Maria Grazia Bellardi

Chromatographic (GC-MS, HPLC) and virological evaluations of Salvia sclarea infected by BBWV-I

Salvia sclarea cultivated at the Herb Garden of Casola-Valsenio (Emilia-Romagna region, Italy) has been found for the first time naturally infected by broad bean wilt fabavirus, serotype I (BBWV-I). Symptomatic plants showed malformed leaves, with chlorotic mosaic followed by yellowing and stunting. BBWV-I was identified by applying virological tests: mechanical inoculations on herbaceous plants, electron microscopy, DAS-ELISA and PAS-ELISA. The essential oil obtained from BBWV-infected material corresponded to 2/3 the quantity of that from healthy material. The GC-MS and HPLC analyses of these oils afforded a comparative analytical profile of the two plant materials attributed to BBWV-I infection. The oils from infected materials showed higher percentages of sesquiterpene hydrocarbons (e.g. germacrene D and beta-caryophyllene), monoterpene alcohols (e.g. alpha-terpineol) and diterpenoids (mainly sclareol). In contrast, lower levels of monoterpene hydrocarbons (e.g. myrcene, limonene and the two ocimene isomers) and the principal components (linalyl acetate and linalool) were observed.

GC-MS analysis of the lipophilic principles of Echinacea purpurea and evaluation of cucumber mosaic cucumovirus infection.

An analytical GC-MS method based on nonpolar fused silica capillary column was developed to analyze the lipophilic constituents, mainly alkamides, from the root extracts of Echinacea purpurea (L.) Moench. In particular, the proposed method was applied to evaluate the phytochemical impacts of cucumber mosaic cucumovirus (CMV) infection on the plants lipophilic marker phytochemicals. Methanolic (70% v/v) extracts, obtained from root materials by ultrasonic treatments, were subjected to liquid-liquid extraction with n-hexane-ethyl acetate (1:1 v/v) to recover the lipophilic, volatile to semivolatile, principles. Seventeen components, including the 11 alkamides known to E. purpurea roots, were identified in the GC-MS traces of the analyzed fractions and efficiently separated in a turnaround time of 25 min. CMV infection was found to be responsible for significant variations in the relative compositions of the major constituents, in particular germacrene D, Dodeca-2E, 4E, 8Z, 10Z(E)-tetraenoic acid isobutylamide cis/trans isomers, Undeca-2Z, 4E-diene-8, 10-diynoic acid isobutylamide and Dodeca-2E, 4Z-diene-8, 10-diynoic acid isobutylamide.

Characterization of the Essential Oils of Healthy and Virus Infected Echinacea purpurea (L.) Moench Plants

Abstract The essential oils hydrodistilled from the aerial parts of Echinacea purpurea (L.) Moench grown at the Herb Garden of Casola Valsenio (Ravenna, Italy) from healthy and infected (cucumber mosaic cucumovirus; CMV) plant materials, were analyzed by GC/MS. The oil from healthy material was rich in germacrene D (57.8%) and was more abundant. The infected materials afforded a lower oil content and significant quantitative variations in the oil composition. In particular, the observed percentage of germacrene D (52.6%) was reduced as were other sesquiterpene hydrocarbons. These variations, tested to be significant for all the compound-class fractions and individual major components, were ascribed to the CMV infection, the only fixed-effect variable that might affect the oil composition.

Optimization and validation of a high-performance liquid chromatography method for the analysis of cardiac glycosides in Digitalis lanata.

In this study, a simple and reliable HPLC method for the qualitative and quantitative analysis of cardiac glycosides in Digitalis lanata Ehrh. raw material was developed and applied to healthy and phytoplasma-infected plants. The target analytes cover a broad range of secondary metabolites, including primary, secondary and tertiary glycosides and the corresponding aglycones. The sample preparation was carried out by sonication of the plant material with 70% (v/v) aqueous methanol at room temperature, followed by reversed-phase solid-phase extraction purification from interfering pigments. The HPLC analyses were performed on a Symmetry C(18) column (75 mm x 4.6mm I.D., 3.5 microm), with a gradient elution composed of water and acetonitrile, at a flow rate of 1.0 mL/min. The column temperature was set at 20 degrees C and the photodiode array detector monitored the eluent at 220 nm. The method was validated with respect to ICH guidelines and the validation parameters were found to be highly satisfactory. The application of the method to the analysis of D. lanata leaves indicated that air-drying was the optimum method for raw material processing when compared with freeze-drying. The analysis of healthy and phytoplasma-infected plants demonstrated that the secondary metabolite mainly affected by the pathogen presence was lanatoside C (153.2 microg/100mg versus 76.1 microg/100mg). Considering the importance of D. lanata plant material as source of cardiac glycosides, the developed method can be considered suitable for the phytochemical analysis and for the quality assurance of D. lanata used for pharmaceutical purpose.

Chromatographic methods for metabolite profiling of virus- and phytoplasma-infected plants of Echinacea purpurea.

This study was focused on the effects of virus and phytoplasma infections on the production of Echinacea purpurea (L.) Moench secondary metabolites, such as caffeic acid derivatives, alkamides, and essential oil. The identification of caffeic acid derivatives and alkamides was carried out by means of high-performance liquid chromatography-diode array detection (HPLC-DAD), HPLC-electrospray ionization-mass spectrometry (ESI-MS), and MS(2). Quantitative analysis of these compounds was carried out using HPLC-DAD. The results indicated that the presence of the two pathogens significantly decreases (P < 0.05) the content of cichoric acid, the main caffeic acid derivative. Regarding the main alkamide, dodeca-2E,4E,8Z,10E/Z-tetraenoic acid isobutylamide, a significant decrease (P < 0.05) in the content of this secondary metabolite was observed in virus-infected plants in comparison with healthy plants, while in the phytoplasma-infected sample the variation of this secondary metabolite was not appreciable. The % relative area of the E/Z isomers of this alkamide was also found to change in infected samples. The gas chromatography (GC) and GC-MS analysis of E. purpurea essential oil enabled the identification of 30 compounds. The main significant differences (P < 0.05) in the semiquantitative composition were observed for three components: limonene, cis-verbenol, and verbenone. The results indicate that the presence of virus and phytoplasma has an appreciable influence on the content of E. purpurea secondary metabolites, which is an important issue in defining the commercial quality, market value, and therapeutic efficacy of this herbal drug.

Population genetics of cucumber mosaic virus infecting medicinal, aromatic and ornamental plants from northern Italy

The genetic variation and evolution of cucumber mosaic virus (CMV) from aromatic, medicinal and ornamental plants in northern Italy was studied by sequence analysis of the movement protein gene and comparison with equivalent sequences of isolates from other countries. Comparison of nonsynonymous and synonymous substitutions suggested that 30% of amino acid sites were under negative selection and only one was under positive selection. Phylogenetic, nucleotide diversity and genetic differentiation analyses suggested that long-distance migration plays a role in the evolution and determination of the genetic structure and diversity of CMV in northern Italy and other areas.

Change in Caco-2 cells following treatment with various lavender essential oils

Abstract Lavender is an aromatic evergreen shrub diffused in the Mediterranean basin appreciated since antiquity. The genus Lavandula is part of Lamiaceae family and includes more than 20 species, among which true lavender (L. vera D.C. or L. angustifolia Miller.) and spike lavender (L. latifolia Medikus); there are also numerous hybrids known as lavandins (L. hybrida Rev.). L. vera, spike lavender and several hybrids are the most intensely used breeding species for the production of essential oils. Lavender and lavandin essential oils have been applied in food, pharmaceutical and other agro industries as biological products. In their chemical composition, terpenes linalool and linalyl acetate along with terpenoids such as 1,8-cineole are mostly responsible for biological and therapeutic activities. This study evaluates cytotoxic activity of essential oils derived from four lavender species on human epithelial colorectal adenocarcinoma cells. Analysis of pre- and post-treatment cell morphology has been performed using scanning electron microscope.

Lavandula x intermedia and Lavandula angustifolia essential oils: phytochemical composition and antimicrobial activity against foodborne pathogens

Abstract Four cultivars (cv) of Lavandula x intermedia (‘Abrialis’, ‘Alba’, ‘Rinaldi Ceroni’ (R.C.) and ‘Sumiens’) were cultivated in Italy and their essential oils (EOs) were distilled from Alfalfa Mosaic Virus-free plants. These EOs and one from L. angustifolia Miller were chemically characterised by GC-MS and GC-FID. Antimicrobial activity was evaluated against Listeria monocytogenes (24 strains) and Salmonella enterica (10 food strains). Minimal inhibitory concentrations (MIC) ≥ 10.0 μL/mL inhibited Salmonella (cv ‘R.C.’ was the most active); MIC of 0.3 μL/mL for cv ‘Abrialis’ and ‘R.C.’ inhibited L. monocytogenes, revealing noticeable activity, especially on clinical strains. This activity appears related to EOs composition. Particularly cv ‘Abrialis’ and ‘R.C.’ showing the highest antimicrobial activity, were rich in the specific constituents: linalool (38.17 and 61.98%), camphor (8.97 and 10.30%), 1,8-cineole (6.89 and 8.11%, respectively). These EOs could find potential applications in food biopreservation and in surface decontamination, even in hospitals, and deserve deeper investigations.

Chemical Composition and Antimicrobial Activity of Essential Oils from Aerial Parts of Monarda didyma and Monarda fistulosa Cultivated in Italy

Abstract Monarda spp. are promising Lamiaceae due to essential oil (EO) composition and antimicrobial activity. To investigate these properties, Monarda didyma and M. fistulosa were grown for two years in two Italian sites for EO extraction (distillation), analysis (GC and GC/MS), and antimicrobial activity (microplate diffusion broth method). EO yield greatly increased from first to second year of cultivation (0.27 and 0.51% in 2013 and 2014, respectively), showing minor differences between M. didyma and M. fistulosa. Conversely, EO composition significantly varied between the two species and years. Of the fifteen major compounds identified, eight were shared by the two species. However, the amounts differed, especially in thymol (62 vs. 31% in M. didyma vs. M. fistulosa) opposed to α- and β-phellandrene (their average, 1% in M. didyma and 16% in M. fistulosa). An increase of thymol was observed between 2013 and 2014 in both species (average, +11%), reflecting juvenile plants showing only vegetative organs (2013) vs. fully flowered plants (2014). Despite such differences, EO’s from the two Monarda species exhibited a similar activity against pathogenic and non-pathogenic strains of fungi and bacteria from human, animal and plant source. In general, Monarda EO’s showed a lower minimum inhibitory concentration, i.e. a stronger activity, against pathogenic (Escherichia coli, Erwinia amylovora and Candida albicans) than beneficial microorganisms (Bifidobacterium animalis and Lactobacillus casei). The higher susceptibility of pathogenic microorganisms supports the use of Monarda EO’s as antimicrobial agents with a favourable profile of selectivity. Applications are envisaged in food preservation, plant protection and human health.

PHYTOPLASMA DISEASES OF MEDICINAL AND AROMATIC PLANTS

Medicinal and aromatic plants include a broad array of wild and cultivated plants which contain many biologically-active compounds, known as phytochemicals, that are of great interest for their ability to promote human and animal health. The present review provides a literature overview of phytoplasma diseases affecting medicinal and aromatic plants, with an emphasis on phytoplasma taxa associated. An overview of studies that examined the effect of phytoplasma infections on phytochemical content and other secondary metabolites of affected plants is also included. Phytoplasma diseases of medicinal and aromatic plants occur worldwide; however, the majority of reports are from Europe and southeastern Asian countries. These diseases affect plant species belonging to over 70 families, mostly to Apiaceae and Asteraceae. They differ considerably in geographic distribution and size of the various taxonomic groups and subgroups of the associated phytoplasmas. Subgroup 16SrI-B phytoplasmas are the prevalent agents occurring mainly in Europe, North America and Asia. Phytoplasma presence induces changes in the amount and composition of secondary metabolites in diseased plants in which, however, the concentrations of valuable phytochemicals are greatly affected. An exception is represented by phytoplasma diseases of periwinkle in which an accumulation of pharmaceutically important compounds occurs upon phytoplasma infections. Prospects for future research are identified and critically discussed as well.