Propolis is rich in bioactive compounds, including polyphenols, with various biological activities. However, its application is limited by low water solubility, instability in aqueous environments and limited absorption within the body. A nanoparticle-based approach represents a promising strategy to overcome these limitations by enhancing the bioavailability, solubility, stability and absorption of these compounds. This study focuses on encapsulating propolis extract in chitosan nanoparticles, PE-NP and evaluating their physical characteristics and antioxidative properties.
Preparation of PE-NP
Chitosan, a weak base composed of D-glucosamine groups, is insoluble in neutral and alkaline pH solutions. However, in acidic environments, its amino groups are protonated, transforming chitosan into a water-soluble polysaccharide and a positively charged polysaccharide with high charge density, as each D-glucosamine unit carries a single positive charge (
Desai, 2016). This polysaccharide is widely used to prepare chitosan-based nanoparticles
(Alishahi et al., 2011; Kim et al., 2022; Kim et al., 2023). Chitosan rapidly forms a gel when interacting with polyanions, a process known as ionic gelation, which involves the establishment of inter and intramolecular cross-linking mediated by polyanions. In this study, chitosan-TPP nanoparticles, as the wall materials, are prepared using the ionic gelation technique. This process occurs through the electrostatic interaction between the polycationic chitosan and an anionic cross-linker, TPP. During nanoparticle preparation, TPP interacts with the amino groups of chitosan to form ionically cross-linked chitosan. Once mixed in dilute acetic acid, chitosan and STTP produced nanoparticles with a positive surface charge
(Alishahi et al., 2011; Kim et al., 2022). The parameters that can affect the characteristics of PE-NP include the molecular weight of the chitosan, the chitosan concentration, the pH and the encapsulant concentration (propolis extract).
Chitosan-TPP nanoparticles were produced as wall materials to encapsulate propolis’s phenolic compounds. These nanoparticles could form ionic and hydrogen bonds with phenolic compounds in propolis extract, as described by
Alishahi et al., (2011), which explained the mechanism of the interaction of vitamin C with chitosan-TPP nanoparticles. Moreover, encapsulants can be incorporated into nanoparticulate systems through three primary mechanisms: electrostatic interaction, physical encapsulation and surface adsorption
(Alishahi et al., 2011; Yang and Hon, 2009).
Physical characteristics
The formation of chitosan-TPP nanoparticles via the ionic gelation technique relies on multiple electrostatic interactions between the positively charged amino groups of chitosan and negatively charged TPP, resulting in an opalescent solution. An opalescent visual indicates nanoparticle synthesis
(Li et al., 2003). An opalescent suspension revealed turbidity of nanoparticle suspensions. The turbidity of nanoparticle suspensions was evaluated by measuring their light absorbance at 750 nm using a spectrophotometer, with higher absorbance values indicating higher suspension turbidity. The absorbance (or turbidity) values of PE-NP are presented in Table 1. The absorbance (or turbidity) values increased with increasing chitosan concentration, at both LMW and MMW chitosan. This increase is attributed to the enhanced electrostatic interactions between chitosan and its polyanion (TPP), which form more cross-links, resulting in a more opalescent solution and higher absorbance value. Nanoparticle suspensions prepared using LMW chitosan have lower absorbance (or turbidity) values than MMW chitosan, as shown in Table 1.
The pH levels of the nanoparticle suspensions in all treatment variations are 3.9-4.8 (Table 1). The pH of nanoparticle suspensions is influenced by chitosan concentration and the addition of propolis extract. A higher chitosan concentration decreases the pH values due to the addition of a more acidic chitosan solution. The pH is influenced by the acetic acid solution used to dissolve the chitosan. As the concentration of chitosan increases, an equivalent increase in the concentration of acetic acid is required, resulting in a proportional increase in the use of acetic acid and a decrease in the pH of the suspension. The addition of higher-volume extracts slightly decreases the pH values. Propolis extract contains caffeic acid, phenolic acid and their esters, which are weak acids (
Gregoris and Stevanato, 2010;
Zullkiflee et al., 2022). Chitosan-based nanoparticles effective at pH 4.5 provide better physical stability, particle size and distribution characteristics (
Kammona and Kiparissides, 2012).
Chitosan-TPP nanoparticles may interact with phenolic compounds present in propolis extracts by means of hydrogen bonding and ionic interactions. Table 1 presents the encapsulation efficiency of phenolic compounds in propolis extract loaded into the nanoparticulate system. The values range from 30.680-41.192% for nanoparticle suspensions using LMW chitosan and 23.937-36.350% for nanoparticle suspensions using MMW chitosan. The encapsulation efficiency values of this study tend to be low compared to other studies using different active compounds. Nanoparticles loaded with quercetin have an encapsulation efficiency of 48.5%
(Tan et al., 2011). Similarly, vitamin C-loaded nanoparticles achieved an encapsulation efficiency in the range of 30 to 40% when formulated with chitosan of MW 65, 90, 250 and 450 kDa and increasing to approximately 70% with chitosan of MW 110 kDa
(Alishahi et al., 2011). In addition, nanoparticles encapsulating polyphenolic compounds derived from
Rosa canina showed an encapsulation efficiency of 46%
(Stoica et al., 2013).
The encapsulation efficiency increases as the chitosan concentration increases, at both LMW and MMW chitosan. The more chitosan and TPP complexes formed, the greater the ability of nanoparticles to entrap or bond interaction with active compounds (
Nastiti and Siahaan, 2015). The encapsulation efficiency of nanoparticle suspensions using LMW chitosan is higher than that of MMW chitosan. These results are the same findings of
Alishahi et al., (2011);
Yang and Hon (2009). Chitosan with a lower molecular weight has shorter polymer chains, facilitating easier protonation of its free amino groups. This increased protonation capacity promotes greater adsorption of the phenolic compounds present in propolis extract, primarily through hydrogen or ionic bonding interactions
(Alishahi et al., 2011; Yang and Hon, 2009). The larger chitosan molecules lead to higher viscosity and thicker solutions, which can reduce the encapsulation efficiency
(Wu et al., 2005).
Antioxidative properties
The total phenolic content of PE-NP was quantified by a colorimetric assay using the Folin-Ciocalteu reagent, with results standardized to gallic acid equivalents (GAE). Fig 1 shows the standard curve plotted to estimate total phenolic content in the PE-NP with regression equation y = 9.6348x - 0.0009 and R² = 0.9978. The results obtained were expressed in µg GAE/ml of sample. Fig 2 displays the total phenolic content of PE-NP with the addition of 0.1, 0.2 and 0.3 ml propolis extracts. The propolis extract used in this study had a total phenolic content of 1.939 mg GAE/ml. The total phenolic content of nanoparticle samples with the addition of 0.1 ml propolis extract ranged from 21.049 to 23.021 µg GAE/ml, the addition of 0.2 ml propolis extract ranged from 37.188 to 40.976 µg GAE/ml and 0.3 mL propolis extract ranged from 54.625 to 59.711 µg GAE/ml, in both LMW and MMW chitosan. The total phenolic content was found to increase with the addition of more propolis extract. Another researcher also reported similar results (
Shahab-Navaei and Asoodeh, 2023), stating that adding more propolis extracts increased phenolic content in nanoparticles. The total phenolic content in nanoparticle samples using LMW and MWM chitosan at the same chitosan concentration have relatively the same values. Total phenolic content decreased slightly with increasing chitosan concentration. Chitosan does not contain phenolic compounds
(Nadia et al., 2014). Zullkiflee et al., (2022) stated that propolis contains many flavonoids and phenolic compounds, including phenolic acid. Crysin, pinocembrin, caffeic acid, caffeic acid phenethyl ester (CAPE), 1,1-dimethylallylcaffeate (DMAC) and galangin have been identified as the main phenolic constituents of propolis, with smaller amounts of apigenin, quercetin, kaempferol and naringenin also detected (
Gregoris and Stevanato, 2010). Propolis
Trigona sp. from Indonesia has a phenolic content of 104-498 μg/g and a flavonoid content of 0.405-129.265 μg/g
(Hasan et al., 2014). Another researcher also stated that propolis extract from Indonesia has a total phenolic content of 140.32 µg GAE/g and a total flavonoid content of 12.088 µg QE/g (
Hariyanto, 2017).
The DPPH assays were used to evaluate the antioxidant capacity of PE-NP, with results expressed as mmol of Trolox equivalent per ml of sample. The antioxidant capacity of nanoparticle samples with the addition of 0.1 ml propolis extract ranged from 0.0030 to 0.0032 mmol TE/ml, the addition of 0.2 ml propolis extract ranged from 0.0047 to 0.0051 mmol TE/ml and 0.3 mL propolis extract ranged from 0.0066 to 0.0072 mmol TE/ml, in both LMW and MMW chitosan (Fig 3). The findings demonstrate that a greater volume of propolis extract enhances the ability to neutralize free radicals, as evidenced by an increase in antioxidant capacity.
Shahab-Navaei and Asoodeh (2023);
do Nascimento et al., (2016) also explained the same results, namely that adding a higher amount of propolis extract increased the antioxidant capacity of nanoparticles. Increased chitosan concentration and using LMW and MMW chitosan resulted in not-too-different antioxidant capacity values.
Propolis is known to be rich in polyphenolic compounds, particularly flavonoids and phenolic acids. Besides flavonoids, propolis also contains aromatic acids and esters, aldehydes and ketones, terpenoids and phenylpropanoids, steroids, amino acids, polysaccharides and many other organic and inorganic compounds
(Zulhendri et al., 2021; Zullkiflee et al., 2022). The antioxidant capacity of PE-NP is attributed to the presence of phenolic compounds, especially flavonoids and phenolic acids. Previous studies have demonstrated that propolis extract or its fractions contain phenolic compounds capable of exerting antioxidant effects
(Cottica et al., 2015; Hasan et al., 2014). Additionally, a positive correlation between total phenolic content and antioxidant activity in propolis samples has been reported (
da Silva et al., 2006). However, other bioactive compounds, such as terpenoids, may also contribute to the overall antioxidant potential of propolis. Therefore, while phenolic compounds play a significant role in antioxidant activity, other compounds like terpenoids may also contribute and their influence should be considered. Antioxidants are generally divided into two categories based on their mechanism of action: preventive and chain-breaking. Phenolic compounds in propolis function as chain-breaking antioxidants by neutralizing peroxyl radicals involved in chain propagation. This reaction shortens the chain length and demonstrates antioxidant efficacy (
Shahab-Navaei and Asoodeh, 2023). Beyond its antioxidant properties, propolis exhibits significant antiviral, antibacterial, antifungal, antiparasitic, antiapoptotic, anticancer, antiinflammatory and antidiabetic activities (
da Silva et al., 2006;
Zulhendri et al., 2021; Zullkiflee et al., 2022).
Particle characteristics
PE-NP suspensions using LMW chitosan (concentrations of 0.1 and 0.2%) and MMW (concentration of 0.1%) with the addition of 3 mL propolis extract are selected for assay particle characteristics (size, PDI and zeta potential). These nanoparticle formulas have a stable appearance and better encapsulation efficiency. The suspensions remained stable and well-dispersed, with no aggregations or precipitation observed after seven days of storage. In addition, 0.3 mL of propolis extract has the highest antioxidant capacity. The results of particle characterization of PE-NP, including size, PDI and zeta potential, are presented in Table 2.
Nanoparticles generally have particle sizes below 1000 nm and particle sizes below 500 nm have better characteristics
(Buzea et al., 2007). The particle size of PE-NP ranged within the nanometric scale, measuring between 350.00 and 488.280 nm, with a particle size distribution or PDI varying from 0.305 to 0.482 (Table 2). Compared with the other research of
Kim et al., (2023), nanoparticles loaded with propolis extract, using chitosan and hyaluronic acid, resulted in particle sizes ranging from 357.5 nm to 378.3 nm, with the PDI ranging from 0.173 to 0.424. The results also revealed that increased chitosan concentration and molecular weight increased particle size and PDI due to nanoparticle suspensions that may produce aggregates. Hence, the particle size is relatively non-uniform and heterogeneous. Moreover,
Yang and Hon (2009) explained that using a higher molecular weight of chitosan resulted in a larger nanoparticle size. This is due to an increase in the viscosity, so particle formation is more difficult and implies the formation of larger particles. PE-NP suspensions have PDI values higher than 0.3 (Table 2) and are heterogeneous dispersion.
Kim et al., (2023) stated that a PDI value greater than 0.3 indicates heterogeneity, which may be attributed to the presence of chitosan aggregates. According to
Lu et al., (2011), the PDI with ranging values of 0-0.25 has a homogeneous size distribution. Moreover, the PDI can range between 0 and 1, with values close to 0 indicating a homogeneous dispersion system and above 0.5 indicating high heterogeneity
(Avadi et al., 2010). Nanoparticles with a particle size of 100-300 nm generally have a PDI of less than 0.3, while nanoparticles with more than 500 nm have a PDI of more than 0.5 (
NanoComposix, 2015). Nanoparticles with a homogeneous particle size distribution are more physically stable, so they do not cause particles to aggregate
(Avadi et al., 2010).
PE-NP suspensions have a positive zeta potential of 39.340 to 48.200 mV (Table 2), revealing that PE-NP is stable. According to
do Nascimento et al., (2016), a zeta potential with high absolute values, either negative or positive (±30 mV), indicates a stable suspension, as the repulsive forces between particles inhibit the aggregation of nanoparticles. The nanoparticle suspensions showed stability and dispersion, with no observed tendency towards aggregation. Moreover, dispersion with low zeta potential makes it easier for particles to interact and form aggregates (
NanoComposix, 2015). The increase in chitosan concentration and molecular weight also increased positive zeta potential. In contrast to previous findings by
Kouchak et al., (2012), insulin-loaded nanoparticles with a larger molecular weight of chitosan resulted in lower zeta potential values.
