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.

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.

For all the analytical studies, absorbance was measured using a Shimadzu UV-Visible Pharmaspec 1700 spectrophotometer.

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.

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.

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.

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:

%  RSC = [ ( A Control − A Sample ) / A Control ] × 100 %

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.

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.

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.

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.

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).

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:

%  cell survival = [ ( A t − A b ) / ( A c − A b ) ] × 100

Where A_t = absorbance of test sample; A_b= absorbance of blank (medium); A_c= absorbance of control (cells).

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).

Ten selected plants regularly prescribed in Unini system of medicine was investigated here (Table 1). 50% methanolic extracts of different parts of the were determined TPC, TFC and ASC were expressed as mg GAE/g extract, mg QEE/g extract and mg AA/g extract, respectively (Table 2).

The mean values of total phenols ranged from 62.89 ± 0.43 to 166.13 ± 0.56 mg GAE/g, flavonoids from 38.89 ± 0.52 to 172.23 ± 0.08 mg QEE/g extract and ASC from 0.14 ± 0.091 to 0.98 ± 0.218 AA/g extract. The highest TPC was observed in C. icosandra (166.13 ± 0.56 mg GAE/g extract), followed by R. damascena (142.23 ± 0.09 mg GAE/g extract) and C. scariosus (128.83 ± 0.32 mg GAE/g extract). For TFC, C. icosandra (172.23 ± 0.08 mg QEE/g extract) showed the highest content, also followed by R. damascena (151.32 ± 0.51 mg QEE/g extract) and C. scariosus (118.93 ± 0.23 mg QEE/g extract). The ASC contents were 0.98 ± 0.21, 0.82 ± 0.092 and 0.39 ± 0.017 mg AA/g extract in C. icosandra, R. damascena and C. scariosus, respectively followed by other extracts.

The free radical scavenging activities of the extracts, as measured by the ability to scavenge DPPH free radicals, were compared with quercetin; the lower the IC₅₀, the stronger the scavenging activity (Table 3). The maximum % inhibition of the following extracts was noted like 85% in C. icosandra at 12.37 ± 1.09 μg/ml, 83% in R. damascena at 17.19 ± 0.23 μg/ml and 80.52% in C. scariosus at 17.71 ± 0.71 μg/ml followed by other extracts.

The IC₅₀ values of the plant extracts were also determined for ABTS^·+ (Table 3). Significant activity was noted with C. icosandra, R. damascena and C. scariosus with 98.23% inhibition at 6.98 ± 0.07 μg/ml, 91.83% inhibition at 8.42 ± 0.13 μg/ml and 72% inhibition at 8.32 ± 0.09 μg/ml, respectively.

The ^·OH scavenging potentials manifested by the different plant extracts were also evaluated by decreased formation of the chromogen in the Fenton reaction. The ^·OH scavenging activities of the 50% methanolic extracts correlated with protection against DNA damage, as shown in Table 3. Best scavenging activity was noted with C. icosandra, R. damascena and C. scariosus which showed 67.08% inhibition at 34.54 ± 0.92 μg/ml, 69.7% inhibition at 23.48 ± 0.85 μg/ml and 67.2% inhibition at 29.33 ± 0.43 μg/ml, respectively.

In all the extracts tested, peroxynitrite-scavenging activity was concentration dependent. The scavenging activities was, 72% inhibition at 766.08 ± 12.23 μg/ml, 78% inhibition at 993.72 ± 52.34 μg/ml and 69% inhibition at 814.2 ± 37.89 μg/ml for C. icosandra, R. damascena and C. scariosus respectively. Hence these three extracts have superior activity than that of gallic acid standard. Howerver, IC₅₀ values of H. antidysenterica (880 ± 9.99 μg/ml) and G. gummifera (890 ± 52.23 μg/ml) were comparable to the standard (i.e. 820.12 ± 47.2 μg/ml) in peroxynitrite scavenging potential (Table 3).

As is evident from Table 3, the extracts of C. icosandra (73% inhibition at 45.20 ± 8.25 μg/ml), R. damascena (81% inhibition at 68.20 ± 7.23 μg/ml) and C. scariosus (78.23% inhibition at 45.23 ± 0.37 μg/ml) also caused considerable scavenging of superoxide anion in comparison to the reference compound quercetin. The IC₅₀ values for the superoxide scavenging activities of extracts and the reference standard are shown in Table 3. As evident from results, C. scariosus (28.85 ± 0.23 μg/ml) was able to quench superoxide radicals more effectively than the reference compound quercetin (41.98 ± 0.95 μg/ml).

C. icosandra showed significant nitric oxide scavenging activity than that of other plant extracts having 69% inhibition at 210.07 ± 18.27 μg/ml. However modest scavenging activity was also noted with R. damascena (73.9% at 398.84 ± 52.1 μg/ml) and C. scariosus (72.24% at 350.85 ± 12.3 μg/ml) respectively. IC₅₀ values were presented in Table 3 along with reference compound quercetin (at 18.23 ± 0.42 μg/ml).

To assess the prevention of oxidative DNA damage by the plant extracts further, the preventive effects were evaluated over Fenton-induced damage of pBluescript II SK (-) supercoiled DNA maintained in E. coli XL-1 Blue strain. Control pBS DNA showed two bands, one of open circular DNA, which was hardly visible, and one of supercoiled DNA. Combined treatment with FeSO₄ and H₂O₂ in the absence of plant extract led to the formation of open circular DNA by strand scission of the supercoiled DNA, whereas the plant extracts at different concentrations showing optimum activity prevented this strand scission. The maximum prevention of DNA damage was shown by C. scariosus at 0.13 μg/ml whereas C. icosandra showed the same activity at 0.16 μg/ml. R. damascena, A. pindrow, G. gummifera, O. mascula, A. pyrethrum, A. tenuifolius, H. antidysenterica and V. wallichii showed the same preventive activity at 0.2 μg/ml, 0.22 μg/ml, 0.53 μg/ml, 1.60 μg/ml, 1.52 μg/ml, 1.85 μg/ml, 0.1 μg/ml and 1 μg/ml, respectively. Among these plant extracts, C. scariosus, C. icosandra, R. damascena and H. antidysenterica provided the most effective prevention of DNA damage, as shown in Figure 1. Densitometric analysis confirmed the experimental data (Figure 2).

Correlation of phenolics content and antioxidant activity of three plant extracts with superior antioxidant activity was determined. Results showed that the correlation coefficients of total phenolics and flavonoid contents of C. icosandra were greater than 0.9 (R = 0.9995; R = 0.9919 respectively), the same of R. damascena R = 0.9830; R = 0.9848) and C. scariosus (R = 0.9604; R = 0.9910) was comparative as shown in Figure 3. This signified that the oxidative DNA damage preventive activity as well as antioxidant potential of these three effective plant extracts could be strictly correlated with their total phenolics and flavonoid contents.

The cytotoxic activities of the extracts of R. damascena, C. scariosus and C. icosandra at different concentrations (0.2 and 2 μg/ml) were determined against U937 cells (Table 4). These extracts showed no cytotoxicity against U937 cells in comparison to doxorubicin.