Characterizing the degradation of cannabidiol in an e-liquid formulation.

The reported characteristics of cannabidiol (CBD) have encouraged significant growth in commercial CBD products. There is limited information on the stability of CBD and some researchers have noted significant reductions of CBD in products. In this study, the chemical profiles of plant-based and chemically synthesized CBD in a prototype e-liquid formulation were assessed during 4 weeks of storage under varying conditions. Samples were analysed on days 1, 8, 15, 22, and 29 by untargeted analysis using ultra-high performance liquid chromatography-trapped ion mobility-time-of-flight mass spectrometry (UHPLC-TIMS-TOF-MS). On day 1, analysis of plant-based and synthetic CBD formulations showed small differences in their composition, with plant-based CBD e-liquid containing trace levels of a higher number of phytocannabinoid-related impurities. Storage for 4 weeks under stress (40 °C, 75% relative humidity, dark) and ambient (25 °C, 60% relative humidity, daylight) conditions led to increases in the number and abundance of cannabinoid-related degradation products, including cannabielsoin (CBE) and CBD-hydroxyquinone (HU-331), which are products of the oxidation of CBD, and other unidentified cannabinoid-related compounds. The unidentified cannabinoid-related compounds were probed by accurate mass measurement and MS2 fragmentation but could not be matched using a mass spectral library derived from 39 commercially available cannabinoid reference standards. Based on elemental composition and MS2 fragmentation patterns, the unidentified cannabinoid-related compounds were classified as hydroxy-CBE, hydroxy-CBD, and dihydroxy-CBD. The analysis of e-liquid formulations protected from light and stored at 4 °C for 4 weeks indicated only very small increases in CBD oxidation products. The results indicate that CBD degrades in e-liquid solution at ambient temperature in dark and light to form potentially undesirable products, including cannabielsoin and cannabidiol hydroxyquinone.

Chemoinformatic analysis of alkaloids isolated from Peganum genus.

Peganum genus is rich with its high phytochemical and botanical variability. Peganum species have been used as a sedative, antitumor, analgesic and antidepressant. This paper aims to study the molecular diversity of Peganum genus and to shed more light on the structure-activity relationship of the alkaloids isolated from Peganum genus. All Peganum alkaloids were grouped according to their structural properties. A chemoinformatic approach (SwissTargetPrediction) was used to determine the molecular targets of these alkaloids. To analyze and visualize the results, R software was used to generate hierarchical clustering heatmaps. The results of this study can help researchers to better understand the structure-activity relationship of Peganum alkaloids and how substitution can affect the biological activity of those alkaloids.

Phenylethanoid glycosides as a possible COVID-19 protease inhibitor: a virtual screening approach.

From the beginning of pandemic, more than 240 million people have been infected with a death rate higher than 2%. Indeed, the current exit strategy involving the spreading of vaccines must be combined with progress in effective treatment development. This scenario is sadly supported by the vaccine's immune activation time and the inequalities in the global immunization schedule. Bringing the crises under control means providing the world population with accessible and impactful new therapeutics. We screened a natural product library that contains a unique collection of 2370 natural products into the binding site of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (Mpro). According to the docking score and to the interaction at the active site, three phenylethanoid glycosides (forsythiaside A, isoacteoside, and verbascoside) were selected. In order to provide better insight into the atomistic interaction and test the impact of the three selected compounds at the binding site, we resorted to a half microsecond-long molecular dynamics simulation. As a result, we are showing that forsythiaside A is the most stable molecule and it is likely to possess the highest inhibitory effect against SARS-CoV-2 Mpro. Phenylethanoid glycosides also have been reported to have both protease and kinase activity. This kinase inhibitory activity is very beneficial in fighting viruses inside the body as kinases are required for viral entry, metabolism, and/or reproduction. The dual activity (kinase/protease) of phenylethanoid glycosides makes them very promising anit-COVID-19 agents.

In silico-based Approach to Investigate the Ability of PEGylated Rapamycin to Inhibit Galectin-3.

AIMS: To utilize in silico-based approach for investigating the ability of PEGylated rapamycin as a competitive inhibitor to Galectin-3 for curing various diseases or that may provide an attractive strategy for treatment of a wide variety of tumors. BACKGROUND: Galectin-3 (Gal-3) signaling protein is a unique member of lectin family present at the cell surface, intracellularly in both the nucleus and cytoplasm and extracellularly in the general circulation. Circulating Gal-3 is present in both normal and cancer cells. High levels of circulating Gal-3 have been proven to be associated with inflammation and fibrosis in several acute and chronic conditions, which include neurological degeneration, inflammatory and immune responses, autoimmune diseases, diabetes, heart failure, atherosclerosis, response to infection, wound healing, liver, lung, and kidney disease. Gal-3 is known to regulate many biological activities including cell adhesion, angiogenesis, growth, apoptosis, migration, and metastasis. Rapamycin has been examined alone or in combination with other drugs for treatment of various cancers in clinical studies. Although it has shown promising therapeutic effects, its clinical development was interrupted by poor aqueous solubility and limited preferential distribution. To overcome these limitations, RA has been chemically modified to hydrophilic analogues, such as everolimus (EV). However, all these approaches can only partially increase the solubility, but has little effect on the blood distribution and pharmacokinetics. Therefore, it is necessary to explore other RA conjugates to improve aqueous solubility and tissue distribution profile. Recently we reported that RP-MPEG inhibits the growth of various cancer cell lines by acting on mammalian target of RP (mTOR) receptor site and it can be used for gastric cancer. OBJECTIVE: To construct various molecular weight RP-MPEG by replacing MPEG chain in 40-O-(2- hydroxyethyl) position of the EV and analyze their binding affinity to Gal-3. METHODS: The chemical structures of various molecular weight RP-MPEG were built using ChemSketch software. The molecular docking study was performed to find the best probable structure of RP-MPEG for competitive inhibition of the CRD, based on the interaction score. For that purpose, the 3D structures of RP and EV were obtained from NCBI PubChem compound database, where the structural protein-co-crystallized ligand complex of Gal-3 (TD2, as a native ligand) was retrieved from RCSB Protein Data Bank. All structures of the selected compounds, served as molecules for molecular modeling, were optimized through MOE.2014 software before docking. Hydrogen atoms and partial charges were added to the protein. Protein minimization was performed in gas solvation with the side chains, keeping it rigid and the ligand flexible. The selected site was isolated and minimized, followed by protonating the protein. The 3D ligands were minimized using MMFF94x with cutoffs of 10 to 12 Å. The hydrogens and charges were fixed, and the RMS gradient was set to 0.001 kcal/mol. The docking results were analyzed to identify and assess the binding affinity of these compounds to CRD using drug discovery software. RESULTS: Our results indicated that RP-MPEG with MW 1178.51 g/mol has a logP value of 3.79 and has possessed the strongest binding affinity toward CRD of Gal-3 with a docking score of -6.87. Compared with TD2, there were additional close contacts for RP-MPEG (MW 1178.51 g/mol), coming from three hydrogen bonds with Asp148, Arg162, and Arg144 which suggest that this ligand is a strong competitive inhibitor among the other molecules for Gal-3. CONCLUSION: RP-MPEG with the MW 1178.51 g/mol could be a promising blocker for various biological action of Gal-3 includes profibrotic activity, modulation of immune responses and inflammatory responses to cancer that contributes to neoplastic transformation, angiogenesis and metastasis. Other: The 95% confidence intervals (CIs) of the binding affinity (according to their mean and standard errors) were estimated with 2.5 and 97.5 percentile as the lower and upper bounds.

Neuroactivity of the naturally occurring aporphine alkaloid, roemerine.

Roemerine is a naturally occurring aporphine alkaloid. In this study, we screened a conformer library of Food and Drug Administration (FDA)-approved drugs to identify similar drugs that can assist in identifying the biological targets of roemerine. To assess the neuroactivity in vitro, we measured the levels of cell metabolites, Brain-Derived Neurotrophic Factor (BDNF) and serotonin (5-HT) in SH-SY5Y cell line. By means of structure-based virtual screening, we identified five drugs that are similar to roemerine; mirtazapine, atomoxetine, epinastine, diphenhydramine and orphenadrine. GC-MS metabolomics study revealed that roemerine has a high impact on alanine-aspartate-glutamate pathway in cell lysate and cultured medium. Additionally, roemerine increased intercellular 5-HT level and intracellular BDNF protein expression at 10 µM. In conclusion, roemerine - a major alkaloid in antidepressant-like effect possessing plants (P. lacerum and P. syriacum) - has a neuronal activity through increasing BDNF protein expression and affecting serotonergic and glutamatergic systems in SH-SY5Y cell line.

Cyclodextrin inclusion complex inhibits circulating galectin-3 and FGF-7 and affects the reproductive integrity and mobility of Caco-2 cells.

Galectin-3 (Gal-3) is a carbohydrate-binding protein, that promotes angiogenesis through mediating angiogenic growth factors such as vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF). There is strong evidence confirming FGF involvement in tumor growth and progression by disrupting cell proliferation and angiogenesis. In this study, we investigated the effect of ß-cyclodextrin:everolimus:FGF-7 inclusion complex (Complex) on Caco-2 cell migration, cell motility and colony formation. In addition, we examined the inhibitory effect of the Complex on the circulating proteins; Gal-3 and FGF-7. Swiss Target Prediction concluded that Gal-3 and FGF are possible targets for ß-CD. Results of the chemotaxis cell migration assay on Caco-2 cell line revealed that the Complex has higher reduction in cell migration (78.3%) compared to everolimus (EV) alone (58.4%) which is possibly due to the synergistic effect of these molecules when used as a combined treatment. Moreover, the Complex significantly decreased the cell motility in cell scratch assay, less than 10% recovery compared to the control which has ~ 45% recovery. The Complex inhibited colony formation by ~ 75% compared to the control. Moreover, the Complex has the ability to inhibit Gal-3 with minimum inhibitory concentration of 33.46 and 41 for ß-CD and EV, respectively. Additionally, ß-CD and ß-CD:EV were able to bind to FGF-7 and decreased the level of FGF-7 more than 80% in cell supernatant. This confirms Swiss Target Prediction result that predicted ß-CD could target FGF. These findings advance the understanding of the biological effects of the Complex which reduced cell migration, cell motility and colony formation and it is possibly due to inhibiting circulating proteins such as; Gal-3 and FGF-7.

Neuroactivity of naturally occurring proaporphine alkaloid, pronuciferine.

Pronuciferine is a naturally occurring proaporphine alkaloid that belongs to isoquinoline alkaloids. The aim of this study is to investigate the neuroactivity of pronuciferine. We assessed the neuroprotective effect of pronuciferine against hydrogen peroxide (H2 O2 )-induced apoptosis in human neuronal SH-SY5Y cells. In addition, we measured the effect of pronuciferine on cell metabolites and brain-derived neurotrophic factor (BDNF) level in SH-SY5Y cells. In vitro result shows that pronuciferine at 10 µM significantly (P < .001) increased the proliferation of SH-SY5Y by 45%, and upon H2 O2 addition, pronuciferine significantly (P < .001) suppressed neuronal death caused by H2 O2 . Gas Chromatography-Mass Spectrometry (GC-MS) metabolomics study revealed that pronuciferine has a high impact on glycine-serine-threonine pathway by changing the intracellular level of serine dimethylglycine, sarcosine, and threonine. Also, pronuciferine increased the intercellular level of aspartic acid, glutamine, and tryptophan. Additionally, pronuciferine significantly (P < .05) increased the intracellular BDNF protein expression at 10 µM. Therefore, pronuciferine is a neuroactive molecule that might act as a neuroprotective agent to prevent apoptosis in neurodegenerative diseases.

Metabolomics and proteomics profiles of some medicinal plants and correlation with BDNF activity.

BACKGROUND: Identification of the low abundance of phytochemicals in plant extracts is very difficult. Pharmacological activity observed in such plants is not due to a single compound. In most cases, plant extracts show activity based on synergistic or antagonistic effects. Therefore, the idea of a holistic approach is more rational. PURPOSE: This study was planned to compare the metabolomics and proteomics profiles of Valeriana officinalis L. (Valerianaceae), Melissa officinalis L. (Lamiaceae), Hypericum perforatum L. (Hypericaceae) and Passiflora incarnata L. (Passifloraceae) used in sedative anxiolytic and sleep disorders. Integrated omics analyses were used to provide a better understanding of the effect of plant extracts on the brain-derived neurotrophic factor (BDNF) expression levels on the SH-SY5Y cell line by a holistic approach. METHODS: Metabolomic profiling of the plants was performed using the GC-MS and LC-qTOF-MS systems, and the proteomics analysis using the LC-qTOF-MS system after trypsin digestion. The Human BDNF Quantikine ELISA kit was utilized to test BDNF expression activity on the SH-SY5Y cell line. RESULTS: The investigated plant extracts showed a significant increase in BDNF expression (p < 0.05). M. officinalis was found as the most active extract. According to the correlation analyses between BDNF activity and metabolomics or proteomics level, 94 metabolites had a positive correlation while 23 metabolites had a highly negative correlation; those for proteins are 24 and 6, respectively. CONCLUSION: The multivariate data analysis revealed a similar metabolomics profile of H. perforatum and P. incarnata, which also had a similar activity profile. Remarkably, all the primary metabolites belonging to the Krebs Cycle (citric acid, fumaric acid, succinic acid, pyruvic acid, malic acid and citramalic acid, an analog of malic acid) were positively correlated with BDNF activity. Secondary metabolites with a high BDNF expression belonged to flavonoids, xanthone, coumarines, tannin, naphtalenes, terpenoids and carotenoid skeleton. Two proteins from the cytochrome P450 family (P450 71B11 and P450 94B3) were positively correlated with BDNF activity. Employing omics technologies in the plant research area will offer a better understanding of the role of plant extracts and may lead to the discovery of new compounds with specific activity.

Molecular Modeling-Based Delivery System Enhances Everolimus-Induced Apoptosis in Caco-2 Cells.

Several studies have shown that the mammalian target of rapamycin (mTOR) inhibitor; everolimus (EV) improves patient survival in several types of cancer. However, the meaningful efficacy of EV as a single agent for the treatment of colorectal cancer (CRC) has failed to be proven in multiple clinical trials. Combination therapy is one of the options that could increase the efficacy and decrease the toxicity of the anticancer therapy. This study revealed that the ß-cyclodextrin (ß-CD):FGF7 complex has the potential to improve the antiproliferative effect of EV by preventing FGF receptor activation and by enhancing EV cellular uptake and intracellular retention. Molecular docking techniques were used to investigate the possible interaction between EV, ß-CD, and FGF7. Molecular docking insights revealed that ß-CD and EV are capable to form a stable inclusion complex with FGF at the molecular level. The aqueous solubility of the inclusion complex was increased (3.1 ± 0.23 µM) when compared to the aqueous solubility of pure EV (1.7 ± 0.16 µM). In addition, the in vitro cytotoxic activity of a FGF7:ß-CD:EV complex on Caco-2 cell line was investigated using real-time xCELLigence technology. The FGF7:ß-CD:EV complex has induced apoptosis of Caco-2 cells and shown higher cytotoxic activity than the parent drug EV. With the multitargets effect of ß-CD:FGF7 and EV, the antiproliferative effect of EV was remarkably improved as the IC50 value of EV was reduced from 9.65 ± 1.42 to 1.87 ± 0.33 µM when compared to FGF7:ß-CD:EV complex activity. In conclusion, the findings advance the understanding of the biological combinational effects of the ß-CD:FGF7 complex and EV as an effective treatment to combat CRC.

The roles of valerenic acid on BDNF expression in the SH-SY5Y cell.

The roots of Valeriana officinalis L. (Valerianaceae) are used for treating sleep disorders and/or mild nerve tension. The effect of valerenic acid on brain-derived neurotrophic factor (BDNF) has not yet been studied, although it is known that gamma-amino butyric acid A (GABAA) receptor is regulated by BDNF, which modulates the depressive-like behavior and neurogenesis. The purpose of this study is to determine the effect of V. officinalis root extract (VO), its main constituents valerenic acid (VA) and acetoxy valerenic acid (AVA) as well as valerenic acid-free (VAF), acetoxy valerenic acid-free (AVAF) extracts and increasing amounts of valerenic acid containing extracts on the BDNF expression in SH-SY5Y cell lines. The effect of methanolic extracts of VO, VA, AVA, VAF, AVAF, and the extracts whose amount of VA were increased gradually, were tested using a Human BDNF ELISA kit with 17ß-estradiol as a positive control. The VO and VA extracts caused a significant (p < 0.001) increase in the BDNF expression in SH-SY5Y cells compared to control. This effect completely disappeared when cells were treated with VAF extract. AVA alone did not show any significant change in the BDNF levels. The extracts with increasing amount of VA led to a concentration- dependent effect on the cells. In conclusion, our findings suggest that the antidepressant-like effect of the VO extract is also related to BDNF expression, and that this is mainly due to the presence of VA in the extract. Removing VA from VO extract leads to a loss of activity. Moreover, the concentration of VA plays a role for BDNF expressions in SH-SY5Y cells, which demonstrates the importance of quality control on the commercially available products.

An LC-ESI-MS/MS method for the simultaneous determination of pronuciferine and roemerine in some Papaver species.

Papaver species, well known for their alkaloids, have been used for the treatment of several diseases, such as inflammation, diarrhea, depression, and sleep disorders in certain parts of Anatolia. In this study, four Papaver species (P. lacerum, P. syriacum, P. glaucum and P. rhoeas) were collected from different localities of Turkey. Methanolic extracts were prepared from the aerial parts of the plants. A rapid analytical method was developed for the simultaneously quantitative analysis of two alkaloids, pronuciferine and roemerine, using liquid chromatography tandem mass spectrometry. Multiple reaction monitoring in the positive ionization mode was used for detection. Pronuciferine and roemerine were analyzed on a C18 column (2.1 × 50 mm, 3 µm) with the mobile phase run in the gradient mode with 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile at a flow rate of 0.3 mL/min. The transitions 312.1→283.1 m/z and 280.0→249.0 m/z were used to monitor pronuciferine and roemerine, respectively. The assay was linear in the concentration range of 0.01 µg/mL to 1 µg/mL (r = 0.996 for roemerine, r = 0.998 for pronuciferine). The validation studies revealed that the method was linear, sensitive, accurate, precise, selective, repeatable, robust, and rugged. Finally, the developed method was applied to quantify pronuciferine and roemerine in the selected species. The amounts of pronuciferine and roemerine were respectively found as 8.5 to 48 µg/g and 4.4 to 43,000 µg/g.