Effects of various extracts from Pistacia eurycarpa Yalt. on growth duration of Mycobacterium tuberculosis

1 Department of Medical Microbiology, Faculty of Medicine, Gazi University, Ankara, Turkey. 2 Department of Pharmacognosy, Faculty of Pharmacy, Gazi University, Ankara, Turkey. 3 Department of Field Crops, Faculty of Agriculture, Selcuk University, Konya, Turkey. 4 Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmacy, Eastern Mediterranean University, Famagusta, the Northern Cyprus.


INTRODUCTION
Tuberculosis control can be achieved if individuals with the disease receive adequate and timely treatment. Currently, the rapid diagnosis of tuberculosis has gained important public health significance due to the increasing incidence of tuberculosis and the advent of multidrugresistant Mycobacterium tuberculosis strains.
In 2011, 1.4 million people died from TB, including almost one million deaths among HIV-negative individuals and 430 thousand among people who were HIV-positive (World Health Organization, 2012;Lawn and Zumla, 2011;Lönnroth et al., 2010). In order to prevent the spread of tuberculosis and start the specific treatment immediately, rapid diagnosis is of a great importance. Presently mycobacterial culture method is the most accepted method in the diagnosis of tuberculosis. However, 3 to 6 weeks are required for the diagnosis by the conventional culture method. It is possible to reduce this period to 10 to 15 days using liquid culture systems (Cruciani et al., 2004;Winn et al., 2006). Diagnostic molecular microbiological methods which provide faster results with higher sensitivity and specificity are also used in the diagnosis of tuberculosis (Kaul, 2001). Unfortunately, there are various problems concerning these techniques (Lucke, 2011). Therefore, the *Corresponding author. E-mail:simsul@yahoo.com Tel: +905305401812. techniques which are more reliable, labor and cost effective, and easier to implement and providing faster results are needed.
Pistacia eurycarpa Yalt. is a member of Anacardiaceae family (Yaltirik, 1976;Kafkas, 2002). Various researches have shown that this plant, an endemic plant in Turkey, is rich in fat content and contains high levels of fatty acids (Demirci et al., 2001;Kafkas et al., 2007). It was determined that the extracts obtained from this plant have antimicrobial and antifungal effects. There are soaps which used in the area of cosmetics are made from P. eurycarpa oils (Kordali et al., 2003;Alma et al., 2001). Mycobacteria possess a complex lipid-rich cell wall therefore this could be a reason for the slow growth. Hence it is considered the extracts of P. eurycarpa that contains high levels of lipid could be increase the growth rate of mycobacteria. This study was an analysis on whether the addition of various extracts of P. eurycarpa into Löwenstein Jensen (LJ) and Middlebrook 7H11 agar (7H11) medium has an effect on the acceleration of growth rate of M. tuberculosis.

MATERIALS AND METHODS
A total of 38 clinical strains defined as M. tuberculosis complex and 2 standard strains of Mycobacterium tuberculosis (H37Rv, H37Ra) were included in this study. Twenty strains were isolated from the clinical specimens submitted to the clinical microbiology laboratory of Gazi Hospital and 18 strains were isolated from the clinical specimens in the Tuberculosis Laboratory of Refik Saydam Hygiene Center in Ankara, Turkey. Clinical specimens like sputum were processed using NALC-NaOH and cultured in BACTEC MGIT 960 tubes with PANTA. The samples taken from the tubes which detected positive for growth were stained. The bacteria identified as acid fast positive were re-cultured with and without NAP. The mycobacteria strains inhibited by NAP were accepted as M. tuberculosis complex (Winn et al., 2006;Babady and Wegnenack, 2012). The growth duration of every strain in LJ medium was measured and recorded. Effects of the plant extracts on the growth rate of M. tuberculosis were determined using LJ Medium (Biomerieux) and 7H11 (BBL Seven H11 Agar Base) with 10% fresh OADC added (Winn et al., 2006). The fruits belonging to the plant selected for the analysis were collected from the trees growing in vicinity of Siirt province in October, 2010. There is no requirement or ethical approval for this in vitro research.

Identifying fatty acid components of the plant
The peels and fruits of the plant were pounded in a mortar and its fixed oils were exposed to extraction for 6 h with n-hexane in the Soxhlet apparatus. After the organic phase was evaporated at 40°C in reduced pressure, the remaining portion was incubated for 10 min after adding 0.5 N methanolic sodium hydroxide reagent and then, was exposed to saponification.
Subsequently, for methyl trans-esterification reaction, 5 ml of 14% BF 3 with methanol was added to the oils obtained and concentrated by boiling in a hot water bath for 2 min. Five milliliter (5 ml) of n-hexane was added to this component and boiled for 1 min. It was left to cool down at room temperature and after addition of saturated NaCl. After cooling, it was put into 25 ml volumetric flask and completed to 25 ml with the addition of saturated NaCl and transmitted to separator funnel and then upper phase was removed for analysis (Kan et al., 2009).

Gas chromatography-mass spectrometry (GC-MS) analysis
GC-MS analysis was carried out in order to determine fatty acid methyl ester contents of the oil (Kan et al., 2009;Blomberg et al., 2002). Fatty acid composition analysis was achieved by Agilent 6890 Network GC system combined with Agilent 5975C VL MSD (Flame ionization detector, FID) detector and DB Waxetr column (60.0 mm × 0.23 mm × 0.25µm) was used. Helium with 3.3 ml/min flow rate and 1 µl injection capacity was used as the carrier gas. FID temperature was 250°C and initial flow rate was 3.1 ml/min. The MS parameters were applied under electron impact ionization (70 eV) with 35-450 atomic mass units.

Identifying the peaks
The retention times of the oils of the fruit and peel samples and mass weights of fatty acids that were obtained with GC, respectively were identified with GC-MS and a comparison was made using the mass spectra in Wiley and Nist databases for the detection of peaks. As an authentic sample, Fame mix Supelco-1891-1AMP that is composed of methyl esters of palmitate, stearate, oleate, linoleate, linolenate, and arachidic acid was used.

Preparation of peels and fruits of P. eurycarpa's water-soluble extract samples and addition to media
The water-soluble extracts of the peels and fruits of P. eurycarpa were obtained using the cold infusion technique. To achieve this, 15 g of each of the peel and fruit of P. eurycarpa were pounded in a sterilized mortar and 15 ml of distilled water was added in each and pounded for 15 min more. Water (45 ml) was added to each extract obtained and mixed for one hour and the upper liquid was sterilized using a 45 µm millipore filter (Singh, 2008). Various concentrations (2, 5, 8, and 10 µl ) of extracts were spread on the surface of the solid media and observed that 5 µl optimally filled the surface without overflowing and thus it was decided to add 5 µl for each application. Each of these extracts was taken for 5 µl and they were spread to surface of 7H11 and LJ media.

Preparation of the fixed oils
The fixed oils obtained with Soxhlet extraction from the fruit and peels of P. eurycarpa were sterilized using millipore filters and 5 µl from each of oil samples were spread onto surface of 7H11 and LJ media.

Addition of individual fatty acids into medium
Fatty acids to be analyzed were used in 0.5 µg/ml concentration which was found to have no antibacterial effect by Alma et al. (2004). Myristic acid (Sigma), palmitoleic acid (Sigma P9417), stearic acid (Sigma S4751), linoleic acid (Sigma L1376), linolenic acid (Sigma L2376), arachidic acid (Sigma A3631), and methyl cis-11-eicosenoic acid (Sigma E6885) were obtained from their corresponding commercial suppliers. In preparation of the solutions, myristic acid was dissolved in ether, while other fatty acids were dissolved in isoamyl alcohol. Final concentrations of 0.5 µg/ml were prepared and 5 µl from each was spread to the surfaces of LJ and 7H11 media. For every strain of bacteria, 12 LJ and 12 7H11 media were used and media were enumerated from 1 to 12. Media numbered as 12 were reserved for growth control media and nothing was added to these media.

Cultivation of bacteria
7H11 medium was poured into 12-well tissue culture test plates (Orange Scientific), 5 ml for each well. Commercially available LJ media were used in the study. The loopful colonies of the bacteria from 3-4 weeks-old M. tuberculosis cultures grown on LJ medium were suspended in sterile tubes with glass beads containing 5 ml of Middlebrook 7H9 broth with OADC (Becton Dickinson). The preparations were vortexed and supernatants were removed after 30 min. After centrifuging at 3200 rpm, the pellets were washed and the optical densities were adjusted to 0.05. An aliquot of 0.005 ml, containing 6x10 4 cfu was inoculated into each medium (Hedegecock, 1970). We obtained good results with these dilutions in the preliminary experiments. Inoculated media were incubated at 37°C and controlled daily for any growth. The exact time of appearance of the colonies were recorded.
Duplicate inoculations were made for each sample, and bacteria were isolated generally with one or two days of deviation.

Statistical analysis
While investigating the effect of each substance on the growth rate of bacteria, in the face of each variable, the difference between the average day of growth of the control strains and all other strains of bacteria was analyzed using the Wilcoxon Signed Ranks Test.

RESULTS
Percentage of the fruit oil yield was calculated as 24.86% and the peel as 54.51% (w/w). 99.98 and 99.96% of the fatty acid compositions of the fruit oil and fruit peel oils, respectively was identified by GC-MS technique. We found that the fruit oil composition was consisted of mostly oleic, palmitic, linoleic and stearic acids (58.55, 22.85, 11.03, and 3.4%, respectively) and to a lesser amount linolenic, palmitoleic, arachidic, eicosenoic and myristic acids.
When the daily growth times for the substance-free LJ and 7H11 media were compared, it was found that the growth in 7H11 medium (in 12.3 days) took place earlier than it was in LJ medium (in 15.9 days) and the difference was found to be in statistically significant levels.
In this study, no fatty acid was found to have a remarkable effect on growth rate of bacteria in terms of growth times measured. In the experiments made in LJ medium, it was found that the average growth time is 15.9 days for the tested strains. The average growth day for bacteria in LJ medium with the oil extracted from the peel was found to be 13.6 days. It was observed that this provides an advantage of 2.3 days in terms of growth time of bacteria.
The growth time was 9.8 days in the presence of the water soluble peel extract, 10.45 days in the presence of the fruit extract, in LJ medium. In each of the two conditions, growth time has accelerated in significant levels in comparison with the control media. It was Simsek et al. 2403 observed that growth time of bacteria has been shortened a more than one third. However, this time was 7.6 days in the presence of water-soluble peel extract, 8.5 days in the presence of fruit extract, in 7H11 medium. Again, in the statistical evaluations, this shortening of the time of growth was found to be significant levels for each of the two factors.
The average growth day of the tested 40 strains of bacteria in LJ and 7H11 media in the presence of various substances are presented in Tables 1 and 2. The data presented in Tables 1 and 2 are summarized in Tables 3  and 4 with some statistically parameters.
The average growth day for bacteria in the presence of each substance was compared with those in substancefree media. It was observed that the water-soluble extracts of the fruit and peel had significantly increased the growth rate of bacteria in two separate media (p<0.05). These extracts are shown as Arabic numbers in Table 1. It was also observed that other substances whose effects were analyzed did not have any remarkable effect on the growth rates of bacteria.

DISCUSSION
The increase of multiple resistant strains to antituberculosis drugs named MDR and XDR have made the infection more dangerous (Nathanson et al., 2010;Marvar et al., 2011). Early diagnosis of tuberculosis makes it possible for specific treatment to begin without delay. Besides, early diagnosis is of vital importance in terms of preventing the spread of the disease. Various molecular techniques that facilitate early diagnosis of tuberculosis have been developed and are presently in use. However, molecular test such as polymerase chain reaction (PCR) and other alternative amplification methods are difficult to apply or have low sensitivity (Fakruddin et al., 2013;Nikam et al., 2013;Derese et al., 2012;Lucke, 2011;Washington State Department of Health, 2011;Levy et al., 1989).

M.tbc
In this study, GC-MS method has demonstrated that there are 8 different fatty acids in fruit peel and 9 fatty acids in the fruit. Myristic acid was found only in the fruit. Composition of fatty acids found within the scope of this research was revealed to be in conformity with the profiles found by Kafkas et al. (2007).
It is determined that M. tuberculosis strains grow 2.3     12.1 11.9 12.1 12.3 95% Confidence Intervals 6.7-8.5 7.5-9.6 8.9-11.3 9.2-11.5 10. 8-13.6 10.5-13.3 10.9-13.7 10.5-13.4 10.6-13.5 10.4-13.4 10.6-13.6  days earlier with peel lipid extracts and 1 day earlier with fruit extracts in LJ medium than the control growth tubes. It was obtained 1-2 day earlier growth time by adding some substances to conventional media (Lu et al., 2002;Anargyros et al., 1990). Nevertheless it does not provide a major benefit to diminish the growth time 2 days in a period of 1-1.5 month-long growth time in LJ medium. The isolation periods of M. tuberculosis was found approximately 13.2 days by Bactec MGIT 960, 15.2 days by Bactec 460 and 25.8 days in LJ medium from clinical samples. It is known that mycobacteria grow rapidly in broth media than solid media (Cruciani et al., 2004).
Since the bacteria strains used in this study isolated earlier, the average growth time was found as 15.9 days in LJ medium. It is known that mycobacteria in clinical samples can be determined within hours by molecular microbiological methods (Lucke, 2011). However, LJ and MBA media are still used for isolation of M.tuberculosis from clinical samples in several laboratories. Consequently, these two media were used in this study. Assuming that a positive effect provided in any medium, it can be also applicable to all media. Some researchers have analyzed accelerating effects of growth of mycobacterium by adding various substances to the tuberculosis media. Kotian et al. (1983) showed that addition of nicotine into the medium has a slight contribution to the acceleration of growth of mycobacterium. Hedgecock (1970) have managed to obtain faster and better rate of growth of mycobacteria by adding triton into the medium. Triton solubilizes turbid fatty acids and, thus, reduces the toxic effects of fatty acids. In another article, it has been argued that mycothiol is present in mycobacterium cell wall, reduces the effects of oxidizing agents and some antibacterial substances, causing to faster and better growth of mycobacteria (Sareen et al., 2003). During the course of the study, it has been observed that some fatty acid combinations prevent growth of bacteria especially in 7H11 medium. It was considered that this prevention effect developed due to the fact that the fatty acids were put into wide-surface medium, exposing them to oxygen, and were kept at 37°C for a long time, leading to oxidization and resulting in a toxic effect on the bacteria. Additionally, it is possible that these lipids formed a thin layer between the air and the surface of the medium, preventing to obtain oxygen requirement of the bacteria. In this type of experiments, adding triton to medium may remove these delays (Hedgecock, 1970). Tested bacteria were inoculated by streaking them onto the media since commercially available solid media were used. We used the solid media because these types of media were employed in most of the mycobacteriological studies in our country.
The fruit and peel water-soluble extracts of P. eurycarpa accelerate the growth and development of mycobacterium. In this sense, it is possible to state that this is a first step in finding the existence of a substance of economic value which can be added to media formulas.
In our future experiments, we will study the effects of the water-soluble extracts on the liquid media. Additionally, we will continue to study the identification of the active components in the extracts that affect the acceleration of the growth time of the mycobacterium.
In conclusion, the fruit and fruit peel water-soluble extracts of P. eurycarpa reduce growth time of mycobacterium approximately by one third. Identification of effective substances in these extracts and addition of these extracts into mycobacterium medium will accelerate growth time of mycobacterium by means of culture method.
The diagnosis of the disease in the tuberculosis patients and the mycobacterial susceptibility tests will thus be quickened. We think that the result obtained is a significant step in solving the problem of late diagnosis of tuberculosis by means of culture.