Preparation, Characterization and Ex vivo Evaluation of Trigonellafoenum-gracium seeds extract-loaded Transferosomal gel for the Treatment of Rheumatoid Arthritis

Vandana¹, Hema Arya¹, Koushal Dhamija², Preeti Singh¹*, Gautam Kumar^1,3*

¹Department of Pharmacy, Sharda University, Knowledge Park III, Greater Noida, Uttar Pradesh-201310, India.

²Department of Pharmaceutics, Lloyd Institute of Management and Technology,

Knowledge Park II, Greater Noida, Uttar Pradesh - 201306, India.

³Department of Pharmacy, ApeejayStya University, Sohna - Palwal Road,

Sohna, Gurugram, Haryana -122103, India.

*Corresponding Author E-mail: preetisingh01144@gmail.com, drgautam9265@gmail.com

ABSTRACT:

Rheumatoid arthritis (RA) is a systemic autoimmune disorder characterized by persistent joint inflammation, leading to severe disability and increased mortality rates. Fenugreek, known for its anti-arthritic effects, demonstrates promise in retarding RA onset and progression. However, challenges like poor bioavailability and limited target specificity hinder its therapeutic utility. To circumvent these limitations, transdermal delivery systems, notably transferosomes, offer a viable solution. This study aimed to formulate a transferosomal gel incorporating Trigonella foenum-graecum (TFG) extract, employing the thin-film hydration technique. Evaluation parameters encompassed stability, zeta potential, particle size, entrapment efficiency, and deformability. The synthesized transferosomes were integrated into Carbopol gel to enhance ease of application and prolong skin retention. Comparative analyses with conventional gel assessed parameters such as consistency, transparency, viscosity, and pH. Experimental findings revealed successful entrapment of TFG extract (63.69±3.20% w/w) within transferosomes, exhibiting favorable particle size (269.20±1.20nm) and deformability (23.71±0.92). Skin penetration kinetics demonstrated a gradual increase in TFG extract concentration over time, with TFG-extract-loaded transferosomal gel exhibiting the highest concentration among formulations at all-time points. The stability studies indicated the prolonged stability of the TFG-transferosomes in gel formulations. In conclusion, the developed TFG extract-loaded transferosomal gel presents a promising strategy for improved transdermal delivery, offering potential therapeutic benefits over conventional formulations in RA management.

KEYWORDS: Rheumatoid arthritis, Transferosomal gel, Transdermal delivery carriers, Methi seeds extract, Improved permeability.

INTRODUCTION:

The modern human race faces many health issues, including diabetes, heart disease, arthritis, pulmonary obstruction, and stroke.The main causes of these conditions include poor food, inactivity, and the production of free radicals, among other things^1,2. On the other hand, most ailments are easily curable with an improved lifestyle³. Incorporating antioxidants into the diet can help inhibit the formation of free radicals and reactive oxygen species (ROS), which have the potential to bind to various cells and tissues, causing damage that may result in serious health conditions.⁴Rheumatoid arthritis (RA) is a multifaceted autoimmune disorder that impacts approximately 0.5–1% of the global adult population⁵. This condition occurs when the immune system mistakenly targets the joints, leading to pain, swelling, and chronic damage that can significantly diminish a person's quality of life⁶. RA affects females majorly compared to males in a ratio of 4:1⁷. Genetic predisposition is important, with particular communities and families holding a higher risk⁸. Lifestyle factors such as smoking and obesity can raise the risk of getting RA, but breastfeeding and moderate alcohol intake may provide some protection⁹. Furthermore, environmental factors such as exposure to specific bacteria or viruses, as well as the impact of stress and mental health, are being explored as potential contributors to the disease¹⁰. According to research findings, plants are a rich source of naturally occurring antioxidants, and people have been using plant materials as medicine for a variety of diseasesfor years^11–13. Herbal constituents like flavonoids, alkaloids, phenolic compounds, and tannins have shown effectiveness in the treatment of rheumatoid arthritis^14–17.

The seeds of Fenugreek (Trigonella foenum-graecum Linn. Family-Fabaceae)are also known as Methi or Methika, amost used spice in India. Moreover, it also possessesseveral medicinal properties such as anti-inflammatory, anti-arthritic, and antioxidant activities. It has been used in the treatment of many diseases including colic, flatulence, dysentery, diarrhea, diabetes, and lipid disorders for years^12,18–21.

Orally administered drug formulations often result in ineffective drug delivery to the targeted site. This lack of selectivity results in low drug concentration in the desired area, necessitating frequent dosing. Such frequent dosing may increase the risk of developing undesirable effects²². Dermal drug delivery has various advantages, including easy application, high patient adherence, minimal systemic side effects, and suitable for long-term disease management. However, the stratum corneum, the skin's outer layer, is a considerable barrier to drug penetration²³.

Several strategies have been developed to improve drug delivery through the skin. These include physical procedures such as iontophoresis, electroporation, and microneedling, as well as chemical ones that employ absorption enhancers. Furthermore, liposomal-based delivery systems, such as transferosomes, have sparked considerable interest in improving drug absorption and distribution within the skin, as well as potential bloodstream penetration²⁴. Transferosomes playa major role in better and sustained pharmacological effects^25,26. Researchers have been more interested in transferosomes during the last few decades. They are composed of an ultra-deformable lipid layer complex around a hydrated core^24,27,28.

Research studies indicated that the transdermal administration of natural antioxidants containing preparation is one of the best alternatives to prevent or reduce free radical formation in the body as compared to other modes. However, strong skin penetration and superior absorption characteristics are necessary for topical medicines used in treatments^29,30.

MATERIAL AND METHODS:

Materials:

Ethanol and Tween-80 were procured from LobaChemie Private Limited, Mumbai, India. Soy phospholipid (Phospholipon® 90 G), Carbopol 934 powder, Propylene glycol, and Triethanolamine were sourced from Merck Life Sciences, India.

Methods:

a. Collection, authentication, processing and preparation of TFG extract:

TFG seeds were obtained from Baghpat, Uttar Pradesh, India, and their authenticity was verified by the Council of Scientific and Industrial Research - National Institute of Science Communication and Policy Research (CSIR-NIScPR), New Delhi, with the authentication number NIScPR/RHMD/Consult/2021/3956-57. TFGseeds were dried at room temperature for ten days. The dried TFG seeds were then grounded into coarse powder using a mechanical grinder. In the Soxhletapparatus, the TFG seeds powder was kept on the extraction chamber and 75% ethanol was filled into the round bottom flask. The Soxhlet apparatus was then kept on a heating mantle and the extraction process was performed at 40-45°C for 3 to 4 days until the complete extraction was done^31,32. After the completion of extraction procedure,the extract was filtered, and the filtrate was evaporated with the help of vacuum-fitted rotary evaporator, the concentrated TFG extract was then completely dried in a vacuum desiccator.Finally, the dried extract was subjected to physicochemical characterization.

b. Pre-formulation studies:

Phytochemical and physicochemical characterization of TFG seed extract:

The phytoconstituent analysis was performed for the presence of flavonoids, alkaloids, Glycosides, polyphenols, tannins, and carbohydrates in the TFG extract^33,34.

Ultraviolet (UV)-spectrophotometric analysis:

The UV spectra of the TGFextract solution was recorded using a UV-visiblespectrophotometer (Shimadzu, 1800-UV) and the lambda max was recorded.

Preparation of calibration curve:

TFG extract sample (1mg/mL) was prepared using phosphate buffer pH 6 and kept at 4±2°C for further analysis. Various dilutions were made to achieve 25, 50, 75, 100 and 125µg/mL concentration from 1mg/mL solution in phosphate buffer pH 6. A calibration curve of TGFseed extract solutions in the range of 25-125 µg/mL concentration wasgenerated at 273nm.

Differential scanning colorimetry (DSC):

DSC analysis was performed to ensure the formation of complex between TFG extract and Soya lecithin, to confirm the integrity and presence of TFG extract in the transferosomes using DSC analyzer (Perkin Elmer).

Fourier-transform infrared spectroscopy (FTIR):

The FTIR analysis of TGF seed extract, soy lecithin, and physical mixture of soy lecithin and TFG extract wereperformed by FTIR (Agilent, cary 630 FTIR-ATR) using the KBr dispersion method for the analysis of drug (extract) compatibility withsoya lecithin.

c. Preparation and Optimization of TFG seed extract-loaded transferosomes:

Thin-layer hydration technique was used for producing TFG extract-loaded transferosomes^35,36. The composition of transferosomes for batches TFG1 - TFG9 is given in Table 1. To summarise, these preparations involved dissolving TFG extract in chloroform, adding phospholipon 90G (phospholipids), and tween 80 (edge activator) to a round-bottom flask followed by shaking to dissolve completely. After that, the organic solvent (chloroform) was evaporated, and a thin film was created using a rotary evaporator operating at a lowered pressure and 60 rpm, above the lipid transition temperature of 55°C. Following the solvent's full evaporation, the lipid films were preserved for the night in a vacuum desiccator. After that, phosphate buffer (pH 6) was used to rehydrate the dried lipid films until the whole thin layer turned into vesicles. The rehydration procedure was run for 30 minutes without a vacuum, at 37±2°C and 100 revolutions per minute of rotation.

d. Characterization of TFG extract-loaded transferosomes:

Particle size and surface charge (zeta potential):

A Zeta Potential Analyzer and Particle Sizer from Malvern Instruments Ltd. (ZSU3100) was used to measure the vesicle size and potential of the transferosomes.

Entrapment efficacy:

The amount of TFG seed extract entrapped within the vesicles was determined by ultracentrifugation method. In brief, 1.5ml of the synthesizedtransferosomal formulations were kept for centrifugation at 14000rpm for 30 mins and the supernatants were collected, diluted, and assessed for drug content using UV-Visible spectrophotometer. The following formula (Eq1) was used for calculating the entrapment efficiency (EE).

%EE

Where Wt is the amount of TFGextract added, and Ws is the amount of TFG detected in the supernatant.

Deformability Index (Vesicle Elasticity Measurement):

Using a custom-built apparatus, the deformability investigation for the transferosomal formulation was carried out. The extrusion method was utilized to assess the flexibility of transferosome vesicles. Applying 2.5 bar of pressure, the prepared transferosomes were extruded through a membrane filter with a pore size width of 100nm, utilizing a 50mm diameter stainless steel filter holder. It was measured how many suspension vesicles were extruded in five minutes ^37,38. The deformability index was recorded using the following equation (Eq2)-

E = J * (rv/rp)²……………………………………..Eq2

Where,

E = Elasticity of vesicles membrane,

J = Amount of suspension extruded in 5 minutes,

rv = Vesicles size, and

rp = pore diameter.

Transmission electron microscopy analysis for morphological evaluation of transferosomes in gel formulations:

Transferosome vesicles were observed using a transmission electron microscope (Hitachi, H-7500, Tokyo, Japan) at a voltage of 100 kV. The samples were negatively dyed using 1% of phosphotungustic acid (PTA) in an aqueous solution.

e. Preparation and evaluation of TFG-transferosomes-loaded gel:

A precisely measured amount of Carbopol 934 (1g for each formulation set) was placed in three separate 200 ml beakers, followed by the addition of 70ml deionized water, and allowed to hydrate for 4 hours. After this period, 20g of Polyethylene glycol 400 and TFG-extract-transferosomes (equivalent to 2.5g extract) were incorporated into the gel mixture and stirred continuously for 4 hours to form a homogeneous gel. Triethylamine was used to adjust the pH of the final gel formulations, which were then assessed based on several parameters^22,39,40.

Homogeneity:

Three distinct Carbopol-940 gel formulations were created and evaluated based on their outward appearance using visual inspection.

pH:

To determine the pH of the formulations, three sets of the developed gel were diluted with water, and the pH was recorded using a digital pH meter. The pH of each formulation was measured three times for accuracy.

Spreadability:

A 500mg sample of the gel formulation was applied to a glass slide, while another slide, holding 5.8±1g of gel, was dropped from a height of 5cm. After one minute, the spread area of the gel was observed, and the diameter of the resulting circle was measured.

Viscosity:

The Brookfield viscometer was employed to determine the viscosity of the transferosomal gel loaded with TFG extract.

Drug Content:

Accurately weighed 1gm of TFG-transferosome-loaded gel formulations (from three sets of gel) were taken and treated with 20ml ethanol to lyse the vesicles usingbath sonicator for 20mins. Later, the sonicated gel solution was centrifugedat 14000rpm for 30minutes at 4±2°C. The supernatants were collected and diluted with 50% ethanol and the drug content of each sample was examinedusing UV-Visible spectrophotometer at 273 nm.

Ex vivo skin penetration study:

A Franz cell diffusion apparatus was employed to evaluate the ex vivo penetration of the enhanced TFG extract-loaded transferosome into freshly obtained hairless goat abdominal skin. The goat skin was placed between the donor and receptor compartments, with the apparatus having a cell volume of 25mL and an effective diffusion area of 2.26cm². The receptor compartment contained 15mL of release medium consisting of phosphate buffer (pH 6) and ethanol (8:2), maintained at 37±0.5°C. The medium was continuously stirred with a magnetic stirrer at 100rpm using a magnetic bead. To assess the ex vivo penetration of the improved TFG extract-loaded transferosome into the skin of freshly hairless goat abdomens, a Franz diffusion cell apparatus was utilized. The setup included goat skin with an effective diffusion area of 2.26cm² and a receptor compartment filled with 25mL of diffusion media, used to measure the permeation rate and kinetics. In summary, the receptor compartment contained 15mL of release medium, composed of phosphate buffer (pH 6) and ethanol (8:2), maintained at 37±0.5°C. The medium was continuously stirred with a magnetic stirrer and bead operating at 100rpm. Skin samples were extracted from the cells, homogenized at 5000rpm in PBS (pH 6), and the amount of extract retained per unit area (µg/cm²) in the skin was calculated. Additionally, graphs for mathematical modeling were plotted^41,42.

Stability studies:

The physicochemical stability of the optimized formulation (TFG-transferosome-loaded gel) was evaluated over three months at temperatures of 4°C±2°C and 25°C±2°C, in accordance with International Council for Harmonisation (ICH) guidelines^42,43.

3. RESULTS AND DISCUSSION:

Pre-formulation studies:

a. Phytochemical analysis of Trigonella Foenum Graecum seed extract:

The hydroethanolic extract of TFG seeds appeared golden yellow. Phytoconstituent analysis identified the presence of flavonoids, alkaloids, glycosides, polyphenols, tannins, and carbohydrates, as detailed in Table 2. The presence of these compounds suggests that the TFG extract possesses anti-inflammatory and antioxidant properties, which may aid in alleviating symptoms of rheumatoid arthritis.

Table 2: Phytochemical analysis of TFG extract.

S. No.	Test	Result
1	Flavonoids	Positive
2	Alkaloid	Positive
3	Glycoside	Negative
4	Saponin	Negative
5	Tannin	Positive
6	Carbohydrate	Positive
7	Polyphenol	Positive

b. Ultraviolet (UV)-spectrometric analysis:

The TFG extract was analyzed and UV-Visible spectra was recorded using UV-Vis spectrophotometer (Shimadzu) at 273nm and UV-Vis spectra is shown in Figure 1.

Figure 1: UV-Vis spectra of TFG extract at 273 nm.

The calibration curve for the TFG extract (see Figure 2) demonstrated linearity with an R² value of 0.9954 across the concentration range of 25-125µg/mL. This indicates that the method is reliable for analyzing and quantifying TFG extract, supporting its use in the formulation and development of TFG extract-loaded transferosomes and gel formulations.

Figure 2: Calibration curve of the TFG extract.

c. Fourier-transform infrared spectroscopy (FT-IR) of TFG seed extract,Soy lecithin and their physical mixture:

FT-IR spectroscopy was employed to investigate the physicochemical interactions between soy lecithin and TFG extract. This technique uses radiation frequencies to identify functional groups and their absorbance values, revealing the primary chemical groups in both the extract and soy lecithin, as well as any new interactions formed during the transferosome manufacturing process (see Table 3). The FT-IR spectra of soy lecithin, TFG extract, and their physical mixture are illustrated in Figure 3. Soy lecithin exhibited characteristic peaks for C-H stretching vibrations at 2921 cm⁻¹ and 2851 cm⁻¹, C=O stretching at 1734 cm⁻¹, C-N stretching at 1056 cm⁻¹, and a peak at 698 cm⁻¹ indicative of a benzene derivative. TFG extract showed peaks for NH stretching at 3383 cm⁻¹, C-H stretching at 2983 cm⁻¹, C=O stretching at 1748 cm⁻¹, C-H bending at 1380 cm⁻¹, C-N stretching at 1033 cm⁻¹, and a peak at 695 cm⁻¹ suggesting a benzene derivative. The physical mixture of TFG extract and soy lecithin displayed peaks for C-H stretching at 2925 cm⁻¹ and 2854 cm⁻¹, C=O stretching at 1737 cm⁻¹, C-H bending at 1380 cm⁻¹, C-N stretching at 1048 cm⁻¹, and a peak at 717 cm⁻¹ indicating a benzene derivative. The observation of similar characteristic peaks in the physical mixture suggests that no significant physicochemical interaction occurs between soy lecithin and TFG extract.

d. Differential scanning colorimetric (DSC) analysis of TFG seed extract, Soy lecithin and their physical mixture:

DSC thermograms of the pure TFG extract, Soy lecithin, and physical mixture of TFG extract and Soy lecithin are shown in Figure 4. DSC thermogram of soy lecithin showed an endothermal peak at 284.481°C, with an area of 11.410mJ, and Delta H value of -5.705 J/g. DSC thermogram of Pure TFG extract showed an endothermal peak at 222.194°C, with an area of 100.393 mJ, and Delta H value of -50.197 J/g. DSC thermogram of physical mixture of TFG extract and soy lecithin showed an endothermal peak at 203.169°C, with an area of 33.253 mJ, and Delta H value of -16.627 J/g. This analysis indicated that there was not much variation between the endothermic peaks and the difference was within ±20°C. This slight variation in the endothermic peaks may be due to the physicochemical interaction by Hydrogen bonding between-OH group of TFG extract and polar part of soy lecithin.

Table 3: Interpretation of FT-IR results.

Frequency Range ⁴⁴	Absorption (cm^-1)			Appearance	Group	Compound Class
Frequency Range ⁴⁴	Soy Lecithin	TFG extract	Physical mixture of Soy lecithin and TFG extract	Appearance	Group	Compound Class
3400-3300	-	3383	-	medium	N-H stretching	aliphatic primary amine
3000-2840	2921; 2851	2983	2925; 2854	medium	C-H stretching	alkane
1750-1735	1734	1748	1737	strong	C=O stretching	Esters/δ-lactone
1690-1640	-	-	1652	medium	C=N stretching	imine / oxime
1390-1380	-	1380	1380	medium	C-H bending	Aldehyde/ alkane
1075-1020	1056	1033	1048	medium	C-N stretching	amine
700 ± 20	698	695	717	-	-	benzene derivative

Figure 3: FT-IR spectra of soy lecithin, TFG extract, and their physical mixture.

Figure 4: DSC thermograms of the pure TFG extract, Soy lecithin, and their physical mixture.

Preparation and characterization of transferosomes:

The TFG-loaded transferosomes were prepared and analysed for particle size, zeta potential, entrapment efficiency, and deformability index. The particle size of the transferosomes was within the range of 269.20±1.20nm to 342.30±2.52nm. The prepared transferosome showed the zeta potential value in the range of -28±2.2 to -40±1.6mV. From the estimations of the percentage entrapment (%EE), the transferosome formulations i.e., TFG1 to TFG9 showed 30.35±3.51% to 63.69±3.20% drug (extract) entrapment. The vesicle elasticity (deformability index) of the transferosomes, ranging from TFG1 to TFG9, varied between 9.42±1.95 and 27.79±1.48. Among these, formulation TFG4 exhibited a particle size of 269.20±1.20nm and a zeta potential of -28±2.2mV, suggesting it is a promising delivery carrier. The entrapment efficiency of TFG4 was approximately 63%, which is favorable for skin permeation and effective drug delivery through topical applications. Detailed characteristics of the transferosomes are provided in Table 4.

Table 4: Preparation and optimization of transferosome.

Formulation code

Particle size(nm)

Zeta potential (mV)

Entrapment efficiency (%)

Deformability index

TFG1

305.73±

4.04

-39±1.6

30.35±

3.51

9.42±

1.95

TFG2

297.34±

2.52

-39±0.5

40.56±

2.96

10.58±

0.95

TFG3

283.70±

4.04

-40±1.6

48.89±

1.20

15.28±

0.83

TFG4

269.20±

1.20

-28±2.2

63.69±

3.20

23.71±

0.92

TFG5

276.72±

7.64

-35±1.2

63.53±

1.28

26.22±

0.34

TFG6

332.33±

2.52

-35±1.4

56.81±

1.67

25.40±

0.89

TFG7

284.73±

3.51

-39±1.6

47.79±

1.24

26.66±

1.23

TFG8

292.71±

2.52

-39±2.2

39.22±

0.72

27.79±

1.48

TFG9

342.30±

2.52

-38±2.4

32.69±

2.67

26.22±

1.79

Values were taken as Mean±SD, n=3.

Morphological evaluation of optimized transferosomes:

Transmission electron microscopic (TEM) analysis of TFG extract-loaded transferosomes (Figure 5) confirmed that the prepared transferosome vesicles were spherical in shape.

Figure 5: Transmission electron microscopic (TEM) analysis of TFG extract-loaded transferosomes.

Preparation and evaluation of TFG-transferosomes-loaded gel:

The TFG-transferosomes-loaded gel was prepared and evaluated for various parameters as given below in Table 5.

Table 5: Characteristics of prepared TFG-transferosomes-loaded gel.

Gel characteristics tested	Initial values (Mean±SD, n=3)
Homogeneity	Good (+++)
pH	6.00±0.00
Spreadability (cm/5min)	4.57±0.21
Viscosity (Pa.s)	642.95±40.92
Drug content (mg/g Gel)	19.96±0.07

All the prepared TFG-transferosomes-loaded gels were appeared to be yellowish translucent colour. The preparedTFG-transferosomes-loaded gels exhibited a pleasant and homogeneous appearance. We did not observe any gritty particles and phase separation in all three sets of gels. Images of prepared gels are shown below (Figure 6).

Figure 6: Images of prepared gels.

Ex vivo skin permeation study of TFG-transferosome-loaded gel:

Using an excised goat's abdomen skin as a permeation membrane, Franz diffusion cells were used to perform ex vivo skin permeation of gels containing transferosomes loaded with TFG extract.The concentration of TFG-extract when administered alone shows a gradual increase over time. At 24 hours, the concentration reaches 897.49±128.97 µg/mL, indicating a slow and steady release of the extract.The TFG-extract-loaded gel exhibits a higher concentration of the extract compared to the TFG-extract alone throughout the entire duration of the study. This suggests that the gel matrix serves as a carrier for the TFG-extract, facilitating its sustained release over time. At 24 hours, the concentration reaches 1056.22±75.95 µg/mL, indicating a more pronounced release compared to the TFG-extract alone.The TFG-extract transferosomal gel demonstrates the highest concentration of the extract among the three formulations at all time points. This indicates that the transferosomal gel formulation enhances the release and bioavailability of the TFG-extract compared to both the TFG-extract alone and the TFG-extract-loaded gel. At 24 hours, the concentration reaches 1750.66±88.50 µg/mL, signifying a significantly enhanced release profile compared to the other formulations. Figure 7and Table 6 shows the permeation profile of the formulations.

Table 7: Release kinetics of the optimized formulated gels.

Formulation	The amount retained in the skin after 24h (µg/cm²)	Correlation coefficient (R²)					Diffusion exponent (n)
Formulation	The amount retained in the skin after 24h (µg/cm²)	Zero-order	First-order	Higuchi model	Korsmeyer-Peppas	Hixson-Crowell model	Diffusion exponent (n)
TFG-Transferosomal Gel	1750.66±88.50	0.8868	0.992	0.9689	0.9423	0.9722	0.72

Figure 8: Ex vivo drug release kinetic and mathematical modelling of optimized Transferosomal gel.

Table 8: Stability evaluation parameters for formulated gels.

Time	Temperature	Homogeneity	pH	Spreadability (cm/5min)	Viscosity (Pa.s)	Drug content (mg/g Gel)
Initial values (Mean±SD, n=3)	4±2°C	Good (+++)	6.00±0.00	4.57±0.21	1111.42±26.60	20.01±0.04
Initial values (Mean±SD, n=3)	25±2°C	Good (+++)	6.00±0.00	4.57±0.21	1109±23.68	19.97±0.07
After 3 months values (Mean±SD, n=3)	4±2°C	Good (+++)	6.00±0.00	4.34±0.12	1124.67±23.18	20.01±0.01
After 3 months values (Mean±SD, n=3)	25±2°C	Good (+++)	6.00±0.00	4.57±0.15	1092.80±22.22	19.97±0.03

Stability studies of TFG-transferosomes-loaded gel:

The physical stability of the optimized gel formulations was evaluated, with the results summarized in Table 8. The stability data indicated that the gel formulations remained stable over a 3-month period at both 4±2°C and room temperature (25±2°C).

4. CONCLUSION:

The results confirm that the TFG-extract-loaded transferosomal gel formulations were successfully prepared and optimized, showing improved permeability through excised goat skin, consistent with the ex-vivo study. These formulations enable the TFG extract to be retained in various skin layers following transdermal application. The stability of the transferosomes was enhanced when incorporated into the gel. The hydrogel, made with carbopol 934 and containing a TFG extract concentration of 2 mg/mL, demonstrated sustained action over 24 hours, making it a key dosage form for managing rheumatoid arthritis with effective permeability.

5. CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

6. ACKNOWLEDGEMENT:

The authors are thankful to CSIR-NIScPR, New Delhi for helping in the authentication of TFG seeds. The authors are also thankful to Sharda University, Greater Noida for providing infrastructure and all other facilities to carry out this project.

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Received on 19.03.2024 Revised on 11.07.2024

Accepted on 20.09.2024 Published on 27.03.2025

Available online from March 27, 2025

Research J. Pharmacy and Technology. 2025;18(3):1118-1127.

DOI: 10.52711/0974-360X.2025.00161

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

Material name	Formulation code
Material name	TFG1	TFG2	TFG3	TFG4	TFG5	TFG6	TFG7	TFG8	TFG9
TFG extract (%w/w)	5	5	5	5	5	5	5	5	5
Tween 80 (%w/v)	0.5	1	1.5	2	2.5	3	3.5	4	4.5
Soya phospholipid (%w/w)	5.5	5	4.5	4	3.5	3	2.5	2	1.5
Phosphate buffer saline (pH 6) for hydration	10mL	10mL	10mL	10mL	10mL	10mL	10mL	10mL	10mL

Time (h)	Cumulative permeation (%), n=3, Mean±SD
Time (h)	TFG-extract (µg/mL)	TFG-extract-loaded Gel (µg/mL)	TFG-extract transferosomal Gel (µg/mL)
0	0.00±0.00	0.00±0.00	0.00±0.00
1	77.38±18.18	78.7±12.12	257.28±29.78
2	158.07±22.56	193.78±21.86	373.68±41.30
4	242.72±23.92	263.89±43.65	539.02±36.01
6	345.90±91.3	353.84±64.15	732.14±75.71
8	458.33±26.02	548.28±45.65	1049.60±71.43
10	598.54±74.91	602.51±64.64	1234.79±118.87
12	679.23±25.51	843.25±111.96	1375.00±67.81
24	897.49±128.97	1056.22±75.95	1750.66±88.50