Fungal Bioactive Anthraquinones and Analogues

This review, covering the literature from 1966 to the present (2020), describes naturally occurring fungal bioactive anthraquinones and analogues biosynthesized by the acetate route and concerning several different functionalized carbon skeletons. Hydrocarbons, lipids, sterols, esters, fatty acids, derivatives of amino acids, and aromatic compounds are metabolites belonging to other different classes of natural compounds and are generated by the same biosynthetic route. All of them are produced by plant, microorganisms, and marine organisms. The biological activities of anthraquinones and analogues comprise phytotoxic, antibacterial, antiviral, anticancer, antitumor, algicide, antifungal, enzyme inhibiting, immunostimulant, antiplatelet aggregation, cytotoxic, and antiplasmodium activities. The review also covers some practical industrial applications of anthraquinones.


Introduction
Anthraquinones are a group of natural compounds with a plethora of biological activities and potential practical applications. Most of them are produced by plant and micro-organisms among the living organisms [1,2]. They are acetate-derivative metabolites biosynthesized starting from a polyketide containing eight C2 units, which generates in turn with three aldol type condensations the carbon skeleton of anthraquinones except for the two carbonyl oxygens of the central ring. The latter are introduced by successive steps with an oxidation process. One example of this kind of biosynthesis is reported in Figure 1 for endocrin, a fungal anthraquinone produced by several Penicillium and Aspergillus species [3].
Among secondary metabolites anthraquinones are the most investigated natural products for their mechanism of action [4]. Plants, microorganisms, lichens, and algae are producers of metabolites possessing diverse biological activities such as phytotoxic, antibacterial, antiviral, anticancer, antitumor, algicide, antifungal, enzyme inhibiting, immunostimulant, antiplatelet aggregation, cytotoxic and antiplasmodium activities. Anthraquinones are frequently reported among the plethora of different classes of natural compounds as alkaloids, hydrocarbons, lipids, sterols, esters, fatty acids, derivatives of amino acids, terpenoids, and aromatic compounds [5][6][7]. The activity of several hydroxyland amino-anthraquinones cannot be exploited due to their weak solubility in water. Thus, some of them are converted into water-soluble analogues by biotransformation [8]. Anthraquinones have This review describes an advanced overview on anthraquinones, and related analogues grouped for the first time according to their natural sources. In particular, in addition to the isolation from fungal sources and their chemical characterization, their potential applications in different fields such as agriculture, medicine and the dyes industry are considered on the basis of their biological activities.
The first section chronologically describes the fungal anthraquinones starting from 1966 to the present day focusing on their sources, structures, and biological activities. The second section treats the industrial application of anthraquinones in a different field essentially as natural dyes. This part is focused on the comparison between natural and synthetic anthraquinone based dyes, their chemical derivatization and classification, and the advanced methods used in the treatment of the relative industrial wastewater to avoid severe negative environmental pollution. Finally, the main points described are summarized in the conclusion.

Fungal Anthraquinones and Analogues
Dothistromin (1, Figure 2, Table 1) was isolated as the main phytotoxin produced by Dothistroma pini (Hulbary), a pathogen inducing necrotic disease characterized by the formation of red bands on the infected needles of Pinus radiata and other pines [13]. The same fungus also produced six other anthraquinones: bisdeoxydothistromin; bisdeoxydehydrodothistromin; This review describes an advanced overview on anthraquinones, and related analogues grouped for the first time according to their natural sources. In particular, in addition to the isolation from fungal sources and their chemical characterization, their potential applications in different fields such as agriculture, medicine and the dyes industry are considered on the basis of their biological activities.
The first section chronologically describes the fungal anthraquinones starting from 1966 to the present day focusing on their sources, structures, and biological activities. The second section treats the industrial application of anthraquinones in a different field essentially as natural dyes. This part is focused on the comparison between natural and synthetic anthraquinone based dyes, their chemical derivatization and classification, and the advanced methods used in the treatment of the relative industrial wastewater to avoid severe negative environmental pollution. Finally, the main points described are summarized in the conclusion.

Fungal Anthraquinones and Analogues
Dothistromin (1, Figure 2, Table 1) was isolated as the main phytotoxin produced by Dothistroma pini (Hulbary), a pathogen inducing necrotic disease characterized by the formation of red bands on the infected needles of Pinus radiata and other pines [13]. The same fungus also produced six other anthraquinones: bisdeoxydothistromin; bisdeoxydehydrodothistromin; 6-deoxyversicolorin C;   Macrosporin and 6-methylxanthopurpurin 3-methyl ether (8 and 9, Figure 2, Table 1) are two anthraquinones produced by Alternaria bataticola, the causal agent of a black spot of sweet potato [16]. Compound 8 was also isolated from other fungi of the same genus as A. porri, A. solani and A. cucumerina while 9 was isolated also from A. solani [16]. Then macrosporin was isolated together with another two anthraquinones, named altersolanols A and J (10 and 11, Figure 2, Table 1), as well as nectriapyrone, an α-pyrone, from the culture filtrates of Diaporthe angelicae (anamorph Phomopsis Macrosporin and 6-methylxanthopurpurin 3-methyl ether (8 and 9, Figure 2, Table 1) are two anthraquinones produced by Alternaria bataticola, the causal agent of a black spot of sweet potato [16]. Compound 8 was also isolated from other fungi of the same genus as A. porri, A. solani and A. cucumerina while 9 was isolated also from A. solani [16]. Then macrosporin was isolated together with another two anthraquinones, named altersolanols A and J (10 and 11, Figure 2, Table 1), as well as nectriapyrone, an α-pyrone, from the culture filtrates of Diaporthe angelicae (anamorph Phomopsis foeniculi), the causal agent of serious disease on fennel (Foeniculum vulgare) in Bulgaria. These four metabolites were tested on detached tomato leaves and only nectriapyrone and altersolanols A and J showed a modulate phytotoxicity while macrosporin was not toxic [17].
Stemphylin, is a phytotoxin (12, Figure 2, Table 1) produced by Stemphyfium botryosum, a fungal pathogen inducing a destructive disease in lettuce. The first structure of compound 12 was wrongly assigned by Barash et al. (1975 and1978) [18,19], and then corrected when it was isolated from the same fungus together with two other phytotoxic anthraquinones, the above cited macrosporin and dactylariol (13, Figure 2, Table 1) [20]. The latter compound (13) showed anti Adenosine TriPhosphate (ATP) catabolism in Erlich I ascite tumor cells while stemphylin showed a weak antitumor activity on the treated animal at a dose of 40 mg/kg [20]. Stemphylium botryosum, inducing leaf spot disease on beet plants, also synthesized macrosporin and dactylariol (8 and 13), together with other anthraquinones identified as stemphyrperylenol (14, Figure 2), alteroporiol, and stemphynols A and B (15-17, Figure 3, Table 1). The phytotoxicity of all the metabolites (8, 13-17) was tested on lettuce and beet evaluating the seeds elongation. Compound 13 was the most active while compound 14 exhibited a moderate inhibition while the other ones were inactive [21].
Rugulosin (18) was isolated together with emodin and skyrin from Hormonema dematioides and showed activity against survival of budworm larvae while the other two anthraquinones were inactive [24].
Aspergillus fumigatus, which is responsible for a lung disease, produced a plethora of secondary metabolites belonging to different classes of natural compounds. Among them, emodin, 2-chloro-emodin (21 and 22 Figure 3), and physcion (23, Figure 3) were also isolated. However, compound 23 appeared not to have a role in the fungal infection [25].
Physcion (23) was also isolated from the organic culture extract of the marine derived fungus Microsporum sp. and showed a cytotoxic effect on human cervical carcinoma HeLa cells and its apoptosis induction was deeply investigated. Physcion also caused the formation of reactive oxygen species (ROS) in the same cells [26].
Many Drechslera species, which are important pathogens on gramineous plants and their seeds, produced colored pigments [27]. The red pigment exudated from Drechslera teres, D. graminea, D. tritici-repentis, D. phlei, D. dictyoides, D. avenae was identified as the anthraquinone catenarin (24, Figure 3, Table 1) while that from D. avenae and Bipolaris sorokiniana were two other anthraquinones recognized as helminthosporin and cynodontin (25 and 26, Figure 3, Table 1). Catenarin (24) showed a total inhibition of Bacillus subtilis (Gram+) growth but had no effect on the Gram-bacterium Ervinia carotova but in part inhibited the mycelium growth of D. teres [28]. In particular, catanerin and emodin (24 and 21) were also found in kernels infected by Pyrenophora tritici-repentis (Died.) Drechs. (anamorph: Drechslera tritici-repentis (Died.) Shoem.), the causal agent of tan spot of wheat. Compound 24 caused the reddish discoloration with red smudge of kernels, while compound 21 indicated that P. tritici-repentis is a mycotoxigenic fungus. Compound 24 also induced non-specific leaf necrosis and appeared moderately active against some of the fungi associated with P. tritici-repentis suggesting its possible role in the life strategy of the pathogen [29]. Cryphonectria (Endothia) parasitica (Murr.) Barr, the causal agent of chestnut (Castanea sativa) canker disease and other species produces diaporthin, a phytotoxic benzopyranone pigment, together with phytotoxic anthraquinones. The hundreds of fungal strains were grouped into virulent, intermediate and hypervirulent and produced, respectively, diaporthin , rugulosin and skirin (18 and 19, Figure 3, Table 1), crysophanol (20, Figure 3, Table 1) and emodin (21, Figure 3, Table 1) [22]. Virulent and hypovirulent strains of C. parasitica also produced a main polysaccharide identified as pullulan (a polymer constituted of α-1,4-and α-1,6-glucan) and a minor fraction which Cytoskyrins A and B (27 and 28, Figure 3, Table 1) are two closely related bisanthraquinones obtained from large-scale cultures of an endophytic fungus, CR200 (Cytospora sp.), isolated from the branch of a Conocarpus erecta tree in the Guanacaste Conservation Area of Costa Rica. Previously, a substituted benzopryrone, named cytosporone, was isolated from the same fungus and showed antibiotic activity [30]. Cytoskyrin A showed strong BIA activity down to 12.5 ng in the standard assay Toxins 2020, 12, 714 6 of 30 while cytoskyrin B was inactive at the concentrations tested (<50 mg) [31]. The biochemical induction assay (BIA) measures the induction of the SOS response in bacteria and is used to identify compounds that inhibit DNA synthesis, either directly by inhibiting the DNA replication machinery or more often indirectly by modifying DNA [32][33][34]. BIA activity is highly dependent on the three-dimensional structure and not a general property of these polyphenolic compounds. In fact, close bisanthraquinones such as luteoskyrin (29, Figure 3) and rugulosin (18) are known to interact with DNA. The structural basis of these differences is not yet clear [31].
Dendryols A-D (30-33, Figure 4, Table 1), four phytotoxic anthraquinones, were produced by Dendryphiella sp. [35], a fungus isolated from an infected sample of the paddy weed Eleocharis kuroguwai (Cyperaceae) in Japan [36]. The dendryols 30-33, when tested for the phytotoxic activity by leaf-puncture assay on weeds (kuroguwai, barnyardgrass, and velvetleaf) and cultivated crops (rice, corn, and cowpea), showed toxicity only against barnyardgrass and the nercrotic area appeared to be dose-dependent. Compound 30 caused similar necrosis only on velvetleaf [35].  Rubellin A (34, Figure 4, Table 1) was isolated from the culture filtrates of Ramularia collocygni, the causal agent of leaf-spot disease of barley in central Europe [37]. From the same fungus rubellins B-F and 14-deydro rubellin D (35-38, Figure 4, Table 1) were also isolated. Biosynthetic studies carried out by the incorporation of both [1-13 C]-acetate and [2-13 C]-acetate into the rubellins Rubellin A (34, Figure 4, Table 1) was isolated from the culture filtrates of Ramularia collocygni, the causal agent of leaf-spot disease of barley in central Europe [37]. From the same fungus rubellins B-F and 14-deydro rubellin D (35-38, Figure 4, Table 1) were also isolated. Biosynthetic studies carried out by the incorporation of both [1-13 C]-acetate and [2-13 C]-acetate into the rubellins demonstrated that such anthraquinone derivatives were biosynthesized via the polyketide pathway. Rubellin A (34) increased photodynamic oxygen activation [38], while rubellins B-E exhibited antibacterial activity, as well as light-dependent, antiproliferative and cytotoxic activity in a series of human tumor cell lines [39]. Closely related anthraquinones were isolated from the same fungus and from Ramularia uredinicola and identified as uridinetubellins I and II, and caeruleoramulin (40-42, Figure 4 and Table I). Both uredinorubellins (40 and 41) showed photodynamic activity comparable to rubellin D, whereas caeruleoramularin did not display such activity [40].
1-Hydroxy-3-methyl-anthraquinone and 1,8-dihydroxy-3-methyl-anthraquinone (43 and 44, Figure 5, Table 1), were isolated together with other bioactive metabolites from Trichoderma harzianum in a study aimed to improve the production and application of novel biopesticides and biofertilizers and thus to help in the management of crop plant diseases. However, the two anthraquinones had no role in this activity [41].
Fungi belonging to the Alternaria genus are also well known as producers of a plethora of bioactive metabolites. In fact, five new hydroanthraquinone derivatives, named tetrahydroaltersolanols C-F (54-57, Figure 6, Table 1) and dihydroaltersolanol A (58, Figure 6, Table 1), and five new alterporriol-type anthranoid dimers, named alterporriols N−R (59-63, Figure 6), were isolated from the culture broth and the mycelia of Alternaria sp. ZJ-2008003.
The fungus also produced seven known analogues as tetrahydroaltersolanol B, altersolanol B, altersolanol C, altersolanol L, ampelanol, macrosporin (8, Figure 2) and alterporriol C. The fungus was isolated from Sarcophyton sp. soft coral collected from the South China Sea. All the compounds were assayed against the porcine reproductive and respiratory syndrome virus (PRRSV) and 54 and 62 showed antiviral activity with IC 50 values of 65 and 39 µM, respectively. Compound 61 exhibited cytotoxic activity against PC-3 and HCT-116 cell lines, with IC 50 values of 6.4 and 8.6 µM, respectively [47].
anthraquinones had no role in this activity [41].
Two new dimeric anthraquinones with a rare chemical skeleton, named torrubiellins A and B (65 and 66, Figure 7, Table 1), were isolated from Torrubiella sp. BCC 28517 (family Clavicipitaceae) belonging to a genus of fungus that attacks spiders, scale-insects, and hoppers. Torrubiellin B (66) exhibited a broad range of biological activities including strong antimalarial (Plasmodium falciparum), antifungal (Candida albicans), antibacterial (Bacillus cereus) activities, and cytotoxicity to cancer cell lines. Its biological activity was always higher than that of torrubiellin A (65) [49].
Two new xanthone-anthraquinone heterodimers, named acremoxanthones C and D (67 and 68, Figure 7, Table 1), were isolated from an unidentified fungus of the Hypocreales order (MSX 17022). The fungus also produced the close and already known acremonidins A and C, benzophenone, and moniliphenone. All the metabolites showed moderate cytotoxic activity in vitro. In addition, acremoxanthone D (68), and acremonidins A and C exhibited moderate 20S proteasome inhibitory activity [50]. Two new dimeric anthraquinones with a rare chemical skeleton, named torrubiellins A and B (65 and 66, Figure 7, Table 1), were isolated from Torrubiella sp. BCC 28517 (family Clavicipitaceae) belonging to a genus of fungus that attacks spiders, scale-insects, and hoppers. Torrubiellin B (66) exhibited a broad range of biological activities including strong antimalarial (Plasmodium falciparum), antifungal (Candida albicans), antibacterial (Bacillus cereus) activities, and cytotoxicity to cancer cell lines. Its biological activity was always higher than that of torrubiellin A (65) [49].
Two new xanthone-anthraquinone heterodimers, named acremoxanthones C and D (67 and 68, Figure 7, Table 1), were isolated from an unidentified fungus of the Hypocreales order (MSX 17022). The fungus also produced the close and already known acremonidins A and C, benzophenone, and moniliphenone. All the metabolites showed moderate cytotoxic activity in vitro. In addition, acremoxanthone D (68), and acremonidins A and C exhibited moderate 20S proteasome inhibitory activity [50].
,6β-triol, and 6β-methoxyergosta-7,22-diene-3β,5α-diol were isolated from the same fungus. Compound 81 exhibited antibacterial activity against Escherichia coli and S. aureus, and lethality against brine shrimp (Artemia salina) with an LC50 value of 0.5 μg/mL [56].  (81), together with three known anthraquinones 1-O-methyl averantin, averufin (9), and versicolorin C were also produced by the endophytic fungus ZSUH-36 isolated from a mangrove collected from the South China Sea. At that time, only the unambiguous structure of 79, being a new anthraquinone, was determined by advanced NMR spectra while no activity was described [54]. Previously, compounds 82 and 85, together with the two xanthones 5-methoxysterigmatocystin and sterigmatocystin (86 and 87, Figure 9, Table 1), had been isolated from the same fungus and only the unambiguous structure of compound 85, which at that time was a new anthraquinone, was determined by advanced NMR spectra [55]. Compounds 80-82 and 84 were previously isolated from the culture of Aspergillus versicolor, an endophytic fungus obtained from the marine brown alga Sargassum thunbergii.
Aspetritones A and B (108 and 109, Figure 10, Table 1) were isolated from the culture of the coral-derived fungus Aspergillus tritici SP2-8-1, together with 4-methyl-candidusin A and fifteen known metabolites belonging to different classes of natural compounds as prenylcandidusin, candidusin, and terphenyllin derivatives and anthraquinones. Bostrocyn (110, Figure 10, Table 1) and other four anthraquinones (111-114, Figure 10, Table 1) were isolated. Aspetritone A (108) showed the most significant activity against methicillin-resistant strains of S. aureus in respect to that of the positive control chloramphenicol and exhibited strong cytotoxicity against human cancer cell lines HeLa, A549, and Hep G2 [63].
The crude organic extract of the fungal culture filtrates showed cytotoxicity, using "Brine Shrimp Lethality Bioassay", antimicrobial and antioxidant activity. Among the isolated metabolites, compound 138 appeared to be the most potent anticancer and antimicrobial metabolite [77].

Industrial Application of Anthraquinones
Since 1869 with the determination of the structure of alizarin (167, Figure 13, Table 1), a yellow anthraquinone, the main industrial application of this anthraquinone was its use as a dye in textile manufacturing [85]. Compound 167 was isolated for the first time from Rubia tinctorum [84]. Thus, over a span of 20 years many analogues with different functionalities were also prepared by synthesis to obtain different dyes such as red, blue, and green mordant. Successively, the first acidic anthraquinone dye used to color wool without pretreatment with mordants was reported. At the beginning of 1900 the sulfonation and nitration of anthraquinone opened up a new era for anthraquinone based dyes.
A new phase in this development occurred with the introduction of synthetic fibers, such as polyester, polyamide, and polyacrylonitrile fibers, with the substitution of anthraquinones with other dyes. The use of acid anthraquinone dyes increased with the discovery of the first fiber-reactive dyes. At the same time, the utilization of natural substances instead of synthetic ones, increased worldwide. This satisfy the request of environmentally friendly sustainable technologies. As reported in Section 1 fungi are a significant source of pigments as several genera can produce pigments in good amounts identified as anthraquinones or analogues. The production of anthraquinones by fungal fermentation had been developed for rapid and easy growth to produce pigments useful in various industrial applications [86]. The natural anthraquinones, as well as other natural pigments, have noteworthy less toxic effects than the synthetic dyes and are easily degradable avoiding the high environmental pollution. Thus, these anthraquinone based dyes are used in medical, textile coloring, food coloring, and cosmetic industries [84,86].
On this basis also plants have been largely used as a source of natural colored anthraquinones. In fact, screening of dyeing plants was carried out for their widespread use in previous centuries. Colorimetric analysis showed that the principal color was yellow-orange shades and could be attributed to flavonoids while the red colors were due to anthraquinones. Colors from plants that contain anthocyanins varied from blue-violet through to red. The nature of the support fibers (wool or cotton) plays an important role in the perceived colors [87].
Recently water-repellent, self-cleaning and stain resistant textiles were obtained by developing anthraquinone reactive dyes which were covalently grafted onto cotton fabric surfaces obtaining bright colors with good wash-fastness properties and giving rise to breathable superhydrophobic textiles with self-cleaning properties [89].
The large number of textile dyes required a method for their classification which was based on the functional groups attached to the typical anthraquinone carbon skeleton. Thus, there are: anthraquinone, azo, phthalocyanine, sulfur, indigo, nitro, nitroso anthraquinone derivatives etc. taking into account their chemical structures. Another classification was based on the method of applying these dyes on an industrial scale, grouped as disperse, direct, acid, reactive, basic, vat dyes etc. [90].
The intensity of research focused on natural compounds has been growing over the past few decades. Anthraquinones have been most studied in China producing several publications which report different advanced extraction methods, analytical techniques, and industrial applications. These publications also describe the most used plants for anthraquinone content as Polygonaceae, Rubiaceae, and Fabaceae and report the best known anthraquinones: rhein aloe emodin, emodin, physcion, chrysophanol which are responsible for their numerous biological properties. Furthermore, the use of natural anthraquinones for industrial applications, has been described as an alternative to synthetic dyes to avoid some unwanted side effects [9].
However, the environmental contamination by wastewater containing dyes is today a severe problem to solve. The application of advanced oxidation processes (AOPs) to industrial wastewater has increased as well as an integrated approach for their biological and chemical treatment. The toxicity of the detergents and the dye have been determined in terms of effective concentration EC 50 using mixed cultures of activated sludge as well as a pure culture of luminescent bacteria Vibrio fischeri NRRLB-11177. However, the dye was not degraded without AOP pretreatment, therefore the degree of its removal (decolorization) by the AOPs is an important preliminary stage of bio-sorption on activated sludge [91].

Conclusions
The sources, structures, and the biological activities of fungal bioactive anthraquinones were reported starting from 1966 to the present day. In the introduction the previous review published on this topic was also cited which did not however treat the topic extensively. The anthraquinones were chronologically described and in some cases their isolation and biological activity was investigated in depth. Furthermore, their industrial application in different fields, essentially as natural dyes, was also reported focusing on the comparison between natural and synthetic anthraquinone based dyes, their chemical derivatization and classification, and the advanced methods used in the treatment of the relative industrial wastewaters to avoid severe negative environmental pollution.
Author Contributions: The authors equally contributed to this work. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.