Andrea Capuzzo

Supercritical Fluid Extraction of Plant Flavors and Fragrances

Supercritical fluid extraction (SFE) of plant material with solvents like CO2, propane, butane, or ethylene is a topic of growing interest. SFE allows the processing of plant material at low temperatures, hence limiting thermal degradation, and avoids the use of toxic solvents. Although today SFE is mainly used for decaffeination of coffee and tea as well as production of hop extracts on a large scale, there is also a growing interest in this extraction method for other industrial applications operating at different scales. In this review we update the literature data on SFE technology, with particular reference to flavors and fragrance, by comparing traditional extraction techniques of some industrial medicinal and aromatic crops with SFE. Moreover, we describe the biological activity of SFE extracts by describing their insecticidal, acaricidal, antimycotic, antimicrobial, cytotoxic and antioxidant properties. Finally, we discuss the process modelling, mass-transfer mechanisms, kinetics parameters and thermodynamic by giving an overview of SFE potential in the flavors and fragrances arena.

Differential expression of CPKs and cytosolic Ca2+ variation in resistant and susceptible apple cultivars (Malus x domestica) in response to the pathogen Erwinia amylovora and mechanical wounding

BackgroundPlant calcium (Ca2+) signals are involved in a wide array of intracellular signalling pathways following pathogen invasion. Ca2+-binding sensory proteins such as Ca2+-dependent protein kinases (CPKs) have been predicted to mediate signalling following Ca2+ influx after pathogen infection. However, to date this prediction has remained elusive.ResultsWe conducted a genome-wide identification of the Malus x domestica CPK (MdCPK) gene family and identified 30 CPK genes. Comparative phylogenetic analysis of Malus CPKs with CPKs of Arabidopsis thaliana (AtCPKs), Oryza sativa (OsCPKs), Populous trichocarpa (PtCPKs) and Zea mays (ZmCPKs) revealed four different groups. From the phylogenetic tree, we found that MdCPKs are closely related to AtCPKs and PtCPKs rather than OsCPKs and ZmCPKs, indicating their dicot-specific origin. Furthermore, comparative quantitative real time PCR and intracellular cytosolic calcium ([Ca2+]cyt) analysis were carried out on fire blight resistant and susceptible M. x domestica apple cultivars following infection with a pathogen (Erwinia amylovora) and/or mechanical damage. Calcium analysis showed an increased [Ca2+]cyt over time in resistant cultivars as compared to susceptible cultivars. Gene expression studies showed that 11 out of the 30 MdCPKs were differentially expressed following pathogen infection.ConclusionsWe studied the genome-wide analysis of MdCPK gene family in Malus x domestica and analyzed their differential gene expression along with cytosolic calcium variation upon pathogen infection. There was a striking difference in MdCPKs gene expressions and [Ca2+]cyt variations between resistant and susceptible M. x domestica cultivars in response to E. amylovora and mechanical wounding. Our genomic and bioinformatic analysis provided an important insight about the role of MdCPKs in modulating defence responses in susceptible and resistant apple cultivars. It also provided further information on early signalling and downstream signalling cascades in response to pathogenic and mechanical stress.

Antioxidant and radical scavenging activities of chamazulene

Essential oils (EOs) of chamomile contain several bioactive compounds, including monoterpenes, sesquiterpenes, triterpenes and fatty acids. Hydrodistillation of chamomile EO induces the formation of chamazulene, a bioactive compound. Chamazulene was isolated from the EO by column chromatography. The total antioxidant capacity confirmed a higher antioxidant activity of chamazulene (IC50 = 6.4 μg mL− 1) than of ascorbic acid (IC50 = 12.8 μg mL− 1), α-tocopherol (IC50 = 20.5 μg mL− 1) and of butylated hydroxytoluene (BHT) (IC50 = 30.8 μg mL− 1). Chamazulene was unable to react with DPPH√. However, when chamazulene was assayed with ABTS√, a strong and significantly (P < 0.05) higher free radical scavenging activity was observed (IC50 = 3.7 μg mL− 1), with respect to BHT (IC50 = 6.2 μg mL− 1) and α-tocopherol (IC50 = 11.5 μg mL− 1). The results of this work show that chamazulene is an important factor for the antioxidant power of chamomile oil.

Chrysolina herbacea modulates jasmonic acid, cis-(+)-12-oxophytodienoic acid, (3R,7S)-jasmonoyl-l-isoleucine, and salicylic acid of local and systemic leaves in the host plant Mentha aquatica

Abstract Little information is available on the interaction between herbivorous insects and plants storing terpenoids. In this work we describe the response of the essential oil plant Mentha aquatica to the specialist herbivore Chrysolina herbacea. Feeding from C. herbacea induced a significant increase of jasmonic acid (JA) in both local and systemic M. aquatica leaves, whereas the content of the JA precursor, cis-(+)-12-oxophytodienoic acid (OPDA), was increased in local leaves and decreased in systemic leaves. The JA conjugate, (3R,7S)-jasmonoyl-l-isoleucine (JA-Ile), was slightly increased in herbivore wounded (HW) local leaves, whereas its content significantly increased in systemic leaves. Herbivory by C. herbacea did not increase the content of salicylic acid (SA); however, SA showed a two-fold increase in HW systemic leaves. Our results indicate that also in plants producing direct defences, such as the essential oil plant M. aquatica, JA, and SA signalling is triggered by herbivory just like in plants that respond with indirect defence.

Comparative analysis of supercritical CO2 extracts and essential oils from an Ocimum basilicum chemotype particularly rich in T-cadinol

Ocimum basilicum (sweet basil) is an important essential oil plant used for different purposes (from food flavoring to pharmaceutical applications) characterized by the presence of several chemotypes. Here we show a comparative analysis between hydrodistillation of essential oils (EO) and supercritical CO2 extraction (SFE) of an O. basilicum chemotype particularly rich in T-cadinol. SFE yielded a higher percentage of 1,8-cineole (10%; 4-fold), linalool (23.2%; 5.8-fold), eugenol (13.3%; 1.2-fold) and germacrene D (5.6%; 28-fold) with respect to EO. On the other hand, EO composition was characterized by higher percentages of T-cadinol (27.5%; 3-fold) and some other sesquiterpenes with respect to SFE. The presence of high percentages of T-cadinol in EO is of great importance owing to the biological activity of this compound in cancer therapy and as an antibiotic.

Comparative analysis of alpha- and beta-thujone in the essential oil and supercritical CO2 extract of sage (Salvia officinalis L.)

Salvia officinalis L. (Sage) is an important industrial plant used both for food and pharmaceutical purposes. The terpene fraction of this plant, which contains thujones and other mono and sesquiterpenoids, is responsible for many of its therapeutic and culinary properties. We used two extraction methods [hydrodistillation (HD) to obtain the essential oil (EO), and supercritical CO2 extraction (SFE)] to analyze by gas chromatography–mass spectrometry (GC–MS) the terpene fraction extracted from sage dried leaves. α-Thujone, β-thujone and other oxygenated monoterpenes (1,8-cineole, linalool, camphor, borneol and bornyl acetate) as well as hydrocarbon (β-caryophyllene and α-humulene) and oxygenated sesquiterpenes (caryophyllene oxide, viridiflorol, humulene epoxide II and III) were found. The EO contained a significantly (p < 0.05) higher percentage of 1,8-cineole (10.4%), α-thujone (17.3%) and camphor (29.2%), whereas supercritical fluid (SF) extracts contained a significantly higher percentage of borneol (8.4%), bornyl acetate (2.2%), α-humulene (6.4%), viridiflorol (22.1%), humulene epoxide II and III (2.4% and 0.4%), and some unidentified sesquiterpene alcohols. Both EO and SF extracts contained equal amounts of β-thujone (4.8%) and β-caryophyllene (~7%). Our results show that HD of EO is a more efficient and economic method for α- and β-thujone extraction.

Comparison of different extraction methods for the determination of α‐ and β‐thujone in sage (Salvia officinalis L.) herbal tea

Salvia officinalis L. (sage) is an important industrial plant used both for food and pharmaceutical purposes. The terpene fraction of this plant is responsible for many of its therapeutic and culinary properties. We used different extraction methods Tenax TA® purge and trap, headspace (HS) solid-phase microextraction, HS sorptive extraction, and stir bar sorptive extraction to analyze the terpene fraction extracted from sage tea by GC-MS. Twenty compounds were identified, including α-, β-thujone, and several other oxygenated monoterpenes (1,8-cineole, linalool, camphor, boneol, and bornyl acetate) and oxygenated sesquiterpenes (caryophyllene oxide, viridiflorol, humulene epoxide I, II, and III). Tenax TA® and HS sorptive extraction extracted a lower number of identified compounds, whereas HS solid-phase microextraction allowed the complete extraction of volatiles with particular reference to α- and β-thujone. The importance of the determination of thujones content in sage herbal tea is also discussed.

Extracellular Self-DNA (esDNA), but Not Heterologous Plant or Insect DNA (etDNA), Induces Plasma Membrane Depolarization and Calcium Signaling in Lima Bean (Phaseolus lunatus) and Maize (Zea mays)

Extracellular self-DNA (esDNA) is produced during cell and tissue damage or degradation and has been shown to induce significant responses in several organisms, including plants. While the inhibitory effects of esDNA have been shown in conspecific individuals, little is known on the early events involved upon plant esDNA perception. We used electrophysiology and confocal laser scanning microscopy calcium localization to evaluate the plasma membrane potential (Vm) variations and the intracellular calcium fluxes, respectively, in Lima bean (Phaseolus lunatus) and maize (Zea mays) plants exposed to esDNA and extracellular heterologous DNA (etDNA) and to etDNA from Spodoptera littoralis larvae and oral secretions. In both species, esDNA induced a significant Vm depolarization and an increased flux of calcium, whereas etDNA was unable to exert any of these early signaling events. These findings confirm the specificity of esDNA to induce plant cell responses and to trigger early signaling events that eventually lead to plant response to damage.

Cultivable gut bacteria provide a pathway for adaptation of Chrysolina herbacea to Mentha aquatica volatiles

BackgroundA chemical cross-talk between plants and insects is required in order to achieve a successful co-adaptation. In response to herbivory, plants produce specific compounds, and feeding insects respond adequately7 to molecules produced by plants. Here we show the role of the gut microbial community of the mint beetle Chrysolina herbacea in the chemical cross-talk with Mentha aquatica (or watermint).ResultsBy using two-dimensional gas chromatography–mass spectrometry we first evaluated the chemical patterns of both M. aquatica leaf and frass volatiles extracted by C. herbacea males and females feeding on plants, and observed marked differences between males and females volatiles. The sex-specific chemical pattern of the frass paralleled with sex-specific distribution of cultivable gut bacteria. Indeed, all isolated gut bacteria from females belonged to either α- or γ-Proteobacteria, whilst those from males were γ-Proteobacteria or Firmicutes. We then demonstrated that five Serratia marcescens strains from females possessed antibacterial activity against bacteria from males belonging to Firmicutes suggesting competition by production of antimicrobial compounds. By in vitro experiments, we lastly showed that the microbial communities from the two sexes were associated to specific metabolic patterns with respect to their ability to biotransform M. aquatica terpenoids, and metabolize them into an array of compounds with possible pheromone activity.ConclusionsOur data suggest that cultivable gut bacteria of Chrysolina herbacea males and females influence the volatile blend of herbivory induced Mentha aquatica volatiles in a sex-specific way.

Molecular fingerprinting of some Mentha species by sequencing and RFLP analysis of the 5S-rRNA non-transcribed spacer region

The genus Mentha is of particular economic importance. The development of new methods for the characterisation of Mentha species is crucial for their unequivocal identification. Amplification of the non-transcribed spacer (NTS) of the 5S-rRNA gene was used to characterise some Mentha species, which revealed a high-specific variability. Cloning and sequencing of all amplified NTS fragments enabled the discrimination among almost all species. In silico and experimental analyses identified specific restriction sites on the amplified 5S-NTS regions, facilitating the rapid and unambiguous discrimination of all the different species by polymerase chain reaction–restriction fragment length polymorphism. A direct comparison between essential oil composition and DNA fingerprinting confirmed a relationship between chemical and molecular data.