Nutritional Profile and Radical Scavenging Capacity of Tubers of Two Dioscorea Species

This study was aimed at evaluating the nutritional and antioxidant properties of raw and boiled tubers of Dioscorea dumetorum and D. hirtiflora. Tubers of D. hirtiflora were characterized by higher total essential amino acids (9576 mg/100 g dry weight (dw)) than those of D. dumetorum (1085 mg/100 g dw). Boiling decreased the total amino acid content of both tubers. Monounsaturated Fatty Acids (MUSFAs) and Polyunsaturated Fatty Acids (PUFAs) accounted for 15.92 and 26.64% in D. dumetorum tubers respectively whereas, MUSFAs and PUFAs accounted for 29.62 and 24.11% in D. hirtiflora tubers respectively. Boiling increased the total unsaturated fatty acids from 42.56 to 56.81% in D. dumetorum tubers and decreased it from 53.73 to 49.08% in D. hirtiflora tubers. The total phenolic content of boiled D. hirtiflora tubers showed 4.5-fold gain which might explain partly the increase in their antioxidant capacity (IC50 22.35 mg/L in DPPH assay). Boiling did not change significantly the antioxidant activity of D. dumetorum tubers (IC50 74.13 mg/L in DPPH assay) although a significant decrease in total phenolic (60%) and vitamin C contents (56.7%) was observed. These results suggest that boiled tubers of D. dumetorum and D. hirtiflora can contribute significantly to human nutrition and health.


INTRODUCTION
In many developing countries people utilize wild edible plants to meet their food needs especially in periods of food shortage (Bussmann et al., 2006;Grivetti and Ogle, 2000;Medley and Kalibo, 2007).
A balanced diet fully meets all the nutritional needs of a person.One of the most common causes of dietary deficiencies and food insecurity appears to be the decreasing diversity of traditional diets (Batal and Hunter, 2007).Several studies suggest that consumption of many different wild edible plants as food provides favorable nutritional effects (Vanderjagt et al., 2000;Cook et al., 2000;Doka et al., 2014).
There are a number of clinical studies suggesting that the antioxidants in fruits, vegetables, tea and red wine have protective effect against many human neurologic disorders, heart disease and some cancers (Miller et al., 2000).Antioxidants present in edible plants involve mainly vitamin antioxidants (vitamin E, vitamin C, carotenoids) and polyphenols (Bouayed and Bohn, 2013).
Local people of Kordofan (Western Sudan) know about the importance and the contribution of wild plants such as yam to their daily diet.Moreover, during the famine of 1988 wild edible plants contributed more than any other food sources in saving the lives of a large number of famine victims.Two species of yam (family Dioscoraceae) were commonly eaten; Dioscorea dumetorum (Kunth) Pax. and D. hirtiflora Benth.People remove the bitter taste of yam by soaking the sliced tubers in water and a meal is prepared by either being cooked with meat or ground into fine flours and cooked with milk or boiled and eaten as chips.
No detailed reports evaluating the nutritional content and beneficial effect of boiled D. dumetorum and D. hirtiflora tubers were presented in the literature.Therefore, the present study aims to analyze the amino acid content, fatty acid profile, vitamin C, total phenolics and radical scavenging capacities of raw and boiled tubers of D. dumetorum and D. hirtiflora.

MATERIALS AND METHODS
Plant materials: Plants were collected from Southern-West Kordofan in July 2009, were identified and voucher specimens (voucher No. of D. dumetorum 1109 KD3 andof D. hirtiflora 1109 KD4) were deposited in the Herbarium of Botany Department, Faculty of Science, University of Khartoum.
Preparation of samples and extracts: Tubers were washed with tap water after removing manually inedible parts, peeled and sliced.Leaching of the bitter taste of tubers was accomplished by steeping in water for three days using fresh water each day.Each sample 263 was divided into two portions.One portion was retained raw, the other was cooked by boiling.Samples (100 g) were added to 150 mL of water that had just reached the boil in a stainless steel pan and cooked for 10 min.The samples were drained off and air dried.Dry raw and processed samples were pounded and were kept at -20°C until analyses.All calculations were made according to dry matter basis.
Ethanolic extracts of raw and processed samples were also prepared for total phenolic and antioxidant capacity determination.The ethanol extract was prepared by soaking 20 g of sample in 200 mL ethanol at ambient temperature for 6 h.The extract was decanted, filtered and concentrated in a rotary evaporator to yield 0.8 and 1.2 g from raw and boiled tubers of D. dumetorum respectively and 1.3 and 1.9 g from raw and boiled D. hirtiflora tubers respectively.

Chemicals:
Ninhydrin, boron trifluoride, metaphosphoric acid, dichloroindophenol sodium salt, Folin-Ciacalteu reagent, L-ascorbic acid, gallic acid, 1, 1-diphenyl-2-picrylhydrazyl (DPPH), 2, 2'-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), potassium persulfate were purchased from Sigma-Aldrich (France).Other chemicals used were all analytical grade.Amino acids analysis: Amino acids composition of samples was measured as hydrolysate using an amino Acid Analyzer (Sykam-S7130) based on high performance liquid chromatography technique.Sample hydrolysis was prepared following the method of Moore and Stein (1963).Two hundred mg of sample were taken into a hydrolysis tube.Five mL 6 N HCl were added to the sample.The tube was tightly closed and incubated at 110°C for 24 h.After incubation period, the solution was filtered and 200 μL of the filtrate were evaporated to dryness at 140°C for an hour.The hydrolysate was diluted with one mL of buffer (citrate buffer pH 2.2).Aliquot of 150 μL of sample hydrolysate was injected in cation separation column at 130°C.Ninhydrin solution and an eluent buffer (the buffer system composed of solvent A of pH 3.45 and solvent B of pH 10.85) were delivered simultaneously into a high temperature reactor coil (16 m length) at a flow rate of 0.7 mL/min.The buffer/ninhydrin mixture was heated at 130°C for 2 min to accelerate chemical reaction of amino acid with ninhydrin.The products of the reaction mixture were detected at wavelength of 570 nm (440 nm for proline) on a dual channel photometer.The amino acids were identified by their retention time and wavelength ratio calculated from the areas of standards obtained from the integrator and expressed as mg/100 g.

Fatty acids:
Fatty acid profiles of total lipids were determined after transesterification with 14% boron trifluoride in methanol (1:1 v/v).Fatty acid methyl esters were analyzed by GC-MS (QP 2010 Shimadzu GC-MS equipment).Supelco equity 1 column with a film thickness of 30 m×0.25 microns was used.The total flow rate was 24 mL/min and column flow rate was 1 mL/min.Ultra high purity Helium was used as the carrier gas with injector split ratio of 20: 1.The ion source and inter-phase temperatures were 200 and 250°C, respectively.The solvent cut time of 4 min and detector gain was 0.70 kv.A Wiley 229 library search was conducted on major peaks of the sample in order to identify the components of the sample.The relative percentage of each compound was determined.

Determination of vitamin C:
The modified method of Bahorun et al. (2004) was used to determine the vitamin C content of Dioscora spp tubers.A 10 g of sample was blended with 40 mL of a solution of 3% metaphosphoric acid in 8% glacial acetic acid, pH 1.5, for 1 min.The extract was then mechanically shaken for 15 min in darkness filtered through glass wool.After filtration the clear extract was stored at -40°C prior to analysis by the 2, 6-dichloroindophenol titrimetric method (AOAC, 1995).Triplicate titration was conducted for all samples.
Determination of total phenolics: Total phenol contents in the extracts of raw and boiled Dioscora spp tubers were determined using modified Folin-Ciocalteu method (Wolfe et al., 2003).Ethanol extracts were resuspended in ethanol to make 50 mg/mL stock solutions.An aliquot of the extract was mixed with 5 mL Folin-Ciocalteu reagent (previously diluted with water at 1:10 v/v) and 4 mL (75 g/L) of sodium carbonate.The tubes were vortexed for 15 s and allowed to stand for 30 min at 40°C for color development.Absorbance was then measured at 765 nm using the SHIMADZU UV-2550 UV-VS spectrophotometer.Total phenolic contents were expressed as gallic acid equivalents (mg/100 g) using the following equation based on the calibration curve: y = 0.0057x, R2 = 0.9315, where x was the absorbance.

DPPH radical-scavenging test:
Antioxidant activity of the extracts of raw and boiled Dioscora spp tubers was estimated using DPPH in vitro method (Mensor et al., 2001).Test samples were dissolved separately in methanol to get test solution of 1 mg/mL.Series of extract solutions of different concentrations (1,5,10,20,40,60,80 and 100 μg/mL) were prepared by diluting with methanol.Assays were performed in 96well, microtiter plates.140 μL of 0.6×10-6 mol/L DPPH was added to each well containing 70 μL of sample.The mixture was shaken gently and left to stand for 30 min in dark at room temperature.The absorbance was measured spectrophotometrically at 517 nm using a microtiter plate reader (Synergy HT Biotek, logiciel GEN5).Blank was done in the same way using methanol and sample without DPPH and control was done in the same way but using DPPH and methanol without sample.Ascorbic acid was used as reference antioxidant compound.Every analysis is done in triplicate.
The ability to scavenge DPPH radical was calculated by the following equation:

ABTS radical-scavenging test:
A second in vitro method was performed to estimate antioxidant potential of the extracts: ABTS assay, based on the method of Re et al. (1999).Test samples were dissolved separately in methanol to get test solution of 1 mg/mL.Series of extract solutions of different concentrations (1,5,10,20,40,60,80 and 100 μg/mL) were prepared by diluting with methanol.The ABTS radical cation (ABTS *+ ) was produced by reacting 7 mM stock solution of ABTS with 2.45 mM potassium persulfate and allowing the mixture to stand in the dark at room temperature for 12 h before use.The obtained ABTS *+ solution was diluted with methanol to an absorbance of 0.700±0.02at 734 nm.190 μL of ABTS *+ solution was added to each well containing 10 μL of sample.The mixture was shaken gently and left to stand for 15 min in dark at room temperature.The absorbance was measured spectrophotometrically at 734 nm using a microtiter plate reader (Synergy HT Biotek ® , logiciel GEN5).The ABTS *+ scavenging capacity of the extract was compared with that of ascorbic acid and the percentage inhibition calculated as: The IC 50 value was calculated from the linear regression of plots of concentration of the test sample against the mean percentage of the antioxidant activity obtained from three replicate assays.The IC 50 values obtained from the regression plots (Sigma PlotsR 2001, SPSS Science) had a good coefficient of correlation, (R2 = 0.9926).

Statistical analysis:
All analyses were performed in triplicate and data reported as mean±standard deviation (SD).Differentiation between data sets was determined by Student's t-test and significant differences were considered when means of compared sets differed at p<0.05.

RESULTS AND DISCUSSION
Amino acids content: The composition and amount of amino acids in D. dumetorum and D. hirtiflora raw and boiled tubers are presented in Table 1.Comparing the total amounts of amino acids in the two Dioscorea species, the amount of the essential and non-essential amino acids in D. hirtiflora was higher.The total amount of essential amino acids was 9576 mg/100 g in raw D. hirtiflora tubers and 1085 mg/100 g in raw D. dumetorum tubers whereas the total amount of nonessential amino acids was 13869 mg/100 g in raw D. hirtiflora tubers and 3075 mg/100 g in raw D. dumetorum tubers.The contents of all amino acids varied significantly (p<0.05)within both Dioscorea species.Boiling decreased the total amino acids of both species.A decrease (23.29%) in total essential amino acids content in D. dumetorum was observed after processing.However, boiling increased significantly (p<0.05)isoleucine from 43 mg/100 g to 265 mg/100 g (13.2% of RDA) and methionine from 18 mg/100 g to 57 mg/100 g (5.7% of RDA) whereas phenylalanine was reduced significantly (p<0.05) from 106 mg/100 g to 67 mg/100 g.Comparison of these data of D. dumetorum tubers from Sudan with previous works on amino acid composition of wild and edible D. dumetorum varieties from Nigeria showed that the Nigerien species contained higher level of amino acids with remarkable high amount of leucine ranged from 2280 to 4900 mg/100 g and lysine ranged from 1140 to 490 mg/100 g (Alozie et al., 2009;Lape and Treche, 1994).Variations in levels of nutrients between Sudanese and Nigerian D. dumetorum tubers could be explained partly by variation in nature of the soil, seasonality, genetic diversity and stage of maturity (Guthrie and Picciano, 1995;Greenfield and Southgate, 2003).

Fatty acids composition:
The composition of fatty acids in D. dumetorum and D. hirtiflora tubers is shown in Table 2. Total saturated fatty acids ranged from 44.72% in D. hirtiflora tubers to 57.30% in D. dumetorum tubers.These values were higher than the values reported for Nigerian edible (36.7%) and wild (36.5%)D. dumetorum (Alozie and Akpanabsatu,  Alozie and Akpanabsatu (2010).Boiling of D. dumetorum tubers increased the Total Unsaturated Fatty Acids (TUSFTs) from 42.56 to 56.81% whereas, the total saturated fatty acids was decreased from 57.30 to 43.1%.However, D. hirtiflora tubers showed an increase in TSFAs from 44.72 to 50.84% and decrease in TUSFTs from 53.73 to 49.08% upon boiling.MUSFT was increased from 15.92 to 31.70% and PUSFAs showed a slight decrease from 26.64 to 25.11% in boiled D. dumetorum tubers.A slight decrease in both MUSFTs and PUSFAs was observed in boiled D. hirtiflora tubers.As in raw materials, palmitic acid and oleic acid were the most abundant saturated and unsaturated fatty acids respectively in both species.The palmitic acid content was significantly (p<0.05)decreased from 42.71 to 31.58% in D. dumetorum tubers and increased from 33.54 to 37.28% in D. hirtiflora tubers, whereas, a significant (p<0.05)increase in oleic acid content in boiled D. dumetorum tubers from 13.71% to 29.13% and a decrease from 27.19 to 21.73% was observed in D. hirtiflora tubers.Linolelaidic acid was the most abundant PUSFA in raw and boiled tubers of the both Dioscorea species.Shajeela et al. (2013) found that the predominant PUSFA of others Dioscorea species from India was its isomer linoleic acid.

Vitamin C:
The content of vitamin C in Dioscorea tubers varied significantly (p< 0.05) within the two species (Table 3).Vitamin C content was twofold higher in D. dumetorum tubers (127 ± 0.01 mg/kg) content than D. hirtiflora tubers (60.5 ± 0.06 mg/kg).This variation might be related to the differences in genotypes.However, boiling caused a significant decrease (p<0.05) in the vitamin C content of the two Dioscorea tubers.Boiling led to a decrease of 56.7 and 52.4% in D. dumetorum and D. hirtiflora tubers respectively.It is well established that vitamin C and thiamine are the nutrient most susceptible to the thermal degradation and leaching from food (Nagy and Smooth, 1977;Bognár, 1998).

Phenols:
The total phenolic contents were expressed as mg Gallic Acid Equivalent (GAE) per 100 g dry sample and are listed in Table 3.The raw D. dumetorum tubers showed higher the total phenol content (602 mg GAE/g) than that of raw D. hirtiflora tubers (131 mg GAE/g).Interestingly, after boiling D. hirtiflora tubers showed 4.5-fold gain in their total phenolic content whereas, D. dumetorum tubers showed 60% loss in their total phenolic content after boiling.Contradicted results on the effect of different cooking process on the total phenolic content were obtained.This difference might depend on type of vegetable used.Blessington et al. (2010) and Burgos et al. (2013) showed that cooked potato samples had greater levels of total phenolic than in uncooked ones.On the other hand, some studies (Ismail et al., 2004;Zhang and Hamauzu, 2004;Turkmen et al., 2005) showed that cooking process like boiling, baking and microwaving reduced both the polyphenol content in selected vegetables.The percent gain in the total phenol content during cooking may be due to the breakdown of tough cell walls and increased extractability of compounds (Adefegha and Oboh, 2011).Dewanto et al. (2002a) found that ferulic acid found in the cell wall of grains such as corn, wheat and oats, doubled after 10 min of cooking and increased by as much as 900% after 50 min of cooking.
Antioxidant activity: In this study, DPPH radical and ABTS radical cation assays were used for evaluation of free radical-scavenging properties of the ethanolic extracts of raw and processed Dioscorea spp.tubers.The results of investigation are shown in Fig. 1 and 2. Raw D. dumetorum tubers showed higher radicalscavenging activity (IC 50 72.351mg/L) than that of D. hirtiflora (IC 50 307.958mg/L).Boiling caused a sharp increase, comparable to the control, in the radicalscavenging properties of D. hirtiflora tubers (IC 50 22.35 mg/L).This increase in antioxidant activity could be correlated to the increase in total phenolic content (4.5fold gain) after boiling.It is worth noting that this increase in antioxidant activity with cooking, agrees with earlier reports on the effect of cooking on the antioxidant properties of maize (Dewanto et al., 2002a), carrots (Talcott et al., 2000) and tomatoes (Dewanto The antioxidant capacity of the two Dioscorea spp.tubers using the ABTS method was lower than that obtained from the DPPH method and very weak when compared with that of ascorbic acid (Fig. 2).The antioxidant capacity using the ABTS method was 6.4 3.6 fold lower for raw D. dumetorum and D. hirtiflora tubers than those determined by the DPPH method.Boiling did not also change the scavenging capacity of the two Dioscorea spp.tubers.The ABTS and DPPH assays are both associated with electron and radical scavenging but have been reported to give different results (Burgos et al., 2013).Gramza et al. (2005) found that Yunan tea extracts showed also different scavenging capacity on using the two different methods of scavenging the stable free radicals ABTS +• and DPPH • .They suggested that variation might be attributed to different action mechanisms of oxidative factors, including free radicals and in addition, they proposed that possible antiradical activity of plant extracts was conditioned by antioxidant structures as well as other component interactions.

CONCLUSION
Boiling decreased the amino acids content of both tubers studied whereas, the total unsaturated fatty acids increased in D. dumetorum tubers and decrease in D. hirtiflora tubers.Boiling enhanced the antioxidant capacity of D. hirtiflora tubers and did not significantly affect that of D. dumetorum tubers.The high phenolic content present in the tubers in raw and boiled state might be the main contributors of this antioxidant activity.Therefore, boiled D. dumetorum and D. hirtiflora tubers could contribute positively for human diets and health.
DPPH radical scavenging activity (%) = 1-[(Abs sample -Abs blank )]/(Abs control )] ×100 where, Abs sample = The absorbance of DPPH radical+sample Abs blank = The absorbance of sample+methanol Abs control = The absorbance of DPPH radical+methanol The IC 50 value was calculated from the linear regression of plots of concentration of the test sample against the mean percentage of the antioxidant activity.The IC 50 values obtained from the regression plots (Sigma PlotsR 2001, SPSS Science) had a good coefficient of correlation, (R2 = 0.998).

Table 2 :
Each value represents mean±S.D. of triplicate (n = 3); Different lowercase letters for in the same row correspond to significant differences by Student's t test (p<0.05)between raw and boiled samples; Different capital letters in the same row correspond to significant differences by Student's t test (p<0.05)between raw samples Composition of fatty acids (dry weight basis, %) of Dioscorea dumetorum and D. hirtiflora tubers

Table 3 :
Vitamin C and total phenolic content of Dioscorea dumetorum and D. hirtiflora tubers