The scaffold was attached to the Franz diffusion cell in such a way that GS was released into the receptor compartment, which was filled with (5 mL) any one of the dissolution mediums such as water, physiological fluid (PSF) and phosphate buffer saline (PBS) solution of pH 7.4 at 37 ± 1 °C. The elution medium was stirred magnetically, and the aliquots (1 mL) were withdrawn at predetermined time intervals up to 16 h and replaced with the same volume of fresh dissolution medium [45 (link)]. The collected samples were diluted with equal volumes of the medium, and the absorbance was recorded depending on the λmax of the medium (water, PSF and PBS) using a UV-spectrophotometer (Agilent Cary, NC, USA).
Uv spectrophotometer
Manufactured by Agilent Technologies
Sourced in United States, Australia, Germany
A UV spectrophotometer is an analytical instrument that measures the absorption of ultraviolet (UV) and visible light by a sample. It is used to quantify the concentration of a specific substance in a sample by analyzing the amount of light absorbed at a particular wavelength.
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Lab products found in correlation
Cfx connect, by Bio-Rad (2 mentions)
2 2 diphenyl 1 picrylhydrazyl (dpph), by Merck Group (2 mentions)
Automated chemistry analyzer, by Beckman Coulter (2 mentions)
Tccnai g2 f20 s twin, by Thermo Fisher Scientific (2 mentions)
Nano zs 90 nanosizer, by Malvern Panalytical (2 mentions)
Powerwave xs microplate reader, by Agilent Technologies (2 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
Rnase free dnase 1, by Qiagen (1 mentions)
Reverse transcription kit, by Takara Bio (1 mentions)
N hexane, by Loba Chemie (1 mentions)
Cooling centrifuge, by Merck Group (1 mentions)
Bhi broth, by Merck Group (1 mentions)
Ethyl acetate, by Loba Chemie (1 mentions)
Whatman, by Cytiva (1 mentions)
F2500 luminescence spectrometer, by Hitachi (1 mentions)
Tobramycin, by AK Scientific (1 mentions)
Ampicillin, by Bio Basic (1 mentions)
Poly ra, by Merck Group (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
48 protocols using uv spectrophotometer
1
In Vitro Drug Release Evaluation
2
Liposomes-in-Hydrogel Anti-Inflammatory Assay
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The in vitro anti-inflammatory activities of formulations were studied by assessing the effects of EPI or RES liposomes-in-hydrogel formulations on the inhibition of NO production in LPS-induced macrophages (RAW 264.7 cells) by similar methods as reported earlier [3 (link)]. In brief, macrophages (1 × 105 cells/mL) were incubated in a 24-wells plate with RPMI 1640 medium supplemented containing 10% serum and glutamine for at 37 °C/5% CO2. After 24 h, old medium was replaced with new media containing 1 µg/mL LPS to induce inflammation. The formulations at various lipid concentrations, namely 1, 10 and 50 µg/mL, or 1 µg/mL LPS-containing media only (control) were added each with 10 µL volume. After 24 h incubation, NO production by the cells in the media was expressed by measuring the nitrite concentration with Griess reagent by measuring the absorbance at 540 nm using UV spectrophotometer (Agilent Technologies, Santa Clara, CA, USA). The effect of the formulations on inhibition of NO production was expressed as percentage inhibition of produced NO, in comparison to 100% NO detected in the control (cells treated with 1 µg/mL LPS).
3
Bacterial Gene Expression Analysis
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Bacteria cultures were the same of the hemolytic activity assay. Bacteria were centrifuged by 5000×g at 4°C for 5 min. The cell pellets were resuspended and lysed in TES buffer. Total RNAs of samples were extracted by using the RNeasy mini kit (Qiagen) according to the manufacturer's instructions. DNA was removed from each RNA preparation using RNase-free DNase I (Qiagen) according to the manufacturers' directions for rigorous DNase treatment. The OD260 of the purified RNA was measured by a UV spectrophotometer (Agilent Technologies). cDNA was synthetized from extracted RNA using iScript cDNA synthesis Kit (Bio-Rad). PCR amplification was assessed by Real-Time System (CFX ConnectTM, Bio-Rad Laboratories, Inc., USA). All samples were analyzed in triplicate. The 16S rRNA was used (housekeeping gene) as a reference gene. In this study, the relative expression of a target gene versus the 16S rRNA gene was utilized to determine the changes in the transcript-level between samples. The results were analyzed with ABI Prism 7000 SDS software. PCRs were performed with the primers that are listed in Table 1 .
4
Cinnamaldehyde Modulates Fimbrial Gene Expression
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The expression levels of fimA, fimZ, fimY, fimH and fimW were detected by real-time reverse transcription PCR. Salmonella was inoculated in TSB medium for 18 h at 37 °C, and cinnamaldehyde was added to make a final concentration of 0, 16, 32 and 64 μg/mL. DMSO was added to the control group. Total bacterial RNA was extracted according to the instructions of the total bacterial RNA extraction kit (Takara). The purity of RNA was measured for purity by a UV spectrophotometer (Agilent Technologies) at 260 nm and then transcribed into cDNA according to the instructions of a reverse transcription kit (Takara). PCR amplification and gene expression were assessed by the Real-Time System (CFX Connect™, Bio-Rad Laboratories, Inc, Hercules, CA, USA). All the above operations were performed on ice. The results were normalized to 16S RNA expression as an internal standard and calculated (Livak and Smith TGEN, 2001). The primer sequences used are shown in Table 1 . The experiment was repeated three times.
5
Streptozotocin-Induced Diabetic Rat Model
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The following drugs, chemicals, and instruments were used in the study. Streptozotocin (Fisco Research laboratories, India), glibenclamide (Julphar pharmaceuticals, Ethiopia), citric acid (Lab tech chemicals, India), ascorbic acid (Blulux Labratories, India), n-hexane (Loba chemie, India), chloroform (Aristar, England), sodium hydroxide (BluluxLabratories, India), DPPH (Sigma-Aldrich, Germany), sodium citrate (Lab tech chemicals, India), Methanol (Nice chemicals, India), 5% glucose solution (Reyoung pharmaceuticals, China), What man filter paper No.1, test tube, beakers, funnels, measuring cylinder, glass rod, spatula, pipettes, gavages (oral feeding syringes), Syringes (1 mL, 3 mL, and 5 mL) with needles, desiccators, digital analytical balance (EPH-400 Abron Exports), pH meter (Bante Instruments, UK), Sensor Card glucometer and strip (Alliance international, Taiwan), Lyophilizer (Labfreez, China), Hot air oven (Medit-Medizin Technik, Germany), Automated chemistry analyzer (Beckman coulter, Germany), UV-spectrophotometer (Agilent Technologies, Malaysia), rotary evaporator (Hamato, Japan), Tween-80 (Avishkar Lab Tech chemicals, India). All chemicals used were of analytical grade.
6
Cultivation and Genetic Manipulation of Cyanobacteria
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Bacterial strains used in this work are in Table 1 . For cloning, E. coli strains were grown in lysogeny broth medium (10 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl) at 37°C. When appropriate, medium was supplemented with 25 μg/mL chloramphenicol, 50 μg/mL kanamycin, 100 μg/mL spectinomycin. S. elongatus PCC 7942 (wild type). Derivatives were cultivated at 30°C in 100 mL glass bottles under continuous fluorescent light (100 μmol photons/m2/s) in BG-11 medium (1.5 g/L NaNO3, 0.006 g/L ferric ammonium citrate, 0.001 g/L Na2EDTA·2H2O, 0.039 g/L K2PO4, 0.075 g/L MgSO4·7H2O, 0.020 g/L Na2CO3, 0.036 g/L CaCl2·2H2O, 0.006 g/L citric acid, 2.860 mg/L H3BO3, 1.810 mg/L MnCl2·4H2O, 0.222 mg/L ZnSO4, 0.39 mg/L Na2MoO4·2H2O, 0.079 mg/L CuSO4·5H2O, 0.0494 mg/L Co[NO3]2·6H2O) supplemented with 10 mM MOPS (pH 7.5). Supplied at a constant flow rate of 10 mL/min into medium was 5% (v/v) CO2 gas and 95% (v/v) air. Selection was with 10 μg/mL spectinomycin, 10 μg/mL kanamycin or 3 μg/mL chloramphenicol. Isopropyl-β-D-1-thiogalactopyranoside (IPTG) was added into culture medium at inoculation for induction. Cell growth was monitored by OD730 with a UV spectrophotometer (Agilent Technology, CA, USA).
7
Protein Purification via Ammonium Sulfate Precipitation
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Precipitation, a method of protein purification [25] was carried out by ammonium sulphate [48] , [11] (link). The culture supernatant was treated with powdered ammonium sulphate (20, 40, 50, and 60% saturation). After sufficient shaking, solution was placed in the cold for one hour and precipitates were collected by centrifugation at 14,000 rpm for 15 min at 4 °C. The precipitates were resuspended in 15 ml of 0.05 M potassium phosphate buffer at pH 6. Dialysis was carried out against the same buffer for 24 h in dialyzing bag and the pallets were freeze dried. Further purification was processed by gel permeation chromatography. The column was carefully loaded with 3% sephadex G-75 gel suspended in 0.05 M phosphate buffer. The dialyzed protein sample was eluted at a flow rate of 36 ml/h and fractions (3 ml each) were collected. The absorption of these fractions was measured at 280 nm by UV-spectrophotometer (Agilent USA). The fraction showing antibacterial activity were pooled and lyophilized. The activity of these fractions were tested against indicator bacterial strains by agar well diffusion method. Finally, the purified sample was analyzed by reverse phase HPLC (C18 column; 5 μm; 4.6 mm × 150 mm) using isocratic elution of 20 mM Na2HPO4/130 mM NaCl at a flow rate of 1.0 ml/min. Polymyxin B was taken as standard peptide antibiotic due to same class of metabolite sample.
8
Evaluating Drug Entrapment Efficiency Using Ultracentrifugation
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TFS (TFS-1-TFS-6) were evaluated for entrapment efficiency (EE) by separating the free drug from TFS using the ultracentrifugation method [22 (link)]. TFS were subjected to ultracentrifugation after placing them in centrifugation tubes and centrifuged at relative centrifugal force of 21952 g for 2 hours at 4°C in the cooling centrifuge (Sigma, Germany). After centrifugation, the supernatant was withdrawn and diluted appropriately with phosphate buffer pH 6.8 and analyzed at 364 nm and 248 nm for MLX and DEX determination, respectively, using a UV spectrophotometer (Agilent USA) [23 ]. All the results were taken in triplicate and the following mathematical expression was used to calculate the amount of drug entrapped.
9
Quantifying Niosomal Drug Entrapment
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Spectroscopic measurements were used to assess entrapment effectiveness. A UV spectrophotometer (Agilent Technologies, Cary 50, USA) was used to determine the concentration of niosomal-encapsulated PTX. The encapsulation efficiency was calculated by the following equation:
10
Polysaccharide UV-vis and FT-IR Analysis
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The UV-vis absorption spectra of polysaccharide samples from 200 nm to 400 nm were recorded with a UV spectrophotometer (Agilent, Santa Clara, CA, USA). The FT-IR spectrum for the polysaccharide in the 4000–400 cm−1 wavelength was obtained with a Bruker vertex spectrometer (Bruker, Karlsruhe, Germany) at room temperature.
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