Chemical Composition and Antibacterial and Antibiofilm Activity of Acetone Extract of Pistacia atlantica Leaf, Fruit, and Gall

Background : Bacterial infectious diseases caused by antibiotic resistance and biofilm formation agents are one of the most important challenges researchers and doctors face. Therefore, many studies have been done on medicinal plants to find phytochemical antibacterial and anti-biofilm agents. Objectives: In this study, the antibacterial and anti-biofilm activities of the acetone and aqueous extract of Pistacia atlantica leaf, fruit, and gall were evaluated against some bacteria. Methods: The leaves, fruits, and galls of P. atlantica were collected from the forests of Lorestan province, Iran. Antibacterial effects of extracts were studied by well diffusion method against Staphylococcus aureus , Bacillus cereus , Enterococcus faecalis , Pseudomonas aeruginosa and Escherichia coli . Microdilution method was used to evaluate the minimum inhibitory concentration and minimum bactericidal concentration of extracts. Anti-biofilm activity of acetone and aqueous extracts in sub-lethal concentration was investigated by crystal violet-stained microtiter method. Results: Acetone extract of P. atlantica had significant antibacterial effects against tested bacteria except for E. coli . There was a significant relationship between antibacterial activity and extract concentration ( P < 0.05). The minimum inhibitory concentration of acetone and aqueous extract of leaf, fruit, and galls ranged from 3-12.5 mg/mL. The acetone extract of P. atlantica galls had a high inhibitory effect on S. aureus and P. aeruginosa biofilm formation at a concentration of 12.5 and 25.0 mg/mL, respectively. Conclusions: The gall extracts of P. atlantica have a significant inhibitory effect against bacteria, which is probably related to certain active compounds. These extracts inhibited biofilm formation of S. aureus and P. aeruginosa .


Background
The use of antibiotics to prevent and control infection is very common, but infection and the prescription of antibiotics have led to the spread of resistant strains. It is estimated that more than 80% of human infections are related to bacterial biofilm formation. Bacterial biofilms are involved in the development of chronic human infections, dental plaque, and catheter infections. Currently, the use of herbal substances for treating diseases and microbial infections has been considered (1).
The Zagros mountain ranges are one of the main areas of wildlife growth. Pistacia atlantica, known as Bennet in Iran, is a tree with 2 to 7 meters' height that grows in the cold and humid weather of the Zagros (2,3). The leaf of the P. atlantica is used to treat hypertension, cough, sore throat, eczema, and stomachache. P. atlantica leaves inhibit colon cancer cells, in vitro (4,5). The nut of P. atlantica contains fatty acids including oleic acid, linoleic acid, alpha linoleic acid and palmitic acid (3,6). Phenolic compounds have been identified in P. atlantica fruits. The galls are induced by the aphid Slavum wertheimae on branches of P. atlantica trees (7). Galls are abnormal growths of some parts of plants that are caused by insects. Galls are very diverse in morphologically and biochemically (8,9). The molecular mechanism of gall formation is still unknown. Ecological studies and phylogenetic analyses have shown that insects control the behavior of galls (10).
It was demonstrated that the essential oil of P. atlantica subsp. Kurdica had broader antibacterial effects against the Gram-positive bacteria than Gram-negative bacteria and this essential oil was suggested to be used as a preservative in food industry to increase safety and reduce food pathogen risks (4). Kordbacheh et alshowed that active components of P. atlantica have high anti-Quorum sensing (QS) activities and may potentially treat chronic infections caused by Pseudomonas aeruginosa (11). The extracts of P. nigra exhibited antimicrobial activity and antibiofilm effects greater than 70% (12).

Objectives
In this study, the antibacterial and anti-biofilm activities of the acetone and aqueous extract of leaf, fruit, and gall of Pistacia atlantica were evaluated against pathogenic bacteria.

Plant Materials
Leaves, fruits, and galls of P. atlantica were collected from forests of Lorestan province, Iran (Oshtaran Kuh-Path Gahar Lake: 49°14'08"E, 33°209'28"N). Samples were approved by experts of the Agricultural and Natural Resources Research Center. The specimens were dried in shade, prepared in powder form and kept in dark glass at 4°C. The dried specimen powder was extracted by Soxhlet procedure for 5-6 hours. The different concentration of extracts was prepared by serial dilution method (final concentrations from 3 to 100 mg/mL).

Screening for Antibacterial Activity
Well diffusion method was used to evaluate the antimicrobial effect of the extracts. Final concentrations of each extract from 3 to 100 mg/mL were added to the wells. Sterile distilled water served as negative control and Imipenem (10 µg/mL) was used as a positive control. After 24 hours of incubation at 37°C, the diameter of the growth inhibition zone around each well was measured (13). The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values were determined by the microdilution method. 100 µL of different concentrations of extracts were added in to each well of a 96-well sterile plate, separately. 100 µL of bacterial inoculum (1.5× 10 7 cfu/mL) were added to the wells. The plates were incubated at 37°C for 24 hours. After incubation, the optical density (OD) of each well was measured at a wavelength of 630 nm by ELISA reader. After incubation, the lowest concentration with no visible growth was defined as the MIC. The MBC was determined by plating 10µl of well samples on nutrient agar. The lowest concentration with no visible growth on plates was the MBC (14-16).

Screening for Anti-biofilm Activity
The acetone and aqueous extracts of P. atlantica were tested for their potential to prevent biofilm formation of S. aureus and P. aeruginosa by the microtiter plate method (17). The extracts were added to the Brain Heart Infusion broth including 1% glucose (3.0, 6.25, and 12.5, mg/mL).
In each well of a sterile 96-well microtiter plate, 150 µL of each concentration was added. All bacterial cultures were adjusted to 10 8 CFU/mL by absorbance (0.08-0.1 at 630 nm) in a spectrophotometer and 150 mL of this bacterial suspension was added to each well (18). After 24 hours of incubation, the plates were washed and the contents of the wells were evacuated. The biofilm created by cells attached to wells were fixed in the plate wells by adding 150 μL of ethanol for 15 minutes. TThen, the ethanol was removed, and 200 μL of a 2% violet crystal solution (for 20 min) were add . After washing and drying the wells, 200 µL of 33% (v/v) glacial acetic acid (Merck, Germany) were added per well. 300 μL of brain heart infusion (BHI) medium containing 1% glucose was used as negative control and 150 μL of BHI medium containing 1% glucose with 150 mL of bacterial suspension was considered as a positive control. The optical density was measured at 495 nm by an ELISA reader (Statfax-2100, USA). The biofilm formation reduction (%) was calculated by the following formula (19,20):

Gas Chromatography/Mass Spectrometry (GC/MS) Analysis
The ingredient of P. atlantica gall were analyzed by GC/ MS. The GC/MS analysis was done with an Agilent 5975 GC-MSD system equipped with HP-5MS capillary column (30 m × 0.25 mm, 0.25 μm film thickness). Helium was used as the carrier gas, at a flow rate of 1.2 mL/min. The oven temperature was maintained at 50°C for 3 minutes and programmed to 200°C at a rate of 5°C/min, and kept constant at 290°C for 3 minutes, at the split mode. The injector temperature was set at 280°C. Identification of the components of the extract was accomplished based on comparison of retention times indices using a MS library.

Statistical Tests
Each experiment was repeated three times. Statistical analysis was performed using SPSS software, version 17.0. The differences between the control and treated groups were analyzed by Kruskal-Wallis test. P≤0.05 was considered to be statistically significant.

Results
The antibacterial activity of the acetone and aqueous extracts of P. atlantica at different concentrations (ranging from 3 to 100 mg/mL) against selected bacteria are shown in Tables 1 and 2. The antibacterial activity was observed http hmj.hums.ac.ir in acetone gall extract. Statistical analysis of the data using the Kruskal-Wallis test indicated that the difference between these concentrations was significant (P < 0.05).
The MIC and MBC values of different extracts of P. atlantica are presented in Table 3 . The MIC and MBC values of acetone extracts were lower than aqueous  Table 3). The reduction of biofilm formation by S. aureus and P. aeruginosa is shown in Figure 1. Evaluation of the anti-biofilm activity of acetone extract of P. atlantica in concentrations lower than the MIC value showed that leaf and fruit extracts inhibited the production of S. aureus biofilm completely at a concentration of 6.25 mg/mL.
The anti-biofilm activity of the extracts decreased by reducing the concentration of acetone and aqueous extracts to 1.5 mg/mL (Figure 1). Also, acetone and aqueous leaf extract at the concentration of 6 mg/ml inhibited biofilm formation of P. aeruginosa completely (Figure 1).
Phytochemical analysis results of different part of P. atlantica are summarized in Table 4. These results show the presence of the different functional organic materials such as pinene.

Discussion
Herbal extracts have been used for the treatment of diseases since ancient times. The antibacterial effects of some herbs used in traditional medicine have been proven. In this study, the acetone and aqueous extracts of the fruit, leaf, and gall of P. atlantica showed a relatively high antibacterial effect against tested bacteria, especially against gram-positive strains.
The diameter of inhibition zone was significantly correlated with the concentration of the acetone and aqueous extracts (P < 0.05). The acetone and aqueous extracts of P. atlantica have a certain antibacterial effect, which is increased by increasing the concentration of extracts. B. cereus and S. aureus were more sensitive to the aqueous and acetone extracts of the fruit. P. aeruginosa was more sensitive to the aqueous and acetone extracts of gall. According to the results of Kruskal-Wallis test, there was a significant difference between the diameter of inhibition zones of all tested bacteria at concentrations of 6.25 to 100 mg/mL in acetone and aqueous extracts. Also, there was a significant difference between the diameter of the inhibition zone aqueous and acetone extract of gall at concentrations of 1 to 100 mg/mL in tested bacteria. On the other hand, the diameter of the inhibition zones caused by the antibacterial activity of the acetone and   Edrah and colleagues showed the ethanol extract of the P. atlantica leaf has antibacterial activity against S. aureus, but S. epidermidis and E. coli were resistant (21). So far, no study has been published on the antibacterial effects of acetone and aqueous extracts of P. atlantica against bacteria. Mortazavi and colleagues evaluated the antibacterial activity of Pistacia khinjuk fruit against S. aureus and E. coli and they found that the sensitivity of the ethanol extract of the P. khinjuk fruit against E. coli is higher than S. aureus. The MIC values were reported in the range of 10-20 mg/mL (3). The results of these studies differ in the present study. The acetone and aqueous extracts of P. atlantica fruit were effective against S. aureus and had no appropriate activity against E. coli.
One of the interesting points in this study was the antibacterial activity of acetone and aqueous extracts of P. atlantica against P. aeruginosa, while the bacterium was resistant to Imipenem. The inhibition zones of acetone and aqueous gall extracts of P. atlantica were 21.0 and 15.3 mm, respectively, at a concentration of 100 mg/ml against P. aeruginosa. Antibacterial activity of acetone and aqueous gall extracts of P. atlantica was higher than the antibacterial activity of Imipenem against E. faecalis. The inhibition zones of acetone and aqueous gall extracts of P. atlantica at a concentration of 100 mg/mL against E. faecalis was 20.7 and 15.7 mm, respectively. Jalayer Naderi and colleagues reported that Streptococcus mutans and P. aeruginosa were not sensitive to Pistacia lentiscus, but S. aureus and Streptococcus sanguinis were sensitive at concentrations of 50, 100, and 1000 mg/mL of methanol extracts. The MIC of P. lentiscus for S. sanguinis was 1000 mg/ml (22).
A few studies are available on the anti-biofilm effects of leaves, fruits, and galls of P. atlantica. Hosseini and colleagues reported that the extracts of P. atlantica resin decreased the S. mutans biofilms. They showed β-pinene (70 %), α-copaene (76%) and α-terpinolene (86%) were found to be the major components P. atlantica resin. Also the extracts of resin has antibacterial activity against S. mutans (4). In this study, extracts of P. atlantica similarly reduced the biofilm production of S. aureus and P. aeruginosa in the range of 3.25 to 25 mg/mL.
Omidi and Sharifi reported that the methanol extracts of Quercus brantii, Pistacia atlantica and Elaeagnus angustifolia inhibited 60, 57 and 72% biofilms formation of P. aeruginosa, respectively (23). According to these findings, acetone and aqueous extracts of P. atlantica have higher ability to inhibit biofilms than methanol extracts.

Conclusions
Fruit and gall extracts of P. atlantica have a significant preventive effect against bacteria, which is probably related to certain active compounds. These extracts inhibited biofilm formation of S. aureus and P. aeruginosa. Therefore, clinical and pharmacological studies on these extracts are recommended.