Biochemical Characterization of Helichrysum italicum (Roth) G.Don subsp. italicum (Asteraceae) from Montenegro: Phytochemical Screening,Chemotaxonomy, and Antioxidant Properties

The chemical composition and antioxidant properties of the essential oil and EtOH extract of immortelle (Helichrysum italicum (Roth) G.Don subsp. italicum, Asteraceae) collected in Montenegro were evaluated. The essential oil was characterized by GC/MS analysis, and the content of total phenolics and flavonoids in the EtOH extract was determined using the Folin? Ciocalteu reagent. The free‐radical‐scavenging capacity (RSC) of both the essential oil and the EtOH extract was assessed with the 2,2‐diphenyl‐1‐pycrylhydrazyl (DPPH) method. Moreover, the inhibition of hydroxyl radical (^.OH) generation by the EtOH extract of immortelle was evaluated for the first time here. Neryl acetate (28.2%) and γ‐curcumene (18.8%) were the main compounds in the essential oil, followed by neryl propionate (9.1%) and ar‐curcumene (8.3%). The chemical composition of the oils of the examined and additional 16 selected Helichrysum italicum taxa described in literature were compared using principal component (PCA) and cluster (CA) analyses. The results of the statistical analyses implied the occurrence of at least four different main and three subchemotypes of essential oils. Considering the antioxidant properties, the EtOH extract of immortelle exhibited similar potential as propyl gallate and quercetin, while the essential oil exhibited relatively weak DPPH^.‐scavenging capacity.     

Essential‐Oil Composition of Helichrysum italicum (Roth) G.Don ssp. italicum from Elba Island (Tuscany,Italy)

The composition of 21 essential‐oil samples isolated from Helichrysum italicum collected in seven locations of Elba Island (Tuscany, Italy), characterized by different soil types, during three different periods (January, May, and October 2010) was determined by GC‐FID and GC/EI‐MS analyses. In total, 115 components were identified, representing 96.8–99.8% of the oil composition. The oils were characterized by a high content of oxygenated monoterpenes (38.6–62.7%), while monoterpene and sesquiterpene hydrocarbons accounted for 2.3–41.9 and 5.1–20.1% of the identified constituents, respectively. The main oxygenated derivatives were nerol (2.8–12.8%) and its ester derivative neryl acetate (5.6–45.9%). To compare the chemical variability of the species within Elba Island and between the island and other localities within the Mediterranean area, studied previously, multivariate statistical analysis was performed. The results obtained showed a difference in the composition of the essential oils of H. italicum from Elba Island, mainly due to the environment where the plant grows, and, in particular, to the soil type. These hypotheses were further confirmed by the comparison of these oils with essential oils obtained from H. italicum collected on other islands of the Tuscan archipelago.     

Genetic variation in Mediterranean Helichrysum italicum (Asteraceae; Gnaphalieae): do disjunct populations of subsp. microphyllum have a common origin?

The yellow‐flowered everlasting daisy Helichrysum italicum (Asteraceae, Gnaphalieae) is widely distributed in the Mediterranean basin, where it grows in continuous and widespread populations in diverse open habitats. Helichrysum italicum subsp. microphyllum has a disjunct distribution in the Balearic Islands (Majorca and Dragonera), Corsica, Sardinia, Crete and Cyprus. Numerous morphological intermediates between subsp. italicum and subsp. microphyllum are known from Corsica, where the two subspecies co‐occur. The aims of the study were to investigate if subsp. microphyllum has a common origin, constituting an independent gene pool from subsp. italicum, or if the morphological differences between subsp. microphyllum and subsp. italicum have arisen independently in different locations from a common wider gene pool. Our analyses of AFLP, cpDNA sequences and morphological characters show that there is geographic structure to the genetic variation within H. italicum, with eastern and western Mediterranean groups, which do not correspond with the division into subsp. microphyllum and subsp. italicum as currently circumscribed. Local selection on quantitative trait loci provides sufficient explanation for the morphological divergence observed and is consistent with genetic data. Within the western Mediterranean group of the species we found considerable polymorphism in chloroplast DNA sequences among and within some populations. Comparison with chloroplast DNA sequences from other Helichrysum species showed that some chloroplast haplotypes are shared across species.     

Chemical Composition of the Essential Oils of Three Endemic Species of Anthemis Sect. Hiorthia (DC.) R.Fern. Growing Wild in Sicily and Chemotaxonomic Volatile Markers of the Genus Anthemis L.: An Update

The chemical composition of the essential oils isolated from the aerial parts of Anthemis pignattiorum Guarino, Raimondo & Domina and A. ismelia Lojac . and the aerial parts and flowers of Anthemis cupaniana Tod . ex Nyman , three endemic Sicilian species belonging to the section Hiorthia, was determined by GC‐FID and GC/MS analyses. (Z)‐Muurola‐4(14),5‐diene (27.3%) was recognized as the main constituent of the A. pignattiorum essential oil, together with isospathulenol (10.6%), sabinene (7.7%), and artemisyl acetate (6.8%), while in the oil obtained from the aerial parts of A. ismelia, geranyl propionate (8.8%), bornyl acetate (7.9%), β‐thujone (7.8%), neryl propionate (6.5%), and τ‐muurolol (6.5%) prevailed. α‐Pinene was the main compound of both the aerial part and flower oils of A. cupaniana (18.4 and 13.2%, resp.). Also noteworthy are the considerable amounts of artemisyl acetate (12.7%) and β‐thujone (11.8%) found in the oil from the aerial parts and those of tricosane (9.8%) and sabinene (7.6%) evidenced in the flower oil. Furthermore, an update on the main compounds identified in the essential oils of all the Anthemis taxa studied so far was presented, and cluster analyses were carried out, to compare the essential oils of these taxa.     

Evaluation of chemical composition and anti-inflammatory,antioxidant, antibacterial activity of essential oil of Sardinian Santolina corsica Jord. & Fourr.

Santolina corsica Jord. & Fourr. Corsican-Sardinian is an endemism almost present all around Corsica; in Sardinia, it can only be found in Monte Albo (calcareous substratum and poor in nutrients). The aim of our study is to investigate the chemical composition of S. corsica essential oils from plants growing in three different stations located at different altitudes and evaluate the biological activity using anti-inflammatory, antioxidant and antimicrobial test. The composition of the essential oils was determined by gas chromatography and gas chromatography/mass spectrometry. The essential oils of the Sardinian-Corsican endemism S. corsica, growing in Monte Albo, showed a great variability, probably due to genetic characters different from the Corsican type. We found three different chemotypes: artemisia ketone-β-fellendrene; myrcene and β-fellandrene-myrcene. Standard microbiological assays demonstrated that the essential oils collected in the selected stations, compared with oil and compound with demonstrated antibacterial activity, don’t have any antibacterial activity. DPPH test carried out on the tree samples, compared with chatechin, demonstrated that the oils don’t have antioxidant activity.Regarding anti-inflammatory activity the study demonstrated that the essential oils have a good anti-inflammatory activity on the bronchial tract.The addition of essential oil make easy the exocytose and the histiocytes can expel the anthracotic pigment into the culture medium, purifying its cytoplasm and restoring its ability to phagocytize more material. With a higher concentration of granulocytes in the sample, the incubation of cells shows a non-specific inflammatory pattern in which the addition of the essential oils has a positive impact on the decrease of granulocytes.More experiments are requested to confirm the data, but on the basis of these first results S. corsica essential oil showed potential activity against respiratory infections.     

Anti-bacterial activity of chitosan loaded plant essential oil against multi drug resistant K. pneumoniae

The development of antibiotic resistant in K. pneumoniae is an emerging thread worldwide due to the poor antimicrobial drugs. To overcome this issue, researchers are focused on plant material and their essential oils to fight against multi drug resistant bacteria. In this context, the current study was concentrated in medicinal plant of guva leaves and their essential oils to combat multi drug resistant bacterial infections. The essential oils were successfully screened and confirmed by HRLC-MS analysis. The anti-bacterial ability of the compounds were loaded into the chitosan nanoparticles and proved by FT-IR analysis. In addition, the chitosan loaded essential oils morphology was compared with chitosan alone in SEM analysis and suggested that the material was loaded successfully. Further, the anti-bacterial ability of the chitosan loaded essential oils were primarily confirmed by agar well diffusion method. At the 100 µg/mL of lowest concentration of chitosan loaded essential oils, the multi-drug resistant K. pneumoniae was inhibited with 96% and confirmed by minimum inhibition concentration experiment. Hence, all the experiments were proved that the essential oils were successfully loaded into the chitosan nanoparticles, and it has more anti-bacterial activity against multi-drug resistant K. pneumoniae.     

Assessment of Daucus carota L. (Apiaceae) subspecies by chemotaxonomic and DNA content analyses

For a clearer distinction between the four subspecies of Daucus carota native from Portugal (subsp. carota, subsp. maximus, subsp. gummifer and subsp. halophilus), morphological features of the fruits, DNA content analyses by flow cytometry, and chemical characterization of the essential oils were undertaken.We found chemotaxonomic evidences to consider D. carota subsp. maximus as a separate species rather than a subspecies of D. carota. This separation is based on the morphometric analysis of the fruits and in the high levels of asarone present only in the essential oil of the subsp. maximus. The remaining subspecies are difficult to distinguish from each other based on the morphology of the fruits and in DNA content. However, based on the essential oils, it was possible to distinguish the subspecies halophilus from the other two (subsp. gummifer and subsp. carota) because of its high content of elemicin, with the other two having high levels of geranyl acetate.Based on these results, the subspecies maximus is proposed as a different species (Daucus maximus Desf.) and the taxonomic status of other three subspecies (subsp. carota, subsp. gummifer and subsp. halophilus) is maintained. Still, the latter three taxa need to be further studied for a more precise taxonomic characterization.     

Essential oil polymorphism of Thymus praecox subsp. arcticus on the British Isles

The essential oils of 732 individual plants of Thymus praecox Opiz subsp. arcticus (E. Durand) Jalas (syn. T. drucei Ronn.) collected in Scotland, Ireland, and in the south of England have been analysed by gas chromatography (GC) and mass spectrometry (GC–MS) in order to elucidate the chemical character of this subspecies on the British Isles. In total, 69 components were identified, most of them monoterpenoids and sesquiterpenoids with hedycaryol, linalyl acetate, linalool, the germacradienols, trans-nerolidol, T-cadinol, and β-caryophyllene being the most important compounds. The analysis of the quantitative essential oil data by means of neural networks revealed that T. praecox subsp. arcticus growing in Britain is highly polymorphous. There were 17 chemotypes with the hedycaryol chemotype as the most frequent (24% of the plants), followed by the linalool/linalyl acetate chemotype (22% of the plants) and germacra-1(10),4-dien-6-ol chemotype (18% of the plants). It seems that each part of the British Isles has its special chemotype pattern with 13 chemotypes in Scotland, 11 in Ireland, and 17 in the south of England. An overview of the North Atlantic region of Europe revealed that the polymorphism of T. praecox subsp. arcticus in the essential oil is more distinctive in the southern than in the northern regions, with only 2, 5, and 1 chemotypes in Greenland, Iceland, and Norway, respectively.     

Achillea filipendulina Lam.: Chemical Constituents and Antimicrobial Activities of Essential Oil of Stem,Leaf, and Flower

In this study, we extracted the essential oils of the stem, leaf, and flower of Achillea filipendulina, analyzed them, and studied their antibacterial properties. Of 16, 53, and 35 compounds identified in the stem, leaf, and flowers, respectively, only five are present in all three segments of the plant. The essential oil of the stem was mainly composed of neryl acetate, spathulenol, carvacrol, santolina alcohol, and trans‐caryophyllene oxide. However, the main identified components of leaf were 1,8‐cineole, camphor, ascaridole, trans‐isoascaridole, and piperitone oxide and the main components of the flower oil were ascaridole, trans‐isoascaridole, 1,8‐cineole, p‐cymene, and camphor. The extracted oil from different segments demonstrated varying antibacterial properties against both Gram‐positive and Gram‐negative bacteria, demonstrated by disk, minimum inhibitory concentration, and minimum bactericidal concentration methods. These suggest that the application of all segments of aerial parts of A. filipendulina may have a better therapeutic effect in fighting pathogenic systems.     

Studies on Leaf Oils of Citrus Species

Leaf oil samples of four Japanese citrus species were analysed by gas chromatography to determine the detailed composition of each leaf oil. The following components were identified: α-pinene, α-thujene, camphene, β-pinene, sabinene, β-myrcene, α-terpinene, limonene, β-phellandrene, trans-2-hexen-1-al, γ-terpinene, p-cymene, terpinolene, cis-2-penten-1-ol, n-hexyl alcohol, cis-3-hexen-1-ol, trans-2-hexen-1-ol, p-α-dimethylstyrene linalool, linalyl acetate, β-elemene, terpinen-4-ol, caryophyllene, humulene, α-terpineol, neryl acetate, geranyl acetate, β-selinene, geraniol and thymol. Most components were contained in common in leaf oils of the four citrus species, but relative contents of some of the components; such as γ-terpinene, linalyl acetate, and thymol differed from species to species. For example, γ-terpinene was the major component (33.8%) of Hassaku, whereas it was only a minor component in Daidai. Daidai is characterized by a very high content of linalyl acetate (35%) which is only a trace in the other three species. Kishu-mikan is characterized by a high content of thymol (15%).     

Essential oil chemical diversity of twenty Iranian Origanum vulgare L. subsp. viride populations

Origanum vulgare L. belonging to the Lamiaceae family, commonly known as oregano, is one of the most traded and consumed medicinal plants worldwide. It is a rich natural source of specialized metabolites including essential oils. The present study is the first comprehensive account of the essential oil profile of wild O. vulgare subsp. viride plants in Iran. Twenty native populations from four provinces of northern Iran were investigated for the content and composition of essential oils. Essential oil contents were found to vary from 0.1 to 0.5% (v/w) based on dry weight. Analysis of these oils by GC-FID and GC-MS resulted in a total of forty-nine constituents, representing 96.2–99.7% of the total compositions. The subsequent classification of the studied populations by multivariate statistical procedures characterized four chemotypes: i) γ-terpinene/p-cymene; ii) linalool/α-phellandrene; iii) sabinene; and iv) carvacrol/trans-caryophyllene. These data may be useful in the future selection of populations comprising specific chemical characteristics for industrial purposes, and furthermore contribute beneficial information for developing conservation strategies, domestication and breeding programs of Iranian O. vulgare subsp. viride germplasm.     

Nanoencapsulation (in vitro and in vivo) as an efficient technology to boost the potential of garlic essential oil as alternatives for antibiotics in broiler nutrition

The addition of essential oil (EO) as chitosan encapsulated can increase the efficiency of these oils in broiler feeding. Therefore, the objective of the current research was to explore the antibacterial and antioxidant potential of garlic essential oil (GEO) (free vs. nanoencapsulated) and their effects on performance, gene expression of mucin2, microbial, and morphology of intestine in broilers. A total of 900 1-day-old male broilers (Ross 308) were assigned to six dietary treatments (0, 100, and 200 mg/kg free GEO and 0 (contain of chitosan), 100 and 200 mg/kg nanoencapsulated GEO) with a 2 × 3 factorial arrangement based on completely randomized design. Garlic essential oil encapsulation with chitosan significantly enhanced antibacterial and antioxidant parameters. At 100 mg/kg nanoencapsulated GEO had significant (P < 0.01) advantages in improving BW gain (BWG) (22–42 and 0–42) and feed conversion ratio (FCR) (0–42). Maximum feed intake (FI) was also associated with the control group (P < 0.05). Broilers fed on 100 mg/kg of nanoencapsulated GEO showed higher villi length and width relative to other treatments and villi length to crypt depth ratio as well (P < 0.01). The nanoencapsulation process of GEO (P < 0.01) affected the Lactobacilli population in the digesta of ileo-caecum and mucin2 gene expression. In broiler chickens, the tested EO, especially nanoencapsulated type, enhanced more evaluated parameters. Because of its ideal properties, nanoencasulating with chitosan may also be an effective and inexpensive way to protect bioactive compounds and improve GEO effects in broiler chickens.     

Chemical Composition,Toxicity and Repellency of Inula graveolens Essential Oils from Roots and Aerial Parts against Stored-Product Beetle Tribolium castaneum (Herbst)

In this work, essential oils extracted from roots and aerial parts of Inula graveolens by hydrodistillation and their fractions obtained by chromatographic simplification were first investigated for their chemical composition by GC/MS and then evaluated for the first time for their repellency and contact toxicity properties against Tribolium castaneumadults. Twenty-eight compounds were identified in roots essential oil (REO), which accounted for 97.9 % of the total oil composition, with modhephen-8-β-ol (24.7 %), cis-arteannuic alcohol (14.8 %), neryl isovalerate (10.6 %) and thymol isobutyrate (8.5 %) as major constituents. Twenty-two compounds were found in the essential oil from aerial parts (APEO), which accounted for 93.9 % of the total oil, with borneol (28.8 %), caryophylla-4(14),8(15)-dien-6-ol (11.5 %), caryophyllene oxide (10.9 %), τ-cadinol (10.5 %) and bornyl acetate (9.4 %) as main compounds.REO and APEO displayed stronger repellency after 2 h of exposure (80.0 and 90.0 %, respectively) against T. castaneum at the concentration of 0.12 μL/cm². After fractionation, fractions R₄ and R₅ exhibited greater effects (83.3 % and 93.3 %, respectively) than the roots essential oil. Furthermore, the fractions AP₂ and AP₃ showed higher repellency (93.3 and 96.6 %, respectively) than the aerial parts oil. The LD₅₀ values of oils from roots and aerial parts topically applied were 7.44 % and 4.88 %, respectively. Results from contact toxicity assay showed that fraction R₄ was more effective than the roots oil with LD₅₀ value of 6.65 %. These results suggests that essential oils of roots and aerial parts from I. graveolens may be explored as potential natural repellent and contact insecticides against T. castaneum in stored products.     

Chemical composition of volatiles from cortical oleoresin of Pseudotsuga menziesii

Pseudotsuga menziesii cortical oleoresin was found to contain 1·7% of oxygenated terpenoids and compounds of similar volatility composed of linalool, methylsalicylate, bornyl acetate, citronellol, geranyl acetate, methylthymol, citronellyl acetate, terpinen-4-ol, borneol, isopulegol, anethole, terpinen-4-ol acetate, camphor, geraniol, neryl acetate, and nerol. Sesquiterpenoid hydrocarbons were low (only 0·07%) and contained sibirene and longifolene as main constituents, with β-caryophyllene, γ-muurolene, γ-cadinene (identified by IR), and 20 additional compounds in small amounts. p-Cymen-8-ene was identified in monoterpene hydrocarbon fraction.     

Chemotaxonomic study on Thymus villosus from Portugal

The composition of the essential oils of four populations of Thymus villosus subsp. lusitanicus (Boiss.) Coutinho from Portugal was investigated by GC and GC-MS. To study the chemical polymorphism the results obtained from GC analyses of the volatile oils from individual plants from four populations were submited to Principal Component and Cluster analyses. A comparision with the essential oil of T. villosus subsp. villosus, previously studied by us was done. Important differences with regard to the major constituents in these two taxa were found. Linalool, geranyl acetate, geraniol and terpinen-4-ol were the main components of the essential oils of T. villosus subsp. lusitanicus, whereas in the oil of T. villosus subsp. villosus p-cymene, myrcene and alpha-terpineol were the major ones. Although, both taxa showed chemical polymorphism, different types of essential oils were characterized in each one: linalool; linalool/ terpinen-4-ol/trans-sabinene hydrate; linalool/1,8-cineole; geranyl acetate/geraniol; geranyl acetate/geraniol/1,8-cineole in T. villosus subsp. lusitanicus and p-cymene/camphor/linalool; p-cymene/borneol; linalool/geraniol/geranyl acetate; alpha-terpineol/camphor/myrcene in T. villosus subsp. villosus. Thus, the two subspecies of T. villosus can be easely differenciated by the composition of their essential oils.     

Biopesticidal value of selected essential oils against pathogenic fungus, termites, and nematodes

The biopesticidal potential of six plant-derived essential oils (mint [Mentha arvensis], ajwain [Carum capticum], lemongrass [Cymbopogon citrates], clove [Eugenia caryophyllata], cedarwood [Cedrus deodara], and eucalyptus [Eucalyptus globulas]) was evaluated against Odontotermes obesus (termites), Fusarium oxysporum (plant pathogenic fungi), and Meloidogyne incognita (nematodes). In the case of termites, a “no-choice” bioassay revealed that the mint oil gave the best results (100% mortality in 30 min with 10% oil and in 10 h with 0.12% oil) followed by the lemongrass and ajwain oils. The disc diffusion method was adopted to test the anti-fungal activity of the essential oils and it was found that the clove oil gave the maximum inhibition measured in terms of the average inhibition zone diameter (5.3 ± 0.2 cm with 10% oil and 6.6 ± 0.9 cm with 20% oil), followed by the ajwain oil. To check the anti-nematicidal activity of the essential oil, in-vitro growth chamber experiments revealed that eucalyptus oil was the most efficient (100% mortality in 6 h with 1000 ??l l⁻¹ oil and in 30 h with 125 ??l l⁻¹ oil), followed by the ajwain oil. The use of the crude oils at low concentrations provided satisfactory results at the laboratory level against these pathogens, and needs further evaluation in field trials.     

Ecotypic variation in plant characteristics for Origanum vulgare subsp. hirtum populations

Origanum vulgare L. subsp. hirtum (Link) Ietsw. is a polymorphic taxon with respect to essential oil production and glandular trichome density. Here it is examined whether the natural populations that are indigenous in continental Greece may be considered as different ecotypes (i.e. populations with different genetic variation) and whether evidence regarding a fitness cost from essential oil production could be obtained. Samples from 30 different natural populations, differing in climatic conditions, were collected across continental Greece in 1998–2000 and were studied for essential oil production and glandular trichome density. Additionally, individual plants, derived from propagated rhizomes, originated from the natural populations, were transplanted in pots in two gardens that varied in terms of mean temperature and aridity. In these experimental populations, the above-ground phytomass and ratio of leaves and inflorescences to above-ground phytomass were additionally measured. The main findings of the study were (a) a strong positive correlation between essential oil concentration and glandular trichome density, (b) a high positive correlation between natural and experimental populations in essential oil concentration (or glandular trichome density), (c) a negative correlation between essential oil concentration and phytomass and (d) a positive relationship between the essential oil concentration and the factors thermal status and summer drought of the location of origin. Results suggest that the examined populations of O. vulgare subsp. hirtum are different ecotypes, the production of essential oils has a cost to plant growth and that the prime target of selection, by producing these oils, is to increase the resistance to drought and heat stresses.     

Essential oil in Betula spp. leaves naturally growing in Estonia

The essential oils of the leaves of Betula pendula Roth., Betula pubescens Ehrh., Betula humilis Schrank and Betula nana L. (Betulaceae), and B. pendula buds were obtained using hydrodistillation and their chemical compositions were analyzed using gas chromatography and gas chromatography – mass spectrometry. The yield of essential oil was the highest (0.27%) in leaves of. B. humilis and the lowest in the leaves of B. pendula (0.11%), B. pubescens (0.05%) and B. nana (traces). Overall, 35–60 compounds were identified representing more than 92% of the total volume of the oils investigated. The bicyclical sesquiterpenoids found in the essential oils extracted from the leaves of B. pendula, B. pubescens and B. humilis and from the buds of B. pendula where α-betulenol (20.4–33.1%), α-betulenol acetate (5,6–28.9%), β-betulenal (5.5–6.2%) and β-betulenol (2.0–5.8%). The buds of B. pendula contained more birkenal, (E)-β-caryophyllene, δ-cadinene and caryophyllene oxide, but less α-betulenol acetate, β-betulenal and aliphatic compounds than the oils extracted from the leaves investigated. The oil composition of B. humilis and B. nana leaves was studied for the first time. The content of essential oil of B. nana leaves was much more varied: the oil contained less bicyclical sesquiterpenoids and more aliphatic compounds than the other Betula species investigated. The findings of our study could be used in chemosystematics of birch species, especially for B. nana.     

Essential oils of five Tanacetum vulgare genotypes

The essential oils of five Tanacetum vulgare genotypes were investigated and their main components identified. Artemisia alcohol, γ-campholenol, davanone, lyratol, lyratyl acetate and 4-thujen-2α-yl acetate have not been reported before as constituents of Tanacetum vulgare. This is the first time that γ-campholenol has been isolated from a natural source. 4-Thujen-2α-yl acetate is a novel compound.     

Glandular trichomes and essential oil characteristics of in vitro propagated <Emphasis Type="Italic">Micromeria pulegium</Emphasis> (Rochel) Benth. (Lamiaceae)

Main conclusion

In vitro conditions and benzyladenine influenced both content and composition of micropropagated Micromeria pulegium essential oils, with pulegone and menthone being the main essential oil components. The content and chemical composition of Micromeria pulegium (Rochel) Benth. essential oils were studied in native plant material at vegetative stage and in micropropagated plants, obtained from nodal segments cultured on solid MS medium supplemented with N⁶–benzyladenine (BA) or kinetin at different concentrations, alone or in combination with indole-3-acetic acid. Shoot proliferation was achieved in all treatments, but the highest biomass production was obtained after treatment with 10 μM BA. Phytochemical analysis identified up to 21 compounds in the essential oils of wild-growing and in vitro cultivated plants, both showing very high percentages of total monoterpenoids dominated by oxygenated monoterpenes of the menthane type. Pulegone and menthone were the main essential oil components detected in both wild-growing plants (60.07 and 26.85 %, respectively) and micropropagated plants grown on either plant growth regulator-free medium (44.57 and 29.14 %, respectively) or BA-supplemented medium (50.77 and 14.45 %, respectively). The percentage of total sesquiterpenoids increased in vitro, particularly owing to sesquiterpene hydrocarbons that were not found in wild-growing plants. Differences in both content and the composition of the essential oils obtained from different samples indicated that in vitro culture conditions and plant growth regulators significantly influence the essential oils properties. In addition, the morphology and structure of M. pulegium glandular trichomes in relation to the secretory process were characterized for the first time using SEM and light microscopy, and their secretion was histochemically analyzed.