Unprecedented Neoverrucosane and Cyathane Diterpenoids with Anti-Neuroinflammatory Activity from Cultures of the Culinary-Medicinal Mushroom Hericium erinaceus

The culinary medicinal mushroom Hericium erinaceus holds significant global esteem and has garnered heightened interest within increasingly ageing societies due to its pronounced neuroprotective and anti-neuroinflammatory properties. Within this study, two novel diterpenes, 16-carboxy-13-epi-neoverrucosane (1) and Erinacine L (2); three known xylosyl cyathane diterpenoids, Erinacine A (3), Erinacine C (4), and Erinacine F (5); and four lanostane-type triterpenoids, and three cyclic dipeptides (10–12), in addition to orcinol (13), were isolated from the rice-based cultivation medium of H. erinaceus. Their structures were determined by NMR, HR-ESI-MS, ECD, and calculated NMR. Compound 1 marks a pioneering discovery as the first verrucosane diterpene originating from basidiomycetes, amplifying the scope of fungal natural product chemistry, and the intricate stereochemistry of Compound 5 has been comprehensively assessed for the first time. Compounds 2–5 not only showed encouraging neurotrophic activity in rat adrenal pheochromocytoma PC-12 cells, but also significantly inhibited lipopolysaccharide (LPS)-induced nitric oxide (NO) production in BV2 microglia cell cultures with IC50 values as low as 5.82 ± 0.18 μM. To elucidate the mechanistic underpinnings of these bioactivities, molecular docking simulation was used to analyze and support the interaction of 1 and 2 with inducible NO synthase (iNOS), respectively. In particular, compound 2, a cyathane-xyloside containing an unconventional hemiacetal moiety, is a compelling candidate for the prevention of neurodegenerative diseases. In summation, this investigation contributes substantively to the panorama of fungal diterpene structural diversity, concurrently furnishing additional empirical substantiation for the role of cyathane diterpenes in the amelioration of neurodegenerative afflictions.


Introduction
Mushrooms, predominantly the fruiting bodies of basidiomycetes, are an important source of natural products [1].Mushrooms produce structurally diverse bioactive compounds that play essential roles in natural medicines, complementary and alternative medicines, and nutraceutical functional foods [1][2][3].Additionally, many mushrooms have nutritional value, with H. erinaceus being a prime example.H. erinaceus is one of the most common edible and medicinal mushrooms, known as "Houtou" in China, "Yamabushitake" in Japan, and Lion's Mane in the West.It has a long history of use in traditional Chinese medicine and as a delicacy due to its proven health benefits.

Structure Elucidation for 1 and 2
Compound 1 was isolated as a white powder.The molecular formula of 1 was determined to be C20H32O3, with five degrees of unsaturation, basis on HRESIMS at m/z 343.2240 [M+Na] + (Figure S1).Compound 1 showed a similar NMR spectroscopic profile to that of 5β-hydroxy-epineoverrucosane [20].The key difference between two compounds was the replacement of the methyl at C-16 (δC 21.3) by carbonyl (δC 181.2), which was implied by HMBC correlation of H-13 (δH 2.33) and 17-Me (δH 1.26) with C-16.Meanwhile, the absolute configuration of 1 was determined combining the results of NMR calculations of the chirality of C13 and C15 (Figure 2B), as well as experimental CD and calculated CD comparisons (Figure 2C).Therefore, 1 was named 16-carboxy-13-epi-neoverrucosane.Its IR and UV spectra were shown on Figures S8 and S9.
Compound 2 was isolated as light yellow powder with a molecular formula of C28H46O8 based on its HR-ESI-MS peak at m/z 533.3091 [M+Na] + (calcd for C28H46O8Na, 533.3084) (Figure S10), indicating six indices of hydrogen deficiency.The UV spectrum displayed absorption maxima at 200 and 230 nm (Figure S4).Analyses of the 1 H and 13 C NMR spectra (Figures S11 and S12) of 2 revealed the presence of seven methyl groups, six methylene groups (one oxygenated sp3 carbons at δC 60.8), ten methines (one sp2 carbon at δC 132.0, and seven oxygenated sp3 carbons), and five quaternary carbons (three sp2 carbons at δC 139.4,139.8, and 141.4 and two quaternary sp3 carbons at δC 43.7 and 49.7).

Structure Elucidation for 1 and 2
Compound 1 was isolated as a white powder.The molecular formula of 1 was determined to be C 20 H 32 O 3 , with five degrees of unsaturation, basis on HRESIMS at m/z 343.2240 [M+Na] + (Figure S1).Compound 1 showed a similar NMR spectroscopic profile to that of 5β-hydroxy-epineoverrucosane [20].The key difference between two compounds was the replacement of the methyl at C-16 (δ C 21.3) by carbonyl (δ C 181.2), which was implied by HMBC correlation of H-13 (δ H 2.33) and 17-Me (δ H 1.26) with C-16.Meanwhile, the absolute configuration of 1 was determined combining the results of NMR calculations of the chirality of C13 and C15 (Figure 2B), as well as experimental CD and calculated CD comparisons (Figure 2C).Therefore, 1 was named 16-carboxy-13-epi-neoverrucosane.Its IR and UV spectra were shown on Figures S8 and S9.
Compound 2 was isolated as light yellow powder with a molecular formula of Comprehensive analysis of the 1D and 2D NMR data (Figures S13-S16) of 2 resulted in the whole assignment of the proton signals to their respective carbons (Table 1).
Molecules 2023, 28, x FOR PEER REVIEW 4 of 18 Comprehensive analysis of the 1D and 2D NMR data (Figures S13-S16) of 2 resulted in the whole assignment of the proton signals to their respective carbons (Table 1).Analysis of the 1 H-1 H COSY spectrum (Figure S14) of 2 revealed the presence of six spin-coupling systems (Figure 2D).In the HMBC spectrum (Figure S15), the correlations from H-17 to C-1/C-9/C-4, H-18 to C-2, H-20 to C-3, H-8 to C-6, H-11 to C-5/C-13, and H-16 to C-14 suggested the existence of a fused 5/6/7 tricarbocyclic cyathane-type scaffold in compound 2 (Figure 2D).In addition, the HMBC correlations from H-14 and C-1 , H-1 to C-5 , H-5 to C-3 suggested that the erinacine-like xyloside was located at the C-14.Further analysis of the NMR data with those of reported Erinacine Z2 suggested that both compounds share the similar structure exception an acetal [-CH(OCH 3 ) 2 ] moiety on 2 instated of an aldehyde in Erinacine Z2 [9,29], which was confirmed by the diagnostic HMBC correlations from H-15 to C-22/C-23 (Figure 2D).Finally, the structure with relative configuration of compound 2 was determined as shown and named Erinacine L.

Structure Revision for 5
Compound 5 was first reported to have been isolated from the mycelium of the H. erinaceus in 1994 [5], and no further isolation has been reported since.In this work, 5 was identified as erinacine F by matching one-dimensional NMR data with literature report [5].Although the planar structure of 5 was determined to be consistent with that of erinacine E [5], its absolute configuration has not been confirmed due to the absence of a two-dimensional correlation of the xylosyl group.The chirality of the C1' of 5 was inferred to be the S configuration based on the absolute configuration of the identified xylosyl cyathane diterpenes.The remaining four C atoms of undetermined chirality are C15, C2', C3', and C4', with hydroxyl modifications.Thus, there are 16 absolute configurations of 5, i.e., I_1-I_16.Chemical shift calculations were carried out for the 16 conformations using the correlation basis set based on density functional theory (DFT) within Gaussian 16.Then the data were disposed with the help of DP4+ application (Dataset_DP4+).The results showed that the absolute configuration of 5 is 98.88% probability of I_9, i.e., four chiral carbons with the configuration 15S, 2'S, 3'S, 4'R (Figure 3).The percentage of configuration I_16, the absolute configuration of erinacine E, is only 0.004%, which proves the reliability of this result to some extent (Figure 3).

Structure Revision for 5
Compound 5 was first reported to have been isolated from the mycelium of the H. erinaceus in 1994 [5], and no further isolation has been reported since.In this work, 5 was identified as erinacine F by matching one-dimensional NMR data with literature report [5].Although the planar structure of 5 was determined to be consistent with that of erinacine E [5], its absolute configuration has not been confirmed due to the absence of a twodimensional correlation of the xylosyl group.The chirality of the C1' of 5 was inferred to be the S configuration based on the absolute configuration of the identified xylosyl cyathane diterpenes.The remaining four C atoms of undetermined chirality are C15, C2', C3', and C4', with hydroxyl modifications.Thus, there are 16 absolute configurations of 5, i.e., I_1-I_16.Chemical shift calculations were carried out for the 16 conformations using the correlation basis set based on density functional theory (DFT) within Gaussian 16.Then the data were disposed with the help of DP4+ application (Dataset_DP4+).The results showed that the absolute configuration of 5 is 98.88% probability of I_9, i.e., four chiral carbons with the configuration 15S, 2'S, 3'S, 4'R (Figure 3).The percentage of configuration I_16, the absolute configuration of erinacine E, is only 0.004%, which proves the reliability of this result to some extent (Figure 3).

Neurotrophic Activity
Compounds 1-9 were selected for evaluation of PC-12 cell-based neurotrophic activity, and at a concentration of 5 µM, 1-9 all showed NGF-dependent growth promotion of PC12 cells.Among them, 2-5 showed significant promotion, while 1 and 6-9 showed weak activity.Compared with the 12.11% promotion rate of 20 ng/mL NGF, the 5 µM concentration of 2 increased the promotion rate to 24.51%, showing the strongest promotion effect (Figure 4A).To investigate the effect of different concentrations on the promotion

Neurotrophic Activity
Compounds 1-9 were selected for evaluation of PC-12 cell-based neurotrophic activity, and at a concentration of 5 µM, 1-9 all showed NGF-dependent growth promotion of PC12 cells.Among them, 2-5 showed significant promotion, while 1 and 6-9 showed weak activity.Compared with the 12.11% promotion rate of 20 ng/mL NGF, the 5 µM concentration of 2 increased the promotion rate to 24.51%, showing the strongest promotion effect (Figure 4A).To investigate the effect of different concentrations on the promotion effect of 2, a concentration gradient experiment for 2 was performed.At a concentration of 1 µM, the promotion rate of NGF-dependent 2 was 20.73%, and when the concentration of 2 reached 9 µM, the promotion rate of 2 increased to 28.14% (Figure 4B,C).This finding suggests that the NGF-dependent promotion of 2 has a dose-dependent effect over a certain range.
effect of 2, a concentration gradient experiment for 2 was performed.At a concentration of 1 µM, the promotion rate of NGF-dependent 2 was 20.73%, and when the concentration of 2 reached 9 µM, the promotion rate of 2 increased to 28.14% (Figure 4B,C).This finding suggests that the NGF-dependent promotion of 2 has a dose-dependent effect over a certain range.

Anti-Neuroinflammatory Activities
To further explore anti-neurodegenerative disease activity, 1-9 was used to test BV2 microglia cell-based anti-neuroinflammatory activity.Preliminary toxicity testing showed that at a concentration of 40 µM, 1-9 showed over 80% cell viability after 24 h of incubation with BV2 cells, which was superior to that (81.06%) of the positive control quercetin (Figure 5A).
In the anti-LPS-induced inflammation assay in BV2 cells, compounds 1-5 inhibited LPS-induced NO production in medium with IC50 values ranging from 5.82 µM to 31.44 µM, while compounds 6-9 showed extremely weak inhibitory activity with IC50 values above 40.00µM.The inhibition of NO production by compounds 1-2 was significantly stronger than that of the positive control quercetin (16.00 µM) (Figure 5B and Table S1).The promising inhibitory activity demonstrated by compound 2 led to the investigation of its influence on the expression of genes involved in the pathway of NO production.In contrast to the upregulation of P-NF-κB P65, iNOS, COX-2, and TLR4 stimulated by LPS, the addition of 2 significantly downregulated the expression of these genes.In addition, the addition of 2 weakly attenuated the expression of NF-κB P65.(Figure 5C).LPS was not significantly shown to induce NF-κB P65 and GAPDH, on which the addition of 2 weakly attenuated NF-κB-P65 expression, although it did not cause changes in GAPDH expression (Figure 5C).

Anti-Neuroinflammatory Activities
To further explore anti-neurodegenerative disease activity, 1-9 was used to test BV2 microglia cell-based anti-neuroinflammatory activity.Preliminary toxicity testing showed that at a concentration of 40 µM, 1-9 showed over 80% cell viability after 24 h of incubation with BV2 cells, which was superior to that (81.06%) of the positive control quercetin (Figure 5A).

Molecular Docking Simulation of iNOS Inhibition
To better understand the anti-neuroinflammatory activity of 1-2, molecular docking simulations of 1-2 with iNOS were performed.The docking analysis showed that 1-2 binds to iNOS by occupying pockets through multiple interactions (Figure 6A,B).Compound 1 interacts directly with Gln263, Arg266, Glu377, Arg381, and Asp382 to bind to iNOS with a docking energy of −22 kcal/mol CDOCKER interaction energy (Figure 6A).Compound 2 interacts directly with Met120, Gln263, Glu377, Arg381, and Trp463, and binds to iNOS with a docking energy of −37 kcal/mol CDOCKER interaction energy (Figure 6B).Compound 2 has a stronger inhibitory activity as reflected by its lower docking energy value.Both putative models share residues Gln263, Glu377, and Arg381, which may be key residues in the inhibition of iNOS activity.In the anti-LPS-induced inflammation assay in BV2 cells, compounds 1-5 inhibited LPS-induced NO production in medium with IC 50 values ranging from 5.82 µM to 31.44 µM, while compounds 6-9 showed extremely weak inhibitory activity with IC 50 values above 40.00µM.The inhibition of NO production by compounds 1-2 was significantly stronger than that of the positive control quercetin (16.00 µM) (Figure 5B and Table S1).The promising inhibitory activity demonstrated by compound 2 led to the investigation of its influence on the expression of genes involved in the pathway of NO production.In contrast to the upregulation of P-NF-κB P65, iNOS, COX-2, and TLR4 stimulated by LPS, the addition of 2 significantly downregulated the expression of these genes.In addition, the addition of 2 weakly attenuated the expression of NF-κB P65.(Figure 5C).LPS was not significantly shown to induce NF-κB P65 and GAPDH, on which the addition of 2 weakly attenuated NF-κB-P65 expression, although it did not cause changes in GAPDH expression (Figure 5C).

Molecular Docking Simulation of iNOS Inhibition
To better understand the anti-neuroinflammatory activity of 1-2, molecular docking simulations of 1-2 with iNOS were performed.The docking analysis showed that 1-2 binds to iNOS by occupying pockets through multiple interactions (Figure 6A,B).Compound 1 interacts directly with Gln263, Arg266, Glu377, Arg381, and Asp382 to bind to iNOS with a docking energy of −22 kcal/mol CDOCKER interaction energy (Figure 6A).Compound 2 interacts directly with Met120, Gln263, Glu377, Arg381, and Trp463, and binds to iNOS with a docking energy of −37 kcal/mol CDOCKER interaction energy (Figure 6B).Compound 2 has a stronger inhibitory activity as reflected by its lower docking energy value.Both putative models share residues Gln263, Glu377, and Arg381, which may be key residues in the inhibition of iNOS activity. .

Discussion
Mushrooms have enjoyed a longstanding dual role as both sustenance and medicinal agents, with their consumption tracing back in tandem with the trajectory of human civilization.The curative attributes and therapeutic potential intrinsic to mushrooms have Furthermore, the stability of the iNOS-2 docking complex was evaluated using Molecular dynamics (MD) simulations.The results show that the root mean square deviation (RMSD) values tend to stabilize on a time scale of 20 ns and at 300 K (Figure 6C).The root-mean-square fluctuation (RMSF) plots of the iNOS-2 docking complex calculated in the MD simulations at 300 K show that some of the residues show less stability.The RMSF plots of the iNOS residues calculated in the MD simulation at 300 K showed a small region with small fluctuations (Figure 6D).Visualization by PyMOL reveals that the fluctuating region contains amino acids 110-118, which form a coil (Figure S52).This region is outside the region where 2 directly interacts with iNOS (Figure S52), and changes in this region do not affect the direct interaction of 2 with iNOS.These results suggest that 2 was able be to stably bind to iNOS through the above five amino acid residues and thus affect iNOS activity.

Discussion
Mushrooms have enjoyed a longstanding dual role as both sustenance and medicinal agents, with their consumption tracing back in tandem with the trajectory of human civilization.The curative attributes and therapeutic potential intrinsic to mushrooms have held paramount significance in the annals of traditional medicine, with Hericium erinaceus emerging as a globally acknowledged medicinal fungus.In contemporary times, the attention lavished upon H. erinaceus has been fueled by its burgeoning reputation as a prolific source of cyathane diterpenes, exhibiting singular promise in the realm of counteracting neurodegenerative ailments [7].Although cyathane diterpene-producing mushrooms encompasses genera such as Hericium [7], Cyathus [10], Sarcodon [8], and various others [8], the genus Hericium possesses both medicinal and culinary values.Therefore, the therapeutic use of Hericium mushrooms for the prevention and alleviation of neurodegenerative disorders is of great significance.The bioactive compounds derived from mushrooms often show species specificity.For example, des-A-ergostane-type compounds with anti-cancer activity, blazeispirols, are unique to Agaricus species [37][38][39].Ganoderma acids, lanosterol-type triterpene derivatives, are the most biologically active components of the Ganoderma species [40].However, the realm of cyathane diterpenoids, renowned for their potent neuroprotective attributes, traverses beyond the confines of the Hericium genus [7] to encompass Cyathus species [10] and other mushrooms [8].This remarkable distribution underscores the pervasive prevalence of specific mushroom compounds.In parallel, the arena of biologically active natural pigments finds expression in styrylpyrone compounds, an illustrious group discerned widely within the fungal domains of Inonotus [2,41] and Phellinus [42] nestled within the Geomycetaceae family.
As the metabolites of cyathane diterpenoid producers continue to be elucidated, the probability of discovering new xyloside-cyathane diterpenes is decreasing.The most recent report is the discovery of eight previously undescribed cyathane-xylosides from Dentipellis fragilis [16].Compound 2, as a new xyloside-cyathane diterpene, was isolated from H. erinaceus together with 3 and 4, which have been isolated several times.Compound 2 contains an unusual hemiacetal group, which is rare in cyathane diterpenes, and even in terpenoids.Although cyahookerin E [43] is the only cyathane diterpene previously found to contain a hemiacetal group, 2 is, to our knowledge, the only xyloside-cyathane diterpene currently found to contain a hemiacetal group.Compound 2 with hemiacetal group has excellent neurotrophic and anti-neuroinflammatory activities, suggesting that the hemiacetal moiety may be an essential pharmacophore for the treatment of neurodegenerative diseases.Using calculational NMR, the absolute configuration of 5 has been determined for the first time.Simultaneously, the anti-neuroinflammatory activity of 5 was tested for the first time [44].Studies targeting neurotrophic and anti-neuroinflammatory activities at the level of signaling pathways are uncommon.Sarcodonin G, a naturally occurring cyathane diterpene, has been found to exert neuroprotective effects through activation of TrkB on the Trk signaling pathway [45].It is proposed that the neurotrophic activity of compounds 2-5 is mediated in a similar manner.Another interesting compound is 13, which is closely related to lichens.Although several chlorinated and oxidized derivatives of 13 have been found in Hericium species [46][47][48], no 13 has been reported from H. erinaceus.The present work reports, for the first time, 13 isolated from H. erinaceus.
Verrucosane diterpenes are a group of terpenoids with a 3,6,6,5 tetracyclic carbon skeleton, whose 6,5-biring portion is highly congruent with that of the cyathane diterpene skeleton.The 5,6,7-tricyclic cyathane diterpene skeleton was proposed to be a precursor structure for the biosynthesis of verrucosane diterpenes [49], which was partially confirmed by a study based on transition state energy calculations [50].Compound 1 identified in this work is the first example of an isopropyl group being oxidized to form a carboxyl group in a verrucosane diterpene, and oxidation at this position in the cyathane diterpene is only seen in cyathin C5 [11].Verrucosane diterpenes have previously been reported to occur in lower plants [19,25], marine sponges [21,28], and bacteria [51], and here, we isolate the first such compounds from a basidiomycete.Furthermore, this is the first time that both neoverrucosane (1) and cyathane (2-5) diterpenes, two different types of diterpenoids, have been found in the same species.Compound 1 was found to have favorable antineurodegenerative disease activity, which is the first evaluation of verrucosane diterpenes for neuroprotective and anti-neuroinflammatory related activities.

General Experimental Procedures
NMR spectra were recorded on a Bruker Avance III 500 MHz NMR spectrometer at 400 MHz ( 1 H) and 100 MHz ( 13 C) using TMS as internal standard and chemical shifts were recorded in parts per million δ (ppm).HR-ESI-MS was performed on an AB Sciex TripleTOF ® 6600 LC/MS system.Circular dichroism and IR were recorded on a Chirascan™ CD spectrometer.Column chromatography was performed using silica gel (100-200 mesh and 300-400 mesh), Sephadex LH-20 (GE Healthcare, Chicago, IL, USA), and reversed phase C18 silica gel (RP-18, GE Healthcare).For thin-layer chromatography, precoated silica gel 60 F254 plates were used and spots were visualized under UV light (210, 254 and 365 nm) or by spraying with vanilin-H 2 SO 4 10% solution and heating for two min.Concentration was performed with a Büchi Rotavapor R-101.

Fungal Material
The fungus H. erinaceus was obtained from the China General Microbiological Culture Collection Center (CGMCC) with the accession number CGMCC 5.579.A copy (No.HE2019) was deposited at the College of Biology Pharmacy & Food Engineering, Shangluo University, Shaanxi, China.The culture medium consisted of glucose 2%, yeast extract 0.2%, peptone 0.5%, MgSO 4 0.05%, and KH 2 PO 4 0.1%.Fermentation was carried out on a shaker at 130 rpm for 30 days at room temperature.

Extraction, Isolation and Purification
A total of 10 kg of rice was divided into 100 500-mL shaking flasks, each containing 100 g of rice.Then, 60 mL of sterile water was added and soaked for 2 h and finally autoclaved to complete the medium preparation.Fermentation was carried out at room temperature for 30 days after the strains were added to the medium.The culture was collected and extracted with ethyl acetate (10 L × 3).The EtOAc extract was concentrated under reduced pressure to give a crude extract (102.2 g).

Structural Identification and Quantum Chemistry Calculations
The theoretical ECD spectra of compounds 1-2 were calculated using Gaussian 16 software (Gaussian Inc., Wallingford, CT, USA) based on the relative configurations determined from the NOESY spectra.Conformational searches were performed using the Monte Carlo Molecular Mechanics (MCMM) method and the OPLS_2005 force field within an energy window of 21 kJ/mol.The resulting conformers were optimized at the B3LYP/6-311+G (d, p) level of theory in Gaussian 16.ECD calculations on the optimized conformers employed B3LYP/6-311+G (d, p) in Gaussian 16, incorporating methanol solvent effects with the PCM.Boltzmann averaged ECD spectra were generated in Microsoft Excel.
NMR chemical shift calculations were performed using DFT in Gaussian 16.Preliminary conformational searches were conducted in Spartan 14, with conformers viewed in GaussView 9.0 to prepare Gaussian input files.Geometries were optimized at the B3LYP/6-311+G (d, p) level, then stable conformations from B3LYP/6-31G (d, p) underwent magnetic shielding constant calculations at B3LYP/6-311++G (2d, p) level by solvent of methanol using PCM.The results of the previous NMR calculations were imported into Multiwfn 3.7 [52] to obtain the shielding values in different conformations.The shielding values are weighted and averaged by calculating the Boltzmann distribution from the energies obtained by conformational optimization and finally converted to chemical shifts by scaling.DP4+ [53] probability analysis compares experimental and calculated shifts for stereochemical assignment.The neurotrophic activity of compounds 1-9 was investigated using PC-12 cells obtained from the China Center for Typical Culture Collection.PC-12 cells were cultured in F-12 (Ham) medium supplemented with 10% heat-inactivated horse serum (HS), 5% heat-inactivated fetal bovine serum, 100 U/mL penicillin G, 100 µg/mL streptomycin, and 2.5 g/L sodium bicarbonate.Cells were maintained at 37 • C in a humidified 5% CO 2 atmosphere.
As previously described, PC-12 cells were analyzed morphologically and quantified for neurite outgrowth using phase contrast microscopy.Cells were seeded in 24-well plates coated with poly-L-lysine at a density of 2 × 10 4 cells/mL in standard serum medium for 24 h.Before exposure to the vehicle (0.1% dimethyl sulfoxide (DMSO)) or test compounds, the F-12 medium containing low serum (1% HS and 0.5% fetal bovine serum (FBS)) was replaced.Cells were treated with 10 µM and 20 ng/mL concentrations of test compound 1.Untreated cells served as a negative control, while 20 ng/mL nerve growth factor (NGF) was used as a positive control.Each concentration was tested in triplicate wells.After 48 h of incubation, neuronal growth of the PC-12 cells was photographed using an inverted microscope with a phase contrast objective and digital camera.Five random images were acquired from each well.The percentage of cells with neurites greater than or equal to one cell body length was considered positive for neurite outgrowth.This was quantified as a percentage of the total cells counted over the five randomly selected fields of view.The experiment was repeated at least three times and the results are presented as mean ± standard deviation.

Evaluation of Anti-Neuroinflammatory Activity
BV-2 cells were obtained from the Cell Resource Center of Peking Union Medical College (Beijing, China).The cells were cultured in DMEM supplemented with 10% heatinactivated FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin at 37 • C under 5% CO 2 .For drug treatment, cells at 70-80% confluence in DMEM with 10% FBS were treated with 40 µM of test compounds for specified times, while control cells received an equal volume of DMSO.
Cell viability was determined by the MTT assay.BV-2 cells were seeded overnight in 96-well plates at 1.5 × 10 5 cells/well.After 24 h of treatment with various compound concentrations, 10 µL of MTT solution (5 mg/mL) was added to each well for four hours.The supernatant was discarded, 150 µL DMSO was added to dissolve the formazan crystals, and absorbance was measured at 570 nm using a Bio-Rad plate reader.Cell viability was expressed as a percentage of the viability of DMSO-treated control cells.
For NO assays, BV2 cells were seeded at 2 × 10 5 cells/well in 96-well plates for 24 h before treatment with 3 µg/mL LPS and various compound concentrations, using DMSO as a solvent control.Quercetin was used as a positive control drug.Equal volumes of cell culture supernatants were assayed for NO production using a Griess reaction-based kit.Briefly, 50 µL supernatant was combined with 100 µL Griess reagent in a new 96well plate, incubated at room temperature for 15 min, and the absorbance was measured at 540 nm.Sodium nitrite standards were used construct a standard curve to calculate nitrite concentrations.The relative iNOS inhibitory activity was calculated as follows: iNOS inhibitory activity (%) = [1 − (Asp − Ab)/(Ast − Ab)] × 100%, where Asp is the absorbance of the sample reaction (containing all reagents), Ab is the absorbance of the blank group (containing all reagents without test compound), and Ast is the absorbance of the standard (containing all reagents).The IC 50 value was defined as the concentration that reduced NO generation by 50%, and calculated via the online tool (www.aatbio.com/tools/ic50-calculator/, accessed on 6 July 2023).

Molecular Docking and Dynamics Simulation
To elucidate potential binding modes, we performed molecular docking simulations using the crystal structure of human iNOS (PDB: 3E7G) in Discovery Studio 2017 R2.Prior to molecular docking, the receptor proteins underwent necessary pre-treatments such as energy minimization.The receptor and ligands 1-2 were prepared and semi-flexible docking simulations were conducted as previously described [54,55].The docking results were analyzed in Discovery Studio, and the 3D complexes from the docking models were visualized using PyMOL v2.5 (Schrodinger Inc., New York, NY, USA).
MD simulations were carried out with GROMACS 2023.2 (GROMACS Development Team, Groningen, The Netherlands) using the CHARMM 36 force field.The iNOS-2 docking complex was validated to confirm all necessary atoms were present.Topology files were generated using the pdb2gmx module, and the SPC216 water model was used to solvate the proteins.The electrically neutral system had ions added based on protein charges for MD simulations.The structures were energy minimized using 50,000 steps and a maximum force of 1000.0 kJ/mol/nm.The temperature was set to 298 K (25 • C) and system temperature was stabilized by a 100 ps NVT equilibrium, followed by a 100 ps NPT equilibrium to stabilize pressure.After equilibration, 20 ns production MD was performed for analysis.Protein stability was assessed by calculating RMSD and RMSF values and visualizing the data in Microsoft Excel 2019.

Conclusions
In conclusion, two new diterpenoids, 16-carboxy-13-epi-neoverrucosane (1) and erinacine L (2), together with eleven known compounds (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13), were isolated from the metabolites of rice medium of H. erinaceus.Compounds 1, 2, and 13 were discovered for the first time from the genus, and, furthermore, 1 was isolated from the fungi for the first time.Bioactivity assay revealed that compounds 2-5 have good neurotrophic activity in PC-12 cells and anti-neuroinflammatory activity in in BV2 cells.Among them, 2 showed the best activity (24.51% promotion rate under 5 µM and IC 50 of 5.82 ± 0.18 µM), probably related to the hemiacetal structure it contains.Molecular docking simulations of compounds 1 and 2 with iNOS, respectively, reflect three amino acid residues, Gln263, Glu377, and Arg381, as key sites that may influence iNOS activity.Further MD simulation confirmed the stability of the iNOS-2 docking complex.These results enrich the structural diversity of diterpenoids derived from mushroom, and provide additional evidence for the ability of xylosylated cycloalkane diterpenes to prevent and treat neurodegenerative diseases.
C 28 H 46 O 8 based on its HR-ESI-MS peak at m/z 533.3091 [M+Na] + (calcd for C 28 H 46 O 8 Na, 533.3084) (Figure S10), indicating six indices of hydrogen deficiency.The UV spectrum displayed absorption maxima at 200 and 230 nm (Figure S4).Analyses of the 1 H and 13 C NMR spectra (Figures S11 and S12) of 2 revealed the presence of seven methyl groups, six methylene groups (one oxygenated sp3 carbons at δ C 60.8), ten methines (one sp2 carbon at δ C 132.0, and seven oxygenated sp3 carbons), and five quaternary carbons (three sp2 carbons at δ C 139.4, 139.8, and 141.4 and two quaternary sp3 carbons at δ C 43.7 and 49.7).

Figure 3 .
Figure 3. Absolute configuration identification of 5 based on calculated NMR.

Figure 3 .
Figure 3. Absolute configuration identification of 5 based on calculated NMR.

Figure 6 .
Figure 6.Molecular docking and molecular dynamics simulations.(A,B) Schematic representation of the molecular docking of 1 and 2 with iNOS, respectively.(C) MD simulates iNOS-2 docking complex at 300 K. (D) RMSF plot of iNOS-2 docking complex.

Figure S25: 1 H
NMR spectra of compound 5 in CD 3 OD.FigureS26:13 C spectra of compound 5 in CD 3 OD.
Figure S32: 13 C spectra of compound 7 in CD 3 OD.

Figure S40: 1 H
NMR spectra of compound 10 in CD 3 OD.
Figure S41: 13 C spectra of compound 10 in CD 3 OD.

Figure S43: 1 H
NMR spectra of compound 11 in CD 3 OD.
Figure S44: 13 C spectra of compound 11 in CD 3 OD.
Figure S46: 1 H NMR spectra of compound 12 in CD 3 OD.
Figure S47: 13 C spectra of compound 12 in CD 3 OD.

Figure S49: 1 H
NMR spectra of compound 13 in CD 3 OD.
Figure S50: 13 C spectra of compound 13 in CD 3 OD.
Figure S52: Region of protein instability identified by RMSF analysis.Table S1: IC 50 data of 1-9 inhibited the LPS-induced NO production in culture medium.