Uv spectrophotometer

DPPH free radical scavenging ability of test samples were tested according to previously reported procedures (Shah et al., 2015 (link); Zahoor et al., 2018 (link)). Samples solutions were prepared ranging from 125 to 1,000 μg/ml and were added to 0.004% methanolic solution of DPPH. Following incubation for 30 min, absorbance values were recorded at 517 nm using UV spectrophotometer (Thermo electron corporation, USA). DPPH scavenging activities were calculated as;
Where A₀ characterizes absorbance of the control and A₁ is the absorbance of test samples. Ascorbic acid was used as a positive control. Where A₀ characterizes absorbance of control and A₁ is the absorbance of test samples. All experiments were performed in triplicate and inhibition graphs were made with the help of GraphPad prism program (GraphPAD, San Diego, California, USA).

The ABTS free radical scavenging potential of Plant samples were tested following previously published procedure (Zafar et al., 2019 (link)). Briefly, solutions of ABTS 7 mM and potassium persulphate (K₂S₂O₄) 2.45 mM were mixed and stored in a dark place at room temperature for about 12–16 h to obtain a dark colored solution. This solution was diluted using Phosphate buffer (0.01 M) pH 7.4 and absorbance value was adjusted to 0.70 at 734 nm. Finally, 300 μl solution of the test sample was added to 3.0 ml of ABTS solution in cuvette and was analyzed using a UV spectrophotometer (Thermo electron corporation, USA) at 734 nm. Reduction in absorbances was determined following 1 min of mixing the solutions and analysis was continued for 6 min. Ascorbic acid was used as a positive control. The assay was repeated in triplicate and percentage inhibition was calculated using the formula;

For the determination of DPPH (2, 20-diphenyl-1-picrylhydrazyl) free radical scavenging ability of the extracts, Brand-Williams assay [26 ] was used with some modification. About 24 mg DPPH was dissolved in 100 mL methanol. Plant sample stock solutions (1 mg/mL) were also prepared in methanol. Using serial dilutions working solutions with the following concentrations: 1000, 500, 250, 125, 62.5 and 31.05 μg/mL were prepared. About 0.1 mL of each working dilution was mixed with DPPH (3.0 mL) and incubated at 23 °C for 30 min. Absorbance was measured at 517 nm via UV-spectrophotometer (Thermo Electron Corporation: USA). Ascorbic acid was used as a standard. Results were presented as Mean ± SEM. % DPPH scavenging potential was calculated by the following formula: $$\mathrm{\%}DPPHScavengingpotential=\frac{controlabsorbance-sampleabsorbance}{controlabsorbance}\times 100$$

Brand-Williams assay [22 (link)] was used with some modification to check the scavenging potential of essential oil of E. umbellata against DPPH (2, 2-diphenyl-1-picrylhydrazyl) free radical. To prepare DPPH solution, 24 mg of it were dissolved in 100 mL methanol. Approximately, 1 mg/mL stock solution of essential oil was also prepared in methanol and serially diluted to obtain the dilution having concentrations; 1000, 500, 250, 125, 62.5 and 31.05 μg/mL. Subsequently 0.1 mL of each dilution was mixed with 3 mL of DPPH solution. The mixtures were incubated for 30 min at 25 °C. Absorbance was measured at wave length 517 nm through UV spectrophotometer (Thermo Electron Corporation; USA) and ascorbic acid was used as a positive control. All the samples were analysed in triplicates and the results are presented as Mean ± SEM. Percent DPPH scavenging potential was calculated using the following equation:
$$\mathit{\text{Percent Scavenging potential}}=\frac{\mathit{\text{Blank sample absorbance}}-\mathit{\text{sampleabsorbance}}}{\mathit{\text{Blank sample absorbance}}}\times 100$$

Free radicals scavenging ability of the essential oil was determined following well established procedures [29 (link), 55 (link)]. Different dilutions (12.5, 50, 100, 200, 400, 800 and 1000 μg/ml) of essential oils (0.1 ml) were added to 0.004 % methanolic solution of DPPH. After 30 min, absorbance was measured at 517 nm using UV spectrophotometer (Thermo electron corporation, USA). Percent DPPH scavenging activity was calculated as; $\frac{{\mathrm{A}}_0-{\mathrm{A}}_1}{{\mathrm{A}}_0}\times 100$
Ascorbic acid was used as positive control. Where A₀ characterize absorbance of control and A₁ is the absorbance of the essential oils. All experiments were performed in triplicate and inhibition graphs were made with the help of GraphPad prism program (GraphPAD, San Diego, California, USA). Median inhibitory concentrations IC₅₀ values were calculated using Microsoft Excel programme.

Brand-Williams assay [34 (link)] was used to find out DPPH free radical scavenging activity of isolated compounds of E. umbellata. About 24 mg, DPPH was dissolved in 100 mL methanol. Compounds solutions (1 mg/mL) were also prepared in methanol. Working solutions were prepared using serial dilutions in the concentrations range of 1000, 500, 250, 125, 62.5 and 31.05 μg/mL. About 0.1 mL of each working dilution was mixed with DPPH (3.0 mL) and incubated at 25 °C for 30 min. Absorbance was measured at 517 nm via UV-spectrophotometer (Thermo Electron Corporation: USA). Ascorbic acid was used as a standard. Results are presented as Mean ± SEM that has been calculated using the formula:
$$\%\mathit{\text{DPPH Scavenging potential}}=\frac{\text{standard absorbance}‐\text{sample absorbance}}{\text{standard absorbance}}\times 100$$

For DPPH free radicals scavenging activity the method of Brand-Williams et al. was followed with some modifications [49 (link)]. DPPH (24 mg) was dissolved in 100 ml of methanol to prepare DPPH solution. The stock solutions of plant samples were prepared in methanol having concentrations of 1 mg/ml and then diluted to the concentrations 1000, 500, 250 μg/ml. DPPH and sample solutions were mixed in a ratio of 1:1 and were incubated at 23°C for 30 min. Finally, absorbance was measured at 517 nm using UV spectrophotometer (Thermo electron corporation USA). Ascorbic acid was used as positive control. Percent radical scavenging activity was measured using the following equation;