Effect of different extraction methods on antioxidant properties and encapsulation efficiency of anthocyanin of pomegranate peel

Abstract This study aimed to measure the efficiency, total anthocyanin content (TAC), and total phenol content (TPC) of pomegranate peel powder (PPP) extract from different extractions. Also, the characteristics of the nanoencapsulated extracts with maltodextrin (MD)/Lepidium perfoliatum (Qodume Shahri) seed gum were investigated. The highest and lowest extraction efficiency was related to solvent ethanol–water extraction (SEWE) (76.35%) and solvent ethanol extraction (SEE) (25.73%), respectively. Extracts obtained from microwave extraction (ME) and ultrasound extraction (UE) methods had the highest and lowest values of TAC (4.00–0.35) (mg C3G/g PPP) and TPC (702.13–232.58) (mg GAE/100 g sample), respectively. Peak 3213 in FT‐IR indicates the O–H bond, which showed the highest content of phenolic compounds in the extract obtained from ME compared with SEE, SEWE, and UE. The nanoencapsulated extracts from SEE, SEE, and UE had the lowest particle size of peak 1, particle distribution in peak 1, and average particle size distribution compared with other extractions, respectively. The highest encapsulation efficiency of anthocyanin (EEA) and encapsulation efficiency of phenol (EEP) were related to UE (96.15%) and SEWE (86.57%), respectively. The EEP and EEA of SEE were not significantly different from ME and SEWE, respectively. On the other hand, the type and amount of extractive compounds in the extract have a great impact on the efficiency of nanoencapsulation and the average size distribution of nanoencapsulated particles. As a result, PPP extract is rich in antioxidant compounds, which can be determined by carefully examining the appropriate method of extraction and preservation of the extracted compounds.


| INTRODUC TI ON
Recently, due to many concerns about the carcinogenicity of synthetic antioxidants, the use of natural antioxidants has been considered. Scientific information on the antioxidant properties of various plants, especially those less commonly used in food and medicine, is still lacking. Therefore, it is practical to evaluate such properties, especially to find new sources of natural antioxidants (Arabshahi-Delouee & Urooj, 2007). On the other hand, the interest in developing food colors from natural sources as an alternative to artificial colors has increased due to legal measures and consumer concerns.
Anthocyanins belong to a broad group of phenolic compounds commonly called flavonoids. Anthocyanins and phenolic compounds can activate antioxidants by donating hydrogen to radicals and preventing the production of more oxidation products (Assous et al., 2014).
Inexpensive resources and food industry remnants are the focus of more attention. Fruit peels are valuable wastes that can be obtained from industrial and domestic use. One of the natural sources of food coloring is anthocyanin extract from pomegranate peel (PP).
Pomegranate fruit (Punica granatum L.) is abundantly produced in Iran. This fruit can be consumed fresh, as a juice, drink, or in other food products (Abid et al., 2017). Health effects of pomegranate fruit: In addition to the edible part, it also includes non-edible parts (especially the peel, which contains biologically active compounds).
PP has more phenolic compounds than meat and seeds. PP can be a good source of high-value antioxidants (Essa & Mohamed, 2018;Shaban et al., 2013).
The extraction process is a critical step and plays an important role in determining the optimal amount of bioactive compounds such as anthocyanins. Parameters affecting the yield of bioactive compounds from plant sources include the following: plant matrix, type of solvent, temperature, pressure, time, amount of solvent, and liquid/solid ratio (Azmir et al., 2013;Ilaiyaraja et al., 2015).
Anthocyanins are usually extracted with acidic solvents under mild conditions (Puértolas et al., 2013). This solvent system destroys cell walls and membranes while dissolving, and stabilizing anthocyanins (Navas et al., 2012). The use of ultrasound in plant extraction has the advantages of increasing mass transfer, better solvent penetration, less dependence on the solvent used, extraction at low temperatures, faster extraction speed, and higher crop yields (Rana & Meena, 2017). Researchers prefer to use the ME method because it is cheaper, requires less time, has higher extraction efficiency, and also has a better yield product (Maran et al., 2013).
Encapsulation is used in the food industry to control the release of perfumes, flavorings, bioactive substances, and foods containing probiotics (Kuang et al., 2010). Encapsulation is a method of coating materials (such as natural dyes) in the form of micro and nanoparticles (Khazaei et al., 2014). The components of the wall, in addition to being natural, must have maximum protection of the active materials against environmental conditions, keep the active materials inside the capsule structure during processing or storage in different conditions, and have good rheological properties (Elsebaie & Essa, 2018).
The most widely used for coating in food are polysaccharides, which contain starch and their derivatives -MD, as well as gums (Wandrey et al., 2010). The use of indigenous gums such as LPSG has been considered because of their cheapness. So far, no research has been done on the nanoencapsulating of anthocyanin extract of PPP with LPSG. The purpose of this work is to study the effect of different extraction methods on antioxidant properties and the combination of LPSG and MD on the encapsulation efficiency of anthocyanin extracts of PP. In this study, solvent, microwave, and ultrasound were used to extract phenolic compounds and anthocyanins from PPP. It was then encapsulated with MD and LPSG. The effects of different extraction methods on the content of anthocyanins, phenolic compounds, and nanoencapsulation properties of PPP extract were evaluated.

| Materials
First, 5 kg of Saveh red pomegranate fruit was first prepared. The PP was removed from the fruit and then dried for 72 h at room temperature, away from sunlight. Then, it was crushed, and a sieve (mesh 18) was used to prepare the powder. It kept in the refrigerator until use. Qadomeh Shahri seeds were purchased from Attarak Company. All the chemical materials were purchased from Sigma-Aldrich.

| Solvent extraction
Extraction was performed by the method of Fuleki and Francis (1968) with some modifications; 20 g of PPP was combined with 150 mL of acidified ethanol (0.01% citric acid) and a mixture of acidified ethanol and water (1:1) for 5 min with an electric mixer soaked at maximum speed (Folki & Francis, 1968).

| Microwave extraction
Extraction was performed by the method of Duan et al. (2015). A quantity of 1 g of PPP was mixed with 20 mL of 96% ethanol and 1% hydrochloride. After mixing, the sample was placed in a microwave (45°C: 360 w: 7 min; Duan et al., 2015).

| Ultrasound extraction
Pomegranate peel powder was extracted by bath ultrasound according to the method of Chranioti et al. (2015). About 1 g of PPP was mixed with distilled water (20 mL) and placed in a bath ultrasound (Elmasonic S) (30°C: 37 kHz: 60 min; Chranioti et al., 2015).
After each extraction, the extracts were filtered with Whatman 42 filter paper by the Buchner funnel. The extracts were centrifuged by refrigerated centrifugation (5 min: 10°C: 7168 g). Then the supernatant was collected and placed in an oven (45°C) to evaporate the solvent. The final solution of each extraction was kept at −18°C (Assous et al., 2014).

| Extraction efficiency
Extraction efficiency was calculated using the following equation:

| Total anthocyanin content
Total anthocyanin content was determined by the pH differential method, as described by Kırca and Cemeroğlu (2003). About 1 mL of anthocyanin extract of PPP was mixed with 9 mL of distilled water and then mixed with potassium chloride buffer solution at pH = 1 and sodium acetate at pH = 4.5. The absorption was recorded at 520 and 700 nm using UV-VIS spectrophotometer after 20 min (Model 1502) (Kırca & Cemeroğlu, 2003).

| Total phenol content
Total phenol content (TPC) was determined by the Folin-Ciocalteu method (Singh et al., 2002). First, 1 mL of the extract at a concentration of 1 mg/mL was mixed with 5 mL of 10-Folin-Ciocalteu reagent.
A volume of 4 mL of 7.5% sodium carbonate was added. It was placed at 27°C for 30 min, and the absorbance was measured at 760 nm using a UV-VIS spectrophotometer. The results were equivalent to mg of GAE/100 g of sample. Gallic acid was used as a standard to make a calibration curve (Kuspradini et al., 2016).

| Extraction of the gum of Qodume Shahri seeds
First, the impurity present in the seed of Qodume Shahri was separated, and then its gum was extracted (seed/water = 1/30 at 48°C and pH = 8) according to the method of Koocheki et al. (2009). The pH of distilled water was adjusted with NaOH solution. Then it was placed in a 48°C water bath. After the bath reached the desired temperature, seeds were added to it. It was stirred continuously (2 h) to increase the absorption of water by the seeds. A juicer was used to extract gum. The resulting extract was dried (70°C) in an oven.
Finally, after grinding and sieving (mesh 40), the gum powder was stored in closed containers in the refrigerator for use in nanoencapsulation (Razavi et al., 2011). 2.2.8 | Pomegranate peel extract nanoencapsulating A combination of 0.5 g of LPSG and 4.5 g of MD was used to nanoencapsulate the anthocyanin extract. MD was mixed with LPSG powder then distilled water was gently added to the solids to 15%. Then anthocyanin extract was added to the coating materials in a ratio of 1:2 (w/w) and homogenized using ultratorax (21952 g: 5 min). To further reduce the particle size, a prop ultrasound was used for 6 min (10 s per cycle and 2 s rest). It was frozen for 24 h (−18°C), then it was dried by a freeze-dryer (48 h) (Vaco 5, Zirbus) (Carneiro et al., 2013). 2.2.9 | Particle size Nanoencapsulated compound particle size was measured by

| Encapsulation efficiency of anthocyanin (EEA)
About 2 mL of distilled water was added to 200 mg of samples. Then 18 mL of ethanol was added to and filtered with Whatman 42 paper.
Total anthocyanin (TA) was calculated using the following equation (Lee et al., 2005): To measure the surface anthocyanin (SA), 100 mg of the nanoencapsulated extract was added to 10 mL ethanol. Then it was centrifuged (15°C: 1792 g: 5 min). The supernatant was removed. SA was calculated through differential pH. EEA was calculated from the following formula (Barbosa et al., 2005):

| Encapsulation efficiency of phenol
Encapsulation efficiency of phenol (EEP) was determined according to the method of Kaderides et al. (2015). To measure the total phenol (TP), 5 mg of the sample was added to 5 mL of distilled water. It was then mixed with a magnetic stirrer for 5 min. It was then filtered with Whatman 42 paper. To measure the surface phenolic compounds (SP), 5 mg of the sample was added to 5 mL of distilled water. The surface of the mixture was quickly removed, and the Folin-Ciocalteu method was used to measure TP and SP (Singh et al., 2002). EEP was calculated using the following equation:

| Statistical analysis
Data in this study were analyzed using SAS software version 9.4 by one-way analysis of variance (ANOVA) and Duncan test (p Value ≤ .05).
The results were expressed as mean with standard deviation.
Experiments were performed in three replications.

| Extraction efficiency
The different extraction conditions are given in Table 1. In Table 2, there is a significant difference in the level of confidence above 95% of the yields obtained from different extracts of PPP. The highest rate of extraction related to SEWE was 76.35 ± 1.44 and the lowest rate of extraction related to SEE was 25.73 ± 1.67. Researchers found in a study that the solvent most commonly used to extract polar flavonoids (anthocyanins) is a mixture of water and organic solvents (Chen et al., 2008). The researchers obtained the best extraction efficiency from the combination of ethanol solvent with different concentrations (35%-90%) and water (Chaves et al., 2020).
Researchers reported the highest yield with water-ethanol (50:50) and the lowest yield with water (Malviya et al., 2014). For this reason, some authors recommend that despite its safety and low cost, water is not suitable for the extraction of phenolic compounds. Recently, researchers have estimated that mixing ethanol: acidic water with acetic acid increases extraction performance (Masci et al., 2016).
Inequality between extraction efficiencies can be explained by the difference between the solubility of phenols between solvents (Li et al., 2006). Table 2 shows the value of TAC in different extracts. The highest amount of TAC measured by differential pH by spectrophotometric colorimetry related to ME extract is equivalent to 4.00 mg/g PPP, and the lowest amount of anthocyanin composition with a large difference from other methods for UE extract is 0.35 mg/g PPP. Some studies have reported that extraction solvents can be acidified to protect sensitive flavonoids (anthocyanins) from oxidative degradation (Dzah, 2014), which produces hydrogen ions (H + ) that are free radicals which may be produced during UE (Dzah et al., 2020). This study actually explains the reason for the low amount of anthocyanin extracted by ultrasound in the current study. In a study by Rababah et al. (2010), the amount of anthocyanin in PP was reported to be 1.7-1.3 mg/g dry weight, which is consistent with the results of this study.

| Total phenol content
The TPC is shown in Table 2. TPC for ME was equal to 702.13 mg GAE/100 g of sample, which was higher than SEE, SEWE, and UE, respectively. The results also showed that ethanol as a solvent is more effective than the combination of water-ethanol and water in extracting phenolic compounds from PPP. A study showed that increasing the ratio of solvent to solid from 1:10 to 1:40 in ME leads to the TPC from PP (Huang et al., 2017). Another report evaluated polyphenols, showing that the extraction of these compounds from PP was affected by the pH of the solvent, better results were obtained in an acidic environment, and at extra pH above 7.0, lower extraction efficiencies were recorded (Motikar et al., 2021). In the study conducted by Kaderides et al. (2015) in extracting the phenolic compounds of PP by ultrasound, they obtained the highest amount of phenolic compounds in pomegranate equivalent to 13.85 mg of gallic acid in 100 g of PP. In a study on Pistacia khinjuk, researchers reported TPC in the extract as 46 mg/g dry weight (Hosseinialhashemi et al., 2021). In a report by Rashid et al. (2022), the TPC of PP was reported to be 217.6 mgGAE/g dry powder.
Also, in another study on PP, the amount of TPC was reported as 234.18 mg of GAE/g of extract (Dundar et al., 2023). In another study, phenolic compounds of PP were extracted by ultrasound (at 40°C, 37 Hz). TPC was reported to be 228 mg GAE/100 g of sample (Machado et al., 2019), which is consistent with the results of this study. TA B L E 3 Average particle size distribution, particle size and percentage of distribution in peak 1 in different nanoencapsulated extracts.
F I G U R E 1 FTIR spectrum for nanoencapsulation samples. A, Microwave extraction; B, Solvent ethanol-water extraction; C, Solvent ethanol extraction; D, Ultrasound extraction.

| Nanoencapsulated compounds particle
The nanoencapsulated compounds' particle size of the anthocyanin extract of PPP was measured by a Scatroscope1. The average particle size distribution, particle size at peak 1, and its distribution percentages from different extractions are given in Table 3. The average particle size distribution in UE (329 nm) and SEE (422 nm) extracts showed the smallest and largest particles size, respectively.
In the SEE, the particle size uniformity was lower. The researchers reported that the mixed emulsion reduced the particle size, which they attributed to the different behavior of biopolymers in reducing the particle size . In a study on PP, Rashid et al. (2022) stated that the particle size of encapsulated PP (MD/soy protein isolate) was 191.12 nm.

| FT-IR
The FT-IR spectra for ME extract ( (Jamshidi et al., 2020;Oliveira et al., 2016). Peaks at 934 to 662 are related to the C-H bond in aromatic compounds, the amount of which is the same in SEWE extract and UE extract. Phenolic compounds in the ME extract are higher than that in other extracts.

| Encapsulation efficiency of anthocyanin
The results Table 4 showed that EEA in SEE extract and SEWE extract had insignificant differences. The ME and UE had the lowest (87.64) and highest (96.15) EEA (%), respectively. In a study by Khazaei et al. (2014) stated that with increasing compounds in the sample, the EE decreased, which is also the case in the study. Also, on encapsulated anthocyanins (core/wall =50%), they reported higher EEA with MD/Arabic gum (89%) than MD/gelatin and MD. The results showed that the EEA with the same core/wall ratio was 96% for UE, 93% for SEE, 92% for SEWE, and 87% for ME. In fact, it shows the superiority of LPSG over Arabic gum for nanoencapsulation of PP anthocyanin compounds. In a report by Robert et al. (2010) on anthocyanin encapsulation, they stated that anthocyanin encapsulation of PP and fruit juice was more efficient than its phenolic compounds with MD and soy protein isolate, which actually indicates that the anthocyanin flavylium cation is bonded with the wall compounds.

| Encapsulation efficiency of phenol (EEP)
The highest EEP (%) was related to the SEWE extract, and the lowest was related to the UE extract, and the amount of EEP (%) of the ME extract and the SEE extract was not significantly different (Table 4).
In both extractions, ethanol was used as a solvent, so the polarity of the compounds was similar. The results showed that the phenolic compounds of the SEWE have the highest EEP. Since the chemical structure of the wall and the type of phenolic compounds are suitable, better nanoencapsulation was obtained as a result. The EEP in the UE is at a lower level, which actually indicates inadequate coverage. In a research, Roshanpour et al. (2021) reported EEP using chitosan and Alyssum homolocarpum gum at 88.7% (Roshanpour et al., 2021). Also, in another study, researchers stated that EEP with a combined coating of chitosan and locust bean gum was 93.3% (Estakhr et al., 2020

| CON CLUS ION
Due to the importance of anthocyanins and phenolic compounds and also for the optimal use of food industry waste in this study, extraction of anthocyanins and phenolic compounds from PPP was performed with the help of solvent, microwave, and ultrasound.
They were then nanoencapsulated with MD and LPSG. The results showed that SEWE had the highest efficiency. However, the ME extract had the highest amount of anthocyanin pigment as well as total phenolic compounds compared with other extraction methods.
FT-IR showed the highest amount of phenolic compounds in the nanoencapsulated extract of ME. In the SEE method, the particle size dispersion was higher. The highest EE of anthocyanins and phenolic compounds were related to UE and SEWE, respectively.

ACK N OWLED G M ENTS
This study was a part of Niloofar Zahed's master's thesis with the support of SANRU (Sari Agriculture and University of Natural Resources).

CO N FLI C T O F I NTE R E S T S TATE M E NT
Authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
Research data are not shared.