Nuova ricerca

Stefania BENVENUTI

Professore Associato presso: Dipartimento Scienze della Vita sede ex Scienze Farmaceutiche Via Campi 103


Home | Curriculum(pdf) | Didattica |


Pubblicazioni

2020 - Propolis affects Pseudomonas aeruginosa Growth, Biofilm Formation, Phenazines and eDNA Release: Potential Involvement of Polyphenols [Articolo su rivista]
Meto, Aida; Colombari, Bruna; Meto, Agron; Boaretto, Giorgia; Pinetti, Diego; Marchetti, Lucia; Benvenuti, Stefania; Pellati, Federica; Blasi, Elisabetta
abstract

Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic pathogen responsible for a wide range of clinical conditions, from mild infections to life-threatening nosocomial biofilm-associated diseases, which are particularly severe in susceptible individuals. The aim of this in vitro study was to assess the effects of an Albanian propolis on several virulence-related factors of P. aeruginosa, such as growth ability, biofilm formation, extracellular DNA (eDNA) release and phenazine production. To this end, propolis was processed using three different solvents and the extracted polyphenolic compounds were identified by means of High Performance Liquid Chromatography coupled to–Mass spectrometry (HPLC-MS) analysis. As assessed by a bioluminescence-based assay, among the three propolis extracts, the ethanol (EtOH) extract was the most effective in inhibiting both microbial growth and biofilm formation, followed by propylene glycol (PG) and polyethylene glycol 400 (PEG 400) propolis extracts. Also, Pseudomonas exposure to propolis EtOH extract caused a decrease in eDNA release and phenazine production. Interestingly, caffeic acid phenethyl ester (CAPE) and quercetin decreased upon propolis EtOH extract exposure to bacteria. Overall, our data add new insights on the anti-microbial properties of a natural compound, such as propolis against P. aeruginosa. The potential implications of these findings will be discussed.


2020 - The effect of polyphenols on pomegranate fruit susceptibility to Pilidiella granati provides insights into disease tolerance mechanisms [Articolo su rivista]
Mincuzzi, A.; Ippolito, A.; Brighenti, V.; Marchetti, L.; Benvenuti, S.; Ligorio, A.; Pellati, F.; Sanzani, S. M.
abstract

Pilidiella granati, also known as Coniella granati, is the etiological agent of pomegranate fruit dry rot. This fungal pathogen is also well-known as responsible for both plant collar rot and leaf spot. Because of its aggressiveness and the worldwide diffusion of pomegranate crops, the selection of cultivars less susceptible to this pathogen might represent an interesting preventive control measure. In the present investigation, the role of polyphenols in the susceptibility to P. granati of the two royalties-free pomegranate cultivars Wonderful and Mollar de Elche was compared. Pomegranate fruit were artificially inoculated and lesion diameters were monitored. Furthermore, pathogen DNA was quantified at 12–72 h post-inoculation within fruit rind by a real time PCR assay setup herein, and host total RNA was used in expression assays of genes involved in host-pathogen interaction. Similarly, protein extracts were employed to assess the specific activity of enzymes implicated in defense mechanisms. Pomegranate phenolic compounds were evaluated by HPLC-ESI-MS and MS2. All these data highlighted ‘Wonderful’ as less susceptible to P. granati than ‘Mollar de Elche’. In the first cultivar, the fungal growth seemed controlled by the activation of the phenylpropanoid pathway, the production of ROS, and the alteration of fungal cell wall. Furthermore, antifungal compounds seemed to accumulate in ‘Wonderful’ fruit following inoculation. These data suggest that pomegranate polyphenols have a protective effect against P. granati infection and their content might represent a relevant parameter in the selection of the most suitable cultivars to reduce the economic losses caused by this pathogen.


2019 - Antibacterial activity of Rosmarinus officinalis L. and Thymus vulgaris L. essential oils and their combination against food-borne pathogens and spoilage bacteria in ready-to-eat vegetables [Articolo su rivista]
Iseppi, Ramona; Sabia, Carla; de Niederhäusern, Simona; Pellati, Federica; Benvenuti, Stefania; Tardugno, Roberta; Bondi, Moreno; Messi, Patrizia
abstract

The antibacterial activity of Rosmarinus officinalis L. and Thymus vulgaris L. essential oils (EOs), and their combination against food-borne and spoilage bacteria (Listeria monocytogenes, Salmonella enteritidis, Yersinia enterocolitica, Escherichia coli and Pseudomonas spp.) was determined. The EOs inhibitory effect was evaluated both in vitro by using the disk diffusion assay and the minimum inhibitory concentration (MIC) determination, and on food by using an artificially contaminated ready-to-eat (RTE) vegetables. The results showed that the lowest MIC values were obtained with R. officinalis and T. vulgaris EOs against E. coli (4 and 8 μL/mL, respectively). The incorporation of the EOs alone or their combination in RTE vegetables reduced the viable counts of all the tested strains. Lastly, in the on food study we simulated the worst hygienic conditions, obtaining results that can be considered a warranty of safety.


2019 - Botanical sources, chemistry, analysis, and biological activity of furanocoumarins of pharmaceutical interest [Articolo su rivista]
Bruni, R.; Barreca, D.; Protti, M.; Brighenti, V.; Righetti, L.; Anceschi, L.; Mercolini, L.; Benvenuti, S.; Gattuso, G.; Pellati, F.
abstract

The aim of this work is to provide a critical review of plant furanocoumarins from different points of view, including their chemistry and biosynthetic pathways to their extraction, analysis, and synthesis, to the main biological activities found for these active compounds, in order to highlight their potential within pharmaceutical science. The limits and the possible improvements needed for research involving these molecules are also highlighted and discussed.


2019 - Chemical Composition and In Vitro Neuroprotective Activity of Fibre-Type Cannabis sativa L. (Hemp) [Articolo su rivista]
Corsi, Lorenzo; Pellati, Federica; Brighenti, Virginia; Plessi, Nicolò; Benvenuti, Stefania
abstract

Background: Fibre-type Cannabis sativa L. (hemp) usually contains cannabidiolic acid and cannabidiol as the main non-psychoactive cannabinoids. Even though there is evidence of the neuroprotective activity of pure cannabidiol, no in vitro studies have reported so far the role of hemp extracts on neuroprotection. The objective of this study was to evaluate the neuroprotective effect of hemp extracts in in vitro cellular models of neurotoxicity. Methods: One extract was obtained from raw hemp inflorescences, while the other was prepared from the same plant material submitted to a decarboxylation process. The composition of both these extracts was evaluated by HPLC-UV/DAD. Human neuroblastoma SH-SY5Y and microglial BV-2 cell lines treated with rotenone were selected as the model of neurodegeneration. The neuroprotection of hemp extracts was assessed also in serum-free conditions both in the presence and in the absence of rotenone as the toxic agent by using the same cell lines. The neuroprotective potential of cannabidiol was tested in parallel. Results: The decarboxylated hemp extract possesses a mild neuroprotective activity on BV-2 cells treated with rotenone, higher than that of pure cannabidiol. As regards serum-free experiments, the nondecarboxylated hemp extract was the most effective neuroprotective agent toward SH-SY5Y cells, while BV-2 cells were better protected from the toxic insult by the decarboxylated extract and cannabidiol. Conclusion: Both hemp extracts and pure cannabidiol displayed a moderate neuroprotective activity in the neurotoxicity models considered in this study; in addition, they showed a trophic effect on SHSY5Y cells.


2019 - Chemical characterization and evaluation of the antibacterial activity of essential oils from fibre-type cannabis sativa L. (Hemp) [Articolo su rivista]
Iseppi, R.; Brighenti, V.; Licata, M.; Lambertini, A.; Sabia, C.; Messi, P.; Pellati, F.; Benvenuti, S.
abstract

Volatile terpenes represent the largest group of Cannabis sativa L. components and they are responsible for its aromatic properties. Even if many studies on C. sativa have been focused on cannabinoids, which are terpenophenolics, little research has been carried out on its volatile terpenic compounds. In the light of all the above, the present work was aimed at the chemical characterization of seventeen essential oils from different fibre-type varieties of C. sativa (industrial hemp or hemp) by means of GC-MS and GC-FID techniques. In total, 71 compounds were identified, and the semi-quantitative analysis revealed that α- and β-pinene, β-myrcene and β-caryophyllene are the major components in all the essential oils analysed. In addition, a GC-MS method was developed here for the first time, and it was applied to quantify cannabinoids in the essential oils. The antibacterial activity of hemp essential oils against some pathogenic and spoilage microorganisms isolated from food and food processing environment was also determined. The inhibitory effects of the essential oils were evaluated by both the agar well diffusion assay and the minimum inhibitory concentration (MIC) evaluation. By using the agar diffusion method and considering the zone of inhibition, it was possible to preliminarily verify the inhibitory activity on most of the examined strains. The results showed a good antibacterial activity of six hemp essential oils against the Gram-positive bacteria, thus suggesting that hemp essential oil can inhibit or reduce bacterial proliferation and it can be a valid support to reduce microorganism contamination, especially in the food processing field.


2019 - Development of a new method for the analysis of cannabinoids in honey by means of high-performance liquid chromatography coupled with electrospray ionisation-tandem mass spectrometry detection [Articolo su rivista]
Brighenti, V.; Licata, M.; Pedrazzi, T.; Maran, D.; Bertelli, D.; Pellati, F.; Benvenuti, S.
abstract

Fibre-type Cannabis sativa L. (hemp) represents a valuable resource in many different fields, including both the pharmaceutical and food ones. This plant contains non-psychoactive cannabinoids, a class of bioactive compounds biosynthesized in both female and male inflorescences. Among them, cannabidiol (CBD) is the most interesting compound from a medicinal point of view. Indeed, several scientific studies have proved its therapeutic potential in a large number of pathologies, in addition to its biological effects attributable to its antioxidant, neuroprotective and anti-inflammatory properties. The analysis of the amount of cannabinoids in food and food supplements represents a critical issue in the ambit of both the quality assurance and the dietary intake control of these biologically active compounds. In this ambit, a particular attention is necessary for apiary products, since they are widely consumed and they can be produced by bees starting from different floral sources. In the light of all the above, the aim of this study was to develop for the first time a new analytical method based on RP-HPLC with ESI-MS/MS detection for the determination of CBD and related cannabinoids in honey. A quick, easy, cheap, effective, rugged and safe (QuEChERS) extraction procedure with an un-buffered method was selected and optimised as the more suitable protocol. As regards detection, it was carried out by using a linear ion trap quadrupole (QTRAP) mass analyser, operated in the multiple reaction monitoring (MRM) mode. Hemp male inflorescences and pollen were analysed in parallel by means of HPLC-UV/DAD, since bees can transfer pollen into their hives and, consequently, into beehive products. The method developed and validated for the first time in this work was finally applied to the analysis of cannabinoids in honey samples, thus demonstrating to be a useful tool for both quality control and safety assurance.


2019 - In vitro effects of tea tree oil (Melaleuca alternifolia essential oil) and its principal component terpinen-4-ol on swine spermatozoa [Articolo su rivista]
Elmi, Alberto; Ventrella, Domenico; Barone, Francesca; Carnevali, Giacomo; Filippini, Gianfranco; Pisi, Annamaria; Benvenuti, Stefania; Scozzoli, Maurizio; Bacci, Maria Laura
abstract

The growing interest towards essential oils stems from their biological capabilities that include antibacterial and antioxidant effects. Such properties may be extremely useful in the reproductive field; nonetheless essential oils show toxic effects that can lead to cell disruption. The present study aimed to evaluate and compare the effects of tea tree oil (TTO) and its principal component terpinen-4-ol (TER) on the morpho-functional parameters of swine spermatozoa. Experimental samples were prepared by suspending 15 × 10 7 spermatozoa in 5 mL of medium with different concentrations of the above-mentioned compounds: from 0.2 to 2 mg/mL at an interval of 0.2 for TTO, while TER concentrations were adjusted according to its presence in TTO (41.5%). After 3 h incubation at 16 ◦ C, samples were analyzed for pH, viability, acrosome status, and objective motility. The results highlighted a concentration-dependent effect of TTO with total motility as the most sensitive parameter. TER was better tolerated, and the most sensitive parameters were related to membrane integrity, suggesting a different pattern of interaction. The study confirms the importance of evaluating the effects of natural compounds on spermatozoa before exploiting their beneficial effects. Spermatozoa seem to be good candidates for preliminary toxicological screenings in the light of their peculiar properties.


2019 - Lavandula x intermedia and Lavandula angustifolia essential oils: phytochemical composition and antimicrobial activity against foodborne pathogens [Articolo su rivista]
Tardugno, Roberta; Serio, Annalisa; Pellati, Federica; D’Amato, Serena; Chaves López, Clemencia; Bellardi, Maria Grazia; Di Vito, Maura; Savini, Vincenzo; Paparella, Antonello; Benvenuti, Stefania
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.


2019 - Use of 1H NMR to detect the percentage of pure fruit juices in blends [Articolo su rivista]
Marchetti, L.; Pellati, F.; Benvenuti, S.; Bertelli, D.
abstract

The consumption of high-nutritional-value juice blends is increasing worldwide and, considering the large market volume, fraud and adulteration represent an ongoing problem. Therefore, advanced anti-fraud tools are needed. This study aims to verify the potential of 1H NMR combined with partial least squares regression (PLS) to determine the relative percentage of pure fruit juices in commercial blends. Apple, orange, pineapple, and pomegranate juices were selected to set up an experimental plan and then mixed in different proportions according to a central composite design (CCD). NOESY (nuclear Overhauser enhancement spectroscopy) experiments that suppress the water signal were used. Considering the high complexity of the spectra, it was necessary to pretreat and then analyze by chemometric tools the large amount of information contained in the raw data. PLS analysis was performed using venetian-blind internal cross-validation, and the model was established using different chemometric indicators (RMSEC, RMSECV, RMSEP, R2CAL, R2CV, R2PRED). PLS produced the best model, using five factors explaining 94.51 and 88.62% of the total variance in X and Y, respectively. The present work shows the feasibility and advantages of using 1H NMR spectral data in combination with multivariate analysis to develop and optimize calibration models potentially useful for detecting fruit juice adulteration.


2018 - Cannabis sativa L. and Nonpsychoactive Cannabinoids: Their Chemistry and Role against Oxidative Stress, Inflammation, and Cancer [Articolo su rivista]
Pellati, Federica; Borgonetti, Vittoria; Brighenti, Virginia; Biagi, Marco; Benvenuti, Stefania; Corsi, Lorenzo
abstract

In the last decades, a lot of attention has been paid to the compounds present in medicinal Cannabis sativa L., such as Δ9-Tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD), and their effects on inflammation and cancer-related pain. The National Cancer Institute (NCI) currently recognizes medicinal C. sativa as an effective treatment for providing relief in a number of symptoms associated with cancer, including pain, loss of appetite, nausea and vomiting, and anxiety. Several studies have described CBD as a multitarget molecule, acting as an adaptogen, and as a modulator, in different ways, depending on the type and location of disequilibrium both in the brain and in the body, mainly interacting with specific receptor proteins CB1 and CB2. CBD is present in both medicinal and fibre-Type C. sativa plants, but, unlike Δ9-THC, it is completely nonpsychoactive. Fibre-Type C. sativa (hemp) differs from medicinal C. sativa, since it contains only few levels of Δ9-THC and high levels of CBD and related nonpsychoactive compounds. In recent years, a number of preclinical researches have been focused on the role of CBD as an anticancer molecule, suggesting CBD (and CBD-like molecules present in the hemp extract) as a possible candidate for future clinical trials. CBD has been found to possess antioxidant activity in many studies, thus suggesting a possible role in the prevention of both neurodegenerative and cardiovascular diseases. In animal models, CBD has been shown to inhibit the progression of several cancer types. Moreover, it has been found that coadministration of CBD and Δ9-THC, followed by radiation therapy, causes an increase of autophagy and apoptosis in cancer cells. In addition, CBD is able to inhibit cell proliferation and to increase apoptosis in different types of cancer models. These activities seem to involve also alternative pathways, such as the interactions with TRPV and GRP55 receptor complexes. Moreover, the finding that the acidic precursor of CBD (cannabidiolic acid, CBDA) is able to inhibit the migration of breast cancer cells and to downregulate the proto-oncogene c-fos and the cyclooxygenase-2 (COX-2) highlights the possibility that CBDA might act on a common pathway of inflammation and cancer mechanisms, which might be responsible for its anticancer activity. In the light of all these findings, in this review we explore the effects and the molecular mechanisms of CBD on inflammation and cancer processes, highlighting also the role of minor cannabinoids and noncannabinoids constituents of Δ9-THC deprived hemp.


2018 - Chemical profile and biological activities of Cedrelopsis grevei H. Baillon bark essential oil [Articolo su rivista]
Tardugno, Roberta; Spagnoletti, A.; Grandini, A.; Maresca, I.; Sacchetti, G.; Pellati, Federica; Benvenuti, Stefania
abstract

Cedrelopsis grevei H. Baillon bark, endemic plant from Madagascar, is used in folk medicine for the treatment of rheumatism, muscular pain, and for its antifungal and antibiotic activities. In this paper, the phytochemical composition, antioxidant, antimicrobial, and cytotoxic activities of C. grevei bark essential oil (EO), its non-polar (I and II) and polar (III) fractions and its main compounds (ishwarane, β-elemene and α-copaene) were investigated. The GC–MS analysis pointed out the presence of 36 components, representing about 80% as semi-quantitative characterization of the total. The presence of ishwarane, β-elemene and α-copaene as the main constituents highlighted its peculiar composition as a sesquiterpene-rich phytocomplex. Moreover, the quantification was performed for the first time by means of the experimental and predicted response factors (ERFs and PRFs, respectively). As regards the biological activity, C. grevei EO and its fractions showed weak antioxidant activity against Trolox. The whole EO demonstrated instead considerable antimicrobial activity against Staphylococcus aureus and Staphylococcus epidermidis, while its polar-fraction evidenced an interesting bioactivity against Pseudomonas aeruginosa and Candida albicans. Finally, C. grevei EO and its fractions exhibited an interesting cytotoxic activity on human lung cancer cells (A549) and human colorectal cancer cells (CaCo-2).


2018 - High-performance liquid chromatography for the analytical characterization of anthocyanins in Vaccinium myrtillus L. (bilberry) fruit and food products [Articolo su rivista]
Benvenuti, S.; Brighenti, V.; Pellati, F.
abstract

Anthocyanins represent the most abundant class of bioactive compounds present in Vaccinium myrtillus L. (bilberry) fruit, conferring it several health-promoting properties. The content of anthocyanins in food products produced from bilberries can be affected by many parameters, making the study of their composition a critical issue. In this ambit, this work was aimed at a comprehensive profiling of anthocyanins in bilberry fruit and derivatives from the Italian Northern Apennines, including jam, juice, and liqueur (“Mirtillino”). Anthocyanins were extracted from the jams by means of a dynamic maceration with acidified methanol, while juice and liqueurs were directly analyzed. The analysis of anthocyanins in the extracts was carried out by means of HPLC-UV/DAD, HPLC-ESI-MS, and MS2, under gradient elution. As a comparison, authentic bilberry fruits were analyzed. The total anthocyanin content was in the range 582.4–795.2 mg/100 g (FW) for the fruit, 2.3–234.5 mg/100 g for the jams, 109.2–2252.2 mg/L for the juice, and 27.9–759.3 mg/L for the liqueurs. To deeper investigate the anthocyanin profile of the liqueurs that exhibited a remarkably different composition in comparison with the other products, an authentic bilberry liqueur was prepared in the lab, following a traditional recipe, and monitored weakly by HPLC. The percentage of degradation of 3-O-galactosides and 3-O-arabinosides of bilberry anthocyanidins was found to be higher than that of 3-O-glucosides. The results of this work demonstrated the importance of a suitable and reliable analysis of bilberry fruit and related food products to ensure their genuineness and quality.


2018 - New methods for the comprehensive analysis of bioactive compounds in Cannabis sativa L. (hemp) [Articolo su rivista]
Pellati, F.; Brighenti, V.; Sperlea, J.; Marchetti, L.; Bertelli, D.; Benvenuti, S.
abstract

Cannabis sativa L. is a dioecious plant belonging to the Cannabaceae family. The main phytochemicals that are found in this plant are represented by cannabinoids, flavones, and terpenes. Some biological activities of cannabinoids are known to be enhanced by the presence of terpenes and flavonoids in the extracts, due to a synergistic action. In the light of all the above, the present study was aimed at the multi-component analysis of the bioactive compounds present in fibre-type C. sativa (hemp) inflorescences of different varieties by means of innovative HPLC and GC methods. In particular, the profiling of non-psychoactive cannabinoids was carried out by means of HPLC-UV/DAD, ESI-MS, and MS2. The content of prenylated flavones in hemp extracts, including cannflavins A and B, was also evaluated by HPLC. The study on Cannabis volatile compounds was performed by developing a new method based on headspace solid-phase microextraction (HS-SPME) coupled with GC-MS and GC-FID. Cannabidiolic acid (CBDA) and cannabidiol (CBD) were found to be the most abundant cannabinoids in the hemp samples analysed, while ß-myrcene and ß-caryophyllene were the major terpenes. As regards flavonoids, cannflavin A was observed to be the main compound in almost all the samples. The methods developed in this work are suitable for the comprehensive chemical analysis of both hemp plant material and related pharmaceutical or nutraceutical products in order to ensure their quality, efficacy, and safety.


2018 - Phytochemical composition and in vitro screening of the antimicrobial activity of essential oils on oral pathogenic bacteria [Articolo su rivista]
Tardugno, Roberta; Pellati, Federica; Iseppi, Ramona; Bondi, Moreno; Bruzzesi, Giacomo; Benvenuti, Stefania
abstract

In this study, the activity of essential oils (EOs) against microorganisms involved in oral diseases was evaluated. Fourteen EOs were selected and subjected to gas chromatographic analysis, including Illicium verum, Eucaliptus globulus, Eugenia caryophyllata, Leptospermum scoparium, Mentha arvensis, Mentha piperita, Myrtus communis, Salvia officinalis, Melaleuca alternifolia, Rosmarinus officinalis, Lavandula x intermedia, Thymus capitatus and Thymus vulgaris. These EOs were tested for their antimicrobial activity on Streptococcus mutans and Lactobacillus species clinically isolated from dental surgery patients. The antibacterial activity was evaluated by means of the disc diffusion and the minimum inhibitory concentration (MIC). Five EOs, having shown an interesting antimicrobial activity, were selected for a second screening in combination between them and with chlorhexidine. From the second assays, two EO-EO and three EO-chlorhexidine associations gave interesting results as potential constituents of mouthwashes, especially for the contribution of oxygenated monoterpenes, including menthol, thymol and carvacrol.


2018 - Testing the influence of digestate from biogas on growth and volatile compounds of basil (Ocimum basilicum L.) and peppermint (Mentha x piperita L.) in hydroponics [Articolo su rivista]
Ronga, Domenico; Pellati, Federica; Brighenti, Virginia; Laudicella, Katia; Laviano, Luca; Fedailaine, Maamar; Benvenuti, Stefania; Pecchioni, Nicola; Francia, Enrico
abstract

In this study, a digestate was evaluated for the first time as a sustainable alternative to the conventional growing medium and the nutrient solution in the hydroponic production of aromatic species. Basil (Ocimum basilicum L.) and peppermint (Mentha x piperita L.) were assessed at crop vegetative stage with five fully expanded leaves. Four combinations, i.e. agriperlite (AG) + standard solution (SS), AG + liquid digestate (LD), solid digestate (SD) + SS and SD + LD, were compared. The SD used as the growing medium increased all the investigated agronomical traits, apart the harvest index and shoot dry weight height ratio, that showed the same values compared to agriperlite, on basil and peppermint, respectively. The LD used as the nutrient solution performed as well as the SS on basil and peppermint production, except for the percentage of emergence and total dry weight of basil, showing lower and higher values compared to SS, respectively. Shoot dry weight was the most important agronomical parameter and both basil and peppermint displayed high values using the SD as the growing medium. However, basil recorded the highest dry matter value using SD + LD, while peppermint showed similar values using SD + SS and SD + LD. As regards the volatile compounds of both species, the percentage relative amount of sesquiterpenes was found to be higher under SD + LD and SD + SS conditions in comparison to AG + SS and AG + LD, where the content of monoterpenes and allyl phenol derivatives was higher. SD and LD appear to be sustainable and suitable growing medium and nutrient solution for basil and peppermint grown in hydroponics. Further studies are needed to investigate the influence of the treatments also in advanced developmental stages of the crops, appropriate for the production of plants with high yield and quality.


2018 - Use of Pinus sylvestris L. (Pinaceae), Origanum vulgare L. (Lamiaceae), and Thymus vulgaris L. (Lamiaceae) essential oils and their main components to enhance itraconazole activity against azole susceptible/not-susceptible Cryptococcus neoformans strains [Articolo su rivista]
Scalas, Daniela; Mandras, Narcisa; Roana, Janira; Tardugno, Roberta; Cuffini, Anna Maria; Ghisetti, Valeria; Benvenuti, Stefania; Tullio, Vivian
abstract

Abstract Background: Cryptococcal infections, besides being a problem for immunocompromised patients, are occasionally being a problem for immunocompetent patients. In addition, the lower susceptibility of this yeast to azoles is a growing problem in health care. To date, there are very few molecules with any activity towards Cryptococcus neoformans, leading to heightened interest in finding new alternatives or adjuvants to conventional drugs for the treatment of mycosis caused by this yeast. Since the essential oils (EOs) are considered as a potential rich source of bioactive antimicrobial compounds, we evaluated the antifungal activity of Origanum vulgare (oregano), Pinus sylvestris (pine), and Thymus vulgaris (thyme red) EOs, and their components (α-pinene, carvacrol, thymol) compared with fluconazole, itraconazole, and voriconazole, against C.neoformans clinical strains. Then, we investigated the effect of EOs and components in combination with itraconazole. Methods: EO composition was analysed by Gas chromatography-mass spectrometry (GC-MS). A broth microdilution method was used to evaluate the susceptibility of C.neoformans to azoles, EOs and components. Checkerboard tests, isobolograms and time-kill assays were carried out for combination studies. Results: C.neoformans isolates were susceptible to azoles, while one C.neoformans exhibited a reduced susceptibility to all test edazole drugs. All EOs exerted a good inhibitory activity against all C.neoformans strains. Pine EO was the most effective. Among components, thymol exerted the most remark able activity. By checkerboard testing and isobolographic analysis, combinations of itraconazole with oregano, pine, or thyme EOs, and carvacrol were found to be synergistic (FICI ≤ 0.5) against azole susceptible C.neoformans. Regarding the azole not susceptible C.neoformans strain, the synergistic effect with itraconazole was observed with thyme EO (chemotype: thymol 26.52%; carvacrol 7.85%), and carvacrol. Time-kill assays confirmed the synergistic effects of itraconazole and oregano or thyme EO against azole susceptible C.neoformans. Binary mixtures of itraconazole/thyme EO or carvacrol yielded additive effects on the azole not susceptible C.neoformans. Conclusions: Our findings highlight the potential effectiveness of thyme, oregano EOs, and carvacrol as natural and cost-effective adjuvants when used in combination with itraconazole. Identification of EOs exerting these effects could be one of the feasible ways to overcome drug resistance, reducing drug concentration and side effects.


2017 - A new method based on supercritical fluid extraction for polyacetylenes and polyenes from Echinacea pallida (Nutt.) Nutt. Roots [Articolo su rivista]
Tacchini, M.; Spagnoletti, A.; Brighenti, V.; Prencipe, F. P.; Benvenuti, S.; Sacchetti, G.; Pellati, F.
abstract

The genus Echinacea (Asteraceae) includes species traditionally used in phytotherapy. Among them, Echinacea pallida (Nutt.) Nutt. root extracts are characterized by a representative antiproliferative activity, due to the presence of acetylenic compounds. In this study, supercritical fluid extraction (SFE) was applied and compared with conventional Soxhlet extraction (SE) in order to obtain a bioactive extract highly rich in polyacetylenes and polyenes from E. pallida roots. The composition of the extracts was monitored by means of HPLC–UV/DAD and HPLC–ESI–MSn by using an Ascentis Express C18 column (150 mm × 3.0 mm I.D., 2.7 μm, Supelco, Bellefonte, PA, USA) with a mobile phase composed of (A) water and (B) acetonitrile, under gradient elution. By keeping SFE time at the threshold of 1 h (15 min static and 45 min dynamic for 1 cycle) with the oven temperature set at 40–45 °C and 90 bar of pressure, an overall extraction yield of 1.18–1.21% (w/w) was obtained, with a high selectivity for not oxidized lipophilic compounds. The biological activity of the extracts was evaluated against human non-small lung A549 and breast carcinoma MCF-7 cancer cell lines. The cytotoxic effect of the SFE extract was more pronounced towards the MCF-7 than the A549 cancer cells, with IC50 values ranging from 21.01 ± 2.89 to 31.11 ± 2.l4 μg/mL; cell viability was affected mainly between 24 and 48 h of exposure. The results show the possibility of a new “green” approach to obtain extracts highly rich in genuine polyacetylenes and polyenes from E. pallida roots. The bioactivity evaluation confirmed the cytotoxicity of E. pallida extracts against the considered cancer cell lines, especially against MCF-7 cells, thus suggesting to represent a valuable tool for applicative purposes in cancer prevention.


2017 - Development of a new extraction technique and HPLC method for the analysis of non-psychoactive cannabinoids in fibre-type Cannabis sativa L. (hemp) [Articolo su rivista]
Brighenti, Virginia; Pellati, Federica; Steinbach, Marleen; Maran, Davide; Benvenuti, Stefania
abstract

The present work was aimed at the development and validation of a new, efficient and reliable technique for the analysis of the main non-psychoactive cannabinoids in fibre-type Cannabis sativa L. (hemp) inflorescences belonging to different varieties. This study was designed to identify samples with a high content of bioactive compounds, with a view to underscoring the importance of quality control in derived products as well. Different extraction methods, including dynamic maceration (DM), ultrasound-assisted extraction (UAE), microwave-assisted extraction (MAE) and supercritical-fluid extraction (SFE) were applied and compared in order to obtain a high yield of the target analytes from hemp. Dynamic maceration for 45min with ethanol (EtOH) at room temperature proved to be the most suitable technique for the extraction of cannabinoids in hemp samples. The analysis of the target analytes in hemp extracts was carried out by developing a new reversed-phase high-performance liquid chromatography (HPLC) method coupled with diode array (UV/DAD) and electrospray ionization-mass spectrometry (ESI-MS) detection, by using an ion trap mass analyser. An Ascentis Express C18 column (150mm×3.0mm I.D., 2.7μm) was selected for the HPLC analysis, with a mobile phase composed of 0.1% formic acid in both water and acetonitrile, under gradient elution. The application of the fused-core technology allowed us to obtain a significant improvement of the HPLC performance compared with that of conventional particulate stationary phases, with a shorter analysis time and a remarkable reduction of solvent usage. The analytical method optimized in this study was fully validated to show compliance with international requirements. Furthermore, it was applied to the characterization of nine hemp samples and six hemp-based pharmaceutical products. As such, it was demonstrated to be a very useful tool for the analysis of cannabinoids in both the plant material and its derivatives for pharmaceutical and nutraceutical applications.


2017 - Metabolite fingerprinting of Punica granatum L. (pomegranate) polyphenols by means of high-performance liquid chromatography with diode array and electrospray ionization-mass spectrometry detection [Articolo su rivista]
Brighenti, Virginia; Groothuis, Sebastiaan Frearick; Prencipe, FRANCESCO PIO; Amir, Rachel; Benvenuti, Stefania; Pellati, Federica
abstract

The present study was aimed at the development of a new analytical method for the comprehensive multi-component analysis of polyphenols in Punica granatum L. (pomegranate) juice and peel. While pomegranate juice was directly analysed after simple centrifugation, different extraction techniques, including maceration, heat reflux extraction, ultrasound-assisted extraction and microwave-assisted extraction, were compared in order to obtain a high yield of the target analytes from pomegranate peel. Dynamic maceration with a mixture of water and ethanol 80:20 (v/v) with 0.1% of hydrochloric acid as the extraction solvent provided the best result in terms of recovery of pomegranate secondary metabolites. The quali- and quantitative analysis of pomegranate polyphenols was performed by high-performance liquid chromatography with diode array and electrospray ionization-mass spectrometry detection. The application of fused-core column technology allowed us to obtain an improvement of the chromatographic performance in comparison with that of conventional particulate stationary phases, thus enabling a good separation of all constituents in a shorter time and with low solvent usage. The analytical method was completely validated to show compliance with the International Conference on Harmonization of Technical Requirements for the Registration of Pharmaceuticals for Human Use guidelines and successfully applied to the characterisation of commercial and experimental pomegranate samples, thus demonstrating its efficiency as a tool for the fingerprinting of this plant material. The quantitative data collected were submitted to principal component analysis, in order to highlight the possible presence of pomegranate samples with high content of secondary metabolites. From the statistical analysis, four experimental samples showed a notable content of bioactive compounds in the peels, while commercial ones still represent the best source of healthy juice.


2017 - Metabolite profiling of flavonols and in vitro antioxidant activity of young shoots of wild Humulus lupulus L. (hop) [Articolo su rivista]
Maietti, Annalisa; Brighenti, Virginia; Bonetti, Gianpiero; Tedeschi, Paola; Prencipe, FRANCESCO PIO; Benvenuti, Stefania; Brandolini, Vincenzo; Pellati, Federica
abstract

Humulus lupulus L., commonly named hop, is well-known for its sedative and estrogenic activity. While hop cones are widely characterized, only few works have been carried out on the young shoots of this plant. In the light of this, the aim of this study was to identify for the first time the flavonoids present in young hop shoots and to compare the composition of samples harvested from different locations in Northern Italy with their antioxidant activity. The samples were extracted by means of dynamic maceration with methanol. The HPLC-UV/DAD, HPLC-ESI-MS and MS2 analysis were carried out by using an Ascentis C18 column (250 × 4.6 mm I.D., 5 μm), with a mobile phase composed of 0.1 M formic acid in both water and acetonitrile, under gradient elution. Quercetin and kaempferol glycosides were the main compounds identified and quantified in hop shoot extracts. Total flavonols ranged from 2698 ± 185 to 517 ± 48 μg/g (fresh weight). The antioxidant activity was determined by means of the radical scavenging activity assay against diphenylpicrylhydrazyl (DPPH[rad]) and by using a photochemiluscence assay with a Photochem® apparatus. The results showed that hop shoots represent a new source of flavonols; therefore, they can be useful for a possible incorporation in the diet as a functional food or applied in the nutraceutical ambit.


2017 - Produzioni castanicole e identità territoriale: parliamone! [Capitolo/Saggio]
Durante, Caterina; Ferrari, Erika; Foca, Giorgia; Benvenuti, Stefania; Tassi, Lorenzo
abstract

Il capitolo del volume tratta, in forma divulgativa, il problema della tracciabilità merceologica di alcune varietà castanicole della provincia di Modena. La caratterizzazione chimico-analitica effettuata su diversi campioni rappresentativi di altrettante cultivar geograficamente localizzate, consente la eventuale discriminazione delle varietà medesime. Il volume editato da Artestampa, appartiene ad una Collana che si sviluppa nel tempo, con l'obiettivo di far conoscere, descrivere e rappresentare alcune eccellenze agroalimentari e produzioni specifiche dei territori modenesi.


2017 - Thymbra capitata (L.) cav. and rosmarinus officinalis (L.) Essential oils: in vitro effects and toxicity on swine spermatozoa [Articolo su rivista]
Elmi, A; Ventrella, D; Barone, F; Benvenuti, Stefania; Scozzoli, M; Bacci, Ml
abstract

In the last years, Essential Oils are commanding the attention of the scientific world in a variety of fields because of their peculiar characteristics. Among the reasons behind this growing interest, there is their capability of interfering with bacteria, viruses and fungi. Moreover, they can also act as effective antioxidant and spermicidal agents. The aim of this preliminary study was to analyze the effects of the Essential Oils extracted by Cuminumcyminum and Coriandrumsativum on the membranes of spermatozoa using porcine ejaculates as model, with the future prospective of possible applications in reproductive medicine. Four different concentrations of the oils mix (1:1) were tested on samples of swine spermatozoa alongside with two controls (one with and one without Penicillin). The prepared samples were incubated at 16°C (± 1°C) in a refrigerated bath, and evaluated for Viability and Acrosome Status at three different time points (24, 72 and 120 h). When compared to the control samples, the two lower tested concentrations (0.1 and 0.2 mg/ml) do not seem to alter viability nor acrosome reaction percentage. On the other hand, the two remaining concentrations impair both parameters in a concentration-dependent manner. Overall, these preliminary results prove how this Essential Oils mix can interact with the spermatozoa membranes, both cytoplasmic and acrosomal


2016 - Chemical composition and the anti-melanogenic potential of different essential oils [Articolo su rivista]
Fiocco, Daniela; Arciuli, Marcella; Arena, Mattia Pia; Benvenuti, Stefania; Gallone, Anna
abstract

Tyrosinase is the key enzyme of melanogenesis, the process that determines skin and hair pigmentation in mammals. In humans, various pigmentary disorders depend on excessive melanin production and accumulation. Moreover, tyrosinase activity can cause the undesirable browning of plant-derived food. For such reasons, ongoing researches aim at developing effective melanogenesis inhibitors for medical, cosmetic and food applications. Diverse biological activities have been observed for certain plant essential oils (EOs) and their components. In this study, the EOs from nine different plant species were investigated for their anti-melanogenic potential. The EOs chemical composition was assessed by gas chromatography-mass spectrometry (GC-MS) revealing the presence of some constituents with known tyrosinase inhibitory ability. Most EOs inhibited significantly and dose-dependently mushroom tyrosinase. Two samples also inhibited the activity of tyrosinase from murine B16 melanoma cells. Our findings indicate that some of the analysed EOs may be promising natural sources for developing novel anti-melanogenic agents, thus having a good potential in medical, cosmetic and food-processing industries.


2016 - Chemical composition, cytotoxicity, antimicrobial and antifungal activity of several essential oils [Articolo su rivista]
Cannas, Sara; Usai, Donatella; Tardugno, Roberta; Benvenuti, Stefania; Pellati, Federica; Zanetti, Stefania; Molicotti, Paola
abstract

Essential oils (EOs) are known and used for their biological, antibacterial, antifungal and antioxidant properties. Numerous studies have shown that EOs exhibit a large spectrum of biological activities in vitro. The incidence of drug-resistant pathogens and the toxicity of antibiotics have drawn attention to the antimicrobial activity of natural products, encouraging the development of alternative treatments. The aim of this study was to analyse the phytochemical and the cytotoxic characteristic of 36 EOs; we then evaluated the antimicrobial activity of the less-toxic EOs on Gram-positive, Gram-negative and fungi strains. The results showed low cytotoxicity in seven EOs and good activity against Gram-negative and Candida spp. strains. Based on our results, EOs could be proposed as a novel group of therapeutic agents. Further experiments are necessary to confirm their pharmacological effectiveness, and to determine potential toxic effects and the mechanism of their activity in in vivo models.


2015 - Essential oils in ocular pathology: an experimental study [Articolo su rivista]
Cannas, S; Usai, D; Pinna, Antonio Daniele; Benvenuti, Stefania; Tardugno, Roberta; Donadu, M; Zanetti, S; Kaliamurthy, J; Molicotti, P.
abstract

Introduction: The antimicrobial activity of essential oils (EOs) has been known for ages; in particular, the EOs of Melaleuca alternifolia, Thymusvulgaris, Menthapiperita, and Rosmarinusofficinalishave been used for the treatment of fungal and bacterial infections. Methodology: This study focused on the in vitrocytotoxicity to normal human conjunctiva cells and antimicrobial activity of 20 EOs. Results: The oils tested showed no cytotoxic effect at very low concentrations. Rosmarinusofficinalis, Melaleucaalternifolia, and ThymusvulgarisL. red thyme geraniol sel oils had good antimicrobial activity against Gram-positive and Gram-negativestrains. Conclusions: The results of this study are of great interest and may have a major impact on public health, providing useful tips to optimize the therapeutic use of some natural drugs. INTRODUCTION: The antimicrobial activity of essential oils (EOs) has been known for ages; in particular, the EOs of Melaleuca alternifolia, Thymus vulgaris, Mentha piperita, and Rosmarinus officinalis have been used for the treatment of fungal and bacterial infections. METHODOLOGY: This study focused on the in vitro cytotoxicity to normal human conjunctiva cells and antimicrobial activity of 20 EOs. RESULTS: The oils tested showed no cytotoxic effect at very low concentrations. Rosmarinus officinalis, Melaleuca alternifolia, and Thymus vulgaris L. red thyme geraniol sel oils had good antimicrobial activity against Gram-positive and Gram-negative strains. CONCLUSIONS: The results of this study are of great interest and may have a major impact on public health, providing useful tips to optimize the therapeutic use of some natural drugs.


2015 - HPLC-UV/DAD and ESI-MSn analysis of flavonoids and antioxidant activity of an Algerian medicinal plant: Paronychia argentea Lam [Articolo su rivista]
Sabrina, Sait; Sabrina, Hamri Zeghichi; Lila, Boulekbache Makhlouf; Khodir, Madani; Peggy, Rigou; Brighenti, Virginia; Prencipe, FRANCESCO PIO; Benvenuti, Stefania; Pellati, Federica
abstract

This study was carried out in order to evaluate the effect of the preparation method (decoction and infusion) on the flavonoid profile, phenolic content and antioxidant activity of the aerial parts of Paronychia argentea Lam. (Caryophyllaceae), also known as Arabic tea. Eleven flavonoids were characterized by using high-performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MSn) and quantified by HPLC-UV/DAD; six of them, including isorhamnetin-3-O-dihexoside, quercetin-3-O-glucoside, quercetin methylether-O-hexoside, quercetin, jaceosidin and isorhamnetin, were described in this plant for the first time in this study. The plant ethanol extract (used as a control) showed the highest content of phenolic compounds, followed by the decoction and infusion. The antioxidant activity of the decoction (reducing power, scavenging capacity on DPPH radical and inhibition of lipid peroxidation) was found to be higher than that of the infusion. The decoction obtained from the aerial part of P. argentea can be considered as a protective beverage against oxidative stress.


2014 - An innovative approach for the phytochemical analysis of bioactive compounds in Humulus lupulus L. [Articolo su rivista]
Prencipe, FRANCESCO PIO; Pellati, Federica; Brighenti, Virginia; Benvenuti, Stefania; Bruni, Renato
abstract

In this work a new method was developed, based on a simple sample preparation procedure and a highly efficient HPLC technique, for the metabolite fingerprinting of the bioactive compounds in Humulus lupulus L. The method was fully validated and applied to commercial cultivars and genotypes.


2014 - Characterization of polyphenols in Vaccinium berries and evaluation of their chemopreventive properties [Abstract in Atti di Convegno]
Pellati, Federica; Prencipe, FRANCESCO PIO; Renato, Bruni; Alessandra, Guerrini; Damiano, Rossi; Benvenuti, Stefania
abstract

This study was aimed at the complete metabolite profiling of polyphenols in Vaccinium floribundum Kunth berries from Ecuador by means of HPLC-UV/DAD, HPLC-ESI-MS and MS2, using an ion trap mass analyzer. As a comparison, Vaccinium myrtillus L. berries from the Italian Northern Apennine were analyzed in parallel. A sequential selective extraction procedure was developed to maximize the yields of the secondary metabolites from the berries, using ethyl acetate for flavonoids and phenolic acids, followed by acidified methanol for anthocyanins. The results of the HPLC analyses indicated several representative differences in both the quali- and quantitative composition of flavonoids and anthocyanins in the berry extracts obtained from V. floribundum and V. myrtillus, which showed typical fingerprints of their phenolic secondary metabolites. Thanks to its unique polyphenol composition, V. floribundum can be easily differentiated from common bilberries using the proposed techniques. The chemopreventive activity of these berries and their fractions was evaluated by the determination of their antioxidant activity using a panel of in vitro tests (DPPH, ABTS) and their antimutagenic properties against dietary and environmental, direct and indirect, mutagens. The results of these biological assays were correlated with the chemical composition of the two investigated Vaccinium species, to establish the potential application of V. floribundum as a source of phytochemicals in the nutraceutical and functional food ambit.


2014 - Chemical composition and cytotoxic activity of Cedrelopsis grevei H. Baillon essential oil on human cancer cells [Abstract in Atti di Convegno]
Tardugno, Roberta; Pellati, Federica; Antonella, Spagnoletti; Gianni, Sacchetti; Benvenuti, Stefania
abstract

In this study, a detailed chemical characterization of Cedrelopsis grevei H. Baillon (Ptaeroxylaceae family) bark essential oil was perfomed. The GC analysis revealed a peculiar composition in terms of sesquiterpenes. The major components were found to be ishwarane, β-elemene, α-copaene, calamenene, β-selinene, γ-muurolene, α-muurolene and β-pinene. In addition to semi-quantification data, essential oil quantification was performed by means of the experimental and predicted relative response factors (RRF), using methyl octanoate as the internal standard. In vitro biological activity of C. grevei essential oil and its main isolated compounds (including ishwarane, β-elemene and α-copaene) was assayed for cytotoxic activity on human colon cancer cells CaCo-2 and human lung carcinoma cells A549 by means of the MTT assay. Furthermore, by combining the three main compounds, it was possible to evaluate the potential synergic effects. The results showed that the C. grevei essential oil exhibited interesting cytotoxic activity on both cancer cell lines.


2014 - Innovative methods based on the fused-core technology for the HPLC analysis of natural products [Abstract in Atti di Convegno]
Pellati, Federica; Prencipe, FRANCESCO PIO; Brighenti, Virginia; Tardugno, Roberta; Benvenuti, Stefania
abstract

Plant extracts are composed of a complex mixture of different phytochemicals (plant secondary metabolites). Frequently, these constituents work “synergistically” and cannot be separated into active parts. Consequently, it is necessary to define all the phytochemical constituents of plant extracts to understand the bioactivity and possible adverse effects of active compounds, and to enhance product quality control. Due to the complex composition of plant extracts, separation methods are highly recommended in phytochemical analysis. HPLC is the most frequently used technique for both quali- and quantitative analysis of natural compounds. However, the HPLC analysis of plant extracts usually requires a long analysis time on conventional fully porous stationary phases. The most recently reported strategy for improving the chromatographic performance in the analysis of complex matrices is based on the fused-core technology (also known as core-shell technology). In this ambit, this work provides several examples of the application of reversed-phase fused-core stationary phases for the metabolite profiling/fingerprinting of natural products widely used in phytotherapy, including propolis (phenolic acids and flavonoids), Humulus lupulus L. (prenylflavonoids and prenylphloroglucinols) and Echinacea pallida (Nutt.) Nutt. (polyacetylenes and polyenes). An additional example related to the phytochemical analysis of steviol glycosides in Stevia rebaudiana Bertoni is also provided. In particular, these examples demonstrate that the fused-core columns offer good chromatographic performance, such as narrow peaks, high resolution and a short analysis time.


2014 - Metabolite profiling of polyphenols in Vaccinium berries and determination of their chemopreventive properties [Articolo su rivista]
Prencipe, FRANCESCO PIO; Bruni, Renato; Guerrini, Alessandra; Rossi, Damiano; Benvenuti, Stefania; Pellati, Federica
abstract

A detailed investigation on the chemical composition and chemopreventive activity of Vaccinium floribundum Kunth berries was carried out in comparison with Vaccinium myrtillus L. Berry polyphenols were extracted by using two sequential dynamic maceration steps, which enabled to maximize the yields of secondary metabolites. In particular, phenolic acids and flavonols were extracted from berries using ethyl acetate (EtOAc), whereas anthocyanins were extracted from the residue with 0.6M HCl in methanol (MeOH). The analysis of secondary metabolites in berry extracts was performed by means of two specific HPLC methods. Phenolic acids and flavonols were analyzed on an Ascentis C18 column (250mm×4.6mm I.D., 5μm), with a gradient mobile phase composed of 0.1M HCOOH in H2O and ACN. Anthocyanin analysis was carried out on a Zorbax SB-C18 column (150mm×4.6mm I.D., 5μm), with a gradient mobile phase composed of H2O-HCOOH (9:1, v/v) and MeOH-H2O-HCOOH (5:4:1, v/v/v). Detection was performed by UV/DAD, MS and MS(2). The polyphenol composition of V. floribundum and V. myrtillus was studied in detail. The samples of V. floribundum analyzed in this study had a much higher content of both phenolic acids and flavonols in comparison with V. myrtillus (mean value 41.6±10.2 and 13.7±0.2mg/100g FW, respectively), while V. myrtillus showed a higher amount of anthocyanins if compared with V. floribundum (568.8±8.8 and mean value 376.2±49.9mg/100gFW, respectively). The extracts gave negative results in antimutagenic assays against carcinogens 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) and 4-nitroquinoline-1-oxide (4-NQO), while they performed similarly in both ABTS(+) and DPPH antioxidant assays.


2014 - Olio essenziale di Katrafay (Cedrelopsis grevei H. Baillon): profilo fitochimico e attività biologica [Abstract in Atti di Convegno]
Benvenuti, Stefania
abstract

Cedrelopsis grevei H. Baillon, noto anche come Katrafay, è una pianta aromatica appartenente alla famiglia delle Ptaeroxylaceae, originaria del Madagascar. L’olio essenziale ottenuto dalla corteccia del fusto è largamente utilizzato nella medicina popolare per curare reumatismi, dolori muscolari, come agente antibatterico ed antimicotico; altri estratti sono stati utilizzati per il trattamento della malaria, della febbre e dello stato di affaticamento. Tuttavia, in letteratura non è stata riportata una descrizione approfondita della composizione dell’olio essenziale di C. grevei, probabilmente per la notevole variabilità fra le diverse zone di produzione. Questo studio è pertanto finalizzato alla caratterizzazione chimica completa dell’olio essenziale proveniente dal Madagascar, insieme alla valutazione delle sue attività citotossiche e antimicrobiche verso batteri fitopatogeni. I campioni di olio essenziale, forniti dalla ditta Ophera srl, sono stati caratterizzati e quantificati rispettivamente mediante tecniche GC-MS e GC-FID. Utilizzando i dati di ritenzione e gli spettri di massa sono stati identificati 35 composti, che rappresentano l’80% della composizione totale dell’olio essenziale. I componenti principali determinati nei campioni analizzati in questo studio sono rappresentati da ishwarane, β-elemene, α-copaene, calamenene, β-selinene, γ-muurolene, α-muurolene e β-pinene. L'olio essenziale di C. grevei è stato poi testato per la sua attività citotossica nei confronti di linee cellulari Caco 2, Hep-2, WKD e antimicrobica nei confronti di batteri fitopatogeni di interesse per l'agricoltura biologica, quali Agrobacterium tumefaciens, Agrobacterium vitis, Clavibacter michiganensis sub. michiganensis e Pseudomonas syringae pv syringae. I risultati ottenuti permettono di giungere ad alcune considerazioni in merito sia alla composizione di un o.e. ottenuto da un’insolita droga, quale la corteccia, al suo significato metabolico e sia sull’attività biologica dei sesquiterpeni, componenti maggiormente rappresentati nell’olio essenziale di C. grevei.


2014 - Phytochemical analysis of Cedrelopsis grevei H. Baillon essential oil and evaluation of its antimicrobial and antifungal activity on plant pathogens [Abstract in Atti di Convegno]
Tardugno, Roberta; Pellati, Federica; Gianni, Sacchetti; Renato, Bruni; Benvenuti, Stefania
abstract

Cedrelopsis grevei H. Baillon is an aromatic and medicinal plant belonging to the Ptaeroxylaceae family from Madagascar. In this work, a detailed chemical characterization of the essential oil from the bark of C. grevei was performed, in combination with the evaluation of its antimicrobial activity. In particular, four samples of C. grevei essential oil were fully characterized by GC-MS and quantified by GC-FID. A total of 35 components were identified, according to their retention time data and mass spectra, representing 80% of the amount obtained by semi-quantification using relative response factors (RRFs) = 1. In the samples analyzed in this study, the major components were found to be ishwarane, β-elemene, α-copaene, calamenene, β-selinene, γ-muurolene, α-muurolene and β-pinene. Moreover, in addition to semi-quantification data, for the first time C. grevei essential oil quantification was performed by means of the experimental and predicted RRFs, using methyl octanoate as the internal standard. The biological activity of the essential oil was tested on plant pathogenic bacteria and fungi of interest in the ambit of biological agriculture and horticulture, including Agrobacterium tumefaciens, Agrobacterium vitis, Clavibacter michiganensis sub. michiganensis, Pseudomonas syringae pv syringae, Alternaria sp. and Fusarium oxysporum.


2013 - An efficient chemical analysis of phenolic acids and flavonoids in raw propolis by microwave-assisted extraction combined with high-performance liquidchromatography using the fused-core technology [Articolo su rivista]
Pellati, Federica; Prencipe, FRANCESCO PIO; Bertelli, Davide; Benvenuti, Stefania
abstract

A closed-vessel microwave-assisted extraction (MAE) technique was optimized for the first time for the extraction of polyphenols from raw propolis. The results obtained by means of response surface experimental design methodology showed that the best global response was reached when the extraction temperature was set at 106°C, the solvent composition close to EtOH-H2O 80:20 (v/v), with an extraction time of 15min. In comparison with other techniques, such as maceration, heat reflux extraction (HRE) and ultrasound-assisted extraction (UAE), the extraction with MAE was improved by shorter extraction time and lower volume of solvent needed.The HPLC analyses of propolis extracts were carried out on a fused-core Ascentis Express C18 column (150 mm×3.0 mm I.D., 2.7 μm), with a gradient mobile phase composed by 0.1% formic acid in water and acetonitrile. Detection was performed by DAD and MS.The method validation indicated that the correlation coefficients were >0.999; the limit of detection was in the range 0.5-0.8. μg/ml for phenolic acids and 1.2-3.0. μg/ml for flavonoids; the recovery range was 95.3-98.1% for phenolic acids and 94.1-101.3% for flavonoids; the intra- and inter-day %RSD values for retention times and peak areas were ≤0.3 and 2.2%, respectively.The quali- and quantitative analysis of polyphenols in Italian samples of raw propolis was performed with the validated method. Total phenolic acids ranged from 5.0 to 120.8. mg/g and total flavonoids from 2.5 to 168.0. mg/g.The proposed MAE procedure and HPLC method can be considered reliable and useful tools for the comprehensive multi-component analysis of polyphenols in propolis extracts to be used in apitherapy.


2013 - Caratterizzazione chimica, attività citotossica e antibatterica in vitro di diversi oli essenziali [Relazione in Atti di Convegno]
S., Cannas; D., Usai; P., Molicotti; Pellati, Federica; Tardugno, Roberta; Benvenuti, Stefania; S., Zanetti
abstract

In questo studio è stata valutata l'azione citotossica di 36 oli essenziali appartenenti a diverse specie vegetali nei confronti di tre linee cellulari, quali Caco2 cellule tumorali umane del colon, WKD cellule umane della congiuntiva e J774 macrofagi di origine murina. L'attività citotossica è stata evidenziata utilizzando il saggio con MTT (Sigma), esponendo le cellule a diluizioni v/v dei diversi oli da 16% a 0.125%. Dal pool iniziale, sono stati selezionati 7 oli essenziali che presentavano la minore citotossicità per valutare l'attività antimicrobica. Gli oli in esame erano: Salvia sclarea, Melaleuca alternifolia, Thymus hyemalis e 4 oli di Thymus vulgaris. Sono stati saggiati 2 ceppi di S. aureus, 3 di E. coli, 2 di P. aeruginosa e 3 specie di Candida. L'olio essenziale di Salvia sclarea ha mostrato scarsa attività su tutti i ceppi testati, mentre l'olio essenziale di Melaleuca alternifolia si è rivelato efficace su E. coli, P. aeruginosa e 2 ceppi di Candida su 3. L'olio essenziale di Thymus hyemalis da dato discreti risultati soprattutto su Candida e i restanti 4 oli essenziali di Thymus vulgaris hanno mostrato buona attività su E. coli e Candida. Ciascun olio essenziale è stato sottoposto ad analisi qualitativa e semi-quantitativa, mediante tecniche GC-MS e GC-FID, rispettivamente. La caratterizzazione qualitativa combinata all’analisi semi-quantitativa, ha permesso di disegnare il profilo caratteristico dei 36 oli essenziali oggetto di questo studio e di valutare la correlazione fra attività antibatterica e componenti presenti.


2013 - Caratterizzazione fitochimica della frazione volatile di propoli mediante tecnica HS-SPME-GC-MS [Relazione in Atti di Convegno]
Benvenuti, Stefania; Pellati, Federica; Prencipe, FRANCESCO PIO; I., Fabretto
abstract

Dato il crescente interesse per i prodotti naturali, è importante riuscire a caratterizzarli per definirne nuove applicazioni per la salute, l'agricoltura e l'ambiente. Alcuni di essi, infatti, costituiscono alternative efficaci o complementari a composti sintetici dell'industria chimica, senza mostrarne gli effetti secondari. Fra questi prodotti di origine naturale è da ricordare la propoli, sostanza resinosa raccolta, trasformata e utilizzata dalle api (Apis mellifera L.) per sigillare i fori degli alveari, levigare le pareti interne e ripararsi dagli agenti esterni. Le api raccolgono queste resine da foglie, germogli e corteccia di alcune piante, quali pioppo (Populus sp.), betulla (Betula sp.), quercia (Quescus sp.), ippocastano (Aesculus hippocastanum), ontano (Alnus sp.) e conifere varie. Questa resina viene poi masticata, arricchita di enzimi salivari, parzialmente digerita e successivamente miscelata a cera d’api per essere usata nell’alveare. Per la propoli sono descritte numerose proprietà biologiche quali quella antibatterica, antimicotica, antivirale, antiossidante, anti-infiammatoria, antiproliferativa, immunostimolante. A dispetto di eventuali differenze di composizione dovute alle diverse fonti vegetali, la maggior parte dei campioni di propoli possiede una notevole somiglianza nella natura chimica complessiva. Essa è costituita da: 50% di resina (composta da polifenoli), 30% di cera, 10% di oli essenziali, 5% di polline, 5% di altri composti organici. I composti di interesse farmacologico si trovano nella frazione volatile e nella frazione polifenolica, le quali includono principalmente terpenoidi, steroidi, flavonoidi e acidi fenolici. Scopo del presente lavoro è la caratterizzazione quali e quantitativa dei componenti della frazione volatile della propoli: a tal fine sono state ottimizzate le condizioni di isolamento mediante tecnica SPME in spazio di testa (Head Space Solid Phase Micro Extraction) e quelle di analisi quali- e quantitativa con tecnica GC-MS (Gas Chromatography-Mass Spectrometry) e GC-MS (Gas Chromatography-Mass Spectrometry). In questo modo è stato possibile un confronto tra i campioni di propoli di origine diversa, sia di tipo qualitativo che semi-quantitativo.


2013 - Caratterizzazione fitochimica e attività antimicrobica dell'olio essenziale di Cedrelopsis grevei H. Baillon [Abstract in Rivista]
Tardugno, Roberta; Pellati, Federica; R., Bruni; G., Sacchetti; L., Pecorari; Benvenuti, Stefania
abstract

Cedrelopsis grevei H. Baillon, noto anche come Katrafay, è una pianta aromatica appartenente alla famiglia delle Ptaeroxylaceae, originaria del Madagascar. L’olio essenziale ottenuto dalla corteccia è largamente utilizzato nella medicina popolare per curare reumatismi e dolori muscolari; è anche impiegato come agente antibatterico ed antimicotico. In letteratura non è stata descritta un’indagine approfondita della composizione dell’olio essenziale di C. grevei e le relative proprietà antimicrobiche non sono state investigate in modo dettagliato. Questo studio è stato pertanto finalizzato alla caratterizzazione chimica completa dell’olio essenziale ottenuto dalla corteccia di C. grevei, insieme alla valutazione delle sue attività antimicrobiche verso batteri fitopatogeni. I campioni di olio essenziale, forniti dalla ditta Ophera s.r.l., sono stati caratterizzati e quantificati rispettivamente mediante tecniche GC-MS e GC-FID. Utilizzando i dati di ritenzione e gli spettri di massa sono stati identificati 35 composti, che rappresentano l’80% della composizione totale dell’olio essenziale. L’analisi semi-quantitativa è stata effettuata utilizzando le aree sottese ai picchi cromatografici e applicando fattori di risposta relativi (RRFs) pari a 1. I componenti principali determinati nei campioni analizzati in questo studio sono rappresentati da ishwarane (25.2%), β-elemene (10.3%), α-copaene (8.8%), calamenene (6.2%), β-selinene (3.4%), γ-muurolene (2.6%), α-muurolene (2.3%) e β-pinene (2.0%). La composizione quantitativa dell’olio essenziale è stata inoltre valutata in modo più approfondito utilizzando i valori di RRFs predetti e i corrispondenti sperimentali, con il metil octanoato come standard interno. In virtù della funzione in genere espletata in natura dagli oli essenziali accumulati nei tessuti tegumentali, l'olio essenziale di C. grevei è stata testato per la prima volta per la sua attività antimicrobica nei confronti di batteri fitopatogeni di interesse per l'agricoltura biologica, quali Agrobacterium tumefaciens, Agrobacterium vitis, Clavibacter michiganensis sub. michiganensis e Pseudomonas syringae pv syringae.


2013 - Gas chromatography combined with mass spectrometry, flame ionization detection and elemental analyzer/isotope ratio mass spectrometry for characterizing and detecting the authenticity of commercial essential oils of Rosa damascena Mill. [Articolo su rivista]
Pellati, Federica; Orlandini, Giulia; K. A., van Leeuwen; G., Anesin; Bertelli, Davide; M., Paolini; Benvenuti, Stefania; F., Camin
abstract

RATIONALE The essential oil of Rosa damascena Mill. is known for its fine perfumery application, use in cosmetic preparations and for several pharmacological activities. Due to its high value, it can be easily adulterated with flavors or cheaper oils. This study is aimed at a detailed phytochemical characterization of commercial samples of R. damascena essential oil and at their authenticity assessment. METHODS Nineteen commercial samples of R. damascena essential oil of different geographic origin and an additional authentic one, directly extracted by hydro-distillation from fresh flowers, were considered. GC-MS and GC-FID techniques were applied for the phytochemical analysis of the samples. EA-IRMS (Elemental Analysis–Isotope Ratio Mass Spectrometry) and GC/C(Combustion)-IRMS were used to determine the delta-13C composition of bulk samples and of some specific components. RESULTS Citronellol (28.7-55.3%), geraniol (13.5-27.3%) and nonadecane (2.6-18.9%) were the main constituents of Bulgarian and Turkish essential oils, while those from Iran were characterized by a high level of aliphatic hydrocarbons (nonadecane: 3.7-23.2%). The delta-13C values of bulk samples were between –28.1 and –26.9‰, typical for C3 plants. The delta-13C values of specific components were in the usual range for natural aromatic substances from C3 plants, except for geranyl acetate, which displayed higher values (up to –18‰). These unusual delta-13C values were justified by the addition of a natural cheaper oil from a C4 plant (Cymbopogon martinii, palmarosa), which was found to occur in most of the essential oils. CONCLUSION GC/C-IRMS, in combination with GC-MS and GC-FID, can be considered as an effective and reliable tool for the authenticity control of R. damascena essential oil.


2013 - Headspace solid-phase microextraction-gas chromatography-mass spectrometry characterization of propolis volatile compounds [Articolo su rivista]
Pellati, Federica; Prencipe, FRANCESCO PIO; Benvenuti, Stefania
abstract

In this study, a novel and efficient method based on headspace solid-phase microextraction (HS-SPME), followed by gas chromatography-mass spectrometry (GC-MS), was developed for the analysis of propolis volatile compounds. The HS-SPME procedure, whose experimental parameters were properly optimized, was carried out using a 100. μm polydimethylsiloxane (PDMS) fiber. The GC-MS analyses were performed on a HP-5 MS cross-linked 5% diphenyl-95% dimethyl polysiloxane capillary column (30 m × 0.25 mm I.D., 1.00 μm film thickness), under programmed-temperature elution.Ninety-nine constituents were identified using this technique in the samples of raw propolis collected from different Italian regions. The main compounds detected include benzoic acid (0.87-30.13%) and its esters, such as benzyl benzoate (0.16-13.05%), benzyl salicylate (0.34-1.90%) and benzyl cinnamate (0.34-3.20%). Vanillin was detected in most of the samples analyzed in this study (0.07-5.44%). Another relevant class of volatile constituents is represented by sesquiterpene hydrocarbons, such as δ-cadinene (1.29-13.31%), γ-cadinene (1.36-8.85%) and α-muurolene (0.78-6.59%), and oxygenated sesquiterpenes, such as β-eudesmol (2.33-12.83%), T-cadinol (2.73-9.95%) and α-cadinol (4.84-9.74%). Regarding monoterpene hydrocarbons, they were found to be present at low level in the samples analyzed in this study, with the exception of one sample from Southern Italy, where α-pinene was the most abundant constituent (13.19%). The results obtained by HS-SPME-GC-MS were also compared with those of hydrodistillation (HD) coupled with GC-MS.The HS-SPME-GC-MS method developed in this study allowed us to determine the chemical fingerprint of propolis volatile constituents, thus providing a new and reliable tool for the complete characterization of this biologically active apiary product.


2013 - Innovative methods for the extraction and chromatographic analysis of honey bee products [Abstract in Atti di Convegno]
Pellati, Federica; Prencipe, FRANCESCO PIO; Tardugno, Roberta; Benvenuti, Stefania
abstract

In this study, new and efficient chromatographic techniques were developed for a complete chemical characterization of raw propolis polyphenols and volatile compounds, which are responsible for the biological activity of the extracts widely used in apitherapy. The analysis of polyphenols, including phenolic acids and flavonoids, was performed by RP-HPLC coupled with UV/DAD and MS detection, using the new fused-core technology. Microwave-assisted extraction (MAE) was carried out for these constituents and its parameters were optimized by means of response surface experimental design methodology. The characterization of propolis volatile compounds was based on GC coupled with MS detection. In this ambit, a suitable HS-SPME extraction procedure was developed for the first time using a PDMS fiber. Both these chromatographic techniques were applied to raw propolis samples collected in different Italian regions to determine their metabolite fingerprinting, thus providing new and reliable tools for the complete chemical characterization of this biologically active material.


2013 - Metabolite profiling of cancer preventive polyphenols in a Terminalia chebula Retzius extract [Relazione in Atti di Convegno]
Righi, Davide; Pellati, Federica; Prencipe, FRANCESCO PIO; R., Bruni; Benvenuti, Stefania
abstract

Terminalia chebula (fam. Combretaceae) is a staple ayurvedic remedy and different drugs are prepared from its leaves, roots and bark; alone or in combination with other herbal drugs it represents an esteemed ayurvedic rasayana. The prolonged oral administration of its decoction is considered as a generic detoxifying agent at intestinal and hepatic level and is reputed as a way to “restore normal function” also under the perspective of a reduced risk of cancer, metabolic and cardiovascular diseases. This includes well-being maintenance and the prevention of a wide range of degenerative diseases involving inflammation, carcinogenesis and oxidative stress. Besides that, traditional uses of T. chebula decoction encompasses digestive, tonic, antipyretic, spasmolytic, astringent, expectorant, antiasthmatic, antiviral and hypoglycemic purposes and such bioactivities are related to the presence of polyphenolic compounds. However, no reports are available on the phytochemical profile of the traditional pharmaceutical form actually employed in the ayurvedic medicine and on its chemopreventive properties. In this study, a metabolite profiling of the polyphenol compounds of a T. chebula decoction was performed using HPLC-UV/DAD, HPLC-ESI-MS and MS2 with an ion trap mass analyzer. The analysis were carried out on an Ascentis C18 column (250 × 4.6 mm, 5 µm), with a mobile phase consisting of H2O and acetonitrile, both containing 0.1% HCOOH. The most relevant constituents were isolated by means of preparative chromatographic techniques from the aqueous and organic fractions obtained from the crude extract; the isolated compounds were characterized by relevant MS and MS2 data. In particular, chebulic acid, chebulanin, punicalagin, 1,6-di-O-galloyl-beta-D-glucose and corilagin were isolated from the aqueous phase; gallic acid, 3,4,6-tri-O-galloyl-beta-D-glucose, chebulagic acid, chebulinic acid, 1,2,3,4,6-penta-O-galloyl-beta-D-glucose were isolated from the organic phase. The crude decoction, enriched fractions and purified compounds were also tested for their antimutagenic properties against direct and indirect mutagens and for their ability to modulate the EphA2-ephrinA1 system.


2013 - Metabolite profiling of polyphenols in Vaccinium berries and determination of their chemopreventive properties [Abstract in Atti di Convegno]
Pellati, Federica; Prencipe, FRANCESCO PIO; R., Bruni; D., Rossi; Benvenuti, Stefania
abstract

Berries and their derived products have demonstrated to possess several positive effects on human health, due to their high content of polyphenols and their chemopreventive activity against many degenerative diseases, involving oxidative stress, inflammation and carcinogenesis. Amongst berry fruits, Vaccinium myrtillus L. (bilberry), which is a species native to Europe, has received special attention, due to its traditional use in folk medicine. Several studies have demonstrated the benefits of bilberry in the inhibition of cancer cell growth and management of visual disorders. Vaccinium floribundum Kunth, also called Mortiño, is a species from South America and belongs to the Ericaceae family. V. froribundum berries are widely consumed in Ecuador as the fresh fruit or as processed products. Local communities have also used this plant to treat various medical conditions, including diabetes and inflammation. The chemical composition of V. floribundum has been reported in the literature, but the polyphenol characterization and the biological properties have not been deeply investigated. In this study, the complete metabolite profiling of polyphenols in V. floribundum berries from Ecuador was carried out my means of HPLC-UV/DAD, HPLC-ESI-MS and MS2, using an ion trap mass analyzer. As a comparison, V. myrtillus berries from Italian Northern Apennine were analyzed in parallel. A sequential selective extraction procedure was developed to maximize the yields of the secondary metabolites from the berries, using ethyl acetate for flavonoids and phenolic acids, followed by acidified methanol for anthocyanins. The HPLC analysis of flavonoids and phenolic acids was carried out on an Ascentis C18 column (250 × 4.6 mm, 5 µm), with a gradient mobile phase consisting of H2O with 0.1 M formic acid and acetonitrile. Anthocyanins were analyzed using a Zorbax SB-C18 column (150 × 4.6 mm, 5 µm), with a gradient mobile phase composed by H2O-formic acid (9:1, v/v) and methanol-H2O-formic acid (5:4:1, v/v/v). The results of the HPLC analyses indicated several representative differences in both the quali- and quantitative composition of flavonoids and anthocyanins in the berry extracts obtained from V. floribundum and V. myrtillus, which showed typical fingerprints of their phenolic secondary metabolites. Thanks to its unique polyphenol composition, V. floribundum can be easily differentiated from common bilberries using the proposed techniques. The chemopreventive activity of these berries and their fractions was evaluated by the determination of their antioxidant activity using a panel of in vitro tests (DPPH, ABTS) and their antimutagenic properties against dietary and environmental, direct and indirect, mutagens. The results of these biological assays were finally correlated with the chemical composition of the two investigated Vaccinium species, to establish the potential application of V. floribundum as a source of phytochemicals in the nutraceutical and functional food ambit.


2013 - Metabolite profiling of polyphenols in a Terminalia chebula Retzius ayurvedic decoction and evaluation of its chemopreventive activity [Articolo su rivista]
Pellati, Federica; R., Bruni; Righi, Davide; A., Grandini; M., Tognolini; Prencipe, FRANCESCO PIO; F., Poli; Benvenuti, Stefania; D., Del Rio; D., Rossi
abstract

Ethnopharmacological relevance: The decoction of Terminalia chebula fruit is an ayurvedic remedy whose prolonged or aladministration is prized as a generic intestinal and hepatic detoxifying agent. Its administration is suggested also under the perspective of a reduced risk of cancer, metabolic and cardiovascular diseases. Aim of the study: To evaluate the phytochemical profile and the chemopreventive potential of Terminalia chebula fruit decoction prepared according to the ayurvedic decoction recipe. Materials and methods: The quali-and quantitative metabolite profiling o fpolyphenols was obtained using HPLC–UV/DAD and HPLC-ESI-MS. The crude decoction and purified compounds were tested for their capability to interact with the EphA2-ephrin-A1 system and for their antimutagenic properties against dietary and environmental mutagens (AA, 2-NF, NaN3, and heterocyclic amines IQ, MeIQ, MeIQx, Glu-P1,Glu-P2) in the Ames–Salmonella/microsome assay, with and without enzymatic induction. Results: The decoction was found to contain 3,4,6-tri-O-galloyl-D-glucose (55.87 mg/g), chebulic acid (54.03 mg/g), β-punicalagin (41.25mg/g), corilagin (40.31mg/g), α-punicalagin (35.55 mg/g), chebulagic acid (29.09 mg/g), gallic acid (27.96 mg/g), 1,3,4,6-tri-O-galloyl-β-D-glucose (24.25 mg/g), chebulinic acid (20.23 mg/g), 1,2,3,4,6-penta-O-galloyl-D-glucose (13.53 mg/g), ellagic acid (8.00 mg/g), 1,6-di-O-galloyl-D-glucose (4.16 mg/g). An inhibitory effect was recorded in both Salmonella typhimurium TA98 and TA100 strains against the mutagenic activity of heterocyclic amines (22–61%), promutagenAA (91–97%) and directly actingmutagen 2-NF (52%) with but not against NaN3 (7%). Galloyl derivatives allowed an inhibition of mutagenicity induced by MeIQ above 80% at 0.01mol/plate. Both decoction and purified compounds were able to modulate the EphA2-ephrin A1 system,suggesting a potential multiple chemopreventive mechanism. Conclusions: The traditional ayurvedic decoction of Terminalia chebula may harbour a potential as a safe and low-costchemopreventive agent at the intestinal level, if administered according to the ayurvedic specifications. Moreover,its recourse may enhance the presence of some polyphenolic constituents.


2013 - Metabolite profiling of propolis polyphenols by microwave-assisted extraction combined with high-performance liquid chromatography using the fused-core technology [Relazione in Atti di Convegno]
Prencipe, FRANCESCO PIO; Pellati, Federica; Benvenuti, Stefania
abstract

The chemical composition of propolis extracts is known to be very complex and influenced by several factors, such as the geographic origin. In propolis from temperate zones, the most representative biologically active compounds are polyphenols, including phenolic acids and flavonoids. Raw propolis can not be used as crude material, but it must be purified by solvent extraction to remove the useless material and preserve the active polyphenol fraction. In this study, a closed-vessel microwave-assisted extraction (MAE) technique was optimized for the first time for the extraction of phenolic acids and flavonoids from raw propolis. The optimal extraction conditions were determined by means of response surface experimental design methodology. In comparison with other extraction techniques, such as maceration, heat reflux extraction (HRE) and ultrasound-assisted extraction (UAE), the extraction with MAE was improved by shorter extraction time and lower volume of solvent needed. The HPLC analyses of propolis extracts were carried out for the first time on a fused-core Ascentis Express C18 column (150 mm × 3.0 mm I.D., 2.7 µm, Supelco), with a mobile phase composed by 0.1% formic acid in water and acetonitrile. Detection was performed by UV/DAD and ion trap mass analyzer. A total of thirty-eight polyphenol compounds were identified in propolis samples, on the basis of the UV, MS and MS2 data. In comparison with a conventional fully porous stationary phase, the fused-core one allowed a good separation of polyphenols and a reduction of both time and solvent usage. The method validation, performed in agreement with ICH guidelines, indicated that the correlation coefficients were > 0.999; the limit of detection (LOD) was in the range 0.5-0.8 ug/mL for phenolic acids and 1.2-3.0 ug/mL for flavonoids; the recovery range was 95.3-98.1% for phenolic acids and 94.1-101.3% for flavonoids; the intra- and inter-day %RSD values for retention times and peak areas were found to be ≤ 0.3 and 2.2%, respectively. The quali- and quantitative analysis of polyphenols in Italian samples of raw propolis was performed with the validated method. Total phenolic acids ranged from 5.0 to 120.8 mg/g and total flavonoids from 2.5 to 168.0 mg/g. The proposed MAE procedure and HPLC method can be considered reliable and useful tools for the comprehensive multi-component analysis of polyphenols in raw propolis extracts to be used in apitherapy.


2012 - Chromatographic techniques for metabolite fingerprinting of propolis polyphenols and volatile compounds [Abstract in Atti di Convegno]
Prencipe, FRANCESCO PIO; Pellati, Federica; Benvenuti, Stefania
abstract

This study was aimed at developing advanced chromatographic methods for a complete phytochemical characterization of raw propolis polyphenols and volatile compounds, which are responsible for the biological activity of the extracts widely used in phytotherapy. The study of polyphenols was based on RP-HPLC coupled with DAD and MS detection, using the new fused-core column technology, which allowed a sensitive reduction in the total analysis time in comparison with conventional particulate stationary phases. The HPLC analyses were carried out on an Ascentis Express C18 column (150 mm × 3.0 mm I.D., 2.7 µm), with a mobile phase composed by 0.1% aqueous formic acid and ACN, under gradient elution. Different extraction methods were compared, including sonication and microwave extraction, in order to obtain a high recovery of polyphenols from raw propolis. The study of volatile compounds was based on GC coupled with MS detection. A suitable HS-SPME extraction procedure, whose experimental parameters were properly optimized, was developed using a 1 cm PDMS fiber. The GC analyses were carried out on an HP-5 MS cross-linked 5% diphenyl-95% dimethyl polysiloxane capillary column (30 m × 0.25 mm I.D., 1.00 µm film thickness), under programmed-temperature elution. The volatile compounds extracted from raw propolis were identified by comparing the MS fragmentation patterns with those of pure compounds and by mass spectrum database search using NIST. The chromatographic methods developed in this study were applied to samples of raw propolis collected in different Italian regions and allowed us to determine their metabolite fingerprinting, thus providing new and reliable tools for the complete phytochemical characterization of this material.


2012 - Development of advanced chromatographic methods for a complete phytochemical characterization of raw propolis polyphenols and volatile compounds [Abstract in Atti di Convegno]
Prencipe, FRANCESCO PIO; Pellati, Federica; Benvenuti, Stefania
abstract

Propolis, or bee glue, is a dark-colored resinous substance collected by honeybees (Apis mellifera L.) from several tree species. Because of its biological and pharmacological properties, such as antibacterial, antiviral, antifungal, anticancer, anti-inflammatory, and immunomodulatory activities, propolis has attracted researchers’ interest in the last decades. Since propolis is used extensively, a rapid and efficient method for the extraction and analysis of propolis is certainly very useful. With the aim to determine polyphenols in propolis we have applied RP-HPLC coupled with DAD and MS detection, using the new fused-core column technology, which allowed a sensitive reduction in the total analysis time in comparison with conventional particulate stationary phases. The HPLC analyses were carried out on an Ascentis Express C18 column (150 mm × 3.0 mm I.D., 2.7 µm), with a mobile phase composed by 0.1% aqueous formic acid and acetonitrile, under gradient elution. Different extraction methods were compared, including sonication and microwave extraction, in order to obtain a high recovery of polyphenols from raw propolis. The other active component of propolis includes volatile compounds and its analysis was performed by GC coupled with MS detection. A suitable HS-SPME extraction procedure, whose experimental parameters were properly optimized, was developed using a 1 cm PDMS fiber. The GC analyses were carried out on an HP-5 MS cross-linked 5% diphenyl-95% dimethyl polysiloxane capillary column (30 m × 0.25 mm I.D., 1.00 m film thickness), under programmed-temperature elution. The volatile compounds extracted from raw propolis were identified by comparing the MS fragmentation patterns with those of pure compounds and by mass spectrum database search using NIST. The chromatographic methods developed in this study were applied to samples of raw propolis collected in different Italian regions and allowed us to determine their metabolite fingerprinting, thus providing new and reliable tools for the complete phytochemical characterization of this material.


2012 - Isolation and characterization of cancer preventive polyphenolic compounds from a Terminalia chebula Retzius extract [Abstract in Atti di Convegno]
D., Righi; Pellati, Federica; Benvenuti, Stefania; Orlandini, Giulia; M., Tognolini; R., Bruni
abstract

In this study, a bioassay-guided fractionation was carried out from a Terminalia chebula Retzius extract to isolate and characterize the polyphenolic compounds that were tested for their cancer preventive activities.


2012 - Isolation, structure elucidation, synthesis and cytotoxic activity of polyacetylenes and polyenes from Echinacea pallida [Relazione in Atti di Convegno]
Pellati, Federica; Benvenuti, Stefania; Prati, Fabio; P., Nieri
abstract

This study focuses on presenting an overview of recent results on the cytotoxic activity of polyacetylenes and polyenes isolated from Echinacea pallida. In a search for biologically active compounds from plants of the genus Echinacea, the lipophilic extract from E. pallida roots was characterized by a higher cytotoxic activity if compared with the other tested species. A subsequent bioassay-guided fractionation allowed the isolation and structure elucidation of ten polyacetylenes and polyenes from E. pallida roots. The isolated secondary metabolites were tested for their cytotoxic activity on selected human cancer cell lines and (8Z,13Z)-pentadeca-8,13-dien-11-yn-2-one was the most active constituent, particularly on the colonic COLO320 cancer cells (IC50 = 2.3 ± 0.3 μM) and breast carcinoma MCF-7 cancer cells (IC50 = 2.5 ± 0.7 μM). Arrest of cell cycle in the G1 phase and induction of apoptosis were found to be involved in its mechanism of action. Due to the difficult purification of this compound from the plant material, its first total synthesis was also described. A HPLC stability study of this natural product finally indicated that its cytotoxic activity can be mainly attributed to the genuine, not oxidized, molecule. These results indicated that polyacetylenes and polyenes from E. pallida are effective in inhibiting cancer cell proliferation and incorporation of enriched fractions of these compounds in the diet may be useful for cancer prevention. The most active compound represents also an interesting lead structure for the development of new antiproliferative agents.


2012 - Metabolite fingerprinting of bioactive compounds in Echinacea pallida by high-performance liquid chromatography with diode array and electrospray ionization-mass spectrometry detection [Abstract in Atti di Convegno]
Pellati, Federica; Orlandini, Giulia; Prencipe, FRANCESCO PIO; Benvenuti, Stefania
abstract

This study was aimed at developing a novel, reliable and fully validated method for the simultaneous analysis of biologically active caffeic acid derivatives and polyacetylenes/polyenes in Echinacea pallida (Nutt.) Nutt. plant material and natural products by HPLC-UV/DAD, HPLC-ESI-MS and MS/MS using ion trap (IT) and triple quadrupole (TQ) mass analyzers. Even if it is more difficult to optimize the separation of such different constituents in one run, a method for the simultaneous determination of both caffeic acid derivatives and polyacetylenes/polyenes in E. pallida is useful because it can reduce both the time and the sample size required for the analysis. In addition, a technique able to provide a complete fingerprint of the secondary metabolites is of great interest in the ambit of phytochemical analysis and quality control of E. pallida raw material and derivatives, widely used in phytotherapy. The HPLC analyses were carried out on an Ascentis C18 column (250 mm × 4.6 mm I.D., 5 um), with a mobile phase composed by H2O and ACN both containing 0.1% formic acid, under gradient elution. A relevant novel aspect of the proposed technique is that the combination of MS and MS/MS data with retention times and UV spectra made the peak identification very reliable. The fragmentation patterns of caffeic acid derivatives and polyacetylenes/polyenes obtained by IT and TQ are discussed in detail for the first time in the present work. The oxidation mechanism of monocarbonylic acetylenes is demonstrated for the first time in this study using MS and MS/MS data. The practical applicability of the technique was demonstrated by the quantitative analysis of E. pallida root extracts and commercially available dietary supplements to provide reliable chromatographic fingerprints of their bioactive secondary metabolites.


2012 - Metabolite fingerprinting of raw propolis from different Italian regions by innovative HPLC-ESI-MS and GC-MS [Abstract in Atti di Convegno]
Prencipe, FRANCESCO PIO; Pellati, Federica; Benvenuti, Stefania
abstract

This study was aimed at a detailed chromatographic analysis of propolis samples by means of the development and application of innovative HPLC-ESI-MS and HS-SPME-GC-MS techniques to provide a complete fingerprinting of the biologically active components.


2012 - Metabolite profiling of polyphenols in a Terminalia chebula Retzius extract and evaluation of cancer preventive activities [Abstract in Atti di Convegno]
Pellati, Federica; Benvenuti, Stefania; D., Righi; Orlandini, Giulia; Prencipe, FRANCESCO PIO; M., Tognolini; R., Bruni
abstract

Medicinal plants have been employed for several centuries as the main source of biologically active compounds for pharmaceutical and health purposes. A valuable research strategy for the discovery of active compounds from plants is represented by the validation of ethnopharmacological data by means of a scientific and rational approach. Following this concept, a decoction of Terminalia chebula Retzius, obtained from the pericarp of ripe fruits (called in Bengali language Haritaki), was selected in this study to evaluate its polyphenol profile and its cancer preventive properties. This herbal drug is used alone or as an ingredient of preparations like Triphala, an esteemed Ayurvedic rasayana formulation whose usual intake through the diet is reputed as a way to restore normal function, also under the perspective of a reduced risk of cancer, metabolic and cardiovascular diseases. Besides that, traditional use of T. chebula decoction encompasses purgative, mild laxative, digestive, tonic, hepatoprotective, antipyretic, spasmolytic, astringent, expectorant, antiasthmatic, antiviral and hypoglycemic purposes. The T. chebula fruit extract can be obtained with different methods, with the consequence of a considerable variability in the chemical composition. In any case, the traditional extraction method is represented by the decoction of the powdered fruits. Many polyphenols have recently been identified in this plant, but no reports are available on the phytochemical profile of the traditional drug, which is the pharmaceutical form actually employed in the ayurvedic medicine. In this study, a metabolite profiling of the polyphenol compounds of a T. chebula decoction was performed using HPLC-UV/DAD, HPLC-ESI-MS and MS2 with an ion trap mass analyzer. The analysis were carried out on a C18 column (250 × 4.6 mm, 5 µm), with a mobile phase consisting of H2O and acetonitrile, both containing 0.1% HCOOH. The most relevant constituents were isolated by means of preparative chromatographic techniques from the aqueous and organic fractions obtained from the crude extract; the isolated compounds were characterized by relevant MS and MS2 data. In particular, chebulic acid, chebulanin, punicalagin, 1,6-di-O-galloyl-beta-D-glucose and corilagin were isolated from the aqueous phase; gallic acid, 3,4,6-tri-O-galloyl-beta-D-glucose, chebulagic acid, chebulinic acid, 1,2,3,4,6-penta-O-galloyl-beta-D-glucose were isolated from the organic phase. The crude decoction, enriched fractions and purified compounds were then tested for their antimutagenic properties against direct and indirect mutagens and for their ability to modulate the EphA2-ephrinA1 system.


2012 - Simultaneous metabolite fingerprinting of hydrophilic and lipophilic compounds in Echinacea pallida by high-performance liquid chromatography with diode array and electrospray ionization-mass spectrometry detection [Articolo su rivista]
Pellati, Federica; Orlandini, Giulia; Benvenuti, Stefania
abstract

In this study, a detailed phytochemical characterization of Echinacea pallida (Nutt.) Nutt. root extracts and dietary supplements was carried out for the first time by developing advanced chromatographic techniques, based on HPLC with diode array (DAD) and electrospray ionization-mass spectrometry (ESI-MS) detection (with ion trap and triple quadrupole mass analyzers), for the simultaneous analysis of hydrophilic and lipophilic secondary metabolites. The HPLC analyses were carried out on an Ascentis C18 column (250 mm × 4.6 mm I.D., 5 um), with a mobile phase composed by H2O and ACN both containing 0.1% formic acid, under gradient elution. The UV spectra, in combination with MS and MS/MS data, allowed the identification of fourteen compounds, including caffeic acids derivatives, polyacetylenes and polyenes, in the analyzed samples. MS and MS/MS data were discussed in detail and the typical fragmentation patterns of each class of secondary metabolites were identified. For the first time, a hydroperoxide intermediate was characterized as an oxidation product of one of E. pallida monocarbonilyc acetylenes, providing a confirmation of the mechanism that leads to the generation of hydroxilated derivatives. The HPLC method was fully validated in agreement with ICH guidelines and then applied to real samples. The quantitative analysis indicated that there was a great variability in the amount of the active compounds in the dietary supplements containing E. pallida root extracts: the content of total caffeic acid derivatives ranged from 2.31 to 11.45 mg/g and the amount of total polyacetylenes and polyenes from 6.38 to 30.54 mg/g. In the analyzed samples, the most abundant caffeic acid derivative was found to be echinacoside. Regarding polyacetylenes and polyenes, the most representative compounds were found to be tetradec-(8Z)-ene-11,13-diyn-2-one, pentedeca-(8Z,11Z)-dien-2-one and pentadec-(8Z)-en-2-one. The developed method can be considered suitable for metabolite fingerprinting and quality control of E. pallida plant material and natural products.


2011 - Antifungal Activity of Some Essential Oils against Multi-Resistant Candida Strains [Abstract in Atti di Convegno]
Rossi, Tiziana; Benvenuti, Stefania; Pellati, Federica; A. I., Ruberto; E., Bortolazzi; Baroni, Lorenza; A., Fabio
abstract

Resistance to oral antifungal drugs, such as fluconazole, ketoconazole, itraconazole for example, becomes a problem when the drugs are used or as prophylaxis or for frequent short-term exposures. Candida is a common fungus that is normally controlled by the immune system, but when immune system is suppressed, Candida can grow on mucous membranes or elsewhere in the body, causing symptoms known as candidiasis. HIV positive and HIV negative people may experience candidiasis and more and more strains become resistant to antifungal drugs. Very recent studies (Luqman et al.,2007) demonstrate that rosemary oil is a potential alternative compound to treat drug-resistant infections. Zambonelli (2004) demonstrated the fungicidal activity of Thymus vulgaris essential oil and the data is confirmed by the Italian Society of Gynecology and Obstetrics. In the present study we performed a screening on a series of essential oils with the aim to find new antifungal agents. Materials and Methods: Essential oils from Anethus graveolens, Mentha piperita, Rosmarinus officinalis and Thymus vulgaris were tested against multi-resistant strains of Candida (albicans, parapsilosis, krusei, glabrata, tropicalis, dubliniensis and guillermondi) from clinical specimens from HIV-seropositive subjects kindly supplied by the Department of Medicine Laboratory-Ospedale Policlinico-Modena. Qualitative analysis of the essential oils was performed by a gas chromatograph 6890-N, quantitative analysis by a gas chromatograph DANI 86-10 and a flame ionization detector (FID). Strains were cultured in Sabouraud Dextrose Liquid Medium (pH 5,6 ± 0,2 at 25°C). Fluconazole was chosen as reference drug. Sensitivity tests were performed in according with the NCCLS method for agar dilution and MIC values (minimal inhibitory concentration) were calculated. Results: Essential oils, characterized by variable percentages of p-cymene, gamma therpinene, tymol, sabinene and 1-8 cyneol, were diluted, added to Sabouraud dextrose liquid medium and evaluated for the antifungal activity in comparison with fluconazole (dilution from 200 to 0.1 mg/l). Thymus, Mentha and Rosmarinus appear to be the most active oils since the antifungal activity is maintained with very low dilution (>1000 of the mother solution) over 24 h and against the most of strains but in particular against C. albicans, C. tropicalis and C. krusei this latter responsible of fungal infections in newborns and small infants. On the same strains, fluconazole MIC is >100mg/l thus showing the presence of strong resistance to the drug. The very high percentage of 1-8 cyneol (14-25%), in Rosmarinus oil could represent a safety index in that the molecule is non toxic on Vero cell cultures, but the data is referred to the isolated compound. Conclusion: At the present we are unable to identify a compound responsible of the anti-fungal activity. No compound is present at the same percentage in all the oils we studied. The results we observed may be due or to the phytocomplex or to a molecule present even at very low concentration. The phytocomplex could reduce the toxicity of isolated compounds as camphora, camphene or alpha pynene. The interesting result is the very good antifungal activity exerted at very low dilutions. Further studies are to be performed to investigate both on the mechanism of action and to the presence of side effects.


2011 - Chromatographic Methods for Metabolite Profiling of Virus- and Phytoplasma-Infected Plants of Echinacea purpurea [Articolo su rivista]
Pellati, Federica; F., Epifano; N., Contaldo; Orlandini, Giulia; L., Cavicchi; S., Genovese; Bertelli, Davide; Benvenuti, Stefania; M., Curini; A., Bertaccini; M. G., Bellardi
abstract

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 MS2. Quantitative analysis of these compounds wascarried 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 therapeuticefficacy of this herbal drug.


2011 - HPLC-DAD and HPLC-ESI-MS/MS methods for metabolite profiling of propolis extracts [Articolo su rivista]
Pellati, Federica; Orlandini, Giulia; Pinetti, Diego; Benvenuti, Stefania
abstract

In this study, the composition of polyphenols (phenolic acids and flavonoids) in propolis extracts was investigated by HPLC-DAD and HPLC-ESI-MS/MS by comparing the performance of ion trap and triple quadrupole mass analyzers. The analyses were carried out on an Ascentis C18 column (250 mm×4.6 mm I.D., 5 um), with a mobile phase composed by 0.1% formic acid in water and acetonitrile. Overall, the UV spectra, the MS and MS/MS data allowed the identification of 40 compounds. In the case of flavonoids, the triple quadrupole mass analyzer provided more collision energy if compared with the ion trap, originatingproduct ions at best sensitivity.The HPLC method was validated in agreement with ICH guidelines: the correlation coefficients were >0.998; the limit of detection was in the range 1.6–4.6 ug/ml; the recovery range was 96–105%; the intra- and inter-day %RSD values for retention times and peak areas were found to be <0.3 and 1.9%, respectively.The developed technique was applied to the analysis of hydroalcoholic extracts of propolis available on the Italian market. Although the chromatographic profile of the analyzed samples was similar, the quantitative analysis indicated that there is a great variability in the amount of the active compounds: the content of total phenolic acids ranged from 0.17 to 16.67 mg/ml and the level of total flavonoids from 2.48 to 41.10 mg/ml. The proposed method can be considered suitable for the phytochemical analysis of propolis extracts used in phytotherapy.


2011 - Lavender and peppermint essential oils as effective mushroom tyrosinase inhibitors: a basic study [Articolo su rivista]
D., Fiocco; D., Fiorentino; L., Frabboni; Benvenuti, Stefania; Orlandini, Giulia; Pellati, Federica; A., Gallone
abstract

Screening and characterization of novel tyrosinase inhibitors is useful for applications in food technology, cosmetics and medicine. The hydrodistilled essential oils from lavender Lavandula spica L. and peppermint Mentha x piperita L.were investigated for tyrosinase inhibitory activity. Their composition was assessed by gas chromatography–massspectrometry. Both oils inhibited mushroom tyrosinase in a dose dependent manner. The IC50 values were estimated,kinetics were analysed and Ki values were determined. Our results indicate that lavender and peppermint essential oilsmay be promising herbal ingredients for developing depigmenting agents in clinical, cosmetic and industrial processes.


2011 - SIMULTANEOUS METABOLITE PROFILING OF HYDROPHILIC AND LIPOPHILIC COMPOUNDS IN ECHINACEA PALLIDA BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY WITH PHOTODIODE ARRAY AND ELECTROSPRAY IONIZATION-MASS SPECTROMETRY DETECTION [Abstract in Atti di Convegno]
Orlandini, Giulia; Pellati, Federica; Prencipe, FRANCESCO PIO; Benvenuti, Stefania
abstract

Echinacea spp. (family Asteraceae) herbal medicines and dietary supplements are traditionally used as immunostimulants in the prevention and treatment of inflammatory and viral diseases . Despite of many studies that have shown the chemical composition of alkamides in medicinally important species such as E. purpurea and E. angustifolia, in this study the attention was focused on secondary metabolites contained in E. pallida. In particular, caffeic acids derivatives, that have demonstrated to possess antioxidant, antiviral and anti-inflammatory activities, have been identified from the hydrophilic fractions of E. pallida extracts, while polyacetylenes and polyenes, that have shown interesting cytotoxic activities against a number of solid and leukemic cancer cell lines, have been isolated and characterized from lipophilic extracts of E. pallida roots.In this study, a detailed phytochemical characterization of E. pallida extracts and herbal medicines was carried out for the first time by developing advanced chromatographic techniques, based on RP-HPLC coupled with diode array (DAD) and electrospray ionization-mass spectrometry (ESI-MS) detection, for the simultaneous analysis of the hydrophilic and lipophilic secondary metabolites. In the context of mass spectrometry detection, the performance of two mass analyzers, such as an ion trap and a triple quadrupole, were compared. The HPLC analyses were carried out on an Ascentis C18 column (250 mm × 4.6 mm I.D., 5 um), with a mobile phase composed by 0.1% aqueous formic acid and ACN with 0.1% of formic acid, under gradient elution. The flow rate was 1.0 mL/min and the column temperature was set at 30 °C. The UV spectra, in combination with MS and MS/MS data, allowed the identification of ten compounds, including caffeic acids derivatives, polyacetylenes and polyenes, in the analyzed samples. MS and MS/MS data were discussed in details and the typical fragmentation patterns of each class of secondary metabolites were identified.The RP-HPLC method was then fully validated in agreement with the ICH (Q2-R1) guidelines and then applied to real samples. The reference compounds used for quantification were isolated from the plant material and their structures were determined on the basis of the analysis of UV, IR, NMR and MS data. The quantitative analysis indicated that there was a great variability in the amount of the active compounds in the herbal medicines: the content of total caffeic acid derivatives ranged from 2.02 to 11.05 mg/g and the level of total polyacetylenes and polyenes from 9.62 to 39.12 mg/g. In the analyzed samples, the most abundant caffeic acid derivative was found to be echinacoside. Regarding polyacetylenes and polyenes, the most representative compounds were found to be pentadec-8-en-2-one, pentadeca-8,11-dien-2-one and tetradec-8-ene-11,13-diyn-2-one .The chemical standardization of E. pallida extracts is necessary to guarantee the quality, efficacy and safety of the corresponding herbal medicines. In this context, the proposed method, allowing the simultaneous determination of both the hydrophilic and lipophilic compounds contained in E. pallida, can be considered a useful and reliable tool for the metabolite profiling of plant material and the quality control of natural products


2010 - Cytotoxic activity and G1 cell cycle arrest of a dienynone from Echinacea pallida [Articolo su rivista]
A., Chicca; B., Adinolfi; Pellati, Federica; Orlandini, Giulia; Benvenuti, Stefania; P., Nieri
abstract

In the present study, a further investigation of the cytotoxic activity of an acetylenic constituent of Echinacea pallida roots, namely, pentadeca-(8 Z,13 Z)-dien-11-yn-2-one, was performed, revealing a concentration-dependent cytotoxicity on several human cancer cell lines, including leukemia (Jurkat and HL-60), breast carcinoma (MCF-7), and melanoma (MeWo) cells. As part of its mechanism of action, the ability of this constituent to arrest the cell cycle in the G1 phase was demonstrated on HL-60 cells. Furthermore, a stability test of the target compound over 72 h was carried out, indicating that the cytotoxic activity can be attributed mainly to the genuine, not oxidized, molecule.


2010 - Gestione della flora infestante con l'utilizzo dell'olio essenziale di menta [Articolo su rivista]
L., Frabboni; F., Cristella; V., Russo; A. M., Tomaiulo; Benvenuti, Stefania; Orlandini, Giulia; L., Ciciretti
abstract

L’accresciuta consapevolezza della necessità di attivaresistemi produttivi in agricoltura più rispettosidell’ambiente stimola l’interesse a rivolgersi alla ricercaper studiare sostanze di origine naturale per ladifesa delle colture.Negli oli essenziali sono contenute molte sostanze allelopatiche.In particolare l’olio essenziale di menta(Menta x piperita L.) risulta essere fitotossico neiconfronti di alcune colture.Per questi motivi si è voluto impostare una prova sperimentalein provincia di Foggia per valutare le proprietàallelopatiche dell’olio essenziale di menta neiconfronti delle malerbe. I risultati hanno confermatoil potere bioerbicida dell’olio in oggetto. Amaranthusretroflexus L., Portulaca oleracea L., Convolvulusarvensis L., Eruca sativa L., Matricaria chamomillaL., Papaver roeas L. sono le specie che hanno subitodi più l’effetto allelopatico dell’estratto di menta.


2010 - Isolation, structure elucidation, synthesis and cytotoxic activity of polyacetylenes and polyenes from Echinacea pallida [Abstract in Atti di Convegno]
Pellati, Federica; A., Chicca; B., Adinolfi; Orlandini, Giulia; Prati, Fabio; P., Nieri; Benvenuti, Stefania
abstract

Lipophilic extracts of Echinacea roots were screened for their cytotoxic activity on human cancer cell lines and E. pallida was found to be the most active. Ten polyacetylenes and polyenes were then isolated by bioassay-guided fractionation from E. pallida, characterized and tested for cytotoxic activity. (8Z,13Z)-Pentadeca-8,13-dien-11-yn-2-one was the most active constituent, particularly on the colonic COLO 320 cancer cells (IC50 = 2.3 ± 0.3 uM). The cytotoxicity of this compound was also tested on breast carcinoma MCF-7 (IC50 = 2.5 ± 0.7 uM), melanoma MeWo (IC50 = 28.6 ± 2.3 uM) and pancreatic MIA PaCa-2 cancer cells (IC50 = 32.2 ± 3.9 uM). Apoptotic cell death was found to be involved in its mechanism of action. The total synthesis of this secondary metabolite was also described. A HPLC stability study of this polyenic compound indicated that the cytotoxic activity can be mainly attributed to the genuine not oxidized molecule.


2010 - La scienza delle piante officinali: passato, presente e futuro [Altro]
Benvenuti, Stefania; P., Paltrinieri; Pellati, Federica; Romagnoli, Carlo; Rossi, Tiziana; Zanoli, Paola
abstract

Il Congresso “La scienza delle piante officinali: passato, presente e futuro” si è svolto presso il Centro Servizi Didattici della Facoltà di Medicina e Chirurgia e si è proposto come un’opportunità di incontro fra la realtà del settore produttivo e quello della formazione, della ricerca e dell’innovazione nello studio delle piante officinali e dei principi biologicamente attivi in esse contenuti. Non sono mancati i riferimenti storici e culturali dal momento che l’Archivio di Stato e la Biblioteca Estense di Modena e hanno aderito a tale iniziativa e hanno ospitato una sezione di questo congresso, in cui saranno presentate opere e documenti relativi al mondo delle piante officinali.La Facoltà di Farmacia, che ha da anni istituito un corso di laurea di primo livello in Tecniche Erboristiche, è attivamente impegnata nella formazione di esperti del settore delle piante officinali e ha laureato numerosi giovani che hanno apportato in questo ambito un notevole contributo culturale e professionale. L’attività didattica è sempre stata accompagnata da una continua e approfondita attività di ricerca dei Docenti, che hanno lavorato in stretta collaborazione con aziende del territorio, nazionali ed internazionali. Ne sono testimonianza le numerose pubblicazioni scientifiche prodotte dai Ricercatori del nostro Ateneo in questo campo di indagine.Patrocinato dalla Società Botanica Italiana, dalla Società di Farmacognosia, da CO.N.P.T.ER e da Comune e Provincia di Modena, oltre che dai Dipartimenti Universitari di Scienze Farmaceutiche e di Scienze Biomediche, che hanno ricevuto il sostegno della Fondazione Cassa di Risparmio di Modena e di aziende e società del settore, il convegno ha riunito docenti ed esperti provenienti da tutta Italia per discutere di aspetti legati alla coltivazione delle piante officinali, al loro utilizzo nella formulazione di prodotti erboristici, fitoterapici e fitocosmetici, nonché dell’importanza del controllo di qualità e della fitovigilanza.


2010 - Metabolic profiling di piante officinali affette da agenti patogeni [Relazione in Atti di Convegno]
Pellati, Federica; R., Bruni; Benvenuti, Stefania
abstract

In questo studio sono stati valutati gli effetti di agenti patogeni quali i fitoplasmi sulla produzione di metaboliti secondari di piante officinali, quali Hypericum perforatum L. e Digitalis lanata Ehrl. L’analisi genetica ha rivelato la presenza di fitoplasmi appartenenti al gruppo 16SrVII nel caso dell’iperico e al gruppo 16SrI-B nel caso della digitale. Nell’ambito del metabolic profiling dell’iperico, estratti metanolici di piante infette e sane sono stati analizzati mediante RP-HPLC al fine di quantificare i componenti appartenenti alle classi dei flavonoidi e dei naftodiantroni, mentre l’olio essenziale è stato analizzato tramite GC e GC/MS [1]. I risultati hanno permesso di evidenziare che nelle piante infette sono diminuiti i contenuti di componenti biologicamente attivi rispetto alle piante sane, quali rutina (1.96 ± 0.23 vs. 4.96 ± 0.02 mg/g), iperoside (2.38 ± 0.21 vs. 3.04 ± 0.05 mg/g), isoquercitrina (1.47 ± 0.04 vs. 3.50 ± 0.08 mg/g), amentoflavone (0.12 ± 0.01 vs. 0.39 ± 0.02 mg/g) e pseudoipericina (1.41 ± 0.23 vs. 2.29 ± 0.07 mg/g), mentre il contenuto di acido clorogenico è risultato raddoppiato (1.56 ± 0.11 vs. 0.77 ± 0.02 mg/g). I contenuti di quercitrina, quercetina e ipericina sono risultati analoghi nelle piante infette e in quelle sane. Come descritto in letteratura, la presenza di agenti patogeni può determinare cambiamenti della biosintesi dei metaboliti secondari nei tessuti infetti delle piante, con inibizione della via biosintetica dei flavonoidi e incremento della biosintesi degli acidi fenolici. Per quanto riguarda l’olio essenziale, la resa è risultata drasticamente ridotta nel materiale vegetale infetto (0.11 vs. 0.75%); l’analisi GC dell’olio essenziale ha mostrato come nelle piante infette si verifichi un incremento del contenuto di sesquiterpeni (in particolare, beta-cariofillene, delta-elemene e germacrene D) e un calo del contenuto di monoterpeni. Anche questo fenomeno è stato attribuito ad un’alterazione delle vie biosintetiche delle piante infette, legate ai processi di necrosi dei tessuti dovute alla presenza di fitoplasmiNel caso della digitale, è stato eseguito metabolic profiling dei glucosidi cardioattivi nelle piante infette e in quelle sane, utilizzando una tecnica RP-HPLC, opportunamente sviluppata e validata [2]. Per eliminare i pigmenti interferenti (quali flavonoidi e clorofilla) dagli estratti idroalcolici, è stata ottimizzata una procedura RP-SPE. Le analisi HPLC hanno mostrato che la presenza del fitoplasma influisce significativamente sul contenuto dei principi attivi della digitale: in particolare, il contenuto di lanatoside C è risultato molto inferiore nelle piante infette rispetto a quelle sane (76.1 ug/100 mg vs. 153.2 ug/100 mg). Un trend differente è stato osservato nel caso della alfa-acetildigossina e del deacetil lanatoside C, entrambi derivanti dall’idrolisi del lanatoside C, la cui presenza è risultata superiore nelle piante infette rispetto a quelle sane (7.1 ug/100 mg vs. 5.8 ug/100mg e 46.1 ug/100 mg vs. 37.6 ug/100 mg, rispettivamente). Queste differenze nel contenuto di glucosidi cardioattivi tra le piante infette e quelle sane è stata attribuita alle profonde modificazioni istologiche indotte dalla presenza del fitoplasma negli organi della pianta, che hanno determinato un’alterazione nella biosintesi dei metaboliti secondari. In aggiunta, la rapida senescenza dei tessuti affetti dall’agente patogeno può avere accelerato i processi di degradazione enzimatica di questi principi attivi, come suggerito dall’aumento del contenuto di alfa-acetildigossina e deacetil lanatoside C, la cui presenza è legata all’attività degli enzimi idrolitici.I risultati degli studi condotti su iperico e su digitale sottolineano che la valutazione della presenza di fitoplasmi rappresenta un aspetto importante nella definizione della qualità commerciale e dell’efficacia terapeuti


2010 - PLGA nanoparticles surface decorated with the sialic acid, N-acetylneuraminic acid [Articolo su rivista]
Bondioli, Lucia; Costantino, Luca; Ballestrazzi, Antonio; Davide, Lucchesi; Diana, Boraschi; Pellati, Federica; Benvenuti, Stefania; Tosi, Giovanni; Vandelli, Maria Angela
abstract

There is a broad interest in the development of nanoparticles (NPs) carrying on their surface carbohydrates such as sialic acids. It is known that these carbohydrates influence the biological and physical properties of biopharmaceutical proteins and living cells. Macromolecular compounds containing these carbohydrates showed an anti-recognition effect, exert an antiviral effect and also are able to be recognized by the cell surface of some kind of cancer cells. Thus, in the present research we performed two different approaches in order to obtain polymeric (poly(d,l-lactide-co-glycolide), PLGA) NPs surface decorated with the sialic acid N-acetylneuraminic acid (Neu5Ac). The first strategy that has been followed is based on the derivatization of the polyester PLGA with the thioderivative of Neu5Ac, starting material for the preparation of the NPs; the second is based on the synthesis of compounds potentially able to insert their lipophilic moiety into the underivatized PLGA NPs during their preparation, and to display their hydrophilic moiety (Neu5Ac) on their surface. The first approach allowed the obtainment of NPs surface decorated with Neu5Ac, as evidenced by ESCA spectroscopy and interaction with the lectin Wheat Germ Agglutinin. Moreover, a formulation of these NPs suitable for in vitro assays showed that they are phagocytosed by human monocytes with an apparently different mechanism with respect of those made of underivatized PLGA. The second strategy led to NPs in which their surface appears to be very different with respect to the NPs obtained following the first strategy, with the carboxylic groups of Neu5Ac markedly shielded. Thus, the new Neu5Ac-modified PLGA polyester represent a useful starting material for the preparation of NPs surface decorated with this sialic acid.


2010 - Study on the racemization of synephrine by off-column chiral high-performance liquid chromatography [Articolo su rivista]
Pellati, Federica; Cannazza, Giuseppe; Benvenuti, Stefania
abstract

In this study, the racemization kinetic parameters of R-(-)-synephrine, the active phenethylamine alkaloid of Citrus aurantium L., were determined by means of an off-column HPLC method. Enantioseparation was carried out in different buffer solutions and solvents on a chiral stationary phase (CSP) with cellobiohydrolase as the chiral selector (Chiral-CBH, 100 mm x 4.0 mm i.d., 5 microm). The mobile phase was 10 mM sodium phosphate buffer (pH 6.0)-2-propanol (95:5, w/w), with 50 microM disodium EDTA, at 0.8 mL/min. The column was thermostatted at 20 degrees C and detection was set at 225 nm. The influence of pH value, ionic strength, temperature and addition of organic modifier on the rate constant, the half-life of racemization and the free energy barrier of racemization of R-(-)-synephrine were determined. Among the different chemical and physical parameters evaluated as affecting the racemization of naturally occurring R-(-)-synephrine, pH, temperature and addition of an organic co-solvent appear to have the strongest effect, while ionic strength does not exert a significant influence on the racemization rate. The results of the present study indicated that synephrine racemization is possible at high temperature at both acidic and basic pH values; therefore, the extraction procedure of R-(-)-synephrine from the plant material should be carried out under specific conditions to preserve the stereochemical integrity and the biological activity of this secondary metabolite.


2009 - In vitro evaluation on the anti-apoptotic and anti-oxidant effect of a series of natural polyphenols on UVB- irradiated normal human keratinocytes and melanocytes [Abstract in Atti di Convegno]
E., Bruni; Giudice, Stefania; Veratti, Eugenia; Magnoni, Cristina; Benassi, Luisa; Pellati, Federica; Benvenuti, Stefania; Rossi, Tiziana
abstract

UV radiation, in particular its UVB component, is an important environmental factor in the pathogenesis of skin aging and cancer.One of the features of UVB-caused DNA damage is the formation of cyclobutane pyrimidine dimers and (6-4) photoproducts. Further, indirect DNA damage is also caused by increase in the level of reactive oxygen species (ROS) (1) that cause oxidative damage reacting with DNA, proteins, fatty acids and saccharides (2). Such injuries result in a number of harmful effects: disturbed cell metabolism, morphological and ultrastructural changes, attack on the regulation pathways and, alterations in the differentiation, proliferation and apoptosis of skin cells (3). These processes can lead to photoaging and skin cancer development. In order to avoid UVB radiation damage, phytocompounds and antioxidants as photoprotectives has been considered (4). In recent years naturally occurring herbal compounds such as phenolic acids, flavonoids, and high molecular weight polyphenols have gained remarkable attention as strong protective agents (5,6). In the present study we considered a series of natural polyphenols with the aim to investigate on their anti-oxidant and anti-apoptotic effect in UVB-irradiated normal human keratinocytes and melanocytes.Methods: growing concentration (from 10 to 100 M) of forty-six polyphenols selected by the Department of Pharmaceutical Sciences of the University of Modena were added to normal human keratinocytes and melanocytes coltures. In the first part of the study we assessed the activity on UVB-irradiated normal human keratinocytes and melanocytes. These substances were submitted to a primary in vitro screening by MTT test in order to valuate proliferation rate. Subsequently, 2’7’- dichlorodihydrofluorescein diacetate (DCF) assay was performed to determine the formation of intracellular ROS. The results showed that glabridin, 18β-glycyrrhetinic acid, kaempferol, quercetin, rutin and butein presented interesting properties and a low toxicity. In the second part of the study the antiapoptotic effects of these six selected compounds in UVB-irradiated (50 mJ/cm2/11’and 44’’) normal human keratinocytes and melanocytes was investigated by the western blot analysis.Results: with regard to analysed polyphenols, the results showed that a pre-treatment of human keratinocytes and melanocytes with these molecules inhibited UVB mediated apoptosis through the activation of p53, down-regulation of bcl-2 and bid full-length, up- regulation of bax and inhibition of PARP cleavage. For these purpose we proposed that some substances could be combined to evaluate their synergistic effects.Conclusion: To further elucidate the molecular mechanism of polyphenol or more generally phytocompounds mediated apoptosis, we decided to investigate cell cycle analysis and the extrinsic and intrinsic apoptotic pathways. Human skin is constantly exposed to the UV radiation present in sunlight. This may induce a number of pathobiological cellular changes. The development of novel preventive and therapeutic strategies depends on our understanding of the molecular mechanism of UV-damage. Phytochemical that were identified may be candidates for prevention of adverse effects of UV radiation and melanoma on the skin and evaluation of there clinical efficacy is awaited.


2009 - Optimization and validation of a high-performance liquid chromatography method for the analysis of cardiac glycosides in Digitalis lanata [Articolo su rivista]
Pellati, Federica; R., Bruni; M. G., Bellardi; A., Bertaccini; Benvenuti, Stefania
abstract

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 ug/100 mg versus 76.1 ug/100 mg). 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.


2009 - Recent advances in the chromatographic analysis of phenethylamine alkaloids in natural products [Relazione in Atti di Convegno]
Pellati, Federica; Benvenuti, Stefania
abstract

Phenethylamine alkaloids are a group of non-heterocyclic compounds widely distributed in plants. The main representative molecules of this class are ephedrine (isolated from Ephedra species) and synephrine (isolated from Citrus species). Products containing E. sinica or C. aurantium have been widely used for the treatment of obesity or for increasing performance in body-building. However, these products have shown side-effects on the cardiovascular system by means of adrenergic stimulation. Considering the variability in the quality composition of the secondary metabolites of Ephedra and Citrus natural products, continued research effort is desired toward the application of fully validated methods to monitor the quality of crude drugs, extracts and dietary supplements. There have been many reports on the HPLC determination of phenethylamine alkaloids in plant material and natural products. RP-HPLC with UV detection is usually employed for the determination of these analytes and most separations are based on ion-pairing. In recent years, RP-HPLC stationary phases that contain polar groups as part of their structure often succeed in retaining and resolving compounds that C18 phases do not, because they can interact with analytes by mechanisms that are not possible with C18 alkyl chains. Of many such phases currently available, the pentafluorophenylpropyl (PFPP) stationary phase has shown wide applicability. In this study, the chromatographic performance of the fluorinated stationary phase was investigated to develop reliable HPLC methods for the separation and quantitative determination of phenethylamine alkaloids in Ephedra and Citrus natural products. The proposed methods were carefully optimised and fully validated in agreement with ICH guidelines. The practical applicability of the developed techniques was demonstrated by the analysis of Ephedra and Citrus natural products. Furthermore, due to the difference in the pharmacological effect between the two enantiomeric forms of synephrine, an efficient method for the enantioselective separation and determination of this compound in Citrus natural products was optimized and validated.


2009 - Root extracts of three species of Echinacea affect the maturation state of monocyte-generated human dendritic cells [Abstract in Rivista]
I., Mucci; A., Legitimo; M., Compagnino; Pellati, Federica; P., Nieri; Benvenuti, Stefania; M. R., Metelli; B., Longoni; F., Mosca; P., Macchia; R., Consolini
abstract

Our study aims at investigating the effects of Echinacea phytocompounds on monocyte derived-dendritic cell (DC) generation, maturation and biological activity. Phenotypical evaluation, endocytic capacity and the dosage of culture surnatant cytokine are performed on immature and mature DCs, treated or not with E. purpurea, E. pallida, E. angustifolia root extracts (100-150-200 μg/mL). Our results demonstrate that Echinacea extracts don’t affect DC generation, while they interfere with their maturation (shown as a reduced expression of CD40, CD80, CD83 and HLA-DR antigens). Moreover, they seem to reduce the endocytic activity and the production of IL-10. No significant differences among the extracts belonging to the three analysed species have been noticed.Introduction. Echinacea represents a perennial plant indigenous to North America, but also cultured in Europe since a lot of years 1. Ten species belong to this genus 2, but only three species show pharmacological properties: E. angustifolia, E. pallida, E. purpurea 3.Up to date, formulations with Echinacea extracts are usually used for their immunomodulatory, antiiflammatory, antiviral and antioxidant activities, even if the mechanism of the action of their component has not yet been investigated.The goal of our study is to investigate the mechanism of action by which Echinacea phytocompounds have an immunoenhancing activity, focusing on the effects of these extracts on dendritic cell (DC) generation, maturation and biological activity. DCs are the most important antigen-presenting cells: they check both the first phase and the maintenance of adaptative immune responses and the induction of tolerance 4-7.Materials and Methods. DCs are in vitro generated from CD14+ circling monocytes, obtained from mononuclear cells of healthy donors, through an immunomagnetic system (Miltenyi Biotec, Germany). Monocytes are cultured in RPMI 1640 medium, added with fetal bovine serum, L-glutamine, streptomycin and penicillin and supplemented with growth factors (GM-CSF and IL-4) 5, in presence or absence of total root extract of the three specie of Echinacea (E. purpurea, E. pallida, E. angustifolia). These extracts are tested at a concentration of 100, 150 and 200 μg/mL. After 6 days of incubation, LPS is added to the culture for further 24 hours, in order to induce maturation. Then, morphological and phenotypical analysis are performed respectively by cytospin, May-Grunwald Giemsa staining and cytometric assays. The markers used are CD14 antigen, present on monocytes surface, but not on DC surface, CD40 and CD80 costimulatory molecules, HLA class II (HLA-DR) antigen and CD83 antigen, expressed by mature DCs 8.Functional studies on DCs generated in presence of Echinacea include cytometric analysis of endocytotic activity mediated by mannose receptor, using the dextran FITC-conjugated as an antigen, and the dosage of culture surnatant cytokines (IL-6, IL-1, IL-10, IL-12, TNF-α, TGF-β) through an enzyme immunoassay.Results and Discussion. Echinacea phytocompounds don’t compromise DC generation from monocytes (loss of CD14 expression on cells surface).Neverthless, total extracts interfere with the maturation, as the reduced expression of CD83 antigen shows if compared to the control. Moreover, in presence of Echinacea, a reduced expression (both as percentage and mean) of the CD80 and HLA-DR is observed, if compared to the control; as far the CD40 antigen is concerned, its percentage remains unchanged, while the mean decreases in presence of the extracts, compared to the control. No significant differences of cell phenotype among the different concentrations used, nor among the extracts belonging to the three analysed species, is noticed (Fig. 1).When on the 6th day we perform endocytosis assay, in presence of total root extract of Echinacea we observe no differences compared with the control: the cells show a marked phagocytic activity. On the


2009 - Stability study on a cytotoxic dienynone isolated from Echinacea pallida [Abstract in Atti di Convegno]
Orlandini, Giulia; Pellati, Federica; Benvenuti, Stefania
abstract

Recent studies have demonstrated that some of the secondary metabolites (polyacetylenes and polyenes) isolated from the raw lipophilic extract of Echinacea pallida roots have good cytotoxic activity on cancer cell lines3. In particular, pentadeca-(8Z,13Z)-dien-11-yn-2-one was found to be the most active constituent, with an IC50 value of 32.17 ± 3.98 uM on pancreatic MIA PaCa-2 cancer cells and 2.34 ± 0.33 uM on colonic COLO320 cancer cells, after 72 h of exposure. This compound is present in the roots in a not-oxidized form but, during the drying process and the subsequent storage, a change in its concentration was observed, which has been attributed to a possible oxidation process. As part of our ongoing research on bioactive metabolites from Echinacea, the aim of this research was the evaluation of the stability of this secondary metabolite by RP-HPLC-DAD2 under the experimental conditions applied during the biological assays. The study was initially carried out on the lipophilic crude extract obtained from E. pallida roots and then it was focused on the individual component isolated and purified from the plant material by preparative chromatographic techniques. The results showed a moderate change in the HPLC profiles of E. pallida crude extract in 72 h, with an increase of hydroxylated derivatives. The oxidation kinetic of the most active compound, pentadeca-(8Z,13Z)-dien-11-yn-2-one, was found to be quite slow, since the % area of the intermediate hydroperoxide was about 25% after 72 h of exposure. The final product of the oxidation process, i.e. the hydroxilated derivative (i.e. 8-hydroxy-pentadeca-(9E,13Z)-dien-11-yn-2-one, available thanks to a synthetic approach), was not observed in the HPLC chromatogram after 72 h. Considering that during the first 24 h of exposure, the % area of the hydroperoxide intermediate is below 15%, the observed cytotoxic activity can be mainly attributed to the genuine not oxidized compound.


2009 - Studio di stabilità dei costituenti biologicamente attivi di Echinacea pallida [Abstract in Atti di Convegno]
Orlandini, Giulia; Pellati, Federica; Benvenuti, Stefania
abstract

Recenti studi hanno dimostrato che i metaboliti secondari (poliacetileni e polieni), isolati dall’estratto grezzo delle radici di Echinacea pallida1,2, possiedono buona attività citotossica su alcune linee cellulari tumorali (MIA PaCa-2 e COLO320)3. Tali composti sono presenti nella radice in forma non ossidata; tuttavia, durante i processi di essiccamento e successiva lavorazione, si nota una variazione della loro concentrazione, che è stata attribuita ad un possibile processo di ossidazione1.Considerata l’importanza dell’attività biologica di tali composti, scopo della presente ricerca è la valutazione della loro stabilità mediante RP-HPLC-DAD2. L’obiettivo più specifico è costituito dalla determinazione della stabilità di suddetti componenti in condizioni simili a quelle cellulari. Lo studio è stato inizialmente condotto su un estratto grezzo lipofilo ottenuto dalle radici di E. pallida ed è stato successivamente focalizzato sui singoli composti isolati e purificati mediante tecniche cromatografiche preparative. I risultati hanno evidenziato che, nel corso delle 72 ore di monitoraggio nelle diverse condizioni sperimentali, l’estratto grezzo subisce una progressiva ossidazione, con incremento dei derivati idrossilati. Le successive prove di stabilità condotte sui tre metaboliti isolati dall’estratto grezzo di E. pallida (tetradec-(8Z)-ene-11,13-diyn-2-one (1), pentadec-(8Z)-ene-11,13-diyn-2-one (2), pentadeca-(8Z,13Z)-dien-11-yn-2-one(3)) hanno confermato che i suddetti composti subiscono un processo di ossidazione, tramite la formazione di un intermedio idroperossido. Dei composti 1-3 è stata quindi studiata la cinetica di ossidazione nelle diverse condizioni sperimentali.


2009 - Study on the enantiomerization of synephrine by chiral high-performance liquid chromatography [Abstract in Atti di Convegno]
Pellati, Federica; Cannazza, Giuseppe; Orlandini, Giulia; Benvenuti, Stefania
abstract

Synephrine, the active phenethylamine alkaloid of Citrus aurantium L., is a biologically active compound that has effects on human metabolism that could help to reduce fat mass in obese people, since it stimulates lipolysis, raises the metabolic rate and promotes the oxidation of fat through increased thermogenesis. In the light of this, C. aurantium extracts are commonly used in the formulation of phytotherapic products employed in the treatment of obesity. It is well known that synephrine is a chiral compound and its enantiomers have shown different pharmacological activity on alpha- and beta-adrenoreceptors. In particular, R-(−)-synephrine is from 1 to 2 orders of magnitude more active than its S-(+)-counterpart. R-(−)-Synephrine has been isolated and identified in Citrus fruits. However, a certain amount of S-(+)-synephrine has been detected in C. aurantium dry extracts and dietary supplements (1). The presence of S-(+)-synephrine in C. aurantium natural products has been attributed to a possible enantiomerization of R-(-)-synephrine during the industrial production of the fruit extracts, using a high temperature and a long period of refluxing. To evaluate the enantiomerization kinetic parameters of R-(-)-synephrine, an off-column HPLC method based on a chiral stationary phase (CSP) with cellobiohydrolase as the chiral selector (Chiral-CBH) was developed. Analyses were carried out on a Chiral-CBH column (100 × 4.0 mm i.d., 5 um), with a mobile phase consisting of 2-propanol (5%, w/w) in sodium phosphate buffer (pH 6.0; 10 mM) and disodium EDTA (50 uM). The flow rate was 0.8 mL/min. The column was thermostatted at 20°C. Detection was set at 225 nm. The individual enantiomers of the studied compound were isolated by fractional crystallization of the diastereomeric salts and subsequent ion-exchange. The rate constants and the free energy barriers of enantiomerization were determined in different solvents and buffer solutions at pH 1-11. The results generated by the off-line method were used to determine the influence of solvents and pH values on the enantiomerization rate of (+) and (-)-synephrine and to gain further insight into the enantiomerization mechanism of chiral phenethylamine type alkaloids in relation to the pKa values.


2008 - Cytotoxic activity of polyacetylenes and polyenes isolated from roots of Echinacea pallida [Articolo su rivista]
A., Chicca; Pellati, Federica; B., Adinolfi; A., Matthias; I., Massarelli; Benvenuti, Stefania; E., Martinotti; A. M., Bianucci; K., Bone; R., Lehmann; P., Nieri
abstract

BACKGROUND AND PURPOSE: The n-hexane extracts of the roots of three medicinally used Echinacea species exhibited cytotoxic activity on human cancer cell lines, with Echinacea pallida found to be the most cytotoxic. Acetylenes are present in E. pallida lipophilic extracts but essentially absent in extracts from the other two species. In the present study, the cytotoxic effects of five compounds, two polyacetylenes (namely, 8-hydroxy-pentadeca-(9E)-ene-11,13-diyn-2-one (1) and pentadeca-(9E)-ene-11,13-diyne-2,8-dione (3)) and three polyenes (namely, 8-hydroxy-pentadeca-(9E,13Z)-dien-11-yn-2-one (2), pentadeca-(9E,13Z)-dien-11-yne-2,8-dione (4) and pentadeca-(8Z,13Z)-dien-11-yn-2-one (5)), isolated from the n-hexane extract of E. pallida roots by bioassay-guided fractionation, were investigated and the potential bioavailability of these compounds in the extract was studied. EXPERIMENTAL APPROACH: Cytotoxic effects were assessed on human pancreatic MIA PaCa-2 and colonic COLO320 cancer cell lines. Cell viability was evaluated by the WST-1 assay and apoptotic cell death by the cytosolic internucleosomal DNA enrichment and the caspase 3/7 activity tests. Caco-2 cell monolayers were used to assess the potential bioavailability of the acetylenes. KEY RESULTS: The five compounds exhibited concentration-dependent cytotoxicity in both cell types, with a greater potency in the colonic cancer cells. Apoptotic cell death was found to be involved in the cytotoxic effect of the most active, compound 5. Compounds 2 and 5 were found to cross the Caco-2 monolayer with apparent permeabilities above 10 x 10(-6) cm s(-1). CONCLUSIONS AND IMPLICATIONS: Compounds isolated from n-hexane extracts of E. pallida roots have a direct cytotoxicity on cancer cells and good potential for absorption in humans when taken orally.


2008 - Determination of ephedrine alkaloids in Ephedra natural products using HPLC on a pentafluorophenylpropyl stationary phase [Articolo su rivista]
Pellati, Federica; Benvenuti, Stefania
abstract

In this study a pentafluorophenylpropyl (PFPP) stationary phase was applied to the fast and reliable qualitative and quantitative analysis of ephedrine alkaloids in Ephedra plant material and derivatives. A Discovery HS F5 column (150 mm × 4.6 mm i.d., 5 um) was used, with an isocratic mobile phase composed of ammonium acetate (7 mM) in acetonitrile–water (90:10, v/v), at a flow rate of 1.0 ml/min. The column temperature was set at 45 °C. UV detection was set at 215 and 225 nm. The total analysis time was 16 min. The validation parameters, such as linearity, sensitivity, accuracy, precision and specificity, were found to be highly satisfactory. Sonication and microwave extractions were compared in order to optimize the yield of the target analytes. Under the optimized extraction conditions (based on two cycles of sonication with methanol at 40 °C for 15 min), different matrices containing Ephedra were successfully analyzed for their alkaloid content. The method was applied to the analysis of standard reference materials (SRMs) containing Ephedra. Furthermore, the developed technique allowed the simultaneous determinationof ephedrine alkaloids and synephrine, the main phenethylamine alkaloid of Citrus aurantium, that has replaced Ephedra in dietary supplements used in the treatment of obesity. The results indicated that this procedure is suitable for the phytochemical analysis of Ephedra plant material and extracts, and can be applied to demonstrate the label claims for product content, including the absence of ephedrine alkaloids in Ephedra-freeproducts.


2008 - Isolation, structure elucidation and total synthesis of a cytotoxic dienone from Echinacea pallida [Articolo su rivista]
S., Morandi; Pellati, Federica; C., Ori; B., Adinolfi; P., Nieri; Benvenuti, Stefania; Prati, Fabio
abstract

The isolation and structure characterization of a dienone from the roots of Echinacea pallida, namely (8Z,11Z)-pentadeca-8,11-dien-2-one, are described here. To assess the configuration of this secondary metabolite, the stereoselective total synthesis of the two isomeric forms, 8Z,11Z)- and (8Z,11E)-pentadeca-8,11-dien-2-one, was undertaken and the structure elucidation of the naturalcompound was unambiguously carried out. The cytotoxic activity of both isomers was also evaluated on a human T cell leukaemia cancer line (Jurkat cells). The results indicated that these compounds exert a dose-dependent cytotoxicity with a medium-level potency on the tested cell line.


2008 - P-Glycoprotein inhibitory activity of lipophilic constituents of Echinacea pallida roots in a human proximal tubular cell line [Articolo su rivista]
N., Romiti; Pellati, Federica; P., Nieri; Benvenuti, Stefania; B., Adinolfi; E., Chieli
abstract

The n-hexane root extracts from Echinacea pallida, Echinacea angustifolia and Echinacea purpurea were evaluated for inhibitionof the multidrug transporter P-glycoprotein (Pgp) activity,the product of the ABCB1 gene, involved in cancer multidrug resistance (MDR) and in herb-drug or drug-drug interactions. Thebiological assay was performed using the human proximal tubule HK-2 cell line that constitutively expresses ABCB1. The n-hexaneextracts of all three species reduced the efflux of the Pgp probe calcein-AM from HK-2 cells two-fold in a concentration-dependent manner, and E. pallida was found to be the most active species. For the first time, two polyacetylenes and three polyenes, isolated from the n-hexane extract of E. pallida roots by a bioassay- guided fractionation, were found to be able to reduce Pgp activity. Pentadeca-(8Z,13Z)-dien-11-yn-2-one was the most efficient compound, being able to decrease the calcein-AM efflux about three-fold with respect to the control at 30 μg/mL.


2008 - Total synthesis of a dienynone from Echinacea pallida [Articolo su rivista]
S., Morandi; Pellati, Federica; Benvenuti, Stefania; Prati, Fabio
abstract

The first total synthesis of (8Z,13Z)-pentadeca-8,13-dien-11-yn-2-one is described. This dienynone was recently isolated from the n-hexane extract of Echinacea pallida roots and displayed a selective cytotoxic activity toward cancer cells, thus featuring as a potential anticancer lead. The product was obtained in 11 steps in 25% overall yield.


2008 - Variability of the active compounds in phytotherapic products with antidepressant activity: the case of Hypericum perforatum L. [Relazione in Atti di Convegno]
Pellati, Federica; Benvenuti, Stefania
abstract

In this presentation, the variability of the active compounds in phytotherapic products containing Hypericum perforatum L. was discussed.


2007 - Chromatographic and electrophoretic methods for the analysis of phenetylamine alkaloids in Citrus aurantium [Articolo su rivista]
Pellati, Federica; Benvenuti, Stefania
abstract

Citrus aurantium (bitter orange) is a plant belonging to the family Rutaceae, whose fruit extracts have been used recently for the treatment of obesity. The most important biologically active constituents of the C. aurantium fruits are phenethylamine alkaloids (i.e. octopamine, synephrine, tyramine, N-methyltyramine and hordenine). Synephrine is a primary synthesis compound with pharmacological activities such as vasoconstriction, elevation of blood pressure and relaxation of bronchial muscle. Synephrine is present in the peel and the edible part of Citrus fruit. Of the adrenergic amines of natural origin, synephrine has been found to be the main constituent of C. aurantium fruits and extracts; the other alkaloids are either absent or present in only low concentrations. It is known that synephrine and the other amines found in C. aurantium have adverse effects on the cardiovascular system, owing to adrenergic stimulation. In light of the great commercial proliferation of C. aurantium herbal medicines in recent years, this review provides an overview of various extraction, separation and detection techniques employed for the qualitative and quantitative determination of the alkaloids in C. aurantium and related species. The application of chromatographic and electrophoretic methodsfor the separation and determination of these active components in C. aurantium plant material and derivatives are described. Since synephrine is a chiral compound, enantioselective chromatographic and electrophoretic techniques for the analysis of synephrine enantiomers in natural products are presented. Furthermore, examples of identification of these active compounds in complex matrices by hyphenated methods, such as gas chromatography–mass spectrometry and high-performance liquid chromatography–mass spectrometry, are described. The advantages and limitations of these separation and identification methods are assessed and discussed.


2007 - Cytotoxic effects of Echinacea root hexanic extracts on human cancer cell lines [Articolo su rivista]
A., Chicca; B., Adinolfi; E., Martinotti; S., Fogli; M. C., Breschi; Pellati, Federica; Benvenuti, Stefania; P., Nieri
abstract

Echinacea is one of the most widely used alternative medicine in the world. Intake of Echinacea preparations is common among patients with advanced malignancies enrolled onto phase I chemotherapy trials; however, to our knowledge, no data are available regarding the possible direct effect of Echinacea species on human cancer cells. The purpose of the present study was to investigate potential in vitro cytotoxic and pro-apoptotic properties of hexanic root extract of the three medicinal Echinacea (Asteraceae) species (Echinacea pallida (Nutt.) Nutt., Echinacea angustifolia DC. var. angustifolia, Echinacea purpurea (L.) Moench.) on the human pancreatic cancer MIA PaCa-2 and colon cancer COLO320 cell lines. We demonstrated, for the first time, that all the three species reduced cell viability in a concentration- and time-dependent manner; Echinacea pallida was the most active species with IC(50)s of 46.41 +/- 0.87 and 10.55 +/- 0.70 ug/ml in MIA PaCa-2 and COLO320 cells, respectively. Echinacea pallida extract was able to induce apoptosis by increasing significantly caspase 3/7 activity and promoting nuclear DNA fragmentation. These results represent the starting point to establish viable scientific evidence on the possible role of Echinacea species in medical oncology.


2007 - Cytotoxic effects of polyacetylenes from E. pallida on human cancer cell lines and their bioavailability through Caco-2 cell monolayers [Abstract in Rivista]
A., Chicca; Pellati, Federica; A., Matthias; B., Adinolfi; Benvenuti, Stefania; K. M., Bone; R. P., Lehmann; P., Nieri
abstract

In previous studies, root hexanic extracts from the three medicinal Echinacea species (E. purpurea, E. pallida and E. angustifolia) exhibited a cytotoxic activity on human cancer cell lines and E. pallida was found to be the most cytotoxic one. Moreover, E. pallida root hexanic extract contains polyacetylenes, which are almost absent in the other two species of Echinacea where alkylamides are the main constituents. In the present study the cytotoxic effects of polyacetylenes single compounds isolated by a bio-guided assay fractionation from an E. pallida hexanic extract and their potential bioavailability were investigated. Antitumoral effects were assessed on human pancreatic MIA PaCa-2 and colonic COLO320 cancer cell lines. Cell viability was evaluated by the colorimetric WST-1 assay and apoptotic cell death was evaluated by an immunoenzimatic analysis of the cytosolic internucleosomal DNA enrichment and by the caspase 3/7 activity test. Bioavailability was studied using the Caco-2 cell monolayer, an in vitro model of intestinal permeability. All the isolated compounds exhibited a concentration- and time-dependent cytotoxicity (1-100 µM; 24-72h) in both cell types and a greater potency on colonic cancer cells was observed. Apoptotic cell death was demonstrated to be involved in the cytotoxic effect of the most active polyacetylene, as revealed by both the assays used. Finally, polyacetylenes were found to cross the Caco-2 monolayer suggesting a likely bioavailability when taken orally. In conclusion, our data demonstrate, for the first time, an anticancer activity of constituents from E. pallida root hexanic extract and suggest their potential in vivo bioavailability.


2007 - Determination of ephedrine alkaloids in Ephedra natural products using HPLC on a pentafluorophenylpropyl stationary phase [Abstract in Atti di Convegno]
Pellati, Federica; Benvenuti, Stefania; M., Melegari
abstract

In this research, the determination of ephedrine alkaloids in Ephedra natural products using HPLC on a pentafluorophenylpropyl stationary phase was carried out.


2007 - Fast high-performance liquid chromatography analysis of phenethylamine alkaloids in Citrus natural products on a pentafluorophenylpropyl stationary phase [Articolo su rivista]
Pellati, Federica; Benvenuti, Stefania
abstract

In this study, the chromatographic performance of a pentafluorophenylpropyl (PFPP) stationary phase was evaluated for the rapid separation of phenethylamine alkaloids (i.e. (±)-octopamine, (±)-synephrine, tyramine, N-methyltyramine and hordenine) in Citrus aurantium plant material (fruits and peel), various Citrus species, extracts and dietary supplements claiming to contain C. aurantium. The problems of phenethylaminealkaloid separation, such as peak tailing, low retention and low resolution, were successfully solved with this stationary phase. The parameters used for the method optimization included the mobile phase counter ion concentration and column temperature. A Discovery HS F5 column (150mm×4.6mm i.d., 5m) was used, with an isocratic mobile phase composed of 10mM ammonium acetate in 90:10 ACN–H2O (v/v), at a flow rate of 1.0 mL/min. The column temperature was set at 20 ◦C. The photodiode array detector monitored the eluent at 225 nm. The total analysis time was 10 min. The validation parameters, such as linearity, sensitivity, accuracy, precision and specificity, were found to be highly satisfactory. With a simple sample preparation procedure, different matrices were successfully analyzed for their alkaloid content. The results indicated that the products on sale, labeled as dietary supplements, vary widely in the quantitative composition of the active constituents: the amount of (±)-synephrine, the major alkaloid, in such products ranged from 0.65 to 27.41 mg/g. The other compounds were either not detected or were present at low levels. The developed method can be considered suitable for the quality control of Citrus plant material and commercial products.


2007 - High-performance liquid chromatography analysis of polyacetylenes and polyenes in Echinacea pallida by using a monolithic reversed-phase silica column [Articolo su rivista]
Pellati, Federica; S., Calò; Benvenuti, Stefania
abstract

In this study, a RP-HPLC method for the analysis of polyacetylenes and polyenes in Echinacea pallida roots and phytopharmaceuticals was developed. The reference compounds used for quantification were isolated from the plant material and their structures were determined on the basis of the analysis of UV, IR, NMR and MS data. The complete structure elucidation of three compounds, namely 8-hydroxy-tetradec-(9E)-ene-11,13-diyn-2-one (1), tetradec-(8Z)-ene-11,13-diyn-2-one (6) and pentadec-(8Z)-en-2-one (9) is described. In the analysis of the n-hexane extracts of E. pallida roots, the comparison between conventional and monolithic columns showed that the elution order in both cases is identical and the selectivity is equivalent. However, the retention times achieved by the monolithic column are shorter, resulting in a faster separation (20 min).Therefore, the analyses were carried out on a Chromolith Performance RP-18e (100 mm× 4.6 mm i.d.), with a gradient mobile phase composed by H2O and ACN at the flow rate of 2 mL/min. The column was thermostatted at 20 ◦C. The photodiode array detector monitored the eluent at 210 nm. The validation procedure confirmed that this technique affords reliable analysis of these components and is appropriate for the quality control of complex matrices, such as E. pallida roots and phytopharmaceuticals.


2007 - In vitro anticancer activity of E. pallida polyacetylenes [Abstract in Atti di Convegno]
B., Adinolfi B; A., Chicca; Pellati, Federica; Benvenuti, Stefania; I., Massarelli; E., Martinotti; P., Nieri
abstract

In this work, the in vitro anticancer activity of E. pallida polyacetylenes was evaluated.


2006 - Development and validation of HPLC methods for the analysis of phenethylamine and indoloquinazoline alkaloids in Evodia species [Articolo su rivista]
Pellati, Federica; Benvenuti, Stefania; F., Yoshizaki; Melegari, Michele
abstract

The aim of this study was to evaluate the chromatographic performance of a PEG stationary phase, in comparison with those of C-18 columns, for the HPLC analysis of phenethylamine ((+/-)-synephrine) and indoloquinazoline (rutaecarpine and evodiamine) alkaloids in methanolic extracts of fruits of Evodia rutaecarpa (Juss.) Benth. and E. rutaecarpa (Juss.) Benth. var. officinalis (Dode) Huang (i.e., E. officinalis Dode) (Rutaceae family). The method was validated and showed good linearity, precision, accuracy, sensitivity, and specificity. The highest content of both phenethylamine and indoloquinazoline alkaloids was found in methanolic fruit extracts of E. rutaecarpa, and it was closely related to the degree of maturity. E. officinalis fruits displayed low amounts of both types of alkaloids. Furthermore, an enantioselective HPLC method for the enantioseparation of (+/-)-synephrine from Evodia fruits was applied, by using a protein-based chiral stationary phase with cellobiohydrolase (CBH) as the chiral selector (Chiral-CBH). Isolation of synephrine from Evodia aqueous fruit extracts was carried out by strong cation-exchange SPE. The results of the application of the method to the analysis of Evodia samples showed that (-)-synephrine was the main component while (+)-synephrine was present in low concentration.


2006 - Fast HPLC analysis of polyacetylenes and polyenes in E. pallida by using a monolithic silica column [Abstract in Atti di Convegno]
Pellati, Federica; S., Calò; Benvenuti, Stefania
abstract

In this work, a fast HPLC method for the analysis of polyacetylenes and polyenes in Echinacea pallida natural products by using a monolithic silica column was optimized and validated.


2006 - High-Performance Liquid Chromatography Analysis of Polyacetylenes and Polyenes in E. pallida Roots [Abstract in Atti di Convegno]
Pellati, Federica; Benvenuti, Stefania; S., Calò; B., Adinolfi; P., Nieri; M., Melegari
abstract

In this study, a HPLC method for the analysis of polyacetylenes and polyenes in E. pallida natural products was developed and validated.


2006 - Isolation and structure elucidation of cytotoxic polyacetylenes and polyenes from Echinacea pallida [Articolo su rivista]
Pellati, Federica; S., Calo; Benvenuti, Stefania; B., Adinolfi; P., Nieri; M., Melegari
abstract

Bioassay-guided fractionation of n-hexane extracts of Echinacea pallida (Asteraceae) roots led to the isolation and structure elucidation of two polyacetylenes (1, 3) and three polyenes (2, 4, 5). Two are known hydroxylated compounds, namely 8-hydroxy-pentadeca-(9E)-ene-11,13-diyn-2-one (1) and 8-hydroxy-pentadeca-(9E,13Z)-dien-11-yn-2-one (2). Two dicarbonylic constituents, namely pentadeca-(9E)-ene-11,13-diyne-2,8-dione (3) and pentadeca-(9E,13Z)-dien-11-yne-2,8-dione (4), were isolated and characterized for the first time. Furthermore, the structure elucidation of pentad eca-(8Z,13Z)-dien-11-yn-2-one (5) is described. The structure of the compounds isolated was determined on the basis of UV, IR, NMR (including 1D and 2D NMR experiments, such as H-1-H-1 gCOSY, gHSQC-DEPT, gHMBC, gNOESY) and MS spectroscopic data. The cytotoxic activity of the isolated constituents against MIA PaCa-2 human pancreatic adenocarcinoma cells was evaluated in the concentration range 1-100 mu g/ml. Results show that the hydroxylated compounds (1, 2) have low cytotoxicity, while the more hydrophobic polyacetylenes (3) and polyenes (4, 5) displayed moderate activity.


2005 - Amine oxidase substrates for impaired glucose tolerance correction [Articolo su rivista]
C., Carpene; S., Bour; V., Visentin; Pellati, Federica; Benvenuti, Stefania; M. C., Iglesias Osma; M. J., Garcia Barrado; P., Valet
abstract

Amine oxidases are widely distributed from microorganisms to vertebrates and produce hydrogen peroxide plus aldehyde when catabolizing endogenous or xenobiotic amines. Novel roles have been attributed to several members of the amine oxidase families, which cannot be more considered as simple amine scavengers. Semicarbazide-sensitive amine oxidase (SSAO) is abundantly expressed in mammalian endothelial, smooth muscle, and fat cells, and plays a role in lymphocyte adhesion to vascular wall, arterial fiber elastic maturation, and glucose transport, respectively. This latter role is detailed and the perspectives opened by the insulin-like actions of amine oxidase substrates are discussed in the present review. Independent studies have demonstrated that SSAO substrates and monoamine oxidase substrates mimic diverse insulin effects in adipocytes: glucose transport activation, lipogenesis stimulation and lipolysis inhibition. These substrates also stimulate in vitro adipogenesis. Acute in vivo administration of amine oxidase substrates improves glucose tolerance in rats, mice and rabbits, while chronic treatments with benzylamine plus vanadate exert an antihyperglycaemic effect in diabetic rats. Dietary supplementations with methylamine, benzylamine or tyramine have been evidenced to influence metabolic control in rodents by increasing glucose tolerance or decreasing lipid mobilisation, without noticeable changes in the plasma markers of lipid peroxidation or protein glycation, despite adverse effects on vasculature. Thus, the ingested amines are not totally degraded at the intestinal barreer and can act on adipose and vascular tissues. In regard with this influence on metabolic control, more attention must be paid to the composition or supplementation in amines in foods and nutraceutics.


2005 - Antiproliferative effects of Echinacea root hexanic extracts on MIA PaCa-2 cells [Abstract in Atti di Convegno]
A., Chicca; B., Adinolfi; S., Fogli; E., Martinotti; M. C., Breschi; P., Nieri; Benvenuti, Stefania; Pellati, Federica; M., Melegari
abstract

In this study, the antiproliferative effects of Echinacea root hexanic extracts on MIA PaCa-2 cells was evaluated.


2005 - Chromatographic performance of a new polar poly(ethylene glycol) bonded phase for the phytochemical analysis of Hypericum perforatum L. [Articolo su rivista]
Pellati, Federica; Benvenuti, Stefania; Melegari, Michele
abstract

The aim of this study was to evaluate the chromatographic performance of a poly(ethylene glycol) (PEG) stationary phase for the HPLC analysis of the secondary metabolites (chlorogenic acid, flavonoids, phloroglucinols and naphthodianthrones) in methanolic extracts of Hypericum perforatum L. (St. John's Wort) flowering tops, herbal medicinal products and dietary supplements. A fast and reliable method was developed. The analyses were carried out on a Supelco Discovery HS PEG column (150 mm × 4.6 mm i.d., 5 μm). A gradient mobile phase, composed of 0.1 M aqueous acetic acid solution (pH 2.8) and methanol–acetonitrile (5:4, v/v), was used. The flow rate was 1 mL/min. The photodiode array detector monitored the eluent at 270 (for chlorogenic acid, flavonoids and phloroglucinols) and 590 nm (for naphthodianthrones). The column was maintained at room temperature. The total running time was 40 min. The method was validated and showed good linearity, precision, accuracy, sensitivity and specificity. Through the above described phytochemical markers, this technique allowed the unequivocal identification and standardization of H. perforatum plant material and phytoproducts. The quantification data highlighted the fact that the products on sale, in particular those labeled as dietary supplements, varied widely in the quantitative composition of the active constituents. The developed method could be considered suitable for the quality control of H. perforatum herb and derivatives.


2005 - Enantioselective LC analysis of synephrine in natural products on a protein-based chiral stationary phase [Articolo su rivista]
Pellati, Federica; Benvenuti, Stefania; Melegari, Michele
abstract

An enantioselective LC method with photodiode array detection (PAD) was developed for the enantioseparation of synephrine from C. aurantium L. var. amara fruits and phytotherapic derivatives by using a protein-based chiral stationary phase with cellobiohydrolase as the chiral selector (Chiral-CBH). Analyses were carried out on a Chiral-CBH column (100 x 4.0 mm i.d., 5 um), with a mobile phase consisting of 2-propanol (5%, w/w) in sodium phosphate buffer (pH 6.0; 10 mM) and disodium EDTA (50 uM). The flow rate was 0.8 mL/min. Detection was set at 225 nm. To identify the order of elution, the racemate was resolved by the preparation of suitable diastereoisomeric salts with antipodes of appropriate organic acids. Isolation of synephrine from C aurantium fruits and phytoproducts was performed by solid-phase extraction (SPE) with a strong cation-exchange phase. The method developed was validated and was found to be linear in the 0.40-40.14 ug/mL range (r(2) = 1.000, P < 0.0001) for both synephrine enantiomers. The limit of detection (LOD) for each enantiomer was 0.04 ug/mL. The limit of quantification (LOQ) for each enantiomer was 0.13 ug/mL. Intra-day precision (calculated as %R.S.D.) ranged from 0.03 to 0.24% for (-)-synephrine and from 0.03 to 0.35% for (+)-synephrine. Inter-day precision (calculated as %R.S.D.) ranged from 0.07 to 1.45% for (-)-synephrine and from 0.06 to 1.26% for (+)synephrine. Intra- and inter-day accuracies (calculated as %recovery) were in the ranges of 97.4-100.6 and 98.0-101.6% for (-)-synephrine, and in the ranges 97.0-101.5 and 98.1-102.8% for (+)-synephrine. The results of the application of the method to the analysis of C aurantium samples showed that (-)-synephrine was the main component. (+)-Synephrine was not detected in C aurantium fruits and was present in low concentration in the phytoproducts.


2005 - Headspace solid-phase microextraction-gas chromatography-mass spectrometry analysis of the volatile compounds of Evodia species fruits [Articolo su rivista]
Pellati, Federica; Benvenuti, Stefania; F., Yoshizaki; Bertelli, Davide; Rossi, Maria Cecilia
abstract

In this study the investigation of the aroma compounds of dried fruits of Evodia rutaecarpa (Juss.) Benth. and E. rutaecarpa (Juss.) Benth. var. officinalis (Dode) Huang (i.e. E. officinalis Dode) (Rutaceae family) was carried out to identify the odorous target components responsible for the characteristic aroma of these valuable natural products. To avoid the traditional and more time-consuming hydrodistillation, the analyses were carried out by means of headspace solid-phase microextraction (HS-SPME) coupled to gas chromatography–mass spectrometry (GC–MS). The SPME headspace volatiles were collected using a divinylbenzene–carboxen–polydimethylsiloxane (DVB–CAR–PDMS) fiber. The extraction conditions were optimized using a response surface experimental design to analyze the effect of three factors: extraction temperature, equilibrium time and extraction time. The best response was obtained when the extraction temperature was around 80 °C, equilibrium time near 25 min and extraction time close to 18 min. Analyses were performed by GC–MS with a 5% diphenyl–95% dimethyl polysiloxane (30 m × 0.25 mm I.D., film thickness 0.25 μm) capillary column using He as the carrier gas and a programmed temperature run. The main components of the HS-SPME samples of E. rutaecarpa (concentration >3.0%) were limonene (33.79%), β-elemene (10.78%), linalool (8.15%), myrcene (5.83%), valencene (4.73%), β-caryophyllene (4.62%), linalyl acetate (4.13%) and α-terpineol (3.99%). As for E. officinalis, the major compounds were myrcene (32.79%), limonene (18.36%), β-caryophyllene (9.92%), trans-β-ocimene (6.04%), linalool (5.88%), β-elemene (7.85%) and valencene (4.62%).


2005 - Herbal drug quality and phytochemical composition of Hypericum perforatum L. affected by ash yellows phytoplasma infection [Articolo su rivista]
R., Bruni; Pellati, Federica; Bellardi, M. G.; Benvenuti, Stefania; S., Paltrinieri; A., Bertaccini; A., Bianchi
abstract

Qualitative/quantitative phytochemical variations were observed in dried flowering tops of cultivated Hypericum perforatum L. cv. Zorzi infected by phytoplasmas of the ash yellows class, identified by direct and nested polymerase chain reaction (PCR); this is the first report of ribosomial group 16SrVII phytoplasmas in St. John's Wort. Methanolic extracts of healthy and infected plants were separated by reversed phase high-performance liquid chromatography to quantify naphthodianthrones and flavonoids, while essential oils were analyzed by means of gas chromatography (GC)-GC/MS. The affected plants exhibited decreased amounts of rutin (1.96 +/- 0.23 vs 4.96 +/- 0.02 mg/g), hyperoside (2.38 +/- 0.21 vs 3.04 +/- 0.05 mg/g), isoquercitrin (1.47 +/- 0.04 vs 3.50 +/- 0.08 mg/g), amentoflavone (0.12 +/- 0.01 vs 0.39 +/- 0.02 mg/g), and pseudohypericin (1.41 +/- 0.23 vs 2.29 +/- 0.07 mg/g), whereas the chlorogenic acid content was doubled (1.56 +/- 0.11 vs 0.77 +/- 0.02 mg/g). Hypericin, quercitrin, and quercetin contents were not severely affected. The essential oil yield was drastically reduced in infected material (0.11 vs 0.75% in healthy material) and revealed an increased abundance of sesquiterpenes (beta-caryophyllene, delta-elemene, and germacrene D, in particular) and a matching decrease in monoterpene hydrocarbons and aliphatics. The consequences that the phytopathological condition of cultivated H. perforatum plants has on the commercial quality, market value, and, therapeutic efficacy are outlined.


2005 - Isolation, structural elucidation and biological activity of potential antiproliferative polyacetylenes from Echinacea pallida [Abstract in Atti di Convegno]
Pellati, Federica; S., Calò; Benvenuti, Stefania; B., Adinolfi; A., Chicca; P., Nieri; E., Martinotti; M., Melegari
abstract

In this work, the isolation, structural elucidation and biological activity of potential antiproliferative polyacetylenes and polyenes from Echinacea pallida was carried out and discussed.


2005 - Optimization and validation of HPLC methods for the analysis of phenethylamine and indoloquinazoline alkaloids in Evodia species [Abstract in Atti di Convegno]
Pellati, Federica; Benvenuti, Stefania; F., Yoshizaki; M., Melegari
abstract

In this work, HPLC methods were developed and validated for the phytochemical analysis of phenethylamine and indoloquinazoline alkaloids in Evodia species.


2005 - Second Joint Italian-Swiss Meeting on Medicinal Chemistry , Italy, Modena 19-20 February 2005. [Relazione in Atti di Convegno]
Brasili, Livio; Benvenuti, Stefania; Costantino, Luca; Costi, Maria Paola; Philipp, Floersheim; Franchini, Silvia; Gamberini, Gianfranco; Parenti, Carlo; Rastelli, Giulio; Rustichelli, Cecilia; Tait, Annalisa; Tondi, Donatella
abstract

The Second Joint Italian-Swiss Meeting on Medicinal Chemistry (ITCHMC2005) was held in Modena, (Italy) from September 12 to 16, 2005, under the auspices of the European Federation of Medicinal Chemistry (EFMC).This year, the annual meeting of the Division of Medicinal Chemistry of the Italian Chemical Society was co-organized with the Division for Medicinal Chemistry of the Swiss Chemical Society and it followed the first, successful one held in Torino in September 1997. This important event in the field of medicinal chemistry brought together scientists from both academia and industry to discuss different aspects of modern medicinal chemistry. Top-ranking scientists from medicinal chemistry and clinical development had the opportunity to meet and discuss the following topics: Carbohydrate Chemistry in Drug Design, Nuclear Receptors, Progress in Design and Development of Protease Inhibitors, Progress in Oncology Research and finally, Pain and Neurodegenerative Diseases.IntroductionThe conference was attended by 300 scientists from 13 countries, with most of the participants from Italy and Switzerland. The meeting allowed an extensive exchange of information and widespread networking. Of the 134 posters on display, 19 were selected for short oral presentations and two were selected for the Farminidustria awards. The meeeting was organized in six sessions with 6 Plenary (PL), 16 Main Lectures (ML) and 19 short communications (SC).The scientific sessions were held at the Forum Guido Monzani, a modern complex with multifunctional facilities; the opening ceremony took place at Accadenia Militare di Modena, housed in the seventeenth century Palazzo Ducale.Modena, city of art, culture and prosperous economy, offered an exciting background for stimulating scientific interactions. Participants were mainly from academia and other research centers together with 46 pharmaceutical companies; among them, six presented their work, namely Novartis Basel , Roche Basel, Novartis East Hannover USA, IRBM Pomezia Italy, Santhera Pharmaceutical AG, Heidelberg, GlaxoSmithKline (GSK) Verona, S-IN Soluzioni Informatiche, Vicenza. The areas covered by the meeting were advanced medicinal chemistry including computational chemistry, established drug targets, libraries and screens, inhibitor design and clinical advances. There were two poster sessions, with presentations given mainly by young scientists.


2005 - Variability in the composition of anti-oxidant compounds in Echinacea species by HPLC [Articolo su rivista]
Pellati, Federica; Benvenuti, Stefania; Melegari, Michele; T., Lasseigne
abstract

A fast and reliable HPLC method for the determination of caffeic acid derivatives (caftaric acid, chlorogenic acid, caffeic acid, cynarin, echinacoside and cichoric acid) in various species of the genus Echinacea has been developed. Extraction of root samples by magnetic stirring with 80% methanol aqueous solution at room temperature allowed the complete recovery of all compounds of interest. Root extracts were analysed on a reversed-phase column with gradient elution and photodiode array detection. Caffeic acid derivatives showed differential qualitative and quantitative distributions in Echinacea species. The total amount of phenolic compounds ranged from 33.95 to 0.32 mg/g. The highest contents of caffeic acid derivatives were found in E. paradoxa var. paradoxa, E. paradoxa var. neglecta and E. purpurea, followed by E. angustifolia var. angustifolia, E. simulata, E. pallida and E. laevigata, whilst E. tennesseensis, E. sanguinea and E. atrorubens had low amounts of phenolic compounds. The radical scavenging activities of methanolic extracts of roots of Echinacea species was evaluated in vitro using the DPPH* radical scavenging method. The EC50 values of the samples ranged from 122 to 1223 mu g/mL. The radical scavenging activities of the root extracts were correlated with the content of phenolic compounds, with a correlation coefficient (r(2)) of 0.923.


2004 - Analysis of phenolic compounds and radical scavenging activity of Echinacea spp. [Articolo su rivista]
Pellati, Federica; Benvenuti, Stefania; L., Magro; Melegari, Michele; F., Soragni
abstract

The aim of this study was to set up and validate an RP-LC method with DAD-detection to quantify caffeic acid derivatives in various Echinacea spp. Samples were extracted with 80% methanol. The analyses were carried out on a Lichrospher RP-18 column (125 mm x 4 mm i.d., 5 mum), with a mobile phase gradient, which increases the acetonitrile level in a phosphoric acid solution (0.1%). The flow rate was 1.5 ml/min. Detection was set at 330 nm. This method allowed the identification and quantification of caftaric acid, chlorogenic acid, caffeic acid, cynarin, echinacoside and cichoric acid in Echinacea roots and derivatives. The total phenolic content was 10.49 mg/g for E. angustifolia, 17.83 mg/g for E. pallida and 23.23 mg/g for E. purpurea. Among Echinacea commercial herbal medicines, a certain variability in the concentrations of phenolic compounds was observed. The radical scavenging activity of Echinacea methanolic extracts was evaluated in vitro with a spectrophotometric method based on the reduction of an alcoholic 2,2-diphenyl-1-picrylhydrazyl (DPPH.) radical solution at 517 nm in the presence of a hydrogen donating antioxidant. As for pure compounds, echinacoside had the highest capacity to quench DPPH. radicals (EC50 = 6.6 muM), while caftaric acid had the lowest (EC50 = 20.5 muM). The average EC50 values for E. purpurea, E. pallida and E. angustifolia were 134, 167 and 231 mug/ml, respectively. The radical scavenging activity of Echinacea root extracts reflected their phenolic composition. The results indicate that Echinacea roots and derivatives are a good source of natural antioxidants and could be used to prevent free-radical-induced deleterious effects.


2004 - Catharanthus roseus L. plants and explants infected with phytoplasmas: alkaloid production and structural observations. [Articolo su rivista]
Favali, M.; Musetti, R.; Benvenuti, Stefania; Bianchi, A.; Pressacco, L.
abstract

The results of several experiments concerning the presence and composition of alkaloids in different tissues (stems, leaves, roots) of Catharanthus roseus L. plants and explants, healthy and infected by clover phyllody phytoplasmas, are reported. The alkaloids extracted and determined by the reverse phase high-pressure liquid chromatography were vindoline, ajmalicine, serpentine, vinblastine, and vincristine. The total alkaloid concentration was higher in infected plants than in the controls, in particular the increase of vinblastine in infected roots was very significant. The ultrastructural observations of infected roots showed alterations of the cell walls and of the nuclei. These results demonstrate that phytoplasmas, detected in all infected tissues by light fluorescence and transmission electron microscopy, play an important role on secondary metabolism of the diseased plants, modifying both the total content of alkaloids and their ratio.


2004 - Chemical composition and fungicidal activity of commercial essential oils of Thymus vulgaris L [Articolo su rivista]
A., Zambonelli; Severi, Aldo; Benvenuti, Stefania; A., Zechini D’Aulerio; L., Maggi; A., Bianchi
abstract

.


2004 - Chromatographic performance of a new polar poly(ethyleneglycol) bonded phase for the phytochemical analysis of H. perforatum L. [Abstract in Atti di Convegno]
Pellati, Federica; Benvenuti, Stefania; M., Melegari
abstract

In this study, the chromatographic performance of a new polar poly(ethyleneglycol) bonded phase was evaluated for the phytochemical analysis of H. perforatum L. plant material and commercial products.


2004 - Enantioselective LC analysis of adrenergic amines of natural origin on a protein-based chiral stationary phase [Abstract in Atti di Convegno]
Pellati, Federica; Benvenuti, Stefania; M., Melegari
abstract

In this research, the enantioselective LC analysis of adrenergic amines of natural origin on a protein-based chiral stationary phase was carried out.


2004 - HS-SPME-GC-MS analysis of the volatile compounds of Evodia species fruits [Abstract in Atti di Convegno]
Pellati, Federica; Benvenuti, Stefania; Bertelli, Davide; F., Yoshizaki; Rossi, Maria Cecilia
abstract

In this study, a HS-SPME-GC-MS method for the analysis of the volatile compounds of Evodia species fruits was developed.


2004 - High-performance liquid chromatography methods for the analysis of adrenergic amines and flavanones in Citrus aurantium L. var. amara [Articolo su rivista]
Pellati, Federica; Benvenuti, Stefania; Melegari, Michele
abstract

Reverse-phase HPLC coupled with photodiode array detection was used for the simultaneous separation and determination of naturally occurring adrenergic amines (octopamine, synephrine and tyramine) in fruits and dry extracts of Citrus aurantium L. var. amara and in herbal medicines derived therefrom. Synephrine was the main component in fruits (0.10-0.35%) and in dry extracts (3.00-3.08%) and was present in the range 0.25-0.99% in herbal medicines. Flavanones were analysed in the same samples using a reverse-phase HPLC technique which allowed the identification and quantification of neoeriocitrin, narirutin, naringin, hesperidin, neohesperidin, naringenin and hesperetin. C. aurantium fruits and derivatives contained mainly glycosylated flavanones: in particular, naringin and neohesperidin were found to be the major flavonoids and their concentrations ranged from 1.80 to 26.30 and from 3.90 to 14.71 mg/g, respectively. The levels of aglycones were very low in all samples tested.


2004 - Poly(ethyleneglycol) stationary phase for the analysis of natural bioactive compounds [Abstract in Atti di Convegno]
Benvenuti, Stefania; Pellati, Federica; M., Melegari
abstract

In this study, the development and validation of a HPLC method for the analysis of bioactive compounds in H. perforatum extracts and natural products using a poly(ethyleneglycol) stationary phase was described.


2004 - Polyphenols, anthocyanins, ascorbic acid, and radical scavenging activity of Rubus, Ribes, and Aronia [Articolo su rivista]
Benvenuti, Stefania; Pellati, Federica; M., Melegari; Bertelli, Davide
abstract

Total polyphenols, total anthocyanins, and reduced ascorbic acid were evaluated in berries belonging to the genera Rubus, Ribes, and Aronia by means of spectrophotometric and HPLC techniques.The 2,2-diphenyl-1-picrylhydrazyl (DPPH•) radical scavenging activity of the fruit extracts was tested. Total polyphenols ranged from 140.6 to 888.5 mg/100 g fresh weight (FW), total anthocyanins ranged from 22.0 to 460.5 mg/100 g FW, and reduced ascorbic acid ranged from 12.4 to 153.8 mg/100 g FW. The average EC50 values for A. melanocarpa, R. nigrum, R. rubrum, R. fruticosus and R. idaeus were 1.8, 2.8, 5.3, 6.4 and 8.2 mg FW, respectively. The results indicate that the fruits tested are good sources of natural antioxidants.


2003 - Analysis of phenolic compounds and radical scavenging activity of Echinacea spp. [Abstract in Atti di Convegno]
Pellati, Federica; Benvenuti, Stefania; H., De Togni; L., Magro; M., Melegari; F., Soragni
abstract

In this study, the phytochical analysis of phenolic compounds and radical scavenging activity of Echinacea spp. was described.


2003 - Caratteristiche fitochimiche, proprietà farmacologiche e metodologie estrattive-analitiche di Vernonia kotchyana: recenti acquisizioni [Articolo su rivista]
Magro, L.; De Togni, H.; Pellati, Federica; Benvenuti, Stefania; Melegari, Michele
abstract

In questo articolo vengono descritte le recenti acquisizioni relative alle caratteristiche fitochimiche, proprietà farmacologiche e alle metodologie estrattive-analitiche applicate alla specie Vernonia kotchyana.


2003 - Caratteristiche fitochimiche, proprietà farmacologiche e metodologie estrattive-analitiche di Vernonia kotschyana: recenti acquisizioni [Relazione in Atti di Convegno]
L., Magro; H., De Togni; Pellati, Federica; Benvenuti, Stefania; M., Melegari
abstract

In questa relazione, sono descritte le caratteristiche fitochimiche e le proprietà biologiche di Vernonia kotschyana. Vengono inoltre presentate le recenti tecniche estrattive ed analitiche per la caratterizzazione dei componenti biologicamente attivi di questa specie.


2003 - Vernonia kotschyana e Pteleopsis suberosa: metodologie estrattive e caratterizzazione fitochimica [Articolo su rivista]
Magro, L.; De Togni, H.; Pellati, Federica; Benvenuti, Stefania; Melegari, M.
abstract

In questo articolo vengono descritte le metodologie estrattive ed analitiche utili alla caratterizzazione fitochimica di Vernonia kotschyana e Pteleopsis suberosa.


2002 - Analisi di componenti attivi e morfologia di Echinacea spp. [Abstract in Atti di Convegno]
Pellati, Federica; Benvenuti, Stefania; M., Melegari; L., Maffi; A., Bianchi
abstract

In questo studio, sono stati messi a punto metodi analitici avanzati e teniche di indagine farmacognostica per la caratterizzazione fitochimica di specie del genere Echinacea.


2002 - Aronia melanocarpa Elliot: un “piccolo frutto” di interesse nutrizionale e farmaceutico [Articolo su rivista]
Benvenuti, Stefania; Pellati, Federica; A., Rotundo; E., Marone; M., Melegari
abstract

In questo articolo vengono descritti i componenti biologicamente attivi di Aronia melanocarpa.


2002 - Determination of adrenergic agonists from extracts and herbal products of Citrus aurantium L. var. amara by LC [Articolo su rivista]
Pellati, Federica; Benvenuti, Stefania; Melegari, Michele; F., Firenzuoli
abstract

The purpose of this study was to set up a HPLC method to separate adrenergic amines (dl-octopamine, dl-synephrine and tyramine) and to determine their content in fruits, extracts and herbal products of Citrus aurantium L. var. amara. A rapid method for the quantitative analysis of these amines is described. based on their separation by RP-HPLC technique with UV detection. The analysis were conducted on a Lichrospher RP-18 column at room temperature, using a mobile phase consisting of 0.02 M citric acid-0.02 M NaH2PO4 (7:3 v/v) and adjusted to a final pH of 3. The detection was at 220 nm. Since some of these amines are chiral compounds and their enantiomers showed different pharmacological activity, the direct separation of synephrine enantiomers was carried out with HPLC on a beta-cyclodextrin stationary phase, The mobile phase consisted of methanol-NaH2PO4 25 mM pH 3.5 (20:80 v/v) and tetrabutylammonium hydrogen sulfate 10 mM in ratio of 30:70 v/v in isocratic condition and the detection was at 220 nm. The two proposed methods were applied to the analysis of fruits, extracts and herbal products of C. aurantium L. var. amara. Taking into account that some authors have reported that l-synephrine may be converted into its d-form by high temperature, this optical isomerization was monitored by the same HPLC method used for the separation of enantiomers.


2002 - Metodologie SCX-SPE e RP-HPLC per l’analisi di ammine biogene chirali da Citrus aurantium L. var. amara [Abstract in Atti di Convegno]
Pellati, Federica; Benvenuti, Stefania; M., Melegari
abstract

In questa presentazione vengono descritte recenti tecniche SCX-SPE e RP-HPLC per l'analisi di ammine biogene in Citrus aurantium.


2001 - Inter-population variability of secondary metabolites in Hypericum spp. (Hypericaceae) of the Northern Apennines, Italy [Articolo su rivista]
L., Maffi; Benvenuti, Stefania; R. B., Fornasiero; A., Bianchi; Melegari, Michele
abstract

Hypericum spp. flowers were collected from different sites of Northern Apennines in Italy and were analysed by HPTLC technique to determine their contents in flavonoids (rutin, hyperosid, quercitrin, isoquercitrin and quercetin) and hypericins (pseudohypericin and hypericin). The H. pet bratum and H. richeri composition was evaluated in order to investigate the influence of ecological conditions on secondary metabolites production. The species Studied showed some differences in the amounts of investigated metabolites and high variability was observed in populations gathered in different places. No significant effects of environmental factors on active compound production were observed, except for the rutin content which is in positive correlation with the altitude of the growing site. Interesting informations are also given about the morphology of secretory structures found in Hypericum richeri.


2001 - Nuove metodiche HPLC per la separazione enantiomerica di agonisti adrenergici di Citrus aurantium L. var. amara [Abstract in Atti di Convegno]
Pellati, Federica; Benvenuti, Stefania
abstract

In questa presentazione vengono descritte tecniche HPLC per la determinazione di agonisti adrenergici in Citrus aurantium L. var. amara.


2001 - Separation and determination of adrenergic agonists from extracts and herbal products of Citrus aurantium L. var. amara by HPLC techniques [Abstract in Atti di Convegno]
Pellati, Federica; Benvenuti, Stefania; M., Melegari; F., Firenzuoli
abstract

Separation and determination of adrenergic agonists from extracts and herbal products of Citrus aurantium L. var. amara by HPLC techniques.


2000 - Morphological and phytochemical features of secretory structures in Hypericum richeri (Clusiaceae) [Articolo su rivista]
Fornasiero, Rb; Maffi, L; Benvenuti, Stefania; Bianchi, A.
abstract

Structural and complementary chemical studies were carried out on Hypericum richeri, a lesser known species amongst those reported for folk medical use. We found only one type of secretory glands consisting of black dots which are present even in early emerging leaves. In the fully expanded leaves the nodular structure appears to be composed by a cluster of cells. These become unfunctional and disassembled towards the end of their development, and are used only as reservoirs of secretion products. HPTLC analyses showed that flower buds and flowers are the plant parts richest in active compounds. However, the spectrum of active compounds accumulated by H. richeri was both quantitatively and qualitatively similar to those reported for the pharmaceutically utilized. H. perforatum, and thus could potentially represent a possible alternative to this species.


2000 - Structural bases for the inhibition of aldose reductase by phenolic compounds [Articolo su rivista]
Rastelli, Giulio; L., Antolini; Benvenuti, Stefania; Costantino, Luca
abstract

Aldose reductase (ALR2) is an enzyme involved in the development of long-term diabetic complications. In the search for aldose reductase inhibitors less acidic than carboxylic acids, phenolic compounds related to benzopyran-4-one and chalcone are particularly interesting because they possess good inhibitory properties. In order to investigate the similarities between these two classes of compounds and to provide a structural basis for their inhibition of ALR2, the existing structure-activity relationships were reconsidered. To this end, the acidity constants of a set of chalcones were measured and compared with those of benzopyran-4-one derivatives. Then, having established the relevant protonation state of these phenolics at physiological pH, a conformational analysis was performed on the most active benzopyran-4-one and chalcone derivatives' and the results were compared with the crystal structures of some analogues. Finally, molecular docking of the most active chalcone into the ALR2 binding site was performed, and the structure of the enzyme-inhibitor complex was compared with that of the complex formed between ALR2 and a previously-obtained benzopyran-4-one derivative. (C) 2000 Elsevier Science Ltd. All rights reserved.


1999 - Melaleuca alternifolia L. ("Tea tree"): indagini analitiche e microbiologiche su oli essenziali e preparati del commercio [Articolo su rivista]
Benvenuti, Stefania; A., Camporese; Melegari, Michele; G., Vampa; A., Bianchi; M., Zandomeneghi
abstract

Melaleuca alternifolia L. ("Tea tree"): indagini analitiche e microbiologiche su oli essenziali e preparati del commercio


1998 - Caratterizzazione fitochimica, proprietà biologiche e nuove prospettive di utilizzo di ribes, lamponi e mirtilli [Articolo su rivista]
Melegari, Michele; Benvenuti, Stefania
abstract

Caratterizzazione fitochimica, proprietà biologiche e nuove prospettive di utilizzo di ribes, lamponi e mirtilli


1998 - Quality and yield of Ribes and Rubus cultivars grown in southern Italy hilly locations [Articolo su rivista]
Rotundo, A; Bounous, G; Benvenuti, Stefania; Vampa, G; Melegari, M; Soragni, F.
abstract

The berry fruit species, belonging to the genera Ribes and Rubus, are important for diversification of production in the mountainous and marginal areas of the Southern Apennines. The fruit, both for food and non-food (pharmaceutical, cosmetic industries) use, is a source of revenue for people living in these marginal areas. These species could be intensively exploited. To improve quality, plantation could be carried out according to the principles of modern genetic, agronomic and technical information. To observe the behaviour of the cultivars tested and to evaluate their bioagronomical performance and the quality of the fruits, trials were made in Melfi and Villa D´Agri (province of Potenza). Concerning the quality of the fruit, among other traits, the polyphenol, vitamin C and anthocyanin contents have been analysed. These compounds have interesting properties such as antiinflammatory, antioxidative, healing, aromatic, vitaminic, capillary vessel protecting. This justifies a broad use of berry fruits not only for food purposes but also as a source for pharmaceutical and cosmetic industries. The results obtained show a good adaptability of the cultivars tested in the different grounds: their productivity and the qualitative traits of the fruits are expected to improve the cultivation.


1998 - Synthesis and activity of a new series of chalcones as aldose reductase inhibitors [Articolo su rivista]
Severi, F; Benvenuti, Stefania; Costantino, Luca; Vampa, G; Melegari, M; Antolini, L.
abstract

A new series of chalcone derivatives has been synthesized and tested in vitro in order to assess their ability to inhibit aldose reductase enzyme (ALR2) and their specificity towards the target enzyme with respect to other oxidoreductases, such as aldehyde reductase, sorbitol dehydrogenase, and glutathione reductase. All the compounds display affinity for ALR2. The X-ray crystal structure of 1-(2,4-dihydroxyphenyl)-3-(2-methoxyphenyl)propen-1-one was determined.


1998 - Synthesis and aldose reductase inhibitory activity of benzoyl-amino acid derivatives [Articolo su rivista]
Benvenuti, Stefania; Severi, F; Costantino, Luca; Vampa, G; Melegari, M.
abstract

A series of N-(4-methoxy, 4-fluoro, 4-trifluoromethyl and 4-nitrobenzoyl)-L-amino acids was synthesized and their inhibitory activity towards bovine lens aldose reductase (ALR2) was tested. (C) 1998 Elsevier Science S.A. AII rights reserved.


1997 - Analytical investigations on active principles in aloe leaf exudate [Abstract in Atti di Convegno]
Benvenuti, Stefania; Rustichelli, Cecilia; Vampa, Gabriella; Ferioli, Valeria; G., Grassi; Melegari, Michele; P., Codeluppi; Gamberini, Gianfranco
abstract

The present work deals with the development of various procedures to estimate aloin and related compounds in aloe products by means of UV, HPTLC and HPLC procedures.


1997 - Synthesis, antimicrobial and genotoxic properties of some benzoimidazole derivatives [Articolo su rivista]
Benvenuti, Stefania; Severi, F; Sacchetti, A; Melegari, M; Vampa, G; Zani, F; Mazza, P; Antolini, L.
abstract

A number of 1H-benzoimidazol-2-ylamine and of 1-methyl-1H-benzoimidazol-2-ylamine derivatives were synthesized and the crystal and molecular structure of N-[4-(2-amino-benzoimidazole-1-sulfonyl)-phenyl] acetamide was determined by X-ray diffraction analysis. The compounds obtained were investigated for antimicrobial and genotoxic activities.


1996 - Identificazione, caratterizzazione e proprietà biologiche di derivati calconici da Glycyrrhiza Glabra L [Articolo su rivista]
Benvenuti, Stefania; F., Severi; Costantino, Luca; Melegari, Michele; Mucci, Adele
abstract

Sono state isolate, purificate e caratterizzate diverse sostanze di natura calconica e ne è stata saggiata l'attività sull'Aldoso Reduttasi.


1996 - Synthesis and description of chalcone-like compounds, inhibitors of aldose reductase [Articolo su rivista]
Severi, F; Costantino, Luca; Benvenuti, Stefania; Vampa, Gabriella; Mucci, Adele
abstract

A series of hydroxy- and hydroxy-methoxychalcones was synthesized and the inhibitory activity and selectivity of the compounds towards bovine lens aldose reductase (AR) were tested. All the compounds display affinity for AR The most active proved to be 1-(2,4-dihydroxyphenyl)-3-(4-hydroxyphenyl)propen-1-one (isoliquiritigenin, IC50 = 7.60 mu M). The selectivity of this compound was also tested, its inhibitory activity being assayed against glutathione reductase and sorbitol dehydrogenase


1995 - Chemical characterization of essential olis of some Origanum vulgare L. sub-species of various origin [Articolo su rivista]
Melegari, Michele; F., Severi; M., Bertoldi; Benvenuti, Stefania; G., Circella; I., Morone Fortunato; A., Bianchi; C., Leto; A., Carrubba
abstract

Chemical characterization of essential olis of some Origanum vulgare L. sub-species of various origin


1995 - Identificazione, caratterizzazione e proprietà biologiche di derivati calconici da GLYCYRRHIZA GLABRA L. [Abstract in Atti di Convegno]
Benvenuti, Stefania; F., Severi; Costantino, Luca; Melegari, Michele; Mucci, Adele
abstract

Sono state isolate, purificate e caratterizzate diverse sostanze di natura calconica e ne è stata saggiata l'attività sull'Aldoso Reduttasi.


1995 - SYNTHESIS, CHARACTERIZATION, AND CRYSTALLOGRAPHIC ANALYSIS OF SOME BENZOIMIDAZOLE DERIVATIVES [Articolo su rivista]
Benvenuti, Stefania; Severi, F; Vampa, G; Malmusi, L; Antolini, L.
abstract

A number of benzenesulfonyl derivatives of benzoimidazol-2-ylamine and 1-methyl-benzoimidazol-2-ylamine were synthesized, their synthesis reactions under different experimental conditions being monitored by hptlc. The crystal and molecular structures of N-(1-benzenesulfonyl-1,3-dihydrobenzoimidazol-2-ylidene)benzenesulfonamide (4) and N-(1-benzenesulfonyl-3-methyl-1,3-dihydro-benzoimidazol-2-ylidene)-benzenesulfonamide (7) were determined by X-ray diffraction analysis. The structure of compound 4 is made up of two crystallographically independent molecules and that of compound 7 of one molecule. In both cases, the structure contains the imido form of the molecules. There are strong conjugative effects between the imido groups and the imidazolic rings. Weak intramolecular C-H ... O hydrogen bonding interactions could influence the molecular conformations.


1995 - Synthesis, characterization and aldose reductase inhibitory of chalcone derivatives [Abstract in Atti di Convegno]
F., Severi; Benvenuti, Stefania; Costantino, Luca; Vampa, Gabriella; Melegari, Michele; Mucci, Adele
abstract

Synthesis, characterization and aldose reductase inhibitory of chalcone derivatives are reported.


1995 - Synthesis, characterization, crystallographic analysis, antifungal and genotoxic properties of some 1-methyl-1H-imidazoles [Articolo su rivista]
Zani, F; Mazza, P; Benvenuti, Stefania; Severi, F; Malmusi, L; Vampa, Gabriella; Antolini, L.
abstract

A number of 1-methyl-1H-imidazole derivatives and some of their oxygenated products were synthesized. An HPTLC technique for following the oxidation reactions in the different experimental conditions used was applied. The X-ray crystal structures of 1-methyl-2-methylsulfanyl-5-nitro-1H-imidazole, 2-methanesulfinyl-1-methyl-5-nitro-1H-imidazole and 2-methanesulfonyl-1-methyl-5-nitro-1H-imidazole were determined. The compounds obtained were investigated for antimycotic and genotoxic activities. The compounds tested were found to exert very low growth inhibition against yeasts and moulds. Moderate antifungal properties against dermatophytes were demonstrated for 5-nitro derivatives. 2-Methanesulfonyl-1-methyl-5-nitro-1H-imidazole was the most active substance. All 5-nitroimidazoles were genotoxic in Bacillus subtilis rec-assay, Salmonella microsome test and in Saccharomyces cerevisiae mitotic segregation assay. Structure-activity relationships are discussed


1995 - THE METHYLATION, OXIDATION AND CRYSTALLOGRAPHIC CHARACTERIZATION OF IMIDAZOLE DERIVATIVES [Articolo su rivista]
Vampa, G; Benvenuti, Stefania; Severi, F; Malmusi, L.
abstract

Compounds 2, 3 and 4 were synthesized and the crystal and molecular structures of 1 and 4 were determined. An hptlc technique for studying the methylation rate of 1 and the oxidation rate of 2 was applied.


1994 - Attivité antifongique in vitro de l’huille essentielle de chemotypes de lavandin [Articolo su rivista]
Costantino, Luca; Benvenuti, Stefania
abstract

Attivité antifongique in vitro de l’huille essentielle de chemotypes de lavandin


1994 - Glycyrrhizin and 18 beta-glycyrrhetinic acid: a comparative study of the pharmacological effects induced in the rat after prolonged oral treatment. [Articolo su rivista]
Rossi, T; Vampa, G; Benvenuti, Stefania; Pini, Luigi Alberto; Galatulas, I; Bossa, R; Castelli, M; Ruberto, Ai; Baggio, G.
abstract

Recent clinical and toxicological studies have investigated the mineralcorticoid-like and hypertensive effects of liquorice, and we therefore set out to identify the active component responsible for these effects. We conducted a 30-day comparative analysis of glycyrrhizin and 18 beta-glycyrrhetinic acid and found that the latter causes significant variations both in systolic blood pressure and in the excretion in the urine of Ca++. The effects were fully reversible on suspension of treatment.


1993 - Inhibition of mutagenicity in Salmonella typhimurium by Glycyrrhiza glabra extract, glycyrrhizinic acid, 18 alpha- and 18 beta-glycyrrhetinic acids [Articolo su rivista]
Zani, F; Cuzzoni, Mt; Daglia, M; Benvenuti, Stefania; Vampa, G; Mazza, P.
abstract

The effects of Glycyrrhiza glabra L. extract, glycyrrhizinic acid, 18 alpha- and 18 beta-glycyrrhetinic acids on the mutagenicity of the ethyl methanesulfonate, N-methyl-N'-nitro-N-nitrosoguanidine, and ribose-lysine Maillard model systems were investigated by using the Salmonella/microsome reversion assay. The protocol used allowed us to detect desmutagenic and antimutagenic activity and to avoid false positive results due to toxicity. For all the compounds tested, no desmutagenic activity was observed against ethyl methanesulfonate and N-methyl-N'-nitro-N-nitrosoguanidine; only Glycyrrhiza glabra extract showed antimutagenic activity against ethyl methanesulfonate. On using the ribose-lysine mutagenic browning mixture, the desmutagenic activities of the Glycyrrhiza glabra extract, glycyrrhizinic acid, 18 alpha- and 18 beta-glycyrrhetinic acids were observed. 18 beta-Glycyrrhetinic acid was the most active compound. Glycyrrhiza glabra extract also exhibited antimutagenic activity against ribose-lysine


1992 - Activity of polyphenolic crude extracts as scavengers of superoxide radicals and inhibitors of xanthine oxidase [Articolo su rivista]
Costantino, Luca; A., Albasini; Rastelli, Giulio; Benvenuti, Stefania
abstract

In view of the pharmacological interest in phenolic substances, we have determined the total amount of anthocyanins and polyphenols present in the berries of several cultivars of Ribes, Rubus, and Vaccinium genera. The in vitro antiradical activity of the crude extracts on chemically-generated superoxide radicals as well as the inhibitory activity towards the enzyme xanthine oxidase were studied. All the crude extracts examined showed a remarkably high activity towards chemically-generated superoxide radicals. The activities were greater than those expected on the basis of the quantities of anthocyanins and polyphenols present in the samples. Furthermore, the extracts showed a certain inhibitory activity towards xanthine oxidase. Ribes nigrum extracts exhibit the highest activity, being the richest in both anthocyanins and polyphenols. On the other hand, Ribes rubrum extracts seem to contain more active substances than the other crude extracts.


1992 - Crystal and molecular structure of 18α-glycyrrhetinic acid [Articolo su rivista]
Antolini, L; Vampa, G; Benvenuti, Stefania; Pecorari, P.
abstract

The crystal structure of 18-alpha-glycyrrhetinic acid (trans junction of the D/E rings) has been determined in order to compare its stereochemical features with those of the known 18-beta form (cis junction of the D/E rings). The two stereoisomers exhibit different physiological actions. A comparison of their common moieties shows significant differences between the conformations of the unsaturated C ring, with some torsion angles differing by more than ten degrees. Further differences involve the convexity of the ABCD region, which in the alpha-form is smaller than that observed in the beta-isomer, and smaller than that reported for the mean steroid configuration.


1992 - DETERMINATION OF THE REACTIVITY OF URACIL DERIVATIVES WITH RESPECT TO METHYL-IODIDE BY HIGH-PERFORMANCE THIN-LAYER CHROMATOGRAPHIC DENSITOMETRY [Articolo su rivista]
Vampa, G; Benvenuti, Stefania; Pecorari, P.
abstract

A high-performance thin-layer chromatographic method is described for the investigation of the methylation rates of uracil, 5-fluoro-, 5-chloro-, 5-bromo-, 5-methyl- and 5-nitrouracil and their N1- and N3-methyl derivatives. The method allowed the amounts of compounds obtained, the time required to complete the reaction and therefore the reactivity of the uracil derivatives to be determined. The N1- and N3-methyl and N1,3-dimethyl derivatives were synthesized and separated by droplet countercurrent chromatography and/or medium-pressure liquid chromatography and their R(F) values were determined in different solvent systems by thin-layer chromatography.


1992 - Determination of glycyrrhizin and 18 alpha-, 18 beta-glycyrrhetinic acid in rat plasma by high-performance thin-layer chromatography. [Articolo su rivista]
Vampa, Gabriella; Benvenuti, Stefania; Rossi, Tiziana
abstract

An HPTLC method is described for the simultaneous determination of glycyrrhizinic acid tripotassium salt (3K-G), 18 alpha- and 18 beta-glycyrrhetinic acid (18 alpha-GA, 18 beta-GA) in rat plasma, after oral administration of 3K-G (30 mg/kg once a day for 30 days). Systolic pressure and the volume of urine excreted in 24 hours were recorded during this period to observe any drug-induced effects.


1992 - HPTLC determination of ascorbic acid in fruits of the genera Ribes, Rubus and Vaccinium [Relazione in Atti di Convegno]
G., Vampa; Benvenuti, Stefania; Melegari, Michele
abstract

analysis of ascorbic acid in Ribes, Rubus, Vaccinium by HPTLC


1991 - Determination of glycyrrhizin in pharmaceutical preparations an in licorice products by high-performance thin-layer chromatographic densitometry [Articolo su rivista]
G., Vampa; Benvenuti, Stefania; Melegari, Michele
abstract

Determination of glycyrrhizin in pharmaceutical preparations an in licorice products by high-performance thin-layer chromatographic densitometry


1991 - SEPARATION OF 18-ALPHA-GLYCYRRHETINIC AND 18-BETA-GLYCYRRHETINIC ACID BY HIGH-PERFORMANCE THIN-LAYER CHROMATOGRAPHIC DENSITOMETRY [Articolo su rivista]
Vampa, G; Benvenuti, Stefania
abstract

A high-performance thin-layer chromatographic method with scanning densitometry was developed for the simultaneous separation and determination of 18-alpha- and 18-beta-glycyrrhetinic acid.


1991 - Studies on the genotoxic properties of essential oils with bacillus subtilis rec-assay and Salmonella/microsome reversion assay [Articolo su rivista]
F., Zani; G., Massimo; Benvenuti, Stefania; A., Bianchi; A., Albasini; Melegari, Michele; G., Vampa; A., Bellotti; P., Mazza
abstract

Genotoxic properties of essential oils from Anthemis nobilis L., Artemisia dracunculus L., Salvia officinalis L., Salvia sclarea L., Satureja hortensis L., Satureja montana L., Thymus capitatus L., Thymus citriodorus Schreb., Thymus vulgaris L., Citrus bergamia Risso, were studied with Bacillus subtilis rec-assay and Salmonella/microsome reversion assay. The essential oil of Artemisia dracunculus L. Piemontese turned out to be active in the rec-assay but not in the Salmonella test. DNA-damaging activity was demonstrated to be due to the estragol component of the oil. Advantages of the combined use of these two short-term microbial assays in genotoxic studies are discussed.


1989 - Caratterizzazione morfologica e chimica di presunti cloni di Castanea sativa Mill.. Atti Conv. "- Bedonia (PR), 15-17 [Relazione in Atti di Convegno]
Plessi, Maria; Benvenuti, Stefania; R., Antonaroli; Vampa, Gabriella; Monzani, Agar
abstract

I frutti di Castanea sativa di diverse coltivar vengono analizzati per il loro contemnuto in componenti nutrizionali e valutati dal punto di vista morfologico.


1987 - Ricerche su piante officinali nell'Appennino modenese - Piante spontanee - IV - Indagini sull'olio essenziale di Juniperus communis L. (ginepro) [Articolo su rivista]
Melegari, Michele; G., Vampa; A., Bianchi; A., Albasini; Benvenuti, Stefania; M., Rinaldi; A., Provvisionato
abstract

Ricerche su piante officinali nell'Appennino modenese - Piante spontanee - IV - Indagini sull'olio essenziale di Juniperus communis L. (ginepro)