Variability in total antioxidant capacity, antioxidant leaf pigments and foliage yield of vegetable amaranth

Umakanta Sarker , Md Tofazzal Islam, Md Golam Rabbani, Shinya Oba

1 Department of Genetics and Plant Breeding, Faculty of Agriculture, Bangabandhu Sheikh Mujibur Rahman Agricultural University,Gazipur 1706, Bangladesh

2 Department of Biotechnology, Faculty of Agriculture, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur 1706,Bangladesh

3 Department of Horticulture, Faculty of Agriculture, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

4 Laboratory of Field Science, Faculty of Applied Biological Science, Gifu University, Gifu 5011193, Japan

1. Introduction

Recently, antioxidants have attracted considerable interest in food technology researches. Antioxidants, their availability in diets and probable roles in countering deadly diseases such as cancer, neurodegenerative and cardiovascular diseases have been highlighted in various studies (Manachet al. 2004; Scalbertet al. 2005; Thaiponget al. 2006;Surangiet al. 2012). Natural antioxidants, particularly in fruits and vegetables, have gained the attention of both researchers and consumers. Leaf pigments, such as β-cyanins, β-xanthins, chlorophyll, carotenoids and vitamins,are the major groups of natural antioxidants that are available in vegetables amaranth and involved in defences against several diseases including cancer, cardiovascular diseases, atherosclerosis, arthritis, cataracts, emphysema,retinopathy, neuro degenerative diseases and in flammation,and preventing aging (Caiet al. 2003; Alvarezjubeteet al.2010; Ritvaet al. 2010; Steffensenet al. 2011; Esatbeyogluet al. 2015).

The interest of consumers in the aesthetic, nutritional and safety aspects of food has increased the demand for natural pigments such as chlorophyll, betalains, and carotene. Betalains are water-soluble compounds found in a limited number of families of the plant order Caryophyllales,likeAmaranthushaving aunique source of betalains and important free radical-scavenging activity (Caiet al. 2003;Dantaset al. 2015). β-Cyanins are colored betalains from red to purple (condensation of betalamic acid and cyclodopa, considering hydroxycinnamic acid derivatives or sugars as residue) and β-xanthins are yellow betalains(imine condensation products between betalamic acid and amines or amino acid residues) (Herbachet al. 2006).Similarly, carotene grouped into α-carotene, β-carotene and xanthophyll.

Pigments and their pharmacological activities include anticancer (Szaeferet al. 2014), antilipidemic (Wroblewskaet al. 2011) and antimicrobial (Canadanovicet al. 2011)activities, indicating that betalains and carotene may be a potential source for the production of functional foods.Presently, the only commercial source of betalains and carotene is the red beet root. The colorant preparations from red beet root labelled as E-162 are exempted from batch certification. E-162 is used in processed foods such as dairy products and frozen desserts (Stintzing and Carle 2007).

Among the naturally occurring vegetable pigments,betalains are rare and limited to a few edible vegetables,such as red beet and amaranth, while chlorophylls are widely distributed in plant species (Schwartz and Von-Elbe 1980).The active ingredients of betalains and carotene provide anti-in flammatory property to our food and act as potential antioxidants, which can reduce the risk of cardiovascular disease and lung and skin cancers and is widely used as additive for food, drugs, and cosmetic products because of natural properties and absence of toxicity (Kanneret al.2001; Buteraet al. 2002).

In Asia and Africa, vegetable amaranth is intaken by boiling and making curries, while in Amricas and a few Asian and European countries, it is freshly intaken by making salad or juice. Recently, we extracted red color juice fromAmaranthusfor natural drink containing pigments chlorophyll, betalains, and carotene. It demands more genotypes enriched with leaf pigments. We found lots of variations in vegetable amaranth germplasm in respect to minerals, vitamins, leaf color, quality, and agronomic traits in our earlier studies (Sarkeret al. 2014, 2015a, b, 2016).Therefore, we studied 20 cultivated genotypes of vegetable amaranth to: i) estimate total antioxidant capacity, amount of antioxidant leaf pigments and foliage yield; ii) select high-yielding genotypes containing high antioxidant leaf pigments for making colorful juice commercially; and (iii)find out possible ways for improving the antioxidant leaf pigments without compromising foliage yield.

2. Materials and methods

2.1. Experimental site

The experiment was conducted at the experimental field of Bangabandhu Sheikh Mujibur Rahman Agricultural University, Bangladesh. The experimental site was located in the center of the Madhupur Tract (AEZ-28) at approximately 24°23′ north latitude and 90°08′ east longitude, having a mean elevation of 8.4 m above sea level. The experimental field is a highland with silty clay soil. The soil conditions were: slightly acidic (pH 6.4), low in organic matter (0.87%),total N of 0.09% and exchangeable K of 0.13 centimol kg–1.The experimental site is in a subtropical zone and has sharp differences in summer and winter temperatures.

2.2. Materials

Twenty distinct and promising selected genotypes of vegetable amaranth were investigated during 2014–2015 and 2015–2016 growing seasons.

2.3. Experiment design, layout, and cultural practices

Vegetable amaranth was sown in a randomized complete block design (RCBD) in triplicate for both years. The unit plot size of each genotype was 1 m2. The spacing was 20 cm between rows and 5 cm between plants.Recommended fertilizer and compost doses and appropriate cultural practices were maintained. Thinning was performed to maintain appropriate plant density within rows. Weeding and hoeing were performed at 7-day intervals. Day time temperatures during the experimental period ranged from 25 to 38.5°C. Irrigation was provided at 5- to 7-day intervals.For foliage yield, the plants were cut at the base of the stem(ground level).

2.4. Data collection of foliage yield

Data were collected 30 days after sowing of seeds. The foliage yield (g plant-1) were recorded on 10 randomly selected plants in each replication.

2.5. Determination of chlorophyll and total carotenoid content

Chlorophylla, chlorophyllb, and total chlorophyll were determined from 96% ethanolic extracts of the fresh-frozen amaranth leaves following the method in Lichtenthaler and Wellburn (1983) and total carotenoid content was determined from acetone: Haxen extract of the fresh-frozen amaranth leaves using spectrophotometer (Hitachi, U-1800,Tokyo, Japan) at 665, 649, and 470 nm for chlorophylla,chlorophyllband total carotenoid contents, respectively.

2.6. Determination of β-cyanins and β-xanthins content

β-Cyanins and β-xanthins were extracted from fresh-frozen amaranth leaves using 80% methanol containing 50 mmol L–1ascorbic acid according to Wyleret al. (1959). β-Cyanins and β-xanthins were measured spectrophotometrically at 540 and 475 nm, respectively. The quanti fications were done using mean molar extinction coef ficients, which were 62×106cm2mol–1for β-cyanins and 48×106cm2mol–1for β-xanthins. The results were expressed as nanograms betanin per gram fresh-frozen weight (FFW) for β-cyanins and nanograms indicaxanthin per gram FFW for β-xanthins.

2.7. Ascorbic acid

Ascorbic acid was analyzed using the method described by Roe (1954). To extract the sample, 5 g of fresh leaves were ground with 5% H3PO3:10% acetic acid (dissolving 50 g of H3PO3in 900 mL of distilled water+100 mL of glacial acetic acid and volume was made up to 1 L with distilled water)for 1–3 min. The amount of extraction fluid was taken such that it should yield 1–10 μg mL–1ascorbic acid. One or two drops of bromine were also added to the solution, which was then stirred until it turned yellow. The excess bromine was decanted onto a bubbler, and the air passed until the bromine color disappeared. The bromine-oxidized solution was placed in two tubes. In the first tube, 1 mL of filtered 2,4-DNP thio-urea reagent (2,4-dinitrophenyl hydrazine-thiourea, prepared by dissolving 2 g of 2,4-DNP in 100 mL of 9 N H2SO4and then dissolving 4 g of thio-urea in this solution) was added, and the tube was placed in a 37°C water bath for 3 h. Then, 5 mL of 85% H2SO4(100 mL of distilled water+900 mL of concentrated H2SO4) was added drop-wise to the tube that was placed in a beaker of ice water.In the second tube, 1 mL of 2,4-DNP thio-urea reagent only was added, and this sample was used as a blank solution.After 30 min, the absorbance reading of the sample was taken at 540 nm using a spectrophotometer. The blank solution was used for setting the zero transmittance of the spectrophotometer. The standard solution was prepared by dissolving 100 mg of high-purity ascorbic acid in 100 mL of 5% H3PO3:10% acetic acid and the solution oxidized with bromine water as described above. A total of 10 mL of this dehydro-ascorbic acid was brought up to 500 mL with the 85% H2SO4solution. Different dilutions were prepared by taking 5, 10, 20, 30, 40, 50 and 60 mL of the above solution into 100 mL volumetric flasks, and the total volume of each was brought up to 100 mL by the addition of 85% H2SO4.This procedure was also used for the experimental samples.The calibration curve was prepared by plotting absorbance values against concentration of vitamin C (in μg).

The amount of vitamin C (μg 100 g–1) was calculated as follows:

Ascorbic acid (μg 100 g–1)=Ascorbic acid obtained from curve (μg)/1 000×10 mL (extract)/4×100/Sample weight (g)

Finally, it was converted in to mg 100 g–1.

2.8. Extraction of samples for chemical analysis

The leaves were harvested at the edible stage, 30 days after sowing, and dried overnight in an oven for chemical analysis. A total of 1 gram of dried leaf from each cultivar was ground and dissolved in 40 mL of 90% methanol. The tightly capped bottle was then placed in an 80°C shaking water bath (Thomastant T-N22S, Thomas Kagaku Co., Ltd.,Japan). After 1 h, the extract was cooled and filtered for further analytical assays of total antioxidant capacity.

2.9. Total antioxidant capacity

Antioxidant activity was measured using the diphenylpicryl-hydrazyl (DPPH) radical degradation method(Alvarez-Jubeteet al. 2010). Briefly, 10 μL of leaf extract solution (in triplicate) was placed in test tubes along with 4 mL of distilled water and 1 mL of 250 μmol L-1DPPH solution. The tubes were mixed and allowed to stand for 30 min in the dark before the absorbance was read at 517 nm using a spectrophotometer (U-1800, HITACHI,Tokyo, Japan). Antioxidant activity was calculated as the percent of inhibition relative to the control using the following equation:

Antioxidant activity (%)=(A blank–A sample/A blank)×100

Where, A blank is the absorbance of the control reaction(10 μL of methanol instead of sample extract) and A sample is the absorbance of the test compound. Trolox was used as the reference standard, and the results were expressed as μg trolox equivalent g–1DW.

2.10. Statistical analysis

The raw data of consecutive two years were compiled by taking the means of all the plants taken for each treatment and replication for different traits. The mean data of consecutive two years were averaged and statistically and biometrically analyzed. Analysis of variance was done according to Panse and Sukhatme (1978) for each character. Genotypic and phenotypic variances, genotypic and phenotypic coefficient of variation (GCV and PCV),heritability (h2b) in broad sense, and genetic advance in percent of mean (GAMP, %) and correlation were estimated according to Singh and Chaudhary (1985).

3. Results

3.1. Mean performance

Mean performance coef ficient of variation (CV, %) and critical difference (CD) for bioactive leaf pigments,antioxidant vitamins, and foliage yield of 20 vegetable amaranth genotypes are presented in Table 1. The analysis of variance revealed significant differences among the genotypes for all the 10 traits which was the indication of the validity of further statistical analysis due to the presence of a wide range of variability among the 20 genotypes of vegetable amaranth.

Antioxidant leaf pigmentsIn statistical analysis, the chlorophyllacontent had significant pronounced variations among the genotypes. Accession VA13 had the highest chlorophyllacontent (636.87 μg g–1), followed by VA19(523.21 μg g–1), VA14 (517.16 μg g–1), and VA16 (504.56 μg g–1). The lowest amount of chlorophyllawas found in VA8 (127.26 μg g–1). Twelve genotypes showed above average mean values for chlorophyllacontent. The mean chlorophyllacontent was 346.21 μg g–1. The estimated CV for chlorophyllawas 2.61%.

Accession VA17 had the highest chlorophyllbcontent(292.19 μg g–1), followed by VA7 (278.21 μg g–1), VA15(271.08 μg g–1) and VA13 (268.34 μg g–1). The lowest amount of chlorophyllbwas found in VA8 (51.67 μg g–1). The mean chlorophyllbcontent was 181.25 μg g–1.Eleven genotypes showed above average mean values for chlorophyllbcontent. The estimated CV for chlorophyllbwas 1.96%.

Table 1 Mean performance of total antioxidant capacity, antioxidant leaf pigments and vitamins, foliage yield in vegetable amaranth genotypes

The total chlorophyll content showed a highly pronounced variation among all the chlorophyll traits. VA13 had the highest total chlorophyll content (906.23 μg g–1), followed by VA14(770.22 μg g–1), VA7 (753.73 μg g–1), VA16 (737.43 μg g–1),VA15 (704.83 μg g–1) and VA19 (703.04 μg g–1). The lowest amount of total chlorophyll was found in VA8(179.94 μg g–1). The mean total chlorophyll content was 528.47 μg g–1. Eleven genotypes showed above-average mean values for total chlorophyll content. The estimated CV for total chlorophyll was 1.53%.

There were significant variations among the genotypes in β-cyanins contents and the average β-cyanins content was 359.53 ng g–1. The highest β-cyanins content was observed in VA18 (538.51 ng g–1), followed by VA3 (537.21 ng g–1) and VA14 (500.40 ng g–1), while the lowest β-cyanins content was observed in VA6 (185.52 ng g–1). The CV of this trait was 2.06%. Out of 20 genotypes, 9 showed above-average values for β-cyanins content.

There were significant variations among the genotypes in β-xanthins contents. The average β-xanthins content was 364.30 ng g–1. The highest β-xanthins content was observed in VA3 (584.71 ng g–1), followed by VA18 (554.31 ng g–1),VA14 (502.79 ng g–1), and VA16 (492.99 ng g–1), while the lowest β-xanthins content was observed in VA6 (181.90 ng g–1). The CV was 2.12%. Nine genotypes showed aboveaverage values for β-xanthins content.

There were significant variations among the genotypes in betalains contents. The average betalains content was 723.97 ng g–1. The highest betalains content was observed in VA3 (1 121.85 ng g–1) followed by VA18 (1 092.74 ng g–1), VA14 (1 003.12 ng g–1), VA16 (977.69 ng g–1), and VA20 (920.87 ng g–1), while the lowest betalains content was observed in VA6 (367.35 ng g–1). The CV was 1.24%.Out of 20 genotypes, 9 showed above-average values for betalains content.

There were significant variations among the genotypes in total carotene contents. The average total carotene content was 69.87 mg 100 g–1. The highest total carotene content was observed in VA20 (105.08 mg 100 g–1), followed by VA19 (96.37 mg 100 g–1), VA17 (96.08 mg 100 g–1), and VA11 (88.29 mg 100 g–1), while the lowest total carotene content was observed in VA13 (32.77 mg 100 g–1). The CV of this trait was 1.25%. Out of 20 genotypes, 9 showed above-average values for total carotene content.

Ascorbic acidThere were significant variations among the genotypes in ascorbic acid contents. The average ascorbic acid content was 92.86 mg 100 g–1. The highest ascorbic acid content was observed in VA14 (184.77 mg 100 g–1),followed by VA1 (175.59 mg 100 g–1), VA11 (135.58 mg 100 g–1), and VA10 (134.61 mg 100 g–1), while the lowest ascorbic acid content was observed in VA2 (11.97 mg 100 g–1). The CV was 2.06%. Out of 20 genotypes, 11 showed above-average values for ascorbic acid content.

Total antioxidant capacityThe variations of total antioxidant capacity were highly pronounced among the genotypes which ranged from 14.99 (VA8) to 32.83 TEAC μg g–1DW(VA3). The highest total antioxidant capacity was found in the genotype VA3 (32.83 TEAC μg g–1DW), VA13 (32.82 TEAC μg g–1DW), and VA20 (32.65 TEAC μg g–1DW) followed by VA12 (32.02 TEAC μg g–1DW), VA19 (31.68 TEAC μg g–1DW), VA2 (30.95 TEAC μg g–1DW), VA11 (29.98 TEAC μg g–1DW), VA18 (29.93 TEAC μg g–1DW) and VA9 (29.85 TEAC μg g–1DW). In contrast, the lowest total antioxidant capacity was observed in VA8 (14.99 TEAC μg g–1DW). The average mean of total antioxidant capacity was 25.74 TEAC μg g–1DW. Twelve genotypes showed above-average performance for total antioxidant capacity. The coef ficient of variation for this trait was 0.102%.

Foliage yieldThere were the significant and highest variations among the genotypes. The highest value was found in VA14 (32.06 g) followed by VA18 (26.40 g), VA16 (26.46 g),VA15 (23.28 g), and VA20 (23.14 g). The lowest value was observed in VA6 (7.32 g) followed by VA1 (8.94 g), VA4(9.14 g) and VA7 (9.20 g). The average was 15.95 g. The CV (1.07) was the lowest among all the traits analyzed.Seven genotypes showed above-average values.

3.2. Variability studies

The genotypic and phenotypic variance, coefficients of variation (GCV and PCV),h2b, GA and GAMP are presented in Table 2. The highest genotypic variance was observed for total chlorophyll (50 937.32), followed by betalains (49 079.42), chlorophylla(23 739.53), β-xanthins(13 122.89), β-cyanins (11 448.54). Chlorophyllb(6 626.21),ascorbic acid (81 881.67) exhibited moderate genotypic variances. On the other hand, the lowest genotypic variance was observed for total antioxidant capacity (42.09). The phenotypic variances for all the traits were slightly higher but close to the genotypic variances. GCV values ranged from 26.59% (total carotene) to 44.91% (chlorophyllb).The PCV values ranged from 27.50% (total carotene) to 48.15% (ascorbic acid). In the present investigation, all the traits had high to moderate genotypic and phenotypic variances along with moderate GCV and PCV values. The heritability estimates were high for all the traits and ranged from 95.59% (foliage yield) to 99.58% (chlorophyllb). The highest expected genetic advance was exhibited for total chlorophyll (464.93%) followed by betalains (456.37%),chlorophylla(317.40%), β-xanthins (235.98%), β-cyanins(220.42%), and chlorophyllb(167.69%). GAMP ranged from 54.77 (total carotene) to 96.23 (ascorbic acid). The highest GAMP was found in ascorbic acid (96.23%), followed by chlorophyllb(92.52%), chlorophylla(91.68%), foliage yield (89.50%), total chlorophyll (87.98%). Total carotene,β-cyanins, β-xanthins, betalains, and total antioxidant capacity showed moderate GAMP (around 50–60%).

3.3. Correlation studies

The phenotypic and genotypic correlations between the various characters are presented in Table 3. In the present investigation, the genotypic correlation coef ficients were very much close to the corresponding phenotypic values for all the traits. The chlorophyllahad a significant positive correlation with all the traits except total carotene and ascorbic acid. Chlorophyllbexhibited significant positive correlation with all the traits except total carotene and ascorbic acid. Similarly, total chlorophyll had a significant positive interrelationship with all the other traits except total carotene and ascorbic acid. β-Cyanins had a significant positive associations with β-xanthins, betalains and foliage yield. β-Xantins showed significant positive associations with betalains and foliage yield. Similarly,betalains showed possitive interrelationships with foliage yield. Total antioxidant capacity showed significant positive associations with all the leaf pigments, ascorbic acid and foliage yield. All the antioxidant leaf pigments and total antioxidant capacity had a significant positive associations with foliage yiled and among each other. The interesting results is that ascorbic acid had an insignificant negative interrelationships among all antioxidant vitamins and leaf pigments, while it exhibited significant positive associations with total antioxidant capacity. Total carotene had an insignificant negative correlations with chlorophylla, β-cyanins, β-xanthins and betalains, ascorbic acid and insingni ficant positive corelations with chlorophyllband total chlorophyll and foliage yield. This trait showed significant positive associations with total antioxidant capacity only.This indicates that selection for antioxidant vitamins content might be possible without compromising yield loss.

4. Discussion

The present investigation revealed that vegetable amaranth is rich in chlorophylla(346.21 μg g–1), chlorophyllb(181.25 μg g–1), total chlorophyll (528.47 μg g–1), β-cyanins (359.73 ng g–1) and β-xanthins (364.30 ng g–1), betalains (723.97 ng g–1),total carotene (69.87 mg 100 g–1), ascorbic acid (92.86 mg 100 g–1) and total antioxidant (25.74 TEAC μg g–1DW).

Five genotypes, VA14, VA16, VA18, VA15, and VA20 showed high foliage yield and also found to be a rich source of antioxidant leaf pigments and vitamins. Selection of these genotypes would be economically useful for antioxidant leaf pigments and vitamins, and high yield aspects. The genotypes VA13 and VA19 had above-average foliage yield along with rich source of the antioxidant leaf pigments and vitamins while the genotypes VA2, VA3, VA9, VA11, VA12 and VA17 had a high amount of the colorant antioxidant leaf pigments and below-average foliage yield yield. These eight genotypes can be used as a donor parent for integration of potential genes of the high antioxidant leaf pigments and vitamins into other genotypes.

The highest genotypic variance was observed for total chlorophyll (50 937.32), followed by betalains (49 079.42),chlorophylla(23 739.53), β-xanthins (13 122.89), and β-cyanins (11 448.54) indicating greater scope of selection for these traits. The phenotypic variances for all the traits were slightly higher but close to the genotypic variances which indicated the predominance of additive gene actions.GCV values ranged from 26.59% (total carotene) to 44.91%(chlorophyllb). In the present investigation, all the traits had high to moderate genotypic and phenotypic variances along with moderate GCV and PCV values, which indicate scope for improvement in these traits through selection dueto predominance of additive gene action for these traits. The heritability estimates were high for all the traits and ranged from 95.59% (foliage yield) to 99.58% (chlorophyllb). The highest expected genetic advance was exhibited for total chlorophyll (464.93%) followed by betalains (456.37%),chlorophylla(317.40%), β-xanthins (235.98%), β-cyanins(220.42%), and chlorophyllb(167.69%). The highest GAMP was found in ascorbic acid (96.23%), followed by chlorophyllb(92.52%), chlorophylla(91.68%), foliage yield(89.50%), and total chlorophyll (87.98%). Total carotene,β-cyanins, β-xanthins, betalains, and total antioxidant capacity showed moderate GAMP (around 50–60%). In the present study, the high heritability and high to moderate genetic advance values were observed for all the traits except foliage yield indicated preponderance of additive gene effects and improvement could be achieved through selection of these traits.

Table 2 Genetic parameter for total antioxidant capacity, antioxidant leaf pigments and vitamins, and foliage yield in vegetable amaranth genotypes

Table 3 Genotypic and phenotypic correlation co-efficient (rg and rp) for total antioxidant capacity, antioxidant leaf pigments and vitamins, and foliage yield in vegetable amaranth genotypes

In the present investigation, the genotypic correlation coefficients were very much close to the corresponding phenotypic values for all the traits that indicating predominance of additive gene action, i.e., less environmental influence of these traits. The chlorophylla, chlorophyllband total chlorophyll had a significant positive correlation with all the traits except total carotene and ascorbic acid. A similar trend of positive associations was observed by earlier work inAmaranthus tricolor(Shuklaet al. 2006; Sarkeret al.2014). β-Cyanins had a significant positive associations with β-xanthins, betalains and foliage yield. β-Xantins showed significant positive associations with betalains and foliage yield. Similarly, betalains showed possitive interrelationships with foliage yield. Total antioxidant capacity showed significant positive associations with all the leaf pigments,ascorbic acid and foliage yield. These indidates that high antioxidant content was closely associated with foliage yield of vegetable amaranth. All the antioxidant leaf pigments and total antioxidant capacity had a significant positive associations with foliage yiled and among each other. These indicate that improvement of foliage yield, total antioxidant content and antioxidant leaf pigments might be possible by improving any of the antioxidant leaf pigments. Shuklaet al. (2010) observed a positive association of foliage yield with β-carotene and ascorbic acid. Interesting results is that, ascorbic acid and total carotene had an insignificant interrelationships among all antioxidant vitamin and leaf pigments while it exhibited significant positive associations with total antioxidant capacity. This indicates that selection for antioxidant vitamin content might be possible without compromising yield loss.

5. Conclusion

Considering high genotypic and phenotypic variances along with GCV and PCV values, high heritability coupled with GAMP, all the traits could be selected for the improvement of 20 vegetable amaranth genotypes under study.However, the correlation study revealed a strong positive association among all the antioxidant leaf pigments, total antioxidant capacity and foliage yield. Selection based on total antioxidant capacity, antioxidant leaf pigments could be economically viable to improve the yield potential of vegetable amaranth genotypes. Insignificant negative genotypic correlation was observed between total carotene versus chlorophylla, β-cyanins, β-xanthins, betalains, and ascorbic acid. In contrast, this trait also showed insignificant positive associations with chlorophyllb, total chlorophyll, and foliage yield. Ascorbic acid had an insignificant negative interrelationships among all antioxidant vitamins and bioactive leaf pigments. This indicates that selection for antioxidant vitamins might be possible without compromising yield loss. Based on mean performance, five vegetable amaranth genotypes VA14, VA16, VA18, VA15 and VA20 were identified as high yielding having substantial amount of antioxidant leaf pigments and vitamins and suitable for extraction of colorful juice. The genotypes VA13 and VA19 had above-average foliage yield and enrich of antioxidant pro files while the genotypes VA2, VA3, VA9, VA11, VA12 and VA17 had a high antioxidant pro files and below-average foliage yield. These eight genotypes can be used as a donor parent for integration of potential genes of the high antioxidant leaf pigments into other genotypes.

Acknowledgements

The authors are thankful to the Research Management Committee (RMC) of Bangabandhu Sheikh Mujibur Rahaman Agricultural University, Bangladesh for providing partial financial support to carry out the present investigation.

Alvarezjubete L, Wijngaard H, Arendt E K, Gallagher E. 2010.Polyphenol composition andin vitroantioxidant activity of amaranth, quinoa buckwheat and wheat as affected by sprouting and baking.Food Chemistry, 119, 770–778.

Butera D, Tesoriere L, Gaudio F. 2002. Antioxidant activities of sicilian prickly pear (Opuntia ficus indica) fruit extracts and reducing properties of its betalains: Betanin and indicaxanthin.Journal of Agriculural and Food Chemistry,50, 6895–6901.

Cai Y, Sun M, Corke H. 2003. Antioxidant activity of betalains from plants of the Amaranthaceae.Journal of Agricultural and Food Chemistry, 51, 2288–2294.

Canadanovic B J M, Savatovic S S, Cetkovic G S. 2011.Antioxidant and antimicrobial activities of beet root pomace extracts.Czech Journal of Food Science, 29, 575–585.

Dantas R L, Silva S M, Brito-Primo D M, Sousa A S B, Brito E S, Macedo E M S. 2015. Changes during maturation in the bioactive compounds and antioxidant activity ofOpuntia stricta(Haw.) fruits.Acta Horticulturae, 1067, 159-165.

Esatbeyoglu T, Wagner A E, Schini-Kerth V B, Rimbach G.2015. Betanin - A food colorant with biological activity.Molecular Nutrition and Food Research, 59, 36–47.

Herbach K M, Stintzing F C, Carle R. 2006. Betalain stability and degradation structural and chromatic aspects.Journal of Food Science, 71, R41–R50.

Kanner J, Harel S, Granit R. 2001. Betalains - A new class of dietary cationized antioxidants.Journal of Agriculural and Food Chemistry, 49, 5178–5185.

Lichtenthaler H K, Wellburn A R. 1983. Determinations of total carotenoids and chlorophyllsaandbof leaf extracts in different solvents.Biochemistry Society Transactions,603, 591–592.

Manach C, Scalbert A, Morand C, Remesy C, Jimenez L. 2004.Polyphenols: Food sources and bioavailability.American Journal of Clinical Nutrition, 79, 727–747.

Panse V G, Sukhatme P V. 1978.Statistical Methods For Agricultural Workers. 3rd ed. ICAR (Indian Council of Agricultural Research), New Delhi.

Ritva R C, Jarkkok H, Juhamatti P, Mattila P H. 2010.Flavonoids and other phenolic compounds in Andean indigenous grains: Quinoa (Chenopodium quinoa), kaniwa(Chenopodium pallidicaule) and kiwicha (Amaranthus caudatus).Food Chemistry, 120, 128–133.

Roe J H. 1954. Chemical determination of ascorbic acid,dehydroasorbic, and diketoguloric acids. In: Glick D, ed.,Methods of Biochemical Analysis. Interscience Publishers,New York. pp. 115–139.

Sarker U, Islam M T, Rabbani M G, Oba S. 2014. Genotypic variability for nutrient, antioxidant, yield and yield contributing traits in vegetable amaranth.Journal of Food Agriculture and Environment, 12, 168–174.

Sarker U, Islam M T, Rabbani M G, Oba S. 2015a. Genotype variability in composition of antioxidant vitamins and minerals in vegetable amaranth.Genetika, 47, 85–96.

Sarker U, Islam M T, Rabbani M G, Oba S. 2015b. Variability,heritability and genetic association in vegetable amaranth(Amaranthus tricolor).Spanish Journal of Agricultural Resserach,13,1-8.

Sarker U, Islam M T, Rabbani M G, Oba S. 2016. Genetic variation and interrelationship among antioxidant, quality and agronomic traits in vegetable amaranth.Turkish Journal of Agriculture and Forestry, 40, 526–535.

Scalbert A, Manach C, Morand C, Remesy C, Jimenez L. 2005.Dietary polyphenols and the prevention of diseases.Critical Review in Food Science and Nutrition,45, 287–306.

Schwartz S J, Von-Elbe J H. 1980. Quantitative determination of individual betacyanin pigments by high-performance liquid chromatography.Journal of Agricultural and Food Chemistry,28, 540–543.

Shukla S, Bhargava A, Chatterjee A, Pandey A C, Rastogi A,Kumar A. 2010. Genetic interrelationship among nutritional and quantitative traits in the vegetable amaranth.Crop Breeding and Applied Biotechnoly, 10, 16–22.

Shukla S, Bhargava A, Chatterjee A, Srivastava J, Singh N,Singh S P. 2006. Mineral pro file and variability in vegetable amaranth (Amaranthus tricolor).Plant Foods for Human Nutrition, 61, 23–28.

Singh R K, Chaudhary B D. 1985.Biometrical Methods in Quantitative Genetic Analysis. 3rd ed. Kalyani Publisher,New Delhi.

Steffensen S K, Rinnan A, Mortensen A G, Laursen B, Troiani R M, Noellemeyer E J, Janovska D, Dusek K, Delano-Frier J, Taberner A, Christophersen C, Inge S, Fomsgaard I S. 2011. Variations in the polyphenol content of seeds of field grownAmaranthusgenotypes.Food Chemistry, 129,131–138.

Stintzing F C, Carle R. 2007. Betalains - Emerging prospects for food scientists.Trends in Food Science and Technology,18, 514–525.

Surangi H, Thilakarathna H P, Rupasinghe V. 2012. Antiatherosclerotic effects of fruit bioactive compounds: A review of current scienti fic evidence.Cananian Journal of Plant Sciences,92,407–419.

Szaefer H, Krajka-Kuzniak V, Ignatowicz E, Adamska T, Baer-Dubowska W. 2014. Evaluation of the effect of beetroot juice on DMBA-induced damage in liver and mammary gland of female Sprague-Dawley rats.Phytotheraphy Research,28, 55–61.

Thaipong K, Boonprakob U, Crosby K, Cisneros-Zevallos L,Byrne D H. 2006. Comparison of ABTS, DPPH, FRAP, and ORAC assays for estimating antioxidant activity from guava fruit extracts.Journal of Food Composition and Analysis,19, 669–675.

Wroblewska M, Juskiewicz J, Wiczkowski W. 2011. Physiological properties of beetroot crisps applied in standard and dyslipidaemic diets of rats.Lipids in Health and Disease,10, 178.

Wyler H, Vincenti G, Mercier M, Sassu G, Dreiding A S.1959. Zur konstitution des randenfarbstoffes betanin.2. (Vorlau fige) mitteilung.Helvetica Chimica Acta,42,1696–1698. (in German)