**1. Introduction**

Pomegranate peel, seeds, juice, and arils are a rich source of several valuable bioactive compounds with considerable nutritional, antioxidant, and other beneficial properties [1–3]. Peel possesses a higher polyphenol content than seeds and juice [4]. These polyphenols include punicalagin, which exhibits high antioxidant activity. Pomegranate also contains other polyphenols, including anthocyanins (delphinidin, cyanidin, and pelargonidin 3- glucosides and 3,5-glucosides) as well as flavonols [5]. Pomegranate peel is known for its healing properties with respect to inflammatory diseases, diabetes, atherosclerosis, oxidative stress, cancer, and microbial infections [6–11]. Moreover, pomegranate fruit is used in the food industry such as dairy products, charcuterie and juice preparation, and conservation. Sweet pomegranates are consumed fresh while sour pomegranates with hard seeds are generally intended for processing [12]. Even if the consumption of

**Citation:** Benchagra, L.; Berrougui, H.; Islam, M.O.; Ramchoun, M.; Boulbaroud, S.; Hajjaji, A.; Fulop, T.; Ferretti, G.; Khalil, A. Antioxidant Effect of Moroccan Pomegranate (*Punica granatum* L. Sefri Variety) Extracts Rich in Punicalagin against the Oxidative Stress Process. *Foods* **2021**, *10*, 2219. https://doi.org/ 10.3390/foods10092219

Academic Editor: Antonello Santini

Received: 28 June 2021 Accepted: 12 September 2021 Published: 18 September 2021

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**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/).

arils leaves less waste, the fact remains that agri-food industry use of the pomegranate generates large amounts of peel-waste and by-products that are usually poorly exploited. Pomegranate peels represent 50% of total fruit weight and are a potential source of bioactive compounds, mainly phenolic compounds with a very broad spectrum of activity [13]. Indeed, several studies have reported the effects (antioxidant, anti-cancer, anti-inflammatory, lipid-lowering, anti-hypertensive) of phenolic extracts from the peel of the pomegranate, which makes them suitable as natural ingredients [13–17]. In addition, the natural product supplementation industry is increasingly interested in peel waste, manufacturing capsules containing concentrated phenolic compounds based on peels or whole fruit. Moreover, pomegranate peels are also used for their dyeing properties [18]. In fact, pomegranate peels may also be used as green antimicrobial agents to reduce inorganic nanoparticle consumption on wool yarns [19]. Pomegranate waste (peels and seeds) is also used in meat preparation and conservation to prevent bacterial development and the oxidation process [17,20,21].

Morocco's annual pomegranate production exceeds 58,000 tones from a total area of 5000 hectares [22,23] and half of this production is grown in central Morocco on the planes of the Middle Atlas (Beni Mellal-Khenifra area) and is mainly represented by the Sefri variety. There are many pomegranate varieties in Morocco. P. granatum also has considerable synonymy, in which the same genotype is known by different names in different regions.

In this study we investigated several beneficial health properties of various bioactive compounds from *Sefri* pomegranates, notably the anti-oxidative and physicochemical properties of pomegranate peel phenolic extract (PPPE) and pomegranate aril phenolic extract (PAPE) and evaluated their effects on atherosclerosis and bladder cancer cells.

LDL oxidation is considered to be a hallmark of early atherogenesis. Nutritional antioxidants such as phenolic compounds can markedly inhibit LDL oxidative damage by reducing free radicals generated during oxidative metabolism, preserving endogenous antioxidants in LDL (vitamin E and carotenoids), chelating transition metal ions, and modulating the oxidative state of the arterial cell wall. These properties act to inhibit cell-mediated oxidation of LDL and increase serum paraoxonase (PON1) activity [24].

Polyphenols and/or their derivatives are used to treat cancer. Cancer initiation may be modulated by an increase in ROS levels, which can damage DNA and stimulate prooncogenic signaling [25]. Oxidative stress regulates the progression of different types of cancer, including breast, liver, lung, colon, prostate, and bladder cancer.

In the Beni Mellal-Khenifra area, all parts of the pomegranate, especially peel, are used for health remedies to treat diarrhea, ulcers, nasal bleeding, and inflammation. Moreover, this fruit is highly consumed by patients with vascular disorders. This variety may have a more powerful health potential regard to its possible richness in phenolic compounds.

To the best of our knowledge, and even though various pomegranates are cultivated in different regions of Morocco, only a few studies have focused on the chemical composition and properties of the Sefri variety of pomegranate and even fewer on its biological properties, particularly in the prevention of diseases linked to oxidative stress such as cardiovascular disease and cancer. From this perspective, we conducted this study firstly to analyze the chemical composition of phenolic compounds from peels and arils of this variety. At the biological level, we took a particular interest in studying the relationship between antioxidant activity of the Sefri cultivar and its impact in preventing lipid peroxidation in human LDLs [26] and in modulating PON 1 activity and enzyme expression. On the other hand, since oxidative stress plays an important role in cancer development and progression [27], we evaluated the effect of sefri pomegranate polyphenols on reactive oxygen species (ROS) production in J82 human bladder cancer cells.

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