Amira S. Wanas

Quantitative Determination of Cannabinoids in Cannabis and Cannabis Products Using Ultra-High-Performance Supercritical Fluid Chromatography and Diode Array/Mass Spectrometric Detection.

Ultra‐high‐performance supercritical fluid chromatography (UHPSFC) is an efficient analytical technique and has not been fully employed for the analysis of cannabis. Here, a novel method was developed for the analysis of 30 cannabis plant extracts and preparations using UHPSFC/PDA‐MS. Nine of the most abundant cannabinoids, viz. CBD, ∆8‐THC, THCV, ∆9‐THC, CBN, CBG, THCA‐A, CBDA, and CBGA, were quantitatively determined (RSDs < 6.9%). Unlike GC methods, no derivatization or decarboxylation was required prior to UHPSFC analysis. The UHPSFC chromatographic separation of cannabinoids displayed an inverse elution order compared to UHPLC. Combining with PDA‐MS, this orthogonality is valuable for discrimination of cannabinoids in complex matrices. The developed method was validated, and the quantification results were compared with a standard UHPLC method. The RSDs of these two methods were within ±13.0%. Finally, chemometric analysis including principal component analysis (PCA) and partial least squares‐discriminant analysis (PLS‐DA) were used to differentiate between cannabis samples.

Determination of 11 Cannabinoids in Biomass and Extracts of Different Varieties of Cannabis Using High-Performance Liquid Chromatography.

An HPLC single-laboratory validation was performed for the detection and quantification of the 11 major cannabinoids in most cannabis varieties, namely, cannabidiolic acid (CBDA), cannabigerolic acid (CBGA), cannabigerol (CBG), cannabidiol (CBD), tetrahydrocannabivarin (THCV), cannabinol (CBN), Δ9-trans-tetrahydrocannabinol (Δ9-THC), Δ8-trans-tetrahydrocannabinol (Δ8-THC), cannabicyclol (CBL), cannabichromene (CBC), and Δ9-tetrahydrocannabinolic acid-A (THCAA). The analysis was carried out on the biomass and extracts of these varieties. Methanol-chloroform (9:1, v/v) was used for extraction, 4-androstene-3,17-dione was used as the internal standard, and separation was achieved in 22.2 min on a C18 column using a two- step gradient elution. The method was validated for the 11 cannabinoids. The concentration-response relationship of the method indicated a linear relationship between the concentration and peak area with r2 values of >0.99 for all 11 cannabinoids. Method accuracy was determined through a spike study, and recovery ranged from 89.7 to 105.5% with an RSD of 0.19 to 6.32% for CBDA, CBD, THCV, CBN, Δ9-THC, CBL, CBC, and THCAA; recovery was 84.7, 84.2, and 67.7% for the minor constituents, CBGA, CBG, and Δ8-THC, respectively, with an RSD of 2.58 to 4.96%. The validated method is simple, sensitive, and reproducible and is therefore suitable for the detection and quantification of these cannabinoids in different types of cannabis plant materials.

Structure elucidation of secondary metabolites isolated from the leaves of Ixora undulate and their inhibitory activity toward advanced glycation end-products formation

Three aromatic glycosides (1-3), two sulfur and nitrogen-containing compound glucosides (4, 5), and one flavonoid glycoside (6) were isolated from the leaves of Ixora undulata. Their structures were established by extensive 1D, 2D NMR, and HRESIMS experiments, and structure 4 was further confirmed by single crystal X-ray diffraction analysis. Of the assayed compounds, 7, 11 and 12 showed strong inhibitory activity toward advanced glycation end-products formation with IC50 values of 86.0 μM, 76.6 μM and 98.6 μM, respectively.

Quantitative Determination of Δ9-THC, CBG, CBD, Their Acid Precursors and Five Other Neutral Cannabinoids by UHPLC-UV-MS

Cannabinoids are a group of terpenophenolic compounds in the medicinal plant Cannabis sativa (Cannabaceae family). Cannabigerolic acid, Δ9-tetrahydrocannabinolic acid A, cannabidiolic acid, Δ9-tetrahydrocannabinol, cannabigerol, cannabidiol, cannabichromene, and tetrahydrocannabivarin are major metabolites in the classification of different strains of C. sativa. Degradation or artifact cannabinoids cannabinol, cannabicyclol, and Δ8-tetrahydrocannabinol are formed under the influence of heat and light during processing and storage of the plant sample. An ultrahigh-performance liquid chromatographic method coupled with photodiode array and single quadruple mass spectrometry detectors was developed and validated for quantitative determination of 11 cannabinoids in different C. sativa samples. Compounds 1:  - 11: were baseline separated with an acetonitrile (with 0.05% formic acid) and water (with 0.05% formic acid) gradient at a flow rate of 0.25 mL/min on a Waters Cortec UPLC C18 column (100 mm × 2.1 mm I. D., 1.6 µm). The limits of detection and limits of quantitation of the 11 cannabinoids were below 0.2 and 0.5 µg/mL, respectively. The relative standard deviation for the precision test was below 2.4%. A mixture of acetonitrile and methanol (80 : 20, v/v) was proven to be the best solvent system for the sample preparation. The recovery of all analytes was in the range of 97 - 105%. A total of 32 Cannabis samples including hashish, leaves, and flower buds were analyzed.

Natural Cannabinoids of Cannabis and Methods of Analysis

Cannabis has gained a lot of popularity in last few years not only because of its use as illicit drug but due to its use as food, fiber and medicine. It is a complex mixture of constituents which contain a unique class of secondary metabolites called phytocannabinoids. In general, so far a total of 565 constituents including 120 phytocannabinoids have been reported in Cannabis sativa. This chapter discusses the chemistry of phytocannabinoids in the plant with particular emphasis on the Δ9-THC type of cannabinoids and different analytical methods available for cannabinoids analysis in cannabis plant and cannabis products.

New α-Pyrone derivatives from the endophytic fungus Embellisia sp

Two new α- Pyrone derivatives (1,2) have been isolated from the endophytic fungus Embellisia sp. The isolated compounds were chemically identified as 5-(3-S-hydroxybutyl)-4-methoxy-6-methyl-2H-pyran-2-one (1) and 4-(4-methoxy-6-methyl-2-oxo-2H-pyran-5-yl)butanoic acid (2). The chemical structures of 1 and 2 were determined based on one and two dimensional NMR spectroscopy and high resolution mass spectrometry. Absolute configuration of compound 1 was determined using modified mosher ester reaction. In vitro antileishmanial, antifungal, antibacterial and antimalarial activities of 1 and 2 were examined but they didn’t show promising activities.

Flavonoids of Alcea rosea L. and their immune stimulant, antioxidant and cytotoxic activities on hepatocellular carcinoma HepG-2 cell line

Abstract Alcea rosea L. is widely cultivated in gardens of Egypt as an ornamental plant and it has a great history of folkloric medicinal uses. In the present work, phytochemical investigation of the alcoholic extract of the flowers of A. rosea L. led to the isolation of six flavonoids (1–6). Dihydrokaempferol-4′-O-β-d-glucopyranoside (1), dihydrokaempferol (2), kaempferol-3-O-[6″-(E-coumaroyl)]-β-d-glucopyranoside (3), kaempferol-3-O-β-d-glucopyranoside (4), Apigenin (5) and kaempferol-3-O-α-l-rhamnopyranosyl-(1′″→6″)-β-d-glucopyranoside (6). Four of the isolated compounds were evaluated for their antioxidant, immunostimulant and cytotoxic activities against HepG-2 cell line. Compound (3) showed potent cytotoxic activity against HepG-2 cell line with high selectivity towards hepatocellular carcinoma in vitro (with IC50 = 3.8 μg/mL). Compounds 1 and 2 exhibited significant antioxidant activity and compound 4 showed a significant immune stimulant activity. Compound 1 is isolated for the first time from genus Alcea and this is the first report for its biological investigation.

Cytotoxic activity evaluation and molecular docking study of phenolic derivatives from Achillea fragrantissima (Forssk.) growing in Egypt

Achillea fragrantissima (Forssk) Sch. Bip. (Asteraceae) is one of the most important medicinal plant species in Egypt. Phytochemical investigation of the methanolic extract of A. fragrantissima led to the isolation of two phenolic compounds Piceol (1) and Veratric acid (2), which are reported for the first time from this plant. In addition, four known flavonoid compounds; Eupatilin 7-methyl ether (3), Chrysosplenol D (4), Cirsiliol (5), and Cirsimaritin (6) were isolated. Structure elucidation was achieved using spectroscopic techniques, including one-dimensional and two-dimensional nuclear magnetic resonance. Potential cytotoxic activities of the isolated compounds were measured by the sulphorhodamine B assay. Compounds 1, 2, 3, 4, 5, and 6 displayed cytotoxic activity against (MCF7) with IC50 values of 18.2, 15.7, 9.5, 8.33, 3.23, and 3.83 µg/ml, respectively, and against (HepG2) with IC50 values of 19.4, 41.2, 28.3, 20.8, 23.8, and 23.8 µg/ml, respectively. Also there was moderate cytotoxic activity against (A549) with IC50 values of 17.8, 13.6, 3.98, and 10.3 µg/ml for the compounds 1, 2, 4 and 5, respectively, and against (HeLa) with IC50 values of 10.1, 10.4, 4.88, and 3.98 µg/ml for the compounds 3, 4, 5, and 6, respectively. In addition, compounds 4 and 5 showed cytotoxic activity against (PC3) with IC50 values of 3.83 and 3.98 µg/ml, respectively. An in silico study was performed, where cirsiliol (5) and piceol (1) were docked into the active sites of the crystal structures of PI3K and Akt, two protein kinases which are involved in prostate and breast cancer proliferation and survival.