The Moringa oleifera stem bark samples were obtained from the Sumampir area, North Purwokerto, Banyumas Regency, Central Java. The samples were thoroughly washed, cut into small pieces, and dried using a drying cabinet at a temperature of 70°C. Once dried, the samples were ground into a fine and uniform powder, then stored in a sealed container (Cholifah et al., 2020).

The extraction was carried out using the maceration method. A total of 100 grams of Moringa oleifera stem bark powder was macerated in 500 mL of methanol for three days, with stirring every 24 hours at room temperature and protected from direct sunlight (Cholifah et al., 2020). The maceration filtrate was then filtered and concentrated using a rotary evaporator at 60°C, followed by evaporation in a water bath at 70°C until a thick extract was obtained. The extract yield was calculated using the following formula ((Dewatisari et al., 2018); (Wahyuni et al., 2024)).

A total of 1 gram of the extract was mixed with concentrated hydrochloric acid (HCl) in a test tube and heated for 15 minutes in a water bath. The appearance of a red or yellow coloration indicated a positive result for the presence of flavonoid compounds such as flavones, chalcones, and aurones (Muthmainnah, 2017).

A total of 2 grams of the extract was mixed with 5 mL of 2N HCl, heated, then cooled, and divided into three test tubes containing 1 mL each. Different reagents were added to each tube: a positive result for alkaloids was indicated by the formation of a white or yellow precipitate with Mayer’s reagent, a brown precipitate with Wagner’s reagent, and an orange precipitate with Dragendorff’s reagent (Muthmainnah, 2017).

A total of 2 grams of the extract was mixed with 2 mL of ethyl acetate in a test tube and shaken. The ethyl acetate layer was separated, dropped onto a spot plate, and allowed to dry. Subsequently, two drops of acetic anhydride and one drop of concentrated sulfuric acid were added. A positive result for terpenoids was indicated by the appearance of a red or yellow color, while a green coloration indicated the presence of steroids (Muthmainnah, 2017).

A total of 1 gram of the extract was mixed with 10 mL of hot water in a test tube, then cooled and vigorously shaken for 10 seconds. The formation of a stable froth measuring 1–10 cm in height that persisted for more than 10 minutes and did not disappear after the addition of one drop of 2N HCl indicated a positive result for saponins (Muthmainnah, 2017).

A total of 1 gram of the extract was mixed with 10 mL of hot water in a test tube and boiled for 5 minutes. The resulting filtrate was then added with 3–4 drops of FeCl₃ solution. The appearance of a bluish-green or dark green coloration indicated the presence of catechol-type tannins, while a dark blue coloration suggested the presence of pyrogallol-type tannins (Muthmainnah, 2017).

The transdermal patch formulations were prepared at three different extract concentrations: 4%, 8%, and 12% (Kalsum et al., 2023).

HPMC was dissolved in hot distilled water and allowed to stand for 15 minutes until fully swollen. PVP K-25 was dissolved separately in hot distilled water, and the two solutions were then combined and stirred until homogeneous. The Moringa oleifera stem bark extract was dissolved in 95% ethanol, followed by the addition of propylene glycol and DMSO. Both mixtures were combined, thoroughly mixed, and the total volume was adjusted to 30 mL. The mixture was poured into silicone molds with a diameter of 2.5 cm, dried at 40°C for 10 hours, and stored in a desiccator for 20 hours (Kalsum et al., 2023).

This test was conducted visually to assess the shape, color, odor, and texture of the patch formulation (Gunarti et al., 2024).

Three patches were randomly selected from each formulation and weighed. The patches were considered uniform if the coefficient of variation (CV) was less than 5% (Wardani & Saryanti, 2021).

The thickness of the patch was measured using a screw micrometer at three different points. An acceptable patch thickness should not exceed 1 mm (Balaji et al., 2012).

The patch was repeatedly folded at the same position until it broke. A satisfactory folding endurance is indicated by more than 200 folds (Jadhav et al., 2009).

The patch was immersed in 2 mL of distilled water for two hours at room temperature, after which the pH was measured using a pH meter. A safe pH range for topical formulations is between 4.5 and 6.5 (Mariadi & Bernardi, 2023).

This test was conducted to evaluate the physical stability of the transdermal patch containing Moringa oleifera stem bark methanolic extract by storing the formulation at 50°C for 12 hours, followed by 35°C for 12 hours, repeated for 6 cycles. Observations indicated no physical changes in the shape, odor, color, thickness, or weight uniformity of the patch (Nurmesa et al., 2019).

A total of 20 panelists evaluated the color, aroma, and skin sensation of the patch using a 5-point scale (1 = dislike, 5 = like very much)(Dira & Dewi, 2022).

The antibacterial activity was evaluated using the well diffusion method. The well diffusion method was selected due to its suitability for evaluating antibacterial activity of extract-based and semi-solid formulations, allowing diffusion of active compounds into the agar medium. A total of 25 mL of Nutrient Agar (NA) was mixed with 25 μL of Staphylococcus aureus suspension, homogenized, and poured into sterile Petri dishes to solidify. The bacterial suspension was standardized to 0.5 McFarland turbidity prior to use. Wells with a diameter of approximately 6 mm were created to accommodate the samples: positive control (Oxy patch), negative control (patch without active ingredient), and transdermal patches containing methanolic extract of Moringa oleifera stem bark at concentrations of 4%, 8%, and 12% (Cholifah et al., 2020). Each sample was tested in triplicate (n=3) in three independent experiments. The plates were incubated for 24 hours at 37°C (Formulasi Sediaan Salep Dan Uji Aktivitas Antibakteri Ekstrak Etanol Daun Bidara Arab (Ziziphus spina-christi (L.) Desf) Terhadap Bakteri Staphylococcus Epidermidis, 2022). The diameter of the inhibition zones was measured in milimeters using a caliper and expressed as mean ± standard deviation (Aprilia et al., 2017).

Based on the results of the organoleptic evaluation, all transdermal patch formulations containing methanolic extract of Moringa oleifera stem bark showed no observable changes in odor, shape, or color during six stability testing cycles (Figure 2). Therefore, the formulations were considered organoleptically stable throughout the stability testing process.

Based on Table 4, the pH of the formulations decreased with increasing extract concentration, ranging from F0 (±6.3) to F3 (±4.6); however, all formulations remained within the safe range of 4.5–6.5 (Mariadi & Bernardi, 2023). This decrease was attributed to the acidic nature of the extract and chemical reactions such as hydrolysis and the formation of acidic compounds during storage ((Nitiariksa & Iskandar, 2021); (Sugiharta & Ningsih, 2021); (Yamamoto et al., 2015)). Components such as HPMC, PVP, propylene glycol, and DMSO contributed to maintaining pH stability (Kalsum et al., 2023). Results from the Repeated Measures ANOVA indicated that F0–F2 remained stable (p > 0.05), whereas F3 (p = 0.007) showed a significant change due to the higher concentration of acidic extract (Banerjee et al., 2014). The folding endurance values increased from F0 (361 ± 1) to F3 (668 ± 3.78), all exceeding the standard threshold of >200 folds, indicating good elasticity and mechanical strength of the patches (Jadhav et al., 2009). This increase corresponds to the higher extract concentration, which enhances the film structure and flexibility ((Latif et al., 2022); (Singh & Bali, 2016)). Formula F3 demonstrated the highest folding endurance, consistent with (Wardani & Saryanti, 2021) who stated that patches with folding endurance ≥200 exhibit strong and elastic matrices. The Repeated Measures ANOVA results showed p-values for F0–F3 greater than 0.05, indicating that all formulations were stable in terms of folding endurance.

Based on Table 4, the six-cycle evaluation showed an increase in the mean values with increasing extract concentration, ranging from 0.21 in F0 to 0.38 in F3. All formulations exhibited coefficient of variation (CV) values between 0.03% and 0.3%, which were well below the acceptable limit of ≤5%, indicating good weight uniformity ((Novia & Noval, 2021); (Wardani & Saryanti, 2021)).

The low CV values demonstrate stable and homogeneous weight distribution among the patches, reflecting a consistent formulation process (Pratasik et al., 2019). Results of the Repeated Measures ANOVA test showed p-values > 0.05 for all formulations (F0–F3), confirming that all transdermal patch formulations were stable in terms of weight uniformity.

All formulations exhibited a thickness of less than 1 mm, meeting the criteria for an ideal transdermal patch (Balaji et al., 2012). The mean thickness increased with extract concentration, ranging from 0.31 mm in F0 to 0.58 mm in F3, and remained stable up to the sixth cycle. The uniform thickness indicates a homogeneous formulation mixture (Pratasik et al., 2019).

The increase in thickness is influenced by film density and the higher concentration of HPMC polymer ((Fakruddin et al., 2019); (Kalsum et al., 2023)). Stable thickness also contributes to comfort during skin application (Wardani & Saryanti, 2021). he Repeated Measures ANOVA results showed p-values greater than 0.05 for all formulations, indicating no significant difference; thus, the patches were considered stable in terms of thickness.

This study has several limitations. The evaluation was primarily limited to the physicochemical properties, hedonic characteristics, and in vitro antibacterial activity of the transdermal patch formulations containing methanolic extract of Moringa oleifera stem bark. In addition, stability assessment was restricted to accelerated testing, while long-term stability under real-time storage conditions was not evaluated.

The antibacterial activity was assessed against only one bacterial strain, which may not fully represent the broader antimicrobial potential of the formulation. Furthermore, drug release kinetics and skin permeation profiles were not investigated, limiting the understanding of active compound delivery through the skin. Therefore, future studies should include long-term stability testing, broader-spectrum antimicrobial evaluations, and in vitro or ex vivo permeation studies to provide a more comprehensive assessment of the efficacy and therapeutic potential of the developed transdermal patch.

The analysis using the Friedman test revealed significant differences in color, aroma, and skin sensation (Asymp. Sig < 0.05). Formula F1 obtained the highest mean ranks for color (3.25) and skin sensation (3.63), indicating an attractive greenish-brown appearance with a smooth and elastic texture ((Supit et al., 2015); (Qamariah et al., 2022)).

Formula F0 showed the highest score for aroma due to its odorless characteristic ((Lamusu, 2018); (Khalisa et al., 2021)). Overall, Formula F1 was the most preferred by the panelists and has the greatest potential to be developed as the optimal transdermal patch formulation containing the methanolic extract of Moringa oleifera Lam. stem bark.

Based on the overall results, formula F3 containing 12% extract exhibited the best physical properties, high stability, favorable acceptability, and the strongest antibacterial activity. These findings indicate that the methanolic extract of Moringa oleifera Lam. stem bark has promising potential to be developed as an active ingredient in transdermal patch formulations for the treatment of skin infections caused by Staphylococcus aureus ((Bukar et al., 2010); (Tilarso et al., 2021)).