Abstract Prasa Mawaeng is a traditional Thai herbal remedy rich in bioactive compounds recognized for their potent antioxidant and antimicrobial properties. As the impact of drying on this remedy remains unexplored, this study aimed to determine the effect of different drying methods on the phytochemical profile and biological activities of the preparation. Prasa Mawaeng was prepared and quality-evaluated according to the Thai Herbal Preparation Pharmacopoeia (THPP). The remedy was extracted by decoction and subsequently dried into spray-dried (SD-PSM) and freeze-dried (FD-PSM) powders. Phytochemical characteristics were evaluated using thin-layer chromatography (TLC) and total phenolic content (TPC), while antioxidant and antimicrobial activities were determined using ABTS/FRAP assays and the macrobroth dilution method. Scanning electron microscope (SEM) revealed distinct differences in particle morphology between drying methods, whereas TLC profiles, TPC, and antioxidant capacity were comparable, and neither powder exhibited inhibitory activity against Staphylococcus aureus, Candida albicans, Escherichia coli, and Streptococcus mutans (0.5–15 mg/mL). Following stability assessment for three months at room temperature and one month under accelerated conditions (40°C/75% relative humidity), SD-PSM maintained a fine powder form while FD-PSM agglomerated, although TPC remained statistically stable in both powders throughout storage. In conclusion, SD-PSM demonstrated better physical stability than FD-PSM under accelerated storage, considering the commercial scale-up cost. These findings support its potential for scalable pharmaceutical manufacturing and innovative herbal health product development.

Keywords: Prasa Mawaeng remedy, Spray drying, Freeze drying, Phytochemical properties, Stability testing

Funding: This research study was financially supported by Naresuan University and the National Science, Research and Innovation Fund (NSRF), Grant No. R2569B067, and was (partially) supported by the Frontier Research and Innovation Cluster Fund, Naresuan University, Grant No. R2569C007.

Citation:  Apirattanajaroensuk, T., Limsirilak, S., Prommee, N., Jongcharoenkamol, J., Sungthongjeen, S., and Kriangkrai, W. 2026. Effects of spray drying and freeze drying on phytochemical composition and stability of Prasa Mawaeng Thai traditional remedy powder. Natural and Life Sciences Communications. 25(4): e2026101.

Graphical Abstract:

INTRODUCTION

Prasa Mawaeng is a traditional Thai herbal remedy that has been used for centuries to alleviate respiratory symptoms and is included in the National List of Essential Medicines as an antitussive and expectorant. Among its components, Solanum trilobatum (S. trilobatum) and Solanum violaceum (S. violaceum) are major ingredients and traditionally used to manage respiratory and febrile conditions, with S. trilobatum applied for bronchitis, asthma, and cough, and S. violaceum for chronic febrile infections, leukoderma, nasal ulcer, and gonorrhea (Quamar and Bera, 2014; Govindarajan et al., 2022). Their respiratory-related effects are largely attributed to phenolic compounds such as caffeic, ferulic, and p-coumaric acids, with chlorogenic acid as a major antioxidant through hydrogen donation and radical stabilization (Liang and Kitts, 2015; Tyagi et al., 2020). Additionally, Ocimum tenuiflorum (O. tenuiflorum) contains eugenol, identified as the key compound contributing to its antimicrobial efficacy by disrupting cell membranes and damaging genomic DNA (Jeyakumar and Lawrence, 2021). Curcumin and curcuminoids from Curcuma spp. have also been reported to exhibit antioxidant and antimicrobial activities through radical scavenging and Nrf2/NF-κB modulation (Rahayu et al., 2018; Burapan et al., 2020).

The Prasa Mawaeng remedy can be prepared in various dosage forms such as a lime juice preparation with salt, tablets, and lozenges (Bunjob, 1992). One common method for preparing Thai traditional medicines is decoction, which is boiling dried or fresh herbs in water (Santianotai et al., 2025). Boiling enhances the extraction of certain active compounds from herbs, particularly phenolics and flavonoids, while preventing glycoside hydrolysis through glucuronidase inhibition (Bitwell et al., 2023). Supporting this, a decoction of S. trilobatum has shown potential in managing acute and chronic bronchitis (Jawahar et al., 2004), and its aqueous extracts exhibit broad-spectrum antibacterial activity against both Gram-positive and Gram-negative pathogens (Dasthageer et al., 2022; Radhakrishnan et al., 2022). Similarly, hot water extracts of Curcuma species possess potent antioxidant and antibacterial properties (Uchio et al., 2017; Khan et al., 2024), while decoction processes in Caesalpinia spp. have been shown to enhance the recovery of active compounds for antibacterial and antibiofilm applications (Chachomchuen et al., 2026). However, a major limitation of this method is that the active substances readily deteriorate and may spoil quickly (Neelapong et al., 2018).

Drying therefore represents a practical strategy to improve the extract's stability, handling, and storage, provided the physicochemical changes and loss of bioactive constituents that occur during processing are well understood (Gopinathan et al., 2020). Among various methods, spray drying and freeze drying were selected as the two industrially dominant processes (Xu et al., 2023). Spray drying rapidly atomizes liquid feeds to improve the solubility and stability of various bioproducts, offering superior scalability, throughput, and reduced packaging weight (Swm et al., 2025), although it may be limited by low yield and thermal degradation (Muzaffar et al., 2016). To avoid thermal degradation, freeze drying is a non-thermal alternative involving freezing, vacuum sublimation, and secondary drying (Adams et al., 2015), enhancing stability and flavor preservation, but limited by high cost, energy demand, and long processing time (Nowak and Jakubczyk, 2020), with scale-up remaining problematic due to trial-and-error parameter setting (Patel and Pikal, 2011). As the impact of drying on the Prasa Mawaeng remedy remains unexplored, this study evaluated the effects of spray and freeze drying on its phytochemical profile and antioxidant, and antimicrobial activities, providing insights to support its pharmaceutical and nutritional development.

MATERIALS AND METHODS

Materials

Prasa Mawaeng powder was sourced from Charoensuk Osoth (Nakhon Pathom, Thailand) and had the ratio of plant ingredients according to the Thai Herbal Preparation Pharmacopoeia (THPP), as shown in Table 1. All chemicals and standards used in this study included Trolox (TCI, Tokyo, Japan), ABTS (TCI, Tokyo, Japan), potassium persulfate (Sigma-Aldrich, St. Louis, MO, USA), 2,4,6-tripyridyl-s-triazine (TPTZ) (TCI, Tokyo, Japan), ferric chloride (Sigma-Aldrich, St. Louis, MO, USA), iron (II) sulfate heptahydrate (Sigma-Aldrich, St. Louis, MO, USA), Folin–Ciocalteu reagent (Merck, Darmstadt, Germany), sodium carbonate (Merck, Darmstadt, Germany), anisaldehyde (Fluka Chemical Company, Buchs, Switzerland), methanol (Labscan, Bangkok, Thailand), butan-1-ol (Labscan, Bangkok, Thailand), ethyl acetate (Labscan, Bangkok, Thailand), glacial acetic acid (Labscan, Bangkok, Thailand), sulfuric acid 98% (Labscan, Bangkok, Thailand). All other reagents were of analytical grade.

Table 1. The proportions of medicinal plants in Prasa Mawaeng remedy according to the THPP.

Quality control assay of Prasa Mawaeng remedy

Microscopic examination

The Prasa Mawaeng remedy was mounted in water and examined under a light microscope model CHS (Olympus Optical Co., Ltd., Tokyo, Japan), for morphological characterization. The observed components were identified according to the THPP (DTAM, 2022) to support the identify of plant materials based on standard herbal characteristics.

Total ash content

Ash content determination involves heating the plant material until their organic matter and its derivatives decompose and evaporate, resulting in residual mineral and inorganic elements. The total ash content of the Prasa Mawaeng remedy was determined according to the THPP to evaluate physiological and non-physiological ash (DTAM, 2022). The Prasa Mawaeng powder was weighed (2 g) into the crucible and burned in a muffle furnace (Nabertherm, Lilienthal, Germany) at 450°C until the ash turned to gray or white, and the total ash content was calculated using the following equation:

Acid-insoluble ash

The ash obtained from total ash determination was boiled with 25 mL of hydrochloric acid for 5 min. The insoluble component was then filtered through an ash-free filter paper and rinsed with hot water until reaching pH 7. The paper was burned at 500°C in a muffle furnace for 24 h (DTAM, 2022). The crucible and sample were weighed, and the acid-insoluble ash content was calculated using the following the equation:

Water content

The water content of Prasa Mawaeng was determined using the Karl Fischer titrator methods. A 150-mg of the remedy was weighted and transferred into the titration chamber containing water, iodine, and sulfur dioxide, and performed automatically using a Karl Fischer titrator, 870 KF Titrino Plus (Metrohm India Private Ltd., Chennai, Tamil Nadu, India).

Loss on drying

The loss on drying was determined as the maximum amount of moisture and volatile components lost during the drying process, using a moisture analyzer (MA40, Sartorius, Göttingen, Germany). The 2-g of Prasa Mawaeng remedy was evenly spread on an aluminum sample pan and heated at 105°C. The instrument measured the loss in sample weight during the drying process, and the results were calculated based on the percentage reduction in weight before and after drying using the following the equation:

Preparation of Prasa Mawaeng decoction extraction method

Decoction is a boiling-based extraction method commonly used in Thai traditional medicine preparation (DTAM, 2021). Prasa Mawaeng powder (200 g) was wrapped in cotton cloth and soaked in 500 g of reverse osmosis water for 30 min at room temperature, corresponding to a powder-to-water ratio of 1:5. An additional 500 g of RO water was then added, and the mixture was boiled for 15 min. The resulting decoction was filtered through cotton and centrifuged at 25°C. The collected water extract was subsequently dried using two methods (spray drying and freeze drying, described below), and the yield of each dried powder was calculated as a percentage using the following equation:

Drying methods

The decoction extract of Prasa Mawaeng was subjected to spray drying and freeze drying using conditions adopted from a previously reported procedure with some modifications (Aryaee et al., 2023). For spray drying, the Yamato spray dryer (ADL311S, Yamato Scientific Co., Ltd., Tokyo, Japan) was operated at an inlet temperature of 120°C and an outlet temperature of 75–77 °C, with a feed rate of 6 mL/min, blower setting of 0.45 m³/min, and spray air pressure of 0.2 MPa. The freeze drying process was performed by using a freeze dryer (CHRIST Gamma 2-16 LSC, Osterode am Harz, Germany). The extract was frozen at −80°C for 24 h and subsequently lyophilized at −50°C under a pressure below 0.05 mbar for 72 h. After drying, both powders were stored in an appropriate desiccator for further analysis.

Field emission scanning electron microscope (FE-SEM)

The surface morphology of SD-PSM and FD-PSM powders was examined using an SEM Apreo S (Thermo Fisher Scientific, Waltham, USA). The powder was mounted on aluminum stubs and coated with a thin layer of gold and observed at 5,000x magnification with an accelerating voltage of 5.00 kV.

Phytochemical screening

Thin-layer chromatography profiling

Phytochemical screening of SD-PSM and FD-PSM was performed using thin-layer chromatography (TLC). For comparison, the parent was extracted according to THPP guidelines (DTAM, 2022). Briefly, 500 mg of the Prasa Mawaeng was extracted with 80% ethanol and sonicated for 40 min. Silica gel plates (Merck TLC Silica gel 60 F254) were used as the stationary phase (20 × 5 cm). The mobile phase consisted of n-butanol, ethyl acetate, and glacial acetic acid (10:7:3, v/v/v). Each sample (SD-PSM, FD-PSM, and parent) was applied at 6 µL per spot, and chromatographic distance was 17 cm. The plates were examined under white light and UV light at 254 and 366 nm, and the spraying of anisaldehyde, which was heated at 105°C, was examined under white light.

Determination of total phenolic content (TPC)

The total phenolic content (TPC) was determined using the Folin–Ciocalteu colorimetric method, modified from the protocol from Sembiring et al. (2018). The 25 μL of 0.5 mg/mL SD-PSM and FD-PSM were added to 96-well plates, followed by 25 μL of Folin-Ciocalteu reagent and 200 μL of water, and the mixture was incubated for 5 min in a dark room. Then, 25 μL of sodium carbonate and incubated for 1 h. and the absorbance was measured at 725 nm using the microplate reader. Measurement was performed in triplicate. The results of determination were expressed in terms of gallic acid equivalent (GAE).

ABTS radical cation decolorization assay

The antioxidant activity of the SD-PSM and FD-PSM was evaluated using the ABTS radical scavenging assay, following the method reported by Kerdphon et al. (2025). The ABTS radical was generated by mixing 1 μL of 7 mM aqueous ABTS with 1 μL of potassium persulfate (K₂S₂O₈). Incubated in a dark room at 4°C for 16 h until the development of a dark blue color developed. The absorbance was then adjusted to 0.70 ± 0.02 at 734 nm with water. SD-PSM and FD-PSM at 0.01–1,000 μg/mL (20 μL) were mixed with 180 μL of ABTS solution in 96-well plates, incubated for 6 min, and measured at 734 nm. Trolox (0.01-100 μg/mL) was used as a positive control. The measurement was performed in triplicate and expressed in terms of IC₅₀ value for inhibition of ABTS radical scavenging capacity.

Ferric reducing antioxidant power (FRAP) assay

The antioxidant activity was evaluated using the ferric reducing antioxidant power (FRAP) assay to assess the ability of the SD-PSM and FD-PSM to reduce ferric (Fe³⁺) to ferrous (Fe²⁺) ions. The FRAP reagent was freshly prepared following the protocol of Kerdphon et al. (2025) by mixing acetate buffer (300 mM, pH 3.6), 10 mM TPTZ in 40 mM HCl, and 20 mM FeCl₃ at a ratio of 10:1:1 (v/v/v). In each well of a 96-well plate, 20 μL of sample solution was mixed with 180 μL of FRAP reagent. The mixture was incubated at room temperature for 30 min. Absorbance was then measured at 595 nm using a microplate reader. FeSO₄·7H₂O was used to prepare the standard curve. All reagents and solutions were freshly prepared on the day of the experiment. Results were expressed as mg Fe²⁺ equivalent per gram of extract (mg Fe²⁺/g extract). Measurements were performed in triplicate.

Determination of anti-microbial activity by using the macrobroth dilution assay

To determine the minimum inhibitory concentration (MIC), the macrobroth dilution assay was used. The tested microorganisms included Staphylococcus aureus (S. aureus), Candida albicans (C. albicans), Escherichia coli (E. coli), and Streptococcus mutans (S. mutans). Each microbe was adjusted to 10⁵ CFU/mL and inoculated into tubes containing various concentrations of the SD-PSM and FD-PSM (0.5, 1, 2.5, 5, 7.5, and 15 mg/mL), with inoculated broth and sterile water acting as positive and negative controls, respectively.  The concentration was selected based on previously reported ranges for antimicrobial evaluation in individual plants (Latha and Kannabiran, 2006; Loganathan et al., 2024). The tubes were incubated at 35–37 °C for 24 h. For S. mutans, incubation was performed under anaerobic conditions. The MIC was defined as the lowest concentration of the test sample that showed no visible turbidity, indicating inhibition of microbial growth.

To determine the minimum bactericidal concentration (MBC) of the SD-PSM and FD-PSM, 10 μL from tubes showing no turbidity was then sub-cultured on tryptic soy agar (TSA) plates for 24 h at 35–37 °C. The lowest concentration of samples that showed no bacterial growth was considered as MBC. Each measurement was performed in triplicate.

Stability testing

The stability of SD-PSM and FD-PSM was evaluated in accordance with the guidelines of the International Council for Harmonisation (ICH). The samples were stored in a desiccator at room temperature (25 ± 2°C) for up to three months. For accelerated stability testing, samples were stored at 40 ± 2°C and 75 ± 5% relative humidity (RH) for one month. The desired humidity condition was maintained using a saturated sodium chloride solution. At each sampling time, samples were evaluated for changes in physical appearance and TPC. Physical characteristics, including color and powder texture, were visually inspected, while TPC was determined as a chemical stability marker. These results were measured in triplicate.

Statistical analysis

All experiments were performed in triplicate, and the results are expressed as mean ± SD (n = 3). Statistical analyses were performed using one-way ANOVA followed by Tukey’s post hoc test in GraphPad Prism version 6.0 (GraphPad Software, Inc., La Jolla, California, USA). Differences were considered significant at P < 0.05.

RESULTS

Identification of Prasa Mawaeng remedy

Plant identification can be achieved using microscopic characteristics, as distinct tissue structures differ characteristically across species. Microscopic identification of the Prasa Mawaeng remedy was performed as shown in Figure 1. The observed photomicrographs were compared with the descriptions provided in the THPP and relevant literature (DTAM, 2022). As observed in Figure 1A, the pericarp exhibited characteristic cuticular undulation, suggesting the possible presence of Solanum spp. components in the remedy (Matias et al., 2016). In Figure 1B, multicellular uniseriate trichomes, which are elongated structures composed of multiple cells arranged in a linear sequence, are suggested to be consistent with Curcuma spp. Additionally, two-armed multicellular hairs were identified in Figure 1C. These structures are characteristic T-shaped trichomes of Tarlmounia elliptica. Therefore, the microscopical characteristics identified in this study may represent reference standards for raw material quality control in future investigations.

Figure 1. Microscopic images of diagnostic structures observed in Prasa Mawaeng powder: Epidermis of the pericarp of Solanum spp. (A), multicellular uniseriate trichome of Curcuma spp. "Khamin Oi" (B), and T-shaped trichome of Tarlmounia elliptica (C). Arrows indicate the diagnostic structures used for identification.

Total ash, acid-insoluble ash, water content, and loss on drying of Prasa Mawaeng remedy

The physicochemical properties of the Prasa Mawaeng remedy were determined as part of quality control evaluation, and the results are presented in Table 2. The total ash, acid-insoluble ash, moisture content, and water content were 8.46 ± 0.05% w/w, 0.48 ± 0.06% w/w, 9.15 ± 0.07% v/w, and 5.96 ± 0.84% w/w, respectively. All evaluated parameters were within the limits specified by the THPP (DTAM, 2022). The pharmacopoeia acceptance criteria stipulate that total ash, acid-insoluble ash, and water content should not exceed 11.0, 1.0, and 10.0% w/w, respectively. Overall, these findings confirm that the Prasa Mawaeng remedy meets the required quality standards and is suitable for further physicochemical and biological analysis.

Table 2. Physicochemical parameters for the quality control assay of the Prasa Mawaeng remedy.

Physical appearance and process yield of SD-PSM and FD-PSM

The yields of SD-PSM and FD-PSM were 3.20% and 3.15%, respectively, suggesting that both drying methods produced comparable yields. These findings are consistent with reports where Rubrivivax gelatinosus also exhibited comparable yields under spray-drying and freeze-drying conditions (Santo et al., 2013). Regarding the extraction process, the aqueous extract of Ageratum conyzoides demonstrated that a shorter extraction time resulted in a lower yield compared to longer durations (Paulina et al., 2017). Moreover, using water as the extraction solvent produced a comparatively low extraction yield (Hlatshwayo et al., 2025). The aqueous extract of chamomile further indicates that this approach may result in relatively low yields (Lee et al., 2022). The low yield may limit initial development. However, industrial feasibility depends not only on yield but also on cost and product quality during optimization and scale-up (Acosta-Esquijarosa et al., 2009). Regarding physical appearance, SD-PSM was observed as a light-yellow, free-flowing powder, whereas FD-PSM was yellow-brown, had a distinct odor and showed a porous, flaky appearance, as shown in Figures 2A and 2B. The intense color and odor of FD-PSM were also observed in Artocarpus integer following freeze drying (Gopinathan et al., 2020). Therefore, these characteristics cannot be conclusively attributed to the drying method based on the current data. Further color and particle size analyses are required.

Figure 2. Physical appearance of SD-PSM (A) and FD-PSM (B), and SEM images of SD-PSM (C) and FD-PSM (D) at 5,000x magnification.

Morphological structure of SD-PSM and FD-PSM

The morphology of SD-PSM and FD-PSM was evaluated using SEM, and representative micrographs are shown in Figures 2C and 2D. The two drying techniques produced distinct powder morphologies. SD-PSM exhibited small, wrinkled, and hollow particles caused by rapid solvent evaporation and late-stage shrinkage (Goula et al., 2004; Gimbun et al., 2018). Conversely, FD-PSM displayed a larger, porous, sheet-like structure due to ice crystal sublimation at −80°C, creating microscopic voids that enhance rehydration (Nowak and Jakubczyk, 2020). Collectively, these results demonstrate that the drying mechanism directly dictates powder structure, aligning with morphological trends observed in Thymus serpyllum and sea cucumber powders (Jovanović et al., 2021; Aatab et al., 2023). Additionally, spray drying yields tenfold smaller particles than freeze drying, which consequently alters downstream handling and processing efficiency (Xin et al., 2022).

Phytochemical profiling of SD-PSM and FD-PSM

The presence of phytoconstituents in SD-PSM and FD-PSM was evaluated by TLC following the conditions described in the THPP (DTAM, 2022), using a mobile phase consisting of n-butanol: ethyl acetate: glacial acetic acid (10:7:3, v/v/v). Representative chromatograms and summary of TLC profiles are shown in Figure 3 and Table 3. The chromatographic profile of the parent was similar to that reported in the THPP. In contrast, SD-PSM and FD-PSM, which were prepared from the decoction method, exhibited chromatographic profiles that differed from that of the parent extract, reflecting the influence of extraction solvent polarity on phytochemical composition. Nevertheless, SD-PSM and FD-PSM displayed similar chromatographic patterns to each other, suggesting that neither spray drying nor freeze drying substantially altered the major phytochemical constituents of the remedy.

Figure 3. TLC chromatograms of the parent (80% ethanol extract of Prasa Mawaeng) (1), SD-PSM (2), FD-PSM (3), visualized under (A) white light, (B) UV irradiation at 254 nm, (C) UV irradiation at 366 nm, and (D) after spraying with anisaldehyde reagent.

Table 3. TLC fingerprint profile of parent (80% ethanol extract of Prasa Mawaeng), SD-PSM, and FD-PSM, showing the observed bands, colors, and corresponding Rf values.

According to the THPP, curcumin is employed as a reference marker for chromatographic identification. The parent, prepared using 80% ethanol following the THPP extraction procedure, exhibited a chromatographic profile consistent with that described in the pharmacopoeia (DTAM, 2022). A band at an Rf value comparable to that reported for curcumin was observed in all samples. The corresponding band appeared less intense in SD-PSM and FD-PSM than in the parent, which may relate to the low solubility of curcumin in aqueous systems compared to hydroalcoholic systems. Furthermore, without a reference standard or densitometric data, these qualitative TLC results do not confirm differences in actual curcumin levels.

Determination of total phenolics in SD-PSM and FD-PSM

Phenolic compounds are widely distributed in plants and are recognized for their potential health-promoting properties, particularly their ability to protect against oxidative stress and free-radical-mediated damage. The TPC was expressed as GAE. As shown in Table 4, SD-PSM and FD-PSM exhibited phenolic contents of 29.82 ± 2.32 and 26.57 ± 1.34 mg GAE/g extract, respectively, suggesting that the drying process did not markedly influence the phenolic content. These findings indicate that phenolic content remains stable under both high-temperature spray drying and low-temperature freeze drying (Xin et al., 2022). While spray drying efficiently preserves TPC at levels comparable to dehydrated plant standards (Warren-Walker et al., 2025), freeze drying is consistently reported to achieve similarly high phenolic retention (Vargas-Madriz et al., 2023). As plants contain hundreds of phenolics with varying reducing capacities, GAE serves as a standardized baseline for sample comparison. However, TPC may be overestimated because certain non-phenolic compounds such as vitamin C and glutathione can also react with the reagent (Pérez et al., 2023).

Table 4. Total phenolic contents and antioxidant activity of SD-PSM and FD-PSM.

Note: Values are presented as mean ± SD (n = 3). No significant differences were observed among the groups by one-way ANOVA followed by Tukey’s test (P > 0.05).

Evaluation of antioxidant activity in SD-PSM and FD-PSM

The radical scavenging activity of SD-PSM and FD-PSM was evaluated using the ABTS radical cation decolorization assay. As presented in Table 4, the IC₅₀ values of SD-PSM and FD-PSM were 246.97 ± 28.65 and 266.83 ± 8.63 μg/mL, respectively. The comparable IC₅₀ values may suggest that both drying methods did not markedly alter antioxidant activity, which appears consistent with the similar TPC after the drying processes. Ferric reducing antioxidant power (FRAP) was additionally determined to assess metal ion chelation capacity as a complementary antioxidant mechanism. SD-PSM exhibited a FRAP value of 129.07 ± 4.84 mg Fe²⁺/g extract, while FD-PSM showed a comparable value of 128.96 ± 6.46 mg Fe²⁺/g extract (Table 4). This consistency across both assays further suggests that neither spray drying nor freeze drying substantially diminished the antioxidant capacity of the remedy. The antioxidant activities observed in both SD-PSM and FD-PSM suggest that the decoction extract of Prasa Mawaeng remedy acts as a hydrogen donor, inhibiting oxidation by stabilizing free radicals (Aatab et al., 2023). The consistency of antioxidant performance across both drying methods supports the suitability of either process for preserving the bioactive potential of the remedy.

Antimicrobial activity of SD-PSM and FD-PSM

The MIC and MBC of SD-PSM and FD-PSM were determined to compare the effects of different drying methods on antimicrobial activity. The tested concentration range (0.5–15 mg/mL) was selected according to previous studies on individual plants, aqueous S. trilobatum exhibited MICs of 0.06–0.5 mg/mL (Latha and Kannabiran, 2006; Megala et al., 2025), and aqueous O. tenuiflorum inhibited S. mutans at 25–100 μg/mL (Loganathan et al., 2024). Furthermore, its key constituent, eugenol, targets S. aureus, E. coli, and C. albicans (Fajdek-Bieda et al., 2025). As presented in Table 5, the turbidity was observed across all tested concentrations (0.5–15 mg/mL) in the macrobroth dilution assay, indicating that samples derived from neither drying process exhibited detectable antimicrobial activity against the tested microorganisms. Assay validity was confirmed as the positive control showed microbial growth and the negative control remained sterile. Subculturing onto TSA plates further confirmed the absence of microbicidal action. Therefore, the MIC and MBC of both SD-PSM and FD-PSM exceeded 15 mg/mL against S. aureus, C. albicans, E. coli, and S. mutans, indicating insufficient antimicrobial activity within this concentration range.

Table 5. MIC and MBC values of SD-PSM and FD-PSM against test microorganisms, including positive and negative controls.

As the Prasa Mawaeng remedy has not been previously evaluated for antimicrobial activity, the findings were compared with those of its individual constituent plants. The aqueous extracts of S. trilobatum and O. tenuiflorum have demonstrated antimicrobial activity in previous studies. (Dasthageer et al., 2022; Loganathan et al., 2024; Megala et al., 2025). However, the present Prasa Mawaeng extract did not show detectable activity within the tested concentration range. This may be related to differences in extraction conditions and the recovery of antimicrobial constituents. (Adzhar et al., 2019). In particular, the relatively short decoction time may have limited the extraction of some potentially antimicrobial compounds such as eugenol and steroidal alkaloids. (da Silva et al., 2022; Sharma et al., 2024). In addition, aqueous extraction may provide lower antimicrobial activity than organic extraction (Witkowska et al., 2013; Sbhatu and Abraha, 2020). These factors may partly explain the lack of detectable antimicrobial activity in the present aqueous Prasa Mawaeng preparation.

Stability of SD-PSM and FD-PSM

The stability of SD-PSM and FD-PSM was evaluated under two storage conditions consisting of room temperature (25°C) for three months and under accelerated conditions (40°C/75% RH) for one month, using TPC as the chemical stability biomarker. The physical appearance after storage is displayed in Figure 4.  Both SD-PSM and FD-PSM showed no observable changes in visual appearance under room temperature conditions for three months (Figures 4A and 4B). In contrast, the two powders showed different physical stability under accelerated conditions (Figures 4C and 4D). SD-PSM maintained its powder form with only minor observable agglomeration. In contrast, FD-PSM showed marked physical changes, transforming into a dense, hardened mass indicative of severe particle aggregation. This difference in physical stability between the two powders may be associated with the highly porous and partially collapsed structure often observed in freeze-dried materials, which may increase their susceptibility to particle caking (Nowak and Jakubczyk, 2020).

Figure 4. Visual appearance of SD-PSM and FD-PSM after storage at room temperature for 3 months (A and B) and under accelerated conditions (40°C, 75% RH) for 1 month (C and D).

Figure 5. Total phenolic content of SD-PSM and FD-PSM under room temperature (25°C) for three months and accelerated conditions (40°C/75% RH) for one month. Values are presented as mean ± SD (n = 3). No significant differences were observed among the groups (P > 0.05).

Both SD-PSM and FD-PSM maintained stable phenolic contents under both tested storage conditions (Figure 5). At the end of the storage period, the room temperature TPC values were 28.12 ± 1.22 and 24.77 ± 0.97 mg GAE/g extract for SD-PSM and FD-PSM, respectively. Under accelerated conditions, the corresponding TPC values were 26.19 ± 3.93 and 25.94 ± 1.44 mg GAE/g extract. Statistical analysis confirmed that no significant changes in TPC occurred in either powder throughout the evaluation. This preservation is attributed to their distinct mechanisms, suggesting that rapid moisture evaporation promotes encapsulation during spray drying, thereby protecting the entrapped phenolics from oxidation (Swm et al., 2025), while freeze drying minimizes thermal degradation (Babaei Rad et al., 2025). Importantly, SD-PSM exhibited better physical stability during storage, suggesting its potential advantage for formulation development. Additionally, the lower production costs of the spray-dried powder potentially provide better processability during manufacturing, highlighting its practical advantages for industrial handling and large-scale development (Swm et al., 2025).

CONCLUSION

This study evaluated and compared the effects of spray drying and freeze drying on the phytochemical properties and biological activities of the Prasa Mawaeng remedy. Both drying methods produced comparable TLC fingerprints and TPC values, which were consistent with the comparable antioxidant activities observed in the ABTS and FRAP assays. These findings suggest that both drying methods are similarly effective in preserving phenolic compounds, thereby maintaining the antioxidant activity. Conversely, neither drying method exhibited antimicrobial activity within the tested range of 0.5–15 mg/mL. During stability testing, both methods exhibited no statistically significant difference in TPC values throughout storage. However, spray-dried powder demonstrated better physical stability under the tested accelerated condition because it retained a fine, free-flowing form, whereas freeze-dried powder clearly aggregated. Therefore, spray drying may be considered a promising drying approach for Prasa Mawaeng when physical stability and potential future scale-up are considered. A current limitation of this study is the short-term stability testing of only three months. Therefore, further studies should extend the storage duration to assess the long-term degradation rate of bioactive constituents. Importantly, the novelty of this study lies in being the first to investigate the biological activities of the Prasa Mawaeng remedy. The evaluation provides a practical basis for selecting the optimal drying process, supporting future industrial scale-up and innovative herbal product development.

ACKNOWLEDGEMENTS

The authors gratefully acknowledge the financial support from Naresuan University, the National Science, Research and Innovation Fund (NSRF) under Grant No. R2569B067, and the Frontier Research and Innovation Cluster Fund of Naresuan University under Grant No. R2569C007. The authors also thank the Faculty of Pharmaceutical Sciences, Naresuan University, for facilitating this research, and the Science Laboratory Center, Faculty of Science, Naresuan University, for assistance with SEM analysis.

AUTHOR CONTRIBUTIONS

Thanapohn Apirattanajaroensuk: Conceptualization (Lead), Methodology (Lead), Validation (Lead), Formal Analysis (Lead), Data Curation (Lead), Writing - Original Draft (Lead); Sasiprapa Limsirilak: Methodology (Equal), Writing - Review & Editing (Supporting); Nuntika Prommee: Methodology (Supporting), Validation (Supporting), Writing - Review & Editing (Supporting); Jira Jongcharoenkamol: Methodology (Supporting), Validation (Supporting), Visualization (Supporting), Formal Analysis (Supporting), Resources (Supporting), Writing - Review & Editing (Supporting); Srisagul Sungthongjeen: Resources (Lead), Supervision (Equal), Funding Acquisition (Equal); Worawut Kriangkrai: Conceptualization (Lead), Methodology (Lead), Resources (Lead), Writing - Review & Editing (Lead), Supervision (Lead), Project Administration (Lead), Funding Acquisition (Lead). All authors have read and agreed to the published version of the manuscript.

CONFLICT OF INTEREST

The authors declare that they have no conflicts of interest.

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