1. Introduction
Enteric Gram-negative bacilli constitute a large group of bacteria which are naturally positioned in intestinal tracts of human and animals. These bacteria include several genera. Some of these bacteria are normal floras which accidentally contribute to disease, while others are always pathogenic to humans [1]. These bacteria are defined as enteric bacteria due to their colonization in intestine or bowel [2].
Past history indicates that plants were served as an important source of medicine. On the other hand, several factors such as incidence of allergies and emergence of antibiotic-resistant strains among bacteria, indicates the importance of medicinal plants [3]. There exist several reports suggesting antimicrobial effects of plant extracts [4-9].
Increasing price of drugs made researchers to follow plants as important sources of potent drugs. Traditional knowledge have vital role in using medicinal plants as a remedy and discovery of potent drugs to address increasing health problems [10]. Otherwise, in developing countries people use medicinal plants against infectious disease because they cannot afford expensive drugs. Due to increasing rate of drug-resistant diseases, there is an urgent need to detect novel antimicrobial compounds from medicinal plants [11-13]. Ephedra pachyclada plant belongs to the Ephedraceae family and its boiled young branches are used to relieve peptic ulcers and stomachache. Ephedra pachyclada is known as traditional medicine among rural residents and the elderlies [14]. According to numerous conducted studies; phenolic compounds is the active ingredient of this plant.
Researchers also found a direct relation between phenolic compounds of this plant and antioxidant and antimicrobial activity. In Ephedra pachyclada plant; phenolic compounds have a crucial role in inhibitory activity of the hydroxyl groups on free radicals. Therefore, these phenolic compounds may be in direct relation with antioxidant activity of flavonoids and phenylpropanoids biosynthesis [15]. Also, several studies examined antimicrobial effects of Ephedra pachyclada plant on microorganisms. In a study conducted by Bonjar and his colleagues in Iran, antimicrobial effects of Ephedra species which is called Ephedra intermedia were examined [16]. Mahdavi Meymand et al. also examined antimicrobial effects of Ephedra procera [9]. Parsaeimehr et al. examined antimicrobial effects of three species of Ephedra namely E. procera,E. strobiliacea, and E. pachyclada [15]. Azarnia and his colleagues studied protective effects of Ephedra pachyclada on animal models with hepatic disease [17]. Also in other countries, Motomura et al. and Lee et al. also, studied antimicrobial effects of Ephedra pachyclada species [18, 19]. Iran is a country with a long history in traditional medicine and use of medicinal plants in treatment of diseases. Rich plant flora of Iran and knowledge of Iranian people about medicinal plants, scientific centers in the cities of Isfahan, Shiraz and Ray, scientific resources such as Abo Ali Sina and Razi’s books (which promoted use of medicinal plants among Iranian people), and Iranian attention and interest toward medicinal plants demonstrate the importance of medicinal plants in Iran. Note that the use of herbal plants has different customs, traditions, and methods, therefore the adopted methods are different in various locations. According to diversity of climate, Kerman has diverse vegetation and so far different regions such as Bidkhon, Bardsir, Bam, Chatrud and Jiroft have been studied by several researchers [10].
2. Methods
In this cross-sectional study, Ephedra pachyclada was collected from Jiroft Heights and then was transported to faculty of biology of Kerman University in order to identify and confirm the genus and species. After confirmation of Ephedra pachyclada; branches and flowers of the plant were dried, crushed, and the powder was used to prepare the extracts.
2.1. Preparation Plant Extraction
The methanolic extract was prepared by dissolving 50 gr of the Ephedra pachyclada powder in 300 mL of 80% methanol. The powder was mixed with methanol in order to provide uniform extract. Then the extract was kept in the dark for 48 hours After this period, the prepared extract was condensed in the incubation at 40°C to be dehydrated. Maceration method; used in this study; do not impose any damage to the ingredients of the extracts [3, 5].
Agar dilution method: Sensitivity of six bacterial species was determined by using agar dilution method in accordance to minimum inhibitory concentration of growth. At first various dilutions of the extracts with the concentrations of 0.128, 0.25, 0.5, 1, 2, 4 and 8 mg/mL were prepared in Mueller Hinton agar media. After that, a suspension equal to 0.5 McFarland concentration was prepared from Pseudomonas aeruginosa, Escherichia coli, Klebsiella pnemoniae, Serratia marcescens. Then inoculation of 10 µL of bacterial suspension on Mueller Hinton agar containing various dilutions of the extract was done by using a sampler. The plates were incubated for 24 hours at 37°C and then were examined.
3. Results
In this study, intestinal Gram-negative bacteria were exposed to 0.128, 0.25, 0.5, 1, 2, 4 and 8 mg/mL of Ephedra pachyclada extract in order to examine its’ antimicrobial effects. It was demonstrated that methanolic extract of Ephedra pachyclada affect intestinal Gram-negative bacteria. All bacteria grew at 0.128 0.25 and 0.5 mg/mL concentrations whereas the minimum inhibitory concentrations (MIC) of methanolic extract on Pseudomonas aeruginosa and other bacteria were observed at 0.5 and 1 mg/mL, respectively. No antimicrobial effects were observed on each of six strains at concentrations of 0.25 and 0.128 mg/mL.
Table 1.The Six Standard Strains
| Standard Strains | |
|---|---|
| Pseudomonas aeruginosa | (ATCC-27853) |
| Klebsiella pneumoniae | (PTCC-1053) |
| Escherichia coli | (PTCC-O157) |
| Escherichia coli | (ATCC-25922) |
| Serratia marcescens | (PTCC-1111) |
| Shigella dysenteriae | (PTCC-1188) |
Table 2.The Effect of Ephedra pachyclada Extract on the Six Standard Strainsa
| Concentration Bacteria | 0.128 | 0.25 | 0.5 | 1 | 2 | 3 | 4 |
|---|---|---|---|---|---|---|---|
| Pseudomonas aeruginosa | + | + | _ | _ | _ | _ | _ |
| Klebsiella pneumonia | + | + | + | _ | _ | _ | _ |
| Escherichia coli (PTCC-O157) | + | + | + | _ | _ | _ | _ |
| Escherichia coli (ATCC-25922) | + | + | + | _ | _ | _ | _ |
| Serratia marcescens | + | + | + | _ | _ | _ | _ |
| Shigella dysentery | + | + | + | _ | _ | _ | _ |
aNote: Sign (-) indicates that the extract is effective while (+) indicates that the extract is not effective at that level of concentration against the bacteria.
4. Discussion
Antibiotics are the first line treatment for bacterial infections. Based on findings of the current study, methanolic extracts of Ephedra pachyclada plant (with defining minimum inhibitory concentration (MIC) by agar dilution method) have antibacterial effects on Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, Serratia marcescens and Shigella dysenteriae at concentrations of 0.5 , 1 , 2 , 4 and 8 mg/mL of. It has been demonstrated that Pseudomonas aeruginosa is the most sensitive bacteria to methanolic extracts of Ephedra pachyclada. However; due to increasing rate of antibiotic-resistant bacteria and side effects of drugs; using a new method to treat these infections is of vital significance. Nowadays, herbal extracts are effective in treatment of bacterial infections and has attracted a lot of attention [12, 20]. Currently, medicinal plants and their derivatives account for 20 percent and 80 percent of medicinal prescriptions in developed and developing countries, respectively. United States of America is the largest herbal medicine market in the world and Japan is the largest importer of medicinal plants in Asia [21].
In a study conducted by Mahdavi et al. on Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae and other bacteria; Ephedra pachyclada had an inhibitory effect on these bacteria which is in agreement with the findings of current study [9]. In the past few years, several effects of Ephedra pachyclada extract on different bacteria have been reported. In 2010, Parsaismehr and his colleagues studied antibacterial effects of three species of Ephedra on Gram negative bacteria and unexpectedly observed that the most antimicrobial effect of these plant was on Pseudomonas aeruginosa, which corresponded with the results of the current study [15]. In 2009, Chi-hoon lee and colleagues examined antimicrobial effect of the Ephedra pachyclada plant on some intestinal bacteria, and observed that Ephedra pachyclada has antibacterial effect on some intestinal bacteria at 1 mg/mL concentration [19]. Finally, some of limitations of current study contain, much data for easy analysis, and the complexity of data is difficult to represent simply.
4.1. Conclusion
In this study, sensitivity of several intestinal bacteria to extracts of Ephedra pachyclada was obvious. Hence, the risk of infections induced by these bacteria can be reduced and treatment of these infections can be increased through using Ephedra pachyclada extracts. According to considerable antimicrobial effects of Ephedra pachyclada extracts on Gram-negative bacteria (especially Pseudomonas), wide spectrum of diseases caused by these bacteria, and increasing rates of antibiotic resistance; effective ingredients of Ephedra pachyclada can be extracted in order to prevent the prevalence of these bacteria in communities and hospitals and treatment of infections.
According to the current study and assessments of Ephedra Pachyclada effects, it is recommended to develop investigations on synergistic effects of this plant with drugs effective on Pseudomonas and other pathogenic Gram-negative bacteria. Finally, it is recommended to use Ephedra pachyclada extract in producing new antibiotics.
