**Natural Additives in Food**

Editors

**Lillian Barros Isabel C.F.R. Ferreira**

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

*Editors* Lillian Barros Centro de Investigac¸ ao˜ de Montanha (CIMO) Instituto Politecnico ´ de Braganc¸a Braganc¸a Portugal

Isabel C.F.R. Ferreira Centro de Investigac¸ ao˜ de Montanha Instituto Politecnico ´ de Braganc¸a Braganc¸a Portugal

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

This is a reprint of articles from the Special Issue published online in the open access journal *Molecules* (ISSN 1420-3049) (available at: www.mdpi.com/journal/molecules/special issues/ natural additives).

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 978-3-0365-4106-8 (Hbk) ISBN 978-3-0365-4105-1 (PDF)**

© 2022 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.

The book as a whole is distributed by MDPI under the terms and conditions of the Creative Commons license CC BY-NC-ND.

### **Contents**


Reprinted from: *Molecules* **2018**, *23*, 2744, doi:10.3390/molecules23112744 . . . . . . . . . . . . . . **145**


### **About the Editors**

#### **Lillian Barros**

Lillian Barros is an assistant researcher at the Centro de Investigac¸ao de Montanha (CIMO), ˜ Instituto Politecnico de Braganc¸a and vice-coordinator of CIMO. She obtained her Ph.D. in Pharmacy ´ (Nutrition and Bromatology) at the University of Salamanca (2008). She has published more than 515 indexed papers in the Food Science and Technology area (61 h-index), edited 4 books, written several book chapters, registered 8 national patents, is principal investigator of several research projects and has supervision several post-doc, Ph.D. and master students. She has reached top positions in the world rankings, such as Highly Cited Researcher Clarivate, since 2016. Her research targets are mainly in the identification, separation and recovery of functional molecules from different natural products.

#### **Isabel C.F.R. Ferreira**

Isabel Ferreira is currently the Secretary of State for Inland Enhancement of Portugal. She was a Vice-President at the Polytechnic Institute of Braganc¸a, the Director of the Mountain Research Centre and Mentor of the Collaborative Laboratory Mountains of Research. She is a Full Professor at the IPB, with a Ph.D. in Biochemistry and one of the world's most cited scientists (top 1%) since 2015 by Clarivate Analytics in the Essencial Science Indicators index. She has published more than 700 papers in the Food Science and Technology area (61h-index), edited 4 books, written 60 book chapters, registered several national and international patents, most focusing on the transfer of technology to the industry.

### **Preface to "Natural Additives in Food"**

The controversy and ambiguity related with chemical additives, allied to sporadic scares, have paved the way for natural additives to gain interest and funding. Today, most consumers prefer foods added with natural additives, rather than artificial ones, which is seen by the food industry as an opportunity to find new and more efficient natural-based solutions, while fighting to reduce the overall use of additives, producing minimally processed goods. The benefits of natural additives are endless, their synergy and effectiveness are a great leap over artificial additives that carry out, in most cases, only one effect over the food.

The plant and fungi kingdoms are great sources of bioactive compounds, that can be used to develop natural food ingredients. These natural compounds can be added as extracts, taking advantage of the synergistic effects between compounds, or as individual molecules, after purification, thus adding the most bioactive ones to the foodstuff. Although quite promising, natural additives still face some drawbacks and limitations, availability of natural resources, exhaustive and not very efficient extraction methods, and several intrinsic and extrinsic factors that can affect the stability, availability, and bioactivity of natural compounds. Therefore, an important research topic is the discovery of new alternative sources of natural additives fulfilling the different classes: preservatives (antimicrobials, antioxidants, and anti-brownings), nutritional additives, coloring agents, flavoring agents, texturizing agents, and miscellaneous agents.

In order to address the advantages and challenges the use of natural additives in foods, an enormous amount of work, divided into three review and 15 original articles, involving diverse expert teams of from different parts of the world, embodied this book "Natural Additives in Foods", where current issues on natural additives are discussed and explored. Briefly, topics on alternatives for sustainable obtaining of bioactive molecules from agro-food byproducts, an important cheap source of added value compounds, which can contribute to total exploration of natural source, and for reduction of environmental impact, are presented and discussed in some studies. Likewise, optimized extraction techniques, aiming at greater extraction efficiency with less use of natural resources, are proposed by for some authors. Several potential natural colorants to replace artificial ones are covered in topics that discuss their main sources and bioactivities; and the sustainable production methods and the chemical stability of these compounds for later commercial use. In addition, interesting studies approach the valorization of wild species to obtain molecules with biological proprieties, both for human health and for addition in foods. And, finally, the production of foodstuff healthier than the traditional, is proposed through the use of natural ingredients rich in phytochemicals and through processes that aim to minimize the absorption of compounds harmful to health.

> **Lillian Barros and Isabel C.F.R. Ferreira** *Editors*

### *Review* **Agro-Food Byproducts as a New Source of Natural Food Additives**

### **Margarida Faustino, Mariana Veiga , Pedro Sousa, Eduardo M. Costa , Sara Silva and Manuela Pintado \***

CBQF–Centro de Biotecnologia e Química Fina–Laboratório Associado, Escola Superior de Biotecnologia, Universidade Católica Portuguesa, Rua Arquiteto Lobão Vital 172, 4200-374 Porto, Portugal; afaustino@porto.ucp.pt (M.F.); mveiga@porto.ucp.pt (M.V.); pedro.miguel.c.s@hotmail.com (P.S.); emcosta@porto.ucp.pt (E.M.C.); snsilva@porto.cup.pt (S.S.)

**\*** Correspondence: mpintado@porto.ucp.pt; Tel.: +351-225-580-097

Academic Editors: Lillian Barros and Isabel C.F.R. Ferreira Received: 4 February 2019; Accepted: 12 March 2019; Published: 18 March 2019

**Abstract:** Nowadays, the agro-food industry generates high amounts of byproducts that may possess added value compounds with high functionality and/or bioactivity. Additionally, consumers' demand for healthier foodstuffs has increased over the last years, and thus the food industry has strived to answer this challenge. Byproducts are generally secondary products derived from primary agro-food production processes and represent an interesting and cheaper source of potentially functional ingredients, such as peptides, carotenoids, and phenolic compounds, thus promoting a circular economy concept. The existing body of work has shown that byproducts and their extracts may be successfully incorporated into foodstuffs, for instance, phenolic compounds from eggplant can be potentially used as a mulfitunctional food additive with antimicrobial, antioxidant, and food colorant properties. As such, the aim of this review is to provide insights into byproducts and their potential as new sources of foodstuffs additives.

**Keywords:** byproducts; food additives; antimicrobial; antioxidant; colorants; texturizing agents; foaming capacity and emulsifiers

#### **1. Introduction**

Food functionalization is an ever-increasing market that requires new bioactive ingredients that can be used by the food industry for the development of innovative functional products with scientifically sustained claims. In this regard, much attention has been paid in recent years to natural compounds and their associated bioactivities. However, natural sources are finite, and new alternatives have to be sought to sustain the ever-growing needs for ingredients and additives of the food industry [1,2].

The European Union (EU) action plan for the circular economy to reduce food waste includes a strategic approach based on the reduction, reuse, recovery, and recycling of materials and energy, enhancing the value and consequently the useful life of products, materials, and resources in the economy. The reuse of agro-industrial byproducts can represent a renewable source for some already in use food additives or even originate new added-value ingredients with functional compounds and properties, which will benefit the entire food system [3]. For instance, byproducts contain polysaccharides, organic acids, proteins, and other compounds, which, at no additional production cost and at a reduced industrial cost, make them a rich source of natural compounds that can potentially be applied in the food industry as food additives sources (summarized in Table 1) [4].

Furthermore, these natural compounds may also be regarded as nutraceutical ingredients or complements, allowing for the development of products with enhanced nutritional value, potential health benefits, longer shelf-life, as well as a good sensory profile [5–7].


#### **Table 1.** Potential applications of agro-food byproducts compounds.


**Table 1.** *Cont.*

Taking this into account, this review aims to provide a broader look into the potential use of byproducts as new sources of food additives (already in use or potential new ones) to be used by the industry.

#### **2. Consumer Perspectives**

In the 1960s, the E number system was introduced to assure consumers that the additives included into their foodstuffs were safe for consumption. However, the use of this code made some consumers reticent in regard to these compounds with false allegations (on their lack of safety) being made in some publications [37–39]. Moreover, with the increase in life expectancy, concerns grew in regard to overall life quality. This, coupled with the widespread link between diet and health, made consumers particularly aware of the foodstuffs they ingested and increased the demand for healthier solutions. One trend associated with this shift in perception is "clean labels", i.e., products that are perceived as "natural", such as "free-range", "less processed", "organic", or "biological" foods [39,40]. Overall, this means that not adding additives has become a differentiating factor for food products, and consequently that the industry has become more interested in new solutions that, while exerting the same technological effect as traditional additives, have no negative perception. Agro-food byproducts present an interesting source of bioactive and technologically relevant compounds that, given their low commercial value, pose as a relevant potential source of new natural additives [41–44].

#### **3. Applicable Legislation**

Food additives have an essential role in the current industry and consumption habits, as they not only make food products more appealing, but they increase their stability and inherent safety. Overall, food additives may be defined as compounds/extracts that are added to a food product in order to accomplish a specific technological goal but are not ingested as a food product themselves. According to the European Food Safety Agency (EFSA), an additive must not pose a safety concern for the consumers health (when ingested) while fulfilling a specific technological need that cannot be satisfied through other reasonable means. Examples of these needs are the enhancement of the sensory quality, the fulfillment of specific dietary needs, or the ease of production, packaging, transport, and/or storage of food products [45]. Overall in the EU, the use of additives (non-enzymatic) is regulated by European Commission (EC) No 1333/2008 with the additives, the list of allowed additives, and subsequent limitations always dependent on the appearance of new evidence regarding their safety. In this legislation, the different groups are defined (Table 2) along with rules on how an additive must be referred to in a product (e.g., the information must be present in the label with the compounds referred to either by their name or their E-number and by the function they play in the final product). Moreover, food additives must follow specific purity criteria that are described in three different directives: Directive 2008/60/EC for sweeteners; Directive 2008/128/EC for colors; and Directive 2008/84/EC for other additives [46–48]. After the inclusion of the list of approved additives and food carriers (and the conditions associated with their use) into Regulation (EC) No 1333/2008, a revision of the purity criteria of food additives was undertaken, resulting in a new regulation, Regulation (EU) No 231/2012, that repealed the previous directives for sweeteners, colors, and other additives [45,48–50].



4

Some of the additives currently allowed under the scope of Regulation (EC) No 133/2008 may be found in agro-food byproducts. Namely, anthocyanins (E163) may be found in grape/winemaking byproducts, chlorophylls (E140) may be found in almost all green leaf vegetable byproducts or mango peels along with all green leaf wastes that result from pruning during agricultural production, and lycopene (E160d) can be found in tomato wastes [51–57]. There is a consensus that as long as the additive compound/molecule is already part of the list of authorized compounds, it can be used [58]. However, if the production process is varied significantly (by using a significantly different raw matter or using new production procedures), the "new" additive must be evaluated again by EFSA. This means that the focus given to the development of new and more efficient green technologies to attain additives from agro-food byproducts may result in potential new additives that must be subjected to a new evaluation in order to ensure their safety. This path starts with a thorough safety (short and long term) evaluation of any potential metabolic, genotoxic, reproductive, and chronic or carcinogenic side effects [59]. Following this, it is possible to define a no observable effect level (NOEL) and then an allowable/acceptable daily intake (ADI). Once all the relevant information is gathered, EFSA or other similar organizations (like the Food and Drug Administration—FDA) can be petitioned to validate the introduction of the additive through an amendment of the legislation in order to add the substance to the list of authorized food additives. If this authorization is granted, the additive will be eligible to be used on the market under direct supervision of the agency that granted the permission [49,59,60]. In the EU, the submission of a potential new additive for validation must start with an application submitted to the EC, who will verify it. If valid, EFSA must then give an opinion within a timeframe of nine months, a period that may be extended if further information is required from whoever submitted the application (for risk management purposes, EC may also require further elucidation). If EFSA gives a positive opinion, EC has nine months to submit a regulatory draft aimed at the inclusion of the substance in the allowed additives list, whose approval is dependent on the votes of member states. If approved, as with all decisions of the EU, it must then pass a three month long period of scrutiny. Overall, this process is very long and, in the new era of circular economy where food byproducts valorization is of the upmost importance, legislation approaches should be reanalyzed to facilitate and speed up the process of new additives approval while still guaranteeing the safety of the final additive [49,61].

#### **4. Preservatives**

Microbiological processes can adversely affect the quality of food, leading to its spoilage. For this to occur, conditions that favor the growth and development of spoilage microorganisms must be met, such as bioavailable nutrients, favorable water activity, adequate pH value, presence/absence of oxygen, and redox potential [62]. The term "food spoilage" is only applied if the changes in the foodstuffs due to the microorganisms' potentially harmful metabolic products become recognizable, thus making the product unsafe for consumption and augmenting the risk of foodborne illness [62–65]. However, not all microbiological change in food is considered harmful (for example, fermentation of grape juice in order to produce wine) [66].

Taking this into account, preservatives are widely used in the food industry in order to prevent microbial contaminations, demonstrating a significant impact upon a product's shelf-life as well as food safety [58,66–68]. There are different antimicrobial compounds that can potentially be used as preservatives ranging from enzymes, bacteriocins, fungicides, and salts to essential oils and other components, some of which may be found agro-food byproducts [7,69–76]. The use of natural compounds to replace traditional additives is an emerging trend that has been driven by the consumer's preferences for "clean labels", with the scientific community striving to provide natural alternatives, some of which may be attained from agro-food byproducts (e.g., phenolic compounds) [7,44,77]. Nitrates (E240-E259) and nitrites (E249-E250) are the most commonly used preservatives in foodstuffs. Both have been associated with the formation of nitrosamine (a carcinogenic compound responsible for the development of gastric and other types of cancer). Therefore, actions have been taken to

reduce their intake [the current daily intake for nitrates is 3.7 milligrams per kilogram of body weight (mg/kg bw/day), while for nitrites it was re-established to 0.07 mg/kg bw/day] [78–80]. However, EFSA determined that there was insufficient evidence to ban the use of nitrates and nitrites as food additives due to health concerns, particularly with them being the only additives capable of exerting antimicrobial activity against *Clostridium botulinum* and preventing botulinic toxin production/accumulation [81].

Agro-food byproducts, particularly fruit peels and seeds, have been regarded as a potential source of preservatives with several reports reporting on the potential antimicrobial activity of different fruit and vegetable byproduct extracts, which could potentially be translated into an industrial application if the appropriate regulatory body gives a positive opinion [7,41,67,82]. For instance, Gul and Bakht [83] reported how an ethanolic turmeric extract possessed antibacterial activity against *Escherichia coli* and *Staphylococcus aureus,* an effect that has been attributed to its phenolic content [84–86]. Additionally, turmeric oil, a byproduct from curcumin manufacture, has also been described as possessing antibacterial and antifungal activity [86–88]. Berries are fruits with high phenolic content, particularly anthocyanins. While by themselves they possess an interesting commercial value, if the fruits fall from the bushes (overly ripe berries), they will not be commercialized [89–91]. However, they remain a phenolic rich fruit that can be used as a source of potential antimicrobial additives. For instance, blueberry and cranberry anthocyanin-rich extracts have been reported as possessing vast antimicrobial activity and could potentially be exploited as new natural food additives [92–97]. Olive leaves are also a good source of phenolic compounds and have been reported as possessing some antimicrobial activity against *Bacillus cereus*, *E. coli*, *S. aureus,* and some fungi such as *Candida albicans* and *Cryptococcus neoformans* [98–101]. Wang, et al. [102] reported how the addition of green tea polyphenols (mainly constituted by catechins) and tocopherol to dry-cured bacon resulted in significantly lower *Enterobacteriaceae* content. Green tea and black tea wastes have been studied for their potential nutritive, antimicrobial, and antioxidant values due to their high tannin and catechin content [103,104].

Wine pomace, a well-known byproduct, also showcases some potential as a new source of antimicrobial food additives, as its activity has also been associated with its high phenolic content and anthocyanins in particular [22,105]. Pomegranate peel extracts, reported to be natural inhibitors of food-borne pathogens such as *Listeria monocytogenes*, *E. coli,* and *Yersinia enterocolitica*, have been added to poultry products with the results showing good antimicrobial activity against *S. aureus* and *B. cereus* and permitting the increase of shelf-life by two weeks [32,33,106–109]. Avocado, a tropical fruit, has also been described as possessing a relevant antimicrobial activity, with several reports focusing on the biological activity of its peel and seed [110,111]. For instance, Calderón-Oliver et al. [112] reported how a nisin (an antimicrobial peptide) avocado peel mixture resulted in an enhancement of nisin's antimicrobial activity against food-borne pathogens such as *Listeria* sp. Overall, the reported results favor the use of natural byproduct extracts as potential new preservatives at an industrial level, helping to reduce costs and environmental impact, although the leap to an industrial setting is limited by a lack of regulatory framework for their use.

Currently, the only animal derived antimicrobial additive used in the EU and United States (US) is lysozyme (E1105). Lysozyme originates from eggs, and while it is mainly used in cheese conservation, studies concerning eggs, milk, and beef have been carried out. However, it does not exert any action against yeasts or fungi [113–115].

#### **5. Antioxidant Additives**

Oxidation is a not a process exclusive to the human body. It occurs in every living organism and biological system, such as food products. Food oxidation may result in altered flavor, color, nutritional value, and texture, as well as create toxic compounds [82,116,117]. As such, antioxidant compounds are one of the most important conservation technologies used by the food industry with their main function being the prevention of oxidative induced degradation of foods, therefore allowing for extended shelf times [82,117,118]. These additives help stabilize lipids (avoiding lipidic peroxidation) as well as other compounds and can neutralize free radicals, avoiding a cascade of oxidative reactions. [117,119].

As previously mentioned, due to a shift in consumer preferences, in recent years there has been an increase in the demand for more natural (i.e., with less additives) food products [120]. As such, there have been studies comparing synthetic and natural antioxidants with results showing that natural phenolic antioxidants are capable of inhibiting oxidation and toxin formation, meaning that they present an interesting natural alternative to the traditionally used antioxidant additives [117,121]. Butylated hydroxy anisole (BHA), butylated hydroxytoluene, ethoxyquin, tert-butylhydroquinone, and propyl gallate are the most common synthetic antioxidants used in foods. Reports on their potential health impact are divided [121–124].

Since plants are one of the main sources of antioxidants compounds, agricultural byproducts are among the most relevant potential sources of natural antioxidants that could be exploited for product quality preservation. Phenolic compounds, besides being associated with antimicrobial activity, are known for their high antioxidant capacity. They are ubiquitous to plants and therefore present one interesting class of antioxidant compounds to be exploited, although other compounds with a strong antioxidant capacity can also be found, such as some vitamins (vitamin C, E, and A), bioactive peptides, polysaccharides, some minerals, and enzymes. Any byproduct with a high content of any of these compounds may be regarded as a possible source of new antioxidant food additives, e.g., overly ripe berries, or citric and exotic fruits, peels, and seeds [77,116,121,125]. Meat byproducts (including blood, bones, meat trimmings, and viscera) can result in protein hydrolysates with a relevant bioactivity, namely antioxidant bioactive peptides [126,127]. Onion byproducts (namely onion peels and stems) have been regarded as potential food additives due to their antioxidant and anti-browning properties [128]. Larrosa et al. [129] reported that adding an artichoke byproduct extract (namely artichoke blanching waters) to a tomato juice resulted in higher antioxidant activity (measured by the DPPH• and ABTS•<sup>+</sup> methods) and consequently a longer shelf life for this product. Similarly, eggplant aqueous acetone extracts have also been studied, with reports describing a high antioxidant potential of its peels (evaluated by FRAP and TEAC) likely due to its rich anthocyanin content [130]. Mango byproducts are an example of a vastly studied tropical fruit with a high antioxidant capacity and a wide range of potential applications [54,131]. An example is the inclusion of mango peel powder in macaroni and bakery products (such as biscuits) to provide some added functional value as well as function as a natural antioxidant (as the supplemented products exhibit a higher capacity to quench DPPH• ) [54,132,133].

The potential for the use of natural alternatives to antioxidant additives has been supported by the work of Caleja, Barros, Antonio, Oliveira, and Ferreira [121], who reported no significant differences between the use of natural extracts (chamomile and fennel) and a synthetic (BHA) antioxidant additive in biscuits, with no significant changes in color or nutritional value observed after 60 days of storage. Similarly, there have been reports on the successful addition of natural antioxidant extracts to bakery, dairy, and meat products, which also confer some added functionality to the foodstuffs [79,121,134–137]. Overall, byproducts of industrial fruit processing consist mainly of seeds, peels, and unused flesh. Some of these residues have been reported as possessing a higher concentration of bioactive compounds than the used fruit flesh [108,111,132,138–140]. Furthermore, the antioxidant compounds of natural origin, when attained using adequate solvents, are considered as generally recognized as safe (GRAS). Moreover, some of the antioxidant compounds naturally found in these byproducts are already approved for use as antioxidant additives and possess an E number, namely ascorbic acid (E300), tocopherol (E306), and β-carotene (E160a) [68,102,113].

#### **6. Food Colorants**

Although the flavor and nutritional value tend to be the most studied and appreciated components of a food product, its appearance is also an important sensory aspect [141,142]. Colorants are food additives used to impart color to foodstuffs to make them look more appetizing and/or help compensate color loss due to exposure to natural elements (light, air, temperature, etc.) [143,144]. Color plays an important role in the consumer's emotional reaction and acceptance of food. Color is appreciated both for its aesthetic and quality indicator role, as an adequate color is frequently used for quality assessment due to its association with flavor, nutritional value, and food safety [145]. Color provides visual suggestions to flavor identification and taste thresholds, influencing food preference, food acceptability, and ultimately, food choice [146]. Current market trends include the substitution of synthetic colorants for natural compounds found in certain foodstuffs (such as fruits) or in food byproducts, a trend that is reinforced by studies regarding possible detrimental effects of synthetic colorant usage in foods [142,144]. Most commercial colorants are produced synthetically, including erythrosine (red), cantaxanthin (orange), amaranth (azoic red), tartrazine (azoic yellow), and annatto bixine (yellow orange) [67]. Nonetheless, a few colorants like carotenoids (β-carotene, astaxanthin, canthaxanthin, and zeaxanthin) are obtained from natural sources, such as tomato, paprika, and algae [147]. However, synthetic colorants are still used due to their stability and low cost [44]. As agro-food byproducts are usually discarded, their use as a new source of these coloring agents could be a means to shift to more natural colors while still maintaining a low production cost (Table 3).


**Table 3.** Food byproducts sources of potential colorant food additives. Adapted from Iriondo-DeHond, Miguel, and del Castillo [142].

As previously mentioned, consumers have been demanding the replacement of synthetic colorants by natural alternatives. Authors like Siegrist and Sütterlin [155] reported that symbolic information such as the E-numbers on the foodstuff's label influences a consumer's perception of different foodstuff and its origin, with consumers being hesitant to accept the addition of synthetic food additives. Additionally, there have been several reports pertaining to synthetic colorants side effects, including hypersensitive and allergic reactions as well as potential toxicity and carcinogenicity claims [144,156,157]. Natural additives have been associated with health promoting benefits, as they are a part of the bioactive compounds present in fruit and vegetable byproducts. However, the use of these natural pigments can be limited by their lower stability and weaker color strength (when compared to their synthetic counterpart). Additionally, natural additives may confer an undesirable flavor or odor to the food

products, which will negatively impact the consumer's acceptance [142,145,158]. Nonetheless, fruit and vegetable byproducts have become an important potential source of natural pigments, as they are colored by green chlorophylls, yellow-orange-red carotenoids, red-blue-purple anthocyanins, and red betanins [158]. Overall, the main groups of coloring substances found in nature are carotenoids, anthocyanins, porphyrins, and chlorophylls [145,158–160].

Anthocyanins are a good example of natural color additives. These compounds are a group of natural pigments responsible for the blue, red, purple, violet, and magenta coloration of several species in the plant kingdom. They can also be found in extracts of their byproducts. Some examples are winery byproducts, radishes, red potatoes, red cabbage, black carrots, purple sweet potatoes, coffee husks, and berries, among others [106,161].

Carotenoids stand as the major group of compounds used as color additives. These natural pigments are responsible for many of the colors seen in edible fruits, vegetables, mushrooms, flowers, and even lobster and trout hues from the animal kingdom [158]. Much like anthocyanins, carotenoids are produced synthetically (β-carotene, astaxanthin, canthaxanthin, and zeaxanthin), although some are obtained from natural sources, namely annatto, marigold, tomato, algae, and microbial fermentation [157]. In addition, these compounds function as sources of provitamin A and are capable of absorbing solar light, oxygen transporters, powerful quenchers of singlet oxygen, as well as other functions not yet studied [160].

The natural pigments were defined in the Regulation (EC) No 1333/2008 of the European Parliament and of the Council of 16 December 2008 and are listed in the Annexes of said regulation [161]. This document includes detailed information on the application of individual pigments in defined food products, their doses, and limitations of use. Presently, 16 natural pigments are permitted: betalains–betanin, quinones–cochineal, flavonoids–anthocyanins, isoprenoids–carotene, annatto (bixin, norbixin), paprika extract, lutein, canthaxanthin, porphyrins–chlorophylls and chlorophyllins, and copper complexes of these compounds, among others, like caramels, curcumin, or plant coal. According to the Regulation (EC) No 1129/2011 [162] of the European Parliament, in the EU, there are 40 color additives legislated for food use.

New technologies such as pulsed-light, high pressure, pulsed-electric, magnetic fields high pressure processing, ionizing radiation, and ultraviolet radiation are being studied and could allow for the use of byproducts as natural source pigments, which could then be exploited as potential new food colorants in the food industry with the advantage of imparting potential health benefits to the consumer as well as contributing to an economical valorization of residues and avoiding waste [163]. For instance, there have been studies regarding the addition of banana peels to biscuits, which resulted in a product with low calories and high dietary fiber content without any significant differences in color, aroma, and taste observed. The banana peel was incorporated at a 10% and 20% concentration into the biscuits [164–166]. The peel is the main byproduct of the banana, rich in phytochemical compounds with high antioxidant capacity, such as phenolic compounds, anthocyanins (delphinidin and cyanidin), carotenoids (β-carotenoids, α-carotenoid, and xanthophylls), catecholamines, sterols, and triterpenes, which, as previously mentioned, could provide different functions as potential food additives besides coloring, namely as antioxidant and antimicrobial components [18,167]. Mango is another example of a fruit with biologically interesting compounds (including phenolic compounds, carotenoids, and dietary fiber) that could be used in the food industry. Most mango byproducts result from the epicarp and endocarp, but it is the mango seed kernel residue with the highest amount of carotenoids in its composition, which is likely due to the amount of fruit pulp left around the kernel by the chopping machine. The carotenoid content was found to be four to eight times higher in ripe mango peels compared to raw fruit peels [133,165]. The high levels of bioactive compounds in the mango peel makes this byproduct a potentially valuable raw material for the formulation of additives and supplements for the food industry [168]. Likewise, tomato peel is the main byproduct resulting from the tomato processing industry [169]. The carotenoid pigment lycopene present is the compound responsible for its red color. Tomato lycopene extract and tomato lycopene concentrate from tomato peels have been approved for use as colorants exempt from certification [170]. Oleoresins, powders, and water-dispersible preparations that can impart colors from yellow to orange to red are commercially available. An example of the utilization of lycopene from tomato byproducts includes dairy foods, where this compound is applied in the coloring of butter and ice-cream, maintaining a stable reddish color for up to four months [146,148,149].

Even though the use of the aforementioned compounds as food colorants could present an ecological solution to current production issues in addition to possessing an added advantage of potential health benefits, their use has been limited. Regulated colorants from natural sources include anthocyanins (E163), betanin (E162), and carotenoids (E160), including β-carotene (E160a), lycopene (E160d) (its obtention from tomato processing byproducts has been optimized), lutein (E161b), canthaxanthin (E161g), chlorophyll and chlorophyllin (E140 and E141), and curcumin (E100) [according to Regulation (EC) No 1129/2011] [163]. However, the list of anthocyanin colorants in the Codex Alimentarius includes only grape skin extract (E163), and in the FDA, "grape color extract" and "grape skin extract" (enocyanin) [146,148,149].

Regardless, to include a new pigment as a food colorant additive according to Regulation (EC) No 1333/2008 of the European Parliament and of the Council of 16 December 2008 [162], these new pigments need to be capable of restoring the original appearance of food whose color has been affected by processing, storage, packaging, and distribution, leading to impaired visual acceptability. Thus, colorants need to make food more visually appealing as well as give color to food that is otherwise colorless.

#### **7. Texturizing Agents**

Texturizing agents, such as emulsifiers, stabilizers, thickeners, and bulking agents, are used in the food industry to modify the overall texture and mouth feel of foodstuffs [171]. Thickeners, when added to the food mixture, increase the viscosity without modifying other food properties, while bulking agents increase the bulk of a food without affecting its nutritional value. Emulsifiers, on the other hand, allow water and oil to remain mixed together in an emulsion. These agents are used to add or modify the texture of food products by modifying the creaminess, thickness, viscosity, or by stabilizing foodstuffs structure [67]. These agents are used in frozen desserts, dairy products, cakes, puddings, gelatin mixes, dressings, jams, jellies, and sauces [172]. An example of the use of these food additives is their incorporation into hydrocolloids like fermented milks, dairy desserts, cream, and ice-cream to stabilize and thicken them. Another example of texture additives are phosphates and coagulation agents that are used in the curdling of milk in cheese production [173]. Most of the hydrocolloids used in the dairy industry result from the isolation of seaweeds and plant cells and are obtained and/or extracted from byproducts such as plant food wastes. The natural agar obtained from algae is the most researched texture agent used as a food additive in bakery products, confectionery, ice cream, peanut butter, and beverages [171]. Table 4 discloses some examples of byproducts used as source of texture additives.


**Table 4.** Food byproducts sources of potential texturizing agents' additives. Adapted from Iriondo-DeHond, Miguel and del Castillo [142].

Citrus fruits and their byproducts (such as peels and seed powders) have been studied as possible sources of texturizing agents due to their natural high pectin content and dietary fiber [142,176,180]. Currently, there are some examples of citrus byproducts being used in the industry. Oranges are being used as a texturizing agents in yogurts and/or ice creams [21,179,181], and lemon byproducts are being used as thickening, texturizing, gelling, and stabilizing agents [182]. Furthermore, citrus byproducts have the added advantage of being rich in bioactive compounds, which possess nutritional and functional benefits including reducing the risk of certain pathologies such as obesity, cardiovascular disease, and colon cancer, as well as preventing neurodegenerative diseases and osteoporosis [139,182–185]. Additionally, their high dietary fiber content is an added bonus, as it can be used as fat replacers and thus functions as a food additive to impart texture to the final product [186–188]. In fact, Crizel et al. [189] showed that incorporation of fiber from orange byproducts into yogurts allowed for the manufacture of low-fat yogurts, and Dervisoglu and Yazici [190] reported that while citrus fiber as a single stabilizer did not improve the viscosity, overrun, and sensory properties of ice cream, it improved the melting resistance of these foodstuffs. Similarly, the industrial processing of tomato leads to high amounts of unused matter (mostly peels and seeds), which are byproducts rich in lycopene and dietary fiber. These byproducts have been incorporated in tomato sauce as a food texturizer, with sensorial tasting panels deeming it as acceptable [149,191]. Another example is the ß-glucans resulting from cereals such as oat and barley, which have also been used as fat replacers in a variety of foodstuff ranging from baked goods and pasta to beverages and soups with promising results [192–194]. Additionally, the presence of β-glucans in foods has also been shown to lead to an increase in fiber intake, which in turn prevents constipation, reduces intestinal transit time, reduces the risk of colorectal cancer, and promotes the growth of beneficial intestinal bacteria [195].

Other potential sources of dietary fiber and pectin are cocoa (Theobroma cacao L.) pod husk (an abundant industrial waste with potential application in the food industry), and oat bran. Studies have shown that cocoa byproducts can be used as a texturizing agents after drying and grinding, while juice resulting from the pods can be used to prepare hydrocolloids [196]. On the other hand, oat bran extract has also been studied as a natural emulsifier, with results showing stability through a different range of pH values, heat treatments, and storage life up to 40 days [197].

On a different note, fat plays an important role in the structural integrity and mouth feeling of foodstuffs (ice-cream and yogurt in particular) due to its interaction with casein micelles [198]. Many different types of fat replacers have been explored in bovine and goat milk yogurts, including the addition of inulin, β-glucan, high milk protein powder, and whey protein concentrates [199–204]. Whey proteins, obtained as a byproduct of the dairy industry, have many functional properties including gelation, thickening, and water-holding capacity [205]. In the study by Wang et al. [206], whey protein isolate was used to produce a goat's milk yogurt of acceptable quality. Milk fortification with whey protein improved the textural and microstructural characteristics and some sensory characteristics of yogurts. In addition, whey protein concentrates caused some interactions between globular proteins and caseins, which led to an improved texture of goat's milk yogurt and higher water retention capacity [36,206–208]. Whey proteins are also present in high amounts in a byproduct of butter-making—buttermilk. This product is now considered valuable because of its high content in fragments of milk fat globule membrane in addition to phospholipids [209,210]. Studies indicated that the moisture content of cheese supplemented with buttermilk remained high due largely to phospholipids improving its texture [211,212].

#### **8. Foaming Capacity**

Foam is a colloidal dispersion in which a gaseous phase is dispersed in a liquid or solid phase. Food foams are dependent on the surface activity and film-forming properties of specific protein compounds [213,214]. Proteins have to be either very hydrophobic or hydrophilic to possess good foaming properties, and therefore their chemical or enzymatic modification can make them more active on the surface. As such, most foaming agents commonly used in the food industry are mainly

natural modified food proteins such as soy, casein, egg white, whey, serum proteins isolated from lactoglobulins, and lysozyme [214–216]. Globulins are excellent foaming agents, but their foaming is significantly affected by interactions of the proteins with ovomucine, lysozyme, and, to a lesser extent, ovomucoid, ovotransferrin, and ovalbumin [217]. A novel source of possible foaming agents is the fishery industry, as fish processing leads to high amounts of byproducts rich in collagen and gelatin. This gelatin foaming capacity has been studied, with reports showing that gelatin from shark cartilage possessed foaming properties similar to those of porcine skin [218]. According to Muzaifa et al. [219], fish byproducts (dark muscle, cut offs, viscera, skin, scales, small bones, and fins) could potentially be used to obtain protein hydrolysates through an enzymatic hydrolysis using Alcalase® 2.4 L and Flavourzyme® 500 L, leading to compounds with foaming capacity. Protein hydrolysates obtained from poultry byproducts (head and leg) and rainbow trout (*Onchorhynchus mykiss*) viscera after an enzymatic hydrolysis using Alcalase® 2.4 L also demonstrated foaming capacity [14]. On another work, Kotlar et al. [220] reported on the use of brewer's spent grain (BSG) hydrolysates (attained using a *B. cereus* extracellular peptidase) to improve the foaming expansion in brewery products. Okara, a byproduct obtained from the soy milk production, was also analyzed for its functional properties, with the authors observing that the isolated proteins from this byproduct could potentially be used as a foaming agent [27]. When it comes to slaughter byproducts, there are several residues, including skin, bones, hooves, muscles, and blood. Blood represents up to 4% of the animal live weight. However, the direct use of blood in foods is not useful due to the dark color given to the food. In practice, blood is separated by centrifugation into cellular and plasma fractions. Plasma proteins have relevant and interesting properties for food processing [221], e.g., they contribute to cross-link proteins and gelling [222], proteins enrichment [223], as well as emulsifying and foaming agents [224].

#### **9. Emulsifiers**

Emulsifiers, molecules such as polysaccharides (e.g., gum arabic) or phospholipids (e.g., lecithins) with a surface activity capable of mixing and stabilizing two immiscible phases like water and oil, are largely used in food technology [225,226].

Emulsifier additives can be obtained from a variety of food products (e.g., milk protein isolates) [227] and byproducts (e.g., okara) [27]. Gbogouri, Linder, Fanni, and Parmentier [11] suggested that salmon (Salmo salar) head hydrolysates treated with the commercial enzyme Alcalase® 2.4 L could potentially be a new source of compounds with great emulsifying capacity and stability. Using the same enzyme mix, Sathivel et al. [13] analyzed the potential of herring (Clupea harengus) byproducts hydrolysates. Although the emulsifying capacity was lower than that of egg albumin and soy protein, the hydrolysates still demonstrated some emulsifying capacity and stability, an effect that was also observed for the protein extracts before hydrolysis [12]. A potential emulsifier additive could be obtained from okara, a byproduct obtained from soymilk production. Even though okara protein isolates had poor solubility, they exhibited other functional properties (emulsification, foaming, and binding properties) that were comparable to those of a commercial soy isolate, further demonstrating the potential use of these isolates as a food ingredient [27]. The Horchata production, a vegetable milk obtained from tiger nuts (Cyperus esculentus), also originates a solid waste byproduct rich in dietary fiber that could potentially be used as a new ingredient for its emulsifying capacity and high emulsifying stability [28]. Emulsifiers can also be found in meat industry byproducts. For example, bovine blood derivate products (plasma and globulin) may be used as a potential new emulsifier agents additive in meat products and others [28,228,229]. As such, compounds obtained directly or indirectly from byproducts could potentially be used as new emulsifying agents in the food industry.

#### **10. Conclusions**

Given the consumer's demand for "clean label" products and the environmental constraints that reinforce the need to change the traditional industrial raw matters with renewable sources, agro-food byproducts have appeared as one of the most relevant potential solutions. In fact, some of the additives used nowadays (like anthocyanins and carotenes) can be found in these materials, which makes their extracts interesting from a consumer's perspective (some would prefer a tomato extract instead of traditional lycopene), particularly when considering the possibilities opened up by green, safe, new extraction methodologies like high pressure extraction, ohmic extraction, pulsed electric field, or supercritical extraction. However, their direct inclusion into commercial products may depend on the limitations posed by the legislation itself because, even if the additive itself is already approved for use, should its production process or raw material differ significantly from the one currently used, its future as an additive will be dependent on a new safety evaluation.

Overall, it is possible to see the potential of byproducts derived food additives and potential new additives for application in the food industry. They are an integrated solution with low cost and reduced environmental impact capable of providing alternatives for an industry that relies heavily upon the chemical synthesis compounds. Thus, the use of byproducts as a source of food additives stands out as an economically and environmentally conscious choice and will promote the new era of circular economy.

**Author Contributions:** M.V., M.F. and P.S. carried out the research and wrote the document, E.M.C. and S.S. were responsible for defining the paper's scope, structure and contributed to its writing and, along with M.P., were responsible for the overall definition and reviewing of the document. All authors reviewed and agreed on the final version.

**Funding:** This work was supported by National Funds from FCT (grant number UID/Multi/50016/2013, POCI-01-0145-FEDER-016403) from FEDER (grant number PDR2020-101-030775) and from QREN-ANI (grant number 17819).

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

#### **References**


© 2019 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* **Green Natural Colorants**

### **Isabel Viera, Antonio Pérez-Gálvez and María Roca \***

Food Phytochemistry Department, Instituto de la Grasa (CSIC), University Campus, Building 46, Carretera de Utrera km. 1, 41013 Sevilla, Spain; iviera@ig.csic.es (I.V.); aperez@ig.csic.es (A.P.-G.) **\*** Correspondence: mroca@ig.csic.es; Tel.: +34-954-611-550

Academic Editors: Lillian Barros and Isabel C. F. R. Ferreira

Received: 6 November 2018; Accepted: 30 November 2018; Published: 2 January 2019

**Abstract:** Although there is no legal and clear definition of the term "natural food colorant", the market trends, and consequently industrial and commercial interest, have turned to foods with added natural pigments. This progressive substitution of artificial colorants has faced chemical complications with some colors, with a lack of stable green hues being one of them. Several strategies have been applied for green color stabilization in processed foods, from the formation of metallochlorophylls to the microencapsulation of green pigments. However, at present, the utilization of green coloring foodstuffs, which are considered an ingredient in the EU, seems to be the more successful solution for the market. Besides those topics, the present review aims to clarify the current confusion between the different chlorophyll compounds that form part of the authorized green food colorants. In this sense, legislations from different countries are compared. Finally, and in line with current concerns, the knowledge gathered so far in relation to the absorption, distribution, metabolism and excretion of all green natural food colorants is reviewed.

**Keywords:** ADME; absorption; chlorophylls; chlorophyllin; green colorant; zinc-chlorophylls; copper-chlorophyll; coloring foodstuff; natural colorants; food colors

#### **1. Introduction**

The first moment of truth is a term coined by Procter & Gamble [1] to describe the 3–7-s gap that makes a customer select a product over the rest of its competitors. It has been established that color is responsible for 62–90% of the consumer's assessment [2]. This fact makes expertise in food colorants a very profitable activity. Specifically, some market research companies have prognosticated a global food colors market size of USD 2.97 billion by 2025, with an estimated USD 1.79 billion in 2016 [3]. In this sense, consumer claims are responsible for the progressive strengthening of the food industry. We want food products to be more delightful, nutritive, attractive and healthy, with "fun-foods" as the maximum expression of this appeal [4]. The response to this worldwide trend is the unavoidable application of food colorants.

Before going further in this review, several concepts should be defined. Colorants can be classified following different parameters [5]. For example, in the USA, food colorants are categorized into whether they require or not the batch certification carried out by the US Food and Drug Administration (FDA). The list with the approved colorants that require certification is published in Title 21 CFR (Code of Federal Regulations) 74, where a Foods, Drugs and Cosmetics (FD & C) number is assigned. Hence, the certified colors commonly defined as synthetic could be classified as dyes (food-grade water soluble colorant [6]) or lakes (oil-dispersible; generally, dye extended on alumina). On the other hand, the list of the colorants exempted from certification is published in the Title 21 CFR 73 document [7]. Colorants within this category are mainly natural, although a few of them are produced by synthesis, but considered "nature-identical". On the other hand, authorized food colorants in the

EU are mainly legislated by the Regulation (EU) No. 1333/2008 [8], which is amended by Regulation (EC) No. 1129/2011. EU legislation includes indistinctly natural or artificial colorants.

Food colorants can be allocated following their origin (vegetal, animal, bacterial, fungal, etc.), their hue (red, yellow, purple, blue, green, etc.) or their chemical structure: flavonoid derivatives (anthocyanins), isoprenoid derivatives (carotenoids), nitrogen–heterocyclic derivatives (betalains), and the subject of this review, pyrrole derivatives or chlorophylls, which are responsible for blue and green hues.

At this point, it is relevant to distinguish between natural colorants and artificial ones. Although we could have an intuitive and clear conception about what each term means, the truth is that there is no official or definitive definition of what a natural or an artificial colorant is [9]. Only natural flavorings, another class of food additives, have a specific definition given by the FDA and EU [10]. Although there have been suggestions to extend the distinction of natural flavorings to food colorants, it seems difficult to reach a consensus between the different interested parties. In fact, neither in the US nor in the EU is there a recognized legal advertisement for "natural color" [11]. Indeed, the limits could be even more ambiguous if we consider, for example, the food colorant copper chlorophyllin, which is considered as natural. As will be noted below, this colorant is extracted from a natural source (generally from edible green leaves), but its manufacture requires additional chemical batch processing. Consequently, the question is as follows: where do we set the limit between natural and synthetic colorants? However, even realizing the hurdle of setting a frontier between natural and artificial colorants, the consumer increasingly demands more information, transparency, naturalness, clarity and trustworthiness in food label specifications. Thus, it is evident that there is an increasing global trend towards the natural side of foods.

Although previous studies linked the consumption of artificial food colorants with behavioral disorders, the inflection point was probably the well-known "Southampton study" [12]. This randomized, double-blinded trial test was performed with 137 3-year-old and 130 8/9-year-old children and concluded that the intake of dietary artificial food colorants increased the hyperactivity of children. Since then, many other research works and meta-analysis studies have confirmed and even amplified the disorders which probably originated from artificial food colors, although the precise physiological mechanism is unknown to date. In any case, the food industry and food legislative authorities have implemented some actions. The consumers' concern about the safety of artificial food colorants, reinforced by the possible health benefits of natural pigments, have induced the food industry to withdraw artificial colorants. In fact, artificial food colorants represent only 16% in the EU and 29% in the North America of the food colorants portfolio [13].

The progressive substitution of synthetic colorants by natural ones faces the challenge of the low stability of some colorants. Probably, green is one of the most complicated colors to both naturally set-up and counteract. The aim of the present review is to specifically address the current status of natural green food colorants, including the legal issues, the chemistry of chlorophylls and the existing alternatives for the stabilization of green color in foods, as well as the acclaimed "coloring foodstuff". For a general study of other food colorants, high-quality reviews have recently been published [4,10,11,13–17].

#### **2. Legal Aspects on Green Natural Colorants**

As has been noted elsewhere [16], one of the main problems affecting food additives is the lack of a globally harmonized legislation. This fact creates problems for wholesalers in this globalized food market. Even the concepts "natural" and "artificial additives" are country-dependent. Consequently, although this review only deals with the natural green colorants, whose main legal considerations are firstly described, a brief outline also shows the authorized synthetic green food colorants in different countries at the end of this section. Generally, the regulations of food additives include the list of authorized standards, their specifications, as well as the conditions of use (limitations on specific foods and maximum amounts). In addition, other requirements that should be considered are the schemes

through which authorization is obtained and the conditions for packaging and labelling, which are different in each country.

European current legislation (Regulation (EC) No 1333/2008 and its amendments) [8] has allowed the use of two natural green colorants (Table 1), E140 and E141, which are structurally related with the chlorophylls, since the first European food colorant legislation was published in 1962 (Council Directive 62/2645/EEC). E140 comprises direct chlorophyll derivative extraction with organic solvent from natural sources: grass, alfalfa, nettles, spinach and other edible plant materials (Figure 1). The end-product could contain other lipids, pigments and waxes, so that the final aspect of the colorant is waxy, and it is marketed according to its solubility. Thus, the E140i or chlorophyll derivative is lipid-soluble, while the alternative for water-soluble foods (marketed as a powder) is E140ii (or chlorophyllins), obtained by the saponification of the solvent-extracted yield from edible plant material. Saponification breaks the ester–phytol bond, as will be detailed below, increasing the polarity of the derived products. However, both colorants are rather unstable, and the color is prone to experience a drastic change from green to brown. Therefore, the food industry favors the use of the colorant E141, which results from the addition of copper to the corresponding lipid or water-soluble chlorophyll solutions. The insertion of copper into the chlorophyll molecule stabilizes the structure, and the green coloration does not change, independent from the processing conditions or the storage time of the colored food. E141 is mainly composed of copper complexes of chlorophyll derivatives, with E141i (or copper chlorophylls) being the lipid-soluble option and E141ii (copper chlorophyllins) the water-soluble alternative [18]. –

**Table 1.** Classification of authorized natural green colorants according to different regulations.


<sup>1</sup> CA: Codex Alimentarius.

**Figure 1.** Scheme of the natural green colorant manufacture process.

In the USA, the regulation of natural food colorants is published in Title 21 CFR 73 [7]. Only copper chlorophyllin (Table 1) is authorized as a natural green colorant (CFR Section 73.125) and only for coloring citrus-based dry beverage mixes, the amount being limited to 0.2% in the dry mix. Current legislation in China [19] recognizes two green colorants: copper chlorophylls (CNS 08.153) and copper–sodium chlorophyllin (CNS 0.009), which is only allowed for use in determined food categories and with fixed maximum levels. At any rate, the China National Center for Food Safety Risk Assessment (CFSA) has published at the beginning of 2018 the first draft for a National Food Safety Standard for the Use of Food Additives, which revises and updates the current version issued in 2014. In Japan, the list of food additives from natural origin was compiled and published by the Ministry of Health and Welfare on 16 April 1996 [20]; in 2018, this accounts for 365 items, with chlorophyll (number 117 in the original Japanese list) and chlorophyllin (116) as the only authorized natural green colorants. In addition, Japanese legislation includes 455 designated food additives to date (obtained by chemical reactions), which include copper chlorophyll (266), sodium–copper chlorophyllin (265) and sodium–iron chlorophyllin (257). In both regulations, the target foods, the maximum limits and other requirements are detailed for each colorant. In India, the regulations are published in the Food Safety and Standards (Food Products Standards and Food Additives) Regulations (2011), which depend on the Food Safety and Standards Authority of India [21]. They only allow the use of chlorophyll as a coloring food matter which has the specification of having both chlorophyll *a* and chlorophyll *b*.

Finally, the Joint Food and Agriculture Organization of the United Nations/World Health Organization (FAO/WHO) Expert Committee on Food Additives (JECFA), as the international body responsible for evaluating the safety of food additives, has published the last version of the Codex Alimentarius general standard of food additives (41st meeting on 2018) [22]. In accordance with European legislation, chlorophylls (INS 140) and copper chlorophylls (INS 141) are registered. It is worthwhile to visit the corresponding websites to have access not only to the specification of each food additive, but also to the complete monographs and evaluations developed for each one.

Along with the colorants generally considered as natural, the legislations also authorize artificial green food colorants (Table 2), green S and fast green FCF, with both compounds being triarylmethane derivatives (Figure 2). In the EU, only green S is legal, under the code E142, while its use is banned in countries such as the USA, Japan, India, and China. It is also recognized by the Codex Alimentarius with the reference number INS 142. This colorant presents the molecular formula C27H25N2NaO7S<sup>2</sup> and a molecular weight equal to 576.63 Da (CAS number 3087-16-9). On the other hand, in the USA (along with other countries, Table 2), the authorized artificial green colorant is the so-called fast green FCF, a compound with the molecular formula C37H34N2O10S3Na<sup>2</sup> and a molecular weight equal to 808.85 Da (CAS number 2353-45-9; PubChem CID 73557432), while its use is banned in the EU.



<sup>1</sup> The Color IndexTM is a classification system for dyes and pigments globally used by manufacturers, researchers and users of dyes and pigments (https://colour-index.com/).

**Figure 2.** Structures of green S and fast green FCF.

#### **3. Chlorophyll Derivatives in the Authorized Natural Green Colorants**

Several chemical (conventional) terms have been commonly confused in the commercial and the industrial environments to tag chlorophyll colorants. We will explicitly detail the chlorophyll compounds, when described, that make up each natural green colorant, along with the conventional terms. To avoid misunderstanding in following the statements, Figure 3 depicts the skeleton of the chlorophyll structure showing the different substituent functions and the numbering system. The difference between chlorophyll *a* and chlorophyll *b* is located at C7, where chlorophyll *a* presents a methyl group while chlorophyll *b* includes a formyl group (Table 3). The central coordinated atom is magnesium in the chlorophylls, while two hydrogen atoms are present in pheophytins and pheophorbides. Otherwise, the phytyl chain (C20H40) is ester-bonded to the C17 3 , which confers the lipophilic properties when present in non-polar derivatives: chlorophylls and pheophytins (Table 3). On the opposite set, the phytyl chain is absent in polar derivatives: chlorophyllides and pheophorbides. Lastly, the isocyclic ring (ring V) presents a carboxymethoxy arrangement at C13 2 . The lack of this group yields the pyroderivatives. Additional unprecedented chlorophyll structures could be present in the colorants [23].

**Figure 3.** Chlorophyll skeleton with the different substituents.

The analysis of the chlorophyll derivatives contained in the green natural colorants started with the development of several chromatographic methods able to separate the different compounds in the mixtures [24,25]. The details of those methods have been recently compiled [23]. Due to the chromatic properties of chlorophylls, DAD is the main detector used, although Raman spectroscopy was also proposed [26]. However, the similarity of the UV-Vis spectrum between several chlorophyll derivatives, made essential the utilization of mass spectrometry to properly identify the different substituents of the chlorophyll compounds [27,28]. Details of the mass spectrometry characteristics of the chlorophyll compounds is not very abundant, although several studies have been developed [23,27].

The colorant named "chlorophyll" or "E140i", as it is marketed after direct solvent extraction from edible green plant materials, is mainly composed of chlorophyll *a* and *b*, along with their corresponding pheophytin derivatives, which are originated during the extraction at low pH, which catalyzes the substitution of the central magnesium by hydrogens (Figure 4). The relative proportion of chlorophyll to pheophytin present in the colorant depends on the conditions of the manufacturing practices, and it is highly variable between the different suppliers. In addition, the chlorophylls present in the colorant product are very labile, and the chemical conditions during the food processing accelerate the degradative process. The transformation causes a change in color from green to brown; a collateral effect which is not desired by the food industry. The colorant named "chlorophyll" or "E140i", as it is


**Table 3.** Main chlorophyll derivatives in function of their substituents.

Chlorophyll derivatives present in the colorant E140i or "chlorophyll". **Figure 4.** Chlorophyll derivatives present in the colorant E140i or "chlorophyll".

The natural green colorant E140ii is commonly named "chlorophyllin". This term is the source of confusion, because currently there is no clear chemical definition of that term, yielding a gap between the genuine chemistry of chlorophylls and the industrial (commercial) application of the nomenclature. Originally, the term [29] referred to the chlorophyll derivatives produced after the saponification of

chlorophyll without a change in color. At that stage, when the definitive structure of chlorophyll was not described yet, chlorophyllin exclusively comprised those chlorophyll derivatives arising after the phytol ester breakdown. This means that chlorophyllins keep the central magnesium ion intact. Later, this term increased its application to other chlorophyll derivatives produced during the saponification; i.e., those arising from the cleavage of the isocyclic ring. However, the exact chemical definition of chlorophyllins covers those chlorophyll derivatives with an intact central magnesium ion. Therefore, the present commercial (industrial and sometimes legislative) use of the term "chlorophyllins" is incorrect, because the colorant products include chlorophyll structures without magnesium [30]. "chlorophyllins" is incorrect,

colorant E140ii is commonly named "chlorophyllin". This term

**Figure 5.** Chlorophyll derivatives present in the colorant E140ii or "chlorophyllins". derivatives present in the colorant E140ii or "chlorophyllins".

Figure 5 describes some of the different chlorophyll structures that have been assigned to the "chlorophyllin *a*" denomination. For example, EU regulation [8] describes a structure with elemental composition C34H32MgN4O<sup>5</sup> and molecular weight 600.9467 Da that chemically corresponds to demethylated chlorophyllide *a*. This compound originates from chlorophyll after the breakdown of the phytyl ester bond at C17 3 (chlorophyllide *a*) and the fragmentation of the methoxy group at C13 1 . Other examples are the compound described in the Chemical Abstract Service (CAS 15611-43-5) or in

PubChem (123798), which chemically corresponds with magnesium chlorin *e*6. This structure with an elemental composition C34H34MgN4O<sup>6</sup> and molecular weight 618.962 Da is formed from chlorophyll *a* after the fragmentation of the phytyl ester bond, loss of the carboxymethoxy group and opening of the ring V. However, during chlorophyll saponification, the reaction required for E140ii manufacturing, the central magnesium ion is replaced by two hydrogens. Consequently, the main chlorophyll compounds that form part of the colorant E140ii are the chlorin *e*<sup>6</sup> which originates from chlorophyll *a* and the rhodin *g*<sup>7</sup> formed from chlorophyll *b*, besides other chlorophyll derivatives such as pheophorbides.

The colorant E141i (Figure 6) is known as "copper chlorophyll", although it would be more accurate to label it as pheophytin with copper. The former nomenclature aims to denote the oily consistency of the colorant, which in chemical terms means that it is constituted by phytylated chlorophyll derivatives and, therefore, is lipid-soluble. This premise, in conjunction with the required copper treatment for its production, means that the main chlorophyll derivatives present in the colorant E141i are copper–pheophytin derivatives. In fact, an HPLC method was developed for the detection and quantification of copper chlorophylls *a* and *b* (copper pheophytins *a* and *b*) [25]. Later, several methods have been settled with the same aim [24,31]. At any rate, the complete characterization of several commercial E141i samples showed not only differences in the chlorophyll profile between them, but also that the common main compound present in all samples is copper–pyropheophytin *a* [32]. The MS characterization of this chlorophyll derivative has allowed us to propose a specific product ion as a probe for tracking the presence of the E141i food where its use is not authorized, particularly in olive oil [28]. Other chlorophyll derivatives that could be present in this colorant are copper–pheophytin *a*, copper–pheophytin *b*, copper–pyropheophytin *b*, and some others in trace amounts [32]. "chlorophyllin " denomination. For example, EU regulation [8] describes a structure with elemen

Chlorophyll derivatives present in the colorant E141i or "copper chlorophylls". **Figure 6.** Chlorophyll derivatives present in the colorant E141i or "copper-chlorophylls".

The colorant E141i (Figure 6) is known as "copper chlorophyll", although it would be mor

–

The water-soluble green food colorant E141ii (Figure 7) has been the most studied as it is one of the most popular within the food industry. Its manufacture requires, besides solvent extraction, saponification and treatment with copper salts, which means a higher degree of transformation from chlorophylls. The first chlorophyll derivative identified in this colorant was the copper chlorin *e*<sup>4</sup> [33].

– – –

–

derivatives present in the colorant E141ii or "copper chlorophyllins". **Figure 7.** Chlorophyll derivatives present in the colorant E141ii or "copper-chlorophyllins".

, in the commercial "sodium copper chlorophyllin" (SCC), by comparison with authentic standards Following this, three additional new chlorophyll derivatives (copper pheophorbide *a*, copper chlorin *e*<sup>6</sup> and copper rhodin *g*7) were identified as the main components in addition to copper chlorin *e*4, in the commercial "sodium copper chlorophyllin" (SCC), by comparison with authentic standards [34]. Authors determined not only absorption maxima but also molar extinction coefficients. Later, in five industrial samples of copper chlorophyllins, copper isochlorin *e*<sup>4</sup> was identified as the major chlorophyll derivative in most of the colorants [35], describing a high variability between commercial preparations. Different manuscripts have dealt with the presence of this colorant in processed foods [18,24,36–39]. At any rate, a further advance was the single HPLC-MS analysis published to date [27] of the chlorophyll derivatives present in several commercial colorants. The authors identified several new chlorophyll structures, such as Cu-chlorin *p*6, although additional assignments were tentatively made. The lack of authentic standards and the fact that most of the substituents in the chlorophyll tetrapyrrole do not modify the features of the UV-Vis spectrum (chlorin/isochlorin, methyl/ethyl, ester or not, etc.) makes the correct identification of chlorophyll derivatives exclusively by their chromatographic properties difficult, or even leads to the misidentification of the chlorophyll compounds being possible.

#### **4. In Vivo and In Vitro Adsorption, Distribution, Metabolism and Excretion (ADME)**

In spite of the widespread presence of natural green colorants in foodstuffs, there is a general lack of information regarding their ADME. Although few studies have been developed either in vitro or in vivo with the aim of stablishing the ADME of E140i (chlorophylls) and E141ii ("copper chlorophyllin"), there are no data regarding the bioavailability of E140ii (chlorophyllins) and E141i (copper chlorophylls). Consequently, there are still several open questions about the in vivo ADME of natural green colorants.

#### *4.1. ADME of E140i*

At the in vitro level, the first study dealing with the digestion of green vegetables and subsequent absorption of the micellarized pigments by human intestinal Caco-2 cells was reported in 2001 [40]. The tested foods included fresh spinach puree, heat and acid-treated spinach puree, and spinach puree treated with ZnCl2. The original chlorophylls were transformed into pheophytins during digestion as a consequence of the gastric pH. However, pheophytins with zinc were relatively stable during the simulated digestion. The authors demonstrated that around 5–10% of the micellarized pheophytins were absorbed by the cells and that micellarization and uptake changed significantly between the different kinds of chlorophyll derivatives. This was the first hint at determining that micellarization and absorption are greatly influenced by the lipophilicity of the compounds. Specifically, the more lipophilic molecules showed the highest accumulation in the intestinal cells.

Subsequently, these results were confirmed by applying the same in vitro protocol to pea preparations [41], pure standards of chlorophyll derivatives isolated from natural sources [42], and seaweeds [43]. Specifically, it has been shown in vitro that pheophorbides (dephytylated chlorophylls) are preferentially absorbed over pheophytins (phytylated chlorophylls) [42,43]. At this point, and by means of comparative absorption experiments with the Caco-2 cellular model at different concentrations of pheophytins, it was hypothesized that the absorption process could correlate with a passive facilitated mechanism [42]. On the contrary, to decipher the kind of cellular transport process for pheophorbides, Viera et al. [44] described the production of micelles rich in pheophorbide *a* to reach physiological micellar concentrations (7 µm). Pre-incubations of cell monolayers with different amounts of one specific inhibitor of the Scavenger Receptor class B type I (SR-BI) transporter (BLT1), significantly inhibited the uptake of pheophorbide *a*, which strongly suggests that SR-BI is involved in the transport of pheophorbide *a*. Consequently, the protein-mediated absorption of pheophorbide explains the preferential absorption of this chlorophyll derivative.

At the in vivo level, the pioneering studies on chlorophyll assimilation were based on the quantification of chlorophyll derivatives (E140i) in the feces of animals and by applying a balance matter (ingested against excreted) approach. Therefore, it has been historically assumed that chlorophylls were not absorbed by the organism, since almost all ingested chlorophylls were excreted. In this line, Brugsch and Sheard [45] estimated the quantitative decomposition of chlorophylls in the human body. Researchers supplied encapsulated crystalline chlorophyll to humans (100 mg per day for 4 days), showing that the highest percentage of degraded chlorophylls matched with fecal pheophytin [46]. Additional experiments [47] found scarce evidence of the subsequent hydrolysis of pheophytin to the water-soluble pheophorbide. The release of phytol by the colonic microflora in humans fed with pheophytin or spinach labelled with <sup>14</sup>C led to the conclusion that the main final digestive products of the ingested chlorophylls were the pheophytins. The significance of this finding lies in the fact that chlorophylls are the single source of phytanic acid, which accumulates in the Refsum disease [47].

After a great period of time, the in vivo absorption of chlorophyll derivatives was investigated in dogs fed with a diet containing 73 mg of chlorophyll (spinach)/kg of diet for 10 days [48], obtaining an apparent absorption of 3.4% of chlorophylls (determination of ingested against excreted). A second experiment was also carried out increasing the dose of chlorophyll; that is, the dogs consumed a diet containing 10% of dried spinach for 10 days. Under these conditions, no chlorophyll derivatives could be detected in the peripheral blood until 150 min after consumption, which suggests to the authors that chlorophylls were hardly absorbed and/or readily metabolized and excreted through the bile. In addition, chlorophylls *a* and *b* were transformed into their corresponding pheophytin derivatives in the gastrointestinal tract, and the authors concluded that beyond pheophytins, no other degradation products were produced.

However, opposite to the classical hypothesis, the in vivo ADME experiments of E140i developed with 30 mice fed with a diet supplemented with spirulina for four weeks [42] showed for the first time the existence of a first-pass chlorophyll metabolism in animals. The analyzed livers accumulated a diverse profile of chlorophyll derivatives, which were identified by HPLC-MS<sup>2</sup> . The study highlighted that chlorophyll derivatives that retain the phytyl chain in their structure (apolar derivatives) are available for absorption from a dietary source and accumulate in the liver. Nevertheless, the explicit enrichment of the liver with pheophorbide *a* is particularly significant. Two possible mechanisms are proposed: that phytylated chlorophylls can be further metabolized in the liver to pheophorbide, or the existence of an intestinal transporter for this metabolite. If the pheophytin is de-esterified in the liver to yield pheophorbide, the authors reveal the enigmatic origin of phytol in the liver [49]. Indeed, as was mentioned above, the authors also presented data on the implication of the intestinal brush border transporter SR-BI in the absorption of pheophorbide. Therefore, independent of the exact mechanism, the present chlorophylls in the colorant E140i are at least absorbed, metabolized, accumulated and excreted in mammals.

#### *4.2. ADME of E140ii and E141i*

There are no scientific data regarding the in vitro or in vivo absorption, distribution, metabolism, excretion and toxicity of chlorophyllins (E140ii) or copper chlorophylls (E141i). This lack of relevant data was also revealed through different Scientific Opinions [50–52] by the European Food Safety Agency (EFSA). According to the conclusions of the panel of experts of the EFSA and in the sight of the great interest of business operators, the European Commission launched on October 2017 a call for scientific and technical data requested by EFSA to complete the risk assessment. The collected data within a three–four-year timeframe will be assessed by the EFSA, and later the European Commission will take the final decision on the status of the revised colorants [53].

#### *4.3. ADME of E141ii*

Following an experimental design similar to the analysis of the ADME of E140i, the Schwartz group [54] showed that part of the chlorophylls present in the E141ii colorant was absorbed through the human intestine. In detail, four chlorophyll derivatives—copper rhodin *g*7, copper chlorin *e*6, copper chlorin *e*<sup>4</sup> and copper pheophorbide *a*—from a commercial-grade sodium copper chlorophyllin were assayed. The copper chlorin *e*<sup>4</sup> (the main component of E141ii) was highly stable to the simulated in vitro digestion process while most of the copper chlorin *e*<sup>6</sup> was lost. However, the integration of the colorant in applesauce significantly improved the recovery of chlorin *e*6, demonstrating the protective role of the food matrix. Moreover, copper chlorophyll derivatives were efficiently absorbed by the intestinal epithelium cells, probably through an active transport. Part of these chlorophyll compounds were even detectable in the basolateral compartment, which means that they are ready to be transported to peripheral tissues.

The first in vivo studies of E141ii were carried out by Henderson and Long in 1941 [55], who orally administered natural chlorophyll and SCC to rats, discovering the existence of uncharacterized derivatives dispersed throughout the liver, lymph nodes and the spleen. Reber and Willigan (in 1954) [56] obtained with their studies the first in vivo indications about the absorption of E141ii. Rats fed with 1% of copper chlorophyllins in their diet for 15 weeks showed, after euthanasia, a greenish tone throughout the skeletal muscle of the body, indicating a systemic distribution of the chlorophyll derivatives. In the same year [57], a study with different doses of copper chlorophyllin in the diet of rats resulted in the transport through the gastrointestinal membrane and accumulation in plasma of copper chlorophyllins in the µg range. The authors did not detect any copper derivative in the organs, and they assumed that copper chlorophyllins are excreted in the feces. Later, the chemopreventive activity against tumorigenesis of copper chlorophyllin was tested in female mice [58]. In this study, the sodium salt of copper chlorophyllin administered orally was rapidly distributed to the heart, liver, skin, kidneys, and lungs. A subsequent study estimated the accumulation of dietary E141ii (10 or 30 mg/kg) in different organs of 30 Wistar rats [59]. The results showed that while copper chlorin *e*<sup>4</sup> was effectively absorbed and accumulated in serum, liver and kidneys, copper chlorin *e*<sup>6</sup> was not detected by HPLC analysis in sera or tissues, according to the data published by Ferruzzi et al. [54].

Indirect evidence in humans suggests that any type of absorption could take place following a copper chlorophyllin diet. For example, urine discoloration has been described with incontinent patients subjected to an oral copper chlorophyllin intake (100–200 mg/day) [60,61]. However, the single evidence of the in vivo absorption and accumulation of E141ii in humans was provided by the studies of Egner et al. [62]. Thus, 182 volunteers ingested 100 mg of copper chlorophyllins for 16 weeks and three times per day (copper chlorin *e*4, copper chlorin *e*<sup>6</sup> and copper chlorin *e*<sup>4</sup> ethyl ester). The study described for the first time the presence of copper chlorin *e*<sup>4</sup> ethyl ester as well as copper chlorin *e*4, but not copper chlorin *e*6, in the sera of all the participants. Therefore, certain components are able to be absorbed through the gastrointestinal membrane. Probably, the instability of copper chlorin *e*<sup>6</sup> to digestion could be responsible for the lack of appearance of this compound in human serum [63].

#### **5. "Coloring Foodstuff"**

The global trend of the replacement of synthetic colors with natural ones has created a new category in the market known as "coloring foodstuffs". This class includes food ingredients such as fruits or vegetables whose secondary effect is coloring. This new conception is bringing regulatory problems as these substances are not covered by the current regulation on food additives in the EU. As the situation is rather unclear and there is an ongoing debate on the distinction between color additives and coloring foodstuffs [16], the European Commission (Standing Committee on the Food Chain and Animal Health) endorsed the Guidance notes providing a tool for classification for when a substance should be considered a color additive or not [64]. The distinction in this guidance is based on the extraction methodology: if the method is a non-selective extraction procedure, then the obtained product is a food ingredient, not a food additive. On the contrary, if the extraction is selective for obtaining a pigment, the compound is considered to be a color additive and consequently covered by the regulation on food additives. Previous to this guidance, nettles and spinach were the preferred coloring foodstuff to provide green hues to foods. However, now these color solutions no longer satisfy the criteria of EU guidelines. Otherwise, blends with spirulina are the alternative to create brilliant shades of green for confectionary products and ice creams; for example, with safflower. Specifically, in the USA, spirulina derived from *Arthrospira platensis* has been recently added to the list of approved color additives exempt from certification in response to a Mars Inc. petition [16]. In relation to spirulina extract, the Joint FAO/WHO Expert Committee has requested, in the meeting held in June 2018, information on the products on the market by December 2019 in order to remove the tentative designation from the specifications. Specifically, it is necessary to provide data on the full compositional characterization of commercial products and regarding the validated analytical methods applied for the identification of the substance and for the determination of the purity of the substance.

In any case, there is a new growing sector in the market, with several different extracts sold by different suppliers. Table 4 list the coloring foodstuffs authorized in the EU and USA, describing the maximum amounts and the foods allowed. As they are considered "natural", to date, no amount limitations have been defined, and in the EU, as they are classified as ingredients, there are no restrictions for their use in any food category.

As a consequence of the natural trend in the coloring food market, recent research has developed looking for new strategies to develop new "natural" green colorants. For example, the utilization of an extract from the leaves of *Centella asiatica* L. after steaming and metal complexations has been proposed [15]. The authors assessed the stability and cytotoxicity in different beverages and food models as an alternative to synthetic colorants. Following the same aim, the properties of spray-dried microalgae have been analyzed (color, sensory and textural qualities) as a natural coloring agent in chewing gum [65]. Although positive results have been obtained with *Isochrysis galbana* and *Nannochloropsis oculata*, the spray-dryer technique requires an optimization in the base of the characteristics of each microalgae specie.


**Table 4.** Classification of "coloring foodstuff" in EU and USA.

<sup>1</sup> GMP: good manufacturing practices.

#### **6. Stabilization of Green Color**

Chlorophylls are stable pigments in their natural environment in physiological conditions. However, once extracted or processed with changes in pH value and temperature during the processing and storage of green foods, chlorophylls are prone to experience modifications in their structure, which consequently change their chromatic properties. Probably, the main reaction that affects chlorophylls is the substitution of the central magnesium ion by two hydrogens. The significance of the reaction for the food industry is related with the drastic change in color, because magnesium-derivatives are green, while magnesium-free derivatives (mainly pheophytins and pheorphorbides) are brown. Consequently, this easy and fast reaction is the principal cause of the loss of the original green color during the processing and storage of green foods. As has been stated before, the reduction or even withdrawal of the original green color is associated by the consumer with a decrease in the quality of the product. Therefore, the food industry has developed several strategies to preserve the initial green coloration. The early attempts consisted of the addition (even in-the-can coating) of alkalinizing agents, but the appearance of negative secondary effects such as a softening of the edible material or an off-flavor release [6] made them to fall into disuse.

A second historical approach has been the substitution of the central hydrogen atoms by zinc or copper ions to form the more stable green metallochlorophylls which consequently "re-green" the corresponding food product. The conditions for the production of zinc and copper complexes of chlorophyll derivatives in vegetables during processing have been optimized through the years [66–68]. Due to the industrial and commercial importance of this process, numerous patents have been published, with the most known being the so-called "Veri-green" method [69]. This patent was developed by the former Continental Can Company and consisted of the blanching of vegetables in brine solutions with some amounts of Zn+2 or Cu+2 salts to form mainly zinc or copper pheophytins [70] and make the processed vegetables greener. However, the patent was unproductive because the maximum limit for zinc concentrations established by the FDA is 75 ppm, and higher amounts of zinc are required to yield an acceptable and desirable green color. To overcome this limitation, new strategies for the encapsulation of zinc-chlorophylls are currently under development using different matrices such as gum Arabic, maltodextrin and OSA modified starch [71] or whey proteins [72]. Indeed, different techniques are being set up to microencapsulate raw chlorophyll extractions from edible vegetables without any salt treatment. For example, spray drying is a dehydration process which has been successfully applied to encapsulate other food compounds, and recently, the physicochemical properties of the process have been optimized to form whey protein isolate–kale leaf chlorophyll microcapsules [73]. A further step in the use of this procedure as an available alternative for the food industry is the enhancement of the stability of green pigments for heated foods. For example, the stability of alfalfa leaves microencapsulated towards temperature regimes and pH conditions has been determined [74], obtaining optimal conditions.

#### **7. Conclusions**

The market of the food colorants is evolving to more natural formulations, which means the appearance of new definitions and consequently, the necessity of new legislations. Green food colorants are one of the most affected. First, because natural green colorants are very labile and secondly, because it is not easy to reproduce green hues naturally. Consequently, in the near future the consumer will face new compounds responsible of the green color in foods. At present, independently of the existence of serious problems due to different legislations and different definitions between countries, authorized green natural colorants are incompletely characterized. This review has summarized the present knowledge of the compounds comprised the authorized green colorants, highlighting the common errors in their daily management. Finally, the scarce research developed on the ADME of green authorized food colorants is also considered but with the confidence that new advances will be shortly achieved.

**Author Contributions:** I.V., A.P.-G. and M.R. have contributed equally to the conceptualization, investigation, writing, review, and editing of this manuscript.

**Funding:** This research was funded by MINECO-CICYT, grant numbers ALI2018-R. The APC was partially funded by CSIC.

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

#### **References**


© 2019 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 Urtica* **spp.: Ordinary Plants with Extraordinary Properties**

#### **Dorota Kregiel ID , Ewelina Pawlikowska and Hubert Antolak \***

Institute of Fermentation Technology and Microbiology, Faculty of Biotechnology and Food Science, Lodz University of Technology, 171/173 Wolczanska, 90-924 Lodz, Poland; dorota.kregiel@p.lodz.pl (D.K.); ewelina.pawlikowska@edu.p.lodz.pl (E.P.)

**\*** Correspondence: hubert.antolak@gmail.com; Tel.: +48-42-631-3475

Received: 5 June 2018; Accepted: 6 July 2018; Published: 9 July 2018

**Abstract:** Nettles (genus *Urtica*, family Urticaceae) are of considerable interest as preservatives in foods for both human and animal consumption. They have also been used for centuries in traditional medicine. This paper reviews the properties of nettles that make them suitable for wider applications in the food and pharmaceutical industries. Nettles contain a significant number of biologically-active compounds. For example, the leaves are rich sources of terpenoids, carotenoids and fatty acids, as well as of various essential amino acids, chlorophyll, vitamins, tannins, carbohydrates, sterols, polysaccharides, isolectins and minerals. Extracts from the aerial parts of nettles are rich sources of polyphenols, while the roots contain oleanol acid, sterols and steryl glycosides. Due to the variety of phytochemicals and their proportions they contain, nettles show noticeable activity against both Gram-positive and Gram-negative bacteria. These properties make nettles suitable for a range of possible applications, including functional food, dietary supplements and pharmacological formulations. Despite these benefits, the nettle is still an underestimated plant source. This paper provides a unique overview of the latest research on nettle plants focusing on the possibilities for transforming a common weed into a commercial plant with a wide range of applications. Special attention is paid to the antimicrobial activity of the active compounds in nettles and to possible uses of these valuable plants in food and feed formulations.

**Keywords:** *Urtica* spp.; bioactive compounds; antioxidant activity; antimicrobial activity; traditional medicine; food industry; animal breeding

#### **1. Habitats of** *Urtica* **spp. Plants**

The genus *Urtica* belongs to the family Urticaceae in the major group Angiosperms (flowering plants). There are 46 species of flowering plant of the genus *Urtica* [1] (Table 1). The most prominent members of the genus are the stinging nettle *Urtica dioica* L. and the small nettle *U. urens* L., which are native to Europe, Africa, Asia and North America. Plants belonging to the genus *Urtica* are herbaceous perennials and can grow up to 2 m tall. Serrated leaves are attached in pairs opposite each other to the stem. The soft leaves and the rest of the plant are coated in hairs, some of which sting. The serrated, hairy leaves and sting are generally recognized characteristics of this plant [2]. However, the European variety *U. galeopsifolia* does not have stinging hairs [3]. The underground roots by which the plant spreads are noticeably yellow. Small flowers, each with four greenish-white petals, sit in dense clusters on elongated inflorescences towards the top of the stem.

The word "nettle" is said to derive from the Anglo-Saxon word "noedl" meaning "needle", while its Latin name "urtica" means "to burn". This refers to the stinging effects of the tiny hairs on the stems and leaves, which when rubbed against the skin cause a burning sensation and temporary rash. The hairs, so small they are almost invisible to the naked eye, are referred to as "trichomes". When touched by the skin, the bulbous tips of the trichomes break off, leaving sharp, needle-like tubes [4]. These can pierce the skin and inject a fluid-containing substances including formic acid, the same chemical in ant and bee stings. Other compounds contained in the fluid include histamine, acetylcholine and serotonin. When these toxins are delivered into the skin, a painful itching and burning sensation occurs that may last up to 12 h. The hairs are naturally designed to protect the plant from insects.

Nettles grow all over the world in mild to temperate climates. They prefer open or partly shady habitats with plenty of moisture and are often found in forests, by rivers or streams and on roadsides. *Urtica* spp. are widespread throughout Europe and North America, North Africa and in parts of Asia. Both species of stinging nettle (*U. dioica* L. and *U. urens* L.) prefer to grow in nitrogen-rich soil and are commonly found in soils high in inorganic nitrates and heavy metals. Heavy metals are poorly processed by the plant and tend to accumulate in the leaves. Long vegetation seasons lead to on-going growth, while harsh winters cause destruction of the plants [5].

Nettles are considered weeds due to their rapid growth and soil coverage. However, there are economic and ecological reasons for cultivating stinging nettles. According to Dreyer and Müssing, nettles can improve soils over-fertilized with nitrogen and phosphate [5]. They can also promote the biodiversity of local flora and fauna [6,7]. Over 40 species of insect are supported by nettles [8]. *U. dioica* can reduce heavy metal content in soil [9]. *Urtica* spp. can be used to produce new high-quality agricultural raw materials for the dyeing, textile and energy sectors [7]. Due to their content of tough fibers, nettles were used in Germany and Austria to make textiles during the First World War [10]. Despite these benefits, most stinging nettles are wild harvested. Primary producers of stinging nettles include Eastern Germany, the former USSR, Bulgaria, the former Yugoslavia, Hungary and Albania [5].



*Molecules* **2018**,

 *23*, 1664




#### **2. Phytochemical Composition of** *Urtica* **spp.**

Different factors affect the chemical composition of nettle plants, such as the variety, genotype, climate, soil, vegetative stage, harvest time, storage, processing and treatment [11–13]. Stinging nettles are a rich source of nutrients. A comprehensive proximate analysis showed that harvested upgrowths contained approximately 90% moisture, up to 3.7% proteins, 0.6% fat, 2.1% ash, 6.4% dietary fiber and 7.1% carbohydrates [10]. On the other hand, nettle leaf powders contain on average 30% proteins, 4% fats, 40% non-nitrogen compounds, 10% fiber and 15% ash (Table 2).


**Table 2.** Chemical composition of nettle leaf powders [10].

In a study of nettles by Rafajlovska et al., higher quantities of proteins were found in the leaves than in the stems and roots. The content of proteins in the leaves ranged from 16.08 ± 0.38–26.89 ± 0.39%, depending on the source of the sample. The highest protein contents in the stem and roots were 14.54 ± 0.27% and 10.89 ± 0.11%, respectively [14]. Other studies of nettle composition have found that the plants contain a significant number of biologically-active compounds. The nettle leaves contain terpenoids [15], carotenoids [16] including β-carotene, neoxanthin, violaxanthin, lutein and lycopene [17,18], fatty acids, especially palmitic, *cis*-9,12-linoleic and α-linolenic acids [17,19], different polyphenolic compounds [20–23], essential amino acids, chlorophyll, vitamins, tannins, carbohydrates, sterols, polysaccharides, isolectins [16,17,24] and minerals [25,26], the most important of which is iron.

The leaves of *Urtica* spp. contain around 4.8 mg/g DM of chlorophyll, depending on the climate and environmental conditions. Interestingly, more chlorophyll and carotenoids are usually found in plants that have been harvested from shady places. Kukri´c and co-workers noted that there were differences in the content of chlorophyll and carotenoids in leaves of different ages [16]. The concentration of chlorophyll increases in growing leaves and decreases during plant aging. The fresh leaves contain high concentrations of vitamins A, C, D, E, F, K and P, as well as of vitamin B-complexes [23]. The leaves are also known to contain particularly large amounts of the metals selenium, zinc, iron and magnesium. Rafajlovska et al. noted that stinging nettle leaves, stems and roots contained larger amounts of calcium than magnesium. These two elements were present at quantities almost three-times higher in the leaves than in the stems and roots. The calcium content expressed in relation to the dry mass ranged from 2.63–5.09% in leaves, from 0.76–1.42% in stems and from 0.61–0.92% in roots. Zinc was found in the highest concentrations in the leaves (27.44 mg/kg of dry mass), followed by copper (17.47 mg/kg) and manganese (17.17 mg/kg). The mean values for cobalt content were significantly higher in leaves than in stems and roots. The contents of cobalt in the leaves, stems and roots, respectively, were in the ranges of 0.11–0.21 mg/kg, 0.10–0.18 mg/kg, and 0.08–0.16 mg/kg, relative to the corresponding dry mass. *Urtica* leaves in addition contain boron, sodium, iodine, chromium, copper and sulfur [14].

The total phenolic content of one gram of nettle powder has been reported as 129 mg GAE (Gaelic Acid Equivalent), which is two-times higher than the phenolic content in 100 mL of cranberry juice (66.61 mg GAE) [27]. Stinging nettles have been shown to be richer in individual polyphenols than other wild plants [13]. Ghaima and co-workers found that the content of phenolic compounds in stinging nettle leaves was significantly higher than in dandelion leaves [28]. Vaji´c et al. reported that the predominant phenolic compound in stinging nettle leaves is rutin [29]. Ðurovi´c and co-workers studied the chemical composition of stinging nettle leaves using different analytical approaches. Soxhlet extraction was performed and qualitative analysis of Ultrasound-Assisted (UA) extracts using the UHPLC-DAD technique with MS/MS. Differences in the chemical profiles were found. For example, after Soxhlet extraction, syringic, cinnamic and protocatechuic acids were detected in the products, which was not the case with the UA extract. On the other hand, ferulic, caffeic, chlorogenic and sinapic acids were detected only after ultrasound-assisted extraction [30].

Orˇci´c and co-workers quantified various plant phenolics in methanol extracts of *U. dioica*, for flowers, roots, stems and leaves separately, harvested from different locations in Serbia [20]. The polyphenol profiles were dependent not only on the parts of the plants, but also on the location of their acquisition. The researchers found that inflorescence extracts were the richest in phenolics. The most abundant compound was 5-*O*-caffeoylquinic acid (chlorogenic acid), followed by quercetin 3-*O*-rhamnosylglucoside (rutin) and 3-*O*-glucoside (isoquercetin).

The quantitative and qualitative composition of the roots differs from that of the aerial parts of the plants [22]. The content of the majority of the phenols in the root extracts is significantly lower, and the only prominent compound is secoisolariciresinol. Therefore, the roots of stinging nettles are considered to be the poorest part in terms of bioactive compounds. The root extracts contain only a few acids and derivatives in significant amounts (Table 3) [20]. They contain starch, gum, albumen, sugars and resins, as well as neurotransmitters and receptors, such as histamine, acetylcholine, choline or serotonin. Methanolic extracts of nettle roots have an inhibitory effect on aromatase, a key enzyme in the biosynthesis of estrogens. The lipophilic fractions usually contain phytosterols, pentacyclic triterpenoids, coumarins, ceramides and hydroxyl fatty acids. In turn, isolectins and some polysaccharides have been isolated from the hydrophilic fractions [31]. Krauss and Spitteler identified eighteen phenolic compounds (including homovanillyl alcohol, vanillin, vanillic acid and phenylpropanes) and nineteen ligands (including isolaric, iresinol, secoisolariciresinol and neoolivil) in root extracts [32]. Scopoletin, a coumarin derivative, has also been identified in nettle roots [24]. All these compounds are considered to be very important in medicine and pharmacology. For example, homovanillyl alcohol has been shown to protect against cardiovascular disease [33], while histamine influences the complex physiology of brain systems, affecting cognitive processes, including learning and memory [34], as well as neurotransmitters involved in neuromodulation processes [35]. Phytosterols reduce the absorption of cholesterol in the gut and thereby lower blood cholesterol levels [36]. Scopoletin is a stimulator of lipoprotein lipase activity and protects against cardiovascular diseases [37]. Lignans improve immune responses [38].

*Molecules***2018**, *23*, 1664


**Table 3.** The phenolic profiles in*U. dioica*extracts (mg per g of dry extract) [20].

\* det: peak observed, but the concentration was too low to evaluate it; \*\* not det: peak not observed. Bolded compounds are those that occur at the highest concentration.

Fresh nettle leaves contain smaller amounts of sterols and higher concentrations of flavonol glycosides. The leaves of the plant also contain carotenoids, mainly β-carotene, violaxanthin, xanthophylls, zeaxanthin, luteoxanthin and lutein epoxide [5]. Terpene diols, terpene diol glucosides, α-tocopherol, as well as five monoterpenoid components have also been detected in nettle leaves [39]. Weglarz and Roslon studied the content of polyphenolic acids in leaves and rhizomes. They found that the level of these compounds was higher in the male forms, but the chemical profiles of polyphenolic acids from the female plants were much more diverse [40,41]. Moreover *U. dioica* is considered the only plant that contains choline acetyl-transferase, an acetylcholine-synthesizing enzyme [42]. Fifteen hydroxycinnamic acid derivatives and sixteen flavonoids, flavones and flavonol-type glycosides were identified in hydroalcoholic extracts from the aerial parts of *U. dioica*, *U. urens* and *U. membranacea* using HPLC-PDA-ESI/MS. Of these, 4-caffeoyl-5-*p*-coumaroylquinic acid and three statin-like 3-hydroxy-3-methylglutaroyl flavone derivatives were identified for the first time in *U. urens* and *U. membranacea*, respectively. *U. membranacea* showed a higher content of flavonoids, mainly luteolin and apigenin glycosides, which are almost absent in the other species studied [43].

The hairs of *Urtica* plants contain an acrid fluid with the active components: acetylcholine, histamine and formic acid, as well as silica, serotonin and 5-hydroxy tryptamine. Many of these chemicals are smooth muscle stimulants [44]. The fresh hairs of *U*. *dioica* also contain a high level of acetylcholine [45]. The results of numerous experiments suggest that each species of nettle, a well as each part of the plant (root, stalk or leaves) have a different content and profile of bioactive compounds. Therefore, different species of nettle may have different uses, depending on their chemical characteristics [21].

Generally, the phenolic composition of plants is affected by different factors, including the variety, genotype, climate, soil, vegetative stage of the plant, harvest time, storage, processing and treatment [46,47]. When and how nettles are harvested strongly determines the final product. For example, for fiber production, stinging nettles should be harvested when the seeds are mature or when the stalks reach 80% of the aboveground biomass, from the second year of planting. During the first year, the stalks are too thin, too ramified and have too many leaves. If the main product is to be the leaves, younger plants are harvested. The time of year for nettle harvesting depends on the purpose. Plants collected in April are used for fodder, medicine or chlorophyll production. Nettles harvested at the end of June are used for fiber production. The second harvest in September may be used for the collection of leaves [7].

#### **3. Antimicrobial Activities of** *Urtica* **spp.**

Nettles possess noticeable antimicrobial activity against Gram-positive and Gram-negative bacteria when compared with standard and strong antimicrobial compounds, such as miconazole nitrate, amoxicillin-clavulanic acid, ofloxacin and netilmicin [48]. Different fractions of various *Urtica* species have been studied to determine their antimicrobial activity. The results indicate the great potential of this plant for the discovery of novel effective compounds (Tables 4 and 5) [49–56].


**Table 4.** In vitro activity of *Urtica* spp. against microorganisms.

#### **Table 4.** *Cont.*



#### **Table 4.** *Cont.*


**Table 5.** In vitro activity of *Urtica* spp. against microorganisms: minimal inhibitory concentrations.

The results presented in Table 5 show the antimicrobial activities of various nettle extracts obtained by different researchers. As can be seen, some nettle extracts show activity at a concentration of 72 mg/mL and others at 1 µg/mL. These differences appear excessive, and the results should therefore be viewed with caution. Such variations may be associated with the location of the plant habitat and climactic conditions, as well as being due to the use of different extraction techniques and evaluation methods. Despite their significant differences, however, the results of these studies show that nettle plants exhibit antimicrobial activity against a wide spectrum of microbial strains, often isolated from foods of low microbiological quality. A study by Kukri´c et al. revealed that nettle extracts had inhibitory effects on various Gram-positive and Gram-negative bacteria including *Bacillus subtilis*, *Lactobacillus plantarum*, *Pseudomonas aeruginosa* and *Escherichia coli* [16]. Mahmoudi et al. reported that all microorganisms tested in their research, Gram-negative and Gram-positive bacteria, as well as *Candida albicans* yeast, were sensitive to alcoholic extract from the nettle stem [62]. In recent studies conducted by Antolak et al., the ethanol extract of *U. dioica* showed inhibitory activity against the growth of acetic acid bacteria belonging to the genus *Asaia*, a beverage spoilage bacteria found in functional drinks [63]. On the other hand, Shale et al. noted that *E. coli* and *P. aeruginosa* were completely

resistant to the ethanol and methanol extracts from stems and leaves of *U. dioica* [64]. Different antimicrobial properties may be the result of the isolation of different compounds in different solvents, of different extraction efficiencies and possibly of chemical degradation by polar and non-polar solvents. The extraction method, the plant type, the geographical and ecological status, the climate, seasonal and experimental conditions, the age of the plant, environmental stress factors, as well as inter-species differences all play a role and may explain the diversity of results in different studies [21,65,66].

#### **4.** *Urtica* **spp. in Traditional and Modern Medicine**

Nettles are one of the most commonly-used medicinal plants in the world, due to their health-enhancing qualities. Because of their high content of nutritive substances, nettles are also used in folk veterinary medicine [48,67,68]. There are many dietary supplements based on *Urtica* spp. now on the market. Their popularity can be explained by their non-toxic chemical composition, relatively low cost and wide availability. The most recognized health benefit of using stinging nettles is activity against Benign Prostatic Hyperplasia (BPH), also known as an enlarged prostate, as well as urinary tract infections. Clinical studies suggest that *Urtica* spp. contain compounds that affect the hormones responsible for BPH. In addition, nettle root extract shows activity against prostate cancer cells. In therapy, nettles are usually used in combination with saw palmetto (*Serenoa repens*) [69,70]. They are also used as a home remedy for bladder infections.

Nettles can help alleviate the symptoms of osteoarthritis and joint pain, typically in the case of hands, knees, hips and spine. Nettles can work in combination with nonsteroidal anti-inflammatory drugs (NSAIDs), allowing patients to decrease their use of NSAIDs. The prolonged use of NSAIDs can increase the risk of heart attack or stroke. In a study by Randall and co-workers, nettles were able to decrease osteoarthritic pain in the base of the thumb when applied to the painful area. In a clinical trial of 37 people with acute arthritis, 50 g of stewed nettle leaves consumed daily, combined with 50 mg of diclofenac, were shown to be as effective as the full 200-mg dose of diclofenac over a two-week period [71]. Studies have also shown that applying nettle leaves directly decreases joint pain and can treat arthritis. In a study by Christensen and Bliddal, it was found that a combination of nettles, fish oil and vitamin E reduced the need for analgesics and other drugs for the symptoms of osteoarthritis [72].

Another study conducted by Klingelhoefer et al. showed the anti-inflammatory benefits of stinging nettles against other autoimmune diseases, such as rheumatoid arthritis [73]. Nettle leaves contain histamine, which may seem inadvisable for allergy medication. However, histamine has been already used to treat strong allergy symptoms [74]. Histamine production causes unwanted allergic reactions, associated with unpleasant nasal congestion, sneezing or itching. Stinging nettles affect numerous receptors and/or enzymes involved in allergic reactions [75]. In addition, because of their anti-histamine and anti-inflammatory properties, stinging nettles can be used as a natural component in eczema medications. Infusions of the plant can be used for nasal and menstrual hemorrhage, diabetes, anemia, asthma, hair loss and to promote lactation [76]. Terpenes and phenols are major groups associated with the inhibition of cancers, as well as with the treatment of headache, rheumatism and some skin diseases [58,77,78]. Phenols also have been associated with the inhibition of atherosclerosis and cancer, as well as age-related degenerative brain disorders [79,80].

The combination of *U. dioica* with common thyme (*Thymus vulgaris*), liquorice (*Glycyrrhiza glabra*), common grape (*Vitis vinifera*) and lesser galangal (*Alpinia officinarum*) has been known in Turkey as an Ankaferd Blood Stopper (ABS). This traditional medicine works on endothelium, blood cells, angiogenesis, cellular proliferation, vascular dynamics and cell mediators to stop bleeding [81]. In a study conducted by Bourgeois et al., nettles were used for cosmetic applications as an anti-aging complex, involving the inhibition of collagenase and elastase activities. These properties could be ascribed to the ursolic acid and quercetin present in the nettle extracts [82].

Herb extract of *Urtica* plants is useful for bladder disorders, reduces postoperative blood loss and prevents hemorrhagic and purulent inflammation following adenomectomy. Aqueous infusions of *U. dioica* exhibit antioxidant activity towards iron-promoted oxidation of phospholipids, linoleic acid and deoxyribose [83]. For a long time, the hypoglycemic effects of *U. dioica* were only speculative. Recent studies show that nettles possess anti-diabetic properties [84]. Thus, nettles could serve as good adjuvant to other oral hypoglycemic agents and seem promising for the development of phytomedicines for diabetes mellitus. In addition, as organic nitrogenous compounds, amino acids from nettles are building blocks in the process of protein biosynthesis [85]. The safety of aqueous extracts of *U. dioica* and their antidiabetic effects have been confirmed with mice models [86].

#### **5. Food and Feed Applications**

Nettles have traditionally been used as a nutritious food, particularly in spring time in rural areas. The Romans are said to have consumed nettles, and a recipe for Saint Columba's broth survives to this day. In Greek and Roman times, nettle roots were used for meat tenderization. Nowadays, nettles are used in a large number of recipes. *Urtica* spp. are blended with fromage blanc, potatoes and nutmeg to make nettle nouvelle [39]. Stinging nettles remain popular, especially in poor countries and among the lower socioeconomic classes [87]. For example, amino acids from dehydrated nettle meal are nutritionally better in comparison to those of alfalfa meal [88]. These plants may be consumed primarily as a boiled or cooked fresh vegetable, which is added to soups, cooked as a pot herb or used in vegetable salads [10]. In the temperate region of the Himalayas, *U. plaviflora* leaves are cooked and eaten as a green vegetable. Upgrowths and leaves are collected with the help of bamboo or iron pincers and cooked as soup. The plants are boiled with maize, millet or wheat flour, with the addition of salt and chili to make a kind of porridge. Due to their seasonality, *U. plaviflora* plants are preserved by lactic acid fermentation [4].

In European countries, nettles are used in soup or as a steamed or wilted vegetable. Since it has a similar flavor and texture, cooked nettle can be used as a substitute for spinach. Raw nettles after blending can be also used in pesto sauces, salad dressings or dips. Boiled nettles with walnuts is a common dish in Georgia, while Romanians make sour soup using fermented wheat bran, vegetables and young nettle leaves [89]. Mature leaves are used in the production of semi-hard Cornish cheese, made from grass-rich milk and wrapped in stinging nettles. The nettle changes the acidity of the outside of the cheese, affecting the way the curd breaks down and matures. It has also been documented that nettle leaves can be used to coagulate milk in the process of fresh cheese making [90]. In some European countries, for example in Serbia and Poland, bread with nettle leaves (up to 1%) is sold as a commercial product [30]. Compared to barley and wheat flour, nettle flour has a much higher content of proteins, crude fibers, fats, ash, calcium and iron and has a low glycemic index. As compared to barley and wheat, nettles have much higher levels of tannins and total polyphenols [10].

Nettle leaves can also be used to make an herbal tea, which is rich in vitamins and minerals. Depending on the amount used, nettle tea has a mild to strong flavor and tastes similar to vegetable broth. Concentrated nettle tea can be used as a soup base or as a component in drinks or green cocktails. Nettle tea can also be used as a nutritional replacement for water. Nettle roots can be used as liquid or powdered extracts, as well as in special decoctions. Nettles are also used in herbal liquors [91]. In the British Isles, *Urtica* plants are used in an alcoholic beverage, which is similar to ginger beer and brewed in the same way. Nettle and oat extracts are the subject of a U.S. Patent describing the use of plant powders as additives in beverages or fruit juice to provide nutritional drinks [92]. Aqueous infusions of *U. dioica* exhibit antioxidant activity towards iron-promoted oxidation of phospholipids, linoleic acid and deoxyribose [83]. The use of such antioxidant and antimicrobial compounds is of considerable interest for the preservation of foods, as well as for improving the shelf-life of food products [53,93].

Despite their beneficial properties, the consumption of nettle teas or juices may cause a skin rash in individual cases. Although it is rare, there have been reports of allergic reactions after ingesting raw nettle leaves in the form of puree or juice [94]. Therefore, stinging nettles need to be correctly prepared by hot water infusion, maceration, drying or tincturing. This pretreatment deactivates the formic acid, allowing safe consumption of this valuable plant.

Oxidation is a serious problem in the food industry. Meat products are particularly susceptible to changes in the oxidation of lipids and heme pigments during storage. Along with meat spoilage bacteria, the oxidation of lipids and myoglobin has a major effect on reducing the shelf-life of meat and meat products. Lipid oxidation decreases the nutritional value of meat through the deterioration of essential fatty acids, causing an unacceptable flavor, generating potentially toxic products and promoting the oxidation of other important molecules, such as myoglobin [95]. Although synthetic antioxidants are available, in recent years, the demand for natural antioxidants has increased, mainly because of the adverse effects of synthetic antioxidants [96]. Iron, the most abundant ion in meat, is released from heme pigments and ferritin and may be an important catalyst in the oxidation of lipids and proteins. Studies have demonstrated that commercially-available polyphenols and extracts rich in polyphenols can inhibit myoglobin oxidation.

Inai et al. studied the inhibition of myoglobin oxidation by some plant polyphenols with activity for flavonols (kaempferol, myricetin and quercetin) [97]. Slightly weaker activity was observed for other polyphenols: sinapic acid, catechin, nordihydroguaiaretic acid, taxifolin, morin and ferulic acid. The use of natural antioxidants from *U. dioica* water extract and dried leaves as a functional ingredient significantly decreased the level of lipid deterioration and increased color stability during storage. Therefore, nettle water extract can be successfully used to reduce lipid oxidation and to enhance the functionality of the final products. Other studies have investigated the use of *U. dioica* water extract in sucuk, a Turkish dry-fermented sausage [98–100], in ground beef [101], meatballs [102], in super-chilled minced meat [103], vacuum-packed beef steaks [104] and in cooked pork sausage [105].

The use of *Urtica* spp. as a dietary supplement may positively affect the health and productivity of poultry and cows. The large number of active compounds in this plant may show stronger antibacterial activities than synthetic antimicrobials. It has been suggested that extracts of *Urtica* spp. may also possess appetite- and digestion-stimulating properties [106–110], which stimulate the growth of beneficial bacteria in the gastrointestinal tract of poultry [111]. In some parts of Europe, fresh leaves are traditionally fed to pigs and poultry. Safamehr and co-workers showed that nettles had no effect on the percentage of breast, thigh and abdominal fat [68]. However, chickens fed with 1% nettles had the highest carcass yield. In contrast, Alçiçek, Bozkurt and Çabuk observed an improvement in the carcass yield of broilers supplemented with a combination of essential oils [109]. This can be explained by more intense protein anabolism [112]. The relative weights of most organs were not affected by the inclusion of nettles to the diet, which is in agreement with the findings by Nobakht [113].

It has been suggested that the antioxidant activity of nettles may induce a decrease in the relative weight of the liver [114]. A study conducted by Safamehr and co-workers showed that 1%–2% supplementation with nettles had a positive effect on the performance, carcass traits and blood biochemical parameters of broilers [68]. A study by Humphries and Reynolds confirmed the usefulness of nettles as a forage crop for cows. Production of milk was maintained when nettles were used to replace dry grass silage in the diet of lactating dairy cows [115]. The addition of nettle haylage to the diet caused changes in rumen pH that were potentially beneficial to lactating dairy cows on high grain diets. It is worth noting that diets based on plant material, rich in immune-promoting bioactive compounds, can avoid the need for antibiotic growth promoters. There is increasing public and government pressure in several countries of the EU and some non-EU nations to find natural alternatives to antibiotics [116,117].

¸Sandru and co-workers noted that *U. dioica* alcoholic extract increased cell-mediated innate immune potential in chickens. Alcoholic nettle plant extract significantly increased total leukocyte numbers, from 15,400–17,125 cells/mm<sup>3</sup> [118]. Similarly, nettle extract treatment significantly enhanced the in vitro functional capacity of phagocytes. This could lead to a higher resistance to diseases and improve the post-vaccination response of broilers, thus reducing economic losses. It was noted that the glucose and total protein content, as well as the heterophil and lymphocyte levels in chickens were not influenced by different quantities of nettle, while the concentrations of cholesterol and triglycerides in the blood were affected significantly. The effect on lowering cholesterol in blood serum may be because of the presence of plant sterols, such as stigmasterol and campesterol. These decrease the cholesterol concentration in micelles [119,120]. Fermont and co-workers and Visioli and co-workers report that the cholesterol levels in blood serum and meat are probably lowered by phenolic compounds [121,122]. However, Khosravi et al. found that the addition of nettle extract to the diet of boilers had no significant positive effect on their total cholesterol [123].

*Urtica* spp. provide animals with nutrients and bioactive components, which support antimicrobial activity, immune enhancement and stress reduction. However, the phytochemical composition is complex, and the mode of action is unclear [124]. Further studies are needed to investigate the bioactive components of nettles and their modes of action. It is worth noting that the World Health Organization (WHO), in its monographs on 'Selected medicinal plants', describes *Urticae* as valuable herbs for many medicinal uses [125]. The European Commission Directorate-General for Health and Food Safety showed that *Urtica* spp. fulfils the criteria of a foodstuff, as defined in Regulation (EC) No. 178/2002. This opinion is supported by the European Food Safety Authority (EFSA). It concluded that *Urtica* spp. has neither an immediate nor delayed harmful effect on human or animal health and has no negative effect on the environment [126].

#### **6. Conclusions**

Stinging nettles can be found all over the world. Plant hairs located on the leaves and stems contain a number of chemicals, which can cause a stinging reaction and uncomfortable irritation when brought into contact with human skin. Nevertheless, stinging nettles have a number of health benefits and have been used medicinally since at least the times of Ancient Greece. Studies have shown that all parts of the nettle have antioxidant, antimicrobial and pro-health capabilities. Most nettle medicines are made from the flowers, stems and leaves, but roots are also used in pharmacology. This valuable plant has been used most commonly as a diuretic and for treating painful muscles and joints, eczema, gout and anemia. Nettles may be used as a vegetable, in juice, tea and as an ingredient in many dishes. The use of *Urtica* spp. as a feed component could also positively affect the health of poultry and animal productivity. However, despite these proven benefits, the nettle is still an undervalued plant.

Research is continuing into this ordinary plant with unique pharmacological and dietary properties. It is worth investigating the possible wider inclusion of nettles in the daily diet to promote well-being and prevent diseases.

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

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

#### **References**


© 2018 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/).

### *Article* **Olive Leaf Addition Increases Olive Oil Nutraceutical Properties**

#### **Imen Tarchoune 1 , Cristina Sgherri 2, \* , Jamel Eddouzi 3 , Angela Zinnai 2,4 , Mike Frank Quartacci 2,4, \* and Mokhtar Zarrouk 1**


#### Academic Editor: Lillian Barros

Received: 17 January 2019; Accepted: 1 February 2019; Published: 2 February 2019

**Abstract:** The aim of the present research was to study the effects of olive leaf addition (0 and 3%) on the major antioxidants and the antioxidant activity of Neb Jmel and Oueslati olive oils. Olives and leaves of the two Tunisian varieties were harvested during the 2016/2017 crop season. Both leaves and oils were characterised for their concentrations in phenolics, tocopherols and antioxidant power. Other parameters such as free acidity, peroxide value, chlorophyll and carotenoid concentrations were also taken into consideration. Compared to Oueslati, the Neb Jmel oil showed a lower free acidity (50%) and peroxide value (5.6-fold), and higher chlorophyll (1.6-fold), total phenolics (1.3-fold), flavonoid (3-fold) and oleuropein derivative (1.5-fold) concentrations, in addition to an increased antioxidant activity (1.6-fold). Leaf addition promoted a significant increment in total chlorophyll, α-tocopherol and phenolics in both varieties, above all in Oueslati oil, due to a higher abundance of bioactive constituents in the corresponding leaves. In particular, chlorophyll and carotenoid concentrations reached values twice higher than in Neb Jmel leaves, and flavonoids and oleouperin derivatives were three-fold higher. This prevented the oxidation and the formation of peroxides, reducing the peroxide value of the fortified oil to the half. The results provide evidence on the performance of the Tunisian Neb Jmel and Oueslati varieties, showing that their oils present a chemical profile corresponding to the extra virgin olive oil category and that, after leaf addition, their nutritional value was improved.

**Keywords:** extra virgin olive oil; leaf addition; Tunisian varieties; phenolics; tocopherols; antioxidant activity

#### **1. Introduction**

In the Mediterranean area, the olive tree is so economically important that oliviculture is one of the most widespread agricultural activities. It is estimated that about 8 million ha are cultivated with olive trees [1,2]. In particular, in Tunisia, an area of 1.7 million ha is covered by olive trees, which results in the production of more than 4% of the global olive oil amount. Due to the 170,000 tons produced per year, Tunisia is the fourth largest producer and exporter of olive oil in the world [3].

The nutritional and health-promoting effects of olives and olive oils are more and more recognized [4]. Indeed, it is well known that olive oil is a potential antioxidant [5–7] showing anti-inflammatory [6,8,9], cardioprotective [6,10], anticancer [6,11], antidiabetic [12] and neuroprotective effects [13].

The importance of olive oil is related to its high amounts of monounsaturated fatty acids and to the presence of low-represented components such as α-tocopherol, phenolics, chlorophyll and carotenoids. Phenols are among the most important nutraceutical compounds because of their nutritional and sensorial characteristics [14]. Phenolic compounds and tocopherols play a protective role against oxidative stress [15] and are able to extend the extra virgin olive oil shelf-life due to their antioxidative properties [16]. Olive oil colour is determined by its pigment composition and concentration, especially with reference to chlorophyll. In the dark, this pigment is also endowed with antioxidant activity, even if under light conditions it can act as a prooxidant, reacting with triplet oxygen to form the excited-state singlet oxygen [17]. Oxidation is the most important process that causes quality deterioration of olive oil because of its high concentration of unsaturated fatty acids. Consequently, synthetic antioxidants have been used as food additives to improve oil stability. As many studies suggest that the use of synthetic antioxidants may lead to health risks [18], considerable attention has been recently focused on plant phenolics due to their healthful and nutritional effects [19]. Recent papers have provided evidence that olive leaves have a high antioxidant activity, originated by the presence of phenolics, thus exhibiting strong preventive effects against oxidation [18,20,21]. Olive leaves are a by-product of olive cultivation and can be easily obtained either from pruning or olive oil industry as a waste product [1].

The present research was designed to verify a possible improvement of the nutritional value of two Tunisian olive oils by adding olive leaves, as a source of natural antioxidants, during oil extraction.

#### **2. Results and Discussion**

#### *2.1. Quality Parameters*

According to the definitions and standards established by the International Olive Oil Council [22], the classification of virgin olive oil into different categories depends on its chemical, physical and sensory parameters, among which the degree of acidity and the peroxide value (Table 1) are the most used. In this study, the differences observed between the two oils could be due, besides variety, to the different geographical location of the plants in terms of climate and soil composition.


**Table 1.** Quality parameters and chlorophyll and carotenoid contents of Neb Jmel and Oueslati olive oils extracted with and without addition of olive leaves (3%).

Data are means of three independent experiments ± SE (*n* = 3). Means followed by different letters are significantly different at *p* ≤ 0.05 as determined by Duncan's multiple range test. SE, standard error; meq, milliequivalent.

#### *2.2. Free Acidity*

Oil acidity is a simple and effective parameter to evaluate and classify a commercial-grade olive oil [23]. The extraction of olive oil from fresh undamaged fruits carried out following a correct crushing procedure gives oils with very low acidity [23]. However, during extraction and storage the olive oil can be altered by the release of free fatty acids as a consequence of the hydrolysis of triglycerides, thus increasing the free acidity.

The acidity values of Neb Jmel and Oueslati olive oils, extracted without (control) and with the addition of 3% olive leaves, are reported in Table 1. The Neb Jmel oils showed very low values (0.6%). The addition of leaves during oil extraction did not affect the free acidity in this variety. Regarding Oueslati, acidity of oils (1%) was higher in comparison with the Neb Jmel ones. However, the addition of leaves influenced Oueslati oil quality, decreasing free acidity to 0.6%. Our results agree with those reported by Ben Mansour et al. [24] for the Neb Jmel variety, whereas the free acidity of Oueslati oils was higher in comparison with the findings of Ouni et al. [25] on the same variety. This behaviour could be related to the use of olives at an advanced stage of maturation. Indeed, a late harvest of olives may alter oil acidity by increasing the lipolytic enzyme activities [26]. However, in our case the addition of olive leaves was responsible for the decrease of free acidity likely due to the presence of antioxidant compounds. Consequently, fortified Oueslati oils could still be classified as extra virgin olive oils as the free acidity value was lower than 0.8%.

#### *2.3. Peroxide Value*

Peroxides are intermediate products of oil oxidation which originate a complex mixture of volatile compounds such as aldehydes, ketones, hydrocarbons, alcohols and esters. These compounds are responsible for the alteration of the organoleptic characteristics [27], dramatically reducing oil shelf-life as well as consumer acceptance. Furthermore, also light and high temperatures are well-known factors generally promoting peroxide formation [28].

Table 1 shows that the peroxide value of Neb Jmel oils was well below the established limit (<20 milliequivalents (meq) O2/kg) for all the categories of olive oil [22]. In contrast, a high peroxide value was observed in the Oueslati control oil (34 meq O2/kg), so that it could no longer be classified as an extra virgin olive oil. However, the addition of leaves during oil extraction and processing prevented the oxidation and the formation of peroxides, reducing the peroxide value to half. Contrary to our results, Malheiro et al. [17] reported that leaf addition increased the peroxide value. Such discrepancy could be explained with differences in the relative presence of additional antioxidants and of leaf residues. These constituents, through gas exchanges occurring during the respiration process, may have increased the availability of oxygen, thus inducing peroxidation.

#### *2.4. Chlorophylls and Carotenoids*

Chlorophylls and carotenoids play important roles in olive oils. They interfere with the oxidative stability, acting as antioxidants in the dark or as prooxidants when exposed to light [29]. Furthermore, these compounds are responsible for the yellow-green pigmentation of olive oils, increasing consumer acceptability. Chlorophyll and carotenoid concentrations of olive oils and leaves are reported in Tables 1 and 2, respectively.


**Table 2.** Chlorophyll, carotenoid and total phenols, flavonoids and tocopherol (α and γ) contents, and ABTS radical cation (ABTS•<sup>+</sup> ) scavenging activity of Neb Jmel and Oueslati olive leaves.

Data are means of six independent experiments ± SE (*n* = 6). Means followed by different letters are significantly different at *p* ≤ 0.05 as determined by Duncan's multiple range test. DW, dry weight; GA, gallic acid; CA, catechin; TE, trolox equivalent; α, alpha tocopherol; γ, gamma tocopherol, tr, trace.

The Neb Jmel oil showed a higher chlorophyll value (Table 1) in comparison with Oueslati. However, Oueslati leaves (Table 2) displayed the highest chlorophyll (829.29 µg/g) and carotenoid (44.3 µg/g) concentrations, highlighting the role that the addition of chlorophyll could have had in the

oxidative stability of the olive oil. This can further explain the difference with the results reported by Malheiro et al. [17] concerning the peroxide value, as in their study olive leaf addition did not affect the chlorophyll content.

In the present work, the addition of leaves (3%) to Neb Jmel and Oueslati oils enhanced chlorophyll and carotenoid concentrations. In fact, chlorophyll reached the same level in both fortified oils, even if Oueslati oil was the one that showed the highest carotenoid concentration. Therefore, significant effects of leaf addition on oil pigments were observed in both varieties (Tables 1 and 2). The addition of leaves also turned the olive oils greener, this visual observation being clear in both oils, and was very likely associated with the increased pigment concentration. The increase in chlorophyll concentrations makes the fortified oils interesting from a nutritional point of view due to the antioxidant activity of chlorophyll and its potential to exert chemopreventive actions against carcinogens [30].

#### *2.5. Total Phenolics*

Phenolics are important components for olive oil quality and organoleptic characteristics. Moreover, they are very effective antioxidants playing an important role in human diet and health [3]. Current evidences strongly support the contribution of phenols to the prevention of cancer, cardiovascular and neurodegenerative diseases. The shelf-life of an oil is also correlated with its natural antioxidant amount [16]. Indeed, phenolics delay the oxidative degradation process, thus extending the shelf-life of the product [3,15].

Figure 1 shows that total phenolic concentration of the Neb Jmel oil (736 mg GA eq/kg oil) was significantly higher than that of Oueslati (528 mg GA eq/kg oil). In the literature, it was reported that the total phenolic concentration of Neb Jmel olive oils varies from 562 to 1167 mg GA eq/kg oil [24], whereas for Oueslati it changes from 100 to 859 mg GA eq/kg oil [25,31]. The effect of the geographic location on phenols can be evidenced from the different behaviour showed by each variety [24,25,31]. Indeed, Neb Jmel oil (from the north of Tunisia) showed a higher phenol concentration than Oueslati (from central Tunisia). The present data confirm previous findings on the effect of the variety on phenol concentration in oils [24,25,31].

Oueslati leaves showed a higher phenolic concentration than Neb Jmel ones (Table 2). It is worth noting that leaf addition, increasing total phenolic concentration by 44 and 10% in Oueslati and Neb Jmel oils, respectively, (Figure 1), determined the same mean value in the fortified products. Similar findings were also confirmed by other studies [32,33].

#### *2.6. Total Flavonoids*

Flavonoids are plant secondary metabolites with different phenolic structures. These compounds are used mostly to generate pigments which play an important role in the colours of plants. During the past decade, many studies have reported their beneficial effects on human health [34,35]. Indeed, flavonoids display important anti-inflammatory, antiallergic and anticancer activities as well as antiviral properties [34,35]. In this study, total flavonoid concentration was determined in both the oils and leaves of the two Tunisian olive cultivars Neb Jmel and Oueslati (Table 2, Figure 1). The results point out that Oueslati leaves showed a concentration of total flavonoids two-fold higher than Neb Jmel. However, in Oueslati oil the total flavonoid content was about half than that found in Neb Jmel one (Figure 1). The effect of leaf addition during oil extraction on these compounds was remarkable. Our results showed an increase in total flavonoids by 22% in Neb Jmel oils and by 160% in Oueslati, thus determining a not significant difference between the two fortified oils. This was likely due to the highest level of total flavonoids of Oueslati leaves. According to the present research, the findings of Ebrahimi et al. [36] reported values ranging from 156 to 361 mg rutin eq/kg for refined and crude olive oils, respectively.

≤ **Figure 1.** Total phenol (**A**) and total flavonoid (**B**) contents and ABTS•<sup>+</sup> scavenging activity (**C**) of Neb Jmel and Oueslati olive oils extracted with and without addition of olive leaves (3%). Data are means of three independent experiments ± SE (*n* = 3). Means followed by different letters are significantly different at *p* ≤ 0.05 as determined by Duncan's multiple range test.

### *2.7. ABTS*•*<sup>+</sup> Scavenging Activity*

ABTS•<sup>+</sup> scavenging activity of the Neb Jmel oil was 66% higher than that of Oueslati (Figure 1). In contrast, Oueslati leaves showed a higher free-radical scavenging activity compared to the Neb Jmel variety (Table 2). Following 3% leaf addition, the antioxidant capacity was increased by 15% and 87% in Neb Jmel and Oueslati oils, respectively. This wide change was probably due to the increases in chlorophyll, carotenoid, total phenolic and flavonoid concentrations. Indeed, some authors found a good correlation between the total phenolic amount and the radical scavenging power [37,38].

#### *2.8. Phenolic Compounds*

The analysis of the phenolic profile of olive leaf extracts and oils from the two varieties is reported in Tables 3 and 4, respectively. Thirteen phenolic compounds, among which phenolic acids, phenolic alcohols and secoiridoids, were identified and quantified. As regards olive leaf extracts, oleuropein derivatives were the most represented, followed by phenolic acids and phenolic alcohols (Table 3).

With minor changes compared to leaves, oleuropein derivatives were still the most abundant compounds in oils, followed by phenolic alcohols and phenolic acids (Table 4). In particular, Oueslati leaves showed a three-fold higher content in oleuropein derivatives than Neb Jmel ones, whereas Neb Jmel oil exhibited the highest concentration of these compounds. With leaf addition, oleuropein derivative concentration increased by 9 and 48% in Neb Jmel and Oueslati oils, respectively, confirming that olive leaves are a source of oleuropein derivatives [39]. Consistent with other reports, we found that oleuropein derivative amounts changed depending on the variety.


**Table 3.** Phenolic compounds (µg/g DW) of Neb Jmel and Oueslati olive leaves.

Data are means of three independent experiments ± SE (*n* = 3). Means followed by different letters are significantly different at *p* ≤ 0.05 as determined by Duncan's multiple range test.



Data are means of three independent experiments ± SE (*n* = 3). Means followed by different letters are significantly different at *p* ≤ 0.05 as determined by Duncan's multiple range test.

During the past few years, the biological activities of olive oil phenolics, namely oleuropein derivatives, have been thoroughly investigated. Several studies have attempted to elucidate the performance of oleuropein derivatives as antioxidant compounds. As the excessive presence of reactive oxygen species has been suggested to participate in the aetiology of several diseases [40], the focus on powerful antioxidants able to counteract the free-radical attack has become increasingly important. The antioxidant actions of oleuropein have been mostly assigned to its free-radical scavenging activity. Considering all together, the addition of olive leaves during oil extraction process suggests that it could be a means for improving oil quality.

Although olive fruits are rich in secoiridoids, hydroxytyrosol and tyrosol represent the two most important phenolic alcohols of both olive leaves [41] and oils [42]. Leaf composition of the Oueslati variety was characterised by a four-fold higher amount of hydroxytyrosol and tyrosol compared to Neb Jmel (Table 3). However, the oils, and likely the fruits, were mostly endowed with these phenolic alcohols in the Neb Jmel variety, showing concentrations of hydroxytyrosol and tyrosol of 3.57 and 17.97 mg/kg, respectively (Table 4). Obviously, the difference in the amounts of phenolic alcohols (hydroxytyrosol and tyrosol) depends not only on the variety, but also on the organ—leaf or fruit—considered. Our findings are in agreement with previous studies on phenolics in olive oil [17,21]. Oueslati oil, after 3% leaf addition, did not show any significant change in the hydroxytyrosol and tyrosol contents in comparison with the control (Table 4). These results suggest that a very high content of phenolics and related compounds could have ended up in olive mill wastewater. Indeed, many investigations clearly showed the occurrence of a high content of phenolic compounds in olive mill wastewater [43–46]. The hydrophilic character of polyphenols was likely responsible for the solubilisation of the most part of phenols into the water phase during oil extraction.

Phenolic acids found at considerable concentrations in Neb Jmel and Oueslati leaves (Table 3) were represented by gallic, protocatechuic, *p*-hydroxybenzoic, chlorogenic, vanillic, caffeic, syringic, vanillin, *p*-coumaric and ferulic acids. In the oils of the two varieties, the identified phenolic acids did not overcome the value of 2 mg/kg (Table 4), which agrees with the results reported by Kelebek et al. [47]. In both olive varieties, no significant effect of leaf addition was registered.

#### *2.9. Tocopherols*

Tocopherols occur in vegetable oils, playing an important role during oxidative processes. Two identified compounds (α- and γ-tocopherol) were quantified in this study. The quantitative profile of α- and γ-tocopherol is shown in Table 2 and Figure 2.

Tocopherol concentration in olive leaves is reported in Table 2. α-Tocopherol was present at higher amounts in Neb Jmel leaves (82.37 µg/g DW) in comparison with Oueslati, which showed an eight-fold lower amount (10.12 µg/g DW); γ-tocopherol was found in trace amounts in the leaves of both varieties.

In control oils (Figure 2), the α-isomer reached values of 257.8 and 283.6 mg/kg in Oueslati and Neb Jmal, respectively, not showing any significant difference between the two cultivars. Following leaf addition, α-tocopherol concentration showed a slight increase in the Neb Jmel oil, where it reached the value of 328.08 mg/kg. This result could be related to the higher presence of this compound in the corresponding leaves (Table 2). In contrast, the lower amount of α-tocopherol detected in Oueslati leaves (10.12 µg/g) did not determine any improvement in the oil following leaf addition (Figure 2). According to Malheiro et al. [17], the amount of α-tocopherol in oils was not significantly influenced when leaf addition was less than 5%.

Concerning γ-tocopherol (Figure 2), the amount was very low compared to the α-isomer (1.17–28.73 mg/kg), being it more represented in the Neb Jmel oil. Leaf addition (3%) did not significantly affect γ-tocopherol concentrations due to the fact that in the leaves of both varieties this isomer was detected in trace amounts (Table 2).

Consistent with previous reports [48], we found that olive leaves can be used as an alternative source to improve the chemical composition of olive oils, mainly the α-tocopherol concentration. Likewise, the present results agree with previous studies on the influence of the cultivar on α-tocopherol concentration. Franco et al. [49] reported very high levels of α-tocopherol in seven varieties of Spanish oils (217–345 mg/kg). In contrast, in the Portuguese olive oil studied by Cunha et al. [50], values ranging from 93 to 260 mg/kg were found. Similar values to those reported in the present experiment were found in some studies performed on different Tunisian oils [51–53].

γ α ≤ **Figure 2.** γ-Tocopherol (**A**) and α-tocopherol (**B**) content of Neb Jmel and Oueslati olive oils extracted with and without addition of olive leaves (3%). Data are means of three independent experiments ± SE (*n* = 3). Means followed by different letters are significantly different at *p* ≤ 0.05 as determined by Duncan's multiple range test.

γ α γ α Antioxidants, such as vitamin E (tocopherols), may prevent the detrimental effects of free radicals. In the Mediterranean diet, olive oil substantially contributes to the daily intake of these antioxidants. The health benefits of vitamin E are evidenced by the fact that the ingestion of fresh fruits and vegetables is inversely related to the extent of some cancers as well as to plasma lipid peroxidation [54]. It should be highlighted that the two vitamin E isoforms have different health-related properties. In fact, γ-tocopherol is the less powerful antioxidant, although being capable of trapping peroxynitrites. For this reason, γ-tocopherol has been acknowledged as the "other" vitamin E important for human health [54].

α

α

#### **3. Materials and Methods**

#### *3.1. Olive Leaves and Fruit Sampling*

The olives of two varieties were collected from different regions: Oueslati, in the centre (Khit El Oued), and Neb Jmel, in the north (Borj El Amri) of Tunisia. The harvest was performed at the same stage of maturity, considering a colour maturity index of about three. The maturity index was evaluated taking into consideration the changes in skin and pulp colours. Samples of 100 fruits were taken randomly and classified into eight groups or categories: green intense (category 0), yellow or yellowish green (category 1), green with reddish spots (category 2), reddish or light violet (category 3), black with white pulp (category 4), black with <50% purple flesh (category 5), black with ≥50% purple flesh (category 6) and black with 100% purple flesh (category 7). The maturity index was calculated as A × 0 + B × 1 + C × 2 + D × 3 + E × 4 + F × 5 + G × 6 + H × 7/100, where A, B, C, D, E, F, G and H are the number of fruits in each class.

The olives were picked by hand from three trees during the 2016/2017 crop season (November). Olive leaves were also collected from the same trees at harvest. The following percentages of olive leaves were added to fruits (*w*/*w*) prior to crushing: 0% (control) and 3% (fortified). The choice of the percentage of olive leaves was based on previous reports [32,33]. According the above studies, 3% was the optimal percentage of olive leaves which can be added to improve oil quality without any negative effects. Only healthy fruits without any kind of infection or physical damage were processed. After harvesting, fresh olives (2.5–3.0 kg) were washed and crushed with a hammer crusher, and the paste, mixed at 25 ◦C for 30 min, was centrifuged without the addition of warm water. The oil yield from each extraction was 200–250 mL/kg. The oil produced was then transferred into dark glass bottles and stored in the dark at 4 ◦C until analysis.

#### *3.2. Methods*

#### 3.2.1. Quality Parameters

Determination of physicochemical quality parameters (free acidity and peroxide values) was carried out following the analytical methods described by Regulation EEC/2568/91 and EEC/1429/92 of the European Union Commission (European Union Commission Regulation, 1991, 1992).

Free acidity, given as percentage of oleic acid, was determined by titration of the oil dissolved in an ethanol–ether solution (1:1, *v*/*v*) with a 0.1 M potassium hydroxide ethanolic solution. The peroxide value, expressed in milliequivalents of active oxygen per kg oil (meq/kg), was determined as follows: a mixture of oil and chloroform–acetic acid was left to react with a solution of potassium iodide (10.5 M) in the darkness. The free iodine was then titrated with a sodium thiosulfate solution (0.01 N).

#### 3.2.2. Pigment Concentration

#### Oil Pigment Determination

One millilitre of oil was diluted ten-fold in *n*-hexane. Chlorophylls and carotenoids were determined colourimetrically as previously described [55]. The maximum absorption at 670 nm was related to the chlorophyll fraction and that at 470 nm to the carotenoid one. The specific extinction coefficients considered for calculation were 613 for pheophytin, as a major component of the chlorophyll fraction, and 2000 for lutein, as a major component of the carotenoid fraction. The pigment concentrations were calculated as follows:

> Chlorophyll (mg/kg) = (A<sup>670</sup> × 10<sup>6</sup> )/(613 × 100 × d)

#### Carotenoids (mg/kg) = (A<sup>470</sup> × 10<sup>6</sup> )/(2000 × 100 × d)

where A is the absorbance and d is the spectrophotometer cell thickness (1 cm).

#### Leaf Pigment Determination

Fresh leaf tissue (0.1 g) was ground in a mortar with sand and 70% ethanol solution. The homogenates were then filtered and washed with 70% ethanol (up to 5 mL). After centrifugation for 10 min at 12,100× *g*, absorbance was read at 646.6 and 663.6 nm for chlorophylls and at 480 nm for carotenoids. Concentrations of total chlorophylls and total carotenoids (µg/g DW) were calculated according to Porra et al. [56].

#### *3.3. Extraction of Phenolic Compounds*

#### 3.3.1. Fresh Leaves

Leaf samples (0.2 g) were ground in a mortar at room temperature with 70% methanol containing 1% HCl. The homogenates were sonicated for 30 min and centrifuged at 12,100× *g* for 30 min at 4 ◦C. The supernatants were stored at −20 ◦C and used to determine both phenolic compounds and antioxidant activity.

#### 3.3.2. Olive Oils

Phenolic compounds of olive oils were extracted according to Rotondi et al. [57]. Two grams of oil were added to 1 mL of *n*-hexane and 2 mL of a methanol/water (70:30, *v*/*v*) solution in a 10 mL centrifuge tube. After vigorous mixing, tubes were centrifuged for 10 min. The hydroalcoholic phase was collected, and the hexane phase was re-extracted twice with 2 mL of a methanol/water (70:30, *v*/*v*) solution. The hydroalcoholic fractions were combined, washed with 2 mL of *n*-hexane to remove the residual oil and vacuum-dried.

#### *3.4. Total Polyphenol and Flavonoid Concentrations*

Total phenolic content was estimated by the Folin Ciocalteu method as described by Singleton and Rossi [58]. To the extract, diluted with distilled water, 1 mL of sodium carbonate (20%) and 1 mL of Folin Ciocalteu reagent were added. The mixture was allowed to stand in a water bath for 30 min at 40 ◦C. The concentration of the total phenolic compounds was expressed as mg of gallic acid equivalents. The absorbance was measured at 765 nm using a UV–vis spectrophotometer (VARIAN, Milan, Italy). The experiments were performed in triplicate, and mean values and standard deviations were calculated using the Microsoft Excel software (Microsoft Corporation, Redmond, WA, USA).

The total flavonoid concentration was determined by the aluminium trichloride method using catechin as reference compound [59]. A 5% NaNO<sup>2</sup> solution was added to the extract, followed after 6 min by 10% aluminium trichloride. The mixture was incubated for further 5 min and then 1 M NaOH was added. The final volume was 2.5 mL. After 15 min of incubation, the absorbance at 510 nm was detected. Total flavonoid concentration was expressed as mg of catechin equivalents.

#### *3.5. Free-Radical Scavenging Ability*

The free-radical scavenging activity of samples was determined by the ABTS radical cation decolourisation assay described by Pellegrini et al. [60]. The radical solution was generated by adding 7 mM ABTS solution to 4.9 mM potassium persulfate. Before use, the radical solution was diluted with ethanol to obtain an absorbance of 0.700 at 734 nm. A control containing ethanol and ABTS•<sup>+</sup> solution was also prepared, and the absorbance was taken as the initial. After a 15 min incubation period at room temperature, the final absorbance was read at 734 nm. Calculations were performed by percentage of inhibition of the ABTS cation radical as follows:

#### % of inhibition = ((initial Abs − final Abs)/initial Abs) × 100

To quantify antioxidant capacity, a calibration curve of the percentage of inhibition against Trolox in the range 2–20 nmol was used.

#### *3.6. HPLC Analysis of Phenolic Compounds*

Qualitative and quantitative analysis were performed by reverse-phase HPLC (RP-HPLC) [61]. Twenty microlitres of extract were injected into a Waters model 515 HPLC system fitted with a 3.9 mm × 150 mm Nova-Pak C18 column (Waters, Milford, MA, USA). Detection was conducted at 280 nm using a Waters 2487 dual λ UV–visible detector. Mobile phase A contained 98% water and 2% acetic acid, and mobile phase B contained 68% water, 30% acetonitrile and 2% acetic acid. A linear gradient

of 10 to 95% mobile phase B was run for 90 min at 1 mL/min. The identity of the phenolic acids was confirmed by cochromatography on HPLC with authentic standards (Sigma Chemical Co., St. Louis, MO, USA), and quantification was performed using a standard curve in the range of 20 to 200 ng of standard mixtures containing gallic, protocatechuic, *p*-hydroxybenzoic, chorogenic, vanillic, caffeic, syringic, *p*-coumaric, ferulic, tyrosol, hydroxytyrosol, vanillin and oleuropein. Chromatogram analysis was performed by the software Millennium 32 (Waters).

#### *3.7. Extraction and Detection of Tocopherols (Vitamin E)*

Tocopherols were determined in the lipid extracts from olive leaves and in oils. Extractions were performed in the dark as previously reported [54] and according to the method of Gimeno et al. [62]. Tocopherol isoforms (α and γ) were determined by isocratic RP-HPLC using a Shimadzu apparatus (model LC-20AD) with an electrochemical detector (Metrohm model 791, Varese, Italy) equipped with a glassy carbon electrode and LC Solution software (Shimadzu) for the integration of peaks. Detection was performed according to Galatro et al. [63] at +0.6 V at 25 ◦C with a Nova Pak C-18 4 µm column (3.9 × 150 mm). The extracts were eluted with 95% methanol containing 20 mM LiClO<sup>4</sup> at a flow rate of 1 mL min−<sup>1</sup> . For identification and quantification of peaks, a calibration curve was prepared using standard mixtures of α-, β-, γ- and δ- tocopherol provided by Sigma (Milan, Italy) in the range of 25 to 75 ng.

#### *3.8. Statistical Analysis*

The results are means from three replicates. All data are reported as mean values ± SE. The significance of differences among mean values was determined by one-way ANOVA. Comparisons among means were performed using Duncan's multiple range test. Means in tables and figures accompanied by different letters are significantly different at *p* ≤ 0.05.

#### **4. Conclusions**

The present study confirms the dependence of olive oil quality on the cultivar, besides geographical location, climate and soil characteristics. The Neb Jmel oil showed the best chemical composition with the lowest free acidity and peroxide values, the highest chlorophyll, total phenol and total flavonoid concentrations as well as antioxidant activity. The addition of olive leaves (3%) to Neb Jmel and Oueslati oils affected both their quality and chemical composition, mainly conferring an increased resistance to oxidation as well as improving the nutritional qualities. In particular, a remarkable increase in oleuropein derivatives was observed, which was responsible for the enhancement in total phenolic amounts. As Oueslati leaves are particularly enriched in chlorophyll, carotenoids, flavonoids and oleouperin derivatives, their addition during oil extraction may have prevented the oxidation and the formation of peroxides. Thus, in the Oueslati variety leaf addition reduced both peroxide values and free acidity of oils, allowing them to be still classified as extra virgin olive oils. The enrichment of the oils with antioxidant compounds from the leaves also led to a remarkable increase in the nutritional quality of the Oueslati oil, which became similar to the Neb Jmel one. In conclusion, the addition of a small percentage of olive leaves could improve the nutraceutical properties of extra virgin olive oil by increasing the phenolic compound content. These compounds, together with tocopherols, play a protective role against oxidative stress, being also able to extend the extra virgin olive oil shelf-life due to their antioxidative properties.

**Author Contributions:** I.T. conceived the experiments. I.T., C.S., and M.F.Q. performed the experiments and wrote the paper. A.Z. analysed and interpreted the data. A.Z., M.F.Q., M.Z. and J.E. revised the paper.

**Funding:** This study was financially supported by the University of Pisa (Fondi di Ateneo, 2017).

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

#### **References**


#### **Sample Availability:** Not available.

© 2019 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/).

### *Article* **Anti-Oxidant and Anti-Melanogenic Properties of Essential Oil from Peel of Pomelo cv. Guan Xi**

**Wanying He 1,2 , Xiaoyan Li 1,2 , Ying Peng 1,2 , Xiaoyan He 1,2 and Siyi Pan 1,2, \***


Received: 20 November 2018; Accepted: 7 January 2019; Published: 10 January 2019

**Abstract:** Here, we investigated the anti-oxidant and anti-melanogenic effects of pomelo peel essential oil (PPEO) from pomelo cv. Guan Xi. The volatile chemical composition of PPEO was analyzed with gas chromatography–mass spectrometry (GC/MS). The most abundant component of PPEO was limonene (55.92%), followed by β-myrcene (31.17%), and β-pinene (3.16%). PPEO showed strong anti-oxidant activities against 2,2-diphenyl-2-picryhydrazyl (DPPH), 2,2 ′ -azinobis-(3-ethylbenzthiazoline-6-sulphonate (ABTS) and superoxide anion free radicals. Based on the B16 melanoma cell system, the effects of PPEO on the viability and morphology of B16 cells and the production of melanin were evaluated. The results revealed that PPEO at concentrations below 50 µg/mL could decrease the melanin content without affecting cell viability and morphology. Intracellular tyrosinase (TYR) activity and Western blot analysis showed that PPEO could down-regulate the expression level of TYR in B16 cells and dose-dependently inhibit TYR activity (by a maximum of 64.54%). In conclusion, PPEO has good anti-oxidant and anti-melanogenic activity, and thus can be widely used as a natural antioxidant in the food, pharmaceutical, and cosmetic industries.

**Keywords:** pomelo peel; essential oil; anti-oxidant; anti-melanogenic; B16 melanoma cell

#### **1. Introduction**

Pomelo, a citrus fruit belonging to the genus Rutaceae, is grown and consumed worldwide due to its unique flavor and high nutritional value [1]. In the production of pomelo juice, jam and other products, pomelo peel (PP) is a major by-product accounting for about 50% of the total weight of the fruit. However, most of the PP is disposed of in landfills, resulting in environmental pollution and loss of economic value [2,3]. Pomelo peel contains many natural chemical ingredients, making it a good source of valuable extracts. Compared with the peel of other fruits, PP has a higher concentration of essential oil (EO). Citrus EO is generally considered to be safe with a broad spectrum of biological activities such as anti-inflammatory and anxiolytic effects [4]. Due to its high content of active substances such as terpenes, sesquiterpene, aldehydes, ketones, and esters, pomelo EO has strong aromatic, antioxidant, bacteriostatic, and antiviral properties [4,5], and thus can be used as a functional ingredient and premium fragrance in the food, cosmetic and pharmaceutical industries. Therefore, the high value-added utilization of PP has important research significance and economic prospects.

Oxidative stress can produce reactive oxygen such as superoxide (O2), hydrogen peroxide (H2O2), and hydroxyl radical (HO). These reactive oxygen species can disrupt the balance of normal metabolic activity in the human body and are associated with many chronic diseases such as aging, cancer, atherosclerosis, and inflammation [6]. As a result, research of natural antioxidants has gradually

become a hot spot. These antioxidants can remove excess free radicals from the body and relieve conditions caused by excessive free radicals [7,8]. There has been extensive research on the antioxidant activities of plant EOs. Most current research on antioxidants is focused on citrus EOs. For example, the antioxidant properties of 34 kinds of citrus EOs have been tested. The results showed that most of the EOs have good inhibitory effects on 2,2-diphenyl-2-picryhydrazyl (DPPH), which are significantly better than the effects of water-soluble vitamin E [9]. Extract of sweet orange peel have significant inhibitory effects on DPPH, with 50% inhibitory concentrations (IC50) of 600 µL/mL [10]. Moreover, the EOs extracted with a cold pressing method also have high antioxidant activity, because cold pressing can better preserve the active ingredients in EOs [11]. However, the above studies are limited to the antioxidant activities of EO mixtures as a whole, while there have been few studies of the antioxidant activities of specific components in EOs.

Melanin is a pigment widely distributed on the surface of skin, hair, retina, and adrenal medulla. It is synthesized from tyrosine under the enzymatic oxidation of tyrosinase (TYR) [12]. However, excessive production and accumulation of melanin can cause pigmentation spots and skin discoloration such as chloasma, freckles, and age spots. Tyrosinase is associated with a variety of diseases and may be a key factor of dopamine neurotoxicity and neurodegeneration associated with Parkinson's disease [13]. Besides, it is also a key rate-limiting enzyme in the initial reaction of melanin production. At present, the application of EOs as a natural enzyme inhibitor has become a research hotspot. Plant EOs have strong biological activities and great application potentials in biology and medicine [14]. Cinnamon EO and clove EO can inhibit the TYR activity in B16 cells by 37% and 10%, respectively [7]. Lemon EO was observed to have significant inhibitory effects on TYR activity, and its major components were determined to be monoterpenoids and oxindoles [15], which are also the main components of pomelo EO. Hence, it can be speculated that pomelo EO might also have inhibitory effects on TYR activity. Mint leaf EO could reduce the synthesis rate of melanin in B16-F10 cells, and β-caryophyllene (main component) can also decrease melanin production by down-regulating the expression of microphthalmia-associated transcription factor protein (MITF), tyrosinase-related protein-1 (Trp-1), tyrosinase-related protein-2 (Trp-2), and TYR [16]. Aromatic or aliphatic compounds such as anisaldehyde and cuminaldehyde are effective TYR inhibitors [13]. Citrus EOs contain a large amount of fatty aldehyde compounds, indicating their potential inhibitory activity against TYR. However, the current research on pomelo mostly focuses on the storage and preservation of fresh fruit and the extraction of pectin from the peel, while little research attention has been paid to the antioxidant and anti-melanogenic effects of pomelo peel EO (PPEO). Therefore, clarifying the effect of PPEO on melanin synthesis is of great significance for improving the high value-added utilization of PP and expanding its application in the health food, pharmaceutical and cosmetics fields.

In this study, we extracted PPEO from pomelo cv. Guan Xi by a cold pressing method and analyzed its main components using gas chromatography–mass spectrometry (GC/MS). The anti-oxidant activities were examined with 2,2-diphenyl-1-picrylhydrazyl (DPPH); 2,2′ -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), and superoxide anion radical scavenging. Furthermore, based on the B16 melanoma cell system, the effect of PPEO on the viability and morphology of B16 cells and the production of melanin was evaluated. Finally, intracellular TYR activity assay and Western blot analysis were performed to validate the inhibitory effect of PPEO on TYR.

#### **2. Results and Discussion**

#### *2.1. Chemical Composition of Pomelo Peel Essential Oil*

The extraction rate of PPEO by the method of cold pressing was 0.82%, which was higher than that in a previous study of citrus essential oils (0.25%) [17]. The obtained PPEO was pale yellow and a clear liquid with a natural aroma. Figure 1 shows the total ion chromatogram obtained from GC/MS analysis of the PPEO 10-fold diluted with absolute ethanol. A total of 21 compounds were detected (Table 1). Among these compounds, 13 were terpenes, which accounted for 94.15% of the

total. The highest content of limonene in PPEO was 55.92%, followed by β-myrcene (31.17%), β-pinene (3.16%), ocimene (1.42%), and β-copaene (1.24%) (Table 1). β β β

**Figure 1.** Total ion chromatogram of aroma components from pomelo peel essential oil (PPEO).



#### *2.2. Antioxidant Activities of Pomelo Peel Essential Oil*

Figure 2 shows the DPPH free radical scavenging rate, superoxide anion radical scavenging rate, and total antioxidant activity of PPEO. PPEO exhibited significant effects on the free radical scavenging rate in a concentration dependent manner. At a low concentration (5 mg/mL), PPEO had no significant effect on DPPH free radical scavenging (Figure 2A). However, with increasing concentration, the DPPH free radical scavenging rate of PPEO reached 68.13% at 150 mg/mL, which was statistically significantly different from that of the control group (*p* < 0.01). TheIC<sup>50</sup> of PPEO was 70.12 mg/mL. The positive control butylated hydroxytoluene (BHT) showed good DPPH free radical scavenging ability at low concentrations. Superoxide anion free radicals can induce lipid peroxidation in the body, thereby accelerating the aging of human skin and even internal organs [18]. As shown in Figure 2B, when the concentration of PPEO was 0.2 mg/mL, the superoxide anion clearance rate was only 11.93% (*p* < 0.05). With increasing PPEO concentration, the clearance rate increased to 44.74% at 1.0 mg/mL (*p* < 0.01). It seems that PPEO could effectively remove the superoxide anion radicals, but with a lower scavenging ability than L-ascorbic acid at the same concentration. Figure 2C shows that the total antioxidant activity of PPEO was slightly lower than that of BHT at the concentrations lower than 0.4 mg/mL. However, as the concentration increased, the antioxidant activity of PPEO exceeded that of BHT. At the concentration of 1.0 mg/mL, the total antioxidant activity of PPEO was 20.35% higher than that of BHT. These results indicated that low-concentration PPEO has no obvious antioxidant effect while high-concentration PPEO has good antioxidant effect. Butylated hydroxytoluene is an industrially synthesized antioxidant that facilitates fast oxidation resistance at low concentrations, while PPEO is a mixture of various compounds with antioxidant activities, which may have complex interactions with each other [19].

**Figure 2.** Antioxidant activities of PPEO. (**A**) 1,1-Dipheny-2-Picryhydrazyl (DPPH) radical scavenging assay; (**B**) superoxide anion radical scavenging activity assay; (**C**) 2,2′ -azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS) total antioxidant activity. \* Indicates samples that are significantly different (*n* = 3; \* *p* < 0.05 and \*\* *p* < 0.01 compared with the positive control group).

μ

μ

μ

μ

#### *2.3. Anti-Melanogenic Effects of Pomelo Peel Essential Oil*

#### 2.3.1. Effect of Pomelo Peel Essential Oil on Cell Viability

B16 melanoma cells were treated with different concentrations of PPEO for 24 h, and the viability of each group was detected by MTT assay (Figure 3). The survival rate of B16 cells treated with a low concentration of PPEO (5 µg/mL) was higher than 100%, indicating that low concentration of PPEO may facilitate the proliferation of B16 cells, which might be related to the active volatile components in PPEO [20]. The cell viability decreased significantly along with increasing PPEO concentration. When the concentration of PPEO was 150 µg/mL, the cell survival rate significantly decreased to 29.35% (*p* < 0.01). The results show that at concentrations lower than 50 µg/mL, PPEO did not affect cell viability. However, high concentrations of PPEO (>50 µg/mL) significantly inhibited cell viability.

**Figure 3.** Effect of PPEO on B16 melanoma cell viability. \* Indicates samples that are significantly different (*n* = 3; \* *p* < 0.05 and \*\* *p* < 0.01 compared with the blank control group).

#### 2.3.2. Effect of Pomelo Peel Essential Oil on Cell Morphology

μ μ μ B16 cells are adherent and mostly fusiform cells with dendrites and relatively more divisions. They are tightly connected monolayers with high transparency (Figure 4). The cells in the control group showed uniform fluorescence, clear cell boundaries, and normal dendritic morphology. When the concentration of PPEO was 10–50 µg/mL, the number of cell deaths was small, the cell boundary was clear, and the fluorescence was relatively uniform. However, at a concentration of 100 µg/mL, the number of cell deaths increased and the boundaries between cells became blurred, accompanied by the appearance of obvious fluorescent spots. At the same time, cells were dispersed and the dendrites were reduced. In the high-concentration PPEO group (100 µg/mL), the cells showed enhanced fluorescence, and were swollen and separated from each other, presenting a typical state of apoptosis in the cells.

2.3.3. Inhibition of Pomelo Peel Essential Oil on Intracellular Tyrosinase Activity and Melanin Content

To determine the anti-melanogenic activity of PPEO, we evaluated its effect on TYR activity and melanin content in B16 melanoma cells. The B16 melanoma cells were treated with various concentrations of PPEO and then co-cultured for 72 h. As shown in Figure 5, PPEO dose-dependently inhibited TYR activity and melanin content. At a concentration of 50 µg/mL, melanin synthesis and TYR activity were inhibited by 48.28% and 64.54%, respectively, and the IC<sup>50</sup> of melanin synthesis in inhibition was 67.64 µg/mL. Kojic acid, the positive control, inhibited TYR activity by 62.09%, which is similar to the inhibitory effect of PPEO at 50 µg/mL (Figure 5B).

μ μ μ **Figure 4.** Optical microscopic morphology of B16 cells. PPEO at concentrations of 0, 10, 20, 50, 100 µg/mL were for (**A**–**E**), respectively, and kojic acid at a concentration of 71 µg/mL was for (**F**). Scale bar: 50 µm

**Figure 5.** Effect of PPEO on melanin content (**A**) and tyrosinase activity (**B**) in B16 cells. \* Indicates samples that are significantly different (*n* = 3; \* *p* < 0.05 and \*\* *p* < 0.01 compared with the blank control group).

2.3.4. Effect of Pomelo Peel Essential Oil on Tyrosinase Expression in B16 Cells

The expression of TYR protein was detected by Western blotting, and the intensity of protein expression was determined by the ratio of the target band to the internal reference band (Figure 6). PPEO down-regulated the expression level of TYR in B16 cells in a concentration-dependent manner.

μ

Compared with that in the blank control group, the TYR expression gradually decreased with increasing PPEO concentration. When the PPEO concentration was 50 µg/mL, the TYR expression was 60.38% lower than that of the blank group and was close to that of the positive group (kojic acid). This result is consistent with the tyrosinase enzyme linked immunosorbent assays (ELISA) test results. μ

**Figure 6.** Effect of PPEO on tyrosinase expression in B16 cells. \* Indicates samples that are significantly different (*n* = 3; \* *p* < 0.05 and \*\* *p* < 0.01 compared with the blank control group). GADPH: glyceraldehyde-3-phosphate dehydrogenase.

#### *2.4. Discussion*

Previous studies have shown that the characteristic aroma of pomelo is mainly attributable to a variety of compounds [2]. In this study, we first extracted PPEO from pomelo cv. Guan Xi and analyzed its main components using GC/MS. Our study demonstrated that the fresh and natural fruit aromas were mainly due to the presence of aldehydes and terpenoids in PPEO. The safety of natural products used in health foods, drug, and cosmetic ingredients is a major concern. Several studies have explored the use of extracts from pulp of guava [21], waste of citrus [6], earthworm [18], oil of *Aquilaria crassna* [8], and oil from *Alpinia zerumbet* [22]. We first determined the antioxidant activities of PPEO. Previous studies have shown that plant EOs have universal antioxidant activities [14]. Meanwhile, it has been reported that terpenes such as limonene, β-myrcene, β-pinene, ocimene, β-copaene, and citral showed anti-oxidant activities [21,23,24]. The strong anti-oxidant activity of PPEOs may be attributable to these components. In our experiment, PPEO was extracted using a cold pressing method under normal temperature conditions, which could better preserve the active components with antioxidant function, resulting in a high total antioxidant capacity. It is known that ultra violet (UV) radiation induces free radical formation in the skin, which is linked directly to the onset of skin photodamage and biological damage. Thus, our results suggest that PPEO may be a useful anti-oxidant source and have the potential to prevent UV-induced damage.

Next, we highlighted the anti-melanogenic effects of PPEO through cell viability, cell morphology, intracellular melanin content, intracellular TYR activity and expression, and compared these results with those of the positive control (kojic acid). Previous studies have shown that the action mechanism of terpenoids in cells is related to the destruction of lipophilic compounds in biofilms [25,26]. Due to the high hydrophobicity of terpenoids, their toxic effects lead to swelling and enhanced fluidity and permeability of the cell membranes [27]. Therefore, terpenoids might cause death of cells at high

concentrations of PPEO. However, when the concentration of PPEO was below 50 µg/mL, the state of the cell was normal. On considering this possibility, concentrations below 50 µg/mL of PPEO were used to evaluate its effects on melanin content and intracellular TYR activity.

To our knowledge, TYR catalyzes the first two steps of mammalian melanogenesis [18], namely the hydroxylation of monophenol to *o*-diphenol and the oxidation of diphenol to *o*-quinones, both of which use molecular oxygen, followed by a series of nonenzymatic steps to finally result in the formation of melanin [12]. Therefore, inhibition of TYR activity may help to avoid abnormal melanin pigmentation in skin. The results of this study preliminarily demonstrated the good antioxidant performance of PPEO. In the co-culture of PPEO and B16 cells, PPEO acted as an antioxidant to inhibit the catalytic reaction of TYR and block the synthesis pathway of melanin, resulting in a decrease in melanin production. Meanwhile, the effect of PPEO on the dendritic morphology of cells might also destroy the normal physiological functions of cells, which in turn affects the formation of melanin in cells. The above results demonstrate that PPEO can decrease the melanin content without affecting the cell viability.

In addition, previous study showed that citral, myrcene, (2E)-alkenal, and terpinolene were popular tyrosinase inhibitors [13]. In another study, citral and myrcene were found to have significant inhibitory effects on TYR, and trans-citral has better inhibitory effect than *cis*-citral. In plant EOs, the content of *trans*-citral is higher than that of *cis*-citral. Meanwhile, citral and myrcene are the main active substances that inhibit TYR in EOs [28]. Therefore, it is most likely that the inhibition of TYR expression by the components (limonene, β-myrcene, β-pinene, ocimene, β-copaene) in PPEO is a synergistic effect, and the main active components could be partly attributed to citral and myrcene.

#### **3. Materials and Methods**

#### *3.1. Materials*

Fully ripe fruits of pomelo cv. Guan Xi were harvested from Fujian province of China. Reagents 2,2-diphenyl-1-picrylhydrazyl (DPPH), dimethyl sulfoxide (DMSO), Hoechst 33258 staining solution, fetal bovine serum (FBS), *n*-paraffins (C7–C30), and tyrosinase enzyme linked immunosorbent assays (ELISA) kit were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Dulbecco's Modified Eagle Medium (DMEM) was from Gibco Chemical Co. (Grand Island, NY, USA). Penicillin-streptomycin double antibody, trypsin, and methyl thiazolyl tetrazolium (MTT) were purchased from Genivew Co. (El Monte, CA, USA). Cell Lysates, tert-butyl hydroxytoluene (BHT), kojic acid, and protein quantification test kit were purchased from Shanghai Yuye biotechnology Co., Ltd. (Shanghai, China). RPMI 1640 medium was purchased from Hyclone Co. (Logan, UT, USA). ECL chemiluminescence detection kit and sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE) Gel Preparation Kit were purchased from Aspentech Co. (Bedford, MA, USA). B16 melanoma cells were purchased from Shanghai Tongpai Biotechnology Co., Ltd (Shanghai, China). All other analytical grade chemicals were bought from Sinopharm chemical reagent Co., Ltd (Shanghai, China).

#### *3.2. Extraction of Essential Oil*

Fresh pomelo was picked and the white peel from the fresh pomelo peel was removed, and then the exocarp chopped in 1.5 cm × 0.5 cm × 0.2 cm sized pieces. Pomelo peel (100 g) was crushed and pressed twice with a cold hydraulic press (Model 6YL-190; Changbai Mountain Technology Limited company, Changchun, China) [28] and then the peel slurry was collected. After filtering through a steel sieve (0.15 mm), saturated sodium chloride (NaCl) solution was added to the sample for the extraction of PPEO for 3 h. Then, the sample was centrifuged at 10,000× *g* for 30 min at 4 ◦C. Supernatants were stored in separate amber bottles at −20 ◦C until use. The extracted PPEO was weighed to determine the extraction yield as follows: extraction yield (%) = [(weight of extracted oil)/(weight of pomelo peel)] × 100%.

#### *3.3. Gas Chromatography-Mass Spectrometry Analysis*

The extracted PPEO was filtered through a 0.45 µm microporous organic membrane. Volatile compounds were analyzed using an Agilent 7890A GC coupled to an Agilent 5975C mass spectrometer (Palo Alto, CA, USA). The components of PPEO were identified using HP-5Ms phenylmethylsiloxane capillary column (30 m × 0.25 mm i.d., 0.25 µm; Agilent Technologies, J & W Scientific Products, Folsom, CA, USA) [24]. The helium was used as a carrier gas with a flow rate of 1 mL/min. Injector temperature was 250 ◦C. The split ratio was 10:1. The temperature program was 45 ◦C (hold for 1 min), increase at 10 ◦C/min to 165 ◦C (hold for 2 min), increase at 1.5 ◦C/min to 180 ◦C (hold for 2 min), and then increase at 10 ◦C/min to 250 ◦C (hold for 2 min). The temperature of both injector and detector was set at 250 ◦C. Mass spectra were scanned from *m/z* 35–350 amu. The electron impact ionization energy was 70 eV. Identification of compounds detected by GC/MS analysis was performed by comparing mass spectra and retention indices (RIs) with published data obtained under similar conditions, as well as by comparing their mass spectra with the MS library of Wiley 7.0 and Nist 05 [29]. A mixture of *n*-paraffins (C7–C30) as standards was used for calculating RIs. Samples were analyzed and identified using an available Retention Time Locked (RTL) database with Deconvolution Reporting Software (DRS) and a database of 926 DRS compounds.

#### *3.4. Antioxidant Activities*

#### 3.4.1. DPPH Radical Scavenging Assay

The DPPH radical scavenging assay was performed according to Boskou et al. [23]. The sample PPEO was prepared with different concentrations with absolute ethanol. A mixture of 50 µL sample and 150 µL 0.1 mmol/L DPPH free radical ethanol solution was taken and placed in a 96-well plate. After vigorous shaking, the mixture was incubated at room temperature in the dark for 30 min. The absorbance at 517 nm was measured. Pomelo peel essential oil was replaced with absolute ethanol to serve as the blank control, and BHT was used as positive control.

#### 3.4.2. Superoxide Anion Radical Scavenging Activity Assay

The superoxide anion radical scavenging activity was measured as described by Zhang et al. [30]. The reaction mixture consisted of 4.5 mL 50 mM Tris-HCl buffer (pH 8.2) and 1 mL sample solution at different concentrations. The mixed solution was pre-incubated at 25 ◦C for 10 min, and then initiated by the addition of 0.45 mL 2.5 mM pyrogallol. After vigorous shaking for 5 min, the reaction was terminated by the addition of 8 mol/L HCl. The absorbance was read at 517 nm.

#### 3.4.3. ABTS Radical Scavenging Assay

The ABTS radical scavenging assay was conducted following the method previously described by Re et al. [31]. Diluted radical solution was prepared by mixing 7 mM ABTS and 2 mM K2S2O<sup>8</sup> in equal amounts, followed by reaction in the dark overnight at room temperature. The samples were prepared in different concentrations with ultrapure water. Aliquots of 10 µL of samples were mixed with 200 µL of the diluted radical solution in 96-well plate and the absorbance was measured at 734 nm after 5 min using an M200 pro enzyme-labeled instrument (Tecan, Ltd., Männedorf, Switzerland). BHT was used as the positive control.

#### *3.5. Cell Culture and Treatment*

The murine metastatic melanoma cell line B16 was cultured in sterile cell culture flasks with RPMI 1640 medium supplemented with 100 U/mL penicillin, 100 U/mL streptomycin, and 10% heat inactivated FBS at 37 ◦C in a humidified incubator containing 5% CO2. Cells in logarithmic growth phase were selected for subsequent experiments [32]. The extracted PPEO was dissolved in Tween 80 and filtered through a 0.45 µm microporous organic membrane. The PPEO was diluted to different concentrations and then was added to the medium.

#### *3.6. MTT Assay for Cell Viability*

Cell viability was evaluated by 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide (MTT) assay according to the method of Satooka et al. [33]. The cell density was 7 × 10<sup>4</sup> cells/mL and the cells were seeded on 96-well cell culture plates at 100 µL per well. After 24 h of culture, the original culture solution was aspirated. Cells were exposed to various concentrations of PPEO or kojic acid (71 µg/mL), with 6 replicates for each concentration (500 µmol/L of kojic acid is equal to 71 µg/mL). After culture for another 24 h, 100 µL of 0.5 mg/mL MTT was added to each well, followed by inoculation for 4 h at 37 ◦C. The liquid was carefully aspirated from the well, and then 150 µL of DMSO was added to each well. After 10 min of shaking, the absorbance was measured at 490 nm using an M200 pro enzyme-labeled instrument (Tecan, Ltd.).

#### *3.7. Immunofluorescence Analysis and Hoechst Staining*

A sterilized coverslip was placed into each hole of the six-hole plate on an ultra-clean workbench. The cell suspension was added to each coverslip and placed in an incubator with a CO<sup>2</sup> concentration of 5% at 37 ◦C until cell fixation (2 h). After the addition of 2 mL culture medium, the culture was continued for about 6 h. The medium was decanted and the cells were washed for 5 min with PBS for 3 times. The cells were fixed with 4% paraformaldehyde for 30 min, and then paraformaldehyde was removed by PBS buffer washing. After the addition of appropriate amounts of Hoechst stain, the coverslips were incubated at room temperature for 15 min in the dark. The coverslips were then rinsed 3 times with PBS for 5 min each time, and the side with the cells was observed under a confocal laser scanning microscope [34].

#### *3.8. Determination of Melanin Content*

Melanin content was determined as described by Huang et al. [35]. B16 cells were plated at a density of 7 × 10<sup>4</sup> cells/well in a 6-well plate. The experimental group was added with 0.2 µmol/L α-MSH (melanocyte-stimulating hormone) to construct a cell model with high melanin expression. After 12 h of culture, cells were exposed to various concentrations of PPEO (10–100 µg/mL). Kojic acid at a concentration of 71 µg/mL was used as a positive control. After 48 h of culture, the supernatant was discarded and the cells were washed 3 times with PBS buffer. After the addition of 200 µmol/L NaOH solution (containing 10% DMSO) to each well, the cells were fully lysed at 80 ◦C for 1 h, and the absorbance was measured at 492 nm. The amount of protein was measured by Micro BCA protein assay kit (Shanghai Yuye biotechnology Co., Ltd, Shanghai, China). The melanin content was calculated by normalization to the total cellular protein (1 g of melanin/mg of protein) and reported as a percentage of the control.

#### *3.9. Intracellular Tyrosinase Activity*

B16 cells were plated at a density of 7 × 10<sup>4</sup> cells/well in a 6-well plate. After 24 h of culture, cells were exposed to various concentrations of PPEO (10–100 µg/mL) or kojic acid (71 µg/mL), and incubated for additional 48 h. The cells were then washed with ice-cold phosphate buffer. The plates were frozen at −80 ◦C for 30 min. After thawing and mixing, the tyrosinase activity was measured by ELISA kit [36].

#### *3.10. Protein Extraction and Western Blot Analysis*

Total protein was extracted from cells lysed by RIPA Lysis buffer with 1% phenylmethylsulfonyl fluoride (PMSF). The amount of protein was measured by Micro BCA protein assay kit (Shanghai Yuye biotechnology Co., Ltd.). The samples were loaded on 10% sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were then placed in blocking solution and blocked at room temperature for 1 h. The membranes were incubated overnight at 4 ◦C with appropriate concentrations of specific antibodies, including rabbit monoclonal antibodies glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:10000 dilution) and rabbit monoclonal antibodies TYR (1:1000 dilution). After five or six times of washing, the blots were then incubated with secondary antibody (HRP-Goat anti Rabbit). BandScan was used to analyze the integrated density of bands.

### *3.11. Statistical Analysis*

All the experiments were performed with freshly prepared samples in triplicate. The results were expressed as means ± standard deviation (SD) and analyzed by one-way analysis of variance (ANOVA) test using SPSS 19.0 (IBM Corporation, Armonk, NY, USA). Differences were considered as statistically significant at the level of *p* < 0.05.

#### **4. Conclusions**

Our study is the first to extract essential oil from the peel of pomelo cv. Guan Xi by a cold pressing method and analyze its main components of limonene, β-myrcene, β-pinene, ocimene, and β-copaene. Our results reveal that PPEO has strong antioxidant activities against DPPH, ABTS, and superoxide anion radicals and the main active components responsible for the effect are terpenes. Besides, the effects of PPEO on the viability of B16 cells and the production of melanin were evaluated based on the B16 melanoma cell system. The results indicate that PPEO down-regulates the expression level of TYR in B16 cells, which inhibits the catalytic reaction of TYR and blocks the synthesis pathway of melanin; and it further reduces melanin production without affecting the cell viability. This study provides data support for expanding the potential application of essential oil from pomelo peel as a natural antioxidant in the food, pharmaceutical and cosmetic industries. Further research in vivo is needed to fully evaluate the potential anti-melanogenic effect of PPEO.

**Author Contributions:** S.P. provided the initial idea for research. W.H. and Y.P. designed the research. X.L. conducted the experimental work and W.H. drafted the manuscript. X.H. edited the paper. All authors discussed and approved the final manuscript.

**Funding:** This work was financially supported by National Natural Science Foundation of China (No. 31571847) and Modern Agricultural Industry Technology System Post Scientist Project of China (No. CARS-26).

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

#### **References**


**Sample Availability:** Not available.

© 2019 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/).

## **Influence of Temperature, Solvent and pH on the Selective Extraction of Phenolic Compounds from Tiger Nuts by-Products: Triple-TOF-LC-MS-MS Characterization**

**Elena Roselló-Soto 1 , Francisco J. Martí-Quijal 1 , Antonio Cilla 1 , Paulo E. S. Munekata 2 , Jose M. Lorenzo 2 , Fabienne Remize <sup>3</sup> and Francisco J. Barba 1, \***


Academic Editors: Lillian Barros and Isabel C.F.R. Ferreira Received: 26 January 2019; Accepted: 18 February 2019; Published: 22 February 2019

**Abstract:** The aim of this study was to assess the effect of temperature, solvent (hydroethanolic mixtures) and pH on the recovery of individual phenolic compounds from "horchata" by-products. These parameters were optimized by response surface methodology and triple-TOF-LC-MS-MS was selected as the analytical tool to identify and quantify the individual compounds. The optimum extraction conditions were 50% ethanol, 35 ◦C and pH 2.5, which resulted in values of 222.6 mg gallic acid equivalents (GAE)/100 g dry matter and 1948.1 µM trolox equivalent (TE)/g of dry matter for total phenolic content (TPC) and trolox equivalent antioxidant capacity (TEAC), respectively. The extraction of phenolic compounds by the conventional solvent method with agitation was influenced by temperature (*p* = 0.0073), and more strongly, by the content of ethanol in the extraction solution (*p* = 0.0007) while the pH did not show a great impact (*p* = 0.7961). On the other hand, the extraction of phenolic acids was affected by temperature (*p* = 0.0003) and by ethanol amount (*p* < 0.0001) but not by the pH values (*p* = 0.53). In addition, the percentage of ethanol influenced notably the extraction of both 4-vinylphenol (*p* = 0.0002) and the hydroxycinnamic acids (*p* = 0.0039). Finally, the main individual phenolic extracted with hydroethanolic mixtures was 4-vinylphenol (303.3 µg/kg DW) followed by spinacetin3-*O*-glucosyl-(1→6)-glucoside (86.2 µg/kg DW) and sinensetin (77.8 µg/kg DW).

**Keywords:** polyphenols; tiger nut; by-products; solvent extraction; horchata de chufa; triple TOF-LC-MS-MS

#### **1. Introduction**

"Horchata de chufa" is a typical beverage from the Valencian Community. It is obtained from tiger nuts (*Cyperus esculentus*), which are tuberous rhizomes that protrude from the tips of the plant's roots under the ground [1]. During "horchata" preparation a large amount of waste and by-products are obtained, representing "horchata" by-products ~60% of the total amount of the raw material used to obtain the beverage [2]. These by-products are a source of polysaccharides, fiber, oil (rich in oleic acid) and antioxidant compounds (e.g., vitamin E and polyphenols), among others [3].

Some previous studies have evaluated the potential application of "horchata" by-products for the preparation of new meat products, due to their high content in fiber [4–6]. However, the exploitation of "horchata" by-products as a source of phenolic compounds for food industries, nutraceuticals and cosmetics has not been widely explored. Some existing studies have evaluated the impact of the use of enzyme pre-treatments alone or combined with high-pressure to extract phenolic compounds from tiger nuts [7]. In this line, a previous study conducted by our research group evaluated the impact of conventional solvent extraction using a combination of binary mixtures consisting of ethanol and water at different percentages, at different temperatures and extraction times to recover total phenolic compounds and total flavonoids with antioxidant capacity from "horchata" by-products, obtaining promising results, particularly with the use of mild heating (up to 60 ◦C) and hydroethanolic solvents (0%–50% ethanol) [8]. Another relevant aspect of phenolic compound extraction is the selection of an appropriate pH that can influence the yield and stability of phenolic compounds. Acidic conditions are associated with higher extraction yields (higher interaction of phenolic compounds with the solvent) on different vegetable sources of phenolic compounds [9,10].

However, only spectrophotometric methods were used, as it was a preliminary study. It is well known that it is not only important to evaluate the total amount of polyphenols but also to characterize their profile as the biological activity differs according to the targeted compound. Accordingly, the use of chromatographic techniques is currently encouraged to establish the structure and activity of bioactive compounds (that can be complemented by less specific but informative spectrophotometric methodologies) to estimate the impact of conventional and non-conventional extraction processes, processing and bioaccessibility outcomes [11]. Therefore, in the present study, the impact of temperature, solvent (hydroethanolic mixtures) and pH on the recovery of individual phenolic compounds from "horchata" by-products was evaluated. For this purpose, a response surface methodology (RSM) approach was used to optimize the extraction. Moreover, triple-TOF-LC-MS-MS was selected as the analytical tool to identify and quantify the individual compounds. In addition, the results were compared to those obtained after using supercritical carbon dioxide and Folch extraction methodology.

#### **2. Results and Discussion**

*2.1. Impact of Temperature, Solvent and pH on the Selective Extraction of Total Phenolic Compounds (TPC) and Trolox Equivalent Antioxidant Capacity (TEAC) from Tiger Nuts by-Products*

The conventional extraction with hydroethanolic mixtures was optimized according to a Box-Behnken design in order to maximize the TEAC and TPC values. The TPC and TEAC values for each extraction are shown in Table 1.

The TPC and TEAC values ranged from 186.52 to 222.58 mg GAE/100 g of dry matter and 617.80 to 1948.07 µM TE/g of dry matter, respectively. The best condition according to the factorial design for TPC and TEAC was 35 ◦C, 50% ethanol and pH 2.5. Our TPC values were higher than those obtained by Ogunlade et al. [12] who found values of 115.70 mg GAE/100 g of tiger nut in roasted tubers. In addition, Oladele et al. [13] noticed TPC values of 351 and 134 mg/100 g for yellow and brown tiger nuts, respectively. Koubaa et al. [14] obtained TPC values of 4.53–6.21 mg GAE/100 g of oil and 4.71–5.29 GAE/100 g of oil, for supercritical fluids (SC-CO2) and mechanical expression (ME) extractions, respectively. Parker et al. [15] found TPC values ranging from 5.63 to 64.9 mg/100 g for tiger nut, whereas Badejo et al. [16] obtained TPC values of 21.67 mg/100 mL for a tiger nut aqueous extract drink.

Moreover, Roselló-Soto et al. [8] reported that TPC values obtained from "horchata" by-products according to the solvent used, temperature and extraction time, showing that the highest TPC values were obtained using 25% ethanol (*v*/*v*), at 60 ◦C with an extraction time of 3 h. The difference in the TPC values could be due to raw material studied but also to the methods of extraction and analysis.



‐

**μ**

The influence of pH (2.5–12), temperature (25–50 ◦C) and volume of ethanol (0%–50%) to obtain phenolic compounds by conventional extraction with an ethanol:water mixtures was analyzed using a response surface methodology (RSM). As can be seen in Figure 1A, the extraction of phenolic compounds by the conventional method was influenced by temperature (*p* = 0.0073), and more strongly, by the content of ethanol in the extraction solution (*p* = 0.0007). On the contrary, the pH did not show a great impact (*p* = 0.7961). Regarding temperature, we appreciated that at pH = 7, the optimum value in an extraction without ethanol was 37 ◦C. However, at the highest studied ethanol concentration (50%), the optimum temperature was increased up to 43.5 ◦C. Furthermore, increasing the ethanol percentage led to a clear increase in extraction yield at temperatures above 40 ◦C, but on the contrary, this improvement was less clear at room temperature (25 ◦C). As proposed elsewhere, when temperature increases, the integrity of the cell wall is altered, and therefore there is a greater contact of the cellular components, among them the polyphenols, with the extraction solution [17]. However, over a certain threshold, despite the increased extraction of polyphenols due to matrix degradation, a decrease of several of these bioactive compounds can happen due to thermal lability, as observed in Figure 1A.

**Figure 1.** *Cont.*

‐ ‐ **Figure 1.** Plot for the influence of extraction condition parameters in total phenolic content (mg gallic acid equivalents ((GAE)/100 g of dry matter) (**A**) and the main effects chart for antioxidant activity (**B**) using solid-liquid extraction. One gram of tiger nuts by-products was extracted using different temperatures (25–50 ◦C), ethanol:water mixture (0%–50% ethanol) and pH (2.5–12).

‐ ‐ ‐ These values fully agree with those obtained by Moreira et al. [18], who observed that using a concentration of 50% ethanol, a 2-fold increase in the values of TPC from apple tree wood was obtained, compared to control samples without ethanol. In addition, they also found that temperature had an important impact on the extraction, getting a greater extraction at 55 ◦C compared to 20 ◦C. Vatai et al. [19] also reached the same conclusions, obtaining the maximum TPC values in Refosk (red grape marc) and lyophilized elderberry with an optimum percentage of 50% ethanol at 60 ◦C. On the contrary, Roselló-Soto et al. [8] observed an 82% increase in the TPC value from tiger nuts by-products with an ethanol volume of 50% at 60 ◦C during 2 h of extraction, but this changed at 3 h, obtaining the maximum TPC values with an ethanol concentration of 25%.

‐ ‐ Figure 1B shows the main effects observed for antioxidant capacity at different temperatures, ethanol concentration and pH. It is clearly observed how an increase in the ethanolic fraction during the extraction of the polyphenols significantly increased the antioxidant capacity of the samples (*p* = 0.0029). Besides, neither the extraction temperature nor the pH influenced the antioxidant capacity (*p* = 0.2328 and 0.3635, respectively). Roselló-Soto et al. [8] also obtained an increase in the antioxidant capacity (TEAC assay) of extracts from "horchata" by-products when they used an ethanol concentration of 50% and an increase in temperature up to 60 ◦C. In our case, there was also an increasing trend (*p* > 0.05) in the antioxidant capacity when the temperature was augmented, as well as when highly acidic and alkaline conditions were used. However, as it was mentioned above, these increases are not significant. Moreover, Li et al. [20] also indicated that the TEAC values of extracts obtained from *Gordonia axillaris* increased with an ethanol volume of 40% and a temperature of 40 ◦C, but decreased when these values were higher. According to the authors, this could be due to the degradation of some thermolabile antioxidant compounds. Moreira et al. [18] studied the extraction of polyphenols and antioxidant capacity from apple tree wood, observing the highest antioxidant capacity (measured using FRAP assay) after using a temperature of 55 ◦C and 50% ethanol volume. Similarly, Rusu et al. [21] indicated that increasing ethanol percentage in the solvent (from 50% to 95%) and extraction temperature (from 20 to 40 ◦C) were associated with higher TPC and TEAC values in walnut septum extract. Interestingly, the authors also obtained higher TPC and TEAC values by carrying out the extraction with 50% ethanol solution at 20 ◦C. In addition, Bamba et al. [22] obtained an increase in the antioxidant capacity (measured using DPPH assay) with augmented temperature, but since the content of phenolic compounds decreased, this increase could be due to the presence of other antioxidant compounds.

The ANOVA results (Table 2) show that not only these parameters by themselves can affect the extraction yield, but the combination of them also can produce significant changes. This is the case of TPC, in which the combination of temperature and ethanol can modify TPC extraction (*p* = 0.0318).


**Table 2.** Analysis of variance (ANOVA) results for each effect in response surface methodology (RSM) of total phenolic compounds (TPC) and trolox equivalent antioxidant capacity (TEAC) values.

Sig: significance; ns: not significant, \* (*p* < 0.05); \*\* (*p* < 0.01); \*\*\* (*p* < 0.001).

#### *2.2. Impact of Temperature, Solvent and pH on the Selective Extraction of Individual Phenolic Compounds from Tiger Nuts by-Products*

The profile and content of specific phenolic compounds extracted with ethanol:water mixtures from tiger nut by-products using the RSM methodology for optimization and after analyzing the extracts by Triple-TOF-LC-MS-MS are shown in Table 3 and Figure 2.

Specifically, as can be seen in Figure 2A, both pH and temperature had a great influence on the extraction of lignans (*p* = 0.0256 and 0.0251, respectively). The maximum yield was observed at pH 10.62 and 43.3 ◦C. However, at room temperature (25 ◦C) the optimum pH dropped to 6.2. This outcome agrees with the data previously obtained by Tu et al. [23] who found an optimum pH in the range 5.5–6 for the extraction of lignans at room temperature for *Fructus forsythiae*.

Phenolic acid extraction was also influenced in a very large amount by temperature (*p* = 0.0003) and by the volume of ethanol (*p* < 0.0001) but not by the pH (*p* = 0.53) (Figure 2B). In this case, it should be noted how the maximum extraction yield was obtained for a temperature of 50 ◦C and an ethanol content of 41.4%. Elez-Garofuli´c et al. [24] also observed an increase in the extraction of phenolic acids by increasing the temperature using the microwave-assisted extraction on sour cherry Marasca, obtaining an optimum temperature of 70 ◦C. A similar trend was also reported by Waszkowiak et al. [25], who studied the influence of the percentage of ethanol used for the extraction of phenolic compounds of flaxseeds extracts, ranging from 60% and 90%, and they observed that the best ratio for phenolic acids corresponded to 60% ethanol in water.

*Molecules***2019**, *24*, 797



Flavonoids:

 Cyanidin;

 Spinacetin

3-*O*-glucosyl-(1→6)-glucoside

 (SGG). ND: not detected.

‐ ‐ ‐ **Figure 2.** Response surface plot for the influence of extraction condition parameters in lignans (**A**) and phenolic acids (**B**). Main effects chart for 4-vinylphenol (**C**) and hydroxycinnamic acids (**D**) using solid-liquid extraction. 1 g of tiger nuts by-products was extracted by varying temperature (25–50 ◦C), ethanol:water mixture (0%–50%) and pH (2.5–12).

Furthermore, the percentage of ethanol influenced notably the extraction of both 4-vinylphenol , and the hydroxycinnamic acids (*p* = 0.0039) (Figure 2C,D). This finding can be explained by the polarity of the solvent, since increasing the volume of ethanol in water increases the polarity. As it is known, these compounds are polar, so a more polar solvent will extract them better. Other authors such as Wo ´zniak et al. [26], Chew et al. [27] and Paini et al. [28] also used mixtures with ethanol to improve the yield of polyphenols extraction. In the case of flavonoids, no statistically significant differences were found on the extraction yield of dihydroxybenzoic acids and flavones when varying these parameters.

In Table 4 the ANOVA results are shown for the influence of temperature, ethanol and pH in the extraction of the compounds explained in Figure 2. As can be seen, for the lignans extraction the combination of temperature and pH had a significant effect (*p* = 0.0344). This could be explained by the increase of solubility, which improved the extraction of these compounds.


**Table 4.** ANOVA results for each effect in RSM of TPC and TEAC values.

Sig: significance; ns: not significant, \* (*p* < 0.05); \*\* (*p* < 0.01); \*\*\* (*p* < 0.001).

Possible beneficial effects of polyphenols on human health are the subject of increasing scientific interest. For example, phenolic acids and lignans have been shown to have a positive hepatoprotective action [29]. The anti-inflammatory action of ferulaldehyde was also found in mice by other authors [30]. For all these reasons, it is necessary to consider the most appropriate extraction conditions depending on the compounds desired to obtain.

#### *2.3. Optimization and Validation of the Extraction Conditions*

The combination of critical parameters (temperature, ethanol and pH), which allowed to obtain the highest TPC yield and TEAC value. To do this, an optimization based on desirability was used. Theoretically, in the case of TPC, optimum values of 229.29 mg GAE/100 g of dry matter were obtained with conditions of 43.7 ◦C, 50% ethanol and pH = 2.5. For the antioxidant capacity, the optimum value obtained was 1846.34 µM TE/g of dry matter at a temperature of 50 ◦C, a volume of ethanol of 50% and a pH = 2.5. As can be seen in Table 1, the maximum extraction of TPC and the maximum TEAC values were obtained experimentally with the conditions of 35 ◦C, 50% ethanol and pH = 2.5 (222.58 ± 2.16 mg GAE/100 g of dry matter and 1948.07 ± 434.18 µM TE/g of dry matter respectively). These results are close to those expected theoretically, so we can affirm that the method has been validated for TPC and TEAC.

### *2.4. Comparison of Hydroethanolic Extraction of Individual Phenolics Compounds from Horchata by-Products with Those of Folchand Supercritical-CO<sup>2</sup> Extraction*

The data on individual phenolics obtained in the present study were compared to those obtained after conventional extraction method (Folch) and an innovative extraction method (supercritical CO<sup>2</sup> extraction). The results for the comparison were obtained from a previously published article about the extraction of phenolic compounds in the oil fraction of "horchata" by-products [31].

It can be noticed that there was a great difference in the compounds obtained with the three different extraction techniques. As for conventional extraction with Folch method, the major compound obtained by far was 4-vinylphenol (216.9 µg/kg DW), followed by p-coumaric acid (25.35 µg/kg DW) and benzoic acid (13.54 µg/kg DW). In contrast, the main compounds extracted by SC-CO2, especially at 40 MPa, were isohydroxymatairesinol (399.44 µg/kg DW), scopoletin (93.24 µg/kg DW) and caffeic acid (30.66 µg/kg DW). In the present study (see Table 2), the main individual phenolic extracted with hydroethanolic mixtures was 4-vinylphenol (303.3 µg/kg DW) followed by spinacetin3-O-glucosyl-(1→6)-glucoside (86.2 µg/kg DW) and sinensetin (77.8 µg/kg DW).

It is worth noting that 4-vinylphenol was the predominant phenolic compound obtained, after using both conventional methods, despite the different polarity of solvents employed, though higher extraction is accomplished with hydroethanolic mixtures. Moreover, a great difference was also observed between the compounds obtained after conventional extraction (Folch and hydroethanolic mixtures) and those obtained by SC-CO2. As already mentioned, a more polar solvent facilitates the extraction of more phenolic compounds, therefore the low yield obtained in the SC-CO<sup>2</sup> extraction is not surprising. We should also keep in mind that the way to prepare the sample is different. In the case of conventional extraction, homogenization is carried out at 10,000× rpm, while for SC-CO<sup>2</sup> extraction the sample is only milled. This protocol differences greatly modify the accessibility of the solvents to the intracellular compounds, since in the case of conventional extraction the samples were homogenized with the solvent, breaking the cellular structures, whereas in the SC-CO<sup>2</sup> extraction only the particle size of the samples was reduced.

These findings agree with the data previously reported by Parker et al. [15] who demonstrated that tiger nuts skins are richer in *p*-coumaric acid than tiger nuts tubers, being this compound the fourth most important (48 µg/kg DW) in "horchata" by-products after hydroethanolic extraction. In addition, Ezeh et al. [7] studied the polyphenols present in tiger nuts, finding mainly *trans*-cinnamic acid. Oladele et al. [13] also determined the phenolic profile of tiger nuts, considering the yellow and brown varieties. They obtained differences between both species, since in yellow tiger nuts the main compounds were ferulic acid (~58 mg/100 g), *p*-hydroxybenzoic acid (~29 mg/100 g), *p*-hydroxybenzaldehyde (~16 mg/100 g) and vanillic acid (~6 mg/100 g), whereas in brown tiger nut, vanillic (~15 mg/100 g), *p*-coumaric (~17 mg/100 g), caffeic (~15 mg/100 g), ferulic (~34 mg/100 g) and sinapinic acids (~21 mg/100 g) were predominant.

#### **3. Material and Methods**

#### *3.1. Chemicals and Reagents*

Sodium hydroxide (NaOH), sodium carbonate (Na2CO3), and acetone were obtained from J.T.Baker (Deventer, Holland). ABTS radical (2,2′ -azino-bis(3-ethylbenzothiazoline-6-sulphonic acid), Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), Folin-Ciocalteau phenol reagent 1N, gallic acid, sodium nitrite (NaNO2), formic acid (HPLC grade), potassium persulfate (K2S2O8) and ethanol p.a. (99.5%) were purchased from Sigma-Aldrich (Steinheim, Germany). Sodium hydrogen carbonate (reagent grade; 99.7%) and methanol (reagent grade; 99.9%) were purchased from Scharlau (Barcelona, Spain). Deionized water was obtained from Millipore (Bedfore, MA, USA).

#### *3.2. Samples*

A conventional process was used for obtaining "horchata" from tiger nuts with a denomination of origin "Chufa de Valencia" (*Cyperus esculentus*). Then "horchata" by-products were taken and provided by the "Consejo Regulador D.O. Chufa de Valencia" (Valencia, Spain). Afterwards, they were dried at 60 ◦C for 72 h using a Memmert UFP 600 air-circulating oven (Schwabach, Germany) and ground for obtaining uniform particle size. Finally, they were vacuum packed until needed.

#### *3.3. Extraction at Different Temperatures, Ethanol:Water Mixtures and pH*

The conditions for solid–liquid extraction were selected based on a previous study [32]. First, one gram of dehydrated "horchata" by-product was weighed and fifteen milliliter of the hydroethanolic mixtures at different ethanol concentrations (0%, 25% and 50%, *v*/*v*) and different pH (2.5, 7.25 and 12), adjusted with NaOH or HCl was used for the extraction. The beakers with the samples were then placed and stirred on a plate with a magnetic stirrer. To avoid the evaporation of the solvent during the extraction, the samples were covered with aluminum foil. The temperature was adjusted to 25, 35 and 50 ◦C in each of the stirring plate's rows. The extraction was carried out for 3 h. The obtained samples were filtered through Whatman No. 1 and used for the determination of phenolic compounds.

#### *3.4. Total Antioxidant Capacity*

TEAC (Trolox equivalent antioxidant capacity) assay was used for the determination of the total antioxidant capacity [8]. Twenty-five milliliter of ABTS (7 mM) was mixed with 440 µL of K2S2O<sup>8</sup> (140 mM) and kept at room temperature for 12–16 h under darkness. For the determination, the absorbance of ABTS•<sup>+</sup> working solution was measured at a wavelength of 734 nm on a Perkin-Elmer UV/Vis Lambda 2 spectrophotometer (Perkin-Elmer, Jügesheim, Germany) to obtain the initial absorbance (A0). When the absorbance of the mixture was 0.700 ± 0.020, 100 µL of the extracts appropriately diluted were added, and the absorbance was measured at 20 min (A<sup>f</sup> ). The following equation was used to calculate the inhibition percentage (%) of the samples:

$$\% \text{ Inhibition} = (1 - \text{A}\_{\text{f}}/\text{A}\_{0}) \times 100\%$$

where A<sup>0</sup> is the absorbance at the initial time and A<sup>f</sup> is the absorbance obtained after 20 min.

The results were expressed as micromolar Trolox equivalent (µM TE)/g of dry matter.

#### *3.5. Determination of Total Phenolic Content (TPC)*

The method previously reported [33], with some modifications [34] was used. To sum up, 500 µL of extract was mixed with 4.5 mL of distilled water and then 1 mL of the 2% Na2CO<sup>3</sup> solution (*w*/*v*), and 0.25 mL of the Folin-Ciocalteau reagent (1N) were added. The mixture was left to stand for one hour under darkness at room temperature. Afterwards, the absorbance was measured at 765 nm. TPC was determined by interpolating the absorbance values in a calibration curve using gallic acid standard (10 µg/mL) at different concentrations between 0 and 5 µg/mL. The results were expressed as mg equivalents of gallic acid (GAE)/100 g of dry matter.

#### *3.6. Triple TOF–LC–MS–MS Characterization of Phenolic Compounds*

The phenolic profile characterization and quantification was performed according to the previously described method [31], using an Agilent 1260 Infinity (Agilent, Waldbronn, Germany) with a Waters UPLC C18 column 1.7 µm (2.1 × 50 mm) Acquity UPLC BEH.C18 (Waters, Cerdanyola del Vallès, Spain) for the separation of the main phenolic compounds in the samples. Moreover, a TripleTOF™ 5600 LC/MS/MS system (AB SCIEX, Foster City, CA, USA) was utilized for the identification. For that purpose, a mobile phase consisting of solvent A (water, 0.1% formic acid) and solvent B (methanol, 0.1% formic acid) was used as follows: 0 min 90% A; 13 min 100% (B); 15 min 90% A. Five microliter and 0.4 mL/min were the injection volume and flow rate, respectively.

MS data were obtained between 80 and 1200 *m*/*z* on negative mode, and the IDA acquisition method was carried out in the survey scan type (TOF-MS) using the dependent scan type (product ion). Ion spray voltage (−4500 V); declustering potential (90 V); collision energy (−50 V); temperature with 25 psi curtain gas (400 ◦C); 50 psi for both ion source gas 1 (GC1) and ion source gas 2 (GS2) were used as the main parameters for the MS analysis.

The IDA MS/MS analysis was carried out with ion tolerance of 50 mDa, 25 V collision energy and activated dynamic background subtract. The software analyst PeakView1.1 (AB SCIEX, Foster

City, CA, USA) and its applications (XIC Manager and Formula Finder) were used for data acquisition and processing. Finally, an external calibration curve using resveratrol as standard was prepared for the quantification of phenolic compounds.

#### *3.7. Experimental Design and Statistical Analyses*

Box-Behnken design with three levels (maximum, minimum, and central) of each independent variable, temperature (25–50 ◦C), the concentration of ethanol (0%–50%), and pH (2.5–12), leading to 15 combinations of these variables. Independent variable levels were selected accounting for the sample and the potential degradation of thermolabile antioxidant compounds after high temperatures (>50 ◦C). The combinations included temperature–ethanol–pH conditions with an intermediate level (central point) of the three variables replicated three times, which was used to check the reproducibility and stability of the results. Experiments were randomized to minimize the systematic bias in the observed responses due to extraneous factors and for higher precision. In addition, we studied whether there were correlations between a pair of variables. The significant differences (*p* < 0.05) between the results were calculated by analysis of variance (ANOVA), using the least significant difference (LSD) test to indicate the samples between which there were differences. All statistical analyses were performed using the software Statgraphics® Centurion XV (Statpoint Technologies, Inc., The Plains, VA, USA).

#### **4. Conclusions**

The optimization of phenolic compounds extraction by response surface methodology, followed by Triple-TOF-LC-MS-MS characterization, indicated that temperature and ethanol content are more important variables than pH. The optimum extraction conditions for total phenolic content and antioxidant activity were 50% ethanol, 35 ◦C and pH 2.5, which could obtain values of 222.6 mg GAE/100 g of dry matter and 1948.1 µM TE/g of dry matter for TPC and TEAC, respectively. The optimized extraction condition also positively influenced the extraction of the main individual phenolic compounds of tiger nuts by-products, particularly 4-vinylphenol and hydroxycinnamic acids. Therefore, tiger nuts by-products can be explored as a valuable source of phenolic compounds with potential applications in food industries, nutraceuticals and cosmetics.

**Author Contributions:** E.R.-S., F.J.B. and J.M.L. conceived, designed and carried out the experiments. E.R.-S., F.J.B., J.M.L., and P.E.S.M. analysed the data and wrote the paper. E.R.-S., F.J.M.-Q., A.C., F.J.B., J.M.L., P.E.S.M. and F.R. wrote and reviewed the paper before submitting.

**Funding:** This work was supported by the project GV/2018/040 "Implementación y optimización de procesos innovadores para la valorización de los subproductos obtenidos a partir del proceso de elaboración de la horchata" for emerging research groups from the Generalitat Valenciana.

**Acknowledgments:** Paulo E. Munekata acknowledges the postdoctoral fellowship support from the Ministry of Economy and Competitiveness (MINECO, Spain) "Juan de la Cierva" program (FJCI-2016-29486). The authors would like to thank the Regulatory Council D.O. Tiger nut of Valencia (Valencia, Spain) for providing "horchata" by-products. Moreover, we are also grateful for the technical support from the "Central Service for Experimental Research (SCSIE)" at the Universitat de València for the help provided in the analysis using the Triple TOF-LC-MS-MS.

**Conflicts of Interest:** The authors have no conflict of interest to disclose.

#### **References**


**Sample Availability:** Samples of the compounds are available from the authors.

© 2019 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/).

*Article*

## **Ultrasound as a Rapid and Low-Cost Extraction Procedure to Obtain Anthocyanin-Based Colorants from** *Prunus spinosa* **L. Fruit Epicarp: Comparative Study with Conventional Heat-Based Extraction**

**Maria G. Leichtweis 1 , Carla Pereira 1 , M.A. Prieto 1,2 , Maria Filomena Barreiro 1,3 , Ilton José Baraldi 4 , Lillian Barros 1, \* and Isabel C.F.R. Ferreira 1, \***


#### Academic Editor: Marcello Locatelli

Received: 13 December 2018; Accepted: 1 February 2019; Published: 5 February 2019

**Abstract:** An ultrasound rapid and low-cost procedure for anthocyanin-based colorants from *Prunus spinosa* L. fruit epicarp was developed, and the advantages were compared with conventional heat-based extraction. To obtain the conditions that maximize anthocyanins' extraction, a response surface methodology was applied using the variables of time, temperature, and ethanol content, in the case of heat extraction, whereas for ultrasound assisted extraction, temperature was replaced by ultrasound power. Two anthocyanin compounds were identified by HPLC-DAD-ESI/MS—namely, cyanidin 3-rutinoside and peonidin 3-rutinoside. The responses used were the extraction yield and the content of the identified anthocyanins. Ultrasound extraction was the most effective method at 5.00 ± 0.15 min, 400.00 ± 32.00 W, and 47.98% ± 2.88% of ethanol obtaining 68.60% ± 2.06% of extracted residue, with an anthocyanin content of 18.17 mg/g (extract-basis) and 11.76 mg/g (epicarp-basis). Overall, a viable green process was achieved that could be used to support pilot-scale studies for industrial production of anthocyanin-based colorants from *P. spinosa* fruit epicarp.

**Keywords:** *Prunus spinosa* L. fruit epicarp; wild fruit valorization; cyanidin 3-rutinoside; peonidin 3-rutinoside; heat and ultrasound assisted extraction; response surface methodology

#### **1. Introduction**

*Prunus spinosa* L. (blackthorn) is a spontaneous wild shrub found in Portugal, Spain, and other European countries. Its fruits are commonly used for liqueur and jam preparations, as well as for medicinal purposes [1]. Nevertheless, no reports were found regarding the industrial, or large scale, use of these fruits, probably because of their bitter and astringent taste.

The valorization of agricultural products has gained much attention in the late years as a mean for a sustainable management, which can concomitantly increase the profit of local economies. In this regard, *P. spinosa* constitutes an underexploited source and can serve as a raw material for

the recovery/production of compounds for food applications [2]. As with other *Prunus* species, anthocyanin compounds can be found in blackthorn fruits at high levels, being responsible for their typical coloration [3,4]. In fact, a complex profile of anthocyanins was previously identified in *P. spinosa* fruits, among which cyanidin 3-rutinoside and peonidin 3-rutinoside were found to be predominant [5,6].

Anthocyanins are natural pigments belonging to the phenolic compounds group and, within that, to the flavonoids class, presenting a range of colors between red, blue, and violet that are characteristic of various fruits and vegetables [7]. Beyond their various physiological benefits, which include effects against cardiovascular diseases, atherosclerosis, and cancer, recently, an increasing interest in these compounds began to arise because of their colorant properties [8,9].

The industrial production of natural-based colorants has been established for years and consists mainly of obtaining colorant-rich extracts through conventional heat assisted extraction (HAE, or maceration) using water as a solvent followed by several isolation/drying steps. This type of conventional process, although used at large-scale, is known for requiring high-energy consumption and long extraction times [10–12]. Alternative extraction processes, able to replace traditional ones, have been established to shorten the needed time, decrease energy requirements, and reduce solvent consumption. Among the non-conventional procedures applied to anthocyanins' extraction, ultrasound, microwave, and supercritical fluid assisted extraction techniques have attracted, in the recent years, the attention of industrials and researchers [10,13]. Regarding ultrasound assisted extraction (UAE), it is considered an inexpensive, simple, and efficient alternative to conventional techniques [14]. The extraction capability of UAE is attributed to mechanical and cavitation phenomena, which lead to cells' disruption, particle size reduction, and enhanced mass transfer across the cell membrane [11,13].

To obtain anthocyanin-rich extracts, it is crucial to consider the factors affecting the stability of these compounds, including structure and concentration, pH, temperature, light exposure, oxygen levels, and used extraction solvents [15]. Thus, the choice of the extraction method, along with the optimization of relevant extraction variables, are essential to guarantee a maximum recovery efficiency [16]. Additionally, the efficiency is also strongly affected by the variability observed among different matrices [17]. Through response surface methodology (RSM), it is possible to optimize the relevant variables simultaneously, obtaining polynomial models capable of describing, within the tested experimental interval, the ideal conditions that maximize the used response criteria [13].

In the present study, the goal was to explore blackthorn anthocyanin composition and promote a higher commercial value of these wild fruits through the development of an anthocyanin-based coloring extract. For that purpose, the fruit epicarp was used because it has a much more intense color than the pulp, and thus a higher concentration of anthocyanins and less interfering compounds in the extraction process (e.g., sugars). To the best of our knowledge, and according to a thorough literature survey, no reference or report on the optimization of anthocyanin compounds extraction from fruit epicarps of *P. spinose* was found. Therefore, the present study aimed to optimize the extraction of these compounds from *P. spinosa* fruit epicarps through HAE and UAE techniques, evaluating the following variables: i) type of solvent (water and green organic solvents); ii) extraction time; iii) solid-to-liquid ratio; and iv) temperature (for HAE) or pressure (for UAE). The most efficient parameters were obtained by response surface methodology (RSM). The identification and quantification of the anthocyanin compounds present in the extracts was assessed by HPLC-DAD-ESI/MS.

#### **2. Results**

#### *2.1. Development of RSM Models to Optimize Responses and Conditions*

The RSM is a valuable instrument to assess the impact of the main extraction factors and their interactions on one or more responses. The technique uses fixed experimental designs with the major goals of minimizing the experimental labor and finding optimal solutions. In this regard, the work presented here uses the *circumscribed central composite design (CCCD)* design plan, which is a popular design among researchers when trying to optimize food processing methods [18], such as the extraction of anthocyanin compounds.

In a previous study [5], authors identified, using HPLC-DAD-ESI/MS, the anthocyanin compounds of cyanidin 3-rutinoside ([M + H]<sup>+</sup> at *m*/*z* 595) and peonidin 3-rutinoside ([M + H]<sup>+</sup> at *m*/*z* 609) in *P. spinosa* fruits, and highlighted that the colorant capacity of these fruits is mainly attributed to these compounds. Although authors quantify the content of those anthocyanin compounds in *P. spinosa* fruits, the conditions of extraction were not optimized. Therefore, based on those preliminary findings, it seems logical to continue to explore the potential of *P. spinosa* fruits as a source of anthocyanin compounds. In this regard, the study applies a RSM technique under a *CCCD* to optimize the operating conditions of the extraction of two common techniques in the industrial environment (HAE and UAE) with the intention of maximizing their extraction. However, because the major quantity of anthocyanin compounds in *P. spinosa* fruits is located in the fruit epicarp, in this study, we ignored the inside parts of the fruit and focused the attention on the fruit epicarps. Additionally, by focusing on the epicarps, we are avoiding a high content of interfering compounds in the extraction process (e.g., sugars) that would require further purification steps. Figure S1 (supplemental material) shows a complete summary of all the steps used for the optimization procedure in order to recover the anthocyanin compounds from the epicarps of *P. spinosa* fruits. Figure S2 (supplementary material) shows a chromatographic example of HPLC-DAD-ESI/MS results for the quantification of anthocyanin compounds in the epicarps of *P. spinosa* fruits.

Table 1 shows the experimental results derived from the *CCCD* used to optimize the extraction of anthocyanins from the fruits epicarps (*Y1*, mg C/g R; *Y2*, mg C/g E dw; and *Yield,* %) for each one of the computed extraction techniques (HAE and UAE). As described, the *CCCD* experimental results are subjected to the mathematical analysis of Equation (1), by applying a fitting procedure coupled with non-linear least-squares estimations. The parametric values of Equation (1) derived from this analytical procedure, the corresponding confidence interval of the parameters (α = 0.05) found after modelling the extraction response values, and basic statistical information of the mathematical procedure are presented in Table 2. The parametric values considered non-significant (*ns*) values were excluded from model construction and the final equations for describing the responses assessed using significant terms are presented in Table S1 (supplementary material).

The significant parametric values in Table 2 are presented as a function of the codification criteria of the *CCCD*. Although they could be presented as the real numerical ranges of the variables assessed (*X<sup>1</sup>* to *X3*), such information would not provide any additional insights of the regression analysis performed or the possible effects that may occur. The key information is the weight of the numerical values of the significant parameters; therefore, it seems logical to present them under a codification mode that allows us to compare the values between them effortlessly. Therefore, based on the numerical values derived, some global conclusions can be deduced as follows:


**Table 1.** Experimental results of the *CCCD* used for the response surface methodology (RSM) optimization of the three main variables involved (*X1*, *X2*, and *X3*) in the heat assisted extraction (HAE) and ultrasound assisted extraction (UAE). Responses comprised three format values assessed (*Y1*, mg C/g R; *Y2*, mg C/g E dw; and *Yield*, %).


In general, positive and highly significant effects of LE, QE, and IE are found to moderately affect the studied responses. In both techniques assessed (HAE and UAE), the variable *S* is the most relevant one. Initial increases of *S* cause an increase of the extraction efficiency until it reaches a maximum, in which case the increase will cause a decrease in the extraction, but its interactive effect with the variable *t* and *T* or *P* causes a more favorable influence.

Additionally, using the significant parametric values of Table 2 coupled with a simplex methodology, it is possible to determine the absolute/relative optimal values of conditions to maximize the responses individually or globally, in order to obtain the most efficient extraction process. Table 3 shows the individual and global optimal response values and the corresponding conditions that produced them. In consequence, the extracting techniques (HAE and UAE) according to the three response value formats (*Y1*, mg C/g R; *Y2*, mg C/g E dw; and *Yield,* %) for each assessed anthocyanin (C1 and C2), as well as for the total anthocyanin content (CT = C1 + C2), are depicted.

**Table 2.** Parametric results of the second-order polynomial equation of Equation (1) for the HAE and UAE techniques assessed and for the three response value formats (*Y<sup>1</sup>* , mg C/g R; *Y<sup>2</sup>* , mg C/g E dw; and *Yield,* %). The parametric subscript 1, 2, and 3 stands for the variables involving *t* (*X<sup>1</sup>* ), *T* or *P* (*X<sup>2</sup>* ), and *S* (*X<sup>3</sup>* ), respectively. Analyses of significance of the parameters (α = 0.05) are presented in coded values. Additionally, the statistical information of the fitting procedure to the model is presented.




### *2.2. Alternative Visual Illustration of the Effects of the Extraction Variables on the Target Responses Used*

Although the parametric values show the behavior of the responses, and could be used to understand their patterns, a more visual way to express the effects of variables on the extraction of any type of response is to generate 3D surface and/or contour plots, by varying two variables in the experimental range under investigation and holding the other one at a fixed level. In this regard, Figures 1 and 2 show the 3D surface and 2D contour plots, respectively, representing the influence of the investigated effects of HAE and UAE parameters on the extraction behavior. The plots enable one to visualize the influence and interaction between the variables. Visual analyses of 3D surface and 2D contour plots are in accordance with parametric values derived from the multiple regression analysis, as described in Table 2 (parametric values) and Table S1 (full mathematical models, supplementary material).

*Molecules* **2019**, *24*, 573

**Figure 1.** Illustration of the graphical results obtained by heat assisted extraction (HAE) and ultrasound assisted extraction (UAE) for the extraction *yield* of the residual content material (R) and the total detected anthocyanin compounds (cyanidin 3-rutinoside and peonidin 3-rutinoside, CT = C1 + C2) in terms of two response formats (*Y1*, mg C/g R and *Y2*, mg C/g E dw). Full results are described in Table 1. Every figure is presented in two parts. Part A shows the 3D net surfaces predicted by Equation (1) when the excluded variable is positioned at the individual optimum (Table 3). Part B describes the statistical analysis in a graphical form to show the goodness of fit of the models applied.

**Figure 2.** The optimized isolines projections for the extraction of C1 (cyanidin 3-rutinoside) and C2 (peonidin 3-rutinoside) as a function of the combination of the three main variables involved (*X<sup>1</sup>* , *X<sup>2</sup>* , and *X<sup>3</sup>* ) in the HAE and UAE. For each compound, the two response value formats (*Y<sup>1</sup>* , mg C/g R and *Y2* , mg C/g E dw) are presented to describe the most favorable conditions. Furthermore, the response projections of the *yield* of the extracted residual material are presented. All the contour graphs were built by the second order polynomial models generated by Equation (1) (Table S1) when the excluded variable is positioned at the individual optimum (Table 3).

The extraction results for HAE and UAE, as function of the combination of the three main involved variables (*X1-3*: *t*, *T* or *P*, and *S*), can be observed in Figures 1 and 2. In this regard, Figure 1 shows the 3D surface plots of the extracted R (*Yield*, %), and CT, in two response formats (*Y1*, mg CT/g R and *Y2*, mg CT/g E dw). On the other hand, Figure 2 shows the optimized isolines projections for C1 (cyanidin 3-rutinoside) and C2 (peonidin 3-rutinoside) extraction, in the two response value formats (*Y1*, mg C/g R and *Y2*, mg C/g E dw). These figures show, respectively, optimized 3D graphical and 2D isolines projection results for the extracted anthocyanins (C1 or C2) as function of the three combined variables (*t*, *T* or *P*, and *S*) in HAE and UAE. The total anthocyanins (C1+C2) are accounted together (CT) in Figure 1, and individually in Figure 2. They are helpful to visualize the tendencies of each response and lead to define of the maximum favorable conditions, considering all together all responses.

Additionally, Figure 1B exemplifies the competence to predict the obtained results. In statistical terms, the distribution of residues (Figure 1) presents, for the majority of the cases, more than 90% of reliability, showing a good agreement between experimental and predicted values. This is also verified by the achieved high *R <sup>2</sup>* values (Table 2), which indicates the percentage of variability explained by the model.

In HAE, small differences between the extraction behavior of the two considered anthocyanins (when comparing C1 and C2, or *Y<sup>1</sup>* and *Y2*) were clearly distinguished. The opposite occurred in UAE, the effects were distinct for each one of the detected anthocyanins, as well as according to the response format. However, for both extraction techniques, the *S* variable was the most significant one, producing a relevant impact on the level of extraction of all anthocyanins assessed. As described above, the LE and the QE of the significant parametric values of the variable *S* can be perceived in all figures. In almost all cases, the variable *S* indicates a maximum level at ~50% of hydroalcoholic mixture (water/ethanol, *v*/*v*). The negative impact of quadratic term of the variable *S* can be explained through the increase of water in the process, which expands the yield of extraction. Other negative effects such as those between *T* or *P* and *S* may suggest that the further use of lower *P*, in combination with higher *S*, will avoid the anthocyanin degradation. The results are in accordance with others recently reported by other authors [19–21], in which inclined surfaces to the side of *T* or *P* may increase the solubility of target compounds by using stronger energies, and consequently improve their release from the sample matrix, while destroying the integrity of connective and structural tissues.

#### *2.3. Conditions That Maximize the Anthocyanins Extraction and Experimental Verification*

The aim of this study was to maximize the extraction yield of targeted anthocyanin compounds from epicarps of *P. spinosa* fruits, in the applied HAE and UAE techniques, within pre-set variable conditions and ranges. The values of the variable conditions that lead to optimal response values for RSM using a *CCCD*, obtained with the aid of *simplex* procedure, for each of the assessed extracting techniques are shown in Table 3. Figure 3 part A shows the individual summary of the effects of all variables assessed for HAE and UAE systems in 2D illustrations, where the variables are positioned at the individual optimal values of the others (Table 3). The dots (⊙) presented alongside each line highlight the location of the optimum value, meanwhile lines are the predicted behavior found by the mathematical analysis of Equation (1) generated by the theoretical second-order polynomial models described in Table S1. Next, some relevant details of the results produced are highlighted:


Considering both the individual and global values, the higher amount of extracted compounds was obtained for the UAE technique. The ideal solvent composition was almost the same, and the two techniques required high energy values, where the highest values of *T* and *P* proposed by the experimental design were the optimal, but the UAE needed less *t* than HAE (~90% less). The obtained results are in accordance with similar conclusions found previously [10,17,22], in which UAE proved to consume less energy because of the lower *t* needed, and provide higher extraction values while increasing the purity and, additionally, aiding to meet the requirements of a green extraction concept.

#### **Figure 3.** Final graphical effects of all variables assessed for HAE and UAE systems. Part A shows the individual 2D responses as a function of all the variables assessed that were positioned at the individual optimal values of the others (Table 2). The points (⊙) presented alongside each line highlight the location of the optimum value. Lines and dots are generated by the theoretical second order polynomial models generated by Equation (1) (Table S1). Part B shows the dose response of *S/L* at the global optimal values of the other three variables (Table 3). The limit value (~150 g/L) shows the maximum achievable experimental concentration until the sample cannot be physically stirred at laboratory scale. RSM—response surface methodology.

#### *2.4. Dose-Response Analysis of the Solid-to-Liquid Ratio Effect at the Optimal Conditions*

The study of *S*/*L* effect was performed at the optimal conditions (Table 3) predicted by the polynomial models obtained for each extraction technique (HAE and UAE) using the total anthocyanin content (CT), as quantified by HPLC analysis, as the response factor. The individual *S*/*L* study for each individual anthocyanin (C1 or C2) was not presented because the behavior was similar to the pattern of the total amount. In both processes, the *S*/*L* was designed to verify the behavior between 5 and 250 g/L. The maximum value of 250 g/L was used as a limit condition because of the impossibility of producing a homogenized extraction when higher values were used.

The obtained dose responses of the *S*/*L* were consistent for both HAE and UAE systems, and could be described by a simple linear relationship (shown in Part B of Figure 3). All experimental points are distributed around the linear equation; consequently, the dose response is explained by the slope (*m*) of the linear relationship. None of the cases showed positive *m* values (the extraction efficiency increases as the *S/L* rate increases), and two cases showed non-significant values or a zero value of *m* (the efficiency doesn't change as the *S*/*L* increases). In all the other cases, the *m* showed negative values (the efficiency decreases as the *S*/*L* increases). The responses from the *Y<sup>1</sup>* value format, for HAE and UAE, were the ones that showed non-significant *m* values, whereas all other responses showed significant negative values of *m* (*Y<sup>1</sup>* and *Yield* for HAE and UAE). The conclusions derived from this analysis are described below:


#### *2.5. Comparison with Other Studies Involving the Extraction of Anthocyanins*

There are few works in the literature dealing with anthocyanins in *P. spinosa* fruits. In one of these studies with *P. spinosa* fruits, Guimarães et al. [5] performed the extraction using methanol with 0.5% TFA added as solvent, and identified eight different anthocyanins, predominantly peonidin 3-rutinoside and cyanidin 3-rutinoside, with 34.47 ± 0.03 µg/100 g fruits dw and 31.12 ± 0.11 µg/100 g fruits dw, respectively. Other authors [6], found 3.5 ± 0.5 mg of anthocyanins/100 g dw of *P. spinosa* fruits. Both authors used the whole fruit, while in this study, only the epicarp was used as the extraction material, a fact that may justify the significant differences between the encountered results, when compared with the present study. Compared with the pulp, the fruit epicarp presents a greater intensity of color and, therefore, a higher concentration of anthocyanins, in addition to less interfering compounds, is obtained. Moreover, another fact that aided the production of large amounts of anthocyanins from the extracted material was the optimization of the extraction process, which led to increased extraction efficiency and yield. In another study that used *P. spinosa* fruits as a source of anthocyanins [6], the total content was quantified by spectrophotometric methods, presenting values that cannot be compared with those found in the present study.

Some examples of other plant-based sources of anthocyanins are *Oryza sativa* L. (var. Glutinosa) bran, which shows 42.00 mg/g [24]; *Phaseolus vulgaris* L. (common beans) fruit coat, presenting 32.00 mg/g [25]; and *Rubus fruticosus* L. (blackberries) fruit, which possess 17.10 mg/g [26]. Although these values are slightly higher than those presented by *P. spinosa* fruits, in general, the referred fruits and vegetables already have a high commercial value and other industrial purposes, unlike *P. spinosa* fruits. On the other hand, residues such as grape vine (*Vitis vinifera* L.) pomace, and mango (*Mangifera indica*) skin presented lower anthocyanin amounts, that is, 6.33 mg/g [27] and 2.03 to 3.60 mg/g [28], respectively. Thus, these wild fruits revealed to be an excellent source of anthocyanins, serving as a base raw-material for the production of natural colorant additives for commercial purposes.

#### **3. Discussion**

The minimalism of using conventional methods (HAE or maceration) versus the compensations of new non-conventional technologies (microwave, ultrasound, cold pressing, squeezing, etc.) to recover compounds from plant materials, as well as by-products, is a principal topic in the list of many industries in order to increase profitability by decreasing energy costs and reducing greenhouse gas emissions to meet legal requirements. Additionally, non-conventional technologies favor the safety of processes and the quality of products, as well as the functionality and product standardization.

Scientific literature shows clear evidence that extraction procedures of target compounds from plant-based products must be assessed individually. Therefore, a nonstop effort needs to be performed, as agro-industrial and food sectors are looking for by-products' valorization into added-value products. However, in order to take full advantage of the technological advances, the extraction conditions need to be optimized. Mathematical solutions, such as RSM tools, could increase the efficiency and profitability of the process and help to change conventional extraction approaches.

Colorants are one of the most important additives in terms of marketing because their presence in food products is considered to influence customers' perceptions, choices, and preferences. *P. spinosa* fruit epicarps have been scarcely explored and, to the best of the authors knowledge, the potential industrial use of their anthocyanin compounds has not been previously investigated. In such a context, the present work presents a new rapid method to extract anthocyanin compounds from *P. spinosa* fruit epicarps. RSM and other mathematical strategies were successfully employed to optimize the extraction conditions that maximize the anthocyanin compounds' recovery to produce a rich extract with potential industrial application as a natural coloring additive.

#### **4. Materials and Methods**

#### *4.1. Plant Material*

Ripe *P. spinosa* fruits were harvested in Bragança (Trás-os-Montes, Northeast Portugal) in September 2017, the epicarp was separated from the rest of the fruit body, frozen, and lyophilized. They were then triturated, to be reduced to a fine powder (~20 mesh), and stored under refrigeration, protected from light until further use.

#### *4.2. Extraction Procedures for P. Spinosa Fruit Epicarps*

#### 4.2.1. Heat Assisted Extraction (HAE)

HAE was performed in a water reactor agitated internally with a CimarecTM Magnetic Stirrer at a constant speed (~500 rpm, Thermo Scientific, San Jose, CA, USA), following a procedure previously performed [13]. The powdered epicarp samples of *P. spinosa* (1.0 g) were extracted with 20 mL of solvent (ethanol/water) acidified with citric acid (pH = 3), under diverse conditions, as previously defined by the established RSM plan (Table 1). The ranges of the experimental design were as follows: time (*<sup>t</sup>* or *<sup>X</sup>1,* 5 to 85 min), temperature (*<sup>T</sup>* or *<sup>X</sup>2,* 20 to 90 ◦C), and ethanol content (*<sup>S</sup>* or *<sup>X</sup>3,* 0% to 100%). The solid-to-liquid ratio (*S/L* or *X4*) was kept at 50 g/L for all conditions.

#### 4.2.2. Ultrasound-Assisted Extraction (UAE)

An ultrasonic device (QSonica sonicators, model CL-334, Newtown, CT, USA) equipped with a water reactor (EUP540A, Euinstruments, France) at a fixed frequency (40 kHz) was used for UAE procedure. The variables considered were as follows: ultrasonic power (*P*, in watts), *S*, and *t*, which were programmed according to the defined RSM plan (Table 1), following a procedure previously performed [29]. The powdered epicarp samples (2.5 g) were placed in a reactor with 50 mL of solvent (ethanol/water) acidified with citric acid (pH = 3), and extracted under diverse conditions, maintaining the *S/L* constant at 50 g/L. The ranges of the experimental design were as follows: *t* (*X1*, 5 to 25 min), *P* (or *X2*, 100 to 400 W), and *S* (or *X3*, 0% to 100%).

#### 4.2.3. Post-Extraction Sample Processing

When all the individual extraction conditions were carried out (for HAE and UAE), the samples were immediately centrifuged (6000 rpm during 20 min at 10 ◦C) and filtered (paper filter Whatman nº 4) to eliminate the non-dissolved material. The supernatant was collected and divided into two portions for HPLC and extraction yield analysis. The portion separated for HPLC analysis (3 mL) was dried at 35 ◦C, re-dissolved in acidified water (citric acid solution with pH 3), and filtered through an LC filter disk (0.22 µm), whereas the portion for the extraction yield determination (5 mL) was dried at 105 ◦C during 48 h and thereafter weighted.

#### *4.3. Identification and Quantification of Anthocyanins by HPLC*

The extract was analyzed using an HPLC-DAD-ESI/MSn (Dionex Ultimate 3000 UPLC, Thermo Scientific, San Jose, CA, USA) system, previously described [30]. The detection was carried out using a DAD (520 nm as the preferred wavelength) and mass spectrometer (Linear Ion Trap LTQ XL mass spectrometer, Thermo Finnigan, San Jose, CA, USA) equipped with an ESI source. The anthocyanins present in the samples were characterized according to their UV and mass spectra. The anthocyanins cyanidin 3-rutinoside (C1) and peonidin 3-rutinoside (C2) were the most relevant compounds found, and were, therefore, quantified using a five-level calibration curve of known concentrations (200–20 µg/mL) of cyanidin 3-glucoside (*y* = 243287 *x* − 1000000; *R <sup>2</sup>* = 0.9953, Polyphenols, Sandnes, Norway) and peonidin 3-glucoside (*y* = 122417 *x* − 447974; *R <sup>2</sup>* = 0.9965, Polyphenols, Sandnes, Norway).

#### *4.4. Response Value Formats for Results Presentation*

The two anthocyanin compounds (C, either C1 or C2) and their sum (C total, CT) were used as responses in each applied technique. The results were presented according to two response formats (*Y*): *Y1*, in mg of C per gram of extracted residue (mg C/g R), which was specifically used to evaluate the C purity in the extracts; and *Y2*, in mg of C per g of fruit epicarp dry weight (mg C/g E dw), which was specifically used to analyses the C extraction yield. Both responses were equally analyzed, but additional considerations regarding the last one (*Y2*, mg C/g E dw) were taken in the results presentation, becuse it is considered as an important guiding response when dealing in terms of optimization for industrial transference. Note that by dividing those responses, *Y2*/*Y1,* the extracted residue quantity (g R/g E dw) is obtained, which provides information regarding the third response criterion expressed (*Yield*, %).

#### *4.5. Experimental Design, Model Analysis, and Statistical Evaluation*

#### 4.5.1. RSM Experimental Design

Trials based on one-at-the-time analysis (analysis of each of the variables for each one of the selected techniques) were conducted, the ranges originating significant changes were selected (Table 1). The joint effects of the three defined variables were studied using a *circumscribed central composite*

*design* (*CCCD*), using five levels for each one with twenty eight response combinations, as described previously [31].

#### 4.5.2. Mathematical Model

The experimental data produced by the RSM design were analyzed mathematically by means of least-squares calculation, using the following second-order polynomial equation with interactive terms [32]:

$$Y = b\_0 + \sum\_{i=1}^{n} b\_i X\_i + \sum\_{\substack{i=1 \\ i = 1}}^{n-1} \sum\_{j=2}^{n} b\_{ij} X\_i X\_j + \sum\_{i=1}^{n} b\_{ii} X\_i^2 \tag{1}$$

where *Y* is the dependent variable (response variable) modelled, *X<sup>i</sup>* and *X<sup>j</sup>* define the independent variables, *b<sup>0</sup>* is the constant coefficient, *b<sup>i</sup>* is the coefficient of linear effect, *bij* is the coefficient of interaction effect, *bii* the coefficient of quadratic effect, and *n* is the number of variables. As responses, the following three value formats were used: *Y<sup>1</sup>* (mg C/g R), *Y<sup>2</sup>* (C/g E dw), and *Yield* (%).

#### 4.5.3. Procedure to Optimize the Variables to a Maximum Response

A simplex algorithm method was used to find the optimum values by solving nonlinear problems in order to maximize the extraction yield and the recovery of anthocyanin compounds, as explained previously [33]. Certain limitations were imposed (i.e., times cannot be lower than 0) to avoid variables with unnatural and unrealistic physical conditions.

#### 4.5.4. Dose-Response Analysis of the Solid-to-Liquid Ratio

Once the optimal conditions (*X1*, *X2*, and *X3*) were found, the following natural optimization step was used to describe the pattern of the *S*/*L* (or *X4*, expressed in g/L). The objective was to achieve more productive conditions as required by industrial applications. In all cases, experimental points are distributed following linear patterns as the *S*/*L* increases, consequently, linear models with intercepts were used to evaluate the responses. The parametric value of the slope (*m*) was used to assess the dose response. Positive values will indicate an increase in the extraction responses, whereas negative values will designate a decrease in the extraction efficiency, as the *S*/*L* increases.

#### *4.6. Mathematical Procedures*

The analytical procedures to model the data, to determine the parametric values, confidence intervals, and statistical calculations, were obtained following the descriptions of other authors [34]. In brief, (a) the parametric values were obtained using the quasi-Newton algorithm (least-square) by running the integrated macro '*Solver*' in Microsoft Excel; (b) the coefficient significance of the parameters produced (α = 0.05) was assessed using the '*SolverAid*' macro to conclude their confidence intervals; and c) the model consistency was proven by means of several statistical criteria, such as (i) the Fisher *F*-test (α = 0.05); (ii) the '*SolverStat*' macro; and (iii) the *R* 2 coefficient.

#### **5. Conclusions**

The efficiency of the UAE was higher than that obtained with HAE. The main anthocyanins identified were cyanidin 3-rutinoside and peonidin 3-rutinoside, being the ones quantified. Through the optimization of the extraction process, it was possible to reach by UAE 18.17 ± 1.82 mg CT/g R (*Y1*), 11.76 ± 0.82 mg CT/g E dw (*Y2*), and 68.60% ± 2.06% (yield of the extracted residue), with the optimal parameters of extraction being 5.00 ± 0.15 min, 400.00 ± 32.00 W, and 47.98% ± 2.88% of ethanol. The used mathematical models (RSM and dose-response models) were statistically significant and allowed the optimization of the anthocyanins extraction. For the *S*/*L* effects, inspected at the optimum conditions, the responses for all assessed criteria followed a decreasing linear relation until 250 g/L.

In conclusion, the present study contributed to the valorization of the wild fruits of *P. spinosa* by exploring anthocyanin-rich extracts that can find potential application as natural colorants in different industrial fields. For that purpose, an optimized extraction method was obtained using advanced and efficient extraction systems.

**Supplementary Materials:** The supplementary materials are available online.

**Author Contributions:** Formal analysis, M.A.P.; Investigation, M.G.L.; Methodology, C.P. and L.B.; Resources, M.F.B.; Supervision, I.J.B. and I.C.F.R.F.

**Funding:** The authors are grateful to the Foundation for Science and Technology (FCT, Portugal) and FEDER under Programme PT2020 for financial support to CIMO (UID/AGR/00690/2013), L. Barros and C. Pereira research contract; to FEDER-Interreg España-Portugal programme for financial support through the project 0377\_Iberphenol\_6\_E.; to European Regional Development Fund (ERDF) through the Regional Operational Program North 2020, within the scope of Project NORTE-01-0145-FEDER-023289: DeCodE and project Mobilizador Norte-01-0247-FEDER-024479: ValorNatural®. This work was also financially supported by the following: Project POCI-01-0145-FEDER-006984, Associate Laboratory LSRE-LCM funded by FEDER through COMPETE2020, Programa Operacional Competitividade e Internacionalização (POCI), and national funds through FCT. The authors thank the GAIN (Xunta de Galicia) for financial support (P.P. 0000 421S 140.08) to M.A. Prieto by a post-doctoral (modality B) grant.

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

#### **References**


**Sample Availability:** Samples of the plant and extracts are available from the authors.

© 2019 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/).

*Article*
