Discovery of a New Compound, Erinacerin W, from the Mycelia of Hericium erinaceus, with Immunomodulatory and Neuroprotective Effects

One new compound with an isoindolinone skeleton, along with erinacines A, C, and S, was isolated from the mycelia of Hericium erinaceus, an edible fungus with a long history of use in traditional Chinese medicine. Based on analysis of MS and NMR spectral data, the structure of the compound was identified as (2E,6E)-8-(2-(1-carboxy-3-methylbutyl)-4,6-dihydroxy-1-oxoisoindolin-5-yl)-2,6-dimethylocta-2,6-dienoic acid. In light of this discovery, we have given this compound the name erinacerin W. Using a co-culture in vitro LPS-activated BV2 microglia-induced SH-SY5Y neuroinflammation model, the results showed that erinacerin W demonstrated protection against the LPS-activated BV-2 cell-induced overexpression of IL-6, IL-1β, and TNF-α on SH-SY5Y cells. This finding may provide potential therapeutic approaches for central nervous disorders.


Introduction
Neurodegenerative diseases are slow processes that cause irreversible damage to specific areas of the brain, resulting in reduced memory and cognitive function loss.The most common neurodegenerative disorders are Alzheimer's disease (AD) and Parkinson's disease (PD) [1].With the rapid increase in the aging population, it can be expected that neurodegenerative diseases will impose a large economic burden on global medical care, the economy, families, and society in the future.Given the lack of curative treatments for neurodegenerative diseases, there has been a growing interest in exploring the potential of natural products for neuroprotection in recent years [2].
One such natural product is Hericium erinaceus, a medicinal and edible fungus with a rich and extensive history of use in traditional medicine and cuisine [3].Several medicinal properties of H. erinaceus have been studied, including its immune-modulatory, antioxidant, cholesterol-lowering, blood sugar-lowering, antimicrobial, and anticancer activities [4][5][6].These effects are attributed to its bioactive components, such as erinacines, hericenones, hericerins, erinacerins, erinaceolactones, alkaloids, steroids, polysaccharides, and glycoproteins [7].However, it is important to note that the expression of bioactive secondary metabolites in the mycelia and fruiting bodies of the fungus can vary significantly due to their distinct structures [8].While it is possible to synthesize some of these compounds, the synthesis of erinacine A, which has a complex structure, requires a lengthy and challenging process with low yield [9]. Apart from erinacines B and E, which also require complex synthesis processes, there has been no reported synthesis of other erinacines or erinacerin series compounds.Therefore, through a controlled manipulation of H. erinaceus mycelia in a bioreactor, such as adjusting the temperature, aeration speed, and pH, it is possible to elicit the production of the active compounds known as erinacines and erinacerins [10,11].Consequently, there has been a remarkable increase in research reports focusing on the potential applications and safety of metabolite-enriched H. erinaceus mycelia over the past decade [12].

Results and Discussion
One new compound with an isoindolinone skeleton (1), along with erinacines A (2), C (3), and S (4), was isolated from the mycelia of Hericium erinaceus.Based on its unique skeleton structure, this new compound was categorized as a member of the erinacerin series and named erinacerin W (1) (Figure 1).Erinacerin W ( 1  compounds, the synthesis of erinacine A, which has a complex structure, requires a lengthy and challenging process with low yield [9]. Apart from erinacines B and E, which also require complex synthesis processes, there has been no reported synthesis of other erinacines or erinacerin series compounds.Therefore, through a controlled manipulation of H. erinaceus mycelia in a bioreactor, such as adjusting the temperature, aeration speed, and pH, it is possible to elicit the production of the active compounds known as erinacines and erinacerins [10,11].Consequently, there has been a remarkable increase in research reports focusing on the potential applications and safety of metabolite-enriched H. erinaceus mycelia over the past decade [12].
Among erinacerins, erinacerin F and compound 1 share similar structural features, as they both possess two hydroxy groups, two carboxyl groups, and two carbonyl groups [13].These functional groups contribute to the high polarity of their overall structures.However, the main point of differentiation between erinacerin F and compound 1 lies in the side chain at the 3 ′′ position on the N atom.In erinacerin F, the side chain consists of an isoleucine, while compound 1 presents a leucine in this position.This slight alteration in the side-chain configuration leads to a distinct structural variation between the two compounds.
Based on their structures, it can be deduced that the polarities of erinacerin F and compound 1 are similar.However, to completely understand the effect of this structural distinction on their chemical properties and potential biological activities.It will be important to determine the absolute configuration and specific rotation of compound 1, as chiral molecules have greatly influenced the development of new drugs.Therefore, for future research, all of these factors should be taken into consideration.
Natural products offer an opportunity to explore new compounds that can be developed into drugs due to their diverse chemical structures.Currently, a total of 22 erinacerins have been discovered, representing a rich pool of compounds for further investigation.While erinacerins A, C, M-N, and U-V are still undergoing rigorous exploration and indepth investigation to uncover their full range of potential applications [14][15][16], previous research has shown that erinacerin B could reduce LPS-induced nitric oxide (NO) and prostaglandin E2 (PGE2) production in RAW 264.7 macrophage cells in a concentrationdependent manner, thus exhibiting anti-inflammatory properties [17].Moreover, erinacerins D-L act as α-glucosidase inhibitors, with IC 50 values in the range of 12.8 to 145.1 µM [13].Additionally, erinacerins O-P have emerged as promising candidates for potential glioma inhibitors, as they have demonstrated the ability to induce apoptosis in U87 cells through the Bax/Capase-2 pathway [18].Furthermore, erinacerins Q-T have exhibited notable inhibitory activity against protein tyrosine phosphatase-1B (PTP-1B), with IC 50 values ranging from 24.9 to 42.1 µM [19].In this study, for the first time, erinacerin W demonstrated anti-neural inflammation and the potential to inhibit the onset and progression of neurodegenerative diseases, distinguishing it from other erinacerin series.
Co-culture modules using SH-SY5Y neuroblastoma and BV2 microglia cells have been successfully utilized to study the harmful effects of microglial activation in neurodegenerative diseases [20].To examine the potential impacts of compounds 1-4 on LPS-activated microglia-mediated cell viability, we investigated the co-culture of BV-2 microglia with SH-SY5Y neurons.After incubating SH-SY5Y cells with a range of concentrations (from 0 to 20 µg of compounds 1-4) for 24 h, it was observed that compound 1 had no cytotoxic effect up to 20 µg/mL, while compounds 1 and 2 above 5 µg/mL and compound 3 above 2.5 µg/mL reduced cell viability by approximately 10% (Figure 4).Therefore, non-toxic doses were selected for further experiments.
However, the main point of differentiation between erinacerin F and compound 1 lies in the side chain at the 3″ position on the N atom.In erinacerin F, the side chain consists of an isoleucine, while compound 1 presents a leucine in this position.This slight alteration in the side-chain configuration leads to a distinct structural variation between the two compounds.
Based on their structures, it can be deduced that the polarities of erinacerin F and compound 1 are similar.However, to completely understand the effect of this structural distinction on their chemical properties and potential biological activities.It will be important to determine the absolute configuration and specific rotation of compound 1, as chiral molecules have greatly influenced the development of new drugs.Therefore, for future research, all of these factors should be taken into consideration.
Natural products offer an opportunity to explore new compounds that can be developed into drugs due to their diverse chemical structures.Currently, a total of 22 erinacerins have been discovered, representing a rich pool of compounds for further investigation.While erinacerins A, C, M-N, and U-V are still undergoing rigorous exploration and indepth investigation to uncover their full range of potential applications [14][15][16], previous research has shown that erinacerin B could reduce LPS-induced nitric oxide (NO) and prostaglandin E2 (PGE2) production in RAW 264.7 macrophage cells in a concentrationdependent manner, thus exhibiting anti-inflammatory properties [17].Moreover, erinacerins D-L act as α-glucosidase inhibitors, with IC50 values in the range of 12.8 to 145.1 µM [13].Additionally, erinacerins O-P have emerged as promising candidates for potential glioma inhibitors, as they have demonstrated the ability to induce apoptosis in U87 cells through the Bax/Capase-2 pathway [18].Furthermore, erinacerins Q-T have exhibited notable inhibitory activity against protein tyrosine phosphatase-1B (PTP-1B), with IC50 values ranging from 24.9 to 42.1 µM [19].In this study, for the first time, erinacerin W demonstrated anti-neural inflammation and the potential to inhibit the onset and progression of neurodegenerative diseases, distinguishing it from other erinacerin series.
Co-culture modules using SH-SY5Y neuroblastoma and BV2 microglia cells have been successfully utilized to study the harmful effects of microglial activation in neurodegenerative diseases [20].To examine the potential impacts of compounds 1-4 on LPSactivated microglia-mediated cell viability, we investigated the co-culture of BV-2 microglia with SH-SY5Y neurons.After incubating SH-SY5Y cells with a range of concentrations (from 0 to 20 µg of compounds 1-4) for 24 h, it was observed that compound 1 had no cytotoxic effect up to 20 µg/mL, while compounds 1 and 2 above 5 µg/mL and compound 3 above 2.5 µg/mL reduced cell viability by approximately 10% (Figure 4).Therefore, nontoxic doses were selected for further experiments.Consistent with a previous study [21], the mRNA levels of TLR4, IL-6, IL-1β, and TNF-α significantly increased in LPS-stimulated BV2 microglia-mediated SH-SY5Y cells, as shown in Figure 5 (p < 0.05).The activation of these signaling proteins, also known as a cytokine storm, not only plays important roles in normal bodily functions and innate immunity [22], but is also associated with neurological diseases [23].In this study, compounds 2 and 3 effectively reduced proinflammatory signaling proteins, such as IL-6 and TNF-α, induced by LPS, which is consistent with earlier research [24,25].Additionally, exposure to a low dose, rather than a high dose, of compound 4 showed a decrease in the neuroinflammatory response, potentially promoting cellular repair and protection.Furthermore, for the first time, compound 1, at a concentration of 20 µg/mL, demonstrated protection against the excessive expression of IL-6, IL-1β, and TNF-α in SH-SY5Y cells induced by LPSactivated BV-2 cells.Previous studies have shown that LPS-induced inflammation in BV-2 microglia cells increases TLR4 expression and activates the NF-κB and MAPKs pathways, leading to the production of TNF-α, IL-6, and IL-1β [26].In line with this, compounds 1-4 may decrease the production of TNF-α, IL-6, and IL-1β, possibly involving pathways such as NF-κB or MAPK.The significant finding of this study is that erinacerin W demonstrated low cell toxicity while effectively mitigating neural inflammation.This indicates its possible use in slowing down, or potentially even halting, the progression of neurodegenerative diseases.However, more studies are needed for further clarification.Overall, considering the importance of the cytokine storm in neurodegeneration, compounds 1-4 may indirectly have protective potential by regulating microglia-mediated inflammatory processes, thus offering promising therapeutic approaches for CNS disorders.
to the untreated controls and represented as the mean ± standard deviation (SD) of three independent experiments.* indicates a significant difference compared to the control group (p < 0.05).
Consistent with a previous study [21], the mRNA levels of TLR4, IL-6, IL-1β, and TNF-α significantly increased in LPS-stimulated BV2 microglia-mediated SH-SY5Y cells, as shown in Figure 5 (p < 0.05).The activation of these signaling proteins, also known as a cytokine storm, not only plays important roles in normal bodily functions and innate immunity [22], but is also associated with neurological diseases [23].In this study, compounds 2 and 3 effectively reduced proinflammatory signaling proteins, such as IL-6 and TNF-α, induced by LPS, which is consistent with earlier research [24,25].Additionally, exposure to a low dose, rather than a high dose, of compound 4 showed a decrease in the neuroinflammatory response, potentially promoting cellular repair and protection.Furthermore, for the first time, compound 1, at a concentration of 20 µg/mL, demonstrated protection against the excessive expression of IL-6, IL-1β, and TNF-α in SH-SY5Y cells induced by LPS-activated BV-2 cells.Previous studies have shown that LPS-induced inflammation in BV-2 microglia cells increases TLR4 expression and activates the NF-κB and MAPKs pathways, leading to the production of TNF-α, IL-6, and IL-1β [26].In line with this, compounds 1-4 may decrease the production of TNF-α, IL-6, and IL-1β, possibly involving pathways such as NF-κB or MAPK.The significant finding of this study is that erinacerin W demonstrated low cell toxicity while effectively mitigating neural inflammation.This indicates its possible use in slowing down, or potentially even halting, the progression of neurodegenerative diseases.However, more studies are needed for further clarification.Overall, considering the importance of the cytokine storm in neurodegeneration, compounds 1-4 may indirectly have protective potential by regulating microgliamediated inflammatory processes, thus offering promising therapeutic approaches for CNS disorders.

Material Preparation
Hericium erinaceus (BCRC 35669) was purchased from the Bioresources Collection, Research, and Development Institute in Hsinchu, Taiwan.A fresh H. erinaceus mycelium block (1 cm 3 ) was added to a 2 L Erlenmeyer flask containing 1.3 L of a modified medium.The modified medium consisted of 0.25% yeast extract, 4.5% glucose, 0.5% soybean powder, 0.25% peptone, and 0.05% MgSO 4 , with the pH adjusted to 4.5.The entire medium was then incubated at 26 • C on a 120 rpm shaker for 5 days.Following this, the fermentation process was scaled up from a 2 L shake flask to 500 L and continued for 5 days.The mycelia grown on the complete medium were harvested, freeze-dried, and ground into a fine powder.

Extraction and Isolation
A schematic diagram of the extraction and isolation of compounds from H. erinaceus mycelia is shown in Figure 6.

Material Preparation
Hericium erinaceus (BCRC 35669) was purchased from the Bioresources Collection, Research, and Development Institute in Hsinchu, Taiwan.A fresh H. erinaceus mycelium block (1 cm 3 ) was added to a 2 L Erlenmeyer flask containing 1.3 L of a modified medium.The modified medium consisted of 0.25% yeast extract, 4.5% glucose, 0.5% soybean powder, 0.25% peptone, and 0.05% MgSO4, with the pH adjusted to 4.5.The entire medium was then incubated at 26 °C on a 120 rpm shaker for 5 days.Following this, the fermentation process was scaled up from a 2 L shake flask to 500 L and continued for 5 days.The mycelia grown on the complete medium were harvested, freeze-dried, and ground into a fine powder.

Extraction and Isolation
A schematic diagram of the extraction and isolation of compounds from H. erinaceus mycelia is shown in Figure 6.The freeze-dried mycelia of H. erinaceus (250 g) were refluxed with 95% ethanol.The ethanol solution was concentrated under vacuum to produce a brown extract, which was then divided into a water layer, ButOH, EA, and a Hexane layer using a H 2 O-EA (1:1) partitioning.The structure of the known compounds, erinacines A (2), C (3), and S (4), was identified by correlating the experimental NMR data with values published in previous studies [27,28].For an additional novel compound, the EA layer was purified by chromatography on a silica gel (70-230 mesh) column, using a gradient system of n-hexane-EA (1:0 to 0:1), resulting in five fractions.Fraction 4 (n-hexane-EA = 1:3 eluate) was further separated using HPLC Fraction Collectors (reversed-phase COSMOSIL 5C 18 -AR-II column, Nacalai Tesque, Kyoto, Japan) and eluted with a gradient system of H 2 O-acetonitrile (4:1 → 1:4) to isolate compound 1, a novel isoindolinone compound (13 mg).

Total RNA Isolation and Quantitative PCR (qPCR) Analysis
After treating the SH-SY5Y cells with the specified agents, they were collected and lysed to assess the expression of TLR4, IL-6, IL-1β, and TNF-α.Total RNA was isolated using an RNA purification kit (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer's instructions.The isolated RNA (1.0 µg) was then reverse-transcribed using the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA) to generate cDNA.The relative mRNA expression level was normalized to GAPDH expression, and the ∆∆Ct method was used for quantification.

Statistical Analysis
All the graphs, calculations, and statistical analyses were performed using the Graph-Pad Prism software, version 8.0 (GraphPad Software, San Diego, CA, USA).The comparison of means between different groups of numerical variables was performed using a one-way ANOVA.A p value less than 0.05 (p < 0.05) was considered as statistically significant.

Conclusions
In conclusion, the newly isolated compound, erinacerin W, exhibited protective effects against inflammation-associated neurotoxicity.Furthermore, erinacines A, C, and S also showed potential in reducing proinflammatory cytokines.These findings suggest that these compounds may have therapeutic potential in the treatment of neurodegenerative diseases by regulating microglia-mediated inflammatory processes.Further research is needed to explore the full potential and mechanisms of action of these compounds and to develop effective therapies for CNS disorders.
) was obtained as a pale-yellow clear oil with no specific odor.It has a molecular formula of C 24 H 31 NO 7 and a molecular weight ion of m/z 444.202 [M − H] −, determined by LC-QTOF MS (Santa Clara, CA, USA).
) was obtained as a paleyellow clear oil with no specific odor.It has a molecular formula of C24H31NO7 and a molecular weight ion of m/z 444.202 [M − H] −, determined by LC-QTOF MS (Santa Clara, CA, USA).

Figure 4 .
Figure 4. Concentration-effect curves of compounds 1-4 in SH-SY5Y cells were assessed after 24 h of exposure using the MTT assay.The results were expressed as a percentage of cell viability relative

Figure 4 .
Figure 4. Concentration-effect curves of compounds 1-4 in SH-SY5Y cells were assessed after 24 h of exposure using the MTT assay.The results were expressed as a percentage of cell viability relative to the untreated controls and represented as the mean ± standard deviation (SD) of three independent experiments.* indicates a significant difference compared to the control group (p < 0.05).

Figure 5 .
Figure 5. Effects of compounds 1-4 and curcumin (positive control) on the expression of TLR4, IL-6, IL-1β, and TNF-α in LPS-stimulated BV2 microglia-mediated SH-SY5Y cells.The mRNA expression levels were calculated relative to a GAPDH control using standard curves.# Significant difference in comparison with the untreated group (p < 0.05).* Significant difference in comparison with LPS-induced group (p < 0.05).The use of double asterisks (**) indicates a significant difference with a p-value of less than 0.01.Results were expressed as mean ± standard deviation (SD).

Figure 5 .
Figure 5. Effects of compounds 1-4 and curcumin (positive control) on the expression of TLR4, IL-6, IL-1β, and TNF-α in LPS-stimulated BV2 microglia-mediated SH-SY5Y cells.The mRNA expression levels were calculated relative to a GAPDH control using standard curves.# Significant difference in comparison with the untreated group (p < 0.05).* Significant difference in comparison with LPS-induced group (p < 0.05).The use of double asterisks (**) indicates a significant difference with a p-value of less than 0.01.Results were expressed as mean ± standard deviation (SD).

Figure 6 .
Figure 6.Schematic diagram of the extraction and isolation of compounds 1-4 from H. erinaceus mycelia.

Figure 6 .
Figure 6.Schematic diagram of the extraction and isolation of compounds 1-4 from H. erinaceus mycelia.