Formulation Development, Characterization and Evaluation of a Topical Microemulgel containing Tinospora cordifolia leaf extract

The materials included were the Tinospora cordifolia leaf extract (authenticated BSI/WRC/IDEN.CER/2024/62), Liquid paraffin oil, Olive oil, Peceol and Eukalyptus oil as the oil phase, Span 20, Tween 80, Stearic acid, and Polyethylene glycol 1500 as surfactants, PEG 400, Polysorbate 80, Span 80 and Span 20 as co-surfactants, and Carbopol 934 as the gelling agent, and methyl as preservative. Triethanolamine (TEA) was employed for pH adjustment, and all solvents were freshly prepared in distilled water before use. Analytical-grade reagents were used throughout the study

 

Formulation Optimization and Development of Tinospora cordifolia Leaf Extract Emulgel

Solubility Studies: Solubility studies were conducted to identify the most suitable oil, surfactant, and co-surfactant for the formulation. These studies ensured maximum solubility of T. cordifolia extract, thereby facilitating higher drug loading and stable microemulsion formation. For surfactant screening, an excess amount of the extract was added to 10 ml of each surfactant-Span 20, Tween 80, Stearic acid, and Polyethylene glycol 1500-contained in stoppered vials. The vials were placed in an incubator shaker at 37 ± 1 °C for 48 hours to attain equilibrium. After equilibration, each mixture was centrifuged at 1500 rpm for 10 minutes, and the supernatant was analyzed colorimetrically at 570 nm to determine the concentration of dissolved extract. Similarly, for co-surfactant selection, the extract was added in excess to 10 ml of each co-surfactant-PEG 400, Polysorbate 80, Span 80, and Span 20-and the same procedure was followed. For oil phase selection, the extract was added to 10 ml of various oils including liquid paraffin, olive oil, Peceol, and eucalyptus oil. After 48 hours of agitation and centrifugation, the concentration of the dissolved extract was measured at 570 nm and plot the graph as shown in fig.1 for surfactant, fig .2 for co-surfactant and fig.3 for oil phase and the values of obtained absorbance were noted in table no.1. Among all tested Tween 80 as surfactant, Span 80 as co- surfactant and olive oil was selected as the oil phase, as per the table no.1 due to its superior solubilising capacity and in-vitro drug release kinetics study by Franz diffusion cell.(6,7)

Table . 1. Solubility data of Tinospora cordifolia leaf extract in oils, surfactants, and co-surfactants (7)
Component Tested	Concentration of Drug (mg / 10 mL)	Absorbance  (λ = 570 nm)
Oils
Liquid Paraffin Oil	100 mg / 10 mL	0.74
Olive Oil	100 mg / 10 mL	1.22
Peceol Oil	100 mg / 10 mL	0.95
Eucalyptus Oil	100 mg / 10 mL	0.88
Surfactants
Span 20	100 mg / 10 mL	0.8
Tween 80	100 mg / 10 mL	1.0
Stearic Acid	100 mg / 10 mL	0.19
PEG 1500	100 mg / 10 mL	0.76
Co-Surfactants
PEG 400	100 mg / 10 mL	0.76
Polysorbate 80	100 mg / 10 mL	0.94
Span 80	100 mg / 10 mL	1.0
Span 20	100 mg / 10 mL	0.8

 

 

             

Figure .1.Solubility studies graph for selection of  surfactant         Figure .2.Solubility studies graph for selection of  Co-surfactant                                                                         

                                                                                                                                            

Figure .3.Solubility studies graph for selection of oil phase

Construction of Pseudo-Ternary Phase Diagrams

To identify the microemulsion region and optimize the ratio of surfactant to co-surfactant (Smix), pseudo-ternary phase diagrams were constructed using the water titration method. The surfactant and co-surfactant were mixed in weight ratios of 1:1, 1:2, 1:3, 2:1, and 3:1, and each Smix was vortexed for 5 minutes to ensure uniform blending. The oil phase was then added to each Smix in different proportions ranging from 1:9 to 9:1. Water was titrated gradually into each mixture with constant stirring until the system turned turbid, indicating phase separation as shown in fig.4. The clear and stable compositions were recorded and plotted using Chemix School 3.60 software to generate the pseudo-ternary phase diagrams as plotted in fig.5. Among the ratios tested, Smix 1:1 produced the largest isotropic region, suggesting its superior emulsification efficiency and stability.(13,16)

Figure  .4. Pseudo-ternary phase Diagrams of surfactant, co-surfactant, oil  and water Phase with the ratio of  1:1, 1:2, 1:3, 2:1 and 3:1.(13,16)

Figure .5 Pseudo ternary phase diagram for 1:1, 1:2, 1:3, 2:1 and 3:1 indicates more emulsion region, microemulsion

Preparation of Tinospora cordifolia Microemulgel

After the Pseudo-ternary phase study (Smix) of surfactant, co-surfactant, oil  and water Phase with the ratio of  1:1, 1:2, 1:3, 2:1 and 3:1, total 45 formulations were prepared  as per given flowchart in fig.5 and prepared 45 formulations of microemulsion among which only four were found to be stable (A-2, B-9, C-9, D-1) which were further  prepared as emulgel by varying the proportions of oil, surfactant, and co-surfactant while keeping the drug and polymer content constant as per the formulation which is given in table no.2.

The formulation of the microemulgel was carried out in a stepwise manner consisting of five key stages:

1. Preparation Gel                                               

2. Preparation Aqueous Phase of Emulsion

3. Preparation Oil Phase of Emulsion               

4. Emulsion Formation

5. Final Emulgel Formation

Carbopol 934 (1 g) was dispersed in cold distilled water (q.s. 50 ml) under continuous stirring until a clear, uniform gel was obtained. The dispersion was left overnight for complete hydration, and the pH was adjusted to 5.0–5.5 using triethanolamine. This ensured that the gel base was compatible with the physiological pH of the skin. For the aqueous phase, Tween 80 was dissolved in distilled water, while methyl paraben was dissolved in propylene glycol. The T. cordifolia extract was dissolved in ethanol and added to the aqueous phase under gentle stirring. For the oil phase, Span 80 was dissolved in olive oil and gently heated to 70 °C to achieve complete mixing. The aqueous and oil phases were each heated separately to 70 ± 2 °C, and the oil phase was added slowly to the aqueous phase with continuous stirring using a mechanical homogenizer at 3000 rpm for 10 minutes. The resulting microemulsion was cooled to room temperature.The preformed microemulsion was incorporated into the Carbopol gel base in a 1:1 ratio with moderate stirring to form the final microemulgel. The formulations were prepared as per given in table.2 and stored in tightly closed containers until further evaluation.(6,7,14)

 

Table no. 2 – Formulation of Emulgel for following four batches A-2, B-9, C-9, D-1 (6,7)

Sr. No	Ingredients	Quantity A-2	Quantity B-9	Quantity C-9	Quantity D-1	Uses
1	T.C. extract	150 mg	150 mg	150 mg	150 mg	Active pharmaceutical ingredient
2	Carbopol 934	1gm	1gm	1gm	1gm	Polymer as gelling agent
3	Tween 80	1ml	3 ml	3 ml	0.65 ml	Emulsifier
4	Span 80	1ml	6 ml	9 ml	0.35 ml	Emulsifier
5	Olive oil	8 ml	1 ml	1 ml	9 ml	Moisturizer
6	Methyl paraben	0.02ml	0.02ml	0.02ml	0.02ml	Preservative
7	Propylene glycol	2ml	2ml	2ml	2ml	Solvent
8	Ethanol	2ml	2ml	2ml	2ml	Permeation Enhancer
9	Triethanolamine	Few drops	Few drops	Few drops	Few drops	Ph modifier
10	Distilled water	q.s. 50gm	q.s. 50gm	q.s. 50gm	q.s. 50gm	To adjust the final volume

 

In-vitro Drug Release and Kinetics

The in-vitro diffusion study using Franz diffusion cells demonstrated a sustained release pattern for the Tinospora cordifolia microemulgel over eight hours. Fill donor compartment with 1gm of prepared Tinospora cordifolia leaf extract emulgel having diffusion area of 1.5 cm². Fill recipient compartment with phosphate buffer (pH 6.0) Maintain system temperature at 37  ± 0.5 °C using water circulation and Magnetic bar was put in recipient chamber and rotated at 75 rpm. Samples (1 ml) were withdrawn at regular time interval and replaced with fresh buffer solution to maintain sink condition. Withdraw samples at 0, 1, 2, 3, 4, 5, 6, 7, 8 hrs. After each withdrawal, replace the sample solution with fresh buffer solution. Measure the absorbance at 250 nm using UV-Visible spectrophotometer and Collect drug release data. Among all batches (A-2, B-9, C-9, D-1) Tinospora cordifolia emulgel A-2 containing Tween 1ml, Span 1ml, Olive oil 8 ml has shown highest drug release (85.76% in 8hrs) than all other emulgel formulations. So, it was selected as a optimized formula and suggesting that this composition provided the most efficient diffusion profile.

 

The drug release kinetics were estimated by fitting the release data into various kinetic models, namely Zero-order, First-order, Higuchi, Korsmeyer–Peppas, and Hixson–Crowell models. The resulting regression plots are shown in Figures 6-10. Among all models, the Korsmeyer–Peppas (power-law) model provided the best fit, with a coefficient of determination (r² = 0.999) as shown in fig-8, indicating that the drug release from the Tinospora cordifolia microemulgel followed a diffusion-controlled mechanism as per given dat in table no.3. The linearity of the plots further confirmed that the release was primarily governed by Fickian diffusion, consistent with the sustained-release characteristics of microemulsion-based gels. (6)

 

Table  3. Drug release kinetic models of optimized Tinospora cordifolia microemulgel formulation(12)

Model	Equation	r² value	Mechanism
Zero-order	Qt = 5.83t + 7.61	0.978	Constant-rate release
First-order	log Qt = 1.35 – 0.128t	0.962	Concentration-dependent
Higuchi	Qt = 12.14√t + 4.87	0.985	Diffusion-controlled
Korsmeyer–Peppas	Qt/Q∞ = 0.44t^0.67	0.999	Fickian diffusion
Hixson–Crowell	(100 – Qt)¹/³ = 0.121t + 4.59	0.971	Erosion mechanism

Figure .6: Drug release profiles of TC emulgel formulation by zero order model (12)

Figure .7: Drug release profiles of TC emulgel formulation by First order model (12)

Figure .8: Drug release profiles of TC emulgel formulation by Korsmeyer Peppas model (12)

Figure .9: Drug release profiles of TC emulgel formulation by Higuchi model (12)

Figure .10: Drug release profiles of TC emulgel formulation by Hixson crowell model (12)

Evaluation of Prepared Microemulgel Formulation (For optimized batch -A2)

The prepared final formulation was subjected to a series of evaluations to determine their physical, physicochemical, and functional characteristics. Visual inspection was performed to assess colour, odour, homogeneity, and phase separation. The pH was measured using a digital pH meter, and viscosity was determined using a Brookfield viscometer (spindle 63 at 50 rpm). Spreadability was assessed by the glass-slide method, while extrudability was evaluated by measuring the force required to extrude the formulation through a collapsible aluminum tube. Drug content was determined by dissolving a known weight of the emulgel in phosphate buffer (pH 6.8), followed by spectrophotometric analysis at 570 nm. The optimized batch (A-2) was selected for further characterization based on its ideal pH, viscosity, spreadability and drug content. (6,7,14,15)

 

Physical Appearance- The physical properties of the emulgel were observed, including the appearance, Colour, Odour, Homogeneity, phase separation, and ease of application and removal.

 

Ph - 1gm of gel was dissolved in 100 ml of distilled water and it was placed for 2 hr and then dip the glass electrode into an emulgel. The measurement of pH of was done using digital Ph meter and the value was noted.

Spreadability - Spreadability refers to the ease and extent to which an emulgel spreads on the skin or affected area upon application. It is a key parameter influencing the bioavailability of the formulation. To determine spreadability, two glass slides of standard size were used. A fixed amount of the herbal emulgel was placed on one slide, and the second slide was placed on top forming a sandwich over a 6 cm area. A 50 g weight was placed on the top slide to evenly spread the emulgel into a thin layer. After removing the weight, excess emulgel adhering to the slides was scrapped off. The two slides in position were fixed to a stand without slightest disturbance and in such a way that only the upper slide to slip off freely by the force of weight tied to it. A 50 gm weight was tied to the upper slide carefully. The time taken for the upper slide to travel the distance of 6 cm and separated away from the lower slide under the influence of the weight was noted. The experiment was done and the time taken to separate the slides was noted for calculation. Spreadability was calculated using formula:

 S = M. L / T

Where M= weight tied to upper slide (50g)

L = length of glass slide (6cm)

T = Time taken to separate the slides

Determination of viscosity:

viscosity of emulgel were determined by Brookfield viscometer (cone and plate). It was done using  viscometer (Spindle no.6) at 6 and 12 rpm. Small amount of the emulgel was taken in the cup and the spindle was dipped in it for about 5 minutes and then the reading was taken.

Excrudability- It is a test to measure the force required to extrude the material from tube. The  emulgel was packed in a collapsible aluminum tube, measuring the length of ribbon extruded from the container in 10 seconds and calculating the extrudability using the formula:

Extrudability = Weight of load (in grams)/Area of ribbon extruded (in cm square)                        

Centrifugation -Selected formulations A2 was centrifuged at 1500 rpm for 15 min. The formulation was observed for any phase separations.

 

Drug content-

1 g of the emulgel formulation (contain 500mg of Tinospora cordifolia extract)   transferred to a beaker. 80 ml of methanol was added to the beaker. The mixture was heated at 50 °C for 30 minutes. After heating, the mixture was filtered, volume was made up to the mark 100mlwith methanol. The resulting solution was sonicated in an ultrasonic bath for 5 minutes to ensure complete dissolution of the drug. The absorbance of the final solution was measured at 250 nm using a UV–visible spectrophotometer.

 

Stability Studies –

Stability studies were carried out for the optimized formulation according to International Conference on Harmonization (ICH) guidelines. The resulting microemulgel was placed  in collapsible tube and tested for three-month stability tests at 5 C ±2°C, 25 C ±2°C  with 60% RH, and 30 C ±2°C with 65% RH, and 40 C ±2°C with 75% RH. After every 15 days, samples were taken out and observed for colour, phase separation, homogeneity, consistency and liquefaction and Ph.

Characterization Studies

 

FTIR –

Fourier Transform Infrared (FTIR) spectroscopy was performed for both the pure extract and the optimized formulation (A2) to evaluate drug–excipient compatibility. The spectra were recorded within the 4000–400 cm⁻¹ range using the KBr pellet method. The characteristic peaks corresponding to O–H, C–H, and C=O groups were retained without significant shifts, indicating no chemical interaction between the extract and excipients.

 

Particle Size –

FESEM is used to visualize and analyze surface structures at very high resolution, particularly useful for understanding the micro scale morphology of materials. FESEM analysis reveals that samples with 1–2 µm to 20 µm particle size, especially those analyzed at 500X to 25.00KX magnification.

The micrographs revealed uniformly distributed spherical particles with smooth surfaces, and particle sizes ranged between 1 and 2 µm. The absence of aggregation confirmed proper dispersion and emulsification of the T. cordifolia extract within the gel matrix.

The formulated Tinospora cordifolia microemulgel exhibited excellent physical characteristics, appearing greenish-white, glossy, and homogeneous with a pleasant herbal odour. The formulation was smooth, easily spreadable, and non-greasy, indicating good patient acceptability as data provided in table no.4. The pH of the optimized batch (A2) was found to be 5.48 ± 0.02, which falls within the normal physiological pH range of the skin, ensuring its compatibility and minimizing irritation potential. The measured viscosity of 67.8 cps confirmed adequate consistency and stability, while the spreadability value of 42.58 g·cm/s indicated that the gel could be applied effortlessly with minimal shear. The extrudability value of 111 g/cm reflected smooth expulsion of the formulation from collapsible tubes, demonstrating its suitability for practical use.

 

The FESEM analysis revealed uniformly distributed spherical particles with sizes ranging from 1–2 µm, as per the data of table no.5, confirming proper emulsification and dispersion of the extract within the gel matrix.

 

Figure.12.–Image of microemulgel having  1 µm particle sizeand magnification at 25.00KX(3,4)

Figure. 11. –Image of microemulgel having 20 µm particlesize and magnification at 500X(3,4)

 

The recorded data of table no.6, showed particle sizes ranging from 1 to 20 µm across different magnifications (100X to 25.00KX), with smaller and more uniform particles observed at higher magnifications. The working distance (WD) varied between 11.7–12.1 mm, and the electron high tension (EHT) was maintained at 3.00 kV, ensuring high-resolution imaging without surface distortion. The micrographs Fig.11 and Fig.12, clearly depicted smooth, spherical particles with no signs of aggregation, indicating proper dispersion of the Tinospora cordifolia extract within the gel matrix. Overall, the FESEM findings confirmed that the optimized formulation possessed desirable morphological characteristics essential for achieving uniformity, stability, and effective topical drug delivery.

 

The small particle size contributes to enhanced drug diffusion and stability of the emulgel. The FTIR spectra of the pure extract and the final formulation displayed all major characteristic peaks of functional groups, such as O–H, C–H, and C=O stretching vibrations, without any significant shifts, confirming that there was no chemical interaction between the extract and excipients as given in table no. 7, ensuring formulation compatibility as shown in fig.13 and fig.14.

 

Fig.ure.13. FTIR spectrum of Tinospora cordifolia microemulgel formulation (5,6)

Figure.14. FTIR spectrum of pure Tinospora cordifolia extract (5,6)

The in-vitro drug release studies using Franz diffusion cells demonstrated a sustained release pattern for the Tinospora cordifolia microemulgel over eight hours. Among all batches, formulation A2 (Tween 1 ml, Span 1 ml, Olive oil 8 ml) showed the highest cumulative drug release of 85.76%, as shown in table no.8, suggesting that this composition provided the most efficient diffusion profile.

 

 

The release kinetics of the optimized formulation followed the Korsmeyer–Peppas model, with an r² value of 0.999 as per table no.9, indicating that the release mechanism was predominantly diffusion-controlled. This finding supports the microemulgel’s potential to maintain prolonged therapeutic levels at the application site.

 

The stability studies conducted under various temperature and humidity conditions (5°C, 25°C, and 40°C) for three months revealed that the optimized formulation remained physically and chemically stable with no signs of phase separation, colour change, or significant alteration in pH. The consistent pH (5.48–5.50) and uniform appearance confirmed that the emulgel retained its integrity throughout the storage period as given values in table no.10.

 

 

Overall, the optimized Tinospora cordifolia microemulgel demonstrated favorable physicochemical characteristics, excellent drug release, and long-term stability, establishing its suitability as a promising topical herbal delivery system.