Formulation of Serum Sprays Containing Leaf Extract and Leaf Extract-Loaded Nanoparticles of Syzygium myrtifolium Walf: Antioxidant Activity and In Vitro Release Using Franz Diffusion Cells

Kosasih Kosasih et al. Formulation of Serum Sprays Containing Leaf Extract and Leaf Extract-Loaded Nanoparticles of Syzygium myrtifolium Walf: Antioxidant Activity and In Vitro Release Using Franz Diffusion Cells. Int. J Med. Pharm. Res., 6 (5): 1717‐1732, 2025 1717 International Journal of Medical and Pharmaceutical Research Online ISSN-2958-3683 | Print ISSN-2958-3675 Frequency: Bi-Monthly Available online on: https://ijmpr.in/ Original Article Formulation of Serum Sprays Containing Leaf Extract and Leaf ExtractLoaded Nanoparticles of Syzygium myrtifolium Walf: Antioxidant Activity and In Vitro Release Using Franz Diffusion Cells Kosasih Kosasih1, Alifah Wahdah Zahroh1, Lilik Sulastri2 1 Faculty of Pharmacy, Universitsas Pancasila, Jakarta 12640, Indonesia 2 Department of Pharmacy, Faculty of Mathematic and Natural Science, Pakuan University, Bogor 16143, Indonesia A B S T R A C T Corresponding Author: Kosasih Kosasih Faculty of Pharmacy, Universitsas Pancasila, Jakarta 12640, Indonesia. Received: 02-08-2025 Accepted: 24-09-2025 Available online: 20-09-2025 The skin serves as the primary body barrier against external threats, with facial skin being particularly exposed and clinically significant. Skincare products— such as moisturizers, cleansers, and serums—are designed to protect, nourish, and rejuvenate the skin. This study aimed to extract Syzygium myrtifolium (SM) leaves, synthesize them into nanoparticles, and formulate antioxidant serum sprays. SM leaves were macerated in 70% ethanol (pH 2) and concentrated using a rotary evaporator. The resulting extract was characterized and converted into nanoparticles via a precipitation method. Nanoparticle characterization included particle size (252.7 nm), polydispersity index (0.459), zeta potential (−27.2 mV), and entrapment efficiency (47.91%). Both the extract and nanoparticles were formulated into serum sprays and evaluated for physicochemical properties, antioxidant activity, and in vitro release using a Franz diffusion cell. The serum sprays appeared as a clear to pale yellow liquid with a characteristic odor, uniform consistency, appropriate viscosity and spreadability, rapid drying time (<3 minutes), and an average pH of 7.20. Antioxidant activity, expressed as IC₅₀, was 15.96 ppm (extract), 32.59 ppm (nanoparticles), 32.29 ppm (extract-based serum, EbSS), and 59.98 ppm (nanoparticle-based serum, NbSS). Franz cell analysis showed flux values of 1.72 μg/cm²/min (EbSS) and 1.79 μg/cm²/min (NbSS), with corresponding permeability coefficients of 2.78 cm/min and 2.90 cm/min, and diffusion coefficients of 3.33 cm²/min and 3.48 cm²/min, respectively. Overall, the SM extract was successfully formulated into nanoparticle and serum spray forms, with NbSS exhibiting superior diffusion performance. Copyright © International Journal of Medical and Pharmaceutical Research Keywords: Syzygium myrtifolium, extract, nanoparticles, serum sprays, Franz diffusion cell. INTRODUCTION The skin serves as the body’s primary defense against external threats through mechanisms such as keratinization, temperature regulation, sweat and sebum secretion, melanin production, and immune responses [1]. Among all regions, facial skin is especially important, often reflecting overall health and being the initial site for many dermatological conditions [2,3]. Skincare products—including moisturizers, cleansers, and serums—are designed to protect, nourish, and rejuvenate the skin [4]. Serums are concentrated wateror oil-based formulations targeting specific skin concerns, often used alongside complementary products [5]. Recently, serums have evolved into spray formats, offering hygienic, hands-free application and enhanced absorption [6]. Nanoparticles, typically <1000 nm, possess unique physicochemical properties that enhance the delivery and stability of active ingredients in cosmetics and pharmaceuticals [7]. One major contributor to skin damage is oxidative stress caused Kosasih Kosasih et al. Formulation of Serum Sprays Containing Leaf Extract and Leaf Extract-Loaded Nanoparticles of Syzygium myrtifolium Walf: Antioxidant Activity and In Vitro Release Using Franz Diffusion Cells. Int. J Med. Pharm. Res., 6 (5): 1717‐1732, 2025 1718 by free radicals—unstable molecules that accelerate aging and cellular degradation. Antioxidants neutralize these radicals, either naturally produced or sourced from flavonoid-rich plants [8]. Syzygium myrtifolium (SM), locally known as pucuk merah, contains flavonoids and anthocyanins with potent antioxidant properties [9,10]. SM thrives in diverse environments and is widely cultivated for landscaping and ecological greening due to its vibrant foliage and environmental benefits [11,12]. These include CO₂ absorption, oxygen production, erosion control, and soil stabilization via robust taproots. SM also supports biodiversity by attracting pollinators and frugivores, aiding seed dispersal and habitat formation. Its compact growth makes it ideal for hedging and pruning [11,13–15]. Antioxidant activity is commonly assessed using the DPPH assay, which measures the IC₅₀ value—the concentration needed to neutralize 50% of DPPH radicals. Lower IC₅₀ values indicate stronger antioxidant potential. SM leaf extract prepared with 70% ethanol has demonstrated high efficacy, with an IC₅₀ of 11.130 ppm [16]. Spray serum evaluation encompasses formulation, physicochemical properties, in vitro performance, user acceptability, and other parameters [17,18]. Key aspects include: 1. Formulation and Physicochemical Properties The formulation was evaluated for stability by monitoring changes in color, texture, and pH under various storage conditions [19]. pH compatibility was assessed to ensure alignment with the skin’s natural pH (~5.5), minimizing the risk of irritation [20]. Spreadability was examined to determine ease of application and absorption [18], while particle size analysis focused on ensuring uniformity and the absence of clumping [18]. 2. In Vitro Performance The formulation was assessed for antioxidant activity using the DPPH assay to determine its radical-neutralizing capacity [17]. Drug release was evaluated to measure how effectively the active ingredients are delivered to the skin [18], while skin irritation testing was conducted using animal models to ensure safety and minimize adverse reactions [18,21]. 3. User Acceptability The evaluation criteria include hydration, which measures moisturizing effects [22]; glow/radiance, assessing the impact on skin luminosity [22,23]; and ease of use, which considers the spray mechanism and overall user experience [22]. Additionally, texture is examined to determine absorption and residue [22], while multi-functionality reviews the product’s performance as a mist, primer, or setting spray [22]. Finally, overall effectiveness gathers user feedback on visible skin improvement [22,23]. 4. Other Parameters The evaluation also includes ingredients, which are screened for potential allergens and the concentration of active components [24,25], as well as packaging, which is assessed based on its design, user convenience, and visual aesthetics [17,25]. The Franz diffusion cell method is a gold-standard in vitro technique for assessing drug permeation through skin-like membranes. It involves placing a membrane between donor and receptor chambers, with the receptor chamber containing a buffered solution. The formulation is applied to the donor chamber, and samples are withdrawn over time to measure permeation via UV–Vis or HPLC. Key metrics include flux, cumulative permeation, and lag time [26-28]. This study aimed to extract SM leaves, synthesize gelatin nanoparticles, and formulate antioxidant spray serums containing both extracts and nanoparticles. These formulations were then evaluated, including diffusion analysis using Franz cells MATERIAL AND METHODS 2.1 Material Research materials included SM leaves, obtained from the gardens at Universitas Pancasila, Jakarta. The chemicals used in this study were of pro-analytical grade, including 70% ethanol, methanol, dimethyl sulfoxide (DMSO), 96% ethanol, poloxamer 188, gelatin, 2% glutaraldehyde, hydroxyethyl cellulose, phenoxyethanol, propylene glycol, and purified water. The following equipment used included: UV–Vis spectrophotometer (UV-1900, Shimadzu, Kyoto, Japan); rotary vacuum evaporator (Heidolph, Schwabach, Germany); analytical balance (Kern, Balingen, Germany); digital homogenizer (RW 20, IKA, Staufen, Germany); microbalance (MT5, Mettler Toledo, Columbus, OH, USA); micropipette (Dragon Lab, Beijing, China); rotational viscometer (RV, Brookfield, Middleboro, MA, USA); pH meter (Hanna Instruments, Woonsocket, RI, USA); and standard laboratory glassware (Pyrex, Iwaki, Japan). Plant Preparations Kosasih Kosasih et al. Formulation of Serum Sprays Containing Leaf Extract and Leaf Extract-Loaded Nanoparticles of Syzygium myrtifolium Walf: Antioxidant Activity and In Vitro Release Using Franz Diffusion Cells. Int. J Med. Pharm. Res., 6 (5): 1717‐1732, 2025 1719 Leaves of SM were from the garden area of Universitas Pancasila (Jakarta, Indonesia). The leaves were thoroughly washed with running water, cleaned to remove debris, air-dried at room temperature, and then ground into a fine powder using a blender. [29] Determining The Degree of Fineness of Simplicia The fineness of the simplicia powder was determined using a #4 sieve and a #18 sieve. A 100 g sample was placed on each sieve and shaken to facilitate separation. The material retained on the sieve and the portion that passed through to the receiving pan were each collected and weighed to evaluate particle size distribution. The same procedure was used with the #18 sieve to determine the particle size distribution [30]. 2.2 Preparation and Evaluation of SM Dry Extract The powdered simplicia of SM were macerated with 70% ethanol in a 1:10 (w/v) ratio for 48 hours at room temperature. After maceration, the macerate was filtered using filter paper, and then concentrated under reduced pressure using a rotary vacuum evaporator (Heidolph, Schwabach, Germany) [31]. Organoleptic Evaluation The organoleptic properties of the thick extract, including color, odor, and taste, were assessed using sensory observation methods [32]. Extract Yield and Drug Extract Ratio (DER-Native) The determination of extract yield was by comparing the weight of the dried extract to the original weight of the simplicia material. Furthermore, the drug-extract ratio or DER-native was determined to quantify the concentration of active constituents present in the extract [33,34]. pH Measurement The pH of the extract was measured using a calibrated pH meter (Hanna Instruments, Woonsocket, RI, USA), calibrated with pH 7.0 and pH 4.0 buffer solutions. The pH meter was always clean before use. The preparation of the sample solution involves dissolving 100 mg of the extract in 100 mL of purified water. After immersing the electrode in the sample solution and obtaining a stable reading, the pH value was recorded [35]. Antioxidant Activity of the Extract Using the DPPH Assay A stock solution of 1000 ppm was prepared and diluted to obtain concentrations of 4, 8, 12, 16, and 20 ppm, in series. To each test tube of 1 mL of a 0.4 mM DPPH solution, add methanol to adjust the final volume to 5.0 mL. The mixtures were incubated in the dark at room temperature for 30 minutes. Absorbance was measured at the maximum wavelength using a UV–Vis spectrophotometer (Shimadzu UV-1900, Kyoto, Japan) [36,37]. 2.3 Preparation and Evaluation of SM Leaf Extract-Loaded Nanoparticles Solvent Phase A quantity of gelatin was dissolved in 1 mL of purified water maintained at 50°C. Separately, SM leaf extract was dissolved in a mixture of 0.2 mL dimethyl sulfoxide (DMSO) and 0.2 mL of 96% ethanol, and stirred until completely dissolved. [16,38] Non-Solvent Phase A total of 700 mg of poloxamer 188 was dissolved in 10 mL of 96% ethanol using a magnetic stirrer until a clear solution was obtained [16,38]. Nanoparticle Synthesis The solvent phase was added dropwise into the non-solvent phase under continuous stirring at 700 rpm. After 15 minutes of stirring, 0.5 mL of 2% glutaraldehyde is dropped into the solution and stirred for the next 17 hours. After that, the nanoparticle suspension was cleaned by centrifuging and purified using purified water twice for 30 minutes. After the centrifuging process, the sediment at the bottom of the tube is collected and freeze-dried [16,38]. Particle Size and Polydispersity Index (PDI) A 1 mL aliquot of the nanoparticle suspension was transferred into a 10 mL volumetric flask and diluted to volume with purified water. The particle size and polydispersity index were measured using a particle size analyzer (Malvern Instruments, Worcestershire, UK). Measurements were performed in triplicate [16,38]. Zeta Potential A 1 mL sample of the nanoparticle suspension was diluted to 10 mL with purified water in a volumetric flask. Zeta potential was measured using a Zetasizer (Malvern Instruments, Worcestershire, UK), and the analysis was conducted in triplicate [16,38]. Kosasih Kosasih et al. Formulation of Serum Sprays Containing Leaf Extract and Leaf Extract-Loaded Nanoparticles of Syzygium myrtifolium Walf: Antioxidant Activity and In Vitro Release Using Franz Diffusion Cells. Int. J Med. Pharm. Res., 6 (5): 1717‐1732, 2025 1720 Entrapment Efficiency (EE) Gelatin nanoparticles are synthesized using the desolvation method with concurrent drug incorporation. Following formulation, the suspension is centrifuged to separate the nanoparticles from the supernatant containing unentrapped (free) drug. The concentration of free drug in the supernatant is quantified using UV-Vis spectrophotometry. Entrapment efficiency (EE) is then calculated using the following equation: EE (%) = (Total drug − Free drug) × 100 Total drug Antioxidant Activity Ten milligrams of the nanoparticles were dissolved in 10 mL of pro-analysis grade methanol to obtain a 1000 ppm stock solution. Aliquots of 50, 100, 150, 200, and 250 µL were transferred into test tubes and diluted with methanol to a final volume of 5.0 mL, yielding concentrations of 10, 20, 30, 40, and 50 ppm, respectively. To each test tube of 1 mL of a 0.4 mM DPPH solution, add methanol to adjust the final volume to 5.0 mL. The mixtures were homogenized, covered with aluminum foil, and incubated at 37 °C for 30 minutes before absorbance was measured [36,37]. 2.4 Formulation and Evaluation of Serum Spray The required ingredients, including SM leaf extract loaded-nanoparticles, hydroxyethyl cellulose, phenoxyethanol, and propylene glycol, were weighed according to the formulation. Hydroxyethyl cellulose was dispersed in 10 mL of purified water and allowed to swell as a gel base. The nanoparticle extract was dissolved in 30 mL of propylene glycol and gradually added to the gel base under continuous stirring using a homogenizer (IKA RW 20, Staufen, Germany). Phenoxyethanol was dissolved in purified water, followed by the addition of purified water to a final volume of 100 mL, and then stirred to homogeneity [16]. Antioxidant Activity of Serum Spray Using the DPPH Assay Ten milligrams of the serum spray were dissolved in 10 mL of pro-analysis grade methanol to obtain a 1000 ppm stock solution. Aliquots of 325, 400, 475, 550, and 625 µL were transferred into test tubes and diluted with methanol to a final volume of 5.0 mL, resulting in concentrations of 65, 80, 95, 110, and 125 ppm, respectively. Each test tube received 1 mL of a 0.4 mM DPPH solution. After that, add methanol to bring the total volume to 5.0 mL. The mixtures were homogenized, covered with aluminum foil, and incubated at 37 °C for 30 minutes before absorbance was measured [16]. Organoleptic Properties The organoleptic properties of the serum spray formulations—such as its color, odor, and physical appearance—were evaluated manually through sensory observation [16] Homogeneity Serum homogeneity was evaluated by spraying it onto a glass slide, ensuring even distribution, and visually inspecting the spread for uniformity [16]. Viscosity Viscosity was determined using a Brookfield rotational viscometer (Model RV; Brookfield Engineering, Middleboro, MA, USA). This instrument operates by rotating a spindle within the fluid and recording the torque necessary to maintain a constant rotational speed. The measured torque, along with the spindle specifications and rotation rate, is used to compute the dynamic viscosity of the fluid, typically reported in centipoise (cP) [16]. Spray Pattern The spray pattern test of the serum was to determine the spray's shape and uniformity during device activation. This analysis helps ensure the drug is delivered effectively to the intended target area and can be used to identify potential issues with the device or formulation. The spray is achieved by dispensing the serum onto a flat surface from varying distances, ranging from 3 to 20 cm. The diameter of the resulting spray and distribution was to determine the uniformity and effectiveness of the spray mechanism [16]. Dry Time The dry time refers to the period required for a spray serum to dry after application to the inner forearm [16]. pH Measurement The pH of the serum spray was measured using a calibrated pH meter. Calibration was at pH 7.0 and pH 4.0. After each measurement, the electrode was rinsed thoroughly with purified water to maintain accuracy and prevent contamination. This essential step removes any residual buffer, preventing contamination and ensuring an accurate reading for further Kosasih Kosasih et al. Formulation of Serum Sprays Containing Leaf Extract and Leaf Extract-Loaded Nanoparticles of Syzygium myrtifolium Walf: Antioxidant Activity and In Vitro Release Using Franz Diffusion Cells. Int. J Med. Pharm. Res., 6 (5): 1717‐1732, 2025 1721 measurement. A 100 mL of serum sample in a glass beaker, the pH value was recorded only after a stable reading, confirming a precise and reliable result. [16] Stability Testing The stability tests of serum spray were determined using a cycling test method. The serum spray was stored at 4 °C for 24 hours, followed by storage at 40 °C for an additional 24 hours. Each cycling test was performed six times for 12 days. The formulations were evaluated for changes in appearance, homogeneity, and pH after each cycle [19,39]. The Franz Diffusion Cell Method [40,41,42] This method is a gold-standard in vitro technique for evaluating drug penetration through skin or membrane models. It is widely used in pharmaceutical, cosmetic, and dermatological research to simulate transdermal or topical delivery. The Franz cell penetration method consists of: a. Setup: The Franz cell consists of two chambers; b. Donor chamber: Contains the drug formulation; c. Receptor chamber: Filled with a buffer solution (e.g., PBS) that mimics physiological conditions; d. A membrane (synthetic or biological, such as rat skin or Strat-M®) separates the two chambers. Procedure: a. The membrane is mounted between the chambers; b. The receptor chamber is stirred and maintained at a controlled temperature (typically 32–37 °C); c. The drug formulation is applied to the donor chamber; d. Samples are withdrawn from the receptor chamber at set intervals to measure drug permeation using UV–Vis spectrophotometry or HPLC. Key Parameters: a. Flux (J): Rate of drug permeation per unit area; b. Cumulative amount permeated: Total drug that has crossed the membrane; c. Lag time: Time before steady-state permeation begins. 3. RESULTS AND DISCUSSION Table 1. Determination of the degree of fineness Simplicia Passed sieve #4 Requirement Passed sieve #18 Requirement SM leaves 100% 100% 35.62% ≤ 40% Table 2. Yield and DER-Native of the extract Simplicia (g) Extract weight (g) Yield (%) DER-Native 1001.70 407.71 40.70 2.46 Table 3. pH of the extract Replication 1 Replication 2 Replication 3 Average + SD 3.49 3.52 3.55 3.52 + 0.03 Table 4. Antioxidant activity (IC50) of SM leaf extract Concentration (ppm) Inhibition (%) IC50 (ppm) 1 2 3 Ave.+SD 4 16.12 16.18 17.14 16.41 15.89 15.57 15.96 ± 0.42 8 24.08 27.36 28.41 12 39.64 40.92 41.97 16 50.31 51.93 52.78 20 58.59 59.53 60.31 Table 5. Particle size (nm, left)) and polydispersity index (right) of the nanoparticles Particle size (nm) Polydispersity index 1 2 3 Ave.+SD 1 2 3 Ave.+SD 222.9 239.9 295.3 252.7+37.9 0.380 0.439 0.558 0.459 ± 0.091 Table 6. Zeta potential of the nanoparticles Replication 1 Replication 2 Replication 3 Average + SD (mV) −24.8 −25.6 −31.3 −27.2 ± 3.5 Table 7. Entrapment efficiency of the nanoparticles Replication 1 Replication 2 Replication 3 Average + SD (mV) 47.91 48.07 47.76 47.91 ± 0.16 Kosasih Kosasih et al. Formulation of Serum Sprays Containing Leaf Extract and Leaf Extract-Loaded Nanoparticles of Syzygium myrtifolium Walf: Antioxidant Activity and In Vitro Release Using Franz Diffusion Cells. Int. J Med. Pharm. Res., 6 (5): 1717‐1732, 2025 1722 Table 8. Antioxidant activity (IC50) of SM leaf extract loaded-nanoparticles Concentration (ppm) Inhibition (%) IC50 (ppm) 1 2 3 Ave.+SD 10 22.34 22.23 22.68 32.79 32.54 32.43 32.59 ± 0,18 20 33.79 33.93 33.49 30 47.08 47.48 47.64 40 59.58 60.07 60.38 50 70.24 70.65 70.84 Table 9. Antioxidant activity (IC50) of serum spray formulations Concentration (ppm) Inhibition (%) IC50 (ppm) 1 2 3 Ave.+SD EbSS (Serum spray containing SM leaf extract) 65 60.02 59.48 59.45 33.53 31.47 31.88 32.29 ± 1.09 80 62.35 62.67 62.69 95 64.34 64.59 64.83 110 68.26 69.16 69.44 125 76.77 75.32 75.47 NbSS (Serum spray containing SM nanoparticles) 65 51.83 51,78 52,07 60.03 60.26 59.66 59.98 ± 0.30 80 57.07 57,03 57,10 95 62.42 62,42 62,73 110 66.76 66,73 66,92 125 73.32 73,44 73,70 Table 10. Organoleptic of serum spray formulations Parameters Blank NbSS EbSS Form Liquid Liquid Liquid Color Colorless Colorless Pale yellow Odor Less odor Less odor Distinctive odor n = 3 Table 11. Homogeneity of the serum spray formulations Formulations Homogeneity (n=3) Blank Homogeneous NbSS Homogeneous EbSS Homogeneous Table 12. Viscosity of the serum spray formulations Formulation Replicate 1 (cP) Replicate 2 (cP) Replicate 3 (cP) Average ± SD (cP) Blank 24.0 19.2 19.2 20.80 ± 2.77 NbSS 19.2 24.0 14.4 19.20 ± 4.80 EbSS 14.4 24.0 28.8 22.40 ± 7.33 Table 13. Spray pattern of the serum spray formulations Parameter Formulation Spraying distance (cm) 3 5 10 15 20 Spray pattern Blank NbSS EbSS Clumped Clumped Clumped Slight clumped Slight clumped Slight clumped Spread Spread Spread Spread Spread Spread Spread Spread Spread Diameter (cm) Blank NbSS EbSS 3.0 3.3 2.5 4.0 4.8 3.7 6.4 7.2 7.7 7.8 8.1 7.7 12.5 13.0 12.6 Spray weight (g) Blank NbSS EbSS 0.12 0.12 0.13 0.14 0.13 0.16 0.16 0.15 0.17 0.15 0.15 0.16 0.17 0.16 0.17 Kosasih Kosasih et al. Formulation of Serum Sprays Containing Leaf Extract and Leaf Extract-Loaded Nanoparticles of Syzygium myrtifolium Walf: Antioxidant Activity and In Vitro Release Using Franz Diffusion Cells. Int. J Med. Pharm. Res., 6 (5): 1717‐1732, 2025 1723 Table 14. Results of Dry Time Test Formulations Dry time 1 (second) Dry time 2 (second) Dry time 3 (second) Average + SD Blank 154 153 155 154 + 1.0 NbSS 149 148 148 148.3 + 0.6 EbSS 163 167 168 166 + 2.6 Table 15. Organoleptic test result before and after stability testing Formulation Condition Form Color Odor Blank Before Liquid Colorless Less odor After Liquid Colorless Less odor NbSS Before Liquid Colorless Less odor After Liquid Colorless Less odor EbSS Before Liquid Pale yellow Distinctive odor After Liquid Pale yellow Distinctive odor n = 3 Table 16. Formulation pHs before and after stability testing Formulation Condition pH Blank Before 7.39 + 0.03 After 7.00 + 0.02 NbSS Before 7.13 + 0.03 After 6.91 + 0.03 EbSS Before 7.09 + 0.08 After 6.91 + 0.07 Table 17. Viscosity test results before and after stability testing Formulation Viscosity (cP) before Viscosity (cP) after Blank n = 3 24.0, 19.2, 19.2 14.4, 14.4, 14.4 Average ± SD 20.8 ± 2.77 14.4 ± 0 NbSS n = 3 19.2, 24.0, 14.4 14.4, 19.2, 9.6 Average ± SD 19.2 ± 4.8 14.4 ± 4.8 EbSS n = 3 19.2, 24.0, 14.4 14.4, 14.4, 19.2 Average ± SD 19.2 ± 4.8 16.0 ± 2.77 Table 18. Comparison of flux, permeability constant, and diffusion coeffient of NbSS and EbSS Parameter EbSS NbSS Flux (J) 1.7171 µg/cm²·min 1.7931 µg/cm²·min Permeability constant (Kp) 2.776 × 10⁻⁵ cm/min 2.899 × 10⁻⁵ cm/min Diffusion coefficient (D) 3.331 × 10⁻⁵ cm²/min 3.479 × 10⁻⁵ cm²/min Kosasih Kosasih et al. Formulation of Serum Sprays Containing Leaf Extract and Leaf Extract-Loaded Nanoparticles of Syzygium myrtifolium Walf: Antioxidant Activity and In Vitro Release Using Franz Diffusion Cells. Int. J Med. Pharm. Res., 6 (5): 1717‐1732, 2025 1724 3. DISCUSSION 3.1 Plant Preparation Determining the Degree of Fineness of Simplicia Determining the degree of fineness of simplicia powder is a critical step in ensuring optimal extraction efficiency and formulation performance. Particle size directly influences the surface area available for solvent interaction, which affects the diffusion rate of analytes during extraction. If the powder is too fine, it may clog filters and slow down the filtration process, leading to operational inefficiencies. Conversely, overly coarse powder reduces the contact surface area, resulting in suboptimal diffusion and lower yield of active constituents. According to standard herbal pharmacognosy practices, a fineness degree of 4/18 is acceptable for many plant-based materials. This specification requires that 100% of the powder passes through sieve no. 4 (4.75 mm), ensuring uniformity, while no more than 40% passes through sieve no. 18 (1.0 mm), preventing excessive fineness that could hinder processing. In this study, the powder from SM leaves met the required specifications, with 100% passing sieve no. 4 and 35.62% passing sieve no. 18, indicating a suitable particle size distribution for further extraction and formulation (Table 1). [30,31] 3.2 Preparation and Evaluation of SM Dry Extract Kosasih Kosasih et al. Formulation of Serum Sprays Containing Leaf Extract and Leaf Extract-Loaded Nanoparticles of Syzygium myrtifolium Walf: Antioxidant Activity and In Vitro Release Using Franz Diffusion Cells. Int. J Med. Pharm. Res., 6 (5): 1717‐1732, 2025 1725 The extraction of SM leaf simplicia was by the maceration method with 70% ethanol. This solvent concentration was selected for its optimal polarity, enabling the selective extraction of bioactive compounds such as flavonoids, tannins, and phenolics, while minimizing the co-extraction of non-target constituents. Ethanol at 70% has been shown to yield extracts with superior purity and enhanced biological activity due to its balanced polarity and selective solubility for key phytochemicals. [30,43-45] Maceration was chosen for its simplicity, cost-effectiveness, and suitability for thermolabile compounds, as it avoids thermal degradation by operating at ambient temperatures. This method is widely applied in phytochemical research to preserve the structural integrity of sensitive secondary metabolites, including flavonoids and phenolics. [29,45] After 48 hours of soaking, the mixture is filtered through filter paper to separate the liquid extract from the plant residue. The resulting filtrate was dried using a rotary vacuum evaporator at 40 °C and 200 rpm, allowing for gentle solvent removal without compromising the stability of heat-sensitive compounds. [44] Organoleptic Examination Organoleptic examination of the thick extract of SM leaves was conducted by evaluating its visual appearance (color), olfactory characteristics (odor), and physical consistency (texture). The extract had a dark red hue, a distinctive tea-like aroma, and a viscous, semi-solid consistency, indicative of the presence of polyphenolic compounds such as flavonoids and tannins, which are known to impart such sensory attributes. [45,46] Extract Yield and Drug Extract Ratio (DER-Native) The extraction process yielded 407.71 g of thick extract from 1001.7 g of dried SM leaf simplicia, resulting in a yield of 40.70%. The calculated Drug Extract Ratio-Native (DER-Native) was 2.46, indicating that 2.46 grams of raw material are required to produce 1 gram of extract (Table 2). Yield is a critical parameter in phytopharmaceutical development, reflecting the efficiency of solvent penetration and compound solubilization during an extraction. A higher yield generally suggests a greater recovery of bioactive constituents, assuming minimal co-extraction of inert materials. [29] The DERNative value is essential for standardizing extract production, ensuring reproducibility, and facilitating dosage calculations in both research and formulation development. It serves as a quantitative benchmark for comparing extraction efficiency across different solvents, methods, and plant matrices. [29,47] pH of the Extract The pH of the thick extract of SM was measured across three replications, yielding an average value of 3.52 ± 0.03, which reflects an acidic profile (Table 3). This acidity is related to the deliberate addition of 1% hydrochloric acid (HCl) during the maceration process. Acidification of the extraction medium is a well-established strategy to enhance the solubility and release of phenolic and alkaloid compounds, many of which exhibit enhanced extractability under low pH conditions due to increased protonation and disruption of plant cell walls. [16,29] The acidic environment also helps stabilize certain polyphenols, preventing oxidative degradation during extraction. [48] Antioxidant Test of Extract with DPPH Assay The antioxidant activity of the SM leaf extract was determined using the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging assay, a widely accepted method for assessing free radical inhibition potential. The extract demonstrated an IC₅₀ value of 15.96 ppm, indicating very potent antioxidant activity, as per established classification criteria where IC₅₀ values below 50 ppm are considered highly potent (Table 4). This strong antioxidant capacity is related to the presence of phenolic compounds, flavonoids, and tannins, which are known to donate hydrogen atoms or electrons to neutralize free radicals. Previous studies have confirmed that SM red leaf extracts contain high levels of these phytochemicals, contributing to their radical-scavenging efficacy. [16,45] 3.3 Evaluation of SM Leaf Extract-Loaded Nanoparticles Nanoparticle Size and Polydispersity Index The physical characterization of SM leaf extract loaded-nanoparticles revealed an average particle size of 252.7 nm and a polydispersity index (PDI) of 0.459 (Table 5). These results indicate that the nanoparticles fall well within the nanometric range (<1000 nm), suitable for enhanced bioavailability and cellular uptake. The PDI value, which reflects the uniformity of particle size distribution, is below the critical threshold of 0.5, suggesting a moderately narrow and acceptable size distribution for pharmaceutical and cosmetic applications. Nanoparticles with PDI values between 0.1 and 0.5 generally have good homogeneity, which is essential for consistent performance, stability, and reproducibility in formulation systems. The particle size and distribution are affected by factors such as polymer type, extract concentration, and crosslinking conditions during the synthesis process. [7,10,49] Zeta Potential The zeta potential of SM leaf extract loaded-nanoparticles was measured across three replications, yielding an average value of −27.2 ± 3.5 mV (Table 6). Zeta potential reflects the surface charge of particles in suspension and is a key indicator of colloidal stability. A value of this magnitude suggests that the nanoparticles exhibit moderate electrostatic repulsion, which helps prevent aggregation and maintains uniform dispersion in the formulation. Zeta potential values greater than ±30 mV are typically associated with high colloidal stability, while values between ±20 and ±30 mV are moderately stable Kosasih Kosasih et al. Formulation of Serum Sprays Containing Leaf Extract and Leaf Extract-Loaded Nanoparticles of Syzygium myrtifolium Walf: Antioxidant Activity and In Vitro Release Using Franz Diffusion Cells. Int. J Med. Pharm. Res., 6 (5): 1717‐1732, 2025 1732 46. Kusriania, R.H., Rosandhya, S.M., and Elfahmi, 2019, Luteolin, a flavonoid from Syzygium myrtifolium Walp, CRBB, 1(1), 31-33. https://doi.org/10.5614/crbb.2019.1.1/FKAN 4064 47. Syafriana, V., et al., 2025, Antibacterial activities of n-hexane and ethyl acetate extracts from young leaves of red lip (Syzygium myrtifolium (Roxb.) Walp.), Res J Pharm Technol, 18(4), 1845-3. https://doi.org/ 10.52711/0974360X.2025.00264 48. Nofita, D., Rahman, D.F., penetapan kadar senyawa fenolik ekstrak etanol 70% daun pucuk merah (syzygium myrtifolium walp.) dengan metode folin-ciocalteu. J Pharma Saintika. 2025; 8(2): 19–26. https://doi.org/10.51225/jps.v8i2.64 49. Hidayati, E.N., Rahayu, A.M., and Azzahra, F., 2023, Physical characterization of chitosan-based Syzygium polyanthum leaves extract nanoparticles, Pharmacon, 20(2), 120-128. Available from: http://journals.ums. ac.id/index.php/pharmacon 50. Biology Insights, 2025, Available from: Zeta potential nanoparticles: Methods, factors, and stability - biology insights [April 29, 2025] 51. Khan, S., Ansari, M.T., and Haider, M.F., 2023, Gelatin nanoparticles: A promising approach for drug delivery, JDDST, 78, 104097. https://doi.org/ 10.1016/ j.jddst.2022.104097 52. Novi, F.U., Rizky, M.S., Chorry, S.I., and Shintia, R., 2025, Analysis of solvent concentration effect and extraction method on the total phenolic of Syzygium myrtifolium Walp leaf extract, Pharmacogn J, 17(4), 461-469. https://doi.org/10.5530/pj.2025.17.59 53. Kosasih, K., Susanti, D. Asianski, I.P.H., and Maryanto, D.I., 2024, Formulation and characterization of antioxidant capsules containing cantigi extract-loaded nanoparticles synthesized via nanoprecipitation method, JNPDD, 1(2), 7985. https://doi.org/ 10.58511/ jnpdd.v1i2.6352 54. Situmorang, C., Butar-Butar, M.E.T., and Sianturi, S., 2025, Effect of sunflower oil and olive oil concentration on the sunscreen activity of Syzygium myrtifolium Walp leaves extract cream, Pharmasipha. 9(2), 34-49. https://doi.org/10.21111/pharmasipha.v9i2.13666 55. Lestari, U., Muhaimin, M., Maimum, M., and Yuliana, Y., 2025, Formulation of soothing gel ethanol extract of red shoot leaves (Syzigium myrtifolium Walp) as an antioxidant, RIK, 14(1), 138-146. https://doi.org/10.30644/rik.v14i1.956 56. Lodén, M., 2005, The clinical benefit of moisturizers, J Eur Acad Dermatol Venereol, 19(6), 672-88. https://doi.org/10.1111/j.1468-3083.2005.01326.x. PMID: 16268870. 57. Mozarafi, M.R., Ed., 2006, Nanocarrier technologies: Frontiers of nanotherapy, Dordrecht: Springer. 58. Alzahra, D.F., Lestari, A.D., Haryadi, E., Malau, N.A., Risanli, V.A., and Apriani, E.F., 2024, Skin penetration of corn silk (Zea mays L.) transdermal patch on Wistar mice skin using Franz diffusion cell, JFIKI, 2024, 11(1), 20–33. https://doi.org/10.20473/jfiki.v11i12024.20-33