Phenolic content, antioxidant capacity and antimicrobial activity of leaf extracts from Pistacia atlantica

Abstract The aim of this study was to investigate the secondary metabolites related to Pistacia atlantica Desf as well as their biological activities. Results show that using ethanol as a solvent exhibited the highest content of total phenolics and flavonoids (68.23 mg GAE g−1 of DW and 44 mg RE g−1 of DW), followed by aqueous one (20.07 mg GAE g−1 of DW and 15 mg RE g−1 of DW). Good correlation (Data not shown) was obtained between the DPPH radical-scavenging activities and total phenolics contents. Many similarities were observed between the results of the DPPH (IC50 = 32 and 200 μg mL−1) and ABTS (IC50 = 42 and 300 μg mL−1) assays. All tested extracts contained phenolic compounds exhibited an antimicrobial effect against Gram-positive and Gram-negative bacteria. Further, researches for identification and purification of phenolic compounds are required.


Introduction
Pistacia genus is widely distributed in the Mediterranean area in three species (Mahmoudi et al. 2016). Pistacia atlantica Desf is well known as a tree located in North Africa, which can reach over 25 m in height and grow in arid and semi-arid areas (Sifi et al. 2015), its vernacular name is 'Butom' . For many centuries, species of the genus Pistacia had a significant place in traditional pharmaceutical medicine. Several different biological properties such as antioxidant, antimicrobial, anti-inflammatory, antipyretic, anti-diabetic, anti-radical and cytotoxic have been reported for species of this genus and some of them are still used in folk medicine (Hatamnia et al. 2016;Uddin et al. 2016). Moreover, previous studies have proven the potential urease inhibitors of two isolated flavonoids as well as the scolicidal effects of P. atlantica fruit extract against protoscoleces of hydatid cysts and also its acute toxicity in mice model (Mahmoudvand et al. 2016;Uddin et al. 2016).
To the best of our knowledge, this study could be considered as the first report on the establishment of total phenolics and total flavonoids and to evaluate antioxidant activities of the two extracts from aerial parts of P. atlantica plants (i.e. leaves) using different in vitro assays. In addition, the extracts were evaluated in terms of their antimicrobial properties using disk diffusion assay.

Polyphenolic composition
The level of phenolic compounds in ethanolic and aqueous extracts measured for the first time according to the Folin-Ciocalteu method of aerial parts of P. atlantica which growing in the north of Tunisia is presented in Table 1. The total amount of phenolic compounds varied significantly between 68.23 ± 0.8 and 20.07 ± 0.2 mg GAE g −1 of dry weight (DW), whereas, the flavonoids content in the ethanolic extract was higher than that found in the aqueous one (~44 vs. 15 mg RE g −1 DW). In recent results, Ben ammar et al. (2010) made a screening of some Algerian medicinal plants for acetylcholinesterase inhibitory activities. Among these plants, they used water as a solvent for extraction of phenolic compounds from the leaves of P. atlantica Desf. and Pistacia lentiscus L. This research team found that the amount of total phenolics varied from 407.68 to 361.59 for P. atlantica Desf. and P. lentiscus L., respectively. These results were not in accordance with those found by our team. Therefore, we assume that these quantitative differences may be related to the geographic origin, genetic variability and harvesting time of the samples.

Antioxidant features
The investigation of antioxidant activity was assessed using two complementary assays: DPPH and ABTS radicals. Among all tested extracts, ethanol extract exhibited the best antioxidant performance against the two tested radicals (IC 50 = 32 and 42 μg mL −1 , for DPPH and ABTS test, respectively) almost near that of ascorbic acid as shown in Table 2. These observations may suggest that both solvent polarity and plant phenolic natures greatly influence the antioxidant activity estimation. Furthermore, correlation between the total phenolics content, DPPH and ABTS radical-scavenging activities of the different solvent extracts was accounted using regression analysis (Data not shown). These findings are consistent with previous reports that evaluated the antioxidant capacity of P. atlantica (Ben ammar et al. 2010).

Antimicrobial activity
The antibacterial potential of P. atlantica organic and aqueous extracts against Gram-positive and Gram-negative bacteria was assessed by the presence or absence of inhibition zones, the minimum inhibitory concentration (MIC) and the minimum bacterial concentration (MBC) values (Table 3). The obtained results showed that both organic and aqueous extracts had great potential for antibacterial against the most evaluated bacteria. The maximal inhibition zones diameters for tested microorganisms were in the range of 14 ± 0.6-16 ± 0.3 mm for ethanolic extract. Blank discs (disc containing only solvent) produced no zone of inhibition indicating that the solvents themselves did not possess any antimicrobial effect. During the assay, ethanol extract was found to have maximum zone of inhibition against both Grampositive and Gram-negative strains including Pseudomonas aeruginosa (16 ± 0.3 mm) and Aeromonas hydrophila EI (15 ± 0.8 mm). Furthermore, Listeria monocytogenes has shown a high sensitivity with an inhibition zone of 14 ± 0.6 mm. Concerning the bacteriostatic and bactericidal results, the MIC and MBC values for different bacterial strains ranged from 6.25 to 25 mg mL −1 and 25 to 50 mg mL −1 , respectively. In fact, the lowest MIC (6.25 mg mL −1 ) and MBC (12.5 mg mL −1 ) values were observed against P. aeruginosa which highlighted the Table 2. antioxidant activity of the extracts of Pistacia atlantica by dPPh radical scavenging, and aBts assays.
Notes: results are expressed as mean ± standard deviation of three determinations. Means with different symbols were significantly different at p < 0.05.
The obtained results clearly showed that P. atlantica ethanolic extracts exhibited interesting antimicrobial activities. This supported their use in traditional medicine in the treatment of infections and encouraged their potential use as alternative antimicrobial agents.

Conclusion
Our results showed that P. atlantica can be a source of polyphenols and flavonoids, confirmed its antioxidant activities and underlined its potential either as natural preservatives or in pharmaceutical applications. In addition, this report can be useful in estimating the efficacy of P. atlantica extracts as valuable plant materials to be used as natural antioxidants in phytotherapy. Additional antimicrobial assays confirmed the potential use of this species in disease treatment.