2.2.11. Antimicrobial Activity

Antimicrobial activities can be defined as the process of inhibiting or destroying the growth of microorganisms, especially pathogenic microorganisms. The antimicrobial properties, including antibacterial and antifungal properties of marine macroalgae, are associated with various groups of bioactive lipids, such as fucosterol. The results of the study by Tyskiewicz et al. [57] showed that fucosterol from *Fucus vesiculosus* at a concentration of 1.0% completely inhibited the germination of macroconidia in *Fusarium culmorum* (Fungi, Ascomycota). Furthermore, when macroconidia were exposed to low doses of fucosterol (0.05–0.2%), their growth was inhibited, and structural degradation occurred. Furthermore, fucosterol from *Sargassum carpophyllum* cultured with *Pyricularia oryzae* (Fungi, Ascomycota) mycelia caused abnormal morphological changes [22]. Previous

studies confirmed the antibacterial and antifungal activity of 3,6,17-trihydroxy-stigmasta-4,7,24(28)-triene, fucosterol, and 14,15,18,20-diepoxyturbinarin compounds from *Turbinaria conoides*, with MICs ranging from 2 to 16 μg/mL, against *Staphylococcus aureus*, *S. epidermidis*, *Escherichia coli*, *Pseudomonas aeruginosa*, *Aspergillus niger*, and *Candida albicans* [58]. The antibacterial properties of *Sargassum longifolium* fucosterol were also tested against the human pathogen *Vibrio parahaemolyticus* and the fish pathogens *V. vulnificus*, *V. harveyii*, and *Aeromonas hydrophililla*. Interestingly, among these bacteria, only *P. fluorescens* was not susceptible to the effect of fucosterol [59]. Overall, fucosterol could potentially be a strong and promising antimicrobial agent.

#### *2.3. Safety and Toxicity of Fucosterol in Bacteria and Fungi*

Several studies on fucosterol in bacteria and fungi have been published [22,57,58]. From these articles, data extraction was performed, as shown in Table 1.



Nd: not determined.

According to Tang et al. [58], fucosterol isolated from *Sargassum carpophyllum* showed low toxicity, with IC50 = 250 μg/mL, and was able to induce morphological changes in *Pyricularia oryzae*. Furthermore, fucosterol extract from *Turbinaria conoides* was used to test the level of growth inhibition in bacteria (*S. aureus*, *S. epidermidis*, *E. coli*, and *P. Aeruginosa*) and fungi (*C. albicans* and *A. niger*). In the tested bacteria, the MIC values ranged from 8 to 16 μg/mL, which indicated that fucosterol was able to inhibit the growth of the tested bacteria well. In addition, fucosterol showed the highest growth inhibition in *C. albicans*, with MIC = 8 μg/mL. Furthermore, research by Tyskiewicz et al. [57] showed that at a concentration of 1.0% fucosterol was able to optimally inhibit the growth of *F. culmorum* macroconidia. Moreover, macroconidia showed shorter length and structural degradation at lower fucosterol concentrations (0.05–0.2%).

From these studies, we conclude that fucosterol can potentially be developed as a new agen<sup>t</sup> for combating the problem of infection due to bacteria and fungi that are pathogenic because of its excellent biological activity as an inhibitor of bacteria and fungi. Based on our literature review, only the genera *Turbinaria* and *Sargassum* have been studied for the treatment of pathogenic bacteria and fungi. No other genera have been reported with respect to their safety and toxicity in bacteria and fungi. Further research on other bacteria and fungi species is required to comprehensively elucidate the safety and toxicity of fucosterol in bacteria and fungi.

#### *2.4. Safety and Toxicity of Fucosterol in Cell Lines*

Several studies have demonstrated safety and toxicity in human cell lines [17,27,28,35– 38,47,53,60–62] and animal cell lines [15,42,43,45,54,55]. The safety and toxicity of fucosterol in human and animal cell lines are summarized in Table 2.

**Table 2.** Studies on safety and toxicity of fucosterol extracted from macroalgae, tested in human and animal cell lines.



**Table 2.** *Cont.*

Nd: not determined.

In a study involving the use of commercial fucosterol for the treatment of RAW 264.7 macrophage cell line stimulated by particulate matter (PM), Jayawardena et al. [43] demonstrated the inhibition of NO production levels by observing inflammatory mediators, such as iNOS, COX-2, and pro-inflammatory cytokines (i.e., IL-6, interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α)), including prostaglandin E2 (PGE2)). Furthermore, the effect of fucosterol was amplified by the decreased expression of mitogen-activated protein kinase (MAPK) and NF-κB signaling pathway molecules and ROS regulation. Fernando et al. [44] showed that increasing concentrations (>100 μg/mL) of Chinese fine dust PM (CPM) in A549 cells significantly increased ROS levels and caused cell death. CPM-induced A549 cells treated with fucosterol of *Sargassum aquifolium* at a concentration of 3.125–50 μg/mL caused an increase in cell viability of up to 94.98 ± 1.26%, and the IC50 value was estimated to be 21.74 ± 0.67 μg/mL [60]. In addition, *Sargassum aquifolium*derived fucosterol was also reported to increase cell viability in HaCaT and HDF cells and yielded non-toxic results.

Another study reported that the addition of fucosterol isolated from *Sargassum fusiforme* in HaCaT cells induced by 500 μM CoCl2 increased cell viability up to 56% at a concentration of 10 μM [61]. Furthermore, as reported by Choi et al. [47], there was no effect on HepG2 cells after treatment with the crude extract of fucosterol up to a concentration of 100 μM. However, in HepG2 cells induced by t-BHP and tacrine, fucosterol showed low toxicity and increased cell viability. In addition, LPS-induced RAW 264.7 macrophages treated with fucosterol of *Undaria pinnatifida* showed an unclear toxic effect because the 3-(4, 5-dimethyl thiazole-2-yl)-2, 5-diphenyltetrazolium bromide MTT test results showed that fucosterol did not affect cell viability at a concentration of 10–50 μM [42]. These findings are similar to those of Wong et al. [54] at concentrations ranging from 12 to 192 μM, fucosterol of *Padina australis* did not have a significant cytotoxic effect on C8-B4 cells when compared with control (untreated cells) (approximately 100% cell viability).

Research conducted by Paudel et al. [55] on activity of fucosterol from *Undaria pinnatifida* and *Eisenia bicyclis*reported that there was no visible effect of the crude fucosterol extract on MAO-A and MAO-B (half-maximal inhibitory concentration (IC50) > 500 μM). MAO is a catecholamine-degrading enzyme with a long-standing therapeutic profile; MAO-A and MAO-B are isoenzymes. In addition, the results of the functional assay showed that

fucosterol did not show agonist activity at any of the receptors tested. Moreover, the crude extract of fucosterol can potentially inhibit PTP1B and α-glucosidase with an IC50 value of 50.58 ± 1.86 μM [28], and BACE1 with an IC50 value of 64.12 ± 1.0 μM [53] without causing side effects.

In a report by Lee et al. [15], treatment with fucosterol from *Ecklonia cava* subsp. *stolonifera* of 3T3-L1 preadipocytes had no effect on inhibiting cell proliferation up to a concentration of 50 μM [15]. This finding is complemented by previous investigations that stated that the survival of HepG2 cells was not affected up to a concentration of 100 μM *Ecklonia cava* subsp. *stolonifera*-derived fucosterol for 24 h. However, cell survival was reduced to 48 h at a concentration of 200 μM. Based on these results, they recommended that additional in vitro studies on the anti-diabetic activity of fucosterol be carried out using non-toxic concentrations of 50, 25, and 12.5 μM [27].

The cytotoxicity of fucosterol in various human cancer cells has been widely published [17,62,64,65]. According to Mao et al. [17], commercial fucosterol inhibited the growth of all human lung cancer cells tested with IC50 values ranging from 15 to 60 μM. However, interestingly, fucosterol showed low toxicity in all normal cells with IC50 > 100 μM, which indicated that fucosterol can selectively inhibit lung cancer cell growth, induce cell cycle arrest, and target the Raf/MEK/ERK signaling pathway. Other studies have shown that commercial fucosterol reduces cell viability and enhances the cytotoxic effect of 5-Fu in HT29 cancer cells without affecting normal colon fibroblasts (CCD-18Co). Studies on the toxicity of fucosterol on HL-60 [63]; ES2 and OV90 [35], HT-29, Caco-2, and T47D [38]; KB, Hep-2, MCF-7, and SiHa [37] yielded low to no toxicity results in DLA cells [36].

In previous studies, the safety and toxicity of fucosterol in human and animal cell lines have been extensively investigated. Most of the studies focused only on the brown seaweeds of genera *Sargassum*, *Undaria*, *Turbinaria*, and *Ecklonia*. Based on the literature reviewed, other genera, such as *Ulva* and *Enteromorpha*, have not been tested in cell lines. Fucosterol from these genera has been reported to have biological activity, but its safety and toxicity levels in cell lines have not been reported. Therefore, the mechanisms of other genera in cell lines should be investigated further.

#### *2.5. Safety and Toxicity of Fucosterol in Animals*

Several studies on the safety and toxicity of fucosterol isolated from marine macroalgae in animals have been published. The brown seaweed *Ecklonia cava* subsp. *stolonifera* [56,57] and *Hizikia fusiformis* or *Sargassum fusiformis* [31,34,56,66] were the most frequently discussed. A summary of the safety and toxicity of fucosterol in animals is presented in Table 3.

The research of Mo et al. [50], complements the data about the anti-diabetic and antiobesity properties of fucosterol that inhibits necrosis and apoptosis in a process mediated by PPARγ activation and inhibition of NF-B, which reduces inflammatory factors. Fucosterol also inhibits apoptosis and autophagy by upregulating Bcl-2 via PPARγ, thereby decreasing functional Bax and Beclin-1. Park et al. [34] reported that administration of 200 mg/kg body weight to mice increased splenocyte proliferation and NO production without cytotoxicity. Based on the research by Oktaviani et al. [56], fucosterol from *Sargassum fusiforme* showed low toxicity because it significantly affected the survival of *C. elegans* (1.54-fold and 1.23-fold increase), at a concentration of 0.05 mg/mL.

Some studies have been conducted to test the effect of fucosterol on the nervous system. Oh et al. [52] reported no toxicological response induced by the administration of fucosterol derived from *Ecklonia stolonifera* when it was injected at 10 mol/h into the dorsal hippocampus for four weeks. After training of aging rats, there was an increase in latency to reach the platform. Furthermore, Zhen et al. [66] showed no neurotoxic effect at the same dose levels administered after 0.5 and 4 h. Conversely, fucosterol from *Sargassum fusiforme*, showed no neurotoxic activity at the doses used in the forced or tail suspension tests (10, 20, 30, and 40 mg/kg). Lee et al. [31] found that all doses of fucosterol from *Sargassum fusiforme* had no toxic effects in OVX mice. The results of the study showed that fucosterol treatment significantly improved the loss of bone density caused by ovariectomy. Furthermore, the research conducted by Choi et al. [47] showed that there were no deaths or gross appearance abnormalities, and no fucosterol-induced abnormal behavioral changes, seizures, or death over 24 h due to *Ecklonia cava* subsp. *stolonifera* and *Eisenia bicyclis*. However, pretreatment with fucosterol at doses of 25, 50, and 100 mg/kg body weight markedly attenuated this cytotoxic effect of tacrine. In addition, the hepatoprotective effect of fucosterol at the highest dose (100 mg/kg body weight) resulted in serum ALT levels similar to those of the control group, suggesting that fucosterol has the potential to reduce tacrine-induced hepatotoxicity. The published results of fucosterol studies indicate that the number of in vivo tests involving algal metabolites is very limited. The experimental model that has been used thus far has focused on mice. Therefore, we propose that future research should focus on determining the full in vivo potency of fucosterol.

**Table 3.** Studies on safety and toxicity of fucosterol extracted from macroalgae, tested in animals.


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

*3.1. Literature Search*

The preferred reporting items for systematic reviews and meta-analyses (PRISMA) method [67] were used for the collection, identification, screening, selection, and analysis of the studies reviewed. A literature search was performed using four databases: PubMed, Science Direct, Wiley, and Web of Science. The search criteria included scientific articles on fucosterol published between 2002 and 2020. The keywords used in the literature search were "fucosterol" and "bioactivites OR "biological activities" OR "safety" OR "toxicity" OR "characteristics" OR "structure" OR "cell lines" OR "microalgae" OR "macroalgae" OR "plant" OR "bacteria" OR "fungi" OR "invertebrates" OR "animals" OR "human." The total number of articles found was 1251, which, upon further screening by checking the title and keywords and removing similar articles, was decreased to 621.
