The stem of E. trigona was collected from local areas of Sirsa, Haryana, India. It was washed thoroughly with tap water and then with distilled water to removes any debris or dust particles and allowed to dry in shade. The dried plant material was ground into fine powder using pulverizer and stored in airtight containers for further use.

Ten grams of dried powder was suspended in 100 ml of respective solvent (water [AqE], acetone [AE] and methanol [ME]) and kept on a rotary shaker for 24 h. The extract was filtered through eight layers of muslin cloth and centrifuged at 5000 g for 15 min. Supernatants were collected and the extracts were concentrated to make the final volume one-fourth of the original volume using rotary vacuum evaporator under reduced pressure at 40℃.

Folin-ciocalteu reagent and organic solvents used for extraction were of analytical grade and purchased from Fischer Scientific. 2,2-Diphenyl-1-picrylhydrazyl (DPPH), 2-thibarbituric acid (2-TBA), deoxyribose, ferrozine, potassium ferricyanide, ferric chloride, EDTA, hydrogen peroxide, L-ascorbic acid, sodium hydroxide, trichloroacetic acid, sodium carbonate, phoaphate buffer, butylated hydroxyanisole (BHA), ferrous chloride and butylated hydroxytoluene (BHT) were from Hi-media Pvt. Ltd., Mumbai.

Total phenolic contents (TPC) of the extracts were determined using Folin-Ciocalteu (FC) reagent following Yu et al. (2002) with slight modifications. Briefly, 100 μl of extracts were mixed with 0.5 ml of FC reagent in a 10 ml volumetric flask. Then 1.5 ml aqueous solution (20%, w/v) of sodium carbonate anhydrous was added, vertexed and incubated for 15 min at room temperature. The flask was filled with distilled water to volume. The absorbance was read at 765 nm, subtracting the value of a control solution consisting of distilled water instead of extracts. The amount of TPC was calculated as gallic acid equivalents (GAE) from the standard calibration curve of gallic acid and expressed as mg gallic acid equivalents per gram of sample.

The extracts were measured for hydrogen donating or radical scavenging ability using the stable DPPH radical according to the modified method of Yen and Chen (1995). The reaction mixture was prepared containing 300 μl of extract of varying concentration (50 - 300 μl/ml) and 2 ml of 100 μM DPPH (4 mg DPPH in 100 ml methanol). The absorbance of mixture was measured at 517 nm against the blank (containing solvent instead of extract) in a spectrophotometer after 10 min of incubation at room temperature. L-ascorbic acid was used as control. The percent DPPH inhibition was calculated by the equation:

% inhibition= A0 - A1 / A0 × 100

Where A0 was the absorbance of control and A1 was the absorbance of reaction mixture.

The site-specific deoxyribose assay was performed following Arouma et al. (1987) and Halliwell et al. (1987) with certain modifications. Briefly, the extracts (from 1 - 100 μg/ml) were mixed with a Haber-Weiss reaction buffer [10mM FeCl₃, 1mM phosphate buffer (pH 7.4), 10 mM H₂O₂, 10 mM deoxyribose, and 1mM L-ascorbic acid] and the final volume of all mixtures was made to 1.0 ml. The mixture was then incubated at 37℃ for 1h and heated at 80℃ for 30 min. with 1 ml of 2-TBA (0.5% 2-TBA in 0.025 M NaOH, 0.02% BHA) and 1ml of 10% trichloroacetic acid (TCA) in water bath for 45 min. After cooling, absorbance of the mixture was measured at 532 nm in a spectrophotometer. The percentage inhibition was calculated employing the formula as given for DPPH scavenging assay and was correlated with total phenolic content.

Reducing power of the extracts was determined following the method of Oyaizu (1986). Briefly, to varying concentrations of extract (1 ml) were added 2.5 ml phosphate buffer (0.2M, pH 6.6) and 2.5 ml potassium ferricyanide (1%). The reaction mixture was incubated at 50℃ for 20 min. Aliquots (2.5 ml) of trichloroacetic acid (10%) were added to the mixture and centrifuged at 9500 rpm for 10 min. The upper layer of solution (2.5 ml) was recovered and mixed with distilled water (2.5 ml) and 2.5 ml FeCl₃ (0.1%) and the absorbance was recorded at 700 nm in a spectrophotometer. Increased absorbance of the reaction mixture indicated increasing reducing power of the extracts.

The chelating effect on ferrous ions was determined according to the method of Dinis et al. (1994). The extracts (0.25 ml) were mixed with 1.75 ml of methanol and 0.25 ml of 250 mM FeCl₂. This was followed by the addition of 0.25 ml of 2 mM ferrozine, which was left to react at room temperature for 10 min. before determining the absorbance of the mixture at 562 nm. The chelating effect (%) was calculated from the formula as given for DPPH scavenging assay.

The mean values and the standard deviations were calculated from the data obtained from three independent experiments. Statistical differences at p < 0.05 were considered to be significant coefficient of determination (R²) to determine the relationship between two variables and were calculated using Microcal Origin 5.0 and Microsoft Excel. One way analysis of variance (ANOVA) with Tukey’s test was used to determine the significant difference (p < 0.05) between the means.

Phenolic compounds are aromatic secondary metabolites that have been extensively investigated in medicinal plants, fruits and vegetables (Djeridane et al., 2006). Phenolics are associated with colour, sensory qualities, nutritional and antioxidant properties (Robbins, 2003). The total phenolic contents of different extracts of E. trigona stem were evaluated using Folin-Ciocalteu method (Table 1). From the investigation, it was observed that TPC of various extract varied from 26.3 to 168.6 mg GAE/g of extracts. Methanolic extracts yielded highest amount of TPC followed by acetone and water extracts that may be responsible for imitation of high antioxidant activity.

DPPH is a stable free radical and is widely used to assess the antioxidant activity of natural extracts or pure compounds to act as free radical scavenger or hydrogen donors. In the present study, all the extracts depicted significantly higher tendency to scavenge the DPPH radicals in a dose dependent manner and steadily increased with increase in extract concentrations (Fig. 1). ME extracts showed strongest antiradical activity (83.25% at 300 μg/ml) which was even higher than the standard (L - ascorbic acid 81.30% at 300 μg/ml). Among the other extracts, AE also showed notably higher quenching ability of the DPPH radical, however AqE were less effective scavenger of the DPPH radical (Fig. 1). A good correlation (R²) between DPPH scavenging activity and TPC was obtained (Table 2) which indicates that phenolic compounds are directly responsible for antioxidant activity.

The results obtained in deoxyribose degradation assay to prevent 2-deoxy-D-ribose oxidation mediated by OHㆍ radicals are shown in Fig. 2. The reduction ability of all tested extracts increased dependently with increasing concentrations in the site-specific assay. In general, it was observed that all the extracts displayed a protective effect depending on the concentration of the extract. Methanol extracts of E. trigona showed significantly higher (p < 0.01) activity (95.55% at 300 μg/ml) compared to control (L - ascorbic acid 74.29% at 300 μg/ml). However, the ability of AE and AqE to bind to Fe in site-specific assay was lesser as compared to control (Fig. 2). The antioxidant potential of all the extracts as displayed by deoxyribose degradation assay was found to be highly correlated with their total phenolic content and the correlation coefficient (R²) varied from 0.833 – 0.981 (Table 2).

Fig. 3 depicts the results of relative reducing power of all the extracts of E. trigona and L - ascorbic acid (standard antioxidant compound). It is clear from the results that ability of AE and AqE was comparable with standard (L – ascorbic acid) in reducing Fe³⁺ to Fe²⁺ and there was no significant difference of activity. The activities of water extracts (AqE) were inferior (0.391 at 300 μg/ml) to L - ascorbic acid (0.647 at 300 μg/ml) and were significantly (p < 0.01) lower. It was suggested that AqE were less effective reducing agent, however, the activity was found to be concentration dependent and was increased with increasing concentrations of extracts and standard antioxidant (Fig. 3). Further, the activity of all extracts correlated (R²) very well with the total phenolic contents of all the extracts (Table 2).

The ferrous state of iron can stimulate lipid peroxidation by the Fenton reaction and is the most powerful pro-oxidant among various species of metal ions. The results of chelating ability of various extracts of E. trigona towards ferrous ion are presented in Fig. 4. In this assay, ME showed significantly (p < 0.01) higher per cent chelating power (88.59% at 300 μg/ml) compared to the standard BHT (48.31% at 300 μg/ml) and was the most potent ferrous ion chelator at all concentrations (Fig. 4). It was also observed that all extracts depicted higher per cent chelating power compared to BHT (Fig. 4). The results also revealed that Fe²⁺-ion chelating activity of extracts did not significantly improved with increasing concentrations of the extracts. Furthermore, the antioxidant potential of acetone extracts evaluated by chelating power assay was also correlated (R²) with the total phenolic contents (Table 2) of extracts of E. trigona.