Paecilomyces cicadae: a systematic overview of the biological activities and potential mechanisms of its active metabolites

ABSTRACT

The microbe Paecilomyces cicadae (Miquel) Samson (P. cicadae) is an asexual strain of Cordyceps cicadae (C. cicadae) used in traditional Chinese medicine. Due to the abundance of secondary metabolites found in cultured metabolites, including polysaccharides, hyaluronic acid, ergosterol peroxide, and active nucleosides, P. cicadae and/or C. cicadae exhibit several therapeutic activities, including immunoregulatory, antioxidant, and anti-diabetic effects. Given the great attention for health care and the desirable medical value of C. cicadae and/or P. cicadae, they are becoming potential alternatives of Cordyceps sinensis (C. sinensis). Although there are many studies on P. cicadae/C. cicadae, most of them are experimental research articles. There are limited reviews to summarize the latest researches on P. cicadae/C. cicadae. This review mainly focuses on the metabolites, physiological activities and mechanisms of action of P. cicadae and/or C. cicadae. This review can serve as a theoretical basis for the more efficient use of this medicinal fungus.

KEYWORDS:

• Paecilomyces cicadae
• Cordyceps cicadae
• metabolic actives
• physiological activities
• mechanisms

1. Introduction

Paecilomyces cicadae (Miquel) Samson (P. cicadae), as the anamorph stage of Cordyceps cicadae (C. cicadae), is an entomogenous and medicinal fungus. C. cicadae is also known as Isaria cicadae Miquel (I. cicadae), Cordyceps zhejianggensis (C. zhejianggensis). Similar to the well-known Cordyceps sinensis (C. sinensis) that selectively infects Hepialus larvae (Zhu et al., 1998a), P. cicadae selectively infects bamboo cicada larvae (Ahn et al., 2000; Chandrashekar, 2006; Li et al., 2021a; Zeng et al., 2014). After the infection, its lifecycle prolonged. The complex of insects and fungi is also known as the cicada flower because its top branches “sprout” like flowers. The formation process of cicada flower has been reported widely. According to the reports (Ke & Lee, 2019; Yan et al., 2014), C. cicadae forms in the manner described below. P. cicadae infect the larvae in the late summer or early autumn, then the bamboo cicada larvae ingest nutrients and proliferate to infect many mycelia. The infected larvae become stiff worms in the winter and eventually branch and blossom at the top of the cicada larvae to form C. cicadae the next spring or summer.

As a traditional Chinese medicine (TCM), C. cicadae has many polysaccharides, cordycepin, cordycepic acid, and other active metabolites (Hur, 2008; Li et al., 2021a), which have effects on anti-oxidation (Olatunji et al., 2016), anti-inflammation (Wang et al., 2019a), anti-diabetes (Yang et al., 2020b), anticancer (Xie et al., 2019), immune regulation (Kuo et al., 2003), and other effects. For centuries, it has been used as tonic and folk medicine to treat malaria, cancer, fever, diabetes, chronic renal diseases, heart palpitations, infantile convulsions, and dizziness (Ke & Lee, 2019; 2015; Kuo et al., 2003; Olatunji et al., 2016; Xie et al., 2019; Yang et al., 2020b). Although C. cicadae has been used for over 1600 years, it is not the most popular amongst the Cordyceps family. C. sinensis is the most explored and widely used, followed by Cordyceps militaris (C. militaris) (Chen et al., 2018; Olatunji et al., 2018; Patterson, 2008; Zhao et al., 2014). Commercial products have been developed from C. sinensis and C. militaris, such as Cobrin, Ningxinbao, Golden Sun Cordyceps, and C. militaris mycelia powder and capsule (Dong et al., 2015). However, the market for C. cicadae derived products in China is still very small (Dong et al., 2015).

C. cicadae related researches done over the past two decades have shown that it poses similar biological properties and bioactive compounds as C. sinensis and C. militaris, and suggested that it can be used as an alternative source of Cordyceps (Nxumalo et al., 2020).

The permission of C. cicadae to be used or sold as food has obtained in China. The fruiting body of C. cicadae (artificial cultivation) has been recognized by the People's Republic of China's National Health Commission as a new type of food raw material since 7 January 2021. In addition to Cordyceps guangdongensis and Cordyceps militaris, it is the third new food raw material belong to genus Cordyceps. It means that C. cicadae can be added into the ordinary food and sells commonly without the permission of health food or drugs.

However, due to the scarcity of naturally occurring C. cicadae and the rise in C. cicadae demand in recent years, the competitive cultivation and fermentation methods are urgently needed. It is possible to culture C. cicadae fruiting bodies artificially, and it has been discovered that P. cicadae, the species’ anamorph, has good and controllable quality. A foundation for the effective use of this medicinal fungus is provided by reports that the chemical constituents of these artificially cultured P. cicadae are essentially the same as those of natural C. cicadae (Ge et al., 2007).

The safety assessment has been drawn close attention by the researchers. The insecticidal and nematicidal beauvericin, a kind of cyclodepsipeptides found in C. cicadae, can induce cytotoxicity and cell apoptosis in a dose-dependent manner (Mallebrera et al., 2016). Traditionally, instead of using pure compounds, C. cicadae mycelium or fruiting bodies samples have been tested for safety assessments in rat, rabbit, pig, mouse, and human models (Chen et al., 2015; Chen et al., 2017a; Fu et al., 2021; Horng et al., 2021; Hsu & Chen, 2021; Li et al., 2017a; Tsai et al., 2021). Excellent safety was demonstrated by the acute toxicity test, subacute toxicity test, and clinical measurement (Chen, 1993; Tsai et al., 2021). These data support the applications of cicada fermentation products in functional foods, medicines, or cosmetics.

Although there are many studies on P. cicadae/C. cicadae, most of them are experimental research articles. There are limited reviews to summarize the latest researches on P. cicadae/C. cicadae. This review mainly focuses on the metabolites, physiological activities and mechanisms of action of P. cicadae and/or C. cicadae. We hope to this paper could provide theoretical basis for future researchers. In addition, cicada flowers were also included in the study.

2. Metabolic actives

The studies of fungal metabolic actives have gathered a lot of attention ever since Byerrum first reported the antitumor activity of polysaccharides in mushrooms in the 1960s (Byerrum et al., 1957). Numerous pieces of literatures have revealed recently that the metabolic actives of some fungi have hypoglycemic (Dai et al., 2015), immunomodulatory (Yu et al., 2018), antiviral (Li et al., 2021c; Verma, 2022; Zhu et al., 2020a), anti-inflammatory (Qi et al., 2021; Xu & Du, 2020), antioxidant (Zhu et al., 2018), and antitumor effects (Tian et al., 2019; Zhang et al., 2021c). The active ingredients of various medicinal fungi are also being used increasingly in medicine, food, biology, cosmetics, and other fields. We summarized some typical outcomes for various polysaccharides, proteins, extracts, and other active substances in fungi (Table 1).

Table 1. Functions and mechanisms of active substances in some fungi.

Active substances	Sources	Research Models	Main active ingredients	Physiological activity	Mechanism of action	Refs
Polysaccharide	Coriolus versicolor	S180 tumor-bearing mouse	β-glucan	Antitumor activity, immunomodulatory properties	Enhancing macrophage function to enhance host immune defense	Ho et al., 2004
	Lentinus edodes	Patients infected with 2019 novel coronavirus	α-glucans	Regulating the immune system	Regulating and protecting immune response	Dietologica, 2020
		In vitro models of lung injury and macrophage phagocytosis	Carbosynth-lentinan (50%β-glucan 12%α-glucan)	Reducing the level of inflammation in the lung	Reducing cytokine induced in human alveolar epithelial A549 cells NF- κ B activation	Murphy et al., 2020
		IHNV	Glucose, glycan, galactose: 19.26:1.20:1.00	Antiviral activity	↑IFN-1, IFN- γ ↓TNF- α,IL-2, IL-11	Ren et al., 2018
		Male Swiss mice	Fucomannogalactan	Antinociceptive and anti-inflammatory effects	↓TNF-a, Interleukin-1B, interleukin-8, glutamate in cerebrospinal fluid	Carbonero et al., 2008
	Ganoderma lucidum	Type 2 Diabetic Rats	/	Antitumor, antioxidation and anti-inflammation.	Decreasing the activities of AST and ALT, increasing hepatic glycogen levels ↑PPAR-γ, GLUT4, PI3K, Akt (p-Akt), SCFA	Ke-Xue et al., 2016
		Normal fasting mice	/	Hypoglycemic effect	Promoting insulin release	Zhang & Lin, 2004
		Human peripheral blood mononuclear cells (PBMCs)	α-glucan β-glucan	Immunomodulatory activity	↑TNF-α, IFN-γ, IL-2, IL-17	Habijanic et al., 2015
		HFD-induced C57BL/6J mice	/	Anti-inflammatory activity,anti-obesity effects	↓TLR4/Myd88/NF-κB ↑SCFA, GPR43 Reducing serum lipopolysaccharide level increasing ileum expression of tight junction proteins and antimicrobial peptides	Sang et al., 2021
		Type 2 diabetic rats	/	Antioxidative activity Anti-inflammation activity	↑IL-1β, IL-6. Decreasing triglycerides, total cholesterol Reducing gut microbiota dysbiosis	Chen et al., 2020
		Japanese big-ear white rabbits	/	Antioxidative activity	↓NOS, MDA, ROS	Li et al., 2021d
	Grifola frondosa	Hyperlipidemia and hypercholesterolemia rats	/	Inhibiting obesity and improving intestinal flora imbalance	Regulating the mRNA expression levels of genes responsible for liver lipid and cholesterol metabolism	Li et al., 2019b
	Poria cocos	Mice immunized by H1N1 influenza and HBsAg vaccines	fucose, mannose, glucose and galactose in molar ration of 1.00:1.63:0.16:6.29	Immune regulation	Improving proliferation of splenocytes ↑IL-12p70, TNF-a	Wu et al., 2016
	Cordyceps sobolifera	S180 sarcoma mouse model	/	Antitumor Activity	Enhanced the immune system adjustment capacity and antioxidant activity.	Xie et al., 2016
	Paecilomyces cicadae	Cyclophosphamide induced mice	/	Immune regulation and improve the structure of intestinal flora	Activating the Nrf2-Keap1 ↓Keap1 ↑NQO1, HO-1	Yao et al. 2023
	Cordyceps cicadae	Glutamic acid induced PC12 cells	/	Antioxidant effect	Increasing cell survival ↑Ca^2 +overload, ROS	Olatunji et al. 2016
Lignin	Lentinus edodes	Human immunodeficiency virus type 1 (HIV-1)	A small amount of protein (3.2%) and sugar (12.2%)	Immunostimulatory activity and anti-HIV effect.	It can inhibit the in vitro cytopathic effect induced by HIV-1 (concentration > 10 μg/ml)	Suzuki et al., 1989
N6-(2-hydroxyethyl)-adenosine	Cordyceps cicadae	Hypertension rat	N6-(2-hydroxyethyl)-adenosine	Antihypertensive effect	Activating adenoptosine A1 recer	Zhang, Chai, et al., 2019
Ergosterol	Cloud ear mushroom	BV2 microglial cell	Ergosterol	Anti-inflammation	Regulating NF-κB ↑SOD-1 enzyme, ROS	Sillapachaiyaporn et al., 2022
	Ganoderma lucidum	Tumor cells and human umbilical vein endothelial cells (HUVEC)	Ergosterols	Antitumor activity	Inhibiting the activity of human tumor cells and HUVECs	Chen et al., 2017b
Ergosterol Peroxide	Ganoderma lucidum	MDA-MB-231 Breast Cancer Cell	Ergosterol Peroxide	Anticancer effect	Inhibiting the activity of cancer cells	El-Sherif et al., 2020a
		Tumor cells of mir-378	Ergosterol Peroxide	Overcome the drug resistance of tumor cells.	Inducing the death of mir-378 transfected cells	Wu et al., 2012
	Cordyceps cicadae	TGF-β1-induced kidney fibroblasts	Ergosterol Peroxide	Renal protective effect	Attenuating TGF-β1-induced renal fibroblast proliferation ↓Cytoskeleton protein, CTGF, ECM	Zhu et al., 2014
	Paecilomyces cicadae	Human RCC cell line 786-0	Ergosterol Peroxide	Anticancer effect	suppressing cell growth, colonization, migration, and invasion, arresting the cell cycle, attenuating β-catenin pathways, and triggering apoptos	He et al., 2018
Triterpene	Ganoderma lucidum	/	Ganoderma acid A, B, C and D, leucine B and Ganoderma triol	Antioxidant activity	Enhancing the antioxidant effect of pyrogallol induced erythrocyte membrane oxidation and Fe (II) – ascorbic acid induced Lipid peroxidation	Zhu et al., 1999
		/	/	Antiviral effect	Inhibiting ACE activity	Rahman et al., 2021a
	Grifola frondosa	/	Triterpenes	Antiviral effect	Inhibiting ACE activity	Rahman et al., 2021a

Numerous studies on P. cicadae and/or C. cicadae have revealed that the mycelia, fermentation broths, and fruiting bodies contain polysaccharides, adenosine, cordycepin, ergosterol, essential amino acids, and alkaloids with biological activities such as immune-enhancing, antitumor, anti-inflammatory, antibacterial, and antiviral effects (Chai et al., 2007; Kim et al., 2011; Kim et al., 2013; Li et al., 2018; Shen et al., 2007; Sun et al., 2017; Tian et al., 2021a; Wang et al., 2012b; Zeng et al., 2014; Zhang et al., 2017a).

Some typical compounds obtained from P. cicadae or C. cicadae are shown in Figure 1. We mentioned the following ingredients in this section to thoroughly define the characteristics, structures, functions, and mechanisms.

Figure 1. Chemical structures of compounds extracted from P. cicadae and/or C. cicadae. The active metabolites of P. cicadae/C. cicadae are classified into six major categories, namely polysaccharides, nucleoside compounds, ergosterol and its peroxides, cordycepin acid, myriocin, and other compounds.

2.1. Polysaccharides

A class of polymeric carbohydrates known as polysaccharides is composed of at least 10 monosaccharides that are linked by glycosidic bonds. Polysaccharides have the general formula C_x(H₂O)_y, where x is often a significant value between 200 and 2500. The general formula can also be written as (C₆H₁₀O₅) _n, where n is usually between 40 and 3000. The repeating units in the polymer backbone are usually six-carbon monosaccharides.

Polysaccharides are one kind of the main active components in mushrooms (Won et al., 2011). They are structurally diverse macromolecules that serve a variety of roles in many biological processes. The distinct advantages of polysaccharides are their excellent safety and easy availability. Furthermore, growing evidences have suggested that natural polysaccharides can directly scavenge reactive oxidative free radicals (ROS) (Asker et al., 2009), increase cell viabilities (Vinderola et al., 2006), and boost immunity in cells (Yamada et al., 1997).

In 1982, researchers discovered for the first time the structure of P. cicadae polysaccharides. The galactomannan in P. cicadae was isolated by Japanese researchers (Ukai et al., 1983). Its relative molecular weight is 27 000, making it a kind of water-soluble polysaccharides. It is composed of D-mannose and D-galactose in a 4:3 ratio. The P. cicadae polysaccharides were subsequently isolated using a chromatographic column by Ren et al. (Ren et al., 2014), who discovered that mannose (43.2%), rhamnose (32.1%), xylose (14.5%), and arabinose (10.2%) made up the majority of its structure. According to Li et al. (Li, He, et al., 2016a), the primary structural characteristics of P. cicadae polysaccharides were rhamnose, arabinose, fucose, xylose, mannose, glucose, and galactose with a molar ratio of 0.63:13.1:1.30:21.3:46.5:6.0:10.22. Although different structures were identified, there are three kinds of monosaccharides, mannose, galactose, and arabinose, are usually existed.

Recently, it has been discovered that a significant amount of hyaluronic acid was detected in the fermentation broth of P. cicadae (Tan et al., 2007), which has gathered people's attention. It may be another source to obtain hyaluronic acid economically, and conveniently. Hyaluronic acid ((C₁₄H₂₁NO₁₁)n, Figure 1, 1) is a polymer, non-protein, acidic mucopolysaccharide, whose structural units are acetylglucosamine and glucuronic acid (Chandrashekar, 2006; Hedén et al., 2020). Hyaluronic hacid exhibits substantial health benefits for the body, such as strong water retention properties. It plays roles in cell proliferation, wound healing, migration, and differentiation, which makes it an important biomedical raw material (Donejko et al., 2015; Mandal et al., 2018). According to Tan et al. (2007), an optimal fermentation medium composition for hyaluronic acid was obtained, those were 6% lactose, 0.5% beef extract, and yeast extract in a 1:1 and 0.1% magnesium sulfate. The yield of hyaluronic acid could reach up to 11.35 mg/mL. Furthermore, the process of extracting hyaluronic acid from P. cicadae has also been studied (Huang & Tian, 2009).

The physical and biological properties of polysaccharides are influenced by many important factors, one of which is molecular weight. It is reported that high-molecular-weight polysaccharides are necessary for their immunomodulatory and antitumor activities (Ren et al., 2012; Suarez et al., 2006). However, some low-weight molecular polysaccharides can also exhibit strong biological functions (He et al., 2020; Li et al., 2021b; Yuan et al., 2020). C. cicadae polysaccharides have strong antioxidant and hypoglycemic properties with a molecular weight of 60.7 kDa (Tang et al., 2022). JCH-3, a relatively low-weight molecular polysaccharide isolated from C. cicadae, had excellent immunoregulatory activities, compared to JCH-1 and JCH-2, according to Xu et al (Xu et al., 2020). A similar result was observed when we discussed the anti-inflammatory properties of Ganoderma lucidum (G. lucidum) polysaccharides of low weight (Liu et al., 2018).

The biological activities may be affected by the high proportion of monosaccharides in polysaccharides. According to Yang et al. (2020d), The antioxidant activity of the polysaccharides was significantly influenced by the content of uronic acid. In general, neutral polysaccharides are thought to have a higher free radical scavenging capacity than acidic polysaccharides. Another literature reported that C. cicadae polysaccharides with high ratios of uronic acid, arabinose, and galactose were considered to have significant contributions to antioxidant activity (Wang et al., 2022).

Furthermore, the composition of monosaccharides in polysaccharides were also closely related to the biological activities (Su & Li, 2020; Xu et al., 2019; Yiyong & Yiting, 2018). Mannose, glucose, and galactose were found to positively impact on the antioxidant activity of cicada polysaccharides (Tang et al., 2022). Additionally, polysaccharide conformations are another element that can influence biological activity. According to Surenjav et al., the antitumor biological activity of polysaccharides with a single flexible chain was significantly lower than that of natural triple helix lentinan (Surenjava et al., 2006).

It is intriguing and important to study the chemical structure of polysaccharides, the composition of monosaccharides, molar ratio of parent compounds and their biological activities. The other parameters that influence the activities are also eager to be found out.

2.2. Nucleosides

The major nucleosides of P. cicadae are uracil (C₄H₄N₂O₂, Figure 1, 2), uridine (C₉H₁₂N₂O₆, Figure 1, 3), thymidine (C₁₀H₁₄N₂O₅, Figure 1, 5), N6-(2-hydroxyethyl) adenosine (HEA) (C₁₂H₁₇N₅O₅, Figure 1, 6), adenosine (C₁₀H₁₃N₅O₄, Figure 1, 7) and cordycepin (C₁₀H₁₃N₅O₃, Figure 1, 8). Nucleoside analogs share adenosine and cordycepin's structural similarities. The absence of 3’ -hydroxy in cordycepin is the only structural difference between it and adenosine. Figure 1 provides detailed information.

Among them, adenosine is frequently used as a typical compound for determining nucleoside levels since it has a base that contains nitrogen. When adenosine is used as a raw material or precursor in synthesizing cordycepin, it serves as a key quality index for measuring cicada flowers. Here, we discussed the typical nucleosides existed in P. cicadae and/or C. cicadae.

2.2.1. Cordycepin

Cordycepin (C₁₀H₁₃N₅O₃, Figure 1, 8) as received a lot of attention for its great therapeutic potential. It is also called 3’-deoxyadenosine (Frederiksen et al., 1965), and is an important secondary metabolite in Cordyceps species such as Cordyceps militaris (C. militaris) (Cunningham et al., 1950) and Cordyceps sinensis (C. sinensis). Additionally, it is present in the broth and mycelium of P. cicadae (Li et al., 2007).

Cordycepin combines with certain adenosine receptor and activates downstream biomarkers to perform biological activities subsequently. According to the reports, Cordycepin can interact with A1, A2A, A2B and A3 adenosine receptors subtypes to exert benefits such as anti-inflammation, anti-virus, cell repair, and protection of liver, kidney, heart, and brain (Cao et al., 2017; Chen et al., 2014; Ravani et al., 2017; Sheth et al., 2014). It can also inhibit protein synthesis and cell adhesion by altering intracellular signal transduction (Varani et al., 2011).

Previously, the fruiting body of C. militaris was mostly used to extract natural cordycepin. However, the needs of the industry could not be met by this production process because it had complicated cultural conditions and a low cordycepin yield. The chemical production of cordycepin has been reported since the 1960s, giving it a preliminary development in the field (Hansske & Robins, 1985; Kwon et al., 2003; Ohno et al., 1984). Adenosine is typically used as a raw material to undergo a sequence of reactions in acidic or alkaline conditions. However, several chemical methods have limitations that make them unsuitable for industrial production, including high costs, environmental pollution, complex processes, and poor yields. Later studies of biological cordycepin synthesis methods, such as enzymatic production and fermentation, have been reported.

Researchers have made great contributions to improving cordycepin yield in C. militaris, such as spore hybridization and mutagenesis technology (Kang et al., 2017; Masuda et al., 2014; Wongsa et al., 2020). P. cicadae can also be found to obtain cordycepin from the mycelium, with a relatively high content (Li et al., 2007). The medium components were closely related to cordycepin yield.

Although the health benefits of cordycepin have been recognized but the commercial products of cordycepin is rare. The fruit bodies or the mycelia of C. militaris have been successfully commercialized for medicinal and health products, and even for direct consumption as edible mushroom in supermarket. Up to 2023, there are 43 health foods made from Cordyceps approved by the Ministry of Health of the People's Republic of China. Among them, there are at least two medicinal products made from C. militaris: C. militaris mycelia powder (Z20030034) and capsule (Z20030035) manufactured by Jilin Zhongsheng Pharmaceutical Co., Ltd. (Dong et al., 2015).

Many different biological activities and pharmacological effects of cordycepin, including antibacterial (Jiang et al., 2019), antiviral (Ryu et al., 2014), antitumor (Khan & Tania, 2020), and anti-inflammatory effects (Ko et al., 2013; Yoou et al., 2017a), have been demonstrated in many investigations. In Figure 2, we summarized some typical underlying mechanisms of antitumor, antiviral, anti-inflammatory and antioxidant activities reported in literatures.

Figure 2. The antioxidant, antitumor, antiviral and anti-inflammatory effects and their mechanisms of cordycepin (COR). Different models or diseases are listed in each aspect. Some biomarkers with the up arrow are accelerated, while, biomarkers with the down arrow are inhibited. Abbreviations: transforming growth factor-β: TGFβ; Superoxide dismutase: SOD; Catalase: CAT; Glutathione Peroxidase: GPX; glutathione reductase: GR; Glutathione S-transferase: GST; Malondialdehyde: MDA; Aspartate Transaminase: AST; Alanine transaminase: ALT; C/EBP homologous protein: CHOP; Reactive oxygen species: ROS; Nuclear factor-κB: NF-κB; Nuclear factor erythroid 2–related factor 2: Nrf2; Heme oxygenase-1: HO-1; Interleukin-1β: (IL)-1β, - 13, -6; Mouse doubleminute 2 homolog: MDM2; B-cell lymphoma-2: Bcl2.

2.2.2. N6-(2-hydroxyethyl)–adenosine (HEA)

N6-(2-hydroxyethyl)–adenosine (HEA, C₁₂H₁₇N₅O₅, Figure 1) is another significant nucleoside compound that can be found in C. cicadae (Ke & Lee, 2016; Ke & Lee, 2019) and C. sinensis (Zhang et al., 2016).

HEA is a potent antioxidant, anti-inflammatory, and hypoglycemic compound with various therapeutic properties, including anti-diabetes, renal protection, anti-inflammatory, antitumor, and sedative effects (Li et al., 2019a; Lu et al., 2015; Meng et al., 2015; Zheng et al., 2018). Studies show that HEA has anti-inflammatory benefits by increasing antioxidant enzyme activity, inhibiting lipid peroxidation, and lowering pro-inflammatory factors like IL-6, IL-1β, TNFα, and NF-kB (Wang et al., 2019a). Additionally, HEA can inhibit H₂O₂-induced cell damage by lowering ROS production to restore or preserve mitochondrial function (Zhang, Wu, et al., 2019a). Studies demonstrated that cicada HEA has significant hypoglycemic and hypolipidemic effects on type 2 diabetic rats (Sun et al., 2021), and can be used as a supplement to diabetes management and reduce the many diabetic complications in genetically obese mice (Li et al., 2019a).

2.3. Ergosterol and its peroxide

The secondary metabolites ergosterol (C₂₈H₄₄O, Figure 1, 9) and its peroxide, ergosterol peroxide (EP, a C28 sterol, Figure 1, 10), are both significant secondary metabolites in numerous medicinal mushrooms, apart from P. cicadae and C. cicadae.

Ergosterol is a precursor to vitamin D2 (Figure 3), which can aid in promoting calcium absorption and digestion in humans (Banlangsawan & Sanoamuang, 2016; Chen et al., 2022; Jasinghe & Perera, 2005). In 1889, ergosterol was first isolated from Xylaria avenae (Weete, 1989). Soon after, scientists extracted ergosterol from P. cicadae/C. cicadae and other fungi (Ge et al., 2007; Kuo et al., 2002a; Kuo et al., 2003; Toh Choon et al., 2012). Ge et al. reported the yield of ergosterol (0.6256 ± 0.0309 mg/g) from P. cicadae, which was higher than that in natural C. cicadae (0.5743 ± 0.018 mg/g) (Ge et al., 2007).

Figure 3. Conversion of ergosterol to vitamin D₂.

Ergosterol has been the subject of many studies, and its biological functions, such as anti-inflammation, antitumor, etc., have gathered a lot of attention. The supplement of ergosterol can been used to prevent vitamin D deficiency and type 2 diabetes (Nutrition et al., 2022; Jasinghe & Perera, 2005; Banlangsawan & Sanoamuang, 2016; Chen et al., 2022). The antitumor properties have also been studied in the lung (Wu et al., 2018), liver (Lin et al., 2017; Sankaran et al., 2017), breast (Li et al., 2015), stomach (Hu et al., 2014b) and prostate (Muñoz-Fonseca et al., 2021) cell lines.

EP was reported to have been isolated from a wide variety of fungi, including C. cicadae, G. lucidum, Paecilomyces tenuipes (P. tenuipes), Trichophyton Schoenleini (T. Schoenleini), and Sarcodon aspratus (S. aspratus) (Bauslaugh et al., 1964; Chen et al., 2009; Nam et al., 2001; Takei et al., 2005). Nowadays, the fermentation broth is another resource to get EP (He et al., 2018).

EP demonstrates physiological activities like immune regulation (Kuo et al., 2003), antioxidation (Kim et al., 1999), anti-inflammation (Kobori et al., 2007), antitumor abilities (Kahlos et al., 1989) and antiviral effects (Lindequist et al., 1989). Numerous cancer cells, including breast cancer (El-Sherif et al., 2020a), prostate cancer (Rhee et al., 2012), myeloma (Li, Wu, et al., 2016), and renal cell carcinoma (He et al., 2018), can be inhibited by EP in vitro. The key antitumor mechanisms are involved in inhibiting cell growth, arresting cell cycle, downregulating oncogene expressions, and upregulating tumor suppressor gene expressions.

2.4. Cordycepic acid

Cordycepic acid (C₆H₁₄O₆, Figure 1, 11) is a polyol-structured isomer of quinic acid, also known as 1,3,4,5-tetrahydroxycyclohexanoic acid. Cordycepic acid was initially isolated from C. sinensis in 1957 (Chatterjee et al., 1957). The composition of the crystalline substance was determined to be D-mannitol (Chatterjee et al., 1957). The content of cordycepic acid is one of the key factors used to evaluate the quality of C. sinensis.

In addition, the fermentation broths, the mycelia, and the fruiting bodies of C. cicadae, P. cicadae, and C. militaris, all contain cordycepic acid (Dong et al., 2012; Dou et al., 2013; Raethong et al., 2020; Shi et al., 2022). Xia et al. (2014) discussed the contents of cordycepic acid in the fruiting bodies of different strains, and found that the fruiting body of P. cicadae had a high concentration of cordycepic acid; it may contain up to 78.19 mg/g, which is higher than that in C. sinensis (77.27 mg/g), C. militaris (43.36 mg/g), and natural C. cicadae (53.6 ± 1.16 mg/g). Apart from the different strains, the contents in fruiting bodies also change as progress through their growth stages and are influenced by various fermentation factors, including carbon sources, nitrogen sources, and inorganic salts (Dou et al., 2013; Raethong et al., 2020; Shi et al., 2022). Additionally, the parameters in solid fermentation, such as the medium, light and humidity were also affected cordycepic acid contents (Curran, 1980).

Cordycepic acid can be used clinically to treat patients with cerebral ischemia and trauma, such as enhancing cerebral microcirculation and cerebral blood flow (Jiang, 1987; Mendelow et al., 1985). Besides, cordycepic acid shows a significant lung tumor inhibitory effect by regulating Nrf-2/HO-1/NLRP3/NF-κB signaling pathway (Wang et al., 2020).

2.5. Myriocin

Myriocin (C₂₁H₃₉NO₆, Figure 1, 12) has been known as either ISP-1 or thermozymocidin and has been produced from Mycelia sterilia, Isaria sinclairii and C. cicadae (Lee et al., 2010). It is a long-chain alpha-amino acid, with unsaturated fatty acids chains in the structure. Its chemical structure was identified as (2S, 3R, 4R, 6E)-2-amino-3,4-dihydroxy-2-hydroxy-methyl-14-oxo-6-eicosenoic acid (St-Jacques, 1973). Myriocin can be produced through fermentation because Yang et al. discovered that yeast extract and sucrose are best for its synthesis (Yang et al., 2008).

As an immunosuppressive agent, myriocin has the potential to be clinically effective. In vivo and in vitro experiments have revealed that it is 10–100 times more potent than the immunosuppressive agent cyclosporine A (CsA), with fewer adverse effects to the kidneys (Fujita et al., 1994). In addition, the antitumor effect of myriocin is proved by Choi et al. (2014). It was shown that ISP-1 can activate DR4 pathway and induce apoptosis of lung cancer cells.

2.5. Other metabolites

Other secondary metabolites, such as amino acids, terpenoids, and flavonoids, can be found in large quantities in the fermentation broth or mycelium of P. cicadae and C. cicadae.

Some cyclodepsipeptides have been identified from P. cicadae and C. cicadae. Kuo et al. (2002a) isolated cyclodepsipeptides with antitumor, anti-convulsant, anti-arrhythmic, and sedative effects from cicada flowers. Later, other cyclodepsipeptides, such as beauvericin (Figure 1, 19), beauvericin A (Figure 1, 20), beauvericin B (Figure 1, 21), beauvericin E (Figure 1, 22), beauvericin J (Figure 1, 23) and cordycecin A (Figure 1, 24) have also been purified (Lin et al., 2005; 2017; 2017; Wang et al., 2014a).

Moreover, furan-containing compounds are purified from the flowers of C. cicadae, those are 5-(2-hydroxyethyl) -2-fluoroacetic acid (Figure 1, 15), 2-(5-(3-hydroxybutyl) furan-2-yl) acetic acid (Figure 1, 16), methyl-2-(5- (3-hydroxybutyl) furan-2-yl) acetate (Figure 1, 17), 2-(5-(3-hydroxybutyl) furan-2-yl) acetic acid (Figure 1, 18), and a-furoic acid (Figure 1, 25) (Chu et al., 2015; Yang et al., 2019c; Zhang, Hu, et al., 2019b).

Several antibiotic compounds such as cephalosporolide E (Figure 1, 26), cephalosporolide J (Figure 1, 27), 3-benzyl-6-isopropyl-2,5-piperazinedione (Figure 1, 28), and 3-isobutyl-6-isobutylpiperazine-2,5-dione (Figure 1, 29) were found in the mycelia of the hybridization of C. cicadae and C. militaris (Yang et al., 2019c). Among them, 3-benzyl-6-isopropyl-2,5-piperazinedione and alpha-furoic acid were both showed moderate inhibitory activities against Panagrellus redivivus (Yang et al., 2019c).

In addition to ergosterol and its peroxides, other sterols have been found, such as 9,11-dehydroergosterol peroxides (Figure 1, 30), 3β, 5α, 6β- (22E, 24R) -ergosta-7,22-dien-3,5,6-triol (Figure 1, 31), 3β, 5α, 6β-6-methoxyergosta-(22E, 24R)-7,22-dien-3,5-diol (Figure 1, 32), 4-hydroxy-17R-methylincisterol (Figure 1, 33), 5α, 6α-epoxy- (22E, 24R) -ergosta-8, 22-diene-3β, and 7α-diol (Figure 1, 35), (22E, 24R) -Ergosta-5,7,trien-3-ol(ergosterol) (Figure 1, 36) (Wang et al., 2017a).

Some aromatic compounds have also been isolated from cicadas, such as 5-methoxycinnamic acid 3- (4’-O-methyl-β-glucopyranoside) (Figure 1, 37), cordycepone (Figure 1, 38), oosporein (Figure 1, 39), fumimycin (Figure 1, 40) and stipitatonic acid (Figure 1, 34) (Zhang & Xuan, 2007; Zhang, Hu, et al., 2019b). Among them, oosporein, fumimycin and stipitatonic acid have been reported to have broad-spectrum in vitro antibacterial, antioxidant and cytotoxic activities (Zhang, Hu, et al., 2019b).

Researches will be performed to continue to discover the unknown secondary metabolites with pharmacologic effects.

3. Biological activities

Metabolites have been reported to have a wide range of biological activities, such as immunoregulatory, antibacterial, antioxidant, and antitumor activities. In the study, we comprehensively summarized the pharmacological effects and their mechanisms of these metabolites derived from P. cicadae and/or C. cicadae (Table 3). Here, we discussed in detail based on each pharmacological effect.

3.1. Immunoregulatory activity

It has been observed that polysaccharides originating from microorganisms or plants can improve immunity by activating macrophages or enhancing the synthesis of cytokines and immunoglobulins (Huang et al., 2016; Li et al., 2017c; Shen et al., 2017). Polysaccharide is one of the most abundant and bioactive components found in mushroom fruit bodies, cultured mycelia, and fermentation broths (Chen et al., 2021). It interacts with receptors on the cell surface, affects the expression of some signaling biomarkers, and then increases the production of immune factors or antibodies, so as to play a role in regulating the immune function of the body (Jiang et al., 2010).

The polysaccharides from P. cicadae and/or C. cicadae have been reported to have immunomodulatory properties (Jin et al., 2008; Tian et al., 2022; Xu et al., 2018). They can regulate the NF-κB/MAPK signaling pathway to improve cellular immunity (Tian et al., 2022) and the TLR4 signaling (Kim et al., 2011) pathway to increase dendritic cell maturation. We are all aware that an excessive increase in inflammatory cytokines can cause inflammation, but an increase in cytokines within a reasonable range can stimulate cellular immunity. High doses of polysaccharides have been shown to significantly increase serum levels of TNF-α, IL-1β, IL-6, IL-10, NO, or immunoglobulin A levels, activating the cellular immune response and enhancing cellular immunity (Jin et al., 2008; Wang et al., 2022; Xu et al., 2018).

Similar to the study on the regulation of intestinal flora by polysaccharides from Grifola frondosa (Li et al., 2019b), polysaccharides from P. cicadae can regulate the systemic immune response by elevating the relative abundance of beneficial bacteria (Lactobacillus) and the concentration of short-chain fatty acids in the intestine (Wan et al., 2022). C. cicadae polysaccharides have been reported to have the abilities in improving metabolic disorders and reducing liver oxidative stress by increasing glutathione peroxidase (GSH-Px), SOD, and catalase (CAT) activities and decreasing lipid peroxidation (MDA) levels (Zhang et al., 2021). In addition, P. cicadae polysaccharides can affect cellular proliferative activities and their immune-enhancing activity. Figure 4 showed the general reported mechanisms of immune regulation of C. cicadae and/or P. cicadae in detail.

Figure 4. The general mechanism of immune regulation of C. cicadae and/or P. cicadae. The immunomodulatory effect exerts in two ways. ① C. cicadae and/or P. cicadae polysaccharides can regulate the systemic immune response by increasing the relative abundance of beneficial bacteria (e. g. Lactobacillus) and the concentration of short-chain fatty acids in the intestine; ② C. cicadae polysaccharides can alleviate metabolic disorders and reduce liver oxidative stress by changing the activities of enzymes and reducing the levels of inflammatory factors.

As a source of polysaccharides, C. cicadae spores are also being researched. The active ingredients found in C. cicadae spores are typically believed to be nearly identical to those found in C. cicadae mycelia and C. cicadae fermentation broth (Sun et al., 2017; Wang et al., 2017a). Polysaccharides in spores have also been reported to have immune-enhancing properties (Fu et al., 2019; Sun et al., 2017; Wang et al., 2017c). Similar to lentinan, C. cicadae spore polysaccharides could protect mice given the immunosuppressive drug cyclophosphamide by increasing macrophage phagocytic activity and regulating the secretion of cytokines and immunoglobulins (Zheng et al., 2022).

3.2. Antitumor activities

Numerous studies have extensively explored the antitumor properties of P. cicadae and C. cicadae. Several tumor cells were used as the assessment model, including renal cancer cells (He et al., 2018), gastric cancer cells (Xie et al., 2019), liver cancer cells (Wang et al., 2014a), and cervical cancer cells (Xu et al., 2021a) to evaluate the mechanisms of the components from P. cicadae or C. cicadae. Several components have been claimed to have antitumor properties, including EP, adenine, uridine, adenosine, HEA, polysaccharides, and certain organic solvent extracts from fruiting bodies, mycelia, and fermentation broth.

How cancer cells are out of the regulation of cell proliferation was the important question to be answered by the researchers. The two most important mechanisms of antitumor action are apoptosis and cell cycle arrest. However, the fundamental driving forces are very diverse depending on the various elements or components.

EP, a secondary metabolite of P. cicadae, was discovered to have antitumor activity by He et al. (2018). It was reported that EP promoted apoptosis, inhibited migration and invasion, blocked the cell cycle, and prevented DNA replication and mitosis (He et al., 2018). The ethanol extract of C. cicadae, with adenine, uridine, adenosine, and HEA presented, showed antitumor effect by the mechanism of cell cycle arrest, MMP depolarization, Ca²⁺ overload, over-expressed Bax, AIF, caspase-8, caspase-6, and caspase-3 and decreased Bcl-2 (Xie et al., 2019).

The polysaccharide extracts from C. cicadae spore powders have antitumor properties comparable to those of Ganoderma lucidum spores (Wang et al., 2012a; Zhu et al., 2012). These extracts can significantly lower the expression of β-catenin and N-cadherin and inhibit Wnt/β-catenin signaling and caspase-mediated mitochondrial apoptosis pathways (Sun et al., 2017). Additionally, C. cicadae may also have an impact on G2/M arrest to obtain the antitumor impact. Besides, C. cicadae polysaccharides could upregulate the expression of P53 (Xu et al., 2021a) and downregulate the expression of cyclin E, cyclin A, and CDK2, which prevented Hela cells from proliferating.

To maximize the antitumor benefits, some researchers have suggested combining natural elements from P. cicadae and C. cicadae with medicines like cisplatin, adriamycin, and cyclophosphamide (Cai et al., 2010; Jin et al., 2008). Various outcomes were obtained. Cai et al. (2010) found that the combination of polysaccharides and adenosine had specific antitumor effects in vitro. While the addition of polysaccharides did not strengthen the antitumor activity of adriamycin or cisplatin. Another study (Jin et al., 2008) found that combining P. cicadae with cyclophosphamide synergistically enhanced antitumor activity.

These studies have demonstrated the potential for P. cicadae and C. cicadae to serve as sources of natural antitumor drugs. Exploring the synergies of the combination of natural extracts and chemical compounds is a meaningful direction.

3.3. Antioxidant and anti-aging activities

Several mechanisms, such as reducing capacity, inhibiting chain initiation, binding transition metal ion catalysts, peroxides decomposition, and radical scavenging, can be used to explain the antioxidant and anti-aging activities. The cellular assessment of antioxidant activities involves the enzyme capacities (SOD, CAT, GSH-Px, etc.), lipid peroxidation products levels (malondialdehyde, lipofuscin, etc.), and the accumulation of ROS. The antioxidant effect, which has an anti-aging impact, is attained by raising or lowering these levels.

Numerous studies have shown that P. cicadae and/or C. cicada have free radical scavenging abilities (He et al., 2010; Ren et al., 2014; Wang et al., 2019b). According to (2019b), the polysaccharides from C. cicada scavenged hydroxyl radicals at various concentrations in a dose-dependent manner. The significant antioxidant properties of P. cicadae polysaccharides and C. cicadae fruiting body polysaccharides were also demonstrated by assessing DPPH free radical, superoxide radical, and hydroxyl radical scavenging capacities in the tube (Ren et al., 2014). He et al. (2010) also documented the ability of P. cicadae polysaccharides to scavenge DPPH, superoxide, and hydroxyl radicals.

The protective and repairing effects on oxidative stress damage models also reflect the antioxidant and anti-aging capacities of the sample. On H₂O₂-induced oxidative damage in PC12 cells, the protective effect of HEA was reported (Zhang, Wu, et al., 2019a). Compared to the damaged model, HEA could boost intracellular antioxidant enzyme activity and cell viability, decrease dehydrogenase release, and limit the ROS production and lipid peroxidation production. In addition, the decrease in inflammatory cytokines such as IL-6, IL-1β, TNF-α, and NF-кB was observed. Animal experiments supported the antioxidant properties of polysaccharides from C. cicadae. It was reported that polysaccharides from C. cicadae spores can boost SOD, CAT, and GSH-Px activities, indicating that they have the antioxidant capacity and hepatoprotective activities (Zhang et al., 2021). Another study found that C. cicadae polysaccharides were crucial in prolonging the lifespan of Drosophila, probably because of the upregulated expression genes associated with antioxidants such as CAT and SOD1 (Zhu et al., 2020b).

Hyaluronan (HA) in cells is a natural component in the skin that helps absorb moisture from the skin surface. The lack of HA results in skin water loss, sagging, increased wrinkles and decreased elasticity. Shao et al. (2023) found that C. cicadae extract promoted HA synthesis in fibroblasts, and the extract was rich in polysaccharides, and five alditols (mainly mannitol).

In addition, it is reported that C. cicadae and P. cicadae showed potential therapeutic benefits in rats with adenine-induced chronic renal failure, increasing SOD activity and decreasing MDA and GSH (Li et al., 2018). Additionally, the serum levels of BUN and Crea were significantly decreased, and TP, ALB, RBC, HCT, and HGB were increased. These results suggested that C. cicadae and P. cicadae can inhibit oxidative stress and enhance antioxidant capacity to exert renal protective effects.

These findings show that P. cicadae and C. cicadae may be investigated as potential natural antioxidants and used as new dietary supplements to delay aging.

3.4. Anti-inflammatory effect

There are lots of factors, such as physical damage, metabolic disorders, infection-related factors, oxidative stress, and chemical substances, can cause inflammation (Salminen et al., 2022; Tilg et al., 2020). It is also well accepted that inflammation is linked with oxidative stress. Oxidative stress refers to elevated intracellular levels of ROS that is considered to be the most potent inflammatory mediators (Shin et al., 2008). Antioxidants play a significant role in reducing inflammation (Waxman K. 1996). The increase of antioxidant enzymes in cells can prevent acute and chronic inflammatory reactions, exert anti-inflammatory effects on the eyes, protect the kidneys, and have anti-diabetic properties.

Lipopolysaccharide (LPS)-induced inflammation, in vitro cell lines and in animals, represents a standard paradigm for studying inflammation. The anti-inflammatory mechanisms of the active substances may be involved in the increased expression of antioxidant enzymes, reduced the deposition of peroxide, and the decrease of the secretion of pro-inflammatory factors (Table 2). Figure 5 displays the general mechanisms of the anti-inflammatory effects of P. cicadae and/or C. cicadae.

Figure 5. Anti-inflammatory effect of P. cicadae and/or C. cicadae reported in the literatures. LPS-induced damage, oxidative damage and renal inflammation can be suppressed by P. cicadae and/or C. cicadae. Pathways and key hubs are exhibited. Inflammatory factors and antioxidant related enzymes are influenced. Abbreviations: Phosphatase and tensin homolog: PTEN; phosphatidylinositol 3-kinase: P13K; protein kinase B, PKB: AKT; Mechanistic Target Of Rapamycin: mTOR; Recombinant Toll Like Receptor 4: TLR4; Type I; of transforming growth factor β receptor: TβR I; Type II of transforming growth factor β receptor: TβR II;Monoclonal Antibody to Mothers Against Decapentaplegic Homolog 2, 3, 4, 7: Smad2, 3, 4, 7; Reactive oxygen species: ROS; Malondialdehvde: MDA; Matrix metalloproteinase: MMP; Lactic dehydrogenase: LDH; Cyclooxygenase 2: COX-2; Tumor necrosis factor alpha: TNF-α; Prostaglandin E2: PGE2; Superoxide dismutase: SOD; Catalase: CAT; Glutathione: GSH.

Table 2. Display the anti-inflammatory mechanisms and pharmacological activities of Cordycepin.

Diseases	Models	Mechanisms of action	Refs
Asthma	Chronic asthmatic rats	↓p38MAPK ↓IL-5, IL-13, TNF-α	Fei et al., 2017
	Ovalbumin-Sensitized Mice	↑IL-10 ↓Lung eosinophils,Th17 Responses,IL-17α	Zhang et al., 2015
	Acute lung injury model in mice	↓p38-MAPK, NF-κB ↓(ICAM-1), IL-4, IL-5 and IL-13	Yang et al., 2015
Stroke	Mice with ischemia following 15 min of the bilateral common carotid artery occlusion and 4 h of reperfusion	↓SOD, MDA, MMP-3	Cheng et al., 2011
	ICH mouse model	↓NLRP3 ↓IL-1β, IL-18	Cheng et al., 2017
Atopic dermatitis	TSLP-stimulated HMC-1 cells	↓IL-6, tumor necrosis factor-α, IL-1β, p53, caspase-3	Yoou et al., 2017a
	2,4-dinitrofluorobenzne AD- like skin lesions	↓TSLP, MIP-2, ICAM-1, TARC, CCR-3, IL-4, IL-6, TNF-α, Caspase-1	Han et al., 2018
Osteoarthritis	Human OA chondrocytes	↓NF-Κb ↓MMP-13, IL-6, iNOS, COX-2	Ying et al., 2014
	Chondrocytes	↓MP-1, MMP-13, cathepsin K, cathepsin S, ADAMTS-4, ADAMTS-5	Hu et al., 2014a
	Sprague Dawley rats	↓TGF-β	Tao et al., 2020
	Intra-articular injection of monosodium iodoacetate in rats and surgical destabilization of the medial meniscus in mice	↓NF-κB ↓MMP-13, ADAMTS-5	Ashraf et al., 2019
Hepatitis	CCL4-induced liver injury	↑Nrf2 ↑SOD ↓ALT, AST, MDA	Qi et al., 2019
	GalN / LPS induced hepatotoxicity in mice	↓TLR4, NF-κB ↓IL-6, TNF-α, ROS, NOX	Li et al., 2017b
Nonalcoholic steatohepatitis	Mice with NASH.	↑AMPK Decreasing hepatic triglyceride accumulation, inflammatory cell infiltration and hepatic fibrosis	Lan et al., 2021
Leukemia	Human THP-1 acute monocytic leukemia cells	↓ERK/Akt ↑BaX↓BCL-2, AKt1, AKt2, AKt3	Wang et al., 2017d
	Leukemia cells	↓MAPK ↑P53 ↓cyclin A2, Cyclin E, CDK2	Liao et al., 2015

Table 3. Pharmacological effects and their mechanisms of the metabolic actives derived from P. cicadae and/or C. cicadae.

Metabolic active substances	Models	Pharmacological effects	Mechanisms of action	Refs
Polysaccharides	Hypertensive rats	Renal protection effect	↑SIRT1/FOXO3a/ ↓ROS, ECM	Cai et al., 2021
	Rats induced by high fructose / high fat diet (HF / HFD)	Reduce metabolic disorders	↓TNF-α, IL-1β, IL-6, ↑MDA, SOD, GSA-Px, CAT	Zhang et al., 2021a
	RAW 264.7	Immune regulation effect	↑NO, TNF-α,IL-1β,IL-6	Cen et al., 2021
	DN rats	Renal protection effect	↓TLR4/NF-κB, TGF-β1/Smad	Yang et al., 2020b
	Diabetes rats induced by intravenous injection of Alloxan Monohydrate (150 mg / kg)	Anti-Diabetic Activity	↑HDL, SOD, GSH ↓TC, TG, LDL, MDA, LDL, ALP, AST	Zhang et al., 2018
	Aged rats	Immune regulation effect	Increasing Proliferative activity and phagocytic rate	Yang et al., 2008
	C3H/HeN and C57BL/6 mice	Immune regulation effect	Degradating of inhibitor of nuclear factor-κB (NF-κB)α/β ↑TLR4	Kim et al., 2011
	Murine macrophage RAW264.7	Immune regulation effect	↑NO	Cheng et al., 2012
	Macrophage Raw 264.7 cells	Immune regulation effect	Degradating of inhibitor of nuclear factor-κB (NF-κB) α/β ↑TLR4- MAPKs, NF-κB NO, IL-1, IL-6, TNF- α	Kim et al., 2012
	Peyer's patches (PP) from C57BL/6J mice	Immune regulation effect	↑IFN- γ ↓IL-2	Takano et al., 2005
	Lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages	Anti-inflammatory effect	↓NO, IL-1β, TNF-α	Yang et al., 2019a
	P12 cells	Neuroprotetive effect	Reducing LDH leakage and alleviating mitochondrial damage ↓ROS	Tian et al., 2021a
HEA	Alloxan-induced diabetic rats	Anti-inflammatory effect; Renal protection effect	↑SOD, CAT, GSH ↓TNF-α, IL-1β, IL-6	Wang et al., 2019a
	Unilateral ureteral obstruction (UUO) was used to induce renal interstitial fibrosis in male C57BL/6 mice	Anti-inflammatory effect; Renal protection effect	Modulating TGF-β1/Smad, NF-κB Decreasing inflammatory cytokine level, TGF-β1-induced fibroblast activation	Zheng et al., 2018
	Type 2 diabetic db/db mice	Anti diabetes effect	↓TC, TG, HDL-C, LDL-C	Li et al., 2019a
	Rat model of type 2 diabetes (T2DM)	Anti diabetes effect	↓TC, TG, HDL-c, LDL-c	Sun et al., 2021
	Renal protection effect	Renal protection effect	Regulating the GRP78/ATF6/PERK/IRE1α/CHOP ↓ROS, ATF-6, PERK, IRE1α, CDCFHOP, IL1β	Chyau et al., 2021
Ergosterol peroxide (EP)	Transforming growth factor- β 1 (TGF- β 1) Stimulated renal fibroblast cell line (NRK-49F) rats	Renal protection effect	Blocking TGF- β1 stimulated phosphorylation of ERK1/2, p38 and JNK pathways ↓CTGF, ECM	Zhu et al. 2014a
	Phytohemagglutinin (PHA) - stimulated primary human T cells	Immune regulation effect	↓Cyclin E, IL-2, IL-4, IL-10, IFN	Kuo et al., 2003
	Renal cell carcinoma (RCC) cells	Antitumor activity	↓β-catenin	He et al., 2018a
Crude extract	CISP induced rats	Protective effect of testicular injury	↓TNF-α, IL-1β	Wang et al., 2020
	SGC-7901 cells	Antitumor activity	↓Bax, AIF, caspase-8, caspase-6, caspase-3, Bcl-2.	Xie et al., 2019
	Diabetes mouse model induced by alloxan	Hematopoietic function	Fighting hemorrhagic anemia and anemia caused by injection of phenylhydrazine hydrochlori	Song et al., 2007
	Human monocytes (hmnc)	Immune regulation effect	↓IL-2, IFN-γ	Weng et al., 2002
	Subtotal nephrectomy (SNx) model	Renal protection effect	↓Col IV, FN, TGF-β1, CTGF	Zhu et al., 2011b
	Optic nerve crush (ONC) rats model	Neuroprotetive effect	↑P1-N2, RGC ↑TUNEL positive cells, ED1 positive cells	Wen et al., 2022
	Adenine-induced chronic renal failure (CRF) rats	Renal protection effect	↑TP, ALB, RBC, HCT ↓BUN, Crea	Li et al., 2018
	Patients with chronic renal failure	Renal protection effect	↑Ccr, ALB, HCT, HB ↓BUN, Scr	Jin & Chen, 2006

It is reported that some therapeutic anti-inflammatory compounds are derived from C. cicadae and P. cicadae. An excellent anti-inflammatory impact of HEA on human bodies is thought to exist. In vivo and in vitro studies (Lu et al., 2015; Wang et al., 2019a; Zheng et al., 2018) have been performed to prove its anti-inflammatory properties. HEA can increase the activity of antioxidant enzymes in kidney tissues, including glutathione (GSH), catalase (CAT), and SOD. Some pro-inflammatory mediators (TNF-α, IL-6, IL-1β, NF-κB, and TGF-β1) (Wang et al., 2019a) can also be decreased in a dose-dependent manner.

Other active compounds in P. cicadae or C. cicadae, such as polysaccharides (Yang et al., 2019a; Zhang et al., 2021) and cordycepin (Lu et al., 2015), have also been demonstrated anti-inflammatory effects. Cordycepin exerts protective effects against inflammatory injury for many diseases including acute lung injury, asthma, rheumatoid arthritis, Parkinson's disease (PD), hepatitis, atherosclerosis, and atopic dermatitis (Ashraf et al., 2019; Cheng et al., 2011; 2020). According to Lu et al. (2015), the inhibition of pro-inflammatory cytokine production by cordycepin in C. cicadae was more efficient than adenosine and HEA in LPS-stimulated RAW 264.7 macrophages.

Additionally, hepatoprotective and renal protective activities of C. cicadae polysaccharides are also partly due to their anti-inflammatory mechanism. It can reduce renal interstitial fibrosis in rats with diabetic nephropathy by reducing inflammation and managing issues with intestinal flora (Yang et al., 2020b). C. cicadae polysaccharides can also play roles in the treatment of liver dysfunction by anti-inflammation and against oxidative damage in a rat model of metabolic syndrome induced by a high-sugar and high-fat diet (Zhang et al., 2021).

The Toll-like receptor (TLR) signaling pathway plays a key role in inflammation. TLR4 is an important transmembrane pattern-recognition receptor and has been extensively documented in a variety of inflammatory conditions. The stimulation of TLR4 initiates a series of signaling cascades that result in the activation of nuclear factor κB (NF-κB), interferon β regulatory factor 3 (IRF3), and mitogen-activated protein kinases (MAPKs) which mediates the upregulation of inflammatory cytokine gene expression leading to an inflammatory response (Shim et al., 2013; Sugiyama et al., 2016). HEA can inhibit TLR4-mediated NF-κB signaling pathway to reduce the pro-inflammatory response induced by LPS (Lu et al., 2015). HEA also obviously decreased LPS-induced inflammatory cytokine level in RAW 264.7 cells and TGF-β1-induced fibroblast activation in NRK-49F cells by modulating NF-κB and TGF-β1/Smad signaling (Zheng et al., 2018).

3.5. Anti-diabetic effect

Diabetes is a global epidemic that caused by either insufficient insulin production from the pancreas (type 1), or when the body cannot effectively utilize the insulin it produces (type 2) (World Health Organization, 1999). Drugs that regulate blood sugar and reduce insulin resistance are the mainstays of treatment for diabetes (Evans & Krentz, 1999; Guo et al., 2016). However, these drugs may cause several adverse reactions, such as hypoglycemia, lactic acidosis, and ketoacidosis (Li et al., 2004). The use of natural ingredients is considered an appropriate alternative medicine for treating diabetes and its associated complications (Day, 1998).

According to Day (1998), C. cicadae exhibited a hypoglycemic impact in a mouse model of type 1 diabetes induced by alloxan. A recent study has shown that HEA in C. cicadae can improve glucose tolerance and exhibits hypoglycemic effects on type 2 diabetes (Li et al., 2019a). A study compared the anti-diabetic benefits of P. cicadae and Astragalus membranaceus, and was reported that P. cicadae was more effective at postponing the onset of diabetic nephropathy than Astragalus membranaceus, in which the PI3K/AKT/mTOR signaling pathway played important roles (Yang et al., 2020a).

Researches will be performed continuedly to understand the underlying mechanisms and advance the application.

3.6. Antibacterial activity

Antibiotics will be used to treat bacteria using traditional antibacterial methods, but overuse of these drugs will result in bacterial resistance (Martínez, 2012; Yang et al., 2020a). Numerous reports have indicated that natural fermentation metabolites have antibacterial activity, including crude proteins and polysaccharides obtained from fungi and traditional Chinese medicines (Keypour et al., 2008; Kim et al., 2001).

According to the report, the crude proteins in the fermentation broth of P. cicadae can destroy the cell membrane structure of E. coli and alter the composition of entire cells or membrane proteins (Cen et al., 2021). Both of the extracellular and intracellular polysaccharides from C. cicadae have been demonstrated to have antibacterial activities. It was discovered that pathogenic microorganisms, including Escherichia coli, Klebsiella pneumoniae, Vivrio choler, Pseudomonas aeruginosa, Vibrio alginolyticus, Staphylococcus aureus, Vibrio parahaemolyticus, and Streptococcus pneumonia were significantly inhibited by both of the extra – and intracellular polysaccharides (Sharma et al., 2015). In addition, the inhibition rate of intracellular polysaccharides was marginally higher than that of extracellular polysaccharides.

Cordycepin was reported to have antibacterial activity by Ahn et al. (2000). It can inhibit the growth of Clostridium paraputrificum and Clostridium perfringens, but have no effect on Bifidobacterium spp. and Lactobacillus spp. (Ahn et al., 2000).

4. Conclusion and future perspectives

The advantages of low toxicity and simple cultivation make natural P. cicadae – the same genus as C. sinensis – a desirable species. It is expected to serve as a replacement for C. sinensis. Due to the limited natural resources and challenging artificial cultivation of P. cicadae, the use of modern biological fermentation technology to cultivate artificial P. cicadae mycelium has almost become the only and fundamental way to solve the problem.

The review primarily described the main active components, physiological functions, and mechanisms of P. cicadae and C. cicadae. We also provided a scientific summary of their typical therapeutic effects.

Recent studies have demonstrated that its extracts have positive therapeutic and health-promoting effects, particularly in inflammation-mediated diseases like diabetes, kidney disease, obesity, and cancer. We hope this will gives us a theoretical basis to use medicinal fungi more effectively.

Future research will be required to determine the exact functional mechanisms of the active ingredients from P. cicadae and/or C. cicadae. The efficacy and safety of single or combined use still need researchers’ attention. In addition, there is a need to advance the industrialization of active substance preparation.

Author contributions

Conceptualization, C.W.; Data curation and software, M.L.; Project administration and Supervision, L.L.; Investigation and Validation, W.C.; Data curation and Investigation, J.Z.; writing of the manuscript, F.D.; editing, J.Z. All authors have read and agreed to the published version of the manuscript.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

Data will be made available on request.