**1. Introduction**

Fucosterol is abundant and one of the dominant sterols in marine macroalgae [1]. The purest form of fucosterol and its potency were first identified and observed in the brown macroalga *Fucus vesiculosus* by Heilbron et al. [2]. Fucosterol is a stigmasterol bond isomer expressed by the empirical formula C29H48O. The fucosterol content in macroalgae ranges from 4 to 95% of the total phytosterol content [3]. Brown macroalgae contain higher levels of fucosterol than green and red macroalgae. The fucosterol content in brown macroalga *Ecklonia radiata* ranged between 312.0 μg/g dry weight in leaves and 378.1 μg/g dry weight in stipes (98.6 and 98.9% of total sterols, respectively) [4]. In the brown macroalgae *Himanthalia elongata*, *Undaria pinnatifida*, and *Laminaria ochroleuca*, fucosterol was observed

**Citation:** Meinita, M.D.N.; Harwanto, D.; Tirtawijaya, G.; Negara, B.F.S.P.; Sohn, J.-H.; Kim, J.-S.; Choi, J.-S. Fucosterol of Marine Macroalgae: Bioactivity, Safety and Toxicity on Organism. *Mar. Drugs* **2021**, *19*, 545. https://doi.org/ 10.3390/md19100545

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

Received: 10 September 2021 Accepted: 24 September 2021 Published: 27 September 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/).

predominantly in 83–97% of the total sterol content [5]. Fucosterol was also reported to be dominant in *Stephanocystis hakodatensis* (formerly *Cytoseira hakodatensis*) and *Sargassum fusiforme*, which contained 65.9% and 67% of fucosterol, respectively [6].

Brown macroalgae are widely used as food and herbal medicine in Southeast Asia and several European countries. In Europe, brown macroalgae have been used to treat goiter and obesity [7]. In East Asia, brown macroalgae from the genera *Laminaria*, *Undaria,* and *Sargassum* (formerly *Hizikia*) are widely consumed daily and used as herb medicine [8–12]. Hence, the bioactive properties of brown macroalgae have drawn the attention of researchers. Previous studies have investigated fucosterol properties and their potential bioactivities. The antioxidant effect of fucosterol was reported by Lee et al. [13]. Furthermore, Jung et al. [14] investigated the anti-inflammatory properties of fucosterol in LPS-stimulated conditions. Fucosterol has the potential to inhibit particulate-induced inflammation and oxidative stress in the alveolar cell line A549. Through regulation of the FoxO signaling pathway, fucosterol exhibits anti-obesity characteristics by suppressing adipogenesis in 3T3-L1 preadipocytes [15]. In addition, fucosterol protects human neuroblastoma cell line SH-SY5Y cells from amyloid-induced neurotoxicity [16] and affects human lung cancer cells by inducing apoptosis and cell cycle arrest and targeting the Raf/signaling mitogen-activated protein kinase/extracellular-signal-regulated kinase (MEK/ERK) pathway [17]. Based on these studies, fucosterol can potentially be developed for use in nutraceutical and pharmaceutical fields. However, before further developing fucosterol properties, information on the safety and toxicity of fucosterol is required to comprehend the optimum and sustainable benefits of fucosterol as a functional agent. This study reviews the current scientific literature regarding the bioactivity, safety, and toxicity of fucosterol extracted from marine macroalgae. In addition to the bioactivity of fucosterol, we investigated the safety and toxicity of fucosterol in various organisms, including bacteria/fungi, animal cell lines, human cell lines, and animals. Through this review article, we express our hope that the applications of fucosterol from marine algae can be further developed in the nutraceutical and pharmaceutical industries.

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

Studies on the bioactivity, safety, and toxicity levels of fucosterol from marine macroalgae conducted from 2002 to 2020 were reviewed, and an increasing trend was observed (Figure 1). This indicates that fucosterol has drawn additional research attention in recent years.

**Figure 1.** Number of publications on the safety and toxicity of fucosterol published in each year.

Articles discussing the bioactivity, safety, and toxicity of fucosterol were categorized with respect to the various organisms or cells whose treatment they describe (Figure 2). Published studies on the safety and toxicity of fucosterol from marine algae have focused on its effects on bacteria and fungi (21%), animal cell lines (14%), human cell lines (38%), and animals (26%). The safety and toxicity of fucosterol have been studied both in vitro and in vivo. No clinical study of fucosterol has been conducted to date. Therefore, the investigation of the safety and toxicity of fucosterol at the clinical stage might be challenging.

**Figure 2.** Numbers of publications on safety and toxicity of fucosterol categorized according to the treated organisms or cells.

The numbers of publications on the safety and toxicity of fucosterol from various sources of macroalgae are shown in Figure 3. Sixteen marine algae species belonging to Dictyotaceae, Sargassaceae, Alariaceae, Lessoniaceae, and Fucaceae families have been studied in relation to their fucosterol bioactivity, safety, and toxicity on bacteria, fungi, animal cells, human cells, and animals. Among the 16 marine algae, *Sargassum fusiforme* (formerly *Hizikia fusiformis*) has become the greatest marine macroalgal source for fucosterol that has been studied. Over the last 10 years, studies on the bioactivities and nutritional and pharmacological properties of *S. fusiforme* have increased steadily. Most of the studies on the bioactivity of *S. fusiforme* have focused on its antioxidant (15.09%), anticancer and antitumor (15.09%), anti-inflammatory (11.32%), photoprotective (11.32%), and neuroprotective (11.32%) properties [18]. Figure 3 shows that the study of fucosterol in *S. fusiforme* has focused on its antioxidant, anti-osteoarthritic, anti-inflammatory, anti-photoaging, antidiabetic, hepatoprotective, and algicidal effects. *Ecklonia cava* subsp. *stolonifera* (formerly *Ecklonia stolonifera*) is the second most frequently reported macroalgal species that has been studied for its fucosterol content. *Ecklonia* species have been known as potential source of bioactive compounds [19]. Studies on the safety and toxicity levels of fucosterol obtained from *Ecklonia stolonifera* were mostly conducted on animal cell lines, human cell lines, and animals. Fucosterol from *Ecklonia stolonifera* has been studied for its antidiabetic, anti-obesity, anti-neurological, and hepatoprotective effects. The safety and toxicity levels of fucosterol obtained from *Ecklonia cava* subsp. *stolonifera* were mostly studied in animals.

**Figure 3.** Numbers of publications on safety and toxicity of fucosterol classified according to sources of macroalgae.

#### *2.1. Characteristics and Structure of Fucosterol*

Generally, fucosterol could be obtained by extracting dry powder of macroalgae using MeOH, EtOH, or *n*-hexane solvents [13,20–24]. The extracts were then partitioned via solvent fractionation [13,25]. After solvent dissolution under reduced pressure, the organic extracts were fractionated using silica gel column chromatography with a mixture of solvents of increasing polarity [14,22,23,26]. The fraction was eluted using a solvent to remove fatty acids, which were then analyzed further [26].

Currently, fucosterol analysis and identification are carried out using physical properties and spectroscopic methods, including 1H-NMR and 13C-NMR, as well as via comparison with published data, and thin-layer chromatography (TLC) analysis [20]. According to the molecular formula of fucosterol, one hydroxy group must be attached to C-28 in an *R* or *S* configuration. An olefin proton signal and two sets of two olefin signals indicate the presence of a tri-substituted double in the fucosterol side chain [22]. Infrared (IR) absorption peaks of fucosterol at 3400 and 1600 cm<sup>−</sup><sup>1</sup> were attributed to the hydroxyl and olefin groups, respectively. Fucosterol derivatives, namely 24*R*,28*R*- and 24*S*,28*R*-epoxy-24- ethylcholesterol, and <sup>24</sup>*R*-saryngosterol have also been used in octadecyl silica gel (ODS) column chromatography [21]. The structure of fucosterol is shown in Figure 4.

**Figure 4.** Chemical structure of fucosterol.

#### *2.2. Bioactivity of Fucosterol*

Fucosterol of marine macroalgae exhibited antidiabetic, anti-obesity, anti-osteoarthritic, immunomodulatory, anticancer, anti-inflammatory, anti-photoaging, hepatoprotective, anti-neurological, antioxidant, and antimicrobial activities (Figure 5).

**Figure 5.** Bioactivities, safety, and toxicity levels of fucosterol derived from macroalgae.

## 2.2.1. Antidiabetic Activity

Diabetes is a chronic disease that occurs when the pancreas does not produce enough insulin or when the body cannot use insulin effectively. One of the antidiabetic effects of fucosterol obtained from *Eisenia bicyclis* and *Ecklonia cava* subsp. *stolonifera* is characterized by inhibition of enzymes, such as rat lens aldose reductase (RLAR), human recombinant aldose reductase (HRAR), α -glucosidase, and PTP1B. The docking simulations clearly demonstrated negative binding energy for fucosterol ( −8.2 kcal mol−<sup>1</sup> for RLAR and −8.5 kcal mol−<sup>1</sup> for HRAR), implying a higher affinity and stronger binding competence for the active site of the enzyme [20]. These results were confirmed by Jung et al. [27], who stated that *Ecklonia stolonifera*-derived fucosterol reduced insulin

resistance by decreasing PTP1B expression and activating insulin signaling pathways. Fucosterol from *Sargassum fusiforme* also showed strong PTP1B inhibition at low concentrations [28]. Streptozotocin-induced diabetic rats treated with fucosterol from *Pelvetia siliquosa* via an orally administered dose of 30 mg/kg showed a decrease in serum glucose levels and inhibition of its accumulation. However, orally administered doses of 300 mg/kg are required for epinephrine-induced diabetic rats to inhibit blood glucose levels and degrade glycogen [29]. These findings illustrate that fucosterol extracts from *Eisenia bicyclis*, *Ecklonia cava* subsp. *stolonifera*, *Sargassum fusiforme*, and *Silvetia siliquosa* have antidiabetic potential that can be developed in the future.
