Development of Transdermal Herbal Formulation with Sustained Release for the Treatment of Mastitis in Dairy Cattle

Introduction

Mastitis is one of the most common source of economic problems encountered by the dairy farmers. Bovine mastitis, inflammation of the mammary gland that is primarily caused by pathogenic microorganisms is a major health hazard in the cattle. Mastitis affects the quality and quantity of milk. Mastitis is a pervasive disease, occurring worldwide, that affects the productivity of a dairy. In the dairy industry, a clinical case of bovine mastitis can cost > 5,000 rupees and up to 20,000 rupees in high-yielding cows, due to milk yield losses, increased mortality, and treatment costs.^[1,2] Mastitis has harmful effects on reproduction in cows, on the yield of milk, and shelf life of dairy products derived from the cow’s milk.^[3] Ethnoveterinary medicines refer to people’s belief, knowledge, skill, and practice relating to the care of their farm.[4] Antibiotics are used in the treatment and control of mastitis, but an intramammary infusion of antibiotics for mastitis therapy was cited as a major reason for milk contamination^[5] and frequent use of antibiotic therapy led to antibiotic resistance.^[6] To address this, alternative therapy to antibiotic therapy for mastitis treatment and control had to be developed such as those using authenticated herbal preparations. Curcumin is derived from Curcuma longa L.^[7] It is a naturally occurring yellow pigment commonly known as turmeric, which is used as a flavouring agent in both vegetarian and non-vegetarian food dishes. Anti-inflammatory,^[8,9] antioxidant^[10,11] and antibacterial^[12] effects of curcumin have been identified. A transdermal patch is a drug delivery system that delivers therapeutic substances through skin for systemic effects at a predetermined and controlled rate. It is made up of a supporting membrane, a matrix incorporated with the drug, and a release liner with/without a rate-controlling membrane. Design-Expert software 12 is a statistical optimization software used to design trial experiments and by analysing response surface plots obtained, an optimised formulation can be formulated. ².

Materials And Methods

2.1 Materials

An authenticated sample of curcumin was procured from Indus Herbs. All chemicals and reagents used were of analytical grade.

2.2 Methods

2.2.1 Drug excipients compatibility studies

The Fourier-transform infrared spectroscopy (FTIR) studies for curcumin alone and along with polymers was done at a resolution of 4 cm-1 over 400-4000 cm-1 wavelength region. The FTIR spectrophotometer (model: Alpha Broker) employed attenuated total reflectance– FTIR technique to determine infrared transmission spectra and record the characteristic peaks.^[13]

2.2.2 Preparation of transdermal patches

The transdermal patches were prepared using the solvent casting technique. The casting solutions for transdermal patches were prepared as per the composition given in Table 1 and Table 2. The required quantities of HPMC and PVP K30 were weighed, dissolved in measured quantities of methanol and chloroform (1:1). Curcumin (20 mg) was dissolved in the solvent mixture and stirred thoroughly to form a homogeneous mixture. This mixture was then poured on the mercury surface placed in the petridish, between the round bangle and then covered using a glass funnel and allowed to dry at room temperature for 24 h by the solvent evaporation technique. The patches were then removed and cut into required dimensions. The prepared patches were kept in the desiccator for 2 days for further drying, wrapped in an aluminium foil and then packed in self-sealing covers.^[14]

2.2.3 Preliminary screening

The preliminary study was carried out to evaluate the effect of various polymer combinations on transdermal patch formulation. The composition of preliminary trial batches F1 to F3 are presented in Table 1.

2.2.4 Optimization of variables using full factorial design.

A 22 -randomized full factorial design was employed in the present study. In this design, two independent factors were evaluated, each one at 2 levels and experimental trials were performed for all the 4 possible combinations. Different concentrations of HPMC (X1) and PVP K30 (X2) were selected as independent variables in 22 full factorial designs. The % cumulative drug release at 24 h (Q1), moisture content (Q2), and folding endurance (Q3) were taken as dependent variables. The formulation layout for the factorial design batches (P1 to P4) are presented in Table 2.

2.2.5 Preparation of Calibration Curve Determination of absorption maximum (λmax)

The wavelength of light in the ultraviolet range at which the compound shows maximum absorbance is λmax. The standard calibration curve of curcumin at 421.0 nm was considered (Figure 1). Correlation Coefficient (r2 ) = 0.992 Equation for regressed line, y = 0.1545x - 0.0083 Slope of regressed line = 0.1545 where x = concentration (μg/ml), y = absorbance

2.2.6. Evaluation

Thickness of the patch

The average thickness of the patch was measured by measuring the thickness of drug loaded patch at different points using a screw gauge. The standard deviation was also calculated for the same to ensure exact thickness of the prepared patch.^[15]

Weight uniformity

The prepared patches were dried at 60°C for 4 hours before testing. A different part of the patch with specified area was cut and weighed on a digital balance. The average weight and standard deviation values were calculated from individual weight.[15,16]

Folding endurance

An evenly cut strip was taken, folded repeatedly at the same place until it broke. The number of times the film can be folded at the same place without breaking was its value of folding endurance.^[17]

Percentage moisture content = 

A desiccator containing fused calcium chloride was taken and the prepared patches were kept inside it at room temperature for 24 hours. Then, the films were weighed and the percentage moisture content was determined from the below mentioned formula.^[15,16,17]

Percentage moisture uptake = 

A desiccator containing a saturated solution of potassium chloride was taken in order to maintain 84% RH. Prepared films were weighed and kept inside the desiccator at room temperature for 24 hours. Then, the films were weighed again and the percentage moisture uptake was calculated using the below mentioned formula.^15,16,17

In vitro drug diffusion studies

The in vitro diffusion study was carried out by using a cellophane membrane with the help of a Franz diffusion cell. The cylinder consisted of two chambers, one being the donor and other being the receptor compartment. The donor compartment was open at the top and was exposed to the atmosphere. Temperature was maintained at 37 ± 0.5o C and the receptor compartment was provided with a sampling port. The diffusion medium used here was phosphate buffer (pH 7.4). A bent stainless-steel pin/ small magnetic bead was used to stir the receptor solution using a magnetic stirrer. The formulated patches were cut into a size of 1cm2 and placed over the drug release membrane and the receptor compartment. Both the compartments were held tightly using clamps. Phosphate buffer with pH 7.4 was the solution used in the receptor. The volume of diffusion cell was 134 ml and temperature was maintained at 37 ± 2°C with the help of a magnetic stirrer. Diffusion was carried out for 24 h and 5 ml sample was withdrawn at an interval of 1, 2, 3, 4, 5, 6, 7, 8, and 24 h. The same volume of phosphate buffer with pH 7.4 was added to the receptor compartment to maintain sink conditions and the samples were analysed at 204 nm using the ultraviolet spectrophotometer.^[15,17]

Results and Discussion

3. Results

3.1 FTIR Study

The FT-IR spectrum of pure curcumin showed absorption bands at 3625 cm-1, which indicated the presence of hydroxyl group, the peak at 2915 cm-1 indicated the presence of C-H stretching, the peak at 1601 cm-1 indicated the presence of C-C stretching, the peak at 1314 cm-1 indicated the presence of enol C-O peak. Mixing curcumin with other polymers in the form of a physical mixture for preparing a formulation did not show any interference with the characteristic drug peaks. This finding confirmed the compatibility of drug with the studied additives.

3.2 Physical evaluation

All the transdermal patches were visually inspected for colour, clarity, flexibility and smoothness. The patches appeared yellow in colour, translucent, flexible, and were smooth to touch.

3.3 Physicochemical evaluation of transdermal patches

Physicochemical evaluation of the prepared formulations was carried out in terms of weight variation, thickness, folding endurance, moisture content, and moisture uptake. Variables such as moisture content and moisture uptake have a large impact on the release behaviour of drug from transdermal patches.^[18] It was concluded that as the concentration of hydrophilic polymer (HPMC) increased in the patches, the moisture content and moisture uptake of patches increased as the hydrophilic part of the polymer blend dissolved and eroded easily on contact with the aqueous medium.

3.4 Analysis of suitability of the model

The essential components of the statistical design comprised of input variables (factors such as the concentration of polymers) and their responses, experiment design, statistical analysis, and optimization of the formulation. The two-square factorial design was used to study the combined effect of process variables such as HPMC concentration and PVP concentration on the dependent variables i.e. drug diffusion, moisture uptake and folding endurance. The adequacy of the models was analysed and the model summary was prepared (Table 5, 6 and Table 7).

Effect of formulation variables on % cumulative drug release at 24 hours

Analysis of variance (ANOVA) was used to evaluate experimental data and the p-value of regression coefficients was used to measure the level of significance (Table 4). Generally, % cumulative drug release (CDR) is a good tool to illustrate the drug release characteristics of the film. A film with low drug release would also have poor bioavailability. The use of proper percentage of a permeation enhancer, type and concentration of hydrophilic/hydrophobic polymer would enhance the drug release from the film. The data presented in Table 4 shows that drug release percent ranged from 59.92 to 81.29%. A linear model was observed to fit the % drug permeation (response) with a p-value of <0.05 and an F value of 199.83. For this model, the coefficients A and B were found to be significant. From the response surface plot, it was evident that the concentration of HPMC and PVP were the major variables influencing the % cumulative drug release at 24 hours in a positive trend, with a p-value of <0.05 (Table 5). As the concentration of HPMC increased, the %CDR at 24 hours also increased significantly. The accuracy of this model was confirmed by the R2 value, 0.9975, as determined by ANOVA.

Effect of formulation variables on folding endurance

ANOVA was used to evaluate experimental data, and p-value of regression coefficients was used to measure the level of significance (Table 4). Generally, folding endurance is a good tool to illustrate the durability of a film. A film with a low folding endurance is very brittle and thin while a film with a high folding endurance is thick. The use of proper percentage of plasticizer and concentration of hydrophilic/hydrophobic polymer enhances the drug release from the film. The data presented in Table 4 shows that folding endurance ranged from 72 to 95. A linear model was observed to fit for response folding endurance with a p and F value of <0.0272 and 673, respectively. For this model coefficients, A and B were found to be significant. The p-value was used to determine the significance of individual coefficients and the interaction strength between variables. From the response surface plot, it was observed that the concentration of HPMC and PVP were the major variables influencing the folding endurance in a positive trend with a p-value of <0.0272 (Table 6). Accuracy of this model was confirmed by the R2 value, 0.9993, as determined by ANOVA.

Effect of formulation variables on moisture content

ANOVA was used to evaluate the experimental data, and p-value of regression coefficients was used to measure the level of significance (Table 4). The moisture content also had an effect on the drug release of the film. A film that has low moisture content is very dry and hard, a film that has high moisture content is too flexible. The use proper concentration of hydrophilic/hydrophobic polymer is important. The data presented in Table 4 demonstrated that moisture content ranged from 10.85 to 14.21%. A linear model was observed to fit for response folding endurance with a p and F value of <0.0443 and 253.92, respectively. For this model coefficients, A and B were found to be significant. P-value was used to determine the significance of individual coefficients and interaction strength between variables. From the response surface plot, it was found that the concentration of HPMC and PVP are major variables influencing the folding endurance in positive trend with a p-value of <0.0272 (Table 7) and the effect of HPMC was profound. Accuracy of this model was confirmed by the R2 value, 0.9993, as determined by ANOVA.

3.5 Optimization

Release of the drug from transdermal film varied with the structure and morphology of the polymer matrix and physicochemical properties of the drug.[19] The hydrophilic nature of HPMC (X1) exerted a positive effect on the release of curcumin owing to its highwater absorbing property. This in turn increased the porosity and pore diameter of the polymer matrix thereby allowing drug molecules to diffuse out easily. ^[20] Moreover, an increase in HPMC loading resulted in faster dissolution of the polymer matrix resulting in the formation of channels for diffusion of the drug from the film.^[21] As a final point, after analysis of experimental variables, an optimum formulation of curcumin film containing acceptable drug release, moisture content and folding endurance was derived, that contained 200 mg HPMC and 50 mg PVP. The desired mechanical and physicochemical properties were ensured by the predicted values of responses for optimized formulation. The predicted values of responses for the optimized transdermal film were very close to the observed values with no considerable prediction error percentage (it was below 6%) and residuals. This outcome confirmed that the optimization technique used is highly reliable and reproducible for the development of curcumin transdermal film with high-quality attributes.^{[22, 23]}

Conclusion

Transdermal patches of curcumin were successfully prepared by using hydroxypropyl methylcellulose and polyvinylpyrrolidone (PVP K30). TDDS of curcumin were prepared by solvent evaporation method and evaluated for different parameters. The two-factorial design was an efficient tool in optimizing the two variables used to prepare curcumin TDDS through studying their effect on the quality attributes and diffusion behaviour of prepared curcumin-loaded transdermal films. The optimized formulations showed good physicochemical and mechanical properties. The in vitro studies reveal that the formulations containing herbal ingredients such as curcumin have the potential to be used as effective alternatives to synthetic current drugs available in the market to treat mastitis. This research work indicates that curcumin may be incorporated into a transdermal drug delivery system. Further research on optimization and in vivo studies of herbal formulations is essential to design products with better features in the future. As these in vitro studies have shown promising results, further pharmacodynamics and pharmacokinetic data should be collected while treating dairy cattle for mastitits.

Conflicts of interest

None.

Acknowledgements

This research project was funded by the Rajiv Gandhi University of Health Sciences under the Short-term Research Grants for Undergraduate Students (Grant No: UGPHA408). The authors are thankful to RGUHS and the Karnataka College of Pharmacy, Bangalore for providing facilities needed to successfully carry out this study.