The method adopted for this study was developed by Blois (1958), which depends on the reduction of purple DPPH to yellow coloured diphenyl picrylhydrazine radical scavenger [29Blois MS. Antioxidant determinations by the use of a stable free radical. Nature 1958; 181: 1199-200.
[http://dx.doi.org/10.1038/1811199a0] ]. A solution of the radical was prepared by dissolving 1mg/m L DPPH in methanol. The determination of the disappearance of the free radical was done using a UV-visible spectrophotometer. Each plant extract sample stock solution (1mg/mL) was diluted in final concentration of 10, 20, 40, 80, 160, 320, 640µg/mL). Ascorbic acid was used as positive control and prepared in the same manner as an extract. As DPPH is sensitive to light, it is exposed to minimum sensitive light. These solutions were allowed to react at room temperature for 30 minutes in the dark [14Upadhyay N, Ahmad G, Agnihotri K, Sharma R. Studies on antioxidant activity and total phenolic content of Tinospora cordifolia stem using in vitro models. Am J Phytomed Clinical Therapeutics 2013; 8: 617-27.].

The absorbance values were measured at 517nm and converted into percentage antioxidant activity using the following equation:

(where Abs =Absorbance)

All the determinations were performed in triplicate.

This method depends on the reduction of sea green coloured ABTS to transparent ABTS [30Saeed N, Khan MR, Shabbir M. Antioxidant activity, total phenolic and total flavonoid contents of whole plant extracts Torilis leptophylla L. BMC Complement Altern Med 2012; 12(1): 221.
[http://dx.doi.org/10.1186/1472-6882-12-221] [PMID: 23153304] ]. ABTS + cation radical was produced by a reaction between 7mM ABTS and 2.45mM potassium persulfate stored in the dark at room temperature for overnight before use. The solution was diluted with 50% methanol. Each plant extract sample stock solution (1mg/mL) was diluted in a final concentration of 10, 20, 40, 80, 160, 320, 640µg/mL). Ascorbic acid was used as positive control and prepared in the same manner as above. Then 2mM ABTS added to different concentrations of extract and incubate in the dark for 30 min at room temperature. As ABTS is sensitive to light, it is exposed to minimum sensitive light at room temperature. The determination of the disappearance of the free radical was done using a UV-visible spectrophotometer. All the measurements were carried out at least three times [20Polu RP, Nayanbhirama U, Khan S, Rajlexmi M. Assessment of free radical Scavenging and anti-proliferative activites of Tinospora cordifolia. BMC Complement Altern Med 2017; 17: 2-12.
[http://dx.doi.org/10.1186/s12906-017-1953-3] ]. Percent inhibition of absorbance at 745nm was calculated using the formula:

(where Abs =Absorbance)

All the determinations were performed in triplicate.

The concentration of any antioxidant, which shows 50% inhibition, is called as IC₅₀ value. In the experiment, IC₅₀ values for extracts and standards were calculated from the regression plot between serial dilutions and the % inhibition.

Total phenolics were estimated by following Folin-Ciocalteu's phenol reagent (FCR) method of Upadhyay et al. with little modification [14Upadhyay N, Ahmad G, Agnihotri K, Sharma R. Studies on antioxidant activity and total phenolic content of Tinospora cordifolia stem using in vitro models. Am J Phytomed Clinical Therapeutics 2013; 8: 617-27.]. Different concentrations (0.5,1, 2, 4, 8mg/mL) of standard and test samples were prepared in methanol from the stock solution. 0.5mL of each concentration was mixed with 0.2 mL of FCR and 0.5 mL Na₂CO₃ (20%). After 30min of incubation at room temperature, absorbance was measured at 750nm against blank (methanol). The concentration of total phenolic content was calculated as mg gallic acid equivalent/g extract from the calibration curve of the standard solution of gallic acid.

Total flavonoid content was estimated by aluminium chloride (AlCl₃) method. Different concentrations (0.5, 1, 2, 4, 8mg/mL) of standard and test samples were prepared in methanol from the stock solution. 0.5ml of each concentration was mixed with 0.2 ml of AlCl₃, 0.5mL NaNo₃ (20%). The concentration of total flavonoid content was calculated as mg quercetin acid equivalent/g extract, from the calibration curve of the standard solution of quercetin acid. Then allow the solution at room temperature for 30 min. The absorbance was measured at 506nm by spectrophotometer [22Garg P, Grag R. Qualitative and quantitative of leaves and stem of Tinospora cordifolia in different solvent extract. J Drug Deliv Ther 2018; 8(5): 259-64.
[http://dx.doi.org/10.22270/jddt.v8i5-s.1967] ].

Both the stem and leaf extracts of Tinospora cordifolia (Wild.) Hook.f. & Thomson showed effective scavenging activity against the free radicals. The DPPH standard (ascorbic acid) had a maximum (96.58%) free radical scavenging activity at 640µg/mL and with the lowest IC₅₀ value of 22.38 µg/mL (Tables 1-3). The methanolic stem extract showed the highest scavenging activity (55.94%) at 640µg/mL and lowest (25.32%) at 10µg/mL (Table 1). It was found that when the concentration of the extract increased, the absorbance value gets decreased regularly. The methanolic leaf extract of T. cordifolia showed comparatively lesser activity with the highest scavenging activity (48.03%) at 640 µg/mL and the lowest (25.01%) at 10µg/mL (Table 1) as compared to the methanolic stem extract. Similar results were also observed by Upadhyay et al. [14Upadhyay N, Ahmad G, Agnihotri K, Sharma R. Studies on antioxidant activity and total phenolic content of Tinospora cordifolia stem using in vitro models. Am J Phytomed Clinical Therapeutics 2013; 8: 617-27.] in the methanolic stem extract of T. cordifolia and found that at a concentration of 10 mg/mL, DPPH free radical scavenging activity was low (44%). In contrast, Praveen et al. [31Praveen N, Thiruvengadam M, Kim HJ, Kumar JKP, Chung IM. Antioxidant activity of Tinospora cordifolia leaf extract through non-enzymatic method. J Med Plants Res 2012; 6(3): 4790-5.] reported that methanolic leaf extract showed the highest scavenging activity. Scavenging effect on DPPH radical was observed to be higher in methanolic leaf extract as compared to the methanolic extract of stem of T. cordifolia procured from the local market of Mysore [11IIaiyaraja N, Khanum F. Antioxidant potential of Tinospora cordifolia extracts and their protective effects on oxidation of biomolecules. Pharmacogn J 2011; 3(20): 56-61.
[http://dx.doi.org/10.5530/pj.2011.20.11] ]. The leaf and stem extracts of the methanolic solvent of T. cordifolia showed greater percentage inhibition with IC₅₀ values of 0.54 mg/mL and IC₅₀ 0.74 mg/mL, respectively, which was more than the present study [11IIaiyaraja N, Khanum F. Antioxidant potential of Tinospora cordifolia extracts and their protective effects on oxidation of biomolecules. Pharmacogn J 2011; 3(20): 56-61.
[http://dx.doi.org/10.5530/pj.2011.20.11] ]. In a previous study, the methanolic stem extract of T. cordifolia from the Philippines exhibited good free radical scavenging activity potential with an IC₅₀ value of 0.861 µg/µL [16Castillo AL, Ysrael MC, Cirna JCD, et al. Free radical scavenging activity of Tinospora cordifolia stem methanolic fraction. Int J Biol Pharm Allied Sci 2015; 4(10): 6241-54.].

All the concentration of the test solution more or less scavenged the free radicals. Both the stem and leaf extracts of Tinospora cordifolia showed effective scavenging activity against ABTS free radical. The ABTS standard (Ascorbic acid) had maximum free radical scavenging activity (91.55%) at 80µg/mL concentration with the least IC₅₀ value of 9.36 µg/mL as represented in Table 2. The methanolic stem extract showed the highest scavenging activity (74.21%) at 640µg/mL concentration and the lowest (4.51%) at 20µg/mL (Table 2). The methanolic leaf extract of T. cordifolia showed comparatively lesser activity with the highest scavenging activity (73.86%) at 640 µg/mL concentration and the lowest (6.33%) at 10µg/mL concentration as compared to the methanolic stem extract (Table 2). The stem extract of T. cordifolia showed better IC₅₀ value in ABTS (126.93 µg/mL) and DPPH (500.40 µg/mL) assays as compared to the leaf extract of the plant with IC₅₀ values if 379.31 µg/mL and 677.78µg/mL in ABTS and DPPH assays respectively (Table 3). With all the extracts (leaf and stem), dose-dependent radical scavenging was observed, i.e., when the concentration of the extract was increased, the free radical scavenging activity also increased. These results are in agreement with reports of Upadhyay et al. [14Upadhyay N, Ahmad G, Agnihotri K, Sharma R. Studies on antioxidant activity and total phenolic content of Tinospora cordifolia stem using in vitro models. Am J Phytomed Clinical Therapeutics 2013; 8: 617-27.]. ABTS scavenging assay of leaf and stem extracts of the methanolic solvent of T. cordifolia from Mysore showed greater inhibition as compared to the present study with the least IC₅₀ values of 95µg/mL and 107 µg/mL [11IIaiyaraja N, Khanum F. Antioxidant potential of Tinospora cordifolia extracts and their protective effects on oxidation of biomolecules. Pharmacogn J 2011; 3(20): 56-61.
[http://dx.doi.org/10.5530/pj.2011.20.11] ]. The ABTS scavenging assay of both leaf and stem extracts was the most potent method as compared to the DPPH scavenging assay with the highest scavenging activity and least IC₅₀ values.

TPC was measured by folin-ciocalteu's phenol reagent method and gallic acid was taken as standard in different concentrations (mg/mL). TPC of methanol extract of Tinospora cordifolia leaves and stem was calculated as gallic acid equivalent (GAE) (mg GAE/g), using the calibration curve, which was based on the following equation: Y= 1.5118x-0.1533 R²=0.9664 shown in Fig. (1). It was clear that the phenolic content in methanolic leaf extract (8.51 mg GAE /g) was lower as compared to the methanolic stem extract (17.44 mg GAE /g) of Tinospora cordifolia as shown in Table 4. The assay supported that there was a positive relationship between total phenolic content and antioxidant activity. The TPC of methanolic stem extract was less than the TPC in the methanolic stem extract (51.86 mg GAE /g), as reported by Upadhyay et al. [14Upadhyay N, Ahmad G, Agnihotri K, Sharma R. Studies on antioxidant activity and total phenolic content of Tinospora cordifolia stem using in vitro models. Am J Phytomed Clinical Therapeutics 2013; 8: 617-27.]. Similar results were reported by IIaiyaraja and Khanum [11IIaiyaraja N, Khanum F. Antioxidant potential of Tinospora cordifolia extracts and their protective effects on oxidation of biomolecules. Pharmacogn J 2011; 3(20): 56-61.
[http://dx.doi.org/10.5530/pj.2011.20.11] ] with total phenolic content of 33.93 mg GAE /g and 52.17 mg GAE /g in the methanolic stem and methanolic leaf extracts of T, cordifolia, respectively. The total phenolic content of methanolic leaf extract of T. crodifolia was reported to be 2.00 mg GAE /g by Shwetha et al. [18Shwetha RJ. Tahareen, Myrene RD. Antioxidant and anti-inflammatory activity of Tinospora cordifolia using in vitro models. J Chem Biol Phys Sci 2016; 6(2): 497-512.], which was less than the present findings.

Table 1
DPPH radical scavenging activity of methanolic stem and leaf extracts of Tinospora cordifolia.

Table 2
ABTS scavenging activity of methanolic stem and leaf extracts of Tinospora cordifolia

Table 3
IC₅₀ values of leaf and stem extracts against DPPH and ABTS activities in, µg/mL.

Fig. (1) Standard curve of gallic acid for total phenolic content.

Table 4
Quantitative analysis of stem and leaf extracts of Tinospora cordifolia.

Fig. (2) Standard curve of quercetin for total flavonoid content.

The TFC was measured by aluminium chloride reagent method and quercetin was taken as standard. TFC of methanol extract of Tinospora cordifolia leaves and stem extracts was calculated as Quercetin Equivalent (QE) (mg/g), using a calibration curve, which was based on the following equation: Y = 0.2216x+ 0.1955 R²=0.9808 as shown in Fig. (2). The flavonoid content of the methanolic leaf extract (20.94 mg QE/g) was lower as compared to the methanolic stem extract (23.82 mg QE/g) of T. cordifolia as shown in Table 4. The high flavonoid content was found to be positively correlated with the antioxidant activity. The TFC in the methanolic leaf extract of T. cordifolia was 0.7 mg QE/g reported by Shwetha et al. [18Shwetha RJ. Tahareen, Myrene RD. Antioxidant and anti-inflammatory activity of Tinospora cordifolia using in vitro models. J Chem Biol Phys Sci 2016; 6(2): 497-512.], which was less than the present findings. The methanolic stem extract showed higher antioxidant activity and higher phenol and flavonoid content as compared to the Methanolic leaf extract. The similar results were observed by Garg and Garg [22Garg P, Grag R. Qualitative and quantitative of leaves and stem of Tinospora cordifolia in different solvent extract. J Drug Deliv Ther 2018; 8(5): 259-64.
[http://dx.doi.org/10.22270/jddt.v8i5-s.1967] ] showing lesser flavonoid content in methanolic stem extract (2.14 mg QE/g) as compared to the methanolic leaf extract (4.53 mg QE/g).