Use of ganoderic acid A in preparation of anti-anxiety drugs

Abstract

The application provides application of ganoderic acid A in preparation of anxiolytic drugs and belongs to the technical field of medicines. The application first proposes that ganoderic acid A has an anxiolytic effect. It is verified through animal experiments that ganoderic acid A with a dose of 0.01 mg / kg-20 mg / kg can effectively improve anxiety of mice. The application further provides an anxiolytic drug. The active ingredient of the drug comprises ganoderic acid A, and the drug can effectively resist anxiety.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine, and particularly relates to application of ganoderic acid A in preparation of anxiolytic drugs. BACKGROUND

[0002] Anxiety disorder is a common mental disorder, a negative emotion, and subjective tension, worry and uneasiness of people when encountering uncertain things, and the autonomic nervous system is also overactive to cope with possible threats. It is divided into generalized anxiety disorder, social anxiety disorder, panic disorder, etc., among which, the generalized anxiety disorder (GAD) is the most common one, which is characterized by excessive worry about various things in life, accompanied by physical discomfort and uneasiness. Current research suggests that anxiety disorder is the mental disorder with the highest prevalence. The increase of life pressure and the acceleration of rhythm also make the number of people with anxiety disorder increasing. However, the pathogenesis of anxiety disorder is unknown, and the treatment drugs are limited. The first-line anxiolytic drugs include benzodiazepines, SSRIs and SNRIs, etc., but these drugs have certain side effects and limitations, such as addiction, slow onset, withdrawal reaction and drug resistance, etc. Therefore, it is necessary to find safer and more effective anxiolytic drugs.

[0003] Ganoderic acid A (GA-A) is a triterpenoid compound mainly isolated from Ganoderma lucidum, and is one of the main active ingredients in Ganoderma lucidum, which can be taken by humans for a long time, and its toxic and side effects have not been reported so far. Ganoderic acid A can also be extracted from cultured red algae and yeast, and has strong industrial practicability. At present, whether ganoderic acid A can be used for effective anxiolytic has not been reported. SUMMARY

[0004] Therefore, the purpose of the present application is to provide application of ganoderic acid A in preparation of anxiolytic drugs, and ganoderic acid A has obvious anxiolytic effect and is expected to become a high-efficiency anxiolytic natural extract.

[0005] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions.

[0006] The present application provides application of ganoderic acid A in preparation of anxiolytic drugs.

[0007] Preferably, the effective dose of ganoderic acid A is 0.01 mg / kg-20 mg / kg.

[0008] The present application also provides an anxiolytic drug, and the active ingredient of the drug comprises ganoderic acid A.

[0009] Preferably, the pharmaceutical dosage form comprises a powder, a granule, a tablet, a capsule, a pill, an oral liquid or an injection.

[0010] Preferably, the content of ganoderic acid A in the medicine is 0.1-99.9wt%.

[0011] Preferably, the medicine further comprises a pharmaceutically acceptable carrier.

[0012] Advantages of the present application:

[0013] The present application first proposes the application of ganoderic acid A in preparing anxiolytic medicine. Through mouse experiments, it is found that the dose of 2.5mg / kg-5mg / kg of ganoderic acid A can effectively improve the anxiety disorder of mice, effectively resist the anxiety of mice, and no tranquilization, epilepsy induction, ataxia and other shortcomings of conventional anxiolytic drugs are found in the test process, indicating that ganoderic acid A has obvious and non-toxic side effects of anxiolytic effect. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 is a structural formula of ganoderic acid A;

[0015] Figure 2 is an experimental technical roadmap of Example 1;

[0016] Figure 3 is the influence of ganoderic acid A injection on the open field experiment of mice;

[0017] Figure 4 is the influence of ganoderic acid A injection on the white and black box experiment of mice;

[0018] Figure 5 is the influence of ganoderic acid A injection on the elevated plus maze experiment of mice;

[0019] Figure 6 is the open field experiment result of the CRS modeling group and the normal control group;

[0020] Figure 7 is an experimental technical roadmap of Example 2;

[0021] Figure 8 is the influence of ganoderic acid A injection on the white and black box experiment of CRS modeling mice;

[0022] Figure 9 is the influence of ganoderic acid A injection on the elevated plus maze experiment of CRS modeling mice. DETAILED DESCRIPTION

[0023] The present application provides the application of ganoderic acid A in preparing anxiolytic medicine.

[0024] The ganoderic acid A described in this invention is a triterpenoid and a major compound in Ganoderma lucidum, which can be extracted from Ganoderma lucidum, red algae, or yeast. The structural formula of the ganoderic acid A described in this invention is as follows: Figure 1 As shown, the chemical formula is C 30 H 42 O7, with a molecular weight of 514.65, is a white crystalline powder. This invention does not specifically limit the source of ganoderic acid A; any commercially available product in the field is acceptable.

[0025] In this invention, the effective dose of ganoderic acid A for anti-anxiety is 0.01 mg / kg to 20 mg / kg, preferably 1 mg / kg to 10 mg / kg, and more preferably 2.5 mg / kg to 5 mg / kg.

[0026] This invention also provides an anti-anxiety drug, wherein the active ingredient of the drug includes ganoderic acid A. The active ingredient in the anti-anxiety drug of this invention may be ganoderic acid A as the sole active ingredient, or ganoderic acid A may be used in combination with other active ingredients that have anti-anxiety effects.

[0027] The drug dosage forms described in this invention can be liquid or solid, including but not limited to true solutions, colloids, microparticles, emulsions, suspensions, tablets, capsules, pellets, aerosols, pills, powders, solutions, suspensions, emulsions, granules, suppositories, and lyophilized powder injections. The drugs described in this invention also include pharmaceutically acceptable carriers. Pharmaceutically acceptable excipients include one or more of the following: diluents, colorants, sweeteners, coating agents, binders, absorbents, disintegrants, releasing agents, dispersants, humectants, solubilizers, buffers, and surfactants. In this invention, "pharmaceutically acceptable" excipients or salts are substances suitable for humans and / or animals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., substances with a reasonable benefit / risk ratio. This invention does not specifically limit other excipient components contained in the drug; commonly used pharmaceutical excipients in the art can be used. The drugs described in this invention can be administered orally, intraperitoneally, or by other means.

[0028] The content of ganoderic acid A in the drug of the present invention is 0.1-99.9 wt%, preferably 35-95 wt%, more preferably 50-85 wt%, and most preferably 65-80 wt%.

[0029] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0030] Unless otherwise specified, the following embodiments are all conventional methods.

[0031] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0032] In specific embodiments of the present application, the CAS number of ganoderic acid A used is: 81907-62-2; the molecular formula is: C 30 H 42 O7; purchased from Shanghai Changwei Industry Co., Ltd.

[0033] In specific embodiments of the present application, the experimental animals used are C57BL / 6 mice, male, weighing 20-26 grams, purchased from Vito Lihua Biotechnology Co., Ltd., license number: SCXK (Jing) 2021-0006. The mice are housed in cages, with a light-dark cycle of 12h / 12h, room temperature of 20-22℃, free access to water, and feed purchased from Hui Linzegu Experimental Animal Center.

[0034] Example 1

[0035] Experimental grouping: After one week of acclimation, the mice were randomly divided into 4 groups, 8 in each group: normal control group (CON), GA-A low-dose group (GA-A 2.5), GA-A high-dose group (GA-A 5), and imipramine group (IMI 15).

[0036] Drug injection and behavioral testing began on day 1: mice were injected intraperitoneally at 0.05 mL / 10g body weight, once a day at 10 am; the normal control group (CON) was injected with 1% DMSO, the GA-A low-dose group (GA-A 2.5) was injected with ganoderic acid A 2.5mg / kg, the GA-A high-dose group (GA-A 5) was injected with ganoderic acid A 5mg / kg, and the imipramine group (IMI 15) was injected with imipramine 15mg / kg. The mice in each group were subjected to the open field test 1h after administration on day 1, the black and white box test 1h after administration on day 4, and the elevated plus maze test 1h after administration on day 6. The brain was taken and stored after the elevated plus maze test. Figure 2 The experimental technical roadmap is shown in the figure, where OFT represents the open field test, LDT represents the black and white box test, and EPM represents the elevated plus maze test.

[0037] 1. Open field test in mice:

[0038] The mouse open field experiment was performed on day 1d. After intraperitoneal administration for 1 h, the mice were subjected to the open field experiment. Each mouse was tested once, and the mouse was placed in the center area of a black open square box (50 cm x 50 cm x 30 cm). The center area (25 cm x 25 cm) was defined as the center area, and the rest was the peripheral area. A camera was fixed above the box, and each mouse was tested for 10 min. After the test, the bottom of the box was cleaned with 75% alcohol to remove the excrement and traces left by the previous mouse, so as to avoid affecting the experimental results of the next mouse. During the experiment, the motion trajectory was recorded by the camera for later analysis of various indicators. The movement distance of the mouse in the center area was negatively correlated with the anxiety emotion, and the shorter the movement distance in the center area, the heavier the anxiety emotion of the mouse. Statistical method: the experimental results were expressed as mean ± SE, and multiple samples were tested by ANOVA.

[0039] The experimental results are shown in Figure 3 . The vertical coordinate in the figure: A represents the distance (m) moved in the center area, and B represents the total movement distance (m); the horizontal coordinate: group, from left to right, normal control group, GA-A low dose group (GA-A 2.5), GA-A high dose group (GA-A 5), imipramine group (IMI 15). *: p<0.05; **: p<0.01. As shown in Figure 3 , there was no statistical difference in the total movement distance and the movement distance in the center area of the 4 groups of mice (N=8).

[0040] 2, Mouse black and white box experiment

[0041] 3d after the end of administration, on the 4d, the black and white box experiment was performed. 1h after intraperitoneal administration, the mice were subjected to the black and white box experiment. The black and white box experiment model takes advantage of the characteristics of rodents that tend to be dark and afraid of light, and at the same time, they are curious about exploring new environments. The black and white box uses two square boxes, the cover above the box is equipped with a light source and a camera to simulate dark and light environments. There is a channel between the black and white boxes for the mouse to shuttle freely. Due to the mouse's tendency to be dark, the mouse tends to stay in the dark. The time spent in the white box can be used as an indicator of the mouse's anxiety state. The longer the time spent in the white box, the less anxious the mouse is. Before the experiment, the partition between the black and white boxes was closed to make the black and white boxes independent of each other, and the mouse could not move between the two boxes. During the experiment, the experimental environment was kept as quiet as possible. The experimental mouse was placed in the black box for 5 min to adapt to the environment. Then the partition between the black and white boxes was opened. The Any-maze software was used to record the video data for 5 min, and the time spent by the mouse in the white box was counted. After the experiment, the feces in the box were wiped clean with paper, and 75% alcohol was used for disinfection to eliminate the odor left by the previous animal. After the alcohol smell was removed, the next experiment was performed. Statistical method: the experimental results were expressed as mean ± SE, and multiple samples were tested by ANOVA.

[0042] The experimental results are shown in Figure 4 . The vertical axis of the graph: time spent in the white box (s), the horizontal axis: group, from left to right: normal control group, GA-A low dose group (GA-A 2.5), GA-A high dose group (GA-A 5), imipramine group (IMI 15). *: p<0.05; **: p<0.01. As shown in Figure 4 , the time spent in the white box by the four groups of mice (N=8), the low dose group of ganoderic acid A (GA-A) (62.20±6.00s) and the high dose group (79.70±11.95s) were longer than the normal control group (40.74±8.31s) (ANOVA, P<0.01, N=32), and the high dose group was longer than the low dose group in the white box (ANOVA Post Hoc Tukey, P<0.01). It shows that the low dose group of ganoderic acid A (GA-A) and the high dose group of ganoderic acid A (GA-A) both have anxiolytic effect, and the high dose group is more effective, better than imipramine.

[0043] 3, Elevated plus maze experiment of mice

[0044] 5d after the end of administration, on the 6th day, the mice were subjected to the elevated plus maze test 1 h after intraperitoneal administration. The mice were placed in the maze from the central square to the open arm at the beginning of the experiment, and the activity within 5 min was recorded using Any-maze software, mainly observing the time of the mice in the open arm. After the experiment, the mice were taken out, the two arms were cleaned, alcohol was sprayed to remove the odor, and the next mouse experiment was continued. The time of entering the open arm is negatively correlated with the anxiety of the mice, and the shorter the time, the more serious the anxiety of the mice. Statistical method: the experimental results are expressed by mean ± SE, and multiple samples are tested by ANOVA.

[0045] The experimental results are shown in Figure 5 . The vertical coordinate in the figure: open arm time (s) of the elevated plus maze experiment, the horizontal coordinate: group, from left to right: normal control group, GA-A low dose group (GA-A 2.5), GA-A high dose group (GA-A 5), imipramine group (IMI 15). *: p<0.05; **: p<0.01. As shown in Figure 5 , four groups of mice (N=8), the GA-A low dose group (38.63±7.95s) and the high dose group (44.77±6.69s) spent more time in the open arm than the normal control group (20.92±7.45s) (ANOVA, P<0.01, N=32), and the high dose group showed a more obvious trend than the low dose group. It shows that the low dose group of ganoderic acid A (GA-A) and the high dose group of ganoderic acid A (GA-A) have the effect of anti-anxiety, and the effect of the high dose group is more obvious, which is better than imipramine.

[0046] During the above experiment, compared with the imipramine control group, the GA-A low dose group and the GA-A high dose group did not show the shortcomings of imipramine such as sedation, epilepsy and ataxia.

[0047] Example 2

[0048] Chronic restraint stress (CRS) anxiety model and grouping treatment:

[0049] All CRS model mice were placed in a well-ventilated 50 ml centrifuge tube (with ventilation holes) for 2 h a day for 14 d, and the mice moved in the limited space in the tube, but no visible injury was caused. After the restraint was over, the mice were put back into the mouse cage for normal activity and diet.

[0050] The normal control group of mice was kept in a normal cage under normal conditions.

[0051] After modeling, the open field test was used to evaluate the anxiety level of the modeling group and the normal control group. The experimental method is the same as in Example 1, and the results are shown in Figure 6 . Figure 6In the figure, the vertical axis represents the time (s) spent in the center area, and the horizontal axis represents the group, from left to right, CON group, ANX group. Figure 6 It can be seen that the time spent in the center area of the modeling group (46.81±26.24s) is less than that of the normal control group (83.63±43.88s) (T-TEST, #: p<0.05), indicating that the CRS modeling is successful.

[0052] The modeling group was randomly divided into an anxiety group (ANX, N=4), a ganoderic acid A treatment group (5mg / kg, GA-A, N=6), a imipramine treatment group (15mg / kg, IMI, N=5), and a normal control group (1% DMSO, CON, N=4). The mice were injected intraperitoneally with 0.05mL / 10g of body weight once a day at 10am. The mice were still modeled for 2h every day during the medication period. The technical route is shown in Figure 7 . Figure 7 In the figure, OFT represents the open field test, LDT represents the black and white box test, and EPM represents the elevated plus maze test.

[0053] 3d After the end of administration, the black and white box test was performed on the 4th day. The mice were injected intraperitoneally with the drug 4h before the black and white box test, and the test method was the same as in Example 1. The test results are shown in Figure 8 . Figure 8 In the figure, the vertical axis represents the time (s) spent in the center area, and the horizontal axis represents the group, from left to right, CON group, ANX group, GA-A group, and IMI group. *: p<0.05; **: p<0.01; #: p<0.05. By Figure 8 It can be seen that the time spent in the center area of the GA-A group (74.13±37.83s), the CON group (73.35±11.52s), and the IMI group (52.22±16.96s) is longer than that of the ANX group (24.35±14.83s) (ANOVA, *: p<0.05; **: p<0.01; #: p<0.05, N=4-6).

[0054] 5d After the end of administration, the elevated plus maze test was performed on the 6th day. The mice were injected intraperitoneally with the drug 4h before the elevated plus maze test, and the test method was the same as in Example 1. The test results are shown in Figure 9 . Figure 9 In the figure, the vertical axis represents the time (s) spent in the center area, and the horizontal axis represents the group, from left to right, CON group, ANX group, GA-A group, and IMI group. *: p<0.05; #: p<0.05. By Figure 9It was found that GA-A group (29.05 ± 14.45 s), CON group (37.33 ± 18.92 s), and IMI group (29.38 ± 13.78 s) spent more time in the open arms than ANX group (9.33 ± 7.43 s) (ANOVA, *: p < 0.05; #: p < 0.05, N = 4-6).

[0055] The above results show that ganoderic acid A can effectively treat anxiety.

[0056] The above only is the preferred embodiment of the present application, it should be pointed out that, for the ordinary skilled in the art, without departing from the principles of the present application, can also make a number of improvements and refinements, these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. Use of ganoderic acid A as the only active ingredient in the preparation of anxiolytic drugs.

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