Complexing the Marine Sesquiterpene Euplotin C by Means of Cyclodextrin-Based Nanosponges: A Preliminary Investigation.

Euplotin C is a sesquiterpene of marine origin endowed with significant anti-microbial and anti-tumor properties. Despite the promising functional profile, its progress as a novel drug candidate has failed so far, due to its scarce solubility and poor stability in aqueous media, such as biological fluids. Therefore, overcoming these limits is an intriguing challenge for the scientific community. In this work, we synthesized ß-cyclodextrin-based nanosponges and investigated their use as colloidal carriers for stably complex euplotin C. Results obtained proved the ability of the carrier to include the natural compound, showing remarkable values of both loading efficiency and capacity. Moreover, it also allowed us to preserve the chemical structure of the loaded compound, which was recovered unaltered once extracted from the complex. Therefore, the use of ß-cyclodextrin-based nanosponges represents a viable option to vehiculate euplotin C, thus opening up its possible use as pharmacologically active compound.

Lavender aromatherapy: A systematic review from essential oil quality and administration methods to cognitive enhancing effects.

Modern society is reviving the practice of aromatherapy, and lavender is reported being the most worldwide purchased plant for essential oil (EO) extraction. Since recent studies reported cognitive enhancing effects of lavender besides the hypno-inducing effects, a literature review is needed. Considering EO quality and diffusion devices, we conducted a systematic review on the effects of lavender EO inhalation on arousal, attention and memory in healthy subjects. Starting from this new multidisciplinary perspective, cognitive effects were reviewed to link outcomes to effective and reproducible protocols. A systematic search on MEDLINE, ERIC, PsycInfo, Google Scholar, and Scopus databases using Cognitive Atlas and plant-related keywords was conducted. Among the 1,203 articles yielded, 11 met eligibility criteria. Subjects administered with lavender EO displayed arousal decrease and sustained attention increase. Controversial results emerged regarding memory. Lack of EO quality assessment and protocols heterogeneity did not allow assessing whether different EO composition differentially modulates cognition and whether placebo effect can be discerned from EO effect itself. However, GABAergic pathway modulation exerted by linalool, a major lavender EO constituent, might explain cognitive functions empowerment. We speculate aromatherapy could be a burgeoning cognition enhancing tool, although further investigation is required to reach robust conclusions.

New insights into the anticancer activity of carnosol: p53 reactivation in the U87MG human glioblastoma cell line.

Glioblastoma multiforme (GBM) is an aggressive brain tumour with high resistance to radio- and chemotherapy. As such, increasing attention has focused on developing new therapeutic strategies to improve treatment responses. Recently, attention has been shifted to natural compounds that are able to halt tumour development. Among them, carnosol (CAR), a phenolic diterpene present in rosemary, has become a promising molecule that is able to prevent certain types of solid cancer. However, no data are available on the effects of CAR in GBM. Here, CAR activity decreased the proliferation of different human glioblastoma cell lines, particularly cells that express wild type p53. The p53 pathway is involved in the control of apoptosis and is often impaired in GBM. Notably, CAR, through the dissociation of p53 from its endogenous inhibitor MDM2, was able to increase the intracellular p53 levels in GBM cells. Accordingly, functional reactivation of p53 was demonstrated by the stimulation of p53 target genes' transcription, the induction of apoptosis and cell cycle blockade. Most importantly, CAR produced synergistic effects with temozolomide (TMZ) and reduced the restoration of the tumour cells' proliferation after drug removal. Thus, for the first time, these data highlighted the potential use of the diterpene in the sensitization of GBM cells to chemotherapy through a direct re-activation of p53 pathway. Furthermore, progress has been made in delineating the biochemical mechanisms underlying the pro-apoptotic effects of this molecule.

Micropropagation of Salvia wagneriana Polak and hairy root cultures with rosmarinic acid production.

Salvia wagneriana Polak is a tropical species native to Central America, well adapted to grow in the Mediterranean basin for garden decoration. Micropropagation has been assessed from axillary shoots of adult plants using a Murashige and Skoog basal medium, with the addition of 1.33-µM 6-benzylaminopurine for shoot proliferation; the subsequent rooting phase occurred in plant growth regulator-free medium. The plants were successfully acclimatised with high survival frequency. Hairy roots were induced after co-cultivation of leaf lamina and petiole fragments with Agrobacterium rhizogenes and confirmed by PCR. The establishment and proliferation of the selected HRD3 line were obtained in hormone-free liquid medium and the production of rosmarinic acid (RA) was evaluated after elicitation. The analysis of RA was performed by LC-ESI-DAD-MS in the hydroalcoholic extracts. The addition of casein hydrolysate increased the RA production, whereas no enrichment was observed after the elicitation with jasmonic acid.

Aromatic profiling of wild and rare species growing in Turkey: Hypericum aviculariifolium Jaub. and Spach subsp. depilatum (Freyn and Bornm.) Robson var. depilatum and Hypericum pruinatum Boiss. and Bal.

The volatile constituents of the rare species of Hypericum, namely Hypericum pruinatum (as one population) and Hypericum aviculariifolium subp. depilatum var. depilatum (endemic, as two populations namely 'Gümüs' and 'Yeniköy') growing wild in the mountainous parts of Northern Turkey were studied for the first time. The essential oils (EOs) were extracted by hydrodistillation of the air-dried aerial parts and analysed by GC-FID and GC-MS. A total of 56, 49 and 50 EO components representing 98.2%, 96.9% and 99.4% of the total composition were identified respectively from one population for H. pruinatum and two populations for Hypericum aviculariifolium subp. depilatum var. depilatum. GC-MS profiles showed significant compositional variations not only between the two Turkish species, but also between the two populations of the same species highlighting the importance of genetic factors affecting secondary metabolite profile.

In vitro production of M. × piperita not containing pulegone and menthofuran.

The essential oils (EOs) and static headspaces (HSs) of in vitro plantlets and callus of Mentha x piperita were characterized by GC-MS analysis. Leaves were used as explants to induce in vitro plant material. The EO yields of the in vitro biomass were much lower (0.1% v/w) than those of the parent plants (2% v/w). Many typical mint volatiles were emitted by the in vitro production, but the callus and in vitro plantelet EOs were characterized by the lack of both pulegone and menthofuran. This was an important difference between in vitro and in vivo plant material as huge amounts of pulegone and menthofuran may jeopardise the safety of mint essential oil. Regarding the other characteristic volatiles, menthone was present in reduced amounts (2%) in the in vitro plantlets and was not detected in the callus, even if it represented the main constituent of the stem and leaf EOs obtained from the cultivated mint (26% leaves; 33% stems). The M. piperita callus was characterized by menthol (9%) and menthone (2%), while the in vitro plantlet EO showed lower amounts of both these compounds in favour of piperitenone oxide (45%). Therefore, the established callus and in vitro plantlets showed peculiar aromatic profiles characterized by the lack of pulegone and menthofuran which have to be monitored in the mint oil for their toxicity.

Production of Curcuminoids in different in vitro organs of Curcuma longa.

Curcuma longa L. (turmeric) is one of the most important spice and safe food additives. Its main constituents, curcuminoids, showed anti-inflammatory, antitumor and antioxidant effects. In the present work, an in vitro propagation method was developed to achieve selected plant organs with quantified curcuminoid content. In vitro plants were obtained from sprouting buds as primary explants. The major curcuminoid constituents, such as curcumin (CUR), demethoxycurcumin (DEM), and bis-demethoxycurcumin (bis-DEM) were examined in different organs by LC-DAD-ESI-MS. A significant production of curcumin (more than 260 microg g(-1) fresh weight) was obtained from in vitro microrhizomes, especially grown in a Murashige and Skoog medium (MS) supplemented with kinetin (0.1 mg L((-1)), alpha-naphthaleneacetic acid (NAA, 1 mg L(-1)), sucrose (6%), agar (5%) and activated charcoal (0.1%). The analyzed microrhizomes showed reduced amounts of DEM and bis-DEM in comparison with CUR levels. In addition a shoot culture line was suitable to biosynthesize curcuminoids, in a ratio very similar to that identified in the fresh rhizomes of parent plants. This study represents the first direct quantification of curcuminoids in turmeric in vitro shoots and microrhizomes to be used in dietary supplements.

Volatile chemical composition and bioactivity of six essential oils against the stored food insect Sitophilus zeamais Motsch. (Coleoptera Dryophthoridae).

Essential oils (EOs) of Achillea millefolium, Myrtus communis, Rosmarinus officinalis, Helichrysum italicum, Foeniculum vulgare and Lavandula angustifolia were analysed with GC-FID and GC-MS in order to define their aromatic profiles and then their toxicity and repellent activity against Sitophilus zeamais Motsch. (Coleoptera Dryophthoridae) with specific bioassays were evaluated. Results from topical applications on insects showed that all EOs had variable and significant insecticidal activity. Mortality rate never exceeded 76%. Results of repellency tests are indicated for M. communis and L. angustifolia EOs, displaying high repellent activity to S. zeamais adults.

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.

Pollen aroma fingerprint of two sunflower (Helianthus annuus L.) genotypes characterized by different pollen colors.

Samples of fresh pollen grains, collected from capitula in full bloom from two genotypes of sunflower (Helianthus annuus L.) and characterized by a different color, i.e., white-cream (WC) and orange (O), were analyzed by the HS-SPME (headspacesolid phase microextraction)/GC/MS technique. This study defined for the first time the fingerprint of the sunflower pollen, separated from the disc flowers, to define its contribution to the inflorescence aroma. In the GC/MS fingerprints of the WC and O genotypes, 61 and 62 volatile compounds were identified, respectively. Monoterpene hydrocarbons (34% in O vs. 28% in WC) and sesquiterpene hydrocarbons (37% in O vs. 31% in WC) were ubiquitous in all samples analyzed and represented the main chemical classes. α-Pinene (21% in O vs. 20% in WC) and sabinene (11% in O vs. 6% in WC) were the dominant volatiles, but also a full range of aliphatic hydrocarbons and their oxygenated derivatives gave a decisive contribution to the aroma composition (10% in O vs. 12% in WC). In addition, dendrolasin (3% in O vs. 4% in WC) and some minor constituents such as (E)-hex-2-en-1-ol (0.4% in O vs. 0.1% in WC) were pointed out not only for their contribution to the pollen scent, but also for their well-known role in the plant ecological relationships. Having evaluated two pollen morphs with different carotenoid-based colors, the study sought to highlight also the presence of some volatile precursors or derivatives of these pigments in the aroma. However, the pollen aroma of the two selected genotypes made a specific chemical contribution to the sunflower inflorescence scent without any influence on carotenoid derivatives.

Morphogenetic changes in essential oil composition of Hypericum perforatum during the course of ontogenesis.

CONTEXT: In the past few years, an increasing interest in the volatile secondary metabolites of Hypericum perforatum L. (Guttiferae) has been arising. OBJECTIVE: The present study is a contribution to better understand the relationship between the morphological variations and volatile composition during the phenological cycle. MATERIALS AND METHODS: Leaves at the stages of vegetative, floral budding, flowering and green capsule, buds, full opened flowers and green capsules were assayed for essential oil (EO) components by gas chromatography-flame ionization detector (GC-FID) and GC-mass spectrometry (MS). RESULTS: Significant amounts of sesquiterpenes (oxygenated 26-50% and hydrocarbons 20-40%) and oxygenated hydrocarbons (13-38%) characterized the all analyzed samples showing peculiar fluctuations during the seven phenological stages. Although monoterpenes were present in much lower amounts (monoterpene hydrocarbons 0.4-6%; oxygenated monoterpenes 0.8-6%) they were considered also important discrimination for several stages. The green capsules and the full opened flowers collected at flowering stage were clearly distinguished in terms of EO compositions from the other samples. DISCUSSION: For the first time, the EO composition of Turkish wild Hypericum perforatum was monitored by the hydrodistillation of different plant organs collected at different seven stages in order to point out the modification of target volatiles related to each phenological step. CONCLUSIONS: Based on the EO composition monitored during these seven morphological stages by GC-MS, principal component analysis and cluster analysis, significant metabolite modifications were observed during the phenological cycle which involved the levels of specific volatile target compounds belonging to the chemical classes of hydrocarbons, monoterpenes and sesquiterpenes.

Essential oil composition and variability of Hypericum perforatum from wild populations of northern Turkey.

CONTEXT: Hypericum perforatum L. (Guttiferae) contains many bioactive secondary metabolites including hypericins, hyperforins, and essential oil. OBJECTIVE: The present study was conducted to determine the variation in composition of essential oil in H. perforatum accessions from Turkey. MATERIAL AND METHODS: At full flowering, aerial parts of 30 plants were collected from 10 sites of northern Turkey and assayed for essential oil components by GC-FID and GC-MS. RESULTS: The chemical analysis revealed that the main constituents of the all analyzed samples were hydrocarbon and oxygenated sesquiterpenes such as beta-caryophyllene (4.08-5.93%), gamma-muurolene (5.00-9.56%), beta-selinene (5.08-19.63%), alpha-selinene (4.12-10.42%), d-cadinene (3.02-4.94%), spathulenol (2.34-5.14%), and caryophyllene oxide (6.01-12.18%). Monoterpenes, both hydrocarbon and oxygenated, were represented by scarce amounts of alpha- and beta-pinene, myrcene, linalool, cis- and trans-linalool oxide, and alpha-terpineol. Principal component analysis was also carried out and, according to the results, the first nine principal components were found to represent 100% of the observed variation. DISCUSSION: The chemical variation among the populations is discussed as the possible result of different genetic and environmental factors. CONCLUSIONS: The wild populations examined here are potentially important sources for breeding and improvement of the cultivated varieties.

Essential oil composition and larvicidal activity of six Mediterranean aromatic plants against the mosquito Aedes albopictus (Diptera: Culicidae).

Laboratory bioassays on insecticidal activity of essential oils (EOs) extracted from six Mediterranean plants (Achillea millefolium, Lavandula angustifolia, Helichrysum italicum, Foeniculum vulgare, Myrtus communis, and Rosmarinus officinalis) were carried out against the larvae of the Culicidae mosquito Aedes albopictus. The chemical composition of the six EOs was also investigated. Results from applications showed that all tested oils had insecticidal activity, with differences in mortality rates as a function of both oil and dosage. At the highest dosage (300 ppm), EOs from H. italicum, A. millefolium, and F. vulgare caused higher mortality than the other three oils, with mortality rates ranging from 98.3% to 100%. M. communis EO induced only 36.7% larval mortality at the highest dosage (300 ppm), a similar value to those recorded at the same dosage by using R. officinalis and L. angustifolia (51.7% and 55%, respectively). Identified compounds ranged from 91% to 99%. The analyzed EOs had higher content of monoterpenoids (80-99%) than sesquiterpenes (1-15%), and they can be categorized into three groups on the basis of their composition. Few EOs showed the hydrocarbon sesquiterpenes, and these volatile compounds were generally predominant in comparison with the oxygenated forms, which were detected in lower quantities only in H. italicum (1.80%) and in M. communis (1%).

Characterization of some Italian ornamental thyme by their aroma.

The aromatic profiles of five commercial thyme cultivars (T. vulgaris 'Silver Poise', T. vulgaris 'Erectus', T. vulgaris 'Faustini', T. x citriodorus 'Anderson's Gold', and T. x citriodorus 'Silver Queen'), cultivated in Italy, were defined both by their static headspaces (HS) and essential oils (EOs). In addition, a botanical garden sample of T. vulgaris was considered as reference material to evaluate the morphological and phytochemical differences from the selected market samples. Extractions of the volatile constituents of the different plant material were carried out by SPME (static headspace, HS) and hydrodistillation (HD) processes. GC-MS analysis provided the separation and identification of approximately 70 components in the HS samples and 50 in the hydrodistilled essential oils, accounting for more than 95% of the total. The typical main constituents of T. vulgaris were detected in all the EO samples, although qualitative and quantitative differences were found among the selected ornamental Thymus varieties. Thymol (50-55%) was the marker constituent for the three T. vulgaris cultivars, while geraniol (61-67%) characterized the essential oils of the two T. x citriodorus varieties. In all the analyzed essential oils, non-oxygenated (16-79%) and oxygenated (5-26%) monoterpenes were the typical volatile constituents. Specific target compounds (thymol, geraniol and 6-methyl-5-hepten-2-one) were selected to characterize the five thyme cultivars that are considered at present only as ornamental plants. A comparative evaluation of their EO quality was carried out in order to propose them as alternative sources of Italian raw plant material for industrial production.

Hairy root cultures for secondary metabolites production.

Hairy roots (HRs) are differentiated cultures of transformed roots generated by the infection of wounded higher plants with Agrobacterium rhizogenes. This pathogen causes the HR disease leading to the neoplastic growth of roots that are characterized by high growth rate in hormone free media and genetic stability. HRs produce the same phytochemicals pattern of the corresponding wild type organ. High stability and productivity features allow the exploitation of HRs as valuable biotechnological tool for the production of plant secondary metabolites. In addition, several elicitation methods can be used to further enhance their accumulation in both small and large scale production. However, in the latter case, cultivation in bioreactors should be still optimized. HRs can be also utilised as biological farm for the production of recombinant proteins, hence holding additional potential for industrial use. HR technology has been strongly improved by increased knowledge of molecular mechanisms underlying their development. The present review summarizes updated aspects of the hairy root induction, genetics and metabolite production.

Plant cell cultures: bioreactors for industrial production.

The recent biotechnology boom has triggered increased interest in plant cell cultures, since a number of firms and academic institutions investigated intensively to rise the production of very promising bioactive compounds. In alternative to wild collection or plant cultivation, the production of useful and valuable secondary metabolites in large bioreactors is an attractive proposal; it should contribute significantly to future attempts to preserve global biodiversity and alleviate associated ecological problems. The advantages of such processes include the controlled production according to demand and a reduced man work requirement. Plant cells have been grown in different shape bioreactors, however, there are a variety of problems to be solved before this technology can be adopted on a wide scale for the production of useful plant secondary metabolites. There are different factors affecting the culture growth and secondary metabolite production in bioreactors: the gaseous atmosphere, oxygen supply and CO2 exchange, pH, minerals, carbohydrates, growth regulators, the liquid medium rheology and cell density. Moreover agitation systems and sterilization conditions may negatively influence the whole process. Many types ofbioreactors have been successfully used for cultivating transformed root cultures, depending on both different aeration system and nutrient supply. Several examples of medicinal and aromatic plant cultures were here summarized for the scale up cultivation in bioreactors.

Analytical methods for the extraction and identification of secondary metabolite production in 'in vitro' plant cell cultures.

The production of plant secondary metabolites by in vitro culture is one of the most challenging and thrilling field of recent scientific researches. In the few last years, pharmaceutical and food industry demand in phytochemicals has increased steadily. Therefore, the establishment of in vitro plant protocols has to be monitored by phytochemical investigation of their selected extracts in order to supply standardized raw material. In this chapter, the advantages and disadvantages of some modem techniques have been described for the sampling, extraction and analysis ofthe invitro plants and derivatives. Depending on the volatile or nonvolatile substances produced by in vitro plant raw material, different kinds of laboratory facilities are needed for the extraction and quali-quantitative analysis. Recent extraction technology such as accelerated solvent extraction or microwave assisted extraction in combination with hyphenated techniques such as gas chromathography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) represent a modern approach to perform fast and reproducible analytical methods for the quality control of secondary metabolite production in 'in vitro' plant material.

Antimicrobial activity of Inga fendleriana extracts and isolated flavonoids.

The EtOAc and n-BuOH extracts of Inga fendleriana inhibited Gram-positive, but not Gram-negative bacteria; a narrow spectrum of activity against Staphylococcus epidermidis was detected. The MIC values of the extracts ranged from 125 to 850 microg/mL. Quercetin 3-methylether, myricetin 3-O-rhamnoside and tricetin showed antibacterial activity against the same bacterial strains with MICs in the range from 31 to 250 microg/mL. In time-kill kinetic studies, the flavonoids showed bactericidal effects at the concentrations corresponding to four times the MICs.

Licoflavone C attenuates the genotoxicity of cancer drugs in human peripheral lymphocytes.

Flavonoids exhibit a wide spectrum of biological activities that can lead to beneficial effects for human health. The search for cytotoxic, genotoxic and/or antimutagenic natural compounds is therefore of great relevance, especially in cancer chemotherapy. In view of this, we screened the potential genotoxicity/antigenotoxicty of licoflavone C (LFLC) - a naturally occurring prenyl-flavone extracted from Genista ephedroides - using the micronucleus (MN) assay on stimulated and cytochalasin B-blocked human lymphocytes. LFLC did not increase the spontaneous MN level up to 600 microM final concentration where a strong toxicity was seen to occur. We therefore performed an antigenotoxicity assay against the two mutagenic anticancer drugs, mitomycin C (MMC) and daunorubicin (DAU), using two non-toxic LFLC concentrations (0.1 microM and 1.0 microM). The MN frequencies induced by 0.025 microg/ml or 0.05 microg/ml DAU were significantly lowered by 45.4% or 46.6% and 41.8% or 44.8% at LFLC 0.1 and 1.0 microM, respectively. After treatment with 0.085 microg/ml or 0.17 microg/ml MMC, we detected a reduction in genotoxicity of 35.1% or 37.0% and of 38.0% or 35.8% at LFLC 0.1 and 1.0 microM, respectively. In conclusion, LFLC was proven to be protective toward the chromosome damage induced by DAU or MMC in cultured human peripheral lymphocytes.

Bioactive constituent production in St. John's Wort in vitro hairy roots. Regenerated plant lines.

A wild strain of Agrobacterium rhizogenes was used to regenerate twelve in vitro plant lines from different hairy roots of H. perforatum (St. John's Wort). The production of the main bioactive constituents was observed even though their yields varied in the different plant lines. Two lines were selected for the hyperoside production (4.9-4.6 mg/gdw) while nine were characterized by significant yields of chlorogenic acid (ranged from 0.47 to 1.09 mg/gdw). Furthermore, one out of twelve lines showed a 10-fold higher hypericin content (0.25 mg/gdw) than that reported for the in vitro shoots in the literature. Morphological and phytochemical features were determined in order to select H. perforatum genotypes enriched in valuable bioactive compounds.