The aerial parts of R. montana were collected in June 2024 from wild populations in the Boulemane region, specifically in the Municipality of Guigou (Figure 1). The harvesting site, the harvested plant part, and its source are detailed in Supplementary Table S1. Botanical identification was carried out through a meticulous morphological analysis of the specimens, comparing them with herbarium material and consulting botanical literature. After the plant was identified by a botanist, represented by Professor Amina Bari from the Biology Department of the Faculty of Sciences (FSDM) at USMBA in Fez, the samples were deposited in the herbarium of the Biology Department under the reference number LRM00252024. After harvesting, the collected plant material was dried to preserve it. The aerial parts were spread out in a single layer and dried at room temperature (protected from direct sunlight) in dry, clean, and well-ventilated areas to minimize the degradation of bioactive substances. The drying process was conducted at a temperature of 25°C for 2 weeks, until the material was completely dehydrated. Each sample of dried plant material was then transferred into a labeled paper bag and stored in a cool, dark place at room temperature for future analysis.

In this study, the antimicrobial activity of the EO and extracts from the flowering tops of R. montana was evaluated against five bacterial strains and five fungal strains. These microorganisms are contaminants and pathogens widely identified in various human pathologies (Table 1). All strains were successively revitalized in Mueller-Hinton and Sabouraud broths, subcultured, and stored in a 20% glycerol stock at −80°C.

In this study, male and female Swiss albino mice (weighing between 30 and 35 g) as well as Wistar rats (weighing between 200 and 250 g) were used as animal models. The animals were initially weighed and then housed individually in polypropylene plastic cages in a controlled temperature environment, with a 12-h light/dark cycle and unlimited access to food and water. All experimental procedures and protocols were meticulously conducted in strict adherence to ethical guidelines and regulations to ensure animal welfare, with the study being reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the Faculty of Sciences Dhar El Mehraz, Sidi Mohamed Ben Abdallah University, Fès, Morocco, under reference #04/2019/LBEAS, in accordance with institutional ethical standards and the European Economic Community (EEC) Directive 86/609/EEC (Council Directive 86/609/EEC of 24 November, 1986).

Phenolic compounds are considered multifunctional agents in medicinal chemistry, pharmaceutical development, and various synthetic processes (Rappoport, 2004; Makarem et al., 2019; 2023; Klika et al., 2021). Thus, studying the phenol content in plant extracts can reveal their biological profile (Alara et al., 2021).

In this section, the extraction of phenolic compounds was carried out using decoction and solid-liquid extraction with the aid of a Soxhlet apparatus. The decoction process was performed by adding 30 g of plant powder to 600 mL of distilled water. The resulting mixture was heated and brought to a boil under stirring for 1 h at 80°C. Subsequently, the decanted mixture was filtered under low pressure, and the decoction extract was recovered in powdered form in a glass vial. After drying the extract in an oven at 70°C, the decoction extract was stored in a tinted glass bottle, protected from light. As for the second extraction method, the Soxhlet apparatus was employed. In this process, 30 g of plant powder were placed in a cartridge and brought into contact with extraction solvents, specifically ethanol (70/30) and methanol (70/30) solutions. The extracts were concentrated using a rotary evaporator after several extraction cycles. The extracts were coded in accordance with Supplementary Table S2, which outlines the codification of R. montana extracts.

The quantification of total phenols in various extracts was performed using the Folin-Ciocalteu method, as described by Singleton and Rossi (1965). The absorbance was recorded using a UV mini-1240 spectrophotometer set at 760 nm and compared to a blank. A calibration curve was established using gallic acid as a positive control. The results were expressed in milligram gallic acid equivalent per gram of extract (mg GAE/g). Each test was performed in triplicate.

The quantification of flavonoids was performed using the method established by Djeridane et al. (2006). The flavonoid content was calculated based on a calibration curve using quercetin as the standard. The results are expressed in milligrams of quercetin equivalent per gram of extract (mg QE/g). Each test was conducted in triplicate.

The quantification of condensed tannins was carried out using the vanillin method, as described by Price et al. (1978). In this technique, a vanillin/methanol solution (4% w/v) was mixed with varying quantities of (+)-catechin solution (2 mg/mL) and manually agitated. Each concentration was then placed in a test tube containing 1.5 mL of hydrochloric acid. The reaction mixture was left at room temperature for 20 min. Absorbance was measured at 499 nm using a UV-visible spectrophotometer, with reference to a blank. The condensed tannin content in our samples was calculated using the calibration curve of catechin as the standard (Y = 0.7421X + 0.0318; R² = 0.998). The tannin content is expressed in milligrams of catechin equivalent per gram of extract (mg CE/g).

The analysis of phenolic compounds in the three extracts of R. montana was conducted using high-performance liquid chromatography coupled with Q Exactive Plus mass spectrometry, employing electrospray as the molecular ionization method (HPLC/UV-ESI-MS). The analysis was performed on an UltiMate 3000 HPLC system (Thermo Fisher Scientific, Sunnyvale, CA, United States) equipped with an autosampler. The autosampler was set to maintain the samples at 5°C. The HPLC system utilized a reversed-phase C18 column with a column temperature of 40°C (Lichro CART, Lichrospher, Merck, Darmstadt, Germany, 250 × 4 mm, ID 5 µm). The mobile phase consisted of solvent A: 0.1% formic acid in water (v/v) and solvent B: 0.1% formic acid in acetonitrile (v/v), with degassing performed ultrasonically. At 20, 25, 26, and 30 min, the gradient composition transitioned from 2% B to 30%, 95%, 2%, and 2% B, respectively. The flow rate was 1 mL/min, and the injection volume was 20 µL. Broadband collision-induced dissociation (bbCID) detection was carried out on a Maxis Impact HD (Bruker Daltonik, Bremen, Germany) following negative electrospray ionization. A diode array detector L-2455 (Merck-Hitachi, Darmstadt, Germany) was also employed for UV detection, with scanning in the range of 190–600 nm and acquisition at three wavelengths: 280, 320, and 360 nm. The parameters used included a capillary voltage of 3,000 V, a drying gas temperature of 200°C, a dry gas flow rate of 8 L/min, a nebulizer gas pressure of 2 bars, and an offset plate voltage of 500 V. Nitrogen was used both as the nebulizing gas and the desolvation gas. The m/z range for MS data acquisition was 100 to 1,500. Data collection and analysis were performed using the Thermo Scientific Chromeleon 7.2 Chromatography Data System (CDS). By examining the mass spectra of the eluted molecules, the eluted compounds were investigated.

The Minimum Inhibitory Concentration (MIC) was determined using 96-well microplates and the reference microdilution method (Balouiri et al., 2016). MIC is defined as the lowest concentration of essential oil required to completely inhibit the visible growth of the tested microorganism during incubation. A series of dilutions were prepared from a stock solution of the essential oil dissolved in 10% DMSO, resulting in concentrations ranging from 5 to 0.93 × 10⁻² mg/mL for each essential oil. These dilutions were prepared in a final volume of 100 µL in Sabouraud broth for fungi and Mueller-Hinton medium for bacteria. Subsequently, 100 µL of microbial inoculum was added to each dilution step, with a final concentration of 10⁶ CFU/mL for bacteria or 10⁴ CFU/mL for fungi. Ten microliters of resazurin were added to each well to measure bacterial growth after a 24-h incubation at 37°C. The color change from purple to pink after a second incubation at 37°C for 2 h indicated microbial growth. The MIC value was determined as the lowest concentration that prevented the color change of resazurin. Growth and sterility controls were included in the 11th and 12th wells, respectively. This test was performed twice for the essential oil. For comparison, 250 mg of terbinafine, a standard antifungal agent used in the study, was ground and dissolved in 2 mL of 10% DMSO. The Minimum Bactericidal Concentration (MBC) and Minimum Fungicidal Concentration (MFC) were determined by transferring 10 µL from wells showing no visible growth onto Mueller-Hinton agar (for bacteria) or Sabouraud broth (for fungi) and incubating for 24 h at 37°C. The lowest concentration of the sample that resulted in a 99.99% reduction in CFU/mL compared to the control was designated as the MBC or MFC. Additionally, the MBC/MIC or MFC/MIC ratio was calculated for each extract to evaluate its antimicrobial potency. If the ratio was less than 4, the essential oil was considered bactericidal/fungicidal, while a ratio greater than 4 indicated a bacteriostatic/fungistatic effect (Bissel et al., 2014). The biological assays were conducted in accordance with globally recognized standardized protocols, following the guidelines of the CLSI (Clinical and Laboratory Standards Institute) for antimicrobial testing.

The anti-inflammatory potential of each extract was evaluated based on Winter et al.’s method (1962) (Winter et al., 1962), with certain modifications. This experiment was repeated twice. Carrageenan at a concentration of 1% was dissolved in saline solution and used as the edema-inducing agent. A total of 35 rats were randomly allocated into 7 groups, each consisting of 5 rats. The difference in footpad circumferences before and after carrageenan injection (Sigma-Aldrich, Ref. 3B-C1804, Barcelona, Spain) was measured in millimeters (mm) at 3, 4, 5, and 6 h using calipers (Küpeli and Yesilada, 2007). The inhibitory activity of each treatment was calculated using the following Formula 7: P e r c e n t   i n h i b i t i o n = 100   1 − a − x b − y (7) where:

a: Mean paw volume after carrageenan administration.

x: Mean paw volume before the injection.b: Mean paw volume of control rats after the injection.

y: Mean paw volume of control rats before the injection.

This formula quantifies the anti-inflammatory effect by comparing the changes in paw volume between treated and control groups over time.

The antinociceptive effect of each extract was assessed by counting the number of writhes induced in mice following the intraperitoneal injection of a 0.7% acetic acid solution (10 mL/kg). The tested extracts were administered orally to the mice 1 h prior to the acetic acid injection. The number of writhes was recorded for 30 min after the acetic acid injection (Hernández-Pérez and Rabanal, 2002). This experiment was repeated twice. The percentage inhibition (PI%) of the writhes was calculated using the following Formula 8: P I % = N C − N T N C * 100 (8) where:

NC: Number of writhes in the negative control group.

NT: Number of writhes in the tested groups.

This formula quantifies the antinociceptive effect by comparing the writhes in treated groups to those in the control group, providing a measure of the extract’s ability to reduce pain-induced behavior.

The results were expressed as mean values ± standard error of the mean (SEM) to ensure accurate representation of data variability. Statistical analyses were performed using one-way analysis of variance (ANOVA), followed by Tukey’s post hoc test to determine significant differences between groups. All analyses were conducted using GraphPad Prism 9 (version 9.5.1, San Diego, CA, United States), a widely recognized statistical software for biological and pharmacological research. A significance threshold of p < 0.05 was applied to establish statistical relevance, ensuring that only robust and meaningful differences were considered in the interpretation of the results. Correlations between phenolic compound content and antioxidant activities were examined using R software (version 4.4.2).

The hydrodistillation extraction of the flowering tops of R. montana yielded an average of 2.07% (v/w) of a yellowish essential oil with a strong aromatic odor (Table 4). This EO yield is higher than those reported in Algeria by Slougui et al. (2023) (0.67%). However, it is lower than those noted in Morocco by Barbouchi et al. (2021) (2.24% ± 0.02%) and in Algeria by Zeraib et al. (2021) (2.5%). Variations in essential oil yields arise from several factors, including the plant’s geographical location, climatic conditions, harvesting period, and the extraction method used. These differences in yield could be attributed to the specific characteristics of the plant material and the extraction conditions employed in each study.

Physicochemical properties such as acid value, peroxide value, and iodine value are among the key methods for verifying and controlling the quality of the essential oil extracted from the flowering tops of R. montana. These parameters are determined according to specific protocols. The results are presented in Table 5 and summarized below.

The essential oil extracted from the studied plant recorded a density of 0.97. The AFNOR standard (2005) recommends a density between 0.906 and 0.990, suggesting that the essential oil extracted from the flowering tops of R. montana meets the standards for a very high-quality essential oil.

The acid value refers to the content of free fatty acids present in the EO. Indeed, fresh essential oils contain very little free acid (Fauconnier, 2006). Moreover, an essential oil is considered well-preserved if its acid value is less than 2. In this study, the EO extracted from the flowering tops of R. montana recorded an acid value of 1.12. Therefore, we conclude that the studied EO is well-preserved against any factors promoting degradation. Among the physicochemical parameters determined in this study is the iodine value, which measures the degree of unsaturation of the EO and directly affects its stability. In this work, the EO extracted from the flowering tops of R. montana. recorded an iodine value of 13 g/100 g of oil. As for the peroxide value, it is a crucial indicator of the quality and stability of the EO, reflecting its level of oxidation. The analyzed EO showed a peroxide value of 13.24 ± 0.98 mEq/kg. By comparing our results with the commercial standards, CODEX STAN 210-1999, we observe that the studied EO complies with the standards, allowing us to classify it as of good quality.

The chromatogram of the essential oil (EO) of R. montana reveals a highly prominent main peak, clearly distinguishable from other less intense peaks (Supplementary Figure S1). This peak, detected at a retention time (RT) of 21.40 min, was identified as corresponding to the major compound 2-undecanone. Furthermore, the use of peak deconvolution enabled the effective separation of co-eluting compounds, thus ensuring a more accurate and detailed representation of the essential oil’s chemical profile.

Based on chromatographic data and mass spectra, the chemical analysis identified 25 components, which together make up the entire composition of the essential oil (Table 6). The five most significant compounds, accounting for 95.55% of the total, are 2-undecanone (81.16%), decyl propanoate (9.33%), 2-dodecanone (1.99%), pulegone (1.86%), and 2-decanone (1.21%). It appears that the essential oil under study lacks monoterpenes (3.21%) and sesquiterpenes (0.49%), but contains other chemical classes (96.3%). These classes are primarily composed of aliphatic ketones (88.6%), followed by esters (9.74%). Meanwhile, alcohols (1.14%) and hydrocarbons (0.52%) are nearly absent (Figure 2).

The chemical composition described in this study shows similarities with the profile of R. montana from Morocco, particularly in terms of the dominance of 2-undecanone: 85.76% reported by Flouchi et al. (2024), and 63.97% by Benali et al. (2020b). In Algeria, comparative studies on the essential oils of R. montana collected from different regions have shown slight variations in the major components. Mohammedi et al. (2019), emphasized the impact of climatic conditions on the chemical composition of the essential oil, with its main components being 2-undecanone (27.2%–81.7%), 2-nonanone (1.9%–39.5%), and 2-nonanyl acetate (trace–24.8%). Zellagui et al. (2012) found that the major components of the first essential oil were 2-undecanone (60.1%), 2-nonanone (8.6%), monoethylhexyl phthalate (6.4%), and decanone (6.2%), while the second essential oil had 2-undecanone (90.4%), 2-nonanone (4%), and decanone (1.4%).

It is equally important to highlight the influence of the developmental stage and the condition of the plants (whether fresh or dried) on the composition of EO. In this context, the fresh aerial parts collected in eastern Algeria are primarily composed of undecan-2-one (37.74%), resorcinol (27.66%), and 2-acetoxytetradecane (9.19%) (Djarri et al., 2013), whereas these compounds were not identified in other Algerian samples. In Tunisia, Hammami et al. (2015) found that the EO extracted from dried leaves was predominantly composed of 1-butene (38.33%), methylcyclopropane (15.47%), 2-butene (22.56%), and caryophyllene oxide (8.18%). Conversely, in the same country, Yosra et al. (2019) reported a chemical profile of EO extracted from dried leaves that was dominated by the same major compounds found in Moroccan and Algerian samples, particularly 2-undecanone (86.77%), followed by 2-decanone (4.91%) and 2-nonanone (23.62%).

It is important to highlight that the abundance of 2-undecanone in the EO of Ruta species has made it a relevant chemotaxonomic marker for this genus (Bennaoum et al., 2017). Thus, 2-undecanone appears to be an indispensable compound in the EO of R. montana, given its presence in all the EOs of the species from Morocco, Algeria, and Tunisia, regardless of the region or the plant’s growth conditions. The variation in cultivation environments is mainly reflected in the differing percentages of this compound depending on the regions.

The results of the phytochemical screening on the plant powder of the flowering tops of R. montana are illustrated in Table 7.

This qualitative analysis revealed the diversity of primary and secondary metabolites in R. montana. The plant is particularly rich in lipids and proteins, and moderately rich in reducing sugars and glycogens. Regarding secondary metabolites, the various extracts showed a moderate presence of alkaloids, flavones, and both gallic and condensed tannins. A positive reaction was also observed for saponosides, sterols, and triterpenes, whereas mucilages showed a negative reaction. The richness of this plant’s extracts in secondary metabolites explains its use in traditional medicine to treat numerous diseases. The results of this study align with those of Drioiche et al. (2020a), who found that the aerial parts of Moroccan R. montana L. contained alkaloids, mucilages, triterpenes, sterols, flavonoids, leucoanthocyanins, and tannins. Similarly, Drioueche et al. (2021) discovered that the polar extract of the aerial parts of mountain rue from Algeria contained alkaloids, flavonoids, tannins, and coumarins.

The chromatographic profile, shown in Figure 3, displays the peaks of phenolic and non-phenolic compounds derived from the decoction and hydroethanolic and hydromethanolic extracts of the flowering tops of R. montana, along with their retention times and relative abundances. The HPLC/UV-ESI-MS technique was used to evaluate the decoction, hydromethanolic, and hydroethanolic extracts of R. montana. By analyzing the mass spectra in parallel with the chromatogram, a total of 35 molecules were identified and are listed in Table 8. The chromatographic profile highlights the diverse richness of the analyzed extracts in various molecules.

The distribution of chemical classes in the extracts of R. montana, as presented in Figure 4, reveals that phenolic compounds predominate across all extracts, with the highest proportion observed in the hydromethanolic extract (36.05%), followed by the decoction (32.33%) and the hydroethanolic extract (31.91%). Phenolic acids are most abundant in the decoction (36.67%), demonstrating the efficiency of water as a solvent for their extraction, whereas flavonoids are better extracted using mixed solvents such as hydromethanolic and hydroethanolic solutions (37.02% and 38.33%, respectively). Coumarins, although present in smaller quantities, are slightly more concentrated in the hydromethanolic extract (0.65%), while compounds classified as “others” are primarily found in the decoction (2.50%). These findings highlight the influence of solvents on the extraction process, with mixed solvents favoring flavonoids and water being more suitable for phenolic acids. From a biological perspective, the phenolic acids and flavonoids, owing to their antioxidant and anti-inflammatory properties, offer promising prospects for therapeutic and nutraceutical applications.

The primary compounds identified in the extracts include rosmarinic acid 3′-glucoside, which is present in significant quantities in all extracts, reaching up to 23.8% in the hydroethanolic extract. p-Coumaroylquinic acid is also highly abundant, with a concentration of 18.4% in the hydroethanolic extract. Ferulic acid stands out due to its dominance in the hydroethanolic extract, where it reaches 8.44%. Embelin is notably present in all extracts, with a maximum concentration of 6.25%. Quercitrin, a major flavonoid, accounts for up to 15.5% in these extracts. Additionally, we recorded a content of Rhamnetin 3-rhamnoside in the hydromethanolic extract with a concentration of 6.27%. Furthermore, certain specific compounds, such as (+)-Gallocatechin, are detected exclusively in the hydroethanolic extract. Figure 5, which presents the chemical structures of the main identified compounds, complements this analysis by providing precise structural details.

The analysis of compounds by mass spectrometry in negative ion mode (ESI-MS) allows for the exploration of their specific fragmentation patterns, thereby providing valuable insights into their molecular structure. The ESI-MS spectrum of Rosmarinic acid 3′-glucoside displays a molecular ion [M−H]⁻ at m/z 521, fragmenting through the loss of a glucose residue (162 u) to m/z 365, followed by the loss of a rosmarinic acid fragment to reach m/z 197. The p-Coumaroylquinic acid, with a [M−H]⁻ ion at m/z 337, suggests the loss of coumaroyl or quinic acid fragments. The fragmentation of Ferulic acid generates fragments at m/z 175 and 147, corresponding to the loss of a water molecule (18 u) and a methoxy group (28 u), respectively. Quercitrin exhibits a [M−H]⁻ ion at m/z 447, with a typical flavonoid fragmentation involving the loss of a rhamnose residue (162 u) to form the aglycone quercetin (m/z 285), followed by an aromatic cleavage at m/z 151. Regarding Embelin, the [M−H]⁻ ion at m/z 293 loses a bulky fragment (109 u) to reach m/z 184. (+)-Gallocatechin fragments into m/z 275 (loss of 30 u, corresponding to a water molecule), then into m/z 125 through cleavage of the flavonoid ring. Finally, Rhamnetin 3-rhamnoside presents a [M−H]⁻ ion at m/z 461, fragmenting through the loss of a rhamnose residue (146 u) to form the aglycone rhamnetin (m/z 315), followed by an aromatic cleavage at m/z 151. These results highlight common fragmentation mechanisms, including the loss of carbohydrate residues, specific functional groups, and cleavages at aromatic rings or side chains, enabling precise characterization of the studied molecules.

In the literature, the phenolic profile of R. montana has been scarcely studied. Khadhri et al. (2017) reported the abundant presence of flavonoid-type polyphenols in the decoction, as well as phenolic acids in the decoction of R. montana originating from Tunisia. Szewczyk et al. (2023) noted the existence of catechin in the methanolic extract of R. montana L. cultivated in vitro. Toker and collaborators (Toker et al., 1998) determined the rutin content in the methanolic extract of the aerial parts of R. montana L. from Turkey, using the HPLC method, to be 6.79 µg/mL.

Moreover, a study conducted by Pacifico et al. (2016) demonstrated that the hydroalcoholic extract of R. graveolens leaves collected in Italy contains rutin, isorhamnetin-3-O-rutinoside, 4-O-p-coumaroylquinic acid, and 4-O-feruloylquinic acid. Additionally, this study highlighted the variation in the chemical composition of this species depending on the harvest season.

All the major compounds quantified in the three extracts of R. montana possess therapeutic properties. For example, rosmarinic acid glucoside exhibits significant anticancer, anti-angiogenic, antioxidant, anti-inflammatory, and antimicrobial properties (Budzianowski et al., 2023). In turn, p-coumaroylquinic acid is distinguished by its numerous biological activities, including antioxidant, anticancer, antimicrobial, antiviral, anti-inflammatory, antiplatelet aggregation, anxiolytic, antipyretic, analgesic, and antiarthritic effects (Pei et al., 2016). Additionally, quercitrin demonstrates potential antioxidant, anti-inflammatory, antiviral, and anticancer properties, as well as the ability to alleviate certain cardiovascular diseases (Ulusoy and Sanlier, 2020). Ferulic acid is also recognized for its antioxidant, anticancer, anti-inflammatory, hepatoprotective, antimicrobial, and antiviral effects (Pyrzynska, 2024). Similarly, rhamnetin possesses various pharmacological properties, including antioxidant, anticancer, anti-inflammatory, antiviral, and antibacterial activities (Medeiros et al., 2022). Finally, embelin exhibits antimicrobial, antioxidant, analgesic, anti-inflammatory, anxiolytic, antifertility, antimalarial, and anticancer properties (Basha et al., 2022).

Figure 6 presents the average extraction yields, as well as the polyphenol, flavonoid, and catechin tannin contents of R. montana, based on the extraction methods employed.

The extraction yields depend on both the solvent and the extraction method. We observe that aqueous methanol yielded the highest extraction yield (17.91%), followed by aqueous ethanol (17.39%). In contrast, decoction recorded the lowest extraction yield (16.16%). Regarding the quantification of polyphenols, flavonoids, and condensed tannins, their contents are expressed in mg GAE/g of extract, mg QE/g of extract, and mg CE/g of extract, respectively. It appears that the flowering tops of R. montana L. are rich in these molecules. Indeed, aqueous methanol is the most effective extractor of polyphenols and flavonoids for R. montana L., with average values of 67.32 mg GAE/g of extract and 26.14 mg QE/g of extract, respectively. This is followed by aqueous ethanol, which shows contents of approximately 55.26 mg GAE/g of extract and 21.39 mg QE/g of extract, respectively. However, the decoction recorded the lowest polyphenol and flavonoid contents, amounting to 40.35 mg GAE/g of extract and 9.92 mg QE/g of extract, respectively.

Unlike polyphenols and flavonoids, the condensed tannins of R. montana were more soluble in aqueous ethanol (58.27 mg CE/g of extract) than in the decoction (58.27 mg CE/g of extract) and aqueous methanol (39.47 mg CE/g of extract).

The contents of Mountain Rue extracts found in this study are significantly higher than those reported by Szewczyk et al. (2023), where the polyphenol and condensed tannin contents of the methanolic extract prepared from in vitro cultivated biomass were 41.61 mg GAE/g of extract and 10.97 mg CE/g of extract, respectively. Similarly, Drioiche et al. (2020a) reported that the polyphenol and flavonoid contents of the hydro-methanolic extract from the aerial parts of Moroccan R. montana L. were 33.684 mg GAE/g of extract and 0.843 mg QE/g of extract, respectively. Merghem and Dahamna (2020) noted that the ethyl acetate extract of R. montana L. from Algeria contained the highest quantities of polyphenols, tannins, and flavonoids, with values of 257.1 µg gallic acid equivalent/mg of extract, 251 tannic acid equivalent/mg of extract, 117.4 µg quercetin equivalent/mg of extract, and 139.5 µg rutin equivalent/mg of extract, respectively. Our results are lower than those reported by Benali et al. (2020a), where the highest phenolic compound and flavonoid contents were found in the methanolic extract of R. montana collected from northeastern Morocco, with values of 117.70 mg GAE/g of extract and 77.00 mg RE/g of extract.

The variation in polyphenol and flavonoid levels among the analyzed extracts can be explained by the difference in the solubility of phenolic compounds, which is influenced by the extraction temperature as well as the structure of these compounds. Additionally, the choice of extraction method can also have a significant impact on the extraction and quantification of polyphenols, flavonoids, and condensed tannins (Alara et al., 2021; Lama-Muñoz and Contreras, 2022; El Mannoubi, 2023).

The results obtained previously demonstrate the relationship between phenolic compounds and the antioxidant activity of the three R. montana extracts. This is illustrated in this context (Table 9).

The linear correlation coefficients range from −0.700 to 0.9997. The antioxidant capacity of the extracts appears to be influenced primarily by the total polyphenol and flavonoid contents. These compounds showed high coefficients in comparison to condensed tannins (0.913 ≤ r ≤ 0.9995). In contrast, a negative correlation was observed between condensed tannins and the three antioxidant activities, namely the DPPH method (r = −0.5), the FRAP method (r = −0.478), and total antioxidant capacity (r = −0.7). This result leads us to suggest that condensed tannins do not influence the antioxidant activity of R. montana L. extracts. Similar results have linked the group of condensed tannins to phenolic compounds (r = −0.795) and flavonoids (r = −0.678).

On the other hand, we observe a very strong correlation between the IC₅₀ and EC₅₀ values from the DPPH and FRAP tests (r = 0.9997), between the IC₅₀ values of DPPH and TCA (r = 0.969), and between the EC₅₀ values of FRAP and TCA (r = 0.962). This indicates that the antioxidant activities demonstrated by these three tests are likely provided by the same active molecules.

Tables 10, 11 present the minimum inhibitory concentrations (MIC), minimum fungicidal concentrations (MFC), and minimum bactericidal concentrations (MBC) expressed in mg/mL for the extracts and EO of R. montana against the tested microorganisms.

According to the results of the antifungal activity illustrated in Table 10, the hydromethanolic extract and the EO of R. montana tested against the four Candida strains and Aspergillus niger revealed promising antifungal activity, while the decoction and hydroethanolic extract showed moderate activity.

For the three extracts, the MIC and MFC values ranged from 3.13 to 50 mg/mL and varied between different fungi; in some cases, they were equal, indicating a fungicidal action. Aspergillus niger was the most sensitive, with MIC and MFC values of 12.5, 6.25, and 3.13 mg/mL for the decoction, hydromethanolic extract, and hydroethanolic extract, respectively. However, against the Candida yeasts, the decoction and hydroethanolic extract exhibited moderate action, with MIC and MFC values of 50 mg/mL, except for Candida albicans and Candida tropicalis, where the activity of both extracts was null.

The hydromethanolic extract also exhibited both fungistatic and fungicidal powers against Candida yeasts at the same concentration of 50 mg/mL, except for C. tropicalis, for which the MIC and MFC were 25 mg/mL.

The essential oil EO, on the other hand, demonstrated a stronger fungicidal effect on the yeasts than the tested extracts. The MIC and MFC values ranged from 2.34 to 37.75 mg/mL. The minimum fungicidal concentration of 4.69 mg/mL was achieved against A. niger. The highest MFC of 37.5 mg/mL was recorded against Candida parapsilosis. Candida tropicalis was inhibited and destroyed at the same concentration of 9.38 mg/mL, while C. albicans and Candida dubliniensis showed MFC values of 18.75 mg/mL.

For the antibacterial activity, the results obtained from Table 11 show that the extracts and the EO of R. montana exhibit activity against bacterial strains. Furthermore, the determination of the MIC of the extracts and EO revealed variable levels of action. Among the three extracts tested, the aqueous ethanolic extract was more active compared to the aqueous methanolic extract and the decoction. The latter was found to be inactive against all the tested bacteria, except for E. cloacae and S. epidermidis, for which the MIC and MBC were 50 mg/mL.

The methanolic and aqueous ethanolic extracts exhibited both bacteriostatic and bactericidal actions at a concentration of 25 mg/mL against E. cloacae and S. aureus. Additionally, both extracts showed bactericidal activity at 50 mg/mL against S. epidermidis. Moreover, the hydroethanolic extract demonstrated bactericidal activity at 50 mg/mL against the Klebsiella pneumoniae strain, while the aqueous methanolic extract was inactive.

Thus, the three extracts tested did not exhibit any effect on wild Escherichia coli. In contrast, the essential oil inhibited and destroyed the strains Enter. cloacae, Kleb. pneumoniae, and wild E. coli at a concentration of 50 mg/mL. S. aureus and S. epidermidis appeared less sensitive, showing higher MIC and MBC values of 100 mg/mL.

The variation in sensitivity of the tested microorganisms to the extracts of R. montana could be explained by the distinct composition of secondary metabolites in these extracts. On the other hand, it may be due to the absence of strongly antimicrobial molecules or the low potency of the antibacterial compounds present in the extracts.

The results of the antimicrobial activity of the EO of R. montana obtained are consistent with those of Drioiche et al. (2020b). These authors described the antibacterial activity of the EO from R. montana from Morocco, primarily containing 2-undecanone (82.62%), as moderate against Staphylococcus epidermidis, K. pneumoniae spp. pneumoniae, E. coli, Enterobacter cloacae, and Staphylococcus aureus STAIML/MRS/mecA/HLMUP/BLACT. However, the same study revealed a significant antifungal potential of this EO against eight yeasts (C. albicans, C. tropicalis, Candida glabrata, C. dubliniensis, Candida sp., Rhodotorula rubra, Trichosporon sp., Cryptococcus neoformans), three molds (A. niger, Fusarium sp., Penicillium sp.), and one dermatophyte (Trichophyton mentagrophytes), with MFC values of 1.8 mg/mL for yeasts, 0.9 mg/mL for molds, and 0.45 mg/mL for dermatophytes, respectively.

Benali et al. (2020a) reported the moderate antimicrobial action of the EO of R. montana from Morocco, predominantly composed of 2-undecanone (63.97%), against eight microbial strains, including Bacillus subtilis, S. aureus, E. coli, Pseudomonas aeruginosa, and C. albicans, with inhibition zone diameters ranging from 9.2 ± 0.5 mm to 18 mm. Flouchi et al. (2024) also revealed the modest antibacterial activity of the Moroccan R. montana EO, which contained 2-undecanone (85.76%), 2-nonanone (3.95%), 2-decanone (3.67%), and 2-dodecanone (1.94%), with MIC values ranging from 4% (v/v) for Pantoea to 8% (v/v) for S. aureus, P. aeruginosa, E. coli, Pantoea spp., K. pneumoniae, Escherichia hermannii, Stenotrophomonas maltophilia, except for K. pneumoniae, where no inhibition was detected.

Another study evaluated the antimicrobial activity of the EO of R. montana isolated from the aerial parts collected in Algeria on eight microbial strains (S. aureus, B. subtilis, Enterococcus faecium, E. coli, Klebsiella pneumonia, and P. aeruginosa) and two yeasts (C. albicans and Saccharomyces cerevisiae) using the agar disc diffusion method. The authors described the antimicrobial activity of this plant as moderate (inhibition zone diameters ≤ 18 mm) (Mohammedi et al., 2019).

Another study compared, using the agar disc diffusion method, the antibacterial activity of R. montana EO alone and in combination with five conventional antibiotics (gentamicin, amoxicillin, cefazolin, tetracycline, and amoxicillin/clavulanic acid against three pathogenic bacteria (S. aureus, P. aeruginosa, and E. coli). The results showed that the EO of R. montana from Algeria, whose main compounds are 2-undecanone (63.39%), 2-nonanone (5.65%), 2-acetoxytetradecane (4.94%), 2-decanone (4.47%), and 2-dodecanone (3.35%), had no or very weak antibacterial activity against E. coli and S. aureus, with MIC values ranging from 125 to 250 mg/mL. However, the combination of R. montana EO with antibiotics, particularly with amoxicillin and cefazolin, induced significant synergistic effects against all the bacterial strains tested. The inhibition zones for amoxicillin and cefazolin were 19.7–34 mm and 21.5–41 mm, respectively (Zeraib et al., 2021).

Generally, the antimicrobial power of EOs is related to their chemical composition. Some previous studies have reported the moderate antimicrobial activity of EOs from the Ruta genus (Coimbra et al., 2020; Nahar et al., 2021). In our study, the modest antimicrobial activity of R. montana L. EO could be attributed to the high percentage of aliphatic ketones, which represent 88.6% of the total chemical compounds, with the main compound being 2-undecanone (81.16%). Indeed, the antibacterial activity of this compound is known to be limited against bacterial strains (Gibka et al., 2009; Kunicka-Styczyńska and Gibka, 2010). On the other hand, this low activity could also be due to the multi-resistance of the strains.

This experiment was conducted to determine the ability of the aqueous extract and the EO to reduce inflammation following carrageenan injections to induce inflammatory pain. Starting from the third hour after the carrageenan injection, doses of the aqueous extract and EO of R. montana (100 and 300 mg/kg, 0.1 and 0.2 mL) showed a significant suppression (p < 0.001) of paw edema compared to the negative control group (Table 12).

However, only the highest concentrations of the aqueous extract and the essential oil (EO) significantly reduced the edema induced by the subplantar injection of carrageenan (p < 0.001 at 6 h). Indeed, the dose of 300 mg/kg of the aqueous extract and the volume of 0.2 mL of the EO provided the greatest protection against paw volume increases, with suppression values of 79% and 71%, respectively, at the sixth hour. In contrast, the doses of 100 mg/kg of the aqueous extract and the volume of 0.2 mL of the EO showed lower protection compared to the 300 mg/kg dose of the aqueous extract and 0.1 mL of the EO, with suppression values of 28% and 23%, respectively. This indicates that the effectiveness of the anti-inflammatory effects of the aqueous extract and EO of R. montana is dose-dependent, and injections at these higher doses are more effective as anti-inflammatory treatments.

When comparing the groups that received indomethacin at a dose of 10 mg/kg, a protective effect of 90% was observed. Although the anti-inflammatory effect of the aqueous extract at a dose of 300 mg/kg and the EO at a dose of 0.2 mL was lower than that of indomethacin (10 mg/kg), both showed comparable anti-inflammatory effects throughout the observation period. Furthermore, after 3 h, the EO and aqueous extract of R. montana, as well as the standard medication, showed significant anti-inflammatory efficacy. This could be attributed to the time required for both substances to reach their site of action.

Carrageenan is a phlogistic agent known for its classic biphasic effect. Its administration triggers an inflammatory process, characterized by pain, heat, redness, and especially by swelling of the rat’s hind paw, i.e., edema. The first phase is vascular and lasts between 90 and 180 min. It is characterized by the release of several mediators such as histamine and serotonin. In contrast, the second swelling phase, known as the cellular phase, extends from 270 to 360 min. It is mediated by the release of substances such as prostaglandins and cytokines (TNF-α and IL-1β) (Di Rosa et al., 1971), during which various cells, including neutrophils and monocytes, infiltrate the inflammatory site (Chen et al., 2018).

There is limited scientific information regarding the anti-inflammatory activity of R. montana L. Previous studies have described the anti-inflammatory effects of certain Ruta species. For example, the methanolic extract of R. graveolens leaves collected in India (400 mg/kg) showed a significant inhibitory effect on edema formation after carrageenan injection, with an inhibition percentage of 47.19% after 5 h, which is lower than our results (Kataki et al., 2014). Another group of researchers found that, in an acute model induced by carrageenan, the anti-inflammatory activity of the alkaloid fraction of Indian R. graveolens at a dose of 10 mg/kg exhibited superior anti-inflammatory activity (83%) compared to polyphenols (64%) and the standard drug used, diclofenac (70%) (Ratheesh et al., 2009). Another study suggested that the anti-inflammatory activity observed in the ethanolic extract of R. chalepensis could be attributed to the presence of flavonoids (Iauk et al., 2004).

Flavonoids, due to their structure, can exert effective anti-inflammatory activity by inhibiting the secretion of factors responsible for inflammation, such as histamine, prostaglandins, serotonin, and lipoxygenases (Oubihi et al., 2020; Abou Baker, 2022; Al-Khayri et al., 2022). Another study revealed that, in the presence of polyphenols, the production of prostaglandins, which are responsible for the onset of inflammation, is reduced (Yahfoufi et al., 2018). HPLC chromatographic analysis of plant extracts showed the presence of the following phenolic compounds: rhamnetin 3-rhamnoside, rosmarinic acid glucoside, quercitrin, p-coumaroylquinic acid, 4-di-O-galloylquinic acid, ferulic acid, and embelin (6.12%). The anti-inflammatory activity of these compounds, such as phenolic acids (Liu et al., 2023), flavonoids (Shahidi and Yeo, 2018), and benzoquinones (Martínez and Bermejo Benito, 2005; Basha et al., 2022), has already been demonstrated in previous studies.

Regarding the EO of R. montana, no previous studies have investigated its anti-inflammatory effect. However, prior research has reported the anti-inflammatory activity of EOs from certain Ruta species. The EO of R. chalepensis from Algeria, composed of 2-undecanone (35.51%), 1-decanol-2-methyl (8.62%), and 2-dodecanone (6.86%), significantly reduced carrageenan-induced edema. The researchers suggested that the anti-inflammatory effect of this essential oil results from the inhibition of inflammation mediators such as serotonin, prostaglandins, and histamine (Boudjema et al., 2018). For the EO of R. graveolens, Nonnenmacher et al. (2016) (Nonnenmacher et al., 2016) found that its oral administration at a dose of 100 mg/kg was effective in reducing the edema index and inflammatory process in mice.

The anti-inflammatory effect of the EO of R. montana observed in this study is primarily associated with its chemical composition. An in vivo study demonstrated that the EO of Houttuynia cordata Thunb., of which 2-undecanone is the major component, possesses anti-inflammatory activity. The study showed that 2-undecanone inhibits the formation of ear edema induced by xylene in a dose-dependent manner. Furthermore, its inhibition percentage of ear swelling is similar to that of aspirin at a dose of 8 mg/20 g, reaching 28% (Chen et al., 2014). Another study by Chen et al. (2014) also confirmed the ability of 2-undecanone to reduce xylene-induced ear edema. Li et al. (2011) reported that 2-undecanone inhibits, in a dose-dependent manner, the induction of TNF-α, NO, and H2O2 production by lipopolysaccharides. Therefore, the anti-inflammatory activity of the EO of R. montana observed in this study can be attributed to its predominant component, 2-undecanone (81.16%).

The abdominal writhing test induced by acetic acid, widely used for its high sensitivity, allows the detection of substances with analgesic effects. In this study, this experimental model is used to determine the analgesic effect of the aqueous extract and essential oil of R. montana. Table 13 illustrates the analgesic activity in mice treated with the essential oil and aqueous extract of R. montana as well as the reference analgesic, Tramadol (10 mg/kg).

The large number of abdominal writhings observed after the injection of acetic acid in the presence of distilled water reveals the algogenic effect of this chemical compound. All tested doses of the aqueous extract and essential oil of R. montana significantly reduced (p < 0.001) the abdominal writhings induced by acetic acid in rats compared to the negative control groups. A preliminary analysis of the results allowed us to conclude that the aqueous extract and essential oil of this plant exert an analgesic effect, the intensity of which varies depending on the dose administered. The aqueous extract (100 and 300 mg/kg), administered orally 1 h before the injection of the stimulus, significantly (p < 0.001) and progressively reduced the writhings of rats exposed to acetic acid (11.621%–23.371%). The analgesic activity of R. montana essential oil was found to be superior to that of the aqueous extract. Indeed, the maximal inhibition was achieved with a volume of 0.2 mL (44.554%), which remained lower than that of Tramadol (77.032%). We also observed that a 0.1 mL volume of the essential oil of this plant produced an analgesic effect close (42.820%) to that of 0.2 mL of the EO.

The abdominal writhings induced by acetic acid result from the activation of local peritoneal receptors (Ouédraogo et al., 2012), as well as the release of mediators such as prostaglandins (PGE2α, PGF2α) and cytokines (TNF-α, IL-1β, IL-8) (Bentley et al., 1983; Negus et al., 2006). The analgesic effect expressed by the aqueous extract and essential oil of R. montana L. indicates the presence of compounds capable of inhibiting the release of these chemical mediators.

In the literature, there is limited scientific data regarding the analgesic properties of Ruta species. The hexane extract of R. graveolens, using the acute tail immersion model, revealed a high analgesic effect of 62.1% (Micael et al., 2015). Similarly, the methanolic leaf extract of this species (100 mg/kg) significantly reduced by 54% the number of writhings induced by acetic acid (Loonat and Amabeoku, 2014). These researchers attributed this effect to the secondary metabolites present, such as flavonoids, tannins, saponins, and triterpenoid steroids.

Moreover, previous studies have demonstrated the analgesic effects of tannins, phenolic acids, and flavonoids (Sengupta et al., 2012; Ferraz et al., 2020; Zouhri et al., 2023). Therefore, the effect of our extract could be attributed to its richness in bioactive compounds, primarily phenolic acids and flavonoids.

Regarding essential oils, previous work suggests that their analgesic effects are due to their main constituents and a possible synergy between these chemical components. Their antinociceptive activity relies on peripheral (anti-inflammatory) and/or central mechanisms (Sarmento-Neto et al., 2016).

R. montana, a plant native to the Mediterranean region, has a long history of medicinal use and is known for its various pharmaceutical activities due to the presence of bioactive compounds. This species contains a significant amount of alkaloids and furocoumarins (Milesi, 2001). Furthermore, ethnobotanical studies have reported cases of poisoning by R. montana, manifested by respiratory diseases, skin toxicity, digestive disturbances, and neurological issues (Mitra et al., 2020; Belhaj et al., 2021). In this context, we investigated the subacute toxicity of the essential oil and aqueous extract at doses previously tested for their anti-inflammatory and analgesic activities. The variation in the relative organ weights, as well as biochemical analyses, highlighted the consequences of oral treatments with the essential oil (at 0.1 and 0.2 mL) and the aqueous extract of R. montana L. (at 100 and 300 mg/kg) on renal and hepatic functions.