Color attributes, betacyanin, and carotenoid profiles, bioactive components, and radical quenching capacity in selected Amaranthus gangeticus leafy vegetables

Four selected A. gangeticus accessions were evaluated in terms of color attributes, phytopigments, including betaxanthin, betacyanin, and carotenoid profiles, proximate, minerals, and antioxidant capacity (AC). Color attributes, phytopigments, proximate, minerals, and AC of A. gangeticus significantly varied across the accessions. For the first time, we identified four betacyanin compounds, such as amaranthine, iso-amaranthine, betanin, iso-betanin. We also identified five carotenoid compounds zeaxanthin neoxanthin, violaxanthin, lutein, and pro-vitamin A in A. gangeticus accessions. A. gangeticus contained adequate carbohydrates, protein, moisture, and dietary fiber. We found adequate iron, manganese, copper, zinc, sodium, molybdenum, boron, potassium, calcium, magnesium, phosphorus, sulfur in A. gangeticus accessions. The accessions LS7 and LS9 had considerable color attributes, betacyanin, and carotenoid compounds, proximate, nutraceuticals, betalain, betaxanthin, and AC that could be used as preferable potent antioxidant varieties for consumption as sources of phytopigments, nutraceuticals, and antioxidants. The correlation study revealed that antioxidant constituents of A. gangeticus accession were strongly associated with AC. The identified components of betacyanin and carotenoid in A. gangeticus demands detail pharmacological study. The baseline data on color attributes, betacyanin, and carotenoid profiles, betaxanthins, betalains, and AC obtained in this present study could contribute to the scientific evaluation of pharmacologically active principles in A. gangeticus.

Carotenoid profiles. Table 2 shows the data on the λmax, molecular ion, retention time, main fragment ions in MS 2 , and identified carotenoid compounds. The values of carotenoid compounds from four accessions (LS3, LS5, LS7, and LS9) separated through LC were compared with standard carotenoid compound masses with respective peaks of the compounds. In A. gangeticus leaves, a total of five carotenoid compounds were identified. Across them, four were identified as xanthophylls such as neoxanthin, violaxanthin, zeaxanthin, and lutein) and one was identified as pro-vitamin A (β-carotene). Figures 3 and 4 show the identified carotenoid profiles including total xanthophylls and total carotenoids and % of xanthophylls (zeaxanthin neoxanthin, violaxanthin, and lutein), pro-vitamin A (β-carotene), total xanthophylls to total carotenoids of four selected A. gangeticus leaves, respectively. Across xanthophylls, the most prominent identified carotenoid was lutein, followed by violaxanthin, while the zeaxanthin and neoxanthin contents were very low in A. gangeticus accessions (Fig. 3). We noticed much higher Zeaxanthin, lutein, β-carotene, total xanthophylls, neoxanthin, and total carotenoid contents of A. gangeticus accession compared to the contents of vegetable amaranth accession of Raju et al. 57 (Fig. 3). The highest total xanthophylls (73.36 mg 100 g −1 ) and lutein (46.72 mg 100 g −1 ) were recorded in the accession LS9, followed by the accession LS7. On the other hand, the accession LS5 exhibited the lowest total xanthophylls (46.81 mg 100 g −1 ) and lutein (26.34 mg 100 g −1 ). The accession LS7 showed the highest β-carotene (67.12 mg 100 g −1 ), violaxanthin (25.30 mg 100 g −1 ), and total carotenoids (135.67 mg 100 g −1 ) followed by the accession LS9. Conversely, the accession LS5 had the lowest total carotenoids (78.45 mg 100 g −1 ) and violaxanthin (17.89 mg 100 g −1 ), whereas the accession LS3 had the lowest β-carotene (30.11 mg 100 g −1 ) (Fig. 3). The accession LS7 exhibited the highest neoxanthin (3.62 mg 100 g −1 ) followed by the accession LS9. In contrast, the accessions LS3 and LS5 showed the lowest neoxanthin (1.58 and 1.75 mg 100 g −1 ). The highest zeaxanthin Table 2. Retention time, wavelengths of maximum absorption in the visible region (λ max ), mass spectral data and tentative identification of carotenoid profiles in four selected A. gangeticus leafy vegetables.     (Fig. 4). The accession LS7 demonstrated the highest percentage of violaxanthin (25.22%), and total xanthophylls (64.52%) to total carotenoids, albeit the accession LS3 and LS9 showed the highest percentage of lutein (37.02 and 36.66%). In contrast, the lowest percentage of lutein (28.24%) and total xanthophylls (50.55%) was observed in the accession LS7, and the lowest percentage of violaxanthin (17.56%) was noticed in the accession LS9. Neoxanthin percentage was the highest in the accession LS7 (2.67%), followed by LS9, while the accession LS3 showed the lowest percentage of neoxanthin (1.85%). The accession LS5 exhibited the highest percentage of zeaxanthin (1.18%), which was statistically similar to the accession LS7 and LS9. Conversely, the lowest zeaxanthin was recorded in the accession LS3 (0.43%). The highest percentage of β-carotene was obtained from the accession LS7 (49.45%) followed by the accession LS9. In contrast, the accession LS3 showed the highest percentage of β-carotene (35.48) (Fig. 4).
The results of total carotenoids of our study corroborated the results of Khanam and Oba 37. They observed higher carotenoids in the red amaranth accession compared to green amaranth. The vegetable amaranth LS7 and LS9 contained higher lutein, violaxanthin, neoxanthin, zeaxanthin, total xanthophylls, β-carotene, and total carotenoids compared to the accession LS3 and LS5. Hence, the carotenoid profiles of vegetable amaranth accession could play a crucial role in the detoxification of ROS in the human body and considered as an essential parameter for consumers as it acts as an antiaging and many degenerative human diseases 17,21 . Our result showed that the vegetable amaranth accession is an excellent source of lutein, violaxanthin, neoxanthin, zeaxanthin, total xanthophylls, β-carotene, and total carotenoids among leafy vegetables that has important free radical-scavenging activity 19 .
In this study, we found considerable pigments profile such as betacyanins, betalains, betaxanthins, and carotenoid profiles such as lutein, violaxanthin, neoxanthin, zeaxanthin, total xanthophylls, β-carotene in A. gangeticus leafy vegetable accession. The results of total carotenoids of our study corroborated with the results of Khanam and Oba 58 and Raju et al. 57 , where they observed higher carotenoids in the red amaranth accession compared to green amaranth and A. gangeticus, respectively. The accession LS7 and LS9 contained higher lutein, violaxanthin, neoxanthin, zeaxanthin, total xanthophylls, β-carotene, and total carotenoid compared to other accessions. Hence, the carotenoid profiles of vegetable amaranth accession could play a crucial role in the detoxification of ROS in the human body and considered as an essential parameter for consumers as it acts as an antiaging and many degenerative human diseases 17,21 . Our result showed that the A. gangeticus accession is an excellent source of lutein, violaxanthin, neoxanthin, zeaxanthin, total xanthophylls, β-carotene, and total carotenoids among leafy vegetables that has important free radical-scavenging activity 19 . A. gangeticus accessions LS7 and LS9 had high carotenoid profiles, such as zeaxanthin, lutein, violaxanthin, neoxanthin, total xanthophylls, β-carotene, and total carotenoids content. The genotypes LS7 and LS9 might be used as carotenoids enriched high-yielding varieties for drink purposes. The present investigation revealed that these two accessions have abundant carotenoids that offered new insight for detail pharmacological study.    ), followed by LS9. In contrast, AC (ABTS + ) was the lowest in LS3. These findings were corroborative to the results of Khanam and Oba 58 , where they observed higher total betaxanthins, betalains content, and AC in the red amaranth accession compared to green amaranth. The A. gangeticus accession LS7 and LS9 contained higher betaxanthins, betalains, and AC than the accession LS3 and LS5. Hence, these antioxidant constituents of A. gangeticus accession played a crucial role in the detoxification of ROS in the human body and are considered an essential parameter for consumers. It acts as an antiaging and many degenerative human diseases 17,21 . The present findings revealed that the A. gangeticus accessions exhibited an excellent source of betalains, betaxanthins, and AC (DPPH & ABTS + ) among leafy vegetables that have important free radical-scavenging activity 19 .
In this study, we found considerable color attributes, betacyanin profiles, carotenoid profiles, betalains, betaxanthins, and AC in the A. gangeticus accessions. The present findings were corroborated by the results of Khanam and Oba 58 , where they observed higher AC, betacyanins, betaxanthins, betalains, total carotenoids in the red amaranth accession compared to green amaranth. Betacyanin, total carotenoids, AC (ABTS + ), and AC (DPPH) obtained in this study corroborated with the results of Khanam et al. 59 in A. tricolor. We found two to threefold greater β-carotene contents in red color accessions compared to the β-carotene contents of A. gangeticus accession of Raju et al. 57 . The leaf β-carotene contents of red color accessions two to three-fold and green color accession were 50% greater than the β-carotene contents of the leaves of A. caudatus 21 . Li et al. 60 noticed the highest total AC (FRAP and ORAC methods) in A. hypochondriacus leaves compared to A. caudatus leaves. They also reported that leaves had the most increased AC (FRAP) than different parts of plants (seed, stalks, sprouts, flowers). It is difficult to compare our present results due to the difference in extraction and estimation methods and standard references. The accessions LS7 and LS9 had high color attributes, betacyanins, carotenoid profiles, betaxanthins, betalains, and AC. The antioxidant profile enriched high-yielding genotypes LS7 and LS9 can be used as drinks. The accessions LS7 and LS9 had high carotenoid profiles that could be used as high carotenoid profiles enriched varieties for drink purposes. The present investigation revealed that these accessions could offer enormous prospects for feeding the antioxidant-deficient community.
Composition of proximate. The composition of proximate of A. gangeticus accessions is shown in Fig. 6.
The range of moisture content of leaves was 81.35 g 100 g −1 to 87.24 g 100 g −1 . LS5 showed the highest moisture content of 87.24 g 100 g −1 ), whereas LS9 exhibited the lowest moisture content (81.35 g 100 g −1 FW). As a higher dry matter of leaf confirm lower moisture contents, two genotypes (19-18% dry matter) had considerable dry biomass. The maturity is strongly associated with the leaf moisture content. The results obtained in this study gangeticus. The highest protein content was obtained from the genotype LS7 (6.24 g 100 g −1 ) followed by LS9. In contrast, the genotype LS3 had the lowest protein content (3.15 g 100 g −1 ). Vegetable amaranth is one of the vital sources of protein for poor people and vegetarians of developing countries. The protein content of A. gangeticus accessions was much higher than A. tricolor (1.26%) in our earlier study 2 . The selected A. gangeticus vegetable amaranths had no significant variations in fat content. The range of fat content was 0.23 to 0.41 g 100 g −1 FW. These results were corroborative to the results of A. tricolor 24 and sweet potato 61 , respectively. The highest carbohydrates were recorded in the genotype LS9 (8.39 g 100 g −1 ) followed by LS3. Conversely, the carbohydrate content was the lowest in LS5 (5.88 g 100 g −1 ) and LS7 (5.97 g 100 g −1 ). The highest energy was recorded in the genotype LS9 (56.68 kcal) followed by LS7. However, the lowest energy was obtained from the genotype LS5 (39.38 kcal). The highest ash content was noticed in LS7 (5.26 g 100 g −1 ) followed by LS9. On the other hand, the lowest ash content was noted in LS5 and LS3 (2.98 and 3.03 g 100 g −1 ). The least variations were noted for dietary fiber across four selected A. gangeticus accessions. The highest dietary fiber was obtained from the accessions LS5 and LS9 (8.22 and 7.85 g 100 g −1 FW) followed by LS3, whereas dietary fiber content was the lowest in LS7 (6.88 g 100 g −1 FW). Dietary fiber had a tremendous contribution to the cure of constipation, the increment of digestibility, and palatability 4 . The current results indicated that leaves of A. gangeticus accessions have abundant moisture, protein, dietary fiber, and carbohydrates. The present study is corroborative to the results of our earlier study 24 . The results of dietary fiber and carbohydrate were corroborative to our previous studies of red morph amaranth 14 , weedy amaranth 10 , green morph amaranth 13 , stem amaranth 11 , and A. blitum 12 . Whereas, dry matter contents of four amaranth accessions were greater than the dry matter contents of red morph amaranth 14 , weedy amaranth 10 , green morph amaranth 13 , stem amaranth 11 , and A. blitum 12 . The protein contents of these four amaranth accessions were greater than the protein contents of red morph amaranth 14 , green morph amaranth 13 , stem amaranth 11 , and A. blitum 12 . Fig. 7. The potassium content ranged from 4.66 mg g −1 to 7.54 mg g −1 . The highest potassium content was recorded in the genotypes LS7. Conversely, the lowest potassium content was observed in the genotype LS5. The range of calcium was 1.68 to 3.25 mg g −1 . The highest calcium content was obtained from the genotype LS7, whereas the lowest calcium content was noted in the genotype LS9. LS3 had the highest magnesium content (3.59 mg g −1 ) followed by LS5 and LS9. On the other hand, the lowest magnesium was recorded in LS7 (2.49 mg g −1 ). The range of phosphorus and sulfur content of vegetable amaranth leaves was 0.65-1.75 and 0.51-1.27 mg g −1 . The highest phosphorus and sulfur content The genotype LS7 exhibited, while the genotype LS5 showed the lowest phosphorus and sulfur content. Adequate calcium (3.25 mg g −1 ), potassium (7.54 mg g −1 ), sulfur (1.27 mg g −1 ), magnesium (3.59 mg g −1 ), and phosphorus (1.75 mg g −1 ) were observed in A. gangeticus accessions. Chakrabarty et al. 6 in A. lividus and Sarker and Oba 24 in A. tricolor also observed similar results in different amaranths. Jimenez-Aguiar and Grusak 62 noted abundant potassium, calcium, magnesium, phosphorus, and sulfur. They also reported pronounced potassium, calcium, magnesium, phosphorus, and sulfur in amaranth compared to spinach, black nightshade, spider flower, and kale.

Minerals composition. Minerals composition of A. gangeticus accessions is shown in
Adequate iron and manganese content was recorded in A. gangeticus accessions. The highest iron content was observed in the genotype LS9 (17.35 µg g −1 ), followed by LS7 and LS5. Conversely, the lowest iron content was recorded in the genotype LS3 (12.99 µg g −1 ). In this study, manganese ranged from 12.25 to 16.77 µg g −1 .  . The highest copper content was observed in LS7, followed by LS9. In contrast, the lowest copper content was obtained from the genotype LS3 and LS5, respectively. Adequate zinc, sodium, and boron were recorded across the A. gangeticus accessions. The range of zinc, sodium, and boron content was 11.33-14.61, 72.24-80.28, and 5.27-7.36 µg g −1 . The highest zinc, sodium, and boron were recorded in LS7, whereas the lowest zinc and sodium were obtained from LS3, and the lowest boron content was recorded in LS5. The range of molybdenum content was 0.26-0.57 µg g −1 . The highest molybdenum content was observed in the genotypes LS7, whereas the lowest molybdenum content was noted in LS3. A. gangeticus accessions contained higher zinc and iron content than the cassava leaves 63 and beach pea 64 . Adequate iron (17.35 µg g −1 ), copper (2.26 µg g −1 ), manganese (16.77 µg g −1 ), sodium (80.28 µg g −1 ), zinc (14.61 µg g −1 ), boron (7.36 µg g −1 ), and molybdenum (0.57 µg g −1 ) were recorded in A. gangeticus accessions. Earlier abundant iron, manganese, copper, zinc sodium, molybdenum, and boron were noted in different amaranths 62 . The leaves of amaranth had pronounced manganese, iron, zinc, and copper in than spinach, black nightshade, spider flower, and kale. The obtained potassium from these accessions was corroborative to previous studies of green morph amaranth 13 , whereas calcium recorded in these accessions was greater than red morph amaranth 14 , stem amaranth 11 , and A. blitum 12 . High phosphorus and sodium were observed compared to weedy amaranth 10 . Likewise, magnesium, zinc, and iron observed in the current study were much pronounced than red morph amaranth 14 , green morph amaranth 13 , stem amaranth 11 , and A. blitum 12 . High copper content was obtained from the present study, which is greater than the earlier study of green morph amaranth 13 , and manganese of the current study was greater than weedy amaranth 10 , green morph amaranth 13 . Hence, these selected advance lines could contribute as high minerals enriched genotypes compared to our previously tested amaranth genotypes.
The correlation coefficient study. The correlation of betacyanins, betaxanthins, betalains, and AC of A. gangeticus leafy vegetables are shown in Table 3. Total betacyanins and betaxanthins, betalains had highly significant positive associations among themselves, with total carotenoids, AC (DPPH and ABTS + ). It revealed  www.nature.com/scientificreports/ that total betacyanins, betaxanthins, and betalains, exhibited strong AC. Total xanthophylls, β-carotene, and total carotenoids had significant positive interrelationships with β-carotene, total carotenoids, AC (DPPH and ABTS + ) that signify that major carotenoids had strong AC. The results of the present study corroborative to the results of our earlier study of drought and salt-stressed A. tricolor 24 .
In conclusion, we identified betacyanin profiles containing amaranthine, iso-amaranthine, betanin, iso-betanin, carotenoid profiles containing zeaxanthin, lutein, violaxanthin, neoxanthin, total xanthophylls, β-carotene, and total carotenoids, betaxanthins, betalains, and AC (DPPH and ABTS + ) in the A. gangeticus accessions. A. gangeticus vegetable amaranth genotypes contained ample proximate, and nutraceuticals, such as protein, carbohydrates, moisture, dietary fiber, iron, manganese, copper, zinc, sodium, molybdenum, boron, potassium, calcium, magnesium, phosphorus, sulfur. The correlation study revealed that all pigments of A. gangeticus had high AC. The present investigation revealed that these accessions exhibited excellent sources of antioxidants components with ROS quenching capability that offered huge prospects for detail pharmacological study. The baseline data on color attributes, betacyanins, carotenoids, betaxanthins, betalains, and AC obtained in the present study could contribute to the scientific evaluation of pharmacologically active principles in A. gangeticus. A. gangeticus accessions LS7 and LS9 had abundant color attributes, betacyanins, and carotenoid profiles, betaxanthins, betalains, proximate, nutraceuticals, and antioxidant potentiality. These two accessions LS7 and LS9 could be recommended as preferable cultivars for consumption as sources of phytopigments, nutraceuticals, and antioxidants.

Methods
Experimental materials. We selected four high yields and antioxidant potential A. gangeticus accessions from few accessions. The seeds of four advance genotypes were collected from the Department of Genetics and Plant Breeding of Bangabandhu Shiekh Mujibur Rahman Agricultural University. It is the first report on color attributes, betacyanin, carotenoid profiles, bioactive components, and antioxidants potentials in A. gangeticus.
Design and layout. The experiment was executed in three replicates following a completely randomized block design (RCBD) at Bangabandhu Sheikh Mujibur Rahman Agricultural University. Each genotype was grown in a 1 m 2 experimental plot following 20 cm and 5 cm distance between rows and plants, respectively. The experimental site is located about 24°23′ N latitude 90°08′ E longitude, in the Agroecological Zones 28 (center of the Madhupur Tract), with an average elevation of 8.4 msl. The site is high land and falls under subtropical climatic conditions with mean winter temperatures of 18 °C and summer temperatures of 29 °C. The soil characteristics of the experimental field are silty clay with low in organic matter (0.87%), slightly acidic (pH 6.4), exchangeable K (0.13 cmol kg −1 ), and total N (0.09%). The soil Zn and P content are above the critical level, while S content is a critical level (Critical levels of Zn, S, and P are 0.2, 14, and 14 mg kg −1 , respectively and the K level is 0.2 cmol kg −1 ).

Intercultural practices.
We applied the recommended compost and fertilizer doses. At the time of land preparation, 10 ton ha −1 compost was applied. Triple superphosphate, urea, gypsum, and murate of potash were applied at 100, 200, 30, and 150 kg ha −1 , respectively. The exact plant spacing in a row was maintained by thinning the row properly. Weeds were regularly removed through proper weeding and hoeing. We provide regular irrigation in the experimental plots for retaining the appropriate growth of vegetable amaranth. We collected the leaf samples at 30 days old plant. Ten randomly selected plants were selected to harvest from each experimental unit. The leaves were immediately sampled from the harvested plants.
Estimation of color attributes. We measured the color attributes C*, L*, b*, and a* using a color meter (TES-135A, Plus, Taiwan) in 15 replicates. The positive value of (+ b*) indicates yellowness, while the negative value of (− b*) indicates blueness. The positive value of (+ a*) suggests the degree of redness, while the negative value of (− a*) indicates greenness. L* indicates lightness, and the C* value indicates leaf color intensity designated as chroma. The chroma value was calculated using the formula, Chroma C* = (a 2 + b 2 ) 1/2 . Samples extraction for HPLC and LC-MS analysis. 10 mL of 80% methanol containing 1% acetic acid was added in 1 g of leaves and homogenized thoroughly, and transferred to a 50 mL tightly capped test tube. The test tubes were placed in a shaker (Scientific Industries Inc., USA) for 15 h at 400 rpm. 0.45 µm filter (MILLEX-HV syringe filter, Millipore Corporation, Bedford, MA, USA) was used to filter the homogenized mixture. The mixture was centrifuged at 10,000 × g for 15 min. Betacyanin components were analyzed from the final filtrate. Betacyanin analysis in the samples could interfere through the precipitation of methanol with the proteins and other insoluble substances in the samples. Strata-X 33 µm Polymeric Reversed-Phase cartridges (Phenomenex, Torrance, CA, USA) were used to purify betacyanin. All extractions were done in triplicate independent samples.
Betacyanin analysis through HPLC. The methods previously used in A. spinosus 54 and A. tricolor 65 were followed to determine betacyanin components in the A. gangeticus leaf sample using HPLC. The high-performance liquid chromatograph Shimadzu SCL10Avp, Kyoto, Japan, was equipped with a degasser (DGU-14A), an LC-10Avp binary pumps, and a detector (Shimadzu SPD-10Avp UV-Vis). A column (CTO-10AC (STR ODS-II, 150 × 4.6 mm I.D., Shinwa Chemical Industries, Ltd., Kyoto, Japan) was used to separate the betacyanin components. Pumping of binary mobile phase was performed with solvent B (acetonitrile) and solvent A (6% (v/v) acetic acid) in the water at the flow rate of 1 mL min −1 for 70 min. The system was run using a gradient program with solvent acetonitrile 0-15% for 45  www.nature.com/scientificreports/ column temperature was maintained at 35 °C with an injection volume of 10 μL. The detector was set at 538 nm for the simultaneous monitoring of betacyanin. For identification of the compound, we compared retention time and UV-Vis spectra with their respective standards. We confirmed the betacyanin components through the mass spectrometry assay method. All samples were prepared and analyzed in triplicates. The results were expressed as mg 100 g −1 FW for betacyanin components. A JEOL AccuTOF (JMS-T100LP, JEOL Ltd., Tokyo, Japan) mass spectrometer fitted with a UV-Vis detector coupled online and an Agilent 1100 Series HPLC system with an ElectroSpray Ionization (ESI) source to analyze the mass spectrometry with negative ion mode with the column elutes in the range of m/z 0-1000 and needle voltage at − 2000 V. Extract constituents were identified by LC-MS-ESI analysis.
Quantification of betacyanin components. Calibration curves of the respective standards were used to quantify individual betacyanin components. The betanin standard was dissolved in 80% methanol as stock solutions to 100 mg mL −1 . Standard curves (10,20,40,60,80, and 100 mg mL −1 ) were prepared and used to quantify the individual betacyanin components. The retention times, UV spectral characteristics, and co-chromatography of samples spiked with commercially available standards were used to identify and match the betacyanin components. Betanin standard was used to prepare standard curves based on the equimolecular conversion for estimating amaranthine and iso-amaranthine in the different samples.
Sample preparation for extraction of carotenoids. The fresh leaf samples were washed thoroughly, blotted dry, lyophilized to dryness. All precautions were taken to prevent any significant losses of carotenoids due to photo-oxidation and isomerization. Sampling was done with subdued lighting and temperature at 20 °C. The dry samples were ground through a mechanical blender. The powdered samples were kept in aluminum foil inside a self-sealing bag and stored below − 20 °C until further use. The samples were stored for one week.
Extraction of carotenoids. Carotenoids were extracted according to the procedure described by Lakshminarayana et al. 66 . Carotenoids were extracted with ice-cold acetone until the samples became colorless. Rapid extraction in cold acetone was employed to reduce the possibility of carotenoid oxidation. The crude extract (50 mL) was taken in a separatory funnel; 100 mL of petroleum ether and 100 mL of aqueous sodium chloride (25%, w/v) were added; after mixing well, the upper layer was separated. The extraction was repeated three times (total volume: 250 mL). The extract was dried over anhydrous sodium sulfate (20 g) and filtered through What- Corporation, Bedford, MA, USA) was used to filter the homogenized mixture. The mixture was centrifuged at 10,000×g for 15 min. The filtered extract was used to determine antioxidant capacity. The antioxidant activity was estimated by the diphenyl-picrylhydrazyl (DPPH) radical degradation method 68 . In a test tube, 10 µL of diluted leaf extract was added to 1 mL of 250 µM DPPH solution and 4 mL of distilled water (in triplicate). In the dark place, the mixture was stood for 30 min. The absorbance was taken at 517 nm using a Hitachi spectrophotometer (Japan). ABTS + assay was carried out using the method of Sarker et al. 69 . In the stock solutions, 2.6 mM potassium persulfate and 7.4 mM ABTS + solution were used. The working solution was prepared by mixing two stock solutions equally. The mixture was allowed to react for 12 h at room temperature in the dark.. 150 μL sample of diluted leaf extract was added to 2850 μL of ABTS + solution ( www.nature.com/scientificreports/ solution mixed with 60 mL methanol) and allowed to react in the dark for two h. The absorbance was taken against methanol at 734 nm using a Hitachi spectrophotometer (Japan). The inhibition percentage of ABTS + and DPPH corresponding to the control was utilized to measure the antioxidant activity following the equation: where A b is the optical density of the control [150 μL methanol for AC (ABTS, 10 µL methanol for AC (DPPH)) instead of leaf extract] and A s is the optical density of the test samples. The reference standard was Trolox. The equations Y = 6.5824X + 5.5298 with R 2 = 0.9998 (Fig. 8) and Y = 20.1598X + 15.6199 with R 2 = 0.9997 (Fig. 9) were obtained from Trolox standard calibration curve for DPPH and ABTS assay, respectively. Finally, the results were expressed as μg Trolox equivalent g −1 DW.
Estimation of proximate composition. AOAC method was followed 70 to estimate the ash, moisture, crude fat, fiber, crude protein contents, and gross energy. The nitrogen was calculated following the Micro-Kjeldahl method. Finally, measure crude protein was estimated by nitrogen × 6.25 (AOAC method 976.05). The ash, total moisture, crude protein, and crude fat (%) were subtracted from 100 for calculating carbohydrate (g 100 g −1 FW).

Estimation of mineral composition.
A. gangeticus accessions leaf samples were dried in an oven at 70 °C for 24 h. Dried samples were ground in a mill. We determined calcium, potassium, magnesium, phosphorus, sulfur, iron, manganese, copper, zinc, sodium, molybdenum, and boron from powdered leaves following the nitric-perchloric acid digestion method 71 . For this digestion, 400 mL HNO 3 (65%), 10 mL H 2 SO 4 (96%), and 40 mL HClO 4 (70%) were poured into a 0.5 g dried leaf sample in the presence of carborundum beads. After digestion, P was measured by diluting the solution appropriately in triplicate following the ascorbic acid www.nature.com/scientificreports/