Pharmaceutical Biology 2002, Vol. 40, No. 02, pp. 135–138
1388-0209/02/4002-135$16.00 © Swets & Zeitlinger
Antimicrobial Activity of the Tar Obtained from the Roots and Stems of Pinus brutia Murat Kızıl, Göksel Kızıl, Murat Yavuz and Çetin Aytekin Department of Chemistry, Faculty of Science and Art, University of Dicle, Diyarbakır, Turkey
Abstract The antimicrobial activity of the crude and methanol, hexane, chloroform, petroleum ether, ethyl acetate and nbutanol extracts of tar obtained from the roots and stems of Pinus brutia Ten., used in the folk medicine of Turkey, were screened in vitro for antimicrobial activity against clinically isolated bacterial strains: Staphylococcus aureus (15 isolates), Streptococcus pyogenes (12 isolates), Escherichia coli (17 isolates) and the yeast fungus Candida albicans (3 isolates) by the paper disc diffusion method. Results revealed that a crude extract of the tar is highly effective against all tested microorganisms by preventing their growth to a significant extent. All the organic extracts showed similar moderate antimicrobial activity against tested microorganisms at a concentration of 80 mg/mL. The results demonstrate that the crude tar extract has a very broad spectrum of activity, and suggests that it may be useful in the treatment of various microbial infections. The results suggest that traditional folk medicine could be used a guide in our continuing search for new natural products with potential medicinal properties.
Introduction The development of microbial resistance towards antibiotics leads to the necessity of searching for new potentially effective compounds against pathogenic microorganisms. One of the most promising targets in the search for new biologically active compounds is plants used in folk medicine, many of which have never been investigated for their chemical composition or pharmacological activity. In the past, medicinal plants were the main source of medicaments for humans. According to the World Health Organisation (WHO), around 80% of the worlds’ population in developing countries rely on traditional plant medicines for their primary health care needs, of which a major portion involves the use of plant
extracts or their active principles (Fransworth & Soejarto, 1985). Many studies have shown that plant extracts have the ability to inhibit the growth of many pathogenic organisms (Mitrokosta et al., 1993; Kelmanson et al., 2000; Yes¸ilada et al., 1999). Pinus brutia Ten., Pinaceae, is a Mediterranean pine belonging to the Slyvestris subsection (section Pinus, subgenus Pinus) of the genus Pinus (Little & Critchfield, 1969). Pinus brutia, strictly localised in the eastern part of the Mediterranean, is found from Greece to Lebnanon and Iraq. Tar is a resinous exudate obtained from the stems and roots of Pinus brutia. The tar obtained from Pinus brutia has been made since the time of Dioscorides by the inhabitants of the inner Taurus Mountains of South Anatolia (Gunther, 1934). In this region, tar is utilised for the relief of upper abdominal discomfort, infections of the skin of humans and animals, respiratory tract diseases and urinary system infections. In this study, extracts made of tar used in traditional medicine for various ailments such as respiratory tract diseases and urinary system infections, which could indicate a possible antimicrobial property, were tested for antimicrobial activity.
Materials and methods Plant materials The collection sites of plant materials used in this study are given below. Pinus brutia Ten. (Pinaceae) stems and roots were collected in June 1999 from the region of (Mersin, Anamur), Turkey (No: DUF 9851). They were botanically identified by Dr. A. Selcuk Ertekin from the same institution. Voucher specimens are deposited in the Herbarium of Dicle
Accepted: September 21, 2001 Address correspondence to: Murat Kızıl, Department of Chemistry, Faculty of Science and Art, University of Dicle, 21280 Diyarbakır, Turkey. Fax: +90 412 2488039, E-mail:
[email protected]
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University, Faculty of Science and Art, Department of Biology (Diyarbakır, Turkey). Test organisms The panel of test organisms developed for the study were well-characterised recent clinical isolates and included the following: Staphylococcus aureus (15 isolates), Streptococcus pyogenes (12 isolates), Escherichia coli (17 isolates) and the yeast fungus Candida albicans (3 isolates). Escherichia coli DH1 was used as a reference strain for control of MullerHinton Agar (Difco laboratories, Detroit, Mich.) and antibiotic discs. The test organisms were obtained from the Microbiology Department at the University of Dicle (Diyarbakır, Turkey). The isolates were held frozen at -70 °C in Muller-Hilton Broth (Difco laboratories, Detroit, Mich.) containing 17% glycerol (Fisher Scientific). Preparation of the extracts Three g of finely powdered tar was kept at room temperature with 75 mL of methanol for 2 h, the insoluble ingredients were filtered and the solvent was evaporated to dryness under reduced pressure to give a brown solid (2.78 g, methanol extract). 0.4 g of the dry methanol extracts were diluted with 30 mL distilled water and extracted with ethyl acetate (2 ¥ 15 mL). The organic extracts were dried over magnesium sulphate, filtered and evaporated to dryness to give a brown solid (37 mg, ethyl acetate extract). The above extraction procedure for ethyl acetate was successively repeated with the same amount of the dry methanol extracts (0.4 g) with petroleum ether, n-hexane, n-butanol and chloroform. The organic extracts were obtained as follows: petroleum ether extract as a brown oil (6.7 mg); n-hexane extracts as a brown oil (8.7 mg); n-butanol extract as a brown oil (19.2 mg) and chloroform extract as a yellow solid (33.8 mg). Preparation of stock tar solutions Stock tar solutions were prepared by dissolving crude tar in ethanol (95%) at a concentration of 4 mg/mL. For each organic solvent extract, 4 mg/mL stock solutions were prepared in ethanol. Stock solutions were sterilised before use by passage through a 0.22 mm-pore size polysulfone membrane filter (Gelman Science, Ann Arbor, Mich.). The stock solutions were tested for antimicrobial activity within 10 days after preparation. Preparation of disks Paper discs (Oxoid) with a diameter of 6 mm containing 40, 60 and 80 mg of samples were prepared as described below. Forty microgram disks were prepared by pipetting 10 mL volumes of stock solutions of crude tar (4 mg/mL) onto sterile blank disk, while 60 and 80 mg disks were similarly prepared by using 15 and 20 mL of stock solution of crude
tar and organic extract solution of tar, respectively. The disks were dried and then stored at 4 °C until use within 5 to 10 days. A disk containing solvent extracts of tar applied to inoculated plates by using flamed forceps. Ethanol was applied to paper discs as a negative control. Antimicrobial activity Antimicrobial activity was assayed by the disc diffusion susceptibility test according to the recommendations of the National Committee for Clinical Laboratory standards (NCCLS) (Appelbaum et al., 1998). The disk diffusion test was performed on Muller-Hinton Agar plates. Plates were dried at 35 to 36 °C for about 30 min in an incubator before inoculation. Three to five freshly grown colonies of bacterial strains were inoculated into 25 mL of Muller-Hinton Broth medium in a shaking water bath for 4 to 6 h until a turbidity of 0.5 McFarland (1 ¥ 108 CFU/mL) was reached. Final inocula were adjusted to 5 ¥ 105 CFU/mL. Three to five colonies of C. albicans were inoculated into 25 mL of Sabouraud Dextrose Broth (Oxoid) in shaking water bath for 8 to 10 h until a turbidity of 0.5 McFarland standard was reached. The final inocula were adjusted to 5 ¥ 105 CFU/mL by using a spectrophotometer (Patterson et al., 1998). The inoculum (100 mL) from the final inocula was applied to each agar plate and uniformly spread with a sterilised cotton spreader over the surface. Absorption of excess moisture was allowed to occur for 10 min before application of dried paper discs with a diameter of 6 mm containing 40, 60 and 80 mg of the crude tar and organic solvent extracts of tar to be assayed. They were deposited on Muller-Hinton agar plates, except for C. albicans, which was deposited on Sabouraud Dextrose Agar plates. The plates were incubated at 37 °C and the zones of inhibition were measured after 24 h for bacteria and 48 h for the yeast fungus C. albicans. Antibiotic discs (all from Oxoid) containing imipenem (IPM, 10 mg/disc), amoxycillin (AML, 25 mg/disc), erythromycin (15 mg/disc) and ampicillin/sulbactan (SAM, 10/10 mg/disc) were used as a positive controls. For each experiment, ethanol was applied to paper discs as a negative control. The crude tar and the organic extracts were dissolved in ethanol.
Results Table 1 shows the results of the antibacterial activity of the standard antibiotics against tested microorganisms. The ethanol (15 mL) negative control showed no inhibiting effect. After each organic solvent extraction, the remaining aqueous extracts were tested against bacteria and fungi and did not show any antibacterial and antifungal activity (data not shown). All the clinical isolates, S. aureus, S. pyogenes, P. aeruginosa and E. coli were susceptible to imipenem. Fifteen (100%) of the S. aureus were resistant to erythromycin. Four (26.6%) isolates of the S. aureus were resistant to amoxycillin. Eleven (73.3%) of the 17 isolates of S. aureus had zone
Antimicrobial activity of Pinus brutia
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Table 1. Results of standard antibiotics.a Zones of inhibition in mm Tested organisms Staphylococcus aureus Streptococcus pyogenes Pseudomonas aeruginosa Escherichia coli Candida albicans a
I (10 mg/paper disc)
E (10 mg/paper disc)
SA (10/10 mg/paper disc)
A (25 mg/paper disc)
30 (100.0) 30 (100.0) 30 (100.0) 28 (100.0) NT
R (100.0) R (41.6) ≥ 23 (46.6) R (100.0) R (88.2) 18 (11.7) NT
14 (20.0) ≥ 15 (80.0) R (16.6) 16 (83.3) R (66.6) 20 (33.3) R (82.3) 18 (17.6) NT
R (26.6) ≥ 18 (73.3) R (50.0) 28 (50.0) R (66.6) 22 (33.3) R (82.3) 20 (17.6) NT
I: imipenem; E: erythromycin; SA: ampicillin/sulbactan; A: amoxycillin; NT: not tested; (%): activity no.; R: resistance.
Table 2. Antimicrobial activity of crude and organic extracts of the tar. Zones of inhibition in mm* Tested organisms Streptococcus pyogenes
mg/paper disc
CR
Em
Ee
Ep
Eh
Ec
Eb
40
12–14 (50.0) 18–20 (33.3) ≥24 (16.6) 14–16 (50.0) 22–24 (41.6) 28 (8.3) 24–26 (66.6) ≥30 (33.3) 12–14 (100.0) 14–16 (100.0) 16–20 (93.0) ≥24 (6.6) – (100.0) 10–12 (100.0) 12–14 (100.0) 12–14 (100.0) 12–14 (88.2) 14–18 (11.7) 18–20 (100.0) 14 (100.0) 16 (100.0) 18 (100.0)
–
–
–
–
–
–
10
10
10
10
10
–
14
16
16
14
15
10
10 15 16
– 14 16
12 12 16
– 14 26
– 12 16
– 10 14
12 14 14 – –
10 10 16 – –
– 10 15 10 12
– 14 15 – –
10 14 14 – 10
– 14 14 – 10
12 – 10 12
12 – – 12
14 – 14 18
12 14 16 22
12 – 10 10
14 – 20 20
60
80 Staphylococcus aureus
40 60 80
Pseudomonas aeruginosa
40 60 80 40 60
Escherichia coli
Candida albicans
80 40 60 80
CR: crude tar; m metanol extract; e ethyl acetate extract; p petroleum ether extract; h hexane extract; c chloroform extract; b n-butanol extract; –: not active; (%): activity no. * Organic extracts were tested on one isolate from each microorganism.
diameters of ≥18 mm with amoxycillin. Of the 15 isolates of S. aureus, 12 (80%) exhibited zone diameters of ≥15 mm with ampicillin/sulbactan. Of the 12 isolates of S. pyogenes, 5 (41.6%) of isolates were resistant to erythromycin, 2 (16.6%) resistant to ampicillin/sulbactan and 6 (50%) resistant to amoxycillin. The antimicrobial studies showed that the crude extract of the tar appeared to be the most active of all the extracts against all tested bacteria and yeast (Table 2). At the 80 mg/mL concentration, the crude tar extracts were found to be highly active against S. pyogenes, S. aureus, P. aeruginosa
and E. coli with zone diameters of ≥30 (33.3%), ≥24 (16.6%), 12–14 (100%) and 18–20 (100%), respectively. The crude tar was also active against C. albicans at 80 mg/mL concentration with zone diameter of 18 mm. The antimicrobial activity of the crude extract of the tar was similar to that of imipenem for S. pyogenes and S. aureus at an 80 mg/mL concentration, but it was more active than erythromycin. Of the 12 isolates of P. aeruginosa, 12 (100%) were found to be resistant to erythromycin, 8 (66.6%) resistant to ampicillin/sulbactan and 8 (66.6%) resistant to amoxycillin. On the other hand, the crude extract inhibited the growth of P.
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aeruginosa with a zone diameter ranging from 12–14 mm. The crude tar extract showed an inhibition diameter ranging from 12 to 14 mm at 40 mg/mL against S. pyogenes, S. aureus, E. coli and C. albicans. No activity was found with the crude tar extract at 40 mg/mL against P. aeruginosa. No significant differences were evident between any of the organic extracts regarding antibacterial activity. The hexane extract of the tar showed the greatest inhibition zone against S. aureus and C. albicans. The zone diameters were 26 and 22 mm, respectively. A similar inhibition zone size was observed for the standard antibiotics imipenen and amoxycillin. Methanol, ethyl acetate, petroleum ether, chloroform and buthanol extracts showed almost the same activity against tested microorganisms with zone diameter ranges of 10–16 mm at 80 mg/mL. All the organic extracts were found to have a low activity against tested microorganisms at concentrations of 40 and 60 mg/mL (inhibition diameters ranged from 10 to 14 mm).
Discussion The current study describes the antimicrobial activity of tar obtained from the stems and the roots of Pinus brutia, on a panel of clinical isolates of bacteria and yeast by the disc diffusion method. This study shows that the crude extracts of the tar of Pinus brutia exhibited high activity and all organic extracts shows similar moderate antimicrobial activity against the tested microorganisms, suggesting that activity could be due to more than one compound. The flavonoids, terpenoids and alkaloids present in the plant appear to be responsible for biological activity (Roussis et al., 1995; Gallis et al., 1998). It is noteworthy that the crude tar was active against clinical isolates of S. aureus, E. coli, P. aeruginose and S. pyogenes which are resistant to erythromycin, ampicillin and ampicillin/sulbactan. These bacteria are major clinical pathogens. Over the past decade, S. aureus has developed resistance to many commonly used antibiotics (Marananan et al., 1997). Many conifers are reported to have antibacterial activities. For example, several parts of Pinus brutia, Juniperus oxycedrus L., Cupressaceae, Abies cilicica (Antoine and Kotschy) Carriere, Pinaceae Cedrus libani A. Rich., Pinaceae and Pinus nigra J. F. Arnold, Pinaceae (Dıgrak et al., 1999). The demonstrated antibacterial activity of Pinus brutia extracts against the tested microorganisms is of particular significance in that these pathogens are primarily responsible for numerous infections and, hence, this provides a rational basis for therapeutic use of these traditional plant medicines. Further investigations on purification, mode of action, stability, safety and effectiveness of the purified compounds
are necessary for the development and clinical use of these plants.
Acknowledgements This work is dedicated to Professor Yavuz Ensari, on the occasion of his death. The authors would like to thank Dr. Selahattin Atmaca for kindly providing some of clinical isolates tested.
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