Abstract

Investigation into the antioxidant properties of plants is a very active field of research. Amaranths are underutilized pseudo-cereals with nutraceutical potentials. The phytochemical and antioxidant activity of five grain amaranth species were evaluated using standard procedures. Highest tannin content (0.14 g/100g) and Fe chelating (66.72%) capacity was recorded in Amaranthus caudatus. Amaranthus cruentus had the highest total flavonoid (9.93 mg CE/100g) content. Amaranthus Hybridus had the highest Phytate (1.58 g/100g), total polyphenol (30.79 mg GAE/100g), DPPH scavenging activity (93.35 %), ferric reducing power (0.19 g/100g), total antioxidant 199.93 mg AAE/100g) and ABTS (201.54 mmol TE/100g) content respectively. Strong correlation was observed between the phytochemicals and antioxidant tested. From the results, grain amaranth species possess antioxidant capacity and polyphenolic content. These qualities in amaranths have promising potential means of food biofortifications.

1. Introduction

Investigations into the antioxidant properties in plants is a very active field of research, especially for the not so well-known and underutilized plants. Phenolic compounds in plants possess antioxidant activity which help protect cells against oxidative damage caused by free radicals ¹. Diet containing whole-grain products and vegetables as primary ingredients has become one of the most important recommendation for reducing the risk of diseases caused by the increased level of free radicals ². Consumption of grains and vegetables has become increasingly popular for improvement and maintenance of heart health status, owing to the associated reduced risks of cardiovascular disease, and other associated chronic diseases such as diabetes and some forms of cancers ^{54, 55}.

Amaranth is a valuable pseudo-cereal, due to its nutritional quality and nutraceutical properties, which contribute to improved human health ^{2, 3}. Grain amaranths contain phytochemicals including flavonoids and antioxidants that help protect cells and tissues from damaging effects of free radicals and oxidative stress ⁴. According to ⁵, the main phenolic compounds found in amaranth seeds are caffeic acid, p-hydroxybenzoic acid and ferulic acid. Some anti-nutritional phytochemicals such as tannins and phytic acid also exhibit some protective effects ⁶ particularly in red wine, and in some ruminants, in which higher retention of nitrogen has been observed in sheep and cattle with low to moderate levels of tannins in forages. Grain amaranth has been shown to exhibit antioxidant activity and this has been attributed to its content of polyphenols, anthocyanins, flavonoids, and tocopherols ^{7, 8}.

Amaranth of the genus Amaranthus L. consist of more than 60 species ⁹. The three main species of grain amaranth widely cultivated are A. caudatus, A. cruentus and A. hypochondriacus and to a lesser extent, the leafy types A. hybridus and A. tricolor ¹⁰. Consumption of A. cruentus products is advised for patients with celiac disease and also for diabetic persons ¹¹. Amaranthus hybridus has been used traditionally for the treatment of liver infections and knee pain. and for its laxative, diuretic and cicatrisation properties ¹²; the products are used particularly for stomach aches, diarrhoea, and dysentery.

Amaranth cultivation remains relatively low and is not even listed in the FAO statistics on production data. Published data on the phytochemical content and antioxidant property of amaranth is limited. However, to assess their relevance as potential sources of dietary antioxidants, information on the phytochemical composition of the various species is essential. Phytochemical content and antioxidant activity of crops vary with species and are affected by environmental conditions and soil factors ^{8, 13}. This study therefore evaluated the phytochemical profile and antioxidant activity of five grain amaranth species: A. cruentus, A. hybridus, A. caudatus, A. hypochondriacus and A. hybrid.

2. Materials and Methods

Potassium ferricyanide, gallic acid, catechin, 3-(2-pyridyl)-5,6-bis-(4-phenylsulfonicacid)-1,2,4-triazine (Ferrozine), 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2’-azinobis (3-ethylobenzothiazoline-6-sulphonic acid diammonium salt) (ABTS) and 6-hydroxy-2,5,7,8-tetramethyl-chroman-2-carboxylic acid (Trolox) were purchased from Sigma-Aldrich. Folin–Ciocalteu reagent was from Merck (Germany). All reagents were of analytical grade.

Seeds of twenty nine grain amaranth accessions belonging to five species obtained from the United States Department of Agriculture, Ames, USA and National Horticultural Research Institute (NIHORT), Ibadan, Nigeria was used in this study. The seeds were planted in the experimental plot of NIHORT and harvested at maturity.

Seeds were milled into flour to obtain a homogenous particle size. Sample weighing 10g was extracted by stirring with 100 ml of methanol at 25°C at 150 rpm for 24h and filtered through Whatman No. 4 paper. The procedure was repeated twice, extracts were pooled together, evaporated at 40°C to dryness and redissolved in methanol at a concentration of 50 mg/ml and stored at 4°C prior to analyses.

Phytic acid was extracted and determined according to the precipitation method of ¹⁴. The conversion factor 3.55 for phosphorus to phytic acid was used. Tannin was determined by the acidified vanillin method of ¹⁵, using tannic acid as the standard.

Total phenolic (TP) was determined colorimetrically using Folin-Ciocalteau reagent as described by ¹⁶. Total phenolic assay was conducted by mixing 2.7 ml of de-ionised water, 0.3 ml of extracts, 0.3 ml 7% Na₂CO₃ and 0.15 ml Folin-Ciocalteu reagent. The mixture was vortexed and incubated at 40°C for 30min. Absorbance of mixture was measured at 725 nm. Results were expressed as gallic acid equivalent (GAE). Flavonoid content was determined by the method of ¹⁶. Aliquots (0.5 ml) of appropriately diluted extracts or standard solutions was pipetted into 15-ml polypropylene conical tubes containing 4.5 ml of double distilled water and mixed with 0.3 ml 5% NaNO₂. After 5min, 0.6 ml 10% AlCl₃.6H₂O solution was added, the mixture was allowed to stand for another 5min, and then 2 ml 1M NaOH was added, followed by 2.1 ml of distilled water. The reaction solution was well mixed, kept for 15 min, and the absorbance was determined at 510 nm. Total flavonoid was expressed as mg catechin equivalent (mg CE) per 100g of dry weight.

The ferric reducing power was determined using the method of ¹⁷. To 1ml of the grain extract in a test tube, 0.5ml of 0.2 M phosphate buffer (pH 6.6) and 2.5ml of 1% potassium ferricyanide were added. The mixture was incubated for 20 min at 50°C. The tube was immediately cooled over crushed ice and then an aliquot of 0.5ml 10% trichloroacetic acid was added. After centrifugation at 3000 g for 10 min, 1 ml of the supernatant was mixed with 1ml of distilled water and 0.Iml of 0.1% ferric chloride. The mixture was left to stand for 10 min and absorbance was measured at 700 nm. Higher absorbance of the reaction indicates higher reducing power. Ascorbic acid was used as standard. The method described by ¹⁸ was used to determine the ferrous ion chelating capacity. Grain flour extract (1 ml), 2mM FeCl₂.4H₂O (0.1 ml), 0.2 ml of 5mM ferrozine (3-(2-pyridyl)-5,6-bis-(4-phenylsulfonic acid)-1,2,4-triazine and 3.7 ml methanol were mixed in a test tube, and were reacted for 10 min. The absorbance at 562 nm was measured; a lower absorbance indicated a higher ferrous ion chelating capacity, which was calculated as follows:

Where A₅₆₂ nm sample is the absorbance of sample at 562nm

A₅₆₂ nm control is the absorbance of control at 562nm.

The assay is based on the reduction of Mo (VI) to Mo (V) by the extract and subsequent formation of a green phosphate/Mo V complex at acid pH ^{19, 20}. To 0.2ml of extract was added 3.8ml reagent solution (0.6 M sulphuric acid, 28mM sodium phosphate and 4mM ammonium molybdate). The mixture was incubated at 95°C for 90 min, cooled to room temperature and the absorbance of the solution was measured at 695 nm against a blank. The total antioxidant capacity was expressed as ascorbic acid equivalent (AAE) per 100g of dry weight.

The capacity to scavenge the ‘‘stable’’ free radical 2,2- diphenyl-1-picrylhydrazyl (DPPH) was monitored according to the method of Hsu et al. (2003). Methanol seed extract (1ml) was mixed with 5ml of freshly prepared methanol solution of DPPH• radical. The mixture was shaken vigorously and left to stand for 50 min in the dark until stable absorption values were obtained. The reduction of the DPPH• radical was measured by monitoring continuously the decrease of absorption at 517nm. Methanol (1ml), replacing the extract was used as the blank. Radical scavenging activity was expressed as inhibition percentage and calculated using the formula:

Where A_C is the absorbance of the solution when extract has been added and A_D is the absorbance of the DPPH solution. ABTS⁺ radical cation was generated by the interaction of 7 mmol/L ABTS and 2.45 mmol/L potassium persulfate ²¹. The solution was held at room temperature in the dark for 12-16h before use. Once the dark green solution was formed it was diluted with 95% ethanol until the absorbance read 0.7 at 734 nm ²². For measurements, 2.5 ml of the resulting solution was mixed with 500µl of the extract. The absorbance was read after 6 min at 734 nm. The percentage decrease of the absorbance at 734 nm was calculated by the formula:

Where I = ABTS % Inhibition

AB = Absorbance of blank sample (t=0)

AA = Absorbance of tested extract solution at the end of the reaction.

A standard curve was obtained by using Trolox solutions (0–250 mmol/mL) with ethanol. The absorbance of the reaction samples was compared to that of the trolox standard and results were expressed in terms of mmol Trolox Equivalent (TE) per 100g dry weight basis.

All analysis was done in triplicate and results were given as means ± SD. The data were analysed by one-way ANOVA using SAS and Tukey’s test was used to find significant differences between the species. Pearson correlation coefficient (r) and p-value were used to show correlations and their significance. Differences of p ≤ 0.05 were considered significant.

3. Results and Discussion

The results of phytochemical composition and antioxidant activity of five species of grain amaranth are presented in (Table 1). The highest tannin (0.14 g/100g) and Fe chelating (66.72%) content was recorded in A. caudatus. Amaranthus cruentus had the highest total flavonoid (9.93 mg CE/100g) content. A. Hybridus had the highest Phytate (1.58 g/100g), total polyphenol (30.79 mg GAE/100g), DPPH scavenging activity (93.35 %), ferric reducing power (0.19 g/100g), total antioxidant 199.93 mg AAE/100g) and ABTS (201.54 mmol TE/100g) content respectively. The lowest value for all the tested parameters was recorded in A. caudatus except tannin and Fe chelating.

Tannin content of the five amaranth species ranged from 0.10 – 0.14 g/100g with A. caudatus > A. cruentus > A. hybrid > A. hypochondriacus > A. hybridus (Table 1). Tannin content recorded in this study was lower than the maximum acceptable tannic acid daily intake for humans ²³. Values are in agreement with results of ²⁴ who recorded tannin content of 0.12 g/100g, 0.06 g/100g and 0.87 g/100g for A. cruentus, A. hypochondriacus and A. hybridus respectively. Values obtained in this study were lower than values recorded for wheat (0.29 g/100g), Barley (0.34 g/100g) and Oat (0.62 g/100g); but higher than 0.07 g/100g obtained for rice ²⁵. Tannin content obtained in this study is similar to value obtained in brown rice (0.1 g/100g); but lower than values obtained in cereals such as wheat (0.4 g/100g), maize (0.4 g/100g), rye (0.6 g/100g), millet (0.6 g/100g), barley (0.7 g/100g), oat (1.1 g/100g) and sorghum (1.5 g/100g) ²⁶. Finger millets have been reported to contain high amounts of tannin ranging from 0.04 to 3.74 g/100g ^{27, 28}. It was also reported by ²⁹ that the tannin content in brown finger millet is 0.36 g/100g, these results is higher than values obtained in this study. Put together, grain amaranth can be seen as a potential source of dietary component for easy digestibility with mild to moderately low tannin content compared to other well-known cereals.

Phytate content of the five grain amaranth species ranged from 1.16 – 1.58 g/100g, with Amaranthus hybridus > A. hypochondriacus > A. hybrid > A. cruentus > A. caudatus (Table 1). Values are higher than 0.03 – 0.80 g/100g obtained for raw amaranth grains ³⁰, 0.00079 g/100g obtained for grain amaranth ³¹; but lower than values obtained for wheat (4.17 g/100g), rice (4.28 g/100g), Barley (4.03 g/100g) and Oat (2.77 g/100g ²⁵. Phytate content obtained in finger millet ranged from 0.679 to 0.693 g/100g ²⁸; and 0.149 to 0.150 g/100g ²⁷. These are lower than values obtained in this present study. Phytic acid is a known anti-nutrient which forms insoluble complexes with minerals such as zinc, calcium, magnesium and iron in the body. ³² reported that the presence of phytic acid below the level of 6 g/100g will not pose any health risk to the body and concluded that dietary intake of low level phytate may protect against a fatty liver resulting from elevated hepatic lipogenesis. The anti-nutrient effect of phytic acid on mineral absorption will only occur at 10 fold higher levels of what was obtained in this study.

Total flavonoid content ranged from 8.91 to 9.93 mg CE/100g (Table 1), with highest value observed for Amaranthus cruentus followed by A. hybridus and A. hypochondriacus then A. Hybrid and A. Caudatus. The result for total flavonoid obtained in this study is lower than 13.4 to 14.3 mg CE/100g obtained for A. hypochondriacus and 17.7 mg/100g obtained for oat ³³. It was reported ⁷ that there were no quantifiable amount of flavonoids in Amaranthus caudatus seeds, only traces of quercetin was found. Low levels of quercetin glycoside, rutin (4.0-10.2 µg/g) was detected in A. hypochondriacus seeds ³⁴. These values corroborate findings from this present study in which low level of flavonoid was obtained in all the five species. The flavonoid content of quinoa (41.7 to 72.6 mg/100g) and Chenopodium (36.2 to 144.3 mg/100g) species ⁷ were also higher than results obtained for amaranth in our study.

Total Polyphenol content of the five grain amaranth species ranged from 27.52 – 30.79 mg GAE/100g (Table 1). Amaranthus hybridus had the highest value (30.97 mg GAE/100g), this is higher than 14.72 to 14.91 mg/100g GAE obtained for A. hypochondriacus and 19.61 mg/100g GAE obtained for oat ³³. Amaranthus caudatus had the lowest total polyphenol content (27.52 mg GAE/100g), this is higher than 21.20 mg/100g GAE obtained for Amaranthus caudatus ³⁵; but comparable to values (16.8 to 32.9 mg GAE/100g) obtained for some A. caudatus genotypes ⁷. Values obtained in this study were lower than values obtained for other pseudocereals like Quinoa (71.7 mg GAE/100g), buckwheat (323.0 mg GAE/100g) and wheat (53.1 mg GAE/100g) ³⁵. They are also significantly lower than values obtained for amaranth v. Rawa (295 mg GAE/100g) and amaranth v. Aztek (300 mg GAE/100g) ². Total polyphenol content of 15.5, 25 and 29 mg GAE/100g was obtained in amaranth, quinoa and buckwheat, respectively ³. The total phenol content of the five amaranth species were also lower than values obtained for Tumeric (119 mg GAE/100g), wheat (53 mg GAE/100g), corn (49 mg GAE/100g), sesame (42 mg GAE/100g), soybean (37 mg GAE/100g), oatmeal (35 mg GAE/100g) and linseed (31 mg GAE/100g) ³⁶. Whole grain flour of rye and wheat have been reported to contain phenolic acids of 137 and 134 mg/100g, respectively ³⁷. The polyphenol content of amaranth seeds is comparable to that of oat, barley, rice; but lower when compared with common cereals like wheat, maize and rye.

DPPH activity ranged from 89.53% – 93.35% with highest value observed in A. hybridus > A. hypochondriacus > A. hybrid > A. cruentus > A. caudatus. The DPPH activities obtained in A. hypochondriacus (91.40%) and A. cruentus (90.15%) in this study are higher than 86.93% obtained in A. hypochondriacus seeds ³⁸ and 84.67% obtained in A. cruentus ³⁹; respectively. Result from this study is similar to values obtained in grain amaranth ⁴⁰ but higher than values obtained for turmeric (31.5%), wheat (10.7%), corn (13%), soybean (12.7%), oatmeal (2.3%) and linseed (5.7%) ³⁶. Ferric reducing power ranged from 0.14 to 0.19 g/100g with A. hybridus and A. Caudatus having the highest and lowest values. High values obtain in this study for all the Amaranth indicates that they are a potent source of antioxidant since the reducing capacity of a compound is usually an indicator of its potential antioxidant activity.

Total antioxidant activity of the five grain amaranth species ranged from 140.22 to 199.93 mg AAE/100g. These are significantly higher than values obtained for certain cereals such as rice (85.49 mg AAE/100g), maize (26.94 mg AAE/100g), wheat (14.17mg AAE/100g) and barley 3.46 mg AAE/100g ⁴¹.

Iron chelating activity of the five amaranth species ranged from 57.52 to 66.72 %. Iron chelating activity obtained in methanol and hexane extract of A. cruentus leaves were 64% and 54% respectively; ⁴². Earlier report had indicated that polyphenols exhibit potent iron chelating ability ⁴³. Data variation in the antioxidant activity are to be expected due to sample type, differences in the methodology employed, standard used to express results and experimental conditions ^{22, 44}.

ABTS antioxidant activity of the five amaranth species ranged from 169.6 to 201.5 mmol TE/100g; with A. hybridus having the highest value. The ABTS activity (179.3 mmol TE/100g) obtained for Amaranthus cruentus is higher than values obtained for Amaranthus cruentus varieties: Aztek (127 mmol TE/100g) and Rawa (114 mmol TE/100g) ². ABTS obtained for Amaranthus caudatus (169.6 mmol TE/100g) is much higher than values obtained for two varieties of A. caudatus: Centenario (82.76 mmol TE/100g) and Oscar Blanco (67.01 mmol TE/100g) ⁴⁵. The ABTS value is lower than value obtained for quinoa seeds (272 mmol TE/100g) ² but higher than values obtained for rye ⁴⁶; barley, oat, buckwheat ⁴⁷ and wheat bran ⁴⁸.

4. Correlation

The result for Pearson correlation analysis is presented in (Table 2) while the linear relationship between individual significant parameters is presented in Figure 1. Pearson’s correlation analysis (Table 2) revealed strong correlation between total polyphenol vs. DPPH (r = 0.678) (Figure 1A) and total polyphenol vs. ABTS (r = 0.688) (Figure 1B). This is in agreement with report of ², who observed strong correlation between total polyphenol content vs. DPPH (r = 0.98); and ABTS (r = 0.98) in amaranth seeds. Strong correlation was also observed between total phenolic content and DPPH (r = 0.99) in A. caudatus seeds ³⁵. These findings suggest that total polyphenols content is a good predictor of in vitro antioxidant activity. Strong correlation was observed between total polyphenol vs. Reducing power (r = 0.88) (Figure 1C). Similar result was observed in some Mexican maize phenotypes in which total polyphenol showed strong correlation with DPPH, ABTS and Reducing power ⁴⁹. Strong correlation was observed between DPPH and ABTS (r = 0.921) (Figure 1D), DPPH vs. ferric reducing power (r = 0.851) (Figure 1E) and ferric reducing power vs. ABTS (r = 0.735) (Figure 1F). Strong correlation was observed between ABTS and DPPH (r = 0.98) ², this corroborates result of this study. Significant (P < 0.05) correlation was also observed between phytate and total polyphenol (0.862), DPPH (0.946), Ferric reducing power (0.930) and ABTS (0.946).

Weak correlation was observed between total polyphenol and total antioxidant activity (r = -0.258). This is not in agreement with result of ⁵⁰ in which strong correlation was observed between total polyphenol and total antioxidant activity (r = 0.97) in amaranth; and between total polyphenol and antioxidant activity (r = 0.96) in rice ^{51, 52}. A significant correlation (r = 0.96) was observed between total phenolic content and antioxidant activity in buckwheat extract ⁵³.

The correlation discrepancies found in literature could be explained on the basis of differences in the interpretation of the results by individual methods. Also, the antioxidant activity of a substance can vary from method to method depending on factors such as antioxidant solubility, oxidation state and medium of pH ^{44, 46}.

5. Conclusion

These results highlight the significance of grain amaranth as potential source of phytochemicals and antioxidants. Amaranthus hybridus had most of the phytochemicals and antioxidant activity in higher levels when compared with the other species examined in the study. The five grain amaranth species showed higher antioxidant activity and phytochemical content when compared to cereals such as oat, barley, wheat, corn, millet and rice. Amaranth is therefore a good substitute for traditional cereals and has potential as source of health-promoting bioactive compounds. As these grains are affordable and widely available, efforts to promote their consumption for health benefits, its use in food biofortification applications and pharmaceutical industries should be encouraged.

References