The reduction of Ag⁺ ions into Ag⁰ nanoparticles was easily observed via the distinct color change from yellow-pale green to reddish and dark brown, respectively, due to the surface plasmon resonance phenomena. The best reaction conditions (the concentration of AgNO₃ solution, the plant extract:AgNO₃ solution ratio, the temperature, and the reaction time) for each plant extract that mediate the AgNPs synthesis should be investigated, and one recent article revealed these conditions for the following five plant extracts: Berberis vulgaris (root extract), Brassica nigra (seed extract), Capsella bursa-pastoris (leaves extract), Lavandula angustifolia (leaves extract), and Origanum vulgare (leaves extract) [40], and other articles presented the results related to Cannabis sativa extracts (hemp herd [37], leaf extracts [36,39], and hemp stem extracts [38]). To examine the formation of AgNPs UV−VIS spectroscopy experiments were conducted (Figure 1). The green synthesis of AgNPs was evaluated at different reaction times and conditions. The green synthesis performed at room temperature for 1 h did not lead to the preparation of AgNPs (Figure 1a–d). Still, it was rapid at 90 °C (about 8 min), a result that is in accordance with the earlier reported studies, where Sing et al. optimized the obtaining method of AgNPs from stem hemp extracts [38]. The best ratio of plant extract:AgNO₃ 5 mM solution of 1:1 (v/v) was the most suitable, and the high temperature (90 °C) lead to the fast formation of AgNPs, a result that was confirmed by our study, and an increase in salt concentration lead to the appearance of a major shift in peak shift [38]. The formation of the AgNPs in suspension was confirmed by UV−VIS spectroscopy, as single, broad and strong surface plasmon resonance peaks, were observed in solutions containing plant extract, as follows: (i) CsDi: 439 nm, (ii) CsDe: 432 nm, (iii) CsSm extract: 436 nm, and (iv) CsSf: 444 nm, and indicated the formation of AgNPs. These results were in good agreement with the 350–500 nm peaks found for the plant extract mediated AgNP preparation suspensions reported [31,35,41].

The specific functional groups from the plant extracts and those bonded with the synthesized AgNPs were determined by FT-IR ATR spectroscopy in order to identify the possible biomolecules responsible for the reduction, capping, and efficient stabilization of AgNPs. In Figure 1a–d, the FT-IR ATR spectra of the Cannabis sativa leaf extracts and that of the mixture of extracts and AgNPs samples are depicted. The analysis of the obtained spectra suggests good similarities between the plant extracts samples and the corresponding mixtures with AgNPs. The bands recorded between 3550–3100 cm⁻¹ are associated with –OH stretching vibration from alcohols and phenols structure [13,38,39,42], and with –NH stretching vibration of amines and amides groups [25,35,43], respectively, from proteins molecules [35,44]. In the 3100–2850 cm⁻¹ range, the C–H and –CH₂ stretching vibrations from the aliphatic structures [24,35,39,45] and =C–H (sp² C) stretching from alkenes and aromatic structures [39] were recorded.

Between 1700–1500 cm⁻¹, a large and strong band was observed. Similar behavior was reported by Singh et. al. [38] for the green synthesis of gold and silver nanoparticles from Cannabis sativa (industrial hemp) and this band was assigned to C=O carbonyl stretching from amides and to C=C stretching (in-ring) from aromatic structures. Some authors [4,43,46] mentioned that this wavenumber range may overlap the –NH bending (in-plane) vibration and C-N stretching vibration from protein structures. It should be noted that all samples containing AgNPs showed a lower intensity for the 1700–1500 cm⁻¹ band than the corresponding leaves of the Cannabis sativa extracts. This behavior suggests that the protein molecules present in the plant extract are involved with functional groups (through the donated electrons pair to the surface of metal nanoparticles) in the reduction of Ag⁺ being an efficient capping agent in nanoparticle production and stabilization. The literature data report that proteins can bind to AgNPs through the free amine groups, and therefore, the stabilization of the nanoparticles by surface-bound proteins occurs [3,24,25,38,47].

The intense band located around 1405 cm⁻¹ (CsDi: 1404.76 cm⁻¹; CsDe: 1407.54 cm⁻¹; CsSf extract: 1405.00 cm⁻¹; CsSm extract: 1405.01 cm⁻¹) was recorded and corresponded to –C–OH bending from alcohols or phenols groups [39]. In the AgNPs containing samples, the intensity at the ~1405 cm⁻¹ band strongly diminished, and only a shoulder was recorded in the FT-IR spectra. At the same time, a new peak appeared at ~1368 cm⁻¹, which, according to Aramwit et al. [48], can arise as a result of –COO^– stretching vibration of carboxylate groups. Abbasi et al. [39] considered that this band was due to the –C–O like phenol groups type. These results indicate that some phytochemicals (i.e., phenols, flavonoids, cannabinoids, polysaccharides, water-soluble biomolecules, etc.) with free hydroxyl groups from the Cannabis sativa extract are responsible for reducing Ag⁺ to Ag⁰, while other biomolecules (i.e., proteins) with –COOH or –OH functional groups rapidly bind the metal ions and are entrapped on the surface of the nanoparticles. Obviously, in this mode, the formation of mostly stable complexes between nanoparticles and effective plant extract functional groups leads to the stabilization of AgNPs, and prevents their aggregation. The obtained results are in good agreement with those reported by Singh et al. [38], which showed that AgNO₃ and (–OH) reaction leads to Ag⁰ particles that undergo aggregation to form clusters. The formatted clusters further act as nucleation centers and catalyze the reduction of residual Ag⁺ ions.

It should be noted that the N-O stretching of the nitro compound gave the FT-IR signal in the 1550–1500 cm⁻¹ and 1372–1290 cm⁻¹ range, and could influence the recorded bands of the AgNPs. The shoulder recorded at ~1268 cm⁻¹ in the plant extracts and the corresponding AgNPs containing mixtures could be attributed to the amide III band [48,49].

The peak recorded at ~1043 cm⁻¹ could be attributed to C–N stretching vibration from amines or amides groups [38,39,43,47] and to –C–O–H bending vibration [1,39]. Therefore, the FT-IR spectra of synthesized NPs containing samples indicated the presence of proteins and hydroxyl compounds on the surface of synthesized AgNPs. The out of plane bending vibrations of =C–H (sp² C) from the aliphatic structures of terpenes, flavonoids, and cannabinoids (i.e., cannabigerol, cannabigerolic acid, cannaflavin, β-myrcene, etc.) from Cannabis sativa extract and entrapped on AgNPs particles surface were recorded at ~923 cm⁻¹, while the =C–H bending from the aromatic groups of protein appeared at ~780 cm⁻¹ and 824 cm⁻¹ [50]. According to Senthil et al. [42], the presence of the –OH and C=C moieties on the surface of the AgNPs is the signature of polyphenols, which are the essential phytochemicals constituents of plant extract.

The –C–N stretching vibration was recorded at ~870 cm⁻¹ [39] and the N–H wagging [48] between 800–850 cm⁻¹. The intensity of this last band increase for the AgNPs containing samples compared with plants extract samples because of the formation of amine salt (NH₂⁺), which, according to Aramwit et al. [48], stabilized the AgNPs and prevented their precipitation or aggregation. The bands recorded between 750–600 cm⁻¹ could be attributed to the stretching vibration of C–Cl and C–Br, which are characteristics of alkyl halides [38].

The FT-IR ATR results (Table 4) and the values of the total phenolic (Table 2) contents for the obtained Cannabis sativa leaves extracts and corresponding AgNPs containing solutions showed that the proteins and other biomolecules with -OH functional groups bind the metallic surface of nanoparticles and stabilized them by preventing their agglomeration. This is shown also in the SEM micrographs (Figure 3) and was confirmed by the DLS data (Table 5). The conformational changes that occurred in the protein molecules lead to more advanced exposure of the hydrophobic groups existing in their structure, relative to the aqueous solvent. This behavior can favor the actions of reducing agents (especially phenolic compounds) from plant extracts and the transformations of entrapped metal into metal nanoparticles, as it was demonstrated by decreasing the values of the total phenolic content by 6.9 times for CsDe to 8.11 times for CsSf (Table 2).

The tentatively qualitative analysis of the secondary metabolites present in Cannabis sativa leaf aqueous extracts was carried out using RP-UHPLC coupled with UV−VIS spectroscopy and mass spectrometry detectors, and the data were compared with that from the literature [50,51]. The presence of ten carboxylic acids (e.g., cannabigerolic acid (CBGA) and cannabidiolic acid (CBDA)) and seventeen decarboxylated constituents, flavonoids (cannflavin A and C), and canniprene was recognized. The data revealed a complex heterogeneous mixture of metabolites extracted in water, and many MS peaks were not assigned to any hemp components extracted in an aqueous mixture of solvents (e.g., methanol, ethanol, and acetone) or other polar solvents (e.g., alcohols and acetone) identified in other studies [52,53,54]. For the phenolic compounds, we identified pyrogallol, gallic acid, quercetin, and kaempferol.

The antioxidant capacity of the extracts and that of extracts + AgNPs were evaluated using the following two assays: DPPH^• and ABTS^•+. There were no correlations between the results obtained, with the values of % inhibition as determined by the ABTS^•+ assay being higher (average 78.2 ± 4.1%) compared with that obtained by the DPPH^• assay (average 18.4 ± 3.9%). The difference in these values arised from the difference in their principles of actions and the solubilisation of the radicals employed: the DPPH^• assay determined the ability of the tested samples that contain phenolic compounds and their derivatives to act as donors of hydrogen to the 2,2-diphenyl-1-picrylhydrazyl free radical, suitable in hydrophobic systems, while in the ABTS^•+ assay, an electron-transfer methodthe, the ABTS^•+ radical can react with more compounds, both in hydrophilic and hydrophobic systems, and lead to obtaining higher values than those calculated for the DPPH^• assay [55,56,57,58].

The surface morphology of the synthesized AgNPs was studied via SEM (Figure 3a–d). The particles were almost spherical and were well dispersed, with less than 65 nm for the AgNPs obtained from the leaf extract of dioecious Silvana variety, and less than 49 nm for the AgNPs formed by the leaf extracts monoecious varieties (Diana and Denise). The biomolecules involved in the Ag⁺ reduction and the stabilization of biogenic AgNPs conferred to the synthesized nanoparticles an eco-friendly nature. Singh et al. determined a similar spherical morphology and size (less than 25 nm) for the synthesized AgNPs using an aqueous extract of fresh leaves of Cannabis sativa harvested in India [35,38]. In comparison, Abassi et al. obtained AgNPs with less than 45 nm in diameter using Cannabis sativa leaf extract harvested in Pakistan [39]. Different parts of plant extracts mediated the production of AgNPs, with sizes that varied from 4 to 100 nm, as was reviewed for bark extracts by Burlacu et al. [59] or for plant leaf, fruit, and root extracts by El-Seedi et al. [60]. The Z-average diameter was greater in solution, as was determined by DLS, compared with the SEM diameter for all samples, which indicated nanoparticle clustering. From the comparison between the AgNPs and extracts + AgNPs data, it was found that the presence of both leaf extracts and AgNPs lowered the aggregation degree in all of the samples, except the CsDe samples.

The antibacterial effect of Cannabis sativa leaf extracts alone and its mixture containing prepared AgNPs was evaluated against both Gram-positive and Gram-negative bacterial strains using the disc diffusion method. Table 6 showed no significant antimicrobial activity for aqueous plant extracts against Pseudomonas fluorescens, and only a moderate effect against E. coli, K. pneumonia, and S. aureus. The synergistic antibacterial effect of mixtures of plant extracts + AgNPs was observed to be less (for P. fluorescens, E. coli, and S. aureus, giving 10–13 mm inhibition zones) compared with the antibacterial positive control (Amikacin: 20 mm for E. coli, and Gentamicin 20 mm for P. fluorescens, and 21 mm for S. aureus). For a mixture of leaf extracts and AgNPs, values between 10–14 mm inhibition zones for K. pneumonia were determined, with values very close to those obtained for the positive control (Amikacin: inhibition zone 14 mm). The present data obtained were in good agreement with that published in earlier reports: AgNPs got from green synthesis mediated by Cannabis sativa leaf extracts harvested in Pakistan have antibacterial effect against K. pneumonia, B. subtilis, E. coli, S. aureus, and P. aeruginosa [39]; the data obtained for the synergic effect of the mixture of Cannabis sativa leaf extracts harvested in India and antibiotics against S. aureus, B. subtilis, E. coli, K. pneumonia, and S. marcescens [35]; the AgNPs prepared using whole stems extracts of Cannabis sativa harvested in France were effective against biofilms of E. coli and P. aeruginosa [38]. The mechanism of action of AgNPs as antibacterial agents includes the damage of the bacterial cell membrane, the influence on the enzymatic system of the respiratory chain, and also DNA fragmentation, which have been earlier reported [61]. Earlier published data confirm that there is a higher inhibitory effect of AgNPs and Ag⁺ ions against the Gram-negative bacteria, because of the thicker peptidoglycan layer of Gram-positive bacteria, which can obstruct the action of Ag⁺ ions [62]. This data confirmed the potential activity of the mixture of Cannabis sativa leaf extract and AgNPs against different bacteria, with the best results shown for Klebsiella pneumonia.