Application of CL in preparation of medicine for preventing or treating depression and anxiety

Abstract

The invention belongs to the technical field of medicines, and particularly discloses application of CL in preparation of medicines for preventing or treating depression and anxiety. Animal experiments prove that the CL can effectively improve depression behaviors of senescence model mice and anxiety behaviors (open field and elevated field) frequently accompanied by depression, can prolong the residence time of the mice in a central area in an open field experiment, and can improve the depression behavior of the mice in an elevated cross maze experiment. The number of times that the anxiety mouse enters the open arm and the time that the anxiety mouse stays in the open arm in a target area are obviously increased; the composition has no toxicity to organs such as heart, liver, spleen, lung, kidney and the like of a mouse, the weight of the mouse is not obviously reduced, and the safety is higher. The compound can be used for preparing medicines for preventing or treating depression and anxiety, and has a wide application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and in particular relates to an application of CL in preparing a drug for preventing or treating depression and anxiety. Background Art

[0002] Depression is a mental illness that seriously endangers human physical and mental health. Its etiology is complex and diverse, and its pathogenesis needs to be further elucidated. Various studies have investigated a variety of hypotheses and mechanisms, such as the monoamine hypothesis, the inflammatory hypothesis, the hypothalamus-pituitary-adrenal (HPA) axis hypothesis, the neuroplasticity and neurogenesis hypothesis, changes in the noradrenergic, dopaminergic and glutamatergic systems, vascular modifications, and genetic and environmental factors. Depression patients mainly show lack of pleasure and excitement, accompanied by anxiety, sleep disorders and other psychological and (or) physical disorders of varying degrees.

[0003] Anxiety disorder is a common mental illness. It is a neurotic disorder characterized by widespread and persistent anxiety or recurrent panic attacks. The patient's anxiety is not always caused by the actual situation. It is often accompanied by autonomic dysfunction and motor restlessness. In severe cases, panic attacks may occur. Because of the fear of current events and the worry about expected events, irrational fears of certain things or times may occur, affecting the patient's thoughts, feelings, abilities and behaviors.

[0004] At present, the drugs used to treat depression can be divided into five categories according to their mechanism of action: A. tricyclic antidepressants; B. monoamine oxidase inhibitors (MAOI); C. selective 5-hydroxylamine reuptake inhibitors (SSR I); D. selective norepinephrine (NA) reuptake inhibitors; E. 5-hydroxytryptamine and norepinephrine reuptake inhibitors. At present, the common A-type antidepressants include imipramine, clomipramine, amitriptyline, doxepin, protriptyline, etc. The B-type antidepressants include moclobemide and toloxatone, etc. The C-type antidepressants include fluoxetine, chlorfluazol, sertraline, citalopram, escitalopram, etc. The D-type antidepressants include maprotiline, amoxapine, reboxetine, etc. The E-type antidepressants include venlafaxine and duloxetine, etc. However, the existing antidepressants still have shortcomings such as slow onset, large individual differences, long efficacy, and large side effects.

[0005] CL is the first cycloartin triterpenoid isolated from the Taxaceae family, and is mainly found in certain species of Taxaceae plants. Studies have found that cycloartin triterpenoids and their glycosides have good pharmacological activities, such as anti-tumor, anti-aging, and antibacterial. There are currently no reports on the anti-depression and / or anxiety effects of CL, a cycloartin triterpenoid isolated from Taxaceae plants. Summary of the invention

[0006] In view of the problems in the background technology, the purpose of the present invention is to provide a new drug use of a cycloartin compound CL, and it is found that CL has a good effect in preparing drugs for preventing or treating depression and anxiety. Animal experiments have shown that CL has obvious pharmacological effects, significantly improves the anxiety-like behavior of AD mice, significantly increases the number of times anxious mice enter the open arm and the time they stay in the open arm; it has no toxicity to the heart, liver, spleen, lungs, kidneys and other organs of mice, and has high safety.

[0007] Specifically, the technical solution of the present invention is as follows:

[0008] Application of CL in the preparation of drugs for preventing and treating depression, anxiety and mental disorders, wherein CL is a compound of the following formula:

[0009]

[0010] The CL of the present invention can be separated and extracted from air-dried natural medicine pond cypress leaves, and can also be prepared by chemical synthesis.

[0011] The extraction method of CL is as follows: using 95% ethanol to reflux extract the air-dried leaves of the pond cypress, removing the solvent, dissolving the crude extract in water, and then extracting in sequence. Using silica gel column chromatography for separation (the eluent is petroleum ether: ethyl acetate with a volume ratio of (100:0 to 0:100)), 4 components are obtained; the obtained component 1 is separated and purified by silica gel column chromatography, and separated and purified by eluting with petroleum ether-ethyl acetate (100:5 to 3:1), and compound CL is obtained at (100:15).

[0012] The volume mass ratio of ethanol to air-dried pond cypress leaves was 1:10, and the extraction times were 3 times; the extraction was performed with petroleum ether, ethyl acetate and n-butanol in sequence.

[0013] The invention discloses an application of CL in preparing a drug for treating depression or anxiety. The open field test and the elevated plus maze test mainly show that CL has anti-depressant and anti-anxiety activities.

[0014] Specifically, CL can significantly increase the frequency of mice entering the central area in the open field test and prolong the time mice spend in the central area of ​​the open field without affecting the total movement distance of the mice;

[0015] CL can significantly increase the number of times mice enter the open arms and the time they stay in the open arms in the elevated plus maze test;

[0016] In addition, H&E staining revealed that CL had no significant effect on the mouse organs (heart, liver, spleen, lungs, and kidneys), had no toxic side effects, and was relatively safe.

[0017] The technical effect of the present invention is as follows: CL has significant anti-depressant and anti-anxiety effects as demonstrated by mouse elevated plus maze test and mouse open field test. The present invention provides a new Chinese medicine monomer component drug for the prevention and treatment of mental disorders such as depression and anxiety, and broadens the range of CL for the prevention and treatment of diseases. Due to the advantages of CL such as wide sources and low toxicity, it has good application development prospects in the preparation of drugs for the prevention, relief or treatment of mental disorders such as depression and anxiety. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Effect of CL on the number of times mice enter the open arm in the elevated plus maze test; ### indicates p < 0.0001, that is, there is a difference compared with the blank group, which is statistically significant. *** indicates p < 0.0001, that is, there is a significant difference between the drug-treated group and the model group, which is statistically significant (CL-L and CL-H refer to low concentration and high concentration, respectively).

[0019] Figure 2 Effect of CL on the time mice stay in the target area in the elevated plus maze test; ### indicates p < 0.0001, which means there is a statistically significant difference compared with the blank group. *** indicates p < 0.0001, which means there is a statistically significant difference between the drug group and the model group.

[0020] Figure 3 Effect of CL on the time mice stay in the center in the open field test; ### indicates p < 0.0001, which means there is a statistically significant difference compared with the blank group. *** indicates p < 0.0001, which means there is a statistically significant difference between the drug group and the model group.

[0021] Figure 4 Effect of CL on the number of times mice enter the central area in the open field test; ### indicates p < 0.0001, which means there is a statistically significant difference compared with the blank group. *** indicates P < 0.0001, which means there is a statistically significant difference between the drug group and the model group.

[0022] Figure 5 H&E staining was used to evaluate the effects of CL on the heart, liver, spleen, lungs, and kidneys of mice. DETAILED DESCRIPTION

[0023] 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.

[0024] Example 1 Preparation process of CL

[0025] 10 kg of air-dried pond cypress leaves were extracted three times with 10 L of 95% ethanol under reflux (2 h). After removing the solvent, the crude extract was dissolved in water (1:1), and then extracted with petroleum ether, ethyl acetate and n-butanol in sequence; the petroleum ether portion (280 g) obtained by extraction was eluted with petroleum ether-ethyl acetate (100:0-0:100) on a silica gel column to obtain 4 components; component 1 (20 g) obtained at 100:25 was separated and purified again by column chromatography using petroleum ether-ethyl acetate (100:5-3:1) as an eluent, and compound CL (120 mg) was obtained under the elution condition of 100:15.

[0026] Example 2 Elevated plus maze test

[0027] 1. Experimental materials:

[0028] Mice: SPF male C57BL / 6 (8 weeks old), weighing about 20-25 g, provided by the Experimental Animal Center of Nantong University.

[0029] 2. Experimental methods: After one week of adaptive feeding, the mice were randomly divided into 4 groups, 8 in each group. One group was a normal blank group without any treatment. The other three groups of mice were induced by aluminum chloride / D-galactose / sodium nitrite (AlCl3 / D-gal / NaNO2) triple induction to establish an aging model, in which the mice were gavaged with AlCl3 (40 mg / kg) solution and subcutaneously injected with D-gal (150 mg / kg) / NaNO2 (70 mg / kg) solution for 4 consecutive weeks; after 4 weeks, the modeling mice were divided into a model group, a low concentration of CL (CL-L 2 mg / kg), and a high concentration (CL-H 8 mg / kg) administration group. The drug administration group was gavaged with the corresponding drugs according to body weight, and the normal group and the model group were gavaged with physiological saline once a day, with a 12h:12h day and night cycle, and behavioral tests were performed after two consecutive weeks.

[0030] 3. Elevated plus maze test:

[0031] (1) Device preparation: The test device consists of four arms, each 25×5 cm, extending outward from a 5×5 cm central cross platform. Two opposite arms are surrounded by a 20 cm opaque high wall to form two closed arms. The entire cross maze has a floor area of ​​40 cm.

[0032] (2) Implementation steps: Place each mouse facing the open arm at the central intersection of the elevated plus maze, and use the ANY-maze analysis system to record the free movement trajectory of the mouse within 5 minutes. Analyze the number of times the mouse enters the open arm and the closed arm, and the time the mouse stays in the open arm and the closed arm, and calculate the open arm retention time, number of entries, etc. The proportion of open arm retention time (%) = open arm retention time / (open arm retention time + closed arm retention time) × 100%, the proportion of open arm retention times (%) = open arm retention times / (open arm retention times + closed arm retention times) × 100%. If the number of times the mouse enters the open arm and the retention time increase, it shows that the drug has an anti-anxiety effect.

[0033] (3) Result analysis: The results of the elevated plus maze test are as follows ( Figure 1 , 2 ) showed that the time the model group mice stayed in the open arm and the number of times they entered the open arm were significantly lower than those in the normal group, which was statistically significant. Compared with the model group, the CL administration group could significantly increase the time the model mice stayed in the open arm and the number of times they entered the open arm in a concentration-dependent manner.

[0034] Example 3 Open field experiment

[0035] The experimental animals and experimental methods are the same as in Example 2

[0036] 1. Open field test:

[0037] (1) The open field test (OFT), also known as the open box test, is a method for evaluating the autonomous movement behavior, exploratory behavior and tension of experimental rodents. It has been widely used in the study of mental diseases such as Parkinson's disease, depression and anxiety. The anxiety level of experimental animals is observed through the open field test. Among them, horizontal activity, central residence time and other indicators reflect anxiety. Generally speaking, mice with anxiety-like behavior have more obvious tactile tendencies (like to stick to the wall), and mice with lower anxiety levels tend to stay more time in the center of the open field, that is, the open area.

[0038] (2) Implementation steps: After the experimental device is installed, the experimental mouse is taken out of the cage and placed in the central area of ​​the open field with its back facing the experimenter. The experimenter leaves quickly, allowing the mouse to move freely in the experimental box. The ANY-maze acquisition and analysis system is used to record and analyze the spontaneous activities of the mouse within 15 minutes, as well as the residence time and movement distance in the central and edge areas. After the experimental monitoring time is over, the mouse is taken out and placed back in the cage. The bottom, placed objects and side walls of the open field reaction box are thoroughly cleaned to prevent the remaining information of the animal from affecting the next test result. Most studies use ethanol for cleaning, and wait for the ethanol to completely evaporate before testing the next animal.

[0039] (3) Result analysis: The results of the open field experiment show that ( Figure 3 , 4 ) showed that compared with the normal group, the number of times the model group mice entered the central area and the time they stayed in the central area were reduced. The CL drug treatment group can significantly increase the number of times the model mice entered the central area and increase the time the depressed mice stayed in the central area in a concentration-dependent manner, and the high-dose group has a better effect.

[0040] Example 4 Pathological staining experiment

[0041] The experimental animals and experimental methods are the same as those in Example 2.

[0042] 1. Experimental principle:

[0043] H&E staining is a commonly used histological staining method that uses two dyes, hematoxylin and eosin, to stain tissues, which can clearly show cell morphology and tissue structure. Among them, hematoxylin can make the cell nucleus appear blue, while eosin can make the cytoplasm appear red, thereby locating and identifying cells.

[0044] 2. Implementation steps:

[0045] (1) Preparation of pathological specimens: After the completion of the previous animal experiment, the mice were starved for 12 h and then killed by dislocation. The heart, liver, spleen, lungs, kidneys and other organs of the mice were collected, rinsed with pre-cooled saline, and dried with filter paper.

[0046] (2) Tissue fixation: Place the tissue block in 4% formaldehyde solution for 24 hours to keep its morphology unchanged.

[0047] (3) Tissue dehydration: Dehydrate the fixed tissue blocks in 50%, 70%, 80%, or 90% alcohol solutions for 1 hour each time.

[0048] (4) Clearing and wax immersion: The dehydrated tissue block was placed in a xylene solution for clearing twice, each time for 15 min, and then placed in a paraffin solution for wax immersion twice, each time for 1 h.

[0049] (5) Sectioning and pasting: The wax-soaked tissue is cut into paraffin blocks with a thickness of 5 μm and the sections are placed on a drying rack for drying.

[0050] (6) H&E staining: Place the sections in H&E staining solution for 10 min, and then rinse off the residual stain with water.

[0051] (7) Sealing and observation: The stained sections were sealed with neutral gum and then observed and recorded using an optical microscope.

[0052] 3. Result analysis: H&E staining results are as follows ( Figure 5), paraffin sections of mouse heart, liver, spleen, lung, kidney and other organs were observed by H&E staining, and the tissue structure and cell morphology of each organ were observed. Among them, the myocardial fibers of the heart are arranged in bundles, the nuclei of hepatocytes are round or oval, the lymphocytes and macrophages of the spleen are densely distributed, the alveolar epithelial cells are arranged in a flat shape in the alveolar cavity, and the glomerular and tubular structures of the kidneys are clearly visible. The above results show that compared with the blank group mice, the model group mice did not have obvious lesions in various organs, and there was no significant difference between the CL administration group and the model group, indicating that CL has no toxicity to the heart, liver, spleen, lung, kidney and other organs, and has a high safety.

Claims

1. A use of CL in the preparation of a drug for preventing or treating depression and anxiety, characterized in that: The chemical structural formula of the CL is:

2. The use according to claim 1, characterized in that The CL is extracted from air-dried pond cypress leaves or synthesized by chemical synthesis.

3. The use according to claim 1, characterized in that The extraction method of CL is as follows: extracting air-dried pond cypress leaves with ethanol reflux, removing the solvent, dissolving the crude extract in water, and then extracting in sequence; separating by silica gel column chromatography to obtain four components; separating and purifying the obtained component 1 by silica gel column chromatography to obtain compound CL.

4. The use according to claim 3, characterized in that The concentration of the ethanol used for the reflux extraction is 95%, the volume mass ratio of the ethanol to the air-dried pond cypress leaves is 1:10, and the extraction times are 3 times.

5. The use according to claim 3, characterized in that The extraction is performed by sequentially extracting with petroleum ether, ethyl acetate and n-butanol; separating by silica gel column chromatography, and eluting with 100:0-0:100 petroleum ether-ethyl acetate when four components are obtained; and the eluent for separation and purification is petroleum ether:ethyl acetate in a volume ratio of 100:5-3:

1.

6. The use according to claim 1, characterized in that The drug prepared by CL has the following properties: (1) In the open field test, it can significantly increase the frequency of mice entering the central area and prolong the time mice spend in the central area of ​​the open field without affecting the total movement distance of the mice; (2) In the elevated plus maze test, it can significantly increase the number of times mice enter the open arms and the time they stay in the target area; (3) In the H&E staining pathological test, there was no obvious effect on the mouse heart, liver, spleen, lungs, kidneys and other organs, and no toxic side effects.

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