Research Advances of Bioactive Sesquiterpenoids Isolated from Marine-Derived Aspergillus sp.

Marine fungi Aspergillus sp. is an important source of natural active lead compounds with biological and chemical diversity, of which sesquiterpenoids are an extremely important class of bioactive secondary metabolites. In this paper, we review the sources, chemical structures, bioactivity, biosynthesis, and druggability evaluation of sesquiterpenoids discovered from marine fungi Aspergillus sp. since 2008. The Aspergillus species involved include mainly Aspergillus fumigatus, Aspergillus versicolor, Aspergillus flavus, Aspergillus ustus, Aspergillus sydowii, and so on, which originate from sponges, marine sediments, algae, mangroves, and corals. In recent years, 268 sesquiterpenoids were isolated from secondary metabolites of marine Aspergillus sp., 131 of which displayed bioactivities such as antitumor, antimicrobial, anti-inflammatory, and enzyme inhibitory activity. Furthermore, the main types of active sesquiterpenoids are bisabolanes, followed by drimanes, nitrobenzoyl, etc. Therefore, these novel sesquiterpenoids will provide a large number of potential lead compounds for the development of marine drugs.


Introduction
More than 70% area of the earth is covered by oceans, which is the largest known habitat for life. The marine environment is characterized by high salinity, high pressure, low oxygen, low temperature, darkness, scarce nutrients, etc. To adapt to the special environment and obtain advantages in the competition of limited resources, marine microorganisms could produce novel secondary metabolites with unique structures and potent biological activities during evolution [1,2]. Rich marine microorganisms, mainly derived from marine actinomycetes and marine fungi, are ubiquitous in the natural environment [3]. Diverse active natural products exist in endophytic fungi from the marine environment, which can be the resources for new lead compounds [4,5].
Aspergillus is a typical filamentous fungus, which is divided mainly into Aspergillus fumigatus, Aspergillus versicolor, Aspergillus flavus, Aspergillus ustus, Aspergillus sydowii, and so on [6]. Fumiquinazolines were isolated by Numata from marine Aspergillus sp. for the first time in 1992, which opened the door to the study of the metabolites of marine Aspergillus [7]. Recent studies have found that many organic compounds with unique structures, which showed a lot of physiological activities, were found in marine Aspergillus sp., including terpenoids, alkaloids, and polyketones [8]. Sesquiterpenoids, the most abundant among all the terpenoids skeletons, exhibit excellent biological activities, such as cytotoxicity, antibacterial, antifungal, antiviral, anti-inflammatory, and enzyme inhibitory activity, and have aroused widespread interest of many scholars [9,10]. This paper attempts to review the sources, bioactivities, biosynthesis, and other studies of sesquiterpenoids discovered from marine fungi Aspergillus sp. in the last 15 years. Marine fungus Aspergillus is a huge community that occupies a great proportion in the fungus family, which is widely distributed in marine plants, marine organisms, marine sediments, and other environments. According to incomplete statistics, there were more than 180 species of fungus Aspergillus, such as Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus, and Aspergillus versicolor [13]. The proportions of the 56 species (Table 1) reviewed in this paper are as follows: Aspergillus versicolor (14.3%), Aspergillus sydowii (12.5%), Aspergillus ustus (10.7%), Aspergillus fumigatus (5.4%), Aspergillus insulicola (3.6%), Aspergillus ochraceus (3.6%), Aspergillus carneus (3.6%), Aspergillus terreus (3.6%), Aspergillus flavus (3.6%), Aspergillus flavipes (3.6%), and Aspergillus unknown (26.8%) (see Figure 2). Marine fungus Aspergillus is a huge community that occupies a great proportion in the fungus family, which is widely distributed in marine plants, marine organisms, marine sediments, and other environments. According to incomplete statistics, there were more than 180 species of fungus Aspergillus, such as Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus, and Aspergillus versicolor [13]. The proportions of the 56 species (Table 1) reviewed in this paper are as follows: Aspergillus versicolor (14.3%), Aspergillus sydowii (12.5%), Aspergillus ustus (10.7%), Aspergillus fumigatus (5.4%), Aspergillus insulicola (3.6%), Aspergillus ochraceus (3.6%), Aspergillus carneus (3.6%), Aspergillus terreus (3.6%), Aspergillus flavus (3.6%), Aspergillus flavipes (3.6%), and Aspergillus unknown (26.8%) (see Figure 2).     In recent years, more and more sesquiterpenoids were found in marine fungi Aspergillus, which consisted of the molecular skeleton structure with three isoprene units and contains 15 carbon atoms [56]. In addition, the number and skeleton types of sesquiterpenoids are the most abundant among all the terpenoids. According to the number of carbon rings, sesquiterpenoids can be divided into acyclic sesquiterpenes, monocyclic sesquiterpenoids, bicyclic sesquiterpenoids, tricyclic sesquiterpenoids, tetracyclic sesquiterpenoids, etc., [57]. Acyclic sesquiterpenes are also known as chain sesquiterpenes but rarely reported in fungi. The monocyclic sesquiterpenes referred mainly to bisabolanes, humaranes, and cybrodins, while the bicyclic sesquiterpenes consist mainly of drimanes, lacticinanes, and eudesmanes. This paper finds that the main types of sesquiterpenoids isolated from marine fungi Aspergillus were bisabolanes (46.6%), drimanes (27.2%), nitrobenzenes (6.3%), and unknown structure (9%) (see Figure 3). In recent years, more and more sesquiterpenoids were found in marine fungi Aspergillus, which consisted of the molecular skeleton structure with three isoprene units and contains 15 carbon atoms [56]. In addition, the number and skeleton types of sesquiterpenoids are the most abundant among all the terpenoids. According to the number of carbon rings, sesquiterpenoids can be divided into acyclic sesquiterpenes, monocyclic sesquiterpenoids, bicyclic sesquiterpenoids, tricyclic sesquiterpenoids, tetracyclic sesquiterpenoids, etc., [57]. Acyclic sesquiterpenes are also known as chain sesquiterpenes but rarely reported in fungi. The monocyclic sesquiterpenes referred mainly to bisabolanes, humaranes, and cybrodins, while the bicyclic sesquiterpenes consist mainly of drimanes, lacticinanes, and eudesmanes. This paper finds that the main types of sesquiterpenoids isolated from marine fungi Aspergillus were bisabolanes (46.6%), drimanes (27.2%), nitrobenzenes (6.3%), and unknown structure (9%) (see Figure 3). Recent studies have indicated that the metabolic pathway of marine fungi-that results in the production of a number of secondary metabolites with various chemical structures and specific physiological activities-is very different from that of terrestrial fungi Recent studies have indicated that the metabolic pathway of marine fungi-that results in the production of a number of secondary metabolites with various chemical structures and specific physiological activities-is very different from that of terrestrial fungi [37]. This article concludes that 131 of the 268 sesquiterpenoids isolated from marine fungi Aspergillus have significant biological activities. Moreover, the structure types of inactive sesquiterpenoids are mostly bisabolanes and drimanes [58][59][60][61][62]. The relatively large number of sesquiterpenoids shows a variety of biological activities such as antitumor, antibacterial, anti-inflammatory, enzyme inhibitory, antioxidant, antiviral, and other activities. Overall, 30.5% of sesquiterpenoids exhibited antibacterial activity, followed by antitumor activity (29%), anti-inflammatory activity (22.9%), enzyme inhibitory activity (8.4%), and other activities (10.7%) (see Figure 4).

Antibacterial Activity
In recent years, inappropriate and irrational use of antibiotics provides favorable conditions for resistant microorganisms to emerge and spread, which has become a global public health problem [63]. Therefore, it is urgent to develop new antibiotics with new structures and significant biological activities. To that end, the secondary metabolites of microorganisms in the marine environment are a great source for new antibacterial agents screening and much attention has been attracted to the relevant studies. This section covers 40 bioactive sesquiterpenoids ( Figure 5) with antibacterial activity described to date from marine-derived Aspergillus sp.
Li et al. [14] isolated four new and one known bisabolane-type sesquiterpenoid from secondary metabolites of Aspergillus sp. from sponge. Compounds 1-5 showed different antibacterial activity against six pathogenic bacteria and two marine bacteria, and compounds 2 and 4 showed selective antibacterial activity. Compound 2 had strong inhibitory effects on Staphylococcus albus and Micrococcus tetragenus, with minimum inhibiting concentrations (MIC) values of 5.00 and 1.25 µM, respectively. The MIC values of compound 4 with S. albus and Bacillus subtilis were 5.00 µM and 2.50 µM, respectively. Notably, compound 1 represents the rare example of a bisabolane-type sesquiterpenoid with a 1, 4disubstituted benzene ring isolated from marine organisms. Compounds 2 and 3 were the enantiomers of (+)-sydonol and (+)-sydonic acid, respectively. This fact suggests that fungi isolated from different marine organisms may produce different stereochemisty compounds. Furthermore, there were three sesquiterpenoids, 6-8, from the sponge-associated fungus Aspergillus sydowii ZSDS1-F6, which has certain antibacterial activities; among them, compound 6 and 7 displayed antibacterial activities against Klebsiella pneumonia, with MIC values of 21.4 and 10.7µM, respectively [15]. In addition, compound 6 showed moderate antibacterial activity against Aeromonas hydrophila (MIC, 4.3 µM), while compound 8 showed moderate antibacterial activity against Enterococcus faecalis (MIC, 18.8 µM). Chen et al. [16] isolated two phenolic bisabolane sesquiterpenoids (PBS) compounds (9-10) from Aspergillus flavipes 297, including a pair of new enantiomers (±)-flavilane A (9). However, compounds 9 and 10 represent the rare PBS-containing methylsulfinyl group and showed selective antibacterial activities against several pathogenic bacteria; their MIC values were 2-64 µg/mL. Furthermore, compound 10 exhibited mild antifungal activity

Antibacterial Activity
In recent years, inappropriate and irrational use of antibiotics provides favorable conditions for resistant microorganisms to emerge and spread, which has become a global public health problem [63]. Therefore, it is urgent to develop new antibiotics with new structures and significant biological activities. To that end, the secondary metabolites of microorganisms in the marine environment are a great source for new antibacterial agents screening and much attention has been attracted to the relevant studies. This section covers 40 bioactive sesquiterpenoids ( Figure 5) with antibacterial activity described to date from marine-derived Aspergillus sp.
Li et al. [14] isolated four new and one known bisabolane-type sesquiterpenoid from secondary metabolites of Aspergillus sp. from sponge. Compounds 1-5 showed different antibacterial activity against six pathogenic bacteria and two marine bacteria, and compounds 2 and 4 showed selective antibacterial activity. Compound 2 had strong inhibitory effects on Staphylococcus albus and Micrococcus tetragenus, with minimum inhibiting concentrations (MIC) values of 5.00 and 1.25 µM, respectively. The MIC values of compound 4 with S. albus and Bacillus subtilis were 5.00 µM and 2.50 µM, respectively. Notably, compound 1 represents the rare example of a bisabolane-type sesquiterpenoid with a 1, 4-disubstituted benzene ring isolated from marine organisms. Compounds 2 and 3 were the enantiomers of (+)-sydonol and (+)-sydonic acid, respectively. This fact suggests that fungi isolated from different marine organisms may produce different stereochemisty compounds. Furthermore, there were three sesquiterpenoids, 6-8, from the sponge-associated fungus Aspergillus sydowii ZSDS1-F6, which has certain antibacterial activities; among them, compound 6 and 7 displayed antibacterial activities against Klebsiella pneumonia, with MIC values of 21.4 and 10.7µM, respectively [15]. In addition, compound 6 showed moderate antibacterial activity against Aeromonas hydrophila (MIC, 4.3 µM), while compound 8 showed moderate antibacterial activity against Enterococcus faecalis (MIC, 18.8 µM). Chen et al. [16] isolated two phenolic bisabolane sesquiterpenoids (PBS) compounds (9-10) from Aspergillus flavipes 297, including a pair of new enantiomers (±)-flavilane A (9). However, compounds 9 and 10 represent the rare PBS-containing methylsulfinyl group and showed selective antibacterial activities against several pathogenic bacteria; their MIC values were 2-64 µg/mL. Furthermore, compound 10 exhibited mild antifungal activity against plant pathogenic fungus Valsa mari. Aromatic bisabolene-type sesquiterpenoids 11-13 were isolated from the marine fungus Aspergillus versicolor SD-330 in the deep-sea sediments [17]. Compounds 11 and 12 had significant inhibitory activities against A. hydrophilia, Escherichia coli, Edwardsiella tarda, and Vibrio harveyi, with MIC values ranging from 2.0 to 8.0 µg/mL. Moreover, compound 13 had significant inhibitory activity against E. coli (MIC value was 1.0 µg/mL), which was better than the positive control chloramphenicol (MIC value was 2.0 µg/mL). A new aromatic bisabolene-type sesquiterpenoid (14) was discovered in Aspergillus sydowii SW9, whose absolute configuration is (S). Compound 14 had significant inhibitory effect on E. coli, and its MIC value was 2.0 µg/mL, which was similar to that of positive control chloramphenicol (MIC 2.0 µg/mL). Compound 14 also exhibited potent activity against S. pneumonise, with an MIC value of 4.0 µg/mL [18]. Wang et al. [19] obtained four sesquiterpenoids 15-18 with antibacterial activity from marine Aspergillus versicolor SD-330. Compounds 15 and 16 showed significant antibacterial activity against E. coli, E. trada, V. harveyi, and Vibrio parahaemolyticus, and the MIC values were less than or equal to 8.0 Aromatic bisabolene-type sesquiterpenoids 11-13 were isolated from the marine fungus Aspergillus versicolor SD-330 in the deep-sea sediments [17]. Compounds 11 and 12 had significant inhibitory activities against A. hydrophilia, Escherichia coli, Edwardsiella tarda, and Vibrio harveyi, with MIC values ranging from 2.0 to 8.0 µg/mL. Moreover, compound 13 had significant inhibitory activity against E. coli (MIC value was 1.0 µg/mL), which was better than the positive control chloramphenicol (MIC value was 2.0 µg/mL). A new aromatic bisabolene-type sesquiterpenoid (14) was discovered in Aspergillus sydowii SW9, whose absolute configuration is (S). Compound 14 had significant inhibitory effect on E. coli, and its MIC value was 2.0 µg/mL, which was similar to that of positive control chloramphenicol (MIC 2.0 µg/mL). Compound 14 also exhibited potent activity against S. pneumonise, with an MIC value of 4.0 µg/mL [18]. Wang et al. [19] obtained four sesquiterpenoids 15-18 with antibacterial activity from marine Aspergillus versicolor SD-330. Compounds 15 and 16 showed significant antibacterial activity against E. coli, E. trada, V. harveyi, and Vibrio parahaemolyticus, and the MIC values were less than or equal to 8.0 µg/mL. However, compound 17 exhibited significant antibacterial effect on E. coli with MIC value of 1.0 µg/mL, which was more potent than that of positive control chloramphenicol (MIC 2.0 µg/mL). Moreover, compound 17 showed strong inhibitory activity against A. hydrophilia, E. tarda, Vibrio anguillarum, and V. harveyi, each with MIC value of 4.0 µg/mL. Compound 17 showed a stronger antibacterial activity than compounds 15 and 16, suggesting that C-15 carboxyl group methyl ester or the methylated C-7 hydroxyl group could reduce their antibacterial activity.
Wei et al. isolated three phenolic bisabolane-type sesquiterpenoids compounds 19-21 from Aspergillus sp., which is the first report of natural metabolites from marine fungus Aspergillus from gorgonian Dichotella gemmacea [20]. All of them exhibited weak antibacterial activity against Staphylococcus aureus, with the diameters of inhibition zones of 11, 7, and 5 mm at 100 µg/mL, respectively. Seven phenolic bisabolane sesquiterpenoids 22-28 were obtained from the endophytic fungus Aspergillus sp. xy02 from a Thai mangrove Xylocarpus moluccensis [21] and displayed moderate inhibitory activities against S. aureus, with IC 50 values ranging from 31.5 to 41.9 µM. Two new phenolic bisabolane sequiterpenes, asperchondols A (29) and asperchondols B (30), were obtained from the sponge-derived fungus Aspergillus sp. and showed antibacterial activity against S. aureus, with the MICs of 50 and 25 µM, respectively [22]. Furthermore, structure-activity relationship found that the coexistence of phenolic bisabolane sesquiterpene and diphenyl ether moieties seems to be very important since the hybrid 30 was more active than phenolic bisabolane sesquiterpenoid 29 and phenyl esters.
A series of phenolic bisabolane-type sesquiterpenoids have been discovered from different marine invertebrates such as sponges [64] and gorgonians [65] in the last century. In addition, such compounds were also found in bacterium CNH-741 and fungus CNC-979 isolated from marine sediments [66]. These results indicate that the real producers of these compounds from marine invertebrates, sponges, and corals may be constituents of microorganisms. Albican-11,14-diol (31) is a sesquiterpene compound isolated from the cultures of the endophytic fungus Aspergillus versicolor, which is isolated from marine green alga Codium fragile [23]. The diameters of inhibition zones of compound 31 against E. coli and S. aureus were 7 and 10.3 mm, respectively, at the concentration of 30 µg/disk. Fang et al. isolated a drimane-type sesquiterpenoid (32) and three unknown-type sesquiterpenoids (33)(34)(35) from the algicolos fungus Aspergillus sp. RR-YLW-12, which exhibited little inhibitory activity against four marine-derived pathogenic bacteria, V. anguillarum, V. harveyi, V. parahaemolytics, and Vibrio splendidus [24]. Zheng et al. isolated and purified three bisabolane sesquiterpenes 36-38 from the fermentation products of Aspergillus versicolor ZJ-2008015, which were obtained from a soft coral Sarcophyton sp. [25]. The results showed that compounds 36-38 exhibited potent antibacterial activity with MICs of 5.3, 6.4, and 5.4 µM against S. albus and 2.6, 6.4, and 5.4 µM against S. aureus, respectively. Cohen et al. [26] isolated two drimane sesquiterpenes (39)(40) from the sponge-derived fungus Aspergillus insuetus (OY-207), which exhibited anti-fungal activity against Neurospora crassa, with the MICs of 140 and 242 µM, respectively.
Insulicolide A (Nitrobenzoyl substituted sesquiterpene, 51) was isolated from the marine-sponge-associated endozoic fungus Aspergillus insulicola MD10-2 [31]. and ACHN (IC 50 of 4.1 and 11 µM, respectively), suggesting that the C-9 hydroxy group may contribute more to the cytotoxic activities against renal carcinoma cells. Additionally, compound 52 showed stronger inhibitory activity at low concentration levels, compared with the positive control sorafenib, a drug approved for the treatment of primary kidney cancer (advanced renal cell carcinoma). Further investigation revealed that the cell cycle was arrested at G 0 /G 1 phase after being treated with compound 52 at 1 µM, whereas after being treated at 2 µM for 72 h, the late apoptosis of 786-O cells were induced. Four nitrobenzoyl sesquiterpenoids (55-58) were isolated from an Antarctica-sponge-derived Aspergillus insulicola by Sun et al. [33], in which compounds 57 and 58 showed selective inhibitory activity against human pancreatic ductal adenocarcinoma (PDAC) cell lines, whereas compounds 55 and 56 were inactive, indicating that hydroxyl groups at C-9 is essential for cytotoxicity. Furthermore, the IC 50 values of compounds 57 and 58 against PDAC cell lines AsPC-1 and PANC-1 were 2.7, 4.6 µM and 2.3, 4.2 µM, respectively. Numerous studies have shown that most of nitrobenzoyl sesquiterpenes were obtained from the marine-derived fungus Aspergillus ochraceus, suggesting that Aspergillus ochraceus may be a good resource for the production of these compounds.

Anti-Inflammatory Activity
Inflammation is a comprehensive array of physiological response to a foreign organism, which has been considered as a major factor for the progression of various chronic diseases/disorders [71]. Therefore, development of effective and economical anti-inflammatory drugs (NSAIDs) is an area of importance in drug discovery while natural anti-inflammatory supplements are becoming more popular and have been the focus of many scientific investigations. This section covers 30 sesquiterpenoids (Figure 7) with anti-inflammatory activity which isolated from marine-derived Aspergillus sp. Cui et al. [45] isolated a sesquiterpene derivative (79) from the mangrove endophytic fungus Aspergillus versicolor SYSU-SKS025, which was found to inhibit nitric oxide (NO) production RAW 264.7 macrophages, with an IC50 value of 12.5 µM (positive control, indomethacin, IC50 = 37.5 µM). Wang et al. [46] found four triketide-sesquiterpenoids A−D (80-83) from the marine-algal-associated fungus Aspergillus sp. ZL0-1B14, which exhibited anti-inflammatory activity in LPS-stimulated RAW264.7 macrophages. In addition, compound 83 inhibited the production of IL-6 with an inhibition rate of 69% at 40 µM. Wu et al. [47] firstly discovered two brasilane sesquiterpenoids (84)(85) with α and β unsaturated ketones from marine-derived fungus Aspergillus terreus, both of which showed moderate inhibitory effects; the inhibitory rates of nitric oxide were 47.7% and 37.3%, respectively, at 40 µM. Chung et al. [48] isolated five sesquiterpenoids (86)(87)(88)(89)(90) with anti-inflammatory activity from Aspergillus sydowii in marine sediments. Among them, compounds 88 and 90 displayed selective inhibition against fMLP/CB-induced superoxide anion generation by human neutrophils, with IC50 values of 5.23 and 6.11 µM, respectively. At the same time, they also exhibited the most potent inhibitory activity against the release of elastase induced by fMLP/CB, with the IC50 values of 16.39 and 8.80 µM, respectively. Interestingly, the anti-inflammatory activity of compound 88 was better than that of compound 86 indicating the important role of hydroxy group on C-7. Moreover, compounds contain- Cui et al. [45] isolated a sesquiterpene derivative (79) from the mangrove endophytic fungus Aspergillus versicolor SYSU-SKS025, which was found to inhibit nitric oxide (NO) production RAW 264.7 macrophages, with an IC 50 value of 12.5 µM (positive control, indomethacin, IC 50 = 37.5 µM). Wang et al. [46] found four triketide-sesquiterpenoids A−D (80-83) from the marine-algal-associated fungus Aspergillus sp. ZL0-1B14, which exhibited anti-inflammatory activity in LPS-stimulated RAW264.7 macrophages. In addition, compound 83 inhibited the production of IL-6 with an inhibition rate of 69% at 40 µM. Wu et al. [47] firstly discovered two brasilane sesquiterpenoids (84)(85) with α and β unsaturated ketones from marine-derived fungus Aspergillus terreus, both of which showed moderate inhibitory effects; the inhibitory rates of nitric oxide were 47.7% and 37.3%, respectively, at 40 µM. Chung et al. [48] isolated five sesquiterpenoids (86)(87)(88)(89)(90) with anti-inflammatory activity from Aspergillus sydowii in marine sediments. Among them, compounds 88 and 90 displayed selective inhibition against fMLP/CB-induced superoxide anion generation by human neutrophils, with IC 50 values of 5.23 and 6.11 µM, respectively. At the same time, they also exhibited the most potent inhibitory activity against the release of elastase induced by fMLP/CB, with the IC 50 values of 16.39 and 8.80 µM, respectively. Interestingly, the anti-inflammatory activity of compound 88 was better than that of compound 86 indicating the important role of hydroxy group on C-7. Moreover, compounds containing methylene alcohol on C-3 (86, 88, and 90) showed more potent anti-inflammatory activity compared with the derivatives with carboxylic acid functional groups (87 and 89). Four Eremophilane sesquiterpenoids (91)(92)(93)(94) were isolated from deep-marine-sediment-derived fungus Aspergillus sp. SCSIOW2, and all showed inhibitory activity of NO production in a dose-dependent manner [49]. Additionally, five sesquiterpenoids (95-99) were isolated from the mangrove endophytic fungus Aspergillus sp. GXNU-MA1 by Zhou et al., which exhibited moderate inhibitory activities against NO production, with IC 50 values ranging from 16.15 to 27.08 µM [50]. Niu et al. isolated six phenolic bisabolane (100-105) and two cuparene sesquiterpenoids (106-107) from Aspergillus sydowii MCCC3A00324 derived from deep sea sediments [51]. Compounds 100, 101, and 103-105 showed anti-inflammatory activity against NO secretion in LPS-activated BV-2 microglia cells, with the inhibition rates of more than 45% at 10 µM, while those of compounds 102, 106, and 107 were 32.8%, 32.6% and 45.4%, respectively. Furthermore, compound 101 exerted an anti-inflammatory effect by inhibiting NF-κB activation pathway in a dose-dependent manner. Tan et al. isolated a new nitrobenzoyl sesquiterpenoid (108) from Aspergillus ochraceus, which could suppress the RANKL-induced osteoclats formation and bone resorption by targeting NF-κB [52]. Additionally, compound 108 attenuated inflammatory bone loss in vivo.

Enzymatic Inhibitory Activity
Enzyme inhibitors are of value in treating many diseases in clinical use, and have become a very attractive target for drug development and discovery. In recent years, the prominence of various enzyme inhibitors has been discussed extensively by many researchers in comprehensive systematic reviews [72]. In this section, the inhibitory activities of sesquiterpenoids ( Figure 8) from marine Aspergillus sp. against three enzymes (α-glucosidase, cholinesterase, and neuraminidase) are briefly reviewed. ing methylene alcohol on C-3 (86, 88, and 90) showed more potent anti-inflammatory ac tivity compared with the derivatives with carboxylic acid functional groups (87 and 89 Four Eremophilane sesquiterpenoids (91)(92)(93)(94) were isolated from deep-marine-sedimen derived fungus Aspergillus sp. SCSIOW2, and all showed inhibitory activity of NO pro duction in a dose-dependent manner [49]. Additionally, five sesquiterpenoids (95-99 were isolated from the mangrove endophytic fungus Aspergillus sp. GXNU-MA1 by Zho et al., which exhibited moderate inhibitory activities against NO production, with IC values ranging from 16.15 to 27.08 µM [50]. Niu et al. isolated six phenolic bisabolan (100-105) and two cuparene sesquiterpenoids (106-107) from Aspergillus sydow MCCC3A00324 derived from deep sea sediments [51]. Compounds 100, 101, and 103-10 showed anti-inflammatory activity against NO secretion in LPS-activated BV-2 microgli cells, with the inhibition rates of more than 45% at 10 µM, while those of compounds 102 106, and 107 were 32.8%, 32.6% and 45.4%, respectively. Furthermore, compound 101 ex erted an anti-inflammatory effect by inhibiting NF-κB activation pathway in a dose-de pendent manner. Tan et al. isolated a new nitrobenzoyl sesquiterpenoid (108) from Asper gillus ochraceus, which could suppress the RANKL-induced osteoclats formation and bon resorption by targeting NF-κB [52]. Additionally, compound 108 attenuated inflammator bone loss in vivo.

Enzymatic Inhibitory Activity
Enzyme inhibitors are of value in treating many diseases in clinical use, and hav become a very attractive target for drug development and discovery. In recent years, th prominence of various enzyme inhibitors has been discussed extensively by many re searchers in comprehensive systematic reviews [72]. In this section, the inhibitory activ ties of sesquiterpenoids ( Figure 8) from marine Aspergillus sp. against three enzymes (α glucosidase, cholinesterase, and neuraminidase) are briefly reviewed. α-Glucosidase is a membrane-bound enzyme present in the small intestinal epithe lium [73], whose role is to promote the absorption of glucose in the small intestine b catalyzing the hydrolysis of oligosaccharides into absorbable glucose. α-Glucosidase in hibitors are the most widely used drugs in the clinical treatment of diabetes in China. B inhibiting the activity of α-glucosidase, the formation and absorption of glucose can b reduced to achieve the goal of lowering blood glucose. At the same time, it can also reduc the stimulation of blood glucose on the pancreas, effectively preventing and relieving d abetic complications [74]. 7-Deoxy-7,14-didehydrosydonol (79) was found from the man grove endophytic fungus Aspergillus versicolor and possessed a significant inhibitory effec on α-glucosidase, with an IC50 value of 7.5 µM (acarbose as 350 µM), and the termina α-Glucosidase is a membrane-bound enzyme present in the small intestinal epithelium [73], whose role is to promote the absorption of glucose in the small intestine by catalyzing the hydrolysis of oligosaccharides into absorbable glucose. α-Glucosidase inhibitors are the most widely used drugs in the clinical treatment of diabetes in China. By inhibiting the activity of α-glucosidase, the formation and absorption of glucose can be reduced to achieve the goal of lowering blood glucose. At the same time, it can also reduce the stimulation of blood glucose on the pancreas, effectively preventing and relieving diabetic complications [74]. 7-Deoxy-7,14-didehydrosydonol (79) was found from the mangrove endophytic fungus Aspergillus versicolor and possessed a significant inhibitory effect on α-glucosidase, with an IC 50 value of 7.5 µM (acarbose as 350 µM), and the terminal ethylene group at C-7 may play a key role in α-glucosidase inhibition activity [45]. Wu et al. [53] isolated four phenolic bisabolane sesquiterpenoids (109-112) from the mangrove endophytic fungus Aspergillus flavus QQSG-3. The inhibitory activity studies of α-glucosidase showed that the compounds (109-112) had strong inhibitory effects, with IC 50 values of 4.5, 3.1, 1.5, and 2.3 µM, respectively (all lower than the positive control drug acarbose).
Alzheimer's Disease (AD) is a degenerative disease with unknown causes, mainly involving cerebral cortical neurons, which is the major cause of dementia [75]. The currently accepted pathogenesis is the cholinergic deficiency hypothesis [76]. Cholinesterase inhibitors (ChEI) are a class of drugs that can bind to cholinesterase (ChE) and inhibit ChE activity; they are also approved as first-line drugs for the treatment of mild-to-moderate AD [77]. Feng et al. firstly isolated the potential reversible cholinesterase inhibitor cyclopentapentalane sesquiterpenoid subergorgic (113) and its analogues 2-deoxy-2β-hydroxysubergorgic (114) from the soft-coral-derived fungus Aspergillus sp. EGF15-0-3 [54].
Neuraminidase (NA) is the most critical enzyme for influenza virus replication and diffusion in host cells and has become an important target for anti-influenza virus drug design [78]. Li et al. [55] isolated four drimane sesquiterpenoids (115-118) from the ascidian endophytic fungus Aspergillus ustus TK-5, which showed significant inhibitory activity against neuraminidase, with IC 50 values of 31.8, 37.3, 28.4, and 36.8 µM, respectively. Further results showed that the degree of unsaturation of 11-OH and C-6 linked side chains, which can improve their neuraminidase inhibitory activity.
Alzheimer's Disease (AD) is a degenerative disease with unknown causes, mainly involving cerebral cortical neurons, which is the major cause of dementia [75]. The currently accepted pathogenesis is the cholinergic deficiency hypothesis [76]. Cholinesterase inhibitors (ChEI) are a class of drugs that can bind to cholinesterase (ChE) and inhibit ChE activity; they are also approved as first-line drugs for the treatment of mild-to-moderate AD [77]. Feng et al. firstly isolated the potential reversible cholinesterase inhibitor cyclopentapentalane sesquiterpenoid subergorgic (113) and its analogues 2-deoxy-2β-hydroxysubergorgic (114) from the soft-coral-derived fungus Aspergillus sp. EGF15-0-3 [54].
Neuraminidase (NA) is the most critical enzyme for influenza virus replication and diffusion in host cells and has become an important target for anti-influenza virus drug design [78]. Li et al. [55] isolated four drimane sesquiterpenoids (115-118) from the ascidian endophytic fungus Aspergillus ustus TK-5, which showed significant inhibitory activity against neuraminidase, with IC50 values of 31.8, 37.3, 28.4, and 36.8 µM, respectively. Further results showed that the degree of unsaturation of 11-OH and C-6 linked side chains, which can improve their neuraminidase inhibitory activity.

Chemically Induced Synthesis
Aspergillus sp. is the important source for the discovery of natural active products with novel and diverse structures. However, in recent years, the continual study of secondary metabolites of marine fungi has led to a high frequency of repeated discovery of known compounds [83]. This encourages us to develop new strategies to obtain new natural products. Studies have found that a large number of secondary metabolite biosynthesis gene clusters exist in the genome of Aspergillus fungi. Furthermore, the genome can be segmented into active and silent clusters, while the silent clusters are inactive under normal environmental conditions [84][85][86]. In order to obtain more active metabolites, researchers have applied a variety of methods to activate silenced biological genetic gene clusters, such as transcription factor regulation, targeted genome mining, heterologous expression of gene clusters, and chemical epigenetic regulation [87][88][89]. Because of its simplicity and effectiveness, chemical epigenetic regulation has been widely used in marine fungi to activate silenced gene clusters, which could lead to the production of new secondary metabolites or known components with a higher concentration. Wang et al. [90] cultivated the gorgonian-derived fungus Aspergillus sp. SC-20090066 with a DNA methyltransferase inhibitor 5-azacytidae (5-AZA) in the culture medium and led to the isolation of six new bisabolane-type sesquiterpenoids ( Figure 10). Among them, compounds (128-130) exhibited broad spectrum activities against S. aureus, Bacillus cereus, Rhizophila, Pseudomonas putida, and Pseudomonas aeruginosa, with MICs of less than 25 µM. In particular, compound 130 exhibited significant antibacterial activity against S. aureus, with MIC value of 3.13 µM, which was close to the positive control ciprofloxacin (MIC value was 2.5 µM). In order to trigger the chemical diversity of marine-derived fungus Aspergillus versicolor XS-2009006, epigenetic agents (histone deacetylase inhibitor SAHA and DNA methyltransferase inhibitor 5-AZA) were added to the culture medium by Wu et al. [91] Interestingly, the secondary metabolites was significantly increased and a new bisabolane sesquiterpene aspergillusene E (131, Figure 10) was isolated, which showed anti-larval attachment activity against bryozoan B. neritina, with the EC 50 and (lethal concentration 50%) LC 50 values of 6.25 µg/mL and 25 µg/mL, respectively. In addition, compound 131 showed certain antibacterial activities against Staphylococcus epidermidis and S. aureus, with MIC values ranging from 8 to 16 µM. By adding DNA methyltransferase inhibitors to the medium of Aspergillus sydowii, the composition of secondary metabolites was further changed and new bisabolane sesquiterpenoids (86)(87) were isolated [48]. In addition, Wang et al. [49] applied chemical epigenetic manipulation to Aspergillus sp. SCSIOW2 and obtained four eremophilane sesquiterpenes with anti-inflammatory activity (91)(92)(93)(94).

Chemical Synthesis and Biosynthesis of Sesquiterpenoids from Marine
Aspergillus sp.

Chemically Induced Synthesis
Aspergillus sp. is the important source for the discovery of natural active products with novel and diverse structures. However, in recent years, the continual study of secondary metabolites of marine fungi has led to a high frequency of repeated discovery of known compounds [83]. This encourages us to develop new strategies to obtain new natural products. Studies have found that a large number of secondary metabolite biosynthesis gene clusters exist in the genome of Aspergillus fungi. Furthermore, the genome can be segmented into active and silent clusters, while the silent clusters are inactive under normal environmental conditions [84][85][86]. In order to obtain more active metabolites, researchers have applied a variety of methods to activate silenced biological genetic gene clusters, such as transcription factor regulation, targeted genome mining, heterologous expression of gene clusters, and chemical epigenetic regulation [87][88][89]. Because of its simplicity and effectiveness, chemical epigenetic regulation has been widely used in marine fungi to activate silenced gene clusters, which could lead to the production of new secondary metabolites or known components with a higher concentration. Wang et al. [90] cultivated the gorgonian-derived fungus Aspergillus sp. SC-20090066 with a DNA methyltransferase inhibitor 5-azacytidae (5-AZA) in the culture medium and led to the isolation of six new bisabolane-type sesquiterpenoids ( Figure 10). Among them, compounds (128-130) exhibited broad spectrum activities against S. aureus, Bacillus cereus, Rhizophila, Pseudomonas putida, and Pseudomonas aeruginosa, with MICs of less than 25 µM. In particular, compound 130 exhibited significant antibacterial activity against S. aureus, with MIC value of 3.13 µM, which was close to the positive control ciprofloxacin (MIC value was 2.5 µM). In order to trigger the chemical diversity of marine-derived fungus Aspergillus versicolor XS-2009006, epigenetic agents (histone deacetylase inhibitor SAHA and DNA methyltransferase inhibitor 5-AZA) were added to the culture medium by Wu et al. [91] Interestingly, the secondary metabolites was significantly increased and a new bisabolane sesquiterpene aspergillusene E (131, Figure 10) was isolated, which showed anti-larval attachment activity against bryozoan B. neritina, with the EC50 and (lethal concentration 50%) LC50 values of 6.25 µg/mL and 25 µg/mL, respectively. In addition, compound 131 showed certain antibacterial activities against Staphylococcus epidermidis and S. aureus, with MIC values ranging from 8 to 16 µM. By adding DNA methyltransferase inhibitors to the medium of Aspergillus sydowii, the composition of secondary metabolites was further changed and new bisabolane sesquiterpenoids (86)(87) were isolated [48]. In addition, Wang et al. [49] applied chemical epigenetic manipulation to Aspergillus sp. SCSIOW2 and obtained four eremophilane sesquiterpenes with anti-inflammatory activity (91)(92)(93)(94).

Biosynthetic Pathways
The skeleton structures of sesquiterpenoids were derived from farnesyl diphosphate (FPP) and underwent a series of reaction steps, including intramolecular rearrangement, cyclysis, and other biosynthetic transformations, leading to their structural diversity [92].
Ingavat et al. [93] studied the proposed biosynthesis of sesquiterpene compound 132 in Aspergillus aculeatus, which starts from a double-bond migration (C1/C2 to C2/C3) of silphineneene intermediate 2, and then the double bond of C2/C3 undergoes oxidative cleavage to generate intermediate 3, which, in turn, undergoes a series of oxidation and lactonizations to finally give 132 ( Figure 10).
Wang et al. [46] proposed a biogenetic pathway for the synthesis of aspertetranones A-D (80)(81)(82)(83). Common drimane-type merosesquiterpene were obtained by cyclization of farnesylated pyrone, followed by oxidation and retro-aldo/aldo rearrangement to produce the unique terpenoid part of aspertetranones. After nucleophilic attack and dehydration, the leaborate preaspertetranone was obtained. Illudalanes derive biosynthetically from a humulene precursor after cyclization, producing a protoilludanes, which is eventually rearranged to form the irudane derivative [94]. According to this report, Orfali et al. speculated a biosynthetic pathway of asperorlactone (41), in which illudol was a key intermediate. The iluane-type sesquiterpene asperorlactone can be synthesized by dehydration, oxidation, and four-membered ring opening [27].

Potency of Sesquiterpenoids from Marine Aspergillus sp.
Secondary metabolites of microorganisms in the marine environment, mainly derived from marine fungi, are a great source for new drug screening. Currently, the marine drug library includes 15 approved drugs (primarily for cancer treatment), 7 phase I compounds, 12 phase II compounds, and 5 compounds in phase III clinical trials, the latter including a recently recommended drug for symptomatic treatment of COVID-19 (Plitidepsin) [95,96]. Compound 13 displayed significant inhibitory activity against E. coli (MIC 1.0 µg/mL), and its antibacterial effect was more potent than that of the positive control chloramphenicol (MIC 2.0 µg/mL), which was expected to be a lead compound for antibiotics [17]. The sesquiterpene compound (79) isolated from Aspergillus versicolor exhibited better inhibitory effect on α-glucosidase than acarbose, while its anti-inflammatory effect was also stronger than that of indomethacin [45]. Compound 88 derived from marine sediments, showed a significant anti-inflammatory effect and hypoglycemic effect. In addition, compound 88 could also inhibit fat accumulation in adipocytes [48]. These results indicated compound 79 and 88 has the potential to be a lead compound targeting the vicious diabetes-inflammation cycle. Feng et al. found that sesquiterpene compound 113, the reversible cholinesterase inhibitor, is a promising new drug candidate for the treatment of Alzheimer's Disease and a preclinical trial is already under way [54].

Conclusions and Perspective
In this paper, the biosources, bioactivities, structural types, biosynthetic, and pharmacogenic potential of sesquiterpenoids found from marine fungi Aspergillus sp. were reviewed. A total of 268 sesquiterpenes were isolated, including 131 bioactive sesquiterpenes, most of which were bisabolanes, followed by drimanes and nitrobenzoyl, etc. Most Aspergillus species derived from sponges, marine sediments, algae, mangroves, corals, etc. The main Aspergillus species involved are as follows: Aspergillus fumigatus, Aspergillus versicolor, Aspergillus flavus, Aspergillus ustus, Aspergillus sydowii, and so on. These sesquiterpenes exhibited excellent pharmacological activities such as antibacterial, antitumor, antiinflammatory, and enzyme inhibitory activities. Additionally, the biosynthesis and total synthesis of sesquiterpenes derived from marine Aspergillus sp. have also promoted the in-depth understanding of these sesquiterpenes. Because of the chemical and biological activity of these sesquiterpenoids, it is worthwhile to find promising lead compounds for the development of marine drugs in further studies from marine fungi.