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2- Department of Biology, Rasht Branch, Islamic Azad University, Rasht, Iran , anvari@iaurasht.ac.ir
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Consumption of medicinal plants for the treatment of ailments has a long history in human life. In recent years, the use of medicinal plants has increased due to lower side effects and costs compared to the chemical drugs with known side effects, and also for the patients' adaptation to these drugs.
Although a large proportion of medicines used today are chemical drugs, it is estimated that at least one-third of all medicinal products are of plant origin or have been modified after extraction from the plant (1,2). Infectious diseases are among the most well-known diseases that have always plagued humans. Lots of efforts have been made to identify their causative agents to help for their treatment and control (1,3).
Chemical drugs, with all their effectiveness, have many adverse effects, and there are fewer pure substances with no adverse effects. In contrast, the active ingredients in medicinal plants have a biological balance due to their association with other substances (6,7).
Due to the fact that Gilan province has a wide range and unique plant diversity in terms of climatic conditions, especially in the case of medicinal species, research on the antimicrobial properties of plant species of this province provides a suitable background and treasure. This study was performed to investigate the antibacterial effect of aqueous and alcoholic extracts of four native medicinal plants of Guilan province (chamomile, fleawort, aquatic pennyroyal and nettle) against the bacterium Klebsiella pneumoniae.
The species were collected from different areas of Gilan province between April to June 2015 and were identified in the herbarium of the Islamic Azad University of Rasht, Gilan, Iran. The parts needed were dried in the air, under shady and dry conditions, and then were ground to extract (Table 1).
Plants Extracts and Bacterial Strain
Ethanol Extract
Maceration method with 70% ethanol was used for ethanol extraction. Thirty gram of the powder of the plant samples was added into the beaker, and then 300 mL of 70% ethanol was added and mixed using a mixer. To extract, the above solution was placed on a shaker at 40°C for 24 hours. In this case, the powdered plant can absorb the solvent and the maximum amount of active ingredients will be dissolved in ethanol. After extraction, the solvent separation from the extract and drying was performed by lyophilization method in Gilan Science and Technology Park and then the weighted powder was dissolved in DMSO and used as an alcoholic extract (8).
| Scientific name of the plant | Plant | Family | Organ used |
| Anthemis austriaca | Chamomile | Asteraceae | Flower |
| Plantago major | Fleawort | Plantaginaceae | Leaf |
| Mentha equatica | Aquatic pennyroyal | Lamiaceace | Leaf |
| Urtica dioica | Nettle | Urticaceae | Leaf |
Distilled water (300 mL) was added to 30 gr of plant powder and placed on the shaker for 24 hours. The solution was passed through 0.45 µm filter to remove larger decanter particles. The resulting extract was dried by freeze dryer (Christ, Germany) and the resulting dry powder was dissolved in distilled water and used as an aqueous extract (8).
Bacterial Strains Studied
The bacterium Klebsiella pneumoniae is among the most common bacteria in urinary tract infections (9). It was isolated from clinical samples of the patients referred to Farabi Laboratory in Ardabil in 2014. This bacterium w::as char::acterized after growing on nutrient agar microbial culture media (Merck, Germany), using biochemical tests such as TSI, SIM, and MR VP citrate. It was sent to the Microbiology Department for anti-bacterial experiments of medicinal plants.
Dilution of Plant Extracts and Preparing Discs Containing Extracts
The aqueous and ethanolic extracts were dissolved in water and DMSO, respectively. The concentrations of 400 and 200 mg/mL were prepared for the aqueous and alcoholic extracts, respectively. Then, blank discs (Padtan Teb, Iran) were placed into the tubes containing the determined dilutions of the extracts. Following three to five minutes immersion, the discs were placed at 37°C to dry completely (8).
Antibacterial Effect of Extracts
Disc Diffusion Method
First, the bacterial suspension of Klebsiella pneumoniae was prepared (0.5 McFarland comparable to a bacterial suspension of 1.5× 108 cfu/mL). Then, uniform culture was performed with 100 μL of prepared suspension on the surface of nutrient agar medium (Christ, Germany).
The extracts-impregnated discs were then placed at a certain distance from the edge of the plate on the surface of the agar culture medium. The test was repeated three times. The diameter of the non-growth halo was finalized measuring the mean diameter after three repetitions (10). The aura diameter of less than 8 mm growth was considered to be resistant, 8 to 9 mm was relatively resistant and more than 10 to 12 mm was considered to be relatively sensitive and more than 12 mm was considered as sensitive (11).
Microdilution Broth
Using microdilution broth, the minimum inhibitory concentration (MIC) of the aqueous and alcoholic extracts of the studied plants was determined. One mL of 1.5×15 cfu/mL bacterial suspension was added to the 0.3-200 mg/mL aqueous extract and 0.1-100 mg/mL of alcohol extract. The optical density (OD) was measured at 680 nm wavelength using the ELISA reader (Update, Belgium(.
The samples were then placed at 37°C, and the OD was re-read in periods of 12 and 24 hours. Finally, the minimum concentration of the extract in which the OD decreased was calculated. It was considered as MIC (12).
| Row | Antibiotics | µg/mL | Disc diffusion | R | I | S |
| 1 | Amikasin | 0.015 | 15 | 14> | 16-15 | 17< |
| 2 | Ampicillin | - | - | 13> | 16-14 | 17< |
| 3 | Ceftazidime | 1 | 12 | 17> | 20-18 | 21< |
| 4 | Cefalotin | - | - | 14> | 17-15 | 18< |
| 5 | Co-trimoxazole | - | - | 10> | 15-11 | 16< |
| 6 | Ciprofloxacin | - | - | 15> | 20-16 | 21< |
| 7 | Gentamicin | - | - | 12> | 14-13 | 15< |
| 8 | Imipenem | 0.007 | 25 | 19> | 22-20 | 23< |
| 9 | Tetracycline | - | - | 11> | 14-12 | 15< |
| 10 | Cefixime | - | - | 15> | 18-16 | 19< |
Table 3. Average diameter of inhibition zone in millimeters of plant extract against bacterium Klebsiella pneumoniae
| Concentration mg/mL Aqueous extracts |
400 |
Concentration mg/mL Alcoholic extracts |
200 |
| Anthemis austriaca | 12 | Anthemis austriaca | 14 |
| Plantago major | - | Plantago major | - |
| Mentha equatica | 8 | Mentha equatica | 9 |
| Urtica dioica | 10 | Urtica dioica | 11 |
| Composition of extracts | 9 | Composition of extracts | 12 |
Table 4. The amount of MIC in mg/mL extract of medicinal plants against Klebsiella pneumoniae
| Aqueous extracts | Concentration mg/mL | Alcoholic extracts | Concentration mg/mL |
| Anthemis austriaca | 0/78 | Anthemis austriaca | 0.39 |
| Plantago major | 5/12 | Plantago major | 1.56 |
| Mentha equatica | 6.25 | Mentha equatica | 0.78 |
| Urtica dioica | 1.5 | Urtica dioica | 0.39 |
| Composition of extracts | 3.12 | Composition of extracts | 0.78 |
Among the four plant species studied, the highest inhibition zone diameter was observed for the ethanolic chamomile extract at concentrations of 200 mg/mL and14 mg/mL. However, the aqueous and alcoholic extract of the fleawort had no effect against the bacterium Klebsiella pneumoniae. Also, the alcoholic extract of the plants in the amount of 200 mg/mL showed the best antimicrobial efficacy. The lowest antimicrobial effect was assessed for the fleawort (Table 3).
Also, the aqueous and alcoholic extracts of chamomile in the concentration of 0/39 and 0/78 mg/mL showed the lowest inhibitory concentration. The results of this study were consistent with the results of Ataei (16), Dadgar (13), NuriZadeh's (14) research outcome.
The results of MIC showed that the ethanolic extract of these plants, even at much lower concentrations, could inhibit the growth of the bacterium Klebsiella pneumoniae. So that, the lowest inhibitory concentrations of chamomile and nettle plants was 0.39 mg/mL. The ethanolic extracts of other plants had also very good effect in their low concentrations (Table 4).
The aqueous and alcoholic extracts of fleawort in 400 and 200 mg/mL concentrations did not develop any inhibition zone against the bacterium Klebsiella pneumoniae. It was found that the MICs of aqueous and alcoholic extracts of fleawort had an inhibitory effect at 12.5 and 1.56 mg/mL concentrations, respectively. The results obtained for the aqueous and alcoholic extracts of fleawort MIC were consistent with the study of Kiai (17) et al. However, the non-inhibitory concentration of Klebsiella pneumoniae was lower than that of the present study. Our results were not consistent with the Chiang study in 2002 and the Eshraghi study (18). This can be due to the type of species and habitat of the plant as the habitat and climate of the plant is effective in the concentration of the active ingredients of the plant (21).
We showed that antibacterial effect of the aqueous and alcoholic extracts of aquatic pennyroyal at concentrations of 400 and 200 mg/mL, made an inhibition zone with a diameter of 8 and 9 mL. The results were consistent with Eshraghi (18) and NuriZadeh (14) studies in terms of antimicrobial effect but different regarding the diameter of the inhibition zone, which can be attributed to the differences in the type of species studied and the concentration of the extract and the type of solvent used (23). In the MIC results of this study, it was found aqueous and alcoholic extracts with the concentrations of 6.25 and 0.78 mg/mL have a minimum inhibitory concentration against Klebsiella. The results of the present study were almost similar to those of the Pajouhi (24) study, but the minimum inhibitory concentration of peppermint was lower in their study, which can be attributed to the differences in the type of strains studied.
In the present study, it was found that aqueous and alcoholic extracts of nettle at concentrations of 400 and 200 mg/mL, created halos of 10 and 11 mm against Klebsiella pneumoniae, respectively. Also, aqueous and alcoholic extracts of nettle against Klebsiella pneumoniae in the present study showed minimum inhibitory concentrations at 1/5 and 0/39 mg/mL. This is consistent with the study of Shariat (26) and Jafari (27), but the amount of MIC in their research is higher, and this discrepancy can be attributed to the type of species studied.
From the 10 antibiotics used, three, including amikacin, ceftazidime, and imipenem had the greatest inhibitory effect on the bacterium Klebsiella pneumoniae. Other antibiotics showed fewer inhibitory effects on this bacterium (Table 2). Comparing the results in Tables 2, 3, and 4 shows the suitability of the studied plants and standard antibiotics for their anti-bacterial effects. Comparing the inhibition zones of the extracts of the studied medicinal plants with a certain concentration was clarified by the diffusion disc method (Table 3).
These results showed the antibacterial effects of the above medicinal plants crude extracts, which is a mixture of the active ingredients. The active ingredients with antimicrobial effect in these extracts should be isolated and purified. They may have better effects compared to antibiotics. Although the present results are laboratory-based, it seems that these findings are justifiable and can be generalized to in vivo experiments and it is useful for further examination on laboratory animals.
The results of this study showed the optimal effect of aqueous and ethanolic extracts of the studied plants (chamomile, fleawort, aquatic pennyroyal and nettle) against Klebsiella pneumoniae. The outcome of this study is important due to the fact that medicinal plants are more compatible with the body, their natural nature, fewer side effects and lower chance in antibiotic resistance. In continue the active ingredients of the extracts with antimicrobial properties can be extracted and their antibacterial effects and clinical conditions can be investigated in vivo.
The authors thank all those who helped them writing this article.
Authors declared no conflict of interests.
Received: 2020/06/28 | Accepted: 2020/07/7 | ePublished: 2020/08/14
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