Characterization of Phytochemicals, Nutrients, and Antiradical Potential in Slim Amaranth

Slim amaranth (A. hybridus) having a C4 photosynthetic pathway with diverse variability is a climate-resilient crop that tolerates abiotic stresses. Owing to the high productivity of the C4 pathway, we have been searching for suitable accessions as preferable high-yielding antioxidant-enriched cultivars with ample bioactive compounds, or for future breeding programs to improve bioactive compounds as a source of natural antioxidants. Twelve slim amaranth accessions were tested for nutraceuticals, phytopigments, radical scavenging capacity (two different assays), vitamins, total flavonoids, and total polyphenols content. Slim amaranth leaves contained ample dietary fiber, protein, moisture, and carbohydrates. The current investigation demonstrated that there was remarkable K, Ca, Mg (8.86, 26.12, and 29.31), Fe, Mn, Cu, Zn, (1192.22, 275.42, 26.13, and 1069.93), TP, TF (201.36 and 135.70), pigments, such as chlorophyll a, ab, and b, (26.28, 38.02, and 11.72), betalains, betaxanthins, betacyanins (78.90, 39.36, 39.53,), vitamin C (1293.65), β-carotene, total carotenoids, (1242.25, 1641.07), and TA (DPPH, ABTS+) (27.58, 50.55) in slim amaranth leaves. The widespread variations were observed across the studied accessions. The slim amaranth accessions, AH11, AH10, and AH12, exhibited high profiles of antioxidants including high potentiality to quench radicals and can be selected as preferable high-yielding antioxidant-enriched cultivars with ample bioactive compounds. Phytopigments, flavonoids, vitamins, and phenolics of slim amaranth leaves showed intense activity of antioxidants. Slim amaranth could be a potential source of proximate phenolics, minerals, phytopigments, vitamins, and flavonoids for gaining adequate nutraceuticals, bioactive components, and potent antioxidants. Moderate yielding accessions having moderate phytochemicals can be used to develop new high-yielding antioxidant-enriched cultivars for future breeding programs to improve bioactive compounds as a source of natural antioxidants.


Introduction
Amaranth is promising grains and vegetables with widespread divergence [1]. In Bangladesh, amaranth may be produced year-round, including in the leafy vegetable gaps, the periods of winter and summer [2,3]. It contains ample protein, including lysine and methionine, minerals, dietary fiber, bioactive pigments, and phytochemicals, including betacyanins and carotenoids betaxanthins, chlorophylls, ascorbic acids and β-carotene, phenolic profiles with sufficient antiradical activity [4][5][6][7][8][9][10][11]. Amaranths are used as folk medicine, especially antimicrobial, anticancer, antidiabetic, antimalarial, and snake antidotes [12]. Therefore, the study was undertaken to achieve the following objectives: (1) To investigate nutraceuticals, phytopigments, bioactive phytochemicals, and the capacity to quench radicals in 12 slim amaranth accessions; (2) To evaluate the variations of these traits in 12 slim amaranth accessions; (3) To select appropriate accession(s) with superior capacity to quench radicals, including nutraceuticals, phytopigments, and bioactive phytochemicals for next-generation high-yielding antioxidant-enriched cultivars, or for future breeding programs to improve bioactive compounds of antioxidants from nature.

Experimental Materials
Seeds of 12 accessions of slim amaranth were collected from the Department of Genetics and Plant Breeding, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur-1706, Bangladesh. The accession numbers of the germplasm are AH1, AH2, AH3, AH4, AH5, AH6, AH7, AH8, AH9, AH10, AH11, and AH12. All accessions have green leaves and stems with high but differential yield potential as leafy vegetables and belong to the same species Amaranthus hybridus. We grew 12 promising slim amaranth accessions to assess nutraceuticals, bioactive compounds, and activity to scavenge radicals.

Design and Layout
The research was implemented in 3 replications following a randomized complete block design (RCBD) at Bangabandhu Sheikh Mujibur Rahman Agricultural University. Individual accession was grown up in a 1 m 2 plot using 5 cm and 20 cm plant and row spacing.

Intercultural Practices
First, 10 t/ha compost was applied during land preparation. Triple superphosphate, urea, gypsum, and murate of potash were applied at 100, 200, 30, and 150 kg/ha, respectively [2]. The spacing of plants in a row was maintained precisely by thinning. Hoeing and weeding were performed regularly to properly eradicate the weeds. Proper growth was maintained by providing regular irrigation in plots. The samples were collected from a 30-day old plant.

Estimation of Proximate Composition
The ash, crude fat, moisture, fiber, protein, and gross energy were measured using the AOAC method. The Micro-Kjeldahl method was followed for nitrogen estimation [30]. Protein was estimated by multiplying nitrogen with 6.25 (AOAC method 976.05). The total protein, ash, moisture, and fat (%) were subtracted from 100 for an estimation of carbohydrate (g 100 g −1 FW). Gross energy was determined using a bomb calorimeter according to the ISO method 9831 [21].

Estimation of Chlorophylls and Carotenoids
Carotenoids and chlorophyll ab, b, and a were measured by extracting the samples in acetone (80%) [32]. The optical density was taken using a Hitachi spectrophotometer (Japan) at 646, 470, and 663 nm. Chlorophylls and carotenoids were measured as mg 100 g −1 and total µg g −1 of fresh weight.

Determination of Betacyanins and Betaxanthins
The samples were extracted in 80% MeOH comprising 50 mM AsA [33][34][35] to measure betaxanthins and betacyanins. The data were measured as µg of betanin and indicaxanthin equivalent to 100 g −1 of fresh weight for betacyanins and betaxanthins.

Estimation of Beta-Carotene
For the assessment of beta-carotene, we followed our previously described method [32]. Data were expressed as micrograms of beta-carotene per gram of fresh weight.

Estimation of Ascorbic Acid
A spectrophotometer (Hitachi, Tokyo, Japan) was set to determine dehydroascorbic acid (DHA) and ascorbic acid (AsA) from the fresh samples of a leaf. Dithiothreitol (DTT) was utilized to pre-incubate the sample. Dithiothreitol (DTT) reduced dehydroascorbic acid to ascorbic acid. As a result of the ascorbic acid reduction, a ferrous ion was formed from the ferric ion. 2,2-dipyridyl reacts to reduced ferrous ions to form complexes [32]. To estimate ascorbic acid, the absorbance of Fe 2+ complexes with 2,2-dipyridyl was read at 525 nm using a spectrophotometric (Hitachi, Japan). The ascorbic acid was calculated in milligrams per 100 g of fresh weight.

Samples Extraction for TP, TF, and TAC Analysis
For TP, TF, and TAC determination, harvested 30-day-old leaves were extracted. The leaves were dried overnight and ground with a mortar and pestle. Leaf powder (0.25 g) was dissolved in 10 mL MeOH (90%) in a bottle capped tightly. Then it was placed in a water bath (Thomastant T-N22S, Thomas Kagaku Co. Ltd., Tokyo, Japan) with shaking. After 1 h, the extract was filtered for further analytical assays of TP, TF, and TAC.

Determination of TP
TP was determined using the Folin-Ciocalteu reagent [36]. The concentration of total phenolic compounds in leaf extracts was determined as µg g −1 of gallic acid equivalent using an equation (Y = 0.009X + 0.019) obtained from a standard gallic acid graph. Results are expressed as the µg g −1 gallic acid equivalent of dry weight (DW).

Estimation of Total Flavonoid Content
The aluminum chloride colorimetric method was used to estimate the total flavonoid content [37]. Rutin was used as a standard compound to make the standard graph (Y = 0.013X). Results are expressed as the µg g −1 rutin equivalent of dry weight (DW).

Radical Quenching Capacity Assay
The antioxidant activity was estimated by the diphenyl-picrylhydrazyl (DPPH) radical degradation method [36]. ABTS + assay was carried out using the method of Khanam et al. [38]. The antioxidant activity was measured following the equation: where A b is the absorbance of the control [150 µL methanol for TAC (ABTS, 10 µL methanol for TAC (DPPH)) instead of leaf extract] and A s is the optical density of the test samples. The results were expressed as µg Trolox equivalent g −1 DW.

Statistical Analysis
Replication-wise data were averaged to obtain the replication mean. Statistix 8 software [39][40][41][42] was used to calculate ANOVA. Tukey's HSD test was used to compare the mean at a 1% level of probability (p ≤ 0.01). The results were expressed as the mean ± SD.

Composition of Proximate
The composition of the proximate of slim amaranth accessions is available in Table 1. AH10 showed the highest moisture (87.58), although AH8 and AH2 displayed the minimum moisture (83.49 and 83.52). The moisture was diverse from 83.49 to 87.58. As lower moisture ensured high dry mass, four accessions (16-17% dry matter) displayed sufficient dry mass. The maturity of the plant is directly associated with the moisture of slim amaranth leaves. The reports of sweet potato [67] and amaranth [21] were corroborated by the present results. Slim amaranth leaves exhibited perceptible differences in protein. The accession AH12 displayed the maximum protein (4.53) although AH6 and AH7 displayed the lowest protein (1.53), which is statistically comparable to the accession AH11 and AH5. Four accessions achieved a better performance over the mean of protein values. The low-income public and vegetarians of low-income nations mostly depend on slim amaranth accessions as a source of protein. The protein of slim amaranth accessions (2.55) was superior to A. tricolor (1.26%) [2].
AH8 displayed the highest fat (0.43 g 100 g −1 ), which showed its statistical similarity to AH11 and AH2. AH5 had the minimum fat content (0.15) with an average of 0.30. Sarker and Oba [21] and Sun et al. [67] observed corroboratory findings in A. tricolor and sweet potato, respectively. They noticed that fat stimuli cell function upheld the temperature of the body and covered the organs. Fats displayed plentiful Ω-6 and Ω-3 fatty acids. Fats performed essential activities in the absorption and transportation of vitamins E, A, D, and K, and digestion. The accession AH8 and AH6 exhibited the highest carbohydrates content (9.47, 9.45), followed by AH9, AH2, AH5, and AH11. The carbohydrates were the lowest in AH12 (5.70) with an average of 7.94. The accession AH9 presented the maximum energy (47.58); thereafter AH8, AH2, AH1, AH6, and AH12, while the accession AH4 displayed the minimum energy (36.27) with an average of 41.97. AH2 revealed the maximum ash (4.65); thereafter AH11, AH8, and AH9, though AH1 unveiled the minimum ash content (2.28) with an average ash content of 3.43.

Composition of Macroelements and Microelements
The macroelements and microelements of slim amaranth accessions are presented in Table 2. In the current study, K varied pronouncedly regarding accessions (5.86 to 10.46). AH1, AH11, AH9, and AH4 demonstrated high K, while AH5 demonstrated the minimum content of K, including the mean K of (8.86). K in six accessions was superior to the mean K. The Ca prominently differed regarding accessions (20.82 to 34.82). AH4, AH11, AH12, AH7, AH5, and AH6 demonstrated high Ca, whereas AH10 revealed the minimum Ca content, including the mean Ca content of 26.12. The Ca of seven accessions was more significant than their mean Ca. AH10 disclosed the maximum Mg (31.13) and AH1 disclosed the minimum Mg (24.51), including a mean Mg of (29.31). AH4, AH8, AH9, AH3, AH7, AH5, and AH2 had more significant Mg content. The Mg displayed the minimum variations regarding accessions (24.51 to 31.13). We noted ample Ca (26.12), K (8.86) and Mg (29.31) in the slim amaranth, although we estimated Ca on dry biomass. Our findings were corroborative of the results of A. lividus [73] and A. tricolor [21], respectively. Jimenez-Aguiar and Grusak [74] noted ample Ca, K, and Mg in several amaranth species. Additionally, they noticed that amaranth's Mg, Ca, and K were much more prominent than in black spider flower, nightshade, kale, and spinach. In the current investigation, we found a much greater Ca, K, and Mg than the Ca, K, and Mg in the A. tricolor of Shukla et al. [75]. The Ca obtained from this study was more prominent than red morph amaranth of our previous studies [68], green morph amaranth [70], stem amaranth [71], and A. blitum [72], while those of weedy amaranth [69] were corroborative of our present findings. The K content of slim amaranth leaves was much more prominent than the K content of our previous studies of green morph amaranth [70] and weedy amaranth [69], while K content obtained from slim amaranth leaves was less than the K content of our earlier studies of red morph amaranth [68], stem amaranth [71], and A. blitum [72]. Mg noticed in slim amaranth leaves was corroborative of our previous findings of red morph amaranth [68], green morph amaranth [70], stem amaranth [71], A. spinosus [69], and A. blitum [72]. In contrast, Mg obtained from slim amaranth leaves was inferior to the Mg of our previous studies of A. viridis [69].  [74], adequate Mn, Fe, Zn, and Cu was found in several amaranth species. Furthermore, they reported that there was a greater preponderance of Mn, Fe, Zn, and Cu in amaranth than in black nightshade, spinach, kale, and spider flower. Our obtained Mn, Fe, Zn, and Cu content was much greater than the Mn, Fe, Zn, and Cu content of several amaranth species [74]. In the current investigation, Mn, Fe, Zn, and Cu in slim amaranth were corroborated by the findings of A. tricolor [21]. Fe content observed in our study was much superior to A. spinosus [69] and inferior to A. viridis, stem amaranth, and A. blitum [69,71,72]. Although Fe in slim amaranth was corroborative of that in red and green morph amaranth [68,69]. The Mn content noticed in our study was much superior to that in red, weedy, stem amaranth, and A. blitum [68,69,71,72], though the present findings of Mn were corroborative of green morph amaranth [70]. The Cu content found in the present study was much better than that found in red and green amaranth and A. spinosus [68][69][70], although slim amaranth Cu content was corroborative of stem amaranth and A. blitum [71,72]. Slim amaranth Zn content was superior to that in red and stem amaranth and A. blitum [68,71,72]. It corroborated with the Zn contents of green amaranth [70], although slim amaranth Zn content was less than that in weedy amaranth [69]. Table 3 shows the pigments of slim amaranth accessions. Chlorophyll a displayed significant and notable variations (12.63 to 47.55). AH8 disclosed the highest chlorophyll a (47.55), whereas the lowest chlorophyll a was recorded in AH6 (12.63). The accessions AH1, AH10, and AH11 showed high chlorophyll a. Four accessions disclosed higher chlorophyll a than the grand mean. Comparable to chlorophyll a, chlorophyll b also demonstrated significant and marked differences across accessions (5.47 to 27.22) in 12 slim amaranth accessions. AH8 displayed the highest chlorophyll b (27.22); thereafter AH10, AH11, and AH1. Contrariwise, AH6 had the minimum chlorophyll b (5.47). Chlorophyll ab had shown significant differences (18.12 to 74.80). AH1, AH10, and AH11 exhibited high chlorophyll ab. AH8 revealed the highest chlorophyll ab; however, AH6 displayed the lowest chlorophyll ab (18.12). Four accessions showed higher chlorophyll ab than the mean values. The notable chlorophyll ab, a, and b (38.02, 26.28, and 11.72) were obtained from slim amaranth; however, in literature [78], comparatively lower chlorophyll was observed in A. tricolor. Chlorophyll a, ab, and b in slim amaranth accessions were much superior to Chlorophyll a, ab, and b of green amaranth [70], although inferior to red, stem, weedy amaranth, and A. blitum [68,69,71,72]. Similarly, AH7, AH12, and AH9 demonstrated good total carotenoids. Seven accessions showed high total carotenoids, which was superior to the mean total carotenoids. In the current investigation, we noted marked betaxanthins (39.53), betacyanins (39.36), total carotenoids (1641.07), and betalains (78.90) in the slim amaranth; these findings of betaxanthin, total carotenoids, betalains, and betacyanins were corroborative to the findings of A. tricolor [78]. The recorded total carotenoid content was much more distinct than the total carotenoids in amaranth [78] and Raju et al. [79]. Betalains, betacyanins, and betaxanthins in slim amaranth were much more preponderant than betalains, betacyanins, and betaxanthins in green and stem [70,71]. At the same time, the pigments were inferior to red, weedy amaranth, and A. blitum [68,69,72]. Total carotenoids of slim amaranth were much superior to the total carotenoids of green and weedy amaranth, and A. blitum [68,69,72]. In contrast, the total carotenoid content of slim amaranth was corroborative of red and stem amaranth [68,71]. The accessions AH8, AH10, and AH1 had abundant chlorophyll a, ab, and b, while the accession AH11, AH10, and AH6 had abundant betacyanins, betaxanthins, betalains, and the accessions AH2, AH7, and AH12 had abundant total carotenoids content as well as the slim amaranth having significant scavenging activity of radicals [80]. The incidence of high betalains, betacyanins, chlorophylls, betaxanthins, and total carotenoids in slim amaranth AH11, AH8, AH10, AH1, AH6, AH2, AH7, and AH12 may make an indispensable contribution to the ROS detoxification of the human body. Hence, these components may prevent many deteriorating human diseases and act as an antiaging agent [18,33] that demands detailed pharmacological study. Table 4 shows the TA, vitamins, TF, and TP of slim amaranth accessions. . AH11 demonstrated the maximum TA. The high TA was noted in AH10, AH12, and AH7. Conversely, TA was at a minimum in AH9 including the mean of TA of 28.57. The TA of four accessions was superior to the mean TA. The beta-carotene of slim amaranth was superior to that in red, weedy, stem amaranth, and A. blitum [68,69,71,72]. Slim amaranth ascorbic acid was superior to that of green weedy and stem amaranth [69][70][71] and lower than in red amaranth and A. blitum [68,72]. TP was superior to A. blitum [72], though it was inferior to red and stem amaranth [68,71]. Total flavonoids in slim amaranth were superior to weedy amaranth [69] and corroborative of green amaranth [70]. In contrast, slim amaranth leaves' TF content was inferior to red, stem, amaranth, and A. blitum [68,71,72]. The antioxidant capacity (DPPH and ABTS + ) of slim amaranth was superior to the survey of green amaranth [70]. In contrast, the TA content of slim amaranth leaves in DPPH and ABTS + was inferior to that of red morph, weedy and stem amaranth, and A. blitum [68,69,71,72].

Bioactive Components and Radical Scavenging Potentiality
We found remarkable amounts of β-carotene (1242.25) and ascorbic acid (1293.65) in slim amaranth which was comparatively superior to A. tricolor [10]. TP (201.36) was also found to be higher than the literature values [38] in A. tricolor. TF (135.70), TA (DPPH) (27.58), and TA (ABTS + ) (50.55) were supported by the earlier findings of A. tricolor [38]. The accessions AH11 and AH10 exhibited a high scavenging activity of radicals, including flavonoids, phenolics, color pigments, and vitamins. The accession AH12 had a high scavenging activity of radicals, including flavonoids, phenolics, total carotenoids, β-carotene, and vitamins. These three accessions had a high scavenging activity of radicals, including flavonoids, phenolics, total carotenoids, β-carotene, and vitamins. Slim amaranth leaves had ample nutraceuticals, phytopigments, bioactive phytochemicals and antioxidants, and presented enormous opportunities for nourishing nutraceuticals and phytopigments bioactive phytochemicals, and antioxidant-deficient communities. Hence, three slim amaranth accessions, AH11, AH10, and AH12, can be selected as preferable high-yielding antioxidant-enriched cultivars with ample bioactive compounds that offer huge prospects for detailed pharmacological study. The data from slim amaranth accessions could make a significant contribution to scientists, nutritionists, and pharmacologists. Moderate yielding accessions that have moderate phytochemicals can be used to develop new high-yielding antioxidantenriched cultivars for future breeding programs to improve bioactive compounds as a source of natural antioxidants.

The Correlation Studies
The associations of the biochemicals in slim amaranth accessions are shown in Table 5. The correlation coefficient values in Table 5 showed encouraging results. TA (DPPH), betacyanins, chlorophyll ab, betaxanthins, chlorophyll a, betalains, TA (ABTS + ), chlorophyll b and TF exhibited significant positive associations among them. The literature on A. tricolor [21] also supports the current findings. Likewise, betalains, betaxanthins, and betacyanins displayed significant positive associations with TA (ABTS + ), TF, chlorophylls, TA (DPPH), and TP [21][22][23][24][25][26][27][28], indicating that the direct increment of any leaf pigment was closely associated with pigments. The positive and significant correlations of TA (DPPH), pigments, TA (ABTS + ), TP and TF indicate that pigments displayed a strong capacity to quench radicals. β-Carotene and total carotenoids (TC) established negative and significant relationships with pigments. In contrast, these parameters displayed positive and significant relationships with TP, TA (ABTS + ) and TA (DPPH), and TF [21][22][23][24][25][26][27][28]. The increment of pigment displayed a drastic decline in TC and β-carotene. Significant positive relationships of β-carotene and TC with TP, TA (ABTS + and DPPH), and TF recommended that β-carotene and TC displayed a strong capacity to quench radicals which were corroborative of the previous amaranths [81][82][83]. β-carotene and TC were associated positively among them. Contrariwise, negligible and insignificant correlations were noted across vitamin C and the rest of the parameters, representing no role in slim amaranth antioxidant activity which was corroborative of an earlier study [74]. TP, TA (ABTS + and DPPH) and TF displayed positive and significant relationships across them; pigments and vitamins showed the participation of antioxidant activity along with flavonoids and phenolics. Correlations of slim amaranth revealed that phytopigments, nutraceuticals and bioactive phytochemicals displayed a significant contribution to the antioxidant capacity of slim amaranth.

Conclusions
Slim amaranth leaves contained ample K, Fe, Ca, Cu, Mg, Mn, Zn, chlorophyll, ascorbic acid, betacyanins, β-carotene, betaxanthins, TA, betalains, carotenoids, protein, digestive fiber, TP, carbohydrates, and TF. Hence, slim amaranth can be utilized as a possible source of pigments, β-carotene, vitamin C, phenolics, nutraceuticals and flavonoids in a regular diet, gaining nutraceuticals and antioxidants sufficiency. The accessions AH11 and AH10 had a high activity for scavenging radicals with flavonoids, phenolics, phytopigments and vitamins. The accession AH12 had a high scavenging activity of radicals with flavonoids, phenolics, total carotenoids, β-carotene, and vitamins. These three accessions had a high activity for scavenging radicals, including flavonoids, phenolics, total carotenoids, β-carotene and vitamins; these can be selected as preferable high-yielding antioxidant-enriched cultivars with ample bioactive compounds and offer huge prospects for detailed pharmacological study. A correlation study revealed that the phenolics, vitamins, flavonoids, and phytopigments of slim amaranth displayed intense activity to scavenge radicals. Slim amaranth could be a potential source of proximate phenolics, minerals, phytopigments, vitamins, and flavonoids for gaining adequate nutraceuticals, bioactive components, and potent antioxidants. The data from slim amaranth accessions could make a significant contribution to scientists, nutritionists, and pharmacologists. Moderate yielding accessions that have moderate phytochemicals can be used to develop new high-yielding antioxidantenriched cultivars for future breeding programs to improve bioactive compounds as a source of natural antioxidants.