Bioactive small secondary metabolites from the mushrooms lentinula edodes and flammulina velutipes

ABSTRACT Mushrooms have been attracting attention as a source of bioactive compounds for the development of dietary supplements and medicines. Many researchers have reported pharmacological

effects of edible mushrooms, and have isolated and identified bioactive substances. _Lentinula edodes_ (shiitake) and _Flammulina velutipes_ (enokitake) are the cultivated edible mushrooms

that are popular throughout the world. In _L. edodes_, polyacetylenes and sulfur compounds have been shown to display antimicrobial activity. In _F. velutipes_, many types of bioactive

terpenes have been reported from mycelium culture filtrate or solid culture substrate. This article reviews the bioactive metabolites of low-molecular weight from _L. edodes_ and _F.

velutipes_. You have full access to this article via your institution. Download PDF SIMILAR CONTENT BEING VIEWED BY OTHERS MEDICINAL POTENTIAL OF MYCELIUM AND FRUITING BODIES OF AN ARBOREAL

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Article Open access 09 October 2023 INTRODUCTION Mushrooms are macroscopic fruiting bodies produced by ascomycete and basidiomycete fungi during their sexual reproduction cycles. Simplified

life cycle of a mushroom is described below: Spores are released from fruiting body and germinate to produce mycelia after landed. The mycelia grow in appropriate substrate such as fallen

leaves, dead branch, wood, etc., and may later form primordia. This primordium then grows to become a fruiting body, namely mushroom. Mushroom have been used as food and traditional medicine

for centuries. More recently, their nutritional and pharmacological properties have become recognized worldwide [1]. The world’s production of edible mushrooms increased from about 1

million tons in 1978 to more than 30 million tons in 2013 [2]. The value of the world mushroom industry in 2013 totaled about $63 billion, with cultivated edible mushrooms accounting for

~$34 billion (54%) [2]. Most of the cultivated edible mushrooms consist of five genera: _Lentinula_ (22%), _Pleurotus_ (19%), _Auricularia_ (17%), _Agaricu_s (15%), and _Flammulina_ (11%)

[2, 3]. Medicinal benefits of edible mushrooms are an active field of research and many published studies describe the screening of fungal extracts for pharmacological properties.

Antibacterial, antifungal, and antiviral activities have been identified in extracts, in addition to antitumor or immunostimulant properties [1, 4]. The identification of individual

compounds that cause a pharmacological response has mostly been focused on substances of high-molecular weight, such as polysaccharides, proteins, or peptides, with only a few reports on

substances of small-molecular weight [1, 4]. This article reviews bioactive secondary metabolites with low-molecular weight derived from the two major cultivated mushrooms, _Lentinula

edodes_ (Berk.) Pegler and _Flammulina velutipes_ (Curt.:Fr.) Sing. _L. edodes_ has a long history of cultivation and there are many reports on its functionality [5]_. F. velutipes_ is the

most cultivated mushrooms in Japan in recent several years [6], and there are relatively many reports on biological active low-molecular metabolites from this species compared with other

cultivated mushrooms. BIOACTIVE SECONDARY METABOLITES FROM _LENTINULA EDODES_ _L. edodes_, commonly known as the shiitake mushroom, has been cultivated in Asian countries for centuries [2,

3]. According to traditional Chinese medicine, _L. edodes_ mushroom was used as a tonic to counter the pain and fatigue associated with aging, and was also thought to have a beneficial

effect on heart health, lung diseases, and intestinal worms [7]. Recently, _L. edodes_ has gained popularity for both its nutritional profile and bioactive compounds. Shiitake mushroom

contains a large amount of dietary fiber and is a good source of vitamins such as provitamin D2, vitamin B1, B2, B12, and niacin [5, 8]. In addition, the extract or ingredients have been

reported to show antitumor effect and antimicrobial, hypolipidemic, antiviral, and immunomodulatory activity. Many scientific studies have been performed to identify the active molecules in

_L. edodes_ that impart the health benefits asserted in traditional medicine. Mostly, compounds of high-molecular weight, such as polysaccharides and peptides, have been reported as

bioactive agents [5, 9, 10]. However, small molecules with pharmacological properties have also been observed (Fig. 1, Table 1). POLYACETYLENES Various polyacetylenes have been observed in

culture filtrates of _L. edodes_. Komemushi et al. found an antimicrobial substance in the culture filtrate of _L. edodes_, which was identified as octa-2,3-diene-5,7-diyne-1-ol ((1):

lentinamycin) [11, 12]. This compound was first reported by Bew et al. from a culture filtrate of _Cortinellus berkeleyanus_ in 1966 [13]. _C. berkeleyanus_ is a synonym of _L. edodes_ [14,

15]. Compound 1 showed antibacterial activity against _Alcaligenes faecalis_, _Bacillus subtilis_, _Escherichia coli_, _Flavobacterium aquatile, Burkholderia_ (_Pseudomonas_) _cepacia,

Pseudomonas fluorescens, Salmonella_ Typhimurium, and _Staphylococcus aureus_ in an antibacterial activity test using the paper disk method (31.3 nmol/disk) [12]. The production of this

compound by _Lentinus_ sp. was also reported by Shiio et al. [16]. According to this study, compound 1 accumulated in the culture filtrate, medium, and wood log, but not in mycelium or

fruiting bodies. Compound 1 also showed antimicrobial activity against multiple filamentous fungi [_Aspergillus niger, Aspergillus oryzae, Mucor mucedo, Moniliella tomentosa_ (_Monilia

tomentosa_)_, Penicillium chrysogenum, Ustilago maydis, Pyricularia setariae, Colletotrichum horii_ (_Gloeosporium kaki_)_, Bipolaris oryzae_ (_Helminthosporium oryzae_)_, Trichophyton

rubrum, Chaetomium globosum_)], yeast [_Saccharomyces cerevisiae, Cyberlindnera jadinii_ (_Torula utilis_)_, Diutina rugosa_ (_Candida rugosa_)], and gram-positive bacteria (_S. aureus, B.

subtilis_) with a minimum inhibitory concentration (MIC) of 0.085–0.42 µM as determined by the agar diffusion assay [16]. During cultivation, _L. edodes_ is frequently affected by

_Trichoderma_ spp. which produce antifungal substances and mycolytic enzymes. However, _L. edodes_ is mostly able to resist _Trichoderma_ attacks. To clarify the detailed interaction between

_L. edodes_ and _Trichoderma_, Tokimoto et al. isolated antifungal substances from the culture filtrate of _L. edodes_ and identified compound 1 and three related polyacetylene molecules

(octa-3,5,7-triyne-1-ol (2), nona-3,5,7-triyne-1-ol (3), nona-7-ene-3,5-diyne-1-ol (4)) which inhibited spore germination of _Trichoderma polysporum_ [17, 18]. Another polyacetylene

(octa-7-ene-3,5-diyne-1-ol (5)) was found only in _L. edodes_ and _Trichoderma_ spp co-culture. The type of growth media in these co-cultures may affect the production of bioactive compounds

with higher concentrations observed in glucose-based medium than in xylose-based medium. _Trichoderma_ attack caused an increase in the total production of antifungal substances up to 4

days after inoculation, although compound 1 in both media and compound 2 in xylose medium decreased after 2 days. Notably, these findings coincide with the fact that the degree of invasion

of _L. edodes_ mycelium by _Trichoderma_ was less in the glucose medium compared with xylose medium [17, 18]. Compound 3 has also been isolated from the culture fluids of _Marasmiellus

peronatus_ (_Collybia peronata_), a basidiomycete, while compounds 2, 4, and 5 seems to be specific to _L. edodes_ [19]. 4-Hydroxyundeca-5,6-diene-8,10-diynoic acid ((6): cortinellin or

nemotinic acid) was isolated in 1962 from a culture filtrate of _Cortinellus shiitake_, another synonym of _L. edodes_ [20, 21], which was previously reported in cultures of three

Basidiomycota, _Oxyporus corticola_ (_Poria corticola)_, _Perenniporia tenuis_ (_Poria tenuis_), and unidentified Basidiomycete B-841 in 1950 [21, 22]. According to Herrmann, cortinellin

showed antimicrobial activity against gram-positive bacteria, gram-negative bacteria, mycobacteria, and fungi (MIC 1.6 µM) [20, 21]. Kavanagh et al. investigated antibacterial and antifungal

activity of this compound [22]. The MIC values of this compound against _B. subtilis_, _S. aureus_, _Mycobacterium smegm_a, _S. cerevisiae_, _P. chrysogenum_ (_Penicillium notatum_), and

_Trichophyton mentagrophytes_ were 1.3, 5.3, 21, 84, 168, and 336 µM, respectively [22]. Compound 6 has been also reported in other several basidiomycetes such as _Perenniporia subacida_

(_Poria subacida_, _Poria colorea_) [23], _Hapalopilus mutans_ (_Poria mutans_) [23], and _Corticium roseum_ (_Aleurodiscus roseus_) [24]. SULFUROUS COMPOUNDS Lenthionine

(1,2,3,5,6-pentathiepane) (7), a characteristic aromatic component of dried _L. edodes_, was isolated from dried fruiting bodies by Morita et al. [25] together with 1,2,4,6-tetrathiepane (8)

and 1,2,3,4,5,6-hexathiepane (9). Antimicrobial activities of compounds 7 and 8 were investigated. Compound 7 showed fairly strong antibiotic effects against a number of micro-organisms

including gram-positive and gram-negative bacteria (_B. subtilis, S. aureus, E. coli, Proteus vulgaris_; MIC of 0.27–1.33 mM), fungi [_Cryptococcus neoformans, Colletotrichum

gloeosporioides_ (_Glomerella cingulata_)_, Pyricularia oryzae, T. rubrum, T. mentagrophytes_; MIC of 16.6–66.4 µM], and yeast (C_andida albicans, S. cerevisiae_; MIC of 33.2 µM) [25]. In

addition, in vitro testing showed that compound 7 inhibited platelet aggregation. Moreover, compound 7 showed inhibitory effect on liver damage caused by carbon tetrachloride (CCl4)-induced

acute liver injury in mice [26, 27]. Overall, the antimicrobial activity of compound 8 was weaker than that of compound 7, with the MIC amounting to 0.73–1.47 mM for many tested

micro-organisms [25]. Chen et al. also identified sulfur compounds (7, 8, 10–16) from chloroform extracts of crushed raw _L. edodes_; however, the biological activity of these compounds has

not been investigated [28]. These sulfurous compounds (7–16) have not been reported from any fungi other than _L. edodes_ as far as I searched. STEROLS Mushrooms including _L. edodes_

contain ergosterol (17), a precursor of vitamin D2, as a cell membrane component. Several other sterols and terpenes have been reported from _L. edodes_. Chen et al. isolated and identified

six sterols including ergosterol (17–22) from acetone extracts of dried fruiting bodies [29]. Compounds 18, 19, and 21 exhibited moderate antimicrobial activity against _E. coli_, _B.

subtilis_, _Bacillus cereus_, _S. aureus_, and _Erwinia carotovora_ (MIC of 46.6–94.0 µM for compound 18 and of 36.0–72.6 µM for compounds 19 and 21). Other compounds, 17, 20, and 22, were

less active against all tested bacteria (MIC above 0.59 mM). Compounds 19 and 21 showed mostly same level of antibacterial activity, and compounds 17 and 20 were much less active than them.

It is suggested that the hydroxyl group at the C-5 position is involved in the antibacterial activity. Further, the hydroxyl group at the C-6 position or C-8 position of compound 19 or 21

might also be important for exhibiting antibacterial activity. Compounds 18–20 and 22 have been isolated from some fungi as follows; compound 18 from _Ganoderma casuarinicola_, _Alternaria

alternata_, etc [30, 31], compound 19 from _Tricholoma matsutake_, _Pleurotus ostreatus_, etc [32,33,34,35], compound 20 from _Pholiota nameko_, _Amanita pantherina_, etc [34, 35], and

compound 22 _Armillaria mellea_ [36]. OTHER COMPOUNDS Lovastatin (23) has been identified in many kinds of mushrooms including _L. edodes_ [37, 38]. Compound 23 is a specific inhibitor of

hydroxymethylglutaryl-CoA (HMG-CoA) reductase, which catalyzes cholesterol synthesis in the liver, thereby lowering blood cholesterol. Several studies have also reported the presence of

lovastatin in the fruiting body of _L. edodes_ at concentrations ranging from 0.27 to 32.73 mg per 100 g of dry weight [37,38,39,40]. Eritadenine (24) is a nucleic acid derivative and was

first isolated as a cholesterol-lowering component from the fruiting body of _L. edodes_. It was previously called lentinacin or lentysine in multiple independent reports [41,42,43]. To

date, this compound has been shown to inhibit angiotensin converting enzyme and _S_-adenosyl-l-homocysteine hydrolase [44,45,46]. Fruiting bodies of _L. edodes_ contains compound 24 at the

concentrations of 40–70 mg per 100 g of dry weight [43]. On the other hand, this compound is rarely isolated from edible mushrooms other than _L. edodes_, and even if it contains this

compound, the content is considerably lower than that of _L. edodes_ [43]. BIOACTIVE SECONDARY METABOLITES FROM _FLAMMULINA VELUTIPES_ _Flammulina velutipes_ (Curt.:Fr.) Sing, also called

enokitake or golden needle mushroom, is one of the most popular edible mushrooms worldwide due to its high nutritional value and flavorful taste. The global production of _F. velutipes_ was

2.7 billion kgs in 2013, ranking 5th in edible mushrooms production. _F. velutipes_ is widely cultivated and consumed in Asian countries, especially in China and Japan [2, 3]. _F. velutipes_

exhibits multiple pharmacological activities, including antitumor, cholesterol-lowering, antioxidant, and immunomodulatory properties. Various high-molecular-weight compounds like

polysaccharides, proteins, and glycoproteins have been isolated from _F. velutipes_ and are classified as important bioactive molecules with beneficial health effects [47]. In addition,

various low molecular bioactive substances have also been isolated from this species, with terpenes being the predominant small molecules (Fig. 2, Table 2). CUPARANE TYPE SESQUITERPENES,

SECO-CUPARANE TYPE SESQUITERPENES Enokipodins are highly oxygenated cuparene-type sesquiterpenes. Enokipodins A–D (25–28) were isolated from a culture filtrate of _F. velutipes_ by Ishikawa

et al. [48, 49]. Enokipodins B (26) and D (28) are oxidized molecules of enokipodins A (25) and C (27), respectively. Since a portion of the enokipodins A and C are converted to enokipodins

B and D by automatic oxidation, even purified enokipodins A or C are contaminated with small amounts of enokipodins B and D. In bioassays using liquid medium, compounds 25–28 showed

antibacterial activity against _B. subtilis_ (IC50 of 13–45 µM), with compound 26 showing the strongest antibacterial activity [50]. Compound 26 also suppressed spore germination of some

plant pathogenic fungi [50]. Compounds 26 and 28 have a stronger antiproliferative activity against several tumor and transformed cell lines (HeLa, HL60, tsFT210, tsNRK; IC50 of 4.9–18.7 µM)

than compounds 25 and 27. Furthermore, these compounds were also tested for exhibiting growth inhibitory activity against the malarial parasite _Plasmodium falciparum_ with compounds 25 and

27 (IC50 of 9.7 µM and 4.2 µM, respectively) showing stronger inhibition than compounds 26 and 28 (IC50 of 21.9 µM and more than 381 µM, respectively) [50]. The concentration of compounds

25–27 in the culture filtrate increases when _F. velutipes_ is co-cultured with _Trichoderma harzianum_, a competitor fungus [50]. In 2012, Wang et al. isolated additional enokipodins E–J

(29–34) from rice substrate fermented by _F. velutipes_, besides compounds 26 and 28 [51]. An enokipodin related compound, 2,5-cuparadinene-1,4-dione (35), was also isolated. Compounds 26,

28, 33–35 had weak antibacterial activity against _B. subtilis_ (IC50 of 140–170 µM), and compounds 30, 31, and 33 showed antifungal activity against _Aspergillus fumigatus_ (IC50 of 229–235

 µM). Compounds 26, 28, 34, and 35 were cytotoxic to a panel of human cancer cell lines (HepG2: IC50 of 23–35 µM, MCF-7: IC50 of 33–59 µM, SGC7901: IC50 of 40–91 µM, A549: IC50 of 25–48 µM).

In addition, these compounds 26, 28, 34, and 35 also showed radical scavenging activities in a diphenylpicryl hydrazine (DPPH) scavenging assay (IC50 of 78–154 µM). Notably, compounds 29–33

did not show cytotoxicity and antioxidant activity even at high concentrations (200 µM), indicating that the benzoquinone moiety is important for these activities. Flamvelutpenoids A–D

(36–39) were also isolated from rice substrates fermented by _F. velutipes_ [52]. The antibacterial activity of these compounds against _B. subtilis_, _E. coli_, and methicillin-resistant

_Staphylococcus aureus_ (MRSA) was weak, and the reported MIC90 was over 100 µM in all tests [52]. Tao et al. isolated flamvelutpenoids E (40) and F (41) having a cuparane skeleton and

flammufuranones A (42) and B (43), which are seco-cuparane sesquiterpene obtained from rice fermented by _F. velutipes_ [53]. The effects on HMG-CoA reductase, dipeptidyl peptidase-4

(DPP-4), and aldose reductase were investigated to evaluate the hypoglycemic and hypolipidemic effect of these compounds, but none of them showed any inhibitory activity [53]. Enokipodins

A–D have been confirmed to be produced by _Flammulina rossica_ [50], but other compounds described above have not been reported in any fungi other than _F. velutipes_. SPIROAXANE, CADINENE,

NOR-EUDESMANE TYPE SESQUITERPENES Flammuspirones A–J (44–53) with a spiroaxane skeletons, 1,2,6,10-tetrahydroxy-3,9-epoxy-14-nor-5(15)-eudesmane (54), 7,13,14-trihydroxy -4-cadinen-15-oic

acid methyl ester (55) were also isolated by Tao et al. from rice fermented by _F. velutipes_ [53]. Compounds 44, 46, 54, and 55 showed inhibitory activity on HMG-CoA reductase with IC50

values of 114.7, 77.6, 87.1, and 55.5 μM, respectively. Moreover, compounds 46–48, 51, 54, and 55 showed DPP-4 inhibitory activity, with IC50 values of 75.9, 83.7, 70.9, 79.7, 74.8, and 80.5

 μM, respectively [53]. STERPURANE TYPE SESQUITERPENES Sterpurane sesquiterpenes, sterpurols A (56) and B (57), and sterpuric acid (58) were isolated by Wang et al. from fermented rice

substrates by _F. velutipes_ [51]. Even at a concentration of 200 µM, these three compounds did not show cytotoxicity against human cancer cell lines HepG2, MCF-7, SGC7901, and A549. Also,

the data suggest that these compounds did not display antioxidative activity. Furthermore, none of these compounds showed antimicrobial activity against _E. coli_, _C. albicans_, MRSA,

_Pseudomonas aeruginosa_, and _A. fumigatus_ [51]. On the other hand, compound 58 inhibited the growth of Jurkat cells with IC50 of 160 µM [54] and showed weak antibacterial activity against

_B. subtilis_ and MRSA with IC50 value larger than 250 µM [55]. Sterpuric acid (58) have been reported as a metabolite from several basidiomycetes such as _Chondrostereum purpureum_

(_Stereum purpureum_) [56] and _Mycoacia uda_ (_Phlebia uda_) [54]. CUCUMANE-TYPE SESQUITERPENE Flamulinol A (59), a cucumane-type sesquiterpene, was isolated by Wang et al. from rice

fermented by _F. velutipes_. The antibacterial activity against _B. subtilis_, and MRSA was weak, and observed IC50 values were higher than 250 µM [55]. ISOLACTARANE SESQUITERPENS,

ISOLACTARANE-RELATED NORSESQUITERPENES Flammulinolide A (60), isolactarane type sesquiterpene, and flammulinolides B–G (61–66), isolactarane-related norsesquiterpenes, were isolated by Wang

et al. from rice substrates fermented by _F. velutipes_ [55]. Compound 60 showed strong cytotoxicity against KB cells, a subline of keratin-forming HeLa cells (IC50 of 3.9 µM) and moderate

cytotoxicity against HepG2 cells (IC50 of 34.7 µM). Compounds 61 and 65 showed a strong cytotoxicity against KB cells (IC50 of 3.6 µM, 4.7 µM, respectively). Compound 62 exhibited a strong

cytotoxicity against HeLa cells (IC50 of 3.0 µM) but only displayed moderate cytotoxicity against KB cells (IC50 of 12.4 µM). Compounds 64 and 66 showed moderate cytotoxicity against HeLa

cells (IC50 of 59.5 µM and 25.8 µM, respectively). Cytotoxicity of compound 63 against all three cell lines was weak and IC50 value was larger than 250 µM. Compound 61 showed cytotoxicity

with IC50 values of 79.0 µM against HepG2 cell line, while IC50 values of compounds 62 were over 200 µM. This indicate that hydroxyl group on the C-1 of cyclopentane ring is preferable to

show cytotoxicity against HepG2 cells than carbonyl group. On the other hand, since compound 63 has a hydroxyl group on C-1 but has low cytotoxicity to HepG2 cells, the hydroxyl group on C-2

also seems to be important for exhibiting activity. On the contrary, since the compound 62 showed strong cytotoxicity against HeLa cells, while the activity of the compound 61 was weak, it

appears that carbonyl group on C-1 is important for its cytotoxicity against HeLa cells. The antibacterial activity of these compounds (60–66) against _B. subtilis_ and MRSA was weak, with

IC50 values higher than 250 µM [55]. NORSESQUITERPE ALKALOID In rice substrates fermented by _F. velutipes_, norsesquiterpe alkaloid,

(_R_)-8-hydroxy-4,7,7-trimethyl-7,8-dihydrocyclopenta[_e_] isoindole-1,3 (2_H_, 6_H_) -dione (67) was identified. This bioactive agent displayed cytotoxicity to KB cells at an IC50 of 16.6 

µM [57]. Total synthesis of this compound and its enantiomer has been reported by Kashinath et al. [58]. MONOTERPENES Though there are few reports on the identification of characteristic

low-molecular weight metabolites from the fruiting body of _F. velutipes_, two monoterpentriols, (1_R_,2_R_,4_R_,8_S_)-(−)-_p_-menthane-2,8,9-triol (68) and its 8-epimer (69), have been

isolated from the stem [59]. These compounds showed growth-promoting activity on the excised stipe with pileus of fruiting bodies at concentrations below 53 µM. However, the growth of these

segments was inhibited at concentration above 530 µM [59]. CONCLUSIONS AND FUTURE PROSPECTS Bioactive metabolites of mushrooms have been attracting attention for their development of dietary

supplements and medicines, since pharmacological effects of multiple mushroom species have been reported [1]. The fruiting bodies of mushrooms are mainly comprised of dietary fibers,

proteins, sugars, amino acids, and fatty acids, but not water [60]. The fruiting bodies of mushrooms also contain various low-molecular-weight secondary metabolites, albeit in very low

amounts, making the latter difficult to isolate. The bioactive secondary metabolites of mushrooms appear to be more readily isolated from the culture filtrate or culture substrate than from

fruiting bodies. The biological activity upon chemical modification and production by organic synthesis are easier to study for low-molecular-weight compounds than for high-molecular-weight

compounds. Therefore, this review focused on the bioactive small molecular secondary metabolites produced by two of the most popular cultivated mushrooms, _L. edodes_ and _F. velutipes_. In

_L. edodes_, sulfur compounds, lovastatin, eritadenine, and several sterols are bioactive secondary metabolites derived from fruiting bodies. Polyacetylene compounds were derived from

culture substrates or culture filtrates. Many unique bioactive terpenes have been reported in _F. velutipes_, with the majority of them derived from culture filtrates and culture substrates

[48,49,50,51,52,53,54,55,56,57,58,59]. Most commercial mushrooms are cultivated on mushroom beds in which the mycelium of mushroom is pure cultured on solid substrate such as saw dust,

leaving behind plentiful spent mushroom substrate after harvesting. To efficiently repurpose the spent substrate as an energy source, livestock feed, compost, or further mushroom cultivation

substrate, various research and tests have been conducted [61]. Alternatively, mixing the substrate with soil without composting is straightforward; however, there is concern about the

effect it can have on plants grown in the soil mixture. Therefore, the effects of compounds isolated from culture substrates or culture filtrate of mushrooms on plants, plant diseases, and

agricultural pests are of great interest. Lastly, mushrooms have been gaining attention for their potential nutraceutical effects, leading to the development of such drugs. However,

investigation of not only their health functionalities but also various other biological activities might lead to more effective utilization of the spent substrate; there is a tantalizing

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Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Fukushima-Sakuno, E. Bioactive small secondary metabolites from the mushrooms _Lentinula edodes_ and _Flammulina velutipes_. _J

Antibiot_ 73, 687–696 (2020). https://doi.org/10.1038/s41429-020-0354-x Download citation * Received: 22 April 2020 * Revised: 17 June 2020 * Accepted: 30 June 2020 * Published: 30 July 2020

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