Total phenolic, condensed tannin and antioxidant activity of four Carya species from China

Different species of functional agricultural crops may vary in antioxidant capacities. In this study, the antioxidant activities of methanol extracts from four species of Carya genus were compared by various antioxidant assays, including the reducing power, 1,1-diphenyl-2-pycrylhydrazyl (DPPH) radical scavenging activity and the superoxide anion scavenging activity. The reducing power of extracts from Carya dabieshanensis, Carya cathayensis, Carya hunanensis and Carya illinoensis were 0.246, 0.237, 0.22 and 0.073 at the concentration of 0.50 mg/ml, respectively. The scavenging effect on the DPPH radical (IC50) were 1.140, 1.364, 1.437 and 3.682 mg/ml, respectively, while the scavenging effect on superoxide anion radical were 27.44, 22.80, 26.15, 1.99 mg AE/g, respectively. Among the four species, C. dabieshanensis possessed the highest antioxidant activity, while C. illinoensis was the lowest. The total phenolic (TP) contents and condensed tannins (CT) were determined in all samples spectrophotometrically. For all species, C. dabieshanensis possessed the highest TP content (80.54 mg GE/g defatted kernel) and C. hunanensis possessed the highest CT content (59.62 mg CE/g defatted kernel). In addition, strong correlations of total phenolic contents and condensed tannins contents with reducing powers, DPPH radical and superoxide anion scavenging activities were also found in this work.


INTRODUCTION
Tree nuts have long been considered as important components of the diet due to several bioactive and health-promoting components.Epidemiological evidence indicates that, the consumption of tree nuts may lower the risk of cardiovascular disease.The European prospective investigation into cancer and nutrition (EPIC) cohort study, conducted in ten European countries also showed that, women consuming more than 6.2 g per day of nuts and seeds reduced their risk of colon cancer Abrreviations: DPPH, 1,1-Diphenyl-2-pycrylhydrazyl; TP, total phenolic; CT, condensed tannins; NBT, nitro blue tetrazolium; BHA, butylated hydroxyanisole; IC50, half maximum inhibitory concentration; EC50, half maximum effective concentration.by 31% (Jenab et al., 2004).The Carya genus (family Juglandaceae) comprises several species and is commercially cultivated in North America and east Asia for over 500 years.Recently, growing interest was developed in the exploitation of Carya (Taipina et al., 2009;Osorio et al., 2010).Pecan, belonging to the Carya genus, has been reported to possess the highest antioxidant capacity and the highest phenolic content among the common fruits and vegetables across the US (Wu et al., 2004).Villarreal-Lozoya et al. (2007) also found that, kernels from different pecan cultivars had high antioxidant capacity and total phenolic content.
Growing evidence suggests that, species and genotype would alter the antioxidant compositions and properties in a selected agricultural crop (Gursoy et al., 2009;Esmaeili et al., 2010).Thus, it is of paramount importance to examine species and genotype for their antioxidant activity, in order to find potential species rich in special healthy functions.The objective of the present study was to characterize four different Carya species for their nutraceutical constituents, including total phenolic content, antioxidant activities and condensed tannins content.

Material and thermal processing
Carya cathayensis seeds were mechanically harvested in early September 2009 from Linan, Zhejiang Province, China.The seeds of Carya dabieshanensis, C. cathayensis, Carya hunanensis and Carya illinoensis were purchased from Hangzhou Donglin Co. Ltd.The seeds were washed with excess water and then sun-dried at about 30°C for three days.The kernels with the brown outer testa or pellicle were separated from the shell by cracking with a small hammer and were ground in a mortar.The kernel powder was defatted with hexane according to the method of Villarreal-Lozoya et al. (2007), then, samples were freeze-dried and stored at -20°C until analyses.

Preparation of methanol extracts
Extraction was done by macerating 1 g of defatted kernel powder with 40 ml of 70% methanol.The mixture was kept in a rotary shaker overnight and centrifuged at 3,000 g for 20 min (SCR20BC, Hitachi, Japan).A working solution (2.5 mg defatted kernels/ml) was prepared by dissolving 1 ml of the supernatant in 10 ml of methanol.

Determination of total phenolic and condensed tannin content
Total phenolic content was determined with Folin-Ciocalteu reagent according to Slinkard and Singleton (1977) using gallic acid as standard.In a volumetric flask, 0.1 ml of the methanol extract (final concentrations were 2.5 mg defatted kernels/ml), 0.9 ml of distilled water and 1 ml Folin-Ciocalteu reagent were mixed thoroughly.After 3 min, 3 ml of 0.188 mol/l Na2CO3 was added, then the mixture was allowed to stand for 2 h with intermittent shaking.The absorbance was measured at 760 nm in a spectrophotometer (UV-2100, Unico, Shanghai, China).The final results were expressed as milligram gallic acid equivalents per gram of defatted kernels (mg GE/g).Condensed tannin (CT) content was evaluated using the vanillin assay (Price et al., 1978).An aliquot of 0.5 g of defatted kernels was placed in centrifuge tubes and 20 ml of 1% HCl in methanol was added to each sample.Each tube was vortexed every 10 min and placed in a water bath at 30°C with constant shaking for 20 min.After incubation, tubes were centrifuged and supernatants were extracted.Aliquots of the supernatants were placed in two separate assay tubes, one for the sample determination and the other for blank determination.Samples and blanks were incubated for exactly 20 min after adding 5 ml of the vanillin reagent (0.5 g of reagent and 200 ml of 4% HCl methanol) to samples and 4% HCl in methanol to the blanks.After 20 min, the absorbance was measured at 500 nm in a spectrophotometer.Results were expressed as milligram catechin equivalents per gram of defatted He et al. 10473 kernels (mg CE/g).

Determination of reducing power
The reducing power of methanol extract was determined by the method of Mao et al. (2006).The 1 ml of methanol extract (0.25, 0.5 and 0.75 mg defatted kernels) prepared from working solution were mixed with 2.5 ml of 0.2 mol/l phosphate buffer (pH 6.6) and 2.5 ml of 0.03 mol/l potassium ferricyanide (K3Fe(CN)6).Aliquots (2.5 ml) of 0.6 mol/l trichloroacetic acid were added to the mixture, which was then centrifuged for 10 min at 1,000 g.The upper layer of the solution (2.5 ml) was mixed with 2.5 ml of distilled water and 0.5 ml of 0.006 mol/l FeCl3 and the absorbance was measured at 700 nm in a spectrophotometer.

Determination of DPPH radical scavenging activity
The model of scavenging the 1,1-diphenyl-2-pycrylhydrazyl (DPPH) radical is a widely used method to evaluate the free radical scavenging ability of various samples (Ebrahimzadeh et al., 2009).Scavenging activity of DPPH free radical was measured based on Lee et al. (1996).Negative control was prepared by mixing 0.125 ml distilled water with 3.875 ml of 0.2 mol/l DPPH.The 0.125 ml of the methanol extract (0.5, 1.0, 1.5, 2.0 and 2.5 mg defatted kernels/ml) was added to 3.875 ml of 0.2mol/ l DPPH.The mixture was gently homogenized and left to stand at room temperature for 30 min.Absorbance was read using a spectrophotometer at 517 nm.The activity of scavenging DPPH radicals was calculated using the equation: where, As is the absorbance of the sample, A(-) is the absorbance values of negative controls, respectively.

Determination of superoxide anion scavenging activity
The superoxide anion scavenging activity was measured using the xanthine/xanthine oxidase method Mao et al., 2006).Working solution of extract (1.25 mg defatted kernels/ml) was separately added to a 1.0 ml mixture of 0.4 mmol/l xanthine and 0.24 mmol/l nitro blue tetrazolium chloride (NBT) in 0.1 mol/l phosphate buffer (pH 8.0).A 1.0 ml solution of xanthine oxidase (0.049 unit/ml), diluted in 0.1 mol/l phosphate buffer (pH 8.0), was added and the resulting mixture incubated in a water bath at 37°C for 40 min.The reaction was terminated by adding 2.0 ml of an aqueous solution of 69 mmol/l sodium dodecylsulphate (SDS) and the absorbance of NBT was measured at 560 nm.A standard curve was prepared using ascorbic acid as reference reagent.Superoxide anion scavenging activity was expressed in milligram ascorbic acid equivalents per gram of defatted sample (mg AE/g).

Statistical analysis
The data were reported as mean ± SD for triplicate determinations.Analysis of variance and the least significant difference tests (SPSS for Windows, 1999, SPSS Inc., Chicago, IL) were conducted to identify differences among means.Statistical significance was declared at P < 0.05.

Total phenolic content
It is well known that phenolic compounds exist in many  2007) also found that, kernels from different pecan cultivars had high antioxidant capacity and total phenolic content.
Table 1 shows the total phenolic (TP) contents of the Carya species, which were significantly (p<0.05)different.Total phenolic content ranged from 14.63 to 80.54 mg GE/g defatted kernel with C. dabieshanensis showing the highest TP value, while the C. illinoensis had the lowest.For all species, the following trend was found; C. dabieshanensis > C. cathayensis > C. hunanensis > C. illinoensis (P < 0.05).The TP value of C. dabieshanensis, C. cathayensis and C. hunanensis were at least 4.9 times higher than that of C. illinoensis (P < 0.05).The TP value of C. illinoensis in this study was in agreement with those of Wu et al. (2004) who found that, the TP value of C. illinoensis ranged from 12.84 to 20.16 mg GE/g.

Condensed tannin content
Most phenolic compounds commonly identified in walnut and hickory are phenolic acids and condensed tannins (Fukuda et al., 2003;Ito et al., 2007;Zhang et al., 2009).The condensed tannins (CT) content evaluated with the vanillin assay showed differences among Carya species, ranging from 13.21 to 59.62 mg CE/g defatted kernel which was similar to the values found by Polles et al. (1981).Also, Prado et al. (2009a) reported that, the CT content of pecan nut (C.illinoinensis (Wangenh.)C. Koch) shell infusion and acetone extracts of kernel cake were 43 ± 7 and 16.4 ± 4.2 mg CE/g, respectively.The CT content of defatted kernel from different pecan cultivars ranged from 23 to 47 mg CE/g (Villarreal-Lozoya et al., 2007).Among the four species, C. hunanensis presented the highest CT values, while C. illinoensis, had the lowest (Table 1).Carya species showed the following in a descending order: C. hunanensis > C. cathayensis > C. dabieshanensis > C. illinoensis (P < 0.05).

Scavenging effect on DPPH radical
1,1-Diphenyl-2-pycrylhydrazyl (DPPH) is a stable nitrogen-centered free radical whose color changes from violet to yellow upon reduction by either the process of hydrogen or electron donation.Substances which are able to perform this reaction can be considered as antioxidants and therefore, radical scavengers (Brand-Williams et al., 1995).The scavenging effects of extracts from Carya kernels tested on the DPPH radical were measured as shown in Figure 1.The scavenging activity of extracts on inhibition of the DPPH radical was related to the concentration of extracts added, the activity increased as a result of increasing concentration for each species.The scavenging effect of extracts from four species of Carya kernels on the DPPH radical followed the order: C. dabieshanensis > C. cathayensis > C. hunanensis > C. illinoensis (P < 0.05) and the half maximum inhibitory concentration (IC 50 ) were 1.140, 1.364, 1.437 and 3.682 mg/ml, respectively, which was low, compared to that reported by Zhu et al. (2008), who stated that, the percentage of DPPH radical scavenging activity at 200 l volume of Chinese Hickory (C.cathayensis) kernel ethanol extracts, -tocopherol and butylated hydroxyanisole (BHA) were 87.8, 82.4 and 87.8%, respectively. Villarreal-Lozoya et al. (2007) reported that, the mean value of the scavenging effect on DPPH radical of different pecan cultivars was 487 ± 42 g Trolox equivalents per gram of defatted sample.The scavenging effect on DPPH radical of acetone extracts of kernel cake from Pecan nut were 68.0 ± 21.0 mg Trolox equivalent antioxidant capacity per gram of defatted sample (Prado et al., 2009b).Futher study on the milligram Trolox equivalent antioxidant capacity per gram of four species in this study should be done.

Reducing capacity
The Fe 3+ -Fe 2+ transformation was determined as the reducing capacity in this study.The presence of reductants (antioxidants) in the samples would result in the reduction of Fe 3+ to Fe 2+ by donating an electron.The amount of Fe 2+ complex can then be monitored by measuring the formation of Perl's Prussian blue at 700 nm.Increasing absorbance at 700 nm indicates an increase in reductive ability (Ebrahimzadeh et al., 2010).The reducing capacity of extracts from the four species of Carya kernels also increased with increasing amount of the extracts and decreased in the order: BHA > C. dabieshanensis > C. cathayensis > C. hunanensis > C. illinoensis (P < 0.05) and the absorbances were 1.19, 0.246, 0.237, 0.22 and 0.073 at the concentration of 0.50 mg/ml, respectively (Figure 2).Zhu et al. (2008) reported that the reducing capacity of Chinese Hickory (C.cathayensis) kernel ethanol extracts, -tocopherol and BHA at 700 nm was 0.76, 0.64, 1.08, at 200 l volume, respectively.It was found that, the reducing capacity of four species of Carya kernels was correlated with the phenolic compounds from the correlation analysis.

Superoxide anion scavenging activity
Compared with other oxygen radicals, the superoxide anion (O 2 •-) has a longer lifetime, can move to to a target cell at a longer distance and thus, is more dangerous.Therefore, it is important to study the ability of the extract to scavenge superoxide anion.Recent researches showed that, scavention of superoxide anion radicals is of importance for protection against early events in oxidative damage (Hu and Skibsted, 2002).In this study, the scavenging effect of extracts from four species of Carya kernels on superoxide anion radical followed the order: C. dabieshanensis > C. hunanensis > C. cathayensis > C. illinoensis (P < 0.05) and were 27.44, 26.15, 22.80 and 1.99 mg AE/g, respectively (Figure 3).Sun et al. (2004) reported that, the scavenging effect on superoxide was related to the number of active hydroxyl groups in the molecules.Therefore, the strong scavenging effect on superoxide by C. dabieshanensis, C. hunanensis and C. cathayensis may be due to the abundant phenolic hydroxyl groups.

Correlation between total phenolic content and antioxidant activity
It is important to examine the correlation between the content of the total polyphenols and antioxidant potential because some authors have reported that, there is no correlation between the content of these main antioxidant compounds and the radical scavenging capacity (Yu et al., 2002).The results obtained by us do not support these claims.In the present study, there is a strong correlation between total phenolic content and reducing power capacity (R 2 = 0.9985).In addition, the content of phenolic compounds was also highly correlated with 1,1-diphenyl-2-pycrylhydrazyl radical scavenging capacity (R 2 = 0.9675) and superoxide anion scavenging capacity (R 2 = 0.9739).These data are in accordance with others, who have shown that high total phenol content increases the antioxidant activity (Villarreal-Lozoya et al., 2007;Prado et al., 2009b).Therefore, the phenolics of Carya kernels may be responsible for its antioxidant properties, but further studies are warranted for the isolation and identification of individual phenolic compounds and also in vivo studies  are needed for a better understanding of their mechanism of action as an antioxidant.
Tannins are water-soluble polyphenols that are present in many plant foods.In this work, data also showed a strong correlation of condensed tannin with reducing power (R 2 = 0.9049), DPPH radical scavenging capacities (R 2 = 0.8544) and superoxide anion scavenging capacity (R 2 = 0.9195).This may be interpreted that, the tannins were the main polyphenolics of Carya kernels.A remarkable radical scavenging effect against DPPH (EC 50 = 0.34-4.72M) of tannins from the n-butanol (n-BuOH) extract of walnuts (the seeds of Juglans regia L.) were also measured by Fukuda et al. (2003).

Conclusions
The results from various free radical scavenging systems Carya species revealed that, the four species of Carya kernels have significant antioxidant activity.The significant variation in antioxidant properties, total phenolic content, condensed tannin content of different species of Carya was observed in this study.C. dabieshanensis possess the highest antioxidant activity and C. illinoensis had the lowest.A correlation between total phenolic content, condensed tannins and the antioxidant properties was also observed.This study can be used as a basis for future breeding programs aiming to develop Carya kernels with improved nutritional profile and health benefits.

Figure 1 .
Figure 1.DPPH radical scavenging effect of methanol extracts from defatted Carya kernels .Each value represents mean ± standard deviation of three replicates.

Figure 2 .
Figure 2. Reducing capapcity of methanol extracts from defatted Carya kernels.Each value represents mean ± standard deviation of three replicates.

Table 1 .
Total phenolic contents and condensed tannin contents of methanol extracts from defatted Carya kernels.