The contents of total polyphenols in SPLE and GCBE samples collected from different phases of gastrointestinal digestion (both soluble and insoluble fractions) were determined with the Folin-Ciocalteau reagent [20 ] using a Hitachi U-2900 spectrophotometer (Hitachi High-Tech, Tokyo, Japan), and the absorption values were determined in 750 nm with gallic acid used as a standard.
U 2900 spectrophotometer
Manufactured by Hitachi
Sourced in Japan, United States
The U-2900 spectrophotometer is a versatile instrument designed for high-performance UV-Vis spectroscopy. It features a wavelength range of 190 to 1100 nm and can be used for a variety of analytical applications.
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Lab products found in correlation
Calf thymus dna ct dna, by Merck Group (3 mentions)
F 7000 fluorescence spectrophotometer, by Hitachi (3 mentions)
Maxis 4g, by Bruker (3 mentions)
K alpha xps system, by Thermo Fisher Scientific (3 mentions)
Classic vortex mixer, by VELP Scientifica (2 mentions)
Heraeus megafuge 8 centrifuge, by Thermo Fisher Scientific (2 mentions)
Extractrna reagent, by Evrogen (2 mentions)
F 7000 spectrofluorimeter, by Hitachi (2 mentions)
F 7000 spectrophotometer, by Hitachi (2 mentions)
J 810 spectropolarimeter, by Jasco (2 mentions)
Gemini c18, by Phenomenex (2 mentions)
Affinity 1 ft ir spectrometer, by Shimadzu (2 mentions)
L 2455, by Phenomenex (2 mentions)
Av 700 mhz nmr spectrometer, by Bruker (2 mentions)
6230 lc tof mass spectrometer, by Agilent Technologies (1 mentions)
Ecz 400s spectrometer, by JEOL (1 mentions)
Quercetin, by Merck Group (1 mentions)
Chlorogenic acid, by Merck Group (1 mentions)
Caffeic acid, by Merck Group (1 mentions)
Sevencompact s220, by Mettler Toledo (1 mentions)
112 protocols using u 2900 spectrophotometer
1
Quantification of Polyphenols in Digestive Fractions
2
In Vitro Drug Release Kinetics
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Throughout the experiment, the powders from nanoformulations were placed with the phosphate-buffered saline (PBS) in glass bottles maintained at 37 °C in a water bath while being shaken at 150 rpm (shaker type 357, Elpin+, Lubawa, Poland). Up to 98 h, we would withdraw an aliquot from each bottle at predetermined timepoints and replace the withdrawn volume with fresh PBS buffer. After the aliquots were centrifuged to separate nanoformulation particles, the resulting supernatants were placed in new vials and stored at 4 °C until the UV–vis analysis. Absorbance was measured at 280 nm using the UV–vis technique (U-2900 spectrophotometer, Hitachi High-Technologies Corporation, Tokyo, Japan). Several measurements were performed for each sample, and the cumulative drug release of PCA from nanoformulations was calculated based on the following Equations (1) and (2):
where mass (t) is the percentage release at the time of measurement and t − 1 is the percentage of the released drug at the previous timepoint.
For kinetic model fitting, we analyzed the data for release profiles using pharmacokinetics software (KinetDS3, developed at Jagiellonian University, Krakow, at the Faculty of Pharmacy’s Department of Pharmaceutical Technology and Biopharmaceutics). The kinetic parameters identified were release rate (RR), release efficiency (RE), and mean dissolution time (MDT) [48 (link)].
where mass (t) is the percentage release at the time of measurement and t − 1 is the percentage of the released drug at the previous timepoint.
For kinetic model fitting, we analyzed the data for release profiles using pharmacokinetics software (KinetDS3, developed at Jagiellonian University, Krakow, at the Faculty of Pharmacy’s Department of Pharmaceutical Technology and Biopharmaceutics). The kinetic parameters identified were release rate (RR), release efficiency (RE), and mean dissolution time (MDT) [48 (link)].
3
Spectroscopic Characterization of Organic Compounds
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UV-vis spectra were recorded on a U-2900 spectrophotometer (HITACHI High-Technologies Co., Ltd, Tokyo, Japan). Absorption measurements over the spectral range of 250 nm to 450 nm were carried out in quartz cells having an optical path length of 1 cm. 1H NMR and 13C NMR spectra were recorded on a JEOL ECZ-400S spectrometer (JEOL Ltd, Tokyo, Japan). The solvent used for NMR measurements was CDCl3. ESI mass spectra were measured on an Agilent Technologies 6230 LC/TOF mass spectrometer (Agilent Technologies Japan, Ltd, Tokyo, Japan).
4
Quantifying Phenolic Compounds in Samples
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Phenolic compounds (total phenolic content—TPC, phenylpropanoids, flavonols and anthocyanins) were determined using UV/VIS spectrophotometry [72 (link)]. Chlorogenic acid (CGA), caffeic acid (CA), quercetin (QC) and cyanidin (CY) (Sigma-Aldrich) were used as standards for TPC, phenylpropanoids, flavonols and anthocyanin content, respectively. Samples were ground with 1 mL of 80% methanol and centrifuged for 15 min at 3000× g and room temperature. The supernatant (0.25 mL) was mixed with 0.25 mL 0.1% HCl (in 96% ethanol) and 4.50 mL 2% HCl (in water) and, after 30 min incubation, the absorbances at 280, 320, 360 and 520 nm were read (U-2900 spectrophotometer, Hitachi High-Tech, Tokyo, Japan). The content of phenolic compounds was expressed in mg of respective standard equivalents per 100 g of FW.
5
Degree of Hydrolysis Measurement in Red Blood Cells
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Degree of hydrolysis was measured by the method described by Thiansilakul et al. (2007) . After the hydrolysis of red blood cells, trichloroacetic acid (1:1 ratio) was added to the hydrolysate and the reaction mixture was centrifuged at 8,000 × g for 30 min. The hydrolysate sample (10–50 μL) was diluted with 0.1 mL of 0.2 M phosphate buffer, pH 8.2, and 0.05 mL of 0.01% 2,4,6-trinitrobenzenesulfonic acid solution was added. The solution was mixed thoroughly and placed in a water bath at 50°C for 30 min in the dark. The reaction was terminated by adding 0.1 mL of 0.1 M sodium sulfite. The mixture was cooled at room temperature for 15 min. The absorbance was read at 420 nm using a Hitachi U-2900 spectrophotometer (Hitachi High-Tech Corporation, Tokyo, Japan), and α-amino acid was expressed in terms of L-leucine. where Lt is the amount of α-amino acid released at time t; L0 is the amount of α-amino acid in red blood cell powder; and Lmax is the total α-amino acid in red blood cells powder after acid hydrolysis (6 N HCl at 100°C for 24 h).
6
DNA Binding Studies of DOX and CP-31398
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For the DNA binding studies, Calf-thymus DNA (CT-DNA) was purchased from Sigma- Aldrich. The lyophilized CT-DNA was dissolved in 10 mM Tris-HCl, pH 7.9, mixed gently and left overnight at 4 °C. The purity of the CT-DNA solution was determined by measuring the ratio of UV absorbance at 260 and 280 nm. A ratio of more than 1.8 indicated that the DNA was sufficiently free of proteins. Then, the concentration of CT-DNA was determined from the absorbance at 260 nm using an extinction coefficient of 6600 M−1cm−1. The tested compounds including DOX and CP-31398 were dissolved to a concentration of 8.35 mM in DMSO, which was then used as the stock solution for preparing the various concentrations (25, 12.5, 6 and 3 µM) in 1 mL in 10 mM of Tris-HCl (pH 7.9). Afterwards, 18 µM CT-DNA was added to the prepared solutions, which were incubated for 1.5 h at 37 °C with occasional vortexing. The absorption spectra were measured using a Hitachi U-2900 spectrophotometer in the range of 200–500 nm. All absorption spectra were imported into OriginPro 8.0 and compared.
7
Antioxidant Activity Determination Protocol
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In test tubes 1 mL of sample from each diluted solution and 2 mL of DPPH solution was added. In the same way, 1 mL of the ascorbic acid was added along with 2 ml of DPPH as control and allowed to stand overnight. The next day, using Hitachi U-2900 spectrophotometer, readings at 517 nm were taken in triplicate and their inhibitory concentration calculated using formula [49 ].
8
Antioxidant Activity Evaluation of HR Compounds
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The antioxidant activities of isolated compounds (HR1-HR5 ) were assessed by utilizing diphenylpicrylhydrazyl (DPPH) as a free
radical.28 Stock solutions of purified
compounds (HR1–HR5 ) with a concentration of 22
mg/mL (0.022 g) were prepared to conduct antioxidant activity. The
stock solution of each compound was then diluted to desired concentrations.
4% solution of DPPH was formed by mixing 4 mg of DPPH in 100 mL of
pure methanol. Ascorbic acid was utilized as a standard and prepared
in water. Sample solution (1 mL) from each dilution and DPPH solution
(2 mL) was added to test tubes. Similarly, ascorbic acid (1 mL) solution
along with DPPH (2 mL) was added in a test tube as a control and kept
undisturbed overnight. The next day, their absorbance was measured
at 517 nm in triplicate utilizing a Hitachi U2900 spectrophotometer.
radical.28 Stock solutions of purified
compounds (
mg/mL (0.022 g) were prepared to conduct antioxidant activity. The
stock solution of each compound was then diluted to desired concentrations.
4% solution of DPPH was formed by mixing 4 mg of DPPH in 100 mL of
pure methanol. Ascorbic acid was utilized as a standard and prepared
in water. Sample solution (1 mL) from each dilution and DPPH solution
(2 mL) was added to test tubes. Similarly, ascorbic acid (1 mL) solution
along with DPPH (2 mL) was added in a test tube as a control and kept
undisturbed overnight. The next day, their absorbance was measured
at 517 nm in triplicate utilizing a Hitachi U2900 spectrophotometer.
9
Quantifying endogenous H2O2 in virus-infected cowpea
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The endogenous H2O2 production was estimated in the GBNV inoculated cowpea plants incubated at four different temperatures (30, 25, 20 and 15 °C), as described by Frew et al. (24 ).
For the measurement H2O2 in the virus infected cowpea leaves, the 0.1 M phosphate buffer (pH 7.2) was used for homogenizing the leaf sample. The extracted homogenate was centrifuged in cooled (4 °C) condition at 10,000 ×g for 10 min. The resulted supernatant was used for estimating H2O2. 3.0 mL of the prepared reagent solution (100 mL contains 0.234 g of phenol, 0.1 g of 4-aminoantipyrine, 1.0 mL of 0.1 M phosphate buffer, pH 7.2) was used for the estimation of the endogenous H2O2. The quantified H2O2 was expressed in μmoL.g−1 fresh weight. The spectrophotometer analysis was carried out by using the HITACHI, U-2900, spectrophotometer.
For the measurement H2O2 in the virus infected cowpea leaves, the 0.1 M phosphate buffer (pH 7.2) was used for homogenizing the leaf sample. The extracted homogenate was centrifuged in cooled (4 °C) condition at 10,000 ×g for 10 min. The resulted supernatant was used for estimating H2O2. 3.0 mL of the prepared reagent solution (100 mL contains 0.234 g of phenol, 0.1 g of 4-aminoantipyrine, 1.0 mL of 0.1 M phosphate buffer, pH 7.2) was used for the estimation of the endogenous H2O2. The quantified H2O2 was expressed in μmoL.g−1 fresh weight. The spectrophotometer analysis was carried out by using the HITACHI, U-2900, spectrophotometer.
10
Emulsifying and Stability Indices of Aquafaba
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The emulsifying activity index (EAI) and the emulsion stability index (ESI) were determined according to the procedure described by Cheung et al. [31 (link)] but developed initially by Pearce and Kinsella [32 (link)]. In brief, 5.0 g of aquafaba or egg yolk was homogenized with 5.0 g of RRO using a homogenizer at a speed of 8000 rpm for 5 min. Then, a 50 µL aliquot of the emulsion was diluted to 7.5 mL of 0.1% sodium dodecyl sulphate (SDS) and vortexed using a classic vortex mixer (Velp Scientifica Srl, Usmate (MB), Italy) for 10 s. The absorbance of the diluted emulsion samples was measured at λ = 500 nm by a Hitachi U-2900 spectrophotometer (Tokyo, Japan). The EAI and ESI were calculated using the following equations:
where, A0 and A10 are the absorbance values measured at an initial time, and after 10 min, respectively, t is the time interval (10 min), N is the dilution factor, c is the protein concentration (g/mL), φ is the oil volume fraction of the emulsion and ϕ is an optical path (1 cm).
where, A0 and A10 are the absorbance values measured at an initial time, and after 10 min, respectively, t is the time interval (10 min), N is the dilution factor, c is the protein concentration (g/mL), φ is the oil volume fraction of the emulsion and ϕ is an optical path (1 cm).
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