Diverse Metabolites and Pharmacological Effects from the Basidiomycetes Inonotus hispidus

Inonotus hispidus mushroom is a popular edible and medicinal mushroom with a long history of use. It is well known as a medicinal fungus with various health benefits for its significant anticancer and immunomodulatory activities. Over the last 60 years, secondary metabolites derived from I. hispidus and their biological activities have been discovered and investigated. Structurally, these compounds are mainly polyphenols and triterpenoids, which have anticancer, anti-inflammatory, antioxidant, antimicrobial, and enzyme inhibitor activities. Here, the secondary metabolites derived from I. hispidus and their activities were systematically and comprehensively classified and summarized, and the biosynthetic pathway of stylylpyrones was deduced and analyzed further. This review contributes to our understanding of I. hispidus and will help with research into natural product chemistry, pharmacology, and the biosynthesis of I. hispidus metabolites. According to this review, I. hispidus could be a promising source of bioactive compounds for health promotion and the development of functional foods.

Since I. hispidus are frequently parasitic on the mulberry tree (pronounced "sang" in Chinese), their fruit bodies are yellow (pronounced "huang" in Chinese) in color, highly similar in morphology to the modern taxonomic definition of the genus Sanghuangporus [5], and relatively close in herbal activity; I. hispidus were described as "Sanghuang" in the ancient Chinese medical classics. In the Aksu region of Xinjiang and Xiajin County of Shandong in China, I. hispidus parasites on mulberry trees has been used as a traditional indigenous medicine "Sanghuang" for the treatment of dyspepsia, cancer, diabetes, and stomach problems [6]. In fact, "Sanghuang" is a traditional collective name including I. hispidus, which has a long history of medicinal use and high economic value in the Southeast Asia region ("Meshimakobu" in Japan, and Sanghwang in South Korea) [7]. In China, the earliest medicinal records on "Sanghuang" can be traced back to Shen Nong's Materia Since I. hispidus are frequently parasitic on the mulberry tree (pronounced "sang" in Chinese), their fruit bodies are yellow (pronounced "huang" in Chinese) in color, highly similar in morphology to the modern taxonomic definition of the genus Sanghuangporu [5], and relatively close in herbal activity; I. hispidus were described as "Sanghuang" in the ancient Chinese medical classics. In the Aksu region of Xinjiang and Xiajin County of Shan dong in China, I. hispidus parasites on mulberry trees has been used as a traditional indig enous medicine "Sanghuang" for the treatment of dyspepsia, cancer, diabetes, and stom ach problems [6]. In fact, "Sanghuang" is a traditional collective name including I. hispidus which has a long history of medicinal use and high economic value in the Southeast Asia region ("Meshimakobu" in Japan, and Sanghwang in South Korea) [7]. In China, the ear liest medicinal records on "Sanghuang" can be traced back to Shen Nong's Materia Medic in the Qin and Han Dynasties. The "Shen Nong's Materia Medica" and "Compendium of Ma teria Medica" have detailed descriptions of the efficacy of "Sanghuang". After the Second World War, the number of cancer cases in Nagasaki, Japan, increased dramatically. Danjo gunto in Nagasaki is rich in "Sanghuang" due to the cultivation of mulberries and silk worms, and the residents were more healthy and had fewer cancer cases due to taking "Sanghuang". It was later found that it was the anti-tumor properties of mulberry tha played a key role [8]. Recent studies in natural product chemistry and pharmacology have shown that I. hispidus contains a diverse spectrum of monomeric compounds and exhibit a diversity of physiological actions in in vitro and in vivo assays.
This review summarizes English and Chinese articles published in international ac ademic journals after 1961, based on retrieving electronic databases PubMed, Wiley Online Library, Science Direct, and Web of Science. Totals of 64 compounds, including polyphenols, terpenoids, fatty acids and other types, were classified and sorted. These metabolites with outstanding medicinal value, and crude extracts of the fruit bodies were then inventoried and summarized according to their antitumor, anti-inflammatory, anti oxidant, antineurodegenerative, and antimicrobial biological activities. The biosynthetic pathways of styrylpyrones derived from I. hispidus and related fungi were deduced and This review summarizes English and Chinese articles published in international academic journals after 1961, based on retrieving electronic databases PubMed, Wiley Online Library, Science Direct, and Web of Science. Totals of 64 compounds, including polyphenols, terpenoids, fatty acids and other types, were classified and sorted. These metabolites with outstanding medicinal value, and crude extracts of the fruit bodies were then inventoried and summarized according to their antitumor, anti-inflammatory, antioxidant, antineurodegenerative, and antimicrobial biological activities. The biosynthetic pathways of styrylpyrones derived from I. hispidus and related fungi were deduced and summarized. Related studies on phenols and sesquiterpenes, and food properties of I. hispidus are discussed. This work will provide a reference for subsequent studies on the natural product chemistry, pharmacological activities, and biosynthesis of secondary metabolites of I. hispidus.

Error in Figure
In the original publication [1], there was a mistake in **Figure 2** as published. ** The correct structure of compound 2 was not listed, and structures 2-24 did not match the corresponding descriptions in the article**. The corrected ** Figure 2** appears below. The authors state that the scientific conclusions are unaffected. This correction was approved by the Academic Editor. The original publication has also been updated.   In the original publication, there was a mistake in

Triterpenoids
Triterpenoids are another important group of secondary metabolites found in I. hispidus, with a total of 15 triterpene compounds isolated (  [15]. Compound 28 is a high-content component in the fruiting bodies of I. hispidus [15].
Antibiotics 2022, 11, x FOR PEER REVIEW 5 of 17 content of total triterpenoids in an orthogonal experiment for liquid fermentation of I. hispidus [24].

Fatty acid Compounds
The fatty acid analogues of I. hispidus were first reported by Yang et al. in 2008, and two monomer compounds hexadecanoic acid (41) and octadecanoic acid (42) were obtained in the ethanolic extract of its fruit bodies [23] (Figure 4). The structure of the former was identified by EI-MS and TLC comparison of the standard, and the identification of the latter was done with additional infrared techniques [23]. Zan et al. published 2012 the detection results of gas mass spectrometry of the yellow oily substance obtained from the petroleum ether extraction part of the methanol extract of fruit bodies. A total of 12 fatty acid compounds were identified by consulting the Wiley Online Library. These compounds are hexadecanoic acid (41), (E)-9-hexadecenoic acid methyl ester (43), hexadeca-  (36), cerevisterol (37), (22E, 24R)-ergosta-7,22-diene-3β,5α,6β,9α-tetrol (38), and Inonoterpene A (39), and 3β-hydroxy-lanosta-8,24-dien-21-al (40). Among them, Inonoterpene A (39) is a new Lanostane triterpenoid, and compound 40 was isolated from the fungus for the first time. Interestingly, the above 15 triterpenoids are all lanosterol-type triterpenoids, all derived from the fruiting bodies of I. hispidus. Only three triterpenes 32, 33, and 39, have been isolated as new compounds for the first time from I. hispidus. Compounds 32 and 40 contain formyl groups at C30 and C21, respectively, while compound 35 possesses glycosylated modifications. It was discovered that specific combinations of four compounds, methyl jasmonate, salicylic acid, oleic acid, and Cu 2+ , and their combinations, can increase the content of total triterpenoids in an orthogonal experiment for liquid fermentation of I. hispidus [24].

Fatty Acid Compounds
The fatty acid analogues of I. hispidus were first reported by Yang et al. in 2008, and two monomer compounds hexadecanoic acid (41) and octadecanoic acid (42) were obtained in the ethanolic extract of its fruit bodies [23] (Figure 4). The structure of the former was identified by EI-MS and TLC comparison of the standard, and the identification of the latter was done with additional infrared techniques [23]. Zan et al. published 2012 the detection results of gas mass spectrometry of the yellow oily substance obtained from the petroleum ether extraction part of the methanol extract of fruit bodies. A total of 12 fatty acid compounds were identified by consulting the Wiley Online Library. These compounds are hexadecanoic acid (41), (E)-9-hexadecenoic acid methyl ester (43), hexadecanoic acid methyl ester (44), hexadecanoic acid ethyl ester (45), 10,13-octadecadienoic acid methyl ester (46), ethyl oleate (47), linoleic acid ethyl ester (48), 9,12-octadecadienoic acid (Z, Z)methyl ester (49), Isopropyl linoleate (50), cis-erucic acid (51), and cis-9,17 octadecadienoic acid methyl ester (52) [15]. Due to their low polarity and insignificant drug properties, fatty acid compounds are not preferred subjects in natural product chemistry studies of fungi. Only two cases of I. hispidus have been reported in this regard. content of total triterpenoids in an orthogonal experiment for liquid fermentation of I. hispidus [24].

Fatty acid Compounds
The fatty acid analogues of I. hispidus were first reported by Yang et al. in 2008, and two monomer compounds hexadecanoic acid (41) and octadecanoic acid (42) were obtained in the ethanolic extract of its fruit bodies [23] (Figure 4). The structure of the former was identified by EI-MS and TLC comparison of the standard, and the identification of the latter was done with additional infrared techniques [23]. Zan

Miscellaneous Compounds
During the constituent analysis of dried fruiting bodies of I. hispidus in 2007, Politi et al. identified a known compound, phenylalaninopine (53), by spectral and mass spectrometric characterization collected by LC-DAD-MS and comparing with the literature, while attempting to develop a phytochemical extraction method for mushrooms using hot water as a solvent [25] ( Figure 5). The compound was isolated initially from Clitocybe acromelalga, a poisonous mushroom [26]. Yang et al. obtained two monosaccharides, D-arabitol (54) and D-glucose (55), along with the isolation of four triterpenoids [23]. In their experiments to examine the fatty acid composition of fruiting bodies, Zan et al. discovered two hydrocarbons, squalene (56) and docosane (57), and one phthalate, 1,2-benzenedicarboxylic acid mono(2-ethylhexyl) ester (58) [15]. In the separation of anticancer active components, Yang et al. isolated a new compound from the methanol extract of fruiting bodies in 2019 and confirmed its structure as (4S,5S)-4-Hydroxy-3,5-dimethoxycyclohex-2-enone (HDE, 59) by LC-MS and NMR [27]. When analyzing the methanol extract composition of I. hispidus fruiting bodies, Yang et al. identified a nucleoside compound, inosine (60), based on a comparison of UV absorption characteristics and retention time with a standard [20]. Kou et al. isolated an unsaturated sesquiterpene, xylaritriol (61) [21], from the methanol extract of fruiting bodies, which had previously been from the metabolites of endophytic fungus Xylaria cubensis (Ascomycete) [28]. Tang et al. isolated compounds, cinnamic acid (62), uridine (63), and Cyclo (L-Leu-L-Phe) (64) from the fermentation broth of I. hispidus mycelium in 2021 [22]. Among these 12 compounds with no shared structural features, compound 59 is the only new compound isolated for the first time from I. hispidus, and compound 61 was the only sesquiterpene isolated from I. hispidus. Cyclic dipeptide compounds are uncommon in mushrooms (Basidomycota), but compound 64 was discovered in I. hispidus. This could be related to the fact that compound 64 is derived from mycelial fermentation broth.
arabitol (54) and D-glucose (55), along with the isolation of four triterpenoids [23] experiments to examine the fatty acid composition of fruiting bodies, Zan et al. dis two hydrocarbons, squalene (56) and docosane (57), and one phthalate, 1,2-benze boxylic acid mono(2-ethylhexyl) ester (58) [15]. In the separation of anticancer act ponents, Yang et al. isolated a new compound from the methanol extract of fruitin in 2019 and confirmed its structure as (4S,5S)-4-Hydroxy-3,5-dimethoxycyclohex-(HDE, 59) by LC-MS and NMR [27]. When analyzing the methanol extract compo I. hispidus fruiting bodies, Yang et al. identified a nucleoside compound, inos based on a comparison of UV absorption characteristics and retention time with ard [20]. Kou et al. isolated an unsaturated sesquiterpene, xylaritriol (61) [21], f methanol extract of fruiting bodies, which had previously been from the metab endophytic fungus Xylaria cubensis (Ascomycete) [28]. Tang et al. isolated com cinnamic acid (62), uridine (63), and Cyclo (L-Leu-L-Phe) (64) from the fermentati of I. hispidus mycelium in 2021 [22]. Among these 12 compounds with no shared st features, compound 59 is the only new compound isolated for the first time from dus, and compound 61 was the only sesquiterpene isolated from I. hispidus. Cycli tide compounds are uncommon in mushrooms (Basidomycota), but compound discovered in I. hispidus. This could be related to the fact that compound 64 is from mycelial fermentation broth.

Biological and Pharmacological Activities
Inonotus hispidus has long been used to treat dyspepsia, cancer, diabetes, and problems in Xinjiang residents [6]. Several studies have been published on the ph logical activities and mechanisms of major compounds from I. hispidus (Table 1 studies have been summarized here, with a special emphasis on polyphenols with inal potential (Figure 6).

Biological and Pharmacological Activities
Inonotus hispidus has long been used to treat dyspepsia, cancer, diabetes, and stomach problems in Xinjiang residents [6]. Several studies have been published on the pharmacological activities and mechanisms of major compounds from I. hispidus (Table 1). These studies have been summarized here, with a special emphasis on polyphenols with medicinal potential (Figure 6).   [20].
NA indicated the data is not available.

Antitumor Activity
As a traditional medicinal fungus with a long-applied history, anti-cancer activity is the principal medicinal value of I. hispidus. Multiple monomers with anticancer activity from I. hispidus have been discovered and preliminarily resolved in modern chemical chemistry and pharmacological investigations. In vitro screening of five compounds including hispidin (1), inonotusin A (7) and inonotusin B (8) against breast cancer cells MCF-7 using the SRB method revealed that inonotusin A (7) had moderate activity against MCF-7 with an IC 50 value of 19.6 µM [16]. The anti-tumor screening study of MBP (21) was carried out by MTT method. The results showed that MBP (21) had a dosage-dependent inhibitory effect on the proliferation of HepG2, MCF-7, Hela and A549 cells. Among them, HepG2 showed the best inhibitory effect, with an IC 50 of 2.3 µg/mL.
According to the results of the apoptosis analysis, it is hypothesized that the compound (21) achieves its anti-tumor activity by inducing apoptosis [20]. A study of compound 26's antitumor activity discovered that it inhibited the growth of lymphoma cells U937, cervical tumor cells Hela, and liver cancer cells QRH-7701 [23]. In vitro screening showed that HDE (59) inhibited the proliferation of HepG2, McF-7, Hela, A549 and H 22 cells, with a greater effect on HepG2 cells (IC 50 = 7.9µg/mL) [27]. Not only did HDE (59) increase the relative activities of Caspase-3 and Caspase-8 and induce HepG2 apoptosis, but it also promoted HepG2 apoptosis by up-regulating Fas expression and down-regulating FasL expression [27] (Figure 7A). In vivo mouse experiments revealed that HDE (59) does not cross the blood-brain barrier and is rapidly metabolized with no adverse effects on organs. Overall, HDE (59) has the potential to be an effective antineoplastic agent [27]. At 10 mmol/L, the compound inotolactone B (34) still showed activity in activating melanogenesis and tyrosinase in B16 melanoma cells, outperforming 8-MOP (a drug used clinically in the treatment of vitiligo). This indicates that inotolactone B (34) has the potential to be developed as an anti-vitiligo agent [18]. The evaluation of the cellular activity of mouse macrophage cells RAW264.7 revealed that compounds 16, 18 and 20 all had anti-inflammatory activity, with compound 16 being the most active. Cytotoxicity studies showed that all three compounds have a low impact on cell proliferation [2]. This result suggests that compound 16 has the potential to be an anti-leukemic agent. In the supernatant of fermented mycelium of a suspected new subspecies of I. hispidus, IH3, a single fraction with high purity of anti-tumor activity, WIH3, was obtained. WIH3 had strong inhibitory against melanoma cells B16 (IC 50 29.32 µg/mL), liver cancer cells Hep-3B (37.39 µg/mL), human cervical cancer cells Hela (47.03 µg/mL), and human breast cancer cells MCF-7 (58.01 µg/mL) [33].  Figure 7A). In vivo mouse experiments revealed that H (59) does not cross the blood-brain barrier and is rapidly metabolized with no adve effects on organs. Overall, HDE (59) has the potential to be an effective antineoplas agent [27]. At 10 mmol/L, the compound inotolactone B (34) still showed activity in a vating melanogenesis and tyrosinase in B16 melanoma cells, outperforming 8-MOP drug used clinically in the treatment of vitiligo). This indicates that inotolactone B (34) h the potential to be developed as an anti-vitiligo agent [18]. The evaluation of the cellu activity of mouse macrophage cells RAW264.7 revealed that compounds 16, 18 and 20 had anti-inflammatory activity, with compound 16 being the most active. Cytotoxic studies showed that all three compounds have a low impact on cell proliferation [2]. T result suggests that compound 16 has the potential to be an anti-leukemic agent. In supernatant of fermented mycelium of a suspected new subspecies of I. hispidus, IH3 single fraction with high purity of anti-tumor activity, WIH3, was obtained. WIH3 h strong inhibitory against melanoma cells B16 (IC50 29.32µ g/mL), liver cancer cells Hep-(37.39µ g/mL), human cervical cancer cells Hela (47.03 µ g/mL), and human breast can cells MCF-7 (58.01µ g/mL) [33]. In vivo antitumor activity experiments with extracts of fruit bodies from differe growth stages of I. hispidus revealed that both petroleum ether extracts (IPE) and aqueo extracts at the mature stage had the best tumor suppressive effect on H22-bearing m [15]. Using non-targeted metabolomics techniques, it was found that the antitumor effe of IPE on H22 tumor-bearing mice were mainly mediated by energy modulation and r In vivo antitumor activity experiments with extracts of fruit bodies from different growth stages of I. hispidus revealed that both petroleum ether extracts (IPE) and aqueous extracts at the mature stage had the best tumor suppressive effect on H 22 -bearing mice [15]. Using non-targeted metabolomics techniques, it was found that the antitumor effects of IPE on H 22 tumor-bearing mice were mainly mediated by energy modulation and regulation of biosynthetic pathways including amino acids and corticosteroids [34]. A series of key regulators of the antitumor activity of IPE were identified in H 22 tumor suppressor model mice through transcriptomic and proteomic approaches, and the results provide a useful reference for the pharmacological study of the antitumor activity of IPE [35]. The feeding experiment of solid fermentation powder of I. hispidus on H 22 -bearing mice demonstrated that it has antitumor effect, whose mechanism may be related to antioxidation, improving immunity and inhibiting tumor tissue angiogenesis [36].

Antioxidant Activity
One of the important properties of mushrooms is their antioxidant function, and antioxidant active molecules derived from I. hispidus have been investigated and studied. Antioxidant assays revealed that compounds 1, 7, and 8 have significant oxidative protective activity against 2,2 -azino-bis (3-ethylbenzthiazoline)-6-sulfonic acid (ABTS) [16]. Compounds 5, 9, 22-25 have remarkable inhibitory activity against 1,1-Diphenyl-2-picrylhydrazyl radical (DPPH) with IC 50 values of 9.82−21.43 µM, with compound 24 being more effective than the positive control [21]. Preliminary structure-activity relationship analysis revealed that catechol unit, double bond on the side-chain, and pyran ring may be important pharmacophores for increasing activity [21]. A study of the antioxidant activity of the compound hispidin (1) to scavenge free radicals discovered that at a low concentration (12.5 mg/L), the scavenging rate of 1 to free radical DPPH was 84.70%. At low concentrations, the DPPH scavenging rate was higher than that of BHA, a commercial antioxidant, and compound 1 has natural antioxidant value [30]. The antioxidant activity of compound 1 for scavenging hydroxyl radicals was found to be superior to that of BHA at concentrations as low as 25 mg/kg [30]. DPPH, ABTS and phosphomolybdenum were used as indicators to determine the antioxidant activity of the ethyl acetate extract of I. hispidus. The extract was found to be more powerful than the commonly tested antioxidant compounds gallic acid and quercetin [31]. The scavenging effect of hydroxyl radical, DPPH radical and superoxide anion of the volatile oil fragments (H-2) obtained by the diethyl ether reflux extraction of the fruiting bodies of I. hispidus was significantly better than that of the volatile oil fragments (H-1) obtained by the steam distillation extraction of the fruiting bodies of I. hispidus [37].

Antimicrobial Activity
The antibacterial activity tests performed on hispidin (1) revealed that compound 1 lacked adequate inhibitory activity against Escherichia coli, Staphylococcus aureus, and Bacillus subtilis [15]. The inhibition activity of a 70% ethanolic extract of fruit bodies was tested, and it was discovered that the ethanolic extract inhibited only E. coli, with the high dosage extract (40.96 mg/mL) achieving a significant level of inhibition. S. aureus and B. subtilis were not inhibited significantly [15]. At a concentration of 1 × 10 −5 g/mL, both the H-1 (water extract) and H-2 (ether extract) fractions of fruit bodies inhibited S. aureus, Candida albicans, Aspergillus niger, and Pseudomonas aeruginosa, with a positive correlation with concentration, but neither inhibited E. coli [15]. The disc diffusion assay of ethanolic extracts of I. hispidus fruit bodies revealed a minor antimicrobial effect against S. aureus, E. coli, and P. aeruginosa [38]. Hispolon (5) and hispidin (1) were discovered to have antiviral activity, inhibiting the growth of influenza A and B viruses [29]. After feeding mice I. hispidus mushroom containing compounds 1 and 5, the anti-influenza virus ability of mice was improved, as was the content of trace elements zinc (194 ± 16.9 mg/kg), selenium, and iron in mouse serum. These metal elements are thought to enhance the antiviral ability of the I. hispidus mushroom [14].

Enzyme Inhibitory Activity
Yousfi et al. discovered in 2013 that the polyphenol-rich ethyl acetate extract of I. hispidus fruit bodies was very effective in inhibiting Candida rugosa lipase, but the specific inhibitory compounds were unknown [39]. In 2015, they isolated hispidin (1) from the fruit bodies and demonstrated that hispidin (1) strongly inhibited C. rugosa lipase, proposing the use of hispidin (1) in the treatment of candidiasis [31]. Hispidin (1) was discovered to have anti-peroxidase activity, with an IC 50 of 23 mg/mL showing a strong competitive inhibition of horseradish peroxidase (HRP) activity. The inhibition mechanism of compound 1 against peroxidases (horseradish and thyroid) was studied using molecular docking simulations in terms of hispidin interaction with amino acid residues, and its drug-forming potential was predicted using ADEMT and Lipinski filtering analyses [40]. According to the combined evaluation, hispidin (1) is a more potent irreversible thyroperoxidase inhibitor than the anti-thyroid drug 6-hropylthiouracil [40]. The discovery of this result, to some extent, revealed the mechanism of the antioxidant activity of hispidin (1). Furthermore, low doses of ethanolic extracts of I. hispidus fruiting bodies increased GST enzyme activity [38]. Compound 34 (IC 50 = 0.24 mM) has excellent α-glycosidase inhibitory activity and is more potent than the clinically used acarbose (IC 50 = 0.46 mM) [40]. At the same concentration, the ethyl acetate extract of fruiting bodies had higher α-glucosidase inhibitory activity than the n-butanol, methanol, and petroleum ether extracts [41].

Immunomodulatory Effects and Other Biological activities
Cellular activity assays on NK cells showed that compounds 1 and 5 both inhibited the activation and proliferation of activated lymphocytes, but the combination of the two did not enhance this effect [17]. This result demonstrates the immunomodulatory activity of these phenolic compounds. In addition, the fermentation broth exopolysaccharide of I. hispidus was shown to attenuate inflammatory responses in mice with acute alcoholic liver disease [42].

Biosynthetic Progresses on Styrylpyrones
Styrylpyrone compounds are a specific class of polyphenols with a wide range of structures and high yields. The majority of I. hispidus-derived yellow polyphenol pigments have a styrylpyrone backbone, and 10 styrylpyrone compounds (1, 3-4, 8, 12, 15-16, 20-21,  24) have been identified from the secondary metabolites of I. hispidus. Nearly hundreds of styrylpyrone pigments have been discovered in a variety of fungi dominated by the Hymenochaetaceae family since the discovery of hispidin (1) as the first naturally occurring styrylpyrone metabolite [43][44][45]. Experiments on light-regulated enzymatic activity revealed that the cinnamic acid (62) pathway involved in phenylalanine metabolism is linked to hispidin synthesis in P. hispidus. Cinnamic acid (62) is thought to be a key intermediate in the biosynthesis of hispidin and other phenylpropanoids [46]. Isotope labeling traces revealed that the styrene unit was incorporated with phenylalanine, tyrosine, cinnamic acid (62), p-coumarate, and caffeic acids, while the pyrone ring was incorporated with acetate and malonate [10]. The isolation of five phenylpropanoids from the fruiting bodies of Inonotus sp., including iso-hispidin (65), inonotic acid methyl ester, and inotilone, provided clues to the biosynthesis of styrylpyrones. The phenylpropanoid polyketide is an important intermediate that is thought to be a side chain elongation of caffeic acid catalyzed by PKS [47]. Integrating metabolomic and proteomic findings that the up-regulation of phenylpropanoid biosynthesis leads to increased levels of hispidin and other polyphenols in P. baumii, genes involved in the up-regulation of expression include PAL, C4H, and 4-coumarate-CoA ligase (4CL) (Figure 8) [48]. PAL, 4CL, C4H, polyketide synthase (PKS), palmitoyl protein thioesterase (PPT), decarboxylase, and dehydrogenase genes were identified in a combined investigation of molecular docking simulations and RNA-seq-based analysis of differentially expressed genes in P. gilvus (Figure 8) [49]. Based on biosynthetic research and the analysis of existing styrylpyrone compounds derived from the Phellinus and Inonotus fungi, the comprehensive biosynthetic pathways of styrylpyrone compounds, including hispidin (1), hispolon (5) and their dimers, as well as inoscavin A (66), have been deduced in this review (Figure 8) [47][48][49]. The natural structural diversity of styrylpyrones suggests that their biosynthetic gene clusters are active and contain multiple post-modification genes. It is speculated that the study of its biosynthetic pathway and related synthetic gene clusters may aid in understanding the biosynthetic network of styrylpyrones and their derivatives in I. hispidus.

Discussions and Perspectives
Triterpenes are characteristic pharmacological components of lignified traditional medicinal fungi, such as Ganoderic acid from Ganoderma species [50], and Eburicoic acid and its derivatives from polypore species [51]; however, they are not monolithic. Phenols, a class of high-value natural products with medicinal potential [44,52], are prominent bioactive components of the highly lignified and well-known medicinal mushrooms of the genus Phellinus and Inonotus belonging to the order of Hymenochaetales [43]. The diversely modified phenolic derivatives yielded by these mushrooms have promising applications in drug discovery and development [43,44]. Hispidin (1) is the first compound isolated and identified from I. hispidus, and these phenolic compounds with styryl and pyrone moieties are gathered here with diverse activities such as antitumor, antioxidant, antimicrobial, and anti-inflammatory activities ( Figure 6). These diverse activities of the natural product reflect the biological activities of its producer. Despite the fact that 5 was initially isolated from I. hispidus in 1996 [12], it is more of a characteristic component of fungi belonging to the genus Phellinus, as it has also been isolated from various species of the genus Phellinus, including P. linteus, P. igniarius, P. lonicerinus and P. merrillii [43]. A summary of the anticancer, antidiabetic, antioxidant, antiviral, and anti-inflammatory properties of compound 5 revealed its potential development value as a complementary and alternative medicine [43].
As one of the most abundant secondary metabolites found in mushrooms, numerous mushroom-derived sesquiterpenes have been isolated and identified [53]. Sesquiterpene synthases are one of the core gene types involved in the biosynthesis of secondary metabolites. Mushroom genomes contain typically at least ten sesquiterpene synthases. I. obliquus is a congener species of I. hispidus. Although the genome of I. obliquus contains at least 4 types of 20 sesquiterpene synthases [54], only 8 sesquiterpenoids of the drimane-type have been reported [55]. So far, only one sesquiterpenoid (61) has been reported for I. obliquus [21], and the type and number of sesquiterpene synthases are also unknown. The identification of sesquiterpene cyclases derived from lots of mushrooms including Coprinus cinereus [56], Omphalotus olearius [57], Stereum hirsutum [58,59], Flammulina velutipes [60], and Agrocybe aegerita [61] have led to the discovery of numerous novel sesquiterpenoids, which are not found in their natural producer instead. As a result, we speculate that in most mushrooms, only one or a few types of sesquiterpene synthases can be expressed to produce specific sesquiterpenoids, while the majority of sesquiterpene synthases are silent and have no corresponding products.
Although I. hispidus has been widely considered a traditional medicinal fungus due to its secondary metabolites with remarkable significant pharmacological value, the food properties of I. hispidus that are rich in health and nutritional functional factors should not be overlooked. The edible fruiting body of I. hispidus is a scarce ingredient. I. hispidus possess a good selenium-enriching function [62], and the antiviral ability of I. hispidus rich in selenium has also been enhanced. The dihydroxy phenylalanine-melanin contained in I. hispidus could effectively remove DPPH radicals and can therefore be used in health food or as a food additive [63]. Furthermore, the extracellular exopolysaccharide of I. hispidus was investigated and found to have antioxidant activity [64] and hepatoprotective functions [42].
Mushrooms are often perceived more for their anticancer or immunomodulatory activity than for their antibacterial activity. In fact, mushrooms with antimicrobial activity are uncommon [65,66], with Pleurotus mutilus and I. hispidus being the few examples. Pleuromutilin was found to have good antifungal inhibitory activity and was used as the lead compound in the development of the human antibiotic retapamulin, which was approved by the FDA in 2007 [67]. Both aqueous and ether extracts of I. hispidus mushrooms inhibit pathogenic microorganisms such as S. aureus [15]; however, monomeric compounds with antibacterial activity have yet to be identified. As a result, the discovery and development of antimicrobial compounds derived from I. hispidus is worth pursuing.
Herein, a total of 64 compounds derived from I. hispidus were collected and classified according to their structural features. Biological and pharmacological activities including antitumor, antioxidant, as well as anti-inflammatory and neurotrophic and neuroprotective activities were summarized. These studies, however, were limited to the isolation, identification, and activity evaluation of compounds. Current research has not paid enough attention to I. hispidus, despite its significant medicinal efficacy and functional food value. Although the genomes of multiple rare medicinal fungi including I. obliquus [54] have been sequenced, the genome of I. hispidus has not been sequenced. The lack of genomic information of I. hispidus severely hampered the biosynthetic pathway elucidation of its active compounds. As a result, it is critical to complete the genome sequencing of I. hispidus [54] using rapidly developing sequencing technology, which will lay a firm foundation for biosynthesis research and synthetic biology production of valuable compounds to meet the needs of the drug development and functional food industries.