Validation and Quantitation of Fifteen Cannabinoids in Cannabis and Marketed Products Using High-Performance Liquid Chromatography-Ultraviolet/Photodiode Array Method.

Background: Cannabis sativa is a psychoactive plant indigenous to Central and South Asia, traditionally used both for recreational and religious purposes, in addition to folk medicine. Cannabis is a rich source of natural compounds, the most important of which are commonly known as cannabinoids that cause a variety of effects through interaction with the endocannabinoid system. Materials and Methods: In this study, a high-performance liquid chromatography-ultraviolet/photodiode array (PDA) method was developed and validated for the analysis of 15 cannabinoids in cannabis plant materials and cannabis-based marketed products. These cannabinoids are cannabidivarinic acid, cannabidivarin, cannabidiolic acid, cannabigerolic acid, cannabigerol, cannabidiol, delta-9-tetrahydrocannabivarin, delta-9-tetrahydrocannabivarinic acid, cannabinol, delta-9-tetrahyrocannabinol, delta-8-tetrahyrocannabinol, cannabicyclol, cannabichromene, delta-9-tetrahyrocannabinolic acid A, and cannabichromenic acid. The separation was carried out using a reversed-phase Luna® C18(2) column and a mobile phase consisting of 75% acetonitrile and 0.1% formic acid in water. A PDA detector was used, and data were extracted at λ=220 nm. Principal component analysis of cannabis four varieties was performed. Results: The method was linear over the calibration range of 5-75 µg/mL with R2>0.999 for all cannabinoids. This method was sensitive and gave good baseline separation of all examined cannabinoids with limits of detection ranging between 0.2 and 1.6 µg/mL and limits of quantification ranging between 0.6 and 4.8 µg/mL. The average recoveries for all cannabinoids were between 81% and 104%. The measured repeatability and intermediate precisions (% relative standard deviation) in all varieties ranged from 0.35% to 9.84% and 1.11% to 5.26%, respectively. Conclusions: The proposed method is sensitive, selective, reproducible, and accurate. It can be applied for the simultaneous determination of these cannabinoids in the C. sativa biomass and cannabis-derived marketed products.

Bio-guided isolation of potential anti-inflammatory constituents of some endophytes isolated from the leaves of ground cherry (Physalis pruinosa L.) via ex-vivo and in-silico studies.

BACKGROUND: Due to the extensive potential of previously studied endophytes in addition to plants belonging to genus Physalis as a source of anti-inflammatory constituents, the present study aimed at isolation for the first time some endophytic fungi from the medicinal plant Physalis pruinosa. METHODS: The endophytic fungi were isolated from the fresh leaves of P. pruinosa then purified and identified by both morphological and molecular methods. Comparative evaluation of the cytotoxic and ex vivo anti-inflammatory activity in addition to gene expression of the three pro-inflammatory indicators (TNF-α, IL-1ß and INF-γ) was performed in WBCs treated with lipopolysaccharide (LPS) for the identified endophytes, isolated compounds and the standard anti-inflammatory drug (piroxicam). For prediction of the binding mode of the top-scoring constituents-targets complexes, the Schrödinger Maestro 11.8 package (LLC, New York, NY) was employed in the docking study. RESULTS: A total of 50 endophytic fungal isolates were separated from P. pruinosa leaves. Selection of six representative isolates was performed for further bioactivity screening based on their morphological characters, which were then identified as Stemphylium simmonsii MN401378, Stemphylium sp. MT084051, Alternaria infectoria MT573465, Alternaria alternata MZ066724, Alternaria alternata MN615420 and Fusarium equiseti MK968015. It could be observed that A. alternata MN615420 extract was the most potent anti-inflammatory candidate with a significant downregulation of TNF-α. Moreover, six secondary metabolites, alternariol monomethyl ether (1), 3'-hydroxyalternariol monomethyl ether (2), alternariol (3), α-acetylorcinol (4), tenuazonic acid (5) and allo-tenuazonic acid (6) were isolated from the most potent candidate (A. alternata MN615420). Among the tested isolated compounds, 3'-hydroxyalternariol monomethyl ether showed the highest anti-inflammatory potential with the most considerable reductions in the level of INF-γ and IL-1ß. Meanwhile, alternariol monomethyl ether was the most potent TNF-α inhibitor. The energy values for the protein (IL-1ß, TNF-α and INF-γ)-ligand interaction for the best conformation of the isolated compounds were estimated using molecular docking analysis. CONCLUSIONS: The results obtained suggested alternariol derivatives may serve as naturally occurring potent anti-inflammatory candidates. This study opens new avenues for the design and development of innovative anti-inflammatory drugs that specifically target INF-γ, IL-1ß and INF-γ.

Development and Validation of a GC-FID Method for the Quantitation of 20 Different Acidic and Neutral Cannabinoids.

For decades, Cannabis sativa had been illegal to sell or consume around the world, including in the United States. However, in light of the recent 2018 Farm Bill and the legalization of hemp across the US, various cannabis preparations have flooded the market, making it essential to be able to quantitate the levels of the different acidic and neutral cannabinoids in C. sativa and to have a complete cannabinoid profile of the different chemovars of the cannabis plant. A GC-FID method was developed and validated for the analysis of 20 acidic and neutral cannabinoids as trimethylsilyl (TMS) derivatives. The analyzed cannabinoids include cannabidivarinic acid (CBDVA), cannabidiolic acid (CBDA), cannabinolic acid (CBNA), cannabielsoic acid (CBEA), cannabicyclolic acid (CBLA), cannabichromenic acid (CBCA), trans-Δ9-tetrahydrocannabivarinic acid (Δ9-THCVA), trans-Δ9-tetrahydrocannabinolic acid A (Δ9-THCAA), cannabigerolic acid (CBGA), cannabidiol (CBD), cannabicyclol (CBL), cannabidivarin (CBDV), trans-Δ9-tetrahydrocannabivarin (THCV), cannabichromene (CBC), trans-Δ8-tetrahydrocannabinol (Δ8-THC), trans-Δ9-tetrahydrocannabinol (Δ9-THC), cannabigerol (CBG), cannabinol (CBN), cannabicitran (CBT), and cannabielsoin (CBE). The method limit of detection (LOD) was as low as 0.1 µg/mL, while the limit of quantitation ranged from 0.25 µg/mL to 0.5 µg/mL. The precision (%RSD) was < 10%, while trueness ranged from 90 - 107%. The developed method is simple, accurate, and sensitive for the quantitation of all 20 acidic and neutral cannabinoids. Finally, the proposed method was successfully applied to the quantitation of the cannabinoids in different cannabis chemovars grown at the University of Mississippi.

Antitrypanosomal, Antitopoisomerase-I, and Cytotoxic Biological Evaluation of Some African Plants Belonging to Crassulaceae; Chemical Profiling of Extract Using UHPLC/QTOF-MS/MS.

In our continuous study for some African plants as a source for antitrypanosomally and cytotoxic active drugs, nine different plants belonging to the Crassulaceae family have been selected for the present study. Sedum sieboldii leaves extract showed an antitrypanosomal activity against Trypanosoma brucei with an IC50 value of 8.5 µg/mL. In addition, they have cytotoxic activities against (HCT-116), (HEPG-2) and (MCF-7), with IC50 values of 28.18 ± 0.24, 22.05 ± 0.66, and 26.47 ± 0.85 µg/mL, respectively. Furthermore, the extract displayed inhibition against Topoisomerase-1 with an IC50 value of 1.31 µg/mL. It showed the highest phenolics and flavonoids content among the other plants' extracts. In order to identify the secondary metabolites which may be responsible for such activities, profiling of the polar secondary metabolites of S. sieboldii extract via Ultra-Performance Liquid Chromatography coupled to High-Resolution QTOF-MS operated in negative and positive ionization modes, which revealed the presence of 46 metabolites, including flavonoids, phenolic acids, anthocyanidins, coumarin, and other metabolites.

Microbial Biotransformation of Cannabidiol (CBD) from Cannabis sativa.

Microbial biotransformation of cannabidiol was assessed using 31 different microorganisms. Only Mucor ramannianus (ATCC 9628), Beauveria bassiana (ATCC 7195), and Absidia glauca (ATCC 22 752) were able to metabolize cannabidiol. M. ramannianus (ATCC 9628) yielded five metabolites, namely, 7,4″ß-dihydroxycannabidiol (1: ), 6ß,4″ß-dihydroxycannabidiol (2: ), 6ß,2″ß-dihydroxycannabidiol (3: ), 6ß,3″α-dihydroxycannabidiol (4: ), and 6ß,7,4″ß-trihydroxycannabidiol (5: ). B. bassiana (ATCC 7195) metabolized cannabidiol to afford six metabolites identified as 7,3″-dihydroxycannabidivarin (6: ), 7-hydroxycannabidivarin-3″-carboxylic acid (7: ), 3″-hydroxycannabidivarin (8: ), 4″ß-hydroxycannabidiol (9: ), and cannabidivarin-3″-carboxylic acid (10: ) along with compound 1: . Incubation of cannabidiol with A. glauca (ATCC 22 752) yielded three metabolites, 6α,3″-dihyroxycannabidivarin (11: ), 6ß,3″-dihyroxycannabidivarin (12: ), and compound 6: . All compounds were evaluated for their antimicrobial and antiprotozoal activity.

IL-33, IL-37, and Vitamin D Interaction Mediate Immunomodulation of Inflammation in Degenerating Cartilage.

Chronic joint inflammation due to increased secretion of pro-inflammatory cytokines, the accumulation of inflammatory immune cells (mainly macrophages), and vitamin D deficiency leads to cartilage degeneration and the development of osteoarthritis (OA). This study investigated the effect of vitamin D status on the expression of mediators of inflammation including interleukin (IL)-33, IL-37, IL-6, tumor necrosis factor (TNF)-α, toll-like receptors (TLRs), damage-associated molecular patterns (DAMPs), and matrix metalloproteinases (MMPs) in degenerating the cartilage of hyperlipidemic microswine. Additionally, in vitro studies with normal human chondrocytes were conducted to investigate the effect of calcitriol on the expression of IL-33, IL-37, IL-6, TNF-α, TLRs, DAMPs, and MMPs. We also studied the effects of calcitriol on macrophage polarization using THP-1 cells. The results of this study revealed that vitamin D deficiency is associated with an increased expression of IL-33, IL-37, IL-6, TNF-α, TLRs, DAMPs, and MMPs, while vitamin D supplementation is associated with a decreased expression of the former. Additionally, vitamin D deficiency is associated with increased M1, while vitamin D-supplemented microswine cartilage showed increased M2 macrophages. It was also revealed that calcitriol favors M2 macrophage polarization. Taken together, the results of this study suggest that modulating expression of IL-33, IL-6, TNF-α, TLRs, DAMPs, and MMPs with vitamin D supplementation may serve as a novel therapeutic to attenuate inflammation and cartilage degeneration in osteoarthritis.

Cannabinoids, Phenolics, Terpenes and Alkaloids of Cannabis.

Cannabis sativa is one of the oldest medicinal plants in the world. It was introduced into western medicine during the early 19th century. It contains a complex mixture of secondary metabolites, including cannabinoids and non-cannabinoid-type constituents. More than 500 compounds have been reported from C. sativa, of which 125 cannabinoids have been isolated and/or identified as cannabinoids. Cannabinoids are C21 terpeno-phenolic compounds specific to Cannabis. The non-cannabinoid constituents include: non-cannabinoid phenols, flavonoids, terpenes, alkaloids and others. This review discusses the chemistry of the cannabinoids and major non-cannabinoid constituents (terpenes, non-cannabinoid phenolics, and alkaloids) with special emphasis on their chemical structures, methods of isolation, and identification.

Phytochemical and pharmacological appraisal of the aerial parts of Lotus corniculatus L. growing in Egypt.

Lotus corniculatus L. (Fabaceae) is widely grown in Egypt. It has a great history of folkloric medicinal uses. All fractions of aerial parts of L. corniculatus L. showed significant antioxidant and immunostimulant activities and could strongly induce lymphoproliferation. However, the light petrol fraction had antifungal activity against C. neoformans with IC50 value (<8 µg/mL) and exhibited strongest in-vitro antiprotozoal activity against protozoan parasites belonging to the genera Trypanosoma with IC50 value (0.98 µg/mL) and Plasmodium (with 100% inhibition using a sample concentration of 15866.7 ng/mL). This is the first study of the immunostimulant and antiprotozoal activities of genus Lotus. By this approach, it was possible to isolate eight compounds (-)-7,2'-dihydroxy-4'-methoxyisoflavan (vestitol) (1), kaempferol (2), kaempferol 3-O-α-L-rhamnoside (afzelin) (3), kaempferol 3, 7-O-α-L-dirhamnoside (kaempferitin) (4), kaempferol-3-O-[ß-D-xylopyranosyl (1″'→2″)-ß-D-galactopyranoside] (5), 3-O-[ß-D-glucuronopyranosyl] soyasapogenol B (6), kaempferol-3-O-[ß-D-xylopyranosyl (1″'→2″)-ß-D-galactopyranoside]-7-O-α-L-rhamnopyranoside (7) and 3-O-[α-L-rhamnopyranosyl (1″'→2″)-ß-D-galactopyranosyl-(1″→2')-ß-D-glucuronopyranosyl] soyasapogenol B (soyasaponin І) (8).

Chemical constituents, Antibacterial and Acetylcholine esterase inhibitory activity of Cupressus macrocarpa leaves.

The chemical constituents of Cupressus macrocarpa were investigated. A new neolignan glycoside (1) in addition to nine known compounds were isolated. The acetylcholinesterase (AChE) inhibitory activity and antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) of different fractions and isolates of C. macrocarpa were evaluated. The light petroleum fraction showed the highest activity in both assays with IC50 value of 88.79 µg/ml and 152.58 µg/ml for the AChE inhibitory activity and MRSA antibacterial activities, respectively. Weak to moderate activity were detected for the isolated compounds.

Analysis of Terpenes in Cannabis sativa L. Using GC/MS: Method Development, Validation, and Application.

Terpenes are the major components of the essential oils present in various Cannabis sativa L. varieties. These compounds are responsible for the distinctive aromas and flavors. Besides the quantification of the cannabinoids, determination of the terpenes in C. sativa strains could be of importance for the plant selection process. At the University of Mississippi, a GC-MS method has been developed and validated for the quantification of terpenes in cannabis plant material, viz., α-pinene, ß-pinene, ß-myrcene, limonene, terpinolene, linalool, α-terpineol, ß-caryophyllene, α-humulene, and caryophyllene oxide. The method was optimized and fully validated according to AOAC (Association of Official Analytical Chemists) guidelines against reference standards of selected terpenes. Samples were prepared by extraction of the plant material with ethyl acetate containing n-tridecane solution (100 µg/mL) as the internal standard. The concentration-response relationship for all analyzed terpenes using the developed method was linear with r2 values > 0.99. The average recoveries for all terpenes in spiked indoor cultivated samples were between 95.0 - 105.7%, with the exception of terpinolene (67 - 70%). The measured repeatability and intermediate precisions (% relative standard deviation) in all varieties ranged from 0.32 to 8.47%. The limit of detection and limit of quantitation for all targeted terpenes were determined to be 0.25 and 0.75 µg/mL, respectively. The proposed method is highly selective, reliable, and accurate and has been applied to the simultaneous determination of these major terpenes in the C. sativa biomass produced by our facility at the University of Mississippi as well as in confiscated marijuana samples.

Vitamin D and macrophage polarization in epicardial adipose tissue of atherosclerotic swine.

Vitamin D functions as a potent immunomodulator by interacting with many immune cells however, its role in regulating inflammation in the epicardial adipose tissue (EAT) is unclear. In the EAT of atherosclerotic microswine that were fed with deficient, sufficient or supplemented levels of vitamin D, we evaluated the phenotype of the macrophages. Vitamin D treatment was continued for 12 months and serum 25(OH)D levels were measured regularly. Infiltration of M1/M2 macrophage was investigated by immunostaining for CCR7 and CD206, respectively in conjunction with a pan macrophage marker CD14. Significant difference in the number of CCR7+ cells was observed in the EAT from vitamin D-deficient swine compared to vitamin D-sufficient or -supplemented swine. Expression of CD206 correlated with high levels of serum 25(OH)D indicating a significant increase in M2 macrophages in the EAT of vitamin D-supplemented compared to -deficient swine. These findings suggest that vitamin D-deficiency exacerbates inflammation by increasing pro-inflammatory M1 macrophages, while vitamin D-supplementation attenuates the inflammatory cytokines and promotes M2 macrophages in EAT. This study demonstrates the significance of vitamin D mediated inhibition of macrophage mediated inflammation in the EAT during coronary intervention in addition to its immunomodulatory role. However, additional studies are required to identify the cellular mechanisms that transduce signals between macrophages and smooth muscle cells during restenosis in the presence and absence of vitamin D.

Bioactivity-Guided Isolation of Potential Antidiabetic and Antihyperlipidemic Compounds from Trigonella stellata.

The in vitro antidiabetic and antihyperlipidemic activities of an alcoholic extract of Trigonella stellata were evaluated in terms of the activation of PPARα and PPARγ in human hepatoma (HepG2) cells. The extract was investigated phytochemically, aiming at the isolation of the most active compounds to be used as a platform for drug discovery. Three new isoflavans, (3 S,4 R)-4,2',4'-trihydroxy)-7-methoxyisoflavan (1), (3 R,4 S)-4,2',4'-trihydroxy-7-methoxy-4'- O-ß-d-glucopyranosylisoflavan (2), and (2 S,3 R,4 R)-4,2',4'-trihydroxy-2,7-dimethoxyisoflavan (3), were isolated and characterized along with the five known compounds p-hydroxybenzoic acid (4), 7,4'-dihydroxyflavone (5), dihydromelilotoside (6), quercetin-3,7- O-α-l-dirhamnoside (7), and soyasaponin I (8). The structures of 1-3 were elucidated using various spectroscopic techniques including HRESIMS and 1D and 2D NMR. The absolute stereochemistry of the new isoflavans (1-3) was determined using both experimental and calculated electronic circular dichroism as well as DP4 calculations. The isolated compounds were tested for their PPARα and PPARγ activation effects in HepG2 cells.

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

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-ß-d-glucopyranoside (1), dihydrokaempferol (2), kaempferol-3-O-[6″-(E-coumaroyl)]-ß-d-glucopyranoside (3), kaempferol-3-O-ß-d-glucopyranoside (4), Apigenin (5) and kaempferol-3-O-α-l-rhamnopyranosyl-(1'″→6″)-ß-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.

Determination of Acid and Neutral Cannabinoids in Extracts of Different Strains of Cannabis sativa Using GC-FID.

Cannabis (Cannabis sativa L.) is an annual herbaceous plant that belongs to the family Cannabaceae. Trans-Δ9-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD) are the two major phytocannabinoids accounting for over 40% of the cannabis plant extracts, depending on the variety. At the University of Mississippi, different strains of C. sativa, with different concentration ratios of CBD and Δ9-THC, have been tissue cultured via micropropagation and cultivated. A GC-FID method has been developed and validated for the qualitative and quantitative analysis of acid and neutral cannabinoids in C. sativa extracts. The method involves trimethyl silyl derivatization of the extracts. These cannabinoids include tetrahydrocannabivarian, CBD, cannabichromene, trans-Δ8-tetrahydrocannabinol, Δ9-THC, cannabigerol, cannabinol, cannabidiolic acid, cannabigerolic acid, and Δ9-tetrahydrocannabinolic acid-A. The concentration-response relationship of the method indicated a linear relationship between the concentration and peak area ratio with R2 > 0.999 for all 10 cannabinoids. The precision and accuracy of the method were found to be ≤ 15% and ± 5%, respectively. The limit of detection range was 0.11 - 0.19 µg/mL, and the limit of quantitation was 0.34 - 0.56 µg/mL for all 10 cannabinoids. The developed method is simple, sensitive, reproducible, and suitable for the detection and quantitation of acidic and neutral cannabinoids in different extracts of cannabis varieties. The method was applied to the analysis of these cannabinoids in different parts of the micropropagated cannabis plants (buds, leaves, roots, and stems).

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.

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.

Cornigerin, a new sesqui-lignan from the hepatoprotective fractions of Cynara cornigera L.

The ethanol extract of Cynara cornigera L. was fractionated and the fractions were subjected to hepatoprotective assays using Wistar albino rats at a dose of 500 and 250mg/kg. The liver injury was induced in rats using carbon tetrachloride. Biochemical parameters such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP) and total bilirubin were estimated as reflections of the liver condition, with silymarin as a positive control. Phytochemical investigation and chromatographic separation of the hepatoprotective fractions led to the isolation of a new sesqui-lignan namely cornigerin (1), along with eight known compounds: apigenin (2), luteolin (3), ß-sitosterol glycoside (4), apigenin 7-O-ß-D-glucopyranoside (5), luteolin-7-O-ß-D-glucopyranoside (6), apigenin-7-O-rutinoside (7), cynarin 1,5-di-O-caffeoylquinic acid (8), and apigenin-7-O-ß-D-glucuronide (9). This is the first report for the isolation of 8 and 9 from this plant.

Vitamin D attenuates inflammation, fatty infiltration, and cartilage loss in the knee of hyperlipidemic microswine.

BACKGROUND: Osteoarthritis (OA) of the knee joint is a degenerative process resulting in cartilage loss. Recent evidence suggests that OA is not merely a disease of cartilage but a disease of the entire knee joint and that inflammation may play an important role. OA has been associated with vitamin D deficiency. Vitamin D as an immunomodulator and anti-inflammatory agent may attenuate inflammation in the knee. The aim of this study was to assess the anti-inflammatory effect of vitamin D on inflammation in the knee. METHODS: This study was conducted with 13 microswine on a high cholesterol diet categorized into three groups of vitamin D-deficient, vitamin D-sufficient, and vitamin D supplementation. After 1 year, microswine were killed, and their knee joint tissues were harvested. Histological and immunofluorescence studies were carried out on the tissue specimens to evaluate the effect of vitamin D status. RESULTS: Histological and immunofluorescence studies of the knee joint tissues showed (1) increased inflammation in the knee joint tissues, (2) fatty infiltration in quadriceps muscle, patellar tendon, and collateral ligaments, and (3) chondrocyte clustering in the vitamin D-deficient and vitamin D-sufficient groups compared with the vitamin D supplementation group. Architectural distortion of the quadriceps muscle, patellar tendon, and collateral ligaments was also seen in the areas of inflammatory foci and fatty infiltration in the vitamin D-deficient group. CONCLUSIONS: Decreased inflammation and fatty infiltration in the vitamin D supplementation group suggest the potential role of vitamin D in attenuating inflammation and fatty infiltration as well as in protecting the architecture of the tissue in the knee joint.

Vitamin D Deficiency Accelerates Coronary Artery Disease Progression in Swine.

OBJECTIVE: The role of vitamin D deficiency in coronary artery disease (CAD) progression is uncertain. Chronic inflammation in epicardial adipose tissue (EAT) has been implicated in the pathogenesis of CAD. However, the molecular mechanism underlying vitamin D deficiency-enhanced inflammation in the EAT of diseased coronary arteries remains unknown. We examined a mechanistic link between 1,25-dihydroxyvitamin D-mediated suppression of nuclear factor-κB (NF-κB) transporter, karyopherin α4 (KPNA4) expression and NF-κB activation in preadipocytes. Furthermore, we determined whether vitamin D deficiency accelerates CAD progression by increasing KPNA4 and nuclear NF-κB levels in EAT. APPROACH AND RESULTS: Nuclear protein levels were detected by immunofluorescence and Western blot. Exogenous KPNA4 was transported into cells by a transfection approach and constituted lentiviral vector. Swine were administered vitamin D-deficient or vitamin D-sufficient hypercholesterolemic diet. After 1 year, the histopathology of coronary arteries and nuclear protein expression of EAT were assessed. 1,25-dihydroxyvitamin D inhibited NF-κB activation and reduced KPNA4 levels through increased vitamin D receptor expression. Exogenous KPNA4 rescued 1,25-dihydroxyvitamin D-dependent suppression of NF-κB nuclear translocation and activation. Vitamin D deficiency caused extensive CAD progression and advanced atherosclerotic plaques, which are linked to increased KPNA4 and nuclear NF-κB levels in the EAT. CONCLUSIONS: 1,25-dihydroxyvitamin D attenuates NF-κB activation by targeting KPNA4. Vitamin D deficiency accelerates CAD progression at least, in part, through enhanced chronic inflammation of EAT by upregulation of KPNA4, which enhances NF-κB activation. These novel findings provide mechanistic evidence that vitamin D supplementation could be beneficial for the prevention and treatment of CAD.

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.