Phenolic content and antioxidant capacity of Philippine potato (Solanum tuberosum) tubers

Journal of Food Composition and Analysis 22 (2009) 546–550

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Journal of Food Composition and Analysis journal homepage: www.elsevier.com/locate/jfca

Original Article

Phenolic content and antioxidant capacity of Philippine potato (Solanum tuberosum) tubers Rowena Grace O. Rumbaoa *, Djanna F. Cornago, Inacrist M. Geronimo Department of Food Science and Nutrition, College of Home Economics, University of the Philippines, 1101 Quezon City, Philippines

A R T I C L E I N F O

A B S T R A C T

Article history: Received 4 June 2008 Received in revised form 14 November 2008 Accepted 17 November 2008

Four potato (Solanum tuberosum) varieties – Bengueta, Ganza, Igorota and 125411.22 – were analyzed for total phenolic content and antioxidant activities to provide baseline data for Philippine potato varieties. Bengueta had the highest phenolic content with 50.0  1.5 mg gallic acid equivalent (GAE)/100 g (dry basis, DB). It also had the highest DPPH radical scavenging activity with an EC50 value of 30.6  3.6 mg/mL (DB). The potato variety125411.22 had the highest reducing power with EC50 equal to 66.2  1.6 mg/mL (DB), while Igorota had the highest iron-chelating capacity with an EC50 of 11.0  3.2 mg/mL (DB) and the best inhibitory action against linoleic acid oxidation at 95.4  2.2% at 50 mg/mL sample concentration. Methanolic potato extracts had better antioxidant activity than a-tocopherol and better iron chelating capacity than ethylenediamine tetraacetic acid (EDTA). Significant (*P < 0.05) negative correlation (R = 0.542) was observed between total phenolic content and the EC50 values for DPPH radical scavenging activity, but none between total phenolic content and reducing power, iron-chelating capacity and total antioxidant activity. ß 2008 Elsevier Inc. All rights reserved.

Keywords: Potato Solanum tuberosum Potato varieties Bengueta, Ganza, Igorota and 125411.22 Phenolic content Antioxidant activity Phillipine potato Radical scavenging Metal chelation Reducing power Food composition

1. Introduction Potato extracts exhibit antioxidant activity as demonstrated by Rodriguez de Sotillo et al. (1994a,b, 1998), Nara et al. (2006), Reyes (2005), Liu et al. (2003), Al-Saikhan et al. (1995), Cao et al. (1996), Halvorsen et al. (2002), Karadeniz et al. (2005), Vinson et al. (1998), Kaur and Kapoor (2002), Lachman et al. (2000) and Singh and Rajini (2004). The antioxidant activity of patatin, the tuber storage protein of potato, had also been investigated (Liu et al., 2003). The antioxidant property of dietary plants have been associated with phytochemicals such as a-tocopherol, ascorbic acid, b-carotene and phenolic compounds (Cao et al., 1996; Kaur and Kapoor, 2002; Kalt, 2005). Current antioxidant researches, however, are primarily focused on polyphenolic compounds, the principal components responsible for antioxidant activity as established by in vitro lipid oxidation models (Vinson et al., 1998; Kaur and Kapoor, 2002). Major phenolics in potato peel are chlorogenic acid, gallic acid, protocatechuic acid, caffeic acid and quercetin (Rodriguez de Sotillo et al., 1994a,b; Nara et al., 2006; Al-Saikhan et al., 1995). Other phenolic compounds in potato include ferulic acid, pcoumaric acid as well as small amounts of rutin, quercetin,

* Corresponding author. Tel.: +63 2 9202091. E-mail address: [email protected] (R.G.O. Rumbaoa). 0889-1575/$ – see front matter ß 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.jfca.2008.11.004

myricetin, kaempferol, naringenin and other flavonoids (Nara et al., 2006; Reyes, 2005). Purple-fleshed potato also contains petunidin- and malvidin-3-rutinoside-5-glycosides acylated with p-coumaric and ferulic acid while red-fleshed potato has pelargonidin- and peonidin-3-rutinoside-5-glycosides acylated with p-coumaric and ferulic acid (Reyes, 2005). According to the study conducted by Vinson et al. (1998), potato had the second best inhibitory action on low-density lipoprotein oxidation among 23 vegetables analyzed despite having the lowest phenolic content. Kaur and Kapoor (2002) classified several Asian vegetables according to total phenolic content and placed potato in the medium category where phenolic content is between 100 and 200 mg catechol/100 g. The oxygen radical absorbance capacity (ORAC) assay, conducted by Cao et al. (1996), ranks potato at 13 among 22 vegetables analyzed for antioxidant activity. Annual production of potato in the Philippines increased from 63,524 metric tons in 2000 to 70,160 metric tons in 2005. Of this, only 70.1% was made available for human consumption in 2000 and 73.8% in 2005 (BAS, 2006). Utilization of potato for its nutritive value and as a source of alternative food antioxidant to synthetic ones, such as butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA) and tertiary butylhydroquinone (TBHQ), may potentially boost consumption. It is the objective of the present study to provide baseline data on the phenolic content and antioxidant activity of Philippine potato varieties. Different varieties may exhibit varying

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total phenolic content and antioxidant capacity due to differences in flesh color and genotype. Antioxidant activities, including hydrogen and electron donation and metal chelation, of four methanolic potato extracts were compared to the activity of a commercially available antioxidant, alpha-tocopherol, and a metal chelator, ethylenediamine tetraacetic acid (EDTA). 2. Materials and methods 2.1. Reagents The reagents used in the study, such as gallic acid (HiMedia, Hi-Media Laboratories Pvt. Ltd., India), ethylenediamine tetraacetic acid (EDTA), disodium salt (Hi-Media, Hi-Media Laboratories Pvt. Ltd., India), ferrozine or 3-(2-pyridyl)-5,6bis(4-phenylsulfonic acid)-1,2,4-triazine, monosodium salt (Fluka, Sigma–Aldrich, U.S.A.) and Folin-Ciocalteu’s phenol reagent were procured from Belman Laboratories (Quezon City, Philippines). Linoleic acid (Cica, Acros Organics, Japan) was purchased from Just-In-One Marketing (Caloocan City, Philippines). All other chemicals, including a-tocopherol (Sigma, Sigma–Aldrich, U.S.A.) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) (Sigma, Sigma–Aldrich, U.S.A.), were purchased from Chemline Scientific Industries (Quezon City, Philippines). Analytical grade reagents were used. 2.2. Sample Potato samples were provided by the Northern Philippines Root Crop Research and Training Center (Benguet, Philippines). Four potato varieties were harvested at 4 months in January of 2007 from Buguias, Benguet, Philippines (168430 5300 N; 1208470 4400 E, elev: 7276 ft). Sampling from the bulk harvest was made by convenient sampling. They were analyzed in the study using completely randomized design. Bengueta, Ganza and Igorota have yellow skin and flesh, while, 125411.22 has red skin and yellow flesh. The samples were weighed, washed, cut into 2 cm slices and steamed at 100 8C for 15 min to prevent browning of flesh. Samples were then cooled, peeled, cut into 2 cm3 cubes, lyophilized and ground to fine powder. The flour was stored in a resealable bag at 4 8C until use. 2.3. Extraction of phenolic compounds Ten grams of potato flour were mixed with 80 mL methanol and kept overnight. The suspension was filtered through Whatman No. 1 filter paper and the filtrate was diluted to 100 mL with methanol. Sample solutions were stored at 4 8C in amber bottles and served as the stock solution (100 mg/mL) for subsequent analyses. 2.4. Determination of total phenolic content A modified method by Slinkard and Singleton (1997) was used to determine the total phenolic content of the samples. Briefly, two hundred microliters of the sample was mixed with 1.4 mL distilled water and 100 mL of Folin-Ciocalteu reagent. After at least 30 s (but not exceeding 8 min), 300 mL of 20% Na2CO3 solution was added and the mixture was allowed to stand for 2 h. The absorbance was read at 765 nm with Lambda 1 UV–Vis Spectrophotometer (PerkinElmer, U.S.A.). Standard gallic acid solutions (10–100 ppm) were prepared similarly for the construction of the calibration curve. Results were expressed as mg gallic acid/100 g dry sample and per 100 g fresh sample. The test was run in duplicate and analysis of the samples was run in triplicate and averaged.

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2.5. Antioxidant activity by ferric thiocyanate method Antioxidant activity was determined using the ferric thiocyanate method used by Huang et al. (2006). One mL aliquot of a 50 mg/mL sample solution was placed in a screw cap container and mixed with 1 mL of 2.51% (v/v) linoleic acid solution in 99.5% (w/v) ethanol, 2 mL of 0.05 M phosphate buffer (pH 7.0), and 1 mL distilled H2O. The reaction mixture was incubated in the dark at 40 8C. For the control solution, the sample was substituted with 1 mL methanol in the reaction mixture. A 0.1 mL aliquot of the reaction mixture was then mixed with 9.7 mL of 75% (v/v) ethanol, 0.1 mL of 30% (w/v) NH4SCN and 0.1 mL of 20 mM FeCl2 in 3.5% (v/v) HCl. The absorbance was read at 500 nm using UVPC-3101 UV–Vis-NIR Spectrophotometer (Shimadzu, Japan) after 3 min. The process was repeated every 24 h until the absorbance of the control solution reached the maximum value. Percent activity was calculated as follows: % Activity ¼

  DASx 1  100 DAblk

(1)

where DA is the absorbance increase. The test was run in duplicate and analysis of the samples was run in triplicate and averaged. 2.6. DPPH radical scavenging activity DPPH radical scavenging activity of potato extracts was determined using the method outlined by Huang et al. (2005b). Sample solutions with concentrations 10, 20, 30, 40 and 50 mg/mL were prepared from the stock solution. A 1-mL aliquot was mixed with 1 mL of freshly prepared 80 ppm DPPH in methanol. The mixture was kept in the dark for 30 min. The absorbance was then read at 517 nm using Lambda 1 UV–Vis Spectrophotometer (PerkinElmer, U.S.A.). The radical scavenging activity of atocopherol (10–50 ppm) was also determined. Percent activity was calculated using the equation: % Activity ¼

  ASx 1  100 Ablk

(2)

The EC50 value, which is the sample concentration at 50% activity, was determined by interpolation. The test was run in duplicate and analysis of the samples was run in triplicate and averaged. 2.7. Reducing power Reducing power of the extracts was determined using the method described by Singh and Rajini (2004). Sample solutions with concentrations 20, 40, 60, 80 and 100 mg/mL were prepared from the stock solution. One mL of 0.2 M phosphate buffer pH 6.6 and 1 mL of 1% (w/v) K3Fe(CN)6 were added to a 1 mL aliquot of the extract. The mixture was incubated at 50 8C for 20 min. Ten percent (10%, w/v) trichloroacetic acid (1 mL) was added to the mixture. The resulting mixture was centrifuged at 3000 rpm for 10 min. One mL of distilled water and 0.2 mL of 0.1% (w/v) FeCl3 solution were added to 1 mL of the supernatant. The absorbance was read at 700 nm using Lambda 1 UV–Vis Spectrophotometer (PerkinElmer, U.S.A.). The reducing power of a-tocopherol (20–100 ppm) was also determined. The EC50 value, the concentration at which the absorbance is 0.500, was determined by interpolation. The test was run in duplicate and analysis of the samples was run in triplicate and averaged. 2.8. Iron-chelating capacity The iron-chelating capacity of the sample was determined based on the procedure described by Hsu et al. (2003). Sample

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solutions of 5, 10, 15, 20 and 25 mg/mL concentrations were prepared from the stock solution. One mL aliquot was mixed with 1 mL of methanol, 0.1 mL of 2 mM FeCl24H2O, and 0.2 mL of 5 mM ferrozine. The mixture was allowed to stand for 10 min; then the absorbance was read at 562 nm using Lambda 1 UV–Vis Spectrophotometer (PerkinElmer, U.S.A.). EDTA, with concentration ranging from 25 to 50 ppm, was used as positive control. Percent activity was calculated using Eq. (2). The EC50 value, which is the sample concentration at 50% activity, was determined by interpolation. The test was run in duplicate and analysis of the samples was run in triplicate and averaged.

Table 1 Total phenolic content of potato samples. Variety

Total phenolic content (mg gallic acid equivalent/100 g sample)e Dry basis

Bengueta Ganza Igorota 125411.22

Wet basis a

9.9  0.3a 6.9  0.2d 8.1  0.3b 7.6  0.3c

50.0  1.5 34.4  0.8d 47.4  1.8b 39.1  1.6c

Means with different letters (a–d) within the same column differed significantly (*P < 0.05). e Each value is expressed as the mean  standard deviation (n = 6).

2.9. Statistical analyses Data were analyzed using univariate analysis of variance and means were compared using Duncan’s multiple range test. The Statistical Analysis Software for Windows (v. 6.12) was employed. A difference was considered to be statistically significant when the P-value is less than 0.05 (*P < 0.05). Correlation tests were done using Microsoft Excel 2007. 3. Results and discussion 3.1. Total phenolic content The total phenolic content of the potato varieties (Bengueta, Ganza, Igorota and 125411.22) were expressed as mg gallic acid equivalent (GAE) per 100 g sample as shown in Table 1. Phenolic content for the potato samples analyzed in the study ranged from 34.4 to 50.0 mg GAE/100 g dry sample (or 6.9–9.9 mg GAE/100 g fresh sample). Among the potato varieties, Bengueta had the highest phenolic content followed by Igorota, 125411.22 and Ganza. All the four potato varieties were found to be significantly (*P < 0.05) different from each other in terms of phenolic content. This discrepancy among varieties may be attributed to genotypes and harvest location which influence the accumulation of phenolic compounds by synthesizing different quantities and/or types of phenolics (Shahidi and Naczk, 1995; Lachman et al., 2008; Sulc et al., 2008). Antioxidant activity values also depend strongly on the preparation of sample (leaching, extended steaming, lyophilisation) and the method used (ferric thiocyanate method) (Sulc et al., 2008). Al-Saikhan et al. (1995) reported that potato contains 237.7–527.2 mg chlorogenic acid equivalent (CAE)/g fresh weight (11.41–27.47 mg GAE/100 g), while, Karadeniz et al. (2005) reported a phenolic content of 553  102.5 mg catechin/kg fresh weight (32.44  6.07 mg GAE/100 g). Higher values for phenolic content of potato were obtained by Kaur and Kapoor (2002) and Vinson et al. (1998), 149.8  6.3 mg catechol/100 g fresh weight (231.46  9.73 mg GAE/100 g) and 5.9  3.9 mmol catechin/g dry sample (100.37  66.35 mg GAE/100 g), respectively. Sample treatment and extraction conditions are important factors in determining the phenolic content of potato. Vigorous extraction methods, including homogenization, heating and hydrolysis, result in higher yield as demonstrated in literatures (Huang et al., 2005b; Vinson

et al., 1998; Kaur and Kapoor, 2002). According to Chu et al. (2002), bound-form phenolics account for as much as 40% of the total phenolic content of the edible part of potato, while, Vinson et al. (1998) puts the amount of conjugated phenolics in potato at 57.9  13.4%. Moreover, the low yield in the present study may be a consequence of steam blanching prior to freeze-drying. Although steam blanching was employed primarily to deactivate degradative enzymes while minimizing losses of phenolic substances due to leaching, extended steaming may result in losses of phenolic compounds due to their susceptibility to leaching from the plant tissue and degradation of heat sensitive phenolic substances (Kalt, 2005). Nara et al. (2006) and Reyes (2005) also cites the tendency of phenolic compounds to accumulate at the peel as the cause of lowphenolic content in the flesh of potatoes. 3.2. Antioxidant activity by ferric thiocyanate method The ability of antioxidants to scavenge peroxyl radicals through hydrogen donation during polyunsaturated fatty acids (PUFA) oxidation was assessed using the ferric thiocyanate method (Lee et al., 2004; Huang et al., 2005a). Thiocyanate reacts with ferric ion, formed through reduction of the ferrous ion by peroxides, to produce a red-colored complex, the absorbance of which is monitored every 24 h until the reaction is complete (Elmastas et al., 2006). In the study, it took 7 days for the reaction to be completed. Inhibition by the methanolic potato extracts at 50 mg dry sample is between 93.5  1.7% and 95.4  2.2% (Table 2). Igorota had the highest antioxidant activity, while, Bengueta had the lowest. However, the difference between the antioxidant activities of the four potato varieties is statistically insignificant (*P > 0.05). Most studies based evaluation of antioxidant activity on the coupled oxidation of b-carotene and linoleic acid (Al-Saikhan et al., 1995; Karadeniz et al., 2005; Kaur and Kapoor, 2002). Al-Saikhan et al. (1995) obtained antioxidant activity between 65.2% and 89.2% at 30 mg sample for yellow- and white-fleshed varieties of potato. Activity of ethanolic and aqueous potato extract at 40 mg is 62.3% and 62.5%, respectively (Kaur and Kapoor, 2002). Karadeniz et al. (2005), on the other hand, reported an activity of 14.2  2.3% at 8 mg sample. The samples had better inhibitory action than a-tocopherol on a mg analyte basis, the latter having an activity of 83.5% at 101 mg. Antioxidants must have a

Table 2 Antioxidant activity of potato samples. Variety

% Inhibition at 50 mg/mL

EC50 value (mg/mL sample, dry basis)e DPPH scavenging capacity

Bengueta Ganza Igorota 125411.22

a

93.5  1.7 93.6  3.0a 95.4  2.2a 95.0  5.1a

d

30.6  3.6 48.6  3.1a 38.2  1.1b 34.6  1.3c

Means with different letters (a–d) within the same column differed significantly (*P < 0.05). e Each value is expressed as the mean  standard deviation (n = 6).

Reducing power b

72.4  3.6 78.7  5.6a 69.2  3.8b,c 66.2  1.6c

Iron-chelating ability 12.4  2.8a,b 14.7  3.2a 11.0  3.2b 11.7  1.2a,b

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lower reduction potential than PUFA (600 mV) for it to be an effective free radical scavenger (Lee et al., 2004). No significant correlation (*P > 0.05; R = 0.014) was found between total phenolic content and antioxidant activity of the potato extracts. Al-Saikhan et al. (1995) reported a low-positive correlation between the two parameters as well. This suggests the presence of other non-phenolic constituents that contribute to antioxidant activity through hydrogen donation. Possible components include ascorbic acid and carotenoids (Kalt, 2005), both of which are present in potato (Al-Saikhan et al., 1995). 3.3. DPPH radical scavenging activity The ability of phenolic compounds to quench reactive species by hydrogen donation was measured through the DPPH radical scavenging activity assay (Singh and Rajini, 2004). Activity is measured as the relative decrease in absorbance at 517 nm as the reaction between DPPH and antioxidant progresses (Huang et al., 2004; Singh and Rajini, 2004). DPPH radical scavenging activity is plotted as a function of mg per mL sample concentration in Fig. 1. Percent activity was observed to increase with sample concentration between 10 and 50 mg per mL, equivalent to about 3–25 mg GAE. Antioxidant activity was evaluated with EC50 values, the concentration at which radical scavenging activity is 50%, as listed in Table 2. The results indicate EC50 values of the potato varieties ranging from 30.6 to 48.6 mg dry sample per mL. Bengueta had the highest radical scavenging activity, while, Ganza had the lowest. EC50 values of the four potato varieties differed significantly (*P < 0.05). Genotype and growth conditions, such as water availability, light quality and temperature, affect the synthesis and accumulation of phenolic compounds in some parts of the plant; and consequently, antioxidant activity (Reyes, 2005; Kalt, 2005). The radical scavenging activity of the potato samples, on a mg phenolic content basis, was higher (EC50 = 13.5–18.1 mg GAE/mL) than that of a-tocopherol (EC50 = 23.0 mg/mL). Alpha-tocopherol acts mainly through hydrogen transfer and scavenging of reactive species (Lee et al., 2004), which accounts for its high-EC50 value. There is evidence that hydrogen abstraction is only a marginal pathway in the reaction between antioxidant and DPPH (Prior et al., 2005). Significant negative correlation (*P < 0.05; R = 0.729) was observed between total phenolic content and EC50 values for DPPH radical scavenging activity, indicating direct contribution of

Fig. 1. DPPH radical scavenging activity of methanolic potato extract. Values are means of six trials.

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phenolics to this activity. This is consistent with the results of a similar study by Nara et al. (2006). Reyes (2005) also reported correlation between phenolic content and antioxidant activity against free radicals although the reactive species used in the study was not specified. 3.4. Reducing power The potassium ferricyanide reduction method was used to measure the ability of phenolic compounds to quench radicals through electron donation. Activity is monitored by measuring the absorbance of Perl’s Prussian blue complex at 700 nm, which increases as antioxidants reduce the ferric ion/ferricyanide complex to the ferrous form (Chou et al., 2003). Fig. 2 shows the plot of reducing power of the methanolic potato extracts as a function of mg per mL sample concentration. A linear increase in reducing power was observed over the concentration range 20– 100 mg sample, equivalent to 3–45 mg GAE. The EC50 value of potato, the concentration at which the absorbance is 0.500, is between 66.2 and 78.7 mg/mL, DB (Table 2). Significant differences (*P < 0.05) were observed between varieties in terms of reducing power although Bengueta was not significantly different from Igorota which was also not significantly different from 125411.22. This may be a consequence of the variability of phenolic compounds present, as affected by genotype and environmental conditions. 125411.22 had the lowest EC50 value, that is, the highest reducing power, while Ganza had the lowest reducing power among the four varieties. The samples had better reducing power (EC50 = 25.1–36.1 mg GAE/mL) than a-tocopherol (EC50 = 94.0 mg/mL), on a mg analyte basis, which is expected since a-tocopherol does not act via electron donation. No correlation was found between reducing power and phenolic content. This indicates the presence of nonphenolic compounds capable of electron donation. Potato also contains carotenoids and vitamin C (Al-Saikhan et al., 1995), both of which can act as electron donors (Kalt, 2005). 3.5. Iron-chelating capacity The ability of antioxidants to form insoluble metal complexes with ferrous ion or to generate steric hindrance that prevent interaction between metal and lipid is evaluated using the ironchelating capacity assay (Hsu et al., 2003). Activity is measured by monitoring the decrease in absorbance of the red Fe2+/ferrozine complex as antioxidants compete with ferrozine in chelating ferrous ion (Elmastas et al., 2006). The plot of iron-chelating capacity as a function of mg per mL sample concentration is shown

Fig. 2. Reducing power of methanolic potato extract. Values are means of six trials.

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References

Fig. 3. Iron chelating capacity of methanolic potato extract. Values are means of six trials.

in Fig. 3. A sigmoidal curve was obtained over the concentration range of 5–25 mg/mL sample concentration, equivalent to 1–13 mg GAE. This indicates that chelating capacity reaches a plateau at high-sample concentrations. EC50 values, the concentration at 50% metal chelation, of the potato samples are listed in Table 2. Values ranged from 11.0 to 14.7 mg/mL, DB. Igorota had the highest chelating capacity among the potato varieties. However, its EC50 value is not significantly different (*P < 0.05) from 125411.22 and Bengueta. Ganza had the lowest chelating capacity among the four varieties. The samples had better chelating capacity (EC50 = 4.55– 6.22 mg GAE/mL) than EDTA (EC50 = 30.0 mg/mL), based on EC50 values in terms of mg analyte. No correlation was found between iron-chelating capacity and phenolic content for potato. This may indicate the presence of other antioxidants responsible for metal chelation. Non-phenolic metal chelators include phosphoric acid, citric acid, ascorbic acid, carnosine, some amino acids, peptides and proteins such as transferrin and ovotransferrin (Lee et al., 2004). Al-Saikhan et al. (1995) reported that potato contains ascorbic acid and reduced tripeptides. 4. Conclusions Methanolic potato extracts have significant antioxidant activity compared to the control samples, a-tocopherol and EDTA. Differences among the four samples – Bengueta, Ganza, Igorota and 125411.22 – in terms of phenolic content, DPPH radical scavenging activity, reducing power and iron chelating capacity may be especially due to genotype and potato cultivar or variety. The correlation between total phenolic content and DPPH radical scavenging activity indicates that phenolic compounds are responsible for antiradical activity. On the other hand, the lack of correlation between phenolic content and reducing power, ironchelating capacity and inhibition of linoleic acid oxidation suggests the presence of non-phenolic components responsible for antioxidant activity. Identification of the antioxidants in potato responsible for hydrogen and electron donation and metal chelation will supplement the findings of the study. The prospect of utilizing potato extracts as a commercial antioxidant will be greatly advanced through optimization of the extraction procedure. Acknowledgements The authors acknowledge the Northern Philippines Root Crop Research and Training Center for providing the rootcrop samples and the University of the Philippines-Office of the Vice Chancellor for Research and Development for financial support.

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