Accepted Article
Received Date: 05-Feb-2014 Revised Date : 28-May-2014 Accepted Date: 28-May-2014 Article type
: Original Article
Antimicrobial activity of selected Iranian medicinal plants against a broad spectrum of pathogenic and drug multi-resistant microorganisms
Running title: Plants against pathogenic resistant microorganisms
Amin Abedinia,b, Vincent Roumya,b, Séverine Mahieuxc, Ahmadreza Goharid, Mahdi Moridi Farimanie, Céline Rivièrea,b, Jennifer Samailliea,b, Sevser Sahpaza,b, François Bailleula,b, Christel Neutc, Thierry Hennebellea,b,*.
a
b
Laboratoire Régional de Recherche en Agro-alimentaire et Biotechnologie: Institut Charles Viollette Université de Lille 2, Laboratoire de Pharmacognosie, EA 4481 GRIIOT, UFR Pharmacie, F-59000
Lille, France c
Université de Lille 2, Laboratoire de Bactériologie, INSERM U995, UFR Pharmacie, F-59000 Lille,
France d
Medicinal Plants Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences,
Tehran, Iran
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/lam.12294 This article is protected by copyright. All rights reserved.
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e
Medicinal Plants and Drugs Research Institute, Department of Phytochemistry, Shahid Beheshti
University, G. C., Evin, Tehran, Iran *corresponding author
Corresponding author: Thierry Hennebelle, Ph.D. 3 rue du Professeur Laguesse 59006 LILLE France Phone: +33 3 20964955 Fax: +33 3 20964039 E-mail: [email protected]
SIGNIFICANCE AND IMPACT OF STUDY This study describes the antimicrobial screening of Iranian plant extracts chosen according to traditional practice against 36 microbial strains, from reference culture collections or recent clinical isolates, and enabled to select 4 candidates for further chemical characterization and biological assessment: Dorema ammoniacum, Ferula assa-foetida, Ferulago contracta (seeds) and Perovskia abrotanoides (aerial parts). This may be useful in the development of potential antimicrobial agents, from easily harvested and highly sustainable plant parts. Moreover, the weak extent of cross-resistance between plant extracts and antibiotics warrants further research and may promote a strategy based on less potent but time-trained products.
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ABSTRACT The antimicrobial activities of 44 methanolic extracts from different parts of Iranian indigenous plant species used in traditional medicines of Iran were tested against a panel of 35 pathogenic and multiresistant bacteria and 1 yeast. The antimicrobial efficacy was determined using Müller-Hinton agar in Petri dishes seeded by a multiple inoculator and minimal inhibition concentration (MIC) method. The 21 most active extracts (MIC < 0.3 mg.ml-1 for one or several micro-organisms) were submitted to a more refined measurement. The best antibacterial activity was obtained by 10 plants. Micro-dilution assays allowed to determinate the MIC and MBC of the 21 most active extracts. The lowest achieved MIC value was 78 µg.ml-1, with 4 extracts. This work confirms the antimicrobial activity of assayed plants and suggests further examination to identify the chemical structure of their antimicrobial compounds.
Key words: antimicrobial activity, Iran, traditional medicine, resistance to antibiotics, plant extract.
INTRODUCTION According to a study conducted by the World Health Organization (WHO), more than 80% of the world population relies on traditional medicine for primary healthcare needs (World Health Organization, 2013). Therefore, plants are a valuable source of therapeutic agents. Moreover, the main components of many drugs are still of natural origin or inspiration (Oskay and Sarı, 2007). The use of medicinal plants in Iran has a major therapeutic role, in keeping with a long history of human interactions with the environment. Iran has a rich flora and a widespread knowledge of its indigenous medicinal herbs. There are some native plants that are still used in remote villages of Iran for their antimicrobial activity without any scientific evidence. That is why the screening of these medicinal plants may result in the discovery of new antimicrobial compounds (Naeini et al., 2009).
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Resistance to antibiotics in numerous pathogens has become a real public health problem. The antimicrobial compounds isolated from plants can inhibit bacterial growth by mechanisms that are different from those of antibiotics in current use. They may also have a significant clinical value in the treatment of resistant microbial strains (Eloff, 1998). The emergence of resistant bacterial strains has been a long-observed problem (May, 2014), but its pace has been increasing constantly since they were introduced in the therapeutic arsenal. Although natural resistance exists, it is currently recognized that bad prescribing practices for humans, as well as overuses in animal farming, have played important parts in this phenomenon and, in spite of continued research (Butler et al., 2013), fewer and fewer new antibiotics are launched (Hede, 2014). In the light of these facts, plant compounds, which have been minor contributors to modern antibacterial therapies, may be reconsidered as potential candidates. Because they have been used for long times, with no documented resistance, it seems sound to try and develop less spectacular, but more liable products. In the present study, methanolic extracts of 36 plant species that had reported uses against infectious diseases in Iranian herbal medicine (Zargari, 2011; Mozafarian, 2009; Rechinger, 1982) were screened for antimicrobial activity against 36 microbial strains belonging to 21 different species. The plants were collected from different regions of Iran (Kordestan, Azarbayejan, Khuzsitan and central regions).
RESULTS AND DISCUSSION The agar dilution method with methanolic extract enabled testing of antimicrobial activity of all plants without any problem of solubility, to obtain standardized numeric value (MIC) for each of the 36 microorganisms. The results of the antimicrobial screening of the methanolic extracts of all plants are shown in Table 2. The data indicated that the extracts displayed a variable degree of antimicrobial activity on different tested micro-organisms and the tested strains exhibited variable
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sensitivity against the 44 extracts. The inhibitory property of the extracts was observed within a range of concentrations from 0.3 to 10 mg.ml-1. Among the plants screened, Salvia species, Satureja khuzistanica, Phlomis anisodonta (roots), Dorema ammoniacum, Ferulago contracta, Ferula assafoetida and Perovskia abrotanoides showed higher activity than the other species. In general, Lamiaceae and Apiaceae species, which predominated in the reported uses, were confirmed as more active. Among the 44 methanolic extracts analyzed (36 plants), 21 extracts showed MIC < 0.3 mg.ml-1 for one or several of the 36 micro-organisms. Antimicrobial activity against all of the microorganisms was obtained by Perovskia abrotanoides (MIC ≥ 1.2 mg.ml-1) and Salvia mirzayanii (MIC ≥ 2.5 mg.ml-1). Values of MIC < 0.3 mg.ml-1, were obtained against 13 bacterial strains for Salvia mirzayanii. The aerial parts of Phlomis olivieri and Ferulago bernardii, the roots of Phlomis anisodonta and Ferula hezarlalezarica were more active than other parts of these plants. The best antifungal activities (against Candida albicans) were obtained by Zataria multiflora (MIC < 0.3 mg.ml-1), Satureja khuzistanica, Dorema ammoniacum, Ferulago contracta and Ferula assa-foetida (MIC = 0.6 mg.ml-1).
In order to determine more precisely the MIC and the MBC of 21 selected extracts (MIC < 0.3 mg.ml1
in the preliminary screening), we have used micro-dilution assays against 6 Gram negative, 9 Gram
positive bacteria and one yeast. According to the results given in table 3, the MICs ranged between 78 and 312 µg.ml-1. The lowest MIC value of 78 µg.ml-1 was achived with Dorema ammoniacum (4 strains), Ferula assa-foetida (3 strains), Ferulago contracta and Perovskia abrotanoides. The best MIC, which were obtained with Salvia mirzayanii and Satureja khuzistanica were 156 µg.ml-1 (on 3 strains). The MBCs of 21 extracts were in general significantly higher than the corresponding MIC values and ranged from 312 to > 1250 µg.ml-1. The best MBC (312 µg.ml-1) were obtained with Dorema ammoniacum (on 4 strains). The MBC of Salvia mirzayanii and Satureja khuzistanica in most cases were 625 and 1250 µg.ml-1, respectively.
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According to Carbomelle et al. (1987), antimicrobial substances are considered as bactericidal agents when the ratio MBC/MIC ≤ 4 and bacteriostatic agents when the ratio MBC/MIC >4. From the 106 boxes of MBC/MIC presented in table 3, 54 were determinated as bactericidal (bold boxes), and the others as bacteriostatic. The methanolic extract of Ferulago contracta was bactericidal (in all cases). The MIC of Zataria multiflora against Candida albicans was 312 µg.ml-1 and with a high MFC (minimum fungicidal concentration) (≥ 1250 µg.ml-1). Plants have evolved through time in the presence of pathogenic microorganisms and show good resistance against bacterial invasion. They constitute a potential weapon for humans in their fight against bacteria that are more and more resistant to antibiotics. Biological in vitro activities of plants should always be considered with caution. Many studies use very high, non-physiological concentrations of drugs which can never be achieved in human tissues (Butterweck and Nahrstedt, 2012). Antimicrobial studies often concern only a few strains from one or two species. We chose to determine the MIC against a large choice of micro-organisms, to try and discover extracts with a large-scale activity. Indeed, for the first time we have evaluated the antimicrobial activity of these plants against a panel of 36 pathogenic and multi-resistant bacteria and fungi which, in most cases, have been recently isolated from human infections. For comparison, we also included some reference strains from the American Type Culture Collection (ATCC). But these strains have often been isolated years ago and subcultured on laboratory culture medium only and they no longer reflect what is encountered today in hospitals. A glance at Tables 2 and 3 shows a greater sensitivity of the Gram positive bacteria in comparison with the Gram negative type (Staphylococcus epidermidis, Stenotrophomonas maltophilia, Mycobacterium smegmatis, Staphylococcus aureus, Staphylococcus lugdunensis, Staphylococcus warneri and Corynebacterium striatum were the most sensitive strains in this study). This is due to their different cell-wall structures. Unlike Gram positive bacteria, the lipopolysaccharide layer along with proteins and phospholipids are the major components in the outer surface of Gram negative
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bacteria (Burn, 1988). Access of most compounds to the peptidoglycan layer of the cell wall and to inner targets of the bacteria is hindered by the outer lipopolysaccharide layer. It results in a stronger resistance of Gram negative strains. The results obtained indicated the existence of antimicrobial compounds in the majority of the crude methanolic extracts tested and showed a good correlation with the reported use of these plants in traditional medicine against infectious diseases. Interestingly, when several strains of a same bacterial species were assayed, the observed activity had no relation whatsoever with the known resistances to antibiotics, which seems to imply that their yet-to-be-identified mechanism of action is different from those that have already found clinical use and may be complementary to them. Some of the assayed plants (e.g. Salvia mirzayanii, Zataria multiflora) have been relatively well studied (Moshafi et al., 2004; Sharififar et al., 2007) but their activity on so many microorganisms had never been evaluated.
According to similar studies performed for the antimicrobial activity of plants, when the extracts displayed a MIC below 100 µg.ml-1, the antimicrobial activity was good; from 100 to 500 µg.ml-1 the antimicrobial activity was moderate; from 500 to 1000 µg.ml-1 the antimicrobial activity was weak; over 1000 µg.ml-1 the extract was considered inactive (Toyang et al., 2012; Holetz et al., 2002). This classification presents 4 plants as good antimicrobial agents against one or several strains: Dorema ammoniacum, Ferula assa-foetida, Ferulago contracta and Perovskia abrotanoides (MIC = 78 µg.ml1
).
Among the plants tested, Dorema ammoniacum was thought to be very promising and showed significant inhibition. In the literature, some biological activities such as antibacterial effects had
been reported, but only for the resin, and on a limited number of bacteria; moreover the seeds had not been tested (Rajani et al., 2002). This plant is listed in the British Herbal Pharmacopoeia as an antispasmodic and expectorant, and it is used occasionally for chronic bronchitis and
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persistent coughs (Langenheim, 2003; British Herbal Medicine Association, 1996). Four compounds isolated from the methylene chloride extract of the resin of this plant, including ammoresinol, dshamirone, and doremone A, were found to display acetylcholinesterase inhibitory activity (Adhami et al., 2013). A recent review paper also presents ammoresinol as the main antibacterial
agent of the resin (Venugopala et al., 2013). Ferula assa-foetida is one of the most important species among the 30 species of the genus Ferula in Iran. There are 236 natural products, including 129 sesquiterpene coumarins, seven monoterpene coumarins, 49 daucane sesquiterpenes, nine polysulfide derivatives, 10 sesquiterpene phenylpropanoids, 21 simple sesquiterpenes, six simple monoterpenes, and five oleanane triterpenes, reported from the genus Ferula to date (Lee et al., 2009). While Ferula assa-foetida has had extensive use in folk medicine for the treatment of influenza, a study evaluated the antiviral activities of 30 isolated compounds of F. assa-foetida resin. Among these compounds, 9 sesquiterpenic coumarins showed great potency against influenza A virus (H1N1) (IC50 = 0.26-0.86 µg.ml-1) (Lee et al., 2009).
Terpenic compounds and more specifically terpenocoumarins, may be tentatively suggested as responsible for the antibacterial activity of Dorema ammoniacum and Ferula assa-foetida, and possibly promising as antibacterial agents. Gum ammoniac and asafetida have been known for centuries for their oleoresin (a concentrated, but typically manpower-consuming form of preparation, relying on a traditional mode of harvest, because plants have to be cut or superficially burnt in order to produce the exudates). Importantly, the discovery of good antimicrobial activity in the seeds of these species, may lead to modern production methods (easily applied to the seeds of Apiaceae) enbling larger scales of production and faster recovery, and thus to a renewal in their valorization.
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The plants of the genus Ferulago are represented by 40 species in the world among which eight grow in Iran and three are endemic. Ferulago contracta is one of these (Mozafarian, 1996). Perovskia abrotanoides is used in traditional Iranian medicine for the treatment of fever. Genus Perovskia has seven species, of which three grow in Iran (Khaliq et al., 2007). In the province of Isfahan, a poultice of this plant is applied with sesame oil and wax to treat leishmaniasis. Jaafari et al. (2007) reported the anti-leishmaniasis activity of phenolic and terpenic compounds from stems and leaves of this species. Phytochemical studies available for the essential oil of this plant demonstrate a high content of monoterpenes and sesquiterpenes such as 1,8-cineole (eucalyptol), myrcene, pinene, camphor, caryophyllene, humulene, camphene and bisabolol (Sajjadi et al., 2005; Semnani, 2004). Another study confirms the presence of seven known abietane diterpenoids and 11O- and 12-O-acetylcarnosic acids from a methanol extract (Aoyagi et al., 2006). According to these previous studies and our own results, further examination by bioautography is required in order to identify the antibacterial compounds of these four plants with view to their use for further studies. As plants also have been shown to produce some efflux pump inhibitors, providing synergy with antibiotics, it is possible that some extracts might be mixtures of antibiotics and pump inhibitors. For this reason, our future studies should include such considerations and look for synergies. Likewise, this may help overcome natural Gram negative bacteria reistance (Fadli et al., 2014).
MATERIALS & METHODS Plant materials. Plants were collected by three herbarium centres in Iran. 17 specimens were supplied by Dr. Gohari from the Medicinal Plants Research Center of the Tehran University of Medical Sciences, 9 specimens by Dr. Farimani from Medicinal Plants and Drugs Research Institute of Shahid Beheshti University and 10 by the Firuzeh Medicinal Herb Garden by Mrs. Mirabadi. The
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taxonomic identifications of plant species were confirmed by every centre and a voucher specimen has been deposited in their herbariums (Table 1).
Preparation of the extracts. Dried and powdered plant materials were extracted by maceration using methanol. The extraction, which consisted in a gentle shaking of 30 g herbal material in 200 mL methanol of analytical grade at ambient temperature (ca 25°C) was repeated thrice (methanol provides a more complete extraction, including less polar compounds, and is more representative of the traditional preparations). The extracts were filtered and the solvent was removed by evaporation under reduced pressure to give the dry residues. Thus, crude extracts were dissolved in methanol to a final concentration of 10 mg.ml-1.
Culture and preparation of microorganisms. The experiments were performed according to internationally recognized guidelines (CLSI, 2006). The investigations used ATCC strains and drug multi-resistant clinical strains, able to grow aerobically in Müller-Hinton Agar (MHA) media. The 36 microbial strains were incubated overnight at 37°C in the tubes containing sloping MHA media. Then, the microorganisms were diluted with Ringer's Cysteine solution (RC) to 106 CFU.ml-1 by means of serial dilution just before the antimicrobial assays. Final concentration of each bacterial suspension was 104 CFU.ml-1. The micro-organisms that were used in this study were: Gram negative: 1: Escherichia coli 8137; 2: Escherichia coli 8138 (penicillin resistance); 3: Escherichia coli 8157 (penicillin – fluoroquinolones resistances); 4: Escherichia coli ATCC 25922 (NA); 5: Klebsiella pneumoniae 11016; 6: Klebsiella pneumoniae 11017 (penicillin resistance); 7: Pseudomonas aeruginosa
8131; 8: Pseudomonas aeruginosa ATCC 27583; 9: Proteus mirabilis 11060; 10:
Providencia stuartii 11038; 11: Salmonella sp. 11033; 12: Salmonella sp. 11037 (CMY 2); 13: Serratia marcescens
11056;
14:
Serratia
marcescens
11057
(cephalosporin
resistance);
15:
Stenotrophomonas maltophilia; 16: Acinetobacter baumanii 9010 (VEB-1); 17: Acinetobacter baumanii 9011 (multiresistant); 18: Citrobacter freundii 11041; 19: Citrobacter freundii 11042
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(cephalosporin resistance); 20: Citrobacter freundii 11043 (TEM 3); 21: Enterobacter cloacae 11050; 22: Enterobacter cloacae 11051 (cephalosporin resistance); 23: Enterobacter cloacae 11053 (NDM 1); 24: Enterobacter aerogenes 9004 (BLSE). Gram positive: 25: Enterococcus faecalis C159-6; 26: Mycobacterium smegmatis 5003; 27: Staphylococcus aureus 8146 (methicillin-kanamycin resistance); 28: Staphylococcus aureus 8147; 29: Staphylococcus epidermidis 5001; 30: Staphylococcus epidermidis10282; 31: Staphylococcus lugdunensis T26A3; 32: Staphylococcus warneri T12A12; 33: Corynebacterium striatum T25-17; 34: Enterococcus sp. 8152; 35: Enterococcus sp. 8153. Yeast: 36: Candida albicans 10286. All strains (except the two ATCC strains) were isolated recently from clinical samples. Antibiotic susceptibility was tested by disk diffusion test in the clinical laboratory. In some cases (when enzymes like NDM-1 or TEM-3 implicated in resistance were indicated), these resistances were genetically defined by the hospital laboratory.
MIC determination by agar macrodilution method. The minimum inhibitory concentration (MIC) was studied using MHA in Petri dishes seeded by a multiple inoculator (Pereira et al., 2004). The methanol extract was tested at six final concentrations (10, 5, 2.5, 1.2, 0.6, and 0.3 mg.ml-1) on 36 micro-organisms. The agar plates containing the extract and bacteria were incubated for 24 hours at 37° C. The activity was then estimated visually by presence or absence of colonies. MIC values were recorded as the lowest concentrations of compounds showing no growth. Solvents used were checked for absence of antibacterial activity. Four regular antibiotics were used as positive controls: gentamicin, vancomycin, amoxicillin and amphotericin B. Antibiotic examination showed that six bacteria were resistant against all of the antibiotics used (gentamicin, vancomycin and amoxicillin) : Pseudomonas aeruginosa ATCC 27583, Salmonella sp. 11037, Serratia marcescens 11057, Acinetobacter baumanii 9011, Enterobacter cloacae 11051 and Enterococcus faecalis C159-6.
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MIC/MBC determinations by broth micro-dilution method. A serial dilution technique using 96-well microtiter plates was used to determine more accurately the MIC of the extracts that have a MIC < 0.3 mg.ml-1 against selected sensitive bacteria (McGaw et al., 2001). Nine concentrations of each extract, from 1250 µg.ml-1 to 4.8 µg.ml-1, were used. They were serially two-fold diluted with RC in nine wells. Two wells were reserved for bacteria culture control (no extract added) and medium sterility control (no inocula added). Then the wells were loaded with MH liquid medium and bacterial suspension (104 bacteria.ml-1) giving a final volume of 200 μL. The plates were incubated overnight at 37°C. Bacterial growth was indicated visually and then by direct spray of 0.2 mg.ml-1 INT (iodonitrotetrazolium chloride) to each well and the plates incubated at 37°C for at least 30 min. Bacterial growth in the wells was indicated by a reddish-pink color. MIC values were determined as the lowest concentrations of extracts showing clear wells. The minimal bactericidal concentration (MBC) was determined by the subculture of 100 µL from samples with no visible bacterial growth before INT spray. After 24 hours incubation, the lowest concentration subcultured and showing no growth was considered as the MBC.
ACKNOWLEDGEMENTS The authors would like to thank Mrs. Samareh Mirabadi, of the Firuzeh Medicinal Herb Garden, Tehran and Ms. Siham Lhafidi for their kind help in this work.
CONFLICT OF INTEREST No conflict of interest declared.
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REFERENCES
Adhami, H.R., Linder, T., Kaehlig, H., Zehl, M. and Krenn, L. (2013) Compounds from gum ammoniacum with acetylcholinesterase inhibitory activity. Sci Pharm 81, 793-805. Aoyagi, Y., Takahashi, Y., Satake, Y., Takeya, K., Aiyama, R., Matsuzaki, T., Hashimoto, S. and Kurihara, T. (2006) Cytotoxicity of abietane diterpenoids from Perovskia abrotanoides and of their semisynthetic analogues. Bioorg Med Chem 14, 5285-5291. British Herbal Medicine Association (1996). British herbal pharmacopeia. 4th (ed). UK; 22-23.
Burn, P. (1988) Amphitropic proteins: A new class of membrane proteins. Trends Biochem Sci 13, 7983.
Butler, M.S., Blaskovich, M.A., Cooper, M.A. (2013). Antibiotics in the clinical pipeline in 2013. J Antibiot 66, 571-591.
Butterweck, V. and Nahrstedt, A. (2012) What is the best strategy for preclinical testing of botanicals? A critical perspective. Planta Med 78, 747-754.
Carbomelle, B., Denis, F., Mannonier, A., Pinon, G. and Vague, R. (1987) Bactériologie médicale: Techniques usuelles, pp. 228-282, Paris : SIMEP.
CLSI (Clinical and Laboratory Standards Institute) (2006) Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 7th ed., vol. M7-A7. Approved Standard. Document M7. CLSI, PA.
Eloff, J.N. (1998) Which extractant should be used for the screening and isolation of antimicrobial components from plants? J Ethnopharmacol 60, 1-8.
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Fadli, M., Chevalier, J., Hassani, L., Mezrioui, N.-E., Pagès, J.-M. (2014). Natural extracts stimulate membrane-associated mechanisms of resistance in Gram-negative bacteria. Lett Appl Microbiol 58, 472-477.
Hede, K. (2014). An infectious arms race. Nature 509, S2-S3.
Holetz, F.B., Pessini, G.L., Sanches, N.R., Cortez, D.A.G., Nakamura, C.V. and Filho, B.P.D. (2002) Screening of some plants used in the Brazilian folk medicine for the treatment of infectious diseases. Mem Inst Oswaldo Cruz 97, 1027-1031.
Jaafari, M.R., Hooshmand, S., Samiei, A. and Hosseinzade, H. (2007) Evaluation of leishmanicidal effect of Perovskia abrotanoides Karel root extract by in vitro leishmanicidal assay using promastigotes of Leishmania major. Pharmacol Online 1, 299-303.
Khaliq, S., Volk, F.J. and Frahm, A.W. (2007) Phytochemical investigation of Perovskia abrotanoides. Planta Med 73, 77-83.
Langenheim, J.H. (2003) Plant resins: chemistry, evolution, ecology and ethnobotany, pp. 412-415. Portland: Timber Press.
Lee, C.L., Chiang, L.C., Cheng, L.H., Liaw, C.C., Abd El-Razek, M.H., Chang, F.R. and Wu, Y.C. (2009) Influenza A (H1N1) antiviral and cytotoxic agents from Ferula assa-foetida. J Nat Prod 72, 1568-1572.
May, M. (2014) Drug development: Time for teamwork. Nature 509, S4-S5.
McGaw, L.J., Rabe, T., Sparg, S.G., Jager, A.K., Eloff, J.N. and Van Staden, J. (2001) An investigation on the biological activity of Combretum species. J Ethnopharmacol 75, 45-50.
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Moshafi, M.H., Mehrabani, M. and Zolhasab, H. (2004) Antibacterial activity studies of Salvia mirzayanii and Salvia atropatana against six standard gram-positive and gram-negative bacteria. J Kerman Univ Med Sci 11, 109-118.
Mozafarian, V. (2009) Dictionary of Iranian plant names (Latin English Persian). Tehran: Farhang Moaser Publication.
Naeini, A., Khosravi, A.R., Chitsaz, M, Shokri H., Kamlnejad, M. (2009) Anti-Candida albicans activity of some Iranian plants used in traditional medicine. J Mycol Med 19, 168-172.
Oskay, M. and Sarı, D. (2007) Antimicrobial Screening of Some Turkish Medicinal Plants. Pharm Biol 45, 176-181.
Pereira, R.S., Sumita, T.C., Furlan, M.R., Jorge, A.O. and Ueno, M. (2004) Antibacterial activity of essential oils on microorganisms isolated from urinary tract infection. Rev Saude Publica 38, 326328.
Rajani, M., Saxena, N., Ravishankara, M.N., Desai, N., and Padh, H. (2002) Evaluation of the antimicrobial activity of ammoniacum gum from Dorema ammoniacum. Pharm Biol 40, 534-541.
Rechinger, K.H. (1982) In Flora Iranica, No. 150, Rechinger KH (ed). Graz, Austria: Akademische Druck-u. Verlagsanstalt.
Sajjadi, S.E., Mehregan, I., Khatamsaz, M. and Asghari, G.H. (2005). Chemical composition of the essential oil of Perovskia abroatanoides Karel growing wild in Iran. Flav Frag J 20, 445-446.
Semnani, M.K. (2004) The essential oil composition of Perovskia abroatanoides from Iran. Pharm Biol 42, 214-216.
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Sharififar, F., Moshafi, M.H., Mansouri, S.H., Khodashenas, M. and Khoshnoodi, M. (2007) In vitro evaluation of antibacterial and antioxidant activities of the essential oil and methanol extract of endemic Zataria multiflora Boiss. Food Control 18, 800-805.
Toyang, N.J., Ateh, E.N., Keiser, J., Vargas, M., Bach, H., Tane, P., Sondengam, L.B., Davis, H., Bryant, J. and Verpoorte, R. (2012) Toxicity, antimicrobial and anthelmintic activities of Vernonia guineensis Benth. (Asteraceae) crude extracts. J Ethnopharmacol 144, 700-704.
Venugopala, K.N., Rashmi, V. and Odhav, B. (2013) Review on natural coumarin lead compounds for their pharmacological activity. Biomed Res Int 2013, 1-14.
World Health Organisation. (2013) Traditional Medicine Strategy 2014-2023, pp. 23-28. Hong Kong SAR, China.
Zargari, A. (2011) Medicinal plants. Tehran: Tehran University of Medical Sciences.
List of tables Table 1. Selected plants for in vitro antibacterial assay and their traditional uses. AP: aerial parts, F: flower, ST: stem, L: leave, R: root, SE: seed, T: thallus. Table 2. Minimum inhibitory concentration (MIC) of 44 methanolic extracts (mg.ml-1) F: flower, AP: aerial parts, ST: stem, L: leave, R: root, SE: seed, T: thallus. MIC (µg.ml-1) of positive controls: gentamicin, S: ≤4, R: >8; vancomycin, S: ≤4, R: >16; amoxicillin, S: ≤4, R: >16; Amphotericin B, S: ≤1, R: >4. Table 3. The MIC and MBC of selected extracts determined by micro-dilution assays (µg.ml-1).
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Table 1.
Family
Extraction
Lamiaceae
Plant species
Local name
Used Parts
Phlomis olivieri Benth.
Mahroo
F, AP, ST, L
Yields (%)
Selected traditional uses in relation with possible anti- Voucher infectious effect
7.5, 6.6, 8.4, Antiseptic
1612-ACECR
5.9 Phlomis persica Boiss.
Goshboreh irani
AP
8.9
Phlomis Boiss.
Givej Belgeh
AP, L, R
14.6, 9.3
Perovskia artemisioides Brazembel Boiss.
ST+L
10.2
Perovskia abrotanoides Brazembel Karel.
AP
8.8
anisodonta
Teucrium Boiss.
persicum
Treatment discomfort
of
digestive
13.2, Treatment discomfort
of
digestive
Anti-leishmaniasis
1610-ACECR
1634-ACECR
271-ACECR
Anti-leshmaniasis, MPH-1964
treatment of fever 9.6
Treatment of sore eyes
397- ACECR
ST+L
8.1
Antiseptic
288-ACECR
ST+L
3.9
Air freshener and purifier
1560-ACECR
Zataria multiflora Boiss. Avhishan Shirazi
L
9.6
Treatment of respiratory FG-1874 and digestive discomfort
Dracocephalum kotschyi Zarringiah Boiss.
AP
12.1
Treatment of fever
MPH-1631
MaryamGoli Banafsh
R
4.1
Treatment of skin diseases
1599-ACECR
MaryamGoli
L
9.4
Antiseptic, disinfection
244-ACECR
Maryam-goli boland
ST+F
18.3
Digestive
MPH-1654
MaryamGoli Sahandi
AP
7.5
Antiseptic, indigestion
MaryamGoli Isphehani
ST+F
6.9
Treatment discomfort
Satureja Jamzad.
Maryam nokhodi AP
khuzistanica Marzeh khuzistani
Hymenocrater longiflorus Benth.
Salvia verticillata L.
Arvaneh
Salvia chloroleuca Rech.f. & Aell Salvia hypoleuca Benth. Salvia sahendica Boiss. & Buhse. Salvia reuteriana Boiss.
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relieving
of
digestive
MPH-1592
MPH-231
Apiaceae
Accepted Article
Salvia Hedge.
lachnocalyx Maryam-goli eghlidi
AP
14.5
Antiseptic, disinfection
MPH-674
Mourporzoo
AP
12.1
Treatment discomfort
FG-1825
Salvia syriaca L.
MaryamGoli Soori
ST+F
9.2
Antiseptic
MPH-343
Salvia ceratophylla L.
MaryamGoli shakhgavazn
AP
11.5
Antiseptic
MPH-1051
Salvia urmiensis Bung.
MaryamGoli urumiehie
AP
8.8
Antiseptic
MPH-1220
Gichoo
R, SE
16.3, 23.5
Treatment of respiratory 6722-TEH discomfort
R
7.8
Boiss. & Buhse.
Kamaye ghalafdar
Treatment of respiratory 42-ACECR disorders
Ferula gummosa Boiss.
Barijeh
SE
30.0
Antiseptic, treatment digestive discomfort
Ferula assa-foetida L.
Anghozeh
SE
18.8
Treatment of influenza
FG-0112
Ferulago Boiss.
Chavil
SE
29.1
Antiseptic
FG-0171
Chenoor
AP, ST, R
13.5, 10.3
Chavir
ST
15.7
Air carminative
Hasaseh
ST
6.8
Antiseptic
1584 -ACECR
Havij vahshi
AP
10.0
Antiseptic
6734-TEH
Khosharizeh
ST
11.5
Anti-mold, freshener
FG-0252
Vashagh
SE
16.2
Treatment of respiratory FG-0125 disorders
Salvia mirzayanii Rech. f. & Esfand.
Ferula hezarlalezarica Ajani. Ferula oopoda
contracta
Ferulago bernardii Tomk. & M. Pimen. Ferulago angulata (Schlecht) Boiss. Zeravschanica pauciradiatum (Tamamsch). M. Pimen.
5.5, Treatment discomfort
of
of
digestive
of
digestive
disinfection,
FG-0115
1562-ACECR
1633-ACECR
Daucus littoralis Smith Subs. Echinophora DC.
platyloba
Dorema ammoniacum D. Don
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Dictyotaceae
Accepted Article
Asteraceae
Achillea tenuifolia Lam.
AP
7.1
Treatment inflammation
SE
7.4
Treatment of hepatitis
FG-0335
Jolbak Ghahveii
T
5.6
Antiseptic
44-14P
Golkhatmi
F
12.1
Treatment of respiratory FG-1152 and digestive discomfort
AP
7.3
Treatment of skin diseases
Bomadaran
Silybum marianum (L.) Kharmaryam Gaertn.
of
skin
1604-ACECR
Padina boergesenii Allender & Kraft
Malvaceae
Alcea rosea L.
Euphorbiaceae
Euphorbia microsciadia Forfion Denaii Boiss.
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FG-1652
Accepted Artic Table 2.
Plant
MIC (mg.ml-1) Gram negative bacteria
Gram positive bacteria
Yeast
Used C. albicans
Enterococcus sp.
Corynebacter -ium
S. warneri
S. lugdunensis
S. epidermidis
S. aureus
M. smegmatis
E. faecalis
E. aerogenes
E. cloacae
C. freundii
A. baumanii
S. maltophilia
S. marcescens
Salmonella sp.
P. stuartii
P. mirabilis
P. aeruginosa
parts
K. pneumoniae
E. coli
species
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
F
10
10
-
-
-
1.2
5
5
2.5
10
2.5
10
10
10
0.6
2.5
5
5
5
5
10
10
10
10
10
1.2
1.2
1.2
1.2
0.6
0.6
1.2
1.2
10
10
5
AP
10
10
10
-
10
1.2
2.5
2.5
2.5
10
2.5
10
10
10
Received Date: 05-Feb-2014 Revised Date : 28-May-2014 Accepted Date: 28-May-2014 Article type
: Original Article
Antimicrobial activity of selected Iranian medicinal plants against a broad spectrum of pathogenic and drug multi-resistant microorganisms
Running title: Plants against pathogenic resistant microorganisms
Amin Abedinia,b, Vincent Roumya,b, Séverine Mahieuxc, Ahmadreza Goharid, Mahdi Moridi Farimanie, Céline Rivièrea,b, Jennifer Samailliea,b, Sevser Sahpaza,b, François Bailleula,b, Christel Neutc, Thierry Hennebellea,b,*.
a
b
Laboratoire Régional de Recherche en Agro-alimentaire et Biotechnologie: Institut Charles Viollette Université de Lille 2, Laboratoire de Pharmacognosie, EA 4481 GRIIOT, UFR Pharmacie, F-59000
Lille, France c
Université de Lille 2, Laboratoire de Bactériologie, INSERM U995, UFR Pharmacie, F-59000 Lille,
France d
Medicinal Plants Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences,
Tehran, Iran
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/lam.12294 This article is protected by copyright. All rights reserved.
Accepted Article
e
Medicinal Plants and Drugs Research Institute, Department of Phytochemistry, Shahid Beheshti
University, G. C., Evin, Tehran, Iran *corresponding author
Corresponding author: Thierry Hennebelle, Ph.D. 3 rue du Professeur Laguesse 59006 LILLE France Phone: +33 3 20964955 Fax: +33 3 20964039 E-mail: [email protected]
SIGNIFICANCE AND IMPACT OF STUDY This study describes the antimicrobial screening of Iranian plant extracts chosen according to traditional practice against 36 microbial strains, from reference culture collections or recent clinical isolates, and enabled to select 4 candidates for further chemical characterization and biological assessment: Dorema ammoniacum, Ferula assa-foetida, Ferulago contracta (seeds) and Perovskia abrotanoides (aerial parts). This may be useful in the development of potential antimicrobial agents, from easily harvested and highly sustainable plant parts. Moreover, the weak extent of cross-resistance between plant extracts and antibiotics warrants further research and may promote a strategy based on less potent but time-trained products.
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ABSTRACT The antimicrobial activities of 44 methanolic extracts from different parts of Iranian indigenous plant species used in traditional medicines of Iran were tested against a panel of 35 pathogenic and multiresistant bacteria and 1 yeast. The antimicrobial efficacy was determined using Müller-Hinton agar in Petri dishes seeded by a multiple inoculator and minimal inhibition concentration (MIC) method. The 21 most active extracts (MIC < 0.3 mg.ml-1 for one or several micro-organisms) were submitted to a more refined measurement. The best antibacterial activity was obtained by 10 plants. Micro-dilution assays allowed to determinate the MIC and MBC of the 21 most active extracts. The lowest achieved MIC value was 78 µg.ml-1, with 4 extracts. This work confirms the antimicrobial activity of assayed plants and suggests further examination to identify the chemical structure of their antimicrobial compounds.
Key words: antimicrobial activity, Iran, traditional medicine, resistance to antibiotics, plant extract.
INTRODUCTION According to a study conducted by the World Health Organization (WHO), more than 80% of the world population relies on traditional medicine for primary healthcare needs (World Health Organization, 2013). Therefore, plants are a valuable source of therapeutic agents. Moreover, the main components of many drugs are still of natural origin or inspiration (Oskay and Sarı, 2007). The use of medicinal plants in Iran has a major therapeutic role, in keeping with a long history of human interactions with the environment. Iran has a rich flora and a widespread knowledge of its indigenous medicinal herbs. There are some native plants that are still used in remote villages of Iran for their antimicrobial activity without any scientific evidence. That is why the screening of these medicinal plants may result in the discovery of new antimicrobial compounds (Naeini et al., 2009).
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Resistance to antibiotics in numerous pathogens has become a real public health problem. The antimicrobial compounds isolated from plants can inhibit bacterial growth by mechanisms that are different from those of antibiotics in current use. They may also have a significant clinical value in the treatment of resistant microbial strains (Eloff, 1998). The emergence of resistant bacterial strains has been a long-observed problem (May, 2014), but its pace has been increasing constantly since they were introduced in the therapeutic arsenal. Although natural resistance exists, it is currently recognized that bad prescribing practices for humans, as well as overuses in animal farming, have played important parts in this phenomenon and, in spite of continued research (Butler et al., 2013), fewer and fewer new antibiotics are launched (Hede, 2014). In the light of these facts, plant compounds, which have been minor contributors to modern antibacterial therapies, may be reconsidered as potential candidates. Because they have been used for long times, with no documented resistance, it seems sound to try and develop less spectacular, but more liable products. In the present study, methanolic extracts of 36 plant species that had reported uses against infectious diseases in Iranian herbal medicine (Zargari, 2011; Mozafarian, 2009; Rechinger, 1982) were screened for antimicrobial activity against 36 microbial strains belonging to 21 different species. The plants were collected from different regions of Iran (Kordestan, Azarbayejan, Khuzsitan and central regions).
RESULTS AND DISCUSSION The agar dilution method with methanolic extract enabled testing of antimicrobial activity of all plants without any problem of solubility, to obtain standardized numeric value (MIC) for each of the 36 microorganisms. The results of the antimicrobial screening of the methanolic extracts of all plants are shown in Table 2. The data indicated that the extracts displayed a variable degree of antimicrobial activity on different tested micro-organisms and the tested strains exhibited variable
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sensitivity against the 44 extracts. The inhibitory property of the extracts was observed within a range of concentrations from 0.3 to 10 mg.ml-1. Among the plants screened, Salvia species, Satureja khuzistanica, Phlomis anisodonta (roots), Dorema ammoniacum, Ferulago contracta, Ferula assafoetida and Perovskia abrotanoides showed higher activity than the other species. In general, Lamiaceae and Apiaceae species, which predominated in the reported uses, were confirmed as more active. Among the 44 methanolic extracts analyzed (36 plants), 21 extracts showed MIC < 0.3 mg.ml-1 for one or several of the 36 micro-organisms. Antimicrobial activity against all of the microorganisms was obtained by Perovskia abrotanoides (MIC ≥ 1.2 mg.ml-1) and Salvia mirzayanii (MIC ≥ 2.5 mg.ml-1). Values of MIC < 0.3 mg.ml-1, were obtained against 13 bacterial strains for Salvia mirzayanii. The aerial parts of Phlomis olivieri and Ferulago bernardii, the roots of Phlomis anisodonta and Ferula hezarlalezarica were more active than other parts of these plants. The best antifungal activities (against Candida albicans) were obtained by Zataria multiflora (MIC < 0.3 mg.ml-1), Satureja khuzistanica, Dorema ammoniacum, Ferulago contracta and Ferula assa-foetida (MIC = 0.6 mg.ml-1).
In order to determine more precisely the MIC and the MBC of 21 selected extracts (MIC < 0.3 mg.ml1
in the preliminary screening), we have used micro-dilution assays against 6 Gram negative, 9 Gram
positive bacteria and one yeast. According to the results given in table 3, the MICs ranged between 78 and 312 µg.ml-1. The lowest MIC value of 78 µg.ml-1 was achived with Dorema ammoniacum (4 strains), Ferula assa-foetida (3 strains), Ferulago contracta and Perovskia abrotanoides. The best MIC, which were obtained with Salvia mirzayanii and Satureja khuzistanica were 156 µg.ml-1 (on 3 strains). The MBCs of 21 extracts were in general significantly higher than the corresponding MIC values and ranged from 312 to > 1250 µg.ml-1. The best MBC (312 µg.ml-1) were obtained with Dorema ammoniacum (on 4 strains). The MBC of Salvia mirzayanii and Satureja khuzistanica in most cases were 625 and 1250 µg.ml-1, respectively.
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According to Carbomelle et al. (1987), antimicrobial substances are considered as bactericidal agents when the ratio MBC/MIC ≤ 4 and bacteriostatic agents when the ratio MBC/MIC >4. From the 106 boxes of MBC/MIC presented in table 3, 54 were determinated as bactericidal (bold boxes), and the others as bacteriostatic. The methanolic extract of Ferulago contracta was bactericidal (in all cases). The MIC of Zataria multiflora against Candida albicans was 312 µg.ml-1 and with a high MFC (minimum fungicidal concentration) (≥ 1250 µg.ml-1). Plants have evolved through time in the presence of pathogenic microorganisms and show good resistance against bacterial invasion. They constitute a potential weapon for humans in their fight against bacteria that are more and more resistant to antibiotics. Biological in vitro activities of plants should always be considered with caution. Many studies use very high, non-physiological concentrations of drugs which can never be achieved in human tissues (Butterweck and Nahrstedt, 2012). Antimicrobial studies often concern only a few strains from one or two species. We chose to determine the MIC against a large choice of micro-organisms, to try and discover extracts with a large-scale activity. Indeed, for the first time we have evaluated the antimicrobial activity of these plants against a panel of 36 pathogenic and multi-resistant bacteria and fungi which, in most cases, have been recently isolated from human infections. For comparison, we also included some reference strains from the American Type Culture Collection (ATCC). But these strains have often been isolated years ago and subcultured on laboratory culture medium only and they no longer reflect what is encountered today in hospitals. A glance at Tables 2 and 3 shows a greater sensitivity of the Gram positive bacteria in comparison with the Gram negative type (Staphylococcus epidermidis, Stenotrophomonas maltophilia, Mycobacterium smegmatis, Staphylococcus aureus, Staphylococcus lugdunensis, Staphylococcus warneri and Corynebacterium striatum were the most sensitive strains in this study). This is due to their different cell-wall structures. Unlike Gram positive bacteria, the lipopolysaccharide layer along with proteins and phospholipids are the major components in the outer surface of Gram negative
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Accepted Article
bacteria (Burn, 1988). Access of most compounds to the peptidoglycan layer of the cell wall and to inner targets of the bacteria is hindered by the outer lipopolysaccharide layer. It results in a stronger resistance of Gram negative strains. The results obtained indicated the existence of antimicrobial compounds in the majority of the crude methanolic extracts tested and showed a good correlation with the reported use of these plants in traditional medicine against infectious diseases. Interestingly, when several strains of a same bacterial species were assayed, the observed activity had no relation whatsoever with the known resistances to antibiotics, which seems to imply that their yet-to-be-identified mechanism of action is different from those that have already found clinical use and may be complementary to them. Some of the assayed plants (e.g. Salvia mirzayanii, Zataria multiflora) have been relatively well studied (Moshafi et al., 2004; Sharififar et al., 2007) but their activity on so many microorganisms had never been evaluated.
According to similar studies performed for the antimicrobial activity of plants, when the extracts displayed a MIC below 100 µg.ml-1, the antimicrobial activity was good; from 100 to 500 µg.ml-1 the antimicrobial activity was moderate; from 500 to 1000 µg.ml-1 the antimicrobial activity was weak; over 1000 µg.ml-1 the extract was considered inactive (Toyang et al., 2012; Holetz et al., 2002). This classification presents 4 plants as good antimicrobial agents against one or several strains: Dorema ammoniacum, Ferula assa-foetida, Ferulago contracta and Perovskia abrotanoides (MIC = 78 µg.ml1
).
Among the plants tested, Dorema ammoniacum was thought to be very promising and showed significant inhibition. In the literature, some biological activities such as antibacterial effects had
been reported, but only for the resin, and on a limited number of bacteria; moreover the seeds had not been tested (Rajani et al., 2002). This plant is listed in the British Herbal Pharmacopoeia as an antispasmodic and expectorant, and it is used occasionally for chronic bronchitis and
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Accepted Article
persistent coughs (Langenheim, 2003; British Herbal Medicine Association, 1996). Four compounds isolated from the methylene chloride extract of the resin of this plant, including ammoresinol, dshamirone, and doremone A, were found to display acetylcholinesterase inhibitory activity (Adhami et al., 2013). A recent review paper also presents ammoresinol as the main antibacterial
agent of the resin (Venugopala et al., 2013). Ferula assa-foetida is one of the most important species among the 30 species of the genus Ferula in Iran. There are 236 natural products, including 129 sesquiterpene coumarins, seven monoterpene coumarins, 49 daucane sesquiterpenes, nine polysulfide derivatives, 10 sesquiterpene phenylpropanoids, 21 simple sesquiterpenes, six simple monoterpenes, and five oleanane triterpenes, reported from the genus Ferula to date (Lee et al., 2009). While Ferula assa-foetida has had extensive use in folk medicine for the treatment of influenza, a study evaluated the antiviral activities of 30 isolated compounds of F. assa-foetida resin. Among these compounds, 9 sesquiterpenic coumarins showed great potency against influenza A virus (H1N1) (IC50 = 0.26-0.86 µg.ml-1) (Lee et al., 2009).
Terpenic compounds and more specifically terpenocoumarins, may be tentatively suggested as responsible for the antibacterial activity of Dorema ammoniacum and Ferula assa-foetida, and possibly promising as antibacterial agents. Gum ammoniac and asafetida have been known for centuries for their oleoresin (a concentrated, but typically manpower-consuming form of preparation, relying on a traditional mode of harvest, because plants have to be cut or superficially burnt in order to produce the exudates). Importantly, the discovery of good antimicrobial activity in the seeds of these species, may lead to modern production methods (easily applied to the seeds of Apiaceae) enbling larger scales of production and faster recovery, and thus to a renewal in their valorization.
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Accepted Article
The plants of the genus Ferulago are represented by 40 species in the world among which eight grow in Iran and three are endemic. Ferulago contracta is one of these (Mozafarian, 1996). Perovskia abrotanoides is used in traditional Iranian medicine for the treatment of fever. Genus Perovskia has seven species, of which three grow in Iran (Khaliq et al., 2007). In the province of Isfahan, a poultice of this plant is applied with sesame oil and wax to treat leishmaniasis. Jaafari et al. (2007) reported the anti-leishmaniasis activity of phenolic and terpenic compounds from stems and leaves of this species. Phytochemical studies available for the essential oil of this plant demonstrate a high content of monoterpenes and sesquiterpenes such as 1,8-cineole (eucalyptol), myrcene, pinene, camphor, caryophyllene, humulene, camphene and bisabolol (Sajjadi et al., 2005; Semnani, 2004). Another study confirms the presence of seven known abietane diterpenoids and 11O- and 12-O-acetylcarnosic acids from a methanol extract (Aoyagi et al., 2006). According to these previous studies and our own results, further examination by bioautography is required in order to identify the antibacterial compounds of these four plants with view to their use for further studies. As plants also have been shown to produce some efflux pump inhibitors, providing synergy with antibiotics, it is possible that some extracts might be mixtures of antibiotics and pump inhibitors. For this reason, our future studies should include such considerations and look for synergies. Likewise, this may help overcome natural Gram negative bacteria reistance (Fadli et al., 2014).
MATERIALS & METHODS Plant materials. Plants were collected by three herbarium centres in Iran. 17 specimens were supplied by Dr. Gohari from the Medicinal Plants Research Center of the Tehran University of Medical Sciences, 9 specimens by Dr. Farimani from Medicinal Plants and Drugs Research Institute of Shahid Beheshti University and 10 by the Firuzeh Medicinal Herb Garden by Mrs. Mirabadi. The
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Accepted Article
taxonomic identifications of plant species were confirmed by every centre and a voucher specimen has been deposited in their herbariums (Table 1).
Preparation of the extracts. Dried and powdered plant materials were extracted by maceration using methanol. The extraction, which consisted in a gentle shaking of 30 g herbal material in 200 mL methanol of analytical grade at ambient temperature (ca 25°C) was repeated thrice (methanol provides a more complete extraction, including less polar compounds, and is more representative of the traditional preparations). The extracts were filtered and the solvent was removed by evaporation under reduced pressure to give the dry residues. Thus, crude extracts were dissolved in methanol to a final concentration of 10 mg.ml-1.
Culture and preparation of microorganisms. The experiments were performed according to internationally recognized guidelines (CLSI, 2006). The investigations used ATCC strains and drug multi-resistant clinical strains, able to grow aerobically in Müller-Hinton Agar (MHA) media. The 36 microbial strains were incubated overnight at 37°C in the tubes containing sloping MHA media. Then, the microorganisms were diluted with Ringer's Cysteine solution (RC) to 106 CFU.ml-1 by means of serial dilution just before the antimicrobial assays. Final concentration of each bacterial suspension was 104 CFU.ml-1. The micro-organisms that were used in this study were: Gram negative: 1: Escherichia coli 8137; 2: Escherichia coli 8138 (penicillin resistance); 3: Escherichia coli 8157 (penicillin – fluoroquinolones resistances); 4: Escherichia coli ATCC 25922 (NA); 5: Klebsiella pneumoniae 11016; 6: Klebsiella pneumoniae 11017 (penicillin resistance); 7: Pseudomonas aeruginosa
8131; 8: Pseudomonas aeruginosa ATCC 27583; 9: Proteus mirabilis 11060; 10:
Providencia stuartii 11038; 11: Salmonella sp. 11033; 12: Salmonella sp. 11037 (CMY 2); 13: Serratia marcescens
11056;
14:
Serratia
marcescens
11057
(cephalosporin
resistance);
15:
Stenotrophomonas maltophilia; 16: Acinetobacter baumanii 9010 (VEB-1); 17: Acinetobacter baumanii 9011 (multiresistant); 18: Citrobacter freundii 11041; 19: Citrobacter freundii 11042
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Accepted Article
(cephalosporin resistance); 20: Citrobacter freundii 11043 (TEM 3); 21: Enterobacter cloacae 11050; 22: Enterobacter cloacae 11051 (cephalosporin resistance); 23: Enterobacter cloacae 11053 (NDM 1); 24: Enterobacter aerogenes 9004 (BLSE). Gram positive: 25: Enterococcus faecalis C159-6; 26: Mycobacterium smegmatis 5003; 27: Staphylococcus aureus 8146 (methicillin-kanamycin resistance); 28: Staphylococcus aureus 8147; 29: Staphylococcus epidermidis 5001; 30: Staphylococcus epidermidis10282; 31: Staphylococcus lugdunensis T26A3; 32: Staphylococcus warneri T12A12; 33: Corynebacterium striatum T25-17; 34: Enterococcus sp. 8152; 35: Enterococcus sp. 8153. Yeast: 36: Candida albicans 10286. All strains (except the two ATCC strains) were isolated recently from clinical samples. Antibiotic susceptibility was tested by disk diffusion test in the clinical laboratory. In some cases (when enzymes like NDM-1 or TEM-3 implicated in resistance were indicated), these resistances were genetically defined by the hospital laboratory.
MIC determination by agar macrodilution method. The minimum inhibitory concentration (MIC) was studied using MHA in Petri dishes seeded by a multiple inoculator (Pereira et al., 2004). The methanol extract was tested at six final concentrations (10, 5, 2.5, 1.2, 0.6, and 0.3 mg.ml-1) on 36 micro-organisms. The agar plates containing the extract and bacteria were incubated for 24 hours at 37° C. The activity was then estimated visually by presence or absence of colonies. MIC values were recorded as the lowest concentrations of compounds showing no growth. Solvents used were checked for absence of antibacterial activity. Four regular antibiotics were used as positive controls: gentamicin, vancomycin, amoxicillin and amphotericin B. Antibiotic examination showed that six bacteria were resistant against all of the antibiotics used (gentamicin, vancomycin and amoxicillin) : Pseudomonas aeruginosa ATCC 27583, Salmonella sp. 11037, Serratia marcescens 11057, Acinetobacter baumanii 9011, Enterobacter cloacae 11051 and Enterococcus faecalis C159-6.
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MIC/MBC determinations by broth micro-dilution method. A serial dilution technique using 96-well microtiter plates was used to determine more accurately the MIC of the extracts that have a MIC < 0.3 mg.ml-1 against selected sensitive bacteria (McGaw et al., 2001). Nine concentrations of each extract, from 1250 µg.ml-1 to 4.8 µg.ml-1, were used. They were serially two-fold diluted with RC in nine wells. Two wells were reserved for bacteria culture control (no extract added) and medium sterility control (no inocula added). Then the wells were loaded with MH liquid medium and bacterial suspension (104 bacteria.ml-1) giving a final volume of 200 μL. The plates were incubated overnight at 37°C. Bacterial growth was indicated visually and then by direct spray of 0.2 mg.ml-1 INT (iodonitrotetrazolium chloride) to each well and the plates incubated at 37°C for at least 30 min. Bacterial growth in the wells was indicated by a reddish-pink color. MIC values were determined as the lowest concentrations of extracts showing clear wells. The minimal bactericidal concentration (MBC) was determined by the subculture of 100 µL from samples with no visible bacterial growth before INT spray. After 24 hours incubation, the lowest concentration subcultured and showing no growth was considered as the MBC.
ACKNOWLEDGEMENTS The authors would like to thank Mrs. Samareh Mirabadi, of the Firuzeh Medicinal Herb Garden, Tehran and Ms. Siham Lhafidi for their kind help in this work.
CONFLICT OF INTEREST No conflict of interest declared.
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Accepted Article
REFERENCES
Adhami, H.R., Linder, T., Kaehlig, H., Zehl, M. and Krenn, L. (2013) Compounds from gum ammoniacum with acetylcholinesterase inhibitory activity. Sci Pharm 81, 793-805. Aoyagi, Y., Takahashi, Y., Satake, Y., Takeya, K., Aiyama, R., Matsuzaki, T., Hashimoto, S. and Kurihara, T. (2006) Cytotoxicity of abietane diterpenoids from Perovskia abrotanoides and of their semisynthetic analogues. Bioorg Med Chem 14, 5285-5291. British Herbal Medicine Association (1996). British herbal pharmacopeia. 4th (ed). UK; 22-23.
Burn, P. (1988) Amphitropic proteins: A new class of membrane proteins. Trends Biochem Sci 13, 7983.
Butler, M.S., Blaskovich, M.A., Cooper, M.A. (2013). Antibiotics in the clinical pipeline in 2013. J Antibiot 66, 571-591.
Butterweck, V. and Nahrstedt, A. (2012) What is the best strategy for preclinical testing of botanicals? A critical perspective. Planta Med 78, 747-754.
Carbomelle, B., Denis, F., Mannonier, A., Pinon, G. and Vague, R. (1987) Bactériologie médicale: Techniques usuelles, pp. 228-282, Paris : SIMEP.
CLSI (Clinical and Laboratory Standards Institute) (2006) Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 7th ed., vol. M7-A7. Approved Standard. Document M7. CLSI, PA.
Eloff, J.N. (1998) Which extractant should be used for the screening and isolation of antimicrobial components from plants? J Ethnopharmacol 60, 1-8.
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Fadli, M., Chevalier, J., Hassani, L., Mezrioui, N.-E., Pagès, J.-M. (2014). Natural extracts stimulate membrane-associated mechanisms of resistance in Gram-negative bacteria. Lett Appl Microbiol 58, 472-477.
Hede, K. (2014). An infectious arms race. Nature 509, S2-S3.
Holetz, F.B., Pessini, G.L., Sanches, N.R., Cortez, D.A.G., Nakamura, C.V. and Filho, B.P.D. (2002) Screening of some plants used in the Brazilian folk medicine for the treatment of infectious diseases. Mem Inst Oswaldo Cruz 97, 1027-1031.
Jaafari, M.R., Hooshmand, S., Samiei, A. and Hosseinzade, H. (2007) Evaluation of leishmanicidal effect of Perovskia abrotanoides Karel root extract by in vitro leishmanicidal assay using promastigotes of Leishmania major. Pharmacol Online 1, 299-303.
Khaliq, S., Volk, F.J. and Frahm, A.W. (2007) Phytochemical investigation of Perovskia abrotanoides. Planta Med 73, 77-83.
Langenheim, J.H. (2003) Plant resins: chemistry, evolution, ecology and ethnobotany, pp. 412-415. Portland: Timber Press.
Lee, C.L., Chiang, L.C., Cheng, L.H., Liaw, C.C., Abd El-Razek, M.H., Chang, F.R. and Wu, Y.C. (2009) Influenza A (H1N1) antiviral and cytotoxic agents from Ferula assa-foetida. J Nat Prod 72, 1568-1572.
May, M. (2014) Drug development: Time for teamwork. Nature 509, S4-S5.
McGaw, L.J., Rabe, T., Sparg, S.G., Jager, A.K., Eloff, J.N. and Van Staden, J. (2001) An investigation on the biological activity of Combretum species. J Ethnopharmacol 75, 45-50.
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Moshafi, M.H., Mehrabani, M. and Zolhasab, H. (2004) Antibacterial activity studies of Salvia mirzayanii and Salvia atropatana against six standard gram-positive and gram-negative bacteria. J Kerman Univ Med Sci 11, 109-118.
Mozafarian, V. (2009) Dictionary of Iranian plant names (Latin English Persian). Tehran: Farhang Moaser Publication.
Naeini, A., Khosravi, A.R., Chitsaz, M, Shokri H., Kamlnejad, M. (2009) Anti-Candida albicans activity of some Iranian plants used in traditional medicine. J Mycol Med 19, 168-172.
Oskay, M. and Sarı, D. (2007) Antimicrobial Screening of Some Turkish Medicinal Plants. Pharm Biol 45, 176-181.
Pereira, R.S., Sumita, T.C., Furlan, M.R., Jorge, A.O. and Ueno, M. (2004) Antibacterial activity of essential oils on microorganisms isolated from urinary tract infection. Rev Saude Publica 38, 326328.
Rajani, M., Saxena, N., Ravishankara, M.N., Desai, N., and Padh, H. (2002) Evaluation of the antimicrobial activity of ammoniacum gum from Dorema ammoniacum. Pharm Biol 40, 534-541.
Rechinger, K.H. (1982) In Flora Iranica, No. 150, Rechinger KH (ed). Graz, Austria: Akademische Druck-u. Verlagsanstalt.
Sajjadi, S.E., Mehregan, I., Khatamsaz, M. and Asghari, G.H. (2005). Chemical composition of the essential oil of Perovskia abroatanoides Karel growing wild in Iran. Flav Frag J 20, 445-446.
Semnani, M.K. (2004) The essential oil composition of Perovskia abroatanoides from Iran. Pharm Biol 42, 214-216.
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Accepted Article
Sharififar, F., Moshafi, M.H., Mansouri, S.H., Khodashenas, M. and Khoshnoodi, M. (2007) In vitro evaluation of antibacterial and antioxidant activities of the essential oil and methanol extract of endemic Zataria multiflora Boiss. Food Control 18, 800-805.
Toyang, N.J., Ateh, E.N., Keiser, J., Vargas, M., Bach, H., Tane, P., Sondengam, L.B., Davis, H., Bryant, J. and Verpoorte, R. (2012) Toxicity, antimicrobial and anthelmintic activities of Vernonia guineensis Benth. (Asteraceae) crude extracts. J Ethnopharmacol 144, 700-704.
Venugopala, K.N., Rashmi, V. and Odhav, B. (2013) Review on natural coumarin lead compounds for their pharmacological activity. Biomed Res Int 2013, 1-14.
World Health Organisation. (2013) Traditional Medicine Strategy 2014-2023, pp. 23-28. Hong Kong SAR, China.
Zargari, A. (2011) Medicinal plants. Tehran: Tehran University of Medical Sciences.
List of tables Table 1. Selected plants for in vitro antibacterial assay and their traditional uses. AP: aerial parts, F: flower, ST: stem, L: leave, R: root, SE: seed, T: thallus. Table 2. Minimum inhibitory concentration (MIC) of 44 methanolic extracts (mg.ml-1) F: flower, AP: aerial parts, ST: stem, L: leave, R: root, SE: seed, T: thallus. MIC (µg.ml-1) of positive controls: gentamicin, S: ≤4, R: >8; vancomycin, S: ≤4, R: >16; amoxicillin, S: ≤4, R: >16; Amphotericin B, S: ≤1, R: >4. Table 3. The MIC and MBC of selected extracts determined by micro-dilution assays (µg.ml-1).
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Accepted Article
Table 1.
Family
Extraction
Lamiaceae
Plant species
Local name
Used Parts
Phlomis olivieri Benth.
Mahroo
F, AP, ST, L
Yields (%)
Selected traditional uses in relation with possible anti- Voucher infectious effect
7.5, 6.6, 8.4, Antiseptic
1612-ACECR
5.9 Phlomis persica Boiss.
Goshboreh irani
AP
8.9
Phlomis Boiss.
Givej Belgeh
AP, L, R
14.6, 9.3
Perovskia artemisioides Brazembel Boiss.
ST+L
10.2
Perovskia abrotanoides Brazembel Karel.
AP
8.8
anisodonta
Teucrium Boiss.
persicum
Treatment discomfort
of
digestive
13.2, Treatment discomfort
of
digestive
Anti-leishmaniasis
1610-ACECR
1634-ACECR
271-ACECR
Anti-leshmaniasis, MPH-1964
treatment of fever 9.6
Treatment of sore eyes
397- ACECR
ST+L
8.1
Antiseptic
288-ACECR
ST+L
3.9
Air freshener and purifier
1560-ACECR
Zataria multiflora Boiss. Avhishan Shirazi
L
9.6
Treatment of respiratory FG-1874 and digestive discomfort
Dracocephalum kotschyi Zarringiah Boiss.
AP
12.1
Treatment of fever
MPH-1631
MaryamGoli Banafsh
R
4.1
Treatment of skin diseases
1599-ACECR
MaryamGoli
L
9.4
Antiseptic, disinfection
244-ACECR
Maryam-goli boland
ST+F
18.3
Digestive
MPH-1654
MaryamGoli Sahandi
AP
7.5
Antiseptic, indigestion
MaryamGoli Isphehani
ST+F
6.9
Treatment discomfort
Satureja Jamzad.
Maryam nokhodi AP
khuzistanica Marzeh khuzistani
Hymenocrater longiflorus Benth.
Salvia verticillata L.
Arvaneh
Salvia chloroleuca Rech.f. & Aell Salvia hypoleuca Benth. Salvia sahendica Boiss. & Buhse. Salvia reuteriana Boiss.
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relieving
of
digestive
MPH-1592
MPH-231
Apiaceae
Accepted Article
Salvia Hedge.
lachnocalyx Maryam-goli eghlidi
AP
14.5
Antiseptic, disinfection
MPH-674
Mourporzoo
AP
12.1
Treatment discomfort
FG-1825
Salvia syriaca L.
MaryamGoli Soori
ST+F
9.2
Antiseptic
MPH-343
Salvia ceratophylla L.
MaryamGoli shakhgavazn
AP
11.5
Antiseptic
MPH-1051
Salvia urmiensis Bung.
MaryamGoli urumiehie
AP
8.8
Antiseptic
MPH-1220
Gichoo
R, SE
16.3, 23.5
Treatment of respiratory 6722-TEH discomfort
R
7.8
Boiss. & Buhse.
Kamaye ghalafdar
Treatment of respiratory 42-ACECR disorders
Ferula gummosa Boiss.
Barijeh
SE
30.0
Antiseptic, treatment digestive discomfort
Ferula assa-foetida L.
Anghozeh
SE
18.8
Treatment of influenza
FG-0112
Ferulago Boiss.
Chavil
SE
29.1
Antiseptic
FG-0171
Chenoor
AP, ST, R
13.5, 10.3
Chavir
ST
15.7
Air carminative
Hasaseh
ST
6.8
Antiseptic
1584 -ACECR
Havij vahshi
AP
10.0
Antiseptic
6734-TEH
Khosharizeh
ST
11.5
Anti-mold, freshener
FG-0252
Vashagh
SE
16.2
Treatment of respiratory FG-0125 disorders
Salvia mirzayanii Rech. f. & Esfand.
Ferula hezarlalezarica Ajani. Ferula oopoda
contracta
Ferulago bernardii Tomk. & M. Pimen. Ferulago angulata (Schlecht) Boiss. Zeravschanica pauciradiatum (Tamamsch). M. Pimen.
5.5, Treatment discomfort
of
of
digestive
of
digestive
disinfection,
FG-0115
1562-ACECR
1633-ACECR
Daucus littoralis Smith Subs. Echinophora DC.
platyloba
Dorema ammoniacum D. Don
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Dictyotaceae
Accepted Article
Asteraceae
Achillea tenuifolia Lam.
AP
7.1
Treatment inflammation
SE
7.4
Treatment of hepatitis
FG-0335
Jolbak Ghahveii
T
5.6
Antiseptic
44-14P
Golkhatmi
F
12.1
Treatment of respiratory FG-1152 and digestive discomfort
AP
7.3
Treatment of skin diseases
Bomadaran
Silybum marianum (L.) Kharmaryam Gaertn.
of
skin
1604-ACECR
Padina boergesenii Allender & Kraft
Malvaceae
Alcea rosea L.
Euphorbiaceae
Euphorbia microsciadia Forfion Denaii Boiss.
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FG-1652
Accepted Artic Table 2.
Plant
MIC (mg.ml-1) Gram negative bacteria
Gram positive bacteria
Yeast
Used C. albicans
Enterococcus sp.
Corynebacter -ium
S. warneri
S. lugdunensis
S. epidermidis
S. aureus
M. smegmatis
E. faecalis
E. aerogenes
E. cloacae
C. freundii
A. baumanii
S. maltophilia
S. marcescens
Salmonella sp.
P. stuartii
P. mirabilis
P. aeruginosa
parts
K. pneumoniae
E. coli
species
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
F
10
10
-
-
-
1.2
5
5
2.5
10
2.5
10
10
10
0.6
2.5
5
5
5
5
10
10
10
10
10
1.2
1.2
1.2
1.2
0.6
0.6
1.2
1.2
10
10
5
AP
10
10
10
-
10
1.2
2.5
2.5
2.5
10
2.5
10
10
10
