Antibacterial Activity of Pistacia vera L . Nuts and Extracted Lipophylic Firas

The present study investigated antibacterial activities of different extracts from Pistacia vera nuts against Gram positive and negative bacteria using disc diffusion method, The Aqueous extract showed moderate effects against test bacteria specially on Bacillus cereus. Ethanol extract showed significant inhibitory effects against most bacteria compared with the standard antibiotics except Serratia marcescens and Salmonella typhimurium which resisted all concentrations of aqueous and ethanol extracts. Acetone extract showed Antibacterial Activity of Pistacia vera .... 60 less activity against test bacteria compared with ethanol extract, and the optimal concentration was 12.5mg/cm 3 against Pseudomonas aeruginosa, The lipophylic extract was active against Staphylococcus aureus and Ps. aeruginosa and also the optimal concentration was 12.5mg/cm 3 against Ps. aeruginosa, meanwhile no inhibitory effect was seen using lipophylic extract against Klebsiella pneumoniae, Salmonella typhimurium and Serr. marcescens.


Introduction
During the last two decades, the development of drug resistance as well as the appearance of undesirable side effects of certain antibiotics [1] has lead to the search of new antimicrobial agents mainly among plant extracts with the goal to discover new chemical structures which overcome the above disadvantages [2,3].Plant materials continue to play a major role in primary health care as therapeutic remedies in many developing countries [4], and almost the exclusive source of drugs for the majority of the world's population [5].
The objective of this study was to elucidate antibacterial activities of P. vera nuts using different extracts, including lipophylic extract among various types of bacteria.

Plant material
Nuts of P. vera L. were collected from Mosul, Ninewa province in June 2006 and was identified at Department of Biology, College of Science / University of Mosul.

Preparation of Extracts
Pistachio nuts were dried at room temperature in dark then grinded to powder using an electrical blender.

Aqueous and Ethanolic extracts
The method for preparing aqueous extracts from plant parts has been already described [15], dried nuts material 25gm was stirred in 250 ml of distilled water for 15 min at 90°C followed by rapid filtration through four layers of gauze and then by a more delicate filtration through Whatman filter paper No.1.The resulting filtrate was frozen and freeze-dried.The extract was stored at −18°C in a desiccant until required.
Ethanol extracts were carried out according to [16], which includes mixing 40gm of plant powder with 400 mL ethanol(concentration 95%), after stirring the extract was kept overnight at room temperature, then filtered using Whatman filter paper No.1, and evaporated under reduced pressure using a rotary evaporator at 40°C .The dried extract was stored in sterile bottles until further use.

Acetone extract
Dried and powdered nuts 80gm were extracted with acetone 300 ml in a soxhlet extractor for 10-12 hours continuously.The solvent was removed in vacuum 40°C to give concentrated extract.

Lipophylic extract
The organic solvent extraction of the grinded nuts of Pistacia vera 80gm dry weight was carried out in a conventional Soxhlet device using n-hexane as solvent, in the presence of anhydrous Na 2 SO4.The cartridges were introduced in the Soxhlet apparatus containing 300 ml of solvent.The total reflux time was 5 hours for the sample.After cooling, the solvent was separated from the solute using a rotary vaccum evaporator with a vacuum controller 40°C.The resulting extracts were weighed in an sensitive scale [17].

Preparation of extract concentrations
Several concentrations (200,100,50,25 and 12.5 mg/cm 3 ) of the different extracts were accomplished by dissolving in DMSO (Dimethylsulphaoxide), then were sterilized via pasturalization, the aqueous extract was dissolved using distilled water and sterilized by membrane filtration, then kept under refrigerated conditions until use.

Test Bacteria
The microorganisms used included Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Proteus vulgaris, Bacillus cereus, Serratia marcescens, Salmonella typhimurium and Klebsiella pneumoniae which were obtained from Department of Biology, College of Education, University of Mosul.

Inoculum preparation
Mueller-Hinton Broth (Difco) was applied for growing and diluting of the microorganism suspensions.strains of bacteria were grown to exponential phase in medium (MHB) at 37°C for 18 hours with aeration.The bacterial suspensions used for inoculation were prepared at 10 8 cfu/ml by diluting fresh cultures by comparing with McFarland density (1.5*10 8 cfu/ml).

Screening of antibacterial activity
Screening plant extracts for their antibacterial activity was conducted using the disc assay described by [18], One hundred micro liters of prepared culture was spread on surfaces of Mueller-Hinton agar.Sterile filter paper discs (Whatman No.1 : 6mm in diameter) were soaked with different concentrations of plant extracts by adding 0.1 mL of extract/10 paper disc, then placed on the surface of the inoculated media plates slightly, antibiotic discs (Bioanalyse) 6mm in diameter of (Gentamycin 10µg, Cefalexin 30µg and Carbencillin 100µg) were used as positive controls.Spread plates were then kept at room temperature for 30 min to allow diffusion and absorption of extracts prior to incubation at 37°C for 24 hours.At the end of the period, inhibition zones formed on the medium were evaluated in mm.

Result and Discussion
The results revealed various inhibitory effects of different extracts against tested bacteria via disc diffusion method, the inhibitory effect depends on extract type and the bacteria strain, in addition to the used concentration progression.Aqueous extract from P. vera nuts showed high inhibitory effect against B. cereus compared with Cefalexin with an inhibition zone of 17.2mm in diameter using the concentration 200mg/cm 3 , the extract showed good inhibitory effect against Pr.vulgaris compared with Gentamycin, meanwhile Ps. aeruginosa, S. aureus, E. coli and Kleb.pneumoniae showed a medium susceptibility compared with the standard antibiotics, the inhibitory effect had a direct relation with extract concentration against all tested bacteria, the concentrations (25, 12.5mg/cm 3 ) were inactive against tested bacteria this may be due to the low concentration of active components in the extract, the results also revealed the complete resistance of Serr.marcescens and Sal.typhimurium against the aqueous extract this may be attributed to the defensive agents of these bacteria such as plasmid resistance which can be obtained through modifications in metabolic pathways of enzymes and receptors of antibacterial materials [3].Many articles have discussed the inhibitory effects of Pistacia species like P. terebinthus and P. lentiscus against gram positive bacteria and proved that the effect is better compared with gram negative bacteria [19], on the other hand it was reported that the isolated gum from P. lentiscus has antifungal activity against the yeast Candida albicans [20].In addition most studies have reported that the aqueous extracts processes moderate inhibitory effects against bacteria, the most close explanation is the inability of water in separating essential oils which have high antibacterial activities [21], on the other hand water is active in separating flavonoids and phenolics which exist in P. vera and are good antimicrobial components.
(Table 1) showed that the minimum concentrations of ethanol extract had inhibitory effects on tested bacteria, E. coli showed high susceptibility to ethanol extract compared with Gentamycin with an inhibition zone 24.1mm in diameter (Figure 1), it was also seen that Pr. vulgaris (Figure 2) and B. cereus were inhibited via ethanol extract with significant variations compared with the standard antibiotics which are used as positive controls, followed by S. aureus and Ps.aeruginosa, as for Serr.marcescens and Sal.typhimurium both bacteria showed complete resistance against all concentrations of ethanol extract.The study results proved the suitability of the ethanol solvent in dissolving active components from plants in addition to natural products, and agrees with [8,22,23].
Our results indicated that acetone extract of Pistachio nuts has potential source of antibacterial effects against all test bacteria except Sal.typhimurium and Serr.marcescens (Table 2), it was also seen that increasing extract concentration didn't increase the antibacterial effect as in Ps. aeruginosa, the optimal concentration was 12.5mg/cm 3 , meanwhile effect against bacteria decreased using the concentration above 12.5mg/cm 3 (Figure 3), the scientific explanation for this situation is that the cytoplasm membrane of bacteria contains pores that prevents high concentrated extracts from insertion, in fact acetone extract was able to breakthrough cell wall and cytoplasm membrane and accomplish antibacterial activity, this was also described by [24] who studied the effect of plant extracts on bacterial cytoplasmic membrane permeability, the membrane permeability changed when incurrence to high concentrations of plant extracts and this also effects on bacterial susceptibility to antibiotics.Its reported that Pistachio trees contain 88 natural chemical compounds among the essential oil these compounds are considered as secondary products and some are secreted as a reaction of Pistachio trees when infected with bacterial, fungal and viral infections such as the secreted gum.This investigation also showed that lipophylic extract had a moderate activity against S. aureus (Figure 4), Ps. aeruginosa and E. coli, meanwhile other types resisted the extract, this can be due to the scanty of active compounds in lipophylic extracts.The active compounds in plants play a cooperating role in showing inhibitory properties, if one or more of these compounds are separated it may decrease extract activity [3].
From (Table 2) it was also seen that E. coli and Ps.aeruginosa were more sensitive to the extract concentrations 25 and 12.5mg/cm 3 respectively than the other used concentrations.The main antibacterial cause of P. vera is due to the existence of compounds such as carvacrol, camphene and limonene which are the main compounds in lipophylic structure (essential oil) [23], and several studies [25, 17, 26 and 7] mentioned that the essential oil of P. vera contains a high rate of fatty acids, like myristic, lignopalmatic, oleic, stearic, elaidic and melissic acids, which are responsible of the plant extract antibacterial activity, meanwhile other studies [27, 28, 29, 30 and 31] reported Pistachio species as a remedy for eczema, paralysis, diarrhea, gingivitis, antiviral and antibacterial which was proved in this study.Figure 1(A):Effect of Lipophylic on S .aureususing different concentration ,1(200 mg/cm 3 )2(100 mg/cm 3 ) 3(50 mg/cm 3 )4(25 mg/cm 3 )5(12.5mg/cm 3 ) Cb(Carbencillin).