Plant collection
Leaves, flowers, stem and root barks of T. pallida were collected from Rajshahi University Campus, Rajshahi, Bangladesh on 11th May, 2013 and were identified by an expert taxonomist at the Department of Botany, University of Rajshahi, where a voucher specimen (Voucher No. MN-03) was deposited. Plant materials were then washed separately with fresh water to remove dirt and other contaminants, and were shade-dried for several days with occasional sun drying. The dried materials were ground into coarse powder by a grinding machine and the materials were stored at room temperature (RT) for future use.
Preparation of the extract
The extraction was performed according to Alam et al. [18]. About 500 g of each powdered plant materials was taken in four amber colored extraction bottles and soaked with 1.5 L of methanol. The sealed bottles were kept for 15 days with occasional shaking and stirring. The extracts were filtered separately through a fresh cotton plug and finally with Whatman No.1 filter papers. The filtrates were concentrated with a rotary evaporator (Bibby Sterlin Ltd, UK) under reduced pressure at 50 °C to afford 30, 35, 45 and 40 g extract of leaves, flowers, stem and root barks extract, respectively.
Chemicals
1,1-diphenyl-2-picrylhydrazyl (DPPH), potassium ferricyanide, catechin (CA), ferrous ammonium sulphate, butylated hydroxytoluene (BHT), gallic acid (GA), ascorbic acid (AA), AlCl3, trichloro acetic acid (TCA), sodium phosphate, sodium nitrate, ammonium molybdate, sodium hydroxide, EDTA and FeCl3 were purchased from Sigma Chemical Co. (St. Louis, MO, USA); potassium chloride, potassium acetate, phosphate buffer, 2-deoxy-d-ribose, thiobarbituric acid (TBA), HCl, H2SO4, H2O2 were purchased from Sigma-Aldrich, folin-ciocalteus’s phenol reagent and sodium carbonate were obtained from Merck (Dam-stadt, Germany).
Determination of total phenolics
Total phenolic contents in the extracts were determined by the modified Folin-Ciocalteu method described by Wolfe et al. [19]. An aliquot of the extract was mixed with 2 mL Folin-Ciocalteu reagent (previously diluted with water 1:10 v/v) and 2 mL (75 g/L) of sodium carbonate. The tubes were vortexed for 15 s and allowed to stand for 20 min at 25 °C for color development. Absorbance was then measured at 760 nm UV-spectrophotometer (Shimadzu, USA). Samples of extract were evaluated at a final concentration of 0.1 and 0.15 mg/mL. Total phenolic contents were expressed in terms of gallic acid equivalent, GAE (standard curve equation: y = 0.091× + 0.167, R2 = 0.994), mg of GA/g of dry extract. The experiment was repeated three times at each concentration.
Determination of total flavonoids
Total flavonoids were estimated using aluminum chloride colorimetric assay described by Zhisen et al. [20]. To 0.5 mL of samples/standard, 150 µL of 5 % sodium nitrate and 2.5 mL of distilled water were added. After 5 min, 0.3 mL of 10 % AlCl3 was added. At 6 min, 1 mL of 0.001 M NaOH and 0.55 mL distilled water was added to the mixture and left at RT for 15 min. Absorbance of the mixtures was measured at 510 nm. Samples of extract were evaluated at a final concentration of 0.1 and 0.15 mg/mL. Total flavonoid contents were expressed in terms of catechin equivalent, CAE (standard curve equation: y = 0.000× + 0.001, R2 = 0.998), mg of CA/g of dry extract. The experiment was repeated three times at each concentration.
Determination of total antioxidant capacity
Total antioxidant capacity (TAC) of samples was determined by the method reported by Prieto et al. [21]. The assay is based on the reduction of Mo(VI) to Mo(V) by samples and formation of green colored phosphate/Mo(V) complex at acidic pH. 0.5 mL of samples/standard at different concentrations (12.5–150 μg/mL) was mixed with 3 mL of reaction mixture containing 0.6 M sulphuric acid, 28 mM sodium phosphate and 1 % ammonium molybdate into the test tubes. The test tubes were incubated at 95 °C for 10 min to complete the reaction. After cooling at RT, sample absorbance was measured at 695 nm using a spectrophotometer against a blank solution. CA was used as standard. A typical blank solution contained 3 mL of reaction mixture and the appropriate volume of the same solvent used for the samples/standard. The blank was incubated at 95 °C for 10 min and the absorbance was measured at 695 nm. Increased absorbance of the reaction mixture indicates increased total antioxidant capacity. We used standard/samples at five different concentrations ranges from 12.5 to 150 μg/mL for each antioxidant assay. Concentrations were selected on the basis of trial and error to fit the range of concentration that can fully represent the rational change of antioxidant activity with the increasing concentration of the samples. Also, we assumed that such range of concentrations is useful for smooth calculation of IC50. The experiment was repeated three times at each concentration.
Ferrous reducing antioxidant capacity assay
The ferrous reducing antioxidant capacity (FRAC) of samples was evaluated by the method of Oyaizu [22]. The Fe2+ can be monitored by measuring the formation of Perl’s Prussian blue at 700 nm. 0.25 mL samples/standard solution at different concentration (12.5–150 μg/mL), 0.625 mL of potassium buffer (0.2 M) and 0.625 mL of 1 % potassium ferricyanide, [K3Fe (CN)6] solution were added into the test tubes. The reaction mixtures were incubated for 20 min at 50 °C to complete the reaction. Then 0.625 mL of 10 % trichloro acetic acid (TCA) solution was added into the test tubes. The total mixture was centrifuged at 3000 rpm for 10 min, after which 1.8 mL supernatant was withdrawn from the test tubes and mixed with 1.8 mL of distilled water and 0.36 mL of 0.1 % ferric chloride (FeCl3) solution. The absorbance of the solution was measured at 700 nm using a spectrophotometer against blank. A typical blank solution contained the same solution mixture without plant extracts/standard and was incubated under the identical conditions. The absorbance of the blank solution was measured at 700 nm. Increased absorbance of the reaction mixture indicates increased reducing capacity. The experiment was repeated three times at each concentration.
DPPH radical scavenging assay
Free radical scavenging ability of the extracts was tested by DPPH radical scavenging assay as described by Blois [23] and Desmarchelier et al. [24]. The hydrogen atom donating ability of the plant extractives was determined by the decolorization of methanol solution of 2,2-diphenyl-1-picrylhydrazyl (DPPH). DPPH produces violet/purple color in methanol solution and fades to shades of yellow color in the presence of antioxidants. A solution of 0.1 mM DPPH in methanol was prepared, and 2.4 mL of this solution was mixed with 1.6 mL of extract in methanol at different concentrations (12.5–150 μg/mL). The reaction mixture was vortexed thoroughly and left in the dark at RT for 30 min. The absorbance of the mixture was measured spectrophotometrically at 517 nm. BHT was used as reference. Percentage DPPH radical scavenging activity was calculated by the following equation:
$$\% {\text{ DPPH radical scavenging activity}} = \left\{ {\left( {{\text{A}}_{0} {-}{\text{ A}}_{ 1} } \right)/{\text{A}}_{0} } \right\} \times 100$$
where A0 is the absorbance of the control, and A1 is the absorbance of the extractives/standard. Then % of inhibition was plotted against concentration, and from the graph IC50 was calculated. The experiment was repeated three times at each concentration.
Hydroxyl radical scavenging activity
Hydroxyl radical scavenging activity of the extractives was determined by the method of Halliwell et al. [25]. Hydroxyl radical was generated by the Fe3+-ascorbate-EDTA-H2O2 system (Fenton reaction). The assay is based on the quantification of the 2-deoxy-d-ribose degradation product, which forms a pink chromogen upon heating with TBA at low pH. The reaction mixture contained 0.8 mL of phosphate buffer solution (50 mmol L−1, pH 7.4), 0.2 mL of extractives/standard at different concentration (12.5–150 μg/mL), 0.2 mL of EDTA (1.04 mmol L−1), 0.2 mL of FeCl3 (1 mmol L−1) and 0.2 mL of 2-deoxy-d-ribose (28 mmol L−1). The mixtures were kept in a water bath at 37 °C and the reaction was started by adding 0.2 mL of ascorbic acid, AA (2 mmol L−1) and 0.2 mL of H2O2 (10 mmol L−1). After incubation at 37 °C for 1 h, 1.5 mL of cold thiobarbituric acid, TBA (10 g L−1) was added to the reaction mixture followed by 1.5 mL of HCl (25 %). The mixture was heated at 100 °C for 15 min and then cooled down with water. The absorbance of solution was measured at 532 nm with a spectrophotometer. The hydroxyl radical scavenging capacity was evaluated with the inhibition of percentage of 2-deoxy-d-ribose oxidation on hydroxyl radicals. The percentage of hydroxyl radical scavenging activity was calculated according to the following formula:
$$\% {\text{ hydroxyl radical scavenging activity}} = [{\text{A}}_{0} {-} \, \left( {{\text{A}}_{ 1} {-}{\text{A}}_{ 2} } \right] \times 100/{\text{A}}_{0}$$
where A0 is the absorbance of the control without a sample. A1 is the absorbance after adding the sample and 2-deoxy-D-ribose. A2 is the absorbance of the sample without 2-deoxy-d-ribose. Then % of inhibition was plotted against concentration, and from the graph IC50 was calculated. The experiment was repeated three times at each concentration.
Lipid peroxidation inhibition assay
The lipid peroxidation inhibition assay was determined according to the method described by Haenen and Bast [26]. Protocol used in this study for the use of rat as animal model for lipid peroxidation inhibition assay was approved by the Institutional Animal, Medical Ethics, Biosafety and Biosecurity Committee (IAMEBBC) for Experimentations on Animal, Human, Microbes and Living Natural Sources (225/320-IAMEBBC/IBSc), Institute of Biological Sciences, University of Rajshahi, Bangladesh. Excised rat liver was homogenized in buffer and then centrifuged to obtain liposome. To make 10 % liver homogenate, excised Wister rat liver (weight of ~150 g) was minced using glass Teflon homogenizer in ice cold phosphate buffered saline (50 mm, pH 7.4). The homogenate was centrifuged at 12,000 rpm for 15 min at 4 °C. The supernatant was used as liposome for in vitro lipid peroxidation assay. The process of homogenization and filtration was carried out in ice cold condition. Firstly, 0.5 mL of supernatant, 1 mL of 0.15 M KCl, and 0.3 mL of extractives or standard at different concentrations (12.5–150 μg/mL) were mixed. Peroxidation was initiated by the addition of 300 μL of 0.5 mM FeCl3. The mixture was incubated at 37 °C for 30 min and the reaction was stopped by adding 2 mL of ice-cold TBA-TCA-HCl-BHT solution. The TBA-TCA-HCl solution was prepared by dissolving 1.68 mg TCA and 41.60 mg TBA in 10 ml of 0.125 M HCl. 1 mL BHT solution (1.5 mg/mL ethanol) was added to 10 mL TBA-TCA HCl solution. The reaction mixture was heated for 60 min at 90 °C and then cooled on ice and centrifuged at 3000 rpm for 5 min. The supernatants were removed and the intensity of the pink colored complex was measured using a spectrophotometer at 532 nm. The degree of lipid peroxidation was assayed by estimating the TBARS (TBA-reactive substances) content. A control experiment was performed in the presence of distilled water without the extract. The percentage of lipid peroxidation inhibition in the samples was calculated using the following formula:
$$\% {\text{ lipid peroxidation inhibition}} = \left[ {\left( {{\text{A}}_{0} {-}{\text{A}}_{ 1} } \right)/{\text{A}}_{0} } \right] \times 100$$
where A0 is the absorbance of the control (300 μl of distilled water), and A1 is the absorbance of extractives/standard. Then % of inhibition was plotted against concentration, and from the graph IC50 was calculated. The experiment was performed in triplicate and repeated three times at each concentration.
Statistical analysis
All tests were carried out in triplicates. Data were presented as mean ± SD. To evaluate significant relationships between experimental parameters by correlation and regression analysis, the F- and t-tests (p-value <0.001) were used. Free R-software version 2.15.1 (http://www.r-project.org/) and Microsoft Excel 2007 (Roselle, IL, USA) were used for the statistical and graphical evaluations.