Nuova ricerca


Personale tecnico amministrativo
Dipartimento di Scienze della Vita sede ex-Scienze Farmaceutiche

Home | Curriculum(pdf) |


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.

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.

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

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 - 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

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.

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 - Formulazione per veicolare principi attivi lipofili [Brevetto]
Maretti, Eleonora; Leo, Eliana Grazia; Brighenti, Virginia

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.

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 - Formulazione per veicolare principi attivi lipofili [Brevetto]
Maretti, Eleonora; Leo, Eliana Grazia; Brighenti, Virginia

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.

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 - 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: 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 - 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.

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

2019 - 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.

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 - 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.

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.

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 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

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

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

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

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

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

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 - 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.

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 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.

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.

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

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 - 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.

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.

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.

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 - 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

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.

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

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

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

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

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.

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

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

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

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 - 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

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 - 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

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.