Use of cl in the preparation of a drug for preventing or treating depression, anxiety

Abstract

The application belongs to the technical field of medicines, and specifically discloses application of CL in preparation of medicines for preventing or treating depression and anxiety. Cycloneolitsol (CL) is a triterpene compound of the cyclalite class which is separated and extracted from the leaves of Podocarpus nryophyllus. Animal experiments prove that CL can effectively improve the depressive behavior of aging model mice and the anxiety behavior (open field and elevated plus maze) often accompanied by depression, can increase the staying time of mice in the center area in the open field experiment, and significantly increase the number of times of entering the open arm and the staying time in the target area open arm of the anxiety mice in the elevated plus maze experiment; and CL has no toxicity to the organs such as heart, liver, spleen, lung and kidney of mice, and the weight of mice does not decrease obviously, and the safety is relatively high. CL 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 application belongs to the technical field of medicine, and particularly relates to an application of CL in preparation of a medicine for preventing or treating depression and anxiety. BACKGROUND

[0002] Depression is a serious mental illness that endangers human physical and mental health, and its pathogenic factors are complex and diverse. Its pathogenesis needs to be further elucidated. Various studies have investigated a variety of hypotheses and mechanisms, such as the monoamine hypothesis, the inflammation hypothesis, the hypothalamic-pituitary-adrenal (HPA) axis hypothesis, the neural plasticity and neurogenesis hypothesis, the changes in norepinephrine, dopamine and glutamate systems, vascular modification, and genetic and environmental factors. Depression patients mainly show anhedonia, loss of excitement, and other different degrees of psychological and / or somatic disorders such as anxiety and sleep disorders.

[0003] Anxiety is a common mental illness, and is a neurotic disorder characterized by persistent anxiety or recurrent panic attacks. The anxiety of patients is not caused by real situations, and is often accompanied by autonomic dysfunction and motor unrest, and severe patients can have panic attacks. Some things or time can cause irrational fear due to fear of current events and worry about expected events, which affects the thoughts, feelings, abilities and behaviors of patients.

[0004] Currently, the drugs for treating depression can be divided into five categories according to the mechanism of action: A. tricyclic antidepressants; B. monoamine oxidase inhibitors (MAOIs); C. selective serotonin reuptake inhibitors (SSRIs); D. selective norepinephrine (NA) reuptake inhibitors; and E. serotonin and norepinephrine reuptake inhibitors. The common A-class antidepressants include imipramine, clomipramine, amitriptyline, doxepin, and protriptyline. The B-class antidepressants include moclobemide and toloxatone. The C-class antidepressants include fluoxetine, fluvoxamine, sertraline, citalopram, and escitalopram. The D-class antidepressants include maprotiline, mianserin, and reboxetine. The E-class antidepressants include venlafaxine and duloxetine. However, the existing antidepressants still have the disadvantages of slow effect, large individual difference, long effect, and large side effects.

[0005] CL is the first cycloartane triterpenoid compound isolated from Taxodiaceae, and mainly exists in some species of Taxodiaceae. Studies have found that cycloartane triterpenoids and their glycosides have good pharmacological activities, such as anti-tumor, anti-aging, and antibacterial activities. The cycloartane triterpenoid compound CL isolated from Taxodiaceae has not been reported to have an anti-depression and / or anti-anxiety effect. SUMMARY

[0006] In view of the problems in the background art, the purpose of the present application is to provide a new drug use of cycloartane compound CL, and it is found that CL has good effects in the preparation of drugs for preventing or treating depression and anxiety. Animal experiments prove that the pharmacological effects of CL are obvious, and CL can significantly improve the anxiety-like behavior of AD mice, significantly increase the number of times of entering the open arm and the time of staying in the open arm of the anxious mice; CL has no toxicity to the hearts, livers, spleens, lungs and kidneys of mice, and is relatively safe.

[0007] Specifically, the technical solutions of the present application are as follows:

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

[0009]

[0010] The CL of the present application can be obtained by separation and extraction from naturally dried medicinal Tsuga leaf, or can be obtained by chemical synthesis.

[0011] The extraction method of CL is as follows: the naturally dried Tsuga leaf is extracted by refluxing with 95% ethanol, and after removing the solvent, the crude extract is dissolved in water and then sequentially extracted. The obtained four components are separated by silica gel column chromatography (eluent is petroleum ether: ethyl acetate with a volume ratio of (100:0~0:100)); component 1 is separated and purified by silica gel column chromatography, and eluted and separated and purified by petroleum ether-ethyl acetate (100:5~3:1) to obtain compound CL at (100:15).

[0012] The volume-to-mass ratio of ethanol to the naturally dried Tsuga leaf is 1:10, and the extraction is performed for 3 times; the extraction is sequentially performed with petroleum ether, ethyl acetate and n-butanol.

[0013] The application of CL in the preparation of drugs for treating depression or anxiety is mainly based on the open field test and the elevated plus maze test, and it is found that CL has anti-depression and anti-anxiety activities.

[0014] Specifically, CL can significantly increase the frequency of mice entering the central region in the open field test, prolong the time of mice staying in the central region of the open field, and has no effect on the total movement distance of mice;

[0015] CL can significantly increase the number of times of mice entering the open arm in the elevated plus maze test, and increase the time of mice staying in the open arm region;

[0016] In addition, it is found by H&E staining that CL has no obvious effect on the organs (heart, liver, spleen, lung and kidney) of mice, has no toxic side effects, and is relatively safe.

[0017] The technical effect of the present application: through the mouse elevated plus maze experiment, mouse open field experiment and the like, it is proved that CL has significant anti-depression, anti-anxiety and the like effects. The present application provides a new Chinese medicine monomer component drug for preventing and treating anti-depression, anxiety and the like mental disorder diseases, and broadens the disease prevention and treatment range of CL. Due to the advantages of wide source and low toxicity of CL, the present application has good application and development prospects in the preparation of drugs for preventing, relieving or treating depression, anxiety and the like mental disorder diseases. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 Influence of CL on the number of times of entering the open arm of mice in the elevated plus maze experiment; ### represents p<0.0001, that is, there is a difference compared with the blank group, and it is statistically significant. *** represents p<0.0001, that is, there is a significant difference compared with the model group, and it is statistically significant (CL-L and CL-H respectively refer to low concentration and high concentration).

[0019] Figure 2 Influence of CL on the time of mice staying in the target area in the elevated plus maze experiment; ### represents p<0.0001, that is, there is a difference compared with the blank group, and it is statistically significant. *** represents p<0.0001, that is, there is a significant difference compared with the model group, and it is statistically significant.

[0020] Figure 3 Influence of CL on the time of mice staying in the center in the open field experiment; ### represents p<0.0001, that is, there is a difference compared with the blank group, and it is statistically significant. *** represents p<0.0001, that is, there is a significant difference compared with the model group, and it is statistically significant.

[0021] Figure 4 Influence of CL on the number of times of entering the center of mice in the open field experiment; ### represents p<0.0001, that is, there is a difference compared with the blank group, and it is statistically significant. *** represents p<0.0001, that is, there is a significant difference compared with the model group, and it is statistically significant.

[0022] Figure 5 H&E staining is used to evaluate the influence of CL on the heart, liver, spleen, lung and kidney of mice. DETAILED DESCRIPTION

[0023] The present application is further illustrated in detail through the following examples, but it should be noted that the scope of the present application is not limited by any of these examples.

[0024] Example 1: Preparation process of CL

[0025] The 10 kg of air-dried leaves of Taxodium ascendens Brongn were extracted with 10 L of 95% ethanol under reflux (2 h) for three times. After removing the solvent, the crude extract was dissolved in water (1 : 1), and then extracted with petroleum ether, ethyl acetate and n-butanol successively. The petroleum ether fraction (280 g) obtained was subjected to silica gel column chromatography with petroleum ether-ethyl acetate (100:0~0:100) as eluent to obtain four fractions. Fraction 1 (20 g) obtained at 100:25 was further purified by column chromatography with petroleum ether-ethyl acetate (100:5~3:1) as eluent to obtain compound CL (120 mg) at 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 Nantong University Experimental Animal Center.

[0029] 2. Experimental method: After one week of adaptive feeding, the mice were randomly divided into four groups, 8 in each group. One group was the normal blank group, without any treatment. The other three groups of mice were induced to establish a senile model by using aluminum chloride / D-galactose / sodium nitrite (AlCl3 / D-gal / NaNO2) triple combination, in which the mice were given AlCl3(40 mg / kg) solution by gavage, and D-gal (150 mg / kg) / NaNO2(70 mg / kg) solution by subcutaneous injection, for 4 weeks. After 4 weeks, the model mice were divided into model group, CL low concentration (CL-L 2 mg / kg) and high concentration (CL-H 8 mg / kg) drug groups. The drug groups were given corresponding drugs by gavage according to body weight, and the normal group and the model group were given normal saline by gavage, once a day, with a 12h:12h day-night cycle, and the behavior was detected after 2 weeks of continuous administration.

[0030] 3. Elevated plus maze test:

[0031] (1) Device preparation: The test device consists of four arms, each 25x5 cm, extending from a 5x5 cm central cross platform. Among them, two opposite arms are surrounded by 20 cm opaque high wall to become two closed arms, and the ground of the whole cross maze is 40 cm.

[0032] (2) Implementation steps: place each mouse facing the open arm at the center intersection of the elevated plus maze, and record the free activity trajectory of the mouse within 5 minutes using the ANY-maze analysis system. Analyze the number of times the mouse enters the open arm and the closed arm, the residence time in the open arm and the closed arm, and calculate the residence time in the open arm, the number of entries, etc. The residence time ratio in the open arm (%) = residence time in the open arm / (residence time in the open arm + residence time in the closed arm) x 100%, and the residence time ratio in the open arm (%) = residence time in the open arm / (residence time in the open arm + residence time in the closed arm) x 100%. If the number of times and the residence time of the mouse entering the open arm increase, it indicates that the drug has anxiolytic effect.

[0033] (3) Results analysis: the results of the elevated plus maze experiment are shown in Figure 1 、 2 ) shows that the residence time of the model group mice in the open arm and the number of times entering the open arm are significantly lower than those of the normal group, which has statistical significance. Compared with the model group, the CL administration group can significantly increase the residence time of the model mice in the open arm and the number of times entering the open arm, and it is concentration-dependent.

[0034] Example 3 Open field test

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

[0036] 1. Open field test detection:

[0037] (1) Open field test (Open field test, OFT) is also called open field test, which is a method for evaluating the voluntary movement behavior, exploratory behavior and tension of rodents. It has been widely used in the research of Parkinson's disease, depression, anxiety and other mental diseases. The anxiety level of experimental animals is observed through the open field test, among which the horizontal activity and central residence time reflect the anxiety situation. Generally speaking, the mice with anxiety-like behavior have more obvious thigmotaxis (like to stick to the wall), and the mice with lower anxiety degree tend to stay in the central area of the open field, i.e. the open area for more time.

[0038] (2) Implementation steps: after the experimental device is ready, the experimental mouse is taken out from the feeding cage, placed in the central area of the open field with its back to the experimenter, and the experimenter quickly leaves, so that the mouse can freely move in the experimental box. The ANY-maze collection and analysis system is used to record and analyze the spontaneous activity of the mouse within 15 minutes, the residence time in the central area and the marginal area, and the movement distance. After the experimental monitoring time is over, the mouse is taken out and put back into the feeding cage. The bottom of the open field reaction box, the placed object and the side wall are thoroughly cleaned to prevent the information left by the animal from affecting the test results of the next test. Most studies use ethanol for cleaning, and the next animal is tested after the ethanol is completely volatilized.

[0039] (3) Result analysis: The open field test results showed that, as shown in ( Figure 3 、 4 ), compared with the normal group, the number of times of entering the central area and the time of staying in the central area of the model group mice were reduced. The CL drug treatment group can significantly increase the number of times of entering the central area of the model mice; increase the time of staying in the central area of the depressed mice, and show a concentration-dependent effect, and the effect of the high-dose group is better.

[0040] Example 4 Pathology Staining Experiment

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

[0042] 1. Experimental principle:

[0043] H&E staining is a commonly used histological staining method, which 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 blue, and eosin can make the cytoplasm red, so as to realize the localization and identification of 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 euthanized by dislocation, and the mouse heart, liver, spleen, lung, kidney and other organs were collected, washed with pre-cooled physiological saline, and the water was absorbed with filter paper.

[0046] (2) Tissue fixation: Place the tissue block in 4% formaldehyde solution for 24 h to fix the shape unchanged.

[0047] (3) Tissue dehydration: Place the fixed tissue block in 50%, 70%, 80%, and 90% alcohol solution for 1 h each time.

[0048] (4) Transparency and wax immersion: Place the dehydrated tissue block in xylene solution for transparency 2 times, 15 min each time, and then place it in paraffin solution for wax immersion 2 times, 1 h each time.

[0049] (5) Sectioning and patching: Paraffin section the wax-impregnated tissue with a thickness of 5 μm, and place the section on the sectioning rack and dry.

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

[0051] (7) Mounting and observation: Mount the stained section with neutral gum, then observe and record the results with an optical microscope.

[0052] 3. Result analysis: The H&E staining results are as shown in ( Figure 5) As shown in Figure 6, paraffin sections of mouse heart, liver, spleen, lung and kidney 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 were arranged in bundles, the nuclei of liver cells were round or oval, the lymphocytes and macrophages of the spleen were densely distributed, the alveolar epithelial cells were arranged in a flat shape in the alveolar cavity, and the glomerular and renal tubular structures of the kidney were clearly visible. The above results show that compared with the blank group mice, the organs of the model group mice did not have obvious pathological changes, and the CL administration treatment group also had no obvious difference compared with the model group, indicating that CL has no toxicity to the heart, liver, spleen, lung and kidney organs, and has high safety.

Claims

1. Use of CL for the preparation of a medicament for preventing or treating senium-related anxiety, characterized in that, The chemical structural formula of the CL is: 。 2. Use according to claim 1, wherein The CL is extracted from air-dried Podocarpus neriifolius leaves or obtained by chemical synthesis.

3. The use according to claim 1, wherein The extraction method of the CL is as follows: air-dried Podocarpus neriifolius leaves are extracted by refluxing with ethanol, after removing the solvent, the crude extract is dissolved in water, and then sequentially extracted; silica gel column chromatography is used for separation to obtain four components; component 1 obtained is separated and purified by silica gel column chromatography to obtain the compound CL.

4. The use according to claim 3, wherein the compound is ###0002### The concentration of ethanol used for reflux extraction is 95%, the volume-mass ratio of ethanol to air-dried Podocarpus neriifolius leaves is 1:10, and the extraction times is 3.

5. The use according to claim 3, wherein the compound is ###0002### The extraction is sequentially performed with petroleum ether, ethyl acetate and n-butanol; when silica gel column chromatography is used for separation to obtain four components, elution is performed with 100:0~0:100 petroleum ether-ethyl acetate; when separation and purification are performed, the eluent is petroleum ether: ethyl acetate with a volume ratio of 100:5~3:

1.

6. The use according to claim 1, wherein The drug prepared by using the CL has the following properties: (1) in the open field experiment, it can significantly increase the frequency of mice entering the central area, prolong the time of mice in the central area of the open field, and does not affect the total movement distance of mice; (2) in the elevated plus maze experiment, it can significantly increase the number of times of mice entering the open arm and increase the time of staying in the target area; (3) in the H&E staining pathological detection, it has no obvious effect on the heart, liver, spleen, lung, kidney and other organs of mice, and has no toxic side effects.

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