Nutraceuticals, antioxidant pigments, and phytochemicals in the leaves of Amaranthus spinosus and Amaranthus viridis weedy species

Six selected weedy Amaranthus genotypes (three accessions from each species of A. viridis and A. spinosus) were evaluated in terms of nutrients, minerals, antioxidant constituents and antioxidant activity for the possibilities of weedy species as a vegetable cultivar in a randomized complete block design with three replications. As leafy vegetable, Weedy Amaranthus has remarkable protein, dietary fiber, carbohydrates, Ca, K, Mg, P, S, Fe, Mn, Cu, Zn, Na, Mo, B, chlorophylls, β-cyanins, β-xanthins, betalains, β-carotene, vitamin C, TPC, TFC, and TAC (DPPH and ABTS+) compared to any cultivated species. The A. viridis genotype WAV7 and A. spinosus genotype WAS13 had the highest nutrients, pigments, vitamins, phenolics, flavonoids, and antioxidant. Hence, these two weedy accessions could be used as an antioxidant profile enriched cultivar with high nutritional and antioxidant activity. Pigments, β-carotene, vitamin C, phenolics, and flavonoids had strong antioxidant activity and played a vital role in the antioxidant activity of weedy Amaranthus genotypes. Weedy species are an excellent source of phenolics, flavonoids, and antioxidants that have many pharmacological and medicinal effects of their traditional applications and detoxify ROS and offered huge prospects for feeding the antioxidant-deficient community to cope with the hidden hunger and attaining nutritional and antioxidant sufficiency.


Results and Discussion
The analysis of variance revealed that all the studied traits differed significantly in terms of the genotypes (Tables 1, 2 Table 1. The moisture content of six selected genotypes of two weedy Amaranthus species ranged from 81.54 Genotypes Moisture (g 100 g −1 ) Protein (g 100 g −1 ) Fat (g 100 g −1 ) Carbohydrates (g 100 g −1 ) Energy (Kcal) Ash (g 100 g −1 ) Dietary fiber (g 100 g −1 FW) www.nature.com/scientificreports www.nature.com/scientificreports/ to 86.26 g 100 g −1 FW. The highest moisture content was noticed in A. spinosus genotype WAS11 (86.26 g 100 g −1 FW) followed by A. spinosus genotype WAS15 (85.42 g 100 g −1 FW) and WAS13 (84.47 g 100 g −1 FW). In contrast, the lowest moisture content was recorded in A. viridis genotype WAV4 (80.35 g 100 g −1 FW). All the genotypes of A. viridis such as WAV4, WAV7, and WAV9 exhibited around 18-20% dry matter could be a promising source of dry matter as higher dry matter ensured with lower moisture contents of leaves. The maturity of the two species   www.nature.com/scientificreports www.nature.com/scientificreports/ could have a vital role in the moisture content of leaves. The moisture contents obtained in our study were fully agreed with the reports of Sun et al. 32 in sweet potato leaves.
As leafy vegetables, leaves of A. viridis and A. spinosus had high protein content with fewer variations which ranged from 4,12 to 5.78 g 100 g −1 FW. The highest protein content was observed in A. spinosus genotype WAS13 (5.78 g 100 g −1 FW) which was statistically similar to A. spinosus genotype WAS11 and WAS15. Conversely, the lowest protein content was exhibited in A. viridis genotype WAV4. Weedy amaranth (A. viridis and A. spinosus) genotypes are the sources of protein for vegetarian and poor people of the third world countries. The protein content of A. viridis and A. spinosus were much higher as compared to amaranth in our earlier study 5 . In this investigation, A. viridis and A. spinosus genotypes showed low-fat content as a leafy vegetable and could be used as a cholesterol free food. A. spinosus genotype WAS13 showed the highest fat content (0.63 g 100 g −1 FW) followed by A. spinosus genotype WAS11. Whereas, A. viridis genotype WAV9 exhibited the lowest fat content (0.28 g 100 g −1 FW) with a range of 0.28 to 0.63 g 100 g −1 FW. Fats help in the digestion, absorption, and transport of fat-soluble vitamins A, D, E, K and source of omega-3 and omega-6 fatty acids. Sun et al. 32 reported similar results in sweet potato leaves. They revealed that fat involved in the insulation of body organs and the maintenance of body temperature and cell function.
A  Tables 2, 3. In this study, the highest K content was observed in A. viridis genotype WAV7 (7.22 16 mg g −1 FW) which was statistically similar to A. viridis genotype WAV9 (6.98 mg g −1 FW) and A. spinosus genotype WAS15 (6.82 mg g −1 FW) with a range of 6.45 mg g −1 to 7.22 mg g −1 FW. Whereas, A. spinosus genotype WAS11 and WAS13 exhibited the lowest K content (6.45, 6.48 mg g −1 FW which was statistically similar to A. viridis genotype WAV4. A. viridis genotypes had higher K content compared to the genotype of A. spinosus, albeit the differences in K content between to weedy species were no pronounced. Albeit there were no prominent variations in Ca content between to weedy species, A. spinosus genotypes had higher Ca content compared to the genotype of A. viridis with a range of 2.44 to 2.84 mg g −1 FW. The highest Ca content (2.84 mg g −1 ) was reported in A. viridus genotype WAV9 which was similar to A. spinosus genotype WAS12 and WAS11. In contrast, the lowest Ca content (2.44 mg g −1 ) was obtained from A. spinosus genotype WAS13. In this investigation, A. viridis and A. spinosus genotypes had no pronounced variations in terms of Mg content (2.88 to 3.78 mg g −1 FW). A. viridis genotype WAV4, WAV7, and WAV9 exhibited the highest Mg content (3.78, 3.65, 3.52 mg g −1 FW), while, A. spinosus genotype WAS11, WAS13, and WAS15 showed the lowest Mg content (2.88, 3.02 and 2.97 mg g −1 FW). Similarly, A. viridis and A. spinosus genotypes had no pronounced variations in terms of P content (0.68 to 0.94 mg g −1 FW). A. viridis genotype WAV7 exhibited the highest P content (0.94 mg g −1 FW), while, A. spinosus genotype WAS13 showed the lowest P content (0.68 mg g −1 FW) which was statistically similar to A. spinosus genotype WAS15, WAS11, and A. viridis genotype WAV4 and WAV9. S content had significant variations in six A. viridis and A. spinosus genotypes which ranged from 1.18 to 1.66 mg g −1 FW. A. viridis genotypes had higher S content compared to the genotype of A. spinosus. A. viridis genotype WAV9 exhibited the highest S content (1.66 mg g −1 FW) followed by WAV7, while, A. spinosus genotype WAS15 and WAS13 showed the lowest S content (1.18 and 1.25 mg g −1 FW). Our investigation revealed that we found remarkable K (7.22 mg g −1 ), Ca (2.74 mg g −1 ), Mg (3.52 mg g −1 ), P (0.94 mg g −1 ), and S (1.66 mg g −1 ) in A. viridis genotype (fresh weight basis). Jimenez-Aguiar and Grusak 33 reported high K, Ca, Mg, P, and S (fresh weight basis) in different A. spp. including A. viridis and A. spinosus. They also reported that spider flower, black nightshade, spinach, and kale had much lower K, Ca, and Mg content than amaranth. Our studied A. viridis and A. spinosus genotype had higher K, Ca, Mg, P, and S (fresh weight basis) compared to studied A. spp of Jimenez-Aguiar and Grusak 33  www.nature.com/scientificreports www.nature.com/scientificreports/ β-cyanins (287.56 ng g −1 FW), and carotenoids content (92.87 mg 100 g −1 FW) in A. viridis genotype, while chlorophyll b (152.42 μg g −1 FW), β-cyanins (286.46 ng g −1 FW), β-xanthins (274.96 ng g −1 FW), and betalains content (561.42 ng g −1 FW) in A. spinosus genotype. Similarly, Khanam and Oba 36 observed similar trend in chlorophyll a, chlorophyll b, chlorophyll ab, β-cyanins, β-xanthins, betalains and carotenoids content of green and red amaranth. A. viridis genotype had the highest chlorophyll a, chlorophyll ab, β-cyanins, and carotenoids content while A. spinosus genotype exhibited the highest chlorophyll b, β-cyanins, β-xanthins, and betalains content.   37 in red amaranth whereas, our obtained results were higher than the results of Khanam et al. 37 in green amaranth. The A. viridis genotype WAV7 and A. spinosus genotype WAS13 had high nutrients, pigments vitamins, phenolics, flavonoids, and antioxidant. These two weedy Amaranthus accessions could be used as antioxidant profile enriched high-yielding varieties with high nutritional and antioxidant activity. The present investigation revealed that weedy Amaranthus is an excellent source of nutritional value, antioxidant phytochemicals, and antioxidant activity offered huge prospects as cultivated vegetable amaranth to feeding the mineral, vitamin, and antioxidant deficient community. correlation studies. Correlation of antioxidant leaf pigments, β-carotene, vitamin C, TPC, TFC, TAC (DPPH) and TAC (ABTS + ) of A. viridis and A. spinosus genotypes are presented in Table 6. Correlation of antioxidant leaf pigments, β-carotene, vitamin C, TPC, TFC, TAC (DPPH) and TAC (ABTS + ) of A. viridis and A. spinosus genotypes showed interesting results. Significant positive associations with TPC, TFC, TAC (DPPH) and TAC (ABTS + ) were observed for all antioxidant leaf pigments. It indicated that the increase in TPC, TFC, TAC (DPPH) and TAC (ABTS + ) were directly related to the increment of chlorophylls, β-cyanins, β-xanthins, betalains, and carotenoids content or vice versa. It meant all leaf pigments had strong antioxidant activity. Similarly, vitamin C had a significant positive interrelationship with TPC, TFC, and TAC, while it exhibited insignificant negative associations among all antioxidant leaf pigments. Sarker and Oba 18,24 in their earlier work in amaranth also observed a similar trend. A significant positive association was exhibited among β-carotene, vitamin C, TPC, TFC, TAC (DPPH), and TAC (ABTS + ). The significant positive interrelationship of β-carotene, vitamin C, TPC, TFC, TAC (DPPH), and TAC (ABTS + ) signify that β-carotene, vitamin C, TPC, TFC had strong antioxidant activity. The validation of the antioxidant capacity of A. viridis and A. spinosus genotypes by two different methods of antioxidant capacity measurements were confirmed with the significant positive associations between TAC (DPPH) and TAC (ABTS + ). Antioxidant phytochemicals such as leaf pigments, β-carotene, vitamin C, TPC, and TFC had strong antioxidant activity, as these showed the significant associations with TAC (DPPH) and TAC (ABTS + ). In the present investigation, all antioxidant leaf pigments, β-carotene, vitamin C, TPC, and TFC played a vital role in the antioxidant activity of A. viridis and A. spinosus genotypes as these compounds had strong antioxidant activity. (2019) 9:20413 | https://doi.org/10.1038/s41598-019-50977-5 www.nature.com/scientificreports www.nature.com/scientificreports/ In conclusion, the present study has demonstrated that leaves of both weedy Amaranthus genotypes exhibited as a good source of potassium, calcium, magnesium, P, S, Fe, Mn, Cu, Zn, Na, B, Mo, protein, dietary fiber, carbohydrates as a leafy vegetable. It is an excellent source of antioxidant leaf pigments, β-carotene, vitamin C, TAC, TPC and TFC and antioxidant that could contribute to human nutrition and health. The A. viridis genotype WAV7 and A. spinosus genotype WAS13 identified as the best accessions and could be cultivated as like as cultivar as a potential source of nutritional value, antioxidant leaf pigments, β-carotene, vitamin C, phenolics, flavonoids and antioxidants in our daily diet to reduce the hidden hunger and accomplishing nutritional and antioxidant sufficiency. Weedy Amaranthus species are the excellent source of phenolics, flavonoids, and antioxidants that have many pharmacological effects of their traditional applications. Finally, the obtained data present a valuable contribution to the scientific evaluation of pharmacologically active principles in weedy species.

experiment materials, design, layout, and cultural practices. Department of Genetics and Plant
Breeding of Bangabandhu Sheikh Mujibur Rahman Agricultural University collected several accessions (genotypes) of weedy amaranth (A. spinosus and A. viridis) from different agro-ecological zones of Bangladesh. We selected six genotypes (three accessions from each species) based on different morphological traits and different agroecological zones. We grew the selected genotypes at the experimental field of Bangabandhu Sheikh Mujibur Rahman Agricultural University, Bangladesh in a randomized complete block design (RCBD) with three replications. The unit plot size of each genotype was one square meter. The spacing of each A. spinosus and A. viridis genotype was 20 cm 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 weed. To maintain the normal growth of the crop proper irrigations were provided. At 30 days after sowing of seed, leaves samples were collected. All the parameters were measured in three replicates. chemicals. Solvent: acetone and methanol. Reagents: H 2 SO 4 , HNO 3 , HClO 3, NaOH, dithiothreitol (DTT), caesium chloride, ascorbic acid, standard compounds of pure Trolox (6-hydroxy-2, 5, 7, 8-tetramethyl-chroman-2-carboxylic acid), gallic acid, rutin, folin-ciocalteu reagent, DPPH (2, 2-diphenyl1-picrylhydrazyl), ABTS + , aluminium chloride hexahydrate, sodium carbonate, potassium acetate, and potassium persulfate. All solvents and reagents used in this study were high purity laboratory products obtained from Kanto Chemical Co. Inc. (Tokyo, Japan) and Merck (Germany).   Determination of β-cyanin and β-xanthin content. The fresh A. spinosus and A. viridis leaves were extracted in 80% methanol containing 50 mM ascorbic acid to measure β-cyanin and β-xanthin following the method of Sarker and Oba 18 . A spectrophotometer (Hitachi, U-1800, Tokyo, Japan) was used to read the absorbance at 540 and 475 nm for β-cyanin and β-xanthin, respectively. The results were expressed as nanogram betanin equivalent to per gram FW for β-cyanin and nanograms indicaxanthin equivalent to per gram FW for β-xanthin.
estimation of β-carotene. Method of Sarker and Oba 18,38 was followed to extract and determine β-carotene content. 500 mg of fresh leaf sample was ground in 10 ml of 80% acetone and centrifuged at 10,000 rpm for 3-4 min to carry out the extraction process. 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 read the absorbance at 510 nm and 480 nm. Data were expressed as mg β-carotene per 100 g fresh weight.
The β-carotene content was calculated using the following formula: Determination of vitamin c. The fresh A. spinosus and A. viridis leaves were used to measure ascorbic acid (AsA) and dehydroascorbate (DHA) acid spectrophotometrically. 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 18,39 . Finally, the absorbance of the sample solution was read. Data were recorded as mg ascorbic acid per 100 g fresh weight (FW). extraction of samples for tpc, tfc and tAc analysis. At the edible stage (30 Days after sowing), A. spinosus and A. viridis leaves were harvested. The leaves were air dried in shade for chemical analysis. 40 ml of 90% aqueous methanol was used to extract 1 g of grounded dried leaves from each cultivar in a tightly capped bottle (100 ml). The extract was then placed in a shaking water bath (Thomastant T-N22S, Thomas Kagaku Co. Ltd., Japan) for 1 h. Then the extract was filtered for further analytical assays of total polyphenol content, total flavonoid content, total antioxidant activity. 18,40 was followed to estimate the total phenolic content of A. spinosus and A. viridis using the folin-ciocalteu reagent with gallic acid as a standard phenolic compound. In a test tube, 1 ml of folin-ciocalteu reagent (previously diluted 1:4, reagent: distilled water) was added to 50 µl of the leaf extract solution and then mixed thoroughly for 3 min. Then, the mixture was allowed to stand for 1 h in the dark by adding 1 ml of Na 2 CO 3 (10%). A Hitachi U1800 spectrophotometer (Hitachi, Tokyo, Japan) was used to read the absorbance was read at 760 nm. An equation obtained from a standard gallic acid graph was used to estimate the concentration of total phenolic compounds in the leaf extracts. The results are expressed as μg gallic acid equivalent (GAE) g −1 FW.

Determination of total flavonoid content (TFC).
The aluminum chloride colorimetric method 38,41 was used to estimate the total flavonoid content of A. spinosus and A. viridis 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. www.nature.com/scientificreports www.nature.com/scientificreports/ total antioxidant capacity (tAc). Diphenyl-picrylhydrazyl (DPPH) radical degradation method 39,42 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 39,43 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. Aa 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.
The results were reported as the average of three measurements (n = 3). The data were also statistically analyzed by ANOVA using Statistix 8 software, and the means were compared by the Duncan's Multiple Range Test (DMRT) at 1% and level of probability.

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