Comprehensive Overview on the Chemistry and Biological Activities of Selected Alkaloid Producing Marine-Derived Fungi as a Valuable Reservoir of Drug Entities

Marine-associated fungal strains act as a valuable reservoir of bioactive diverse secondary metabolites including alkaloids which are highly popular by their biological activities. This review highlighted the chemistry and biology of alkaloids isolated from twenty-six fungal genera associated with marine organisms and marine sea sediments. The selected fungi are from different marine sources without focusing on mangroves. The studied fungal genera comprises Acrostalagmus, Arthrinium, Chaetomium, Cladosporium, Coniothyrium, Curvularia, Dichotomomyces, Eurotium, Eutypella, Exophiala, Fusarium, Hypocrea, Microsphaeropsis, Microsporum, Neosartorya, Nigrospora, Paecilomyces, Penicillium, Pleosporales, Pseudallescheria, Scedosporium, Scopulariopsis, Stagonosporopsis, Thielavia, Westerdykella, and Xylariaceae. Around 347 alkaloid metabolites were isolated and identified via chromatographic and spectroscopic techniques comprising 1D and 2D NMR (one and two dimensional nuclear magnetic resonance) which were further confirmed using HR-MS (high resolution mass spectrometry) and Mosher reactions for additional ascertaining of the stereochemistry. About 150 alkaloids showed considerable effect with respect to the tested activities. Most of the reported bioactive alkaloids showed considerable biological activities mainly cytotoxic followed by antibacterial, antifungal, antiviral, antioxidant; however, a few showed anti-inflammatory and antifouling activities. However, the rest of the compounds showed weak or no activity toward the tested biological activities and required further investigations for additional biological activities. Thus, alkaloids isolated from marine-associated fungi can afford an endless source of new drug entities that could serve as leads for drug discovery combating many human ailments.


Introduction
Nowadays fungi isolated from marine resources serve as promising tools for the alleviation of a large number of hazardous diseases that adversely affect human health such as bacterial and viral infections as well as cancers [1,2]. These prominent effects are greatly relied upon their richness by large categories of secondary metabolites represented by peptides, steroids, terpenoids, lactones, and alkaloids [3,4].These activities are mainly anti-inflammatory, antibacterial, anticancer, and antiviral activities [5,6]. A vast variation in the function and structure of the abundant metabolites in the marine-derived fungal strains is undoubtedly based upon the considerable diversity in the environment where these organisms exist regarding its chemical and physical formation [7].
In addition, these fungal metabolites showed an acceptable oral-bioavailability and physico-chemical manner offering a safer biomedical alternative relative to synthetic entities that constitute a crucial importance in the process of formulation of various dosage Luteoalbusins A (1) and B (2) are two new alkaloids of indole diketopiperazine type which were isolated from Acrostalagmus luteoalbus obtained from deep-sea marine sediments existing in the South China Sea in addition to eight previously isolated alkaloids (3)(4)(5)(6)(7)(8)(9)(10) as illustrated in Figure 1. Compounds (1-5) exhibited a potent cytotoxic activity against HepG-2, MCF-7, SF-268, as well as NCI-H460 cell lines. Noteworthy to mention that luteoalbusins A (1) and B (2) showed superior cytotoxic activity comparable to other isolated compounds and to cisplatin showing IC 50 ranging between 0.23 and 1.29 µM [14].

Chaetomium
In depth phytochemical investigation of Chaetomium globosum derived from a deepsea marine sediment from the Indian oceans led to the isolation of a series of cytoglobosins comprising chaetoglobosin B (23), chaetoglobosin C (24), isochaetoglobosin D (25), chaetoglobosin E (26), chaetoglobosin F (27), chaetoglobosinFex (28) in addition to the new alkaloids, cytoglobosins H (29) and I (30). In the evaluation of the cytotoxicity of the previously mentioned compounds on MDA-MB-231, B16F10, and LNCaP, only chaetoglobosin E (26) exhibited a pronounced antiproliferative activity via induction of apoptosis on LNCaP as well as B16F10 cancer cells displaying IC 50 of 0.62 and 2.78 µM, respectively [17]. Noteworthy to highlight that chaetoglobosin Fex (28) showed a wide array of biological activities showing an immunosuppressive effect thus it could serve as a good candidate in the treatment of autoimmune inflammatory disorder as it effectively inhibited the stimulation of bone marrow-derived dendritic cells which was induced by poly(I:C). This was achieved via attenuation of the production of IFN-β in both mRNA as well as protein level in addition to inhibiting the phosphorylation of IκB-α, IRF-3, p38, and JNK, exerting no effect on ERK1/2 for p38 and JNK. [18]. Chaetomium globosum, marine-derived endophytic fungus, also prohibits the stimulation of the inflammatory mediators through Toll-like receptor 4 signaling present in macrophages. Pre-incubation of LPS-stimulated macrophages cells with chaetoglobosin Fex (28) (0.5 mg/mL) significantly inhibited LPS-induced intracellular TNF-α production (15.2% inhibition by 0.5 mg/mL, 21.3% inhibition by 1 mg/mL, and 56.7% inhibition by 2 mg/mL). Treatment with different concentration of chaetoglobosin Fex (28) also blocked IL-6 secretion induced by LPS for 20 h [19]. Additionally, three azaphilone alkaloids possessing glutamine moieties were newly isolated from the deep sea sediment associated Chaetomium globosum which are N-glutarylchaetoviridins A-C (31)(32)(33). N-glutarylchaetoviridin C (33) exerted a significant activity versus human gastric cancer cell line (MGC-803) and human ovarian cancer cell line (HO8910) displaying IC 50 values equal to 6.6 and 9.7 µM, respectively ( Figure 3) [20].

Cladosporium
In depth phytochemical investigation of the mycelium extract of the marine-derived fungus Cladosporium, fungal strain isolated from the surface of the marine red alga Chondria crassicualis collected at Yokji Island, Kyeongnam Province, Korea, resulted in the isolation of benzodiazepine alkaloid, circumdatin A (34). It shows no antibacterial activity versus methicillin-resistant S. aureus, S. aureus, or multidrug-resistant S. aureus. However, it showed a powerful antioxidant activity evidenced by its UV-A protection potential displaying ED 50 = 82.0 µM which in turn highlights its superior activity comparable to oxybenzone, positive control widely used in sunscreen formulation, that showed ED 50 = 350 µM ( Figure 3) [21].

Curvularia
Curvularia, the marine-associated fungus, was isolated from the gut of Argyrosomus argentatus collected from the Yellow Sea yielded the isolation of an alkaloid of a novel skeleton that is termed curvulamine (37) that showed a potent antimicrobial activity ( Figure 3) [23].
An additional study was performed on one of Eurotium sp. to assess the antifouling activity of its isolated indole alkaloids, neoechinulin A (99) and echinuline (95). The two compounds showed a notable inhibition to barnacle larval settlement with EC 50 values equal to 15.0 and 17.5 µg/mL, respectively. Meanwhile, dihydroxyisoechinulin A (98) displayed a weak antifouling activity, however, variecolortide B (126), variecolortide C (127), and 7-O-methylvariecolortide A (89) isolated from the species were inactive [33].

Eutypella
In depth phytochemical investigation of deep-sea-derived fungus Eutypella, deep sea marine sediment, collected with TV-multicore from South Atlantic Ocean, resulted in the isolation and structural elucidation of thirteen new thiodiketopiperazine-type alkaloids, eutypellazines A-M (137-149) ( Figure 8). Their structures were further confirmed via Mosher's reaction, ECD data, and X-ray single-crystal diffraction for actual determination of the absolute configuration. The isolated compounds were assessed for their anti-HIV potential (human immunodeficiency virus type (1) using pNL4.3.Env-.Luc co-transfected 293T cells. Most of the new compounds revealed significant anti-HIV effect with IC 50 ranging between 3.2 and 18.2 µM with eutypellazine E (141) revealing the highest potency (IC 50 = 3.2 µM) [36].

Exophiala
A new benzodiazepine alkaloid namely circumdatin I (150) in addition to circumdatin C (151) and G (152) were isolated from the marine-associated fungus Exophiala. They were examined for their UV-A protective behavior where they all showed a potent activity with EC 50 values equal to 98, 101, and 105 µM, respectively showing higher potency when compared to oxybenzone (ED 50 , 350 µM), which was used as a positive control being a commonly used sunscreen agent ( Figure 9) [37].

Fusarium
Phytochemical investigation of the crude extract of marine-associated fungus, Fusarium oxysporum, isolated from the marine mudflat collected at Suncheon Bay, Korea, resulted in the isolation of a new polycyclic quinazoline alkaloid, oxysporizoline (153) that revealed an antibacterial activity against MRSA and MDRSA with MIC equal to 6.25 µg/mL in addition to notable antioxidant potential manifested by its observable radical scavenging effect versus DPPH with IC 50 equals to 10 µM ( Figure 9) [38].

Microsphaeropsis
Bioassay-guided fractionation of a marine sediment-derived fungus, Microsphaeropsis, which was collected from the Huanghua in the Bohai Sea, resulted in the isolation of three alkaloids which are fumiquinazolines L (155) and N (156) and notoamide D (157) (Figure 9) [40].

Microsporum
Neoechinulin A (99), a prenylated indole alkaloid, was isolated from the extract of the culture broth of marine-derived Microsporum sp., isolated from the surface of a marine red alga Lomentaria catenata, collected at Guryongpo, NamGu, PoHang in Republic of Korea. The alkaloid revealed a potent cytotoxic effect on HeLa cells inducing apoptosis manifested by the p21, p53, Bax, Bcl-2, caspase 3, and caspase 9 expressions. Neoechinulin A (99) effectively enhances cell apoptosis via the downregulation of Bcl-2 expression with concomitant upregulation of Bax expression and enhancement of caspase-3 as evidenced by the Western blot ( Figure 9) [41].

Neosartorya
Phytochemical investigation of the ethyl acetate extract obtained from the fermentation broth of marine-derived fungus Neosartorya fischeri, isolated from marine mud in the intertidal zone of Hainan Province of China, resulted in the characterization of three alkaloids, two of which are new namely, tryptoquivaline T (158), tryptoquivaline U (159) in addition to fiscalin B (160). All the tested compounds showed notable cytotoxic potential evidenced by induction of HL-60 cells apoptosis displaying IC 50 values of 82.3, 90.0, and 8.88 µM respectively [42]. Furthermore, harmane (161) was isolated from the sponge derived fungus Neosartorya tsunodae, isolated from the marine sponges Aka coralliphaga, collected at the coral reef of Similan Islands, Phang Nga Provice [43]. Besides, four new alkaloids were isolated from a marine-associated fungus, Neosartorya sp. which are tryptoquivalines P-S (162-165) ( Figure 9) [44].

Nigrospora
Two new alkaloids namely nigrospine (166) and nigrospin A (167) were isolated from Nigrospora oryzae, a marine-derived fungus isolated from a marine gorgonian Verrucella umbraculum collected in the South China Sea near Sanya City. The former is a pyrrolidinone alkaloid meanwhile the latter is indole type alkaloid. The absolute configuration of both compounds were determined employing Mosher reaction ( Figure 9) [45].
In depth phytochemical investigation of P. raistrickii, a marine-derived fungus, led to the isolation of four alkaloids, two of which, raistrickindole A (291) and raistrickin (292) represent new alkaloids, the former possess indole diketopiperazine group meanwhile the latter contains benzodiazepine moiety. Additionally, two new alkaloids, sclerotigenin (293) and haenamindole (294) were isolated. Both compounds showed potent antiviral activity versus hepatitis C virus [78]. Furthermore, P. vinaceum, isolated from the marine sponge Hyrtios erectus collected from Yanb, contains a lot of alkaloids comprising a new one, penicillivinacine (295), in addition to other known compounds including terretrione A (296), indol-3-carbaldehyde (297), brevianamide F (298), α-cyclopiazonic acid (299) ( Figure 14). Penicillivinacine (295) and terretrione A (296) showed a considerable antimigratory potential versus the greatly metastatic MDA-MB-23 cells (human breast cancer cells) displaying IC 50 values of 18.4 and 17.7 µM, respectively. In addition all the isolated compounds were tested for their antimicrobial activity against a panel of micro-organism including Staphylococcus aureus, Escherichia coli, and Candida albicans; Brevianamide F (298) showed antibacterial activity against Staphylococcus aureus displaying 19 mm as diameter of inhibition zone; meanwhile α-cyclopiazonic acid showed effect versus E. coli and with zone of inhibition equals to 20 mm. Both terretrione A (296) and brevianamide F (298) displayed inhibition zones of 27 and 25 mm against Candida albicans, respectively [79].

Pleosporales
A deep phytochemical exploration of the ethyl acetate extract obtained from a marinederived fungus isolated from marine sediment collected from the Huanghua in the Bohai Sea, Pleosporales led to the isolation of three alkaloids of diketopiperazine type namely, fructigenine A (300), fructigenine B (192), and brevicompanine G (211). None of the isolated compounds revealed any antimicrobial activity when tested versus 16 pathogenic microbial strains ( Figure 15) [80].

Scedosporium
In depth phytochemical screening of the ethyl acetate extract of the marine fungus S. apiospermum fed by different amino acids led to the isolation of various new alkaloids comprising scedapins A-E (320-324) in addition to scequinadoline D (325). Both scedapin C (322) and scequinadoline D (325) showed promising antiviral potential versus hepatitis C ( Figure 16) [84].

Scopulariopsis
In depth phytochemical investigation of Scopulariopsis, isolated from the fresh crushed inner tissues of the Red Sea hard coral Stylophora sp., led to the isolation of three alkaloids, one of which is new, scopulamide (326) in addition to two known alkaloids, lumichrome (327) and WIN 64,821 (328). They showed weak cytotoxic effect versus e L5178Y mouse lymphoma cell line [85]. In addition, six dihydroquinolin-2-one containing alkaloids namely, aflaquinolone A, D, F, G (329-332), and 6-deoxyaflaquinolone E (333) were also isolated from Scopulariopsis. All the isolated alkaloids showed antifouling effect against larval settlement of barnacle Balanus amphitrite additionally aflaquinolone A (329) showed a significant activity with EC 50 value of 17.5 pM (Figure 16) [86].
A pie chart representing the percentages of the biological activities exerted by the different bioactive alkaloids is represented in Figure 18. Additionally, a table summarizing the reported alkaloids, their biological activities and resources is illustrated in Table 1.

Conclusions
Thus, it can be concluded that marine-derived fungal strains are a very rich source of alkaloids. About 347 alkaloid metabolites were isolated from about twenty-six genera of fungi. About 150 alkaloids showed considerable effect with respect to the tested activities. Most of the reported bioactive alkaloids showed considerable biological activities mainly cytotoxic followed by antibacterial, antifungal, antiviral, antioxidant; however, a few showed anti-inflammatory and antifouling activities. However, the rest of the compounds showed weak or no activity toward the tested biological activities. Thus, alkaloids isolated from marine-associated fungi can afford an endless source of new drug entities that could serve as leads for drug discovery combating many human ailments. However, further investigations for additional biological activities for alkaloids that revealed no activity should be performed.