Antioxidant activity and DNA protection against oxidative damage of bambara groundnut seeds (Vigna subterranea (L.) Verdc.) as affected by processing methods

ABSTRACT This research investigated the effects of processing by dry heating, boiling, and steaming on the antioxidant activity and DNA protection against oxidative damage of bambara groundnut seeds (Vigna subterranea (L.) Verdc.). Comparing raw and processed samples of bambara groundnut seeds, dry heating caused a significant (P < 0.05) reduction of ferric-reducing antioxidant power (FRAP), metal chelating activity, DPPH• and ABTS•+ radical scavenging activity. The boiling process did not cause a significant difference in FRAP and metal chelating activity and caused smaller losses in DPPH• and ABTS•+ radical scavenging activity than the dry heating and steaming processes. The steaming process caused a significant (P < 0.05) reduction of FRAP, DPPH• and ABTS•+ radical scavenging activity. For DNA protection against oxidative damage, boiled and steamed bambara groundnut seed samples were more effective with a lower minimum concentration (50 µg/mL) than raw and dry heated samples. These results indicated that the boiling process caused smaller losses antioxidant activity than dry heating and steaming. Therefore, boiling was recommended as processing method for bambara groundnut seeds to preserve antioxidant components and antioxidant activity.


Introduction
Antioxidants are substances that have the ability to protect the body from damage caused by free radical-induced oxidative stress. They are derived from many dietary sources such as fruits, vegetables, and cereals. Properties of antioxidants are important in biological activity. Plant-derived antioxidants have been shown to function as free radical scavengers, singlet and triplet oxygen quenchers, metal chelators, chain-breakers, synergists, and enzyme inhibitors. [1] These antioxidants include vitamins, carotenoids, phenols, flavonoids, and endogenous metabolites. [2] Phenolic compounds are known to respond to metal chelation and offer anti-inflammatory, anti-cancer, and DNA protection. [3,4] Bambara groundnut (Vigna subterranea (L.) Verdc.) is a legume species of African origin and is widespread south of the Sahara. However, it is also cultivated in Southeast Asian countries such as Malaysia, Indonesia, and Thailand. The seeds of the bambara groundnut must be processed prior to consumption to destroy antinutritional factors such as protease inhibitors, lectins, and phytic acid. Changes in physical and biochemical properties of the food induced during processing could affect overall acceptability by consumers and the nutritional quality of the product. [5] The methods for processing bambara groundnut seeds prior to consumption vary by region. Previous study found that processing by roasting of Zimbabwean bambara groundnut had no effect on the nutrient content, while sifting significantly reduced the phenolic content and antioxidant activity of roasted samples. [6] However, no information is available about the effects of processing by dry heating, boiling, and steaming on antioxidant activity and DNA protection of bambara groundnut seeds.
Therefore, the objectives of this study were to evaluate the effect of processing methods (dry heating, boiling, and steaming) of bambara groundnut seeds on antioxidant compounds (total phenolic, tannin, and flavonoid content), antioxidant activity (ferric-reducing antioxidant power (FRAP), metal chelating activity, DPPH • and ABTS •+ radical scavenging activity), and DNA protection against oxidative damage.

Seed samples and processing
Bambara groundnut seeds, the Songkhla 1 variety (red seed coat) were purchased from a farmer in Trang Province, Thailand. Immature and damaged seeds were removed. Bambara groundnut seeds were randomly divided into four batches. The first batch of seeds was kept raw without any treatment. The second batch of seeds was dry heated in a hot-air oven at 120 o C for 90 min. The third batch of seeds was boiled using the following conditions: water in a ratio of 1:10 (w/v) at 100 o C for 30 min. The fourth batch of seeds was steamed at 120 o C at 15 pounds of pressure for 10 min. After decanting the water, the boiled and steamed seed samples were rinsed with distilled water and dried at 55°C for 20 min. All seed samples were ground to a fine powder using a coffee grinder and stored in separate screw-cap bottles at −20°C.

Sample extraction
The raw and processed ground seed samples were extracted by stirring with 80% methanol (1:5 w/v) at room temperature for 48 h. After extraction, the mixture was centrifuged at 12000 g for 15 min. The residues were re-extracted with an additional 50 mL of 80% methanol for 5 h. The solvent from both 80% methanol extractions was combined. To remove methanol, the solvent was evaporated under reduced pressure using a rotary vacuum-evaporator at below 40 o C. The remaining water was frozen at −80 o C and freeze-dried to obtain a powdered extract.

Determination of total phenolic content
The total phenolic content was determined according to the Folin-Ciocalteu method using gallic acid as a standard. [7] Each 50 mg of the powdered extract was placed in a test tube and made up to the volume of 1 mL with distilled water. A 25-µL aliquot of the dilution was transferred to the tube with the addition of 975 µL of distilled water and 0.5 mL of the Folin-Ciocalteu phenol reagent (1:1 with distilled water).
The reaction was mixed and incubated at room temperature for 5 min. Next, 2.5 mL of 20% sodium carbonate solution was added to each tube. The reaction was mixed using a vortex mixer and incubated in the dark at room temperature for 40 min. The absorbance was measured at 725 nm with UV-Vis spectrophotometer. The analysis was performed in triplicate. The total phenolic content was expressed in micrograms of gallic acid equivalent per gram of extract (µg GAE/g extract).

Determination of tannin content
The powdered extract of samples was prepared using the same method described for determination of total phenolic content. A 1-mL aliquot of the sample was mixed with 1 mL of 100 mg/mL PVPP and incubated at 4°C for 4 h. The mixture was centrifuged at 5000 g for 20 min at room temperature. The tannins were precipitated along with the PVPP. Then, the supernatant with only phenolics other than tannins was collected. The supernatant was measured for phenolics other than tannins using the same method described for determination of total phenolic content. The analysis was performed in triplicate. The tannin content was expressed in micrograms of gallic acid equivalent per gram of extract (µg GAE/g extract). The tannin content of the sample was calculated as:

Determination of total flavonoid
The total flavonoid content was determined according to the method of Zhishen et al. [8] using rutin as a standard. Each 50 mg of the powdered extract was added to a test tube, and made up to the volume of 1 mL with distilled water. A 100-µL aliquot of the dilution was transferred to the tube and 1.9 mL of distilled water and 0.15 mL of 5% sodium nitrite were added. The reaction was mixed and incubated at room temperature for 5 min. Next, 0.15 mL of 10% aluminum chloride solution was added to each tube. The reaction was mixed and incubated at room temperature for 6 min. Then, 1 mL of 1 M sodium hydroxide was added to the mixture, and the volume was immediately increased to 5 mL with distilled water. The absorbance was measured at 510 nm with a UV-Vis spectrophotometer. The total flavonoid content was expressed in micrograms of rutin equivalent per gram of extract (µg RE/g extract). The analysis was performed in triplicate.

Ferric-reducing antioxidant power (FRAP) assay
FRAP assay was determined according to the method described by Sasipriya and Siddhuraju. [9] This assay was used to determine the ferric-reducing antioxidant power of raw and processed bambara groundnut seed samples. The amount of Fe 2+ produced from the reduction of Fe 3+ was calculated from the ferrous sulfate standard curve. Each 50 mg of the powdered extract was added to a test tube, and made up to the volume of 1 mL with distilled water. A 5-µL aliquot of the dilution was transferred to the tube with the addition of 95 µL of distilled water and 900 µL of prepared freshly FRAP reagent (2.5 mL of 20 mM of TPTZ in 40 mM of HCl, 2.5 mL of 20 mM of ferric chloride, 25 mL of 0.3 M of acetate buffer, pH 3.6, incubated at 37°C for 30 min) before use. The mixture was incubated in the dark at 37 o C for 30 min in a water bath. After incubation, absorbance was measured at 593 nm with a UV-Vis spectrophotometer. The antioxidant activity was expressed as micromole of Fe (II) per gram extract (µmol Fe(II)/g extract). BHA, BHT, gallic acid, and rutin were used as positive controls. The analysis was performed in triplicate.

Metal chelating activity assay
The ability of raw and processed bambara groundnut seed samples to chelate ferrous ions was estimated using the method described by Dinis et al. [10] The 700 µL of the samples was added to 100 µL of 2 mM ferrous chloride solution. The mixture was incubated at room temperature for 30 s. Next, 200 µL of 1 mM ferrozine was added to the mixture, which was incubated for 10 min at room temperature. The absorbance was measured at 562 nm with a UV-Vis spectrophotometer. Chelating activity was calculated using the following formula: where A control is the absorbance of control reaction and A sample is the absorbance of the sample reaction. The result is expressed in terms of EC 50 value, which is the effective concentration at which the chelating activity is 50%. EDTA was used as a positive control.

DPPH • radical scavenging activity assay
The radical scavenging ability of raw and processed bambara groundnut seed samples was determined according to the method described by Blios. [11] The 50 µL of the samples was added to 2.5 mL of 0.1 mM methanolic solution of DPPH • . The mixture was incubated in the dark at 27 o C for 20 min. The absorbance was measured at 517 nm with a UV-Vis spectrophotometer. Radical scavenging activity was calculated using the formula: DPPH free radical scavenging activity % ð Þ¼ A control ÀA sample À Á =A control x100 The result was expressed in terms of EC 50 value, which is the effective concentration at which DPPH • radicals are scavenged by 50%. BHA, gallic acid, rutin, and trolox were used as positive controls.

ABTS •+ radical scavenging activity assay
For ABTS •+ assay, the method of Re et al. [12] was followed. ABTS •+ was dissolved in distilled water to make a concentration of 7 mM. ABTS •+ stock solution was produced by mixing the 7 mM ABTS •+ solution with 2.45 mM potassium persulfate (1:1). The mixture was allowed to stand in the dark at room temperature for 12 to 16 h. The ABTS •+ stock solution was diluted with distilled water to an absorbance of 0.70 ± 0.02 at 734 nm before use. To test the samples, 20 µL of the samples was added to 980 µL diluted ABTS •+ solution. The mixture was incubated at 30°C for 6 min before absorbance was read. The absorbance was measured at 734 nm with a UV-Vis spectrophotometer. Radical scavenging activity was calculated using the formula: ABTS þ free radical scavenging activity % ð Þ¼ A control ÀA sample À Á =A control x100 The result is expressed in terms of EC 50 value, which is the effective concentration at which ABTS •+ radicals are scavenged by 50%. BHA, BHT, gallic acid, rutin, and trolox were used as positive controls.

DNA protection study
Two microliters of 0.1 mg/mL pBR 322 plasmid DNA was mixed with 2 µL of the extracted samples. The reaction was incubated at room temperature for 10 min. After incubation, 1 µL of freshly made 100 mM AAPH was added. Distilled water was added to increase total volume in each tube to 20 µL. The reaction was incubated at 37 o C for 10 min. Ten microliters of the reaction were mixed with 1 µL of 4X loading dye and analyzed on 1% agarose gel.

Statistical analysis
The sample concentration providing 50% inhibition (EC 50 ) was analyzed by the Graphpad Prism 7 program. The EC 50 results were expressed as mean ± standard deviation (SD) of the responses of three replicates per sample. Statistical significance between the groups was determined by one-way ANOVA followed by Tukey's post hoc test. Differences were considered to be significant at P < 0.05. Correlation was determined by two-tailed Pearson correlation analysis. All statistical analyses were performed using SPSS 17.0.

Results and discussion
Total phenolic and tannin content The total phenolic and tannin content of the raw and processed bambara groundnut seed samples was calculated from the calibration curve of gallic acid (R 2 = 0.999). The results from Table 1 show that bambara groundnut seed samples processed by all methods (dry heating, boiling, and steaming) exhibited significant (p < 0.05) reduction in total phenolic and tannin content compared to the raw sample (about 8.47-11.86% reduction for total phenolic and 9.78-11.96% reduction for tannin).
These results indicate that processing caused degradation of phenolics and tannins. This could be due to phenolic breakdown during processing. [13] Xu and Chang [14] reported that thermal processing may cause degradation of polyphenols and the release of bound phenolic compositions. In other legumes, processing caused degradation of phenolics, with 30-40% of phenolics potentially removed from common beans (Proteus vulgaris) by cooking and discarding the cooking water. [15] The raw seed extract of Vigna vexillata (L.) A. Rich contains higher levels of total phenolics and tannins than processed seed extracts (i.e., dry heating, soaking followed by steaming with 0.1% sodium bicarbonate, soaking followed by autoclaving, and autoclaving without soaking). [5] Entada scandens seed kernels and Canavalia gladiata seeds have reduced phenolic, tannin, and flavonoid content after autoclaving and soaking followed by autoclaving. [9] According to Spanos and Wrolstad [16] processing and storage of grapes resulted in decrease of phenolic levels as measured by HPLC.

Total flavonoid content
The total flavonoid content of raw and processed bambara groundnut seed samples was calculated from the calibration curve of rutin (R 2 = 0.999). Total flavonoid content is presented in Table 1. Comparing the raw sample and the processed samples of bambara groundnut seeds, total flavonoid content was not significantly different among the raw, dry heated, and boiled samples. However, steaming caused a significant reduction (p < 0.05) in total flavonoid content of about 20.81% when compared to the raw bambara groundnut seed sample (Table 1).
Sasipriya and Siddhuraju [9] reported that leaching of soluble compounds into the cooking medium causes a decrease in flavonoid content. Moreover, the stability of flavonoids during heating may be due to the formation of antioxidant compounds like Maillard reaction products such as hydroxymethylfurfuraldehyde (HMF). [17]

FRAP assay
The FRAP activity of raw and processed bambara groundnut seeds is presented in Table 2. The reducing power was no significant differences between raw and boiled samples of bambara groundnut seeds. Dry heated and steamed samples of bambara groundnut seeds had significantly lower (p < 0.05) FRAP values compared to raw samples. There was a 17.58% reduction in FRAP values in the dry heated and steamed samples of bambara groundnut seeds. These results indicate that the dry heating and steaming processes cause reduced FRAP activity in bambara groundnut seeds compared to raw samples. The correlation analysis in Table 3 shows a significant positive correlation between FRAP and total phenolic content (r 2 = 0.919, P < 0.05) for bambara groundnut seeds. This indicates that total phenolic content of bambara groundnut seeds contributed to reduced activity. This result is similar to that of Sasipriya and Siddhuraju [9] , who found that phenolics in Entada scandens and Canavalia gladiate were the main contributors to their reducing activity. Benzie and Szeto [18] found that FRAP assay results correlate with total phenolic content. Moreover, Rice-Evans et al. [19] reported that phenolic compounds play a key role in antioxidant potential due to their redox properties (reducing agents, hydrogen donators, and singlet oxygen quenchers).

Metal chelating activity
The metal chelating activity EC 50 values of raw and processed bambara groundnut seed samples are presented in Table 2. There were no significant differences in metal chelating activity EC 50 values between the raw, boiled, and steamed samples of bambara groundnut seeds. The dry heated sample had significantly higher (p < 0.05) metal chelating activity EC 50 values than the raw sample. Dry heated sample had a 314.87% increase in metal chelating activity EC 50 values. These results are similar to those of Siddhuraju [17] , who found that metal chelating capability was higher for raw Table 2. FRAP, metal chelating, DPPH, and ABTS activity of raw and processed bambara groundnut seeds.  Vigna aconitifolia seeds than dry heated seeds. From the correlation analysis, total phenolic, tannin and flavonoid content in bambara groundnut seeds was not correlated with metal chelating activity (Table 3).

DPPH • radical scavenging activity
The DPPH • radical scavenging activity EC 50 values of raw and processed bambara groundnut seeds are presented in Table 2. A low EC 50 value indicates strong ability of the sample to act as a DPPH • radical scavenger. Dry heating, boiling, and steaming processes caused increased DPPH • EC 50 values of bambara groundnut seeds by about 8.51%, 5.67%, and 22.70%, respectively compared to the raw sample. However, there was no significant difference between the DPPH • EC 50 values of dry heated and boiled samples. These results indicate that the steamed sample had the highest increase in DPPH • EC 50 value. Among the positive controls, gallic acid exhibited the maximum scavenging effect at a very low concentration, with an EC 50 value of 0.52 µg/mL. These results indicate that bambara groundnut seeds reduced DPPH • radical scavenging activity for all types of processing.
A significant correlation coefficient (r 2 = 0.983, P < 0.01) was found between flavonoid and DPPH • radical scavenging activity for bambara groundnut seeds ( Table 3). The correlation coefficient for bambara groundnut seeds showed that the loss of DPPH • radical scavenging activity in processed samples was due to the leaching of flavonoids.

ABTS •+ radical scavenging activity
The results are presented in Table 2. A low EC 50 value indicates high antioxidant activity. The dry heated, boiled, and steamed samples had significantly lower (p < 0.05) ABTS •+ radical scavenging activity, with 24.59%, 14.75% and 22.30% increases in EC 50 values, respectively, compared to the raw sample. Among the positive controls, gallic acid showed the maximum scavenging effect at a very low concentration, with an EC 50 value of 0.51 µg/mL. The study results show that processing by dry heating, boiling, and steaming reduces ABTS •+ radical scavenging activity in bambara groundnut seeds.
A significant correlation coefficient confirms that phenolic (r 2 = 0.997, P < 0.01) and tannin (r 2 = 0.984, P < 0.01) content is correlated with ABTS •+ radical scavenging activity in bambara groundnut seeds (Table 3). Phenolics can act as free radical scavengers by donating their alcoholic hydrogen or one of their delocalized electrons to radicals. Hagerman et al. [20] reported that tannin has the ability to quench free radicals of ABTS •+ . In vitro studies have shown that flavonoids can directly scavenge molecular species of active oxygen such as superoxide, hydrogen peroxide, hydroxyl radical, single oxygen, or peroxyl radical. [21] DNA protection against oxidative aamage The results show that all the raw and processed bambara groundnut seed samples have the capacity to protect plasmid DNA against AAPH-induced oxidative damage. The DNA protective ability of raw and dry heated samples against AAPH was effective with minimum concentrations of 100 µg/mL and 50 µg/mL for the boiled and steamed samples (Figure 1). Cao et al. [22] reported that scavenging of peroxyl radicals is the ability of flavonoid. Moreover, flavonoids are effective inhibitors of lipid peroxidation and display pro-oxidant activity.
These results indicated that in general, boiling and steaming are the best method for processing bambara groundnut seeds to enhance DNA protection ability against peroxyl radicals. In addition, this suggests that consumption of bambara groundnut seeds may protect humans from peroxyl radical-induced diseases such as neurodegenerative diseases, carcinogenesis, and cytotoxicity.

Conclusion
In conclusion, the results obtained in the present study clearly demonstrate that the seed extracts of raw and processed bambara groundnut seeds contain a number of antioxidant compounds, including phenolics, tannins, and flavonoids, which offer effective antioxidant activity and DNA protection against oxidative damage. Processing by boiling caused smaller losses of antioxidant activity than dry heating and steaming. Therefore, boiling is recommended for processing bambara groundnut seeds to preserve antioxidant components and activity. Furthermore, boiling and steaming are the best methods for processing bambara groundnut seeds to enhance DNA protection against oxidative damage. Figure 1. DNA protection of raw and processed bambara groundnut seeds. Lane 1 is pBR 322 plasmid DNA. Lane 2 is pBR 322 plasmid DNA + AAPH. Lanes 3-16 are pBR 322 plasmid DNA + AAPH + bambara groundnut seed extract. The bambara groundnut seed extract concentrations of lanes 3-16 are 10,000, 5,000, 2,000, 1,000, 500, 300, 100, 50, 10, 5, 1, 0.5, 0.1, and 0.05 µg/mL, respectively. The arrows indicate the minimum concentration of extract that can protect DNA against oxidative damage.