**Antioxidants in Foods**

Editors

**Isabel Seiquer Jos ´e M. Palma**

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

*Editors* Isabel Seiquer Estacion Experimental del ´ Zaid´ın, CSIC Spain

Jose M. Palma ´ Estacion Experimental del ´ Zaid´ın, CSIC Spain

*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 *Antioxidants* (ISSN 2076-3921) (available at: https://www.mdpi.com/journal/antioxidants/special issues/Antioxidants 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 978-3-0365-0578-7 (Hbk) ISBN 978-3-0365-0579-4 (PDF)**

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© 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**




Phytochemical Characterization of *Dillenia indica* L. Bark by Paper Spray Ionization-Mass Spectrometry and Evaluation of Its Antioxidant Potential Against t-BHP-Induced Oxidative Stress in RAW 264.7 Cells


#### **Luana Izzo, Yelko Rodr´ıguez-Carrasco, Severina Pacifico, Luigi Castaldo, Alfonso Narv ´aez and Alberto Ritieni**


#### **Sabina Lachowicz, Michał Swieca and Ewa Pejcz ´**

Improvement of Health-Promoting Functionality of Rye Bread by Fortification with Free and Microencapsulated Powders from *Amelanchier alnifolia* Nutt Reprinted from: *Antioxidants* **2020**, *9*, 614, doi:10.3390/antiox9070614 ................ **365**

#### **Paulina Keska, Sascha Rohn, Michał Halagarda and Karolina M. W ´ojciak**


### **About the Editors**

**Isabel Seiquer** is a senior researcher at Estacion Experimental del Zaid ´ ´ın, a research center from the Spanish National Research Council (CSIC). Dr. Seiquer has more than 30 years of professional experinece dedicated to the study of diverse topics related to the areas of Nutrition and Food Science and Technology. Her research activity has been focused on the effects of food components on the metabolism of minerals and proteins, the bioavailability of nutrients and the antioxidant properties of new compounds formed during food processing and digestive processes. She has addressed different aspects related to oxidative stress, through in vitro, cell cultures and in vivo assays. Moreover, her research interest also deals with animal nutrition and the study of obtaining food products with high quality preserving the environment. She is coauthor of more than 100 publications in international peer-reviewed journals.

**Jos´e M. Palma** Palma is Research Professor of the Spanish National Research Council (CSIC), with expertise in antioxidants and free radicals in plant systems. With more than 150 peer-reviewed research papers published, he has also been editor of five books and several Special Issues of a number of international impact journals. At present, he is involved in the investigation of the interactions between nitric oxide, hydrogen sulfide and antioxidants during fruit ripening and pot-harvest. He leads the research group "Antioxidants, Free Radicals and Nitric Oxide in Biotechnology, Food and Agriculture" at Estacion Experimental del Zaid ´ ´ın (EEZ), CSIC, Granada, Spain. He was also Deputy Director and Acting Director of the EEZ (CSIC) during the period 2007–2014.

### *Editorial* **To Be or Not to Be... An Antioxidant? That Is the Question**

#### **José M. Palma 1,\* and Isabel Seiquer 2,\***


Received: 25 November 2020; Accepted: 2 December 2020; Published: 5 December 2020

The concept of antioxidants refers to a substance with the capacity to either directly scavenge or indirectly prevent the formation of pro-oxidant molecules, basically associated to the so called reactive oxygen species (ROS). Considering the cell/tissue target, the picture is quite different. Thus, in animal tissues, the main source for ROS production is the mitochondria, whereas in plants, chloroplast is the most important organelle to generate ROS, including singlet oxygen (1O2), superoxide radicals (O2 •−) or hydrogen peroxide (H2O2). Accordingly, and due to the respective peculiarities, each organelle/cell/tissue has its own antioxidant machinery to overcome the deleterious effects of their internal ROS levels and production.

The real situation is that our cells are continually threatened by ROS, that disturb their normal and pacific living. Oxidative stress arises from an augmented ROS generation, but also from a decay of the antioxidant defense system, leading to the imbalance between the occurrence of reactive species and the organism's ability to counteract them. Oxidative stress is responsible for cell damage, sometimes irrecoverable, which is further implicated in a cascade of degenerative diseases, cardiovascular diseases, cancer and aging. Therefore, in this dangerous situation, the endogenous defense system (which includes antioxidant enzymes and non-enzymatic compounds such as glutathione, vitamins, coenzyme Q and others) needs external help to lower/modulate the negative effects of excessive ROS. This exogenous help is represented by the dietary antioxidants, i.e., food antioxidants, most of them present in fruits and vegetables or other sources from the plant kingdom.

Oxidative stress is also suffered by plants, in which illumination conditions and excess of light may lead to ROS formation. Chloroplasts contain a well-equipped battery of both enzymatic and non-enzymatic antioxidants to reestablish the initial balanced state and to avoid a situation of oxidative stress. In this response mainly participate the molecular antioxidants α-tocopherol, carotenoids and ascorbate, the enzyme superoxide dismutase and the ascorbate glutathione cycle (AGC) which involves ascorbate, glutathione, NADPH and four redox enzymes. Some of these antioxidants, which in plants are required in great amounts to cope against the stressful conditions, have vitamin properties and may function as dietary antioxidants for animal and human beings. Thus, α-tocopherol is the chemical nature of vitamin E, whereas β-carotene is precursor of vitamin A once the plant carotene is assimilated by our metabolism; ascorbate is vitamin C, one of the most powerful antioxidants and has the ability to directly scavenge most ROS. Besides this antioxidant team, plants contain a huge amount of secondary metabolites with potential antioxidant activity.

As indicated above, the antioxidants are usually required in high quantities to neutralize the oxidative effects of ROS in plants. This is in contradiction with the concept of vitamins in the animal/human scenario, which is considered as an organic molecule that is essential in small quantities for the proper functioning of the organism's metabolism. Commonly, in animals and humans these essential micronutrients are insufficiently or not synthesized and they need to obtain them through the diet. Whereas in plant systems, α-tocopherol and carotenoids are basically used as singlet oxygen scavengers, thus protecting cells against this ROS, in animal cells vitamins A and E play important roles regulating the redox homeostasis in a series of physiological processes. Thus, vitamin A is crucial for vision, growth and development, and for protecting epithelium and mucus integrity in the respiratory, urinary and gastro intestinal tract. Likewise, vitamin E seems to prevent cardiovascular episodes, neurodegenerative disease, macular degeneration and cancer. Little amounts of such lipophilic substances are necessary to carry out their antioxidant role, but the excessive intake may lead to unwanted redox imbalances which may provoke some disorders, and their use as complements is sometimes under debate.

Ascorbic acid (vitamin C) is synthesized in the majority of living beings, excepting primates (including humans), guinea pigs, bats and some birds. This makes our dependency of external vitamin C provision (basically plant products) strictly necessary. In animals and humans, vitamin C stimulates enzymes involved in the biosynthesis of collagen, catecholamines, L-carnitine, cholesterol, and amino acids, as well as in the hormonal activation, histamine detoxification, and phagocytosis by leukocytes, among others. In addition, vitamin C is linked to the reduction of incidence of a series of pathologies related to cancer, blood pressure and cardiovascular diseases prevention, immunity dysfunction, tissue regeneration, nervous system, etc. A lack of vitamin C causes scurvy, which leads to fragility of blood vessels and damage of connective tissue. Then, a failure in the collagen production takes place that may end in death as a consequence of the general collapse.

In plants, ascorbate is synthesized in mitochondria but its main target as powerful antioxidant is the chloroplast, where besides scavenging diverse ROS, as referred above, it is integrated in the AGC for hydrogen peroxide removal. Besides, ascorbate can regenerate α-tocopherol and participates in the xanthophylls' cycle, one type of carotenoids. Thus, due to its functionality, ascorbate is necessary in high amounts (as α-tocopherol and carotenoids are), not only in chloroplasts but also in other plant organelles. An example of the dualism antioxidant/vitamin and regarding ascorbate is as follows: (i) one pepper fruit from the California type (about 250 g) contains 350–375 mg [1]; (ii) one pepper plant is able to yield about 10 Kg fruits, which means around 14 g vitamin C, only in fruits, without considering leaves and other organs (pepper is a factory for vitamin C production); (iii) however, our daily requirements are about 80 mg of this vitamin; (iv) thus, one such pepper plant can satisfy the daily requirements of about 175 people. Why such a difference? The common physiological conditions imposed by light in plants, from an antioxidant-consuming perspective, are much more stressful than those operating in the majority of our physiological dysfunctions and diseases.

Following this rationale about the different viewpoints depending on the target organism, either human, animal or plant, this Special Issue will cover the same question posed in the title and developed on above for antioxidant/vitamin concepts. Thus, the concept of antioxidants in the transition from the field to the body, either human or animal, is exemplified using antocyanins as the case study [2]. Several plant materials have been used as vehicle to provide antioxidants for our diet. Thus, pepper fruits have been shown as one of the most important ascorbate sources, although the global provision of vitamin C upon the pepper intake depends on the variety and the ripening stage of fruits [1,3]. Moreover, in some cases, this antioxidant provision is also complemented by some functional compounds which, in the case of pepper, includes capsaicin, an alkaloid exclusive of this species with diverse therapeutic properties [1]. Variety and ripening are also relevant cues in the antioxidant content (ascorbate, polyphenols, flavonoids) of other noteworthy fruits in our diet, such as the *Citrus* species (Mandarin, Kumquat and Clementine), apples and grapes, and this antioxidant capacity is extensive to any of their parts, either pulp, seeds and peel/skin [4–6]. Likewise, fruits are also sources of other important antioxidants, i.e., α-tocopherol (vitamin E) and tocochromanols. It was proved that avocados are very rich in these compounds, always depending on the variety and the storage conditions [7]. Plants are also used to brew antioxidant-enriched beverages like tea. The type of tea, either white, green, black and red has been found to be essential for the preparation of Kombucha, a beverage obtained from fermented tea [8].

Many plant products are used for the food industry, giving rise to a huge diversity of manufactured goods undergoing a series of processes, which do not necessarily imply transformation. This is the case for stevia, which is a sweetener directly extracted from the original plant without any transformation. In this Special Issue, it is reported that the antioxidant metabolism of stevia is influenced by the acclimation of plants when they are exposed to ex vitro conditions [9].

Virgin olive oil deserves a special mention as a particular source of antioxidants [10], greatly attributed to its high content in polyphenols. Although the phenolic profile of extra virgin olive oil (EVOO) is modified when cooking, EVOO maintains its nutritional parameters and properties within the EU's health claims [11].

In the chapter of transformed products, pasta is one of the most paradigmatic foods obtained from wheat. Pasta can be used as a functional product since it is often consumed and it contains certain levels of phenolic compounds. Studies of bioaccesibility and bioavailability of such antioxidants can help formulating pasta where by-products could be even used as functional ingredients after the application of some pre-processing technologies [12].

Undoubtedly, all screening and quantification assays of antioxidants contained in natural or manufactured foods should be updated to gain accuracy, liability and reproducibility, so the nutritional standards can be fit through the use of consensual methods and approaches. Such a critical review of existing assays and a prospect analysis has been achieved in this Special Issue [13].

This volume also includes a series of articles focused on the role of food antioxidants in the prevention of chronic diseases and health disorders. Thus, the relevance of lycopene, a carotenoid which is abundant in tomato, has been highlighted in certain diseases including diabetes mellitus, cardiac complications, cancer insurgences, oxidative stress-mediated malfunctions, inflammatory, skin and bone diseases, as well as reproductive, hepatic and neural disorders [14]. It has been also reported that carotenoid derivatives inhibit osteoclast differentiation through the partial inhibition of the NF-ÎB pathway. Additionally, they can synergistically inhibit osteoclast differentiation as well in the presence of curcumin and carnosic acid [15].

This goodness of natural food for our health is in some cases undermined by processing. Thus, in the analysis of the antioxidant activity of green tea once it was processed, it was found that food processing does not always have positive effect on products destined for human consumption [16]. In this line, a gamma-conglutin found in narrow-leafed lupin showed anti-inflammatory properties through the promotion of insulin resistance and amelioration of the potential oxidative stress underwent in PANC-1 pancreatic cells [17]. Likewise, using Raw 264.7 murine, it was found that root cell extracts from curly dock (*Rumex crispus*) displayed anti-inflammatory and anticancer activities, possibly due to the anthraquinone present in this plant material [18]. In the same animal model, it was also demonstrated the antioxidant potential of elephant apple (*Dillenia indica* L.) bark against *tert*-butyl-hydroperoxide)-induced oxidative stress [19].

Strategies of (micro)encapsulation of materials and how they can contribute to benefit human health are another trend which is gaining attention nowadays. The use of encapsulating red cabbage extracts to improve the bioaccesibility of their polyphenols has been reported to be a good choice to counteract the diseases associated to oxidative stress episodes [20]. Similarly, it was reported that microencapsulation of Saskatoon Berry fruit (*Amelanchier alnifolia*) might be a promising practice with positive impact in the functionality of rye bread [21].

Research on antioxidant capacity of foods coming from animal sources has been also included in this Special Issue. By making comparisons of the meat origin, either conventional or organic, from selected elements of the pork carcass (ham, loin and shoulder), it was observed that meat products from conventional rearing systems had the best antioxidant properties with respect to antioxidant peptides. This discovery could be addressed to the human health field but also to the related industry [22].

Globally, this volume compiles a series of articles focused on the potential contribution of consuming certain food products for the human welfare. The basis of such benefits is mainly related to the antioxidant contribution of those products, but their content of other health promoting compounds cannot be discarded. This drives our attention to investigate those products and their chemical composition to convert them into functional foods with nutraceutical properties. Much research is necessary to understand the specific mechanisms by which many secondary plant metabolites act over our physiology in a beneficial manner and how they interact to prevent a number of human disorders.

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

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

### **References**


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### *Article* **Antioxidant Profile of Pepper (***Capsicum annuum* **L.) Fruits Containing Diverse Levels of Capsaicinoids**

**José M. Palma 1,\*, Fátima Terán 1,2, Alba Contreras-Ruiz 1,3, Marta Rodríguez-Ruiz <sup>4</sup> and Francisco J. Corpas <sup>1</sup>**


Received: 30 July 2020; Accepted: 14 September 2020; Published: 17 September 2020

**Abstract:** *Capsicum* is the genus where a number of species and varieties have pungent features due to the exclusive content of capsaicinoids such as capsaicin and dihydrocapsaicin. In this work, the main enzymatic and non-enzymatic systems in pepper fruits from four varieties with different pungent capacity have been investigated at two ripening stages. Thus, a sweet pepper variety (Melchor) from California-type fruits and three autochthonous Spanish varieties which have different pungency levels were used, including Piquillo, Padrón and Alegría riojana. The capsaicinoids contents were determined in the pericarp and placenta from fruits, showing that these phenyl-propanoids were mainly localized in placenta. The activity profiles of catalase, total and isoenzymatic superoxide dismutase (SOD), the enzymes of the ascorbate–glutathione cycle (AGC) and four NADP-dehydrogenases indicate that some interaction with capsaicinoid metabolism seems to occur. Among the results obtained on enzymatic antioxidants, the role of Fe-SOD and the glutathione reductase from the AGC is highlighted. Additionally, it was found that ascorbate and glutathione contents were higher in those pepper fruits which displayed the greater contents of capsaicinoids. Taken together, all these data indicate that antioxidants may contribute to preserve capsaicinoids metabolism to maintain their functionality in a framework where NADPH is perhaps playing an essential role.

**Keywords:** ascorbate; ascorbate–glutathione cycle; capsaicin; catalase; dihydrocapsaicin; glutathione; NADP-dehydrogenases; superoxide dismutase

#### **1. Introduction**

Pepper (*Capsicum annuum* L.) fruits are one of the most consumed vegetables worldwide. Pepper fruits are mainly characterized by their high vitamin C and A and mineral contents [1–8]. Thus, about 60–80 g intake of fruits per day can provide 100 and 25% of recommended daily amounts of vitamin C and A, respectively [5,9]. Besides, this horticultural product contains important levels of other health-promoting substances with antioxidant capacity, and they include carotenoids, flavonoids and other polyphenols, among others [1,10–12].

The diversity of pepper varieties is quite high and they are basically differentiated by shape, size, pulp (pericarp) thickness and final color at the ripe stages. This diversity is also mirrored by the number of common names to designate pepper fruits which, in most cases, are used very locally. From culinary and gastronomic points of view, pepper fruits are mainly classified as sweet and hot

depending on the absence or presence of capsaicin, respectively [4,5,12,13]. Within the sweet pepper (also amply known as bell pepper) varieties, three main types are distinguished according to their shape and size: California, Lamuyo and Dulce italiano. Hot peppers include the highest number of varieties and names including chili, habanero, jalapeño, paprika, chipotle and the Spanish Alegría riojana, Padrón and Piquillo used in this work, among others.

Capsaicin is exclusive to the genus *Capsicum* and is responsible for the pungency trait. According to the pungent level, pepper fruits are ranked on the so-called Scoville scale which assigns a score to each fruit variety. In this scale, the highest value for the most pungent pepper fruit variety is around <sup>3</sup> <sup>×</sup> <sup>10</sup>6, pure capsaicin being 16 <sup>×</sup> <sup>10</sup><sup>6</sup> [14–18]. Capsaicin is an alkaloid with a phenyl-propanoid nature which has given rise to a family of capsaicinoids composed of at least 22 primary compounds. Out of them, capsaicin and dihydrocapsaicin contribute to about 90–95% of total capsaicinoids present in most hot pepper varieties [19,20]. These compounds are mainly localized in the epidermal vacuoles of the placenta and the septum from fruits, and they can be separated and identified through the use of high-performance liquid chromatography associated with electrospray ionization mass spectrometry (HPLC-ESI/MS) [19,21,22]. Capsaicin is useful for pepper plants to avoid biting by insects and other animals since this chemical has repellent/insecticide capacity [23–27]. From a pharmacological perspective, the research carried out so far has shown that capsaicinoids, particularly capsaicin, have a diversity of biological and physiological functions in vitro, so they play roles as antioxidants, stimulants of the energetic metabolism, fat accumulating suppressors, anti-inflammatories, neurostimulants and apoptosis-alleviating agents in neurodegenerative disorders [20,28–30]. Regarding the mechanism of action, capsaicinoids act on a family of ion channels known as transient receptor potential (TRP) channels which, in mammals, are framed within the subtype TRP Vanilloid (TRPV1) [31,32]. It has been also found that in many types of cancers, the proapoptotic activity of capsaicin is also mediated by this TRPV, and the activation of the p53 tumor suppressing protein by a phosphorylation process is induced by capsaicin [33,34].

Another relevant feature of pepper fruits is the ripening process, visibly characterized by a shift in the fruit color from green to red, yellow, orange or purple depending on the variety. This event implies chlorophyll breakdown and synthesis of new carotenoids and anthocyanins, emission of organic volatiles, new protein synthesis and cleavage of existing ones and cell wall softening, among others [5,7,35–39]. Relevant differences between the transcriptomes from green immature and ripe pepper fruits have been also reported, involving thousands of genes [8,40] and references therein. From a redox viewpoint, it has been found that reactive oxygen species (ROS) metabolism is also affected during fruit ripening, leading to major changes in total soluble reducing equivalents and the antioxidant capacity in fruits [41]. The profile of the major non-enzymatic antioxidants, including ascorbate, glutathione, carotenoids and polyphenols, has been followed during ripening in pepper fruits [4,11,12,42–46], but less is known on how enzymatic antioxidants evolve with this physiological process. These enzyme systems basically include superoxide dismutase (SOD), catalase (CAT) and the ascorbate–glutathione cycle as the primary defense barriers against ROS, and some NADP-dehydrogenases as a secondary system to help the antioxidative enzymatic block. The profile of these enzymes throughout fruit ripening has been mostly carried out in sweet pepper [4,11,45,46], but scarce references have been reported on how those antioxidant enzymatic systems behave in the ripening of hot varieties [47–49]. Accordingly, using pepper varieties containing increasing capsaicin and dihydrocapsaicin contents, this work was aimed at characterizing the profile of the main antioxidants and their potential interaction with capsaicinoids during fruit ripening. This could provide a biochemical support and an added value for the particular features of each Spanish autochthonous cultivar that is included in the European Register of protected designations of origin for these horticultural products.

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

#### *2.1. Plant Material*

Fruits from four pepper (*Capsicum annuum* L.) varieties were used in this work: California-type (sweet), obtained from plants grown in plastic-covered greenhouses (Zeraim Iberica/Syngenta Seeds, Ltd., El Ejido, Almería, Spain); Padrón (mild hot), provided by the Regulatory Council of Denomination of Origin "Pemento de Herbón" (Herbón, Coruña, Spain) and Piquillo (slightly hot) and Alegría riojana (quite hot), both provided by the Regulatory Council of Denomination of Origin "Pimiento del Piquillo-Lodosa" (Navarra, Spain). Padrón, Piquillo and Alegría riojana (onwards Alegría) fruits were obtained from plants grown in orchards under the local conditions. In all varieties, fruits at both green and red ripe stages were analyzed. Both stages were set according to marketing and consuming preferences as indicated by the growers. Green fruits did not show any ripening symptoms, mainly color shift, and they were totally green. Red fruits were harvested after several days they underwent the color change *in planta*. In all cases, fruits did not display any apparent damages. Figure 1A shows representative pictures of the different varieties used in this work, and in Table 1, comparative data on the mean fresh weight (g) of each type of fruit are given. Fresh weight data were obtained from 10 fruits of each variety at both maturation stages. After harvesting, in all fruits set for analyses, the pericarp and placenta (once seeds were discarded) were separated (Figure 1B), and each one was cut into small cubes (approximately 3–5 mm/side), frozen under liquid nitrogen and then stored at −80 ◦C until use. All biochemical parameters were determined thrice from 5 fruits in each variety and at the two ripening stages. As a whole, each mean was obtained from 15 assays.

**Figure 1.** Representative pictures of plant materials used in this work. (**A**) Fruits from the four varieties at two ripening stages: green and ripe red. Melchor is a variety of California-type sweet pepper fruit. Piquillo Padrón and Alegría riojana contain different levels of capsaicin with the sequence Piquillo <<< Padrón < Alegría riojana. (**B**) Different parts of the pepper fruit.


**Table 1.** Fresh weight (FW) of whole fruits from four pepper varieties at two ripening stages.

Data are the means ± SEM of ten fruits from each variety and ripening stage.

#### *2.2. Determination of Capsaicin and Dihydrocapsaicin by High-Performance Liquid Chromatography-Electrospray Mass Spectrometry (HPLC-ES*/*MS)*

Samples were ground into a powder under liquid N2 and using an IKA® A11 Basic mill (IKA Laboratories Inc., Tirat Carmel, Israel). For each sample, three extractions were made as follows. Plant materials (0.5 g powder) were suspended into 2.0 mL acetonitrile (AcN) containing 100 ppm N-[(3,4-dimethoxyphenyl)methyl]-4-methyl-octanamide (DMBMO), as an internal standard. Mixtures were incubated in the following sequence: 1 h at room temperature and darkness with continuous shaking; 65 ◦C and darkness for 1 h and short shakings every 15 min; and 1 h at room temperature in the dark. Then, samples were centrifuged at 16,000× *g* and room temperature for 15 min. Supernatants were passed through 0.22 μm pore size polyvinylidene fluoride filters and were used for analysis through HPLC-ESI/MS with mode multiple reaction monitoring (MRM). An XBridge 2.1 × 10 mm pre-column and an XBridge 2.1 × 100 mm C18 3.5 μm column (Waters Corporation, Milford, MA, USA) were used connected to an Allience 2695 HPLC system coupled to a Micromass Quattro micro API triple quadrupole mass spectrometer both obtained from the Waters Corporation. The chromatography was run at a flux of 0.3 mL/min with temperatures of 35 ◦C for the column and 5 ◦C for the auto-injector, with 5 μL being injected per sample. The gradient used was: 6 min with AcN:H2O (60:40) containing 0.1% (*v*/*v*) formic acid; 10 + 5 min with AcN:H2O (90:10); and 20 + 4 min with AcN:H2O (60:40). Standard curves were prepared using pure capsaicin and dihydrocapsaicin (Cayman Chemical, Ann Arbor, MI, USA). Under these conditions, the retention time for capsaicin and dihydrocapsaicin was 1.88 and 2.24 min, respectively. The concentration of capsaicinoids was expressed as μg g−<sup>1</sup> of fresh weight (FW).

#### *2.3. Detection and Quantification of Ascorbate, GSH and GSSG by High-Performance Liquid Chromatography-Electrospray Mass Spectrometry (LC-ES*/*MS)*

Pericarps and placentas were ground under liquid N2 with a pestle and a mortar. Then, 0.4 g of powdered tissues was suspended into 1 mL of 0.1 M HCl and spun for 20 min at 15,000× *g* and 4 ◦C. Supernatants were passed through polyvinylidene fluoride filters (0.22-μm pore size) and analyzed immediately. All procedures were performed at 4 ◦C with protection from light to prevent potential degradation of the metabolites. Samples were analyzed by liquid chromatography–electrospray/mass spectrometry (LC-ES/MS) using the HPLC system and mass spectrometer indicated above. HPLC runs were performed with an Atlantis® T3 3 <sup>μ</sup>m 2.1 <sup>×</sup> 100 mm column (Waters Corporation). The MassLynx 4.1 software package (Waters Corporation) was used for the instrument control and collection, analysis and management of data. With this method, the simultaneous detection and quantification of ascorbate, reduced (GSH), and oxidized (GSSG) glutathione is achieved [7,50]. The analytes concentration was calculated with the use of external standards and expressed with reference to fresh weight (FW).

#### *2.4. Preparation of Crude Extracts for Enzyme Activity*

Protein extracts from pericarps and placentas were powdered under liquid nitrogen and then suspended in 0.1 M Tris-HCl buffer, pH 8.0, containing 1 mM EDTA, 0.1% (*v*/*v*) Triton X-100 and 10% (*v*/*v*) glycerol, in a final 1:1 (*w:v*) plant material/buffer ratio. Crude extracts were centrifuged at 15,000× *g* for 30 min and the supernatants were used for enzymatic assays.

#### *2.5. Enzyme Activity Assays*

All enzyme activities were determined using an Evolution 201 UV–visible spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Catalase (EC 1.11.1.6) activity was determined by following the H2O2 breakdown at 240 nm [51]. Ascorbate peroxidase (APX; EC 1.11.1.11) was monitored at 290 nm by plotting the initial ascorbate oxidation by H2O2 [52]. Monodehydroascorbate reductase (MDAR; EC 1.6.5.4) activity was assayed by following the monodehydroascorbate-dependent NADH oxidation. In these assays, monodehydroascorbate was generated through the ascorbate/ascorbate oxidase system as reported earlier [53]. The monodehydroascorbate-independent NADH oxidation rate (without ascorbate oxidase and ascorbate) was deducted from the monodehydroascorbate-dependent reaction. Dehydroascorbate reductase (DHAR, EC 1.8.5.1) activity was measured by monitoring at 265 nm the increase in ascorbate formation, with the use of a N2-saturated buffer. The reaction rate was corrected by the non-enzymatic dehydroascorbate reduction through reduced glutathione (GSH). A 0.98 factor was also considered, due to the little contribution to the absorbance by oxidized glutathione (GSSG) [54]. Glutathione reductase (GR; EC 1.6.4.2) activity was analyzed by following at 340 nm the NADPH oxidation associated with the reduction of GSSG to GSH [55]. The GR reaction rate was corrected for the very small, non-enzymatic NADPH oxidation by GSSG.

Total SOD (EC 1.15.1.1) activity was determined by the ferricytochrome *c* reduction method, with the system xanthine/xanthine oxidase as a superoxide radical (O2 ·−) source. One activity unit was defined as the amount of protein necessary to inhibit 50% of the cytochrome *c* reduction [56]. For the analysis of the SOD isoenzyme profile, proteins from crude extracts were separated by vertical non-denaturing PAGEs on 10% acrylamide gels, using a Mini-Protean III Tetra Cell system (Bio-Rad Laboratories, Hercules, CA, USA). SOD isozymes were detected in the gels as acromatic bands over a purple background by a specific staining based in the photochemical reduction method of nitroblue tetrazolium (NBT) [57]. For the identification of the different SOD isozymes, before the staining procedure, pre-incubation of gels was carried out in the presence of specific inhibitors, either 5 mM KCN or 5 mM H2O2. Copper- and zinc-containing SODs (CuZn-SODs) are inhibited by both KCN and H2O2; iron-containing SODs (Fe-SODs) are inactivated by H2O2; and Mn-SODs are resistant to both inhibitors [58,59].

NADP-dependent dehydrogenase (NADP-DHs) activities were determined by recording the NADPH formation at 340 nm and 25 ◦C. The assay was performed in a reaction medium containing 50 mM HEPES [(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)], pH 7.6, 2 mM MgCl2 and 0.8 mM NADP. Each enzymatic reaction was initiated by the addition of the respective specific substrates [46]. For the glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) activity, the reaction started after the addition of 5 mM glucose-6-phosphate. To monitor 6-phosphogluconate dehydrogenase (6PGDH, EC 1.1.1.44) activity, 5 mM 6-phosphogluconate was used as the substrate. NADP-isocitrate dehydrogenase (NADP-ICDH, EC 1.1.1.42) activity was triggered with 10 mM 2R,3S-isocitrate [60,61]. For the NADP-malic enzyme (NADP-ME, EC 1.1.1.40) activity, the reaction was initiated with 1 mM L-malate [62].

Protein concentration in samples was determined by the Bradford method [63], using the Bio-Rad protein assay solution (Bio-Rad Laboratories) and bovine serum albumin as the standard.

#### *2.6. Immunoblot Analysis*

Proteins separated by native-PAGE (10% acrylamide) and SDS-PAGE (12% acrylamide) were transferred onto polyvinylidene difluoride (PVDF) membranes, using a Trans-Blot SD equipment (Bio-Rad Laboratories). The transfer buffer used was 10 mM N-cyclohexyl-3-aminopropanesulfonic acid (CAPS), pH 11.0, 10% (*v*/*v*) methanol. Runs were developed at 1.5 mA/cm<sup>2</sup> membrane for 2 h [64]. After the protein transfer, membranes were processed for further blotting assays. An antibody against Fe-SOD from pepper fruits (dilution 1:5000) was used. The antibody-recognizing proteins were visualized using the ClarityTM Western ECL Substrate kit (Thermo Fisher Scientific) following the manufacturer's instructions.

#### *2.7. Statistical Analysis*

One-way ANOVA was used for the comparisons between means of capsaicin and dihydrocapsaicin contents using the Statgraphics Centurion program (Statgraphics Technologies, Inc., Madrid, Spain). For other parameters, the *t*-student test was used to detect differences between the two ripening stages of each variety. In both the ANOVA and *t*-student, values for *p* < 0.05 were considered different with statistical significance.

#### **3. Results**

In this work, pepper fruits from four varieties with different pungency tastes were investigated. Thus, the concentration of the main capsaicinoids, capsaicin and dihydrocapsaicin, was analyzed. As shown in Table 2, Melchor, which is a sweet variety, did not contain any of the capsaicinoids, and Piquillo only displayed little values both in green and red fruits, with placenta being the tissue where both metabolites were present in higher amounts. Regarding Padrón and Alegría, both varieties showed high capsaicinoid contents, with less amounts in the pericarp and the major levels being clearly observed in the placenta. In these two last varieties, the concentration of capsaicin and dihydrocapsaicin was remarkably increased in ripe red fruits.

**Table 2.** Content of capsaicin and dihydrocapsaicin in pericarp and placenta from fruits of four pepper varieties at two ripening stages.


Placenta tissue was used once seeds were discarded. Data are the means ± SEM of three replicates determined from five fruits of the four varieties and at the two ripening stages. Different letters after each value indicate that differences were statistically significant (ANOVA, *p* < 0.05). FW, fresh weight.

As shown in Figure 2, the higher ascorbate concentration was found in Melchor, and this parameter only changed due to ripening in the two varieties with higher capsaicinoid levels, Padrón and Alegría. In both, ascorbate was significantly enhanced after fruits ripened. Likewise, this tendency also occurred with GSH, which only increased significantly in Padrón and Alegría after ripening, whereas it lowered in Melchor after this physiological process took place (Figure 3A). The oxidized form of glutathione (GSSG) diminished in Melchor and Piquillo ripened fruits and no changes were observed in Padrón and Alegría. As indicated in Table 3, total glutathione content (GSH + GSSG) increased in Padrón and Alegría and lowered in Melchor after ripening. The ratio GSH/GSSG was enhanced by ripening in the four varieties, thus indicating a shift to a higher reducing environment (Table 3).

**Figure 2.** Ascorbate content in pericarp from fruits of four pepper varieties at two ripening stages. Data are the means ± SEM of three replicates determined from five fruits of the four varieties and at the two ripening stages. Asterisks indicate significant differences of red fruits with respect to green fruits for each variety (*t*-student, *p* < 0.05). FW, fresh weight.

**Figure 3.** Reduced (GSH) and oxidized (GSSG) glutathione contents in pericarp from fruits of four pepper varieties at two ripening stages. (**A**) GSH. (**B**) GSSG. Data are the means ± SEM of three replicates determined from five fruits of the four varieties and at the two ripening stages. Asterisks indicate significant differences of red fruits with respect to green fruits for each variety (*t*-student, *p* < 0.05). FW, fresh weight.

**Table 3.** Total glutathione (GSH + GSSG) and the ratio GSH/GSSG from fruits of four pepper varieties at two ripening stages.



**Table 3.** *Cont*.

GSH, reduced glutathione. GSSG, oxidized glutathione. Data are the means ± SEM of three replicates determined from five fruits of the four varieties and at the two ripening stages. Asterisks indicate significant differences of red fruits with respect to green fruits for each variety (*t*-student, *p* < 0.05). FW, fresh weight.

The activity of the main enzymatic antioxidants was studied. Catalase was significantly lower in ripe fruits from all varieties except for Padrón, where the activity increased after ripening (Figure 4A). SOD activity increased as a consequence of ripening but only significantly in Padrón and Alegría. No changes were observed in the Piquillo variety at the two stages (Figure 4B). This SOD activity pattern was partially confirmed by the analysis of the isoenzymatic profile. Thus, in the Padrón variety, no Fe-SOD activity was detected in green fruits, whereas this isozyme appeared in red fruits (Figure 5A). Additionally, CuZn-SOD I and II were also higher in ripe fruits than in green ones. Regarding the Alegría variety, it was observed that Fe-SOD and CuZn-SOD II were more prominent in red fruits than in green fruits (Figure 5A). To seek for the possible reason of the absence of Fe-SOD activity in the Padrón variety, immunoblot assays were performed under native and denaturing conditions. Thus, after native PAGE and Western blotting analysis using an antibody against an Fe-SOD from pepper fruits, no cross-reacting bands were observed in green fruits from the Padrón variety. Additionally, the use of this approach confirmed that the activity pattern observed in Alegría was due to a higher Fe-SOD protein amount in red fruits (Figure 5B). To further check that Padrón did not contain the Fe-SOD protein, SDS-PAGE and Western blotting was achieved. In all samples, a cross-reacting band, characteristic of the plant Fe-SOD monomeric size (23 kDa), was detected, including green fruits from the Padrón variety, although with a very low quantity (Figure 5C). This indicates that this isozyme is present in this variety, but in such a little amount that its contribution to the total SOD activity is possibly irrelevant.

**Figure 4.** Catalase and superoxide dismutase (SOD) activity in pericarp from fruits of four pepper varieties at two ripening stages. (**A**), catalase. (**B**), SOD. Data are the means ± SEM of three replicates determined from five fruits of the four varieties and at the two ripening stages. Asterisks indicate significant differences of red fruits with respect to green fruits for each variety (*t*-student, *p* < 0.05).



**Figure 5.** Isoenzymatic superoxide dismutase (SOD) pattern in pericarp from fruits of four pepper varieties at two ripening stages. (**A**) Native PAGE on 10% acrylamide gels and further in-gel SOD activity staining by the NBT reduction method; 34 μg protein per well was loaded. (**B**) Immunoblotting after native PAGE on 10% acrylamide gels. (**C**) Immunoblotting after SDS-PAGE on 12% acrylamide gels. In both immunoblotting assays, an antibody against Fe-SOD from pepper fruits (dilution 1:5000) was used. In panel B, the mobility of the detected bands was similar to the one observed for Fe-SOD in panel A. The monomeric molecular size of the cross-reacting bands (23 kDa) is indicated on the left in panel C. Data are representative of at least three independent experiments where different samples from each variety and ripening stage were used. G, green fruits. R, red fruits.

The enzymatic side of the AGC was analyzed, following the activity of APX, MDAR, DHAR and GR. APX was little, but significantly enhanced in ripe fruits with respect to green fruits in Melchor and Piquillo, and lower in red fruits from Padrón (Figure 6A). Regarding MDAR, this enzyme did not show significant changes upon ripening in the four varieties (Figure 6B). DHAR was only significantly lower in red fruits from those varieties with a high capsaicinoid content, Padrón and Alegría (Figure 6C). Finally, all varieties displayed significant enhanced GR activity after ripening (Figure 6D).

Regarding the activity profile of NADP-dependent dehydrogenases (NADP-DHs), four eznymes were studied: G6PDH, 6PGDH, NADP-ICDH and NADP-ME. G6PDH and NADP-ICDH displayed parallel profiles with lower activities in red than in green fruits in the varieties Melchor and Alegría, but enhanced activity after fruits from the Padrón variety ripened (Figure 7A,C). No changes in those enzymatic systems were observed in fruits from the Piquillo variety. With respect to 6PGDH, this activity only changed in Padrón, with enhancement after ripening (Figure 7B). NADP-ME showed disparate evolution depending on the varieties. Thus, it increased in Melchor and Piquillo upon ripening and lowered in Padrón, with no changes in Alegría (Figure 7D).

**Figure 6.** Activity of enzymes from the ascorbate—glutathione cycle in pericarp from fruits of four pepper varieties at two ripening stages. (**A**) Ascorbate peroxidase (APX). (**B**) Monodehydroascorbate reductase (MDAR). (**C**) Dehydroascorbate reductase (DHAR). (**D**) Glutathione reductase (GR). Data are the means ± SEM of three replicates determined from five fruits of the four varieties and at the two ripening stages. Asterisks indicate significant differences of red fruits with respect to green fruits for each variety (*t*-student, *p* < 0.05).

**Figure 7.** Activity of NADP-dehydrogenases in pericarp from fruits of four pepper varieties at two ripening stages. (**A**) Glucose-6-phosphate dehydrogenase (G6PDH). (**B**) 6-Phosphogluconate dehydrogenase (6PGDH). (**C**) NADP-dependent isocitrate dehydrogenase (ICDH). (**D**) NADP-dependent malic enzyme (ME). Data are the means ± SEM of three replicates determined from five fruits of the four varieties and at the two ripening stages.

#### **4. Discussion**

#### *4.1. The Experimental Design Provided a Gradual Capsaicin Concentration Depending on the Pepper Variety and the Ripening Stage*

Pepper varieties used in this work were selected because of their different pungency levels according to consumers taste, which is the basis where the Scoville scale resides. All four varieties are common in Spanish food markets and their culinary uses are diverse. Melchor is a type of sweet pepper characterized by its consistency and appropriateness for different purposes. This variety, along with other sweet pepper varieties, provides the high production figures in Spain. Its tasting features in either green or red frame this variety in the non-pungent fruits' group. Piquillo is an autochthonous variety from northern Spain and its main phenotypic feature is its triangle shape with a sharp-peaked extreme. Upon intake, it is characterized by a very slight pleasant pungency, but it is only consumed in its ripe red stage. Padrón is characteristic of and originally from northwestern Spain, although lately it is also cultivated in many other lands in the Mediterranean area. These fruits are small, and they are usually consumed as green after cooking. Commonly, in the green stage, they show a very slight spicy taste, but it is in the red stage that it is not consumed due to its strong pungency. Finally, Alegría riojana (which might be translated as Riojan joy) is also autochthonous from northern Spain and it is usually used as spice in the red stage. Both green and red, but mainly red, fruits from this variety are extremely pungent.

With this tasting background and considering the antioxidant quality attributed to capsaicinoids [20,28,29], we aimed, in this work, to investigate the potential influence of these compounds (capsaicin plus dihydrocapsaicin; Cap+DiCap) in the profile of the main enzymatic and non-enzymatic antioxidants of pepper fruits containing different levels of these alkaloids. Our experimental design established a gradual scale from null values of Cap+DiCap, both in green and red ripe stages (Melchor), to red Alegría which contained high levels of the two capsaicinoids. The content of the Cap+DiCap couple matched with the tasting scale and the higher values, as expected, were found in the placenta in the three pungent varieties. Based on these data, we found quite the appropriate selection of these varieties and ripening stages to target our objective.

Except for Alegría which has been scarcely used for research purposes so far, reports on the other three varieties can be found in the literature. Thus, the Melchor variety has been used to decipher the mechanisms involved in fruit ripening [18,38,65,66], where some of their antioxidant systems have been reported to be involved [67]. The Piquillo variety was used as a model to address the effects triggered by infection with *Verticillum* [68–71] and how to protect pepper plants against it through diverse practices [72,73], as well as to investigate the effect of sanitized sewage sludge on the growth, yield, fruit quality, soil microbial community and physiology of pepper plants [74,75]. On the other hand, the Padrón variety was set, for example, to investigate either how wounding induces local resistance but systemic susceptibility to *Botrytis cinerea* in pepper plants [76], as a reference to assess real-time PCR as a method for determining the presence of *Verticillium dahliae* in distinct solanaceae species [77], or to study the virulence and pathogenesis issues of *Phytophthora capsici* [78], among others.

#### *4.2. The Ripening Stage and the Capsaicinoids Content Alter the Metabolism of Enzymatic Antioxidants*

The profile of antioxidant enzymes during the ripening process has been investigated in pepper fruits previously but, to our knowledge, no comparisons have been made between varieties with different capsaicinoid contents. Thus, for example, in California-type pepper fruits, it has been reported that the catalase activity decreases as the fruit ripens [79,80] and this event is due to the post-translational modification (PTMs) underwent by the enzyme and promoted by ROS and reactive nitrogen species (RNS) derived from nitric oxide (NO) [41,81]. In fact, it has been proved that the ripening of pepper fruits is controlled by NO [8,40,80]. This inhibitory effect of ripening in the catalase activity also occurred in the same California-type fruits subjected to storage at 20 ◦C [80], in other sweet pepper varieties from Lamuyo and Dulce italiano types [4], and during the ripening of hot pepper Kulai [49]. Our data on the Melchor, Piquillo and Alegría varieties confirm this activity pattern of the catalase activity, although, interestingly, this profile is opposite in the Padrón variety where catalase activity increases in ripe fruits. This same increasing catalase activity was reported in hot pepper varieties either under saline stress conditions [47] or in preventing seed browning during low-temperature storage [48].

Regarding SOD, the total activity was higher in ripe fruits from those varieties which contained a higher capsaicinoids content, namely Padrón and Alegría. In Alegría, this higher activity seems basically to be due to an enhancement of the isozymes Fe-SOD and CuZn-SOD II, whereas in Padrón, the presence of Fe-SOD (nearly absent in green fruits) and the higher activity of both CuZn-SODs could be responsible for such changes. Due to this interesting behavior of the Fe-SOD isozyme in the Padrón variety, complementary immunoblot analyses were performed using an antibody against the isozyme from pepper fruits. Thus, by Western blotting after both non-denaturing- and SDS-PAGEs, it was confirmed that the negligible Fe-SOD activity in ripe Padrón fruits was due to the little amounts of its corresponding protein, whose monomer (23 kDa) could only be detected after SDS-PAGE. This issue needs to be further investigated at the molecular level (gene and protein expression) since it means that it might be an identity feature of this pepper variety. The SOD activity has been also studied earlier in pepper varieties including some of those included in the present work. So, recently, it has been reported that the SOD isoenzyme pattern and gene expression of California-type pepper fruits are regulated by ripening and NO [67], and this enzymatic system from sweet pepper is also involved in the response against low temperatures [4] and the storage of fruits at 20 ◦C [79], as well as in the "accommodation" of fruits to nitrogen deprivation during plant growth [82]. The isoenzymatic SOD pattern was also investigated in the plastid population from sweet pepper fruits of different California-type varieties, and a protective role of these organelles by the different SOD internal isozymes during ripening was reported [45]. In the Piquillo variety, it was found that SOD is involved in the association of pepper plants with arbuscular mycorrhizal fungi (AMF) to avoid the negative effects promoted by *Verticillum* [73]. A number of studies have reported the involvement of SOD from hot pepper in diverse processes including ripening and post-harvest [49,83], salt stress [47,84], storage at low temperature [48] and iodine bio-fortification practices to improve fruit quality [85].

The activity of the four enzymes of the ascorbate–glutathione cycle (AGC), APX, MDAR, DHAR and GR, were analyzed in this work. APX is responsible for the direct scavenging of hydrogen peroxide (H2O2) using ascorbate as the electron donor, whereas MDAR and DHAR restore the reduced status of ascorbate using NAD(P)H and GSH, respectively. The last step of the AGC is carried out by GR, an enzyme which converts the oxidized form of glutathione (GSSG) to the reduced form (GSH) with the use of NADPH as the reducing power. In our experimental design, the most remarkable response of this cycle was found at the GR side which was significantly enhanced in ripe fruits from all four varieties. The profile of these AGC enzymes has been investigated in pepper fruits from diverse varieties, both sweet and hot, and different trends have been reported depending on the experimental conditions, including ripening, post-harvest, salt stress, defense mechanisms or bioremediation practices [4,11,45,49,75,82,83,86]. In our case, it is remarkable that APX behaved oppositely in sweet and hot varieties, with the activity increasing in ripe fruits from Melchor and Piquillo and decreasing in hot ripe fruits from Padrón and Alegría. MDAR and DHAR shared a similar trend with lower values in ripe fruits, but only significant in MDAR from Padrón and Alegría. According to the activity profile of APX, MDAR and DHAR from green to red stages, it could be hypothesized that the cycle seems to be operative in the first steps which involve direct ascorbate metabolism, but more research at different levels is necessary to obtain a whole picture of this antioxidant metabolic pathway. According to our results, it seems that hot peppers have less capacity to recycle ascorbate but all varieties showed a great potentiality to provide GSH.

The activity pattern displayed by the NADP-dehydrogenases (NADP-DHs) can be framed in three main features: (i) the behavior of the two varieties with less Cap+DiCap levels (Melchor and Piquillo) was quite similar with slight, although not strongly significant, decreases in G6PDH and little, although not significant enough, decreases in 6PGDH and NADP-ME in ripe fruits; (ii) except for NADP-ME, all other NADP-DHs rose after ripening in the Padrón variety (high capsaicinoids content), and this suggests a higher NADPH availability for different purposes in ripe fruits from this variety; and (iii) interestingly, the behavior of these enzymatic systems in the other variety with high capsaicinoids content was different to that showed by Padrón. Thus, green fruits from Alegría seemed to have higher capacity to generate NADPH. To our knowledge, no reports on NADP-DHs from hot pepper fruits have been published previously, and the only data concerning these NADP/NADPH systems in pepper refer to sweet varieties. Our data mostly confirm those previously found for other California-type pepper varieties [46]. Recent data report that pepper fruit NADP-DHs are not only influenced by ripening in the Melchor variety [8], but also by NO through diverse PTMs [87,88]. Moreover, it was also found that NADP-DHs are involved in the response of sweet pepper plants to stress exerted by high Cd levels [89].

#### *4.3. The Higher Capsaicinoids Level the Higher Ascorbate and Glutathione Content*

Capsaicinoids, specially capsaicin, have been reported to have, among others, antioxidant properties [20,28–30]. In pepper fruits containing these alkaloids, this feature is quite interesting since these horticultural products are one of those with the highest ascorbate levels [5], with ascorbate being perhaps the most paradigmatic molecular antioxidant for living beings. In fact, ascorbate is one of the parameters which is commonly determined in (sweet and hot) pepper fruit research including either ripening and post-harvest, any type of stress (biotic, abiotic and environmental) or culture practices [4,5,7,11,14,18,45,47,49,75,84,90]. As an appraisal of the potential roles attributed to ascorbate in pepper fruits, it was proposed that in the sweet varieties, ascorbate functions as a redox buffer to balance the great metabolic changes which undergo during ripening [5,7]. Regarding the hottest varieties (Padrón and Alegría), the pattern observed in this work, where ascorbate levels increased in those fruits, was also reported earlier for diverse hot pepper varieties [14,18,49,90]. Perhaps, the redox stabilizing role of ascorbate indicated above for sweet pepper could be also applicable to hot varieties to assure the capsaicinoids level. In fact, it was proved that during the capsaicinoids oxidation catalyzed by peroxidases, capsaicinoid radicals are formed, and ascorbate rapidly reduces capsaicinoid radicals, this being an important cue for capsaicinoid content and preservation in pepper fruits [91].

Glutathione is a ubiquitous and powerful antioxidant in eukaryotes [92]. In spite of its relevant role in many biological processes, this tripeptide has been less investigated in pepper fruits, mainly associated to ripening, or bioremediation purposes [49,75,82,93]. However, not much information is available on glutathione metabolism in capsaicinoids-containing pepper varieties. This work provides the first comparison of the levels of both GSH and GSSG in different pepper varieties containing gradual amounts of capsaicinoids. It is noteworthy that, whereas the total glutathione content (GSH + GSSG) did not change or decrease after ripening in the varieties with no or very few capsaicinoids (Merlchor and Piquillo), in the hot varieties, this parameter augmented in mature fruits. This was due to the evolution of the reduced form of GSH during those physiological processes. This higher content of GSH in ripe fruits found in the hottest pepper varieties could be due to an enhanced GR activity. In these cases, the enzyme GR is perhaps playing a role not linked to the AGC. GSH could be used, in cooperation with ascorbate, to preserve the capsaicinoids functionality in these hot varieties. However, more research is necessary to bring light to this emerging subject. Besides, GSH could be also driven to signaling processes by either glutathionylation events (another PTM), or as *S*-nitrosoglutathione, a chemical form which allows transporting NO among cells and tissues [50,94–96]. GR uses NADPH to achieve the reduction of glutathione. NADPH is also essential in intermediate steps of capsaicin biosynthesis [97]. These eventualities point towards the necessity of investigating the interaction capsaicinoids–ascorbate–glutathione–NADPH in more detail, especially after the perspective of considering NADPH as a quality footprint in horticultural crops, as it has been proposed recently [98].

#### **5. Conclusions and Future Prospects**

The obtained data in this work point towards a close relationship among capsaicinoids and the antioxidant systems in pepper fruits. This interaction seems to maintain a redox and functional homeostasis to preserve the role of capsaicinoids with the cooperation of antioxidants, basically ascorbate and glutathione. However, some antioxidant enzymatic systems are also involved. The exclusivity of capsaicinoid metabolism in *Capsicum* species makes this research more attractive to look for an exclusive model that could provide interesting information at the plant physiological level, but also considering the pharmacological and nutraceutical uses of hot pepper fruits, based mainly on the content of capsaicinoids but also on vitamins C and A. On the other hand, another interesting cue is opened. The role of Fe-SOD needs to be investigated in pepper fruit physiology due to the diverse behavior of this isozyme among varieties. Fe-SOD has been localized in peroxisomes from pepper fruits [5,99] and lately its gene expression profile has been reported in sweet pepper during ripening and under NO treatment. Overall, the interaction of NO in pungent pepper fruits is another issue that deserves to be investigated. Furthermore, this characterization contributes to providing a biochemical antioxidant pattern for each pepper cultivar which could be part of the particular features of these cultivars that are included in the European Register of protected designations of origin for these Spanish agricultural products. Besides, this provides an added value to these autochthonous products and may have some incidence at the marketing and economical levels in their respective producing sectors.

**Author Contributions:** F.T., A.C.-R. and M.R.-R. carried out all biochemical experiments. F.J.C. and J.M.P. designed the work, drove and coordinated the tasks and wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the ERDF-cofinanced grants AGL2015-65104-P from MINECO, PID2019-103924GB-I00 from MICIT, and P18-FR-1359 from the Plan Andaluz de Investigación, Desarrollo e Innovación, Spain.

**Acknowledgments:** The technical support of the Scientific Instrumental Service from the Estación Experimental del Zaidín (CSIC, Granada, Spain) is acknowledged. The valuable assistance of Carmelo Ruiz-Torres, Tamara Molina-Márquez and María Jesús Campos is also appreciated. Authors are thankful to the different companies and Regulatory Councils of Denomination of Origin for their valuable and generous collaboration providing the different pepper cultivars: Melchor by the Zeraim Iberica/Syngenta Seeds, Ltd., El Ejido, Almería, Spain; Padrón by the Regulatory Council of Denomination of Origin "Pemento de Herbón" (Herbón, A Coruña, Spain), and Piquillo and Alegría riojana, both provided by the Regulatory Council of Denomination of Origin "Pimiento del Piquillo-Lodosa" (Navarra, Spain).

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

#### **References**


© 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 (http://creativecommons.org/licenses/by/4.0/).

### *Review* **Anthocyanins: From the Field to the Antioxidants in the Body**

**Vidmantas Bendokas 1,\*, Vidmantas Stanys 1, Ingrida Mažeikiene˙ 1, Sonata Trumbeckaite 2,3, Rasa Baniene 2,4 and Julius Liobikas 2,4,\***


Received: 22 July 2020; Accepted: 29 August 2020; Published: 2 September 2020

**Abstract:** Anthocyanins are biologically active water-soluble plant pigments that are responsible for blue, purple, and red colors in various plant parts—especially in fruits and blooms. Anthocyanins have attracted attention as natural food colorants to be used in yogurts, juices, marmalades, and bakery products. Numerous studies have also indicated the beneficial health effects of anthocyanins and their metabolites on human or animal organisms, including free-radical scavenging and antioxidant activity. Thus, our aim was to review the current knowledge about anthocyanin occurrence in plants, their stability during processing, and also the bioavailability and protective effects related to the antioxidant activity of anthocyanins in human and animal brains, hearts, livers, and kidneys.

**Keywords:** anthocyanin metabolites; antioxidants; cardioprotection; hepatoprotection; nephroprotection; neuroprotection

#### **1. Introduction**

Anthocyanins are pigments belonging to the flavonoid group, which is widely distributed in plants. They are responsible for blue, purple, and red colors in flowers, fruits, and vegetables and protect plants from environmental stresses such as high sunlight irradiance [1] or low nitrogen [2]. Chemically, anthocyanins are produced when anthocyanidins are glycosylated. The most abundant anthocyanidin in plants is cyanidin. Other anthocyanidins are less abundant, and their frequency decreases in this order: delphinidin, peonidin, pelargonidin, petunidin, and malvidin [3]. Anthocyanidins are flavylium ion derivatives that vary in terms of their substituent groups: –H, –OH, or –OCH3. Usually, anthocyanidins are glycosylated at the C3 or C3 and C5 sites, but the glycosylation of other sites has also been reported [4]. The biological activity of anthocyanins depends on their structure; however, all samples, including those with different compositions and amounts of anthocyanins, extracted from various berries and vegetables, are biologically active [5]. Azevedo et al. [6] established that the radical scavenging activity and reducing properties of anthocyanins strongly depend on the chemical structures of particular anthocyanins; this effect increases with the presence of catechol and pyrogallol groups in ring B of cyanidin-3-glucosides and the respective aglycones. Some studies have shown that delphinidin has the highest antioxidant activity compared with the other five anthocyanidins due to the three hydroxyl groups on the B-ring [5,7]. An increasing body of evidence shows that

anthocyanin intake can have a protective effect on human and animal brains, hearts, livers, and kidneys, and many of the therapeutic effects may be purported to the antioxidant activities of anthocyanins and their metabolites [8–12]. The antioxidant activity of these compounds manifests through direct and indirect methods of action. Thus, anthocyanins can directly scavenge reactive oxygen species (ROS) [13,14], whereas the indirect pathways involve stimulation of the synthesis or activity of antioxidant enzymes (catalase, superoxide dismutase (SOD), glutathione peroxidase) [15]; inhibition of ROS-forming enzymes, such as nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and others [16,17]; or even mild uncoupling of mitochondrial respiration preventing ROS generation [9,18]. It can also be assumed that for effective therapeutic action of anthocyanins, both the ROS scavenging activity and the modulation of cellular antioxidant systems are required [14]. Here, we review the current knowledge about anthocyanin occurrence in plants, their stability during processing, and the health benefits to humans and animals.

#### **2. Natural Sources of Anthocyanins**

Anthocyanins are natural, water-soluble plant pigments that are responsible for blue, red, or purple colors in plants. Plant genotypes, agro-climatic conditions, and fruit or vegetable maturity are significant factors in the composition and quantity of anthocyanins [19]. Therefore, the main sources of anthocyanins in human diet are fruits and vegetables, which accumulate anthocyanins in both the peel and flesh; however, their content varies greatly (Table 1).


**Table 1.** Maximum amount of anthocyanins (mg 100 g−<sup>1</sup> of fresh weight (FW)) in fruits and vegetables.

Cy—cyanidin; Dp—delphinidin; Mv—malvidin; Pg—pelargonidin; Pn—peonidin; Pt—petunidin; ara—arabinoside; gal—galactoside; glc—glucoside; rut—rutinoside; sam—sambubioside; xyl—xyloside.

Fruits, especially dark blue berries, accumulate a large total amount of anthocyanins, while vegetables tend to have lower anthocyanin concentrations. One of the highest anthocyanin concentrations was identified in bilberries, with delphinidins being the dominant anthocyanins making up over 57.6% of the total, with cyanidins representing 23.7% and malvidins representing 14.1% of the total [20]. Berries of the golden currant cultivar 'Corona' are the richest in anthocyanins among *Ribes* spp., with cyanidins being the dominant type [21]. Blackcurrants have a higher proportion of delphinidins, ranging from 66.7% to 70.2%, as shown in various studies [20,21]. In contrast, cyanidins dominate in redcurrant, elderberry, sweet cherry, and sour cherry, and sometimes, they are the only anthocyanins [11,20,22,23] (Mikulic-Petkovsek et al., 2014; Veberic et al., 2015; Bendokas et al., 2017, Blackhall et al., 2018). Malvidins are the main anthocyanin in grapes with a relative content ranging from 35.8% to 67.1% [25].

In terms of vegetables, red chicory has been shown to have the highest concentration of anthocyanins; however, it is 2–20 times lower than that in berries. Cyanidins are major anthocyanins in most vegetables; however, eggplant only accumulates delphinidins.

The consumption of anthocyanins varies from 9 mg/day on average in the United States to 19 mg/day in Europe, but daily consumption may reach 28 mg in some European countries [33]. The main sources of anthocyanins are berries (39% in the US and 43% in Europe), wine (18% in the US and 22% in Europe), and fruits (9% in the US and 19% in Europe), with other sources being vegetables and other foods [34].

#### **3. Stability of Anthocyanins in Foods and Beverages**

Anthocyanins are natural plant compounds that are increasingly being used in the food and pharmaceutical industry due to their effects on human health. However, the low stability of anthocyanins is still an obstacle to their use [35]. The stability of anthocyanins depends on the pH, temperature, light, presence of solvents and oxygen, and other factors [36]. Anthocyanins are more stable under acidic conditions. At a pH of 1.0, flavylium cations are the predominant species and contribute to the development of purple and red pigments, while at pH 2.0–4.0, the blue quinoidal species predominates. When the pH value reaches 7.0, anthocyanins are usually degraded [36]. The storage temperature affects the concentration of anthocyanins in extracts; for instance, 11% of rosella anthocyanins were lost after storage for 60 days at 4 ◦C, while 99% of anthocyanins were degraded in the same extracts stored at 37 ◦C for the same period [37].

The stability of anthocyanins from various *Ribes* species was reported to depend on their composition and storage conditions [24]. Anthocyanins in redcurrant berry extract have been shown to be more stable at room temperature and in the presence of light than extracts from berries of golden currant and gooseberry. After the storage of anthocyanin extracts under dark and cold conditions (+4 ◦C) for 84 days, up to 90% of redcurrant, 80% of gooseberry and golden currant, and up to 50% of blackcurrant anthocyanins remained intact [24].

Thermal food processing negatively affects the nutritional value of anthocyanin-rich juices as it results in anthocyanin degradation [38]. Thermal processing is responsible for the loss of up to 35% of anthocyanins. Even short-term thermal treatment (5 s at 85 ◦C) resulted in a loss of 9% of anthocyanins from strawberry juice, while pasteurization for 15 min resulted in a loss of 21% [39]. Boiling of red cabbage resulted in a loss of 41.2% of anthocyanins, while the anthocyanin concentration remained the same after steaming or stir-frying. Possibly, as highly water-soluble pigments, anthocyanins may be lost by leaching in the case of boiling [30].

Various anthocyanin stabilization methods are being developed. For instance, the improvement of anthocyanin thermal stability by yeast mannoproteins at pH 7.0 has been studied. The complexes were found to effectively protect anthocyanins from degradation during heating at 80 and 126 ◦C [40]. Mixing of clarified acerola juice with montmorillonite resulted in 50% more anthocyanins, regardless of time or pH changes [41]. Copigmentation is another natural tool that can be used to enhance anthocyanin stability. The most studied copigments are phenolic acids such as hydroxycinnamic and hydroxybenzoic acids [42]. Babaloo and Jamei [43] established that caffeic acid provides more stability for anthocyanins than benzoic, tannic, and coumaric acids. Encapsulation with polysaccharides, such as β-cyclodextrin, maltodextrin, or Arabic gum, is also important for the stabilization of anthocyanins. The protective effect of β-cyclodextrin was evident for all blackberry anthocyanins after thermal treatment at 90 ◦C for 2 h [44].

Novel techniques for phenolic compound isolation from natural products that avoid the degradation of these compounds have been developed. Block freeze concentration has been employed to extract anthocyanins from strawberries and enrich yogurt with the obtained concentrated strawberry juice. As a result, yogurt with a high anthocyanin content and greater antioxidant activity was produced; however, it had a short shelf life [45]. The foam mat drying technique is now considered to be an effective dehydration method to produce powder from juices or pulp. Only a small reduction in

anthocyanins (7–9%) was observed after the storage of jambolana juice powder produced by foam mat drying for 150 days [46]. When foam-mat freeze-drying was used for powder production from blueberry juice, 80–100% of anthocyanins remained after processing; the most stable was Cy3glc [47].

#### **4. Bioavailability of Anthocyanins**

The beneficial health effects of anthocyanins strongly depend on their bioavailability, as only a small fraction of anthocyanins is absorbed by human body [48,49]. The concentration of anthocyanins in the plasma reaches a maximum value within 0.5–2 h after the consumption of anthocyanin-rich foods [50]. A low concentration of anthocyanins in plasma and urine has been observed in several studies. Less than 2% of original anthocyanins may be found in the plasma or urine after consumption; however, anthocyanins go through several transformations in the small and large intestines; thus, only a small fraction of the anthocyanins remains nonmetabolized or catabolized [51]. Mueller et al. [52] stated that a complex mixture of anthocyanin metabolites in the plasma rather than a single type of anthocyanins may cause beneficial effects in humans.

Gamel et al. [32] evaluated the absorption of anthocyanin metabolites after the consumption of purple wheat crackers and bars. They established that the total concentration of anthocyanin metabolites in urine peaked at 0–2 and 2–4 h, with 18–22 ng/mL excreted on average. For both products, the total amount of accumulated anthocyanins reached 13 μg in 24 h, representing 0.19%. A significant difference between male and female participants was also observed: males excreted 17.1 μg, while females excreted 11.6 μg of anthocyanin metabolites in a 24-h period. Krga and Milenkovic [53] conducted a comprehensive analysis of 20 studies on the anthocyanin concentration in human plasma after the ingestion of food, extracts, and drinks. They found that anthocyanin adsorption was the fastest after drinking red wine and red grape juice. The maximum concentration in the plasma was reached in 0.3 and 0.5 h, respectively, while the maximum concentration of anthocyanins from blueberry powder was reached in 4 h. Interestingly, the maximum anthocyanin concentration in plasma varied from 1.4 to 591.7 nM and did not depend on the administered dose, which was possibly due to differences in the anthocyanin composition in various foods. Recently, a human pilot study was performed by Rohrig and ˙ colleagues [54] on five healthy male volunteers who were consuming anthocyanin-rich blackcurrant extract. They studied the kinetics of the dominant anthocyanins—Dp3rut and Cy3rut—in plasma and urine after the consumption of blackcurrant extract. The peak concentrations of both anthocyanins in plasma were reached within 2 h after consumption. The maximum concentrations were 8.6 ± 5.8 nmol/L for Dp3rut and 9.8 ± 3.1 nmol/L for Cy3rut, which later gradually decreased. Similarly, urinary excretion rates of both studied anthocyanins peaked within 0−2 h of ingestion and reached 20.0 ± 2.6 nmol/h (Dp3rut) and 21.2 ± 3.8 nmol/h (Cy3rut). The total excreted amount was calculated: 0.040% (Dp3rut) and 0.048% (Cy3rut) of the ingested doses. In addition, after consumption of the anthocyanin-rich extract, the concentration of the main metabolite, protocatechuic acid, increased significantly [54].

In humans, anthocyanins consumed with food may be digested in the gastrointestinal tract, absorbed into the blood, and later metabolized [55]. Some studies have stated that minute amounts of anthocyanins are absorbed in the small intestine. The majority of ingested anthocyanins reach the large intestine where they are metabolized [56] and where structural modifications (deglycolysation, degradation, hydroxylation, etc.) take place due to the changing physiological conditions. Anthocyanins can also be further modified by various enzymes in the small intestine before entering the bloodstream [57]. Twenty-two metabolites of Cy3glc were identified using an isotopic approach [58]. Among them, phloroglucinaldehyde, 3,4-dihydroxybenzaldehyde, and hydroxybenzoic acid were found to be produced as phase I metabolites, while the phase II metabolites identified were mainly glucuronidated and methylated cyanidins. Colonic anthocyanin metabolites included hydroxybenzoic acid, hippuric acid, phenylpropenoic acid, ferulic acid, and phase II protocatechuic acid conjugates [58–60]. Bresciani and colleagues [61] analyzed the catabolism of anthocyanin-rich elderberry extract with different gut microbial strains in vitro and established that their metabolic pathways were different. Some common metabolites were found among all studied strains; however, each of them had several phenolic metabolites produced specifically by that strain. These in vitro results could provide new knowledge of the variability in anthocyanin metabolism in different organisms. After absorption, the fast transport of anthocyanins and their metabolites to the liver, heart, lungs, brain, and kidneys may be observed [55,62].

Sandoval-Ramírez and colleagues [63] summarized studies on the anthocyanin concentration in various animal tissues and stated that in short-term experiments, after a single dose of bilberry extract, the highest concentration of cyanidin-3-*O*-glucoside (40.46 pmol/g) was found in the brain after 0.25 min [62]. In addition, Cy3glc was identified in other tissues, such as the gastrointestinal tract (8 × 105 pmol/g), lungs (5.19 × 104 pmol/g), and prostate (4 × 104 pmol/g), with peak concentrations occurring at different times after a single oral dose of the extract [63,64]. In long-term studies, the highest concentration of Mv3glc (4.43 pmol/g) was established in the brains of pigs, while the concentration of Pt3glc reached 6.66 pmol/g [63]. Cy3ara from tart cherry was identified in the hearts, brains, livers, kidneys, and bladders of Wistar rats; however, its concentration was low and ranged from 2.28 <sup>×</sup> <sup>10</sup>−<sup>4</sup> to 1.16 <sup>×</sup> <sup>10</sup>−<sup>3</sup> pmol/g [65]. In general, Cy3glcand its metabolites are the most abundant anthocyanins in animal tissues, and Cy3glcmay be one of the most promising bioactive molecules for human health [63]. It is also worth noting that care is needed to avoid artefacts when studying and evaluating antioxidant effects of bioavailable anthocyanins in model organisms and the human body, as findings can depend on the chosen marker, the sensitivity of method to detect ROS and to measure oxidative damage, or even on the changes in plasma urate concentrations [66]). It is not the aim of the present review to analyze the suitability of methods to evaluate the antioxidant activity of anthocyanins; thus, for the complexity of the matter, one could consult several excellent reviews [66–70].

#### **5. Biological E**ff**ects Related to the Antioxidant Activity of Anthocyanins—In Vivo Studies in Model Organisms**

#### *5.1. Neuroprotection*

At the animal level, oral administration of anthocyanin-rich berry extract of *Vaccinium myrtillis* L. (100 mg/kg for 7 days) has been shown to suppress psychological stress-induced cerebral oxidative stress and dopamine abnormalities in distressed mice [71]. Anthocyanins extracted from black soybeans have also been demonstrated to reverse D-galactose- or lipopolysaccharide (LPS)-induced oxidative stress, neuroinflammation, and neurodegeneration in adult murine models [72–74]. Likewise, the same anthocyanins were shown to reduce elevation of the ROS level and consequent oxidative stress induced by the amyloid beta oligomer (Aβ 1-42) through stimulating the intracellular antioxidant system (the transcription factor E2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) pathways). These anthocyanins also prevented apoptosis and neurodegeneration by suppressing the apoptotic and neurodegenerative markers in the amyloid precursor protein/presenilin-1 (APP/PS1) mouse model of Alzheimer's disease (AD) [75].

Positive findings have also been reported by Qin et al. [76] in Cy3glc-treated (10 mg/kg for 30 days) rats injected with Aβ 1-42, which showed an attenuation of Aβ- and oxidative stress-induced GSK-3β hyperactivation and hyperphosphorylation of the tau protein. These findings are in agreement with a study in which rats were injected with Aβ 1-42 bilaterally into the hippocampal CA1 area in order to produce an animal model of AD [77]. It was observed that the memory impairment was reduced in rats that received 80 mg/kg of *Lycium ruthenicum* Murr. anthocyanin-rich extract in comparison to other AD model rats. Moreover, the anthocyanin extract enhanced the activities of total SOD and catalase and increased the glutathione concentration in serum and brain tissues.

Chen et al. [78] also confirmed that *Lycium ruthenicum* Murr. anthocyanins can exert neuroprotective effects in D-galactose (D-gal)-treated rats. Anthocyanins reduce the level of receptor for advanced glycation end products (RAGE), suppress oxidative stress, and reduce levels of inflammation markers such as nuclear factor kappa B (NF-κB), interleukin-1-β (IL-1β), cyclooxygenase-2 (COX-2), and tumor necrosis factor-α (TNF-α), among others. Sustained levels of antioxidant enzymes (SOD and catalase), a decreased concentration of the lipid peroxidation product malondialdehyde, and significantly decreased expression of aging-associated monoamine oxidase-B in D-gal-treated mice were also demonstrated after the administration (30 mg/kg and 60 mg/kg) of black rice anthocyanins [79]. Anthocyanins from fruits of *Aronia melanocarpa* (Michx.) Elliot (30 mg/kg) retained the levels of total SOD and glutathione peroxidase and inhibited the excessive accumulation of inflammatory cytokines in the brain tissue of D-gal-treated mice [80]. Furthermore, recently, a study using a model of streptozotocin-induced dementia of sporadic AD [81] proposed that pretreatment with a commercial anthocyanin extract from grape skins (200 mg/kg for 25 days) can prevent behavioral alterations and protect against the changes in ROS and antioxidant enzyme (SOD, catalase, and glutathione peroxidase) levels.

Notably, purified anthocyanins and anthocyanidins have also been found to exert neuroprotective activities. For instance, pre-treatment with pelargonidin (Pg) (20 mg/kg) significantly suppressed the formation of thiobarbituric acid reactive substances, indicating reduced lipid peroxidation in 6-hydroxydopamine-lesioned rats (an experimental model or Parkinson's disease) [82]. Furthermore, orally consumed Cy3glc (2 mg/kg) reduced brain superoxide levels, infarct size, and improved neurological functions, thus revealing a neuroprotective effect in the cerebral artery occlusion model of ischemia in mice [83]. A recent report [84] also showed that an intravenous injection of Cy3gal and Cy3glc (0.025 mg/kg and 0.05 mg/kg), but not Cy3rut, in rats protected against ischemia-induced caspase-3 activation and necrotic cell death, as well as reducing the infarct size in the cerebral cortex and cerebellum. In contrast, 0.025 mg/kg of Pg3glc had no effect of the activity of caspase-3 but reduced the infarct size. The effects of anthocyanins have been found to correlate with the cytochrome c reducing capacity, and Pg3glc has been shown to have the smallest effect among tested anthocyanins. Thus, authors have proposed that under certain conditions, such as ischemic brain damage, the reducing properties rather than antioxidant properties of anthocyanins might be important in providing neuroprotection.

#### *5.2. Cardioprotection*

Since it is known that bilberries and blackcurrant berries are rich in anthocyanins, Brader et al. [85] investigated the effects of a berry-enriched diet (5 g of berry powder containing 172 mg of anthocyanins per day) on lipid profiles and other biomarkers in Zucker diabetic fatty rats. The results after eight weeks of supplementation demonstrated reduced levels of total and LDL-cholesterol, which was partly due to the altered expression of hepatic liver X receptor-α. Using an in vivo model of coronary occlusion and reperfusion, another study showed that the infarct size was reduced in the hearts of rats that received a long-term purple maize anthocyanin-enriched diet (with Cy and Pg glycosides as the main components) [86]. The authors proposed that the observed cardioprotection could be associated with increased myocardial glutathione levels and thus an improved endogenous cardiac antioxidant defense system. Moreover, Ziberna et al. [87] demonstrated that bilberry anthocyanins could exert concentration-dependent responses on whole rat hearts under ischemia–reperfusion (I-R) conditions. Thus, at low concentrations (0.01–1 mg/L, expressed as Cy3glc equivalents), the extent of I-R injury was significantly reduced, whereas at 5–50 mg/L, anthocyanins showed cardiotoxic activity despite having an intracellular antioxidant capability that increased in a concentration-dependent manner.

In addition, recent evidence from an LPS-induced myocardial injury model in mice [88] showed that pure anthocyanins such as Cy3glc could restore the activity of the mitochondrial electron transport chain (namely, the activity of complexes I and II) and thus significantly attenuate ROS production. Furthermore, the anthocyanins ameliorated cardiac injury, cell death, and improved cardiac function. It is worth noting that Cy3glc also suppressed the expression of endotoxin-induced pro-inflammatory cytokines and the level of protein nitration while elevating the intracellular level of reduced glutathione.

In general, these observations suggest that the cardioprotective activities of anthocyanins may not be solely attributed to their antioxidant properties; therefore, a broader view should be implemented [18,87,89,90].

#### *5.3. Hepatoprotection*

The liver plays an important role in the metabolic elimination of drugs and toxic compounds known to cause injury and reduce the function of the liver. Arjinajarn et al. [91] observed that rice bran extract (250–1000 mg/kg) rich in Cy3glc and Pn3glc (13.24 and 5.33 mg/g of crude extract, respectively) significantly prevented gentamicin-induced intoxication of the liver. It has been shown that an anthocyanin-rich extract significantly reduced the hepatic malondialdehyde level (a biomarker for oxidized lipids), increased expression of the antioxidant enzyme SOD, and prevented elevation of levels of liver injury markers, namely alanine and aspartate aminotransferase. These effects were related to the suppression of both the oxidative pathway regulated by the transcription factor Nrf2 and the inflammatory pathway regulated by NF-κB. The authors proposed that Cy3glc, the major anthocyanin in this extract, is responsible for these antioxidant and anti-inflammatory properties [91]. Moreover, Hou et al. found that black rice bran extract, which is rich in anthocyanins (mainly Cy3glc and Pn3glc), protected mice liver intoxicated with carbon tetrachloride (CCl4) [87]. It was found that mice treated with black rice extract (200–800 mg/kg) showed increased SOD and glutathione peroxidase activities and increased levels of reduced glutathione as compared with a CCl4-intoxicated model group [92]. Similar results about the protective effects of blueberry anthocyanin extract (anthocyanin content up to 25%) on CC14-induced liver injury in mice in vivo were presented by [93]. The extract had reduced concentrations of alanine aminotransferase, aspartate aminotransferase, and malondialdehyde in a dose-dependent manner, while the activities of SOD, catalase, and glutathione reductase increased [93]. Blackberry fruit extract was similarly shown to attenuate lipid peroxidation and to recover the activity of antioxidant enzymes in CC14-treated rats [94]. Recently, Sun et al. observed that anthocyanins from blueberry (100 mg/kg and 200 mg/kg per day) protected mouse liver from CCl4-induced hepatic fibrosis [95]. It was detected that blueberry anthocyanins reduced ROS generation and tissue oxidative damage, decreased inflammation, and suppressed the activity of hepatic stellate cells. Interestingly, the activity of mitochondrial electron transport chain complexes I and II was also restored after treatment with anthocyanins [95].

It is also known that liver inflammation and an excessive accumulation of lipids play critical roles in the pathogenesis of alcoholic liver diseases. Accordingly, in a recent study, Cy3glc extracted from *Lonicera caerulea* L. was shown to exert a hepatoprotective effect on alcoholic steatohepatitis in mice [96]. Zuo et al. detected substantial decreases in serum aminotransferases and triglycerides and found increased albumin levels after treatment. In addition, anthocyanidins significantly suppressed the expression of SREBP1 (a transcription factor involved in lipogenesis) and enhanced the phosphorylation of AMPK as compared with chronic ethanol administration. Cy-3-g suppressed inflammasome activation, thereby preventing activated macrophages from producing pro-inflammation cytokines [96]. Moreover, another study demonstrated that a phenolic fraction of *Lonicera caerulea* L. ameliorated inflammation and lipid peroxidation by upregulating Nrf2 and SOD and downregulating the transcription factor forkhead box protein O1 and HO-1 in a mouse model of nonalcoholic steatohepatitis (NASH) induced by a high-fat diet in combination with CCl4 [97]. Prokop et al. also showed in vivo that a blue grain bread wheat-based diet (genotype UC66049, containing 121 mg of Cy3glc per kg of wheat) increased the activity of the microsomal xenobiotic-metabolizing system cytochrome P450 by 20–50% in rats after 72 days of intake. In addition, an anthocyanin-rich diet significantly increased the antioxidant power of plasma as shown by a FRAP assay and the level of total –SH groups in plasma when compared with the control [98].

#### *5.4. Nephroprotective E*ff*ects*

Anthocyanins are considered to be a functional food factor and to play an important role in the prevention of kidney diseases. For example, a recent study [99] investigated the effects of anthocyanin-rich bilberry extract (200 mg/kg daily) on the antioxidant status of animals intoxicated with CCl4. Rats received the extract orally for 7 days, and on the last day, a single dose of CCl4 was intraperitoneal (i.p.) injected. It was found that pretreatment with the anthocyanin-rich extract resulted in a significant reduction in pro-oxidative (H2O2, oxidized glutathione, xanthine oxidase) and pro-inflammatory markers (myeloperoxidase, nitric oxide, and TNF-α), and a substantial increase of antioxidant enzyme levels (catalase, SOD, glutathione peroxidase, and S-transferase). Moreover, the anthocyanins significantly reduced the degree of damage to the proximal and distal tubules in the kidney cortex [99].

Positive findings have also been reported by another study [100]. It showed that the administration of *Malva sylvestris* L. extract (200 or 400 mg/kg) rich in anthocyanins reduced the renal toxicity induced by gentamicin and thus led to (i) an improvement in kidney function, (ii) a decrease in the expression levels of pro-inflammatory markers (TNF-α), (iii) a reduction in oxidative stress (levels of malondialdehyde and total antioxidant capacity), and (iv) a decrease in tissue injuries.

Similar beneficial effects of the bilberry diet (100 mg/kg daily) on the levels of serum malondialdehyde, catalase, and advanced oxidation protein products were demonstrated in a rat model of gentamicin-induced nephrotoxicity, and the effects correlated well with the antioxidant activity (assessed in vivo and in vitro) as well as with high anthocyanin levels [101]. It is worth noting that anthocyanin-rich fruits of *Panax ginseng* Meyer have also been shown to attenuate cisplatin-induced elevations in blood urea nitrogen and creatinine levels as well as the prevalence of histopathological injuries in mice [102]. The positive outcomes are related to reduced levels of malondialdehyde, HO-1, cytochrome P450 E1, 4-hydroxynonenal, TNF-α, and IL-1β, as well as concomitantly increased levels of reduced glutathione, catalase, and SOD.

In addition, Lee et al. [103] recently revealed that intravenously administered pure Pg (0.4 mg/kg) could modulate renal function in a mouse model of sepsis. Treatment with Pg reduced renal tissue injury, plasma nitrite and nitrate production, and TNF-α, IL-6, myeloperoxidase, and malondialdehyde levels. The total glutathione content as well as the activity of antioxidant enzymes such as SOD, glutathione peroxidase, and catalase in kidney tissues were also found to be restored after Pg injection.

#### **6. Biological E**ff**ects Related to the Antioxidant Activity of Anthocyanins—In Vivo Studies in Humans**

Epidemiological and clinical studies suggest that an anthocyanin-enriched diet may lower levels of certain oxidative stress biomarkers in humans, and this could be associated with reduced risk of cognitive decline and the development of neurodegenerative and cardiovascular diseases, as well as having sustained hepatic function and kidney protecting activities [12,104–115].

#### *6.1. Antioxidant and Anti-Atherosclerogenic E*ff*ects*

A randomized clinical trial [116] evaluated the effects of a standardized maqui berry (*Aristotelia chilensis* (Mol.) Stuntz) extract (containing 162 mg of anthocyanins) on products of lipid peroxidation in healthy, overweight, and smoker adults. The results suggested that supplementation with the extract can be related to a limited term (max for 40 days) reduction in oxidized low-density lipoprotein (LDL) levels and a decrease in urinary F2-isoprostanes. Another study [117] concluded that the acute consumption of anthocyanin-rich red *Vitis labrusca* L. grape juices could be related to decreased levels of thiobarbituric acid reactive substances and lipid peroxides in the serum of healthy subjects. It has also been demonstrated that regular (for 30 days) anthocyanin-rich sour cherry consumption could suppress the formation of ROS by circulating phagocytes and decrease the risk of systemic imbalance between oxidants and antioxidants [118]. It is worth noting that a portion (300 g) of blueberries, the dietary source of anthocyanins provided to young volunteers involved in a randomized cross-over study, significantly reduced H2O2-induced DNA damage in blood mononuclear cells [119]. In another human pilot intervention study, the consumption of anthocyanin-rich bilberry (*Vaccinium myrtillius* L.) pomace extract was found to modulate transcription factor E2-related factor 2 (Nrf2)-dependent gene expression in peripheral blood mononuclear cells [120].

A single-blind randomized placebo-controlled intervention trial, which lasted for 8 weeks and involved 72 unmedicated subjects, revealed that the administration of various berries (including bilberries, chokeberries, and blackcurrants) increased both the concentration of high-density lipoprotein (HDL) cholesterol and the plasma antioxidant capacity [121]. Higher dietary anthocyanin and flavan-3-ol intake was associated with anti-inflammatory effects in 2375 Framingham Heart Study Offspring Cohort participants [122]. Interestingly, the consumption of 300 mL of red wine (a total dose of anthocyanins was 304 μM, which was the highest amount among detected compounds) with a meal was shown to prevent the postprandial increases in plasma lipid hydroperoxides and cholesterol oxidation products and therefore protect against a potential pro-atherosclerogenic effect [123]. Similar findings were obtained in a randomized cross-over trial, which concluded that a moderate consumption of red wine decreases erythrocyte SOD activity [124]. In another randomized double-blind trial, 150 subjects with hypercholesterolemia consumed a purified anthocyanin mixture derived from bilberries and blackcurrants (320 mg/day) for 24 weeks [125]. It was found that anthocyanin consumption significantly decreased the levels of inflammatory biomarkers (C-reactive protein, soluble vascular cell adhesion molecule-1, and plasma IL-1β) and increased the HDL cholesterol level. Recently, it was shown that a daily intake of 150 g of anthocyanin-rich blueberries resulted in clinically relevant improvements in endothelial function and systemic arterial stiffness, which was probably due to the improved nitric oxide bioactivity and HDL status [126].

#### *6.2. Hepatoprotective Benefits*

Nonalcoholic fatty liver disease (NAFLD), defined by excessive lipid accumulation in the liver, is the hepatic manifestation of insulin resistance and metabolic syndrome. NAFLD encompasses a wide spectrum of liver diseases ranging from simple uncomplicated steatosis to steatohepatitis, cirrhosis, and hepatocellular carcinoma [106]. Zhang et al. [127] reported that anthocyanins extracted from bilberry and blackcurrant (320 mg/day) and administered for 12 weeks ameliorated liver injury in patients with NAFLD. It was observed that a so-called "anthocyanin group" exhibited significant decreases in the plasma alanine aminotransferase, cytokeratin-18 M30 fragment, and myeloperoxidase levels. It was also found that consumption of *Myrica rubra* Sieb. and Zucc. juice (250 mL for 4 weeks) protected young adults (18–25 years old) against NAFLD by improving the plasma antioxidant status and inhibiting the inflammatory and apoptotic responses involved in this disease [128].

#### *6.3. Nephroprotection*

In another study, red fruit juice (40% red grape juice, 20% blackberry juice, 15% sour cherry juice, 15% blackcurrant juice, and 10% elderberry juice) with high polyphenol and anthocyanin contents was tested for its preventive potential in hemodialysis patients [129]. For this purpose, 21 subjects consumed 200 mL/day of juice according to the following protocols: 3-week run-in; 4-week juice uptake; 3-week wash-out. The results revealed a significant decrease in DNA oxidation damage and protein and lipid peroxidation and an increase in the reduced glutathione level; the effects were attributed to the high anthocyanin and polyphenol contents of the juice [129]. Another study [130] demonstrated that the regular consumption of concentrated red grape juice by hemodialysis patients could be associated with the reduced neutrophil NADPH oxidase activity and plasma concentrations of oxidized LDL and inflammatory biomarkers.

#### **7. Concluding Remarks**

In conclusion, anthocyanins are valuable biomolecules with a broad variety of biological effects on human health, and we suggest adding more anthocyanin rich fruits, vegetables, and their products to the daily diet. Numerous studies indicate that bilberry, blackcurrant, elderberry, and other berries have the highest total concentrations of anthocyanins; therefore, the consumption of fresh berries and their processed products may have greater beneficial effects on humans. Various anthocyanin stabilization methods have been developed e.g., copigmentation with other phenolic acids, encapsulation with polysaccharides, block freeze concentration, and powder production using the foam mat drying technique. All of them enable anthocyanins to be preserved during processing, thus increasing their bioavailability and delivery to target tissues in the human body. However, long-term studies on the impacts of anthocyanin-rich product consumption on human health are still rare. Further studies could focus on the identification and tracking of individual anthocyanin metabolites and on the determination of the exact dosage and delivery platforms sustaining the antioxidant properties of anthocyanins in vivo. In addition, as an alternative to natural sources, synthesis of the most bioactive anthocyanins in bioreactors should be considered.

**Author Contributions:** Original idea and conceptualization V.S. and J.L., original draft preparation V.B., V.S., I.M., S.T., R.B. and J.L., review and editing V.B., I.M. and J.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Research Council of Lithuania (V.B., V.S. and J.L.; No. SVE-11-008). J.L. was supported by the COST Action FA0602 «Bioactive food components, mitochondrial function and health». J.L. and S.T. are supported by the program «2014-2020 Investment of EU Funds in Lithuania: Intellect. Common Scientific and Business Projects» (project No J05-LVPA–K-03-0117).

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

#### **References**


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