**1. Introduction**

Among seaweeds, the brown macroalgae (Phaeophyceae) have been identified as an outstanding source of phenolic compounds, from simple phenolic acids to more complex polymers such as tannins (mainly phlorotannins). Algal phlorotannins, a group of phenolic compounds restricted to the polymers of phloroglucinol, present a heterogeneous and high molecular weight group of compounds (from 126 Da to 650 kDa) which are verified in terrestrial plants [1,2]. The phlorotannins play an important role in the cellular and ecological growth and tissue healing of alga but also show strong antioxidant, antimicrobial, cytotoxic, and antitumor properties [3–6].

Brown fucoid algae of the genus *Cystoseira sensu lato* (Sargassaceae) consist of 40 species of large marine canopy-forming macroalgae found along the Atlantic–Mediterranean coasts [7,8]. So far, a total of 214 compounds have been isolated from sixteen *Cystoseira*

**Citation:** Cagalj, M.; Skroza, D.; ˇ Razola-Díaz, M.d.C.; Verardo, V.; Bassi, D.; Frleta, R.; Generali´c Mekini´c, I.; Tabanelli, G.; Šimat, V. Variations in the Composition, Antioxidant and Antimicrobial Activities of *Cystoseira compressa* during Seasonal Growth. *Mar. Drugs* **2022**, *20*, 64. https://doi.org/ 10.3390/md20010064

Academic Editors: Marco García-Vaquero and Brijesh K. Tiwari

Received: 17 December 2021 Accepted: 7 January 2022 Published: 11 January 2022

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

species, and the chemical constituents of *Cystoseira* spp. were found to contain fatty acids and derivatives, terpenoids, steroids, carbohydrates, phlorotannins, phenolic compounds, pigments and vitamins [7]. The chemical composition of the alga depends on numerous ecological factors such as temperature, salinity, UV irradiation, collecting season, depth, geographic location, thallus development, etc. However, their individual and synergistic effect on the brown alga chemical profile and biological activity is still relatively unknown. Recent studies showed that seasonality and thallus vegetative parts significantly affect the nutritional and chemical profile of alga [9]. It is considered that higher nutritional and phenolic content, higher polyunsaturated fatty acid (PUFA) content, higher vitamin and mineral content, as well as the antiproliferative properties of brown algae from brown fucoid algae were obtained during hot and dry summer seasons and higher sea temperatures [10–13]. On the other hand, no seasonal effect was recorded for the pigment profile and fucoxanthin content, nor total phenolic content and antimicrobial activity of the genera *Padina*, *Colpomenia*, *Saccharina* or *Dictyota* [14,15]. So far, there are no reports on seasonal variations in chemical profile nor the biological activity of *Cystoseira* spp.

*Cystoseira* spp. composition suggests their high nutritional value with potential applications in the nutraceutical industry. A range of 29–46% of PUFA, a low *n-6* PUFA/*n-3* PUFA ratio as well as favorable unsaturation, atherogenicity, and thrombogenicity indices were observed in several *Cystoseira* species [16]. Compounds from *Cystoseira* species are important sources of nutraceuticals and may be considered as functional foods, such as extracts of *C. tamariscifolia* and *C. nodicaulis* that were able to protect a human dopaminergic cell line from hydrogen peroxide-induced cytotoxicity and inhibit cholinesterases, while those from *C. crinita* showed significant cytotoxic activity against human breast adenocarcinoma (MCF-7 cells), inducing apoptosis and autophagy [17,18]. Besides non-volatiles, the essential oil constituents of *C. compressa* and their seasonal changes have been identified and among them for a large number of compounds a broad range of biological activities have been already proved [19]. So far, over 50 biological properties have been attributed to compounds found in genus *Cystoseira*, and the most reported are antioxidant, antiinflammatory, cytotoxic, anticancer, cholinesterase inhibition, antidiabetic, and antiherpetic activities [7,20–24]. Phlorotannins are regarded as responsible for high antioxidant activity (e.g., free radical scavenging ability) [1,25–27]. Besides, there is little information on the antimicrobial activity of *Cystoseira* spp. extracts against major foodborne Gram-positive and Gram-negative bacteria [28].

The aim of this study was to investigate the chemical composition of *C. compressa*, one of the most widely distributed algae in the Adriatic Sea, to determine changes in its antioxidant and antimicrobial activity over the seasonal growth (May–September) when the algae are in the growing and reproductive phases, and the development of dense thallus occurs.

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

#### *2.1. Total Phenolic Content, Total Tannin Content and Antioxidant Activity*

Seaweed extracts were screened for total phenolic content (TPC), total tannin content (TTC) and antioxidant activity measured by ferric reducing/antioxidant power (FRAP), 2,2-diphenyl-1-picrylhydrazyl radical scavenging ability (DPPH) and the oxygen radical absorbance capacity (ORAC).

The results of TPC and TTC for *C. compressa* are shown in Figure 1. The results for TPC varied from 48.2 ± 0.5 to 83.4 ± 4.0 mg GAE/g. The highest TPC was found in June samples. On the other hand, the TTC values ranged from 2.0 ± 0.3 to 8.8 ± 0.8 mg CE/g with the highest value found also in June, followed by the extract from May. The FRAP values, shown in Figure 2, ranged from 1.0 ± 0.0 to 2.7 ± 0.1 mM TE. Similar to TPC and TTC, the highest FRAP result was obtained for June, showing the reducing activity of >2.5 mM TE. TPC and FRAP results were in high correlation (0.956; *p* < 0.01). The ORAC results are shown in Figure 2. The seaweed extracts were 200-fold diluted for ORAC assay. Among the investigated samples, the highest ORAC value of 72.1 ± 1.2 μM TE was found

in the August extract, with extracts from May having the second best. June and July extracts had the lowest ORAC values, more than 3-fold lower in comparison to the August extract. The DPPH radical inhibitions (in percentages) are shown in Figure 2. The extract from May had the highest inhibition (90.2%) while the August extract had the lowest inhibition (77.3%). The activity of other extracts was similar, around 85%. In the growing season, the sea temperature was the lowest in May (18.3 ◦C) and it rose every month till August when it peaked at 26.9 ◦C. Finally, a decrease in the temperature by 2.2 ◦C was observed in September (Table 1).

**Figure 1.** Total phenolic content (TPC) and total tannin content (TTC) of *C. compressa* extracts from May to September. a–d different letters denote statistically significant difference (*n* = 4).

**Figure 2.** FRAP, ORAC and DPPH inhibition results for *C. compressa* extracts from May to September. a–d different letters denote statistically significant difference (*n* = 4).


**Table 1.** Sea temperature and salinity recorded during the harvest of the algal samples.

The TPC of alga varies with seasonal changes of sea temperature, salinity, light intensity, geographical location and depth, as well as other biological factors such as age, size, the life cycle of the seaweed, presence of herbivores [1]. In this study, the geographical location and depth were eliminated as a factor as samples were collected from the same area and depth each month. The TPC, TTC and antioxidant activity results showed no correlation to the sea temperature and salinity. If the growth of alga is considered, in June when the TPC, TTC and FRAP were the highest, *C. compressa* had a fully developed, densely ramified thalli with aerocysts. In May the thalli are not ye<sup>t</sup> fully developed, while in July–September it is less dense, aerocysts appear in fewer numbers [29].

The TPC of *Cystoseira* species was previously investigated and researchers reported a strong effect of harvesting location and seasonal changes, especially temperature. Mancuso et al. [12] investigated TPC in *C. compressa* from eight locations along the Italian coast and confirmed the change of TPC with geographical location. The TPC ranged between different locations from 0.1 to 0.5% of algal dry weight (DW). The authors observed the increase in TPC with the rise of sea temperature (measured at different locations). Accordingly, the highest TPC of 0.53% DW was recorded at 28 ◦C. In contrast, Mannino et al. [30] investigated the effect of sea temperature seasonal variation on the TPC of *C. amentacea*. They harvested algae once in every season (winter, spring, summer and autumn) and measured the sea temperature. The authors observed the highest TPC in winter (0.8% DW) when the sea temperature was the lowest. In summer and autumn, when the sea temperatures were above 20 ◦C, the TPCs were the lowest, 0.4 and 0.37% DW, respectively. In their study, the TPC values showed a negative correlation with sea temperature. *Cystoseira compressa* extracts, from Urla (Turkey) [31], were screened for TPC, total flavonoid content (TFC), antioxidant and antimicrobial activity. The highest TPC of 1.5 mg GAE/g and TFC of 0.8 mg QE/g were found for hexane extract while the antioxidant activity of the hexane extract measured by DPPH radical inhibition was only 21.2%, more than four-fold lower than results in our study for hydroalcoholic extracts. In comparison, the methanolic extracts (similar polarity like ethanol) showed the TPC and TFC of 0.2 mg GAE/g and 0.3 mg QE/g, respectively and two-fold lower DPPH inhibition.

Abu-Khudir et al. [18] evaluated the antioxidant, antimicrobial, and anticancer activities of cold methanolic extract, hot methanolic extract, cold aqueous extract, and hot aqueous extract from *C. crinita* and *Sargassum linearifolium*. The highest TPC was found for the cold methanolic extract of *C. crinita*, 15.0 ± 0.58 mg GAE/g of dried extract, which is more than two-fold lower than the amount detected in the September extract from our study which contained the lowest TPC. The authors also found a high content of fatty acids (44%) and their esters in *C. crinita* cold methanolic extract. Both seaweeds showed similar DPPH and ABTS radical scavenging activity with *C. crinita* cold methanolic extract having IC50 of 125.6 μg/mL and 254.8 μg/mL, respectively. De La Fuente et al. [32] extracted *C. amentacea* var. *stricta* with dimethyl sulfoxide (DMSO) and 50% ethanol for determining TPC, TFC and antioxidant activity of extracts by DPPH radical scavenging, FRAP, OH scavenging, and nitric oxide (NO) scavenging methods. The TPC and TFC of DMSO extracts were 65.9 μg GAE/mg and 15.8 μg QE/mg, 3.2- and 5.1-fold higher than ethanolic extracts. Similar to our results, both investigated extracts had DPPH radical scavenging activity higher than 90%. Furthermore, the DMSO extracts showed a reducing activity of almost 90% while ethanolic extract showed a higher OH radical scavenging activity. Both extracts showed very low cytotoxicity, enabling their possible use as nutraceuticals.

Oucif et al. [20] screened six seaweed species (including *C. compressa* and *C. stricta*) for TPC, DPPH radical scavenging activity and reducing power. The highest TPCs were found for *C. compressa* methanolic and ethanolic extracts, 10.24 ± 0.09 and 15.70 ± 0.72 mg GAE/g DW, respectively. *Cystoseira compressa* ethanol extract had over 90% inhibition activity for DPPH radical and the highest reducing power, which can be compared with our results. Mhadhebi et al. [24] determined TPC, DPPH and FRAP in *C. crinita*, *C. sedoides* and *C. compressa* extracts. Among the three alga, *C. compressa* extract had the highest TPC of 61.0 mg GAE/g, which is comparable to our results, the lowest DPPH IC50 of 12.0 μg/mL, and the highest FRAP value, 2.6 mg GAE/g.
