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2023 - Enantioseparation of chiral phytocannabinoids in medicinal cannabis [Articolo su rivista]
Russo, Fabiana; Tolomeo, Francesco; Vandelli, Maria Angela; Biagini, Giuseppe; Laganà, Aldo; Laura Capriotti, Anna; Cerrato, Andrea; Carbone, Luigi; Perrone, Elisabetta; Cavazzini, Alberto; Maiorano, Vincenzo; Gigli, Giuseppe; Cannazza, Giuseppe; Citti, Cinzia

The evaluation of the chiral composition of phytocannabinoids in the cannabis plant is particularly important as the pharmacological effects of the (+) and (-) enantiomers of these compounds are completely different. Chromatographic attempts to assess the presence of the minor (+) enantiomers of the main phytocannabinoids, cannabidiolic acid (CBDA) and trans-Δ9-tetrahydrocannabinolic acid (trans-Δ9-THCA), were carried out on heated plant extracts for the determination of the corresponding decarboxylated species, cannabidiol (CBD) and trans-Δ9-tetrahydrocannabinol (trans-Δ9-THC), respectively. This process produces an altered phytocannabinoid composition with several new and unknown decomposition products. The present work reports for the first time the stereoselective synthesis of the pure (+) enantiomers of the main phytocannabinoids, trans-CBDA, trans-Δ9-THCA, trans-CBD and trans-Δ9-THC, and the development and optimization of an achiral-chiral liquid chromatography method coupled to UV and high-resolution mass spectrometry detection in reversed phase conditions (RP-HPLC-UV-HRMS) for the isolation of the single compounds and evaluation of their actual enantiomeric composition in plant. The isolation of the peaks with the achiral stationary phase ensured the absence of interferences that could potentially co-elute with the analytes of interest in the chiral analysis. The method applied to the Italian medicinal cannabis variety FM2 revealed no trace of the (+) enantiomers for all phytocannabinoids under investigation before and after decarboxylation, thus suggesting that the extraction procedure does not lead to an inversion of configuration.

2023 - Synthesis and pharmacological activity of the epimers of hexahydrocannabinol (HHC) [Articolo su rivista]
Russo, Fabiana; Vandelli, Maria Angela; Biagini, Giuseppe; Schmid, Martin; Luongo, Livio; Perrone, Michela; Ricciardi, Federica; Maione, Sabatino; Laganà, Aldo; Capriotti, Anna Laura; Gallo, Alfonso; Carbone, Luigi; Perrone, Elisabetta; Gigli, Giuseppe; Cannazza, Giuseppe; Citti, Cinzia

Cannabis is a multifaceted plant with numerous therapeutic properties on one hand, and controversial psychotropic activities on the other hand, which are modulated by CB1 endocannabinoid receptors. Δ9-Tetrahydrocannabinol (Δ9-THC) has been identified as the main component responsible for the psychotropic effects, while its constitutional isomer cannabidiol (CBD) has shown completely different pharmacological properties. Due to its reported beneficial effects, Cannabis has gained global popularity and is openly sold in shops and online. To circumvent legal restrictions, semisynthetic derivatives of CBD are now frequently added to cannabis products, producing "high" effects similar to those induced by Δ9-THC. The first semi-synthetic cannabinoid to appear in the EU was obtained through cyclization and hydrogenation of CBD, and is known as hexahydrocannabinol (HHC). Currently, there is limited knowledge regarding HHC, its pharmacological properties, and its prevalence, as it is not commonly investigated in routine toxicological assays. In this study, synthetic strategies were explored to obtain an excess of the active epimer of HHC. Furthermore, the two epimers were purified and individually tested for their cannabinomimetic activity. Lastly, a simple and rapid chromatographic method employing a UV detector and a high-resolution mass spectrometer was applied to identify and quantify up to ten major phytocannabinoids, as well as the HHC epimers, in commercial cannabis samples.

Puja, G.; Ravazzini, F.; Losi, G.; Bardoni, R.; Battisti, U. M.; Citti, C.; Cannazza, G.

The majority of excitatory neurotransmission in vertebrate CNS is mediated by glutamate binding to different types of receptors. Among them, a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) and kainate receptors (KAR) are ionotropic receptors playing important pathophysiological roles. A number of small molecules acting as positive allosteric modulators (PAM) of AMPAR have been proposed as drugs for neurological disorders, however, there is no such abundance of ligands capable of modulating KARs activity. We investigated the ability of IDRA21 and of its derivative, compound 2 (c2), to modulate glutamate-evoked currents at native and recombinantly expressed AMPA and KA receptors. By using the patch clamp technique we analyzed the activity of the two compounds in primary cultures of cerebellar granule neurons and in HEK293 cells transiently transfected with KARs and AMPAR subunits. It resulted that both benzothiadiazine derivatives potentiate AMPAR and KAR mediated currents in native and recombinant receptors, c2 being always more potent and efficacious than IDRA21. The potency of both compounds was higher in native receptors than in recombinant receptors. In HEK293 cells transfected with AMPAR subunits, the efficacy of IDRA21 and c2 was much higher in GluA1 than in GluA2 homomeric receptors while their potency did not change. In recombinant KAR, both compounds had a potency in the high micromolar range, while the efficacy reached a maximum in the GluK2 expressing cells. The benzothiadiazine effect, both in native and recombinant receptors, was detected mainly on plateau current, involving a decrease in AMPAR and KAR desensitization. Our study demonstrates for the first time that two positive allosteric modulators of AMPAR, IDRA21 and its derivative, c2, potentiate KAR activity. Furthermore, we highlighted their subunit selectivity that may enable the design of potent and selective PAMs, which could be relevant for the development of new drugs and for a better understanding of KAR functions in the CNS.

2022 - Cis-Δ9-tetrahydrocannabinolic acid occurrence in Cannabis sativa L. [Articolo su rivista]
Tolomeo, Francesco; Russo, Fabiana; Kaczorova, Dominika; Vandelli, Maria Angela; Biagini, Giuseppe; Laganà, Aldo; Laura Capriotti, Anna; Paris, Roberta; Fulvio, Flavia; Carbone, Luigi; Perrone, Elisabetta; Gigli, Giuseppe; Cannazza, Giuseppe; Citti, Cinzia

Cannabidiolic acid (CBDA) and trans-Δ9-tetrahydrocannabinolic acid (trans-Δ9-THCA) are known to be the major phytocannabinoids in Cannabis sativa L., along with their decarboxylated derivatives cannabidiol (CBD) and trans-Δ9-tetrahydrocannabinol (trans-Δ9-THC). The cis isomer of Δ9-THC has been recently identified, characterized and quantified in several Cannabis sativa varieties, which had been heated (decarboxylated) before the analysis. Since decarboxylation alters the original phytocannabinoids composition of the plant, this work reports the identification and characterization of the carboxylated precursor cis-Δ9-THCA. The compound was also synthesized and used as analytical standard for the development and validation of a liquid chromatography coupled to high resolution mass spectrometry-based method for its quantification in ten Cannabis sativa L. samples from different chemotypes. The highest concentrations of cis-Δ9-THCA were found in CBD-rich varieties, lower levels were observed in cannabigerol (CBG)-rich varieties (chemotype IV) and in those varieties with a balanced level of both CBD and THC (chemotype III), while its levels were not detectable in cannabichromene (CBC)-rich varieties (chemotype VI). The presence of the cis isomer of THC and THCA raises the question on whether to include or not this species in the calculation of the total amount of THC to classify a cannabis variety as a drug-type or a fiber-type (hemp).

2022 - HPLC-MS/MS method applied to an untargeted metabolomics approach for the diagnosis of “olive quick decline syndrome” [Articolo su rivista]
Di Masi, S.; De Benedetto, G. E.; Malitesta, C.; Saponari, M.; Citti, C.; Cannazza, G.; Ciccarella, G.

Olive quick decline syndrome (OQDS) is a disorder associated with bacterial infections caused by Xylella fastidiosa subsp. pauca ST53 in olive trees. Metabolic profile changes occurring in infected olive trees are still poorly investigated, but have the potential to unravel reliable biomarkers to be exploited for early diagnosis of infections. In this study, an untargeted metabolomic method using high-performance liquid chromatography coupled to quadrupole-time-of-flight high-resolution mass spectrometry (HPLC-ESI-Q-TOF-MS) was used to detect differences in samples (leaves) from healthy (Ctrl) and infected (Xf) olive trees. Both unsupervised and supervised data analysis clearly differentiated the groups. Different metabolites have been identified as potential specific biomarkers, and their characterization strongly suggests that metabolism of flavonoids and long-chain fatty acids is perturbed in Xf samples. In particular, a decrease in the defence capabilities of the host after Xf infection is proposed because of a significant dysregulation of some metabolites belonging to flavonoid family. Moreover, oleic acid is confirmed as a putative diffusible signal factor (DSF). This study provides new insights into the host-pathogen interactions and confirms LC-HRMS-based metabolomics as a powerful approach for disease-associated biomarkers discovery in plants. Graphical abstract: [Figure not available: see fulltext.]

2022 - Kynurenine and kynurenic acid: two human neuromodulators found in Cannabis sativa L [Articolo su rivista]
Russo, Fabiana; Tolomeo, Francesco; Vandelli, Maria Angela; Biagini, Giuseppe; Paris, Roberta; Fulvio, Flavia; Laganà, Aldo; Capriotti, Anna Laura; Carbone, Luigi; Gigli, Giuseppe; Cannazza, Giuseppe; Citti, Cinzia

L-Kynurenine (KYN) and kynurenic acid (KYNA) are products of the metabolism of L-tryptophan (TRP) in the central nervous system of animals, but they are not commonly found in plants. In particular, KYNA is known for its interesting pharmacological properties (anti-oxidative, anti-inflammatory, hypolipidemic, and neuroprotective), which suggest a potential functional food ingredient role. The three compounds were identified in samples of Cannabis sativa L. by means of high-performance liquid chromatography coupled to high-resolution mass spectrometry using an untargeted metabolomics approach. Their concentrations were evaluated using a targeted metabolomics method in three organs of the plant (roots, stem, and leaves) in soil at two different growth stages and in hydroponics conditions. The distribution of TRP, KYN and KYNA was found tendentially higher in leaves compared to stem and roots and changed over time. Moreover, the levels of KYNA found in this study are unprecedentedly high compared to those found so far in other plant species, suggesting that Cannabis sativa L. could be a promising alternative source of this metabolite.

2021 - Analysis of sequence variability and transcriptional profile of cannabinoid synthase genes in cannabis sativa l. Chemotypes with a focus on cannabichromenic acid synthase [Articolo su rivista]
Fulvio, F.; Paris, R.; Montanari, M.; Citti, C.; Cilento, V.; Bassolino, L.; Moschella, A.; Alberti, I.; Pecchioni, N.; Cannazza, G.; Mandolino, G.

Cannabis sativa L. has been long cultivated for its narcotic potential due to the accumulation of tetrahydrocannabinolic acid (THCA) in female inflorescences, but nowadays its production for fiber, seeds, edible oil and bioactive compounds has spread throughout the world. However, some hemp varieties still accumulate traces of residual THCA close to the 0.20% limit set by European Union, despite the functional gene encoding for THCA synthase (THCAS) is lacking. Even if some hypotheses have been produced, studies are often in disagreement especially on the role of the cannabichromenic acid synthase (CBCAS). In this work a set of European Cannabis genotypes, representative of all chemotypes, were investigated from a chemical and molecular point of view. Highly specific primer pairs were developed to allow an accurate distinction of different cannabinoid synthases genes. In addition to their use as markers to detect the presence of CBCAS at genomic level, they allowed the analysis of transcriptional profiles in hemp or marijuana plants. While the high level of transcription of THCAS and cannabidiolic acid synthase (CBDAS) clearly reflects the chemical phenotype of the plants, the low but stable transcriptional level of CBCAS in all genotypes suggests that these genes are active and might contribute to the final amount of cannabinoids.

2021 - Analytical Methodologies for Lipidomics in Hemp Plant [Capitolo/Saggio]
Cerrato, A.; Capriotti, A. L.; Montone, C. M.; Aita, S. E.; Cannazza, G.; Citti, C.; Piovesana, S.; Aldo, L.

The chemical composition of Cannabis sativa L. has been extensively studied for tens of years, but little is known about its lipidome. This chapter describes an analytical workflow for polar lipid determination in hemp. After extraction, lipids are enriched and isolated by graphitized carbon black sorbent, and the isolated lipid is analyzed by liquid chromatography (LC) coupled with high resolution mass spectrometry, leading to identification of many lipid species. We have developed a semi-automated platform using commercially available Lipostar software for lipid identification. Our approach affords the identification of 189 polar lipids in hemp extract, including sulfolipids and phospholipids. The number of the identified lipid species is by far the highest ever reported for Cannabis sativa.

2021 - HPLC-UV-HRMS analysis of cannabigerovarin and cannabigerobutol, the two impurities of cannabigerol extracted from hemp [Articolo su rivista]
Tolomeo, Francesco; Russo, Fabiana; Vandelli, Maria Angela; Biagini, Giuseppe; Capriotti, Anna Laura; Laganà, Aldo; Carbone, Luigi; Gigli, Giuseppe; Cannazza, Giuseppe; Citti, Cinzia

A sensitive and straightforward HPLC-UV method was developed for the simultaneous quantification of the two main impurities in "pure" commercial cannabigerol (CBG) samples. The identification of such impurities, namely cannabigerovarin (CBGV) and cannabigerobutol (CBGB), the propyl and butyl homologs of CBG, respectively, was accomplished employing the high-resolution mass spectrometry (HRMS) technique, and subsequently confirmed by comparison with the same compounds obtained by chemical synthesis. Complete spectroscopic characterization (NMR, FT-IR, UV, and HRMS) of both impurities is reported in the present work. The method was validated in terms of linearity, which was assessed in the range 0.01-1.00 μg/mL, sensitivity, selectivity, intra- and inter-day accuracy and precision, and short-term stability, which all satisfied the acceptance criteria of the ICH guidelines. Application of the method to the analysis of four commercial CBG samples highlighted a certain variability in the impurity profile that might be ascribed to the hemp variety of the starting plant material. With these new analytical standards in hand, it would be interesting to investigate their concentrations in different hemp varieties and expand the scope of a phytocannabinomics approach for a comprehensive profiling of this remarkable class of natural compounds.

2021 - Oxidative stress and multi-organel damage induced by two novel phytocannabinoids, cbdb and cbdp, in breast cancer cells [Articolo su rivista]
Salbini, M.; Quarta, A.; Russo, F.; Giudetti, A. M.; Citti, C.; Cannazza, G.; Gigli, G.; Vergara, D.; Gaballo, A.

Over the last few years, much attention has been paid to phytocannabinoids derived from Cannabis for their therapeutic potential. ∆9-tetrahydrocannabinol (∆9-THC) and cannabidiol (CBD) are the most abundant compounds of the Cannabis sativa L. plant. Recently, novel phytocannabinoids, such as cannabidibutol (CBDB) and cannabidiphorol (CBDP), have been discovered. These new molecules exhibit the same terpenophenolic core of CBD and differ only for the length of the alkyl side chain. Roles of CBD homologs in physiological and pathological processes are emerging but the exact molecular mechanisms remain to be fully elucidated. Here, we investigated the biological effects of the newly discovered CBDB or CBDP, compared to the well-known natural and synthetic CBD (nat CBD and syn CBD) in human breast carcinoma cells that express CB receptors. In detail, our data demonstrated that the treatment of cells with the novel phytocannabinoids affects cell viability, increases the production of reactive oxygen species (ROS) and activates cellular pathways related to ROS signaling, as already demonstrated for natural CBD. Moreover, we observed that the biological activity is significantly increased upon combining CBD homologs with drugs that inhibit the activity of enzymes involved in the metabolism of endocannabinoids, such as the monoacylglycerol lipase (MAGL) inhibitor, or with drugs that induces the activation of cellular stress pathways, such as the phorbol ester 12-myristate 13-acetate (PMA).

2021 - Recent applications of mass spectrometry for the characterization of cannabis and hemp phytocannabinoids: From targeted to untargeted analysis [Articolo su rivista]
Capriotti, A. L.; Cannazza, G.; Catani, M.; Cavaliere, C.; Cavazzini, A.; Cerrato, A.; Citti, C.; Felletti, S.; Montone, C. M.; Piovesana, S.; Lagana, A.

This review is a collection of recent applications of mass spectrometry studies for the characterization of phytocannabinoids in cannabis and hemp plant material and related products. The focus is mostly on recent applications using mass spectrometry as detector, in hyphenation to typical separation techniques (i.e., liquid chromatography or gas chromatography), but also with less common couplings or by simple direct analysis. The papers are described starting from the most common approach for targeted quantitative analysis, with applications using low-resolution mass spectrometry equipment, but also with the introduction of high-resolution mass analyzers as the detectors. This reflects a common trend in this field, and introduces the most recent applications using high-resolution mass spectrometry for untargeted analysis. The different approaches used for untargeted analysis are then described, from simple retrospective analysis of compounds without pure standards, through untargeted metabolomics strategies, and suspect screening methods, which are the ones currently allowing to achieve the most detailed qualitative characterization of the entire phytocannabinoid composition, including minor compounds which are usually overlooked in targeted studies and in potency evaluation. These approaches also represent powerful strategies to answer questions on biological and pharmacological activity of cannabis, and provide a sound technology for improved classification of cannabis varieties. Finally, open challenges are discussed for future directions in the detailed study of complex phytocannabinoid mixtures.

2021 - Targeted and untargeted characterization of underivatized policosanols in hemp inflorescence by liquid chromatography-high resolution mass spectrometry [Articolo su rivista]
Montone, C. M.; Aita, S. E.; Cannazza, G.; Cavaliere, C.; Cerrato, A.; Citti, C.; Mondello, L.; Piovesana, S.; Lagana, A.; Capriotti, A. L.

The paper describes the development of a targeted quantitative method for the analysis of policosanols in hemp inflorescence. Policosanols are long chain aliphatic alcohols, with carbon chains typically in the range 20–36, with interesting biological activities. These compounds are typically separated by gas chromatography and only a few methods employ liquid chromatography for policosanols. In both cases, methods always include the derivatization of policosanols. In this study, policosanols were separated by ultra-high performance liquid chromatography without any derivatization and detected using high resolution mass spectrometry by formation of lithiated adducts. The procedure was optimized and a quantitative method was validated for the most abundant policosanols (with C24, C26, C27, C28, and C30 chain lengths) in industrial hemp inflorescence extracts. The method was used for the quantitative analysis of policosanols in five hemp types. Hemp wax was found rich in these compounds, especially C26 and C28 policosanols, which may prove useful for revalorization of wax by-products. Finally, the acquired data were also used to expand the search to the untargeted qualitative analysis of policosanols using Compound Discoverer. The untargeted method allowed the annotation of underivatized policosanols up to C33.

2021 - Techno-economic study of a small scale gasifier applied to an indoor hemp farm: From energy savings to biochar effects on productivity [Articolo su rivista]
Pedrazzi, S.; Santunione, G.; Mustone, M.; Cannazza, G.; Citti, C.; Francia, E.; Allesina, G.

The hemp market is fast growing due to demand for cannabidiol, nutraceutical and hemp fiber products. This work demonstrates the economical advantage of biomass gasification application to indoor hemp production. Gasifiers provide electrical energy, heat and biochar: these are highly valuable products for indoor growers where lights and thermal management are key costs of the business. Energy produced in an autonomous and renewable way increases the sustainability and in the facility. In this paper a small scale gasifier is fueled with certified “A1 plus” wood pellets to test its behavior and its biochar production rate. Biochar is used for hemp growing tests in an indoor hemp production facility. Results show how a 22 kW power plant is sufficient to guarantee almost complete sustainability in a 80 m2 facility. In the best case scenario where energy saving, biochar and thermal energy selling are considered, the gasifier investment has a payback time of about 3.5 years. At the end of the gasifier lifespan, the Net Present Value reaches 249 k€ considering a discount rate of 6%. Consequential results were also obtained from biochar application to pot growing substrates: there was a 7.7% increase in dry flower production and a 33.9% increase in total plant fresh biomass. Cannabinoids profiles resulted not affected by biochar application.

2021 - The novel heptyl phorolic acid cannabinoids content in different Cannabis sativa L. accessions [Articolo su rivista]
Linciano, Pasquale; Russo, Fabiana; Citti, Cinzia; Tolomeo, Francesco; Paris, Roberta; Fulvio, Flavia; Pecchioni, Nicola; Vandelli, Maria Angela; Laganà, Aldo; Capriotti, Anna Laura; Biagini, Giuseppe; Carbone, Luigi; Gigli, Giuseppe; Cannazza, Giuseppe

The recent discovery of the novel heptyl phytocannabinoids cannabidiphorol (CBDP) and Δ9-tetrahydrocannabiphorol (Δ9-THCP) raised a series of questions relating to the presence and abundance of these new unorthodox compounds in cannabis inflorescence or derived products. As fresh inflorescence contains mainly their acid precursors, which are not commercially available, an ad hoc stereoselective synthesis was performed in order to obtain cannabidiphorolic acid (CBDPA) and Δ9-tetrahydrocannabiphorolic acid (THCPA) to be used as analytical standards for quantitative purposes. The present work reports an unprecedented targeted analysis of both pentyl (C5) and heptyl (C7) CBD- and THC-type compounds in forty-nine cannabis samples representing four different chemotypes. Moreover, the ultrahigh performance liquid chromatography coupled to highresolution mass spectrometry-based method was applied for the putative identification of other heptyl homologs of the most common phytocannabinoid acids, including cannabigerophorolic acid (CBGPA), cannabichromephorolic acid (CBCPA), cannabinophorolic acid (CBNPA), cannabielsophorolic acid (CBEPA), cannabicyclophorolic acid (CBLPA), cannabitriophorolic acid (CBTPA), and cannabiripsophorolic acid (CBRPA).

2020 - A new software-assisted analytical workflow based on high-resolution mass spectrometry for the systematic study of phenolic compounds in complex matrices [Articolo su rivista]
Cerrato, A.; Cannazza, G.; Capriotti, A. L.; Citti, C.; La Barbera, G.; Lagana, A.; Montone, C. M.; Piovesana, S.; Cavaliere, C.

Polyphenols are a broad class of plant secondary metabolites which carry out several biological functions for plant growth and protection and are of great interest as nutraceuticals for their antioxidant properties. However, due to their structural variability and complexity, the mass-spectrometric analysis of polyphenol content in plant matrices is still an issue. In this work, a novel approach for the identification of several classes of polyphenol derivatives based on ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry was developed. First, mass-spectrometric parameters were optimized in order to obtain a large set of diagnostic product ions for their high-confidence identification. The software Compound Discoverer 3.0 was then implemented with a comprehensive database of 45,567 polyphenol derivatives and with mass-spectrometric data for their building blocks, resulting in a specific tool for the semi-automatic identification of flavonoids, anthocyanins, ellagitannins, proanthocyanidins and phenolic acids. The method was then applied to the identification of polyphenols in industrial hemp (Cannabis sativa), a matrix whose use is recently spreading for pharmaceutical and nutraceutical purposes, resulting in the identification of 147 compounds belonging to the classes of flavonoids, proanthocyanidins and phenolic acids. The proposed method is applicable to the polyphenol profiling of any plant matrix and it is not dependent on data in the literature for their identification, allowing the discovery of compounds which have been never identified before.

2020 - Application of calcium carbonate nanocarriers for controlled release of phytodrugs against Xylella fastidiosa pathogen [Articolo su rivista]
Baldassarre, F.; De Stradis, A.; Altamura, G.; Vergaro, V.; Citti, C.; Cannazza, G.; Capodilupo, A. L.; Dini, L.; Ciccarella, G.

Calcium carbonate-based hollow or porous particles are one of the preferred carriers for fabrication of drug delivery systems. We have developed an eco-friendly method to produce calcium carbonate nanocrystals, which have shown biocompatibility and optimal capacity to across cell membrane in human cell lines providing new tools in cancer therapy. The success of drug delivery systems has paved the way for the development of systems for controlled release of agrochemicals. In this work, we exploited calcium carbonate nanocrystals as carriers for targeted release of phytodrugs investigating a potential control strategy for the pathogen Xylella fastidiosa. This pathogen is the causal agent of the Olive Quick Decline Syndrome that is an unprecedented emergency in Italy and potentially in the rest of Europe. We studied nanocrystals interactions with bacteria cells and the application in planta to verify olive plants uptake. Ultrastructural analysis by electron microscopy shown an alteration of bacteria wall following nanocrystals interaction. Nanocrystals were adsorbed from roots and they translocated in plants tissues. Calcium carbonate carriers were able to encapsulate efficiently two types of antimicrobial substances and the potential efficacy was tested in experiment under greenhouse conditions.

2020 - Identification of a new cannabidiol n-hexyl homolog in a medicinal cannabis variety with an antinociceptive activity in mice: cannabidihexol [Articolo su rivista]
Linciano, P.; Citti, C.; Russo, F.; Tolomeo, F.; Lagana, A.; Capriotti, A. L.; Luongo, L.; Iannotta, M.; Belardo, C.; Maione, S.; Forni, F.; Vandelli, M. A.; Gigli, G.; Cannazza, G.

The two most important and studied phytocannabinoids present in Cannabis sativa L. are undoubtedly cannabidiol (CBD), a non-psychotropic compound, but with other pharmacological properties, and Δ9-tetrahydrocannabinol (Δ9-THC), which instead possesses psychotropic activity and is responsible for the recreative use of hemp. Recently, the homolog series of both CBDs and THCs has been expanded by the isolation in a medicinal cannabis variety of four new phytocannabinoids possessing on the resorcinyl moiety a butyl-(in CBDB and Δ9-THCB) and a heptyl-(in CBDP and Δ9-THCP) aliphatic chain. In this work we report a new series of phytocannabinoids that fills the gap between the pentyl and heptyl homologs of CBD and Δ9-THC, bearing a n-hexyl side chain on the resorcinyl moiety that we named cannabidihexol (CBDH) and Δ9-tetrahydrocannabihexol (Δ9-THCH), respectively. However, some cannabinoids with the same molecular formula and molecular weight of CBDH and Δ9-THCH have been already identified and reported as monomethyl ether derivatives of the canonical phytocannabinoids, namely cannabigerol monomethyl ether (CBGM), cannabidiol monomethyl ether (CBDM) and Δ9-tetrahydrocannabinol monomethyl ether (Δ9-THCM). The unambiguously identification in cannabis extract of the n-hexyl homologues of CBD and Δ9-THC different from the corresponding methylated isomers (CBDM, CBGM and Δ9-THCM) was achieved by comparison of the retention time, molecular ion, and fragmentation spectra with those of the authentic standards obtained via stereoselective synthesis, and a semi-quantification of these cannabinoids in the FM2 medical cannabis variety was provided. Conversely, no trace of Δ9-THCM was detected. Moreover, CBDH was isolated by semipreparative HPLC and its identity was confirmed by comparison with the spectroscopic data of the corresponding synthetic standard. Thus, the proper recognition of CBDH, CBDM and Δ9-THCH closes the loop and might serve in the future for researchers to distinguish between these phytocannabinoids isomers that show a very similar analytical behaviour. Lastly, CBDH was assessed for biological tests in vivo showing interesting analgesic activity at low doses in mice.

2020 - Improved identification of phytocannabinoids using a dedicated structure-based workflow [Articolo su rivista]
Montone, C. M.; Cerrato, A.; Botta, B.; Cannazza, G.; Capriotti, A. L.; Cavaliere, C.; Citti, C.; Ghirga, F.; Piovesana, S.; Lagana, A.

Phytocannabinoids are a broad class of compounds uniquely synthesized by the various strains of Cannabis sativa. Up to date, most investigation on phytocannabinoids have been addressed to the most abundant species, Δ9-tetrahydrocannabinol and cannabidiol, for their well-known wide range of pharmaceutical activities. However, in the recent years a large number of minor constituents have been reported, whose role in cannabis pharmacological effects is of current scientific interest. With the purpose of gaining knowledge on major and minor species and furnishing a strategy for their untargeted analysis, in this study we present an innovative approach for comprehensively identifying phytocannabinoids based on high-resolution mass spectrometry in negative ion mode, which allows discrimination of the various isomeric species. For a faster and more reliable manual validation of the tandem mass spectra of known and still unknown species, an extensive database of phytocannabinoid derivatives was compiled and implemented on Compound Discoverer software for the setup of a dedicated data analysis tool. The method was applied to extracts of the Italian FM-2 medicinal cannabis, resulting in the identification of 121 phytocannabinoids, which is the highest number ever reported in a single analysis. Among those, many known and still unknown unconventional phytocannabinoids have been tentatively identified, another piece in the puzzle of unravelling the many uncharted applications of this matrix.

2020 - Is cannabidiol a scheduled controlled substance? Origin makes the difference [Articolo su rivista]
Citti, C.; Linciano, P.; Cannazza, G.

Cannabidiol (CBD) is the main cannabinoid naturally occurring in hemp. It has recently attracted the attention of the scientific community because of its numerous pharmacological activities. However, its legal status changes depending on whether it is chemically synthesized or extracted from the plant: extracted CBD is a scheduled controlled substance, whereas synthetic CBD is not under control. In Europe, extracted CBD is excluded from the cosmetic ingredients of the CosIng database. Given the confusion surrounding these different forms of CBD, there is an urgent need for clarity to shed light from both a regulatory and a chemical point of view. The impurity profiles of synthetic and natural CBD are different and could currently represent the only means to distinguish the origin of this substance.

2020 - Isolation of a High-Affinity Cannabinoid for the Human CB1 Receptor from a Medicinal Cannabis sativa Variety: Δ9-Tetrahydrocannabutol, the Butyl Homologue of Δ9-Tetrahydrocannabinol [Articolo su rivista]
Linciano, P.; Citti, C.; Luongo, L.; Belardo, C.; Maione, S.; Vandelli, M. A.; Forni, F.; Gigli, G.; Lagana, A.; Montone, C. M.; Cannazza, G.

The butyl homologues of Δ9-tetrahydrocannabinol, Δ9-tetrahydrocannabutol (Δ9-THCB), and cannabidiol, cannabidibutol (CBDB), were isolated from a medicinal Cannabis sativa variety (FM2) inflorescence. Appropriate spectroscopic and spectrometric characterization, including NMR, UV, IR, ECD, and HRMS, was carried out on both cannabinoids. The chemical structures and absolute configurations of the isolated cannabinoids were confirmed by comparison with the spectroscopic data of the respective compounds obtained by stereoselective synthesis. The butyl homologue of Δ9-THC, Δ9-THCB, showed an affinity for the human CB1 (Ki = 15 nM) and CB2 receptors (Ki = 51 nM) comparable to that of (-)-trans-Δ9-THC. Docking studies suggested the key bonds responsible for THC-like binding affinity for the CB1 receptor. The formalin test in vivo was performed on Δ9-THCB in order to reveal possible analgesic and anti-inflammatory properties. The tetrad test in mice showed a partial agonistic activity of Δ9-THCB toward the CB1 receptor.

2020 - New insights in hemp chemical composition: a comprehensive polar lipidome characterization by combining solid phase enrichment, high-resolution mass spectrometry, and cheminformatics [Articolo su rivista]
Antonelli, M.; Benedetti, B.; Cannazza, G.; Cerrato, A.; Citti, C.; Montone, C. M.; Piovesana, S.; Lagana, A.

The chemical composition of Cannabis sativa L. has been extensively investigated for several years; nevertheless, a detailed lipidome characterization is completely lacking in the literature. To achieve this goal, an extraction and enrichment procedure was developed for the characterization of phospholipids and sulfolipids. Firstly, a study on the solid-liquid extraction was performed, to maximize the recovery of the considered lipids; the best procedure consisted of a simple extraction with a mixture of methanol and chloroform (1:1, v/v). The hemp extracts were analyzed by ultra-high-performance liquid chromatography coupled to high-resolution mass spectrometry and lipids were tentatively identified by Lipostar. To improve the number of identifications, an enrichment method, based on graphitized carbon black solid phase extraction, was evaluated to fractionate phospholipids and sulfolipids into separate eluates. Recovery and matrix effects of the procedure were determined on a mixture of standard lipids, containing representative compounds for each considered lipid class. The optimized method allowed the tentative identification of 189 lipids, including 51 phospholipids and 80 sulfolipids, in the first and second fractions, respectively. The detection of only 6 sulfolipids in the first fraction and 9 phospholipids in the second fraction proved the efficacy of the fractionation method, which also allowed the number of lipid identifications to be increased compared to the same procedure without enrichment, which scored 100 lipids. Finally, a semi-quantitative analysis permitted the hemp polar lipidome to be characterized. The results of this study allow knowledge of the hemp chemical composition to be improved with a detailed description of its phospho- and sulfolipid profiles. [Figure not available: see fulltext.]

2020 - Pitfalls in the analysis of phytocannabinoids in cannabis inflorescence [Articolo su rivista]
Citti, Cinzia; Russo, Fabiana; Sgrò, Salvatore; Gallo, Alfonso; Zanotto, Antonio; Forni, Flavio; Vandelli, Maria Angela; Laganà, Aldo; Montone, Carmela Maria; Gigli, Giuseppe; Cannazza, Giuseppe

The chemical analysis of cannabis potency involves the qualitative and quantitative determination of the main phytocannabinoids: Δ9-tetrahydrocannabinol (Δ9-THC), cannabidiol (CBD), cannabigerol (CBG), cannabichromene (CBC), etc. Although it might appear as a trivial analysis, it is rather a tricky task. Phytocannabinoids are present mostly as carboxylated species at the aromatic ring of the resorcinyl moiety. Their decarboxylation caused by heat leads to a greater analytical variability due to both reaction kinetics and possible decomposition. Moreover, the instability of cannabinoids and the variability in the sample preparation, extraction, and analysis, as well as the presence of isomeric forms of cannabinoids, complicates the scenario. A critical evaluation of the different analytical methods proposed in the literature points out that each of them has inherent limitations. The present review outlines all the possible pitfalls that can be encountered during the analysis of these compounds and aims to be a valuable help for the analytical chemist. Graphical abstract.

2019 - A novel phytocannabinoid isolated from Cannabis sativa L. with an in vivo cannabimimetic activity higher than Δ9-tetrahydrocannabinol: Δ9-Tetrahydrocannabiphorol [Articolo su rivista]
Citti, C.; Linciano, P.; Russo, F.; Luongo, L.; Iannotta, M.; Maione, S.; Lagana, A.; Capriotti, A. L.; Forni, F.; Vandelli, M. A.; Gigli, G.; Cannazza, G.

(-)-Trans-Delta(9)-tetrahydrocannabinol (Delta(9)-THC) is the main compound responsible for the intoxicant activity of Cannabis sativa L. The length of the side alkyl chain influences the biological activity of this cannabinoid. In particular, synthetic analogues of Delta(9)-THC with a longer side chain have shown cannabimimetic properties far higher than Delta(9)-THC itself. In the attempt to define the phytocannabinoids profile that characterizes a medicinal cannabis variety, a new phytocannabinoid with the same structure of Delta(9)-THC but with a seven-term alkyl side chain was identified. The natural compound was isolated and fully characterized and its stereochemical configuration was assigned by match with the same compound obtained by a stereoselective synthesis. This new phytocannabinoid has been called (-)-trans-Delta(9)-tetrahydrocannabiphorol (Delta(9)-THCP). Along with Delta(9)-THCP, the corresponding cannabidiol (CBD) homolog with seven-term side alkyl chain (CBDP) was also isolated and unambiguously identified by match with its synthetic counterpart. The binding activity of Delta(9)-THCP against human CB1 receptor in vitro (K-i = 1.2 nM) resulted similar to that of CP55940 (K-i = 0.9 nM), a potent full CB1 agonist. In the cannabinoid tetrad pharmacological test, Delta(9)-THCP induced hypomotility, analgesia, catalepsy and decreased rectal temperature indicating a THC-like cannabimimetic activity. The presence of this new phytocannabinoid could account for the pharmacological properties of some cannabis varieties difficult to explain by the presence of the sole Delta(9)-THC.

2019 - Analysis of impurities of cannabidiol from hemp. Isolation, characterization and synthesis of cannabidibutol, the novel cannabidiol butyl analog [Articolo su rivista]
Citti, C.; Linciano, P.; Forni, F.; Vandelli, M. A.; Gigli, G.; Lagana, A.; Cannazza, G.

Cannabidiol (CBD), one of the two major active principles present in Cannabis sativa, is gaining great interest among the scientific community for its pharmaceutical, nutraceutical and cosmetic applications. CBD can be prepared either by chemical synthesis or extraction from Cannabis sativa (hemp). The latter is more convenient from several points of view, including environmental and economic, but mainly for the absence of harmful organic solvents generally employed in the chemical synthesis. Although CBD produced by hemp extraction is the most widely employed, it carries two major impurities. The first one is the already known cannabidivarin (CBDV), whereas the second one is supposed to be the butyl analog of CBD with a four-term alkyl side chain. In this work, we report the isolation by semi-preparative liquid chromatography and the unambiguous identification of this second impurity. A comprehensive spectroscopic characterization, including NMR, UV, IR, circular dichroism and high-resolution mass spectrometry (HRMS), was carried out on this natural cannabinoid. In order to confirm its absolute configuration and chemical structure, the stereoisomer (1R,6R) of the supposed cannabinoid was synthesized and the physicochemical and spectroscopic properties, along with the stereochemistry, matched those of the natural isolated molecule. According to the International Nonproprietary Name, we suggested the name of cannabidibutol (CBDB) for this cannabinoid. Lastly, an HPLC-UV method was developed and validated for the qualitative and quantitative determination of CBDV and CBDB in samples of CBD extracted from hemp and produced according to Good Manufacturing Practices regulations for pharmaceutical and cosmetic use.

2019 - Cannabinoid profiling of hemp seed oil by liquid chromatography coupled to high-resolution mass spectrometry [Articolo su rivista]
Citti, Cinzia; Linciano, Pasquale; Panseri, Sara; Vezzalini, Francesca; Forni, Flavio; Vandelli, Maria Angela; Cannazza, Giuseppe

Hemp seed oil is well known for its nutraceutical, cosmetic and pharmaceutical properties due to a perfectly balanced content of omega 3 and omega 6 polyunsaturated fatty acids. Its importance for human health is reflected by the success on the market of organic goods in recent years. However, it is of utmost importance to consider that its healthy properties are strictly related to its chemical composition, which varies depending not only on the manufacturing method, but also on the hemp variety employed. In the present work, we analyzed the chemical profile of ten commercially available organic hemp seed oils. Their cannabinoid profile was evaluated by a liquid chromatography method coupled to high-resolution mass spectrometry. Besides tetrahydrocannabinol and cannabidiol, other 30 cannabinoids were identified for the first time in hemp seed oil. The results obtained were processed according to an untargeted metabolomics approach. The multivariate statistical analysis showed highly significant differences in the chemical composition and, in particular, in the cannabinoid content of the hemp oils under investigation.

2019 - Chemical and spectroscopic characterization data of ‘cannabidibutol’, a novel cannabidiol butyl analog [Articolo su rivista]
Citti, C.; Linciano, P.; Forni, F.; Vandelli, M. A.; Gigli, G.; Lagana, A.; Cannazza, G.

Cannabidibutol (CBDB), a novel butyl analog of cannabidiol, was identified as impurity of commercial cannabidiol (CBD) extracted from hemp (for full data and results interpretation see “Analysis of impurities of cannabidiol from hemp. Isolation, characterization and synthesis of cannabidibutol, the novel cannabidiol butyl analog” Citti et al, 2019). The compound was isolated from a CBD sample and subject to a full characterization. First, a complete spectroscopic characterization was performed by Nuclear Magnetic Resonance (NMR): in particular, 1H-NMR, 13C-NMR, COSY, HSQC and HMBC, which were followed by UV absorption and circular dichroism (CD) spectra. In order to confirm the structural identity and stereochemistry of the compound, a stereoselective synthesis of the trans isomer (1R,6R) was carried out and all the chemical and spectroscopic properties were analyzed. The synthesized compound was characterized by NMR (1H-NMR, 13C-NMR, COSY, HSQC and HMBC), Infra-Red spectroscopy (IR), UV and CD absorption, matching the results obtained for the natural isolated compound. With the analytical standard in hand, a simple high-performance liquid chromatography method coupled to UV detection (HPLC-UV) was developed and validated in house in terms of linearity, accuracy, precision, dilution integrity and stability. The present data might be useful to any researcher or industry that may run into a very common impurity of CBD extracted from hemp, so it can be easily compared with their own experimental data.

2018 - A Metabolomic Approach Applied to a LiquidChromatography Coupled to High-ResolutionTandem Mass Spectrometry Method (HPLC-ESI-HRMS/MS): Towards the ComprehensiveEvaluation of the Chemical Composition ofCannabis Medicinal Extracts [Articolo su rivista]
Citti, Cinzia; Battisti, Umberto Maria; Braghiroli, Daniela; Ciccarella, Giuseppe; Schmid, Martin; Vandelli, Maria Angela; Cannazza, Giuseppe

Introduction – Cannabis sativa L. is a powerful medicinal plant and its use has recently increased for the treatment of several pa-thologies. Nonetheless, side effects, like dizziness and hallucinations, and long-term effects concerning memory and cognition,can occur. Most alarming is the lack of a standardised procedure to extract medicinal cannabis. Indeed, each galenical prepara-tion has an unknown chemical composition in terms of cannabinoids and other active principles that depends on the extractionprocedure.Objective – This study aims to highlight the main differences in the chemical composition of Bediol® extracts when the extractionis carried out with either ethyl alcohol or olive oil for various times (0, 60, 120 and 180 min for ethyl alcohol, and 0, 60, 90 and120 min for olive oil).Methodology.Cannabis medicinal extracts (CMEs) were analysed by liquid chromatography coupled to high-resolution tandem mass spec-trometry (LC–MS/MS) using an untargeted metabolomics approach. The data sets were processed by unsupervised multivariateanalysis.Results – Our results suggested that the main difference lies in the ratio of acid to decarboxylated cannabinoids, which dramat-ically influences the pharmacological activity of CMEs. Minor cannabinoids, alkaloids, and amino acids contributing to this differ-ence are also discussed. The main cannabinoids were quantified in each extract applying a recently validated LC–MS and LC-UVmethod.Conclusions – Notwithstanding the use of a standardised starting plant material, great changes are caused by different extractionprocedures. The metabolomics approach is a useful tool for the evaluation of the chemical composition of cannabis extracts.

2018 - Analysis of cannabinoids in commercial hemp seed oil and decarboxylation kinetics studies of cannabidiolic acid (CBDA) [Articolo su rivista]
Citti, Cinzia; Pacchetti, Barbara; Vandelli, Maria Angela; Forni, Flavio; Cannazza, Giuseppe

Hemp seed oil from Cannabis sativa L. is a very rich natural source of important nutrients, not only polyunsaturated fatty acids and proteins, but also terpenes and cannabinoids, which contribute to the overall beneficial effects of the oil. Hence, it is important to have an analytical method for the determination of these components in commercial samples. At the same time, it is also important to assess the safety of the product in terms of amount of any psychoactive cannabinoid present therein. This work presents the development and validation of a highly sensitive, selective and rapid HPLC-UV method for the qualitative and quantitative determination of the main cannabinoids, namely cannabidiolic acid (CBDA), tetrahydrocannabinolic acid (THCA), cannabidiol (CBD), tetrahydrocannabinol (THC), cannabinol (CBN), cannabigerol (CBG) and cannabidivarin (CBDV), present in 13 commercial hemp seed oils. Moreover, since decomposition of cannabinoid acids generally occurs with light, air and heat, decarboxylation studies of the most abundant acid (CBDA) were carried out in both open and closed reactor and the kinetics parameters were evaluated at different temperatures in order to evaluate the stability of hemp seed oil in different storage conditions.

2018 - Cell-penetrating CaCO3nanocrystals for improved transport of NVP-BEZ235 across membrane barrier in T-cell lymphoma [Articolo su rivista]
Vergaro, Viviana; Civallero, Monica; Citti, Cinzia; Cosenza, Maria; Baldassarre, Francesca; Cannazza, Giuseppe; Pozzi, Samantha; Sacchi, Stefano; Fanizzi, Francesco Paolo; Ciccarella, Giuseppe

Owing to their nano-sized porous structure, CaCO3nanocrystals (CaCO3NCs) hold the promise to be utilized as desired materials for encapsulating molecules which demonstrate wide promise in drug delivery. We evaluate the possibility to encapsulate and release NVP-BEZ235, a novel and potent dual PI3K/mTOR inhibitor that is currently in phase I/II clinical trials for advanced solid tumors, from the CaCO3NCs. Its chemical nature shows some intrinsic limitations which induce to administer high doses leading to toxicity; to overcome these problems, here we proposed a strategy to enhance its intracellular penetration and its biological activity. Pristine CaCO3NCs biocompatibility, cell interactions and internalization in in vitro experiments on T-cell lymphoma line, were studied. Confocal microscopy was used to monitor NCs-cell interactions and cellular uptake. We have further investigated the interaction nature and release mechanism of drug loaded/released within/from the NCs using an alternative approach based on liquid chromatography coupled to mass spectrometry. Our approach provides a good loading efficiency, therefore this drug delivery system was validated for biological activity in T-cell lymphoma: the anti-proliferative test and western blot results are very interesting because the proposed nano-formulation has an efficiency higher than free drug at the same nominal concentration.

2018 - Development of a simple and sensitive liquid chromatography triple quadrupole mass spectrometry (LC–MS/MS) method for the determination of cannabidiol (CBD), Δ9-tetrahydrocannabinol (THC) and its metabolites in rat whole blood after oral administration of a single high dose of CBD [Articolo su rivista]
Palazzoli, Federica; Citti, Cinzia; Licata, Manuela; Vilella, Antonietta; Manca, Letizia; Zoli, Michele; Vandelli, Maria Angela; Forni, Flavio; Cannazza, Giuseppe

The investigation of the possible conversion of cannabidiol (CBD) into Δ 9 -tetrahydrocannabinol (THC) in vivo after oral administration of CBD is reported herein since recent publications suggested a rapid conversion in simulated gastric fluid. To this end, single high dose of CBD (50 mg/kg) was administered orally to rats and their blood was collected after 3 and 6 h. A highly sensitive and selective LC–MS/MS method was developed and fully validated in compliance with the Scientific Working Group of Forensic Toxicology (SWGTOX) standard practices for method validation in forensic toxicology. This method also involved the optimization of cannabinoids and their metabolites extraction in order to remove co-eluting phospholipids and increase the sensitivity of the MS detection. Neither THC nor its metabolites were detected in rat whole blood after 3 or 6 h from CBD administration. After oral administration, the amount of CBD dissolved in olive oil was higher than that absorbed from an ethanolic solution. This could be explained by the protection of lipid excipients towards CBD from acidic gastric juice.

2018 - Inihibition of glycolysis by using a micro/nano-lipid bromopyruvic chitosan carrier as a promising tool to improve treatment of hepatocellular carcinoma [Articolo su rivista]
Hanafy, Nemany A.; Dini, Luciana; Citti, Cinzia; Cannazza, Giuseppe; Leporatti, Stefano

Glucose consumption in many types of cancer cells, in particular hepatocellular carcinoma (HCC), was followed completely by over-expression of type II hexokinase (HKII). This evidence has been used in modern pharmacotherapy to discover therapeutic target against glycolysis in cancer cells. Bromopyruvate (BrPA) exhibits antagonist property against HKII and can be used to inhibit glycolysis. However, the clinical application of BrPA is mostly combined with inhibition effect for healthy cells particularly erythrocytes. Our strategy is to encapsulate BrPA in a selected vehicle, without any leakage of BrPA out of vehicle in blood stream. This structure has been constructed from chitosan embedded into oleic acid layer and then coated by dual combination of folic acid (FA) and bovine serum albumin (BSA). With FA as specific ligand for cancer folate receptor and BSA that can be an easy binding for hepatocytes, they can raise the potential selection of carrier system.

2018 - Polymeric nano-micelles as novel cargo-carriers for LY2157299 liver cancer cells delivery [Articolo su rivista]
Hanafy, Nemany Abdelhamid Nemany; Quarta, Alessandra; Ferraro, Marzia Maria; Dini, Luciana; Nobile, Concetta; De Giorgi, Maria Luisa; Carallo, Sonia; Citti, Cinzia; Gaballo, Antonio; Cannazza, Giuseppe; Rinaldi, Rosaria; Giannelli, Gianluigi; Leporatti, Stefano

LY2157299 (LY), which is very small molecule bringing high cancer diffusion, is a pathway antagonist against TGFβ. LY dosage can be diluted by blood plasma, can be captured by immune system or it might be dissolved during digestion in gastrointestinal tract. The aim of our study is to optimize a "nano-elastic" carrier to avoid acidic pH of gastrointestinal tract, colon alkaline pH, and anti-immune recognition. Polygalacturonic acid (PgA) is not degradable in the gastrointestinal tract due to its insolubility at acidic pH. To avoid PgA solubility in the colon, we have designed its conjugation with Polyacrylic acid (PAA). PgA-PAA conjugation has enhanced their potential use for oral and injected dosage. Following these pre-requisites, novel polymeric nano-micelles derived from PgA-PAA conjugation and loading LY2157299 are developed and characterized. Efficacy, uptake and targeting against a hepatocellular carcinoma cell line (HLF) have also been demonstrated.

2018 - Untargeted rat brain metabolomics after oral administration of a single high dose of cannabidiol [Articolo su rivista]
Citti, Cinzia; Palazzoli, Federica; Licata, Manuela; Vilella, Antonietta; Leo, Giuseppina; Zoli, Michele; Vandelli, Maria Angela; Forni, Flavio; Pacchetti, Barbara; Cannazza, Giuseppe

Cannabidiol (CBD), for long time considered as a minor cannabinoid of Cannabis sativa, has recently gained much attention due to its antioxidant, anti-inflammatory, analgesic and anticonvulsant properties. A liquid chromatography coupled to mass spectrometry based method was developed for the quantitative determination of CBD and other cannabinoids (Δ9-tetrahydrocannabinol (THC), 11-hydroxy-THC and 11-nor-9-carboxy-THC) in rat brain samples after oral administration of a single high dose (50 mg/kg) of CBD. The main challenge of the present work was to study CBD pharmacokinetics in rat cortex: the identification of its metabolites and pharmacodynamics through the study of variations in endogenous compounds’ concentrations following CBD administration. An untargeted metabolomics approach revealed the formation of some CBD metabolites that are not commonly found in other body tissues or fluids. Lastly, the changes in some endogenous compounds’ concentrations were correlated with some of the pharmacological properties of this cannabinoid.

2016 - Biocatalytic Synthesis of Phospholipids and Their Application as Coating Agents for CaCO3Nano-crystals: Characterization and Intracellular Localization Analysis [Articolo su rivista]
Baldassarre, Francesca; Allegretti, Chiara; Tessaro, Davide; Carata, Elisabetta; Citti, Cinzia; Vergaro, Viviana; Nobile, Concetta; Cannazza, Giuseppe; D'Arrigo, Paola; Mele, Andrea; Dini, Luciana; Ciccarella, Giuseppe

Inorganic nanoparticles are widely investigated as drug delivery systems. In particular micro and nanoparticles of CaCO3 offer smart features for different biomaterials applications. In this work we exploit the phospholipids coating of nano-CaCO3. We prepare modified phospholipids through a chemo-enzymatic approach using Phospholipase D to efficiently transform the most abundant natural phospholipids in two products, snglycero-3-phosphocholine (GPC) and sn-glycero-3-phosphoserine (GPS). We have investigated and modified the Spray Drier process to obtain nano-crystals with specific phase, surface zeta-potential and morphology. The intracellular localization of coated nano-crystals was achieved by ultrastructural microscopy analysis. The synthetized phospholipids, GPC and GPS, improve nano-CaCO3 proprieties and promote a specific targeting as opposed to a widespread and nonspecific localization in the cell.