Protein, dietary fiber, minerals, antioxidant pigments and phytochemicals, and antioxidant activity in selected red morph Amaranthus leafy vegetable

Amaranth has two morphological types (morphs), one is red and another is green morph. Red morph amaranth is a marvelous source of nutrients, antioxidant pigments, minerals, and phytochemicals compared to green morph amaranth. For this purpose, we selected 25 red morph genotypes to evaluate in terms of proximate, minerals, antioxidant pigments and phytochemicals and antioxidant activity in RCBD design in three replicates. The leaves of red morph amaranth are an excellent source of dietary fiber, carbohydrates, moisture, and protein. We found remarkable potassium, calcium, magnesium (24.96, 10.13, 30.01 mg g-1), iron, manganese, copper, zinc (1089.19, 243.59, 25.77, 986.61 μg g-1), chlorophyll a, chlorophyll b (31.79, 16.05 mg 100 g-1), β-cyanins, total flavonoids (102.10 RE μg g-1 DW), β-xanthins, betalains (33.30, 33.09, 66.40 μg 100 g-1), carotenoids, total phenolics (172.23 GAE μg g-1 DW), β-carotene (1225.94, 1043.18 μg g-1), vitamin C (955.19 μg g-1), and antioxidant activity (DPPH and ABTS+) (19.97 and 39.09 TEAC μg g-1 DW) in the red morph amaranth leaves. We can select the genotype RA5, RA8, RA18, RA22, and RA25 as antioxidant-enriched red morph amaranth. It revealed that amaranth β-cyanins, phenolics, betalains, flavonoids, β-xanthins, carotenoids, vitamin C, and β-carotene had strong antioxidant activity. These phytochemicals contributed significantly in the antioxidant potentials of red morphs amaranth. Red morph amaranth could be a potential source of nutrients, antioxidant pigments, minerals, and phytochemicals as these compounds scavenged ROS and served as potential antioxidants in our daily diet to attaining nutritional and antioxidant sufficiency.


Introduction
The acceptability of foods largely depends on the color of the food products. Recently, the demand for natural pigments such as carotenoids, β-xanthins, β-cyanins, anthocyanin, betalains, and chlorophylls have increased the interest in consumers in the safety, nutritional, and a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 distance from row to row and 5 cm distance from the plant to plant. Recommended fertilizer, compost doses, and appropriate cultural practices were maintained. Thinning was done to maintain appropriate spacing between plants of a row. As a necessity, weeding and hoeing were done to remove the weeds. To maintain the normal growth of the crop proper irrigations were provided. At 30 days after sowing of seed, leaf samples were collected.

Estimation of proximate composition
AOAC method was followed [26] to estimate the ash, moisture, crude fat, fiber, crude protein contents, and gross energy. Micro-Kjeldahl method was followed to determine crude protein multiplying nitrogen by 6.25 (AOAC method 976.05). The sum of crude protein, moisture, crude fat, and ash percentage was subtracted from 100 to estimate carbohydrate (g kg -1 FW).

Determination of mineral composition
Leaves of red amaranth were dried at 70˚C in an oven for 24 hours. Dried leaves were grounded finely in a mill. Nitric-perchloric acid digestion method [26] was followed to determine calcium, potassium, magnesium, iron, manganese, copper, and zinc from powdered leaves. For this digestion, in the presence of carborundum beads 40 ml HClO 4 (70%), 400 ml HNO 3 (65%), and 10 ml H 2 SO 4 (96%) were added to 0.5 g dried leaf sample. After digestion, the solution was appropriately diluted in triplicate for measuring phosphorus following ascorbic acid method. Addition of ascorbic acid and antimony to the yellow-colored complex solution converted to a blue-colored phosphomolybdenum complex. Sarker and Oba [26] method was followed to read the absorbance by atomic absorption spectrophotometry (AAS) (Hitachi, Tokyo, Japan) at a wavelength of 285.2 nm (magnesium), 76 6.5 nm (potassium), 248.3 nm (iron), 422.7 nm (calcium), 279.5 nm (manganese), 213.9 nm (zinc), 324.8 nm (copper).

Determination of chlorophylls and carotenoids
The leaves of red amaranth were extracted in 80% acetone to estimate chlorophyll ab, chlorophyll b, total carotenoids, and chlorophyll a according to the method of Sarker and Oba [26]. A spectrophotometer (Hitachi, U-1800, Tokyo, Japan) was used to read the absorbance at 663 nm for chlorophyll a, 646 nm for chlorophyll b, and 470 nm for total carotenoids, respectively. Data were calculated as mg chlorophyll per 100 g fresh weight (FW) and μg total carotenoids per g FW.

Determination of β-cyanins and β-xanthins content
The leaves of red amaranth were extracted in 80% methyl alcohol having 50 mM ascorbate to measure β-cyanins and β-xanthins according to the method of Sarker and Oba [26]. A spectrophotometer (Hitachi, U-1800, Tokyo, Japan) was used to measure the absorbance at 540 nm for β-cyanins and 475 nm for β-xanthins, respectively. The results were expressed as microgram betanin equivalent per 100 gram FW for β-cyanins and micrograms indicaxanthin equivalent per 100 gram FW for β-xanthins.

Estimation of β-carotene
Method of Sarker and Oba [26] was followed to extract and determine β-carotene content. In a mortar and pestle, 10 ml of 80% acetone was added in 500 mg of fresh leaf sample and ground thoroughly. The extract was centrifuged at 10,000 rpm for 3-4 min. The final volume was brought up to 20 ml after removing the supernatant in a volumetric flask. A spectrophotometer (Hitachi, U-1800, Tokyo, Japan) was used to take the absorbance at 510 nm and 480 nm. Data were expressed as μg β-carotene per g fresh weight (FW).

Estimation of Vitamin C
The fresh red amaranth leaves were used to measure ascorbate (AsA) and dehydroascorbic acid (DHA) acid through a spectrophotometer. For pre-incubation of the sample and reduction of DHA into AsA Dithiothreitol (DTT) was used. AsA reduced Fe 3 + to Fe 2 + and estimation of AsA was made by the spectrophotometric (Hitachi, U-1800, Tokyo, Japan) measuring Fe 2 + complexes with 2, 2-dipyridyl [26]. Finally, the absorbance of the sample solution was read. Data were recorded as μg vitamin C per g fresh weight (FW).

Sample extraction for TPC, TFC and TAC analysis
30 DAS red amaranth leaves were harvested. For chemical analysis, the leaves were dried in the air in a shade. 40 ml of 90% aqueous methanol was used to extract 1 g of grounded dried leaves from each cultivar in a bottle (100 ml) capped tightly. A shaking water bath (Thomastant T-N22S, Thomas Kagaku Co. Ltd., Japan) was used to the extract for 1 h. The extract was filtered for determination of polyphenols, flavonoids, total antioxidant capacity.

Determination of polyphenols
Method of Sarker and Oba [27] was followed to estimate the total phenolic content of red amaranth using the folin-ciocalteu reagent with gallic acid as a standard phenolic compound. Folin-ciocalteu reagent was previously diluted 1:4, reagent: distilled water. In a test tube, 1 ml of diluted folin-ciocalteu was added to 50 μl extract solution and then mixed thoroughly for 3 min. 1 ml of Na 2 CO 3 (10%) was added to the tube and stand for 1 h in the dark. A Hitachi U1800 spectrophotometer (Hitachi, Tokyo, Japan) was used to read the absorbance at 760 nm. 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.

Determination of flavonoids
The AlCl 3 colorimetric method [28,29] was used to estimate the total flavonoid content of red amaranth extract. In a test tube, 1.5 ml of methanol was added to 0.1 ml of 10% aluminum chloride, 0.1 ml of 1 M potassium acetate, 2.8 ml of distilled water and 500 μl of leaf extract for 30 min at room temperature. A Hitachi U1800 spectrophotometer (Hitachi, Tokyo, Japan) was used to take the absorbance of the reaction mixture at 415 nm. TFC is expressed as μg rutin equivalent (RE) g -1 dry weight (DW) using rutin as the standard compound.

Antioxidant capacity (TAC)
Diphenyl-picrylhydrazyl (DPPH) radical degradation method [30] was used to estimate the antioxidant activity. In a test tube, 1 ml of 250 μM DPPH solution was added to 10 μl of leaf extract solution (in triplicate) and 4 ml of distilled water and allowed to stand for 30 min in the dark. A Hitachi U1800 spectrophotometer (Hitachi, Tokyo, Japan) was used to read the absorbance at 517 nm. Method of Sarker and Oba [31] was followed for ABTS + assay. 7.4 mM ABTS + solution and 2.6 mM potassium persulfate were used in the stock solutions. The two stock solutions were mixed in equal quantities and allowing them to react for 12 h at room temperature in the dark for preparation of the working solution. 2850 μl of ABTS + solution (1 ml ABTS + solution mixed with 60 ml methanol) was mixed with 150 μl sample of leaf extract and allowed to react for 2 h in the dark. A Hitachi U1800 spectrophotometer (Hitachi, Tokyo, Japan) was used to read the absorbance against methanol at 734 nm. The percent of inhibition of DPPH and ABTS + relative to the control were used to determine antioxidant activity using the following equation: Antioxidant activity (%) = (Abs. blank-Abs. sample/Abs. blank) × 100 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
At first, sample data of each trait were averaged replication-wise. The mean data of three replications for all traits were also statistically analyzed by ANOVA using Statistix 8 software, and the means were compared by the Tukey's HSD test at 1% level of probability. The results were reported as the average of three replications ± SD.

Results
The analysis of variance demonstrated that all the traits significantly varied between the different studied genotypes (Tables 1, 2, 3 and 4). Proximate and mineral compositions, antioxidant leaf pigments, vitamins, TAC (DPPH), TFC, TPC, and TAC (ABTS + ) of the 25 tested red morph amaranth genotypes are presented in Tables 1, 2, 3 and 4.

Proximate compositions
Proximate compositions of red morph amaranth are presented in Table 1. The genotype RA11 exhibited the highest moisture content (884.73 g kg -1 FW), while the genotype RA3 and RA18 had the lowest moisture content (814.64 and 814.83 g kg -1 FW). The moisture content ranged from 814.64 to 884.73 g kg -1 FW. Red morph amaranth leaves exhibited noticeable variations in protein content. The genotype RA3 had the highest protein content (62.26 g kg -1 ) followed by RA11 and RA15, whereas the genotype RA6 exhibited the lowest protein content (11.38 g kg -1 ). For protein content, ten genotypes performed better over their mean value. Among them, eight genotypes RA3, RA8, RA11, RA15, RA5, RA9, RA18, and RA19 showed higher protein content as leafy vegetables. The highest fat content was observed in the genotype RA7 (4.35 g kg -1 FW) against the lowest content recorded for the genotype RA25 (1.42 g kg-1 FW) with an average of 2.93 g kg -1 FW.
The highest carbohydrates content were noted in the genotype RA16 and RA6 (98.54 and 97.51 g kg -1 FW) followed by RA1, RA25, and RA20, while the lowest carbohydrates content was observed in RA19 (15.48 g kg -1 FW) with an average of 71.41 g kg -1 FW. The genotype RA18 and RA3 had the highest energy (56.07 and 55.33 Kcal 100 g -1 FW) followed by RA3, RA8, RA15, and RA25, while the lowest energy was obtained from the genotype RA19 (26.95 Kcal 100g -1 FW) with an average of 43.40 Kcal 100 g -1 FW. Ash content was the highest in the genotype RA3 (56.55 g kg -1 FW) followed by RA8, RA5, RA19, RA15, and RA18, while the lowest ash content was noted in RA24 (20.57 g kg -1 FW) with an average of 38.86 g kg -1 FW. Dietary fiber content exhibited remarkable variations in 25 red morph amaranth studied. The dietary fiber content was the highest in RA25 and RA2 (91.94 and 91.66 μg g -1 FW) followed by RA2, RA4, RA22, RA21, RA9, RA1, RA16, RA8, and RA20, while RA23 exhibited the lowest dietary fiber content (59.96 μg g -1 FW) with an average of 78.27 μg g -1 FW.

Mineral compositions
Mineral compositions of red morph amaranth are presented in Table 2. In the present investigation, K content ranged from 6.55 mg g -1 to 16.28 mg g -1 DW. The genotypes RA13, RA15, RA3, RA11, and exhibited high K content, while genotype RA17 and RA23 showed the lowest K content with an average of 10.13 mg g -1 DW. Thirteen genotypes performed The significant and remarkable variations were detected for iron content (195.12 μg g -1 DW in RA24 to 2057.02 μg g -1 DW in RA18). The genotypes RA18, RA15, RA13, RA14, and RA23 exhibited the highest iron content. Conversely, the genotype RA24 showed the lowest iron content, with an average value of 1089.19 μg g -1 DW. Nine genotypes had higher iron content over their average performance. In this study, the manganese content ranged between 132.65 μg g -1 DW and 356.84 μg g -1 DW, with an average of 243.59 μg g -1 DW. The genotype

Correlation coefficient analysis
Correlation of bioactive compounds of red morph amaranth is presented in Table 5. The analysis of correlation coefficients presented in Table 5

Discussion
Color, flavor, and taste predominantly influenced the acceptability of foods products. Considering the safety, nutritional, and aesthetic aspects of food, the demand for natural pigments have been increased in consumers day by day. Red morph amaranth was a unique and inexpensive source of color pigments such as β-cyanins, β-xanthins, betalains, anthocyanin, amaranthine, carotenoids, and chlorophylls that have potential free radical detoxifying ability and act as potent antioxidants [1,3]. The active ingredients of these pigments have significant contributions to human health as they provide protection against lung and skin cancers, cardiovascular and inflammatory disease [4]. Red morph amaranth also had abundant natural antioxidant phytochemicals such as flavonoids, β-carotene, phenolics, and vitamin C along with protein, dietary fiber, and minerals. These natural antioxidants protect cancer, emphysema, cardiovascular diseases, atherosclerosis, diabetes, retinopathy, osteoporesis, neurodegenerative diseases, arthritis, cataracts, inflammation, and prevent aging [23][24][25]. It is a widely distributed leafy vegetable in Bangladesh, Asia, Africa and South America with great variability and phenotypic plasticity [22]. In Bangladesh, red morph amaranth was grown year-round and in the hot summer, a gap period of foliage vegetables [11][12]. Its attractive leaf color, nutritional value, and taste make it popular in the rest of the continent and elsewhere. Its production and consumption have been remarkably increased due to the presence of excellent nutritional and natural antioxidants such as antioxidant leaf pigments, vitamins, phenolics, and flavonoids. So, red morph amaranth could significantly contribute to local, regional and international nutritional and antioxidants security challenges by reducing the hidden hunger and attaining nutritional and antioxidant sufficiency in the world. In this study, we comprehensively evaluated 25 red morph amaranth genotypes in terms of proximate and mineral compositions, antioxidant pigments, phytochemicals, and antioxidant activity and their variability. Our results demonstrated that red morph amaranth had abundant natural pigments, phytochemicals with high antioxidant activity along with nutritional components. However, the components varied significantly in terms of genotypes. One of the interesting findings of our study was that we obtained abundant antioxidant pigments and phytochemicals with high antioxidant activity along with nutritional components in the red morph amaranth genotypes which could contribute to local, regional, and international nutritional and antioxidant sufficiency by reducing the hidden hunger and attaining antioxidant sufficiency in the world. We found remarkable chlorophyll a (31.79 mg 100 g -1 ), chlorophyll b (16.05 mg 100 g -1 ) and chlorophyll ab (47.83 mg 100 g -1 ) content in the red morphs amaranth, whereas, Khanam and Oba [32] observed comparatively lower chlorophyll content in red morphs amaranth. We observed remarkable β-cyanins (56.78 μg 100 g -1 ), βxanthins (58.12 μg 100 g -1 ), betalains (114.89 μg 100 g -1 ), and total carotenoids (1677.26 μg g -1 ) in the red morphs amaranth, similarly, Khanam and Oba [32] observed more or less similar trend in β-cyanins, β-xanthins, betalains, and total carotenoids content of red morphs amaranth. Regarding phytochemicals, we found remarkable β-carotene (1524.41 μg g -1 ), vitamin C (1990.58 μg g -1 ) in the red morphs amaranth, which exhibited comparatively higher values in terms of our previous studies in A. tricolor [12]. TPC (260.84 GAE μg g -1 DW) obtained in this study were also higher than the results of Khanam and Oba [32] in A. tricolor. TFC (171.26 RE μg g -1 DW), TAC (DPPH) (31.70 TEAC μg g -1 DW), and TAC (ABTS + ) (62.03 TEAC μg g -1 DW) obtained in this study were more or less similar to the results of Khanam et al. [33] in A. tricolor. The genotypes RA25 had the highest TAC (DPPH, ABTS+), chlorophylls, high betalains, total carotenoids, β-carotene, phenolics, and flavonoids. The genotype RA18 had high TAC (DPPH, ABTS+), chlorophylls, betalains, total carotenoids, β-carotene, phenolics, and flavonoids. The genotype RA15 exhibited high TAC (DPPH, ABTS+), chlorophylls, betalains, total carotenoids, β-carotene, and the highest phenolics and flavonoids. The genotype RA5 and RA3 had high TAC (DPPH, ABTS+), chlorophylls, betalains, total carotenoids, moderate β-carotene, phenolics, and flavonoids. These five genotypes could be used as antioxidant profile enriched high-yielding varieties. We can conclude that red amaranth has abundant phenolics, pigments, flavonoids, vitamins, and antioxidant that offered enormous prospects for nourishing the vitamin and antioxidant scarce people.
As lower moisture content was desirable to confirm the higher dry matter, six genotypes such as RA3, RA18, RA15, RA8, RA5, and RA25 showed 18-19% dry matter might be selected for dry matter. The moisture content of red morph amaranth leaves directly related to the maturity of the plant. Similar results were reported by Sun et al. on sweet potato leaves [34]. Eight genotypes RA3, RA8, RA11, RA15, RA5, RA9, RA18, and RA19 showed higher protein content as leafy vegetables. Vegetarian and poor people of the low-income countries mainly depend on red amaranth for their protein source. So, red morph amaranth might be an excellent source of protein for vegetarian and poor people. The protein content of red morph amaranth (33.99 g kg -1 ) was much higher as compared to A. tricolor (1.26%) in our earlier study [2]. The fat content in the present study agreed to the results of Sun et al. [34] in sweet potato leaves. They mentioned that fat involved in the insulation of body organs and maintenance of body temperature and cell function. Fats have abundant omega-3 and omega-6 fatty acids. Fats play a significant role in absorption, digestion, and transport of fat-soluble vitamins A, D, E, and K. Dietary fiber has a significant role in palatability, digestibility, and remedy of constipation [14]. From the results, we observed that red morph amaranth leaves have abundant carbohydrates, protein, moisture, and dietary fiber. As red morph amaranth had low energy content, this may not impact significantly on energy contribution to the human body as low amounts of this vegetable consumed in a day. Like other leafy vegetables, the low carbohydrate content of red morph amaranth may not have a significant impact on carbohydrate contribution to the human body considering the low amount of vegetable uptake per day and a very high daily requirement for the human body.
Amaranth had higher mineral contents than commonly consumed leafy vegetables, such as spinach, lettuce, and kale [35]. In our present study, we found remarkable K (10.13 mg g -1 ), Ca (24.96 mg g -1 ), and Mg (30.01 mg g -1 ) in the red morph amaranth, albeit we estimated in dry weight basis. Jimenez-Aguiar and Grusak [35] noted abundant K, Ca, and Mg in different amaranths including red amaranth. They also found amaranth K, Ca, and Mg was much higher than spinach, spider flower, kale, and black nightshade. Zinc and iron content of red morph amaranth is higher than the cassava leaves [36] and beach pea [37]. In this study, we found remarkable Fe (1089.19 μg g -1 ), Mn (243.59 μg g -1 ), Cu (25.77 μg g -1 ), and Zn (996.61 μg g -1 ) in red morph amaranth, although we estimated in dry weight basis. Similarly, Jimenez-Aguiar and Grusak [35] noted abundant Fe, Mn, Cu, and Zn in different amaranths including red amaranth. They also found amaranth Fe, Mn, Cu, and Zn were much higher than spinach, spider flower, kale, and black nightshade. The U.S. Department of Agriculture's National Nutrient Database for Standard Reference [35] lists a serving size of spinach as 30 g fresh weight FW (1 cup). As red amaranth has higher mineral concentrations than spinach so, a serving size of leaves of 30 g FW is enough for nutritional sufficiency.
The chlorophyll ab, chlorophyll b, and chlorophyll b exhibited positive and significant associations among each of them and with β-cyanins, β-xanthins, betalains, TAC (ABTS + ), TFC, TPC, and TAC (DPPH). It revealed that increment of one leaf pigment straightly associated with an increase of another leaf pigment. The significant positive correlation of all leaf pigments with TAC (ABTS + ), TFC, TPC, and TAC (DPPH) signifies that all the leaf pigments had strong antioxidant activity. Significant positive association of carotenoids and β-carotene with TAC (ABTS + ). TFC, TFC, TPC, and TAC (DPPH) suggested that carotenoids and β-carotene had strong antioxidant activity. Ascorbic acid exhibited insignificant interrelationships with all the traits indicating negligible contribution in the antioxidant potentiality of red morphs amaranth. Jimenez-Aguilar and Grusak [35] reported similar findings for ascorbic acid in amaranth. A similar trend of the insignificant association was also observed by Shukla et al. [38] in their earlier work in amaranth. TAC (ABTS + ), TFC, TPC, and TAC (DPPH) exhibited substantial positive association among each other, all leaf pigments, and vitamins representing the involvement of these phytochemicals in antioxidant activity. In the present investigation, it revealed that phenolics, pigments, flavonoids, and vitamins contribute significantly in the antioxidant potentiality of red morphs amaranth.

Conclusions
Red morphs amaranth leaves have abundant antioxidant pigments and phytochemicals such as β-carotene, chlorophyll, vitamin C, β-cyanins, carotenoids, β-xanthins, TAC, betalains, TPC, and TFC. It also has abundant protein, dietary fiber, and minerals such as Ca, K, Mg, Fe, Cu, Zn, and Cu compared to leafy vegetables. Correlation study revealed that natural antioxidant pigments and phytochemicals had strong antioxidant capacity. It could be a potential leafy vegetable as a source of natural antioxidant pigments and phytochemicals having strong antioxidant activity along with nutritional components in our daily diet to combat with the hidden hunger and attaining nutritional and antioxidant sufficiency. Hence, red morphs amaranth with rice, wheat, and maize in our daily diet could contribute to attaining nutritional and antioxidant sufficiency.