Investigation of the Antioxidant Activity and Phenolic Compounds of Andricus quercustozae Gall and Host Plant

: Andricus quercustozae (Bosc, 1792) is a cynipid gall wasp, which induces gall on oaks ( Quercus spp.). It is known that both cynipid galls and oaks are used in traditional medicine. In this study, some biological characteristics of various extracts (acetone and ethanol) of A. quercustozae asexual gall and its host plant, Quercus infectoria Olivier, were investigated. The antioxidant capacities of the extracts were evaluated using radical scavenging activity (ABTS and DPPH assays), the β-carotene-linoleic acid method, the phosphomolybdenum method, and the reducing power (CUPRAC method). Total phenolics, flavonoid and tannin contents were measured in the gall and the oak leaf extracts. Moreover, ethanol extracts of the gall and the host plant were evaluated using HPLC for the composition of phenolics. Generally, the gall extracts (acetone and ethanol, respectively) exhibited the strongest radical scavenging (DPPH, IC 50 value of acetone extract: 11.00 μg/mL and IC 50 value of ethanol extract: 8.67 μg/mL; ABTS, 52.27 μg/mL and 44.97 μg/mL) and antioxidant activities with the highest level of phenolics. The antioxidant activity of the gall extracts was in the range of 80.74 to 87.49 % for β-carotene-linoleic acid method, while and it was ranged from 75.68 to 78.20 mgAEs/g for phosphomolybdenum method. In the results of some antioxidant methods (ABTS and β-carotene-linoleic acid), it is observed that the host plant extract has values close or high to the gall extract. In this context, our results suggested that the cynipid gall extracts could be used as a natural agent in food, medicinal and pharmaceutical applications.


INTRODUCTION
Quercus infectoria belonging to the Fagaceae family is a small tree or a shrub widely grown in Turkey (Anatolia), Syria, Iran, and Greece. The oak is known as one of the medicinal plants, which has been traditionally used in oriental folks [1,2]. The gall wasps or cynipids (Cynipidae), which are known as the gall inducers, is a large group with roughly 1400 species mL, 2%, Na2CO3) was added. The absorbance of the mixture was measured at 760 nm after the incubation (in the dark, 2 hours, room temperature). The total phenolic content was expressed as equivalents of gallic acid (mgGAEs/g).

Quantification of Total Flavonoid Content
The total flavonoid contents of each extract were analysed according to the method [38]. Briefly, aluminium trichloride (1 mL, 2% AlCl3) was mixed with the same volume of extract solution (2 mg/mL). The absorbance was measured at 415 nm after the incubation (10 min, room temperature). The total flavonoid content was expressed as equivalents of quercetin (mgQEs/g).

Quantification of Total Tannin Content
The vanillin method [39] with slight modification was used for analysing the total tannin content. The solution (0.5 mL) was mixed with vanillin reagent (1.5 mL, 1% in 7 M H2SO4) in an ice bath. The solution absorbance was measured at 500 nm after the incubation (15 min, room temperature). The total tannin content was expressed as equivalents of (+)-catechin (mgCEs/g).

Determination of DPPH Radical-Scavenging Activity
The radical scavenging activity of the extracts was determined using the method [40]. Different concentration (5 to 25 μg/mL) of the extracts (1 mL) was mixed with 4 mL of DPPH (2,2-diphenyl-1-picrylhydrazyl) radical methanolic solution. The absorbance was measured at 517 nm after 30 min. Synthetic antioxidant BHT (butylated hydroxytoluene) was used as a positive control. The results were expressed as IC50 values.

Determination of ABTS Radical-Scavenging Activity
The method [41] with slight modification was used to determine the radical scavenging activity of the extracts. ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) cation was produced by reacting ABTS solution (7 mM) with potassium persulfate (2.45 mM) and then the mixture to stand in dark (12)(13)(14)(15)(16) hours, at the room temperature). The mixture was diluted with ethanol to give an absorbance of 0.700 ± 0.02 units at 734 nm for the study. The extract solution (1 mL) and ABTS solution (2 mL) were mixed. The mixture absorbance was read at 734 nm after the incubation period (15 min, at room temperature). The results were expressed as IC50 values.

β-Carotene-Linoleic Acid Method
The total antioxidant activity of the extracts was analyzed using the β-carotene-linoleic acid method [42] with slight modifications. β-Carotene (0.2 mg) was dissolved in chloroform (1 mL) and added linoleic acid (20 μL) and Tween-20 (200 mg). The chloroform was evaporated using a rotary evaporator. The mixture was diluted with dH2O (100 mL). As soon as the emulsion (4.8 mL) and 1 mL extracts (1 mg/mL) were placed into test tubes, initial absorbance was measured at 470 nm. The measurement was carried out at 30 min intervals for 2 hr. BHA and BHT were used as standards. The antioxidant activity was calculated using the equation below: Where A0 and Aº0 are the absorbance values measured at the initial incubation time for samples and control, respectively. While At and Aºt are the absorbance values measured in the samples or standards and control at 2 hr.

Phosphomolybdenum Method
The phosphomolybdenum method [43] with slight modification was used to determine the antioxidant activity of the extracts. Extract solution (0.3 mL) was mixed with 3 mL of reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). The mixture absorbance was read at 695 nm after the incubation period (90 min, at 95 °C). The total antioxidant capacity was expressed as equivalents of ascorbic acid (mgAEs/g).

Activity Cupric Ion Reducing (CUPRAC) Method
The method [44] was used to determine the cupric ion reducing activity (CUPRAC). Extract solution (0.5 mL) was added to premixed reaction mixture containing CuCl2 (1 mL, 10 mM), neocuproine (1 mL, 7.5 mM) and NH4Ac buffer (1 mL, 1 M, pH 7.0). Similarly, a blank was prepared by adding sample solution (0.5 mL) to the premixed reaction mixture (3 mL) without CuCl2. The mixture and blank absorbances were measured at 450 nm after the incubation period (30 min, at room temperature). The absorbance of the blank was subtracted from that of the sample. CUPRAC activity was expressed as equivalents of Trolox (mgTEs/g).

Quantification of the Phenolic Compounds by HPLC
Phenolic compounds were analyzed by high performance liquid chromatography (HPLC) according to the method [45] with some modification. Detection and quantification were performed with a diode array detector (SPD-M20A), a LC-20AT pump, a CTO-10ASVp column heater, SIL-20ACHT auto sampler, SCL-10Avp system controller and DGU-14A degasser. The mobile phases were A: 3.0% formic acid in distilled water and B: methanol. Methanol was used to dissolve samples, and then 20 μL of this solution was injected into the column. Gallic acid, 3,4-dihydroxybenzoic acid, 4-hydroxybenzoic acid, 2,5-dihydroxybenzoic acid, chlorogenic acid, vanillic acid, epicatechin, caffeic acid, p-coumaric acid, ferulic acid, rutin, ellagic acid, naringin, quercetin, and cinnamic acid were used as standards. The differentiation and quantitative analysis were made by comparing the standards. The quantity of each phenolic compound was expressed as μg per gram of the extract.

Statistical Analysis
The SPSS Statistical Package program were used to analyze the results. The results were presented as mean ± std. Differentiations between the extracted groups were tested using Analysis of Variance and Tukey method were performed (p < 0.05).

Determination of Total Bioactive Components
The calibration curve generated from the analysis of the standard (gallic acid) was linear with y=0.0016x+0.0407; r 2 =0.983. The A. quercustozae gall extracts showed the highest phenolic content, acetone (479.56 ± 45.36 mgGAE/g) and ethanol (437.27 ± 3.14 mgGAE/g), while Q. infectoria extracts showed the lowest contents. Among the studied extracts, total phenolic content ranged from 79.35 to 479.56 mgGAE/g (Table 1). Phenolic contents of the gall extracts were found to be more than the extracts of host oak (Q. infectoria). In addition, there were no differences between the acetone and ethanol extracts for each species (p>0.05), but the gall extracts and host plant extracts were found to be statistically different from each other (p<0.05).
In the present study, total flavonoid contents were analysed with the spectrophotometric method [38], and the flavonoid contents ranged from 33.19 to 110.28 mgQE/g (Table 1). Statistical differences among each group were found (p<0.05). Tannin content was analysed using the vanillin method and these results were evaluated as catechin equivalents. The total tannin content of the extracts varies from 7.03 to 34.04 mgCE/g ( Table 1). There were no differences (p>0.05) between the acetone and ethanol extracts of the host plant, but the gall extracts and host plant extracts were found to be statistically different from each other (p<0.05). Unlike phenolic compounds, Q. infectoria extracts showed the highest flavonoid and tannin content while the gall extracts showed the lowest contents. These results suggested that the phenolic, flavonoid and tannin contents were best extracted with acetone for both the gall and the host plant (Table 1).

Radical Scavenging Activity (DPPH and ABTS Assays)
The free radical scavenging activities of the samples of A. quercustozae gall and Q. infectoria leaves were tested by DPPH and ABTS assays. The DPPH and ABTS are known as radicals, they can be readily undergone scavenging by an antioxidant [54,55]. The lower IC50 reflected a higher antioxidant activity in both assays ( Table 2). In DPPH assay, the gall extracts more than the leaf extracts, have stronger scavenging activity. Among all the extracts, only ethanolic gall extract exhibited the highest radical scavenging capacity with IC50: 8.67 μg/mL, followed by acetonic gall extract. The lowest scavenging activity was observed in ethanolic leaf extract with a very high IC50 value of 54.37 μg/mL. The ABTS scavenging capacity of the extracts was determined and IC50 values are given in Table 2.
The extracts showed scavenging activities in the range of 19.75 to 52.27 μg/mL. The acetonic leaf extract showed significantly stronger ABTS scavenging capacity (IC50: 19.75 ± 0.92 μg/mL) than that of all other extracts (p<0.05). In compared extracts, A. quercustozae gall extracts had higher antioxidant capacity than Q. infectoria leaf extracts. The results of the strong radical scavenging capacity of the gall extracts are related to the high concentration of phenolic compounds in the gall extracts (Tables 1, 2).

Total Antioxidant Activity (β-carotene-linoleic Acid and Phosphomolybdenum
Methods) The total antioxidant capacities of the extracts were evaluated by using β-carotenelinoleic acid assay. The extracts of both samples exhibited generally high antioxidant activities (Table 2). However, the leaf extracts had strong antioxidant activity (91.05 ± 1.29% and 89.45 ± 1.72%) more than the gall extracts (80.74 ± 7.39% and 87.49 ± 1.27%). When compared with the inhibition values of all extracts, the acetone extract of the leaf (91.05 ± 1.29%) had a higher value than others, but lower than synthetic antioxidants (BHA and BHT). In phosphomolybdenum assay, the extracts obtained from the gall had a higher antioxidant capacity than leaf extracts (p<0.05). Furthermore, the acetone extracts showed the highest antioxidant activity while the ethanol extracts showed the lowest activity (Table 2). Therefore, both the gall and the leaf extracts can be considered as natural inhibitors in the food industry. It must be the first study to compare the antioxidant capacities of the gall and the host plant.

Reducing Power (CUPRAC Method)
Cu 2+ reduction is used to determine electron donation activity which is known an important mechanism of antioxidant. Therefore, in order to analyse extracts' electron-donating power, their ability to reduce Cu (II) was tested. The high values of TEs reflected a high reducing activity. The reducing power activities of the extracts are presented in Table 2. When compared to the cupric reducing ability of all extracts, the ethanol extracts had a high cupric reduction potential for both samples. Moreover, the gall extracts exhibited higher values than the leaf extracts (p<0.05). The high reducing power of the gall extracts might relate to the high phenolic compounds that act as electron donors.
There are investigations on the antioxidant activities of both the host plant [51] and the cynipid gall [17-20, 22, 56, 57]. For the first time, a different species (A. quercustozae) and its host plant (Q. infectoria) were compared with this study. This and similar studies should reveal the important biological characteristics of many cynipid galls and host plant, which has been used since ancient times against many diseases, in terms of human health.

Phenolic Composition (HPLC)
In the study, the phenolic components of the ethanolic extracts of A. quercustozae asexual gall and Q. infectoria leaf were determined using the HPLC method (Table 3). For both species' extracts, caffeic acid has the highest concentration followed by epicatechin. Caffeic acid and epicatechin are abundant in medicinal plants and possess many biological effects such as antioxidant, anti-aging [58,59]. Moreover, other phenolic compounds found in the extracts such as gallic acid also possess beneficial effects on human health. Radical scavenging activity can thus be explained by the presence of epicatechin and caffeic acid.

CONCLUSION
The results reported in this study revealed that tested total phenolic compounds were significantly found more in the gall extracts. However, the gall extracts were strong radical scavenging because of the highest level of phenolics. The non-galled leaves extracts have the highest flavonoid and tannin contents while the gall extracts showed the lowest contents. These results suggested that the phenolic, flavonoid and tannin contents were best extracted with acetone for both the gall and the host plant. Due to the high antioxidant activities were observed, it is suggested that they can be used as a natural agent in food, medicinal and pharmaceutical applications. This study clearly indicates that the gall derived remedies may have distinct therapeutic effect as compared with analogues produced from other parts of the host plant. The galls can also be used for the prevention and treatment of various diseases. Although cynipid diversity is rich in worldwide, only gall extracts of a few species have been studied so far. Further studies are necessary to determine some biological characteristics of other cynipid galls extract for the food industry and medicine.