Microsoft Word - EJBR2022v12i4art330 ISSN 2449-8955 European Journal of Biological Research Research Article European Journal of Biological Research 2022; 12(4): 330-338 DOI: http://dx.doi.org/10.5281/zenodo.7448940 Investigation of antibacterial and antioxidant properties of three medicinal plants from Gaziantep, Turkey Sinem Aydin *, Mustafa Sümbül Giresun University, Faculty of Arts and Sciences, Department of Biology, Giresun, Turkey * Corresponding author e-mail: sinem.aydin@giresun.edu.tr Received: 26 August 2022; Revised submission: 16 November 2022; Accepted: 12 December 2022 https://jbrodka.com/index.php/ejbr Copyright: © The Author(s) 2022. Licensee Joanna Bródka, Poland. This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/) ABSTRACT: Current research aimed to reveal antibacterial and antioxidant properties of acetone and ethyl acetate extracts of Phlomis armeniaca, Echinophora tenuifolia subsp. sibthorpiana and Moringa oleifera plants obtained from herbalists in Gaziantep. Extracts of P. armeniaca, E. tenuifolia subsp. sibthorpiana and M. oleifera plants have antibacterial effect at varying degrees against test bacteria. Both ethyl acetate and acetone extracts of P. armeniaca plant exhibited higher antibacterial activity than studied other plant extracts. It was also found that the antioxidant activity increased with increasing concentrations. Since antioxidant and antibacterial activities were observed in almost all of the tested plant extracts, it was concluded that P. armeniaca, E. tenuifolia subsp. sibthorpiana and M. oleifera plants could be natural sources of antioxidant and antibacterial. Keywords: Medicinal plant; Antibacterial activity; Bacteria; Antioxidant activity; Free radical. 1. INTRODUCTION Infectious diseases are responsible for about 50,000 deaths worldwide each year. This situation has become even more serious with the increase of bacterial strains with multidrug resistance. Infectious illnesses influence every people. Due to solving the problem of resistance, researchers are in the race to find brand antibiotics [1]. Medicinal plants have some bioactive compounds such as coumarins, terpenoids, tannins, essential oils and alkaloids and they utilized as a starting point for antibiotic synthesis [2]. Imbalance with free radical activity and antioxidant activity cause oxidative stress. Antioxidants prevent oxidation which produce free radicals. They can start a chain reaction which produce more free radicals, leading to death of cells and tissues. Antioxidants convert these radicals into less reactive species [3]. Because of their natures, plants are known as natural antioxidant sources. They have many phytochemicals which reduce the risks of brain dysfunction, cardiovascular diseases, cataracts and cancers. When synthetic antioxidants and natural antioxidants are compared, synthetic antioxidants have many carcinogenic effects [4]. Phlomis armeniaca is a perennial plant which belongs to Lamiaceae [5]. It has many medicinal properties. It is an expectorant and provides healing by softening the chest in cough and bronchitis. It plays a role in curing respiratory tract mucosal infections. It is good for hoarseness. It is good for stomach aches caused by stomach cold and has diuretic properties [5]. Aydin & Sümbül Antibacterial and antioxidant properties of three medicinal plants 331 European Journal of Biological Research 2022; 12(4): 330-338 Echinophora tenuifolia subsp. sibthorpiana is a perennial plant. The leaves and flowers of the plant are used. It is good for cold, cough, bronchitis and asthma. It also has gas-digesting, stimulating and stomach- relieving effects [6]. E. tenuifolia subsp. sibthorpiana is used both as a fungicide and added to pickles to give fragrance after the aboveground part of this plant is dried in Turkey, especially Isparta [7]. Moringa oleifera is a plant originating from India and generally grows in tropical climates. It is usually a small perennial tree. It is also called the miracle tree, because almost every part of the M. oleifera plant has a separate value [8]. In Malaysia, M. oleifera seeds are consumed as peanuts. The roots are edible like horseradish. The leaves can be eaten as greens, in salads, in vegetable dishes. In the Philippines, the leaves are used in soup, etc. Its seeds contain Ben oil which is used as a paint ingredient in painting and fine arts, as well as in the lubrication of sensitive machines like watches [9]. The target of the current research is to reveal antibacterial and antioxidant properties of ethyl acetate and acetone extracts of Phlomis armeniaca, Echinophora tenuifolia subsp. sibthorpiana and Moringa oleifera. 2. MATERIALS AND METHODS 2.1. Collecting of the plant materials Phlomis armeniaca, Echinophora tenuifolia subsp. sibthorpiana and Moringa oleifera were bought from a herbal shop in Gaziantep, Turkey. 2.2. Preparation of the extracts 30 g of P. armeniaca, E. tenuifolia subsp. sibthorpiana and M. oleifera were extracted in a shaker for 24 h utilizing 300 ml acetone and ethyl acetate, separately. The extracts were filtered and residues were evaporated (40°C) with a rotary evaporator [10]. 2.3. Antibacterial activity 2.3.1. Microorganisms Ten bacteria were used in the study as follows: Listeria monocytogenes ATCC 7644, Salmonella enterica serovar typhimirium ATCC 14028, Staphylococcus aureus subsp. aureus ATCC 25923, Bacillus cereus 702 ROMA, Yersinia pseudotuberculosis ATCC 911, Enterococcus faecalis ATCC 29212, Bacillus subtilis IMG 22, Enterobacter aerogenes CCM 2531, Gordonia rubripertincta (lab isolate) and Proteus vulgaris (lab isolate). 2.3.2. Disc diffusion method The antibacterial properties of extracts of P. armeniaca, E. tenuifolia subsp. sibthorpiana and M. oleifera were determined with utilizing disc diffusion method. Each plant extract was dissolved in dimethyl sulfoxide (DMSO) at 30 mg/ml concentration. Acetone extracts and ethyl acetate extracts were studied in different petri dishes. 20 µl plant extracts added to the discs (5 mm diameter), separetely. 20 µl DMSO added to the disc for negative control. Gentamycine was used as positive control. Plates were incubated at 37°C overnight. Diameter of zones were measured with a ruler [11,12]. The tests were carried out two times. 2.3.3. Determination of Minimum Inhibition Concentration (MIC) Acetone and ethyl acetate extracts were prepared 30 mg/ml concentration in DMSO. MIC values of the extracts were determined with 96 well plates by the method of Yiğit et. [13]. Aydin & Sümbül Antibacterial and antioxidant properties of three medicinal plants 332 European Journal of Biological Research 2022; 12(4): 330-338 2.4. Antioxidant activity 2.4.1. Total Phenolic Content The quantity of the total phenolic content was denoted as μg of gallic acid equivalent (GAE)/ml. The tests were carried out three times [14]. 2.4.2. Total Flavonoid Content The quantity of the total flavonoid content was denoted as μg of catechin equivalent (CE)/ml. The tests were carried out three times [15]. 2.4.3. Total Antioxidant Capacity The quantity of the total antioxidant capacity was denoted as μg of ascorbic acid equivalent (AAE)/ml. The tests were carried out three times [16]. 2.4.4. 1,1‐Diphenyl‐2‐picryl‐hydrazyl (DPPH) radical scavenging activity Plant extracts were prepared at 250-1000 μg/ml concentrations. DPPH radical scavenging activity of the extracts was determined by the method of Brand-Williams et al. [17]. The tests were carried out three times. BHT and rutin were used as standards. The DPPH radical scavenging activity was calculated using the following equation: DPPH radical scavenging activity (%) = [(A0 – A1) / A0] x 100 A0 is the absorbance of the control A1 is the absorbance of the sample 2.4.5. Cupric reducing antioxidant capacity (CUPRAC) CUPRAC activity of the extracts was studied by the method of Özyürek et al. Absorbance was measured at 450 nm. BHT was utilized as standard antioxidant substance [18]. 3. RESULTS AND DISCUSSION 3.1. Antibacterial activity Antibiotic resistance creates an important health problem by increasing health costs and mortality rates. Recently, important works have been carried out to control the spread of resistant pathogens and plants are investigated as new antibiotic resources [19]. Inhibition zones which were created by test extracts were demonstrated in Table 1. Both acetone and ethyl acetate extracts of P. armeniaca exhibited higher activity than tested other plant extracts. The weakest antibacterial activity generally was found in M. oleifera. DMSO which was used as negative control didn’t show any activity against test bacteria. Gentamycine which was used as positive control showed higher activity than tested plant extracts except for acetone extract of P. armeniaca against E. faecalis. The weakest antibacterial effect was found in acetone extracts of E. tenuifolia subsp. sibthorpiana against S. enterica (6.5±0.70) and acetone extract of M. oleifera against P. vulgaris (6.5±0.70). The highest antibacterial effect was detected in acetone extract of P. armeniaca against E. faecalis (21±1.41). MIC is defined as the lowest concentration which inhibits microorganisms growth [20]. In MIC assay, extracts which showed inhibition zones ≥10 mm were studied [21]. MIC values of the extracts were given in Table 2. Aydin & Sümbül Antibacterial and antioxidant properties of three medicinal plants 333 European Journal of Biological Research 2022; 12(4): 330-338 Table 1. Inhibition zones which was created by plant extracts and gentamycine (mm). Bacteria EAE PAE MAE EEE PEE MEE DMSO CN S. enterica serovar typhimirium 6.5±0.70 14.5±0.70 6.5±0.70 - 8±1.41 8±0.00 - 17.5±0.70 P. vulgaris 10±0.00 12±1.41 6.5±0.70 6.5±0.70 7±0.00 - - 16±1.41 Y. pseudotuberculosis 7.5±0.70 11±1.41 11±1.41 9±0.00 8.5±0.70 9.5±0.70 - 18±0.00 B. subtilis 8.5±0.70 13.5±0.70 8±1.41 - - - - 15.5±0.70 B. cereus 7±1.41 8±1.41 - 11±1.41 12±1.41 7±1.41 - 20±1.41 E. aerogenes 11±1.41 12±1.41 - 12.5±0.70 11±1.41 9±0.00 - 18.5±0.70 S. aureus subsp. aureus 11.5±0.70 12.5±0.70 11.5±0.70 14±0.00 20.5±0.70 11±1.41 - 19.5±0.70 G. rubripertincta 11.5±0.70 12.5±0.70 - 16±0.00 10.5±0.70 9±0.00 - 22±1.41 L. monocytogenes 9±1.41 8.5±0.70 7.5±0.70 13.5±0.70 11.5±0.70 11.5±0.70 - 21.5±0.70 E. faecalis 17±1.41 21±1.41 - 8±1.41 8.5±0.70 8.5±0.70 - 20±0.00 EAE: Acetone extract of E. tenuifolia subsp. sibthorpiana; PAE: Acetone extract of P. armeniaca; MAE: Acetone extract of M. oleifera; EEE: Ethyl acetate extract of E. tenuifolia subsp. sibthorpiana; PEE: Ethyl acetate extract of P. armeniaca; MEE: Ethyl acetate extract of M. oleifera; CN 10: Gentamycine 10 µg/ml. Table 2. MIC values of the extracts (µg/ml). Bacteria EAE PAE MAE EEE PEE MEE S. enterica serovar typhimirium - 93.75 - - - P. vulgaris 187.5 375 - - - - Y. pseudotuberculosis - 46.88 375 - - - B. subtilis - 187.5 - - - - B. cereus - - - 187.5 93.75 - E. aerogenes 187.5 187.5 - 187.5 93.75 - S. aureus subsp. aureus 187.5 375 375 375 187.5 187.5 G. rubripertincta 93.75 23.44 - 375 93.75 - L. monocytogenes - - - 375 187.5 187.5 E. faecalis 93.75 375 - - - - EAE: Acetone extract of E. tenuifolia subsp. sibthorpiana; PAE: Acetone extract of P. armeniaca; MAE: Acetone extract of M. oleifera; EEE: Ethyl acetate extract of E. tenuifolia subsp. sibthorpiana; PEE: Ethyl acetate extract of P. armeniaca; MEE: Ethyl acetate extract of M. oleifera. Aydin & Sümbül Antibacterial and antioxidant properties of three medicinal plants 334 European Journal of Biological Research 2022; 12(4): 330-338 While acetone extracts of the plants are ranges from 23.44 µg/ml to 375 µg/ml; ethyl acetate extracts of the plants are ranges from 93.75 µg/ml to 375 µg/ml. Lower MIC values show higher antibacterial activity. The lowest MIC value was exhibited by acetone extract of P. armeniaca against G. rubripertincta as 23.44 µg/ml. There are studies about the antibacterial activities of P. armeniaca. E. tenuifolia subsp. sibthorpiana and M. oleifera. For example, Aybey investigated antibacterial activity of ethyl acetate extract of P. armeniaca and it was found that this extract created 12 mm and 14 mm inhibition zones on Bacillus subtilis and Salmonella typhimurium, respectively. Also, MIC values were found as 0.625 mg/ml and 1.25 mg/ml against these bacteria [22]. In our study, it was found ethyl acetate extract of P. armeniaca showed no activity against B. subtilis and created 8 mm inhibition zone against S. typhimurium. These differences arised from collecting samples from different locations and using different extraction methods. In literatures, there are studies generally about antibacterial activity of essential oils of E. tenuifolia subsp. sibthorpiana. Gökbulut et al. found MIC values of essential oil of E. tenuifolia subsp. sibthorpiana as 125 µg/ml. 62.5 µg/ml and 500 µg/ml against Staphylococcus aureus. Bacillus cereus and Enterococcus faecalis, respectively [23]. In our current study, MIC values of extract of E. tenuifolia subsp. sibthorpiana found at 187.5 µg/ml and 93.75 µg/ml against S. aureus and E. faecalis, respectively. These different results might be arised from different secondary metabolites which in our extract and essential oil. Fouad et al. searched antibacterial effect of M. oleifera leaf extract against pyogenic bacteria isolated from camel abscess [24]. 3.2. Antioxidant activity 3.2.1. Total phenolic content The total phenolic content of the extracts was presented in Table 3. While the highest total phenolic content was found in acetone extract of P. armeniaca (434.21±0.011 μg GAE/ml), the lowest total phenolic content was found in ethyl acetate extract of M. oleifera (25.66±0.003 μg GAE/ml). In a study which was carried out by Yumrutaş and Saygıdeğer, total phenolic content of methanol and hexane extracts of P. armeniaca was found as 320.37±6.97 mg GAE/g and 55.90±1.01 mg GAE/g, respectively [25]. Using different extraction methods and solvents cause different results between our study and Yumrutaş and Saygıdeğer’s study. Table 3. Total phenolic content of the tested plant extracts (μg GAE/ml). Plant Extract Total Phenolic Content (μg GAE/ml) Acetone extract of P. armeniaca 434.21±0.011 Acetone extract of E. tenuifolia subsp. sibthorpiana 46.97±0.001 Acetone extract of M. oleifera 27.48±0.003 Ethyl acetate extract of P. armeniaca 120.06±0.009 Ethyl acetate extract of E. tenuifolia subsp. sibthorpiana 141.6±0.006 Ethyl acetate extract of M. oleifera 25.66±0.003 3.2.2. Total flavonoid content Total flavonoid content of the extracts were given in Table 4. Total flavonoid content of ethyl acetate extracts of the plants were found higher than acetone extracts of the plants except for ethyl acetate extract of M. oleifera. The highest and the lowest total flavonoid content was found in ethyl acetate extract of E. tenuifolia Aydin & Sümbül Antibacterial and antioxidant properties of three medicinal plants 335 European Journal of Biological Research 2022; 12(4): 330-338 subsp. sibthorpiana (739.17±0.010 µg QE/ml) and acetone extract of P. armeniaca (177.49±0.011 µg QE/ml), respectively. Table 4. Total flavonoid content of the tested plant extracts (µg QE/ml). Plant Extract Total Flavonoid Content (μg QE/ml) Acetone extract of P. armeniaca 177.49±0.011 Acetone extract of E. tenuifolia subsp. sibthorpiana 344.64±0.044 Acetone extract of M. oleifera 354.27±0.037 Ethyl acetate extract of P. armeniaca 228.95±0.049 Ethyl acetate extract of E. tenuifolia subsp. sibthorpiana 739.17±0.010 Ethyl acetate extract of M. oleifera 320.92±0.089 3.2.3. Total antioxidant capacity Total antioxidant capacity of the tested plant extracts were presented in Table 5. Total antioxidant capacity of ethyl acetate extracts of the plants were detected higher than acetone extracts of the plants except for ethyl acetate extract of P. armeniaca. The highest total antioxidant capacity was found in ethyl acetate extract of E. tenuifolia subsp. sibthorpiana (373.50±0.033 µg AAE/ml) and the lowest total antioxidant capacity was found in acetone extract of M. oleifera (85.23±0.010 µg AAE/ml). Table 5. Total antioxidant capacity of the tested plant extracts (µg AAE/ml). Plant Extract Total Antioxidant Capacity (μg AAE/ml) Acetone extract of P. armeniaca 162.26±0.014 Acetone extract of E. tenuifolia subsp. sibthorpiana 100.32±0.020 Acetone extract of M. oleifera 85.23±0.010 Ethyl acetate extract of P. armeniaca 119.56±0.025 Ethyl acetate extract of E. tenuifolia subsp. sibthorpiana 373.50±0.033 Ethyl acetate extract of M. oleifera 169.41±0.023 3.2.4. DPPH radical scavenging activity Figure 1 shows DPPH radical scavenging activity of extracts and standards. DPPH radical scavenging activity of acetone extract of M. oleifera is higher than BHT and Rutin which were synthetic antioxidants at 1000 µg/ml concentration. When the activities of extracts, BHT and Rutin are compared at 1000 µg/ml concentration, we can make a ranking as follows: Acetone extract of M. oleifera > BHT > Rutin > Acetone extract of E. tenuifolia subsp. sibthorpiana > Ethyl acetate extract of P. armeniaca > Ethyl acetate extract of E. tenuifolia subsp. sibthorpiana > Ethyl acetate extract of M. oleifera >Acetone extract of P. armeniaca. Abdulkadir et al. found DPPH radical scavenging activity (% inhibition) of methanol extract of M. oleifera ranges from 58.62±1.13 and 83.62±1.32. Moreover, DPPH radical scavenging activity (% inhibition) of hexane extract ranges from 15.98±1.24 and 32.91±1.63 [26]. Aydin & Sümbül Antibacterial and antioxidant properties of three medicinal plants 336 European Journal of Biological Research 2022; 12(4): 330-338 Figure 1. DPPH scavenging activity extracts and standards. Table 6. CUPRAC activity of extracts and BHT. Plant Extract Concentration (µg/ml) Absorbance (nm) PEE 250 0.6856±0.024 500 1.4135±0.021 750 1.8411±0.018 1000 2.048±0.026 EEE 250 0.8741±0.018 500 1.4926±0.023 750 1.970±0.016 1000 2.127±0.030 MEE 250 0.3248±0.028 500 0.4933±0.016 750 0.7494±0.024 1000 1.0246±0.038 PAE 250 0.6213±0.007 500 0.8782±0.003 750 0.8842±0.004 1000 0.9676±0.019 EAE 250 0.068±0.011 500 0.2171±0.002 750 0.4048±0.012 1000 0.5634±0.027 MAE 250 0.074±0.006 500 0.2245±0.018 750 0.4269±0.031 1000 0.5799±0.0001 BHT 250 0.6635±0.023 500 0.7016±0.021 750 0.8283±0.024 1000 0.9716±0.014 PEE: Ethyl acetate extract of P. armeniaca; EEE: Ethyl acetate extract of E. tenuifolia subsp. sibthorpiana; MEE: Ethyl acetate extract of M. oleifera; PAE: Acetone extract of P. armeniaca; EAE: Acetone extract of E. tenuifolia subsp. sibthorpiana; MAE: Acetone extract of M. oleifera. Aydin & Sümbül Antibacterial and antioxidant properties of three medicinal plants 337 European Journal of Biological Research 2022; 12(4): 330-338 3.2.5. CUPRAC activity CUPRAC activity of the extracts was demonstrated in Table 6. When CUPRAC activity of extracts are compared at 1000 µg/ml concentration, the highest activity was detected in ethyl acetate extract of E. tenuifolia subsp. sibthorpiana and the lowest activity was detected in acetone extract of E. tenuifolia subsp. sibthorpiana. Ethyl acetate extracts exhibited higher activity than acetone extracts of extracts. Moreover. ethyl acetate extracts of the plants showed higher CUPRAC activity than BHT which was used standard antioxidant agent. Sarıkürkçü et al. found CUPRAC activity of ethyl acetate extract of P. armeniaca was higher than methanol and water extracts [27]. In our study, we also found CUPRAC activity of ethyl acetate extract of P. armeniaca higher than acetone extract. 4. 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