Novel fluorescence spectroscopy method coupled with N-PLS-R and PLS-DA models for the quantification of cannabinoids and the classification of cannabis cultivars.

INTRODUCTION: Cannabis sativa L. inflorescences are rich in secondary metabolites, particularly cannabinoids. The most common techniques for elucidating cannabinoid composition are expensive technologies, such as high-pressure liquid chromatography (HPLC). OBJECTIVES: We aimed to develop and evaluate the performance of a novel fluorescence spectroscopy-based method coupled with N-way partial least squares regression (N-PLS-R) and partial least squares discriminant analysis (PLS-DA) models to replace the expensive chromatographic methods for preharvest cannabinoid quantification. METHODOLOGY: Fresh medicinal cannabis inflorescences were collected and ethanol extracts were prepared. Their excitation-emission spectra were measured using fluorescence spectroscopy and their cannabinoid contents were determined by HPLC-PDA. Subsequently, N-PLS-R and PLS-DA models were applied to the excitation-emission matrices (EEMs) for cannabinoid concentration prediction and cultivar classification, respectively. RESULTS: The N-PLS-R model was based on a set of EEMs (n = 82) and provided good to excellent quantification of (-)-Δ9-trans-tetrahydrocannabinolic acid, cannabidiolic acid, cannabigerolic acid, cannabichromenic acid, and (-)-Δ9-trans-tetrahydrocannabinol (R2 CV and R2 pred  > 0.75; RPD > 2.3 and RPIQ > 3.5; RMSECV/RMSEC ratio < 1.4). The PLS-DA model enabled a clear distinction between the four major classes studied (sensitivity, specificity, and accuracy of the prediction sets were all ≥0.9). CONCLUSIONS: The fluorescence spectral region (excitation 220-400 nm, emission 280-550 nm) harbors sufficient information for accurate prediction of cannabinoid contents and accurate classification using a relatively small data set.

Hydroponic versus soil-based cultivation of sweet basil: impact on plants' susceptibility to downy mildew and heat stress, storability and total antioxidant capacity.

BACKGROUND: In recent years, hydroponically cultivated basil has gained extensive popularity over soil-based cultivation. Evidence for potential differences between both cultivation methods, in terms of resistance to biotic and abiotic stress factors, storage properties and shelf-life, is still lacking and the potential effect of cultivation method on the antioxidant capacity has not yet been fully explored. This study aimed to determine which of the two basil cultivation methods produces plants that are more resilient to downy mildew and external heat treatment and that exhibit better storage and shelf-life performance. RESULTS: Hydroponically grown basil was significantly more affected by browning than the soil-grown basil at the end of the storage and end of the shelf-life period. Under both cultivation methods, the extent of browning increased significantly between the end of the storage and end of the shelf-life period, by a factor of 1.4. Moreover, hydroponically grown plants were significantly more sensitive to heat treatment than soil-grown basil. However, the soil-grown basil exhibited significantly greater susceptibility to downy mildew than the hydroponically grown basil. At harvest, and at the end of the storage period, the antioxidant capacity of hydroponically cultivated basil was significantly greater than that of soil-grown basil. CONCLUSIONS: Hydroponically cultivated basil exhibited greater resistance to downy mildew, but less resilience to heat and browning during storage and a shelf-life period, resulting in poorer storage and shelf-life performance as compared to soil-cultivated basil. The greater total antioxidant capacity of the hydroponically cultivated basil seems to be the major cause for the observed phenomena. © 2023 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Optimization of sweet basil harvest time and cultivar characterization using near-infrared spectroscopy, liquid and gas chromatography, and chemometric statistical methods.

BACKGROUND: Terpene, eugenol and polyphenolic contents of basil are major determinants of quality, which is affected by genetics, weather, growing practices, pests and diseases. Here, we aimed to develop a simple predictive analytical method for determining the polyphenol, eugenol and terpene content of the leaves of major Israeli sweet basil cultivars grown hydroponically, as a function of harvest time, through the use of near-infrared (NIR) spectroscopy, liquid/gas chromatography, and chemometric methods. We also wanted to identify the harvest time associated with the highest terpene, eugenol and polyphenol content. RESULTS: Six different cultivars and four different harvest times were analyzed. Partial least square regression (PLS-R) analysis yielded an accurate, predictive model that explained more than 93% of the population variance for all of the analyzed compounds. The model yielded good/excellent prediction (R2 > 0.90, R2 cv and R2 pre > 0.80) and very good residual predictive deviation (RPD > 2) for all of the analyzed compounds. Concentrations of rosmarinic acid, eugenol and terpenes increased steadily over the first 3 weeks, peaking in the fourth week in most of the cultivars. Our PLS-discriminant analysis (PLS-DA) model provided accurate harvest classification and prediction as compared to cultivar classification. The sensitivity, specificity and accuracy of harvest classification were larger than 0.82 for all harvest time points, whereas the cultivar classification, resulted in sensitivity values lower than 0.8 in three cultivars. CONCLUSION: The PLS-R model provided good predictions of rosmarinic acid, eugenol and terpene content. Our NIR coupled with a PLS-DA demonstrated reasonable solution for harvest and cultivar classification. © 2021 Society of Chemical Industry.

Comparative Risk Assessment of Three Native Heliotropium Species in Israel.

Pyrrolizidine alkaloids (PAs) are genotoxic carcinogenic phytotoxins mostly prevalent in the Boraginaceae, Asteraceae and Fabaceae families. Heliotropium species (Boraginaceae) are PA-producing weeds, widely distributed in the Mediterranean region, that have been implicated with lethal intoxications in livestock and humans. In Israel, H. europaeum, H. rotundifolium and H. suaveolens are the most prevalent species. The toxicity of PA-producing plants depends on the PA concentration and composition. PAs occur in plants as mixtures of dozens of various PA congeners. Hence, the risk arising from simultaneous exposure to different congeners has to be evaluated. The comparative risk evaluation of the three Heliotropium species was based on recently proposed interim relative potency (iREP) factors, which take into account certain structural features as well as in vitro and in vivo toxicity data obtained for several PAs of different classes. The aim of the present study was to determine the PA profile of the major organ parts of H. europaeum, H. rotundifolium and H. suaveolens in order to assess the plants' relative toxic potential by utilizing the iREP concept. In total, 31 different PAs were found, among which 20 PAs were described for the first time for H. rotundifolium and H. suaveolens. The most prominent PAs were heliotrine-N-oxide, europine-N-oxide and lasiocarpine-N-oxide. Europine-N-oxide displayed significant differences among the three species. The PA levels ranged between 0.5 and 5% of the dry weight. The flowers of the three species were rich in PAs, while the PA content in the root and flowers of H. europaeum was higher than that of the other species. H. europaeum was found to pose a higher risk to mammals than H. rotundifolium, whereas no differences were found between H. europaeum and H. suaveolens as well as H. suaveolens and H. rotundifolium.

Pharmacokinetic and pharmacodynamic evaluation of 5-methoxy-2-aminoindane (MEAI): A new binge-mitigating agent.

5-Methoxy-2-aminoindane (MEAI) is a novel psychoactive aminoindane derivative, exerting euphoric, alcohol-like tipsy experience and reduced desire to consume alcoholic beverages. Our previous toxicological evaluation of MEAI in rats, clearly indicated MEAI's potential to be further evaluated as a promising binge mitigating agent due to its favorable safety profile. In the light of these observations, we have determined MEAI's pharmacokinetic (PK) profile in rats and evaluated in-vitro its pharmacodynamics (PD) profile. Following oral and intravenous administration of MEAI, two metabolites were identified, namely, N-acetyl-MEAI and 5-hydroxy-N-acetyl-AI, arising from N-acetylation and oxidative demethylation. The PK-parameters of MEAI and N-acetyl-MEAI were derived from single i.v. bolus (10 mg/kg) and single oral doses (10 and 90 mg/kg) of MEAI to rats. MEAI displayed extensive total clearance (2.8 L/h/kg) and a very short plasma and brain half-life (0.5-0.7 h). At 10 mg/kg, MEAI displayed low oral bioavailability (25%) and a plasma to brain ratio in the range of 3-5.5, with brain MEAI peak levels attained rapidly. Non-linear pharmacokinetic behavior was observed in the 90 mg/kg oral group, in which the bioavailability increased by 500%. The non-linear behavior was also evident by the significant increase in plasma half-life of MEAI and its metabolite, N-acetyl-MEAI. N-acetyl-MEAI levels in plasma and brain were about ten times lower than the parent compound, indicative of its minor contribution to MEAI's pharmacological effect. MEAI displayed weak to moderate ligand binding inhibition at the 5-HT2B receptor, while the remaining neurochemical targets were unaffected. Further studies, in non-rodent species are required, in-order to assess MEAI's PK and PD profile adequately.

Toxicological evaluation of 5-methoxy-2-aminoindane (MEAI): Binge mitigating agent in development.

5-Methoxy-2-aminoindane (MEAI) is a psychoactive compound of the aminoindane class, which in recent years has been recreationally used by many people, who reported of a mild euphoric, alcohol-like tipsy experience and reduced desire to consume alcoholic beverages. In the light of these observations it was decided to progress MEAI through a preliminary drug development route and evaluate the acute and subacute toxicity of MEAI administrated orally to Sprague Dawley rats, as well as to determine potential in-vitro cytotoxic and mutagenic effects using state-of-the-art protocols. Furthermore, the interaction of MEAI at the highest non-toxic concentration (100mg/L) with ethanol at cytotoxic levels of 6% and 7.5% was explored, in order to identify possible additive or synergistic effects. MEAI showed a good safety profile in rats at 10 and 30mg/kg body weight, corresponding to the human doses of 1.6mg/kg and 4.8mg/kg body weight, respectively. Cytotoxic effect was demonstrated using concentrations of 500 and 1000mg/L with calculated IC50 value of 368.2mg/L for rat brain striatum primary neurons and 403.1mg/L for human primary healthy hepatocytes. The combination of 6% or 7.5% ethanol with 100mg/L MEAI revealed no statistically significant increase of cytotoxic effect. Further studies, especially long term chronic and addictive behavior studies, are required in-order to assess MEAI safety profile.

Detection of Pyrrolizidine Alkaloid DNA Adducts in Livers of Cattle Poisoned with Heliotropium europaeum.

Pyrrolizidine alkaloids are among the most common poisonous plants affecting livestock, wildlife, and humans. Exposure of humans and livestock to toxic pyrrolizidine alkaloids through the intake of contaminated food and feed may result in poisoning, leading to devastating epidemics. During February 2014, 73 mixed breed female beef cows from the Galilee region of Israel were accidently fed pyrrolizidine alkaloid contaminated hay for 42 days, resulting in the sudden death of 24 cows over a period of 63 days. The remaining cows were slaughtered 2.5 months after the last ingestion of the contaminated hay. In this study, we report the histopathological analysis of the livers from five of the slaughtered cows and quantitation of pyrrolizidine alkaloid-derived DNA adducts from their livers and three livers of control cows fed with feed free of weeds producing pyrrolizidine alkaloids. Histopathological examination revealed that the five cows suffered from varying degrees of bile duct proliferation, fibrosis, and megalocytosis. Selected reaction monitoring HPLC-ES-MS/MS analysis indicated that (±)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP)-derived DNA adducts were formed in all five livers. The livers from the three control cows did not have any liver damage nor any indication of DHP-DNA adduct formed. These results confirm that the toxicity observed in these cattle was caused by pyrrolizidine alkaloid poisoning and that pyrrolizidine alkaloid-derived DNA adducts could still be detected and quantified in the livers of the chronically poisoned cows 2.5 months after their last exposure to the contaminated feed, suggesting that DHP-derived DNA adducts can serve as biomarkers for pyrrolizidine alkaloid exposure and poisoning.

The new Israeli feed safety law: challenges in relation to animal and public health.

The Israeli feed safety legislation, which came to prominence in the early 1970s, has undergone a major change from simple feed safety and quality regulations to a more holistic concept of control of feed safety and quality throughout the whole feed production chain, from farm to the end user table. In February 2014, a new law was approved by the Israeli parliament, namely the Control of Animal Feed Law, which is expected to enter into effect in 2017. The law is intended to regulate the production and marketing of animal feed, guaranteeing the safety and quality of animal products throughout the production chain. The responsibility on the implementation of the new feed law was moved from the Plant Protection Inspection Service to the Veterinary Services and Animal Health. In preparation for the law's implementation, we have characterized the various sources and production lines of feed for farm and domestic animals in Israel and assessed the current feed safety challenges in terms of potential hazards or undesirable substances. Moreover, the basic requirements for feed safety laboratories, which are mandatory for analyzing and testing for potential contaminants, are summarized for each of the contaminants discussed. © 2016 Society of Chemical Industry.

Induction of CYP26A1 by metabolites of retinoic acid: evidence that CYP26A1 is an important enzyme in the elimination of active retinoids.

All-trans-retinoic acid (atRA), the active metabolite of vitamin A, induces gene transcription via binding to nuclear retinoic acid receptors (RARs). The primary hydroxylated metabolites formed from atRA by CYP26A1, and the subsequent metabolite 4-oxo-atRA, bind to RARs and potentially have biologic activity. Hence, CYP26A1, the main atRA hydroxylase, may function either to deplete bioactive retinoids or to form active metabolites. This study aimed to determine the role of CYP26A1 in modulating RAR activation via formation and elimination of active retinoids. After treatment of HepG2 cells with atRA, (4S)-OH-atRA, (4R)-OH-atRA, 4-oxo-atRA, and 18-OH-atRA, mRNAs of CYP26A1 and RARß were increased 300- to 3000-fold, with 4-oxo-atRA and atRA being the most potent inducers. However, >60% of the 4-OH-atRA enantiomers were converted to 4-oxo-atRA in the first 12 hours of treatment, suggesting that the activity of the 4-OH-atRA was due to 4-oxo-atRA. In human hepatocytes, atRA, 4-OH-atRA, and 4-oxo-atRA induced CYP26A1 and 4-oxo-atRA formation was observed from 4-OH-atRA. In HepG2 cells, 4-oxo-atRA formation was observed even in the absence of CYP26A1 activity and this formation was not inhibited by ketoconazole. In human liver microsomes, 4-oxo-atRA formation was supported by NAD(+), suggesting that 4-oxo-atRA formation is mediated by a microsomal alcohol dehydrogenase. Although 4-oxo-atRA was not formed by CYP26A1, it was depleted by CYP26A1 (Km = 63 nM and intrinsic clearance = 90 µl/min per pmol). Similarly, CYP26A1 depleted 18-OH-atRA and the 4-OH-atRA enantiomers. These data support the role of CYP26A1 to clear bioactive retinoids, and suggest that the enzyme forming active 4-oxo-atRA may be important in modulating retinoid action.

Improved Long-Term Preservation of Cannabis Inflorescence by Utilizing Integrated Pre-Harvest Hexanoic Acid Treatment and Optimal Post-Harvest Storage Conditions.

The effort to maintain cannabinoid and terpene levels in harvested medicinal cannabis inflorescence is crucial, as many studies demonstrated a significant concentration decrease in these compounds during the drying, curing, and storage steps. These stages are critical for the preparation and preservation of medicinal cannabis for end-use, and any decline in cannabinoid and terpene content could potentially reduce the therapeutic efficacy of the product. Consequently, in the present study, we determined the efficacy of pre-harvest hexanoic acid treatment alongside four months of post-harvest vacuum storage in prolonging the shelf life of high THCA cannabis inflorescence. Our findings indicate that hexanoic acid treatment led to elevated concentrations of certain cannabinoids and terpenes on the day of harvest and subsequent to the drying and curing processes. Furthermore, the combination of hexanoic acid treatment and vacuum storage yielded the longest shelf life and the highest cannabinoid and mono-terpene content as compared to all other groups studied. Specifically, the major cannabinoid's-(-)-Δ9-trans-tetrahydrocannabinolic acid (THCA)-concentration decreased by 4-23% during the four months of storage with the lowest reduction observed following hexanoic acid pre-harvest treatment and post-harvest vacuum storage. Hexanoic acid spray application displayed a more pronounced impact on mono-terpene preservation than storage under vacuum without hexanoic acid treatment. Conversely, sesqui-terpenes were observed to be less prone to degradation than mono-terpenes over an extended storage duration. In summation, appropriate pre-harvest treatment coupled with optimized storage conditions can significantly extend the shelf life of cannabis inflorescence and preserve high active compound concentration over an extended time period.

In Pursuit of Optimal Quality: Cultivar-Specific Drying Approaches for Medicinal Cannabis.

A limited number of studies have examined how drying conditions affect the cannabinoid and terpene content in cannabis inflorescences. In the present study, we evaluated the potential of controlled atmosphere drying chambers for drying medicinal cannabis inflorescence. Controlled atmosphere drying chambers were found to reduce the drying and curing time by at least 60% compared to traditional drying methods, while preserving the volatile terpene content. On the other hand, inflorescences subjected to traditional drying were highly infested by Alternaria alternata and also revealed low infestation of Botrytis cinerea. In the high-THC chemovar ("240"), controlled N2 and atm drying conditions preserved THCA concentration as compared to the initial time point (t0). On the other hand, in the hybrid chemovar ("Gen12") all of the employed drying conditions preserved THCA and CBDA content. The optimal drying conditions for preserving monoterpenes and sesquiterpenes in both chemovars were C5O5 (5% CO2, 5% O2, and 90% N2) and pure N2, respectively. The results of this study suggest that each chemovar may require tailored drying conditions in order to preserve specific terpenes and cannabinoids. Controlled atmosphere drying chambers could offer a cost-effective, fast, and efficient drying method for preserving cannabinoids and terpenes during the drying process while reducing the risk of mold growth.

Stereoselective formation and metabolism of 4-hydroxy-retinoic Acid enantiomers by cytochrome p450 enzymes.

All-trans-retinoic acid (atRA), the major active metabolite of vitamin A, plays a role in many biological processes, including maintenance of epithelia, immunity, and fertility and regulation of apoptosis and cell differentiation. atRA is metabolized mainly by CYP26A1, but other P450 enzymes such as CYP2C8 and CYP3As also contribute to atRA 4-hydroxylation. Although the primary metabolite of atRA, 4-OH-RA, possesses a chiral center, the stereochemical course of atRA 4-hydroxylation has not been studied previously. (4S)- and (4R)-OH-RA enantiomers were synthesized and separated by chiral column HPLC. CYP26A1 was found to form predominantly (4S)-OH-RA. This stereoselectivity was rationalized via docking of atRA in the active site of a CYP26A1 homology model. The docked structure showed a well defined niche for atRA within the active site and a specific orientation of the ß-ionone ring above the plane of the heme consistent with stereoselective abstraction of the hydrogen atom from the pro-(S)-position. In contrast to CYP26A1, CYP3A4 formed the 4-OH-RA enantiomers in a 1:1 ratio and CYP3A5 preferentially formed (4R)-OH-RA. Interestingly, CYP3A7 and CYP2C8 preferentially formed (4S)-OH-RA from atRA. Both (4S)- and (4R)-OH-RA were substrates of CYP26A1 but (4S)-OH-RA was cleared 3-fold faster than (4R)-OH-RA. In addition, 4-oxo-RA was formed from (4R)-OH-RA but not from (4S)-OH-RA by CYP26A1. Overall, these findings show that (4S)-OH-RA is preferred over (4R)-OH-RA by the enzymes regulating atRA homeostasis. The stereoselectivity observed in CYP26A1 function will aid in better understanding of the active site features of the enzyme and the disposition of biologically active retinoids.

Cuticular fatty acid profile analysis of three Rhipicephalus tick species (Acari: Ixodidae).

Cuticular fatty acids (CFA) are important constituents of the arthropod exoskeleton, serving as structural and defense components, and participating in intra-species communication. Here we describe for the first time a comparative analysis of the CFA profiles of three tick species of the genus Rhipicephalus: R. annulatus, R. bursa and R. sanguineus. CFA profiles were determined for R. bursa and R. sanguineus grown both on rabbit or calf, and for R. annulatus grown on calf. CFA composition was compared for each species before and after ethanol treatment, for different hosts of each species, and between the different species. Our data suggest that adsorption of the host's fatty acids changes the apparent CFA composition. Ethanol treatment efficiently removed the unbound fatty acids from the ticks and revealed the actual composition. Comparison between ticks grown on rabbit versus calf showed significant difference in the relative abundance of fatty acids C14 and 9,12-C18:2 for R. bursa, and a difference in the relative abundance of C14 for R. sanguineus. Comparison of the CFA between the three species revealed significant differences in the abundance of fatty acids C16, 9,12-C18:2, 9-C18:1, C18 and C20. Our results show that while the host had a minor effect on CFA composition within each species, significant differences were observed in the CFA profiles of different species. We suggest that CFA profiles may be used to distinguish between related species. CFA analysis can also be used in studies of communication and defense mechanisms in ticks and other arthropods.

Valproate uncompetitively inhibits arachidonic acid acylation by rat acyl-CoA synthetase 4: relevance to valproate's efficacy against bipolar disorder.

BACKGROUND: The ability of chronic valproate (VPA) to reduce arachidonic acid (AA) turnover in brain phospholipids of unanesthetized rats has been ascribed to its inhibition of acyl-CoA synthetase (Acsl)-mediated activation of AA to AA-CoA. Our aim was to identify a rat Acsl isoenzyme that could be inhibited by VPA in vitro. METHODS: Rat Acsl3-, Acsl6v1- and Acsl6v2-, and Acsl4-flag proteins were expressed in E. coli, and the ability of VPA to inhibit their activation of long-chain fatty acids to acyl-CoA was estimated using Michaelis-Menten kinetics. RESULTS: VPA uncompetitively inhibited Acsl4-mediated conversion of AA and of docosahexaenoic (DHA) but not of palmitic acid to acyl-CoA, but did not affect AA conversion by Acsl3, Acsl6v1 or Acsl6v2. Acsl4-mediated conversion of AA to AA-CoA showed substrate inhibition and had a 10-times higher catalytic efficiency than did conversion of DHA to DHA-CoA. Butyrate, octanoate, or lithium did not inhibit AA activation by Acsl4. CONCLUSIONS: VPA's ability to inhibit Acsl4 activation of AA and of DHA to their respective acyl-CoAs, when related to the higher catalytic efficiency of AA than DHA conversion, may account for VPA's selective reduction of AA turnover in rat brain phospholipids, and contribute to VPA's efficacy against bipolar disorder.

Lithium salts: assessment of their chronic and acute toxicities to honey bees and their anti-Varroa field efficacy.

BACKGROUND: Varroa control is essential for the maintenance of healthy honey bee colonies. Overuse of acaricides has led to the evolution of resistance to those substances. Studies of the short-term acaricidal effects and safety of various lithium (Li) salts recently have been reported. This study examined the long-term in vitro and in vivo bee toxicities, short-term motor toxicity to bees and long-term anti-Varroa field efficacy of several Li salts. RESULTS: In an in vitro chronic-toxicity assay, lithium citrate (18.8 mm) was the most toxic of the examined salts, followed by lithium lactate (29.5 mm), and lithium formate (32.5 mm). In terms of acute locomotor toxicity to bees, all of the Li salts were well-tolerated and none of the treatment groups differed from the negative control group. In an in vitro survival study, all of the Li treatments significantly reduced bee life spans by a factor of 1.8-7.2, as compared to the control. In terms of life expectancy, lithium citrate was the most toxic salt, with no significant differences noted between lithium formate and lithium lactate. In the bee-mortality field study, none of the examined treatments differed from the negative control. Amitraz and lithium formate exhibited similar acaricide effects, which were significantly different from those observed for lithium lactate and the negative control. CONCLUSION: In light of lithium formate's honey bee safety and efficacy as an acaricide, additional sublethal toxicity studies in brood, drones and queens, as well as tests aimed at the optimization of administration frequency are warranted. © 2022 Society of Chemical Industry.

Use of near-infrared spectroscopy for the classification of medicinal cannabis cultivars and the prediction of their cannabinoid and terpene contents.

Cannabis sativa L. is used to treat a wide variety of medical conditions, in light of its beneficial pharmacological properties of its cannabinoids and terpenes. At present, the quantitative chemical analysis of these active compounds is achieved through the use of laborious, expensive, and time-consuming technologies, such as high-pressure liquid-chromatography- photodiode arrays, mass spectrometer detectors (HPLC-PDA or MS), or gas chromatography-mass spectroscopy (GC-MS). Hence, we aimed to develop a simple, accurate, fast, and cheap technique for the quantification of major cannabinoids and terpenes using Fourier transform near infra-red spectroscopy (FT-NIRS). FT-NIRS was coupled with multivariate classification and regression models, namely partial least square-discriminant analysis (PLS-DA) and partial least squares regression (PLS-R) models. The PLS-DA model yielded an absolute major class separation (high-THC, high-CBD, hybrid, and high-CBG) and perfect class prediction. Using only three latent variables (LVs), the cross-validation and prediction model errors indicated a low probability of over-fitting the data. In addition, the PLS-DA model enabled the classification of chemovars with genetic-chemical similarities. The classification of high-THCA chemovars was more sensitive and more specific than the classifications of the remaining chemovars. The prediction of cannabinoid and terpene concentrations by PLS-R yielded 11 robust models with high predictive capabilities (R2CV and R2pred > 0.8, RPD >2.5 and RPIQ >3, RMSECV/RMSEC ratio <1.2) and additional 15 models whose performance was acceptable for initial screening purposes (R2CV > 0.7 and R2pred < 0.8, RPD >2 and RPIQ <3, 1.2 < RMSECV/RMSEC ratio <2). Our results confirm that there is sufficient information in the FT-NIRS to develop cannabinoid and terpene prediction models and major-cultivar classification models.

Multivariate classification of cannabis chemovars based on their terpene and cannabinoid profiles.

Cannabis is used to treat various medical conditions, and lines are commonly classified according to their total concentrations of Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD). Based on their ratio of total THC to total CBD, cannabis cultivars are commonly classified into high-THC, high-CBD, and hybrid classes. While cultivars from the same class have similar compositions of major cannabinoids, their levels of other cannabinoids and their terpene compositions may differ substantially. Therefore, a more comprehensive and accurate classification of medicinal cannabis cultivars, based on a large number of cannabinoids and terpenes is needed. For this purpose, three different chemometric-based classification models were constructed using three sets of chemical profiles. We examined those models to determine which provides the most accurate "chemovar" classification. This was done by analyzing profiles of cannabinoids, terpenes, and the combination of these substances using the partial least square-discriminant analysis multivariate (PLS-DA) technique. The chemical profiles were selected from the three major classes of medicinal cannabis that are most commonly prescribed to patients in Israel: high-THC, high-cannabigerol (CBG), and hybrid. We studied the correlations between cannabinoids and terpenes to identify major bio-indicators representing the plant's terpene and cannabinoid content. All three PLS-DA models provided highly accurate classifications, utilizing six to nine latent variables with an overall accuracy ranging from 2 to 11% CV. The PLS-DA model applied to the combined cannabinoid-and-terpene profile did the best job of differentiating between the chemovars in terms of misclassification error, sensitivity, specificity, and accuracy. The combined cannabinoid-and-terpene PLS-DA profile had cross-validation and prediction misclassification errors of 4% and 0%, respectively. This is the first study to demonstrate the highly accurate classification of samples of medicinal cannabis based on their cannabinoid and terpene profiles, as compared to cannabinoid profiles alone. Furthermore, our correlation analysis indicated that 11 cannabinoids and terpenes might serve as bio-indicators for 32 different active compounds. These findings suggest that the use of multivariate statistics could assist in breeding studies and serve as a tool for minimizing the mislabeling of cannabis inflorescences.

Substrate specificity and ligand interactions of CYP26A1, the human liver retinoic acid hydroxylase.

All-trans-retinoic acid (atRA) is the active metabolite of vitamin A. atRA is also used as a drug, and synthetic atRA analogs and inhibitors of retinoic acid (RA) metabolism have been developed. The hepatic clearance of atRA is mediated primarily by CYP26A1, but design of CYP26A1 inhibitors is hindered by lack of information on CYP26A1 structure and structure-activity relationships of its ligands. The aim of this study was to identify the primary metabolites of atRA formed by CYP26A1 and to characterize the ligand selectivity and ligand interactions of CYP26A1. On the basis of high-resolution tandem mass spectrometry data, four metabolites formed from atRA by CYP26A1 were identified as 4-OH-RA, 4-oxo-RA, 16-OH-RA and 18-OH-RA. 9-cis-RA and 13-cis-RA were also substrates of CYP26A1. Forty-two compounds with diverse structural properties were tested for CYP26A1 inhibition using 9-cis-RA as a probe, and IC(50) values for 10 inhibitors were determined. The imidazole- and triazole-containing inhibitors [S-(R*,R*)]-N-[4-[2-(dimethylamino)-1-(1H-imidazole-1-yl)propyl]-phenyl]2-benzothiazolamine (R116010) and (R)-N-[4-[2-ethyl-1-(1H-1,2,4-triazol-1-yl)butyl]phenyl]-2-benzothiazolamine (R115866) were the most potent inhibitors of CYP26A1 with IC(50) values of 4.3 and 5.1 nM, respectively. Liarozole and ketoconazole were significantly less potent with IC(50) values of 2100 and 550 nM, respectively. The retinoic acid receptor (RAR) γ agonist CD1530 was as potent an inhibitor of CYP26A1 as ketoconazole with an IC(50) of 530 nM, whereas the RARα and RARß agonists tested did not significantly inhibit CYP26A1. The pan-RAR agonist 4-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid and the peroxisome proliferator-activated receptor ligands rosiglitazone and pioglitazone inhibited CYP26A1 with IC(50) values of 3.7, 4.2, and 8.6 µM, respectively. These data demonstrate that CYP26A1 has high ligand selectivity but accepts structurally related nuclear receptor agonists as inhibitors.

Design and pharmacological activity of glycinamide and N-methoxy amide derivatives of analogs and constitutional isomers of valproic acid.

A series of glycinamide conjugates and N-methoxy amide derivatives of valproic acid (VPA) analogs and constitutional isomers were synthesized and evaluated for anticonvulsant activity. Of all compounds synthesized and tested, only N-methoxy-valnoctamide (N-methoxy-VCD) possessed better activity than VPA in the following anticonvulsant tests: maximal electroshock, subcutaneous metrazol, and 6-Hz (32-mA) seizure tests. In mice, the ED(50) values of N-methoxy-VCD were 142 mg/kg (maximal electroshock test), 70 mg/kg (subcutaneous metrazol test), and 35 mg/kg (6-Hz test), and its neurotoxicity TD(50) was 118 mg/kg. In rats, the ED(50) of N-methoxy-VCD in the subcutaneous metrazol test was 36 mg/kg and its protective index (PI=TD(50)/ED(50)) was >5.5. In the rat pilocarpine-induced status epilepticus model, N-methoxy-VCD demonstrated full protection at 200mg/kg, without any neurotoxicity. N-Methoxy-VCD was tested for its ability to induce teratogenicity in a mouse strain susceptible to VPA-induced teratogenicity and was found to be nonteratogenic, although it caused some resorptions. Nevertheless, a safety margin was still maintained between the ED(50) values of N-methoxy-VCD in the mouse subcutaneous metrazol test and the doses that caused the resorptions. On the basis of these results, N-methoxy-VCD is a good candidate for further evaluation as a new anticonvulsant and central nervous system drug.

Depletion kinetics and concentration- and time-dependent toxicity of a tertiary mixture of amitraz and its major hydrolysis products in honeybees.

Although amitraz is one of the acaricides most commonly applied within beehives, to date, its time-dependent oral toxicity in honeybees has not been investigated, due to amitraz's instability in aqueous media. In aqueous media such as honey, amitraz rapidly forms a continuously changing tertiary mixture with two of its major hydrolysis products, DMF and DMPF. The contribution of each hydrolysis product to the overall oral toxicity of this acaricide is not known. Therefore, we aimed to characterize the depletion and formation kinetics of amitraz and its hydrolysis products in 50% sucrose solution provided to caged honeybees, including the calculation of the 50% lethal oral concentration (LC50) of amitraz. We sought to determine the contribution of each component of the mixture to the overall observed toxicity. We also investigated the time- and concentration-dependent toxicity of the amitraz mixture and its hydrolysis products. A novel approach based on the analysis of the areas under the depletion and formation curves of amitraz and its hydrolysis products revealed that DMPF, amitraz and DMF accounted for 92%, 7% and 1% (respectively) of the overall toxicity of the mixture. The chronic oral LC50 of amitraz was 3300 µmol/L, of similar magnitude as that of the non-toxic hydrolysis product DMF. The toxicity of DMPF and the mixture decreased over time; whereas the toxicity of DMF increased over time. Amitraz's instability in aqueous media and the highly toxic profile of DMPF, suggest that DMPF is the actual toxic entity responsible for amitraz's toxicity toward honeybees.