1. Background
Evidence indicates that the antibiotic resistance of bacteria has increased (1). Acinetobacter baumanii and Staphylococcus aureus are two particularly ubiquitous bacteria. A. baumanii is a Gram-negative and non-glucose fermenting coccobacillus that is one of the main common causes of nosocomial infections because of high levels of drug resistance (2). In Chile, Carbapenem resistance of A. baumanii in the ICU was reported to be up to 70% (3). A study in 2012 showed that the resistance of A. baumanii to Imipenem and Meropenem in the ICU-admitted patients was 76% and 80.2%, respectively (4). A study in Ohio, USA showed that the mortality was 70% because of resistant A. baumanii infection and the mortality only 25% because of non-resistant A. baumanii infection (5). Moreover, Staphylococcus aureus is one of the life-threatening pathogens for the human that can cause a wide spectrum of diseases, ranging from skin infections to respiratory and urinary tract infections. This bacterium can cause certain life-threatening diseases such as endocarditis, toxic shock syndrome toxin, and osteomyelitis (6). Asia is addressed as one of the regions with the highest prevalence of methicillin-resistant S. aureus (MRSA) worldwide, and vancomycin-resistant S. aureus strains have been reported in certain Asian countries as well (7). The latest findings have shown that the prevalence of MRSA is 73% of the clinical samples collected from the hospitals in Taiwan (8).
Given the reported extensive drug resistance, there is a need for developing and using new antibacterial drugs that do not lead to drug resistance and also have efficient therapeutic effects. In this regard, the use of medicinal plants is an approach to discover new drugs (9-11). In addition, the medicinal plants that are collected from the regions with different climates may have different amounts of active compounds and exert biological activities of different degrees (12,13). In this regard, the aim of this study was to investigate the phytochemical properties and antibacterial effects of Salvia multicaulis Vahl., Euphorbia microsciadia Boiss., and Reseda lutea, collected from Chaharmahal and Bakhtiari province, against A. baumanii and S. aureus.
To date, 1000 plant species from the Salvia genus of the subfamily Nepetoideae and the family Lamiaceae have been identified, of which 56 are native to Iran (14). Salvia multicaulisVahl. grows mainly in Central and East Asia (15). The main compounds of this plant include 1,8-cineole, α-pinene, and camphor (16). The pharmaceutical effects of this plant include anti-inflammatory, antimicrobial, and analgesic effects (15). Euphorbia microsciadia Boiss is from the family of Euphorbiacea that is one of the largest families of flowering plants, including over 300 genera and 5000 species (17). In relevant textbooks, E. microsciadia has been reported to have anti-anxiety, analgesic, antipyretic, and antimicrobial effects (18). Reseda lutea L., also known as Reseda vulgaris, is a member of the family Resedaceae (19). R. lutea has been reported to have cytotoxic, antitumor, anti-HIV, antibacterial, and anti-inflammatory effects (19, 20). This plant contains benzyl isothiocyanate and 2-(α-L-rhamnopyranosyloxy) benzyl isothiocyanate that has cytotoxic effects (19).
2. Methods
2.1. Collecting Plants
The plants (E. microsciadia, S. multicaulis Vahl. and R. lutea) were collected from different regions of Chaharmahal and Bakhtiari Province (in Iran) such as Saman, Shahrekord, and Teshniz between late March 2016 and late September 2016 and then identified as the plants of interest by a botanist (Dr. Shirmardi) at the Research Center of the Agricultural Jihad Organization of Chaharmahal and Bakhtiari province.
2.2. Extraction
Extraction was conducted by maceration of the aerial parts of E. microsciadia and S. multicaulis Vahl. and the seeds of R. lutea in triplicate (each time for 72 hours). In this method, water and butyric acid-free bitter ethanol at 30/70 ratio were used (ethanol 70%). The resulting extract was filtered using a filter paper and evaporated under next-to-vacuum pressure and 40°C by a rotary evaporator to concentrate. The resulting solution was stored at -20°C until later use.
2.3. Preparing Different Dilutions of Extract
Ninety-six mg of each extract was weighed using a digital scale and stock solutions of the extracts prepared using dimethyl sulfoxide (DMSO; Sigma, USA) and distilled water. Then different dilutions (0.25, 0.5, 1, 2, 4, 8, and 16 mg/mL) of the extracts were prepared using Mueller-Hinton agar. The maximum concentration of DMSO was 0.2% in the final concentration (21, 22).
2.4. Preparing Standard Bacterial Strains
S. aureus strain (ATCC 12923) and A. baumanii strain (PTCC 1855) were purchased as lyophilized from Iranian Research Organization for Science and Technology.
2.5. Preparing Microbial Suspension
To prepare a microbial suspension equivalent to 0.5 McFarland standard (1.5 × 108 CFU/mL), a 24-hour culture of the bacteria was performed on blood agar, and then a suspension with 0.5 McFarland turbidity prepared in normal saline.
2.6. Determining Minimum Inhibitory Concentrations (MICs) and Minimum Bactericidal Concentrations (MBCs)
The antimicrobial effects of the extracts were determined by broth microdilution in a sterile 96-well plate (SPL life science; Korea) with reference to 0.5 McFarland standard (1.5 × 108 CFU/mL). In this method, the first well was considered negative control (culture medium + the extract) and the second well positive control (culture medium + the bacterium). After adding 95 µL culture medium and 100 µL extracts to microplate wells and diluting them, we incubated the samples at 37°C for 24 hours. The concentration of the last (most diluted) well without turbidity was considered MIC (23). To determine MBC, we subsequently performed a culture of the samples of each well at 10 µL on Mueller-Hinton agar and incubated them at 37°C for 24 hours. The lowest concentrations of the extract in which the bacteria could not grow were considered MBCs. The tests to determine the MICs and MBCs were conducted in triplicate (24).
2.7. Determining Total Phenolic and Flavonoid Content
Total phenolic content was measured by Folin-Ciocalteu colorimetric assay and expressed in terms of gallic acid equivalent and total flavonoid content by aluminum chloride colorimetric method and in terms of rutin equivalent (25-27).
For total phenolic content, 0.1 mL of extract was transferred to a test tube, and 0.5 mL of Folin-Ciocalteu reagent was added and mixed gently. After 5 minutes incubation, 0.4 mL of 7.5% (w/v) sodium carbonate was added to the mixture. The mixture was allowed to stand at room temperature for 30 minutes. UV-vis absorption measurements were carried out at 765 nm using a spectrophotometer (UNICO 2100: USA). The standard calibration curve was plotted using gallic acid in methanol. The TPC was expressed as gallic acid equivalents (mg GAE/g dry weight of the extract).
For flavonoid content, 0.5 mL of the extract was transferred to a test tube and mixed with 1.5 mL of methanol. Then 0.5 mL of 2% aluminum chloride (AlCl3) and 3 mL of 5% potassium acetate were added to the extract. After 40 minutes incubation, UV-vis absorption measurements were carried out at 415 nm using a spectrophotometer (UNICO 2100: USA). The standard calibration curve was plotted using rutin in methanol.
4. Results
The S. aureus was susceptible to S. multicaulis and inhibited at 1 mg/mL, but was resistant to 0.5 mg/mL of this extract. Therefore, the most acceptable MIC of this plant against S. aureus was determined 1 mg/mL. On the other hand, this bacterium was completely eliminated by S. multicaulis and could not grow at 4 mg/mL concentration. Therefore, the MBC of this plant against S. aureus was determined 4 mg/mL. The results on the antimicrobial effect of hydroalcoholic S. multicaulis extract on A. baumanii showed that this extract at 2 mg/mL could inhibit this bacterium, with 16 mg/mL determined as its MBC for A. baumanii. Furthermore, the MIC and MBC of E. microsciadia for S. aureus were determined 4 mg/mL and 16 mg/mL, respectively, and the MIC and MBC of this plant for A. baumanii 8 mg/mL and 32 mg/mL, respectively; the MIC and MBC of R. lutea for S. aureus were determined 1 mg/mL and 2 mg/mL, respectively, and the MIC and MBC of this plant for A. baumanii 8 mg/mL and 32 mg/mL, respectively (Tables 1 and 2).
Table 1.The Minimum Inhibitory Concentrations and Minimum Bactericidal Concentrations of the Hydroalcoholic Extracts of Studied Plants for Staphylococcus aureus
a(+), Lack of microorganism growth in culture medium and the antimicrobial activity of the hydroalcoholic (ethanol 70%) extracts of plants; (-), Microorganism growth in culture medium and lack of antimicrobial activity of the hydroalcoholic extracts of plants.
bThe extraction of aerial parts.
cThe extraction of seeds.
Table 2.The Minimum Inhibitory Concentrations and Minimum Bactericidal Concentrations of the Hydroalcoholic Extracts of Studied Plants for Acinetobacter baumanii
a(+), Lack of microorganism growth in culture medium and the antimicrobial activity of the hydroalcoholic (ethanol 70%) extracts of plants; (-), Microorganism growth in culture medium and lack of antimicrobial activity of the hydroalcoholic extracts of plants.
bThe extraction of aerial parts.
cThe extraction of seeds.
According to Table 3, all three plants contained phenols and flavonoids. E. microsciadia and S. multicaulis contained the highest total phenolic and flavonoid contents, respectively (Table 3).
Table 3.Total Phenolic and Flavonoid Content of the Studied Plantsa
| Plants | Used Organ | Total Phenolic Content mg GAE/g DW | Flavonoid Content mg RU/g DW |
|---|---|---|---|
| Euphorbia microsciadia Boiss | Shoot | 69.56 ± 6.75 | 24.39 ± 1.38 |
| Salvia multicaulisVahl. | Shoot | 63.96 ± 5.11 | 26.74 ± 2.24 |
| Reseda lutea L. | Seeds | 65.32 ± 3.72 | 21.93 ± 2.67 |
aValues are expressed as mean ± SD.
5. Discussion
This study was conducted to investigate the phytochemical properties and antibacterial effects of S.multicaulis, E. microsciadia, and R. lutea against A. baumanii and S. aureus, showing that these plants can be considered alternatives for developing new antibiotics against some microorganisms that are resistant to routine drugs, owing to phenols and flavonoids and exerting antibacterial effects.
Our results showed that S. multicaulis could exert inhibitory and bactericidal effects on both S. aureus and A. baumanii but these effects on S. aureus were more potent. The flavonoid content of this plant was also higher than the other two plants. The study of Akin et al. to investigate the antimicrobial effects of Salvia cryptantha and Salvia heldreichiana, revealed that these two species did not exert any inhibitory effect on S. aureus (28). Inconsistently, our study with S. multicaulis showed that this plant exerted a potent inhibitory effect on this bacterium. The study of Alim et al. indicated that methanol Salvia cedronella extract had inhibitory effects on S. aureus and B. cereus, with an MIC of 31.25 μg/mL, which is in agreement with our study. Alim et al. attributed their observations about the inhibitory properties to the high concentrations of phenols and flavonoids in S. cedronella (29). Fazly Bazzaz et al. reported that Salvia chloroleuca exerted a partial inhibitory effect on S. aureus, while Salvia ceratophylla, Salvia lourifolia, Salvia limbata, and Salvia macrosiphone had no inhibitory effect on this bacterium (30).
Our results were promising regarding the antimicrobial effects of E. microsciadia to inhibit S. aureus and A. baumanii thus its MIC and MBC for S. aureus were determined 4 mg/mL and 16 mg/mL and for A. baumanii 8 mg/mL and 32 mg/mL, respectively. Also, E. microsciadia contained comparatively higher concentrations of phenols and flavonoids. The study of Eshraghi et al. investigated the effects of 10 plant species on the Nocardia genus, showed that Euphorbia denticulate could inhibit the growth of Nocardia asteroides and Nocardia brasiliensis (31). Kaveh et al. observed that the species of Euphorbia were rich sources of flavonoids, and E. microsciadia contained abundant phenols such as kaempferol (32).
Moreover, R. lutea was found to exert antimicrobial effects on S. aureus and A. baumanii. This plant was also found to contain phenols and flavonoids. The study of Boroumand et al. showed that R. lutea-assisted synthesis of silver nanoparticles led to the inhibition of Escherichia coli in vitro (33). Benmerache et al. reported that chloroform Reseda phyteuma L. extract had inhibitory effects on S. aureus, Proteus mirabilis, and Pseudomonas aeruginosa so that its MIC for all three bacteria was 80 μg/mL and the inhibition zone diameters for P. mirabilis, S. aureus, and P. aeruginosa were 13 mm, 12 mm, and 11 mm, respectively (34), while the study of Yildirim et al. in Turkey showed that R. lutea showed no antimicrobial effect on S. aureus. That study also demonstrated that this plant could not exert any antimicrobial effect on Staphylococcus epidermidis, Streptococcus pyogenes, Serratia marcescens, Salmonella typhimurium, P. aeruginosa, Proteus vulgaris, and E. coli, meanwhile had a partial inhibitory effect on Klebsiella pneumoniae (35).
The concentrations of bioactive compounds in a medicinal plant may vary and, therefore, exhibit various biological effects depending on the location and time of its collection as well as its part of use (12,13). The inconsistency in the findings of our study and others’ can be attributed to different concentrations of the bioactive compounds due to the difference in the occurrence locations of the studied plants as well as their parts of use. The plants that were investigated in the current study and collected from Chaharmahal and Bakhtiari province could pave the way for producing antibiotics due to their phenols and flavonoids and exerting antibacterial effects on the studied bacteria.
