Investigation of Antibacterial and Antioxidant Activity of Citrus medica L Extract on Human Pathogenic Bacteria

Medicinal plants are a rich source of lead compounds for traditional and modern medicines (1). Currently, clinical effectiveness of many antibiotics is compromised by the emergence of resistant pathogens. Therefore, there is a continuing and urgent need for the discovery of new antimicrobial sources. Herbal drugs are widely used in ethnomedicine due to their unknown bioactive compounds, low side effects, and relatively low costs (2). Accordingly, several studies have focused on medicinal plants to find more effective drugs against microbial infections (3,4). Citrus medica L. is a valuable medicinal plant used in Iranian ethnomedicine. It is a small plant, having short thorns and large and rectangular leaves with elliptical fruits. It is reported that C. medica extract plays a role in the treatment of diabetes and Alzheimer disease (5). The root extract of C. medica is used for its anthelmintic and antilithic properties in the treatment of urinary calculi in India (6). It is known that plant oriented compounds such as carotenoids, phenolics, flavonoids, and ascorbic acid eliminate free radicals and have antioxidant and antimutagenic properties (7). Antimicrobial activity of saponin and tannins (8), alkaloids (9), and flavonoids have been reported (10). Therefore, differences in the sensitivity of bacteria to plant extracts could be either due to the intrinsic sensitivity of microorganisms, or the nature of compounds with antimicrobial properties such as alkaloids, tannins, saponins, phenols, glycosides, and flavonoids (11,12). Moreover, several parameters affecting the plant extract efficacy are extraction method, plant genotypes (13), the moisture content of the plant, and the temperature of the extraction time (1). As a result, the extraction method, extraction time, solvent and tissue type, as well as the plant species affect the presence and the amount of desired compounds in the extracts (14). To our knowledge, no research has been done on the antimicrobial activity of Iranian C. medica. Therefore, here we aimed at investigating antibacterial and antioxidant activities of various extracts of C. medica Avicenna Journal of Clinical Microbiology and Infection

against some human pathogenic bacteria in vitro. We further investigated the presence of flavonoids, saponins, and tannins in methanol extracts of C. medica in order to find a possible mechanism for such an effect.

Preparation of the Plant Extracts
The plant samples of C. medica were collected from Mazandaran province, north of Iran. The samples were immediately transferred to the laboratory and dried at room temperature in the shade and far from direct sunlight. Dried samples were broken into small pieces. Ethanol (96%), methanol (80%), and distilled water extracts were obtained by soxhlet extractor (15). To this end, 30 g of dried powder was separately added to 350 mL of each solvent used. After 4 hours, the extracts were filtered through filter paper and centrifuged at 10 000 rpm for 8 minutes (16). The extract solvent was evaporated by a rotary evaporator and was transferred to an oven at 37˚C for complete drying. The residue was stored in the dark at -22˚C. Agar Well Diffusion Assay for Assessing the Antibacterial Activity Agar well diffusion assay was used to determine the antibacterial activity of plant extracts (17). The concentrations of 400 mg/mL (root, seed, or leaf extract) of ethanol (96%), methanol (80%) and distilled water were prepared. A volume of 200 mL of bacterial suspension (1.5 × 10 8 CFU) was poured onto MHA medium and uniformly spread with a swab. Then, 5 mm diameter wells were created in Petri plates, and 50 µL of the extract was poured into each well. Petri plates were incubated at 37˚C for 24 hours (18). Gentamicin (10 µg) and ciprofloxacin (0.005 µg) were used as positive controls (19). The inhibitory zone formed around each well was measured (cm). The results from three replicates of the experiment were statistically analyzed.
Determination of MIC and MBC Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of ethanol, methanol and distilled water extracts were determined by serial dilution method (20). Dilution series of 400, 200, 100, 50, 25, 12.5, and 6.25 mg/mL were prepared for MIC. A volume of 15 μL of each bacterial suspension (0.5 McFarland) was added to each test tube. The positive (300 μL of the extract with 285 μL of culture medium) and negative (285 μL of the extract with 15 μL of bacteria) controls were considered. The tubes were incubated at 37°C for 24 hours. The lowest dilution of the extract with no growth of bacteria was considered as MIC. To measure MBC, 5 µL of the tubes with no bacterial growth was added on MHA culture medium. Plates were incubated at 37°C for 24 hours. The minimum concentration with no bacterial growth on the plates was considered as MBC.
Determination of Total Phenolic Content Total phenolics content was estimated according to the Folin-Ciocalteu method (21). Accordingly, 100 mg of extract was dissolved in 1 mL of methanol. Then, 2 mL of sodium carbonate (Na2CO3) was poured into a tube and vortexed. Afterwards, 2.5 mL of 10% Folin solution was added to the solution and remixed. After 15 minutes at 25˚C in darkness, the absorbance of samples was measured at 765 nm using a spectrophotometer and determined as mg of gallic acid per gram of dry extract weight (mg GA/g).

Determination of Total Flavonoids Content
Total flavonoids content was determined by aluminum chloride method (22). Accordingly, 100 μL of 10% aluminum chloride and 100 μL of potassium acetate were poured into a tube and mixed. Then, 2.8 mL of distilled water and 0.5 mL of extract solution were added. After 30 minutes at room temperature, the absorption of samples was measured at 415 nm using a spectrophotometer and determined as mg of quercetin per gram of dry extract weight (mg Q/g).
Determination of DPPH for Free Radical Scavenging Activity Free radical activity was investigated according to Stojičević et al (23). Different concentrations (0.2, 0.4, 0.6, 0.8, and 1 mg/mL) of methanol extracts of root, seed, and leaf were prepared and ascorbic acid was used as the standard. The samples were placed in darkness for 30 minutes, and then, solvent absorption was recorded with a spectrophotometer at 517 nm. Methanol (99%) was used as the blank. The free radical scavenging activity (%) was calculated by the following formula: RSAC (%) = 100(1 -(As -Ab)/Ac Where As indicated sample; Ab indicated blank; and Ac indicated control.

Identification of Tannin, Saponin, and Alkaloids
To investigate the presence of alkaloids, 0.5 g of methanol extract was dissolved in 5 mL of 1% HCl and kept for 5 minutes in a warm distilled water bath. Then, the solution was passed through a filter paper, and a few drops of Mayer's reagent were added to it (24). The sedimentation or turbidity indicated the presence of alkaloids (25). To track tannin, 0.5 g of methanol extract was dissolved in 5 mL of distilled water and the solution was passed through filter paper. Then, a few drops of FeCl 3 chloride (10%) were added to it. The appearance of the black-green color indicated the presence of tannin (26).
To track saponin, 20 mL of distilled water was added to 0.25 g methanol extract and boiled. The solution was passed through a filter paper, and 5 mL of it was mixed with 20 mL of distilled water and shaken. The formation of the stable foam indicated the presence of saponin (27).

Statistical Analysis
The experiments were performed in a completely randomized design with factorial test. The average comparisons were done using Duncan test at P<0.05 using SPSS software version 16.0.

Antibacterial Activity
The inhibitory effects of different alcoholic and aqueous extracts of root, seed, and leaf of Citrus medica were evaluated against human pathogenic bacteria (Table 1). Negative control (50 μL of used solvents) and positive controls (gentamicin and ciprofloxacin) were included. After incubation, diameters of zone of inhibition around the wells were measured. Data indicated that the methanol extracts of leaves showed a better inhibitory effect on B. cereus, S. aureus, M. luteus, and E. coli. Inhibitory activity of leaf methanol extract on B. cereus, E. coli, and E. aerogenes was more potent than gentamicin. Furthermore, methanol extracts of roots showed the highest inhibitory activity against M. luteus. The inhibitory activity of root methanol extract on M. luteus and E. aerogenes and that of root ethanol extract on E. aerogenes was more potent than gentamicin. The methanol extract of seeds showed the highest inhibitory activity against M. luteus; however, S. pyogenes, S. boydii, and P. aeruginosa showed resistance against aqueous extracts. Additionally, Shigella boydii showed resistance against the methanol, ethanol, and aqueous extracts of seeds. In total, seed extracts showed less inhibitory activity than the leaves and roots extracts ( Table 1).
The MIC of the methanol extract of leaves against E. coli was 6.25 mg/mL. MIC of root, leaf, and seed methanol extract against B. subtilis, S. aureus, and S. typhi were 12.5 mg/mL. However, no inhibitory effect was observed against S. pyogenes and S. boydii; therefore, these bacteria were deleted from Table 2. The leaf aqueous extract did not show inhibitory activity on all tested bacteria. According to the results, methanol extract showed more inhibitory activity compared to ethanol and aqueous extracts. In addition, gram-positive bacteria such as B. subtills, B. cereus, and M. luteus were more sensitive than gram-negative bacteria. The tested bacteria showed better sensitivity against root ( Table 2). The aqueous extract did not show any effect on E. faecalis and K. pneumoniae.
Antioxidant Activity of Citrus medica Extracts As seen in Table 3, the amount of DPPH free radicals inhibition was increased by increasing the concentration of plant extracts. A significant difference was observed between the IC50 values of methanol extracts and ascorbic acid as the control (Table 3). Seed and root methanol extracts of C. medica showed strong radical scavenging activities.

Total Phenolic and Flavonoid Content
The results of total phenolic and flavonoid content of root, seed and leaf methanol extracts of C. medica have been shown in Table 4. Total phenolic contents of root, seed, and leaf were determined as 106.1, 103.8, and 102.7 mgGA/g, respectively. The contents of flavonoids were determined as 3.24, 3.02, and 3.96 mgQ/g, respectively.
The Presence of Alkaloids, Tannins, and Saponins The methanol extract, which showed the highest antimicrobial activity, was used to study the presence and absence of alkaloids, saponins, and tannins. The results of the presence and absence of alkaloids, saponins, and tannins have been shown in Table 5. Alkaloids, saponins, and tannins were observed in the methanol extract of leaf, while the methanol extracts of root and seed showed only the presence of alkaloids.

Discussion
According to the obtained results, the methanol extract showed better inhibitory activity on tested bacteria compared to ethanol and aqueous extracts. The highest inhibitory zone diameter was observed against M. luteus against the root methanol extract. The MIC and MBC of root, seed, and leaf tissues were not reported against S. pyogenes and S. boydii. Secondary metabolites such as phenol and flavonoids have a strong potential for clearing free radicals that are present in all parts of plants such as leaves, fruits, seeds, roots, and peels (28). The emergence of new bacterial strains with multiple resistances, unavailability, and high costs of chemical drugs has led to an increase in mortality in the world (28,29). Therefore, it is necessary to find compounds with antimicrobial  Leaf  M  MIC  50  25  12.5  50  50  100  -6.25  100  200   MBC  100  50  25  ----25  200  -E  MIC  50  50  50  50  25  50  -25  50  100   MBC  100  -100  -50  100  -50  100  200   A  MIC  --100  100  -100  ----MBC  --------- properties from medicinal plant sources (14). The ethanol and methanol (polar solvents) can better dissolve the polar compositions in the plant and have antibacterial effects on pathogenic microorganisms (30). Sah et al (13) tested antimicrobial properties of root and leaf ethanol extract of C. medica against human pathogenic bacteria. The MIC of the root was 0.5 mg/mL against B. subtillis, which is not in line with our results. Kabra et al (3) investigated the antimicrobial effect of ethanol extract of C. medica peel on human pathogenic bacteria and reported that S. aureus and P. aeruginosa had more sensitivity against the tested extract. In our research, the seed ethanol extract did not show any inhibitory effect against P. aeruginosa, which is contrary to the results of the mentioned group. These differences probably could be due to compound extraction methods, plant genotype, type of organ and the time and stage of plant growth.
Menichini et al (31) reported that IC50 of hexane extract of C. medica skin was 0.471 mg/mL, which was almost similar to our results. Reddy et al (32) reported the highest percentage of free radical scavenging for the leaf methanol and hydro methanol extracts of C. aurantifolia, equal to 93.53. Ghasemi et al (33) reported that IC50 values of methanol extract of 13 Iranian Citrus species ranged from 0.6 to 2.9 mg/mL. Differences in the chemical composition and secondary metabolites with antimicrobial properties such as phenol, ascorbic acid, and carotenoids, as well as differences in species can affect the level of antioxidant changes (2).
Choudhury et al (12) related the antimicrobial properties of plant extracts to the compounds such as phenolic, flavones, tannins, terpenoids, alkaloids, flavonoids, and saponin. Tannin showed a good in vitro antimicrobial activity (34). Some flavonoids with anticancer properties including hesperidin, narirutin, naringin, and eriocitrin were reported (35). The antimicrobial activity of saponins has been shown on some microorganisms (36). Gorinstein et al (37) determined the total phenolic content of lemon and orange peel as 1.9 and 1.8 (mgGA/g). The total flavonoid contents of ethanol extract of C. sinensis, C. maxima, and C. reticula were estimated as 0.13, 0.20, and 0.14 (mgQ/g), respectively (38), which are different from our results. These differences could be probably due to differences in species type, tissue type, and extraction method. Ghasemi  Karou et al (39) reported the presence of alkaloids and the absence of saponins and tannins in ethanolic extract of C. medica by phytochemical methods. Bairagi et al (40) showed the presence of alkaloid and the absence of saponin and tannin in the leaf extract of C. medica. Wu et al (41) reported the presence of alkaloid in root acetone extract of C. grandis, and the presence of tannin, alkaloids, and saponin was reported in leaf diethyl ether extract of C. grandis (42). Pandey et al (16) reported the presence of tannin and the absence of saponin in the seed methanol extract of C. limon. Pathan et al (43) reported the presence of tannin in leaf and the presence of alkaloids in the leaf and root hydroalcoholic extract of C. aurantium. Reddy et al (32) by phytochemical analysis showed the presence of alkaloid, saponin, and tannin in the leaf methanol extract of C. aurantium.

Conclusions
Our results showed that the alcoholic extracts of C. medica contain antibacterial compounds. These results could be useful in the identification of active compounds that can be formulated into antibacterial herbal drugs. This study suggests that C. medica may be used to discover natural bioactive products which might lead to the development of new drugs with antibacterial and antioxidant properties in the field of medicine.