Nutrients, minerals, pigments, phytochemicals, and radical scavenging activity in Amaranthus blitum leafy vegetables

A. blitum is good sources of abundant natural antioxidant phytopigments such as anthocyanin, betalain, betaxanthin, and betacyanin and antioxidant phytochemicals of interest in the food industry. The chances of utilizing amaranth pigments and phytochemicals had been evaluated for extracting colorful juice as drink purposes. Hence, the presence of nutrients, phytopigments, phytochemicals, and radical scavenging activity of selected A. blitum leafy vegetables were evaluated. Leaves of A. blitum have considerable fiber, moisture, protein, and carbohydrates. It has considerable magnesium, calcium, potassium (30.42, 24.74, 10.24 mg g−1), zinc, iron, copper, manganese, (878.98, 1153.83, 26.13, 207.50 µg g−1), phytopigments such as chlorophyll a, chlorophyll ab, chlorophyll b, (63.69, 90.60, 29.32 mg 100 g−1), betalain, betaxanthin, betacyanin (112.01, 58.38, 53.63 µg 100 g−1), vitamin C (1848.15 µg g−1), total carotenoids, β-carotene (1675.38, 1281.66 µg g−1), TPC, TFC (253.45 GAE and 162.97 RE µg g−1 DW), and TAC (29.46, 55.72 µg g−1 DW in Tolax equivalent DPPH and ABTS+ radical scavenging capacity) in A. blitum. The accessions DS3, DS6, DS8, and DS12 exhibited the highest TAC in Trolox equivalent DPPH and ABTS+ radical scavenging capacity, flavonoids, and considerable phytopigments. These accessions had excellent antioxidant profiles along with high yielding potentiality. Hence, A. blitum provides an excellent source of proximate, phenolics, minerals, flavonoids, vitamins, and phytopigments to address the nutritional and antioxidant deficiency in daily diet.


DS1
85.46 ± 1.87d 1.13 ± 0.03k 0.25 ± 0.03e 9.71 ± 0.07b 46.32 ± 0.35 g 3.45 ± 0.03 g 73. 40  The considerable variations were observed in 16 A. blitum genotypes in terms of dietary fiber. The accession DS13 showed the highest content of fiber (97.88 µg g −1 ) followed by DS12, DS15, and DS8 whereas the lowest content of fiber was noted in DS9 (59.96 µg g −1 ) with a mean value of 76.60 µg g −1 . Dietary fiber significantly contributed to the cure of constipation, digestibility, and palatability 6 . Our results exhibited that the leaves of A. blitum were a considerable amount of dietary fiber, moisture, carbohydrates, and protein. The results of this study corroborated with the results of Sarker and oba 25 . The genotype DS4 could be used as dry matter, protein, and ash enrich leafy vegetables. The genotype DS15 could be used as carbohydrates enrich leafy vegetables, while The genotype DS13 could be used as dietary fiber and DS6 as calories enrich leafy vegetables. composition of minerals. Manganese, potassium, copper, magnesium, iron, calcium, and zinc content of A. blitum are shown in Table 2. In this study, the range of potassium content was 0.95 to 16.28 mg g −1 . The accessions DS2, DS4, DS14, DS12, DS15, DS5, and DS1 showed good content of potassium, while the lowest potassium content was reported in the accession DS10, with mean potassium content of 10.24 mg g −1 . The potassium content of nine genotypes was much higher than their grand mean. The range of calcium content was 15.22-32.82 mg g −1 DW. The accessions DS1, DS7, DS14, DS10, DS4, DS11, and DS13 had good calcium content, while the lowest calcium content was recorded in the accession DS16 with a mean calcium content of 24.74 mg g −1 . High calcium content was noted in seven accessions which were better than the respective average value. The accession DS2 had the highest magnesium content. In contrast, the accessions DS8, DS9, and DS16 showed the lowest magnesium content with a mean value of 30.42 mg g −1 . The accessions DS2, DS5, DS1, DS13, DS6, DS7, DS10, and DS12 had considerable magnesium content. Magnesium content did not exhibit pronounced variations in 16 A. blitum genotypes (28.63 to 35.43 mg g −1 ). Our study revealed that we noted a considerable amount of potassium (10.24 mg g −1 ), calcium (24.74 mg g −1 ) and magnesium (30.42 mg g −1 ) in the leaf of A. blitum, albeit we determined based on the dry weight. Chakrabarty et al. 27 in stem amaranth and Sarker and Oba 25 in A. tricolor also observed similar results. Jimenez-Aguiar and Grusak 28 reported a good amount of Mg, K, and Ca in different species of amaranth. They reported that Mg, Ca, and K content of different species of amaranth was much greater than kale, black nightshade, spider flower, and spinach.
Iron content showed prominent variations in terms of genotypes (195.12 to 2057.02 µg g −1 ). The highest iron content was observed in the genotypes DS6. In contrast, the lowest iron content was obtained from the genotype DS11, with a mean iron content of 1153.83 µg g −1 . Six accessions exhibited higher content of iron than their mean iron content. The range of manganese content was 132.65 to 356.84 µg g −1 , with a mean value of 207.50 µg g −1 . The accessions DS2, DS1, DS5, DS9, and DS7 had considerable content of manganese, while the lowest manganese content was recorded in the genotype DS6 (132.65 µg g −1 ). Copper content exhibited considerable variations in terms of accessions (16.09-45.12 µg g −1 ). The accession DS3 showed the highest copper content (45.12 µg g −1 ), followed by DS7, DS9, DS10, DS5, and DS2. Seven accessions showed better copper content than the average value (26.13 µg g −1 ). The accession varied considerably in the content of zinc (680.41, 681.07, 681.38 µg g −1 in DS10, DS13, and DS8, respectively to 1473.54 µg g −1 in DS2). High zinc content was observed in seven genotypes which were higher than the grand mean value (878.98 µg g −1 ). Three genotypes DS2, DS3, and DS7 exhibited excellent zinc content (1082.09 to 1473.54 µg g −1 DW). Leaves of A. blitum contained higher zinc and iron compared to beach pea 29  www.nature.com/scientificreports www.nature.com/scientificreports/ (1153.83 µg g −1 ), manganese (207.50 µg g −1 ), copper (26.13 µg g −1 ), and zinc (878.98 µg g −1 ), albeit it was measured based on the dry weight. Jimenez-Aguiar and Grusak 28 reported a good amount of iron, manganese, copper, and zinc in the different species of amaranth. They reported that iron, manganese, copper, and zinc content of different species of amaranth were much greater than kale, black nightshade, spider flower, and spinach. The genotype DS2 could be used as potassium, magnesium, iron, manganese, and zinc enrich leafy vegetables. The genotype DS1 could be used as calcium enrich leafy vegetable, while DS3 could be used as copper and DS6 as iron enrich leafy vegetables. composition of antioxidant phytopigments. Table 3 represents the composition of antioxidant phytopigments of 16 A. blitum genotypes. Chlorophyll a content differed remarkably in A. blitum genotypes (13.17 to 63.69 mg 100 g −1 ). The highest chlorophyll a content was obtained from the genotype DS8 (63.69 mg 100 g −1 ), while the accessions DS3 and DS2 showed the lowest chlorophyll a (13.17 and 13.26 mg 100 g −1 ). Chlorophyll a content was high in the genotypes DS13, DS4, DS6, and DS15. The chlorophyll a content of six genotypes was higher than the average value. There were prominent variations in chlorophyll b content of 16 A. blitum genotypes (4.97 to 29.32 mg 100 g −1 ). The highest chlorophyll b content was observed in DS15 (29.32 mg kg −1 ), followed by DS8, DS4, DS12, and DS6. Conversely, DS7 had the lowest chlorophyll b (4.97 mg 100 g −1 ). Prominent variations were also observed in chlorophyll ab (19.72 to 90.60 mg 100 g −1 ). DS8, DS4, DS13, DS15, and DS6 showed good content of chlorophyll ab, while DS2 had the lowest chlorophyll ab content (19.72 mg 100 g −1 ). Eight accessions exhibited higher content of chlorophyll ab than the mean chlorophyll ab content. Our study revealed that stem amaranth genotypes had a considerable amount of chlorophyll ab (90.60 mg 100 g −1 ), chlorophyll a (63.69 mg 100 g −1 ), and chlorophyll b (29.32 mg 100 g −1 ), whereas, chlorophylls content of A. tricolor reported by Khanam and Oba 31 were relatively lower.
In this study, we reported considerable β-carotene (1281.66 µg g −1 ) and vitamin C (1848.15 µg g −1 ) in A. blitum, which was relatively higher than A. tricolor 3 of our earlier studies. Our obtained TPC (253.45 GAE µg g −1 FW) was higher than the TPC of A. tricolor reported by Khanam et al. 32 . Our reported TFC (162.97 RE µg g −1 DW) and TAC (ABTS + and DPPH) (55.72 and 29.46 TEAC µg g −1 DW) were corroborative to the results of A. tricolor of Khanam et al. 32 . The accessions DS14, DS7, and DS4 could be used as beta-carotene, vitamin C, and TPC enrich leafy vegetables, respectively. The accession DS3 showed the highest TAC (ABTS + and DPPH), flavonoids, and copper, as well as DS6, exhibited the highest TAC (ABTS + and DPPH), flavonoids, and iron. Similarly, The accession DS8 contained the highest TAC (ABTS + and DPPH), chlorophylls, flavonoids, and polyphenols, as well as DS12, showed the highest TAC (ABTS + and DPPH), flavonoids, betacyanin, betalain, and betaxanthin. These four accessions had excellent antioxidant profiles along with high yielding potentiality. Hence, A. blitum provides an excellent source of proximate, phenolics, minerals, flavonoids, vitamins, and phytopigments to address the nutritional and antioxidant deficiency in daily diet.   www.nature.com/scientificreports www.nature.com/scientificreports/ correlation studies. The coefficient of correlation of biologically active compounds of A. blitum is shown in Table 5. The coefficient of correlation of biologically active compounds shown in Table 5 had interesting results. We observed a significant positive correlation among TAC (DPPH), chlorophyll ab, betacyanin, chlorophyll a, betaxanthin, betalain, TAC (ABTS + ), chlorophyll b, and TFC. Shukla et al. 33 also reported positive associations in their earlier work in A. tricolor. Similarly, betacyanin, betaxanthin, and betalain showed positive and significant interrelationships among each of them and with TAC (ABTS + ), chlorophylls, TFC, TAC (DPPH), and TPC which was corroborated with the results of our earlier studies 8,9 indicating an increase in any phytopigment was directly related to increment of another phytopigment. The positive and significant interrelationships of TAC (DPPH), all phytopigments, TAC (ABTS + ), TFC, and TPC indicated that phytopigments, TFC, and TPC exhibited strong antioxidant potential. The significant negative association was observed between phytopigments vs. total carotenoids and phytopigments vs. beta-carotene, while total carotenoids and beta-carotene exhibited a significant positive association with TAC (ABTS + ), TAC (DPPH), TPC, and TFC which was corroborated with the results of our earlier studies in amaranth [20][21][22][23][24] . It indicated that the increment of any phytopigment had a direct decrement of total carotenoids and beta-carotene. The positive and significant interrelationship of total carotenoids and beta-carotene with TPC, TAC (ABTS + and DPPH), and TFC signifies that β-carotene and total carotenoids had excellent antioxidant potentiality. There were positive associations between beta-carotene and total carotenoids. In contrast, the negligible insignificant association was observed between vitamin C and all the characters indicating that vitamin C had no contribution to the antioxidant activity of A. blitum. Jimenez-Aguilar and Grusak 28 reported a negligible insignificant association for ascorbic acid in amaranth. The positive and significant associations were observed among TAC (ABTS + ), TPC, TAC (DPPH), and TFC as well as all phytopigments, and vitamins indicating the contribution of these compounds in the antioxidant potentiality of A. blitum genotypes. Our reported results revealed that phytopigments, vitamins, phenolics, and total flavonoids played a significant contribution to the antioxidant capacity of A. blitum.
In conclusion, A. blitum leaves were good sources of K, Ca, Mg, iron, manganese, copper, zinc, chlorophyll, vitamin C, betacyanin, betaxanthin, TAC, betalain, carotenoids, β-carotene, dietary fiber, carbohydrates, protein, TPC, and TFC. It could be used as leafy vegetables for potential sources of antioxidant phytopigments, vitamin C, β-carotene, phenolics, minerals and proximate, flavonoids in the human diet to address the nutritional deficiency and gaining antioxidant and nutritional sufficiency. Details studies on animal models and humans are prerequisites to confirm nutrition and pharmacology before promoting the use of the leaves for health purposes.  oven. We ground the dried leaves in a mill finely. Calcium, potassium, magnesium, iron, manganese, copper, and zinc were determined following nitric-perchloric acid digestion method 36 . Exactly 0.5 g dried leaf sample was digested with 40 ml HClO 4 (70%), 400 ml HNO 3 (65%), and 10 ml H 2 SO 4 (96%) in the presence of carborundum beads. After digestion, the ascorbic acid method was followed to measure P in triplicate from an appropriately diluted solution. Ascorbic acid and Sb were added to the yellow-colored complex solution to convert a blue-colored phosphomolybdenum complex. estimation of carotenoids and chlorophylls. Method of Sarker and Oba 35,37 was followed to estimate chlorophyll ab, chlorophyll b, total carotenoids, and chlorophyll a through extracting the fresh leaves of A. blitum in 80% acetone. The absorbance was read at 663 nm for chlorophyll a, 646 nm for chlorophyll b, and 470 nm for total carotenoids, respectively using a spectrophotometer (Hitachi, U-1800, Tokyo, Japan). Data were expressed as mg chlorophyll per 100 g and µg total carotenoids per g fresh weight.
estimation of betacyanin and betaxanthin composition. Method of Sarker and Oba 35,38 was followed to estimate betacyanin and betaxanthin through extracting the leaves of A. blitum in 80% methyl alcohol having 50 mM ascorbate. Betacyanin and betaxanthin were estimated using a spectrophotometer (Hitachi, U-1800, Tokyo, Japan) at 540 nm for betacyanin and 475 nm for betaxanthin, respectively. The results were expressed as microgram betanin equivalent per 100 gram fresh weight (FW) for betacyanin and micrograms indicaxanthin equivalent per 100 gram FW for betaxanthin. estimation of β-carotene β-carotene content was extracted following the method of Sarker and Oba 35 . Exactly 500 mg of fresh leaf sample was ground thoroughly in a mortar and pestle with 10 ml of 80% acetone. After removing the supernatant in a volumetric flask, the extract was centrifuged at 10,000 × g for 3-4 min. The final volume was brought up to 20 ml. The absorbance was taken at 510 nm and 480 nm using a spectrophotometer (Hitachi, U-1800, Tokyo, Japan). Data were expressed as µg β-carotene per g fresh weight.
The following formula was used to estimate the β-carotene content:  35,39 . Finally, the absorbance of the sample solution was read at 525 nm using a spectrophotometer (Hitachi, U-1800, Tokyo, Japan) and data were expressed as µg vitamin C per g fresh weight. The solution was read at 525 nm and data were expressed as µg vitamin C per g fresh weight.
Extraction of samples for TAC, TFC, and TPC analysis. The leaf samples were dried in the air in a shade for chemical analysis. Exactly 1 g of grounded dried leaves was extracted in 40 ml of 90% aqueous methanol in a tightly capped bottle (100 ml). We placed the bottles in a shaking water bath (Thomastant T-N22S, Thomas Kagaku Co. Ltd., Japan) for 1 h. The extract was filtered for measuring total antioxidant capacity, flavonoids, and polyphenols. total polyphenols estimation. The method described by Sarker and Oba 35,40 was followed to estimate the total phenolic content of A. blitum leaf samples. The gallic acid was used as a standard phenolic compound. We diluted the Folin-ciocalteu reagent in the ratio of 1:4, reagent: distilled water. Exactly 1 ml Na 2 CO 3 (10%), and 1 ml diluted folin-ciocalteu solution were added to a test tube containing 50 µl extract and mixed thoroughly for 3 min.
www.nature.com/scientificreports www.nature.com/scientificreports/ The tube was allowed to stand for 1 h in the dark. The absorbance was read at 760 nm using a Hitachi U1800 spectrophotometer (Hitachi, Tokyo, Japan). A standard gallic acid graph was made to determine the concentration of phenolics in the extracts. The results are expressed as μg gallic acid equivalent (GAE) g −1 DW.
Estimation of total flavonoids. The total flavonoid content of A. blitum extract was estimated following the AlCl 3 colorimetric method 35,41 . Exactly 1.5 ml methanol, 0.1 ml 1 M potassium acetate, 0.1 ml 10% aluminum chloride, and 2.8 ml distilled water was added to a test tube containing 500 µl leaf extract and allowed to stand for 30 min at room temperature. The absorbance of the reaction mixture was taken at 415 nm using a Hitachi U1800 spectrophotometer (Hitachi, Tokyo, Japan). TFC is expressed as μg rutin equivalent (RE) g −1 dry weight (DW) using rutin as the standard compound.
Estimation of total antioxidant capacity (TAC). The antioxidant capacity was determined through diphenyl-picrylhydrazyl (DPPH) radical degradation method 35 . Exactly 1 ml 250 µM DPPH solution was added to a test tube containing 10 µl of leaf extract (in triplicate) with 4 ml distilled water and allowed to stand for 30 min in the dark. The absorbance was read at 517 nm using a Hitachi U1800 spectrophotometer (Hitachi, Tokyo, Japan). The method described by Sarker and Oba 35 was followed to estimate TAC (ABTS + ) assay. Exactly 2.6 mM potassium persulfate and 7.4 mM ABTS + solution were used in the stock solutions. For the preparation of the working solution, the two stock solutions were mixed in equal quantities and allowed them to react for 12 h at room temperature in the dark. Exactly 150 μl sample of leaf extract was mixed with 2850 μl of ABTS + solution (1 ml ABTS + solution mixed with 60 ml methanol) and allowed to react for 2 h in the dark. The absorbance was read at 734 nm using a Hitachi U1800 spectrophotometer (Hitachi, Tokyo, Japan) against methanol. The percent of inhibition of DPPH and ABTS + relative to the control were used to determine antioxidant activity using the following equation:
− . . × Where, Abs. blank is the absorbance of the control reaction [10 µl methanol for TAC (DPPH), 150 μl methanol for TAC (ABTS + ) instead of leaf extract] and Abs. 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 −1 DW. Statistical analysis. Mineral, phytopigments, chlorophylls, carotenoids, beta-carotene, vitamin C, polyphenols, flavonoids, and antioxidant activity (ABTS + & DPPH) analysis were evaluated in three independent samples per replication (each sample was prepared from a combined sample of leaves from multiple plants) and nine samples per genotype 41 . Results were expressed as mean value ± standard deviation per genotype. Every mean represents the average of all measurements for the same genotype (Tables 1-4). ANOVA was performed using Statistix 8 software and the means were compared by Tukey's HSD test at 1% and level of probability.

Data availability
Data used in this manuscript will be available to the public.