INTRODUCTION:

Tinea pedis is the most prevalent infection with superficial dermatophytes in the developing world, affecting as much as 10 percent of the total population at any given time¹. Tinea pedis is a dermatophytosis of the foot's plantar surface and interdigital areas, distinguished by inflammatory and non-inflammatory lesions².

Tolnaftate is widely used to handle various forms of tinea and pityriasis versicolor topically as a paste, mist, aerosol, cream and/or gel. Tolnaftate is selective by preventing ergosterol biosynthesis in the fungus cell walls and inhibiting fungal microsomal squalene epoxidase.

Tolnaftate antifungal action is responsible for the deposition of squalene and ergosterol deficiency³.

Oral antifungal agents have disadvantages like headache, gastrointestinal disturbance, urticaria, diarrhoea, nausea, anorexia, vomiting and photosensitivity. Because of its benefits, including the ability to target medications at the infection site and lower the possibility of systemic side effects, topical treatment is a suitable option for treating cutaneous infections. Regarding permeability, bioavailability, and therapeutic effect, the earlier methods of treating these conditions, such as creams, ointments, or jellies, were ineffective.To overcome this issue, various carriers have been shown to work best when applied topically to treat fungal illnesses. These carriers include transfersomes, liposomes, niosomes, ethosomes, microemulsions and solid lipid nanoparticles.⁴

Liposomes are tiny artificial spherical vesicles which can be produced from cholesterol and normal, non-toxic phospholipids⁵. Liposomes, safe and effective carriers, have been the subject of extensive research due to their structural flexibility and ability to improve skin penetration of incorporated drugs.⁶Liposomes act as a local drug reservoir with minimal penetration to deeper skin layers as their penetration is confined to the stratum corneum and upper skin layers.⁷

Another exciting form of enhanced conventional liposomes is ethosomal systems, new vesicular lipid carriers containing phospholipids, ethanol, propylene glycol, and water. Ethosomes have a reasonably small concentration of ethanol, a vital permeation enhancer that lets the medication pass into the stratum corneum with great ease⁸. Theycan facilitate drug delivery to the skin, including their in-depth penetration through the skin layers⁹. The vesicular membrane of ethosomes has a high degree of flexibility due to the presence of ethanol. ^10,11.

Transfersomes areultra-deformable vesicular systems with high elasticity and flexibility andcomprise phospholipids, surfactants, and water.Squeezing through the intracellular sealing lipid of the stratum corneum, they can enter the deepest layers of the skin in an intact form, thus localising high concentrations of incorporated drugs.^12,13.

The DoE method is primarily used to help evaluate the relationship between the input factors that influence output responses (dependent variables) by developing mathematical models. Therefore, the present study aims to formulate and optimise the liposome by 3² factorial designs and perform a comparative study of liposomes,ethosomes and transfersomes as a carrier for improving topical delivery of tolnaftate for treating Tinea pedis.

MATERIALS AND METHODS:

Material:

Tolnaftate was purchased from Yarrow Chem Product, Mumbai, India. Soya lecithin was obtained from Hi Media Laboratories, in Mumbai, India. Cholesterol was procured from Merck, Mumbai, India.Span 80, Chloroform, disodium hydrogen phosphate, carbopol 934, potassium dihydrogen orthophosphateand triethanolamine wereprocured from Lobachemie, Mumbai, India. Ethanol and methanol were purchased from Nice Chemicals, Kerala, India.

Methodology:

Design of experiments:

As described in the literature, soy phosphatidylcholine and cholesterol are essential factors which affect the vesicle size, PDI, zeta potential and % entrapment efficiency (EE) of liposomal. Hence, 3² factorial designs were applied for liposome formation, where SPC (600, 900 and 1200 mg) and Cholesterol (200, 300 and 400 mg) were taken as independable variables to obtain the optimised formulation with minimum vesicle size and PDI and highest zeta potential and% EE The response from the ten runs was subjected to multiple regression analysis using the Design Expert® software (version 11.0.3.0 64-bit, Stat- Ease, Inc. Minneapolis, MN, USA). The model derived from regression analysis is expressed as the following equation,

Preparation of vesicular systems:

Preparation of tolnaftate-loaded liposome was performed using a rotary flash evaporator (Super fit rota Vap (series 6-BU) Continental Pvt Ltd, R/185., India) utilising a thin-film hydration process as shown in Table 1. The liposomal suspension was prepared by dissolving the drug in a 1:2 ratio of chloroform (15ml) and methanol (25ml) solution. Add soy phosphatidylcholine and cholesterol to this solution and dissolve. Then, the solution in a round bottom flaskwas fully vacuum-dried under a rotary evaporator at 40ºC to create a lipid layer on the flask surface. Allow the film to dry overnight and then hydrate with the buffer mixture (25 ml). The preparation was then sonicated at 4ºC in 3 intervals of 5 min and 5 min in each period using a probe sonicator (CV 18 Sonics and Materials INC, USA)¹⁴.

The transpersonal formulation was prepared using the same procedure as liposome with an extra 5ml span 80 ¹⁴.

Preparation of tolnaftate-loaded ethosomes was conducted using the cold method. Ethosomal suspension was prepared on a magnetic stirrer by mixing soya phosphotidyl choline, cholesterol and drugin 30% v/v ethanol.The ethanolicsolution was introduced into the water bath at a temperature of 30°C, following which 10 ml of propylene glycol was added. Subsequently, 60 ml of water was incorporated into theethanolic solution while stirring at 700 rpm. To facilitate the formation of ethosomal vesicles, the solution was stirred continuously for 15 minutes.¹¹.

Optimisation of the vesicular systems:

The optimisation of liposomes was carried out by fixing the goal with minimum vesicle size and PDI, maximum entrapment efficiency and zeta potential in the range (-45 to -51mV). The software generated the optimised formulation with a desirability of ≥0.8. According to the software, 1200 mg SPC and 269.019 mg of cholesterol were utilised to create the optimal formulation.

Further ethosomes and transfersomes were prepared using the same optimised amount of SPC and cholesterol with the addition of Ethanol and Span 80, respectively.

Characterisation of vesicular system:

Vesicle size, size distribution and zeta potential:

The dynamic light scattering (Nano ZS, Malvern Instruments, UK), Zeta sizer determines the size distribution and particle size of liposomes, ethosomes, and transfersomes. Potential Zeta values help to determine formulation stability.

Percentage entrapment efficiency:

Transfer 10ml of the liposomal, ethosomal, and transfersomal suspension into a 15ml centrifuge tube, then subject it to centrifugation at 10,000 rpm for 90 minutes at 4°C using a cold centrifuge (REMI Elecktrotechnik Ltd, model CM8P105, located in Vasai).The sediment and supernatant are separated during centrifugation. The tolnaftate concentration present in the supernatant was analysed at 257 nm using UV spectrometry (Jasco, V-630, Japan). The % entrapment efficiency is calculated using the following formula¹⁵.

Amount of durg entraped

Entrapment efficiency (%) = ------------------------- × 100

Total amount of drug used

Scanning Electron Microscopy (SEM.):

This approach was utilised to assess the distribution of particle size, the surface's topography, and shape and even to investigate the morphology of broken or sectioned surfaces. SEM studies were performed using an analytical electron scanning microscope (JEOL-JSM 6380LA). SEM samples were made by gently sprinkling the liposomal, transfersomal, and ethosomal powder on a double-sided adhesive tape placed on an aluminium stub. The photomicrographs were taken with an SEM¹⁶.

Preparation of vesicular and conventional gels:

Vesicular and conventional gels were prepared by dissolving 1 g of carbopol 934 gel in 100 ml of distilled water and maintaining it for 2 hours for magnetic stirring. Hold it in for swelling overnight. Then Liposomal (FL), ethosomal (FE) and transfersomal (FT) suspension (containing 1 g of drug) and 1 g of tolnaftate was added to 1% carbopol 934 gels and stirred homogeneously by introducing a small amount of triethanolamine to achieve GFL, GFE, GFT and GFC gel, respectively ¹⁷.

Characterisation of vesicular gels:

Organoleptic Characteristics:

The formulations were tested for their physiological rheological properties, such as colour, odour, texture, the distinction of phases and feeling after application (grittiness, greasiness)¹⁸.

Measurement of pH, Measurement of viscosity, Washability:

Weigh about 1 g of ethosomal gel, which uses distilled water to dilute it to 100 ml. By immersing the electronic pH metre (Eutech Instruments) in the solution and allowing it to equilibrate for one minute, the pH of both formulations is ascertained¹⁶.

The viscosity measurement was conducted using a Brookfield viscometer (model DV-II+pro D220) with spindle number T-94 set at 10 rpm. To determine viscosity, 50 grams of gel were placed in a 50 ml beaker, and the spindle was submerged into the formulation, with the speed set at 10 rpm to obtain the dial reading. A small amount of gel was applied to the skin's surface. After rinsing with water, the washability of the gel was tested to determine if it could be completely washed off¹⁹.

Spreadability:

The spreadability of the gel was measured using modified wooden blocks and glass slide equipment. Spreadability was measured as per the equation,

S=(M×L)/T

Where S = is the spreadability,

M = is the weight in the pan (attached to the upper slide),

L = is the length transferred by the glass slide

T = reflects the time required to remove the slide entirely from each other¹⁵.

A surplus of gel (about 1 gm) was measured on one slide. Then, the gel was sandwiched and fitted with the hook between this slide and another glass slide with a set ground slide length. A 100gm weight was placed on the two slides for 5 minutes to eliminate air and provide a transparent gel film between the slides. The top plate was subsequently subjected to a pull of 30 g. To cover a distance of 5 cm, using the string connected to the handle, the time (in seconds) taken by the top slide is noted ²⁰.

Drug content:

A precisely weighed amount of gel (about 1 g) was dissolved in approximately 100 ml of pH 5.5 phosphate buffer that contained 50% methanol. After quantitatively transferring these solutions to volumetric flasks, the same buffer solution was used to make the necessary dilutions. The solution was further filtered, and by using UV spectrophotometric drug was analysed at 257nm. Drug content was determined from the standard curve of tolnaftate²¹.

Ex vivo drug permeation studies using goat skin:

The goat ear skin was obtained from a slaughterhouse, where the hair was removed and the skin was placed in a phosphate solution with a pH of 5.5, containing alcohol. Ex vivo skin experiments were conducted using Franz Diffusion cells consisting of two compartments. The donor compartment had two open ends, with one side covered by the previously treated goat skin soaked in the pH 5.5 phosphate buffer with alcohol. In each dermal side of the skin, 0.5ml (equivalent to 2mg of the drug) of liposomal, transfersomal, and ethosomal suspension, as well as 0.5gm (equivalent to 2mg of the drug) of liposomal, transfersomal, and ethosomal gel, were added to the donor compartment. The reservoir compartment was loaded with 12 ml pH 5.5 phosphate buffer containing alcohol comprising a small rotating magnetic bead at a steady 50 rpm speed. The study was conducted for 8 hours at 37± 0.5 °C. At a fixed time, 5 ml of the sample was withdrawn from the reservoir compartment, suitably diluted, and its absorbance measured spectrophotometrically at 257 nm.²¹.

Calculation of Skin Permeation Parameters:

The cumulative drug penetration per unit area was calculated over time. From the linear portion of the slope, the flux was determined. Using the formula derived from the first law of Fick's diffusion, the tolnaftate permeability coefficient (Kp) with goat skin was calculated. This formula is represented as:

𝐾𝑝 =𝐽/𝐶

Where 𝐽 is the flux and

𝐶 is the drug concentration in the donor compartment¹⁴

In-vitro Antifungal Activity:

The cup plate technique was used to evaluate antifungal action. A standard was used for the branded drug (ketoconazole). The standard and tolnaftate-loaded liposomal, ethosomal, and transfersomal gel (test) was taken into sterile sabouraud dextrose agar cups previously seeded with Candida albicans obtained from the Nitte Science Education and Research Center. The plate was incubated at 25˚C for 48 hours after enabling the formulation to expand for 2 hours. The inhibition zones were assessed in mm after 48 hrs22 for test and standard.

RESULTS AND DISCUSSION:

The liposomes containing tolnaftate were formulated by the thin-film hydration method. 3²-level factorial designs were applied to analyse the effects of the liposomal constituent, i.e., the SPC and cholesterol, on the responses.

Vesicle size of liposomes:

The degree to which drugs penetrate the skin is mainly dependent on the size of vesicles, as per the literature. As the concentration of SPC increases, there is a slight decrease in the vesicle size of the vesicular systems (Table 1). As the concentration of cholesterol increased, it showed an increase in the vesicle size. Cholesterol increases the phospholipid packaging and the thickness of the bilayer's hydrophobic portion, resulting in increased vesicle size. The highest vesicle size was 436.9 nm, and the lowest was 132.0 nm.

Regression analysis offers a more comprehensive understanding of how formulation variables collectively influence vesicle size. The polynomial model showed significance, with a model F-value of 29.49. The small difference of less than 0.2 between the Adjusted R-squared value of 0.8636 and the Predicted R-squared value of 0.7498 suggests a reasonable agreement. The polynomial equation derived from the analysis is as follows:

Vesicle size= +288.34-69.02(A)* +67.13(B)*

In this equation, where A represents the SPC and B represents the cholesterol concentration, the coefficients reflect the standardized beta coefficient, and significance of variables is denoted by an asterisk symbol. The obtained regression model was found to be statistically significant (p<0.05), with a notably high adjusted R-squared value of 0.8636(Fig. 1a).

PDI of liposomes:

With a p-value of less than 0.05 and an F-value of 104.78, the model generated for the PDI was considered significant. The value of 0.8251 indicates a non-significant lack of fit, implying that the model is appropriate for calculating the PDI. The Predicted R2 of 0.9501 is in rational agreement with the Adjusted R² of 0.9830; i.e. the difference is less than 0.2. The amount of soya phosphatidylcholine significantly affected the liposome's PDI, as shown in the equation. The generated equations for the response i.e., PDI based upon the quadratic model, are:

PDI=+0.3157 - 0.0183(A)* + 0.0043(B)* + 0.0180(AB)* + 0.0186(A2)* + 0.0096(B2)*

Where A is the SPC, B is the cholesterol concentration, the coefficient in this equation reflects the standardised beta coefficient and the asterisk symbol shows the variable's importance. It was observed that the regression model obtained was statistically relevant (p<0.05), with a high adjusted R² value of 0.9830 (Table 1 and Fig. 1b).

Effect on Zeta potential:

An integral parameter that may impact the vesicle stability is zeta potential. During storage, the electrostatic repelling force generated by charged particles prevents vesicles from aggregating. The cholesterol prevents or at least delays the formation of vesicle aggregates by providing the polar head region of SPC with a concentration-dependent surface negative charge at all SPC concentrations. The increased SPC concentration showed no significant change in the zeta potential. (Table 1).

The polynomial model demonstrated significance, as indicated by a model F-value of 12.82. The Predicted R² of 0.6226 aligns reasonably well with the Adjusted R² of 0.7242, with a difference of less than 0.2. The polynomial equation derived from the analysis exhibited a linear relationship, suggesting that the variables have a predominantly linear effect.

Zeta potential= -48.77+2.27(A) +5.15(B)*

Where A is the SPC, B is the cholesterol concentration, the coefficient in this equation reflects the standardised beta coefficient, and the asterisk symbol shows the importance of the variable. It was found that the regression model obtained is statistically significant (p<0.05), with a high adjusted R² value of 0.7242 (Fig.1c).

Effect on % Entrapment efficiency:

The study observed that as cholesterol concentration increased, the entrapment efficiency of liposomes improved across all concentrations of SPC. This enhancement is attributed to cholesterol's structure and hydrophobic nature, facilitating its integration into the phospholipid bilayer. Consequently, cholesterol reinforces the mechanical rigidity of the bilayer, thereby promoting formulation stability and increasing entrapment efficiency.However, with increasing SPC concentration at all levels of cholesterol, there was a slight decline in entrapment efficiency. This decrease is attributed to the heightened risk of vesicle aggregation at higher SPC concentrations, which compromises the formation of a stable film surface. Consequently, this aggregation leads to drug leaching and subsequently reduces entrapment efficiency.

The effect of formulation factors simultaneously on the entrapment efficiency (% EE) was then further understood by applying regression analysis. The polynomial model implied significance with a model F-value of 27.75. The Predicted R² of 0.7930 is in rational agreement with the Adjusted R² of 0.9370; the difference is less than 0.2. The polynomial equation which was obtained from the analysis results showed a quadratic model:

% EE= +91.21-0.8567(A) +8.77(B)*+0.9325 (AB)-1.48(A²)-8.99(B²)*

It was observed that the regression model obtained is statistically relevant (p<0.05), with a high adjusted R² value of 0.937 (Fig.1d).

Table 1: Effect of independent process variables on responses of liposomes as per 3² Full Factorial Designs

Form. Code	A: SPC mg	B: Cholesterol mg	Y1: Vesicle Size (nm)	Y2:PDI	Y3:EE (%)	Zeta potential mV
FP1	600	200	275.5±2.51	0.377±0.12	-57.3±3.26	75.32±1.23
FP2	900	200	232.2±1.26	0.321±0.21	-53.7±3.24	71.29±1.69
FP3	1200	200	132.8±3.02	0.302±0.13	-52.2±2.09	70.76±2.04
FP4	600	300	325.2±1.45	0.35±0.2	-51.5±2.54	88.23±2.84
FP5	900	300	309.1±1.05	0.313±0.14	-45±2.78	91.66±3.15
FP6	1200	300	252.8±2.34	0.318±0.11	-50.4±1.65	88.48±1.64
FP7	600	400	436.6±3.12	0.35±0.17	-48.6±2.98	90.21±3.56
FP8	900	400	369.1±1.09	0.329±0.21	-42.5±4.26	90.4±2.49
FP9	1200	400	237.6±1.55	0.347±0.25	-41.2±3.57	89.38±1.64
FP10	900	300	312.5±2.48	0.319±0.22	-45.3±4.12	930.5± 3.47

*Mean of 3 replications ± SD

Fig. 1: Response surface curve representing the effect of SPC and Cholesterol on a) vesicle size, b) PDI, c) Zeta Potential, d) % Entrapment efficiency of liposomes

Formulation and characterisation of optimised batch:

Liposome was optimised based on constraints such as minimum vesicle size, PDI, and maximum zeta potential and entrapment efficiency, and desirability of more than 0.8. The vesicle size, PDI, zeta potential, and % EE values given by the software were 198.52 nm, 0.309, - 48.09 mV and 84.99 %, respectively. In contrast, the observed value was 201.0 nm, 0.313, -45mV and 84.2%, respectively. It was determined that the observed values were within an acceptable ± 5% error of the expected value.

The ethosomes and transfersomes had a vesicle size of 214 and 193.4 nm, respectively, a zeta potential of -49.5 and -51.5mV, respectively and a % entrapment efficiency of 75.0 and 77.2%, respectively.

Scanning Electron Microscopy (SEM):

Scanning Electron Microscopy is used to obtain the surface morphology of the formulated liposomes, ethosomes and transfersomes. Images are shown in Fig 2.

Fig. 2. SEM of optimised (a) liposomal (b) ethosomal, and (c) transfersomal vesicle

Characterisation of vesicular gels:

Liposomal(GFL), transfersomal (GFT) and Ethosomal gel(GFE) was prepared by using carbapol as the polymer and the prepared gel was off-white in colour, smooth, free from grittiness and homogenous. These prepared gels are characterised by various parameters Table 6).The pH of the gels was observed to be in the 6.41-7.00 range, closer to the skin's pH, and avoided any irritation or damage to the skin. All gels have been found to be quickly washable without leaving any residues on the skin surface. The spreadability of the liposomal gel (GFL) ethosomal gel (GFE), and transfersomal gel (GFT), were found to be 15.26g/cm², 14.94g/cm2 and 13.75g/cm^2, respectively. Thus, the gel that was developed has good spreadability because it spreads easily with a small amount of shear. The viscosity of the formulation ranged between 598-71.6cps for liposomal gel, 367-40.8cps for ethosomal gel and 150- 602 cps for transfersomal gel. Based on the data collected, a rheogram was developed, and it was revealed that each formulation exhibited shear-thinning effects. An improvement in the shear rate lead to a lowering of viscosity of the formulation, i.e. pseudoplastic behaviour has been observed (Figure 7). Drug content of the optimised liposomal, ethosomal and transfersomal gel was estimated, and the result was found to be 85.61±1.63%, 87.56±1.34% and 89.45±1.12%, respectively. The assessment of drug content also suggested that the drug was distributed uniformly throughout the formulation.

Table 2: Characterisation parameter of gels

Form. Code	Measurement of pH	Washability	Spreadability (g/cm²)	% Drug content
GFL	6.63	Good	15.26	85.61±1.63
GFE	7.00	Good	14.94	87.56±1.34
GFT	6.41	Good	13.75	89.45±1.12
GFC	7.20	Good	18.46	85.05±1.28

GFL: Liposomal gel, GFE: Ethosomal gel. GFT: Transfersomal gel, GFC: Conventional gel:

Ex vivo permeation study using goat skin:

An ex vivo study has been performed to evaluate the amount of drug permeated through the skin of goats. The obtained release profile is seen in Figure 9. The drug permeation adopted a different trend at the end of 480mins than the in vitro drug release model. It thus revealed that the release of the medication from the transfersomal gel had a higher permeation of the drug through the skin at the end of 480 min compared to other gel.

Calculation of Skin permeability parameter:

In comparison to vesicular systems, especially transfersomal formulations (1233.46 μg / cm²), the total amount of drug that permeated goat skin after 480 minutes for both carbopol gel and hydroethanol solution (515.53 and 479.03 μg / cm²) was significantly less. This suggests that vesicular systems could enhance the delivery of hydrophobic drugs like tolnaftate to the skin. On the contrary, after 480 minutes, the cumulative amount of drugs that had permeated from liposomal formulations was considerably less than that which had permeated from ethosomal or transfersomal formulations (P <0.01). The increased tolnaftate permeability from the transfersomal formulation may be due to the high degree of flexibility of transfersomes, which enable them to easily pass through the intracellular lipid of stratum corneum to penetrate the skin and bypass the barrier function. Additionally, after applying transfersomes to the skin, the osmotic gradient causes them to move from the dry stratum corneum to a deeply hydrated layer. The surfactant included in the structure of the transferosome aids in this process by solubilising the lipid in the stratum corneum, allowing for a high level of vesicle penetration. (Figure 10).

Fig 3. Ex vivo permeation of tolnaftate from variation formulation

All the vesicles had higher steady-state flux when compared to the drug solution,as shown in Table 8. A direct relationship between permeability coefficients and steady-state flux was found. The permeability coefficient of transfersomal suspension also was higher than drug solution. The ability of transfersomes to overcome the properties of the skin barrier may have been due to their considerable deformability and flexibility.

The cumulative amount of drug permeated, permeability coefficient and steady-state flux were determined to be less in the case of all the gel formulations when compared to vesicular suspensions. It may be due to viscous nature of the gel; retarded the release of the drug from the formulation. The cumulative amount of drug permeated from the transfersomal gel was significantly higher than that from the ethosomal or liposomal gel (P <0.05).

Table 3: The permeated amount of Tolnaftate at 480 mins, flux and permeability coefficient

Form. Code	Permeated amount at 480 mins (µg/cm²)	Flux (µg/cm².min)	Permeability constant (K_p) × 10⁻³(cm/h)
FL	1145.36	2.277	0.6777
FE	1171.71	2.412	0.8040
FT	1233.46	2.420	0.8067
Pure drug	515.53	1.164	0.2910
GFL	1015.75	2.075	0.3053
GFE	1047.42	2.149	0.3070
GFT	1080.24	2.176	0.3109
GFC	479.03	0.966	0.1380

In vitro Antifungal Studies:

Using the Candida albicans culture, the antifungal activity of tolnaftate liposomal, ethosomal and transfersomal gels is determined against the standard ketoconazole using cup plate method. The report shows that liposomal, ethosomal and transfersomal gels have a zone of inhibition of 20.6 mm, 21.2 mm and 22.5 mm,respectively,compared to standard ketoconazole of 24.3 mm. The result shows that all vesicular gels' antifungal activity was significantly similar to standard.

CONCLUSION:

The present study highlights a strategy of a comparative study of three types of vesicular systems of a specific drug to know which of the three shows better results for treating Tenia pedis. Compared to liposomal and ethosomal gel, the transfersomal gel displaysmore excellent skin permeability. Thus, the current study proved that tolnaftate-loaded transfersomal gel was better than liposomal and ethosomal gel for treating Tenia pedis.

ACKNOWLEDGEMENT:

The authors are thankful to Nitte (Deemed to be University) and NGSM Institute of Pharmaceutical Sciences, for providing necessary facilities to carry out the study.

CONFLICT OF INTEREST:

The authors declared no conflict of interest.

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Received on 30.06.2023 Modified on 23.12.2023

Research J. Pharm. and Tech 2024; 17(6):2661-2668.

DOI: 10.52711/0974-360X.2024.00417