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Phytochemical Screening, Antimicrobial and Antioxidant Activity of Methanolic Extract of Juniperus phoenicea Leaves

A
A. Derradjia1,2,*
S
S. Deboucha3
Z
Z. Meskini4
1Department of Biological Sciences, Faculty of Natural and Life Sciences, University of Ahmed Zabana Relizane, Algeria.
2Laboratory of Valorization and Bioengineering of Natural Resources, Department of Natural and Life Sciences, Faculty of Sciences, University of Algiers 1, Algeria.
3Department of Natural and Life Sciences, Faculty of Sciences, University of Algiers 1, Algeria.
4Department of Agricultural Sciences, Laboratory of Environment, Natural Plant Substances and Food Technology, University of Ahmed Zabana Relizane, Algeria.

ABSTRACT

Background: As part of the valorization of Algerian medicinal flora, Juniperus phoenicea has attracted attention due to its diverse pharmacological properties. The current investigation aims to evaluate the phytochemical profile, antimicrobial potential and antioxidant activity of the methanolic extract of J. phoenicea leaves.

Methods: J. phoenicea’s methanolic extract was subjected to various phytochemical tests. Antimicrobial activity was tested against six conventional pathogenic bacterial strains and one fungal strain using the disc-diffusion technique. The minimum inhibitory concentration was determined using the agar dilution method. Antioxidant activity was estimated by DPPH radical scavenging assay.

Result: The phytochemical screening of the methanolic extract indicated the presence of many secondary metabolite categories, including tannins, flavonoids, saponins and terpenoids. Antimicrobial activity was evaluated using the disc diffusion method and the bacterial strains Staphylococcus aureus and Listeria monocytogenes were shown to be the most sensitive. However, no antimicrobial activity was detected against Pseudomonas aeruginosa, Escherichia coli or the fungal strain Candida albicans. The minimal inhibitory concentration (MIC) was determined using the solid medium dilution technique and the results ranged from 1.56 to 12.5 mg/mL. The DPPH scavenging capability indicated that the methanolic extract has modest antioxidant activity, with an IC50 value of 0.82 mg/mL.

INTRODUCTION

For thousands of years, human have used various plant species found in its natural environment to treat and cure different diseases (Amrati et al., 2021; Mottaghipisheh et al., 2024). Plants contain a large variety of secondary metabolites, making them an immense source of chemically diverse substances with a wide range of biological activities (Erenler et al., 2019; Divekar et al., 2022; Gbenou et al., 2024). Among these bioactive constituents, alkaloids, terpenes and phenolic compounds are particularly noteworthy for their pharmacological importance and multiple health benefits (Kebede et al., 2021). Numerous epidemiological studies have highlighted that a diet rich in plant polyphenols may help reduce the risk of developing cancer, cardiovascular diseases, diabetes, osteoporosis and neurodegenerative disorders (Chang et al., 2022; Omidfar et al., 2023). Some of them are also used as ingredients in the food, pharmaceutical and cosmetic industries (Mayyas et al., 2021).
       
The alarming rise of antimicrobial resistance has intensified the global search for new, effective and safe antimicrobial agents from natural sources. Medicinal and aromatic plants have thus emerged as a promising alternative for developing novel bioactive molecules with fewer side effects (Khameneh et al., 2019; Aici and Benmehdi, 2021; Haile et al., 2022; Mishra et al., 2025).
       
The genus Juniperus (family Cupressaceae) is among the most taxonomically diverse conifer groups, comprising approximately 67 species and 28 varieties distributed mainly across subtropical and temperate regions of the Northern Hemisphere, with some extending into the Southern Hemisphere (Chelouati et al., 2023; Liu et al., 2024). Juniperus phoenicea is a resinous, aromatic shrub or small tree with a wide distribution across southern Europe, temperate and subtropical Asia, the Atlantic coast and North Africa (Ennajar et al., 2009; Mehira et al., 2021). This species is valued for its medicinal properties and traditional uses, leaves of Juniperus phoenicea are commonly prepared as a decoction to treat diabetes, diarrhea, rheumatism and digestive disorders. Furthermore, a combination of leaves and berries is traditionally employed as an oral hypoglycemic agent (Chelouati et al., 2023).
       
Algeria, with its remarkable diversity of climatic zones, harbors an exceptionally rich flora that includes numerous aromatic and medicinal species (Walas and Taib, 2022). This biodiversity offers a valuable reservoir of bioactive compounds with significant pharmacological potential.
               
In this context, the present study aims to evaluate the phytochemical composition, antimicrobial activity and antioxidant potential of the methanolic extract of Juniperus phoenicea leaves collected from northwestern Algeria. The results are expected to contribute to the valorization of this native species as a potential source of natural therapeutic agents.

MATERIALS AND METHODS

Plant material
 
Leaves of J. phoenicea were collected in March 2022 in the Sidi Khatab district of Relizane, Algeria. The plant was used after being identified and verified by a taxonomist from the Department of Natural and Life Sciences, University of Algies 1, Algeria. The leaves were air-dried at ambient temperature in a dark, well-ventilated environment, ground into a fine powder and stored in sterile amber glass bottles until further use.
 
Preparation of methanolic extract
 
The plant material was extracted using a soxhlet under continuous agitation for 24 hours. 30 mg of plant powder were dissolved in 100 mL of methanol. The obtained extract was concentrated under reduced pressure using a rotary evaporator and stored at 4°C until analysis (Zarei Yazdeli et al., 2021). The extraction yield was calculated according to the formula:


Where,
WD= The weight of the dried extract.
PP= The weight of the plant powder utilized.
 
Phytochemical screening
 
The methanolic extract was subjected to qualitative phytochemical analysis for the detection of major secondary metabolites including flavonoids, tannins, saponins, terpenoids and glycosides using standard procedures (Ingle et al., 2017; Berreghioua and Ziane, 2025a). These methods are based on colorimetric or precipitation reactions characteristic of each compound class.
 
Test for flavonoids
 
 An aqueous solution of the methanolic extract was treated with 10% ammonium hydroxide. Yellow fluorescence showed the presence of flavonoids.
 
Test for tannins
 
A 0.5 mg sample of the extract was boiled in 20 mL of distilled water then filtered. The addition of a few drops of 0.1% ferric chloride produced a brownish-green or blue-black colouration, confirming the presence of tannins.

Test for terpenoids
 
Five millilitres of extract were mixed with 2 mL of chloroform, followed by the careful addition of concentrated sulfuric acid. The formation of a brownish-red interface indicated the presence of terpenoids.
 
Test for saponins
 
Ten millilitres of the diluted extract were vigorously shaken in a graduated cylinder for 15 minutes. A stable 2 cm foam layer persisting for 15 minutes indicated the presence of saponins.
 
Test for glycosides
 
In total, 50 mg of extract was hydrolyzed with strong hydrochloric acid in a water bath for 2 hours before being filtered. The hydrolysate underwent the Borntrager test. To 2 mL of filtered hydrolysate was added 3 mL of chloroform. The chloroform layer was separated after shaking the mixture,then ammonia solution (10%) was added to it. Pink colouration suggests glycosides presence.
 
Antimicrobial activity
Preparation of standard strains
 
Six strains from the American type culture collection (ATCC) were kindly provided from the Laboratory of Valorization and Bioengineering of Natural Resources, University of Algiers 1, Algeria, to evaluate the antimicrobial properties of methanolic extract of J. phoenicea: Staphylococcus aureus 6538, Bacillus subtilis 6051, Enterococcus faecalis 29212, Pseudomonas aeruginosa 9027, Escherichia coli 8739, Listeria monocytogenes 19117 and Candida albicans 10231. Strains were revived in sterile Brain Heart Infusion broth (BHIB) and incubated at 37°C overnight.
 
Disc-diffusion method
 
Antimicrobial activity was evaluated using the disc diffusion method (Raoof et al., 2019). Bacterial suspensions (108 CFU/mL) and yeast suspension (106 CFU/mL) were evenly spread on Mueller-Hinton agar and Sabouraud dextrose agar plates, respectively. Sterile Whatman paper discs (6 mm diameter) were impregnated with 10 µL of the methanolic extract at concentrations of 100, 50, 25 and 12.5 mg/mL (dissolved in DMSO) and placed on the inoculated agar surfaces. Plates were incubated at 37°C for 24 hours for bacterial strains and 48 hours for the fungal strain. Antimicrobial activity was expressed as the diameter (mm) of the inhibition zone, including the disc diameter. Discs containing only DMSO served as negative controls.
 
Agar dilution method
 
The minimum inhibitory concentration (MIC) of the methanolic extract was determined by the agar dilution method (Zarei Yazdeli et al., 2021). Different concentrations of the extract (0.39 to 12.5 mg/mL) were incorporated into agar media. Standardized microbial inocula were applied to the agar surface and incubated under appropriate conditions. The MIC was defined as the lowest extract concentration that completely inhibited visible microbial growth.
 
Antioxidant activity
 
The antioxidant activity was carried out using the DPPH radical scavenging assay (Bougandoura and Bendimerad, 2013). 0.5 µL of each methanolic extract solution at various concentrations (from 0.0125 to 5 mg/mL) were added to 1.95 mL of DPPH methanolic solution (0.025 g/L). After 30 minutes of incubation in the dark at room temperature, absorbance was measured at 515 nm against a corresponding blank. The positive control was a solution of ascorbic acid, a standard antioxidant, whose absorbance was measured under identical circumstances as the sample. The percentage of inhibition (I%) was calculated and the IC50 value (concentration required to inhibit 50% of DPPH radicals) was determined by linear regression analysis.
 
Statistical analysis
 
The data obtained were analyzed using SPSS Statistical Software for Windows. All assays were performed in triplicate and quantitative variables were expressed as mean ± standard deviation (SD).

RESULTS AND DISCUSSION

Phytochemical screening
 
The methanolic extract of J. phoenicea leaves was characterised by a brownish-green colour. The extraction yield is around 19.61%, which is  considerably lower than the values reported by Medini et al., (2013) (28.77 and 39.23 %).  However, Farahat (2020) reported that methanolic extract showed a yield of 9.8% for J. phoenicea leaves collected from Egypt. The yield of plant extracts can be affected by several parameters, it can vary depending on the chemical composition and physical characteristics of the plant material as well as the method and conditions under which the extraction was carried out (Dai and Mumper, 2010; Elsherif et al., 2023; Berreghioua and Ziane, 2025b). Phytochemical analysis of J. phoenicea identified a range of bioactive compounds, including flavonoids, saponins, tannins and terpenoids. Likewise, the phytochemical screening of J. phoenicea from two different locations in Tunisia revealed the presence of tannins, flavonoids and saponins (Medini et al., 2013). These phytochemicals have essential biological functions, saponins can lower cholesterol, modulate the immune system and  inhibit tumor growth (Shraim et al., 2021). However, a study by Makhloufi et al., (2014) did not detect tannins in J. phoenicea leaves, which contradicts the findings of the current study. Additionally, no glycosides were found in J. phoenicea leaves.
 
Antimicrobial activity
 
The antimicrobial screening of the methanolic extract of Juniperus phoenicea leaves against six bacterial strains and one Candida albicans strain revealed varying degrees of sensitivity. The inhibition zone diameters ranged from 9 mm to 19.5 mm (Table 1). Listeria monocytogenes and Staphylococcus aureus were the most sensitive strains, exhibiting the largest inhibition zones. In contrast, Bacillus subtilis and Enterococcus faecalis showed moderate inhibitory responses. However, Escherichia coli, Pseudomonas aeruginosa and Candida albicans were resistant to the extract’s antimicrobial effect. These results indicate that Gram-positive bacteria are generally more sensitive than Gram-negative bacteria, as previously reported by Bouzouita et al., (2008) and Ait-Ouazzou et al. (2012). According to established criteria, an extract is considered active when it produces an inhibition zone ≥10 mm, whereas inhibition zones <7 mm are classified as inactive (Tekwu et al., 2012; Draoui et al., 2022). The higher sensitivity of Gram-positive bacteria can be explained by structural differences in the cell envelope. Gram-negative bacteria possess a complex, rigid outer membrane rich in lipopolysaccharides (LPS), which acts as an effective barrier limiting the diffusion of hydrophobic compounds. In contrast, Gram-positive bacteria lack this outer membrane and have a peptidoglycan layer whose relatively permeable structure does not provide sufficient protection against bioactive compounds (Burt, 2004).

Table 1: Antimicrobial activity of methanolic extract of J. phoenicea leaves.


       
Overall, our results are consistent with several previous studies. Similar to the findings of the present investigation, Draoui et al., (2022) reported that methanolic extract of J. phoenicea exhibited moderate to strong antibacterial activity against pathogenic microorganisms, with inhibition zone diameters ranging from 9 to 19 mm. Likewise, a study conducted in Libya in 2021 demonstrated that methanolic extract of J. phoenicea leaves showed a strong inhibitory effect against S. aureus (Alhadad et al., 2023). An additional study conducted in Libya reported a strong antibacterial activity of methanolic extract of J. phoenicea leaves against S. aureus and various wild strains of human pathogenic bacteria. In contrast to the findings of the present work, Elmhdwi et al., (2015) observed that the methanolic extract was also effective against E. coli and P. aeruginosa. On the other hand, C. albicans was found to be resistant, as previously reported by Zubi et al., (2025).
       
The minimum inhibitory concentration (MIC), defined as the lowest extract concentration that reduces microbial colony growth by 90%. The MIC was determined for strains that showed sensitivity using the disc diffusion assay. The results obtained show that L. monocytogenes and S. aureus had the lowest MIC values of 1.56 mg/mL and 3.12 mg/mL, respectively, while E. faecalis and B. subtilis exhibited higher MICs of 6.25 and 12.5 mg/mL, respectively. These results are consistent with previous findings reported in North Africa and the Mediterranean region. Draoui et al., (2022) reported MIC values between 0.78 and 6.25 mg/mL for methanolic extract of J. phoenicea against various Gram-positive and Gram-negative bacteria. Similarly, Zerrouki and Riazi (2021) observed a MIC of 10 mg/mL against S. aureus for hydromethanolic extract.
 
Antioxidant activity
 
The antioxidant capacity of the methanolic extract of J. phoenicea was determined using the DPPH technique. The results showed that the methanolic extract of J. pheonicea had a percentage of inhibition of around 90.59% (Fig 1). The synthetic molecule, ascorbic acid, showed a strong reduction at the first concentrations (Fig 2).

Fig 1: Percentage inhibition of DPPH by methanolic extract of J. phoenicea.



Fig 2: Percentage inhibition of DPPH by ascorbic acid.


       
In a previous study, the percentage of inhibition for the methanolic extract of J. pheonicea leaves harvested in Tunisia was 95.89%, which is slightly higher than that obtained in our research (Medini et al., 2013). In another study, the maximum antiradical power of the methanolic extract of J. pheonicea was 62.41% (Al-Mustafa et al., 2021). This disparity might result from various extraction techniques, plant components, solvents or sample origins (Elsherif et al., 2023).
               
The concentration required to block 50% of the DPPH radicals was also estimated. The IC50 value of the methanolic extract was determined to be 0.82 mg/mL. This indicates a moderate antioxidant activity, which remains lower than that of ascorbic acid (IC50≈0.05 mg/mL). Antioxidant potency can vary depending on extraction methods and plant origin, as reported by Ennajar et al., (2009), J. phoenicea leaves showed a much lower IC50  (8.5±0.3 mg/L), highlighting these variations.

CONCLUSION

Medicinal plants continue to represent a reliable source of natural bioactive compounds with significant therapeutic potential. The present study demonstrated that the methanolic extract of Juniperus phoenicea leaves from northwestern Algeria exhibits notable antibacterial activity particularly against Gram-positive bacteria and moderate antioxidant capacity.
       
These findings support the traditional uses of J. phoenicea and highlight its potential as a natural source of antimicrobial and antioxidant agents. Future investigations should focus on quantitative phytochemical profiling, isolation of active compounds and elucidation of their mechanisms of action to better understand the pharmacological value of this species.

CONFLICT OF INTEREST

The authors declare that they have no conflicts of interest regarding the content of this Manuscript. They report no financial, commercial, academic, or personal relationships that could be perceived as influencing the work presented. All authors have reviewed and approved this declaration.

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    Full Research Article

    Phytochemical Screening, Antimicrobial and Antioxidant Activity of Methanolic Extract of Juniperus phoenicea Leaves

    A
    A. Derradjia1,2,*
    S
    S. Deboucha3
    Z
    Z. Meskini4
    1Department of Biological Sciences, Faculty of Natural and Life Sciences, University of Ahmed Zabana Relizane, Algeria.
    2Laboratory of Valorization and Bioengineering of Natural Resources, Department of Natural and Life Sciences, Faculty of Sciences, University of Algiers 1, Algeria.
    3Department of Natural and Life Sciences, Faculty of Sciences, University of Algiers 1, Algeria.
    4Department of Agricultural Sciences, Laboratory of Environment, Natural Plant Substances and Food Technology, University of Ahmed Zabana Relizane, Algeria.

    ABSTRACT

    Background: As part of the valorization of Algerian medicinal flora, Juniperus phoenicea has attracted attention due to its diverse pharmacological properties. The current investigation aims to evaluate the phytochemical profile, antimicrobial potential and antioxidant activity of the methanolic extract of J. phoenicea leaves.

    Methods: J. phoenicea’s methanolic extract was subjected to various phytochemical tests. Antimicrobial activity was tested against six conventional pathogenic bacterial strains and one fungal strain using the disc-diffusion technique. The minimum inhibitory concentration was determined using the agar dilution method. Antioxidant activity was estimated by DPPH radical scavenging assay.

    Result: The phytochemical screening of the methanolic extract indicated the presence of many secondary metabolite categories, including tannins, flavonoids, saponins and terpenoids. Antimicrobial activity was evaluated using the disc diffusion method and the bacterial strains Staphylococcus aureus and Listeria monocytogenes were shown to be the most sensitive. However, no antimicrobial activity was detected against Pseudomonas aeruginosa, Escherichia coli or the fungal strain Candida albicans. The minimal inhibitory concentration (MIC) was determined using the solid medium dilution technique and the results ranged from 1.56 to 12.5 mg/mL. The DPPH scavenging capability indicated that the methanolic extract has modest antioxidant activity, with an IC50 value of 0.82 mg/mL.

    INTRODUCTION

    For thousands of years, human have used various plant species found in its natural environment to treat and cure different diseases (Amrati et al., 2021; Mottaghipisheh et al., 2024). Plants contain a large variety of secondary metabolites, making them an immense source of chemically diverse substances with a wide range of biological activities (Erenler et al., 2019; Divekar et al., 2022; Gbenou et al., 2024). Among these bioactive constituents, alkaloids, terpenes and phenolic compounds are particularly noteworthy for their pharmacological importance and multiple health benefits (Kebede et al., 2021). Numerous epidemiological studies have highlighted that a diet rich in plant polyphenols may help reduce the risk of developing cancer, cardiovascular diseases, diabetes, osteoporosis and neurodegenerative disorders (Chang et al., 2022; Omidfar et al., 2023). Some of them are also used as ingredients in the food, pharmaceutical and cosmetic industries (Mayyas et al., 2021).
           
    The alarming rise of antimicrobial resistance has intensified the global search for new, effective and safe antimicrobial agents from natural sources. Medicinal and aromatic plants have thus emerged as a promising alternative for developing novel bioactive molecules with fewer side effects (Khameneh et al., 2019; Aici and Benmehdi, 2021; Haile et al., 2022; Mishra et al., 2025).
           
    The genus Juniperus (family Cupressaceae) is among the most taxonomically diverse conifer groups, comprising approximately 67 species and 28 varieties distributed mainly across subtropical and temperate regions of the Northern Hemisphere, with some extending into the Southern Hemisphere (Chelouati et al., 2023; Liu et al., 2024). Juniperus phoenicea is a resinous, aromatic shrub or small tree with a wide distribution across southern Europe, temperate and subtropical Asia, the Atlantic coast and North Africa (Ennajar et al., 2009; Mehira et al., 2021). This species is valued for its medicinal properties and traditional uses, leaves of Juniperus phoenicea are commonly prepared as a decoction to treat diabetes, diarrhea, rheumatism and digestive disorders. Furthermore, a combination of leaves and berries is traditionally employed as an oral hypoglycemic agent (Chelouati et al., 2023).
           
    Algeria, with its remarkable diversity of climatic zones, harbors an exceptionally rich flora that includes numerous aromatic and medicinal species (Walas and Taib, 2022). This biodiversity offers a valuable reservoir of bioactive compounds with significant pharmacological potential.
                   
    In this context, the present study aims to evaluate the phytochemical composition, antimicrobial activity and antioxidant potential of the methanolic extract of Juniperus phoenicea leaves collected from northwestern Algeria. The results are expected to contribute to the valorization of this native species as a potential source of natural therapeutic agents.

    MATERIALS AND METHODS

    Plant material
     
    Leaves of J. phoenicea were collected in March 2022 in the Sidi Khatab district of Relizane, Algeria. The plant was used after being identified and verified by a taxonomist from the Department of Natural and Life Sciences, University of Algies 1, Algeria. The leaves were air-dried at ambient temperature in a dark, well-ventilated environment, ground into a fine powder and stored in sterile amber glass bottles until further use.
     
    Preparation of methanolic extract
     
    The plant material was extracted using a soxhlet under continuous agitation for 24 hours. 30 mg of plant powder were dissolved in 100 mL of methanol. The obtained extract was concentrated under reduced pressure using a rotary evaporator and stored at 4°C until analysis (Zarei Yazdeli et al., 2021). The extraction yield was calculated according to the formula:


    Where,
    WD= The weight of the dried extract.
    PP= The weight of the plant powder utilized.
     
    Phytochemical screening
     
    The methanolic extract was subjected to qualitative phytochemical analysis for the detection of major secondary metabolites including flavonoids, tannins, saponins, terpenoids and glycosides using standard procedures (Ingle et al., 2017; Berreghioua and Ziane, 2025a). These methods are based on colorimetric or precipitation reactions characteristic of each compound class.
     
    Test for flavonoids
     
     An aqueous solution of the methanolic extract was treated with 10% ammonium hydroxide. Yellow fluorescence showed the presence of flavonoids.
     
    Test for tannins
     
    A 0.5 mg sample of the extract was boiled in 20 mL of distilled water then filtered. The addition of a few drops of 0.1% ferric chloride produced a brownish-green or blue-black colouration, confirming the presence of tannins.

    Test for terpenoids
     
    Five millilitres of extract were mixed with 2 mL of chloroform, followed by the careful addition of concentrated sulfuric acid. The formation of a brownish-red interface indicated the presence of terpenoids.
     
    Test for saponins
     
    Ten millilitres of the diluted extract were vigorously shaken in a graduated cylinder for 15 minutes. A stable 2 cm foam layer persisting for 15 minutes indicated the presence of saponins.
     
    Test for glycosides
     
    In total, 50 mg of extract was hydrolyzed with strong hydrochloric acid in a water bath for 2 hours before being filtered. The hydrolysate underwent the Borntrager test. To 2 mL of filtered hydrolysate was added 3 mL of chloroform. The chloroform layer was separated after shaking the mixture,then ammonia solution (10%) was added to it. Pink colouration suggests glycosides presence.
     
    Antimicrobial activity
    Preparation of standard strains
     
    Six strains from the American type culture collection (ATCC) were kindly provided from the Laboratory of Valorization and Bioengineering of Natural Resources, University of Algiers 1, Algeria, to evaluate the antimicrobial properties of methanolic extract of J. phoenicea: Staphylococcus aureus 6538, Bacillus subtilis 6051, Enterococcus faecalis 29212, Pseudomonas aeruginosa 9027, Escherichia coli 8739, Listeria monocytogenes 19117 and Candida albicans 10231. Strains were revived in sterile Brain Heart Infusion broth (BHIB) and incubated at 37°C overnight.
     
    Disc-diffusion method
     
    Antimicrobial activity was evaluated using the disc diffusion method (Raoof et al., 2019). Bacterial suspensions (108 CFU/mL) and yeast suspension (106 CFU/mL) were evenly spread on Mueller-Hinton agar and Sabouraud dextrose agar plates, respectively. Sterile Whatman paper discs (6 mm diameter) were impregnated with 10 µL of the methanolic extract at concentrations of 100, 50, 25 and 12.5 mg/mL (dissolved in DMSO) and placed on the inoculated agar surfaces. Plates were incubated at 37°C for 24 hours for bacterial strains and 48 hours for the fungal strain. Antimicrobial activity was expressed as the diameter (mm) of the inhibition zone, including the disc diameter. Discs containing only DMSO served as negative controls.
     
    Agar dilution method
     
    The minimum inhibitory concentration (MIC) of the methanolic extract was determined by the agar dilution method (Zarei Yazdeli et al., 2021). Different concentrations of the extract (0.39 to 12.5 mg/mL) were incorporated into agar media. Standardized microbial inocula were applied to the agar surface and incubated under appropriate conditions. The MIC was defined as the lowest extract concentration that completely inhibited visible microbial growth.
     
    Antioxidant activity
     
    The antioxidant activity was carried out using the DPPH radical scavenging assay (Bougandoura and Bendimerad, 2013). 0.5 µL of each methanolic extract solution at various concentrations (from 0.0125 to 5 mg/mL) were added to 1.95 mL of DPPH methanolic solution (0.025 g/L). After 30 minutes of incubation in the dark at room temperature, absorbance was measured at 515 nm against a corresponding blank. The positive control was a solution of ascorbic acid, a standard antioxidant, whose absorbance was measured under identical circumstances as the sample. The percentage of inhibition (I%) was calculated and the IC50 value (concentration required to inhibit 50% of DPPH radicals) was determined by linear regression analysis.
     
    Statistical analysis
     
    The data obtained were analyzed using SPSS Statistical Software for Windows. All assays were performed in triplicate and quantitative variables were expressed as mean ± standard deviation (SD).

    RESULTS AND DISCUSSION

    Phytochemical screening
     
    The methanolic extract of J. phoenicea leaves was characterised by a brownish-green colour. The extraction yield is around 19.61%, which is  considerably lower than the values reported by Medini et al., (2013) (28.77 and 39.23 %).  However, Farahat (2020) reported that methanolic extract showed a yield of 9.8% for J. phoenicea leaves collected from Egypt. The yield of plant extracts can be affected by several parameters, it can vary depending on the chemical composition and physical characteristics of the plant material as well as the method and conditions under which the extraction was carried out (Dai and Mumper, 2010; Elsherif et al., 2023; Berreghioua and Ziane, 2025b). Phytochemical analysis of J. phoenicea identified a range of bioactive compounds, including flavonoids, saponins, tannins and terpenoids. Likewise, the phytochemical screening of J. phoenicea from two different locations in Tunisia revealed the presence of tannins, flavonoids and saponins (Medini et al., 2013). These phytochemicals have essential biological functions, saponins can lower cholesterol, modulate the immune system and  inhibit tumor growth (Shraim et al., 2021). However, a study by Makhloufi et al., (2014) did not detect tannins in J. phoenicea leaves, which contradicts the findings of the current study. Additionally, no glycosides were found in J. phoenicea leaves.
     
    Antimicrobial activity
     
    The antimicrobial screening of the methanolic extract of Juniperus phoenicea leaves against six bacterial strains and one Candida albicans strain revealed varying degrees of sensitivity. The inhibition zone diameters ranged from 9 mm to 19.5 mm (Table 1). Listeria monocytogenes and Staphylococcus aureus were the most sensitive strains, exhibiting the largest inhibition zones. In contrast, Bacillus subtilis and Enterococcus faecalis showed moderate inhibitory responses. However, Escherichia coli, Pseudomonas aeruginosa and Candida albicans were resistant to the extract’s antimicrobial effect. These results indicate that Gram-positive bacteria are generally more sensitive than Gram-negative bacteria, as previously reported by Bouzouita et al., (2008) and Ait-Ouazzou et al. (2012). According to established criteria, an extract is considered active when it produces an inhibition zone ≥10 mm, whereas inhibition zones <7 mm are classified as inactive (Tekwu et al., 2012; Draoui et al., 2022). The higher sensitivity of Gram-positive bacteria can be explained by structural differences in the cell envelope. Gram-negative bacteria possess a complex, rigid outer membrane rich in lipopolysaccharides (LPS), which acts as an effective barrier limiting the diffusion of hydrophobic compounds. In contrast, Gram-positive bacteria lack this outer membrane and have a peptidoglycan layer whose relatively permeable structure does not provide sufficient protection against bioactive compounds (Burt, 2004).

    Table 1: Antimicrobial activity of methanolic extract of J. phoenicea leaves.


           
    Overall, our results are consistent with several previous studies. Similar to the findings of the present investigation, Draoui et al., (2022) reported that methanolic extract of J. phoenicea exhibited moderate to strong antibacterial activity against pathogenic microorganisms, with inhibition zone diameters ranging from 9 to 19 mm. Likewise, a study conducted in Libya in 2021 demonstrated that methanolic extract of J. phoenicea leaves showed a strong inhibitory effect against S. aureus (Alhadad et al., 2023). An additional study conducted in Libya reported a strong antibacterial activity of methanolic extract of J. phoenicea leaves against S. aureus and various wild strains of human pathogenic bacteria. In contrast to the findings of the present work, Elmhdwi et al., (2015) observed that the methanolic extract was also effective against E. coli and P. aeruginosa. On the other hand, C. albicans was found to be resistant, as previously reported by Zubi et al., (2025).
           
    The minimum inhibitory concentration (MIC), defined as the lowest extract concentration that reduces microbial colony growth by 90%. The MIC was determined for strains that showed sensitivity using the disc diffusion assay. The results obtained show that L. monocytogenes and S. aureus had the lowest MIC values of 1.56 mg/mL and 3.12 mg/mL, respectively, while E. faecalis and B. subtilis exhibited higher MICs of 6.25 and 12.5 mg/mL, respectively. These results are consistent with previous findings reported in North Africa and the Mediterranean region. Draoui et al., (2022) reported MIC values between 0.78 and 6.25 mg/mL for methanolic extract of J. phoenicea against various Gram-positive and Gram-negative bacteria. Similarly, Zerrouki and Riazi (2021) observed a MIC of 10 mg/mL against S. aureus for hydromethanolic extract.
     
    Antioxidant activity
     
    The antioxidant capacity of the methanolic extract of J. phoenicea was determined using the DPPH technique. The results showed that the methanolic extract of J. pheonicea had a percentage of inhibition of around 90.59% (Fig 1). The synthetic molecule, ascorbic acid, showed a strong reduction at the first concentrations (Fig 2).

    Fig 1: Percentage inhibition of DPPH by methanolic extract of J. phoenicea.



    Fig 2: Percentage inhibition of DPPH by ascorbic acid.


           
    In a previous study, the percentage of inhibition for the methanolic extract of J. pheonicea leaves harvested in Tunisia was 95.89%, which is slightly higher than that obtained in our research (Medini et al., 2013). In another study, the maximum antiradical power of the methanolic extract of J. pheonicea was 62.41% (Al-Mustafa et al., 2021). This disparity might result from various extraction techniques, plant components, solvents or sample origins (Elsherif et al., 2023).
                   
    The concentration required to block 50% of the DPPH radicals was also estimated. The IC50 value of the methanolic extract was determined to be 0.82 mg/mL. This indicates a moderate antioxidant activity, which remains lower than that of ascorbic acid (IC50≈0.05 mg/mL). Antioxidant potency can vary depending on extraction methods and plant origin, as reported by Ennajar et al., (2009), J. phoenicea leaves showed a much lower IC50  (8.5±0.3 mg/L), highlighting these variations.

    CONCLUSION

    Medicinal plants continue to represent a reliable source of natural bioactive compounds with significant therapeutic potential. The present study demonstrated that the methanolic extract of Juniperus phoenicea leaves from northwestern Algeria exhibits notable antibacterial activity particularly against Gram-positive bacteria and moderate antioxidant capacity.
           
    These findings support the traditional uses of J. phoenicea and highlight its potential as a natural source of antimicrobial and antioxidant agents. Future investigations should focus on quantitative phytochemical profiling, isolation of active compounds and elucidation of their mechanisms of action to better understand the pharmacological value of this species.

    CONFLICT OF INTEREST

    The authors declare that they have no conflicts of interest regarding the content of this Manuscript. They report no financial, commercial, academic, or personal relationships that could be perceived as influencing the work presented. All authors have reviewed and approved this declaration.

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