A reducing triterpenoid compound with neuroprotective effect and a preparation method and application thereof

Abstract

The application discloses a hypotriper compound with a nerve protection effect and a preparation method and application thereof, and belongs to the technical field of natural medicines. The hypotriper compound with the nerve protection effect has a structure shown in formula 1 or formula 2, and is two new hypotriper compounds first discovered in tea disease ganoderma fruiting bodies. The nerve protection effect of the compound is evaluated by using a SH-SY5Y cell damage model induced by H2O2, and the result shows that the two hypotriper compounds have good nerve protection effects and have obvious dose-effect relationships. The hypotriper compound with the nerve protection effect can be applied to the preparation of medicines for treating neurodegenerative diseases.

Description

Technical Field

[0001] This invention relates to the field of natural medicine technology, and in particular to a triterpenoid compound with neuroprotective effects, its preparation method and application. Background Technology

[0002] Neurodegenerative diseases are one of the serious medical problems facing the 21st century. They are characterized by impaired cognitive function, behavioral abnormalities, memory impairment, and worsening symptoms with age. Common diseases include Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), and Huntington's disease. With the increasing aging of my country's population, the incidence of neurodegenerative diseases is rising year by year, seriously endangering the health of the elderly and placing a heavy burden on society and families.

[0003] Neurodegenerative diseases are closely related to oxidative stress. Protecting nerve cells from oxidative damage can effectively inhibit the development of neurodegenerative diseases, thereby achieving the goal of preventing and treating neurodegenerative diseases. Therefore, it is crucial to conduct in-depth research on the mechanisms of action of these diseases, identify effective drugs for treating oxidative stress, and develop lead compounds with potential therapeutic effects on neurodegenerative diseases. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide a neuroprotective triterpenoid compound, its preparation method and application. The neuroprotective triterpenoid compound is one of two novel triterpenoid compounds first discovered in the fruiting body of *Ganoderma lucidum*, which exhibit significant SH-SY5Y neuroprotective effects and are non-toxic.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] This invention provides a triterpenoid compound with neuroprotective effects, having the structure shown in Formula 1 or Formula 2:

[0007]

[0008] The structures shown in Formula 1 and Formula 2 are two novel triterpenoid compounds, ganothenoids B and ganothenoids D, which were first discovered in the fruiting body of Ganoderma lucidum with tea disease. These compounds, ganothenoids B and ganothenoids D, have a good protective effect against H2O2-induced SH-SY5Y cell damage and can significantly reduce oxidative damage and inhibit cell apoptosis.

[0009] The present invention also provides a method for preparing the above-mentioned triterpenoid compound with neuroprotective effect, wherein the fruiting body of Ganoderma lucidum is subjected to heating and reflux extraction, extraction, separation by normal phase silica gel column chromatography, separation by reverse phase ODS column chromatography, and separation by semi-preparative high performance liquid chromatography to obtain the above-mentioned triterpenoid compound with neuroprotective effect.

[0010] Preferably, the present invention further includes removing the solvent after the heating and reflux extraction to obtain an extract paste, then mixing the extract paste with water to obtain a suspension, and finally extracting the suspension.

[0011] Preferably, the solvent used for the heating and reflux extraction is selected from ethanol or methanol; more preferably, it is ethanol.

[0012] The ethanol is preferably 95% ethanol.

[0013] The preferred number of heating and reflux extraction cycles is 3.

[0014] Preferably, the solvent for extraction is selected from ethyl acetate or chloroform; more preferably, it is ethyl acetate.

[0015] Preferably, in this invention, the normal-phase silica column chromatography separation is performed by separating the extract obtained after extraction with petroleum ether and ethyl acetate at a volume ratio of 10:1 to 1:1, resulting in 11 components denoted as Fr.1 to Fr.11.

[0016] Preferably, in this invention, the component Fr.4 is separated by reversed-phase ODS chromatography to obtain components Fr.4-1 to Fr.4-9;

[0017] Preferably, the reversed-phase ODS column separation is performed using methanol and water in sequential volume ratios of 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, and 100:0, with an elution volume of 800 mL for each volume ratio.

[0018] Preferably, the component Fr.4-6 is separated by semi-preparative high performance liquid chromatography to obtain the compound shown in Formula 1.

[0019] Preferably, the semi-preparative high-performance liquid chromatography separation uses methanol and water at a volume ratio of 9:11 to 11:9. In some specific embodiments of the present invention, it is preferred to use methanol and water at a volume ratio of 1:1.

[0020] Preferably, the component Fr.4-3 is separated by semi-preparative high performance liquid chromatography to obtain the compound shown in Formula 2.

[0021] Preferably, the semi-preparative high-performance liquid chromatography separation is performed using acetonitrile and water at a volume ratio of 2:3 to 2:5. In some specific embodiments of the present invention, it is preferred to use acetonitrile and water at a volume ratio of 3:7.

[0022] The preferred method for preparing the above-mentioned triterpenoid compounds with neuroprotective effects includes the following steps:

[0023] 1) The dried Ganoderma lucidum fruiting bodies were crushed and extracted by heating and reflux with 95% ethanol. The extract was then extracted with ethyl acetate and separated by normal phase silica gel chromatography to obtain the fractions Fr.1~Fr.11.

[0024] 2) Fr.4 was separated by reversed-phase ODS column chromatography to obtain Fr.4-1 to Fr.4-9;

[0025] 3) Take Fr.4-6, and after high performance liquid chromatography (HPLC) detection and analysis, use semi-preparative HPLC for gradient elution separation to obtain the compound ganothenoid B shown in Formula 1;

[0026] 4) Take Fr.4-3, and after high performance liquid chromatography (HPLC) detection and analysis, use semi-preparative HPLC for gradient elution separation to obtain the compound ganothenoid D shown in Formula 2.

[0027] The present invention also provides a pharmaceutical composition comprising the above-described triterpenoid compound with neuroprotective effects or the triterpenoid compound with neuroprotective effects prepared by the above-described preparation method.

[0028] The present invention also provides the use of the above-mentioned neuroprotective triterpenoid compound or the neuroprotective triterpenoid compound prepared by the above-mentioned preparation method or the above-mentioned pharmaceutical composition in the preparation of a drug for treating neurodegenerative diseases.

[0029] Compared with existing technologies, the neuroprotective triterpenoid compounds provided by this invention have the structures shown in Formula 1 or Formula 2, and are two novel triterpenoid compounds discovered for the first time in the fruiting bodies of Ganoderma lucidum. The neuroprotective effects of the compounds described in this invention were evaluated using an H2O2-induced SH-SY5Y cell injury model. The results showed that the two triterpenoid compounds have good neuroprotective effects and exhibit a clear dose-response relationship. The neuroprotective triterpenoid compounds described in this invention can be used in the preparation of drugs for treating neurodegenerative diseases. Attached Figure Description

[0030] Figure 1 For ganothenoid B 1 H NMR spectrum;

[0031] Figure 2 For ganothenoid B 13 C NMR spectrum;

[0032] Figure 3 The DEPT spectrum of ganothenoid B;

[0033] Figure 4 The HSQC spectrum of ganothenoid B;

[0034] Figure 5 The HMBC spectrum of ganothenoid B;

[0035] Figure 6 For ganothenoid B 1 H- 1 H COSY spectrum;

[0036] Figure 7 The ROESY spectrum of ganothenoid B;

[0037] Figure 8 For ganothenoid D 1 H NMR spectrum;

[0038] Figure 9 For ganothenoid D 13 C NMR spectrum;

[0039] Figure 10 The DEPT spectrum of ganothenoid D;

[0040] Figure 11 The HSQC spectrum of ganothenoid D;

[0041] Figure 12 The HMBC spectrum of ganothenoid D;

[0042] Figure 13 For ganothenoid D 1 H- 1 H COSY spectrum;

[0043] Figure 14 The ROESY spectrum of ganothenoid D;

[0044] Figure 15 The effects of ganothenoid B and ganothenoid D on the survival rate of SH-SY5Y cells;

[0045] Figure 16The effects of ganothenoid B and ganothenoid D on H2O2-induced survival of SH-SY5Y cells;

[0046] Figure 17 The effect of ganothenoid D on the relative ROS content in H2O2-induced SH-SY5Y cells. Detailed Implementation

[0047] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, describes the neuroprotective triterpenoid compounds provided by the present invention, their preparation methods, and their applications.

[0048] Example 1

[0049] Isolation and purification of ganothenoid B or ganothenoid D, which are triterpenoid compounds

[0050] The fruiting body samples of Ganoderma lucidum with tea disease were dried and pulverized, and extracted three times by heating and reflux with 95% ethanol. The extracts were concentrated under reduced pressure and combined to obtain an ethanol extract, which was then extracted with ethyl acetate. The extracts were combined to obtain a total ethyl acetate extract.

[0051] The total ethyl acetate extract of Ganoderma lucidum was separated by normal-phase silica gel chromatography. Gradient elution was performed using a petroleum ether and ethyl acetate system with specific volume ratios of 10:1, 8:1, 6:1, 4:1, 2:1, and 1:1 (V:V = 10:1 to 1:1). The fractions were collected, analyzed by thin-layer chromatography and high-performance liquid chromatography, and then combined to obtain 11 fractions, Fr.1 to Fr.11.

[0052] Fr.4 was separated by an ODS column using a gradient elution with 30%–100% methanol. After high-performance liquid chromatography (HPLC) analysis and merging fractions under the same conditions, nine fractions, Fr.4-1 to Fr.4-9, were obtained. Fr.4-6 was purified by semi-preparative HPLC with isocratic elution in a methanol-water system (V:V = 50:50) at a flow rate of 4 mL / min, yielding compound ganothenoid B (5.0 mg). Fr.4-3 was analyzed by HPLC and thin-layer chromatography, followed by semi-preparative HPLC with isocratic elution in an acetonitrile-water system (V:V = 30:70) at a flow rate of 4 mL / min, yielding compound ganothenoid D (4.0 mg).

[0053] The structures of the isolated compounds ganothenoids B or D were identified using a variety of modern spectroscopic techniques, including nuclear magnetic resonance (NMR), infrared spectroscopy (IR), and mass spectrometry, combined with chemical methods. Compounds ganothenoids B or D were identified as novel Ganoderma lucidum triterpenoid compounds.

[0054] The structural formula of ganothenoid B (Equation 1) is as follows:

[0055]

[0056] The structural formula of ganothenoid D (Equation 2) is as follows:

[0057]

[0058] Regarding the structural analysis of the above compound ganothenoid B, we obtain... Figures 1-7 The results are shown. Among them, Figure 1 For ganothenoid B 1 H NMR spectrum Figure 2 For ganothenoid B 13 C NMR spectrum Figure 3 The DEPT spectrum of ganothenoid B. Figure 4 The HSQC spectrum of ganothenoid B is shown. Figure 5 The HMBC spectrum of ganothenoid B. Figure 6 For ganothenoid B 1 H- 1 H COSY spectrum, Figure 7 The ROESY spectrum of ganothenoid B.

[0059] The compound ganothenoid B is a white powder. HR-ESI-MS shows a peak [M+Na] at 469.2544 m / z (theoretical value 469.2561). + The molecular formula is C. 26 H 38 O6 has an unsaturation degree of 7.

[0060] 13 The C10 NMR (Table 1), DEPT, and HSQC spectra showed 26 carbon signals, containing two ketone carbonyl groups (δ¹⁸). C 200.4, 217.2), 1 carboxylic acid (δ C 175.0), 2 sps 3 oxygen-containing methine (δ C 78.9, 67.9), 6 sps 3 Methylene, 5 sp 3 Methione, 6 sps 3 Quaternary carbon (1 double bond) and 6 methyl groups (δ) C17.8, 18.9, 19.8, 28.7, 16.2, 24.9). 1 HNMR, 13 C NMR (Table 1), DEPT and HSQC spectra indicate that this compound has a lanostane-lowering triterpenoid skeleton.

[0061] The above data are very similar to the NMR data of ganothenoid B in the literature (XRZhao,XKHuo,PPDong, et al., Inhibitory effects of highly oxygenated lanostane derivatives from the fungus *Ganoderma lucidum* on P-glycoprotein and α-glucosidase, J.Nat.Prod.78(2015)1868-1876. https: / / doi.org / 10.1021 / acs.jnatprod.5b00132). The main difference is that ganothenoid B has an additional methylene group (-CH2) at the C-20 position. 1 H- 1 The correlation between the H-20 / H2-22 signal in the HCl COSY spectrum and the H3-21 signal in the HMBC spectrum with the C-20, C-22, and C-17 signals confirms this. Furthermore, the ketone carbonyl signal at the C-3 position of ganodernoid B is replaced by an oxygen-containing methine signal in ganodernoid B, indicating that the ketone carbonyl group at the C-3 position in ganodernoid B is reduced to a hydroxyl group (-OH).

[0062] 1 H- 1 The correlation between the H2-1 / H2-2 / H-3 signals in the H COSY spectrum and the signals H3-28 and H3-29 in the HMBC spectrum and the C-3, C-4 and C-5 signals confirms the above speculation.

[0063] Furthermore, the presence of relevant signals H-3 / H-5 / H3-29, H3-30 / H-17, and H3-18 / H3-19 / H3-28 in the ROESY spectrum confirmed that the compound ganothenoid B has a lanostane-neotriterpenoid stereoconfiguration.

[0064] Based on the above, the structure of compound ganothenoid B (Formula 1) was identified, and it was found to be a new triterpenoid compound.

[0065] Regarding the structural analysis of the above compound ganothenoid D, we obtain... Figures 8-14 The results are shown. Among them, Figure 8 For ganothenoid D 1 H NMR spectrum Figure 9 For ganothenoid D 13 C NMR spectrum Figure 10 The DEPT spectrum of ganothenoid D. Figure 11 The HSQC spectrum of ganothenoid D is shown. Figure 12 The HMBC spectrum of ganothenoid D is shown. Figure 13 For ganothenoid D 1 H- 1 HCOSY spectrum Figure 14 The ROESY spectrum of ganothenoid D.

[0066] The compound ganothenoid D is a yellow powder, and HR-ESI-MS gives a peak [M+Na] at 443.2437 m / z (theoretical value 443.2428). + The molecular formula is C. 26 H 35 O6 has an unsaturation degree of 9.

[0067] The compound 1 H NMR and 13 The C10 NMR data are very similar to those of ganothenoid B, suggesting that they share the same lanostane triterpenoid structural skeleton. The main difference is that the oxymethyl signal at the C-7 position of ganothenoid B is replaced by the ketone carbonyl signal of ganothenoid D. This indicates that the hydroxyl group (-OH) at the C-7 position of ganothenoid B is oxidized to a ketone carbonyl group by ganothenoid D. This inference can be confirmed by the correlation between H2-6 and C-7 and C-5, and the correlation between H3-30 and C-8 and C-7 in the HMBC spectrum.

[0068] In addition, this structure has a Δ at position C-20 on the side chain. 20(22) The above inferences regarding double bonds can be determined by the correlation signals in the HMBC spectra of H3-21 and H-17 with C-20 and C-22.

[0069] The correlation between H-22 and H-17 in the ROESY spectrum proves Δ 20(22)The double bond configuration is E. Furthermore, ganothenoid D exhibits similar H-3 / H-5 / H3-29, H3-30 / H-17, and H3-18 / H3-19 / H3-28 related signals in the ROESY spectrum as ganothenoid B, indicating that ganothenoid D also possesses the stereoconfiguration of lanostane-triterpenoids. This, combined with comparisons with similar compounds... 1 H NMR and 13 C NMR data.

[0070] Therefore, the chemical structure of compound ganothenoid D (Formula 2) was identified, and it was found to be a new triterpenoid compound.

[0071] Table 1. Ganothenoid B and Ganothenoid D 13 C NMR (125MHz) and 1 1H NMR (500MHz) data, solvent DMSO

[0072]

[0073]

[0074]

[0075] ganothenoid B (Formula 1) is a white powder, soluble in DMSO; [α]25D+99(c 0.1,MeOH); UV(MeOH)λmax(logε):257(3.73)nm; 1 H NMR (DMSO, 500MHz) and 13 C NMR (DMSO, 125MHz) data are shown in Table 1; IR (KBr)ν max (cm -1 ):3431,2969,1723,1664,1383,1273,1178, and 1031; HRESIMS m / z[M+Na] + 469.2544 (Theoretical value C) 26 H 38 NaO6 469.2561).

[0076] ganothenoid D (Formula 2) is a yellow powder, soluble in DMSO; [α]25D+47(c 0.1,MeOH); UV(MeOH)λmax(logε):230(3.77),251(3.58),265(3.54)nm; 1 H NMR (DMSO, 500MHz) and13 C NMR (DMSO, 125MHz) data are shown in Table 1; IR (KBr)ν max (cm -1 ):3442,2935,1744,1682,1382,1198, and 1037; HRESIMSm / z[M+Na] + 443.2437 (Theoretical value C) 26 H 35 NaO6 443.2428).

[0077] Application Example 1

[0078] The pharmacological effects of the compound described in this invention, composed of common pharmaceutical excipients, were tested in SH-SY5Y nerve cells and H2O2-damaged SH-SY5Y nerve cells to demonstrate its neuroprotective activity.

[0079] 1) Test the protective activity of the compounds of this invention against SH-SY5Y nerve cells.

[0080] 1.1) Cytotoxic effects of compounds ganothenoid B and ganothenoid D on SH-SY5Y cells

[0081] Human neuroblastoma SH-SY5Y cells were cultured in MEM / F12 medium containing 10% fetal bovine serum and 1% penicillin antibiotics at 37°C and 5% CO2.

[0082] A control group and a drug group were set up. The control group did not contain the drug. The drug group was divided into a low concentration group (12.5 μM), a medium concentration group (25 μM), and a high concentration group (50 μM) of ganothenoid B (GTD-B) and a low concentration group (12.5 μM), a medium concentration group (25 μM), and a high concentration group (50 μM) of ganothenoid D (GTD-D).

[0083] Log-grown SH-SY5Y cells were used at a ratio of 2.5 x 10. 4 The cells were seeded in 96-well plates. When the nerve cells reached 80% confluence, the supernatant was discarded, and pre-prepared concentrations of GTD-B and GTD-D compounds at 12.5 μM, 25 μM, and 50 μM were added for incubation. After observing at 37°C for 24 h, 20 μL of MTT was added and incubation continued for 4 h. The supernatant was then discarded, and 150 μL of DMSO was added and shaken for 10 min. The absorbance was measured at 490 nm using a microplate reader, and the cell viability was calculated.

[0084] This experiment used the MTT assay to determine the effects of compounds ganothenoid B and ganothenoid D on cell viability. Different concentrations of ganothenoid B and ganothenoid D were selected, and their effects on the viability of SH-SY5Y cells were measured. Figure 15 The effects of ganothenoid B and ganothenoid D on the survival rate of SH-SY5Y cells were investigated by... Figure 15 It was found that the cell survival rate of each group of compounds at different concentrations was above 90%, and they also had a certain proliferative effect. This indicates that compounds ganothenoid B and ganothenoid D have no toxic effect on SH-SY5Y cells.

[0085] 1.2) Protective effects of compounds ganothenoid B and ganothenoid D on SH-SY5Y cells damaged by H2O2.

[0086] Following method 1.1), when SH-SY5Y neurons reached 80% confluence, the supernatant was discarded, and H2O2 with final concentrations of 300 μM, 400 μM, 500 μM, 600 μM, 700 μM, 800 μM, and 1000 μM was added and the cells were treated for 12 h. The absorbance was measured at 490 nm, and the half-maximal inhibitory concentration (MCI) of the cells was calculated based on the experimental results as the modeling concentration (800 μM).

[0087] The cell culture system included a control group, an H2O2 model group, and groups containing Trolox (6-hydroxy-2,5,7,8-tetramethyltryptane-2-carboxylic acid), GTD-B, and GTD-D. After culturing the cells for 36 hours, the supernatant was discarded. The drug groups (GTD-B and GTD-D) were treated with different concentrations of the prepared compound (GTD-B at 12.5 μM, 25 μM, and 50 μM, and GTD-D at 12.5 μM, 25 μM, and 50 μM). The control and model groups were cultured in cell culture incubators for 12 hours. Then, H2O2 at a final concentration of 800 μM was added, and the culture was continued for another 12 hours. The absorbance was measured at 490 nm using a microplate reader, and cell viability was calculated.

[0088] After modeling oxidative damage using 800 μM H2O2 cells, intervention experiments were conducted by adding 12.5 μM, 25 μM, and 50 μM concentrations of ganothenoid B (GTD-B) and ganothenoid D (GTD-D) to investigate the protective effects of the triterpenoid compounds ganothenoid B and ganothenoid D on oxidatively damaged SH-SY5Y cells. Figure 16The effects of ganothenoid B and ganothenoid D on H2O2-induced SH-SY5Y cell survival, such as Figure 16 As shown, compared with the control group, the cell survival rate of the model group treated with H2O2 was significantly reduced to 49.37±1.92%, which was statistically significant (P<0.01). Compared with the model group, the positive control drug trolox had a significant protective effect, with a cell survival rate of 75.39±2.26% (50μM), which was statistically significant (P<0.01). Compared with the H2O2 model group, the survival rate of oxidatively damaged SH-SY5Y cells significantly increased after adding different concentrations of ganothenoid B and ganothenoid D, with cell survival rates of 71.13±0.18% (ganothenoid B, 50μM) and 86.89±1.24% (ganothenoid D, 50μM), respectively. Furthermore, the cell survival rate generally increased gradually with increasing drug concentration (P<0.01). This indicates that ganothenoid B and ganothenoid D have a good protective effect against H2O2-induced SH-SY5Y cells.

[0089] 1.3) Detection of H2O2-induced SH-SY5Y cell cycle

[0090] Log-grown SH-SY5Y cells were harvested at a concentration of 3.5 x 10⁻⁶. 4 Cells were seeded in 6-well plates. When the cell confluence reached 80%, grouping and treatment time were set according to method 1.2), and drugs were administered. To compare the activities of lanosterol triterpenoids and lanosterol triterpenoids and to investigate the effect of side chains on neuroprotection, a control group, ganoderenic acid H (structure shown below, differing only from the side chain structure of ganothenoid D), was added. Control group, model group, Trolox (50 μM) group, ganoderenic acid H (50 μM) control group, and GTD-B and GTD-D groups were set up. After incubation with the drugs for 12 h, H2O2 at a final concentration of 800 μM was added for 12 h of damage. Cells from each group were collected, fixed overnight with pre-cooled 70% ethanol, washed with PBS after fixation, and then incubated at 37°C in the dark for 30 min with 500 μL RNase and 25 μL PI (propidium iodide) staining working solution. Cells were then analyzed by flow cytometry to detect the cell content distribution in the G0 / G1, S, and G2 phases.

[0091]

[0092] The structure of ganoderenic acid H

[0093] As shown in Table 2, compared with the control group, the proportion of cells in the G0 / G1 phase was significantly increased in the model group, while the proportion of cells in the S and G2 / M phases was significantly decreased (P<0.01), indicating that H2O2 can induce significant cell arrest in the S and G2 / M phases of SH-SY5Y cells. Compared with the model group, except for the ganoderenic acid H control group, the proportion of cells in the G0 / G1 phase was significantly decreased in all other treatment groups (P<0.01), while the proportion of cells in the S and G2 / M phases was significantly increased (P<0.01), indicating that ganothenoid B and ganothenoid D can significantly alleviate the H2O2-induced cell arrest in the S and G2 / M phases of SH-SY5Y cells, thereby alleviating the H2O2-induced changes in the SH-SY5Y cell cycle and demonstrating a protective effect against H2O2-induced SH-SY5Y cells.

[0094] Table 2 Effects of ganothenoid D on H2O2-induced SH-SY5Y cell cycle

[0095]

[0096]

[0097] Note: Compared with the model group: # P<0.05, **P<0.01, ***P<0.001; compared with the control group, # P<0.05, ## P<0.01, ### P<0.001

[0098] 1.4) Detection of H2O2-induced apoptosis in SH-SY5Y cells

[0099] Log-grown SH-SY5Y cells were harvested at a concentration of 3.5 x 10⁻⁶. 4 / wells were seeded in 6-well plates. When the cell confluence reached 80%, the groups and treatment time were set according to the method in 1.3), and the drugs were administered. The cells were collected and washed with pre-cooled PBS (phosphate buffered saline). The cells were centrifuged at 1000 r / min for 10 min at 4 °C. The cells were resuspended in 500 μL of 1× Binding Buffer. 5 μL of Annexin V-FITC and 5 μL of propidium iodide (PI) staining solution were added. After mixing, the cells were incubated in the dark for 10 min. Apoptosis of each group was detected by flow cytometry.

[0100] As shown in Table 3, compared with the control group, the apoptosis rate (the sum of early and late apoptotic cells) in the model group was significantly increased (21.82±1.41%). Compared with the model group, except for the ganoderenic acid H control group, the apoptosis rates in the high, medium, and low concentration groups of GTD-B and GTD-D were significantly decreased (P<0.01), showing a dose-dependent effect. Therefore, ganothenoid B and ganothenoid D can slow down H2O2-induced apoptosis in SH-SY5Y cells.

[0101] Table 3. Effects of ganothenoid D on H2O2-induced apoptosis in SH-SY5Y cells.

[0102]

[0103]

[0104] Note: Compared with the model group: #P<0.05, **P<0.01, ***P<0.001; compared with the control group, # P<0.05, ## P<0.01, ### P<0.001

[0105] 1.5) Detection of intracellular ROS content in SH-SY5Y cells after H2O2 damage

[0106] Log-grown SH-SY5Y cells were harvested at a ratio of 3.5 x 10. 4 Cells were seeded in 6-well plates. When cell confluence reached 80%, grouping and treatment time were set according to method 1.3), and the drugs were administered. Cells were collected, centrifuged, and the supernatant was discarded. Cells were washed twice with PBS. Cells in each group were stained, and DCFH-DA was diluted 1:1000 with serum-free medium. A final concentration of 10 μM DCFH-DA was added to each well. Cells were incubated at 37°C in the dark for 20 min. Cells were washed twice with pre-cooled PBS, collected, centrifuged, and the supernatant was discarded. Cells were resuspended in PBS, and changes in ROS content in each group were detected by flow cytometry.

[0107] Excessive ROS can disrupt the balance between ROS and the antioxidant defense system, causing oxidative stress damage to biomolecules such as proteins, lipids, and DNA in the body's cells, which in turn induces apoptosis and accelerates cell death. This is an important cause of cell and organ damage, decreased immunity, and various chronic diseases.

[0108] Therefore, detecting changes in intracellular ROS production, such as... Figure 17As shown, compared with the control group, the intracellular ROS level in the model group was significantly increased (2.28±0.03%). After treatment with high, medium, and low concentrations of GTD-D compound, the ROS levels were 0.98±0.39%, 1.43±0.33%, and 1.66±0.25%, respectively, which were significantly lower than those in the model group. This indicates that ganothenoid D can significantly alleviate the increase in intracellular ROS levels in SH-SY5Y cells damaged by H2O2 and reduce cellular oxidative damage.

[0109] The above description of the embodiments is only for the purpose of helping to understand the method and core ideas of the present invention. It should be noted that those skilled in the art can make several improvements and modifications to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

1. A triterpenoid compound with neuroprotective effects, characterized in that, It has the structure shown in Equation 1 or Equation 2: ； 。 2. The method for preparing the neuroprotective triterpenoid compound according to claim 1, characterized in that, The fruiting body of Ganoderma lucidum with tea disease was subjected to heating and reflux extraction, extraction, separation by normal phase silica gel column chromatography, separation by reverse phase ODS column chromatography, and semi-preparative high performance liquid chromatography to obtain the triterpenoid compound with neuroprotective effect as described in claim 1. The solvent for the heating and reflux extraction is selected from ethanol or methanol; The process after heating and reflux extraction also includes removing the solvent to obtain an extract paste, then mixing the extract paste with water to obtain a suspension, and finally extracting the suspension with ethyl acetate or chloroform. The normal-phase silica column chromatography separation was performed by separating the extract obtained after extraction with petroleum ether and ethyl acetate at a volume ratio of 10:1 to 1:1, resulting in 11 components denoted as Fr.1 to Fr.11; The component Fr.4 was separated by reversed-phase ODS chromatography to obtain components Fr.4-1 to Fr.4-9; The reversed-phase ODS column separation was performed using methanol and water in sequential volume ratios of 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, and 100:0, with an elution volume of 800 mL for each volume ratio. The component Fr.4-6 was separated by semi-preparative high performance liquid chromatography to obtain the compound shown in Formula 1, and the semi-preparative high performance liquid chromatography separation was performed using methanol and water at a volume ratio of 9:11 to 11:

9. The component Fr.4-3 was separated by semi-preparative high performance liquid chromatography to obtain the compound shown in Formula 2, and the semi-preparative high performance liquid chromatography separation was performed by separating acetonitrile and water in a volume ratio of 2:3 to 2:

5.

3. A pharmaceutical composition, characterized in that, This includes the neuroprotective triterpenoid compound of claim 1 or the neuroprotective triterpenoid compound prepared by the method of claim 2.

4. The use of the neuroprotective triterpenoid compound of claim 1, the neuroprotective triterpenoid compound prepared by the preparation method of claim 2, or the pharmaceutical composition of claim 3 in the preparation of a drug for treating neurodegenerative diseases.

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