Application of CL in medicine for treating and preventing Alzheimer's disease and Parkinson's disease

Abstract

The invention belongs to the technical field of medicines, and particularly discloses application of CL in medicines for treating and preventing Alzheimer's disease and Parkinson's disease. Researches confirm that CL can significantly improve the athletic ability and chemical tendency ability of Alzheimer's disease caenorhabditis elegans; the service life of the Alzheimer's disease caenorhabditis elegans can be obviously prolonged; the CL can significantly recover damaged neurons of the Parkinson's Caenorhabditis elegans, which prompts that the CL has the potential of preparing drugs for treating 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 (ND) are neurological diseases characterized by progressive loss of the central nervous system or peripheral nervous system, including the common Alzheimer's disease (AD), Parkinson's disease (PD), and multiple sclerosis (MS). With the increasing global population aging, the incidence of ND continues to rise, bringing great burdens to society and families. Therefore, many researchers have developed animal models to screen potential new drugs for the treatment of ND. Caenorhabditis elegans (nematodes) is widely used in the screening of potential clinical drugs for ND because of its clear genetic background, small size, short life cycle, unique pathological behavior, 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, but these two proteins have been identified as the key to treating AD. Therefore, researchers used human Aβ gene and human Tau protein gene to establish a transgenic AD nematode model to evaluate AD disease in a direct or indirect way, such as directly observing the loss process of neurons, impaired movement and shortened lifespan by mediating fluorescent proteins.

[0004] PD is a common disease in middle-aged and elderly people. With increasing age, the incidence rate increases and is slightly higher in men than in women. The recognized causes of PD are mainly the reduction of dopaminergic neurons (DA) and the aggregation of Lewy bodies (LB). Dopamine neuron damage models caused by chemical drugs and transgenic Lewy body aggregation models have been successfully prepared in nematodes. 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 also one of the main targets for the treatment of PD today. 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 view of the technical problems in the background technology, the purpose of the present invention is to provide a new drug use of the pool cypress extract CL, 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 of the pool cypress extract CL is as follows: the air-dried pool cypress leaves are crushed, and refluxed with 95% ethanol for 3 times (ethanol: pool cypress leaves = 10:1), each time for 2 hours, to obtain the total extract. The total extract is extracted three times with petroleum ether, ethyl acetate, and n-butanol. The petroleum ether layer extract is loaded on a silica gel column, and gradient eluted with petroleum ether / ethyl acetate (100 / 0 to 0 / 100) to obtain Fr.1 to Fr.4; the obtained Fr.1 (100:25, 20g) is loaded on an ODS column, and gradient eluted with 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 of the present invention is mainly based on the discovery that CL has a neuroprotective effect through 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 movement ability of nematodes, extend lifespan, and restore damaged neurons in nematodes. The concentration range of CL is: 0.75-3μM.

[0015] Technical effects of the present invention: Through the basic slow reaction experiment of Caenorhabditis elegans, tropism experiment, lifespan experiment, fluorescence quantitative experiment, etc., it is proved that CL has significant efficacy in treating and preventing neurodegenerative diseases. The present invention provides a new Chinese medicine monomer component drug for treating and preventing neurodegenerative diseases, which broadens the scope of CL in preventing and treating diseases. Due to the advantages of CL such as wide sources and low toxicity, it has good application development prospects in the treatment and prevention of neurodegenerative diseases such as AD and PD. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0017] Figure 2 Effect 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 Restoration effect of CL on 6-OHDA-induced DA neurons in BZ555 nematodes. DETAILED DESCRIPTION

[0022] The present invention is further described 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 of the pool cypress extract CL is as follows: 10 kg of air-dried pool cypress leaves are crushed and extracted with 95% ethanol reflux for 3 times (ethanol: pool cypress leaves (volume-to-mass ratio) = 10:1), each time for 2 hours, to obtain 2 kg of total extract. The total extract is extracted three times with petroleum ether, ethyl acetate, and n-butanol. The petroleum ether layer extract (280 g) is loaded on a silica gel column and gradient eluted with petroleum ether / ethyl acetate (100 / 0 to 0 / 100) to obtain Fr.1 to Fr.4; the obtained Fr.1 (100:25, 20 g) is loaded on an ODS column and gradient eluted with methanol / water (80:20 to 100:0) to obtain 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] (II) Reagents

[0029]

[0030]

[0031] (III) Implementation steps

[0032]

[0033] (4) Nematode growth cycle and synchronization

[0034] Caenorhabditis elegans is a hermaphroditic model organism. After its individual eggs are laid and hatched, they go through four larval stages (L1-L4). When the population is crowded or food is insufficient, the nematodes will enter another larval stage, called dauer larvae. Dauer larvae can resist adversity and do not age. Generally, eggs are laid during the adult stage, and drugs are used for treatment in the L2 stage.

[0035] Synchronization of nematodes: In order 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 a NGM medium with sufficient food and no pollution, then place the culture dish in a 16°C constant temperature incubator until they lay eggs. After most of the adults lay eggs, rinse the nematodes three times with nematode buffer M9 (4500rpm, 2min), remove the supernatant, add 1mL of lysis solution prepared according to the above reagent formula, shake with a vortex shaker to mix thoroughly, centrifuge (4500rpm, 2min), 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 a clean NGM medium with sufficient food, and place it in a 16°C incubator until the eggs hatch, and you can get larvae of the same period.

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

[0037] Experimental method: The present invention uses the BR5270 strain of nematodes transfected with human Tau protein as an AD model for drug activity screening, and BR5271 as a positive control. According to the above synchronization method, BR5270 larvae grown to the L2 stage are collected, added to the above prepared drug-containing plates (0.375μM, 0.75μM, 1.5μM, 3μM, 6μM), placed in a 16°C incubator for 72h to observe the on-site growth. When grown to adults, 30 nematodes are selected from each group to observe the head swing frequency of the nematodes within 30s.

[0038] Experimental results: Compared with the blank group, after treatment with different concentrations of CL, the movement ability 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) Effect of CL on the chemotaxis of benzaldehyde in nematodes of strain CL2355

[0040] Experimental method: According to the above experimental results, the present invention finally selected CL (0.75μM, 1.5μM, 3μM) as the final concentration for experimental verification and mechanism research. The CL2355 strain of nematodes causes the transfer of human Aβ genes and is often used in the study of AD-related diseases. According to the above scheme, the present invention collects CL2355 larvae at the same period and gives the final selected different concentrations of CL intervention. Since the Aβ gene is only expressed at a specific temperature, the larvae need to be transferred to a 25℃ constant temperature incubator for 24h after CL is given to induce Aβ expression and detect the therapeutic effect of CL. Before statistical results, draw a tropism plate, take the center of the plate as the intersection, draw two perpendicular lines at the bottom, and divide the entire plate into four quadrants. Then draw a circle with a radius of 10mm with the center intersection as the dot (excluding the influence of nematodes with poor movement that have not been out of the circle). Rinse the above-mentioned cultured nematodes and drip about 50 nematodes into the center of the culture dish. When the plate is dry and the nematodes can crawl freely, drop 1 μL of sodium azide in the center of each quadrant to anesthetize the nematodes, and drop 1 μL of 0.1% benzaldehyde ethanol solution in the center of quadrants 1 and 3, and drop 1 μL of anhydrous ethanol in the center of quadrants 2 and 4. The drop position should be about 30 mm away from the center of the circle. Incubate at 20°C for 1 hour, and count the number of nematodes in each quadrant (excluding the nematodes in the small circle in the center) ( Figure 2 A).

[0041] Experimental results: Normal nematodes will be attracted by the smell of benzaldehyde and tend to move towards it. Aβ toxicity damages the normal neurons of nematodes, resulting in defects in the nematode behavior controlled by neurons - chemotaxis. 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 it was concentration-dependent ( Figure 2 B). The nematodes with normal neuronal function were close 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) / the total number of nematodes.

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

[0043] Experimental method: The present invention uses the double transgenic nematode UM0001 with human Tau protein and Aβ as the research object, and the specific steps are as follows: collect the synchronized L2 larvae according to the implementation step 4, add them to the prepared drug-containing plate, ensure that each group of nematodes is ≥100, put them in 20℃ for incubation for 24h, and then transfer them to 25℃ for incubation for 24h. After they grow to adults, pick 60 nematodes from each group to count the number of surviving nematodes until all nematodes die. When the nematodes are lightly touched with the end of the inoculation loop, they are recorded as dead if they do not move and cannot shrink. Loss, escape or other causes of death are 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 method: This experiment uses autophagy fluorescent DA2123 nematodes to detect the degree of activation of autophagy by CL. The nematodes stably express LGG-1::GFP in muscle cells. When autophagy occurs in the body, the LGG-1::GFP protein in the nematodes aggregates to form fluorescent spots, reflecting the occurrence of autophagy in the nematodes. Collect the L2 larvae obtained in the above-mentioned step 4, place them in a drug-containing plate and culture them at 20°C until they become adults. Pick 10 nematodes from each group on a slide containing an agar pad, drop a drop of NaN3 to anesthetize them, cover them with a coverslip and place them under an inverted microscope to observe the number of autophagy fluorescent spots in the nematodes.

[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] (II) Reagents

[0052] Same as Example 2

[0053] (III) 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: According to Example 2, larvae of the same period were collected, and the larvae were placed in a blank control group and plates containing different concentrations of CL, and cultured at 20°C for 48 h. After that, 10 larvae were selected from each group and placed on a glass slide. The green fluorescence aggregation in the muscle tissue of the nematodes was observed under an inverted microscope, and statistical analysis was performed.

[0057] Experimental results: Figure 5 As shown, 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 with dopaminergic neurons marked by GFP, and its neuronal structure can be observed under a fluorescence microscope. Usually, there are 4 pairs of DA neurons in the nematode, of which there are three pairs in the head area. Therefore, the changes in its neuronal structure can be observed through fluorescence examination. The present invention induces damage to its neurons through 6-OHDA, and then gives different concentrations of CL to restore the treatment. The nematodes are treated according to the method mentioned in Example 2 to obtain larvae of the same period, and are 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 on a slide containing an agar pad, covered with a coverslip and observed for the integrity of the neurons of each group of nematodes.

[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 of 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 had recovered to varying degrees, and the recovery effect became more obvious as the concentration of the drug increased.

Claims

1. A use of CL in 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, characterized in that The CL improves the locomotor ability of nematodes, prolongs lifespan, and restores damaged neurons of nematodes. The concentration range of CL is 0.75-3 μM.

4. The use according to claim 1, characterized in that The extraction process of CL is as follows: the air-dried leaves of pond cypress are crushed, and reflux extracted with 95% ethanol for 3 times to obtain a total extract; the total extract is extracted with petroleum ether, ethyl acetate and n-butanol for 3 times; the petroleum ether layer extract is loaded on a silica gel column, and gradient eluted with petroleum ether / ethyl acetate to obtain Fr.1 to Fr.4; the obtained Fr.1 is loaded on an ODS column, and gradient eluted with methanol / water to obtain compound CL.

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