New application of twenty-ingredient nutmeg pill

Abstract

The invention belongs to the technical field of medical application, particularly relates to new application of a twenty-ingredient nutmeg pill and application of the twenty-ingredient nutmeg pill in preparation of a medicine for treating and / or preventing anxiety disorder, especially anxiety caused by pentyltetrazole, and discloses the new application of the twenty-ingredient nutmeg pill, the application range of the twenty-ingredient nutmeg pill is widened, application taboos are few, and the application cost is low. No adverse reaction report exists.

Description

Technical Field

[0001] The invention belongs to the technical field of medical use, and particularly relates to a new use of twenty-flavor nutmeg pills. Background Art

[0002] Tibetan medicine twenty flavors nutmeg pill (national medicine standard Z20023260) has the effect of calming and tranquilizing the mind, and is used for "Ninglong" disease. "Ninglong" disease is a term used in Tibetan medicine. In traditional Chinese medicine, it belongs to the category of insomnia, which is what we often call insomnia; in Western medicine, it is classified as palpitations, neurasthenia, and hysteria. Twenty flavors nutmeg pill is composed of nutmeg, agarwood, agarwood, travertine, jujube, safflower, fennel, cloves, garlic (charcoal), cardamom, asafoetida, grass fruit, terminalia chebula, frankincense, terminalia chebula, catechu, emblica, legadu, sandalwood, and artificial bezoar. Modern research shows that Twenty Flavor Nutmeg Pill can also treat dragon-type angina pectoris, is used for myocardial ischemia and arrhythmia, has the effects of opening up the mind, improving memory and concentration, and is also effective in treating gynecological diseases (menstrual headache, perimenopausal syndrome), cardiovascular and cerebrovascular diseases (cardiovascular neurosis, cholecardiac syndrome), endocrine diseases (insomnia) and Ningsai disease in Tibetan medicine.

[0003] Anxiety disorder is a group of mental disorders with anxiety syndrome as clinical manifestation, which often include two groups of symptoms: 1. Mental symptoms: mainly characterized by fear and worry, accompanied by nervous and uneasy inner performance. There can be many objects of fear, such as a scene, an environment or an animal. The content of worry is more, which can be related to reality or have no obvious object; 2. Physical symptoms: refers to the manifestation of autonomic nervous function excitement under the background of mental symptoms. Patients usually experience palpitations, headaches, dizziness, sweating, hand tremors, and physical discomfort of self-conscious anxiety. The discomfort and the above mental symptoms occur in one person at the same time. The two groups of symptoms may wax and wane. Sometimes the mental symptoms may be more severe, and sometimes the physical symptoms may be more severe. At present, with the increase of life pressure, the prevalence of anxiety disorder continues to rise, seriously affecting the physical, psychological and social functions of patients.

[0004] At present, modern medicine mainly uses benzodiazepines (estazolam) and tricyclic drugs (doxepin) to treat generalized anxiety disorder. In clinical treatment, there is a certain effect, but the toxicity and side effects of these drugs are also obvious. Such as daytime sleepiness, lack of concentration, impaired learning ability, memory loss, severe drug dependence and physical withdrawal symptoms, as well as strong anticholinergic and cardiovascular side effects. Therefore, it is necessary to find a safer and more effective drug for the treatment of anxiety disorders. Summary of the invention

[0005] The invention provides a new use of the twenty-flavor nutmeg pill, broadens the indications of the twenty-flavor nutmeg pill, and has fewer adverse reactions.

[0006] The inventors of the present invention, through creative relevant experimental research, found that 28 days after the administration of Twenty Flavors Nutmeg Pill, the residence time in the light box and the percentage of the light box time of mice in the high, medium and low dose groups of Twenty Flavors Nutmeg Pill were significantly increased; 45 days after the administration of Twenty Flavors Nutmeg Pill, the total movement distance, the number of entries into the central area, the movement distance in the central area, and the percentage of the movement distance in the central area of ​​mice in the high, medium and low dose groups of Ten Flavors Nutmeg Pill were significantly increased. Twenty Flavors Nutmeg Pill can prevent and / or treat anxiety-like behavior caused by PTZ, and therefore can be used to prepare drugs for treating and / or preventing anxiety.

[0007] The anxiety disorder is anxiety caused by pentylenetetrazol.

[0008] The dosage of the twenty-flavor nutmeg pill is 0.039-0156 g / kg / d, once a day.

[0009] Compared with the prior art, the present invention discloses the use of Twenty Flavors Nutmeg Pill in preparing a drug for treating and / or preventing anxiety disorder. The Twenty Flavors Nutmeg Pill has achieved remarkable effects in preventing and improving PTZ-induced anxiety-like behavior in mice, can be used for preparing a drug for treating and / or preventing anxiety disorder, broadens the application scope of the Twenty Flavors Nutmeg Pill, has few application contraindications, and has no adverse reaction reports. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 Experimental process for studying the effect of Ershiwei Roudoukou Wan on improving the anxiety-like behavior of mice induced by pentylenetetrazol;

[0011] Figure 2 is the result of the O-maze experiment, where A1 is the total distance of movement, A2 is the distance of movement in the open arm %, A3 is the number of times of entering the open arm %, A4 is the number of times of entering the open arm %, A5 is the time of staying in the open arm, and A6 is the time of staying in the open arm %;

[0012] Figure 3 is the result of the elevated plus maze experiment, where B1 is the number of times entering the open arm, B2 is the number of times of open arms %, B3 is the time spent in the open arm, and B4 is the time of open arms %;

[0013] Figure 4 is the result of the light-dark box experiment, where C1 is the number of times entering the light box, C2 is the number of times in the light box %, C3 is the time spent in the light box, and C4 is the time in the light box %;

[0014] Figure 5 is the result of the elevated plus maze experiment, where D1 is the number of times entering the open arm, D2 is the number of times of open arms %, D3 is the time spent in the open arm, and D4 is the time of open arms %;

[0015] Figure 6is the result of the open field test, where E1 is the total movement distance, E2 is the number of times entering the central area, E3 is the time entering the central area, E4 is the distance entering the central area, and E5 is the central area movement distance%. DETAILED DESCRIPTION

[0016] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0017] With respect to the new use and effect of the twenty-flavor nutmeg pills proposed in the technical solution of the present invention, further experimental confirmation and summary will be carried out below:

[0018] The experimental instruments and materials used in the following experiments are commercially available.

[0019] 1 Materials and methods

[0020] 1.1 Experimental animals

[0021] SPF grade 5-week-old male ICR mice, Beijing Weitonglihua Experimental Animal Co., Ltd., license number SCXK (Beijing) 2016-0006. Mice were kept at 22±2℃, humidity 40-50%, 12h / 12h (7:00pm-7:00am) day and night cycle, free access to food and water. All behavioral experiments were scheduled to be carried out on the day of the experiment from 9:00 to 17:00 (SDUTCM20210606002).

[0022] 1.2 Drugs and reagents

[0023] Twenty Flavors Nutmeg Pills (National Medicine Standard No. Z20023260), Tibet Shenhou Pharmaceutical Co., Ltd.; Diazepam Tablets (National Medicine Standard No. H20237049), Shandong Xinyi Pharmaceutical Co., Ltd.; Pentylenetetrazol (PTZ) (74404A), Shanghai MacLean Biochemical Technology Co., Ltd.; Mouse gonadotropin-releasing hormone ELISA kit (CSB-E08152m), mouse follicle-stimulating hormone kit (CSB-E06871m) and mouse luteinizing hormone kit (CSB-E12770m), mouse androgen ELISA kit (CSB-E05101m), mouse estrogen ELISA kit (CSB-E07280m), Wuhan Huamei Bioengineering Co., Ltd.; Mouse γ-aminobutyric acid ELISA kit (H168-1-2), Nanjing Jiancheng Bioengineering Institute; BCA protein quantification kit (ZJ102), Shanghai Yamei Biotechnology Co., Ltd.

[0024] 1.3 Experimental instruments

[0025] O-maze system (XR-XZR209), elevated plus maze system (XR-XG201), light-dark box system (XR-XG201), autonomous activity box and open field system (Tracking MasterV3.0) Shanghai Xinruan Company; full-wavelength microplate reader (MT90126728), Thermo Scientific; laboratory constant temperature refrigerator (FYL-YS-280L), Beijing Fuyi Lian Electric Co., Ltd.; centrifuge (ST8R), Thermo Scientific; ultrapure water machine (EKUP-Ⅱ-40E), Sichuan Yike Pure Water Equipment Co., Ltd.; microplate constant temperature oscillator (L-MPI-SA), Changzhou Langbo Instrument Manufacturing Co., Ltd.; tissue grinder (BD-GDB1), Wuxi Bofred Biotechnology Co., Ltd.

[0026] 1.4 Grouping, modeling and drug administration

[0027] After one week of feeding, 5-week-old male ICR mice were divided into normal control group, model group, positive drug group, and high-, medium-, and low-dose groups of Twenty Flavors Nutmeg Pills according to their spontaneous activity and body weight. The experimental groups and dosages are shown in Table 1. The clinical dosage of Twenty Flavors Nutmeg Pills is 2-3g once, twice a day, so the clinical daily dosage of the drug is 6g / d. In this experiment, the medium dose administered orally to mice was set as the clinical equivalent dose. The O maze, elevated plus maze, light-dark box, and open field tests were used to detect its effect on PTZ-induced anxiety-like behavior. The specific experimental procedures are shown in Figure 1 .

[0028] Table 1 Experimental groups of the study on the effect of Twenty Flavor Nutmeg Pills on improving anxiety

[0029]

[0030] 1.5 Behavioral Experiments

[0031] On the day of behavioral testing, the animals in the drug group and the control group were subjected to the elevated plus maze test 1.5 hours after the last oral gavage, and the animals in the positive drug group were subjected to the elevated plus maze test 1 hour after the last oral gavage. All animals were subjected to behavioral testing 1.5 hours after the last oral gavage. 0.5 hours before the behavioral experiment, the control group was intraperitoneally injected with normal saline, and the anxiety model was injected with PTZ (30 mg / kg) once intraperitoneally. Adult female Lewis rats were injected with PTZ intravenously. According to pharmacokinetic studies, the total clearance of 20 mg / kg PTZ was 5.36±0.34 mL / min / kg, the terminal half-life was 116±25 min, and the apparent volume distribution was 896±134 ml / kg. Serum protein binding was negligible (less than 10%). It is speculated that the washout period of PTZ in rats is about 5 hours, so each behavioral test is separated by more than 5 days.

[0032] O-maze test: Randomly place mice in the middle of any open arm area of ​​the O-maze, with their heads facing the center of the circle, and record the time and number of entries into the two arm areas. After release, the following indicators are recorded: number of open arm entries (open arm entry, OE), open arm stay time (open arm time, OT). The above indicators are calculated as: percentage of open arm times, percentage of open arm time. The total time of behavioral testing is 5 minutes.

[0033] Elevated plus maze test: During the experiment, the mouse was placed in the center of the central platform, with its head facing the open arm into the elevated plus maze. After release, the following indicators were recorded: number of open arm entries (OE) and open arm stay time (OT). The above indicators were used to calculate: percentage of open arm times and percentage of open arm time. The total time for behavioral testing was 5 minutes.

[0034] Light-dark box experiment: Randomly put the experimental mice from the light box into the dark box, with their heads facing the dark box, and then remove the partition. Observation indicators: number of entries into the light box, time spent in the light box, number of entries into the light box / total number of entries, time spent in the light box / total time. The total time for behavioral testing was 5 minutes.

[0035] Open field test: A mouse was randomly placed in the open field for behavioral testing. The mouse was placed in the center of the box during the experiment. After release, the following indicators were recorded: number of central area shuttles, total movement distance, central area residence time, central area movement distance, and total movement distance. The total time for behavioral testing was 10 minutes.

[0036] After each mouse test was completed, both arms were cleaned and sprayed with 75% alcohol to eliminate odor before proceeding to the next experiment.

[0037] 1.7 Statistical analysis

[0038] All data were expressed as "Mean±SD". The two groups were compared using Student's t-test, and multiple groups were compared with the model group using One-way ANOVA followed by Dunnett's multiple comparisons test. GraphPad Prism 8.0.1 software was used for analysis, and P < 0.05 was considered statistically significant.

[0039] 2 Results

[0040] 2.1 Effect of Ershiwei Roudoukou Pill on the improvement of PTZ-induced anxiety-like behavior

[0041] After 7 days of administration of Ershiwei Roudoukou Pill, the O-maze test was used to detect its improvement effect on PTZ-induced anxiety-like behavior in mice. The results showed that compared with the control group, the total movement distance, percentage of open arm movement distance, number of open arm entries, open arm stay time, and percentage of open arm stay time in the model group were significantly reduced (Figure A1-6, P < 0.05). This result suggests that PTZ at a dose of 30 mg / kg can induce anxiety-like behavior in ICR mice. Compared with the model group, the total movement distance, percentage of open arm movement distance, open arm stay time, and percentage of open arm stay time in the diazepam group were significantly increased (Figure A1, A2, A5, A6, P < 0.05), and there was no statistical difference in the Ershiwei Roudoukou Pill administration group compared with the model group. This result suggests that diazepam has an improvement effect on PTZ-induced anxiety-like behavior after 7 days of administration.

[0042] After 15 days of administration of Ershiwei Roudoukou Pill, the elevated plus maze test was used to detect its improvement effect on PTZ-induced anxiety-like behavior in mice. The results showed that compared with the control group, the model group had a significantly reduced number of open arm entries (Figure B1, P < 0.05), which suggested that PTZ at a dose of 30 mg / kg could induce anxiety-like behavior in ICR mice. Compared with the model group, the diazepam group had a significant increase in the number of open arm entries (Figure B1, P < 0.05), while there was no statistical difference in the Ershiwei Roudoukou Pill administration group. The results suggested that diazepam had an improvement effect on PTZ-induced anxiety-like behavior after 15 days of administration.

[0043] After 28 days of administration, the light-dark box test was used to detect the improvement effect of Twenty Flavors Roudoukou Pill on PTZ-induced anxiety-like behavior in mice. The results showed that compared with the control group, the light box residence time and percentage of light box time of the mice in the model group were significantly reduced (Figure C3, C4, P < 0.05). Compared with the model group, the number of times of entering the light box, the percentage of the number of times of entering the light box, the residence time of the light box, and the percentage of the light box time in the diazepam group were significantly increased (Figure C1-4, P < 0.05), and the time of entering the light box and the percentage of the light box time in the high, medium, and low dose groups of Twenty Flavors Roudoukou Pill were significantly increased (Figure C3, C4, P < 0.05). The above results show that after 28 days of administration, diazepam and Twenty Flavors Roudoukou Pill both have an improvement effect on PTZ-induced anxiety-like behavior.

[0044] After 39 days of administration of Ershiwei Roudoukou Pill, the elevated plus maze test was used to detect its improvement effect on PTZ-induced anxiety-like behavior in mice. The results showed that compared with the control group, the model group had significantly reduced open arm stay time and open arm stay time percentage (Figure D3, D4, P < 0.05). Compared with the model group, the diazepam group had significantly increased open arm stay time and open arm stay time percentage (Figure D3, D4, P < 0.05), and there was no statistical difference in the Ershiwei Roudoukou Pill administration group.

[0045] After 45 days of administration of Ershiwei Roudoukou Pill, the open field test was used to evaluate its improving effect on PTZ-induced anxiety-like behavior in mice. The results showed that compared with the control group, the total movement distance, the number of times entering the central area, the distance entering the central area, and the percentage of central area movement distance in the model group were significantly reduced (Figure E1, E2, E4, E5, P < 0.05). Compared with the model group, the total movement distance, the number of times entering the central area, the central area residence time, and the central area movement distance in the diazepam group were significantly increased (Figure E1, E2, E3, E4, P < 0.05), and the total movement distance, the number of times entering the central area, the central area movement distance, and the percentage of central area movement distance in the high, medium, and low dose groups of Ershiwei Roudoukou Pill were significantly increased (Figure E1, E2, E4, E5, P < 0.05). The above results indicate that after 45 days of administration, both diazepam and Ershiwei Roudoukou Pill have an improving effect on PTZ-induced anxiety-like behavior.

[0046] In summary, the results of the light-dark box and open field tests showed that the twenty-flavor nutmeg pills administered 7 days in advance could prevent the anxiety-like behavior caused by PTZ.

Claims

1. Use of Twenty Flavor Nutmeg Pills in preparing medicines for treating and / or preventing anxiety disorders.

2. The use of the twenty-flavor nutmeg pill according to claim 1 in preparing a drug for treating and / or preventing anxiety, characterized in that: The anxiety disorder is anxiety caused by pentylenetetrazol.

3. The use of the twenty-flavor nutmeg pill according to claim 1 in preparing a medicament for treating and / or preventing anxiety, characterized in that: The dosage of the twenty-flavor nutmeg pill is 0.039-0156 g / kg / d, once a day.

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