Chitosan, food grade (average viscosity molecular weight of 796 kDa, degree of deacetylation 92%), was purchased from Merine Bio Resources Co., Ltd., Samutsakhon, Thailand. Mannitol, USP, was purchased from P.C. Drug Center Company Limited, Bangkok, Thailand. Curcumin, demethoxycurcumin, bisdemethoxycurcumin, ar-turmerone, sodium nitroprusside, and 1, 1-diphenyl-2-picrylhydrazyl (DPPH) were purchased from Sigma-Aldrich, Missouri, USA. All other chemicals used in the experiments were of analytical grade and purchased from RCI Labscan, Bangkok, Thailand. Turmeric extract, extracted with ethanol (96%), was obtained from the Pharmaceutical Laboratory Service Center, Prince of Songkla University, Songkhla, Thailand.

Spray-dried microparticles containing turmeric extract were prepared following the method described by Goëlo et al. (2020) with some modifications. The microparticles were designed with a theoretical curcuminoids loading of 20% (w/w). A wall material solution was prepared by dissolving chitosan or chitosan and mannitol, at a weight ratio of 1:3, in 1% v/v of acetic acid solution. A solution of turmeric extract in ethanol (96%) was dispersed into the encapsulating agent solution prior to being fed into a spray dryer. The microparticles were produced by using a Pilotech YC-015A Mini inert loop spray dryer (Shanghai Pilotech Instrument & Equipment Co. Ltd., China) with a standard 0.7 mm nozzle. The operating conditions used include a 15 mL/min solution flow rate, a 150 °C or 160 °C inlet temperature and air blower 35 Hz. Dry particles were packaged in airtight low-density polyethylene bags, covered with aluminum foil at 4 °C before testing.

The product yield was determined as the ratio of the weight of spray-dried microparticles to the weight of raw materials (turmeric extract, chitosan, and mannitol) by Equation 1 (Goëlo et al. 2020).

$\text{Product yield}(\%)=\frac{\text{Weight of microparticles}}{\text{Weight of raw materials}}\times 100$ (1)

The characteristics of the spray-dried microparticles, including particle size and surface morphology, were analyzed using a scanning electron microscope (SEM) (Quanta 400, FEI Company, USA). By assuming the particles were spherical, their diameters were measured using ImageJ software. This analysis was conducted by randomly choosing 150 particles from three distinct SEM images per sample (O’Toole et al. 2012). In addition, the volume diameter and zeta potential of the microparticles dispersed in Milli-Q water were determined using a Spraytech instrument (Malvern Instrument, UK) and a Zetasizer (Malvern Instrument, UK), respectively. The size distribution, or SPAN, was calculated using Equation 2, with D10, D50, and D90 obtained from cumulative size distribution at 10%, 50%, and 90%, respectively.

$\text{SPAN}=\frac{D90-D10}{D50}\times 100$ (2)

Infrared spectra of the microparticles and ingredients were recorded using FT-IR spectrophotometry (Spectrum One, Perkin Elmer Ltd., USA) and DSC curves were analyzed using a differential scanning calorimeter (DSC 8000, Perkin Elmer Ltd., USA).

Four biomarkers, including curcumin, demethoxycurcumin, bisdemethoxycurcumin, and ar-turmerone, in the spray-dried microparticles were quantified by high-performance liquid chromatography (HPLC) following the method described by Tanmanee et al. (2023). The samples were extracted using methanol and sonicated for 30 min, then centrifuged at 6,000 rpm for 10 min. The supernatants were collected and filtered through a 0.20 µm membrane filter (Whatman, USA). The HPLC analysis was performed on a Shimadzu LC-20AD system equipped with an Inertsil^® ODS-3 C18 column (4.00 mm × 3.0 mm, 5 µm, Phenomenex security guard® ODS). The column temperature was maintained at 40 °C, and the UV detector was set at 254 nm. The flow rate was 1.0 mL/min, and the injection volume was 20 µL. The mobile phase consisted of a binary eluent of 0.1% formic acid solution (A) and CH₃CN (B) under flowing gradient conditions.

The total curcuminoids content was calculated as the sum of the three individual curcuminoids, while ar-turmerone served as a biomarker representing the essential oil content in turmeric. Encapsulation efficiency was calculated using Equation 3.

$\text{Encapsulation efficiency}(\%)=\frac{\text{Practical biomarker content}}{\text{Theoretical biomarker content}}\times 100$ (3)

The quality of the product was assured by assessing its moisture content and microbial attributes, including total aerobic microbial count, total yeast and mold count, bile-tolerant gram-negative bacteria, Salmonella species, Escherichia coli, Staphylococcus aureus, and Clostridium species, as described by Thai Herbal Phamacopoeia (THP) (Thai Pharmacopoeia Committee 2021). Heavy metals such as arsenic, cadmium, and lead were analyzed using an Inductively Coupled Plasma Mass Spectrometer (Perkin Elmer, NexION2000, USA), while mercury levels were measured by the Direct Mercury Analyzer (NIC MA3000, Japan). Additionally, the active phytoconstituents of the microparticles were determined after storing them at 30 °C with 75% relative humidity for 6 months in an airtight, light-resistant container.

The release profiles of spray-dried microparticles and turmeric extract were examined using the USP Dissolution Apparatus II paddle method at a rotation speed of 100 rpm, according to Wan et al. (2012). For the experiments, samples containing the equivalent of 10 mg of curcuminoids were immersed in 900 mL of 0.1 M hydrochloric acid solution with 0.05% w/v polysorbate 80, maintained at 37 ± 0.5 °C. At specific intervals, 2 mL of samples were withdrawn and immediately replaced with an equal volume of the medium. These samples were then filtered through a 0.45 µm filter. The concentration of curcuminoids in the samples was determined by measuring absorbance at 420 nm using UV-Vis spectrophotometry, following the protocol outlined by the THP (Thai Pharmacopoeia Committee 2021). The results, expressed as cumulative release of curcuminoids over time, were plotted to illustrate the in vitro release pattern, with data presented as mean ± SD (n = 6). Additionally, the release data were analyzed using the zero-order equation (Q = k₀t), the first-order equation (Q = 1 − exp (−k₁t)), the Higuchi equation (Q = k_Ht ½), and Korsmeyer-Peppas equation (Q = k_KPtⁿ) where Q refers to the fraction of drug released at a particular time, t. The release kinetic constants obtained from the linear curves of the zero-order, first-order, Higuchi, and Korsmeyer-Peppas models are represented by k₀, k₁, k_H, and k_KP, respectively (Dash et al. 2010).

The acute toxicity study followed the guidelines of the Organization for Economic Co-operation and Development (OECD) 425, using the up-and-down method (Organization for Economic Co-operation and Development 2008). Ten female Wistar rats (Rattus norvegicus), weighing 200–250 g and aged 8 weeks, were used for this study. The rats were procured from Nomura Siam International, Thailand, and housed at the Laboratory Animal Center, Prince of Songkla University, Thailand. All rats were acclimated for 7 days before the start of the experiment. The conditions for raising the rats included a temperature of 22 ± 3 °C, a relative humidity of 55% ± 10%, light intensity ranging from 130 to 325 lux, a lighting period of 12/12 h (bright/dark), and being provided with filtered water through a reverse osmosis system. After acclimation, the rats were orally administered a single dose of 2,000 mg/kg of microparticles. The microparticles were dispersed in a mixture of water and glycerol with a ratio of 3.6:1.4. The concentration of microparticles was adjustable; if rat mortality occurred, the dose would be reduced to 1,750, 550, 175, and 55 mg/kg, respectively. Following microparticle administration, rat mortality and clinical toxic signs were closely monitored for 14 days. On the final day of the experiment, all rats were euthanized using 120 mg/kg of sodium thiopental after a period of fasting. Blood samples were collected through cardiac puncture for biochemical evaluation, including complete blood count (CBC), liver function (alanine aminotransferase (ALT), alkaline phosphatase (ALP), aspartate aminotransferase (AST)), and kidney function (creatinine, blood urea nitrogen (BUN)). Organs, including the liver, kidney, and heart, were weighed and collected in 10% formalin for histopathological analysis using H&E staining to assess potential side effects or changes induced by the microparticles in vital organs.

Ethical authorization on the use of experiment animals for this study was issued by the Institutional Animal Care and Use Committee, Prince of Songkla University, Thailand, with Ref. AR004/2023.

The spray-dried microparticles appeared as an orange-fine dry powder with a mild turmeric odor. The wall material and operating conditions notably influenced the product yield. Utilizing a blend of chitosan and mannitol as wall materials at an inlet temperature of 150 °C successfully encapsulated turmeric extract, resulting in a higher product yield (56.40%) compared to using chitosan alone (40%). The product yield of microparticles employing a blend of chitosan and mannitol at an inlet temperature of 160 °C was 31.72%. The decrease in yield at a higher inlet temperature could be attributed to the evaporation of turmeric essential oil during the spray-drying process. These findings corroborate previous studies indicating the product yield and characteristics of microparticles depend on the wall materials and operating parameters of spray drying (O’Toole et al. 2012; Cano-Higuita et al. 2015; Goëlo et al. 2020). In earlier investigations, the inlet temperature for the preparation of spray-dried powder containing turmeric, turmeric oleoresin, and curcumin varied in the range of 100 °C to 180 °C. Remarkably, the blending of wall materials significantly increased the percentage yield compared to using a single wall material (Cano-Higuita et al. 2015).

The optimized spray-dried microparticles, utilizing a blend of chitosan and mannitol as wall materials, exhibited a spherical shape, a non-smooth surface, and an average size of 4.02 ± 0.24 µm, as shown in Fig. 1.

Figure 1. 

Morphology of spray-dried turmeric extract powder, SEM images 1,000× (A); 10,000× (B).

The volume diameter and size distribution (SPAN) of the optimized microparticles (18.62 ± 0.76 µm and 3.31 ± 0.70, respectively) were smaller than those of the microparticles made with chitosan alone (23.32 ± 1.10 µm and 4.46 ± 0.68, respectively). A lower SPAN value indicates a more uniform particle size distribution. These findings suggest that the optimized microparticles have a broad size distribution, though it is narrower than that of microparticles using only chitosan. The microparticles had a zeta potential of +30.5 ± 0.2 mV, indicating a significant energy barrier against particle coalescence, thereby ensuring stability.

The FT-IR spectra of the microparticles and their ingredients are shown in Fig. 2. The FT-IR spectra of the microparticles exhibited strong peaks in the 1500–1600 cm⁻¹ range and a weak peak near the 1400 cm⁻¹ region, attributed to asymmetric and symmetric carboxylate anion stretching, respectively. The disappearance of the carbonyl stretching peak at 1651 cm⁻¹ (amide I peak) of chitosan and the appearance of a new peak at 1625 cm⁻¹, attributed to asymmetric NH₃⁺ bending, were observed (Parize et al. 2012). The FT-IR spectra of the turmeric extract revealed the hydroxyl group stretching region between 3200–3600 cm⁻¹. The change in this absorbing band in the microparticles suggested hydrogen bonding interactions (Azkia et al. 2020), confirming the successful encapsulation of the turmeric extract within the microparticles.

Figure 2. 

FT-IR spectrum of turmeric extract, chitosan, mannitol and spray-dried microparticles of turmeric extract.

The DSC thermograms of the microparticles and their ingredients are shown in Fig. 3. The glass transition temperature of chitosan was not observed in the thermogram. The curve of the turmeric extract showed two endothermic peaks at 112 °C and 155 °C, which might be attributed to curcuminoids and other phytoconstituents present in the turmeric extract. Endothermic peaks of curcuminoids around 166–176 °C have been reported (Opustilová et al. 2023). Mannitol exhibited a sharp peak at 170 °C due to its α and β polymorphs (Milenkova et al. 2024). The appearance of a small peak at 165 °C in the DSC curve of the microparticles indicated mannitol in a low percent crystallinity form. The disappearance of the turmeric extract peaks suggested that the encapsulated phytoconstituents in the microparticles exist in an amorphous state.

Figure 3. 

DSC curves of turmeric extract, chitosan, mannitol and spray-dried microparticles of turmeric extract.

The microparticles were produced with encapsulation efficiencies of 85.25% for curcuminoids and 38.71% for ar-turmerone. The blending of chitosan and mannitol as wall materials successfully encapsulated both curcuminoids and ar-turmerone in the microparticles at a ratio of approximately 4:1. The physical, chemical, and microbial analysis results for the spray-dried microparticles containing turmeric extract used in the present study are presented in Table 1. The rising global consumption of natural food products and herbal medicines has heightened safety concerns in several regions. These concerns are primarily related to microbial contamination and the presence of heavy metals. They may be exposed to microorganisms from the soil, water, and inappropriate handling or storage. Heavy metals are commonly found in herbal medicine products in concentrations exceeding the permitted limits (Alharbi et al. 2024). Notably, lead, a potent neurotoxin, was detected as a contaminant in turmeric from Bangladesh and South Asia due to the addition of lead chromate (PbCrO4), a yellow pigment used to enhance brightness (Forsyth et al. 2019). In this study, the moisture content, microbial levels, and heavy metal contamination in the microparticles were found to be within the acceptable limits for herbal preparation for oral administration as per the standard of the Thai FDA.

The microparticles were physicochemically stable after storage at 30 °C with 75% relative humidity for 6 months, with the remaining percentage of curcuminoids, and ar-turmerone at 93.54 ± 1.73% and 94.98 ± 4.43%, respectively. These findings suggest that using a blend of chitosan and mannitol as wall materials enhances the stability of turmeric extract more effectively than using chitosan alone, where the content of curcuminoids was lower than 90%.

The correlation coefficients for the zero-order, first-order, and Higuchi models were 0.8688, 0.8794, and 0.9320, respectively. The release profile of the spray-dried microparticles was fitted to the Korsmeyer-Peppas equation, with a correlation coefficient of 0.9598, indicating a diffusion and erosion mechanism. Analysis of the release profile in simulated gastric fluid revealed that the microparticles increased the release rate and curcuminoid content by about 6 times compared to the turmeric extract, as shown in Fig. 4. This finding indicates that curcuminoids within the microparticles were in an amorphous state, consistent with the DSC findings. This finding aligns with previous research emphasizing the synergistic effects between turmeric oil and curcuminoids in turmeric formulations with enhanced bioavailability (Antony et al. 2008; Henrotin et al. 2013; Aggarwal et al. 2016). Specifically, the inclusion of turmeric oil has been shown to increase the solubility of curcumin in duodenal conditions by about 7.5 times compared to native curcumin (Henrotin et al. 2013).

Figure 4. 

Release profiles of spray-dried microparticles containing turmeric extract and turmeric extract. (Mean ± SD, n = 6).

The antioxidant capacities of the spray dried powder, turmeric extract, and curcumin, expressed as half maximal inhibitory concentration (EC₅₀) values, are shown in Table 2. The scavenging of nitric oxide and DPPH by the spray-dried microparticles and turmeric extract increased in a dose-dependent manner and demonstrated lower inhibitory efficacy compared to pure curcumin. These may be attributed to individual curcuminoids, essential oils, and other constituents in turmeric and microparticles that affect free radical scavenging activity (Sreejayan and Rao 1997; Zhang et al. 2017).

Although chitosan and mannitol are generally used as excipients in oral pharmaceutical formulations and food supplements, the spray-dried microparticles encapsulated with turmeric extract possess new properties such as increasing the dissolution of curcuminoids, which may impact their safety.

The weights of rats on days 1, 3, 7, and 14, which were 204.50 ± 2.83 g, 213 ± 3.00 g, 230 ± 4.41 g, and 245 ± 4.59 g, respectively (Fig. 5A). The average growth rate over the 14 days increased by 8.89% (Fig. 5B), and there were no significant differences in the variance of the average body weight among the rats (p > 0.05). A comparison of the growth rate data with the data from Nomura Siam International, Thailand, showed a growth rate of 6.34%. This indicates that the administered microparticles did not induce abnormalities in the growth rate.

Figure 5. 

Effects of spray-dried microparticles containing turmeric extract on bodyweight (A) and growth rate (B). (Mean ± SEM, n = 10).

After administering the microparticles to the rats and raising them for 14 days, internal organs including the heart, kidneys, liver, spleen, and ovaries did not exhibit any abnormalities. The organ-to-bodyweight ratio (relative organ weight) is presented in Table 3. There were no significant differences in the variance of the organ-to-bodyweight ratio among the rats (p > 0.05). Therefore, the microparticles did not induce abnormalities in internal organs. On the final day, no mortality was observed among the rats. The microparticles were classified as category 5, indicating low-dose toxicity according to the Globally Harmonized System for Classification and Labelling of Chemicals (GHS). The 50% lethal dose (LD50) of the microparticles was estimated to be between 2,000 and 5,000 mg/kg, suggesting their safety.

Liver injury associated with turmeric appears to increase when combined with other ingredients, such as black pepper, that enhance the absorption of curcumin (Halegoua-DeMarzio et al. 2023). Importantly, turmeric products with high bioavailability may potentiate liver injury. Table 4 illustrates the liver and kidney functions of rats treated with microparticles. Microparticles did not affect liver function, as indicated by ALT, ALP, and AST levels within the reference range, according to the study by Giknis and Clifford (2008). However, at a dose of 2,000 mg/kg, microparticles affected the creatinine level, which was 0.52 ± 0.04 mg/dL, exceeding the reference range by 4.0% (Giknis and Clifford 2008). The histopathology results of rats administered 2000 mg/kg of microparticles (Fig. 6) were observed under a light microscope at magnifications of 4×, 10×, and 40×. The liver, kidney, and heart tissues were stained using H&E staining, and no abnormalities were found in any of these tissues. Creatinine level is a normal waste product that accumulates in the blood due to muscle use, indicating the kidney’s healthy status (Khorsandi and Orazizadeh 2008). Kidney issues lead to increased creatinine levels and decreased urine output. Upon considering both the creatinine levels and the renal histopathology of rats, no abnormalities were observed in the renal architecture, including the glomeruli and tubules. Therefore, the administration of 2000 mg/kg of microparticles mildly increased creatinine levels but did not significantly impact renal architecture. It is suggested to further focus on kidney function and extend the study period for more comprehensive insights.

Figure 6. 

The histology and specimen of the organ changed in rats treated with spray-dried microparticles.

Table 5 illustrates that rats administered 2000 mg/kg of microparticles did not significantly affect hematology, except for a mild reduction in platelet count to 672.63 ± 52.07 × 10¹²/L, which decreased by approximately 1.17% from the reference range. A decrease in platelet count may result from bone marrow dysfunction or other causative factors, leading to decreased platelets in specific diseases such as idiopathic thrombocytopenic purpura (ITP) and systemic lupus erythematosus (SLE) (Rasmy et al. 2015). The severity of thrombocytopenia is classified into five levels according to the National Cancer Institute Common Terminology Criteria for Adverse Events (2010), ranging from 1 to 5, representing normal to the most serious conditions. In the treated rats, the platelet count was at level 2, indicating a condition that does not pose significant harm to the body (Sekhon and Roy 2006). Hence, it is recommended to further investigate the platelet count in subsequent studies.