The UV spectrum (200–500 nm) was measured using a PerkinElmer Lambda-25 UV spectrophotometer. IR spectra (200–4,000 cm-1) were recorded on an FT–IR PerkinElmer RX-1 spectrometer. The mass spectrum was recorded using a micromass Quattro II triple quadrupole mass spectrometer. 1H and 13C nuclear magnetic resonance (NMR) spectra were obtained using a 300 MHz Varian Inova instrument in deuterated water (D2O). Elemental analysis was performed using a Carlo Erba 1106 CHNO/S elemental analyzer. The melting point was determined using a WRS-1B digital melting-point apparatus (Singh et al., 2013 (link)).
Lambda 25 uv spectrophotometer
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The PerkinElmer Lambda 25 UV spectrophotometer is a compact and reliable instrument designed for routine absorbance measurements in the ultraviolet and visible wavelength ranges. It features a simple and intuitive user interface, allowing for efficient data collection and analysis.
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10 protocols using lambda 25 uv spectrophotometer
1
Characterization of Organic Compound
2
Polysaccharide UV Spectral Analysis
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The polysaccharides were made into 1 mg/mL aqueous solutions. UV spectra were generated using a Lambda 25 UV spectrophotometer (Perkin-Elmer, USA) in the range of 200–700 nm.
3
Extraction and Purification of Bioactive Compounds
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Fermented culture was centrifuged at 10,000 rpm for 20 min to separate the biomass. The active metabolite was recovered from the fermented broth using two phase solvent extraction system with organic solvent. Solvents containing the active compounds were concentrated under vacuum to get “dried crude.” The obtained “crude” was treated with non-polar solvents like hexane or chloroform to separate the polar and non-polar components. The active components were purified by adsorption chromatography using silica gel (pore size 60 Å, mesh size: 230–400, particle size 40–63 μm) as a stationary phase and gel filtration chromatography using sephadex LH-20. The eluted fractions were assayed for their bioactivity against B. subtilis ATCC 6633 and C. albicans ATCC 24433 by disc diffusion method (Wu, 1984 ). The purity of the active fractions was further checked by high pressure liquid chromatography (HPLC) using reverse phase silica column (RP18). Finally, the UV spectra (Perkin Elmer Lambda-25 UV spectrophotometer) of various antibacterial and antifungal compounds were determined in methanol at 200–500 nm wave length.
4
Isolation and Characterization of Antifungal Compound
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Biomass was separated from the fermented culture by centrifuging it at 11,110xg for 20 min. The supernatant was extracted with chloroform and the organic layer containing active metabolites was collected and evaporated under reduced pressure. The residue was purified by silica gel (mesh size: 230–400) column chromatography using increasing gradient of methanol-chloroform (0–50%) as an elutant. The collected fractions were tested for antifungal activity by disc diffusion method against Candida albicans ATCC 24433. The fractions showing similar antifungal activity were pooled together and again subjected to silica gel column chromatography using mesh size 230–400. Purity of the fractions showing significant antifungal activity was further examined by high pressure liquid chromatography (HPLC) using analytical C-18 silica column (Lachrom) at a flow rate of 1 ml/min with Merck system at 254 nm. Finally the chemical structure of the compound was established with the help of UV (Perkin Elmer Lambda- 25 UV spectrophotometer), IR (FT-IR Perkin Elmer RX-1 spectrometer), ESMS (Micromass Quattro II triple quadrupole mass spectrometer) and 1H and 13C NMR spectra (600 MHz VARIAN INOVA instrument).
5
Extraction and Characterization of Oenothera paradoxa Polyphenols
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Defatted and crushed seeds (100 g) of O. paradoxa (obtained from Agropharm, Poland) were processed by extraction with 60% ethanol (500 mL) with stirring and shaking during four hours at 51°C. Subsequently, the mixture was concentrated in a rotary evaporator (Heidolph), dried, and lyophilized.
Total polyphenol content was determined using modified Folin-Ciocalteu method. Folin-Ciocalteu reagent (10 × diluted; 2.5 mL) was added to ethanolic evening primrose extract (0.5 mL), and in the next step 7.5% sodium bicarbonate solution was added (2 mL) and all was mixed well. The entire mixture was left for 2 hours in room temperature. A gallic acid standard curve was obtained for the calculation of polyphenolic content which amounted to 55% as determined spectrophotometrically at 765 nm (LAMBDA 25 UV spectrophotometer, Perkin Elmer, UK) [15 ].
The O. paradoxa stock solution (3 mg/mL) was prepared by solving 30 mg of the extract in 10 mL of phosphate-buffered saline (PBS) containing 1% ethanol and was stored at 4°C. Under these conditions the solution was stable at least for a month, as confirmed by HPLC analysis. All experiments in this study were performed with O. paradoxa defatted seed extract at the final concentrations of 0.1 and 0.25 mg/mL.
Total polyphenol content was determined using modified Folin-Ciocalteu method. Folin-Ciocalteu reagent (10 × diluted; 2.5 mL) was added to ethanolic evening primrose extract (0.5 mL), and in the next step 7.5% sodium bicarbonate solution was added (2 mL) and all was mixed well. The entire mixture was left for 2 hours in room temperature. A gallic acid standard curve was obtained for the calculation of polyphenolic content which amounted to 55% as determined spectrophotometrically at 765 nm (LAMBDA 25 UV spectrophotometer, Perkin Elmer, UK) [15 ].
The O. paradoxa stock solution (3 mg/mL) was prepared by solving 30 mg of the extract in 10 mL of phosphate-buffered saline (PBS) containing 1% ethanol and was stored at 4°C. Under these conditions the solution was stable at least for a month, as confirmed by HPLC analysis. All experiments in this study were performed with O. paradoxa defatted seed extract at the final concentrations of 0.1 and 0.25 mg/mL.
6
Measuring Polyphenols in Evening Primrose
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The total polyphenol content (TPC) was calculated using the Folin–Ciocalteu method [2 (link)] and expressed as gallic acid equivalents (GAE) [28 ]. The EPE concentration range 0.01–1000 µg/mL was equivalent to 0.00208–208.24 µg GAE/mL, that means 0.01224–1224 µM GAE. GAE is a commonly used determinant in assessing TPC reported in previous studies [29 (link),30 (link)]. To calculate TPC in O. paradoxa extract, the gallic acid standard curve was determined for the concentration range from 12.5 to 200 µg/mL of gallic acid. Briefly, 2.5 mL of 10 times diluted Folin–Ciocalteu reagent was added to 0.5 mL of evening primrose extract solution in the concentrations: 100 µg/mL and 50 µg/mL and incubated with 2 mL of 7.5% (w/v) sodium carbonate solution for 2 h at room temperature. The absorbance was measured at 765 nm on a LAMBDA 25 UV Spectrophotometer (Perkin Elmer, UK). All determination was performed from three independent experiments. In this study, the concentration of extract used for in vitro MPM cells treatment was presented in µg/mL of extract as well as µM GAE to enable further discussion of the results.
7
Spectrophotometric Analysis of Chlorophenols
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A PerkinElmer Lambda 25 UV spectrophotometer (Waltham, Massachusetts, US) was used to analyze both 2,4-DCP (λmax: 284.6 nm) and 2,4-DNP (λmax: 358.5 nm). An orbital shaker (Orbitron, INFORS HT) from Bottmingen, Switzerland was used to shake the samples during the adsorption process. The pH of the solution was adjusted using a pH meter from Hanna Instruments (Rhode Island, USA).
8
Solubility of IMC Polymorphs in SGF
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To determine the solubility of the of the non-ionized species of polymorphs α-IMC, γ-IMC and δ-IMC, an excess of each powdered polymorph was dispersed in simulated gastric fluid (SGF, HCl 0.1 M) and the dispersion was stirred in an oven at 37 °C for 96 hours, after which thermodynamic equilibrium was reached. The solution was then analyzed with a Perkin Elmer Lambda 25 UV spectrophotometer at a wavelength of 263 nm after filtration through a cellulose acetate filter (0.45 µm).
9
Quantification of Total Phenolics
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To determine the total phenolics, the most known method was applied to plant samples by using Folin-Ciocalteu (F-C) reagent. The details were reported in our previous studies [35, 36] . The concentration of the extracts was prepared as 1 mg/mL. Ferulic acid was used for preparing a standard calibration curve. Total phenolic contents of the extracts were measured at 725 nm by spectrophotometrically using the Perkin Elmer lambda 25 UV spectrophotometer. The data was expressed as mg ferulic acid/100 g extract.
10
Hydrogel Drug Absorption and Release
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The mechanical properties of the dried membrane and hydrogel samples were determined using E Z Test machine (SM-50N-168, Shimadzu) which was set in tensile mode. The samples were cut into 5 cm x 1 cm size and stretched at a crosshead speed of 50 mm/min. Average values obtained from four measurements were used to calculate the tensile strength and Young's modulus from the stress-strain data.
Drug absorption and release studies: Dried samples of each hydrogel (about 4 x 4 cm) was immersed into Quetiapine fumarate solution of concentration of 2 mg/mL for 24 hrs to allow for maximum absorption of the drug into the hydrogel matrix. The amount of drug absorbed was calculated from the difference in the concentration of the Quetiapine fumarate solution before and after absorption. The swollen hydrogels containing the drug was dried under vacuum at 40 o C until constant weight was achieved. The dried hydrogels were placed in distilled water and at 10 min intervals; the drug release was monitored by recording the absorbance at 298 nm using the Perkin Elmer Lambda 25 UV spectrophotometer. Average from three trials was considered. Data obtained were fitted into zero order (5) (link), first order (6) and Higuchi (7) kinetic models as well as the Korsmeyer-Peppas power law (8) to ascertain the drug release kinetics and transport mechanisms (n), respectively.
Drug absorption and release studies: Dried samples of each hydrogel (about 4 x 4 cm) was immersed into Quetiapine fumarate solution of concentration of 2 mg/mL for 24 hrs to allow for maximum absorption of the drug into the hydrogel matrix. The amount of drug absorbed was calculated from the difference in the concentration of the Quetiapine fumarate solution before and after absorption. The swollen hydrogels containing the drug was dried under vacuum at 40 o C until constant weight was achieved. The dried hydrogels were placed in distilled water and at 10 min intervals; the drug release was monitored by recording the absorbance at 298 nm using the Perkin Elmer Lambda 25 UV spectrophotometer. Average from three trials was considered. Data obtained were fitted into zero order (5) (link), first order (6) and Higuchi (7) kinetic models as well as the Korsmeyer-Peppas power law (8) to ascertain the drug release kinetics and transport mechanisms (n), respectively.
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