Hydroxyethyl adenosine analogs

Hydroxyethyl Adenosine Analogs (N6-(2-Hydroxyethyl)adenosine / HEA)

1. Identity: Chemical Names, Sources, and Common Forms

1.1 Nomenclature and Chemical Identity

N6-(2-Hydroxyethyl)-adenosine (HEA) is a natural adenosine derivative widely present in various fungi, with many biological and pharmacological activities. The compound is classified formally under several synonymous names in the chemical and pharmacological literature: N6-(2-hydroxyethyl)adenosine, N-(2-hydroxyethyl)adenosine, and 6-((2-hydroxyethyl)amino)purine riboside. Its molecular formula is C₁₂H₁₇N₅O₅ and the molecular weight is 311.29 g/mol. The compound is registered under CAS number 4338-48-1. HEA is a bioactive nucleoside identified from the butanolic fraction of Cordyceps cicadae.

N6-(2-hydroxyethyl)-adenosine (HEA) is a derivative of adenosine that was the first identified calcium ion channel antagonist from biological sources. As a purine nucleoside analog, it belongs to the broader class of adenosine analogs — molecules that share the core adenosine scaffold (an adenine nucleobase attached to a ribose sugar) but bear structural modifications at the N6 position of the purine ring or the ribose moiety. In HEA's case, the modification is a 2-hydroxyethyl substituent at the N6 (exocyclic amine) position of adenine.

1.2 Natural Sources and Producing Organisms

As one of the main biological components of Cordyceps species, N6-(2-Hydroxyethyl)-adenosine (HEA) was first isolated from Cordyceps and Isaria species in 1983. The initial isolation was performed by Furuya and colleagues, who tested for the presence of Ca²⁺ antagonists and inotropic agents in the water–ethanol extracts from mycelia of 17 Cordyceps species and 6 Isaria species using the left atrium of a guinea pig heart in an in vitro system.

The primary natural sources of HEA are entomopathogenic fungi — fungi that parasitize insects. The principal producing species include:

• Cordyceps cicadae (Isaria cicadae, "Cicada Flower") — an entomopathogenic fungus belonging to the family Clavicipitaceae which grows mostly on the larvae of Cicada flammata Dist. This is the species most intensively studied for HEA content.
• Cordyceps militaris — a species that contains a variety of bioactive compounds, including polysaccharides, cordycepin (3′-deoxyadenosine), cordycepic acid (mannitol), ergosterol, adenosine, and N6-(2-hydroxyethyl) adenosine, among other nucleosides.
• Cordyceps pruinosa — HEA, a primary biological component of Cordyceps species, was first isolated from Cordyceps pruinosa.
• Isaria tenuipes — noted among confirmed HEA-producing species alongside C. pruinosa and C. militaris. Up to now, the production of HEA has been detected only in a few species such as Cordyceps pruinosa, C. militaris, and Isaria tenuipes — prior to the discovery of HEA in Beauveria bassiana.
• Beauveria bassiana — a new HEA-producing fungus, identified based on morphological and phylogenetic characteristics. The HEA production was verified by reversed-phase HPLC/mass spectrometry, and HEA was also detected in the mycelia of two other B. bassiana strains from different origins, but not in the culture medium of all tested strains; the maximum production of HEA (0.8483 ± 0.0439 mg/g mycelia dry weight) was achieved on day 7.

Different nucleosides, including Adenosine, Adenine, Cordycepin (3′-deoxyadenosine), 2′-Deoxyadenosine, 2′3′-Dideoxyadenosine, 2′-Methoxyadenosine, N6-(2-hydroxyethyl)-Adenosine, 3′-Amino-3′-deoxyadenosine, Hypoxanthine, Uridine, and others have been reported to be found in different species of Cordyceps. Among this broad nucleoside profile, HEA, adenosine, and cordycepin are regarded as the principal bioactive nucleosides.

1.3 Co-occurring Constituents in Source Fungi

The main active constituents of C. cicadae are adenosine, 3′-deoxyadenosine (cordycepin), and N6-(2-hydroxyethyl)-adenosine (HEA). Profiling of nucleosides and their associated molecules is used as a chemical marker for Cordyceps and derived products, and is quite common practice for quality assurance. Other major constituents present in source fungi include polysaccharides, D-mannitol (cordycepic acid), ergosterol, and various amino acids.

1.4 Common Forms and Preparations

In traditional and commercial contexts, HEA is typically encountered in the following forms:

• Whole dried fruiting bodies or mycelia of C. cicadae or C. militaris, used in decoctions or powdered extracts following centuries-old preparation methods.
• Standardized ethanolic or aqueous-ethanolic extracts of Cordyceps mycelia, in which HEA is one of the quantified chemical markers. N6-(2-Hydroxyethyl) adenosine has been purified from C. cicadae by adding powdered fungus to ethanol, soaking overnight, centrifuging, filtering, and concentrating under reduced pressure.
• Submerged-fermentation-derived mycelia — due to the low yield of wild C. cicadae, artificial cultivation approaches are needed to meet increasing market demand; bioreactor culture can increase mass production and the abundance of HEA.
• Isolated/purified HEA — for research purposes, three nucleosides including 15.6 mg of cordycepin, 16.9 mg of HEA, and 23.2 mg of adenosine were obtained from 500 mg of crude sample in one-step separation using macroporous resin and high-speed counter-current chromatography from Cordyceps militaris.

2. Traditional and Historical Use

2.1 Traditions, Time Periods, and Geographic Extent

The cicadae flower has been used as traditional Chinese medicine — not only in China but also in Japan and Taiwan — for at least 1,600 years to treat fatigue, night perspiration, fever, childish convulsion, palpitation, epilepsy, and many eye diseases. This positions the medicinal use of C. cicadae (the primary HEA source) as one of the earliest-documented uses of entomopathogenic fungi in any medical tradition, predating modern awareness of HEA's molecular identity by many centuries.

The fruiting bodies of Cordyceps cicadae, an entomopathogenic fungus, have been used as a natural medicine over the centuries to treat various conditions including childhood palpitations, epilepsy, and convulsions. C. cicadae has been documented in many Chinese medicine prescriptions for the treatment of palpitations, epilepsy, convulsions, and several eye diseases.

2.2 Preparations and Methods of Traditional Use

In traditional Chinese medicine (TCM), C. cicadae was principally prepared as decoctions from the dried fruiting bodies (the fungal stroma growing from the buried larva). The whole preparation, rather than any isolated constituent, was the therapeutic vehicle. Cordyceps cicadae is a well-known traditional Chinese medicine for treating palpitations and eye diseases; it contains several bioactive compounds such as adenosine, N6-(2-hydroxyethyl)-adenosine (HEA), and polysaccharide.

It is important to note that HEA as an isolated molecule was not known to traditional practitioners; rather, it was one of many active constituents in the whole fungal preparation. Only following Furuya et al.'s 1983 isolation of HEA from Cordyceps and Isaria mycelia did researchers begin to attribute specific pharmacological actions to this specific compound.

2.3 Relationship to Cordyceps sinensis

C. cicadae has similar medical properties and effects to C. sinensis, the more globally recognized medicinal Cordyceps species widely used in TCM to tonify the lungs and kidneys and enhance energy. The shared nucleoside chemistry — including HEA — is part of the basis for this pharmacological overlap.

3. Key Constituents and Biosynthetic Origin

3.1 Chemical Structure

HEA is a purine nucleoside analog in which the N6 amino group of adenosine bears a 2-hydroxyethyl substituent. Its spectroscopic signature has been characterized: melting point 194–196°C; ESI-MS m/z 312 [M+H]⁺; and a characteristic ¹H-NMR spectrum showing H-2 at δ 8.19 (s), H-8 at δ 8.32 (s), and H-1′ at δ 5.85 (d, J = 6.4 Hz), among other resonances consistent with a riboside nucleoside bearing an N-hydroxyethyl side chain.

3.2 Biosynthetic Pathway in Fungi

Similar to the conversion of IMP and L-aspartate into N6-(1,2-dicarboxyethyl)-AMP by adenylosuccinate synthetase, IMP and ethanolamine can be converted into N6-(2-hydroxyethyl)-AMP by adenylyl hydroxyethyl synthetase, and N6-(2-hydroxyethyl)-AMP is subsequently dephosphorylated to yield HEA. This enzymatic pathway distinguishes HEA biosynthesis from that of other adenosine analogs in Cordyceps, such as cordycepin (which arises from a distinct set of biosynthetic genes designated Cns1 and Cns2).

3.3 Relative Abundance Among Nucleosides

Nucleosides, including adenosine, cordycepin, and N6-(2-hydroxyethyl)-adenosine (HEA), represent the major active components in Cordyceps sensu lato. The relative quantities of these three nucleosides vary by species, cultivation conditions, and extraction method. Across experiments with C. militaris, HEA has been isolated in amounts comparable to or slightly lower than adenosine and cordycepin: from the fruiting bodies of C. militaris, macroporous resin NKA-II absorbed more than 3.8 mg cordycepin, 4.7 mg HEA, and 4.7 mg adenosine per gram of resin.

4. Established and Proposed Mechanisms of Action

4.1 Calcium Channel Antagonism (Ca²⁺ Antagonism)

HEA was the first calcium antagonist derived from biological sources, has been intensively investigated because of its ability to inhibit tumour cell proliferation, restrain inflammation, protect kidneys, and function as a sedative and insecticide. HEA was the first calcium antagonist derived from biological sources, and can be used as an inotropic agent. This mechanism — modulation of intracellular calcium dynamics — is central to several of its downstream effects, including cardiovascular activity, sedation, and cell death pathways in cancer cells.

4.2 NF-κB Signaling Inhibition

HEA attenuated the LPS-induced pro-inflammatory responses by suppressing the toll-like receptor (TLR)4-mediated nuclear factor-κB (NF-κB) signaling pathway. This mechanism accounts for much of HEA's anti-inflammatory activity and is shared with several other natural nucleoside analogs from Cordyceps species. HEA also attenuated lipopolysaccharide-induced pro-inflammatory responses by suppressing the toll-like receptor 4-mediated nuclear factor-κB signalling pathway.

4.3 TGF-β1/Smad Pathway Suppression

In vitro, HEA obviously decreased lipopolysaccharide-induced inflammatory cytokine levels in RAW 264.7 cells and TGF-β1-induced fibroblast activation in NRK-49F cells by modulating NF-κB and TGF-β1/Smad signaling. The TGF-β1/Smad axis is a master regulator of fibrosis; HEA's suppression of this pathway underlies its antifibrotic actions in kidney tissue.

4.4 Endoplasmic Reticulum (ER) Stress Regulation

HEA induced reactive oxygen species (ROS) production and mitochondrial membrane potential depolarization; it could trigger caspase-dependent apoptosis, promoting intracellular Ca²⁺-related endoplasmic reticulum (ER) stress and autophagy. In the context of cytoprotection (e.g., in renal cells exposed to NSAIDs), HEA acts in the opposite direction: HEA (20 µM) effectively prevented ER stress by attenuating ROS production and gene expression of ATF–6, PERK, IRE1α, CHOP, IL-1β, and NF-κB within 24 h; HEA reversed the increase of GRP78 and CHOP protein expression levels induced by diclofenac and meloxicam, and restored the ER homeostasis.

4.5 Apoptosis Induction in Tumor Cells

HEA could trigger caspase-dependent apoptosis, promoting intracellular Ca²⁺-related endoplasmic reticulum (ER) stress and autophagy. In osteosarcoma cells specifically, HEA inhibited cell migration and invasion and modulated epithelial-mesenchymal transition (EMT) markers by upregulating E-cadherin and downregulating N-cadherin and vimentin; it downregulated IGF1 at both the mRNA and protein levels and reduced IGF1 secretion; furthermore, HEA inhibited the PI3K-AKT signaling pathway, which is activated by IGF1.

4.6 Adenosine Receptor Interactions

As a structural adenosine analog, HEA interacts with adenosine receptors — a family of G protein-coupled receptors (A1, A2A, A2B, A3) that mediate adenosine's endogenous signaling. HEA is an adenosine derivative associated with control of the brain and coronary circulation and has anti-inflammatory activity. Its insecticidal activity has been directly linked to adenosine receptor engagement: HEA exhibited insecticidal activity against Plutella xylostella larvae by targeting an adenosine receptor, suggesting an environmentally friendly pesticide.

4.7 Interaction with Human Serum Albumin

The binding constants between human serum albumin (HSA) and HEA were 27.102, 19.409, and 13.002 × 10³ M⁻¹ at 17, 27, and 37°C respectively; HEA can quench the intrinsic fluorescence of HSA via static quenching, and it can bind with HSA to form complexes with a single binding site. This binding to plasma albumin has implications for HEA's distribution and bioavailability in vivo.

4.8 JunB/PGC-1α Axis in Lipid Metabolism

More recently described mechanistic work in the context of non-alcoholic fatty liver disease (NAFLD) found that HEA significantly reduced the population of JunB⁺ adipocytes, which exert lower thermogenic capacity; further studies verified that HEA interacted with ASP-12, ASP-13, and TYR-15 of JunB subunits through hydrogen bonding, leading to activated PGC-1α activity and governing thermogenic adipocyte heterogeneity and consequent biological responses.

5. Scientific Evidence by Area of Use

Overall evidentiary note: As of the current literature, all published studies on HEA's specific pharmacological effects are preclinical — conducted in cell culture (in vitro) or in animal models (in vivo). No published registered clinical trials in humans evaluating isolated HEA have been identified. The host organism (C. cicadae and related fungi) has a long history of human medicinal use, but human clinical evidence specific to HEA as an isolated compound does not yet exist in the peer-reviewed literature. The evidence summarized below should therefore be read as exploratory and mechanistic, rather than confirmatory for human health applications.

5.1 Anti-inflammatory Activity

Body of evidence: In vitro and animal studies; no human clinical data specific to HEA.

In a study published in the Journal of Natural Products, investigators cultured the fruiting bodies of C. cicadae and investigated the anti-inflammatory activities of water and methanol extracts of wild and artificially cultured C. cicadae fruiting bodies; they determined the amount of three bioactive compounds (adenosine, cordycepin, and HEA) in the extracts and evaluated their synergistic anti-inflammatory effects. The results indicated that cordycepin was more potent than adenosine and HEA in suppressing LPS-stimulated release of pro-inflammatory cytokines by RAW 264.7 macrophages, and no synergistic effect was observed; however, HEA attenuated LPS-induced pro-inflammatory responses by suppressing the TLR4-mediated NF-κB signaling pathway. This result provides evidence for a defined mechanism of action (TLR4/NF-κB suppression) but represents a single preclinical cell-culture study with standard macrophage lines.

Pharmacological studies have demonstrated that HEA exhibits a wide range of effects, including anti-inflammatory, anticonvulsive, neuroprotective, antioxidant, antiangiogenic, hypolipidemic, immunoregulatory, and myocardium protective effects. These properties are supported across multiple in vitro and animal models but have not been tested in human subjects for HEA specifically.

5.2 Renal Protection and Anti-fibrotic Effects

Body of evidence: Multiple in vitro and animal studies; no human clinical data specific to HEA.

HEA, a physiologically active compound in C. cicadae, has been identified as a Ca²⁺ antagonist; renal interstitial fibrosis is characterized by inflammation and an excessive accumulation of extracellular matrix, which leads to end-stage renal failure. In a mouse UUO model, unilateral ureteral obstruction (UUO) was used to induce renal interstitial fibrosis in male C57BL/6 mice; different doses of HEA (2.5, 5, and 7.5 mg/kg) were given by intraperitoneal injection 24 h before UUO, and the treatment was continued for 14 days post-operatively. The findings indicated that HEA had a beneficial effect on UUO-induced tubulointerstitial fibrosis by suppression of inflammatory and renal fibroblast activation, which may be a potential therapy in chronic conditions such as renal interstitial fibrosis.

In studies of NSAID-induced renal tubular cell injury, the MTT assay revealed HEA to be nontoxic up to 100 µM toward HK–2 cells; HK–2 cells were pretreated with HEA (10–20 µM) and then insulted with diclofenac (DCF, 200 µM) and meloxicam (MXC, 400 µM) for 24 h; HEA (20 µM) effectively prevented ER stress by attenuating ROS production and gene expression of ATF–6, PERK, IRE1α, CHOP, IL-1β, and NF-κB within 24 h. This was a study in human proximal tubular cells (HK-2 cell line), representing in vitro work with human-derived cells — a step above animal cell lines but still not a clinical study.

Regarding cisplatin-induced nephrotoxicity, a study using doses of 10 mg/kg and 20 mg/kg body mass of HEA in cisplatin-challenged mice assessed markers of renal function and oxidative stress, including blood urea nitrogen, serum creatinine, and antioxidant enzymes (SOD, CAT, GSH-Px) and malondialdehyde (MDA) in kidney tissue. This evidence is animal-model only.

For diabetic kidney disease, N6-(2-Hydroxyethyl)adenosine (20–40 mg/kg, ip, once daily for 6 weeks) reduces the blood glucose level and diabetes-induced kidney damage, increases the activities of antioxidant enzymes (such as SOD, catalase, and GSH) in renal tissue, reduces the level of malondialdehyde (MDA), and ameliorates alloxan-induced diabetes in rat models. Again, this is rodent pharmacology.

Strength of evidence summary for renal effects: Consistent and mechanistically convergent across multiple preclinical models (fibrosis, diabetic nephropathy, NSAID injury, cisplatin toxicity), but entirely preclinical. No human data are available.

5.3 Antitumor / Anti-cancer Activity

Body of evidence: In vitro cell-line studies and one xenograft mouse study; no human clinical data.

Gastric carcinoma: HEA was assessed for its antineoplastic effect on gastric carcinoma. HEA exerted cytotoxic effects against gastric carcinoma cells (SGC-7901 and AGS) in a dose- and time-dependent manner. HEA induced reactive oxygen species production and mitochondrial membrane potential depolarization; it triggered caspase-dependent apoptosis, promoting intracellular Ca²⁺-related endoplasmic reticulum (ER) stress and autophagy. HEA significantly inhibited the growth of transplanted tumors in nude mice and induced apoptosis of tumor tissue cells in vivo. In conclusion, HEA induced apoptosis of gastric carcinoma cells in vitro and in vivo.

In the xenograft arm of this study, N6-(2-Hydroxyethyl)adenosine (75–150 mg/kg, po, once daily for 19 days) exhibited antitumor effects in SGC-7901 xenograft mouse models.

Lewis lung cancer and K562 erythroleukemia: HEA can inhibit the proliferation of tumour cells in vitro, including Lewis lung cancer and K562 erythroleukemia cells. These findings are in vitro only.

Osteosarcoma: In a 2024 study, IGF1 silencing and recombinant IGF1 treatments were used to explore HEA's mechanisms; HEA significantly decreased osteosarcoma cell viability and induced apoptosis in a dose- and time-dependent manner; it inhibited cell migration and invasion, and modulated EMT markers by upregulating E-cadherin and downregulating N-cadherin and vimentin; HEA downregulated IGF1 at both the mRNA and protein levels and reduced IGF1 secretion; furthermore, HEA inhibited the PI3K-AKT signaling pathway, which is activated by IGF1. This represents in vitro work in Saos2 and MG63 osteosarcoma cell lines.

Strength of evidence: Preclinical only. Mechanistic data from multiple cancer cell lines are internally consistent, and one xenograft study demonstrates in vivo effect in immunocompromised mice. However, xenograft models do not replicate the complexity of human oncology, and no Phase I or later trials are reported in the literature for HEA.

5.4 Antidiabetic and Antihyperglycemic Activity

Body of evidence: Animal models (rodent diabetes); one study in db/db mice.

Research published in the Journal of the Science of Food and Agriculture (2019) reported that Cordyceps cicadae mycelia and its active compound HEA exert beneficial effects on blood glucose in type 2 diabetic db/db mice. The db/db mouse is a leptin-receptor-deficient model of genetic obesity and type 2 diabetes, conferring somewhat higher translational relevance than chemically-induced rodent models.

Additionally, at 20–40 mg/kg ip, once daily for 6 weeks, HEA reduces the blood glucose level in alloxan-induced diabetic rat models, and exhibits anti-inflammatory and antioxidant activities in renal tissue. The mechanism in this setting involves restoration of antioxidant enzyme activities (SOD, catalase, GSH) and reduction of MDA.

Strength of evidence: Weak-to-moderate for antihyperglycemic effects; consistent across rodent models but no human data exist.

5.5 Hepatoprotective Activity / Non-Alcoholic Fatty Liver Disease

Body of evidence: Mouse models and in vitro liver cell lines (2026 study).

HEA effectively alleviated the progression of NAFLD by regulating glucolipid metabolism and insulin resistance both in vitro and in vivo; lipidomic data suggested that HEA markedly reduced triglyceride levels by blocking hepatic de novo lipogenesis. The mouse NAFLD models were induced by high-fat diet feeding or methionine-choline-deficient diet feeding, and AML12 and HepG2 cells were used for the in vitro study. The mechanism was traced to HEA significantly reducing the population of JunB⁺ adipocytes and interacting with JunB subunits through hydrogen bonding, leading to activated PGC-1α activity.

Strength of evidence: Preliminary. This work is recent (2026 publication), using established preclinical NAFLD models, and offers a novel mechanistic pathway. No human data are available.

5.6 Neuroprotection and Central Nervous System Effects

Body of evidence: In vitro (PC12 cells) and pharmacological observation in rodents.

Cordyceps bioactives including N6-(2-hydroxyethyl)-adenosine exert potential antioxidant, anti-inflammatory, and anti-apoptotic activities and display beneficial effects in the management and/or treatment of neurodegenerative disorders in vitro and in vivo. However, although a considerable list of compounds is available from Cordyceps, only a few have been evaluated for their neuroprotective potential and still lack information for clinical trials.

HEA is associated with control of the brain and coronary circulation and has anti-inflammatory activity; HEA has also been reported to have sedative–hypnotic activity. The mechanism underlying the analgesic activity of HEA differs from opioids, a widely used drug for analgesia. More importantly, HEA is neither addictive nor affected by pepsin, which is the advantage over opioids — this observation, while intriguing, is based on pharmacological test findings and has not been validated in clinical settings.

In a neuroprotective in vitro model, the butanol fraction of C. cicadae protected rat adrenal pheochromocytoma (PC12) cells against glutamate-induced oxidative damage.

Strength of evidence: Very preliminary. Sedative and circulatory effects were noted in early pharmacological screening; neuroprotective effects in PC12 cells are in vitro. No clinical human neurology or sleep trials exist for isolated HEA.

5.7 Cardiovascular Effects

Body of evidence: Pharmacological in vitro and early animal tests; identified as an inotropic agent.

HEA has been identified as a Ca²⁺ antagonist and shown to control circulation and possess sedative activity in pharmacological tests. As the first naturally derived calcium antagonist identified from a biological source (in 1983), HEA's cardiovascular relevance was recognized early. HEA is a Ca²⁺ antagonist and anti-inflammatory agent that is associated with the control of cerebral and coronary circulation and is thought to possess sedative activity.

Strength of evidence: Based on classical pharmacological tests rather than controlled cardiovascular clinical studies. No human clinical cardiology data are available for isolated HEA.

5.8 Insecticidal Activity

Body of evidence: In vitro and larval bioassay in Plutella xylostella.

HEA, at a dose of 1 mg/mL, was found to be lethal to the second-instar larvae of P. xylostella, and a significant reduction of mortality and growth inhibition ratio were obtained when HEA was administered along with PxAdoR-dsRNA or antagonist of AdoR (SCH58261) for 36, 48, or 60 h; at 48 h, the rate of growth inhibition of the PxAdoR knockdown group was 3.5-fold less than that of the HEA group, and the corrected mortality of the SCH58261 group was reduced almost 2-fold compared with the HEA group. This mechanism — acting via an insect adenosine receptor — is distinct from its mammalian pharmacology and has relevance for potential biopesticide applications rather than human supplementation.

6. Body Systems and Health Areas Associated with HEA

Based on the preclinical literature reviewed, HEA has been studied across the following organ systems:

• Renal system: Antifibrotic effects in UUO models; protection against diabetic nephropathy, NSAID-induced tubular injury, and cisplatin-induced acute kidney injury.
• Oncology: Cytotoxicity and pro-apoptotic effects in gastric, lung (Lewis), leukemia (K562), and osteosarcoma cell lines; in vivo tumor suppression in gastric carcinoma xenograft models.
• Cardiovascular system: Identified as a Ca²⁺ antagonist with cerebral and coronary circulatory effects; inotropic properties.
• Central nervous system: Sedative-hypnotic activity in pharmacological tests; neuroprotection against oxidative damage in PC12 cells; analgesic effect by non-opioid mechanism.
• Metabolic/endocrine: Antihyperglycemic and antidiabetic effects in alloxan-induced and db/db rodent models.
• Hepatic system: Alleviation of hepatic steatosis and insulin resistance in NAFLD mouse models via JunB/PGC-1α axis.
• Immune/inflammatory: Suppression of NF-κB and TGF-β1/Smad pathways; reduction of pro-inflammatory cytokines in macrophage models.

7. Dosage Forms and Dosages Reported in Studies

The following dosages are reported in the cited preclinical literature. As there are no human clinical trials for isolated HEA, these data describe animal or cell-culture experimental conditions only.

• Renal interstitial fibrosis (mouse, UUO model, intraperitoneal): Different doses of HEA (2.5, 5, and 7.5 mg/kg) were given by intraperitoneal injection 24 h before UUO, and the treatment was continued for 14 days post-operatively.
• Diabetic kidney disease / antihyperglycemic (rat, alloxan model, intraperitoneal): N6-(2-Hydroxyethyl)adenosine (20–40 mg/kg, ip, once daily for 6 weeks) reduces blood glucose level and diabetes-induced kidney damage.
• Gastric carcinoma xenograft (mouse, oral): N6-(2-Hydroxyethyl)adenosine (75–150 mg/kg, po, once daily for 19 days) exhibited antitumor effects in SGC-7901 xenograft mouse models.
• NSAID-induced renal tubular injury (HK-2 cells, in vitro): The MTT assay revealed HEA to be nontoxic up to 100 µM toward HK–2 cells; the cells were pretreated with HEA (10–20 µM) and then insulted with NSAIDs for 24 h; HEA (20 µM) effectively prevented ER stress.
• HEA-enriched mycelia safety study (mouse, oral gavage): The study evaluated the CNS, cardiovascular system, and respiratory system in ICR male mice via oral gavage administration; two batches of eight mice were tested on a vehicle and C. cicadae mycelia (1,000 mg/kg).
• Cisplatin-induced acute kidney injury (mouse, intraperitoneal): HEA was tested at 10 mg/kg and 20 mg/kg body mass in the cisplatin-challenged group.

N6-(2-Hydroxyethyl)adenosine is noted to inhibit the NF-κB/Smad signaling pathway and is stated to be orally active. The oral bioavailability in animal studies supports the feasibility of oral administration, but human pharmacokinetic data have not been published for isolated HEA.

8. Safety Considerations

8.1 In Vivo Safety Pharmacology

Many studies have confirmed the safety of C. cicadae mycelia; however, the acute safety pharmacology of the C. cicadae enriched with the high HEA (3.90 mg/g) compound had not been previously evaluated; one study evaluated the central nervous system, cardiovascular system, and respiratory system in ICR male mice via oral gavage administration. In the cardiovascular assessment of this safety study, the heart rate at 60 min for the vehicle and C. cicadae mycelium treatment was 700.3 ± 55.4 and 603.0 ± 42.3 bpm, respectively (p = .4279), indicating no statistically significant effect on heart rate at the tested dose of 1,000 mg/kg mycelial material.

8.2 Oral Activity and Non-Opioid Analgesic Profile

The mechanism underlying the analgesic activity of HEA differs from opioids; HEA is neither addictive nor affected by pepsin, which is an advantage over opioids. The resistance to pepsin digestion implies that oral HEA is not readily degraded in the stomach, supporting its oral bioavailability in animal studies.

8.3 Interaction with Human Serum Albumin

Studies confirmed that HEA and human serum albumin interacted to form a complex by hydrophobic interaction. High plasma protein binding, as quantified by the binding constants reported in fluorescence spectroscopy studies, has implications for the apparent volume of distribution, half-life, and potential for displacement interactions with other highly albumin-bound drugs. However, specific clinically significant drug–drug interactions mediated by this mechanism have not been studied for HEA.

8.4 Cytotoxicity Profile in Normal vs. Tumor Cells

In the NSAID-renal protection study, the MTT assay revealed HEA to be nontoxic up to 100 µM toward HK–2 cells (human proximal tubular cells), suggesting a selectivity for tumor vs. non-transformed cells at lower concentrations. In the gastric cancer cytotoxicity study, HEA exerted cytotoxic effects against gastric carcinoma cells (SGC-7901 and AGS) in a dose- and time-dependent manner, while normal HEK293 cells were also included as a control in the same dose-response assays. However, no systematic toxicology, genotoxicity, or reproductive toxicology studies for isolated HEA have been published.

8.5 Absence of Human Safety and Interaction Data

No published pharmacokinetic studies in humans, no maximum tolerated dose studies in humans, and no human drug–drug interaction studies exist for isolated HEA at the time of writing. The traditional safety record of C. cicadae-based preparations across 1,600 years of use in East Asian medicine provides contextual reassurance for whole-fungus preparations, but does not directly address the safety profile of isolated, concentrated HEA. Additionally, HEA is a potent antioxidant with glucose-lowering, hepatoprotective, cardioprotective, sedative, antitumor, eye-protective, and anti-inflammatory properties — its calcium channel antagonism and potential circulatory effects suggest that caution may be warranted in individuals taking antihypertensive agents, calcium channel blockers, or other cardiovascular medications, though this has not been formally investigated.

9. Current Research Status and Knowledge Gaps

Limited literature is available on the neuroprotective activities of HEA. More broadly, while HEA's preclinical pharmacology spans multiple organ systems with mechanistic consistency, the compound remains at an early translational stage. Both HEA and cordycepin (3′-deoxyadenosine) are main components of Cordyceps species and are also analogues of adenosine; the antitumor effect of cordycepin has been widely reported, but studies on HEA antineoplastic activity are relatively few.

Key open questions in the field include the following: (1) Human pharmacokinetic and pharmacodynamic data for isolated HEA are entirely absent. (2) Optimal dosing, route of administration, and formulation for human use have not been defined. (3) The relative contribution of HEA, cordycepin, and adenosine to the biological activity of whole Cordyceps extracts remains incompletely characterized — cordycepin is more potent than adenosine and HEA in suppressing the LPS-stimulated release of pro-inflammatory cytokines by RAW 264.7 macrophages, and no synergistic effect was observed with these three compounds in at least one anti-inflammatory assay, although effects may vary by tissue and endpoint. (4) Long-term safety at supplemental doses has not been evaluated. (5) Human clinical trials are needed to validate the renal-protective, antidiabetic, antitumor, and neuroprotective signals observed preclinically.

References

• Xie H et al. N6-(2-hydroxyethyl)-Adenosine Induces Apoptosis via ER Stress and Autophagy of Gastric Carcinoma Cells In Vitro and In Vivo. Int J Mol Sci. 2020;21(16):5815. PMC7461581
• Zheng R et al. N6-(2-Hydroxyethyl) Adenosine From Cordyceps cicadae Ameliorates Renal Interstitial Fibrosis and Prevents Inflammation via TGF-β1/Smad and NF-κB Signaling Pathway. Front Physiol. 2018;9:1229. PMC6131671
• Zheng R et al. N6-(2-Hydroxyethyl) Adenosine From Cordyceps cicadae Ameliorates Renal Interstitial Fibrosis and Prevents Inflammation via TGF-β1/Smad and NF-κB Signaling Pathway. Frontiers in Physiology. 2018.
• Lu Y et al. N6-(2-Hydroxyethyl)adenosine in the Medicinal Mushroom Cordyceps cicadae Attenuates Lipopolysaccharide-Stimulated Pro-inflammatory Responses by Suppressing TLR4-Mediated NF-κB Signaling Pathways. J Nat Prod. 2015.
• Liu K et al. Beauveria bassiana: a new N6-(2-hydroxyethyl)-adenosine–producing fungus. Mycology. 2017;8(4):259-266. PMC6059059
• Meng X et al. Cordycepin and N6-(2-Hydroxyethyl)-Adenosine from Cordyceps pruinosa and Their Interaction with Human Serum Albumin. PLOS ONE. 2015.
• Fang M et al. N6-(2-Hydroxyethyl)-Adenosine Exhibits Insecticidal Activity against Plutella xylostella via Adenosine Receptors. PLoS ONE. 2016;11(9):e0162859. PMC5036850
• Li IC et al. Safety assessment of HEA-enriched Cordyceps cicadae mycelia on the central nervous system, cardiovascular system, and respiratory system in ICR male mice. PMC8441276
• Zhu J et al. Preparative isolation of cordycepin, N6-(2-hydroxyethyl)-adenosine and adenosine from Cordyceps militaris by macroporous resin and purification by recycling high-speed counter-current chromatography. Biochem Syst Ecol. 2017.
• Unique Bioactives from Zombie Fungus (Cordyceps) as Promising Multitargeted Neuroprotective Agents. Nutrients. 2023;16(1):102.
• New Insights Into the Biosynthesis of Typical Bioactive Components in the Traditional Chinese Medicinal Fungus Cordyceps militaris. Front Bioeng Biotechnol. 2021.
• Chen CC et al. Potential Protection Effect of ER Homeostasis of N6-(2-Hydroxyethyl)adenosine Isolated from Cordyceps cicadae in Nonsteroidal Anti-Inflammatory Drug-Stimulated Human Proximal Tubular Cells. Int J Mol Sci. 2021;22(4):1577. PMC7913954
• N6-(2-hydroxyethyl)-adenosine (HEA) exhibits antitumor activity for osteosarcoma progression by regulating IGF1 signaling. Fitoterapia. 2024.
• N6-(2-Hydroxyethyl) Adenosine Alleviates Hepatic Steatosis and Insulin Resistance by Inhibiting JunB+ Adipocytes. Phytother Res. 2026.
• Ke BJ et al. Using submerged fermentation to fast increase N6-(2-hydroxyethyl)-adenosine, adenosine and polysaccharide productions of Cordyceps cicadae NTTU 868. AMB Express. 2019. PMC6901654
• Secondary metabolites from Hypocrealean entomopathogenic fungi: Novel bioactive compounds. PMC7529686
• N6-(2-hydroxyethyl)-adenosine from Cordyceps cicadae attenuates hydrogen peroxide induced oxidative toxicity in PC12 cells. Metab Brain Dis. 2019.
• Telomere-to-Telomere Assembly of the Cordyceps militaris CH1 Genome and Integrated Transcriptomic and Metabolomic Analyses. PMC12194794