|Iranian Journal of Pharmaceutical Research
((2008), 7 (4): 291-295
Received: March 2008
Accepted: May 2008
Copyright ? 2008 by School of Pharmacy
Antibacterial Activity of Thymus pubescens Methanolic Extract
Hadi Mehrgana, Faraz Mojabb*, Shima Pakdamanc and Mahshid Poursaeedc,d
a,bSchool of Pharmacy and Pharmaceutical Sciences Research Center, Shaheed Beheshti University (M.C.), Tehran, Iran. cSchool of Pharmacy, Islamic Azad University, Tehran, Iran. dDeputy of Food and Drug Affairs, Shaheed Beheshti University (M.C.), Tehran, Iran.
During the last two decades, various medicinal plants have been studied for their possible antimicrobial activity to discover new antimicrobial agents capable of resolving problems such as the development of drug resistance in pathogenic microorganisms as well as the side effects of some present antibiotics. In this study, the antibacterial activity of methanolic extract of T. pubescens (rich in flavonoids) was investigated.
The aerial parts of the plant were collected from Alvand mountainside (Hamadan, Iran) in May 2005 and identified by Hamadan Natural Sources Organization. The air-dried plant materials were ground to fine powder and then extracted by soxhelet apparatus using methanol. The extract was tested at a concentration of 100 mg/ml against a panel of Gram-positive and Gram-negative bacteria using the disk diffusion technique. This methanolic extract demonstrated antibacterial activity against Gram-positive bacteria including Staphylococcus aureus, Methicillin-resistant S. aureus (MRSA), Streptococcus pyogenes, Enterococcus faecalis, Vancomycin-resistant E. faecalis (VRE) and Micrococcus luteus, and produced inhibition zones with 8-16 mm diameters. However, it showed no activity against Gram-negative bacteria, such as Escherichia coli, Pseudomonas aeruginosa and Salmonella spp. Minimum concentrations (MC) of the extract forming a clear zone were determined against susceptible bacteria. MC90 of the extract against 10 MRSA strains tested was 1.56 mg/ml, indicating its good activity against this important nosocomial pathogen. T. pubescens seems to be a good candidate for further phythochemical studies in an attempt to find new chemical entities combating resistant bacteria.
Infectious diseases are the second leading cause of death worldwide. Treatment of infections continues to be problematic in modern time because of the severe side effects of some drugs and the growing resistance to antimicrobial agents. Hence, search for newer, safer and more potent antimicrobials is a pressing need. Herbal medicines have received much attention as a source of new antibacterial drugs since they are considered as time-tested and comparatively safe both for human use and the environment (1).
Iran has a great deal of ecological diversity and has a rich herbal flora which is still much unstudied regarding phytochemistry and bioactivity. Lamiaceae (formerly Labiatae) is one of the most important plant families in which Thymus with about 215 species, is a significant genus (2).
Thymus species are commonly used as tonic, carminative, digestive, antitussive, expectorant and for the treatment of cold in Iranian traditional medicine. Recent studies imply that these species have strong antibacterial activities (3). Extracts of Thymus vulgaris of Greek origin were examined as potential sources of phenolic compounds and showed antimicrobial activity against selected microbes (4). In another research, aqueous and ethanolic extracts (10-200 mg/ml) of Thymus capitatus inhibited the growth of several bacteria and fungi (5). It is believed that flavonoids are responsible for these activities (6-9). They are known to be synthesized by plants in response to microbial infection (10). Analysis of the essential oil obtained from the aerial parts of T. pubescens revealed the main components to be carvacrol, thymol, α-terpineol and p-cymene (11). Abundantly distributed among the edible plants, flavonoids seem to include some of the most potent next generation drugs for the treatment of infections, since some of them possess unique antibacterial potency. Therefore, in the present study, T. pubescens methanolic extract, being rich of flavonoids (12), was evaluated for its antibacterial activities against the standard and clinical isolates of various Gram-positive and Gram-negative bacteria.
The aerial parts of T. pubescens Boiss. & Kotschy ex Celak (syn. T. xylorrhizus Boiss. & Kotschy) (Lamiaceae) were collected from the Alvand mountainside (Hamadan, Iran) in May 2005 and identified by Hamadan Natural Sources Organization. The plant parts were air-dried and finally ground to a fine powder.
Extraction of plant material
The dried ground material was extracted by Soxhelet apparatus using methanol (Merck). The extract was filtered and its solvent removed under reduced pressure to produce a thick dark green paste. The resulting extract was kept in a sterile vial in a dark and cold place for further tests.
Various Gram-positive and Gram-negative bacteria including both standard and clinical isolates were used as test strains. Standard strains were obtained from MAST (MAST Ltd., UK) or Persian Type Culture Collection, Iranian Research Organization for Science and Technology (PTCC, Iran). Clinical methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VRE) were isolated from patients in Milad Hospital (Tehran, Iran). The organisms were maintained on soybean casein digest agar (SCDA) and transferred onto fresh slants on a regular basis.
Antibacterial activity of the crude extract was investigated against 14 bacterial strains by the paper disk diffusion technique (13). The extract was redissolved in methanol to make a 100 mg/ml solution and then filtered. From this solution, 40-?l aliquots were transferred onto blank paper disks with a diameter of 6 mm (3). Dried disks were placed onto Mueller Hinton agar medium (Merck) previously inoculated with a bacterial suspension (ca. 108 CFU/ml) and incubated at 35?1 ?C for 24 h. Plates were then examined for the presence of growth inhibition zones, and diameters were measured, if any. Oxacillin disks (1 ?g) and vancomycin disks (30 ?g) (Padtan Teb, Iran) as well as ciprofloxacin disks (5 ?g) were used as positive controls, where appropriate. A disk loaded by 40 ?l methanol instead of the extract solution served as the negative control. The experiments were carried out four times and the results were presented as mean?SD.
Minimum concentration producing growth inhibition zone (MC)
MC of the extract was determined by testing 7 concentrations of the extract against Gram-positive bacteria, including 11 strains of MRSA, by the paper disk diffusion technique. The reconstituted extract was diluted to give concentrations of 50 mg/ml, 25 mg/ml, 12.5 mg/ml, 6.25 mg/ml, 3.12 mg/ml and 1.56 mg/ml. The lowest concentration of the extract that could inhibit the bacterial growth was considered as MC (3).
Results and Discussion
The inhibitory effects of methanolic extract of T. pubescens against different test organisms are shown in Tables 1 and 2. The extract indicated significant antibacterial activity (growth inhibition zone diameters ranging from 8 to 16 mm) against Gram-positive bacteria including S. aureus, MRSA, S. pyogenes, E. faecalis and VRE. Similar results have been reported for essential oils from T. pubescens (14) and aqueous-methanolic extracts of T. vulgaris (15). This strong antibacterial effect could be due to flavonoids, which have been shown to be active against MRSA (16, 17). On the other hand, the extract had no activity against Gram-negative bacteria. This could be related to the outer membrane acting as a permeability barrier in these bacteria (18). Inversely, essential oil of T. pubescens has been shown to have strong inhibitory effects on Gram-negative bacteria even in diluted forms (14).
Using the disk diffusion technique, minimum concentration of the extract producing a clear zone was also determined using the disk diffusion technique, which was 100 mg/ml. For E. faecalis ATCC 29212 and VRE, methanolic extract could inhibit the growth only at 100 mg/ml; however, the extract could effectively inhibit other test bacteria even at concentrations as low as 1.56-12.5 mg/ml. It was interesting to note that the MRSA strains were the most sensitive species to the investigated extract, with the MC90 against these isolates being 1.56 mg/ml. T. pubescens contains various compounds including terpenoids, tannins and polyphenolic compounds, as well as flavonoids. The latter being more likely to possess antimicrobial activity. Flavonoids? activity is probably due to their ability to form complex with extra-cellular and soluble proteins, as well as bacterial cell wall. lipophilic flavonoids may also disrupt bacterial membranes (19). Negative control did not show any growth zone. It is concluded that, separation of components of this extract and evaluation of their antimicrobial activity may lead into new agents against important nosocomial pathogens, such as MRSA.
This work was supported by Pharmaceutical Sciences Research Center (PSRC), Shaheed Beheshti University (M.C.), Tehran, Iran.
(1) Fazly-Bazzaz BS, Khajehkaramadin M and Shokooheizadeh HR.
In vitro antibacterial activity of Rheum ribes extract obtained from various
plant parts against clinical isolates of Gram-negative pathogens. Iranian J. Pharm. Res. (2005) 2: 87-91
(2) Zaidi MA and Crow SA. Biologically active traditional medicinal herbs from Balochistan, Pakistan. J. Ethnopharmacol. (2005) 96: 331-4
(3) Vila R. Flavonoids and further polyphenols in the genus Thymus. In: Stahl- Biskup E and Saez F (eds.) Thyme, Taylor and Francis, London (2002) 144-75
(4) Proestos C, Chorianopoulos N, Nychas GJ and Komaitis M. RP-HPLC analysis of the phenolic compounds of plant extracts: Investigation of their antioxidant capacity and antimicrobial activity. J. Agric. Food Chem. (2005) 53: 1190-5
(5) Kandil O, Radwan NM, Hassan AB, Amer AM, el-Banna HA and Amer WM. Extracts and fractions of Thymus capitatus exhibit antimicrobial activities. J. Ethnopharmacol. (1994) 44: 19-24
(6) Özç elik B, Orhan I and Toker G. Antiviral and antimicrobial assessment of some selected flavonoids. Z. Naturforsch. [C] (2006) 61: 632-38
(7) Maheswara M, Rao YK, Rao VM and Rao CV. Antibacterial activity of acylated flavonol glycoside from Waltheria indica. Asian J. Chem. (2006) 18: 2761-65
(8) Friedman M.Overview of antibacterial, antitoxin, antiviral, and antifungal activities of tea flavonoids and teas. Mol. Nutr. Food Res. (2007) 51: 116-34
(9) Cowan MM. Plant products as antimicrobial agents. Clin. Microbiol. Rev. (1999) 12: 564-82
(10) Dixon RA, Dey PM and Lamb CJ. Phytoalexins: enzymology and molecular biology. Adv. Enzymol. (1983) 55: 1-69
(11) Morteza-Semnani K, Rostami B and Akbarzadeh M. Essential oil composition of Thymus kotschyanus and T. pubescens from Iran. J. Essen. Oil Res. (2006) 18: 272-274
(12) Markham KR. Techniques of Flavonoid Identification. Academic Press, London (1982) 24-27
(13) Bauer AW, Kirby WMM, Sherris JC and Turck M. Antibiotic susceptibility testing by a standardized single disk method. Am. J. Clin. Pathol. (1966) 45: 493-96
(14) Rasooli I and Mirmostafa SA. Antibacterial properties of Thymus pubescens and Thymus serphyllum essential oils. Fitoterapia (2002) 73: 244-50
(15) Essawi T and Srour M. Screening of some Palestinian medicinal plants for antibacterial activity. J. Ethnopharmacol. (2000) 70: 343-49
(16) Sato Y, Shibata H, Arakaki N and Higuti T. 6, 7-dihydroxyflavone dramatically intensifies the susceptibility of methicillin-resistant or -sensitive Staphylococcus aureus to beta-lactams. Antimicrob. Agents Chemother. (2004) 48: 1357-60
(17) Xu HX and Lee SF. Activity of plant flavonoids against antibiotic-resistant bacteria. Phytother. Res. (2001) 15: 39-43
(18) Denyer SP and Russell AD. Non-antibiotic antibacterial agents: mode of action and resistance. In: Denyer SP, Hodges NA and Gorman SP. (eds.) Hugo & Russell?s Pharmaceutical Microbiology. 7th ed. Blackwell, Oxford (2004) 306-22
(19) Tsuchiya H, Sato M and Miyazaki T. Comparative study on antibacterial activity of phythochemical flavanones against methicillin-resistant Sthaphylococcus aureus. J. Ethnopharmacol. (1996) 50: 27-34