Abstract
Ethanol extracts of 19 Turkish medicinal plants, used in the traditional system of medicine, were investigated for their antimicrobial activity against 14 pathogenic bacterial species and a yeast, Candida albicans., using the agar well diffusion method. Anticandidal activity was detected in 10 plant extracts. Extracts of Eucalyptus camuldulensis. (leaves), Rosmarinus officinalis. (leaves), Ecballium elaterium. (leaves, fruits; 2:1, v/v), Liquidambar orientalis. (leaves), Cornus sanguinea. (leaves, flowers, stems; 2:1:1, v/v/v), Vitis vinifera. (leaves, raw fruits, young branches; 2:1:1, v/v/v), Inula viscosa. (leaves), Hypericum perforatum. (leaves, flowers, stems; 2:1:1, v/v/v), and Punica granatum. (leaves, flowers; (2:1, v/v) showed broad-spectrum antimicrobial activity with inhibition zones ranging from 4 to 34 mm. The most resistant organisms were Escherichia coli., Candida albicans., Pseudomonas fluorescens., Bacillus subtilis. ATCC 6683, and Enterobacter faecalis. ATCC 29212, and the most susceptible species were Proteus vulgaris. ATCC 6997, Salmonella typhimurium. CCM 5445, Staphylococcus epidermidis. ATCC 12228, and Serratia marcescens. CCM 583, respectively. The minimum inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were determined for the seven highly active plants that showed antimicrobial activity against methicillin-resistant Staphylococcus aureus. ATCC 95047 (MRSA), E. coli., and C. albicans.. The MICs of active extracts ranged from 8 to 14.2 mg/mL while the MBCs were 14.2 to 24.4 mg/mL.
Introduction
Turkey has an extraordinarily rich flora and a wide knowledge of its indigenous medicinal plants. Medicinal plants constitute an important component of flora and are widely distributed in different floristic regions of Turkey because of its geographic location, climate, and the presence of nearly 10,000 natural plant species (Baytop, Citation1999; Ateş & Erdoğrul, Citation2003).
Human infections, particularly those involving the skin and mucosal surface, constitute a serious problem, especially in tropical and subtropical developing countries (Falahatiı et al., Citation2005); methicillin-resistant Staphylococcus aureus. (MRSA), Staphylococcus aureus., Escherichia coli., and Candida albicans. being the most frequent pathogens. MRSA gained much attention in the past decade, as it is a major cause of hospital-acquired infections. The drug-resistant bacteria and the fungal pathogen have further complicated the treatment of infectious diseases in immunocompromised, AIDS, and cancer patients. In the current scenario of emergence of multiple drug resistance to human pathogenic organisms, this has necessitated a search for new antimicrobial substances from other sources including plants (Ahmad & Beg, Citation2001).
It is expected that plant extracts showing target sites other than those used by antibiotics will be active against drug-resistant microbial pathogens. The use of medicinal plants still plays a vital role to cover the basic health needs in developing countries. In this connection, plants continue to be a rich source of therapeutic agents. The active principles of many drugs are found in plants or are produced as secondary metabolites. The remarkable contribution of plants to the drug industry was possible because of the large number of phytochemical and biological studies all over the world. Herbal remedies used in folk medicine provide an interesting and still largely unexplored source for the creation and development of potentially new drugs for chemotherapy, which might help overcome the growing problem of resistance and also the toxicity of the currently available commercial antibiotics. Therefore, it is of great interest to carry out a screening of these plants in order to validate their use in folk medicine (Kıanbakht & Jahanıanı, Citation2003).
Similar studies with crude plant extracts were reported for antimicrobial activity in Turkey (Keleş et al., Citation2001; Erdoğrul, Citation2002; Dülger & Gönüz, Citation2004; Uzun et al., Citation2004); yet, the information, particularly of medicinal plants active against some bacteria and C. albicans., until recently has not been studied. Therefore, we have selected 19 Turkish medicinal plants to be screened against multidrug-resistant bacteria including MRSA, Staphylococcus aureus., Salmonella typhimurium. and Escherichia coli..
Materials and Methods
Plant materials
Plants were collected at different sites of Manisa province and aroun Turkey. Voucher specimens were deposited in the Herbarium of Botany, Department of Biology, Celal Bayar University. The parts used were leaves, stems, flowers, roots, young branches, and in some cases, fruits ().
Table 1.. List of plants screened for antimicrobial activity.
Microorganisms and growth conditions
Test microorganisms included the following bacteria: Staphylococcus aureus. ATCC 6538P, methicillin-resistant Staphylococcus aureus. ATCC 95047 (MRSA), Escherichia coli., Micrococcus luteus. ATCC 9341, Bacillus cereus. CM 99, Bacillus subtilis. ATCC 6683, Salmonella typhimurium. CCM 5445, Pseudomonas fluorescens., Proteus vulgaris. ATCC 6997, Serratia marcescens. CCM 583, Staphylococcus epidermidis. ATCC 12228, Enterococcus faecalis. ATCC 29212, Enterobacter cloaceae. ATCC 13067, Enterobacter aerogenes. ATCC 13048, and for yeast Candida albicans.. Cultures of these bacteria were grown in Mueller-Hinton broth (Oxoid Ltd., Basingstoke, England) at 37°C for 24 h, and the studied yeast was incubated in glucose yeast extract broth at 30°C for 48 h. All the microorganisms were obtained from the Department of Biology, Ege University (Izmir, Turkey).
Preparation of the crude ethanol extracts
The plant parts were separated, washed with distilled water, dried, and powdered finely using a blender. Thirty grams of ground air-dried plant material was shaken in 150 mL 96% (w/w) ethanol (EtOH) at room temperature for 60 h (180 cycles/min). The insoluble material was filtered by filter paper (Whatman no. 4) and evaporated to dryness in a water bath at 50°C. The extract was weighed and dissolved in EtOH at a concentration of 200 mg/mL and stored at + 4°C for further experiments.
Antimicrobial assays
Agar well diffusion assay
The assay was conducted as described by Perez et al. (Citation1990) with slight modification according to the current experimental conditions. Briefly, 50 µL inoculum (containing approximately 108 bacteria per milliliter and 107 yeast per milliliter) was added to 25 mL melted Mueller-Hinton agar (MHA) and potato dextrose agar (PDA) medium cooled at 50°C. This was then poured into 90-mm-diameter Petri dishes and maintained for 1 h at room temperature. Small wells (6 mm) were cut in the agar plate using a cork borer; 100 µL of extract concentration (4 mg/mL) with a negative control (EtOH, 100 µL) were loaded in the wells. The dishes were preincubated at 4°C for 2 h to allow uniform diffusion into the agar. After preincubation, for bacteria, the plates were incubated aerobically at 37°C for 24 h, and at 28°C for 48 h for yeast. The antimicrobial activity was evaluated by measuring the inhibition zone diameter observed. In addition, commercial antibiotics [penicillin G (10 IU), nalidixic acid (30 µg), novobiocin (30 µg), ampicillin (10 µg), imipenem (10 µg), erythromycin (15 µg), vancomycin (30 µg), chloramphenicol (30 µg), and nystatin (10 µg)] were used as positive control to determine the sensitivity of the strains. These studies were performed in triplicate.
Determination of minimum inhibitory concentration and minimal bactericidal concentration
The minimum inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were determined for the seven highly active plants that showed antimicrobial activity against methicillin-resistant Staphylococcus aureus., E. coli., and C. albicans.. The broth macrodilution method (Nakamura et al., Citation1999) with slight modification was used to determine MIC and MBC of extracts against selected test microorganisms; for bacteria, in broth media Mueller-Hinton, and in glucose yeast extract broth for yeast. In these experiments, 0.5 mL of a bacterial suspension containing 1 × 108 colony forming units (CFU)/mL and 1 × 107 CFU/mL yeast was added to 4.5 mL of susceptibility test broth containing serial two-fold dilutions of the extract in glass test tubes. All tubes were incubated in air at 37°C for 24 h for bacteria and at 28°C for 48 h for C. albicans. before being read. The MIC was considered the lowest concentration of the sample that prevented visible growth. MBCs were determined by subculturing 10 µL from each negative tube and from the positive growth control. MBC was defined as the lowest concentration yielding negative subcultures or only one colony. All samples were examined in duplicate in three separate experiments.
Statistical analysis
The mean values were statistically analyzed with the MINITAB Release 13.20 program by the general one-way (unstacked) analysis of variance (ANOVA) to find out the most effective plants and the most sensitive test organisms. Similarity (%) of microorganisms in relation to their susceptibility to the plant extracts was analyzed by the multivariate cluster analysis according to the data obtained from well diffusion assay.
Results and Discussion
Antimicrobial activity of 19 plants belonging to 17 botanical families () has been evaluated in vitro. against 14 bacterial species and one yeast (C. albicans.) that are known to cause dermic and mucosal infections besides other infections in humans.
All plants, except the Pyracantha coccinea., studied in this work showed antimicrobial activity against at least one of the test microorganisms, with inhibition zones ranging from 2 to 34 mm (). This result showed that most of the studied plants are potentially a rich source of antimicrobial agents. However, the plants differ significantly in their activity against test microorganisms. The most active plants were Eucalyptus camuldulensis., Rosmarinus officinalis., Ecballium elaterium., Liquidambar orientalis., Cornus sanguinea., Vitis vinifera., Inula viscosa., Hypericum perforatum., and Punica granatum., which showed broad-spectrum antimicrobial activity against Gram-positive and Gram-negative bacteria that are resistant to some antibiotics such as nalidixic acid, penicillin G, novobiocin, imipenem, erythromycin, vancomycin, and chloramphenicol (), whereas the least active plants were Conyza canadensis., Citrus reticulate., Nerium oleander., Carpobrotus acinaciformis., and Euphorbia peplus.. However, Artemisia arborescens., Thuja orientalis., Mirabilis jalapa., and Lonicera japonica. were somewhat active plants. On the other hand, negative control (EtOH, 100 µL) inhibited test microorganisms ranged from 2 to 10 mm and not included calculations if the inhibition equal to EtOH inhibitions or under ().
Table 2.. Antimicrobial activity of the ethanol extracts of the plants against bacteria and C. albicans..
Table 3.. Inhibitory activity of some standard antibiotics against various bacteria and C. albicans..
Maximum inhibition was shown by extract of Rosmarinus officinalis. against S. aureus. and MRSA, 26 and 20 mm, respectively. A high inhibition zone diameter (22 mm) against Escherichia coli. was obtained by extract of Ecballium elaterium.. Ten plants, namely, Eucalyptus camuldulensis., Punica granatum., Conyza canadensis., Euphorbia peplus., Vitis vinifera., Inula viscose., Rosmarinus officinalis., Lonicera japonica., Ecballium elaterium., and Cornus sanguinea., demonstrated anticandidal activity; the first one was highly active (22 mm).
Sensitivity of test strains was, in decreasing order: P. vulgaris. > S. typhimurium. > S. epidermidis. > S. marcescens. > E. aerogenes. > M. luteus. > S. aureus. > B. cereus. > MRSA > E. cloaceae. > E. faecalis. > B. subtilis. > P. fluorescens. > C. albicans. > E. coli. (). The last one was least sensitive compared with the other test bacteria, which may be due to their differences in cell wall composition. It was interesting to note that antibiotic-resistant bacteria showed more sensitivity to the investigated extracts. This has clearly indicated that antibiotic resistance does not interfere with the antimicrobial action of plant extracts, and these extracts might have different modes of action on test organisms.
Figure 1 Mean values of microorganisms in relation to their susceptibility to the plant extracts. *Means are indicated by solid circles. aSee for abbreviations of test microorganisms.
Significant antimicrobial effects, expressed as MIC and MBC (MFC for C. albicans.) of crude extracts against MRSA, E. coli., and C. albicans., are shown in (). Extracts of selected plants were among the most active with the MIC values ranging from 8 to 14.2 mg/mL. Among the plants tested, ethanol extract of Cornus sanguinea. and Liquidambar orientalis. showed very strong activity against MRSA with the best MIC (8 mg/mL). The MBC values of seven plants ranged from 14.2 to 24.4 mg/mL; the lowest MBC for MRSA was obtained with Eucalyptus camuldulensis. extract and was 14.2 mg/mL, whereas the highest MBC was 24.4 mg/mL for C. albicans.. MBC values for E. coli. from Liquidambar orientalis. and Ecballium elaterium. were 22.2 and 23.6 mg/mL, respectively.
Table 4.. MIC and MBC values of selected plant ethanol extracts against SA, MRSA and CA.
summarizes the similarity of microorganisms in relation to their susceptibility to the plant extracts. This has clearly indicated that Gram-positive bacteria were more sensitive to plant extracts in the same cluster, whereas Gram-negatives resistant existing in different cluster groups because of their differences in the cell wall composition, metabolism, nature, and resistance to antibiotics. On the other hand, susceptibility of C. albicans. (eukaryotic) to the plant extracts was found in similar to E. faecalis..
Figure 2 Similarity (%) of microorganisms in relation to their susceptibility to the plant extracts. *See for abbreviations of test microorganisms.
According to Erdoğrul (2002), the antibacterial activities of ethyl acetate, methanol, chloroform, and acetone extracts of the leaves of Rosmarinus officinalis. showed various inhibitory effects (7–16 mm inhibition zone), on B. subtilis., E. coli., S. aureus., and P. fluorescens.. Our similar results confirm this situation (8–26 mm). Also, Dülger and Gönüz have reported a 10% aqueous dimethylsulfoxide (DMSO) extract of R. officinalis. inhibited the growth of S. aureus. and B. cereus. (15–22 mm) but not E. coli., P. vulgaris., and C. albicans.. Our results obtained with R. officinalis. for E. coli., P. vulgaris., and C. albicans. were 10, 15, and 8 mm, respectively. It is thought that the observed dissimilar results may be attributed to differences in techniques and extracts because different methods were used and the variable sensitivity of different microorganisms to chemical substances relates to different resistance levels between the strains.
A study reported that Hypericum perforatum. showed no inhibitory effect against E. coli. and P. aeruginosa. except for S. aureus. (16 mm) (Keleş et al., Citation2001). It was found that H. perforatum. was effective against S. aureus., MRSA, M. luteus., B. cereus., S. typhimurium., P. vulgaris., S. marcescens., S. epidermidis., E. cloaceae., and E. aerogenes. with inhibition zones of 20, 18, 16, 22, 28, 30, 6, 16, 16, and 6 mm, respectively.
The results of the current investigation clearly indicate that the antibacterial and anticandidal activity vary with the species of the plants and support a good correlation with the reported traditional medical uses of these plants, especially Ecballium elaterium., Vitis vinifera., Hypericum perforatum., and Punica granatum., in Turkey (Baytop, Citation1999; Karaman & Kocabas, Citation2001; Uzun et al., Citation2004) and treatment of infectious diseases caused by C. albicans. and some pathogenic bacteria such as MRSA, S. aureus., E. coli., and S. typhimurium.. Further, the active phytocompounds of these plants against multidrug-resistant bacteria and C. albicans. should be characterized and their toxicity should be evaluated in vivo..
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