Antioxidant constituents of three selected red and green color Amaranthus leafy vegetable

Red color (A. tricolor) genotypes are an excellent source of pigments, such as betalain (1122.47 ng g−1 FW), β-xanthin (585.22 ng g−1 FW), β-cyanin (624.75 ng g−1 FW), carotenoids (55.55 mg 100 g−1 FW), and antioxidant phytochemicals, such as vitamin C (122.43 mg 100 g−1 FW), TFC (312.64 RE µg g−1 DW), TPC (220.04 GAE µg g−1 DW), TAC (DPPH and ABTS+) (43.81 and 66.59 TEAC µg g−1 DW) compared to green color (A. lividus) genotype. Remarkable phenolic acids, such as salicylic acid, vanillic acid, protocatechuic acid, gallic acid, gentisic acid, β-resorcylic acid, p-hydroxybenzoic acid, syringic acid, ellagic acid, chlorogenic acid, sinapic acids, trans-cinnamic acid, m-coumaric acid, caffeic acid, p-coumaric acid, ferulic acid, and flavonoids, such as rutin, hyperoside, isoquercetin, myricetin, quercetin, apigenin, kaempferol, and catechin were observed in the red color amaranth genotypes, which was much higher compared to the green color amaranth genotype. We newly identified four flavonoids such as quercetin, catechin, myricetin, and apigenin in amaranth. Among the three selected advanced genotypes studied the red color genotype VA13 and VA3 had abundant antioxidant pigments, phytochemicals, phenolic acids, flavonoids, and antioxidant activity could be selected for extracting colorful juice. Correlation study revealed that all antioxidant constituents of red color amaranth had strong antioxidant activity. The present investigation revealed that two red color genotypes had an excellent source of antioxidants that demand detail pharmacological study.

Antioxidants leaf pigments. Antioxidant leaf pigments of three selected red and green color Amaranthus leafy vegetables are shown in Fig. 2. Betalain, β-xanthin, and β-cyanin varied significantly and remarkably with the genotypes and ranged from 384.39 to 1122.47, 144.11 to 585.22, and 240.28 to 624.75 ng g −1 FW, respectively. The red color genotype VA13 exhibited the highest β-cyanin content, followed by VA3. Among genotypes, significant and remarkable variations were observed in β-xanthin content. Betalain and β-xanthin content were the highest in the red color genotype VA3 followed by VA13. In contrast, the green color genotype GRA1 exhibited the lowest β-cyanin, β-xanthin, and betalain content. Pronounced variations were observed in the carotenoids content of the genotypes. Carotenoids content ranged from 23.02 to 55.55 mg 100 g −1 FW. The red color genotype VA3 exhibited the highest carotenoids, while the lowest carotenoids were recorded in the green color genotype GRA1. The red color genotype VA13 and VA3 had high carotenoids pigments compared to the green color genotype GRA1. In this study, we found remarkable carotenoids, β-cyanin, betalain, and β-xanthin in the red color genotype compared to the green color genotype, which was corroborated with the results of red and green amaranth of Khanam and Oba 32 . We obtained two to three fold greater carotenoids contents in red color genotypes compared to the carotenoids contents of A. gangeticus genotype of Raju et al. 33 . The leaf carotenoids contents of red color genotypes two to three fold and green color genotype were 50% greater than the carotenoids contents of the leaves of A. caudatus 15 . The betalain content of our study was also corroborated with the betalain content of Li et al. 34 where they reported the highest betalain content in A. caudatus leaves compared to A. hypochondriacus leaves. They also reported that leaves had the highest betalain compared to different parts of plants (seed, stalks, sprouts, flowers). Generally, the red genotype with darker or deeper red-violet color showed higher pigments (betalain, carotenoids, β-cyanin, β-xanthin) as well as greater antioxidant activity 31 . In the present study, color parameters of three red and green color Amaranthus leafy vegetables were also related to antioxidant pigments and phytochemical contents. Specifically, relative lower lightness (L*) values, higher redness value (a*), were found in red color Amaranthus leafy vegetable that had higher pigments (betalain, carotenoids, β-cyanin, β-xanthin). The genotype VA13 and VA3 had abundant antioxidant pigments with free radical-scavenging activity 17 . Hence, the red color genotype VA13 and VA3 could be consumed in our daily diet for human health benefits as these pigments played an essential role in detoxification of ROS in the human body and preventing many degenerative human diseases and antiaging 15,16 . Antioxidant phytochemicals. Total phenolic compounds (TPC), vitamin C, total flavonoid compounds (TFC) and total antioxidant activity (TAC) varied significantly among the studied genotype (Fig. 3   . Vitamin C, total phenolics, total flavonoids, and free radical scavenging capacity in three selected red and green color Amaranthus leafy vegetable, vitamin C (mg 100 g −1 FW), TPC, Total polyphenol content (GAE µg g −1 DW); TFC, Total flavonoid content (RE µg g −1 DW); TAC (DPPH), Total antioxidant capacity (DPPH) (TEAC µg g −1 DW); TAC (ABTS + ), Total antioxidant capacity (ABTS + ) (TEAC µg g −1 DW); different letters in the bar are differed significantly by Duncan Multiple Range Test (P < 0.001), (n = 3).
C, TPC, TFC, and TAC compared to green color genotype GRA1. Our results were fully agreed to the results of Khanam and Oba 32 where they observed higher TPC, TFC, and TAC content in the red color amaranth genotype compared to green color amaranth. Similarly, our observed vitamin C was much greater than vitamin C reported by Jiminez-Aguilar and Grusak 35 in fifteen different species of Amaranthus leafy vegetables. Li et al. 34 noticed the highest total polyphenol content, total flavonoids content and total antioxidant activity (FRAP and ORAC methods) in A. hypochondriacus leaves compared to A. caudatus leaves. They also reported that leaves had the highest total polyphenol content, total flavonoids content and total antioxidant activity (FRAP) compared to different parts of plants (seed, stalks, sprouts, flowers). Kraujalis et al. 36 reported the highest TPC and antioxidant capacity (ABTS+, DPPH, and ORAC) in the leaves compared to other parts (Stem, flower, seed) of Amaranthus hybridus.
It is difficult to compare with our present results due to the difference in extraction and estimation methods and standard references. Color parameters of red and green color genotypes were also related to antioxidant pigments and antioxidant activity. Red Amaranthus leafy vegetables with darker or deeper red-violet color having higher antioxidants pigments and phytochemicals such as vitamin C, TPC, and TFC showed greater antioxidant activity 31 . In the present study, antioxidant phytochemicals such as TPC and TFC were measured by spectrophotometric methods to compare with HPLC detected total polyphenols, flavonoids and total phenolic index (TPI). In this study, an accurate analysis of individual compounds was done through HPLC. Therefore, the total phenolic index (TPI) has been proposed as an alternative and complementary approach to the TPC 34 .
In this study, we found remarkable pigments such as carotenoids, β-cyanin, betalain, β-xanthin, and antioxidant phytochemicals such as TFC, vitamin C, TPC, and antioxidant potential in the red color amaranth genotypes, which was much higher compared to green color amaranth genotype. Our results were fully agreed to the results of Khanam and oba 32 where they observed higher TAC, carotenoids, β-cyanin, TFC, betalain, β-xanthin, and TPC content in the red amaranth genotype compared to green amaranth. Pigments such as betalain (1122.47 ng g −1 FW), β-cyanin (624.75 ng g −1 FW), carotenoids (55.55 mg 100 g −1 FW), β-xanthin (585.22 ng g −1 FW), and antioxidant phytochemicals such as TAC (ABTS + ) (66.59 TEAC µg g −1 DW), TFC (312.64 RE µg g −1 DW), and TAC (DPPH) (43.81 TEAC µg g −1 DW) obtained in this study were corroborated with the results of Khanam et al. 37 in A. tricolor. The red color genotypes VA13 and VA3 had abundant pigments such as carotenoids, β-cyanin, betalain, β-xanthin, and antioxidant phytochemicals such as TAC, vitamin C, TFC, and TPC. The genotypes VA13 and VA3 could be used as antioxidant profile enriched high-yielding varieties. The present investigation revealed that these two genotypes have an excellent source of phenolics, flavonoids, vitamins, antioxidant leaf pigments, and antioxidants which were much greater than the results of Khanam and Oba 32 and Khanam et al. 37 that offered huge prospects for feeding the vitamin and antioxidant deficient community. flavonoids and phenolic acids. Table 1 represents the data on the molecular ion, main fragment ions in MS 2 , λmax, retention time, and identified compounds. The LC separated flavonoids and phenolic acid values from three genotypes (VA13, VA3, and GRA1) were compared with standard flavonoids and phenolic acid masses through the corresponding peaks of the compounds. A total of twenty-four phenolic compounds were identified. Among them, nine benzoic acids, seven cinnamic acids, and eight flavonoids compounds. We identified four flavonoids (quercetin, catechin, myricetin, and apigenin) compounds in red and green color Amaranthus leaves for the first time. Except for these nine flavonoids and phenolic acids, Khanam and Oba 32 , Khanam et al. 37 in red and green amaranths reported the rest 15 flavonoids and phenolic acids. Li et al. 34 reported 11 phenolic compounds such as gallic acid, protocatechuic acid, chlorogenic acid, gentisic acid, β-resorcylic acid, ferulic acid, salicylic acid, ellagic acid, rutin, quercetin, and kaempferol in different parts (Leaf, seed, stalks, sprouts, flowers) of A. hypochondriacus, A. cruentus, and A. caudatus. Pasko et al. 38 identified 8 phenolic acids such as gallic acid, p-hydroxybenzoic acid, vanillic acid, p-coumaric acid, syringic acids, ferulic acid, caffeic acids, cinnamic acids and 3 flavonoids such as rutin, vitexin, isovitexin in the sprouts and seeds of A. cruentus (Aztec and Rawa). Figures 4-7 represent the identified phenolic compounds of three selected red and green color Amaranthus leaves. Among three major groups of compounds, benzoic acids were the most abundant compounds followed by flavonoids in three studied genotypes (Figs. 4-7).

Correlation coefficient analysis. Correlation of antioxidant pigments and phytochemicals of red and
green Amaranthus leafy vegetables are presented in Table 2. Highly significant positive associations of betalain, β-xanthin, and β-cyanin were exhibited among pigments and with TAC (ABTS + ), TAC (DPPH), and TPC. Pigments of red and green amaranth (betalain, β-xanthin, and β-cyanin) showed strong antioxidant activity as all the pigments exhibited significant associations with TAC (ABTS + ) and TAC (DPPH). Carotenoid pigments had significant correlation coefficients with TAC (ABTS + ), TAC (DPPH), TFC, and vitamin C, while it showed insignificant associations with betalain, β-xanthin, and β-cyanin. Vitamin C exerted significant associations with TAC (ABTS + ) and TAC (DPPH), whereas it exhibited negligible insignificant associations with betalamic pigments, TPC, and TFC. Our results corroborate with the results of Jimenez-Aguilar and Grusak 35 for vitamin C in different amaranth species. The significant positive associations of carotenoids and vitamin C with TAC (ABTS + ) and TAC (DPPH) also suggested a strong antioxidant activity. The significant associations of TPC and TFC were observed with TAC (ABTS + ) and TAC (DPPH) indicating the strong antioxidant capacity of phenolics and flavonoids in red and green Amaranthus leafy vegetable. Alam et al. 45 also reported corroborative results of TPC, carotenoids, TFC with TAC (FRAP) in salt-stressed purslane. Similarly, TAC (ABTS + ) significantly associated with TAC (DPPH) that validated the measurement of antioxidant activity of two different methods in red and green Amaranthus leafy vegetables.
Red color Amaranthus genotypes are an excellent source of pigments, phytochemicals such as β-TPC, β-xanthin, TFC, cyanin, betalain, carotenoids, and vitamin compared to green color genotype. We also identified 24 phenolic acids and flavonoids such as vanillic acid, salicylic acid, protocatechuic acid, gallic acid, gentisic acid, β-resorcylic acid, p-hydroxybenzoic acid, syringic acid, ellagic acid, chlorogenic acid, sinapic acids, trans-cinnamic acid, m-coumaric acid, caffeic acid, p-coumaric acid, ferulic acid, hyperoside, rutin, isoquercetin, www.nature.com/scientificreports www.nature.com/scientificreports/ myricetin, quercetin, apigenin, kaempferol and catechin in the red color amaranth genotypes, which was much higher compared to green color amaranth genotype. Among them, we newly identified four flavonoids quercetin, catechin, myricetin, and apigenin in Amaranthus leaves. The red color genotypes VA13 and VA3 had abundant phenolic acids, pigments, flavonoids, antioxidant phytochemicals, and antioxidant could be used for extracting the juice. Correlation study revealed that all antioxidant constituents of red color amaranth had strong antioxidant activity. The present investigation revealed that two red color genotypes are an excellent source of antioxidants that offered huge prospects for detail pharmacological study. In the present study, the baseline data obtained from red and green amaranth could contribute to the scientists for the evaluation of pharmacologically active constituents.
Methods experiment materials, design, layout, and cultural practices. We selected three high yields and potentially antioxidant red color A. tricolor genotypes VA13 and VA3 as well as green color A. lividus genotype GRA1 (Fig. 8) from 120 genotypes [2][3][4][5][6][12][13][14] . Selected genotypes were grown in Bangabandhu Sheikh Mujibur Rahman Agricultural University in a randomized complete block design (RCBD) with four replications. The unit plot size of each genotype was 1 square meter. The spacing of each red and green color amaranth genotype was 20 cm between rows and 5 cm between plants. Recommended compost doses, fertilizer, and appropriate cultural practices were maintained. Thinning was done to maintain appropriate spacing between plants of a row. As a necessity, weeding and hoeing were done to remove the weeds. To maintain the normal growth of the crop proper irrigations were provided. At 30 days after sowing (DAS) of seed, leaves samples were collected. We measured all the parameters in four replicates.     www.nature.com/scientificreports www.nature.com/scientificreports/ intensity. The chroma and L* values were calculated using the formula, Chroma C* = (a 2 + b 2 ) 1/2 and Lightness, L* = 116 f (Y/Yn) − 16, respectively. β-cyanin and β-xanthin content measurement. The leaves of red and green amaranth were extracted in 80% methyl alcohol having 50 mM ascorbate to measure β-cyanin and β-xanthin according to the method of Sarker and Oba 21 . A spectrophotometer (Hitachi, U-1800, Tokyo, Japan) was used to measure the absorbance at 540 nm for β-cyanin and 475 nm for β-xanthin, respectively. The results were expressed as the nanogram betanin equivalent per gram fresh weight (FW) for β-cyanin and nanograms indicaxanthin equivalent per gram FW for β-xanthin. estimation of carotenoids. The leaves of red and green amaranth were extracted in 80% acetone to estimate carotenoids according to the method of Sarker and Oba 21 . A spectrophotometer (Hitachi, U-1800, Tokyo, Japan) was used to read the absorbance at 663 nm for chlorophyll a, 646 nm for chlorophyll b, and 470 nm for carotenoids, respectively. The data were calculated as mg carotenoids per 100 g FW. estimation of vitamin c. The fresh red and green amaranth leaves were used to measure ascorbate (AsA) and dehydroascorbic acid (DHA) acid through a spectrophotometer. For pre-incubation of the sample and reduction of DHA into AsA Dithiothreitol (DTT) was used. AsA reduced Fe 3 + to Fe 2 + and estimation of AsA was made by the spectrophotometric (Hitachi, U-1800, Tokyo, Japan) measuring Fe 2 + complexes with 2, 2-dipyridyl 21,46 . Finally, the absorbance of the sample solution was read. The data were recorded as mg vitamin C per 100 g of fresh weight (FW).
Samples extraction for tpc, tfc, and tAc analysis. Thirty days after sowing (DAS) red and green amaranth leaves were harvested. For chemical analysis, the leaves were dried in the air in a shade. Forty ml of 90% aqueous methanol was used to extract 1 g of grounded dried leaves from each cultivar in a bottle (100 ml) capped tightly. A shaking water bath (Thomastant T-N22S, Thomas Kagaku Co. Ltd., Japan) was used to the extract for 1 h. The extract was filtered for determination of polyphenols, flavonoids, total antioxidant capacity. estimation of phenolics. Method of Sarker and Oba 21,47 was followed to estimate the total phenolic content of red and green amaranth using the folin-ciocalteu reagent with gallic acid as a standard phenolic compound. The folin-ciocalteu reagent was previously diluted 1:4, reagent: distilled water. In a test tube, 1 ml of diluted folin-ciocalteu was added to 50 µl extract solution and then mixed thoroughly for 3 min. One ml of Na 2 CO 3 (10%) was added to the tube and stand for 1 h in the dark. A Hitachi U1800 spectrophotometer (Hitachi, Tokyo, Japan) was used to read the absorbance at 760 nm. A standard gallic acid graph was made to determine the concentration of phenolics in the extracts. The results are expressed as μg gallic acid equivalent (GAE) g −1 DW.
Estimation of flavonoids. The AlCl 3 colorimetric method 46,48 was used to estimate the total flavonoid content of red and green amaranth extract. In a test tube, 1.5 ml of methanol was added to 0.1 ml of 10% aluminum chloride, 0.1 ml of 1 M potassium acetate, 2.8 ml of distilled water and 500 µl of leaf extract for 30 min at room temperature. A Hitachi U1800 spectrophotometer (Hitachi, Tokyo, Japan) was used to take the absorbance of the reaction mixture at 415 nm. TFC is expressed as μg rutin equivalent (RE) g −1 dry weight (DW) using rutin as the standard compound.
Antioxidant capacity assay. Diphenyl-picrylhydrazyl (DPPH) radical degradation method 21,49 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 47,50 was followed for ABTS + assay. Exactly 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.
Samples extraction for HpLc and Lc-MS analysis. 10 ml of 80% methanol containing 1% acetic acid was added in 1 g of fresh leaves and homogenized thoroughly. 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. Flavonoids and phenolic acids were analyzed from the final filtrate.
flavonoids and phenolic acids analysis through HpLc. Sarker and Oba 27 method was followed to determine flavonoids and phenolic acids using HPLC in red and green Amaranthus leaf samples. A variable Shimadzu SPD-10Avp UV-vis detector, LC-10Avp binary pumps, and a degasser (DGU-14A) were equipped with the HPLC system (Shimadzu SCL10Avp, Kyoto, Japan). A CTO-10AC (STR ODS-II, 150 × 4.6 mm I.D., Shinwa Chemical Industries, Ltd., Kyoto, Japan) column was used to separate phenolic acids and flavonoids.