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Getting Weed in Meknes, Morocco – Quick Guide

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Official websites use. Share sensitive information only on official, secure websites. Correspondence: m. This study investigated the molecular, phytochemical, and biological aspects of ten local Moroccan traditional landrace Cannabis seeds. Furthermore, a new sequence was identified by sequencing of the THCA synthase coding gene. These results contribute to a thorough comprehension of Moroccan Cannabis seeds, illuminating their molecular, phytochemical, and biological features. Furthermore, they highlight the seeds as a potential source of nutritious components with antioxidant properties, offering valuable insights for future research. For centuries, Cannabis sativa L. However, the Moroccan government has recently legalized and decriminalized its medical use under the law no. This has prompted an increased interest among scientists and the pharmaceutical industry. Cannabis is a subcosmopolitan dioecious flowering plant belonging to the Canabaceae family \[ 2 \]. The number of species in the genus Cannabis has been controversial. Some authors have proposed Cannabis as a polytype that includes three species namely the Cannabis sativa , C. Indica , and C. Ruderalis \[ 3 \]. Both of these subspecies encompass both wild and domesticated varieties \[ 4 \]. The Cannabis sativa var. Some of these cannabinoids, such as tetrahydrocannabinol THC , are known to have psychotropic activities \[ 5 \]. According to two separate studies realized by Hillig and Clarke, the Moroccan local cultivated variety is considered rare because it has undergone an introgressive hybridization with other varieties of Cannabis plants \[ 6 , 7 \]. Cannabis is currently recognized for its medicinal properties. Since the Neolithic era, hemp has been cultivated for its fibers used in textiles, and its seeds, which are used for food \[ 9 \]. This ratio offers various benefits for cardiovascular health, skin, hormonal balance, and insulin resistance \[ 10 \]. Additionally, Cannabis contain tocopherols, which have both antioxidant and anticancer activities. These seeds also have sufficient and effective amounts of terpenes, cannabinoids, and certain phenolics, including methyl salicylate, which offers various health benefits \[ 11 , 12 \]. The seeds of Cannabis sativa possess a diverse range of properties, making them highly promising for the development of various therapeutic, cosmetic, functional foods, and industrial nutraceuticals products. The seeds of Moroccan Cannabis sativa L. They are also rich in nutrients and contain various bioactive compounds including antioxidants, antimicrobials, and probably cytotoxic and antiviral agents. The primary objective of this study is to conduct a thorough investigation and analysis of the genetic and chemical variations present in a set of Cannabis seeds from the northern Morocco. This will be achieved by employing a combination of the high-performance liquid chromatography-electrospray ionization-full scan mass spectrometry combined with the gas chromatography—tandem mass spectrometry TQ chemical profiling and genomic fingerprinting methodologies. Through the utilization of in vitro and in silico analyses, our primary aim is to elucidate the functional characteristics of these compounds as bioactive constituents in para-pharmaceutical and nutraceutical formulations. Consequently, the findings not only contribute to the progression of our understanding regarding the genomic and phytochemical resources of Moroccan Cannabis , but also provide substantial prospects for future breeding endeavors and the discovery of new bioactive compounds with distinct properties. All standards, reagents, and solvents used in this study were purchased from Sigma-Aldrich Saint-Quentin-Fallavier Cedex, France and were of analytical grade. The Cannabis seed samples were collected from five different regions in the northern of province of Al-Hoceima, Morocco. The collection took place between June and September , after the grain had matured. The data of all the coordinates are listed in Figure 1. A total of ten Cannabis seeds were evaluated. During the harvesting stage, the samples were collected from the farmers. The plant that had been collected was meticulously positioned on Kraft paper, subjected to compression, and subsequently dispatched to the Scientific Institute of Mohammed V University of Rabat, Morocco for the purpose of botanical identification by proficient botanists. Subsequently, the plant specimen was subjected to scanning using the HerbScan system, employing the Epson Expression 10, XL flatbed scanner renowned for its exceptional resolution capabilities of DPI. The scanning procedure was carried out using two standardized dimensions. Ultimately, the botanical specimen underwent a process of thermal drying within a specifically designed apparatus, which serves the purpose of eliminating any potential impurities before it is stored. The genetic identification was conducted in order to discern and differentiate the gathered seed samples. Initially, a quantity of mg of Cannabis seeds samples was subjected to grinding using the Tissulyser II. The process of sequencing was conducted through the amplification of various fragments of the THCA synthase gene, with the objective of assembling the complete coding region of the THCA synthase gene. The PCR amplification procedure was executed in accordance with the established standard protocol. The reaction mixture comprised The rbcl and ITS universal primers, which have been previously referenced, were chosen to amplify the barcode regions. This selection was made in order to assess genetic variation and facilitate the identification and classification of the various Cannabis seed samples available to us. In order to conduct genetic identification, a singular sample was subjected to the utilization of Cannabis -specific primers targeting THCA synthase. Following this, sequence reactions were executed using the Big Dye terminator cycle sequencing kit version 3. The sequencing data obtained from the two markers for each sample were analyzed using the software Unipro Ugene version The phylogenetic analysis was performed using the software MEGA11 version Bootstrap support BS values for individual clades were calculated by running bootstrap replicates of the data. For sample preparation, 0. The mixture was processed under controlled pressure and temperature for 35 min. The reaction mixture was filtered through a 0. A blank was prepared in the same conditions. The H 2 O value was established by calculating the dry weight before and after the lyophilization process at 0. The fat content was determined by Soxhlet extraction with petroleum ether organic solvent, according to AOAC The extraction procedure included a 24 h dynamic maceration at room temperature with seven different organic solvents arranged in ascending order of polarity hexane, ethyl ether, chloroform, acetone, ethanol, methanol, and distilled water in a ratio, respectively. The procedure was carried out with the aid of controlled magnetic stirring. Total polyphenol content was determined by the Folin—Ciocalteu colorimetric method as described by Singleton et al. Then, 2 mL of Na 2 CO 3 solution 7. Each extract 0. After a 30 min incubation at room temperature, absorbance was measured at nm. A blank was prepared under the same conditions, and Quercetin was served as the standard. After centrifugation at rpm for 20 min to obtain a clear solution, absorbance was measured at nm. The total anthocyanin content was assessed using the differential pH method involving the measurement of absorbance at pH values of 1. Specifically, 0. The mixture was stirred and incubated for 15 min, then measured by spectrophotometer at and nm. For the second pH 4. The results are expressed as milligrams of cyanidinglucoside equivalents per gram of extract and calculated according to the following standard equation:. Before injection, the samples underwent filtration using a disposable LC syringe filter disc with a pore size of 0. The mobile phases consisted of A acidified water with citric acid adjusted to an appropriate pH of 3. The analysis was conducted in gradient mode, employing different pillars with suitable polarity for 45 min. The molecules detected in each extract chromatogram were screened against the Nist MS Search 2. This method was designed specifically for Cannabis seed extracts, and it is still undergoing optimization and validation. To convert the fixed compounds in the extracts into low-molecular-weight volatile compounds suitable for gas chromatography analysis, a trimethylsilylation derivatization was performed on all seven extracts. An aliquot of the extract equivalent to one eq. Subsequently, 0. Helium was used as the carrier gas. The pressure was set to This method was specifically developed to detect and quantify even trace amounts of substances. The antioxidant capacity of the seven extracts was evaluated in vitro using three assays. Absorbance was assessed at nm against a blank after a 30 min incubation at room temperature. Methanol replaced the sample for the negative control. The experiments were conducted in triplicate, and the results are presented as the percentage of DPPH inhibition, calculated using the following equation:. The IC 50 was calculated from the graph plotted of inhibition percentage against extract concentration. The absorbance was determined at nm against the blank. The antibacterial activity was first evaluated qualitatively by the disc diffusion method \[ 26 \] to screen and select only the active extracts. The active extracts were studied by the microdilution method at concentration from 0. In addition, and for a complementary purpose, the resazurin test was also realized. The positive controls used in this test are ampicillin and penicillin. The tests were carried out in triplicate. Trypan blue staining Sigma-Aldrich, St. After 72 h of incubation, cytotoxicity was determined by microscopic examination of the cell morphology. All tests were performed in triplicate \[ 27 \]. The examination of cell toxicity and the appearance of cytopathic effect CPE were performed every 24 h \[ 28 \]. The 2D configuration of all molecules identified in Cannabis sativa L. Epik was employed to predict the potential ionization states at a target pH range of 7. To ensure the preservation of specified chiralities, up to the maximum of 32 stereoisomers per ligand were produced. The force field OPLS3 was employed in these computations \[ 29 \]. Additionally, the following proteins were employed to evaluate antimicrobial activity: beta-ketoacyl-\[acyl carrier protein\] synthase from E. The default pH of 7. To evaluate the docking results, the XP GScore was used. The scoring of docking outcomes involved considering energy-minimized poses, and the evaluation relied on the Glide score. The top-docked pose, characterized by the lowest Glide score value for each ligand, was identified and recorded for subsequent analysis \[ 33 \]. Statistical analyses were performed using GraphPad Prism version 7. Statistical significance was acknowledged when p was less than 0. The botanical assessment primarily involved the examination of the morphological attributes of each component of the plant, followed by a comparative analysis with reference species. The ten samples that were examined have been classified as Cannabis sativa. Nevertheless, it seems that relying exclusively on botanical identification may be insufficient for establishing significant differentiation. This led us to conduct a more comprehensive genetic study. The ITS gene facilitated the identification of subspecies and the differentiation between varieties. Similar results were reported by Mello et al. Based on the obtained results, the studied samples were classified into two major groups. Therefore, this molecular study supports the hypotheses proposed by Clarke and Merlin \[ 8 \], and Bachir et al. The statistical significance of the tree was tested and evaluated through bootstrap analysis to measure the robustness and confidence level of the phylogenetic tree obtained. The results obtained exhibited distinct sequences that were determined to be comparable to universally recognized references for Cannabis of the NLD drug type Figure 1. Specifically, subdomain Ia extends from position 28 to aa, subdomain Ib covers positions to aa and to aa, while domain II spans positions to aa \[ 38 \]. These sites were identified at the same locations as described earlier. The OR The OQ The JQ Humulus lupulus Common Houblon is a plant species belonging to the family Cannabaceae native to Europe, Asia, and North America in their temperate region. The analysis of the proximity of this plant GenBank accession number: LA However, we observed that cultivars in Morocco are not homogeneous populations and that they are organized into lineages that are closely related to each other and are close to or away from other varieties of the other paid varieties. As there is a radical difference in the nucleotide sequence between exotic Moroccan, American, European, and Asian accessions, this explains the mixing and genetic diversity experienced by the Moroccan varieties. A previous study on the analysis of the sequence of THCA synthase by Kojoma showed that the types of exotic drugs showed little or no variation indicating that the analysis of the sequence of the THCA synthase can distinguish the accessions of Morocco from those of other countries \[ 17 \]. Moreover, the distribution of Moroccan varieties of the Moroccan drug type placed in the tree in last position compared to other accessions is consistent with the hypothesis that Cannabis was newly introduced by the Arab invasions and subsequently by the European colonization, and subsequently by the introduction of foreign varieties of traffickers from Pakistan and Afghanistan, for use as donors in hybridization and selection, to obtain cultivars adapted to Moroccan climatic and geographical conditions with a high level of THC \[ 39 , 41 \] while keeping the originality of Moroccan Cannabis offered by its climate and geographical position that give Morocco its first position as a producer of Cannabis in the world \[ 42 \]. It is necessary to develop a database of sequences of Cannabis plants of different geographical origins to facilitate the analysis and identification of varieties specific to Morocco seized from trafficking or used for biomedical applications. This database will help national and international agencies identify the geographical origin and route of seizures used by smugglers as well as the types of varieties used by Moroccan growers and biomedical associations. Hemp seed is now a foodstuff under the EU Regulation on nutrition and health claims made on foods \[ 43 \]. Its proximate nutritional quality including its energy value was elucidated by several recent works \[ 11 , 19 , 44 \]. The selection of an element menu was based on the major and minor constituents in the seed materials analyzed. Eleven elements i. The elemental analysis of our sample revealed high contents of Ca However, Cu and Cr were not detected. As shown, the levels of the intake of the different inorganic elements are generally lower in AMSD1 seeds, compared to the recommended daily intake conformed by FDA. The studied seeds also represent a comparable mineral composition with those of the Romanian variety of a Cannabis seeds studied by Menezes et al. Therefore, these results confirm the good nutritional quality of the studied Moroccan Cannabis seed but at moderately lower mineral quantities than the compared European varieties. Concerning the rate of H 2 O is about 3. These values agree with those data reported to date \[ 11 , 46 \]. However, concentrations of Shapiro—Wilk test p greater than 0. Consistent with previous studies \[ 46 , 47 \], our analysis revealed the presence of significant bioactive compounds. Additionally, the identification of specific polyphenols, such Cannabisin A, B, and C, in the ethanol extract, agrees with previous research \[ 48 , 49 \] and underscores the unique chemical diversity within different extracts. The presence of phosphatidylinositol and tetrasaccharides hydrate, also supports previous reports on the diverse functional compounds present in Cannabis seeds \[ 48 \]. Moreover, the detection of isomers, such as Cannabisin B \[isomer 1\], echoes findings from other studies emphasizing the intricate chemical complexity of Cannabis seeds \[ 50 \]. Collectively, our results align with and build upon prior research, providing valuable insights into the functional potential of Moroccan Cannabis sativa L. The most important results are presented below. Other chromatograms and mass spectra are detailed in the Supplementary Material attached to this article. Cannabis seeds are rich in nutritional constituents and non-cannabinoid organic compounds, renowned for their bioavailability and bioreactivity. A qualitative analysis was performed on the seven extracts obtained from the AMSD1 sample, and the resulting chromatograms were processed and compiled see attached sup. This comprehensive analysis revealed the presence of over 60 compounds, with the most significant 24 compounds listed in Table 4. Notably, this GC-MS-MS TQ analysis unveiled seven molecules that were identified for the first time in Cannabis seed: 4,5,7-tris 1,1-dimethylethyl -3,4-dihydro-1,4-epoxynaphthalene-1 2 H -methanol, oleamide, 2-palmitoylglycerol, benzenepropanoic acid, Lowinox , 2,4-DBAL, and 2,4-di-tert-butylphenol. The composition of these extracts is characterized by a balanced presence of essential nutritional elements, such as fatty acids and fatty nitriles, as well as antioxidant elements Table 4. These compounds exhibit various properties, including antioxidant, anti-inflammatory, antimicrobial, and potential health benefits. The presence of these compounds underscores the potential of Cannabis seeds as a valuable functional food source. Evaluating the biological activities of Cannabis seeds is about discovering their potential health benefits and as a functional food. The data showed that each extract exhibited an interesting antioxidant potency, particularly the ethanolic and methanolic extracts with IC 50 values of Acetone, chloroform, diethyl ether, and ethanol extracts showed good antioxidant activity with EC 50 values of For the total antioxidant capacity TAC test, the ethanolic extract showed better results Nevertheless, these activities were almost the same than that found for the synthetic antioxidant BHT, Quercetin, and ascorbic acid Table 4. This antioxidant effectiveness of our extracts may be attributed primarily to the presence of the new detected compounds such as benzenepropanoic acid and 2,4-DBAL in the volatile fraction and the Cannabisin A, B, and C in the non-volatile part of studied Cannabis seeds. Concerning the assessing of the antibacterial and antifungal activity, unfortunately, none of the seven Cannabis seed extracts exhibited an inhibitory effect on the tested bacterial and fungal strains see attached sup. Surprisingly, these extracts were found to have a stimulatory effect on bacterial growth during the entire 24 h period of spectrophotometer monitoring. This unexpected result suggests that the Cannabis seed extracts may contain compounds that promote bacterial growth rather than inhibiting it. Further investigation is necessary to understand the mechanisms behind this stimulatory effect and to explore the potential implications. For the preliminary cytotoxicity test on the Vero cell line, the obtained results Table 4 show that the Cannabis seed extracts studied are not cytotoxic to Vero cells \[ 51 , 52 , 53 \]. Nevertheless, special attention should be given to the aqueous extract, warranting further additional studies for toxicity and safety, especially if intended for incorporation into nutritional and cosmetic formulations. Concerning the antiviral activity anti-SARS-CoV-2 , the obtained results showed that all extracts revealed no significant antiviral effect. The prediction of protein—ligand interactions can be studied by combining methodologies such as virtual screening and computer-aided design. Currently, molecular docking methods are a widely used tool in selecting powerful molecules as a part of virtual screening of large databases. In this work, conducting docking analyses of the identified phytochemicals from volatile fraction of Cannabis seeds against specified targets and their binding affinities for the proteins were evaluated based on the docking scores provided in Table 5 a. Molecular docking analysis results a , the two-dimensional viewer of target ligands in active sites b , d , and the three-dimensional viewer of target ligands in active sites c , e , for antioxidant and antimicrobial activities evaluation of volatile and non-volatile compounds detected from studied Cannabis sativa L. The non-volatile fraction of Cannabis sativa L. These findings suggest that these molecules serve as potent NADPH inhibitors, indicating their potential as robust antioxidant compounds. In Table 5 b,c, a comprehensive breakdown is provided regarding the quantity and nature of bonds established between the volatile compounds originating from Cannabis sativa L. Additionally, a hydrogen bond was created with the ASN residue in the active site of S aureus nucleoside diphosphate kinase. To the best of our knowledge, this study is the first research to highlight the genetic polymorphism of the Moroccan Cannabis sativa L. The genetic profiles of ten seed strains from different regions in the northern Morocco revealed two distinct molecular profiles, namely Cannabis , sp. The volatile part of the seed, analyzed by GC-MS-MS, revealed 24 metabolites of significant nutritional and biological importance, including 7 molecules which were discovered for the first time within Cannabis seeds. The elemental nutritional composition of the studied Moroccan Cannabis seeds revealed a good nutritional quality, with essential minerals, fatty acids, and proteins contributing to their potential health benefits. Antioxidant activity tests showed that the extracts from Cannabis seeds, mainly ethanol and methanol, exhibit interesting antioxidant effects. In silico analysis provided insights into the binding affinities of identified phytochemicals with selected protein targets, indicating their potential as potent antioxidants and moderate to low antimicrobials. In vitro testing did not reveal significant antibacterial activity for any of the extracts. The Cannabis seeds can be utilized as alternative food source rich in nutritional compounds and antioxidants, with potential applications in the food, nutricosmetic, and cosmetic industries. We would like to express our sincere gratitude to Euromed University of Fes and CNRST of Morocco for their invaluable support and resources provided during the course of this project. Conceptualization, A. All authors have read and agreed to the published version of the manuscript. Data are contained within the article and supplementary materials. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. This section collects any data citations, data availability statements, or supplementary materials included in this article. As a library, NLM provides access to scientific literature. Plants Basel. Find articles by Amira Metouekel. Find articles by Fadwa Badrana. Find articles by Rabie Kachkoul. Find articles by Mohamed Chebaibi. Find articles by Mohamed Akhazzane. Find articles by Abdelfattah El Moussaoui. Find articles by Nadia Touil. Find articles by Hamid El Amri. Find articles by Elmostafa El Fahime. Find articles by Nabil El Brahmi. Ain Raal : Academic Editor. Open in a new tab. Similar articles. Add to Collections. Create a new collection. Add to an existing collection. Choose a collection Unable to load your collection due to an error Please try again. Add Cancel. Positive controls. Tetrasaccharide hydrate. Cannabisin B. Cannabisin C. Cannabisin A. Cannabielsoic Acid A. Cannabinolic Acid,. Cannabisin B \[isomer 1\]. Cannabidiolic acid, CBDA. Benzenepropanoic acid Or Methyl 3- 3,5-di-tert-butylhydroxyphenyl propionate. Alkylbenzene class of phenolic esters. Tris 2,4-di-tert-butylphenyl phosphite. Alkylbenzene class of phosphites. Carbonyl class of aromatic aldehydes. Palmitic Acid. Saturated fatty acid. Palmitamide or Hexadecanamide. Fatty acid amide. Fatty amide. Satureted fatty acid ester: a triglyceride. Organic compound. Carbonic acid, monoamide, N-octadecyl-, 2-ethylhexyl ester. Alkyl nonane. Important compunds Structure Interest. Antioxydant Food additive. Cannabis sativa L. Seeds Fractions. Methyl 3- 3,5-di-tert-butylhydroxyphenyl propionate. Cannabielsoic acid A.

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