Medicinal Properties and Bioactive Compounds from Wild Mushrooms Native to North America

Mushrooms, the fruiting bodies of fungi, are known for a long time in different cultures around the world to possess medicinal properties and are used to treat various human diseases. Mushrooms that are parts of traditional medicine in Asia had been extensively studied and this has led to identification of their bioactive ingredients. North America, while home to one of the world’s largest and diverse ecological systems, has not subjected its natural resources especially its diverse array of mushroom species for bioprospecting purposes: Are mushrooms native to North America a good source for drug discovery? In this review, we compile all the published studies up to September 2020 on the bioprospecting of North American mushrooms. Out of the 79 species that have been investigated for medicinal properties, 48 species (60%) have bioactivities that have not been previously reported. For a mere 16 selected species, 17 new bioactive compounds (10 small molecules, six polysaccharides and one protein) have already been isolated. The results from our literature search suggest that mushrooms native to North America are indeed a good source for drug discovery.


Introduction
Natural products have been used as medicines for centuries, however, only in the last century have researchers begun to diligently characterize their biological and chemical properties. Mushrooms are natural reservoirs of potent pharmaceuticals and are now the new interface for drug discovery. Mushrooms belong to ascomycetes and basidiomycetes phyla of the kingdom Fungi, and have been defined as "epigeous and hypogeous fruiting bodies of macroscopic fungi" [1]. According to the recent estimates, fungi constitute 2.2-3.8 million species worldwide [2]. These fungal estimates include all the different types of fungi including mushrooms, the fruiting bodies of fungi. Another study provides estimates on the total number of mushrooms to be 140,000-160,000 where only 10% have been explored [3,4].
Mushrooms have a long history of medicinal use in various cultures across the globe. It has earned medicinal status long ago in China and other parts of Asia, including Japan and Korea. The scope of medicinal mushrooms has expanded to other countries such as USA and the eastern European countries such as Russia [5][6][7][8][9]. Initially, mushrooms became popular as a folklore remedy. For example, in the sixteenth century in Russia and Europe, Inonotus obliquus became famous as a folklore medicine for the treatment of cancer [10]. Later on, scientists became interested in finding the evidence behind the diverse bioactive potential that is contained in mushrooms. This has led to the exploration of untapped mushroom resources for their potential medicinal benefit and the bioactive compounds that impart these properties. There are many excellent recent reviews written on the topic of medicinal mushrooms and the bioactive compounds that derived from them [11][12][13][14]. Unlike in Asia and parts of Eastern Europe, there are relatively less studies on the exploration of mushrooms native to North America for medicinal properties. Anti-inflammatory (a), antioxidant (b), antituberculosis (c) [25] 95% ethanol (a,c), 70% ethanol (b) 50 µg/mL (a) (++), IC 50

Medicinal Properties of Mushrooms Native to North America
Mushrooms are known to possess medicinal properties that provide benefits against a large number of diseases. Some of the important medicinal benefits reported include antimicrobial, antioxidant, anticancer, immune system enhancer, antiviral, anti-hyperlipidemia, radical scavenger, anti-parasitic and anti-inflammatory [42]. Amongst these, the most common medicinal properties reported from mushrooms native to North America are anticancer, immuno-stimulatory, anti-inflammatory, antimicrobial and antioxidant as shown in Table 1 41].

Anti-Bacterial and Anti-Viral Activities
Antimicrobial resistance is a major healthcare problem worldwide. A recent landmark report indicated that bacterial infections resistant to treatment are likely to grow from 26% in 2018 to 40% By 2050, and such increases are expected to cost thousands of lives, billions of dollars in hospital expenses and gross domestic product and have a negative social impact on people worldwide [43]. Therefore, it is recommended that efforts to discover new antimicrobial drugs to combat specific antibiotic-resistant pathogens should be strengthened and should include innovative strategies [43][44][45]. Many studies around the world had focused on isolating antibacterial compounds and extracts from basidiomycete mushrooms [46]. For instance, the small molecules enokipodins A-D, isolated from Flammulina velutipes had activity against Bacillus subtilis and Staphylococcus aureus [47], while others including emodin, physcion, and erythroglaucin, austrocortilutein, torosachrysone, and 6-methylxanthopurpurin-3-O-methyl ether isolated from Cortinarius species were effective against S. aureus [48]. Small molecules from Lentinus edodes (Shiitake mushroom) have also shown antibacterial effects against S. aureus, Streptococcus faecalis and Bacillus cereus [49].
Not surprisingly, researchers have also focused on mushrooms native to North America, as avenues to discover novel antimicrobial metabolites. Grifolin, neogrifolin and confluentin isolated from Albatrellus flettii collected in California were found to have strong activity against gram positive bacteria B. cereus and Enterococcus faecalis [21]. Another lanostane-type tripterpene isolated from Jahnoporus hirtus also inhibited the growth of B. cereus and E. faecalis [21]. Other anti-bacterial small molecules isolated include lanostane triterpenoids and ergostane steroid from Fomitopsis pinicola collected in Oregon [30], new subclass of bisnaphthalenes from Urnula craterium [41] collected in La Crosse (WI, USA) and 2-aminoquinoline from Leucopaxillus albissimus [36]. 2-aminoquinoline showed modest antibacterial activity against multidrug resistant clinical isolates with an MIC of 8-64 µg/mL and weak antibacterial activity against A. baumannii, using tetracycline as a reference antibacterial agent [36]. The antibacterial activity of 2-aminoquinoline was not very convincing due to its high MIC, therefore, it was not suggested for further drug screening as an antibacterial agent in its current form. The antibacterial activity of small molecules from F. pinicola was compared with tetracycline [30]. Vancomycin and tetracycline were used as positive controls to compare the antimicrobial activity of urnucratin A-C isolated from Urnula craterium [41]. Amongst these compounds, urnucratin A (26) was the most promising with MIC values of 2, 1, and 0.5 µg/mL against S. aureus, E. faecium and S. pyogenes respectively [41]. It had an IC50 value greater than 100 µM against J774A.1 murine monocyte cells, indicating that it lacks non-specific toxicity [41]. Supernatants from culture of Lenzites betulina and Haploporus odorus [33] as well as extracts from Pleurotus ostreatus and P. levis [39] also have antimicrobial activity. However, the responsible antimicrobial compounds have not been isolated from these mushrooms. In another study using extracts from 75 mushrooms collected in Oxford (OH, USA), it was found that a total of 25 samples had antibacterial activity against at least one of the bacterial strains assessed [26]. From this study, extracts from Ganoderma lucidum and Laetiporus sulphureus were found to have the strongest antibacterial activity [26]. MIC was determined for these mushroom extracts using kanamycin as a reference antibacterial agent which has an MIC of 0.01 mg/mL against S. epidermidis [26]. Four small molecules Ramariolides A-D (22)(23)(24)(25) were isolated from methanol extracts of Ramaria cystidiophora [50]. The major metabolite ramariolide A (21) was found to be active against Mycobacterium smegmatis and Mycobacterium tuberculosis with MIC of 8 µg/mL and 64−128 µg/mL respectively. This compound appears to be promising because isoniazid, a first-line antituberculosis bactericidal antibiotic also has an MIC of 8 µg/mL against M. smegmatis [50]. The authors did not report any antibacterial from ramariolides B-D (22)(23)(24), and therefore it is currently unknown whether these compounds have any biological activity.
Extracts of polypore mushrooms native to North America have been shown to have antiviral activities against viruses that attack honeybees [51]. Mycelium extracts from Fomes fomentarius collected in Ithaca (NY, USA) and Ganoderma resinaceum culture from Ontario (Canada) were found to reduce the levels of honeybee deformed wing virus and Lake Sinai virus in vivo in both laboratory and field studies [51].

Anti-Proliferative Activity In-Vitro
Mushrooms native to North America have been explored for anti-proliferative activity against cancer cell lines. For instance, out of 29 species of mushrooms examined from northcentral BC [24] and Haida Gwaii, BC [23], 27 species exhibited anti-proliferative activity. Sixteen out of the 27 species (59%) had their anti-proliferative activity reported for the first time [23,24]. These species include Amanita augusta, Cantharellus cibarius, Chroogomphus tomentosus, Guepinia helvelloides, Gyromitra esculenta, Hydnellum sp., Inocybe sp., Laetiporus conifericola, Leucocybe connata, Phellodon atratus, Pleurotus ostreatus, Ramaria cystidiophora, Russula paludosa, Trichaptum abietinum, Tricholomopsis rutilans, and Tyromyces chioneus. It would be of interest to further investigate what the anti-proliferative compounds from these mushrooms are. Another study was conducted on 38 species of mushrooms collected from the greater Seattle area (WA, USA). Out of 38 species, aqueous extracts from three species were identified as anti-proliferative in human estrogen receptor negative (MDA-MB-231, BT-20) and estrogen receptor positive (MCF-7) breast cancer cells. These included Coprinus comatus, Coprinellus sp., and Flammulina velutipes [27]. The anti-proliferative effects of these 3 species were compared with 5-fluorouracil, a known anticancer drug [27]. Elsewhere, the ethanol and water extracts of Pleurotus tuber-regium from Washington State displayed anti-proliferative effects in HCT-116 colon and HeLa cervical cancer cell lines [52]. Although in most parts the identity of anti-proliferative compounds from the mushroom species described above remains unknown, there were detailed structural elucidation and mechanistic studies of both bioactive small molecules and polysaccharides isolated from selected species. For example, a growth-inhibitory polysaccharide GIPinv that caused growth inhibition in several cancer cell lines and induced apoptosis in HeLa cancer cells was isolated from Paxillus involutus [37]. It has an IC50 of 0.05 mg/mL and 0.04 mg/mL against HeLa cells and MCF-7 cells respectively. In the future, GIPinv should be evaluated for its anticancer activity in animal models. Small molecules grifolin, neogrifolin and confluentin were found to be the major growth-inhibitory compounds in the ethanol extracts of Albatrellus flettii [22]. It was also discovered that confluentin can inhibit the RNA-binding function of the oncogenic protein insulin-like growth factor 2 mRNA-binding protein 1 (IMP1) [22]. This is potentially a novel inhibitory pathway leading to the suppression of KRAS expression in human colon cancer cells. To determine the significance of this finding, it will be important to evaluate the inhibitory function of confluentin using in vivo models. Elsewhere, an exopolysaccharide isolated from P. tuber-regium inhibited the growth of chronic myelogenous leukemia K562 cells [40]. Another example is orenalline, a small molecule isolated from Cortinarius armillatus that inhibited renal carcinoma in a dose-dependent manner [20].

Anti-Proliferative/Anti-Cancer Activity in Animal Models
To the best of our knowledge, to date there has been no animal studies for anticancer activity of any compounds isolated from North American mushroom. There are numerous animal studies reporting the anti-cancer activity of extracts, small molecules and large molecules isolated from mushrooms collected in different parts of the world. Several excellent reviews have already dealt with this topic [11][12][13][14]. Here, we only highlight some of the key findings from popular medicinal mushrooms. A mushroom mixture named as Micotherapy U-care consisted of G. lucidum, Lentinula edodes, Agaricus blazei, Ophiocordyceps sinensis, and Grifola frondosa was orally administered in mice implanted with triple-negative breast cancer. The results showed reduced pulmonary metastases density as well as decreased expression of interleukin-6, NOS, and COX-2 [53]. In another study, the anticancer effect of β-glucans along with radiation therapy was evaluated against lung cancer. The β-glucan isolated from G. lucidum caused inhibition of primary lung cancer metastasis in C57BL/6 mice [54]. In another study, the antitumor effects of a βglucan isolated from G. frondosa combined with cisplatin were evaluated in a BALB/cA mouse model. The β-glucan was able to enhance the antitumor and antimetastatic effects of cisplatin as well as reduced the side effects; myelosuppression and nephrotoxicity, associated with cisplatin treatment [55]. The ethanol extracts from F. pinicola has been shown to significantly inhibit xenograft sarcoma-derived tumor growth in mice and prolong their survival time when administered as a food supplement [56].
Hericium erinaceus is one of the medicinal mushrooms that has been most extensively studied in animal models [11]. For example, its ethanol extract was able to inhibit gastric, liver and colon cancer in mouse xenograft tumors [57] while its water extracts has been shown to possess anti-metastatic activity by inhibiting the migration of colon carcinoma cells to lungs [58]. Two compounds isolated from H. erinaceus were demonstrated to possess anti-cancer activity in pre-clinical mouse models. The cyanthine diterpenoid Erinacine A has shown inhibition of DLD-1 human colorectal adenocarcinoma xenograft tumor growth in nude mice [59]. A protein isolated from H. erinaceus called HEP3 also showed ability to inhibit growth of colon cancer cells in mouse xenograft tumors [60]. Animal studies have also been conducted with I. obliquus, Trametes versicolor and Phellinus linteus. A proteoglycan isolated from P. linteus was able to potentiate immune system as well as inhibit cancer in HT-29-bearing BALB/c-nu/nu mice by inhibiting Reg IV/EGFR/Akt signaling pathway [61]. As for I. obliquus, its extracts were capable of inhibiting tumor growth in human melanoma B16-F10 cells-and sarcoma-180 cells-derived xenografts in mice [62,63]. Specific compounds isolated from I. obliquus have shown anti-cancer activity in vivo. Two unique lanostane-type, inonotodiol and inonotsuoxides, have demonstrated anti-carcinogenic effects on mouse skin and human leukemia-derived mouse xenograft tumors [64,65]. For T. versicolor, the β-glucan-based polysaccharopeptide fraction PSP exhibited growth-inhibitory effect on a number of cancer cells in mouse xenograft tumors [11]. In addition, a new glucan isolated from the water extract of T. versicolor was able to inhibit the xenograft sarcoma growth in mice [66].

Anti-Proliferative/Anti-Cancer Activity in Humans
To date, there has been no clinical studies evaluating the anti-cancer potential of mushrooms native to North America. However, there are 600 published papers and reports of clinical therapeutic effects of medicinal mushrooms collected from different parts of the world. Here, we highlight some of the key clinical studies. Perhaps, the most clinically-relevant anti-cancer compounds from mushroom are PSP and polysaccharide-K (PSK). These immunotherapeutic anti-cancer compounds had been extensively studied clinically and have been in use in Japan since 1977 and in China since 1987 [11]. Another mushroom that has been studied extensively on humans is G. lucidum, commonly known as Reishi or Ling Zhi in Asia [42]. One such clinical study was conducted on Ganopoly, a polysaccharide isolated from G. lucidum. A randomized controlled double blinded clinical trial on 68 patients with advanced lung cancer was conducted to evaluate the efficacy and safety of Ganopoly. Patients were evaluated after 12 weeks of administration of Ganopoly. Ganopoly was found to enhance the host immune function by increasing the activity of macrophages, T lymphocytes and natural killer cells, suggesting its role as an adjunct [67]. Another study was conducted on 47 patients with advanced colorectal cancer in which 5.4 g/day doses of Ganopoly was administered to patients for 12 weeks. Ganopoly was found to stimulate the immune response in the cancer patients [68].
A phase I/II dose escalation trial has been carried out using polysaccharide extract from G. frondosa, also known as Maitake mushroom. The efficacy of the polysaccharide extract was evaluated at multiple doses ranging from 0.1-6 mg/kg two times daily for 3 weeks. Thirty postmenopausal breast cancer patients were enrolled and were sub-divided into 5 cohorts with 6 patients in each cohort. The polysaccharide extract stimulated the immune response at a dose of 6mg/kg [69]. A hospital-based case controlled study was conducted to assess the combined effect of edible mushrooms and green tea on breast cancer. The study showed a reduced risk of breast cancer in both pre-and postmenopausal breast cancer patients due to high intake of A. bisporus, L. edodes and green tea [70]. Another hospital-based case-controlled study showed a reduced risk of stomach cancer from higher intake of mushrooms including Flammulina velutipes, Hypsizigus marmoreus, Corrinellus shiitake and Pholiota nameko [71]. Lastly, a superfine dispersed lentinan (SDL) oral formulation was evaluated on advanced colorectal cancer. Lentinan is an immunomodulatory β-glucan isolated from L. edodes. Eighty patients with advanced colorectal cancer were administered with 15 mg dose per day of SDL for 12 weeks. The SDL formulation, found to be safe and efficacious, reduced the adverse effects of chemotherapy and was associated with increasing the quality of life of patients with advanced colorectal cancer [72].

Anti-Inflammatory Activity In-Vitro
Mushrooms native to North America have also been explored for anti-inflammatory activity. Out of 29 species examined from north-central BC [24] and Haida Gwaii [23], 26 species (69%) had their anti-inflammatory activity reported for the first time. These species include A. augusta, C. tomentosus, Clavulina cinerea, G. helvelloides, G. esculenta, Hydnellum repandum, Hygrophoropsis aurantiaca, Hypholoma fasciculare, Inocybe sp., L. conifericola, L. connate, Phellinus nigricans, P. atratus, P. ostreatus, R. cystidiophora, R. paludosa, T. abietinum, T. rutilans, and T. chioneus. Extracts from I. obliquus collected in BC, like those found in other parts of the world, showed strong anti-inflammatory activity in vitro. Polymyxin B was used as positive control to assess anti-inflammatory activity [23,24]. In another study conducted on North American mushrooms, 95% ethanol extracts of Elaphomyces granulatus, Rhizopogon nigrescens, and Scleroderma laeve possessed strong anti-inflammatory activity whereas E. muricatus, Hymenogaster subalpinus, Melanogaster tuberiformis, Rhizopogon couchii, R. subaustralis, and R. subgelatinosus were also active against inflammation. COX-2 activity was assessed to determine the anti-inflammatory potential and NS-0398 was used as a positive control [25]. A limited number of anti-inflammatory compounds have been isolated from mushrooms native to North America. A 5 kDa polysaccharide isolated from the NaOH extract of North American mushroom E. tinctorium was able to induce antiinflammatory response in Raw264.7 macrophage cells [18]. A polysaccharide called CDP from Gymnopus dryophilus [31], and CDP-like polysaccharides from L. edodes and Marasmius oreades [32], can inhibit nitric oxide production in Raw264.7 macrophage cells. Elsewhere, the ethanolic extract as well as two small molecules, syringaldehyde and syringic acid, isolated from E. granulatus inhibited COX-2 enzyme in Raw264.7 cells [29]. The extract caused 68% inhibition of COX-2 at 50 µg/mL, whereas syringaldehyde and syringic acid were effective with IC50 of 3.5 µg/mL and 0.4 µg/mL respectively. The COX-2 inhibition of small molecules was compared with a positive control, NS-398, a known COX-2 inhibitor with IC50 of 0.2 µg/mL (0.64 µM) [29].

Anti-Inflammatory Activity in Animal Models
Currently, there are very limited studies examining the anti-inflammatory potential of North American mushrooms using animal models. In one study, the polysaccharide AIPetinc isolated from E. tinctorium showed anti-inflammatory activity on histamineinduced inflammatory mouse microcirculation model (n = 10 mice) [18]. An intra-vital mouse model was used to determine acetylcholine-induced vasodilation, which is one of the events in inflammation. AIPetinc was able to induce nitric oxide production, reversed the response to histamine and restored the vascular integrity, thereby resulting in antiinflammatory response [18]. To determine the significance of the anti-inflammatory activity of AIPetinc, it should be further evaluated in other animal models. In another study, the methanol extracts of I. obliquus was found to attenuate histamine-induced inflammation conducted vasodilation in arterioles in the gluteus muscle of mice (n = 41) [34]. Antiinflammatory compounds ergosterol, ergosterol peroxide and trametenolic acid had been isolated from the sclerotia of I. obliquus available from Haerbin, China [73]. It is unknown whether these compounds are present in the wild I. obliquus from B.C. and were responsible for attenuating histamine-induced inflammation in the gluteus muscle mouse model [34].

Anti-Inflammatory Activity in Clinical Studies
Given that there had been only two animal studies as described above, it is not surprising that there are currently no clinical studies examining anti-inflammatory potential of mushrooms North America. However, we wish to draw readers'attention to another review article that has summarized clinical studies alongside in-vitro and animal studies on investigating anti-inflammatory properties of mushrooms [74].
To date, most of the immuno-stimulatory compounds from mushrooms are polysaccharides or polysaccharide-protein complexes [76,77]. Due to problems such as: (i) high variability of polysaccharides that is further exacerbated during extraction and purification, (ii) impurities and contaminants, and (iii) difficulty in defining the structure and chemical fingerprint to understand structure-function relationships [78], there has been little interest amongst scientists in North America to study and isolate immuno-stimulatory polysaccharides from mushrooms native to North America.

Immuno-Stimulatory Activity in Animal Models
There is currently no animal studies examining the immuno-stimulatory activity of mushrooms native to North America. However, there were many animal studies evaluating the immuno-stimulatory effects of mushrooms collected elsewhere [11]. For instance, there were animal studies examining immunomodulatory activity of G. lucidum and its related species, G. tsugae and G. applanatum [79]. In another study, Agarikon 1 and Agarikon Plus which contains medicinal mushroom mixtures, were found to activate and maintain the immune system equilibrium in an advanced colorectal mouse model [80]. Elsewhere, the hot water, methanol and polysaccharide extracts of Lentinula edodes induced immune response against eimeriasis in chicken [81].

Immuno-Stimulatory Activity in Clinical Studies
As with animal studies, there is currently no clinical studies evaluating the immunostimulatory effect of North American mushrooms. There are also limited clinical studies evaluating immuno-potentiating effects of mushrooms collected from elsewhere. Two of the most notable medicinal mushrooms. T. versicolor and Grifola frondosa (Maitake mushroom), had been clinically investigated for such purposes. As described in 3.2.3, both PSP and PSK are immuno-potentiating anticancer polysaccharides isolated from T. versicolor that had undergone many clinical studies and this has been critically reviewed recently [11]. For G. frondosa, its polysaccharide extract was administered to 34 breast cancer patients on a phase I/II clinical trial. Cytokine analysis in patients' peripheral blood showed signs of both immuno-stimulatory and -inhibitory effects [82]. There was no dose-limiting toxicity observed in patients, suggesting that the extract is safe. The authors concluded that the G. frondosa polysaccharide should be classified as immuno-modulatory rather than immuno-stimulatory.

Anti-Oxidant Activity In-Vitro
There has been relatively less study on anti-oxidant activity of mushrooms native to North America. One study reported ethanolic extracts from 11 species of mushrooms collected from Idaho, Oregon, and New Jersey, to have moderate to weak anti-oxidant activity. In the study, 70-95% ethanolic extracts of various mushrooms were assessed and Vitamin C was used as a positive control [25]. Scleroderma laeve showed strong antioxidant activity with an IC50 of less than 20 µg/mL whereas Rhizopogon pedicellus, R. couchii, Leucogaster rubescens, and Geopora clausa had moderate anti-oxidant effect with IC50 of 20-50 µg/mL. Elaphomyces granulatus, E. muricatus, Gautieria monticola, Melanogaster tuberiformis, Rhizopogon nigrescens, and R. subaustralis had weak anti-oxidant activity with IC 50 values of more than 50 µg/mL [25].
The ethanolic extract as well as syringic acid isolated from the fruiting bodies of Elaphomyces granulatus showed potent antioxidant effect on myelomonocytic HL-60 cells with an IC50 of 41 µg/mL for the extract and 0.7 µg/mL for syringic acid. Vitamin C was used as a positive control with IC50 of 0.5 µg/mL (2.84 µM) to compare the anti-oxidant activity of syringic acid and ethanol extracts [29]. This study shows the promising effects of syringic acid as a potent anti-oxidant and can be considered for drug development. Two edible mushrooms, P. ostreatus from USA and P. levis from Mexico, also showed antioxidant activity. The effective concentration (EC-50) was also determined via DPPH free radical scavenging assay, ABTS scavenging assay, and β-carotene linoleic acid assays where BHA, BHT, α-tocopherol, and ascorbic acid were used as standards [39]. The anti-oxidant activity was also determined by oxygen radical absorbance capacity assay and the anti-oxidant effect was compared with a known anti-oxidant, Trolox, a vitamin E analog [39].

Anti-Oxidant Activity in Animal Models
As with the immuno-stimulatory activity, there is currently no study on the evaluation of North American mushrooms for anti-oxidant activity in animal models. However, there were animal studies investigating the anti-oxidant effect of medicinal mushrooms collected elsewhere. For instance, since oxidative stress plays important role in the etiology of diabetes, the impact of mushrooms on antioxidant enzymes involved in streptozotocininduced diabetes were investigated in mouse models [83,84]. The effect of administration of G. lucidum and Agaricus brasiliensis were studied in rat leukocytes. Streptozotocin was administered intraperitoneally at 50 mg/kg body weight to induce diabetes whereas mushrooms were given orally at a dose of 1 g/kg every day for 2 weeks. Mushroom preparations were found to increase the activity of anti-oxidant enzymes [83]. Another study also determined the anti-oxidant enzymes protection by mycelia of the two mushrooms on rat erythrocytes after induction of diabetes by streptozotocin [84]. In another separate study, anti-oxidant effect and regulation of lipid metabolism by F. velutipes extracts was studied. Forty eight hamsters were divided into eight groups, six hamsters/group, where one group was given normal diet while others were given high fat diet. 1-3% of mushroom preparation was added to their diet for 8 weeks, which resulted in significant effect on lipid metabolism of hamsters with high fat diet [85].

Anti-Fungal Activity
Many mushrooms are known to possess antifungal activity [86]. However, there has been only one study on anti-fungal activity of mushroom native to North America. A study shows that a chlorinated orcinol derivative, 2-chloro-1,3-dimethoxy-5-methyl benzene isolated from mycelial cultures of Hericium sp. collected in Minnesota (USA), showed inhibitory effect on Candida albicans and Candida neoformans, suggesting its role as an antifungal agent [17]. Hericium sp. is an edible mushroom containing multiple small molecules, making it an attractive option for further screening.

Other Bioactivities
In addition to the bioactivities described above, mushrooms from North America have also been investigated for antiparasitic, antimalarial and antituberculosis activities. A study conducted in Mexico showed antiparasitic effects of hydroalcoholic extracts from Pleurotus djamor against Haemonchus contortus eggs [38], suggesting its metabolites can act as anthelmintics. In the same study, few small molecules with antiparasitic activity were also isolated [38]. Stanikunaite and co-workers assessed 22 species of mushrooms native to North America and found Rhizopogon subareolatus to have antimalarial activity [25]. They also found the following species to have antituberculosis activity: Astraeus pteridis, Barssia oregonensis, Elaphomyces granulatus, Elaphomyces muricatus, Hymenogaster subalpinus, Melanogaster tuberiformis, Rhizopogon couchii, Rhizopogon pedicellus, Rhizopogon subareolatus, and Scleroderma leave [25].

Bioactive Compounds from Mushrooms Native to North America
Since there is relatively limited number of studies on mushrooms native to North America, it is not surprising to find relatively limited number of bioactive compounds isolated from the mushrooms. Here, we summarize all the bioactive compounds that have been isolated from mushrooms native to North America into two general groups, large molecules and small molecules (Table 2) (Figure 1).
As mentioned briefly in Section 3.4, there has been a lack of interest amongst scientists in the West, especially in North America, in studying bioactive polysaccharides for use as medicinal compounds [78]. However, with recent advances in identifying biosynthetic gene clusters and transcriptomic studies including those in fungi, it is now possible to produce compounds including large polysaccharides using heterologous expression systems by genetic engineering [88][89][90][91]. Such efforts are expected to enable large scale production of pure bioactive polysaccharides, thereby overcoming some if not all of the problems previously encountered [78].

Small Molecules
Small molecules that had been isolated from mushrooms include quinones, cerebrosides, amines, catechols, sesquiterpenes, triacylglycerols, steroids, organic germanium, and selenium ( Figure 1) [87]. Amongst the handful of small molecules isolated from North American mushrooms, few are terpene derivatives as shown in Table 2 and Figure 2.
Two small molecules, syringaldehyde and syringic acid with molar mass of 182 g/mol and 198 g/mol respectively, were isolated from ethanol extract of the fruiting bodies of E. granulatus [29]. The possible toxicity of both compounds was evaluated on macrophage Raw 264.7 cells and HL-60 cells. The authors found that both syringaldehyde and syringic acid had no effect on Raw 264.7 and HL-60 cells viability of up to 25 µg/mL and 31.25 µg/mL respectively [29]. Grifolin (328 g/mol), neogrifolin (328 g/mol) and confluentin (326 g/mol) with growth-inhibitory and antibacterial activities were isolated from the ethanol extract of the fruiting bodies of A. flettii [21,22]. A lanostane-type triterpene named 3, 11-dioxdanosta-8,24(Z)-diene-26-oic acid with molar mass of 468 g/mol was isolated from J. hirtus. This triterpene effectively inhibited the growth of two Gram positive bacteria; Bacillus cereus and Enterococcus faecalis [21]. Three new bisnaphthalene subclass compounds called urnucratins A-C were isolated from the North American cup fungus U. craterium [41]. Urnucratin A (348 g/mol) was found to be active against methicillin-resistant Staphylococcus aureus, vanocymin-resistant Enterococcus faecium and Streptococcus pyogenes [41]. A new lanostane triterpenoid named 3-oxo-24-methyl-5α-lanost-8,25-dien-21-oic acid (468 g/mol) with activity against B. cereus was isolated from F. pinicola native to North America [30]. The same study also reported isolation of four known lanostane triterpenoids and one known ergostane steroid with anti-bacterial activity [30].

Mushrooms from North America as a Source for Drug Discovery
As shown in Table 1, to date 79 species of mushrooms native to North America had been investigated and exhibited medicinal properties. Out of these, 48 species (60%) have bioactivities that have not been previously reported (Table 1). These include A. augusta with strong anti-proliferative activity, G. dryophilus with anti-inflammatory activity, G. esculenta with strong immunostimulatory activity, and H. coralloides with strong immunostimulatory activity [23,24]. This also includes C. tomentosus, G. helvelloides, L. conifericola, L. connata, T. abietinum with strong activity in antiproliferation, immunostimulation and anti-inflammation [23,24]. There were specimens that could only be identified to the genus level, Inocybe sp. and Hydnellum sp., suggesting that they are new species. Inocybe sp.
As shown in Table 2, there has been very limited number of studies exploring bioactive compounds from North American mushrooms. Despite the limited number of studies, 10 new small molecules, six new polysaccharides and a new protein with bioactivity have been discovered (Table 2).
Such observations and the fact that a large number of mushrooms native to North America having bioactivities that have never been previously described, strongly suggests that North American mushrooms are indeed a good source for drug discovery.

Edibility of Mushrooms Native to North America
North American mushrooms offer a wide variety of medicinal benefits and some have been appreciated for their exquisite flavors. While some are unique and edible, others are toxic for human consumption. Edible mushrooms are valuable as functional foods. Nonetheless, both edible and non-edible mushrooms offer great benefits against diseases. Edibility of mushrooms is an important attribute that allows them to be used as functional foods and potential phytomedicines. Like elsewhere, many edible mushrooms are found in North America. Here, we only highlight some of the popular ones. Some of the North American edible mushrooms [92,93] with medicinal properties are A flettii, I obliquus (Chaga), G. dryophilus, C. comatus, L. edodes (Shiitake), Marasmius oreades (fairy ring mushroom), Boletus curtisii, and Amanita augusta. Most mushrooms that belong to the Pleurotus genus are also edible and they include P. tuberregium, P. djamor, P. ostreatus, and P. levis. G. helvelloides is edible but it is rather bland and is therefore not much preferred in culinary. One of the most popular wild edible mushrooms in North America is chanterelle [85]. There are over 70 species of chanterelles (Cantharellus spp.) described worldwide and the most sought-after in North America is the Pacific golden chanterelle (Cantharellus formosa) found from northern California to northern BC [94]. Another popular culinary mushroom in North America as well as in Europe is Morchella, the true morels. At least 19 species of Morchella had been identified in North America [95] with black morels (Morchella elata and related species) found in Washington State and BC being one of the most highly demand culinary mushrooms. Lastly, the wild edible lobster mushroom (Hypomyces lactifluorum/Russula brevipes), uniquely confined to North America, is not a species but the result from infection of Russula (mostly Russula brevipes) or Lactarius genera by Hypomyces lactifluorum parasite [96]. The lobster mushroom, widely distributed in northern parts of United States and in Canada, has a seafood-like flavor and is enjoyed by many locals.
North American medicinal mushrooms that are considered toxic or non-edible [92,93] for general human consumption can be tailored as drugs after sufficient toxicity testing and targeted dosage form development. Some of the features that make mushrooms inedible are the presence of toxic metabolites in the whole mushroom, its taste and toughness. E. tinctorium is a hard, inedible conk. P. atratus and J. hirtus are also tough mushrooms and apparently J. hirtus has bitter taste. P. involutus is considered poisonous for human use. E. granulatus is also non-edible. At high doses, C. armillatus is considered toxic due to the presence of orenalline [19], a potent nephrotoxin originally isolated from Cortinarius orellanus [97]. Therefore, it is possible that orenalline is present at low amount in C. armillatus.

Conclusions
Having extensively reviewed the literature on the bioactivities and compounds isolated from mushrooms native to North America, we can now attempt to answer the questions posed at the beginning of this review. (i) Do similar species found in North America and elsewhere exhibit similar bioactivities and produce similar group of bioactive compounds? Based on subjective approach of bioactivity-guided investigations on selective species, similar species found in North America and elsewhere indeed produce similar bioactivities. For example, this is true for the commonly studied I. obliquus and P. ostreatus. Furthermore, compounds such as grifolin, neogrifolin and confluentin produced in North American A. flettii, are also made by other Albatrellus genus found elsewhere [21,22]. However, a better answer can only be obtained by using more advanced and objective method such as Quadruple Time-of-Flight Mass Spectrometry to globally examine the metabolites produced and using multiple biological screening assays to simultaneously monitor different bioactivities. (ii) Do similar species found in North America and elsewhere exhibit distinct bioactivity and produce distinct compounds? Again, the answer to this question would come from more advanced approaches described above. (iii) Do new species found in North America produce new compound(s)? Due to limited number of studies on mushrooms native to North America, it is currently unknown whether new species found in North America produce new compound(s). However, based on very limited study (Table 2), it is likely that new medicinal compounds are produced by new species found in North America. (iv) Based on the answers to (i) to (iii) above, is it worth exploring mushrooms native to North America for medicinal properties and for new compounds? We believe that North American mushrooms are worth further exploration. This is based on the fact that there are many species, including new species, in North America whose bioactivities have never been previously reported (Table 1). Furthermore, novel compounds have been isolated from North American mushrooms despite the very limited number of studies (Table 2).
In summary, to date only 79 mushroom species in North America that have been studied and reported to possess medicinal properties. Of these, 48 species (60%) exhibited bioactivities that have never been previously reported (Table 1). To date, only 16 mushroom species in North America that had been subjected to bioactivity-guided compound isolation studies. Out of this limited number of studies, already 10 new small molecules, 6 new polysaccharides and a new protein with medicinal properties have been discovered ( Table 2). For the majority of these compounds, studies are still at an early stage using cell lines, and their significance needs to be demonstrated in animal models and beyond.