**Preface to "Antioxidant Capacity of Anthocyanins and other Vegetal Pigments: Modern Assisted Extraction Methods and Analysis"**

"Hence we must believe that all the Sciences are so inter-connected, that it is much easier to study the all together than to isolate one from the others."Rene Descartes (1596–1650), in Rules for ´ the Direction of the Mind (1701).

The investigation of phenolic compounds from the chemical, biochemical and biological points of view during the last few decades has focused intensely on the study of anthocyanins. Interest in understanding the medicinal, therapeutic, and nutritional values of these naturally present phytochemical compounds has increased notably, with the number of papers published in this regard experiencing a significant increase. It is important to highlight the interdisciplinary nature of the research that is currently being carried out in this prolific area of work.

The highly bioactive anthocyanin compounds present in all plant tissues including leaves, stems, flowers, roots and fruits, e.g., berries, impart the colors orange, red, purple, and blue to many fruits, vegetables, grains, flowers, and plants. Anthocyanins are part of a group of secondary metabolites known as flavonoids, a subclass of the polyphenol family, being common components of the human diet. The identification and quantification of anthocyanins in natural products, as well as the elucidation of their effects in vivo and in vitro, requires the development of analytical techniques, and constitutes a challenge.

Anthocyanins also show an antioxidant activity largely dependent on their chemical structure. Numerous epidemiological studies have shown the beneficial effects of a diet rich in fruits and vegetables on human health, and in the prevention of various disorders related to oxidative stress, such as cancers and cardiovascular diseases. On the other hand, extracts rich in anthocyanins are highly valued in the food industry, given their coloring properties, as an alternative to the use of dyes and synthetic lakes. This is because various adverse effects on human health are attributed to synthetic dyes.

Anthocyanins are found in solution as a mixture of different secondary structures: a flavylium ion, a quinoid base, a carbinol base, and a chalcone pseudobase. Various stabilization mechanisms occur through self-association, and intermolecular and intramolecular copigmentation of anthocyanins, leading to the formation of tertiary structures. Anthocyanins show enormous potential for the food, pharmaceutical and cosmetic industries, although their use is limited by their relative instability (the Achilles heel) and low extraction percentages. Much progress has been made in the pretreatment of samples, having also developed some novel extraction techniques, as well as in stabilization techniques.

Thus, this reprint shows studies (Urbonaviciene et al.; Carmona et al.; Fanyuk et al. and Urbstaite et al.) applied to exotic or wild fruits, in which the aim is to elucidate how different external factors, such as climatic and/or geographical factors, affect the production, antioxidant capacity and physicochemical properties of anthocyanins and polyphenols. Moreover, other studies (Gonzalez-de-Peredo et al.; Salazar-Bermeo et al.; Carrera et al. and Ortiz et al.) show the feasibility of ´ applying certain extraction methods to obtain anthocyanins from different wild fruits or by-products. Finally, other authors (Tena et al.; Enaru et al.; Morata et al. and Roca et al.) review the state of the art of extraction methods for these precious compounds, not only polyphenols and anthocyanins, but also carotenes and chlorophyll pigments. These studies provide, among other things, interesting information on the experimental parameters to be applied for each type of extraction, including material preparation, extraction procedures, different recommendations and information on recovery rates. A critical discussion on the effects of biotic and abiotic stressors on living organisms, in which chlorophylls and carotenoids are involved, was also included in the last revision of this reprint.

In short, the possibilities of these vegetable chameleons, as Mikhail Tsvet (father of chromatography) baptized them, are enormous, and some interesting applications can be seen in this reprint published today by *MDPI*, which covers a fairly wide space, honoring the interdisciplinary nature of this polyhedric subject.

Last but not least, we would like to thank the authors for their brilliant contributions, which have made this reprint possible. We would also like to thank *Antioxidants* for trusting us as editors with the publication of this reprint. In addition, we cannot fail to mention the reviewers for the important work they have done, thus ensuring that the results shown in this reprint have sufficient rigor and scientific quality, and for which we also show our gratitude.

> **Agust´ın G. Asuero and Noelia Tena** *Editors*

## *Editorial* **Antioxidant Capacity of Anthocyanins and Other Vegetal Pigments: Modern Assisted Extraction Methods and Analysis**

**Noelia Tena and Agustin G. Asuero \***

Departamento de Química Analítica, Facultad de Farmacia, Universidad de Sevilla, Prof. García González 2, 41012 Sevilla, Spain; ntena@us.es

**\*** Correspondence: asuero@us.es

Anthocyanins [1,2], chlorophylls, and carotenoids [3] are pigments responsible for the colour of many fruits, flowers, and plant tissues. In particular, anthocyanins show potential utility as natural dyes and for having a beneficial impact on health (as demonstrated by many epidemiological studies), as in addition to being safe and innocuous molecules, they have antioxidant properties and exert an effect on the gut microbiome. This special issue of Antioxidants contains twelve contributions—eight research articles and four reviews—including recent advances in the field. The interdisciplinary nature of the subject and the breadth of the content presented by the authors make this special issue very interesting and comprehensive. Modern methods of analysis of anthocyanins, their geographical variability, the improvement of their antioxidant properties, the valorisation of by-products, stability studies, and the metabolomics of chlorophylls and carotenoids are the subject of research or review. "Today, science has few boundaries and collaboration is the name of the game" [4].

Urbonaviciene et al. [5] carried out an evaluation of the antioxidant capacity and physicochemical properties of biologically active compounds (total polyphenol and total anthocyanin content) of wild blueberries by developing chemometric tools that make it possible to relate authenticity and quality control to geographical origin. This is the first study of its kind carried out on wild blueberries from several northern European countries.

The production of anthocyanins from blood oranges requires low temperature conditions, thus being a useful postharvest strategy to be applied in hot climates. Peña et al. [6] found a different response in the case of pear and Moorish oranges at the biochemical and molecular level, the changes being more prominent in the latter case. Blood orange has found use in traditional Asian medicine due to the vital bioactivity of the polyphenols it contains.

The red skin colour of some fruits, such as mango and apple, is vital for marketing and consumer acceptance. Alkan et al. [7] sought to determine whether external pre-harvest treatment of apples and mangoes with phenylalanine can promote red skin colouring of the fruit, and experimentally proved the hypothesis to be true, especially when combined with exposure to sunlight. The level of anthocyanin content of the treated peel of Cripps pink or May Kent apples, as determined by HPLC, increased in both cases.

Janulis et al. [8] performed a qualitative and quantitative analysis of anthocyanin and anthocyanidin composition in a variety of cultivars and genetic clones of American blueberries. The novelty is the growth under Lithuanian climatic conditions. Chemometric tools, such as hierarchical cluster analysis and principal component analysis, indicate that the Woodman cultivar is different from other cranberry cultivars, as its samples contain twice the average total amounts of anthocyanins. A correlation was observed between the total anthocyanin content and the anti-radical and reducing activity of the in vitro extracts.

*Allium cepa* L. has a wide abundance worldwide, its versatility being a feature in culinary uses with the bulbs also showing many interesting medicinal uses, due to their high content of bioactive compounds. Barbero et al. [9] developed assisted extraction methods for the phenolic and anthocyanin compounds present, making use of a Box–Behnken design

**Citation:** Tena, N.; Asuero, A.G. Antioxidant Capacity of Anthocyanins and Other Vegetal Pigments: Modern Assisted Extraction Methods and Analysis. *Antioxidants* **2022**, *11*, 1256. https://doi.org/10.3390/ antiox11071256

Received: 22 June 2022 Accepted: 23 June 2022 Published: 26 June 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

for their optimisation. Both methods show high repeatability and intermediate precision, short extraction times, and good recoveries.

Food by-products with high content of dietary fibre and free and bound bioactive compounds are usually discarded. Persimmon by-products are an interesting source of fibre and bioactive compounds, as demonstrated by Valero et al. [10]. The effects of solvent extraction of persimmon dietary fibre by-products after in vitro gastrointestinal digestion and probiotic bacterial fermentation on techno- and physico-functional properties were evaluated.

The purple potato variety is not well known although it is as rich in nutrients, amino acids, and starches as other potato varieties. In addition, it has a high anthocyanin content and its consumption is attractive in relation to human health. Barbero et al. [11] developed a methodology based on ultrasound-assisted extraction to achieve a higher anthocyanin yield. The method has been applied to successfully extract and quantify anthocyanins found in Vitelotte, Double Fun, Highland, and Violet Queen potatoes.

Native Chilean berries (rich in total polyphenols and anthocyanins) were studied by Alcudia et al. [12], and a large-scale extract of maqui berries was tested on intestinal epithelial and immune cells and shown to have potential as a nutraceutical agent with health benefits for the treatment of inflammatory bowel disease (IBD). Total polyphenol content (Folin–Cioculteau) and antioxidant capacity (DPPH, FRAP, and ORAC) were estimated, and the anthocyanin profile was assessed by ultra-high-performance liquid chromatography (UHPL-MS/MS).

Non-conventional extraction techniques meet the requirements of the food industry in terms of legal aspects, waste policy, safety, and environmental protection. However, the selection of a particular process is not an easy task and multiple factors are involved in planning the choice of the most suitable one. Tena et al. [13] provided an overview of recent applications in the field of anthocyanins extracted from different natural matrices, both by conventional and non-conventional techniques. Aspects such as the principles of the techniques involved, optimisation, technical progress, and industrial applications were considered and some useful recommendations were made.

The Achilles heel of anthocyanins is their lack of stability, which is affected by a number of factors such as pH, light, co-pigmentation, sulphites, ascorbic acid, oxygen, and enzymes. Diaconeasa et al. [14] reviewed all these factors affecting anthocyanin stability and degradation, assessing the impact of each parameter in order to minimise negative behaviour and consequently enhance the beneficial health effects.

The unstable nature of anthocyanins, which are affected as we have seen by changes in pH, oxidation, or high temperatures, requires the application of gentle non-thermal technologies for their extraction. Morata et al. [15] reviewed the characteristics, advantages, and disadvantages in the extraction of anthocyanins from grapes by applying non-thermal technologies such as Hugh hydrostatic pressure (HHP), ultra-high pressure homogenisation (UHPH), pulsed electric fields (PEF), ultrasound (US), irradiation, and pulsed light (PL). These techniques significantly increase extraction capacity while reducing extraction times and maintaining antioxidant capacity.

Chlorophylls and carotenoids are two families of antioxidants that include a large and complex number of compounds, present in daily food intake, with added value ingredients and functional properties. Their extraction and analysis require more powerful, precise, and accurate methods at hand, as well as a better understanding of the technical and biological context. Roca and Pérez-Gálvez [16] reviewed recent advances in the metabolomics of chlorophylls and carotenoids (pigmentomics), including material preparation and extraction procedures, and the use of instrumental techniques, e.g., spectroscopic and spectrometric (mass spectrometry to pigment metabolomics). The review also covered a critical account of studies showing the effects of biotic and abiotic stressors on living organisms, in which chlorophylls and carotenoids are involved.

Many thanks to the authors for their brilliant contributions, which have made this special issue possible. Many thanks also to Antioxidants for having us as guest editors for this special issue. Finally, we cannot leave out the reviewers for the important work they have done, which is worthy of mention and praise, and for which we also show our gratitude.

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

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

## **References**

