2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) was obtained from Roche Diagnostics, Mannheim, Germany. 6-hydroxy-2,5,7,8-tetramethychroman-2-carboxylic acid (Trolox) was obtained from Fluka, Buchs, Switzerland. Potassium persulfate (K2S2O8), ethylenediamine tetraacetic acid (EDTA), ascorbic acid, 2-deoxy-2-ribose, trichloroacetic acid (TCA), mannitol, nitro blue tetrazolium (NBT), reduced nicotinamide adenine dinucleotide (NADH), phenazine methosulfate (PMS), sodium nitroprusside (SNP), sulfanilamide, naphthylethylenediamine dihydrochloride (NED), L-histidine, lipoic acid, sodium pyruvate, quercetin and ferrozine were obtained from Sisco Research Laboratories Pvt. Ltd, Mumbai, India. Hydrogen peroxide, potassium hexacyanoferrate, Folin-Ciocalteu reagent, sodium carbonate (Na2CO3), butylated hydroxytoluene (BHT), sodium hypochlorite (NaOCl), aluminium chloride (AlCl3), ammonium iron (II) sulfate hexahydrate ((NH4)2Fe(SO4)26H2O), potassium nitrite (KNO2), N, N-dimethyl-4-nitrosoaniline and xylenol orange were obtained from Merck, Mumbai, India. Gallic acid and curcumin were obtained from MP Biomedicals, France. Ferrous sulfate and catalase were obtained from HiMedia Laboratories Pvt. Ltd, Mumbai, India. Evans Blue was purchased from BDH, England. Manganese dioxide was obtained from SD Fine Chemicals, Mumbai, India. Diethylene-triamine-pentaacetic acid (DTPA) was obtained from Spectrochem Pvt. Ltd, Mumbai, India. Thiobarbituric acid (TBA) was obtained from Loba Chemie, Mumbai, India. Sodium nitrite was obtained from Qualigens Fine Chemicals, Mumbai, India.
The bark of the S. pinnata plant was collected from the Bankura district of West Bengal, India and authenticated through the Central Research Institute of Ayurveda, Kolkata, India.
The stem bark of S. pinnata was dried at room temperature for 7 days, finely powdered and used for extraction. The powder (100 g) was mixed with 500 ml methanol:water (7:3) using a magnetic stirrer for 15 hours, then the mixture was centrifuged at 2850 × g and the supernatant was decanted. The pellet was mixed again with 500 ml methanol-water and the entire extraction process was repeated. The supernatants collected from the two phases were mixed in a round bottom flask and concentrated under reduced pressure in a rotary evaporator. The concentrated extract was then lyophilized. The residue was kept at -20°C for future use.
Total antioxidant activity
The ability of the test sample to scavenge ABTS.+ radical cation was compared to trolox standard . The ABTS.+ radical cation was pregenerated by mixing 7 mM ABTS stock solution with 2.45 mM potassium persulfate (final concentration) and incubating for 12–16 h in the dark at room temperature until the reaction was complete and the absorbance was stable. The absorbance of the ABTS.+ solution was equilibrated to 0.70 (± 0.02) by diluting with water at room temperature, then 1 ml was mixed with 10 μl of the test sample (0.05–10 mg/ml) and the absorbance was measured at 734 nm after 6 min. All experiments were repeated six times. The percentage inhibition of absorbance was calculated and plotted as a function of the concentration of standard and sample to determine the trolox equivalent antioxidant concentration (TEAC). To calculate the TEAC, the gradient of the plot for the sample was divided by the gradient of the plot for trolox.
Hydroxyl radical scavenging
This was assayed as described by Elizabeth and Rao  with a slight modification. The assay is based on quantification of the degradation product of 2-deoxyribose by condensation with TBA. Hydroxyl radical was generated by the Fe3+-ascorbate-EDTA-H2O2 system (the Fenton reaction). The reaction mixture contained, in a final volume of 1 ml, 2-deoxy-2-ribose (2.8 mM); KH2PO4-KOH buffer (20 mM, pH 7.4); FeCl3 (100 μM); EDTA (100 μM); H2O2 (1.0 mM); ascorbic acid (100 μM) and various concentrations (0–200 μg/ml) of the test sample or reference compound. After incubation for 1 h at 37°C, 0.5 ml of the reaction mixture was added to 1 ml 2.8% TCA, then 1 ml 1% aqueous TBA was added and the mixture was incubated at 90°C for 15 min to develop the color. After cooling, the absorbance was measured at 532 nm against an appropriate blank solution. All tests were performed six times. Mannitol, a classical OH. scavenger, was used as a positive control. Percentage inhibition was evaluated by comparing the test and blank solutions.
Superoxide radical scavenging
This activity was measured by the reduction of NBT according to a previously reported method . The non-enzymatic phenazine methosulfate-nicotinamide adenine dinucleotide (PMS/NADH) system generates superoxide radicals, which reduce nitro blue tetrazolium (NBT) to a purple formazan. The 1 ml reaction mixture contained phosphate buffer (20 mM, pH 7.4), NADH (73 μM), NBT (50 μM), PMS (15 μM) and various concentrations (0–20 μg/ml) of sample solution. After incubation for 5 min at ambient temperature, the absorbance at 562 nm was measured against an appropriate blank to determine the quantity of formazan generated. All tests were performed six times. Quercetin was used as positive control.
Nitric oxide radical scavenging
At physiological pH, nitric oxide generated from aqueous sodium nitroprusside (SNP) solution interacts with oxygen to produce nitrite ions, which may be quantified by the Griess Illosvoy reaction . The reaction mixture contained 10 mM SNP, phosphate buffered saline (pH 7.4) and various doses (0–70 μg/ml) of the test solution in a final volume of 3 ml. After incubation for 150 min at 25°C, 1 ml sulfanilamide (0.33% in 20% glacial acetic acid) was added to 0.5 ml of the incubated solution and allowed to stand for 5 min. Then 1 ml of napthylethylenediamine dihydrochloride (NED) (0.1% w/v) was added and the mixture was incubated for 30 min at 25°C. The pink chromophore generated during diazotization of nitrite ions with sulphanilamide and subsequent coupling with NED was measured spectrophotometrically at 540 nm against a blank sample. All tests were performed six times. Curcumin was used as a standard.
Hydrogen peroxide scavenging
This activity was determined according to a previously described method  with minor changes. An aliquot of 50 mM H2O2 and various concentrations (0–2 mg/ml) of samples were mixed (1:1 v/v) and incubated for 30 min at room temperature. After incubation, 90 μl of the H2O2-sample solution was mixed with 10 μl HPLC-grade methanol and 0.9 ml FOX reagent was added (prepared in advance by mixing 9 volumes of 4.4 mM BHT in HPLC-grade methanol with 1 volume of 1 mM xylenol orange and 2.56 mM ammonium ferrous sulfate in 0.25 M H2SO4). The reaction mixture was then vortexed and incubated at room temperature for 30 min. The absorbance of the ferric-xylenol orange complex was measured at 560 nm. All tests were carried out six times and sodium pyruvate was used as the reference compound .
Peroxynitrite (ONOO-) was synthesized by the method described by Beckman et al. . An acidic solution (0.6 M HCl) of 5 ml H2O2 (0.7 M) was mixed with 5 ml 0.6 M KNO2 on an ice bath for 1 s and 5 ml of ice-cold 1.2 M NaOH was added. Excess H2O2 was removed by treatment with granular MnO2 prewashed with 1.2 M NaOH and the reaction mixture was left overnight at -20°C. Peroxynitrite solution was collected from the top of the frozen mixture and the concentration was measured spectrophotometrically at 302 nm (ε = 1670 M-1 cm-1).
An Evans Blue bleaching assay was used to measure peroxynitrite scavenging activity. The assay was performed by a standard method  with a slight modification. The reaction mixture contained 50 mM phosphate buffer (pH 7.4), 0.1 mM DTPA, 90 mM NaCl, 5 mM KCl, 12.5 μM Evans Blue, various doses of plant extract (0–200 μg/ml) and 1 mM peroxynitrite in a final volume of 1 ml. After incubation at 25°C for 30 min the absorbance was measured at 611 nm. The percentage scavenging of ONOO- was calculated by comparing the results of the test and blank samples. All tests were performed six times. Gallic acid was used as the reference compound.
Singlet oxygen scavenging
The production of singlet oxygen (1O2) was determined by monitoring N, N-dimethyl-4-nitrosoaniline (RNO) bleaching, using a previously reported spectrophotometric method [17, 18]. Singlet oxygen was generated by a reaction between NaOCl and H2O2, and the bleaching of RNO was monitored at 440 nm. The reaction mixture contained 45 mM phosphate buffer (pH 7.1), 50 mM NaOCl, 50 mM H2O2, 50 mM histidine, 10 μM RNO and various concentrations (0–200 μg/ml) of sample in a final volume of 2 ml. It was incubated at 30°C for 40 min and the decrease in RNO absorbance was measured at 440 nm. The scavenging activity of sample was compared with that of lipoic acid, used as a reference compound. All tests were performed six times.
Hypochlorous acid scavenging
Hypochlorous acid (HOCl) was prepared immediately before the experiment by adjusting the pH of a 10% (v/v) solution of NaOCl to 6.2 with 0.6 M H2SO4, and the concentration of HOCl was determined by measuring the absorbance at 235 nm using the molar extinction coefficient of 100 M-1 cm-1. The assay was carried out as described by Aruoma and Halliwell  with minor changes. The scavenging activity was evaluated by measuring the decrease in absorbance of catalase at 404 nm. The reaction mixture contained, in a final volume of 1 ml, 50 mM phosphate buffer (pH 6.8), catalase (7.2 μM), HOCl (8.4 mM) and increasing concentrations (0–100 μg/ml) of plant extract. The assay mixture was incubated at 25°C for 20 min and the absorbance was measured against an appropriate blank. All tests were performed six times. Ascorbic acid, a potent HOCl scavenger, was used as a reference .
The ferrous ion chelating activity was evaluated by a standard method  with minor changes. The reaction was carried out in HEPES buffer (20 mM, pH 7.2). Briefly, various concentrations (0–120 μg/ml) of plant extract were added to 12.5 μM ferrous sulfate solution and the reaction was initiated by the addition of ferrozine (75 μM). The mixture was shaken vigorously and incubated for 20 min at room temperature, then the absorbance was measured at 562 nm. All tests were performed six times. EDTA was used as a positive control.
The Fe3+-reducing power of the extract was determined by the method of Oyaizu  with a slight modification. Different concentrations (0.0–0.4 mg/ml) of the extract (0.5 ml) were mixed with 0.5 ml phosphate buffer (0.2 M, pH 6.6) and 0.5 ml potassium hexacyanoferrate (0.1%), followed by incubation at 50°C in a water bath for 20 min. After incubation, 0.5 ml of TCA (10%) was added to terminate the reaction. The upper portion of the solution (1 ml) was mixed with 1 ml distilled water, and 0.1 ml FeCl3 solution (0.01%) was added. The reaction mixture was left for 10 min at room temperature and the absorbance was measured at 700 nm against an appropriate blank solution. All tests were performed six times. A higher absorbance of the reaction mixture indicated greater reducing power. Butylated hydroxytoluene (BHT) was used as a positive control.
Determination of total phenolic content
Total phenolic content was determined using Folin-Ciocalteu (FC) reagent according to the method of Singleton and Rossi  with a slight modification. Briefly, the plant extract (0.1 ml) was mixed with 0.75 ml of FC reagent (previously diluted 1000-fold with distilled water) and incubated for 5 min at 22°C, then 0.06% Na2CO3 solution was added. After incubation at 22°C for 90 min, the absorbance was measured at 725 nm. All tests were performed six times. The phenolic content was evaluated from a gallic acid standard curve.
Determination of total flavonoid content
The total flavonoid content was determined with aluminium chloride (AlCl3) according to a known method  using quercetin as a standard. The plant extract (0.1 ml) was added to 0.3 ml distilled water followed by NaNO2 (0.03 ml, 5%). After 5 min at 25°C, AlCl3 (0.03 ml, 10%) was added. After a further 5 min, the reaction mixture was treated with 0.2 ml 1 mM NaOH. Finally, the reaction mixture was diluted to 1 ml with water and the absorbance was measured at 510 nm. All tests were performed six times. The flavonoid content was calculated from a quercetin standard curve.
All data are given as the mean ± SD of six measurements. Statistical analysis was performed using KyPlot version 2.0 beta 15 (32 bit). The IC50 values were calculated by the formula Y = 100*A1/(X + A1), where A1 = IC50, Y = response (Y = 100% when X = 0), X = inhibitory concentration. The IC50 values were compared by paired t tests. p < 0.05 was considered significant.