Medicinal plants have been utilized throughout human history as effective remedies for various illnesses (Michel et al. 2020). These plants harbour bioactive compounds, including antioxidants and antimicrobial agents, which are integral components of many therapeutic interventions (Craig 1999; Lourenço et al. 2019; Rastogi et al. 2016). Currently, there is a global surge in the consumption of medicinal plants due to their documented therapeutic efficacy and safety (Perez et al. 2009). These plants are capable of synthesizing significant quantities of secondary metabolites by bypassing primary metabolic pathways (Bandyopadhyay et al. 2022). Secondary metabolites such as flavonoids and tannins exhibit diverse biological activities and possess potent antioxidant, antibacterial, and antifungal properties (Guerriero et al. 2018). Morocco boasts a rich botanical diversity owing to its strategic geographical position. With approximately 4,500 plant species distributed among 940 genera, Morocco showcases considerable botanical richness, including numerous endemic species, particularly in the Atlas Mountains and the Rif area (Chaachouay et al. 2022). In terms of the abundance of medicinal and aromatic plants in Morocco, there are approximately 500 to 600 species, thus representing a significant heritage of MAPs (Idm'hand et al. 2020; Kachmar et al. 2021).

Antioxidants play a crucial role in safeguarding living organisms against oxidative stress by counteracting the detrimental effects of free radicals and radical ions, which possess unpaired electrons within their structure. These compounds are adept at efficiently scavenging radical ions generated through oxidation processes within the system, while preserving the integrity of biological processes (Çalişkan et al. 2021). Significant natural sources of antioxidants exist in various forms, predominantly derived from plants, encompassing pure bioactive compounds, fruit extracts, leaves, roots, seeds, and essential oils (Manzoor et al. 2023).

The extensive utilization of antibiotics has significantly contributed to the escalation of bacterial tolerance towards specific antibiotics. Subsequent investigations have elucidated the remarkable adaptability of pathogenic bacteria, enabling them to develop resistance to particular antibiotics or even multiple antibiotics. In response to this challenge, many researchers are motivated to explore alternative approaches for combating these bacteria, including the utilization of plant extracts. Antimicrobial activity mediated by plant extracts often involves the disruption of cell membranes, which constitutes a primary mode of action of these extracts (Álvarez-Martínez et al. 2020; Bouyahya et al. 2017; Sayout et al. 2020).

The objective of this investigation is to conduct a comparative evaluation of selected Moroccan medicinal and aromatic plants (MAPs) to elucidate their potential antioxidant and antibacterial properties, thus substantiating their efficacy as novel therapeutic agents against bacterial infections and oxidative ailments.

Plant material

Six medicinal and aromatic plant species were chosen for inclusion in our investigation (Table 1). The leaves of these plants were meticulously harvested and subjected to drying at 37°C for a period ranging from 48 to 72 hours. Subsequently, the dried leaves were finely ground into powder to facilitate their use in the extraction process.

Table 1 . Data on the selected medicinal and aromatic plants for the study.

Plant species	Sampling time	Sampling biomass (kg)	Place of sampling
Eucalyptus torquataLuehm.	March 2022	3	Taounate (34.5361°N, -4.6411°W)
Thymus broussonetiiBoiss.	March 2022	3	Taounate (34.5361°N, -4.6411°W)
Lavandula angustifoliaMedik.	March 2022	3	Taounate (34.5361°N, -4.6411°W)
Rosmarinus officinalisL.	March 2022	3	Taounate (34.5361°N, -4.6411°W)
Ziziphus lotusLahm.	May 2022	3	Sahel Boutaher (34°30.4718'N, 4°47.8572'W)
Acacia raddianaSavi.	May 2022	1	Assa (28° 36′ 31″ N, 9° 25′ 37″ W)

Preparation of extracts

Hydro-ethanol extracts (with a ratio of 8:2) were formulated for each plant species. Concentrations of 40 mg of finely powdered dry material, from each plant, per 10 mL of solvent were subjected to ultrasound treatment at 35 kHz for a duration of 40 minutes. Following this, the mixtures were centrifuged at 3000 revolutions per minute (rpm) for 30 minutes. The resultant supernatants, containing the extracted samples, were carefully preserved at 4°C for subsequent utilization.

Phytochemical evaluation

Total phenolic content (TPC)

TPC was determined using the Folin-Ciocalteu method, as described by Cheng et al. (2004). In this procedure, 200 µl of the extract was mixed with 1.5 mL of 10% Folin-Ciocalteu reagent, followed by the addition of 1.5 mL of 5% sodium carbonate solution. After an incubation period of 2 hours, the optical density was measured at 725 nm. TPC values were calculated using a calibration curve constructed with gallic acid standards. The results were expressed as milligrams of Gallic Acid Equivalents per gram of Dry Matter (mg GAE/g of DM).

Total flavonoids contents (TFC)

TFC was assessed using the Aluminum Chloride method, as outlined by Barros et al. (2011). Initially, 0.3 mL of 5% NaNO2 solution was added to 1 mL of the extract, followed by the addition of 0.3 mL of 10% AlCl3 solution after 5 minutes. Subsequently, 2 mL of 1 M NaOH solution was added, and the final volume was adjusted to 10 mL with distilled water. The absorbance was measured at 510 nm, and TFC was determined using a calibration curve prepared with quercetin standards. The results were expressed in milligrams of Quercetin Equivalents per gram of Dry Matter (mg QE/g of DM).

Condensed tannins content (CTC)

CTC was determined using the Hydrochloric Acid method as described by Ribéreau (1968). In this procedure, 1 mL of the extract was mixed with 1.5 mL of hydrochloric acid (37%) and 0.5 mL of distilled water, and the mixture was incubated in a 95°C water bath for 30 minutes. Simultaneously, another sample was subjected to the same process but at ambient temperature, followed by incubation in the dark for 30 minutes. The absorbance of both samples was measured at 550 nm. The concentration of condensed tannins was calculated using the formula: CTC=19.33×(D2 - D1), where D1 represents the absorbance of the assay at room temperature and D2 represents the absorbance of the assay at 95°C.

Antioxidant activity testing

DPPH radical activity scavenging assay

The method proposed by Brand-Williams et al (1995) was employed to evaluate the antioxidant activity, and it involved the use of the original extract at a concentration of 4 mg/mL, from which subsequent dilutions were prepared. Dilutions were made in the order of 1/2, 1/4, 1/8, and 1/16. Subsequently, 1 mL of each dilution was mixed with 1 mL of DPPH solution (0.004%) and incubated in darkness for 30 minutes. Ascorbic acid served as the standard molecule for comparison. The absorbance of the resulting solutions was measured at 517 nm. Antioxidant activity was calculated using the equation:

Antioxidant Activity (%) = (Abs DPPH - Abs of Extract) / (Abs DPPH)×100

Where: Abs DPPH: Absorbance of the DPPH solution, Abs of the extract: Absorbance value of the extract.

A regression curve analysis was conducted to determine the concentration required to achieve 50% inhibition (IC₅₀).

Total antioxidant capacity (TAC)

TAC was determined following the method described by Prieto et al. (1999). In this procedure, 200 µL of the extract was mixed with 3 mL of a reagent medium composed of 6 M sulfuric acid, 280 mM sodium phosphate, and 40 mM aluminum molybdate. The mixture was then incubated at 95°C for 90 minutes. Subsequently, after quenching, the absorbance was measured at 695 nm. The TAC results were expressed as micrograms of Ascorbic Acid Equivalents per gram of Dry Matter (µg AAE/g of DM).

ABTS

The procedure involved the preparation of the ABTS reagent using Potassium peroxydiphosphate and ABTS, following the method outlined by Re et al. (1999). The reagent was vigorously shaken and then allowed to incubate in darkness for 16 hours. Following the incubation period, 40 mL of methanol was added to 0.78 mL of the prepared reagent, resulting in an optical density of 0.7 when measured at 734 nm. Subsequently, the original extract, at a concentration of 4 mg/mL, was subjected to sequential dilutions of 1/2, 1/4, 1/8, and 1/16. Next, 0.5 mL of each dilution was combined with 1 mL of the ABTS reagent and incubated in darkness for 7 minutes at room temperature. The absorbance of the resulting solutions was measured at 734 nm.

Ferric reducing antioxidant power assay (FRAP)

The fundamental principle of this methodology is centred on the reduction of ferric ions (Fe³⁺) to ferrous ions (Fe²⁺), enabling the assessment of the reducing potential of compounds, as elucidated by Ou et al. (2001).

Initially, 1 mL of each sample was combined with 2.5 mL of phosphate buffer (0.2 M, pH=7), followed by the addition of 2.5 mL of potassium hexacyanoferrate solution (1%). The mixture was then incubated at 50°C for 20 minutes. Subsequently, 2.5 mL of trichloroacetic acid (10%) was added to the mixture, which was then centrifuged at 3000 revolutions per minute for 10 minutes. Finally, 2.5 mL of the supernatant was mixed with 2.5 mL of distilled water and 0.5 mL of FeCl₃ solution (1%). The absorbance of the resulting solution was measured at 700 nm. The values obtained were expressed as milligrams of Ascorbic Acid Equivalents per gram of Dry Matter (mg AAE/g DM).

Antibacterial activity testing

Bacterial species

The antimicrobial efficacy of the plant extracts was assessed against four bacterial strains, encompassing two Gram⁺ bacteria, specifically Bacillus subtilis and Staphylococcus aureus, and two Gram^‒ bacteria, namely Pseudomonas aeruginosa and Escherichia coli. These bacterial strains were obtained as pure cultures from Laboratory of Functional Ecology and Environmental Engineering, in Faculty of Sciences and Techniques of Fez.

Bacterial culture suspension preparation

One or two bacterial colonies were selected from early-stage cultures on nutrient agar and suspended in physiologically sterile water. The turbidity of the suspension was standardized by comparison to a 0.5 McFarland solution.

Antibacterial activity assay

The antibacterial activity of the extracts was evaluated using the broth microdilution method, as described by (Ouedrhiri 2017). Briefly, 50 µl of Lysogeny Broth (LB) solution and 50 µl of each extract were dispensed into wells of a 96-well microplate. The solutions were then serially diluted, and 50 µl of bacterial suspension was added to each well. The microplates were then incubated at 37°C overnight, and the results were read after 24 hours to determine the minimum inhibitory concentrations (MIC). An additional 24 hours of incubation were required to determine the minimum bactericidal concentrations (MBC), following the protocol outlined by (Abedini 2013).

Bactericidal effect

The MBC/MIC ratio was employed to ascertain the bactericidal effect of each plant extract. A ratio of ≤ 4 indicates a bactericidal effect, whereas a ratio of > 4 signifies a bacteriostatic effect, following the criteria outlined by (Mogana et al. 2020).

Statistical evaluation

The data were subjected to analysis of variance (ANOVA) for statistical evaluation. Specifically, a two-way ANOVA was conducted, supplemented with Tukey's test to identify statistically significant differences. Statistical analyses were performed utilizing "SYS-TAT 12" software. Additionally, a comparison of means test was conducted in instances where the ANOVA indicated a statistically significant impact of the studied factor.

Phytochemical analysis

Ziziphus lotus and Eucalyptus torquata demonstrated the highest phenolic content, with respective values of 87.55 mg GAE/g of DM and 86.75 mg GAE/g of DM. In contrast, Acacia raddiana showed the lowest phenolic content at 1.56 mg QE/g of DM (Fig. 1, Table 2). Statistical analysis of the TPC assay results revealed a highly significant difference between the plant species used (F=1305.275; df=5; p≤0.001). Thymus broussonetii exhibited the highest TFC at 297.81 mg QE/g of DM, followed closely by Lavandula angustifolia with 272.19 mg QE/g of DM (Fig. 2, Table 2). The TFC assay data indicated a highly significant variation among the plant species tested (F=2646.574; df=5; p≤0.001). Rosmarinus officinalis displayed the highest CTC with a value of 5.10 g/L, followed by Eucalyptus torquata and Ziziphus lotus with values of 3.38 and 2.54 g/L, respectively (Fig. 3, Table 2). The variance analysis for CTC among the six plant species revealed a profoundly significant distinction (F=2893.342; df=5; p≤0.001)

Table 2 . Polyphenols, flavonoids and condensed tannins content of the studied plants.

Plant species	Polyphenols (mg GAE/g of DM)	Flavonoids (mg QE/g of DM)	Condensed tannins (g/L)
E. torquata	86.75a±0.083	129.38a±2.52	3.38a±0.02
T. broussonetii	51.21b±1.21	297.81b±1.77	0.73b±0.001
L. angustifolia	27.75c±1.83	272.19c±2.78	2.18c±0.01
R. officinalis	42.87d±0.625	247.81d±0.25	5.10d±0.005
Z. lotus	87.58a±0.208	169.38e±1.01	2.54e±0.016
A. raddiana	1.3e±0.09	-	-

Mean values labelled with different letters (superscripts) indicate very highly significant differences at p<0.001

Figure 1. The total polyphenol content of the six plants studied. Columns with different letters indicate very highly significant differences with p<0.001.

Figure 2. The total flavonoid content of the six plants studied. Columns with different letters indicate very highly significant differences with p<0.001.

Figure 3. Condensed tannin content of the six plants studied. Columns with different letters indicate very highly significant differences with p<0.001.

Evaluation of antioxidant activity

All six plant extracts demonstrated notable efficacy in scavenging free radicals as assessed by the DPPH assay (Table 3). Among the extracts, Eucalyptus torquata exhibited the most favorable IC₅₀ value (0.048 mg/mL), signifying robust antioxidant activity comparable to that of Ascorbic acid (IC₅₀=0.01 mg/mL). Thymus broussonetii followed closely with an IC₅₀ value of 0.089 mg/mL. Statistical analysis of IC₅₀ values revealed a highly significant variance among the studied plant species (F=407219.786; df=5; p≤0.001). Rosmarinus officinalis exhibited the highest antioxidant capacity, measuring at 1,033 µg AAE/g of DM, succeeded by Thymus broussonetii with a value of 779.82 µg AAE/g of DM, whereas Acacia raddiana displayed the least impact at 20.61 µg AAE/g of DM (Fig. 4, Table 4). Statistical evaluation of TAC results unveiled a notably significant influence (F=5482.577; df=5; p≤0.001) across the examined plant species. Among the six extracts, Eucalyptus torquata demonstrated the highest iron-reducing capacity, recording a value of 209.375 mg AAE/g of DM, followed by Thymus broussonetii with 145.75 mg AAE/g of DM (Fig 5, Table 4). Statistical analysis of FRAP results indicated a highly significant effect attributed to the diverse plant species studied (F=4592.327; df=5; p≤0.001). Table 5 presents the outcomes of the ABTS assay, wherein the antioxidant capacity of the various tested extracts was determined through the IC₅₀ value. Notably, the IC₅₀ value for the Eucalyptus torquata plant extract was 0.11 mg/mL. Statistical analysis of the ABTS assay findings revealed a markedly significant effect across the six studied plant species (F=1013.797; df=5; p≤0.001)

Table 3 . Plant extracts and ascorbic acid with their respective IC₅₀ values for DPPH assay.

Plant species	IC50 (mg ml-1)
E. torquata	0.048a±0.00002
T. broussonetii	0.089b±0.0001
L. angustifolia	0.181c±0.0029
R. officinalis	0.105b±0.004
Z. lotus	0.053a±0.0004
A. raddiana	4.99d±0.0056
Ascorbic Acid	0.01

Mean values labelled with different letters (superscripts) indicate very highly significant differences at p<0.001

Table 4 . Total antioxidant capacity and ferric reducing antioxidant power assays results for the studied plants.

Plant species	TAC (µg AAE/g of DM)	FRAP (mg AAE/g DM)
E. torquata	744.30a±3.95	209.38a±1.125
T. broussonetii	779.82b±10.53	145.75b±0.75
L. angustifolia	776.75b±0.44	74.75c±0.5
R. officinalis	1033.77c±3.07	97.88d±0.875
Z. lotus	444.74d±0.88	124.69e±3.56
A. raddiana	20.61e±0.44	17.06f±0.56

Mean values labelled with different letters (superscripts) indicate very highly significant differences at p<0.001

Table 5 . Plant extracts and ascorbic acid with their respective IC₅₀ values for ABTS assay.

F=1013.797; ddl=5; p≤0.001
Plant	IC50 (mg ml-1)
E. torquata	0.11a±0.00305
T. broussonetii	0.37b±0.0131
L. angustifolia	0.46c±0.0026
R. officinalis	0.33d±0.0016
Z. lotus	0.36b±0.00025
A. raddiana	2.71e±0.0301
Ascorbic Acid	0.01

Mean values labelled with different letters (superscripts) indicate very highly significant differences at p<0.001

Figure 4. Total antioxidant capacity of the six plants studied. Columns with different letters indicate very highly significant differences with p<0.001.

Figure 5. Ferric reducing antioxidant power of the six plants studied. Columns with different letters indicate very significant differences with p<0.001.

Evaluation of antibacterial activity

All extracts exhibited significant MIC effects, except for Acacia raddiana, which displayed no activity against any of the four bacterial strains. Rosmarinus officinalis showcased the most favorable MIC across all bacterial strains tested, with values of 0.625 mg/mL for E. coli (S1) and S. aureus (S2), 1.25 mg/mL for P. aeruginosa (S3), and 5 mg/mL for B. subtilis (S4) (Fig. 6, Table 6). Regarding the four bacterial strains, Rosmarinus officinalis demonstrated the highest potency in terms of MBC, with values of 1.25 mg/mL, 10 mg/mL, 5 mg/mL, and 5 mg/mL, respectively (Table 7). Each of the extracts demonstrated a bactericidal effect, as shown in Table 8, with the exception of Acacia raddiana.

Table 6 . MIC results for all hydro-ethanol extracts on the 4 bacterial strains.

	Plant extract	[MIC] mg/ml
		20	10	5	2.5	1.25	0.625	0.3125	0.15625	0.078125	0.03906	0.01953
S1	E. torquata	-	-	-	-	+	+	+	+	+	+	+
	T. broussonetii	-	-	-	-	+	+	+	+	+	+	+
	L. angustifolia	-	+	+	+	+	+	+	+	+	+	+
	R. officinalis	-	-	-	-	-	-	+	+	+	+	+
	Z. lotus	-	-	-	+	+	+	+	+	+	+	+
	A. raddiana	+	+	+	+	+	+	+	+	+	+	+
S2	E. torquata	-	-	-	-	-	+	+	+	+	+	+
	T. broussonetii	-	-	-	+	+	+	+	+	+	+	+
	L. angustifolia	-	-	-	+	+	+	+	+	+	+	+
	R. officinalis	-	-	-	-	-	-	+	+	+	+	+
	Z. lotus	-	-	-	+	+	+	+	+	+	+	+
	A. raddiana	+	+	+	+	+	+	+	+	+	+	+
S3	E. torquata	-	-	-	+	+	+	+	+	+	+	+
	T. broussonetii	-	-	-	+	+	+	+	+	+	+	+
	L. angustifolia	-	+	+	+	+	+	+	+	+	+	+
	R. officinalis	-	-	-	-	-	+	+	+	+	+	+
	Z. lotus	-	-	+	+	+	+	+	+	+	+	+
	A. raddiana	+	+	+	+	+	+	+	+	+	+	+
S4	E. torquata	-	-	-	+	+	+	+	+	+	+	+
	T. broussonetii	-	-	+	+	+	+	+	+	+	+	+
	L. angustifolia	-	+	+	+	+	+	+	+	+	+	+
	R. officinalis	-	-	-	+	+	+	+	+	+	+	+
	Z. lotus	-	-	-	-	+	+	+	+	+	+	+
	A. raddiana	+	+	+	+	+	+	+	+	+	+	+

S1: Escherchia coli, S2: Staphyloccocus aureus, S3: Pseudomonas aeruginosa, S4: Bacillus subtilis

(+): Bacterial growth // (-): No Bacterial growth

Table 7 . MBC results for the no bacterial growth on the 4 bacterial strains.

	Plant extract	[MBC] mg/ml
		20	10	5	2.5	1.25	0.625
S1	E. torquata	-	-	+	+	+	+
	T. broussonetii	-	-	+	+	+	+
	L. angustifolia	+	+	+	+	+	+
	R. officinalis	-	-	-	-	-	+
	Z. lotus	-	-	-	+	+	+
	A. raddiana	N/A	N/A	N/A	N/A	N/A	N/A
S2	E. torquata	+	+	+	+	+	+
	T. broussonetii	-	-	+	+	+	+
	L. angustifolia	-	+	+	+	+	+
	R. officinalis	-	-	+	+	+	+
	Z. lotus	-	+	+	+	+	+
	A. raddiana	N/A	N/A	N/A	N/A	N/A	N/A
S3	E. torquata	-	+	+	+	+	+
	T. broussonetii	-	-	+	+	+	+
	L. angustifolia	-	+	+	+	+	+
	R. officinalis	-	-	-	+	+	+
	Z. lotus	-	+	+	+	+	+
	A. raddiana	N/A	N/A	N/A	N/A	N/A	N/A
S4	E. torquata	-	+	+	+	+	+
	T. broussonetii	-	-	+	+	+	+
	L. angustifolia	-	+	+	+	+	+
	R. officinalis	-	-	-	+	+	+
	Z. lotus	-	+	+	+	+	+
	A. raddiana	N/A	N/A	N/A	N/A	N/A	N/A

(+): Bacterial growth // (-): No Bacterial growth. N/A: Not Applicable

Table 8 . Bactericidal effect of the extracts on the 4 bacterial strains.

	Plant extract	MBC/MIC ratio
S1	E. torquata	4
	T. broussonetii	4
	L. angustifolia	-
	R. officinalis	2
	Z. lotus	1
	A. raddiana	-
S2	E. torquata	-
	T. broussonetii	2
	L. angustifolia	4
	R. officinalis	16
	Z. lotus	4
	A. raddiana	-
S3	E. torquata	4
	T. broussonetii	2
	L. angustifolia	1
	R. officinalis	4
	Z. lotus	2
	A. raddiana	-
S4	E. torquata	4
	T. broussonetii	1
	L. angustifolia	1
	R. officinalis	1
	Z. lotus	8
	A. raddiana	-

Figure 6. Evaluation of antibacterial effect of the 6 studied plants on 4 bacterial strains using micro-dilution method. The determination of antibacterial activity was conducted using the microdilution method. Samples of plant extracts were tested against four different bacterial strains, designated S1, S2, S3 and S4. The concentrations of the extracts were arranged from left to right in the image, with a progressive decrease in concentration. Each vertical line represents a specific plant, with repetitions on the following line. For example, for Ziziphus lotus, Z1 signifies the effect of this plant on strain S1, Z2 on strain S2, and so forth... Observation of the results reveal that bacterial growth is indicated by a pink coloration, facilitated by the addition of resazurin, a chemical compound serving as an indicator of bacterial presence. Areas without bacterial growth are observed by purple or green colorations.

The study on phytochemical composition revealed distinct levels of TPC among the investigated plant species. Z. lotus had the highest TPC at 87.58 mg GAE/g of DM, followed by E. torquata with 86.75 mg GAE/g of DM. Other species showed descending TPC values: T. broussonetii (51.21 mg GAE/g of DM), R. officinalis (42.87 mg GAE/g of DM), L. angustifolia (27.75 mg GAE/g of DM), and A. raddiana (1.56 mg GAE/g of DM).

Bouhlali et al. (2020 and 2021) reported TPC values of 73.48 and 76.68 mg GAE/g of DM for E. torquata extracts from powdered waste, slightly differing from our findings likely due to their use of aqueous extraction. For R. officinalis, Kabubii et al. 2023) found a TPC of 39.71 mg GAE/g of DM, close to our results, potentially influenced by geographical factors in Kenya, whereas Bouloumpasi et al. (2024) found a higher TPC value of 138 mg GAE/g of DM, this difference may be due to the usage of a different approach in preparing the plant material by the authors, where they obtained the extract from post-distillation solid residues of the plant. For L. angustifolia, Spiridon et al. (2011) reported nearly double the TPC (54.9±2.14 mg GAE/g of DM) compared to our findings, while Tsakni et al. (2023) observed a much lower value of 428 µg GAE/g of DM. Dhibi et al. (2022) documented a significantly higher TPC of 468.57 mg GAE/g of DM for Z. lotus, attributing differences to plant location in Sidi Aich, Tunisia, and ethanol extraction. (Petrović et al. 2017) found a TPC of 178.83±1.09 mg GAE/g of DM for T. broussonetii, over three times higher than our results, possibly due to dichloromethane extraction. No prior studies have assessed the phytochemical composition of A. raddiana exudate, with Elshamy et al. (2023) and Lakhera et al. (2017) focusing on leaves and exudate monosaccharides, respectively, precluding direct comparison with our data.

Regarding TFC results, T. broussonetii had the highest value at 297.81 mg QE/g of DM, followed by L. angustifolia (272.19 mg QE/g of DM) and R. officinalis (247.81 mg QE/g of DM). Z. lotus and E. torquata had lower TFC values of 169.375 mg QE/g of DM and 129.375 mg QE/g of DM, respectively. Notably, A. raddiana did not yield measurable TFC results. For L. angustifolia, Adaszyńska-Skwirzyńska et al. (2017) reported a TFC of 3.51 mg QE/g of DM, with discrepancies likely due to different solvents, extraction methods, and growing locations. Dhibi et al. (2022) found a TFC of 12.96 mg QE/g of DM for Z. lotus, significantly lower than our findings.

CTC results varied among the plant species, with R. officinalis showing the highest value at 5.1 g/L, followed by E. torquata (3.38 g/L), Z. lotus (2.54 g/L), L. angustifolia (2.18 g/L), and T. broussonetii (0.73 g/L). Notably, A. raddiana did not yield measurable CTC results. Kabubii et al. (2023) reported a CTC value of 15.02 g/L, significantly higher than our observed value for R. officinalis.

Several studies indicate a positive correlation between flavonoids and phenolic compounds with a strong antioxidant activity (Kozlowski 2007; Moussa et al. 2022; Popovici et al. 2009; Sandhar et al. 2011; Zhao et al. 2015). Our findings from the DPPH assay for antioxidant activity show the following IC₅₀ values in descending order: 0.049 mg/mL (E. torquata), 0.053 mg/mL (Z. lotus), 0.089 mg/mL (T. broussonetii), 0.105 mg/mL (R. officinalis), 0.181 mg/mL (L. angustifolia), and 4.99 mg/mL (A. raddiana). Ashour et al. (2023) reported an IC₅₀ of 5.8 µg/mL, higher than ours, using E. torquata flowers. Kontogianni et al. (2013) found an IC₅₀ of 0.0406 mg/mL for R. officinalis, slightly above our result, likely due to different extraction methods and solvents, including hexane and ethyl acetate in a 6-hour Soxhlet extraction. El Hachlafi et al. (2023) documented an IC₅₀ of 0.163 mg/mL for L. angustifolia, similar to our findings. Dhibi et al. (2022) reported an IC₅₀ of 1.28 mg/mL for Z. lotus, while Khouchlaa et al. (2018) found an IC₅₀ of 5 mg/mL. Our results are comparatively superior, potentially due to geographic factors, harvest season, and different solvents. Jamali et al. (2012) observed an IC₅₀ of 0.097 mg/mL for T. broussonetii, aligning with Ouariachi et al. (2014) who reported 0.090 mg/mL. Labiad et al. (2022) reported a much higher IC₅₀ of 0.007 mg/mL for T. broussonetii, using its essential oil.

Concerning TAC, the quantified values were 1033.77 µg/g (R. officinalis), 779.82 µg/g (T. broussonetii), 776.75 µg/g (L. angustifolia), 744.30 µg/g (E. torquata), 444.74 µg/g (Z. lotus), and 20.61 µg/g (A. raddiana).

In the FRAP assay, our results were 209.37 mg/g (E. torquata), 145.75 mg/g (T. broussonetii), 124.69 mg/g (Z. lotus), 97.88 mg/g (R. officinalis), 74.75 mg/g (L. angustifolia), and 17.06 mg/g (A. raddiana). Bouhlali et al. (2021) reported a much higher FRAP value of 474.04 mg/g for E. torquata, more than double our result. This discrepancy might be due to their use of aqueous extraction, highlighting the influence of solvent polarity on extract composition, as discussed by Herrera-Pool et al. (2021).

In the ABTS assay, our results were 0.11 mg/mL (E. torquata), 0.33 mg/mL (R. officinalis), 0.36 mg/mL (Z. lotus), 0.37 mg/mL (T. broussonetii), 0.46 mg/mL (L. angustifolia), and 2.71 mg/mL (A. raddiana). Labiad et al. (2022) reported a much lower IC₅₀ of 0.007 mg/mL for T. broussonetii, using its essential oil. Ashour et al. (2023) documented an IC₅₀ of 21.4 µg/mL in ABTS assays. Jamali et al. (2012) found an IC₅₀ of 0.168 mg/mL for T. broussonetii, and El Bouzidi et al. (2013) reported 0.132 mg/mL. These findings are consistent with our results.

E. torquata showed the highest antioxidant activity in the DPPH, FRAP, and ABTS assays, while R. officinalis had the highest total antioxidant activity. Although similar activities have been reported in other studies, comparisons are difficult due to differences in extraction methods and result expression (Carrasco et al. 2015; Dobros et al. 2022; Duda et al. 2015).

In terms of antibacterial activity, all plants showed significant effects except for A. raddiana, which had no effect on any bacterial strains. E. torquata had varying MIC and MBC values: MIC=2.5 mg/mL and MBC=10 mg/mL (S1), MIC=5 mg/mL and MBC=20 mg/mL (S3), MIC=1.25 mg/mL with no MBC (S2), and MIC=5 mg/mL and MBC=20 mg/mL (S4). R. officinalis showed MIC values of 0.625, 0.625, 1.25, and 5 mg/mL for S1, S2, S3, and S4, with MBC values of 1.25, 10, 5, and 5 mg/mL respectively. L. angustifolia had MIC values of 20, 5, 20, and 20 mg/mL for S1, S2, S3, and S4, with MBC values of 20 mg/mL for S2, S3, and S4. Z. lotus was effective against all strains, with MICs from 2.5 to 10 mg/mL and MBCs from 5 to 20 mg/mL. T. broussonetii showed MICs of 2.5, 5, 5, and 10 mg/mL for S1, S2, S3, and S4, with a consistent MBC of 10 mg/mL against all strains.

Regarding E. torquata, Ashour (2008) found that Gram (-) bacteria were generally more resistant to inhibition than Gram (+) bacteria, except for E. coli, which aligns with our observations. However, our results for R. officinalis differ from those of Soulaimani et al. (2021), who reported an MIC of 4.502 mg/mL and MBC of 9.004 mg/mL against E. coli (S1), and an MIC and MBC of 72.032 mg/mL against P. aeruginosa (S3), both lower than our findings. Conversely, Karadağ et al. (2019) and Becer et al. (2023) reported stronger antibacterial activity, with MICs of 125 μg/mL and 62.5 μg/mL against S. aureus and E. coli respectively for Karadağ, and MICs ranging from >1000 to 78 μg/mL against E. coli, S. aureus, and P. aeruginosa for Becer. These variations may be due to differences in extraction solvents affecting the phenolic compound composition.

For L. angustifolia, Messaoudi Moussii et al. (2020) reported activity against S1 and S2, with slightly better MIC and MBC values than ours (MIC=3.33 mg/mL, MBC=10.67 mg/mL against E. coli; MIC=1.33 mg/mL, MBC=6.67 mg/mL against S. aureus) but lower activity against S3 (MIC=42.67 mg/mL, MBC=85.33 mg/mL). Our results for Z. lotus align somewhat with Tlili et al. (2021), who reported MICs of 250-1000 µg/mL and MBCs of 500-2000 µg/mL against S2, and with Zazouli et al. (2022), who observed MICs of 1024-2048 µg/mL against S1 and S2. In contrast, Chaimae et al. (2019) reported MICs of 50-200 mg/mL against S1, S2, and S3, indicating significantly lower antibacterial activity than our findings, possibly due to their use of plant seeds.

Regarding T. broussonetii, Fadli et al. (2012) reported MIC values of 0.339, 5.48, 0.085, and 0.339 mg/mL, and MBC values of 0.339, 5.48, 0.339, and 0.685 mg/mL against S1, S3, S4, and S2 respectively. El Bouzidi et al. (2013) observed MIC values of 0.90, 0.23, and 0.90 mg/mL and MBC values of 0.90, 0.23, and 0.90 mg/mL against S2, S4, and S1 respectively. However, Boukhira et al. (2016) reported different results, with an MIC of 0.2, 1.3, and 20 μl/mL and MBC=0.2, 1.3, and 80 μl/mL against S2, S1, and S3 respectively. These differences may arise from variations in extraction solvents, plant harvest seasons, and other influencing factors. Ultimately, R. officinalis exhibited the most potent antibacterial activity, followed by E. torquata, while A. raddiana showed no activity against the four bacterial strains tested.

Through our investigation, we have uncovered significant properties exhibited by the plants under study. Our quantitative analysis of hydroethanolic extracts revealed notable concentrations of polyphenols, flavonoids, and tannins. Particularly, we observed compelling antioxidant activity in these plants, notably in E. torquata as assessed by DPPH, FRAP, and ABTS assays, and in R. officinalis as determined by TAC assay. Moreover, we unveiled potent antibacterial activity of the extracts against four distinct bacterial strains (Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Bacillus subtilis), with R. officinalis demonstrating the highest efficacy. This study not only highlights the therapeutic potential of these plants but also positions them as promising candidates for mitigating oxidative stress and preventing diseases instigated by free radicals, as well as for managing bacterial infections. Thus, further exploration encompassing all facets of this study would be invaluable for maximizing the utilization of these plants.

We would like to express our sincere gratitude to the PRIMA leaders and members for their valuable contribution to this scientific work that is part of the project. Their expertise and collaboration greatly enriched our research and were essential to the completion of this project. Their continuous support and insightful advice have been of invaluable value, and we warmly thank them for their commitment and contribution for the realization of this work.