1. Background
Infectious diseases caused by various microorganisms are very common worldwide (1). Acinetobacter spp. are hospital pathogens that have spread due to the widespread use of antibiotics. These bacteria are Gram-negative, oxidase-negative, obligately aerobic, and non-motile coccobacilli and a common cause of nosocomial infections (2-4). The bacteria from this genus are also resistant to disinfectants and can survive in natural environments such as water and soil for a long time and lead to infections, especially in immunodeficient individuals (5). Acinetobacter baumannii, the most important species of this genus, is responsible for respiratory, blood, urinary tract, and wound infections (6). The ICU environment and ICU patients can contribute to the spread of this microorganism. Generally, this bacterium is harmless to healthy people, but in some cases, is strongly resistant to antibiotics and can spread to patients. In addition, the treatment of such organisms is difficult and can lead to increased mortality (7). Infectious disease specialists have intended to use herbal medicines and natural compounds in recent years due to the numerous side effects of chemical drugs, as well as significant advances in the production of herbal medicines (7).
Nowadays, to discover new therapeutic methods that are more economical with fewer side effects, the study of medicinal plants and their properties is particularly important so that over 30% of medicinal plants are currently being used in hospitals and clinics (8, 9). Herbal drugs are usually a complex of hundreds of different chemical compounds, but only a few of them are responsible for the beneficial or adverse effects of these drugs (10).
Rumex acetosella L. is a perennial herb that widely germinates in East Asia (11). This plant grows mainly in wet valleys and can reach up to one meter in height. Its leaves are green and fleshy and contain large amounts of chlorophyll. The chemical compounds isolated from it include flavonoids, phenolic compounds, and terpenoids (12). Rumex acetosella extract has diuretic, insecticidal, anti-inflammatory, antioxidant, antimicrobial, and anticancer properties, and its aqueous extract is used for gastritis and gastric ulcers in traditional medicine (12-15). Various species of the Rumex genus have been studied, and it has been observed that different extracts of the aerial parts and root of this plant have antimicrobial effects on certain bacteria such as Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Bacillus cereus (16, 17).
Pumpkin (Cucurbita maxima L.) is an herbaceous and annual plant that is used for treating earache, fever, and bronchitis, as well as diseases and infections of the urinary tract and prostate due to its antibacterial properties (18, 19). Anti-diabetic, antioxidant, anticancer, and anti-inflammatory effects of C. maxima have also been demonstrated (20). In a study that examined the oil derived from C. maxima seeds, the oil was observed to contain essential fatty acids that could maintain the health of blood vessels, nerves, and tissues (21).
2. Objectives
Concerning the effective antibacterial properties of R. acetosella and C. maxima, lower costs, and fewer complications of herbal drugs, and recent advances in the production of plant-derived natural compounds in the treatment and prevention of bacterial infections, the present study was conducted to investigate the antimicrobial effects of these two plants on A. baumannii and compare them with conventional antibiotics in vitro.
3. Methods
An experimental study was carried out to investigate the antimicrobial effects of hydroalcoholic and aqueous extracts of R. acetosella and C. maxima on A. baumannii. To achieve this purpose, maceration was used for extraction; in addition, to measure the extracts’ antibacterial effects, the lyophilized A. baumannii standard strain (ATCC no.: 747) was obtained from the Iranian Research Organization for Science and Technology (IROST). Then, the antibacterial effects of the hydroalcoholic and aqueous extracts of the plants on A. baumannii were investigated by broth microdilution and agar disk diffusion.
3.1. Determination of Sample Size
We investigated the effects of two different extracts from two plants and five antibiotics on one bacterium, and each experiment was conducted in triplicate. Thus, the total number of experiments (sample size) was 60 (7).
3.2. Collection and Authentication of Studied Species
The plants were purchased from groceries and fruit stores and identified as the plants of interest by botanists according to the botanical and floral keys of Iran and available resources on plants. Also, they were adapted to the herbarium specimens available at the Medical Plants Research Center of Shahrekord University of Medical Sciences and Shahrekord Research Center for Agriculture and Natural Resources and assigned the herbarium vouchers of 184 (R. acetosella) and 1400 (C. maxima).
3.3. Extraction Method
For the preparation of hydroalcoholic extracts, 300 g of each plant was ground in a mill, and extraction was conducted by the maceration method. For this purpose, 70% alcohol was added to 300 g of each plant to a final volume of one liter; the resulting solution was stored for four days and then filtered through a Whatman filter paper. The filtrate was then concentrated in a rotary evaporator at 40°C. After distillation, the concentrated filtrate was incubated at 37°C to obtain the extract, and a stock solution was prepared by dimethyl sulfoxide (DMSO). The aqueous extracts were prepared by boiling. For this purpose, 200 g of powder of each plant was mixed with 300 mL of distilled water, and the mixture was heated for 20 min while it was constantly being stirred. The resulting extract was filtered through a filter paper (7).
3.4. Preparation of Microbial Suspension
To prepare a microbial suspension, 24 hours before the experiments, a fresh (24-h) culture medium was prepared using culture media. Before starting the inoculation, some colonies were transferred from the surface of the Mueller-Hinton broth culture medium to the tube containing the physiological serum by using a sterile swab, and then the turbidity of microbial suspension was measured and compared with that of 0.5 McFarland standard (McFarland standard is a chemical solution with turbidity comparable to that of microbial suspension) (7).
3.5. Determining Minimum Inhibitory Concentration (MIC)
The minimum inhibitory concentration (MIC) was determined in a 96-well sterile plate by using the broth microdilution method and Mueller-Hinton broth culture medium according to specific instructions for the use of Merck (Germany) products. In this method, the first well was selected as a negative control, and the second well was selected as the positive control. After adding culture media, stock solution, and bacterial solution to the microplate wells and diluting them, the samples were incubated at 37°C for 24 h. The first well in which the turbidity was absent was considered for MIC determination (7).
3.6. Determining Minimum Bactericidal Concentration (MBC)
To determine the minimum bactericidal concentration (MBC), all wells without turbidity were cultured separately on blood agar, and after 24 hours, the lowest concentration of extract at which the bacterium could not grow was considered for MBC determination (7).
3.7. Assessment of Antimicrobial Effects
To perform the disk diffusion test, after isolating the bacteria, some colonies of the bacteria were dissolved in sterile physiological serum by using an inoculating needle. After preparing the homogeneous solution and shaking it with a sterile swab, it was transferred to Mueller-Hinton agar. Then, the sterile blank paper disks (Padtan Teb Co., Iran) were left in the extracts at prepared concentrations for 24 h so that the extracts were completely absorbed into the disks. Then, the discs containing the extracts at different concentrations were incubated at 37°C for one hour to dry, and then placed on the plates and cultivated at appropriate distances. They were incubated at 37°C for one hour to examine the antibacterial properties of the extracts. The diameters of the inhibition zones were measured with a ruler. For this purpose, the zero of the ruler was placed next to a diameter of the inhibition zone, and the distance between zero and the end of the inhibition zone was measured and expressed in mm. Then, the results were compared with standard disks containing amikacin, imipenem, ceftazidime, gentamicin, and ciprofloxacin (Padtan Co., Iran) in tables provided by the Clinical and Laboratory Standards Institute (CLSI) (7).
3.8. Statistical Analysis
The tests were repeated in triplicate, and the mean and standard deviation (SD) of the growth inhibition zone diameters in the cup-plate method and the MIC and MBC of the extracts were determined.
4. Results
This study was carried out using the broth microdilution method to determine the antibacterial effects of hydroalcoholic and aqueous extracts of R. acetosella and C. maxima. The results obtained from the hydroalcoholic extracts indicated the higher levels of MIC and MBC of R. acetosella extract (Table 1).
Table 1.The MIC and MBC Of R. acetosella and C. maxima Extracts on A. Baumannii By Broth Microdilution
| Plant | Hydroalcoholic Extract | Aqueous Extract | ||
|---|---|---|---|---|
| MIC, µg/mL | MBC, µg/mL | MIC, µg/mL | MBC, µg/mL | |
| Rumexacetosella L. | 128 | 256 | 64 | 128 |
| Cucurbita maxima L. | 64 | 128 | 128 | 256 |
Abbreviations: MBC, minimum bactericidal concentration; MIC, minimum inhibitory concentration.
The findings on the aqueous extract of the plants showed that the MIC and MBC of C. maxima extract were higher. The highest MICs against A. baumannii were obtained for the C. maxima aqueous extract and R. acetosella hydroalcoholic extract, and the lowest MICs were obtained for C. maxima hydroalcoholic extract and R. acetosella aqueous extract.
The highest MBCs were obtained for C. maxima aqueous extract and R. acetosella hydroalcoholic extract, and the lowest MBCs were obtained for C. maxima hydroalcoholic extract and R. acetosella aqueous extract. The MICs and MBCs of the C. maxima hydroalcoholic extract were lower than those of its aqueous extract (Table 1).
In the disk diffusion method, the diameters of the inhibition zone in different groups are shown in Tables 2 and 3. The results showed that the R. acetosella hydroalcoholic extract exhibited the strongest antibacterial effect with a mean inhibition zone diameter of 24.83 ± 0.29 mm and C. maxima aqueous extract at 512 µg/mL concentration exhibited the lowest antibacterial effect with a mean inhibition zone diameter of 8 mm. There was a direct correlation between the inhibition zone and antibacterial activity of the extracts. No inhibition zone was observed around the blank disk and ethanol disk as negative controls.
| Extract | Different Concentrations, µg/mL | ||||||
|---|---|---|---|---|---|---|---|
| 8 | 16 | 32 | 64 | 128 | 256 | 512 | |
| Rumexacetosella L. | R | R | R | R | R | 21.5 ± 0.5 | 24.83 ± 0.29 |
| Cucurbita maxima L. | R | R | 1.67 ± 0.58 | 4.17 ± 0.29 | 5.5 ± 0.5 | 7.67 ± 0.58 | 10.33 ± 0.58 |
Abbreviation: R, resistant.
aValues are expressed as mean ± SD.
bGentamicin: 14.5 ± 0.5 mm, imipenem: 15.67 ± 0.58 mm, amikacin: 16.83 ± 0.29 mm, ceftazidime: 17.83 ± 0.29 mm, ciprofloxacin: 21 ± 0.5 mm.
| Extract | Different Concentrations, µg/mL | ||||||
|---|---|---|---|---|---|---|---|
| 8 | 16 | 32 | 64 | 128 | 256 | 512 | |
| Rumexacetosella L. | R | R | R | R | 14.5 ± 0.5 | 19.33 ± 0.58 | 21.83 ± 0.29 |
| Cucurbita maxima L. | R | R | R | 2.83 ± 0.29 | 4.83 ± 0.29 | 7 | 8 |
Abbreviation: R, resistant.
aValues are expressed as mean ± SD.
bGentamicin: 14.5 ± 0.5 mm, imipenem: 15.67 ± 0.58 mm, amikacin: 16.83 ± 0.29 mm, ceftazidime: 17.83 ± 0.29 mm, ciprofloxacin: 21 ± 0.5 mm.
5. Discussion
Acinetobacter baumannii is highly resistant to antimicrobial agents, which can be inherited or acquired through genetic resistance factors. Resistance to antimicrobial agents among clinical isolates may make it difficult to treat infections and may negatively affect clinical outcomes and treatment costs (22). Nowadays, due to changes in the pattern of resistance of bacteria and their resistance to common antibiotics, there is a tendency to replace them with new antibiotics. Meanwhile, the products of plant origin with antimicrobial activity have recently attracted special attention (7). In this study, the hydroalcoholic extracts of C. maxima and R. acetosella had the highest inhibitory effects on A. baumannii, which prevented the growth of this bacterium at a concentration of 64 µg/mL. The study of Qian showed that C. maxima polysaccharides had a very strong antibacterial effect against Bacillus subtilis, S. aureus, and E. coli at a concentration of 100 mg/mL (23).
In addition, the study by Ravishankar et al. showed that the ethanolic extract of C. maxima seed, due to active biological compounds such as carbohydrates, steroids, proteins, and amino acids, exhibited antibacterial activity against S. aureus, Staphylococcus warneri, B. subtilis, P. aeruginosa, Proteus mirabilis, Klebsiella pneumoniae, and E. coli (21). However, in the study by Elzaawely et al. (17), the antioxidant and antibacterial activities of aerial parts of a species from the Rumex genus was studied, showing that the ethyl acetate extract of aerial parts of the plant had the most potent antibacterial effect among the ethanol, hexane, chloroform, and aqueous extracts against Bacillus and E. coli. It has been reported that, in general, phenolic compounds in plant extracts act as active compounds against Gram-negative Acinetobacter bacteria (24). Many of the isolated compounds from plants whose activity has been demonstrated are secondary compounds that have been isolated by using certain solvents such as methanol, ethanol, water, and acetone by a variety of techniques. Antimicrobial agents are certain polyphenols, such as simple phenols, phenolic acids, quinones, flavones, flavonoids, flavonols, tannins, and coumarins. Besides, terpenoids, essential essences, alkaloids, lectins, polypeptides, and other compounds have also been reported. The action mechanism of these compounds varies depending on their type. These mechanisms include the enzymatic activity inhibition, the reaction of herbal extract active compounds with extracellular proteins, and the solution of microbial cell or bacterial cell wall (25). Some extracts can also interact with the microorganisms’ DNA, which may create ionic channels in the microbial membrane, or may compete with microbial proteins to bind to host polysaccharide receptors (26). As known, R. acetosella and C. maxima are among the plants that have phenolic compounds.
In the present study, the inhibitory and bactericidal effects of R. acetosella aqueous extract were stronger than those of its hydroalcoholic extract, while in the study by Jimoh et al. (27) on the antibacterial effects and phenolic content of methanolic, aqueous, and acetonic extracts of a species from the Rumex genus, the phenolic content and antibacterial effects of the methanolic and acetonic extracts were stronger than those of the aqueous extract.
Therefore, essences and herbal extracts can be used in pharmacology, microbiology, phytopathology, and food preservation. Nowadays, due to chemical material misuse and acquisition of antibiotic resistance due to the improper use of antibiotics, it is proposed to replace these substances with natural compounds such as herbal extracts and essences, including the plants used in this study, to control and prevent diseases.
5.1. Conclusions
This study, for the first time, showed the moderate-to-strong antibacterial effect of hydroalcoholic and aqueous extracts of R. acetosella and C. maxima altering in a concentration-dependent manner. The aqueous extracts had the strongest effect and can be, therefore, used as antimicrobial agents, as well as complementary and alternative therapies. However, laboratory and clinical studies are necessary to investigate the inhibitory effect of these extracts against A. baumannii.
