Of all Cordyceps species, only 35 have been characterized ^[1][23], of which C. militaris and C. sinensis are the two most widely studied. C. sinensis is a rare and expensive species that is difficult to cultivate, whereas C. militaris is a successful commercially grown species and is considered an alternative to C. sinensis ^[2][57]. By changing the culture conditions, the concentrations of bioactive compounds can be manipulated.

Several extraction methods and solvents have been employed for the isolation of selective bioactive compounds ^[3][4][50,58], with each extract exhibiting specific activity. As polar molecules, the aqueous extract contains functional concentrations of nucleosides and polysaccharides. In contrast, alcoholic extracts are rich in nucleosides, polysaccharides, and proteins with a high antioxidant potential.

C. militaris is a valuable TCM that grows on moth larvae (Lepidoptera). This fungus has been reported to treat respiratory, renal, hepatic, and cardiovascular diseases and has antiaging, antiviral, anti-inflammatory, and antitumor potentials ^[5][59]. Recently, C. militaris has become an economical alternative to C. sinensis in TCM because it can be easily cultivated under artificial conditions using diverse media ^[2][57]. Analyses of the compositions revealed that the concentrations of cordycepin and polysaccharides in the media of cultured C. militaris were higher than those in C. sinensis from the natural site ^[6][60]. The major bioactive components of C. militaris are nucleosides (adenosine, uridine, and cordycepin), myriocin, ergosterol, polysaccharides, L-arginine, and L-proline ^[2][7][8][43,57,61]. Previous studies have shown the presence of GABA (γ-aminobutyric acid), ergothioneine, D-mannitol (cordysepic acid), glycolipids, glycoproteins, xanthophylls (like carotenoids), sterols, statins, phenolic compounds, vitamins, and biominerals in C. militaris ^[6][9][60,62]. A previous study reported differences in the concentrations of cordycepin, cordycepic acid, and ergothioneine between fruiting bodies and mycelial biomass. The concentrations of cordycepin, cordycepic acid, and carbohydrates are higher in mycelial biomass, whereas those of ergothioneine and total amino acids are higher in fruiting bodies ^[9][62]. The reported optimal drying temperature for C. militaris is 60 °C, over which, cordycepin and phenolic compounds are lost ^[10][63]. Pentostatin, used as an antileukemia drug, is also produced by C. militaris through the same biosynthetic gene cluster for cordycepin production ^[11][64]. Similar to other chemotherapeutic drugs, it also has side effects such as diarrhea, nausea, and neurological toxicities ^[12][65]. Cordymin is an antifungal peptide that inhibits the mycelial growth of various fungi, including Candida albicans, Bipolaris maydis, and Rhizoctonia solani ^[13][66]. Ergosta-7,9(11),22-trien-3β-ol isolated from C. militaris shows anti-inflammatory and antioxidative activity ^[14][67].

Selective deterioration of cholinergic neurons in AD diminishes acetylcholine (ACh) levels, contributing to cognitive decline ^[15][68]. In addition to acting as a neurotransmitter, ACh also induces neurite outgrowth ^[16][17][69,70]. The methanolic extract of C. militaris promoted neurite outgrowth and ACh expressions in Neuro 2A mouse neuroblastoma cells in a dose-dependent manner (5–20 µg/mL). It also reversed scopolamine-induced memory deficits in rats and increased central cholinergic function at a dose of 300 mg/kg ^[18][71]. The ethanolic extract has been known to promote neurite outgrowth in Neuro 2A cells ^[19][72], provide protection from amyloid beta (Aβ)-induced toxicity ^[20][73], reduce the expression of inflammatory markers (cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS)), and downregulate mitogen-activated protein kinases/c-Jun N-terminal kinase/extracellular signal-regulated kinase (MAPK/JNK/ERK) pathway in C6 glial cells ^[21][74], which helped to reduce stress, inflammation, and apoptosis ^[22][75]. In addition, the extract restores recognition and memory functions by inhibiting oxidative stress (nitric oxide (NO) and lipid peroxidation) caused by toxic peptides ^[23][76]. Moreover, it upregulated the dopaminergic system in vivo and in vitro by upregulating tyrosine hydroxylase, an enzyme that catalyzes the rate-limiting steps in the biosynthesis of dopamine and other catecholamines ^[24][77].

One of the most conspicuous age-related diseases is ischemia, which is a common form of neurodegeneration that leads to cognitive impairment in the elderly ^[25][78]. The post-ischemic brain induces hippocampal neuronal death, neuroinflammation, and neuropathy, similar to AD ^[26][79]. Post-ischemic treatment with the butanolic extract of the fungus (WIB-801C: 50 mg/kg) decreased the inflammatory cell infiltration into ischemic lesions by inhibiting chemotaxis through adenosine receptor A3 (A3AR), thus providing neuroprotection in the middle cerebral artery occlusion (MCAO) rat model ^[27][80]. Moreover, after spinal cord injury (SCI), it mitigated blood–spinal cord barrier (BSCB) disruption by inhibiting matrix metalloprotease-9 (MMP-9), downregulating the expression of chemokine and promoting that of pro-nerve growth factor (NGF) in microglia (MG) ^[28][81]. The fungus also improved memory impairment caused by global cerebral ischemia and memory deterioration by delaying neuronal death, decreasing MG expression in the CA1 region of the hippocampus in rats ^[29][82], and increasing the expression of brain-derived neurotrophic factor (BDNF) and tyrosine kinase B (TrkB) in gerbils ^[30][83].

C. militaris aqueous extract showed beneficial effects in a D-galactose (Gal)-induced aging mouse model by improving memory ^[31][84]. Extract supplementation improved the levels of antioxidants (superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione (GSH)) and reduced malondialdehyde (MDA) and monoamine oxidase (MAO), which play important roles in the progression of aging. These results suggest a role for the antioxidant action of the fungus in recovering memory impairments in mice with D-Gal-induced aging ^[31][84].

In a recent study, nanoencapsulated C. militaris extract relieved neuronal pathology in SH-SY5Y cells (human neuroblastoma cells) by significantly improving dopamine secretion and the expression of dopaminergic-specific genes such as leucine-rich repeat kinase 2 gene (LRRK2), LIM homeobox transcription factor 1 beta (LMX1B), Forkhead Box (FOXA2), engrailed homeobox 1(EN1), and nuclear receptor-related 1 protein (NURR1) ^[32][85]. In line with this, C. militaris treatment enhanced the expression of neuronal protein paired box 6 (PAX6), a crucial player in brain development and function ^[33][86], and neuron-specific class III beta-tubulin (nestin), a marker of neuronal progenitor cells in the adult brain ^[34][87], indicating the role of C. militaris in enhancing neuronal maturation. Furthermore, it reduced amyloid precursor protein (APP) secretion by promoting autophagy ^[32][85]. As autophagy helps clear Aβ and tau aggregates in brain cells ^[35][88], C. militaris is considered important in AD treatment. The downregulated expression of AD-related genes presenilin 1 (PSEN1), presenilin 2 (PSEN2), and APP and the increased expression of the non-amyloidogenic pathway, ADAM metallopeptidase domain 10 (ADAM10), and sirtuin1 (SIRT1) by nanoencapsulated C. militaris extract suggest its potential in improving AD pathology at both the gene and protein levels ^[32][85].

These results suggest that the fungus is highly effective in protecting against memory-related neuronal degeneration in the brain and in retarding the progression of memory deficits associated with various NDs by its antioxidant, anti-inflammatory, and anti-apoptotic properties.

C. ophioglossoides, commonly known as the “golden thread Cordyceps”, is colonized on fruiting bodies of truffle-like Elaphomyces ^[36][89]. The fungus contains a variety of polysaccharides (antioxidant nature), ophiocordin (antibiotic), peptibiotics (antibiotic and antifungal properties), sesquiterpenes (antitumor activity), balanol (a protein kinase inhibitor with antitumor activity), and arsenocholine-O-sulfate (a nontoxic form of arsenic) ^{[36][37][38][39][40]}[89,90,91,92,93].

Aβ_(25–35) represents the biologically active region of Aβ, since it is the shortest fragment that displays large β-sheet aggregated structures, keeping the toxicity of the full-length peptide ^[41][94]; hence, it is often used as a model for inducing toxicity and memory deficits. The neuroprotective effect of C. ophioglossoides (methanolic extract) has been observed in vitro (extract: 100 μg/mL) and in vivo (extract:100 mg/kg) in Aβ_(25–35) AD models, where the fungal extract protected SK-N-SH human neuroblastoma cells from cell death and helped in the restoration of spatial memory loss in induced memory deficit by Aβ_(25–35) in rats probably by suppressing Aβ-induced oxidative stress ^[42][95].

C. sinensis is the most popular Cordyceps, which parasitizes the larva of Hepialus armoricanus. This fungus has long been used in TCM to promote longevity and has anti-inflammatory and antitumor activities ^[43][24]. The major biochemical markers of nucleosides are adenosine and cordycepin ^[3][44][50,96], with immunomodulatory and antioxidant activities. In 2008, Yuan et al. reported the presence of other nucleosides (thymine, adenine, cytosine, uracil, uridine, hypoxanthine, ionosine, guanosine, and thymidine) in aqueous extracts of C. sinensis ^[45][97]. Polysaccharides are major contributors to the biological activities of C. sinensis. Guan et al. identified several monosaccharides (fructose, mannitol, galactose, arabinose, ribose, rhamnose, mannose, xylose, glucose, and sorbose) using GC-MS ^[46][98]. Ergosterol is the main identified sterol ^[47][99] and is present either as free or esterified ergosterol ^[3][48][49][50,100,101] with antitumor activity ^[50][102]. Other compounds, such as polyamines and free fatty acids, have also been identified in C. sinensis extracts ^[51][103]. Two peptides (cordymin and cordycedipeptide) and an ergosterol (H1-A) with biological activities were also isolated from the fungus ^[52][104].

Aqueous and different alcoholic extracts (CSEs) from the fungus revealed the presence of the antioxidants hesperidin, rutin, and ascorbic acid by high-performance thin-layer chromatography (HPTLC). Hesperidin, rutin, and ascorbic acid were present at high concentrations in the aqueous extract. However, the highest hesperidin content was observed in the 25% alcoholic extract in comparison to others ^[53][105]. Additionally, adenosine, adenine, and uracil are present at higher concentrations in the aqueous extract than in the other extracts ^[54][106]. The protective effects of the extracts against hypoxia-induced oxidative stress and inflammation were studied in mouse hippocampal (HT22) cells. CSEs (250 μg/mL) show neuroprotection by increasing the expression of endogenous antioxidants (GSH, GPx, and SOD), limiting lipid oxidation by decreasing MDA levels and reducing the level of inflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) as well as transcription factor nuclear factor-κB (NF-κB) to various extents. The aqueous extract is more effective as an antioxidant in hypoxia, whereas the alcoholic extract prevented oxidative stress and inflammation ^[53][105], owing to the presence of more phenolics and flavonoids ^[54][106].

The aqueous ^[55][107] and ethanolic ^[56][108] extracts of the fungus were also evaluated for anti-inflammatory effects in an experimental middle cerebral artery occlusion/reperfusion (MCAO/R) model, as ischemic brain injury is associated with inflammatory reactions. In addition, neuronal apoptosis is triggered by increased expression of Bcl2-associated X (Bax), an apoptosis regulator, which in turn activates caspase-3 and inhibits B-cell lymphoma-2 (Bcl-2) expression ^[57][109]. The extract provided neuroprotection by downregulating the expression of inflammatory cytokines and other inflammatory mediators (IL-1β, TNF-α, Myeloperoxidase (MPO), and adhesion molecules ICAM-1, COX-2, and iNOS), blocking polymorphonuclear cell (PMNC) infiltration, thereby subsiding neurological deficits and infarct volume. Moreover, the aqueous extract also revealed the decreased Bax, cytochrome c (Cyt c), and caspase-3 protein expressions, which in turn improved mitochondrial membrane potential (ψ_m), thus modulating the electron transport chain in the mitochondria in vivo and in vitro ^[58][110].

Multiple sclerosis (MS) is an autoimmune demyelinating disease of the CNS that involves a variety of immune cells ^[59][111]. Inflammation resulting from MS is mediated by the infiltration of autoreactive T cells into the CNS through the blood–brain barrier (BBB) ^[60][112]. Amongst the T cells, primarily interferon-gamma (IFN-γ)-producing T-helper 1 (Th1) cells and IL-17-producing Th17 cells had an important role in the pathogenesis of the disease ^[61][113]. Effector molecules secreted by Th1 cells directly affect the phenotype, function, and recruitment of MG, whereas Th17 cells upregulate chemokines during the inflammatory process ^[62][114]. C. sinensis extract was reported to reduce the number of Th1 cells in a mouse model of MS/experimental autoimmune encephalomyelitis (EAE), thus relieving EAE severity and the associated pathology ^[63][115].

The neuroprotective effect of fermented fungus powder (Cs-C-Q80 or ‘corbrin capsule’) was evaluated in subcortical ischemic vascular dementia induced in a mouse model of right unilateral common carotid artery occlusion (rUCCAO) ^[64][116], which damaged the white matter region in the brain, resulting in myelin loss, glial activation, neuroinflammation, and dementia ^[65][117]. However, both the prophylactic and therapeutic administration of corbrin (1 g/kg) significantly reduced white matter lesions and improved learning and memory loss through anti-inflammatory actions ^[64][116]. A lower dose of corbrin (1 mg/kg) was effective in reducing the pro-inflammatory cytokines (TNF-α, IL-1β, IL-6), improving the levels of oxidative stress parameters (SOD, MDA), increasing ATP concentration, and alleviating neurological deficits in an MCAO mice model ^[66][118].

C. cicadae is the oldest known therapeutic fungus that feeds on Lepidoptera species larvae ^[67][119]. It has been used in TCM for the treatment of asthma, cancer, convulsions, dizziness, palpitations, and chronic renal disease. Natural C. cicadae is a slow-growing fungus in high demand, whereas its anamorph, Paecilomyces cicadae, can be cultured easily and used as a substitute for C. cicadae to accommodate market requirements ^[68][120]. Various bioactive compounds, such as cyclopeptides, myriocins, polysaccharides, nucleosides, and mannitol have been identified in C. cicadae ^[68][69][120,121]. LC-MS analyses have detected adenosine and adenosine analogs, N6-(2-hydroxyethyl)-adenosine (HEA), a Ca²⁺ antagonist, and an anti-inflammatory agent ^[70][71][72][122,123,124]. HEA is a major bioactive compound in C. cicadae that exhibits antidiabetic, sedative, analgesic, antitumor ^[73][125], and renoprotective activities ^[74][75][126,127]. Another isolated bioactive compound, ergosterol peroxide, exhibits immunomodulatory and anti-inflammatory effects ^[76][77][128,129].

Trauma to the CNS and NDs initiate a torrent of cellular and molecular reactions that result in neuronal loss and regenerative failure. To understand the associated mechanisms, the rodent optic nerve crush (ONC) model can be used and later extrapolated to NDs ^[78][130]. C. cicadae mycelium extract provided neuroprotection in the ONC rat model through anti-apoptotic and anti-inflammatory effects by improving retinal ganglion cell (RGC) density and P1-N2 amplitude ^[79][131], which intensified with visual–spatial attention in the visual cortex. The butanol fraction protected rat adrenal pheochromocytoma (PC12) cells against glutamate-induced oxidative damage. Additionally, the extract restored the mitochondrial function, suppressed ROS accumulation, upregulated the antioxidant enzymes (GPX and SOD), increased cell viability, decreased lactase dehydrogenase (LDH) release, and reduced apoptosis ^[80][81][132,133]. Subsequently, adenosine was identified as the main nucleoside responsible for this neuroprotective action ^[81][133]. The anti-inflammatory activities of three bioactive nucleosides (adenosine, cordycepin, and HEA) isolated from wild-type and artificially cultured C. cicadae were evaluated. Cordycepin was found to be more potent than other nucleosides in limiting the release of pro-inflammatory cytokines by lipopolysaccharide (LPS)-stimulated RAW 264.7; however, no synergistic effect of the three compounds was observed. LPS-induced pro-inflammatory responses were attenuated by HEA through the suppression of the toll-like receptor (TLR)-4-mediated NF-κB signaling pathway ^[82][134]. The effects of the hydroalcoholic fungal extract on cisplatin toxicity have also been evaluated. Cisplatin is an anticancer agent involved in multi-organ toxicity, including neurotoxicity. It accumulates in the dorsal root ganglion (DRG) and causes oxidative stress, neuronal apoptosis, and inflammation ^[83][135]. The nucleoside-rich extract of C. cicadae ameliorated memory impairment and neuropathy by reducing oxidative stress, acetylcholinesterase enzyme (AChE) levels, and inflammation in cisplatin-treated rats ^[84][136].

In a recent study, increased levels of bioactive compounds were obtained from cultured C. cicadae in deep ocean water (DOW) and minerals, thus increasing their therapeutic value ^[85][137]. The effect of DOW-cultured fungus (DCC) was investigated on D-Gal-induced brain damage and memory impairment in rats. DCC (100–500 mg/kg), in turn, improved cognition by alleviating the expressions of antioxidants and inflammatory genes (iNOS, TNF-α, IL-6, IL-1β, COX-2), along with reduced expressions of the aging-related proteins (glial fibrillary acidic protein (GFAP) and PS1) ^[85][137].