Pharmaceutical Biology 2007, Vol. 45, No. 3, pp. 176–181
Antimicrobial Screening of Some Turkish Medicinal Plants Mustafa Oskay and Dilek Sarı Biology Department, Faculty of Sciences and Arts, Celal Bayar University, Manisa, Turkey
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. Keywords: Antimicrobial, drug resistant, medicinal plants, MRSA, Turkish.
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, 1999; Ates¸ & Erdog˘rul, 2003). Human infections, particularly those involving the skin and mucosal surface, constitute a serious problem, especially in tropical and subtropical developing countries (Falahatı et al., 2005); 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, 2001). 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
Accepted: October 18, 2006. Address correspondence to: Mustafa Oskay, Biology Department, Faculty of Sciences and Arts, Celal Bayar University, Campus of Muradiye, 45030 Manisa, Turkey. Tel.: +90 236 2412151-122; Fax: +90 236 2412158; E-mail:
[email protected] DOI: 10.1080/13880200701213047 # 2007 Informa Healthcare
Antimicrobial activity of Turkish plants a screening of these plants in order to validate their use in folk medicine (Kıanbakht & Jahanıanı, 2003). Similar studies with crude plant extracts were reported for antimicrobial activity in Turkey (Keles¸ et al., 2001; Erdog˘rul, 2002; Du¨lger & Go¨nu¨z, 2004; Uzun et al., 2004); 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 multidrugresistant 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
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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). 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
Table 1. List of plants screened for antimicrobial activity. Scientific name=code
Family
Local name
Used plant part(s)
Nerium oleander L.=1 Pyracantha coccinea M. Roem.=2 Cornus sanguinea L.=3 Artemisia arborescens L.=4
Apocynaceae Rosaceae
Zakkum NI
LF,FL LF,RF
Cornaceae Compositae
NI Pelin otu
LF,FL,S LF,FL,S
Thuja orientalis L.=5
Cupressaceae
NI
Carpobrotus acinaciformis (L.) L. Bolus=6 Punica granatum L.=7
Aizoaceae
Conyza canadensis (L.) Cronquist.=8 Mirabilis jalapa L.=9
Preparation rate
Collection time
2:1 2:1
June 24, 2005 June 24, 2005
2:1:1 2:1:1
June 25, 2005 June 25, 2005
LF,RF
2:1
June 24, 2005
Makas otu
LF,S
2:1
June 27, 2005
Punicaceae
Nar
LF,FL
2:1
June 28, 2005
Compositae
NI
LF
Nyctaginaceae
Aks¸am sefası
LF,FL,S
Euphorbia peplus L.=10
Euphorbiaceae
Su¨tleyen
LF,FL,S,RT
Citrus reticulata L.=11
Rutaceae
Mandalina
LF,RF
Vitis vinifera L.=12
Vitaceae
LF,RF,S,YB
Liquidambar orientalis Mill.=13
Hamamelidaceae
Inula viscosa (L.) Aiton.=14 Rosmarinus officinalis L.=15 Hypericum perforatum L.=16
Compositae Lamiaceae Guttiferae
¨ zu¨m SultaniyeU Asma Gu¨nlu¨k ag˘acı, sıg˘ala NI Kus¸dili SarıKantaron
June 30, 2005
Campus Botanical garden Campus Botanical garden Botanical garden Botanical garden Botanical garden Botanical garden Botanical garden Campus, Yagcılar Botanical garden Botanical garden Botanical garden Muradiye Campus Campus, Yagcılar Botanical garden Yagcılar
Lonicera japonica Thunb.=17
Caprifoliaceae
Hanımeli
LF
Ecballium elaterium A. Richard=18
Cucurbitaceae
LF,FR
Eucalyptus camaldulensis Dehnh.=19
Myrtaceae
Acıdu¨velek, es¸ek hıyarı Viks otu Ekaliptus
July 05, 2005
Campus
July 07, 2005 2:1:1
June 26, 2005
2:1:1:1
July 08, 2005
2:1
July 06, 2005
2:1:1:1
June 24, 2005
LF
June 24, 2005
LF LF LF,FL,S
July 10, 2005 June 27, 2005 June 27, 2005
LF
2:1:1
June 27, 2005 1:1
Origin
LF, leaves; FL, flowers; FR, fruits; RF, raw fruits; RT, roots; S, stems; YB, young branches; NI, not informed.
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in Mueller-Hinton broth (Oxoid Ltd., Basingstoke, England) at 37C for 24 h, and the studied yeast was incubated in glucose yeast extract broth at 30C 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 50C. The extract was weighed and dissolved in EtOH at a concentration of 200 mg=mL and stored at þ4C for further experiments. Antimicrobial assays Agar well diffusion assay The assay was conducted as described by Perez et al. (1990) with slight modification according to the current experimental conditions. Briefly, 50 mL 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 50C. 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 mL of extract concentration (4 mg=mL) with a negative control (EtOH, 100 mL) were loaded in the wells. The dishes were preincubated at 4C for 2 h to allow uniform diffusion into the agar. After preincubation, for bacteria, the plates were incubated aerobically at 37C for 24 h, and at 28C 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 mg), novobiocin (30 mg), ampicillin (10 mg), imipenem (10 mg), erythromycin (15 mg), vancomycin (30 mg), chloramphenicol (30 mg), and nystatin (10 mg)] 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., 1999) 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 37C for 24 h for bacteria and at 28C 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 mL 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 oneway (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 (Table 1) 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 (Table 2). 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 (Table 3), 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 mL)
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Table 2. Antimicrobial activity of the ethanol extracts of the plants against bacteria and C. albicans. Inhibition zone (mm)a SAa N.oleander P. coccinea C. sanguinea A. arborescens T. orientalis C.acinaciformis P. granatum C. canadensis M. jalapa E. peplus C. reticulata V. vinifera L. orientalis I. viscosa R. officinalis H. perforatum L. japonica E. elaterium E. camuldulensis NCd
b
0 0 12 0 0 0 16 0 12 0 0 16 20 18 26 20 0 20 18 10
MRSA
EC
ML
BC
BS
STYP
PF
PV
SM
SE
EF
ECLO
EA
CA
0 0 13 0 10 0 10 0 10 0 0 14 20 14 20 18 14 10 16 8
0 0 0 0 0 4 0 10 0 0 3 8 12 0 10 0 0 22 10c 2
10 0 18 12 12 0 0 0 8 8 8 16 12 12 16 16 10 14 18 6
10 0 14 10 16 0 12 0 0 0 0 12 0 20 14 22 8 18 14 6
0 0 18 0 0 0 10 6 6 0 10 10 14 10 18 0 8 16 10 4
8 0 10 8 0 12 0 18 8 0 10 22 22 20 14 28 14 16 24 6
0 0 8 0 0 0 10 0 4 2 0 16 22 10 8 0 0 10 28 0
0 0 22 9 22 0 14 0 10 12 0 28 34 28 15 30 14 12 28 8
11 0 16 10 6 10 9 0 12 6 10 10 18 6 18 6 10 30 18 4
12 0 22 12 0 10 14 0 10 10 0 16 10 10 20 16 14 22 16 8
0 0 12 0 8 8 12 0 8 8 0 8 10 10 12 0 22 6 14 4
0 0 15 0 11 6 14 0 5 8 0 12 10 11 14 16 0 12 16 4
0 0 18 8 6 8 10 0 12 10 6 20 18 10 26 6 10 16 10 4
0 0 16 0 0 0 14 6 0 4 0 4 0 14 8 0 10 4 22c 2
Microorganisms: SA, Staphylococcus aureus; MRSA, methicillin-resistant Staphylococcus aureus; EC, Escherichia coli; ML, Micrococcus luteus; BC, Bacillus cereus; BS, Bacillus subtilis; STYP, Salmonella typhimurium; PF, Pseudomonas fluorescens; PV, Proteus vulgaris; SM, Serratia marcescens; SE, Staphylococcus epidermidis; EF, Enterococcus faecalis; ECLO, Enterobacter cloaceae; EA, Enterobacter aerogenes; CA, Candida albicans. a Inhibition zone diameter in millimeters, not including well diameter (6 mm), and if equal to negative control inhibitions or under recorded as 0. b Mean values, n ¼ 3; 0, no inhibitory activity. c Partial inhibition. d NC, negative control, 100 mL 96% ethanol.
inhibited test microorganisms ranged from 2 to 10 mm and not included calculations if the inhibition equal to EtOH inhibitions or under (Table 2). 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 (Fig. 1). 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. 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 (Table 4). 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. Figure 2 summarizes the similarity of microorganisms in relation to their susceptibility to the plant extracts. This has clearly indicated that Gram-positive bacteria
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Table 3. Inhibitory activity of some standard antibiotics against various bacteria and C. albicans. Inhibition zone (mm)b Standard antibiotics Tested microorganismsa S. aureus ATCC 6538P MRSA ATCC 95047 E. coli M. luteus ATCC 9341 B. cereus CM 99 B. subtilis ATCC 6683 S. typhimurium CCM 5445 P. fluorescens P. vulgaris ATCC 6997 S. marcescens CCM 583 S. epidermidis ATCC 12228 E. faecalis ATCC 29212 E. cloaceae ATCC 13067 E. aerogenes ATCC 13048 C. albicans
NA 20 24 26 10R 28 32 6R 30 12R 30 6R 30 12 R 26 ND
NV
AMP
32 34 6R 28 25 13R 40 20 26 6R 26 28 22 17R ND
R
15 16R 6R 26 6R 10R 6R 6R R 10 6R 16 16 15 6R 6
P 24 6R 6R 20R 10R 8 R 6R 6R R 10 6R 11R 24 12R 6R 6
ERY
IPM
VA
CLH
NYS
20 6R
34 15 30 40 36 34 30 24 22 26 26 40 30 30 ND
12 14 6R 15 15 6R 28 6R 22 6R 15 16 16 6R 6
20 25 26 30 28 30 40 12R 15 30 25 30 26 12R ND
ND ND ND ND ND ND ND ND ND ND ND ND ND ND 22
12 R 26 26 15 28 28 20 12R 12R 30 15R 24 6
NA, nalidixic acid (30 mg=disk); NV, novobiocin (30 mg=disk); AMP, ampicillin (10 mg=disk); P, penicilline G (10 IU=disk); ERY, erythromycin (15 mg=disk); IPM, imipenem (10 mg=disk); VA, vancomycin (30 mg=disk); CHL, chloramphenicol (30 mg=disk); NYS, nystatin (10 mg=disk); R, resistant; , partial inhibition; 6, no activity; ND, not determined. a Bacteria tested in MHA medium, yeast in PDA. b Diameter of inhibition zone in millimeters, including disk diameter (6 mm).
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. According to Erdog˘rul (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
Figure 1. Mean values of microorganisms in relation to their susceptibility to the plant extracts. Means are indicated by solid circles. aSee Table 2 for abbreviations of test microorganisms.
results confirm this situation (8–26 mm). Also, Du¨lger and Go¨nu¨z 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) (Keles¸ et al., 2001). 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, 1999; Karaman & Kocabas, 2001; Uzun et al., 2004) and treatment of infectious diseases caused by C. albicans and some pathogenic bacteria
Antimicrobial activity of Turkish plants
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Table 4. MIC and MBC values of selected plant ethanol extracts against SA, MRSA and CA. MIC (mg=mL)
Cornus sanguinea L. Punica granatum L. Vitis vinifera L. Liquidambar orientalis Mill. Rosmarinus officinalis L. Ecballium elaterium A. Richard Eucalyptus camuldulensis Dehnh.
MBC (mg=mL)
MRSA
EC
CA
MRSA
EC
CAa
8 10.2 8.6 8 9.4 ND 8.6
NA NA ND 14.2 ND 11.8 ND
12.6 14.2 NA NA ND ND 13.4
16.6 16.6 15 17.4 18.2 ND 14.2
NA NA ND 22.2 ND 23.6 ND
20.6 24.4 NA NA ND ND 23
MRSA, methicillin resistant Staphylococcus aureus; EC, Escherichia coli; CA, Candida albicans. MIC, minimum inhibitory concentration; MBC, minimal bactericidal concentration. a Minimal fungicidal concentration (MFC); ND, not determined; NA, not applicable.
Figure 2. Similarity (%) of microorganisms in relation to their susceptibility to the plant extracts. See Table 2 for abbreviations of test microorganisms.
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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