Classification, structure and mechanism of antiviral polysaccharides derived from edible and medicinal fungus.

The deficiency of chemical-synthesized antiviral drugs when applied in clinical therapy, such as drug resistance, and the lack of effective antiviral drugs to treat some newly emerging virus infections, such as COVID-19, promote the demand of novelty and safety anti-virus drug candidate from natural functional ingredient. Numerous studies have shown that some polysaccharides sourcing from edible and medicinal fungus (EMFs) exert direct or indirect anti-viral capacities. However, the internal connection of fungus type, polysaccharides structural characteristics, action mechanism was still unclear. Herein, our review focus on the two aspects, on the one hand, we discussed the type of anti-viral EMFs and the structural characteristics of polysaccharides to clarify the structure-activity relationship, on the other hand, the directly or indirectly antiviral mechanism of EMFs polysaccharides, including virus function suppression, immune-modulatory activity, anti-inflammatory activity, regulation of population balance of gut microbiota have been concluded to provide a comprehensive theory basis for better clinical utilization of EMFs polysaccharides as anti-viral agents.

Introduction

Virus is a filterable vector and is sub-microscopic, with most animal viruses physically ranging in size from 20 to 200 nm in diameter [1]. As is well-known, about 60% of epidemic infectious diseases are caused by viruses and viral infections have always been a worldwide problem that jeopardizes human health. The current common antiviral drugs on the market are mainly the following: 3CLpro inhibitors (generated immature or weakly infectious virus by inhibiting 3CLpro activity), such as lopinavir/ritonavir [[2], [3], [4], [5]], and darunavir/cobicistat [[6], [7], [8]]; RdRp inhibitors, (inhibited viral nucleic acid synthesis by intervening), such as remdesivir [[9], [10], [11]], favipiravir [12,13], and ribavirin [14,15]; Arbidol (blocking virus entry by interfering with clathrin) [[16], [17], [18]] and ribavirin combined with PegIFN-α2a [[19], [20], [21], [22]], glucocorticoid [[23], [24], [25]], chloroquine and hydroxychloroquine [9,[26], [27], [28]], camostat mesylate [29] and nitazoxanide [[30], [31], [32]]. However, the use of these drugs is frequently restricted as a result of toxic side-effects (such as headache, nausea, vomiting, decreased appetite, skin rash, fever, fatigue, osteoporosis, metabolic disorders, heart damage, leukocyte reduction leading to reversible anemia and so on), weak oral bioavailability and the occurrence of drug resistance [9,15,33,34].

Coronavirus disease 2019 (COVID-19) caused by a new emerging coronavirus, has been pronounced as a Public Health Emergency of International Concern (PHEIC) by WHO [35]. By early October 2020, more than 36,382,001 patients had been diagnosed with the disease worldwide, with 1,056,131 deaths. The number of infected and dead people is still increasing rapidly every day. However, according to the data collected by the National Health Commission of the People's Republic of China, clinical practice in common antiviral drugs such as oseltamivir, arbidol, and lopinavir/Ritonavir failed to cure these patients of this epidemic. Until now, no specific therapeutic strategies or medicine has been developed to cure the new emerging virus yet. Hence, screening possible anti-viral drug candidate attract more and more attentions.

Traditional Chinese medicine has been used in the prevention, treatment, and rehabilitation of virus for a long history and plays a critical role in the batting of COVID-19 [36]. It is worth noting that the main components of the “Lung Cleansing and Detoxifying Decoction” widely used in the prevention and treatment of COVID-19 includes edible and medicinal fungus (EMFs) (Poria cocos, Polyporus umbellatus) [36]. EMFs commonly known as mushrooms, are large fungi that can be employed to food and medicine and have fleshy, gelatinous, woody and leathery proton entities. In China, mushrooms that occur in wood are called “fungus” and “mushroom”, while those that emerge from soil are called “蕈”. In Japan, the two terms “蕈 and fungus” are combined as a synonym for mushroom. More than 2000 species of EMFs have been identified to date [37]. EMFs as a branch of traditional Chinese herbal medicine, also played a vital role in the prevention and control of virus for a long history, such as, this COVID-19 outbreak [36]. As early as the Eastern Han Dynasty (25–220 CE) period of Chinese history, the classic Chinese medicine book “Shennong's Herbal Classic (Shennong baicao jing)” contains records on the treatment of plagues with edible and medicinal fungus, such as Ganoderma lucidum (Lingzhi), Polyporus umbellatus (Zhu ling) and Omphalia lapidescens (Lei wan). In addition, the classic book of Chinese medicine “Compendium of Materia Medica (Bencao gangmu)” also contains records of the treatment of plague with edible and medicinal fungus. With in-depth research, the efficacy of EMFs polysaccharides in suppressing viruses has attracted the attention of many scholars (Fig. 1 ). Although the direct antiviral edible fungus polysaccharides were previously summarized by He et al. [38]. Here we add recent studies on the direct or indirect antiviral activity of medicinal fungus polysaccharides and more comprehensively summarize the structure, mechanism, and structure-activity relationships of EMFs polysaccharides with antiviral effects from a classification perspective so that people can be better utilization of them.

Fig. 1

Statistical analysis of the literature on the antiviral activity of edible and medicinal fungus polysaccharides. (a) Antiviral polysaccharides derived from EMFs related papers published between 2000– 2020 (based on Web of Science). (b) Antiviral EMFs polysaccharides effect related biological activity papers. (c) The categories of antiviral EMFs polysaccharides. (d) Antiviral EMFs polysaccharides investigate the relationship among immunomodulatory activity, anti-inflammatory activity, improve gut function, and antiviral activity.

Abbreviations: BHV (BoHV)-1: Bovine herpesvirus 1; BIDV (IBDV)-1: Infections bursal disease virus 1; EV 71: Human enterovirus 71; FCV: Feline calicivirus; H1N1: Influenza A virus; HBV: Viral hepatitis type B; HIV: Human immunodeficiency virus; HPV: Human papillomavirus; HSV: Herpes simplex virus; MDRV: Muscovy duck reovirus; NDV: Newcastle disease virus; PV-1: Poliovirus 1).

Types and structural characteristics of EMFs antiviral polysaccharides

Ever since polysaccharides were first found to have bioactivities and applied to clinical practice, many scholars have devoted research to polysaccharides from different sources. Also, exploration of EMFs polysaccharides and the development of related medicines has always been the research of hot topics. To date, the China Food and Drug Administration (CFDA) has approved some patent drugs, such as Coriolus versicolor polysaccharides capsules, Poria cocos polysaccharides oral liquid, Polyporus umbellatus polysaccharides capsules, Lentinula edodes polysaccharide injection, Lentinula edodes polysaccharides medicinal tablet and Lentinula edodes polysaccharides capsules (Table S1) which can be applied for the treatment of chronic hepatitis, and as adjuvant drugs help to regulate the immune system and to enhance the efficacy and thereby reducing the side effects of chemotherapeutic agents.

The classification of Table 1 is shown by the mushroom taxonomy “Subphylum, Class, Order, Family, Genus, Species” and alphabetical order according to Colored illustrations of mushrooms of China. So as Table S2. Specifically, it generally divides into two subphylum (Ascomycotina and Basidiomycotina). The reported antiviral EMFs and its polysaccharides over the past decade and a piece of comprehensive information (classification, molecular weight, monosaccharide composition, and antiviral-biological activity) have been shown in Fig. 2 and listed in Table 1, and the structures of common EMFs polysaccharides with direct or indirect antiviral effects was shown in Fig. 3 . Based on the available references, most of the antiviral EMFs polysaccharides source from Basidiomycotina Heterobasidiomycete EMFs, such as Holobasidiomycetidae Aphyllophorale (Cantharellaceae, Ganodermataceae, Hericiaceae, Polyporaceae, Steccherinaceae etc.) EMFs, and Phragmobasidiomycetidae Auriculariales (Auriculariaceae), Tremellaceae (Tremellaceae) EMFs. The number of Hymenomycetes Gasteromycetidae and Holobasidiomycetidae antiviral EMFs polysaccharides occupied second, specifically such as Phallales (Phallaceae), Agricales (Agaricaceae, Bolbitiaceae, Boletaceae, Pleurotaceae, Pluteaceae, Russulaceae, and Tricholomataceae) EMFs polysaccharides. Additionally, there is part of Ascomycotina Discomycetes and Pyrenomycetes EMFs polysaccharides with directly or indirectly antiviral activity, such as Pezizales (Morchellaceae), Clavicipitales (Clavicipitaceae), Sphaeriales (Hypocreaceae) and Xylariales (Xylariaceae) EMFs polysaccharides. Interestingly, only 15 EMFs polysaccharides (Cordyceps militaris, Ganoderma lucidum, Hericium erinaceus, Fomitiporia punctate, Grifola frondosa, Inonotus obliquus, Auricularia auricular, Tremella, Agaricus blazei, Lentinus edodes, Pleurotus abalonus, Pleurotus ostreatus, Pleurotus pulmonarius, Pleurotus tuber-regium, and Flammulina velutipes) could directly exhibit anti-virus effect but others just have antiviral effects through indirect antiviral-biological activity such as improving immunity, eliminating inflammation, and regulating intestinal function.

Table 1

The composition of monosaccharides, molecular weight, antiviral activity, and cytotoxicity of polysaccharides from the edible mushroom.

Classification	Polysaccharides name	Monosaccharide composition	MW (kDa)	Structural feature	Biological activity	Ref.
Ascomycotina Discomycetes Pezizales
Morchellaceae
Morchella conica	MCP	Only Man	81.2	→6)-α-D-Manp-(1 → 6)-[α-D-Manp-(1 → 6)-]n-α-D-Manp-(1→	Immunomodulatory activity	[39]
Morehella esculenta	MIPW50-1	GlcNAc:Gal:Glc:Man = 1.00:14.95:1.53:10.51	28.5	→2,3,6)-α-D-Manp-(1→,→3,6)-α-D-Manp-(1→,→2)-α-D-Galp-(1→,→6)-α-D-Manp-(1→, →4)-β-D-Glcp-(1→, →4)-α-D-GlcpNAc-(1→, →6)-α-D-Glcp-(1→, α-D-Galp-(1→, α-D-GlcpNAc-(1 → and β-DGlcp-(1→	Immunomodulatory activity	[40]
Ascomycotina Pyrenomycetes Clavicipitales
Clavicipitaceae
Cordyceps militaris	CMP-III	Glc:Man:Gal = 8.09:1.00:0.25	47,960	1 → 4)-α-D-Glcp-(1→, →4,6)-α-D-Manp-(1→, α-D-Manp-(→1 and →2,6)-α-D-Galp-(1→	Immunomodulatory activity	[41]
	SDQCP-1	Man:Glc:Gal = 13.3:1.0:9.7	19.3	Backbone composed of (1 → 2)-α-D-Manp and (1 → 4)-β-D-Glcp residues. Branched chains at O-6 position of (1 → 2)-α-D-Manp mainly by (1 → 2)-β-D-Galf or (1 → 6)-α-D-Manp residues	Immunomodulatory activity	[42]
Cordyceps sinensis	UM01 PS	Glc:Man:Gal = 100:59:17	610,86,25	(1 → 6)-α-D-glucosidic linkages	Immunomodulatory activity	[43]
Hirsutella sinensis	HS002-II	Man:Rib:Rha:GlcAc:GalAc:Glu:Gal:Xyl:Ara = 6.47:2.27:1.25:0.69:0.42:65.89:16.17:2.13:4.26	44	Backbone composed of (1 → 3)-α-D-Rib (1 → 4)-α-D-Xyl and (1 → 4)-β-D-Glc, which was substituted at C-6.The two branches were mainly composed of (1 → 4)-D-Galp, and terminated with D-Galp	Immunomodulatory activity	[44]
Hypocreaceae
Shiraia bambusicola	SB2-1	Glc:Gal:Man = 2.0:1.5:1.0	22.2	The Man core was composed of (1 → 2)-Manp as the main chain. Glucose with (1 → 4)-D-Glcp, (1 → 2)-D-Glcp and (1 → 6)-D-Glcp at different degrees of polymerization were linked at C-6 and C-3 of the (1 → 2)-Manp as the side chains.	Immunomodulatory activity	[45]
Basidiomycotina Heterobasidiomycetes Holobasidiomycetidae Aphyllophorales
Cantharellaceae
Cantharellus cibarius	WCCP-N-b	Gal:3-O-Me-D-Gal:Glc:Man = 14.4:4.6:1.0:1.2	18	A linear methylated galactan which was composed of α-(1 → 6)-α-D-Galp and 3-O-Me-D-Galp	Immunomodulatory activity	[46]
	CC-1	Glc:Xyl = 5:1	61,056	Backbone composed of (1 → 4)-β-D-Glcp which branched at O-6 and the branches were mainly composed of 6 → 1)-α-D-Xylp	Immunomodulatory activity	[47]
Craterellus cornucopioides	CCP	Man:Gal:Glc:Xyl = 48.73:17.37:15.97:17.93	1970	(1 → 3)-β-D-Manp-(1 → 6)-α-D-Galp backbone distributed by(1 → 4)-α-D-Xylp-t-α-D-Manp and t-β-D-Glup units at O-6.	Immunomodulatory activity	[48,49]
Ganodermataceae
Ganoderma atrum	PSG-1	Man:Gal:Glc = 1:1.28:4.91	1013	The main glycosidic linkage types composed of→3)-Glcp-(1→3)-Glcp-(1 → 3)-Glcp-(1 → , which was heavily substituted via (1 → 6) glucosidic bonds with -R, where -R would be one of these fragments: T-Glcp-(1-[→6)-Glcp-(1]n→, T-Glcp-(1 → 4)-GlcAp-(1→, or T-Glcp-(1 → 4)-GlcAp-(1-[→6)-Glcp-(1]n→	Immunomodulatory activity	[[50], [51], [52], [53], [54], [55], [56], [57]]
Ganoderma lucidum	GSG	ND	8	GSG is a branched glucan that contains (1 → 3)-linked, (1 → 3,6)-linked, (1 → 6)-linked and (1 → 4) linked d-glucopyranosyl residues of either β or α configuration.	Immunomodulatory activity	[58]
Ganoderma sinense	GSP-2	Only Glc	32	Backbone composed of (1 → 4)-and (1 → 6)-β-D-Glcp, bearing the side chains of (1 → 3)-and terminal β-D-Glcp at O-3 position of (1 → 6)-β-D-Glcp, as well as trace amounts of Gal and Man residues	Immunomodulatory activity	[59]
Hericiaceae
Hericium erinaceus	HEP-S	Rha:Fuc:Man:Glc:Gal = 1.47:0.93:1.36:8.68:4.08	18.3	The main glycosidic linkage types composed of (1→)-α-D-Glc, (1 → 3,4)-α-D-Glc, (1 → 6)-α-D-Gal, (1 → 3,4)-β-D-Man, (1 → 3,6)-α-Rha and (1 → 2)-β-L-Fuc	Immunomodulatory activity	[60]
	HEP-W	Rha:Fuc:Man:Glc:Gal = 0.98:1.59:0.89:5.60:7.06	15.9	The main glycosidic linkage types consisted of (1→)-α-D-Glc, (1 → 3,6)-α-D-Glc, (1 → 2,6)-α-D-Gal, T-β-Gal, (1 → 3,4)-β-D-Man, (1 → 3)-α-Rha, and (1 → 2)-β-L-Fuc	Immunomodulatory activity	[61]
Polyporaceae
Bjerkandera fumosa	DBFM3	Man:GlcAc:Gal = 1:18.16:0.702	180	(1 → 6), (1 → 3,6), (1 → 3) linkages and pyranose conformation	Immunomodulatory activity	[62]
Cryptoporus volvatus	PRA-1	2,3,4,6-Me4-Glc:2,4,6-Me3-Glc:2,4-Me2-Glc = 5.72:1.00:2.41	24,190;109.6;4.12	Hyperbranched (1 → 3), (1 → 6)-β-D-glucan with a degree of branching of 0.89, backbone composed of →1)-D-Glcp-(3 → linkages and side chains having the major branching points at O-6 positions	Immunomodulatory activity	[63]
Fomitiporia punctate	G1	Ara:Fru:Gal:Glc = 1.6:3.8:19.7:19.7	151	Furanoid rings; β-glycosidic bonds between the sugar units	Anti-HIV activity	[64]
Grifola frondosa	GF-P	Fuc:Gal:Glc:Man:Rib:GlcAc = 5.8:10.5:72.2:7.8:2.2:1.2	19.6;722.7	Contained the highest amount of (1 → 3,6)-β-D-glucans.The degree of branching (0.38) of (1 → 3,6)-β-D-glucans	Immunomodulatory activity	[65]
	GFP1	Glc:Fuc = 2.3:0.5	40.5	(1 → 6)-β-D-Glcp backbone with a single (1 → 3)-α-D-Fucp side-branching unit	Anti-EV 71 activity	[66]
Inonotus obliquus	IOI-WN	Ara:Rha:Fuc:Xyl:Man:Gal:Glc:GlcAc:GalAc:3-O-methyl Gal = 1.4:1.3:1.7:6.9:6.8:17.5:53.1:1.0:1.8:8.7	60	(1) The neutral polysaccharides were heterogeneous and branched and consisted of a (1 → 3)-linked β-Glc backbone with (1 → 6)-linked kinks in the chain at approximately every fifth residue, with branches of (1 → 6)-linked β-Glc in addition to substantial amounts of (1 → 6)-linked α-Gal with 3-O-methylation at about every third Gal residue.	Immunomodulatory activity	[67]
	IOI-WAC	Ara:Rha:Fuc:Xyl:Man:Gal:Glc:GlcAc:GalAc:3-O-methyl Gal = 2.1:5.1:1.3:8.8:8.2:11.2:37.9:5.1:15.7:4.6	15
Phellinus igniarius	P. igniarius polysaccharides	Rha:Man:Ara:Gal:Xyl:Glc = 1.31:14.51:2.63:20.65:3.32:57.58	18.518	The glycosidic linkages were mostly 1 → 3, 1 → 6 or 1 → 3,6. main chain of →3)-β-D-Glcp-(1 → with →6)-β-D-Glcp-(1 → side chain	Immunomodulatory activity	[68]
Phellinus pini.	PPE	Man:Gal:Glc = 38.40:1.00:1.76	38	Backbone of (1 → 2)-linked Man, which was heavily substituted via (1 → 6) glucosidic bonds with (1 → 3)-linked Man, and terminated mainly with Man, as well as a small amount of Gal and Glc.	Immunomodulatory activity	[69]
Polyporus umbellatus	ZPS	>90% Glc	227	(1 → 6, 1 → 4)-linked β-D-Glcp backbone, substituted at O-3 position of (1 → 6)-linked β-D-Glcp by (1 → 3) linked β-D-Glcp branches	Immunomodulatory activity	[70]
Steccherinaceae
Sarcodon aspratus	HBP	Only Glc	430	Backbone of (1 → 6)-β-D-Glcp, which occasionally branched at O-3 position on along the backbone and substituted by the side chains that consisting of (1 → 3)-β-D-Glcp, (1 → 4) linked-β-D-Glcp and non-reducing end β-D-Glcp	Immunomodulatory activity	[71]
Basidiomycotina Heterobasidiomycetes Phragmobasidiomycetidae Auriculariales
Auriculariaceae
Auricularia auricular	CEPSN-1	Glc:Man:Gal:GlcAc = 98.90:0.11:0.38:0.61	4.6	Backbone chain composed of (1 → 4)-α-D-Glcp in glucopyranose type	Immunomodulatory activity	[72]
	CEPSN-2	Glc:Man:Gal:Ara:Fuc:GluAc:GalAc = 97.56:0.14:1.04:0.11:0.45:0.50:0.20	6.7
Basidiomycotina Heterobasidiomycetes Phragmobasidiomycetidae Tremellaceae
Tremellaceae
Tremella	TP	Man:Xyl:GlcAc = 57.50:32.09:10.14	1500	Backbone was composed of (1 → 3)-β-D-Manp, and the side chain composed of (1 → 6)-β-D-Xylp was attached to the C2 of the backbone Manp	Immunomodulatory activity	[73]
Tremella aurantialba	TAP-3	Man:Xyl:GlcAc = 3.0:1.0: 1.0.	624	Backbone was composed of (1 → 3) and (1 → 2)-α-Manp, side chains formed by β-Xylp and β-GlcpA linked to the C-2 position of α-Manp, and acetyl groups connected to the sixth hydroxyl positions of Manp	Immunomodulatory activity	[74]
Basidiomycotina Hymenomycetes Gasteromycetidae Phallales
Phallaceae
Dictyophora indusiata	DIP	Man:Rib:Rha:GlcAc:Glc:Gal:Xyl:Ara:Fuc = 23.55:0.46:0.043:1.014:59.84:12.95:0.36:0.17:1.58	650	β-(1 → 3)-D-Glc with side branches of β-(1 → 6)-Glc units	Immunomodulatory activity	[75]
Basidiomycotina Hymenomycetes Holobasidiomycetidae Agricales
Agaricaceae
Agaricus bisporus	ABS	Glc:Man:Gal:Gal-Me:Fuc:Ribe = 51.4:6.8:33.9:3.0:1.6:3.3	ND	(1 → 6), (1 → 4)-α-Glc, (1 → 6)-β-Glc, and mannogalactan	Anti-inflammatory activity	[76]
Agaricus blazei	FR-S	ND	3.5	(1 → 6)-(1 → 3)-β-D-glucan	Anti-HSV activity	[77]
Bolbitiaceae
Agrocybe aegerita	AAPS	Rha:Fuc:Man:Glc = 2.90:10.25:3.70:38.27	18.1	α-LRhap-(1 → 6)-β-D-Glcp-(1 → 2)-α-L-Fucp-(1 → 6)-α-D-Glcp-(1 → 5)-α-L-Araf-(1 → 4)-β-D-GlcpA-(1 → 5)-α-L-Araf-(1 → 6)-α-D-Manp-(1 → 6)-α-D-Manp-(1 → 2)-α-L-Fucp-(1 → 6)-β-D-Glap-(1 → 2)-α-L-Rhap-(1 → 6)-β-D-Galp-(1→, which linked with two side chains α-L-Fucp-(1 → 6)-β-D-Glcp-(1 → 6)-β-D-Manp-(1 → and α-D-Xylp-(1 → 2)-α-L-Fucp-(1 → 5)-α-D-Araf-(1 → 6)-β-D-Galp-(1 → at OH2 at H-4-Ara and the terminal Galp residues, respectively.	Immunomodulatory activity	[78]
Boletaceae
Boletus speciosus	BSF-X	Glc:Gal = 2:1	141.309	Backbone of 1, 4-β-D-Glc of which branches were mainly composed of two 1, 6-α-D-Gal residue and a 4-β-D-Glc at the end of the branches	Immunomodulatory activity	[79]
	BEP	Glc:Gal:Rha:Ara = 2.9:3.2:1.3:1.6	113.432	Backbone consisting of (1 → 6)-linked-α-D-Glcp, (1 → 2,6)-α-D-Galp, (1 → 6)-α-D-Galp, and (1 → 3)-α-D-Rhap residues, which were branched at O-2 position of (1 → 2,6)-α-D-Galp residue with a single terminal (1→)-α-L-Araf residue	Immunomodulatory activity	[80]
Leccinum rugosiceps	LRP-1	Gal:Rha:Fuc:Man:Glc:Gala = 25.77:1.98:4.99:27.45:39.13:0.68	18.82	Backbone mainly composed of →1-α-D-Fucp-3→, →1-α-D-Rhap-3→, →1-β-D-Glcp-6→, →1-β-D-Glcp-3→, →1-α-D-Glcp-4→, →1-β-D-Galp-6→ and →1-α-D-Manp-6→.	Immunomodulatory activity	[81]
Pleurotaceae
Lentinus edodes	RPS	ND	3.08	→3)-L-Rhap-(1→, →6)-D-Glcp(1→, →3,6)-D-Manp-(1→, →3)-L-Arap-(1→, D-Manp-(1→, and →6)-D-Galp-(1→,	Anti-inflammatory activity	[82]
	ERPS	ND	1.16	→3)-L-Rhap-(1→, →6)-D-Glcp-(1→, →3)-D-Manp-(1→, →6)-D-Galp-(1→, and L-Arap-(1→
Pleurotus abalonus	LA	Glc:Rha:GlcA:Xyl:Gal = 26.3:2.7:1.0:1.4:1.8:1.2	120	[→6)-α-D-Glcp(1→]n	Anti-HIV activity	[83]
Pleurotus citrinipileatus	PCPS	1, 5, 6-Tri-O-acetyl-2, 3, 4-tri-O-methyl glucitol:1, 3, 5, 6-tetra-O-acetyl-2, 4-di-O-methyl glucitol = 2:1	450	Backbone mainly composed of β-(1 → 6)-Glcp withβ-(1 → 6)-Glcp residues branched	Immunomodulatory activity	[84,85]
Pleurotus eryngii	WPEP-N-b	Gal:Man:3-O-Me-D-Gal:Glc = 43.8:39.3: 11.7:9.20	21.4	Backbone mainly composed of α-(1 → 6)-D-Galp and 3-O-Me-D-Galp, branched at O-2 with single t-β-D-Manp, and β-(1 → 6)-D-Glcp residues are present as minor components either in side-chains or backbone	Immunomodulatory activity	[86]
	EPA-1	Man:Glc:Gal = 2.20:1.00:3.20	99.7	Backbone mainly composed of (1 → 6)-Gal residue	Immunomodulatory activity	[87]
Pleurotus florida	PfloVv5FB	1,5-Di-O-acetyl-2,3,4,6-tetra-Omethyl-D-glucitol:1,5,6-tri-O-acetyl-2,3,4-tri-O-methyl-D-glucitol:1,3,5,6-tetra-O-acetyl-2,4-di-O-methyl-D-glucitol = 1:2:1	187	Repeating unit of the polysaccharide has a backbone of three (1 → 6)-linked β-D-Glcp units, where one of them is substituted at O-3 position with the β-D-Glcp moiety	Immunomodulatory activity	[88]
Pleurotus ostreatus	Heteropolysaccharide	Glc:Gal = 7:1	187	Backbone mainly composed of →3)-β-D-Glcp-(1→6)-β-D-Glcp-(1 → 6)-β-D-Glcp-(1 → 3)-β-D-Glcp-(1 → 6)-β-D-Glcp-(1 → 6)-β-Glcp-(1 → The branches consisted of (1 → 3)-α-D-Galp, and (1 → 3)-α-D-Glcp	Immunomodulatory activity	[89]
Pleurotus pulmonarius	β-Glucan	Xyl:Man:Gal:Glc = 2:5:3:90	ND	(1 → 3)β-D-Glcp main chain substituted at O-6 of every third unit by single β-D-Glcp nonreducing end units	Anti-inflammatory activity	[90]
	PEIsR	Man:Gal:3-Omethyl-Gal =37.0:39.7:23.3	64	Main chain of (1 → 6)-α-D-Galp and 3-O-methyl-α-D-Galp units. Some of the α-D-Galp units were substituted at O-2 by non-reducing end units of β-D-Manp	Anti-inflammatory activity	[91]
Pluteaceae
Volvariella volvacea	VGPI-a	Only Glc	1435.6	(1 → 4)-D-Glcp backbone with the substitution at C-6 with 1-D-Glcp residue	Immunomodulatory activity	[92]
Tricholomataceae
Armillaria mellea	AMPS-1-1	Glc:Gal:GlcA = 89.06:9.59:1.34	123	(1→)-β-D-Glcp, (1 → 3,6)-α-D-Glcp and (1 → 3)-β-D-Glcp residues	Immunomodulatory activity	[93]
	AMPS-2-1	Glc:Gal:GlcA:Man = 65.28:22.87:2.87:8.98	676	(1 → 3,6)-α-D-Glcp and (1 → 6)-β-D-Glcp residues
Flammulina velutipes	FVP2	Gal:Glc:Man = 19.96:60.66:19.38	18.3	(1 → 3)-β-D-Gal, (1 → 6)-β-D-Gal, (1 → 6)-α-D-Glc and (1 → 3,6)-α-D-Man	Improves gut microbiota function	[94]
	FVPB2	Gal:Man:Fuc:Glc = 1.9:1.2:1:2.5	15	Backbone mainly composed of →2)-α-D-Galp-(1 → 4)-α-D-Galp-(1 → 6)-α-D-Glcp-(1 → 3)-β-D-Glcp-(1→ The branches consisted of (1 → 3)-α-D-Manp, and (1 → 6)-α-L-Fucp	Immunomodulatory activity	[95]
Tricholoma crassum	PS	Glc:Gal:Man = 3:1:1	200	Backbone mainly composed of →6)-β-D-Glcp-(1→6)-α-D-Glcp-(1 → 6)-α-D-Glcp-(1 → The branches consisted of (1 → 3)-α-D-Galp-(1 → 4)-β-D-Manp	Immunomodulatory activity	[96]

Abbreviations: glucose (Glc), xylose (Xyl), rhamnose (Rha), mannose (Man), fucose (Fuc), fructose (Fru), galactose (Gal), arabinose (Ara), ribose (Rib), glucuronic acid (GlcA), and galacturonic acid (GalA).

Due to table typesetting, the classification of “subphyla, class, order” in the table and Table S2 is presented in the form of “no indentation”, “family” is indented by 1 character, “genus and species” is indented by 2 characters.

Fig. 2

Common EMFs with direct or indirect antiviral effects.

Fig. 3

Structures of common EMFs polysaccharides with direct or indirect antiviral effects. (a) Proposed structure of MIPW50–1 (Morchella importuna polysaccharide); (b) Putative structure of HBP (Sarcodon aspratus (Berk.) polysaccharide); (c) NOESY and HMBC correlations of TAP-3 (Tremella aurantialba Bandoni et Zang glucuronoxylomannan); (d) The structure of polysaccharide BSF-X (Boletus speciosus Frost polysaccharide); (e) Proposed structure of the polysaccharides from the fruiting bodies of G. sinense; (f) The proposed main structure of the galactoglucomannan SB1–1 (Shiraia bambusicola polysaccharide); (g) One possible structure of Pleurotus eryngii polysaccharide (EPA-1) (m = 5, n = 2).

In Tables 1 and S2, most of the polysaccharides are heteropolysaccharides consisting of 2 or more kinds of monosaccharides like glucose (Glc), xylose (Xyl), rhamnose (Rha), mannose (Man), fucose (Fuc), fructose (Fru), galactose (Gal), arabinose (Ara), ribose (Rib), glucuronic acid (GlcA), and galacturonic acid (GalA). It is worth noting that Morchella conica polysaccharides (MCP) and Agaricus bisporus polysaccharides (TJ3) consist only of Man. At the meantime, their polysaccharide structure is made up of Man repeating units linked by α (1 → 6) glycosidic bonds. Sarcodon aspratus polysaccharides (HBP) consist only of Glc, and backbone of (1 → 6)-β-D-Glcp, which occasionally branched at O-3 position on along the backbone and substituted by the side chains that consisting of (1 → 3)-β-D-Glcp, (1 → 4) linked-β-D-Glcp and non-reducing end β-D-Glcp. Volvariella volvacea polysaccharides (VGPI-a, PS) consist only of Glc, and backbone of (1 → 4)-D-Glcp with the substitution at C-6 with 1-D-Glcp residue. These EMFs polysaccharides composed of a single monosaccharide do not appear to have a paradigm that can be systematically summarized in their structure. However, there are many studies on EMPs polysaccharides that do not characterize the composition of monosaccharides and molecular weight but only explain the biological activity. Next, we will try to summarize the structure-activity relationships of these EMFs polysaccharides from the perspective of the same classification.

In Ascomycotina Discomycetes Pezizales Morchellaceae EMPs polysaccharides, Mw is substantially in the range in the range of 6.9–192 kDa [39,40,[97], [98], [99], [100], [101], [102], [103], [104], [105]]. Based on Tables 1 and S2, there is no directly antiviral ability in Morchellaceae polysaccharides, and more is the ability to increase the immunity of indirect anti-virus. Besides that, there are some anti-inflammatory polysaccharides in Morehella esculenta, such as EMP-1, SEMP-1, PEMP, Ac-PMEP1-3 [97,99], which may be related to the sulfate and acetylation of morel polysaccharides.

In Ascomycotina Pyrenomycetes Clavicipitales Clavicipitaceae EMPs polysaccharides, Mw is approximately in the range of 20.2–853.8 kDa [41,43,44,[106], [107], [108], [109], [110], [111], [112], [113], [114], [115], [116], [117], [118]]; expect a special high-molecular-weight (47,960 kDa) polysaccharide from Cordyceps militaris, CMP-III, and predicted structural feature is 1 → 4)-α-D-Glcp-(1→, →4,6)-α-D-Manp-(1→, α-D-Manp-(→1 and →2,6)-α-D-Galp-(1→ [42]. In addition to the anti-NDV and anti-H1N1 activity of Cordyceps militaris polysaccharides [111,117] (CMP40, 50, and APS respectively). Unfortunately, no research has been done to analyze the structure of these three polysaccharides. Other Clavicipitaceae polysaccharides are indirectly anti-virus by improving immunity. In Basidiomycotina Heterobasidiomycetes Holobasidiomycetidae Aphyllophorales, there are few antiviral-biological activity studies on the Cantharellaceae polysaccharides (Tables 1 and S2), and most of these polysaccharides could improve the immune function, to achieve an indirect anti-virus effect, but their molecular weights vary enormously [[46], [47], [48], [49],119]. The smallest molecular weight is WCCP-N-b isolated and purified from Cantharellus cibarius, only 18 kDa, and a linear methylated galactan which was composed of α-(1 → 6)-α-D-Galp. CC-1 also isolated from Cantharellus cibarius has the highest molecular weight of 61,056 kDa, and the backbone composed of (1 → 4)-β-D-Glcp which branched at O-6 and the branches were mainly composed of 6 → 1)-α-D-Xylp. Ganodermataceae (Ganoderma atrum, Ganoderma lucidum, and Ganoderma sinense) is a kind of rare EMFs, in recent years, many scholars on the composition and function of its polysaccharides (Tables 1 and S2). As a result of the difference between the different varieties, their Mw (from 8 to 1860) also makes a significant difference [[50], [51], [52], [53], [54], [55], [56], [57], [58], [59],65,[120], [121], [122], [123], [124], [125], [126], [127], [128], [129], [130]]. Among these polysaccharides from Ganodermataceae, except for APBP (Ganoderma lucidum), which has anti-HSV properties [130], but the precise structure of the polysaccharide is unclear. Other polysaccharides reach indirectly by boosting the immunity of anti-viral capabilities. A polysaccharide (PSG-1) was isolated and purified from Ganoderma atrum, and the main glycosidic linkage types composed of →3)-Glcp-(1→3)-Glcp-(1 → 3)-Glcp-(1 → , which was heavily substituted via (1 → 6) glucosidic bonds with -R, where -R would be one of these fragments: T-Glcp-(1-[→6)-Glcp-(1]n→, T-Glcp-(1 → 4)-GlcAp-(1→, or T-Glcp-(1 → 4)-GlcAp-(1-[→6)-Glcp-(1]n→. GSG isolated from Ganoderma lucidum is a branched glucan that contains (1 → 3)-linked, (1 → 3,6)-linked, (1 → 6)-linked and (1 → 4) linked d-glucopyranosyl residues of either β or α configuration. In Hericiaceae Hericium erinaceus polysaccharides, Mw are approximately in the range of 16.15–46 kDa [60,61,[131], [132], [133], [134], [135], [136], [137], [138]], and it's worth noting that HEP could directly anti-MDRV, composed of Glc:Gal:Man:Ara (51.02:42.24:4.5:2.2), molecular weight of 16.18 kDa [132,133]. As showed in Tables 1 and S2, most of the EMFs polysaccharides with antiviral-biological activity are collected in Polyporaceae polysaccharides. For instance, Fomitiporia punctate polysaccharide (G1) could be anti-HIV [64], with furanoid rings and β-glycosidic bonds between the sugar units; Grifola frondosa polysaccharide (GFP1) could anti-EV 71 [66], which (1 → 6)-β-D-Glcp backbone with a single (1 → 3)-α-D-Fucp side-branching unit; and Inonotus obliquus polysaccharide (IOPs) could anti-FCV [139], unclear structure. Therefore, we can try to speculate that β-glycosidic bonds play a critical role in the antiviral efficacy of EMFs polysaccharides. In addition, other Polyporaceae EMFs polysaccharides often through the enhancement of immune or anti-inflammatory effect, and achieve tacit anti-virus effect. In Basidiomycotina Heterobasidiomycetes Phragmobasidiomycetidae Auriculariales polysaccharides, interestingly, the polysaccharides (AAPt, AAP1, and AAP2) and sulfate derivatives (sAAPt, sAAP1, and sAAP2) of the widely consumed Auricularia auricular have anti-NDV activity [140]. In Tremellaceae (Tremella and Tremella aurantialba) polysaccharides, Mw is approximately in the range of 4–1500 kDa [73,74,[141], [142], [143], [144], [145], [146], [147], [148], [149], [150], [151], [152]]. Among these polysaccharides from Tremellaceae, only Tremella polysaccharides (sTPStp and sTPS70c) could anti-NDV [149]. Other polysaccharides reach indirectly by improving the immunity of anti-viral properties. The Basidiomycotina Hymenomycetes Holobasidiomycetidae Agricales as another important classification of EMFs polysaccharides against viruses, most of antiviral-biological activities of EMFs polysaccharides are gathered here (Tables 1 and S2). Taking several typical EMFs as examples, Agaricus blazei polysaccharides (FR-S, MI-S, PLS, SPLS), have anti-HSV activity with (1 → 6)-(1 → 3)-β-D-glucan and the sulfate group generally occurs at the C4 of configuration β (1 → 6) and preferably at the C6 of configuration α (1 → 4) in F3; PLS isolated from Agaricus blazei have anti-PV-1 activity and PLS, β-glucan also derived from Agaricus blazei could anti-BoHV-1 with β (1 → 6)-D-Glc with branches made of β (1 → 3)-D-Glc [77,[153], [154], [155], [156]]. On the other hand, Lentinus edodes polysaccharides, PLWM and LEP (structure unknown), could be anti-HBV and PV-1 respectively [157,158]; Efs (structure unclear) derived from Pleurotus ostreatus could anti-BIDV [159]; Pleurotus pulmonarius polysaccharides (PBG, structure unknown), also have anti-HPV activity [160]; Flammulina velutipes polysaccharides (FVP1, structure unknown) could also anti-HBV [120]. It is important to note that Pleurotus tuber-regium polysaccharides S-TM8-1–6 (structure unknown) have the ability of anti-HSV and Flu A [161]. Therefore, it can be speculated that the polysaccharides of EMFs after chemical modification (sulfation, phosphorylation, and methylation modification) may have better antiviral effects and the antiviral ability of chemical modified polysaccharides increases with the degree of modification within an optimal scope. Besides the EMFs polysaccharides described above, other polysaccharides of Agricales often fight viruses indirectly by enhancing immunity, anti-inflammatory activity and modulating the active function of the gut microbiota.

Unfortunately, we were unable to derive a specific paradigm to summarize the antiviral activity from the structure of EMFs polysaccharides owing to the relatively large differences in the classes of EMFs. Therefore, more studies on the Structure-activity relationships of the polysaccharides of EMFs are still needed (Fig. 3).

Antiviral mechanisms of EMFs polysaccharides

It is well known that binding to cell surface through electrostatic interactions is the first step for a virus to invade a cell, while unstable reversible binding grows into stable irreversible adsorption to accomplish the subsequent invasion process (Nigel Dimmock et al., 2006). Numerous studies have demonstrated that the sulfate polysaccharides could interfere with the viral adsorption process on cells by blocking the positive charge of the cell surface through the negative charge of the sulfate group [162]. For example, HIV-1conserved domains (gp120), defined as an envelope glycoprotein mediates virus attachment and entry, bind to CD4+ T cells. On the other hand, different variable domains (in particular V3), as the third variable part of the HIV-1 gp120, could bind to the chemokine co-receptor [163]. These interactions described above clarify the mechanisms of virus attachment, uptake, intracellular transport, and eventual penetration into the cytoplasm [1]. In the past decades, natural EMFs polysaccharides exhibit antiviral activity through four core strategies (Fig. 4 ): suppressing virus function, improving immunomodulatory activity, regulating anti-inflammatory activity, and population balance of gut microbiota.

Fig. 4

Review on Antiviral mechanisms of EMFs polysaccharides.

Suppress virus function

There is a relatively small body of literatures that is devoted to direct antiviral activity from EMFs polysaccharides. EMFs polysaccharides are structurally diverse and heterogeneous and the antiviral activities of these polysaccharides are also determined by their structure, especially their advanced structures (Tables 1 and S2). Also, there is a growing body of literatures that recognize the sulfate EMFs polysaccharides often have better antiviral activity. Although the S-TM8 fractions were inactive in inhibiting virus replication for Flu A, they showed a different degree of inhibitory effect toward the three enveloped viruses [161]. Besides, S-TM8s showed relatively higher anti-HSV-1 and HSV-2 activity, which IC50 values were 10-fold lower than those for RSV [161]. In 2004, a seminal article was issued entitled evaluation of sulfate fungal β-glucans of Pleurotus tuber-regium as a potential water-soluble anti-viral agent, which had a major impact on the study of the antiviral activity of sulfate EMFs polysaccharides, the anti-virus (HSV-1, HSV-2, RSV and Flu A) activity of sulfate Pleurotus tuber-regium polysaccharides (S-TM8-1 to S-TM8-6) were evaluated [161]. In a study conducted by Gomes and colleagues, it was shown that sulfate derivative Agaricus blazei polysaccharides (MI-S) demonstrated a potential inhibitory activity against HSV-1 [KOS and 29R (acyclovir-resistant) strains] and HSV-2 strain 333, with selectivity indices (SI=CC50/IC50) higher than 439, 208, and 562, respectively [154]. However, in a follow-up study, Gomes et al. also found that FR-S, sulfate derivative also derived from Agaricus blazei polysaccharides, presented no in vitro antiherpetic action at 1 mg/mL, FR-S displayed potential anti-HSV-1 and anti-HSV-2 activities in both simultaneous and post-infection treatments, resulting in SI higher than 393. The reduction of viral adsorption upon cell pretreatment with FR-S also suggested its interaction with cellular components. FR-S inhibited HSV-1 (EC50 = 8.39 μg/mL) and HSV-2 (EC50 = 2.86 μg/mL) penetration more efficiently than heparin [77]. Similarly, Zhao and his team reported that sulfate Tremella polysaccharides (sTPStp and sTPS70c) could be substantially anti-NDV activity. And the antiviral activity in discrete processing stages of polysaccharide has been tested in their research [149]. As a pre-adding polysaccharide, the virus inhibitory rate of sTPStp (1.563 μg/mL) was the highest (97.1%) and considerably higher than those of the other four groups. Next was sTPS70c group (85.99%) and significantly higher than the non-sulfated TPStp and TPS70c groups. As simultaneously adding polysaccharide and NDV after mixed, the virus inhibitory rate of sTPStp (1.563 μg/mL) group was the highest (94.42%) and significantly higher than those of the other four groups (0.0782 μg/mL sTPS70c, TPStp, TPS70c, and TPStc), and the following were 0.782 μg/mL TPStp (81.55%) group and significantly higher than those of the other three groups. As a post-adding polysaccharide, the virus inhibitory rate of sTPStp (1.563 μg/mL) group was the highest (83.14%) and the following was 6.25 μg/mL sTPS70c (73.26%), which were significantly higher in comparison with other three non-sulfated polysaccharide groups (1.563 μg/mL TPStp, 3.907 μg/mL TPS70c and TPStc) [149]. To determine the effects of sulfate on the antiviral activity of polysaccharides, in a follow-up study, Hu and colleagues compared the anti-NDV activity of Auricularia auricula polysaccharides (AAPt, AAP1, and AAP2) and sulfate polysaccharides (sAAPt, sAAP1, and sAAP2) [140]. As pre-adding polysaccharide, the virus inhibitory rate in sAAP1 group was the highest (43.38%), and the following was the AAP1 (36.71%), sAAPt (35.40%), sAAP2 (24.60%), AAP2 (22.90%) and AAPt (16.78%), respectively. Nguyen [140] argued that the virus inhibitory rate of sAAP1 group was the highest and the following was sAAPt group in post-adding polysaccharide, in which two groups were significantly greater than that of AAP2 group. As simultaneous-adding polysaccharide and NDV, the virus inhibitory rate in sAAP1 and sAAPt group was the highest, 70.90%, and 65.75%, respectively, which was markedly higher than that in APPs groups. The following was sAAP2 (51.95%) which was also significantly higher than the non-sulfate group, AAP2 (24.74%).

In addition to the above sulfate EMFs polysaccharides, CMP40 derived from Cordyceps militaris was found to significantly improve the immune activity of NDV vaccination, which could be as the candidate of a new-type immune adjuvant [111]. The Cordyceps militaris polysaccharide (APS) also was reported to anti-H1N1 [117]. In a study investigating Ganoderma lucidum polysaccharide (APBP) by Oh et al., it was shown that combinations of APBP with acyclovir (ACV) on HSV-1 and HSV-2 were measured potent synergistic effects [130]. A recent study by Wu and colleagues' involved Hericium erinaceus polysaccharide (HEP) on the lymphocyte homing in Muscovy duck reovirus (MDRV) infected ducklings [132,133]. Results showed that HEP dramatically improved intestinal morphological structure and related indexes, and significantly inhibited the reduction of colonic mucosal IELs, goblet cells, and mast cells caused by MDRV infection [132,133]. Furthermore, Fomitiporia punctate polysaccharide (G1) [64] and Pleurotus abalonus polysaccharide (LA) [83] could inhibit HIV activity.

Immunomodulatory activity

Since the outbreak of COVID-19, there has been recognized that improved immunity plays an indispensable role in the prevention and elimination of the virus, as well as disease recovery [[165], [166]]. A growing number of researches are also continuing to explore effective immune boosting foods in people's daily diets to achieve indirect anti-virals effect. It is universally known that viruses Cells are non-cellular microorganisms that usually need to be parasitized in living cells to reproduce, and cellular immunity plays an important part in obliterating viral infections [[166], [167], [168]]. From the traditional perspective of viral infections and individual immune responses, immunomodulatory drugs have attracted attention for their ability to rapidly enhance cellular immunity and help fight viral infections. Immune cells including macrophages, neutrophils, monocytes, lymphocytes, and NK cells are the primary targets of coupling between polysaccharides of activating or stimulating immunization and specific proteins, which can directly or indirectly enter into dialogue with the host immune system, initiate a series of molecular interactions, lead to the activation of the immune system [165,166,169]. Immunomodulatory activity is one of the most expressive abilities of EMFs polysaccharides. Pharmacological tests showed that EMFs enhanced humoral immunity and nonspecific (or innate) immunity even at low doses. The immunomodulatory activity of EMFs polysaccharide is considered a critical basis for the antiviral effects mentioned above. The immunoreactivity of separate families (Morchellaceae, Clavicipitaceae, Cantharellaceae, Ganodermataceae, Hericiaceae, Polyporaceae, Tremellaceae, Pleurotaceae, Pluteaceae, Russulaceae, and Tricholomataceae) of EMFs polysaccharides will be described in the following section.

Morchellaceae (Morchella conica and Morehella esculenta) polysaccharides

In vivo and in vitro experiments during the past decades have elucidated some mechanisms by which the MCP, EPMC, and IPMC derived from Morchella conica were found to significantly improve the immune activity though modulating NO production, stimulating splenocyte proliferation, and inducible nitric oxide synthase (iNOS) expression in RAW264.7 cells [39,103]. Meng et al. illustrated that a novel polysaccharide (MSP-II) extracted from Morehella esculenta could stimulate NO production, proliferation, and phagocytosis of RAW 264.7 [98]. Moreover, Yao et al. discovered that a polysaccharide fraction (MIPW50-1) from Morehella esculenta could significantly improve NO production, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and phagocytosis of RAW264.7, rising immunomodulatory activity through the TLR4/JNK and Akt/NF-κB signaling pathways in RAW264.7 cells [40]. Additionally, MIPW50-1, MEEP, MCP, MEP-SP2 and MEP-SP3 derived from Morehella esculenta were capable of stimulating macrophage function, NO production, and the proliferation of T and B lymphocytes [39,40,102,105]. However, interestingly, Cui and colleagues reported that Morehella esculenta polysaccharide just selectively activated T cells and macrophages, but not B cells [104].

Clavicipitaceae (Cordyceps militaris, Cordyceps sinensis, and Hirsutella sinensis) polysaccharides

Most recently, He et al. discovered that a polysaccharide (CMP-III) from Cordyceps militaris could significantly promote macrophage phagocytosis and secretion of NO, TNF-α and IL-6, which involved MAPKs and NF-κB signaling pathways [41]. Similarly, other studies indicated that PLCM could enhance NO production, ROS, TNF-α, and RAW264.7 macrophages phagocytic though activated MAPK and NF-κB. Meanwhile, antibodies specific for the extracellular domain of TLR2, TLR4, or the macrophage receptor Dectin-1 significantly attenuated PLCM-induced secretion of TNF-α [109]. In addition, several other polysaccharides (CMP—W1, CMP—S1, CP2c2-S2, CM, and CPMN Fr III) isolated from Cordyceps militaris also can enhance immunity and thus indirectly play an anti-viral role [106,107,113,114].Meng et al. found a novel Cordyceps sinensis polysaccharides UM01 PS, which promoted mouse RAW 264.7 macrophages proliferation, phagocytic activity, the release of NO and multiple cytokines and chemokines. UM01 PS not only induced differentiation of RAW 264.7 macrophages into dendritic-like cells but also promoted phenotypic and functional maturation of mouse JAWS II dendritic cells [43]. Besides, Cheung and colleagues found UST 2000 could induce cell proliferation and the secretion of IL-2, IL-6 and IL-8. In addition, the phosphorylation of extracellular signal-regulated kinases (ERK) was induced transiently by the treatment of cordysinocan [116]. Additional in vitro studies further illuminated HS002-II derived from Hirsutella sinensis showed that the NO, TNF-α, IL-1β, and NF-κB using murine macrophages cell line (RAW264.7), which exhibited significant immunomodulatory activity by stimulating the IκB-NF-κB pathway [44].

Cantharellaceae (Cantharellus cibarius and Craterellus cornucopioides) polysaccharides

Yang et al. reported a polysaccharide (WCCP-N-b) of Cantharellus cibarius could significantly increase macrophage phagocytosis, the release of NO and secretion of TNF-α, IL-6, and IL-1β [46]. On a cellular mechanistic level, WCCP-N-b activated MAPKs and NF-κB signaling pathways via TLR2. CC-1 and JBP-1 were found that could encourage the proliferation of B cells and T cells [47,119]. Guo and colleagues found a novel Craterellus cornucopioides polysaccharides CCP [48,49], it was found that the BALB/c mice models in the preventive groups treated with CCP had better immunoregulatory activity by spleen and thymus weight indices evaluation and histopathological analysis, indicating a protective function of CCP against the immunosuppression induced by cyclophosphamide (CTX). Moreover, CCP displayed definite and positive synergistic effects on the T- or B-lymphocyte proliferation induced by ConA or LPS, respectively, promoted the natural killer (NK) cell activity and markedly increased phagocytic activity to activate peritoneal macrophages in immunosuppressive mice. Besides, CCP could increase the protein expression of the G-protein coupled cell membrane receptor TLR4 and the production of its downstream protein kinases (TRAF6, TK1, p-IKKα/β, and NF-κB p50), while enhancing the production of cytokines (IL-2, IL-6, TNF-α, and IFN-α) through both preventive and therapeutic treatments via regulation of the TLR4-NFκB pathway in the peritoneal macrophage of immunosuppressive mice [48,49].

Ganodermataceae (Ganoderma atrum, Ganoderma lucidum and Ganoderma sinense) polysaccharides

In the past decade, there are a large number of published studies from Xie Mingyong's team that described the immunomodulatory effects of Ganoderma atrum polysaccharides (PSG-1) [[51], [52], [53], [54], [55], [56], [57],121,122,170]. Li et al., found PSG-1 treatment could boost basal lymphocyte proliferation (T and B cells) as well as enhance IL-2 production [52,53]. Zhang et al., also demonstrated that PSG-1 treatment could significantly increase the thymus and spleen index and the phagocytosis of macrophages, while the production of TNF-α, IL-1β, and NO also grew [56]. Furthermore, it was reported that PSG-1 acted on TLR4, signaled via the p38 MAPK pathway, and then activated NF-κB and stimulated TNF-α production, in the meantime increased the expression of TLR4 and NF-κB, the degradation of IjBa and the phosphorylation of p38 MAPK. Ganoderma atrum polysaccharide also dose-dependently motivated the release of TNF-α and IL-1β and induced NF-κB activation by elevation of p65 nuclear translocation. Moreover, Yu et al. indicated that PSG-1 plays a major role in the protection against myelosuppression and immunosuppression and oxidative stress in cyclophosphamide (Cy)-treated mice as a potential immunomodulatory agent [170]. It is worth noting that the signaling pathways underlying the immunomodulatory effects of reishi polysaccharide on splenic lymphocytes from Ca2+ and calcineurin (CaN) activity perspective [121]. PSG-1 was first evaluated to increase the nuclear factor activity of activated T cells (NFAT), but this activity could be attenuated by treatment with CaN inhibitors (cyclosporine A and FK506), which provides a new perspective of research for subsequent studies on the immunoreactivity of EMFs polysaccharides.

Interestingly, Ming-Yong Xie's team conducted a study on mannose receptor (MR) mediated macrophage immune responses to Ganoderma atrum polysaccharides in 2017, which is not covered by many authors conducting studies on EMFs polysaccharide immunoreactivity [52]. They reported that MR was essential for the immune response to PSG-1 since enhanced phagocytosis in normal macrophages and the increased concentrations of IL-1β and TNF-α when the concentration of MR was raised. Interestingly, anti-MR antibody at least debilitated PSG-1-mediated anti-inflammatory responses, nevertheless it could not affect TNF-α secretion, suggesting that another receptor might be associated with PSG-1-triggered immunomodulation [52]. Xiang et al., reported that PSG-1 could improve the Th17 cell-specific production of IL-17A and IL-6, the expression of transcription factors (STAT3, RORγt), and phosphorylation of STAT3 in cyclophosphamide-induced immunosuppressed mice [51]. Besides, the Treg cell-specific cytokines (TGF-β1, IL-10) expression, and the transcription factor (Foxp3) were increased. It also raised the ratio of Th17 cells to Treg cells and restored the Th17/Treg ratio.

In the study of Ganoderma lucidum polysaccharides, GSG was just as an effective inducer of MAPKs- and Syk-dependent secretion (TNF-α and IL-6) in peritoneal macrophages, while dectin-1 could effectively recognize GSG and partially intervene its biological activities [58]. In other vitro studies, cy-treated mice compared to low-dose (2.5 mg/kg, i.p), Gl-PS could advance recovery of bone marrow cells, red blood cells, and white blood cells, as well as splenic natural killer T cells, and improved the proliferation responses of T and B cell (day 8), cytotoxic T lymphocyte activity (day 5), also NK cell and lymphokine-activated killer cell activity (days 7–9). Additionally, macrophage phagocytosis was increased (day 12). Meanwhile, Gl-PS turned out to promote the maturation of bone marrow-derived DC in vitro and also increase the initiation of DC-induced immune response [127,129].

In the study of Ganoderma sinense polysaccharides, GSP-4 was reported that could considerably stimulate the production of TNF-α, IL-1β, IL-12, and granulocyte-macrophage colony-stimulating factor (GM-CSF) in human peripheral blood mononuclear cells (PBMCs). GSP-4 also improved the iNOs mRNA expression in a dose-dependent manner [125]. Han et al. concluded that GSP-6B could also significantly induce the production of IL-1β and TNF-α in PBMC and exhibited no toxicity to either PBMC or a human macrophage cell line THP-1, while activating dendritic cells (DC) by encouraging the secretion of IL-12 and IL-10 [126].

Hericiaceae (Hericium erinaceus) polysaccharides

A large number of in vitro and in vivo studies have demonstrated that Hericium erinaceus polysaccharides could improve immunity. Sheng et al. found that ConA-stimulated proliferation of splenic lymphocytes was considerably increased after HEP treatment [134]. In another study described by Wu et al. illuminated that the polysaccharide HEP-W was able to stimulate proliferation of T and B lymphocytes and secretions of IL-2, IL-4, and IFN-γ [61]. Recent pieces of evidence have reported that HEP holds macrophage activation activity through enhancing capacities of phagocytic, production of NO, and proinflammatory cytokines. Meanwhile, the mRNA and protein expressions of iNOS, IL-6, and TNF-α needed to be improved [60,61,131,134,136]. Furthermore, although the action mechanism of HEP isolated from the fruiting body in activating RAW 264.7 cells has been affirmed, mycelium or culture broth was not clear [131]. Wu et al. [61] asserted that a heteropolysaccharide (HEP—W) could active the macrophage activity through myeloid differentiation protein 88 (MyD88)/IRAK-1/TRAF6/PI3K/protein kinase B (Akt)/MAPKs signaling pathways, and TLR2 synergism with mannose receptor (MR) to co-regulate the immunomodulatory response in RAW 264.7 cells. However, it is worth noting that the possible mechanism of macrophage, induced elution with 0.05 M NaCl solution, immunomodulatory activity was chiefly through PI3K/Akt/MAPKs/NF-κB and MyD88/IRAK/TRAF-6/IKKs/IΚBs/NF-κB signaling pathways, which might be ascribed to their distinctions in structural features caused by different elution solvent [60]. In another study, HEP from the fruiting body was reported as induce DCs maturation and suppress DCs endocytosis. Meanwhile, exposure stimulated DCs was significantly stimulated secretion of IL-12 and IFN-γ cytokines that promoted TH1 responses after polysaccharide (sHEP and HEP) treatments. Further study demonstrated that the HEP could up-regulate the MAPK and down-regulate NF-κB signaling pathways of TLR4 [136,137].

Polyporaceae polysaccharides

Polyporaceae is the most widely used EMPs of the classification (family), such as Bjerkandera fumosa, Coriolus versicolor, Cryptoporus volvatus, Grifola frondosa, Inonotus obliquus, Phellinus igniarius, Phellinus pini, Polyporus umbellatus, Poria cocos, and Trametes orientalis. There is a large amount of literature has confirmed that Polyporaceae polysaccharide has a significant effect on improving immunity. A novel polysaccharide (DBFM3) isolated from Bjerkandera fumosa was found that could increase the lymphocyte proliferation in the presence of mitogen A or lipopolysaccharide [62]. Coriolus versicolor polysaccharide's immune function is widely known. Also, a study carried out to evaluate the antiviral effect of Coriolus versicolor polysaccharide CV-S2-Fr.I combined with INF-γ suggested that they could co-induce the production of NO. However, CV-S2-Fr.I used alone was invalid on NO proliferation [171]. A separately published report revealed evidence that Coriolus versicolor polysaccharide (CVP) markedly accelerated the mouse splenocytes proliferation. Besides, CVP-induced B cell proliferation may be considerably constrained by anti-mouse immunoglobulin (Ig) blocking antibody (Fab) or in cells from TLR4-mutant mice (C3H/HeJ), as well as phosphorylation of ERK-1/2 and p38 MAPK distinctly rose in a time-dependent manner, which was the nuclear translocation of the cytosolic NF-κB p65 subunit after CVP-treatment stimulation [172]. Furthermore, the polysaccharide (mPRSon) isolated from Polyporus rhinocerus was also demonstrated to boost immunity on bone marrow dendritic cells, while could also activate functional maturation of BMDCs [173]. Meanwhile, the expression of membrane phenotypic marker CD86 was increased by mPRSon treatment, while binding to the dectin-1 receptor and encourage the release of macrophage inflammatory protein 1α (MIP-1α), MIP-2, and IL-2 [173].

In research of Grifola frondosa polysaccharides, Ma et al. revealed that GFP-A prompted the function of phagocytes [174]. Other in vitro studies have shown that GFP-A exhibited the same splenic cell proliferation as ConA or LPS, while the indices of thymus and spleen were increased, the levels of LDH and ACP in the spleen were rose, and the mRNA levels of IL-1β, IL-2, IL-6, and IFN-γ in splenocyte were also elevated. It is worth noting that GFP-A can significantly improve the expression of CD4+ and CD8+ T lymphocytes, which were restrained by the CTX in peripheral blood [174]. In research of Inonotus obliquus polysaccharides, PFIO was in a position to enhance NO, ROS production, TNF-α secretion, and phagocytosis of phagocytes, while cell proliferation, and IFN-γ/IL-4 secretion in mouse splenocytes [175]. The phosphorylation of three MAPKs as well as the nuclear translocation of NF-κB, resulting in the activation of RAW264.7 macrophages was induced after PFIO treatment. PFIO also induced the inhibition secretion of TNF-αthrough the TLR2 receptor [175]. Similarly, other studies reported that the polysaccharide fraction (DEPS) could not only tremendously produce the secretion of TNF-α, IFN-γ, IL-1β, and IL-2 in PBMCs but also showed no toxicity to PBMCs [176]. Chen et al. found that the proliferation of spleen cells and lymphocytes induced by ConA and LPS was enhanced in a dose-dependent manner after IP3a treatment [177]. At the same time, IP3a could promote cytokine secretion (IL-2, IL-6, IL-12 and TNF-α) and macrophage phagocytosis in mice. It is worth noting that IP3a could increase Bax expression and inhibit Bcl-2 expression significantly [177]. However, other recent studies have not measured the expression of these two metrics (Bax and Bcl-2). Furthermore, the polysaccharide isolated from Phellinus igniarius was also illuminated to improve the cellular immunity as evidenced by the fact that a heteropolysaccharide (PISP1) accelerated the proliferation of mouse spleen lymphocytes [178].

In research of Polyporus umbellatus polysaccharides, the treatment of BMDCs with PPS demonstrated effective activity of improving the cell-surface expression of CD86, and increasing production of both IL-12 p40 and IL-10 in a dose-dependent manner via monoclonal antibodies to TLR4. Additionally, the same treatment of BMDCs with PPS was found that could increase T cell-stimulatory ability and reduce phagocytosis [179,180]. PPS (f-PPS) was bound specifically to BMDCs. Interestingly, binding intercepted by unlabeled PPS and anti-TLR4, but not by anti-TLR2 and anti-CR3 monoclonal antibodies, which has not been previously described by others. Similarly, in vitro studies further demonstrated that PPS was able to effectively promote the NO production and cytokine expression in macrophages. The evaluation of C3H/HeN mice treated with PPS showed that the proliferation of splenocytes and the production of TNF-α, IL-1β, and NO of peritoneal macrophages were markedly alleviated after treatment, as well as function-blocking antibodies to TLR-4, but not TLR-2 and CR3, obviously restrained production of TNF-α and IL-1β after PPS treatment [179,180]. On the other hand, a separately published report revealed evidence that a polysaccharide named ZPS isolated from Polyporus umbellatus was an effective activator of B cell, macrophages, and dendritic cells. Interestingly, a substantial reduction caused by ZPS branches was not only to motivate B cells in vitro but also to induce specific IgM production in vivo [70].

In research of Poria cocos polysaccharides, PCPs were reported to improve the function of the mononuclear phagocyte system, antigen-presenting cells and humoral immunity [181,182]. The immunomodulatory activity of two polysaccharides, P1 and P2, from Poria cocos was investigated [183]. In another animal study, polysaccharides fractions from Poria cocos were considered to boost immunity by activating T cells in Balb/c mice. After the treatment of ovalbumin-immunized mice given PRF (200 mg/kg), the proportion of cytotoxic T cells among splenocytes was remarkably heightened, suggesting that PRF had the potentiality to modulate the specific immune response by activating T cells [184]. In another interesting study, similarly, a polysaccharide (PCSC) was illuminated that could substantially promote NO production and activate NF-κB/Rel that upregulated the transcription of iNOS [185]. It was universally known that p38 kinase was the basis of LPS-induced signal transduction through activating the synthesis of some cytokines [166]. So, it is worth noting that p38 kinase was overexpression in RAW 264.7 cells after PCSC treatment. Besides, when p38 kinase inhibitor was added to the culture medium of RAW 264.7 cells, overexpression levels of NF-κB/Rel and NO production were reduced. However, it could not happen when using inhibitors of mitogen-activated protein kinase/extracellular signal-regulated kinase 1. Beyond that, NO production could also be prevented by treating cells using anti-CD14, anti-TLR4, and anti-CR3 antibodies, suggesting PCSC could interact with these cell membrane receptors and consequently upregulate p38 kinase pathway [185].

Tremellaceae (Tremella and Tremella aurantialba) polysaccharides

Jiang et al. illuminated that Tremella polysaccharides (TP) improved the number of leukocytes in the peripheral blood which were significantly decreased by cyclophosphamide [73]. Zhou et al. found that Tremella polysaccharides (TP) could significantly enlarge the TI and SI (thymus and spleen index), moderate pathological features of immunosuppression (the arrangement of the liver sinusoid, disordered hepatic plates, infiltrated massive inflammatory cells and fatty degeneration of hepatocytes in the liver, intermixed red and/or white pulp, demolished and/or disappeared splenic corpuscles, extended splenic sinusoid, and decreased lymphocytes of the spleen in the spleen) [142]. TP exhibited a preventive effect for cyclophosphamide-induced immunosuppressed mice, which could also up-regulate serum levels of immune factors (IL-2, IL-12, INF-γ, and IgG), reduce the level of TGF-β in serum, noticeably raise mRNA expression of IL-1β, IL-4 and IL-12 in liver and spleen, and restrain mRNA expression of TGF-β [142].

In the research of Tremella aurantialba polysaccharides, TAPA1 could stimulate the proliferation of mouse spleen lymphocytes in vitro [147]. Du et al. compared with TAPA1, TAPA1-ac indicated considerable immunostimulation effects on the proliferation of mouse spleen lymphocytes (MSLs) and the production of NO by macrophages RAW264.7, while TAPA1-deac exhibit significant lower effects [147]. Additionally, it's worth noting that the immune activity above may be related to the content and concentration of acetyl groups, suggesting that acetylation of TAPA1 was an effective way of improving immuno-stimulating activities [147]. Other in vitro animal experiments show that TAP-3 as an immunopotentiator could produce strong immune enhancement effects, such as promoting the production of NO, IL-1β, and TNF-α secretion by macrophages. Further research indicated that the crucial membrane receptor of TAP-3 was identified to be TLR4, and the chain length was indispensable for its immunoregulatory activity [74].

Pleurotaceae polysaccharides

Pleurotaceae is the other most widely used EMPs of the classification (family), which were commonly known as mushrooms, for instance, Lentinus edodes, Pleurotus albidu, Pleurotus eryngii, Pleurotus ferulae, Pleurotus florida, Pleurotus nebrodensis, Pleurotus ostreatus, Pleurotus pulmonarius, and Pleurotus tuber-regium. In some of the earlier studies, the Lentinus edodes polysaccharide (L-II) could significantly increase the concentration of TNF-α, IFN-γ in serum in the polysaccharide groups compared with the model control group, but IL-2 not [186]. Moreover, L-II could raise the production of NO and catalase activity in macrophages [186]. Lo et al. researched Lentinula edodes polysaccharides in vitro macrophage stimulated activities from 10 regionally different strains [187]. Additional in vitro studies further illuminated the potential mechanism of action that stimulated immune activates treatment by Lentinus edodes polysaccharides associated with the up-regulation of MHC II, CD80/CD86, and TLRs in spleen dendritic cells (DCs) [187]. Zhou et al. found that α-(1–3)-β glucan (lentinan) from Lentinus edodes could enhance the production of IL-12, IFN-γ, and NO in spleen cells of infected mice [188]. Additional animal studies further illuminated that lentinan could enhance expression of MHC II, CD80/CD86, and TLRs (TLR2/TLR4), and increase the production of IL-12 in spleen dendritic cells (DCs) co-cultured with parasitism red blood cells (pRBCs). Moreover, both the amount of CD4+, CD25+ regulatory T cells, and the levels of IL-10 secreted dropped by pre-treatment with lentinan in the spleen of malaria-infected mice. Meanwhile, apoptosis of CD4+T cells in the spleens of mice pretreated with lentinan was considerably reduced [188]. Kojima et al. found that IA-a and IA-b could potently phagocytosis and cytokine production in RAW264.7 cells [189]. Similarly, other studies indicated that LEP1 and LEP2 could dramatically improve by LEP1/-2 treatment. Production of NO, TNF-α, and IL-6 were higher in LEP1/-2-treated groups than in the cLEP-treated group. Also, LEP1/-2 had a greater improving effect on mRNA transcription of iNOS, TNF-α, and IL-6 genes. On the other hand, the phosphorylation of kinases ERK and JNK was heavily promoted by LEP1/-2 treatment, suggesting this improving immunocompetence via MAPK signaling pathway [190].

In the research of Pleurotus eryngii polysaccharides, Xu et al. found that a water-soluble polysaccharide EPA-1 could significantly induce macrophage to release the lever of NO, TNF-α, IL-1, and IL-6 through up-regulating signal protein of p38, ERK, JNK in MAPKs and translocation of nuclear NF-κB [87]. Similarly, other studies also indicated that WPEP-N-b boosted the degradation of IκB-α, and increased phosphorylation of MAPKs and the NF-κB p65 subunit, which demonstrated this polysaccharide activates RAW264.7 cells through MAPK and NF-κB signaling pathways and the TLR2 [86]. In the research of Pleurotus florida polysaccharides, PS and glucan were found that could stimulate macrophages, splenocytes, and thymocytes. PfloVv5FB could exhibit strong immune activation of macrophages, splenocytes as well as thymocytes [[191], [192], [193], [194]].

Additionally, other studies in research of Pleurotus nebrodensis polysaccharides reported a novel polysaccharide (PN50G) [195]. The phagocytosis of macrophages was significantly enhanced, and remarkable changes were noted in the morphology of PN50G-treated cells. Compared with the control group, the production of TNF-α, IL-6, IL10, and iNOS in the macrophages, as well as the RNA expressions were strongly induced after PN50G treatment. Pro-/anti-inflammatory cytokine secretion ratios (IL-6/IL-10, TNF-α/IL-10, NO/IL-10) by lipopolysaccharide-stimulated RAW264.7 macrophages were significantly declined by PN50G in a dose-dependent manner under an immoderate immune experimental model [195]. Most recently, Cui and colleagues demonstrated that the phagocytosis of macrophages was markedly improved after exposure to PN-S, with observed remarkable changes in morphology. PN-S was reported to improve RAW264.7 cells to progress via S and G2/M phases [196,197]. Additionally, PN-S could enhance the production of IL-6, NO, INF-γ, and TNF-α in the macrophages, with up-regulating mRNA expressions of IL-6, iNOS, INF-γ, and TNF-α being monitored in a dose-dependent manner. Additional animal studies further illuminated that PN-S treatments considerably changed the CY-in reduced weight loss, boosted the TI and SI, and promote prolife action of T lymphocyte, B lymphocyte, and macrophages. PN-S also raised the activity of natural killer cells and increased the IgM and IgG levels in the serum [196,197].

In research of Pleurotus ostreatus polysaccharides, POP was able to improve concanavalin A (ConA)-or lipopolysaccharide (LPS)-induced lymphocyte proliferation [198]. The heteroglycan (WPOP-N1) isolated from Pleurotus ostreatus was reported that could stimulate macrophages, splenocytes, and thymocytes, and markedly be growing in secretion level of TNF-α in the serum [199]. Additionally, WPOP-N1 raised the phagocytic capability of peritoneal macrophages in vitro, as well as the secretion of TNF-α, NO, and the amount of TNF-α and iNOS transcript increased substantially when the peritoneal macrophages were susceptible to WPOP-N1. Meanwhile, further molecular experiments showed that the stimulation of peritoneal macrophages by WPOP-N1 induced the phosphorylation of p65 and a marked down-regulation of IκB expression [199]. In the research of Pleurotus pulmonarius polysaccharides, purified β-glucan was able to effectively increase NO, TNF-α and IL-1β production in macrophages, while these effects being very similar to those of Escherichia coli serotype lipopolysaccharide (LPS), while not modifying the response of LPS-activated macrophages [200].

Tricholomataceae (Armillaria mellea, Flammulina velutipes, Tricholoma crissum, Tricholoma lobayense, and Tricholoma matsutake) polysaccharides

In the research of Armillaria mellea polysaccharides, the water-soluble Armillaria mellea polysaccharide (AMP) was reported which could stimulate lymphocyte proliferation induced by concanavalin A or lipopolysaccharide in a dose-dependent manner [201]. Similarly, Chen et al. reported that a polysaccharide fraction (AMPs-2-1) isolated from Armillaria mellea could promote proliferation of splenocyte lymphocytes and phagocytosis of macrophages RAW264.7, exhibited significant immunomodulatory activities [93].

A polysaccharide (FVSPs) was extracted from the base of Flammuliana Velutipes. In this research, FVSPs was shown a high ability to significantly increase the proliferation and phagocytic activity of macrophage [202]. Interestingly, another study revealed an enhancing-immune polysaccharide (PFVM) derived from Flammuliana Velutipes in terms of the number of T lymphocyte subsets (CD3+, CD4+, and CD8+) [203]. The percentage of CD3+ and CD4+ T lymphocyte, the ratio of CD4+/CD8+, and the levels of IL-2 and TNF-α were significantly enlarged in polysaccharide of PFVM. On the other hand, the percentage of CD8+ T lymphocyte was reduced in polysaccharide of PFVM dose-dependent manner indicated that the T lymphocyte immune function was stimulated after a long term exposure of PFVM [203].

In the research of other Tricholomataceae polysaccharides, Tricholoma crissum polysaccharide (PS) were reported that could exhibit splenocyte, thymocyte as well as macrophage activations [96]. Zhang et al. found that polysaccharide TLH-3 extracted from Tricholoma lobayense could significantly enhance the phagocytic activity, production of NO, and secretion of the cytokine TNF-α, IL-6 [204]. Furthermore, TNF-α and IL-6 were blocked by the inhibitor of TLR4 (Toll-like receptor4), which indicated TLR4 was a receptor of TLH-3 as well as immunomodulatory activity of TLH-3 was mediated by TLR4 [204]. Moreover, immunofluorescence suggested that TLH-3 leads to the nuclear translocation of NF-κB subunit p65, which demonstrated that NF-κB levels in nuclei increased and cytoplasmic IκB-α degraded after TLH-3 treatment [204]. Similarly, other studies reported that TMF-II derived from Tricholoma matsutake would comparably increase and/or highly upregulate the production of NO production and expression of IL-1β, IL-6, IL-12, and TNF-α to LPS [205]. On the other hand, TMF-II provoked the phosphorylation of IκBα, a vital step for NF-κB activation and translocation. It is worth noting that the upstream signaling enzymes (SRC and AKT) were observed the responsible upstream signaling components in the induction of NO production, even though TMF-II strongly upregulated the phosphorylation of all MAPK pathways [205]. In another study, TMC-2 also derived from Tricholoma matsutake was demonstrated that could strongly increase the production of NO and TNF-α [206]. Additionally, phagocytic uptake and ROS generation were also improved by TmC-2 treatments. Interestingly, TmC-2 stimulated CD29-mediated cell-cell or cell-fibronectin adhesions in monocytes, while CD43-mediated cell adhesion was down-regulated. The enhancement of proliferation and IFN-γ production was strikingly reported in TmC-2-treated splenic lymphocytes. Further molecular experiments showed that the activation mediated by up-regulating intracellular signaling cascades such as PI3K/Akt and MAPK (ERK and p38) and by the involvement of surface receptors (dectin-1 and TLR-2) [206].

Anti-inflammatory activity

Inflammation is a defense reaction produced by the living tissues of the vascular system after the organism is stimulated by various injury factors, and the pathological process is the exudation, degeneration, and hyperplasia of tissues in the inflamed area [166]. The clinical data from this novel coronavirus study revealed that pathogenic coronavirus infection initially presents as a mild influenza-like illness with fever, dyspnea, and cough, which progresses to atypical interstitial pneumonia and diffuse alveolar damage, with more severe cases of the disease dyspnea and/or hypoxemia develops 1 week after the onset of symptoms, with rapid progression to acute respiratory distress syndrome in severe cases (ARDS), septic shock, intractable metabolic acidosis and coagulopathy, COVID-19 infection [33,34]. The pathogenesis of severe lung injury caused by a severe inflammatory response and acute lung injury may be through an acute inflammatory response and the cytokines IL-1, IFN-γ, interferon-inducible protein 10 (IP-10), monocyte chemotactic protein-1 (MCP-1), IL-4 and IL-10 secretion is increased, and the patients' strength of the cytokine storm following infection correlates with the severity of the disease [166].

Numerous previous studies have demonstrated that anti-inflammatory activity is very common in diverse sources of EMFs polysaccharides. Its mechanism of action was determined by the following anti-inflammatory mechanisms, the foremost being inflammatory factors that promote inflammatory responses, such as TNF-α, and interleukins (IL-1β, IL-2, IL-6, IL-8, and IL-12). Among them, TNF-α, as an important inflammatory factor, can induce lymphocytes and epithelial cells to produce a variety of adhesion molecules, induce the production of chemokines, prompting inflammatory cells to localize to inflammation, further causing inflammation, tissue damage, and other pathological changes [166]. The second category is anti-inflammatory factors that reduce or inhibit the inflammatory response, such as IL-4, IL-5, and IL-10. Among them, IL-10 is the most popular and prominent anti-inflammatory cytokine, which can exert its anti-inflammatory effects through multiple pathways. The role of MAPK in the production of pro-inflammatory cytokines, such as antagonizing the secretion of pro-inflammatory cytokines, enhances the effect of 5-HT, modulating MAPK, and enhancing the production of pro-inflammatory cytokines [166]. Other anti-inflammatory cytokines such as IL-4 and IL-13 may also have similar characteristics to IL-10. The anti-inflammatory details in separate families (Morchellaceae, Xylariaceae, Ganodermataceae, Polyporaceae, Phallaceae, Agaricaceae, Pleurotaceae, Russulaceae, and Tricholomataceae) of EMFs polysaccharides will be described as followed.

Morchella esculenta polysaccharide (EMP-1) and sulfated polysaccharide (SEMP-1) were found that the best-protecting effect in decreasing PM2.5-induced cell death, cell apoptosis and production of TNF-α and IL-1β, as well as accompanied by a vitiated level in ROS formation, caused by PM2.5 in rat alveolar macrophage NR8383 cells [99]. Moreover, SEMP-1 could decrease the expression of iNOS and COX-2 at both mRNA and protein levels with PM2.5 treatment. Besides, the PM2.5-induced phosphorylation of NF-κB was also decreased via suppressing nuclear translation of the NF-κB and inhibiting the degradation and phosphorylation of IκBα [99]. Similarly, other studies reported that acetylation of polysaccharide (PEMP and Ac-PMEP1 – 3) extracted from Morchella angusticeps compared with the control group, PMEP and AcPMEP1 – 3 boosted cell proliferation and the production of NO and TNF-α of RAW264.7 macrophages (cultured without lipopolysaccharide). On the other hand, compared with PMEP, Ac-PMEP3 improved cell viability and NO production by inducing the degradation of cytoplasmic IκBα and nuclear translocation of NF-κB subunit p65 as well as the expression of iNOS and phosphorylated-p38 [97]. The intensive search for alleviating PM2.5-induced lung injury in mice has resulted in the discovery of Trametes orientalis polysaccharide (TOP-2) intervention could moderate PM2.5-induced lung injury (pulmonary edema) in mice through its antioxidant and anti-inflammatory activities [207]. PM2.5 could notably raise the number of inflammatory cells and proportion of neutrophils in bronchoalveolar lavage fluid (BALF), and remarkably declined the percentages of macrophages in BALF, however, TOP-2 eliminated these effects. Additionally, TOP-2 could inhibit increasing levels of total protein, albumin, C-reactive protein (CRP), myeloperoxidase (MPO), lactate dehydrogenase (LDH), alkaline phosphatase (AKP), acid sphingomyelinase (ASM), TNF-α, IL-1β and IL-6 in BALF after PM2.5 exposure. On the other hand, TOP-2 up-regulated the expressions of nuclear factor-erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) and inhibited the activation of NLR family pyrin domain-containing 3 (NLRP3) inflammasome in the lung tissue [207].

Furthermore, the polysaccharide isolated from the Dictyophora indusiata (DIP) was also shown to be anti-inflammatory activity as evidenced by the facts that DIP restrained NF-κB signal pathway through regulating TLR4 expression, phosphorylation of IκBα and nuclear translocation of NF-κB-p65 subunit [75]. Similarly, DIP decreased inflammasome activation through down-regulating NLRP3 expression in cytoplasmic pools, confining self-assembly of NLRP3 inflammasome, meanwhile the subsequent triggering of caspase-1 and the secretion of IL-1β and IL18 [75]. In another study, a glucogalactomanan polysaccharide (TJ3) isolated from Agaricus bisporus induced an inflammatory response through the ERK/MAPK and IjB/NFjB pathways in macrophages [208]. Huijeong et al. found that Lentinan (LNT) from shiitake could selectively attenuate AIM2 and non-canonical inflammasome activation as well as inducing pro-inflammatory cytokine production, which could selectively inhibit lacking in melanoma 2 (AIM2) inflammasome activation [209]. In addition, LNT improved pro-inflammatory cytokines and induced expression of inflammasome-related genes via toll-like receptor 4 signalings, which indicated that could as an anti-AIM2 and anti-non-canonical inflammasome candidate even if its enhancing of cytokine expression [209].

Ken-ichiro et al. reported that Pleurotus citrinopileatus polysaccharide (PCPS) was able to regulate the monocyte-to-macrophage differentiation early at the monocyte stage [84]. PCPS could inhibit the levels of secreted pro-inflammatory cytokines (TNF and IL-6), increase the secreted levels of the anti-inflammatory cytokine IL-10, and the expression levels of CCL2 and CCL8 mRNAs, similarly constrained mRNA expression of CCR2 in the IFNγ/LPS activated macrophages [84]. In previous study, Ken-ichiro et al. [85] found that PCPS improved the surface maturation markers (CD80, CD86, and HLA-DR) on DCs, which suggested its potential to induce DC maturation. Additionally, PCPS was a DCs activator to secrete the pro-inflammatory cytokines (TNF, IL-1β, IL-6, and IL-12), as well as the anti-inflammatory cytokine IL-10. At the same time, PCPs also enhanced mRNA levels of the chemokines (CCL2, CCL3, CCL8, CXCL9, CXCL10, and LTA). The secretion of TNF and IL-12 by PCPS-activated DCs could significantly be eased by an anti-Dectin-1 antibody, as well as by a Syk kinase and a Raf-1 inhibitor, suggesting that PCPS motivated Dectin-1 signaling at least partly via the Syk- and the Raf1-dependent pathways in DCs [85]. Song et al. reported that anti-inflammatory and hepatoprotective effects of exopolysaccharides (EPS) extracted and purified from Pleurotus geesteranus on alcohol-induced liver injury [210]. Changes in anti-inflammatory factors are essentially consistent with the other EMFs polysaccharides mentioned above. [210].

Marcia L.L. Silveira et al. found that an exopolysaccharide (PEIsR) produced by Pleurotus sajor-caju has anti-inflammatory activity. In vivo could be as a potent and effective antinociceptive and anti-inflammatory candidate [91]. On the other hand, Marcia et al. reported the LPS from P. sajor-caju demonstrated an immunomodulatory activity on THP-1 macrophages [91]. Further animal experiments showed that LPS significantly inhibited the inflammatory phase of pain sensation in mice induced by formalin, and effectively reduced the total number of leukocytes and the level of myeloperoxidase in mice induced by LPS [91].

Regulation of population balance of gut microbiota

The intestinal tract is an important digestive organ of the body and is also the main host site for symbiotic microflora, which plays an essential role in the maintenance of normal life activities such as immune and endocrine functions [211]. Normally, intestinal flora, host, and external environment maintain a dynamic balance, but due to drug metabolism, changes in the flora, abnormal intestinal dynamics, age, dietary habits, and immune dysfunction, this balance can be disrupted, causing an imbalance in intestinal flora and pathological changes in host organism [211,212]. In this novel coronavirus, it has also been shown that there is a correlation between changes in the intestinal flora of patients with neo-corneal pneumonia and the neo-corneal virus [35]. Many studies have shown that EMFs polysaccharide has the function of regulating intestinal function, including participating in the immune process by acting on the intestinal mucosa, protecting the integrity of the intestinal barrier structure and function, regulating the composition of intestinal flora and stimulating the intestinal endocrine, and so on.

A polysaccharide extracted and purified from Dictyophora indusiata was reported to advance recovery from antibiotic-driven intestinal dysbiosis and promote gut epithelial barrier function in a mouse model [213]. Among controls, daily oral administration of clindamycin and metronidazole for two weeks was evaluated to reduce bacterial diversity and richness and disorder rates of microflora at different taxonomic levels (altered Firmicutes/Bacteroidetes ratio and rose relative abundance of harmful flora (Proteobacteria, Enterococcus, and Bacteroides), however, DIP administration recovered the dysbiosis and improving beneficial flora, such as lactic acid-producing bacteria (Lactobacillaceae), and butyrate-producing bacteria (Ruminococaceae). Additionally, endotoxemia was reduced by DIP treatment via LPSs and pro-inflammatory cytokine (TNF-α, IL-6, IL-1β) levels, with the increased expression of tight-junction associated proteins (claudin-1, occludin, and zonula occludens-1), which indicated a comprehensive perception of the protective effects of a DIP in the restoration or rebuilding of gut microbiota and emphasized its vital function in the enrichment of intestines barrier integrity, decrease of inflammation, and lowering of endotoxin levels in mice [213]. On the other hand, Zhang et al. [214,215] systematically investigated that Lentinula edodes polysaccharide (L2) refreshed mice in terms of immune responses and gut microbiota. L2 was reported to restore the aging immune responses by improving cytokine levels in peripheral blood and partly alter the aging composition of intestines microbiota. The results of further group animal experiments suggest the advantageous effects of L2 on boosting immunity and enhancing gut health. Besides, analysis of Caco-2 cells and a Caco-2/RAW264.7 co-culture system indicated that Lentinula edodes-derived polysaccharide (L2) remarkably intensified immune responses by differentially affecting gene expressions of small intestine (55 genes), cecum (26 genes) and colon (25 genes), recognized 3 core regulation networks (in the small intestine, cecum, and colon) for assorted parts of the gut [214,215]. In another study, the fecal microbiota in PEP (a homogeneous Pleurotus eryngii polysaccharide) treatment was evaluated structural differences compared to the control group, while the Porphyromonadaceae, Rikenellaceae; Bacteroidaceae and Lactobacillaceae abundances were entirely boosted at the family level [216]. It is worth noting that the immune response produced by oral administration of high dose PEP also changed significantly. In summary, PEP intake could play a positive role in gastrointestinal health [216]. Also, a separately published report revealed evidence that a novel polysaccharide (FVP2) isolated from Flammulina velutipes, impact on gut microbiota in rats was reported by Jufeng Ye [94]. On the one hand, the concentrations of two short-chain fatty acids (isobutyric acid and butyric acid) and the abundance of beneficial bacteria in the caecum after the FVP2 treatment were significantly higher than those in control group, as well as butyric acid level was effectively improved, thus increasing the intestinal beneficial bacteria. On the other hand, FVP2 can maintain the integrity of intestinal mucosa by improving intestinal barrier function [94].

Conclusion and future perspective

Nowadays, there is a restricted range of antiviral drugs with certain toxic side effects, but with the emergence of new mutant and resistant strains of viruses, the availability of highly effective, low-toxicity, antiviral drugs have been increasingly important. Research on antiviral drugs for resistant viruses is imminent. EMPs polysaccharide has the characteristics of multi-component, multi-pathway, multi-target effect, and has unique advantages in antiviral, not easy to generate drug resistance. The unique advantage of EMPs polysaccharides against viruses is their ability to operate directly on viruses, inhibit their proliferation and, more importantly, modulate the host immune response, alleviate inflammatory damage, and fully exploit antiviral effects through multiple pathways and targets, thus seeking out natural EMPs polysaccharides is an important way to develop highly effective and low-toxicity novel antiviral drugs, in vivo. As a potential antiviral drug, its antiviral effect still has to be validated through clinical practice. EMPs polysaccharide species are numerous, and their absorption and distribution in the body were varied greatly. Why the EMPs polysaccharide were still only in the laboratory stage due to the following reasons:

The EMFs polysaccharide molecules are very large, and existing studies suggest that polysaccharides containing β-glycosidic bonds have some antiviral activity (especially β-glucan), in addition to which some degree of sulphation modification may also enhance the antiviral activity of polysaccharides, but the analysis of the structure-antiviral relationship of most polysaccharides is not yet clear. Also, there is no way to control the quality of each batch, limiting its industrial production;

The most existing industrialized production methods of EMFs polysaccharide are water extraction and alcohol precipitation, so the products are mostly crude polysaccharides and the amount of refined polysaccharides were limited which cannot meet the requirements for industrialization. Even if there are some EMFs polysaccharides pharmaceutical products on the market, basically their functional ingredients are crude polysaccharides, so there are still big controversies in terms of safety and stability.

As the mode of action of EMFs polysaccharides is multifaceted and coordinated, their effect is slow and not immediate, and the changes in their biological activity after oral and gastrointestinal digestion have to be taken into account, so they cannot meet clinical requirements for the time being;

Most current researches related to EMFs polysaccharides are based on cellular and/or animal levels. Unfortunately, there are few preclinical and/or clinical studies on the effect and mechanism of EMFs polysaccharides. As a result, there is still a long way to go from laboratory to market. Therefore, further research is urgently needed on its constitutive relationship and action mechanism.

CRediT authorship contribution statement

Yuxi Guo: Writing original draft, Conceptualization. Xuefeng Chen: Visualization, Investigation, Writing review & editing. Pin Gong.

Declaration of competing interest

The authors declare no conflicts of interest.