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Federica PELLATI

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

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Pubblicazioni

2024 - Therapeutic potential of stilbenes in neuropsychiatric and neurological disorders: A comprehensive review of preclinical and clinical evidence [Articolo su rivista]
Socała, Katarzyna; Żmudzka, Elżbieta; Lustyk, Klaudia; Zagaja, Mirosław; Brighenti, Virginia; Costa, Anna Maria; Andres-Mach, Marta; Pytka, Karolina; Martinelli, Ilaria; Mandrioli, Jessica; Pellati, Federica; Biagini, Giuseppe; Wlaź, Piotr
abstract

: Neuropsychiatric disorders are anticipated to be a leading health concern in the near future, emphasizing an outstanding need for the development of new effective therapeutics to treat them. Stilbenes, with resveratrol attracting the most attention, are an example of multi-target compounds with promising therapeutic potential for a broad array of neuropsychiatric and neurological conditions. This review is a comprehensive summary of the current state of research on stilbenes in several neuropsychiatric and neurological disorders such as depression, anxiety, schizophrenia, autism spectrum disorders, epilepsy, traumatic brain injury, and neurodegenerative disorders. We describe and discuss the results of both in vitro and in vivo studies. The majority of studies concentrate on resveratrol, with limited findings exploring other stilbenes such as pterostilbene, piceatannol, polydatin, tetrahydroxystilbene glucoside, or synthetic resveratrol derivatives. Overall, although extensive preclinical studies show the potential benefits of stilbenes in various central nervous system disorders, clinical evidence on their therapeutic efficacy is largely missing.

2023 - An electrochemical approach for the prediction of Δ9-tetrahydrocannabinolic acid and total cannabinoid content in Cannabis sativa L [Articolo su rivista]
Monari, A.; Cantalu, S.; Zanfrognini, B.; Brighenti, V.; Verri, P.; Zanardi, C.; Pellati, F.; Pigani, L.
abstract

Two electrochemical sensors are proposed here for the first time for the fast screening of cannabinoids in Cannabis sativa L. plant material (inflorescences). The accurate control of cannabinoid content is important for discriminating between recreational, i.e. illegal, and fibre-type C. sativa samples, which differ mainly according to the amount of Δ9-tetrahydrocannabinol (Δ9-THC) and Δ9-tetrahydrocannabinolic acid (Δ9-THCA). Two screen printed electrodes obtained using different electrode materials were tested for the analysis of extracts from recreational and fibre-type C. sativa and their performance was compared with a consolidated method based on high-performance liquid chromatography (HPLC). The voltammetric responses recorded in the different samples reflected the compositional differences of the recreational and fibre-type extracts in accordance with the results of HPLC analyses. Moreover, the quantification of Δ9-THCA and the total cannabinoid content on the basis of the intensity of the peaks of the voltammograms was possible through a simple and fast electrochemical procedure.

2023 - Cannabidiol-rich non-psychotropic Cannabis sativa L. oils attenuate peripheral neuropathy symptoms by regulation of CB2-mediated microglial neuroinflammation [Articolo su rivista]
Borgonetti, Vittoria; Anceschi, Lisa; Brighenti, Virginia; Corsi, Lorenzo; Governa, Paolo; Manetti, Fabrizio; Pellati, Federica; Galeotti, Nicoletta
abstract

Neuropathic pain (NP) is a chronic disease that affects the normal quality of life of patients. To date, the therapies available are only symptomatic and they are unable to reduce the progression of the disease. Many studies reported the efficacy of Cannabis sativa L. (C. sativa) on NP, but no Δ9-tetrahydrocannabinol (Δ9-THC)-free extracts have been investigated in detail for this activity so far. The principal aim of this work is to investigate the potential pain-relieving effect of innovative cannabidiol-rich non-psychotropic C. sativa oils, with a high content of terpenes (K2), compared to the same extract devoid of terpenes (K1). Oral administration of K2 (25 mg kg−1) induced a rapid and long-lasting relief of pain hypersensitivity in a mice model of peripheral neuropathy. In spinal cord samples, K2 reduced mitogen-activated protein kinase (MAPKs) levels and neuroinflammatory factors. These effects were reverted by the administration of a CB2 antagonist (AM630), but not by a CB1 antagonist (AM251). Conversely, K1 showed a lower efficacy in the absence of CB1/CB2-mediated mechanisms. In LPS-stimulated murine microglial cells (BV2), K2 reduced microglia pro-inflammatory phenotype through the downregulation of histone deacetylase 1 (HDAC-1) and nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor (IKBα) and increased interleukin-10 (IL-10) expression, an important antiinflammatory cytokine. In conclusion, these results suggested that K2 oral administration attenuated NP symptoms by reducing spinal neuroinflammation and underline the important role of the synergism between cannabinoids and terpenes.

2023 - Helichrysum stoechas (L.) Moench reduces body weight gain and modulates mood disorders via inhibition of silent information regulator 1 (SIRT1) by arzanol [Articolo su rivista]
Borgonetti, V.; Caroli, C.; Governa, P.; Virginia, B.; Pollastro, F.; Franchini, S.; Manetti, F.; Les, F.; López, V.; Pellati, F.; Galeotti, N.
abstract

The prevalence of obesity is steadily rising, making safe and more efficient anti-obesity treatments an urgent medical need. Growing evidence correlates obesity and comorbidities, including anxiety and depression, with the development of a low-grade inflammation in peripheral and central tissues. We hypothesized that attenuating neuroinflammation might reduce weight gain and improve mood. We investigated the efficacy of a methanolic extract from Helichrysum stoechas (L.) Moench (HSE), well-known for its anti-inflammatory properties, and its main constituent arzanol (AZL). HPLC-ESI-MS2 and HPLC-UV were used to characterize the extract. HSE effects on mood and feeding behavior was assessed in mice. The mechanism of action of HSE and AZL was investigated in hippocampus samples and SH-SY5Y cells by western blotting and immunofluorescence. Oral administration of HSE for 3 weeks limited weight gain with no significant decrease in food intake. HSE produced an anxiolytic-like and antidepressant-like phenotype comparable to diazepam and amitriptyline, respectively, in the absence of locomotor and cognitive impairments and induced neuroprotective effects in glutamate-exposed SH-SY5Y cells. A dose-dependent reduction of SIRT1 expression was detected in SH-SY5Y cells and in hippocampal samples from HSE-treated mice. The inhibition of the SIRT1-FoxO1 pathway was induced in the hypothalamus. Molecular docking studies proposed a mechanism of SIRT1 inhibition by AZL, confirmed by the evaluation of inhibitory effects on SIRT1 enzymatic activity. HSE limited weight gain and comorbidities through an AZL-mediated SIRT1 inhibition. These activities indicate HSE an innovative therapeutic perspective for obesity and associated mood disorders.

2022 - Application of experimental design in HPLC method optimisation for the simultaneous determination of multiple bioactive cannabinoids [Articolo su rivista]
Durante, C.; Anceschi, L.; Brighenti, V.; Caroli, C.; Afezolli, C.; Marchetti, A.; Cocchi, M.; Salamone, S.; Pollastro, F.; Pellati, F.
abstract

The scientific interest in Cannabis sativa L. analysis has been rapidly increasing in recent years, especially for what concerns cannabinoids, plant secondary metabolites which are well known for having many biological properties. High-performance liquid chromatography (HPLC) is frequently used for both the qualitative and quantitative analysis of cannabinoids in plant extracts from C. sativa and its derived products. Many studies have been focused on the main cannabinoids, such as Delta(9)-tetrahydrocannabinolic acid (Delta(9)-THCA), cannabidiolic acid (CBDA), cannabigerolic acid (CBGA) and their decarboxylated derivatives, such as Delta(9)-tetrahydrocannabinol (Delta(THC)-T-9), cannabidiol (CBD) and cannabigerol (CBG). In addition to the abovementioned compounds, the plant produces other metabolites of the same chemical class, and some of them have shown interesting biological activities.In the light of this, it is important to have efficient analytical methods for the simultaneous separation of cannabinoids, which is quite complex since they present similar chemical-physical characteristics. The present work is focused on the use of the Design of Experiments technique (DoE) to develop and optimise an HPLC method for the simultaneous separation of 14 cannabinoids. Experimental design optimisation was applied by using a Central Composite Face-Centered design to achieve the best resolution with minimum experimental trials. Five significant variables affecting the chromatographic separation, including ammonium formate concentration, gradient elution, run time and flow rate, were studied. A multivariate strategy, based on Principal Component Analysis (PCA) and Partial Least Squared (PLS) regression, was used to define the best operative conditions. The developed method allowed for the separation of 12 out of 14 cannabinoids. Due to co-elution phenomena, HPLC coupled with a triple quadrupole mass analyser (HPLC-ESI-MS/MS) was applied, monitoring the specific transitions of each compound in the multiple reaction monitoring (MRM) mode. Finally, the optimised method was applied to C. sativa extracts having a different cannabinoid profile to demonstrate its efficiency to real samples.The methodology applied in this study can be useful for the separation of other cannabinoid mixtures, by means of appropriate optimisation of the experimental conditions.

2022 - Chemical characterization of non-psychoactive Cannabis sativa L. extracts, in vitro antiproliferative activity and induction of apoptosis in chronic myelogenous leukaemia cancer cells [Articolo su rivista]
Anceschi, L.; Codeluppi, A.; Brighenti, V.; Tassinari, R.; Taglioli, V.; Marchetti, L.; Roncati, L.; Alessandrini, A.; Corsi, L.; Pellati, F.
abstract

In this study, extracts from non-psychoactive Cannabis sativa L. varieties were characterized by means of ultra high-performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC-HRMS) and their antiproliferative activity was assessed in vitro. The human chronic myelogenous leukaemia cell line K562 was chosen to investigate the mechanism of cell death. The effect on the cell cycle and cell death was analysed by flow cytometry. Proteins related to apoptosis were studied by western blotting. Mechanical properties of cells were assessed using the Micropipette Aspiration Technique (MAT). The results indicated that the cannabidiol (CBD)-rich extract inhibited cell proliferation of K562 cell line in a dose-dependent manner and induced apoptosis via caspase 3 and 7 activation. A significant decrease in the mitochondrial membrane potential was detected, together with the release of cytochrome c into the cytosol. The main apoptotic markers were not involved in the mechanism of cell death. The extract was also able to modify the mechanical properties of cells. Thus, this hemp extract and its pure component CBD deserve further investigation for a possible application against myeloproliferative diseases, also in association with other anticancer drugs.

2022 - Disclosing the Antioxidant and Neuroprotective Activity of an Anthocyanin-Rich Extract from Sweet Cherry (Prunus avium L.) Using In Vitro and In Vivo Models [Articolo su rivista]
Filaferro, M.; Codeluppi, A.; Brighenti, V.; Cimurri, F.; Gonzalez-Paramas, A. M.; Santos-Buelga, C.; Bertelli, D.; Pellati, F.; Vitale, G.
abstract

In this study, an autochthonous variety of sweet cherry (Prunus avium L.), namely “Moretta di Vignola”, was processed to prepare extracts rich in polyphenols, which were characterized by high-performance liquid chromatography (HPLC) separation coupled to UV/DAD and ESI-MSn analysis. Then, a sweet cherry anthocyanin-rich extract (ACE) was prepared, fully characterized and tested for its activity against Parkinson’s disease (PD) in cellular (BV2 microglia and SH-SY5Y neuroblastoma) and in Drosophila melanogaster rotenone (ROT)-induced model. The extract was also evaluated for its antioxidant activity on Caenorhabditis elegans by assessing nematode resistance to thermal stress. In both cell lines, ACE reduced ROT-induced cell death and it decreased, alone, cellular reactive oxygen species (ROS) content while reinstating control-like ROS values after ROT-induced ROS rise, albeit at different concentrations of both compounds. Moreover, ACE mitigated SH-SY5Y cell cytotoxicity in a non-contact co-culture assay with cell-free supernatants from ROT-treated BV-2 cells. ACE, at 50 µg/mL, ameliorated ROT (250 µM)-provoked spontaneous (24 h duration) and induced (after 3 and 7 days) locomotor activity impairment in D. melanogaster and it also increased survival and counteracted the decrease in fly lifespan registered after exposure to the ROT. Moreover, heads from flies treated with ACE showed a non-significant decrease in ROS levels, while those exposed to ROT markedly increased ROS levels if compared to controls. ACE + ROT significantly placed the ROS content to intermediate values between those of controls and ROT alone. Finally, ACE at 25 µg/mL produced a significant increase in the survival rate of nematodes submitted to thermal stress (35 °C, 6–8 h), at the 2nd and 9th day of adulthood. All in all, ACE from Moretta cherries can be an attractive candidate to formulate a nutraceutical product to be used for the prevention of oxidative stress-induced disorders and related neurodegenerative diseases.

2022 - In vitro bioactivity evaluation of mulberry leaf extracts as nutraceutical for the management of diabete mellitus [Articolo su rivista]
Marchetti, Lucia; Truzzi, Eleonora; Frosi, Ilaria; Papetti, Adele; Cappellozza, Silvia; Saviane, Alessio; Pellati, Federica; Bertelli, Davide
abstract

2022 - Investigating the effect of polarity of stationary and mobile phases on retention of cannabinoids in normal phase liquid chromatography [Articolo su rivista]
De Luca, C.; Buratti, A.; Krauke, Y.; Stephan, S.; Monks, K.; Brighenti, V.; Pellati, F.; Cavazzini, A.; Catani, M.; Felletti, S.
abstract

This work reports about a screening of four adsorbents with different polarity employed for the separation of the main phytocannabinoids contained in Cannabis sativa L., under normal phase liquid chromatography (NPLC). The effect of polarity and type of interaction mechanisms of the adsorbents (namely Si-, CN-, Diol-, and NH2-based SPs) on retention has been investigated under a variety of conditions either by using different combinations of apolar solvents (heptane or hexane) and alcohols (ethanol or isopropanol). The columns have also been employed for the separation of a real cannabis sample. [Figure not available: see fulltext.]

2022 - Non-psychotropic Cannabis sativa L. phytocomplex modulates microglial inflammatory response through CB2 receptors-, endocannabinoids-, and NF-κB-mediated signaling [Articolo su rivista]
Borgonetti, V.; Benatti, C.; Governa, P.; Isoldi, G.; Pellati, F.; Alboni, S.; Tascedda, F.; Montopoli, M.; Galeotti, N.; Manetti, F.; Miraldi, E.; Biagi, M.; Rigillo, G.
abstract

Cannabis sativa L. is increasingly emerging for its protective role in modulating neuroinflammation, a complex process orchestrated among others by microglia, the resident immune cells of the central nervous system. Phytocannabinoids, especially cannabidiol (CBD), terpenes, and other constituents trigger several upstream and downstream microglial intracellular pathways. Here, we investigated the molecular mechanisms of a CBD- and terpenes-enriched C. sativa extract (CSE) in an in vitro model of neuroinflammation. We evaluated the effect of CSE on the inflammatory response induced by exposure to lipopolysaccharide (LPS) in BV-2 microglial cells, compared with CBD and β-caryophyllene (CAR), CB2 receptors (CB2r) inverse and full agonist, respectively. The LPS-induced upregulation of the pro-inflammatory cytokines IL-1β, IL-6, and TNF-α was significantly attenuated by CSE and only partially by CBD, whereas CAR was ineffective. In BV-2 cells, these anti-inflammatory effects exerted by CSE phytocomplex were only partially dependent on CB2r modulation and they were mediated by the regulation of enzymes responsible for the endocannabinoids metabolism, by the inhibition of reactive oxygen species release and the modulation of JNK/p38 cascade with consequent NF-κB p65 nuclear translocation suppression. Our data suggest that C. sativa phytocomplex and its multitarget mechanism could represent a novel therapeutic strategy for neuroinflammatory-related diseases.

2022 - Preface - Analytical issues realted to cannabinoids [Articolo su rivista]
Ganzera, M.; Pellati, F.
abstract

2021 - Antifungal Activity and DNA Topoisomerase Inhibition of Hydrolysable Tannins from Punica granatum L. [Articolo su rivista]
Brighenti, V.; Iseppi, R.; Pinzi, L.; Mincuzzi, A.; Ippolito, A.; Messi, P.; Sanzani, S. M.; Rastelli, G.; Pellati, F.
abstract

Punica granatum L. (pomegranate) fruit is known to be an important source of bioactive phenolic compounds belonging to hydrolysable tannins. Pomegranate extracts have shown antifungal activity, but the compounds responsible for this activity and their mechanism/s of action have not been completely elucidated up to now. The aim of the present study was the investigation of the inhibition ability of a selection of pomegranate phenolic compounds (i.e., punicalagin, punicalin, ellagic acid, gallic acid) on both plant and human fungal pathogens. In addition, the biological target of punicalagin was identified here for the first time. The antifungal activity of pomegranate phenolics was evaluated by means of Agar Disk Diffusion Assay and minimum inhibitory concentration (MIC) evaluation. A chemoinformatic analysis predicted for the first time topoisomerases I and II as potential biological targets of punicalagin, and this prediction was confirmed by in vitro inhibition assays. Concerning phytopathogens, all the tested compounds were effective, often similarly to the fungicide imazalil at the label dose. Particularly, punicalagin showed the lowest MIC for Alternaria alternata and Botrytis cinerea, whereas punicalin was the most active compound in terms of growth control extent. As for human pathogens, punicalagin was the most active compound among the tested ones against Candida albicans reference strains, as well as against the clinically isolates. UHPLC coupled with HRMS indicated that C. albicans, similarly to the phytopathogen Coniella granati, is able to hydrolyze both punicalagin and punicalin as a response to the fungal attack. Punicalagin showed a strong inhibitory activity, with IC50 values of 9.0 and 4.6 µM against C. albicans topoisomerases I and II, respectively. Altogether, the results provide evidence that punicalagin is a valuable candidate to be further exploited as an antifungal agent in particular against human fungal infections.

2021 - Antioxidant power on dermal cells by textiles dyed with an onion (Allium cepa L.) skin extract [Articolo su rivista]
Volpi, C.; Bartolini, D.; Brighenti, V.; Galli, F.; Tiecco, M.; Pellati, F.; Clementi, C.; Sardella, R.
abstract

In this study, the phenol loading and antioxidant activity of wool yarn prepared with the aqueous extract of onion (Allium cepa L.) skin was enhanced by implementing the dyeing process with the use of alum as a mordant. Spectrophotometric and chromatographic methods were applied for the characterization of polyphenolic substances loaded on the wool yarn. The antioxidant/anti-inflammatory properties were evaluated by determining the level of intra-and extra-cellular reactive oxygen species (ROS) production in keratinocytes and dermal fibroblasts pre-treated with lipopolysac-charide put in contact with artificial sweat. An elevated dye uptake on wool was observed for the pre-mordanted sample, as demonstrated by high absorbance values in the UV-Visible spectral range. Chromatographic results showed that protocatechuic acid and its glucoside were the main phenolic acid released in artificial sweat by the wool yarns, while quercetin-4′-glucoside and its aglycone quercetin were more retained. The extract released from the textile immersed in artificial sweat showed a significant reducing effect on the intra-and extracellular ROS levels in the two cell lines considered. Cytofluorimetric analyses demonstrated that the selected mordant was safe at the concentration used in the dyeing procedure. Therefore, alum pre-mordanted textiles dyed with onion-skin extracts may represent an interesting tool against skin diseases.

2021 - Antiseizure Effects of Fully Characterized Non-Psychoactive Cannabis sativa L. Extracts in the Repeated 6-Hz Corneal Stimulation Test [Articolo su rivista]
Costa, Anna-Maria; Senn, Lara; Anceschi, Lisa; Brighenti, Virginia; Pellati, Federica; Biagini, Giuseppe
abstract

Compounds present in Cannabis sativa L. preparations have recently attracted much attention in the treatment of drug-resistant epilepsy. Here, we screened two olive oil extracts from a non-psychoactive C. sativa variety, fully characterized by high-performance liquid chromatography and gas chromatography. Particularly, hemp oils with different concentrations of terpenes were administered at the same dose of cannabidiol (25 mg/kg/day orally), 1 h before the 6-Hz corneal stimulation test (44 mA). Mice were stimulated once a day for 5 days and evaluated by videoelectrocorticographic recordings and behavioral analysis. Neuronal activation was assessed by FosB/DFosB immunoreactivity. Both oils significantly reduced the percentage of mice experiencing convulsive seizures in comparison to olive oil-treated mice (p < 0.050; Fisher’s exact test), but only the oil enriched with terpenes (K2) significantly accelerated full recovery from the seizure. These effects occurred in the presence of reduced power of delta rhythm, and, instead, increased power of theta rhythm, along with a lower FosB/deltaFosB expression in the subiculum (p < 0.050; Duncan’s method). The overall findings suggest that both cannabinoids and terpenes in oil extracts should be considered as potential therapeutic agents against epileptic seizures and epilepsy.

2021 - Chemical composition, antifungal and antiproliferative activities of essential oils from Thymus numidicus L [Articolo su rivista]
Elaissi, A.; Elsharkawy, E.; El Mokni, R.; Debbabi, H.; Brighenti, V.; Nardoni, S.; Pellati, F.; Hammami, S.
abstract

In this study, Thymus numidicus Poir. plant material was collected from two different locations in north-western Tunisia and the aerial parts essential oils (EOs) were extracted via hydro-distillation. Gas chromatography coupled to mass spectrometry (GC-MS) and flame ionisation detection (GC-FID) were used for the qualitative analysis and quantification of the volatile constituents. Thymol (50.1–52.8%) was identified as the main compound of both EOs. To evaluate the potential application of the EOs as antifungal agents, the in vitro inhibitory effects were tested against six fungal strains; a strong antifungal activity of one sample was observed (MIC = 40–400 µg/mL). The in vitro antiproliferative activity was investigated on two human cancer cell lines, i.e. the colonic (HCT116) and breast adenocarcinoma (MCF7) using the colourimetric MTT assay. Again, the same sample demonstrated to possess good antiproliferative activity against both cancer cell lines, with IC50 values of 26.9 and 11.7 µg/mL, respectively.

2021 - Coupling a microwave-assisted process with HPLC-ELSD for the extraction, purification and analysis of bioactive policosanols from beeswax [Abstract in Atti di Convegno]
Venturelli, Alberto; Brighenti, Virginia; Tagliazucchi, Lorenzo; Costi, Maria Paola; Pellati, Federica
abstract

2021 - Determination of 1-deoxynojirimycin (1-dnj) in leaves of italian or italy-adapted cultivars of mulberry (morus sp.pl.) by hplc-ms [Articolo su rivista]
Marchetti, L.; Saviane, A.; Monta, A. D.; Paglia, G.; Pellati, F.; Benvenuti, S.; Bertelli, D.; Cappellozza, S.
abstract

Recently, 1-DNJ has been widely studied by scientists for its capacity to inhibit α-gluco-sidase and reduce postprandial blood glucose and fat accumulation. To the best of our knowledge, this is the first analytical determination of 1-DNJ in Morus sp.pl. leaves carried out on Italian crops, and it could be used as a reference to assess the quality of the plant material in comparison to Far Eastern Asia cultivations. The effects of two thermal treatments were compared to test the incidence of the drying process on the 1-DNJ extractability. In addition, two harvesting seasons in the same year (2017) and two subsequent harvesting years (2017–2018) were considered. The amount of 1-DNJ herein found was comparable to that reported in the scientific literature for Asian cultivations. The increase in 1-DNJ along the summer and the higher level of this compound in the apical leaves also complies with previous findings. However, a strong implication for the climatic conditions in the different years and a significant interaction between climate and genotypes suggest exploring very carefully the agronomic practices and selecting cultivars according to different environmental conditions with a view to standardize the 1-DNJ amount in leaves.

2021 - Discriminating different Cannabis sativa L. chemotypes using attenuated total reflectance - infrared (ATR-FTIR) spectroscopy: A proof of concept [Articolo su rivista]
Cirrincione, M.; Saladini, B.; Brighenti, V.; Salamone, S.; Mandrioli, R.; Pollastro, F.; Pellati, F.; Protti, M.; Mercolini, L.
abstract

An original, innovative, high-throughput method based on attenuated total reflectance - Fourier's transform infrared (ATR-FTIR) spectroscopy has been developed for the proof-of-concept discrimination of fibre-type from drug-type Cannabissativa L. inflorescences. The cannabis sample is placed on the instrument plate and analysed without any previous sample pretreatment step. In this way, a complete analysis lasts just a few seconds, the time needed to record an ATR-FTIR spectrum. The method was calibrated and cross-validated using data provided by liquid chromatography - tandem mass spectrometry (LC–MS/MS) analysis of the different cannabis samples and carried out the statistical assays for quantitation. During cross-validation, complete agreement was obtained between ATR-FTIR and LC–MS/MS identification of the cannabis chemotype. Moreover, the method has proved to be capable of quantifying with excellent accuracy (75–103 % vs. LC–MS/MS) seven neutral and acidic cannabinoids (THC, THCA, CBD, CBDA, CBG, CBGA, CBN) in inflorescences from different sources. The extreme feasibility and speed of execution make this ATR-FTIR method highly attractive as a proof-of-concept for a possible application to quality controls during pharmaceutical product manufacturing, as well as on-the-street cannabis controls and user counselling.

2021 - Emerging challenges in the extraction, analysis and bioanalysis of cannabidiol and related compounds [Articolo su rivista]
Brighenti, V.; Protti, M.; Anceschi, L.; Zanardi, C.; Mercolini, L.; Pellati, F.
abstract

Cannabidiol (CBD) is a bioactive terpenophenolic compound isolated from Cannabis sativa L. It is known to possess several properties of pharmaceutical interest, such as antioxidant, anti-inflammatory, anti-microbial, neuroprotective and anti-convulsant, being it active as a multi-target compound. From a therapeutic point of view, CBD is most commonly used for seizure disorder in children. CBD is present in both medical and fiber-type C. sativa plants, but, unlike Δ9-tetrahydrocannabinol (THC), it is a non-psychoactive compound. Non-psychoactive or fiber-type C. sativa (also known as hemp) differs from the medical one, since it contains only low levels of THC and high levels of CBD and related non-psychoactive cannabinoids. In addition to medical Cannabis, which is used for many different therapeutic purposes, a great expansion of the market of hemp plant material and related products has been observed in recent years, due to its usage in many fields, including food, cosmetics and electronic cigarettes liquids (commonly known as e-liquids). In this view, this work is focused on recent advances on sample preparation strategies and analytical methods for the chemical analysis of CBD and related compounds in both C. sativa plant material, its derived products and biological samples. Since sample preparation is considered to be a crucial step in the development of reliable analytical methods for the determination of natural compounds in complex matrices, different extraction methods are discussed. As regards the analysis of CBD and related compounds, the application of both separation and non-separation methods is discussed in detail. The advantages, disadvantages and applicability of the different methodologies currently available are evaluated. The scientific interest in the development of portable devices for the reliable analysis of CBD in vegetable and biological samples is also highlighted.

2021 - HR‐1H NMR spectroscopy and multivariate statistical analysis to determine the composition of herbal mixtures for infusions [Articolo su rivista]
Marchetti, Lucia; Rossi, Maria Cecilia; Pellati, Federica; Benvenuti, Stefania; Bertelli, Davide
abstract

This study aims to verify the potential of proton nuclear magnetic resonance (1H‐NMR) combined with partial least square regression to determine the composition of herbal mixtures. NMR one‐dimensional nuclear Overhauser effect spectroscopy (1D‐NOESY) spectra were analyzed to extract significant information from the raw data. The best performing model included four factors explaining 88.70 and 83.77% of the total variance in X and Y, respectively. These promising results have laid the basis for further development of the method, useful also for the analysis of commercial herbal infusions.

2021 - Micronized / ultramicronized palmitoylethanolamide (PEA) as natural neuroprotector against COVID-19 inflammation [Articolo su rivista]
Roncati, L.; Lusenti, B.; Pellati, F.; Corsi, L.
abstract

Coronavirus Disease 2019 (COVID-19) is upsetting the world and innovative therapeutic solutions are needed in an attempt to counter this new pandemic. Great hope lies in vaccines, but drugs to cure the infected patient are just as necessary. In the most severe forms of the disease, a cytokine storm with neuroinflammation occurs, putting the patient's life at serious risk, with sometimes long-lasting sequelae. Palmitoylethanolamide (PEA) is known to possess anti-inflammatory and neuroprotective properties, which make it an ideal candidate to be assumed in the earliest stage of the disease. Here, we provide a mini-review on the topic, pointing out phospholipids consumption in COVID-19, the possible development of an antiphospholipid syndrome secondary to SARS-CoV-2 infection, and reporting our preliminary single-case experience concerning to a 45-year-old COVID-19 female patient recently treated with success by micronized / ultramicronized PEA.

2021 - Separation and non-separation methods for the analysis of cannabinoids in Cannabis sativa L [Articolo su rivista]
Brighenti, V.; Marchetti, L.; Anceschi, L.; Protti, M.; Verri, P.; Pollastro, F.; Mercolini, L.; Bertelli, D.; Zanardi, C.; Pellati, F.
abstract

Cannabis sativa L. is a plant known all over the world, due to its history, bioactivity and also social impact. It is chemically complex with an astonishing ability in the biosynthesis of many secondary metabolites belonging to different chemical classes. Among them, cannabinoids are the most investigated ones, given their pharmacological relevance. In order to monitor the composition of the plant material and ensure the efficacy and safety of its derived products, extraction and analysis of cannabinoids play a crucial role. In this context, in addition to a conventional separation method based on HPLC with UV/DAD detection, a new strategy based on a non-separation procedure, such as 13C-qNMR, may offer several advantages, such as reduced solvent consumption and simultaneous acquisition of the quali/quantitative data related to many analytes. In the light of all the above, the aim of this work is to compare the efficiency of the above-mentioned analytical techniques for the study of the main cannabinoids in different samples of cannabis inflorescences, belonging to fibre-type, recreational and medical varieties. The 13C-qNMR method here proposed for the first time for the quantification of both psychoactive and non-psychoactive cannabinoids in different cannabis varieties provided reliable results in comparison to the more common and consolidated HPLC technique.

2020 - Assessment of the in vivo antioxidant activity of an anthocyanin-rich bilberry extract using the Caenorhabditis elegans model [Articolo su rivista]
Gonzalez-Paramas, A. M.; Brighenti, V.; Bertoni, L.; Marcelloni, L.; Ayuda-Duran, B.; Gonzalez-Manzano, S.; Pellati, F.; Santos-Buelga, C.
abstract

Anthocyanins have been associated with several health benefits, although the responsible mechanisms are not well established yet. In the present study, an anthocyanin-rich extract from bilberry (Vaccinium myrtillus L.) was tested in order to evaluate its capacity to modulate reactive oxygen species (ROS) production and resistance to thermally induced oxidative stress, using the nematode Caenorhabditis elegans as an in vivo model. The assays were carried out with the wild-type N2 strain and the mutant strains daf-16(mu86) I and hsf-1(sy441), which were grown in the presence of two anthocyanin extract concentrations (5 and 10 µg/mL in the culture medium) and further subjected to thermal stress. The treatment with the anthocyanin extract at 5 µg/mL showed protective effects on the accumulation of ROS and increased thermal resistance in C. elegans, both in stressed and non-stressed young and aged worms. However, detrimental effects were observed in nematodes treated with 10 µg/mL, leading to a higher worm mortality rate compared to controls, which was interpreted as a hormetic response. These findings suggested that the effects of the bilberry extract on C. elegans might not rely on its direct antioxidant capacity, but other mechanisms could also be involved. Additional assays were performed in two mutant strains with loss-of-function for DAF-16 (abnormal DAuer Formation factor 16) and HSF-1 (Heat Shock Factor 1) transcription factors, which act downstream of the insulin/insulin like growth factor-1 (IGF-1) signaling pathway. The results indicated that the modulation of these factors could be behind the improvement in the resistance against thermal stress produced by bilberry anthocyanins in young individuals, whereas they do not totally explain the effects produced in worms in the post-reproductive development stage. Further experiments are needed to continue uncovering the mechanisms behind the biological effects of anthocyanins in living organisms, as well as to establish whether they fall within the hormesis concept.

2020 - Effect-directed analysis of bioactive compounds in Cannabis sativa L. by high-performance thin-layer chromatography [Articolo su rivista]
Corni, G.; Brighenti, V.; Pellati, F.; Morlock, G. E.
abstract

The scientific interest on the plant Cannabis sativa L., and in particular on its non-psychoactive or fibre-type variety (hemp), has been highly increasing in recent years, due to the pharmaceutical and nutraceutical potential of its bioactive compounds. This plant is indeed characterized by a very rich chemical composition, which encompasses different classes of constituents, such as cannabinoids and terpenes. In this context, the bioanalytical testing of hemp extracts can be difficult and time-consuming. Effect-directed analysis (EDA) by the combination of high-performance thin-layer chromatography (HPTLC) with biological and enzymatic assays represents one of the latest tools available for the rapid bioprofiling of complex matrices, such as plant extracts. In this ambit, the aim of this project was the non-targeted screening of inflorescence extracts from ten different hemp varieties for components exhibiting radical scavenging, antibacterial, enzyme inhibiting and estrogen-like effects. By HPTLC-EDA, the hemp samples exhibited strong antibacterial activities against both Gram-positive Bacillus subtilis and especially Gram-negative Aliivibrio fischeri bacteria, and also estrogen-like activity. They also inhibited α- and β-glucosidase, tyrosinase and acetylcholinesterase. The characterization of two prominently multipotent bioactive compound zones was finally achieved by HPTLC-HRMS and preliminary assigned as cannabidiolic acid and cannabidivarinic acid.

2020 - Hippocampal synaptic and membrane function in the DBA/2J-mdx mouse model of Duchenne muscular dystrophy [Articolo su rivista]
Bianchi, Riccardo; Eilers, Wouter; Pellati, Federica; Corsi, Lorenzo; Foster, Helen; Foster, Keith; Tamagnini, Francesco
abstract

Dystrophin deficiency is associated with alterations in cell physiology. The functional consequences of dystrophin deficiency are particularly severe for muscle physiology, as observed in Duchenne muscle dystrophy (DMD). DMD is caused by the absence of a 427 kDa isoform of dystrophin. However, in addition to muscular dystrophy symptoms, DMD is frequently associated with memory and attention deficits and epilepsy. While this may be associated with a role for dystrophin in neuronal physiology, it is not clear what neuronal alterations are linked with DMD. Our work shows that CA1 pyramidal neurons from DBA/2J-mdx mice have increased afterhyperpolarization compared to WT controls. All the other electrotonic and electrogenic membrane properties were unaffected by this genotype. Finally, basal synaptic transmission, short-term and long-term synaptic plasticity at Schaffer collateral to CA1 glutamatergic synapses were unchanged between mdx and WT controls. These data show that the excitatory component of hippocampal activity is largely preserved in DBA/2J-mdx mice. Further studies, extending the investigation to the inhibitory GABAergic function, may provide a more complete picture of the functional, network alterations underlying impaired cognition in DMD. In addition, the investigation of changes in neuronal single conductance biophysical properties associated with this genotype, is required to identify the functional alterations associated with dystrophin deficiency and clarify its role in neuronal function.

2020 - Innovative methods for the preparation of medical Cannabis oils with a high content of both cannabinoids and terpenes [Articolo su rivista]
Ternelli, M.; Brighenti, V.; Anceschi, L.; Poto, M.; Bertelli, D; Licata, M.; Pellati, F.
abstract

Cannabis-based medications are being increasingly used for the treatment of different clinical conditions. Among all galenic formulations, olive oil extracts from medical Cannabis are the most prescribed ones for their easy preparation and usage. A great variety of methods have been described so far for the extraction of medical Cannabis oils to reach a high yield of Δ9-tetrahydrocannabinol (Δ9-THC), but poor attention has been paid to the preservation of the terpene fraction from the plant, which may contribute to the overall bioactivity of the extracts. In this context, the present study was aimed at the chemical characterization of different medical Cannabis oils prepared by following both innovative and existing extraction protocols, with particular attention to cannabinoids and terpenes, in order to set up a suitable method to obtain an extract rich in these chemical classes. In particular, six different extraction procedures were followed, based on different techniques, of which all but one included a decarboxylation of the plant material. The profile of cannabinoids was studied in detail by means of HPLC-ESI-MS/MS, while terpenes were characterized by means both GC-MS and GC-FID techniques coupled with solid-phase microextraction operated in the head-space mode (HS-SPME). An innovative method that is based on the extraction of the oil by dynamic maceration at room temperature from plant inflorescences, which were partially decarboxylated in a closed system at a moderate temperature and partially pre-extracted with ethanol, produced similar yields of bioactive compounds as that obtained by using a microwave-assisted distillation of the essential oil from the plant material, in combination with a maceration extraction of the oil from the residue. Both these new methods provided a higher efficiency over already existing extraction procedures of medical Cannabis oils and they can be applied to obtain a product with a high therapeutic value.

2020 - New insights into bioactive compounds from the medicinal plant Spathodea campanulata P. Beauv. and their activity against Helicobacter pylori [Articolo su rivista]
Ngnameko, C. R.; Marchetti, L.; Zambelli, B.; Quotadamo, A.; Roncarati, D.; Bertelli, D.; Njayou, F. N.; Smith, S. I.; Moundipa, P. F.; Costi, M. P.; Pellati, F.
abstract

The medicinal plant Spathodea campanulata P. Beauv. (Bignoniaceae) has been traditionally applied for the prevention and treatment of diseases of the kidney and urinary system, the skin, the gastrointestinal tract, and inflammation in general. The present work shows for the first time how chemical components from this plant inhibit Helicobacter pylori growth by urease inhibition and modulation of virulence factors. The crude extract and the main fractions of S. campanulata bark were tested on H. pylori isolated strains and the active ones were further fractionated. Fractions and sub-fractions of the plant crude extract were characterized by ultra-high-performance liquid chromatographic tandem high resolution-mass spectrometry detection (UHPLC-HRMS). Several phenolics and triterpenoids were identified. Among the sub-fractions obtained, SB2 showed the capacity to inhibit H. pylori urease in a heterologous bacterial model. One additional sub-fraction (SE3) was able to simultaneously modulate the expression of two adhesins (HopZ and BabA) and one cytotoxin (CagA). The flavonol kaempferol was identified as the most interesting compound that deserves further investigation as a new hit for its capacity to modulate H. pylori virulence factors.

2020 - Phytochemical Composition and In Vitro Antimicrobial Activity of Essential Oils from the Lamiaceae Family against Streptococcus agalactiae and Candida albicans Biofilms [Articolo su rivista]
Iseppi, Ramona; Tardugno, Roberta; Brighenti, Virginia; Benvenuti, Stefania; Sabia, Carla; Pellati, Federica; Messi, Patrizia
abstract

Abstract: The antimicrobial activity of different essential oils (EOs) from the Lamiaceae family was evaluated on Streptococcus agalactiae, Candida albicans, and lactobacilli. S. agalactiae is the main cause of severe neonatal infections, such as sepsis, meningitis, and pneumonia. C. albicans is a primary causative agent of vulvovaginal candidiasis, a multifactorial infectious disease of the lower female reproductive tract. Lactobacilli represent the dominant bacterial species of the vaginal flora and constitute the natural defense against pathogens. On the basis of the preliminary results, the attention was focused on the EOs from Lavandula x intermedia Emeric ex Loisel. and Mentha arvensis L. By using gas chromatography (GS) retention data and mass spectra, it was possible to identify more than 90% of the total composition of the EO samples. The minimal inhibitory concentration (MIC) and anti-biofilm activity of the two EOs were determined against all isolated strains, using the EOs by themselves or in combination with each other and with drugs (erythromycin and fluconazole). The results showed a good antimicrobial and anti-biofilm activity of both EOs and a synergistic effect, leading to the best results against all the strains, resulted using the combinations EOs/EOs and antimicrobials/EOs.

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 - Repositioning natural products in drug discovery [Articolo su rivista]
Rastelli, G.; Pellati, F.; Pinzi, L.; Gamberini, M. C.
abstract

No abstract available

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.

2020 - Use of a zwitterionic surfactant to improve the biofunctional properties of wool dyed with an onion (Allium cepa L.) skin extract [Articolo su rivista]
Puri, C.; Pucciarini, L.; Tiecco, M.; Brighenti, V.; Volpi, C.; Gargaro, M.; Germani, R.; Pellati, F.; Sardella, R.; Clementi, C.
abstract

To improve the loadability and antioxidant properties of wool impregnated with onion skin extract, the introduction of SB3-14 surfactant in the dyeing process was evaluated. A preliminary investigation on the surfactant–quercetin interaction indicated that the optimal conditions for dye solubility, stability, and surfactant affinity require double-distilled water (pH = 5.5) as a medium and SB3-14 in a concentration above the c.m.c. (2.5 × 10−3 M). The absorption profile of textiles showed the flavonoid absorption band (390 nm) and a bathochromic feature (510 nm), suggesting flavonoid aggregates. The higher absorbance for the sample dyed with SB3-14 indicated greater dye uptake, which was further confirmed by HPLC analysis. The Folin–Ciocalteu method was applied to evaluate the total phenol content (TPC) released from the treated wool, while the assays FRAP, DPPH, ABTS, and ORAC were applied to evaluate the corresponding total antioxidant activity (TAC). Higher TPCs (about 20%) and TACs (5–55%) were measured with SB3-14, highlighting textiles with improved biofunctional properties. Spectrophotometric analyses were also performed with an artificial sweat. The potential cytotoxic effect of SB3-14 in both monomeric and aggregated forms, cell viability, and induction of apoptosis were evaluated in RAW 264.7 cells. These analyses revealed that SB3-14 is safe at concentrations below the c.m.c.

2020 - Zingiber officinale Roscoe rhizome extract alleviates neuropathic pain by inhibiting neuroinflammation in mice [Articolo su rivista]
Borgonetti, V.; Governa, P.; Biagi, M.; Pellati, F.; Galeotti, N.
abstract

Background: Current therapies for neuropathic pain are generally symptomatic and possess several side effects, limiting their prolonged usage. Hypothesis/Purpose: Thus, it is urgent to develop novel and safe candidates for the management of this chronical condition. For this purpose, we investigated the analgesic effect of a standardized extract from Zingiber officinale Roscoe rhizomes (ZOE) obtained by CO2 supercritical extraction, in a mice model of peripheral neuropathy. We also explored the mechanism of action of ZOE and its main constituents using an in vitro model of neuroinflammation. Methods: Peripheral mono-neuropathy was induced in mice, by spared nerve injury (SNI). The analgesic effect of ZOE after oral administration was assessed by measuring mechanical and thermal allodynia in SNI mice. The mechanism of action of ZOE and its main constituents were investigated using spinal cords samples and in an in vitro model of neuroinflammation by ELISA, western blotting and immunofluorescence techniques. Results: Oral administration of ZOE 200 mg kg−1 ameliorated mechanical and thermal allodynia in SNI mice, with a rapid and a long-lasting effect. ZOE did not alter locomotor activity. In BV2 cells and spinal cord samples, ZOE, 6-gingerol and 6-shogaol reduced pERK levels, whereas ZOE and terpene fraction reduced HDAC1 protein levels, inhibited NF-κB signalling activation and decreased IL-1β, TNF-α and IL-6 release. ZOE and each tested constituent had a positive effect on inflammation-impaired SH-SY5Y cell viability. Conclusions: The oral administration of ZOE attenuated SNI-induced neuropathic pain symptoms by reducing spinal neuroinflammation, suggesting ZOE as a novel and interesting candidate for the management of neuropathic pain.

2019 - A new strategy based on microwave-assisted technology for the extraction and purification of beeswax policosanols for pharmaceutical purposes and beyond [Articolo su rivista]
Venturelli, A.; Brighenti, V.; Mascolo, D.; Pellati, F.
abstract

Policosanols (PCs) are a mixture of long chain primary aliphatic alcohols mainly known for their ability to reduce cholesterol level. Due to this property, there is an increasing interest in the extraction process of these compounds. In this context, beeswax, a natural product produced by honey bees of the genus Apis, is a promising source for their extraction and purification. The present research work was aimed at the development of a new procedure for the extraction and purification of PCs from yellow beeswax by using microwave-assisted technology, which hitherto has never been applied to this mixture. The developed process comprises three main steps: 1) microwave-assisted trans-esterification; 2) microwave-assisted hydrolysis; 3) final purification by means of preparative liquid chromatography. The final step is responsible for the increased purity of PCs, thanks to the removal of undesired compounds, such as natural paraffins. The predominant alcohols investigated in this work are tetracosanol (C 24 OH), hexacosanol (C 26 OH), octacosanol (C 28 OH), triacontanol (C 30 OH) and dotriacontanol (C 32 OH). Compound identification was performed using GC-EI-MS, while GC-FID analysis was chosen for the quantification of the main fatty alcohols present in the product. This new method represents a useful tool for the production of PCs from beeswax to be used in pharmaceuticals and nutraceuticals for human use, feed and veterinary supplements.

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 - BetaSweet carrot extracts have antioxidant activity and in vitro antiproliferative effects against breast cancer cells [Articolo su rivista]
Jayaprakasha, G. K.; Chidambara Murthy, K. N.; Pellati, F.; Patil, B. S.
abstract

Fruits and vegetables have long been studied for their potential roles in reducing certain cancer risks. Herein, lyophilized BetaSweet carrots were extracted with hexane, methanol, and water and their chemical constituents were identified by UPLC-QTOF-HR-MS. Methanol extracts exhibited significantly higher radical scavenging activity compared with hexane and water extracts. Hexane extracts showed significantly higher (88.4%) inhibition of breast cancer cells proliferation after 24 h at 100 µg/mL, followed by water and methanol extracts, likely due to the presence of polyacetylenes. Cells treated with hexane extracts showed the highest cytochrome-c release, indicating induction of apoptosis, as supported by DNA fragmentation and Bax2 and bcl2 expression and confirmed by staining with propidium iodide and acridine orange. The methanol extract showed the highest antioxidant activity and was purified to obtain cyanidin-3-2″-xylosyl-6″-(ferulosyl-glucosyl)-galactoside. In conclusion, BetaSweet carrots exhibits multiple health benefits to use as functional foods.

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 - Cannabinoids from Cannabis sativa L.: A New Tool Based on HPLC-DAD-MS/MS for a Rational Use in Medicinal Chemistry [Articolo su rivista]
Protti, M.; Brighenti, V.; Battaglia, M. R.; ANCESCHI, LISA; Pellati, F.; Mercolini, L.
abstract

Cannabis sativa L. represents one of the most widely used source of drugs and drugs of abuse worldwide. Its biologically active compounds are mainly cannabinoids, including 9 -Tetrahydrocannabinol (THC), which is responsible for the psychoactive effects, tetrahydrocannabinolic acid (THCA), cannabinol (CBN), cannabidiol (CBD), and cannabidiolic acid (CBDA). Together with recreational and drug-Type (or medicinal) Cannabis, some new products have been recently released into the market as fiber-Type Cannabis variants (also known as hemp or industrial hemp) with low THC content and high content of nonpsychoactive CBD. In this research work, the aim was to characterize Cannabis recreational and drug-Type samples by quantifying their active principles, after the development and validation of a suitable analytical method. In addition to the Cannabis samples described above, fiber-Type plant varieties were also analyzed to monitor their content of nonpsychoactive compounds for both pharmaceutical and nutraceutical purposes. To do this, a highly efficient HPLC-DAD-MS/MS method, with an electrospray ionization (ESI) source and a triple-quadrupole mass analyzer acquiring in the multiple reaction monitoring (MRM) mode also coupled to a diode array detector (DAD), was developed and applied. Satisfactory validation results were obtained in terms of precision (RSD < 6.0% for all the analytes) and accuracy (>92.1% for all the compounds). The proposed methodology represents a versatile and reliable tool to assess both psychoactive and nonpsychoactive cannabinoid levels in Cannabis samples for a more rational use in both medicinal chemistry and nutraceutics.

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 - 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 - 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 - Identification and determination of bioactive phenylpropanoid glycosides of Aloysia polystachya (Griseb. et Moldenke) by HPLC-MS [Articolo su rivista]
Marchetti, Lucia; Pellati, Federica; Graziosi, Riccardo; Brighenti, Virginia; Pinetti, Diego; Bertelli, Davide
abstract

Aloysia polystachya (Griseb. et Moldenke) has not been deeply investigated in past years and currently data about its chemical composition are limited. Phenolic compounds characterization can be very difficult in vegetable matrices, owing to bonds to sugar moieties or conjugation, giving rise to complex structures. In this work, methanolic extracts of Aloysia polystachya leaves were analyzed by HPLC-ESI-MS, the favourite technique for the separation and quantification of their polyphenols. To assess the complete characterization and quantification of the phenylpropanoid fraction, three different MS techniques have been coupled to HPLC: ion trap mass spectrometry (Ion Trap LC/MS), quadrupole-time of flight high resolution mass spectrometry (Q-TOF HRMS) and triple-quadrupole (TQ LC/MS) for the quantification. Eleven phenylpropanoid glycosides were identified and quantified and, among them, the compounds forsythoside A, plantainoside C, purpureaside D, martynoside and its two isomers were detected for the first time to the best of our knowledge. The results presented here could be helpful to assess the quality of this plant and could further contribute to the chemotaxonomy of the genus.

2019 - In silico repositioning of cannabigerol as a novel inhibitor of the enoyl acyl carrier protein (ACP) reductase (INHA) [Articolo su rivista]
Pinzi, L.; Lherbet, C.; Baltas, M.; Pellati, F.; Rastelli, G.
abstract

Cannabigerol (CBG) and cannabichromene (CBC) are non-psychoactive cannabinoids that have raised increasing interest in recent years. These compounds exhibit good tolerability and low toxicity, representing promising candidates for drug repositioning. To identify novel potential therapeutic targets for CBG and CBC, an integrated ligand-based and structure-based study was performed. The results of the analysis led to the identification of CBG as a low micromolar inhibitor of the Enoyl acyl carrier protein (ACP) reductase (InhA) enzyme.

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 - Monitoring of chlorogenic acid and antioxidant capacity of Solanum melongena L. (eggplant) under different heat and storage treatments [Articolo su rivista]
Šilarová, P.; Boulekbache-Makhlouf, L.; Pellati, F.; Česlová, L.
abstract

Solanum melongena L., also known as eggplant, is a widely consumed vegetable and it is well-known for its beneficial antioxidant properties, due to phenolic compounds. In this work, the influence of different cooking procedures on the content of chlorogenic acid was evaluated on eggplant samples of different geographic origin by high-performance liquid chromatography (HPLC). An easy and quick extraction procedure with 50% methanol as the extraction solvent was optimized for the first time by means of a design-of-experiment and applied to heat treated samples of eggplant. The antioxidant capacity of eggplant extracts was also evaluated by using the ABTS assay and it was correlated with the data obtained by the HPLC method. The content of chlorogenic acid was different in each heat-treated eggplant sample and it depended on the temperature applied during the cooking procedure. In particular, an increase of chlorogenic acid content with rising temperature was observed. Conversely, a very high temperature (250◦C) caused a decrease of chlorogenic acid amount. The influence of storage on the content of chlorogenic acid was also monitored. While the level of chlorogenic acid in fresh samples decreased during four weeks of storage, an increase in its content in heat treated eggplant was observed within the same period. Multivariate data analysis was used to classify eggplant samples into different groups, according to the country of origin and heat treatment procedure. This study provides new insights to preserve the antioxidant properties of eggplant phenolics during different thermal and storage treatments in order to highlight their health promoting effects.

2019 - Onion (Allium cepa L.) Skin: A rich resource of biomolecules for the sustainable production of colored biofunctional textiles [Articolo su rivista]
Pucciarini, L.; Ianni, F.; Petesse, V.; Pellati, F.; Brighenti, V.; Volpi, C.; Gargaro, M.; Natalini, B.; Clementi, C.; Sardella, R.
abstract

The aqueous extract of dry onion skin waste from the ‘Dorata di Parma’ cultivar was tested as a new source of biomolecules for the production of colored and biofunctional wool yarns, through environmentally friendly dyeing procedures. Specific attention was paid to the antioxidant and UV protection properties of the resulting textiles. On the basis of spectrophotometric and mass spectrometry analyses, the obtained deep red-brown color was assigned to quercetin and its glycoside derivatives. The Folin–Ciocalteu method revealed good phenol uptakes on the wool fiber (higher than 27% for the textile after the first dyeing cycle), with respect to the original total content estimated in the water extract (78.50 ± 2.49 mg equivalent gallic acid/g onion skin). The manufactured materials showed remarkable antioxidant activity and ability to protect human skin against lipid peroxidation following UV radiation: 7.65 ± 1.43 (FRAP assay) and 13.60 (ORAC assay) mg equivalent trolox/g textile; lipid peroxidation inhibition up to 89.37%. This photoprotective and antioxidant activity were therefore ascribed to the polyphenol pool contained in the outer dried gold skins of onion. It is worth noting that citofluorimetric analysis demonstrated that the aqueous extract does not have a significative influence on cell viability, neither is capable of inducing a proapoptotic effect.

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.

2019 - Use of 13C-qNMR Spectroscopy for the Analysis of Non-Psychoactive Cannabinoids in Fibre-Type Cannabis sativa L. (Hemp) [Articolo su rivista]
Marchetti, L.; Brighenti, V.; Rossi, M. C.; Sperlea, J.; Pellati, F.; Bertelli, D.
abstract

Cannabis sativa L. is a dioecious plant belonging to the Cannabaceae family. The discovery of the presence of many biologically-active metabolites (cannabinoids) in fibre-type Cannabis (hemp) has recently given rise to the valorisation of this variety. In this context, the present study was aimed at the multi-component analysis and determination of the main non-psychoactive cannabinoids (cannabidiol, cannabidiolic acid, cannabigerol and cannabigerolic acid) in female inflorescences of different hemp varieties by means of 13C quantitative nuclear magnetic resonance spectroscopy (qNMR). The method proposed here for the first time for the determination of cannabinoids provided reliable results in a competitive time with respect to the more consolidated HPLC technique. In fact, it gave sufficiently precise and sensitive results, with LOQ values lower than 750 µg/mL, which is easily achievable with concentrated extracts, without affecting the quality of 13C-qNMR spectra. In conclusion, this method can be considered as a promising and appropriate tool for the comprehensive chemical analysis of bioactive cannabinoids in hemp and other derived products in order to ensure their quality, efficacy and safety.

2018 - Analytical methods for the study of bioactive compounds from medicinally used Echinacea species [Articolo su rivista]
Bruni, R.; Brighenti, V.; Caesar, L. K.; Bertelli, D.; Cech, N. B.; Pellati, F.
abstract

Echinacea purpurea (L.) Moench, Echinacea angustifolia DC. var. angustifolia and Echinacea pallida (Nutt.) Nutt. are frequently used as medicinal plants and their preparations are among the most widely used herbal medicines. The extracts from these species have shown a highly complex chemical composition, including polar compounds (caffeic acid derivatives, CADs), non-polar compounds (alkylamides and acetylenic secondary metabolites; essential oil) and high molecular weight constituents (polysaccharides and glycoproteins). All these chemical classes of compounds have demonstrated to possess interesting biological activities. In the light of all the above, this paper is focused on the analytical techniques, including sample preparation tools and chromatographic procedures, for the chemical analysis of bioactive compounds in medicinally used Echinacea species. Since sample preparation is considered to be a crucial step in the development of analytical methods for the determination of constituents present in herbal preparations, the strength and weakness of different extraction techniques are discussed. As regards the analysis of compounds present in Echinacea plant material and derivatives, the application of different techniques, mainly HPLC, HPLC-ESI-MS, HPLC-ESI-MS/MS, HPCE, HPTLC and GC, is discussed in detail. The strength, weakness and applicability of the different separation tools are stated.

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 - Nuclear magnetic resonance and high-performance liquid chromatography techniques for the characterization of bioactive compounds from Humulus lupulus L. (hop) [Articolo su rivista]
Bertelli, D.; Brighenti, V.; Marchetti, L.; Reik, A.; Pellati, F.
abstract

Humulus lupulus L. (hop) represents one of the most cultivated crops, it being a key ingredient in the brewing process. Many health-related properties have been described for hop extracts, making this plant gain more interest in the field of pharmaceutical and nutraceutical research. Among the analytical tools available for the phytochemical characterization of plant extracts, quantitative nuclear magnetic resonance (qNMR) represents a new and powerful technique. In this ambit, the present study was aimed at the development of a new, simple, and efficient qNMR method for the metabolite fingerprinting of bioactive compounds in hop cones, taking advantage of the novel ERETIC 2 tool. To the best of our knowledge, this is the first attempt to apply this method to complex matrices of natural origin, such as hop extracts. The qNMR method set up in this study was applied to the quantification of both prenylflavonoids and bitter acids in eight hop cultivars. The performance of this analytical method was compared with that of HPLC-UV/DAD, which represents the most frequently used technique in the field of natural product analysis. The quantitative data obtained for hop samples by means of the two aforementioned techniques highlighted that the amount of bioactive compounds was slightly higher when qNMR was applied, although the order of magnitude of the values was the same. The accuracy of qNMR was comparable to that of the chromatographic method, thus proving to be a reliable tool for the analysis of these secondary metabolites in hop extracts.

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.

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 - Discriminant biomarkers of acute respiratory distress syndrome associated to H1N1 influenza identified by metabolomics HPLC-QTOF-MS/MS platform [Articolo su rivista]
Ferrarini, A.; Righetti, L.; Martínez, M. P.; Fernández-López, M.; Mastrangelo, ANNA LAURA; Horcajada, J. P.; Betbesé, A.; Esteban, A.; ROJAS ORDOÑEZ, JIMMY ALEXANDER; Gea, J.; Cabello, J. R.; Pellati, F.; Lorente, J. A.; Nin, N.; Rupérez, F. J.
abstract

Acute respiratory distress syndrome (ARDS) is a serious complication of influenza A (H1N1) virus infection. Its pathogenesis is unknown and biomarkers are lacking. Untargeted metabolomics allows the analysis of the whole metabolome in a biological compartment, identifying patterns associated with specific conditions. We hypothesized that LC-MS could help identify discriminant metabolites able to define the metabolic alterations occurring in patients with influenza A (H1N1) virus infection that developed ARDS. Serum samples from patients diagnosed with 2009 influenza A (H1N1) virus infection with (n = 25) or without (n = 32) ARDS were obtained on the day of hospital admission and analyzed by LC-MS/MS. Metabolite identification was determined by MS/MS analysis and analysis of standards. The specificity of the patterns identified was confirmed in patients without 2009 influenza A(H1N1) virus pneumonia (15 without and 17 with ARDS). Twenty-three candidate biomarkers were found to be significantly different between the two groups, including lysophospholipids and sphingolipids related to inflammation; bile acids, tryptophan metabolites, and thyroxine, related to the metabolism of the gut microflora. Confirmation results demonstrated the specificity of major alterations occurring in ARDS patients with influenza A (H1N1) virus infection.

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.

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.

2016 - Novel and less explored chemotypes of natural origin for the inhibition of Hsp90 [Articolo su rivista]
Pellati, Federica; Rastelli, Giulio
abstract

The Hsp90 chaperone is a promising target for the treatment of cancer. Well known inhibitors, e.g. geldanamycin, radicicol, novobiocin and their derivatives, have been extensively investigated and optimized as inhibitors of Hsp90. More recently, new chemotypes of natural origin were reported to modulate Hsp90 function via various and often “non-conventional” mechanisms. This review focuses on these novel classes of natural products whose structures have not yet been thoroughly explored for medicinal chemistry purposes. These novel chemotypes may constitute interesting starting points for future drug discovery campaigns.

2016 - Profiling of Flavonol Derivatives for the Development of Antitrypanosomatidic Drugs [Articolo su rivista]
Borsari, Chiara; Luciani, Rosaria; Pozzi, Cecilia; Poehner, Ina; Henrich, Stefan; Trande, Matteo; Cordeiro da Silva, Anabela; Santarem, Nuno; Baptista, Catarina; Tait, Annalisa; Di Pisa, Flavio; Dello, Iacono; Lucia, Landi; Giacomo, ; Gul, Sheraz; Wolf, Markus; Kuzikov, Maria; Ellinger, Bernhard; Reinshagen, Jeanette; Witt, Gesa; Gribbon, Philip; Kohler, Manfred; Keminer, Oliver; Behrens, Birte; Costantino, Luca; Tejera Nevado, Paloma; Bifeld, Eugenia; Eick, Julia; Clos, Joachim; Torrado Santiago, Juan; Jiménez Antón, María D.; Corral, María J.; Alunda, José Ma; Pellati, Federica; Wade, Rebecca C.; Ferrari, Stefania; Mangani, Stefano; Costi, Maria Paola
abstract

Flavonoids represent a potential source of new antitrypanosomatidic leads. Starting from a library of natural products, we combined target-based screening on pteridine reductase 1 with phenotypic screening on Trypanosoma brucei for hit identification. Flavonols were identified as hits, and a library of 16 derivatives was synthesized. Twelve compounds showed EC50 values against T. brucei below 10 μM. Four X-ray crystal structures and docking studies explained the observed structure-activity relationships. Compound 2 (3,6-dihydroxy-2-(3-hydroxyphenyl)-4H-chromen-4-one) was selected for pharmacokinetic studies. Encapsulation of compound 2 in PLGA nanoparticles or cyclodextrins resulted in lower in vitro toxicity when compared to the free compound. Combination studies with methotrexate revealed that compound 13 (3-hydroxy-6-methoxy-2-(4-methoxyphenyl)-4H-chromen-4-one) has the highest synergistic effect at concentration of 1.3 μM, 11.7-fold dose reduction index and no toxicity toward host cells. Our results provide the basis for further chemical modifications aimed at identifying novel antitrypanosomatidic agents showing higher potency toward PTR1 and increased metabolic stability.

2015 - Enantiomeric resolution of [(2,2-diphenyl-1,3-dioxolan-4-yl)methyl](2-phenoxyethyl)amine, a potent α1and 5-HT1Areceptor ligand: an in vitro and computational study [Articolo su rivista]
Franchini, Silvia; Baraldi, Anna Maria; Sorbi, Claudia; Pellati, Federica; Cichero, Elena; Battisti, UMBERTO MARIA; Angeli, Piero; Cilia, Antonio; Brasili, Livio
abstract

In this paper, the enantiomers of (±)-1, previously studied as α1 and 5-HT1A ligands, were prepared both by resolution of the racemate and asymmetric synthesis. The enantiomeric purity and absolute configuration were determined by means of HPLC and polarimetric analysis. Enantiomers were evaluated for in vitro 5-HT1A and α1 receptor affinity by binding and functional assays. Results indicate that the two enantiomers are almost equally potent at 5-HT1A and α1 receptor systems and, contrary to WB 4101, the stereoselectivity is poor. As further support to these experimental findings, molecular docking studies on the two enantiomers of (±)-1 have been performed and a comparison with those obtained for 5-HT1A potent agonist (R)-flesinoxan and α1d antagonist (S)-WB 4101 has been drawn.

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.

2015 - Optimization and validation of a chiral GC-MS method for the determination of free d-amino acids ratio in human urine: application to a gestational diabetes mellitus study [Articolo su rivista]
Lorenzo, M. P.; Dudzik, D.; Varas, E.; Gibellini, M.; Skotnicki, M.; Zorawski, M.; Zarzycki, W.; Pellati, Federica; García, A.
abstract

Gestational Diabetes Mellitus (GDM) is defined as glucose intolerance with onset or first recognition during pregnancy. It is affecting approximately up to 14% of all pregnancies with an increasing tendency. GDM has been related to relevant short-term and long-term health complications for both mother and offspring. Recent studies strongly emphasized the role of several essential amino acids in the pathogenesis of obesity and highlighted their strong correlation with insulin resistance, but there are no references related to modifications in d-AAs in biological fluids. As d-AA elimination proceeds mainly by renal excretion, urine was the selected sample to evaluate the alterations in free d-AAs ratio in a GDM study. Only 1. mL of first void urine or standard solution was required for purification, by using a Discovery DSC-SCX SPE cartridge (500. mg/3. mL) and derivatization into their N(O)-pentafluoropropionyl amino acid 2-propyl esters. Enantiomeric separation was carried out by GC-MS on a Chirasil-. l-Val N-propionyl-. l-valine-tert-butylamide polysiloxane fused-silica capillary column (25. m. ×. 0.25. mm I.D., 0.12. μm film thickness, Agilent Technologies, Waldbronn, Germany), under programmed temperature elution. Detection was performed with an ion trap mass analyzer, operating in the full scan mode in the m/. z 50-350 range. 14 pairs of derivatives of d-and l-AAs were separated.The steps of sample preparation, derivatization and GC-MS conditions were optimized for both urine and standards. Several conditions affecting the SPE procedure, such as sorbent mass/volume ratio of the cartridge, sample dilution and pH, were optimized. Volume of reagents and solvents and reaction temperature and time were also tested for the derivatization. Regarding the GC-MS parameters, split ratio, temperature program and mass range were optimized.The final method was validated in terms of linearity, sensitivity, accuracy and precision for d-Ala, d-Pro, d-Ser, d-Met, d-Phe, d-Glu, d-Orn and d-Lys. Identification of AAs in urine samples was based on retention time and mass spectra.Urine from 20 women with GDM and 20 pregnant women with normal glucose tolerance (after 2-h 75-g oral glucose tolerance test), matched according to the week of gestation and age (22-28 week of gestation and age 24-37 years), were enrolled into the study. %. d-Relative amounts were determined for Ala, Val, Thr, Ser, Leu, Asx (Asp+Asn), Glx (Glu+Gln), Met, Phe, Tyr, Orn and Lys. Statistically significant differences (. p<. 0.05) were observed only for d-Phe and higher values were found in the GDM group. It is possible that d-Phe could be involved in metabolic/signaling pathways to compensate early stages of insulin resistance, although further work is necessary to confirm this hypothesis.

2015 - The effect of desflurane on neuronal communication at a central synapse [Articolo su rivista]
Mapelli, Jonathan; Gandolfi, Daniela; Giuliani, Enrico; Prencipe, FRANCESCO PIO; Pellati, Federica; Barbieri, Alberto; D'Angelo, Egidio; Bigiani, Albertino
abstract

Although general anesthetics are thought to modify critical neuronal functions, their impact on neuronal communication has been poorly examined. We have investigated the effect induced by desflurane, a clinically used general anesthetic, on information transfer at the synapse between mossy fibers and granule cells of cerebellum, where this analysis can be carried out extensively. Mutual information values were assessed by measuring the variability of postsynaptic output in relationship to the variability of a given set of presynaptic inputs. Desflurane synchronized granule cell firing and reduced mutual information in response to physiologically relevant mossy fibers patterns. The decrease in spike variability was due to an increased postsynaptic membrane excitability, which made granule cells more prone to elicit action potentials, and to a strengthened synaptic inhibition, which markedly hampered membrane depolarization. These concomitant actions on granule cells firing indicate that desflurane re-shapes the transfer of information between neurons by providing a less informative neurotransmission rather than completely silencing neuronal activity.

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 - Development of a new high-performance liquid chromatography method with diode array and electrospray ionization-mass spectrometry detection for the metabolite fingerprinting of bioactive compounds in Humulus lupulus L [Articolo su rivista]
Prencipe, FRANCESCO PIO; Brighenti, Virginia; Rodolfi, Margherita; Mongelli, Andrea; Dall'Asta, Chiara; Ganino, Tommaso; Bruni, Renato; Pellati, Federica
abstract

The study was aimed at developing a new analytical method for the metabolite fingerprinting of bioactive compounds in Humulus lupulus L. (hop), together with a simple extraction procedure. Different extraction techniques, including maceration, heat reflux extraction (HRE), ultrasound-assisted extraction (UAE) and microwave-assisted extraction (MAE), were compared in order to obtain a high yield of the target analytes. Dynamic maceration for 30min with MeOH-HCOOH (99:1, v/v) as the extraction solvent provided the best result in terms of recovery of secondary metabolites. The analysis of hop constituents, including prenylflavonoids and prenylphloroglucinols (bitter acids), was carried out by means of HPLC-UV/DAD, HPLC-ESI-MS and MS(2), using an ion trap mass analyzer. An Ascentis Express C18 column (150mm×3.0mm I.D., 2.7μm) was used for the HPLC analysis, with a mobile phase composed of 0.25% formic acid in both water and acetonitrile, under gradient elution. The method validation was performed to show compliance with ICH guidelines. The validated technique was successfully applied to the phytochemical analysis of ten commercial cultivars and twenty-three wild Italian hop genotypes, thus demonstrating to be a reliable and useful tool for the comprehensive multi-component analysis of hop secondary metabolites.

2014 - Development of flavonoids derivatives as leads against Leishmaniasis and Trypanosomiasis-invited lecture [Altro]
Borsari, Chiara; Luciani, Rosaria; Pozzi, Cecilia; Tait, Annalisa; Costantino, Luca; Pellati, Federica; Ferrari, Stefania; Costi, Maria Paola
abstract

Parasites of the family of Trypanosomatidae are the agents of serious human diseases, including African sleeping sickness, Chagas' disease and leishmaniasis. There is an urgent requirement for new effective drugs since current treatments show severe side effects, develop toxicity and drug resistance. Enzymes involved in the folate cycle, e.g. dihydrofolate reductase (DHFR)-thymidylate synthase (TS) bifunctional proteins are important drug targets for the treatment of bacterial infections, cancer and certain parasitic diseases, notably malaria. Trypanosomatids are auxotrophic for folates and pterins, and the inhibition of the enzymes involved in the salvage pathways should provide effective treatments. However, antifolates are currently not yet employed in the therapy of trypanosomatids infections also because of the pteridine reductase (PTR1) activity in the target organisms. PTR is a short-chain dehydrogenase/reductase involved in the biopterin reduction pathway, able to carry out subsequent reductions of both conjugated (folate) and unconjugated (biopterin) pterins. When classical antifolate drugs inhibit DHFR-TS, PTR1 can be over-expressed ensuring the parasite survival and causing resistance to typical antifolate. The treatment of trypanosomatidic infections through the simultaneous inhibition of DHFR and PTR1 may provide a successful treatment for leishmanisis infections and Tbrucei/cruzi. Previous work in our laboratories has contributed to demonstrate this principle. Computational studies were performed to screen through a docking approach a library of 90 natural compounds from plants of different origins and target-based screening delivered flavans as one of the interesting compound class to be explored. The flavonoid structure however has been largely explored and often pleiotropic properties can be observed leading to promiscuous inhibition. The aim of the presented work is to identifiy novel chemically tractable scaffolds that will be further developed through medicinal chemistry approaches. X-ray crystallographyc structure of some of the studied compounds have been obtained and used for further structure-based drug design. While the initial compounds showed moderate detectable biological activities even related to their low solubility, recent derivatives were more soluble and showed improved enzyme inhibitory activity. The design, synthesis and biological evaluation of the flavan derivatives will be discussed. Reference 1.Ong HB et al. J Biol Chem.2011 Mar 25;286(12):10429-38. Cavazzuti A et al. PNAS. 2008 Feb 5;105(5):1448-53. 3. Ferrari S. et al, J Med Chem. 2011 Jan 13;54(1):211-21 Acknowledgement This work was supported by “PRIN 2009-2011 to MPC and NMTrypI (New medicine for Trypanosomatidic Infections) This project has received funding from the European Union’s Seventh Framework Programme for research, technological development and demonstration under grant agreement no.603240.

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 - Innovative Methods for the Extraction and Chromatographic Analysis of Honey-Bee Products [Relazione in Atti di Convegno]
Pellati, Federica
abstract

The aim of this study was to provide an overview of recent techniques developed for the chemical characterization of raw propolis, based on the determination of 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. As regards the extraction of polyphenols from raw propolis, 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 using a PDMS fiber. These chromatographic techniques were applied to raw propolis samples collected from different Italian regions to determine their fingerprinting, thus providing new and reliable tools for the complete chemical characterization of this biologically active material.

2014 - Metabolite profiling of polyphenols in the Tunisian plant Tamarix aphylla (L.) Karst [Articolo su rivista]
Mahfoudhi, Adel; Prencipe, FRANCESCO PIO; Mighri, Zine; Pellati, Federica
abstract

In this study, a detailed investigation on the composition of polyphenols of Tamarix aphylla (L.) Karst., consisting of phenolic acids and flavonoids, was carried out. In order to optimize the yield of secondary metabolites, three extraction techniques were compared, including dynamic maceration, ultrasound-assisted extraction and Soxhlet extraction. The latter technique provided the best results in terms of both recovery and selectivity, using ethyl acetate as extraction solvent for 2h. The analysis of T. aphylla polyphenols was performed by means of HPLC-UV/DAD, HPLC-ESI-MS and MS(2), using an ion trap mass analyzer. Phenolic acids and flavonoids were separated on an Ascentis C18 column (250mm×4.6mm I.D., 5μm), with a mobile phase composed of 0.1M formic acid in water and acetonitrile, under gradient elution. The proposed method was fully validated in agreement with ICH guidelines and then applied to the analysis of T. aphylla leaves and stems. A total of 14 phenolic compounds were characterized for the first time in this plant extracts by using UV, MS and MS(2) data. The amount of total phenolics was found to be 993.1±22.5μg/g in the leaves and 113.1±25.8μg/g in the stems, respectively. The most abundant constituents found in the leaves include ellagic acid (211.4±10.8μg/g), quercetin (125.7±4.7μg/g) and gallic acid (120.6±1.2μg/g), whereas those in the stems were ellagic acid (44.4±3.9μg/g), gallic acid (24.3±3.3μg/g) and kaempferol (16.3±1.6μg/g). The developed method can be considered a useful tool for the metabolite profiling of T. aphylla, which represents a potential source of bioactive compounds to be used in phytotherapy.

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 - Nuovo trattamento assistito da microonde per la produzione di miscele arricchite in policosanoli da matrici cerose [Brevetto]
Pellati, Federica; Mascolo, Danilo; Venturelli, Alberto; Barbato, Amedeo
abstract

Il brevetto riguarda un processo di estrazione e purificazione tramite tecnologia assistida da microonde di alcoli alifatici con catena di lunghezza da 20 a 36 atomi di carbonio, denominati policosanoli, da matrici cerose.

2014 - NUTRASCIENCE s.r.l. [Spin Off]
Bertelli, Davide; Plessi, Maria; Pellati, Federica; Forni, Flavio; Papotti, Giulia; Graziosi, Riccardo
abstract

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.

2014 - Preface [Capitolo/Saggio]
Jayaprakasha, G. K.; Patil, B. S.; Pellati, F.
abstract

2013 - Advanced chromatographic techniques for metabolite fingerprinting of natural compounds with antioxidant and antiproliferative activity [Abstract in Atti di Convegno]
Pellati, Federica
abstract

Considering the relevant antiproliferative activity of polyacetylenes/polyenes isolated from lipophilic root extracts of Echinacea pallida Nutt. (Nutt.) (Asteraceae family) and the chemopreventive action of caffeic acid derivatives identified in the hydrophilic fraction of this plant, this study provides an innovative and reliable method for the complete fingerprinting of these secondary metabolites by means of HPLC-UV/DAD, HPLC-ESI-MS and MS/MS. A relevant novel aspect of the proposed technique is that MS data, obtained by ion trap and triple quadrupole mass analyzers, made the peak identification very reliable. The fragmentation patterns of the bioactive compounds are discussed in detail in the present work. The oxidation mechanism of monocarbonyl acetylenes is shown for the first time using MS data. The practical applicability of the technique is demonstrated by the quantitative analysis of E. pallida root extracts and natural products commercially available to provide reliable chromatographic fingerprints of their bioactive secondary metabolites.

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 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 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 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.

2013 - Optimization and validation of a GC-MS method for determination of free D-amino acids in human urine in a diabetes type 1 study [Abstract in Atti di Convegno]
M. P., Lorenzo; Gibellini, Manuel; Pellati, Federica; C., Barbas; A., García
abstract

Type 1 Diabetes Mellitus is one of the most common chronic disorders of childhood. Amino acids play a major role in energy metabolism, neurotransmission, and lipid transport. In humans, D-AAs originate primarily from bacterial metabolism and food intake. As D-AA elimination proceeds mainly by renal excretion and its easy collection, urine samples were selected. Amino acids were isolated from 1 ml of urine or standard solution using a cation exchanger Discovery DSC-SCX tube (500 mg/3 ml) and converted into their N(O)-pentafluoropropionyl amino acid 2-propyl esters. Enantiomers were separated and quantified by GC-IT-MS on a Chirasil-L-Val (N-propionyl-L-valine-tert-butylamidepolysiloxane) capillary column. The isolation (i), the derivatization process (ii), and the separation conditions (iii) were optimized for both urine and standards. Conditions such as cartridge sorbent mass/column volume for SPE (100 mg/1 ml and 500 mg/3 ml), pH (1-7), dilution of the loaded urine sample (0, 2, 5, 10 times diluted) (SPE) and solvent volumes (methanol, 10-2 M HCl and 4 M ammonia in methanol) were optimized for the isolation process (i). Volume of reagents and solvents (40-200 µl) and reaction time (30-60 min) were tested for the derivatization (ii). Regarding to the separation conditions (iii), split ratio (1:1-1:70), temperature program and mass range were optimized. The final method was validated in terms of linearity, accuracy and precision for D-Ala, D-Pro, D-Ser, D-Met, D-Phen, D-Glu, D-Orn and D-Lys. Identification of the amino acid was based on retention time and mass spectra. The results for the pool urine, obtained with healthy volunteers, were confirmed with previous literature. Urine samples from seven type 1 diabetic children and seven control, 6 to 11 years old, for boys and girls were analyzed. All of them were collected in La Paz Hospital, Madrid (Spain). D- Relative amounts (%D-AA) were determined for D-Ala, D-Val, D-Ser, D-Leu, D-Asp, D-Glu, D-Orn and D-Lys. Statistically significant differences were found for D-Ala, D-Val and D-Glu.

2013 - Phytochemical analysis and authenticity control of commercial essential oils and absolutes of Rosa damascena Mill. [Abstract in Atti di Convegno]
F., Camin; K. A., van Leeuwen; Orlandini, Giulia; M., Paolini; Bertelli, Davide; Pellati, Federica
abstract

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. In this study we considered 24 commercial samples of R. damascena essential oil and absolute of different geographic origin. GC-MS and GC-FID techniques were applied for the phytochemical analysis of the samples. EA-IRMS (Elemental Analyser–Isotope Ratio Mass Spectrometry) and GC/C(Combustion)-IRMS were used to determine the δ13C composition of bulk samples and of some specific components. 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. The samples from Iran were characterized by a high level of aliphatic hydrocarbons (nonadecane: 3.7-23.2%), while in the Turkish absolute rose and in the oils from Morocco and Ukraine phenylethyl alcohol was the main constituent (47.6-75.4%). The δ13C values of bulk samples were in the range between –29 and –26.9‰, typical for C3 plants. For most of the analyzed compounds in the essential oils and absolutes, the δ13C values 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 δ13C values could be justified by the addition of a natural cheaper oil from a C4 plant (Cymbopogon martinii, palmarosa). A synthetic origin was moreover postulated for geranyl acetate in the absolute from Ukraine. The results indicated that GC/C-IRMS, in combination with GC-MS and GC-FID, represents an effective and reliable tool for the authenticity control of R. damascena essential oil.

2013 - Protein-protein interaction inhibitors: case studies on Small Molecules and Natural Compounds. [Capitolo/Saggio]
Costi, Maria Paola; Pellati, Federica; Ferrari, Stefania
abstract

The large size and variety of the human protein interactome and the obvious relevance of the protein–protein interactions in every physiological function, render protein–protein interactions at the same time an extremely challenging and attractive target for developing of new therapeutic substances. A further interesting aspect of targeting protein–protein interactions (PPI) for drug discovery is that, at least in some cases, molecules directed against PPI may provide a way to overcome the resistance mechanisms encountered for active site binding enzyme inhibitors. The considerations above can be extended to bacterial and viral interactome, further expanding the vastness and complexity of the subject of PPI inhibition and modulation. For these reasons, there is no surprise that targeting PPI has become a subject of intense research activity in both industry and academia over the past decade. Reviewing the literature and the available databases shows that over 150 small molecule compounds have been found to inhibit about 20 PPI targets and that some of these molecules have already reached, or are about to reach, the drug market. As usual, these figures can have a double reading: the optimistic interpretation is that these successes demonstrate the validity of the approach, while the critics might say that the huge effort dedicated to find PPI inhibitors has resulted in scarce results. The fact is that exploring PPI is a stimulating new subject of study that is relevant for the basic knowledge on the chemistry of living organisms with the important outcome to offer the possibility of opening a new era of drug discovery. This book is a collection of essays from Italian research groups from Industry and University involved in drug discovery and, although the book presents different subjects in each chapter, the unifying idea comes from our belief that only an integrated approach of the different techniques nowadays available, may overcome the challenges presented by this new frontier in drug discovery. For this reason, the book opens with reviews about the current status of the research on PPI in drug discovery and goes on by presenting the state of the art in basic techniques like computational tools, NMR, X-ray crystallography and FRET that, integrated, may give the opportunity of success in this field.

2013 - Stable nitrogen isotopic composition of wine: a preliminary investigation [Abstract in Atti di Convegno]
M., Paolini; M., Valentinotti; L., Ziller; Pellati, Federica; F., Camin
abstract

The 15 N/14 N ratio has been measured in several foods to trace geographical origin and agricultural systems, but not yet in wine. In this work, we developed a method for measuring 15 N/14 N in bulk wine, following dealcoholisation and dehydratation. The delta15N of wine did not change after freeze-drying the sample or evaporating it under vacuum, obtaining adequate repeatability. The addition of nitrogen rich substances to must can affect the delta15N of wine. Delta15N analysis of 170 wine samples from 17 Italian regions showed differences between red and white wines and between regions.

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 - Development of chromatographic methods for the determination of genotoxic impurities in cloperastine fendizoate [Articolo su rivista]
A., García A; F. J., Rupérez; F., Ceppa; Pellati, Federica; C., Barbas
abstract

The classification of an impurity of a drug substance as genotoxic means that the “threshold of toxicological concern” (TTC) value of 1.5 ug/day intake, considered to be associated with an acceptable risk, should be the admissible limit in the raw material and that leads to new analytical challenges. In this study, reliable chromatographic methods were developed and applied as limit tests for the control of three genotoxic impurities (GTIs) in cloperastine fendizoate, drug widely used as an antitussive active pharmaceutical ingredient (API). In particular, GC–MS was applied to the determination of one alkyl halide (2-chloroethanol, 2-CE), while HPLC-DAD was selected for the analysis of two sulfonate esters (methyl p-toluenesulfonate, MPTS, and 2-chloroethyl p-toluenesulfonate, CEPTS).Regarding GC–MS, strong anion-exchange (SAX)-SPE was applied to remove fendizoate from the sample solutions, due its low volatility and its high amount in the raw material. The GC–MS analysis was performed on a Factor Four VF-23ms capillary column (30 m × 0.25 mm I.D., film thickness 0.25 um, Varian). Single ion-monitoring (SIM) detection mode was set at m/z 80.In the case of HPLC-DAD, a suitable optimization of the chromatographic conditions was carried out in order to obtain a good separation of the impurity peaks from the drug substance peaks. The optimized method utilizes a SymmetryShield RP8 column (250 mm × 4.6 mm, 5 um, Waters) kept at 50 ◦C, withphosphate buffer (pH 3.0; 10 mM)–methanol (containing 10% ACN) (45:55, v/v) as the mobile phase, at the flow-rate of 1.7 mL/min and UV detection at 227 nm. The required sensitivity level was achieved by injecting 80 uL of sample solution, purified from fendizoate by SAX-SPE, followed by a 1:1 (v/v) dilutionof the SPE eluate with water.For both GC–MS and HPLC-DAD, the method validation was performed in relation to specificity and limit of detection (LOD), as required by ICH guidelines in relation to limit assays. The developed methods were successfully applied for the determination of GTIs in five different batches of cloperastine fendizoate. In all the analyzed batches, the three target GTIs were below the concentration limit.

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 - METABOLOMIC ASSESSMENT WITH LC-QTOF-MS OF THE NUTRACEUTICAL EFFECT OF ROSMARINUS OFFICINALIS EXTRACTS ON PLASMA SAMPLES OF DIABETIC RATS [Abstract in Atti di Convegno]
Prencipe, FRANCESCO PIO; F. J., Rupérez; J., Godzien; Pellati, Federica; C., Barbas
abstract

Diabetes mellitus is one of the major worldwide health problems. The peculiarity of this disease is to have high levels of oxidative stress, which plays a crucial role in the pathogenesis of other complications, such as nephropathy, neuropathy and retinopathy. Rosmarinus officinalis L. extracts, processed by supercritical fluid extraction (SFE), have long been recognized as having antioxidant properties. To investigate the potential nutraceutical properties of these extracts on type 1 diabetes, a metabolomic fingerprinting study was carried out using LC-QTOF-MS. Metabolomic fingerprinting is a technique which study as many analytes as possible to get a complete view of a disease and of the different levels of metabolites involved, and finally to identify possible biomarkers.In this work, Sprague-Dawley male rats (12 ± 2 weeks of age), treated with streptozotocin, were used in controlled conditions in which they received, by intragastric administration, five doses of rosemary extract containing 10% folic acid (used to improve the endothelial progenitor cell function) dispersed in 1 mL vehicle or only 1 mL of vehicle. There were also healthy rats that received the same treatment, thus generating four groups of samples: treated or non-treated (only vehicle) controls, treated and non-treated diabetics.Plasma fingerprints of control and diabetic rats, focused on the most polar constituents, were obtained by LC-QTOF-MS. Retention of polar compounds from plasma in hydrophilic interaction liquid chromatography (HILIC) mode and use of solvents readily compatible with mass spectrometry is a new approach that was initially explored by testing different stationary phases. The HILIC columns used in this study included an Ascentis Si (15 cm × 2.1 mm, 5 µm, Supelco), a TSK gel Amide-80 (5 cm × 2.0 mm, 3 µm, Tosoh), a Luna HILIC (10 cm × 2.0 mm, 5 µm, Phenomenex) and a ZIC-HILIC (10 cm × 2.1 mm, 3.5 µm, Merck). Unfortunately, the results were not as good as it was expected and this was mainly due to the lack of precision. It was therefore decided to analyze the plasma samples under reversed-phase (RP) conditions, using a Discovery HS C18 column (15 cm × 2.1 mm, 3 µm, Supelco), with a mobile phase composed by water (with 0.1% of formic acid) and ACN (with 0.1% of formic acid), under gradient elution. The injection volume was 10 µL; the flow rate was 0.6 mL/min and the column temperature was set at 30 °C. Data were collected in the positive ion mode with a QTOF mass analyzer, operated in the full scan mode from 50 to 1000 m/z. Experimental data from RP-HPLC were further processed by multivariate analysis to find compounds with statistically significant changes. Then, MS/MS experiments were performed under the same experimental conditions and identifications were carried out by studying the fragmentation pattern of the target analytes or using reference standards. Finally, the biochemical interpretation of potential biomarkers associated with the disease was performed. The compounds that were found to be affected by the nutraceutical treatment include:•Lysophosphatidylcholines (LPCs), represent the first group of identified compounds. LPCs are generated from ox-LDL or from inflammatory cells, and are very important intermediates of different metabolic pathways. These compounds were found to increase in diabetic rats after the treatment.•Pipecolic acid, the major metabolite of lysine degradation. This compound was found to increase in diabetic rats after the treatment.•Lipoic acid, an antioxidant synthesized in mitochondria. It is a necessary co-factor for mitochondrial α-ketoacid dehydrogenases, and thus plays a critical role in mitochondrial energy metabolism. This metabolite was found to increase in diabetic rats after the treatment.•Carnitine and derivates, which is important to transport fatty acids into the mitochondria. Also these compounds were found to be affected by the treatment.In con

2011 - Microparticulate polyelectrolyte complexes for gentamicin transport across intestinal epithelial [Articolo su rivista]
Iannuccelli, Valentina; Montanari, Monica; Bertelli, Davide; Pellati, Federica; Coppi, Gilberto
abstract

Polysaccharide microparticles for the oral administration of gentamicin were designed in order to obtainan increased drug absorption by means of microparticle transport across the intestinal epithelia. Alginate/chitosan microparticles with a size of ∼ 2 μm were developed by spray-drying a water solution containingthe drug complexed with the polyanionic alginate and subsequent alginate cross-linking process bycalcium ions and chitosan. The pre-formulation study, performed by changing the concentration of bothcross-linkers, led to the selection of the most suitable formulation which was assayed for its capacity to be translocated across intestinal epithelia, via both M cells contained in Follicle Associated Epithelium (FAE) ofPeyer’s patches and enterocytes of the mucosal epithelium. An ex vivo perfusion technique of rabbit andrat intestinal tissues containing Peyer’s patches combined with an in vitro method by using Caco-2 cellmonolayers demonstrated the microparticulate carrier ability to be taken up by both M cells and enterocytes.However, only the endocytosis by M cells appeared to provide the microparticle transport from theepithelium toward deeper sub-epithelial regions.

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 - Chronic benzylamine administration in the drinking water improves glucose tolerance, reduces body weight gain and circulating cholesterol in high-fat diet-fed mice [Articolo su rivista]
Z., Iffiú Soltész; E., Wanecq; A., Lomba; M. P., Portillo; Pellati, Federica; E., Szökő; S., Bour; J., Woodley; F. I., Milagro; J., Alfredo Martinez; P., Valet; C., Carpéné
abstract

Benzylamine is found in Moringa oleifera, a plant used to treat diabetes in traditional medicine. In mammals, benzylamine is metabolized by semicarbazide-sensitive amine oxidase (SSAO) to benzaldehyde and hydrogen peroxide. This latter product has insulin-mimicking action, and is involved in the effects of benzylamine on human adipocytes: stimulation of glucose transport and inhibition of lipolysis. This study examined whether chronic, oral administration of benzylamine could improve glucose tolerance and the circulating lipid profile without increasing oxidative stress in overweight and pre-diabetic mice. The benzylamine diffusion across the intestine was verified using everted gut sacs. Then, glucose handling and metabolic markers were measured in mice rendered insulin-resistant when fed a high-fat diet (HFD) and receiving or not benzylamine in their drinking water (3600micromol/(kgday)) for 17 weeks. HFD-benzylamine mice showed lower body weight gain, fasting blood glucose, total plasma cholesterol and hyperglycaemic response to glucose load when compared to HFD control. In adipocytes, insulin-induced activation of glucose transport and inhibition of lipolysis remained unchanged. In aorta, benzylamine treatment partially restored the nitrite levels that were reduced by HFD. In liver, lipid peroxidation markers were reduced. Resistin and uric acid, surrogate plasma markers of metabolic syndrome, were decreased. In spite of the putative deleterious nature of the hydrogen peroxide generated during amine oxidation, and in agreement with its in vitro insulin-like actions found on adipocytes, the SSAO-substrate benzylamine could be considered as a potential oral agent to treat metabolic syndrome.

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 - Development and validation of a HPLC method for the determination of sertraline and three non-chiral related impurities [Articolo su rivista]
A., Ferrarini; A. L., Huidobro; Pellati, Federica; C., Barbas
abstract

In this study, a screening on reversed-phase stationary phases (including C(8), C(18), CN, PEG and amide) was carried out in order to obtain an efficient HPLC method for the determination of sertraline and three of its more closely related synthetical and non-chiral impurities, without using ion-pair reagents. The best results in terms of both retention time and resolution of the target analytes were obtained with a Zorbax Bonus-RP column, which contains a polar amide group embedded in a C(14) alkyl chain. Once the most suitable stationary phase was chosen, the HPLC method was optimized by using a factorial design, evaluating three quantitative factors (column temperature, buffer pH and buffer concentration) in order to find the best conditions which maximize the resolution between impurities A and B (positional isomers) and minimize the total run time. The final HPLC conditions were set by means of a second experimental design, which allowed optimizing the effects of the buffer pH and the proportion of methanol in the mobile phase. The optimal conditions for simultaneously determining sertraline and its impurities, being baseline separated in less than 10min, were finally obtained with Zorbax Bonus-RP column (150mmx4.6mm, 5 um), under isocratic conditions with phosphate buffer (pH 2.8; 10mM)-methanol (63:37, v/v) at 50 degrees C, at the flow-rate of 1.0mL/min. UV detection was set at 220nm. This method was successfully validated following ICH guidelines and it proved to be reliable for the determination of sertraline and related impurities in tablets as pharmaceutical forms.

2010 - High-performance liquid chromatoghraphy/tandem mass spectrometry methods in the phytochemical analysis of hydro-alcoholic propolis extracts. [Abstract in Atti di Convegno]
Orlandini, Giulia; Pellati, Federica
abstract

Polyphenolic compounds are the major active constituents of propolis extracts. Due to its phytochemical composition, propolis extracts are widely used in phytotherapy, particularly as anti-bacterial, anti-inflammatory and antioxidant.The interest in propolis compounds and their biological activity is still increasing. In this ambit, the development and validation of suitable analytical methods aimed at the phytochemical analysis and quality control of propolis is very important.In this study, a detailed characterization of propolis hydro-alcoholic extracts was carried out by developing advanced chromatographic techniques, based on RP-HPLC coupled with diode array and mass spectrometry detection and analyzing commercially available hydro-alcoholic propolis extracts. 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, under gradient elution. The flow rate was 1.2 mL/min and the column temperature was set at 30°C. In view of UV spectra, MS and MS/MS data, forty polyphenolic compounds, including phenolic acids and flavonoids, were identified as typical components of the analyzed samples. The comparison of the performance of the selected mass analyzers indicated that the triple quadrupole system provided more structural information on target analytes than the ion trap.The HPLC method was then fully validated in agreement with the guidelines of the International Conference on Harmonization of Technical Requirements for the Registration of Pharmaceuticals for Human Use and then applied to real samples.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 16.72 mg/mL and the level of total flavonoids from 2.51 to 42.72 mg/mL. In the analyzed samples, the most abundant flavonoids were found to be chrysin, galangin, pinocembrin and pinobanksin (and its esters). Regarding phenolic acids, the most representative compounds were found to be caffeic acid derivatives.The chemical standardization of propolis extracts is necessary to guarantee its quality, efficacy and safety. In this context the proposed method, allowing the determination of the complete phenolic profile characterized by both phenolic acids and flavonoids, can be considered as a useful tool for metabolite profiling and quality control of propolis natural products.

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; Lucchesi, Davide; Boraschi, Diana; 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 - Development, optimization and validation of a stability indicating HPLC method for sertraline hydrochloride [Abstract in Atti di Convegno]
A., Ferrarini; A. L., Huidobro; Pellati, Federica; C., Barbas
abstract

Sertraline HCl is an antidepressant compound for oral administration, belonging to the group of selective serotonin reuptake inhibitors (SSRI) in the brain. Chemically, sertraline is a secondary amine with a pKa value of 9.47.The analytical method employed for the determination of sertraline and its impurities (A, Band C) throughout the stability assays is generally based on reversed phase with ion-paring chromatography at acidic pH, employing 1-octane sulphonate and acetonitrile as organic modifier. The drawbacks of ion-pairing chromatography are generally known. One of them, the poor retention time reproducibility, is a source of problems during stability indicating assays. The availability of new stationary phases with new selectivities in reverse phase HPLC gave us the possibility of new separations avoiding these drawbacks. During the development also the replacement of acetonitrile with methanol was tested. Moreover, to our knowledge, there is no validated HPLC method including sertraline and these impurities described in the literature.A screening on RP stationary phases was conducted to explore different selectivities, including a wide range of polarities: C8, C18, cyano, PEG and amide. The best result was obtained with a Zorbax Bonus-RP column, which contains a polar amide group embedded in a C14 alkyl chain and has a triple end-capping. These characteristics yield in a reduction of the interactions between basic compounds and the silica support, improving peak shape and reducing the analysis time. Once the best stationary phase was chosen, a HPLC method was developed by means of an experimental design with four quantitative factors (organic proportion in the mobile phase, temperature, pH and buffer concentration) in order to find the best conditions which maximize the resolution between impurities A and B (positional isomers) and minimize the total run time.Optimal conditions for simultaneously determining sertraline and its impurities, being baseline separated in less than 10 min, were found with Zorbax Bonus-RP column (150 x 4.6 mm, 5 mm, Agilent Technologies), under isocratic conditions with 10 mM phosphate buffer (pH=2.8) and MeOH (63:37, v/v) at 50 ºC.This method has been successfully validated following ICH guidelines and it has proved to be reliable for the determination of sertraline and related impurities in tablets as pharmaceutical forms.

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.

2009 - Synergistic immunomopharmacological effects of N-alkylamides in Echinacea purpurea herbal extracts [Articolo su rivista]
A., Chicca; S., Raduner; Pellati, Federica; T., Strompen; K. H., Altmann; R., Schoop; J., Gertsch
abstract

Echinacea purpurea extracts are used in the production of standardized herbal medicines for the prevention and treatment of upper respiratory infections. Unsaturated N-alkylamide lipids, the main constituent of E. purpurea and E. angustifolia preparations capable of activating the cannabinoid receptor type-2 (CB2) have been suggested to play a role as potential anti-inflammatory and immune-modulatory principles. Here we show that ethanolic E. purpurea radix and herba extracts produce synergistic pharmacological effects on the endocannabinoid system in vitro. Superadditive action of N-alkylamide combinations was seen at the level of intracellular calcium release as a function of CB2 receptor activation. Likewise, synergism of the radix and herba tinctures was observed in experiments measuring LPS-stimulated cytokine expression from human PBMCs. While the expression of the anti-inflammatory cytokine IL-10 was significantly superstimulated, the expression of the pro-inflammatory TNF-alpha protein was inhibited more strongly upon combination of the extracts. We show that N-alkylamides act in concert and exert pleiotropic effects modulating the endocannabinoid system by simultaneously targeting the CB2 receptor, endocannabinoid transport and degradation.

2009 - Synergistic immunopharmacological effects of N-alkylamides in Echinacea purpurea herbal extracts [Abstract in Rivista]
A., Chicca; S., Raduner; Pellati, Federica; T., Strompen; K. H., Altmann; R., Schoop; J., Gertsch
abstract

Echinacea purpurea (L.) Moench extracts are used in the production of standardized herbal medicines for the prevention and treatment of upper respiratory infections. Unsaturated N-alkylamide lipids, the main constituents of E. purpurea and E. angustifolia preparations capable of activating the cannabinoid receptor type-2 (CB2) have been suggested to play a role as potential anti-inflammatory and immune-modulatory principles. Here we show that ethanolic E. purpurea radix and herba extracts produce synergistic pharmacological effects on the endocannabinoid system in vitro. Superadditive action of N-alkylamide combinations were seen at the level of intracellular calcium release as a function of CB2 receptor activation. Likewise, synergism of the radix and herba tinctures was observed in experiments measuring LPS-stimulated cytokine expression from human PBMCs. While the expression of the anti-inflammatory cytokine IL-10 was significantly superstimulated, the expression of the pro-inflammatory TNF-alpha protein was inhibited more strongly upon combination of the extracts. We show that N-alkylamides act in concert and exert pleiotropic effects modulating the endocannabinoid system by simultaneously targeting the CB2 receptor, endocannabinoid transport and degradation.

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 - Erratum to "Chromatographic and electrophoretic methods for the analysis of phenethylamine alkaloids in Citrus aurantium" [J. Chromatogr. A 1161 (2007) 71-88] (DOI:10.1016/j.chroma.2007.05.097) [Articolo su rivista]
Pellati, F.; Benvenuti, S.
abstract

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 - Microparticulate carrier for gentamicin oral administration [Abstract in Atti di Convegno]
Sala, Nicoletta; Iannuccelli, Valentina; Pellati, Federica; Coppi, Gilberto
abstract

In this study, a microparticulate carrier for gentamicin oral administration was developoed.

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 - 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 - 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 - 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 - 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 - 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 - Development and application of HPLC methods for the analysis of biologically active compounds of vegetal origin [Relazione in Atti di Convegno]
Pellati, Federica
abstract

In this presentation, the development and application of HPLC methods for the analysis of biologically active compounds of vegetal origin in Citrus aurantium was discussed.

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 - 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.