Application of kauriane-type diterpenoids in the preparation of drugs for the prevention or treatment of neurodegenerative diseases

Abstract

This invention discloses the application of kauriane-type diterpenoids in the preparation of drugs for the prevention or treatment of neurodegenerative diseases. The invention also relates to the application of these compounds in the preparation of drugs for improving cognitive impairment, β-amyloid inhibitors, and neuroinflammatory response inhibitors. Experiments show that these compounds can significantly improve spatial learning and memory abilities and recognition memory abilities in Alzheimer's disease model mice; reduce β-amyloid plaque deposition in the mouse brain; inhibit excessive activation of microglia; and reduce the mRNA expression levels of pro-inflammatory factors TNF-α, IL-6, and IL-1β. This invention is the first to discover that these kauriane-type diterpenoids possess multi-target neuroprotective activity and can be used to prepare drugs for the prevention or treatment of neurodegenerative diseases (especially Alzheimer's disease) characterized by β-amyloid deposition and / or neuroinflammation, showing broad clinical application prospects.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically to the use of kauriane-type diterpenoids in the preparation of medicaments for the prevention or treatment of neurodegenerative diseases. Background Technology

[0002] Alzheimer's disease (AD) is a neurodegenerative disease of the central nervous system with insidious onset and progressive development, primarily affecting the elderly. Its core pathological features include β-amyloid (Aβ) plaque deposition, neurofibrillary tangles (Tau), brain atrophy, and neuroinflammatory responses. Persistent neuroinflammatory activity is considered the third major core pathological feature after Aβ plaques and Tau tangles. Recent studies have found that long-term abnormal activation of microglia can significantly exacerbate Aβ deposition and play a crucial role in the development and progression of the disease. Aβ accumulation can induce neuroinflammatory responses, and these inflammatory responses often precede obvious Aβ accumulation; the interaction between the two is a significant driving factor in the disease's progression.

[0003] Currently, clinical interventions mainly utilize cholinesterase inhibitors, N-methyl-D-aspartate (NMDA) receptor antagonists, and anti-Aβ monoclonal antibodies. Existing technologies, such as Chinese patent CN202110090090.8, disclose the application of diphenyl ether compounds in the preparation of acetylcholinesterase inhibitors or drugs for treating Alzheimer's disease. Compounds isolated from the fermentation culture of the marine fungus *Aspergillus sp. BH4-11* exhibit significant inhibitory activity against acetylcholinesterase, providing candidate compounds for the development of new acetylcholinesterase inhibitors. Chinese patent CN202110721170.9 discloses the application of a flavonoid compound in the preparation of cholinesterase inhibitors and drugs for the prevention of Alzheimer's disease. The flavonoid compound *Eriosematin* exhibits highly efficient inhibitory activity against cholinesterase and can be used to prevent Alzheimer's disease caused by damage to the cholinergic system. Chinese patent CN202080063538.0 discloses a neutralizing anti-Aβ amyloid antibody for treating Alzheimer's disease, which specifically binds to one or more types of soluble AD brain-derived synaptic toxic β-amyloid protein (Aβ) without binding to classical monomers, primary fibrils, or fibrillary Aβ-binding peptides (e.g., antibodies and their antigen-binding fragments). It also provides a method for alleviating one or more symptoms of Alzheimer's disease using binding peptides that specifically bind to one or more types of soluble synaptic toxic Aβ. While these drugs can slow disease progression to some extent, their overall efficacy is limited and they cannot yet achieve fundamental prevention and treatment of the disease.

[0004] Therefore, there is an urgent need to develop AD treatment drugs with better efficacy and higher safety. Due to the holistic approach and focus on treating both the root cause and symptoms of AD, traditional Chinese medicine (TCM) offers a significant practical advantage and research necessity for developing novel TCM preparations with clear mechanisms of action for treating AD. Summary of the Invention

[0005] In view of this, the present invention provides a novel application of the kaurane-type diterpenoid compound Caesalactone K in the preparation of drugs for treating Alzheimer's disease.

[0006] In a first aspect, the present invention provides the use of a kauriane-type diterpenoid compound of formula (I) or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention or treatment of neurodegenerative diseases, wherein the kauriane-type diterpenoid compound is 13,19-dihydroxy-8,15-open-enanthracycline-16-ene-8,15-lactone (Caesalactone K), and the structural formula of the kauriane-type diterpenoid compound is as follows:

[0007]

[0008] (I).

[0009] Furthermore, the neurodegenerative disease is Alzheimer's disease.

[0010] Preferably, the drug further comprises pharmaceutically acceptable excipients.

[0011] Preferably, the dosage form of the drug includes, but is not limited to, injections or oral formulations.

[0012] Secondly, the present invention provides the use of kauriane-type diterpenoids or pharmaceutically acceptable salts thereof in the preparation of medicaments for improving cognitive impairment. The kauriane-type diterpenoid shown is 13,19-dihydroxy-8,15-open-enanthracycline-16-ene-8,15-lactone (Caesalactone K), and the structural formula of the kauriane-type diterpenoid is as follows:

[0013]

[0014] (I).

[0015] Preferably, the cognitive impairment is an Alzheimer's disease-related cognitive impairment, which includes, but is not limited to, spatial learning and memory impairment and recognition and memory impairment. This invention has demonstrated through animal behavioral experiments that the compound of formula (I) can significantly shorten the escape latency of AD model mice in the water maze experiment, increase the time spent in the target quadrant and the number of times they cross the platform, and improve the recognition index in the new object recognition experiment, indicating that it can improve cognitive function in multiple dimensions.

[0016] Thirdly, the present invention provides the use of kauriane-type diterpenoids or pharmaceutically acceptable salts thereof in the preparation of β-amyloid inhibitors, wherein the kauriane-type diterpenoid is 13,19-dihydroxy-8,15-open-enantiokauri-16-ene-8,15-lactone (Caesalactone K), and the structural formula of the kauriane-type diterpenoid is as follows:

[0017]

[0018] (I).

[0019] Fourthly, this invention provides the use of kauriane-type diterpenoids or pharmaceutically acceptable salts thereof in the preparation of inhibitors of neuroinflammatory responses. The kauriane-type diterpenoid shown is 13,19-dihydroxy-8,15-open-enantiokauri-16-ene-8,15-lactone (Caesalactone K), and the structural formula of the kauriane-type diterpenoid is as follows:

[0020]

[0021] (I).

[0022] This invention also provides a method for extracting the above-mentioned kaurane-type diterpenoid compounds, characterized by comprising the following steps:

[0023] (1) Take the branches and leaves of nine-winged pheasant, crush them, extract them with methanol, combine the extracts, concentrate them under reduced pressure, and obtain crude extract.

[0024] (2) Dissolve the crude extract in water, extract with ethyl acetate, combine the ethyl acetate extracts, concentrate under reduced pressure to obtain ethyl acetate extract;

[0025] (3) The petroleum ether-ethyl acetate mixed solvent was used for gradient elution, the gradient being 20:1, 15:1, 10:1, 5:1, 2:1, 1:1, 0:1, v / v. The eluents of each gradient were collected and concentrated under reduced pressure. The petroleum ether-ethyl acetate 2:1 eluent was collected as fraction E. The silica gel column chromatography column was 110 mm × 1200 mm.

[0026] (4) The fraction E is separated by MCI column chromatography, and gradient elution is performed sequentially with methanol-water mixed solvent. The gradients are 5:5, 6:4, 7:3, 8:2, and 9:1, v / v. The eluents of each gradient are collected, concentrated under reduced pressure, and the methanol-water 5:5 elution fraction is collected as fraction E1. The MCI column has a column size of 70 mm × 800 mm.

[0027] (5) Separate fraction E1 by silica gel column chromatography, using a dichloromethane-methanol mixed solvent for gradient elution, wherein the gradients are 45:1, 30:1, 25:1, 15:1, 8:1, and 5:1, v / v, respectively. Collect each gradient eluent, concentrate under reduced pressure, and collect the dichloromethane-methanol 25:1 eluent fraction as fraction E14; the silica gel column has a column size of 15 mm × 600 mm.

[0028] (6) The fraction E14 was separated by gel column chromatography with chloroform-methanol 3:2, v / v elution. The eluent was collected and concentrated under reduced pressure to obtain compound 11, which is the kaurane-type diterpenoid compound. The gel type of the gel column chromatography was Sephadex LH-20, the column size was 15 mm × 800 mm, and the elution flow rate was 0.5 mL / min.

[0029] As can be seen from the above technical solution, compared with the prior art, the present invention discloses the application of kauriane-type diterpenoid compounds in the preparation of drugs for the prevention or treatment of neurodegenerative diseases, which has the following beneficial effects:

[0030] This invention is the first to discover that the kaurane-type diterpenoid compound represented by formula (I) possesses multiple neuroprotective activities: it can significantly improve cognitive dysfunction, reduce abnormal Aβ deposition, and inhibit microglial activation and neuroinflammatory responses. Therefore, this compound can be used as a multi-target drug to treat neurodegenerative diseases (especially Alzheimer's disease) with Aβ deposition and / or neuroinflammation as the core pathology, and has significant clinical application value. Attached Figure Description

[0031] 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 embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0032] Figure 1 This diagram illustrates the effect of compound 171 on spatial learning and memory abilities in AD mice and its effect on recognition and memory abilities; among which, Figure 1 A represents the typical swimming trajectory of mice during the MWM training (day 5) and exploration phases (day 6). Figure 1 B represents the time it takes for the mouse to reach the hidden platform (escape latency). Figure 1 C represents the time when the mouse reaches the hidden platform on day 5. Figure 1 D represents the time the mouse spent in the target quadrant on day six. Figure 1 E represents the number of times the mouse crossed the platform within 60 seconds on day 6. Figure 1 F is a representative trajectory from the new object recognition experiment. Figure 1 G is the identification index RI;

[0033] Figure 2 The experiment investigated the effects of compound 171 on Aβ deposition and microglial activation in the brains of AD mice, as well as its influence on the expression of anti-inflammatory factors; among which, Figure 2 A represents the result of thiamine S staining; Figure 2 B represents the result of immunofluorescence staining; Figure 2 C is the IBA-1 statistical chart; Figure 2 D is a 6E10 statistical chart; Figure 2 E represents the TNF-α expression level; Figure 2 F represents the IL-6 expression level; Figure 2 G represents the expression level of IL-1β. Detailed Implementation

[0034] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0035] Example 1

[0036] Extraction and separation of kauriane-type diterpenoids as described in formula (I):

[0037] The leaves and branches of *Anemarrhena asphodeloides* (28.0 kg) were air-dried and pulverized. They were then extracted four times (once every seven days) with industrial methanol (75 L) at room temperature. The extracts were combined and concentrated under reduced pressure to obtain 2.6 kg of crude extract of *Anemarrhena asphodeloides*. The crude extract was dissolved in water (6 L), and then extracted five times with ethyl acetate (6 L). The extracts were combined and concentrated under reduced pressure to obtain 640 g of ethyl acetate extract.

[0038] Ethyl acetate extract was mixed with 1.5 times its volume of silica gel (80-100 mesh), and then subjected to gradient elution using a silica gel column (200-300 mesh). The elution solvent system was petroleum ether:ethyl acetate (20:1, 15:1, 10:1, 5:1, 2:1, 1:1, 0:1, v / v). Each gradient elution consisted of 4-6 column volumes. The eluents from each gradient were collected separately, concentrated under reduced pressure, and the 2:1 petroleum ether-ethyl acetate eluent was collected as fraction E. The silica gel column used in the chromatography was 110 mm × 1200 mm.

[0039] After fraction E was subjected to MCI column chromatography (methanol:water, 5:5, 6:4, 7:3, 8:2, 9:1, v / v), the eluent was collected in fractions. The methanol-water 5:5 elution fraction was collected as fraction E1. The MCI column chromatography column had a size of 70 mm × 800 mm.

[0040] E1 was eluted by silica gel column chromatography (dichloromethane:methanol, 45:1, 30:1, 25:1, 15:1, 8:1, 5:1, v / v), and the eluent was collected in fractions. The dichloromethane-methanol 25:1 eluent was collected as fraction E14; the silica gel column used in the chromatography was 15 mm × 600 mm.

[0041] After E14 was subjected to gel column chromatography (CHCl3 / MeOH 3:2), the eluent was collected and concentrated under reduced pressure to obtain compound 11, which is the kaurane-type diterpenoid compound described in formula (I); the gel type of the gel column chromatography was Sephadex LH-20, the column size was 15 mm × 800 mm, and the elution flow rate was 0.5 mL / min.

[0042] The compound described in formula (I) is a white powder, soluble in methanol, EI-MS: m / z 357 [M+Na] + The molecular formula is C 20 H 30 O4, 1 H-NMR and 13 CNMR data:

[0043] 1H-NMR (500 MHz, CD3OD): δ 6.45 (1H, d, J = 1.7 Hz, H-17a), 6.05 (1H,d, J = 1.7 Hz, H-17b), 3.69 (1H, d, J = 11.2 Hz, H-19a), 3.39 (1H, d, J = 11.2 Hz, H-19a) =11.2 Hz, H-19b), 2.36 (1H, d, J = 13.5 Hz, H-14a), 2.04 (1H, m, H-7a), 1.99(1H, m, H-2a), 1.97(2H, m, , H-1a, 11a), 1.99 (1H, m, H-2b), 1.83 (3H, m, H-1b, H-6a, 12a), 1.81 (2H, m, H-3a, 14b), 1.48 (4H, m, H-12b, 6b, 9, 11b), 1.16 (1H, m, H-5), 1.10 (3H, s, H-20), 1.06 (1H, m, H-6b), 1.03 (1H, m, H-7b), 1.01 (3H, s, H-18), 1.00 (1H, m, H-3b);

[0044] 13 C-NMR (125 MHz, CD3OD): δ 167.9 (C-15), 145.3 (C-16), 126.4 (C-17), 87.1 (C-8), 70.7 (C-13), 65.4 (C-19), 57.4 (C-5), 53.0 (C-9), 43.4 (C-14), 43.3 (C-1), 41.9 (C-7), 40.6 (C-10), 39.7 (C-4), 39.4 (C-12), 36.6 (C-3), 28.1 (C-18), 20.3 (C-6), 19.8 (C-11), 19.5 (C-2), 18.7 (C-20).

[0045] Example 2

[0046] Experimental study on the therapeutic effect of the kauriane-type diterpenoid compound (hereinafter referred to as compound 171) of formula (I) on Alzheimer's disease:

[0047] I. Experiment on the effects of compound 171 on spatial learning and memory in AD mice. The specific steps are as follows:

[0048] 1. Experimental Methods

[0049] APP / PS1 transgenic mice (male, weighing 22–25g) were used as AD model mice. The APP / PS1 transgenic mice were purchased from Hangzhou Ziyuan Experimental Animal Technology Co., Ltd.

[0050] The experimental animals were housed in a standardized animal facility with a room temperature maintained at 22–25°C and suitable relative humidity, under a 12-hour / 12-hour light-dark cycle, and with free access to food and water. The APP / PS1 transgenic mouse is a classic animal model widely used in Alzheimer's disease research. Its brain exhibits abnormal Aβ deposition forming typical amyloid plaques, accompanied by pathological features such as abnormal microglial activation, neuronal degeneration, and synaptic damage. Behavioralally, this model demonstrates significant cognitive and memory decline, particularly in spatial learning and memory impairment, and is therefore widely used to evaluate the in vivo efficacy of potential AD treatment strategies and candidate drugs.

[0051] Dosing regimen: Thirty-two 6-month-old male APP / PS1 mice were randomly divided into three groups:

[0052] (1) APP / PS1 group (APP / PS1 transgenic mice, without drug intervention);

[0053] (2) Group 171 (APP / PS1 transgenic mice, given compound 171 at a dose of 5 mg / kg per day).

[0054] (3) Donepezil group (APP / PS1 transgenic mice, given 5 mg / kg of donepezil daily).

[0055] Six-month-old male C57BL / 6J mice were used as a normal control group (WT). All treatment groups received intraperitoneal injection for 30 consecutive days. After treatment, all mice underwent systematic behavioral tests to assess changes in cognitive function.

[0056] 2. The effect of compound 171 on improving spatial learning and memory in AD mice was evaluated using the Morris water maze test.

[0057] The Morris water maze experiment was conducted in a circular pool (120 cm in diameter and 50 cm in height). Water containing titanium dioxide was added to conceal the platform, and the water temperature was maintained at 22–23°C. The pool was divided into four quadrants of equal area. The circular escape platform was fixed in one quadrant and hidden 1 cm below the water surface, preventing the mice from visually identifying its location.

[0058] The experiment consisted of a spatial learning training phase (days 1–5) and a spatial exploration testing phase (day 6). During the training phase, mice were randomly placed into the pool from four different quadrants each day, with a maximum search time of 60 seconds each time. If a mouse failed to find the platform within the allotted time, the experimenter guided it onto the platform and allowed it to stay there for 30 seconds to learn spatial skills; if it successfully found the platform, it was also allowed to stay on it for 30 seconds.

[0059] The training process was conducted continuously for 5 days. On the 6th day, during the spatial exploration experiment, the hidden platform was removed, and the mice were randomly placed into a pool from one quadrant, allowed to swim freely for 60 seconds, to assess their spatial memory retention ability. Behavioral indicators such as escape latency, time spent in the target quadrant, number of times the mice crossed the original platform location, and swimming speed were recorded and analyzed using an automated image acquisition and analysis system (TopScan, Virginia, USA). Results are as follows: Figure 1 As shown in AE.

[0060] Compared to the WT group, the APP / PS1 group mice exhibited significantly prolonged escape latency throughout the training process, demonstrating marked spatial learning impairment. In contrast, the escape latency of the 171 group and the donepezil group was significantly shorter than that of the APP / PS1 group. Figure 1 AC), and this improvement effect was particularly pronounced in the later stages of training, suggesting that 171 can effectively promote the recovery of spatial learning ability in AD mice. In subsequent spatial exploration experiments, mice in the 171-treated group and the donepezil group significantly increased the number of times they crossed the original platform location and stayed in the target quadrant for a longer time, showing better spatial memory retention (AC). Figure 1 DE). The above results indicate that 171 has a significant therapeutic effect in improving spatial learning and memory impairment in AD mice.

[0061] 3. Evaluate the impact of 171 on recognition and memory abilities through a new object recognition experiment.

[0062] The new object recognition experiment was conducted in a non-reflective cube-shaped experimental setup (40×40×40cm). The day before the formal experiment, the mice were placed in an empty experimental box and allowed to explore freely for 10 minutes to acclimatize to the experimental environment and reduce interference from novel stimuli.

[0063] The first day of the experiment was the familiarization phase: two identical objects were placed diagonally opposite each other in the experimental chamber. Mice were placed inside and allowed to explore freely for 10 minutes, and the exploration time was recorded. Exploration behavior was defined as the mouse pointing its nose or forepaws within 2 cm of the object or making direct contact with the object. After the experiment, the mice were immediately removed, and the experimental chamber and objects were thoroughly cleaned with 75% ethanol to eliminate interference from olfactory cues. The second day of the experiment was the testing phase: one of the familiar objects was replaced with a new object, and the mouse was placed back in the experimental chamber and allowed to explore freely for 10 minutes. The exploration time for the new object and the old object was recorded separately. Finally, the recognition index (RI) was calculated to assess the mice's recognition and memory abilities. The calculation formula is as follows:

[0064] RI (%) = (New object exploration time / (New object exploration time + Old object exploration time)) × 100%

[0065] The results are as follows Figure 1 As shown in FG.

[0066] from Figure 1 FG shows that mice treated with 171 or donepezil exhibited a significantly enhanced tendency to explore new objects, demonstrating greater sensitivity to novel stimuli. This result further demonstrates that 171 can effectively improve the recognition and memory abilities of AD mice.

[0067] All experimental results were statistically analyzed using GraphPad Prism 9.5.0. All data underwent analysis of variance (ANOVA). Two-way ANOVA was used to compare escape latency, while one-way ANOVA was used for other data. Data are expressed as mean ± SD. A p-value < 0.05 was considered statistically significant. * indicates p ≤ 0.05, ** indicates p ≤ 0.01, *** indicates p ≤ 0.001, and **** indicates p ≤ 0.0001.

[0068] II. Effects of Compound 171 on Aβ Deposition and Microglial Cell Activation in the Brain of AD Mice

[0069] To assess whether compound 171 administration affected abnormal Aβ deposition, mice in each group were sacrificed after the behavioral experiments, and brain tissue was rapidly harvested for thiamine S staining and immunofluorescence staining. Thiamine S staining was used to detect Aβ plaques in the mouse brain tissue, while immunofluorescence staining was used to analyze the distribution of Aβ plaques and the activation status of microglia in the hippocampus. The specific steps are as follows:

[0070] 1. Thioflavin S (ThS) staining

[0071] After the behavioral experiments, mice were euthanized and brain tissue was rapidly harvested. The brain tissue was fixed overnight in 4% paraformaldehyde, then transferred to phosphate buffer containing 30 wt.% sucrose and subjected to sucrose precipitation treatment at 4°C for 24 h. After thorough dehydration, 30 μm thick frozen sections of brain tissue were prepared using a cryostat. The brain tissue sections were incubated in 0.5% thiamine S solution (CAS#1326-12-1; Shanghai Yuanye Biotechnology Co., Ltd.) in the dark for 8 min to label amyloid protein deposition. After staining, the sections were sequentially dehydrated using 70 v / v%, 80 v / v%, 90 v / v% aqueous ethanol solution, and anhydrous ethanol (2 min per stage). Finally, fluorescence images were acquired using a Nikon confocal microscope (Nikon Ti2-E), and the results are shown below. Figure 2 As shown in Figure A.

[0072] from Figure 2 As shown in Figure A, compared with the WT group, the Aβ deposition in the brains of mice in the APP / PS1 group was significantly increased. Compared with the APP / PS1 group, the Aβ deposition levels in the brains of mice in the 171 group and donepezil group were significantly decreased, indicating that 171 or donepezil can reduce the Aβ deposition level in the brains of AD mice.

[0073] 2. Immunofluorescence staining

[0074] Brain tissue sections were first washed three times with PBS (5 min each time), then placed in blocking solution containing 0.3% Triton X-100 and 10% sheep serum for 2 h at room temperature for blocking and permeabilization. Afterwards, the sections were incubated overnight at 4°C with the corresponding primary antibodies (6E10-labeled Aβ, Iba-1-labeled microglia). The next day, after washing with PBS, the sections were incubated for 1 h at room temperature with the corresponding fluorescently labeled secondary antibodies, washed again, and mounted using a DAPI-containing anti-fluorescence quenching mounting medium. Finally, images were acquired using a Nikon confocal microscope (Nikon Ti2-E) for subsequent quantitative analysis, and the results are as follows: Figure 2 As shown in BD.

[0075] from Figure 2 BD analysis revealed abundant Aβ plaque deposition in the hippocampus of APP / PS1 mice, accompanied by significantly enhanced microglial activation. In contrast, 171 treatment significantly reduced the Aβ plaque burden in the hippocampus and significantly weakened microglial activation. The 6E10 and IBA-1 statistical plots showed that, compared to the APP / PS1 group, 171 significantly reduced Aβ content and microglial activation.

[0076] 3. Detect the expression levels of pro-inflammatory factors.

[0077] The expression levels of pro-inflammatory factors were detected using qPCR. The primer sequences used in the method are shown in Table 1. The detection results are as follows: Figure 2 As shown in EG.

[0078] Table 1. Primer sequences

[0079]

[0080] from Figure 2 The results from EG showed that administration of 171 significantly downregulated the mRNA expression levels of pro-inflammatory factors TNF-α, IL-6, and IL-1β in AD mice, suggesting that it has a clear anti-inflammatory effect.

[0081] In summary, the above results indicate that 171 exerts a synergistic effect on regulating and improving the pathological process of Alzheimer's disease by reducing Aβ deposition, reversing the abnormal activation state of microglia, and inhibiting neuroinflammatory responses.

[0082] The various embodiments described in this specification are presented in a progressive manner, with each embodiment focusing on its differences from other embodiments. Similar or identical parts between embodiments can be referred to interchangeably. The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The use of kauriane-type diterpenoids or pharmaceutically acceptable salts thereof in the preparation of medicaments for the prevention or treatment of neurodegenerative diseases, wherein the structural formula of the kauriane-type diterpenoid is as follows: 。 2. The application according to claim 1, characterized in that, The neurodegenerative disease mentioned is Alzheimer's disease.

3. The application according to claim 1 or 2, characterized in that, The drug also contains pharmaceutically acceptable excipients.

4. The application according to claim 1 or 2, characterized in that, The drug is available in either injectable or oral formulation.

5. The use of kauriane-type diterpenoids or pharmaceutically acceptable salts thereof in the preparation of medicaments for improving cognitive impairment, wherein the structural formula of the kauriane-type diterpenoid is as follows: 。 6. The application according to claim 5, characterized in that, The cognitive impairment mentioned refers to cognitive impairment related to Alzheimer's disease.

7. The use of kauriane-type diterpenoids or pharmaceutically acceptable salts thereof in the preparation of β-amyloid inhibitors, wherein the structural formula of the kauriane-type diterpenoid is as follows: 。 8. The use of kauriane-type diterpenoids or pharmaceutically acceptable salts thereof in the preparation of inhibitors of neuroinflammatory responses, wherein the structural formula of the kauriane-type diterpenoid is as follows: 。 9. A method for extracting kauriane-type diterpenoids according to any one of claims 1-8, characterized in that, Includes the following steps: (1) Take the branches and leaves of nine-winged pheasant, crush them, extract them with methanol, combine the extracts, concentrate them under reduced pressure, and obtain crude extract. (2) Dissolve the crude extract in water, extract with ethyl acetate, combine the ethyl acetate extracts, concentrate under reduced pressure to obtain ethyl acetate extract; (3) The petroleum ether-ethyl acetate mixed solvent was used for gradient elution, the gradient being 20:1, 15:1, 10:1, 5:1, 2:1, 1:1, 0:1, v / v. The eluents of each gradient were collected and concentrated under reduced pressure. The petroleum ether-ethyl acetate 2:1 eluent was collected as fraction E. The silica gel column chromatography column was 110 mm × 1200 mm. (4) The fraction E is separated by MCI column chromatography, and gradient elution is performed sequentially with methanol-water mixed solvent. The gradients are 5:5, 6:4, 7:3, 8:2, and 9:1, v / v. The eluents of each gradient are collected, concentrated under reduced pressure, and the methanol-water 5:5 elution fraction is collected as fraction E1. The MCI column has a column size of 70 mm × 800 mm. (5) Separate fraction E1 by silica gel column chromatography, using a dichloromethane-methanol mixed solvent for gradient elution, wherein the gradients are 45:1, 30:1, 25:1, 15:1, 8:1, and 5:1, v / v, respectively. Collect each gradient eluent, concentrate under reduced pressure, and collect the dichloromethane-methanol 25:1 eluent fraction as fraction E14; the silica gel column has a column size of 15 mm × 600 mm. (6) The fraction E14 was separated by gel column chromatography with chloroform-methanol 3:2, v / v elution. The eluent was collected and concentrated under reduced pressure to obtain compound 11, which is the kaurane-type diterpenoid compound. The gel type of the gel column chromatography was Sephadex LH-20, the column size was 15 mm × 800 mm, and the elution flow rate was 0.5 mL / min.

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