Secondary Metabolites from the Genus Eurotium and Their Biological Activities

Eurotium is the teleomorph genus associated with the section Aspergillus. Eurotium comprises approximately 20 species, which are widely distributed in nature and human environments. Eurotium is usually the key microorganism for the fermentation of traditional food, such as Fuzhuan brick tea, Liupao tea, Meju, and Karebushi; thus, Eurotium is an important fungus in the food industry. Eurotium has been extensively studied because it contains a series of interesting, structurally diverse, and biologically important secondary metabolites, including anthraquinones, benzaldehyde derivatives, and indol diketopiperazine alkaloids. These secondary metabolites have shown multiple biological activities, including antioxidative, antimicrobial, cytotoxic, antitumor, insecticidal, antimalarial, and anti-inflammatory activities. This study presents an up-to-date review of the phytochemistry and biological activities of all Eurotium species. This review will provide recent advances on the secondary metabolites and their bioactivities in the genus Eurotium for the first time and serve as a database for future research and drug development from the genus Eurotium.


Introduction
Eurotium (Eurotiaceae), now renamed Aspergillus, is the sexual generation of the genus Aspergillus.Despite the renaming, the majority of mycologists prefer adhering to the established and commonly used nomenclature [1][2][3].Eurotium is characterised by its golden cleistothecia, lenticular ascospores, uniseriate conidial heads in shades of green or blue, and yellow-, orange-or red-encrusted hyphae [2,4].The genus Eurotium comprises approximately 20 species [2], of which Eurotium amstelodami, Eurotium cristatum, and Eurotium repens have received the most attention [5,6].All species of Eurotium are hypertonic fungi, which are widely distributed in nature and human environments, especially in environments of high salt, high sugar, and low water.Eurotium species are generally considered to be benign fungi without mycotoxins [7][8][9][10][11].Therefore, Eurotium species are widely used in the food processing industry.Katsuobushi is a traditional Japanese food made from tuna fermented by Eurotium.During the fermentation process, the Eurotium reduces the fat content of the tuna and turns it to a deep red colour, giving it a milder taste and a unique flavour [12].Meju is a traditional Korean fermented soybean product, and

Secondary Metabolites from Eurotium
Nearly 180 compounds have been isolated and identified from Eurotium species using nuclear magnetic resonance (NMR) spectroscopy.These compounds mainly include anthraquinones, benzaldehyde derivatives, and indol diketopiperazine alkaloids.These secondary metabolites are not only derived from food but also produced by some Eurotium species in other environments, and we have included them in this review for future use in the fermented food industry.
The endophytes derived from saline-alkali plants are attracting increasing attention due to the extreme environment of high osmolarity and nutrient deprivation.The chemical investigation of saline-alkali plant-derived endophytic fungi has just begun compared with those of marine mangrove plant-derived endophytes.Zhang et al. [27] found a new anthraquinone named rubrumol (25), as well as emodin, catenarin, rubrocristin, and 2-O-methyleurotinone, in a halo-tolerant endophytic fungus E. rubrum.This fungus is derived from the salt-tolerant wild plant Suaeda salsa.These anthraquinones displayed topoisomerase inhibitory activity, which implied that endophytic Eurotium fungi from saline-alkali plants may be one new reservoir for natural products in the future (Figure 1).

Benzaldehyde Derivatives
Benzaldehyde derivatives constitute a class of polyketides synthesised via the combination of polyketone and terpenoid pathways [40].It has been reported that benzaldehyde derivatives have various bioactivities, including antioxidative, antibacterial, antifungal, antitumor, antimalarial, and antileishmanial activities [33,36,38].Benzaldehyde derivatives, which are a kind of natural pigments, are a class of main metabolites in the genus Eurotium [14].Over 20 benzaldehyde derivatives have been identified in Eurotium.
Four new and seven known benzaldehyde derivatives were identified from E. rubrum, an endophytic fungus isolated from the inner tissue of stems in the mangrove plant Hibiscus tiliaceus by Li et al. [33] These four benzaldehyde derivatives were 2-(2′,3-

Benzaldehyde Derivatives
Benzaldehyde derivatives constitute a class of polyketides synthesised via the combination of polyketone and terpenoid pathways [40].It has been reported that benzaldehyde derivatives have various bioactivities, including antioxidative, antibacterial, antifungal, antitumor, antimalarial, and antileishmanial activities [33,36,38].Benzaldehyde derivatives, which are a kind of natural pigments, are a class of main metabolites in the genus Eurotium [14].Over 20 benzaldehyde derivatives have been identified in Eurotium.

Indole Diketopiperazine Alkaloids
Indole diketopiperazine alkaloids constitute a crucial class of important secondary metabolites, and they are widely distributed in filamentous fungi, especially in the genus Eurotium [18].Indole diketopiperazine alkaloids are formed via the condensation of certain amino acids, including tryptophan, proline, and leucine [76].Due to their significant biological activities, including antimicrobial, antiviral, anticancer, immunomodulatory, antioxidative, and insecticidal activities, indole diketopiperazine alkaloids in the genus Eurotium are attracting increasing attention [41,42].
Swine-rejected feed was found to have a high propagule density of Eurotium sp.Additionally, echinulin (48) was both detected in this feed and isolated from the E. repens derived from it [43].Although significant differences in the metabolite composition were observed between the feed-derived and marine-derived E. repens, the biosynthesis of

Indole Diketopiperazine Alkaloids
Indole diketopiperazine alkaloids constitute a crucial class of important secondary metabolites, and they are widely distributed in filamentous fungi, especially in the genus Eurotium [18].Indole diketopiperazine alkaloids are formed via the condensation of certain amino acids, including tryptophan, proline, and leucine [76].Due to their significant biological activities, including antimicrobial, antiviral, anticancer, immunomodulatory, antioxidative, and insecticidal activities, indole diketopiperazine alkaloids in the genus Eurotium are attracting increasing attention [41,42].
Swine-rejected feed was found to have a high propagule density of Eurotium sp.Additionally, echinulin (48) was both detected in this feed and isolated from the E. repens derived from it [43].Although significant differences in the metabolite composition were observed between the feed-derived and marine-derived E. repens, the biosynthesis of echinulin was conserved in E. repens regardless of its origin [24].Kimoto et al. [48] isolated neoechinulin A (49) from marine fungus E. rubrum Hiji 025, and further synthesised this compound according to its natural configuration.Slack et al. [44] investigated the metabolites in E. herbariorum, E. amstelodami, and E. rubrum, which are common in the built environment of Canadian homes.Neoechinulin B (50) and neoechinulin A were the major metabolites, but preechinulin (51), neoechinulin E (52), and echinulin were the minor metabolites in E. amstelodami and E. rubrum.E. herbariorum also produced a small amount of neoechinulin E. In addition, a new spirocyclic diketopiperazine alkaloid, 7-O-methylvariecolortide A (53; yellow amorphous powder), was isolated from the mangrove plant Hibiscus tiliaceus-derived E. rubrum, along with variecolortides A-C (54)(55)(56).Structurally, compounds 53-56 represent the unique spiro-anthronopyranoid diketopiperazine skeleton with a stable hemiaminal functional group.Further, a hydroxyl group in compound 54 is replaced by a methoxyl group at C-7 in compound 53 [52].Fructigenine A (57) bearing a reverse-prenyl group was isolated from Eurotium sp.SF-5130 [54].
Zhong et al.

Bioactivities of Secondary Metabolites from Eurotium
Pharmacological investigations have affirmed that the structurally distinctive compounds extracted from Eurotium species exhibit a spectrum of biological activities, encompassing antioxidative, antimicrobial, cytotoxic, antitumor, insecticidal,

Bioactivities of Secondary Metabolites from Eurotium
Pharmacological investigations have affirmed that the structurally distinctive compounds extracted from Eurotium species exhibit a spectrum of biological activities, encompassing antioxidative, antimicrobial, cytotoxic, antitumor, insecticidal, antimalarial, and anti-inflammatory properties.We provide a review of these functional secondary metabolites to provide a scientific basis for the development of functional foods using Eurotium as a fermentative strain.

Antioxidative Activity
Numerous studies have demonstrated the exceptional antioxidative activity of metabolites isolated from Eurotium species.Further, the absolute and stereoscopic configurations affect the antioxidative activity of these compounds [46,56].Ishikawa et al. [77] discovered that flavoglaucin (31) was an excellent antioxidant and synergist with tocopherol.The antioxidative and synergistic effects of flavoglaucin and its derivatives largely depend on their hydroxy group, which does not form hydrogen bonds with the formyl group in the molecule.These compounds are found in a variety of foods fermented by Eurotium and contribute to their functional activity [78].Li et al. [51] assessed the antioxidative activity of metabolites isolated from a marine mangrove plant-derived endophytic fungus E. rubrum using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay.They found that neoechinulin E (52) showed a strong radical scavenging activity with half maximal inhibitory concentration (IC 50 ) values of 46.0 µM, which were stronger than that of the well-known synthetic antioxidant butylated hydroxytoluene (IC 50 = 82.6 µM).Eurorubrin ( 19) and 2-O-methyleurotinone (10) also displayed strong radical scavenging activity with IC 50 values of 44.0 and 74.0 µM, respectively, while 2 (18), asperflavin (15), and questin (1) only showed weak or moderate activity [23].In 2009, a study by Miyake et al. [35] demonstrated that isodihydroauroglaucin (34), auroglaucin (39), dihydroauroglaucin (41), tetrahydroauroglaucin (40), and flavoglaucin exhibited the high radical scavenging capacities of DPPH and superoxide when compared to α-tocopherol (a standard antioxidant for the scavenging capacity).The structures of 1 -monoene or 1 ,3 -diene in the substituent formed by the seven-carbon aliphatic chain of dihydroauroglaucin and tetrahydroauroglaucin may be related to their high radical scavenging activity.Subsequently, Miyake et al. [16] found that isoechinulin A (70) exhibited higher radical scavenging activity than α-tocopherol.Asperflavin, isoechinulin B, neoechinulin B (50), and variecolorin O (83) were found to have a similar activity to α-tocopherol in respect to DPPH radical scavenging.

Antimicrobial Activity
Microbial interference poses a significant threat to human health, and the search for antimicrobial compounds from Eurotium species represents a promising strategy to combat the escalating challenges posed by human and plant pathogens, particularly drugresistant strains.Further, the antimicrobial activity of Eurotium species may be related to anthraquinones [79][80][81].As early as 1980, erythroglaucin (3) was found to have slight antibacterial activity against Bacillus brevis, Bacillus subtilis, and Streptomyces viridochromogenes.However, rubrocristin (6) and physcion (2) had no significant antimicrobial activity, indicating that the number and location of the hydroxyl groups might play an important role in the antibacterial activity of polyhydroxyanthraquinones [22].Chevalone C (102), eurochevalierine (148), and CJ-12662 (106) demonstrated antimycobacterial activity against Mycobacterium tuberculosis with minimal inhibitory concentration (MIC) values of 6.3, 50.0, and 12.5 µg/mL, respectively [58].In 2012, Du et al. [41] evaluated the antimicrobial activities of compounds isolated from E. cristatum against two bacteria (Staphylococcus aureus and Escherichia coli) and five plant-pathogenic fungi (Valsa mali, Sclerotinia miyabeana, Alternaria brassicae, Physalospora obtuse, and Alternaria solania).The MIC value of the positive control chloramphenicol against E. coli and S. aureus was 4 µg/mL.Cristatumin A (66) and tardioxopiperazine A (71) displayed potent inhibitory activity against E. coli and S. aureus with MIC values of 64 and 8 µg/mL, whereas cristatumin D (69) and echinulin showed weak activity against S. aureus, each creating an inhibition zone of 8 mm at 100 µg/disk (the MICs were not determined).In addition, the compound 9-dehydroxyeurotinone (23) isolated from E. rubrum showed weak antibacterial activity against E. coli with an inhibition zone of 7.0 mm at 100 µg/disk, while amphotericin B had an inhibition zone of 11.0 mm at 20 µg/disk as the control [28].
Dihydroauroglaucin was active against the Gram-positive bacteria with MIC values of 128 µg/mL, 64 µg/mL, and 8 µg/mL on S. aureus, E. faecalis, and S. pneumoniae, respectively.Compound 41 was previously considered inactive against reference and MRSA S. aureus strains [25].Neoechinulin A, L -alanyl-L -tryptophan anhydride (81), dihydroxyisoechinulin A (78), and questin showed obvious antibacterial activity against B. cereus and P. vulgaris with MIC values of 1.56 to 25 µM when ciprofloxacin (MIC values of 0.78 and 0.20 µM, respectively) was used as the positive control and DMSO (25 µM) was used as the negative control [11].Asperglaucins A (129) and B (130) exhibited potent antibacterial activities against Pseudomonas syringae pv.actinidae and B. cereus, with all having MIC values of 6.25 µM.Compound 129 also exhibited a weak inhibitory effect against MRSA with an MIC value of 25 µM.The activity of compounds 129 and 130 is probably due to their heterocyclic fraction [17].Notably, the above intriguing new compounds, which exhibit excellent antimicrobial properties, could be used as the leading compounds for the development of new drugs in the future.

Anti-Inflammatory Activity
Kim et al. [50] demonstrated that neoechinulin A had an anti-inflammatory effect on lipopolysaccharide-stimulated RAW264.7 macrophages.Further, compound 49 blocked the activation of nuclear factor-kappa B (NF-κB) by inhibiting the phosphorylation and degradation of inhibitor kappa B-α, and decreased p38 mitogen-activated protein kinase (MAPK) phosphorylation.The anti-inflammatory effect of compound 49 was thus attributed to the inhibition of the NF-κB and p38 MAPK pathways.In addition, the compounds flavoglaucin, isotetrahydroauroglaucin (33), and asperflavin were found to inhibit the production of pro-inflammatory mediators and cytokines, including tumor necrosis factor-α, interleukin-1β, interleukin-6, nitric oxide (NO), prostaglandin E2, nitric oxide synthase, and cyclooxygenase-2 [30,32,36].Cristaldehyde A (46) and cristaquinone A (176) inhibited the NO production in lipopolysaccharide-induced RAW264.7 cells, with IC 50 val-ues of 12.26 and 1.48 µM when paclitaxel was used as a positive control, with an IC 50 value of 41.00 µM [38].

Other Activities
Several isolated compounds have certain unique biological activities, including a good binding affinity for human opioid or cannabinoid receptor activity, inhibiting protein tyrosine phosphatase 1B activity, alleviating insulin resistance activity, inhibiting caspase-3 activity, inhibiting α-glucosidase activity, and antiviral activity.

Conclusions
Eurotium, a crucial genus within the Aspergillus family, has emerged as a significant source of bioactive compounds.Several factors contribute to its importance, including its widespread distribution, its role as a key microorganism in the fermentation of traditional foods and beverages (e.g., Fuzhuan brick tea), and its abundant production of secondary metabolites with promising bioactivities.Approximately 180 chemical components have been isolated from Eurotium species, spanning anthraquinoes, benzaldehyde derivatives,

Conclusions
Eurotium, a crucial genus within the Aspergillus family, has emerged as a significant source of bioactive compounds.Several factors contribute to its importance, including its widespread distribution, its role as a key microorganism in the fermentation of traditional foods and beverages (e.g., Fuzhuan brick tea), and its abundant production of secondary metabolites with promising bioactivities.Approximately 180 chemical components have been isolated from Eurotium species, spanning anthraquinoes, benzaldehyde derivatives, indol diketopiperazine alkaloids, and some other compounds.Various pharmacological activities, including antioxidative, antimicrobial, cytotoxic, antitumor, insecticidal, antimalarial, and anti-inflammatory activities, have been demonstrated in Eurotium species using numerous test models.However, further research employing in vivo models is imperative.In addition, secondary metabolites with health benefits should be introduced into the food industry to develop new functional foods.Most of the research has focused on three Eurotium species-E.amstelodami, E. cristatum, and E. repens-and should be further expanded to discover other species in the genus Eurotium from natural environments, such as the sea, with a view to introducing new strains for food fermentation.The other species in genus Eurotium the should be further studied, and this study will also provide information on the taxonomic relationships between Eurotium species.In addition, more attention should focus on the discovery of new secondary metabolites and their biological activities from fermented food/drink-derived and marine-derived Eurotium species.Delving into the pathways responsible for the formation of these metabolites is equally crucial for advancing our understanding of their potential applications.
C-NMR of compound 35 are replaced by two methylene signals at C-3 and C-4 in compound 26.The structures of compounds 27 and 30 are similar, except that the signals at H-6 and C-6 in compound 30 are replaced by a carbonyl signal at C-6 in compound 27.

Figure 5 .
Figure 5. Overview of main biological activities.

Table 1 .
Secondary metabolites from the genus Eurotium and their biological activities.