Aspergillus ochraceus: Metabolites, Bioactivities, Biosynthesis, and Biotechnological Potential

Fungus continues to attract great attention as a promising pool of biometabolites. Aspergillus ochraceus Wilh (Aspergillaceae) has established its capacity to biosynthesize a myriad of metabolites belonging to different chemical classes, such as isocoumarins, pyrazines, sterols, indole alkaloids, diketopiperazines, polyketides, peptides, quinones, polyketides, and sesquiterpenoids, revealing various bioactivities that are antimicrobial, cytotoxic, antiviral, anti-inflammatory, insecticidal, and neuroprotective. Additionally, A. ochraceus produces a variety of enzymes that could have variable industrial and biotechnological applications. From 1965 until June 2022, 165 metabolites were reported from A. ochraceus isolated from different sources. In this review, the formerly separated metabolites from A. ochraceus, including their bioactivities and biosynthesis, in addition, the industrial and biotechnological potential of A. ochraceus are highlighted.


Introduction
Recently, a dramatic shift has increased towards the utilization of eco-friendly and sustainable sources for discovering therapeutic agents against various health concerns to promote human health and wellbeing. Fungi have a prolonged and close relationship with human beings, especially at the chemical level. Particularly, they have drawn great interest for their capacity to biosynthesize a variety of structurally unique metabolites that possess promising bioactivities [1][2][3][4][5]. Additionally, the current advances in genetics, synthetic biology, natural product chemistry, and bioinformatics have considerably reinforced the capability to mine their genomes for discovering novel drugs [6].
The genus Aspergillus (Aspergillaceae) is one of the most widespread, diversified genera, comprising 400 filamentous species of substantial pharmaceutical, biotechnological, and commercial values [7][8][9][10]. Some of its species have promising enzyme production capacity, as well as causing various illnesses in humans and animals [7,11,12]. They cause various clinical infections that range from allergic and chronic infections to acute invasive aspergillosis [13]. On the other hand, its species are renowned, prolific producers of various metabolites, such as butyrolactones, polyketides, xanthones, sterols, anthraquinones, terpenoids, peptides, and alkaloids, which demonstrate various bioactivities [7,14,15].

Enzymes of A. ochraceus and Their Applications
Fungi are fascinating producers of various enzymes that have beneficial contributions in the industrial field. A. ochraceus produces a variety of enzymes, which are reviewed in this work, along with their possible biotechnological and industrial values.

Glycoside Hydrolases
Lignocellulosic biomass is one of the alternative energy sources to fossil fuel that is composed of hemicelluloses, cellulose, and lignin [4,5,43,44]. Cellulases are the principal catalytic enzymes for lignocellulosic biomass hydrolysis, involving β-D-glucosidase, cellobiohydrolase, and endoglucanase, that synergistically hydrolyze cellulose into glucose [4,5,43,44]. They are applicable in the fermentation industry, which needs stability under extreme bioprocessing conditions and high yield. Coir pith or coconut pith is a byproduct of the coir industry with 25% cellulose that is possibly utilized as substrate for saccharification.
Xylan is a major component of the plant cell wall. It consists of 1,4-connected β-Dxylopyranose residues [4,5,43,44]. Xylanases facilitate xylan hydrolysis, primarily utilized in the kraft operation for removing the generated LCC (lignin-carbohydrate complex), which is a physical barrier towards bleaching chemicals entry [45]. Chemical bleaching relies on the utilization of a large quantity of chlorine and chlorine-related chemicals that results in bio-accumulating, mutagenic, toxic, and bio-harmful byproducts [46]. Alternatively, xylanases used in the paper and pulp industry is an eco-friendly method that minimizes pulp fibers' damage and generates superior quality dissolving pulps [47]. The production Molecules 2022, 27, 6759 3 of 46 of microbial xylanases using various lignocellulosic residues as growth substrates have received great attention because of its low cost and high yield [45][46][47]. Betini et al. reported the production of xylanases from A. ochraceus under SSF (solidstate fermentation) using agro-industrial residues (e.g., wheat bran, rice straw, oatmeal, corncob, and Eucalyptus grandis sawdust) [39]. It was found that xylanase production (20%) was favored when a mixture of corncob and wheat bran was utilized. The biobleaching assay of these enzymes revealed twice to thrice times increased brightness and maintained viscosity, indicating that their use could assist the reduction of chlorine compound concentration in cellulose pulp treatment [39]. In another study, A. ochraceus produced high levels of cellulase-free xylanase in oat spelt or birchwood xylan media using wheat bran residue. The enzyme had maximal activity at 65 • C and 5.0 pH [48]. It caused the bleaching of eucalyptus kraft pulp. The results could improve the economic characteristics of bio-bleaching technology and minimize the pollutant compounds used in the process [48]. In 2012, Michelin et al. studied the production of xylanase by A. ochraceus utilizing wheat straw autohydrolysis liquor as a carbon source [49]. It was found that the best yield of β-xylosidase and xylanase was obtained when A. ochraceus was cultivated with 1% wheat bran added to 10% wheat straw liquor in a stirred tank bioreactor, suggesting the possibility of scaling up this process for commercial production [49]. The enhancement of xylanase and p-xylosidase productivity by A. ochraceus using various chemical and physical mutagenesis was assessed [40]. It was found that the NG-13 (N-methyl-N -nitro-Nnitrosoguanidine) mutant strain secreted high levels of β-xylosidase and xylanase during growth on agricultural waste and commercial xylan, which were stable with optimal activity at pH 5-10 and temperature 45-50 • C [40].
Invertases (β-D-fructo-furanosidases) hydrolyze polysaccharides and sucrose to produce glucose and fructose [50]. The resulting glucose and fructose mixture is called inverted sugar. Invertases are substantial in the food industry, particularly in confectionery for artificial sweetener preparation and increasing sweetening properties [50]. Ghosh et al. (2001) purified invertase enzymes from A. ochraceus that was thermotolerant with high sucrose and raffinose affinity [41]. A. ochraceus also produced high levels of an extracellular thermostable β-D-fructofuranosidase using sugar cane bagassesupplemented Khanna medium at 40 • C [51]. This enzyme had a hydrolytic activity with no trans-fructosylating potential. Furthermore, it was positively affected by glucose, which distinguished it from the other β-d-fructofuranosidases, supporting its application for fructose syrup production and sucrose hydrolysis [51].

Proteolytic Enzymes
Medical interest has been drawn to the thrombolytic enzymes of microbial origin. These enzymes act directly by dissolving blood clots as plasmin or as blood plasminogen tissue activators [52]. Protein C prevents blood hyper-clotting in hemostasis [53]. Protein C activator's addition to the blood inactivates clotting factors VIII and V, which are necessary for thrombin formation, resulting in the prolongation of the partial thromboplastin time [54]. Discovering protein C activators from microbes could be beneficial for clinical practice use because of the low cost.
From A. ochraceus 513, proteinase belonging to protein C activator types was separated and assessed for anti-coagulant and fibrinolytic potential [38]. This enzyme was efficient as Agkistrodon snake venom protein C activator in thrombin formation time prolongation [38]. Osmolovskiy et al. stated that extracellular proteinases produced by submerged cultures of a micromycete A. ochraceus L-1 exhibited specific fibrinogenolytic and fibrinolytic potential, whereas the highest effectiveness was observed at pH 7.0 and 28 • C [55].

Tannases
Tannases (tannin acyl hydrolase) catalyze the hydrolysis of depside and the ester bonds of hydrolyzable tannins [56]. They have been utilized as clarifying agents in the industrial processing of coffee-flavored soft drinks and fruit juices, instant teas manufacture, gallic acid production, and treatment of polyphenolics-contaminated wastewaters, as well as the removal of tannin from foodstuffs and animal feeds [57,58].
A. ochraceus was reported to yield extracellular thermostable tannase with distinctive monomeric structural characteristics. This enzyme was activated by manganese, revealing its biotechnological potential for gallic acid production [42]. Furthermore, it was found that A. ochraceus biofilm produced tannase in Khanna medium containing tannic acid (1.5% w/v, carbon source), which was higher than that obtained using conventional submerged fermentation. This enzyme exhibited potent effectiveness at pH 6.0 and 30 • C and was not affected by detergent and surfactant addition [36]. It had different biotechnological applications in propyl gallate production, tannin-rich leather effluent treatment, and sorghum feed formulation [36]. Thus, fungal biofilm is an interesting alternative to produce high levels of tannase with the biotechnological potential to be applied in different industrial sectors.

Oxidases
Alcohol oxidases catalyze the oxidation of alcohols to the corresponding carbonyl compounds with a concomitant release of hydrogen peroxide [59]. They have potential applications in biosensors and the biocatalytic production of different carbonyl compounds that are beneficial in pharmaceutical, flavor, and clinical industries [59]. A. ochraceus AIU031 secreted alcohol oxidase (AOD), belonging to the same group as methylotrophic yeast AOD. It oxidized short-chain primary alcohols and ethylene glycol [60]. It was found to possess an optimal ethanol oxidation capacity at 50-55 • C and pH 5-7 [60].

Bioethanol Production
Bioethanol is a liquid biofuel that could be produced from various biomass resources (corn, wheat, paddy straw, sugar beet, wood, municipal waste, forestry byproducts, etc.) through microbial fermentation [61]. This process provides an economically competitive source of energy for biofuel production from cellulosic and lignocellulosic materials.
A. ochraceus cellulase demonstrated high hydrolyzing potential of sawdust and its ethanol yield in shaking fermentation than in stationary fermentation. The results supported the use of sawdust as a potential substrate for bioethanol production [62].

Dye Decolorization
Kadam et al. developed a consortium of A. ochraceus NCIM-1146 and Pseudomonas sp. SUK1 to decolorize adsorbed dyes from textile effluent wastewater under solid-state fermentation [63]. This consortium had a remarkable potential to decolorize adsorbed textile dyes from CPTDE (chemical precipitate of textile dye effluent) and textile wastewater on rice bran. This influence was strongly referred to as the synergism of the excreted extracellular enzymes, such as laccase, azoreductase, NADH-DCIP reductase, and tyrosinase [63]. This approach could be effective in reducing effluent volume through adsorbing textile dye on available agricultural waste residues at a low cost, as well as its disposal or bioremediation to nontoxicity by solid-state fermentation [63]. Saratale et al. reported that A. ochraceus remarkably decolorized cotton blue and malachite green dyes, while it had less decolorization capacity on methyl violet and crystal violet [64]. This effect was found to be due to microbial metabolism, not biosorption. Therefore, A. ochraceus could be used for bioremediation of cotton blue and malachite green dye-containing wastewater [64]. In addition, the intracellular blue laccase produced by A. ochraceus NCIM1146 had maximum substrate specificity toward ABTS (2,2 -azinobis, 3-ethylbenzothiazoline-6-sulfonic acid) and decolorized azo dyes with an optimal effect at 60 • C and 4.0 pH [65].

Kerosene Biodegradation
Accidental spillage of petroleum and its byproducts represents an environmental problem as it causes acute toxicity in some forms of aquatic life [66]. Fungi and bacteria are known for their capacity for petroleum hydrocarbon consumption [67]. Due to their degrading potential, they can be a promising alternative for reducing the environmental impacts of oil spills.
A. ochraceus was found to possess kerosene-degrading potential when the previously grown mycelium was incubated in kerosene-containing broth due to its alkane-oxidizing capacity [68]. It also revealed high levels of kerosene biodegradation, aminopyrine Ndemethylase, and NADPH-DCIP reductase. The production of acetaldehyde could be utilized as an indicator for kerosene biodegradation [68]. In another study, A. ochraceus also showed a noticeable ability to utilize and degrade gasoline and crude oil [69].

Nanoparticles (NPs) of A. ochraceus
Intracellular silver nanoparticles synthesized by exposure of Ag + ions to A. ochraceus were heat-treated in an N 2 environment to yield AgNPs embedded in carbonaceous supports. These carbonaceous AgNPs had an antibacterial capacity versus B. subtilis and E. coli, and an antiviral potential towards the M-13 phage virus [70].
Ochratoxins are toxic metabolites, biosynthesized by this fungus, that contaminate a variety of foodstuffs, resulting in serious effects on both animals and humans. Ochratoxin A (14) is the highly toxic one of this group. It is implicated in nephrotoxic syndromes in various animals, as well as its hepatotoxic, carcinogenic, genotoxic, immune-toxic, and teratogenic effects on animal species and potent carcinogenic effects on humans. It is worth mentioning that ochratoxins A (14) and B (18) are well-known mycotoxins that are associated with diverse animal and human diseases, including porcine nephropathy, poultry ochratoxicosis, and human endemic nephropathies [17]. Ochratoxin A (14) features 7-carboxy-5-chloro-8-hydroxy-3,4-dihydro-3R-methyl isocoumarin, connected by an amide bond to L-β-phenylalanine at 7-carboxy group and, together with its dechloro and ethyl ester derivatives (ochratoxins B (18) and C (12), respectively) these were purified by SiO 2 and ion exchange (Dowex I) CC from the fungal CHCl 3 -MeOH extract and characterized by NMR and chemical method [74] (Figure 2). Moreover, 14 was found as a major metabolite,  (2), along with 1 from the CHCl 3 extract utilizing SiO 2 CC, pTLC (acetone/CHCl 3 7:93), and crystallization (CHCl 3 :hexane) [16]. Compound 18 was reported to show strong cytotoxicity versus A2780 (IC 50 3.0 µM) compared to cisplatin (IC 50 2.2 µM) [17]. The substitution of proton in 18 by chlorine in 14 decreased its cytotoxic effect versus A2780 cells [17]. Ochratoxins are toxic metabolites, biosynthesized by this fungus, that contaminate a variety of foodstuffs, resulting in serious effects on both animals and humans. Ochratoxin A (14) is the highly toxic one of this group. It is implicated in nephrotoxic syndromes in various animals, as well as its hepatotoxic, carcinogenic, genotoxic, immune-toxic, and teratogenic effects on animal species and potent carcinogenic effects on humans. It is worth mentioning that ochratoxins A (14) and B (18) are well-known mycotoxins that are associated with diverse animal and human diseases, including porcine nephropathy, poultry ochratoxicosis, and human endemic nephropathies [17]. Ochratoxin A (14) features 7-carboxy-5-chloro-8-hydroxy-3,4-dihydro-3R-methyl isocoumarin, connected by an amide bond to L-β-phenylalanine at 7-carboxy group and, together with its dechloro and ethyl ester derivatives (ochratoxins B (18) and C (12), respectively) these were purified by SiO2 and ion exchange (Dowex I) CC from the fungal CHCl3-MeOH extract and characterized by NMR and chemical method [74] (Figure 2). Moreover, 14 was found as a major metabolite, in addition to 18 in the CHCl3 extract of A. ochraceus NRRL-3174 [75]. In In 1995, ochratoxins 12, 13, 15, 17, and 20 were purified from A. ochraceus NRRL3174 culture and structurally elucidated by HPLC, MS, and NMR (

Pyrazine Derivatives
These metabolites possess pyrazin-2(1H)-one core that are biosynthesized from two amino acid molecules, such as isoleucine, leucine, norvaline, and valine. These compounds were reported from bacteria and fungi and revealed cytotoxic, antibacterial, and brine shrimp toxicity capacities.
Using the OSMAC technique through cultivating Mediterranean sponge Agelas-oroidesderived A. ochraceus on white beans yielded a new diketopiperazine, waspergillamide B (39), featuring an uncommon p-nitrobenzoic acid unit and diketopiperazine moiety that was originated from 2-hydroxy leucine and 3-hydroxy valine residues ( Figure 4). It was characterized by NMR and its 9R configuration was established by Marfey's analysis [17].

Benzodiazepine Derivatives
Benzodiazepines are psychoactive metabolites that are naturally reported from Streptomyces, Aspergillus, and Penicillium [91,92]. These metabolites are renowned as a chemotaxonomic marker for this fungus [26,86].
New benzodiazepine-related metabolites, compounds 68-70, in addition to the known 64-66, were separated from an A. ochraceus terrestrial strain obtained from milosorghum seeds using UV-vis/HPLC analysis and characterized using various spectral analyses ( Figure 6). Compound 68 is a C-5 methoxy circumdatin E (69), while 70 lacks an OH group at C-5 in circumdatin C (66) [92]. The benzodiazepine core of 66 and 70 was formed from L-proline and substituted anthranilic acid, while that of 64, 65, 68, and 69 has L-alanine instead of L-proline [91]. Their ADMET prediction revealed the appropriate features for CNS drugs, better druglikeness characteristics, and preferable safety scores of toxicities [24]. Therefore, 56 could be an advantageous lead compound for anti-Parkinson's therapeutic drugs.

Benzodiazepine Derivatives
Benzodiazepines are psychoactive metabolites that are naturally reported from Streptomyces, Aspergillus, and Penicillium [91,92]. These metabolites are renowned as a chemotaxonomic marker for this fungus [26,86].
New benzodiazepine-related metabolites, compounds 68-70, in addition to the known 64-66, were separated from an A. ochraceus terrestrial strain obtained from milosorghum seeds using UV-vis/HPLC analysis and characterized using various spectral analyses ( Figure 6). Compound 68 is a C-5 methoxy circumdatin E (69), while 70 lacks an OH group at C-5 in circumdatin C (66) [92]. The benzodiazepine core of 66 and 70 was formed from L-proline and substituted anthranilic acid, while that of 64, 65, 68, and 69 has L-alanine instead of L-proline [91]. Moreover, a new alkaloid, circumdatin G (71) and 66 and 70 were isolated from a sediment-derived strain by Sephadex LH-20 CC and HPLC. Compound 71 is a 15-OH derivative of 70 based on spectroscopic analysis. They exhibited no HCV-NS3 protease inhibition in the scintillation proximity assay (SPA) [20]. In 2005, circumdatin H (72), a new derivative from the culture broth of soil-derived A. ochraceus, in addition to 69, was isolated by SiO2 CC and HPLC and established by the spectral and chemical method by López-Gresa et al. [86]. Their mitochondrial respiratory chain inhibition capacity was estimated by measuring NADH oxidase potential [86]. These metabolites prohibited (IC50 1.50 and 2.50 μM, respectively) NADH oxidase activity (integrated electron transfer chain), whereas 72 was slightly more potent than 69, suggesting that the C-15 hydroxy group in 69 might lead to a more difficult interaction with the respiratory chain [86]. Thus, these compounds may act as leads in developing new tools for controlling insects [86]. Compound 64 exhibited antioxidant effectiveness (IC50 32 μM) in comparison to ascorbic acid (IC50 20.0 μM) [72].
The marine brown alga Sargassum kjellmanianum-derived A. ochraceus produced a new benzodiazepine analog, 2-hydroxycircumdatin C (67), as well as the known 66, 68, and 70 that were isolated from culture broth and mycelia extract by RP-18, SiO2, and Se- Moreover, a new alkaloid, circumdatin G (71) and 66 and 70 were isolated from a sediment-derived strain by Sephadex LH-20 CC and HPLC. Compound 71 is a 15-OH derivative of 70 based on spectroscopic analysis. They exhibited no HCV-NS3 protease inhibition in the scintillation proximity assay (SPA) [20]. In 2005, circumdatin H (72), a new derivative from the culture broth of soil-derived A. ochraceus, in addition to 69, was isolated by SiO 2 CC and HPLC and established by the spectral and chemical method by López-Gresa et al. [86]. Their mitochondrial respiratory chain inhibition capacity was estimated by measuring NADH oxidase potential [86]. These metabolites prohibited (IC 50 1.50 and 2.50 µM, respectively) NADH oxidase activity (integrated electron transfer chain), whereas 72 was slightly more potent than 69, suggesting that the C-15 hydroxy group in 69 might lead to a more difficult interaction with the respiratory chain [86]. Thus, these compounds may act as leads in developing new tools for controlling insects [86]. Compound 64 exhibited antioxidant effectiveness (IC 50 32 µM) in comparison to ascorbic acid (IC 50 20.0 µM) [72].
The marine brown alga Sargassum kjellmanianum-derived A. ochraceus produced a new benzodiazepine analog, 2-hydroxycircumdatin C (67), as well as the known 66, 68, and 70 that were isolated from culture broth and mycelia extract by RP-18, SiO 2 , and Sephadex LH-20 CC and structurally established by spectroscopic and CD tools. Only 67 revealed marked antioxidant potential (IC 50 9.9 µM) that was 8.9-fold more powerful than BHT (butylated hydroxytoluene, IC 50 88.2 µM); however, 66 and 68 (IC 50 > 100 µg/mL) had weak potential in the DPPH assay. On the other hand, none of them had antibacterial potential in the well diffusion method [26]. Hu   Their structures and absolute configuration were assured by spectral and chemical methods. These compounds are epimers, differing only in configurations at C-8'. Their cytotoxic potential versus 2 normal and 26 human cancer cell lines was evaluated in the CTG (Cell Titer Glo) assay. Compound 117 had the most broad-spectrum cytotoxic capacity against 16 cancer cells (IC50 ranged from 2.54 to 9.79 μM) because the epoxide could alkylate DNA by a nucleophilic addition with the sulfhydryl of protein or the base of DNA. Compound 76 also displayed a wide spectrum cytotoxic potential versus 10 cancer cells (IC50 ranged from 3.10 to 11.32 μM). In addition, 67, 77, and 79 exhibited a selective cytotoxic capacity versus U251, while 78 selectively prohibited U87, A673, and Hep3B. Interestingly, the conjugation among 67 and 117 promoted the inhibitory potential on U87 and U251. Compounds 76 and 78 with 8′S configuration revealed cytotoxic potential versus U87 cells; however, 77 and 79 with 8′R configuration were active versus U251 cells. On the other hand, 76-79 exhibited low cytotoxicity on the normal human cells, HEK-293F and L02. Thus, these metabolites could represent leads in anticancer drugs against human glioblastoma cells with non-cytotoxic action to human normal cells [31]. Their structures and absolute configuration were assured by spectral and chemical methods. These compounds are epimers, differing only in configurations at C-8 . Their cytotoxic potential versus 2 normal and 26 human cancer cell lines was evaluated in the CTG (Cell Titer Glo) assay. Compound 117 had the most broad-spectrum cytotoxic capacity against 16 cancer cells (IC 50 ranged from 2.54 to 9.79 µM) because the epoxide could alkylate DNA by a nucleophilic addition with the sulfhydryl of protein or the base of DNA. Compound 76 also displayed a wide spectrum cytotoxic potential versus 10 cancer cells (IC 50 ranged from 3.10 to 11.32 µM). In addition, 67, 77, and 79 exhibited a selective cytotoxic capacity versus U251, while 78 selectively prohibited U87, A673, and Hep3B. Interestingly, the conjugation among 67 and 117 promoted the inhibitory potential on U87 and U251. Compounds 76 and 78 with 8 S configuration revealed cytotoxic potential versus U87 cells; however, 77 and 79 with 8 R configuration were active versus U251 cells. On the other hand, 76-79 exhibited low cytotoxicity on the normal human cells, HEK-293F and L02. Thus, these metabolites could represent leads in anticancer drugs against human glioblastoma cells with non-cytotoxic action to human normal cells [31].

Indole and Other Alkaloids
Chromatographic investigation using SiO2 and Sephadex LH-20 CC of anti-insectan A. ochraceus NRRL3519 sclerotia EtOAc extracts yielded new prenylated bis-indolyl benzenoid metabolites, ochrindoles A-D (80)(81)(82)(83), and a new bis-indolyl quinone, ochrindole D (83) (Figure 8). These metabolites display a central ring-linked prenyl sidechain. In feeding assays, they had moderate effectiveness (concentration of 200 ppm w/w) towards Carpophilus hemipterus (dried fruit beetle) and H. zea, whereas 80 caused a 20% and 30% weight gain reduction, respectively [29]. These compounds also had potential versus B. subtilis (inhibition zone 15-18 mm, concentration of 100 μg/disk) in the disk assay. It is worth mentioning that these compounds were found to be the main components of A. ochraceus NRRL3519 sclerotia; therefore, they could be accountable for the anti-insectan potential of the A. ochraceus extracts [29]. Perlolyrine (84), a β-carboline alkaloid with a 5hydroxymethyl substituent in the C-1 linked furan ring, was reported from A. ochraceus MCCC3A00521 culture by Hu et al. [24]. Furthermore, A. ochraceus ATCC22947 yielded a novel pyrrolidine antibiotic, L-657,398 (86), that was separated from mycelia EtOAc extract by SiO2 and Sephadex LH-20 CC and elucidated by various NMR spectroscopic data. This metabolite is related to anisomycin, an anti-protozoal and antifungal metabolite produced by Streptomyces roseochromogenes and S. griseolus [111]. Compound 86 also possessed marked broader efficacy towards yeasts and filamentous fungi than anisomycin using the disk diffusion method [93]. Ochracesol A (85), a new alkaloid, featured an oxazole ring linked to p-OH-disubstituted benzene. Its 17R configuration was assigned by Xray and ECD analyses. This compound had weak anti-Parkinson's potential (EC50 17.84 μM) [24].

Indole and Other Alkaloids
Chromatographic investigation using SiO 2 and Sephadex LH-20 CC of anti-insectan A. ochraceus NRRL3519 sclerotia EtOAc extracts yielded new prenylated bis-indolyl benzenoid metabolites, ochrindoles A-D (80)(81)(82)(83), and a new bis-indolyl quinone, ochrindole D (83) (Figure 8). These metabolites display a central ring-linked prenyl sidechain. In feeding assays, they had moderate effectiveness (concentration of 200 ppm w/w) towards Carpophilus hemipterus (dried fruit beetle) and H. zea, whereas 80 caused a 20% and 30% weight gain reduction, respectively [29]. These compounds also had potential versus B. subtilis (inhibition zone 15-18 mm, concentration of 100 µg/disk) in the disk assay. It is worth mentioning that these compounds were found to be the main components of A. ochraceus NRRL3519 sclerotia; therefore, they could be accountable for the anti-insectan potential of the A. ochraceus extracts [29]. Perlolyrine (84), a β-carboline alkaloid with a 5-hydroxymethyl substituent in the C-1 linked furan ring, was reported from A. ochraceus MCCC3A00521 culture by Hu et al. [24]. Furthermore, A. ochraceus ATCC22947 yielded a novel pyrrolidine antibiotic, L-657,398 (86), that was separated from mycelia EtOAc extract by SiO 2 and Sephadex LH-20 CC and elucidated by various NMR spectroscopic data. This metabolite is related to anisomycin, an anti-protozoal and antifungal metabolite produced by Streptomyces roseochromogenes and S. griseolus [111]. Compound 86 also possessed marked broader efficacy towards yeasts and filamentous fungi than anisomycin using the disk diffusion method [93]. Ochracesol A (85), a new alkaloid, featured an oxazole ring linked to p-OH-disubstituted benzene. Its 17R configuration was assigned by X-ray and ECD analyses. This compound had weak anti-Parkinson's potential (EC 50 17.84 µM) [24].

Peptides
Aspochracin (88), a cyclotripeptide with N-methyl L-valine, N-methyl L-alanine, and L-ornithine residues and an octatrienoic acid side chain, was separated as a pale-yellow powder from A. ochraceus EtOAc extract by SiO2 CC and characterized using NMR and chemical methods. It was tested for its insecticidal, antimicrobial, and cytotoxic activities. It demonstrated insecticidal potential by injection of silkworm and fall webworm, with 17 μ/g minimal concentration, on larvae, causing paralysis followed by death into the final instar larvae of silkworm and 170 μ/g for the final instar larvae of fall webworm. Additionally, it demonstrated the silkworm's first instar larva and egg contact toxicity using the dipping method. Its hydrogenation completely minified the effect, suggesting the potential of the side chain's conjugated triene in its action. Its 165 mg/kg IV dose did not kill mice and had no antimicrobial efficacy towards various microbes in agar dilution, paper disc, and liquid dilution methods [95].

Peptides
Aspochracin (88), a cyclotripeptide with N-methyl L-valine, N-methyl L-alanine, and L-ornithine residues and an octatrienoic acid side chain, was separated as a pale-yellow powder from A. ochraceus EtOAc extract by SiO 2 CC and characterized using NMR and chemical methods. It was tested for its insecticidal, antimicrobial, and cytotoxic activities. It demonstrated insecticidal potential by injection of silkworm and fall webworm, with 17 µ/g minimal concentration, on larvae, causing paralysis followed by death into the final instar larvae of silkworm and 170 µ/g for the final instar larvae of fall webworm. Additionally, it demonstrated the silkworm's first instar larva and egg contact toxicity using the dipping method. Its hydrogenation completely minified the effect, suggesting the potential of the side chain's conjugated triene in its action. Its 165 mg/kg IV dose did not kill mice and had no antimicrobial efficacy towards various microbes in agar dilution, paper disc, and liquid dilution methods [95].

Polyketides
A. ochraceus M#1129-85 EtOAc extract yielded the toxic metabolites penicillic acid (97) and 5(6)-dihydropenicillic acid (98). It was found that 97 was only produced on potato dextrose agar medium; however, A. ochraceus in rice flour yielded 98, suggesting that 97 was transformed into 98 in high-nutritive circumstances ( Figure 10). Interestingly, 98 was not mutagenic in the Ames and umu tests [98]. Compounds 99 and 117 were isolated from the mycelia of A. ochraceus collected from an apple-packing house environmental sample. It is noteworthy that 117 displayed better antimicrobial influence than 99 versus 22 bacterial strains and 13 fungal strains in the paper disc assay [99].
Co-cultivation of A. ochraceus with Streptomyces lividans resulted in an increase in the yields of 97 and 98; however, its co-cultivation with B. subtilis yielded two new penicillic acid derivatives, ochraspergillic acids A (123) and B (124), which are new penicillic acidaminobenzoic acid hybrids. Interestingly, it was suggested that the aminobenzoic acid moiety of 123 is originated by bacteria [17] (Figure 11).  Aspinonene (128), a branched pentaketide with uncommon oxygenation, was separated from A. ochraceus DSM-7428 culture broth using SiO2 and Sephadex LH-20 CC [100]. Its biosynthesis was proposed utilizing [ 18 O2]-rich atmosphere fermentation and [ 13 C]-labelled acetate feeding experiments (Scheme 3). It was proved that 128 was biosynthesized via the polyketide pathway and post-polyketide modification is a key step that directed aspinonene biosynthesis [100].  Aspinonene (128), a branched pentaketide with uncommon oxygenation, was separated from A. ochraceus DSM-7428 culture broth using SiO 2 and Sephadex LH-20 CC [100]. Its biosynthesis was proposed utilizing [ 18 O 2 ]-rich atmosphere fermentation and [ 13 C]labelled acetate feeding experiments (Scheme 3). It was proved that 128 was biosynthesized via the polyketide pathway and post-polyketide modification is a key step that directed aspinonene biosynthesis [100]. Aspinonene (128), a branched pentaketide with uncommon oxygenation, was separated from A. ochraceus DSM-7428 culture broth using SiO2 and Sephadex LH-20 CC [100]. Its biosynthesis was proposed utilizing [ 18 O2]-rich atmosphere fermentation and [ 13 C]-labelled acetate feeding experiments (Scheme 3). It was proved that 128 was biosynthesized via the polyketide pathway and post-polyketide modification is a key step that directed aspinonene biosynthesis [100].  New C9 polyketides asperochratides A-J (107-116) and 102, 103, 121, and 122 were isolated from EtOAc extract of the fermentation broth of deep-sea water-derived A. ochraceus using SiO 2 , RP-18, Sephadex LH-20, pTLC, and HPLC and characterized by NMR, MS, Mosher's method, ICD, ECD, and specific rotation. Structurally, they share the same polyketide skeleton and belong to aspyrone-related metabolites. Compounds 103 and 107-111 exerted a significant cytotoxic effect on the BV-2 cell line with inhibitions ranging from 50.29 to 72.81% in the MTT assay. In the anti-inflammation assay, 103 (1 µM) produced a marked decline (44.87%) of NO concentration in the LPS-stimulated BV-2 cells using Griess reagent. However, none of them exhibited significant anti-H1N1 virus, anti-food allergic, and antimicrobial capacities in vitro [80].

Xanthine and Quinone Derivatives and Flavonoids
Xanthomegnin (131) and viomellein (132) are structurally related quinones that were produced by the A. ochraceus group (Figure 12). These quinones could induce cholangiohepatitis and hepatic lesions in mice toxicity assay [105], as well as nephrotoxicity in pigs and swine [107] and mycotoxicosis in mice [17].
FOR PEER REVIEW 32 of 42 ranging from 50.29 to 72.81% in the MTT assay. In the anti-inflammation assay, 103 (1 μM) produced a marked decline (44.87%) of NO concentration in the LPS-stimulated BV-2 cells using Griess reagent. However, none of them exhibited significant anti-H1N1 virus, antifood allergic, and antimicrobial capacities in vitro [80].

Xanthine and Quinone Derivatives and Flavonoids
Xanthomegnin (131) and viomellein (132) are structurally related quinones that were produced by the A. ochraceus group (Figure 12). These quinones could induce cholangiohepatitis and hepatic lesions in mice toxicity assay [105], as well as nephrotoxicity in pigs and swine [107] and mycotoxicosis in mice [17]. Additionally, 132 exhibited potent cytotoxic capacity versus A2780 and L5178Y (IC50 5.0 and 5.3 μM, respectively) compared to cisplatin (for A2780, IC50 2.2 μM) and kahalalide F (for L5178Y, IC50 4.3 μM), while it was inactive towards human Jurkat T and Ramos B cell lines. Compound 130 was obtained from A. ochraceus isolated from Chinese potato by the solid phase extraction method. The optimal yield of 130 was accomplished when the fermentation was done at pH 7.0 and 32°C [104]. Secalonic acid A (135), a C-4-C-4` dimer xanthone, was isolated from the EtOAc fraction of A. ochraceus Wilh IFM4443. This metabolite was formerly reported from Claviceps purpurea [113]. It was not lethal to mice (concentration of 250 mg/kg, orally) and not toxic to the chicken embryos; however, it displayed antibacterial activity towards B. subtitis and piricularia oryzae in the disc assay [103]  Additionally, 132 exhibited potent cytotoxic capacity versus A2780 and L5178Y (IC 50 5.0 and 5.3 µM, respectively) compared to cisplatin (for A2780, IC 50 2.2 µM) and kahalalide F (for L5178Y, IC 50 4.3 µM), while it was inactive towards human Jurkat T and Ramos B cell lines. Compound 130 was obtained from A. ochraceus isolated from Chinese potato by the solid phase extraction method. The optimal yield of 130 was accomplished when the fermentation was done at pH 7.0 and 32 • C [104]. Secalonic acid A (135), a C-4-C-4 dimer xanthone, was isolated from the EtOAc fraction of A. ochraceus Wilh IFM4443. This metabolite was formerly reported from Claviceps purpurea [113]. It was not lethal to mice (concentration of 250 mg/kg, orally) and not toxic to the chicken embryos; however, it displayed antibacterial activity towards B. subtitis and piricularia oryzae in the disc assay [103]. Compound 137 is a rare 7-norsteroid with an uncommon pentalactone B ring system [27]. It demonstrated selective cytotoxic potential towards SW1990, NCI-H460, and SMMC-7721 cell lines (IC50 28.0, 5.0, and 7.0 μg/mL, respectively), while 138 revealed selectivity (IC50 28.0 µg/mL) towards SMMC-7721 in the MTT assay and both had no antimicrobial potential in the agar diffusion test [27]. 22E,24R-Ergosta-7,22-diene-3β,5ɑ,6β,9ɑtetraol (149), reported from A. ochraceus derived from fresh sea water, displayed no notable anti-H1N1 virus, anti-food allergic, and antimicrobial activities [80]. On the other hand, 143-154 isolated by Hu (Figure 14) [24].

Other Metabolites
The new compounds 155 and 163 were produced by mutant strain A. ochraceus NRRL3174 due to UV irradiation of its conidia during the active and stationary growth phases, respectively ( Figure 15). They were purified by TLC and HPLC. Compound 155 is a phenylacetic acid derivative. They were tested for antimicrobial potential versus M. luteus, B. subtilis, S. aureus, M. smegmatis, L. monocytogenes, K. pneumoniae, P. syringae, and A. tumefaciens, M. ramannianus, and S. cerevisiae in the agar diffusion assay. Compound 163 showed marked effectiveness versus S. aureus and B. subtilis and no effect on the other strains. However, 155 did not display any effects on any strains [109]. Clavatol (156), an acetophenone derivative, had mild antioxidant capacity (IC50 30 μM) in the DPPH assay compared to L-ascorbic acid (IC50, 20.0 μM) [72].

Other Metabolites
The new compounds 155 and 163 were produced by mutant strain A. ochraceus NRRL3174 due to UV irradiation of its conidia during the active and stationary growth phases, respectively ( Figure 15). They were purified by TLC and HPLC. Compound 155 is a phenylacetic acid derivative. They were tested for antimicrobial potential versus M. luteus, B. subtilis, S. aureus, M. smegmatis, L. monocytogenes, K. pneumoniae, P. syringae, and A. tumefaciens, M. ramannianus, and S. cerevisiae in the agar diffusion assay. Compound 163 showed marked effectiveness versus S. aureus and B. subtilis and no effect on the other strains. However, 155 did not display any effects on any strains [109]. Clavatol (156), an acetophenone derivative, had mild antioxidant capacity (IC 50 30 µM) in the DPPH assay compared to L-ascorbic acid (IC 50 , 20.0 µM) [72]. Dichotella gemmacea coral-derived A. ochraceus produced AW1 (165), a novel galactomannan extracellular polysaccharide, that was characterized using chemical and spectroscopic methods. AW1 could be a marked source for galacto-furanose oligosaccharides and could have food and pharmacology applications [35].

Conclusions
Fungi have been extensively investigated because of their importance as wealth producers for diverse biometabolites and enzymes, in addition to their industrial, agricultural, and pharmaceutical potential. A. ochraceus can biosynthesize diverse biometabolites that may be utilized as leads in the discovery of therapeutic agents for several health concerns. In this review, 165 metabolites belonging to diverse chemical classes (isocoumarins, pyrazines, diketopiperazines, benzodiazepine, indoles, peptides, sesquiterpenoids, polyketides, quinones, xanthines, flavonoids, sterols, and other metabolites) have been reported from A. ochraceus isolated from various source in the period from 1965 to June 2022 (Figures 16 and 17). These sources include soil, culture, marine (sediment, water, sponge, coral, and algae), plants, fresh water, and other sources ( Figure 17). In Figure 17, it is clear that the major number of metabolites have been reported from the fungus strains collected from diverse marine sources. Among these metabolites, isocoumarins, diketopiperazines, and polyketides represent the major reported constituents. Additionally, sesquiterpenoids with nitrobenzoyl moiety, a rare class of metabolites, are mainly reported from Aspergillus species, including A. ochraceus. Therefore, these sesquiterpenoids deserve more attention because of their marked antitumor potential. Compounds 158 and 160, purified from sponge-derived A. ochraceus MP2, exhibited antimicrobial capacity versus potential pathogens K. pneumonia ATCC-15380, S. aureus ATCC-25923, and P. aeruginosa ATCC-27853 [108].
Dichotella gemmacea coral-derived A. ochraceus produced AW1 (165), a novel galactomannan extracellular polysaccharide, that was characterized using chemical and spectroscopic methods. AW1 could be a marked source for galacto-furanose oligosaccharides and could have food and pharmacology applications [35].

Conclusions
Fungi have been extensively investigated because of their importance as wealth producers for diverse biometabolites and enzymes, in addition to their industrial, agricultural, and pharmaceutical potential. A. ochraceus can biosynthesize diverse biometabolites that may be utilized as leads in the discovery of therapeutic agents for several health concerns. In this review, 165 metabolites belonging to diverse chemical classes (isocoumarins, pyrazines, diketopiperazines, benzodiazepine, indoles, peptides, sesquiterpenoids, polyketides, quinones, xanthines, flavonoids, sterols, and other metabolites) have been reported from A. ochraceus isolated from various source in the period from 1965 to June 2022 (Figures 16 and 17). These sources include soil, culture, marine (sediment, water, sponge, coral, and algae), plants, fresh water, and other sources ( Figure 17). In Figure 17, it is clear that the major number of metabolites have been reported from the fungus strains collected from diverse marine sources. Among these metabolites, isocoumarins, diketopiperazines, and polyketides represent the major reported constituents. Additionally, sesquiterpenoids with nitrobenzoyl moiety, a rare class of metabolites, are mainly reported from Aspergillus species, including A. ochraceus. Therefore, these sesquiterpenoids deserve more attention because of their marked antitumor potential.  Despite the great number of metabolites reported from this fungus, bioactivities were investigated for a limited number of them. The tested bioactivities were antimicrobial, anti-Parkinson's, antiviral, antioxidant, cytotoxic, neuroprotective, and insecticidal. Some of the reported metabolites possessed potent effectiveness similar to or higher than the positive control, for example, 4 displayed a selectivity towards V. harveyi compared to that of chloramphenicol. Compounds 6 and 67 demonstrated more potent antioxidant potential than BHT. In addition, 6 had cytoprotective potential H2O2-induced oxidative damage in SHSY5Y cells without toxicity, suggesting its possible protective role in neurodegenerative illnesses with oxidative stress. Additionally, 56 featured prominent anti-Parkinson's effectiveness compared to levodopa, which could be an advantageous lead compound for anti-Parkinson's therapeutic drugs. Moreover, some metabolites were reported to show strong cytotoxic capacity (e. g., 18, 76-79, and 91).
In addition, there is a lack of pharmacological studies that focus on exploring the possible mechanisms of the active metabolites. In addition, the untested metabolites should be further explored for their possible bioactivities.  Despite the great number of metabolites reported from this fungus, bioactivities wer investigated for a limited number of them. The tested bioactivities were antimicrobia anti-Parkinson's, antiviral, antioxidant, cytotoxic, neuroprotective, and insecticidal. Som of the reported metabolites possessed potent effectiveness similar to or higher than th positive control, for example, 4 displayed a selectivity towards V. harveyi compared to tha of chloramphenicol. Compounds 6 and 67 demonstrated more potent antioxidant poten tial than BHT. In addition, 6 had cytoprotective potential H2O2-induced oxidative damag in SHSY5Y cells without toxicity, suggesting its possible protective role in neurodegener ative illnesses with oxidative stress. Additionally, 56 featured prominent anti-Parkinson' effectiveness compared to levodopa, which could be an advantageous lead compound fo anti-Parkinson's therapeutic drugs. Moreover, some metabolites were reported to show strong cytotoxic capacity (e. g., 18, 76-79, and 91).
In addition, there is a lack of pharmacological studies that focus on exploring th possible mechanisms of the active metabolites. In addition, the untested metabolite should be further explored for their possible bioactivities. Despite the great number of metabolites reported from this fungus, bioactivities were investigated for a limited number of them. The tested bioactivities were antimicrobial, anti-Parkinson's, antiviral, antioxidant, cytotoxic, neuroprotective, and insecticidal. Some of the reported metabolites possessed potent effectiveness similar to or higher than the positive control, for example, 4 displayed a selectivity towards V. harveyi compared to that of chloramphenicol. Compounds 6 and 67 demonstrated more potent antioxidant potential than BHT. In addition, 6 had cytoprotective potential H 2 O 2 -induced oxidative damage in SHSY5Y cells without toxicity, suggesting its possible protective role in neurodegenerative illnesses with oxidative stress. Additionally, 56 featured prominent anti-Parkinson's effectiveness compared to levodopa, which could be an advantageous lead compound for anti-Parkinson's therapeutic drugs. Moreover, some metabolites were reported to show strong cytotoxic capacity (e. g., 18, 76-79, and 91).
In addition, there is a lack of pharmacological studies that focus on exploring the possible mechanisms of the active metabolites. In addition, the untested metabolites should be further explored for their possible bioactivities.
Using the OSMAC technique on the sponge-derived A. ochraceus yielded waspergillamide B (39), a new metabolite with uncommon structural features. In addition, modification of culture media-for example by adding NaBr or NaI-resulted in the production of new metabolites, e.g., (R)-(-)-5-bromomellein (5) and ochramides A-D (31-34), respectively, while mutation of the A. ochraceus strain by UV irradiation produced new metabolites (e. g., 155 and 163). In the same line, the co-cultivation with bacterial strains, such as S. lividans, increased the yield (e.g., 97 and 98); however, B. subtilis yielded new derivatives (e.g., ochraspergillic acids A (123) and B (124)). This suggests further studies on utilizing these techniques could be performed.
Furthermore, the few reports on the biosynthesis of these metabolites promote further study for exploring the biosynthetic pathways of other metabolites and the associated genes that support the potential of A. ochraceus for novel metabolite production using metabolic engineering. Only one study reported the synthesis of NPs using this fungus; further research on synthesizing various types of NPs and its bio-evaluation represents an interesting area for more expected valuable impacts. Additionally, the enzyme production capacity of this fungus has been approved in various studies, suggesting its potential for industrial and biotechnological applications. Thus, A. ochraceus could represent an eco-friendly tool for exchanging hazardous waste into valuable products. Despite the considerable published studies, this fungus still needs more in-depth research from mycologists, biologists, and chemists to shed light on the unexplored potential of this fungus and its metabolites.