Phytomorphological and Essential‐Oil Characterization in situ and ex situ of Wild Biotypes of Oregano Collected in the Campania Region (Southern Italy)

Oregano is an aromatic species of great interest, which spreads spontaneously over the Mediterranean area, but its genetic resources are not yet adequately developed. Moreover, the results of studies of wild strains of different origin – although quite numerous – are not always comparable, and, therefore, the influence of the environment and genotype on the variability cannot be distinguished. Hence, the plant morphology and the essential‐oil composition of three wild, white‐flowering biotypes of oregano collected in the Campania region (southern Italy) were characterized, and the effects of genotype and environment were assessed by comparison in situ and ex situ. This allowed deducing that the biotypes belonged to two different subspecies, i.e., Origanum vulgare ssp. virens and O. vulgare ssp. viridulum. The essential‐oil yield was higher for the biotype belonging to ssp. virens, and it was significantly correlated with the glandular and stomatal density. The chemical composition of the oils obtained by hydrodistillation was found to be influenced by the genotype and the conditions of plant growth.     

Chemical Diversity in Indian Oregano (Origanum vulgare L.)

The terpenoid composition of the essential oils of 17 different populations of Origanum vulgare L., collected from wild populations and subsequently grown under similar conditions in the sub‐temperate region of the Western Himalaya, was studied. Analysis by GC (RI) and GC/MS allowed the identification of 51 components, representing 90.15 to 99.94% of the total oil. The two classes of the phenolic compounds and the monoterpenoids were predominant in all the essential oils. On the basis of the major constituents, i.e., marker compounds, and by comparison of the results with previous reports, new chemotypes could be identified. Principal component analysis was performed to determine the chemical variability within the different populations of O. vulgare collected and grown under similar conditions. Based on the marker compounds, six chemotypes with significant variations in their terpenoid profile were noticed within the 17 populations.     

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.     

Characterization of Secondary Metabolites,Biological Activity and Glandular Trichomes of Stachys tymphaea Hausskn. from the Monti Sibillini National Park (Central Apennines,Italy)

Stachys tymphaea (Lamiaceae) is a perennial herb growing in forest openings and dry meadows of central and southern Italy. It was investigated for the first time here, determining the content of secondary metabolites, the micromorphology of glandular trichomes, the histochemical localization of secretion, and the biological activity of the volatile oil, namely, the cytotoxic, antioxidant, and antimicrobial properties. The plant showed a peculiar molecular pattern, being rich of biophenolic compounds as flavonoids, phenylethanoid glycosides, and caffeoylquinic acid derivatives, but poor of iridoids, which are known as marker compounds of the genus Stachys. The essential oil was characterized by GC‐FID and GC/MS analyses, revealing a high percentage of sesquiterpene hydrocarbons (54.6%), with germacrene D (30.0%) and (E)‐β‐farnesene (12.4%) as the most abundant compounds, while other main components were representatives of the diterpenes (19.2%), represented mainly by (E)‐phytol (11.9%). This composition supported the taxonomic relationships in the genus Stachys, which comprises oil‐poor species producing essential oils rich in hydrocarbons, with germacrene D as one of the predominant components. The micromorphological study revealed three types of glandular hairs, i.e., Type A peltate trichomes, being the primary sites of essential oil biosynthesis, Type B short‐stalked trichomes, typical mucopolysaccharide producers, and Type C long capitate trichomes, secreting a complex mixture of both lipophilic and hydrophilic substances, with a major phenolic fraction. Moreover, the MTT assay revealed the potential of the volatile oil to inhibit A375, HCT116, and MDA‐MB 231 tumor cells lines (IC₅₀ values of 23.9–34.4 μg/ml).     

Glandular trichomes as a barrier against atmospheric oxidative stress: Relationships with ozone uptake,leaf damage,and emission of LOX products across a diverse set of species

There is a spectacular variability in trichome types and densities and trichome metabolites across species, but the functional implications of this variability in protecting from atmospheric oxidative stresses remain poorly understood. The aim of this study was to evaluate the possible protective role of glandular and non‐glandular trichomes against ozone stress. We investigated the interspecific variation in types and density of trichomes and how these traits were associated with elevated ozone impacts on visible leaf damage, net assimilation rate, stomatal conductance, chlorophyll fluorescence, and emissions of lipoxygenase pathway products in 24 species with widely varying trichome characteristics and taxonomy. Both peltate and capitate glandular trichomes played a critical role in reducing leaf ozone uptake, but no impact of non‐glandular trichomes was observed. Across species, the visible ozone damage varied 10.1‐fold, reduction in net assimilation rate 3.3‐fold, and release of lipoxygenase compounds 14.4‐fold, and species with lower glandular trichome density were more sensitive to ozone stress and more vulnerable to ozone damage compared to species with high glandular trichome density. These results demonstrate that leaf surface glandular trichomes constitute a major factor in reducing ozone toxicity and function as a chemical barrier that neutralizes the ozone before it enters the leaf.     

Chemotaxonomic and Micromorphological Traits of Satureja montana L. and S. subspicata Vis. (Lamiaceae)

Satureja montana and S. subspicata are used as spice, pepper substitute, for preparing tea, juice, and as a medicine. Fourteen populations (seven per species) of Satureja montana L. and S. subspicata Vis . growing in Croatia were examined to determine the chemical composition of the essential oil (analyzed by GC‐FID and GC/MS), the content of macroelements (Na, K, Ca, Mg) and trace elements (B, Fe, Cu, Mn, Zn, Al, Pb, Cr, Cd, Ni, Hg, As) analyzed by ICP‐AES, antioxidant compounds (analyzed by UV/VIS spectrophotometer), and the types and distribution of trichomes (analyzed by scanning electron microscopy). The main constituents of the essential oil were carvacrol and thymol in S. montana (all populations belong to one phenol chemotype), while α‐eudesmol, β‐eudesmol, and spathulenol dominated in S. subspicata (three chemotypes could be distinguished). Both species possess considerably higher quantities of Ca and Mg, and moderate concentrations of K and Na, while Hg and As levels were below the limit of quantification. Non‐glandular trichomes, peltate trichomes, and three types of capitate trichomes were observed on leaves, stem, calyx, and corolla.     

The Essential Oils and Glandular Hairs in Different Chemotypes of Origanum vulgare L.

Leaves and flowers of four chemotypes of Origanum vulgare L.were examined for the main components of their essential oiland for the types and distribution of their glandular hairs.Two varieties have high phenol content, one thymol and the othercarvacrol, in their essential oils; one has a moderate thymolcontent and the fourth has a low phenol content and a high alcoholcontent. The percentage of essential oil and the number of peltatehairs were higher in the flowers than in the leaves, the highestbeing in the flowers of a chemotype with a high phenol (thymol)concentration. While there were no differences in structureof the peltate and two types of capitate hairs between chemotypes,the density of the peltate hairs varied and appeared to be correlatedwith the total essential oil content. Origanum vulgare L., essential oils, glandular hairs     

Glandular trichomes and essential oil constituents ofCalamintha menthifolia (Lamiaceae)

Plant material ofCalamintha menthifolia was collected from two populations grown in the Vikos-Aoos area (NW. Greece). The structure and ontogeny of the glandular trichomes and the chemical composition of the secreted essential oil were studied. The leaves ofC. menthifolia have numerous glandular trichomes of three morphological distinct types: 1) glandular scales with a unicellular foot and stalk, and a 12-celled head, 2) glandular hairs with a unicellular foot and stalk, and a unicellular pear-shaped head and 3) glandular hairs with a unicellular foot, a bicellular stalk and a unicellular elongated head. Qualitative and quantitative GC-MS analyses of the essential oils revealed piperitone oxide as the main constituent in both populations. In the light of these results the relationships beweenC. menthifolia and other members of theSatureja group as well as its taxonomy are discussed.     

Micromorphological and chemical characterisation of Stachys recta L. subsp. serpentini (Fiori) Arrigoni in comparison to Stachys recta L. subsp. recta (Lamiaceae)

Stachys recta L. is a very polymorphous species in which numerous subspecies were recognised. S. recta L. subsp. serpentini (Fiori) Arrigoni is a typical endemism growing on serpentine soils in northern Apennines and particularly in Tuscany (Italy). In order to contribute to a better knowledge of this plant and to its differentiation with respect to S. recta L. subsp. recta, the micromorphological characters (non-glandular and glandular trichomes) and the essential oil composition of the two subspecies were investigated. Micromorphological characters were studied using scanning and transmission electron microscopy, while light microscopy was used for histochemical observations. Essential oil analysis was carried out by gas chromatography and mass spectrometry.In the two examined taxa, the morphology and distribution of glandular and non-glandular trichomes, and the different essential oil composition, may be considered distinctive characters at subspecies level. This is consistent with the taxonomic classification considering S. recta subsp. serpentini a subordinate taxon of S. recta.     

Ficus carica L. leaf anatomy: Trichomes and solid inclusions

Ficus carica L., a typical plant of the Mediterranean environment, presents leaves covered by an extensive indumentum, and a mesophyll full of solid inclusions. The morphology and ultrastructure of the trichomes, calcium carbonate cystoliths and silicified structures of leaves of F. carica cv Dottato were investigated with light, confocal, scanning electron microscopy, and energy-dispersive X-ray spectroscopy. At the same time, histochemical reactions were also employed to analyse the indumentum composed by glandular and non-glandular trichomes by applying chemical reagents and fluorescence microscopy. Non-glandular and glandular trichomes, capitate, are described. Non-glandular trichomes are unicellular simple, spine-like and present different morphology and sizes. The capitate glandular trichomes are present on leaf adaxial and abaxial surface and consist of one-celled stalk and 3/4 cells spherical head. Histochemical characterisation of leaf hairs revealed the presence of flavonoids, while glandular trichome head cells showed a complex mixture of alkaloids, essential oil and flavonoids. Cu and Al were found in the constitutive structures, spike and dome, of the cystoliths. Several epidermal cells and non-glandular trichomes were silicified. Leaf hairs, trichomes secretions, solid inclusions and silicification of F. carica leaf have significant roles to play in relation to leaf protection from external factors, including high-intensity radiation, herbivores or pathogens.     

A Comparative Study on Essential Oil Intraspecific and Seasonal Variations: Melissa romana Mill. and Melissa officinalis L. from Sardinia

Two species of Melissa are currently present in Sardinia: Melissa officinalis L. and Melissa romana Mill . Our research can only count on a few supported evidences (as reported in Flora Italiana and Moris) and some notes on new stations in Sardinia that give us some information about morphology, distribution, bioecological, and ethnobotanical characteristics of both species. In this paper, we present the results of the research about morphological aspects of M. romana vs. M. officinalis, and their essential oils in different stations at different phenological periods. Moreover, we compared the essential oil of M. romana with the one obtained from M. officinalis growing in the few naturalized stations still present in Sardinia. The most evident morphological differences between the two entities are the long‐stalked capitate glandular trichomes, shorts and inclined capitate trichomes, and peltate hairs. The chemical composition of essential oil presents several significant differences between the species. In fact, oils show that in none of the phenological stages, M. romana recalls in its composition M. officinalis. Major distinctions are also evident between dry and fresh plants, and among essential oils distilled in different seasons.     

Humulus lupulus L. cv. Cascade grown in Northern Italy: morphological and phytochemical characterization

Abstract

Several aroma hops (Humulus lupulus L.) were recently introduced in Northern Italy as a small-scale production of excellence. In this preliminary study, the American cultivar Cascade was investigated in a combined morphological and phytochemical survey. Morphological investigation on trichome structure, density and distribution was performed by scanning electron microscopy and light microscopy. Essential oil composition, α/β-acid and polyphenol profiles over 3 years were determined by GC-MS and HPLC analyses. Two types of non-glandular (simple and cystolithic) and glandular (peltate and bulbous) trichomes were observed on leaves and female inflorescences. The peltate trichomes resulted as the main sites of terpene production and accumulation. The essential oil profiles showed myrcene, β-caryophyllene, (E)-β-farnesene and humulene epoxide II as the dominant compounds over the three collection times, although with different relative abundances. The presence of two exclusive compounds, γ-muurolene and trans-γ-cadinene, characterized the investigated cv. Cascade, potentially enhancing herbal, woody and spicy aroma traits of this cultivation in Northern Italy. The bitter acid composition showed quantitative values consistent with literature data only for the second and third monitoring year. Qualitative differences in polyphenol content were also recorded, for the presence of quercetin-3-O-malonylglucoside and kaempferol-3-O-rutinoside, which may characterize this peculiar Italian cultivation.     

A New Histochemical Method for Localization of the Site of Monoterpene Phenol Accumulation in Plant Secretory Structures

A new method is reported for the histochemical localizationof monoterpene phenols in essential oil secretory structures.The method was adapted from a spot test originally devised forin vitro detection of phenolic compounds in organic analyses.Plant subjects were the Lamiaceae species Thymus vulgaris L.,Oreganum vulgare L. and Mentha x piperita L., which accumulateessential oil in glandular trichomes. A reagent consisting of4-nitrosophenol in conc. H₂SO₄was applied to sample leaves ofeach species. A positive test for phenol was indicated by theproduction of coloured indophenols. Using this method, monoterpenephenols were identified in the trichomes of T. vulgaris(thymol)and O. vulgare(carvacrol), indicated by colour changes to redand green respectively. No phenol was detected in trichomesof M. x piperita. Results were confirmed by GC-MS analysis ofleaf volatile extracts from each species, and in vitro testswith thymol and carvacrol. The method could be used in fieldsurveys for rapid identification of potential medicinal plantsand bioactive compounds. Copyright 2001 Annals of Botany Company Histochemistry, secretory structures, glandular trichomes, Lamiaceae, Thymus vulgaris, Oreganum vulgare, Mentha x piperita, essential oil, aromatic monoterpenes     

Volatiles of Geranium purpureum Vill. and Geranium phaeum L.: Chemotaxonomy of Balkan Geranium and Erodium Species (Geraniaceae)

The essential oils obtained by hydrodistillation of Geranium purpureum and G. phaeum were characterized by GC‐FID and GC/MS analyses (the former for the first time in general). In total, 154 constituents were identified, accounting for 89.0–95.8% of the detected GC peak areas. The investigated essential oils consisted mainly of fatty acids and fatty‐acid‐derived compounds (45.4–81.3%), with hexadecanoic acid and (E)‐phytol as the major components. The chemotaxonomic significance of the variations in the essential‐oil composition/production of the presently and previously investigated Geranium and highly related Erodium taxa from Serbia and Macedonia was assessed by multivariate statistical analyses. The main conclusions drawn from the high chemical similarity of the two genera, visible from the obtained dendrograms and biplots, confirm the close phylogenetic relationship between the investigated Geranium and Erodium taxa, i.e., that there is no great intergeneric oil‐composition variability. Changes in the composition and production of essential oils of the herein investigated taxa and 60 other randomly chosen species belonging to different plant genera were also statistically analyzed. The results put forward pro arguments for the oil‐yield–oil‐composition correlation hypothesis.     

Comparative Chemical and Molecular Variability of Cananga odorata (Lam.) Hook.f. & Thomson forma genuina (Ylang‐Ylang) in the Western Indian Ocean Islands: Implication for Valorization

Cananga odorata (Lam.) Hook .f. & Thomson forma genuina (Annonaceae) is a tropical tree, grown for the production of ylang‐ylang essential oil, which is extracted from its fresh and mature flowers. Despite its economic and social importance, very little information is available on its variability and the possible factors causing it. Therefore, the relationship between the genetic structure, revealed by amplified fragment length polymorphism (AFLP), and the essential oil chemical composition, determined by GC/MS analysis, of ylang‐ylang grown in semi‐managed systems in three Indian Ocean islands (Grande Comore, Mayotte, and Madagascar) was investigated. Our results revealed a low genetic variation within plantations and contrasted situations between islands. Variations of the chemical composition could be observed within plantations and between islands. The genetic differentiation pattern did not match the observed pattern of chemical variability. Hence, the chemical variation could not be attributed to a genetic control. As Grande Comore, Madagascar, and Mayotte present different environmental and agronomic conditions, it can be concluded that the influence of these conditions on the ylang‐ylang essential oil composition is consistent with the patterns observed. Finally, several strategies were proposed to valorize the chemical composition variations.     

Anthelmintic Effects of the Essential Oil of Fennel (Foeniculum vulgare Mill., Apiaceae) against Schistosoma mansoni

Foeniculum vulgare Mill . (Apiaceae), known as fennel, is a widespread aromatic herbaceous plant, and its essential oil is used as additive in the food, pharmaceutical, cosmetic, and perfume industries. The in vitro antischistosomal activity and cytotoxic effects against V79 cells of the essential oil of F. vulgare cultivated in southeastern Brazil (FV‐EO) was investigated. The FV‐EO was obtained by hydrodistillation and characterized by GC‐FID and GC/MS analyses. (E)‐Anethole (69.8%) and limonene (22.5%) were identified as the major constituents. Its anthelmintic activity against Schistosoma mansoni was evaluated at concentrations of 10, 50, and 100 μg/ml, and it was found to be active against adult S. mansoni worms, although it was less effective than the positive control praziquantel (PZQ) in terms of separation of the coupled pairs, mortality, and decreased motor activity. However, FV‐EO elicited an interesting dose‐dependent reduction in the number of S. mansoni eggs. On their own, (E)‐anethole and the limonene enantiomers were much less effective than FV‐EO and PZQ. An XTT‐cytotoxicity‐based assay evidenced no FV‐EO cytotoxicity against V79 cells. In summary, FV‐EO displayed moderate in vitro schistosomicidal activity against adult S. mansoni worms, exerted remarkable inhibitory effects on the egg development, and was of low toxicity.     

Developmental plasticity in birch leaves: defoliation causes a shift from glandular to nonglandular trichomes

The structures on leaf surfaces, e.g. trichomes, can act as effective antiherbivory mechanisms as chemical repellents. Structural defences usually represent constitutive resistance, but there are also a few cases of inducible morphological defences. We tested whether defoliation may induce changes in trichome production in white birch (Betula pubescens). The studied birches were either 0, 50 or 100% defoliated during the previous or current summer, and we measured the alterations in the production of glandular vs. nonglandular leaf trichomes, developmental instability (fluctuating asymmetry, FA) and leaf and shoot growth. We detected a clear shift from glandular to nonglandular leaf trichomes following previous‐year defoliation but not after current‐year defoliation. Furthermore, the density of nonglandular trichomes around the mid‐vein of leaves increased following previous‐year defoliation but decreased after current‐year defoliation. While leaf and shoot growth showed a distinct decrease in response to defoliation, FA turned out to be less sensitive. Consequently, previous‐year defoliation can induce the production of nonglandular trichomes in birch leaves. Because this response was accompanied by a reduction in glandular trichomes, the present results may suggest a trade‐off between the different trichome types of birch leaves.     

Role of leaf glandular trichomes of melon plants in deterrence of Aphis gossypii Glover

External characteristics of the leaf epidermis and their effects on behaviour of Aphis gossypii Glover were evaluated in two Cucumis melo L. genotypes, ‘Bola de Oro’ (aphid susceptible) and TGR‐1551 (aphid resistant) in order to explore their role in the early rejection of TGR‐1551 by this aphid. No differential effects of epicuticular waxes on aphid behaviour were observed. The type, distribution and number of trichomes on melon leaves were also studied. Pubescence in melon, measured as the number of non‐glandular trichomes per cm², was not sufficient to prevent aphid settling. However, there was a high density of type I glandular trichomes on leaves of the aphid‐resistant genotype. According to microscopic observations and stain testing, these trichomes store and secrete phenols and flavonoids. Free‐choice tests were conducted to determine the effect of these glandular trichomes on A. gossypii preference, revealing that aphids reject leaf disks of TGR‐1551 from the onset of the experiment. Additional experiments after removal of leaf type I glandular trichome exudates showed that A. gossypii preferred washed TGR‐1551 leaf disks over unwashed disks, while this effect was not observed in experiments using washed and unwashed ‘Bola de Oro’ leaf disks. These results suggest that a high density of glandular trichomes and chemicals secreted by them deter A. gossypii and disturb aphid settling on TGR‐1551.     

Essential Oil Composition of Phagnalon sordidum (L.) from Corsica,Chemical Variability and Antimicrobial Activity

The chemical composition of Phagnalon sordidum (L.) essential oil was investigated for the first time using gas chromatography and chromatography/mass spectrometry. Seventy‐six compounds, which accounted for 87.9% of the total amount, were identified in a collective essential oil of P. sordidum from Corsica. The main essential oil components were (E)‐β‐caryophyllene (14.4%), β‐pinene (11.0%), thymol (9.0%), and hexadecanoic acid (5.3%). The chemical compositions of essential oils from 19 Corsican locations were investigated. The study of the chemical variability using statistical analysis allowed identifying direct correlation between the three populations of P. sordidum widespread in Corsica and the essential oil compositions they produce. The in vitro antimicrobial activity of P. sordidum essential oil was evaluated and it exhibited a notable activity on a large panel of clinically significant microorganisms.