*Article* **Nutritional Value and Biofunctionalities of Two Edible Green Seaweeds (***Ulva lactuca* **and** *Caulerpa racemosa***) from Indonesia by Subcritical Water Hydrolysis**

**Ratih Pangestuti 1,\*, Monjurul Haq 2, Puji Rahmadi <sup>3</sup> and Byung-Soo Chun 4,\***


**Abstract:** *Caulerpa racemosa* (sea grapes) and *Ulva lactuca* (sea lettuces) are edible green seaweeds and good sources of bioactive compounds for future foods, nutraceuticals and cosmeceutical industries. In the present study, we determined nutritional values and investigated the recovery of bioactive compounds from *C. racemosa* and *U. lactuca* using hot water extraction (HWE) and subcritical water extraction (SWE) at different extraction temperatures (110 to 230 ◦C). Besides significantly higher extraction yield, SWE processes also give higher protein, sugar, total phenolic (TPC), saponin (TSC), flavonoid contents (TFC) and antioxidant activities as compared to the conventional HWE process. When SWE process was applied, the highest TPC, TSC and TFC values were obtained from *U. lactuca* hydrolyzed at reaction temperature 230 ◦C with the value of 39.82 ± 0.32 GAE mg/g, 13.22 ± 0.33 DE mg/g and 6.5 ± 0.47 QE mg/g, respectively. In addition, it also showed the highest antioxidant activity with values of 5.45 ± 0.11 ascorbic acid equivalents (AAE) mg/g and 8.03 ± 0.06 trolox equivalents (TE) mg/g for ABTS and total antioxidant, respectively. The highest phenolic acids in *U. lactuca* were gallic acid and vanillic acid. Cytotoxic assays demonstrated that *C. racemosa* and *U. lactuca* hydrolysates obtained by HWE and SWE did not show any toxic effect on RAW 264.7 cells at tested concentrations after 24 h and 48 h of treatment (*p* < 0.05), suggesting that both hydrolysates were safe and non-toxic for application in foods, cosmeceuticals and nutraceuticals products. In addition, the results of this study demonstrated the potential of SWE for the production of high-quality seaweed hydrolysates. Collectively, this study shows the potential of under-exploited tropical green seaweed resources as potential antioxidants in nutraceutical and cosmeceutical products.

**Keywords:** *Caulerpa racemosa*; *Ulva lactuca*; nutritional; potential; SWE

### **1. Introduction**

Seaweed, also known as marine macroalgae, comprises photosynthetic organisms and includes more than 12,000 species [1,2]. Based on photosynthetic pigment, they can be categorized into: Rhodophyceae (red seaweeds), Phaeophyceae (brown seaweeds) and Chlorophyceae (green seaweeds) [3]. Seaweeds play an important ecological, socioeconomic role for coastal communities and are also used for many purposes such as food, medicinal, building materials, feed and many others. Seaweeds are rich in bioactive materials such as polysaccharides, proteins, peptides, amino acids and secondary metabolites including polyphenolic compounds and natural pigments. These bioactive materials have

**Citation:** Pangestuti, R.; Haq, M.; Rahmadi, P.; Chun, B.-S. Nutritional Value and Biofunctionalities of Two Edible Green Seaweeds (*Ulva lactuca* and *Caulerpa racemosa*) from Indonesia by Subcritical Water Hydrolysis. *Mar. Drugs* **2021**, *19*, 578. https://doi.org/10.3390/md19100578

Academic Editors: Bill J. Baker, María Lourdes Mourelle, Herminia Domínguez and Jose Luis Legido

Received: 30 August 2021 Accepted: 13 October 2021 Published: 15 October 2021

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**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/).

been demonstrated to possess various biological activities and medicinal and health beneficial effects. In addition, many studies have found that countries where seaweeds are consumed on a daily basis have significantly fewer diet-related diseases and longer life expectancy [1]. Seaweeds' bioactive compounds have become a driving factor for their increased demand in food, nutraceutical and cosmeceutical products [4].

Seaweeds are widely distributed and can be found in all zones on the Earth from polar, temperate to tropical regions. Indonesia is an archipelagic country with a long coastline and lies within the heart of the Coral Triangle, the center of the highest marine biodiversity on Earth [5]. The earliest documentation of seaweeds diversity in Indonesia is reported by Rumphius (1750), who established the botanical foundations of the flora of Indonesia. Further, in 1912, van Bosse documented 782 seaweed species in Indonesia, which consisted of 196 species of green seaweeds, 452 species of red seaweeds and 134 species of brown seaweeds. Recently, it was reported that around 1000 seaweeds species can be found in Indonesia [4,6]. Despite the great diversity of seaweed species in Indonesia, only a few species have been used for foods, supplements, nutraceutical and cosmeceutical industries. Among tropical seaweeds species, *Caulerpa racemosa* (known as sea grapes or green caviar) and *Ulva lactuca* (known as sea lettuces) belong to the green algae (Chlorophyta) represent under-exploited seaweed resources in Indonesia. Sulfated polysaccharides from *Ulva* spp. have beneficial effects for cancer chemoprevention, anti-hypertensive and immune-modulating activities [7–9]. In addition, the aqueous extract of *Caulerpa* spp. showed anti-photoaging activity in UVB-irradiated mice [10]. Unfortunately, bioactive compounds, as well as biofunctionalities of *C. racemosa* and *U. lactuca* from Indonesia, are not well characterized. In addition, fewer studies were conducted concerning the bioactive compounds from green seaweeds and their biological activities compared to other seaweed classes [11].

Generally, hot water extraction (HWE), organic solvents' extraction and acid/base extraction were used to extract bioactive compounds from seaweeds [4]. However, exposure to organic solvents and strong acids/bases can lead to deleterious effects on human health and environmental concerns. Therefore, environmentally friendly technologies such as microwave-assisted extraction (MAE), ultrasound-assisted extraction (UAE), enzymatic hydrolysis (EAE), ultrasound-assisted extraction (UAE) and subcritical water hydrolysis (SWE) are gaining more attention for development in many sectors. Bioactive compounds such as polysaccharides, carotenoids and phenolic compounds have been extracted from seaweeds by SWE [12,13]. During the SWE process, solvents were maintained in a subcritical state, between boiling point (100 ◦C; 0.10 MPa) and critical point (374 ◦C; 22 MPa), where they remain as a liquid due to the high pressure [4]. Temperature is one of the crucial factors that affect the efficiency and selectivity in the SWE process [5]. Previous studies have demonstrated that the seaweed hydrolysates obtained from SWE have better biological activities as compared to hydrolysates obtained by the conventional HWE process [14]. Considering its high productivity, effectiveness, extraction time, low cost and environmental friendliness, the SWE process has shown many benefits over conventional HWE and other extraction methods.

The main objective of this work was to characterize bioactive compounds in two edible under-exploited tropical seaweeds. First, proximate compositions and fatty acid profiles of *U. lactuca* and *C. racemosa* were analyzed. These two green seaweeds species were further hydrolyzed by conventional HWE at 100 ◦C and SWE at four different temperature conditions (110 ◦C, 150 ◦C, 190 ◦C and 230 ◦C). The hydrolysates from HWE and SWE were further analyzed for biochemical compositions, including total protein, sugar, phenolic, flavonoid and saponin contents. Biological activities of *C. racemosa* and *U. lactuca* were tested using radical scavenging assays, and cytotoxic potentials were studied to gain insight into the potential toxicity of seaweeds hydrolysates. The data obtained and presented in this research on the chemical composition of two edible seaweeds can provide the foundations for the explorations of under-exploited seaweeds in Indonesia and fill the gaps for future

research in the development of functional foods, nutraceuticals and cosmeceuticals from *U. lactuca* and *C. racemosa*.

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

*2.1. Proximate and Fatty Acid Composition of C. racemosa and U. lactuca*

Carbohydrates were the major component of *C. racemosa* and *U. lactuca*, accounting for 38.62 ± 0.01 and 61.83 ± 0.01% of the proximate content, respectively (Table 1). Both green seaweeds also contained protein (7.60 ± 0.01 and 10.0 ± 0.01%), ash (38.41 ± 1.90 and 17.86 ± 0.87%), lipids (0.71 ± 0.01 and 0.13 ± 0.01%) and moisture contents (14.66 ± 0.43 and 10.18 ± 0.04%). When *C. racemosa* and *U.lactuca* were compared directly, the protein and carbohydrates contents of *U. lactuca* were higher than those of *C. racemosa*. The carbohydrate contents of *U. lactuca* found in this study were slightly higher than reported in other studies [15–17]. For example, Rasyid et al. (2017) reported that carbohydrate contents of *U. lactuca* from Pamengpeu, and West Java–Indonesia were 58.1% [15]. The carbohydrates contents in seaweeds are likely to be dependent on geographic location, the season of harvest and algal maturity [18]. Many studies have reported that seaweeds contain high carbohydrates and/or protein but low lipid contents. High carbohydrate contents in *U. lactuca* suggest that these green seaweeds could be an important source of polysaccharides for industrial uses. One of the major sulfated polysaccharides found in the genera *Ulva* spp. is ulvan, which may constitute 8 to 40% of the seaweed biomass [19]. Although the industrial applications based on ulvans are still limited, these sulfated polysaccharides have been demonstrated to possess a broad range of bioactivities such as immunomodulating, antiviral, antioxidant, antihyperlipidemic and anticancer activities [20]. Ulvan has been demonstrated to promote gastrointestinal health and has been linked to a reduction in the incidence of non-communicable diseases (NCD) [21,22]. Ulvan has the potential to be applied as bioactive compounds in foods, nutraceuticals and cosmeceuticals; however, the structural and biological properties of ulvan from *U. lactuca* require thorough investigation.

**Table 1.** Proximate composition of seaweeds.


In terms of lipid content, the values found in both species were relatively low, indicating that both *C. racemosa* and *U. lactuca* are an ideal choice for people who require a low-fat diet [23]. The differences in proximate contents of seaweeds could be attributed to the differences in species, biological conditions, postharvest treatment and preparative methods. The fatty acids composition (area%) of *C. racemosa* and *U. lactuca* are given in Table 2. The Table 2 shows that 11 of 37 types of authentic standard fatty acids were identified in *U. lactuca,* while 24 of 37 types of authentic standard fatty acids were identified in *C. racemosa*. In *C. racemosa* and *U. lactuca*, palmitic acid (C16:0) showed maximum quantities of 50.73 ± 1.41 and 46.64 ± 1.12%, respectively. The proportion of PUFAs in *C. racemosa* found in this study was higher compared to *U. lactuca,* with linolenic acid (C18:3) found as the major omega-3 PUFAs in both species. Our results show that EPA (C20:5n3), DHA (C22:6n3) and AA(C20:4n6) were not detected in both seaweeds, but they contained essential fatty acids such as linoleic acid (C18:2n6) and linolenic acid (C18:3). In contrast, previous studies reported the presence of EPA and DHA in *C. racemosa* and *U. lactuca* [17,24]. It has been reported by Nelson et al. (2002) that variations in fatty acid compositions are attributable to environmental and genetic differences. Ratios of omega-6/omega-3 fatty acids found in this study were relatively low, at 2.71 and 1.18 for *C. racemosa* and *U. lactuca*, respectively. As regards international organizations, the World Health Organization recommends that the ratio of omega-6/omega-3 fatty acids should not exceed 10 in the daily diet [17], since high omega-6/omega-3 fatty acids ratios will increase risk of many diseases [25]. Hence, the low omega-6/omega-3 fatty acids ratios found in *C. racemosa* and *U. lactuca* suggest that both seaweeds are a good source of omega-3 fatty

acids and also an important source of supply of omega-3 fatty acids for homeostasis and maintaining human health [26].


**Table 2.** Fatty acid compositions of two green seaweeds from Indonesia.

alues are means±standard deviations (*n* = 3). Abbreviations: ND: not detected, ω-3: omega-3; ω-6: omega-6; PUFAs: polyunsaturated fatty acids; SFAs: saturated fatty acids; MUFAs: monounsaturated fatty acids.

### *2.2. Extraction Yield of C. racemosa and U. lactuca*

During the hydrolysis process, HWE was maintained at 100 ◦C and SWE at temperatures of 110 ◦C, 150 ◦C, 190 ◦C and 230 ◦C. The reaction times taken were 2 h and 10 min for HWE and SWE, respectively. The pressure of the SWE process was monitored using the pressure gauge and maintained at 5–7 MPa. The extraction yields ranged from 16.37 to 36.38% and 41.49 to 52.08% (dry weight) for *C. racemosa and U. lactuca,* respectively (Figure 1A). Compared to the conventional HWE, the SWE process showed higher extraction yields. The temperature and SWE process directly affected the extraction yield of green seaweed and reached the highest yield at 190 ◦C. It has been reported that temperature is one of the important parameters during the SWE process. The increased extraction yield in SWE at higher temperatures can be correlated with the change in the dielectric constant of water. As the temperatures during SWE process increase, the dielectric constant will also increase; hence, bioactive materials would also increase significantly. Higher temperatures in SWE led to increases in mass transfer, rapid extraction, lower surface tension and higher solubility of bioactive materials [27]. However, some compounds will also be degraded at elevated temperatures. The change in pH value can be related to those processes (Figure 1B). The solvent pH prior to hydrolysis was 7.2, and following the SWE process, seaweed hydrolysate tended to be acidic. The pH reached the lowest value at hydrolysis temperatures of 230 ◦C, with the value of 4.32 ± 0.01 and 4.24 ± 0.01 for *C. racemosa and U. lactuca,* respectively. The low pH value might correlate with the degradation of sugar into organic acids, which further increased the acidity of green seaweed hydrolysates. In accordance with the findings of our study, Park et al. (2019) found that the pH value of red seaweeds *Porphyra yezoensis* hydrolysates following SWE process ware decreased from 7.15 ± 0.01 to 4.16 ± 0.06 at hydrolysis temperatures of 210 ◦C [28].

**Figure 1.** Chemical characteristics of *C. racemosa* and *U. lactuca*. Yield (**A**), pH (**B**), UV-absorbance spectra of *C. racemosa* (**C**) and *U. lactuca* (**D**) obtained by subcritical water hydrolysis. Abbreviations: HWE: hot water extractions; SWE: subcritical water extractions; UL: *Ulva lactuca*; CR: *caulerpa racemosa*. Different letters (a–e) denote a statistically significant difference (*p* < 0.05).

The UV absorption spectra of *C. racemosa and U. lactuca* hydrolysates are shown in Figure 1C,D. A peak observed at 235 nm was attributed to n–π\* transition; the absorption peak near 275 nm was attributed to n→σ\* transition for the amino groups; and the spectral absorption at 300 nm was assigned to n→π\* transition for the carbonyl or carboxyl groups [29]. When *C. racemosa and U. lactuca* were hydrolyzed by SWE at temperatures of 190 ◦C and 230 ◦C, the intensities of the absorption peaks significantly increased, probably because the total protein and other bioactive compounds such as polyphenolic compounds were higher compared to HWE and SWE at lower temperatures. In addition, *C. racemosa* and *U. lactuca* showed strong absorption in the ultraviolet (UV)-B region around 280 to 320 nm (Figure 1C,D) indicating that both green species were rich in UVB-absorbing compounds. In marine environments, light variations occur on much shorter timescales, ranging from seconds to minutes, hours and even days. As a result, seaweeds including *C. racemosa* and *U. lactuca* must avoid the contradiction between effective light absorption on the one hand and a quick photoprotective response to photoinhibitory light intensities on the other [30]. In addition, Wiraguna et al. (2018) has reported UVB-protective activity of *Caulerpa* sp from Indonesia [31]. The presence of UVB-absorbing compounds in *C. racemosa* and *U. lactuca* will allow for future perspectives to understand the photoprotective mechanisms in these tropical green seaweeds. Further, these UVB-absorbing compounds can be used as UVB filters to absorb the entire spectrum of UVB radiation, and these potential compounds can be delivered for the development in the cosmeceutical applications [4].

Analysis of the seaweed hydrolysates' color is shown in Table 3, in which the HWE and SWE at low extraction temperature gave the highest lightness (L\*) value. One possible reason for the lighter color observed in HWE and SWE at 110 ◦C is a shorter exposure to the heat treatment as compared to the higher reaction temperatures. The L\* value then decreased when the reaction temperature increased [32]. The L\* values of hydrolysates obtained in this study ranged from 32.43 to 54.47 and 23.64 to 56.18 for *C. racemosa* and *U. lactuca*, respectively. It can be seen that L\* values were remarkably lower in hydrolysates obtained by SWE at higher temperatures (*p* < 0.05), which showed the significant effect of the temperature and hydrolysis process on L\* values. Accordingly, redness (a\*) and blueness (b\*) values were higher as the temperature of SWE increased, and the lowest values were obtained from the HWE process. There was a significant difference due to the hydrolysis process (*p* < 0.05). The chroma (C\*) value indicates the degree of saturation of color and is proportional to the strength of the color. In this study, we found changes in variations in C\* values between HWE and SWE. In addition, the C\* values also varied at different hydrolysis temperatures (*p* < 0.05). The highest C value was found for seaweed hydrolysates obtained by HWE. In addition, hue angle (H\*) is another parameter often used to determine the color of hydrolysates. In our study, we found that H\* values of seaweed hydrolysates obtained by SWE at higher temperatures (190 ◦C and 230 ◦C) were higher than those of HWE and SWE at lower temperatures (*p* < 0.05). Our results showed that the hydrolysis process especially by SWE at higher temperatures gives greater a\*, b\*, C\* and H\* to the seaweed hydrolysates. Pourali et al. (2010) reported that dark color following the SWE process might be correlated with the formation of 5-hydroxymethyl-2-furfural (HMF) and soluble polymers from the decomposition of the produced soluble sugars in a subcritical medium [33]. In addition, the dark color observed at higher temperatures is also attributed to the formation of undesired materials undergoing the Maillard reaction products (MRPs). The UV absorbance at 420 nm is often used to monitor the browning intensity caused by brown polymeric substances, such as melanoidins, which are formed at the final phase of MRPs [34]. Temperature is an important parameter of MRPs, as increasing the temperature could reduce the surface tension and viscosity of water, which resulted in an enhanced solubility of the analytes in the solvent, which further increased reaction rate. As demonstrated in Table 4, compared to the HWE, the MRPs levels were increased under the SWE process. The MRPs product level was the highest under SWE extraction conditions of 230 ◦C (*p* < 0.05). This subset of MRPs contributes to the coloration of many processed products. The intensity of brown color of these extracts increased with elevation

in temperature, supporting the occurrence of MRPs during the SWE process. The MRPs provide a unique aroma and changes in food quality parameters. The process could be indicated from the appearance of the extract, as the color of extracts turned dark brown at temperatures above 150 ◦C. Interestingly, in our study, we noticed a burning odor in the seaweed hydrolysates obtained by SWE at an extraction temperature above 150 ◦C. Similar observations (in terms of solution color and odor) have been reported in several studies [34,35].


**Table 3.** Color characteristics of green seaweed hydrolysates.

Abbreviations: HWE: hot water extraction; SWE: subcritical water extraction; L\*: lightness; a\*: red/green coordinate; b\*: yellow/blue coordinate; C\*: chroma; H\*: hue. Values correspond to mean ± SD from three independent experiments. Different letters (a–d, *a*–*d*) denote a statistically significant difference (*p* < 0.05).



Abbreviations: MRPs: Maillard Reaction Products; HWE: hot water extraction; SWE: subcritical water extraction. Values correspond to mean ± SD from three independent experiments. Values correspond to mean ± SD from three independent experiments. Different letters (a–d, *a*–*d*) denote a statistically significant difference (*p* < 0.05).

### *2.3. Total Protein, Sugars and Reducing Sugar Contents of C. racemosa and U. lactuca Hydrolysates*

The total protein contents in *C. racemosa* and *U. lactuca* hydrolysates obtained by HWE and SWE at various temperatures are provided in Figure 2A. In this study, we found that the protein contents of green seaweeds were not significantly different in HWE and SWE processes at extraction temperatures of up to 150 ◦C (*p* < 0.05). Interestingly, at higher temperatures (above 150 ◦C), the protein contents were increased dramatically. The highest protein yield (330.37 mg/g ± 5.46) was obtained from the *U. lactuca* hydrolyzed at 230 ◦C. Protein has low solubility at low temperature, due to robust aggregation via hydrophobic interactions [36,37]. When the temperature rises, the water ionization constant rises, increasing the protein yield observed in *C. racemosa* and *U. lactuca.*

**Figure 2.** Total protein (**A**), sugar (**B**) and reducing sugar (**C**) of green seaweed hydrolysates obtained by HWE and SWE. CR: *C. racemosa*; UL: *U. lactuca*. Values correspond to mean ± SD from three independent experiments. Different letters (a–e) denote a statistically significant difference (*p* < 0.05).

The total sugar values of *C. racemosa* and *U. lactuca* hydrolysates are shown in Figure 2B. The proportions of total sugars of *C. racemosa* and *U. lactuca* ranged from 56.88 to 93.04 mg/g and 51.67 to 258.95 mg/g, respectively. Total sugar of *C. racemosa* and *U. lactuca* with the highest content was obtained by SWE at 150 ◦C, with values of 93.04 ± 2.13 mg/g and 258.93 ± 2.71 mg/g, respectively. Compared to *C. racemosa, U. lactuca* showed higher total sugar contents. The sugar contents of *U. lactuca* found in this study are comparable to the total sugar contents of *U. lactuca* from Tunisia and Israel, which were 272 mg/g and 68.10 to 159.29 mg/g, respectively [38,39]. However, compared to Arctic *U. lactuca*, the sugar contents found in this study were slightly lower [40]. It was reported that the variations in total sugar contents could be affected by temporal or spatial variations in sugar contents of particular seaweed species, and also by methodological differences. In addition, we found that the levels of total sugars in both green seaweeds were decreased at temperatures above 150 ◦C. The hydrolysis of poly- or oligosaccharides and the degradation of monosaccharides caused by the high ionic product of solvent at elevated temperature under SWE conditions were thought to be the cause of the decrease in total sugar content [41]. Both *C. racemosa* and *U. lactuca* produce low amounts of reduced sugars when hydrolyzed by HWE or SWE at temperatures of up to 150 ◦C. The highest reducing sugar levels of both *C. racemosa* and *U. lactuca were* obtained by SWE at temperatures of 190 ◦C with values of 53.06 ± 3.65 mg/g 73.00 ± 5.15 mg/g, respectively. It was

demonstrated that reducing sugar content from the seaweeds polysaccharides by SWE increased up to certain reaction temperatures and then decreased [42]. The lower levels of reducing sugar may be correlated to the decomposition of sugar into other products, such as ketones and aldehydes, from which organic acids can be produced.

### *2.4. Phenolics, Saponins and Flavonoid Contents of C. racemosa and U. lactuca Hydrolysates*

Polyphenols are naturally present in plants such as seaweeds, which help them to eliminate free radicals. In this study, results for total phenolic (TPC), saponin (TSC) and flavonoid (TFC) *C. racemosa* and *U. lactuca* hydrolysates are shown in Figure 3. The values of TPC, TSC and TFC were represented as gallic acid equivalent (GAE), diosgenin equivalent (DE) and quercetin equivalent (QE), respectively. The values of TPC, TSC and TFC of *C. racemosa* and *U. lactuca* hydrolysates extracted by HWE and SWE at 110 up to 150 ◦C are low. However, as temperatures increased from 190 to 230 ◦C, the TPC, TSC and TFC of both *C. racemosa* and *U. lactuca* were significantly increased (*p* < 0.05). The highest TPC, TSC and TFC of both green seaweeds were obtained at reaction temperatures of 230 ◦C. The TPC, TSC and TFC values were obtained from *U. lactuca* hydrolyzed at 230 ◦C with the value of 39.82 ± 0.32 GAE mg/g, 13.22 ± 0.33 DE mg/g and 6.5 ± 0.47 QE mg/g, respectively. It has been reported that temperature is one of the most important factors affecting TPC, TSC and TSC in SWE process. In addition, it was reported that when the dielectric constant SWE decreases as the temperature rises, more nonpolar phenolics are being extracted [43].

**Figure 3.** Total phenolic (**A**), flavonoid (**B**) and saponin (**C**) contents of green seaweed hydrolysates were obtained by HWE and SWE. CR: *C. racemosa*; UL: *U. lactuca*. Values correspond to mean ± SD from three independent experiments Different letters (a–e) denote a statistically significant difference (*p* < 0.05).

Total phenolic contents of *C. racemosa* and *U. lactuca* hydrolysates are higher as compared to the TSC and TFC. Therefore, phenolic acid constituents from both green seaweed hydrolysates were quantified by HPLC. The contents of phenolic compounds in *C. racemosa* and *U. lactuca* hydrolysates were estimated based on the reference phenolic acid standards calibration curves. The main constituents of the phenolic acids present in *C. racemosa* and *U. lactuca* are summarized in Table 5. The phenolic acids with the highest levels in *U. lactuca* were gallic acid and vanillic acid. Interestingly, phenolic acids in both green seaweeds hydrolysates obtained by SWE generally increased at elevated temperatures up to 230 ◦C. However, a previous study reported a loss of phenolic acids, which were hydrolyzed using SWE at high temperatures (above 200 ◦C). Decreased phenolic acid levels in the SWE hydrolysates at elevated temperatures may be related to the conversion of phenolic acid into decarboxylation products and other gaseous products [44]. At elevated temperatures, phenolic compounds degraded much faster. Khuwijitjaru et al., (2014) demonstrated that only the chlorogenic, p-hydroxybenzoic, protocatechuic and syringic acids were present at 200 ◦C after 1h of SWE treatments. Notably, in this study, we found an increment in chlorogenic, p-hydroxybenzoic and protocatechuic acid at temperatures of 190 and 230 ◦C. It was reported that substituent groups on the ring structure of phenolic acids, such as amino, hydroxyl and methoxyl, acted as an activating group in the SWE process, assisting the thermal decarboxylation of benzoic acid derivatives [45].

**Table 5.** Phenolic acid constituents of green seaweed hydrolysates obtained by HWE and SWE (mg/g dry material).


Abbreviations: HWE: hot water extraction; SWE: subcritical water extraction; L: lightness; a: red/green coordinate; b: yellow/blue coordinate; C: chroma; H: hue. Values correspond to mean ± SD from three independent experiments. Different letters (a–d, *a*–*e*) denote a statistically significant difference (*p* < 0.05).

### *2.5. Potential of Cytotoxic and Antioxidant Activities of C. racemosa and U. lactuca Hydrolysates*

Potential cytotoxic effects *C. racemosa and U. lactuca* hydrolysates were tested at 50 μg/mL using MTT cell viability assay in cultured macrophage (RAW 264.7 cells). As shown in Figure 4, all green seaweed hydrolysates obtained by HWE as well as SWE did not show any toxic effect on RAW 264.7 cells at tested concentrations after 24 h and 48 h of treatment (*p* < 0.05). These results showed that *C. racemosa* and *U. lactuca* hydrolysates were safe and non-toxic. Similar non-toxic properties of *C. racemosa* and *U. lactuca* aqueous extracts have been reported by previous studies [46,47]. These results showed the potential of *C. racemosa* and *U. lactuca* to be developed in nutraceutical and cosmeceutical products.

Seaweeds, including green seaweeds, have been continuously demonstrated to possess a wide range of bioactive materials as well as biological activities [10]. In this study, the antioxidant potential of green seaweed hydrolysates obtained by HWE and SWE was tested using ABTS radical scavenging and total antioxidant assays, which are represented as ascorbic acid equivalents (AAE) and trolox equivalents (TE), respectively. The antioxidant potentials of *C. racemosa and U. lactuca* hydrolysates are shown in Table 6. The antioxidant activity of both *C. racemosa* and *U. lactuca* reaches a maximum value with SWE at 230 ◦C. During the subcritical process at certain temperatures, solvents could extract more bioactive compounds that could not be extracted at lower temperatures and/or by conventional HWE. A temperature of 230 ◦C was found to be the most optimal condition to obtain

bioactive materials from *C. racemosa* and *U. lactuca* using SWE. It was reported that the potential antioxidant activity of *U. lactuca* could be attributed to the higher content of polyphenols, flavonoids, saponins and sulfated polysaccharides compounds, with a known ability to scavenge synthetic radicals in in vitro tests (i.e ABTS) [48]. In addition, in our previous study, we found that the antioxidant activities of seaweeds are strongly correlated with their phenolic contents [5]. The results of the present study demonstrated that green seaweed hydrolysates obtained by SWE could be effective and safe alternatives to fight against radicals. In addition, green seaweed hydrolysates could be used as effective sources for antioxidative nutraceutical and cosmeceutical ingredients. Furthermore, more attention has been raised about the use of natural antioxidants as "natural" entities in nutraceutical and cosmeceutical products [49,50]. These will increase the potency of seaweeds extracts obtained by green extraction methods in various industries since it is of natural origin and environmental friendly.

**Figure 4.** Effects of *C. racemosa* (**A**) and *U. lactuca* (**B**) on the viability of RAW 264.7 cells. HWE: hot water extraction; SWE: subcritical water extraction. Results are the percentage of three independent experiments and are shown as the percentage of viable cells compared with the viability of untreated cells. Values correspond to mean ± SD from three independent experiments.


**Table 6.** Antioxidant activity of green seaweed hydrolysates.

Abbreviations: HWE: hot water extraction; SWE: subcritical water extraction; AAE: ascorbic acids equivalents; TE: trolox equivalents. Values correspond to mean ± SD from three independent experiments. Different letters (a–d, *a*–*e*) denote a statistically significant difference (*p* < 0.05).

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

### *3.1. Materials*

Two under-exploited green seaweed species (*C. racemosa* and *U. lactuca*) were collected from Tual, Southeast Maluku, in June 2018. A voucher specimen was deposited in Balai

Bioindustri Laut (BBIL), Lembaga Ilmu Pengetahuan Indonesia (LIPI) West Nusa Tenggara with the accession numbers of GSW-CR-180601 and GSW-UL-180602 for *Caulerpa racemosa* and *Ulva lactuca*, respectively. All the chemicals utilized in this study were obtained from Merck and Junsei Chemical Co., Ltd. (Tokyo, Japan) and were of analytical grade.

### *3.2. C. racemosa and U. lactuca Sample Preparation*

Both *C. racemosa* and *U. lactuca* were washed with clean water; sand debris and other dirt were gently removed. The green seaweeds were further oven-dried at 45 ◦C for 120 h. In the next step, dried green seaweeds were further freeze-dried and then powderized into a very fine particle (passed through a 0.71 mm siever). The green seaweeds were further kept at −20 ◦C prior to analysis.

### *3.3. Proximate Analysis of C. racemosa and U. lactuca*

The protein, ash, lipid, protein and moisture contents of *C. racemosa* and *U. lactuca* were measured according to the Association of Official Analytical Chemists methods [51]. Further, total carbohydrate content was estimated by subtracting the total mass of green seaweeds from the sum of other proximate contents.

### *3.4. Fatty Acid Composition Analysis of C. racemosa and U. lactuca*

The fatty acid composition of *C. racemosa* and *U. lactuca* were determined using a Fatty Acid Composition Analysis (Agilent Technologies, Wilmington, NC, USA) gas chromatograph with a fused silica capillary column (Supelco, Bellefonte, PA, USA). Methylation of fatty acids (fatty acid methyl esters; FAMEs Supelco, Bellefonte, PA, USA) were prepared according to The American Oil Chemists' Society's protocols. The oven temperature was turned on at 130 ◦C and run for 180 s, and then the temperatures were increased up to 240 ◦C at a rate of 4 ◦C/min and then maintained at 240 ◦C for 600 s. Both the injector and the detector were set to 250 ◦C. The FAMEs were identified by comparison of retention time with a standard fatty acid methyl ester mixture (Supelco, Bellefonte, PA, USA).

### *3.5. Sample Extraction*

### 3.5.1. Hot Water Extraction of Green Seaweeds

Fine powder of *C. racemosa* and *U. lactuca* was mixed with distilled water at normal pH (7.2) with the sample to solvent ratios of 1:40 (*w/v*). The mixtures were kept at 100 ◦C and agitated (200 rpm) for 2 h. The hydrolysate obtained after HWE processes was filtered and freeze-dried.

### 3.5.2. Subcritical Water Extraction (SWE) of Green Seaweeds

The SWE was operated in a continuous-type subcritical water system (Phosentech, South Korea). Fine powder of *C. racemosa* and *U. lactuca* was added into the reactor with distilled water at normal pH (7.2) at 1:40 ratios (*w/v*). The chamber was sealed tightly, purged with nitrogen gas and kept at the desired reaction temperature, pressure and speed (200 rpm) for 10 min. Hydrolyzed green seaweeds were immediately collected after the reaction was terminated and filtered with 0.45 μm membrane filter. The hydrolysate obtained after SWE processes was freeze-dried.

$$\text{Yield } (\%) = \frac{\text{Whyd}}{\text{W0}} \times 100 \,\% \tag{1}$$

where Whyd is the weight of freeze-dried hydrolysate and W0 is the initial weight of green seaweeds.

### *3.6. Physical Properties of C. racemosa and U. lactuca (Color, pH and Maillard Reaction Products (MRPs))*

Color properties of *C. racemosa* and *U. lactuca* hydrolysates were measured using a chromameter (Lovibond RT Series, Amesbury (Wiltshire), UK) [5]. The color characteristics of *C. racemosa* and *U. lactuca* were distinguished based on lightness value (*L*\*), redness

value (*a*\*) and yellowness value (*b*\*). Chroma meter was standardized each time with black and white references prior to analysis. The hue angle (*h\*ab*) and chroma (*C\*ab*) of *C. racemosa* and *U. lactuca* hydrolysates were calculated based on the following equations:

$$H^{\circ} = \tan^{-1} \left( \frac{b\*}{a\*} \right) \tag{2}$$

$$C \ast ab = \sqrt{\left(a \ast\right)^2 + \left(b \ast\right)^2} \tag{3}$$

Following the hydrolysis process, *C. racemosa* and *U. lactuca* were filtered and cooled down, and then pH was measured by using a pH meter (Mettler-Toledo, Greifensee, Switzerland). The MRPs were determined through the UV absorbance of samples, as described previously [52]. After the hydrolysis processes, 0.2 mL of filtered *C. racemosa* and *U. lactuca* (1 mg per mL) was measured at 294 and 420 nm.

### *3.7. Total Protein, Total Sugar and Reducing Sugar of C. racemosa and U. lactuca*

The protein concentrations of *C. racemosa* and *U. lactuca* hydrolysates were determined following Lowry's method. The *C. racemosa* and *U. lactuca* hydrolysates (0.2 mL) were mixed with CuSO4 reagent at 1:10 ratios (*v/v*) and vortexed. After incubation for 600 s at RT, 0.2 mL of 0.2 N Folin–Ciocalteu reagent (FCR) was loaded into the mixture and incubated for another 0.5 h. Total protein was determined through the UV absorbance of samples at 660 nm, and bovine serum albumin was used as the reference standard.

The total sugar value of *C. racemosa* and *U. lactuca* hydrolysates was measured based on the phenol sulfuric acid method. The *C. racemosa* and *U. lactuca* (0.2 mL) were mixed with 5% phenol (0.2 mL) and sulfuric acid (H2SO4; 1 mL) and 0.5 h at 100 ◦C. The total sugar was determined through the UV absorbance of samples at 490 nm, and C6H12O6 was used as the reference standard.

Reducing sugar analyses of *C. racemosa* and *U. lactuca* hydrolysates were measured by using the 3,5-dinitrosalicylic (DNS) acid method with slight modifications. *C. racemosa* and *U. lactuca* hydrolysates (0.5 mL) were mixed with DNS reagent solution at 1:1 ratios. The mixtures were then incubated at 95 ◦C for 15 min. After incubation, 0.5 mL of KNaC4H4O6·4H2O (40%) was added. Reducing sugar was determined through the UV absorbance of samples at 575 nm, and C6H12O6 was used as the reference standard.

### *3.8. Total Flavonoid Content (TFC), Total Phenolic Content (TPC) and Total Saponin Content (TSC) of C. racemosa and U. lactuca*

The TFC of *C. racemosa* and *U. lactuca* hydrolysates were measured according to previous methods [53]. Green seaweed hydrolysates (0.2 mL) were mixed with 0.4 mL of H2O and 0.2 mL of 5% NaNO2 and incubated at RT for 10 min. Following incubation periods, 10% AlCl3 (0.03 mL) and 1 M NaOH (0.4 mL) were added. The mixtures were loaded onto 96-well plates, and the absorbance was measured at 510 nm using multimode microplate readers. Quercetin (Q) was used as the reference standard for flavonoids. The original reaction solution was used to convert the value of the diluted samples. The final results were given in mg Q equivalent/g dry weight (mg Q/g DW).

The TPC of *C. racemosa* and *U. lactuca* hydrolysates was measured using FCR methods [54]. The *C. racemosa* and *U. lactuca* (0.5 mL) were mixed with 0.2 N FCR solution (0.5 mL) and kept in the dark at RT for 10 min. A 7.5% mixture of Na2CO3 was added (0.5 mL) and kept in the dark at RT for 2 h. The TPC was determined through the UV absorbance of samples at 765 nm, and the final values were expressed as mg phloroglucinol equivalent/g dry weight (mg/g DW).

The TSC of *C. racemosa* and *U. lactuca* hydrolysates was measured using the methods described previously with slight modifications. *C. racemosa* and *U. lactuca* were placed into tubes, at volumes with MeOH at 80% and 0.25 mL; 0.25 mL of 8% vanillin reagent and 2.5 mL H2O4S (72%) were added. The mixtures were mixed properly and kept at 60 ◦C for 10 min. After 10 min, the mixtures were transferred into ice. The TSC was determined through the UV absorbance of samples at 544 nm, and the final values were expressed as mg diosgenin equivalent/g dry weight (mg/g DW).

### *3.9. High-Performance Liquid Chromatography (HPLC) Analysis of C. racemosa and U. lactuca Hydrolysates*

The *C. racemosa* and *U. lactuca* hydrolysates were further analyzed for phenolic acid compositions using the HPLC system (Hitachi America Ltd., White Plains, NY, USA) on a Nucleosil C8 column (Macherey-Nagel, Düren, Germany) with linear gradients of solvent A (H2O with 0.1% CH3COOH) and solvent B (C2H3N with 0.1% CH3COOH) at a flow rate of 1 mL per min. The elution peaks were detected at 280 nm. The HPLC peak was confirmed with the reference phenolic acids and expressed as mg/g DW.

### *3.10. Antioxidant Activity*

3.10.1. 2,2-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) Scavenging Assay

The 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) (7 mmol/L) and K2S2O8 (2.45 mmol/L) were prepared in a separate bottle, and both solutions were kept in the dark at RT. After 24 h, both solutions were mixed. The radical mixtures were diluted with MeOH to obtain absorbance values of 0.72. The *C. racemosa* and *U. lactuca* hydrolysates were mixed with ABTS radical mixture at 1:5 ratios (*v/v*). The ABTS scavenging activity was determined through the UV absorbance of samples at 734 nm, and MeOH was used as the negative control. In comparison, different concentrations of ascorbic acid were used as standard and evaluated. The results are expressed in terms of AAE.

### 3.10.2. Total Antioxidant Capacity (TAC)

The *C. racemosa* and *U. lactuca* hydrolysates (0.1 mL) were mixed with 3 mL of radical mixture consist of 0.6 M H2SO4, 28 mM Na3PO4 and 4 mM (NH4)6Mo7O24. The mixtures were maintained at 95 ◦C for 180 min. The total antioxidant activity was determined through the measurements of UV absorbance at 695 nm, and MeOH was used as negative control. In comparison, different concentrations of trolox were used as standard and evaluated. The results are expressed in terms of TE.

### *3.11. Effects of Seaweeds Hydrolysates on Cell Viability*

Cytotoxic effects of *C. racemosa* and *U. lactuca* hydrolysates were determined by MTT reduction assay [55]. First, macrophage (RAW 264.7) cells were seeded into cell culture plates at a cell density of 2 × <sup>10</sup><sup>4</sup> cells/well in serum-free DMEM. The *C. racemosa* and *U. lactuca* hydrolysates (50 μg/mL) were then loaded in the cell culture and then incubated for 24 h. One hundred microliters of an MTT (0.5 mg/ml) solution was loaded into the cultures, and incubation was continued for another 240 min. MTT was used as an indicator of cell viability through its mitochondrial reduction to formazan [56]. The absorbance was measured at 540 nm by using a microplate reader. RAW 264.7 cell viability was calculated by comparison of the absorbance of the control group with treated groups.

### *3.12. Statistical Analysis*

The data were presented as means ± SD (*n* = 3). Differences between the means of the individual groups were assessed by one-way ANOVA with Duncan's multiple range tests. Differences were considered significant at *p* < 0.05. The statistical software package, SPSS v.16 (SPSS Inc., Chicago, IL, USA), was used for the analysis.

### **4. Conclusions**

Green seaweed hydrolysates, *C. racemosa* and *U. lactuca,* were prepared via HWE and SWE. Compared to HWE, the SWE process showed higher extraction yields, bioactive compounds and antioxidant activities of seaweed hydrolysates. In addition, six phenolic acids, including gallic acids, chlorogenic acid, gentisic acid, procatechuic acid, *p*-hydroxybenzoic acid and vanillic acid were identified in *U. lactuca* hydrolysates obtained by SWE at 230 ◦C. The SWE-enabled recovery of bioactive compounds from *C. racemosa* and *U. lactuca* with hydrolysis temperature at 230 ◦C was found to be the most optimum conditions to obtain bioactive materials with good radical scavenging activities, making it a potential candidate for antioxidant compounds. Collectively, this study provides the foundations for exploring under-exploited tropical green seaweeds and filling the gaps for future research in the development of nutraceuticals and cosmeceuticals from sea grape and sea lettuces.

**Author Contributions:** Conceptualization, R.P.; methodology, R.P., M.H.; analysis, R.P., M.H.; resources, P.R.; writing—original draft preparation, R.P.; writing—review and editing, R.P., P.R., M.H., B.-S.C.; supervision, B.-S.C.; project administration, B.-S.C., P.R.; funding acquisition, B.-S.C., P.R. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was supported by the National Research Foundation (Rep. of Korea) and National Research Priority (PRN)-MALSAI from the Indonesian Institute of Sciences (LIPI), National Research and Innovation Agency (BRIN) and Indonesia Endowment Fund for Education (LPDP), Rep. of Indonesia.

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

**Informed Consent Statement:** Not applicable.

**Acknowledgments:** The authors would like to thank the National Research Foundation (South Korea) for the Postdoctoral fellowship awards and Pukyong National University for the research supports. The authors also acknowledge Deputy for Earth Science LIPI (2015-2019) Zainal Arifin, and Chairman of Research Organizations for Earth Sciences BRIN Ocky K. Radjasa for all the support.

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

### **References**


## *Article* **Marine Ingredients for Sensitive Skin: Market Overview**

**Marta Salvador Ferreira 1,2,†, Diana I. S. P. Resende 3,4,†, José M. Sousa Lobo 1,2, Emília Sousa 3,4 and Isabel F. Almeida 1,2,\***


**Abstract:** Marine ingredients are a source of new chemical entities with biological action, which is the reason why they have gained relevance in the cosmetic industry. The facial care category is the most relevant in this industry, and within it, the sensitive skin segment occupies a prominent position. This work analyzed the use of marine ingredients in 88 facial cosmetics for sensitive skin from multinational brands, as well as their composition and the scientific evidence that supports their efficacy. Marine ingredients were used in 27% of the cosmetic products for sensitive skin and included the species *Laminaria ochroleuca*, *Ascophyllum nodosum* (brown macroalgae), *Asparagopsis armata* (red macroalgae), and *Chlorella vulgaris* (microalgae). Carotenoids, polysaccharides, and lipids are the chemical classes highlighted in these preparations. Two ingredients, namely the *Ascophyllum nodosum* extract and *Asparagopsis armata* extracts, present clinical evidence supporting their use for sensitive skin. Overall, marine ingredients used in cosmetics for sensitive skin are proposed to reduce skin inflammation and improve the barrier function. Marine-derived preparations constitute promising active ingredients for sensitive skin cosmetic products. Their in-depth study, focusing on the extracted metabolites, randomized placebo-controlled studies including volunteers with sensitive skin, and the use of extraction methods that are more profitable may provide a great opportunity for the cosmetic industry.

**Keywords:** marine ingredients; algae; sensitive skin; cosmetics

### **1. Introduction**

The largely unexplored marine environment harbors unique biodiversity and represents the vastest resource for the discovery of novel chemical entities with novel modes of action that cover a biologically relevant chemical space. These new scaffolds derived from various marine organisms offer valuable bioactive properties with great relevance in medical, pharmaceutical, and cosmetic fields [1–6]. Although synthetic strategies towards natural products have evolved tremendously over the last years, natural marine products are still preferred against their synthetic counterparts since they have better physicochemical, biochemical, and rheological characteristics, maintaining their stability at different pH and temperature ranges [7]. Among marine organisms, algae are recognized as one of the richest sources of new bioactive compounds [7]. The unique diversity of bioactive compounds contained in algae, such as vitamins, minerals, amino acids, sugars, lipids, and other biologically active compounds, is translated into numerous attractive properties for various industries [8], including the food, pharmaceutical, and cosmetic industries, as evidenced by the appearance in the market of various cosmetic products derived from these

**Citation:** Ferreira, M.S.; Resende, D.I.S.P.; Lobo, J.M.S.; Sousa, E.; Almeida, I.F. Marine Ingredients for Sensitive Skin: Market Overview. *Mar. Drugs* **2021**, *19*, 464. https://doi.org/10.3390/md19080464

Academic Editors: María Lourdes Mourelle, Herminia Domínguez and Jose Luis Legido

Received: 2 August 2021 Accepted: 14 August 2021 Published: 17 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/).

compounds [9]. Cosmetic products are stable substances or substance mixtures intended to clean, protect, perfume, and/or change the appearance of the external parts of the human body, teeth, and mucous membranes of the oral cavity, keeping them in good condition or correcting body odors [10]. They result from a formulation of raw materials which are categorized as active ingredients, excipients, and additives [11]. Cosmetic products may be categorized as body care, hair care, sun care, decorative cosmetics, oral care, and skin care, which is the largest cosmetic product category worldwide [12,13]. Skin care comprises a wide variety of products that should meet expectations of consumers with different skin types and organoleptic preferences. Sensitive skin is a condition characterized by multiple symptoms such as tightness, stinging, burning, or pruritus, which affects about 71% of the general adult population, being more frequent in the facial area [14–16]. Erythema, dryness, and desquamation are typically absent, but they may also occur [17]. Therefore, the sensitive skin segment allows meeting the needs of consumers who suffer from this condition [18]. Sensitive skin manifests in the presence of stimuli such as cold, heat, sun, pollution, cosmetics, or moisture which are not expected to produce unpleasant sensations, and the pathophysiological mechanisms involved in sensitive skin remain unknown [18]. Genetics, poor mental health, and microbiome imbalances have been proposed as contributing factors for this condition [19–21]. There are three hypotheses appointed in scientific literature for explaining the pathophysiology of this condition, namely the hyperactivity of the somatosensory and vascular systems, increased stratum corneum permeability, and an exacerbated immune response [22]. The hypothesis of an abnormal response from the somatosensory system is gaining increasing relevance. The skin contains sensory nerve fibers which are activated upon contact with physical and chemical stimuli such as heat, low pH solutions, or known irritants such as capsaicin, resulting in the release of neuropeptides, namely substance P or calcitonin gene-related peptide (CGRP). These neuropeptides cause a burning pain sensation through the activation of keratinocytes, mast cells, antigen-presenting cells, and T cells [23]. Neurosensory defects may lead to abnormalities in the communication with the central nervous system, resulting in a lower sensitivity threshold [15,16]. For example, an overexpression of transient receptor potential vanilloid type 1 (TRPV1), which is activated by heat and capsaicin, is thought to be involved in the pathophysiology of sensitive skin by increasing neuronal excitability [24,25]. Moreover, vascular hyperreactivity has been proposed in the pathophysiology of sensitive skin despite the absence of skin erythema [26]. The immune system is also associated with sensitive skin due to its interaction with nerve fibers, producing neurogenic inflammation. Neuropeptides activate keratinocytes, mast cells, antigen-presenting cells, and T cells, resulting in an inflammatory response [25]. Conversely, defects in the skin barrier function may be due to a derangement of intercellular lipids, due to a decrease in the ceramide content, a thinner stratum corneum with smaller corneocytes, as well as lower levels of pyrrolidone carboxylic acid (PCA) from the natural moisturizing factor (NMF), bleomycin hydrolase (BH), which is responsible for profilaggrin conversion, and transglutaminase (TG), which is essential for catalyzing the cross-linking between proteins and lipids during the corneocyte maturation process [22,27,28]. This may result in an increased permeability of the stratum corneum, which allows for the penetration of environmental aggressors [20]. More recently, this hypothesis has been questioned due to a study which failed to find significant differences in stratum corneum thickness, fatty acids, and the ceramide content, transepidermal water loss, or natural moisturizing factors between individuals with or without sensitive skin [29].

Recently, we have characterized the trends in the use of peptides in the sensitive skin care segment, reviewing their synthetic pathways and the scientific evidence that supports their efficacy [30]. We were able to conclude that three out of seven peptides have a neurotransmitter-inhibiting mechanism of action, while another three are signal peptides. As an example, palmitoyl tripeptide-8 may be a prime candidate for the development of pharmaceuticals aimed at alleviating the signs and symptoms of rosacea [30].

Given the variety of molecular targets involved in the sensitive skin pathophysiology, the chemical diversity of the marine ecosystem is a promising source for cosmetic ingredients for managing its symptoms. Our group has previously analyzed the market impact of marine ingredients in anti-aging cosmetics from multinational brands [31]. However, the use of these active ingredients in cosmetic products for sensitive skin remains unexplored. This study aims to unveil the state-of-the-art of marine ingredients in this segment by documenting their prevalence, as well as the most relevant species, their composition, and the scientific evidence that supports their efficacy for sensitive skin care.

### **2. Trends in the Use of Marine Ingredients in Cosmetic Formulations for Sensitive Skin**

The analysis of the presence of marine ingredients in all of the studied 88 cosmetic formulations for sensitive skin (19 multinational brands) indicated that 27% of them contain marine-derived ingredients. Interestingly, a more detailed analysis regarding the origin of these ingredients (Table 1) revealed that they all derive from algae, mainly macroalgae. Although over the last decades mariculture and aquaculture techniques have been developed towards the sustainable supply of other marine organisms, such as fish, sponges, corals, mollusks, echinoderms, *Artemia*, plankton, and microorganisms [5,11,32–38], their potential is not translated in the number of cosmetic formulations for sensitive skin that have been commercialized in the Portuguese market and contained the referred ingredients. The constraints related to reduced biomass availability of these marine organisms and difficulties regarding their production/cultivation at larger scales still represent a major bottleneck in the sustainable supply of the desired natural ingredients for the cosmetic industry [32].

**Table 1.** Analysis of the prevalence and categorization of marine ingredients from the analyzed cosmetic products for sensitive skin (2019).


<sup>1</sup> INCI—International Nomenclature of Cosmetic Ingredients.

On the other hand, algae are emerging as one of the most promising long-term, sustainable sources of bioactive ingredients to be used in the formulation of cosmetic and skin care products, with a large number and wide variety of benefits associated with their secondary metabolites [2]. Their biodiversity, easy cultivation, and growth modulation are the main reasons for their increased use in a variety of industries [2]. Depending on their size, they can be divided into macroalgae, which are seaweeds and other benthic marine algae that are generally visible to the naked eye, and microalgae, which require a microscope to be observed [39]. Additionally, macroalgae can be divided into three groups based on their dominant pigments: Rhodophyceae (red algae), Phaeophyceae (brown algae), and Chlorophyceae (green algae) [40]. Bioactive substances derived from these algae have diverse functional roles as a secondary metabolite and these properties can be applied to the development of novel cosmetic products. Brown algae account for approximately 59% of the total macroalgae cultivated in the world, followed by red algae at 40% and green algae at less than 1% [40]. Hence, it is interesting to notice that the wider availability of brown and red algae is clearly translated to their use as ingredients amongst the 88 studied cosmetic formulations for sensitive skin (Figure 1). Additionally, red algae are represented in 17% of the cosmetic formulations, and none of them contained green algae, probably due to their limited availability and, therefore, associated cost to the cosmetic industry. Microalgae are also well-represented, with 13% of the studied formulations containing these marine ingredients (Figure 1).

**Figure 1.** Categorization of the marine ingredients present in cosmetic formulations for sensitive skin commercialized in the Portuguese market (2019).

For several years, the use of the designation "algae extract" as a marine ingredient present in several cosmetic formulations with no specification of the species was permitted and included in the European Commission database for information on cosmetic ingredients contained in cosmetics (CosIng) [41]. Nowadays, the Commission requires that the new name assignment should be based on the current genus and species name of the specific alga. However, for an interim period of time, trade name assignments formerly published with the INCI name "algae extract" were retained. In this study, 2.3% of the 88 studied cosmetic formulations contained algae extract as a marine ingredient (Table 1, marked in Figure 1 as "undefined"); since the type of algae was not specified, a further detailed analysis could not be performed.

### **3. Efficacy of Algae-Containing Formulations on Sensitive Skin**

**Figure 2.** Flowchart of the selected articles according to four different parts of the search process: identification, screening, eligibility, and inclusion.

### *3.1. Brown Macroalgae*

Brown seaweeds belonging to two different taxonomic orders, Fucales (*Ascophyllum Nodosum*) and Laminariales (*Laminaria ochroleuca*), were used as ingredients in 17% of the studied cosmetic formulations. The brown color presented by these species results from the dominance of the pigment fucoxanthin (Figure 3), which masks the other pigments (chlorophyll *a* and *c*, β-carotene, and other carotenoids) and, as reserve substances, oils and polysaccharides [31]. The main polysaccharide found in the brown seaweeds is alginic acid, while laminarins (up to 32–35% dry weight) and fucoidans appear as sulfated polysaccharides (Figure 3).

**Figure 3.** Bioactive constituents of brown seaweeds.

Although these main constituents are common to both taxonomic orders Fucales and Laminariales, studies focused on the discovery of other secondary metabolites of *Ascophyllum nodosum* and *Laminaria ochroleuca* complemented with studies on the biological activity of these metabolites have also been developed and are analyzed below. The scientific and marketing evidence of the application of active ingredients from *Ascophyllum nodosum* and *Laminaria ochroleuca* in cosmetic formulations for sensitive skin was also compiled and analyzed.

*Laminaria ochroleuca* is a yellow brown digitate kelp presently distributed from Morocco to southwest England in the United Kingdom [42]. This species is highly sensitive to temperature, which models their growth and performance, and the recent ocean warming has led to a proliferation of *Laminaria ochroleuca* by extension of their geographical ranges to new habitats [43]. Due to these temperature and geographical changes, along with other variables such as habitat, season of harvesting, and environmental conditions (light, temperature, and salinity), this species experiences major shifts in their composition. An interesting study on the effect of different harvesting times, depths, and growth conditions of *Laminaria ochroleuca* revealed considerable differences in both qualitative and quantitative pigment profiles [44]. Significant seasonal variations in the photosynthetic pigment composition of *Laminaria ochroleuca* were observed which point to the occurrence of a photoprotective mechanism in the algae that deflects energetic resources to pigment biosynthesis. The samples collected in months with higher sun exposure (June–October) exhibited higher amounts of zeaxanthin, β-carotene, and chlorophyll *c* (Figure 4), with some species presenting nearly twice the levels of pigments, amongst which carotenoids were the most prevalent (56.1% of the total quantified) [44]. Another study dealing with the determination of phenolic compounds in *Laminaria ochroleuca* for human consumption revealed epigallocatechin (Figure 4) as the main polyphenol (760.2 ± 5.2 μg/g dry weight), followed by epicatechin (28.7 ± 2.0 μg/g dry weight), catechin gallate (21.4 ± 5.7 μg/g dry weight), epicatechin gallate (11.2 ± 1.6 μg/g dry weight), and epigallocatechin gallate (9.7 ± 1.3 μg/g dry weight) [45]. These polyphenols have been shown to provide an antioxidant, anti-inflammatory, and UVB protective action [46,47]. Other phenolic derivatives include linear phlorethols, containing either ortho, meta-, or para-oriented (or even a com-

bination) C–O–C oxidative phenolic couplings (Figure 4) as exemplified in tetraphlorethols A and B [48].

**Figure 4.** Bioactive metabolites of *Laminaria ochroleuca*.

A similar study was performed regarding fatty acid patterns of *Laminaria ochroleuca* [49]. This species exhibits a complex fatty acid profile, characterized mainly by the presence of medium and long fatty acyl chains (14–22 carbon atoms), with different degrees of unsaturation. The specimens from winter exhibited the lowest fatty acid concentrations (1255–1477 mg/kg of dry algae) whereas those harvested in warmer months presented higher fatty acid levels (1760 mg/kg of dry algae) [49].

Extracts of *Laminaria ochroleuca* have been incorporated in makeup, cleansers, moisturizers, and self-tanners, among other cosmetic products [50]. This extract is considered a natural skin soothing ingredient on several levels since it acts as an anti-inflammatory agent for skin irritations by boosting the skin's immune response and protects the DNA from UV damage [51].

One raw materials supplier performed a transcriptomic analysis by mRNA extraction and evaluation of expression makers by RT-qPCR on reconstituted human epidermis (RHE model, 11 days old), after a single application of a lipidic *Laminaria ochroleuca* extract (3 mg/cm2) for 24 h [52]. An increase in the expression of proteins from the innate immune system was found, namely for toll-like receptor 4 (TLR 4), psoriasin (S 100 A7), RNAse 7, as well as upregulation of the enzymes linked to cellular homeostasis and oxidative stress, metallothioneins 1 (MT-1) and extracellular superoxide dismutase (SOD). Moreover, there was downregulation in the expression of proinflammatory cytokines IL-1α and IL-6, metalloproteinases 1, 3, and 9 (MMPs), as well as in plasminogen activator urokinase (PLAU), which are involved in the dermis' extracellular matrix degradation.

The same supplier also performed a clinical study including 10 volunteers, who were exposed to a fixed irradiation dose of the minimal erythema dose (minimum dosage of radiation that produces skin erythema) × 1.5. Then, a gel formulation containing 2% *Laminaria ochroleuca* extract was applied to the test area, and another irradiated area was left untreated. The test area and the amount of product which was applied are not disclosed. Skin erythema was measured after product application and in the next 30, 60, and 120 min. The gel reduced skin erythema by 6.07% after 30 min, presenting the greatest difference in comparison to the control, and it kept reducing skin erythema over time. Statistical significance was not assessed.

Another lipidic *Laminaria ochroleuca* extract was evaluated by a distinct raw materials supplier regarding its biological activity in in vitro studies using reconstituted skin which was subject to epidermal trauma. After the extract application, an anti-inflammatory effect was observed through the inhibition of IL-1α and IL-6, as previously stated [52], but also through PGE2 release by epidermal cells and corneocyte degradation reduction, improving epidermal quality. Moreover, there was an increase in epidermal lipid content through phosphatidylcholine deposition, which contributes to reinforcing the epidermal barrier, thus reducing the penetration of environmental aggressors [53].

The anti-inflammatory activity of a lipidic *Laminaria ochroleuca* extract (Antileukine 6), which is mainly composed of phosphatidylcholine (Figure 4) derivatives, was evaluated in a murine model (C57BL/6 mice) [53,54]. Both ears were pretreated for 3 days twice a day with a *Laminaria ochroleuca* extract (2% in acetone/olive oil (4:1)) or the vehicle alone. Then, skin inflammation was induced by the application of 0.3% 2,4-dinitro-fluorobenzene (DNFB, hapten) in mouse ears, and the inflammatory response in terms of ear swelling (in μm) was scored at 0, 3, 6, 9, and 24 h in comparison with the vehicle applied at the other ear. The *Laminaria ochroleuca* extract reduced the inflammatory response as early as after 3 h, reaching the maximum effect at 6 h, with statistical significance, and showing a lasting effect up to 24 h. This anti-inflammatory effect may be due to the reduced DNFB penetration and/or a decrease in epidermal cytokines synthesis [54]. Having these results in mind, a *Laminaria ochroleuca* extract may be useful for reducing the symptoms associated with sensitive skin by improving the skin barrier function while modulating the neurogenic inflammation cascade by reducing the release of proinflammatory cytokines by mast cells, namely of IL-1 and prostaglandin E2 (PGE2). The metabolites which are responsible for these biological actions remain undisclosed.

*Ascophyllum nodosum* is an intertidal species characterized by its olive-brown fronds commonly detected around the periphery of the North Atlantic Ocean [55]. This intertidal fucoid has been extensively analyzed and studied for its chemical composition [55]. The most important constituents are the polysaccharides alginic acid, laminarins, and fucoidans (Figure 3), while other significant constituents like lipids, mannitol, ascophyllan, proteins, fibers, pigments, and phenols (Figure 5) [56–58], as well as vitamins, hormones, and enzymes are also present [55]. *Ascophyllum nodosum* has been used in bath oils, tablets, salts, as well as in skin cleansing and moisturizing cosmetics [50]. It has been shown to provide an antioxidant and photoprotective activity while inhibiting elastase and lipase [59–62]. While alginic acid or alginates have several applications in cosmetic formulations thanks to their thickening, gelling, emulsifying, and stabilizing abilities, fucoidans have been shown to reduce the intensity of the inflammatory response and promote a more rapid tissue healing, especially after wound or surgical trauma [55]. Fucoidans are fucans, sulfated polysaccharides with a fucose backbone, originating from seaweeds [39]. They have been shown to reduce the production of IgE by B cells which have been stimulated by allergens, thus blocking signals mediated by NFκB-p52. Furthermore, they have a free radical scavenging capacity, which may contribute to ameliorating skin inflammation [63,64]. Together, these properties make fucoidans a promising active ingredient for cosmetics intended to aid in the management of itching, stinging, and rashes [63]. One study evaluated the ability of this compound to reduce the inflammatory response using BALB/c mice as the murine model for atopic dermatitis and a DNFB solution (acetone/olive oil, 4:1) as the hapten [65]. Atopic dermatitis was induced in BALB/c mice by sensitization of the pre-shaved abdomen, with further challenge on the abdomen and ears after four, five, and nine days. Then, the treatment group received 50 μL of 0.2% fucoidan (from *Fucus vesiculosus*), while the negative control group received an acetone/olive oil vehicle, and the positive control group was given 0.1% dexamethasone. Fucoidan has been shown to ameliorate atopic dermatitis by decreasing inflammatory cell infiltration, splenocytes proliferation, and the CD4+ T cell response. However, fucoidans from *Ascophyllum nodosum* are distinct from those from *Fucus vesiculosus*, and their mechanisms of action are not expected to be effective on sensitive skin, based on what is known regarding the pathophysiology of this condition.

**Figure 5.** Bioactive metabolites of *Ascophyllum nodosum*.

Ascophyllan has been shown to inhibit MMP expression, reduce the production of NO, tumor necrosis factor-α (TNF-α), and granulocyte colony-stimulating factor (G-CSF) more markedly than fucoidan and provide an antioxidant action [66,67]. No studies were found regarding its benefits for sensitive skin or inflammatory conditions.

One raw materials supplier evaluated the efficacy of a cosmetic formulation containing *Ascophyllum nodosum* and *Asparagopsis armata* (red algae) extracts [68]. Keratinocytes were exposed to phorbol myristate acetate (PMA), a tumor promoter and proinflammatory substance, and incubated with 0.2% of the active ingredient (methods are not further described) [69]. The incubated keratinocytes have shown a very significant reduction both in the vascular endothelial growth factor (VEGF) and PGE2 levels. VEGF stimulates the growth and dilation of capillaries, which may result in increased skin redness [17]. This combination also inhibited MMP-2 activity in a dose-dependent manner, reaching 37% inhibition at the concentration of 0.5%. Additionally, a clinical study was performed by the same supplier. Fifty-six volunteers presenting wrinkles and dry sensitive skin applied a formulation with 0.4% of this ingredient twice a day for 28 days. Then, their perception of the products was registered. Reduced tingling sensations, improved resiliency, immediate relief, and skin comfort were reported by 58%, 59%, 70%, and 71% of the volunteers, respectively. Although these results reveal a potential application of this ingredient for sensitive skin, it is not possible to conclude that *Ascophyllum nodosum* can be useful for this purpose as the tested ingredient also contains the algae *Asparagopsis armata*.

### *3.2. Red Macroalgae*

*Asparagopsis armata* is a red seaweed which can be found in European coasts and in the Northeast Atlantic [70]. The main photosynthetic pigments of red algae are chlorophyll *a*, carotenoids (lutein, zeaxanthin, β-carotene) and phycobilins (phycocyanin and phycoerythrin), water-soluble pigments localized in the phycobilisomes, which give red algae their distinctive color [71]. Phycocyanin has been shown to provide anti-inflammatory, antioxidant, and wound-healing properties [72]. Besides photosynthetic pigments, red algae are

also constituted of other interesting bioactive compounds (Figure 6), including agar [39], sulfated polysaccharides (carrageenans and porphyrans) [73,74], and mycosporine-like amino acids (MAA) [75–78].

The wide practical uses of these polysaccharides are based on their ability to form gels in aqueous solutions and act as a stabilizer, being generally used in creams, sticks, soaps, shampoos, lotions, foams, and gels [79]. On the other hand, MAAs are used in cosmetic formulations due to their photoprotective potential, antioxidant and skin protective properties [9,80,81]. They constitute a group of low-molecular-weight watersoluble molecules that can absorb UV radiation and disperse the absorbed energy as heat without generating reactive oxygen species (ROS), being a natural promising UV-absorbing alternative [82]. Examples of the most abundant MAAs in red macroalgae are mycosporineglycine, shinorine, and porphyra-334 (Figure 6) [83]. The anti-inflammatory effects of these MAAs on the expression of genes associated with inflammation in response to UV irradiation was investigated using the human fibroblast cell line, HaCaT [82]. Mycosporineglycine was able to suppress the expression of an inflammation marker gene, COX-2, in a concentration-dependent manner [82,83].

One raw materials supplier reported the cytostimulatory action of an *Asparagopsis armata* extract on human fibroblasts (WI 38), reaching the maximum level at 0.1%. No further details are provided [84].

Other studies including the use of an *Asparagopsis armata* extract in cosmetic formulations for sensitive skin were already disclosed [68,69]. However, these formulations contain not only *Asparagopsis armata*, but also *Ascophyllum nodosum*, and were previously described in Section 3.1.

### *3.3. Microalgae*

Marine microalgae also constitute an innovative source of bioactive compounds such as polyunsaturated fatty acids, tocopherols and sterols, vitamins and minerals, antioxidants, and pigments (e.g., chlorophyll and carotenoids), with great relevance in medical, pharmaceutical, and cosmetic fields [85]. Due to their unicellular or simple multicellular structure, they can grow rapidly and live under harsh conditions and environmental stressors such as heat, cold, anaerobiosis, salinity, photooxidation, osmotic pressure, and exposure to ultraviolet radiation [85]. The microalgae usually commercialized and used in biotechnology belong to the green algae, Chlorophyceae (such as *Chlorella vulgaris*, *Haematococcus pluvialis*, *Dunaliella salina*, and cyanobacteria) [85]. Their composition varies according to species and culture environments such as light intensity, temperature, pH, salinity, and medium [86]. *Chlorella vulgaris* is mainly constituted by proteins (43–58%), lipids (5–58%), carbohydrates (12–55%), pigments (chlorophyll (1–2%) and carotenoids (0.4%, astaxanthin, lutein, β-carotene, lycopene, canthaxanthin, see Figure 7 for examples)), vitamins (vitamins A, B, C, and E), and minerals (calcium, potassium, magnesium, and zinc) [86].

**Figure 7.** Pigments of *Chlorella vulgaris*.

Sulfated polysaccharides from *Chlorella vulgaris* exhibited a capacity to prevent the accumulation and activity of free radicals and reactive chemical species, acting as protecting systems against these oxidative and radical stress agents [87]; in addition, peptides have been shown to reduce the matrix metalloproteinase-1 (MMP-1) expression in human skin cell fibroblasts, responsible for the breakdown of collagen [88]. The fact that a *Chlorella vulgaris* extract is able to stimulate collagen synthesis in the skin makes it suitable to be used in anti-aging cosmetics, as well as in wound-healing products [89,90].

Several studies report beneficial effects of extracts of *Chlorella vulgaris* for skin health. One study found that a *Chlorella vulgaris* extract was able to attenuate *Dermatophagoides farinae* (DFE)-induced atopic dermatitis (AD) in NC/Nga mice by oral administration [91], reducing 12-dimethylbenz[a]anthracene (DMBA)-induced tumor size and number by upregulating the sulfhydryl (-SH) and glutathione S-transferase (GST) levels in skin tissues [92]. These findings indicate *Chlorella vulgaris* could be useful as a preventive and therapeutic agent for various inflammatory skin diseases.

The evidence of the use of extracts of this microalga in cosmetic formulations for sensitive skin is limited. One cosmetic product was tested for its angiogenic inhibiting ability against positive and negative controls (suramide and VEGF, respectively) by using the in vitro model AngioKitTM (TCS Cellworks), which allows following the development of the angiogenic process [93]. The formulation contained rhamnose, shea butter, argan oil, polyphenols, dextran sulfate, *Laminaria digitata*, caprapenols, *Chlorella vulgaris*, glycosaminoglycans, and UV filters (SPF 20). The formulation presented an antiangiogenic effect comparing to the positive control in the concentration range of 0.7–0.8 mg/mL, thus being useful for patients presenting rosacea. In spite of these results and *Chlorella vulgaris*' potential to modulate the inflammatory response involved in sensitive skin, the composition of this formulation does not allow drawing conclusions regarding *Chlorella vulgaris*' efficacy for treating this condition.

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

### *4.1. Data Collection*

The composition of a pool of skin care facial cosmetic products from multinational manufacturers marketed in Portuguese parapharmacies and pharmacies was collected in 2019 in order to access the most used active ingredients in formulations for sensitive skin. Skin care products were included in the study if they exhibited in the label one of the following expressions: "sensitive skin" or "reactive skin" or "intolerant skin". All the information available in the product labels was collected, along with the information available on the manufacturers' websites.

### *4.2. Data Analysis*

The marine ingredients contained in cosmetic products for sensitive skin were listed according to the International Nomenclature of Cosmetic Ingredients (INCI). Afterwards, the data were analyzed with respect to the following parameters:

### 4.2.1. Marine Ingredients Use

The relative amount of cosmetic products for sensitive skin containing marine ingredients was evaluated and expressed in percentage.

### 4.2.2. Top Marine Ingredients for Sensitive Skin

Marine ingredients were identified from INCI lists and ranked in the descending order of occurrence to disclose the top. Their categorization was also performed based on their marine organism species.

### 4.2.3. Scientific Evidence Supporting the Efficacy of Marine Ingredients in Sensitive Skin Care

The efficacy data for each marine ingredient were searched in the online databases PubMed, Scopus, KOSMET, and SciFinder. Due to the lack of studies regarding the applicability of active ingredients in cosmetics for sensitive skin, a broader search was performed, using the keywords ("INCI name" OR "synonyms" when applicable) AND ("skin" OR "topical).

### **5. Conclusions**

Sensitive skin affects a significant proportion of the population worldwide, making it an appealing segment for the cosmetics industry. Marine organisms possess unique chemical pathways that are able to produce unprecedented scaffolds.

Marine ingredients were present in 27% of the analyzed cosmetic products for sensitive skin. Noteworthy, macroalgae are the prime marine ingredient used probably due to the easiness of cultivation allied with the development of a cutting-edge technology. These are easily cultivated either in a pond or a photobioreactor, in nonarable lands with minimal use of freshwater, or even in seawater or wastewater. It is also worth highlighting that among macroalgae, brown algae represent the main type of algae used in the analyzed cosmetic formulations.

Two preparations from brown algae (a *Laminaria ochroleuca* extract and an *Ascophyllum nodosum* extract), one—from red algae (an *Asparagopsis armata* extract), and one—from green microalgae (an *Chlorella vulgaris* extract) were found. The scientific evidence regarding the efficacy of these ingredients on sensitive skin is limited, especially due to the lack of clinical studies including volunteers with this condition. Noteworthily, there is one study that meets these requirements referring to a combination of an *Ascophyllum nodosum* extract and an *Asparagopsis armata* extract, which was found to reduce a tingling sensation, resiliency, and skin comfort in volunteers with sensitive skin. On the other hand, an *Laminaria ochroleuca* extract has a potential for improving the skin barrier function due to its lipid content and for reducing neurogenic inflammation by decreasing the release of pro-inflammatory cytokines by mast cells while increasing the production of antioxidant enzymes such as MT-1 and SOD. As for a *Chlorella vulgaris* extract, the in vivo evidence supporting its use in inflammatory conditions is still preliminary.

It is interesting to notice that efforts amongst the scientific community towards the identification of the active ingredient responsible for a certain property in the analyzed cosmetic formulation are still scarce. Usually, the entire extract is applied without further understanding of which chemical entity is associated with the bioactivity. Hence, research and development strategies should be employed both to identify the specific compounds responsible for the observed activities and determine their mechanisms of action. Among the chemical substances that can be found in these ingredients, carotenoids, sulfated polysaccharides, amino acids, and lipids are the most abundant. Of those, certain compounds usually isolated from marine organisms could be of interest for managing the symptoms of sensitive skin. Fucoidans from brown algae present evidence for managing inflammatory conditions, and they have been proposed to reduce itching and stinging symptoms. Additionally, mycosporine-like amino acids provide an antioxidant and antiinflammatory activity by modulating the expression of the fibroblasts' genes associated with inflammation. New strategies to increase the profitability of the extraction process are also needed in order to increase the cosmetic industry interest. Biotechnology may present advantages in this regard by reducing the environmental impact from the exploitation of these resources. The preliminary studies described herein are a major step towards the design of more innovative target-oriented ingredients by the cosmetic industry, providing efficacious products for sensitive skin. Overall, marine ingredients are already used in the sensitive skin segment, and they have a great potential to keep growing. Their in-depth study and the further investigation of other organisms, such as fish, sponges, corals, mollusks, echinoderms, *Artemia*, plankton, and microorganisms, constitute a great opportunity for formulators, cosmetic companies with R&D departments, and raw materials suppliers from the cosmetic industry.

**Author Contributions:** Conceptualization: I.F.A.; Data collection and analysis: M.S.F.; Writing original draft preparation and final manuscript: M.S.F. and D.I.S.P.R.; Supervision: J.M.S.L.; Writing review and editing: I.F.A. and E.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work is financed by national funds from FCT—Fundação para a Ciência e a Tecnologia, I.P., in the scope of the project UIDP/04378/2020 and UIDB/04378/2020 of the Research Unit on Applied Molecular Biosciences—UCIBIO and the project LA/P/0140/2020 of the Associate Laboratory Institute for Health and Bioeconomy—i4HB." This research was also supported by national funds through FCT (Foundation for Science and Technology) within the scope of UIDB/04423/2020, UIDP/04423/2020 (Group of Natural Products and Medicinal Chemistry—CIIMAR), and under the project PTDC/SAU-PUB/28736/2017 (reference POCI-01–0145-FEDER-028736), co-financed by COM-PETE 2020, Portugal 2020 and the European Union through the ERDF and by FCT through national funds, as well as structured program of R&D&I ATLANTIDA (NORTE-01-0145-FEDER-000040), supported by NORTE2020, through ERDF, and CHIRALBIO ACTIVE-PI-3RL-IINFACTS-2019.

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

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data sharing not applicable.

**Acknowledgments:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

**Limitations:** This study was performed for the Portuguese cosmetic market, which is dominated by multinational cosmetic brands. Therefore, this may result in discrepancies when comparing the data with other markets. Many ingredients found in cosmetic products from the market lack scientific literature regarding their efficacy. Therefore, some of the information used in this study was collected in technical documents and patents from suppliers.

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