Photodegradation of cannabidiol (CBD) and Δ9-THC in cannabis plant material.

Δ9-THC, the psychotropic cannabinoid in Cannabis sativa L., for many years has been the focus of all the pharmacological attention as the main promising principle of the plant. Recently, however, cannabidiol (CBD) has brought a sudden change in the scenario, exponentially increasing the interest in pharmacology as the main non-psychotropic cannabinoid with potential therapeutic, cosmetical and clinical applications. Although the reactivity of CBD and Δ9-THC has been considered, little attention has been paid to the possible photodegradation of these cannabinoids in the vegetal matrix and the data available in the literature are, in some cases, contradictory. The aim of the present work is to provide a characterization of the photochemical behaviour of CBD and Δ9-THC in three cannabis chemotypes, namely I (Δ9-THC 2.50%w/w), II (CBD:Δ9-THC 5.82%w/w:3.19%w/w) and III (CBD 3.02%w/w).

A new HPLC method with multiple detection systems for impurity analysis and discrimination of natural versus synthetic cannabidiol.

Cannabidiol (CBD) is the main non-psychoactive phytocannabinoid derived from Cannabis sativa L. It is now an active pharmaceutical ingredient (API), given its usage in treating some types of pediatric epilepsy. For this reason, this compound requires a deep characterization in terms of purity and origin. Previous research work has shown two impurities in CBD samples from hemp inflorescences, namely, cannabidivarin (CBDV) and cannabidibutol (CBDB), while abnormal-cannabidiol (abn-CBD) has been described as the primary by-product that is generated from CBD synthesis. Both natural and synthetic CBD samples exhibit the presence of Δ9-tetrahydrocannabinol (Δ9-THC) and Δ8-THC. This study aimed to develop a new analytical method based on high-performance liquid chromatography (HPLC) with different detection systems to study the purity of CBD and to define its origin based on the impurity profile. In addition to the above-mentioned cannabinoids, other compounds, such as cannabigerovarin (CBGV), cannabigerol (CBG), cannabichromevarin (CBCV), and cannabichromene (CBC), were examined as potential discriminating impurities. Qualitative and quantitative analyses were carried out by UHPLC-HRMS and HPLC-UV/Vis, respectively. Principal component analysis was applied for statistical exploration. Natural CBD samples exhibited purities ranging between 97.5 and 99.7%, while synthetic samples were generally pure, except for three initially labeled as synthetic, revealing natural-derived impurities. To further confirm the origin of CBD samples, the presence of other two minor impurities, namely cannabidihexol (CBDH) and cannabidiphorol (CBDP), was assessed as unequivocal for a natural origin. Finally, an enantioselective HPLC analysis was carried out and the results confirmed the presence of the (-)-trans enantiomer in all CBD samples. In conclusion, the HPLC method developed represents a reliable tool for detecting CBD impurities, thus providing a clear discrimination of the compound origin.

Arzanol, a natural phloroglucinol α-pyrone, protects HaCaT keratinocytes against H2O2-induced oxidative stress, counteracting cytotoxicity, reactive oxygen species generation, apoptosis, and mitochondrial depolarization.

Skin oxidative stress results in structural damage, leading to premature senescence, and pathological conditions such as inflammation and cancer. The plant-derived prenylated pyrone-phloroglucinol heterodimer arzanol, isolated from Helichrysum italicum ssp. microphyllum (Willd.) Nyman aerial parts, exhibits anti-inflammatory, anticancer, antimicrobial, and antioxidant activities. This study explored the arzanol protection against hydrogen peroxide (H2O2) induced oxidative damage in HaCaT human keratinocytes in terms of its ability to counteract cytotoxicity, reactive oxygen species (ROS) generation, apoptosis, and mitochondrial membrane depolarization. Arzanol safety on HaCaT cells was preliminarily examined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and microscopic observation. The arzanol pre-incubation (5-100 µM, for 24 h) did not induce cytotoxicity and morphological alterations. The phloroglucinol, at 50 µM, significantly protected keratinocytes against cytotoxicity induced by 2 h-incubation with 2.5 and 5 mM H2O2, decreased cell ROS production induced by 1 h-exposure to all tested H2O2 concentrations (0.5-5 mM), as determined by the 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) assay, and lipid peroxidation (thiobarbituric acid reactive substances [TBARS] method). The 2-h incubation of keratinocytes with H2O2 determined a significant increase of apoptotic cells versus control cells, evaluated by NucView® 488 assay, from the dose of 2.5 mM. Moreover, an evident mitochondrial membrane potential depolarization, monitored by fluorescent mitochondrial dye MitoView™ 633, was assessed at 5 mM H2O2. Arzanol pre-treatment (50 µM) exerted a strong significant protective effect against apoptosis, preserving the mitochondrial membrane potential of HaCaT cells at the highest H2O2 concentrations. Our results validate arzanol as an antioxidant agent for the prevention/treatment of skin oxidative-related disorders, qualifying its potential use for cosmeceutical and pharmaceutical applications.

Protective Effect of Arzanol against H2O2-Induced Oxidative Stress Damage in Differentiated and Undifferentiated SH-SY5Y Cells.

Oxidative stress can damage neuronal cells, greatly contributing to neurodegenerative diseases (NDs). In this study, the protective activity of arzanol, a natural prenylated α-pyrone-phloroglucinol heterodimer, was evaluated against the H2O2-induced oxidative damage in trans-retinoic acid-differentiated (neuron-like) human SH-SY5Y cells, widely used as a neuronal cell model of neurological disorders. The pre-incubation (for 2 and 24 h) with arzanol (5, 10, and 25 µM) significantly preserved differentiated SH-SY5Y cells from cytotoxicity (MTT assay) and morphological changes induced by 0.25 and 0.5 mM H2O2. Arzanol reduced the generation of reactive oxygen species (ROS) induced by 2 h oxidation with H2O2 0.5 mM, established by 2',7'-dichlorodihydrofluorescein diacetate assay. The 2 h incubation of differentiated SH-SY5Y cells with H2O2 determined a significant increase in the number of apoptotic cells versus control cells, evaluated by propidium iodide fluorescence assay (red fluorescence) and NucView® 488 assay (green fluorescence). Arzanol pre-treatment (2 h) exerted a noteworthy significant protective effect against apoptosis. In addition, arzanol was tested, for comparison, in undifferentiated SH-SY5Y cells for cytotoxicity and its ability to protect against H2O2-induced oxidative stress. Furthermore, the PubChem database and freely accessible web tools SwissADME and pkCSM-pharmacokinetics were used to assess the physicochemical and pharmacokinetic properties of arzanol. Our results qualify arzanol as an antioxidant agent with potential neuroprotective effects against neuronal oxidative stress implicated in NDs.

Bitter Taste Receptor 46 (hTAS2R46) Protects Monocytes/Macrophages from Oxidative Stress.

Bitter taste receptors (TAS2Rs) are not only responsible for taste perception in the oral cavity, but are spread throughout the body, generating a widespread chemosensory system. In humans, 25 subtypes have been identified and are differentially expressed in tissues and organs, including in the immune system. In fact, several TAS2R subtypes have been detected in neutrophils, lymphocytes, B and T cells, NK cells, and monocytes/macrophages, in which they regulate various protective functions of the innate immune system. Given its recognized anti-inflammatory and antioxidant activity, and the generally protective role of bitter taste receptors, in this work, we studied TAS2R46's potential in the protection of human monocyte/macrophage DNA from stress-induced damage. Through both direct and indirect assays and a single-cell gel electrophoresis assay, we demonstrated that absinthin, a specific TAS2R46 agonist, counteracts the release of reactive oxygen species (ROS) and reactive nitrogen species (RNS) and reduces DNA damage in both cell types. Even though the release of ROS from monocytes/macrophages is fundamental for contrast pathogen agents, supraphysiological ROS production impairs their function, finally leading to cell death. Our results highlight TAS2R46 as a novel player involved in the protection of monocytes and macrophages from oxidative stress damage, while simultaneously supporting their antimicrobial activity.

Antiviral Activity of Cannabidiolic Acid and Its Methyl Ester against SARS-CoV-2.

In the present study, the antiviral activity of cannabinoids isolated from Cannabis sativa L. was assessed in vitro against a panel of SARS-CoV-2 variants, indicating cannabidiolic acid (CBDA) was the most active. To overcome the instability issue of CBDA, its methyl ester was synthesized and tested for the first time for its antiviral activity. CBDA methyl ester showed a neutralizing effect on all the SARS-CoV-2 variants tested with greater activity than the parent compound. Its stability in vitro was confirmed by ultra-high-performance liquid chromatography (UHPLC) analysis coupled with high-resolution mass spectrometry (HRMS). In addition, the capacity of both CBDA and its derivative to interact with the virus spike protein was assessed in silico. These results showed that CBDA methyl ester can be considered as a lead compound to be further developed as a new effective drug against COVID-19 infection.

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.

Δ8-THC Induces Up-Regulation of Glutamatergic Pathway Genes in Differentiated SH-SY5Y: A Transcriptomic Study.

Cannabinoids, natural or synthetic, have antidepressant, anxiolytic, anticonvulsant, and anti-psychotic properties. Cannabidiol (CBD) and delta-9-tetrahydrocannabinol (Δ9-THC) are the most studied cannabinoids, but recently, attention has turned towards minor cannabinoids. Delta-8-tetrahydrocannabinol (Δ8-THC), an isomer of Δ9-THC, is a compound for which, to date, there is no evidence of its role in the modulation of synaptic pathways. The aim of our work was to evaluate the effects of Δ8-THC on differentiated SH-SY5Y human neuroblastoma cells. Using next generation sequencing (NGS), we investigated whether Δ8-THC could modify the transcriptomic profile of genes involved in synapse functions. Our results showed that Δ8-THC upregulates the expression of genes involved in the glutamatergic pathway and inhibits gene expression at cholinergic synapses. Conversely, Δ8-THC did not modify the transcriptomic profile of genes involved in the GABAergic and dopaminergic pathways.

Δ8-THC Protects against Amyloid Beta Toxicity Modulating ER Stress In Vitro: A Transcriptomic Analysis.

Alzheimer's disease (AD) represents the most common form of dementia, characterized by amyloid ß (Aß) plaques and neurofibrillary tangles (NFTs). It is characterized by neuroinflammation, the accumulation of misfolded protein, ER stress and neuronal apoptosis. It is of main importance to find new therapeutic strategies because AD prevalence is increasing worldwide. Cannabinoids are arising as promising neuroprotective phytocompounds. In this study, we evaluated the neuroprotective potential of Δ8-THC pretreatment in an in vitro model of AD through transcriptomic analysis. We found that Δ8-THC pretreatment restored the loss of cell viability in retinoic acid-differentiated neuroblastoma SH-SY5Y cells treated with Aß1-42. Moreover, the transcriptomic analysis provided evidence that the enriched biological processes of gene ontology were related to ER functions and proteostasis. In particular, Aß1-42 upregulated genes involved in ER stress and unfolded protein response, leading to apoptosis as demonstrated by the increase in Bax and the decrease in Bcl-2 both at gene and protein expression levels. Moreover, genes involved in protein folding and degradation were also deregulated. On the contrary, Δ8-THC pretreatment reduced ER stress and, as a consequence, neuronal apoptosis. Then, the results demonstrated that Δ8-THC might represent a new neuroprotective agent in AD.

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.