Isoprenylated polyketide compound, and preparation method and application thereof

Abstract

The application discloses a prenylated polyketide compound, a preparation method and application thereof, and the prenylated polyketide compound is separated from rice fermented with Xylaria nigriplicata. The neuroprotective effect of the prenylated polyketide compound on glutamate-induced excitotoxicity of PC12 cells is clarified through comprehensive evaluation of cell viability, apoptosis and key oxidative stress markers.

Description

Technical Field

[0001] This invention belongs to the field of natural product chemistry technology, and particularly relates to an isopentenylated polyketide compound, its preparation method and application. Background Technology

[0002] *Anthracis niger*, known as "Wuling Shen" in traditional Chinese medicine, is a fungus with a long history of medicinal use, particularly effective for insomnia, depression, and other neurological and mental disorders. Its clinical application has been demonstrated through commercially available fermented mycelium capsules, which are widely used to improve sleep and cognitive function. Modern pharmacological studies have confirmed these traditional uses, indicating that crude extracts of *Anthracis niger* and related preparations possess various biological activities, including sedation, anti-anxiety, antidepressant, antioxidant, neuroprotective, and anti-inflammatory effects, demonstrating its significant potential as a source of neuroactive drugs. In terms of chemical composition, this fungus has been identified with various secondary metabolites, including alkaloids, sterols, chromophores, terpenes, and isocoumarin derivatives. Our research group has long been dedicated to the study of bioactive components of *Anthracis* fungi, and has reported the isolation of novel naphthone derivatives, resorcinol derivatives, and brasilane-type sesquiterpenes from *Anthracis niger*, some of which have shown good neuroprotective activity. These research findings fully reveal the chemical diversity of *Anthracis niger* and its potential value in the treatment of neurological diseases.

[0003] However, systematic studies on isopentenylated polyketides from *Anthracis niger* are still relatively limited in the current technology, with a small number of reported compounds, and the understanding of their structural types, structure-activity relationships, and specific neuroprotective mechanisms is still insufficient. Therefore, developing novel isopentenylated polyketides and conducting in-depth research on their neuroprotective activities has significant theoretical and practical value. Summary of the Invention

[0004] The purpose of this invention is to further explore the potential of neuroactive components in *Anthracis niger* and develop novel compounds with good neuroprotective effects, providing a new class of isopentenylated polyketides, their extraction and separation methods, and their application in neuroprotective drugs. This invention isolates isopentenylated polyketides from *Anthracis niger* rice fermentation products, which exhibit significant neuroprotective activity in a glutamate-induced PC12 cell model.

[0005] To achieve the above objectives, the present invention provides an isopentenylated polyketide compound having the structure shown in formula (I): Formula (I) Among them, R 1Selected from -C(=O)-CH2-CH2-C(=O)-CH3, -C(=O)-CH2-CH2-CH(OH)-CH3 or -C(=O)-CH2-CH(OH)-CH2-CH3; R 2 Selected from hydroxyl groups; Or R 1 and R 2 Together with one or more atoms to which they are attached, they form a five-membered ring, which contains an ester group as a ring member; R 3 Selected from -CH2-CH(OH)-C(OH)(CH3)2 or -CH2-C(=O)-OCH3.

[0006] This invention isolates isopentenylated polyketide compounds from rice fermentation products of *Carya niger*, containing both isopentenyl side chains and a polyketide backbone, forming a unique molecular skeleton. Experimental results show that these compounds exhibit significant protective effects against glutamate-induced excitotoxicity in PC12 cells within the concentration range of 0.01–10 μM, significantly improving cell viability, inhibiting apoptosis, and effectively alleviating oxidative stress. Furthermore, within the effective concentration range, compounds 1–5 show no significant toxicity to normal PC12 cells, demonstrating good safety profiles, providing important assurance for further development into neuroprotective drugs.

[0007] The compounds of this invention are derived from the medicinal fungus *Carya nigricans* (also known as *Carya nigricans*), obtained through rice fermentation combined with modern separation and purification techniques. The raw material source is clear, the preparation process is relatively simple, and it has good potential for industrial application. These compounds can be used to prepare drugs for treating neurological diseases related to excitotoxicity, providing new candidate compounds for the prevention and treatment of insomnia, depression, cognitive impairment, and neurodegenerative diseases, and have significant clinical application value.

[0008] According to an embodiment of the present invention, R 1 and R 2 Together with the carbon atoms they are attached to, they form a five-membered lactone ring; R 3 It is -CH2-CH(OH)-C(OH)(CH3)2.

[0009] According to a second aspect of the present invention, a method for preparing isopentenylated polyketide compounds is provided, comprising the following steps: S1. The strain of *Caryopsis niger* was inoculated into rice culture medium and cultured to obtain fermentation product; S2. The fermented product is extracted with a solvent, then concentrated, extracted, and concentrated again to obtain a total extract; S3. The total extract is separated and purified to obtain a polyketide compound containing isopentenylated.

[0010] According to an embodiment of the present invention, in step S1, the preparation of the *Carobstroemia indica* strain includes the following steps: after disinfecting the surface of the *Carobstroemia indica* fruiting body with alcohol, a tissue block is cut under aseptic conditions, inoculated onto PDA medium, and cultured until mycelia grow to obtain strain No. CGBWSHF00611.

[0011] According to an embodiment of the present invention, in step S1, the culture temperature is 20~30℃ and the culture time is 20~40 days.

[0012] According to an embodiment of the present invention, the method for preparing the rice culture medium includes mixing rice and water, and then sterilizing it at 115~125℃ for 10~20 minutes before use.

[0013] According to an embodiment of the present invention, in step S2, the solvent in the extraction step includes at least one of acetone and ethanol.

[0014] According to an embodiment of the present invention, in step S2, the preparation of the total extract includes: soaking the fermented product in acetone for 20 to 28 hours each time, centrifuging and then concentrating the supernatant under reduced pressure, extracting the crude extract with ethyl acetate, and concentrating under reduced pressure to obtain the extract; repeating the above extraction, centrifugation, concentration and extraction process on the precipitate for a total of 3 to 6 times, and combining all the extracts to obtain the total extract.

[0015] According to an embodiment of the present invention, the separation method includes: S3.1 The total extract is subjected to gradient elution by silica gel column chromatography to obtain multiple crude components; S3.2 Separate the crude component by gel column chromatography to obtain the finer components.

[0016] According to an embodiment of the present invention, in step S3.1, the step of gradient elution by silica gel column chromatography includes: passing the total extract through a 200-300 mesh normal phase silica gel column for gradient elution with a mixed solvent of petroleum ether / ethyl acetate volume ratio of 1:0 to 0:1, with each gradient elution volume being 2-4 column volumes, to obtain 10 crude components, which are sequentially denoted as components A to J.

[0017] According to an embodiment of the present invention, step S3.1 further includes taking component H and performing gradient elution using normal phase silica gel column chromatography with petroleum ether / ethyl acetate as the eluent to obtain 10 subcomponents, denoted as H1 to H10.

[0018] According to an embodiment of the present invention, step S3.2 further includes taking H5 and performing Sephadex LH-20 gel column chromatography with methanol as the eluent to separate it into 11 sub-components, denoted as H5a~H5k.

[0019] According to an embodiment of the present invention, the purification method includes: purifying the subdivided components by high performance liquid chromatography to obtain the isopentenylated polyketide compound.

[0020] According to an embodiment of the present invention, the purification method includes: taking H5d, H5f or H5g and subjecting them to preparative high performance liquid chromatography, using acetonitrile / water as the mobile phase for gradient elution, with a flow rate of 2 to 4 mL / min and a detection wavelength of 254 nm, collecting the target retention time peak, concentrating under reduced pressure, redissolving in acetonitrile and evaporating to dryness to obtain isopentenylated polyketide compounds.

[0021] According to an embodiment of the present invention, the purification method includes: taking H5d and purifying it by preparative high performance liquid chromatography, using an acetonitrile / water system as the mobile phase, with a volume ratio gradient from 15 / 85 to 20 / 80, an elution time of 25 min, a flow rate of 3 mL / min, and collecting peaks with retention times of approximately 16.0 min and 20.0 min, respectively, to obtain the isopentenylated polyketide compound.

[0022] According to an embodiment of the present invention, the purification method includes: taking H5f and purifying it by preparative high performance liquid chromatography, using an acetonitrile / water system as the mobile phase, with a volume ratio gradient from 16 / 84 to 20 / 80, an elution time of 25 min, a flow rate of 3 mL / min, and collecting peaks with retention times tR of approximately 17.1 min and 21.5 min, respectively, to obtain the isopentenylated polyketide compound.

[0023] According to an embodiment of the present invention, the purification method includes: taking 5g of H and purifying it by preparative high performance liquid chromatography, using an acetonitrile / water system as the mobile phase, with a volume ratio gradient from 15 / 84 to 20 / 80, an elution time of 20 min, a flow rate of 3 mL / min, and collecting the peak with a retention time tR of approximately 17.5 min to obtain the isopentenylated polyketide compound. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0025] Figure 1The chemical structure of isopentenylated polyketide compound 1 is shown. Figure 2 The chemical structure of isopentenylated polyketide compound 2; Figure 3 The chemical structure of isopentenylated polyketide compound 3 is shown. Figure 4 The chemical structure of isopentenylated polyketide compound 4 is shown. Figure 5 The chemical structure of isopentenylated polyketide compound 5; Figure 6 For compound 2 1 H NMR spectrum; Figure 7 For compound 2 3 C NMR spectrum; Figure 8 Bar graph showing the cell viability of compound 1 in response to the protective effect of compound 1 against glutamate-induced PC12 cell viability; Figure 9 Bar graph showing the cell viability of compound 2 in response to glutamate-induced PC12 cell viability protection. Figure 10 The effects of compounds 1–5 on the release of LDH, a marker of oxidative stress induced by glutamate, in PC12 cells; Figure 11 The effects of compounds 1–5 on the activity of SOD, a marker of oxidative stress induced by glutamate in PC12 cells; Figure 12 The effects of compounds 1-5 on the content of MDA, a marker of glutamate-induced oxidative stress, in PC12 cells; Figure 13 The effects of compounds 1–5 on the content of GSH, a marker of glutamate-induced oxidative stress, in PC12 cells; The realization of the objective, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0026] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] Furthermore, the technical solutions of the various embodiments of the present invention can be combined with each other, but only if they are based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention.

[0028] Example 1 This embodiment provides an isopentenylated polyketide compound 1 and its preparation method, specifically: S1. Place the fruiting bodies of *Caryopsis buergerianum* in an inoculation box, disinfect the surface of the mushroom with 75% alcohol, sterilize the scalpel, make a longitudinal cut in the middle of the stem, and use an inoculation spatula to cut 5 square pieces at the cap and stem. Pick one piece of tissue and transfer it to PDA (Potato Dextrose Agar) medium. Incubate at 24℃. Once mycelium grows from the tissue piece, strain No. CGBWSHF00611 is obtained. Cut the above-mentioned strain cultured on PDA into small pieces and inoculate them into rice medium. Incubate at 25℃ in the dark for 30 days to obtain the fermentation product. The preparation process of the rice culture medium is as follows: 100g of rice and 100mL of water are put into a 500mL Erlenmeyer flask and sterilized in an autoclave at 121℃ for 15min. S2. The fermented product obtained after being cultured on rice culture medium was first soaked in acetone for 24 hours each time. Then, the acetone extract and precipitate were separated by centrifugation. The acetone extract was evaporated and concentrated by rotary evaporator, leaving an appropriate amount of water in the extract to obtain a crude acetone layer. This crude extract was extracted with ethyl acetate to obtain an ethyl acetate layer, which was then concentrated under reduced pressure to obtain an extract. The above soaking-centrifugation-concentration-extraction-reduced pressure concentration process was repeated on the precipitate obtained by centrifugation for a total of 5 times. Finally, the extracts from the five times were combined to obtain a total extract of 95.5g. S3.1 The total extract was passed through a 200-300 mesh normal-phase silica gel column, with petroleum ether / ethyl acetate mixed solvent (1 / 0, 20 / 1, 10 / 1, 5 / 1, 2 / 1, 1 / 1, 0 / 1, v / v) as the eluent. The elution was carried out in a gradient, with each gradient having a solvent elution volume of 3 column volumes (column volume of 2100 cm3). A total of 10 crude fractions were obtained, which were designated as fractions A, B, C, D, E, F, G, H, I, and J, respectively. Finally, the residual sample on the silica gel column was washed off with methanol. S3.2 and H components were further separated by a 200-300 mesh normal-phase silica gel column (petroleum ether / ethyl acetate, gradient elution: 10:1, 5:1, 2:1, 1:1, v / v) to obtain 10 sub-components (H1-H10). Sub-component H5 (300 mg) was separated by a Sephadex LH-20 gel column (methanol elution) to obtain 11 further sub-components (H5a-H5k). S3.3. Take the fraction H5f (40 mg) and purify it by preparative high performance liquid chromatography (acetonitrile / water, volume ratio from 16 / 84 to 20 / 80, 25 min, flow rate 3 mL / min) to obtain compound 1 (4.3 mg, tR = 17.1 min).

[0029] Compound 1 is a dark red powder with the molecular formula C. 16 H 22 O6 was determined from HRESIMS data ( m / z 309.1343 [MH] － Calculated value C 16 H 22 O6309.1344), indicating that its degree of unsaturation is 6. 1 H NMR and HSQC spectra showed: a group of adjacent coupled proton signals δ H 6.76 (1H, d, J = 8.3 Hz, H-5) and 7.35 (1H, d, J = 8.3 Hz, H-6) are attributed to a 1,2,3,4-tetrasubstituted benzene ring; in addition, three methylene proton signals were observed. δ H 3.25 (2H, t, J = 5.7, 12.1 Hz, H-8), 2.82 (2H, t, J = 5.7, 12.1 Hz, H-9), 2.95 (1H, dd, J = 1.5, 13.6 Hz, H α -12) and 2.44 (1H, dd, J = 10.4, 13.6 Hz, H β -12), a hydroxymethyl proton signal δ H 3.44 (1H, dd, J = 1.5, 10.4 Hz, H-13), and three methyl proton signals. δ H 2.16 (3H, s, H-11), 1.10 (3H, s, H-15) and 1.11 (3H, s, H-16).13 C10 NMR and DEPT spectra show that the compound contains 16 carbons, including two ketone carbonyl groups (C10 NMR and DEPT spectra). δ C 205.5 and 207.6), six aromatic carbons ( δ C 118.1, 120.1, 121.5, 135.5, 144.4 and 150.4), three methylene carbons ( δ C 32.7, 33.0 and 37.0), one oxygen-containing methylene carbon ( δ C 77.8), a quaternary carbon ( δ C 72.2), and three methyl carbons ( δ C 25.1, 26.8 and 30.2). 1 H- 1 The correlation signals of H-5 / H-6 and H-12 / H-13 in the HCl COSY spectrum, combined with HMBC correlations: H-3 correlates with C-13 / C-4, H-12 with C-3 / C-4 / C-5 / C-14, CH3-15 with C-13 / C-16, and CH3-16 with C-14 / C-13, indicating that a dihydroxyisopentenyl substituent is attached to the C-4 position of the benzene ring. Furthermore, the HMBC correlations of CH3-11 with C-10 / C-9, H-8 / 9 with C-10 / C-7, and H-6 with C-1 / C-7 / C-2 / C-4, combined with the COSY correlation of H-8 / H-9, reveal a hexane-2,5-dione side chain attached to the C-1 position. Therefore, the planar structure of compound 1 is determined as follows: Figure 1 As shown. Through comparative experiments, electronic circular dichroism (ECD) and (13...) R The calculated weighted spectrum of C-13 was used to determine the absolute configuration of C-13.

[0030] Example 2 This embodiment provides an isopentenylated polyketide compound 2 and its preparation method. The difference between this embodiment and Example 1 is that in step S3.3, 5g (50 mg) of finely divided H is taken and purified by preparative high performance liquid chromatography (acetonitrile / water, volume ratio from 15 / 84 to 20 / 80, 20 min, flow rate 3 mL / min) to obtain compound 2 (5.5 mg, tR = 17.5 min).

[0031] The molecular formula of isopentenylated polyketide compound 2 was determined by HRESIMS data to be C 13 H 16 O5 ( m / z 251.0932 [MH]－ Calculated value C 13 H 16 O5251.0925). 1 H NMR ( Figure 6 The spectrum shows two 1,2,3,4-tetrasubstituted aromatic proton signals. δ H 6.35 (1H, d, J = 7.6 Hz, H-5) and 7.30 (1H, d, J = 7.6 Hz, H-6), a hydroxymethylene signal δ H 5.29 (2H, s, H-8), a set of methylene proton signals δ H 3.05 (1H, dd, J = 1.9, 14.1 Hz, H α -9) and 2.80 (1H, dd, J = 10.2, 14.1 Hz, H β -9), a hydroxymethyl signal δ H 3.60 (1H, dd, J = 1.9, 10.2 Hz, H-10), and two methyl signals. δ H 1.25 (6H, s, H-12 / H-13). 13 C NMR ( Figure 7 The NMR spectra and HSQC spectra show that this compound contains 13 carbons, belonging to two methyl groups, one ester carbonyl group, six aromatic carbons, two methylene groups, and one methine group. Comparison with the NMR data of compound 1 confirms that compound 2 contains a dihydroxyisopentenyl unit. Furthermore, H-6 and ester carbonyl carbon C-7 (… δ C The presence of a five-membered lactone ring was confirmed by the HMBC correlation of 173.9 and the correlation signals between H-8 and C-7 / C-1 / C-3. Based on this, the planar structure of compound 2 was established. Figure 2 Finally, by comparing experimental and calculated ECD spectra, the absolute configuration of C-10 was determined to be... R .

[0032] Example 3 This embodiment provides an isopentenylated polyketide compound 3 and its preparation method. The difference between this embodiment and Example 1 is that in step S3.3, the fractionated H5f (40 mg) is purified by preparative high performance liquid chromatography (acetonitrile / water, volume ratio from 16 / 84 to 20 / 80, 25 min, flow rate 3 mL / min) to obtain compound 3 (4.5 mg, tR = 21.5 min).

[0033] Isoprenelated polyketide compound 3 is a dark red powder. Its molecular formula, determined based on HRESIMS data, is C3. 14 H 18 O6 ( m / z 305.09943 [M + Na] + Calculated value C 14 H 18 O6Na 305.09956). By comparing the NMR data with those of compound 1, the differences were found to be: in compound 3, the dihydroxyisoprenyl unit was replaced by a methyl acetate group, and the carbonyl group at the C-5 position was replaced by a hydroxyl group. This structural deduction yielded key results. 1 H- 1 Support for H COSY related signals (H-8 / H-9 / H-10 / CH3-11) and HMBC related signals: H3-11 and C-10 ( δ C 65.6) and C-9 ( δ C Related to 33.9), H-10 and C-8 ( δ C 35.1) is related. Furthermore, OCH3-14 is related to C-13 ( δ C The HMBC correlation of 171.4) and the correlation signals between H-12 and C-3, C-4, C-5, and C-13 confirmed that the methyl acetate group is located at the C-4 position. Based on the above data, the planar structure of compound 3 was determined. Figure 3 By comparing the experimental ECD spectrum with (10) S )-3 and (10 R The calculation curve of -3 determines its absolute configuration as 10. S , among which (10 S The excellent fit of )-3 verifies the attribution of this configuration.

[0034] Example 4 This embodiment provides an isopentenylated polyketide compound 4 and its preparation method. The difference between this embodiment and Example 1 is that in step S3.3, the fraction H5d (60 mg) is taken and purified by preparative high performance liquid chromatography (acetonitrile / water, volume ratio from 15 / 85 to 20 / 80, 25 min, flow rate 3 mL / min) to obtain compound 4 (4.5 mg, retention time tR = 16.0 min).

[0035] HRESIMS analysis of isopentenylated polyketide compound 4 showed an ion peak of m / z 283.11755 [M + H] + (Calculated value C) 14 H 19 O6283.11761), thus its molecular formula is determined to be C. 14 H 19 O6, the same as compound 3. By comparing compounds 4 and 3... 1 H and 13 C10 NMR data revealed that the two compounds have highly similar skeletons, the difference being that the hydroxyl group shifts from the C-10 position in compound 3 to the C-9 position in compound 4. This assignment is... 1 H- 1 Support for H COSY related signals (H-8 / H-9 / H-10 / CH3-11) and HMBC related signals: H3-11 and C-10 ( δ C 30.6) and C-9 ( δ C Related to 69.5), H-10 and C-8 ( δ C 46.4) and C-9 are related, as are H-6, H-8, H-9 and C-7 ( δ C 206.9) is related. By comparing experimental ECD data with (9 R The calculated curves for compound 4 and 4 show a high degree of agreement, thus confirming the absolute configuration of compound 4 as 9. R Based on the above data, the planar structure of compound 4 was determined. Figure 4 ).

[0036] Example 5 This embodiment provides an isopentenylated polyketide compound 5 and its preparation method. The difference between this embodiment and Example 1 is that: 60 mg of fraction H5d was purified by preparative high performance liquid chromatography (acetonitrile / water, volume ratio from 15 / 85 to 20 / 80, 25 min, flow rate 3 mL / min) to obtain compound 5 (4.0 mg, tR = 20.0 min).

[0037] Isoprene-modified polyketide compound 5 is a dark red powder, and its molecular formula is obtained by HRESIMS ion peaks. m / z 311.1508 [MH] － (Calculated value C) 16 H 23 O6311.1500) was determined to be C 16 H 23 O6 corresponds to five degrees of unsaturation. Detailed analysis of one-dimensional and two-dimensional NMR data revealed that its structure is highly similar to compound 1. The main difference lies in one ketone carbonyl carbon in compound 1. δ C In compound 5, 207.6, C-10 is replaced by a hydroxymethyl group ( δ H / C 3.66 / 65.6, C-10). This structural modification is consistent with the observed increase in molecular weight and is further facilitated by... 1 H- 1 The H-COSY spin system H-8 / H-9 / H-10 / CH3-11 and key HMBC correlation signals (CH3-11 correlated with C-10 / C-9, H-8 / 9 correlated with C-10 / C-7) were elucidated. To determine the absolute configuration of compound 5, theoretical ECD calculations were performed on its four possible stereoisomers (5a-5d). The experimental ECD spectra of compound 5 are compared with those of (10... S ,13 R The calculated curve for )-5a is the best match. Furthermore, biosynthetic considerations support designating the hydroxyl groups at C-10 and C-13 as )-5a. S Configuration and R The configuration is consistent with that of compounds 1-4. Therefore, the absolute configuration of compound 5 is determined to be 10. S ,13 R Its structure is as follows Figure 5 As shown.

[0038] Effect Measurement 1. Cytotoxicity assay: Before conducting the neuroprotective experiment, the cytotoxicity of compounds 1-5 to normal PC12 cells was first investigated. The results showed that within the concentration range of 0.01-10 μM, compounds 1-5 had no significant cytotoxicity to PC12 cells, and the cell viability was not significantly different from that of the blank control group, indicating that the above compounds have good safety at the tested concentrations.

[0039] 2. Protective effect against glutamate-induced PC12 cell viability: Exposure of PC12 cells to 12.5 mM glutamate for 24 h significantly reduced cell viability (P<0.01). Pretreatment of cells with compounds 1–5 at concentrations of 0.01–10 μM significantly improved cell viability after glutamate-induced damage, exhibiting a clear dose-dependent effect. At a concentration of 10 μM, the protective effects of compounds 1–5 were comparable to those of the positive control drug fluoxetine, with significantly higher cell survival rates than the model group (P<0.01). Among them, compound 2 showed the strongest neuroprotective activity, especially at a low concentration (0.01 μM), where its cell survival rate was significantly higher than that of other compounds at the same dose (P<0.01), more effectively reversing the glutamate-induced decrease in cell viability and thus more significantly maintaining the normal physiological function of PC12 cells, demonstrating optimal neuroprotective potential.

[0040] Figure 8 and Figure 9 Bar charts showing the protective effects of compounds 1 and 2 against glutamate-induced PC12 cell viability are displayed. The cell viability of the normal control group (100%) was used as a baseline to visually represent the survival status of PC12 cells. The x-axis represents the control group, the model group (damaged group treated with only 12.5 mM glutamate for 24 h), and the treatment groups at different concentrations of the compounds (0.01, 0.1, 1, 10 μM). The height of the bars represents the mean cell viability value (Mean) for each group, and the error bar above represents the standard deviation (± SD). Each group was independently replicated (n=3). The bars above the bars... or The symbol indicates that the group has a significant (P<0.05) or highly significant (P<0.01) difference compared with the control group; # or ## indicates that the group has a significant (P<0.05) or highly significant (P<0.01) difference compared with the model group, which is used to prove whether the compound truly has a neuroprotective effect.

[0041] 3. Protective effect against glutamate-induced apoptosis in PC12 cells: The protective effect of compounds 1-5 against glutamate-induced apoptosis in PC12 cells was analyzed using TUNEL staining (100×). Fluorescence intensity as determined by TUNEL analysis. Blue: PC12 cell nuclei counterstained with DAPI; Green: Cell nuclei showing DNA fragmentation as indicated by TUNEL staining. Glutamate treatment significantly increased the apoptosis rate of PC12 cells, and the number of TUNEL-positive cells (green fluorescence) increased significantly. Pretreatment with 10 μM compounds 1-5 significantly reduced the number of apoptotic cells and decreased the fluorescence intensity compared to the model group, indicating that compounds 1-5 have significant anti-apoptotic effects. Among them, compounds 2 and 3 showed the strongest anti-apoptotic protective effects, with the most significant decrease in TUNEL fluorescence intensity compared to the model group (P<0.01), and significantly fewer apoptotic cells than the groups treated with compounds 1, 4, and 5. These compounds were able to more effectively reverse glutamate-induced neuronal cell apoptosis damage, thus exerting a more prominent neuroprotective effect.

[0042] 4. Effects on Oxidative Stress Markers: Glutamate induction leads to significant oxidative stress in PC12 cells, manifested by a significant decrease in intracellular superoxide dismutase (SOD) and glutathione (GSH) levels, and a significant increase in lactate dehydrogenase (LDH) release and malondialdehyde (MDA) production. Pretreatment with 10 μM compounds 1–5 effectively reversed these changes: SOD and GSH activities significantly recovered, while LDH release and MDA levels significantly decreased (P<0.05 or P<0.01), indicating that compounds 1–5 exert neuroprotective effects by enhancing cellular antioxidant defense and mitigating lipid peroxidation damage. Among them, compound 2 showed the strongest regulatory effect, with its high-dose group significantly reducing SOD activity (… Figure 11 ), GSH Figure 13 The content of ) was close to that of the normal control group, while LDH () was significantly reduced. Figure 10 ) release amount and MDA ( Figure 12 The production of compound 2 was significantly better than that of other compounds at the same dosage (P<0.05), indicating that compound 2 has a significant advantage in inhibiting glutamate-induced oxidative stress damage in PC12 cells.

[0043] The above results indicate that compounds 1-5 all exhibit significant neuroprotective activity in an in vitro glutamate-induced PC12 cell excitotoxicity model. Their mechanism of action is at least partly related to anti-oxidation and anti-apoptosis. Therefore, compounds 1-5 of the present invention are applicable to the treatment of neurological diseases related to excitotoxicity, including insomnia, depression, cognitive impairment, and neurodegenerative diseases, and have good clinical application prospects.

[0044] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. An isopentenylated polyketide compound, characterized in that, It has the structure shown in equation (I): Formula (I) Among them, R 1 Selected from -C(=O)-CH2-CH2-C(=O)-CH3, -C(=O)-CH2-CH2-CH(OH)-CH3 or -C(=O)-CH2-CH(OH)-CH2-CH3; R 2 Selected from hydroxyl groups; or R 1 and R 2 Together with one or more atoms to which they are attached, they form a five-membered ring, which contains an ester group as a ring member; R 3 Selected from -CH2-CH(OH)-C(OH)(CH3)2 or -CH2-C(=O)-OCH3.

2. The isopentenylated polyketide compound according to claim 1, characterized in that, R1 and R2, together with one or more atoms they are attached to, form a five-membered ring, which includes an ester group as a ring member; R 3 It is -CH2-CH(OH)-C(OH)(CH3)2.

3. A method for preparing the isopentenylated polyketide compound according to claim 1 or 2, characterized in that, Includes the following steps: S1. The strain of *Caryopsis niger* was inoculated into rice culture medium and cultured to obtain fermentation product; S2. The fermented product is extracted with a solvent, then concentrated, extracted, and concentrated again to obtain a total extract; S3. The total extract is separated and purified to obtain a polyketide compound containing isopentenylated.

4. The method for preparing isopentenylated polyketide compounds according to claim 3, characterized in that, In step S1, the culture temperature is 20~30℃ and the culture time is 20~40 days.

5. The method for preparing isopentenylated polyketide compounds according to claim 3, characterized in that, In step S2, the solvent includes at least one of acetone and ethanol.

6. The method for preparing isopentenylated polyketide compounds according to claim 3, characterized in that, The separation includes the following steps: S3.1 The total extract is subjected to gradient elution by silica gel column chromatography to obtain multiple crude components; S3.2 The crude component is separated by gel column chromatography to obtain the finer components.

7. The method for preparing isopentenylated polyketide compounds according to claim 6, characterized in that, The purification process includes the following steps: purifying the subdivided components by high performance liquid chromatography to obtain the isopentenylated polyketide compound.

8. The method for preparing isopentenylated polyketide compounds according to claim 7, characterized in that, The mobile phase of the high-performance liquid chromatography includes acetonitrile and water. The flow rate of the mobile phase is 2–4 mL / min.

9. The use of an isopentenylated polyketide compound as described in claim 1 or 2 in the preparation of a medicament for treating nervous system diseases.

10. The application according to claim 9, characterized in that, The neurological disorders include insomnia, depression, cognitive impairment, or neurodegenerative diseases.

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