Türkiye 3. Cam Recınesı Kimyasal Prosesleri ve Isletmeciligi Uluslararası Sempozyumu, Ege Univ. 19-21 Kasim 1998, Izmır

Antimicrobial Activities of Several Parts of Pinus brutia and Pinus nigra

M. Hakki ALMA

Department of Industrial Eng. of Forestry, Kahramanmaras Sütçü Imam University Faculty of Forestry, 46100-Kahramanmara?, TURKEY

Metin DIGRAK Ahmet ILCIM

Department of Biology, Faculty of Arts and Science, Kahramanmaraş Sütçü İmam University, 46045-Kahramanmaraş, TURKEY

ABSTRACT

In this study, the antimicrobial activities of several parts of two pine species (Pinus brutia Ten. and Pinus nigra Arn.) grown in Kahramanmaraş region (Turkey) were investigated by using disc diffusion method. Chloroform, acetone and methanol extracts of leaves, resins, barks and cones of P. brutia Ten. and P. nigra Arn. were prepared and tested against Bacillus megaterium DSM 32, Bacillus subtilis IMG 22, Bacillus cereus FMC 19, Escherichia coli DM Klebsiella pneumoniae FMC 3, Enterobacter aerogenes CCM 2531, Staphylococcus aureus Cowan 1, Mycobacterium smegmatis RUT, Proteus vulgaris FMC 1, Listeria monocytogenes Scoot A, Pseudomonas aeruginosa DSM 5007, Candida albicans CCM 314, Candida tropicalis MDC 86, and Penicillium italicum K.

The results showed that antifungal effects were not observed for the whole extracts E. coli could not be inhibited by any of the plant extracts except the chloroform extracts of the bark of P. brutia and methanol extracts of the leaf of P. nigra which showed inhibition zones of 8-10 mm, respectively. All the plant extracts used in this study inhibited the development of the other bacteria studied. When compared the results found in this study to ampicillin standard, it was determined that microorganisms studied were, generally, susceptible, intermediate and resistant to the extracts of species when compared to ampicillin standard.

Keywords: Pinus brutia, Pinus nigra, antimicrobial activity

INTRODUCTION

A number of plants in the flora of Turkey have been used as traditional medicines. However, most of them have not been evaluated scientificall, and their effects have not been explained experimentally yet. About 25% of the medicines used for people’s health have been obtained from these plants so far (1).

In Turkey, five pine species (Pinus nigra, P. sylvestris, P. brutia, P. pinae and P. halepensis) are indigenous. Different parts of these species are used for the same purposes regardless of species. Due to their tannin contents, the dried barks have been used as a tanning agent and as an infusion to produce constipation effect. An essence obtained from the leaves of pines contain compounds such as pinene, cadinene, terpineol, and bornyl acetate. They can also be used as a mucus remover and an antiseptic. Pinus brutia and P. nigra species contain compounds such as a -pinene, ????????????????????????????????????????????????In this study, the microbial properties of several parts of various tree species peculiar to Kahramanmaraş region were investigated.

MATERIAL AND METHODS

Materials

The several parts (leaves, cones, resins and barks) of two pine species (Pinus brutia Ten,and Pinus nigra Arn.) used in this study were collected from the region of Başkonuş (Kahramanmaraş-Turkey). The species collected were authenticated according to conventional method (3). The collected parts were ground by using a blender. The resins from P. brutia were directly extracted with several solvents. The solvents selected (chloroform, acetone, and methanol; Merck, Darmstadt) were used without further purification (4,5).

Microorganisms tested in this study were provided from the culture collections of Microbiology Laboratory of Science & Art Faculty of the University of Fırat, in Elazığ-Turkey. In the study, Bacillus megaterium DSM 32, Bacillus subtilis IMG 22, Bacillus cereus FMC 19, Escherichia coli DM Klebsiella pneumoniae FMC 3, Enterobacter aerogenes CCM 2531, Staphylococcus aureus Cowan 1, Mycobacterium smegmatis RUT, Proteus vulgaris FMC 1, Listeria monocytogenes Scoot A, and Pseudomonas aeruginosa DSM 50071 bacteria, and Candida albicans CCM 314, Candida tropicalis MDC 86 and Penicillium italicum K fungi were used.

Methods

The collected parts of the species mentioned above were identified and broken into pieces, and aliquats (20 g) were separately extracted with chloroform, acetone, and methanol solvents (150 ml) for 24 h using soxhlet equipment (6,7). The extracts thus obtained were injected into empty sterilized antibiotic discs having a dimeter of 6 mm (Schleicher & Shüll No: 2668, Germany) in the concentration of 483 mcg/disc. The discs injected with only chloroform, acetone and methanol alone were used as control (8,9,10). Moreover, standard antibiotic discs such as ampicillin and nystatin used for comparison were provided from Microbiology Division of Medicine Faculty of Sütçü İmam University in Kahramanmaraş-Turkey.

Preparation of Microorganism Cultures

All the bacteria mentioned above were incubated at 30±0.1 °C for 24 h in Nutrient Broth (Difco), and the yeasts studied were incubated in Malt Extract Broth (Difco) for 48 h. Müeller Hinton Agar (oxoid) sterilized in a flask and cooled to 45-50 ⁰C was distributed (15 ml aliquats) to the sterilized petri dishes having a diameter of 9 cm, after inoculating with (0.01 ml) cultures of bacteria [The dilution plate method was used to enumerate microorganisms] (10⁵ bacteria per ml) and yeasts (10³-10⁴ yeasts per ml) for 24 h (11). The discs injected with extracts were located on the solid agar medium by pressing slightly. The petri dishes were left at 4 ⁰C for 2 h, and then plates injected were incubated at 30 ± 0.1 ⁰C for 24 h (8,9,12,13). At the end of the period, inhbition zones were measured in millimeter (mm).

RESULTS AND DISCUSSION

Table 1 depicts the antibacterial and antifungal activities of the chloroform, acetone, and methanol extracts of the bark, resin, and cone of Pinus brutia. As shown in this table, the extracts of the parts of the tree have no antifungal effect. The chloroform extract of the bark inhibited the growth of E. coli, with an inhibition zone of 8 mm. All the extracts tested inhibited growth of all the other bacteria tested with zones of inhibition between 9-19 mm. These results are in good agreement with those found in this study. Some differences determined in the results are thought to result from existing some different chemical components in the plant species and bacteria studied.

Table 1. The antimicrobial activities of chloroform, acetone, and methanol extracts of barks, resins, and cones of Pinus brutia (Inhibition zone, mm)

^aCh: Chloroform; ^bAc: Acetone; ^cMe: Methanol; ^dA 10: Ampicillin 10 mcg; ^eNST: Nystatin 30 mcg; ^fNT: Non Tried; ^g(-): No inhibition zone

Control (Acetone, Methanol and Chloroform) : - (No inhibition zone)

Bagci and Digrak (14) showed that the essential oils of A. nordmanniana subsp. nordmanniana and A. nordmanniana subsp.equ-i trojani had antimicrobial effects on the B. megaterium, B. subtilis, B. brevis, P. aeruginosa, L. monocytogenes, except for S. aureus and E. coli, at different ratios. As well as, they didn’t inhibit the developments of S. cerevisiae and C. albicans. Moreover, Digrak and Bagci (15) reported that the essential oils of Picea pungens cv. glauca and Cedrus libani had no antibacterial effect against E. coli. They were also found to be effective on the above-mentioned bacteria at different ratios.

Table 2 shows the antimicrobial effects of several solvent-based extracts of leaves, cones, and barks of Pinus nigra, consisting of the most important parts of Turkey’s Forests, on various bacteria and fungi. As can easily be seen from this table, the leaf and cone extracts of this species don’t inhibit the gorwth of E. coli. Yet, chloroform extract of cone is found to inhibit the growth of E. coli having an inhibition zone of 10 mm. It is also determined that chloroform and acetone extracts inhibit the growth of L. monocytogenes and P. aeruginosa. Table 3 shows the antimicrobial activities of chloroform, acetone and methanol solvents used as control.

Table 2. The antimicrobial activities of chloroform, acetone, and methanol extracts of leaves, cones and barks of Pinus nigra (Inhibition zone, mm).

* Zone Diameter Interpretive Standards

____________________________________________________________________

Standard Antibiotics Resistant (mm) Intermediate Susceptible (mm)

or less (mm) or more

____________________________________________________________________

¹Ampicillin 10 mcg 11 12-13 14

²Nystatin 30 mcg 12 13-15 16

____________________________________________________________________

¹The bacterium studied is Staphylococcus aureus ATCC 25923

²The fungi studied is Candida albicans CCM 314

As compared with the results shown in Table 1, it was seen that the chloroform and acetone extracts of P. brutia are, generally, moderately sensitive and resistant. The bacteria or fungi studied are found to be susceptible, intermediate and resistant to the extracts of P. nigra in comparison to ampicillin standard.

The results showed that like the similar pine species studied before, the species studied in this work have not any antifungal activities, however, they have do have antibacterial activities. The differences observed can arise from the differences observed in the chemical composition of the species. It is shown that different parts of the species possess antibacterial activities.

Table 3. The antimicrobial activities of chloroform, acetone, and methanol (Control) Inhibition zone, mm.

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