Mentha spicata (Lamiaceae) chemotypes growing wild in Greece

Mentha spicata is the commonest mint species growing wild in Greece, exhibiting great morphological and chemical variability. The oil content from different wild populations examined ranged from 0.3% to 2.2%; the most common value being ca.1%. Though commercially exploited M. spicata plants are always rich in carvone and dihydrocarvone, wild populations are very variable; four different chemotypes were distinguished within the species. These chemotypes are characterized by the high contribution of the following compounds: (1) linalool, (2) piperitone oxide or piperitenone oxide, (3) carvone-dihydrocarvone and (4) pulegone-menthone-isomenthone. This chemical variability suggests the possibility of further developing and exploiting the full potential of the species.     

Genetic and Chemical Diversity in Perovskia abrotanoides Kar. (Lamiaceae) Populations Based on ISSRs Markers and Essential Oils Profile

Genetic and the essential oil composition variability among twelve Perovskia abrotanoides populations (PAbPs) growing wild in Iran were assessed by ISSR markers, GC‐FID and GC/MS, respectively. Nine selected ISSR primers produced 119 discernible bands, of them 96 (80.7%) being polymorphic. Genetic similarity values among populations ranged between 0.07 and 0.79 which indicated a high level of genetic variation. Polymorphic information content, resolving power and marker index generated by ISSR primers were, 0.31, 6.14, and 3.32, respectively. UPGMA grouped PAbPs into four main clusters. Altogether, 38 chemical compounds were identified in the oils, and a relatively high variation in their contents was found. Camphor (11.9 – 27.5%), 1,8‐cineole (11.3 – 21.3%), α‐bisabolol (0.0 – 13.1%), α‐pinene (5.9 – 10.8%), and δ‐3‐carene (0.1 – 10.5%) were the major compounds. Oxygenated monoterpenes (32.1 – 35.8%) and monoterpene hydrocarbons (25.7 – 30.4%) were the main groups of compounds in the oils studied. Cluster analysis and principal‐component analysis were used to characterize the samples according to oil components. Four main chemotypes were found to be Chemotype I (camphor/1,8‐cineol), Chemotype II (1,8‐cineole/camphor), Chemotype III (camphor/1,8‐cineol/α‐bisabolol), and Chemotype IV (camphor/δ‐3‐carene/α‐bisabolol). The information, provided here on P. abrotanoides populations, will be useful to introduce this plant into agricultural systems.     

Variation in Essential Oil Yield and Composition of Dorema aucheri Boiss., an Endemic Medicinal Plant Collected from Wild Populations in Natural Habitats

In the present research, variability in essential oil (EO) composition of five Dorema aucheri populations collected from natural habitats in different regions of Iran, were investigated. The EO content of populations varied from 0.28 to 0.68%. According to gas chromatography/mass spectrometry analysis, β‐caryophyllene (7.17 – 35.73%), thymol (23.45 – 29.64%), β‐gurjunene (2.58 – 5.89%), carvacrol (1.32 – 2.67%) and cuparene (1.97 – 2.98%) were the major components. Hierarchical cluster, principal component and canonical correspondence analyses classified the studied populations into three groups based on major EO components. The environmental parameters of the collected sites were also evaluated. According to the results, it might be suggested that sandy soils with high mean annual precipitation were major environmental factors influencing the amount of β‐caryophyllene, while thymol, cuparene and caryophyllen oxide increased in silty and clay soils. Finally, the population collected in high altitudes and clay soils had higher amount of β‐gurjunene.     

Essential‐Oil Diversity of Three Calamintha Species from Greece

The aim of the present study was to contribute to the knowledge of the essential‐oil composition of the Calamintha officinalis‐nepeta complex in Greece and to clarify the main patterns of its variation. The oils obtained from 22 wild‐growing populations of C. glandulosa, C. nepeta, and C. menthifolia were studied. They could be classified into two different chemotypes, which correspond to the main biosynthetic routes of the C(3)‐oxygenated p‐menthane compounds. Chemotype I includes oils rich in trans‐piperitone oxide, cis‐piperitone oxide, and piperitenone oxide, while Chemotype II comprises oils rich in pulegone and menthone or menthone and isomenthone. Within both chemotypes, quantitative fluctuations of the main components were observed. Comparison with published data showed that the presence of Chemotype II has not been observed before in C. menthifolia, while Chemotype I has been reported in C. nepeta plants from Greece for the first time.     

Yield,Composition and Antioxidant Capacity of the Essential Oil of Sweet Basil and Holy Basil as Influenced by Distillation Methods

The profile and bioactivity of essential oil (EO) depends on genetic, environmental, and other factors. We hypothesized that the basil EO may be influenced by the distillation methods. Hence, a study was conducted to evaluate the effect of steam distillation (SD) and hydrodistillation (HD) extraction method on the yield, composition, and bioactivity of EO of sweet basil (Ocimum basilicum) and holy basil (Ocimum tenuiflorum). In both basil species, the EO yield (content) was significantly higher from SD than from HD. There were significant differences in the compounds’ concentrations of EO obtained from SD and HD as well, however, the same compounds were identified in the EO from HD and SD. In the EO of O. basilicum, the concentration of 74% of the identified compounds were higher in SD than HD, whereas in the EO of O. tenuiflorum, the concentration of 84% of identified compounds were higher in SD than in HD. However, the concentrations of two of the major compounds of O. basilicum EO (estragole and methyl cinnamate) and a major compound of O. tenuiflorum EO (methyl eugenol) were significantly higher in HD than in SD. The type of distillation did not affect the antioxidant capacity of basil EO within the species. This study demonstrated that the type of distillation may significantly affect oil yield and composition but not the antioxidant capacity of the EO from sweet and holy basil.     

Chemical Diversity among the Essential Oils of Wild Populations of Stachys lavandulifolia Vahl (Lamiaceae) from Iran

The variation of the essential‐oil composition among ten wild populations of Stachys lavandulifolia Vahl (Lamiaceae), collected from different geographical regions of Iran, was assessed by GC‐FID and GC/MS analyses, and their intraspecific chemical variability was determined. Altogether, 49 compounds were identified in the oils, and a relatively high variation in their contents was found. The major compounds of the essential oils were myrcene (0.0–26.2%), limonene (0.0–24.5%), germacrene D (4.2–19.3%), bicyclogermacrene (1.6–18.0%), δ‐cadinene (6.5–16.0%), pulegone (0.0–15.1%), (Z)‐hex‐3‐enyl tiglate (0.0–15.1%), (E)‐caryophyllene (0.0–12.9), α‐zingiberene (0.2–12.2%), and spathulenol (1.6–11.1%). For the determination of the chemotypes and the chemical variability, the essential‐oil components were subjected to cluster analysis (CA). The five different chemotypes characterized were Chemotype I (germacrene D/bicyclogermacrene), Chemotype II (germacrene D/spathulenol), Chemotype III (limonene/δ‐cadinene), Chemotype IV (pulegone), and Chemotype V (α‐zingiberene). The high chemical variation among the populations according to their geographical and bioclimatic distribution imposes that conservation strategies of populations should be made appropriately, taking into account these factors. The in situ and ex situ conservation strategies should concern all populations representing the different chemotypes.     

Chemometric Tools to Highlight the Variability of the Chemical Composition and Yield of Lebanese Origanum syriacum L. Essential Oil

This study deals with the variation in the yield and composition of Lebanese Origanum syriacum L. essential oil (EO) according to harvesting time, drying methods used, and geographical location. Plant material was harvested twice a month all over 2013 and 2014 from Qartaba and Achkout located at high altitude and from Byblos at low altitude. EOs of the aerial parts were obtained by hydrodistillation. The highest yields were obtained at full flowering stage and slightly reduced after flowering. The GC/MS analysis revealed the presence of 50 components representing 90.49 – 99.82%, 88.79 – 100%, and 95.28 – 100% of the total oil extracted from plants harvested from Qartaba, Achkout, and Byblos, respectively. The major components in the oils were: carvacrol (2.1 – 79.8%), thymol (0.3 – 83.7%), p‐cymene (2.8 – 43.8%), thymoquinone (0.4 – 27.7%), γ‐terpinene (0.4 – 10.0%), octan‐3‐ol (0.3 – 4.9%), caryophyllene oxide (0.2 – 4.7%), oct‐1‐en‐3‐ol (0.3 – 3.7%), β‐caryophyllene (0.7 – 3.2%), cis‐sabinene hydrate (0.1 – 2.8%), terpinen‐4‐ol (0.1 – 2.8%), and α‐terpinene (0.2 – 2.2%). Independent components analysis (ICA) revealed that two groups were discriminated, reflecting compositional differences in the EOs profiles of the Lebanese oregano samples: O. syriacum grown in Qartaba and Achkout belongs to carvacrol chemotype, while O. syriacum grown in Byblos belongs to thymol chemotype. The flowering phase was the most productive period in terms of yield, bringing marked changes in the EO composition by increasing the amounts of carvacrol or thymol, and decreasing those of thymoquinone and p‐cymene.     

Variations in Essential Oil Composition and Antioxidant Activity in Perovskia abrotanoides Kar. Collected from Different Regions in Iran

Essential oil (EO) composition, phenolic content, and antioxidant activity were investigated in 17 P. abrotanoides populations collected from different geographical regions in Iran. The highest (3.61%) and lowest (1.25%) essential oil yields were measured in populations from Semnan Province (PSESM₂) and PISKS from Isfahan Province, respectively. GC/MS analysis identified camphor (4.05 – 35.94%), 1,8‐cineole (7.15 – 24.34%), borneol (0 – 21.75%), and α‐pinene (2.05 – 10.33%) as the main constituents of Perovskia essential oil. Cluster analysis classified the studied populations into four different groups: (I) high camphene, (II) high camphor/1,8‐cineole, (III) high borneol/δ‐3‐carene, and (IV) high α‐cadinol/trans‐caryophyllene. The highest flavonoid and phenolic contents were detected in PISAK from Isfahan Province (4.09 ± 0.05 mgQE/gDW, 58.51 ± 1.63 mgGAE/gDW) and PKRGS from Khorasan Province (3.80 ± 0.002 mgQE/gDW, 66.86 ± 0.002 mgGAE/gDW). DPPH and reducing power activity model systems identified PMASA and PKRKL as the populations with the highest antioxidant activity. Finally, the data obtained represented valuable information for introducing elite populations with EO components favorable to pharmaceutical and industrial applications.     

Chemical Composition and Antimicrobial Activity of Origanum libanoticum,Origanum ehrenbergii,and Origanum syriacum Growing Wild in Lebanon

The essential oils (EOs) of the aerial parts of Origanum libanoticum and Origanum ehrenbergii, endemic to Lebanon, and Origanum syriacum, endemic to the Levantine, were obtained by distillation with a Clevenger apparatus. GC and GC/MS allowed identification of 96.4%, 93.5%, and 95.2% of their constituents, respectively. Carvacrol was the major component of both O. syriacum EO (79%) and O. ehrenbergii EO (60.8%). This compound was absent in O. libanoticum EO and the major compounds were β‐caryophyllene (26.8%), caryophyllene oxide (22.6%), and germacrene D (17.2%). The assessment of their antimicrobial activity against Candida albicans and six pathogenic bacteria revealed that O. libanoticum EO was inactive, while O. syriacum and O. ehrenbergii showed moderate antimicrobial activity with minimal inhibitory concentrations varying from 400 to 1200 μg/ml. These results support the traditional use of these last two species in traditional herbal preparations in Lebanon.     

Genetic resources of Tunisian Artemisia arborescens L. (Asteraceae), pattern of volatile metabolites concentration and bioactivity and implication for conservation

Artemisia arborescens L. (Asteraceae) is an endemic and a valuable medicinal and ornamental species in the Mediterranean regions. Despite the interest given to A. arborescens L. as promising medicinal crop, this species is becoming threatened of extinction in Tunisia as a result of the fragmentation of its habitat and the absence of any specific measures for its conservation and valorisation. Based on the last considerations, extent information about their volatile metabolites profiling, contents and antimicrobial potential were undertaken based on fourteen populations originated from various geographical and bioclimatic habitats of Tunisia. The obtained results reveal an essential oil content mean value of 1.47%. The leaves of Tunisian tree wormwood populations were dominated by camphor (15.05–58.05%), chamazulene (11.72–45.26%) and β-thujone (0–36.53%). Based on the three first major compounds detected for each population, four different oil types were defined (Oil type I: camphor-chamazulene-β-thujone; Oil type II: camphor-chamazulene-terpinen-4-ol; Oil type III: camphor-chamazulene-bornyl acetate; Oil type IV: camphor-chamazulene-linalool). PCA, HCA and Heatmap chemometric analyses revealed three classification clusters defined mainly by minor compounds. These minor volatile metabolites succeed to discriminate among the fourteen studied populations which promote their chemotaxonomic interest. Our findings confirm previous reports concerning this species in the world and show chamazulene, camphor and β-thujone as the major compounds of this species which define their well known chemotypes independently of the geographical, bioclimatic and genotypic factors. This makes of these three components promising chemosystematic tool for the discrimination of A. arborescens L. in the genus Artemisia. The results showed that the four tested oils were effective against the nine tested microbial strains. The highest antimicrobial potential was revealed for the population AAr_MB characterized by camphor, chamazulene and β-thujone as the dominant components. The pattern of essential oils concentration offers wide range of possibilities for the initiation of breeding programs according to the desired bioactive compounds. Both in situ and ex situ conservation strategies were suggested for the maintenance of the highest chemical polymorphism of this valuable medicinal species.     

Essential-oil diversity of three Calamintha species from Greece

The aim of the present study was to contribute to the knowledge of the essential-oil composition of the Calamintha officinalis-nepeta complex in Greece and to clarify the main patterns of its variation. The oils obtained from 22 wild-growing populations of C. glandulosa, C. nepeta, and C. menthifolia were studied. They could be classified into two different chemotypes, which correspond to the main biosynthetic routes of the C(3)-oxygenated p-menthane compounds. Chemotype I includes oils rich in trans-piperitone oxide, cis-piperitone oxide, and piperitenone oxide, while Chemotype II comprises oils rich in pulegone and menthone or menthone and isomenthone. Within both chemotypes, quantitative fluctuations of the main components were observed. Comparison with published data showed that the presence of Chemotype II has not been observed before in C. menthifolia, while Chemotype I has been reported in C. nepeta plants from Greece for the first time.     

Barks Essential Oil,Secondary Metabolites and Biological Activities of Four Organs of Tunisian Calligonum azel Maire

This study is the first to investigate the chemical composition of barks essential oil (EO), secondary metabolites and biological activities of the MeOH and infusions extracts of seeds, leaves, barks and roots of Calligonum azel Maire (Polygonaceae) harvested from Tunisian desert. The gas chromatography/mass spectrometry (GC/MS) results showed the presence of fifty‐four compounds in barks EO. The major components were: viridiflorol (14.6%), α‐eudesmol (8.65%), trans‐caryophyllene (6.72%), elemol (6.63%), β‐eudesmol (6.21%). The obtained results showed that C. azel is a very rich plant in secondary metabolites. High contents in polyphenols, flavonoids and tannins were observed in both extracts of all studied organs. Significant differences were found between both extracts of the four organs. Thus, polyphenols and tannins were more abundant in leaves infusion extract, while, flavonoids showed a high level in barks extract. The antioxidant activity data demonstrated that all extracts showed strong antioxidant and radical scavenging activities. The MeOH extracts presented potential for antibacterial and antifungal activities against all tested microorganisms. The inhibition zones diameters and minimal inhibitrice concentration values were in the range of 9 – 15 mm and 2.5 – 20 μg/ml, respectively. This study demonstrated that C. azel can be regarded as an excellent plant source for natural antimicrobial agents.     

Essential Oil Variation among Natural Populations of Lavandula multifida L. (Lamiaceae)

Volatiles from twelve wild Tunisian populations of Lavandula multifida L. growing in different bioclimatic zones were assessed by GC (RI) and GC/MS. Thirty‐six constituents, representing 83.48% of the total oil were identified. The major components at the species level were carvacrol (31.81%), β‐bisabolene (14.89%), and acrylic acid dodecyl ester (11.43%). These volatiles, together with α‐pinene, were also the main compounds discriminating the populations. According to these dominant compounds, one chemotype was revealed, a carvacrol/β‐bisabolene/acrylic acid dodecyl ester chemotype. However, a significant variation among the populations was observed for the majority of the constituents. A high chemical‐population structure, estimated both by principal component analysis (PCA) and unweighted pair group method with averaging (UPGMA) cluster analysis based on Euclidean distances, was observed. Both methods allowed separation of the populations in three groups defined rather by minor than by major compounds. The population groups were not strictly concordant with their bioclimatic or geographic location. Conservation strategies should concern all populations, because of their low size and their high level of destruction. Populations exhibiting particular compounds other than the major ones should be protected first.     

Insecticidal Activity of the Leaf Essential Oil of Peperomia borbonensis Miq. (Piperaceae) and Its Major Components against the Melon Fly Bactrocera cucurbitae (Diptera: Tephritidae)

The essential oil from the leaves of Peperomia borbonensis from Réunion Island was obtained by hydrodistillation and characterized using GC‐FID, GC/MS and NMR. The main components were myristicin (39.5%) and elemicin (26.6%). The essential oil (EO) of Peperomia borbonensis and its major compounds (myristicin and elemicin), pure or in a mixture, were evaluated for their insecticidal activity against Bactrocera cucurbitae (Diptera: Tephritidae) using a filter paper impregnated bioassay. The concentrations necessary to kill 50% (LC₅₀) and 90% (LC₉₀) of the flies in three hours were determined. The LC₅₀ value was 0.23 ± 0.009 mg/cm² and the LC₉₀ value was 0.34 ± 0.015 mg/cm² for the EO. The median lethal time (LT₅₀) was determined to compare the toxicity of EO and the major constituents. The EO was the most potent insecticide (LT₅₀ = 98 ± 2 min), followed by the mixture of myristicin and elemicin (1.4:1) (LT₅₀ = 127 ± 2 min) indicating that the efficiency of the EO is potentiated by minor compounds and emphasizing one of the major assets of EOs against pure molecules.     

Allelopathic Activity and Chemical Composition of Rhynchosia minima (L.) DC. Essential Oil from Egypt

Aromatic plants attract the attention of many researchers worldwide due to their worthy applications in agriculture, human prosperity, and the environment. Essential oil (EO) could be exploited as effective alternatives to synthetic compounds as it has several biological activities including allelopathy. The EO from the aerial parts of Rhynchosia minima was extracted by hydrodistillation and investigated by gas chromatography/mass spectrometry (GC/MS). Different concentrations (50, 100, 150 and 200 μL L⁻¹) of the EO were prepared for investigation of their allelopathic potential on two weeds; Dactyloctenium aegyptium and Rumex dentatus. Twenty‐eight compounds, mainly sesquiterpenes (69.13%) were determined. The major compounds are α‐eudesmol, 2‐allyl‐5‐tert‐butylhydroquinone, caryophyllene oxide, trans‐caryophyllene, and τ‐cadinol. The EO from the R. minima showed a significant inhibition of D. aegyptium and R. dentatus germination, while the seedling growth was stimulated. Therefore, it is not recommended to treat these noxious weeds with the EO of R. minima before the germination. In contrast, the apparent stimulatory effect on the seedling growth offers further studies to use the EO of R. minima to enhance the fitness of different economic crops. However, characterization of green bio‐herbicides such as EO (allelochemicals) from wild plants raises a new opportunity for the incorporation of new technology of bio‐control against the noxious weeds.     

Chemical Profiling and Antifungal Activity of Ziziphora clinopodioides Leaf Essential Oil against the Pathogen Verticillium dahliae

Essential oils (EOs) are often used as natural antifungal agents to control the growth of phytopathogenic fungi. The aim of this study was to determine the effect of Ziziphora clinopodioides leaf EO against Verticillium dahliae, a pathogenic fungus of cotton. Gas chromatography-mass spectrometry (GC/MS) analysis revealed the presence of 15 compounds of the total of extracted oil, which was consisted of 98.79 % monoterpenes and 0.61 % sesquiterpenes. The major constituents were pulegone (62.17 %), isomenthone (18.42 %), l-menthone (5.55 %) and piperitenone (3.99 %). The mycelial growth of Verticillium dahliae was completely inhibited at 0.24 μL/mL air under vapor phase condition. Considerable morphological variations were also observed in the fungal sclerotia at the contact phase at 3 μL/mL. This study demonstrated for the first time that Z. clinopodioides EO can effectively inhibit the growth of V. dahliae, implying that it has the potential to be explored as an antifungal agent against Verticillium Wilt of cotton.     

Essential Oils from Anthemis maritima Flowers: Infraspecific Variability along the Adriatic Coast (Italy)

The hydrodistilled essential oils (EOs) from flowers of five Adriatic populations of Anthemis maritima were analyzed by GC‐FID and GC/MS. Anthemis maritima is a psammophilous plant living generally on coastal sand dunes but occasionally on sea cliffs and shingle beaches. A total of 163 chemical compounds were identified, accounting for 90.5% of the oils. The main classes of compounds represented in the EOs were monoterpene hydrocarbons, oxygenated monoterpenes, sesquiterpene hydrocarbons, oxygenated sesquiterpenes, and terpene esters.The multivariate chemometric techniques, in particular cluster analysis and principal coordinate analysis, used to classify the samples, highlighted three different chemotypes linked to a geographic origin. One group living in northern Italy was characterized by the highest content of β‐pinene, γ‐terpinene, and β‐caryophyllene, a second chemotype was in central Italy with the highest amount of trans‐chrysanthenyl acetate and a third group living in southern Italy with a more heterogeneous volatile profile was characterized by the highest values of cis‐chrysanthenyl acetate, trans‐chrysanthenyl isobutyrate, cis‐carveol propionate, α‐zingiberene, and cubenol. Moreover, the comparison of the Adriatic populations with the Tyrrhenian samples, analyzed in a previous research, showed that cubenol (absent in all the Tyrrhenian populations) and (E)‐β‐farnesene (absent in all the Adriatic samples) play a crucial role in discriminating the Italian populations.     

Essential Oils of Micromeria dalmatica Benth., a Balkan Endemic Species of Section Pseudomelissa

The essential oils of 13 Greek populations of Micromeria dalmatica, a Balkan endemic species and member of the section Pseudomelissa, were examined for the first time. Among the studied populations, two main oil types could be distinguished. Type I was found to be rich in β‐pinene, limonene, and germacrene D (accounting for 55.6–70.2% of the total oil), and Type II was characterized by the preponderance of p‐menthane compounds (accounting for 64.2–89.9% of the oil). The latter oil type could be further divided into two subtypes, one comprising oils with predominance of piperitenone and piperitenone oxide and another composed of oils containing high proportions of pulegone, menthone, and isomenthone. The abundance of p‐menthane compounds is a common feature of the oils of all members of the section Pseudomelissa studied to date. However, the existence of oils of Type I has not been previously reported for M. dalmatica, neither for other members of the section Pseudomelissa.     

Mentha longifolia in vitro cultures as safe source of flavouring ingredients

In vitro plantlets and callus of M. longifolia were established and their volatile constituents characterized by GC-MS analysis of their headspaces (HSs) and essential oils (EOs). Significant quali-quantitative differences were found in the aromatic fingerprints in comparison with the M. longifolia parent plants. In fact, limonene and carvone were the main constituents in the EOs of the mother plants, while the aroma of the in vitro plant material were especially enriched in oxygenated terpenes. In particular, huge amounts of piperitenone and piperitenone oxide (75 %) were found for in vitro plantlets, while trans-carvone oxide (19 %) and trans-piperitone epoxide (9 %) were found in callus EO. However, the established in vitro plant material showed lack of pulegone and menthofurane, thus preserving an important feature observed in the volatile fingerprint of the parent plants. In fact, because of their well-known toxicity significant amounts of pulegone and menthofurane may compromise the safety using of mint essential oil. Therefore the in vitro M. longifolia plantlets and callus may be regarded as a potential source of a safe flavouring agent.     

Essential oil variation and antioxidant capacity of Mentha pulegium populations and their relation to ecological factors

Mentha pulegium L. is an aromatic herb belonging to the Lamiaceae family, a wild plant which is distributed in different areas of Iran. In this research, we evaluated the variability of essential oil content and compositions of 12 M. pulegium populations. Essential oils were analyzed using gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS) methods. The essential oils content varied from 0.22 to 1.63% w/w within different populations. Twenty-nine compounds were identified which represent 83.4–98.7% of the total essential oil. The most significant essential oil compounds among the studied population were identified using the principal components analysis (PCA-biplot). According to the PCA-biplot, the major compounds were pulegone (2.5–51.7%), menthone (0.2–25.3%), limonene (0.0–35.4%), 1,8-cineol (0.0–33.4%), piperitenone oxide (0.2–55.2%), and trans-piperitone epoxide (0.0–28.5%). Besides, hierarchical cluster analysis indicated that the studied populations were classified into two main clusters based on the essential oil components. The canonical correspondence analysis (CCA) indicated that some environmental factors could influence the phytochemical constituents as well as the antioxidant activity. The temperature and altitude were effective environmental factors with regards to 1–8 cineol, limonene and menthone content, while average rainfall was the most effective factor with respect to trans-piperitone epoxide, piperitenone oxide, and pulegone content. Our results consequently showed that environmental factors had a significant effect on the essential oil content and its antioxidant activity in M. pulegium populations.