Effect of microencapsulation on antioxidant activities of Eugenia punicifolia (Kunth) DC hydroethanolic extracts

Abstract

Eugenia punicifolia (Kunth) DC (Myrtaceae) is a folk medicinal plant in the Brazilian Cerrado with antioxidant, anti-inflammatory, antinociceptive, antiulcerogenic activities, etc., usually attributed to its phenolic compounds. Since these compounds are sensitive to heat and light, and to increase their applications, Hydroethanolic Extracts E. punicifolia (HEEP, EtOH:H₂O 70% v/v) were encapsulated by freeze-drying in xanthan gum (mesh 80, HEEPX80; mesh 200, HEEPX200) in ratio 1:1(w/w). Flavonoids had the highest encapsulation efficiency in HEEPX80, with a total flavonoid content of 55.56%. The release profile at different pH levels showed that pH = 4.5, a relevant antioxidant activity for HEEPX80 and HEEPX200. Also, in HEEP-modified release, higher antioxidant activity was observed in more acidic media (pH 4.5) than in a more neutral medium (pH 7.4). From these results, we could infer that HEEP encapsulations with Xanthan gum could be a good alternative for preserving antioxidants in these extracts.

Key words Eugenia punicifolia; freeze-drying; microencapsulation; xantham gum

INTRODUCTION

Eugenia punicifolia (Kunth) DC. (Myrtaceae), known as “pedra-ume caa”, is a shrub widely distributed in the Brazilian Cerrado, and their leaves have been traditionally used to treat diseases such as diabetes, fever, flu, etc. (Santos et al. 2023). Hydroethanolic extracts E. punicifolia (HEEP, EtOH:H₂O 70% v/v) have already been described by our research group as antioxidant, anti-inflammatory, antinociceptive, and gastroprotective (Basting et al. 2014, Périco et al. 2022). HEEP bioactive properties have been correlated to the phenolic compounds, such as phenolic acids, quercetin, myricetin, and hexahydroxydiphenoyl derivatives (HHDP) (Basting et al. 2014, Costa et al. 2016, dos Santos et al. 2020, Périco et al. 2022, Ribeiro et al. 2022). This phenolic compound set in HEEP is attractive since it may raise other pharmaceutical, food, and cosmetic uses. Phenolic compounds, as found in HEEP, are also known for their poor solubility in water and low permeability due to the absence of specific receptors at the small intestinal epithelial cells’ surface (Dias et al. 2015, Heleno et al. 2015). Also, these compounds are oxidizable and unstable to light, heat, oxygen, or processing conditions (pH, temperature, storage time, etc.). Moreover, HEEP extracts solubility, stability, bioactivity, and bioavailability will determine whether they can have industrial applications. For these reasons, various technologies have been established to stabilize these phenols; among them, encapsulation stands out (Cao et al. 2021, Esfanjani & Jafari 2017).

Encapsulation can improve phenolics’ stability and preserve their bioactivity. Microcapsules can provide a controlled phenolic release in different pH values, increase solubility in water, and cover unwanted odors and tastes (Pérez-Córdoba et al. 2018, Tessaro et al. 2021). The choice of encapsulation technique focuses on defining how bioactive compounds will be released, the places where the bioactive need to start their action, the time it will take to start, and the duration of the release. For instance, microcapsules can ensure bioactive components will be targeted to a specific part of the digestive tract, and their release can be controlled (Grgić et al. 2020). Polysaccharides represent the largest category of materials used as encapsulating agents due to their many advantages, such as their biocompatibility, biodegradability, and low toxicity and gelling capacity (Yahoum et al. 2023). Among these biopolymers, Xanthan gum is one of the most common materials used to encapsulate phenolic compounds (Grgić et al. 2020) because this polysaccharide is a nontoxic, abundant, biocompatible, and biodegradable polymer (Raschip et al. 2023). Xantham gum is an extracellular anionic polysaccharide produced by Xanthomonas campestris. It has high water solubility, broad pH (2.5 to 11), temperature stabilities (10°C to 90°C), and the viscosity is little affected by the presence of salts. This gum is used in the food industry as a thickening and stabilizing agent (Kumar et al. 2018). It has also been used to encapsulate enzymes, microorganisms, and phenolic compounds (Grgić et al. 2020, Zabot et al. 2022). Among the available encapsulation techniques, freeze-drying is the most efficient technique to preserve phenolic compounds from chemical decomposition due to its low processing temperature. This technique also allows better maintenance of the activities of the extracts of interest despite this process’s time and energy consumption (da Rosa et al. 2013).

Considering this, this work aimed to present the results of the assessment of hydroethanolic extract E. punicifolia (HEEP) EtOH:H₂O 70% (v/v) microparticles by freeze-drying, using xanthan gum of two different size particles (80 and 200 Mesh) as encapsulating agent. It also evaluated the content of phenols, flavonoids, Encapsulation Efficiency (EE%), antioxidant activity, morphology by Scanning Electron Microscopy (SEM), and functional groups by Fourier Transform Infrared Spectroscopy (FTIR) of the microencapsulated material.

MATERIALS AND METHODS

Drugs and chemicals

HPLC analytical standards, such as gallic acid, ellagic acid, quercetin, and myricetin, as well as 2,2-diphenyl-1-picrylhydrazyl (DPPH), were purchased from Sigma-Aldrich (St. Louis, MO, USA). Xanthan gum (80 and 200 Mesh, food grade) was provided by Cotia Foods (Cotia-SP, Brazil). Other analytical-grade reagents were purchased locally.

E. punicifolia hydroethanolic extracts (HEEP) characterizations and activity

Eugenia punicifolia leaves were collected in December 2015 at the Instituto Florestal e Estações Experimentais—Floresta Estadual de Assis at 22°33’ to 22°37’ Lat. S–50°21’ to 50°24’ Long W, Assis, State of São Paulo, Brazil. Dr. Antônio C. G. Melo identified the specimen, and a voucher (no. 43.522) was deposited in the Herbarium D. Bento Pickel, São Paulo, SP, Brazil, in compliance with legal norms and registered as Cotec 206108-005.298/2009.

For extract preparation, 10 g of crushed, dry leaves were moistened with EtOH: H₂O 70% v/v (1:10 plant/solvent ratio) and dynamically macerated for 2h at room temperature (25±2°C). After this time, the solvent was filtered off. The maceration was repeated two more times from the residue. After filtration, these combined solutions were concentrated in a rotary evaporator, followed by freeze-drying (Liotop, L-101, Liobras, Brazil) of the aqueous residue to provide the extracts studied in this manuscript.

HEEP Polyphenol (TPC) and Flavonoid Content (TFC)

HEEP total polyphenol content was determined by using Folin-Ciocalteu’s assay. Thus, Folin–Ciocalteau (2.5 mL, 10% v/v) and sodium carbonate (2.0 mL, 4% w/v) solutions were added to 0.5 mL of ethanolic solution of HEEP (1.0 mg/mL w/v), followed by thorough mixing, keeping for 120 min in the dark, at room temperature (rt), and measure of absorbance at 750 nm. A calibration curve (0.5–40 µg/mL) of gallic acid was used to express the results as mg gallic acid equivalent (GAE)/g dry extract (Kalia et al. 2008).

HEEP total flavonoid content was determined by the aluminum chloride colorimetric assay with modifications. 1.5 mL of absolute ethanol, 0.1 mL 10% (w/v) aluminum chloride, 0.1 mL potassium acetate 1M, and 8.0 mL distilled water were added to 0.5 mL ethanolic solution of HEEP (1.0 mg/mL). After homogenization, the solution was kept for 30 minutes in the dark at rt. So, the absorbance was measured at 425 nm. Quercetin was used as the standard, and a calibration curve (0.5–40 µg/mL) was used to express the results expressed mg of quercetin equivalent (QE)/g of dry extract (Kalia et al. 2008). All experiments were carried out in triplicate.

Quantification of phenolics from HEEP by UPLC-PDA

The chromatographic analysis was performed in a Waters ACQUITY UPLC Photodiode Array (PDA) chromatograph controlled by Empower 2 Chromatography Data Software (Waters Corporation, Milford, MA, USA). The chromatographic conditions were adapted from the methodology validated in the literature (Pires et al. 2017).

The phenolic compounds were separated using a reverse-phase gradient system using ultra-performance liquid chromatography (UPLC). A chromatographic column (250 mm x 4.6 mm) with C18 reverse phase (4 μm particle diameter, Phenomenex) and a guard column were used. The mobile phase consisted of orthophosphoric acid solution (0.1% w/w) as solvent A and acetonitrile as solvent B, in the elution conditions: 90-80% A and 10-20% B (0-5 min); 80-75% A and 20-25% B (5-35 min); 75-0% A and 25-100% B (35-60 min), a flow rate of 0.8 mL/min, at 25 °C, and injected volume of 10 μL. The wavelengths scanned were 220 nm and 254 nm, and room temperature (20 ± 2^oC) was monitored during all chromatographic runs.

The standard curves for gallic acid, ellagic acid, myricetin, and quercetin were calculated by linear regression from 0.1000 to 1000 µg/mL. A stock solution for each standard was prepared at 0.1 mg/mL in MeOH: water (1:1 v/v), diluted (0.01 to 1.0 µg/mL), and filtered through the PTFE membrane filter (0.22 μm). 2.7 µg of HEEP dry extract dissolved at 1.0 mL methanol HPLC grade was used for HEEP injection. The studied compounds’ HEEP samples were identified by comparing their retention times and UV absorption.

Spectrum with those of the standards.

The limits of detection (LOD) and quantification (LOQ) were determined by the signal-to-noise ratio (S/N) ratios of 3:1 and 10:1, respectively. The extraction efficiency and the analytical method were evaluated by performing recovery tests (triplicate, with the optimized parameters).

HEEP Antioxidant Activity

HEEP antioxidant activity was measured through the scavenging ability radical DPPH (2,2-diphenyl-1-picrylhydrazyl) according to Enujiugha et al. with modifications (Enujiugha et al. 2012).

To 2.0 mL of the ethanolic solution of HEEP (1.56 to 200 µg /mL) was added 0.5 mL DPPH 0.03% in methanol, in triplicate. After that, the samples were stirred and kept in darkness at rt for 30 min. The absorbance was determined at 517 nm. Methanol was the negative control, and quercetin and gallic acid were the positive controls. The percentage of DPPH radical scavenging activity of the extract was calculated according to the following Equation (1):

$$\mathrm{\%}\text{Inhibition}=(1-\frac{Ab{s}_{sample}}{Ab{s}_{control}})\ast 100$$ (1)

Abs_sample is the HEEP’s absorbance measured to the different concentrations, and Abs _control is the absorbance of methanol in DPPH (blank).

Equation 1 results were plotted as a graphic of % DPPH inhibition versus concentration using MATLAB (R2012, The MathWorks, Inc., Boston, MA, USA). From this graphic, the HEEP concentration necessary to decrease the initial concentration of the DPPH radical by 50% (EC₅₀) was measured and expressed in µg/mL.

E. punicifolia hydroethanolic extracts encapsulated with Xanthan gum (HEEPX) characterizations and activity.

The HEEP/Xanthan gum microencapsulations (Mesh 200 and Mesh 80, Cotia Foods) were done in a 1:1 (w/w) ratio according to the method described in the literature (Laine et al. 2008, Pralhad & Rajendrakumar 2004).

A solution of 100 mg of HEEP, dissolved in 20 mL of water, was dripped with a burette into a xanthan gum solution (100 mg of gum/20 mL of water), with constant stirring for three h and the protection of light. After this time, subsequently being subjected to freeze-drying (Liotop, L-101).

Encapsulation Efficiency (%EE)

For calculating encapsulation efficiency (EE%) (Equation 2), the first step was quantifying the total phenolic content of the HEEPX microcapsule. Aliquots of 25 mg of HEEPX80 or HEEPX200 in 10 mL of water were sonicated for 15 min, 20 Hz (Quimis 3335D, Diadema, Brazil), and centrifugated (Kasvi k14-0815C, São Jose dos Pinhais, Brazil) at 3420 g for 10 min. The supernatant was filtered through a 0.45 μm PTFE filter, and its phenolics quantification was measured by Folin-Ciocalteu’s assay in triplicate to express the results as mg gallic acid equivalent (GAE)/g xanthan gum (Kalia et al. 2008).

Similarly to the first step, the surface phenolic compounds on the HEEPX were also quantified. 25 mg of HEEPX80 or HEEPX200 was added to 10 mL methanol, vortexed, and centrifuged at 3420 g for 10 min. The solution was not sonicated, only filtrated. In this step, Folin-Ciocalteu’s assay was used to quantify the phenolics in the filtrate in triplicate to express the results as mg gallic acid equivalent (GAE)/gxanthan gum (Kalia et al. 2008).

Both TPC results were applied to Equation 2 to calculate the encapsulation efficiency (Laine et al. 2008).

$$\mathrm{\%}EE=\frac{\text{total phenolic compounds}-\text{surface phenolic compounds}}{\text{total phenolic compounds}}\ast 100\mathrm{\%}$$ (2)

HEEPX total flavonoid content quantification

Aliquots of 20 mg HEEPX80 or HEEPX200 dissolved in 10 mL of methanol were sonicated for 15 min 20 Hz (Quimis 3335D, Diadema, Brazil), centrifugated at 3420 g for 10 min (Kasvi k14-0815C, São Jose dos Pinhais, Brazil), and filtrated on PTFE 0.45μm. For 3 mL of this filtrate, 400 μL of methanolic aluminum chloride 5% w/v was added, and the volume was adjusted to 10 mL with methanolic acetic acid solution 2.5% v/v. This solution was kept for one and half hours, in the dark, at rt, and the absorbance measurements were measured at 425 nm, and quercetin was used as the standard. Encapsulated flavonoid content was expressed as a percentage (%TF). All experiments were in triplicate (Equation 3).

$$\mathrm{\%}TF=A\ast \frac{DF}{v\ast E_{1cm}^{1\mathrm{\%}}}\ast 100\mathrm{\%}$$ (3)

Where, TF = Total flavonoids content percentage; A = absorbance; DF = dilution factor (10 mL/0.002g); v = sample volume (mL); E 1cm 1% = specific absorption of the complex quercetin - aluminum chloride (560) (Marques et al. 2012).

The dilution factor was calculated based on the ratio between the volume and the mass of the stock solution dilution (Equation 3). That is, from 1 mL, a stock solution containing 2500 μg of dry extract (2500 μg extract/mL of methanol) was diluted to 10 mL, which corresponds to a dilution factor (DF) of 4000 mL/g (10 mL/0.0025 g). 3 mL from this diluted solution was used in the analysis, corresponding to a total mass of 7500 μg of extract. For , the value used was 560, according to the literature (Marques et al. 2012).

HEEPX Antioxidant Activity

HEEPX80 or HEEPX200 antioxidant activity was also measured through the scavenging ability of radical DPPH (2,2-diphenyl-1-picrylhydrazyl) (Enujiugha et al. 2012).

The 300 μL solution, prepared from 100 mg of microcapsules/10 mL of water (w/v), was added to 3.7 mL of the DPPH 0.03% in methanol. After 30 minutes, the absorbance measurements were measured at 517 nm in the dark and rt. The antioxidant activity was expressed as the percentage of inhibition of the DPPH radical/mg of the sample (Equation 1).

Measure of Antioxidant Activity HEEPX by Release Profile

The antioxidant capacity of the compounds released from HEEPX80 and HEEPX200 microcapsules was also measured through the scavenging ability radical DPPH radical. 1g of microcapsules were immersed at times 0, 10, 30, and 50 h in aqueous solutions and acetate buffer solutions (pH 4.5 and 7.4) at 25 °C, simulating physiological conditions according to the method by Rutz (Rutz et al. 2013).An aliquot was measured by % inhibition of the DPPH radical in the same way described previously.

Scanning Electron Microscopy

Scanning Electron Microscopy (SEM) was used to analyze the surface morphology of the HEEPX microcapsules. The micrographs for HEEPX80 and HEEPX200 were obtained from a Carls Zeiss benchtop scanning electron microscope, model EVO LS15 (Jena, Germany), equipped with secondary electron detectors (SE), high vacuum detectors (HV), variable pressure detectors (VP), backscattered electron detectors (BSE) and X-ray dispersive energy detectors (EDS). The HEEPX80 or HEEPX200 samples were previously metalized under vacuum from a Quorum gold and carbon vaporizer, model Q150R ES (East Sussex, United Kingdom), and their images were taken with amplitudes of 200, 1000, and 2000 times.

Infrared Spectroscopy (FTIR)

The infrared spectra with Fourier Transform (FTIR) of HEEPX80 and HEEPX200 were obtained from a Bruker spectrophotometer, model Vertex 70 (Billerica, USA) with scanning in the region of 4000-500 cm^-1, the spectral resolution of 4 cm^-1, using the Total Attenuated Reflection (ATR) module. A total of 254 scans were made.

Statistical analysis

The results were expressed as means referring to the determinations made in triplicate and submitted to analysis of variance. One-way ANOVA (ρ ≤ 0.05) and Tukey post hoc evaluated the encapsulation efficiency, comparing the encapsulated compounds. Three-way ANOVA test (ρ ≤ 0.05) and Tukey post hoc compared 80 and 200 Mesh release groups in aqueous media and buffers pH 4.5 and 7.4. The results and the standard curves were analyzed and elaborated using Excel® and Origin(Pro)® 8.6 (OriginLab Corporation, Northampton, MA, USA). Data reliability was determined by calculating the relative standard deviation (RSD ≤ 5%).

RESULTS AND DISCUSSION

HEEP chemical characterizations

HEEP results found for yield (49.20%), Total Phenolic Content (TPC, 74.56 ± 0.02 mg gallic acid/g extract), and Total Flavonoid Content (TFC, 57.93 ± 0.05 mg quercetin acid/g extract) were similar to the previously published results (dos Santos et al. 2020, Costa et al. 2016, Basting et al. 2014). The literature describes the ellagic and gallic acids as frequent in Myrtaceae (Silva et al. 2023). On the UPLC-PAD analysis, the ellagic acid was the main component found on the HEEP (1.83 µg/mL). Gallic acid, also easily found in genera, is below the LOD (Silva et al. 2023, Ferraz Bezerra et al. 2020). Flavonols like quercetin and myricetin are also described, but these phenolics are found mostly in glycosylated form (Peixoto Araujo et al. 2021, Ferraz Bezerra et al. 2020, Costa et al. 2016, Braga et al. 2023).

For this reason, free quercetin was slightly above your limit of quantification (LOQ), and myricetin was under the limit of detection (LOD) in HEEP (Table I). Conversely, for Caesalpinia decapetala, the most suitable condition for gallic acid extraction was found to use Ethanol:H₂O (70:30 v/v) with an increasing temperature (65-70^oC) and extraction time (48 hours) (Pawar & Surana 2010). Similarly, Ethanol:H₂O (70:30), temperature (50–60 °C), and frequency (37 kHz) were shown to be determinants on the extraction of polyphenols α-punicalagin, β-punicalagin and ellagic acid from pomegranate peel (Gil-Martín et al. 2022).

Table I
Linear regression data for analytical calibration and quantification of patterns in HEEP.

	Gallic Acid	Ellagic Acid	Quercetin	Myricetin
Precision	99.4 ± 1.9	99.8 ± 0.8	100.0 ± 0.6	100.1 ± 2.1
Slope	21.5	152.7	63.5	20.5
Intercept	0.11	0.51	0.46	0.27
r	0.999	0.999	0.999	0.999
LOD	1.17 x 10-2	1.05 x 10-3	1.52 x 10-3	1.01 x 10-2
LOQ	3.54 x 10-2	3.19 x 10-3	4.60 x 10-3	3.06 x 10-2
HEEP*	<LOD	1.83 ± 0.01	7.18x10-3± 1.0x10-4	<LOD

• * LOD (Limit of Detection in µg/mL) and LOQ (Limit of Quantification in µg/mL). Linearity range (1.00-10.00 µg/mL).

HEEP Encapsulation and Release Profile

Encapsulations between xanthan gum and polyphenols could also be explained by particle size, interactions between gum-polyphenols, and the chemical structures of phenolic compounds (Jakobek 2015, Liu et al. 2020).

The results revealed that the coating used for encapsulation had an important role in retaining antioxidant phenolic compounds. Xanthan gum 80 mesh has a larger particle size (180µm) than 200 mesh (75µm) and, consequently, a larger surface area (Liu et al. 2020). For this reason, HEEP phenolics EE% was significantly greater in Xantham Gum 80 (HEEPX80, 36.27%) than in 200 mesh (HEEPX200, 23.47%) (F=360.41, ρ<0.05). HEEPX80 also showed the highest TFC values and TFC percentage because of better interaction between Xanthan gum mesh 80 and HEEP flavonoids (Table II). So, it can be inferred that HEEPX80 may have more contact points with flavonoids, such as glycosylated myricetin and quercetin. Gum-polyphenol ion-permanent interactions with phenolic compounds could occur since xanthan gum is partially anionic (Costa et al. 2016, Jakobek 2015, Rutz et al. 2013). Moreover, favored interactions occur when hydrogen bonds and dipole-dipole are formed from the polyphenol’s hydroxyl groups and the oxygen atoms of the glycosidic bonds of polysaccharides (Renard et al. 2017).

Table II
Total phenolic content (TPC), total flavonoid content (TFC), and Encapsulation efficiency of HEEP with Xanthan gum mesh 80 and mesh 200.

Microcaps		TPC mgGA/g**	TFC %	EE %
HEEPX80	Total	3.03 ± 0.05 a*	55.56 ± 0.00ª*	36.27± 0.02ª*
	Surface	1.93 ± 0.07 b
HEEPX200	Total	2.93 ± 0.03 c	52.54 ± 0.01b	23.47± 1.09b*
	Surface	2.24 ± 0.07 d

• *Mean values ± standard deviation (n=3 replicates). Means followed by different letters indicate significant differences (ρ ≤ 0.05). ** g Xanthan Gum.

The other Eugenia spp. have higher EE% values compared to the literature. Eugenia uniflora L. (pitanga) juice had an encapsulation efficiency of 76.37% of extracts with xanthan gum, which was richer in gallic acid and anthocyanins (Rutz et al. 2013). Eugenia stipitata pulp microparticles with maltodextrin (1:9) had the best bioactivity conservation after in vitro gastrointestinal digestion, conserving 61% of total polyphenols and 31% of antioxidant capacity according to the DPPH test methods. (Iturri et al. 2021). However, the HEEPX microcapsule’s potential could be assessed by analyzing its antioxidant activity. Even though the encapsulation efficiency is low, if the antioxidant capacity is preserved, it indicates that its components were properly preserved.

HEEP, HEEPX80 and HEEPX200 antioxidant activity

HEEP antioxidant activities, measured by antiradical capacity in the DPPH method, are compatible with our previous research group’s results (dos Santos et al. 2020).

In this manuscript, HEEP showed an EC₅₀ (6.23±0.02 μg/mL), which is near to positive result controls: gallic acid (1.98±0.01 μg/mL) and quercetin (4.73±0.02 μg/mL). Conversely, we also consider that 1mg of HEEP provided percentage was 92.32±0.04% antioxidant inhibition.

It’s well known that HEEP antioxidant activity is provided by ellagic acid, quercetin, and myricetin derivatives (Michala & Pritsa 2022, dos Santos et al. 2020, Costa et al. 2016). These phenolics’ antioxidant activity is due to their proton donor ability, which correlates with the number of hydroxyl groups in these phenolics. For example, quercetin has greater antioxidant activity than rutin, a quercetin glycosylated molecule, because of the available hydroxyl groups. For ellagic acid, a correlation between antiradical activity is also attributed to hydroxy functions, which exhibit a greater ability to donate a hydrogen atom and support the unpaired electron. Still, a sugar moiety would decrease this effect (dos Santos et al. 2020, Michala & Pritsa 2022). So, encapsulation with Xanthan gum-HEEP was expected to protect HEEP antioxidant phenolics and deliver a better-releasing control with improved antioxidant activity.

Table III shows that Xanthan-HEEP showed good antioxidant activity values when compared to the HEEP extracts.

Table III
HEEP Antioxidant Activities (% AA) in Xanthan Gum (Mesh 80 and 200) Microcapsules with H₂O and buffered solutions at pH 4.5 and 7.4.

HEEPX80	H2O	pH 4.5	pH 7.4
t(h)	%AA
0	81.97±0.19a*,a**	79.80±0.42e,b	32.53±1.83i,c
10	83.81±0.11bc,a	94.39±0.26f,b	40.45±0.18j,c
30	85.70±0.11bd,a	94.43±0.29f,b	49.26±0.32k,c
50	86.78±0.40bd,a	94.81±0.11f,b	50.02±0.33k,c
HEEPX200	H 2 O	pH 4.5	pH 7.4
t(h)	%AA
0	82.04±0.40ac,a	79.62±0.27e,b	42.54±0.62j,d
10	83.50±0.50b,a	94.27±0.20f,b	52.08±1.70kl,d
30	85.39±0.19d,a	94.50±0.50f,b	54.46±0.22lm,d
50	86.21±0.11d,a	94.62±0.11f,b	55.04±0.32m,d

• *Averages followed by at least one equal letter indicate non-significant difference (ρ ≤ 5%).** The letters before the comma indicate the difference (or not) between the medium and the release time. The letters after the comma indicate the difference between the medium and the mesh (80 and 200). HEEPX80, HEEPX200 (HEEP encapsulated with xanthan gum 80 or 200 mesh). Water (pH=5.0, artesian well), pH=4.5, and 7.4 buffered solutions.

HEEPX80 and HEEPX200 did not significantly differ in the release profile of antioxidants in the water (pH=5.0, artesian well, non-buffered). Both microparticles showed a significant difference from t=10h until 50h h compared to t=0h. Time releases t=10h to 30h showed a significant difference between them, but after t=30h, there is no significant difference in the antioxidant release. (Table III). Similarly, HEEPX80 and HEEPX200 did not show significant differences in the release profile of antioxidants at pH=4.5. HEEPX80 and HEEPX200 showed a significant difference in the antioxidants-release profile from time t=10 to 50h compared to the t=0h. However, the antioxidant activity at this pH is significantly superior to water results (pH=5,0). At pH 7.4, HEEPX200 and HEEPX80 antioxidant activity significantly differ at t=0h. But at t=10-30h, a better antioxidant activity was achieved for HEEPX200 (Table III).

So, Table III shows that the drug release rate in the Xanthan-gum microencapsulated systems was controlled by changing the pH of the release medium. It is well known that Xanthan gum possesses higher stability at low pH due to its double helix conformation and hydrogen bonds between their chains (Garcı́a-Ochoa et al. 2000, Jadav et al. 2023, Oh et al. 1999).

Also, for pH>4.5, Xanthan gum behaves as a polyanion because of the deprotonation of O-acetyl and pyruvyl residues, increasing negative charge density. Their carboxylic groups can become partially protonated (pH 4) or completely deprotonated for pH> 6. Deacetylation of Xanthan gum occurs in an extremely alkaline medium (pH≥9) and forms a gel at pH>10 (Jadav et al. 2023).

So, as the pH increased, the xanthan gum was modified its conformation from a double helix to an unfolded and disorderly serpentine state (Brunchi et al. 2016, Bueno & Petri 2014).

Additionally, as related in the literature, at pH=5, a reduced viscosity of Xanthan-polyphenol solutions was observed. This low viscosity is caused by repulsing electrostatic forces between the carboxyl groups in the side chain (Brunchi et al. 2016).

Other types of gum, such as Bitter Almond Gum (BAG), had similar behavior, i.e., with increasing pH, acid groups of coils are gradually ionized and expanded due to an increase in electrostatic repulsion between functional groups, leading to more intermolecular interactions among the coils and consequent higher viscosity of solution (Hosseini et al. 2017).

From Table III, HEEPX200 displayed a better antioxidant activity than HEEPX80 for pH=7.4. Both Xanthan gum microparticles have an unfolded conformational structure at this pH, with deprotonated carboxylic groups and increased viscosity. In such conditions, phenolics are more efficiently retained, decreasing antioxidant activity (Table III). However, since HEEPX200 phenolics are mostly on the surface (Table II), this gum presented a greater antioxidant activity regardless of the smaller size.

So, it is concluded that the HEEP-Xanthan microparticles modify the drug release profile, especially under acidic conditions (Jadav et al. 2023).

Microparticle structure analysis

In addition to providing information about chemical bonds and molecular structures, FTIR spectroscopy also allowed the detection of intermolecular interactions in HEEP phenolics and Xanthan gum. The FTIR spectrum of xanthan gum shows characteristic absorption bands at 1024 cm−¹, 1419 cm−¹, 1603 cm−¹, and 1719 cm−¹, corresponding, respectively, to the elongation of the ether function C–O–C, the C–H bonds of methyl groups and the asymmetric vibrations of COO–, as well as the elongation of CO esters (acetyls). The ν(CO) vibrations of the alcoholic COH groups, the rings skeletal ones, and those characteristic to the glycosidic COC linkages are all covered in the broad 1170–970 cm−¹ band (Brunchi et al. 2016, Yahoum et al. 2023).

The FTIR analysis of xanthan gum, HEEPX80, and HEEPX200 confirmed the compatibility of HEEP phenolics with Xanthan gum in both mesh sizes. The band above 3500 cm−¹ shifted to 3200 cm−¹ (OH stretching vibration) in the presence of HEEP extracts. The variations in the shape and peak positions of the –CO group and –OH bending vibration (1603 to 1590 cm−¹ cm−¹) and (1024 to 1017 cm^-1) were observed. These observations clearly show the interaction of HEEP phenolics with the OH group of Xanthan (Brunchi et al. 2016).

HEEPX80 and HEEPX200 microscopy

The HEEP encapsulation with the two particle sizes of xanthan gum (80 and 200 mesh) formed particles with a smooth structure but an irregular shape, characteristic of freeze-drying-obtained capsules. Crystalline depositions of extract compounds (Figure 2 a-d) spread on the surface, similar to the results found by other authors (Cilek et al. 2012, da Rosa et al. 2013, Sosa et al. 2011, Visentin et al. 2012). Also, the extract encapsulation occurred with both meshes, as related similarly in the literature, and there were no observed differences between them (da Rosa et al. 2013, Wang et al. 2011) (Figure 2a-d, Figure 3a-d).

Figure 1
FTIR spectra of Xanthan Gum (XG) and their microcapsules HEEPX80 and HEEPX200.

Figure 2
a. Scanning Electron Microscopy (SEM) HEEPX80 Microcapsules. b. Magnification 0.4x, c. Magnification 2x. d. Magnification 4x.

Figure 3
a. Scanning Electron Microscopy (SEM) HEEPX200 Microcapsules. b. Magnification 0.4x. 3c. Magnification 2x. d. Magnification 4x.

CONCLUSIONS

HEEP encapsulation with Xanthan gum by lyophilization preserved and protected the phenolic compounds responsible for their antioxidant activity. HEEP’s phenolic extract encapsulated in xanthan was confirmed by FTIR and scanning electron microscopy techniques. Also, encapsulation efficiency depended on the gum particle size used. The encapsulation showed higher efficiency for HEEPX80 due to its larger particle size and surface area, which increased the interaction of the phenolic compounds with the matrix. The highest antioxidant activity was related to the equitable presence of phenolic compounds in microcapsules coated with xanthan. Flavonoids were the highest fraction of these encapsulated compounds due to the interactions between the hydrophilic ligands of these compounds and the gum. The release profile of phenolic extract from the capsules was affected by pH. In HEEPX80 and HEEPX200, antioxidant activity was preserved, especially at pH 4.5, similar to the extract. At pH 7.4, the release was impaired due to changes in the electrostatic forces that modify the gum conformation. These results may lead to a future application of HEEP formulations, which requires a release profile with constant antioxidant activity, contributing to product preservation and consumer health.

ACKNOWLEDGMENTS

This study was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) - grants number 2015/22528-2 and 2017/15610-0.

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Publication Dates

• Publication in this collection
  15 Nov 2024
• Date of issue
  2024

History

• Received
  27 Feb 2024
• Accepted
  9 July 2024