Application of CL in the treatment and prevention of Alzheimer's disease and Parkinson's disease

Abstract

This invention belongs to the field of pharmaceutical technology and specifically discloses the use of CL in a drug for the treatment and prevention of Alzheimer's disease and Parkinson's disease. Studies have demonstrated that CL can significantly improve the motility and chemotaxis of Caenorhabditis elegans with Alzheimer's disease, significantly extend the lifespan of Caenorhabditis elegans with Alzheimer's disease, and significantly restore damaged neurons in Caenorhabditis elegans with Parkinson's disease, suggesting that CL has the potential to be used in the development of therapeutics for neurodegenerative diseases.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and specifically discloses an application of CL in drugs for treating and preventing Alzheimer's disease and Parkinson's disease. Background Art

[0002] Neurodegenerative diseases (NDs) are neurological disorders characterized by progressive loss of the central or peripheral nervous system, including the common Alzheimer's disease (AD), Parkinson's disease (PD), and multiple sclerosis (MS). With the increasing aging of the global population, the incidence of NDs continues to rise, placing a significant burden on society and families. Consequently, many researchers have developed animal models to screen for potential new drugs to treat NDs. Caenorhabditis elegans (C. elegans) is widely used to screen potential clinical drugs for NDs due to its well-defined genetic background, small size, short life cycle, unique pathological behaviors, pathological defects, and simple nervous system.

[0003] Although the pathological characteristics of AD are mainly extracellular senile plaques formed by Aβ deposition and intracellular neurofibrillary tangles formed by hyperphosphorylation of Tau protein, its main pathogenesis is still unclear. However, these two proteins have been identified as key to the treatment of AD. Therefore, researchers have used human Aβ genes and human Tau protein genes to establish a transgenic AD nematode model, and assess AD disease directly or indirectly, such as directly observing the process of neuronal loss, impaired movement, and shortened lifespan through the mediation of fluorescent proteins.

[0004] PD is a common disease in middle-aged and elderly people. The incidence rate increases with age and is slightly higher in men than in women. The generally recognized causes of PD are mainly the reduction of dopaminergic neurons (DA) and the aggregation of Lewy bodies (LB). In nematodes, models of dopamine neuron damage caused by chemical drugs and transgenic Lewy body aggregation models have been successfully prepared. When nematodes are exposed to 6-OHDA, they will show a DA damage phenotype. α-synuclein is the most important protein in Lewy bodies and is one of the main targets for current research on the treatment of PD. Because nematodes cannot express α-synuclein themselves, it is necessary to artificially introduce the α-synuclein gene to establish an ideal transgenic α-synuclein nematode PD model.

[0005] Currently, drugs used clinically to treat neurodegenerative diseases, such as donepezil for the treatment of Alzheimer's disease, can quickly increase the concentration of acetylcholine in the brain and improve cognitive function; levodopa for the treatment of Parkinson's disease can quickly replenish the missing dopamine in the brain, and the patient's motor symptoms can be improved in a short period of time. However, these drugs can often only relieve symptoms and have many adverse reactions. Summary of the Invention

[0006] In response to the technical problems in the background technology, the purpose of the present invention is to provide a new pharmaceutical use of the pool cypress extract CL, and to discover the application of CL in the treatment and prevention of neurodegenerative diseases. CL has a good therapeutic effect on neurodegenerative diseases, provides a new treatment idea for neurodegenerative diseases, and has broad application prospects.

[0007] To achieve the above object, the technical solution of the present invention is as follows:

[0008] The present invention provides a use of the pool cypress extract CL in a drug for treating and preventing neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. The structural formula of the CL is as follows:

[0009]

[0010] The specific extraction process for the pool cypress extract CL is as follows: air-dried pool cypress leaves are pulverized and extracted three times with 95% ethanol (ethanol:pool cypress leaves = 10:1) at reflux for 2 hours each time to obtain the total extract. The total extract is then extracted three times with petroleum ether, ethyl acetate, and n-butanol. The petroleum ether extract is loaded onto a silica gel column and eluted with a gradient of petroleum ether / ethyl acetate (100 / 0 to 0 / 100) to obtain Fr.1 to Fr.4. Fr.1 (100:25, 20 g) is loaded onto an ODS column and eluted with a gradient of methanol / water (80:20 to 100:0) to obtain compound CL (methanol:water = 100:15).

[0011] Furthermore, the application of CL in the treatment and prevention of neurodegenerative diseases described in the present invention is mainly based on the discovery that CL has a neuroprotective effect in the model organism Caenorhabditis elegans.

[0012] Specifically, CL can significantly improve the movement disorder of BR5270 nematodes, enhance the chemotaxis ability of CL2355 nematodes, prolong the lifespan of UM0001 nematodes, and promote the autophagy level of DA2123 nematodes;

[0013] CL can significantly restore 6-OHDA-induced damaged neurons in BZ555 nematodes and promote the clearance of α-Syn deposition in NL5901 nematodes.

[0014] Improve the motility of nematodes, extend lifespan, and restore damaged neurons in nematodes. The concentration range of CL is: 0.75-3μM.

[0015] The technical effects of this invention include demonstrating that CL has significant efficacy in treating and preventing neurodegenerative diseases through experiments using Caenorhabditis elegans, including basic slow reaction tests, tropism tests, lifespan tests, and fluorescence quantitative tests. This invention provides a new, single-ingredient Traditional Chinese Medicine drug for treating and preventing neurodegenerative diseases, broadening the range of diseases CL can prevent and treat. Due to its wide availability and low toxicity, CL has promising potential for development as a drug for treating and preventing neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Effects of CL on the motility of nematodes BR5270 and BR5271.

[0017] Figure 2 Effects of CL on the chemotaxis of nematodes CL2355 and CL2122.

[0018] Figure 3 Effects of CL on the lifespan of UM0001 nematodes.

[0019] Figure 4 Effect of CL on the autophagy level of DA2123 nematodes.

[0020] Figure 5 Effects of CL on α-Syn in NL5901 nematodes.

[0021] Figure 6 Restorative effect of CL on 6-OHDA-induced DA neurons in BZ555 nematodes. DETAILED DESCRIPTION

[0022] The present invention is further illustrated in detail by the following examples, but it should be noted that the scope of the present invention is not limited by these examples.

[0023] Example 1

[0024] The extraction process for the pool cypress extract, CL, was as follows: 10 kg of air-dried pool cypress leaves were crushed and extracted three times with 95% ethanol (ethanol:pool cypress leaves (volume-to-mass ratio) = 10:1) at reflux for 2 hours each time, yielding 2 kg of total extract. The total extract was then extracted three times with petroleum ether, ethyl acetate, and n-butanol. The petroleum ether extract (280 g) was loaded onto a silica gel column and eluted with a gradient of petroleum ether / ethyl acetate (100 / 0 to 0 / 100) to obtain fractions 1 to 4. Fr. 1 (100:25, 20 g) was loaded onto an ODS column and eluted with a gradient of methanol / water (80:20 to 100:0) to yield 120 mg of compound CL (methanol:water = 100:15).

[0025] Example 2 Effects of CL on AD Model Caenorhabditis elegans

[0026] (1) Biomaterials

[0027]

[0028] (2) Reagents

[0029]

[0030]

[0031] (3) Implementation steps

[0032]

[0033] (4) Nematode growth cycle and synchronization

[0034] Caenorhabditis elegans is a hermaphroditic model organism. After hatching, individual worms undergo four larval stages (L1-L4). When the worms are overcrowded or food is scarce, they enter another larval stage, called the dauer larvae. Dauer larvae are resilient to adversity and do not age. They typically lay eggs during the adult stage, and drug treatment is initiated during the L2 stage.

[0035] Synchronization of nematodes: To obtain nematodes of the same growth period, a large number of nematodes need to be synchronized. First, pick 100 sexually mature adults and place them in NGM medium with sufficient food and no contamination. Then place the culture dish in a 16°C constant temperature incubator until they lay eggs. After most of the adults have laid eggs, rinse the nematodes three times with nematode buffer M9 (4500 rpm, 2 min), remove the supernatant, add 1 mL of lysis buffer prepared according to the above reagent formula, shake thoroughly with a vortex shaker, centrifuge (4500 rpm, 2 min), remove the supernatant, and then wash three times with M9 buffer. Remove the supernatant, collect the nematode fragments and eggs at the bottom, drop them into clean NGM medium with sufficient food, and place in a 16°C incubator until the eggs hatch. This will obtain larvae of the same period.

[0036] (5) Screening of appropriate CL activity concentration

[0037] Experimental Methods: This study used the human Tau protein-transfected BR5270 nematode strain as an AD model for drug activity screening, with BR5271 as a positive control. BR5270 larvae grown to the L2 stage were collected according to the aforementioned synchronization method and added to the prepared drug-containing plates (0.375μM, 0.75μM, 1.5μM, 3μM, and 6μM). The larvae were incubated in a 16°C incubator for 72 hours and their growth was observed. Upon reaching adulthood, 30 nematodes were selected from each group and their head movement frequency within 30 seconds was observed.

[0038] Experimental results: Compared with the blank group, after treatment with different concentrations of CL, the motility of BR5270 nematodes was improved to varying degrees. Among them, 0.75μM, 1.5μM, and 3μM had the best effect, and were concentration-dependent and statistically significant (p < 0.001). Although low and high concentrations had some relief, the effect was not very obvious and had no statistical significance (p > 0.05). Figure 1 .

[0039] (6) Effects of CL on the chemotaxis of nematodes CL2355 to benzaldehyde

[0040] Experimental Methods: Based on the above experimental results, the present invention ultimately selected CL (0.75μM, 1.5μM, and 3μM) as the final concentration for experimental validation and mechanism studies. The CL2355 strain of nematodes, transduced with a human Aβ gene, is commonly used in the study of AD-related diseases. According to the above protocol, CL2355 larvae from the same age were collected and treated with the final selected concentrations of CL. Because Aβ gene expression is only observed at specific temperatures, after CL administration, the larvae were transferred to a 25°C incubator and cultured for 24 hours to induce Aβ expression and test the therapeutic effects of CL. Before statistical analysis, a tropism plate was constructed. Two perpendicular lines were drawn at the bottom of the plate, with the center of the plate as the intersection point, dividing the plate into four quadrants. A circle with a radius of 10mm was then drawn at the center intersection point (to account for the influence of nematodes that were not moving well and did not escape the circle). The cultured nematodes were rinsed and approximately 50 nematodes were dripped into the center of the culture dish. Once the plate is dry and the nematodes are free to crawl, drop 1 μL of sodium azide in the center of each quadrant to anesthetize the nematodes. Also, drop 1 μL of 0.1% benzaldehyde ethanol solution in the centers of quadrants 1 and 3, and 1 μL of absolute ethanol in the centers of quadrants 2 and 4. The drop position should be approximately 30 mm from the center of the circle. Incubate at 20°C for 1 hour, and count the number of nematodes in each quadrant (excluding those in the small circle in the center). Figure 2 A).

[0041] Experimental results: Normal nematodes are attracted to the smell of benzaldehyde and tend to move towards it. Aβ toxicity damages the nematode's normal neurons, resulting in defects in the nematode's taxis, a neuron-controlled behavior. This defective behavior can be quantified by the chemotaxis index (CI). Compared with the control group, the reduced chemotaxis of CL2355 nematodes caused by Aβ peptide expression after treatment with different concentrations of CL was improved to a certain extent, and in a concentration-dependent manner ( Figure 2 B). Nematodes with normal neuronal function were closer to the benzaldehyde area and were more sensitive. Chemotaxis index (CI) = (the sum of the number of nematodes in quadrants 1 and 3 - the sum of the number of nematodes in quadrants 2 and 4) / total number of nematodes.

[0042] (7) Effect of CL on the lifespan of UM0001 strain of nematodes

[0043] Experimental Methods: This study used the double-transgenic nematode UM0001, transgenic for both human Tau protein and Aβ, as the research subject. The specific steps were as follows: L2 larvae, synchronized according to step 4, were collected and added to prepared drug-containing plates, ensuring that each group contained ≥100 nematodes. The larvae were incubated at 20°C for 24 hours, then transferred to 25°C for another 24 hours. After reaching adulthood, 60 nematodes were selected from each group and counted for survival until all nematodes died. Nematodes were considered dead if they did not move or retract when gently touched with the tip of an inoculating loop. Those that were lost, escaped, or died due to other reasons were not counted.

[0044] Experimental results: According to the experimental results, compared with the control group, the average lifespan of nematodes was prolonged after treatment with different concentrations of CL ( Figure 3 ), suggesting that CL has a certain therapeutic effect on nerve-damaged nematodes.

[0045] (8) Effect of CL on autophagy fluorescent spots in DA2123 strain of nematodes.

[0046] Experimental Methods: This experiment used the autophagy-fluorescing DA2123 nematode to examine the extent of CL-induced autophagy activation. This nematode stably expresses LGG-1::GFP in its muscle cells. When autophagy occurs in the nematodes, the LGG-1::GFP protein aggregates to form fluorescent spots, reflecting the occurrence of autophagy. L2 larvae obtained in step 4 above were collected and placed on drug-containing plates at 20°C until they became adults. Ten worms per group were placed on a glass slide containing an agar pad and anesthetized with a drop of NaN3. The worms were then covered with a coverslip and placed under an inverted microscope to observe the number of autophagic fluorescent spots.

[0047] Experimental results: Figure 4Compared with the blank control group, after treatment with different concentrations of CL, the autophagy fluorescent spots in DA2123 nematodes increased in a dose-dependent manner, indicating that CL can induce the occurrence of autophagy in DA2123 nematodes.

[0048] Example 3 Effects of CL on PD Model Caenorhabditis elegans

[0049] (1) Biomaterials

[0050]

[0051] (2) Reagents

[0052] Same as Example 2

[0053] (3) Implementation steps

[0054] The implementation steps (1)(2)(3)(4) are the same as those shown in Example 2.

[0055] (5) Fluorescence analysis of α-syn::YFP aggregation in NL5901 nematodes by CL

[0056] Experimental method: Larvae of the same age were collected according to Example 2, and the larvae were placed in a blank control group and plates containing different concentrations of CL, and incubated at 20°C for 48 h. After that, 10 larvae from each group were selected and placed on a glass slide. The green fluorescence accumulation in the nematode muscle tissue was observed under an inverted microscope, and statistical analysis was performed.

[0057] Experimental results: Figure 5 As shown in the figure, compared with the blank group, CL reduced the fluorescence intensity of YFP in the muscle cells of nematodes in a dose-dependent manner, indicating that CL can inhibit the aggregation of α-Syn in nematodes.

[0058] (6) CL fluorescence analysis of 6-OHDA-induced DA neuron degeneration in BZ555 nematodes

[0059] Experimental method: BZ555 nematode is a transgenic nematode whose dopaminergic neurons are marked with GFP, and its neuronal structure can be observed under a fluorescence microscope. Normally, there are 4 pairs of DA neurons in the nematode, of which there are three pairs in the head area. Therefore, changes in its neuronal structure can be observed through fluorescence examination. The present invention induces damage to its neurons through 6-OHDA, and then administers different concentrations of CL to restore the treatment. The nematodes were treated according to the method mentioned in Example 2 to obtain larvae of the same period, and added to the corresponding blank plates, drug-containing plates and 6-OHDA positive control plates respectively. After three days of treatment at 16°C, 10 nematodes from each group were picked and placed on a slide containing an agar pad, covered with a coverslip, and the integrity of the neurons of each group of nematodes was observed.

[0060] Experimental results: The results are as follows Figure 6 As shown in the figure, after three days of exposure to 6-OHDA, the DA neurons in the nematodes were damaged and the GFP fluorescence intensity decreased. After treatment with different concentrations of CL, the fluorescence showed that the damaged neurons in the nematodes recovered to varying degrees, and the recovery effect became more obvious with increasing drug concentration.

Claims

1. A use of CL in the preparation of a drug for treating and preventing Alzheimer's disease and Parkinson's disease, characterized in that: The structural formula of the CL is shown below: 。 2. The use according to claim 1, characterized in that The CL treats and prevents Alzheimer's disease and Parkinson's disease by improving the nematode's movement ability, extending lifespan, and restoring damaged neurons in the nematode.

3. The use according to claim 2, wherein The CL improves the nematode's locomotion ability, prolongs its lifespan, and restores damaged neurons in the nematode. The concentration range of CL is 0.75-3 μM.

4. The use according to claim 1, wherein The extraction process of CL is as follows: air-dried pond cypress leaves are crushed and extracted with 95% ethanol reflux three times to obtain a total extract; the total extract is extracted three times with petroleum ether, ethyl acetate, and n-butanol; the petroleum ether layer extract is loaded onto a silica gel column and eluted with a petroleum ether / ethyl acetate gradient to obtain Fr.1 to Fr.4; the obtained Fr.1 is loaded onto an ODS column and eluted with a methanol / water gradient to obtain compound CL.

5. The use according to claim 4, wherein During the reflux extraction process, the volume mass ratio of ethanol to pond cypress leaves was 10:1, each extraction lasted 2 h, the gradient elution volume ratio of petroleum ether / ethyl acetate was 100 / 0~0 / 100, and the volume ratio of methanol / water was 80:20~100:

0.

6. The use according to claim 4, characterized in that The volume ratio of petroleum ether / ethyl acetate for gradient elution was 100:25; the volume ratio of methanol / water was 100:15.

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