Design And Development of Ethosomes Containing Extract of Curcuma Caesia Roxb for the Treatment of Acne Infection

Acne vulgaris is a common chronic inflammatory skin disorder of the pilosebaceous unit, affecting nearly 85% of adolescents and young adults. It results from multiple factors including excessive sebum production, follicular hyperkeratinization, bacterial colonization by Cutibacterium acnes, and inflammation. Clinically, acne manifests as comedones, papules, pustules, and in severe cases, nodulocystic lesions. Conventional treatments such as topical retinoids, benzoyl peroxide, antibiotics, and systemic agents like isotretinoin often cause irritation, resistance, or systemic side effects, highlighting the need for safer, more effective alternatives using herbal actives and advanced delivery systems.^[1]

The skin, comprising the epidermis, dermis, and hypodermis, serves as a barrier that limits drug permeation due to the tightly packed lipids of the stratum corneum. However, drug delivery through the skin can be optimized using novel vesicular carriers such as ethosomes, which enhance penetration by combining phospholipids with high ethanol content. Ethosomes are soft, deformable vesicles that allow improved transdermal and dermal drug delivery, increasing therapeutic efficacy while minimizing systemic exposure. Their small vesicle size, flexibility, and ethanol-induced lipid fluidization make them suitable for the delivery of both hydrophilic and lipophilic molecules, particularly in topical therapy.^[2]

Curcuma caesia Roxb (Black Turmeric), a member of the Zingiberaceae family, is a perennial herb with bluish-black rhizomes known for its diverse pharmacological properties. Traditionally used for treating wounds, skin infections, respiratory and digestive ailments, and inflammation, it is rich in curcuminoids, flavonoids, essential oils (camphor, ar-turmerone), and phenolic compounds. These bioactive exhibit potent antimicrobial, antioxidant, anti-inflammatory, analgesic, and wound-healing activities. In acne management, C. caesia demonstrates inhibitory action against Staphylococcus aureus and Propionibacterium acnes, reduces oxidative stress, and promotes tissue repair. Compared to Curcuma longa, black turmeric possesses stronger antimicrobial and antioxidant activities, making it a novel and underexplored candidate for topical acne formulations.^[3]

MATERIALS AND METHODOLOGY

MATERIALS

Curcuma caesia Roxb was purchased from local market of Mysuru, Karnataka. Soya lecithin was obtained from Oxford Lab Fine Chem LLP (Palghar, Maharashtra), and ethanol from Bio Liqua Research Pvt. Ltd. (Bangalore). Cholesterol was purchased from Rolex Chemical Industries (Mumbai), propylene glycol and triethanolamine from Sisco Research Laboratories Pvt. Ltd. (Mumbai), and Carbopol 934 from SD Fine Chem Ltd. (Mumbai). Methyl paraben was supplied by MERCK Specialities Pvt. Ltd. (Mumbai), while ethyl acetate and methanol were procured from Isochem Laboratories (Palakkad) and Central Drug House (P) Ltd. (New Delhi), respectively. All chemicals and solvents used were of analytical grade.

METHODOLOGY

Preparation of Extracts

Rhizomes of Curcuma caesia Roxb were collected from Mysuru, Karnataka, in February and authenticated by Dr. M. Devika, Principal, Sarada Vilas College, Mysuru. The rhizomes were shade-dried, coarsely powdered, and extracted with methanol, water, and ethyl acetate by maceration for 48 hours. The extracts were filtered, concentrated to dryness, and stored at 4 °C until further use.^[4]

Determination of percentage yield of plant extract ^[5]

The percentage yield was obtained using this formula,

% yield=Weight of dried extract obtained (g)Weight of plant material taken (g)×100

Phytochemical screening of extracts

Preliminary phytochemical tests were performed on the crude extracts to identify constituents such as carbohydrates, proteins, alkaloids, glycosides, terpenes, steroids, flavonoids, tannins, and saponins using standard methods.^[5]

FTIR Analysis of Curcuma Caesia Roxb ^[6]

The IR spectrum of Curcuma caesia Roxb extract was obtained to assess its compatibility with excipients used in the formulation. A physical mixture of the extract and various polymers was prepared for this purpose. Approximately 2-3 mg of the sample was mixed with potassium bromide, dried at 40-50°C, and compressed into a transparent pellet under 8-10 tonnes of pressure using a hydraulic press. The prepared pellet was analysed in an FTIR spectrophotometer, scanning within the 4000-400 cm?¹ range.

Determination of Absorption Maxima (λ Max) ^[7]

An accurately weighed 10 mg of Curcuma caesia Roxb extract was dissolved in 10 ml of methanol in a volumetric flask, yielding a stock solution of 1000 μg/ml. From this stock, 1 ml was transferred to another 10 ml volumetric flask and diluted to the mark with a methanol, forming the working solution. From this, dilutions ranging from 10 to 50 μg/ml were prepared. The absorbance of each solution was scanned between 200-600 nm using UV spectrophotometer against a blank to determine the absorption maxima (λmax).

Preparation of Standard Calibration Curve ^[7]

10 mg of Curcuma caesia Roxb extract was accurately weighed, dissolved in methanol, and the volume was made up to 10 mL to obtain a 1000 μg/mL stock solution. From this, 1 mL was diluted to 10 mL to prepare a 100 μg/mL working solution. Standard solutions ranging from 1-10 μg/mL were prepared using methanol as the diluent. The absorbance of each solution was measured at 425 nm using a UV-Visible spectrophotometer, with methanol as the blank. A calibration curve was plotted between absorbance and concentration, and the regression equation was calculated.

Preparation of Ethosomes ^[8]

Ethosomes were prepared using the cold method. Curcuma caesia Roxb extract was dissolved in ethanol, followed by the addition of soya lecithin and cholesterol, and mixed thoroughly using a mechanical stirrer. Propylene glycol was incorporated into the lipid mixture with continuous stirring. In a separate container, purified water heated at 30°C, was slowly added to the lipid phase as a fine stream under constant stirring at 700 rpm. The mixture was further stirred for 10-15 min to form a uniform ethosomal suspension, which was then sonicated for 1-3 min to reduce vesicle size. The prepared ethosomes were stored at 4-8°C until further use.

Ethosomes were evaluated for particle size, zeta potential, PDI, and entrapment efficiency. The formulation with the smallest vesicle size, highest entrapment, stable zeta potential, and low PDI was selected as the optimized formulation for gel preparation.

Physical examination ^[9]

Ethosomal formulations were visually evaluated for color, appearance, clarity, and phase separation under uniform daylight conditions.

Microscopic study ^[9]

An ethosomal suspension was examined under a light microscope (100×) to observe vesicle morphology and distribution.

Vesicle size determination ^[10]

Vesicle size of ethosomes were analysed by Scanning Electron Microscopy (SEM). The lyophilized sample was gold-coated and imaged under high vacuum at 3-10 kV, and vesicle diameters were measured using ImageJ software.

Surface morphology ^[11]

Surface morphology of ethosomes was examined by Scanning Electron Microscopy (SEM).

Zeta potential ^[12]

Zeta potential of the ethosomal suspension was analysed using a Zetasizer to assess surface charge and stability. Measurements were performed after appropriate dilution, and values beyond ±30 mV indicated stable vesicular dispersion.

Particle size ^[10]

Particle size and polydispersity index (PDI) of ethosomes were determined using a Zetasizer (DLS) at 25°C.

Entrapment efficiency (EE%) ^[13]

The entrapment efficiency of ethosomal formulations was determined by ultracentrifugation at 15,000 rpm for 30 min at 4°C. The unentrapped drug in the supernatant was measured using UV spectrophotometry at 425 nm (placebo as blank), and EE% was expressed as the percentage of drug encapsulated. EE% is then calculated using the formula:

EE%=Total drug added-amount of free drugTotal drug added×100

pH determination ^[14]

The pH of the ethosomal suspension was measured using a digital pH meter at room temperature (25°C).

Determination of Drug content ^[13]

Drug content of the ethosomal suspension was determined using a UV spectrophotometer at 425 nm. A precisely weighed amount of suspension was transferred into a 10 mL volumetric flask, diluted with methanol, and mixed thoroughly. The solution was filtered through a 0.45 μm membrane filter, and 1 mL of the filtrate was further diluted to 10 mL with methanol. The absorbance of the final solution was measured, and drug content was calculated using a standard calibration curve.

% Drug load=WDWB×100

Where, WD is the amount of drug loaded in beads and WB is the weight of beads.

Preparation of Ethosomal Gel of Optimized Formulation ^[15]

The optimized ethosomal formulation was incorporated into a gel base using a Carbopol 934 as the gelling agent. Initially, the required quantity of Carbopol 934 was dispersed in distilled water at 300 rpm and allowed to swell overnight to ensure complete hydration. The drug-loaded ethosomal suspension was then incorporated dropwise into the swollen polymer base with continuous mixing to obtain a uniform dispersion. Preservatives such as methyl paraben (0.05% w/v) and propyl paraben (0.05% w/v) were incorporated slowly with gentle stirring and the volume of gel was adjusted to 100 ml with distilled water. Triethanolamine was carefully added to adjust the pH and facilitate the formation of a transparent gel. The final preparation yielded a smooth, homogeneous ethosomal gel suitable for further evaluation.

Table 2: Formulation trials of Ethosomal gel of optimized formulation

Characterization of Ethosomal Gel

Physical parameters ^[13]

The prepared formulation was systematically evaluated for their physical properties, including color, overall appearance, homogeneity, and any signs of phase separation.

pH determination ^[14]

The pH of ethosomal gel was evaluated using a digital pH meter at room temperature (25°C).

Viscosity determination ^[16]

Viscosity of the ethosomal gel was measured using a Brookfield RV viscometer (spindle RV-7) at 25 ± 0.5°C.

Spreadability ^[17]

The spreadability of the ethosomal gel was determined using the glass-slide method. A measured amount of gel (0.5-1 g) was placed between two glass slides and compressed with a 100 g weight for 5 min. The upper slide was then attached to a string with a standard weight, and the time required to travel was recorded. Spreadability was calculated using the formula:

S=M×LT

Where,

M- weight applied (g)

L- distance moved (cm)

T- time taken in sec (s).

Determination of Drug content ^[13]

Drug content of the ethosomal gel was assessed using a UV spectrophotometer. An accurately weighed amount of 500 mg gel was transferred into a 10 ml volumetric flask and the volume was made up to the mark with methanol. The solution was mixed thoroughly, filtered through a 0.45 μm membrane filter, and from the filtrate, 1 ml was withdrawn and further diluted to 10 ml with the same solvent mixture. The absorbance of the prepared solution was measured at 425 nm using a UV spectrophotometer.

% Drug load=WDWB×100

Where, WD is the amount of drug loaded in beads and WB is the weight of beads.

In Vitro Drug Release Study ^[18]

In vitro drug release from the ethosomal gel was studied using a Franz diffusion cell with a pre-soaked dialysis membrane. The receptor compartment contained phosphate buffer (pH 7.4) at 37 ± 0.5°C under continuous stirring. Gel samples were placed in the donor compartment, and 1 mL aliquots were withdrawn at predetermined intervals (0.5-6 h), filtered, and analysed spectrophotometrically. Cumulative drug release was calculated and plotted against time.

In Vitro Drug Release Kinetics ^[19]

The in vitro drug release data of Curcuma caesia Roxb ethosomal gel (Carbopol 934-based) were analysed using various kinetic models Zero-order, First-order, Higuchi, and Korsmeyer-Peppas to determine the release mechanism. The model with the highest correlation coefficient (R) and suitable release rate constant (k) was considered the best fit to describe the release behaviour.

Antibacterial Activity ^[20]

The antibacterial potential of the formulations was evaluated using the agar well diffusion method. A 24-hour bacterial culture was suspended in sterile distilled water to prepare the inoculum. Sterile nutrient agar medium was inoculated with 0.1 mL of this suspension at 40°C to ensure uniform microbial distribution and poured into sterile petri dishes. After solidification, 6 mm diameter wells were aseptically made, and the test samples methanolic extract and optimized ethosomal gel were added. The plates were allowed to stand for 30 minutes for pre-diffusion, followed by incubation at 37°C for 24 hours. The antibacterial activity was determined by measuring the diameter of the inhibition zones around each well.

RESULT AND DISCUSSION

Preparation of Extracts

The rhizomes of Curcuma caesia Roxb were subjected to maceration using three different solvents such as methanol, water and ethyl acetate. The choice of solvents was based on their respective extractive values, ensuring maximum recovery of phytoconstituents. The results are showed in Table 3.

Table 3: Characteristics of Curcuma caesia Roxb extract

The methanolic extract showed a dark brown colour, strong aromatic odour, and viscous consistency, indicating efficient extraction of active constituents. The aqueous extract appeared brownish-yellow with a mild odour and semi-solid texture, while the ethyl acetate extract was yellowish-brown with a slightly pungent taste and sticky consistency.

Determination of percentage yield of plant extract

The percentage yield indicates the efficiency of the extraction process. In this study, extracts prepared with methanol, water, and ethyl acetate were evaluated for their respective yields. The results are presented in Table 4.

Table 4: % yield of rhizomes of Curcuma caesia Roxb

The percentage yield of the extract varied with the type of solvent used, reflecting differences in their extraction efficiency. Methanol and water yielded the highest amounts of extract (23.43% and 23.26%, respectively), indicating that the majority of the phytoconstituents in the sample are polar and readily soluble in these solvents. In contrast, ethyl acetate gave a much lower yield (8.56%), suggesting that fewer non-polar compounds are present in the sample. This demonstrates that solvent choice significantly affects the amount of extract obtained.

Phytochemical screening of extracts

The phytochemical screening of the extracts revealed the presence of a variety of bioactive constituents, which varied with the solvent used. Alkaloids, terpenoids, steroids, and phenolics were detected in all three extracts, indicating that these compounds are widely present in the sample and soluble in both polar and non-polar solvents. Glycosides and flavonoids were present in methanol and ethyl acetate extracts but absent in the water extract, suggesting their moderate polarity. Saponins were detected only in the water extract, reflecting their high polarity and water solubility. Tannins were present in methanol and water extracts but not in ethyl acetate, consistent with their polar nature. Proteins and carbohydrates were absent in all extracts, indicating that these macromolecules are either not present in significant amounts or were not efficiently extracted. Overall, these results demonstrate that the choice of solvent strongly influences the profile of phytoconstituents extracted, with polar solvents like methanol and water favouring the extraction of most bioactive compounds. The results are tabulated in Table 5.

Table 5: Phytochemical screening of extracts of rhizomes of Curcuma caesia Roxb

(“+ve” present, “_ve” absent)

FTIR Analysis of Curcuma Caesia Roxb

The FTIR spectra of Curcuma caesia, its physical mixture with soya lecithin, and the mixture with other excipients were analysed to assess possible interactions and confirm the presence of characteristic functional groups. The FTIR spectrum of curcuma caesia reveal the presence of peaks at 3332.63 cm-1 due to the presence of O-H stretching, 2922.34 cm-1 due to the presence of C-H asymmetric stretching, 2853.25 cm-1 due to the presence of C-H symmetric stretching, 1731.51 cm-1 due to the presence of C=O stretching, 1615.36 cm-1 due to the presence of C=C stretching, 1455.34 cm-1 due to the presence of C-H bending, 1320.91 cm-1 due to the presence of O-H bending (phenol), 1215.51 cm-1 due to the presence of C-O stretching, 625.92 cm-1 due to the presence of Aromatic C-H bend. There were no changes in the existing peaks and there were no appearance or disappearance of peaks, indicating that the excipients used were compatible with the drug. The results are showed in Fig 1.

Determination of Absorption Maxima (λ Max)

The λmax of Curcuma caesia Roxb was determined by scanning the drug solution in the UV spectrophotometer over the wavelength range of 200-600 nm against a blank. The maximum absorbance was observed at 425 nm. This wavelength was chosen for further analysis. The findings are illustrated in Fig 2.

Preparation of Standard Calibration Curve

Extract solutions with concentrations ranging from 2 μg/ml to 10 μg/ml were prepared in methanol, and their absorbance was measured at the absorption maximum (λmax) of 425 nm using a UV spectrophotometer against methanol as the blank. A calibration curve was subsequently constructed by plotting concentration (x-axis) versus absorbance (y-axis). The results are shown in Fig 3.

The absorbance data obtained for the drug solution were plotted against concentration, and the calibration curve was found to be linear, obeying Beer-Lambert’s law in the range of 0-10 μg/mL. The regression equation was found to be y = 0.0728x + 0.0081 with a correlation coefficient (R²) of 0.9993, indicating excellent linearity.

Preparation of Ethosomes

Ethosomes were prepared by the cold method using ethanol and phospholipids. The influence of formulation composition on particle size, zeta potential, vesicle morphology, and entrapment efficiency was systematically evaluated. All prepared formulations were subjected to a series of characterization tests in order to identify the most suitable formulation for incorporated into an ethosomal gel.

Characterization of Ethosomes

Physical examination

The ethosomal formulations were visually inspected for their physical characteristics such as color, appearance, clarity, and phase separation. The results are shown in Table 6.

Table 6: Physical examination of ethosomal formulation

The physical evaluation of the formulations showed clear variations in their characteristics.  F1 appeared pale brown with slight turbidity and poor clarity, and mild separation was observed during storage. F2 was brownish-yellow to deep orange, smooth and uniform in texture, with a translucent appearance and no phase separation, making it visually the most acceptable formulation. F3 had a light yellowish-brown colour, grainy and less uniform appearance, with a hazy look and slight separation. F4 was dark brown, non-uniform, slightly viscous, and opaque, with a clear tendency toward phase separation.

Microscopic study

The prepared Ethosome samples, when observed under a light microscope at 100× magnification, revealed spherical and well-dispersed vesicles with uniform distribution and no aggregation, indicating successful Ethosome formation and good physical stability. The findings are depicted in Fig 4.

Fig 4: Microscopic observation of ethosomes (F1-F4)

Vesicle size determination

All formulations (F1-F4) formed nanosized vesicles suitable for dermal/transdermal delivery, with sizes ranging from 185 ± 1.6 nm to 234 ± 2.1 nm. F2 had the smallest vesicles (185 nm), favouring better skin penetration, while F3 had the largest (234 nm). F1 and F4 showed moderate sizes of 223 nm and 206 nm, indicating stable vesicle formation.  PDI values ranged from 0.20 ± 0.02 to 0.28 ± 0.03, showing good homogeneity. F2 had the lowest PDI (0.20), confirming uniformity, whereas F1 had a slightly higher PDI (0.28) but still within the acceptable monodisperse range (<0.3). The results are shown in Fig 5&6.

Surface morphology

SEM analysis of F1-F4 showed predominantly spherical to slightly oval vesicles with smooth surfaces and occasional wrinkling, indicating stable structures. F2 had the most uniform and well-dispersed vesicles, while F3 and F4 showed minor irregularities and slight aggregation. Overall, all formulations exhibited intact vesicles, minimal aggregation, and consistent morphology, supporting their stability and suitability for topical application. Fig 7 illustrates the obtained results

Zeta potential

All formulations (F1-F4) had negative zeta potentials, ranging from -15.5 ± 2.1 to -28.8 ± 1.7 mV, indicating negatively charged vesicles and electrostatic stability. F2 showed the highest negative value (-28.8 mV), suggesting the best stability, while F3 and F4 had moderate stability (-22.2 and -24.6 mV). F1 had the lowest zeta potential (-15.5 mV), indicating a higher risk of aggregation. The results are illustrated in Fig 8.

Particle size

The particle size of all formulations F1-F4 was within the nanometre range 211-256 nm, confirming successful formation of nanosized vesicles suitable for efficient skin delivery. F2 showed the smallest particle size of 211 ± 1.4 nm, which is desirable for enhanced penetration and uniform distribution. F3 exhibited the largest particle size, 256 ± 2.1 nm. F1 and F4 showed intermediate particle sizes, 240 nm and 225 nm, indicating stable vesicle formation. Among all batches, F2 showed the smallest particle size (211 ± 1.4 nm). The results are shown in Fig 9.

Entrapment efficiency (EE%)

Drug entrapment efficiency was determined by using UV spectrophotometer for all trial batches. The results are depicted in Fig 10.

The entrapment efficiency of all formulations F1-F4 ranged between 51.65 ± 0.51% and 73.65 ± 0.76%, indicating successful incorporation of drug within the vesicles. F2 exhibited the highest entrapment efficiency of 73.65%, which may be due to optimized phospholipid concentration and ethanol content, resulting in better drug loading. F1 showed the lowest entrapment efficiency of 51.65%, possibly due to inadequate vesicle formation or drug leakage. F3 and F4 demonstrated moderate entrapment of 64.65% and 70.54%, suggesting good drug incorporation.

pH determination

The pH of ethosomal formulation F1-F4 was found to range between 6.31 to 6.74. The results are shown in Fig 11.

The pH of all formulations F1-F4 was found to be within the range of 6.31 ± 0.04 to 6.74 ± 0.05, which lies within the acceptable range for topical formulations (5.5-7). F1 showed the highest pH of 6.74, whereas F2 exhibited the lowest pH of 6.31, but all formulations remained close to neutrality, indicating good stability of the preparation.

Determination of Drug content

The Drug content of ethosomes formulation (F1-F4) was analysed using UV spectrophotometry to determine the percentage of drug incorporated. The results are shown in Fig 12.

The drug content of all formulations F1-F4 ranged from 87.4 ± 2.8% to 96.4 ± 1.9%, indicating good incorporation of the drug within the vesicles. F2 showed the highest drug content of 96.4%, suggesting optimal formulation parameters and minimal drug loss during preparation. F3 exhibited the lowest drug content of 87.4%, which may be due to inefficient entrapment or slight drug leakage during processing. F1 and F4 showed drug content values of 94.2% and 95.0%, respectively, which are within the acceptable range. Among the batches, F2 showed the highest drug content (96.4 ± 1.9%).

Preparation of Ethosomal Gel of Optimized Formulation

The optimized ethosomal formulation (F2) containing Curcuma caesia Roxb was successfully incorporated into a gel base using Carbopol 934 as the gelling agent at various concentrations. Based on the procedure described in the methodology section, a total of three ethosomal gel formulations were successfully prepared.

Characterization of Ethosomal Gel

Physical parameters

The formulations were evaluated for their color, appearance, homogeneity and phase separation.  The physical evaluation of the ethosomal gel formulations (EG1-EG3) showed satisfactory results for all parameters. EG1 exhibited a pale-yellow colour with a smooth, semi-gel appearance, while EG2 was golden yellow with a creamy, uniform, and stable gel texture, and EG3 appeared light yellow with a thicker, slightly sticky consistency. All formulations demonstrated excellent homogeneity, indicating uniform distribution of components without any signs of grittiness or lumps. No phase separation was observed in any formulation, confirming their physical stability. These results suggest that the prepared ethosomal gels possess acceptable aesthetic and physical characteristics suitable for topical application. The results are illustrated in Table 7 and Fig 13.

pH determination

The pH of ethosomal gel formulation EG1-EG3 was found to range between 6.33 to 6.73. The results are shown in Fig 14.

The pH values of the ethosomal gel formulations EG1, EG2, and EG3 were found to be 6.44 ± 0.02, 6.33 ± 0.03, and 6.73 ± 0.01, respectively. These values fall within the acceptable range for topical formulations (5.5-7.0), ensuring compatibility with the skin’s natural pH and minimizing the risk of irritation upon application. EG2 exhibited the lowest pH, which is still suitable for maintaining skin health.

Viscosity determination

The viscosity of the ethosomal gel formulation EG1-EG3 was measured using a Brookfield RV viscometer. The results are shown in Fig 15.

Viscosity of EG1, EG2, and EG3 was 13,650 ± 150, 11,567 ± 153, and 10,083 ± 275 cP, respectively. EG1 showed the highest viscosity with good retention, while EG3 had the lowest and was slightly sticky. EG2 exhibited ideal viscosity, offering smooth spreadability, good consistency, and excellent retention, making it the most suitable formulation for topical application.

Spreadability

The results are illustrated in Fig 16.

Spreadability values of EG1, EG2, and EG3 were 14.2 ± 0.3, 10.8 ± 0.4, and 7.6 ± 0.2 g·cm/sec, respectively. Among all, EG2 exhibited the most desirable Spreadability, offering smooth application, uniform distribution, and adequate consistency for topical use. EG1 showed higher but slightly runny Spreadability, while EG3, with the lowest value, was thicker and less spreadable.

Determination of Drug content

The drug content of EG1-EG3 ethosomal gels was determined using UV spectrophotometry. Fig 17 summarizes the results.

The drug content of ethosomal gel formulations EG1, EG2, and EG3 was found to be 95.4 ± 1.2%, 98.1 ± 0.8%, and 96.6 ± 1.0%, respectively. All values lie within the pharmacopeial acceptance range of 95-105%, indicating uniform distribution of drug in the formulations. The highest drug content was observed in EG2 (98.1 ± 0.8%), suggesting better entrapment efficiency and minimal drug loss during preparation.

In Vitro Drug Release Study

In vitro drug release studies of ethosomal gels were carried out using a Franz diffusion cell apparatus using 7.4 pH phosphate buffer. Samples were withdrawn at predetermined time intervals of 0.5, 1, 2, 3, 4, 5, 6, 7 and 8 hours and analysed using a UV spectrophotometer at 425nm. The findings are depicted in Table 8 and Fig 18.

Table 8: Cumulative % drug release of C. caesia Ethosomal Gel (EG1-EG3)

The In vitro drug release study was conducted for all three ethosomal gel formulations EG1, EG2, and EG3 over a period of 8 hours. The results demonstrated a sustained and gradual release pattern for all formulations, indicating the ability of ethosomes to prolong drug release. Among the three, EG2 exhibited the highest cumulative drug release of 87.7 ± 0.72% at the end of 8 hours, which reflects its optimal vesicle size, entrapment efficiency, and appropriate gel consistency that favour enhanced drug diffusion. EG1 showed moderate release (82.4 ± 0.48%), while EG3 displayed the lowest release (78.8 ± 0.64%), which may be attributed to its higher viscosity, leading to slower drug diffusion. These results suggest that EG2 is the most efficient formulation for sustained and enhanced drug delivery.

In Vitro Drug Release Kinetics

In vitro drug release data of ethosomal gel formulations were subjected to various mathematical models to understand the release kinetics. The results of these analyses are summarized in Table 26. The selection of the most appropriate release model was based on the regression coefficient (R²), with values closer to 1 indicating a better fit. An R² value equal to 1 signifies a perfectly linear relationship, meaning the drug is released at a constant rate as time progresses. Among all tested models, formulations EG2 (R² = 0.9874) exhibited the highest R² values in the Zero-order kinetics, indicating that these formulations follow this release mechanism. This suggests that the drug release from the optimized formulation is governed by the Zero-order kinetics, which represents a constant drug release rate over time, independent of the remaining drug concentration. The subsequent graphs illustrate the plots used for determining drug release kinetics. The results are presented in Table 9 and Fig 19.