**Natural Antioxidants: Innovative Extraction and Application in Foods**

Editors

**Monica Rosa Loizzo Ana Sanches Silva**

MDPI • Basel • Beijing • Wuhan • Barcelona • Belgrade • Manchester • Tokyo • Cluj • Tianjin

*Editors* Monica Rosa Loizzo University of Calabria Italy

Ana Sanches Silva National Institute of Agrarian and Veterinary Research Portugal

*Editorial Office* MDPI St. Alban-Anlage 66 4052 Basel, Switzerland

This is a reprint of articles from the Special Issue published online in the open access journal *Foods* (ISSN 2304-8158) (available at: https://www.mdpi.com/journal/foods/special issues/Natural Antioxidants Extraction Application Foods).

For citation purposes, cite each article independently as indicated on the article page online and as indicated below:

LastName, A.A.; LastName, B.B.; LastName, C.C. Article Title. *Journal Name* **Year**, *Volume Number*, Page Range.

**ISBN 978-3-0365-2556-3 (Hbk) ISBN 978-3-0365-2557-0 (PDF)**

© 2021 by the authors. Articles in this book are Open Access and distributed under the Creative Commons Attribution (CC BY) license, which allows users to download, copy and build upon published articles, as long as the author and publisher are properly credited, which ensures maximum dissemination and a wider impact of our publications.

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## **Contents**

## **About the Editors**

**Monica Rosa Loizzo** graduated cum laude in pharmacy at the University of Calabria (Italy) in 1999. She obtained a PhD with her thesis "Methodology for the Development of New Molecules of Pharmacological Interest" in 2005 at the University of Calabria, and also obtained a master's in clinical pathology from the same institution. From 2002 to 2004 she spent time as a researcher at the 'Centre for Bioactivity Screening of Natural Products' at King's College London (UK). In 2008, she spent a period of time in the 'Area of Food Industries and Technologies' laboratory of Politecnica delle Marche University (Ancona, Italy). From 18.12.2008 to 29.12.2017 she was a researcher in food science technology at the Department of Pharmacy, Health Science, and Nutrition of the University of Calabria. In 2017 she become an associate professor at the University of Calabria.

Prof. Loizzo is the author of 204 research papers, 17 book chapters, and 10 editorials. Her h index is 45 and she has over 6805 citations.

Monica Rosa Loizzo developed specific abilities for main separative techniques using different detector systems to determine the chemical composition of different food matrices. She developed specific abilities for the main spectrophotometric methods used to evaluate the health properties via different in vitro methods (e.g., antioxidant, hypoglycemic, hypolipidemic, etc.) of phytochemicals rich-food. Moreover, her research interests include the influence of technological processes on the overall quality of food, with particular reference to compounds capable of prolong the shelf-life (e.g., antioxidant, antimicrobial, anti-browning) of food items She has also studied the chemical composition, sensory evaluation, and health properties of extra virgin olive oil, wines, liquor, fish products and sausages. She is referee of several national and international institutions and was a member of the Management Committee of COST Action "Eurocaroten" until 2020.

Editorial board Member of Antioxidants (2019-today); FOODS (2019-today); Italian Journal Food Science (2019-today); The Open Nutraceuticals Journal (2008-today); International Journal of Nutrition and Dietetics (2012-today); American Journal of Experimental Agriculture (2012-today); Foods (2014-today); Annals of Nutrition and Food Science (2017-today); Food Science and Nutrition Research (2018-today).

**Ana Sanches Silva** obtained a degree in pharmaceutical sciences at the Pharmacy Faculty of the University of Coimbra (FFUC), Portugal, and received her Ph.D. with honors in pharmacy from the University of Santiago de Compostela (USC), Spain. In addition, she was awarded with two awards for best Ph.D. thesis. She is a member of the executive board of the Animal Science Studies Center and was invited to be professor at the FFUC. Ana has a remarkable track record, namely as co-author of papers in peer-reviewed journals with high impact factors, numerous book chapters, and as a co-editor of scientific books in the field of food science. Ana's research has focused on antioxidants, namely natural antioxidants, and their potential for use in active food packaging. In addition, she has a special interest in the development and validation of analytical methodologies (especially related to mass spectrometry) to determine food and food packaging components and contaminants.

## *Editorial* **Natural Antioxidants: Innovative Extraction and Application in Foods**

**Monica Rosa Loizzo 1,\* and Ana Sanches Silva 2,3**

Research has devoted great attention to the study of the biological properties of plants, animal products, microorganisms, marine species, and fungi, among others, often driven by the need to discover new medicines. Many times, in order to enhance biological activities, extracts are prepared. One of the most well-studied biological activities is antioxidant capacity related to anticancer and antiaging properties, improvement of immune function, and protection against cardiovascular diseases and neurological disorders. Moreover, in foods, antioxidants allow for delayed oxidation onset and enhancing food shelf life.

Changes in lifestyle patterns and world population growth demand safe, nutritious, flavourful, colourful, affordable, and convenient food, and high-quality standards have increased the use of food additives, especially antioxidants. The effects of some food additives on human health are controversial, and synthetic food additives are often associated with potential public health risk. Therefore, there is a tendency to substitute synthetic food additives with natural compounds.

We have organized a Special Issue titled "Natural Antioxidants: Innovative Extraction and Application in Foods" in the Foods (ISSN 2304-8158; CODEN: FOODBV; https://www. mdpi.com/journal/foods, accessed on 24 April 2021). This thematic issue focused on the application of innovative extraction techniques for the recovery of natural antioxidants from foods and their possible application in food industries. This Special Issue, now converted into a book, includes 11 chapters, which are important contributions to this topic made by distinguished experts in this area. Ten of these chapters are research papers, and one is a review paper.

Chapter 1 is titled "The Effect of Blanching on Phytochemical Content and Bioactivity of *Hypochaeris* and *Hyoseris* Species (Asteraceae), Vegetables Traditionally Used in Southern Italy" [1]. This chapter regards the effect of blanching on the bioactivity and phytochemical content of *Hypochaeris* and *Hyoseris* species, traditionally used in Southern Italy. The results of this study indicated that these wild plants are a good source of bioactive compounds; however, their antioxidant capacity decreased after blanching. In fact, blanching water presented higher antioxidant capacity than the blanched samples. Therefore, the reuse of blanching water is recommended in food preparation because it is a good source of bioactives, and its consumption can increase the uptake of micronutrients.

Chapter 2 ("Effect of Microwave Pretreatment of Seeds on the Quality and Antioxidant Capacity of Pomegranate Seed Oil") [2] is a very interesting study on the consequences of microwave pretreatment on pomegranate seeds and on the antioxidant capacity of pomegranate seed oil.

A considerable number of quality attributes were evaluated in three different pomegranate cultivars, including yellowness index, refractive index, oil yield, *p*-anisidine value, total oxidation value, conjugated dienes, total phenolic content, peroxide value, total carotenoid content, phytosterol composition, fatty acid composition, and antioxidant capacity through

**Citation:** Loizzo, M.R.; Silva, A.S. Natural Antioxidants: Innovative Extraction and Application in Foods. *Foods* **2021**, *10*, 937. https://doi.org/ 10.3390/foods10050937

Received: 19 April 2021 Accepted: 22 April 2021 Published: 25 April 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

ferric reducing antioxidant power (FRAP) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging capacity. Most of the parameters were increased after microwave pretreatment; however, punicic acid and beta-sitosterol were decreased.

Pomegranate seed oil may be enhanced if microwave pretreated seeds are used, although oil quality varies with cultivar.

Chapter 3 ("Chemical Composition and Antioxidant Activity of Thyme, Hemp, and Coriander Extracts: A Comparison Study of Maceration, Soxhlet, UAE, and RSLDE Techniques") [3] is a valuable chapter comparing different extraction techniques for the obtainment of thyme, hemp, and coriander extracts. The selected techniques were maceration, Soxhlet, ultrasound-assisted extraction, and rapid solid–liquid dynamic extraction (RSLDE). Several parameters were measured in order to compare the extraction techniques. ABTS˙+, FRAP, and DPPH˙ assays were used to evaluate the antioxidant capacity. The total phenolic content by Folin–Ciocalteu method was also evaluated. The results revealed that all the evaluated techniques are valid extraction methods to extract bioactives and preserve their activity. However, extracts obtained by RSLDE showed to have slightly higher antioxidant capacity, and the technique is easy to use besides allowing the standardization of the extraction process.

Chapter 4 regards the paper "Organic Selenium as Antioxidant Additive in Mitigating Acrylamide in Coffee Beans Roasted via Conventional and Superheated Steam" [4]. In this chapter, the effect of coffee beans pretreated with selenium in the formation of acrylamide was evaluated in coffee beans roasted using two different methods (conventional versus superheated steam). The results showed that the antioxidant capacity of the organic selenium suppressed the formation of acrylamide during coffee roasting by 73%. Superheated steam roasting increased antioxidant activity and significantly reduced acrylamide (up to 32%), which was only noticed in the untreated coffee beans.

Chapter 5 is titled "Antioxidant Compounds for the Inhibition of Enzymatic Browning by Polyphenol Oxidases in the Fruiting Body Extract of the Edible Mushroom *Hericium erinaceus*" [5]. This noteworthy chapter focused on the identification of the cause of the dark brown pigmentation via the enzymatic reaction of the polyphenol oxidase (PPO) family with oxidation activity, and the reduction of the occurrence of this pigmentation. The mushroom contained relatively high amounts of natural antioxidant compounds for the inhibition of tyrosinase and the scavenging of free radicals. These antioxidants could diminish the browning reaction via PPO inhibitory mechanisms in the fruiting body of the *H. erinaceus* mushroom.

These results of this chapter allow for understanding the metabolites and PPO enzymes responsible for the enzymatic browning reaction of *H. erinaceus*.

Chapter 6 comprises the paper "Impact of Stability of Enriched Oil with Phenolic Extract from Olive Mill Wastewaters" [6]. This notable chapter evaluated the effect of phenolic extract addition in the oxidative deterioration of sunflower oil. XAD-7-HP resin was used to recover phenolic compounds from olive mill wastewaters. The extract was evaluated in terms of single phenol concentration by ultra-high-performance liquid chromatography. The highest amount was found for hydroxytyrosol. The oxidation state of fortified sunflower oil was evaluated during 90 days for different physicochemical parameters (refractive index, peroxide value, and oxidative resistance to degradation) and antioxidant assays (DPPH, ABTS, and ORAC). The study revealed that there was an increase of 50% in the oxidative stability of fortified oil compared with control. This indicates that olive mill wastewaters can be valorized through an efficient extraction method.

Chapter 7 concerns the paper "A Novel and Simpler Alkaline Hydrolysis Methodology for Extraction of Ferulic Acid from Brewer's Spent Grain and Its (Partial) Purification through Adsorption in a Synthetic Resin" [7].

This chapter interestingly developed a simple method to extract ferulic acid (FA) from brewer's spent grain (BSG), produced by brewing companies. The method includes an autoclave step to perform the alkaline hydrolysis, which allows for simplifying the

postextraction process and increasing the ferulic acid yield. Finally, the extracted ferulic acid carries out a partial purification in a synthetic resin.

Chapter 8 is titled "Radical Scavenging and Antimicrobial Properties of Polyphenol Rich Waste Wood Extracts" [8]. This chapter evaluated the radical scavenging and antimicrobial capacities of wood waste extracts from black locust (*Robinia pseudoacacia* L.), mulberry (*Morus alba* L.), myrobalan plum (*Prunus cerasifera* Ehrh.), wild cherry (*Prunus avium* L.), and different species of oaks (*Quercus petraea* (Matt.) Liebl., *Q. robur* L., and *Q. cerris* L.) in order to conclude about their potential use in the food and pharmaceutical industries. Phenolic compounds were separated by using high-performance thin-layer chromatography (HPTLC), while radical scavenging activity was determined using DPPH-HPTLC. DPPH-HPTLC identified gallic, ferulic, and/or caffeic acids as the compounds with the highest contribution to antioxidant capacity. Regarding antimicrobial capacity, mulberry extract showed the lowest minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values against methicillin-resistant *Staphylococcus aureus*. The growth rate of *Listeria monocytogenes* was significantly inhibited by extracts of myrobalan plum, wild cherry, and mulberry. *Candida albicans* showed poor sensitivity to the action of all extracts, with the exception of the wild cherry extract. *Escherichia coli* was less sensitive to the tested extracts.

The study concluded that due to their antimicrobial activities, cherry and mulberry wood extracts can be useful in preserving short-shelf-life foods.

Chapter 9 comprises the paper "Extract from Broccoli Byproducts to Increase Fresh Filled Pasta Shelf Life" [9]. This is a very interesting chapter that aims to find an alternative to chemical/conventional preservation strategies for fresh filled pasta. The idea was to evaluate the suitability of an extract from broccoli by-products for this purpose. The study monitored microbiological and sensory qualities besides phenolic compound content before and after in vitro digestion of pasta samples.

Results revealed that the shelf life of the natural extract increased by 18 days in comparison with control. The addition of the by-products' extract to pasta increased phenolic content after in vitro digestion. Consequently, it was concluded that broccoli byproducts could be valorized for obtaining extracts able to enhance shelf life and improve the nutritional content of fresh filled pasta.

Chapter 10 is titled "Bioactive Compounds from Norway Spruce Bark: Comparison among Sustainable Extraction Techniques for Potential Food Applications" [10]. This is a great chapter comparing different techniques to extract antioxidants from Norway spruce bark (*Picea abies* (L.) Karst), a wood industry waste. Supercritical fluid extraction (SFE), pressurized liquid extraction (PLE), and ultrasound-assisted extraction (UAE) were compared, and results showed that PLE, using ethanol as solvent, was the most effective method for extracting total flavonoid compounds, with the highest antioxidant capacity according to ABTS˙+. On the other hand, UAE extract contained the maximum phenolic concentration and the highest antioxidant capacity by FRAP. UAE revealed the greatest efficiency in the extraction of *trans*-resveratrol with ethanol 70% (*v*/*v*), therefore suggesting its potential to be used to record antioxidants to be further applied in the food and pharmaceutical industries.

Chapter 11 comprises the review titled "A New Insight on Cardoon: Exploring New Uses besides Cheese Making with a View to Zero Waste" [11]. This chapter is the only review of this Special Issue/book. It regards cardoon (*Cynara cardunculus* L.), which is a plant native to the Mediterranean area whose flowers are used in cheese making, as vegetal rennet. The aim of the review was to address the properties of cardoon leaves, considered a by-product of this crop, and discuss their potential uses. The findings indicated that cardoon leaves are recognized for their potential health benefits, (e.g., diuretic, hepatoprotective, choleretic, hypocholesterolemic, anticarcinogenic, and antibacterial properties), and they can have new potential uses. In particular, they can be used for the preparation of extracts to be incorporated into active food packaging. In sum, the chapter concluded that the new uses of cardoon leaves will contribute to zero waste of this crop.

The chapters of this book address the unequivocal importance of natural antioxidants. There is a plethora of matrices that can be used to obtain natural antioxidants, including different parts of plants (e.g., leaves, bark, seeds), food by-products, and fungi. In conclusion, the choice of extraction technique is critical in order to improve the biological properties, especially the antioxidant and antimicrobial capacities, of the extracts and is strictly related to their potential application in the food industry. Nowadays, the food industry is looking for environmentally friendly extraction procedures as well as extraction procedures that allow for upscaling from lab to industry. Finally, the choice of pretreatment and processing methods can also have a great influence on the antioxidant capacity of the extracts.

**Funding:** This research received no external funding.

**Conflicts of Interest:** The authors declare no conflict of interest.

## **Abbreviations**

## **References**

## *Article* **The Effect of Blanching on Phytochemical Content and Bioactivity of** *Hypochaeris* **and** *Hyoseris* **Species (Asteraceae), Vegetables Traditionally Used in Southern Italy**

**Vincenzo Sicari 1, Monica R. Loizzo 2,\*, Ana Sanches Silva 3,4, Rosa Romeo 1, Giovanni Spampinato 1, Rosa Tundis 2, Mariarosaria Leporini 2 and Carmelo M. Musarella 1**

**Abstract:** The impact of blanching on the phytochemical content and bioactivity of *Hypochaeris laevigata* (HL), *Hypochaeris radicata* (HR), *Hyoseris radiata* (HRA), and *Hyoseris lucida* subsp. *taurina* (HT) leaves was studied and compared to fresh plant materials and residual blanching water. For this purpose, total phenols, flavonoids, carotenoids, and chlorophyll contents were quantified. The antioxidant effect was investigated by using different in vitro tests (β-carotene, ferric reducing ability power (FRAP), 2,2- -Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH), whereas the potential inhibitory activity of key enzymes linked to obesity was screened against lipase, α-amylase, and α-glucosidase. Generally, the phytochemical content followed the trend: fresh > blanching water > blanched samples. The same trend was observed in the antioxidant activity independently of the applied test as well as in the inhibition of lipase and carbohydrates-hydrolysing enzymes. In particular, fresh *Hypochaeris laevigata* (HL1) showed the lowest inhibitory concentration 50% (IC50) values of 31.3 and 42.7 μg/mL, against α-glucosidase and α-amylase, respectively, whereas fresh *Hyoseris radiata* (HRA1) showed the most promising hypolipidemic activity (IC50 value of 39.8 μg/mL). Collectively, these results support the health effect of these wild plants and demonstrated that blanching water should be reused in food preparation since it is a good source of bioactive compounds and its consumption should be recommended in order to increase the uptake of micronutrients.

**Keywords:** *Hyoseris radiata*; *Hyoseris taurina*; *Hypochaeris laevigata*; *Hypochaeris radicata*; phytochemicals; antioxidants; obesity; diabetes type 2

## **1. Introduction**

*Hyoseris* L. and *Hypochaeris* L. species are largely used in Italy not only to prepare salads, omelettes or boiled in soups, but also for medicinal use through the use of infusions using blanching water. *Hyoseris radiata* L. and *Hypochaeris radicata* L. are the most studied species and have several ethnobotanical uses in Italy [1–8]. *Hyoseris lucida* L. subsp. *taurina* (Pamp.) Peruzzi & Vangelisti and *Hypochaeris laevigata* (L.) Ces., Pass. & Gibelli have not been much studied, probably due to their relatively narrow geographical distribution [9,10]. Guarrera and Savo [11,12] reported the use of leaves from *H. radicata* and *H. radiata* as boiled vegetables or salad in Piedmont, Liguria, Marche, Latium, Sardinia, Sicily, and Calabria. In this last region, these species are preserved in olive oil to be consumed as vegetable side dish.

**Citation:** Sicari, V.; Loizzo, M.R.; Sanches Silva, A.; Romeo, R.; Spampinato, G.; Tundis, R.; Leporini, M.; Musarella, C.M. The Effect of Blanching on Phytochemical Content and Bioactivity of *Hypochaeris* and *Hyoseris* Species (Asteraceae), Vegetables Traditionally Used in Southern Italy. *Foods* **2021**, *10*, 32. https://dx.doi.org/10.3390/foods 10010032

Received: 27 November 2020 Accepted: 22 December 2020 Published: 24 December 2020

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/ licenses/by/4.0/).

Metabolic syndrome is a cluster of conditions that occur together.  The World Health Organization (WHO) Global Health Observatory estimated through Prophet models that the prevalence of diabetes and obesity in 2030 is likely to increase by 10.1% [14].

Oxidative stress plays a key role in the development of Diabetes Mellitus Type 2 (DMT2) complications [15]. In fact, the metabolic irregularities of diabetes cause an overproduction of superoxide in endothelial cells of both large and small vessels, and, also in the myocardium. Moreover, Găman et al. [16] demonstrated that these irregularities impair tissue glucose uptake and reduces β cell insulin secretion.

The consumption of wild plants represents a key part of the Mediterranean diet, recently recognized by United Nations Educational, Scientific and Cultural Organization UNESCO (UNESCO) as an Intangible Cultural Heritage of Humanity (UNESCO). Although several plants of *Hyoseris* and *Hypochaeris* species have been consumed daily for centuries, there is still no in-depth study on their micronutrient content and health properties after preparation prior to consumption. In the food industry and at a domestic level, blanching is a pretreatment largely used to inactivate enzyme activity, which can affect micronutrient content and preserve vegetables [17]. However, if it is effective in reducing degradation during shelf-life, on the other hand it produces modifications in cell structure and composition with a consequent significant loss of micronutrients in the food matrix [18].

In this context, the evaluation of the impact of blanching on Calabrian traditional vegetables, namely *H. laevigata*, *H. radicata*, *H. radiata,* and *H. lucida* subsp. *taurina,* was investigated. For this purpose, basal leaves were screened for their Total Phenols Content (TPC), Total Flavonoids Content (TFC), chlorophylls and Total Carotenoids Content (TCC) before and after blanching process. β-Carotene bleaching, FRAP, ABTS, and DPPH tests were applied to test the antioxidant activity. The inhibitory effects of enzymes linked to obesity and DMT2 such as lipase, α-glucosidase, and α-amylase were assessed. In order to evaluate the possible loss of bioactive compounds after blanching, the residual blanching water was also investigated.

## **2. Materials and Methods**

#### *2.1. Chemicals and Reagents*

Chemicals and reagents used in this research were purchased from Sigma-Aldrich Chemical Co. Ltd. (Milan, Italy) and VWR International (Milan, Italy). Acarbose from *Actinoplanes* spp. was obtained from Serva (Heidelberg, Germany) whereas β-carotene was from Extrasynthese (Genay-France).

#### *2.2. Plant Materials*

The collection (Table 1) of the basal rosettes of *H. radicata* and *H. radiata* was carried out at 1157 m a.s.l. in the area of the Aspromonte Massif known as "Cucullaro" (38.171987◦ N–15.815650◦ E), a mountain resort in the municipality of Santo Stefano in Aspromonte. The collection of the basal rosettes of *H. lucida* subsp. *taurina* was carried out in the municipality of Scilla (40 m a.s.l., 38.253987◦ N–15.716493◦ E), while those of *H. laevigata* at the mountain resort of Trepitò, located at 952 m a.s.l. (38.284906◦ N–16.046640◦ E) in the municipality of Molochio. The collection of the basal rosettes was carried out before flower stem development, as traditionally these and similar plants are collected by local people in this way [8,19]. All the collection sites are located in the province of Reggio Calabria (South Italy)

**Table 1.** Sampling locations.

For each of the 4 taxa examined, a minimum quantity of 1000 g of basal rosettes was collected and, afterwards, in the laboratory, a cleaning and separation of the edible leaves was performed manually to obtain a minimum quantity of 200 g of leaves. These were washed several times with distilled water in order to ensure the elimination of any type of residue. Finally, they were placed on absorbent paper and gently dabbed several times, so as to eliminate as much water as possible and thus avoid problems of contamination.

#### *2.3. Blanching*

To blanche, leaves were placed on a low heat in 1000 mL of hot water (90 ◦C) for 10 min. The ratio of sample to water was 1:5 (*w*/*w*). After blanching, the samples were cooled to room temperature under running tap water. Finally, the edible parts were drained and the blanching water collected.

## *2.4. Extraction Procedure*

Extracts were prepared by mixing 10 g of each sample (fresh and blanched, both ground) with 50 mL of water. The mixture was shaken using a Ultraturrax T-25 (Ika Labortechnik, Janche & Kunkel, Milan, Italy). It was subsequently centrifuged by refrigerated centrifuge Nᑂve NF 1200R (Saracalar Kümeevleri, Ankara, Turkey), 10 min at 5000 rpm. Then, the supernatant was filtered through a 0.45 mm Millipore filter (GMF Whatman, Carlo Erba, Milan, Italy) before analysis.

#### *2.5. Determination of pH, Titratable Acidity and Total Soluble Solid Content*

The pH was determined by direct measurement in a digital potentiometer (Crison Instruments S.A., Milan, Italy). The Total soluble solids (TSS) were measured at 20 ◦C using a digital Atago Model PR-101 α refractometer (Atago Co. Ltd., Milan, Italy), results were reported as Brix degrees (◦Brix). The titratable acidity (TA) was determined using 0.1 N NaOH to pH 8.1. Results were expressed as percentage of monohydrate citric acid.

#### *2.6. Total Phenol Content (TPC) and Total Flavonoid Content (TFC)*

TPC was determined as described by Sicari et al. [20]. An aliquot of 350 μL of aqueous extract was mixed with Folin-Ciocalteu reagent (1 mL) and 20% Na2CO3 solution (10 mL). The absorbance was measured at 760 nm in a spectrophotometer (UV-VIS-Agilent 8453) and the results were expressed in mg of gallic acid equivalent (GAE)/100 g fresh weight (FW).

A spectrophotometric method was used to measure the total flavonoid content. TFC was determined using a method based on the formation of a flavonoid-aluminium complex [21]. The extract was mixed with 2% aluminium chloride solution. The samples were incubated at room temperature for 15 min and then measured against a blank at 510 nm. TFC was calculated based on a standard curve and expressed as mg quercetin equivalents (QE)/100 g FW.

#### *2.7. Spectrophotometric Determination of Carotenoids*

For the carotenoid determination, the spectrophotometric analysis was carried out after extraction [22]. The analyses were carried out in darkness to prevent carotenoid degradation and isomerisation. Before chemical extraction, leaves were homogenised in a blender and an aliquot of 5 g of the sample was weighed into a 50 mL amber coloured flask wrapped with aluminium foil. Then, 100 mL of the solvent mix (hexane/acetone/methanol 2:1:1 *v*/*v*/*v*) was added to the flask and sonicated continuously for 10 min (Misonix Ultrasonic Liquid Processor, Misonix, Inc. 1938, New Highway, Farmingdale, NY, USA).

The extraction was repeated until the sample became colorless. The combined extract was transferred to a separating funnel and 5 mL of distilled water was added to separate polar and nonpolar phases. The nonpolar hexane layer containing carotenoids was collected and concentrated in a rotary evaporator (Heidolph, Schwabach, Germany) until dry. The residue was dissolved in 10 mL of hexane. The total carotenoid content was determined by a spectrophotometric method using a UV-Vis spectrophotometer (Agilent 8453 Technologies, Agilent, Milan, Italy). The absorbance was read at 450 nm. All analyses were performed in triplicate and the results were expressed as mean ± standard deviation (SD).

The results were expressed as g β-carotene/100 g fresh weight of sample.

#### *2.8. Chlorophyll Determination*

The chlorophyll content was determined spectrophotometrically. The leaves were placed into a mortar and then ground in the dark until the green colour disappeared.

Ground leaf material was extracted with ethanol, filled to 10 mL, centrifuged at 5000 rpm for 3 min and the absorbance of the supernatant solution in a 1 cm cell was read at 440, 649 and 665 nm. Chlorophyll was calculated using the equations previously reported by Lichtenthaler and Buschmann [23].

#### *2.9. Radical Scavenging Activity Assays*

In ABTS radical scavenging ability test, potassium persulphate solution and ABTS solution were mixed to obtain ABTS radical cation solution [21]. After 12 h, this solution was stabilized (absorbance of 0.70) at 734 nm employing a UV-Vis spectrophotometer (Jenway 6003, Carlo Erba, Milan, Italy). ABTS+ solution was mixed with different concentrations of aqueous extracts (from 1 to 400 μg/mL) and the absorbance was read at 734 nm after 6 min.

Another test used to evaluate the radical scavenging activity of our samples is the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay [21]. The DPPH radical solution was mixed with aqueous extracts (at concentrations in the range from 1 to 1000 μg/mL) and after 30 min the absorbance was read at 517 nm.

#### *2.10. Ferric Reducing Ability Power (FRAP) Assay*

In the ferric reducing ability power (FRAP) assay, FRAP reagent, tripyridyltriazine (TPTZ), HCl, FeCl3, acetate buffer (pH 3.6) were mixed and added to the sample (2.5 mg/mL) [24]. The absorption reaction was read at 595 nm after 30 min of incubation.

#### *2.11. β-Carotene Bleaching Test*

The ability of samples to inhibit lipid peroxidation was evaluated by using the βcarotene bleaching test [21]. The aqueous extracts (from 2.5 to 100 μg/mL) were mixed with linoleic, Tween 20 acid and β-carotene solution. The absorbance was read at t = 0, and after 30 and 60 min of incubation at 470 nm.

## *2.12. Pancreatic Lipase Inhibitory Activity*

The hypolipidemic potential of aqueous extracts was studied by the inhibition of pancreatic lipase [25]. A mixture of lipase (1 mg/mL), 4-nitrophenyl octanoate (substrate), and samples (from 25 to 4000 μg/mL) was prepared. After 30 min of incubation at 37 ◦C, the Tris-HCl buffer (pH 8.5) was added and the absorbance was measured at 405 nm.

#### *2.13. Carbohydrate-Hydrolysing Enzyme Inhibitory Activity*

The hypoglycaemic activity of samples was evaluated through inhibition of α-amylase and α-glucosidase enzymes [21]. For α-amylase assay, aqueous extracts (from 25 to 1000 μg/mL) and starch solution were added to the enzyme at room temperature for 5 min, and the absorbance at 540 nm was measured. For α-glucosidase inhibitory activ-

ity test, samples extracts (from 25 to 1000 μg/mL) were mixed with enzyme solution, a maltose solution, peroxidase/glucose oxidase (PGO) system-colour reagent solution and *o*-dianisidine (DIAN) solution. This mixture was incubated for 30 min and the absorbance was measured.

## *2.14. Statistical Analysis*

All the investigations were performed in triplicate and results were expressed as means of three different experiments ± standard deviation (S.D.). They were processed by analysis of variance (ANOVA). Differences among the samples were analyzed by Turkey's test using SPSS statistics software (version 17.0, SPSS Inc., Chicago, IL, USA). All the *p* values at < 0.05 were observed as significant.

Principal Component Analysis (PCA) was applied using SPSS software for Windows, version 17.0 (SPSS Inc., Chicago, IL, USA).

## **3. Results and Discussion**

In this work the phytochemical content and bioactivity of traditionally consumed wild plants namely *H. laevigata*, *H. radicata*, *H. radiata,* and *H. lucida* subsp. *taurina* were investigated. The edible portions (leaves) were studied, both fresh and after blanching, to assess the impact of processing on these food matrices. Among bioactive phytochemicals, TPC, TFC, lycopene, β-carotene, and chlorophylls were quantified. Samples were studied for their antioxidant potential using different approaches and as inhibitors of enzymes linked to obesity and hyperglycaemia.

#### *3.1. Phytochemical Content*

Used in the experiment procedures had a significant effect on changes of total soluble solids (TSS), titratable acidity (TA), and pH. The blanching effect of leaves is shown in Table 2. Fresh *Hypochaeris radicata* (HR) and *H. laevigata* (HL) leaves showed a higher TSS contents than the values obtained in the leaves after blanching and in the blanching water. The same trend was observed in the titratable acidity values.

**Table 2.** Total soluble solid content (TSS), Titratable acidity (TA), and pH of *Hypochaeris* and *Hyoseris* extracts.

**Table 2.** *Cont.*

Data are reported to mean ± Standard Deviation (SD) (*n* = 3). 1: Extract fresh plant materials; 2: Extract plant materials after blanching; 3: Blanching water extract. \*\* Significance at *p* < 0.01. Results followed by different letters in a same column are significantly different (*p* < 0.05) by Tukey's multiple. range test.

Carotenoid content in raw and cooked leaf samples are shown in Table 3. The reported values show a greater concentration of total carotenoids (expressed in β-Carotene equiv.) in the fresh leaves than in the blanched ones. The blanching water had a significantly higher (*p <* 0.05) carotenoid content than the blanched leaves, but lower than the fresh leaves. A similar trend was found in leaves of both *Hypochaeris* (*leavigata* and *radicata*) and *Hyoseris* (*radiata* and *lucida* subsp. *taurina*). Compared to the initial concentration in the fresh leaves (HL1, HR1, HRA1 and HT1), the lowest carotenoid concentration in blanching water was found in sample HT3, with a value of 205.2 g/100 g, whereas the highest value was found in sample HRA3 (298.1 g/100 g). The presence of carotenoids in blanching water was reported by Parmar et Rupasinghe [26], who observed that an infusion of wild berry stems in hot water had a carotenoid content in the range of 275–417 mg/L. They further showed a high carotenoid content in commercial green tea with average values of 410 and 1332 mg/L for extraction in methanol and hot water respectively. In a study carried out by Loranty et al. [27] on 25 infused fruit and herbal teas, only lutein was present in the infusions, other carotenoids not being found. The highest level of lutein (24.3 mg/200 mL) was found in a Tilias infusion (Edward Tea). Suzuki et Shioii [28] found chlorophyll and carotenoids in seven teas infused from *Camellia sinensis* leaves.

**Table 3.** Phytochemical content of *Hypochaeris* and *Hyoseris* extracts.

**Table 3.** Phytochemical content of *Hypochaeris* and *Hyoseris* extracts.

Data are reported to mean ± Standard Deviation (SD) (*n* = 3). Data are expressed as mean ± S.D. (*n* = 3). 1: Extract fresh plant materials; 2: Extract plant materials after blanching; 3: Blanching water extract. \*\* Significance at *p* < 0.01. Results followed by different letters in a same column are significantly different (*p* < 0.05) by Tukey's multiple range test.

A drastic reduction in chlorophyll was also observed between fresh and blanched samples (from 170.2 to 212.3 for HL1 and HRA1, vs. from 89.3 to 118.4 for HRA2 and HR2, respectively). Blanching reduced the chlorophyll content independently of the species (Table 3).

The concentration of total polyphenols and flavonoids was shown in Table 3. Phenolic compounds are known to be responsible for the antioxidant activity of the food matrix. All fresh samples were characterized by a high TPC content. Leaves of *hypochaeris* (*leavigata* e *radicata*) and *hyoseris* (*radiata* e *lucida* subsp. *taurina*), showed a significantly higher (*p* < 0.05) total polyphenol content in their raw samples compared with their cooked samples [29,30]. The aqueous extract of fresh *H. radicata* showed the highest TPC value of 1103.6 mg GAE/100 g FW followed by *H. lucida* subsp. *taurina* (997.6 mg GAE/100 g FW) (Table 3). Fresh *H. radiata* leaves (HRA1) showed the highest TFC with a value of 855.6 mg QE/100 g FW. It is interesting to note that the blanching process significantly affects both TPC and TFC and that the water in which the leaves are cooked retained a great amount of these bioactive phytochemicals.

The total polyphenol content decreased significantly (*p* < 0.05) after blanching as reported by [31–34]. In addition, Gawlik-Dziki [35] demonstrated that boiling significantly reduced the polyphenol content of fresh broccoli. Similarly, Sikora et al. [36] reported a significant decrease in total polyphenol and antioxidant components in boiled broccoli. Abu-Ghannam and Jaiswal [37] described a reduction in the total phenolic content of up to 45% at lower blanching temperatures (80–90 ◦C) within 2 min of blanching and reduction of the total polyphenol content continued within 6 min at high blanching temperatures. Furthermore, as some authors have observed, the degree of leaf fragmentation may be a factor in a greater diffusion of the bioactive compounds from the leaves to the water.

Medina et al. [38] compared infusions obtained from samples at various degrees of fragmentation. Levels of oleuropein were found to be 103 mg/kg in an infusion from a whole sample used as control, and 466 mg/kg in another sample passed through a blender and subsequently an Ultra-Turrax. The greater the degree of leaf fragmentation, the greater the quantities of phenols in the infusions. The total flavonoid content dropped significantly (*p* < 0.05) after boiling. Interestingly, despite heating, the concentration of flavonoids in the blanching water remained high. This may be due to the fact that after boiling there was a greater availability of flavonoids, and a more efficient extraction from the softened cell walls [39].

Moreover, several studies have reported that the increase in bioactive compounds in boiled vegetables may be partly due to the breakdown of cell walls and subcellular structures by boiling, which allows the release of antioxidants [40]. Thus, it is probable that the structural matrix of the cell walls is the factor that determines the cell's ability to hold onto or breakdown phytochemical compounds.

## *3.2. Antioxidant Activity*

The antioxidant activities of *H. laevigata* (HL), *H. radicata* (HR), *H. radiata* (HRA), and *H. lucida* subsp. *taurina* (HT) fresh and blanched leaves were assessed employing in vitro methods: β-carotene bleaching, FRAP, ABTS, and DPPH tests. The resulting blanching water was also screened. To our knowledge this is the first report to evaluate the antioxidant potential of *H. laevigata*, and *H. lucida* subsp. *taurina* leaves, both fresh and processed. Generally, the following antioxidant trend was observed fresh samples > blanching water > blanched samples (Table 4).

**Table 4.** Antioxidant activity of *Hypochaeris* and *Hyoseris* extracts.

Data are expressed as mean ± Standard Deviation (SD) (*n* = 3). 1: Extract fresh plant materials; 2: Extract plant materials after blanching; 3: Blanching water extract. Ferric Reducing Antioxidant Power (FRAP); Antioxidant Capacity Determined by Radical Cation (ABTS+); DPPH Radical Scavenging Activity Assay. a: [100 μg/mL]. Propyl gallate (IC50 = 0.09 ± 0.04 μg/mL after t = 30 min and t= 60 min of incubation) was used as control positive in β-carotene bleaching test, BHT (IC50 = 63.2 ± 2.3 μMFe (II)/g) in FRAP assay and ascorbic acid in ABTS and DPPH radical scavenging test (IC50 = 5.0 ± 0.8 and 1.7 ± 0.1 μg/mL, respectively). One-way ANOVA followed by Tukey's multiple range test was applied for statistical analysis. Different letters in the same column are significantly different \*\* at *p* < 0.01.

A great variability of results was observed in the β-carotene bleaching test. In this assay, the presence of antioxidant compounds minimized the oxidation of β-carotene by hydro-peroxides, which were counteracted by bioactive compounds in the extract. In the present study, both *H. radicata* and *H. laevigata* fresh leaves exerted a greater activity than the other investigated species (IC50 = of 46.7 and 54.6 μg/mL after 30 min of incubation, respectively). Blanched samples were less active with a percentage of inhibition of 39.6% and 48.1% at maximum concentration tested (100 μg/mL). A low activity was observed, also with HT1 sample (IC50 = 84.7 and 99.1 μg/mL after 30 and 60 min of incubation, respectively).

In FRAP assay, a great ferric reducing power higher than that found for BHT (Butylated Hydroxytoluene) was observed with both HL3 and HL1 samples with values of 92.6 and 84.4 μM Fe(II)/g, respectively (Table 4). A promising ability was observed also with HRA1 (73.5 μM Fe(II)/g).

A different result was observed using ABTS radical cation. In fact, in this test fresh *H. lucida* subsp. *taurina* leaves (HT1) showed a comparable radical scavenging activity to that reported for ascorbic acid (IC50 = 1.8 and 1.7 μg/mL, respectively). A promising ABTS radical scavenging potential was noted with the blanching water from the blanching of the leaves of the same species (HT3, IC50 = 2.9 μg/mL).

The DPPH free radical scavenging method is based on electron-transfer reaction that produces a violet solution. The DPPH radical is stable at 25 ◦C. Among the investigated species, aqueous extract of fresh leaves of HR and HL exerted a higher radical scavenging potential (IC50 = 18.7 and 24.7 μg/mL, respectively) (Table 4). In addition, interest activity was found for HR3 and HRA3 (IC50 = 23.7 and 29.7 μg/mL, respectively).

Previously, Senguttuvan et al. [41] studied the radical scavenging potential of an infusion from the dried leaves of *H. radicata* and found IC50 values of 595.23 μg/mL and 2143.1 μmol of Trolox equivalent (TE)/dried weight (DW). Dried leaves are also able to exert their antioxidant activities through other mechanisms, including the protection of lipid peroxidation and ferric reducing power.

Values of 97.99% at 250 mg/mL and 38.69% at 5 mg/mL were recorded by Senguttuvan et al. [41] for Indian *H. radicata* dried leaves in DPPH and ABTS test, respectively. Successively the same research group showed that the oral administration of *H. radicata* methanolic leaf and root extracts and isolated compounds proved to be significant, promising candidates able to quench free radicals. The antioxidant effect was more pronounced in the animals treated with root extract, probably due its high content in alkaloids, flavonoids, saponins and terpenoids that could act as antioxidant compounds [41]. Lower antioxidant activity was recorded for *H. radicata* collected in Latium [42] where IC50 average values of 2.02 and 2.33 mg/mL were found for fresh and boiled leaves, respectively in DPPH assay. Average values of 6.2 and 2.3 mmol/kg FW were found for fresh and boiled leaves in ABTS test, respectively.

Compared to the Calabria sample HRA1, a lower ferric reducing power was recorded for *H. radiata* fresh leaves collected in Liguria that showed FRAP value of 31.1 mM Fe (II) /Kg. More recently, Souilah et al. [43] investigated the antioxidant effect of *n*-butanol, dichloromethane and ethyl acetate fractions of the aerial parts of *Hypochaeris laevigata* var. *hipponensis*. The highest DPPH radical scavenging activity was exhibited by *n*-Butanol extract (IC50 = 8.12 μg/mL) followed by ethyl acetate extract (IC50 = 8.70 μg/mL). This last extract was also able to exert a potent ABTS radical cation inactivation (IC50 = 4.32 μg/mL). The following rank dichloromethane, *n*-butanol and ethyl acetate in protection of lipid peroxidation was found.

## *3.3. Inhibition of Enzymes linked to Obesity*

The study of bioactive foods useful in the prevention and management of metabolic diseases including obesity and diabetes is a topic of great interest for researchers in the area of food science. To our knowledge, no previous studies have investigated the species *Hypochaeris* and *Hyoseris* for their ability to inhibit carbohydrate-hydrolyzing and lipase enzymes. All investigated samples exerted inhibitory activity on enzymes linked to obesity and DMT2 in a concentration dependent manner (Table 5).

**Table 5.** Lipase, α-amylase, and α-glucosidase inhibitory activity [IC50 (μg/mL)] of *Hypochaeris* and *Hyoseris* extracts.

Data are expressed to mean ± Standard Deviation (SD) (*n* = 3). 1: Extract fresh plant materials; 2: Extract plant materials after blanching; 3: Blanching water extract. Orlistat used as positive control in lipase test (IC50 = 37.4 ± 1.0). Acarbose used as positive control in α-amylase and α-glucosidase tests (IC50 = 50.1 ± 1.3 and 35.5 ± 0.9 respectively for α-amylase α-glucosidase). One-way ANOVA followed by Tukey's multiple range test was applied for statistical analysis. Different letters in the same column are significantly different \*\* at *p* < 0.01.

Fresh *H. laevigata* leaves (HL1) exerted a promising α-amylase inhibitory activity with IC50 value of 42.7 μg/mL which is lower compared to positive control acarbose (IC50 = 50.1 μg/mL). A notable activity was observed, also in the blanching water (HL5, IC50 = 56.8 μg/mL). The HL1 sample showed the greatest α-glucosidase inhibitory activity followed by *H. radicata* (HR1) with IC50 values of 31.3 and 37.6 μg/mL, respectively. Both results are comparable with those found for acarbose (IC50 = 35.5 μg/mL). Also, in this case, blanching significantly affected the bioactivity of the samples. Fresh *H. radiata* leaves (HRA1) showed the highest lipase inhibitory activity (IC50 = 39.8 μg/mL) whereas, values of 52.4 and 58.2 μg/mL were found for HR1 and HL1, respectively. Unlike what happened for the carbohydrate hydrolysing enzymes, the blanching water has only a minimal inhibitory activity (IC50 values ranged from 62.8 to 78.8 for HR3 and HT3, respectively) testifying that compounds able to inhibit lipase enzyme are retained in the matrix also after blanching.

Among our investigated samples, only *H. radicata* had been previously investigated for its hypoglycaemic activity. However, our data disagree with Ko et al. [44], who did not find α-glucosidase inhibitory activity at the maximum concentration tested of 1000 μg/mL. Probably, this lack of activity was due the relatively low content of TPC and TFC found by the authors.

## *3.4. Principal Component Analysis (PCA)*

PCA was performed to identify accession groups and to determine the axes and the characters significantly contributing to the variation. In this procedure, the similarity matrix was used to generate eigenvalues and scores for the accessions. The first two principal components, which accounted for the highest variation, were then used to plot two-dimensional scatter plots [45].

PCA was applied to differentiate the four different taxa of *Hypochaeris* and *Hyoseris.* By choosing eigenvalues greater than one (>1), the dimensionality was reduced from 16 variables to two principal components (PC). PCA results revealed that the first two principal components explained total variance completely 78.4%. The loadings of first and second principal components (PC1 and PC2) accounted for 45.25 and 33.15% of the variance, respectively (Figure 1). The first component (PC1) is highly positively correlated with Chlorophylls, and FRAP. The second component (PC2) is positively correlated with TSS, β-carotene bleaching test t = 30 min, β-carotene bleaching test t = 60 min, while citric acid, β-Carotene, TPC, and TFC) are positively correlated with component 1 and component 2. pH, DPPH, α-glucosidase, and α-amylase show a negative correlation for PC1 and PC2.

**Figure 1.** Factor loadings for principal components (PC) PC1 and PC2 and scatter plot of all. samples for principal component analysis.

The bi-dimensional PCA analysis clearly classifies the similarities or differences of the botanic species and the treatments performed. The score plot analysis clearly classifies the species HL1, HR1, HRA1, and HR3, HL3, HRA3 in the lower right region of the PCA score plot. This shows that fresh plants and blanching water maintain a higher bioactivity than blanched leaves.

#### **4. Conclusions**

The present study assessed for the first time the impact of the blanching process on the phytochemical content and bioactivity of the spontaneous plants namely *Hypochaeris laevigata*, *H. radicata*, *Hyoseris radiata,* and *H. lucida* subsp. *taurina.* Traditionally, these species are widely consumed in Central and South Italy, both fresh and after blanching. For this purpose, fresh and blanched samples as well as residual blanching water were studied. The blanching process determined a reduction in the content of all investigated phytochemical classes. At the same time, the analysis of the data showed that the blanching water retains most of the bioactive compounds and for this reason it is characterized by a good antioxidant and inhibitory activity against enzymes linked to obesity and related diseases such as diabetes type 2. For this reason, the consumption of the fresh spontaneous plant and the reutilization of residual blanching water should be promoted in order to

ensure the right amounts of healthy micronutrients able to counteract oxidative stress and related diseases.

**Author Contributions:** Methodology, V.S. and M.R.L.; software, G.S.; validation, V.S., M.R.L., and R.T.; formal analysis, M.L. and R.R.; investigation, M.L. and R.R.; data curation, M.R.L., V.S., M.L., and A.S.S.; writing—original draft preparation, M.R.L. C.M.M., and V.S.; writing—review and editing, M.R.L., V.S., and R.T. project administration, V.S. and M.R.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Conflicts of Interest:** No potential conflict of interest was reported by the authors.

## **References**

## *Article* **E**ff**ect of Microwave Pretreatment of Seeds on the Quality and Antioxidant Capacity of Pomegranate Seed Oil**

## **Tafadzwa Kaseke 1, Umezuruike Linus Opara 1,2,\* and Olaniyi Amos Fawole 3,\***

Received: 19 August 2020; Accepted: 6 September 2020; Published: 14 September 2020

**Abstract:** Microwave pretreatment of oilseeds is a novel technique used to enhance oil nutraceutical properties. In this study, the effect of microwave pretreatment of seeds was investigated on pomegranate seed oil quality attributes including oil yield, yellowness index, refractive index, peroxide value, ρ-anisidine value, total oxidation value, conjugated dienes, total phenolic content, total carotenoids content, phytosterol composition, fatty acid composition, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging capacity, and ferric reducing antioxidant power (FRAP). The seeds of three different pomegranate cultivars ('Acco', 'Herskawitz', and 'Wonderful') were microwave heated at 261 W for 102 s. Pomegranate seeds microwave pretreatment enhanced oil yield, yellowness index, total carotenoids content, total phenolic content, FRAP and DPPH radical scavenging capacity, despite an increase in conjugated dienes, and peroxide value. Palmitic acid, oleic acid, linoleic acid, saturated, and monosaturated fatty acids were increased after pomegranate seeds microwave pretreatment, whilst the levels of punicic acid and β-sitosterol were reduced. Nevertheless, the refractive index, the ratio of unsaturated to saturated fatty acid of the extracted oil were not significantly (*p* > 0.05) affected by pomegranate seeds microwave pretreatment. Principal component analysis and agglomerative hierarchical clustering established that 'Acco' and 'Wonderful' oil extracts from microwave pretreated PS exhibited better oil yield, whilst 'Herskawitz' oil extracts showed higher total carotenoids content, total phenolic content, and antioxidant capacity.

**Keywords:** pomegranate seeds; oil; microwave pretreatment; total phenolic content; antioxidant capacity

## **1. Introduction**

The demand for the use of natural products in preventing chronic and degenerative diseases has increased in recent decades, driven by increased consumer health awareness [1]. Attention has been given to functional foods that provide both nutritional functions and health benefits. Pomegranate fruit is rich in both nutritional and biological properties [2]. The fruit has been cultivated since ancient times throughout the Mediterranean region, mainly for its nutritional and pharmacological value [3]. Pomegranates have been used in the treatment of sore throats, coughs, ulcers, urinary infections, intestinal worms, digestive disorders, skin disorders, and arthritis for centuries [4]. Apart from being consumed as fresh fruits, pomegranates may be processed into various products such as juice, jam, wine, syrup, among other products. The fruit contains seeds that range between 37 and 143 g/kg

of the total fruit weight depending on factors such as cultivar, growing region, growing conditions, and maturity stages [5,6]. Pomegranate seeds (PS) have oil that range between 12% to 20% (dry weight basis) and is a good source of bioactive compounds such as punicic acid, tocopherols, phenols, sterols, and carotenoids [3,7].

Epidemiological studies have revealed that pomegranate seed oil (PSO) has biological properties related to the prevention of microbial growth, lipoperoxidation, skin photoaging, cancer, diabetes, and obesity that are linked to the bioactive compounds [7,8]. In line with the biological activities, the oil can be used as a functional ingredient in nutraceutical, pharmaceutical, and functional foods preparations [9]. Despite the wealth of health benefits in PSO, the seeds are still regarded as waste after processing the fruits into juice and other products, thereby increasing the total pomegranate fruit postharvest losses. In some instances, the seeds are used as stock feed [10]. From an industrial and health perspective, valorization of the PS into oil presents a more valuable alternative utilization of the postharvest waste.

Among other conventional seed oil extraction techniques, such as cold pressing and supercritical carbon dioxide, extraction using organic solvents is the best technique with regards to oil extraction efficiency [11]. Needless to say, the use of organic solvents such as hexane has become unpopular due to its hazardous effects on humans and the environment [11,12]. Consequently, the avoidance or reduction in the use of hexane as an extraction solvent has become a requirement for the food industry [13]. Alternatively, short-chain alcohols such as ethanol are promising solvents in seed oil extraction. Ethanol is a less hazardous, bio-renewable, and cheaper organic solvent [14]. The high polarity of ethanol provides it with the ability to extract polar bioactive compounds, such as the phenolic compounds, and therefore oil extracted with ethanol has better biological activities [15]. The main drawback in using ethanol is that it produces low oil yield [16,17]. Therefore, treatment of the oilseeds before oil extraction is important for improving the oil extraction efficacy of ethanol.

The treatment of seeds with microwaves before oil extraction has received great interests due to obvious advantages including uniform energy delivery, high thermal conductivity to the interior of the material, energy saving, and precise process control [18]. The application of microwave radiation to seeds result in direct interaction of the electromagnetic waves with the polar oxygen group from the seeds moisture [19]. This results in rapid heating and evaporation of the moisture in the seeds, thereby creating an internal pressure that causes the rapture of seed matrices [20]. The seeds microstructure alterations facilitated by microwave heating increases the interaction of the extraction solvent with the intracellular materials and enhances the lipids and bioactive compounds mass transfer into the extraction solvent [21]. Zhang and Jin [22], Li et al. [23], Porto et al. [24], and Güne¸ser and Yilmaz [25] have reported an improvement in oil yield and bioactive compounds recovery after microwave pretreatment of the camellia oleifera, yellow horn, moringa, and orange seed, respectively.

In order to establish whether microwave pretreatment adds value or not to pomegranate seed oil, it is important to investigate different cultivars. Cultivar significantly influences the quality of seed oil from the perspective of genetic characteristics variation [26]. Therefore, the effect of microwave pretreatment on the quality of the extracted oil may also vary with cultivar. To optimize the economic benefits, cultivars with better oil quality after seeds microwave pretreatment are valuable to food processors. However, information about the application of microwave irradiation on seed from different cultivars to establish variation in the quality of the extracted oil is limited.

Therefore, this study aimed to investigate the effect of microwave pretreatment of seeds on the quality and antioxidant capacity of PS oil extracted from three pomegranate cultivars.

#### **2. Materials and Methods**

## *2.1. Experimental Material*

Pomegranate fruits (cv. Wonderful, Herskawitz, Acco) free of quality defects were harvested at commercial maturity stage from a farm (33◦48 0 S, 19◦53 0 E) in Western Cape Province, South Africa between February and April during the 2019 season. Pomegranate seeds (PS) extracted from the fruits were thoroughly cleaned before drying in an oven at 55 ± 2 ◦C for 24 h [27]. The dried seeds were stored at 4 ± 2 ◦C before use [28].

#### *2.2. Sample Moisturizing*

The moisture content of PS was measured in a moisture analyzer at 100 ◦C (DBS60-3, KERN, Balingen, Germany). The procedure of PS moisturizing was done following the method described by Rekas et al. [29]. PS (200 g) was sprayed with the pre-calculated amount of water, thoroughly mixed, sealed in zipped polyethylene bags, and equilibrated at 4 ± 2 ◦C for 48 h. This procedure was applied to moisturize the seeds to obtain a moisture content of 6% before microwave pretreatment. Water is a polar molecule and an important heat transfer medium during seeds microwave pretreatment and therefore PS moisturizing was vital [30]. A mass balance was used to calculate the amount of water to be added to the PS to obtain 6% moisture content. After 48 h of equilibration, the PS moisture content was checked in order to verify moisture homogeneity in the samples.

#### *2.3. Microwave Pretreatment*

#### 2.3.1. Equipment Calibration

A 2450 MHz domestic microwave oven (Model: DMO 351, Defy Appliances, Cape Town, South Africa) with a nominal power of 900 W was used in the present study. The microwave power calibration was performed following the procedure described by Rekas et al. [29]. Briefly, 500 g of water was heated in a glass beaker (80 mm diameter) and the time of 10 ± 2 ◦C elevation of water temperature was measured. The procedure was done in triplicate. The microwave power absorbed by the water during the heating was calculated as:

$$\mathcal{W} = m\_w \mathcal{C}\_{pw} \frac{\Delta T}{\Delta t} \tag{1}$$

where *W* is the power absorbed by the water (*W*), *mw* is the mass of water (kg), *Cpw* is the specific heat (J/C kg), Δ*T* is the difference in temperature (◦C), and Δ*t* is the time (s). The absorbed power by the water was 261 W for the applied 40% microwave power.

#### 2.3.2. Pretreatment

Ground PS (30 g) of uniform particle size (<1 mm) were evenly spread in a glass petri dish (190 mm in diameter) inside the calibrated microwave. The seed powder was exposed to microwave irradiation at 2450 MHz and 261 W for 102 s. This condition was established in preliminary experiments using response surface methodology (RSM), which confirmed 261 W and 102 s as the optimum microwave conditions for higher oil yield and antioxidant activity (unpublished). The microwave treated PS powder was allowed to cool to ambient temperature and thoroughly mixed to ensure sample homogeneity. Each experiment was performed in triplicate.

## *2.4. Oil Extraction*

An ultrasonic bath (Separation Scientific, Cape Town, South Africa) (700 W, 40 kHz and 25 L capacity) was used to extract the oil. The PS powder (20 g) was mixed with 100 mL ethanol in 500 mL plastic capped glass bottles. The samples were sonicated at 700 W, 40 ± 5 ◦C for 40 min before filtration through Whatman No. 1 filter paper and vacuum evaporation to recover the solvent (G3 Heidolph, Schwabach, Germany). Unmicrowaved PS powder was used as the control samples. Oil extractions were done twice on triplicated samples (*n* = 3). The yield of pomegranate seed oil (PSO) was calculated using Equation (2).

$$\text{PSO yield } (\%) = \frac{\text{M}\_1}{\text{M}\_2} \times 100 \tag{2}$$

where M1 and M2 are the mass of PSO and dry weight (dw) of the pomegranate seed powder, respectively. The extracted PSO samples were packed in brown bottles and stored at 4 ± 2 ◦C to minimize oxidation during analyses [31].

## *2.5. Pomegranate Seeds Microstructures Analysis*

Scanning electron microscopy (SEM) studies assess changes in the PS morphology due to microwave treatment and were conducted using the field emission scanning electron microscope (FESEM) (Thermo Fisher Apreo, Hillsboro, OR, USA). The samples were mounted on aluminum stubs using a double-sided carbon tape before sputter-coating with a thin layer of gold (10 nm thick) using a gold sputter coater (EM ACE200, Leica, Wetzlar, Germany) to induce conductivity within the sample. A voltage of 2 kV was used to collect the images, which were recorded digitally.

## *2.6. Determination of PSO Quality Indices*

## 2.6.1. Refractive and Yellowness Index

A calibrated Abbe 5 refractometer (Bellingham + Stanley, Kent, United Kingdom) was used to measure refractive index (RI) at ambient condition (25 ◦C). PSO colour properties including L\* (lightness) and b\* (yellowness) measured using a calibrated Chromameter CR-410 (Konica Minolta, INC, Tokyo, Japan) were used to calculate yellowness index (YI).

$$\text{YI} = \frac{142.86 \text{b}^\*}{\text{L}^\*} \tag{3}$$

#### 2.6.2. Peroxide Value, Conjugated Dienes, ρ-Anisidine Value and Total Oxidation Value

PSO peroxide value (PV) was determined using the modified ferrous oxidation-xylenol orange (FOX) method [32]. Conjugated dienes (K232) and trienes (K270) were analyzed according to the standard [33]. The ρ-anisidine value (AV) was measured in accordance with [34]. Total oxidation (TOTOX) value was calculated from the PV and AV using the equation [35].

$$\text{TOTOX} = 2\text{PV} + \text{AV} \tag{4}$$

## *2.7. Determination of Bioactive Compounds and Antioxidant Capacity*

## 2.7.1. Total Carotenoids Content and Total Phenolic Content

Total carotenoids content (TCC) was measured following the method described by Ranjith et al. [36]. Briefly, PSO (0.2 g) was dissolved in hexane (5 mL) and 0.5 mL of 0.5% (*w*/*v*) sodium chloride (NaCl) was added. The mixture was vortexed and centrifuged (Centrifuge 5810R, Eppendorf, Germany) at 4000 rpm for 5 min. The absorbance of the supernatant was measured at 460 nm using a UV spectrophotometer (Helios Omega, Thermo Scientific, Waltham, MA, USA). The results were reported as mg β-carotene/100 g of PSO. Total phenolic content (TPC) was determined using the Folin–Ciocalteu method [37]. The reaction mixture contained 200 μL of PSO methanol extracts, 250 μL of the Folin–Ciocalteau reagent and 750 μL of 2% (*w*/*v*) sodium carbonate, and 3 mL of distilled water. The reaction mixtures were incubated in the dark for 40 min after which their absorbances were measured at 760 nm using a UV spectrophotometer (Helios Omega, Thermo Scientific, Waltham, MA, USA), and the final results were expressed as milligram gallic acid equivalent per g PSO (mg GAE/g PSO).

## 2.7.2. Phytosterol Composition

The phytosterol composition was determined following the method described by Fernandes et al. [6] with some modifications. PSO (100 mg) samples weighed in 15 mL glass vials were mixed with 2.5 mL of saponification reagent (94 mL of absolute ethanol, 6 mL of 33% (*w*/*v*) potassium hydroxide, 500 μL of 20% (*w*/*v*) ascorbic acid). A hundred microliters of 5α-Cholestane (1000 mg/L) in chloroform (internal standard) was added and the mixture vortexed before saponification in an oven at 60 ◦C for 1 h. After saponification, the samples were cooled in ice for 10 min, followed by the addition of 5 mL of distilled of water and 2 mL of chloroform. The mixture was vortexed before centrifugation at 3000 rpm for 4 min. The chloroform extracts (500 μL) were concentrated with a gentle stream of nitrogen to ± 200 μL. To 100 μL of the concentrated chloroform extracts, pyridine (100 μL), and N,O-Bis (trimethylsilyl) trifluoroacetamide (30 μL) were added, and the mixture was vortexed before derivatization at 100 ◦C for 1 h in an oven. The derivatized sterol fractions were analyzed using gas chromatography connected to mass spectrometry (GC-MS) (Thermo Scientific Co. Ltd., Milan, Italy). The samples were injected 100 ◦C and held for 2 min before they were heated to 250 ◦C at the speed of 7 ◦C/min. The temperature was maintained for 2 min. A split ratio of 5:1, and an injection volume of 1.0 μL were used. The flow rate of helium, the carrier gas was maintained at 1 mL/min. The detector was operated under electron impact mode at ionization energy of 70 eV, scanning between 40 and 650 m/z. For peak identification, a standard containing a mixture of sterols (β-sitosterol, stigmasterol and ergosterol) was used. Phytosterol compounds identification was done by comparing the retention times. The results were reported as mg/100 g of PSO.

## 2.7.3. Radical Scavenging Ability

PSO antiradical activity was evaluated using 2,2-Diphenyl-1-picryl hydrazyl (DPPH) assay [38]. Briefly, PSO methanol extracts (100 μL) were added to 2.5 mL of 0.0004% (*w*/*v*) freshly prepared DPPH in 80% (*v*/*v*) methanol. The mixture was vortexed before incubation in the dark for 60 min. The absorbance of the remaining DPPH was measured using a UV spectrophotometer (Helios Omega, Thermo Scientific, Waltham, MA, USA) at 517 nm. The absorbance of DPPH in 80% methanol was measured as the negative control. The final result was expressed as mmol Trolox/g of PSO.

## 2.7.4. Ferric Reducing Antioxidant Power

The ferric reducing antioxidant power (FRAP) of PSO methanol extracts was determined following the method described by Benzie and Strain [39]. Freshly prepared FRAP reagent consisting of 2.5 mL of 10 mM 2,4,6-Tri(2-pyridyl)-s-triazine (TPTZ) solution in 40 mM HCl, 2.5 mL of 20 mM FeCl3 and 25 mL of 0.3 M acetate buffer, pH 3.6 was warmed at 37 ◦C for 10 min. In total, 40 microliters of PSO methanol extracts were mixed with 200 μL distilled water and 1.8 mL FRAP reagent. The samples were incubated at 37 ◦C for 30 min before the absorbances were measured at 593 nm using a UV spectrophotometer (Helios Omega, Thermo Scientific, Waltham, MA, USA). Trolox was used to prepare the standard curve (5-100 mM), and the final results were expressed as mmol Trolox/g of PSO.

## *2.8. Fatty Acid Composition*

Gas chromatography–mass spectrometry (GC–MS) was used to determine the fatty acid composition of PSO following a procedure described in a previous study [40]. PSO (100 mg) was weighed into 15 mL glass vials after which 2.0 mL hexane, 50 μL heptadecanoic acid (1000 mg/L, internal standard), and 1.0 mL of 20% (*v*/*v*) H2SO4 in methanol were successively added. The samples were thoroughly vortexed before incubation at 80 ◦C for 1 h in an oven. To the cooled the samples, 3 mL of saturated NaCl was added, and the mixture vortexed and centrifuged at 3000 rpm for 3 min. The hexane extracts were transferred into vials for analysis with GC-MS (6890N, Agilent technologies network, Palo Alto, CA, USA) coupled to an Agilent technologies inert XL EI/CI Mass Selective Detector (MSD) (5975B, Agilent Technologies Inc., Palo Alto, CA, USA). Separation of the FAMEs was performed on a polar RT-2560 (100 m, 0.25 mm ID, 0.20 μm film thickness) (Restek, Bellefonte, Pennsylvania, USA) capillary column. Helium was used as the carrier gas at a flow rate of 0.017 mL/s. One microliter (1 μL) of the sample was injected in a split ratio of 10:1. The oven temperature was run as: 100 ◦C/min, 180 ◦C at 25 ◦C/min, and held for 3 min; 200 ◦C at 4 ◦C/min and held for 5 min; 280 ◦C at 8 ◦C/min, 310 ◦C at 10 ◦C/min, and held for 5 min. The PSO fatty acids profiles were identified using the NIST library. Results were expressed as a percentage of the total and calculated by dividing the area peak of each fatty acid by the total area peaks of all the fatty acids.

### *2.9. Statistical Analysis*

The results of all the studied variables are presented as mean ± SD (standard deviation). One-way analysis of variance (ANOVA) was performed to compare the means using Statistica software (Statistical v13, TIBC, Palo Alto, CA, USA) after which the means were separated using Duncan's multiple range test. Graphs were prepared using Microsoft Excel (Version: 16.0.13029.20344, Microsoft Cooperation, Washington, USA). The relationship between the PSO quality attributes and cultivars was determined by performing the principal component analysis (PCA) and agglomerative hierarchical clustering (AHC) and using Microsoft Excel software (XLSTAT 2<IP_ADDRESS>305, Addinsoft, New York, NY, USA).

## **3. Results and Discussion**

## *3.1. Oil Yield and Seeds Microstructures*

The results in Figure 1 shows that pretreating pomegranate seeds (PS) with microwaves significantly enhanced the oil yield between 10% and 14%. 'Acco' exhibited significantly higher oil yield (17.10%) (dw) than 'Wonderful' (15.77%) (dw) and 'Herskawitz' (13.10%) (dw) after PS microwave pretreatment, a phenomenon that can be explained by the differences in their genetic material [41]. Previously, Durdevic et al. [19] reported that microwave (100, 250 and 600 W for 2 and 6 min) pretreatment of PS could enhance oil yield between 23% and 32%. Compared to the current study, the difference in oil yield could be explained by variation in cultivar, seeds pretreatment conditions, oil extraction methods, and fruit growing region, among other factors. Depending on the concentration of cellulose and lignin, PS rheological properties such as hardness and toughness may vary with cultivar [42]. This could have affected the cultivars' response to microwave pretreatment and oil extraction. The scanning electron microscopy (SEM) images in Figure 2 confirmed that PS microwave pretreatment significantly deformed the cell walls. As shown in Figure 2b microwave pretreated PS were characterized by conspicuous perforations on the cell walls. Microwaves generate heat by interacting with polar substances and, therefore, water as a polar molecule is an essential heat transfer medium during seeds microwave pretreatment [30]. The heat energy causes a rapid increase in the seed temperature and vaporization of the water in the seeds creating an intracellular pressure that ruptures the oilseeds cell walls and membranes [20]. Figure 2c shows parenchymal cells from unmicrowaved PS with intact cell walls, which could have created a major resistance to solvent penetration into the seeds cells and could be the reason for the low oil yield observed from unmicrowaved PS [43]. On the other hand, Figure 2d shows extensively damaged PS parenchymal cells due to microwave treatment. Similar findings have been reported from microwave pretreatment of hazelnuts [44]. In addition to damaging the cell walls, microwave pretreatment could have deformed the lipoprotein membranes surrounding the individual lipid bodies [45]. These microstructural changes could have enhanced porosity of the PS cell walls and membranes that led to the improved efficiency of oil extraction with ethanol.

## *3.2. Refractive and Yellowness Index*

Thermal treatment of the oilseeds could result in fatty acids conjugation and an increase in the oil refractive index [46]. Therefore, RI could be used as an indirect quality measure of oil. Neither cultivar nor PS microwave pretreatment significantly (*p* > 0.05) affected the RI of the oil extracts, despite the significant cell walls and membranes deformation (Table 1). The pomegranate seed oil RI values (1.5180–1.5181) in the current study were comparable to those reported by Costa et al. [47] (1.5091–1.5177) from cold pressed PSO further demonstrating that PS microwave pretreatment did not cause significant negative effect on the oil quality.

**Figure 1.** Oil yield from unmicrowaved and microwaved (261 W for 102 s) pomegranate seeds of three pomegranate cultivars. Within the same cultivar (unmicrowaved and microwave), columns followed by different letters are significantly different (*p* < 0.05) according to Duncan's multiple range test. Vertical bars indicate the standard deviation of the mean.

**Figure 2.** Representative scanning electron microscopy (SEM) micrographs show the effect of microwave pretreatment (261 W/102 s) on the pomegranate seeds microstructures. (**a**) Unmicrowaved pomegranate seeds, (**b**) microwaved pomegranate seeds, (**c**) parenchymal cells from unmicrowaved pomegranate seeds, and (**d**) parenchymal cells from microwaved pomegranate seed.

**Table 1.** Physicochemical characteristics of oil from unmicrowaved and microwave pretreated (261 W/102 s) pomegranate seeds of three pomegranate cultivars.

Values represent mean ± SD of triplicate determinations. Different superscript letters in the same column indicate statistical significance (*p* < 0.05) according to Duncan's multiple range test. RI= index (25 ◦C), PV = Peroxide value (meqO2/kg PSO), meqO2/kg = milli-equivalents of active oxygen per kg), AV = Anisidine value, TOTOX = Total oxidation value, RI = Refractive, K232 = Conjugated dienes, K270 = Conjugated triene.

Color is a valuable parameter that influences the consumer's preference and decision to purchase a food product. Yellowness index can be used to measure the influence of processing, including seeds microwave pretreatment on oil color [48]. The results in Figure 3 show that PS microwave pretreatment significantly improved the YI of 'Herskawitz' and 'Acco' oil extracts by 1.5 and 1.7 fold, respectively. The significant increase in the YI after PS microwave pretreatment could be ascribed to the improved extraction of the oil color pigments such as carotenoids as facilitated by the extensively damaged cell walls and membranes (Figure 2). In the study of Rekas et al. [49], YI significantly increased by 13% and 63% when dehulled rape seeds were microwaved at 800 W for 2 and 4 min, respectively. However, PS microwave pretreatment insignificantly (*p* > 0.05) changed the YI of 'Wonderful' oil extracts suggesting that the effect of pretreating PS with microwaves on oil color compounds differed among the cultivars.

**Figure 3.** Yellowness index (YI) of pomegranate seed oil from unmicrowaved and microwave pretreated (261 W/102 s) seeds of three pomegranate cultivars. Within the same cultivar (unmicrowaved and microwaved), columns followed by different letters are significantly different (*p* < 0.05) according to Duncan's multiple range test. Vertical bars indicate the standard deviation of the mean.

## *3.3. Peroxide Value, Conjugated Dienes and Trienes,* ρ*-Anisidine Value and Total Oxidation Value*

Peroxide value indicates the extent of fats and oils oxidation and therefore is one of the most widely used quality indicators in the food industry. As shown in Table 1, PS microwave pretreatment significantly increased the PV from 'Herskawitz' and 'Acco' oil extracts by 29% and 30%, respectively. The significant increase in PV could be explained by significant heat penetration into the seeds matrices during microwave pretreatment that could have induced lipid oxidation and hydroperoxides formation. Despite the significant increase in PV of 'Herskawitz' and 'Acco' oil extracts after seeds microwave pretreatment, the values (0.17–0.35 meqO2/kg PSO) conformed to the Codex Alimentarius Commission standard for seed oil that permits a maximum of 15 meqO2/kg in unrefined seed oils [50]. Microwave pretreatment of PS did not significantly oxidize 'Wonderful' oil extracts. Our PV results

were lower than those reported by Basiri [51] (0.79 meqO2/kg PSO) from petroleum ether extracted PSO, further demonstrating that microwave pretreatment of PS may not cause significant oil degradation.

During hydroperoxides formation, the non-conjugated double bonds of fatty acids may be converted to conjugated double bonds through isomerization [52]. Therefore, fatty acids conjugation can also be used as a quick indirect quality measure of oil. Conjugated dienes are part of fatty acid oxidation initial products. The results in Table 1, demonstrate that the level of K232 significantly increased in 'Wonderful' (27%) and 'Acco' (45%) oil extracts, whilst it significantly decreased in 'Herskawitz' (37%) oil extracts after PS microwave pretreatment. Although there was a significant increase in conjugated dienes in 'Wonderful' and 'Acco' oil extracts due to PS microwave pretreatment, the K232 values (0.19–0.30) were lower than the K232 values (4.15) reported by Amri et al. [53] from 'Tounsi' hexane PSO extracts, indicating that the oil from the present study was of higher quality.

A low PV is not the only marker for good oil quality because hydroperoxides are unstable and quickly decompose into secondary oxidation products. For this reason, the analysis of secondary products of seed oil oxidation is equally important. Unlike the K232 values, PS microwave pretreatment did not significantly influence the levels of K270 values in the oil from all the cultivars. ρ-Anisidine value measures the secondary products of fatty acids oxidation, such as aldehydes formed due to further hydroperoxides decomposition [52]. Despite the AV of oil extracted from 'Acco' significantly increasing by 3 fold after PS microwave pretreatment, the values (2.00–5.90) were lower than those from 'Wonderful' (14.22–12.50) and 'Herskawitz' (13.06–12.90) oil extracts that insignificantly changed after PS microwave pretreatment (Table 1). The observation that AV from 'Wonderful' and 'Herskawitz' oil extracts did not significantly change after PS microwave pretreatment suggests that there was minimum decomposition of hydroperoxides to form carbonyl compounds. In a previous study, Costa et al. [47] reported AV ranging from 13.8 to 18.6 from cold pressed PSO that were higher than the AV (5.90–14.22) results in the present study. High AV in freshly processed seed oil could indicate interference by other substances, leading to false positive overestimation of AV values.

Total oxidation value is a summation of the primary and secondary oxidation products and provides a better indication of fats and oils overall oxidative deterioration. As can be seen in Table 1, PS microwave pretreatment significantly increased the TOTOX value of 'Acco' oil extracts by 2.6 fold and this could be linked to either increased heat penetration into the seed matrices or increased lipolytic enzyme activity in the microwave damaged cells [54]. The result that the level of TOTOX value from 'Wonderful' and 'Herskawitz' oil extracts did not significantly change after PS microwave pretreatment suggests resistance to oxidation by the oil from the two cultivars, which could be attributed to enhanced total phenolic compounds [43].

#### *3.4. Total Carotenoids Content, Total Phenolic Content, and Antioxidant Capacity*

Epidemiological studies suggest that the consumption of carotenoid-rich foods such as seed oil is associated with the prevention of cancers, cardiovascular diseases, age-related cataracts, and immune system function improvement [55]. Therefore, the maximum extraction of these antioxidative compounds from plant materials such as seeds is important. The results in Table 2 indicate that PS microwave pretreatment significantly increased the total carotenoids content of 'Herskawitz' and 'Acco' oil extracts by 11% and 19%, respectively. Previously, Mazaheri et al. [56] also observed a significant improvement in carotenoids after microwave pretreatment of black cumin seeds. The extensive damage of the PS cell walls and membranes by microwave pretreatment could have increased the dissociation of carotenoids from the carotenoprotein complexes enhancing their mass transfer into the extraction solvent (Figure 2) [57]. On the other hand, the finding that TCC of oil extracted from 'Wonderful' did not significantly change after PS microwave pretreatment, whilst that of 'Herskawitz' and 'Acco' oil extracts significantly changed after seeds microwave pretreatment, indicating that the response of carotenoids compounds to PS microwave pretreatment was cultivar dependent.

**Table 2.** TCC, TPC and antioxidant capacity (DPPH, FRAP) of oil extracted from unmicrowaved and microwave pretreated (261 W/102 s) seeds of three pomegranate cultivars.

Values represent mean ± SD of triplicate determinations. Different superscript letters in the same column indicate statistical significance (*p* < 0.05) according to Duncan's multiple range test. TPC = Total phenolic content (mg GAE/g PSO, TCC = Total carotenoids content (mg β-carotene/100 g PSO), FRAP = Ferric reducing antioxidant power (mmol Trolox/g PSO), DPPH = 2,2-Diphenyl-1-picryl hydrazyl (mmol Trolox/g PSO), Trolox = 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, PSO = Pomegranate seed oil, GAE = Gallic acid equivalence.

Phenolic compounds have been implicated in the anti-inflammatory and antioxidant properties of potential functional foods [26]. In this respect, polyphenol-rich foods intake may be associated with decreased risk of chronic diseases. PS microwave pretreatment significantly enhanced the total phenolic compounds of oil extracted from 'Wonderful' and 'Acco' by 25% and 17%, respectively, but did not significantly change the TPC of 'Herskawitz' oil extracts (Table 2). The results demonstrate that cultivar is an invaluable factor in PSO value addition. In addition, it has been reported that in plant materials phenolic compounds exist as either glycosylated, non-glycosylated, esterified, or free compounds, which could vary with cultivar and significantly influence their extraction [58]. The TPC (1.67–3.12 mg GAE/g PSO) results from the current study were higher than those reported by Pande and Akoh [59] (0.85–0.91 mg/g PSO) and Costa et al. [47] (0.00–0.17 mg/g PSO) from solvent extracted, and cold pressed PSO an indication that pomegranate cultivars from the current study could be valuable sources of phenolic compounds. Besides, factors such as cultivar, oil extraction technique, and fruit growing region could also be sources of variation in the TPC results among the studies.

Phytochemicals are complex; no single assay accurately reflects all antioxidants in a complex system such as seed oil. In this study, the antioxidant capacity of PSO was assessed using the DPPH and FRAP assays. PS microwave pretreatment significantly enhanced the DPPH radical scavenging capacity of 'Herskawitz' (7%) and 'Acco' (4%) oil extracts but did not significantly change the DPPH radical scavenging capacity of oil extracted from 'Wonderful'. FRAP significantly increased in the oil extracted from 'Wonderful' and 'Herskawitz' by 47 and 82%, respectively, after PS microwave pretreatment. In contrast to this finding, the reducing potential of 'Acco' oil extracts did not significantly change after PS microwave pretreatment. The finding that cultivar significantly influenced the antioxidant capacity of oil from microwave pretreated PS agrees with the results from Xi et al. [60], who reported significant variation in DPPH radical scavenging capacity and FRAP of seed oil from different lemon cultivars. The significant increase in the oil antioxidant capacity after PS microwave pretreatment, particularly from 'Herskawitz' could be related to the improved TCC and TPC. However, it is worth mentioning that PSO is also a good source of tocopherols, which have been reported to be associated with the oil antioxidant capacity in previous studies [61].

#### *3.5. Phytosterol Composition*

The ability of phytosterols to lower blood cholesterol may reduce the risk of coronary heart disease. Optimum extraction of these valuable compounds during seed oil processing is therefore essential to enhance the extracted oil health benefits. The effect of PS microwave pretreatment on phytosterol composition is presented in Figure 4. Three different phytosterols, including β-sitosterol (455.91–683.37 mg/100g PSO), stigmasterol (9.04–45.74 mg/100g PSO), and ergosterol (2.06–2.53 mg/100g PSO) were quantified in PSO from the studied cultivars. The levels of β-sitosterol and stigmasterol

were consistent with the findings of Pande and Akoh [59] and Caligiani et al. [62] from PSO extracted using hexane and ethyl ether, respectively. In addition, the concentration of phytosterols was higher than those reported from other fruit seed oils such as apple, strawberry, and raspberry but comparable to those from sour cherry [63,64]. The level of β-sitosterol significantly decreased in 'Acco' and 'Wonderful' oil extracts by 26% and 29%, respectively, after PS microwave pretreatment, whilst it did not significantly change in 'Herskawitz' oil extracts, regardless of the PS cell walls and membranes extensive damage by microwave pretreatment (Figure 2). The finding that β-sitosterol significantly decreased in 'Wonderful' and 'Acco' oil extracts after PS microwave pretreatment suggests that the applied microwave pretreatment conditions thermally degraded this low-density lipoprotein (LDL) reducing phytosterol. Likewise, the levels of stigmasterol and ergosterol significantly decreased between 8 and 13% in 'Wonderful' and 'Herskawitz' oil extracts after treating the seeds with microwaves. Unlike in the present study, Azadmard-Damirchi et al. [21] and Fathi-Achachlouei et al. [43] reported significant improvement in phytosterols from microwave pretreated rape and milk thistle seed, respectively. The dissimilarity of our results with previous studies indicates that microwave pretreatment conditions are seed specific.

**Figure 4.** Phytosterol composition of oil extracted from unmicrowaved and microwave pretreated (261 W/102 s) seeds of three pomegranate cultivars. Within the same cultivar (unmicrowaved and microwaved), columns representing the same phytosterol and followed by different letters are significantly different (*p* < 0.05) according to Duncan's multiple range test. Vertical bars indicate the standard deviation of the mean.

On the other hand, microwave pretreatment of PS significantly improved the concentration of ergosterol and stigmasterol (9 and 111%, respectively) in 'Acco' oil extracts. Naturally, phytosterols exist as free compounds or conjugates in which they are either esterified to fatty acids or glycosylated with sugars [65]. The form in which they exist may therefore influence their dissociation and isolation from the seed matrix.

## *3.6. Fatty Acid Composition*

The GC chromatogram shows that the primary fatty acids identified in PSO from the studied cultivars were palmitic acid, stearic acid, oleic acid, linoleic acid and punicic acid (Figure 5), which accounted for 5.64–7.74%, 2.34–3.08%, 7.43–9.62%, 11.59–16.54%, and 62.75–70.51%, respectively. The fatty acid composition was comparable to the findings of Tian et al. [66] and Aruna et al. [67]. However, the values of punicic acid were lower when compared with the findings of Khoddami et al. [5] and Fernandes et al. [4], which could be attributed to differences in fruit ripening index, seed oil

extraction method, cultivar, and geographical location, among other factors. As can be seen in Table 3, PS microwave pretreatment significantly increased palmitic acid between 6% and 20%. Moreover, treatment of PS with microwaves significantly increased the stearic acid from 'Acco' oil extracts by 7%. Among the saturated fatty acids, stearic acid has unique properties, as it has been associated with a decrease in LDL cholesterol, cancer, and atherosclerosis risk [68]. The levels of stearic acid did not significantly change in 'Wonderful' and 'Herskawitz' oil extracts after PS microwave irradiation, which was comparable with the results reported by Durdevic et al. [69]. Oleic acid, the main monosaturated fatty acid in PSO significantly improved in 'Herskawitz' and 'Acco' oil extracts by 9% and 10%, respectively, after seeds microwave pretreatment. Improvement in oleic acid after seeds microwave pretreatment is desirable in oil oxidative stability as monosaturated fatty acids are less susceptible to oxidation. More so, PS microwave pretreatment significantly increased linoleic acid from 'Herskawitz' (37%) and 'Acco' (12%) oil extracts. The finding that PS microwave pretreatment enhanced the oil oleic acid and linoleic acid is essential to human health since oleic acid is associated with lowering low density lipoprotein (LDL) blood cholesterol, and linoleic acid has an important role in balancing fatty acid content proportions in body cells [20]. However, the concentration of punicic acid, the primary bioactive lipid with several biological properties significantly decreased in 'Herskawitz' and 'Acco' by 10% and 5%, respectively (Table 3). PSO health benefits potential is mostly attributed to punicic acid and therefore, its decrease after PS microwave pretreatment was not desirable. Arachidic acid significantly increased in 'Herskawitz' (13%) oil extracts, whilst it significantly decreased in 'Acco' (14%) oil extracts. Saturated fatty acids (SFA) significantly increased in 'Acco' (11%) and 'Herskawitz' (15%) oil extracts after PS microwave pretreatment. However, the polyunsaturated fatty acids (PUFA) and the ratio of unsaturated to saturated fatty acid (UFA: SFA) in 'Herskawitz' oil extracts decreased by 3% and 4%, respectively, after PS microwave pretreatment, indicating a loss in nutritional quality despite the significant increase in the antioxidant capacity (Tables 1 and 2). The decrease in the ratio of UFA: SFA could be attributed to the decline in punicic acid, the main polyunsaturated fatty acid in PSO. The amount of monosaturated fatty acids (MUFA) was insignificantly changed after microwave pretreatment of PS from all the cultivars. The levels of PUFA and UFA: SFA ratio in oil extracted from 'Acco' were not significantly affected by PS microwave pretreatment. Except for palmitic acid, PS microwave pretreatment had no significant effect (*p* > 0.05) on the fatty acid content of oil extracted from 'Wonderful'. Insignificant effect of seeds microwave pretreatment on the oil fatty acids content has also been reported in prior researches. For example, Wroniak et al. [20] and Guneser and Yilmaz [25] observed no significant change in the fatty acids content of oil extracted from orange and rape seeds, respectively, after microwave pretreatment.

#### *3.7. Principal Component Analysis and Agglomerative Hierarchical Clustering Analysis*

Principal component analysis (PCA) and agglomerative hierarchical clustering (AHC) were performed in order to provide an overview of the relationship between pomegranate cultivars, seeds microwave pretreatment, and the oil quality attributes. According to Kaiser's rule, only eigenvalues greater than 1 are considered significant descriptors of data variance [70]. The first two factors with the highest eigenvalues (F1 = 5.0, F2 = 3.1) accounted for 62.27% (F1: 38.10% and F2: 24.10, respectively) of the total variance in the original data and were considered more important (Figure 6). The first factor (F1), which was contributed by 'Acco' and 'Wonderful' oil extracts, was positively correlated with oil yield and PV, but negatively correlated with RI, AV, TOTOX, punicic acid, and FRAP. This points out that cultivars, which exhibited higher oil yield after seeds microwave pretreatment such as 'Acco', were associated with low FRAP. Although extensive damage of the PS cell walls and membranes by microwave pretreatment facilitated increased extraction of lipids, it could have exposed the oil to thermal degradation (Figure 2). The second factor (F2) that was contributed by 'Herskawitz' oil extracts from microwave pretreated seeds and 'Acco' oil extracts from unmicrowaved seeds was positively correlated with TCC, TPC, and DPPH radical scavenging capacity, but negatively correlated with β-sitosterol and YI. As shown in Table 2. microwave pretreatment of 'Herskawitz' PS improved

the extraction of TCC and TPC that could have enhanced the oil DPPH radical scavenging capacity. The agglomerative hierarchical clustering (AHC) of PSO extracts from the different cultivars clustered the 'Acco' and 'Wonderful' oil extracts from microwaved seeds together that were correlated with higher oil yield and YI (Figure 7). These results concurred with the PCA analysis results. Like the PCA results, AHC separated 'Herskawitz' oil extracts from 'Acco' and 'Wonderful', illustrating that 'Herskawitz' was higher in oil quality attributes that were lower in 'Acco' and 'Wonderful', such as TCC, TPC, and DPPH radical scavenging capacity. Although microwave pretreatment of 'Acco' and 'Wonderful' PS may enhance oil yield, It was established that it also increases the oil oxidative degradation. On the other hand, microwave pretreatment of 'Herskawitz' PS may not produce oil yield results comparable to 'Wonderful' and 'Acco', but the oil has better bioactive compounds and antioxidant capacity.

**Figure 5.** *A* typical gas chromatography-mass spectrometry (GC-MS) chromatograph of the major fatty acids identified in pomegranate seed oil and their retention times. C16:0 = palmitic acid, C17:0 = heptadecanoic acid (internal standard), C18:0 = stearic acid, C18:1 = oleic acid, C18:2 = linoleic acid, C18:3 = punicic acid, C20:0 = arachidic acid.

**Table 3.** Fatty acid composition (% relative area) of pomegranate seed oil from unmicrowaved and microwave pretreated (261 W/102 s) seeds of three pomegranate cultivars.

Values represent mean ± SD of triplicate determinations. Different superscript letters in the same row indicate statistical significance (*p* < 0.05) according to Duncan's multiple range test. SFA = Saturated fatty acid, MUFA = Monounsaturated fatty acid, PUFA = Polyunsaturated fatty acid, UFA = Unsaturated fatty acid.

**Figure 6.** Principal component analysis data of pomegranate seed oil (PSO) quality attributes from unmicrowaved and microwaved (261 W/102 s) pomegranate seeds of three pomegranate cultivars. A = 'Acco', H = 'Herskawitz', W = 'Wonderful', AV = ρ-anisidine value, TOTOX = Total oxidation value, K232 = Conjugated dienes, RI = Refractive index, TPC = Total phenolic content, TCC = Total carotenoids content, PA = punicic acid, FRAP = Ferric reducing antioxidant power, DPPH = 2,2-diphenyl-1-picryl hydrazyl.

**Figure 7.** Agglomerative hierarchical clustering (AHC) of PSO extracts from unmicrowaved and microwaved (261 W/102 s) seeds. A = 'Acco', H = 'Herskawitz', W = 'Wonderful', TOTOX = Total oxidation value, TPC = Total phenolic content, TCC = Total carotenoids content, DPPH = 2,2-diphenyl-1-picryl hydrazyl, FRAP = Ferric reducing antioxidant power.

### **4. Conclusions**

The present study established that PSO quality may be enhanced by seeds during microwave pretreatment, although oil quality varies with cultivar. Microwave pretreatment of PS improved oil yield, YI, TCC, TPC, DPPH radical scavenging capacity, and FRAP. This is a desirable development to the food industry given the increasing consumers' demand for natural and healthier foods. Moreover, the enhancement of bioactive compounds and antioxidant capacity after PS microwave pretreatment is valuable for the oil oxidative stability and storability. Despite an increase in K232 and PV, PS microwave pretreatment slightly decreased the oil TOTOX value.

Regarding fatty acid composition, PS microwave pretreatment increased palmitic acid, oleic acid, linoleic acid, SFA, and MUFA, but reduced the level of punicic acid. Pretreating PS with microwaves did not significantly affect the RI, PUFA, ratio of UFA: SFA, and phytosterol composition of the extracted oils. According to the PCA and AHC, 'Acco' and 'Wonderful' oil extracts from microwave pretreated PS exhibited better oil yield, whilst 'Herskawitz' oil extracts showed higher TCC, TPC, and DPPH radical scavenging valuable in functional foods formulation. In conclusion, 'Herskawitz' is a desirable cultivar for exploitation in nutraceutical and functional foods formulations.

**Author Contributions:** Conceptualization, O.A.F. and U.L.O.; Formal analysis, T.K.; Funding acquisition, O.A.F. and U.L.O.; Investigation, T.K.; Methodology, T.K.; Supervision, O.A.F. and U.L.O.; Validation, O.A.F. and U.L.O.; Visualization, T.K.; Writing-original draft, T.K.; Writing-review and editing, T.K.; O.A.F. and U.L.O. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the National Research Foundation of South Africa (grant number 64813) and APC was partly funded by Stellenbosch University.

**Acknowledgments:** This work is based on the research supported wholly or in part by the National Research Foundation of South Africa (Grant Numbers: 64813). The opinions, findings and conclusions or recommendations expressed are those of the author(s) alone, and the NRF accepts no liability what so ever in this regard.

**Conflicts of Interest:** The authors declare that they have no conflict of interest.

## **References**

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