Helichrysum stoechas (L.) Moench reduces body weight gain and modulates mood disorders via inhibition of silent information regulator 1 (SIRT1) by arzanol.

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

Regulation of Energy Metabolism and Anti-Inflammatory Activities of Mastiha Fractions from Pistacia lentiscus L. var. chia.

Pistacia lentiscus L. var. chia resin (Chios Mastiha), the first natural chewing gum, is widely used in Mediterranean cuisine and has been used in traditional medicine from ancient times. Regarding its chemical composition, Chios Mastiha is known to be rich in triterpenes. Triterpenes have a similar structure to glucocorticoids (GCs), the steroid hormones that exert strong anti-inflammatory activities and play crucial roles in the regulation of cellular metabolism. To simplify the characterization of the bioactive compounds of Mastiha resin, three different polarity fractions were isolated and were further analyzed regarding their main chemical composition and an assessment of their biological activities. The biological assessment focused on the evaluation of the potential anti-proliferative, anti-inflammatory, and apoptotic activities as well as the possible interference of the three different polarity Mastiha fractions with the glucocorticoid receptor signaling, with the aim of characterizing the biochemical mechanisms of the actions of the Mastiha fraction. Applying MTT cell viability assay, luciferase/ß-galactosidase assay, and Western blot analysis showed that Chios Mastiha apolar, medium-polar, and polar fractions reduced the HEK293 cell viability in a dose-dependent manner, possibly by mitochondrial-mediated induction of apoptosis. Medium-polar and polar Mastiha fractions also suppressed the GR and NF-κΒ transcriptional activation and the p65 protein levels. These activities were accompanied by the modulation of protein levels of regulatory molecules playing a crucial role in cellular energy homeostasis, such as GR, phosphoenolpyruvate carboxykinase (PEPCK), and/or peroxisome proliferator-activated receptor alpha (PPARα), and by the induction of phosphorylation and the activation of the AMP-activated protein kinase (AMPK). The medium-polar fraction was found to be enriched in triterpenes, such as lupeol, 24Z-masticadienonic acid methyl ester, and 24Z-isomasticadienonic acid methyl ester, and it was the most active one, so we propose that triterpenes in medium-polar fraction are possibly the bioactive compounds responsible for Mastiha's regulatory actions on energy metabolism and anti-inflammatory activities via interference with GR, NF-κΒ, and AMPK signaling. This highlights its potential applications in many fields of pharmaceutical, cosmetic, and nutraceutical interest.

Xanthomicrol Activity in Cancer HeLa Cells: Comparison with Other Natural Methoxylated Flavones.

The methoxylated flavone xanthomicrol represents an uncommon active phenolic compound identified in herbs/plants with a long application in traditional medicine. It was isolated from a sample of Achillea erba-rotta subsp. moschata (musk yar-row) flowering tops. Xanthomicrol promising biological properties include antioxidant, anti-inflammatory, antimicrobial, and anticancer activities. This study mainly focused on the evaluation of the xanthomicrol impact on lipid metabolism in cancer HeLa cells, together with the investigation of the treatment-induced changes in cell growth, morphology, and apoptosis. At the dose range of 5-100 µM, xanthomicrol (24 h of incubation) significantly reduced viability and modulated lipid profile in cancer Hela cells. It induced marked changes in the phospholipid/cholesterol ratio, significant decreases in the levels of oleic and palmitic acids, and a marked increase of stearic acid, involving an inhibitory effect on de novo lipogenesis and desaturation in cancer cells. Moreover, marked cell morphological alterations, signs of apoptosis, and cell cycle arrest at the G2/M phase were observed in cancer treated cells. The bioactivity profile of xanthomicrol was compared to that of the anticancer methoxylated flavones eupatilin and artemetin, and structure-activity relationships were underlined.

Drug affinity-responsive target stability unveils filamins as biological targets for artemetin, an anti-cancer flavonoid.

Artemetin is a valuable 5-hydroxy-3,6,7,3',4'-pentamethoxyflavone present in many different medicinal plants with very good oral bioavailability and drug-likeness values, owing to numerous bioactivities, such as anti-inflammatory and anti-cancer ones. Here, a multi-disciplinary plan has been settled and applied for identifying the artemetin target(s) to inspect its mechanism of action, based on drug affinity-responsive target stability and targeted limited proteolysis. Both approaches point to the disclosure of filamins A and B as direct artemetin targets in HeLa cell lysates, also giving detailed insights into the ligand/protein-binding sites. Interestingly, also 8-prenyl-artemetin, which is an artemetin more permeable semisynthetic analog, directly interacts with filamins A and B. Both compounds alter filamin conformation in living HeLa cells with an effect on cytoskeleton disassembly and on the disorganization of the F-actin filaments. Both the natural compound and its derivative are able to block cell migration, expectantly acting on tumor metastasis occurrence and development.

Application of experimental design in HPLC method optimisation for the simultaneous determination of multiple bioactive cannabinoids.

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

Effect of the natural polymethoxylated flavone artemetin on lipid oxidation and its impact on cancer cell viability and lipids.

The biochemical class of the polymethoxylated flavonoids represents uncommon phenolic compounds in plants presenting a more marked lipophilic behavior due to the alkylation of its hydroxylic groups. As a polymethoxylated flavone, which concerns a different bioavailability, artemetin (ART) has been examined in vitro against lipid oxidation and its impact on cancer cells has been explored. Despite this flavone only exerted a slight protection against in vitro fatty acid and cholesterol oxidative degradation, ART significantly reduced viability and modulated lipid profile in cancer Hela cells at the dose range 10-50 µM after 72 h of incubation. It induced marked changes in the monounsaturated/saturated phospholipid class, significant decreased the levels of palmitic, oleic and palmitoleic acids, maybe involving an inhibitory effect on de novo lipogenesis and desaturation in cancer cells. Moreover, ART compromised normal mitochondrial function, inducing a noteworthy mitochondrial membrane polarization in cancer cells. A dose-dependent absorption of ART was evidenced in HeLa cell pellets (15.2% of the applied amount at 50 µM), coupled to a marked increase in membrane fluidity, as indicate by the dose-dependent fluorescent Nile Red staining (red emissions). Our results validate the ART role as modulatory agent on cancer cell physiology, especially impacting viability, lipid metabolism, cell fluidity, and mitochondrial potential.

Agathadiol, a labdane diterpenoid from juniper berries, is a positive allosteric modulator of CB1R.

The neutral fraction of a juniper (Juniperus communis L.) berries acetone extract could positively modulate the activity of type 1 - cannabinoid receptor (CB1R). Bioactivity-directed fractionation identified the labdane diterpenoid agathadiol (4) as a positive allosteric modulator of CB1R, while closely related analogues were inactive. Agathadiol (4) is a minor constituent of juniper, but could be more conveniently obtained by semisynthesis from agathic acid (8), a major constituent of Manila copal.

Separation and non-separation methods for the analysis of cannabinoids in Cannabis sativa L.

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.

Anti-inflammatory Activity of Absinthin and Derivatives in Human Bronchoepithelial Cells.

Bitter taste receptors (hTAS2R) are expressed ectopically in various tissues, raising the possibility of a pharmacological exploitation. This seems of particular relevance in airways, since hTAS2Rs are involved in the protection of the aerial tissues from infections and in bronchodilation. The bis-guaianolide absinthin (1), one of the most bitter compounds known, targets the hTAS2R46 bitter receptor. Absinthin (1), an unstable compound, readily turns into anabsinthin (2) with substantial retention of the bitter properties, and this compound was used as a starting material to explore the chemical space around the bis-guaianolide bitter pharmacophore. Capitalizing on the chemoselective opening of the allylic lactone ring, the esters 3 and 4, and the nor-azide 6 were prepared and assayed on human bronchoepithelial (BEAS-2B) cells expressing hTAS2R46. Anti-inflammatory activity was evaluated by measuring the expression of MUC5AC, iNOS, and cytokines, as well as the production of superoxide anion, qualifying the methyl ester 3 as the best candidate for additional studies.

Cannabidiol exerts protective effects in an in vitro model of Parkinson's disease activating AKT/mTOR pathway.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the degeneration of the nigrostriatal dopaminergic pathway with loss of substantia nigra pars compacta neurons and dopamine depletion. Various natural compounds showed protective actions against PD. In this work, the protective effects of cannabidiol (CBD), obtained from Cannabis sativa, were evaluated in retinoic acid differentiated SH-SY5Y cells exposed to 1-methyl-4-phenylpyridinium (MPP+), an in vitro PD model. In order to evaluate which receptor is involved in CBD actions CB1, CB2 and TRPV1 receptor antagonists were used. CBD counteracted the loss of cell viability caused by MPP+, reducing apoptosis as demonstrated by the reduction of Bax and caspase 3. Moreover, CBD reduced the nuclear levels of PARP-1. The protective effects of CBD seem to be mediated by the activation of ERK and AKT/mTOR pathways. The treatment with AKT1/2 inhibitor and the mTOR inhibitor rapamycin abolished CBD protective effects. The CBD-induced ERK activation may be mediated by CBD interaction with CB2 and TRPV1. We also investigated the protein levels of the autophagic proteins LC3 and beclin 1. CBD reduced the MPP+-induced increase of LC3 by CB2 and TRPV1 receptors. These data suggested the involvement of ERK in the modulation of autophagy. However, beclin 1 levels were not modified neither by MPP+ nor by CBD. These results indicated that CBD may exert preventive and protective actions in PD.

Non volatile constituents of the vermouth ingredient Artemisia vallesiaca.

The Alpine wormwood Artemisia vallesiaca All. was considered the most valuable ingredient of vermouth, a celebrated aromatized wine. A. vallesiaca has a very limited geographical distribution, and the booming market of vermouth decimated its natural population, resulting in the eventual replacement of this rare species with more common and less expensive wormwoods like A. absinthium L.. Over the past years, attempts to revive the original recipe(s) of vermouth have fostered the establishment of cultivations of A. vallesiaca in pre-montane settings. In order to assist these projects, the phytochemical profile of cultivated plants and of several native populations of A. vallesiaca from the Swiss Valais were comparatively evaluated, focusing on sesquiterpene lactones and on lipophilic flavonoids, the hallmark constituents of Artemisia species. Remarkably, no significant difference was detected between the samples, despite the different origins. The lipophilic flavonoids of A. vallesiaca were similar to those of related species used in the production of vermouth, but the presence of C-9 oxygenated 11ß-methyl germacranolides and eudesmanolides (herbolides) made its sesquiterpene lactone profile peculiar. In addition to known compounds, two novel germacranolides were also characterized (herbolides J and K), and the major sesquiterpene lactone from the plant, the bitter germacranolide herbolide D (4), was detected and quantified by 1H NMR in a bitter liqueur aromatized with A. vallesiaca. Taken together, these observations qualify herbolides as marker to identify A. vallesiaca in aromatized alcohol matrixes.

O-Methyl Phytocannabinoids: Semi-synthesis, Analysis in Cannabis Flowerheads, and Biological Activity.

A general protocol for the selective mono-O-methylation of resorcinyl phytocannabinoids was developed. The availability of semisynthetic monomethyl analogues of cannabigerol, cannabidiol, and cannabidivarin (1A: -3A: , respectively) made it possible to quantify these minor phytocannabinoids in about 40 different chemotypes of fiber hemp. No chemotype significantly accumulated mono-O-methyl cannabidiol (2B: ) or its lower homologue (3B: ), while at least three chemotypes containing consistent amounts (≥ 400 mg/kg) of O-methylcannabigerol (1B: ) were identified. O-Methylation of alkyl phytocannabinoids (1B: -3B: ) does not significantly change the activity on peroxisome proliferator-activated receptors in contrast to what was reported for phenethyl analogues.

An Artemisia-derived natural product-based fluorescent probe for the bitter taste receptor hTAS2R38.

The discovery of taste receptors hTAS2Rs expression in extra oral tissue, especially in the gastrointestinal tract and in the respiratory system, has endowed bitter receptors of functionalities that exceed the simple perception of taste and flavour. In particular, stimulation of hTAS2Rs by bitter agents in the airway smooth muscle triggers bronchodilation of possible pharmacological relevance. To study the receptor localization in pulmonary smooth muscle cells and to investigate their biological response to hTAS2R38 activation, we have developed a fluorescent probe for hTAS2R38 starting from the sesquiterpene lactone costunolide, available in multigram amounts from Artemisia umbelliformis Lam. The N-methylanthranilate-containing probe demonstrated a very low cytotoxicity compared to the natural product toward human airway smooth muscle cells and epithelial bronchial cells, but fully retained its binding to hTAS2R38, making it possible the fluorescent detection of cells expressing this bitter receptor.

Cannabis Phenolics and their Bioactivities.

BACKGROUND: Although Cannabis sativa L. is one of the most versatile plant species with multipurpose use both as medical, alimentary source and as psychoactive abuse, its biomedical relevance focused the attention on major cannabinoids. Phytochemical characterization of cannabis highlights the presence of various non-cannabinoids constituents including flavonoids, spiroindans, dihyrostilbenes, dihydrophenanthrenes, lignanamides, steroids and alkaloids. This review aims to identify polyphenols present in this plant, their biosynthesis, their bioactivities and their synthesis, when this occurred. METHODS: We undertook a systematic research focused on bibliographic databases including all noncannabinoids phenolics in various C. sativa strains from their isolation, structural elucidation, their biological activity to their synthesis. RESULT: Nevertheless, attention has so far been focused only on cannabinoids (more than one hundred isolated), cannabis is a complex plant able to produce more than 480 chemical entities that represent almost all of the different biogenetic classes. Regarding phenolic compounds, the plant biosynthesises a plethora of unique non-cannabinoids second metabolites, such as prenylated flavonoids, stilbenoids derivatives and lignanammides. CONCLUSION: Cannabis is a plant with high pharmacological and nutrition values, its potentialities and applications are not only circumscribed to cannabinoids biological activities, but also defined by noncannabinoid compounds. The combination of other cannabinoids together with noncannabinoid components could enhance the beneficial effects of THC and could reduce undesirable side effects.

The anti-migraine component of butterbur extracts, isopetasin, desensitizes peptidergic nociceptors by acting on TRPA1 cation channel.

BACKGROUND AND PURPOSE: The mechanism of the anti-migraine action of extracts of butterbur [Petasites hybridus (L.) Gaertn.] is unknown. Here, we investigated the ability of isopetasin, a major constituent of these extracts, to specifically target TRPA1 channel and to affect functional responses relevant to migraine. EXPERIMENTAL APPROACH: Single-cell calcium imaging and patch-clamp recordings in human and rodent TRPA1-expressing cells, neurogenic motor responses in rodent isolated urinary bladder, release of CGRP from mouse spinal cord in vitro and facial rubbing in mice and meningeal blood flow in rats were examined. KEY RESULTS: Isopetasin induced (i) calcium responses and currents in rat/mouse trigeminal ganglion (TG) neurons and in cells expressing the human TRPA1, (ii) substance P-mediated contractions of rat isolated urinary bladders and (iii) CGRP release from mouse dorsal spinal cord, responses that were selectively abolished by genetic deletion or pharmacological antagonism of TRPA1 channels. Pre-exposure to isopetasin produced marked desensitization of allyl isothiocyanate (AITC, TRPA1 channel agonist)- or capsaicin (TRPV1 channel agonist)-evoked currents in rat TG neurons, contractions of rat or mouse bladder and CGRP release from mouse central terminals of primary sensory neurons. Repeated intragastric administration of isopetasin attenuated mouse facial rubbing, evoked by local AITC or capsaicin, and dilation of rat meningeal arteries by acrolein or ethanol (TRPA1 and TRPV1 channel agonists respectively). CONCLUSION AND IMPLICATIONS: Activation of TRPA1 channels by isopetasin results in excitation of neuropeptide-containing nociceptors, followed by marked heterologous neuronal desensitization. Such atten uation in pain and neurogenic inflammation may account for the anti-migraine action of butterbur.

Target regulation of PI3K/Akt/mTOR pathway by cannabidiol in treatment of experimental multiple sclerosis.

This study was aimed to investigate whether treatment with purified cannabidiol (CBD) may counteract the development of experimental multiple sclerosis (MS), by targeting the PI3K/Akt/mTOR pathway. Although the PI3K/Akt/mTOR pathway was found to be activated by cannabinoids in several immune and non-immune cells, currently, there is no data about the effects of CBD in the PI3K/Akt/mTOR activity in MS. Experimental Autoimmune Encephalomyelitis (EAE), the most common model of MS, was induced in C57BL/6 mice by immunization with myelin oligodendroglial glycoprotein peptide (MOG)35-55. After EAE onset, which occurs approximately 14days after disease induction, mice were daily intraperitoneally treated with CBD (10mg/kg mouse) and observed for clinical signs of EAE. At 28days from EAE-induction, mice were euthanized and spinal cord tissues were sampled to perform immunohistochemical evaluations and western blot analysis. Our results showed a clear downregulation of the PI3K/Akt/mTOR pathway following EAE induction. CBD treatment was able to restore it, increasing significantly the phosphorylation of PI3K, Akt and mTOR. Also, an increased level of BNDF in CBD-treated mice seems to be involved in the activation of PI3K/Akt/mTOR pathway. In addition, our data demonstrated that therapeutic efficacy of CBD treatment is due to reduction of pro-inflammatory cytokines, like IFN-γ and IL-17 together with an up-regulation of PPARγ. Finally, CBD was found to promote neuronal survival by inhibiting JNK and p38 MAP kinases. These results provide an interesting discovery about the regulation of the PI3K/Akt/mTOR pathway by cannabidiol administration, that could be a new potential therapeutic target for MS management.

Anti-inflammatory and antioxidant effects of a combination of cannabidiol and moringin in LPS-stimulated macrophages.

Inflammatory response plays an important role in the activation and progress of many debilitating diseases. Natural products, like cannabidiol, a constituent of Cannabis sativa, and moringin, an isothiocyanate obtained from myrosinase-mediated hydrolysis of the glucosinolate precursor glucomoringin present in Moringa oleifera seeds, are well known antioxidants also endowed with anti-inflammatory activity. This is due to a covalent-based mechanism for ITC, while non-covalent interactions underlie the activity of CBD. Since these two mechanisms are distinct, and the molecular endpoints are potentially complementary, we investigated in a comparative way the protective effect of these compounds alone or in combination on lipopolysaccharide-stimulated murine macrophages. Our results show that the cannabidiol (5µM) and moringin (5µM) combination outperformed the single constituents that, at this dosage had only a moderate efficacy on inflammatory (Tumor necrosis factor-α, Interleukin-10) and oxidative markers (inducible nitric oxide synthase, nuclear factor erythroid 2-related factor 2, nitrotyrosine). Significant upregulation of Bcl-2 and downregulation of Bax and cleaved caspase-3 was observed in cells treated with cannabidiol-moringin combination. Treatment with the transient receptor potential vanilloid receptor 1 antagonist was detrimental for the efficacy of cannabidiol, while no effect was elicited by cannabinoid receptor 1 and cannabinoid receptor 2 antagonists. None of these receptors was involved in the activity of moringin. Taken together, our in vitro results testify the anti-inflammatory, antioxidative, and anti-apoptotic effects of the combination of cannabidiol and moringin.

A new formulation of cannabidiol in cream shows therapeutic effects in a mouse model of experimental autoimmune encephalomyelitis.

BACKGROUND: The present study was designed to investigate the efficacy of a new formulation of alone, purified cannabidiol (CBD) (>98 %), the main non-psychotropic cannabinoid of Cannabis sativa, as a topical treatment in an experimental model of autoimmune encephalomyelitis (EAE), the most commonly used model for multiple sclerosis (MS). Particularly, we evaluated whether administration of a topical 1 % CBD-cream, given at the time of symptomatic disease onset, could affect the EAE progression and if this treatment could also recover paralysis of hind limbs, qualifying topical-CBD for the symptomatic treatment of MS. METHODS: In order to have a preparation of 1 % of CBD-cream, pure CBD have been solubilized in propylene glycoland basic dense cream O/A. EAE was induced by immunization with myelin oligodendroglial glycoprotein peptide (MOG35-55) in C57BL/6 mice. After EAE onset, mice were allocated into several experimental groups (Naïve, EAE, EAE-1 % CBD-cream, EAE-vehicle cream, CTRL-1 % CBD-cream, CTRL-vehicle cream). Mice were observed daily for signs of EAE and weight loss. At the sacrifice of the animals, which occurred at the 28(th) day from EAE-induction, spinal cord and spleen tissues were collected in order to perform histological evaluation, immunohistochemistry and western blotting analysis. RESULTS: Achieved results surprisingly show that daily treatment with topical 1 % CBD-cream may exert neuroprotective effects against EAE, diminishing clinical disease score (mean of 5.0 in EAE mice vs 1.5 in EAE + CBD-cream), by recovering of paralysis of hind limbs and by ameliorating histological score typical of disease (lymphocytic infiltration and demyelination) in spinal cord tissues. Also, 1 % CBD-cream is able to counteract the EAE-induced damage reducing release of CD4 and CD8α T cells (spleen tissue localization was quantified about 10,69 % and 35,96 % of positive staining respectively in EAE mice) and expression of the main pro-inflammatory cytokines as well as several other direct or indirect markers of inflammation (p-selectin, IL-10, GFAP, Foxp3, TGF-ß, IFN-γ), oxidative injury (Nitrotyrosine, iNOS, PARP) and apoptosis (Cleaved caspase 3). CONCLUSION: All these data suggest an interesting new profile of CBD that could lead to its introduction in the clinical management of MS and its associated symptoms at least in association with current conventional therapy.