Biological Activities of Secondary Metabolites from the Edible-Medicinal Macrofungi

Macrofungi are well-known as edible-medicinal mushrooms, which belong mostly to Basidiomycota, with a few from Ascomycota. In recent years, macrofungi have been recognized as a rich resource of structurally unique secondary metabolites, demonstrating a wide range of bioactivities, including anti-tumor, antioxidant, anti-inflammatory, antimicrobial, antimalarial, neuro-protective, hypoglycemic, and hypolipidemic activities. This review highlights over 270 natural products produced by 17 families of macrofungi covering 2017 to 2023, including their structures, bioactivities, and related molecular mechanisms.


Introduction
Macrofungi, a kind of large and visible fungi, can form fleshy or gelatinous macroscopic fruiting bodies or sclerotia, which are usually known as mushrooms [1].Most mushrooms belong to the Basidiomycete (such as Lentinus edodes), and a small portion belong to the Ascomycete (such as morels and truffles) [2].These fruiting bodies contain a variety of nutrients, including protein, essential fatty acids, vitamins, and minerals [1].Therefore, mushrooms are deeply loved by people due to their nutritional value.In order to protect the fruiting body from harmful organisms, including viruses, bacteria, and insects, mushrooms can secrete various secondary metabolites (such as terpenoids and phenolics) with antiviral, antioxidant, and other biological activities as chemical weapons.Mushrooms contain abundant active proteins, such as lectins with an antiviral effect [3], deoxyribonuclease with an anti-tumor function [4], and ribotoxin with an anti-proliferative effect [5].Similarly, mushrooms are also one of the main sources of antioxidants ergothioneine and glutathione [6].Modern pharmacology found that the secondary metabolites produced by mushrooms have certain medicinal value, including anti-Alzheimer [7], antidiabetic [8], and antitumor [9].Lentinan is a type of glucan isolated from the fruiting body of shiitake mushrooms.It has detoxification activity and can significantly alleviate side effects such as leukocyte suppression, nausea, and vomiting caused by cancer chemotherapy.Currently, lentinan is used as injections for adjuvant treatment of cancer in clinical practices [10].Additionally, lentinan can be used in combination with rabies vaccines.It not only resolves vaccine-induced side effects, but it also enhances therapeutic effects [11].The mushroom resources that can be developed and utilized are very abundant.At the present time, approximately 2000 species of edible mushrooms and 650 species of medicinal mushrooms have been found in forests around the world [12], which have attracted the interest of food and pharmacy scholars due to their rich nutritional and medicinal values, as well as the abundant resources.

Mushroom Mushroom Family Secondary Metabolites
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Anti-Tumor Activity
The cancer incidence rate and cancer-related mortality are rising in the whole world.Based on cancer prevalence data, it is estimated that 28.4-million cancer cases will be diagnosed by 2040 [75].At present, there are various methods of cancer treatment (such as chemotherapy, radiotherapy, immunotherapy, and others), among which chemotherapy is the most widely used [76].Although chemotherapy drugs exhibit significant therapeutic effects, they have the disadvantages of low bioavailability, prominent side effects, and high susceptibility to drug resistance.In the last few years, it

Biological Activities of Secondary Metabolites 2.1. Anti-Tumor Activity
The cancer incidence rate and cancer-related mortality are rising in the whole world.Based on cancer prevalence data, it is estimated that 28.4-million cancer cases will be diagnosed by 2040 [75].At present, there are various methods of cancer treatment (such as chemotherapy, radiotherapy, immunotherapy, and others), among which chemotherapy is the most widely used [76].Although chemotherapy drugs exhibit significant therapeutic effects, they have the disadvantages of low bioavailability, prominent side effects, and high susceptibility to drug resistance.In the last few years, it has been reported in the literature that natural active ingredients exhibit significant activity and few side effects [77], which have the potential as therapeutic drugs for inhibiting and treating cancer [13].
The following is a systematic review of the direct cytotoxic effect of secondary metabolites obtained from macrofungi fruiting bodies or mycelium against cancer cells.These studies mainly validated the inhibitory activity of secondary metabolites on malignant tumors such as lung cancer, liver cancer, and cervical cancer through 3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) experiments.
According to the above analysis, it is found that secondary metabolites derived from mushrooms have a significant anticancer effect, especially in inhibiting the growth of lung cancer cells (Calu-6), colorectal cancer cells (SW480), and hepatoma cells (SMMC-7721).Furthermore, it is worth mentioning that 27, 28, and 65 exhibit remarkable inhibitory activity against various cancer cells.

Antioxidant Activity
The pathogenesis of many human diseases (cancer, atherosclerosis, Alzheimer's disease, and others) is related to oxidative stress.The occurrence of oxidative stress is caused by the imbalance between oxidation and antioxidation in the body [78].Therefore, maintaining oxidative balance in the body helps to suppress the progression of diseases, and it is necessary to explore new drugs with antioxidant activity.The following is a general overview of studies on secondary metabolites with antioxidant activity of large edible and medicinal fungi in recent years.These studies mainly validated the antioxidant activity through 1,1-dipheny-1-2-picrylhydrazyl (DPPH) and 2,2-azinobis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), the free-radical scavenging assay, the cupric-reducing antioxidant capacity (CUPRAC), and the oxygen radical-absorbance capacity [79].

Anti-Inflammatory Activity
Inflammation is a protective response against injury and infection by secreting nitric oxide (NO) and pro-inflammatory cytokines [80].A moderate inflammatory response helps to resist external harmful stimuli.However, excessive inflammation can easily lead to acute diseases such as enteritis and arthritis.And prolonged inflammation can easily cause chronic diseases [81].Drugs including steroids and nonsteroidals are commonly used in clinical to alleviate inflammation even though their side effects (hypertension, hepatotoxicity, and others) are non-negligible [82].Therefore, it is imperative to search for potential low-toxic and effective anti-inflammatory drugs.The following is a summary of studies on secondary metabolites with the anti-inflammatory activity of large edible and medicinal fungi in recent years.These studies mainly validated the anti-inflammatory activity through constructing inflammation models using lipopolysaccharide (LPS).
As to the analysis above, it is found that these compounds exert an anti-inflammatory effect by inhibiting the secretion of NO and the expression of IL-6 and TNF-α and the NF-κB-signaling pathway.Meanwhile, Compounds 69-72, 126, 129-131, 134, and 143 have remarkable anti-inflammatory effects.Therefore, it is speculated that these secondary metabolites have the potential to become highly effective anti-inflammatory agents.

Antimicrobial Activity
Invasive fungal and bacterial infections are key causes of the incidence rate and mortality in immune-compromised populations [83,84].Antibiotics are often used for the treatment of microbe infections.However, the emergence of antibiotic resistance has greatly reduced the effectiveness of antimicrobial drugs and will increase the severity of infection, morbidity, and treatment costs [85].Therefore, newer and more effective antibiotics are urgently needed.Similarly, the treatment cycle for fungal infection is long, which means there is an urgent need to explore more effective antifungal drugs.The following is a systematic review of studies on secondary metabolites with the antimicrobial activity of mushrooms in recent years.These studies mostly evaluated the inhibitory activity of secondary metabolites on various fungi (Candida albicans and Cryptococcus neoformans) and bacteria (Mycobacterium tuberculosis and Staphylococcus aureus) through the minimum inhibitory concentration (MIC) index.Moreover, in order to facilitate the comparison of the strength of activity of the compounds, the MIC values were categorized into three classes according to Kuete's work [86]: significant (MIC < 10 µg/mL), moderate (10 < MIC < 100 µg/mL), and low or negligible (MIC > 100 µg/mL).
Based on the above analysis, compounds 147 and 148 are the potential drugs for the treatment of tuberculosis.

Antimalarial Activity
Malaria is a disease caused by the infection of protozoan parasites in the genus Plasmodium, which is prevalent in tropical regions (especially in Africa, Southeast Asia, and South America) and has a significant mortality rate [87].It is necessary to explore effective antimalarial drugs.The following is a brief review of studies on secondary metabolites with antimalarial activity of mushrooms in recent years.Compounds 149-162 (IC 50 = 5.1−19 µM) from the fruiting body of the wood-rot Tomophagus sp.showed antimalarial activity, of which 150 (IC 50 = 5.1 µM) had the strongest antimalarial activity (dihydroartemisinin as positive drug, IC 50 = 0.0028 µM) [47].Compound 176 (IC 50 = 257.8nM) had an inhibitory effect on chloroquine-sensitive strain P. falciparum (chloroquine as positive drug, IC 50 = 22.66 nM) [52].The antimalaria test results showed that 177 (IC 50 = 0.05 µM) and 178 (IC 50 = 0.45 µM) had significant inhibitory effects on P. falciparum, while chloroquine (IC 50 = 0.50 µM) was used as positive control [53].Overall, compounds 150, 177, and 178 exhibit potential antimalarial activity effects.It is speculated that these compounds have the potential to become more effective antimalarial drugs.

Neuro-Protective Activity
In recent years, the incidence rate of nervous system diseases (Alzheimer's disease, stroke, and others) has increased continuously and has gradually become the main cause of global disability [88].Neuroglial cells are the most important cells in the central nervous system, which maintain homeostasis and the operation of the central nervous system through interactions with neurons, immune cells, and other factors [89].The following is a systematic review of studies on secondary metabolites with the neuroprotective effect of large edible and medicinal fungi in recent years.These studies mainly validated the neuroprotective effect by constructing neural system injury models using the PC12 cell line and BV-2 microglia.
Compounds 73-75 could alleviate H 2 O 2 , and amyloid β-protein-induced SH-SY5Y cells damaged through the reduction of the production of reactive oxygen species (ROS) or free radicals [29].Compounds 128 and 129 could exert neuroprotective effects by reducing the production of NO in BV-2 microglia [41].Compounds 130-136 exerted neuroprotective effects by reducing the production of NO in BV-2 microglia, with 130, 131, and 134 exhibiting superior neuroprotective activity compared to positive drugs (quercetin) [42].Compounds 179 and 180 could enhance nerve growth factor-induced neurite outgrowth in PC12 cells through the tyrosine kinase A (TrkA) and kinase1/2 (ERK1/2) pathway to exert neuroprotective effect [54].Compounds 181 and 182 could alleviate hydrogenperoxide (H 2 O 2 )induced PC12 cell damage by stimulating neurite activity.Through electron microscopy observation, it was found that 181 and 182 showed neurotrophic effects on undifferentiated PC12 cells [55].Compounds 183-186 exerted neuroprotective effects by promoting the release of neurotrophic factors in astrocytic cells [56].Compounds 187-192 exhibited neurotrophic activity and could promote axonal growth [57].Compounds 193-199 could exert neuroprotective effects by reducing the production of NO in BV-2 microglia, and 193 had the most significant effect.Further research found that the neuroprotective effect of 193 could be related to the reduction of the levels of IL-1β, IL-6, and TNF-α and the mitigation of abnormal changes in mitochondrial membrane potential and reduction in ROS generation.Mechanism studies showed that the neuroprotective effect of 193 was related to its inhibition of TLR-4/NF-κB and MAPK-signaling pathways and activation of the Akt/GSK-3β/Nrf2-signaling pathway [58].Compound 200 reduced the production of ROS and alleviated mitochondrial damage to protect SH-SY5Y cells from H 2 O 2 -induced damage, which could be related to the Nrf2-and BDNF/TrkB/ERK/CREB-signaling pathways [59].Compounds 201-203 could considerably enhance neurite outgrowth in PC-12 cells [60].Compounds 204-206 exerted a neuroprotective effect by reducing the levels of inflammatory cytokines.Compound 201 could markedly reduce the level of TNF-α in BV-2 microglia, and 205 significantly reduced the level of IL-6 in BV-2 microglia.Compound 206 noticeably reduced the levels of NO and IL-1β in BV-2 microglia, as well as the expression of phosphorylated nuclear factor-kappa B inhibitor-α and the activity of iNOS [61].

Hypoglycemic Activity
The elevation of blood glucose levels mainly causes diabetes, which also causes metabolic disorders in the organism.Moreover, high blood glucose can easily induce cardiovascular diseases, obesity, kidney diseases, and other diseases [90].Therefore, maintaining blood glucose balance in the body is of great importance.The α-glucosidase, aldose reductase (AR), maltase, sucrase, and protein tyrosine phosphatase 1b (PTP1B) play important roles in lowering blood glucose levels [91][92][93][94].The α-glucosidase and AR can mainly act on hyperglycemia to lower blood glucose levels [95,96].PTP1B can reduce blood glucose levels by negatively regulating the insulin metabolism pathway [97].This section reviewed promising drugs derived from mushrooms that can suppress α-glucosidase, AR, maltase, sucrase, and PTP1B enzyme activities.
Based on the above analysis, it is found that 210, 223, and 224 have a significant suppression of α-glucosidase, AR, maltase, and sucrase.Additionally, compounds 35, 38, 40, 82, 123, 124, and 127 effectively inhibit cell absorption of glucose.Therefore, they have great potential in the treatment of diabetes.

Other Bioactivities
Edible and medicinal mushrooms can effectively produce useful bioactive metabolites.Due to their anti-tumor, anti-inflammatory, and antioxidant activities, these secondary metabolites have protective effects on organs such as the liver and kidneys.In addition, these secondary metabolites also have immunomodulatory activity and inhibitory effects on some cholinesterase and tyrosinase.

Discussion
As early as prehistoric times, there were records of mushrooms as medicines [101].Due to the rich nutritional content of mushrooms, they have always attracted scholars to deeply research the mechanisms behind their medicinal properties [102].At present, there is considerable research on mushroom polysaccharides, and it has been reported that the immune regulation and anti-tumor effect of mushrooms mainly stem from mushroom polysaccharides [103].With the continuous deepening of research on the composition of mushrooms, the secondary metabolites produced from mushrooms have also attracted scholars' attention.
According to further data analysis, 61 secondary metabolites show hypoglycemic activity, 42 metabolites exhibit anti-tumor function, 40 metabolites have neuroprotective effects, 19 metabolites are able to inhibit microorganisms, 13 metabolites have hypolipidemic activity, 18 metabolites display inhibitory tyrosinase activity, 11 metabolites exhibit enzyme-inhibitory activity, 3 metabolites possess insecticidal activity, and 6 metabolites exhibit organ-protective functions.Furthermore, it is worth mentioning that oxidative stress and inflammatory responses are often key pathological processes in some acute and chronic diseases (Alzheimer's disease, heart failure, and others) [104][105][106].There are 35 and 37 secondary metabolites with antioxidant and anti-inflammatory effects, respectively.And their mechanisms may relate to TrkA/ERK1/2, Nrf2, BDNF/TrkB/ERK/CREB, TLR-4/NF-κB, and MAPK pathways.Summarizing the above, it can be hypothesized that mushrooms are important drug resources used to treat diabetes, cancer, neurological diseases, fungal and bacterial infectious diseases, and hyperlipidemia.However, it is undeniable that although these secondary metabolites reviewed in this paper have certain medicinal potential, further exploration is still needed to determine whether they produce side effects and have sufficient yield.
In addition, most of the edible and medicinal mushrooms included in this article come from Ganodermataceae, Hymenochaetaceae, Polyporaceae, and Hericiaceae, indicating that mushrooms from these families produce a wide range of pharmacologically active secondary metabolites.Overall, macrofungi is a rich source of bioactive secondary metabolites with a high potential for exploitation.

Conclusions
In recent years, relevant scholars have elaborated on the types of medicinal and edible mushrooms, the extraction techniques of bioactive components from mushrooms, and the pharmacological effects of active metabolites.The research on active ingredients focuses on primary metabolites (polysaccharides, proteins, polyunsaturated fatty acids) and nutritional components of mushrooms themselves, along with their development in food, medicine, and other fields [107][108][109].This review mainly aims at the pharmacological activity and action mechanism of secondary metabolites derived from mushrooms.274 secondary metabolites (Figures 1-9) from 17 families of mushrooms, including 116 terpenoids, 71 sterols, 22 phenols, 14 polyketides, 9 alkaloids, and some aromatics and pyridines, as well as their biological activities, are reviewed.Among them, terpenoids and sterol-active metabolites accounted for the highest percentage.For example, Terpenoids 27, 28, and 65 possess significant anti-tumour activity; 69-72, 126, 129-131, and 134 show primary anti-inflammatory activity; compounds 35, 38, 40, 82, 123, 124, and 127 possess a significant hypoglycaemic effect.Sterols 147 and 148 exhibit anti-microbial activity, while 176-178 exhibit better anti-malarial activity.
In summary, the purpose of the current review is to provide a valuable theoretical reference for researchers to reasonably develop and utilize edible and medicinal mushrooms.