The field trials were set up in El Kalâa, a small village in the region of Chefchaouen (35° 13’ 110’’ N, 5° 14’ 42’’ W), 873(m) in altitude. The village has a mountainous morphology with the famous Jbel el Kalâa summit (1721 m). The climate is typically mountainous, with frequent rainfall, cold in winter and mild or hot in summer. Rainfall is generally between 800 and 1400, but sometimes it can exceed 2000 mm/year. During the summer (July, to mid-August) a dry period occurs with scarce rainfall and high temperatures which sometimes reach or exceed 40 °C, (Benabid, 1982).

Four Cannabis sativa L. commercial cultivars namely; CA, Cannabis Sativa L. Cultivar Amnesia; CB, Cannabis Sativa L. Cultivar Beldia; CM, Cannabis Sativa L. Cultivar Mexicana; CK, Cannabis Sativa L. Cultivar Khardala, were studied. Certified seeds were sown according to the local traditional method of hemp cultivation, then subjected to the same processes of harvesting, drying and production of resin and achenes. Sowing was carried out between March and April (2020) and harvesting between August and September (2020). At the maturity phase, during which more than 90% of brown seeds appeared, the female plants were harvested and then subjected to sun-drying for 4 days. A total of four samples (1 kg for each sample) of hemp seeds were collected from the harvested and dried plants. The collected seed samples were cleaned and excluded from the unripe and the empty seeds, then stored at 4 °C. Herbarium specimens of the Cannabis Sativa seeds used in this study were deposited in the herbarium of the Applied Botany Laboratory, Department of Biology, Faculty of Sciences of Tetouan, Abdelmalek Essaâdi University.


The seeds were air-dried beforehand and had an average moisture content of 7.614 ± 1.623 % according to the AOAC Official Method 925.40 (2000), such that 5 grams of each seed variety were placed in a temperature-stabilized oven at 60 ºC until constant mass. Moisture content was calculated as percent (%) of the loss in recorded weight.


Total lipids were extracted from dry Achenes samples according to the method of Folch et al. (1957). Deuterated N-acylethanolamine (NAEs) and congeners were added to the samples as internal standards before extraction for quantification by isotope dilution. Aliquots of the lipid fraction were used for their quantification. Internal deuterated standards N- arachidonoylethanolamine [2H]⁸AEA, N-oleoylethanolamine [2H]²OEA, N-palmitoylethanolamine [2H]⁴PEA, N-stearoylethanol- amine [2H]³SEA, 2-arachidonoyl-glycerol-d5 [2H]⁵2AG, were purchased from Cayman Chemicals (MI, USA).


NAE quantification was carried out using an Agilent 1260 UHPLC system (Agilent, Palo Alto) equipped with a mass spectrometry (MS) Agilent Technologies QQQ triple quadrupole 6420 with an electrospray ionization (ESI) source, using positive mode (ESI+).


A Poroshell 120 EC-C-18 column (Agilent, Palo Alto, CA, USA) with 2.7 μm particle size and 3 × 100 mm was used with a mobile phase of CH3OH/H2O/HCOOH (80/20/0.1, v/v/v) at a flow rate of 0.5 mL/min. N₂ was used as a nebulizing gas with a pressure of 50 psi, a drying gas temperature of 300 ºC, a flow of 11 L/min, and 4000 V capillary voltage. For each standard, the precursor ion [M+ H]⁺ was determined during a full scan (SCAN) in MS, and subsequently, the obtained product ion (PI) was monitored for each transition in MRM mode in MS/MS. The parameters of source, such as cone voltage or fragmentor (CV) and collision energy (CE), were optimized for each MRM transition (Table 1), (Murru et al., 2021).


The data was acquired by the MassHunter workstation acquisition software (version B.08.02), analyzed with MassHunter software for qualitative analysis (version B.08.00 SP1) and by the MassHunter workstation software for quantitative analysis (version B.09.00).


All measurements were taken on dry samples, and values were expressed as means ± SD of two replicates from each independent experiment. The differences among the four cultivars were assessed using One-way Anova and Tukey’s test. Statistical analyses were made using the SPSS package version 23 (IBM, Armonk, NY, USA). Differences were deemed significant at a probability level of 5%. Principal component analysis (PCA) was done for cultivars based on the major compounds. The XLSTAT software was used for different data processing.


The test for homogeneity of variance revealed a p-value < 0.05 for the variable “total NAE rate’’ in the four achene varieties: CA, Cannabis Sativa L. Cultivar Amnesia; CB, Cannabis Sativa L. Cultivar Beldia; CM, Cannabis Sativa L. Cultivar Mexicana; CK, Cannabis Sativa L. Cultivar Khardala. According to Tukey’s test, the four cultivars were categorized into two homogeneous subsets. The NAE average levels in our achene samples were 921.38 ± 260.22 pmol/g dry weight. Figure 1 shows significant differences in total NAE levels only between CA and CM (p-value < 0.05); CM achenes had the lowest total NAE levels (751.83 ± 2.15 pmol/g dry weight), while the highest levels were contained in CA achenes, at greater than 1.75-fold (1309.23 ± 132.61 pmol/g dry weight).


We reported the total NAE levels identified in the seeds and tissues of different plant taxa in Table 2, such as Soybean, Peanut, Castor oil, Tomato, Okra, Cotton and Corn, which ranged from 490 ng/g in Peanut to 1608 ng/g in Cotton. NAE levels in the CA achenes of our sample (416.66 ± 41.49 ng/g dry weight), were comparable to Peanut seeds.


The different NAE species of the four cultivars studied are presented in Table 3. LEA, SEA and PEA displayed significant differences (p-value < 0.05) in our achene samples. The NAE profile of the four varieties of achenes was similar to the patterns observed in non-leguminous seeds, (Arias-Gaguancela et al., 2022), in which the most abundant were NAE-18:2, NAE-18:1 and NAE-16:1 species followed by NAE-18:0 and NAE-16:0.

In a study conducted on plant species, the variability of NAEs among varieties of species such as cotton, was greater than the variability in our analyzed species, (Chapman et al., 1999). Similar data was obtained from a species of the legume family, the Medicago Sativa. L species, (Venables et al., 2005). Furthermore, the chemical composition of NAEs can vary due to several factors, such as genotype (variety), plant section used for extraction analysis, extraction method, and conditions (times and temperature). These differences in NAE composition appear upon seed germination and seedling growth, such that, in young seedlings it is similar to that in the leaves of mature plants, (Wang et al., 2006). In seeds, the proportion of NAE-PUFA decreased more substantially than NAE-SFA species and the oxidative metabolism may contribute significantly to the overall changes during seedling growth, (Chapman et al., 1999).


PEA (NAE-16:0) presented significant differences (p-value < 0.05) among the four achene varieties. The average PEA levels in all four varieties of achenes were 78.03 ± 51.23 pmol/g dry weight; CB achenes showed the lowest levels (44.54 ± 1.96 pmol/g dry weight), while the highest were in the CA achene species (153.86 ± 6.21 pmol/g dry weight), representing 11.75% of the total NAEs.


In addition, SEA (NAE-18:0) showed significant differences (p-value < 0.05). The average SEA of the four species of achenes was higher by 23% than PEA levels. Similar to PEA, CB and CA achenes showed the minimum and maximum levels, 51,84 ± 7,76 pmol/g dry weight and 144,10 ± 17,68 pmol/g dry weight, respectively. In CA achenes, SEA accounted for 10.83 - 11.16% of the total NAEs.

These differences are in accordance with the NAE profile reported for seeds of the legume taxa, (Venables et al., 2005); PEA levels ranged from 0.38 ± 0.03 µg/g lipid weight in the seeds of Bauhinia congesta species to 99.7 ± 6.38 µg/g of lipid in the seeds of Medicago truncatula cv. Jemalong. SEA levels represented 0.08 ± 0.1 µg/g lipid weight in the seeds of the Bauhinia congesta species and 15.9 ± 1.03 µg/g lipid weight in the seeds of Medicago truncatula cv. Jemalong.


These two NAE-SFA molecules (PEA, SEA) possess promising therapeutic potential. PEA shows an autocoid negatively modulating mast cell behavior in response to inflammatory noxious stimuli in vivo, (Hesselink et al., 2017). PEA acts as a potent anti-inflammatory and anti-neuroinflammatory agent via activation of the nuclear peroxisome proliferator receptor (PPAR)-α, (Costa et al., 2008), which regulates the activation of genes which are responsible for the synthesis of inflammatory cascades and pro-inflammatory mediators such as cytokines and the tumor necrosis factor alpha (TNF-α), (Hesselink et al., 2017). In addition, the targeting of the transient receptor potential vanilloid-1 (TRPV-1) by PEA is another aspect of its physiotherapeutic importance which confers its anti-allodynic and anti-hyperalgesic effects.

Similarly, SEA is known for its therapeutic effect in modulating immune and inflammatory responses in allergic diseases, in synaptic dysfunction and acute and late neurodegeneration, (Kasatkina et al., 2020).

We found other NAE subtypes biosynthesized by monounsaturated FA (MUFA), such as oleic acid (18:1) for NAE-18:1 (OEA), and palmitoleic acid (16:1) for the NAE-16:1 (POEA) production. We observed no significant changes in NAE-18:1 (OEA) levels in the four achene varieties (p-value > 0.05). The highest levels were in CA achenes (355.33 ± 65.20 pmol/g dry weight), which ranged from 25.44 to 28.61% of the total NAEs. Also, NAE-16:1 (POEA) presented no significant difference (p-value > 0.05), with the highest amount in the CB achenes (161.45 ± 104.25 pmol/g dry weight), ranging from 14.44 to 23.48% of the total NAEs.


Nutritionally, OEA is considered a promising therapeutic agent for weight control, obesity and associated diseases. OEA induces hypophagia and reduces fat mass in rodents and PPAR-α has been shown to be the most widely accepted mediator of the hypophagic action of OEA via signaling homeostatic brain centers, (Brown et al., 2017). Recent studies have revealed that OEA reduces food intake via effects on dopamine and endocannabinoid signaling in the brain, (Sihag et al., 2018). OEA also binds with two other known receptors with moderate potency, namely the G protein-coupled receptor 119 (GPR119), and the capsaicin receptor, transient receptor potential vanilloid-1 (TRPV1) (Im, 2021).

POEA is a palmitoleic acid derivative, and is a well-characterized agonist of GPR119 receptors capable of counteracting the metabolic syndrome associated with complicated obesity. It has pharmacological activity which is similar to that of the oleic acid derivative OEA in the regulation of energy intake through insulin reléase, (Bandres-Meriz et al., 2023). POEA is among the plasma NAEs which act on brain connectivity in homeostatic and reward circuits through hunger and satiety states to maintain homeostasis in humans, (DiPatrizio, 2021). In addition, POEA is a vital and effective nutritional ingredient that can be used against metabolic disorders associated with diet-induced obesity.


Among the polyunsaturated FA (NAE-PUFA), we observed only NAE-18:2, derived by LA which varies significantly (p-value < 0,05) among achene varieties, with an average of 345.42 ± 112.17 pmol/g dry weight. CM achenes had the lowest levels (259.31 ± 89.88 pmol/g dry weight), while CA achenes possessed about twice the CM amount (509.15 ± 10.51 pmol/g dry weight). LEA accounted for 35.76 to 42.50% of the total NAEs quantified in CA achenes.


In plant tissue, LEA is a signaling molecule which regulates seed germination and plant growth, (Keereetaweep et al., 2015). According to our data, it is the most abundant molecular species of NAEs identified in the dry seeds of Pea, Soybean, Peanut, Castor, Tomato, Okra, Cotton and Maize, with levels exceeding 800 ng/g fresh seed weight, (Chapman et al., 1999). In another study, LEA ranged from 28.8 ± 5.7 ng/g fresh seed weight in the seeds of the species Phaseolus vulgaris cv. Amarillo del Norte to 12740 ± 995 ng/g fresh seed weight in the seeds of the species Glycine max cv. Dare, (Venables et al., 2005).

The NAE relations found in the achenes were studied by principal component analysis (PCA). The first factorial plane, made up of the axes (F1) and (F2), represented 98.12% of the total inertia. The projection of the variables on the first two axes of the PCA (Figure 2), allows us to highlight groupings, oppositions and directional tendencies. The first axis (F1) explains 72.18% of the total variance, while the axis (F2) explains 25.94%. The PEA, LEA and OEA variables are closely linked and evolve in the same direction while positively differentiating on the axis (F2) and on the axis (F1). On the other hand, they are opposed according to the axis F1 to SEA, while the latter is positively correlated with F2. However, POEA shows a strong positive correlation at the F1 axis, and a negative correlation at F2. The projection of the observations (achenes variety) on the two axes of the PCA (F1 and F2) shows a strong separation and a fairly clear differentiation between cultivars. For example, the F2 axis opposes the CA of all the three varieties, while giving it an extreme positive coordinate. Furthermore, the CA is positively correlated to F1, and the achenes corresponding to the CA show a strong affinity for the variables PEA, LEA, OEA, SEA. Conversely, CB and CM are positively correlated to F1 with an extreme positive coordinate for CB, while the latter shows a strong affinity for POEA. CK exhibits a negative correlation at both F1 and F2. The cultivars CA and CB appear to be significant, given their richness in different NAE species which has been identified in this study.