Antioxidant activity of different parts of Pistacia khinjuk Stocks fruit and its correlation to phenolic composition

Abstract The fruits of Pistacia khinjuk Stocks were collected from Ilam province, Iran. The aim of this study was to analyse antioxidant capacity and phenolic composition of different parts of P. khinjuk fruit. The antioxidant capacity of extracts was measured using different assays: ferric reducing ability of plasma, 2,2-diphenyl-1-picrylhydrazyl and nitric oxide radical scavenging. The phenolic composition of P. khinjuk fruit is reported for the first time. Amongst different parts of the fruit analysed in this study, hull extract contained the highest total phenolic and flavonoid contents. We observed a high correlation between different antioxidant activity and total phenolic and flavonoid contents. Therefore, antioxidant capacity can be related to total phenolic and flavonoid contents. A correlation analysis revealed that ascorbic acid, gallic acid, rutin, caffeic acid, ferulic acid and sinapic acid were the phenolic compounds mainly responsible for antioxidant power of the fruit extracts. Graphical abstract


Introduction
Pistacia khinjuk Stocks is a deciduous tree from Anacardiaceae family that is found naturally alongside Pistacia atlantica and various Quercus species in the west regions of Iran (Zagros mountains). Plant antioxidant compounds play an important role as health protection factors. The main characteristic of an antioxidant is its ability to trap free radicals. Antioxidants such as phenolic compounds including flavonoid, phenolic acids, tannins and phenolic diterpenes scavenge free radicals such as peroxide, hydroproxide or lipid peroxyl, and thus inhibit the oxidative mechanisms led to degenerative diseases and are of great benefits to our health (Chung et al. 1998;Pietta 2000;Amarowicz et al. 2004). Therefore, many studies were carried out on antioxidant and free radical scavenging activities of phenolic compounds in genus Pistacia L. (Hossein Goli et al. 2005;Gourine et al. 2010;Tomaino et al. 2010;Farhoosh et al. 2011;Hatamnia et al. 2014Hatamnia et al. , 2015.
P. khinjuk fruit is used by local people after grinding and mixing with other ingredients as snack food, and the fruit oil is used as frying oil by natives. Different parts of P. khinjuk fruit has been used by natives as useful remedies for different diseases. For example, the fruit of P. khinjuk is used for treatment of stomach, heart and respiratory system disorders. Also, the gum resin obtained from P. khinjuk is used for wound healing activity and treatment of stomach and gastrointestinal disorders.
There are few studies on P. khinjuk regarding to the chemical composition of essential oils (Tavakoli & Haddad Khodaparast 2013) and flavonoids (Kawashty et al. 2000;Pietta, 2000). However, antioxidant activity and phenolic profile of different parts of P. khinjuk fruit has not already been studied. Hence, the main objectives of this study were to unveil the above-mentioned specificities included in P. khinjuk plant. Also, in order to obtain more details of phenolic compounds of fruit extracts, high performance liquid chromatography (HPLC) was used to separate phenolic compounds of different parts of the fruits.

Determination of total phenolic and flavonoid
Total phenolic content of hull, shell and kernel of P. khinjuk fruit is shown in Table S1 (Supplementary material). Amongst different analysed parts of fruit, hull contained the highest total phenolic content (25.9 mg GAEs/g) followed by shell and kernel (4.1 and 3.1 mg GAEs/g, respectively). Indeed, the total phenolic content of hull extract was significantly (p < 0.05) higher than those of other parts. Furthermore, Table S1 shows flavonoid content in different parts of P. khinjuk fruit. The content of flavonoid in different parts of fruits was ranged from 1.4 mg CEs/g (kernel) to 12.2 mg CEs/g (hull). The flavonoid content of hull extract was approximately 4.5-and 8.7-fold higher than that of shell and kernel extracts, respectively. However, hull with high phenolic and flavonoid contents was further investigated for their antioxidant activity and phytochemical characteristics. A positive correlation between phenolics and antioxidant activity has been previously established for different nuts (Akbari et al. 2012;Hatamnia et al. 2014). In a manner consistent with our results, total phenolic and flavonoids contents of hull in P. atlantica subsp. Kurdica (Hatamnia et al. 2014) and Pistacia vera L., variety Bronte (Tomaino et al. 2010) were higher than those of other parts.

DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging assay
The assay is based on the reduction of DPPH solutions in the presence of a hydrogen donating antioxidant, which is recorded as a change in colour. Table S1 shows the statistical analysis of DPPH radical scavenging activity and the values of EC 50 obtained from antioxidant activity of extracts and BHA (butylated hydroxyanisol = as a reference compound). On DPPH assay, EC 50 values were obtained for samples and BHA, so that a lower value of EC 50 indicated a higher antioxidant activity and vice versa. The extract concentration providing 50% of radicals scavenging activity (EC 50 ) was calculated from the graph of RSA percentage against extract concentration. DPPH radical scavenging activity of extracts was as follows: hull > shell > kernel. The extract of hull with an EC 50 equal to 0.151 mg/mL was almost three times less active than BHA, while the extracts of shell and kernel were nearly 17-25 times less active than BHA. Comparing to BHA, the extracts of hull revealed a good DPPH free radical scavenging activity, but shell and hull extracts with higher EC 50 had a weak activity (Table S1).
It can be detected that DPPH radical scavenging activity of different parts of fruit extracts was phenol and flavonoid contents dependent, so that there was a positive correlation coefficient between DPPH radical scavenging activity and phenolic (r = 0/95) and flavonoid (r = 0/96) contents [Table S2 (Supplementary material)]. A positive relation between phenolic compounds as potent hydrogen donors and DPPH radical scavenging activity has been reported by Chirinos et al. (2010), Myoda et al. (2010), and Hatamnia et al. (2014Hatamnia et al. ( , 2015. However, the present results indicated that hull extract with high phenolic content may play a major role in scavenging of free radicals.

FRAP (ferric reducing ability of plasma) assay
The FRAP assay is a simple direct test for measuring antioxidant capacity. The principle of the assay is based on the reduction of ferric ion Fe 3+ to ferrous Fe 2+ . The antioxidant activity was expressed as AEAC (ascorbic acid equivalent antioxidant capacity) and the antioxidant power of extracts was compared with BHA as a reference antioxidant. The results of FRAP assay is shown in Table S1. Amongst the three parts of fruit, hull extract had the greatest antioxidant activity (mean value of AEAC = 3.58 mg/mL) followed by kernel and shell (mean value of AEAC = 0.58 and 0.55 mg/mL, respectively). The extracts of hull were almost 5.7 times more active than BHA, while activity of shell and kernel extracts was almost equal to BHA (Table S1). There was a strong correlation between FRAP assay and phenolic (r = 0/99) and flavonoid (r = 0/98) contents (Table S2). A high positive correlation between phenolic compounds and FRAP assay has been reported by Alañón et al. (2011). Some authors have suggested the fact that the FRAP assay was one of the major trials to confirm antioxidant activity (Gourine et al. 2010;Farhoosh et al. 2011;Hatamnia et al. 2014Hatamnia et al. , 2015.

Nitric oxide radical scavenging activity
Statistical analysis of the nitric oxide radical scavenging assay data exhibited significant differences between the antioxidant capacity of different parts of fruit. Maximum nitric oxide radical scavenging percentage was 61.8% in hull extract followed by shell (mean value = 56.4%) and kernel (mean value = 46.6%) extracts (Table S1). A positive correlation coefficient between nitric oxide radical scavenging activity and phenolic (r = 0/79) and flavonoid (r = 0/83) contents was observed in this study (Table S2). These results supported by Hatamnia et al. (2014), who proposed that there is a direct relationship between phenolic content and nitric oxide radical scavenging activity. The correlation analysis was made between antioxidant capacity values estimated by FRAP, DPPH and nitric oxide radical scavenging assays (Table S2). The results indicated that, there were significant correlations between three different assays (0.76 < r < 0.94). These high correlations showed that the three different assays provided comparable values when they were used to evaluate the antioxidant capacity of P. khinjuk fruit.

Identification and quantification of phenolic compounds by HPLC
Ten phenolic compounds (ascorbic acid, gallic acid, rutin, caffeic acid, p-hydroxybenzoic acid, vanillic acid, p-coumaric acid, syringic acid, ferulic acid and sinapic acid) are reported here for the first time as P. khinjuk fruit components. The phenolic compounds were identified by comparing their retention times and UV-vis spectra properties to those of reference standards. Phenolic compounds of different parts of fruit identified by HPLC are shown in Table S3 (Supplementary material). A 20 μL aliquot of sample solution was separated using HPLC equipped with UV-vis detector. By comparing their retention times and UV-vis spectra properties with those of reference standards, we identified 7, 9 and 6 phenolic compounds in shell, hull and kernel, respectively.
Caffeic acid is a predominant phenolic compound in all extracts prepared from different parts of the fruit, with 230.1 μg/g d.m. determined in hull extract. The major phenolic compounds in hull extract were caffeic acid (230.1 μg/g d.m.), followed by sinapic acid (44.2 μg/g d.m.) and ferulic acid (42.1 μg/g d.m.) and rutin (1.98 μg/g d.m.) in which the later one was the lowest amount of identified phenolic compounds (Table S3). In shell extract, caffeic acid with 95.4 μg/g d.m. and vanillic acid with 5.66 μg/g d.m. were observed in the highest and the lowest amount of identified phenolic compounds, respectively. In kernel extract, the largest and smallest amounts of detected phenolic compounds were caffeic acid (17.3 μg/g d.m.) and ascorbic acid (2.33 μg/g d.m.), respectively (Table S3).
With the exception of p-coumaric acid, all of the other phenolic compounds used in this study were identified in hull extract. All phenolic compounds reported above were detected in shell extract, with the exception of rutin, p-coumaric acid and syringic acid. In kernel extract we found most of the compounds other than rutin, p-hydroxybenzoic acid, vanillic acid and ferulic acid. Ascorbic acid, gallic acid, caffeic acid and sinapic acid were found in the three parts of fruit (Table S3). The results indicated that amongst all extracts analysed by HPLC, the hull extract showed the highest concentration of phenolic compounds (383.7 μg/g d.m.) followed by shell (184.4 μg/g d.m.) and kernel (54.3 μg/g d.m.) extracts (Table S3). It shows a, trend similar to that of total phenolic content determined by Folin-Ciocalteu method (Table S1). Thus, a direct relationship is detectable between the amount of phenolic compounds identified by HPLC and total phenolic content. Furthermore, the amount of identified phenolic compounds of different parts of P. khinjuk fruit is lower than those of P. atlantica subsp. kurdica described by Hatamnia et al. (2014). However, this results is consistent with the finding of Hatamnia et al. (2014), who showed the highest concentration of phenolic compounds observed in hull extract. The phenolic composition of extracts from genus Pistacia L. has been described before (Tomaino et al. 2010;Hatamnia et al. 2014Hatamnia et al. , 2015, but this is the first report describing the phenolic profile of different parts of P. khinjuk fruit. Unfortunately, it is difficult to compare the phenolic profile results obtained by others authors because different species, different parts of plant or experimental conditions were used.

Correlation between antioxidant capacity and phenolic composition of different parts of P. khinjuk fruit
In order to reveal the contribution of each phenolic compound to the antioxidant activity, the correlation between the antioxidant capacity estimated by different assays (FRAP, nitric oxide radical scavenging activity and DPPH radical scavenging) and detected phenolic profile was investigated [Table S4 (Supplementary material)]. A high correlation was observed between antioxidant capacity and some phenolic compounds including ascorbic acid, gallic acid, rutin, caffeic acid, ferulic acid and sinapic acid. However, this high correlation shows that these compounds can greatly contribute to the overall antioxidant power of extracts. These results supported by Sroka and Cisowski (2003), who proposed that gallic acid with strong radical scavenging activity can greatly contribute to the overall antioxidant capacity. However, the rest of phenolic compounds do not make a major contribution to the antioxidant power of P. khinjuk fruit (Table S4).

Conclusions
As far as our literature review is concerned, this is the first research that investigated the phenolic profile for different parts of P. khinjuk fruit. Hull extract showed antioxidant activities stronger than those of extracts from other parts of fruit, which can be due to a higher phenolic content of hull. Also, it was specified that there is a correlation between total phenolic content with HPLC data, so that hull extract with higher total phenolic content in comparison with extracts obtained from other parts showed higher quantitative and qualitative phenolic compounds identified by HPLC. The results revealed not only the number of phenolic compounds, but also their contents that were significantly different between all parts of fruit. As a conclusion, it was shown that hull extract represents a high antioxidant activity and total phenolic content, suggesting their possible application in pharmaceutical and food technology.