Phenolic profiles and antioxidant activities in selected drought-tolerant leafy vegetable amaranth

Four selected advance lines of drought-tolerant leafy vegetable amaranth were characterized for phenolic profiles, vitamins, and antioxidant activities. The selected advance lines exhibited differences in terms of genotypes with remarkable phenols, vitamins, flavonoids content, and potential radical quenching capacity. We identified twenty-five phenolic and flavonoid compounds including protocatechuic acid, salicylic acid, gentisic acid, gallic acid, β-resorcylic acid, vanillic acid, p-hydroxybenzoic acid, chlorogenic acid, ellagic acid, syringic acid, ferulic acid, kaempferol, m-coumaric acid, trans-cinnamic acid, quercetin, p-coumaric acid, apigenin, caffeic acid, rutin, sinapic acid, isoquercetin, naringenin, myricetin, catechin, and hyperoside. The selected advance lines VA14 and VA16 had abundant phenols, vitamins, flavonoids, and antioxidants potentiality. The selected drought-tolerant leafy vegetable amaranth showed high antioxidant potentiality as phenols, vitamins, flavonoids of these lines had a significant positive correlation with antioxidant capacities equivalent to Trolox using 2,2-diphenyl-1-picrylhydrazyl and ABTS+. Therefore, drought-tolerant leafy vegetable amaranth VA14 and VA16 can be grown in semi-arid and drought-prone areas in the world to attaining vitamins and antioxidant sufficiency. The phenolic and flavonoids compounds identified in drought-tolerant leafy vegetable amaranth demand a comprehensive pharmacological study. The baseline data on phenolic and flavonoids compounds obtained in the present study will contribute to the scientist forum for the scientific evaluation of these compounds in vegetable amaranth.

. Retention time (Rt), wavelengths of maximum absorption in the visible region (λ max ), mass spectral data and tentative identification of phenolic compounds in selected four drought-tolerant leafy vegetables amaranth. Benzoic acids. The most preponderant benzoic acids were identified as salicylic acids. Rest of the benzoic acids were identified in the order: gallic acid ˃ vanillic acid ˃ protocatechuic acid ˃ p-hydroxybenzoic acid ˃ gentisic acid ˃ β-resorcylic acid ˃ syringic acid ˃ ellagic acid (Fig. 1). It revealed from the present study that droughttolerant leafy vegetable amaranth VA14 and VA16 exhibited much higher benzoic acid content in comparison with the results of the benzoic acid content of green amaranth of our previous study 11 and the results of Khanam et al. 54 in A. tricolor. The reason for higher benzoic acids obtained from our drought-tolerant vegetable amaranth genotypes in comparison with the results of our previous study and Khanam et al. 54 1). The genotype VA16 exhibited the highest vanillic acid (15.36 µg g −1 FW), p-hydroxybenzoic acid (12.33 µg g −1 FW), and ellagic acid (6.23 µg g −1 FW) followed by VA14, while the genotype VA11 showed the lowest vanillic acid (11.06 µg g −1 FW) and the genotype VA6 exhibited the lowest p-hydroxybenzoic acid (2.58 µg g −1 FW), and ellagic acid (1.24 µg g −1 FW). The highest syringic acid was noticed in the genotype VA14 (7.63 µg g −1 FW) followed by VA16 and the lowest syringic acid was observed in the genotype VA14 (3.05 µg g −1 FW) (Fig. 1). Nine benzoic acids obtained in the current study were much higher than the results of nine cinnamic acids of green amaranth of our previous  Cinnamic acids. Trans-cinnamic acid was identified as the most prominent component within cinnamic acids followed by chlorogenic acid. (Fig. 2). Selected drought-tolerant leafy vegetable amaranth had considerable cinnamic acids. The cinnamic acids obtained from the leafy vegetable amaranth genotype VA14 and VA16 were much higher in comparison with the results of cinnamic acids of green amaranth of our previous study 11 (Fig. 2). The highest caffeic acid and m-coumaric acid (12.42 µg g −1 FW and 12.53 µg g −1 FW) were reported in the genotype VA16 followed by the genotype VA14. In contrast, the genotype VA6 had the lowest caffeic acid and m-coumaric acid (2.36 µg g −1 FW and 4.57 µg g −1 FW). Seven cinnamic acids obtained in the current study were much higher than the results of seven cinnamic acids of green amaranth of our previous study 11 . The differences in species may be the reason for obtaining higher cinnamic acids from our drought-tolerant vegetable amaranth genotypes in comparison with the results of our previous study.
Flavonoids. In the current investigation, selected drought-tolerant leafy vegetable amaranth had abundant flavonoids such as rutin, isoquercetin, quercetin, myricetin, naringenin, kaempferol, catechin, apigenin, and hyperoside which were much higher than the results of nine flavonoid compounds of green amaranth of our previous study 11    ). In contrast, the genotype VA11 showed the lowest apigenin and hyperoside (5.52 and 3.33 µg g −1 FW). (Fig. 3). Quercetin and hyperoside of our selected drought-tolerant leafy vegetable amaranth were higher than the content of quercetin and hyperoside reported by Khanam et al. 54 in A. tricolor genotypes. The varietal differences, and differential geographic locations, climatic and edaphic conditions, and cultural managements may be played a major contribution in securing higher quercetin and hyperoside in our drought-tolerant vegetable amaranth genotypes in comparison with the results of Khanam et al. 54 .
Phenolic fractions. Total flavonoids, total phenolic index, total phenolic acids, total benzoic acids, and total cinnamic acids of selected drought-tolerant leafy vegetable amaranth varied from 107. 37 4). The highest total phenolic acids (220.39 µg g −1 FW), total benzoic acids (114.75 µg g −1 FW), total phenolic index (443.82 µg g −1 FW), total cinnamic acids (105.64 µg g −1 FW), and total flavonoids (223.43 µg g −1 FW) were recorded in the genotype VA14 followed by the genotype VA16. In contrast, the lowest total cinnamic acids (55.61 µg g −1 FW), total benzoic acids (51.76 µg g −1 FW), total phenolic index (204.33 µg g −1 FW), total phenolic acids 107.37 µg g −1 FW), and total flavonoids (96.96 µg g −1 FW) were noticed in the genotype VA6 (Fig. 4). We noticed much greater total flavonoids total phenolic acids, and total phenolic index in selected leafy vegetables in comparison with the results of A. tricolor reported by Khanam et al. 54 . The varietal differences, and differential geographic locations, climatic and edaphic conditions, and cultural managements may be played a major contribution in securing higher phenolic fractions in our drought-tolerant vegetable amaranth genotypes in comparison with the results of Khanam et al. 54 . Cinnamic acid was synthesized in plant tissues from the most extensively distributed phenolic acids phenylalanine 58 . In the tissue of plants, although glycoside derivatives are the most common forms of flavonoids, occasionally these compounds occur as aglycone. Flavonoids represent approximately 60% of total dietary phenolic compounds 59 . The most predominant flavonoids in the plants are flavonols and quercetin glycosides are naturally occurring most prominent flavonols 59 . Significance differences in phenolic acids and flavonoids profiles among different Cichorium spinosum species were reported by Petropoulos et al. 60 .
In the current investigation, we observed abundant phenols and flavonoid compounds such as protocatechuic acid, salicylic acid, vanillic acid, gallic acid, β-resorcylic acid, p-hydroxybenzoic acid, naringenin, gentisic acid, myricetin, ellagic acid, chlorogenic acid, isoquercetin, syringic acid, m-coumaric acid, quercetin, caffeic acid, trans-cinnamic acid, rutin, sinapic acid, p-coumaric acid, catechin, ferulic acid, kaempferol, apigenin, and hyperoside in selected drought-tolerant leafy vegetable amaranth. We found corroborative results with the results of Khanam and Oba 55 where they observed higher syringic acid, salicylic acid, p-hydroxybenzoic acid, vanillic acid, gallic acid, isoquercetin, ferulic acid, ellagic acid, rutin, trans-cinnamic acid, chlorogenic acid, m-coumaric acid, caffeic acid, and p-coumaric acid in red amaranth in comparison with green amaranth. p-hydroxybenzoic acid, rutin, m-coumaric acid, hyperoside, salicylic acid, chlorogenic acid, ferulic acid, ellagic acid, vanillic acid, gallic acid, syringic acid, caffeic acid, trans-cinnamic acid, and p-coumaric acid obtained from this study were higher than the results of Khanam et al. 54 in A. tricolor. The selected drought-tolerant leafy vegetable amaranth VA14 and VA16 had high vitamins along with high flavonoids and phenols, such as protocatechuic acid, salicylic acid, gentisic acid, vanillic acid, gallic acid, p-hydroxybenzoic acid, β-resorcylic acid, ellagic acid, syringic   55 where they noticed higher total flavonoids, total antioxidant capacity, and total polyphenols in red amaranth in comparison with green amaranth. The selected drought-tolerant leafy vegetable amaranth VA14 and VA16 contained higher vitamin C, total flavonoids, total polyphenols, and antioxidant capacity in comparison with VA11 and VA6. Hence, these antioxidant constituents of leafy vegetable amaranth could be crucial attributes for consumers due to the high detoxifying capacity of ROS in the human body and preventing many degenerative human diseases and anti-aging activity 18,20 . It suggested from the present results of vitamin C, total flavonoids, total polyphenols, and antioxidant capacity in selected drought-tolerant leafy vegetable amaranth that leafy vegetables have important free radical-scavenging activity 21 .
In the current investigation, we observed remarkable vitamin C, total flavonoids, total polyphenols, and antioxidant capacity in selected drought-tolerant leafy vegetable amaranth. The current results were corroborative with the results of total polyphenols, total flavonoids, and antioxidant capacity of Khanam and Oba 55 . They noticed higher antioxidant capacity, total flavonoids, and total polyphenols content in A. tricolor genotypes in comparison with green amaranth genotype. Antioxidant capacity (ABTS + ), total flavonoids and antioxidant capacity (DPPH) obtained in the current study were corroborated with the results of A. tricolor reported by Khanam et al. 54 , while total phenols noticed in this investigation was much pronounced than total phenols in A. tricolor reported by Khanam et al. 54 . Vitamin C obtained from our study was much greater than vitamin C reported by Jiminez-Aguilar and Grusak 61 in Amaranthus species. The varietal differences, and differential geographic locations, climatic and edaphic conditions, and cultural managements may be played a key role in accumulating higher phenols and vitamin C in our drought-tolerant vegetable amaranth genotypes in comparison    54 and Jiminez-Aguilar and Grusak 61 . The higher total antioxidant activity (FRAP and ORAC methods), total flavonoids, and total phenols were reported in the leaves of A. hypochondriacus than A. caudatus leaves 56 . They reported the highest total antioxidant activity (FRAP), total flavonoids, and total phenols in the leaves than stalks, seed, flowers, and sprouts. All the extraction and estimation methods and standard references differed to our methodology, hence, it is tedious to compare our present results with their results. The genotypes VA14 and VA16 had high phenolic profiles, antioxidant constituents such as vitamin C, total polyphenols, total flavonoids, and antioxidant capacity. The selected drought-tolerant leafy vegetable amaranth VA14 and VA16 could be used as antioxidant profiles enrich high-yielding varieties. It revealed from the study that these two genotypes could offer greatly contributed to feeding the antioxidant-deficient community.
Correlation coefficient study. The correlation of antioxidant constituents and antioxidant capacity of selected drought-tolerant leafy vegetable amaranth are shown in Table 2. Vitamin C had significant positive interrelationships with total flavonoids, total polyphenols, and antioxidant capacity (DPPH and ABTS + ) that signify that vitamin C had high antioxidant activity. The results of the present study corroborated with the results of our earlier study of drought and salt-stressed A. tricolor [22][23][24]28 . The significant correlation among total polyphenols, total flavonoids, antioxidant capacity (DPPH and ABTS + ) were observed indicating potential antioxidant activity of total polyphenols and total flavonoids of selected drought-tolerant leafy vegetable amaranth. The findings for total antioxidant capacity (FRAP), total flavonoids, and total polyphenols in salt-stressed purslane 62 were corroborative to our present findings. Similarly, AC (ABTS + ) significantly associated with AC (DPPH) that validated the estimation of antioxidant activity of two different methods in selected leafy vegetables.
In conclusion, we identified twenty-five phenols and flavonoid compounds such as protocatechuic acid, p-hydroxybenzoic acid, vanillic acid, salicylic acid, gentisic acid, β-resorcylic acid, gallic acid, ellagic acid, www.nature.com/scientificreports/ chlorogenic acid, syringic acid, m-coumaric acid, caffeic acid, trans-cinnamic acid, ferulic acid, p-coumaric acid, sinapic acid, naringenin, isoquercetin, rutin, kaempferol, hyperoside, catechin, apigenin, myricetin, and quercetin in selected drought-tolerant leafy vegetable amaranth. The selected leafy vegetable amaranth VA14 and VA16 exhibited remarkable phenols, vitamins, flavonoids, antioxidant constituents, and antioxidant potentiality. It revealed from the correlation study that all antioxidant compositions of selected drought-tolerant leafy vegetable amaranth exhibited high antioxidant potentiality. It revealed from the study that two selected drought-tolerant leafy vegetable amaranth showed excellent sources of antioxidants components including high ROS quenching capacity that offered huge prospects for attaining antioxidant sufficiency in the world. It revealed from this study that data reported from selected drought-tolerant leafy vegetable amaranth greatly contributed to the scientists to evaluate pharmacologically active constituents.

Methods
Experimental materials. It is the first report on phenolic profiles, antioxidant compositions, and antioxidant capacity in drought-tolerant leafy vegetable amaranth. We previously evaluated 43 genotypes for antioxidant and yield potentiality to select the best four high yielding and antioxidant enrich genotypes for this experiment.
Design and layout. We executed the experiment in three replicates following a completely randomized block design (RCBD) at Bangabandhu Sheikh Mujibur Rahman Agricultural University. Each genotype was grown in 1 m 2 experimental plot following 20 cm and 5 cm distance between rows and plants, respectively.
Intercultural practices. Recommended compost doses, fertilizer, and appropriate cultural practices were maintained 63 . For maintaining the exact spacing of plants in a row, proper thinning was executed. Weeds of experimental plots were regularly removed through proper weeding and hoeing. We provide regular irrigation in the experimental plots for maintaining the proper growth of vegetable amaranth. We collected the leaf samples at 30 days old plant.

Samples extraction for HPLC and LC-MS analysis.
The leaf samples were extracted by adding 10 ml methanol (80%) containing acetic acid (1%) in 1 g leaves. The mixture was thoroughly homogenized. Then the mixture was kept to a test tube (50 ml) and capped tightly. The test tube was shaken in a shaker (Scientific Industries Inc., USA) for 15 h at 400 rpm. Exactly 0.45 µm filter (MILLEX-HV syringe filter, Millipore Corporation, Bedford, MA, USA) was used to filter the homogenized mixture. We centrifuged the mixture at 10,000 × g for 15 min. The phenolic compounds were analyzed from the final filtrate. We performed all extractions in triplicate independent samples.
Flavonoids, and phenolic acids analysis through HPLC. The method previously described by Sarker and Oba 11,28 was followed to phenolic profile, respectively in leaf sample using HPLC. We equipped the Shimadzu SCL10Avp (Kyoto, Japan) HPLC with a binary pump (LC-10Avp), DGU-14A degasser, and a Shimadzu SPD-10Avp UV-vis detector. A CTO-10AC (STR ODS-II, 150 × 4.6 mm I.D., (Shinwa Chemical Industries, Ltd., Kyoto, Japan) column was used for the separation of flavonoids and phenolic acids 11 . The binary mobile phase was pumped with solvent A (6% (v/v) acetic acid) in water and solvent B (acetonitrile) at the flow rate of 1 ml/ min for 70 min. 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 11 . We set the detector at 360, 370, 280, and 254 nm, respectively for continuous monitoring of flavonoids, cinnamic acids, and benzoic acids. For identification of the compound, we compared retention time and UV-vis spectra with their respective standards. We confirmed the flavonoids, and phenolic acids through the mass spectrometry assay method. HPLC detected total compounds were represented as a total phenolic index (TPI). The previously described method of Sarker and oba 11,28 was used to TPI from the HPLC data. All samples were prepared and analyzed in duplicate. We estimated phenolic compounds as µg g −1 FW. A mass spectrometer (AccuTOF JMS-T100LP, JEOL Ltd., Tokyo, Japan) fitted with an Agilent 1100 Series HPLC system and a UV-vis detector coupled on-line 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 0-1,000 and needle voltage at -2,000 V. Extract constituents were identified by LC-MS-ESI analysis.
Quantification of phenolic compounds. We used the respective standards of calibration curves to quantify each phenolic compound. We dissolved 25 phenolic compounds in 80% methanol as stock solutions to the final concentration of 100 mg/ml. Respective standard curves (10,20,40,60,80, and 100 mg/ml) were used to quantify the individual phenolic compounds with external standards. UV spectral characteristics, retention times and co-chromatography of samples spiked with commercially available standards were utilized for identification and match the phenolics.
Scientific Reports | (2020) 10:18287 | https://doi.org/10.1038/s41598-020-71727-y www.nature.com/scientificreports/ Estimation of vitamin C. A Hitachi spectrophotometer (U-1800, Tokyo, Japan) was utilized to estimate ascorbic acid (AsA) and dehydroascorbic acid (DHA) from the fresh amaranth leaves. Dithiothreitol (DTT) was used for the sample pre-incubation and reduction of dehydroascorbic acid into ascorbic acid. Ascorbic acid reduced ferric ion to ferrous ion. Reduced ferrous ion forms complexes with 2, 2-dipyridyl 8 . We read the absorbance of Fe 2 + complexes with 2, 2-dipyridyl at 525 nm for estimation of vitamin C through the spectrophotometric (Hitachi, U-1800, Tokyo, Japan). We calculated vitamin C in mg 100 g −1 FW. Estimation of total polyphenols. Extraction of total polyphenols was carried out according to Sarker and Oba 64 using 25 mg of sample in 2.5 mL of 1.2 M HCl containing methanol (90%) at 90 °C for 2 h in a water bath. With readjusting the volume (2.5 mL), the leaf extract was centrifuged at 7,500 rpm for 20 min. The leaf extracts (100 µL) were added to the Folin-Ciocalteau reagent (2 N, 50 µL). After 5 min, 2 N Na 2 CO 3 (400 µL) and water (1 mL) was added. The leaf extracts were incubated for 90 min at 37 °C. Finally, it was removed to a microplate (flat bottom). In a microplate reader, the absorbance was detected at 740 nm. We estimated the results in equivalent to gallic acid (GAE) standard µg g −1 of FW.
Estimation of total flavonoids. Total flavonoids were extracted and quantified according to the method described by Sarker and Oba 65 . Samples (100 mg) were mixed with 5 mL methanol (50%) in water and placed for 1 h with ultrasound. The leaf extracts were centrifuged for 10 min at 13,000 g (4 °C). The supernatants were then recovered. Flavonoid extracts (400 µL) were homogenized with water (500 µL), 5% NaNO 2 (60 µL), 10% AlCl3 (140 µL). After 10 min, 1 mM NaOH (400 µL) was added. The leaf extracts were incubated for 10 min at a normal temperature. Finally, it was removed to a flat bottom microplate. The absorbance was read at 500 nm in a microplate reader. Results are expressed in µg of rutin equivalents (RE) per gram of sample DW.
Radical quenching capacity assay. Thirty days old amaranth leaves were harvested. Antioxidant capacity assay, the leaves were dried in the air in a shade. 40 ml aqueous methanol (90%) was utilized to extract grounded dried leaves (1 g) from each cultivar in a capped bottle (100 ml). A Thomastant T-N22S (Thomas Kagaku Co. Ltd., Japan) shaking water bath was utilized to extract leaf samples for 1 h. Exactly 0.45 µm filter (MILLEX-HV syringe filter, Millipore Corporation, Bedford, MA, USA) was used to filter the homogenized mixture. After centrifugation for 15 min at 10,000 × g, the antioxidant capacity was estimated from the filtered extract.
Diphenyl-picrylhydrazyl (DPPH) radical degradation method [66][67][68] was used to estimate the antioxidant activity. We added 1 ml DPPH solution (250 µM) to 10 µl extract (in triplicate) in a test tube. After adding 4 ml distilled water the extract was placed in the dark for 30 min. A Hitachi U1800 spectrophotometer (Hitachi, Tokyo, Japan) was used to measure the absorbance at 517 nm. Method of Sarker and Oba 28 was followed for ABTS + assay. To prepare two stock solutions separately ABTS + solution of 7.4 mM and potassium persulfate of 2.6 mM were used. We mixed both solutions in equal proportion to prepare the working solution at room temperature. The working solution was allowed to react in the dark for 12 h. One hundred fifty μl extract was added to 2.85 ml of ABTS + solution and allowed to react in the dark for 2 h. For the preparation of the solution, one ml of ABTS + solution was mixed with sixty ml of methanol. A Hitachi spectrophotometer (U1800, Tokyo, Japan) was utilized to take the absorbance against methanol at 734 nm. The inhibition (%) of DPPH and ABTS + corresponding with control was used to determine antioxidant capacity using the equation as follows: 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. Statistix 8 software was used to analyze the data for analysis of variance (ANOVA) 69,70 .
Duncan's Multiple Range Test (DMRT) at a 1% level of probability was used to compare the means. The results were reported as the mean ± SD of three separate replicates.