Bioactive Phytochemical Constituents of Wild Edible Mushrooms from Southeast Asia.

Mushrooms have a long history of uses for their medicinal and nutritional properties. They have been consumed by people for thousands of years. Edible mushrooms are collected in the wild or cultivated worldwide. Recently, mushroom extracts and their secondary metabolites have acquired considerable attention due to their biological effects, which include antioxidant, antimicrobial, anti-cancer, anti-inflammatory, anti-obesity, and immunomodulatory activities. Thus, in addition to phytochemists, nutritionists and consumers are now deeply interested in the phytochemical constituents of mushrooms, which provide beneficial effects to humans in terms of health promotion and reduction of disease-related risks. In recent years, scientific reports on the nutritional, phytochemical and pharmacological properties of mushroom have been overwhelming. However, the bioactive compounds and biological properties of wild edible mushrooms growing in Southeast Asian countries have been rarely described. In this review, the bioactive compounds isolated from 25 selected wild edible mushrooms growing in Southeast Asia have been reviewed, together with their biological activities. Phytoconstituents with antioxidant and antimicrobial activities have been highlighted. Several evidences indicate that mushrooms are good sources for natural antioxidants and antimicrobial agents


Introduction
Popularly, the term mushrooms (or higher fungi) is used to identify fungi producing macroscopic fruiting bodies. This rather inaccurate definition mostly refers to species belonging to the phyla Basidiomycota and Ascomycota. The total number of species of the kingdom fungi is far from being exactly known. It was believed to be around 1.5 millions [1], but more recent estimates have increased the number to a range of 2.2-3.8 millions, worldwide [2]. With 120,000 currently accepted About fungal species, 93% of the fungi growing in northern Thailand appeared to be novel [30]. Therefore, the biodiversity of Southeast Asia is likely to be a vast reservoir for finding new mushroom species, since fungi occurring in Myanmar, Laos, Vietnam, and Cambodia have barely been studied so far [30]. Moreover, it is worth noting that numerous ethnic groups living in Southeast Asian countries resort to several wild mushrooms for obtaining food and medicines; however, very few studies have been carried out on the nutritional value and biological activities of these mushrooms. This review has collected the information available in the literature on the phytochemical constituents of selected wild edible mushrooms occurring in Southeast Asian countries, namely Agaricus silvaticus, Ampulloclitocybe clavipes, Butyriboletus roseoflavus, Cantharellus cibarius (Figure 2a The most important biological/pharmacological activities reported for extracts and isolated compounds are also described, with special attention to antioxidant, antimicrobial and cytotoxic properties. They have been summarized in Table 1. The chemical structures of new compounds or compounds that are specific to the collected musroom species are depicted in . It is worth noting that a great number of data reported herein have been collected through investigations conducted on mushroom samples collected outside Asia, especially in Europe. Therefore, even if the same species is reported to grow in different continents, varieties or sub-varieties may exist for the same species. Thus, possible differences may exist for the phytochemical contents of mushrooms growing in different ecosystems.

Antioxidant Activity
Reactive oxygen (ROS) and nitrogen (RNS) species, which are extremely reactive with most organic compounds, are products of the normal cellular metabolism [31] and may have either harmful or beneficial effects on living systems [32]. Free radicals are atoms or molecular fragments containing one or more unpaired electrons in atomic or molecular orbitals [33]. They are formed naturally in the body, especially in mitochondria, as necessary intermediates in a variety of normal biochemical reactions, thus playing a positive role in many normal cellular processes. However, at

Antioxidant Activity
Reactive oxygen (ROS) and nitrogen (RNS) species, which are extremely reactive with most organic compounds, are products of the normal cellular metabolism [31] and may have either harmful or beneficial effects on living systems [32]. Free radicals are atoms or molecular fragments containing one or more unpaired electrons in atomic or molecular orbitals [33]. They are formed naturally in the body, especially in mitochondria, as necessary intermediates in a variety of normal biochemical reactions, thus playing a positive role in many normal cellular processes. However, at high concentrations, ROS and RNS are responsible for the oxidative damage to biological macromolecules, including DNA, proteins, and lipids in cell membranes. The damage to cells caused by free radicals, especially the damage to DNA, may contribute to the development of many diseases, including cancer [34,35]. Free-radical scavengers or free-radical quenchers are chemicals that react with free radicals and neutralize them, thus helping stop or limit damages caused by those reactive species. Most cells in our body produce antioxidant and repair systems which protect them against oxidative damage; however, these systems are often insufficient to prevent or repair the damage entirely [36]. Therefore, the introduction in the body of additional antioxidant agents from the diet is believed to be critical for maintaining cell homeostasis and thus a healthy organism [37]. Although synthetic antioxidants such as butylhydroxyanisole (BHA), butylhydroxytoluene (BHT), propyl gallate (PG) and tert-butylhydroquinone (TBHQ) have commonly been used as antioxidant additives in foods for years, their safety has long been questioned [38]. This finding has led to an increased interest in natural antioxidants. Antioxidant activities of extracts and isolated compounds from edible mushrooms have been determined by several research groups that used different tests in vitro to measure the reducing power ability, the total antioxidant activity, the 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity, the lipid peroxide inhibitory activity, the ferric reducing antioxidant power, the nitric oxide (NO) scavenging activity, and the ABTS radical scavenging, superoxide radical, and hydroxyl radical scavenging properties. Thus, a large number of results in the literature clearly indicates that several edible mushrooms have significant antioxidant properties due to their bioactive compounds, such as polyphenols, polysaccharides, vitamins, carotenoids and minerals [18,[39][40][41].

Antimicrobial Activity
Infectious diseases produced by organisms such as bacteria, viruses, fungi or parasites, are among the most serious causes of morbidity and mortality worldwide [42]. Nowadays many infections are often caused by multi-resistant microorganisms resulting in difficult to treat diseases; as a very well-known example, coronavirus Covid-19 is killing thousands of people worldwide. Consequently, healthcare costs are increasing substantially every year, becoming a serious problem in many countries [43][44][45]. This situation has led to an increasing search for new antimicrobial agents from different sources. Several researches have been conducted to explore the antimicrobial potential of natural or synthetic compounds [46,47]. Thus, natural sources, including mushrooms, have been investigated for finding novel antimicrobial compounds [48][49][50][51].
In food industry, contamination of food products by bacteria and fungi may be the result of exposure to sources of contamination during harvesting, processing and/or packaging process [52]. Therefore, chemical additives have been extensively used in food industries to increase the shelf life of food and to prevent the proliferation of microorganisms. In this regard, natural antimicrobials, including those isolated from mushrooms, are gaining an increasing importance as potential alternatives to synthetic preservatives, whose safety and impact on human health are still questionable [53][54][55]. Instead, the safety of many natural antimicrobials have been generally recognized in EU and USA [56].

Ampulloclitocybe clavipes (Pers.) Redhead, Lutzoni, Moncalvo and Vilgalys
Ampulloclitocybe clavipes (Pers.) Redhead, Lutzoni, Moncalvo & Vilgalys, formerly known as Clitocybe clavipes (Pers.) P. Kumm, belongs to the family Hygrophoraceae. It is a wild mushroom growing in China, Thailand, and Japan [4,58,65]. Clavilactones A-C (4-6, Figure 4) were isolated in an Italian laboratory from a culture of the fungus and exhibited antifungal and antibacterial activities [66]. The structures of clavilactones D and E (7), were initially inferred by 1-and 2-D NMR data [67]. However, the subsequent total synthesis of clavilactones A, B, and D led to a revision of the original structure of clavilactone D which was established to be as formula (8) in Figure 4 [68]. Clavilactone A, B, and D displayed potent inhibitory activity in kinase assays against the Ret/ptc1 and epidermal growth factor receptor (EGFR) tyrosine kinases [67,69]. Subsequently, Sun et al. [70] isolated from a fungal strain of A. clavipes, clavilactone F (12) together with three novel meroterpenoids, named clavipines A-C (9-11), which exhibit a benzoquinone ring fused to an azepine ring and a tenmembered carbocycle bearing an α,β-epoxy/unsaturated-γ-lactone. Compound 9 exhibited significant antiproliferative activity against HepG2 and A549 cells with IC50 values of 4.28 ± 0.26 and 7.49 ± 0.41 μM, respectively [70]. Subsequently, clavipols A-B (13-14) containing a 12-membered ether ring and clavilactones G-I (15)(16)(17) were isolated from the fruiting bodies of A. clavipes collected in China. Compound 16 exhibited moderate cytotoxic activity against Hela and SGC-7901 cancer cell lines, with IC50 values of 23.5 and 14.5 μM, respectively [71]. Five fatty acid derivatives, isolated from A. clavipes have been reported to have potent strong inhibitory activity against aldehyde dehydrogenase [65]. Ampulloclitocybe clavipes (Pers.) Redhead, Lutzoni, Moncalvo & Vilgalys, formerly known as Clitocybe clavipes (Pers.) P. Kumm, belongs to the family Hygrophoraceae. It is a wild mushroom growing in China, Thailand, and Japan [4,58,65]. Clavilactones A-C (4-6, Figure 4) were isolated in an Italian laboratory from a culture of the fungus and exhibited antifungal and antibacterial activities [66]. The structures of clavilactones D and E (7), were initially inferred by 1-and 2-D NMR data [67]. However, the subsequent total synthesis of clavilactones A, B, and D led to a revision of the original structure of clavilactone D which was established to be as formula (8) in Figure 4 [68]. Clavilactone A, B, and D displayed potent inhibitory activity in kinase assays against the Ret/ptc1 and epidermal growth factor receptor (EGFR) tyrosine kinases [67,69]. Subsequently, Sun et al. [70] isolated from a fungal strain of A. clavipes, clavilactone F (12) together with three novel meroterpenoids, named clavipines A-C (9-11), which exhibit a benzoquinone ring fused to an azepine ring and a ten-membered carbocycle bearing an α,β-epoxy/unsaturated-γ-lactone. Compound 9 exhibited significant antiproliferative activity against HepG2 and A549 cells with IC 50 values of 4.28 ± 0.26 and 7.49 ± 0.41 µM, respectively [70]. Subsequently, clavipols A-B (13-14) containing a 12-membered ether ring and clavilactones G-I (15)(16)(17) were isolated from the fruiting bodies of A. clavipes collected in China. Compound 16 exhibited moderate cytotoxic activity against Hela and SGC-7901 cancer cell lines, with IC 50 values of 23.5 and 14.5 µM, respectively [71]. Five fatty acid derivatives, isolated from A. clavipes have been reported to have potent strong inhibitory activity against aldehyde dehydrogenase [65].  Butyriboletus roseoflavus is an Asian species which was previously named Boletus speciosus Frost. It belongs to the family Boletaceae and to the genus Butyriboletus, which has recently been created to accommodate the former section Appendiculati within the large genus Boletus. This edible mushroom Butyriboletus roseoflavus is an Asian species which was previously named Boletus speciosus Frost. It belongs to the family Boletaceae and to the genus Butyriboletus, which has recently been created to accommodate the former section Appendiculati within the large genus Boletus. This edible mushroom grows abundantly in Southern China (Yunnan) and Thailand, and it is commonly sold in street markets [4,58]. A group of Chinese researchers reported the isolation, from the fruiting bodies of a novel heteropolysaccharide, which has a backbone of (1→4)-α-l-mannopyranose residues, which branched at O-6. The branches are mainly composed of one with →1)-α-d-galactopyranose residue [72]. In addition to a strong antioxidant activity [72], this polysaccharide with a unique structure activates the secretion of cytokines from immune cells and inhibits the growth of Hep-2 cells. The concentration of 400 µg/mL has the highest inhibitory rate [73,74]. A new water-soluble polysaccharide, having a backbone of 1,4-linked β-d-glucose, with branches mainly composed of two 1,6-linked α-d-galactose residues and bearing a 4-linked β-d-glucose unit at the end of the branches, has been reported to exhibit unique antitumor and immunoregulatory properties [75]. Sun et al. [76] reported that hemagglutinin isolated from B. speciosus, showed antiproliferative activity towards hepatoma Hep G2 cells and mouse lymphocytic leukemia cells (L1210) in vitro, with an IC 50 of 4.7 µM and 7.0 µM, respectively. It also exhibited HIV-1 reverse transcriptase inhibitory activity with an IC 50 of 7.1 µM.

Fistulina hepatica (Schaeff.)
Fistulina hepatica (Schaeff.), commonly known as beefsteak fungus, is a wild edible fungus belonging to the family Fistulinaceae [95]. It is distributed in temperate and subtropical hardwood forests of China, Thailand, Hungary, Portugal [4,58,115,116], and other European countries. Two novel triacetylene derivatives have been isolated from the fruiting bodies and named cinnatriacetins A (37) and B (38) [117]. Compounds 37 and 38 (see structures in Figure 7) showed antimicrobial activity against gram-positive bacteria, but no activity towards gram-negative bacteria [117]. Caffeic acid, p-coumaric acid, ellagic acid, hyperoside, quercetin, oxalic acid, aconitic acid, citric acid, malic acid, ascorbic acid and fumaric acid were also isolated from F. hepatica, and an aqueous extract showed a significant scavenger activity of DPPH • and superoxide radicals [118]. A sample of F. hepatica collected in Portugal contained tocopherols and showed strong antioxidant activity [115,119]. Ribeiro and his co-workers extensively studied the free amino acid and fatty acid composition of F. hepatica, comparing their contents with those of other wild edible mushrooms [120,121]. Wu et al. [122] studied the volatile compounds from the fruiting bodies and 11 compounds were identified as

Termitomyces albuminosus (Berk.) R. Heim
Termitomyces albuminosus (Berk.) R. Heim, belonging to the family Lyophyllaceae is a very wellknown wild edible mushroom, which is commonly distributed in Asia in China, Indonesia, Malaysia, and Singapore [4,228]. It cannot be cultivated, because a symbiotic relationship with termites is necessary [229,230]. The mushroom has been reported to contain water-soluble polysaccharides with a great variety of biological activities, including antioxidant, anti-inflammatory, hepatoprotective, hypolipidemic activities [230][231][232][233][234]. In addition, T. albuminosus has been reported to contain many other bioactive components, such as chitin-glucan complex, alkaline protease, saponins, melanin, lipids and ergosterol; some of which possess analgesic and anti-inflammatory activities [235][236][237][238]. Mau et al. studied a methanolic extract of T. albuminosus mycelia, reporting an interesting reducing power, scavenging activity and chelating effects of ferrous ions. [239]. Qi, et al. described the chemical structures of six novel cerebrosides, named termitomycesphins A-F (see structures 90-95 in Figure  18), together with known cerebroside 96, and reported their neuritogenic activities [240,241]. Other two new cerebrosides, named termitomycesphins G (97) and H (98) were later isolated from this mushroom by the same research group [242]. Four new selinane-type sesquiterpenoids, named teucdiol C-F (see structures 99-102 in Figure 18), together with the known compounds teucdiol B (103) and epi-guaidiol A (104) were isolated by from a fermentation broth of T. albuminosus [243]; epiguaidiol A (104) showed potent anti-acetylcholinesterase activity in a dose-dependent manner [243]. Termitomyces albuminosus (Berk.) R. Heim, belonging to the family Lyophyllaceae is a very well-known wild edible mushroom, which is commonly distributed in Asia in China, Indonesia, Malaysia, and Singapore [4,228]. It cannot be cultivated, because a symbiotic relationship with termites is necessary [229,230]. The mushroom has been reported to contain water-soluble polysaccharides with a great variety of biological activities, including antioxidant, anti-inflammatory, hepatoprotective, hypolipidemic activities [230][231][232][233][234]. In addition, T. albuminosus has been reported to contain many other bioactive components, such as chitin-glucan complex, alkaline protease, saponins, melanin, lipids and ergosterol; some of which possess analgesic and anti-inflammatory activities [235][236][237][238]. Mau et al. studied a methanolic extract of T. albuminosus mycelia, reporting an interesting reducing power, scavenging activity and chelating effects of ferrous ions. [239]. Qi, et al. described the chemical structures of six novel cerebrosides, named termitomycesphins A-F (see structures 90-95 in Figure 18), together with known cerebroside 96, and reported their neuritogenic activities [240,241]. Other two new cerebrosides, named termitomycesphins G (97) and H (98) were later isolated from this mushroom by the same research group [242]. Four new selinane-type sesquiterpenoids, named teucdiol C-F (see structures 99-102 in Figure 18), together with the known compounds teucdiol B (103) and epi-guaidiol A (104) were isolated by from a fermentation broth of T. albuminosus [243]; epi-guaidiol A (104) showed potent anti-acetylcholinesterase activity in a dose-dependent manner [243].

Termitomyces eurhizus (Berk.) R. Heim
Termitomyces eurhizus (Berk.) R. Heim, belonging to the family Lyophyllaceae, is a wild edible mushroom, which grows in association with termites in China, India, Myanmar, Malaysia, Nepal and Thailand [4,58,228,244]. Two water-soluble polysaccharides, whose structures were established to be (1→3)-D-Glcp and (1→6)-D-Glcp, and (1→6)-D-Glcp were isolated from a hot aqueous extract of fruiting bodies [244]. On the other hand, a water-insoluble (1→3)-β-D-glucan was isolated from a hot alkaline extract of the mushroom collected in India [245]. The biological activity of a water-soluble polysaccharide-rich fraction of T. eurrhizus was investigated by an Indian research group. The fraction revealed healing properties against indomethacin-induced stomach ulceration in mice [246]. Pharmacological studies on mushroom polysaccharides have highlighted other biological properties such as anticarcinogenic, antimicrobial, antioxidant and anti-inflammatory activities etc. [21]; therefore, T. eurhizus deserves further in-depth pharmacological investigations.

Termitomyces heimii Natarajan
Termitomyces heimii Natarajan, (family-Lyophyllaceae) is a wild edible mushroom which grows in nature in symbiosis with termites in China, Malaysia, Thailand, and India [4,58,228,247]. Manna et al. reported the structure of a water-soluble β-glucan from this mushroom, together with its antioxidant activity [248]. Termitomyces eurhizus (Berk.) R. Heim, belonging to the family Lyophyllaceae, is a wild edible mushroom, which grows in association with termites in China, India, Myanmar, Malaysia, Nepal and Thailand [4,58,228,244]. Two water-soluble polysaccharides, whose structures were established to be (1→3)-d-Glcp and (1→6)-d-Glcp, and (1→6)-d-Glcp were isolated from a hot aqueous extract of fruiting bodies [244]. On the other hand, a water-insoluble (1→3)-β-d-glucan was isolated from a hot alkaline extract of the mushroom collected in India [245]. The biological activity of a water-soluble polysaccharide-rich fraction of T. eurrhizus was investigated by an Indian research group. The fraction revealed healing properties against indomethacin-induced stomach ulceration in mice [246]. Pharmacological studies on mushroom polysaccharides have highlighted other biological properties such as anticarcinogenic, antimicrobial, antioxidant and anti-inflammatory activities etc. [21]; therefore, T. eurhizus deserves further in-depth pharmacological investigations.

Thelephora ganbajun M. Zang
Thelephora ganbajun M. Zang, belonging to the family Thelephoraceae, is one of the most favorite edible mushrooms. It widely grows in symbiosis with pine trees in China and the Greater Mekong region [260], where it is highly prized for its unique taste and flavor [260,261]. A novel ribonuclease, showing potent inhibitory activity toward HIV-1 reverse transcriptase, was isolated from dried fruiting bodies of the mushroom by Wang and Ng [262]. Two new polysaccharide fractions isolated from the fruiting bodies were characterized by Gong's group [263]. They exhibited strong inhibitory effects on HeLa cells and moderate inhibitory effect on α-amylase and α-glucosidase. Separation of an EtOAc-partitioned MeOH extract of T. ganbajun fruiting bodies collected in China afforded, in addition to 3-O-methylatromentin, five new poly(phenylacetyloxy)-substituted 1,1 :4 ,1"-terphenyl derivatives, called ganbajunins A-E (see structures 105-109 in Figure 19) [264]. Subsequently, ganbajunin F and G (see structures 110-111 in Figure 19), together with cycloleucomelone were isolated from fresh fruiting bodies by the same research group [265]. The extracts obtained under optimized conditions by an ultrasonic-assisted extraction procedure, possessed significant antiproliferative activities towards human lung and liver cancer cells [266]. Moreover, ganbajunins A-C (105-107) and 3-O-methylatromentin possessed potent lipid peroxidation inhibitory activity, SOD activity in rat liver homogenate, and DPPH radical scavenging activity [261,267,268].      Polysaccharide (immunoregulatory activity [73][74][75]). Hemagglutinin (HIV-1 reverse transcriptase inhibitory activity [76]) Cantharellus cibarius Fr.

Conclusions
Southeast Asia is one of the biodiversity hot-spots in the world and has an outstanding rate of species discovery. In fact, hundreds of new species are described annually. However, regional biological resources are currently threatened by climatic changes and human activity-related factors such as the high rate of mining in the tropics, the construction of a great number of hydropower dams, and an indiscriminate consumption of plants in traditional medicines [274][275][276]. Therefore, access to biodiversity resources of Southeast Asia must be done paying great attention to their conservation or renovation. In this context, mushrooms play important roles in different ecosystems; however, they are often obtained in artificial cultures, thus avoiding the collection in the wild.
Although the variety of higher mushroom (Basidiomycetes) growing in Southeast Asia is calculated to be very high, only few scientific mycological investigations have been conducted, and most species growing in countries such as Myanmar, Laos, and Cambodia, have not been identified so far.
We believe that this review clearly demonstrates that edible mushrooms are a rich source of various bioactive substances having antimicrobial, antioxidant, anti-inflammatory, anti-proliferative, cytotoxic, anti-HIV, anti-diabetic properties, among other ones. Therefore, edible mushrooms must be considered not only culinary delicacies but also functional foods and, in some cases, even therapeutic agents. Of course, mushroom edibility is a proof of their non-acute toxicity. Therefore, edible mushrooms containing bioactive compounds can have high potential as sources of medicinal remedies.