Nutritional and bioactive constituents and scavenging capacity of radicals in Amaranthus hypochondriacus

A. hypochondriacus leaves contained ample phytopigments including betalain, anthocyanin, β-xanthin, β-cyanin, and bioactive phytochemicals of interest in the industry of food. We have been evaluating the possibility of utilizing phytopigments of amaranth and bioactive constituents for making drinks. Therefore, we evaluated bioactive phytopigments and compounds including the potentiality of antioxidants in A. hypochondriacus leaves. A. hypochondriacus leaves have abundant protein, carbohydrates, and dietary fiber. We found considerable levels of inorganic minerals including magnesium, calcium, potassium (3.88, 3.01, 8.56 mg g−1), zinc, manganese, copper, iron (16.23, 15.51, 2.26, 20.57 µg g−1), chlorophyll b, chlorophyll ab chlorophyll a (271.08, 905.21, 636.87 μg g−1), scavenging capacity of radicals (DPPH, ABTS+) (33.46, 62.92 TEAC μg g−1 DW), total polyphenols (29.34 GAE μg g−1 FW), β-xanthin, betalain, β-cyanin (584.71, 1,121.93, 537.21 ng g−1), total flavonoids (170.97 RE μg g−1 DW), vitamin C, β-carotene, carotenoids (184.77, 82.34, 105.08 mg 100 g−1) in A. hypochondriacus leaves. The genotypes AHC6, AHC4, AHC11, AHC5, and AHC10 had a good scavenging capacity of radicals. Polyphenols, phytopigments, flavonoids, and β-carotene of A. hypochondriacus had potential antioxidant activity. Extracted juice of A. hypochondriacus can be an ample source of phytopigments and compounds for detoxification of reactive oxygen species (ROS) and attaining nutritional and antioxidant sufficiency.

. The composition of proximate (g kg −1 fresh weight) and dietary fiber (g 100 g −1 FW) of 11 A. hypochondriacus genotypes. CV coefficient of variation; n = 3; **Significant at 1% level. Genotypes Moisture (g kg −1 ) Protein (g kg −1 ) Fat (g kg −1 ) Carbohydrates (g kg −1 ) Energy (kcal) Ash (g kg −1 ) www.nature.com/scientificreports/ of 46.90 g kg −1 FW. The highest energy (58.47 kcal 100 g −1 ) was found in genotype AHC4, followed by that of AHC8, AHC1, AHC6, and AHC7, while the genotype AHC5 and AHC3 (37.26, 38.15 kcal 100 g −1 ) showed the lowest energy including an average of 45.74 kcal 100 g −1 FW. AHC4 (56.22 g kg −1 FW) exhibited the highest ash content followed by that of AHC11, AHC8, and AHC1, while AHC9, AHC5, and AT17 had the lowest ash content (36.44, 36.46, 37.28 g kg −1 FW, respectively) including a mean value of 42.85 g kg −1 FW. The remarkable variations were noticed in the content of the digestive fiber of 11 A. hypochondriacus studied. The highest dietary fiber was noticed in AHC2 and AHC6 (10.27, and 10.03 g 100 g −1 ) followed by AHC4, while AHC11 had the lowest dietary fiber contents (7.26 g 100 g −1 ) which was statistically similar to the genotype AHC8 and AHC7 with an average of 8.73 g 100 g −1 . For mitigation of palatability, food digestibility, and remedy of constipation, dietary fiber has a crucial role 9 . In this study; we observed that A. hypochondriacus has abundant protein, carbohydrates, dietary fiber, and moisture. The moisture contents observed in A. hypochondriacus leaves was greater than our previous studies of red morph amaranth 35 , weedy amaranth 36 , green morph amaranth 37 , stem amaranth 38 , while the moisture contents of A. hypochondriacus leaves were corroborated with our previous studies of A. blitum 39 . In contrast, the protein contents observed in A. hypochondriacus leaves were greater than our previous studies of red morph amaranth 35  Macro and microelements compositions. The macro and microelements composition of A. hypochondriacus is presented in Table 2. In this study, the K content ranged from 5.19 mg g −1 to 8.56 mg g −1 FW. The lowest K content was recorded in the genotypes AHC3, AHC6, AHC1, AHC9, AHC4, AHC11, AHC7, AHC2, and AHC10, while the highest K content was exhibited in the genotype AHC8 followed by that of AHC5, with a grand mean value of 5.74 mg g −1 FW. Two genotypes had higher K contents than the mean K content. The highest Ca content was noted in the genotype AHC8 (3.01 mg g −1 ), followed by that of AHC2, whereas, the lowest Ca content was reported in the genotypes AHC4 (1.89 mg g −1 ) with an average of 2.43 mg g −1 FW. Six accessions exhibited high Ca contents than the corresponding mean. The highest magnesium content was recorded in the genotype AHC8 (3.88 mg g −1 ) and the lowest magnesium content was observed in AHC4 (3.20 mg g −1 ), including an average of 3.41 mg g −1 FW. In the current investigation, Mg content had not varied considerably among the genotypes (3.20 to 3.88 mg g −1 FW). Our results showed that A. hypochondriacus genotypes (FW basis) had remarkable Ca (3.01 mg g −1 ), K (8.56 mg g −1 ), and Mg (3.88 mg g −1 ) which was more pronounced than Mg, Ca, and K content of different amaranth species including A. hypochondriacus of Jimenez-Aguiar and Grusak 40 . Jimenez-Aguir and Grusak 40 observed that different amaranth species including A. hypochondriacus had abundant Mg, Ca, and K in comparison with spider flower, kale, black nightshade, and spinach. K content of A. hypochondriacus leaves was greater than the K content of our previous studies of green morph amaranth 37 , while K content obtained from A. hypochondriacus leaves was lower than the K content of our previous studies of weedy amaranth 36 . Ca content observed in the current investigation were corroborated with green morph amaranth 37  36 .
The iron content of A tricolor had the significant and pronounced variations regarding genotypes (7.48 µg g −1 FW in AHC7 to 20.57 µg g −1 FW in AHC10). High iron content was recorded in the genotypes AHC10, AHC5, AHC8, and AHC6. On the contrary, the lowest content of iron was recorded in the genotype AHC3, AHC1, AHC9, and AHC4, including an average of 12.75 µg g −1 FW. Four accessions exhibited higher iron contents than their corresponding average value. The highest manganese content was noted in the genotype AHC8 (15.51 µg g −1 FW), while the lowest manganese content was noted in the genotype AHC10 (5.77 µg g −1 FW) with an average of 9.58 µg g −1 FW. The genotypes AHC5, AHC8, AHC3, and AHC1 had high manganese content. The genotypes differed remarkably in copper content (0.90-2.26 µg g −1 FW). The highest copper content was noted in AHC9 and AHC5 (2.26 and 2.11 µg g −1 FW) followed by AHC10. Four accessions exhibited greater Cu contents than the mean value. The significant and remarkable variations were noted in the zinc content of the studied genotypes (6.40 µg g −1 FW (AHC10) to 16.23 µg g −1 FW (AHC5). Four genotypes showed better zinc contents than the corresponding mean (9.98 µg g −1 FW). Iron and zinc content of A. hypochondriacus was greater than the cassava leaves 41 and beach pea 42 . We observed considerable Mn (15.51 µg g −1 ), Fe (20.57 µg g −1 ), Zn (16.23 µg g −1 ), and Cu (2.26 µg g −1 ) in A. hypochondriacus (fresh weight basis). Similarly, Jimenez-Aguiar and Grusak 40 noted abundant Cu, Fe, Zn, and Mn (FW basis) in various amaranth species with A. hypochondriacus. They reported higher Cu, Fe, Mn, and Zn in different amaranth species including A. hypochondriacus than spider flower, spinach, black nightshade, and kale. Mn content noticed in the current investigation was much lower than our previous studies of green morph amaranth 37 and A. spinosus weedy amaranth 36 , while it corroborated with our previous findings of A. viridis weedy amaranth 36 . Fe content observed in the current investigation were much greater than our previous studies of green morph amaranth 37 and lower than our previous studies of weedy amaranth 36 . Cu and Zn contents observed in the current investigation was corroborated with our previous studies of green morph amaranth 37 , while Cu and Zn contents obtained from A. hypochondriacus leaves were lower than our previous studies of weedy amaranth 36 . Table 3 represents the composition of phytopigments of A. hypochondriacus genotypes studied. The chlorophyll a content (131.07-636.87 μg g −1 FW) exhibited prominent variations among genotypes. The highest content of chlorophyll a (636.87 μg g −1 FW) was noted in the genotype AHC4, while the genotype AHC9 exhibited the lowest content of chlorophyll a (131.07 μg g −1 FW). The high content of chlorophyll a was noted in the genotypes AHC2 and AHC11. Six genotypes had a higher content of chlorophyll a than the mean value. Similar to chlorophyll a, chlorophyll b content also had significant and progressive variations among 11 A. hypochondriacus genotypes studied (71.27-271.08 μg g −1 FW). The highest chlorophyll b content (271.08 μg g −1 FW) was recorded in AHC7, followed by that of AHC4 and AHC11. Conversely, the lowest chlorophyll b content was noticed in the genotype AHC9 (71.27 μg g −1 FW). The content of chlorophyll ab showed significant and remarkable variations (193.98 to 905.21 μg g −1 FW). The genotypes AHC4, AHC11, AHC5, and AHC6 exhibited high chlorophyll ab content, whereas, the genotype AHC8 exhibited the lowest chlorophyll ab content (193.98 μg g −1 FW). Six accessions had greater chlorophyll ab contents than the mean value. Our studied A. hypochondriacus genotypes had considerable chlorophyll ab (905.21 μg g −1 FW), chlorophyll a (636.87 μg g −1 FW), and chlorophyll b (271.08 μg g −1 FW) contents which were greater than the content of chlorophylls of red and green amaranth of Khanam and Oba 43 . Chlorophyll a, chlorophyll ab, and chlorophyll b content in the present study were much greater than chlorophyll a, chlorophyll ab, and chlorophyll b content Table 3. Mean performance for phytopigments of 11 A. hypochondriacus genotypes. CV coefficient of variation; n = 3; **Significant at 1% level.  www.nature.com/scientificreports/ of green morph amaranth 37 , red morph amaranth 35 , stem amaranth 38 , and weedy amaranth 36 of our earlier study, while these photosynthetic pigments content of this study were lower corroborated with our earlier studies of A. blitum 39 .

Composition of phytopigments.
The highest content of β-cyanin was noted in the genotype AHC6 (537.21 ng g −1 FW) followed by that of AHC11 and AHC5, with an average value of 384.23 ng g −1 FW. On the contrary, the lowest β-cyanin content was observed in the genotype AHC9 (233.87 ng g −1 FW). The β-xanthin content had significant and considerable variations regarding genotypes which ranged from 181.90 to 584.71 ng g −1 FW. The genotype AHC6 exerted the highest β-xanthin content (584.71 ng g −1 FW) and the genotypes AHC11, AHC5, and AHC10 had high β-xanthin content. In contrast, the lowest β-xanthin content was recorded in the genotype AHC8 (181.90 ng g −1 FW). Six accessions exerted greater β-xanthin contents than the mean value. There were pronounced variations in the betalain content of the genotypes studied. The highest betalain content was observed in the genotype AHC6 (1,121.93 ng g −1 FW) and ranged from 367.42 to 1,121.93 ng g −1 FW. The high betalain content was noted in the genotype AHC11, AHC5, and AHC10. Whereas, the lowest content of betalain was recorded in the genotype AHC8 (367.42 ng g −1 FW). Seven accessions exerted greater betalain contents than the mean value. The highest carotenoid content was found in the genotype AHC10 (105.08 mg 100 g −1 ) and ranged from 32.77 mg 100 g −1 to 105.08 mg 100 g −1 . The high carotenoids were recorded in the genotype AHC2, AHC1, and AHC3. Five accessions exerted greater carotenoids than the mean value. The considerable β-xanthin (584.71 ng g −1 FW), chlorophyll a (636.87 μg g −1 FW), chlorophyll ab (905.21 μg g −1 FW), chlorophyll b (271.08 μg g −1 FW), β-cyanin (537.21 ng g −1 FW), betalain (1,121.93 ng g −1 FW), and carotenoids (105.08 mg 100 g −1 ) were recorded in A. hypochondriacus, which were fully corroborative to the findings of Khanam and Oba 43 of green and red amaranth. Betalain, betacyanin, and betaxanthin content in the current investigation were much pronounced than betalain, betacyanin, and betaxanthin content of red morph amaranth 35 , green morph amaranth 37 , stem amaranth 38 , and weedy amaranth 36 , while the content of these color pigments of this study was corroborated with A. blitum 39 of our earlier studies. Total carotenoids content in the current investigation was much greater than the total carotenoids content of green morph amaranth 37 , A. spinosus weedy amaranth 36 and total carotenoids content of this study were corroborated with A. viridis weedy amaranth 36 , while total carotenoids content in the current investigation was lower than the total carotenoids content of red morph amaranth 35 , stem amaranth 38 and A. blitum 39 of our earlier studies.
Bioactive components and radical scavenging capacity. The total polyphenols (TP), vitamin C, β-carotene, total antioxidant activity (TA), and total flavonoids (TF) contents of A. hypochondriacus are presented in Table 4. The highest content of β-carotene was recorded in the genotype AHC2 (82.34 mg 100 g −1 ), while the genotype AHC4 exhibited the lowest β-carotene content (48.33 mg 100 g −1 FW) with an average of 58.26 mg 100 g −1 FW. Five accessions had greater β-carotene contents than the mean value. The content of β-carotene was high in the genotypes AHC3, AHC5, and AHC8. The vitamin C exhibited pronounced variations regarding genotypes, which ranged from 11.97 mg 100 g −1 in the genotype AHC3 to 184.77 mg 100 g −1 in the genotype AHC11 with an average of 84.44 mg 100 g −1 FW. Six accessions had higher vitamin C contents than the grand mean value. High vitamin C content was recorded in the genotypes AHC1, AHC2, AHC9, and AHC4. The total polyphenols (TP) ranged from 13.23 GAE μg g −1 FW (AHC8) to 29.34 GAE μg g −1 FW (AHC11) with an average TP content of 24.04 GAE μg g −1 FW. High TP was noted in the genotypes AHC4, AHC2, AHC1, and AHC6. Six accessions exerted greater TP contents than the mean TP content. The pronounced variability was recorded in TF content regarding genotypes, which ranged from 106.80 RE μg g −1 DW ( AHC8) to 170.97 RE   www.nature.com/scientificreports/ μg g −1 DW) ( AHC9). The average value of TF content was 147.26 RE μg g −1 DW. The highest TF content was recorded in the genotype AHC9 with the order of AHC9 ˃ AHC4 = AHC5 ˃ AHC10 = AHC1 ˃ AHC2 = AHC6. Six accessions had higher TF content than the mean TF content. The TA (DPPH) content was high and variability was not pronounced, which ranged from 16.27 Trolox equivalent antioxidant capacity (TEAC) μg g −1 DW (AHC8) to 33.46 TEAC μg g −1 DW (AHC6). The highest TA (DPPH) content was recorded in the genotypes AHC6, AHC4, AHC11, AHC5, and AHC10 (DPPH). Conversely, the lowest TA (DPPH) content was found in the genotype AHC8 with a mean TA (DPPH) value of 30.52 TEAC μg g −1 DW. Eight accessions exerted much greater TA (DPPH) contents than the mean TA (DPPH) content. Similar to TA (DPPH), TA (ABTS + ) showed a similar trend, which validated the measurement of the quenching capacity of radicals in two methods. The TA (ABTS + ) was high and variations were not pronounced, which ranged from 28.54 TEAC μg g −1 DW (AHC8) to 62.92 TEAC μg g −1 DW (AHC6). The highest TA (ABTS + ) was noted in the genotypes AHC6, AHC4 AHC11, AHC5, and AHC10. Conversely, the lowest TA (ABTS + ) was recorded in the genotype AHC8 with an average of 56.88 TEAC mg g −1 DW. Nine genotypes exhibited much higher TA (ABTS + ) contents than the mean TA (ABTS + ) value. We observed considerable vitamin C and β-carotene (184.77 and 82.34 mg 100 g −1 ) in the accessions of A. hypochondriacus, which were greater than the findings of red amaranth of our previous studies 10 .
The TP (29.34 GAE μg g −1 FW) recorded in this investigation was much greater than the findings of green and red amaranth of Khanam et al. 44  Analysis of correlation coefficient. The correlation of TA (DPPH), β-carotene, phytopigments, vitamin C, TF, TA (ABTS + ), and TP of A. hypochondriacus are presented in Table 5. The above-mentioned correlation coefficients exhibited interesting results. All phytopigments exerted positive and significant interrelationships with TA (DPPH), TF, TA (ABTS + ), and TP. It indicated that the increase in chlorophylls, carotenoids, β-xanthin, betalain, and β-cyanin content was directly associated with the increment of TA (ABTS + ), TP, TA (DPPH), and TF. Similarly, vitamin C had positive and insignificant correlations with TA, TF, and TP, whereas it exerted negative and insignificant correlations with all phytopigments. In the earlier works of our research group in A. tricolor 9,11,12,16 ; in red morph amaranth 35 , in green morph amaranth 37 and Jiménez-Aguilar and Grusak 40 in 15 amaranth species observed the similar results which were corroborative to our present study. TA (ABTS + ), TF, TA (DPPH), TPC, and β-carotene showed a positive and significant correlation among each other. It indicated that TF, TP, and β-carotene had strong quenching capacity of radicals. Similarly, positive and significant associations versus TA (DPPH) and TA (ABTS + ) ensured the confirmation of the quenching capacity of radicals of A. hypochondriacus by two methods. All TP, phytopigments, TF, and β-carotene had strong quenching capacity of radicals as these antioxidant phytochemicals showed significant correlations with TA (DPPH) and TA (ABTS + ).
In this study, we observed that TP, phytopigments, TF, and β-carotene of A. hypochondriacus had strong quenching capacity of radicals. In conclusions, It is the first report on A. hypochondriacus leaves. We grew eleven A. hypochondriacus genotypes to evaluate nutraceuticals, bioactive components, and quenching capacity of radicals. A. hypochondriacus leaves contained ample iron, K, zinc, Ca, copper, Mg, manganese, TF, protein, TP, dietary fiber, phytopigments, β-carotene, TA, vitamin C, carbohydrates, and antioxidants. Thus we can make drinks from A. hypochondriacus containing plentiful nutraceuticals, flavonoids, β-carotene, phytopigments, phenolics, vitamin C, and antioxidants to combat hidden hunger and achieve nutritional and antioxidant sufficiency. Based on correlation coefficients, all bioactive compounds of A. hypochondriacus leaves exhibited strong antioxidant activity. Hence, A. hypochondriacus leaves with staple foods (e.g., rice, wheat, maize) could significantly contribute to reducing the occurrence of hidden hunger in the globe. The genotypes AHC6, AHC4, AHC11, AHC5, and AHC10 can be utilized for making drinks. Intercultural practices. Recommended fertilizer doses, such as triple super phosphate, murate of potash, gypsum, and urea, at the rate of 100, 150, 30, and 200 kg/ha, respectively were applied 45 . Cultural practices were maintained appropriately 45 . Compost (10 ton/ha) was applied during preparation of lands 10 . 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. Estimation of mineral composition. A. hypochondriacus 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, iron, manganese, copper, zinc, from powdered leaves following nitric-perchloric acid digestion method 22 . For this digestion, in the presence of carborundum beads 40 ml HClO 4 (70%), 400 ml HNO 3 (65%), and 10 ml H 2 SO 4 (96%) were added to 0.5 g dried leaf sample. After digestion, the ascorbic acid method was followed to measure P through dilution of the solution appropriately in triplicate. We added ascorbic acid and antimony to the yellow-colored complex solution for converting it to a blue-colored phosphomolybdenum complex. Sarker and Oba 22 method Finally, chlorophylls were calculated as micrograms per gram and carotenoids milligrams per 100 g of fresh weight, respectively.

Solvent and reagents.
Betacyanins and betaxanthins content measurement. The leaves were extracted in 80% methyl alcohol containing 50 mM ascorbate to measure betacyanins and betaxanthins according to the method of Sarker and Oba 22 . A spectrophotometer (Hitachi, U-1800, Tokyo, Japan) was used to measure the absorbance at 540 nm for betacyanins and 475 nm for betaxanthins, respectively. The data were calculated as nanograms betanin equivalent per gram of fresh weight for betacyanins and nanograms indicaxanthin equivalent per gram of fresh weight for betaxanthins.

Estimation of beta-carotene.
For the estimation of beta-carotene, we followed our previously described method 22 . Exactly 500 mg of fresh leaf sample was added with 10 ml of 80% acetone and ground thoroughly in a mortar and pestle. The extract was centrifuged at 10,000 × g for 3-4 min. The final volume was marked up to 20 ml after removing the supernatant in a volumetric flask. A Hitachi (U-1800, Tokyo, Japan) spectrophotometer at 510 nm and 480 nm, respectively was set to take the absorbance. Data were expressed as milligrams betacarotene per 100 g of fresh weight.
Estimation of ascorbic acid. A Hitachi spectrophotometer (U-1800, Tokyo, Japan) was utilized to estimate ascorbic acid (AsA) and dehydroascorbic acid (DHA) from the fresh 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 22 . 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 milligrams per 100 g of fresh weight.
Estimation of total polyphenols. Extraction of total polyphenols was carried out according to Jiménez-Aguilar and Grusak 40 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. Note that results could be slightly overestimated because these samples contained ascorbic acid 47 .
Estimation of total flavonoids. Total flavonoids were extracted and quantified according to the method described by Jiménez-Aguilar and Grusak 40 . 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.
Scientific Reports | (2020) 10:19962 | https://doi.org/10.1038/s41598-020-71714-3 www.nature.com/scientificreports/ 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: 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 replication mean was obtained by averaging the replication-wise sample data. We performed the analysis of variance (ANOVA) using Statistix 8 software. The mean separation was performed by Tukey's HSD test at a probability level of 1%. We reported the results as the mean ± SD. Ethical statement. The lab and field experiment in this study was carried out following guidelines and rec-