Exploring The Potential Of Icelandic Seaweeds Extracts Produced By Aqueous Pulsed Electric Fields-Assisted Extraction For Cosmetic Applications

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Abstract: A growing concern for overall health is driving a global market of natural ingredients not only in the food industry but also in the cosmetic field. In this study, a screening of potential cosmetic applications of aqueous extracts from three Icelandic seaweeds produced by pulsed electric fields (PEF) was performed. Produced extracts by PEF from Ulua Lactuca, Alaria esculenta, and Palmaria palmitate were compared with the traditional hot water extraction in terms of polyphenol, flavonoid, and carbohydrate content. Moreover, antioxidant properties and enzymatic inhibitory activities were evaluated by using in vitro assays. PDF exhibited similar results to the traditional method, showing several advantages such as its non-thermal nature and shorter extraction time. Amongst the three Icelandic species, Alaria esculenta showed the highest content of phenolic (mean value 8869.7 μg GAE/g do） and flavonoid （mean value 12，098.7 μg QE/g DW） compounds， also exhibiting the highest antioxidant capacities. Moreover, Alaria esculenta extracts exhibited excellent anti-enzymatic activities (76.9,72.8, 93.0, and 100% for collagenase, elastase, tyrosinase, and hyaluronidase, respectively)for their use in skin whitening and anti-aging products. Thus, our preliminary study suggests that Icelandic Alaria esculenta-based extracts produced by PEF could be used as potential ingredients for natural cosmetic and cosmeceutical formulations.

Keywords: macroalgae; Ulloa Lactuca; Alaria esculenta; Palmaria palmata; PEF-assisted extraction;bioactive compounds; green extraction; natural ingredients; cosmeceuticals

1. Introduction

In recent years, the demand for new bioactive compounds with potential health benefits has undergone a substantial increase. Many research groups have placed emphasis on research on marine organisms, such as macroalgae, to find novel and sustainable sources of natural compounds for applications in the agri-food industry, pharmacology, foods, and, more recently, in the field of cosmetics [1,2]. Macroalgae are a large and heterogeneous group of photosynthetic organisms characterized by huge biodiversity and complex biochemical composition. According to their chemical structure and pigment content, macroalgae can be divided into three lineages including brown algae (Phaeophyceae), red algae (Rhodophyta), and green algae (Viridiplantae). Algal compounds are stored inside the cell cytoplasm or bound to cell membranes; thus, cell disruption is crucial for the valorization of algal biomass. Additionally, the cell wall composition is highly variable between algae species ranging from tiny membranes to multi-layered complex structures, making the recovery of algal products a challenge [3]. In general, seaweeds are excellent sources of polysaccharides, proteins, lipids, and a wide variety of secondary metabolites such as phenolic compounds, terpenoids, carotenoids, pigments, and nitrogen derivatives [4-6]. Although primary metabolites have crucial importance, recent data have shown that the content of secondary metabolites determines the biological activities of seaweed extracts[7].

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A growing concern for overall health and wellness, as well as awareness of harmful chemicals in everyday products, is driving a global market of natural and organic ingredients [8]. Over the past years, consumer consciousness towards the preference for natural ingredients and eco-friendly products has extended from the food industry to the cosmetic and personal care industry [9]. Furthermore, in the current context of global warming and ecological issues, there has been increasing public awareness of environmental issues. In light of these current concerns, consumers have turned their interests toward green, healthy and chemical-free products. As a result, the cosmetic industry is currently replacing toxic chemicals and harmful ingredients with novel and natural high-value compounds to produce"chemically-clean" beauty products [10].

Cosmetics have traditionally been defined as products to be applied to the human body for cleansing, beautifying, or promoting attractiveness without affecting body structure or functions. However, new trends and recent consumer demands have promoted the development of novel products that supply multiple benefits with minimal effort. The term cosmeceutical is now frequently used to describe cosmetic products with bioactive ingredients claiming to have medical or drug-like benefits [1]. Cistanche Extract Anti Radiation Cosmeceuticals usually contain functional ingredients such as vitamins, phytochemicals, enzymes, antioxidants, and/or essential oils [12]. Since a wide range of these bioactive compounds has been found in macroalgae, the investigation of new seaweeds and marine algae-derived extracts has proven to be a promising area of cosmeceutical and cosmetics studies [13,14].

A number of secondary metabolites derived from seaweeds are known for their valuable health beneficial effects on the skin, such as photo-protective, moisturizing, antioxidant, anti-inflammatory, and regenerative properties [15]. Based on these beneficial effects, algae are incorporated in cosmeceutical products such as sunscreen, and anti-aging products, as well as for the prevention of hyperpigmentation, while polysaccharides are used for keeping the skin moisturized and prevent dryness[16]. During aging, the extracellular matrix proteins are susceptible to excessive activity of r proteolytic enzymes such as collagenases and elastases, resulting in visible changes in the skin, such as wrinkles or the loss of skin elasticity. A promising approach to prevent extrinsic skin aging is the inhibition of collagenase and elastase activities by natural compounds. Plant extracts have been widely investigated and found to possess anti-collagenase and anti-elastase activities [17]. However, there is little information on the inhibitory enzymatic activities of seaweed extracts.

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The most frequently applied extraction methods for the isolation of bioactive from seaweeds are based on conventional techniques. Nevertheless, the utilization of traditional methods has several drawbacks, such as the use of high volumes of organic solvents, longer extraction times, high temperatures, selectivity problems, high energy requirements, and coextraction of untargeted or interfering compounds [18]. Hence, new extraction techniques based on green chemistry principles have a potential interest [19].

Pulsed electric field (PEF) is an emerging, nonthermal, and energy-efficient for pro-cessing technology [20]. PDF involves the application of electric field pulses usually at high voltages (kV range) and short durations (micro or nanoseconds) to a product placed between two electrodes [21]. The application of electric pulses produces the formation of reversible or irreversible pores in the cell membranes, defined as electroporation or electro-permeabilization, which consequently facilitates the rapid diffusion of the solvents and the mass transfer enhancement of intracellular compounds[22]. Recent applications have focused on the use of pulsed electric energy as an extraction technique (PEF-assisted extraction) from bio-, food, and agricultural products [23]. With PEF treatment it is feasible to obtain extracts with higher purity, increase the extraction rate of bioactive compounds such as polyphenols, carotenoids, or anthocyanins, eliminate the use of organic solvents, and shorten the extraction time [24,25]. cistanche herba PEF treatment has been successfully applied for the extraction of valuable compounds from different marine sources, such as proteins [26-28], carbohydrates [29,30], lipids [31,32], and pigments such as carotenoids, chlorophylls or phycocyanins [22,33,34] from microalgae and seaweeds.

Thus, the main objective of the present study was to assess the potential cosmetic applications of PEF extracts from three macroalgae species growing in Iceland: U. Lactuca (green macroalgae), A. esculenta(brown macroalgae), and P. palmitate(red macroalgae). In an effort, to develop organic and natural ingredients for green formulations, PEF-assisted extraction was proposed as an eco-friendly alternative to the traditional organic solvent extraction. After the extraction process, aqueous seaweed extracts were characterized in terms of polyphenol, flavonoid, and carbohydrate content. Moreover, antioxidant properties and enzymatic inhibitory activities were evaluated by using in vitro activity assays. The results reported herein will provide the basis for improving the understanding of brown, red, and green macroalgae to produce active ingredients for innovative formulations in cosmetic products containing biologically active compounds isolated from natural and sustainable sources.

2. Results and Discussion

2.1.PEF-Assisted Extraction for the Processing of Icelandic Seaweed Biomass

The results show that the electrical conductivity was highest in suspension prepared from A.esculenta followed by P.palmata and U.lactuca (p<0.05)(Table 1). However, the effect of treatment type was not identified as significant (p>0.05). Electrical conductivity measurement has been successfully used by other authors to evaluate the efficacy of PEF treatment in biological tissues for the release of intracellular ionic substances, as a result of the increased cell membrane permeabilization [35-37].

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In our study, the results did not indicate a stronger release of these substances by PEF, since the changes in conductivity induced by extraction treatments tended to be highest in HW suspensions. Previous studies have concluded that the initial conductivity of the extracellular medium influences the electroporation efficacy but there is a lack of agreement on whether they're a positive or negative relationship between these two factors [38]. Variations in conductivity and characteristics of the material may make the comparison complicated. In our study, there was a large difference between the conductivity of A.esculenta suspensions and the other two species, which was not reflected in the degree of conductivity changes during extraction treatment. It has been stated that the ash content of brown seaweed can account for over 50 % of its dry weight [39], consisting largely of ions, which may partly explain the high conductivity in A.esculenta suspensions compared to the other two species.

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The results show that the pH in U. Lactuca suspension was lower than for the other two species, but no clear effects from extraction type were produced. The temperature was increased from 22 ± 1°C before treatment, to 95℃C by HW(for all species), to 36.0±1.0°C,46.3±0.6°C and51.0±1°Cby PEF, in A.esculenta, P.palmata, and U. Lactuca suspensions. The same trend was seen for the groups treated with PEF, which were then further heated by HW. The rise in temperature was caused by the conversion of electric energy to thermal energy (ohmic heating), in the suspension during PEF treatment. The level of temperature increase is known to be in proportion to the applied current but in inverse proportion to the conductivity. This could explain why P. palmate and U.lactuca reached higher temperatures during the PEF treatment although they have lower conductivity than A. esculent.

2.2.UV-VIS Absorption Spectra of Icelandic Seaweed Extracts

The studied seaweeds differ in the spectral profiles(Figure 1), suggesting that the composition and the UV-absorbance potential vary between species. However, the type of extraction technique did not exhibit a remarkable effect in the UV absorption spectra; seaweed extracts showed similar absorption profiles regardless of the extraction method.

The UV absorption spectra of the green alga U. Lactuca showed a prominent peak in the UV-B range (280-320 nm)(Figure la), while the extracts from the brown alga A.esculenta showed no clear formation of absorption zone(Figure c). However, results indicated a stronger absorbance at 220 nm in A. esculenta extracts compared to U. Lactuca and P. palmata which was presumed to result from the high content of phenolic compounds in A. esculenta (Table2). An absorption maximum within this range has been related to a linkage between phenolic compounds and alginates. This relationship is presumed to preserve the UV absorption capability of phenolic compounds over time [40].

A more interesting finding was that the results obtained for the red algal extracts, P. palmata absorbed part of UV-A radiation (320-400 nm). It is known that red algae accumulate photoprotective compounds with ultraviolet radiation absorption capabilities such as mycosporine-like amino acids(MAAs), which absorb in this specific UV region [41]. P. palmata excelled in the UV absorption spectrum with prominent peaks between 320 and 340 nm in accordance with the presence of MAAs absorbing in this range [42], such as polyphenol (peak absorption at 332 nm), asteria-330 (absorption peak at 330 nm), Porphyra-334 (peak absorption at 334 nm) and others [43]. Because extraction conditions, such as type of solvent, are known the influence the efficiency of extraction, the results in the present study were compared to previous studies on the extraction of MAAs with water from P.palmata. In these studies, the absorption maximum peaks were detected at 325 to 330 nm[44], as in the present study. Therefore, it is possible to assume that the peaks observed between 320 and 340 nm may be due to the presence of MAAs.

Differences in the absorption spectra between 350 and 700 nm have been explained by the presence of different accessory pigments in respective photosystems of the green, brown and red macroalgae, chlorophyll-b(450-500 nm), fucoxanthin (400-500 nm), and PHY erythrin (600-650 nm) respectively [45]. The concentration of the water-soluble compounds in the extracts had stronger effects. Consequently, the pattern reflecting the difference in pigments between algae species was not apparent in the present study.

2.3.Total Phenolic, Flavonoid, and Carbohydrate Content of Icelandic Seaweed Extracts

The total phenolic content in the seaweeds ranged from 1592 to 9368 μg GAE/g (Table 2). The brown alga A.esculenta showed the highest quantity (p<0.05) of phenolic compounds(mean value 8869.7 ugs GAE/g do), followed by P. palmitate (mean value 1806.2 μg GAE/g do） and U. Lactuca （mean value 1750.7 ug GAE/g dw）（there were no significant differences between P. palmata and U.lactuca extracts)). For each seaweed species, the content of polyphenols did not differ among extraction methods except for U. Lactuca, which results showed that HW was the most efficient technique (p<0.05). However, the advantages of PEF including its non-thermal nature, shorter extraction time (10 min vs. 45 min), and green process, should be highlighted.

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Amongst the three algal groups, brown macroalgae contain a higher number of polyphenols than red and green macroalgae. Results were in agreement with early studies 46,47| which reported that brown (e.g., A.esculenta and Saccharina platysma) algae species had higher phenolic content than red(P. palmitate) and green species(e.g., U. Lactuca). This was supported by other authors [48] who concluded that the mean polyphenol content was species-specific(A.esculenta>S.latissma>P. palmitate) and the phenolic content was more than three-time higher in A.esculenta than in the other species (A. esculenta:37 mg phloroglucinol equivalents (PGE)/g DW; S.latissma:8 mg PGE/g do; P. palmata:5 mg GAE/g do). Furthermore, in the same study, the authors reported that the polyphenol content varies with season, while the spatial variations(algae were harvested in Norway, France, and Iceland) showed a marginal effect. For example, Gager et al.(2020) found that there was a significant effect of seasonal variations in polyphenol content of A.esculenta, with more than 300 mg GAE/g DWin autumn compared to under 20 mg GAE/g DW in springtime. Phlorotannins from seven brown seaweeds commercially harvested in Brittany(France) detected by 1 H NMR and in vitro assays: temporal variation and potential valorization in cosmetic applications. Our samples were collected in July (U.lactuca and A.esculenta) and in November (P. palmitate). In Roleda's study [48], the average content in A.esculenta from Trondheim, Norway (not collected in Iceland) in summer was 40 mg PGE/g DW and P.palmata from Iceland but was 4 mg GAE/g in the autumn. The higher values reported in comparison with our study can be explained by the extraction media used (80:20 acetone: water), likely to result in higher extraction yields. Higher polyphenol content was also found for A. esculenta extracts using a mixture of ethanol and water (50:50) with ultrasound [49]. However, using the same extraction medium and the classic solvent extraction, A.esculenta was reported to contain 44.1 mg GAE/100 g DW in aqueous extracts [50], relatively similar to that observed in the present study. Mean flavonoid content was species-specific (A. esculenta > U. lactuca > P. palmata;(p<0.05)(Table 2). The highest amount of flavonoids was observed for A.esculenta extracts （mean value 12098.7 μg QE/g do）， while lower content was found for UI. Lactuca （mean value 4152.4 ugs QE/g do), and a minimum content were determined for P. palmata extracts (mean value 905.8 ugs QE/g do). Similar to the behavior found for the total phenolic content, the type of extraction technology did not have significant effects on the flavonoid content (p > 0.05), with the exception of U. Lactuca. Results showed that HW and the combination of both techniques (PEF+ HW) were the most efficient techniques for the extraction of flavonoids in U.lactuca (p <0.05).

There are numerous studies on the flavonoid content in terrestrial plants, but flavonoid content studies in algae are scarce [51] and especially in the species studied in the present work. Namely, the study of Ummat et al. [49] reported that ultrasound-assisted extraction enhanced the recovery of flavonoids in all 11 seaweeds investigated (including A.esculenta)compared to conventional solvent extractions using a mixture of 50% ethanol. In another study, flavonoids were quantified in the methanolic extracts of four Ulua species (Ulloa clathrate, Ula Linza, Ulloa flexuosa, and Ulva intestinalis)grown at different parts of the northern coasts of the Persian Gulf in the south of Iran; the flavonoid content of algal extracts varied from 8 to 33 mg RE/g do [52]. However, previous studies by the same research group found marked changes in the chemical constituents with changes in seasons and environmental conditions [53]. Thus, it is a little hard to have a full overview of the bibliography of these bioactive compounds in seaweeds, due to the lack of available published research, but also because of the changes in the flavonoid content influenced by the growing conditions and geographic location.

Mean carbohydrate content of produced extracts was also species-specific(P. palmata > U.lactuca>A.esculenta;p<0.05)(Table2).Contents ranged from 44.8 to 510 mg GluE/g do depend on algae species. Seaweed contains a large number of polysaccharides with important functions for the macroalgal cells including structural support and energy storage. For instance, the main part of red and brown seaweed cell walls is represented by sulfated galactans, which are known as agar, alginate, and carrageenan [54]. The red algae P. palmata showed the highest amount of carbohydrate content (mean value 441 mg GluE/g do). Results were in agreement with previous studies that reported the highest polysaccharide concentration in Palmaria species [55]. Moreover, Mutripah et al. [56]described a total carbohydrate content of P. palmata of 469 mg/g of dry seaweed, relatively similar to that observed in the present study.

The green macroalgae U. Lactuca showed contents of up to 249.5 mg GluE/g do depend on the extraction technique used (Table 2). Based on the literature, U. Lactuca has water-soluble and insoluble cellulose corresponding to structural polysaccharides with a major component called Ivan, which contributes from 9 to 36%dry weight to the biomass [57]. Ryan is mainly composed of sulfated rhamnose, uronic acids (glucuronic acid and iduronic acid), and xylose. Due to its polar nature, the solubility of Ivan in aqueous solutions is enhanced by extraction at high temperatures(80-90°C)[58]. The extraction temperature could be the reason why the total carbohydrate content of U. Lactuca extracts produced by the traditional hot water extraction and the combination of both methods(PEF+ HW) was higher(p<0.05) than the content achieved using only PEF.

On the other hand, other authors highlight the importance of the seasonal variation in the polysaccharide content. For instance, Schiener et al., claim to identify seasonal variations and predict the best harvest times for kelp. The seasonal composition analysis of A.esculenta demonstrated that maximum values of carbohydrates coincided with reduced concentrations of protein, ash, polyphenols, and moisture [39]. According to the authors, these relationships, which vary between seasons and species, can be used by industries to maximize the yields of targeted seaweed components.

This article is extracted from Mar. Drugs 2021, 19, 662. https://doi.org/10.3390/md19120662 https://www.mdpi.com/journal/marinedrugs