Wild Mushrooms: A Hidden Treasure of Novel Bioactive Compounds

Secondary metabolites are hidden gems in mushrooms. Understanding these secondary metabolites' biological and pharmacological effects can be aided by identifying them. The purpose of this work was to profile the mycochemical components of the extracts of Auricularia auricula judae, Microporus xanthopus, Termitomyces umkowaani, Trametes elegans, and Trametes versicolor to comprehend their biological and pharmacological capabilities. Mushroom samples were collected from Kenya's Arabuko–Sokoke and Kakamega National Reserved Forests and identified using morphological and molecular techniques. Chloroform, 70% ethanol, and hot water solvents were used to extract the mycochemical components. Gas chromatography mass spectrometry (GC-MS) was used to analyze the chloroform, 70% ethanol, and hot water extracts of all the species examined. A total of 51 compounds were isolated from all extracts and classified as carboxylic acids, esters, phenols, fatty acids, alcohol, epoxides, aldehydes, fatty aldehydes, isoprenoid lipids, and steroids. Tetracosamethyl-cyclododecasiloxane (18.90%), oleic acid (72.90%), phenol, 2, 6-bis (1, 1-dimethylethyl)-4-methyl-, and methylcarbamate (26.56%) were all found in high concentrations in A. auricular judae, M. xanthopus, T. umkowaani, T. elegans, and T. versicolor, respectively. Fatty acids make up the majority of the compounds isolated from the T. elegans chloroform extract and the T. umkowaani 70% ethanol extract, respectively. Particularly, these fatty acids play crucial roles in the anti-inflammatory, hypocholesterolemic, anticancer, and antibiofilm formation activities. These bioactive elements indicate that the extracts of five wild mushrooms may be reliable sources of secondary metabolites for therapeutic development. Therefore, additional research is required to comprehend the usefulness of these chemicals in many functional areas and to improve the present understanding of macrofungi.

Auricularia auricular judge (Bull.)belongs to phylumbasidiomycota, class: agaricomycetes, order: auriculariales, family: auriculariaceae, and genus: Auricularia.A. auriculajudae, also known as black fungus, wood ear, Jew's ear, or jelly ear, is a species of edible mushroom that is very nutrient-dense [28,29].It contains a variety of nutrients that are worthy for our health, including polysaccharides, melanin, polyphenols, favonoids, amino acids, carbs, vitamins, and trace minerals.Furthermore, it has a variety of chemical combinations with antioxidant, anticoagulant, and anticancer properties [30].
Microporus xanthopus (Fr.) kuntze is a member of the genera Microporus and phylum-basidiomycota, class: agaricomycetes, order: polyporales, and family: polyporaceae.It is a polypore medicinal mushroom that cannot be eaten.It contains a variety of chemical components, including alkaloids, favonoids, steroids, triterpenoids, and coumar, which have the potential to have positive pharmacological efects with applications in agriculture, medicine, and other felds [31].According to reports, it has anthelmintic [32], antibacterial [33], anticancer [1], and antiangiogenic properties.
Trametes elegansis a member of the phylumbasidiomycota, class: agaricomycetes, order: agaricales, and family: polyporaceae.T. elegans is an endophytic and saprotrophic fungus that brought white rot on wood [38].Due to its extensive use in the culinary and pharmaceutical industries, T. elegans (also known as Turkey tail) has become incredibly well-known [39].Its capacity to break down dead organic matter and utilize a variety of substrates has resulted in a diversity of its biological and metabolic processes [40].It is also well-known for its therapeutic benefts, commercial applications (such as in the food sector), and roles in bioremediation and the biodegradation of cellulosic waste [41,42].
Numerous biologically active polysaccharides found in Trametes versicolor (L) Lloyd (family: polyporaceae) are used to treat a variety of ailments, including rheumatoid arthritis, chronic hepatitis, infections of the respiratory tract, urinary tract, and digestive system, and tumors.It consists of 18 diferent amino acids, including aspartic acid, threonine, serine, glutamic acid, glycine, alanine, valine, and leucine, as well as a wide range of other substances, including proteins, fatty acids, polysaccharides, polysaccharopeptides, glucans, vitamins, and inorganic salts [43,44].All of these amino acids are necessary for growth and repair because they play various roles in cellular, tissue, and organ structure [45,46].
Only a small or nonexistent amount of research has been carried out on the identifcation of bioactive chemicals that confer these therapeutic capabilities on the very few species of Kenyan wild mushrooms that have been claimed to have therapeutic potential.Determining the bioactive substances in wild mushroom extracts that are responsible for their therapeutic benefts is crucial.Terefore, the objective of this study was to investigate the bioactive substances found in the chloroform, ethanol, and hot water extracts of fve wild mushrooms and to ascertain their biological and pharmacological therapeutic capabilities that may shed light on their usage in both traditional and contemporary medicine.

Wild Mushrooms Collection and Identifcation.
In the national reserved forests of Kakamega and Arabuko-Sokoke, mushrooms were collected.Tey were randomly collected from tree bark or other substrates (wood, soil, or leaf litter).To keep them structurally sound and wet, they were wrapped in aluminum foil and put in an icebox.Following that, they were recognized using both morphological and molecular techniques.Specimens were identifed using spore print color (white, black, brown, pink, purple, etc.) and macroscopic and microscopic methods (form and size of basidiospores, basidia, cystidia, and generative hyphae) [47].In addition, Species Fungorum and associated literature were used to compare the morphological traits of the specimens [48].Te size and form of the gill, the color and shape of the cap, the color and shape of the stipe, and other morphological characteristics of the mushroom are some of the parameters utilized for identifcation.For the sake of identifcation, the gill margin, stipe location, stipe base, and pileal margin and surface were also applied.Other morphological details of the mushrooms, such as the cap's structure, the gills' margin and placement, the stipe's surface and form, and the pileus' margin and surface, were also noted.Te ornamentation of the pileus and stipe surfaces, the presence or absence of an annulus on the stipe, and the presence or absence of a volva at the base of the stipe were additional characteristics that were utilized to describe and identify the mushrooms (Table 1).Te samples were then preserved for additional analyses after being dried in an electric drying oven at 50 °C for 168 h [49].
Te dried fruiting body of mushrooms was used to extract gDNA using the cetyl trimethyl ammonium bromide (CTAB) technique [12].Highly conserved portions of the ITS1 and ITS4 of the mushroom rDNA genes were amplifed using the PCR amplifcation technique by using specialized markers [50].PCR products that had been amplifed were separated using gel electrophoresis and seen under UV illumination.Each PCR product's presence and quantity were calculated by contrasting it with the control (1 kb DNA ladder).

Extraction of Bioactive
Compounds.Chloroform, 70% ethanol, and hot water solvents were used to extract bioactive chemicals, with a few changes from other investigations [51,52].A 100 g of powdered mushroom was combined with 1L of each of distilled hot water (heated at 2 International Journal of Analytical Chemistry  International Journal of Analytical Chemistry  International Journal of Analytical Chemistry
By regulating pancreatic insulin secretion, mushroom polysaccharides have demonstrated antidiabetic properties that help to maintain blood glucose homeostasis [114].A prior study claimed that polysaccharides derived from AAJ extracts signifcantly reduced the risk of diabetes in streptozotocin-induced diabetic rats.After giving AAJ polysaccharides to streptozotocin and high-fat diet-induced diabetic rats, low-density lipoprotein, and total cholesterol levels in the blood were markedly decreased [115].In addition, streptozotocin-induced diabetic mice improved the insulin resistance islet damage in diabetes-induced rats treated with AAJ polysaccharides, which changed glucose metabolism, elevated insulin levels, and decreased blood glucose levels [116,117].Tese results supported the idea that AAJ-derived polysaccharides could be employed as possible diabetic treatment agents by modifying blood glucose levels [28].
High concentrations of insoluble fbers can be found in the HWE of AAJ [118].Trough the modifcation of gut microbiota, these fbers may have positive efects on health [119,120].Insoluble fbers have a crucial role in regulating the environment of the gut microbiota and infuencing their metabolic activities [121,122].Tey also serve as prebiotics.By fghting for food and preventing adhesion to the gut wall, the benefcial gut microbiota is essential in defending our body against a variety of disease-causing pathogenic bacteria [123].Tese gut bacteria also aid in the formation of shortchain fatty acids, such as acetate, propionate, and butyrate, which are crucial for our epithelial cells during their digestion and fermentation processes [124,125].By upholding a healthy gut environment and acting as the only carbon source for intestinal bacteria during fermentation, β-glucans derived from HWE of AAJ have many health-promoting efects.Tey also raised the quantity of good bacteria, such as Bifdobacteria and Lactobacillus, which aid in the synthesis of short-chain fatty acids in our intestines [126].During the oral treatment of mice, they also saw an increase in serum IgA and IgG levels [127].Furthermore, they stop the development of harmful bacteria in our gut, which may eventually shield our bodies against a variety of disorders linked to the gut [128,129].
Biological defenses against cardiovascular disease exist in edible mushrooms.Tere have been claims that certain Auricularia species contain substances that decrease cholesterol [130].AAJ extracts have reportedly been shown to lower low-density lipoprotein cholesterol levels, which are the cause of cardiovascular disease [121].AAJ extract effectively decreased serum and liver total cholesterol (TC), total triglyceride (TG), and serum lactate dehydrogenase C (LDH-c) levels in mice using hyperlipidemic mice as a model [131].
Natural immunomodulators can be found in large quantities in medicinal mushrooms.Tey contain a variety of immune-regulatory substances, including immunomodulatory proteins, lectins, polysaccharides, and terpenes.Immunomodulators can act as immunological adjuvants, immune stimulants, or immune suppressants [132].For instance, an active substance from AAJ called AF1 with a 1, 3-d-glucan main chain and two 1, 6-d-glucosyl residues has been shown to cause apoptosis in cancer cells [133].
In line with the current fndings, HWE of MX, numerous mushroom extracts including Agaricus bisporus, Cyclocybe aegerita, Cyclocybe cylindracea, and Tremella fuciformis have been investigated for the treatment or prophylaxis of type-2 diabetes, which develops when insulin production is unbalanced as a result of the dysfunction of insulin-secreting beta cells in the pancreas [134,135].Mushrooms assist patients in avoiding excessive blood sugar levels since they are the food with the least quantity of digestible carbohydrates [136].Diabetes can be treated with bioactive compounds that are extracted from medicinal mushrooms [137,138].Extracts from Inocutis levis and Antrodia cinnamomea have been suggested as treatments for diabetes because they improve insulin resistance, insulin sensitivity, and tissue uptake of glucose, which helps to regulate blood sugar levels [135,139].
Most of the substances discovered in the HWE of MX in the current results demonstrated antibacterial action.Another investigation confrmed that HWE of MX-derived oligosaccharides, polysaccharides, and polyphenols had antibacterial efects on S. aureus strains that were resistant to methicillin and E. coli strains that produced Shiga toxin [33].Similar to this, MX's CE has shown increased antibacterial activity against S. aureus (ATCC 25923), MRSA (ATCC 33591), and K. pneumoniae (ATCC 13883) [52].
Te majority of mushrooms are known to produce several bioactive compounds that are employed as potential treatments for cardiovascular disorders [140,141].Although the mechanism of action/treatment of these bioactive compounds is still unknown, it may be related to changes in phospholipid metabolism, bile acid secretion, and LDL receptor expression [142].Te presence of chemicals in mushrooms that can alter cholesterol metabolism, absorption, and gene expression has also been noted in other investigations [141,143].For example, Grifola frondosa, Hypsizigus marmoreus, and Pleurotus ostreatus extracts have been shown to alter the gene expression patterns in mouse livers [135,144].
Te EE of TU contains hexadecanoic acid, and ethyl ester (palmitic acid ester), which has antioxidant, hypocholesterolemic, nematicide, pesticide, antiandrogenic, antibacterial, anti-infammatory, antitumor, immunostimulant, hemolytic 5-reductase inhibitor, and lipoxygenase inhibitor efects.Dietary fats contain palmitic acid (PLA), which ensures an intake of 20 g on average per day.Its important nutritional function [149] justifes the relatively high need for these fatty acids in the human body (20-30% of total fatty acids).It was discovered through transcriptomic research that palmitic acid afected many signaling pathways, including lipid metabolism in neurons.On the other hand, excessive ingestion of palmitic acid has been linked to neurodegenerative conditions, such as Parkinson's disease [150,151].To prevent harmful efects such as oxidative stress, PLA has a critical role in low levels of stress, which can activate the stress response pathway.
Te EE of TU contained the 9, 12-octadecadienoic acid (Z, Z)-, that is also known as conjugated linoleic acid.Omega-3 and omega-6 fatty acids are present in linolenic acid (LA).LA can minimize risk factors for arthritis and heart disease as well as assist in reducing bodily infammation.Prostaglandin E1, a product of omega-3 fatty acids, increases immunity and lowers blood cholesterol [152,153].Te heart's health is improved by omega-3 fatty acids, which also lower the risk of stroke, heart attack, and high blood pressure [153,154].In general, mushrooms have more unsaturated fatty acids than saturated ones [155].Tese polyunsaturated fatty acids preserve the liver's ability to produce bile acids, prevent hormonal imbalance, and afect prostaglandin synthesis [156].
LA currently exhibits antibacterial action.In support of the present fndings, Termitomyces species extract in methanol and ethanol demonstrated strong antibiotic action against pathogenic microorganisms such as E. coli, B. cereus, S. aureus, P. aeruginosa, S. typhimurium, and C. albicans [157].Te dichloromethane extract of Termitomyces striatus also demonstrated antibacterial action against fungi (C.albicans and S. cerevisiae) and bacteria (P.aeruginosa, E. coli, B. subtilis, and S. aureus) [158].Numerous species of Termitomyces have notable antibacterial action against various harmful pathogens.For instance, the water extract of T. clypeatus demonstrated antibacterial and antifungal activities against C. albicans, E. coli, S. typhi, and S. aureus.Te water extract of T. heimiialso demonstrated antibacterial and antifungal activities against E. coli, K. pneumoniae, Pseudomonas sp., S. aureus, S. pyogenes, and Ralstonia sp.
Fatty acids from the EE of TU, such as octadecanoic acid, ethyl ester, h-hexadecanoic acid, 9, 12octadecadienoic acid (Z, Z), 9, 12-octadecadienoic acid, and ethyl ester, have demonstrated hypocholesterolemic action.Edible mushrooms include large levels of dietary fber and other nutrients such as eritadenine, guanylic acid, and ergosterol that can prevent diseases associated with nutrition, including atherosclerosis, by reducing hypocholesterolemic levels [159,160].Total and LDL cholesterol levels in the blood were found to decrease with dietary TU intake [161].Rats that were fed diets containing a combination of mushrooms saw a decrease in their triglyceride and total cholesterol levels [156].By changing lipid metabolism and preventing both the buildup of liver lipids and the rise of serum lipids, polysaccharides and fbers extracted from aqueous extract of edible mushrooms also reduced the serum triglyceride concentration inhypertensive and hyperlipidaemic rats [162].

GC-MS Analysis of Chloroform Extract of Trametes elegans (TRE).
Tree substances were found in the T. elegans chloroform extract (CE), as shown in Figure 1(d).Nhexadecanoic acid (16.89%), oleic acid (72.90%), and octadecanoic acid (10.21%) are among the chemicals that have been discovered.Tese substances fall under the category of essential fatty acids, which are required for their antiinfammatory, antioxidant, and hypocholesterolemic properties.In rats, a lack of the normal necessary fatty acid linoleic acid results in hair loss [21], minor skin scaling, and slow wound healing [22].
According to Table 5, the majority of the discovered compounds show antimicrobial, antioxidant, anticancer, antiandrogenic, hypocholesterolemic, nematicide, pesticide, and antibioflm formation capabilities.Numerous substances found in the extract, including tocopherols, favonoids, polyphenols, tannins, and lignins, may be associated with these wide-ranging activities [163].Te oxidizing cascade of free radical reactions in molecules is blocked by the TRE extract's antioxidant activity, which also lessens oxidative damage brought on by oxidative stress [164].Antioxidants shield our bodies from serious health problems such as diabetes, cancer, aging, atherosclerosis, and others [165].
From the CE of TRE, three isolated essential fatty acids have shown antibioflm-forming activity.By preventing pathogenic microorganisms from forming bioflms, fungal metabolites have antiquorum-sensing properties that have the potential to reduce the development of drug resistance.Numerous secondary metabolites with bioflm-inhibitory properties are found in many edible mushrooms, according to earlier investigations.For instance, active antibioflm inhibitory activity against Pseudomonas, S. aureus, and C. albicans has been demonstrated by coprinuslactone, roussoellenic acid, and microporenic acid A obtained from Coprinus comatus, Roussoella sp, and Kenyan basidiomycete, respectively [166,167].By facilitating antibiotics' capacity to enter bioflms, bioflm inhibitors improve the efectiveness of the antibiotics [168].
Te CE of TRE possesses anticancer properties.Te extracts combat cancer cells in a variety of ways, such as immune system regulation and cell death [169].Preclinical and clinical testing for several promising anticancer drugs based on fungi is now underway [170].Irofulven, for instance, is a semisynthetic medication made from illudin S, a toxin discovered in the Omphalotus illudens.Irofulven has been tested in phases I and II clinical studies, showing promise in its ability to treat malignancies of the breast, 12 International Journal of Analytical Chemistry blood, colon, sarcoma, prostate, lungs, ovary, and pancreas, as well as the brain and central nervous system [171,172].Another anticancer substance discovered in fungi Akanthomyces muscarius and Nigrospora sphaerica is called aphidicolin.Aphidicolin has not yet been marketed as an anticancer medication, even though it specifcally targets the binding site on the DNA polymerase and enzymes [135].
One of the fatty acids, n-hexadecanoic acid, found in the CE of TRE showed nematicidal action as shown in Table 5.Even though chemical nematicides (like methyl bromide) are efective and have been commercialized, they can have detrimental efects on the environment by destroying all soil life and thinning the ozone layer.Finding ecologically sound substitutes has recently been the focus of intense research in both academia and industry [135].Edible mushrooms have been found to contain several nematotoxic substances, including fatty acids, alkaloids, peptide compounds, terpenes, condensed tannins, phenolic compounds, and proteases [173].One of the nematicidal substances that have been identifed from Arthrobotrys species and other fungi is linoleic acid [174].Pleurotus pulmonarius and Hericium coralloides, on the other hand, are two basidiomycetes that have demonstrated potent nematicidal actions against Caenorhabditis elegans [175].From a Sanghuangporus species obtained in Kenya, metabolites (3, 14′-bihispidinyl and hispidin and phelligridin L) with mild nematicidal activity against Caenorhabditis elegans have been identifed [176].Recent studies have shown that chaetoglobosin A and its derivative 19-O-acetylchaetoglobosin A, which were isolated from Ijuhya vitellina, have nematicidal activity against the eggs of Heterodera flipjevi [177].
In TRV, a polyunsaturated fatty acid called 9, 12-Octadecadienoic (Z, Z)-has demonstrated antitumor action [107].Te TRV extract contains substances that fght cancer and boost the immune system, such as polysaccharides, β-glucans, lignins, and ergosta-7, 22-dien-3 beta-ol [178].Cytotoxic action against cancer cells was shown by polysaccharides extracted from the TRV extract [45].In addition to signifcantly improving the quality of life of cancer patients receiving chemotherapy or radiation therapy, polysaccharides containing peptides also help patients with hepatitis, hyperlipidemia, and other chronic diseases live longer and have better quality of life [178,179].In mice bearing xenografts, an aqueous extract of TRV inhibited the migration and invasion of 4T1 breast cancer cells and downregulated the activity of the xenograftinducing molecules tumor necrosis factor, interferon, interleukin-2, interleukin-6, and interleukin-12 [180].Te polysaccharides linked to the TRV protein displayed tumor necrosis factor-dependent antiproliferative activity towards MCF-7 cells and enhanced the proliferative response of blood lymphocytes, which was connected to the upregulation of interleukin-6 and interleukin-1 mRNA [181].

Conclusion
Bioactive compounds identifed from the fve wild mushroom extracts possess anti-infammatory, antioxidant, nematicide, antimicrobial, anticancer, hypocholesterolemic, antihypertensive, pesticide, and antibioflm formation properties.Te wild mushroom extracts are rich in essential fatty acids and other many bioactive compounds that could have high industrial potential and biological activities.Phenol, 2, 6-bis (1, 1-dimethylethyl)-4-methyl-, methylcarbamate (A.auricular-judae), 1monolinoleoylglycerol trimethylsilyl ether (Microporus xanthopus), tetracosamethyl-cyclododecasiloxane (T.umkowaani), oleic acid (T.elegans), and phenol, 2, 6-bis (1, 1-dimethylethyl)-4-methyl, methylcarbamate (T.versicolor) are the most abundant compounds.Tese compounds can be deployed to discover novel drugs against various noninfectious diseases such as cancer, hypertension, and diabetes.Te identifed compounds shall be subjected to further studies to utilize their usefulness in the prevention and treatment of infectious and noninfectious human diseases.To understand the mechanisms of action of the active ingredients, rigorous chemical analyses as well as in vivo pharmacokinetics and pharmacodynamics of individual compounds are needed.Future investigation is needed to clarify the long-term efects of taking medicinal mushroom products with other drugs.

Free
Note.N/A-not applicable, -denotes the absence of annulus and volva.

Table 1 :
Morphological characters and keys used for the identifcation of the wild mushrooms.

Table 2 :
GC-MS analysis of A. auricula judae hot water extract.

Table 3 :
GC-MS analysis of M. xanthopus hot water extract.

Table 5 :
GC-MS analysis of T. elegans chloroform extract.

Table 6 :
[53]S analysis of T. versicolor hot water extract.MS Analysis of Extracts.Te GC-MS analysis was conducted using a silica capillary column (30 × 0.25 mm ID × 1 µm, composed of 100% dimethylpolysiloxane) and operated in an electron impact mode at 70 eV (Agilent Scientifc, Palo Alto, CA).Helium (99.999%) was a carrier gas at a constant fow of 1 mL/min.Extracts were dissolved in dichloromethane and 1 µL solution was injected into the column at 250 °C and ion-source temperature 280 °C.Te oven temperature was programmed at 110 °C for 2 min.Te temperature was increased from 110 °C to 200 °C (10 °C/min) then to 280 °C (5 °C/min) and fnally ended at 280 °C for 9 min.Te total run time was 28 min.Te compounds were identifed from the MS data, by comparing the spectra of known compounds stored in the National Institute of Standards and Technology (NIST) library with the mass spectrometry (MS) of unknown compounds.Te relative % amount of each compound was calculated by comparing its average peak area to the total areas.Measurement of peak areas and data processing were carried out by the Turbo-Mass-OCPTVS-Demo SPL software[53].
[52]molecular formula; RT: retention time; PA: peak area.2.4.Statistical Analysis.All the tests, experiments, and measurements were carried out in triplicate.Microsoft Excel software package was used to analyze quantitative data[52].