Antioxidant capacity and phenolic contents of three Quercus species

. The antioxidant capability and phenolic contents of ethanol extracts (free phenolics) and ethyl acetate extracts (bound phenolics) of three Quercus species were estimated in this work. The antioxidant activities were examined by 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) free radical, reducing power and  -carotene bleaching methods. HPLC was employed to detect major phenolic acids. The leaf extract of Q. salicina contained maximum total phenolics while the highest total flavonoid content was found in the leaf extract of Q. serrata . The antioxidant activities varied among three species. Bark extract of Q. salicina was the most potential and it was closed to levels of the standard antioxidative dibutyl hydroxytoluene (BHT). The bark extract of Q. serrata also showed promising antioxidant activities despite their eminence was negligibly lower than Q. salicina . Stronger antioxidant activities of free phenolics than those of the bound phenolics may be attributed to higher quantities of free phenolics in the barks of Quercus species, however total flavonoids may not contribute a critical role. By HPLC analysis, thirteen phenolic acids were detected in the leaf and bark extracts. Of them, Q. salicina showed maximum in number (ten compounds) and quantities of detected phenolic acids. Ellagic, chlorogenic and benzoic acids were dominant in Quercus species. Findings of this study revealed that leaves and barks of three Quercus species are rich source of antioxidants, and Q. salicina is the most promising and should be elaborated to exploit its pharmaceutical properties.


INTRODUCTION
In recent years, plant phenolic compounds have a great deal of attention because of multifunctional properties and beneficial effects on human health [1,2]. Phenolic including phenolic acids and flavonoids, which are characterized by at least one aromatic ring (C6) bearing one or more hydroxyl groups [3][4][5][6], are naturally synthesized via the shikimate pathway [7]. It has been estimated that there are over six thousand phenolic compositions identified and still a large percentage remains unexplored [4,8]. Phenolics are powerful antioxidants due to their beneficial properties to neutralize free radicals effectively by providing a hydrogen or an electron [2]. Antioxidants act as an inhibitor in the initiation of deleterious free radical reactions because they can inhibit or delay the oxidation of an oxidizable substrate in a chain reaction [8]. Antioxidant compounds are widely distributed in vegetables, fruits, herbs and many food sources [9][10][11]. Thus, natural antioxidants need to be explored in supporting human health.
Quercus (Oak) trees belong to Fagaceae family and they are dominant species in forest ecosystem with 450 species distributed worldwide [12]. Oaks are a major source of hardwood for use in construction, furniture, veneer barrels and other purposes because the wood of Quercus is durable and tough, and wood grain is really attractive [13]. It was reported that there were 20 rotary evaporator at 30 °C. The precipitates were weighted, dissolved in methanol and kept in the dark at 4 °C for further analysis.

Extraction of bound phenolics
Bound phenolics were extracted following the method described previously by Xuan et al. [22] with some modifications. The residue from above free phenolic extraction was hydrolyzed with 50 mL of 4 M NaOH and stirred at 50 °C for 4 h. This suspension was centrifuged at 5000 rpm for 10 min followed by filtrating through the Advantec No. 1 filter paper (Toyo Roshi Kaisha, Ltd., Japan) and then neutralized to pH 1.5 with 37% hydrochloric acid. The filtrate was extracted four times with ethyl acetate. Subsequently, the extracts were combined and evaporated to dryness on a rotary evaporator at 30 °C. The dried extract was dissolved in methanol and then stored in the dark at 4 °C to further use.

Determination of total phenolic content
The total phenolic contents of the extracts were determined using the Folin-Cicalteau method described by Medini et al. [23]. Briefly, an amount of 0.125 mL of the extracts (free or bound) was mixed with 0.5 mL of distilled water and 0.125 mL of Folin-Cicalteau's reagent was added. After 6 min, 1.25 mL of 7.5% aqueous Na 2 CO 3 solution was added. The solution was then adjusted to a final volume of 3 mL with distilled water and mixed vigorously. The mixture was incubated for 90 min at room temperature. The absorbance at 760 nm was recorded using a spectrophotometer (HACH DR/4000U-USA). The total phenolic content was expressed as mg of gallic acid equivalents (GAE)/g dry weight (DW).

Determination of total flavonoid content
The flavonoid content in extracts was determined using the method described by Djeridane et al. [24]. Sample extract (0.5 mL) was mixed with 0.5 mL of 2% aluminum chloride methanol solution. After 15 min at room temperature, the absorption was measured at 430 nm using a spectrophotometer (HACH DR/4000U-USA). The total flavonoid content was expressed as mg of rutin equivalents (RE)/g DW.
Antioxidant properties DPPH radical scavenging activity DPPH scavenging activity was evaluated according to the method described by Elzaawely et al. [10]. The various concentrations of 0.5 mL sample extracts were mixed with 0.25 mL of 0.5 mM DPPH and 0.5 mL of 0.1 M acetate buffer (pH 5.5). The mixtures were shaken vigorously and left at room temperature in the dark for 30 min. The reduction of the DPPH radical was determined by reading absorbance at 517 nm using a spectrophotometer (HACH DR/4000U-USA). BHT concentrations of 0.01 mg/mL to 0.05 mg/mL were used as a positive control. The percentage of DPPH radical scavenging was calculated as follows: Radical scavenging (%) = [(abs control -abs sample )/abs control ] x 100 The abs control is the absorbance of reaction without sample and abs sample is the absorbance of reaction with sample. IC 50 (inhibitory concentration) values were determined as inhibitory concentration of the extract necessary to decrease the initial DPPH radical concentration by 50% and were expressed in mg/mL. Lower IC 50 value indicates higher DPPH radical scavenging activity. All measurements were performed in triplicate.

ABTS radical scavenging activity
The ABTS radical cation decolorization assay was used according to method of Re et al. [25]. Briefly, ABTS radical solution was prepared by mixing 7 mM ABTS and 2.45 mM potassium persulfate in water and kept at room temperature in the dark for 16 h. Prior to use in the assay, ABTS radical solution was diluted with 80% methanol to give an absorbance of 0.70 ± 0.03 at 734 nm using a spectrophotometer (HACH DR/4000U-USA). Free radical scavenging activity was International Letters of Natural Sciences Vol. 54 assessed by mixing 30 µL of test sample or standard with 1 mL of methanolic ABTS radical solution and mixed thoroughly. The mixture was left in the dark at room temperature for 30 min and the absorbance was recorded at 734 nm. BHT standard solutions (concentrations 0.1 to 0.5 mg/mL) were prepared and assayed at the same conditions. All measurements were performed in triplicate. The percentage inhibition was calculated according to the formula: Radical scavenging (%) = [(abs control -abs sample )/abs control ] x 100 The abs control is the absorbance of reaction without sample and abs sample is the absorbance of reaction with sample. The antioxidant property of test sample was defined by IC 50 as the inhibitory concentration of the extract necessary for 50% reduction of ABTS and was expressed in mg/mL.

Reducing power
The reducing power was assayed by using the method described previously [2] with some slight modifications. Two hundred microliters of each extract or BHT was mixed with 0.5 mL phosphate buffer 0.2 M (pH 6.6) and 0.5 mL potassium ferricyanide [K 3 Fe(CN) 6 ] (10 g/L) in a test tube. The mixture was then incubated at 50 °C for 30 min. Afterwards, 0.5 mL of trichloroacetic acid (100 g/L) was added to the mixture, then centrifuged at 4000 rpm for 10 min. Finally, 0.5 mL of the supernatant was diluted with 0.5 mL of distilled water and 0.1 mL FeCl 3 solution (1 g/L) was added. The mixture was mixed thoroughly and the absorbance was measured at 700 nm using a spectrophotometer (HACH DR/4000U-USA). The assays were carried out in triplicate. The IC 50 values were calculated at which the absorbance was 0.5. Lower IC 50 indicates higher reducing power.

Antioxidant assay using the β-carotene bleaching system
The antioxidant activity of sample extracts was evaluated by the β-carotene linoleate bleaching system described by Elzaawely et al. [26]. β-carotene (2 mg) was dissolved in 10 mL of chloroform and 1 mL of the chloroform solution was mixed with 20 µL linoleic acid and 200 mg Tween-40. The chloroform was evaporated under vacuum at 45 °C. Afterward, 50 mL oxygenated water was added, the emulsion was vigorously shaken until complete homogenization was achieved. The obtained emulsion was freshly prepared before each experiment. A methanolic solution 0.12 mL of sample extract or BHT (1 mg/mL) were mixed with 1 mL of the emulsion. An equal amount of methanol was used for negative control. The solutions were incubated at 50 °C and recorded at 492 nm using a spectrophotometer (HACH DR/4000U-USA). All extracts were measured at zero time and every 15 min up to 180 min. The assays were carried out in triplicate for each extract. Lipid peroxidation inhibition (LPI) was calculated using the following equation: LPI (%) = A 1 /A 0 x 100. Where A 0 is the absorbance value measured at zero time for the test sample, while A 1 is the corresponding absorbance value measured after incubation for 180 min. Higher LPI value shows the higher antioxidant activity.

Statistical analysis
The data were analyzed by one -way ANOVA using the Minitab 16.0 software for Window. Upon significant differences, means were separated using Tukey's test at p < 0.05 with three replications and expressed as the mean ± standard errors (SE).

Total phenolics and flavonoids
The phenolic and flavonoid contents in both free and bound forms from leaves and barks varied among Q. crispula, Q. salicina and Q. serrata (Table 1). In general, amounts of free phenolics and flavonoids were much higher than the bound forms. The leaf and bark extracts of Q. salicina contained significant higher amount of total phenolics than other extracts. Meanwhile, the leaf extract of Q. serrata showed the highest value of total flavonoids.

Antioxidant activities
In DPPH scavenging activity, the free phenolics in barks of Q. salicina and Q. serrata showed similar strength (IC 50 = 0.031 and 0.026 mg/mL, respectively) to that of BHT (p > 0.05), which was used as a positive control in all assays of antioxidant activity trials. Both free and bound extracts in leaves of Q. salicina were potential (IC 50 = 0.067 and 0.079 mg/mL, respectively) ( Table 2). In the ABTS assay, none of the extracts showed similar levels as the BHT did, but the maximum activities were the free extracts of barks of Q. salicina and Q. serrata (IC 50 = 0.287 and 0.286 mg/mL, respectively ( Table 2).
In the reducing power assay, the antioxidant activity of free phenolic extracts in bark of Q. serrata was the most potential (IC 50 = 0.227 mg/mL) and it was not significantly different from that of BHT (p > 0.05), followed by free phenolic extracts in bark of Q. salicina (IC 50 = 0.274 mg/mL). The other free phenolic extracts were much lower magnitudes of antioxidant activities, as compared to that of BHT (Table 3). Regarding bound phenolics, the leaf extract of Q. salicina showed the strongest antioxidant activity, which was not significantly different from that of BHT (p > 0.05); other bound phenolic extracts did not show any potential reducing power activity (Table 3).
In the β-carotene bleaching method, all the extracts inhibited β-carotene oxidation. The free phenolic extracts exhibited a superior inhibition compared to BHT ( Figure 1A) whereas bound phenolic extracts showed a lower inhibition than BHT ( Figure 1B). Additionally, as showed in Table 3, the LPI% of the free phenolics in leaves of Q. salicina and Q. serrata, and barks of Q. crispula indicated the strongest activity comparable to that of BHT (p > 0.05). Beside, the free extract in bark of Q. salicina was also potential. In contrast to the free phenolic extracts, none of the bound phenolic extracts showed similar antioxidant activity as BHT did (Table 3).

Correlation between phenolic, flavonoid contents and antioxidant activities
As shown in Table 4, highly significant and positive correlation (R 2 = 0.532 -0.730) were observed between phenolic contents and DPPH, ABTS, reducing power and β-carotene/linoleic acid inhibition, while non significant correlation was found between flavonoid contents and antioxidant activities. These results may confirm that phenolic contents are responsible for the antioxidant activities of extracts.

Identification and quantification of phenolic compounds
Phenolic compounds of extracts were identified by comparing retention times of fifteen standard phenolic acids separated by HPLC (Figure 2A). The results showed that thirteen phenolic acids including gallic, protocatechuic, chlorogenic, p-hydroxybezoic, vanillic, syringic, ferulic, sinapic, p-coumaric, benzoic, ellagic, cinnamic acids and vanillin in free and bound phenolic extracts were identified (Table 4). Ellagic, chlorogenic and benzoic acids were the major phenolic compounds and their amounts varied among Quercus species, plant organs and extracting solvents. Gallic and vanillic acids were solely detected in Q. salicina whereas syringic and cinnamic acids were only found in Q. serrata. In general, the free phenolic extracts obtained higher quantities as well as greater numbers of phenolic acids as compared to that of bound phenolic extracts, with exception of Q. salicina leaves (Table 4). In the free phenolics in leaf extracts of Q. serrata had the highest amount of phenolic acids (3.99 mg/g DW), followed by bark and leaf extracts of Q. salicina (3.63 mg/g 3.24 DW, respectively) ( Table 4). The bound phenolics in leaf extract of Q. salicina was detected eight phenolic acids ( Figure 2B) followed by the free phenolics in bark extract of Q.

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Volume 54 serrata (six phenolic acids), whereas other extracts detected either three or four phenolic acids among the fifteen standard constituents (Table 4). Qse Values represent means ± SE (n = 3). Different letters in the same column indicate significant differences (p < 0.

DISCUSSION
Plants are rich in phenolics and flavonoids that produce beneficial effects by protecting cells against the oxidative damage caused by free radicals [27]. They are considered as one of the most potent and therapeutically useful natural components because of the antioxidant, anticancer, antimicrobial, antiviral and anti-inflammatory activities [28]. Total phenolic contents of plants depend on species and different organs [23]. Comparing with previous results reported in some other Quercus species, total phenolic contents of all extracts in this study showed higher quantities. Santos et al. [29] noted that cork of Quercus super obtained 2.4 -10.6 mg GAE/g DW while twigs and acorns extracts of Quercus robur and Quercus petraea showed 7.44 and 16.25 mg catechin/g DW respectively. Other reports included the total phenolic contents in leaf extracts of Q. robur and Q. petraea ranged from 32 -35 mg catechin/g DW [30] and Quercus resinosa, Quercus laeta, and Quercus obtusata (5.7 -7.1 mg GAE/g DW) [31]. In addition, total phenolic contents in leaf and bark extracts of Q. salicina were higher than some medicinal plants in previous studies such as Aristolochia mollissima, Lobelia chinensis, Dianthus superbus and Centipeda minima (3.9 -5.9 mg/g DW) [32]. Flavonoids are the chief of phenolic compounds and they also have potent antioxidant activities with positive effects on human health [33]. The total flavonoid content in leaves of Q. salicina and Q. serrata were much higher than that of Launaea procumbens which as a food supplement in Pakistan (4 -13 mg RE/g DW) [34]. From the results, this study concluded that leaves of Q. salicina and bark of Q. serrata may obtain rich phenolics and should be further elaborated to exploit their biological properties.

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Volume 54 Antioxidant capacity is widely used as a parameter for checking the activity of plant extracts [2]. Natural bioactive compounds are considered as an important alternative to the synthetic antioxidants, which are used as functional food to prevent human diseases [35]. Many previous studies showed a direct correlation between the phenolic contents and the antioxidant activity [36,37]. This study showed a highly significant and positive correlation between phenolic contents and DPPH, ABTS. Therefore, free extracts contained a higher amounts of phenolic contents which may be the reason for stronger radical scavenging activities. However, the antioxidant activity of plant extracts may relate to the presence of some individual phenolic compounds and their structures [24]. In addition, the position and quantitation of hydroxyl groups are possibly one of the main factors [38]. If an additional hydroxyl group is added to ortho or para position of the benzen ring, the antioxidant activity will be significantly increased, principally the ortho position can create the intramolecular hydrogen bond, so such position is proposed more effective than para and meta one [39,40]. As can be seen in the free phenolic extract of Q. serrata bark (Table 1), this species showed moderate phenolic contents but it had the highest antioxidant activity in DPPH assay. Moreover, syringic and cinnamic acids were only found in bark extract of Q. serrata. These compounds may have an important contribution to antioxidant activity. Comparing to previous studies, results of the DPPH assay revealed that the free phenolic extracts from Q. salicina and Q. serrata were superior to stem, flower and leaf extracts of Alpinia zerumbet [41]. Moreover, the antioxidant activity in term of DPPH scavenging in bark extracts of Q. salicina and Q. serrata were higher than all Limnophila aromatic extracts which were obtained by vigorously stirring with different aqueous solvents including methanol, ethanol and acetone [42]. Similarly, in ABTS assay we found that both bark and leaf of Q. salicina extracts contained higher concentration of total identified phenolics. From the results we stated that Q. salicina extracts are the most potential sources of natural antioxidant. Reducing power represents a crucial mechanism of phenolic antioxidant action because the total phenolic content and ferric reducing power have high relation [43]. Furthermore, an amino group and different substituents on the phenyl of chalcone moiety play an important role in the reducing power compounds [44]. The phenolic compounds obtained from three Quercus species might contain a number of reductants which may react with the free radicals to stabilize and terminate from free radical chain reaction. The reducing power of free phenolic compounds were more pronounced than those of bound phenolics probably because the free phenolics act as strong reducing agents by donating electrons and converted free radicals to more stable molecules [2,45]. The β-carotene bleaching method is based on loss of yellow color of βcarotene because the free radical was generated from linoleic acid can highly attack unsaturated βcarotene. The rate inhibition of linoleic acid oxidation by free phenolics was high compared to standard BHT and the values from previous research [46]. In this study, leaves of Q. salicina and Q. serrata, and barks of Q. crispula were the most potent in β-carotene assays (Table 3). By comparing the antioxidant activities using DPPH radical scavenging, ABTS radical scavenging, reducing power, and β-carotene-linoleate bleaching methods, it is observed that their activities varied among solvents and plant parts (leaves and barks). In general, the free phenolic extracts of all samples showed stronger antioxidative levels than the bound phenolic extracts (Tables 2,3).
Results of HPLC analysis demonstrated that amounts and types of phenolic compounds varied among oak species, leaves and barks (Table 4). In previous reports, several phenolic acids were identified in other Quercus species such as chlorogenic, cinnamic, gallic, gentisic acids and (+) catechin [47]; gallic, protocatechuic, ellagic acids and esculetin were the major compounds in corks of Q. super [29] or quercetin in stems of Q. salicina [16]. Ellagic acid also was identified as the major constituent of Q. suber from Spain and Portugal [35]. In this study, ellagic acid was the predominant composition, followed by chlorogenic and benzoic acids. Ellagic acid was a compound has four hydroxyl groups with multi-functions for anti-mutagenic, antimicrobial and antioxidant activities. Moreover, this compound was an inhibitor of human immune deficiency virus (HIV) [48,49]. As a result of inhibiting cell cycle progression and inducing apoptosis, ellagic acid may be considered as a product of cytotoxicity in tumor cells [50]. Chlorogenic acid is an important antioxidant in plants to ameliorate the damaging effects of oxidative stress in plants and hinder degenerative diseases in animals when supplied in their diet [51]. Benzoic acid is extensively used as preservatives and flavor enhancers, analgesics, antiseptics, chemotherapeutics for chemical syntheses [52,53].

CONCLUSIONS
This study proposed that Q. crispula, Q. salicina, Q. serrata possessed high total phenolic contents and appeared to be strong radical scavengers. Thirteen phenolic compounds were identified from bark and leaf extracts. Ellagic, chlorogenic and benzoic acids were the dominant phenolic compounds in Quercus species. Evidences obtained in this study also suggested that among three species of oak, Q. salicina was the most potential and should be considered as a rich source of natural antioxidants. Further searches for potent and novel antioxidants in these Quercus species should be needed.