Experimental work:
Preformulation studies:
Analysis of honey:
Materials:
Materials selected for the analysis of honey were procured from College of Pharmacy, IPS Academy, and Indore. Samples of Honey (Dabur honey) of different batches were selected and analysed where (n=3).
Parameters studied:
Sensory evaluation
Foreign matter
Ash value
pH
Refractive index
Moisture content
Acidity
Standard procedure was followed for the analysis of different samples of honey.
Determination of λmax:
For determination of λmax, stock solution of drug (concentration 1000μg/mL) in water was prepared by dissolving 10 mg curcumin in 10 mL of distilled water .The working solutions in the concentration range of 2-10 μg/mL were prepared. Resulting solutions were scanned in the range of 400 to 800 nm with help of UV-visible spectrophotometer, and the maximum wavelength was determined. The λmax of curcumin was found to be 420 nm.
Preparation of Calibration Curve by UV-visible Spectroscopy:
A. Preparation of Calibration Curve in distilled water: The stock solution of curcumin was prepared by dissolving 10 mg of curcumin in 10 mL methanol to produce concentration of 1000μg/mL.
Preparation of standard solutions: Standard solutions were prepared in the concentration range of 2-10μg/mL by suitable dilutions of the stock solution in methanol and absorbance were taken at 420nm in visible spectrum (Shimadzu 1800).
B. Calibration curve in PBS 6.8
Preparation of stock solution: The stock solution of curcumin was prepared by dissolving 10 mg of curcumin in 10 mL Phosphate Buffer Saline to produce concentration of 1000μg/mL.
Preparation of standard solutions: Standard solutions were prepared in the concentration range of 2-10μg/mL by suitable dilutions of the stock solution in PBS 6.8 and absorbance were taken at 420 nm in visible spectrum (Shimadzu 1800).
Formulation and optimization of gel:
Materials:
Carbopol 934p NF, triethanolamine, honey, glycerin, methyl and propyl Parabens and all other chemicals were procured from college of pharmacy IPS Academy, Indore are of analytical grade and used without further purification.
Curcumin were procured as a gift sample from Ajmera Pharmaceuticals Pvt. Ltd., Indore, India.
Preparation of gel:
The topical gel was prepared by soaking the Carbopol 934 in water for 24 h. Drug was first dispersed in small quantity of glycerin with gentle heating and then preservatives were dissolved in glycerin and then added to Carbopol solution with stirring the remaining ingredients were added to it and triethanolamine was added to the neutralize the Carbopol gel base.
Preparation of topical gel base:
S.No.
Ingredients
Quantity (w/w)
1
Carbopol 934P NF
3%
2
Honey
50%
3
Curcumin
0.5%
4
Triethanolamine
q.s.
5
Glycerin
5 mL
6
Methyl paraben
q.s.
7
Propyl paraben
q.s.
8
Distilled water
q.s.
Composition for the medicated formulation:
S.No.no
Ingredients
G1
G2
G3
G4
G5
G6
G7
G8
G9
G10
1
Carbopol (%)
1.5
1.7
2
2.5
3
1.5
1.7
2
2.5
3
2
Honey (%)
25
25
25
25
25
50
50
50
50
50
3
Curcumin
0.5g
0.5g
0.5g
0.5g
0.5g
0.5g
0.5g
0.5g
0.5g
0.5g
4
Glycerin
5 mL
5 mL
5 mL
5 mL
5 mL
5 mL
5 mL
5 mL
5 mL
5 mL
5
Triethanolamine
q.s.
q.s.
q.s.
q.s.
q.s.
q.s.
q.s.
q.s.
q.s.
q.s.
6
Methyl paraben
0.2g
0.2g
0.2g
0.2g
0.2g
0.2g
0.2g
0.2g
0.2g
0.2g
7
Propyl paraben
0.1g
0.1g
0.1g
0.1g
0.1g
0.1g
0.1g
0.1g
0.1g
0.1g
8
Distilled water
q.s.
q.s.
q.s.
q.s.
q.s.
q.s.
q.s.
q.s.
q.s.
q.s.
Evaluation of gel formulation:
pH:
The pH of prepared gel formulation was determined by using digital ph meter. 1 g of gel was dissolved in 100 mL freshly prepared distilled water and stored it for 2 hours. The measurement of pH of each formulation was done in triplicate and average values were calculated.
Viscosity:
Brookfield digital viscometer was used to measure the viscosity of prepared gel. The T shaped spindle was selected (T3) was rotated different ppm range. The reading, near to 100% torque was noted down. A sample was measured at 30±1°C.
Spreadability:
Spreadability was determined by wooden block and glass slide apparatus. Weight of about 2 g was selected and added to the pan and the time was noted for upper slide to separate completely from the fixed slide.
Spreadability was calculated by the given formula:
S= M.L/T
Where;
S= Spreadability
M= weight tied to the movable upper slide
L= length of a glass slide
T= time taken to separate the slide completely from each other.
Homogeneity:
All the formulations were tested for this parameter by visual inspection after the gel have been set in the container. They are observed for any aggregation or their appearance.
Drug content:
A specific quantity of gel generally 1 g of gel was taken and dissolved completely in 100 ml of phosphate buffer 6.8. The volumetric flask containing gel was shaked for 2 h on a mechanical shaker in order to get uniform solution. The solution was filtered by 0.45µm membrane filter and estimated spectrophotometrically at 420nm using phosphate buffer 6.8 as a blank solution.
Invitro release profile:
In- vitro release studies was performed by using a diffusion cell with a receptor compartment capacity of about 20 ml. the egg membrane was mounted between the donor and receptor compartment of the assembly.
The formulated preparation was weight up to 1g was placed over the membrane and the receptor compartment of the diffusion cell was filled with phosphate buffer 6.8. the whole assembly was fixed on magnetic stirrer, and the solution in the receptor compartment was constantly and continuously stirred using magnetic beads at 50 rpm and the temperature was maintained at 37±0.50 °C the samples of 1 ml was withdrawn at time interval of 15, 30, .60, 90, 120, 150, 180, 210, 240, 270 and 300 min., analysed for drug content spectrophotometrically at 420nm against blank. The receptor compartment was replaced with an equal volume of phosphate buffer at each time of the sample withdrawn. The cumulative graph was plotted against time.
Determination of antimicrobial activity:
Preparation of inoculums:
For evaluation of antibacterial activity, 24 hours fresh culture of bacteria was suspended in sterile water to obtain a uniform suspension of microorganism.
Determination of zone of inhibition:
Antibacterial activity is checked by agar well diffusion method. in this method a previously liquefied medium was inoculated with 0.2ml of bacterial suspension having a uniform turbidity at temperature of 40°C. 20 ml of culture medium was poured into the sterile petri dish having a internal diameter of 8.5 cm. care was taken for the uniform thickness of the layer of medium in different plates.
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After complete solidification of liquefied inoculated medium, the wells were made aseptically with cork borer having 6mm diameter. In each of the plates gel solution was placed carefully. Plates was kept for pre-diffusion for 30 min. after that plates were incubated at 37 °C for 24 hr. after incubation period was over, the zone of inhibition was measured with the help of Hi-media.
Stability studies:
It is the most important component of any formulation the acceptance and the rejection of the particular preparation depends on this study. The international conference on harmonization (ICH) guidelines titled stability testing of new drug substance and product (QIA) defines the stability test requirement for drug requirement for drug registration application in the European, USA and Japan.
Long term stability testing: 25 ± 2 °C /60 % RH ±5 % for 12 months.
Accelerated testing: 40 ± 2 °C / 75 % RH ± 5%for 6 months.
Stability studies were carried out at 40 ±2 °C /75 ± 5 % RH for the selected formulation for one month.
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