Formulation And Evaluation Of Micro-Emulsion Based Hydrogel Of Hydrocortisone For Anti-Inflammatory Acticity

Figure no 1: Ratio at 1:1                                     Figure no 2: Ratio at 2:1

Figure no 3 Ratio at 3:1

2.2.2  Preparation of Microemulsion based Hydrogel:

Table No. 1Preparation of Microemulsion based hydrogel

Hydrocortisone was dissolved in the selected oil phase. The surfactant and co-surfactant were mixed separately to prepare Smix and added to the oil phase with continuous stirring. Distilled water was added dropwise under constant stirring until a clear and transparent microemulsion was formed.Carbopol 934 was dispersed in distilled water and allowed to swell completely. Propylene glycol and preservatives were added, followed by adjustment of pH using triethanolamine to obtain a smooth gel base. microemulsion was slowly incorporated into the prepared gel base with gentle stirring until a homogeneous microemulsion-based hydrogel was obtained. The final formulation was stored in airtight containers for further evaluation.¹⁸

2.2.3  Characterization of Microemulsion

2.2.3.1 Appearance:

The appearance of the prepared microemulsion was evaluated by visual inspection under normal light conditions. The formulation was observed for color, clarity, homogeneity, phase separation, and consistency. A good microemulsion should appear transparent or slightly translucent with a uniform texture and should not show any signs of precipitation or phase separation.^19-24

2.2.3.2 pH Measurement:

The pH of the prepared hydrocortisone microemulsion was determined using a calibrated digital pH meter at room temperature. Before measurement, the instrument was standardized using buffer solutions of pH 4.0 and 7.0. The electrode was immersed into the formulation and the pH value was recorded after stabilization.The pH of the formulation was maintained within the skin-compatible range to avoid irritation and ensure suitability for topical application. Appropriate pH also plays an important role in maintaining drug stability and formulation performance.

2.2.3.3Viscosity:

The viscosity of the hydrocortisone microemulsion was measured using a Brookfield viscometer at room  temperature. An adequate quantity of formulation was placed in the sample container and analyzed using a suitable spindle at a fixed rotational speed.Viscosity is an important parameter that affects the spreadability, consistency, stability, and drug release behavior of the formulation. The prepared microemulsion showed suitable viscosity for topical application with smooth flow characteristics. ^25-30

2.2.3.4Particle Size Analysis:          

Particle size determination is an essential parameter in the evaluation. as it influences the stability, drug release pattern, and overall therapeutic performance of the system. The particle size distribution of the prepared Formulation was analyzed by using the Dynamic Light Scattering (DLS) technique.This method measures the average diameter of particles suspended in a dispersion medium and also provides information about particle uniformity. Formulation with smaller and uniformly distributed particle size are generally considered more stable and effective for drug delivery applications. Hence, particle size analysis is important for assessing the quality and performance of the developed formulation.³¹

2.2.3.5Polydispersity index(PDI):

The PDI value is commonly determined using Dynamic Light Scattering (DLS) analysis. In this technique, the scattering behavior of light caused by suspended particles is measured to evaluate particle size distribution. PDI serves as an important indicator for assessing the quality, consistency, and stability of colloidal systems.PDI value below 0.1 indicates excellent homogeneity of particles.

7.4.6. Surface Charge Potential:

The surface charge potential of the hydrocortisone microemulsion was measured using a zeta sizer. This study was carried out to determine the electrical charge present on the surface of the droplets and to evaluate  the physical stability of the formulation.A sufficient zeta potential value helps in preventing droplet aggregation by providing electrostatic repulsion between particles. The obtained results indicated good stability of the prepared microemulsion formulation.

2.2.3.6 Stability study:

The stability study of the prepared microemulsion was carried out to evaluate the physical and chemical stability of the formulation during storage. The optimized formulation was stored in tightly closed containers under different storage conditions such as room temperature and accelerated temperature conditions for a specified period.During the study, the formulation was periodically examined for changes in appearance, color, homogeneity, phase separation, pH, viscosity, and drug content. ^31-34

 2.2.3.6 Drug content:

The drug content of the prepared microemulsion was determined to evaluate the amount of drug present in the formulation. Accurately weighed quantity of Microemlsion was dissolved in phosphate buffer pH 7.4 and mixed properly using a magnetic stirrer or shaker to obtain a uniform solution. The prepared solution was then filtered to remove any undissolved particles.A suitable quantity of the filtrate was withdrawn and further diluted with phosphate buffer pH 7.4. The absorbance of the diluted solution was measured using a UV–Visible spectrophotometer at the selected wavelength against phosphate buffer as blank. The drug content of the formulation was calculated using the standard calibration curve of the drug.

2.2.4 Characterization of Microemulsion based hydrogel

2.2.4.1 pH

The pH of the prepared microemulsion-based hydrogel was determined using a calibrated digital pH meter at room temperature. The pH of topical formulations should be compatible with the skin to avoid irritation and discomfort during application. The prepared formulation showed pH within the acceptable skin-friendly range, indicating its suitability for topical administration and good formulation stability.³⁵

2.2.4.2Spreadability

The spreadability of the developed gel formulation was assessed by employing the slip and drag method using two glass slides. A known quantity of gel was carefully placed between the slides, and a uniform film was formed by applying a fixed weight on the upper slide. Care was taken to ensure even distribution of the gel without trapping air bubbles.After removing the excess formulation, an additional weight was applied to the upper slide, and the time taken for the slide to move a certain distance over the lower slide was measured with the help of a stopwatch. This method is commonly preferred for semisolid formulations because it is simple, accurate, and easy to perform. The spreadability of the gel was then calculated by using the standard formula.

7.5.3 Viscosity

The viscosity of the hydrocortisone microemulsion-based hydrogel was measured using a Brookfield viscometer at room temperature. The viscosity of the formulation influences its consistency, stability, drug release, and ease of application.The prepared hydrogel showed appropriate viscosity with smooth and homogeneous consistency. Suitable viscosity helps the formulation remain at the site of application for a longer duration and provides better topical performance.

7.5.4 Scanning Electron Microscopy (SEM)

Scanning Electron Microscopy (SEM) analysis was performed to study the surface morphology and structural properties of the prepared hydrocortisone microemulsion-based hydrogel. SEM helps in examining the surface appearance, texture, and internal structure of the formulation at high resolution.For the analysis, a small quantity of dried hydrogel sample was fixed on an aluminum stub using adhesive tape and coated with a thin gold layer to improve conductivity. The prepared sample was then observed under a Scanning Electron Microscope at suitable magnification.³⁶

2.2.4.2 In vitro anti-inflammatory activity by Protein denaturation method

The reaction mixture (10 mL) consisted of 0.4 mL of egg albumin, 5.6 mL of phosphate buffered saline (PBS, pH 6.4) and 100 µL of different concentration sample. Similar volume of double-distilled water served as control. Then the mixtures were incubated at (37⁰c ±2) in a incubator for 15 min and then heated at 70^oc for 5 min. After cooling, their absorbance was measured at 660 nm by using vehicle as blank. Diclofenac sodium at the concentration was used as reference drug and treated similarly for determination of absorbance. The percentage inhibition of protein denaturation was calculated by using the following formula,

% Inhibition = C –T/ C

Where,

T = absorbance of test sample

2.2.4.3 In vitro drug relase study

The in vitro drug release study of the hydrocortisone-loaded microemulsion based hydrogel was carried out using the dialysis membrane diffusion technique. Phosphate buffer solution of suitable pH was used as the dissolution medium to maintain sink conditions and ensure proper diffusion of the drug throughout the study. A measured quantity of the prepared microemulsion based hydrogel was placed carefully inside a pre-soaked dialysis membrane, and both ends of the membrane were tied securely to prevent leakage of the formulation.The prepared dialysis bag was then suspended in the receptor compartment containing the dissolution medium and maintained at a temperature of 37±2°C under continuous stirring at 50 rpm to simulate physiological conditions. At predetermined time intervals, a specific volume of sample was withdrawn from the receptor medium and immediately replaced with an equal quantity of fresh dissolution medium in order to maintain constant volume and sink conditions during the experiment.The withdrawn samples were suitably diluted and analyzed using a UV-visible spectrophotometer at the selected wavelength of hydrocortisone. The cumulative percentage drug release from the microemulsion based hydrogel formulation was calculated and recorded for evaluation of the drug release behavior.^38-40

3.RESULTS AND DISCUSSION:

3.1 Preformulation Studies and Characterization of Drugs

3.1.1. Organoleptic Properties

Hydrocortisone was observed as a white to slightly creamy crystalline powder with no characteristic odour. The appearance and texture confirmed the purity and identity of the drug sample.

Table .2 Identification tests for Hydrocortisone

3.1.2Solubility:

The solubility behaviour of Hydrocortisone was evaluated in different solvents used for Microemulsion preparation. The drug showed varying degrees of solubility depending on the solvent system.

Table 8.2: Solubility profile of Hydrocortisone in different solvents

3.1.3 Melting point:

The melting point of Hydrocortisone was determined and found to be approximately 215°C–220°C, which is in agreement with the reported standard values. The obtained result indicates good purity and stability of the drug.

Table .3 Melting point of Hydrocortisone

3.1.4Analytical studies by UV-Spectrophotometry:

Determination of λmax of Hydrocortisone:

The maximum wavelength was found to be 242nm from the Hydrocortisone spectra which was chosen for the study.

Standard calibration curve Hydrocortisone:

The graph of absorbance versus concentration for pure Hydrocortisone was obtained at 223 nm using UV–Visible spectroscopy. The calibration curve was plotted by using the values of concentration and corresponding absorbance data.

Table No.4 Absorbance value at various concentration of Hydrocortisone in methanol

Fig. No. 4Calibration curve of Hydrocortisone in methanol

3.1.5 Identification by FTIR spectroscopy:

FTIR of Hydrocortisone:

The FTIR spectrum of Hydrocortisone was recorded using an FTIR spectrophotometer. The spectrum was obtained in the range of 4000 to 400 cm⁻¹. The IR spectrum showed the characteristic peaks corresponding to the functional groups present in hydrocortisone. From the observed characteristic peaks, it was concluded that hydrocortisone was present in pure form and exhibited all the characteristic functional groups of the drug.

Fig. No. 5 FTIR Spectrum of Hydrocortisone Drug

The FTIR spectrum of Hydrocortisone was recorded using an FTIR spectrophotometer (Alpha II Bruker). The spectrum was obtained in the range of 4000 to 400 cm⁻¹ and is shown in Figure. The observed peaks at 3432.56 cm⁻¹ indicated O–H stretching of the hydroxyl group, while the peaks at 3011.76 cm⁻¹ and 2967.16 cm⁻¹ corresponded to C–H stretching vibrations. A strong characteristic peak observed at 1719.17 cm⁻¹ confirmed the presence of the carbonyl (C=O) functional group. The spectrum also showed characteristic peaks for alkane, alcohol, and aromatic groups. From the obtained FTIR spectrum, it was concluded that hydrocortisone was present in pure form and showed all characteristic functional groups of the drug.

3.1.6 Drug-excipient compatibility studies by IR spectroscopy:

FTIR of Hydrocortisone with oleic acid

Fig. No. 6 FTIR Spectra of Hydrocortisone and Oleic acid

The FTIR spectrum of the physical mixture of Hydrocortisone and oleic acid was recorded using an FTIR spectrophotometer (Alpha II Bruker). The spectrum was obtained in the range of 4000 to 400 cm⁻¹ and is shown in Figure. The characteristic peaks observed at 3005.86 cm⁻¹, 2929.44 cm⁻¹, and 2853.98 cm⁻¹ indicated C–H stretching vibrations of alkane and aliphatic chains. A strong peak observed at 1708.05 cm⁻¹ confirmed the presence of the carbonyl (C=O) functional group. Peaks at 1459.10 cm⁻¹ and 1283.64 cm⁻¹ corresponded to C–H bending and C–O stretching vibrations respectively. The characteristic peaks of hydrocortisone and oleic acid were retained without significant shifting, indicating the compatibility of the drug with the excipient and absence of chemical interaction.

FTIR of Hydrocortisone with Tween 80

Fig. No.7  FTIR Spectra of Hydrocortisone and Tween 80

The FTIR spectrum of the physical mixture of Hydrocortisone and Tween 80 was recorded using an FTIR spectrophotometer (Alpha II Bruker). The spectrum was obtained in the range of 4000 to 400 cm⁻¹ and is shown in Figure. The broad peak observed at 3499.32 cm⁻¹ indicated O–H stretching vibration of the hydroxyl group. The peaks at 2920.13 cm⁻¹ and 2860.45 cm⁻¹ corresponded to C–H stretching vibrations of aliphatic chains. A characteristic peak at 1734.94 cm⁻¹ confirmed the presence of the carbonyl (C=O) functional group. The peaks observed at 1247.50 cm⁻¹ and 1093.65 cm⁻¹ indicated C–O stretching vibrations. The characteristic peaks of hydrocortisone and Tween 80 were retained without major shifting, indicating good compatibility and absence of chemical interaction between the drug and excipient.

FTIR of prepared Microemulsion

Fig. No. 8 FTIR of Microemulsion

The FTIR spectrum of the prepared microemulsion containing Hydrocortisone was recorded using an FTIR spectrophotometer. The spectrum was obtained in the range of 4000 to 400 cm⁻¹ and is shown in Figure. The broad peak observed at 3355.69 cm⁻¹ indicated O–H stretching vibration of the hydroxyl group. The peaks at 2925.73 cm⁻¹ and 2868.46 cm⁻¹ corresponded to C–H stretching vibrations of aliphatic chains. A characteristic peak observed at 1708.91 cm⁻¹ confirmed the presence of the carbonyl (C=O) functional group. The peaks at 1290.01 cm⁻¹ and 1131.32 cm⁻¹ indicated C–O stretching vibrations. The characteristic peaks of hydrocortisone and excipients were retained without significant shifting, indicating compatibility of the drug with excipients and successful formulation of the microemulsion without any chemical interaction.

3.1.7 Differential Scanning Calorimetry (DSC):

 DSC study Hydrocortisone

Fig. No.9DSC of Liranaftate Drug:

The DSC thermogram of Hydrocortisone was recorded using a Differential Scanning Calorimeter under nitrogen atmosphere at a heating rate of 10 °C/min. The DSC curve showed a sharp endothermic peak at 223.48 °C, which corresponds to the melting point of hydrocortisone. The onset temperature was observed at 221.09 °C with an enthalpy value of 113.15 J/g. The sharp and well-defined endothermic peak indicated the crystalline nature and purity of the drug. The obtained DSC thermogram confirmed that hydrocortisone was present in stable crystalline form without any sign of decomposition or impurity.

3.2 Experimental Design

ANOVA for Quadratic model

Response 1: Particle Size

Table No.5 Response Table of Particle Size

The Model F-value of 1820.14 implies the model is significant. P-values less than 0.0500 indicate model terms are significant. In this case A, B, A², B² are significant model terms.

Fit Statistics

Table No. 6 Fit Statistics of Particle size

The Predicted R² of 0.9961 is in reasonable agreement with the Adjusted R² of 0.9991; i.e. the difference is less than 0.2.

Adeq Precision measures the signal to noise ratio. A ratio greater than 4 is desirable. Your ratio of 118.812 indicates an adequate signal. This model can be used to naviFinal Equation in Terms of Coded Factors

Response 1: Particle Size, Y1

Y1 = 146.42+7.83X₁-2.47X₂+0.2250X₁X₂-0.8333X₁²-1.43X₂²  …. Eq (1)gate the design space.

Fig no 10 2D Contour plots and 3D Response surface plots  for evaluating influence of Oleic acid (X1) and Smix (X2) on Particle size (Y1)

Effect of Independent Variables on Particle Size

The particle size of the prepared microemulsion-based hydrogel was strongly affected by the concentration of oleic acid (X1) and Smix (X2). It was observed that increasing the amount of oleic acid led to an increase in particle size because a higher oil content produced larger dispersed droplets within the system. On the other hand, increasing the concentration of Smix decreased the particle size due to effective reduction of interfacial tension and improved stabilization of droplets. The obtained contour and response surface plots cleay demonstrated that both formulation variables significantly influenced particle size and overall formulation stability.

 ANOVA for Quadratic model

Response 2: Viscosity

Table No. 7 Response of Viscosity

The Model F-value of 14100.68 implies the model is significant. P-values less than 0.0500 indicate model terms are significant. In this case A, B, B² are significant model terms.

Fit Statistics

Table -8

The Predicted R² of 0.9996 is in reasonable agreement with the Adjusted R² of 0.9999; i.e. the difference is less than 0.2.

Adeq Precision measures the signal to noise ratio. A ratio greater than 4 is desirable. Your ratio of 325.202 indicates an adequate signal. This model can be used to navigate the design space

Response 2: Viscosity, Y2

Y2 = 6063.11+699.67X₁+188.00X₂+0.5000X₁X₂+0.3333X₁²+35.33X₂² ……. Eq (2)

Figure .11 2D Contour plots and3D Response surface plots for evaluating influence of Oleic acid (X1) and Smix (X2) on Viscosity (Y2)

Effect of Independent Variables on Viscosity

The viscosity of the developed formulation was significantly influenced by both oleic acid (X1) and Smix (X2). An increase in oleic acid concentration resulted in higher viscosity because of the increase in internal phase volume and thicker consistency of the formulation. Similarly, higher concentration of Smix also contributed to improved viscosity by enhancing the structural arrangement and uniformity of the microemulsion system. The response surface analysis confirmed that both variables showed a positive effect iscosity and helped in obtaining a stable formulation with suitable consistency for topical application.

3.3 Characterization of Microemulsion

3.3.1 Appearance:The microemulsion appeared translucent to slightly opalescent with no phase sepration or creaming,indicating stable dispersion and successful emulsification.

3.3.2 Viscosity

The viscosity of the prepared microemulsion formulations ranged from 52 cP to 98 cP, indicating low to moderate viscosity suitable for topical microemulsion application.

 3.3.3 Particle size

The particle size of the prepared microemulsion formulations ranged from 133.6 nm to 148.0 nm, indicating the successful formation of nano-sized globules. Among all formulations, batch F3 showed the smallest particle size of 133.6 nm, which suggested better emulsification efficiency and uniform droplet distribution.

Fig .12 Particle size Analysis

    8.3.4 Polydispersity Index

The polydispersity index (PDI) values of the prepared nanoemulgel formulations were found to be in the range of 0.286 to 0.417, indicating satisfactory uniformity in particle size distribution. Lower PDI values represent better homogeneity and stability of the formulation. Among all the batches, formulation F4 exhibited the lowest PDI value (0.286), suggesting a more uniform particle distribution and improved formulation stability.

3.4.4 Zeta potential

Zeta potential analysis was performed to evaluate the stability of the prepared microemulsion system under stress and storage conditions according to ICH stability guidelines. The optimized formulation having nanosized globules showed a zeta potential value of –23.4 mV, which indicated good stability of the colloidal system due to sufficient electrostatic repulsion between the particles. The negative zeta potential value suggested lower chances of particle aggregation and confirmed uniform dispersion of the formulation.

Fig 8.9:Zeta Potential

3.4.5 Stability study:

The formulation was also observed no any signs of creaming, cracking, or microbial growth. The obtained results indicated that the prepared microemulsion-based hydrogel remained stable throughout the study period without showing any significant changes in its physicochemical properties.The stability study confirmed that the developed formulation possessed good stability and was suitable for topical application.

3.4.6 Drug content:

The drug content of the prepared microemulsion formulations was found to be in the range is 91.24% indicating uniform distribution of drug within the formulation.  showed satisfactory drug content, which confirms proper mixing and good incorporation of the drug into the Microemulsion. the highest drug content of 98.63%, suggesting better drug entrapment and formulation uniformity. The obtained results indicate that the prepared microemulsion formulations possessed acceptable drug content suitable for topical application.

3.5 Characterization of Microemulsion based hydrogel:

3.5.1 Visual Inspection

The prepared Hydrocortisone loaded of microemulsion based hydrogel  was found to be transperent,smooth,uniform in apperance there is no sign of phase sepration

3.5.2 pH Determination

The pH of the prepared microemulsion-based hydrogel formulations was measured using a digital pH meter. About 1 g of hydrogel was dispersed in distilled water and stirred properly to obtain a uniform solution before measurement. The pH values of all formulations were found to be in the range of 5.4 to 6.4, which is considered suitable for topical application and compatible with normal skin pH. The obtained pH values indicated that the formulations may minimize the possibility of skin irritation and are appropriate for dermal drug delivery.

3.5.3 Spredability

The spreadability of the prepared nanoemulgel formulations was evaluated to determine the ease of application on the skin surface. The obtained spreadability values were found in the range of 13.58 to 18.14, indicating satisfactory consistency and smooth spreading characteristics of the formulations. Good spreadability ensures uniform distribution of the gel over the affected area and enhances patient acceptability. Among all the formulations, batch F3 exhibited the highest spreadability value of 18.14, suggesting better applicability and suitability for topical administration.

3.5.4Viscosity:

The viscosity of the prepared microemulsion-based hydrogel formulations was determined using a Brookfield viscometer at controlled shear rate conditions. It was observed that the viscosity of the formulations increased gradually with an increase in polymer concentration. The obtained viscosity values indicated good consistency, satisfactory spreadability, and suitable rheological behavior for topical application. The results suggested that the polymer concentration significantly influenced the viscosity and stability of the hydrogel formulations.

3.5.5 SEM of microemulsion based hydrogel:

Fig 8.10:Scanning electron microscopy

The scanning electron microscopy (SEM) image of the Microemulsion based hydrogel is presented in the figure. The SEM analysis showed that the droplets were nearly spherical or globular in shape and present within the micrometer size range. The micrograph also indicated slight aggregation of particles, which may have occurred due to the removal or evaporation of water during the sample preparation process before SEM examination.

3.5.7 Ant inflammatory activity:

Fig 8.11:Anti inflammatory activity by PDA method

Table No.9 Anti-inflammatory activity

anti-inflammatory activity of the microemulsion-based hydrogel was evaluated using the Protein Denaturation Assay (PDA) method and compared with the standard drug at different concentrations (20–100 µg/ml). The results revealed a concentration-dependent increase in the percentage inhibition of protein denaturation for both the standard and the formulated hydrogel. The standard drug exhibited strong anti-inflammatory activity with inhibition values of approximately 22%, 41%, 58%, 75%, and 88% at increasing concentrations. The microemulsion-based hydrogel also demonstrated appreciable inhibition, showing around 8%, 17%, 25%, 39%, and 54% inhibition at the respective concentrations. Although the activity of the hydrogel formulation was lower than that of the standard, the steady increase in inhibition with concentration indicates significant anti-inflammatory potential. The ability of the formulation to inhibit protein denaturation suggests that the microemulsion-based hydrogel may be effective in reducing inflammatory responses and could serve as a promising topical anti-inflammatory delivery system

3.5.7 in vitro drug release study:

Table No. 10. in vitro Drug release study

Fig. No. 12 in vitro Drug Release

Fig. No. 13 Zero-Order Kinetics

Fig. No. 14 First-Order Kinetics

Fig. No. 15  Higuchi model

Fig no.16:Hixcon crowell model

Fig no .17:Korsemeyer peppas model

Table 8.21:Release kinetic Models