# **Advances on Applications of Bioactive Natural Compounds**

Edited byAna M. L. Seca and Eugenia Gallardo Printed Edition of the Special Issue Published in *Applied Sciences*

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## **Advances on Applications of Bioactive Natural Compounds**

## **Advances on Applications of Bioactive Natural Compounds**

Editors

**Ana M. L. Seca Eugenia Gallardo**

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

*Editors* Ana M. L. Seca cE3c-GBA, FCT- Universidade dos Ac¸ores Portugal

Eugenia Gallardo Universidade da Beira Interior (CICS-UBI) 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 *Applied Sciences* (ISSN 2076-3417) (available at: https://www.mdpi.com/journal/applsci/special issues/Bioactive Natural Compounds).

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 978-3-0365-3975-1 (Hbk) ISBN 978-3-0365-3976-8 (PDF)**

Cover image courtesy of Ana M. L. Seca

© 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.

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## **Contents**

Reprinted from: *Appl. Sci.* **2021**, *11*, 11417, doi:10.3390/app112311417 ................ **167**

Mexican Propolis

## **About the Editors**

**Ana M. L. Seca** has been an Assistant Professor at the University of Azores since 2000, and she conduts research a the Center for Ecology, Evolution and Environmental Changes—cE3c (ABG) as full member and at LAQV-REQUIMTE as a collaborator. She has a degree in chemistry and an MSc. in the science and technology of paper and forest products, both of which were obtained at the University of Aveiro, Portugal, where she also received her PhD in chemistry in 2000. She has published 58 SCI papers and 8 book chapters. Her current research interests cover the isolation and identification of secondary metabolites with potential pharmacological applications and the synthesis of pharmaceutically relevant natural compound analogues.

**Eugenia Gallardo** has been an Assistant Professor of Toxicology at the Faculty of Health Sciences of the University of Beira Interior since 2009 and conducts her research at the Health Sciences Research Centre at the University of Beira Interior. She has a degree in pharmaceutical sciences (2000), a Master's of advanced studies in toxicology (2002), and a European PhD in toxicology (2006) that she obtained from the University of Santiago de Compostela (Spain). Since 2011, she has been the Head of the Laboratory of Farmaco-Toxicologia at UBImedical (Portugal). She has published 100 SCI ´ papers and 16 book chapters and has guest-edited 1 book. Her main areas of interest are bioanalysis, toxicology, and drug monitoring, namely the development of analytical methods for the analysis of environmental substances, pesticides, drugs, and drugs of abuse with a special focus on biofluids as well as other bioactive compounds in non-biological specimens (plants and extracts).

### *Editorial* **Secondary Metabolites and Their Applications**

**Eugenia Gallardo 1,2,\* and Ana M. L. Seca 3,4,\***

#### **1. Introduction**

The identification of secondary metabolites present in both terrestrial and marine species continues to be a fundamental and privileged path for the emergence of new and fundamental natural products available on the market with very different applications. For example, aplidine is a new natural anticancer agent, and it was approved in Australia in 2018 to treat multiple myeloma [1] and was isolated from the first time from tunicate *Aplidium albicans* Milne Edwards. (+)-Nootkatone is a natural sesquiterpene compound, found in very small amounts in several species such as *Chamaecyparis nootkatensis* (D. Don) Sudworth, *Chrysopogon zizanioides* (L.) Roberty and *Citrus paradisi* Macfad., which exhibits highly appreciated organoleptic properties and is, therefore, highly demanded as a flavoring agent or adjuvant in the food, pharmaceutical and perfumery industries [2,3]. This same natural secondary metabolite is applied as insecticide and acaricide, and it was very recently authorized by United Sates Environmental Protection Agency to be include on formulations to control the spread of mosquitoes that transmit infectious diseases as dengue and zika [4]. And who does not know the application of ascorbic acid, found for example in acerola and lemon fruits, as an antioxidant agent [5], widely used by the food industry?

The successful application of secondary metabolites in diverse requests often involves the use of, more or less, complex mixtures of metabolites, extracted by different methods and from different natural sources, but whose chemical composition and active principles are perfectly established. For example, there are the defined botanical mixtures, called natural product botanicals, which are recognized by the FDA as medicinal entities, successfully used in clinical therapy for the treatment of various diseases. This is the case of the mixture of the secondary metabolites of *Solanum sodomaeum* L., solamargine, solasonine, and monoand di-glycosides derivatives of solasodine, approved by the European Medicine Agency (trade name Curaderm) for the treatment of basal cell carcinoma of the skin [6].

One area in which the application of secondary metabolites mixtures rather than of pure compounds is common is in the formulation of biopesticides. For example, the insecticide Grandevo® is a mixture of metabolites produced in the fermentation of *Chromobacterium subtsugae*, which includes pigments from the violacein family and proteins that are repellent and antifeeding [7].

Despite the great successes already achieved regarding the secondary metabolites' identification and the development of new applications for these metabolites, this is an area of research that should not slow down. Research must continue identifying and isolating secondary metabolites in unexplored natural sources; new methodologies for extracting secondary metabolites should be tested and optimized to develop greener and more

**Citation:** Gallardo, E.; Seca, A.M.L. Secondary Metabolites and Their Applications. *Appl. Sci.* **2022**, *12*, 2317. https://doi.org/10.3390/ app12052317

Received: 15 February 2022 Accepted: 17 February 2022 Published: 23 February 2022

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

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

1

efficient processes; pure secondary metabolites and chemically characterized mixtures must be tested in different biological activities in order to enhance new applications responding to the growing needs of humanity.

The Special Issue of *Applied Sciences*, "Advances in Applications of Natural Bioactive Compounds", aims to contribute to the desired continuous advance in this scientific field, bringing together publications focused on the most recent advances in the identification of secondary metabolites from terrestrial and marine sources, in new extractive methodologies, and in proposals for applications that add value to natural resources and contributes to a more sustainable development.

#### **2. Contributions**

This Special Issue brings together seven research articles and four review articles presenting the latest advances in the study of secondary metabolites from different natural sources and their potential applications. The scientific results are presented that led to the identification of secondary metabolites in samples of terrestrial and marine origin, obtained by classic and innovative extractive methodologies, as well as studies to evaluate their effect in different areas such as health, biopesticides, etc.

Invasive alien species are recognized for triggering various impacts, such as habitat modification, alteration of community structure, and affecting ecosystem processes. The characterization of secondary metabolites in invasive species could be a way of valuing and increasing the search for available biomass promoting its harvest and contribute to the conservation of biodiversity and sustainability of the ecosystem. This is one of the objectives of the studies carried out by Paula et al. [8] and Pinto et al. [9], in which the secondary metabolite composition of two invasive species, one terrestrial (*Acacia dealbata* L.) and one marine (seaweed *Asparagopsis armata* Harvey), respectively, are presented. The results show that the hydroethanolic extract of non-mature flowers of *Acacia dealbata* exhibits the greatest potential as an antioxidant and acetylcholinesterase inhibitor, these activities being correlated with the higher content of chalcones present [8]. These authors suggest the extract as a source of compounds capable of improving brain functions, while collecting the flower at an early stage of flowering contributes to interrupting the plant's reproduction, which will help to control its invasive potential. Pinto et al. [9] address new perspectives for the valuation of *Asparagopsis armata*, an invasive alga in Europe, and have determined the profile of the secondary metabolites of this species using GC-MS and UHPLC-MS techniques. The authors established the species as a source of halogenated compounds and fatty acids, with 1,4-dibromobuten-1-ol and palmitic acid being the two most abundant compounds identified in the lipophilic fraction, while in the polar fraction, brominated phenolic compounds derived from cinnamic acid are predominant. This work also shows that changing the extraction method modifies the metabolomic profile of the obtained extract. The use of ethanol under microwave energy extracted almost exclusively one compound, the 2,3-diphenylpropanoic acid derivative with one hydroxyl group and two bromine atoms in each aromatic ring [9].

The Martí-Quijal group [10] also uses samples of marine origin, in this case the cyanobacterium *Arthrospira platensis* Gomont, and optimizes the extraction with pulsed electric fields to propose a more selective, sustainable, and efficient methodology to extract chlorophylls, carotenoids, and phenolic compounds, which have applications as nutritional and antioxidants agents.

In recent years, natural phytotoxic compounds derived from plants have been extensively investigated for herbicidal properties and have been shown to suppress weeds. These compounds can then be used as promising models for standard bioherbicides. In this sense, Rob et al. [11] have demonstrated in their manuscript for the first time the presence of methyl phloretate in *Garcinia xanthochymus* Hook.f. ex T.Anderson, as well as its phytotoxic properties. Vanillic acid was also identified. Both compounds have significant growth inhibitory activities in watercress and timothy, demonstrating as such the potential of the leaves of *Garcinia xanthochymus* in weed management.

The leaves of *Afzelia xylocarpa* (Kurz) Craib. were investigated as a natural source of herbicides. Krumsri et al. [12] have identified vanillic acid and *trans* ferulic acid as the major bioactive compounds, both being responsible for the phytotoxic activity of *Afzelia xylocarpa*. The concentration of those compounds and differences in the sensitivity of the plant models are key to their phytotoxic action. Additionally, a mixture of both compounds at low concentrations had synergistic effects concerning phytotoxic activity.

Shedding light on the mechanisms of action that justify the observed secondary metabolite bioeffects is an essential point where more research is needed before it can advance towards clinical therapy. The secondary metabolite thymoquinone can be extracted from the seed oil of *Nigella sativa* L. (black seed oil) and exhibits several biological activities [13], among which are relevant antitumor properties, and has already been the subject of clinical studies (ClinicalTrials.gov Identifier: NCT03208790). However, its mechanism of action is not yet fully understood. Alhosin et al. [14] make a very significant contribution to clarifying this point. They show that thymoquinone, unlike doxorubicin, specifically targets the oncogene UHRF1. Probably through the downregulation of HAUSP, thymoquinone induces the rapid ubiquitination of UHRF1, an essential event for the induction of cancer cell apoptosis without, however, affecting the Bcl-2 oncogene [14].

The total synthesis of natural secondary metabolites contributes not only to confirm the proposed chemical structure of the natural compound but also to satisfy the demand for more compounds for different applications without degrading the environment. Thus, the work by Kim et al. [15] is a great contribution to the development of new applications for the secondary metabolite Aaptoline B, a pyrroloquinoline alkaloid extracted from the marine sponge *Aaptos aaptos* Schmidt and considered a privileged scaffold in drug discovery. The authors suggest a short but comprehensive and efficient synthetic process for Aaptolin B and demonstrate, using *Caenorhabditis elegans* as a model of the Parkinson's disease-like neuronal environment, that this compound exhibits a neuroprotective effect [15]. Thus, the door is open for investigation into a new application for this secondary metabolite.

Laboratory synthesis is not the only strategy applied to obtain new and greater amounts of bioactive secondary metabolites. *Streptomyces* is a group of Gram-positive bacteria capable of biosynthesizing a wide range of natural compounds from different families, with different commercial applications and high economic value [16]. Doxorubicin, an antitumor antibiotic, and avermectin, used to treat parasitic worms and insect pests, are just two well-known examples. However, the potential of these microorganisms to produce secondary metabolites is much greater than achieved so far. The problem is, under laboratory conditions, many of the genes responsible for this biosynthesis remain silent. Heterologous biosynthesis has been an applied approach to overcome this limitation. Pham et al. [17] publish in this Special Issue a review of the literature on the latest advances in heterologous biosynthesis to produce natural compounds from *Streptomyces*, which include the overexpression/deletion of regulatory genes, promoter replacement strategies, and engineering of ribosomes.

On the other hand, many natural secondary metabolites, semi-synthesized or synthesized in the laboratory or by a biotechnological approach, exhibit excellent pharmacodynamics but very poor pharmacokinetic properties. The development of nano delivery systems has been a successful way to overcome the pharmacokinetic limitations of many natural compounds, allowing them to broaden their medicinal application [18]. Sindhu et al. [19] present in this Special Issue a review of the literature on the development of nano formulations involving natural compounds with application in the treatment of cardiovascular diseases, highlighting those with the most significant results for the prevention and treatment of this type of disease, that is, cases involved in clinical studies.

As referred to before, invasive species are considered one of the most significant global threats to biodiversity. However, these species can also be quite helpful to humans, providing a complex number of services, which makes it difficult to assess their positive and negative effects. Caramelo et al. [20] review the knowledge of the chemical composition and the discovery of the biological properties of one such invasive alien species*—Ailanthus*

*altissima* (Mill.) Swingle*—*and its potential use as medicine and additive by the pharmacy and food industries. Methods for the extraction and detection to know the chemical composition of extracts have been discussed in detail by the authors.

Finally, Silva-Beltrán et al. [21] evaluate the chemical profiles of propolis produced in Brazil and Mexico. Both countries' geographic and ecosystem differences provide propolis with different chemical profiles. However, data in both countries indicate that more research is still needed to determine the optimal characteristics of propolis and its components, such as period of intake or type of extract to use before it can be administered to humans as medicine The authors have also review the use of propolis as environmental bioindicator, concluding that more studies are necessary.

In the guest editors' opinion, this Special Issue of *Applied Sciences* brings together great contributes to the desired continuous advance in secondary metabolites and the development of new applications for these metabolites. In fact, the published papers are focused on the most recent advances in the identification of secondary metabolites from terrestrial and marine sources obtained by classical and greener extractive methodologies. They are also focused on proposals for new applications that enhance the natural resources and their secondary metabolites, contributing to more sustainable development.

**Author Contributions:** A.M.L.S. and E.G. conceived, designed, and wrote the editorial. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was financed by Portuguese National Funds, through FCT—Fundação para a Ciência e Tecnologia, the European Union, QREN, FEDER, and COMPETE, through funding the cE3c center (UIDB/00329/2020), the LAQV-REQUIMTE (UIDB/50006/2020) and the CICS-UBI (UIDB/00709/2020 and UIDP/00709/2020).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** Thanks are due to all the authors and peer reviewers for their valuable contributions to this Special Issue.

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

#### **References**

## *Article* **Special Bioactivities of Phenolics from** *Acacia dealbata* **L. with Potential for Dementia, Diabetes and Antimicrobial Treatments**

**Vanessa Paula 1,†, Soraia I. Pedro 2,3,4,†, Maria G. Campos 5,6,\*, Teresa Delgado 2, Letícia M. Estevinho 1 and Ofélia Anjos 2,4,7,\***

**Abstract:** Some diseases still need better therapeutic approaches, including the prevention of development. Natural resources are investigated with this purpose; among them, we decided to use an invasive plant as a main strategy. This will help in two ways: screening new compounds in flowers prevents the plant from causing widespread damage by controlling the dissemination and also obtains crude material for further applications. In the present study, flower extracts from *Acacia dealbata* Link harvested in Portugal were studied during three stages of flowering. Phenolic compounds were evaluated using HPLC/DAD and the total phenolics as the total flavonoids content was determined. The bioactivities screened were antioxidant potential, inhibitory activities of some enzymes (acetylcholinesterase, lipase and α-glucosidase) and, to complete the screening, the inhibition of microbial growth was determined against Gram-negative and Gram-positive bacteria, as well as for yeasts. The data obtained suggested that the hydroethanolic extracts gave good results for all these biological activities and varied according to the maturation status of the flowers, with the early stage being the most active, which can be related to the chalcones content. This new approach will lead to the possible control of the invasive plant and also future perspective research for therapeutic purposes.

**Keywords:** invasive species; flowers; antioxidant; microbial activity; bioactivity; enzyme inhibitory potential; acetylcholinesterase; lipase; α-glucosidase

#### **1. Introduction**

In drug discovery, screening for potential bio-active structures is often started with natural product extracts. The present work reflects the preliminary study carried out with extracts of an invasive plant, *Acacia dealbata* L., which may be used in a win–win relationship, that is, as a raw material to supply bioactive molecules, while the harvest itself will contribute to controlling its invasiveness by decreasing the proliferation of this plant.

Among the possible bioactivities to explore that are believed to be linked to the compounds expected to be in the Acacia extracts we chose, the acetylcholinesterase inhibitors (AChEIs) can be explored further for the treatment of dementia symptoms [1] or even

**Citation:** Paula, V.; Pedro, S.I.; Campos, M.G.; Delgado, T.; Estevinho, L.M.; Anjos, O. Special Bioactivities of Phenolics from *Acacia dealbata* L. with Potential for Dementia, Diabetes and Antimicrobial Treatments. *Appl. Sci.* **2022**, *12*, 1022. https://doi.org/ 10.3390/app12031022

Academic Editor: Ana M. L. Seca

Received: 21 December 2021 Accepted: 14 January 2022 Published: 19 January 2022

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

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

to delay the disease. The current extracts used to slow the progression of many of these pathologies still have modest outcomes.

Acacia species are aggressive invaders that affect ecosystem integrity worldwide [2]. *Acacia dealbata* Link is considered to be one of the most aggressive in Portugal. These plants are distributed throughout all Portuguese provinces [3–5] and may invade farmland and autochthonous forests, establishing monocultures, modifying the ecosystem structure and impacting the economy. Their flowers play a key role in the colonizing capacity, limiting the growth of other species due to the allopathic interference caused by vegetal material decomposition in the soil, which maintains the toxicity of soil percolates for a long time [6]. Extracts from the flowers are already explored as hydrogels for personal care products, cosmetics or pharmaceuticals, as well as perfumes based on their antiradical and anti-proliferative potential [7].

Along with this research, other possibilities were explored based on previous studies on ethanol extracts of leaves and flowers of Acacia species that suggested the presence of secondary metabolites, such as coumarins, cyanogenic glycosides, tannins, alkaloids and steroids [8]. Notwithstanding, this genus is recognized as a source of phenolic compounds [9–11], with flavonoids being the predominant chemical group [12]. In the study performed by Farid et al. [13] on the leaves, flowers and pods of *A. seyal*, *A. nilotica* and *A. laeta,* the antioxidant activity was promising. Furthermore, according to Ziani et al. [14], *A. tortilis* extracts exhibited various biological activities and their phenolic compounds were studied for the development of cytotoxic and anti-inflammatory drugs.

Antimicrobial activity was also identified in several Acacia species against *Escherichia coli*, *Staphylococcus aureus* and *Salmonella typhi* bacteria and the fungal strain *Candida albicans* and *Aspergillus niger* [15–17].

According to the aforementioned studies, the bark and pods contain secondary metabolites, such as flavonoids, alkaloids, saponins and tannins, which may contribute to the bioactivities. Among the most relevant potential applications in health, the inhibitory activities of the enzymes α-glucosidase, acetylcholinesterase and lipase will have an important impact on the development of new drugs. For instance, lipases, which are present in pancreatic secretions and are responsible for fat digestion, are able to break down triglycerides into free fatty acids and glycerol. Acetylcholinesterase inhibitors are relevant for Alzheimer's disease treatment strategies.

For the possible applications cited above, especially regarding the antioxidant potential, the need to screen new molecules for the development of, for example, new drugs for dementia-related diseases, and the need to use a crude material that will leave a low environmental impact, are all good reasons to explore *A. dealbata* flowers. This reflection leads to the goal of the present work, which consisted of obtaining more robust data for possible future research using selected assays to evaluate the inhibitory activities of the enzymes acetylcholinesterase, α-glucosidase and lipase. To complete the screening, the inhibition of microbial growth was determined against Gram-negative and Gram-positive bacteria, as well as for the yeasts *Candida albicans* ATCC 10231TM and amphotericin-B-resistant *Candida albicans* ESA100.

In addition, phenolic compounds and flavonoids were screened using HPLC/DAD to characterize the extracts.

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

#### *2.1. Plant Samples*

The flowers of *A. dealbata* Link used in this study were harvested during three flowering stages (early flower—EF, mid flower—MF and late flower/ripening—LF), as shown in Figure 1, in 2018. After each harvest, the flowers were manually separated from the thick stems and leaves, and then lyophilized in dark conditions and milled. The fraction lower than 60 mesh was frozen at −20 ◦C until the extraction procedures. To study the characteristics and biological properties of the flowers, samples from two locations (A and

B) in the central region of Portugal were analyzed and compared. These two regions were selected because there are large stands of this invasive species.

**Figure 1.** Different stages of *A. dealbata* flower used in this study (EF—early flower, MF—mid flower and LF—late flower/ripening). Photo from Ofélia Anjos.

#### *2.2. Extraction Conditions*

The extraction procedure was performed according to Paula et al. [18]. Briefly, 10 g of flower samples were extracted with 100 mL of solvent with 50/50 ethanol/water following 15 h of stirring at 120 rpm at room temperature. Afterward, the flower samples were centrifuged at 5000 rpm for 10 min and the supernatant was collected and stored at −20 ◦C until further analysis. MilliQ water was used for the extraction and 96% ethanol was purchased from Merck. Therefore, in this study, the extracts were prepared via mixing. All extractions were performed in duplicate and all subsequent measurements and analyses were performed in triplicate. The final concentration of the extract was 100 mg/mL, which involved a calculation based on the differences between the dried weight before and after the extraction of plant material. Then, all the dilutions were corrected so that these presented the same concentration, namely, 100 mg/mL.

#### *2.3. HPLC/DAD Conditions*

Chromatographic analyses were carried out using HPLC/DAD [19]. Briefly, 50 μL of each extract prepared were submitted to analysis in a Gilson 170 and Waters Spherisorb ODS2 (5 mm) column (4.6 × 250 mm) using water acidified with *o*-phosphoric acid to achieve pH = 2.4 (A) and an acetonitrile (B) gradient with a 0.8 mL/min flow rate and a column temperature of 24 ◦C. All samples were analyzed in triplicate. Standard chromatograms were plotted at λmax values of 260 and 340 nm. Spectral data for all peaks were accumulated in the range 220–500 nm using DAD (Gilson 170; World Headquarters, Middleton, WI, USA), and the software Unipoint was used to analyze each of them. The suitability of the method was previously evaluated by Campos [19]. All the extracts were submitted to a results analysis of the main phenolic constituents. Structural determination of the phenolic compounds was performed according to the theoretical rules presented in Campos and Markham [20].

#### *2.4. Total Phenol (TPC) and Flavonoid (TFC) Contents*

The total phenolic compounds were determined using the Folin–Ciocalteu method [21]. A volume of 0.5 mL of the sample or standard was diluted with 2.5 mL of Folin–Ciocalteu (10% *v/v*) reagent, and afterward, 2 mL of sodium carbonate (Na2CO3, 75 g/L) was added. The absorbance was measured at a wavelength of 760 nm after incubation for 2 h in the dark. The calibration curve was prepared using standard solutions of gallic acid (AG) with concentrations between 50 and 350 mg/L.

The flavonoid content was estimated according to Woisky et al. [22]. Different extract concentrations (2.5 mL) were diluted with 2.5 mL of ethanol solution of AlCl3 (2%). After 1 h of incubation in the dark at room temperature, the absorbance of the mixture was read at 420 nm.

#### *2.5. Antioxidant Activity*

The original ferric reducing antioxidant power (FRAP) assay (without incubation) was monitored according to a method reported by Berker et al. [23]. A sample extract concentration (0.1 mL) was diluted with 0.3 mL distilled water and 3 mL FRAP reagent. The mixture was shaken and the absorbance was read at 595 nm after 6 min in the dark.

Previous to this assay, a DPPH screening was performed following the methodology described in Alencar et al. [24].

#### *2.6. Acetylcholinesterase Inhibition Assay*

The acetylcholinesterase inhibition was assessed spectrophotometrically as described by Sukumaran et al. [25]. The acetylcholinesterase activity was quantified using 5,5 dithiobis-(2-nitrobenzoic acid) (15 mM, DTNB, Sigma Chemical, St. Louis, MO, USA); *Electrophorus electricus* acetylcholinesterase (electric eel Type-VI-S, Sigma Chemical, St. Louis, MO, USA) was used as the substrate. The hydrolysis of acetylcholine iodide was monitored through the formation of the anion 2-nitro-5-thiobenzoate (yellow) at 412 nm for 15 min. The percentage of inhibition was determined by comparing the rates of reaction of the samples with those of the blank (ethanol in phosphate buffer 0.2 M, pH = 8) using the expression ((E − S)/E) × 100, where E is the enzymatic activity without the extract and S is the activity of the sample. IC50 values were estimated from the graphic of inhibition percentage versus sample concentration. Eserine was used as a control.

#### *2.7. Lipase Inhibitory effect*

The inhibition of pancreatic lipase was assessed as described by Habib et al. [26]. In brief, 25 μL of the sample or the lipase inhibitor (Orlistat, Sigma Chemical, St. Louis, MO, USA) were mixed with 25 μL of lipase solution (16.7 U/mL in Tris-HCl, pH 8.0). For a 30 min incubation at 25 ◦C, the 4-methylumbelliferone product was released and the reaction was stopped by adding sodium citrate (0.1 mL of 0.1 M, pH 4.2). The measurements were performed with a fluorescence microplate reader (at 460 nm emission and 355 nm excitation). The results are expressed as percentages of α-lipase inhibition.

#### *2.8. Inhibition Assay for α-Glucosidase Activity*

The α-glucosidase activity was measured as reported by Tadera et al. [27]. The α-glucosidase-mediated transformation of the substrate pNPG into α-D-glucose and pnitrophenol at 405 nm was monitored. In brief, the enzyme (0.05 U/mL, Sigma Chemical, St. Louis, MO, USA) was dissolved in 100 mM phosphate buffer (pH 6.8) in a 96-well plate. It was pre-incubated at 37 ◦C for 5 min with the samples (10–60 μg/mL); p-nitrophenyl-α-D-glucopyranoside (600 μM, Sigma Chemical, St. Louis, MO, USA) was then added and the mixture was incubated at 37 ◦C for 30 min. The enzymatic reaction was monitored spectrophotometrically by measuring the absorbance at 405 nm. The obtained values corresponded to the slope measured between 5 and 20 min. Acarbose (0–200 μM, Sigma Chemical, St. Louis, MO, USA) was used as a positive control.

#### *2.9. Antimicrobial Potential*

The in vitro antimicrobial activity was evaluated through a broth microdilution assay in 96 multi-well microtiter plates, as recommended by the Clinical and Laboratory Standards Institute (CLSI) Guidelines. The minimum inhibitory concentration (MIC) of the samples, defined as the lowest concentration of extract necessary to inhibit microbial growth, was determined against Gram-negative bacteria (*Escherichia coli* ATCC 29998TM and *Escherichia coli* ESA37 cephalosporins-resistant), Gram-positive bacteria (*Staphylococcus aureus* ATCC 43300TM and *Staphylococcus aureus* ATCC ESA111) and the yeasts *Candida albicans* ATCC 10231TM and amphotericin B-resistant *Candida albicans* ESA100. Gentamicin and amphotericin B were used as controls. The cell mass was diluted with 0.85% NaCl solution to reach 0.5 on the "MacFarland scale", which was checked at 580 nm for bacteria and 640 nm for yeast. Muller–Hinton broth was used for bacteria or Yeasts Peptone Dextrose broth for yeast. Samples were diluted with broth to reach a concentration ranging from 5 to 30 mg/mL. The cell mass (106 colony-forming units (CFU)/mL) was added to all wells and incubated at 37 ◦C for 24 h and 25 ◦C for 48 h for bacteria and yeast, respectively. The antimicrobial activity was then determined by adding 20 μL of 2,3,5-triphenyl-2H-tetrazoliumchloride (TTC, 5 μg/mL) solution. In each assay, a negative control (containing only broth), a positive control (inoculated broth) and a DMSO control (DMSO with inoculated broth) were analyzed. The results were expressed in micrograms per milliliter.

#### *2.10. FT-Raman Spectral Acquisition*

The spectra of the plant extracts were obtained using the methodology proposed by [28] using an FT-Raman spectrometer (BRUKER, MultiRAM, Bruker Portugal Unipessoal, Lisbon, Portugal), equipped with a 180◦ high-throughput collecting lens, an ultra-high sensitivity liquid nitrogen cooled Ge Diode detector and an integrated 1064 nm diodepumped Nd:YAG laser with a maximum output power of 500 mW. The spectra were collected with 50 scans per spectrum at a spectral resolution of 8 cm−1 using a scanner velocity of 5 kHz in the wavenumber range from 4000 to 70 cm−1. The measurements were performed in duplicate in a 5 mm optic space quartz cell with the opposite face mirrored. The spectra were collected at constant room temperature (22 ◦C).

#### *2.11. Statistical Analysis*

The analysis of variance (ANOVA) was performed to identify the significance of the results and to determine the percentage of the variance of each factor using STATISTICA 7 (StatSoft Inc., Tulsa, OK, USA) software. Whenever the *p*-value was below 0.05, Tukey's test was applied. Mean values and standard deviations were calculated and are presented in the figures as error bars.

Principal component analysis (PCA) was performed to identify the relationship between the factor (site and flower maturation) and the different variables measured.

For ANOVA and PCA, the StatSoft software was used; for spectral data analyses OPUS OPUS®, version: 7.5.18 (Bruker Optik, Ettlingen, Germany) and UnscramblerX 10.5 (CAMO, Oslo, Norway) were applied.

#### **3. Results and Discussion**

*3.1. Characterization of the Extract*

#### 3.1.1. HPLC/DAD

From the HPLC/DAD chromatograms, six compounds were identified (Figure 2) and the corresponding ultraviolet absorption spectra of the most concentrated are provided in Figure 3. The minor compounds were related to the nucleus of flavonols (compounds **1**–**4**) and the major were related to chalcones (compounds **5** and **6**).

**Figure 2.** Example of an HPLC/DAD profile of the early maturation stage flowers from region A obtained with the most active *Acacia dealbata* L. hydroethanolic extract (50% (*v/v*)). Compounds **1**–**4** were flavonols and **5**–**6** were chalcones.

**Figure 3.** Ultraviolet absorption spectra obtained using HPLC/DAD analysis of the flavonoid content. Compounds (**a**,**b**) were chalcone derivatives.

These latter components also belong to the flavonoid group of polyphenols; however, the C-ring remained open between the oxygen in position 1 and the carbon in position 2 (for a better understanding, please check the structure in Figure 4). These two compounds were chalcone derivatives that were previously identified by Campos [19].

**Figure 4.** Basic structures of flavone (**a**) and chalcone (**b**).

Chalcones are polyphenols are structured as *α*,*β*-unsaturated aromatic ketones and can be found in various plants. They have been targeted in various studies that provide data about their potential in various therapeutic purposes, attributing bioactivities to them, such as helpfulness regarding oncology, infections and even in neurodegenerative diseases, including Alzheimer's [29]. For the latter, the antioxidant and anti-inflammatory effects are also very important, and the data obtained with the present extract was found to be promising for some of these applications, as shown below.

#### 3.1.2. Total Phenol (TPC) and Flavonoid (TFC) Contents

Nevertheless, phenolic acid derivatives were not found in significate amounts in the hydroethanolic extracts, where its full quantification was performed using the fast, cheap and conventional Folin–Ciocalteu assay; chalcones can also be quantified with this methodology [30]. These results are shown in Figure 5, as well as the total content for flavonoids. The high TPC and TFC for both region extracts corresponded to the hydroethanolic solutions (50%) of the EF. However, even with lower differences, the highest concentration corresponded to EF from plants in region A.

**Figure 5.** Total phenol and flavonoid contents ((**a**) TPC and (**b**) TFC) of *A. dealbata* flower extracts with different maturation stages from two central regions of Portugal. A—region A, B—region B; EF—early flower, MF—mid flower, LF—late flower. Different lowercase letters (a, b, c) denote significant differences between values (Tukey's test; *p* < 0.05).

The amount of TPC in flowers from the two different regions ranged from 201.28 to 313.04 μg gallic acid/mL for region B and region A, respectively, among the various statuses of maturation. The results obtained are in agreement with other authors, who obtained, for example, a concentration of 206.4 mg gallic acid/g sample in the Acacia flowers [31]. Wu et al. [32] obtained higher results for TPC with an ethanol extraction for *A. confusa* (544.4 mg/g of flower). In the aforementioned study, it was also found that the TPC was higher when ethanol was used as extract solvent. They also obtained high values for the antioxidant bioactivity due to this content in phenolic compounds.

The flowers harvested in region A presented a higher value of total phenol and flavonoid content, where this difference represented 24.4% of the total variation for the TPC and 37.4% for the TFC (Table 1). The maturation status was also of key importance where the higher values were obtained for the early flowering status (55.5% of the total variation for the TPC and 27.3% of the total variance for the TFC). This data was in accordance with the function of chalcones as precursors of flavonoids.

**Table 1.** Percentage of variance obtained in a two-way ANOVA of the total phenol and flavonoid contents of the flowers extracts.

MS—maturation status, EF—early flower, MF—mid flower, LF—late flower; n.s. for *p* > 0.05, \* 0.01 < *p* < 0.05, \*\* 0.001 < *p* < 0.01, \*\*\* *p* < 0.001.

One of the goals in this work was to understand whether the early flowering status had better potential as a source of antioxidant compounds, or at least equal to the bioactivity of the compounds in the maturated flowers in the ripening stage, in order to collect them earlier and not give the possibility for the pollen to disperse. In this case, the results showed better activity than was expected, certainly due to the highest amounts of chalcones in this phase of the vegetative cycle of Acacia. These values were in line with the data obtained for the antioxidant activities. Studies from various authors with other *Acacia* spp. also showed that the TPC can vary according to the plant organ, solvent used in the extraction procedure and season [33,34], which were in accordance with the data here discussed for *A. dealbata* extracts.

#### *3.2. Bioactivities*

#### 3.2.1. Antioxidant Activity

Among many other bioactivities associated with polyphenolic compounds, the antioxidant activity is one of the most reported [35,36] and can be explored for various purposes. The concerns about the increase in neurodegenerative diseases have increased the interest in some of these most promising molecules [29]. In the present work, our main goal was to verify its previous potential for the evaluation of the freshness of the matrix, for further standardization of the extracts and its potential for future research regarding anti-inflammatory drugs or to help in the prevention of degenerative diseases in accordance with its inhibitory activity for the enzyme acetylcholinesterase.

Various methodologies were chosen to evaluate this potential, as presented below. One of the most used to obtain preliminary data is the DPPH scavenging assay, which determines the capacity of a compound or group of compounds to stabilize the 2,2-diphenyl-1-picrylhydrazyl (DPPH˙) radical. The amount of extract required to scavenge 50% of DPPH solution (and for FRAP reagent) is defined as the IC50 (mg/mL). The antiradical free scavenging radical activity is measured through the stable radical DPPH, which shows the ability of substances present in the extracts to sequester free radicals from the existing medium. The extracts under evaluation showed free radical scavenging activity with EC50 values ranging from 6.79 to 9.93 mg/mL (Figure 6), which are good activities. The hydroethanolic extracts prepared with the EF collected in both locations showed similar results between the analyzed samples. Nevertheless, the data collected presented a decrease in this bioactivity with increasing maturation status. The results could be correlated to the content of phenols and flavonoids quantified in the samples. These first results suggested that *A. dealbata* could be a good source of natural antioxidants, which may be used to stabilize free radicals, which induce oxidative stress.

The other analysis performed to validate this data was a FRAP (ferric reducing antioxidant power) assay, which measures the ability of antioxidants to reduce the Fe(III)–TPTZ complex to the intensely blue Fe(II)–TPTZ complex in an acidic medium. The antioxidant activity can be measured spectrophotometrically by the change in color of the solution to blue resulting from the ferric to ferrous ion reduction, i.e., Fe(II) to Fe(III), while coordinated by 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ). The data on the ferric reducing potential of the analyzed extracts also indicated better values for the EF extracts in both locations. All other extracts recorded moderate FRAP activity.

**Figure 6.** Antioxidant activity ((**a**) FRAP EC50 and (**b**) DPPH EC50) of *A. dealbata* flowers extracts with different maturation stages with a mixture of 50/50% (*v/v*) ethanol/water from two central regions of Portugal. A—region A, B—region B; EF—early flower, MF—mid flower, LF—late flower. Different lowercase letters (a, b, c) denote significant differences between values (Tukey's test; *p* < 0.05).

The data acquired with the extracts under study are shown in Figure 6.

Table 2 depicts that the material region harvested was not significant for the FRAP assay; however, the maturation status represented nearly 83% of the total variation in DPPH and nearly 54% for FRAP with a high significance.

**Table 2.** Percentage of variance obtained in a two-way ANOVA (factor 1: site (S) and factor 2: maturation status (M)) of the antioxidant activity parameters for the flower's extracts.

MS—maturation status, EF—early flower, MF—mid flower, LF—late flower; n.s. for *p* > 0.05, \* 0.01 < *p* < 0.05, \*\* 0.001 < *p* < 0.01, \*\*\* *p* < 0.001.

#### 3.2.2. Enzyme Inhibitory Activity (Acetylcholinesterase, Lipase and α-Glucosidase Enzymes)

The enzyme inhibitory activity of *A. dealbata* flower extracts with different maturation stages was evaluated due to the potential application in drug discovery in different possible applications. The data shown here correlates with the three enzymes studied. First, the most relevant of them, which can be directly associated with neurodegenerative diseases, is the effect of acetylcholinesterase. This enzyme is able to metabolize acetylcholine, and its inhibition will increase this neurotransmitter in the synaptic cleft. The activity is expected to increase cerebral functions [37], which can contribute to delaying the development of neurodegenerative diseases.

All the flower extracts showed the ability to inactivate acetylcholinesterase, lipase and α-glucosidase enzymes (Figure 7). Despite all the concentrations of the extracts being able to inhibit the acetylcholinesterase enzyme, the most effective, with values between 44.1 and 49.16 μg/mL, was correlated with the EF maturation stage. Even using different *Acacia mearnsii* material (bark rich in tannin compounds), Ogawa and Yazaki [38] also verified similar results for lipase and glucosidase inhibition.

**Figure 7.** Enzyme inhibitory activity of *A. dealbata* flowers' hydroethanolic extracts with different maturation stages for (**a**) acetylcholinesterase, (**b**) lipase and (**c**) α-glucosidase activities. A—region A, B—region B; EF—early flower, MF—mid flower, LF—late flower. Different lowercase letters (a, b, c) denote significant differences between values (Tukey's test; *p* < 0.05).

The flowers harvested in region A had a higher value of inhibition capacity for all the enzymes and this difference represented 51.9% of the total variation for acetylcholinesterase inhibition, 25.7% for lipase and without effect for 13.6% for α-glucosidase (Table 3). The maturation status (Table 3), once again, was also of key importance, with a higher variability attributed to these factor (27.7% of the total variation for acetylcholinesterase, 52.6% of the total variation for lipase and 68.5% of the total variation for α-glucosidase).

**Table 3.** Variance percentages obtained in a two-way ANOVA of enzyme inhibitory activity of *A. dealbata* flowers.

GLUC—α-glucosidase, ACET—acetylcholinesterase; MS—maturation status, EF—early flower, MF—mid flower, LF—late flower; n.s. for *p* > 0.05, \* 0.01 < *p* < 0.05, \*\* 0.001 < *p* < 0.01, \*\*\* *p* < 0.001.

The importance associated with these three enzymes is the added value in the bioactivity, for instance, for acetylcholinesterase inhibition.

#### 3.2.3. Antimicrobial Bioactivity

This bioactivity can be explored alone or even in parallel to others. For instance, if the target patients with neurodegenerative diseases need to be medicated with corticosteroids, which induce an immunodepression effect that makes them more vulnerable to infections, a molecule that accumulates acetylcholinesterase inhibition and activity against the most common bacteria and yeasts will improve the therapy [39]. Therefore, to complete the bioactivities for these extracts, we chose this last bioactivity, which will contribute better performance to further studies of the compounds in the extracts.

The extracts were screened for the antimicrobial activity for various microorganisms *E. coli* ATCC 29998 TM, *Staphylococus aureus* ATCC 43300TM, *Candida albicans* ATCC 10231 TM, *E. coli* ESA37, *S. aureus* ATCC ESA111 and amphotericin-B-resistant *Candida albicans* ESA 100 were tested.

The minimum inhibitory concentration of the *A. dealbata* flower extracts with different maturation stages is shown in Table 4.

**Table 4.** Antimicrobial bioactivity activity of the *A. dealbata* flower extracts with different maturation stages.

A—region A, B—region B; EF—early flower, MF—mid flower, LF—late flower; EcoliA—*E. coli* ATCC 29998 TM, Saur—*S. aureus* ATCC 43300TM, Calbi—*C. albicans* ATCC 10231 TM, EcoliESA—*E. coli* ESA37, SaurESA—*S. aureus* ATCC ESA111, AB\_r—amphotericin-B-resistant *Candida albicans* ESA 100. Different lowercase letters (a, b, c, d, e) in the same column denote significant differences between values (Tukey's test; *p* < 0.05).

The results shown in Table 5 report the higher effects of the region and maturation status on the antimicrobial bioactivity. Once again, the extracts from the EF harvested in region A gave the best activity. It seems that maybe a possible change occurred due to the different climatic conditions, soils or other factors. The effect of the harvest place should be explored more in future research designed with this purpose.

**Table 5.** Variance percentages obtained in a two-way ANOVA of the antimicrobial bioactivity of *A. dealbata* flowers.

EF—early flower, MF—mid flower, LF—late flower; EcoliA—*E. coli* ATCC 29998 TM, Saur—*S. aureus* ATCC 43300TM, Calbi—*C. albicans* ATCC 10231 TM, EcoliESA—*E. coli* ESA37, SaurESA—*S. aureus* ATCC ESA111, AB\_r—amphotericin-B-resistant *Candida albicans* ESA 100; n.s. for *p* > 0.05, \* 0.01 < *p* < 0.05, \*\* 0.001 < *p* < 0.01, \*\*\* *p* < 0.001.

To summarize, in Figure 8, it is possible to see that all the data collected clearly highlighted that for all the bioactivities explored in these extracts, the EF status of maturation was the most significant. The principal component analysis explained 79.2% of the total variability observed for the different parameters analyzed concerning the two factors (site and maturation status). It is possible to note that regions A and B showed different behaviors concerning the studied properties, highlighting an influence of the harvest place regarding the properties of the plants given to the extracts. PC1 (which explained 64.3% of the total variation) clearly separated the three maturation stages, with EF showing the better results, which could be an advantage because collecting the flower in an early stage inhibits the possibility of the reproduction of the plant, which prevents further reproduction and invasion of other lands.

**Figure 8.** Principal component analysis of ethanol:water (50:50) extracts from the *A. dealbata* flowers in different maturation stages from two central regions of Portugal. A—region A, B—region B; EF—early flower, MF—mid flower, LF—late flower; TPC—total phenol content, TFC—total flavonoid content; GLUC—α-glucosidase, ACET—acetylcholinesterase; EcoliA—*E. coli* ATCC 29998 TM, Saur—*S. aureus* ATCC 43300TM, Calbi—*C. albicans* ATCC 10231 TM, *Ecoli*ESA—*E. coli* ESA37, SaurESA—*S. aureus* ATCC ESA111, AB\_r—amphotericin-B-resistant *Candida albicans* ESA 100.

#### *3.3. FT-Raman Spectral Acquisition*

The spectra obtained with the hydroethanolic flowers extract (50:50) shown in Figure 9 display the strong influence of the compounds in this matrix [40]. The intense bands around 2975, 2931 and 2883 cm−1 were assigned to C–H stretching of some compounds present in the plant extracts, but also of the CH2 and CH3 stretching of the ethanol [18], and were influenced by the other substances from the plant extracts. The peak at 1450 cm−1 corresponded to the C–OH bending of ethanol, but also originated from the combination of bending vibration of CH2 and the vibration of the COO– group in the flavanol and organic acids [41,42].

**Figure 9.** Raman spectrum, baseline corrected spectrum for Acacia flowers samples.

The band at 1270 cm−1 was found to be due to the deformation vibration of O–C–H, C–C–H and C–O–H.

The peaks at 1090 and 1050 cm−1 were from the in-plane movement of the carbon ring and the substituents, which are regions linked to the characterization of alcohols, ethers, esters, carboxylic acids and anhydrides [43].

The bands around 705 cm−1 corresponded to the C–O and C–C–O stretching and O–C–O bending, and the bands in the 880 cm−1 region were assigned to the C–H, C–O–H and CH2 bending.

Concerning the results of the PCA (mean centered) made with the spectral data (Figure 10) the separation between regions as observed for the analytical data is clear, as well as the separation between the maturation stages of the flowers.

**Figure 10.** Score plot of the first two principal components after the PCA performed on the Raman spectra recorded from Acacia flowers samples using the first derivative Savitzky–Golay spectra transform with 15 smoothing points.

These results showed that Raman spectroscopy is a promising technique for discriminating between flower raw materials to identify possible differences in the biological activities of their extracts.

To our knowledge, this is the first time that these extracts have been studied and these bioactivities were explored together to provide a possible new drug strategy in neurodegenerative treatments, among other possibilities, and to help to contribute to the control of an invasive plant.

#### **4. Conclusions**

One of the challenges to be faced soon is the green chemistry and development of new drugs with low environmental impacts. The compounds of *A. dealbata* in the EF stage were found to have the necessary bifocality that fulfilled these two requirements. The antioxidant activity and the acetylcholinesterase inhibition of the compounds in the extracts enrolled in this work are presented for the first time as promising for further potential research for some therapeutic purposes, for instance, neurodegenerative diseases. These compounds can also be redesigned in new structures for new drugs, for instance, to enhance memory and other brain functions by influencing chemical activity in the brain. From the data presented in this work, all the assays revealed the best potential for the non-mature flowers of *A. dealbata*. This allows for the possibility to harvest them at this preliminary status and stop the reproduction of the plant, which will help to control its invasive potential.

In this study, it was also shown that Raman spectroscopy is a promising technique for identifying the differences between flower extracts in order to better select raw materials based on their biological activities.

**Author Contributions:** Conceptualization, O.A., M.G.C. and L.M.E.; methodology, V.P., M.G.C., S.I.P., T.D., L.M.E. and O.A.; software, O.A.; validation, V.P., M.G.C., S.I.P., T.D., L.M.E. and O.A.; formal analysis, V.P., M.G.C., S.I.P., T.D., L.M.E. and O.A.; investigation, V.P., M.G.C., S.I.P., L.M.E. and O.A.; writing—original draft preparation, V.P., M.G.C., S.I.P., L.M.E. and O.A.; writing—M.G.C., L.M.E. and O.A.; supervision O.A., M.G.C. and L.M.E.; project administration, O.A., M.G.C. and L.M.E.; funding acquisition, M.G.C., L.M.E. and O.A. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by project PCIF/GVB/0145/2018 (Acacia4fireprev). This work is also funded by National Funds through the FCT—Foundation for Science and Technology under the Projects UIDB/00239/2020 (CEF), (UI0204): UIDB/00313/2020 and UIDB/00690/2020 (CIMO).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** We choose to exclude this statement because the study did not report any data.

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

#### **References**

## *Article* **GC- and UHPLC-MS Profiles as a Tool to Valorize the Red Alga** *Asparagopsis armata* **†**

**Diana C. G. A. Pinto 1,\*, Marie L. Lesenfants 2, Gonçalo P. Rosa 1,2,3, Maria Carmo Barreto 2,3, Artur M. S. Silva 1 and Ana M. L. Seca 1,2,3**

**Abstract:** *Asparagopsis armata* Harvey is a red alga native from the southern hemisphere and then introduced in the Mediterranean Sea and the Atlantic Ocean, including the Azores Archipelago, where it is considered an invasive alga. Some studies show that the extracts exhibit antimicrobial and antifouling activities, and it is incorporated in some commercialized cosmetic products. (e.g., Ysaline®). However, knowledge of this species chemical composition is scarce. The GC-MS and UHPLC-MS profiles of both the nonpolar and polar extracts were established to contribute to this problem solution. According to the results, *A. armata* is rich in a great structural variety of halogenated lipophilic and aromatic compounds, some of them identified here for the first time. In the lipophilic extract, 25 compounds are identified, being the halogenated compounds and fatty acids, the two major compound families, corresponding to 54.8% and 35.7% of identified compounds (224 and 147 mg/100 g of dry algae, respectively). The 1,4-dibromobuten-1-ol and the palmitic acid are the two most abundant identified compounds (155 and 83.4 mg/100 g of dry algae, respectively). The polar extract demonstrated the richness of this species in brominated phenolics, from which the cinnamic acid derivatives are predominant. The results obtained herein open new perspectives for valuing the *A. armata* as a source of halogenated compounds and fatty acids, consequently improving its biotechnological and economic potential. Promoting this seaweed and the consequent increase in its demand will contribute to biodiversity conservation and ecosystem sustainability.

**Keywords:** *Asparagopsis armata*; Rhodophyta; GC-MS; UHPLC-MS; dibrominated compounds; 1,4-dibromobuten-1-ol; palmitic acid; brominated phenolics; red seaweed; invasive seaweed

#### **1. Introduction**

Marine organisms live in complex habitats and are often at the mercy of too variable environmental conditions. To protect themselves from predators, competitors, and many other threats, sea creatures were forced to develop protective mechanisms that include producing a vast number of bioactive compounds, many of which are unique in chemical structure and biological function [1,2]. Seaweeds are among the most explored marine organisms due to their richness in bioactive compounds [3]. However, there are still several species for which the chemical profile is unknown, and *Asparagopsis armata* is one example.

*Asparagopsis* species belong to the phylum Rhodophyta (red seaweeds), order Bonnemaisoniales, genus *Asparagopsis* with three taxonomically accepted species: *A. armata* Harvey, *A. svedelii* Taylor, and *A. taxiformis* Trevisan [4]. *Asparagopsis taxiformis* has been used as food by Hawaiians [5] while *A. armata* is commercially farmed in northern Europe

**Citation:** Pinto, D.C.G.A.; Lesenfants, M.L.; Rosa, G.P.; Barreto, M.C.; Silva, A.M.S.; Seca, A.M.L. GC- and UHPLC-MS Profiles as a Tool to Valorize the Red Alga *Asparagopsis armata*. *Appl. Sci.* **2022**, *12*, 892. https://doi.org/10.3390/ app12020892

Academic Editor: Magdalena Biesaga

Received: 26 December 2021 Accepted: 13 January 2022 Published: 16 January 2022

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

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

to extract bioactive molecules [6]. *Asparagopsis armata* is native to Australia and is now widely distributed from the North Atlantic to the coast of Senegal and in the Mediterranean basin [7]. This seaweed seems to exhibit an ecological advantage due to its toxic metabolites that affect the competing algae fixation, forming monospecific coverages, reducing the habitat's species richness, and causing an indirect economic impact by negatively affecting fishing and aquaculture [8]. Despite its negative impact, *A. armata* is commercially farmed in northern Europe to extract bioactive molecules such as sulphated polysaccharides with iodine and bromine groups [6], that are applied as a natural preservative in cosmetics and products to anti-acne treatment (e.g., Invincity®). In Azores, this invasive seaweed presents an enormous rate of biomass production for which there is no use. Uses such as food or technological application that increase their commercial value and thus create a dynamic that mitigates the negative effects mentioned above are essential.

Although several studies have been published on the biological activity of *A. armata* extracts showing their potential as a source of natural compounds with antiviral, antimicrobial, and antifouling activity [9–11], there are few published works dedicated to the characterization of secondary metabolites existing in the extracts of this species, apart from sterols and volatile halogenated compounds [12–14]. Herein we present and discuss the composition of the polar and lipophilic extracts of *A. armata*, obtained by greener and more efficient methodologies and using GC-MS and UHPLC-MS as tools for separation and identification of the metabolites present.

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

#### *2.1. Chemicals*

The following chemicals are used as standards for UHPLC-MS analysis: 2-bromopropionic acid, 2-bromo-5-methoxybenzoic acid and methyl 2-bromo-5-methoxybenzoate were purchased from Alfa Aesar (Kandel, Germany). 2-Bromobenzaldehyde and 3-bromobenzaldehyde were purchased from Sigma-Aldrich GmbH (Darmstadt, Germany). 2 -Bromoacetophenone was obtained from TCI (Oxford, UK). Caffeic, *p*-coumaric, and chlorogenic acids were supplied by Acros Organics (Geel, Belgium).

UHPLC mobile phase eluents: formic acid and acetonitrile HPLC-grade solvents were purchased from Panreac (Barcelona, Spain), and ultrapure water was obtained by a Direct-Q® water purification system (Merck Life Science, Darmstadt, Germany). All other chemicals were of analytical grade.

Solvents used for GC–MS analysis were purchased from Panreac and Acros Organics and were of analytical grade. Chemicals such as *N,O*-bis(trimethylsilyl)trifluoroacetamide (BSTFA) (99%) and trimethylsilyl chloride (TMSCl) (99%), purchased from Sigma-Aldrich, were used as derivatization agents. Octadecane (99%), eicosane (99%), palmitic acid (99%), stearic acid (>97%), (*Z*,*Z*) 9,12-octadecadienoic acid (99%), 1-bromononane (98%), 1,8-dibromooctane (98%), (3β) cholest-5-en-3-ol (99%), 1-octadecanol (99%), and 1-mono palmitin (99%) were purchased from Sigma-Aldrich and used as standards to qualitative and quantitative GC-MS analysis.

#### *2.2. Plant Material and Extracts Preparation*

*Asparagopsis armata* Harvey (on gamethophyte phase) was collected in June 2017, in the intertidal zone of S. Miguel Island coast, in Ponta Delgada, Azores. Prof. Ana Isabel Neto, expert on seaweeds from University of Azores, was responsible for the taxonomic identification of the harvested alga. A voucher specimen (SMG-2017-06) was deposited in Ruy Telles Palhinha Herbarium (AZB).

*Asparagopsis armata* sample was cleaned from salt and epiphytes, drained between sheets of absorbent paper, and cut into pieces approximately 1 cm long. Fresh algae after draining contains 92.9% (±0.97%) of water.

Dried *A. armata* (231.06 g) were obtained from fresh material by air drying, protected from light, and then dried in an oven with forced ventilation at 40 ◦C, until constant weight.

The lipophilic extract (extract A) was prepared from 229.2 g of dried seaweed by maceration (1:10 algae:dichloromethane) at room temperature, under stirring and in the dark, for 3 cycles of 3 days each, with solvent renewal at the end of each cycle. The solvent from the combined extraction steps was first evaporated with a rotary vacuum evaporator at 40 ◦C and then dryed under vacuum at room temperature (yield 1.25%).

From fresh *A. armata*, collected from the same place and date, several polar extracts were prepared, using different polar solvents (ethanol, ethanol:water (50%), and water) and different methods of extraction (maceration, ultrasounds, and microwave) in order to optimize the extraction procedure according to the Table 1.

**Table 1.** Experimental conditions used in the preparation of the *A. armata* polar extracts.

1 The proportion algae:solvent was 1 g:4 mL. 2 The proportion algae:solvent was 1 g:10 mL.

The dried polar extracts were obtained after evaporation and/or lyophilization. The yields obtained are shown in the Table 2.

**Table 2.** Extraction yield of the polar *A. armata* extracts prepared.

#### *2.3. Biologic Activities*

The following paragraphs present the main experimental details of each of the bioactivity assays such as range of concentrations tested, and the positive controls used. A more detailed description of each of the methods employed can be found in Zárate et al. [15].

#### 2.3.1. DPPH Scavenging Activity

Antioxidant activity was assayed by the DPPH (1,1-diphenyl-2-picryl-hydrazyl) radical scavenging assay [16]. The extracts were tested at concentrations ranging between 0.244 μg/mL and 250 μg/mL in methanol. Trolox, at concentrations ranging from 0.100 to 100 μg/mL, was used as the positive control.

#### 2.3.2. ABTS Scavenging Activity

A microplate adaptation of the method by Re et al. [17] was adopted to perform the ABTS radical scavenging assay. The extracts were tested in a range of concentrations of 0.244 to 250 μg/mL, and the positive control was Trolox (0.100–100 μg/mL).

#### 2.3.3. Ferric Chelating Activity

The Fe2+ chelating ability of the extracts was assayed by the ferrous iron-ferrozine complex method [18]. The positive control used was EDTA (0.195–100 μg/mL). Samples were assayed at serial concentrations between 0.244 and 250 μg/mL.

#### 2.3.4. Anticholinesterasic Activity

A modification of the method described by Ellman et al. [19] and Arruda et al. [20] was used for measuring the extracts' ability to inhibit the activity of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). The algal extracts were tested at concentrations ranging from 0.293 to 150 μg/mL and compared to the positive control donepezil (0.010–5.0 μg/mL).

#### 2.3.5. Inhibition of Tyrosinase Activity

The extracts were assayed by adapting the tyrosinase inhibition method described by Shimizu et al. [21] and modified by Manosroi et al. [22]. Serial concentrations of the extracts (0.488 μg/mL to 250 μg/mL dissolved in 100 mM phosphate buffer, pH 6.8 were tested. Kojic acid at 0.293–150 μg/mL was used as positive control.

#### 2.3.6. Inhibition of Collagenase Activity

The anti-collagenase activity was assayed through an adaptation of the method of Mandl et al. [23]. The extracts and the reference compound EDTA were tested with concentrations ranging between 15.6–250 μg/mL.

#### 2.3.7. Inhibition of Elastase Activity

A modified version of the described by Ndlovu et al. [24] was used to measure the inhibition of elastase activity by the extracts. The concentration of extract used in the assay ranged from 0.488 μg/mL to 250 μg/mL. *N*-Methoxysuccinyl-Ala-Ala-Pro-chloromethyl ketone at 0.019–20 μg/mL was used as a positive control.

#### *2.4. UHPLC-DAD-ESI-MS/MSn Characterization of A. armata Aqueous Extract*

A methanolic solution of *A. armata* aqueous extract (prepared under the experimental conditions described in Table 1 for extract **2** and then lyophilized) was analyzed using an UHPLC-ESI-DAD/MS apparatus. The column used was a thermo scientific hypersil gold column (1000 mm × 20 mm) with a part. size of 1.9 μm at 30 ◦C. The mobile phase was composed of (A) 0.1% formic acid aqueous solution (*v/v*) and (B) acetonitrile, and the flow rate was 0.2 mL/min. The solvent gradient started with 5% of solvent A over 14 min followed by 40% of solvent A for 2 min, 100% over 7 min and finally 5% over 10 min. The injection volume was 2 μL, and the chromatographic profiles were documented at 280 nm. The mass spectrometer used was an LTQ XL linear ion trap 2D equipped with an orthogonal electrospray ion source (ESI). The equipment was operated in negative-ion mode with electrospray ionization source of 5.00 kV and ESI capillarity temperature of 275 ◦C. The full scan covered a mass range of 50–2000 *m/z*. The identification of individual phenolic compounds by UHPLC-MS was achieved by comparing with data available on the literature and interpretation of the MS fragmentation.

#### *2.5. GC-MS Analysis of A. armata Dichloromethan Extract*

Before GC-MS analysis, four replicates of dried dichloromethane extract of *A. armata* (extract A, nearly 20 mg each), was dissolved in 1 mL of dichloromethane, added the internal standard (octadecane) and was silylated using 250 μL of pyridine, 250 μL of BSTFA, and 50 μL of TMSCl in a screw glass tube at 70 ◦C during 30 min. This derivatization procedure is well known [25,26] and allows the hydroxyl groups that may exist in the extract constituents to be transformed into silyloxyl groups. Thus, compounds with -OH substituents will be detected by GC-MS analysis as silylated derivatives which are more volatile, stable, and less polar [25,26].

Each replicate was analyzed by GC–MS twice using a QP2010 Ultra Shimadzu apparatus with Xcalibur software, equipped with a DB-5- J &W capillary column (30 m × 0.25 mm i.d. and a film thickness of 0.25 μm). The injection was in split mode. The temperature of the column was 70 ◦C during 2 min, at 3.5◦/min until 120 ◦C, at 9◦/min until 170 ◦C, 170 ◦C during 10 min, at 9◦/min until 300 ◦C, which was maintained for 6 min. Injector temperature was at 320 ◦C, and the transfer-line temperature was at 200 ◦C. The mass spectrometer was operated in the EI mode with energy of 70 eV, and data were collected at a rate of 1 scan s−1 over a range of *m/z* 50–1000. The carrier gas was helium (purity of 99.995%) at 1.2 mL/min.

The compounds were identified, from total ion chromatogram, by comparing the retention times and by comparing their mass spectra with (i) those existing in the MS databases of Wiley229, NIST14, and Shimadzu Pesticide Library; (ii) those published in the literature and by analyzing the fragmentation pattern; (iii) those obtained in the same experimental conditions to standard compounds.

Quantitative analysis was carried out using internal standard method (octadecane, 28.68 min), by intrapolation on the calibration curves obtained with at least 4 distinct but known concentrations of representative standards of each family of organic compounds identified in the dichloromethane extract (namely, palmitic acid, stearic acid, (*Z*,*Z*) 9,12-octadecadienoic acid, 1-octadecanol, monopalmitin, cholesterol, sitosterol, phenyl acetic acid, 1,8-dibromooctane, 1-bromononane) relative to octadecane. The correlation coefficient (r2) of each calibration curve was greater than 0.99. All standards with hydroxyl substituents were subjected to the silylation procedure described above.

#### **3. Results and Discussion**

#### *3.1. Asparagopsis armata Polar Extracts Yield and Chemical Diversity*

Extracts from fresh *A. armata* were prepared using polar solvents (water, ethanol) and different energy sources according to the data present in Table 1. The yields of extractions are presented in Table 2. Preparing polar extracts from fresh instead of dry seaweed has the additional advantage of circumventing the difficulties of drying seaweed and grinding after drying. The alga has a high-water content (92.9%), and the drying process is prolonged. However, no signs of chemical degradation were evident (no changes in color or observed microorganism development). After drying, the seaweed becomes extremely hard, requiring very robust equipment for its grinding.

The different methods and solvents tested reveal identical performance in terms of extraction yield.

Considering the yields obtained and the more sustainable experimental conditions (economically and environmentally "greener"), the profiles of the most interesting extracts were qualitatively analyzed by UHPLC-MS, with the most relevant results shown in Figure 1.

The chemical profiles of the ethanol extracts (extracts 7 and 8) are very similar, being both ultrasound and microwave the most selective extraction method. The use of ethanol under microwave energy extracted almost exclusively one compound. Although it was not our aim to identify this compound, its isotopic pattern and *m/z* in the mass spectra suggests that it is a 2,3-diphenylpropanoic acid derivative, having two hydroxyl groups and four bromine atoms distributed in the aromatic rings ([M-H]− *m/z* 568/570/572/574/576). Due to the fragment with *m/z* 319/321/323, we suggest that each aromatic ring possesses one hydroxyl group and two bromine atoms.

The chemical profile of the water extracts (extracts **1** and **2**) showed greater chemical diversity. Thus, the extraction method must be chosen according to the intended application to the extracted compounds that are different according to the method used. If the extraction time is the most relevant, it should choose ultrasound extraction since it is cheaper than microwave extraction, which requires a more expensive apparatus. Using water extraction by maceration at room temperature will be cheaper and allow chemically richer extracts.

**Figure 1.** UHPLC chromatograms of *greener A. armata* polar extract with higher yield, recorded at 280 nm.

As this work intends to study the chemical profile of the polar extract of *A. armata*, the characterization of the chemical composition of the aqueous extract obtained by maceration at room temperature (extract **2**) constitutes a significant scientific contribution to knowledge about new polar secondary metabolites in this alga.

#### *3.2. Biological Activities of Asparagopsis armata Extracts*

Considering the potential application of *A. armata* extracts in the cosmetic industry, the dichloromethane extract (extract A) and the aqueous extract (extract **2**), which are in our perspective the most interesting ones, were evaluated for their potential as anti-aging agents. In fact, if we consider some green chemistry aspects, such as extraction yields and energy consumption, these extracts are the best option. Moreover, their chemical profile characterization was achieved.

In this regard, the antioxidant, antiacetylcholinesterase, and antibutyrylcolinesterase activities of extract A were evaluated. Regarding the antioxidant activity, this extract demonstrated an interesting ability to scavenge both the DPPH and ABTS radicals (23.6 ± 2.6% and 31.4 ± 2.8%, respectively), if we compare with the positive control values (Troloxinhibition of DPPH 89.7 ± 0.5% and inhibition of ABTS 92.4 ± 0.2%). However, these values were obtained at concentrations of 250 μg/mL, and it was not possible to calculate EC50 value for extract A. Concerning the cholinesterases inhibition, the extract A showed, at a concentration of 150 μg/mL, inhibition values of 17.1 ± 2.3% and 18.9 ± 2.0%, respectively for acetylcholinesterase and butyrylcholinesterase. Again, it is imperative to highlight that it was impossible to obtain the EC50 and the positive control value for donepezil at a concentration of 2 μg/mL was 95.2 ± 0.4%.

Regarding the extract **2** biological assays, we evaluated the ferric chelating ability, the antioxidant, anticollagenase, and antielastase activities. All the activities were evaluated using 250 μg/mL extract concentration, and the EC50 could not be obtained. The extract ability to scavenge the DPPH radical and to chelate iron(III) can be considered low and moderate, with percentage inhibition values of 1.47 ± 0.42% and 7.75 ± 0.44%, respectively for DPPH (trolox 89.71 ± 0.50%, concentration of 250 μg/mL) and ferric chelating (EDTA 93.73 ± 0.24%, concentration of 100 μg/mL) assays. In the case of anticollagenase, the same extract showed an inhibition percentage of 7.42 ± 1.79%, which was much lower than the value for positive control EDTA (96.31 ± 1.20%). Finally, the extract inhibited elastase by 43.20 ± 2.58% at a 250 μg/mL concentration, an interesting value, although not comparable to the positive control, *N*-methoxy succinil-Ala-Ala-Pro-Val-chloromethyl ketone which at a 20 μg/mL inhibited the enzyme by 95.51 ± 3.96%.

Although the results of the bioassays were not exceptional, it is possible to conclude that *A. armata* produce bioactive metabolites, and we can infer that their isolation will improve the activity.

#### *3.3. UHPLC-MS Characterization of A. armata Aqueous Extract*

Given the above discussed, the aqueous extract obtained at room temperature (extract **2**) was analyzed by UHPLC-MS in the negative mode and showed the presence of several phenolic compounds (Figure 2 and Table 3).

**Figure 2.** UHPLC chromatogram of *A. armata* aqueous extract, recorded at 280 nm.

The most noticeable aspect of the UHPLC-MS profile is the richness in compounds having bromine atoms (Table 3 and Figure 3), aspect that it is not completely new in view of the publications concerning *A. armata* [11,27] and other red algae [28–30].

Tentativelyidentifiedcompounds*A.*by **Figure 3.** (**a**) Proposed structures for monobromide derivatives; (**b**) proposed structures for dibromide derivatives; (**c**) proposed structure for tetrabromide derivative.

(**a**) (**b**) (**c**)

Among the identified compounds are four cinnamic acid derivatives, specifically from caffeic and *p*-coumaric acids (Table 3), prevalent compounds produced by natural resources, including seaweeds and other marine organisms [31,32]. The fragments observed in MS2, *m/z* 179, and *m/z* 163 are an essential clue to establish that the compounds are respectively caffeic and *p*-coumaric acid derivatives (Table 3).

The other metabolites present in the extract are brominated derivatives, and the compound with a retention time of 12.34 min could not be identified, but the isotopic pattern suggests that it has four bromine atoms (Table 3). A structure was proposed for the remaining five metabolites (Figure 3) based on the pseudomolecular ion, isotopic pattern, and fragment ions observed. However, we should highlight that we did not establish the aromatic rings substitution pattern, and in the proposed structures, the substituents order was made considering the most common ones described in the literature [28,30].

Compounds eluted at 1.43 min and 13.10 min were identified as cinnamic acid derivatives bearing one bromine atom (Figure 3a) due to their pseudomolecular ion isotopic pattern (Table 3). Moreover, brominated polyphenols and polymethoxybenzenes are commonly found in seaweeds [30]. The main fragments ions suggest that the compound eluted at 1.43 min is the methyl 3-(2-bromo-3,4,5-trihydroxyphenyl)acrylate or one isomer, being the fragment ion *m/z* 254 obtained through the loss of the bromine atom and the fragment ion *m/z* 195 obtained through an extra loss of CO2CH3. In the case of compound eluted at 13.10 min 3-(3-bromo-4,5-dimethoxyphenyl)acrylic acid, it can be observed the typical loss of CO2 and also the loss of the bromine atom to give the fragment ion *m/z* 241 (Figure 3a).

Next, we identified two compounds, methyl 3-(5-bromo-3-(2-bromo-3,4,5- trihydroxyphenoxy) -2,4-dihydroxyphenyl)acrylate eluted at 3.69 min and 2-(2-bromo-3,4-dimethoxy-5 methylphenyl)-3-(3-bromo-4,5-dimethoxyphenyl)propanoic acid eluted at 14.51 min, both having an ion cluster for two bromine atoms. The main fragment ions confirm the proposed structures (Figure 3b); however, we should again stress that the bromine atoms' position in the aromatic rings was not demonstrated. The fragments just suggest that they are one in each ring.

Finally, we have methyl 2,3-dibromo-5-(2,5-dibromo-3,4-dihydroxyphenoxy)-4 hydroxybenzoate (Figure 3c), eluted at 5.08 min, showing an ion cluster for four bromine atoms (Table 3) and presenting fragment ions consistent with the proposed structure. Once more, it is important to highlight that some fragment ions establish that the bromine atoms are equally distributed in the aromatic rings, but their position can be interchanged.

#### *3.4. GC-MS of Lipophilic Extract*

To prepare the lipophilic extract, in a preliminary study, hexane and dichloromethane solvents were used, and the results (unpublished results) showed that dichloromethane extraction yield was superior to that obtained with hexane under the same experimental conditions. Furthermore, dichloromethane is a widely used non-polar and unspecific solvent capable of dissolving a wide variety of less polar metabolites. Thus, to obtain broader knowledge, the dichloromethane extract (extract A) was selected for analysis by GC-MS to determine the chemical composition of the less polar fraction of *A. armata*. The results of qualitative and quantitative analysis are summarized in Table 4.

**Table 4.** Tentative identified compounds on dichloromethane extract (extract A) from dried *A. armata* by GC-MS.

Rt—Retention time. SD—Standard deviation. \* All the compounds possessing hydroxyl groups are identified as the corresponding TMS derivatives. Compounds were identified by: a comparison with pure standards; b comparison with the GC-MS spectral libraries with similarity index higher than 94% and confirmation of the peaks at *m/z* values corresponding to the characteristic fragmentation pattern of the family in question; c interpretation of pattern fragmentation of MS spectrum according to published literature.

The results in Table 4, graphically summarized in Figure 4, clearly show the predominance, in this lipophilic extract, of halogenated compounds (54.8%), being 40.6% dibromine mono-unsaturated alcohols and 14.2% mono-bromine and mono-chloride compounds.

**Figure 4.** Graphical presentation of the percentage of each class of compounds dichloromethane extract of *A. armata*.

The most abundant compound in the extract (1,4-dibromobutenol, Rt 15.5 min) belongs to this class (68.8% of the total halogenated compounds and 37.8% of the total identified compounds). The presence of one or more halogen atoms, chlorine, or bromine, in the chemical structure of several of the identified compounds is assumed, with a high degree of certainty, from the characteristic isotopic pattern of each chemical element. The bromine exists as two isotopes in approximately equal amounts of atomic mass 79 and 81, while chlorine has 2 isotopes of atomic mass 35 and 37 in a ratio of approximately 3:1.

In the case of the most abundant compound, at Rt 15.5 min, the presence of 2 bromine atoms is deduced from the peak corresponding to molecular ion [M]+• at *m/z* 300/302/304 (a very weak signal) in the ratio of 1:2:1. The mass spectrum of this compound also shows the base peak at *m/z* 75, corresponding to [HOSi(CH3)2] +, and the second most intense peak at *m/z* 285/287/289 (also in a 1:2:1 ratio), corresponding to [M-15]+ (loss of a methyl group from the TMS ether moiety). These signals support the hypothesis that the compound at Rt 15.5 min is a silylated primary alcohol [33], with two bromine atoms as substituents. Thus, it can be deduced that the compound in the extract will have a molecular mass of 230 corresponding to the chemical formula C4H6OBr2, suggesting the presence of a double bond in the basic structure of dibromo butanol. The greatest diagnostic value is the fragments with *m/z* at 131/133 and 137/139. The presence of these peaks, with identical intensity in each pair, shows that the fragmentation of the molecule gives rise to fragments containing one bromine atom each, being a strong indication of bromine atoms on different carbons. The peak at *m/z* 131/133 will correspond to the fragment [C4H4Br]+, resulting from loss of HBr by the molecular ion, following a δ-chain loss, indicating that the bromine atoms are found in the carbons 1 and 4. The peak at *m/z* 137/139 could result from loss of a methyl from the silyloxyl group, followed by bonding of the resulting siliconium ion to the terminal bromine atom, and a final rearrangement [33] to originate the fragment [BrSi(CH3)2] +.

Comparing the mass spectrum of 1,4-dibromobuten-1-ol (15.5 min) with that exhibited by the compound at 23.7 min, it is concluded that both are dibrominated mono-unsaturated alcohols with molecular formula C4H6OBr2, which mean they are isomers. They have a very similar fragmentation pattern [M-CH3] + at *m/z* 285/287/289, [BrSi(CH3)2] + at *m/z* 137/139, and the base peak at *m/z* 73 ([Si(CH3)3] +, being the last one ubiquitous to the spectra of TMS derivatives. The most significant differences are the absence of signals at *m/z* 131/133 and

the presence of the peak at *m/z* 197/199/201 corresponding to the fragment [C3H3Br2] +, resulting from an α-cleavage with retention of the 2 bromine atoms and the double bond on the cation. These fragments suggest that the difference between the compounds at 15.5 and 23.7 min is the position of the bromo substituents. These two compounds are the only di-halogenated compounds identified in the extract and correspond to 166.2 mg/100 g of dry algae (40.6% of identified compounds).

The peaks at 19.1 and 19.3 min also correspond to two unsaturated alcohols silylated, but in this case mono-brominated, as deduced from the isotopic pattern analysis of fragments [M-15]+ at *m/z* 333/335 (1:1); [C2H6SiBr]+ at *m/z* 137/139 (1:1). The peak at *m/z* 179 on the mass spectra of both isomers, corresponding to the non-brominated fragment [C13H23] + ([M-90-Br]+), suggests the same bromine substitution pattern on both isomers. The molecular formula of the two compounds on the extract is C13H25BrO, named bromotridecenol. The difference between them should be the position of the double bond, difficult to determine by MS.

The second most abundant halogenated compound (7.8%) corresponds to the peak at 8.5 min, identified as 3-bromoprop-2-enoic acid TMS derivative. The mass spectrum shows the signals at *m/z* 207/209 (~1:1), corresponding to the fragment C5H8BrO2Si+ ([M-15]+) and 137/139 (~1:1) corresponding to [(CH3)2SiBr]+, both mono-bromine silylated fragments typical from halocarboxylic acids pattern fragmentation [33]. The *m/z* value of these two fragments differs by 2 units from those observed for 3-bromopropanoic acid, evidencing the existence of a double bond at C2-C3. Moreover, the three signals observed in the spectrum of 3-bromoprop-2-enoic acid TMS derivative at *m/z* 143 [M-Br]+, 75 (base peak, [HOSi(CH3)2] +) and 53 (loss of trimethylsilanol and HBr resulting the fragments [C3H3O]+), all corresponding to non-brominate fragments, confirm the proposed identification of compound at 8.5 min. This compound was previously identified on the *A. armata* as ethyl ester [14].

The presence of halogenated alcohols and carboxylic acids in *Asparagopsis* species was already reported in the literature [11,14,34]. The results of several studies show that these are toxic metabolites that, in addition to exhibiting antimicrobial activity, confer a competitive advantage over predators and other species [35,36]. Moreover, to our best knowledge, this is the first time that 1,4-dibromobuten-1-ol and its isomer have been reported and quantified in the *Asparagopsis* genus.

The second most abundant family of compounds was the fatty acids (35.8%), being the content of saturated acids 5.1 times higher than the content of unsaturated acids (Figure 4). This can be an asset since saturated fatty acids seem to have a beneficial effect on the modulation of the immune system on food-borne bacterial infection [37].

The results of Table 4 show that the second most abundant compound is palmitic acid (20.3%), being the most plentiful fatty acid (57.0%). In comparison, the second most abundant fatty acid is myristic acid (16.1%). These results are in line with those published by Pereira et al. [38] for the same species, where palmitic and myristic acids are, by far, the most abundant fatty acids. Interestingly, in line with this result, the monoacylglycerols of these two acids, monomyristin and monopalmitin, were identified and quantified in the extract (Table 4; Rt = 41.5 and Rt = 43.3 min, respectively), representing about 2.1% of the compounds detected.

As far as unsaturated fatty acids are concerned (Figure 4), the most abundant is oleic acid ((9*Z*) 9-octadecenoic acid). It is the third most abundant fatty acid (9.83%), representing only 3.50% of the identified compounds.

From the point of view of beneficial effects on health, the presence of oleic acid can be considered an asset, while the high content of palmitic acid can be seen as an obstacle to seaweed consumption. However, we draw readers' attention to the fact that data in the literature suggest that the potential adverse effect of saturated acids is controversial and should be reviewed [39,40]. Moreover, the results show that *A. armata* can be valued as a source of palmitic acid. In fact, this compound is described as modulating the immune response [37] and it is an interesting antifouling agent due to its action against the first bacterial colonies on submerged surfaces [41].

Neither polyunsaturated acid nor unsaturated acids with *trans* configuration were detected, contrary to what is reported by Pereira et al. [38], where *trans*-oleic acid is detected in greater amounts than *cis*-oleic acid, and small amounts of arachidonic and eicosapentaenoic (EPA) acids are also noticed. These discrepancies may be related to geographical, seasonal, and/or environmental factors, but above all, due to the significant differences between sample preparation procedures before GC-MS analysis.

Only one compound from the hydrocarbon family has been identified, the heptadecane (Table 4, Rt = 25.4 min, [M]+• at *m/z* 240;). This alkane represents 2.7% of the total content of compounds, being the seventh most abundant. Although some hydrocarbons have been identified in the genus *Asparagopsis* [42], they have a chain of up to 5 carbons, while in this work, a long-chain alkane (17 carbons) is identified for the first time.

Cholesterol was the only sterol detected in the dichloromethane extract of *A. armata*, corresponding to 7.71 mg/100 g of dry seaweed (1.8% of identified compounds). This result aligns with the literature [12,13] that describes cholesterol as the most abundant sterol in this species. Lopes et al. [13] indicate a total cholesterol content of 28.92 mg/100 g of dry material, a value higher than the one reported here. This difference is justified because cholesterol exists mainly in non-free form as a constituent of the cell membrane, being released during the alkaline hydrolysis to which the sample was submitted in work by Lopes et al. [13]. It should be noted that the sterol content of *Asparagopsis* species is variable and depends on several factors (e.g., geographic, environmental, and seasonal) but mainly dependent on the life cycle phase [12]. Despite its "bad reputation" as a constituent of food products, cholesterol is a valuable natural product due to its chemical skeleton being easily transformable into new derivatives with diverse applications, such as drug delivery, production of liquid crystals and gelling agents, hormone precursor, and bioactive compounds [43].

#### **4. Conclusions**

The initial study on applying different methodologies to obtain polar extracts of *A. armata* showed that the choice of the extraction method would have to consider which compounds are intended to be extracted and the intended application for the obtained extract. The chemical profile of the water extract obtained by maceration (extract **2**) has greater chemical diversity and is economically cheaper.

Herein, for the first time, the aqueous and lipophilic *A. armata* extracts were tested as anti-aging agents with antioxidant and/or enzymatic inhibitors action. The polar extract was the most active, exhibiting the ability to inhibit 43.20 ± 2.58% of elastase activity at a 250 μg/mL concentration. On the other hand, although the lipophilic extract is a weak cholinesterase inhibitor, it is a dual inhibitor (it inhibits acetyl and butyryl-cholinesterase identically), which constitutes an advantage concerning its potential as an agent against Alzheimer's Disease.

Qualitative analysis by UHPLC-MS demonstrates that *A. armata* water extract is rich in phenolic compounds bearing bromine atoms; furthermore, the cinnamic acid scaffold is a prominent nucleus. The secondary metabolites herein described may explain the seaweed persistence and absence of predators. Furthermore, the anti- elastase activity mentioned above may be related to these phenolic compounds present in the water extract; nevertheless, more studies are essential.

The analysis of the most lipophilic fraction of *A. armata* (dichloromethane extract) by GC-MS allowed to identify and quantify 25 compounds, seven of them for the 1st time in this species, such as two dibrominated unsaturated alcohols, the 1,4-dibromobuten-1-ol and its isomer with the bromine atoms in different positions (a total of 166.2 mg/100 g of dry algae, 40.6% of identified compounds); two monobromine unsaturated alcohols, the bromotridecenol and its isomer with double bond on different position (a total of 20.6 mg/100 g of dry algae, 5.0% of identified compounds); one alkane, the heptadecane (a

total of 11.2 mg/100 g of dry algae, 2.7% of identified compounds); and two monoacylglycerols, the monopalmitin and the monomyristin (a total of 8.85 mg/100 g of dry algae, 2.1% of identified compounds).

The halogenated compounds and fatty acids are the two major compound families in the extract, being the 1,4-dibromobuten-1-ol and the palmitic acid the two most abundant identified compounds (155 and 83.4 mg/100 g of dry algae, respectively) and the myristic acid the third most abundant compound (23.5 mg/100 g of dry algae). The unsaturated fatty acids family, mainly 9*Z*-octadecenoic acid (14.4 mg/100 g of dry algae), corresponds to nearly 6% of the identified compounds.

As it is already known that no completely satisfactory method allows the analysis of the entire range of halogenated compounds of the *Asparagopsis* species, as they have a wide range of solubilities and boiling points [44], the work presented here is innovative as it allows the identification of a variety of lipophilic and halogenated aromatic compounds structurally distinct from those identified so far in *A. armata*.

Considering what is already known about the ecotoxicological effect of several halogenated compounds identified in *Asparagopsis* species [11,45], which give them a competitive advantage, and the commercial value of several scaffold phytochemicals identified in this species, the results obtained herein open new perspectives for valuing the *A. armata* as a source of halogenated compounds and fatty acids with high biotechnological and economic potential.

Valuing invasive species as a source of value-added natural products is an approach that promotes biodiversity conservation and ecosystem sustainability. It increases the algae harvest, allowing the assisted recolonization of free space by native species, because it increases the demand for existing and available biomass.

**Author Contributions:** Conceptualization, D.C.G.A.P., M.C.B. and A.M.L.S.; formal analysis and investigation, D.C.G.A.P., M.L.L., G.P.R., M.C.B. and A.M.L.S.; writing—original draft preparation, D.C.G.A.P. and A.M.L.S.; writing—review and editing, D.C.G.A.P., G.P.R., M.C.B., A.M.S.S. and A.M.L.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was financed by ASPAZOR project (DRCT: ACORES-01-0145-FEDER-00060- AS-PAZOR); Portuguese National Funds, through FCT—Fundação para a Ciência e a Tecnologia, and as applicable co-financed by the FEDER within the PT2020 Partnership Agreement by funding the LAQV-REQUIMTE (UIDB/50006/2020) and the cE3c centre (FCT Unit funding (Ref. UID/BIA/00329/2013, 2015–2018) and UID/BIA/00329/2019).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** Thanks are also due to the University of Aveiro and the University of Azores, to Mónica S.G.A. Válega and her support in UHPLC-MS analysis, and to Carlota Ferro for her support in biological activities.

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

#### **References**

## *Article* **Extraction of Antioxidant Compounds and Pigments from Spirulina (***Arthrospira platensis***) Assisted by Pulsed Electric Fields and the Binary Mixture of Organic Solvents and Water**

**Francisco J. Martí-Quijal 1, Francesc Ramon-Mascarell 1, Noelia Pallarés 1, Emilia Ferrer 1, Houda Berrada 1, Yuthana Phimolsiripol 2,3 and Francisco J. Barba 1,4,\***

**Citation:** Martí-Quijal, F.J.; Ramon-Mascarell, F.; Pallarés, N.; Ferrer, E.; Berrada, H.; Phimolsiripol, Y.; Barba, F.J. Extraction of Antioxidant Compounds and Pigments from Spirulina (*Arthrospira platensis*) Assisted by Pulsed Electric Fields and the Binary Mixture of Organic Solvents and Water. *Appl. Sci.* **2021**, *11*, 7629. https://doi.org/ 10.3390/app11167629

Academic Editors: Claudio Medana, Ana M. L. Seca and Eugenia Gallardo

Received: 16 July 2021 Accepted: 17 August 2021 Published: 19 August 2021

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

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

**Abstract:** The application of pulsed electric fields (PEF) is an innovative extraction technology promoting cell membrane electroporation, thus allowing for an efficient recovery, from an energy point of view, of antioxidant compounds (chlorophylls, carotenoids, total phenolic compounds, etc.) from microalgae. Due to its selectivity and high extraction yield, the effects of PEF pre-treatment (3 kV/cm, 100 kJ/kg) combined with supplementary extraction at different times (5–180 min) and with different solvents (ethanol (EtOH)/H2O, 50:50, *v*/*v*; dimethyl sulfoxide (DMSO)/H2O, 50:50, *v*/*v*) were evaluated in order to obtain the optimal conditions for the extraction of different antioxidant compounds and pigments. In addition, the results obtained were compared with those of a conventional treatment (without PEF pre-treatment but with constant shaking). After carrying out the different experiments, the best extraction conditions to recover the different compounds were obtained after applying PEF pre-treatment combined with the binary mixture EtOH/H2O, 50:50, *v*/*v*, for 60–120 min. PEF extraction was more efficient throughout the study, especially at short extraction times (5–15 min). In this sense, recovery of 55–60%, 85–90%, and 60–70% was obtained for chlorophylls, carotenoids, and total phenolic compounds, respectively, compared to the maximum total extracted amount. These results show that PEF improves the extraction yield of antioxidant bioactive compounds from microalgae and is a promising technology due to its profitability and environmental sustainability.

**Keywords:** pulsed electric fields; green extraction; microalgae; antioxidants; pigments

#### **1. Introduction**

Over the last decade, several research studies have evaluated the use of microalgae as a source of nutrients and bioactive compounds. This is due to a growing interest in the development of new foods that provide health benefits and that meet basic energy and nutritional requirements [1,2]. It is preferred that functional foods have a natural origin, such as from plants, algae and/or microalgae. In this sense, proteins obtained from microalgae have been used to replace proteins of animal origin in meat-like preparations such as turkey burgers [3] or in the fortification of vegan foods such as kefir produced from soy and almond based beverages [4].

Microalgae are becoming increasingly relevant, especially for their composition, since they are a source of high-added-value compounds [5], such as carotenoids, chlorophylls, and other pigments (antioxidants) [6], and polyunsaturated fatty acids [7,8].

Conventional extraction of antioxidant bioactive compounds from microalgae is often carried out using solvents and using dry biomass [9]. However, this conventional extraction method is very slow, involves the extraction of unwanted compounds, and can promote the degradation of some thermolabile compounds. Therefore, there is a need for innovative approaches such as pulsed electric fields (PEF) that affect the quality of the extracted compounds to a lesser extent and can be applied in a continuous flow to obtain higher extraction efficiency rates, minimize the use of solvents, and thus be a more efficient and sustainable extraction alternative [10,11].

In recent years, several studies have applied PEF technology on microalgae for extraction purposes [12–15]. This non-thermal technique consists of applying high-voltage electrical pulses between two electrodes in the treatment chamber [16]. Short electric pulses (1–100 μs) at field intensities of 0.1–1 kV cm−1 are employed for reversible permeabilization in plant cells for stress induction, at 0.5–3 kV cm−1 for irreversible permeabilization of plant and animal tissues, and at 15–40 kV cm−1 for irreversible permeabilization of microbial cells.

The external electric field increases the transmembrane potential and promotes membrane pore formation of the biological cell. High-intensity electric pulses can be generated by the switched discharge of a suitable capacitor bank. The properties of the discharge circuit determine the shape of the time-dependent potential at the treatment chamber. Parallel plate electrodes or colinear type treatment chambers constitute the most commonly used circuits [17]. The PEF technique can be effectively applied in many food processing applications such as microorganism/enzyme inactivation, recovery of bioactive compounds, drying and freezing processes, and to promote the enhancement of some selected properties of food macromolecules and some chemical reactions [16]. It has several advantages compared to conventional techniques as it favours the extraction of bioactive compounds by generating reversible or irreversible micropores in the plasma membrane of the cells, promoting the migration of interesting compounds into the cytoplasm through the membrane with high selectivity/purity, no thermal effect, and short extraction times [18]. The efficiency of PEF to permeabilize cell membranes differs according to process parameters such as electric field strength, treatment time, specific energy, pulse shape, pulse width, frequency, temperature, the properties of the treated food sample such as its pH and conductivity, and the characteristics of the target cells [19].

An example of its usefulness is observed in the extraction of lipids from microalgae. In this regard, a first PEF-assisted extraction of water-soluble bioactive compounds that cross the membrane through the formed micropores can be performed and then a second extraction with ethanol (EtOH) can be applied to obtain lipids or lipid-soluble metabolites that have remained inside the cell, which provides advantages in subsequent purification processing [20]. The use of PEF is possible in aqueous solutions with a low dry matter content, facilitating the more energy-efficient isolation of high-added-value compounds extracted from microalgae directly from the culture without dehydration or drying [20,21].

Most of the studies evaluating the application of PEF focus on the use of aqueous suspensions of microalgae, which mainly allows for the extraction of water-soluble compounds [22,23], while the extraction of non-polar pigments (e.g., chlorophylls and carotenoids) is very low under these conditions due to their low solubility in water [24]. Some previous studies have evaluated the use of complementary extractions with EtOH (96%), obtaining higher yields in the extraction of pigments from microalgae pre-treated with PEF [25]. Studies combining the use of PEF in suspensions of microalgae *(Nannochloropsis)* in water and the subsequent addition of an organic solvent to improve pigment extraction efficiency have been also carried out [12,13].

PEF-assisted extraction yields of nutritionally valuable compounds (lipids, pigments, and proteins) can vary depending on the microalgae used [26]. Therefore, specific studies are needed to evaluate the extraction in different microalgae species at different times and with different solvents to obtain the necessary information and thus be able to scale

up the process to an industrial level in an efficient, sustainable way and with a high extraction yield.

Several studies are currently evaluating PEF-assisted extraction from microalgae biomass such as Chlorella and *Nannochloropsis* [12,22,25,27]. However, there is a lack of data on the impact of PEF on the extraction of high-added-value compounds from other microalgae species such as spirulina (*Arthrospira platensis*). Spirulina is an undifferentiated filamentous cyanobacterium [28,29] whose cells are 3–12 μm wide and can reach 16 μm [30]; it has been used as food for centuries by various cultures as it has a biochemical profile rich in bioactive molecules and there are studies that support its benefits for human health [31].

However, considering the gap that exists regarding different extraction levels of antioxidant compounds according to the different microalgae species, the main aim of this study was to evaluate how PEF pre-treatment combined with the binary mixtures of ethanol (EtOH)/H2O, 50:50, *v*/*v* and dimethyl sulfoxide (DMSO)/H2O, 50:50, *v*/*v* at different extraction times can affect to the recovery of antioxidant bioactive compounds from spirulina.

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

#### *2.1. Sample*

The spirulina, in noodle form, was produced by the company Ecospirulina (Serra, Comunitat Valenciana, Spain). The cultured biomass comes from the species *Artrospira platensis* (more recently *Limnospira platensis*), strain *paracas 15016*. The Paracas reference refers to the lake from which it originated, Lake Paracas, south of Lima, Peru.

In Ecospirulina, cultivation is carried out in ponds in a greenhouse under natural sunlight and without the use of artificial light. Shading is applied to partially cover the culture ponds, which allows pigment production to be controlled. During the production of the sample used in this trial, the average temperature during the day was 32 ◦C, while the average temperature during the night was 24 ◦C. The pH of the culture varied between 9.8 and 10.4, being regulated by the addition of CO2 at the time of each harvest.

The biomass was filtered through a drum filter with 30-micron mesh. The culture substrates were returned to the culture pond, while the biomass was vacuum pressed and then converted into noodle form. In this format, it was air-dried at low temperature (40 ± 2 ◦C) to reduce the degradation of poorly resistant bioactive compounds at higher temperatures.

#### *2.2. Extraction Procedure*

Four samples of 2% (*w*/*v*) aqueous suspensions were made from the spirulina dry matter (DM). For this, 198 mL of deionised water were added to 4 g of dry biomass. In two of four samples, the same volume (198 mL) of EtOH or DMSO was subsequently added to finish with a 1% suspension (Figure 1). These two mixtures only received a conventional shaking treatment under stirring. The other 2% suspensions in water were treated with PEF before mixing with the organic solvents. As can be observed in Figure 1, the extracts were obtained from the 1% suspensions at different times and centrifuged to obtain the supernatant to be analysed. A total of 24 extracts were obtained, 12 of which were obtained by conventional extraction and the other 12 by PEF-assisted extraction.

#### 2.2.1. Pulsed Electric Fields (PEF) Extraction

For the PEF pre-treatment of the spirulina 2% (*w*/*v*) solution was used with the PEF-Cellcrack III equipment (German Institute for Food Technology (DIL)) (ELEA, Germany). A treatment chamber of 900 mL capacity was used, the distance between the electrodes was fixed at 10 cm, and the total mass added to the treatment chamber was 202 g (198 g of water + 4 g of microalgae). A 100 kJ/kg treatment was applied with an electric field of 3 kV/cm according to a previous work [24]. Before and after the treatment, the temperature and conductivity of each sample was measured with a portable conductivity meter, ProfiLine Cond 3310 (WTW, Xylem Analytics, Weilheim in Oberbayern, Germany). The minimum electric field strength needed to produce changes in the cells is 1 kV/cm, and it

has been shown that with a pulse duration of milliseconds, an electric field of 3–4 kV/cm is sufficient to create electroporation [14,32].

**Figure 1.** A schematic representation of the extraction process.

#### 2.2.2. Solvent Extractions

First, conventional extraction (Control) was performed on one set of samples. The solvents dimethyl sulfoxide (DMSO) or ethanol (EtOH) (1:1, *v*/*v*) in deionised water were added up to a final volume of 400 mL to the dry spirulina samples for nutrient extraction. Once the solvents were added, the samples were shaken at 400 rpm for 5, 15, 30, 60, 60, 120, or 180 min at room temperature to test the effect of shaking time on the extraction of compounds from the processed biomasses. Subsequently, the samples were centrifuged for 10 min at 4000 rpm using a 5810R centrifuge (Eppendorf Ibérica, Madrid, Spain). The extract obtained was kept at −20 ◦C for further analysis.

Next, the remaining samples were pre-treated with PEF under the conditions described above, and then an extraction process was carried out following the same methodology used for the conventional method.

Figure 2 shows the extracts obtained after an extraction using (a) DMSO/H2O or (b) EtOH/H2O at different extraction times (5–180 min) and the extracts obtained after pre-treatment with PEF using (c) DMSO/H2O and (d) EtOH/H2O.

#### *2.3. Chemical Analysis*

2.3.1. Total Phenolic Compounds (TPC)

For the determination of total phenolic compounds (TPC) (mg gallic acid equivalents (GAE)/g DM), the Folin–Ciocalteu method was used, using the procedure described by Parniakov et al. [12]. This technique is based on the property of phenols to react against oxidising agents. The Folin–Ciocalteu reagent contains molybdate and sodium tungstate, which react with the phenolic compounds present in the medium to form phosphomolybdic and phosphotungstic complexes. In a basic medium, electron transfer reduces these complexes to tungsten oxide (W8O23) and molybdenum oxide (Mo8O23), which are chromogens with an intense blue colour, proportional to the concentration of phenolic groups present in the sample. Gallic acid (Sigma-Aldrich, Steinheim, Germany) was used as a standard. First, Folin–Ciocalteu reagent at 50% *v*/*v*, 2% Na2CO3, and diluted

gallic acid standards were prepared. To carry out the analysis, 3 mL of Na2CO3 was added to a test tube, then 100 μL of standard or sample extract was added, and finally 100 μL of Folin–Ciocalteu reagent was added to this mixture. The samples were incubated for 60 min at room temperature under darkness. Finally, the samples were measured at 750 nm wavelength using a Perkin-Elmer UV/Vis Lambda 2 spectrophotometer (Perkin-Elmer, Jügesheim, Germany). All analyses were performed in triplicate.

**Figure 2.** Conventional extraction with (**a**) DMSO/H2O or (**b**) EtOH/H2O and their respective extracts using PEF pre-treatment with (**c**) DMSO/H2O and (**d**) EtOH/H2O.

#### 2.3.2. Trolox Equivalent Antioxidant Capacity (TEAC)

To determine the total antioxidant capacity (TAC), the Trolox equivalent antioxidant capacity (TEAC) assay was used. The TEAC value (micromolar Trolox equivalents, μM TE) measures the antioxidant capacity of a substance, compared to the standard, Trolox (Sigma-Aldrich, Steinheim, Germany). The TEAC value was measured using the method described by Safafar et al. [33] based on the discolouration of the ABTS radical.

The radical cation ABTS•+ (chromophore) was produced by reacting a 7 mM ABTS stock solution with 440 μL of an oxidant such as 140 mM potassium persulphate (K2S2O8). The mixture was kept in the dark at room temperature for 16 h before use. The solution was then diluted with 96% EtOH until its absorbance at 734 nm was 0.70 ± 0.02 at 30 ◦C.

Once the initial absorbance was reached, 2 mL of ABTS•+ was mixed with 100 μL of extract and the sample was measured after 3 min. The reaction produced a discolouration due to neutralisation of the radical cation of ABTS•+, which depended on the concentration of antioxidants in the sample. The absorbance was measured at a wavelength 734 nm in a Perkin-Elmer UV/Vis Lambda 2 spectrophotometer (Perkin-Elmer, Jügesheim, Germany). All analyses were performed in triplicate.

#### 2.3.3. Oxygen Radical Absorbance Capacity (ORAC)

The method used was previously described by Khawli et al. [34]. This assay measures the oxidative degradation of a fluorescent molecule such as fluorescein (Sigma-Aldrich, St. Louis, MO, USA) after the addition of a free radical generator such as 2,2 -azobis(2aminodinopropane) dihydrochloride (AAPH). The determination measures the degree of antioxidant protection of the sample with respect to the Trolox standard and is expressed in micromolar Trolox equivalents (μM TE).

The automated ORAC assay was performed on a Wallac 1420 VICTOR 2 plate reader (Perkin-Elmer, Jügesheim, Germany). The measurements were carried out in 96-well plates in which only the inner 60 wells were used. For the determination, a phosphate buffer solution (7.5 mM and pH 7–7.4) was prepared. For this purpose, 22.72 g of Na2HPO4 and 22.16 g of KH2PO4 were weighed and then each dissolved in 200 mL of deionised water. A volume of 61.6 mL of the first solution and 38.9 mL of the second solution were mixed and made up to 1000 mL with deionised water.

The standard (Trolox 100 μM) was prepared each day by adding 12.5 mg of Trolox to 50 mL of the previously prepared phosphate buffer. From this solution, 1 mL was taken and made up to 10 mL with the phosphate buffer to obtain the desired concentration. For fluorescein, 44 mg were weighed and made up to 100 mL with phosphate buffer. The working solution of 78 nM fluorescein was prepared daily. For this, 0.167 mL of the first solution was taken and made up to 25 mL with phosphate buffer.

Finally, the working dilution of the 221 mM AAPH radical was prepared daily by taking 600 mg of AAPH and bringing it up to 10 mL with phosphate buffer. As the ORAC assay is extremely sensitive, the microalgae extracts were adequately diluted prior to analysis to avoid interferences. In this case, the microalgae samples were diluted between 1:100 and 1:200, *v*/*v*.

For plate preparation, 50 μL of fluorescein (78 nM) and 50 μL of sample, blank (phosphate buffer), or standard (Trolox, 100 μM) were placed in each well and finally 25 μL of AAPH (221 mM) was added. As measurement variations may occur from well to well due to the low conductivity of polypropylene plates, the plates were pre-warmed at 37 ◦C for 10 min after the addition of fluorescein and before the addition of AAPH to avoid this problem. The plates were analysed immediately after the addition of AAPH and measurements were taken every 5 min until the relative fluorescence intensity of the standard (Trolox) was less than 5% of the initial reading value. All analyses were performed in triplicate.

#### 2.3.4. Chlorophyll a, Chlorophyll b, and Carotenoids

Chlorophyll a, chlorophyll b, and carotenoids contents were estimated spectrophotometrically according to the study by Parniakov et al. [35]. They were calculated using the equations of Lichtenthaler and Wellburn [36] for EtOH and Wellburn [37] for DMSO. This method is based on the determination of carotenoid and chlorophyll content based on the maximum absorbances of chlorophyll a (Ca), chlorophyll b (Cb), and total carotenoids (Cx+c). Using EtOH as a solvent, the maximum absorbances were found at the wavelengths of 664 nm, 648 nm, and 470 nm for chlorophyll a (Ca), chlorophyll b (Cb) and total carotenoids (Cx+c), respectively. For DMSO, the wavelengths were 665 nm, 649 nm, and 480 nm, respectively. Aliquots of the extracts obtained were diluted and the absorbances (*A*) were measured at the wavelengths listed above according to the solvent used. All analyses were performed in triplicate.

EtOH equations:

$$\text{C}\_{\text{a}} \left( \mu \text{g/mL} \right) = 13.36 \, A\_{664} - 5.19 \, A\_{648} \tag{1}$$

$$\text{C}\_{\text{b}} \left( \mu \text{g/mL} \right) = 27.43 \ A\_{648} - 8.12 \ A\_{664} \tag{2}$$

$$\mathbf{C\_{x+c}} \text{ (\mu g/mL)} = (1000 \, A\_{470} - 2.13 \, \mathbf{C\_a} - 97.64 \, \mathbf{C\_b}) / 209 \tag{3}$$

DMSO equations:

$$\mathcal{C}\_{\text{a}}\left(\mu\text{g/mL}\right) = 12.47\ A\_{665} - 3.62\ A\_{649} \tag{4}$$

$$\text{C}\_{\text{b}} \left( \mu \text{g/mL} \right) = 25.06 \, A\_{649} - 6.5 \, A\_{665} \tag{5}$$

$$\mathbf{C\_{x+c}}\ (\mu\mathbf{g}/m\mathbf{L}) = (1000\ \mathbf{A\_{480}} - 1.29\ \mathbf{C\_a} - 53.78\ \mathbf{C\_b})/220\tag{6}$$

#### *2.4. Statistical Analysis*

Data were analysed using analysis of variance (ANOVA), where PEF pre-treatment, solvents, and extraction time were the factors and chlorophyll, carotenoids, TPC, TEAC, and ORAC concentrations were the variables. Data were expressed as mean ± standard deviation in all cases. A value of *p* < 0.05 was considered significant. In addition, the LSD (Least Significant Differences) test was performed to determine the differences between the means of the values obtained. All analyses were performed with STATGRAPHICS Centurion XVI 16.1.03 (Statgraphics Technologies Inc., Princeton, NJ, USA).

#### **3. Results and Discussion**

In the present study, the effect of extraction time, the use of polar solvents such as DMSO and EtOH (aprotic and protic, respectively) combined with H2O (50:50, *v*/*v*), and the application of a PEF pre-treatment to improve the extraction yield and efficiency of pigments and antioxidant compounds were evaluated.

#### *3.1. Conventional Extraction*

#### 3.1.1. Chlorophyll a, Chlorophyll b, and Carotenoids

Figure 3 shows the chlorophyll a, chlorophyll b, and carotenoid content of the extracts obtained after conventional extraction with respect to the extraction time and solvent used. After performing a 2-factor analysis of variance (ANOVA) (time and solvent), it was observed that both extraction time and solvent had a significant effect (*p* < 0.05) on the extraction of chlorophyll a, chlorophyll b, and carotenoids, observing a higher extraction of all these compounds with the longer extraction times, and in general, observing higher values of chlorophyll a, chlorophyll b, and carotenoids when an EtOH/H2O mixture was used compared to that seen with the DMSO/H2O mixture. Thus, at 180 min, the highest values of chlorophyll a (0.57 ± 0.01 mg/g DM), chlorophyll b (0.55 ± 0.01 mg/g DM), and carotenoids (0.50 ± 0.01 mg/g DM) were obtained after using the EtOH/H2O mixture, representing an increase of 34%, 54%, and 84.2%, respectively, compared to those obtained with the DMSO/H2O solvent at equivalent extraction times.

**Figure 3.** Chlorophyll a, chlorophyll b, and total carotenoids. Conventional extraction (5–180 min) using two binary mixtures (EtOH or DMSO in 50% deionised water). Different lower-case letters in the same parameter and solvent indicate statistical differences as a function of extraction time. Different capital letters in the same parameter and time indicate statistical differences as a function of solvent.

However, it should be noted that this was not verified for all the compounds evaluated. For example, when using the solvent EtOH/H2O, no significant differences were found in the chlorophyll a content after 5 and 15 min of extraction. Moreover, no statistically significant differences in chlorophyll b content were observed after 15 and 30 min of extraction with the solvent DMSO/H2O. When analysing the statistical results between solvents, no differences were found in chlorophyll a extraction after 5 and 15 min of extraction.

These results are in agreement with those obtained by other authors, who observed a higher extraction of chlorophyll a, chlorophyll b, and carotenoids when the EtOH/H2O mixture was used [13]. They attributed this effect to a change in the polarity of the medium, which made it easier for the compounds to cross the lipid membrane of the cell, favouring the extraction of the compounds studied.

#### 3.1.2. Total Phenolic Compounds (TPC) and Total Antioxidant Capacity (TAC)

Figure 4 shows the results obtained for total phenolic compounds (TPC) and total antioxidant capacity (TAC) determined by TEAC and ORAC. After performing a 2-factor analysis of variance (ANOVA) (time and solvent), it was observed that both time and solvent had a significant effect (*p* < 0.05) on the extraction of phenolic compounds.

**Figure 4.** Total phenolic compounds, TEAC and ORAC. Conventional extraction (5–180 min) using two binary mixtures (EtOH or DMSO in 50% deionised water). Different lower-case letters in the same parameter and solvent indicate statistical differences as a function of extraction time. Different capital letters in the same parameter and time indicate statistical differences as a function of solvent.

ORAC values ranged from 295.87 ± 19.01 to 393.24 ± 15.28 μmol TE/g DM after using EtOH/H2O and from 72.45 ± 5.95 to 155.95 ± 10.78 μmol TE/g DM when the mixture DMSO/H2O was used. In addition, a greater effect on the extraction of antioxidant compounds was observed at 30 and 180 min, respectively. On the other hand, the TEAC values ranged from 4.52 ± 0.02 to 19.05 ± 1.25 μmol TE/g DM after using the EtOH/H2O mixture and from 9.19 ± 0.62 to 16.19 ± 1.37 μmol TE/g DM with the DMSO/H2O mixture. The maximum value for the TEAC assay (19.05 μmol TE/g DW) was obtained after 180 min extraction and using EtOH 50% as the solvent. The highest ORAC value was also obtained after using EtOH, but after 30 min of extraction (393.24 μmol TE/g DW). Regarding DMSO, the best values for TEAC and ORAC were obtained at 60 min (16.19 μmol TE/g DW) and 15 min (155.95 μmol TE/g DW), respectively. The higher ORAC values are due to other

antioxidant compounds not measured in this study, especially fat-soluble compounds such as vitamin E that may have an impact on the antioxidant capacity [38–40]. Moreover, ORAC sensitivity for other antioxidant compounds should also be taken into account [34].

In the determination of total phenolic compounds (TPC), concentrations between 3.64 ± 0.07 and 6.04 ± 0.28 mg GAE/g DM were obtained for the EtOH/H2O extraction and between 1.91 ± 0.07 and 5.921 ± 0.175 mg GAE/g DM for the DMSO/H2O extraction and 5.921 ± 0.175 mg GAE/g DM in the extraction with DMSO/H2O: For both solvents, the highest values were obtained at 120 min, the phenolic content at that time was similar to that obtained by Shanti et al. [41] for spirulina.

Regarding the mean values of TAC (TEAC + ORAC), it is worth noting that the values obtained with DMSO/H2O are relatively lower compared to those of the EtOH/H2O mixture, independent of the extraction time (Figure 5). This may be due to a lower extraction of chlorophylls, carotenoids, and phenolic compounds, as seen above, suggesting a clear contribution of these compounds to TAC. This same correlation has been previously observed by other authors after evaluating the use of these solvents for the extraction of these compounds from the microalgae *Nannochloropsis* [13].

**Figure 5.** Total antioxidant capacity (TAC) values obtained by adding the mean values of TEAC and ORAC for each time and solvent.

To evaluate the possible correlations between the different antioxidant compounds (chlorophyll a, chlorophyll b, carotenoids, and total phenolic compounds) and the determination methods used for total antioxidant capacity (TEAC and ORAC), a Pearson's test was performed for 36 samples (Table 1). The main correlations were observed between TEAC and carotenoids (R = 0.9094, *p* < 0.05), chlorophyll a (R = 0.7986, *p* < 0.05), chlorophyll b (R = 0.8419, *p* < 0.05), and total phenolic compounds (R = 0.7114, *p* < 0.05), obtaining moderate to high positive correlation coefficients. These results are in agreement with those of other authors [42,43] who evaluated the existing correlations between the different antioxidant compounds and the methods used for the joint evaluation of the total antioxidant capacity.

**Table 1.** Correlation between antioxidant compounds and total antioxidant capacity (TEAC and ORAC).

TPC: Total phenolic compounds; TEAC: Trolox equivalent antioxidant capacity; ORAC: Oxygen radical antioxidant capacity.

It should be noted that there was no correlation between antioxidant compounds (chlorophyll a and chlorophyll b) and ORAC. This is because the ORAC method is a more sensitive technique and some authors consider it better than TEAC [44] when measuring total antioxidant compounds, as ORAC measures the antioxidant capacity of compounds other than phenolics. A significant correlation was observed, but with a low correlation coefficient between ORAC and carotenoids (R = 0.3586, *p* < 0.05).

#### *3.2. Pulsed Electric Fields (PEF)-Assisted Extraction*

#### 3.2.1. Ethanol/Water

Figure 6 shows the results obtained after applying both treatments (conventional and PEF) using the mixture EtOH/H2O. After performing a two-way ANOVA analysis (time and treatment), it was observed that both the extraction time and the use of the PEF pre-treatment had a significant effect (*p* < 0.05) on the extraction of chlorophylls, carotenoids, (Figure 6a), and total phenolic compounds (Figure 6b). This effect is due to the electroporation produced by the PEF, which facilitates the extraction of these compounds more efficiently and with less agitation time.

**Figure 6.** *Cont.*

**Figure 6.** Chlorophyll a, chlorophyll b, and carotenoid content (**a**); TPC, TEAC, and ORAC (**b**) after conventional extraction and PEF-assisted extraction with EtOH/H2O. Different lower-case letters in the same parameter indicate statistical differences depending on the extraction time or treatment used.