All ingredients were purchased from regional distributors and from LOBA Chemie Pvt. Ltd, Mumbai, India. Melting points were found to be uncorrected.
Ethical Clearance: The protocol operated in this study regarding the use of rats as an animal model for acute oral and dermal toxicity was affirmed by the Institutional Animal Ethical Committee, GIPER, Limb, Satara.
2.1 Synthesis36
Preparation of Chloroacetyl Xanthan gum:
Chloroacetyl xanthan gum was formulated by treating 10 gm xanthan gum with 100 ml pyridine. 5 ml chloroacetyl chloride was added to above mixture slowly with stirring. Stirring was done for further 2 hours and leave the reaction mixture overnight for digestion. Filtered residue was washed using rectified spirit and dried.
Preparation of acetyl amine xanthan gum:
10 gm of chloro-acetyl xanthan gum was dispersed in a flask containing in 50 ml rectified spirit. The 10 ml ammonia solution was added with shaking. After 2 hours stirring mixture was filtered and washed with rectified spirit. The solid residue was dried and used for further activity.
2.2 Characterization
2.2.1.1 Fourier transform infra- red spectroscopy: BRUKAR FT-IR alpha ATR spectrometer was used for spectral determination of MXG and XG.
2.2.1.2 Proton Nuclear Magnetic Resonance: VARIAN MERCURI YH-300 NMR Spectroscopy machine used for analysis of 1H-NMR spectra of MXG and XG. TMS was used as Internal standard. The solvent for xanthan gum and amino acetyl xanthan gum is D2O.
2.2.1.3 Differential Scanning Calorimetry (DSC): Thermogram for XG and MXG were reported in 400C – 3000C temperature range at a heating rate 10 0C/min.
2.2.1.4 X-ray diffraction: X-ray diffractometer (Bruker AXS D8 Advance) along with copper Kα-radiation produced at35 mA and 40 K v in the differential angle range of 3-60 (2θ) were used for recording X-ray diffraction of MXG.
2.2.1.5 Scanning Electron Microscopy: XG and MXG surface morphology and shape were analyzed using scanning electron microscope. The gold coated samples were mounted on aluminum stub having double layer carbon adhesive tape. The electron micrographs were measured at10 kv accelerated voltage.
2.3 Physiochemical Evaluation
2.3.1 Evaluation of mucoadhesive strength of gel37:
Modified physical balance method was used to evaluate comparative mucoadhesive strength of MXG gel. The goat buccal mucosa was collected from the slaughter house and cleaned to remove the underlining fat and loose tissue. The membrane was washed with distilled water and then with simulated saliva having pH 6.8 at 37 oC. On the vial with rubber closure was coated by immersing into standard and test mucoadhesive agent solution (1% w/v). One end of the vial was attached with nylon thread and provision was given to raise the weight at other end. Weight was put on at specific time intervals to separate mucous from glass plate. The time and weight required to detach from the buccal mucosa was recorded as the ex vivo bio adhesion time and detachment force respectively. The result was reported as an average of six observations. The force of adhesion was calculated as
Force of adhesive= 0.00981/2
2.3.2 Viscosity38: Brookfield viscometer (Brookfield DV-E Vis-cometer) were used for determination of the viscosity of xanthan gum and modified xanthan gum viscosity using spindle L4 at 50 and 100rpm speeds.
2.3.3 Gelling Property36: Gelling property was determined as previously described method. The solution of Acetyl amine xanthan gum and xanthan gum (0.2–1%, w/v) in water were prepared in test tube and left for overnight. The consistency of gel in test tubes were examined by tilting at 90o and categorized them as solutions, viscous solution or gels on the basis of visual inspection.
2.3.4 Gelling Capacity36: XG and MXG were examined for gelling capacity by incorporating a solution drop in a vial having newly prepared 2 ml mimicked tear fluid. The time required for its gelling was inspected visually.
2.3.5 Swelling Index39: Gravimetrically determined water uptake of modified xanthan gum. 30 mg disc (prepared by a hydraulic single punch press, diameter 5.0-mm) was fixed on a small mesh and immersing them into saliva solution (pH 6.75 at 37±0.5ºC) in a beaker, weighing of the disc at preplanned time points. Excess water from the swollen discs were evacuated before measuring, Water-uptake was calculated using following formula:
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Where, Wt=Disc Wt. at given time.
Wo=Initial Wt.
2.3.6 Toxicological Study
2.3.6.1 Acute Oral toxicity40: It was performed as perOECD-423 guidelines. In brief, the healthy young adult female rats ranging between 8- 12 weeks and having 140 ± 10 gm weight were used. The temperature and the relative humidity of room where maintained at 22±30C and 50% respectively. Artificial lighting with 12 hours light, 12 hours dark condition and plenty of drinking water along with laboratory diets were provided. The animals were kept for 10 days under laboratory conditions prior to begin of the study. Rats were selected randomly for the study and marked them for individual identification. Animals were fasted before dosing. The dose was decided on the basis of charts. The test substance was administered in lesser quantity for a period not more than 6 hours. Each step involved three animals. The dose level to be used as the starting dose is selected as 2000 mg/kg body weight as per flow charts of Annex 2. Animals were observed immediately after dosing during the first 30 minutes, and periodically during the first 24 hours. Special attention was given during the first 4 hours, and daily thereafter, for a total of 14 days. All observations were systematically recorded and individual records being maintained for each animal. Additional observations such as changes in skin and fur, eyes and mucous membranes, tremors, convulsions, salivation, diarrhea, lethargy and coma, and also respiratory, circulatory and behavior pattern also being recorded. Individual weights of animals were determined on the day of, or immediately prior to the administration of the test substance and at least weekly thereafter.
2.3.6.2 Acute dermal toxicity41:
It was performed as perOECD-434guidelines.The healthy young adult female rats ranging between 8- 12 weeks and having 140 ± 10 gm weight were used. With the help of shaving or clipping fur of test animals were removed from the dorsal regionof the trunk before 24 hours of the study. While removing fur of animals, area was decided on basis of weight of individual animal. The test sample along with porous gauze dressing and non-irritating tape was applied on an area which is about 10 % of the total body surface area for 24 hours. Covering of test site was done in a manner to retain test sample and the gauze dressing, further sample was removed after exposure by using water. On the basis of chart, 2000 mg/kg fixed starting dose was decided. All animals were normally observed for 14 days. Observation of animals were done within first 30 minutes, including particular attention during the first 4 hours, and every day later on for 14 days. All observations were systematically recorded, with individual records being maintained for each animal. Additional observations such as changes in skin and fur, eyes and mucous membranes, and also respiratory, circulatory, autonomic and central nervous systems, and somatomotor activity and behaviour pattern also being recorded. Individual weights of animals were determined on the day of, or immediately prior to the administration of the test substance and at least weekly thereafter.