Potential Beneficial Effects and Pharmacological Properties of Ergosterol, a Common Bioactive Compound in Edible Mushrooms

Ergosterol is an important sterol commonly found in edible mushrooms, and it has important nutritional value and pharmacological activity. Ergosterol is a provitamin. It has been well established that edible mushrooms are an excellent food source of vitamin D2 because ergosterol is a precursor that is converted to vitamin D2 under ultraviolet radiation. The pharmacological effects of ergosterol, which include antimicrobial, antioxidant, antimicrobial, anticancer, antidiabetic, anti-neurodegenerative, and other activities, have also been reported. This review aims to provide an overview of the available evidence regarding the pharmacological effects of ergosterol and its underlying mechanisms of action. Their potential benefits and applications are also discussed.


Introduction
Natural bioactive compounds are widely used as complementary medicines, including in dietary supplements. Their potential applications have been investigated for several decades. Researchers have identified novel biomolecules with desirable pharmacological properties from natural products, optimized their efficacy and safety using medicinal chemistry and pharmaceutical science, and developed them into pharmaceutical or nutraceutical products for the treatment or prevention of several diseases. Most natural compounds have been isolated from edible sources or used in traditional medicines worldwide for many years; therefore, they are supposed to be relatively safe.
Edible mushrooms are rich in proteins, dietary fiber, vitamins, minerals, and many bioactive substances such as polysaccharides, terpenoids, polyphenols, alkaloids, lactones, and sterols [1,2]. The major type of sterol in edible mushrooms is ergosterol [3]. Ergosterol has become an increasingly popular topic in pharmacological research because of its potential health benefits and widespread presence in various foods and dietary products. Its pharmacological effects on oxidation, immune function, diabetes, cancer, and other lactones, and sterols [1,2]. The major type of sterol in edible mushrooms is ergosterol [3]. Ergosterol has become an increasingly popular topic in pharmacological research because of its potential health benefits and widespread presence in various foods and dietary products. Its pharmacological effects on oxidation, immune function, diabetes, cancer, and other diseases have been previously reported. However, there is hitherto no review summarizing these effects and discussing their potential implications.
This review is aimed at outlining an overview of the available evidence proving the pharmacological effects of ergosterol and its underlying mechanisms of action. Its potential benefits and therapeutic applications are also discussed. The information inside this review is based on the scientific literature searched and retrieved from the PubMed and Google Scholar databases.

Overview of Ergosterol Structure
Ergosterol, or ergosta-5,7,22-trien-3β-ol, is the most abundant sterol in the cell membrane of fungi, including edible mushrooms. It maintains fungal cell membrane integrity, similar to cholesterol in animal cell membranes [4]. This compound contains three double bonds and β-hydroxy groups at positions 5, 7, and 22 with a 1,2cyclopentanoperhydrophenanthrene ring nucleus (Figure 1), making it behave like an amphipathic lipid [5]. Ergosterol exists in free and esterified forms. When exposed to ultraviolet light (280-320 nm), it undergoes photolysis. During photolysis, it is transformed into pre-vitamin D2 (pre-ergocalciferol), followed by thermal conversion into vitamin D2 (ergocalciferol), which is essential for human nutrition. After consumption, vitamin D2 is converted to 25-hydroxyvitamin D through hepatic metabolism [6]. It is then transported to the kidney, where it is further transformed into 1,25-dihydroxyvitamin D, which is known as calcitriol, the active form in living organisms that plays important roles in calcium homeostasis and bone health [7].

Natural Sources of Ergosterol
Ergosterol is found in various sources but is abundant in fungi, particularly mushrooms [8]. Ergosterol content varies among different species and parts of mushrooms [9,10]. The contents of ergosterol are 3.52 mg/g and 0.43 mg/g in button mushrooms and morel mushrooms, respectively [11]. The contents of ergosterol in fresh white and brown button mushrooms are 0.446 mg/g and 0.394 mg/g, respectively [12], whereas the contents of ergosterol in the caps of dry white and brown button mushroom are 3.30-3.76 mg/g and 2.71-4.56 mg/g, respectively [9]. Ergosterol content is reduced with aging of the mushrooms. The mushroom cap contains higher ergosterol content than the stem did at the same growth stage [9]. Although ergosterol is found in many edible mushrooms, it has been reported to be present in some plant extracts including Chinese hickory, hazelnut, lupin, cactus, and firethorn [13][14][15][16]. However, it is unclear whether the ergosterol is a real metabolite in these plants or if its presence in plant extracts is due to

Natural Sources of Ergosterol
Ergosterol is found in various sources but is abundant in fungi, particularly mushrooms [8]. Ergosterol content varies among different species and parts of mushrooms [9,10]. The contents of ergosterol are 3.52 mg/g and 0.43 mg/g in button mushrooms and morel mushrooms, respectively [11]. The contents of ergosterol in fresh white and brown button mushrooms are 0.446 mg/g and 0.394 mg/g, respectively [12], whereas the contents of ergosterol in the caps of dry white and brown button mushroom are 3.30-3.76 mg/g and 2.71-4.56 mg/g, respectively [9]. Ergosterol content is reduced with aging of the mushrooms. The mushroom cap contains higher ergosterol content than the stem did at the same growth stage [9]. Although ergosterol is found in many edible mushrooms, it has been reported to be present in some plant extracts including Chinese hickory, hazelnut, lupin, cactus, and firethorn [13][14][15][16]. However, it is unclear whether the ergosterol is a real metabolite in these plants or if its presence in plant extracts is due to the contamination of fungi so further study is required. The dietary sources of ergosterol from mushrooms are summarized in Table 1.

Pharmacokinetics, Drug-Likeness, and Toxicity of Ergosterol
The pharmacokinetic characteristics of ergosterol were determined using a rat model [38]. In the rats that were orally administered ergosterol for 36 h, the area under the plasma drug concentration time curve at 0-36 h was 22.29 ± 5.08 µg·h/mL, the half-life was 5.90 ± 1.41 h, the maximum observed concentration (C max ) was 2.27 ± 0.19 µg/mL, and the time to C max was 8.00 ± 1.18 h. Approximately 62.5% of the administered ergosterol was excreted in feces, whereas 3.2% of the ergosterol was eliminated in urine.
The oral bioavailability of ergosterol is not known because the intravenous injection of ergosterol has not been carried out in a pharmacokinetic study for the calculation of bioavailability. However, the oral bioavailability is expected to be low, as reflected from the long amount of time required for achieving C max and because of the poor aqueous solubility of ergosterol. However, the bioavailability and effectiveness of ergosterol can be further enhanced using a special drug delivery system. Interestingly, one study demon-strated that the use of nanostructured lipid carriers (NLCs) can improve the solubility and bioavailability of ergosterol. A pharmacodynamic study has also confirmed that the inhibitory effects of ergosterol-loaded NLCs on high-glucose-stimulated mesangial cell proliferation and extracellular matrix (ECM) accumulation were stronger than those of raw ergosterol [39]. Similarly, the oral bioavailability of ergosterol can be increased after encapsulation in PGLA nanoparticles [40].
The drug-likeness and toxicity of several bioactive compound from mushrooms, including ergosterol, were previously assessed using an in silico approach by Rangsinth et al. [41]. According to Lipinski's rule, ergosterol exhibited drug-like properties, including a molecular weight ≤ 500, number of hydrogen bond acceptors ≤ 10, number of hydrogen bond donors ≤ 5, and Log P o/w ≤ 5. Additionally, ergosterol did not cause hepatotoxicity or skin allergy, and its predicted median lethal dose in rats was 2.05 mol/kg [41]. Moreover, without the functional groups which confer as carcinogenic or mutagenic as indicated by the Benigni-Bossa rule, ergosterol was assumed to be non-carcinogenic and non-mutagenic.

Antioxidant Activity
The imbalance between the generation and removal of free radicals leads to increased levels of reactive oxygen species (ROS) and reactive nitrogen species (RNS), which play crucial roles in oxidative stress, and hence, the aging process [42]. ROS and RNS such as superoxide (O2 •− ), hydroxyl (OH • ), nitric oxide (NO • ), and nitrogen dioxide (NO2 • ) radicals are byproducts of biological metabolism [43]. Excessive oxidative stress results in damage to macromolecules, including DNA, cell membranes, and proteins, leading to the development of various diseases such as cancer, atherosclerosis, kidney disease, diabetes, and neurodegenerative diseases [42,43].

Anti-Inflammatory Activity
Inflammation is a biological reaction that occurs when body parts are exposed to harmful or irritating stimuli. Various cytokines are generated at inflammatory sites as mediators that remove damaged cells, pathogens, or harmful stimuli. Therefore, the initiation of the healing process is important. However, exaggerated and uncontrolled inflammation is a key characteristic and risk factor related to the pathogenesis of many diseases, including atherosclerosis, obesity, metabolic syndrome, diabetes, cancer, and neurodegenerative disorders [47].
Ergosterol-containing mushrooms suppress the expression of inflammatory mediators through several signaling pathways. One such pathway is the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway, which is involved in cell division, cell death, tumor formation, immunomodulation, and inflammation. Ergosterol interferes with the expression of JAK3/STAT3 genes in cigarette-smoke-induced chronic obstructive pulmonary disease (COPD) in ICR mice. As a result, ergosterol effectively ameliorates COPD severity by inhibiting the pro-inflammatory cytokines [48]. Similar results were observed in the same COPD models using 16HBE cells and Balb/c mice [49]. Ergosterol peroxide, purified from the edible mushroom Sarcodon aspratus, exerts anti-inflammatory responses in RAW264.7 macrophages by downregulating the expression of low-density lipoprotein receptors and suppressing STAT1-mediated inflammatory responses in HT29 cells [50]. In addition, ergosterol and ergosterol peroxide inhibit LPS-induced inflammation, along with the phosphorylation of p38, c-Jun amino-terminal kinase (JNK), extracellular signal-regulated protein kinase (ERK), and mitogen-activated protein kinases (MAPKs) [51]. Ergosterol acetate also suppresses the phosphorylation of ERK in LPS-induced inflammation in RAW246.7 cells [52]. Molecular docking studies have shown that both ergosterol and ergosterol peroxide interact with the ATP-binding site of p38, resulting in the blockade of p38 MAPK phosphorylation, but have no effect on ERK and JNK [53].
Ergosterol exerts its anti-neuroinflammatory activity via the TLR4/NF-κB-dependent pathway. Therefore, exploring the potential for developing ergosterol into a novel drug for the treatment of Alzheimer's disease is viable [64]. It has been reported that the antiinflammatory functions of ergosterol are mediated by the suppression of transcriptional activity in propidium monoazide-treated human chondrosarcoma (SW1353) cells [65]. Ergosterol and ergosterol peroxide also inhibit NF-κB luciferase activity in RAW246.7 macrophages [27]. Moreover, both ergosterol and ergosterol peroxide bind directly to the active site of NF-κB p65 to restrain the phosphorylation and degradation of IκB-α and thus block the phosphorylation of NF-κB p65 [53]. Furthermore, ergosterol peroxide displays a significant anti-inflammatory effect on LPS-induced human monocytic cells through the inhibition of MyD88 (which is a central node of the inflammatory signaling pathway), VCAM-1 expression, and cytokine (IL-1β, IL-6, and TNF-α) production. This compound also inhibits NF-κB p65 activation effectively [66].
Ergosterol attenuates the symptoms of dextran sulfate sodium-induced colitis in mice, as indicated by the disease activity index, which is reflected in weight loss, severity of diarrhea, and shortening of the colon [67]. The effect of ergosterol on dextran sulfate sodium-induced colitis was also mediated through the suppression of the NF-κB signaling pathway [67]. Furthermore, it was recently found that Hericium erinaceus, a medicinal mushroom which contains ergosterol, can cause a significant decrease in the MDA, NO, MPO, NF-κB, IL-6, and TNF-α levels in trinitrobenzene sulfonic acid-induced colitis in mice [68]. Additionally, the pathogenesis of ulcerative colitis is related to mucosal inflammation due to the accumulation of mast cells in the colonic mucosa. Under physiological conditions, the surface of mast cells expresses high-affinity immunoglobulin E (IgE) receptors (FcεRI). The binding of IgE to FcεRI can trigger mast cells to release many inflammatory mediators, including β-hexosaminidase and histamines, which destroy the colonic mucosa. Ergosterol inhibits the aggregation of FcεRI, which is the first step in mast cell activation, and reduces IL-4 and TNF-α mRNA expression in IgE-sensitized RBL-2H3 basophilic leukemia cells [24,25]. Moreover, ergosterol significantly inhibits the activities of β-hexosaminidase and mucosal-type murine bone-marrow-derived mast cells [69]. Similarly, both the ergosterol-rich extract of Grifola frondosa and pure ergosterol inhibit histamine release in air pouch-type allergic inflammation and reduce vascular permeability and edema in mice [24,25]. The extract of edible mushroom Grifola frondosa and its ergosterol also prevent pollen-allergy-induced ocular itching in mice by suppressing leukotriene B 4 production in mast cells [70].

Anticancer Activity
Although the overall survival rate of cancer patients has increased, cancer remains one of the most common causes of morbidity, mortality, and economic burden. Advanced stage cancer and lethal malignancies may not be completely eradicated by current clinical interventions, such as surgery, radiotherapy, chemotherapy, and targeted therapy. The potent anticancer activities of some natural compounds from terrestrial and marine sources have been identified. Paclitaxel, which is isolated from the Pacific yew, is one of the best examples of a natural compound that has been successfully used in clinical practice [71]. Other examples include vinca alkaloids, such as vinblastine, vincristine, and podophyllotoxin [72]. The chemopreventive and chemotherapeutic properties of natural compounds have led to a new era in anticancer drug research. The isolation and characterization of these natural compounds may be more economical than the development of synthetic compounds [73].
In vitro studies on LNCap and DU-145 prostate cancer cell lines further confirmed the androgen-receptor-mediated action of ergosterol. In addition, ergosterol showed antiproliferative activity against MCF7, NCI-H460, and HeLa cells [76]. Ergosterol inhibits the estrogen receptor in MCF7 breast cancer cell lines [77], probably by inducing apoptosis and S-phase cell cycle arrest [78]. Ergosterol also inhibits Ras expression, the ERK-NADPH oxidase-dependent pathway, and ROS-mediated chromosomal and oxidative damage caused by carcinogen-induced breast cancer [79]. Interestingly, ergosterol may produce synergistic anticancer effects with other drugs such as amphotericin B [80,81]. Ergosterol enhances the anticancer effects of cisplatin. Liposomes modified with arginine-glycineaspartic acid and octa arginine peptide-mediated drug delivery of ergosterol and cisplatin exhibited strong inhibitory activity against A549 cells [75].
The anticancer effects of ergosterol have also been demonstrated in vivo. Ethanol and chloroform extracts of mushroom Amauroderma rude, which contain ergosterol as the main bioactive component, suppress tumor growth in Balb/c mice inoculated with B16 melanoma cancer cells. These effects may be mediated through the activation of FOXO-3, which induces the upregulation of various tumor suppressors, including pro-apoptotic genes, such as FasL, Bad, Bim, and Trail [82]. The oral administration of ergosterol efficiently inhibits bladder cancer in rats by suppressing cyclin D1 expression and, subsequently, COX2 expression; COX2 is a key player involved in the development of bladder cancer [59]. Ergosterol can be converted to brassicasterol, a metabolite that can be retained in the circulatory system for a longer period. The suppression of bladder carcinogenesis may be attributed to the antagonistic effect of brassicasterol on androgen receptors, owing to its structural similarity to testosterone [83]. In silico analysis revealed that ergosterol shows an interaction similar to that of 5α-dihydrotestosterone and testosterone in the binding mode with androgen receptors during a molecular dynamic simulation [84]. Therefore, ergosterol is particularly effective against sex-hormone-dependent cancers.
In a pioneering study, oral administration of ergosterol inhibited Matrigel-induced neovascularization in female C57BL/6 mice, suggesting that it may be an antiangiogenic compound [85]. Ergosterol also downregulates positive modulators of angiogenesis such as VEGFC and STAT3 [86]. These antiangiogenic effects may also contribute to the anticancer effects of ergosterol.

Antidiabetic Effects
Diabetes mellitus is a chronic health condition in which blood glucose levels are persistently elevated due to insufficient secretion of insulin from the pancreas or a decreased tissue response to insulin [87]. Nephropathy is the most common and severe complication of diabetes mellitus. It is characterized by irreversible damage to kidney function resulting from hyperglycemia-induced oxidative stress and ECM deposition, which causes renal interstitial fibrosis [88].
Ergosterol derived from the edible mushroom Pleurotus ostreatus exhibited promising antidiabetic activity [89]. Ergosterol treatment was able to lower blood glucose in type 2 diabetes mellitus mice and stimulated GLUT4 translocation via the PI3K/Akt pathway and PKC pathway [89]. Ergosterol alleviates diabetic nephropathy in streptozotocininjected mice with significant correction of biochemical parameters such as plasma levels of glucose, uric acid, creatinine, triglyceride, and total cholesterol. Furthermore, the levels of proteins related to PI 3 K/Akt/NF-κB p65 signaling and cytokines (including IL-6, TNF-α, and MCP-1) [63], as well as to renal pathological changes, were restored by ergosterol treatment [90]. PI 3 K/Akt signaling pathway is controlled by NF-κB and has important functions in metabolic balance [91,92]. In another study involving streptozotocin-induced diabetic mice, ergosterol treatment attenuated mesangial cell proliferation and increases the matrix metalloproteinase-2 and -9 [90]. Furthermore, ergosterol alleviates ECM deposition by increasing transforming growth factor-β1 expression and Smad2 phosphorylation [93]. Ergosterol also increases glucose uptake by upregulating the expression and translocation activity of glucose transporter 4 and the phosphorylation of Akt and PKC in L6 muscle myoblast cells and different mouse tissues [89].

Neuroprotective Effects
Neuronal degeneration is a brain pathology characterized by protein aggregation and decline in the neuronal population, finally leading to neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, and amyotrophic lateral sclerosis [94]. Several risk factors are involved in neurodegeneration including oxidative stress, glial cell activation, neurotransmitter imbalance, and neuronal death [95]. Accumulating data demonstrate that ergosterol potentially serves as a neuroprotective agent by attenuating neuroinflammation and neuronal death. Ergosterol, at a concentration of 10 µg/mL (cal. 25.21 µM), showed an anti-neuroinflammatory effect on LPS-induced BV2 microglial cell activation by reducing the production of NO, a pro-inflammatory agent [57]. Moreover, ergosterol (50 nM), isolated from Auricularia polytricha edible mushroom, could suppress bisphenol-A-induced BV2 microglial cell inflammation by down-regulating the NF-κB signaling and enhancing the antioxidant pathway [96]. Furthermore, 100 nM of ergosterol exhibited a neuroprotective property against TNF-α-induced HT-22 hippocampal cell damage by promoting Nrf2/SOD-1 and RICTOR/Akt/GSK-3β signaling pathways and inhibiting ionotropic glutamate receptor overexpression via EGR-1 regulation [97]. Therefore, ergosterol seems to provide therapeutic effects that benefit neurodegenerative patients.

Antimicrobial Activity
The increased incidence of new microbial infections and the evolution of antibioticresistant strains have led scientists to develop new bioactive compounds from natural resources as alternatives for treating infectious diseases [98]. Ergosterol and ergosterol peroxide exhibit a broad range of antimicrobial activities [99]. For instance, the growth of Helicobacter pylori is inhibited by ergosterol; the minimum inhibitory concentrations are 10-20 µg/mL [100], suggesting that ergosterol has the potential to serve as an effective drug for gastritis. Ergosterol also exhibits antifungal activity against Aspergillus flavus, Pencillium digitatum, and Fusarium vericilloides [99]. Trypanosoma cruzi infection is one of the causes of the high incidence of Chagas disease, which is a life-threatening disease. Ergosterol causes rapid plasma membrane and mitochondrial permeabilization owing to alterations in membrane composition, resulting in the disruption and death of trypomastigotes [101].
Other studies have also revealed that the antibacterial effect of ergosterol is mediated by the disruption of the electron transport chain and oxidative phosphorylation via the disorganization of membrane fluidity, leading to enhanced membrane permeability [102][103][104]. These mechanisms may be advantageous and useful in the treatment of infectious diseases. In a recent study on the anti-HIV-1-protease activity of the hexane extract of the edible mushroom Auricularia polytricha, a phytochemical investigation revealed the presence of ergosterol, apart from other components such as linoleic acid and two other lipopolysaccharides, as the principal component in the extract [19]. Additionally, an in silico study suggested that ergosterol may be a potential candidate for treatment of COVID-19 because of its potential to inhibit SAR-CoV-2 main protease [41]. Nevertheless, further study is required to prove this hypothesis.

Anti-Hepatic Steatosis Effect
In addition to the beneficial effects mentioned above, ergosterol may have therapeutic potential for hepatic steatosis, which is the first step in the development of nonalcoholic fatty liver disease, steatohepatitis, fibrosis, cirrhosis, and hepatic carcinoma. Ergosterol reduces intracellular triglyceride accumulation in HepG2 cells treated with fatty acid. Its mechanism of action involves the direct induction of AMPK and acetyl-CoA carboxylase phosphorylation [35], which regulates lipid metabolism and cellular lipid homeostasis [105].

Future Perspective and Conclusions
Ergosterol is a versatile compound found in natural sources, especially in edible mushrooms. Continuous pharmacological research on ergosterol may lead to the development of new drugs and nutraceuticals for therapeutic use and promoting health. For example, ergosterol can be formulated as a novel antioxidant or anti-inflammatory agent. It may also be used in combination with other drugs, especially chemotherapeutic agents, to enhance drug efficacy and reduce side effects. Ergosterol-containing nutraceuticals can promote immune function, liver health, and overall well-being. Further studies are required to fully explore its mechanisms of action, and to understand its effects on the human body. In particular, animal studies and clinical trials must be encouraged. Formulation optimization should also be considered in order to increase ergosterol bioavailability.