Antioxidant and antimicrobial activities of four medicinal plants from Algeria European Journal of Chemistry 14 (1) (2023) 121-128 European Journal of Chemistry ISSN 2153-2249 (Print) / ISSN 2153-2257 (Online) – Copyright © 2023 The Authors – Atlanta Publishing House LLC – Printed in the USA. This work is published and licensed by Atlanta Publishing House LLC – CC BY NC – Some Rights Reserved. https://dx.doi.org/10.5155/eurjchem.14.1.121-128.2358 European Journal of Chemistry View Journal Online View Article Online Antioxidant and antimicrobial activities of four medicinal plants from Algeria Yuva Bellik 1,* and Nasreddine Mekhoukh 2 1 Department of Biology, Faculty of Nature and Life Sciences, University of Mohamed El Bachir El Ibrahimi, Bordj Bou Arreridj, 34000, Algeria 2 Department of Physico-Chemical Biology, Faculty of Nature and Life Sciences, University of Bejaia, 06000, Algeria * Corresponding author at: Department of Biology, Faculty of Nature and Life Sciences, University of Mohamed El Bachir El Ibrahimi, Bordj Bou Arreridj, 34000, Algeria. e-mail: y.bellik@univ-bba.dz (Y. Bellik). 10.5155/eurjchem.14.1.121-128.2358 Received: 17 November 2022 Received in revised form: 17 January 2023 Accepted: 25 January 2023 Published online: 31 March 2023 Printed: 31 March 2023 Medicinal plants are used in folk medicine to cure several human diseases. This work was designed to evaluate the antioxidant and antimicrobial activities of different extracts of Globularia alypum, Dittrichia viscosa, Juniperus oxycedrus, and Retama sphaerocarpa. The total phenolic content (TPC), the total flavonoid content (TFC), and the condensed tannin content (CTC) were determined spectrophotometrically. The antioxidant activity was tested using TAC, DPPH and reducing power assays. The agar diffusion method was used to determine antimicrobial activity against four bacteria (Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa) and one fungus (Candida albicans). J. oxycedrus acetone extract showed the highest extraction yield (35.56±0.45%), TPC (504.96±14.82 mg GAE/g DE) and TFC (43.91±0.87 mg QE/g DE). The same extract exhibited the highest TAC (350.67±6.05 mg GAE/g DE) and was the most effective against the DPPH free radical (IC50 = 0.21±0.01 mg/mL). In contrast, the J. oxycedrus methanol extract showed the highest reducing power (A0.5 = 0.39 ± 0.09 mg/mL). All extracts tested showed antibacterial and anticandidal activities at different concentrations. The best antimicrobial effect was also observed with the acetone extract of J. oxycedrus against P. aeruginosa (26.77±0.06 mm), B. cereus (17.16±0.08 mm), E. coli (15.84±0.04 mm), and C. albicans (21.36±0.11 mm), while the ethanol extract of D. viscosa was the most active against S. aureus (24.54±0.03 mm). The results of this study provide a scientific basis for the traditional use of these local plants and demonstrate their potential as sources of natural antioxidant and antimicrobial bioactive compounds. Dittrichia viscosa Globularia alypum Juniperus oxycedrus Antioxidant activity Retama sphaerocarpa Antimicrobial activity Cite this: Eur. J. Chem. 2023, 14(1), 121-128 Journal website: www.eurjchem.com 1. Introduction The use of plant substances for medical purposes is asso- ciated with earlier times of humankind. Today, plants are still the new primary source of structurally important chemical substances that lead to the development of innovative drugs [1,2]. Currently, approximately 500,000 to 800,000 plant species are used for medical purposes worldwide. There is an increase in the consumption of medicinal plants due to the proven safety and efficacy of medicinal plants in preventing and curing some diseases [3,4]. Plants continue to be a rich source of therapeutic agents, particularly secondary metabolites such as polyphenols, flavonoids, tannins, saponins, glucosides, etc., involved in ecological functions to improve plant survival during environmental stress. These same plant molecules provide appreciable healing properties in humans [5,6]. In fact, these compounds can interact with reactive oxygen species (ROS) to protect different biological macromolecules (Proteins, lipids, and nucleic acids) from oxidative damage [7]. ROS are produced mainly by enzymatic and non-enzymatic reactions during physiological and pathological conditions. Oxidative stress results from an imbalance between ROS production and antioxidant defenses. In recent years, it has become apparent that oxidative stress plays a central role in various acute and chronic pathological processes such as cancer, neurodege- nerative, cardiovascular, and inflammatory diseases [8]. In addition, a growing number of infections are becoming a major health problem because many bacteria and fungi have become increasingly resistant to multiple antibiotics [9]. Many studies have been conducted to investigate the therapeutic effects of antioxidants from natural sources for their wide variety of pharmacological activities, particularly antioxidant and anti- microbial properties. Phytochemical studies identified different compounds, such as quercetin, gallic acid, rutin, resveratrol, and caffeic acid, responsible for antioxidative reactions [10]. In addition, compounds from medicinal plants have been shown to inhibit the growth of a wide range of Gram-positive and Gram-negative bacteria, fungi, viruses, and protozoa through different mechanisms [1]. Algeria benefits from various climates that favour the development of a rich and diversified flora. Indeed, the Algerian territory has a diversified vegetation cover distributed on the coasts, plains, mountains, steppe, Sahara, and around the water points. Globularia alypum L., also called globular, is a plant belonging to the Globulariaceae family, which includes the two genera Globularia alypum L. and Poskea Vatke and about thirty species widespread in Europe and North Africa. Species of the genus Globularia have been used as agents for various ailments. According to ethnopharmacological studies, G. alypum is one of ABSTRACT RESEARCH ARTICLE KEYWORDS https://dx.doi.org/10.5155/eurjchem.14.1.xxxxx-128.2358 https://www.eurjchem.com/ https://dx.doi.org/10.5155/eurjchem.14.1.121-128.2358 mailto:y.bellik@univ-bba.dz http://www.eurjchem.com/ https://crossmark.crossref.org/dialog/?doi=10.5155/eurjchem.14.1.121-128.2358&domain=pdf&date_stamp=2023-03-31 122 Bellik and Mekhoukh / European Journal of Chemistry 14 (1) (2023) 121-128 2023 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.14.1.121-128.2358 the plants most commonly used to prevent and heal different chronic diseases [11]. Juniperus oxycedrus L. is a plant species of the Cupressaceae family. It is a small dioecious tree highly valued for its richness in secondary metabolites and, in particular, essential oils [12]. This plant is widely used in traditional medicine. It is common in Mediterranean coastal regions and has remarkable resistance to hostile environments such as drought and cold [13]. This plant is used in traditional medicine to treat diseases such as hyperglycemia, obesity, tuberculosis, bronchitis, and pneumonia [14]. Dittrichia viscosa (L.) Greuter is a perennial plant of the Asteraceae (compositeae) family. It is common in the Mediterranean region, where it flowers in late summer and early fall, and is also present in temperate and cold regions of the world. It is considered the queen of medicinal plants because of its multiple biological and pharmacological properties. It is used in traditional medicine for its anti- inflammatory, antipyretic, astringent, and vulnerary properties [15]. Retama sphaerocarpa (L.) Boiss. is a broom-like shrub that occurs in the Mediterranean area of North East Africa and the Iberian Peninsula [16], growing on a wide variety of soil types and under an extensive range of climatic conditions [17]. Plants of the genus Retama Park. of the Mediterranean basin are used in traditional medicine to treat many disorders, including rabies, diabetes, hepatitis, jaundice, sore throat, skin diseases, joint pain, rheumatism, fever, and inflammation [18]. Several medicinal plants and pure compounds have been reported that are being investigated for preventive and therapeutic activities. However, many species of higher plants have not yet been fully described, much less surveyed for chemical or biologically active constituents. Therefore, the present study aimed to determine the phytochemical compo- sition, antioxidant, and antimicrobial properties of four local Algerian plants. 2. Experimental 2.1. Chemicals Folin-Ciocalteu, sodium carbonate, vanillin, 2,2-diphenyl-1- picrylhydrazyl (DPPH), 2,6-di-tert-butyl-4-methylphenol (BHT), ascorbic acid (Vitamin C), iron chloride (II), aluminum chloride, trichloroacetic acid (TCA), dimethylformamide (DMF), gallic acid, quercetin, catechin and other chemicals were purchased from Sigma Aldrich. All organic solvents and chemicals used in the present study were of analytical grade. 2.2. Plant material Fresh leaves of J. oxycedrus, D. viscosa, G. alypum L., and stems of R. sphaerocarpa were collected during the flowering stage in March 2021 in the region of Teffreg, the city of Bordj Bou Arreridj, North-East Algeria. The plants were dried at 37 ° C in an oven prior to extraction. The dried plants were ground to a fine powder (<200 µm particles) using an electric mill (Siyolux, Italy). 2.3. Preparation of plant extracts Briefly, 10 g of each plant powder were extracted with 150 mL of different solvents (Methanol 70%, acetone 70%, ethanol 70%, and ethyl acetate 70%) using a magnetic stirrer for three hours in the dark at room temperature. Subsequently, the extracts were filtered with a filter paper. A second extraction was performed under the same conditions to obtain a maximum amount of active substances. The filtrates obtained were then evaporated on a rotary evaporator to remove the solvent. The residue was dried in an oven at 40 °C. Finally, the dried extracts were stored at -4 °C until analysis. The extraction yield (%) was determined as the ratio between the amount of dry extract obtained and the initial amount of powder used for extraction. 2.4. Phytochemical study 2.4.1. Total phenolic content The total phenolic content of the different extracts was estimated spectrophotometrically using the Folin-Ciocalteu test following the Singleton and Rossi protocol [19]. An aliquot of 1 mL of Folin’s reagent (10 times diluted) was added to 200 µL of sample or standard with suitable dilutions. After 4 min, 800 µL of a sodium carbonate solution (7.5%) was added. The mixture was left to react for 2 h in darkness at room temperature, and then the absorbance was measured at 760 nm against a blank using a double beam UV-vis spectrophotometer (Shimadzu UV- 1601, Japan). All operations were performed in triplicate. The total polyphenols concentration was calculated from the regression equation of the calibration range established with gallic acid. It was expressed in milligram gallic acid equivalent per gram of dry extract (mg GAE/gDry extract). 2.4.2. Total flavonoid content The flavonoid content was evaluated according to the aluminum chloride colorimetric method [20]. A volume of 1 mL of each extract or standard solution (quercetin) at different concentrations was reacted with 1 mL of aluminum chloride (2%). The mixture was vigorously stirred and incubated for one hour at room temperature. The absorbance was read at 430 nm using a double beam UV-vis spectrophotometer (UV-1800 Shimadzu). All samples were analyzed in triplicate and the content of flavonoids was determined using the calibration curve realized using quercetin. Total flavonoid content was expressed as milligram quercetin equivalent per gram of dry extract (mg QE/gDry extract). 2.4.3. Determination of condensed tannins The acidified vanillin method was used to determine the content of condensed tannins [21]. Briefly, 0.5 mL of each extract (1 mg/mL) was mixed with 3 mL of vanillin solution (4%) in methanol and 1.5 mL of hydrochloric acid (37%). The mixture was shaken well and incubated for 15 min at room temperature. The absorbance was read at 500 nm. The experiment was carried out in triplicate. The condensed tannin content was calculated using a standard catechin calibration curve and the concentration was expressed in milligram catechin equivalent per gram of dry extract (mg CatE/gDry xtract). 2.5. Antioxidant activities 2.5.1. Total antioxidant capacity (TAC) The total antioxidant capacity of the extracts was evaluated using the phosphomolybdenum test described by Prieto et al. [22] with specific modifications. A volume of 0.3 mL of each extract or standard was mixed with 3 mL of the reaction mixture (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). The mixture was then incubated at 95 °C for 90 min and the absorbance was recorded at 695 nm. The antioxidant activity was expressed as milligram gallic acid equivalent per gram of dry extract (mg GAE/gDry xtract). 2.5.2. DPPH free radical scavenging activity The free radical scavenging activity of the different extracts against 2,2-diphenyl-1-picrylhydrazyl (DPPH) was evaluated as previously described [23]. DPPH was dissolved in 100 mL of Bellik and Mekhoukh / European Journal of Chemistry 14 (1) (2023) 121-128 123 2023 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.14.1.121-128.2358 methanol to prepare a stock solution which was then diluted to give an absorbance of 0.98±0.02 at 517 nm. A volume of 100 μL of each extract at different concentrations or standards (ascorbic acid and BHT) was added to 2.5 mL of DPPH. The mixture was then allowed to stand in the dark for 30 min and the decolorization compared to the negative control containing only the DPPH solution was measured at 517 nm. The free radical scavenging activity was calculated according to Equation (1): 517-Control 517-Sample 517-Control (Abs -Abs ) I % = ×100 Abs (1) where I is the inhibition percentage. Acontrol represents the absorbance of the control reaction (containing all reagents except the tested compound), and Asample represents the absorbance of the sample. The values were expressed as IC50 (mg/mL), the concentration of samples that causes a 50% scavenging of the DPPH radical. 2.5.3. Reducing power activity The reducing power of the extracts of both plants was determined using the methodology developed by Oyaizu [24]. A volume of 200 µL of each extract at different concentrations was mixed with 500 µL of phosphate buffer (0.2 M; pH = 6.6) and 2.5 mL of 1% potassium ferricyanate solution (K3Fe(CN)6) pre- pared in distilled water. The mixture was incubated at 50 °C for 20 min. Subsequently, 2.5 ml of 10% trichloroacetic acid (TCA) was added, followed by centrifugation at 650 rpm for 10 min. A volume of 500 µL of supernatant was taken to which 2.5 mL of distilled water and 0.5 mL of 0.1% FeCl3 were added. The absorbance was measured at 700 nm. Ascorbic acid and BHT were used as antioxidant standards. Values were expressed as A0.5 (mg/mL), the concentration of the sample that gives an absorbance of 0.5 at 700 nm (A0.5) calculated from a linear regression analysis. 2.6. Antimicrobial activity 2.6.1. Microorganisms The antimicrobial activity of plant extracts was evaluated against the following pathogens: four strains of bacteria (Bacillus subtilis ATCC 6633, Streptococcus aureus ATCC 25923, Escherichia coli ATCC 25922, and Pseudomonas aeruginosa ATCC 27853), and one fungus (Candida albicans). 2.6.2. Inoculums preparation Before the experiment, the bacterial strains were inoculated on the surface of the nutrient agar medium. Inoculum suspensions were obtained by selecting 4-5 well- isolated colonies from 24-hour cultures. The colonies were suspended in nutrient broth (or sterile physiological water with 0.9% NaCl). The optical density was adjusted to a McFarland standard turbidity of 0.5 (equivalent to 1-5×108 CFU/mL). For C. albicans, the inoculum was prepared from a 48-hour culture of isolated fungi grown on potato dextrose agar (PDA). The optical density was also adjusted to 0.08-0.10 to obtain a solution corresponding to ×106 spores/mL. 2.6.3. Antimicrobial screening To evaluate the antibacterial activity of different extracts, the agar well diffusion method was used as previously described [25]. Wells (6 mm in diameter) were punched on Mueller-Hinton agar plates pre-inoculated with young bacteria (1×108 CFU/mL) or yeast (5×106 cells/mL) and filled with 80 μL of each extract (prepared in DMF) at a concentration of 128 mg/mL. Gentamicin 40 mg/mL was used as standard antibiotic due to its wide spectrum of inhibition. After cooling at 4 °C for 2 hours, the plates were incubated at 37 °C for 24 hours (bacteria) or 48 hours (yeast). Antimicrobial activity was deter- mined by measuring the diameter of the inhibition zone around the well. 2.6.4. Determination of the minimum inhibitory concentration (MIC) To determine the minimum inhibitory concentration (MIC) of the different extracts, the macroscopic dilution method was used. Müller-Hinton agar was poured into Petri dishes and inoculated by the smear method with bacterial strains to be tested at a certain density (0.08-0.10 at λ = 628 nm). The sterile Pasteur pipettes were then used to form 6 mm wells in the agar. Extracts at different concentrations (128, 64, 32, 16, 8, 4, 2, and 1 mg/mL) were prepared in DMF by double dilution and then autoclaved at 121 °C for 15 minutes. Eighty microliters of each extract were added to each well. After incubation for 18 hours at 37 °C, the MIC was determined. The MIC is the concentration of the extract that causes the lowest zone of inhibition greater than 8 mm [26]. 2.6.5. Determination of minimum bactericidal and fungicidal concentration (MBC/MFC) The MBC is the concentration of an antibacterial substance with a bactericidal effect and can be obtained after 18-24 hours of incubation at 37 °C. To determine MBC and MFC, the surface of each inhibition zone recorded with different concentrations of extracts (128, 64, 32, 16, 8, and 4 mg/mL) was scraped using a Pasteur pipette. The nutrient broth tubes were then inoculated with the scraped agar pieces. The tubes were incubated at 37 °C for 24 hours. The absence of growth (no turbidity) in the inoculated tubes indicated a bactericidal/fungicidal effect and was declared to contain MBC/MFC, which is equal to the concentration where it was initially scraped [27]. 2.7. Statistical analysis All assays were performed in triplicate and the results were expressed as mean±SD. Statistical comparisons were made with the Student’s t test or the ANOVA test. Differences were considered significant at p < 0.05. 3. Results 3.1. Extraction yields and total polyphenols contents Figure 1 shows the extraction yields, total phenolics, flavonoids, and condensed tannins of extracts of J. oxycedrus, D. viscosa, G. alypum leaves, and R. sphaerocarpa stems from various extraction solvents. The yields of the bioactive compounds varied significantly according to the plant and solvent. The highest extraction yield was detected in the ethanol and acetone extract of J. oxycedrus (35.70±0.35% and 35.56±0.45%, respectively), while the lowest yield was recorded in the G. alypum ethyl acetate extract (12.30±0.45%) (Figure 1a). Similarly, the results of phenolics content revealed that the acetone extract of J. oxycedrus exhibited the highest level of polyphenols (504.96±14.82 mg GAE/g DE), followed by the acetone extract of D. viscosa (454.60±18.06 mg GAE/g DE) and the acetone extract of R. sphaerocarpa (333.01±2.61 mg GAE/g DE). In the case of G. alypum, the highest level of polyphenol compounds was detected in the ethanol extract (197.51±3.21 mg GAE/g DE) (Figure 1b). 124 Bellik and Mekhoukh / European Journal of Chemistry 14 (1) (2023) 121-128 2023 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.14.1.121-128.2358 (a) (b) (c) (d) Figure 1. Extraction yields (a), polyphenols (b), flavonoids (c), and condensed tannins (d) contents of the four plant extracts. The results are reported per 1 g of extract and are presented as mean±SD (n=3). GA: Globularia alypum, DV: Dittrichia viscosa, JO: Juniperus oxycedrus, RS: Retama sphaerocarpa. Ac. E: Acetone extract, Eth. E: Ethanol extract, Ethact. E: Ethyl acetate extract, Meth. E: Methanol extract. Cat E = Catechin equivalent; DE = Dry extract; GAE = Gallic acid equivalent; QE = Quercetin equivalent. According to the data in Figure 1c, the studied plant extracts showed the same flavonoids evolution profile as for phenolics content. The acetone extract of J. oxycedrus had the highest flavonoids concentration with an average value of 43.91±0.87 mg QE/g DE, followed by the acetone extract of D. viscosa (41.28±0.75 mg QE/g DE) and the acetone extract of R. sphaerocarpa (21.69±0.47 mg QE/g DE). Bellik and Mekhoukh / European Journal of Chemistry 14 (1) (2023) 121-128 125 2023 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.14.1.121-128.2358 Table 1. Antioxidant activity of different extracts *. Plants Standards and extracts DPPH, IC50 (mg/mL) RP, A0.5 (mg/mL) TAC (mg GAE/g DE) J. oxycedrus Meth. E Eth. E Ac. E Ethact. E 0.32±0.01 0.23±0.01 0.21±0.01 1.99±0.06 0.39±0.09 0.94±0.03 0.59±0.05 1.80±0.06 340.23±4.13 344.44±5.89 350.67±6.05 222.03±3.74 D. viscosa Meth. E Eth. E Ac. E Ethact. E 0.21±0.06 0.27±0.01 0.36±0.01 0.86±0.01 0.46±0.08 0.44±0.08 0.54±0.05 2.08±0.07 247.32±4.92 282.53±4.07 347.99±8.75 155.88±2.41 R. sphaerocarpa Meth. E Eth. E Ac. E Ethact. E 0.89±0.02 0.48±0.01 0.39±0.02 2.18±0.05 5.22±0.12 3.01±0.17 3.66±0.15 5.68±0.19 247.89±3.62 316.89±0.09 299.87±3,09 103.27±1.53 G. alypum Meth. E Eth. E Ac. E Ethact. E 0.89±0.03 0.83±0.04 0.82±0.02 0.90±0.01 5.31±0.11 4.42±0.03 4.02±0.09 4.73±0.10 116.72±0.79 141.33±3.54 65.02±3.13 29.70±2.84 - Ascorbic acid 2,6-Di-tert-butyl-4-methylphenol 0.11±0.02 2.21±0.10 0.28±0.01 1.09±0.09 403.32±6.66 214.45±5.32 * Ac. E: Acetone extract, Eth. E: Ethanol extract, Ethact. E: Ethyl acetate extract, Meth. E: Methanol extract. RP: reducing power, TAC: Total antioxidant capacity. Table 2. Antimicrobial activity of different plant extracts at a concentration of 128 mg/mL *. Plants Standard and extracts Micro-organisms / Diameter of inhibition zone (mm) P. aeruginosa S. aureus E. coli B. cereus C. albicans J. oxycedrus Ac. E Meth. E Eth. E Ethact. E 26.77±0.06 22.33± 0.04 24.45±0.04 12.70±0.07 12.01±0.03 12.03±0.05 14.04±0.05 14.31±0.04 15.84±0.04 15.37±0.02 15.85±0.02 13.77±0.05 17.16±0.08 16.34±0.12 16.28±0.09 15.86±0.11 21.36±0.11 20.66±0.12 16.56±0.07 14.23±0.08 D. viscosa Ac. E Meth. E Eth. E Ethact. E 22.78±0.04 24.44±0.06 20.13±0.04 18.91±0.02 22.42±0.11 19.13±0.02 24.54±0.03 24.25±0.04 09.22±0.02 09.85±0.06 08.65±0.03 07.35±0.07 13.11±0.03 13.93±0.04 21.91±0.02 13.42±0.04 15.35±0.11 18.34±0.04 20.44±0.06 19.74±0.11 R. sphaerocarpa Ac. E Meth. E Eth. E Ethact. E 24.47±0.12 24.60±0.20 23.24±0.11 29.12±0.04 19.50±0.02 20.18±0.10 20.25±0.06 20.70±0.03 11.42±0.11 11.10±0.09 10.17±0.07 11.51±0.02 11.24±0.11 14.10±0.11 14.60±0.06 08.13±0.11 14.00±0.13 11.16±0.34 24.02±0.17 15.03±0.07 G. alypum Ac. E Meth. E Eth. E Ethact. E 25.68±0.07 23.63±0.06 22.23±0.04 24.90±0.03 20.86±0.05 21.09±0.10 22.08±0.07 22.27±0.09 08.58±0.05 15.11±0.10 10.10±0.02 08.42±0.02 12.05±0.04 12.83±0.05 13.06±0.05 14.33±0.14 10.73±0.43 15.11±0.11 15.10±0.19 19.70±0.34 Antibiotic Gentamycine 37.01±0.96 35.33±1.27 35.78±0.04 38.21±0.06 38.63±0.09 * Ac. E: Acetone extract, Eth. E: Ethanol extract, Ethact. E: Ethyl acetate extract, Meth. E: Methanol extract. In the case of G. alypum, the highest level of flavonoids was detected in the ethanol extract (15.21±1.25 mg QE/g DE). Analysis of condensed tannins showed that the highest concentrations for all plants studied were obtained with ethyl acetate extracts: 78.01±1.02 mg CatE/g DE for J. oxycedrus, 55.13±1.22 mg CatE/g DE for D. viscosa, 28.61±1.28 mg CatE/g DE for R. sphaerocarpa and 25.55±1.26 mg CatE/g DE for G. alypum (Figure 1d). 3.2. Antioxidant activity In the present work, the antioxidant activity of plant extracts was evaluated by measuring the total antioxidant capacity (TAC), the free radical DPPH and the reducing power. The results are given as IC50 and A0.5 values. In general, acetone extracts exhibited higher antioxidant activity. The results in Table 1 showed that J. oxycedrus acetone extract displayed the highest TAC (350.67±6.05 mg GAE/g DE), followed by D. viscosa acetone extract (347.99±8.75 mg GAE/g DE), R. sphaerocarpa ethanol extract (316.89±0.09 mg GAE/g DE), and G. alypum ethanol extract (141.33±3.54 mg GAE/g DE). With regard to DPPH scavenging activity, the acetone extract of J. oxycedrus and methanol extract of D. vicosa were the most active extracts against DPPH free radical with IC50 values of 0.21±0.01 and IC50 = 0.21±0.06 mg/mL, respectively. Furthermore, all extracts tested exhibited higher antioxidant activity than BHT (IC50 = 2.21 ± 0.10 mg/mL) (Table 1). Analysis of metal iron-reduction revealed that J. oxycedrus methanol extract and D. vicosa ethanol extract displayed very potent antioxidant powers with A50 values of 0.39±0.09 and 0.44±0.08 mg/mL, respectively. The ethanol extract of R. sphaerocarpa and the acetone extract of G. alypum exhibited slight antioxidant activity with A50 values of 3.01±0.17 and 4.02±0.09 mg/mL, respectively (Table 1). 3.3. Antimicrobial activity The bacteria and fungus used in this study are associated with various forms of diseases [28]. The antimicrobial activities of the different plant extracts are shown in Table 2. The extracts tested exhibited different degrees of antimicrobial activity. The acetone extract of J. oxycedrus was the most active against almost all strains tested strains (26.77±0.06 mm for P. aeruginosa, 21.36±0.11 mm for C. albicans, 17.16±0.08 mm for B. cereus, 15.84±0.04 mm for E. coli), except S. aureus, which was more sensitive to the action of the ethyl acetate extract (14.31±0.04 mm) (Table 2). In the case of D. viscosa, methanol and ethanol extracts were the most active against the bacteria and fungi tested. Consequently, the mean diameter of the growth inhibition zone of methanol extract against P. aeruginosa was equal to 24.44±0.06 mm. The ethanol extract showed inhibition zones of diameters of 24.54±0.03, 21.91±0.02, and 20.44±0.06 mm against S. aureus, B. cereus, C. albicans, respectively (Table 2). The ethyl acetate extract of R. sphaerocarpa showed the strongest antibacterial activity on P. aeruginosa, S. aureus, and E. coli (29.12±0.04, 20.70±0.03, and 11.51±0.02 mm, respect- tively), while the ethanol extract exhibited high antimicrobial activity against C. albicans and B. cereus (24.02±0.17 and 14.60±0.06 mm, respectively) (Table 2). 126 Bellik and Mekhoukh / European Journal of Chemistry 14 (1) (2023) 121-128 2023 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.14.1.121-128.2358 Table 3. Minimum inhibitory concentration (MIC) *. Microorganisms MIC (mg/mL) Meth. E Eth. E Ac. E Ethact. E JO DV RS GA JO DV RS GA JO DV RS GA JO DV RS GA P. aeruginosa 16 8 8 8 16 32 8 8 8 16 8 8 16 16 8 8 B. cereus 16 8 8 32 16 32 32 8 8 16 16 16 8 16 32 8 S. aureus 8 8 8 8 8 8 8 8 8 8 8 16 8 8 8 8 E. coli 64 64 64 16 64 64 32 16 64 128 64 16 64 128 32 32 C. albicans 8 16 64 16 16 32 16 16 8 32 32 64 16 16 16 6 * DV: D. viscosa, GA: G. alypum, JO: J. oxycedrus, RS: R. sphaerocarpa. Table 4. Minimum bactericidal/fungicidal concentrations (MBC/MFC) *. Microorganisms MBC and MFC (mg/mL) Meth. E Ethact. E Ac. E Ethact. E JO DV RS GA JO DV RS GA JO DV RS GA JO DV RS GA P. aeruginosa 128 64 32 32 128 128 64 128 128 128 64 64 16 128 16 32 B. cereus 128 64 32 64 128 128 128 64 64 128 64 64 64 128 32 128 S. aureus 64 128 64 64 32 64 32 64 128 128 32 128 64 64 64 16 E. coli 128 128 128 64 128 128 128 64 128 128 128 64 128 128 128 128 C. albicans 64 128 128 64 128 128 64 64 32 128 64 128 64 128 64 64 * DV: D. viscosa, GA: G. alypum, JO: J. oxycedrus, RS: R. sphaerocarpa. Regarding G. alypum, all extracts showed antimicrobial activity against the bacteria and fungi tested. The minimum inhibitory concentration (MIC) and the minimum bactericidal/ fungicidal concentrations (MBC and MFC) of the plant extracts are displayed in Tables 3 and 4. 4. Discussion In this study, we investigated the phytochemical composition, antioxidant and antimicrobial activities of differ- rent extracts prepared from different plants including G. alypum, J. oxycedrus, D. viscosa and the stems of R. sphaerocarpa using various solvents of differing polarities. Quantitative analysis revealed that the extraction yields and contents of the phenolic compounds differed significantly depending on the plant and the extraction solvent [29]. In the present study, ethanol and acetone of J. oxycedrus recorded the highest extraction yields (35.70±0.35 and 35.56±0.45%, respectively). The results of this study are higher than those obtained by Chaouche et al. [30] who reported an extraction yield of 12.1%, but lower than that of Živić et al. [31], who reported an extraction yield of 49.80%. The extraction yield is mainly influenced by the chemical nature of the solvent used, the extraction time, the sample size, and the presence of interference substances [32]. Furthermore, the combined use of water and organic solvents can facilitate the extraction of soluble chemicals in water and/or organic solvents [33]. The polyphenols content ranged from 73.75±2.11 to 504.94±3.36 mg GAE/g (Figure 1b). The acetone extract of J. oxycedrus recorded the highest phenolic compounds among all extracts tested. Our results are significantly higher than those of of Taviano et al. [34] Chaouche et al. [30], and Živić et al. [31] with values of 253.29±3.16 mg GAE/g DW, 76.1±2.8 mg GAE/g DW, and 58.73±0.14 mg GAE/g DW, respectively. The acetone extract of D. viscosa also showed a high level of phenolic compounds (454.60±18.06 mg GAE/g DE). The result is much higher than that reported by Gökbulut et al. [35] on D. viscosa from Turkey (177.1±3.6 mg GAE/g DE) and Rhimi et al. [36] on D. viscosa from Tunisia (117.58±1.29 mg GAE/g). Phyto- chemicals are mainly extracted using an organic solvent. Several studies reported that acetone is the best solvent for polyphenols extraction in comparison to methanol, ethanol, and water [37]. The antioxidant activity of medicinal plant is attributed mainly to its richness in phenolic compounds, but also to other bioactive molecules [38,39]. Evaluation of the antioxidant activity of plant extracts using different assays, including TAC, DPPH, and reducing power, showed interesting antioxidant effects. TAC indicates the general antioxidant activity of a food or plant and its ability to neutralise free radicals in the body. The results obtained showed that the TAC decreased in the following order: J. oxycedrus acetone extract (350.67±6.05 mg GAE/g DE) > D. viscosa acetone extract (347.99±8.75 mg GAE/g DE) > R. sphaerocarpa ethanol extract (316.89±0.09 mg GAE/g DE) > G. alypum methanol extract (141.33±3.54 mg GAE/g DE). The difference in total antioxidant capacity from one extract to another can be explained by the ability of substances to transfer electrons in the reaction medium [40]. The results of TAC regarding D. viscosa are much higher than those obtained by Rhimi et al. [36]. The antiradical activity results showed that the J. oxycedrus acetone extract was also the most active against the free radical DPPH with IC50 = 0.21±0.01 mg/mL, followed by the D. viscosa methanol extract (IC50 = 0.21±0.06 mg/mL) R. sphaerocarpa acetone extract (IC50 = 0.39±0.02 mg/mL) and the G. alypum acetone extract (IC50 = 0.82±0.02 mg/mL). Our results for J. oxycedrus are much higher compared to those obtained with Juniperus thurifera L extracts (IC50 = 29.348 mg/mL with ethyl acetate extract, IC50 = 37.538 mg/mL with aqueous extract, and IC50 = 52.573 mg/mL with methanol extract) [41]. The results of D. viscosa are lower than those recorded by Rhimi et al. [36] with value of 0.056±1.2 mg/mL. Regarding R. sphaerocarpa, the results of this study are higher than those of Edziri et al. [42] who reported an IC50 of 0.5 mg/mL. Our results are much higher than those of Khlifi et al. [43] who reported an IC50 of 15.58 mg/L with G. alypum methanol extract. The reducing power of the studied extracts is classified in the following order: J. oxycedrus methanol extract (A0.5 = 0.39±0.09 mg/mL) > D. viscosa ethanol extract (A0.5 = 0.44±0.08 mg/mL) > R. sphaerocarpa ethanol extract (A0.5 = 3.01±0.17 mg/mL) > G. alypum acetone extract (A0.5 = 4.02±0.09 mg/mL). This difference between the different extracts can be explained by the differences in the polarities of the bioactive compounds of each extract, which can influence their solubility and their reducing power [44]. All plant extracts exhibited antibacterial and anticandidal activities, with MIC values ranging from 8 to 128 mg/mL (Table 2). All extracts were more active against Gram-positive bacteria and yeasts than Gram-negative ones. Several studies have shown that Gram-positive bacteria are much more sensitive than Gram-negative ones. It has also been reported that the resistance of Gram-negative bacteria such as E. coli to antimicrobial agents is linked to the presence of an envelope that includes a cell membrane rich in lipopolysaccharides and a wall that limits access to antimicrobial agents, unlike gram- positive bacteria which are less protected against external agents (detergents and antibiotics). According to Akiyama et al. Bellik and Mekhoukh / European Journal of Chemistry 14 (1) (2023) 121-128 127 2023 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.14.1.121-128.2358 [45] and Funatogawa et al. [46], the inhibitory effect of phenolic compounds and flavonoids on the growth of bacteria can be associated with several mechanisms of action, among which the chelation of iron which is necessary for microbial growth, and the action on the membrane of microorganism leading to the loss of its structural integrity. 5. Conclusions This work aimed to investigate the physicochemical, phytochemical, antioxidant and antimicrobial activities of various plant extracts, including D. viscosa, J. oxycedrus, R. sphaerocarpa, and G. alypum, prepared from different solvents. The screening of the phenolic compounds of the different extracts showed that the investigated plants were rich in antioxidant and antimicrobial compounds, including poly- phenols, flavonoids, and condensed tannins. On the other hand, the study of antioxidant activity using complementary methods (total antioxidant capacity, DPPH free radical scavenging activity, and reducing power) showed that most of the extracts exhibited significant antioxidant potency. The antimicrobial activity results showed that all extracts tested have anti- bacterial and anticandidal activities at different concentrations. Our results demonstrate that the studied plants could be a consistent source of antioxidant and antimicrobial compounds and support the data from the previous literature and the widespread usage of the medicinal plants tested. Acknowledgements This work was funded by the Algerian Ministry of Higher Education and Scientific Research (PRFU project grant no D01N01UN340120180001). Disclosure statement Conflict of interest: The authors declare that they have no conflict of interest. Ethical approval: All ethical guidelines have been adhered to. Sample availability: Samples of the compounds are available from the author. CRediT authorship contribution statement Conceptualization: Yuva Bellik; Methodology: Yuva Bellik, Nasreddine Mekhoukh; Software: Yuva Bellik; Validation: Yuva Bellik; Formal Analysis: Yuva Bellik; Investigation: Yuva Bellik, Nasreddine Mekhoukh; Resources: Yuva Bellik; Data Curation: Yuva Bellik; Writing - Original Draft: Yuva Bellik, Nasreddine Mekhoukh; Writing - Review and Editing: Yuva Bellik; Visualization: Yuva Bellik; Funding acquisition: Yuva Bellik; Supervision: Yuva Bellik; Project Administration: Yuva Bellik. ORCID and Email Yuva Bellik y.bellik@univ-bba.dz https://orcid.org/0000-0002-5596-2771 Nasreddine Mekhoukh n.mekhoukh@univ-bba.dz https://orcid.org/0000-0001-6677-4381 References [1]. Gurib-Fakim, A. Medicinal plants: traditions of yesterday and drugs of tomorrow. Mol. Aspects Med. 2006, 27, 1–93. [2]. Newman, D. J.; Cragg, G. M. 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This work is published and licensed by Atlanta Publishing House LLC, Atlanta, GA, USA. The full terms of this license are available at http://www.eurjchem.com/index.php/eurjchem/pages/view/terms and incorporate the Creative Commons Attribution-Non Commercial (CC BY NC) (International, v4.0) License (http://creativecommons.org/licenses/by-nc/4.0). By accessing the work, you hereby accept the Terms. This is an open access article distributed under the terms and conditions of the CC BY NC License, which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited without any further permission from Atlanta Publishing House LLC (European Journal of Chemistry). No use, distribution, or reproduction is permitted which does not comply with these terms. Permissions for commercial use of this work beyond the scope of the License (http://www.eurjchem.com/index.php/eurjchem/pages/view/terms) are administered by Atlanta Publishing House LLC (European Journal of Chemistry). http://www.eurjchem.com/index.php/eurjchem/pages/view/terms http://creativecommons.org/licenses/by-nc/4.0 http://www.eurjchem.com/index.php/eurjchem/pages/view/terms 1. Introduction 2. Experimental 2.1. Chemicals 2.2. Plant material 2.3. Preparation of plant extracts 2.4. Phytochemical study 2.4.1. Total phenolic content 2.4.2. Total flavonoid content 2.4.3. Determination of condensed tannins 2.5. Antioxidant activities 2.5.1. Total antioxidant capacity (TAC) 2.5.2. DPPH free radical scavenging activity 2.5.3. Reducing power activity 2.6. Antimicrobial activity 2.6.1. Microorganisms 2.6.2. Inoculums preparation 2.6.3. Antimicrobial screening 2.6.4. Determination of the minimum inhibitory concentration (MIC) 2.6.5. Determination of minimum bactericidal and fungicidal concentration (MBC/MFC) 2.7. Statistical analysis 3. Results 3.1. Extraction yields and total polyphenols contents 3.2. Antioxidant activity 3.3. Antimicrobial activity 4. Discussion 5. Conclusions Acknowledgements Disclosure statement CRediT authorship contribution statement ORCID and Email References PrintField10: PrintField11: PrintField12: PrintField13: PrintField14: PrintField15: PrintField16: PrintField17: PrintField20: PrintField21: PrintField22: PrintField23: PrintField24: PrintField25: PrintField26: PrintField27: