are native to Iran. The aims of this study were 1) to determine the chemical composition of EOs of ve species, and 2) to compare chemical compositions, yield variations and antimicrobial activities of EOs of 5 species.

Plant material
To select the sampling region, at rst, habitats of plants (T. daenensis, N. sessilifolia, H. incanus, S. in ate, and R. o cinalis) were identi ed through eld surveys. Then, Daran region, located in Isfahan, Iran was selected (longitude: E 46˚ 49 02 and latitude: N 36˚ 54 170 ). To sample of each plant, in June 2018, coinciding with the owering, three points were selected randomly from Daran region. In each point aerial parts of each plant randomly from different plants (50-100 plants in each point) were collected. The specimens were transferred to the laboratory after being harvested and then exposed to free air to dry. One sample of the whole plant was also collected and pressed. The specimens was identi ed and recorded in the herbarium of the University of Kashan

Extraction of EOs
After complete drying, the samples were grinded by using a small electric mill. Then from each sample of plant was weighed (100 g) and subjected to the extraction process by means of water distillation by using a Clevenger apparatus (Made in Germany) for ve hours. The essential oil was dried by anhydrous sodium sulfate and after ltration, were stored in dark bottles at 4°C until their use for further studies. Essential oil yield was calculated based on weight percent (w/w). This process was repeated three times for each plant.

Gas chromatography-mass spectrometry (GC-MS) analysis
The main bioactives contained in the essential oils have been determined by means of GC-MS, using a chromatograph (Model 6890 Chromatography) coupled with an Agilent Mass Spectrometer (Model N-5973). A capillary column (HP-5MS) with 5% Methylphenylsiloxane Static Phase (Length 30 m, Internal Diameter 0.25 mm, Layer Static Thickness 0.25 μm) and ionization energy of 70 eV was used. The temperature for the analyses was rst set at 60°C and then it was increased, at a rate of 3°C up to 246°C. The injector and detector temperatures were maintained at 250°C, the volume of the injected sample was 1 µl and the helium carrier gas was maintained at a ow rate of 1.5 ml/min. The identi cation of chemical components was based on the analysis of the chromatograms obtained for each oil, by means of the retention indices (RI) evaluation in comparison with standards of n-alkane mixtures (C8-C20) and mass spectral data of each peak by using a computer library (Wiley-14 and NIST-14 Mass Spectral Library), and comparison of the obtained results with those contained in the literature (Adams, 2007).

Agar diffusion method of Well diffusion
This procedure was performed according to CLSI standards. 6.0 mm in diameter well plates containing Müller Hinton agar were prepared and 100 µl of bacterial suspensions with a half-McFarland turbidity equivalent in culture medium were cultured. The essential oils were dissolved in dimethyl-sulfoxide (DMSO) at a concentration of 30 mg/mL. 10 μL (equivalent to 300 μg) of each oil was poured into the wells. The plates were incubated at 37°C for 24 h for bacterial strains and 48 h and 72 h at 30°C for yeast and its antimicrobial activity was evaluated for each microorganism by measuring the diameter of the inhibition halo (in millimeters), according to the antibiogram ruler. To evaluate the repeatability of the results, three replicates for each essential oils and each strain were performed. DMSO was used as negative control. Gentamicin (10 lg/disk), and rifampin (5 lg/disk) for bacteria and nystatin (100 I.U.) for yeast were used as standard drugs for positive control in the same conditions to tested oils.

Determine the Minimum inhibitory concentration (MIC)
The minimum concentration able to inhibit the growth of bacteria was calculated by means of microdilution method. Essential oils (2000 μg/ml), were dissolved in a mixture of tryptic soy broth medium and DMSO and then were opportunely diluted, by using the same mixture, to reach different concentrations (1000, 500, 250, 125, 62.5, 31.25 and 15.63 mg/ml).
Sterile 96-well microplates were lled with 95 µl of culture medium, 5 µl of bacterial suspension with 0.5 McFarland dilution, and 100 µL of the essential oils at different concentrations, then plates were incubated at 37°C for 24 h for bacterial strains and 48 h and at 30°C for yeast. The MIC was determined by means of the improvement of opacity or the change in color. The MIC was the lowest concentration of an antimicrobial that inhibited the visible growth (absence of turbidity).

Determination of Minimum bactericide concentration (MBC)
To determine the minimum concentration able to kill the bacteria the same microdilution method described above was used. After 24 h of incubation with both bacteria and oils at different concentrations, 5 µl of the content of each well were inoculated with neutrin agar medium and incubated at 37°C for 24 h for bacterial strains. After incubation, the colony-forming units were enumerated. The MBC was the lowest concentration able to effectively reduce the growth of microorganisms (99.5%).

Statistical analysis
The statistical analysis was performed using SPSS software. First, the normality of the statistical variables was investigated using a Kolmogorov-Smirnov test, and after ensuring the normality of the data, the variance of the data (essential oil and Antimicrobial activity) was analyzed using an F-test and a comparison of the means using a Duncan test with a probability level was 1 % error was performed.

EOs content
The EOs color varied from pale yellow to dark yellow with yields ranging from 1.88% -0.02% (w/w). Based on the results of the analysis of variance, the yield of essential oil of ve species was signi cantly different. The effect of the species on the yield of essential oil was signi cant (P≤ 0.01) ( Table 1) (Bajalan et al., 2017), that were larger than the present study. The synthesis of secondary compounds in plants is one of the most important defense mechanisms against pathogens, and its quantity varies depending on habitat, organ, species. These differences are most likely due to the differences in chemotyp, which are themselves due to the environmental conditions and climate (Yavari et al., 2010).
In NSEO, most of the compounds belonged to acids and mainly to non-terpenes ( Table 3 ). The predominant components in the EO of this plant were oleic Acid (62.09%), Stearic acid (8.16%) and linoleic acid (6.06%). These three compounds were rst observed in this plant and so far no reports of these compounds have been reported even to a minor extent.
Based on the results of analysis of variance the effect of the species on the amount of 1,8-cineole, oleic acid, (-)-caryophyllene oxide, transcaryophyllene and linalool was signi cant (P≤ 0.01) ( Table 1). The data presented in Table 1 show that 1,8-cineole and oleic Aacid were found in the essential oils of 5 plant species. The highest amount of 1,8-Cineole was observed in ROEO with 15.40% and the lowest with 0.22% in HIEO ( Figure 1). The highest and lowest amount of oleic acid was in NSEO (62.09%) and TDEO (0.49%), respectively ( Figure 2). Linalool was found in all essential oils except HIEO, with the highest amount being 2.97% in ROEO. Also, trans-caryophyllene and (-)caryophyllene oxide were found in all essential oils except SIEO with the highest amount being 3.68% and 24.81% in HIEO, respectively. Some high-value compounds such as thymol (67.71) and p-cymene (5.16), only in TDEO, linoleic acid (6.06) only in NSEO, caryophyllenol-II (5.06) only in HIEO, (+) spathulenol (11.16) only in SIEO and β-myrcene (5.38) existed only in ROEO.

Inhibition-zone
The results of analysis of variance showed that there was a signi cant difference between the mean of the inhibition halo obtained treating the different microorganisms with the essential oil of ve Lamiaceae species (P≤ 0.01) ( Table 4). Similar trend was observed by Luo et al., (2019) for six other species of lamiaceae. The highest inhibition zone was belonged to TDEO (39.33±0.58 mm) against Gram-positive S. aureus compared to the inhibition zone rifampin (~21 mm) and gentamicin (~27 mm) is a signi cant activity (Table 5). Other essential oils such as NSEO (9.67 ± 0.58 mm), SIEO (8.50±0.50 mm) and ROEO (9.00±0.00 mm) had little activity on this bacterium and HIEO did not produce any diameter. Reduction of monoterpenes in HIEO seems to be one of the factors affecting the lack of inhibition halo against this S. aureus. In addition, this antibacterial activity of TDEO, NSEO, SIEO, and ROEO can be attributed to other oxygenated compounds such as linalool.
Secondly, was the highest inhibition zone (25.00 ± 0.00 mm), belonging to TDEO against A. brasiliensis, which is a signi cant activity compared to the Nystatin (~30 mm) control antibiotic growth zone against A. brasiliensis (Table 5). On the other hand, the effect of TDEO on the fungi A. sniger and C. albicans also caused an inhibition zone diameter of 12.00 ± 0.00 mm and 11.67 ± 0.58 mm, respectively, in comparison with the inhibition zone of Nystatin control antibiotic (~27 and ~33 mm), had low activity on these fungi.
On the other hand, TDEO with inhibition zone diameter of 17.67 ± 0.58 mm and 16.33±0.58 mm against two Gram-negative bacteria K. pneumonia and Sh. dysenteriae showed good activity in comparison with inhibition zone of antibiotic rifampin (~8 mm and ~9 mm) and antibiotic control gentamicin (~17 mm). so far no report of the effect of TDEO on these two bacteria has been reported and therefore, the present study is important. Also TDEO against B. subtilis and P. vulgaris had a low activity, with inhibition zone diameter of 14.00 ±0.50 mm compared to inhibition zone rifampin (~19 and ~8 mm) and gentamicin (~30 and ~24 mm).
TDEO also had little effect against two Gram-negative bacteria E. coli and S. paratyphi-A with inhibition zone 11.33 ± 0.58 and 11.67 ± 0.58 mm, respectively, compared to inhibition zone diameter rifampin (~10 and ~8 mm) and gentamicin (~23 and ~18 mm) low effect is quite obvious.
Inhibition zone of NSEO was only present against three Gram-positive B. subtilis, S.epidermidis (9.00 ± 0.00 mm), and S. aureus (9.67 ± 0.58 mm), which had little activity compared to rifampin and gentamicin. The NSEO did not create inhibition zone against other microorganisms.
Inhibition zone of HIEO was created only against two Gram-positive bacteria, B. subtilis (10.00 ± 0.00 and S.epidermidis (11.00 ± 0.00 mm), which had little activity compared to rifampin and gentamicin. HIEO did not create inhibition zone against other microorganisms. The SIEO was exhibited little activity against Gram-positive bacteria B. subtilis (inhibition zone diameter 9.00±0.00 mm) and S. aureus (inhibition zone diameter 8.50 ± 0.50 mm). The present results are remarkable when compared to the report of Ebrahimabadi et al., (2009), for the lack of SIEO inhibition zone diameter against these micro-organisms. Inhibition zone diameter of ROEO against three gram-positive bacteria B. subtilis, S. aureus (9.00 ± 0.00 mm) and S. epidermidis (9.33 ± 0.58 mm), which had little activity compared to control antibiotics rifampin and gentamicin.

Minimal inhibitory concentrations (MIC)
The values of MIC varied from 15.63> μg/mL to >2000 as a function of the organism tested and the used oil (Table 6). The lowest inhibitory concentration was found against P. aeruginosa (MIC = 31.25 μg/mL) by HIEO, SIEO, and ROEO, which was very signi cant compared to rifampin (~31.25 μg/mL) and gentamicin (~7.81 µg/mL). It has been previously reported that P. aeruginosa is highly sensitive to EOs (De Martino et al., 2009). Other EOs with a MIC value of 125 µg/mL had a much weaker inhibitory effect against P. aeruginosa. All EOs (except TDEO) with MIC value of 2000 µg/mL showed the highest resistance to A. brasiliensis and A. niger. On the other hand, SIEO with a MIC value of >1000 μg/mL showed the highest resistance to S. aureus, which was two to four times weaker than other EOs studied.
The MIC value of TDEO against all the different bacteria varied between 125 and 500 μg/mL, with its weakest inhibitory effect against Sh. dysenteriae in compared with rifampin (MIC =15.63 µg/mL) and gentamicin (MIC =3.90 µg/mL), which contradicts the results of Golkar et al. (2020) for TDEO. The MIC value of TDEO against all fungi varied from 31.25 to 250 μg/mL. C. albicans showed the least inhibitory resistance to TDEO among all microorganisms, which is signi cantly more potent and three times more potent than nystatin (~125 μg/mL). C. albicans is one of the most common pathogenic fungi in nature that causes serial infectious diseases and has become a serious threat to human health. The MBC value of TDEO also varied from 62.50 µg/mL to 500 µg/mL ( Table 4). The results indicate that the MBC value of TDEO on all microorganisms (except S. aureus, K. pneumonia, and C. albicans) was always equal to the MIC value (Table 6 and 7), indicating that growth and lethal inhibitory power of TDEO are the same.
The MIC value of NSEO against all microorganisms varied between 125 and 2000 μg / mL. Its strongest inhibitory effect was against Gramnegative bacteria Sh. dysenteriae, K. pneumonia and P. aeruginosa, that it is three to four times weaker than control antibiotics. The weakest inhibitory effect of this essential oil was against A. sniger and A. brasiliensis. The results showed that the MBC value of NSEO was the same as MIC value of NSEO in all microorganisms (except S. aureus, B. subtilis, Sh. dysenteriae, and P. vulgaris). This indicates that NSEO's growth and lethal inhibitory potency against many of microorganisms is the same. The lowest MBC value of NSEO belonged to Sh. dysenteriae and K. pneumonia, which was equivalent to MIC value of NSEO, con rming the importance of this EO in inhibiting and killing Sh. dysenteriae and K. pneumonia. The absence of terpenes predominantly in the EO of this plant may be due to its poor antimicrobial effect, although the relatively weak effect of NSEO on some bacteria may be due to the presence of fatty acids (Oleic acid ~62.09%). However, the mechanism of the antibacterial activity of fatty acids is not yet known. But it is believed that their functional nature is related to the permeability and membrane disruption and fatal alterations in the cytoplasmic membrane of the bacterium, thereby disrupting the membrane-dependent conduction systems.
The MIC value of HIEO was against all microorganisms varied between 15.63> ->2000 µg/mL. The strongest inhibitory effect was against Gram-negative P. aeruginosa (MIC 15.63> µg/mL), which is very signi cant in comparison with rifampin (MIC = 31.25 µg/mL). HIEO had a good effect against Sh. dysenteriae, K. pneumonia, and S. paratyphi-A with MIC value of HIEO 62.50 μg/mL (compared with rifampin MIC value of 15.63 μg/mL against Sh. dysenteriae, K. pneumonia, and S. paratyphi-A), that was consistent with results of Fazly Bazzaz and Haririzadeh, (2003) for H. calycinus. Also the MIC value of HIEO against C. albicans was 62.50 µg/mL, which was weaker than nystatin (MIC =125 µg/mL). This is evident in the results of the MBC value of HIEO and the lethality of this against C. albicans has been increased to 250 µg/mL. Also the MIC value of SIEO was against all microorganisms varied between15.63>->2000 μg /mL, that the strongest inhibitory effect was against the Gram-negative bacterium P. aeruginosa. ROEO had the strongest inhibitory effect against P. aeruginosa (with MIC value 15.63> µg/mL), which was twice as potent as the MIC value of rifampin. . Inhibition zone TDEO was recorded against Gram-positive S. epidermidis with 9.00 ± 0.001. However, there is no reported effect of this EO on S. paratyphi-A and S. epidermidis. The difference of antimicrobial activities between species from the same genus might be due to the difference of their major compounds and also to the complexity of EOs chemical pro les. In the present study EOs against P. aeruginosa did not produce any inhibition zone. The reverse process has been developed by Luo et al., (2019) for six species of lamiaceae against P. aeruginosa, so that ROEO has created the highest inhibition zone against this bacterium. This difference in antibacterial activity can be due to the difference in the composition of the essential oil of this species in different regions. The biological activities of the essential oils depend on chemical constituents (Popović-Djordjević et al., 2019).

Discussion
These results are inconsistent with the ndings of Ezzatzadeh et al., (2014) for Nepeta asterotricha. Since the major constituents of NSEO were, oleic acid, stearic acid, and linoleic acid, one might argue that the microbial effect is due to these fatty acids. Fatty acids have antifungal and antibacterial activities against many microorganisms whose spectrum of activity varies based on the degree of saturation, Essential oils of different specie due to the large number of chemical compounds, which work simultaneously, have unparalleled antibacterial potentials and prevent bacterial resistance mechanisms. In addition, synergistic interactions between EOs can enhance their natural antimicrobial effect. Therefore, antimicrobial potential cannot be associated with a single component or mechanism of action (Candy et al., 2018). Meanwhile, antibiotic abuse exacerbates the harm of C. albicans (Delcour, 2009). So TDEO as a natural alternative against this fungus has signi cant and promising potential. The presence of phenolic monoterpenes, and especially p-cymene and Thymol, is a major cause of the strong antifungal activity of TDEO. Phenolic compounds have high antimicrobial properties and in fact, phenolic compounds in plant essential oils have the most effect on the development of the antimicrobial activity (Lemos et al., 2017;Mahboubi et al., 2017). These compounds both penetrate the cell membrane and can contribute to the clotting of cell contents (Burl & Coote 1999 and  Dorman & Dean 2000). Also, although α-pinene is present in small amounts in TDEO (∼1.09%), it can be another factor in uencing this antifungal activity. Research shows the antifungal effect of this compound (Dorman, 2000). In general, the synergistic effects of the diversity between the main and minor components of essential oils in their biological and antimicrobial activity should be considered (Raut and Karuppayil, 2014). The lowest MBC value of TDEO has belonged to C. albicans, that was consistent with results of Dadashpour et al., (2011). In the study of Ebrahimabadi et l., (2009), SIEO had no inhibitory effect on any of the microorganisms, so the plant sample of this study is valuable. Differences in habitat conditions are among the most important causes of these biological traits in the plant, which has led to the dominance of acidic compounds and Sesquiterpenes, followed by the ability of this essential oil to control some bacteria. The HIEO MBC value against various bacteria (except S. epidermidis and S. aureus) was always higher than its MIC value, indicating that the lethal potency of this essential oil was lower than its inhibitory potency. Another report showed that aqueous extract from Aquilaria crassna (Wetwitayaklung et al., 2009) rich of the some sesquiterpene compound was active against some microorganisms.
Some differences in the amount of antimicrobial effects observed in this study and similar studies could be due to differences in the growth locations of the EO producing plants, the use of different methods for extraction, harvesting time and even the concentration of EOs (Has-Szymanczuk et al., 2011; Lopez et al., 2005 andToyoshi et al., 2006). Differences in antimicrobial effects indicated differences in essential oil composition. The ROEO also showed a good inhibitory effect (31.25 µg/mL) against Sh. dysenteriae, E. coli and K. pneumonia, which was weaker than control antibiotics. The lowest MIC value of ROEO was recorded against fungi. The MBC value against the associated microorganisms was also higher than the MIC value, indicating a lower lethal force than the inhibitory power, which is consistent with the results of Messaoudi Moussi et al. (2019). The major compounds that have been attributed to the antimicrobial properties of ROEO include α-pinene, 1,8-cineole, verbenone, and linalool (Okoh, 2010 andOluwatuyi et al., 2004), among these compounds, three conditions may occur: Synergetic, Antagonist, and Additive. Occurrence of any of these conditions affects the antimicrobial properties of essential oil (Marzouk et al., 2006). There is ample evidence that the essential compounds of essential oils also play an important role in antimicrobial properties by creating a synergistic effect between the main compounds and their easier transfer into the bacterial cell (Burt, 2004 andTavassoli et al., 2011).
Essential oils (EOs) produced by plants as secondary metabolites, usually composed of different compounds with different ratios. The present study was conducted for the rst time on ve important species of the Lamiaceae family in Iran which after analysis by chromatography included a variety of compounds such as thymol, oleic acid, (-)-caryophyllene oxide, α-pinene, 1,8-cineole, palmitic acid, (+)spathulenol, germacrene D, bicyclogermacrene, phytol, camphor, and borneol. 1,8-cineole and oleic acid were found in the EOs of ve plant species. The diversity of compounds of different species resulted in a variety of antibacterial and antifungal activity. TDEO had the highest inhibitory zone against various microorganisms compared to other essential oils, which had a particularly signi cant effect against S. aureus and A. brasiliensis. Based on the MIC value results, essentail oils of ve species of Lamiaceae had a relatively good effect against Sh. dysenteriae, P. aeruginosa, E. coli, K. pneumonia, C. albicans. Overall, the MIC value of ROEO against all bacteria was signi cantly more effective than other EOs. These essential oils from ve species of Lamiaceae can have potential applications in the food and pharmaceutical industries as natural food preservatives, natural fungicides and a potential natural alternative to some antibiotics. In future research, it is important to identify and separate the main components or general compounds of these essential oils to con rm the usefulness of these compounds.

Declarations
Ethical Approval Not applicable.

Consent to Participate
Not applicable.

Consent to Publish
Not applicable.

Authors Contributions
Mansureh Ghavam was the supervisor, designer of the hypotheses, and responsible and functor for all the steps and wrote the text of the article.

Funding
No funding

Competing interests
The authors declare no competing interests.

Availability of data and material
Not applicable.   Comparison of the mean effect of species on 1,8-Cineole content of essential oil of ve Lamiaceae species Comparison of the mean effect of species on Oleic acid content of essential oil of ve Lamiaceae species