Preparation, physical properties, and evaluation of antioxidant capacity of aqueous grape extract loaded in chitosan‐TPP nanoparticles

Abstract Grape extract is reportedly rich in phenolic compounds that possess strong antioxidant activities. Encapsulation of such extracts in nanoparticles (NPs) is an effective way to preserve various food products. In the present study, grapes were first extracted, and the amount of total phenolic content and different types of phenolic acids was determined. The extracts at different chitosan/extract weight ratios (1:0.25, 1:0.5, 1:0.75, and 1:1) were then encapsulated in chitosan nanoparticles (NPs) using the ionic gelation method. The extract‐loaded chitosan nanoparticles were characterized by their physicochemical properties using the dynamic light scattering (DLS) technique, chemical properties using Fourier‐transform infrared (FTIR) spectroscopy, and X‐ray powder diffraction technique (XRD), the morphological properties using scanning electron microscopy (SEM), and the antioxidant activity using the 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) test. The encapsulation efficiency (EE) and loading capacity (LC) were also assessed. Our findings showed that the free radical inhibition effect of NPs significantly increased with an increase in extract concentration. Chitosan NPs presented acceptable encapsulation efficiency and loading capacity (LC), and the encapsulation process enhanced the antioxidant activity of the free grape extracts. At the weight ratio of 1:0.5, the particle size and zeta potential of the NPs were 177.5 ± 2.12 nm and 32.95 ± 0.49 mV, respectively. FTIR and XRD analyses verified the credibility of the encapsulated grape extract in chitosan NPs. These NPs can be an efficient way to increase the shelf‐life of food products.


| INTRODUC TI ON
For centuries, techniques in food research have evolved, and the use of conventional techniques has been systematically replaced by the use of complex mixtures and plant extracts (Zorzi et al., 2015). Grape extract is one of the richest sources of polyphenol compounds such as catechin, epicatechin, gallic acid, and pro anti cyanidin, as well as dimer, trimer, and tetramer compounds such as procyanidins. These compounds are also known for their antioxidant, antimicrobial, anticancer, and antiviral properties. Gallic acid in grape seed extract contains various flavonoids such as monomeric flavan-3-ols such as catechin, epicatechin, gallocatechin, epigallocatechin, epicatechin 3-O-gallate, procyanidin dimers and trimers, and highly polymerized procyanidin. It has been reported that cardiac diseases, diabetes, and cancer may occur due to an antioxidant imbalance in the body, so the recommended intake of grape seed extract to prevent cardiovascular risk is between 100 and 300 mg/day (Gibis et al., 2013). However, including these nutraceutical compounds into food products in their pure form results in poor solubility and bioavailability, decreased shelflife, and activity loss during processing. Therefore, an effective delivery system should be engineered to overcome these disadvantages.
Since the beginning of the twentieth century, the food industry has exploited nanotechnology to synthesize food products to improve safety, prolong shelf-life, and include the benefits of bioactive compounds. Nanoencapsulation offers several advantages over microencapsulation. The objectives of encapsulation are to preserve the material against environmental conditions such as light humidity and oxygen, increase the shelf-life of products, and create a controllable condition of effective compounds. Such a nanoparticle (NP) system can encapsulate a more effective extract and generate higher thermodynamic stability. NPs have sizes ranging from 1 to a few hundred nanometers (nm). The selection of the nanoencapsulation technique usually depends on the nature of the bioactive compounds and encapsulating polymer. Selecting the suitable polymer will allow the synthesis of NPs with desired physicochemical properties, morphology, stability, and release kinetics (Čalija et al., 2017).
To date, several encapsulating materials have been used to encapsulate bioactive compounds to manufacture functional foods. Among all these materials, chitosan has been widely investigated and considered a promising industry.
Chitosan is a widely used natural polysaccharide with antimicrobial and antioxidant activities (Omara et al., 2019). Owing to its nontoxic, biodegradable, and biocompatible nature, it is commonly used in different industrial sectors such as food, pharmaceuticals, agriculture, and environmental industries (Alves et al., 2018).
Chitosan-based materials are excellent candidates for the encapsulation of bioactive compounds to prolong the shelf-life of industrially important food products. It has been proven that the encapsulation of herbal extracts such as grape seed extract with carriers like chitosan significantly improves the effectivity of the extract (Zarei et al., 2015). Compared to their normal form, chitosan NPs have a higher total volatile basic nitrogen (TVB-N) inhibition effect. Ionic gelation is a more suitable method to prepare chitosan NPs to encapsulate bioactive compounds. The basis of ionic gelation relies on electrostatic interactions between the positively charged primary amino group of chitosan and negatively charged polyanion group like tripolyphosphate (TPP) (Chandrasekaran et al., 2020). This nontoxic process does not contain an organic solvent, and it is easy and controllable (Rasaee et al., 2016).
In the present study, we synthesized chitosan NPs containing grape extract at different concentrations for food preservation.
The physicochemical, morphological, and chemical properties of the produced NPs were evaluated. The phenolic acid content and the total phenol content of the extract-loaded chitosan NPs were determined. The characterized particles were then analyzed for their antioxidant properties. The chitosan/extract ratio of 1:0.5 was selected as the optimum value in our study due to the desired properties, encapsulation efficiency as well as loading capacity obtained. Our findings showed that grape extract-loaded chitosan NPs are excellent candidates to prolong the shelf-life of the food products and preserve foods from oxidation.

| Extraction of grape
A previously published method was used with slight modifications (Rajaei et al., 2010). The grapes were first dried in an oven for 30 min at 50°C, and then the dried grapes were ground to obtain the powder, which was then passed through a mesh (No. 35). Afterwards, the obtained powder was dissolved in deionized water and placed in an ultrasonic bath (UP200H, Germany) at 55°C for 15 min exposed to 35 kHz waves. Then the extract was filtered using Whatman filter paper No. 5 to remove impurities. The obtained extract was then placed in a sealed container away from light and humidity.

| Encapsulation of grape extract in chitosan NPs
Chitosan NPs were synthesized following the ionic gelation technique, which takes place through an electrostatic interaction be-  Haider et al., 2017;Zarei et al., 2015). Briefly, chitosan was dissolved in acetic acid (1% v/v), followed by sonification for 60 min to obtain a clear chitosan solution (2% w/v). The pH of the solution was stabilized between 3 and 4 using 4N NaOH solution.
The TPP was dissolved in deionized water using a magnetic mixer at ambient temperature to obtain a TPP solution (2%). Afterwards, 4 ml of TPP was added to 100 ml of chitosan solution using a magnetic stirrer for 60 min at 700 rpm (revolutions per minute) to obtain a homogeneous solution. Different volumes of the extract in deionized water (0, 0.05, 0.75, 0.1, 0.125 v/v) were added to chitosan NPs and mixed with a magnetic mixer for 30 min to obtain different concentrations of chitosan-to-extract ratios (1:0, 1:0.25, 1:0.5, 1:075, and 1:1). After adding the extract, mixing was continued for an additional 30 min to achieve complete gelation.

| Physicochemical characterization
The physicochemical properties of the grape extract-loaded chitosan NPs (particle size, polydispersity index (PDI), and zeta potential) were measured using the Dynamic Light Scattering (DLS) technique (Zetasizer Nano ZS, Malvern Instruments, the UK). The analyses were performed in triplicate in water at 25°C. The backscattering configuration was set with a detection angle of 90°. The helium-neon (He/Ne) laser emission and power source were 633 nm and 4.0 mW, respectively.

| Morphological characterization
To visualize the morphological properties of the NPs, Scanning Electron Microscopy (SEM) (JEOL, Japan, JSM 6400) was used.
Images were developed for a chitosan/extract concentration ratio of 1:0.5.

| Encapsulation efficiency of chitosan NPs
The encapsulation efficiency of the chitosan NPs was determined according to the method previously described by Zhang et al. (2016), Tang et al. (2013), Shah et al. (2016 and Dube et al. (2010) with minor modifications. For quantitative examination of the amount of the extract loaded in chitosan NPs, they were first centrifuged for 1 h at 12,000 g at 4°C so that the sedimented NPs became floated.
Glycerol was added to the solution to prevent particle coagulation.
The amount of grape extract in the supernatant was determined by spectrophotometer at 250-400 nm (Cary 600, Agilent, the USA).
The HPLC was used to determine the concentration of the floated extract that represents the loading capacity of the chitosan NPs. Different concentrations of the extract (µg/ml) were prepared, and the adsorption level was read at 275 nm wavelength for each concentration using Perkin-Elmer (series 600) equipped with a ultraviolet (UV) detector and reversed-phase column (Pak C18, ODS1 4.5 mm * 150 mm). The injection volume was 10 ml with a flow discharge rate of 1.2 ml/min, system pressure of 10-15 Mpa, and operating temperature of 25°C. Before each injection, the device was calibrated, and the extract was measured at 280 nm wavelength.
The percentage of nanoencapsulation efficiency and loading capacity (LC) of the chitosan NP were obtained using Equations (1) and (2), respectively.

| Antioxidant activity
Antioxidant activity of the grape extract in free and encapsulated forms was measured following the method described by Tang  NPs were mixed. Then the mixtures were added into 2.7 ml of ethanol solution (6 × 10 −5 mol/L) and then placed in a dark room for 30 min for the DPPH assay. Afterwards, the adsorption capacity of the mixture was measured at 517 nm against a blank (ethanol) using a UV spectrometer. To minimize the error coefficient, each measurement was repeated three times (Table 1). Free radical suppression percentage (DPPH) was obtained using Equation (3): A blank is a light absorbed by the negative control without the extract, and A sample is light adsorbed with different concentrations of the extract.

| Phenolic acid content
The extraction and measurement of phenolic acid were carried out using the method previously described by Koponen et al. (2007) and Vekiari et al. (2008). To this end, 0.5 g of the extract was dis-

| Phenol content assessment
Phenol content of the extract-loaded chitosan NPs was assessed using the method previously described by Roostaee et al. (2017) and Zarei et al. (2015). Total phenol content was measured using the Folin-Ciocalteu assay. Briefly, 20 ml of the grape extract was mixed with 1.6 ml of distilled water and 100 ml of the Folin-Ciocalteu reagent (FCR). After 1 min, 300 ml of sodium carbonate solution (20%) was added. Test tubes were placed in a water bath at 40°C) after shaking, and after 30 min, the adsorption level was read at 760 nm using a spectrophotometer.
Standard gallic acid was used to create the standard curve. First, a gallic acid-based solution was prepared at concentrations ranging between 10 and 100 mg/ml, and the standard curve was created based on the adsorption value against different concentrations of the acid. Total phenolic compounds in the extract were expressed as gallic acid in each gram of the extract. The measurements were carried out in triplicate.

| Statistical method
Data analyses were done in SPSS (ver. 19) and Excel 2020.
Kolmogorov-Smirnov test was used to determine the normality of the data. Tukey test was used to compare the mean score of treatments. One-way analysis of variance (ANOVA) (p < .05) was used to determine if the differences between each measured value were significant.
In an aqueous medium, an increase in the size of extract-loaded chitosan NPs can be less than empty NPs as the empty NPs can be hydrated as the extract with hydrophilic properties occupies the interior of chitosan NPs. Additionally, the degree of degradability of the chitosan NPs may vary during the encapsulation process, which results in the formation of particles of smaller size (Ghaderi-Ghahfarokhi et al., 2017;Barzegar et al., 2016). Prior to measuring particle size using DLS, the samples were sonicated, and ultrasonic waves possibly broke the glycosidic bonds 1 and 4. Ultrasound cavitation destroys and decomposes long chitosan chains and converts

TA B L E 1
The physicochemical properties of the grape extract-loaded chitosan NPs at different chitosan/extract weight ratios them into smaller particles. Through this, the formation of bigger particles during ionic gelation is suppressed (Tang et al., 2013).
Polydispersity index (PDI) indicates the homogeneity of particles in a colloidal suspension (Cho et al., 2013). The particle size and PDI are affected by several factors, such as the molecular weight of the encapsulated compound, polymer material, and mixing method (Barzegar et al., 2016). As shown in Table 1, the decrease in the chitosan/extract ratio leads to the generation of polydisperse NPs.
The PDI of empty chitosan NP is equal to 0.36 ± 0.008. Compared to empty NPs, the increase in PDI of the extract-loaded NPs was statistically significant (p < .05). However, no significant difference was found between the ratios of 1:

| Zeta potential
Zeta potential indicates the surface charge of the particles, the degree of electrostatic repulsion, and their stability in a colloidal system. The particles with the zeta potential of less than −30 mV or greater than +30 mV are considered colloidally stable (Zarei et al., 2015). In our study, empty chitosan NPs had the zeta potential of 36.4 ± 0.14 mV. As shown in Table 1

| Encapsulation efficiency and loading capacity of chitosan NPs
Encapsulation efficiency is defined as the percentage of the grape extract entrapped in chitosan NPs. The efficiency strongly depends on the ratio between chitosan/grape extract. Moreover, the efficiency varies as the solubility of the extract in the matrix material or polymer changes (Kumari et al., 2010). In our study, the encapsulation efficiency was found to be between 51.90 ± 1.33 and 33.56 ± 0.41% (Table 2) The molecular weight of the compound to be encapsulated significantly affects the encapsulation efficiency (Cai & Lapitsky, 2017).
High-molecular-weight molecules usually have limited movement and lead to lower encapsulation efficiency. This is also highly related to the preparation method of the grape extract.
Loading capacity (LC) is the amount of the grape extract encapsulated per unit weight of the chitosan NP. It is calculated by the amount of total entrapped grape extract divided by the total chitosan NP weight. The loading capacity (LC) of the chitosan NPs ranged from 4.2 ± 0.4 to 6.7 ± 0.8% (Table 1). The lowest loading capacity was obtained at a chitosan/extract weight ratio of 1:0.25. As the ratio decreased, the loading capacity showed an increasing trend. However, after reaching the ratio of 1:0.75, the increase was insignificant. Previous studies have reported that an enormous concentration of the compounds (i.e., essential oils or plant extracts) leads to an increase in loading capacity (Ghaderi-Ghahfarokhi et al., 2017;Barzegar et al., 2016;Haider et al., 2017;Tang et al., 2013;Keawchaoon & Yoksan, 2011;Yoksan et al., 2010;).

| Determination of the antioxidant activity
As shown in Figure 1, the antioxidant activity of the grape extract in free form ranged from 15.6% to 51.01%, whereas the encapsulated extract ranged from 21.2% to 62.8%. It can be observed that the increasing amount of encapsulated grape extract in chitosan NPs significantly enhanced the antioxidant activity ( Figure 1). Additionally, a higher chitosan-to-extract ratio leads to a higher antioxidant activity. This result was also correlated with the encapsulation efficiency. There may be different reasons behind this finding. Phenolic compounds in the grape extract are highly volatile and can easily evaporate from the system. However, nanoencapsulation can preserve the phenolic compounds by decreasing the evaporation rate to suppress free radicals (Barzegar et al., 2016;Ghaderi-Ghahfarokhi et al., 2017). As a control group, the antioxidant activity of the chitosan NPs was also evaluated. As shown in Figure 1, chitosan had an antioxidant activity of less than 10%. This is due to the hydroxyl and amino groups present in the chitosan backbone that can react with free radicals. During the ionic gelation process, amino groups of chitosan undergo cross-linking with the polyanions present in TTP.
Using different concentrations of chitosan leads to a varying level of cross-linking, which results in an antioxidant activity ranging be-

| Phenolic acid profile and total phenol content of extract-loaded chitosan NPs
The grape was ground to obtain an extract, and the phenolic acid content of the extract was analyzed. As shown in Figure 2, the amounts of gallic acid, catechin, epicatechin, rutin, resveratrol, quercetin, ellagic acid, and caffeic acid were 10. 395, 25.335, 10.26, 9.675, 5.445, 15.39, 43.605, and 5.985 µg/g, respectively. The highest and lowest phenolic acid contents in the grape extract were ellagic acid and resveratrol, respectively.
The total phenol content of the grape extract is demonstrated in Figure 3 for different chitosan/extract ratios (mg/g). The total phenol content significantly increased as the chitosan/extract ratio decreased (from 1:0.25 to 1:1). The highest total phenol content was obtained with a ratio of 1-1. The total phenol content of chitosan/extract ratios of 1:0.25, 1:0.5, 1:0.75, and 1:1 was determined to be 13.92 ± 1, 23.96 ± 1.8, 52.575 ± 6.04, and 73.095 ± 2.8 mg/g, respectively. The increase in phenol content between the ratios of 1:0.25 and 1:0.5 was not statistically significant (p > .05), while a significant difference was found between the ratios of 1:0.5 and 1:0.75, and between 1:0.75 and 1:1 (p < .05). The phenol content significantly varies depending on the plant extract. Roostaee et al. (2017) reported that the total phenol content of microencapsulated green pistachio-loaded chitosan NP was 10 ± 0.05 mg/ml. Zarei et al. (2015) demonstrated that pomegranate and orange peel phenolic content in chitosan NPs was 364 ± 10.3 and 350 ± 6.4 mg TAE (tannic acid equivalent)/g. In a previous study, total phenol content in the grape peel extract and grape seed extract loaded with chitosan NPs was measured to be 42.064 ± 1.01 and 45.652 ± 3.74 mg/g, respectively (Silva et al., 2020).

| Morphological characterization of NPs
Morphological characterization of the empty (Figure 4a) and extractloaded chitosan NPs (Figure 4b) was conducted utilizing the SEM technique ( Figure 4). For morphological characterization, the chitosan/extract ratio of 1:0.5 was selected, as the physicochemical properties, encapsulation efficiency, and loading capacity were favorable. It can be seen in Figure 4 that the NPs are spherical with the particle size ranging between 100 and 200 nm, as confirmed by the DLS technique, and no particle accumulation can be observed.

| Fourier-transform infrared (FTIR) spectrometry
In the FTIR spectrum of grape extract, the critical absorption

| X-ray diffraction (XRD) analysis
The XRD method is used to analyze and determine the crystal structure of the chitosan NPs. The method is based on X-ray radiation on the sample from different angles and analyzing the diffraction or reflection. The XRD patterns of the grape extract, chitosan, empty chitosan NPs, and extract-loaded chitosan NPs are shown in Figure 6.
Chitosan exhibits two reflection falls: the reflection at 2θ = 11.95°, which confirms the formation of crystal I, and powerful reflection at 2θ = 23.4°, indicating crystal II formations. Notably, chitosan is always covered with hydrated bonds (5%), even when dried out.
Crystal peak at the center around (peak II) indicates the formation of crystalline structures that are formed uniformly with moderation.
The peak at about 2θ = 34.4° indicates a high level of crystallization in chitosan powder. In the XRD pattern of chitosan NPs, the peak at 2θ = 11.95° is eliminated, and the peak at 2θ = 23.3° is extended; therefore, the strength of the last peak is lowered.
Based on these observations, the amorphous nature of chitosan NPs chains is notable, which means that cross-linking agents (acetic acid molecules) in chitosan chains are dissolved during the formation of NPs. Therefore, creating cross-linking prevents the reformation of chitosan chains in crystalline bulk structures.
The extract demonstrated a broad peak at 2θ = 21.57°, which is indicative of the amorphous nature of the extract. Compared

| CON CLUS ION
Nanoencapsulation of the grape extract in chitosan NPs was successfully performed through the ionic gelation technique.
Chitosan/extract concentration of 1:0.5 was the optimum ratio as the desired physicochemical properties, encapsulation efficiency, and loading capacity were obtained. Chitosan NPs loaded with the extract had a higher antioxidant activity compared to the free extract. The highest antioxidant activity was observed with the chitosan/extract ratio of 1:0.5. Although encapsulation of reactive agents within chitosan NPs was challenged with issues such as instability and insolubility, the findings indicated that the encapsulated grape extract with chitosan NPs had a higher antioxidant property and, in turn, could provide a higher shelf-life and stability to the various food products.