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Algal Research 76 (2023) 103293 Contents lists available at ScienceDirect algal Algal Research ELSEVIER journal homepage: www.elsevier.com/locate/algal Check for Pressurized liquid extraction of bioactive extracts with antioxidant and updates antibacterial activity from green, red and brown Algerian algae Badria Keramane Andrea del Pilar Sánchez-Camargo Lidia Montero Firdousse Laincer Fatiha Bedjou a Elena Ibañez f a Laboratoire de Biotechnologies Végétales et Ethnobotanique, Faculté des Sciences de la Nature et de la Vie, Université de Bejaia, 06000 Bejaia, Algeria b Grupo de Diseño de Productos y Procesos (GDPP), Departamento de Ingeniería Química y de Alimentos, Universidad de los Andes, Carrera 1 Este No. 18A-12, Edificio Mario Laserna, Piso 7, Bogotá 110111, Colombia Applied Analytical Chemistry, University of Duisburg-Essen, Universitaetsstrasse 5, Essen 45141, Germany d Teaching and Research Center for Separation, University of Duisburg-Essen, Universitaetsstrasse 5, Essen 45141, Germany e Laboratoire de Biochimie appliqué, Faculté des Sciences de la Nature et de la Vie, Université de Bejaia, 06000 Bejaia, Algeria f Laboratory of Foodomics, Institute of Food Science Research, CIAL, CSIC, Nicolás Cabrera 9, 28049 Madrid, Spain ARTICLEINFO ABSTRACT Keywords: In this work, the protein, sugar, phenolic and phlorotannin contents, as well as the antioxidant and antibacterial Pressurized liquid extraction activity of hydroethanolic extracts of six algae species from the west cost of Algeria (Padina pavonica, Halopteris Seaweeds scoparia, Cystoseira mediterranea, Ulva lactuca, Ulva intestinalis, and Ceramium virgatum), obtained by pressurized Phlorotannins liquid extraction (PLE) were investigated. Ceramium virgatum PLE extracts showed the highest content of protein Antioxidant activity Antibacterial activity (4.36 ± 0.13 %), while the total sugar content was maximum in U. lactuca extract (23.89 ± 0.21 %). Likewise, P. pavonica PLE extract presented a relevant total phenolic content (49.82 ± 1.40 mg GAE/g extract). On the other hand, phlorotannins were only found in brown algae in a range of 1.84-2.93 mg PGE/g. Furthermore, P. Pavonica extract presented considerably higher antioxidant capacity of inhibiting ABTS radical (1.16 ± 0.023 mmol TE/g) and 50 % of DPPH (1,1-diphenyl-2-picryl hydrazyl) (IC₅₀ = 57.03 ± 1.28 µg/mL) in comparison with the other studied algae. Regarding to the potential antimicrobial activity, the results showed that Cystoseira Mediterranea produced the lowest minimal inhibitory concentration on Salmonella typhi (0.83 ± 0.14 mg/mL), Escherichia coli (4.66 ± 0.57 mg/mL), Vibrio cholera (1.08 ± 0.14 mg/mL), and Candida albicans (2.16 ± 0.28 mg/mL). Besides, different carotenoids and phenolic compounds were tentatively identified by LC-DAD in the algae extracts which could be the responsible of the tested bioactivities. These findings show the potential of using PLE as green solvent extraction technique and the prolific bioactivities of Algerian seaweeds. 1. Introduction because of its possible genotoxic effect [3]. However, it was allowed in 2005 in the EU with some conditions of use, and since then its use has Nowadays, the habits of the population are being modified to been highly controversial in the scientific community [4,5]. Moreover, improve human health through natural medicines and functional foods the presence of synthetic additives in several food products leads to an [1]. In the food industry, synthetic preservatives such as antimicrobials accumulating effect that can exceed the authorized daily intake, which is (e.g. potassium sorbate, sodium nitrite, or sorbic acid) and antioxidants not taken into consideration by the corresponding authorities [2]. (e.g. butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), Recent efforts have been concentrated for replacing synthetic pre- and tertbutyl hydroquinone (TBHQ)) are widely used. Some reports servatives with natural ones. To find these alternative natural pre- have indicated that certain synthetic antioxidants such as BHA and BHT servatives, the marine environment has been recognized to have may possess weak carcinogenic effects in some animals at high levels limitless potential [1]. Since the last two decades, >10,000 compounds [2]. In addition, the use of TBHQ which is allowed in USA since 1972, have been isolated from several marine organisms such as seaweeds [6]. was restricted by the European Economic Union, Japan and Canada, In Algeria, algae are not appreciated at their fair value. Their * Corresponding author. E-mail address: ad.sanchez@uniandes.edu.co (A.P. Sánchez-Camargo). Received 30 August 2022; Received in revised form 13 July 2023; Accepted 11 October 2023 Available online 13 October 2023 2211-9264/© 2023 Elsevier B.V. All rights reserved.B. Keramane et al. Algal Research 76 (2023) 103293 exploitation is still marginal despite the potential of the 1200 km long Gallic acid, phloroglucinol dihydrate, 6-hydroxy-2,5,7,8-tetrame- Mediterranean coastline. The use of algae is not a part of Algerian tra- acid (Trolox), 2,2-diphenyl-1-picrylhydrazyl ditions that are nevertheless very oriented toward the traditional food (DPPH), acid) (ABTS), and therapies, and local plants remain in the Algerian mind as the only acetic acid,2,4-dime-thoxybenzaldehyde (DMBA), trichloroacetic acid source to exploit [7]. Some studies related to Algerian seaweeds activ- (TCA), bovine serum albumin (BSA) and glucose were purchased from ities are rising [8-10], but there is still a lack of documentation on the Sigma-Aldrich (Madrid, Spain). The Folin-Ciocalteu reagent and sodium subject. In the rest of the world, seaweeds have been recognized as an carbonate (Na₂CO₃) were provided by Merck (Darmstadt, Germany). under-exploited reservoir of functional food and became the focus for The solvents employed were HPLC-grade. Acetonitrile, was acquired extensive researches of new molecules with interesting biological ac- from VWR Chemicals (Barcelona, Spain) and ultrapure water was ob- tivities [11]. The biological activities associated to algae and/or their tained from a Millipore system (Billerica, MA, USA). extracts are various. For instance, Halopteris scoparia, Padina pavonica, Ulva intestinalis and Ulva lactuca, have been reported to display antiox- 2.1.1. Microbial strains idant, antibacterial, antitumoral, anti-inflammatory and antifouling Escherichia coli ATCC 25922, Salmonella typhi ATCC13311, Vibrio activities [12-19]. On the other hand, Ceramium virgatum, a red alga, is cholerae ATCC 14035, methicillin resistant Staphylococcus aureus more associated with anti-parasitic activities as anti-plasmodium and (MRSA) ATCC 43300 and Candida albicans ATCC 10231 were provided leishmaniosis [20,21], though some studies reported its antibacterial by the laboratory of applied microbiology of the University of Bejaia. and cytotoxic activities [20,22]. Taskin et al. [19] reported the antitu- moral activity and Salvador Soler et al. [23] the antibacterial activity of 2.2. Pressurized liquid extraction Cystoseira mediterranea, however, there is a lack of information available about this seaweed. Among the algae-related functional ingredients, Extractions were performed using an accelerated solvent extractor both, the primary and secondary metabolites have been described as (ASE 200, Dionex, Sunnyvale, CA, USA), equipped with a solvent responsible for the aforementioned activities. For the first type, poly- controller unit. PLE parameters were chosen according to earlier studies saccharides, mainly sulfated [24] and lipids like polyunsaturated fatty on algae [38], in which temperature, pressure, extraction time and acids (PUFAs) [25] have been related to potential bioactivities. For the composition of the solvent had been defined. The conditions selected for secondary metabolites, carotenoids such fucoxanthin, an oxygenated the extractions were 120 °C and a mixture of ethanol-water of 75:25 (% polyenic carotenoid, have lately raised a strong interest for its multi- v/v) was used as extraction solvent. A pressure of 1,500 psi was applied, functional properties, as well as phenolic compounds, especially phlor- during 20 min of extraction. The solvent mixture was degassed in an otannins, a group of compounds found only in brown algae [26]. In ultrasound bath to prevent oxidation during the extraction process order to obtain functional ingredients from natural sources, the use of before it was placed in the solvent controller. The extractions were done modern eco-friendly techniques stands self-evident. Pressurized liquid using 11 mL extraction cells, where 2 g of algae were mixed homoge- extraction (PLE) is one of these techniques, which presents important neously with 4 g of sea sand to prevent the clogging of the system. The advantages over traditional ones. This technique is based on the use of obtained extracts were protected from light and the solvent was solvents at high temperatures and pressures enough to maintain their removed by rotary evaporation followed by freeze-drying. All PLE ex- liquid state during the extraction process [27]. This method has already periments were done by triplicate with relative standard deviation proven its great extraction capacity for a variety of bioactive compounds (RSD) values for the extraction yields lower than from diverse natural samples [28-32]. Moreover, ethanol has a great advantage, compared to other organic solvent (as acetone for instance), 2.3. Determination of protein content in the PLE extracts since it has been reported to be the most efficient solvent for the extraction of phlorotannins' compounds [33-35]. Moreover, ethanol is a The precipitation and quantification of the protein fraction present in GRAS (Generally recognized as safe) solvent and therefore it can be used the PLE extracts was carried out following the procedure described by as a safe solvent for the food industry [36,37]. According to the above, [39] with minor modifications. Briefly, extract (10 mg/mL) were mixed the aim of this work was to obtain hydro-ethanolic extracts from six with a solution of trichloroacetic acid (TCA) 25 % (v/v) in a solvent-to- Algerian macroalgae species using PLE as green extraction method as extract solution ratio of 2.5:1.0 (v/v). After 30 min of precipitation in an well as to evaluate the potential of the PLE extracts as preservative in- ice-bath, the tubes were centrifuged during 20 min at 15,000 and gredients. The PLE obtained extracts were characterized in terms of the °C. Then, the pellet was washed with 150 µL of cold acetone, followed protein, sugar, phenolic and phlorotannins contents, together with the by a centrifugation during 10 min at 10,000 at room temperature. antioxidant and antimicrobial activities, as well as their chemical Acetone washes were repeated until a colorless supernatant was ob- characterization was carried out by liquid chromatography coupled to tained. Finally, the pellet was redissolved in 200 µL NaOH 0.1 M, in diode-array detector (LC-DAD). order to neutralize the pH of the proteins. After that, Bradford assay procedure was used to determine the precipitated protein content from 2. Material and methods the PLE extracts, as follow: a standard calibration curve using BSA (Bovine Serum Albumin) was prepared within the range 0.05-0.5 mg/ 2.1. Samples and reagents mL in 0.5 mL protein LoBind eppendorf tubes. Then, 10 µL of blank, calibration standard or extract were mixed with 200 µL diluted Coo- Six species of seaweeds were harvested on the west coast of the city massie blue dye reagent and after 5 min of reaction, the absorbance was of Bejaia (Algeria) in May 2013. Based on the microscopic analysis, determined at 595 nm in a microplate spectrophotometer reader (Syn- morphometric features and ecological distribution pattern, the collected ergy HT, BioTek Instruments, Winooski, VT, USA). Results were specimens were taxonomically identified by the botanic laboratory of expressed as mg equivalent BSA per gram of extract (mg/g EBSA the University of Bejaia. The identified species were classified in brown extract). The analyses were done in triplicate. algae Padina pavonica, Halopteris scoparia, Cystoseira mediterranea, green algae Ulva lactuca, Ulva intestinalis and red algae Ceramium virgatum. 2.4. Total sugar content: phenol-sulfuric acid method After harvesting, the samples were ventilated to remove moisture, and they were dried in ventilated place for 20-30 days protected from light. Phenol-sulfuric acid method was carried out according to [40]. Cryogenic grinding of the sample under liquid nitrogen was performed Briefly, the method consists on mixing 100 µL of the extract solution at to reduce the particle size of the samples under 5 and then they were 2.5 mg/mL with 300 µL of concentrated H₂SO₄ and 90 µL of 5% phenol; stored at 4 °C until their use. that mixture was heated at 95 °C for 5 min in a Thermomixer R 2B. Keramane et al. Algal Research 76 (2023) 103293 (Eppendorf AG, Hamburg, Germany). After cooling to room temperature was completed after 4 h at 25 °C in the dark and absorbance was for 5 min, 100 µL of the mixture were transferred into a microplate well measured at 516 nm after transferring 300 µL of each mixture to a 96- and the absorbance was measured at 490 nm. A comparison to a glucose well microplate. The blank absorbance of each extract was deduced calibration curve (0.25-2.0 mg/mL) was carried out following the same from absorbance obtained by the corresponding mixture. A calibration procedure. Results were expressed as g of glucose per 100 of extract (g/ curve that associates the different concentrations of DPPH to their 100 g). All analyses were done by triplicate. absorbance at 515 nm was done. The percentage of remaining DPPH against the extract concentration (expressed in µg/mL) was then plotted 2.5. Total phenols content (Folin-Ciocalteu method) to obtain the amount of antioxidant necessary to decrease the initial DPPH concentration by 50 % or EC₅₀. The lowest EC₅₀ corresponds to Total phenols content of the PLE extracts were determined using the the higher antioxidant power. All analyzes were done triplicate. procedure described by [41] with some adjustments. 10 µL of each extract (at concentrations of 10 mg/mL) and 600 µL water were mixed 2.8.2. Trolox equivalents antioxidant capacity assay (TEAC) with 50 µL of undiluted Folin-Ciocalteu reagent (2 N). After 1 min, 150 The antioxidant activity of the extracts was measured by the µL of 20 % (w/v) Na₂CO₃ were added, and the volume was made up to improved Trolox equivalent antioxidant capacity (TEAC) assay, per- 1.0 mL with water. The same procedure steps were repeated with gallic formed essentially as described by Re et al. [44]. Briefly, radical acid as a standard, performing a calibration curve within a range of was generated by reacting 7 mM ABTS and 2.45 mM potassium per- 0.03125 to 2 (0.03125, 0.0625, 0.125, 0.25, 0.5, 1, 2) mg/mL. The sulfate, after incubation at 25 °C for 16 h in the dark. The radical mixture was incubated at 25 °C in the darkness for 2 h, afterwards 300 solution was diluted with 5 mM phosphate buffer (pH = 7.4) to give an µL of each reaction mixture were transferred to microwell plate. The absorbance of 0.7(±0.02) at 734 nm. The reaction was initiated by the absorbance was measured at 760 nm in a microplate spectrophotometer addition of 10 µL of each seaweed extract at 5 different concentrations reader (Synergy HT, BioTek Instruments, Winooski, VT, USA). The re- (within a range of 0.25 to 2 mg/mL) to 1 mL of solution. 300 µL sults were expressed as mg of gallic acid (GAE) per g of extract. All of the mixture were transferred into a 96-well microplate. The absor- analyses were done by triplicate. bance was measured at 734 nm every 5 min during 45 min using a microplate spectrophotometer reader (Synergy HT, BioTek Instruments, 2.6. Total phlorotannin content: DMBA assay Winooski, VT, USA). Trolox was used as reference standard, and results were expressed as TEAC values (mmol Trolox equivalents per g extract). The quantification methodology used was carried out adapting the All analyses were done in triplicate. procedure described by [42]. Equal volumes of DMBA 2 % (w/v) and hydrochloric acid 6 % (v/v) prepared in glacial acetic acid were mixed. 2.9. Antimicrobial activity: determination of minimum inhibitory Then, 250 µL of this working reagent and 50 µL of each extract (5 mg/ concentrations (MIC) mL) were mixed in 96 wells microplate. The reaction was performed at room temperature, in the dark during 60 min. The absorbance was The minimal inhibitory concentration (MIC) was determined by the determined at 515 nm in a microplate spectrophotometer reader (Syn- broth microdilution method by using the method of Santoyo et al. [45]. ergy HT, BioTek Instruments, Winooski, VT, USA). Phloroglucinol was All tests were performed in Mueller-Hinton broth supplemented with used as reference standard and a calibration curve was performed using 0.5 % Tween 20. The bacterial strains inoculated were prepared from series of six phloroglucinol concentrations ranging between 0.98 and overnight Mueller-Hinton broth cultures at 37 °C. Test strains were 62.50 µg/mL. Results were expressed as mg equivalent phloroglucinol suspended in Mueller-Hinton broth to give a final density 10⁷ CFU/mL. per g of extract (mg EPG/g extract). All analyses were done in triplicate. Different concentrations of the algae extracts ranging from 0.5 mg/mL to 20 mg/mL were prepared. The 96-microwellplates were prepared by 2.7. HPLC-DAD analysis of the PLE extracts dispensing 165 µL of Muller Hinton broth, 5 µL of the inoculum and 30 µL of the different extract dilutions, into each well. After that, plates The chromatographic separation of the extracts was performed with were incubated at 37 °C for 18 h. Negative controls were prepared an Agilent 1100 series liquid chromatograph (Agilent Technologies, adding 30 µL of ethanol, the solvent used to dissolve the extracts. Santa Clara, CA), consisting of a binary pump and an autosampler ACE- Chloramphenicol and nystatin were used as positive references stan- 3C18 (4.6 X 150 mm, 5 µm particle diameter, Agilent Technologies, dards to determine the sensitivity of the bacterial and fungal species Santa Clara, CA) column was employed using (A) water (0.1 % acetic used, respectively. After incubation, the MIC of each extract was acid) and (B) acetonitrile (0.1 % acetic acid) as mobile phases. The determined by visual inspection of the well bottoms, since bacterial following elution gradient was used: 0 min, 5 % B; 5 min, 5 % B; 10 min, growth was indicated by the presence of a white "pellet" on the well 10 B; 30 min, 60 % B 35 min, 80 % B; 50 min, 95 % B; 55 min, 95 % B; bottom. The lowest concentration of the extract that inhibited growth of 56 min; 65 min, 5 % B. A flow rate of 0.7 mL and an injection the microorganism, was designated the minimum inhibitory concen- volume of 10 were employed. Detection was achieved by using a tration. All analyses were done in triplicate. diode array system (DAD). Wavelengths at 280 nm and 450 nm were selected for monitoring the separation, although the range 220-600 nm 2.10. Statistical analyses was recorded. All extracts were analyzed in triplicate. Measurement values are 2.8. Antioxidant activity presented in means (±) their standard deviation. Tukey test was carried out to test significant differences in antioxidant activity, TPC, phlor- 2.8.1. DPPH assay otannins, sugar and protein content between macroalgal extracts using The DPPH assay was determined using the procedure described by Excel Stat. Differences were considered as significantly different at a Brand-Williams et al. [43]. This method consists on the neutralization of value of pB. Keramane et al. Algal Research 76 (2023) 103293 3. Results and discussion fact depends in a greater extent of algal composition and it differs among the brown, red and green algae. Indeed, the presence of phlorotannins in As mentioned, marine sources, specially seaweeds have demon- brown algae, phycobiliproteins in red algae, and carotenoids which strated to be a potential source of bioactive compounds and, nowadays, prefers non polar solvents, influences the rate of extraction without most species do not possess the commercial value that they could have being strictly representative of the composition of the algae. [27]. This is the case of Algerian algae. Macro- and microalgae produce Regarding chemical composition (proteins and sugars) of the PLE some valuable metabolites such as proteins, polyphenols, pigments or extracts, results are also presented in Table 1. The highest concentration polysaccharides with relevant biological activities (antioxidant, anti- of carbohydrates was detected in U. lactuca (23.89 % ±0.21) and inflammatory, antiviral or antimicrobial activities) [46]. The applica- U. intestinalis (21.81 % ± 0.1). The red and brown algae showed quite tion of green extraction techniques such as PLE has brought a new lower quantities of carbohydrates ranging from 9.04 % ± 0.36 for alternative for obtaining those compounds in a sustainable way. Besides, Ceramium Virgatum to 5.34% ±0.32 for H. scoparia. Several studies have due to safety concerns regarding the use of some organic solvents, already reported that polysaccharides in algae are one of the main hydro-ethanolic solutions can be used for extraction purposes as food components [52-54]. Green, brown and red algae are rich in sulfated grade solvents. Among the most important PLE advantages over the polysaccharides such as ulvan, fucoidan and floridean, respectively conventional solid-liquid extraction, the extraction is completed in a [24,55,56]. According to Pérez et al. [57], those compounds can shorter period of time with less solvent and achieving higher yields [47]. contribute to the macroalgae bioactivities depending on their sulfate In the present work, extractions were performed using the mixture of content. Regarding protein content, algae extracts range from 1.15 ± ethanol and water (75:25 % v/v) as extracting solvent, at 120 °C during 0.07 to 4.36 ± 0.13 mg EBSA/100 g extract. Significant differences (p 0.05). EBSA: Equivalent Bovine Serum Albumin; GE: Values presented are mean ± SD (n = 3). Equal superscripts mean groups not glucose equivalent. statistically different (p > 0.05). 4B. Keramane et al. Algal Research 76 (2023) 103293 C. mediteranea and H. scoparia, therefore, a direct comparison with other Table 3 results cannot be done. In the study carried by Sánchez-Camargo et al. Characteristic parameters of the compounds detected in the U. lactuca and [34] the PLE extract of the brown macroalga Sargassum muticum U. intestinalis PLE extract analyzed by LC-DAD. exhibited a TPC about 47.55 mg GAE/g. This value is quite similar to the Ulva lactuca TPC of P. pavonica extract obtained in our study under the same con- Peak no. tR max ditions. Otero et al. [63] reported the TPCs in PLE extracts of the brown 450 nm alga L. ochroleuca at 80, 120, and 160 °C with four different polarity 1' 50.034 423, 446, 476 solvents. The highest content was obtained using ethanol at 160 °C with 173.65 mg GAE/g demonstrating the impact of solvent polarity and 280 nm temperature on the TPC content. Boisvert et al. [14] found that PLE 1 20.926 240, 260, 272, 336, 365 ethanol extracts of U. lactuca yield relatively low TPC content (6.9 mg 2 21.666 250, 290, 292 GAE/g) compared to the brown alga Ascophyllum nodosum (50.2 mg 4 23.205 287, 310, 320 GAE/g) obtained under the same conditions, which is in accordance 5 23.692 289, 318 with the results presented in the present work. It is known that brown 6 24.063 288, 330 7 33.956 260, 270, 280 seaweeds mostly exhibit higher TPC than green and red ones [57]. This is due to the presence of a large amount of phlorotannins in the brown algae [64]. Phlorotannins are a great class of marine secondary metab- Ulva intestinalis olites generally considered to act as chemical defense against herbi- vores. They also possess primary functions such as contributing to cell Wavelength Peak no. tR wall structure and reproduction [65]. They consist on polymers of 450 nm phloroglucinol (1,3,5-trihydroxybenzene) [66], which is used as stan- 1' 43.981 410,435, 465 2' dard compound to assess phlorotannins content (DMBA assay) in the 50.022 423, 446,476 obtained extracts. Phlorotannins possess high antioxidant power when they are in contact with oxidizing agents, generating phenoxyl radical 280 nm 1 22.237 species stabilized by conjugation by the relocation of the unpaired 280, 345 2 24.249 280, 330 electron, and by hydrogen bond with the adjacent hydroxyl group [55]. 3 25.491 240, 274, 323 The DMBA assay revealed that P. pavonica, which showed the highest 4 48.269 372, 396, 410 TPC value, also displays the highest amount of phlorotannins with 2.93 ± 0.08 mg phloroglucinol equivalent per g of extract (Table 2). This value was followed by Cystoseira mediterranea containing 2.36 ± 0.16 eluted at 48.9 min could be tentatively identified as astaxanthin thanks mg PGE/g and H. scoparia 1.90 ± 0.06 mg PGE/g. Interestingly, to the comparison of retention time and UV-Vis spectra with the stan- H. scoparia has a relevant TPC value but Cystoseira mediterranea showed dard (Fig. 4). Peak 2', eluted at 49.60 min has max at 423, 446, 473 nm, a higher amount of phlorotannins. A recent research work reported a could correspond to loraxanthin or antheraxanthin according to Maoka phlorotannins content of 0.056 mg PGE/g for a Mediterranean Padina et al. (2002). The peak 3' at tR 41.19 min with max at 416, 440, and pavonica when extracts were obtained using solid liquid extraction and 470 nm could be α-carotene according to Rodriguez-Amaya and Kimura ethanol water 50:50 (v/v) as solvent [12]. Those results support the [91]. potential of use pressurized liquid extraction and the selected parame- The HPLC-DAD spectrum recorded at 280 nm allowed the detection ters in extracting these compounds. of polyphenols including flavonoids [87,92] however, neither our standards nor the bibliography allowed us to identify them. Besides, HPLC-DAD chromatograms of the three brown algae PLE 3.3. HPLC-DAD analysis extracts, recorded at 450 nm for the detection of carotenoids, showed The study of the UV-visible spectra of each peak, recorded at 280 one common and intense peak with a retention time of 40.98 min for and 450 nm by HPLC-DAD, allowed a preliminary characterization of Halopteris scoparia Padina pavonica and Cystoseira mediterranea. The the compounds. The retention times and absorption maxima of each three maximum absorption spectra, characterized by absorption bands peak were recorded and compared with standards when available or between 400 and 470 nm with 3 shoulders at 420-448-469 nm overlap (Fig. 5). As can be observed in the chromatograms, this is the main with data reported in the literature. The results indicate the presence of different phenolic compounds and carotenoids in the extracts. compound present in the PLE extracts of the three algae. In particular, For Ulva lactuca, peaks 1-7 (Table 3, Fig. 1) showed UV absorption this compound was detected in a high intensity in the algae H. scoparia. The retention time and the UV-Vis spectra of this peak can be used to maxima at wavelengths between 285 and 290 nm with absorption bands tentatively identify this compound as fucoxanthin accordingly with at 300-340 nm which is consistent with spectra corresponding to fla- Rajauria et al. [93] Numerous other compounds were detected with vones structure [87]. Concerning U. intestinalis, (Table 3, Fig. 2) the spectrum recorded at UV-Vis spectra characteristic of carotenoids; however, we were not able 280 nm, highlighted polyphenols which were related to flavones (peak to identify them by our standards nor by the data present in the bibliography. #1 at 22.24 min) and to flavonol (peak 2 at tR 24.25 min) according to In comparison to the chemical characterization of carotenoids in red Wekre et al. [88]. Flavonoids such as flavonols contain a conjugated system of double bonds that modify their absorption to wavelengths and green algae, brown algae showed higher concentration of carot- enoid compounds. On the other hand, the spectra recorded at 280 nm, longer (200-400 nm) than those of simple flavonoids. However, different substitutions of the hydroxyl groups lead to alteration in showed the presence of polyphenols with max at 210-270 and 345-375 nm [89]. Some of the detected peaks have maximum absor- wavelength and relative intensities of these maxima [89]. The UV-vis absorption maxima, of the different peaks recorded from bance bands at wavelengths of 190-380 nm, which generally corre- sponds to phenolic acids as well as hydroxybenzoic acid derivatives, the PLE extract of Ceramium virgatum, as well as their respective reten- most of which show absorbance maxima at 255-280 nm [93]. tion times, are listed in Table 4. The chromatogram recorded at 450 nm showed its richness in carotenoids and in particular, the presence of astaxanthin (Fig. 3; Fig. 4). 3.4. Antioxidant activity of PLE extracts Peaks 1', 2', 3' and 4' recorded at 450 nm show UV-Vis spectra with three characteristic shoulders typical for carotenoids [90]. The first one PLE extracts of the selected macroalgae were analyzed by radical 5B. Keramane et al. Algal Research 76 (2023) 103293 280 nm mAU 6 10 3 4 2 1 5 0 16 18 20 22 24 26 28 30 min mAU 280 nm 40 20 7 0 1' 40 450 nm 20 0 10 20 30 40 50 60 min Fig. 1. HPLC-DAD chromatogram, at the wavelength of 280 and 450 nm, of U. lactuca PLE extracts. Peak data as in Table 4. scavenging activity (RSA) using the DPPH as radical. The lowest EC₅₀ DPPH radical. These results could be explained by its protein content value was found by the extract obtained from P. Pavonica with 57.03 ± (4.36 ± 0.13 mg g⁻¹, Table 1), because red algae are known to contains 1.28 µg/mL, which is translated in the highest antioxidant activity high concentrations of phycoerythrin. This type of phycobilin com- (Table 2). Significant disparities are seen between algae groups as the pounds forms a covalent bond with a water-soluble protein [70]. In brown algae showed the highest activity. We noticed that the red alga addition, phycoerythrin has been reported to possess antioxidant ac- Ceramium virgatum needs an extract concentration of 123.52 ± 5.91 µg/ tivity [71], and a proton donation or metal ion chelation has been mL to scavenge of DPPH radical, and there is not significant dif- suggested as reaction mechanism and in consequence, can contribute to ference with the EC₅₀ value obtained by the brown alga H. scoparia (p 100 µg/mL) when comparing to the brown 1.16 ± 0.023 mmol Trolox Equivalent/g. Nevertheless, the lowest value algae tested (between 7.17 and 64.89 µg/mL). Other studies confirmed was found for red alga C. virgatum with 0.25 mmol Trolox Equivalent/g the high EC₅₀ values of U. intestinalis and U. lactuca with EC₅₀ of 1333.3 (Table 2). As a way to associate the presence of phenolic compounds as µg/mL and 1266.7 µg/mL, respectively [49]. However, the high DPPH the possible responsible compounds for the high antioxidant capacity of radical scavenging activity of brown algae, has been reported by many the extracts, their concentrations were correlated against TEAC and studies, for aqueous or ethanolic extracts of P. pavonica (5.59 µg/mL) DPPH radical scavenging value. The Pearson's correlation showed a high [13], Ascophyllum nodosum (10.4 µg/mL), Fucus vesiculosus (4 µg/mL), correlation between TPC with TEAC and DPPH assays (r = -0.745 and r Cystoseira nodicaulis (28.0 µg/mL), Fucus serratus (19 µg/mL), and = 0.969 respectively), revealing the influence of phenolic compounds on Himanthalia elongata (14 µg/mL) [69]. On the other hand, the red alga the antioxidant activity. These promising antioxidant results confirm Ceramium virgatum was more efficient than green algae to scavenge that brown algae contain great potential for the development of new 6B. Keramane et al. Algal Research 76 (2023) 103293 280 nm mAU 3 10 1 2 0 15 20 25 30 35 40 min mAU 280 nm 4 20 10 0 450 nm 20 1' 2' 10 0 10 20 30 40 50 60 min Fig. 2. HPLC-DAD chromatogram, at the wavelength of 280 and 450 nm, of U. intestinalis PLE extracts. Peak data as in Table 4. P. pavonica and Ceramium virgatum (Fig. 6). There was no considerable Table 4 variation in MICs obtained among the different bacteria studied, how- Characteristic parameters of the compounds detected in the Ceramium virgatum ever S. typhi showed higher sensitivity to the different extracts. PLE extract analyzed by LC-DAD. Regarding the yeast Candida albicans, Cystoseira mediterranea extract Ceramium virgatum showed the best antifungal activity followed by P. pavonica with Peak no. tR Amax inhibitory concentrations of 2.16 ± 0.28 mg/mL and 5 ± 0.00 mg/mL, 450 nm respectively. On the other hand, green algae showed the lowest anti- 49.604 420, 446, 472 fungal activity. Concerning Gram-negative bacteria, Cystoseira medi- 2' 51.192 422, 442, 470 terranea was the most active with minimum inhibitory concentrations of 3' 51.728 416, 440, 470 0.83 ± 014 mg/mL, 2.16 ± 0.28 mg/mL and 4.66 ± 0.57 mg/mL for 4' 52.881 (330) 420, 440, 467 S. typhi, V. cholerae and E. coli, respectively. Concerning the Gram-positive methicillin-resistant Staphylococcus 280 nm aureus tested, the red alga Ceramium virgatum showed the best activity 1 4.540 270 2 18.322 270, 370, 445 with an MIC of 2.50 mg/mL, moreover, this red alga, exerts its highest 3 21.871 262, 269, 395 activity toward S. typhi with a MIC of 1.16 ± 0.14 mg/mL. This activity 4 22.351 260 could be due to phycoerythrin which is the most abundant protein- 5 47.901 499, 545, 568 pigment complex in red algae [59], knowing that its protein content 6 48.747 400, 505, 538 7 50.589 400, 505, 538 namely 4.36 ± 0.13 mg EBSA/100 g extract is the highest of the tested 8 54.815 230, 263, 269 algae. Indeed, the antibacterial activity of purified phycoerythrin has already been reported by Hemlata et al. [75]. Our results agree with those of Kumar et al. [76] who found that antioxidant products of great commercial interest especially for food brown algae had the highest antibacterial activity compared to green industries. and red algae. They are also in agreement with those of Salvador Soler et al. [23] who screened the antimicrobial activity of 82 species of macroalgae, and observed that the highest antibacterial activity of 3.5. Antibacterial activity of PLE extracts Phaeophyceae were exhibited the genera Cystoseira. The same obser- vation was reported by El Wahidi et al. [77], they reported that the The antibacterial activity of the seaweed extracts was tested on three ethanol PLE extracts of C. compressa and C. brachycarpa tested among ten bacterial strains involved in food spoilage and in food poisoning: Gram- other species, were the most active against all microbial strains. negative E. coli, Salmonella typhi, Vibrio cholerae. In addition, a Gram- Regarding the results of anti-MRSA activity of the different algae, as positive methicillin resistant Staphylococcus aureus (MRSA), and a explained above, in this study, red algae showed the higher activity, pathogenic yeast Candida albicans, both associated with the various in- however, other authors showed a higher anti-bacterial activity for fections in immune-compromised people, were tested. In an overall way, brown algae. For instance, Chandrasekaran et al. [78] studied the anti- all the extracts exhibited a good antimicrobial activity, however, Cys- MRSA activity of brown and red algae and reported that solvent PLE toseira mediterranea showed the best antibacterial activity with con- extracts of different polarities (hexane, chloroform, ethyl acetate, centrations ranging between 0.83 and 9.66 mg/mL, followed by 7B. Keramane et al. Algal Research 76 (2023) 103293 Table 5 Characteristic parameters of the compounds detected in the Padina pavonica, Halopteris scoparia and Cystoseira mediterranea PLE extract analyzed by LC-DAD. Padina pavonica Peak no. tR Amax 450 nm 1' 37.765 (267) 423, 446, 476 2' 39.397 (268) 420, 449, 470 3' 40.978 (268) 421, 448, 470 4' 43.595 420, 444, 468 5' 44.51 409, 437,462 280 nm 1 18.378 229, 267 2 20.423 225, 270, 370, 445 3 21.592 226, 283 4 25.559 218, 256 5 40.603 290, 305,320 6 40.982 (268) 420, 448, 470 7 42.09 265, 272, 284 Halopteris scoparia Peak no. tR Amax 450 nm 37.486 (268) 421, 448, 470 2' 39.397 (267) 422, 448,476 3' 40.978 (267) 421,448, 470 4' 43.595 420, 444, 468 5' 50.018 428 6' 50.815 (200) 435 280 nm 1 25.56 228, 268 2 31.973 212, 330 3 40.978 420, 448, 470 4 46.298 371,398, 407 5 48.259 375, 396, 410 Cystoseira mediterranea Peak no. tR Amax 450 nm 1' 39.9 438, 473 2' 40.99 422, 447, 469 3' 43.612 425, 443, 466 4' 44.527 410, 435, 461 5' 44.851 418, 441, 461 6' 50.559 425, 452, 480 7' 51.233 331 8' 55.307 393, 413, 434 280 nm 1 29.226 293 2 32.836 290 3 40.99 419, 448, 469 4 48.28 373, 398, 410 5 54.155 420, 460, 480 6 55.299 372, 413, 434 acetone and methanol) from the brown algae Sargassum wightii and showed the higher percentage of inhibition compared to U. intestinalis Stoechospermum marginatum had higher anti-MRSA activity than red and it was more active against E. coli. Boisvert et al. [14] also observed algae. Eom et al. [79] studied and characterized the anti MRSA activity that ethanol extract of U. lactuca obtained with PLE, exerted the of the brown algae and suggested that those activities were mediated by maximum activity toward E. coli. As shown in Table 1, U. lactuca extract phlorotannins compounds. was more concentrated in polysaccharides and proteins. Higher con- Regarding green algae, U. lactuca demonstrated higher antibacterial centrations of polysaccharides such as sulfated ones, could explain the activity than U. intestinalis extracts, and Ulva lactuca was more active higher antibacterial activity of the extracts [80]. There was no consid- against S. typhi and E. coli (2.08 ± 0.72 and 5.16 ± 0.05 mg/mL, erable variation in the MIC found among the different bacteria studied, respectively. Similar findings have been reported by Otero et al. [18], however S. typhi showed a higher susceptibility to the different extracts. who tested the antibacterial activity of ethanolic PLE extracts of six The brown algae showed a better antifungal activity against C. albicans seaweeds against E. coli and S. aureus. They reported that U. lactuca than the red and green ones. Cystoseira mediterranea extract does not 8B. Keramane et al. Algal Research 76 (2023) 103293 mAU 1 280 nm 2 3 4 0 5 10 15 20 25 min mAU 280 nm 40 5 6 20 7 0 2' 450 nm 40 3' 4' 20 1' 5' 0 10 20 30 40 50 60 min Fig. 3. HPLC-DAD chromatogram, at the wavelength of 280 and 450 nm, of Ceramium virgatum PLE extracts. Peak data as in Table 5. mAU mAU mAU 100 800 800- A 400 0 80 250 350 450 550 nm 0 800 mAU 60 800- B 400- 400 40 0 250 350 450 550 nm 0 800 mAU 20 800- 400 400 0 0 250 350 450 550 nm 0 0 10 20 30 40 50 60 min 0 10 20 30 40 50 60 min Fig. 4. HPLC-DAD chromatogram, at the wavelength 450 nm, of Ceramium Fig. 5. HPLC-DAD chromatogram, at the wavelength 450 nm, of H. scoparia (A) virgatum PLE extracts (solid line) and astaxanthin standard (dotted line) at P. pavonica (B) and C. mediterranea (C) PLE extracts. 48.9 min. antimicrobial activity of phlorotannins is due to the interaction with contain the best amounts of TPC or phlorotannins among the brown bacterial proteins as enzymes and cell membranes, inhibiting the bac- algae studied, but has the best antimicrobial activity. Moreover, as terial growth [35]. Phlorotannins bind to the amine function of amino shown in the correlation matrix in Table 6, the antimicrobial activity is acids residues. The linkage is then strengthened by hydrogen bonds not correlated to the TPC nor to the phlorotannin content, suggesting the between carbonyl oxygens of the peptide bonds, of the linked amino presence of other bioactive components such as carotenoids and ter- acid, and the phenolic hydroxyl groups of the phloroglucinol. Another penes, which have already demonstrated their antimicrobial activity mechanism of action has been reported concerning low molecular [81-83]. In addition, it should be borne in mind that the antimicrobial weight phlorotannins, they seem to have the ability to damage the activity could be due to a combination of metabolites acting together. membrane and bacterial wall, leaking cytoplasm and destabilizing the The literature has described that phlorotannins are multifunctional in cell balance [85]. All the PLE extracts tested showed a relatively good brown seaweeds, with ostensive roles as primary cell wall components antibacterial activity toward the methicillin resistant Staphylococcus also implicated in its biosynthesis and defense toward natural antago- aureus with MIC ranging from 2.5 ± 0.0 to 9.66 ± 057 mg/mL. The anti nists, acting as a repellent for herbivores, and as antifouling and anti- MRSA activity of phlorotannins has already been studied. It has been bacterial agents [84]. In addition, it has been reported that supported that some phlorotannins suppressed the genes regulating the expression of methicillin resistance in bacteria mec I, mec R1, and mec 9B. Keramane et al. Algal Research 76 (2023) 103293 18 16 14 12 10 MIC (mg/ml) 8 6 4 2 0 MRSA S. tiphi E. coli V. cholerae C. albicans P. pavonica C. mediterranea H. scoparia C. virgatum U. intestinalis U. lactuca Fig. 6. Minimal inhibitory concentration MIC (mg/mL) of the PLE extracts on five bacterial strains. Values represented by bars are mean ± SD (n = 3). MIC values are expressed in mg/ml. MRSA: Methicillin resistant Staphylococcus aureus. Table 6 Pearson correlation matrix for variables studied. Variables E. Yield TPC DPPH EC50 TE TSC TProtC PC MRSA S. typhi E. coli V. cho C. albicans E. Yield 1 -0,016 0,116 0,085 0,234 -0,647 -0,135 0,137 -0,110 0,670 0,411 0,385 TPC -0,016 1 -0,745 0,969 -0,607 0,154 0,933 0,303 0,308 -0,501 -0,026 -0,736 0,116 -0,745 1 -0,682 0,917 -0,403 -0,828 -0,011 -0,196 0,447 0,205 0,757 TE 0,085 0,969 -0,682 1 -0,511 0,124 0,881 0,343 0,111 -0,513 -0,041 -0,690 TSC 0,234 -0,607 0,917 -0,511 1 -0,530 -0,733 0,160 -0,271 0,355 0,192 0,690 TProtC -0,647 0,154 -0,403 0,124 -0,530 1 0,143 -0,644 -0,007 -0,530 0,039 -0,231 DMBA -0,135 0,933 -0,828 0,881 -0,733 0,143 1 0,420 0,258 -0,568 -0,326 -0,903 SARM 0,137 0,303 -0,011 0,343 0,160 -0,644 0,420 1 -0,162 -0,262 -0,615 -0,475 S. typhi -0,110 0,308 -0,196 0,111 -0,271 -0,007 0,258 -0,162 1 0,230 0,421 -0,055 E. coli 0,670 -0,501 0,447 -0,513 0,355 -0,530 -0,568 -0,262 0,230 1 0,562 0,733 V. cho 0,411 -0,026 0,205 -0,041 0,192 0,039 -0,326 -0,615 0,421 0,562 1 0,647 C. albicans 0,385 -0,736 0,757 -0,690 0,690 -0,231 -0,903 -0,475 -0,055 0,733 0,647 1 TPC: total phenolic content; PC: phlorotannins content; TSC: total sugar content; TE: Trolox equivalent; TProtC: total protein content; MRSA: methicillin-resistant Staphylococcus aureus; V. cho: Vibrio cholerae; S. typhi: Salmonella typhi; C. albicans: Candida albicans. A, resulting the stop of production of penicillin-binding protein 2a, of both metabolites in the antioxidant activity of seaweeds. The HPLC- which is believed to be the main mechanism of MRSA strains resistance DAD analyses revealed the presence of a great amount of carotenoids [80]. There are no numerous studies on the antimicrobial activity of and phenolic compounds in all the samples. Interestingly, brown algae fucoxanthin, though; some of them have proved its antibacterial activ- were the species which showed a higher concentration of carotenoids, in ity. However, the mechanism of action has not yet been solved, particular they were rich in the carotenoid tentatively identify as fuco- [82,83,86]. The scientific literature has suggested a connection between xanthin. All the PLE extracts exhibited potential antimicrobial activity; the antioxidant and antibacterial properties of natural chemical com- however, Cystoseira mediterranea extract was the most active against S. pounds [80]. According to this concept, fucoxanthin, which has anti- typhi, V. cholera, E. coli and C. albicans. Those results were not correlated oxidant activity, could exert its antibacterial activity by three basic to the polyphenols content and perhaps the presence of terpenes could ways: outer membrane permeability, cytoplasm leakage, and inhibition explain that results as meroditerpenoids constitute the most common of nucleic acid formation [86]. metabolites in the Cystoseiracae. 4. Conclusion CRediT authorship contribution statement In the present study, we have assessed the chemical composition in Badria Keramane: Investigation, Writing - original draft. Andrea terms of protein and sugar content, as well as the antioxidant and del Pilar Sánchez-Camargo: Investigation, Formal analysis, Writing - antimicrobial potential of brown, green and red macroalgae from original draft, Writing - review & editing. Lidia Montero: Investigation, Algeria. The PLE extracts from brown macroalgae showed the higher Writing - original draft, Writing - review & editing. Firdousse Laincer: phenolic contents compared to green and red ones, which seem to be Conceptualization, Methodology, Supervision. Fatiha Bedjou: correlated with their phlorotannins valuable amount. The antioxidant Conceptualization, Methodology, Supervision. Elena Ibanez: Method- activity showed relevant values in brown algae, and the red and green ology, Supervision, Resources, Writing - review & editing. ones exhibited limited antioxidant properties. Those last were deficient in phlorotannins content and carotenoids contents, supporting the role 10B. Keramane et al. Algal Research 76 (2023) 103293 Declaration of competing interest potential of twenty-three British and Irish red algae, Phytother. Res. 24 (2010) 1099-1103, https://doi.org/10.1002/ptr.3094. [21] J. Spavieri, A. Allmendinger, M. Kaiser, M.A. Itoe, G. Blunden, M.M. 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