Antioxidant and Antibacterial Activity of the Aqueous and Alcoholic Extracts of the Plant Citrus maxima Merr

Background: The resistance of pathogenic bacteria against synthetic drugs led scientists to conduct research on medicinal plants. The present study investigated the antioxidant and antibacterial activity of the aqueous, methanol, and ethanol alcoholic extracts of the plant Citrus maxima Merr. (Syn. Citrus grandis) against some human pathogenic bacteria. Then, the presence of secondary metabolites was evaluated in vitro, including alkaloid, saponin, and tannin. Methods: The samples (i.e., root, stem, and seed) of C. maxima were collected at Babolsar, Mazandaran province, Iran. The agar well diffusion assay was used to determine antibacterial activity. In addition, several bacteria were applied based on the aim of the study, including Streptococcus pyogenes (PTCC1447), Bacillus subtilis (PTCC-1156), Bacillus cereus (PTCC-1247), Enterococcus faecalis (PTCC-1185), Micrococcus luteus (ATCC-10987), and Staphylococcus aureus (PTCC-1189). Further, some Gramnegative bacteria were used, encompassing Escherichia coli (ATCC-25922), Shigella boydi(-), Salmonella typhi (PTCC-1609), Pseudomonas aeruginosa (PTCC-1181), Enterobacter aerogenes (PTCC-1221), and Klebsiella pneumoniae (PTCC-1139). Next, the minimum inhibitory and bactericidal concentrations were determined by the serial dilution method. Furthermore, free radical activity was identified by 2,2-diphenyl-1-picrylhydrazyl. Moreover, the total phenolic and total flavonoid contents were conducted by Folin-Ciocalteu and aluminum chloride methods, respectively. Finally, the phytochemical compounds were investigated as well. Results: The highest sensitivity was observed on M. luteus against the root methanol extract. Additionally, the total phenolic content of root, seed, and leaf was determined as 98.22, 89.66, and 77.51 (mgGA/g), respectively. Similarly, the flavonoid content was determined as 3.52, 3.43, and 3.56 (mgQ/g), respectively. In addition, the IC50 of the root, seed, leaf, and ascorbic acid were calculated as 0.129, 0.135, 0.113, and 0.109 mg mL-1, respectively. Eventually, the methanol extract of the leaf and root showed the presence of alkaloid, saponin, and tannin. Conclusions: In general, C. maxima is suggested for producing natural drugs with antibiotic properties in the pharmaceutical industry due to the presence of secondary metabolites in its different parts.

secondary metabolites (9). The other evidence is related to the investigation of the antibacterial and antioxidant activity of the root, stem, and seed of C. medica L extracts (10).
Phenolic and flavonoid compounds exhibit antioxidant activities by inactivating active oxygen species (11). The evidence indicates that the absorption of plant flavonoids in human beings reduces the risk of cardiovascular diseases (12). Another study reported the total phenolic and flavonoid contents of the color and the white skin of C. medica (9), along with the total phenolic and flavonoid contents of the root, stem, and seed of C. medica (10). Given the above-mentioned explanations, the aim of this study was to investigate the antibacterial and antioxidant activity of various extracts of C. maxima against 12 human pathogenic bacteria in vitro. Furthermore, the presence of alkaloid, saponin, and tannin was checked in the root, stem, and seed of the methanol extract.

Chemical Materials
Mueller-Hinton agar (MHA) and nutrient broth (NB) culture media, 2,2-diphenyl-1-picrylhydrazyl (DPPH), quercetin and gallic acid from Merck Company (Darmstadt, Germany), as well as gentamicin and ciprofloxacin antibiotics from Paten Tab Company (Tehran, Iran) were prepared based on the aim of the study.

Plant Extracts
The samples (i.e. root, stem, and seed) of C. maxima Merr. (Syn. C. grandis) were collected from Babolsar, Mazandaran province, Iran and then transferred to the Biotechnology Laboratory and dried under shadow at Bu-Ali Sina University. In addition, methanol (80%), ethanol (96%), and distilled water extracts were obtained according to the method by Fuselli et al (13). Next, 25 g of the dried powder was added to 250 mL of the solvent and shaken, and then the extracts were filtered and centrifuged at 10 000 rpm for 8 minutes after 8 hours. Finally, the obtained crude extract was transferred to an oven at 37 ˚C for complete drying (9), and the residue was stored in a dark glass bottle at -22 º C freezer until further use (14).

Agar Well Diffusion Assay
The antibacterial activity was used from crude extracts by agar well diffusion assay (16). Furthermore, ethanol (96%), methanol (80%), and distilled water extracts (200 mg mL -1 ) were prepared from the roots, seeds, and leaves of C. maxima. Moreover, a volume of 250 mL of bacterial suspension (1.5 × 10 8 CFU) was poured onto the MHA medium. The wells (5 mm diameter) were created, and 50 µL of each extract solution was poured into individual wells and then the plates were incubated at 37°C for 24 hours (15). Gentamicin (10 µg) and ciprofloxacin (0.005 µg) were applied as positive controls (17). Then, the inhibitory zone around each well was measured (cm), and the results were analyzed by SAS software in triple.

Determination of Minimum Inhibitory Concentration and Minimum Bactericidal Concentration
The Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of ethanol, methanol, and distilled water extracts were determined by the serial dilution method (15). The dilution series of 100, 50, 25, 12.5, 6.25, and 3.125 mg mL -1 were prepared for the MIC experiment. Additionally, a volume of 185 µL of the fresh culture medium was poured into each tube and then 200 µL of the extract was added to the first tube. Afterward, 200 µL of which was transferred to the second tube, and the process continued. Finally, 15 µL of bacterial suspension (1.5 × 10 8 CFU) was added to all tested tubes. Next, the tubes were incubated for 24 hours at 37°C. The lowest extract dilution with no growth was considered as MIC. To measure MBC, a volume of 5 µL of the plates with no human bacterial growth was added to the MHA culture medium. Eventually, the plates were incubated for 24 hours at 37°C, and the minimum concentration with no bacterial growth was considered as MBC.

Determination of Total Phenolic and Flavonoid Contents
The total phenolic content was performed according to the Folin-Ciocalteu method (18). In addition, the absorbance of samples was measured at 765 nm by a spectrophotometer as mg of the gallic acid per gram of dry extract weight (mgGA/g) after 15 minutes at 25°C in darkness. Then, the total flavonoid content was determined using the aluminum chloride method (19).
Finally, sample absorption was measured at 415 nm using a spectrophotometer as the mg of quercetin per gram of dry extract weight (mgQ/g) after 30 minutes at room temperature.

DPPH
The free radical activity was determined according to Stojičević et al (20). Different concentrations (i.e., 0.2, 0.4, 0.6, 0.8, and 1 mg mL -1 ) of the root, seed, and leaf methanol extracts were prepared, and the ascorbic acid was used as the standard. Then, sample absorption was measured at 517 nm by a spectrophotometer after 30 minutes in darkness. The free radical scavenging activity (%) was calculated as follow: RSA (%) = 100 (1 -(As -Ab)/Ac As: Sample Ab: Blank (methanol 99%) Ac: Control

Identification of Phytochemical Compounds
To detect the presence of alkaloids, 0.5 g of methanol extract was dissolved in 5 mL HCl (1.0%) and kept in a warm distilled water bath for 5 minutes. Then, the solution was passed through the filter paper, and few drops of Mayer's reagent were added to it. The sediment or turbidity was considered as an indication of the presence of alkaloids (21,22). To detect the presence of tannin, 0.5 g of the methanol extract was dissolved in 5 mL distilled water and passed through a filter paper. Then, a few drops of FeCl 3 chloride (10%) were added as well. The observation of the black-green color indicated the presence of tannin (23). Further, 20 mL of distilled water was added to 0.25 g of the methanol extract and boiled to detect the presence of saponin. Then, it was passed through a filter paper, and 5 mL of it was mixed with 20 mL of distilled water and shaken. The stable foam on the paper represented the presence of saponin (23).

Statistical Analysis
The experiment was performed by a factorial test using a completely randomized design. The average comparisons were analyzed by the Duncan test at (P<0.01) with three replications by SPSS software, version 16.

Antibacterial Activity
The diameter of the bacteria growth-inhibitory zone around the wells was measured as well. Table 1 presents the inhibitory effects of different alcoholic and aqueous extracts of the root, seed, and leaf of Citrus maxima against human pathogenic bacteria. Gentamicin and ciprofloxacin antibiotics were used as a positive control. Based on the results, ciprofloxacin showed the highest inhibitory effect on Shigella boydii (Table 1) while negative controls demonstrated no inhibitory effect on bacteria growth.
The methanol extract of the C. maxima root exhibited the most potent activity against M. luteus. Conversely, the root aqueous extract revealed no inhibitory effect on the growth of M. luteus, S. boydi, and P. aeruginosa. Furthermore, the methanol and ethanol extracts of the root The aqueous extract of C. maxima had no effect on E. faecalis and K. pneumonia. Table 3 provides the results of the total phenolic and flavonoid content of the root, seed, and leaf methanol extracts of C. maxima. The total phenolic content of the root, seed, and leaf was determined as 98.22, 89.66, and 77.51 (mgGA/g), respectively. Additionally, the flavonoid content was measured as 3.52, 3.43, and 3.56 (mgQ/g), respectively. Finally, the methanol extracts of C. maxima showed a significant difference in phenolic content.

Antioxidant Activity
The antioxidant activity of the root, seed, and leaf methanol extract using DPPH is presented in Table 4.
The ascorbic acid was used as the standard, and it was observed that the amount of the free radical inhibition of DPPH increased by increasing extract concentration. The IC 50 of the root, seed, leaf, and ascorbic acid was measured Based on the obtained data, a significant difference was observed between the IC50 values of the root and the seed methanol extract with ascorbic acid.

Investigation of Phytochemical Compounds
The presence of alkaloid, saponin, and tannin were tested in the methanol extract. The leaf and root methanol extract showed the presence of alkaloid, saponin, and tannin. Based on the results, alkaloid was only observed in the methanol extract of the seed (Table 5).

Discussion
Due to the resistance of microbes, especially the pathogenic bacteria against synthetic antibiotics which cause infectious diseases, medicinal plants with antimicrobial effects are suggested to be used (24). In most cases, the extracts had higher inhibitory activity, especially on gram-positive bacteria. Once a bacteriostatic agent can be selected as a drug, its MBC value is three times higher than its MIC value (25). There has been no research on the antimicrobial activity of the root, seed, and leaf extracts of C. maxima. Therefore, our results were compared to the, reports related to the plant extracts which are close to this species. According to the results of Mokbel and Suganuma (8), the methanol (80%) extract of C. grandis on S. aureus demonstrated a better inhibitory effect, which is similar to our results. In their study, Kabra et al (26) reported that P. aeruginosa was more susceptible whereas resistance was observed against different extracts from the seeds in the present study. In another study (9), the highest inhibitory activity was related to the methanol extract of C. medica color skin against B. cereus (24±0.57 mm) while the root methanol extract of C. maxima exhibited the most potent activity against M. luteus (26.67±1.76 mm). Based on the results of Shojaemehr et al (10), the methanol extract of the root showed the highest inhibitory activity against M. luteus, which is in line with our results. The MIC of the leaf methanol extract from C. medica against E. coli was obtained in 6.25 mg mL -1 (10) although, in our study, the MIC of the C. maxima methanol extract was observed on M. luteus in 6.25 mg mL -1 . Differences in the degree of bacteria growth sensitivity against the plant extract are related to the intrinsic tolerance of microorganisms, the type of solvent, and the present of antimicrobial compounds in plant extracts including alkaloids, tannins, saponins, phenols, glycosides, and flavonoids, and the like (27).
An antioxidant is a substance that can prevent or delay the oxidative damage to the target molecule and is a major factor in the neutralization of free radicals. In addition, antioxidants reduce the risk of developing cardiovascular diseases and prevent the progression of cancers (28). The results of an empirical study demonstrated that polyphenols play an important role in the prevention of cardiovascular disease, cancer, osteoporosis, diabetes, and neurological diseases (29).
Likewise, Mokbel and Suganuma (8) reported the percentage of the inhibition of the free radicals of the C. maxima methanol extract as 74.5%. The IC 50 value of the C. medica hexane extract was measured as 0.147 mg mL -1 , which is consistent with the findings of the present study (30). The IC 50 of the methanol extract of the color and white skin and ascorbic acid were calculated as 0.1505, 0.1738, and 0.1095 mg mL -1 . On other hand, the total phenolic content and flavonoid content were reported as 109.5 and 105.6 (mgGA/g), as well as 3.53 and 3.02 (mgQ/g) from color and white skin, respectively (9), which is proximately similar to our results. Similarly, Ghasemi et al (31) measured the total phenolic content and flavonoid content from the methanol extract of 13 Iranian citrus species in the range of 66.5-396.8 (mgGA/g) and 0.3-31.1 (mgQ/g), respectively, which contradicts the results of the present study. Further, the total phenolic content and total flavonoid content from the root, seed, and leaf methanol extract of C. medica were determined as 106.1, 103.8, and 102.7 (mgGA/g), as well as 3.24, 3.02, and 3.96 (mgQ/g), respectively (10), which is inconsistent with the result of our study.   (32) demonstrated the presence of alkaloids in the root acetone extract of C. maxima. The presence of tannin, alkaloids, and saponin was reported in the leaf diethyl ether extract of C. maxima (33). According to the results of Reddy et al (34), the presence of alkaloids, saponins, and tannins was observed in the leaf methanol extract of C. aurantium. In another study, Mishra et al (35) indicated the presence of tannin and saponin in the skin methanol extract of C. limetta. Similarly, another study confirmed the presence of saponin and alkaloids from the skin ethanol extract of C. reticula, C. sinensis, and C. maxima (36). Moreover, Shojaemehr and Alamholo (9) reported the presence of alkaloids while the absence of saponin and tannin from the color skin methanol extract of C. medica, which is similar to our findings. In their study, Sheikhlar et al (37) showed the presence of tannin and alkaloid while the absence of saponin in the skin methanol extract of C. limon. As reported in (9), the leaf methanol extract of C. medica revealed the presence of alkaloid, saponin, and tannins. Finally, the difference in the presence and the absence of secondary metabolites in plants rely on the type of species, extract type, solvent, and testing methods (38).

Conclusions
Overall, the highest sensitivity was observed on M. luteus against the root methanol extract of C. maxima. Based on the findings, the antibacterial activity of the C. maxima extract on some human pathogenic bacteria was stabilized, and the results of this research showed better antiradical and antioxidant activity. In addition, the phytochemical analysis of this plant extract represented the presence of some secondary metabolites. Accordingly, it is suggested to create antimicrobial drugs in medicinal and pharmaceutical sciences based on the chemical composition analysis from the extract of this ethnomedicinal plant.