INTRODUCTION:

Systemic fungal infections constitute a major global health issue, with more than 1 billion patients affected each year and a high mortality rate among all clinical conditions affecting various populations. Modern antifungal management of the disease has been greatly challenged by the realization that resistance has risen to 41% in the latest surveillance from the previous decade¹. The existing topical antifungal drugs, while having efficacy, possess severe side effects and interactions making them useful in geriatric patients. Currently, the cost of managing fungal infections amounts to over $7.2 billion in healthcare expenses coupled with the loss of $3.9 billion in productivity. This is especially true in the areas of development where people do not have access to the right health care provisions making such a burden worse². New trends in antifungal drugs have been evaluated in this study; however, more effective, safer and considerably cheaper drugs are urgently required due to the increasing prevalence of immunocompromised patients most of whom are likely to be prone to fungal infections³. A prominent medicinal plant from the Acanthaceae family named Ruellia tuberosa receives significant interest because of its strong antifungal properties. Ruellia tuberosa possesses bioactive compounds made up of flavonoids alkaloids terpenes which exhibit antimycotic activities⁴. Recent phytochemical examinations have revealed that two crucial active elements within Ruellia tuberosa are lupeol and β-sitosterol which demonstrate powerful antifungal properties by damaging membranes and blocking ergosterol synthase activity. Many in vitro tests confirm that the compound shows effective antifungal activity against different fungal organisms and shows equal MIC values like standard antifungal medicines. Due to its traditional medicinal value in indigenous systems and its safe nature the plant demonstrates great potential for contemporary antifungal drug development⁵.Shatadhaut Ghrita-based delivery systems are innovative systems to obtain higher therapeutic action of the extract from the herbal drugs. This base is prepared by hundred-fold processing of milk solid non-fat and known as clarified butter and this base has its own merits in delivering the drug and quality absorption. The latest improvements in the pharmaceutical stability have paved way to the development of highly effective oil in water emulsions for skin penetration. The features mentioned above highlighted the fact that the system speaks to the shortcomings that have for long been associated with herbal products such as lack of stability and bioavailability. HPLC and DLS in particular have been used in detailed formulation characterization of such system, which showed enhanced bioavailability and sustained release profile over typical formulations⁶. The objective of the current study is to prepare and standardise the Ruellia tuberosa Poir extract in Shatdhaut Ghrita base for its stability, efficiency and safety profile. More specifically, at an operational level, the work aims at identifying the means of manipulating formulation parameters to increase the efficacy against antifungal strains that offer a high degree of resistance, coupled with stability and enabling physicochemical properties. The research encompasses comprehensive quality control parameters and efficacy assessment through rigorous in vitro and stability studies⁷.

MATERIALS AND METHODS:

Materials:

Ruellia tuberosa (purity 98%) pure extract and Shatadhaut Ghrita were procured from Sciquaint Innovations Private Limited, Pune India. Tween 80 (Polysorbate 80) and Span 60 (Sorbitan monostearate) were procured from Merck Life Sciences Pvt. Ltd., India. Stearic acid, acetyl alcohol, and propylene glycol were purchased from S.D. Fine Chemicals Ltd., Mumbai, India. Methyl paraben and propyl paraben were obtained from Loba Chem Pvt. Ltd., Mumbai, India. The fungal strains Candida albicans (ATCC 10231) and Aspergillus niger (ATCC 16404) were procured from Hi Media Laboratories, Mumbai, India. Fluconazole (standard) was obtained from Sigma-Aldrich, USA. All other chemicals and reagents used were of analytical grade. Double-distilled water was used throughout the study⁸.

Methods:

QbD approach for formulation design: A 3² full factorial design was employed to optimize the Ruellia tuberosa loaded Shatadhaut Ghrita-based cream formulation. Two independent variables were selected: X₁ (concentration of Shatadhaut Ghrita, 2-6% w/w) and X₂ (concentration of emulsifier blend, 3-7% w/w, consisting of Tween 80 and Span 60 in 2:1 ratio). The design yielded 9 experimental runs with different combinations of the variables⁹. Design-Expert® software (Version 13.0, Stat-Ease Inc., Minneapolis, USA) was used for experimental design generation and statistical analysis. The following polynomial equation was used to analyze the responses:

…(1)

Where

Y represents the response variable, b₀ is the intercept, and b₁ to b₂₂ are the regression coefficients. Analysis of variance (ANOVA) was performed to evaluate the significance of the model terms.

Table 1: 3² Factorial Design showing independent factors and Levels.

Independent Variables
Label	Factors	Level (%w/w)
		Low (-)	Medium	High (+)
A	Shatadhaut Ghrita	2	4	6
B	Emulsifier blend (Tween: Span, 2:1)	3	5	7
Dependant Variables
Y₁	Viscosity (cP)
Y₂	Spreadability (g.cm/sec)

Table 2: Formulation Batches for Preparation of Cream.

Ingredients (% w/w)	MF1	MF2	MF3	MF4	MF5	MF6	MF7	MF8	MF9
Ruellia tuberosa	1	1	1	1	1	1	1	1	1
Shatadhaut Ghrita	2.0	2.0	2.0	4.0	4.0	4.0	6.0	6.0	6.0
Tween 80	2.0	3.33	4.67	2.0	3.33	4.67	2.0	3.33	4.67
Span 60	1.0	1.67	2.33	1.0	1.67	2.33	1.0	1.67	2.33
Stearic Acid	2.0	2.0	2.0	2.0	2.0	2.0	2.0	2.0	2.0
Cetyl Alcohol	1.0	1.0	1.0	1.0	1.0	1.0	1.0	1.0	1.0
Propylene Glycol	5.0	5.0	5.0	5.0	5.0	5.0	5.0	5.0	5.0
Methyl Paraben	0.18	0.18	0.18	0.18	0.18	0.18	0.18	0.18	0.18
Propyl Paraben	0.02	0.02	0.02	0.02	0.02	0.02	0.02	0.02	0.02
Purified Water	q.s.	q.s.	q.s.	q.s.	q.s.	q.s.	q.s.	q.s.	q.s.

q.s. = Quantity sufficient to make 100%

Preparation of herbal cream:

The Ruellia tuberosa -loaded Shatadhaut Ghrita-based antifungal cream was formulated using the hot-emulsification technique. First, the ethanolic extract of Ruellia tuberosa was prepared by macerating the dried plant material (leaves and stems) in 70% ethanol for 72 hours, followed by filtration and vacuum evaporation to obtain a concentrated extract. The oil phase was prepared by combining Shatadhaut Ghrita (2-6% w/w), stearic acid (2% w/w), cetyl alcohol (1% w/w), and Span 60 (1-2.33% w/w) in a borosilicate glass beaker and heating to 70±2°C using a controlled hot plate (IKA C-MAG HS7, Germany). The Ruellia tuberosa extract (2% w/w) was incorporated into the oil phase during heating. Simultaneously, the aqueous phase containing Tween 80 (2-4.67% w/w), propylene glycol (5% w/w), methylparaben (0.18% w/w), propylparaben (0.02% w/w), and purified water was warmed to the same temperature. The aqueous phase was slowly added to the oil phase under high-speed homogenization at 2000 rpm for 15 minutes using IKA T25 digital ULTRA-TURRAX® high shear homogenizer (Germany). The emulsion was cooled to room temperature (25±2°C) for 30 minutes with continuous stirring at 500 rpm using a mechanical stirrer¹⁰. The prepared cream was stored in sealed glass jars at room temperature for further evaluation (n=3 batches).

Evaluation of Herbal cream:

FTIR Spectroscopy Analysis:

To evaluate the molecular compatibility of Shatadhaut Ghrita with the excipients Fourier Transform Infrared (FTIR) spectroscopy was carried out. Analysis was done on a Shimadzu FTIR-8400S spectrophotometer produced by Shimadzu Corporation of Kyoto, Japan and the system included an attenuated total reflectance (ATR) component. All spectra were obtained in the region of 4000 – 400 cm⁻¹ with a spectral resolution of 4 cm⁻¹, and each spectrum was accumulated for 64 scans. The samples were prepared by placing them on the golden surface of the ATR crystal and the analysis was done at ambient temperature (25±2°C). It should be noted that background spectra were obtained before each measurement¹¹. The spectra so obtained were analyzed using a software known as IR Solution (Version 1.50) belonging to Shimadzu Corporation.

Differential Scanning Calorimetry (DSC): Differential scanning calorimetry measurement was carried out on a DSC 214 Polymer Thermograph from NETZSCH of Germany. For sample preparation, 5 to 10mg of the samples were weighed into aluminum pans and sealed hermetically. The aluminium pan with a thermocouple inside was used as reference or sample pan without any substance inside it. The samples were heated from ambient temperature of 25°C up to 300°C with a scanning rate of the temperature at 10°C/minute and the nitrogen gas flow rate of 50 mL/min. The onset temperature and the peak temperature were determined, while the enthalpy change values were obtained using Proteus® software (Version 8.0, NETZSCH).

Physical Examination:

According to the methodology based on USP <1151>, physical examination of the prepared cream formulations was carried out. Sticking to the text, the written content was a brief description of the quality assessment in writing where the appearance, color and homogeneity were observed by holding the cream sample against natural light against a white background. Light microscopy was done using an Olympus BX53 microscope from Japan at 100× magnification in order to assess the size and distribution of the globules¹². All the experiments were carried out three times with similar results at 25±2°C.

pH Measurement:

The pH determination was done using a calibrated digital pH meter from Mettler Toledo Seven Compact™ S220, Switzerland with a glass electrode. For measurement using the pH meter, the pH meter was standardized with buffer solutions of pH 4.0, pH 7.0 and pH 9.2. The cream samples in which they contain 10% w/v cream was prepared in purified water that was boiled and then cooled, and all measurements were done at 25±1°C. For each formulation, three determinations were made; the electrode was rinse with distilled water and then dried before using. Data was pretreated and statistics were shown as Mean ± standard derivation.

Spread ability:

Spreadability was determined using the parallel plate method in accordance to these protocols. An amount of 1 g of cream was applied to the surface area between two glass plates of size 20cm × 20cm while the upper glass plate measured 100g. More mass (100g) was incorporated and kept on the subject for 5minutes. The spread diameter was further measured to the nearest centimetre using a measuring scale. Using the following formula, the spreadability (S) was determined:

S = M × –––– (2)

where M is the weight (g) tied to the upper plate, L is the length (cm) moved by the glass slide, and T is the time (seconds). Measurements were performed in triplicate at room temperature (25±2°C), and results were expressed as mean±standard deviation.

Viscosity: The viscosity measurements were done using Brookfield Viscometer (LVDV-II+Pro Brookfield Engineering Labs Co, USA) having spindle number 64. To minimize experimental error during viscosity measurements, the instrument was first standardized using standard viscosity oils. Five hundred grams of the samples were put inside beaker and allowed to reach room temperature of 25±1°C for 30minutes before performing any tests. Alternatively, using various measurements, the flow behavior was observed at different rotational speeds of 100rpm. The viscosity values were taken after 30seconds after roller speed reached the respective value. Brookfield Rheocalc version 3.3 software was used to compute rheological parameters for the findings. All measuring was done thrice^13,14.

In vitro drug release studies: Drug release studies were done using Franz diffusion cells (PermeGear, USA); the receptor volume was 20mL and the area was 1.77 cm². The selected donor compartment was separated from the receptor compartment with a synthetic cellulose membrane MWCO 12-14 kDa (Sigma-Aldrich)¹⁵. The receptor chamber was filled with phosphate buffer having a pH of 5.5°C with the help of a circulating water bath having temperature stability of 32±0.5°C. The donor chamber contained cream samples (500 mg) while the receptor chamber had 500μL of aliquots removed at specific time intervals (0, 1, 2, 4, 6, 8, 12, 24h) replenishing it with fresh buffer immediately. The content of the extracts was determined using a UV spectrophotometer of Shimadzu 1600 make from Japan, with UV detection at 254nm. The total amount of the drug permeated was determined and depicted against the time domain. Studies were performed in sextuplicate (n=6).

Stability studies:

The stability studies were carried out according to the ICH Q1A(R2) guidelines of stability testing of new drug substances and new drug products. The cream samples were placed in sample tubes that were sealed with a glass stopper and taped with parafilm then stored under two different conditions; long-term and accelerated conditions in the stability incubators (Thermo Scientific™ Stability Chamber, USA) for a period of 6 months. The gel characterized in respect to its physical appearance, pH, viscosity, spreadability and drug content, at 0, 1, 3 and 6 months. All statistical analyses were conducted using one-way Analysis of variance (ANOVA) followed by Tukey’s post hoc test using GraphPad PRISM® version 9.0 software with p < 0.05 considered significant. All the stability parameters were determined in triplicate and the mean values were reported¹⁶.

In-vitro antifungal activity:

Antifungal activity in vitro was done with the agar well diffusion method using Candida Albicans ATCC 10231 & Aspergillus Niger ATCC 16404. Sour cream agar SDA plates were prepared and autoclaved at 150°C for fifteen minutes. The fungal suspensions were prepared in sterile normal saline, and turbidity was adjusted to 1-5 × 10⁶ CFU/mL using McFarland standard. 135°C) sterilized cork borer, Wells of 6 mm diameter were made respectively, in the solidified agar. The test samples (100μL) to be tested in this study were applied into the wells which are Ruellia tuberosa (pure), optimized formulation MF5 and Fluconazole with a concentration of 10μg/mL. The plates were incubated at 28±2°C for 48 hours for A niger and 37±2°C for 24 hours for C albicans. The area of the zone of inhibition was measured using the help of the digital vernier caliper with 3 attempts. The results were analyzed by one way Anova followed by Tukey’s test with a significance level of 0.05¹⁷.

RESULTS:

FTIR analysis: The FTIR spectral analysis revealed characteristic functional group patterns for both Ruellia tuberosa and its physical mixture with excipients (Figure 1 and Figure 2). In Ruellia tuberosa, prominent peaks were observed at 3335.37 cm⁻¹ indicating O-H stretching vibrations, while symmetric C-H stretching was evident at 2877.91 cm⁻¹. The spectrum also showed characteristic peaks at 1637.81 cm⁻¹ (C=C stretching/N-H bending), 1402.76 cm⁻¹ (C-H bending), 1323.70 cm⁻¹ (C-H rocking), and 1041.16 cm⁻¹ (C-O stretching of alcohol). The physical mixture demonstrated similar yet distinct spectral patterns, with the O-H stretching peak slightly shifted to 3328.40 cm⁻¹. Additional peaks were observed at 2976.59cm⁻¹ and 2824.13cm⁻¹, representing asymmetric and additional C-H stretching respectively. Notable new peaks appeared at 1719.50 cm⁻¹ (C=O stretching), 1241.13 cm⁻¹ (C-O stretching), 990.70 cm⁻¹ (= C-H bending), and 820.25 cm⁻¹ (C-H out-of-plane bending), indicating successful incorporation of excipients.

Evaluation of herbal Cream:

Nine batches of Ruellia tuberosa loaded Shatadhaut Ghrita cream were used in the study and data analysed showed some changes in physical characteristics and physicochemical parameters. As presented in table 3 below, all the formulations starting with MF1 MF2 MF3, had a colour of off white and semi solidified texture while formulation MF4 to MF6 had pale yellow colour and soft texture. MF7 through MF9 stained light yellow color predominantly and it was seen that MF7 had slight graininess and phase separation whereas other formulations remained homogenous. The physicochemical characterization (Table 4) showed the pH of the formulations between 5.9 to 6.4 indicating that all formulations meet the requirement for a topical drug delivery system. All the formulations revealed differences in spreadability, and it was higher (15780 ± 368 g.cm/sec) in MF7 and the least spreadability was recorded in MF2 (10902±284 g.cm/sec). Measurements confirmed at 10 rpm ranged from 13.4±0.3 to a maximum of 18.5±0.6 cP; MF2 with the highest viscosity and MF7 with the least viscosity. The spreadability decreased progressively with an increase in the viscosity and peak emerged the correlation between the viscosity and the spreadability. This pattern was seen more in the MF2 which had the highest viscosity of 18.5 ±0.6 cP equivalent to the least spreadability. On the other hand, MF7 exhibited the highest spreadability with the least viscosity, but it was also the least homogeneous and exhibited signs of phase separation implying instability¹⁹.

Table 3: Physical Characteristics and Appearance of Ruellia tuberosa loaded Shatadhaut Ghrita Cream Formulations.

Batch Code	Color	Consistency	Homogeneity	Phase Separation
MF1	Off-white	Semi-solid	Homogeneous	No separation
MF2	Off-white	Semi-solid	Homogeneous	No separation
MF3	Off-white	Semi-solid	Homogeneous	No separation
MF4	Pale yellow	Soft	Homogeneous	No separation
MF5	Pale yellow	Soft	Homogeneous	No separation
MF6	Pale yellow	Soft	Homogeneous	No separation
MF7	Light yellow	Very soft	Slightly grainy	Slight separation
MF8	Light yellow	Soft	Homogeneous	No separation
MF9	Light yellow	Soft	Homogeneous	No separation

Table 4: Physicochemical Parameters of Ruellia tuberosa loaded Shatadhaut Ghrita Cream Formulations.

Batch Code	pH	Spreadability (g.cm/sec)	Viscosity (cP at 10 rpm)
MF1	6.2 ± 0.2	12450 ± 325	15.2 ± 0.4
MF2	6.3 ± 0.1	10902 ± 284	18.5 ± 0.6
MF3	6.4 ± 0.2	12240 ± 298	16.5 ± 0.5
MF4	6.1 ± 0.1	14747 ± 342	14.6 ± 0.3
MF5	6.2 ± 0.2	12358 ± 276	18.2 ± 0.5
MF6	6.3 ± 0.1	13680 ± 315	15.8 ± 0.4
MF7	5.9 ± 0.2	15780 ± 368	13.4 ± 0.3
MF8	6.0 ± 0.1	13450 ± 292	16.9 ± 0.5
MF9	6.1 ± 0.2	14120 ± 328	14.2 ± 0.4

Results are expressed in mean ± SD, n=3

Optimization of concentration of Ruellia tuberosa loaded Shatadhaut Ghrita and Emulsifier blend:

Effect of variables on Viscosity (R1): The viscosity response demonstrated significant correlation with both independent variables as evidenced by the quadratic model (Table 5), showing high adjusted R² (0.9890) and predicted R² (0.9675) values. ANOVA results (Table 6) confirmed the model's significance (p=0.0009), with all terms including linear, interaction, and quadratic effects showing significant impact (p<0.05). The contour and 3D surface plots (Figure 4A and 4B) illustrated that viscosity increased with higher concentrations of Shatadhaut Ghrita (positive coefficient: +1293.00) and decreased with increasing emulsifier blend (negative coefficient: -489.50). The quadratic equation revealed a complex relationship between variables, with significant interaction effects (AB: -362.50) influencing the final viscosity^20,21.

Final Equations in Coded Factors for Viscosity (R1):

Viscosity (10 rpm) = +12528.67 + 1293.00A - 489.50B - 362.50AB438.00A² + 1599.50B² (3)

Table 6: ANOVA for Quadratic Model for Viscosity and Spreadability

Source	Sum of Squares	df	Mean Square	F-value	p-value	Significance
Viscosity (R1)
Model	1.749E+07	5	3.499E+06	144.93	0.0009	Significant
A - Shatadhaut Ghrita	1.003E+07	1	1.003E+07	415.50	0.0003	Significant
B - Tween 80:Span 60 (2:1)	1.438E+06	1	1.438E+06	59.55	0.0045	Significant
AB	5.256E+05	1	5.256E+05	21.77	0.0186	Significant
A²	3.837E+05	1	3.837E+05	15.89	0.0283	Significant
B²	5.117E+06	1	5.117E+06	211.94	0.0007	Significant
Spreadability (R2)
Model	24.655	5	4.931	80.51	0.0006	Significant
A - Shatadhaut Ghrita	5.421	1	5.421	98.24	0.0008	Significant
B - Tween 80:Span 60 (2:1)	1.811	1	1.816	6.45	0.0039	Significant
AB	0.0625	1	0.0625	2.29	0.2276	Not Significant
A²	0.3472	1	0.3472	12.71	0.0377	Significant
B²	17.011	1	17.016	22.88	0.0001	Significant

Figure 4: Contour (A) and 3D surface plot (B) for the effect of independent variables on viscosity (10 rpm). Contour (C) and 3D surface plot (D) for the spreadability.

Statistical validation of model:

The statistical validation of the optimized formulation revealed promising results on the comparison of the predicted and the experimental values which has been illustrated in the Table-7. Percentage error and bias standards were largely narrow and low: in a range of 0.0002% to 0.90%. More specifically, the viscosity was particularly sensitive with a desirable experimental value of 12358.000 CP against the theoretical 12357.976 CP with a minute error estimate of only- -0.0002%. Similarly as with the spreadability, was rather precise and had the experimental value of 18.200 g.sec./cm while the predict value was 18.269 g.sec./cm which made it possible to calculate the percentage of an error of 0,38%. The concentrations of Shatadhaut Ghrita and emulsifier blend (Tween 80:Span 60) were also close to the given values and the percentage errors were found to be 0.25 and 0.90% respectively, hence the optimization model was rock solid^23,24.

Cumulative Percentage Drug Release:

The in vitro drug release study was performed for 12 hours and the release profiles of all the nine formulations of Ruellia tuberosa loaded Shatadhaut Ghrita cream are presented in Figure 5. The release profiles showed an ideal trend were there was fast release at the initial stage then slow and continuous release of drugs. Among all, MF5 has the highest drug release with value of 95.8% at 12 hours followed by, MF6 and MF2 with the value of 93.2 and 92.6 respectively. Therefore, during the first hour MF5 had the highest release of 16.5% meaning that the drug diffused freely at the beginning. Intermediate formulations: The intermediate formulations MF3 and MF9 had a moderate release the release profile and it was observed that both of them had only 88. 9% and 89. 6% release of the same at 12hours of the study. Cumulative release and burst release data for MF7 was the lowest at 12hours and 1 hour respectively, which indicates strong interaction between the drug and the excipient or higher viscosity of the system affect the diffusion of the drug. An increasing trend was seen with regards to the amount of emulsifier incorporated (MF2, MF5 and MF6) which depicted more drug release than the ones with least emulsifier concentration (MF1, MF4 and MF7) Thus the emulsifier system plays a vital role in the release of drug from the cream base. The release profiles is a bi-phasic one for all the formulations: a fast first phase up to 0-4h and a slow second phase from 4-12h. Such release behavior clearly manifests the development of a controlled release formulation used in the topical drug delivery in extended release²⁵.

Results of Stability study:

The stability study of the optimized formulation (MF5) conducted at accelerated conditions (40°C± 2°C/75% ± 5% RH) over 6 months revealed gradual changes in various parameters, as presented in Table 8. The physical appearance transformed from off-white to light yellowish, with the emergence of slight graininess and phase separation by the end of 6 months. The pH showed a moderate decline from 6.2±0.12 to 5.8±0.16, remaining within the acceptable range for topical applications. The rheological properties demonstrated expected variations, with viscosity decreasing from 12358±245 cP to 11842±312 cP, while spreadability increased from 18.2±0.42 to 19.2±0.52g.cm/sec over the study period. Drug content remained relatively stable with minimal degradation, showing a decrease from 99.8 ±0.86% to 97.2±1.24%. The cumulative drug release at 12 hours exhibited only marginal reduction from 88.9± 1.84% to 86.2±2.28%, indicating good stability of the active ingredients.

Figure 5: Cumulative Percentage Drug Release from Ruellia tuberosa loaded Shatadhaut Ghrita Cream Formulations.

In-vitro antifungal activity:

The in-vitro antifungal activity study demonstrated significant inhibitory effects against both test organisms, as shown in Table 9 and illustrated in Figures 6 and 7. The optimized formulation (MF5) exhibited superior antifungal activity compared to Ruellia tuberosa alone, though slightly lower than the standard fluconazole. Against Candida albicans, MF5 produced a zone of inhibition of 21.4±0.8mm, showing considerable improvement over Ruellia tuberosa (20.8±0.6mm) and approaching the efficacy of fluconazole (24.2±0.9mm). Similar trends were observed against Aspergillus niger, where MF5 demonstrated a zone of inhibition of 18.6±0.7mm, compared to 18.2±0.5mm for Ruellia tuberosa and 20.8±0.8mm for fluconazole. The photographic evidence (Figure 6) clearly depicts the zones of inhibition, with MF5 showing same clear zones compared to Ruellia tuberosa. The bar graph representation (Figure 7) statistically confirms these observations, with significant differences (p<0.05) noted between the test samples and standard. The enhanced antifungal activity of MF5 compared to Ruellia tuberosa suggests successful formulation development, with the cream base potentially improving the delivery and efficacy of the active ingredients.

Table 9: Zone of Inhibition Against Test Organisms (mean ± SD, n=3).

Test Sample	Zone of Inhibition (mm)
Test Sample	Candida albicans	Aspergillus niger
Ruellia tuberosa	20.8 ± 0.6	17.2 ± 0.5
Optimized Formulation (MF5)	21.4 ± 0.8	18.6 ± 0.7
Standard (Fluconazole)	24.2 ± 0.9	20.8 ± 0.8

Results are expressed in mean ± SD, n=3

Figure 6: Photographic images showing zones of inhibition on SDA plates after 24-48 hours of incubation. (A) C. albicans: F1-Ruellia tuberosa, F2-MF5, F3-Standard; (B) A. niger: F1-Ruellia tuberosa, F2-MF5, F3-Standard. Scale bar = 10 mm.

Figure 7: Bar graph showing the zone of inhibition (mm) of Ruellia tuberosa, optimized formulation (MF5), and standard (Fluconazole) against C. albicans and A. niger. Values represented as mean±SD (n=3). *p<0.05 compared to standard.

DISCUSSION:

Spectroscopic and thermal analyses provided essential data regarding the compatibility and stability conditions of the Ruellia tuberosa loaded Shatadhaut Ghrita-based formulation. Traditional Ghrita preparations matched the functional groups that the FTIR spectral analysis showed in Figures 1 and 2., The additional peaks in the physical mixture supported the successful incorporation of excipients without chemical interferences with the active compound. The analytical findings align with previous studies analyzing herbal creams because they displayed comparable spectral patterns which confirmed excipient integration. The DSC analysis (Figure 3) also affirmed these observations; Thermal profiles of physical mixture were similar to Ruellia tuberosa analyzed in this study are in accordance with the reported range of other Ghrita formulations. The changes of 0.4°C in the melting point from 64.4°C to 64.8°C and the extra endotherm at 132.5°C indicated for excipients.

According to the physicochemical assessment made on the cream formulations, it has been observed that the formulations (Table 3 and 4) are suitable to be used as topical dosage form. The results falls within the range of pH of 5.9-6.4 this is in agreement with literature reports on skin compatibility, Whereas the viscosity and spread ability of the formulations demonstrated a strong relationship with the stability of the cream. The findings evident from this study regarding the inverse relationship between viscosity and spreadability is consistence with previous studies on other herbal cream formulations^. Notably, formulation MF5 exhibited ideal balance between viscosity (18.2±0.5cP) and Spreadability (12358±276g.cm/sec), comparable to commercially successful topical formulation. The stability profile of MF5 thus supports the practicality of the MF5 as a potential antifungal formulation, which did not show significant variability in essential factors during the accelerated stability period, which is in line with the stability studies made on other herbal formulations.

The optimization process comprised of a sequential optimization of formulation factors utilizing response surface methodology to model the interaction of formulation factors with the critical product attributes. model showed excellent fit for both in terms of viscosity and spreadability response and very high adjusted R² value, as presented in Table 5(0.9890 and 0. 9912) respectively which is higher than the value reported in similar optimization studies of herbal formulations. The ANOVA results (Table 6) revealed significant effects of both Shatadhaut Ghrita concentration and emulsifier blend ratio on the rheological properties, consistent with previous findings in topical cream optimization studies. Notably, the interaction effects between variables showed stronger influence on viscosity (p=0.0186) compared to spreadability (p=0.2276), a phenomenon also observed in related emulsion-based formulations. The statistical validation of the optimized formulation (Table 7) demonstrated remarkable precision, with percentage errors consistently below 1% for all parameters. The minimal deviation between predicted and experimental values, particularly for viscosity (-0.0002%) and spreadability (0.38%), validates the robustness of the optimization model. These results align with acceptable validation criteria reported in pharmaceutical optimization studies and surpass the prediction accuracy observed in similar herbal cream formulations. The contour and response surface plots (Figure 4) effectively visualized the relationship between variables and responses, facilitating the identification of optimal formulation parameters that balanced both rheological properties. This optimization approach successfully established a formulation space that ensures consistent product quality, comparable to modern pharmaceutical development strategies.

The in vitro release studies demonstrated a biphasic release pattern across all formulations, with MF5 exhibiting superior drug release characteristics (Figure 5). The initial burst release followed by sustained release behavior aligns with previous studies on herbal cream formulations, where similar release patterns were attributed to optimal emulsifier concentrations. The enhanced drug release observed in formulations with higher emulsifier content (MF5: 95.8% at 12hours) compared to those with lower concentrations (MF7: 78.6%) corroborates findings from related studies, where surfactant systems played a crucial role in drug diffusion. The biphasic release pattern, characterized by rapid initial release (0-4hours) followed by sustained release (4-12hours), suggests successful development of a controlled release system, comparable to established topical formulations.the accelerated stability studies of the optimized formulation MF5(Table 8) revealed acceptable changes in physicochemical parameters over 6 months, consistent with ICH guidelines for topical formulations. The observed pH decline from 6.2 to 5.8 remained within the skin-compatible range reported in literature while the minimal drug degradation (97.2% retention) and sustained release capability (86.2% at 12 hours) after 6 months demonstrate robust formulation stability. The slight changes in appearance and rheological properties are comparable to stability profiles reported for similar herbal formulations, though the emergence of minor phase separation at 6 months suggests potential for further optimization of the preservative system. These findings indicate that the formulation maintains acceptable stability for up to 3 months under accelerated conditions, with minor physical changes emerging thereafter.

The in vitro antifungal evaluation demonstrated significant enhancement of antimicrobial efficacy through optimized formulation development, as evidenced by the comparative zones of inhibition (Table 9). The optimized formulation MF5 exhibited markedly improved antifungal activity against both test organisms compared to Ruellia tuberosa loaded alone, with inhibition zones approaching those of standard fluconazole. This enhancement aligns with previous studies on herbal formulations where proper vehicle selection and optimization significantly improved the biological activity of traditional medicines. The superior activity against C. albicans (21.4±0.8mm) compared to A. niger (18.6±0.7mm) follows patterns observed in related studies on natural antifungal agents, possibly due to differences in cell wall composition and penetration characteristics between yeasts and filamentous fungi.

The comparative analysis with standard fluconazole (Figures 6 and 7) revealed that MF5 achieved approximately 88.4% and 89.4% of the standard's efficacy against C. albicans and A. niger respectively, surpassing the typical efficacy ratios reported for herbal formulations . The significant improvement in antifungal activity of MF5 compared to pure Ruellia tuberosa (p < 0.05) suggests successful enhancement of active ingredient bioavailability through optimized delivery system design, consistent with findings from similar studies on traditional medicine optimization. The cream base appears to facilitate better contact and penetration of active components, potentially through improved spreading and retention characteristics, a mechanism previously documented in successful herbal formulation developments. These results validate the rationale behind the formulation approach and suggest potential clinical applicability²⁶.

CONCLUSION:

The present study was able to standardize and fine-tune the antifungal cream formulation of Ruellia tuberosa loaded Shatadhaut Ghrita based cream using an appropriate quality by design strategy. The optimized formulation revealed satisfactory preservation profile and found to be stable for up to 3 months under accelerated stability condition. The FTIR analysis showed no interaction of drug with the excipients, and from the DSC study a phase transition was detected, making it suitable for drug delivery by topical route. The formulation was found to possess moderate antifungal activity against both the test fungi, and the results were as promising as those reported for the standard fluconazole, pointing towards it as a possible substitute to synthetic antifungal reagents. These positive features of higher therapeutic effects and stability have proved the potential of using the compound in the treatment of topical fungal infections. Nevertheless, subsequent studies in vivo and clinical trials are needed to uncover the complete therapeutic application and safety of the product.

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Received on 26.02.2025 Revised on 11.06.2025

Accepted on 27.08.2025 Published on 10.02.2026

Available online from February 16, 2026

Research J. Pharmacy and Technology. 2026;19(2):501-511.

DOI: 10.52711/0974-360X.2026.00073

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Response	Model Type	Sequential p-value	Adjusted R²	Predicted R²	Suggested Model
Viscosity (R1)	Linear	0.0418	0.5371	0.2921	-
	2FI	0.5228	0.4924	0.1862	-
	Quadratic	0.0015	0.9890	0.9675	Suggested
	Cubic	0.9512	0.9702	0.3201	-
Spreadability (R2)	Linear	0.3545	0.0564	-0.4823	-
	2FI	0.8987	-0.1283	-1.9361	-
	Quadratic	0.0003	0.9912	0.9609	Suggested
	Cubic	0.2911	0.9978	0.9488	-

Parameters	Initial	1 Month	3 Months	6 Months
Appearance	Off-white, smooth	Off-white, smooth	Slight yellowish, smooth	Light yellowish, smooth
Phase Separation	None	None	None	Slight
Homogeneity	Homogeneous	Homogeneous	Homogeneous	Slightly grainy
pH	6.2 ± 0.12	6.1 ± 0.14	5.9 ± 0.15	5.8 ± 0.16
Viscosity (cP)	12358 ± 245	12245 ± 268	12086 ± 284	11842 ± 312
Spreadability (g.cm/sec)	18.2 ± 0.42	18.4 ± 0.45	18.8 ± 0.48	19.2 ± 0.52
Drug Content (%)	99.8 ± 0.86	99.2 ± 0.92	98.4 ± 1.12	97.2 ± 1.24
Cumulative Drug Release at 12 hrs (%)	88.9 ± 1.84	88.2 ± 1.92	87.4 ± 2.14	86.2 ± 2.28

Response Parameters	Predicted Value	Experimental Value	Residual	% Error	Bias (%)
Shatadhaut Ghrita (%)	4.01	4.00	0.01	0.25	0.25
Tween 80:Span 60 (2:1) (%)	5.045	5.00	0.045	0.90	0.90
Viscosity (cP at 10 rpm)	12357.976	12358.000	-0.024	-0.0002	-0.0002
Spreadability (g.cm/sec)	18.269	18.200	0.069	0.38	0.38