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Phytonutrients, Colorant Pigments, Phytochemicals, and Antioxidant Potential of Orphan Leafy Amaranthus Species

The underutilized Amaranthus leafy vegetables are a unique basis of pigments such as β-cyanins, β-xanthins, and betalains with radical scavenging capacity (RSC). They have abundant phytonutrients and antioxidant components, such as pigments, vitamins, phenolics, and flavonoids. Eight selected genotypes (four genotypes from each species) of underutilized Amaranthus leafy vegetables were evaluated for phytonutrients, pigments, vitamins, phenolics, flavonoids, and antioxidants in a randomized complete block design under ambient field conditions with three replicates. The studied traits showed a wide range of variations across eight genotypes of two species of Amaranthus leafy vegetables. The highest fat, β-xanthins, K, dietary fiber, Mg, β-cyanins, Mn, chlorophyll ab, Zn, TP, TF, betalains, chlorophyll a content, and (RSC) (DPPH) and RSC (ABTS+) were obtained from A. tricolor accessions. Conversely, the highest protein, Cu, carbohydrates, Ca, and chlorophyll b content were obtained from A. lividus accessions. The highest dry matter, carotenoids, Fe, energy, and ash were obtained from A. tricolor and A. lividus. The accession AT2 confirmed the highest vit. C and RSC (DPPH) and RSC (ABTS+); AT5 had the highest TP content; and AT12 had the highest TF content. A. tricolor accessions had high phytochemicals across the two species, such as phytopigments, vitamins, phenolics, antioxidants, and flavonoids, with considerable nutrients and protein. Hence, A. tricolor accessions can be used as high-yielding cultivars comprising ample antioxidants. The correlation study revealed that vitamin C, pigments, flavonoids, β-carotene, and phenolics demonstrated a strong RSC, and showed a substantial contribution to the antioxidant potential (AP) of A. tricolor. The investigation exposed that the accessions displayed a plentiful origin of nutritional values, phytochemicals, and AP with good quenching ability of reactive oxygen species (ROS) that provide enormous prospects for nourishing the mineral-, antioxidant-, and vitamin-threatened community.


Management Practices
We applied 150 kg MP, 200 kg urea, 100 kg TSP, 60 kg gypsum, and 10 compost (cowdung:straw = 1:2 w/w) per ha of land. We maintained suitable cultural management. Thinning was performed to keep the particular space of plants in a row. The experimental plots were kept free from weeds by applying appropriate and regular weeding and hoeing. We applied regular irrigation in the plot for appropriate growth of the crop. Leaf samples were collected from 30-day-old plants.

Determination of Proximate Composition
Crude fat, ash, moisture, crude protein, fiber, and energy (gross) were estimated according to the Association of Official Agricultural Chemists (AOAC) method [164]. We followed the Micro-Kjeldahl method to calculate the nitrogen, and finally, crude protein was measured by multiplying nitrogen by 6.25 (AOAC method 976.05). The crude protein, total moisture, ash, and crude fat (%) were subtracted from 100 for calculating carbohydrate (g 100 g −1 FW).

Estimation of Carotenoids and Chlorophylls
The leaf samples were added in acetone (80%) to determine carotenoids, and chlorophyll a, ab, and b [165]. Carotenoids, chlorophyll b, and chlorophyll a were calculated by measuring the absorbance at 470, 663, and 646 nm, respectively, using a spectrophotometer (Hitachi, Japan).

Estimation of β-Carotene
We used our described method for the estimation of β-carotene [168,169]. In a mortar and pestle, 500 mg of fresh leaves was added with 10 mL acetone (80%), and ground precisely. The extract was centrifuged for 3-4 min at 10,000× g. After removing the supernatant in a volumetric flask, the final volume was marked up to 20 mL. A spectrophotometer (Hitachi, Tokyo, Japan) was used to take the absorbance at 510 nm and 480 nm. Data were expressed as milligrams of β-carotene per 100 g of fresh weight. β-Carotene = 7.6 {(Abs. at 480 nm) − 1.49 (Abs. at 510 nm) × Final volume}/(1000 × fresh weight of leaf).

Determination of Ascorbic Acid
The fresh leaf samples were pre-incubated, and dehydroascorbic acid (DHA) was reduced to ascorbic acid (AsA) using Dithiothreitol (DTT). AsA reduced Fe 3+ to Fe 2+ . 2, 2-dipyridyl forms complexes with reduced Fe 2+ [170]. The optical density was taken using a spectrophotometer (Hitachi, Japan) at 525 nm to estimate ascorbic acid (AsA). We calculated ascorbic acid in milligrams per 100 g of fresh weight.

Samples Extraction and Determination of TP, TF, and RSC
The extraction was performed from the fresh and dried ground leaves (30 d) with a mortar and pestle for total polyphenols (TP) content, total flavonoids (TF) content, and RSC determination. In a tightly capped bottle, 0.25 g of leaves were added in 10 mL MeOH (90%), and placed in a shaking water bath (Tokyo, Japan). The extract was filtered after 1 h and stored for TP, TF, and RSC. Phenolic content was determined by the Folin-Ciocalteu reagent [170]. The optical density was taken at 760 nm using a spectrophotometer (HITACHI, Japan). TP was calculated as gallic acid equivalent µg GAE g −1 of FW using an equation (Y = 0.009X + 0.019) obtained from a standard gallic acid graph. The aluminum chloride colorimetric method was followed to estimate the total flavonoid content [170]. The optical density was measured at 415 nm using a spectrophotometer (HITACHI, Japan). A rutin standard graph (Y = 0.013X) was made to estimate TF as µg RE g −1 DW. The RSC was estimated by the diphenyl-picrylhydrazyl (DPPH) radical degradation method [171], and ABTS + assay was carried out using the method of Sarker and Oba [172]. Percentage of inhibition of ABTS + and DPPH equivalent to the control was applied to measure the RSC using following the equation: where AC is the absorbance of the control (150 µL and 10 µL MeOH for RSC (ABTS), RSC (DPPH) instead of leaf extract), and AS is the absorbance of the samples. The results were calculated as µg Trolox equivalent g −1 DW.

Statistical Analysis
The replication mean was obtained by averaging the replication-wise row data. Analysis of variance (ANOVA) of the mean data was analyzed using Statistix 8 software [173][174][175][176]. Duncan Multiple Range Test at a 1% level of probability was followed to compare means data. The results were expressed as the mean ± SD. The correlation was analyzed using Statistix 8 software.

Results and Discussion
The variance analysis revealed that all the parameters significantly differed regarding the accessions, indicating a wide range of variations across the genotypes of two species of Amaranthus leafy vegetables. An extensive range of variability was also stated in red and green color amaranth [167].

Proximate Contents
The composition of the proximate (g 100 g −1 FW) of eight selected accessions of two underutilized species of Amaranthus leafy vegetables is shown in Table (82.85), which was statistically similar to SA14, SA3, SA21, and AT5. Across eight accessions of two underutilized species, AT5, SA3, SA10, SA14, and SA21 showed low moisture content (18% dry matter). As lower moisture ensures a higher dry weight of leaves, these accessions could be used as superior dry biomass. The moisture content is directly related to the maturity of the leaves. The results obtained from underutilized species were corroborated by the reports of sweet potato leaves [177]. In comparison to leafy vegetables, the leaves of eight selected accessions of two underutilized species of Amaranthus leafy vegetables had good protein content that significantly varied regarding accessions (3.66 to 6.52). The highest protein content was recorded in SA14 (6.52). On the contrary, SA21 displayed the least protein content (3.66). Compared to leafy vegetables, higher protein content was obtained from AT2, AT5, AT9, AT12, SA3, and SA10. Vegetable amaranth is the primary source of protein for vegetarians and poor people in developing countries. The protein content of the accessions of two underutilized species was much more prominent as compared to A tricolor (1.26%) [20]. In this investigation, the selected accessions of two underutilized species displayed lesser fat, owing to leafy vegetables, and they might be utilized as foods free from cholesterol. AT12 confirmed the highest fat (0.35), although SA21 had the least fat (0.16) that had a statistical similarity with SA14. The results of sweet potato [177] were corroborated with our current results. They stated that fat covering the body's organs influences cell function and perpetuation body temperature. Vegetable fats are the primary sources of essential fatty acids, such as omega-6 and omega-3. Fats play a significant role in the absorption, digestion, and transport of vitamins E, D, A, and K, which are soluble in fats. The selected accessions of two underutilized species confirmed good carbohydrate content with ample variations regarding accessions (0.38 to 9.56). SA21 confirmed the highest carbohydrates (9.56), though AT2 had the least carbohydrates (0.38). The accessions of two underutilized species were principally diverse for energy (28.95 to 58.47). The accession AT5, SA3, and SA14 demonstrated the highest energy (53.80, 53.81, and 53.82, respectively). On the other hand, AT2 displayed the least energy (25.11). AT5 and SA14 exhibited the highest ash (5.62 and 5.56, respectively), whereas AT9 showed the least ash (2.91). Fiber was largely diverse among accessions (6.66 to 10.24). AT9 confirmed the highest fiber (10.24) after AT5 and SA21. Inversely, SA14 had the least fiber (6.66). Fiber had a noteworthy involvement in the cure of constipation, the augmentation of digestibility, and palatability [19]. Our results displayed that those leaves of selected accessions of two underutilized species have copious protein, moisture, carbohydrates, and digestible fiber. The highest fat and fiber were obtained from A. tricolor accessions. Similarly, the highest protein and carbohydrates were obtained from A. lividus accessions. The highest dry matter, energy, and ash were obtained from both A. tricolor and A. lividus accessions. The moisture and protein contents received from accessions were superior to the moisture and protein contents of the green, red, stem, and weedy amaranth and A. blitum [178][179][180][181][182]. The carbohydrates of advanced line AT7 were greater than red, green amaranth, A. spinosus, and A. blitum [178][179][180]182], although the carbohydrates of AT7 were corroborative with stem amaranth [181]. The fiber of AT3 and AT7 were superior to red, stem, and green amaranth and A. blitum [178,[180][181][182], although corroborated by weedy amaranth [179].

Mineral Content
Macroelements (mg g −1 FW) and microelements (µg g −1 FW) of eight selected accessions of two underutilized species of Amaranthus leafy vegetables are shown in Table 2. The selected accessions of two underutilized species demonstrated good K content. AT12 had the highest K content (6.72) after AT2, AT5, and AT9. On the contrary, the highest K content was recorded in SA14 and SA21 (3.77 and 3.72, respectively). SA3 showed the highest Ca content (3.45) after AT2, AT9, and AT12. Inversely, SA21 displayed the least Ca (1.56). The accessions of two underutilized species demonstrated good Mg content with prominent variations regarding accessions (2.65 to 3.74). AT12 confirmed the highest Mg (3.62). Inversely, AT7 confirmed the lowest Mg (3.74) after AT2, AT5, and AT9. It exposed that sufficient K (6.72), Mg (3.74), and Ca (3.45) were noted in the selected accessions of two underutilized species (based on fresh weight). Several species of amaranth literature [183] stated sufficient, Mg, Ca, and K. Furthermore, they detected that Ca, Mg, and K of amaranth were much more protuberant than nightshade, black spider flower, kale, and spinach. Mg, Ca, and K obtained from the accessions of two underutilized species were superior to Mg, Ca, and K of literature [183]. A. tricolor had the highest K and Mg. In contrast, the A. lividus demonstrated the highest Ca. K content of advanced lines was more than K of green amaranth [180], though K obtained from these advanced lines was inferior to K of weedy amaranth [179]. Ca content observed in the study was corroborative to the results of green and weedy amaranth [179,180]. Mg noticed in these accessions was greater than our previous green and weedy amaranth [179,180]. The protuberant differences were observed in Fe content regarding accessions (10.72 to 18.34). AT12 and SA10 displayed the highest Fe (18.34 and 18.24, respectively), though the least Fe was displayed in SA21 and AT5 (10.72 and 10.76, respectively). Our study revealed that preponderant variations were noticed.  04 and 1.05, respectively). Zn of the accessions of two underutilized species differed significantly and markedly (6.02 to 17.12). AT12 confirmed the highest Cu (17.12), whereas SA14 exerted the least Cu (6.02). The Fe and Zn content of the accessions of two underutilized species was superior to the leaves of cassava [184] and beach pea [185].
We documented sufficient Fe (18.34), Mn (15.12), Zn (17.12), and noteworthy Cu (2.98) (based on fresh weight) in the accessions of two underutilized species. Similarly, different amaranths in literature [183] observed satisfactory Fe, Mn, Cu, and Zn. They also stated that Fe, Zn, Mn, and Cu in the leaves of amaranth were superior to spinach, spider flower, black nightshade, and kale. Mn, Fe, Zn, and Cu in the study were superior to Mn, Fe, Zn, and Cu in the literature [183]. A. tricolor accessions confirmed the highest Mn and Zn. Similarly, A. lividus accessions confirmed the highest Cu. A. tricolor and A. lividus accessions confirmed the highest Fe. In the study, Fe and Mn of all advanced lines were superior to green amaranth [180], although Fe and Mn of AT15 were superior to weedy amaranth [179]. Cu observed in the study was superior to green and weedy morph amaranth [179,180] and A. spinosus amaranth [179]. AT15 displayed much greater Zn than green and weedy amaranth [179,180].
The accessions of two underutilized species established good β-cyanins content with significant variability among accessions (192. 44 (123.28), in the accessions of two underutilized species, were corroborated to green and red amaranth [186]. β-cyanins, β-xanthins, and betalains pigments were the highest in A. tricolor accessions. Inversely, the highest carotenoids were obtained from both A. tricolor and A. lividus. Betalains, β-cyanins, and β-xanthins in the study were superior to red, green, stem amaranth, and A. blitum [178,[180][181][182]. Carotenoids in the study were superior to green amaranth [180], and verified to weedy amaranth [179], although the carotenoids of this study were inferior to red and stem amaranth and A. blitum [178,181,182].

Phytochemical Contents and Scavenging Activity
TP, β-carotene, TF, ascorbic acid, and RSC of the selected accessions of two underutilized species are shown in Table 4. Pronounced variability was recorded in the β-carotene content of the selected accessions of two underutilized species (27.67 in AT5 to 68.82 in AT12). Table 4. The performance of TP (µg GAE g −1 FW), vitamin C (mg 100 g −1 FW), β-Carotene (mg 100 g −1 FW), RSC (DPPH) (µg g −1 TEAC DW), TF (µg RE g −1 DW), and RSC (ABTS + ) (µg TEAC g −1 DW) of two underutilized species of Amaranthus leafy vegetables. AT2 and SA10 confirmed the high β-carotene. The accessions of two underutilized species demonstrated prominent variations in ascorbic acid (18.54 to 192.75). AT2 confirmed the highest ascorbic acid (192.75), and the least in AT9 (18.54). Noteworthy variations were noted in TP of the accessions of two underutilized species (15.66 to 32.88 (24.45, 42.44). A parallel tendency of RSC (DPPH) and RSC (ABTS + ) methods authenticated the measurement of two different methods of AP. The accessions of two underutilized species exhibited outstanding ascorbic acid and β-carotene (192. 75 and 68.82). TP (32.88), TF (176.88), RSC (DPPH) (36.27), and RSC (ABTS + ) (68.87) found in this study were superior to red and green amaranth [187]. The β-carotene of these lines was confirmative to weedy amaranth [179]. The ascorbic acid of AT11 was superior to green, weedy, stem, amaranth, and A. blitum [179,181,182], and corroborative to red morph amaranth [178]. TP content was greater than green and weedy amaranth [179,180]. TF, RSC (DPPH), and RSC (ABTS + ) documented in the accessions of two underutilized species were greater than the red, green, stem, and weedy amaranth and A. blitum morph [178][179][180][181][182]. The accession AT2 had the highest vitamin C and RSC (DPPH) and RSC (ABTS + ); AT5 had the highest TP content; and AT12 had the highest TF content. A. tricolor accessions had high phytochemicals across the two species, such as phytopigments, vitamins, phenolics, flavonoids, and antioxidants, including considerable nutrients and protein. A. tricolor accessions can be used as high-yielding cultivars comprising ample antioxidants. The accessions confirmed an immense foundation of phytochemicals, and nutritional values and AP presented enormous prospects for feeding the mineral-, vitamin-, and antioxidantdeficient community.

Conclusions
The selected accessions of two underutilized species demonstrated leafy vegetables as abundant sources of K, Fe, Mn, Ca, dry matter, Cu, Zn, protein, Mg, dietary fiber, and carbohydrates. It is an excellent origin of antioxidant pigments and phytochemicals, including TP, β-carotene, TF, ascorbic acid, and antioxidants. The accessions of A. tricolor had abundant phytochemicals and RSC, including considerable proximate, pigments, and nutraceuticals compared to the accessions of A. lividus. The interrelationship exposed that phytochemicals and pigments of leafy vegetable amaranth accessions confirmed good RSC of 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) and 2,2-Diphenyl-1picrylhydrazyl equivalent to Trolox. Although A. tricolor and A. lividus are underutilized, these are promising leafy vegetables. Enormous bioactive phytochemicals and antioxidants of A. tricolor and A. lividus enable growing them as preferable cultivars, and they can be used in the daily diet as fresh salad, boiled, leafy vegetables, and other culinary dishes. Based on their nutritional status, they are comparable to spinach, and can be grown throughout the year, including a gap period of leafy vegetables in summer. They are a potential source of nutritional value, antioxidant phytopigments, β-carotene, ascorbic acid, phenolics, flavonoids, and antioxidants in our daily diet to accomplish nutritional and antioxidant sufficiency.