1. INTRODUCTION
Malaria remains a major health challenge in tropical and subtropical regions, with 229 million cases reported globally in 2019, of which 94% occurred in Africa [1]. In Indonesia, malaria cases are predominantly concentrated in Papua, West Papua, and East Nusa Tenggara, which together accounted for 95% of the total cases in 2020 [2]. By 2023, approximately 75% of districts and cities in Indonesia were declared malaria-free; nevertheless, Papua continues to record the highest transmission rates, with more than 16,000 cases reported in 2023, despite 90% of the population residing in malaria-free zones [3]. The current first-line antimalarial therapy in Indonesia consists of a combination of artesunate, amodiaquine, and primaquine. However, the use of these drugs is limited by their adverse side effects [4,5]. This situation underscores the urgent need for alternative therapeutic agents. Natural products have gained increasing attention as potential sources of antimalarial compounds. One such example is the fruit of Zanthoxylum acanthopodium (locally known as andaliman) from North Sumatra, which contains bioactive compounds such as alkaloids, flavonoids, and coumarins that have demonstrated promising pharmacological activity [6–8]. Nevertheless, the clinical application of natural products remains hampered by poor bioavailability, posing a significant challenge for drug development and therapeutic efficacy [9].
Nanotechnology has emerged as a promising strategy in drug delivery systems, offering the ability to enhance the stability, bioavailability, and intracellular penetration of active substances [10–13]. This approach is specifically designed to address challenges commonly encountered in drug formulation, including poor solubility and limited bioavailability [14–16]. Among the nanocarriers developed, poly(lactic-co-glycolic acid) (PLGA) nanoparticles have shown substantial potential for biomedical applications, particularly in controlled drug release with minimal cytotoxicity [17,18]. PLGA nanoparticles, composed of biodegradable and biocompatible polymers, play an essential role in diverse biomedical applications. One of their most significant uses is in controlled-release drug delivery systems, including antimicrobial therapy [19–21]. These nanoparticles have been demonstrated to effectively inhibit and disrupt microbial biofilms, which are critical contributors to persistent infections and disease progression [22]. Furthermore, combining PLGA nanoparticles with natural polymers such as chitosan and sodium alginate offers an innovative strategy to improve drug delivery efficiency. Chitosan, a cationic polymer, enhances mucoadhesion and prolongs drug retention at the target site [23]. In contrast, sodium alginate, an anionic polymer, interacts electrostatically with chitosan to form a stable hydrogel matrix. This synergistic combination not only enhances nanoparticle stability but also provides improved control over drug release kinetics [24].
Microbial resistance to multiple drugs is an escalating global concern. The effectiveness of synthetic antibiotics is increasingly compromised due to the growing resistance of microorganisms to these agents. This phenomenon poses a serious threat to global health by limiting therapeutic options for infectious diseases. Accordingly, the discovery of new antimicrobial agents, particularly those derived from natural sources, has become crucial to overcoming antibiotic resistance and supporting the management of complex infections. One promising strategy in drug delivery involves the modification of nanoparticles with a synergistic combination of PLGA, chitosan, and sodium alginate. This modification can enhance encapsulation efficiency (EE), facilitate controlled and sustained drug release, and improve the physical and chemical stability of nanoparticles. Moreover, these modifications have the potential to reduce toxicity and enhance biocompatibility, which are essential considerations for pharmaceutical applications, particularly in therapies requiring prolonged drug release. Previous studies have demonstrated the advantages of PLGA-based nanoparticle systems for natural product delivery. Haider et al. [25] reported that incorporating chitosan into drug delivery systems enhances nanoparticle stability and provides controlled release of therapeutic agents. Similarly, Adeyemi et al. [26] showed that PLGA improves the bioactivity while reducing the toxicity of essential oils derived from plant extracts in antimicrobial applications. Alghareeb et al. [27] further highlighted the potential of PLGA-based systems in nasal delivery, where nanoparticle design and surface modifications reduced compound degradation and improved therapeutic efficacy. Additionally, Lu et al. [28] demonstrated that PLGA nanoparticles modified with chitosan can serve as efficient drug carriers in antitumor therapy by increasing drug loading capacity, reducing initial burst release, and prolonging sustained release. Despite the demonstrated potential of nanoparticle-based delivery systems, the application of the PLGA–chitosan–alginate platform for Z. acanthopodium extract remains limited. Zanthoxylum acanthopodium, commonly known as andaliman, contains bioactive compounds with promising antimalarial activity; however, its therapeutic application is constrained by poor bioavailability. Addressing this challenge, the present study seeks to fill an important research gap by focusing on Z. acanthopodium extract as a natural product with untapped antimalarial potential. In this study, the formulation of NANO-PLGA-Cs/NaA-ZAF-DC: PLGA nanoparticles modified with a synergistic combination of chitosan and sodium alginate, used as a carrier for Z. acanthopodium fruit extract in antimalarial delivery and care with high sensitivity and low toxicity (Fig. 1). This innovation represents a strategic advancement in the development of natural product-based drug formulations, contributing to the pharmaceutical field and enhancing the competitiveness of products derived from local resources. The objective of this research was to design and evaluate a PLGA nanoparticle formulation modified with chitosan and sodium alginate as a delivery system for Z. acanthopodium fruit extract with antimalarial activity. The study aimed to improve EE, enhance physicochemical stability, and increase the antimalarial efficacy of the extract through nanoparticle technology, thereby supporting the development of safer and more effective natural-based antimalarial therapies.
 | Figure 1. Modified andaliman (Zanthoxylum acanthopodium) fruit extract carrier system with NANO-PLGA-Cs/NaA-ZAF-DC delivery. [Click here to view] |
2. MATERIALS AND METHODS
2.1 Tools and materials
This study utilized Z. acanthopodium (andaliman) fruit as the primary material, obtained directly from farmers in the highland region of North Tapanuli Regency, North Sumatra, Indonesia. Chitosan, sodium alginate (Na-alginate), PLGA, and polyvinyl alcohol (PVA) were purchased from Sigma-Aldrich (St. Louis, Missouri), a well-established supplier of chemicals, reagents, and research-grade materials for biotechnology, pharmaceutical, and chemical applications. Additional reagents used in this study included 2 N hydrochloric acid (HCl), bromocresol green, dimethyl sulfoxide, ethyl acetate, 70% ethanol, lactose, and chloroform, along with Dragendorff, Bouchardat, and Wagner reagents. Compounds such as magnesium sulfate, 5% potassium hydroxide (KOH), and ferric chloride (FeCl3) were obtained from Merck (Darmstadt, Germany), a company specializing in advanced chemicals and reagents. Water for injection (WFI) was supplied by PT Ikapharmindo Putramas (Bandung, West Java, Indonesia). The Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) enzyme, used for antimalarial activity testing, was sourced from Invilab Bio Indonesia (Bogor, West Java, Indonesia). A range of laboratory instruments was employed in this study. Particle size and zeta potential were measured using a particle size analyzer (Malvern Zetasizer Advance Pro Blue Label, Malvern Panalytical, Malvern, Worcestershire, UK). Solvent removal and sample processing were conducted with a Rotary Evaporator and Vortex Mixer (Heidolph Instruments GmbH & Co. KG, Schwabach, Bavaria, Germany). UV-Vis spectrophotometric analysis was carried out using the SHIMADZU UV-1800 Spectrophotometer (Shimadzu Corporation, Kyoto, Japan). Morphological characterization of nanoparticles was performed with a transmission electron microscope (TEM, JEM 1400; JEOL Ltd., Akishima, Tokyo, Japan). Homogenization was conducted using a T 10 basic ULTRA-TURRAX® disperser (IKA-Werke GmbH & Co. KG, Staufen, Baden-Württemberg, Germany) and an Ultrasonic Homogenizer (Model 150VT, BioLogics, Inc., Manassas, Virginia).
2.2. Extraction of andaliman (Z. acanthopodium) fruit
In this study, 200 mg of Z. acanthopodium fruit powder was subjected to extraction using the maceration method with a solvent mixture of 70% ethanol and ethyl acetate (1:1, v/v). The maceration process was conducted at a controlled temperature of 2°C–8°C for 24 hours in a refrigerated environment. The low-temperature condition was deliberately selected to minimize the degradation of thermolabile compounds and to preserve the bioactivity of the extract. Cooling also reduces enzymatic activity and other chemical reactions that may negatively impact extract quality. Following maceration, the mixture was filtered, and the resulting filtrate was collected and stored at 4°C until further processing. The filtrate was then concentrated by rotary evaporation at 50°C until a viscous extract was obtained. Finally, the extraction yield was calculated to evaluate the efficiency of the process [29].
2.3. Quantitative analysis of andaliman (Z. acanthopodium) fruit extraction tube reaction
Phytochemical screening was conducted to identify the presence of bioactive compounds in the Z. acanthopodium extract. For the alkaloid test, 1 mg of extract was placed into three separate test tubes, followed by the addition of two drops each of Bouchardat, Dragendorff, and Wagner reagents. The presence of polyphenols was examined by adding two drops of FeCl3 solution to a test tube containing 1 mg of extract. The steroid test was performed using the same procedure, but with the addition of two drops of Liebermann–Burchard reagent instead of FeCl3. For the flavonoid test, 1 mg of extract was combined with magnesium powder, followed by the addition of 2 N HCl and 70% ethanol. The mixture was shaken thoroughly and then allowed to stand until phase separation occurred. The tannin test was conducted using a procedure similar to that of polyphenols, namely by adding two drops of FeCl3 solution to a test tube containing 1 mg of extract. Finally, the quinone test was carried out by adding two drops of 5% KOH solution into a test tube containing 1 mg of extract. [30–32].
2.4. Preparation of NANO-PLGA-Cs/NaA-ZAF-DC
PLGA nanoparticles were modified with chitosan (0.1% w/v; pH 5.0; degree of deacetylation 75%–85%) and sodium alginate (0.1% w/v; pH 6.8) using a layer-by-layer technique to form a polyelectrolyte complex (PEC). This modification was intended to enhance mucoadhesion, physicochemical stability, and controlled release of Z. acanthopodium extract. In this system, PLGA serves as a biodegradable matrix with high EE, chitosan provides a cationic surface that facilitates mucoadhesive interactions, and sodium alginate forms an anionic hydrogel layer that reduces burst release and enables pH-sensitive release [33–35]. Nanoparticles were prepared using the solvent evaporation method with slight modifications, consisting of two phases: a non-polar phase and a polar phase (Table 1). The non-polar phase was prepared by mixing 0.5 ml of PLGA, 2 ml of ethyl acetate, and 50 mg of Z. acanthopodium extract, followed by homogenization using a vortex mixer for 1–3 minutes. The mixture was transferred into a 3 ml syringe. The polar phase consisted of 2 ml PVA, 1 ml Tween 80, and 0.5 ml chitosan/sodium alginate solution, which was homogenized in the same manner and placed in a flat-bottom flask. The non-polar phase was then added dropwise into the polar phase using a syringe pump at a rate of one drop every 20 seconds, while stirring at 200 rpm for 1 hour. After nanoparticle formation, particle size reduction was carried out by ultrasonication (130 W, 20 kHz, US) at 70% amplitude in an ice bath for 2.5 minutes. The resulting emulsion was diluted with 50 ml of WFI, transferred into a flat-bottom flask, and subjected to solvent evaporation for 24 hours under stirring at 500 rpm to remove ethyl acetate [36–38]. The obtained suspension was dispersed in cold distilled water (4°C) containing 8% lactose at a 1:1 ratio and homogenized using a stirrer at 750 rpm for 1 hour. The dispersion was then stored at −20°C. Finally, the nanoparticles NANO-PLGA-Cs/NaA-ZAF-DC were dried using a freeze-drying method to obtain the final powdered formulation (Fig. 2) .
 | Figure 2. Flowchart of research procedures. [Click here to view] |
Table 1. NANO-PLGA-Cs/NaA-ZAF-DC formulation.
| Description | Mixture | Material | Concentration (v/w) |
|---|---|---|---|
| PLGA/Chitosan | Non-polar phase | PLGA | 0.5 ml |
| Ethyl acetate | 2 ml | ||
| Extract | 50 mg | ||
| Polar phase | PVA | 2 ml | |
| Chitosan | 1 ml | ||
| Tween 80 | 1 ml | ||
| PLGA/Na-Alginate | Non-polar phase | PLGA | 0.5 ml |
| Ethyl acetate | 2 ml | ||
| Extract | 50 mg | ||
| Polar phase | PVA | 2 ml | |
| Na-Alginate | 1 ml |
2.5. Evaluation of physical characteristics of NANO-PLGA-Cs/NaA-ZAF-DC preparations
Organoleptic evaluation was performed to assess the basic physical properties of the samples, focusing on color, shape, and odor. The samples were placed in 100 ml beaker glasses and observed visually using human sensory perception [39]. The particle size, polydispersity index (PDI), and zeta potential of the nanoparticles were analyzed using a particle size analyzer (PSA). For particle size measurement, 1 ml of sample was placed into a cuvette and inserted into the PSA holder for analysis. For zeta potential measurement, the sample was transferred into a specialized zeta cuvette, which was subsequently placed on the PSA holder and analyzed [40,41]. The morphological characteristics of the NANO-PLGA-Cs/NaA-ZAF-DC nanoparticles were examined using transmission electron microscopy (TEM). For TEM analysis, approximately 500 μl of nanoparticle suspension was dropped onto a carbon-coated copper grid and allowed to adsorb. Excess liquid was removed using filter paper under vacuum assistance before imaging [42,43].
2.6. Evaluation of EE of andaliman fruit extract containing alkaloids in NANO-PLGA-Cs/NaA-ZAF-DC
A total of 2 mg of NANO-PLGA-Cs/NaA-ZAF-DC was dissolved in 10 ml of WFI and homogenized using a vortex mixer for 5 minutes. Subsequently, 5 ml aliquots of the suspension were centrifuged at 5,000 rpm for 30 minutes at 4°C to separate the supernatant from the precipitated NANO-PLGA-Cs/NaA-ZAF-DC nanoparticles [44]. From the supernatant, 500 µl of sample was mixed with 3 ml of 2 N HCl and filtered through a 0.45 µm microfilter. The filtrate was then combined with 5 ml of phosphate buffer (pH 4.7) and 5 ml of 10-4 M bromocresol green solution, followed by shaking to allow complex formation. The resulting complex was extracted twice with 5 ml of chloroform. The chloroform phase was collected and transferred into a 10 ml volumetric flask, with additional chloroform added to reach the calibration mark [45]. Quantitative analysis was conducted using a UV-Vis spectrophotometer after method validation, in order to determine the concentration of encapsulated and non-encapsulated alkaloids. The absorbance spectra were recorded in the range of 250–300 nm, with maximum detection at 280 nm. EE was calculated using the following equation, expressed as the percentage of alkaloids successfully encapsulated in the NANO-PLGA-Cs/NaA-ZAF-DC formulation [46]:
2.7. Stability test of NANO-PLGA-Cs/NaA-ZAF-DC
The heat–cold cycle test was conducted by placing the NANO-PLGA-Cs/NaA-ZAF-DC in a refrigerator at 4°C for 8 hours and then transferring it to an oven at 40°C for 8 hours. The freeze–thaw cycle test was conducted by placing the NANO-PLGA-Cs/NaA-ZAF-DC in a refrigerator at −20°C for 8 hours and then transferring it to a room temperature of 25°C for 8 hours. These two tests were performed for six cycles within 2 days. After the cycle was completed, 2 mg of the tested NANO-PLGA-Cs/NaA-ZAF-DC was dissolved in 10 mL of WFI and homogenized using a vortex for 5 minutes. Next, evaluations were conducted to observe changes in particle size, PDI, zeta potential, and EE. For the durability test, 1 mg of NANO-PLGA-Cs/NaA-ZAF-DC was dissolved with WFI solvent at a ratio of 2.5, 5, 10, and 25 times and tested to assess changes in particle size, PDI, zeta potential, and EE. Furthermore, for storage tests, NANO-PLGA-Cs/NaA-ZAF-DC was sealed in trilaminate bags and stored in a refrigerator at 2°C–8°C for 1 month. At intervals of every 1 week, the samples were evaluated to assess changes in particle size, PDI, zeta potential, and EE [44,47].
2.8. NANO-PLGA-Cs/NaA-ZAF-DC antimalarial PfDHODH method in vitro assay
The solvent control was prepared by diluting 1 ml of pure dimethyl sulfoxide (DMSO) into 99 mL of phosphate-buffered saline (PBS, pH 7.4) to obtain a final concentration of 1%. The NANO-PLGA-Cs/NaA-ZAF-DC sample was weighed and dissolved in 100% DMSO to produce a 10 mg/ml stock solution. Chloroquine sulfate was used as the positive control at a final concentration of 1 µM. The positive control was prepared from a 1 mM stock solution (0.5159 g dissolved in 1 l DMSO) by diluting 1 µL of the stock solution into 999 µl of DMSO. For the assay, 2 µl of each test sample was added to wells containing 192 µl of the reaction mixture, which consisted of 5 mM HEPES buffer (pH 7.6), 36 µM decylubiquinone, 120 µM dichlorophenolindophenol (DCIP), and 0.1 µM PfDHODH. The enzymatic reaction was initiated by the addition of L-dihydroorotate to a final concentration of 200 µM. The reaction mixtures were incubated for 20 minutes at room temperature, and the activity was monitored by measuring the decrease in DCIP absorbance at 600 nm, which corresponds to the visible color change of the solution from blue to clear [48–50].
3. RESULTS AND DISCUSSION
3.1. Results of qualitative analysis of andaliman (Z. acanthopodium) fruit extract
In this study, the extraction of Z. acanthopodium fruit was conducted using 198.78 grams of dried fruit powder. The maceration process was carried out with a 1:1 mixture of 70% ethanol and ethyl acetate, totaling 5 l of solvent. After 24 hours of maceration, a concentrated extract weighing 24.20 g was obtained. The extraction yield was calculated by dividing the weight of the extract by the weight of the raw material, and the yield was expressed as a percentage, resulting in 12.17%. This yield surpasses the minimum acceptable threshold of 10%, indicating an efficient extraction process.
- Weight of dry powder : 198.78 g
- Weight of condensed extract : 24.20 g
= 12,17 %
Qualitative phytochemical analysis of the ethyl acetate fraction of Z. acanthopodium (andaliman) fruit extract, as shown in Figure 3, revealed the presence of multiple bioactive compounds. The alkaloid test produced distinct color changes: a blackish-brown color with Bouchardat reagent, a red-to-orange precipitate with Dragendorff reagent, and a yellowish-orange cloudy precipitate with Wagner reagent, all confirming the presence of alkaloids. The polyphenol test using FeCl3 produced a blue–black coloration, indicating a positive reaction for polyphenols. Steroids were identified by a greenish color change upon addition of the Liebermann–Burchard reagent. Flavonoids were detected through the appearance of yellow discoloration and precipitate formation following treatment with magnesium powder, 2N HCl, and 70% ethanol. The tannin test with FeCl3 yielded a dark blue–black coloration, while the quinone test with 5% KOH showed a color transition from yellow to reddish. Overall, these findings confirm that the andaliman fruit extract contains diverse classes of bioactive compounds, including alkaloids, polyphenols, steroids, flavonoids, tannins, and quinones, thereby supporting its potential for further pharmacological investigation [30–32].
 | Figure 3. The results obtained from the qualitative analysis of andaliman (Zanthoxylum acanthopodium) Fruit extract. (A) Alkaloid test with Bouchardat reagent, (B) Alkaloid test with Dragendorff reagent, (C) Alkaloid test with Wagner reagent, (D) Polyphenol test with FeCl3 reagent, (E) Steroid test with Liebermann–Burchard reagent, (F) Flavonoid test with Magnesium powder + HCl 2N + 70% ethanol, (G) Tannin test with FeCl3 reagent, (H) Quinone test with 5% KOH reagent. [Click here to view] |
3.2. Results of evaluation of the physical characteristics of NANO-PLGA-Cs/NaA-ZAF-DC
The formation of PLGA nanoparticles modified with chitosan and sodium alginate as a delivery system for Z. acanthopodium (andaliman) fruit extract containing alkaloids was achieved using a bottom-up technique, assisted by an ultrasonic probe, followed by solvent evaporation to remove the organic solvent employed during synthesis [36,37]. The bottom-up approach enables nanoparticle formation from the molecular level through precipitation or ionic gelation, in which PLGA is dissolved in an appropriate organic solvent, such as acetone or dichloromethane, and subsequently mixed with a chitosan–sodium alginate solution in the aqueous phase [37]. The application of ultrasonic probes at this stage was critical, as the high-frequency ultrasonic waves generated cavitation and strong shear forces. These forces facilitated homogenization of the solution, reduced particle aggregation, and contributed to the production of nanoparticles with smaller particle sizes and a more uniform distribution [52].
Following nanoparticle formation, the process was continued with the solvent evaporation method, which aimed to eliminate residual organic solvents. This step was performed by stirring the nanoparticle suspension under vacuum or controlled temperature conditions to allow gradual evaporation of the solvent without compromising nanoparticle integrity. The solvent evaporation step was essential to ensure the removal of potentially toxic organic solvent residues and to enhance the overall stability of the final nanoparticle formulation [36].
The organoleptic evaluation of NANO-PLGA-Cs/NaA-ZAF-DC revealed that the freshly prepared formulation appeared as a cloudy, milky-green suspension with a faint odor of ethyl acetate Figure 4A. The suspension was characterized by the presence of dispersed particles and visible sedimentation when allowed to stand for more than 48 hours. This suspension form was obtained following the evaporation of the organic solvent during a 24-hour solvent evaporation process. Furthermore, when the NANO-PLGA-Cs/NaA-ZAF-DC formulation underwent solvent removal by freeze-drying, the resulting product was obtained in the form of a slightly sticky colloidal gel-like powder. This powder also exhibited a milky-green coloration and retained a characteristic organic odor (Fig. 4C).
 | Figure 4. The evaluation results of the physical characteristics of the NANO-PLGA-Cs/NaA-ZAF-DC preparation show several key stages. In panel (A), the NANO-PLGA-Cs/NaA-ZAF-DC preparation is shown after the evaporation of residual organic solvent, followed by the NANO-PLGA-Cs/NaA-ZAF-DC powder after freeze-drying treatment. Panel (B) illustrates the particle size distribution of the preparation. Next, panel (C) displays the NANO-PLGA-Cs/NaA-ZAF-DC colloidal gel powder after freeze-drying treatment. Finally, panel (D) presents the morphological image of the NANO-PLGA-Cs/NaA-ZAF-DC preparation, providing insight into its structure and physical form. [Click here to view] |
The characterization results, as shown in Table 2, showed that the average particle size of the nanoparticles was 205.90 nm ± 1.79, which falls within the optimal range for drug delivery applications. Nanoparticles of this size can improve bioavailability and facilitate penetration into target tissues. The particle size distribution, as shown in Figure 4B, ranging from 100 to 300 nm, further supports these findings and is considered ideal for enhancing circulation time and promoting cellular uptake [53]. The use of an ultrasonic probe during synthesis played a crucial role in minimizing aggregation and ensuring a uniform particle size distribution. The PDI was 0.25 ± 0.02, indicating a relatively narrow size distribution. A low PDI value reflects a monodisperse system, which is essential for achieving controlled drug release and enhancing nanoparticle stability [54,55]. In general, PDI values range from 0 to 1; values closer to 0 indicate uniform particle distribution, whereas values approaching one suggest a heterogeneous system [44].
Table 2. Results of particle size, PDI, and zeta potential testing.
| Particle size (nm) | PdI | Zeta potential (mV) | |
|---|---|---|---|
| Test results* | 205.90 ± 1.79 | 0.25 ± 0.02 | −13.90 ± 1.43 |
Note: *Average of three replications ± SD.
Nanoparticle stability was further evaluated by measuring zeta potential, which yielded a value of –13.90 ± 1.43 mV Table 2. This negative charge is primarily attributed to the intrinsic properties of PLGA and sodium alginate, which provide electrostatic repulsion to reduce aggregation [56,57]. The incorporation of chitosan, a positively charged polymer, reduced the overall negative charge due to electrostatic interactions [58]. Although this value does not reach the ±30 mV threshold typically associated with highly stable colloidal systems, it suggests moderate stability. While some aggregation may occur, the nanoparticles remained stable in suspension [59]. The combined effect of electrostatic and steric stabilization from PLGA, chitosan, and sodium alginate was sufficient to maintain particle dispersion, a balance that is critical to ensure effective drug delivery [56–59]. The relationship between zeta potential and stability is significant, as higher electrostatic repulsion reduces aggregation, whereas lower repulsion may lead to sedimentation [44,60].
Morphological characterization using TEM provided detailed insights into the shape, size, and surface structure of the nanoparticles. TEM analysis confirmed that the nanoparticles exhibited a spherical morphology with a monodisperse distribution, which is critical for consistency in biomedical applications. As shown in Figure 4D, the NANO-PLGA-Cs/NaA-ZAF-DC nanoparticles displayed a smooth surface and uniform size distribution, indicating precise control over fabrication. The observed contrast between the nanoparticle core and surface layers suggests the successful incorporation of chitosan and sodium alginate, which encapsulated the PLGA matrix to enhance surface stability and modulate surface charge. This surface modification is particularly beneficial, as it promotes favorable interactions with cell membranes and improves drug delivery efficiency. Furthermore, TEM allowed confirmation of the thickness and uniformity of the surface layers, parameters that influence nanoparticle–cell interactions. Overall, TEM analysis demonstrated clear morphology and effective surface modification, supporting the suitability of these PLGA-based nanoparticles for biomedical applications, particularly in controlled drug release and enhanced bioavailability [61,62].
3.3. Results of evaluation of EE of andaliman (Z. acanthopodium) fruit extract containing alkaloids in NANO-PLGA-Cs/NaA-ZAF-DC
The EE% results presented in Table 3 demonstrate the extent to which the Z. acanthopodium (andaliman) fruit extract, with alkaloids as marker compounds, was successfully incorporated into the NANO-PLGA-Cs/NaA-ZAF-DC system. The EE% value of 68.52% ± 2.40% indicates that the majority of alkaloids were successfully retained within the nanoparticle matrix, while only a small fraction remained in the supernatant. This relatively high level of encapsulation highlights the synergistic role of PLGA, chitosan, and sodium alginate in the nanoparticle system. PLGA, as a biodegradable and biocompatible polymer, provides the primary structural framework and is widely utilized in drug delivery applications. Chitosan, a cationic polymer, enhances electrostatic interactions with the negatively charged alkaloids, thereby improving both EE and nanoparticle stability [58]. Sodium alginate, an anionic polymer, contributes to the formation of a hydrogel matrix that not only stabilizes the nanoparticle surface but also facilitates controlled release of the encapsulated compounds [63]. Together, the combined properties of PLGA, chitosan, and sodium alginate provide a robust encapsulation system capable of maintaining alkaloid stability while supporting controlled drug release, thereby underscoring the potential of this nanoformulation for antimalarial applications.
Table 3. Results of evaluation of EE of andaliman (Zanthoxylum acanthopodium) fruit extract containing alkaloids in NANO-PLGA-Cs/NaA-ZAF-DC.
| Sample | Concentration in sample (mg/ml) | Concentration in supernatant (mg/ml) | EE (%) |
|---|---|---|---|
| NANO-PLGA-Cs/NaA-ZAF-DC* | 0.299 ± 0.010 | 0.094 ± 0.008 | 68.52 ± 2.40 |
Note: *Average of three replications ± SD.
The alkaloid concentration detected in the nanoparticle suspension was 0.299 ± 0.010 mg/ml, confirming that the majority of the active compound was successfully encapsulated within the NANO-PLGA-Cs/NaA-ZAF-DC system. In contrast, the supernatant contained 0.094 ± 0.008 mg/ml, indicating that only a limited fraction of the alkaloid remained unencapsulated. These findings further support the efficiency of the encapsulation process. EE is known to be influenced by multiple factors, including the technique employed such as emulsification—solvent evaporation or ionotropic gelation—as well as the physicochemical interactions among the polymers used in the formulation. Variations in these interactions, combined with the specific formulation parameters applied, can significantly affect the degree of encapsulation achieved and ultimately determine the stability and release profile of the active compound [64–66].
3.4. Results of Stability test of NANO-PLGA-Cs/NaA-ZAF-DC
Stability testing of the NANO-PLGA-Cs/NaA-ZAF-DC formulation was conducted using three approaches: heating–cooling cycles, freeze–thaw cycles, and multilevel dilution. The evaluation parameters included changes in particle size, PDI, zeta potential, and EE. Each method demonstrated distinct effects on nanoparticle stability, particularly with respect to aggregation behavior and retention of the active compound (Table 4). In the heating–cooling test, both particle size and PDI increased significantly, indicating nanoparticle aggregation caused by temperature-induced structural changes in PLGA and enhanced interparticle interactions [67]. The zeta potential value −9.43 ± 0.64 mV suggested poor colloidal stability, while the EE decreased substantially to 41.49% ± 1.66%, reflecting a reduction in alkaloid retention. This instability was attributed to thermal stress, disruption of the PLGA structure, and weakened polymer–drug interactions [68,69]. In contrast, the freeze–thaw test resulted in smaller particle sizes and lower PDI values, suggesting a more uniform size distribution. The zeta potential was more negative (−12.97 ± 0.38 mV), indicating improved electrostatic stabilization. EE also increased significantly to 61.18% ± 7.12%, suggesting better retention of the active compound. The absence of thermal stress during this test likely preserved nanoparticle integrity, thereby maintaining EE [70]. The multilevel dilution test (2.5–25×) showed that particle size and PDI progressively increased with higher dilution levels, indicating aggregation and greater heterogeneity in particle distribution. Zeta potential values initially became more negative, reflecting enhanced electrostatic repulsion; however, at higher dilutions, the values decreased, indicating loss of stability. EE remained stable at 2.5–5× dilutions but declined markedly at 25× dilution, suggesting leakage of encapsulated alkaloids. These changes were attributed to alterations in electrostatic balance and reduced polymer–drug interactions caused by the increased medium volume [44].
Table 4. Results of heating-cooling, freeze–thaw, and resistance to dilution of NANO-PLGA-Cs/NaA-ZAF-DC stability test.
| Sample | Treatment | PS (nm) | PdI | ZP (mV) | EE (%) |
|---|---|---|---|---|---|
| NANO-PLGA-Cs/NaA-ZAF-DC* | Heating–cooling | 536.74 ± 1.56 | 0.68 ± 0.03 | −9.43 ± 0.64 | 41.49 ± 1.66 |
| Freeze–thaw | 231.77 ± 1.94 | 0.34 ± 0.05 | −12.97 ± 0.38 | 61.18 ± 7.12 | |
| 2.5 times dilution | 310.97 ± 1.03 | 0.40 ± 0.01 | −11.18 ± 1.06 | 64.32 ± 5.03 | |
| 5 times dilution | 345.80 ± 1.15 | 0.46 ± 0.02 | −10.82 ± 0.86 | 63.43 ± 3.45 | |
| 10 times dilution | 353.01 ± 1.24 | 0.54 ± 0.03 | −10.29 ± 0.16 | 63.39 ± 4.77 | |
| 25 times dilution | 410.39 ± 1.43 | 0.57 ± 0.01 | −10.05 ± 0.16 | 58.69 ± 1.28 |
Note: *Average of three replications ± SD.
Based on the observations presented in Figure 5, storage stability testing revealed that particle size remained unchanged during the first week but increased significantly from week 2 onward (p < 0.001), continuing to rise in weeks 3 and 4 (p < 0.0001). This trend indicates nanoparticle aggregation during prolonged storage. Similarly, PDI values remained stable during week 1 but showed a significant increase beginning in week 2 (p < 0.05), reflecting greater heterogeneity in particle size distribution. Zeta potential values exhibited no significant changes in weeks 1 and 2; however, a significant increase was observed from week 3 (p < 0.01), suggesting reduced electrostatic repulsion and a decline in colloidal stability. EE%, however, remained stable throughout the 4-week storage period, with no significant changes detected. These results indicate that although some physical alterations occurred, namely particle growth and reduced zeta potential—the encapsulated alkaloid content from Z. acanthopodium was retained under refrigerated storage at 2°C–8°C. The integrity of the nanoparticles was preserved, with no evidence of significant PLGA matrix degradation or premature drug release. Strong interactions between the alkaloid compounds and the PLGA matrix likely contributed to the limited diffusion of the active constituents [71]. Refrigerated storage conditions further contributed to the stability of EE% by slowing the hydrolysis of PLGA, thereby maintaining nanoparticle structure and preventing alkaloid leakage [72]. In addition, the chitosan–alginate coating formed PECs that stabilized the nanoparticle surface and minimized the loss of active compounds [58,63]. Low temperatures also reduced nanoparticle aggregation, thus supporting EE [72]. The synergistic interaction between cationic chitosan and anionic sodium alginate enhanced overall system stability by increasing nanoparticle integrity, preventing premature alkaloid release, and forming a gel-like barrier that protected the particles during storage. Moreover, this combination reduced interactions with environmental moisture, which could otherwise accelerate PLGA degradation under less optimal storage conditions [58].
 | Figure 5. Stability test observation graph of NANO-PLGA-Cs/NaA-ZAF-DC during 2°C–8°C storage period from week 0 to week 4. (A) particle size, (B) PDI, (C) zeta potential. (D) EE. Samples were analyzed using one-way ANOVA with a 95% confidence interval, followed by a post-hoc test to determine significant differences in each group. n: 5; ns: not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. [Click here to view] |
Instability within the PLGA-Cs/NaA-ZAF-DC system can be attributed to several physicochemical factors. Thermal fluctuations during heating–cooling cycles disrupt the PLGA matrix through repeated expansion and contraction, which can compromise structural integrity. These changes promote particle aggregation and drug leakage, accompanied by a reduction in zeta potential, which decreases interparticle repulsion and enhances sedimentation [67–69]. In the multilevel dilution test, increasing dilution weakened interparticle electrostatic forces, resulting in larger particle sizes, higher PDI values, and decreased EE% due to the release of active compounds [44]. Moreover, the stability of chitosan–alginate interactions may be reduced under conditions of altered pH or ionic strength, further affecting particle stability [73]. During refrigerated storage at 2°C–8°C, particle size increases were likely due to aggregation, PLGA hydrolysis, and reduced surface charge stability. Hydrolytic degradation of PLGA produces charged fragments that disrupt nanoparticle surface balance, thereby increasing the tendency toward agglomeration. Additionally, the hydrophilic nature of sodium alginate may facilitate water absorption, hydration, and swelling, all of which contribute to particle growth [74,75]. The observed increase in PDI indicates greater irregularity in particle morphology, potentially altering extract–polymer interactions and affecting both nanoparticle shape and zeta potential [76]. A decline in zeta potential during storage reflects surface charge changes caused by PLGA hydrolysis, ion interactions within the medium, and nanoparticle aggregation. Reduced surface charge diminishes repulsive forces between particles, thereby increasing the likelihood of agglomerate formation [44]. Nevertheless, as long as the zeta potential remains within the dispersion stabilization range (approximately −10 to −15 mV), the system may still be considered relatively stable for short-term storage [59]. Although EE% was not significantly affected, increases in particle size, higher PDI, and reduced zeta potential suggest potential impacts on formulation performance. Therefore, further optimization is warranted. Adjustments to the PLGA–chitosan–alginate ratio or the incorporation of stabilizing agents such as Tween 80 are recommended to improve nanoparticle stability during storage and under thermal stress conditions [77].
3.5. Results of NANO-PLGA-Cs/NaA-ZAF-DC antimalarial PfDHODH method in vitro assay
In this study, an in vitro antimalarial assay was performed using the PfDHODH method to evaluate the inhibitory activity of compounds against PfDHODH. PfDHODH is a key enzyme in the pyrimidine biosynthesis pathway of P. falciparum, and its inhibition disrupts DNA and RNA synthesis, ultimately suppressing parasite growth. The primary objective of this assay was to assess the effectiveness of PLGA-based nanoparticles modified with chitosan and sodium alginate in enhancing the bioavailability and stability of Z. acanthopodium (andaliman) fruit extract as an antimalarial candidate [48,49].
The in vitro assay results demonstrated that PLGA nanoparticles containing andaliman extract at a concentration of 100 ppm inhibited PfDHODH activity by 19.60% ± 2.50%. Although this level of inhibition confirmed the ability of the formulation to suppress enzymatic activity, the effect was lower compared with the standard PfDHODH inhibitor (Table 5). These findings suggest that the formulation possesses measurable antimalarial potential, but its effectiveness remains limited at the tested dose. Several factors may have contributed to the relatively low inhibitory activity. First, the active compound loading in the nanoparticles may not have been optimal, leading to incomplete encapsulation and reduced bioavailability. Second, heterogeneity in particle size distribution and variations in EE could have limited the availability of active compounds in the reaction phase. Finally, the concentration tested may have been insufficient to achieve a significant inhibitory effect against PfDHODH. These limitations indicate the need for further optimization of the nanoparticle formulation. Strategies such as increasing the extract loading, improving nanoparticle stability, and expanding the dose range should be explored to enhance inhibitory performance and allow calculation of dose–response relationships, including IC50 values.
Table 5. Results of NANO-PLGA-Cs/NaA-ZAF-DC antimalarial PfDHODH method in vitro assay.
| Sample | Inhibition of PfDHODH (%) |
|---|---|
| Solvent control (DMSO 1%)* | 0.00 ± 1.20 |
| NANO-PLGA-Cs/NaA-ZAF-DC 100 ppm* | 19.60 ± 2.50 |
| Positive control (Chloroquine 1 µM)* | 91.30 ± 1.05 |
Note: *Average of three replications ± SD.
While PfDHODH inhibition demonstrates preliminary antimalarial activity, it is important to emphasize that the inhibition of a single enzyme does not provide a comprehensive understanding of the overall therapeutic potential. Additional studies, including in vitro assays of P. falciparum growth inhibition in cell culture and cytotoxicity testing on mammalian cells, are necessary to further evaluate the efficacy and safety of this formulation. Such investigations will be crucial for determining the therapeutic relevance of PLGA-based nanoparticles containing Z. acanthopodium extract and their suitability for future clinical development.
PLGA nanoparticles are extensively utilized in drug delivery systems because of their biocompatibility and biodegradability, which enable controlled and sustained release of active compounds. In this study, modification with chitosan and sodium alginate was employed to increase the nanoparticle surface charge, potentially enhancing adsorption and uptake by Plasmodium parasites. This modification is expected to improve cellular penetration and prolong the antimalarial activity of Z. acanthopodium (andaliman) extract. The antimalarial potential of andaliman extract is strongly supported by the presence of secondary metabolites, including flavonoids, alkaloids, terpenoids, and tannins [78]. These bioactive compounds have been widely reported to possess antiparasitic activities [79]. Flavonoids and alkaloids, for instance, inhibit PfDHODH, thereby disrupting pyrimidine biosynthesis and impairing DNA and RNA synthesis. Alkaloids, particularly quinoline and isoquinoline derivatives, are also known to interfere with hemoglobin metabolism in the parasite and induce oxidative stress. Terpenoids contribute to antimalarial effects by damaging parasite membranes and disrupting energy metabolism, whereas tannins exert astringent activity that can inhibit key enzymes involved in the Plasmodium falciparum lifecycle [80]. In this study, PfDHODH inhibition of 19.60% ± 2.50% demonstrated measurable antimalarial activity of the NANO-PLGA-Cs/NaA-ZAF-DC formulation. However, this effect remains modest and requires further improvement. Optimization strategies may include reducing nanoparticle size to <200 nm to enhance cellular uptake, increasing extract loading concentration, and refining sustained release properties to prolong drug action [49–81]. Furthermore, comprehensive pharmacokinetic and toxicity evaluations are essential to establish the safety and therapeutic feasibility of this nanoparticle system. Such studies will provide critical insights for advancing this formulation toward clinical application as a natural product-based antimalarial therapy.
4. CONCLUSION
The NANO-PLGA-Cs/NaA-ZAF-DC nanoparticles were successfully formulated with a spherical morphology and a narrow, monodisperse particle size distribution, averaging 205.90 ± 1.79 nm. The EE reached 68.52% ± 2.40%, confirming the effective incorporation of bioactive compounds from Z. acanthopodium extract. Stability evaluations demonstrated that the formulation preserved its physicochemical integrity during freeze–thaw cycles as well as after 30 days of storage at 2°C–8°C. In addition, dilution testing indicated favorable dispersion behavior, further supporting the robustness of the system under varying conditions. The successful modification with chitosan and sodium alginate was evident in the enhanced EE, improved physicochemical stability, and controlled release properties of the nanoparticles. These findings emphasize the strength of the formulation and its potential as a drug delivery platform. In vitro antimalarial evaluation using the PfDHODH inhibition assay demonstrated measurable inhibitory activity at a concentration of 100 ppm, indicating that the formulation possesses antimalarial potential. Collectively, these results highlight the successful development of a biopolymer-based nanoparticle system that provides a solid foundation for further optimization and advancement as a natural product-derived antimalarial therapy.
5. ACKNOWLEDGMENTS
The authors would like to thank the Ministry of Education and Culture and the Ministry of Research and Technology for their support in this research through the funding of the “Student Creativity Program” 2024 and the Nano-Pharmaceutical Research Centre, Pharmaceutics and Pharmaceutical Technology Laboratory, Faculty of Pharmacy, Sultan Agung Islamic University, as well as those who have supported the publication of this paper. There is no conflict of interest.
6. AUTHOR CONTRIBUTIONS
All authors made substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; took part in drafting the article or revising it critically for important intellectual content; agreed to submit to the current journal; gave final approval of the version to be published; and agree to be accountable for all aspects of the work. All the authors are eligible to be author as per the International Committee of Medical Journal Editors (ICMJE) requirements/guidelines.
7. CONFLICTS OF INTEREST
The authors report no financial or any other conflicts of interest in this work
8. ETHICAL APPROVAL
This study does not involve experiments on animals or human subjects.
9. DATA AVAILABILITY
The datasets that were used and analyzed during this study are not publicly available due to confidentiality reasons. This restriction is imposed by the Med Chula Institutional Review Board. Data requests can be sent to the corresponding author.
10. PUBLISHER’S NOTE
All statements presented in this article are the sole responsibility of the authors and do not necessarily reflect the views of the publisher, editors, or reviewers. This journal maintains a neutral stance with regard to jurisdictional claims in published institutional affiliations. The authors would also like to express their sincere appreciation to the Ministry of Education and Culture and the Ministry of Research and Technology for their support in this research through funding provided by the 2024 Student Creativity Program.
11. USE OF ARTIFICIAL INTELLIGENCE (AI)- ASSISTED TECHNOLOGY
The authors declare that they have not used artificial intelligence (AI)-tools for writing and editing of the manuscript, and no images were manipulated using AI.
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