Preparation of chitosan microspheres49 In this method, the polymer chitosan was initially dissolved 1% acetic acid, by magnetic stirring for about 2-3 hrs, to the prepared chitosan solution, diltiazem hydrochloride was added, stirred for 1hr, the resulted polymer solution was added to liquid paraffin containing span 60 as surfactant. The resultant emulsion was stirred for one hr further cross linking agent gluteraldehyde was added stirred over night for rigidisation of coating. Microsphers were filtered, washed with petroleum ether, and air dried. Formulation code Diltiazem hydrochloride (mg) Chitosan Gluteraldehyde (ml) Surfactant Conc. FCD1 90 90 3 1 FCD2 90 180 3 2 FCD3 90 240 3 3 FCD4 90 360 3 4 FCD5 90 450 3 5 FCD6 90 90 3 5
The minimum inhibitory concentration of abietic acid was studied by broth micro dilution method using 96-well microtitre plates.9 Test compound was dissolved in DMSO (1%) with the addition of Tween-80(0.5%) and diluted in Muller Hinton Broth to get a concentration range of 100-1000μg/mL. The solution was then two fold diluted in Muller Hinton Broth (100 μL), inoculated with bacterial strains and then incubated at 37°C for 24 h. The bacterial growth was measured as turbidity with a Cyberlab microplate reader at 405 nm. The minimum inhibitory concentration was defined as the lowest concentration of test compound that inhibits the growth of the test bacteria. DMSO assayed as the negative control at a concentration of 1% did not inhibit any of the strains tested. All tests were assayed in triplicate in three independent experiments and median values were used for MICs calculation.
Heat the Agarose gel in a 65 °C water-bath to melt the agarose. After it melted, maintain its temperature at 55 °C until it is ready for use. 2. Transfer the spheroids from the 96-well plate to a 15 mL centrifugal tube using a 1000 μL pipette 3. centrifuge the tube for 5 min at 1000 rpm to form a pellet. 4.
Subsequently extracted by Microwave at power level 70 for 16 minutes and then filtered. The filtrate obtained, was added 10% HCl (until pH 2-3). Then do the bleaching with NaOCl diluted with water 1: 1 to white. Then converted to sodium alginate by adding 20 g of Na2CO3 and stirred in a mixer. The resulting solution is then etched with ethanol to form sodium alginate fibers.
Into round bottom flask taken 180 ml nHexane . Extracted at 75°C for 24 h until the solvent leached colourless. Dry solvent from sample. Followed by transesterification Transesterification process Transesterification is the process by which the glycerides present in fats or oils react with an alcohol in the presence of a catalyst to form esters and glycerol Chemical-Catalyzed Transesterification. In a 150 mL Erlenmeyer flask with a magnetic stir bar placed 0.25 g of anhydrous sodium hydroxide and dissolved it with 10 mL of methanol.
Then, the flask is put on shaker table and mixed at 150 rounds per minute before allowing them to settle for 10 minutes. After settling, the water sample is poured from side spout which is connected to the bottom of the flask. Some researchers reported better reproducibility with modified flask where stopcock is installed at the bottom of the flask, instead of side spout to pour the water sample (Blondina, et al., 1997) (Sorial, et al., 2004a) (Sorial, et al., 2004b). The dispersed oil in the removed water sample is extracted into methylene chloride for further analysis. Then, the oil concentration is evaluated using 340, 370 and 400 nm light absorbance (Environmental Protection Agency,
They were as follows i) N hexane ii) Petroleum ether iii) Methanol iv) Chloroform The powdered plant material (50 gm) was successively extracted in a Soxhlet extractor with an elevated temperature using 250 ml of n-hexane, followed by petroleum ether, methanol and chloroform, according to increasing solubility. All extracts were filtered individually and poured on petri dishes to evaporate the solvents from the extracted material. After drying, crude extracts were stored in stock vials and kept in refrigerator for further use. 3.2 Extraction of the Terminalia bellirica plant material Here hot solvent extraction process was used for extraction of the plant material. Four solvents were used for the extraction of the plant material.
Determination of antioxidant activity Scavenging DPPH radicals DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) radical is used free radical method is an in antioxidant assay forwas used to evaluate measured the free radical scavenging activity of the lichen extract . Two millilitreers of 0 .05 mg/mL methanol solution of DPPH radical in the concentration of (0 .05 mg/mL) and 1 mL of the lichen extract (1 mg/mL) were placed in cuvettes. The mixture is storewas stored stand at room temperature for 30 min. Then, the absorbance was measured at 517 nm in a spectrophotometer (Jenway, UK). Ascorbic acid was used as a positive control.
The substrate was hydrolyzed separately using various concentrations (10, 25, and 50 mg/mL) of Alcalase, Flavourzyme, Neutrase and papain. The samples were activated with the individual enzyme conditions listed in Table 1. After the hydrolysis process, the SBP were immediately heated at 80°C for 20 min to inactivate the enzyme, followed by centrifugation at 5,000×g for 15 min and collection of the supernatants for further use. DPPH radical scavenging assay The antioxidant activity of SBP was measured with DPPH radicals according to the method of Chantaranothai et al.  Appropriate concentrations of SBP hydrolysate were mixed 1: 4 (v/v) with 200 M DPPH solution in anhydrous methanol for 30 min in the dark.
Preparation of the Plant Extract The leaves were washed thoroughly with distilled water. Then, it was oven-dried and pulverized into fine powder. 100g of the powdered leaves were weighed and macerated in 600mL ethanol in a stoppered flask for 24h. The solution was filtered and concentrated by a rotary evaporator. 2.3.