The total content of flavonoids of the different extracts of the T. polium plant material was determined according to the colorimetric assay described by Sharifi-Rad et al. [86 (link)] with some modifications. The extracts were prepared at the same concentration of 1 mg/mL for analysis. In summary, 0.5 mL of each extract, 0.1 mL of an AlCl3 solution (10%), 0.1 mL of a potassium acetate solution (1 mol/L), and 4.3 mL of distilled water were mixed, and the reaction mixtures were incubated for 30 min at room temperature. The absorbance of the resulting reaction mixtures was measured at 510 nm by the spectrophotometer (UV-1800 240 V, Shimadzu Corporation, Kyoto, Japan). Quercetin was applied for preparing the standard calibration curve (10–100 mg/mL, y = 0.0093x − 0.0413, R2 = 0.994). The total content of flavonoids of the examined samples was expressed as the quercetin equivalent (mg QE/g dry weight).
Uv 1800 240 5
Manufactured by Shimadzu
Sourced in Japan
The Shimadzu UV-1800 240 V is a UV-Visible spectrophotometer. It is designed to measure the absorption of ultraviolet and visible light by samples.
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Polyhydroxybutyrate, by Merck Group (1 mentions)
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44 protocols using uv 1800 240 5
1
Quantifying Flavonoids in T. polium Extracts
2
Antioxidant Capacity of Millet Extract
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In this analysis, DPPH (2,2-diphenyl-1 picryl hydrazyl) was used to assess the antioxidant capacity of millet extract following the method of Braca et al. [30 (link)]. The stock solution was prepared by dissolving 5.914 mg of DPPH powder in 100 mL of methanol solvent, and the absorbance range was maintained from 1.1–1.3 by a spectrophotometer. Briefly, 1 mL of millet extract was mixed with 3 mL of DPPH solution, and the mixture was left in a dark environment (at room temperature, for 30 min) after shaking vigorously. The DPPH solution (3 mL) with distilled water (1 mL) was considered the blank sample. The absorbance was measured at 517 nm through a UV-Vis spectrophotometer (UV-1800 240 V, Shimadzu Corporation, Kyoto, Japan). The scavenging capacity of the millet sample was calculated using the following formula and result expressed as a percentage:
3
Characterization of Kappa Selenite-Coated Gold Nanoparticles
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The reaction conditions of KS@GNPs were investigated by UV-visible spectrophotometer (UV-1800 240 V, Shimadzu Corporation, Koyoto, Japan) with 300 to 700 nm wavelength scanning range. The formation of KS@GNPs was confirmed by the Fourier transform infrared spectrophotometer (FTIR) (Perkin-Elmer Model 1600; Norwalk, CT, USA). Here, a pure KBr pellets (2 mg of GNPs were mixed with KBr) was used with the scanning range from 400 to 4000 cm−1. The crystallinity of KS@GNPs was analyzed by X-ray diffractometer (X’pert PRO MPD, PANalytical BV, Almelo, The Netherlands) using an operating voltage 45 kV; a current of 40 mA, Cu radiation (1.54430 Å), and at a scanning rate 0.388/min within the region of 2 thetas between 5 and 90 °. The average particle size distribution and the surface charge were determined through a dynamic light scattering (DLS) equipment (Zeta plus 90, Brookhaven Instrument Co., Holtsville, NY, USA). The size, morphology, Energy Dispersive X-ray (EDX) spectrum, and selected electron diffraction (SAED) of KS@GNPs were evaluated by using HR-TEM (LEO-912AB OMEGA, LEO, Freising, Germany). A thin coat of the sample was prepared for TEM analysis by diffusing a drop of KS@GNPs solution on the cupper grid (operating voltage 200 eV).
4
Spectrophotometric Determination of Total Flavonoids
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The total flavonoid content (TF) was determined according to Ghimeray et al. [23 ] with slight modifications. In brief, a 0.5 mL aliquot of the sample (1 mg/mL) was mixed with 0.1 mL of 10% aluminum nitrate and 0.1 mL of potassium acetate (1 M). To this mixture, 3.3 mL of distilled water was added to make the total volume of 4 mL. The mixture was vortexed and incubated for 40 min. The total flavonoids were measured using a spectrophotometer (UV-1800 240 V, Shimadzu Corporation, Kyoto, Japan) at 415 nm. The TF was expressed as mg/100 g coumarin equivalents on a dry mass basis (dmb).
5
Antioxidant Activity of Kenaf Extracts
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The antioxidant activity of Kenaf leaves and seed extracts was evaluated using DPPH free radical and H2O2, following the method of Braca et al. [19 (link)] and Adnan et al. (2020) [5 (link)]. For the DPPH, 3 mL of freshly prepared DPPH (0.004% w/v in methanol) was added to the 0.5 mL of stock solution. The reaction mixture was vortexed and placed in the dark ambience for incubation (30 min at room temperature). For H2O2 scavenging, 0.6 mL of H2O2 solution (4 mM prepared with 0.1 M phosphate buffer pH 7.4) was mixed with 0.4 mL of stock solution and then incubated for 10 min. The scavenging of DPPH and H2O2 were measured at 517 nm and 230 nm, respectively by the spectrophotometer (UV-1800 240 V, Shimadzu Corporation, Kyoto, Japan). The percentage of scavenging capacity was calculated against negative control (methanol + DPPH) and (stock solution without H2O2) expressed by the following equation: Scavenging effect (%) = [(Absc − Abss)/Absc] × 100, where Absc is the absorbance of control; Abss is the absorbance of DPPH radical/H2O2 + sample (extract/standard).
6
Proline Quantification in Plant Tissues
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Approximately 25 mg of freeze-dried plant material was used to estimate proline concentration. The concentration of proline was determined by following the method described by Bates [33 ]. The absorbance was taken at 520 nm with a UV-Vis spectrophotometer (UV-1800 240 V, Shimadzu Corporation, Kyoto, Japan), and calculations used an appropriate proline standard curve.
7
Antioxidant Capacity by DPPH Assay
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The antioxidant capacity was determined on the basis of the scavenging activity of the stable 2, 2-diphenyl-1 picryl hydrazyl (DPPH) free radical according to methods described by Braca et al. [17 (link)]. The DPPH solution was prepared (5.914 mg of DPPH powder dissolved in 100 mL of methanol) to maintain an absorbance range of 1.1–1.3 by spectrophotometer. Briefly, 1 mL of stock solution was added to 3 mL of DPPH solution. The blank sample was prepared with 1 mL of distilled water instead of stock extract in 3 mL of DPPH solution. The mixture was shaken vigorously and left to stand at room temperature in the dark for 30 min. The absorbance was measured at 517 nm using a spectrophotometer (UV-1800 240 V, Shimadzu Corporation, Kyoto, Japan). The percent inhibition activities of the treated and control AGN samples were calculated against a blank sample using the following equation:
Inhibition (%) = [(blank sample − extract sample)/blank sample] × 100.
Inhibition (%) = [(blank sample − extract sample)/blank sample] × 100.
8
Determining Caffeine Encapsulation Efficiency in Hyalurosomes
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Ultra-filtration was adopted to determine the percentage encapsulation efficiency (% EE) of CAF. Precisely 2.5 ml aliquots of the prepared caffeinated hyalurosomes formulations were transferred into centrifuge tubes fitted with an ultrafilter (Centrisarts®, MWCO 100000, Sartorius, Bohemia, NY, USA). Tubes were centrifuged for 20 min at 3000 rpm at room temperature (Sigma Laboratory Refrigerated Centrifuge, Model 3 K-30, Germany) (Jain et al., 2009 (link)). The amount of CAF in the supernatant was determined spectrophotometrically (UV-1800 240 V, Shimadzu Corporation, Kyoto, Japan) at λmax 273 nm (Abosabaa et al., 2021 (link); Khazaeli et al., 2007 (link)). All measurements were conducted in triplicates. The amount of encapsulated CAF was calculated as follows: (Gaafar et al., 2021 ).
Where A1 is the amount of free CAF and A2 is the total amount of CAF in the formulation.
Where A1 is the amount of free CAF and A2 is the total amount of CAF in the formulation.
9
DPPH Antioxidant Capacity Determination
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The antioxidant capacity was determined on the basis of the scavenging activity of the stable 2,2-diphenyl-1 picryl hydrazyl (DPPH) free radical, according to methods described by Braca et al. [24 (link)]. The DPPH solution was prepared with 5.914 mg of DPPH powder in 100 mL of 100% methanol in the dark. Then, 1 mL of sample extract (1 mg/mL) was added to 3 mL of DPPH solution. The blank sample was prepared with 1 mL of distilled water instead of sample extract in 3 mL of DPPH solution. The mixture was shaken vigorously and left to stand at room temperature in the dark for 30 min. The absorbance was measured at 517 nm using a spectrophotometer (UV-1800 240 V, Shimadzu Corporation, Kyoto, Japan). The percent inhibition activities of the SFC and control were calculated against a blank sample using the following equation:
10
Quantifying Phenolic Content in Kenaf
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The total content of phenol in Kenaf leaves and seed extracts was assessed following the method of Folin-Ciocalteau [16 ]. The Folin-Ciocalteau reagent (200 µL, 1 N) was added in the test tube containing 1 mL of the sample (10 mg/mL). The volume of the mixture was increased by the addition of deionized water (1.8 mL) and kept (3 min at room temperature) for the reaction after the vortex. Afterwards, 400 µL of sodium carbonate (10% v/v) was added to the reaction mixture. Finally, the volume was adjusted up to 4 mL by adding deionized water (600 µL). The mixture was placed in dark ambience for incubation (1 h at room temperature), and the test was done in triplicates. The absorbance was measured against the blank (water) at 725 nm by the spectrophotometer (UV-1800 240 V, Shimadzu corporation, Kyoto, Japan). The TPC was calculated from a calibration curve (plotting the value of absorbance vs. concentration) using gallic acid and expressed as mg of GAE (gallic acid equivalent) per 100 g of extract.
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