Plant materials and extraction procedure
Plants were collected from, and authenticated by, a Unani medical practitioner in Kolkata, India who regularly prescribes these materials. The different plant parts were shade-dried at room temperature (25°C) with occasional turning of the plants upside down for 5-7 days, and then ground to coarse powder with a mechanical grinder. The powdered plant materials (2 g) were extracted with 50 ml of aqueous methanol (50:50) for three consecutive days with intermittent stirring (1 h stirring at every 12 h interval) using magnetic stirrer until the extracts were light colored. The combined extracts were filtered and evaporated under reduced pressure in a rotary vacuum evaporator (Eyela NVC-2100--Rotary Evaporator, water bath temperature maintained at 40°C and 356 mm Hg, Eyela NCB-1200 Chiller unit temperature maintained at 7.5°C). The aqueous layer was lyophilized (at -45°C) and the dry powder was stored at -20°C for future use.
Reagents used
2, 2-Diphenyl-1-picrylhydrazyl (DPPH), thiobarbituric acid (TBA), Folin--Ciocalteu's phenol reagent, butylated hydroxytoluene, agarose and ethidium bromide were purchased from Sigma-Aldrich, USA. 2,2'-Azinobis-(3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS), potassium persulphate, aluminium chloride, iron (III) chloride, and iron (II) sulphate were obtained from MP Biomedicals, USA. 2-Deoxy-D-ribose and ascorbic acid were procured from Himedia Laboratories Pvt. Ltd., Mumbai, India. The QIAprep Spin Miniprep Kit was purchased from Qiagen, Germany. All other chemicals and reagents used were of analytical grade.
Analytical studies
For all the analytical studies, absorbance was measured using a Shimadzu UV-Visible Pharmaspec 1700 spectrophotometer.
Determination of total phenolic content (TPC)
The total phenolic contents of the 50% methanolic plant extracts were determined with gallic acid as a positive standard [21]. Aliquots of test samples (100 μl) were mixed with 2 ml 2% Na2CO3 and incubated at 25°C for 2 min. After incubation, 1:1 (v/v) Folin-Ciocalteu's phenol reagent was added and the contents were mixed vigorously. The mixture was allowed to stand at 25°C for 30 min and the absorbance was measured at 720 nm. The same procedure was repeated with all standard gallic acid solutions and a standard curve was obtained. The total polyphenolic contents of the extracts were expressed in terms of gallic acid equivalents (GAE) of the plant sample.
Determination of total flavonoid content (TFC)
The total flavonoid content was determined using quercetin as a positive standard and expressed in terms of quercetin equivalents (QEE) in mg/g plant sample [22]. NaNO2 (150 μl, 5% w/v) was added to tubes containing plant extracts in 2.5 ml distilled water. The contents were mixed thoroughly and allowed to stand for 5 min at ambient temperature, then 1.5 ml of 10% (w/v) AlCl3 were added and the mixture was allowed to stand for another 6 min. The solution was immediately mixed after addition of 1 ml 1 M NaOH. After 10 min, the absorbance was measured at 510 nm.
Determination of total ascorbic acid (ASC)
ASC of plant extracts were determined according to Roe and Kuether [23] after brief modification. Blanks, standards and samples were prepared in triplicate to measure ASC. Ascorbic acid (AA) standards (0-10 mM) or samples were precipitated with 10% trichloroacetic acid followed by centrifugation. In 500 μL of supernatant, 100 μL of DTC reagent (2,4-dinitrophenylhydrazine 3%, thiourea 0.4%, and copper sulfate 0.05%) prepared in 9N sulfuric acid, was mixed and incubated at 37°C for 3 h. After the addition of 750 μL of 65% (v/v) sulfuric acid, the absorbance was recorded at 520 nm. A standard curve was prepared with AA standards, and ASC was expressed as mg AA/g of plant sample.
Determination of free radical scavenging activity
The radical scavenging activities of the plant extracts in the range 0-200 μg/ml were evaluated using DPPH·. Stock solutions of plant extracts were prepared at a concentration of 10 mg/ml and a freshly-prepared DPPH solution (100 mM) was used as described previously [7].
Scavenging of ABTS·+ was assayed to assess the antioxidant capacities of the 50% methanolic plant extracts according to the method of Re et al. [24]. The ABTS stock solution was prepared by reacting ABTS (7 mM) and potassium persulphate (2.45 mM) and allowing the mixture to stand for at least 16 h to generate ABTS·+ free radicals. The working solution was prepared by diluting the stock solution with methanol such that its absorbance reached 0.7 ± 0.02 at 734 nm (A
Control). For the assays, 1 ml of ABTS·+ working solution was mixed with 10 μl extracts of different concentrations (0-100 μg/ml). Their absorbance (A
Sample) was noted at 734 nm exactly 6 min after the reaction mixture was prepared. In both assays, quercetin was used as positive control. The control reaction contained no test sample. The percentage radical scavenging activity (% RSC) was calculated using the formula:
Determination of hydroxyl radical scavenging activity
The ·OH scavenging assay was performed as standardized before [8]. The reaction mixture consisted of different concentrations (0-100 μg/ml) of plant extract, 3.6 mM deoxyribose, 0.1 mM EDTA, 0.1 mM L-ascorbic acid, 1 mM H2O2 and 0.1 mM FeCl3.6H2O, and the volume was made up to 500 μl with 25 mM phosphate buffer, pH 7.4. This mixture was incubated for 1 h at 37°C, 500 μl of 1% TBA and 500 μl of 1% TCA were added, and the mixture was heated in a boiling water-bath for 15 min and then cooled. The absorbance was measured at 532 nm. The control reaction contained no test sample, and quercetin (20 μg/ml) was used as a standard. Percentage RSC was calculated as described above.
Determination of peroxynitrite scavenging activity
Peroxynitrite was synthesized by the method of Beckman et al. 1994 [25]. Briefly, an acidic solution of 0.7 M H2O2 was mixed with an equal volume of 0.6 M potassium nitrite in an ice bath and an equal volume of ice cold 1.2 M NaOH was added. Granular MnO2 prewashed with 1.2 M NaOH was used to remove excess H2O2 and the reaction mixture was left at -20°C. The concentration of peroxynitrite generated was measured spectrophotometrically at 302 nm (ε = 1670 M-1 cm-1).
Peroxynitrite scavenging activity was measured according to Hazra et al. 2010 [26]. The reaction mixture consisted of 0.1 mM DTPA, 90 mM NaCl, 5 mM KCl, 12.5 μM Evans Blue, plant extracts at various doses ranging from 0-300 μg/ml, and 1 mM peroxynitrite adjusted to a final volume of 1 ml with 50 mM phosphate buffer (pH 7.4). The reaction mixture was incubated at 25°C for 30 min and the absorbance was measured at 611 nm. The percentage peroxynitrite scavenging activity was calculated by comparing the results of the test and blank samples; gallic acid served as the reference compound. All tests were conducted six times. The IC50 values of the extracts were calculated by regression analysis.
Determination of non-enzymatic superoxide radical scavenging activity
Superoxide radical was generated in vitro by a non-enzymatic method involving the nicotinamide adenine dinucleotide-nitro blue tetrazolium-phenazine methosulphate (NADH-NBT-PMS) system following the procedure of Nishikimi et al. [27]. NBT (150 μM in 0.02 M Tris buffer, pH 8.0) was added to 1 ml of NADH solution (50 μM of NADH in 0.02 M Tris buffer, pH 8.0) in the presence of various concentrations (0-50 μg/ml) of extracts. The reactions were initiated by adding PMS (15 μM) and the absorbance was at 560 nm was measured exactly 1 min later. Results were recorded as percentage inhibition. Quercetin at various concentrations was used as standard. All tests were performed six times.
Nitric oxide scavenging activity: concentration dependence
The scavenging activity against nitric oxide was assayed by the method of Marcocci et al. [28]. Sodium nitroprusside (0.5 ml, 5 mM in 20 mM phosphate buffer, pH 7.4, previously bubbled with argon) was added to tubes containing 0.5 ml of different plant extracts of various concentrations (0-300 μg/ml) and incubated at 25°C for 150 min. At the end of the incubation, 1 ml of Griess reagent (equal volumes of 2% w/v sulphanilamide in 5% phosphoric acid and 0.2% w/v naphthylethylenediamine dihydrochloride) was added to each sample and the absorbance was measured at 546 nm against control samples (extracts incubated with only 20 mM phosphate buffer, pH 7.4) and referred to the absorbance of standard solutions of sodium nitrite treated in the same way with Griess reagent. Results were recorded as percentage nitrite formed. Quercetin at various concentrations was used as standard.
Prevention of oxidative DNA damage
This was determined as described previously [8]. Plasmid DNA was isolated using a QIAprep Spin Miniprep Kit according to the manufacturer's instructions. Plasmid pBluescript II SK (-) (250 ng) was treated with FeSO4, H2O2 and phosphate buffer (pH 7.4) in final concentrations of 0.5 mM, 25 mM and 50 mM, respectively, and test extracts at different concentrations (0-2 μg/ml). The total reaction volume was set to 12 μl and the mixture was incubated at 37°C for 1 h. After the incubation, the extent of DNA damage and the preventive effect of the test samples were analyzed on 1% agarose gels at 70 V at room temperature. Quercetin (1 mM) was used as positive control.
Gels were scanned on a Gel documentation system (GelDoc-XR, Bio-Rad, Hercules, CA, USA). Bands were quantified using discovery series Quantity One 1-D analysis software (Bio-Rad).
In vitro cytotoxicity activity (MTT assay)
The cytotoxicity of the plant extracts against the U937 cells was determined using the MTT (thiazolyl blue tetrazolium bromide) assay adapted from Kim et al. [29]. Cells were seeded into 96-well plates at 5,000-10,000 cells/well and treated with different concentrations of the plant extracts. After 48 h, MTT was added to each well and the formazan crystals were dissolved in DMSO. The absorbance was measured at 570 nm using a microplate ELISA reader. All experiments were performed in eight replicates. Percentage cell survival was calculated using the following formula:
Where At = absorbance of test sample; Ab= absorbance of blank (medium); Ac= absorbance of control (cells).
Statistical analysis
All data were expressed as mean ± SD. Statistical analyses were performed using Microsoft Excel. The IC50 values were calculated by regression analysis. Values with p < 0.05 were considered statistically significant. The IC50 values were compared by paired t test (two-sided).