Double beam spectrophotometer

The formation of silver nanoparticles is monitored by measuring the UV-vis spectra of the reaction mixture (Aqueous silver nitrate solution with P. nigrum leaf and stemextracts in separate conical flask). The UV-vis spectra measurements are carried out on Perkin-Elmer double-beam spectrophotometer operated with a resolution of 2 nm. Further, the reaction mixture is centrifuged at 10,000 rpm for 10 min and washed with double-distilled water. Repeat the centrifugation process for 4-5 times and allow the pellet to dry in hot air oven. The dried powder is used for further characterization studies. For XRD analysis, the powdered nanoparticles are coated on the amorphous silica substrate. The spectra are recorded by using XDL 3000 powder X-ray diffractometer with 40 kV and a current of 30 mA with Cu Kα (1.5405 Å) radiation. The shape of the silver nanoparticles is examined by SEM experiments using Philips Scanning Electron Microscope. The presence of elemental silver is analyzed by energy dispersive spectroscopy attached to SEM. For TEM experiment, the silver nanoparticles are coated on copper grids and analyzed by Philips CM200 operated at 200 kV. The FTIR measurements are carried out using the Perkin-Elmer instrument at wavelength ranges from 4000 to 400 cm⁻¹ at a resolution of 4 cm⁻¹.

Lipid peroxidation was measured to estimate the damaging effects of Cd in the cellular membrane. Approximately 0.25 g of fresh leaves were homogenized in 8 ml of 0.1% trichloroacetic acid (TCA) and was centrifuged for 30 min at 16000 rpm. After centrifugation, the supernatant was mixed with TBA (3 ml) prepared in a 20% solution of TCA. This mixture was placed at 95°C for 1 h in a water bath the following cooling on ice for almost 4–5 min. The absorbance of the mixture was taken at 600 nm with a double beam spectrophotometer (PerkinElmer Ltd., United Kingdome). MDA content was measured by an extinction coefficient of 155 mm⁻¹ cm⁻¹ (Dhindsa et al., 1981 (link)).

Lipid peroxidation inhibition was adapted from Kumazawa et al. [24 (link)] based on the bleaching of β-carotene with slight modifications. Six mg of β-carotene was dissolved in 50 mL of chloroform, and 4 mL of the solution was mixed with 40 mg of linoleic acid and 400 mg of Tween 40 emulsifier in a conical flask. Nitrogen gas was then used to evaporate the chloroform. Then, 100 mL of oxygenated Milli-Q water was added and the flask was shaken for the mixture to be fully dissolved. Immediately after water was added, the absorbance of the mixture was measured at 470 and 700 nm using Perkin-Elmer double-beam spectrophotometer. Next, 3 mL of the emulsion was added into test tubes with different volume of sample (10, 50 and 100 μL). The tubes were sealed with parafilm and incubated at 50 °C water bath for an hour before the absorbance was measured again. For control, 100 μL of methanol was used instead of the sample. The blank was prepared by adding the same volume of sample with the emulsion of 400 mg Tween 40 emulsifier and 100 mL of water but without linoleic acid and β-carotene solution. The absorbance (A) at 700 nm was taken in order to correct the haze present in the solution. The LPI was calculated using the formula: $$\text{Degradation Rate}\left(\text{DR}\right)=\left[\text{Ln}\left({\text{A}}_{\text{Initial}}/{\text{A}}_{\text{Sample}}\right)\right]/60$$ $$\text{Antioxidant Activity}\left(\%\text{AOA}\right)=\left[1-\left({\text{DR}}_{\text{Sample}}/{\text{DR}}_{\text{Control}}\right)\right]\times 100.$$