Unearthing the Potential Fungal Bioactive Secondary Metabolites from the Red Sea: A Comprehensive Review

The Red Sea represents a potential ecosystem with very rich biodiversity in the production of unique natural compounds. Recently, fungal secondary metabolites have been a major domain of research in the meadow of natural compounds with special emphasis on fungal strains isolated from marine ecosystems. More than 150 interesting secondary metabolites going with different classes of natural compounds had been reported with potential biological actions as cytotoxic activity with emphasis on the application of different approaches for example OSMAC ( one strain many compounds ) approach. The current review presents a comprehensive report for fungal secondary metabolites isolated from the Red Sea ecosystems either as endophytes from marine invertebrates (for example corals and sponges) and plants for example (algae and mangroves) or as free-living strains collected from sea sediments from different locations of Red Sea. Different databases had been utilized including the EKB (Egyptian Knowledge Bank), SciFinder, and MarinLit. In no doubt, the richness of published data directs to the marine fungi's medicinal potential. It will continue to be challenging to incorporate marine fungus into traditional therapy. The clinical environment demonstration of therapeutic activity is a fundamental task for medical research. For the profit of patients all through the world, it is to be wished that this significant barrier will be removed in the years to come.


Introduction
The creation of new drugs may benefit from the abundant supplies of bioactive secondary metabolites found in marine habitats, thousands of outstanding novel bioactive molecules have been identified from different marine resources [1], many of them acting as drug leads in the course of discovery of anti-cancer, antimicrobial, immunosuppressive, anti-biofilm, antiinflammatory drug candidates, among others [2].
Research in the field of Sea bioactive secondary compounds, mainly from marine invertebrates, has been confronted with many obstacles, mainly, the scarcity of the organisms, and the limitations of collections of endangered species added to the very low yield of active metabolites [3].Although the chemical synthesis of interesting metabolites can provide a solution to these obstacles, however, in many cases, it is not the ideal solution.Endosymbionts could be regarded as an important alleviation of these obstacles, taking into consideration the cross interaction between the biosynthetic pathways between the host and the endosymbionts, where in many cases the host organism is not the actual producer of the metabolite of interest, or at least the symbiotic micro-organism can provide the host with valuable chemical defense means with interesting biological activities [4].
As a result of their potential to create a huge variety of bioactive secondary metabolites, marine-derived fungi have drawn a lot of attention for drug discovery during the past 20 years [5].with thousands of interesting secondary metabolites being identified revealing potential biological and pharmacological properties [6,7].Anthraquinones, terpenes, peptides, and steroids are the interesting molecules yielded from marine-derived fungi [8][9][10][11].
The Red Sea has a distinctive maritime ecosystem with numerous varied coral reefs displaying high variety along its almost 2,000kilometer length and nearly 16 degrees of latitude [12].The Red Sea is home to a wide variety of species that have yet to be properly characterized and categorized.This biodiversity has inspired chemists and others to investigate the connections and interactions between corals and other species and to create new marine biotechnology applications, such as the potential future discovery of novel medications, enzymes, and nutraceuticals from cultivable marine microbes [13].
In this report, bioactive secondary metabolites isolated from the Red Sea-derived fungi spanning the period from 2000 to 2022 have been highlighted with emphasis on the interesting biological activities of the identified metabolites.Many databases were screened in writing this manuscript, including scifinder®, Egyptian knowledge bank "EKB", and MarinLit ® , in addition to relevant review articles [14, 18-21].
Fractionation of the ethyl acetate extract of the endophyte A. flavipes culture broth DY001 selected from the internal parts of the Red Sea tunicate Didemnum sp.resulted in the isolation of two diketopiprazine alkaloids (17)(18) and two Isocoumarin derivatives (19)(20).The isolated compounds were identified as Asperopiprazines A and B (17)(18), +-Citreoisocoumarin (19), and (-)-6,8-di-O-methylcitreoisocoumarin (20).Compounds 17-20 were evaluated for their antimicrobial as well as cytotoxic activities, where compounds 17-18 revealed adequate antibacterial action against E.coli and S.aureas, whereas compounds 17-20 showed potent to adequate cytotoxic action against HCT-116 cancer cell lines that have IC 50 values 15.1, 16.2, 19.3, 17.2 µM, respectively together with moderate activity against MDA-MB-113 cell lines and no activity against Hela cell lines, suggesting selectivity against human colon cancer cell lines [27].Marine sediments from the Canyons of Dahab, Red Sea had yielded the fungus A. falconensis, and chromatographic examination of the ethyl acetate extract of its solid rice fermentation media had yielded 11 azaphilone derivatives (21)(22)(23)(24)(25)(26)(27)(28)(29).Interestingly, applying the OSMAC approach (One strain with many compounds) resulted in the diversity of the isolated compounds, where the fungal strain was fermented on Rice medium supplied with either 3.5% NaCl or 3.5% NaBr.NaCl supplied medium had resulted in the isolation of new chlorinated derivative Falconensins O and P (21)(22), together with the identified Falconensins (23-24) A, M, N, and H, respectively, on the other hand NaBr enriched media had given rise to in the isolation of two new brominated derivatives Falconensin Q (25) and Falconensin R (26), and the new non-halogenated Falconensin S (27) together with the known derivatives Falconensin K (28) and Falconensin I (29).Compounds 21, 23-27, and 29 were tested for their ability to reduce inflammation in a triplenegative cell line of breast cancer.were estimated for their anti-inflammatory action on the triplenegative breast cancer cell line NF-kB-MDA-MB-231 revealing potent to moderate antiinflammatory activity, with Falconensin Q (25) being the most potent showing IC 50 11.9µM without cytotoxicity [28].The same research group had informed additional metabolites from the same fungal strain A. falconensis cultivated on solid rice medium with the addition of salts 3.5 % NaCl or (NH 4 ) 2 SO 4 , where eight diverse polyketides had been isolated from the NaClcontaining medium including the new dibenoxepin derivative Aurgosin O (30) and the new Isocoumarin 2-(8-Hydroxy-6methoxyisochromen-3′-yl) acetic acid (31) along with the six known polyketides Arugosin C (32), Dichlorodiaportin (33), Desmethyldiaportinol (34), Question (35), Diorcinol (36), and 4-Hydroxybenzaldehyde (37).On the other hand, the (NH 4 ) 2 SO 4 -containing medium had directed the fungal strain to produce additional metabolites namely, Sulochrin (38), Monochlorosulochrin (39) and Dihydrogeodin (40).Isolated compounds 30-40 has been evaluated for their cytotoxicity using human DNA topoisomerase II (TOP-2), in silico (molecular modelling) utilizing matrix metalloproteinase 13 (MMP-13), and human cyclin-dependent kinase 2 (CDK-2) where compounds 36-39 revealed significant stability and activity within the active pocket of CDK-2, in vitro cytotoxic assay using mouse lymphoma cell lines compound 38 showed potent cytotoxicity at IC 50 of 5.1µM [29].
The calcium chelating complex Coumamarin (41) along with 4 known compounds Diorcinol (42), Violaceol I (43), and Hydroxysydonic acid (44) were selected from the methanolic extract of A. sydowii fungus ASTI derived from a sample of marine water taken from Tiran Island, the Red Sea.Compound 41 represents the first calcium coumarin complex to be isolated from nature, showing potent antibacterial activity against Bacillus subtilis with no cytotoxicity against the cell line of human cervix carcinoma (KB-3-1) The same research group had stated the isolation and identification of two illudalane sesquiterpenes 46-47 from the same fungal strain A.oryzae.The compounds were identified as Asperorlactone (46) and Echinolactone D (47).two identified pyrones, 4-(hydroxymethyl)-5hydroxy-2H-pyran-2-one (48) and its acetate (49).Compounds 46 and 47 exhibited small cytotoxic actions against A-549 cancer cell lines.It is noteworthy to mention that this was the first report for illualane type sesquiterpenes from Ascomycetes
The same research group had reported additional metabolites from F.equiseti applying the OSMAC approach using different culture media, the metabolites were identified as Cyclo-l-Ala-l-Leu (95), Cyclo(l-Pro-l-Val) (100), Uracil (101), Thymine   In an interesting study, Elhady et al. reported the therapeutic potential of the alkaloid Meleagrin (117) selected from the liquid culture ethyl acetate extract of the fungus P. Chrysogynum S003 derived from the sea sediment collected from the Red Sea, Saudia Arabia.The authors reported the potent protective effect of meleagrin against Bleomycin induced lung fibrosis in rats with the restoration of the antioxidant markers balance and activation of the antioxidant signaling pathways, moreover, it revealed the ability to decrease the release of pro-inflammatory cytokines and to delay the bleomycin-induced apoptosis [43].
A new cerebroside, LAMA-1 (118), along with Kojic acid (119) had been selected and identified from the liquid culture ethyl acetate extract broth of P. chrysogynum S003, resulting from deep-sea sediment collected from the Red Sea.The isolated compounds were verified for their cytotoxicity against a board of cancer cell lines including A-549, DU-145, MCF-7, and HepG-2 cell lines, where they revealed weak cytotoxic activity against all the tested cell lines [44].
Additional cerebrosides, namely, Penicillosides A and B (122-123), had been reported by Youssef et al.Compounds 122-123 were selected from the organic extract of the fungal endophyte Penicillium sp.isolated from the inner parts of the Red Sea tunicate Didemnum sp.122 and 123 were assessed for their antimicrobial actions against a Gram-positive bacterium (Staphylococcus aureus ATCC 25923), a Gram-negative bacterium (Escherichia coli ATCC 25922), and yeast (Candida albicans ATCC 14053) using agar diffusion method.Accurately measured 0.1 ml (100 µg dissolved in DMSO) of each compound were inserted in the cups and then incubated at 37 °C for 24 h.The inhibition zones were measured and compared with the reference antibiotics and antifungal drugs; ampicillin, imipenem, and clotrimazole (each of 10 µg/disc giving 30, 30, and 40 mm inhibition zone respectively).Both compounds revealed no cytotoxicity when evaluated against Hela cancer cell lines [45].
The fungal strain Penicillium sp.MMA had been cultured from the internal tissues of the soft coral Sarcophyton sp. of the fungus ethyl acetate extract had led to the selection of the quinoline alkaloid veridicatol 124, the tricyclic diketopiperazine alkaloid Aurantiomide C 125, along with the sesquiterpene Aspterric acid 126.The isolated compounds had been subjected to variable biological screening.The antimicrobial evaluation revealed that compound 126 was the most effective against B. subtilis and P. aeruginosa.Regarding the antioxidant activity compound 125 showed the most potent activity with maximum DPPH scavenging activity (75%), meanwhile, both compounds 124 and 125 revealed potent cytotoxicity against MCF-7, HepG-2, and HCT 116 cancer cell lines [46] (Fig. 7.).

Scopulariopsis sp
The Scopulariopsis sp fungal strain ST-F1 selected from the internal parts of the Stylophora sp.hard coral collected from the Gulf of Suez, the Red Sea, demonstrated to be a fertile home of various secondary metabolites.In two different reports, El-Naggar et al. described the isolation and identification of more than 40 compounds belonging to different classes of natural products including polyketides, sesquiterpenes, triterpenes, alkaloids, and depsipeptides.The authors applied the OSMAC approach changing the culture media with significant variation in the metabolic profiles between the rice and white beans culture media.
Utilizing rice media, 27 different natural products had been identified including eleven   The summary of the biological activities of secondary metabolites isolated from Red Sea-derived fungi is shown in Table 1.

Conclusion
A thorough literature review on the Red Sea fungal revealed that it produces a wide range of chemical components.The main groups of bioactive metabolites produced by marine fungi are flavonoids, alkaloids, phenolic acids, and triterpenoids.Marine fungus whole extracts, fractions, and isolated pure substances have exhibited a variety of biological and pharmacological effects.Therefore, marine fungus species might potentially produce promising aspirants for examination in medical trials.
Entire extracts or fractions rich in bioactive substances, such as polysaccharides or polyphenolics, may be applied topically or as therapy in the treatment of cancer and serious inflammatory diseases.Aspergillus sp., Penicillium sp., Fusarium sp., Epicoccum sp., Cladosporium sp., Alternaria sp., Scopulariopsis sp., and Chrysosporium sp. are the most studied species biologically, sendoff a large area for more studies on other species that have not yet been completely studied.
The current review contains widespread updated information on the pharmacology and phytochemistry of many marine fungal species, which may stand in the development and discovery of innovative drugs for the cure of various disorders.Without a doubt, the richness of published data directs to the marine fungi's medicinal potential.The clinical efficacy of the marine fungal species is still uncertain, despite years of intensive research.It will continue to be challenging to incorporate marine fungus into traditional therapy.This is a major hindrance not only to marine fungus species but also to many additional therapeutic marine creatures.The clinical environment demonstration of therapeutic activity is a fundamental task for medical research.For the profit of patients all through the world, it is to be wished that this significant barrier will be removed in the years to come.

Fig. 1 .
Fig. 1. illustrates the chemical structures of the isolated secondary metabolites from different Red Sea strains of Aspergillus sp

Fig. 3 . 2 . 5 .
Fig. 3. illustrates the chemical structures of the isolated secondary metabolites from different Red Sea strains of Cladosporium sp 2.4.Chrysosporium sp Le Goff et al. studied the effect of different culture media on the assembly and yield of secondary metabolites from the endophytic fungus C. lobatum TM-237-S5 secluded from the internal tissues of the Sponge Acanthella cavernosa gained from the Gulf of Aqaba, the Red Sea.The authors applied a recent technique in the cultivation enhancing the yield of secondary metabolites from the culture media through pairing solid-state extraction (SSF/SSE) and solid-state fermentation with XAD Amberlite resin, applying different culture media including potato dextrose agar (PDA), potato dextrose broth (PB), marine broth (MB) and marine agar (MA).PDA revealed the most suitable medium

Fig. 6 .
Fig. 6. illustrates the chemical structures of the isolated secondary metabolites from different Red Sea strains of Fusarium sp.

Fig. 7 .Fig. 8 .
Fig. 7. illustrates the chemical structures of the isolated secondary metabolites from different Red Sea strains of Penicillium sp

Fig. 9 .
Fig. 9. illustrates the chemical structures of the isolated secondary metabolites from different Red Sea strains of Unidentified fungal sp Orfali et al. reported an unprecedented cytochalasan alkaloid, Asporychalasin (45) from the rice fermentation medium of A.oryzae isolated from marine sediment from the Red Sea, Jeddah.Compound 45 revealed a unique 6/6/11 tricyclic nucleus and the 14,16,18-trimethylated Cycloundecane ring C not known for the previously isolated Cytochalasan.Compound 45 was assessed contrary to a board of cancer cell lines, where it revealed cytotoxicity against cancer cells A549, MCF-7 & and HepG2 with IC 50 8.8, 7.4 & and 8.3 µg/mL [31].