All of the chemicals and reagents utilised were analytical reagent grade . Hi-media Pvt. Ltd., India, provided DPPH (2,2-diphenyl-1-picrylhydrazyl), ascorbic acid, gallic acid, aluminium chloride, catechin, sodium carbonate, and the Folin–Ciocalteu reagent. Sigma-Aldrich provided the solvents acetone, ethyl acetate, methanol, and hexane (Mumbai, India). Prior to analysis, distilled water was utilised for sample preparation, dilution, and rinsing apparatus.

In the analysis, a FTIR, a UV-VIS spectrophotometer (Shimadzu Model UV 1900), a refrigerator, a remi centrifuge, a scalpel, a grinder, a measuring cylinder, Whatman filter paper (No. 42) micropipettes, an electronic balance, a separatory funnel, and aluminium foil were employed.

The stem, leaves, and flowers of A. mexicana (Mexican poppy) were picked in the semi-arid region of Haryana and delivered to the lab within 2 hours. Before being ground into powder, the samples were shade dried. An electric grinder was used to finely ground the dried stem, leaves, and flower components. After that, the powdered form was kept in airtight containers for assay of total phenols, flavonoids, and antioxidant activity. The Soxhlet extraction device was used to prepare the extracts 17. Then, for sequential extraction, hexane, ethyl acetate, acetone, methanol, and distilled water were utilised in that order (from non-polar to polar). 5g of powdered stem, leaf, and flower samples were placed in Whatman No. 1 thimble filter paper in a 250 mL round bottom flask of a standard soxhlet setup to prepare these extracts. 150 mL of the solvents (distilled water, methanol, acetone, ethyl acetate, and hexane) were added up to one and a half syphons. The process was kept running for 5-6 hours using a syphon mechanism after completing 7-8 cycles with methanol, acetone, ethyl acetate, and hexane as solvents. However, extracting distilled water by the syphon mechanism takes longer, taking more time to complete the 7-8 cycles because each cycle takes longer. The volume of each filtered solvent was measured after extraction. All extracts were kept at 4°C in the refrigerator for further used to estimate total phenols, total flavonoids and antioxidant activity.

The total phenolic content of extracts was determined using the Folin-Ciocalteu method 21. After diluting each extract with the appropriate solvent, 1.0 mL of 1mol/L Folin-Ciocalteu reagent was applied. Following that, 2.0 mL of Na2CO3 (20% w/v) was added. The solution was agitated on a stirrer, and water was added to bring the total volume to 10.0 mL. After 8 minutes, the mixture was centrifuged at 6000 rpm for 10 minutes. A UV-VIS spectrophotometer (Model UV 1900, Shimadzu) was used to compare the absorbance of the supernatant solution to a blank made in the same fashion but without the extracts at 730 nm. A calibration curve was built using gallic acid as a standard. The results were expressed as mg GAE/g on a dry weight basis.

The total flavonoids content of extracts was determined using the colorimetric assay 18. In test tubes containing 1.0 mL of each extract, 4.0 mL of double distilled water and 0.3 mL of NaNO2 (5 percent, w/v) were added. After 5 minutes, 0.3 mL (10% w/v) AlCl3 was added. The entire volume was brought up to 10.0 mL with distilled water after immediately adding 2.0 mL 1M NaOH. The solution was thoroughly mixed, and the absorbance was measured using a UV-VIS spectrophotometer (Model UV 1900, Shimadzu) at 510 nm against a blank prepared similarly but without extracts. A calibration curve was built using catechin as a standard. The data were expressed as mg CE/g on a dry weight basis.

The antioxidant activity of the extracts was assessed using the 2,2'-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity assay 9. The weight of dry mass of A. mexicana leaves, stems, and flowers was calculated using acetone, methanol, aqueous, ethyl acetate, and hexane extracts. To make the stock solution (5000 g/mL), the dry mass of all the different extracts was redissolved in a suitable volume of methanol. The degree of discolouration indicates an antioxidant's scavenging potential in terms of hydrogen donating ability 5. Using appropriate solvent dilutions, various concentrations were obtained from stock solution (i.e. methanol for acetone, methanol, ethyl acetate & hexane extracts, and with methanol: water for water extracts). 3.0 mL of 2,2'-diphenyl-1-picrylhydrazyl radical (DPPH; 0.1 mM in 100% methanol) was added to 0.2 mL of extracts (different concentrations) and rapidly agitated on a stirrer for 5 minutes to test antioxidant activity. A DPPH stock solution was produced in 50 percent (v/v) methanol: water for antioxidant activity in water extracts (different concentrations), and the rest of the technique was the same. Instead of extract, 0.2 mL of each solvent was used as a control. The absorbance of the sample and control was measured at 517 nm using a UV-VIS spectrophotometer (Model UV 1900, Shimadzu) against a blank containing the matching solvent after 30 minutes of incubation in the dark at room temperature. The percent DPPH free radical scavenging activity (y-axis) was plotted versus extract concentration (x-axis) to generate a graph. A quadratic regression equation (y = ax2+bx+c) was then generated using Microsoft Excel. On inserting y = 50% to the equation y = ax2+bx+c, it was changed to the form ax2+bx+c = 0. The following formula was used to derive the IC50 from the equation ax2+bx+c = 0:

Where, x = IC50 (µg/mL)

Calculation- The percentage of DPPH scavenged (% DPPH*sc) was estimated using the following formula:

Where,

A control is the absorbance of the control,

A sample is the absorbance of the sample.

The total phenolic content of aerial parts of A. mexicana varied greatly, as indicated in Table 1. Flowers had the highest phenolic content, with 35.75 mg GAE/g, compared to 26.59 mg GAE/g in the stem and 21.44 mg GAE/g in the leaves. When compared to solvent extracts (aqueous (32.16 mg GAE/g), methanol (31.08 mg GAE/g), acetone (26.46 mg GAE/g), ethyl acetate (26.02 mg GAE/g), and hexane (23.89 mg GAE/g) ) stem, leaves, and flowers of A. mexicana revealed the greatest phenolic content, i.e. 32.16 mg GAE/g). Our findings are consistent with Khan et al. (2019), who found 28.5± 1.15 mg GAE/g of plant extract in stems, 22.23 ±0.61 mg GAE/g in flower, and 20.89 ±0.89 mg GAE/g in leaves of A. Mexicana. Chang et al. (2002), found 106.65 mg GAE/g phenolic content in an ethyl acetate extract of A. mexicana leaves and 70.19 mg GAE/g in a methanol extract.The results vary due to the numerous methods of extract preparation, which include utilising a Soxhlet extraction device and then adding 7 percent H2SO4 and ether then with help of a separatory funnel the ether layers were separated and evaporated at room temperature after being filtered.

The total flavonoids content of various portions of A. mexicana varied greatly, as indicated in Table 2. Flowers (31.20 mg CE/g) had the maximum flavonoid concentration followed by leaves (29.58 mg CE/g) and stems (28.40 mg CE/g). When comparing among solvent extracts of various polarity, it was discovered that methanol extract (36.98 mg CE/g) had the maximum flavonoid concentration followed by aqueous (34.44 mg CE/g), ethyl acetate (31.76 mg CE/g), hexane (31.20 mg CE/g) and acetone (24.19 mg CE/g). Goswami et al., 2014 reported that flowers had the highest flavonoid concentration in A. mexicana methanolic extract (41.76 0.74 mg QE/g), followed by leaf (30.59 1.27 mg QE/g), and stem (22.83 0.83 mg QE/g). Variation in the result due to modified method used by researcher and quercetin was used as the standard flavonoid. Datkhile et al., 2020 estimated 32.5mg QE/g flavonoid content in leaves, 6.25mg QE/g in the stem, and 34.50mg QE/g in flowers.

Other researchers calculated the IC50 value of different portions of A. mexicana extracts. Datkhile et al. (2020), calculated the IC50 value of the whole plant to be 68.76g/ml in aqueous extract, 50.66g/ml in methanol extract, and 39.39g/ml in ethanol extract. A. mexicana Linn. leaf ethanol extract demonstrated significant free radical scavenging action 6.

DPPH free radical scavenging activity (%) of methanol, acetone, aqueous, ethyl acetate, and hexane extracts of leaves of A. mexicana

Different leaf extracts displayed a wide range of DPPH free radical scavenging activity, which increased with increasing concentration levels. Methanol extract scavenging activity against DPPH free radicals ranged from 88.7% to 29.4%. It ranged from 86.2 percent to 11.1 percent in the case of acetone extract. It ranged from 80.3 percent to 33 percent in the case of aqueous. Similarly, DPPH activity ranged from 75.6 percent to 19.8 percent in the case of ethyl acetate extract, and from 99.5 percent to 33.6 percent in the case of hexane extract. The IC50 value of the ethyl acetate extract was 68.91g/ml, followed by 62.29g/ml for the acetone extract, 45.95g/ml for the methanol extract, 37.28g/ml for the aqueous extract, and 36.29g/ml for the hexane extract as in Table 3.

DPPH free radical scavenging activity (%) of methanol, acetone, aqueous, ethyl acetate, and hexane extracts of stem of A.mexicana

Different stem extracts displayed a wide range of DPPH free radical scavenging activity, which increased with increasing concentration levels. Methanol extract's DPPH free radical scavenging activity ranged from 87.9% to 21.5 percent. It ranged from 99.3 percent to 46.7 percent in the case of acetone extract and from 71.6 percent to 23.7 percent in the case of aqueous extract. Similarly, it ranged from 78.4 percent to 26.6 percent in the case of ethyl acetate, and from 97.7 percent to 27.1 percent in the case of hexane extract. The IC50 value of the aqueous extract was 58.88 g/ml, followed by 48.70 g/ml for methanol, 48.02 g/ml for ethyl acetate, 41.81 g/ml for hexane, and 24.98 g/ml for acetone, showing that the aqueous extract has the highest activity, as shown in Table 4.

DPPH free radical scavenging activity (%) of methanol, acetone, aqueous, ethyl acetate, and hexane extracts of flowers of A. mexicana

Different flower extracts displayed a wide range of DPPH free radical scavenging activity, which increased with increasing concentration levels. Methanol extract's DPPH free radical scavenging activity ranged from 82.5 to 21.5 percent. It ranged from 99.2 percent to 36.4 percent in the case of acetone extract and from 78 percent to 21.2 percent in the case of aqueous extract. Similarly, ethyl acetate extract ranged from 92.4 percent to 28.3 percent, and hexane extract ranged from 79.8% to 28.7 percent. The methanol extract had the highest activity, with an IC50 value of 55.18g/ml, followed by 45.74g/ml for the hexane extract, 42.37g/ml for the ethyl acetate extract, 41.92g/ml for the aqueous extract, and 38.33g/ml for the acetone extract, as shown in Table 5.

As shown in Figure 1, FTIR bands associated to A. mexicana's aerial parts revealed the presence of phenols and flavanoids. FTIR analysis showed bands at 3421 cm-1 ,3290 cm-1, 3234 cm-1,3146 cm-1, probably related to -NH and bonded -OH groups of carboxylic acids. The band at 1727cm-1, 1699 cm-1 could be related to C=C stretching vibration of aromatic rings and to the vibration of N-H of amines, C=O of amides and carboxylic groups , in addition, this band could be related to flavonoids and amino acids. The band at 1341 cm-1 could be related to C-O stretching of acid groups or to bending vibrations of -CH3 or -CH2 groups.