In vivo profiling of phytocannabinoids in Cannabis spp. varieties via SPME-LC-MS analysis.

BACKGROUND: In vivo solid-phase microextraction (SPME) is a minimally invasive, non-exhaustive sample-preparation technique that facilitates the direct isolation of low molecular weight compounds from biological matrices in living systems. This technique is especially useful for the analysis of phytocannabinoids (PCs) in plant material, both for forensic purposes and for monitoring the PC content in growing Cannabis spp. plants. In contrast to traditional extraction techniques, in vivo SPME enables continuous tracking of the changes in the level of PCs during plant growth without the need for plant material collection. In this study, in vivo SPME utilizing biocompatible C18 probes and liquid-chromatography coupled to quadrupole time-of flight mass spectrometry (LC-Q-TOF-MS) is proposed as a novel strategy for the extraction and analysis of the acidic forms of five PCs in growing medicinal cannabis plants. RESULTS: The SPME method was optimized by testing various parameters, including the extraction phase (coating), extraction and desorption times, and the extraction temperature. The proposed method was validated with satisfactory analytical performance regarding linearity (10-3000 ng/mL), limits of quantification, and precision (relative standard deviations below 5.5 %). The proposed method was then successfully applied for the isolation of five acidic forms of PCs, which are main components of growing medicinal cannabis plants. As a proof-of-concept, SPME probes were statically inserted into the inflorescences of two varieties of Cannabis spp. plants (i.e., CBD-dominant and Δ9-THC-dominant) cultivated under controlled conditions for 30 min extraction of tetrahydrocannabinolic acid (Δ9-THCA), cannabidiolic acid (CBDA), cannabigerolic acid (CBGA), cannabiviarinic acid (CBVA), and tetrahydrocannabivarinic acid (THCVA). SIGNIFICANCE AND NOVELTY: The results confirmed that the developed SPME-LC-Q-TOF-MS method is a precise and efficient tool that enables direct and rapid isolation and analysis of PCs under in vivo conditions. The proposed methodology is highly appealing option for monitoring the metabolic pathways and compositions of multiple PCs in medicinal cannabis at different stages of plant growth.

Sex Determination of Human Nails Based on Attenuated Total Reflection Fourier Transform Infrared Spectroscopy in Forensic Context.

This study reports on the successful use of a machine learning approach using attenuated total reflectance Fourier transform infrared (ATR FT-IR) spectroscopy for the classification and prediction of a donor's sex from the fingernails of 63 individuals. A significant advantage of ATR FT-IR is its ability to provide a specific spectral signature for different samples based on their biochemical composition. The infrared spectrum reveals unique vibrational features of a sample based on the different absorption frequencies of the individual functional groups. This technique is fast, simple, non-destructive, and requires only small quantities of measured material with minimal-to-no sample preparation. However, advanced multivariate techniques are needed to elucidate multiplex spectral information and the small differences caused by donor characteristics. We developed an analytical method using ATR FT-IR spectroscopy advanced with machine learning (ML) based on 63 donors' fingernails (37 males, 26 females). The PLS-DA and ANN models were established, and their generalization abilities were compared. Here, the PLS scores from the PLS-DA model were used for an artificial neural network (ANN) to create a classification model. The proposed ANN model showed a greater potential for predictions, and it was validated against an independent dataset, which resulted in 92% correctly classified spectra. The results of the study are quite impressive, with 100% accuracy achieved in correctly classifying donors as either male or female at the donor level. Here, we underscore the potential of ML algorithms to leverage the selectivity of ATR FT-IR spectroscopy and produce predictions along with information about the level of certainty in a scientifically defensible manner. This proof-of-concept study demonstrates the value of ATR FT-IR spectroscopy as a forensic tool to discriminate between male and female donors, which is significant for forensic applications.

The Main Therapeutic Applications of Cannabidiol (CBD) and Its Potential Effects on Aging with Respect to Alzheimer's Disease.

The use of cannabinoids (substances contained specifically in hemp plants) for therapeutic purposes has received increased attention in recent years. Presently, attention is paid to two main cannabinoids: delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD). With respect to the psychotropic effects and dependence potential of THC (though it is very mild), its use is associated with certain restrictions, and thus the therapeutic properties of CBD are frequently emphasized because there are no limitations associated with the risk of dependence. Therefore, this review covers the main pharmacodynamic and pharmacokinetic features of CBD (including characteristics of endocannabinoidome) with respect to its possible beneficial effects on selected diseases in clinical practice. A substantial part of the text deals with the main effects of CBD on aging, including Alzheimer's disease and related underlying mechanisms.

Phytocannabinoid-rich galenic preparations for topical administration: extraction and stability testing.

Although medical cannabis was legalized in Czechia in 2013 and its use in topical treatments of skin disorders is now allowed, galenic formulations prepared from medical cannabis have not been widely implemented in the Czech healthcare system. One of the main reasons is the lack of a straightforward standardized protocol for their preparation. Cannabinoids, e.g., cannabidiol (CBD) and tetrahydrocannabinol (THC), have been shown to have therapeutic effects on various skin conditions, such as atopic dermatitis, psoriasis, scleroderma, acne and skin pigmentation. Recognizing the potential of dermatological treatment with medical cannabis, the present study aimed to evaluate the extraction capacity of various pharmaceutical bases for cannabinoids and the stability of prepared galenic formulations for dermatological applications with respect to cannabinoid content. The results showed that the stability of cannabinoids in formulations depended on the bases' physical and chemical properties. The highest THC decomposition was observed in cream bases and Vaseline, with estimated percentage loss of total content of up to 5.4% and 5.6% per week, respectively. In contrast, CBD was more stable than THC. Overall, the tested bases were comparably effective in extracting cannabinoids from plant material. However, olive oil and Synderman bases exhibited the highest cannabinoid extraction efficiencies (approximately 70%) and the best storage stabilities in terms of the content of monitored compounds. The proposed preparation protocol is fast and easily implementable in pharmacies and medical facilities.

Attenuated Total Reflection Fourier Transform Infrared Spectroscopy for Forensic Screening of Long-Term Alcohol Consumption from Human Nails.

Fourier transform infrared (FT-IR) spectroscopy is used throughout forensic laboratories for many applications. FT-IR spectroscopy can be useful with ATR accessories in forensic analysis for several reasons. It provides excellent data quality combined with high reproducibility, with minimal user-induced variations and no sample preparation. Spectra from heterogeneous biological systems, including the integumentary system, can be associated with hundreds or thousands of biomolecules. The nail matrix of keratin possesses a complicated structure with captured circulating metabolites whose presence may vary in space and time depending on context and history. We developed a new approach by using machine-learning (ML) tools to leverage the potential and enhance the selectivity of the instrument, create classification models, and provide invaluable information saved in human nails with statistical confidence. Here, we report chemometric analysis of ATR FT-IR spectra for the classification and prediction of long-term alcohol consumption from nail clippings in 63 donors. A partial least squares discriminant analysis (PLS-DA) was used to create a classification model that was validated against an independent data set which resulted in 91% correctly classified spectra. However, when considering the prediction results at the donor level, 100% accuracy was achieved, and all donors were correctly classified. To the best of our knowledge, this proof-of-concept study demonstrates for the first time the ability of ATR FT-IR spectroscopy to discriminate donors who do not drink alcohol from those who drink alcohol on a regular basis.

Cannabidiol and the possibilities of its use in veterinary medicine of dogs and horses: A brief review.

In connection with the use of cannabinoids for therapeutic purposes in human medicine, there is increased attention for their use in veterinary medicine, particularly by the owners of companion animals and horses. Therefore, veterinarians are expected to face this interest and have the corresponding knowledge on these substances. Presently, it is not possible to use medical marijuana (in terms of the dried cannabis flowers) for veterinary purposes in many countries, but there is increasing evidence that isolated cannabinoids also have beneficial effects (namely cannabidiol - CBD). Thus, this review summarises the possible therapeutic implications of CBD within the scope of evidence-based medicine, particularly in dogs and horses in association with the treatment of pain, epilepsy and anxiety in order to provide veterinarians with a concise overview of scientific findings in this field.

Effects of copper and arsenic stress on the development of Norway spruce somatic embryos and their visualization with the environmental scanning electron microscope.

Somatic embryogenesis is an important biotechnological technique which can be used in studies associated with environmental stress. Four embryogenic cell lines of Norway spruce were grown on media enriched with copper and arsenic in concentration ranges 50-500 µM and 10-50 µM, respectively. The effects were observed during subsequent stages of somatic embryogenesis, the characteristics evaluated being proliferation potential, average number of somatic embryos obtained per g/fresh weight, morphology of developed somatic embryos, metal uptake, and microanalysis of macro- and micronutrients uptake. Copper and arsenic at higher concentrations significantly reduced the growth of early somatic embryos. In almost all treatments, the cell line V-1-3 showed the best performance compared with the other lines tested. Environmental scanning electron microscopy was used to visualize and identify morphological abnormalities in the development of somatic embryos. Abnormalities observed were classified into several categories: meristemless somatic embryos, somatic embryos with disrupted meristem, reduced number of cotyledons, single cotyledon and fused cotyledons. With the application of a low temperature method for the environmental scanning electron microscope, samples were stabilized and whole meristems could be investigated in their native state. As far as we are aware, this is the first report of the effect of copper and arsenic during the process of somatic embryogenesis and the first to evaluate the content of macro and micronutrients uptake in Norway spruce.

Identification of Alleles of Puroindoline Genes and Their Effect on Wheat (Triticum aestivum L.) Grain Texture.

Grain hardness is one of the most important quality characteristics of wheat (Triticum aestivum L.). It is a significant property of wheat grains and relates to milling quality and end product quality. Grain hardness is caused by the presence of puroindoline genes (Pina and Pinb). A collection of 25 genotypes of wheat with unusual grain colour (blue aleurone, purple and white pericarp, yellow endosperm) was studied by polymerase chain reaction (PCR) for the diversity within Pina and Pinb (alleles: Pina-D1a, Pina-D1b, Pinb-D1a, Pinb- -D1b, Pinb-D1c and Pinb-D1d). The endosperm structure was determined by a non-destructive method using light transflectance meter and grain hardness by a texture analyser. Genotype Novosibirskaya 67 and isogenic ANK lines revealed hitherto unknown alleles at the locus for the annealing of primers of Pinb-D1. Allele Pinb-D1c was found to be absent from each genotype. The mealy endosperm ranged from 0 to 100% and grain hardness from 15.10 to 26.87 N per sample.