Use of a silver radix bupleuri n-butanol extract in the preparation of anxiolytic drugs

Abstract

The application discloses application of a n-butanol extract of radix stellariae dichotomae in preparation of anxiolytic drugs and belongs to the technical field of crude drugs. The application discloses the application of the n-butanol extract of radix stellariae dichotomae in preparation of anxiolytic drugs, taking the n-butanol extract of radix stellariae dichotomae as a research object, and comprehensively applying pharmacology and biology methods, so that the n-butanol extract of radix stellariae dichotomae is shown to have obvious anxiolytic effects.

Description

Technical Field

[0001] This invention relates to the field of pharmacognosy, and more specifically to the application of a butanol extract of *Stellaria media* in the preparation of anti-anxiety drugs. Background Technology

[0002] Anxiety disorder is a mental illness that affects human health and daily life. Common clinical classifications include Social Anxiety Disorder (SAD), Generalized Anxiety Disorder (GAD), Specific Phobias, Obsessive-Compulsive Disorder (OCD), and Post-Traumatic Stress Disorder (PTSD). In severe cases, it can co-occur with other mental health disorders, such as depression. Furthermore, anxiety disorder increases the risk of premature death and cardiovascular disease. Studies have found that the lifetime prevalence of anxiety disorder is approximately 7.6%, ranking first among mental illnesses. With the accelerating pace of society, the incidence of anxiety disorder is increasing year by year, harming both physical and mental health and impacting quality of life. Currently, medications for treating anxiety disorder mainly include benzodiazepines, non-benzodiazepines, and antidepressants. However, these medications often have adverse reactions such as drowsiness, forgetfulness, and gastrointestinal disturbances. In addition, anxiety disorder can trigger many physical symptoms, leading to misdiagnosis or overtreatment, which not only wastes medical resources but also increases the burden on patients. Therefore, using safe and low-toxicity traditional Chinese medicine to treat anxiety disorder has become an inevitable trend.

[0003] Stellaria dichotoma L. var. Lanceolata Bge., a plant in the Caryophyllaceae family, is recorded in traditional Chinese medicine classics such as *Shennong Bencao Jing Shu*, *Benjing Fengyuan*, and *Bencao Gangmu Shiyi*. It is used to clear deficiency heat and eliminate infantile malnutrition-related fever. Modern pharmacological studies have shown that Stellaria dichotoma has anti-neuroinflammatory, anti-cancer, vasodilatory, and antioxidant effects. Current research indicates that Stellaria dichotoma has neuroprotective effects, but no reports of anti-anxiety effects have been found.

[0004] Therefore, providing an application of the n-butanol extract of *Stellaria media* in the preparation of anti-anxiety drugs is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides an application of the n-butanol extract of *Stellaria media* in the preparation of anxiolytic drugs.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] Application of a butanol extract of *Stellaria media* in the preparation of anxiolytic drugs.

[0008] Furthermore, the method for preparing the *Stellaria media* n-butanol extract is as follows:

[0009] The dried roots of *Stellaria dichotoma* were pulverized and passed through a 40-mesh sieve to obtain coarse root powder. The powder was then soaked overnight in 8 times its volume of 70% ethanol under sealed conditions. It was then extracted three times by reflux at 60°C for 1 hour each time. The extract was filtered, and the filtrate was collected, concentrated under reduced pressure, and the solvent was evaporated to obtain an extract. The extract was dissolved in ultrapure water at a material-to-liquid ratio of 3:5 to form a suspension. This suspension was extracted three times with dichloromethane at a volume ratio of 1:1, shaking for 10 minutes each time and allowing to stand until clear layers were formed. The dichloromethane extract and the aqueous layer were combined. The aqueous layer was extracted three times with n-butanol at a volume ratio of 1:1, shaking for 10 minutes each time and allowing to stand until clear layers were formed. The n-butanol extract layers were combined, and the solvent was evaporated to obtain the *Stellaria dichotoma* n-butanol extract.

[0010] Furthermore, the application of the *Stellaria media* n-butanol extract in the preparation of drugs that reduce 5-HT and DA content.

[0011] Furthermore, the application of the *Stellaria media* n-butanol extract in the preparation of drugs that reduce ACTH, CORT, and CRF levels.

[0012] As can be seen from the above technical solution, compared with the prior art, the present invention discloses the application of the n-butanol extract of Stellaria dichotoma in the preparation of anti-anxiety drugs. Taking the n-butanol extract of Stellaria dichotoma as the research object, the invention comprehensively uses pharmacological and biological methods to show that it has significant anti-anxiety effects. Attached Figure Description

[0013] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0014] Figure 1 The attached figure shows the effect of the present invention, *Stellaria media*, on rats under restraint stress in an elevated cruciate maze.

[0015] Where A: the percentage of the number of times the arm is opened to the sum of the number of times the arm is opened and closed (OE%); B: the percentage of the time spent moving within the arm opening to the total time spent within the arm opening and closed (OT%).

[0016] Figure 2 The attached figure shows the effect of the present invention, *Stellaria media*, on rats under restraint stress in an open field test;

[0017] Where A: Number of times entering the Central Zone; B: Duration of stay in the Central Zone;

[0018] Figure 3The attached figure shows the effect of the present invention, *Stellaria media*, on rats under restraint stress in a light-dark chamber;

[0019] Where A: Number of times the box was passed through; B: Time spent in the exposed box;

[0020] Figure 1-3 In the group, the n-butanol group, the dichloromethane group, and the total extraction group correspond to SB, SD, and SE, respectively;

[0021] Figure 4 The attached figure shows the effect of the n-butanol extract of *Stellaria media* of this invention on the behavior of restraint-induced anxiety rats in an elevated cruciate maze.

[0022] Figure 5 The attached figure shows the effect of the n-butanol extract of *Stellaria media* of this invention on the behavior of restraint-induced anxiety rats in an open field experiment.

[0023] Figure 6 The attached figure shows the effect of the n-butanol extract of *Stellaria media* of this invention on the behavior of restraint-induced anxiety rats in a light-dark chamber experiment.

[0024] Figure 7 The attached figure shows the effect of the n-butanol extract of *Stellaria media* of this invention on the morphology of neurons in the CA1 region of the brain tissue of restraint-induced anxiety rats.

[0025] Wherein, A: blank group; B: model group; C: diazepam positive control group; D: low-dose group; E: medium-dose group; F: high-dose group; 50μm, 20×; black arrows indicate loose and irregular neurons; red arrows indicate shrunken and deeply stained neurons;

[0026] Figure 8 The attached figure shows the effects of the n-butanol extract of *Stellaria media* of this invention on 5-HT and DA in the hippocampus of restraint-induced anxiety rats; wherein, A: 5-HT; B: DA;

[0027] Figure 9 The attached figure shows the effects of the n-butanol extract of *Stellaria media* of this invention on serum ACTH, CORT, and CRF in restraint-induced anxiety rats; where A: ACTH; B: CORT; C: CRF;

[0028] Figure 10 The attached figure shows the effect of the n-butanol extract of *Stellaria media* of this invention on the behavior of empty bottle-induced anxiety rats in an elevated cross maze.

[0029] Figure 11 The attached figure shows the effect of the n-butanol extract of *Stellaria media* of this invention on the behavior of rats with empty bottle-induced anxiety in an open field experiment.

[0030] Figure 12 The attached figure shows the effect of the n-butanol extract of *Stellaria media* of this invention on the behavior of rats with empty bottle-induced anxiety in a light-dark chamber experiment.

[0031] Figure 13 The attached figure shows the effect of the n-butanol extract of *Stellaria media* of this invention on the morphology of neurons in the CA1 region of the brain tissue of empty bottle-induced anxiety rats.

[0032] Wherein, A: blank group; B: model group; C: diazepam positive control group; D: low-dose group; E: medium-dose group; F: high-dose group; 50μm, 20×; black arrows indicate loose and irregular neurons;

[0033] Figure 14 The attached figure shows the effects of the n-butanol extract of *Stellaria media* of this invention on 5-HT and DA in the hippocampus of empty-bottle-induced anxiety rats; wherein, A: 5-HT; B: DA;

[0034] Figure 15 The attached figure shows the effects of the n-butanol extract of *Stellaria media* of this invention on serum ACTH, CORT, and CRF in empty-bottle-induced anxiety rats; where A: ACTH; B: CORT; C: CRF;

[0035] Compared with the blank group, # P<0.05, ## P<0.01; compared with the restraint group, * P<0.05, ** P<0.01; n=6. Detailed Implementation

[0036] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0037] Example 1

[0038] The preparation methods of the total extract of Stellaria dichotoma, the dichloromethane extract of Stellaria dichotoma, and the n-butanol extract of Stellaria dichotoma are as follows:

[0039] The dried roots of *Stellaria dichotoma* were pulverized and passed through a 40-mesh sieve to obtain coarse root powder. The powder was then soaked overnight in 8 times its volume of 70% ethanol under sealed conditions. It was then extracted three times by reflux at 60°C for 1 hour each time. The extract was filtered, collected, concentrated under reduced pressure, and the solvent evaporated to obtain an extract (the total extract of *Stellaria dichotoma*). The extract was dissolved in ultrapure water at a material-to-liquid ratio of 3:5 to form a suspension. This suspension was extracted three times with dichloromethane at a volume ratio of 1:1, shaking for 10 minutes each time and allowing to stand until clear layers were formed. The dichloromethane extract and the aqueous layer were combined, and some of the dichloromethane solvent was evaporated to obtain the dichloromethane extract of *Stellaria dichotoma*. The aqueous layer was further extracted three times with n-butanol at a volume ratio of 1:1, shaking for 10 minutes each time and allowing to stand until clear layers were formed. The n-butanol extract layers were combined, and the solvent was evaporated to obtain the n-butanol extract of *Stellaria dichotoma*.

[0040] Example 2: Screening of the anti-anxiety activity sites of Stellaria dichotoma extract

[0041] 1) Experimental animals: 72 SPF-grade male SD rats, weighing 160-180g, were purchased from the Experimental Animal Center of Ningxia Medical University (Experimental Animal License No.: SCXK(Ning)20220-0001). The rearing conditions were a temperature of 25±1℃ and a relative humidity of 50±5%, and the rats were acclimatized for one week before the experiment.

[0042] 2) Drug preparation: The total extract of Stellaria dichotoma (SE), the dichloromethane extract of Stellaria dichotoma (SD), the n-butanol extract of Stellaria dichotoma (SB), and diazepam were all dissolved in 0.5% CMC-Na aqueous solution. The high, medium, and low dose groups of SE were prepared with concentrations of 100 mg / ml, 50 mg / ml, and 25 mg / ml, respectively. The high, medium, and low dose groups of SD were prepared with concentrations of 0.88 mg / ml, 0.44 mg / ml, and 0.22 mg / ml, respectively. The high, medium, and low dose groups of SB were prepared with concentrations of 3 mg / ml, 1.5 mg / ml, and 0.75 mg / ml, respectively. The concentration of diazepam solution was 0.1 mg / ml.

[0043] 3) Animal grouping: 72 male SD rats were randomly divided into 12 groups: control group, model group, positive drug diazepam group (DZP), high, medium and low dose groups of total extract of Stellaria dichotoma, high, medium and low dose groups of dichloromethane extract of Stellaria dichotoma, and high, medium and low dose groups of n-butanol extract of Stellaria dichotoma, with 6 rats in each group. The rats were housed in individual cages.

[0044] 4) Animal modeling and drug administration

[0045] Restraint stress model: The DZP group and each of the Yin Chai Hu (Radix Stellariae) administration groups were administered the corresponding drug solution by gavage for 7 consecutive days, with a gavage volume of 10 mL / kg (diazepam daily dose 1 mg / kg; SE low, medium, and high daily doses 250 mg / kg, 500 mg / kg, and 1000 mg / kg, respectively; SD low, medium, and high daily doses 2.2 mg / kg, 4.4 mg / kg, and 8.8 mg / kg, respectively; SB low, medium, and high daily doses 7.5 mg / kg, 15 mg / kg, and 30 mg / kg, respectively). The Control and Stress groups were administered the corresponding volume of 0.5% CMC-Na aqueous solution by gavage. Starting from the 5th day of gavage, except for the Control group, the rats in the other groups who had completed administration were placed in a restraint device for restraint stress for 3 consecutive days, once a day for 30 minutes each time. During this period, the rats were not fed or allowed to drink water, and only their activity was restricted, not their respiration; the Control group had free movement.

[0046] 5) Behavioral testing

[0047] Behavioral tests included the elevated cross maze test, open field test, and light-dark box test. The restraint stress model rats underwent behavioral tests 30 minutes after the end of restraint stress on day 7.

[0048] (1) Elevated Cross Maze Test: The elevated cross maze test is often used to evaluate anxiety-like behaviors in rodents. The elevated cross maze consists of two open arms, each 50 cm long and 10 cm wide; two closed arms, each 50 cm long, 10 cm wide, and 30 cm high; and four cross-shaped structures 70 cm above the ground. A central area, 10 cm long and 10 cm wide, is located at the intersection of the four arms. A camera is installed at the top to observe the test. Before the test, the rats to be tested are brought into the test environment to acclimatize for 30 minutes. During the test, the rats are placed in the central area with their backs to the open arms, and their activities are recorded within 5 minutes. The number of times the rats enter the open arms (OE), the number of times they enter the closed arms (CE), the number of times they enter the central area, and the time spent in the open and closed arms are recorded within 5 minutes. The final anxiety assessment indicators were the percentage of entries to the open arms (OE%) and the percentage of time spent in the open arms (OT%). All tests were conducted in a quiet environment. After each test, feces were removed, and the testing equipment was wiped with 75% alcohol to eliminate odor.

[0049] (2) Open Field Test: The open field test is commonly used to study the neurobiological basis of anxiety and to screen anti-anxiety compounds. The open field test was conducted the day after the elevated cross maze test. The length, width, and height of the open field were 100cm, 100cm, and 40cm, respectively, with a central area measuring 25cm in length and 25cm in width. A camera was installed at the top of the open field to observe the rats' activities over 5 minutes. The test must be conducted in a quiet environment. Before the test, the rats were brought into the test environment for 30 minutes to acclimatize. During the test, the rats were placed in the peripheral area facing the central area, and the number of times they entered the central area and the time they spent in the central area were recorded. After each test, feces were removed, and the testing equipment was wiped with 75% alcohol to eliminate odor.

[0050] (3) Light-Dark Chamber Test: The light-dark chamber setup consisted of an opaque cardboard box measuring 75cm long, 25cm wide, and 20cm high. Two-thirds of the box was a light chamber (covered with white PVC paper on all sides), and one-third was a dark chamber (covered with black PVC paper on all sides). The two chambers were separated by a 25cm x 20cm partition with a 5cm x 5cm opening at the bottom center, allowing the rat to move freely between them. A camera was installed above the setup to record the rats' activities. Before the test, the rats were brought into the testing environment for 30 minutes to acclimatize. During the test, the rats were placed in the central area of ​​the light chamber with their backs to the dark chamber, allowing them to explore freely. The number of times the rats moved through the chamber and the time they spent in the light chamber were recorded. The environment was kept quiet during this period. After each measurement, the feces were removed, and the inner walls of the light-dark chamber were wiped with 75% alcohol to eliminate odor.

[0051] 6) Behavioral test results

[0052] (1) Test results of the elevated cross maze

[0053] The results are as follows Figure 1 As shown, compared with the control group, restraint stress significantly reduced the percentage of rats entering the open arm and the percentage of time spent in the open arm (P<0.01). Compared with the restraint stress group, the diazepam group, the high-dose n-butanol extract group, and the medium and high-dose total extract groups significantly increased the percentage of rats entering the open arm and the percentage of time spent in the open arm (P<0.01); the medium-dose n-butanol extract group significantly increased the percentage of time rats spent in the open arm (P<0.01); and the low-dose total extract group significantly increased the percentage of rats entering the open arm (P<0.01). This indicates that restraint stress can induce anxiety in rats during the elevated cross maze test, and that high doses of the n-butanol fraction of *Stellaria dichotoma* exert a significant anti-anxiety effect, while medium and high doses of the total extract also exert significant anti-anxiety effects.

[0054] (2) Open field test results

[0055] The results are as follows Figure 2As shown, compared with the control group, restraint stress significantly reduced the number of times rats entered the central area and the time spent in the central area (P<0.01). Compared with the restraint stress group, the diazepam group, the medium and high dose groups of n-butanol extract, and the medium and high dose groups of total extract significantly increased the number of times rats entered the central area and the time spent in the central area (P<0.01); the medium dose group of dichloromethane extract significantly increased the number of times rats entered the central area (P<0.05); the low dose group of n-butanol extract (P<0.01) and the low dose group of total extract (P<0.05) significantly increased the time spent in the central area. This indicates that in the open field test, restraint stress can induce anxiety in rats, and the medium dose of the n-butanol fraction of *Stellaria dichotoma* exerts a significant anti-anxiety effect, as does the medium dose of the total extract.

[0056] (3) Results of the dark and light chamber test

[0057] The results are as follows Figure 3 As shown, compared with the control group, restraint stress significantly reduced the number of times rats entered the box and the time spent in the bright box (P<0.01). Compared with the restraint stress group, the diazepam group, the medium and high dose groups of n-butanol extract, the medium dose group of dichloromethane extract, and the high dose group of total extract significantly increased the number of times rats entered the box and the time spent in the bright box (P<0.01); the high dose group of dichloromethane extract and the low and medium dose groups of total extract significantly increased the time spent in the bright box (P<0.01). This indicates that restraint stress can induce anxiety in rats in the light-dark box experiment, and the medium dose of the n-butanol fraction of *Stellaria dichotoma* can exert a significant anti-anxiety effect. The medium dose of dichloromethane extract and the high dose of total extract can also exert a significant anti-anxiety effect.

[0058] In summary, a rat anxiety model was successfully established using restraint stress. Based on relevant behavioral test indicators, it was preliminarily confirmed that the total extract of Stellaria dichotoma (1000 mg / Kg) and the n-butanol extract of Stellaria dichotoma (30 mg / Kg) can effectively combat restraint stress-induced anxiety in rats. The n-butanol fraction of Stellaria dichotoma is the active anti-anxiety fraction of Stellaria dichotoma.

[0059] Example 3: Application of the anti-anxiety activity of *Stellaria media* n-butanol extract

[0060] 1) Experimental animals: 72 SPF-grade male SD rats, weighing 160-180g, were purchased from the Experimental Animal Center of Ningxia Medical University (Experimental Animal License No.: SCXK(Ning)20220-0001). The rearing conditions were a temperature of 25±1℃ and a relative humidity of 50±5%, and the rats were acclimatized for one week before the experiment.

[0061] 2) Drug preparation: The butanol extract of Stellaria dichotoma (SB) and diazepam were dissolved in 0.5% CMC-Na aqueous solution. The high, medium and low dose groups of SB were prepared with concentrations of 3 mg / ml, 1.5 mg / ml and 0.75 mg / ml, respectively, and the concentration of diazepam solution was 0.1 mg / ml.

[0062] 3) Animal grouping: 72 male SD rats were divided into two batches of 36 rats each, using different modeling methods. Each batch of rats was randomly divided into 6 groups: control group, model group, diazepam positive control group (DZP), low-dose group (SBL), medium-dose group (SBM), and high-dose group (SBH), with 6 rats in each group. The rats were housed in individual cages.

[0063] 4) Animal modeling and drug administration

[0064] (1) Restraint stress model: The DZP, SBL, SBM, and SBH groups were given the corresponding drug solutions by gavage for 7 consecutive days, with a gavage volume of 10 mL / kg (the daily doses of SB low, medium, and high were 7.5 mg / kg, 15 mg / kg, and 30 mg / kg, respectively; the daily dose of diazepam was 1 mg / kg). The Control and Model groups were given the corresponding volume of 0.5% CMC-Na aqueous solution by gavage. Starting from the 5th day of gavage, except for the Control group, the rats in the other groups who had completed the administration were placed in a restraint device for restraint stress for 3 consecutive days, once a day for 30 minutes each time. During this period, the rats were not allowed to eat or drink, and only their activity was restricted, but their respiration was not restricted; the Control group had free movement.

[0065] (2) Empty bottle stress model: Days 1-7 were the training phase. The Control group had free access to water. Except for the Control group, the other groups were given water for 10 minutes at fixed times in the morning and afternoon, and no water was given at other times. Days 8-21 were the stress phase. Except for the Control group, the other groups were given uncertain empty water bottle stimulation. From day 15 to day 21, the DZP, SBL, SBM, and SBH groups were given the corresponding drug solution by gavage daily, with a gavage volume of 10 mL / kg (the daily doses of SB low, medium, and high were 7.5 mg / kg, 15 mg / kg, and 30 mg / kg, respectively; the daily dose of diazepam was 1 mg / kg). The Control group and the Model group were given the corresponding volume of 0.5% CMC-Na aqueous solution by gavage.

[0066] 5) Behavioral testing

[0067] Behavioral tests included the elevated cross maze test, open field test, and light-dark box test, using the same methods as step 5 of Example 2. For the restraint stress model rats, behavioral tests were performed 30 minutes after the end of restraint stress on day 7; for the empty bottle stress model rats, behavioral tests were performed 30 minutes after drug administration on day 21.

[0068] 6) Observation of brain tissue morphology

[0069] After behavioral testing, three rats from each group were anesthetized, their hearts were perfused, and their whole brains were harvested and fixed in 4% paraformaldehyde. After paraffin embedding, the brains were sectioned and baked in a 65°C oven for 2 hours. Dewaxing and hydration: Sections were immersed in xylene I and II for 20 minutes, then in anhydrous ethanol I and II, and 75% alcohol for 5 minutes, and rinsed with tap water. Sections were then immersed in hematoxylin for 5 minutes and rinsed with tap water. Hematoxylin and eosin staining: Sections were differentiated using hematoxylin differentiation solution, rinsed with tap water, and then blued with hematoxylin blueing solution, and rinsed with running water. Sections were dehydrated in 85% and 95% graded alcohol solutions for 5 minutes each, and then stained with eosin for 5 minutes. Dehydration and mounting: Sections were sequentially immersed in anhydrous ethanol I, II, and III, and xylene I and II for 5 minutes for clearing, and then mounted. Images were examined under a microscope.

[0070] 7) ELISA method for detecting the hypothalamic-pituitary-adrenal axis and monoamine neurotransmitters

[0071] The HPA axis, a classic closed-loop neurotransmitter (NE), is a key mechanism for anxiety-like behavior. The HPA axis comprises the hypothalamus, pituitary gland, and adrenal glands, all three being the body's primary stress responses. Stressful stimuli can lead to HPA axis hyperactivity, causing anxiety-like behavior. Specifically, the hypothalamus secretes excessive corticotropin-releasing factor (CRF), which in turn causes the pituitary gland to secrete excessive adrenocorticotropic hormone (ACTH), ultimately leading to excessive secretion of corticosteroids (CORT), which has a negative feedback regulatory effect on the HPA axis. Serotonin (5-HT), a monoamine neurotransmitter, is widely distributed in anxiety-related brain regions and participates in individual emotion regulation; both insufficient and excessive 5-HT levels can lead to anxiety. Dopamine (DA), secreted by the substantia nigra, is a neurotransmitter primarily involved in excitation transmission and participates in emotion regulation.

[0072] (1) Detection of serum ACTH, CRF, and CORT levels: After the behavioral test, each group of rats (except for rats that underwent whole-brain testing) was anesthetized and placed on an ice plate to collect blood from the abdominal aorta. After standing for 20 minutes, the supernatant was collected by centrifugation and aliquoted. The levels of ACTH, CRF, and CORT in rat serum were measured according to the ELISA kit instructions.

[0073] (2) Detection of 5-HT and DA levels in the hippocampus: (1) After blood was collected from rats, the hippocampus tissue was quickly separated by decapitation and brain removal. The tissue was placed in liquid nitrogen for quenching, and the hippocampus was thawed on ice. The 5-HT and DA levels in the rat hippocampus were measured according to the instructions of the ELISA kit.

[0074] 8) Effects of the n-butanol extract of *Stellaria media* on restraint-induced anxiety in rats

[0075] I. Behavioral Test Results

[0076] (1) Test results of the elevated cross maze

[0077] As shown in Table 1, Figure 4 As shown, compared with the blank group, the percentage of times rats entered the open arm (OE%) and the percentage of open arm time (OT%) in the model group were significantly reduced. ## P<0.01. Compared with the model group, the positive control group (diazepam) (1 mg / kg), low-dose group (7.5 mg / kg), medium-dose group (15 mg / kg), and high-dose group (30 mg / kg) significantly increased the percentage of rats entering the open arm and the percentage of rats in the open arm position. * P<0.05, ** (P<0.01). The above results indicate that restraint stress can induce anxiety in rats and that the n-butanol extract of *Stellaria media* has a significant anti-anxiety effect.

[0078] Table 1. Effects of the n-butanol extract of *Stellaria dichotoma* on the behavior of restraint-induced anxiety rats in the elevated cross maze. n=6)

[0079]

[0080] Note: Compared to control, # P < 0.05 indicates a significant difference. ## P < 0.01 indicates a highly significant difference; compared with the model, * P < 0.05 indicates a difference. ** P<0.01 indicates a highly significant difference.

[0081] (2) Open field test results

[0082] As shown in Table 2, Figure 5 As shown, compared with the control group, the number of times rats in the model group entered the central area of ​​the open field and the time spent in the central area of ​​the open field were significantly reduced. ## P<0.01. Compared with the model group, the positive control group (diazepam) (1 mg / kg), medium-dose group (15 mg / kg), and high-dose group (30 mg / kg) showed a significant increase in the number of times the drug entered the central zone and the duration of residence in the central zone. **(P<0.01). The above results indicate that the n-butanol extract of *Stellaria media* can alleviate anxiety induced by restraint stress in rats at doses of 15 mg / kg and 30 mg / kg.

[0083] Table 2. Effects of the n-butanol extract of *Stellaria dichotoma* on the behavior of restraint-induced anxiety rats in the open field experiment. n=6)

[0084]

[0085] Note: Compared to control, # P < 0.05 indicates a significant difference. ## P < 0.01 indicates a highly significant difference; compared with the model, * P < 0.05 indicates a difference. ** P<0.01 indicates a highly significant difference.

[0086] (3) Results of the dark and light chamber test

[0087] As shown in Table 3, Figure 6 Compared with the control group, the number of times rats in the model group passed through the box and the time spent in the box were reduced. ## P<0.01. Compared with the model group, the positive control group (diazepam) (1 mg / kg), medium-dose group (15 mg / kg), and high-dose group (30 mg / kg) showed increased central zone frequency and time spent in the test chamber. ** (P<0.01). The above results indicate that acute restraint stress can induce an anxiety model in rats, and the n-butanol extract of *Stellaria media* showed significant anti-anxiety effects at concentrations of 15.0 mg / kg and 30.0 mg / kg.

[0088] Table 3. Effects of the n-butanol extract of *Stellaria media* on the behavior of restrained anxiety-inducing rats in the light and dark chamber experiment. n=6)

[0089]

[0090] Note: Compared to control, # P < 0.05 indicates a significant difference. ## P < 0.01 indicates a highly significant difference; compared with the model, * P < 0.05 indicates a difference. ** P<0.01 indicates a highly significant difference.

[0091] II. Morphological observation results of neurons in the CA1 region of the brain tissue

[0092] HE staining was performed on rat brain tissue to observe pathological changes in the CA1 region. Results are as follows: Figure 7As shown, compared with the blank group, the model group showed loose and irregular neuronal arrangement, wrinkled and deeply stained neurons, shrunken and deformed cell bodies, and unclear boundaries between the nucleus and cytoplasm. However, compared with the pathological changes in the brain tissue of the model group rats, the neuronal damage in the drug-treated group was improved, the number of normal and intact nerve cells increased significantly, and the arrangement was orderly.

[0093] III. Results of 5-HT and DA level measurements in hippocampal tissue

[0094] As shown in Table 4, Figure 8 Compared with the control group, the levels of 5-HT and DA in the restraint-induced stress group were significantly increased (P<0.05, P<0.01), indicating that the model produced anxiety-like behavior. Compared with the model group, the levels of 5-HT in the positive control drug diazepam group and the low, medium and high dose groups of Yinchaihu n-butanol were significantly decreased (P<0.05, P<0.01), and the levels of 5-HT and DA in the positive control drug diazepam group and the high dose group of Yinchaihu n-butanol were significantly decreased (P<0.05, P<0.01). All treatment groups could alleviate anxiety.

[0095] Table 4. Effects of n-butanol extract of *Stellaria dichotoma* on 5-HT and DA levels in the hippocampus of restraint-induced anxiety rats. n=6)

[0096]

[0097] Note: Compared to control, # P < 0.05 indicates a significant difference. ## P < 0.01 indicates a highly significant difference; compared with the model, * P < 0.05 indicates a difference. ** P<0.01 indicates a highly significant difference.

[0098] IV. Results of serum levels of hypothalamic-pituitary-adrenal axis hormones ACTH, CORT, and CRF

[0099] As shown in Table 5, Figure 9 As shown, compared with the blank group, the model group showed significantly increased levels of ACTH, CORT, and CRF (P<0.01), indicating that the HPA axis was hyperactive in the model group. Compared with the model group, the positive drug diazepam group and the medium and high dose groups of the *Stellaria media* n-butanol extract significantly reduced the levels of ACTH, CORT, and CRF (P<0.05, P<0.01), indicating that the hyperactivity of the HPA axis was inhibited and anxiety symptoms were relieved in the positive drug diazepam group and the medium and high dose groups of the *Stellaria media* n-butanol extract.

[0100] Table 5. Effects of the n-butanol extract of *Stellaria dichotoma* on serum ACTH, CORT, and CRF levels in restraint-induced anxiety rats. n=6)

[0101]

[0102] Note: Compared to control, # P < 0.05 indicates a significant difference. ## P < 0.01 indicates a highly significant difference; compared with the model, * P < 0.05 indicates a difference. ** P<0.01 indicates a highly significant difference.

[0103] 9) Effects of the n-butanol extract of *Stellaria media* on empty bottle stress-induced anxiety in rats

[0104] I. Behavioral Test Results

[0105] (1) Test results of the elevated cross maze

[0106] As shown in Table 6, Figure 10 As shown, compared with the blank group, the percentage of times rats entered the open arm and the percentage of time spent in the open arm were significantly reduced in the model group. ## P<0.01. Compared with the model group, the positive control group (diazepam) (1.0 mg / kg), medium-dose group (15.0 mg / kg), and high-dose group (30.0 mg / kg) significantly increased the percentage of rats entering the open arm and the percentage of rats in the open arm position. ** P<0.01). The n-butanol extract of *Stellaria dichotoma* showed significant anti-anxiety effects at concentrations of 15 mg / kg and 30 mg / kg.

[0107] Table 6. Behavioral effects of the n-butanol extract of *Stellaria dichotoma* on empty bottle-induced anxiety in rats during the cross maze ( n=6)

[0108]

[0109]

[0110] Note: Compared to control, # P < 0.05 indicates a significant difference. ## P < 0.01 indicates a highly significant difference; compared with the model, * P < 0.05 indicates a difference. ** P<0.01 indicates a highly significant difference.

[0111] (2) Open field test results

[0112] As shown in Table 7, Figure 11 As shown, compared with the control group, empty bottle stress significantly reduced the number of times and the time spent by rats entering the central region. ##P < 0.01. Compared with the empty bottle stress group, the diazepam group and the medium and high dose groups significantly increased the number of times rats entered the central area and the duration of stay. * P < 0.05 ** P < 0.01. This indicates that the medium and high doses of the butanol extract of *Stellaria media* have a certain anti-anxiety effect.

[0113] Table 7. Effects of the n-butanol extract of *Stellaria dichotoma* on the behavior of rats with empty bottle-induced anxiety in the open field experiment. n=6)

[0114]

[0115] Note: Compared to control, # P < 0.05 indicates a significant difference. ## P < 0.01 indicates a highly significant difference; compared with the model, * P < 0.05 indicates a difference. ** P<0.01 indicates a highly significant difference.

[0116] (3) Results of the dark and light chamber test

[0117] As shown in Table 8, Figure 12 As shown, compared with the control group, the number of times rats with empty bottle stress-induced anxiety passed through the box and the time spent in the open box were significantly reduced. ## P<0.01. Compared with the model group, the positive control group (diazepam) (1 mg / kg), medium-dose group (15 mg / kg), and high-dose group (30 mg / kg) significantly increased the number of times rats entered the test box and the time spent in the test box. * P<0.05, ** (P<0.01). The above results indicate that chronic empty bottle stress can induce anxiety-like behavior in rats, and the n-butanol extract of *Stellaria media* has significant anti-anxiety effects at 15 mg / kg and 30 mg / kg.

[0118] Table 8. Effects of the n-butanol extract of *Stellaria media* on the behavior of rats with empty bottle-induced anxiety in the light and dark chamber experiment. n=6)

[0119]

[0120]

[0121] Note: Compared to control, # P < 0.05 indicates a significant difference. ## P < 0.01 indicates a highly significant difference; compared with the model, * P < 0.05 indicates a difference. **P<0.01 indicates a highly significant difference.

[0122] II. Morphological observation results of neurons in the CA1 region of the brain tissue

[0123] like Figure 13 As shown, compared with the blank group, the CA1 region of the brain tissue in the empty bottle-induced anxiety model group showed a greater number of neurons arranged in a scattered, loose, and irregular manner. Compared with the model group, the CA1 region neurons in the positive drug diazepam group, medium-dose group, and high-dose group had regular morphology, were neatly and densely arranged, had large and round nuclei, were uniformly light blue or blue, had clear nucleoli, and abundant cytoplasm.

[0124] III. Results of 5-HT and DA level measurements in hippocampal tissue

[0125] As shown in Table 9, Figure 14 As shown, compared with the blank group, the levels of 5-HT and DA in the hippocampus of rats in the empty bottle anxiety model group were significantly increased (P<0.01), indicating that the model produced anxiety-like behavior. Compared with the model group, the positive drug diazepam group, the medium and high dose groups of the n-butanol extract of Stellaria dichotoma significantly reduced the levels of 5-HT and DA in the hippocampus of rats in the empty bottle anxiety model group (P<0.05, P<0.01), and the anxiety of the model was significantly improved in the positive drug diazepam group, the medium and high dose groups of the n-butanol extract of Stellaria dichotoma.

[0126] Table 9. Effects of the n-butanol extract of *Stellaria dichotoma* on 5-HT and DA levels in the hippocampus of empty-bottle-induced anxiety rats. n=6)

[0127]

[0128] Note: Compared to control, # P < 0.05 indicates a significant difference. ## P < 0.01 indicates a highly significant difference; compared with the model, * P < 0.05 indicates a difference. ** P<0.01 indicates a highly significant difference.

[0129] IV. Results of serum levels of hypothalamic-pituitary-adrenal axis hormones ACTH, CORT, and CRF

[0130] As shown in Table 10, Figure 15As shown, compared with the blank group, empty bottle stress significantly increased the levels of ACTH, CORT, and CRF in the serum of rats (P<0.01), indicating that the HPA axis was hyperactive in the model group. Compared with the model group, the positive drug diazepam group, medium-dose group, and high-dose group significantly reduced the levels of ACTH, CORT, and CRF in the serum of rats in the empty bottle-induced anxiety model group (P<0.05, P<0.01), indicating that the HPA axis hyperactivity was inhibited in the positive drug diazepam group and the medium and high-dose groups of the butanol extract of *Stellaria media*, and the anxiety symptoms were relieved.

[0131] Table 10. Effects of the n-butanol extract of *Stellaria dichotoma* on ACTH, CORT, and CRF in empty-bottle-induced anxiety rats. n=6)

[0132]

[0133] Note: Compared to control, # P < 0.05 indicates a significant difference. ## P < 0.01 indicates a highly significant difference; compared with the model, * P < 0.05 indicates a difference. ** P<0.01 indicates a highly significant difference.

[0134] In conclusion, the n-butanol extract of *Stellaria media* can significantly improve anxiety-like behavior induced by restraint stress and empty bottle stress in rats. Its main mechanism may be related to 5-HT and DA monoamine neurotransmitters, and it may exert its anti-anxiety effect by regulating the levels of HPA axis hormones.

[0135] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The application of a butanol extract of *Stellaria media* in the preparation of anxiolytic drugs, characterized in that, The method for preparing the n-butanol extract of *Stellaria media* is as follows: The dried roots of *Stellaria dichotoma* were pulverized and passed through a 40-mesh sieve to obtain coarse root powder. The powder was then soaked overnight in 8 times the volume of 70% ethanol under sealed conditions. It was extracted three times by reflux at 60°C for 1 hour each time. After filtration, the filtrate was collected, concentrated under reduced pressure, and the solvent was evaporated to obtain an extract. The extract was dissolved in ultrapure water at a material-to-liquid ratio of 3:5 to prepare a suspension. The suspension was extracted three times with dichloromethane at a volume ratio of 1:1, shaking for 10 minutes each time and allowing to stand until clear layers were formed. The dichloromethane extract and the aqueous layer were combined. The aqueous layer was extracted three times with n-butanol at a volume ratio of 1:1, shaking for 10 minutes each time and allowing to stand until clear layers were formed. The n-butanol extract layers were combined, and the solvent was evaporated to obtain the n-butanol extract of *Stellaria dichotoma*.

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