Salt Eustress Induction in Red Amaranth (Amaranthus gangeticus) Augments Nutritional, Phenolic Acids and Antiradical Potential of Leaves

Earlier researchers have highlighted the utilization of salt eustress for boosting the nutritional and phenolic acid (PA) profiles and antiradical potential (ARP) of vegetables, which eventually boost food values for nourishing human diets. Amaranth is a rapidly grown, diversely acclimated C4 leafy vegetable with climate resilience and salinity resistance. The application of salinity eustress in amaranth has a great scope to augment the nutritional and PA profiles and ARP. Therefore, the A. gangeticus genotype was evaluated in response to salt eustress for nutrients, PA profile, and ARP. Antioxidant potential and high-yielding genotype (LS1) were grown under four salt eustresses (control, 25 mM, 50 mM, 100 mM NaCl) in a randomized completely block design (RCBD) in four replicates. Salt stress remarkably augmented microelements, proximate, macro-elements, phytochemicals, PA profiles, and ARP of A. gangeticus leaves in this order: control < low sodium chloride stress (LSCS) < moderate sodium chloride stress (MSCS) < severe sodium chloride stress (SSCS). A large quantity of 16 PAs, including seven cinnamic acids (CAs) and nine benzoic acids (BAs) were detected in A. gangeticus genotypes. All the microelements, proximate, macro-elements, phytochemicals, PA profiles, and ARP of A. gangeticus under MSCS, and SSCS levels were much higher in comparison with the control. It can be utilized as preferential food for our daily diets as these antiradical compounds have strong antioxidants. Salt-treated A. gangeticus contributed to excellent quality in the end product in terms of microelements, proximate, macro-elements, phytochemicals, PA profiles, and ARP. A. gangeticus can be cultivated as an encouraging substitute crop in salt-affected areas of the world.


Samples Extraction and Determination of Phenolic Acids (PAs) by HPLC
Fresh leaves (1 g) were extracted in MeOH (10 mL, 80%) containing CH 3 COOH (1%). The thoroughly homogenized mixture was kept in a 50 mL tightly capped test tube and placed in a shaker (Scientific Industries Inc., New York, NY, USA) for 15 h at 400 rpm. It was filtered in a 0.45 µm filter (MA, New York, USA) and centrifuged for 15 min at 10,000× g. The filtrate was used to estimate PAs. All extractions were repeated 3 times. The method of Sarker and Oba [112] was followed to determine PAs using HPLC. Shimadzu HPLC (Kyoto, Japan) was furnished with a binary pump, degasser, and detector. A column (150 × 4.6 mm, 5 µm; Shinwa Chemical Industries, Ltd., Kyoto, Japan) was used for the separation of PAs. Solvent B and solvent A (acetonitrile and 6% (v/v) acetic acid in water, respectively) were pumped for 70 min at 1 mL min −1 . HPLC system was run using a gradient program with 0-15% acetonitrile for 45 min, 15-30% for 15 min, 30-50% for 5 min, and 50-100% for 5 min; 35 • C temperature in the column was maintained with a 10 µL volume of injection. For monitoring Pas continuously, the detector was set at 254 and 280 nm. The retention time and UV-vis spectra with their respective standards were compared for the identification of the compound. Pas was estimated as µg g −1 FW.
Each PA was quantified using the corresponding standards of calibration curves. A total of 16 PAs were dissolved in MeOH (80%) 100 mg mL −1 as stock solutions. Individual PAs were quantified using corresponding standard curves (10,20,40,60,80, and 100 µg mL −1 ) with external standards. Retention times, co-chromatography of samples spiked with commercially available standards, and UV spectral characteristics were utilized for identification and matching the PA.

Statistical Analysis
All the sample data of a trait were averaged for each treatment to obtain a replication mean [113][114][115]. The mean data of various traits were statistically and biometrically analyzed [116][117][118]. Data analysis and ANOVA were performed using Statistix software version 8.0, Tallahassee FL 32312, USA [119][120][121]. The means were compared at a 1% level of probability using Duncan's Multiple Range Test (DMRT). The results were reported as the mean ± SD of four separate replicates [122][123][124].

Results and Discussion
3.1. The Response of Proximate Compositions to Sodium Chloride Stress Figure 1 represents the nutritional compositions of A. gangeticus under different salinity stresses. A. gangeticus leaves had a high moisture content like most leafy vegetables. Nevertheless, our study revealed that A. gangeticus leaves have copious ash, carbohydrates, dietary fiber, moisture, and protein. The constituents of these components were several times greater than C. spinosum [99]. The maximum moisture and fat were exhibited under the control treatment, whereas the minimum moisture and fat were observed under SSCS. Petropoulos et al. [99] reported a similar reduction in fat with the increase in salinity stress in C. spinosum. Moisture and fat were significantly reduced in the order: (control > LSCS > MSCS > SSCS) and (control > LSCS > MSCS = SSCS), respectively. Higher leaf dry matter obtained from leaves ensure lower moisture content. Hence, saltstressed A. gangeticus leaves confirmed greater dry matter in comparison to the control. The maximum dietary fiber, ash, carbohydrates, energy, and protein were recorded at SSCS, while the minimum dietary fiber, ash, carbohydrates, energy, and protein were noticed under the control. Similarly, Petropoulos et al. [99] reported higher ash and protein at the maximum and 8.0 and 6.0 dS m −1 , than the control and minimum salinity in C. spinosum. Energy, protein, and dietary fiber contents were sharply augmented in the following order: control < LSCS < MSCS < SSCS, whereas ash and carbohydrates contents were statistically similar in the control and LSS levels and progressively augmented from MSCS to SSCS levels. In LSCS, MSCS, and SSCS, dietary fiber, energy, carbohydrates, ash, and protein were increased by 17%, 2%, 2%, 9%, and 4%; 6%, 10%, 8%, 14%, and 19%; and 23%, 14%, 9%, 16%, and 29%, respectively, in comparison with the control condition ( Figure 2). In LSCS, MSCS, and SSCS, dietary fiber, energy, carbohydrates, ash, and protein were increased by 17%, 2%, 2%, 9%, and 4%; 6%, 10%, 8%, 14%, and 19%; and 23%, 14%, 9%, 16%, and 29%, respectively, in comparison with the control condition ( Figure 2). Dietary fiber has significantly acted in the remedy of constipation, increased digestibility, and palatability. Vegetarians and deprived rural communities in underdeveloped countries mostly trust A. gangeticus for protein. Since the low amounts consumed in a daily diet, the increments of energy content in the order of control < LSCS < MSCS < SSCS had no substantial influence on the energy balance in humans. The findings of A. gangeticus conformed with the outcomes of AT [91] and leaves of Ipomoea batata [125], respectively. They specified that it influences cell function, the fat covering the body's organs, and continues the temperature of the body. The fats of vegetables are prime sources of crucial fatty acids, such as Ω-6 and Ω-3. Fats perform a noteworthy contribution to the absorption, digestion, and transportation of vitamins A, E, K, and D.

Sodium Chloride Impact on Minerals (Macroelements and Microelements) Composition
A. gangeticus has abundant minerals (macroelements and microelements) (Figure 3). High levels of minerals were observed and corroborated with A. tricolor under normal cultivation practice in an open field [126]. A. gangeticus had higher Fe and Zn than Manihot esculenta leaves [127] and Lathyrus japonicus [128]. Jimenez-Aguiar and Grusak [129] also found abundant Zn, Cu, Mn, and Fe in different A. spp. They also found higher iron and copper compared with kale and higher Zn compared with leaf cabbage, Spinacia oleracea, and Solanum nigrum. The maximum Zn, Ca, Mo, Mg, Na, S, Cu, B, Mn, and Fe was noticed under the SSCS level, while the minimum levels Zn, Ca, Mo, Mg, Na, Cu, B, Mn, and Fe were reported under control conditions, and the lowest sulfur content was observed under the LSCS level. Zn, Ca, Mo, Mg, Na, Cu, B, and Mn were progressively augmented in the order control < LSCS < MSCS < SSCS. In contrast, potassium and phosphorus contents were drastically reduced in the order control > LSCS > MSCS > SSCS. Dietary fiber has significantly acted in the remedy of constipation, increased digestibility, and palatability. Vegetarians and deprived rural communities in underdeveloped countries mostly trust A. gangeticus for protein. Since the low amounts consumed in a daily diet, the increments of energy content in the order of control < LSCS < MSCS < SSCS had no substantial influence on the energy balance in humans. The findings of A. gangeticus conformed with the outcomes of AT [91] and leaves of Ipomoea batata [125], respectively. They specified that it influences cell function, the fat covering the body's organs, and continues the temperature of the body. The fats of vegetables are prime sources of crucial fatty acids, such as Ω-6 and Ω-3. Fats perform a noteworthy contribution to the absorption, digestion, and transportation of vitamins A, E, K, and D.

Sodium Chloride Impact on Minerals (Macroelements and Microelements) Composition
A. gangeticus has abundant minerals (macroelements and microelements) ( Figure 3). High levels of minerals were observed and corroborated with A. tricolor under normal cultivation practice in an open field [126]. A. gangeticus had higher Fe and Zn than Manihot esculenta leaves [127] and Lathyrus japonicus [128]. Jimenez-Aguiar and Grusak [129] also found abundant Zn, Cu, Mn, and Fe in different A. spp. They also found higher iron and copper compared with kale and higher Zn compared with leaf cabbage, Spinacia oleracea, and Solanum nigrum. The maximum Zn, Ca, Mo, Mg, Na, S, Cu, B, Mn, and Fe was noticed under the SSCS level, while the minimum levels Zn, Ca, Mo, Mg, Na, Cu, B, Mn, and Fe were reported under control conditions, and the lowest sulfur content was observed under the LSCS level. Zn, Ca, Mo, Mg, Na, Cu, B, and Mn were progressively augmented in the order control < LSCS < MSCS < SSCS. In contrast, potassium and phosphorus contents were drastically reduced in the order control > LSCS > MSCS > SSCS.

Impact of Salinity on Phytochemicals and ARP
Polyphenols, beta-carotene, AsA, flavonoids, and ARP varied significantly under different sodium chloride stresses ( Figure 5). Sodium chloride stress progressively augmented polyphenols, beta-carotene, AsA, flavonoids, and ARP in the following order: control < LSCS < MSCS < SSCS. Most of the minerals increased under different salt levels compared with control conditions, which were corroborated with minerals of C. spinosum under salinity stress [99]. Petropoulos et al. [99] reported sharp augmentation in calcium, magnesium, iron, manganese, zinc, and sodium content and a reduction in potassium content in C. spinosum. They stated that the application of fertilizer and treatments of salinity could be the reason for the amelioration of sodium content and suggested that the species utilized accumulated sodium to cope with the adverse effects of salinity. Iron content was statistically similar to the value of the control and LSCS levels, while iron content was progressively augmented under MSCS and SSCS levels by 12% and 62%, respectively. The lowest sulfur content was obtained from the LSCS levels, which differed significantly from the control condition. The sulfur content was gradually augmented under MSCS and SSCS levels by 20% and 51%, respectively ( Figure 4).

Impact of Salinity on Phytochemicals and ARP
Polyphenols, beta-carotene, AsA, flavonoids, and ARP varied significantly under different sodium chloride stresses ( Figure 5). Sodium chloride stress progressively augmented polyphenols, beta-carotene, AsA, flavonoids, and ARP in the following order: control < LSCS < MSCS < SSCS.

Response of Salinity on PA Profiles
The HPLC-identified PA values of A. gangeticus (accession LS11) under four sodium chloride stress were compared with PAs using the respective peaks of the compounds (Table 1). Sixteen PAs, including seven CAs and nine Bas, were confirmed in A. gangeticus. Three BAs [protocatechuic acid (PCA), β-resorcylic acid (β-RA), and gentisic acid (GA)] were identified as new compounds for the first time in Amaranthus leaves. Table 1. Wavelengths of maximum absorption in the visible region (λ max ), mass spectral data, retention time (Rt), and tentative identification of PAs in A. gangeticus. BAs were the amplest among the two categories of acids, thereafter CAs in A. gangeticus (Figures 7 and 9). Salicylic acid (SA) was the most copious PAs across BAs thereafter GAA, GA, PCA, vanillic acid (VA), p-hydroxybenzoic acid (p-HBA), β-RA, and syringic acid (SYA) (Figure 7). BA contents in the A. gangeticus genotype under control conditions were superior to the BA content of A. tricolor [130]. Chlorogenic acid (CHA) was the most noticeable compound across CAs thereafter ferulic acid (FA), sinapic acid (SIA), m-coumaric acid (m-COA), trans-cinnamic acid (Trans-CA), and caffeic acid (CFA) (Figure 7). A. gangeticus had abundant CAs under control conditions. Seven CAs obtained were confirmed superior to CAs of A. tricolor [130]. Phenylalanine is the most extensively distributed PA in plant tissues, which are finally synthesized into CAs [131]. Identified Benzoic acids (BAs) have important biological activities. For instance, gallic acid and its ester derivatives ARE flavoring agents and preservatives in the food industry. There are diverse scientific reports on the biological and pharmacological activities of these phytochemicals, with emphasis on antioxidant, antimicrobial, anti-inflammatory, anticancer, cardioprotective, gastroprotective, and neuroprotective effects [132]. Vanillic acid exerts diverse bioactivity against cancer, diabetes, obesity, neurodegenerative, cardiovascular, and hepatic diseases by inhibiting the associated molecular pathways. Its derivatives also possess the therapeutic potential to treat autoimmune diseases, as well as fungal and bacterial infections [133]. Syringic acid shows a wide range of therapeutic applications in the prevention of diabetes, CVDs, cancer, and cerebral ischemia, as well as antioxidant, antimicrobial, anti-inflammatory, antiendotoxic, neurologic, and hepatoprotective activities [134]. High salicylate in diets has proven health benefits, such as lower risks of cancer, heart disease, and diabetes. Ellagic acid has been reported to have antimutagenic on bacteria and in mammalian systems as well. It has also shown strong antioxidant, anti-inflammatory, and anticarcinogenic activities, as well as a better preservative effect against oxidative stress when compared with vitamin E [135]. PCA is a major metabolite of anthocyanin. The pharmacological actions of PCA have been shown to include strong in vitro and in vivo antioxidant activity. In in vivo experiments using rats and mice, PCA has been shown to exert anti-inflammatory as well as antihyperglycemic and antiapoptotic activities [136]. β-resorcylic acid has antimicrobial activity [137]. Finally, gentisic acid possesses fibro growth factor inhibition, antimicrobial, antioxidant, anti-inflammatory, hepatoprotective, and neuroprotective activities [138].
SA, GAA, and PCA contents had no statistical variations at the control and LSCS level; however, three acids were augmented remarkably from LSCS to SSCS with a range from 23.83 to 45.82, 15.76 to 22.46, and 11.95 to 23.42 µg g −1 FW, respectively (Figure 7). In MSCS and SSCS, SA, GAA, and PCA contents were augmented by (37% and 92%), (12% and 43%), and (41% and 96%), respectively (Figure 8). GA and ellagic acid (EA) ranged from 12.68 to 22.58 and 5.08 to 6.55 µg g −1 FW. GA and EA had no statistical variations between control and LSCS levels and between MSCS and SSCS levels; however, the contents of these acids were augmented remarkably from control condition or LSCS to MSCS or SSCS level (26% and 77%) (Figures 7 and 8).
All the CA contents were sharply augmented under sodium chloride levels. All the CAs showed the highest contents under the SSCS level, whereas the control treatment exhibited the lowest CA contents. From control to SSCS, CHA, m-COA, and p-coumaric acid (p-COA) ranged from 14.38 to 27.35, 7.87 to 21.36, and 4.16 to 8.75 µg g −1 FW, respectively, (Figure 9). Identified Cinnamic acids (CAs) have important biological activities. For instance, Caffeic acid (CA) and its derivatives have antioxidant, anti-inflammatory, and anticarcinogenic activity [139]. Chlorogenic acid was effective in preventing weight gain, inhibiting the development of liver steatosis, and blocking insulin resistance induced by a high-fat diet [140]. p-coumaric acid decreases low-density lipoprotein (LDL) peroxidation, shows antioxidant and antimicrobial activities, and plays an important role in human health [141]. Ferulic acid has low toxicity and possesses many physiological functions (anti-inflammatory, antioxidant, antimicrobial activity, anticancer, and antidiabetic effects). It has been widely used in the pharmaceutical, food, and cosmetics industries [142]. Sinapic acid shows antioxidant, antimicrobial, anti-inflammatory, anticancer, and anti-anxiety activity [143]. Cinnamic acids have been identified as interesting compounds with antioxidant, anti-inflammatory, and cytotoxic properties [144]. Antioxidants 2022, 11, x FOR PEER REVIEW 12 of 21 SA, GAA, and PCA contents had no statistical variations at the control and LSCS level; however, three acids were augmented remarkably from LSCS to SSCS with a range from 23.83 to 45.82, 15.76 to 22.46, and 11.95 to 23.42 µg g −1 FW, respectively (Figure 7). In MSCS and SSCS, SA, GAA, and PCA contents were augmented by (37% and 92%), (12% and 43%), and (41% and 96%), respectively (Figure 8). GA and ellagic acid (EA) ranged from 12.68 to 22.58 and 5.08 to 6.55 µg g −1 FW. GA and EA had no statistical variations between control and LSCS levels and between MSCS and SSCS levels; however, the contents of these acids were augmented remarkably from control condition or LSCS to MSCS or SSCS level (26% and 77%) (Figures 7 and 8).
All the CA contents were sharply augmented under sodium chloride levels. All the CAs showed the highest contents under the SSCS level, whereas the control treatment exhibited the lowest CA contents. From control to SSCS, CHA, m-COA, and p-coumaric acid (p-COA) ranged from 14.38 to 27.35, 7.87 to 21.36, and 4.16 to 8.75 µg g −1 FW, respectively, (Figure 9). Identified Cinnamic acids (CAs) have important biological activities. For instance, Caffeic acid (CA) and its derivatives have antioxidant, anti-inflammatory, and anticarcinogenic activity [139]. Chlorogenic acid was effective in preventing weight gain, inhibiting the development of liver steatosis, and blocking insulin resistance induced by a high-fat diet [140]. p-coumaric acid decreases low-density lipoprotein (LDL) peroxidation, shows antioxidant and antimicrobial activities, and plays an important role in human health [141]. Ferulic acid has low toxicity and possesses many physiological functions (anti-inflammatory, antioxidant, antimicrobial activity, anticancer, and antidiabetic effects). It has been widely used in the pharmaceutical, food, and cosmetics industries [142]. Sinapic acid shows antioxidant, antimicrobial, anti-inflammatory, anticancer, and antianxiety activity [143]. Cinnamic acids have been identified as interesting compounds with antioxidant, anti-inflammatory, and cytotoxic properties [144]. CHA, m-COA acid, and p-COA were progressively augmented in the order of control < LSCS < MSCS < SSCS (Figure 9). In LSCS, MSCS, and SSCS, CHA, m-COA acid, and p-COA were predominately augmented by 13%, 42%, 90%; 25%, 74%, 171%; and 23%, 65%, 110% compared with the control condition, respectively ( Figure 10). Trans-CA and SIA contents at control condition were statistically similar to the LSCS level; however, these two acids' contents were remarkably augmented from LSCS to SSCS with a range from 9.85 to 18.62 and 11.35 to 12.56 µg g −1 FW, respectively (Figure 9). In MSCS and SSCS, Trans-CA and SIA contents were augmented by 41% and 89%; and 6% and 11%, respectively ( Figure 10). FA and CFA ranged from 8.20 to 20.45 and 6.56 to 7.62 µg g −1 FW, respectively. FA and CFA contents at the control condition were statistically similar to the LSCS level, and at the MSCS level were statistically similar to the SSCS level. However, the contents of these acids were remarkably augmented from the control condition or LSCS to MSCS or SSCS level (47% and 16%) (Figures 9 and 10). CHA, m-COA acid, and p-COA were progressively augmented in the order of control < LSCS < MSCS < SSCS (Figure 9). In LSCS, MSCS, and SSCS, CHA, m-COA acid, and p-COA were predominately augmented by 13%, 42%, 90%; 25%, 74%, 171%; and 23%, 65%, 110% compared with the control condition, respectively ( Figure 10). Trans-CA and SIA contents at control condition were statistically similar to the LSCS level; however, these two acids' contents were remarkably augmented from LSCS to SSCS with a range from 9.85 to 18.62 and 11.35 to 12.56 µg g −1 FW, respectively (Figure 9). In MSCS and SSCS, Trans-CA and SIA contents were augmented by 41% and 89%; and 6% and 11%, respectively ( Figure 10). FA and CFA ranged from 8.20 to 20.45 and 6.56 to 7.62 µg g −1 FW, respectively. FA and CFA contents at the control condition were statistically similar to the LSCS level, and at the MSCS level were statistically similar to the SSCS level. However, the contents of these acids were remarkably augmented from the control condition or LSCS to MSCS or SSCS level (47% and 16%) (Figures 9 and 10). COA were predominately augmented by 13%, 42%, 90%; 25%, 74%, 171%; and 23%, 65%, 110% compared with the control condition, respectively ( Figure 10). Trans-CA and SIA contents at control condition were statistically similar to the LSCS level; however, these two acids' contents were remarkably augmented from LSCS to SSCS with a range from 9.85 to 18.62 and 11.35 to 12.56 µg g −1 FW, respectively (Figure 9). In MSCS and SSCS, Trans-CA and SIA contents were augmented by 41% and 89%; and 6% and 11%, respectively ( Figure 10). FA and CFA ranged from 8.20 to 20.45 and 6.56 to 7.62 µg g −1 FW, respectively. FA and CFA contents at the control condition were statistically similar to the LSCS level, and at the MSCS level were statistically similar to the SSCS level. However, the contents of these acids were remarkably augmented from the control condition or LSCS to MSCS or SSCS level (47% and 16%) (Figures 9 and 10).  Total BAs, total CAs, and total PAs were progressively augmented in the order control < LSCS < MSCS < SSCS (Figure 11). In LSCS, MSCS, and SSCS, total BAs, total CAs, and total PAs were predominately augmented by 7%, 52%, and 89%), (8%, 52%, and 87%), and (7%, 52%, and 89%), compared with control condition, respectively ( Figure 12). Total BAs, total CAs, and total PAs were progressively augmented in the order control < LSCS < MSCS < SSCS ( Figure 11). In LSCS, MSCS, and SSCS, total BAs, total CAs, and total PAs were predominately augmented by 7%, 52%, and 89%), (8%, 52%, and 87%), and (7%, 52%, and 89%), compared with control condition, respectively ( Figure 12). Figure 11. Increase of PA fractions (µg g −1 FW) (total BAs, total CAs, and total PAs) under control, LSCS, MSCS, and SSCS in A. gangeticus accession; (n = 6); different letters in columns are varied significantly by DMRT (p < 0.01).
The cost is very low to maintain salt stress by adding sodium chloride to the plants. Furthermore, we suggested cultivating in salt-prone areas where there are no salt susceptible crops grown successfully. So, those areas will be efficiently utilized for amaranth leafy vegetable cultivation to meet the demand for the leafy vegetable of that locality, as leafy vegetables are too susceptible to salinity stress as amaranth is a salinity-tolerant leafy vegetable with up to 200 mM salt concentration. It can produce enough biomass and perform optimal photosynthesis at 100 mM saline stress. Amaranth is highly tolerant to salinity. It can tolerate 200 mM NaCl [147]. As amaranth is salt tolerant, it increases all enzymatic and non-enzymatic antioxidants, and metabolites, to detoxify ROS and cope with salt stress.

Conclusions
Sodium chloride stress remarkably augmented the energy, ash, carbohydrates, protein, calcium, dietary fiber, magnesium, S, Fe, Mo, Mn, Na, Cu, B, Zn, and ARP of A. gangeticus leaves. All the nutrients, phytochemicals, PA profiles, and ARP of A. gangeticus leaves under MSCS and SSCS levels were superior to the control. It can be utilized as a valued product for human consumption and health benefits. Salt-treated A. gangeticus leaves had abundant nutrients, phytochemicals, PA profiles, and ARP. Phytochemicals, PA profiles, and ARP scavenge ROS that would be advantageous for human health benefits as these bioactive compounds have potent antioxidants. Furthermore, sodium chloride-stressed A. gangeticus contributed with excellent quality in the end users for nutrients, phytochemicals, PA profiles, and ARP. It can be cultivated as a promising substitute crop in sodium chlorideaffected areas of the world.