Application of pellitorine in the preparation of anti-anxiety drugs

Abstract

The present invention belongs to the field of biomedicine technology and relates to the use of pellitoryline in the preparation of antianxiety drugs. Pelitoryline can increase the exploratory behavior and spontaneous activity of mice in a new environment and reduce tension. After pellitoryline intervention, the total number of times mice entered the arms and the percentage of time entering the open arms in the elevated plus maze experiment increased, while the percentage of time entering the closed arms decreased. After pellitoryline intervention, the number of times mice entered the light box increased. After pellitoryline intervention, the MDA content in the hippocampal tissue of mice decreased, and the activities of SOD and GSH‑Px increased. Pelitoryline can reduce CRS-induced mouse hippocampal tissue and inhibit oxidative stress. After pellitoryline intervention, the expression of Bcl‑2 in the hippocampal tissue of mice increased, and the expression of Bax decreased. Pelitoryline can inhibit CRS-induced mouse hippocampal tissue cell apoptosis.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine and relates to the application of pellitorine in the preparation of antianxiety drugs. Background Art

[0002] Anxiety disorders are common clinical psychiatric diseases that often lead to emotional disorders and central nervous system disorders, and are causing an increasing global health burden[1]. According to epidemiological reports, anxiety disorders are one of the most common psychiatric diseases affecting all age groups in the general population, and the incidence rate is increasing year by year[2]. According to relevant statistics, the prevalence of anxiety disorders is the highest in my country, with an annual prevalence of 4.98% and a lifetime prevalence of 7.57%[3]. At present, the drugs commonly used in the treatment of anxiety in clinical practice include benzodiazepines, such as diazepam; selective 5-HT and norepinephrine reuptake inhibitors (SNRIs), such as venlafaxine and duloxetine; and selective 5-HT reuptake inhibitors (SSRIs), such as fluoxetine and sertraline[4]. Although these drugs have shown certain efficacy in the treatment of anxiety disorders, unfortunately, long-term use of these drugs is prone to drug resistance, addiction, and relapse after discontinuation[5]. In addition, patients often experience adverse reactions such as dizziness, headache, gastrointestinal dysfunction, and sexual dysfunction[6]. Therefore, it is necessary to develop anti-anxiety drugs with good clinical efficacy and few side effects.

[0003] Traditional Chinese medicine has developed a series of treatments for anxiety disorders and plays an important role in the field of mental illness. It has certain advantages over Western medicine. Compared with Western medicine, traditional Chinese medicine is safer, more effective and has fewer side effects in treating anxiety. Suanzaoren Decoction, Xiaoyao Powder, Chaihu Jia Longgu Muli Decoction and Banxia Houpu Decoction are considered important alternative therapies due to their significant efficacy in treating anxiety disorders [7-8]. With the deepening of research on molecular Chinese medicine, the anti-anxiety effects of active components of traditional Chinese medicine have received more and more attention. Research on the anti-anxiety mechanism of valerian, jujube saponin A, asiatica glycoside, quercetin, etc. has opened up new strategies for the treatment of anxiety [9].

[0004] Pellitorine (PEL) is an active ingredient in the traditional Chinese medicine Achillea alpina L.

[10] . Achillea alpina L. is the whole herb of the Asteraceae plant, also known as Artemisia alpina, Chaotic Hair, and Thousand Centipedes. It has been recorded as a folk medicine in Guizhou, Jiangxi, Inner Mongolia, Northeast China and other places in my country. The structural formula of pellitorine is as follows:

[0005]

[0006] As a traditional Chinese medicine, Achillea has a long history of use. It was first recorded in Shennong's Herbal Classics. Achillea is included in the 2020 edition of the Chinese Pharmacopoeia. Achillea tastes bitter, sour, and flat, and is in the lung, spleen, and bladder meridians. It has the functions of detoxification, dampness, blood circulation, and blood stasis. Modern studies have shown that Achillea has anti-inflammatory, antioxidant, antibacterial, blood pressure lowering, liver protection, and liver treatment effects, but there is very little research on the chemical composition of Achillea. So far, the anti-anxiety effects and applications of pellitorine have not been reported at home and abroad.

[0007] References:

[0008] [1]Liu J, Shi JL, Guo JY, et al. Anxiolytic-like effect of Suanzaoren-Wuweizi herb-pair and evidence for the involvement of the monoaminergic system in mice based on network pharmacology[J]. BMC complementary medicine and therapies, 2023, 23(1):7.

[0009] [2] Zhao CBW, Liu J, Shi JL, et al. Anxiolytic Effect of Alcohol-WaterExtracted Suanzaoren-Wuweizi Herb-Pair by Regulating ECS-BDNF-ERK SignalingPathway Expression in Acute Restraint Stress Male Rats[J]. Evidence-basedComplementary and Alternative Medicine, 2020, 2020(12):1-13.

[0010] [3] She Kaijie, Yang Jingwen, Meng Danhua, et al. Molecular mechanism of Erxian Decoction in treating anxiety disorders based on network pharmacology and experimental verification[J]. Chinese Journal of Experimental Traditional Chinese Medicine, 2022, 28(19): 185-193.

[0011] [4] Liu Sijie, Guo Weifeng, Wang Junjun, et al. Exploring the potential anti-anxiety mechanism of Coptis chinensis based on network pharmacology[J]. Western Chinese Medicine, 2023, 36(1): 12-17.

[0012] [5]Deplanque D, Machuron F, Waucquier N, et al. Etifoxine impairs neitheralertness nor cognitive functions of the elderly: A randomized, double-blind, placebo-controlled crossover study[J]. European Neuropsychopharmacology, 2018, 28(8):925-932.

[0013] [6] Feng Chaoying. Research progress on drug treatment of anxiety disorders[J]. Medical Herald, 2006, 25(5):4.

[0014] [7]Baloch Z,Ma K,Wang X,et al.From the perspective of TraditionalChinese Medicine:treatment of mental disorders in COVID-19survivors[J].Biomedicine&

[0015] Pharmacotherapy,2020,132:110810.

[0016] [8] Peter K, Schinnerl J, Felsinger S, et al. A novel concept fordetoxification:complexation between aconitine and liquiritin in a Chinese herbal formula ('Sini Tang'). [J]. Journal of Ethnopharmacology, 2013, 149 (2): 562-9.

[0017] [9] Zhao Wan, Wang Yanyan, Huang Lili, Zhao Hang, Li Tingli. Research progress on the effective ingredients and mechanisms of action of traditional Chinese medicine for anxiolysis[J]. Shanghai Journal of Traditional Chinese Medicine, 2021, 55(9): 96-100.

[0018]

[10] Zhang Ruize, Xu Yanjie, Xiong Juan, et al. Study on the chemical components of Achillea millefolium[J]. Chinese Herbal Medicine, 2013, 44(20): 2812-2815. Summary of the invention

[0019] The purpose of the present invention is to provide the use of pellitorine in the preparation of antianxiety drugs.

[0020] The implementation process of the present invention is as follows:

[0021] Application of pellitoryline in the preparation of antianxiety drugs.

[0022] Furthermore, pellitoryline can increase the exploratory behavior and spontaneous activity of mice in a new environment and reduce stress.

[0023] Furthermore, after intervention with pellitoryrine, the total number of arm entries and the percentage of open arm entry time in the elevated plus maze test increased, while the percentage of closed arm entry time decreased.

[0024] Furthermore, after intervention with pellitorycin, the number of times mice entered the light box increased.

[0025] Furthermore, after intervention with pellitoryrine, the MDA content in the hippocampal tissue of mice decreased, and the activities of SOD and GSH-Px increased.

[0026] Furthermore, pellitoryrine could reduce CRS-induced oxidative stress in the hippocampus of mice.

[0027] Furthermore, after intervention with pellitoryrine, the expression of Bcl-2 in the hippocampus of mice increased, while the expression of Bax decreased.

[0028] Furthermore, pellitoryrine can inhibit CRS-induced apoptosis in mouse hippocampal tissue cells.

[0029] Furthermore, the antianxiety drug at least contains pellitoryrine.

[0030] Furthermore, the antianxiety drug comprises pellitorine and a drug carrier, and the antianxiety drug is in the form of tablets, capsules, pills, injections, sustained-release preparations, controlled-release preparations or various microparticle delivery systems.

[0031] Positive effects of the present invention:

[0032] (1) The results of the open field test showed that the PEL (10 mg / kg) group had a better effect, with significant statistical differences, indicating that PEL can improve the anxiety-like behavior of mice.

[0033] (2) The results of the elevated plus maze test showed that after DIA and PEL (high and low doses) intervention, the total number of arm entries and the percentage of time spent in the open arms increased, while the percentage of time spent in the closed arms decreased, indicating that PEL can improve the anxiety-like behavior of mice.

[0034] (3) The results of the light-dark box experiment showed that the number of times mice in the DIA group and the high-dose PEL group entered the light box increased, indicating that PEL can improve the anxiety-like behavior of mice.

[0035] (4) The MDA content in the hippocampus of mice in the DIA group and the high-dose PEL group was significantly reduced, and the activities of SOD and GSH-Px were significantly increased. High-dose PEL (10 mg / kg) can reduce CRS-induced oxidative stress in the hippocampus of mice. This indicates that PEL can improve the anxiety-like behavior of mice.

[0036] (5) Western blot results showed that the expression of Bcl-2 in the hippocampus of mice in the DIA group, low-dose PEL group, and high-dose PEL group was significantly increased, and the expression of Bax was significantly decreased, which was statistically significant. PEL can significantly inhibit CRS-induced apoptosis of hippocampal cells in mice. This indicates that PEL can improve the anxiety-like behavior of mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The results of the open field test of each group of mice, n = 6, compared with the Control group, ## P<0.01, ### P<0.001; compared with the CRS group, *P<0.05, **P<0.01, ***P<0.001;

[0038] Figure 2 The results of the elevated plus maze test of mice in each group, n = 6, compared with the Control group, ## P<0.01, ### P<0.001; compared with the CRS group, *P<0.05, **P<0.01, ***P<0.001;

[0039] Figure 3 Results of the light-dark box experiment of each group of mice, n=6, compared with the Control group, ### P<0.001; compared with the CRS group, **P<0.01, ***P<0.001;

[0040] Figure 4 MDA content and SOD and GSH-Px activities in hippocampal tissue of mice in each group, n=6, compared with the Control group, # P < 0.001, ### P<0.001; compared with the CRS group, *P<0.05, **P<0.01, ***P<0.001;

[0041] Figure 5 The expression of Bcl-2 and Bax in the hippocampus tissue of mice in each group, n=4. DETAILED DESCRIPTION

[0042] The present invention will be further described below in conjunction with the embodiments.

[0043] 1. Experimental Animals

[0044] Fifty male C57BL / 6 mice, weighing 22-24 g, were purchased from the Animal Center of Air Force Medical University (certificate number: 0009241) and maintained at a temperature of 25±1°C and a relative humidity of 50±5%. They were adaptively maintained for one week before the experiment.

[0045] 2. Drug preparation

[0046] Both pellitoryline and diazepam were dissolved in purified water.

[0047] Pellitorine (PEL) dosage concentration: 5mg / kg, 10mg / kg

[0048] Diazepam tablets (DIA) concentration: 1.3 mg / kg

[0049] 3. Animal Grouping

[0050] The experimental animals were randomly divided into Control (blank control group), CRS (acute restraint stress group), DIA (acute restraint stress + diazepam group), PEL-L (acute restraint stress + low-dose pellitorycin group), and PEL-H (acute restraint stress + high-dose pellitorycin group).

[0051] 4. Establishment of CRS model

[0052] The restraint stress (Chronic restraint stress, CRS) model has been shown to be an effective anxiety stress model. The specific operation is as follows: the experimental mice were placed in a well-ventilated centrifuge tube (25mL capacity) for 2 hours a day for 7 consecutive days to induce acute restraint stress, while the control group mice were placed in a home cage that moved freely during the same period. After 30 minutes of restraint stress, the DIA group was given diazepam (Diazepam, DIA) 1.3mg / kg; the PEL-L and PEL-H groups were given pellitoryne (5mg / kg, 10mg / kg), respectively; the Control (blank control group) and CRS group (model group) were given an equal volume of normal saline. Behavioral tests were performed 30 minutes after the last administration.

[0053] 5. Behavioral Experiments

[0054] 5.1 Open Field Test (OFT)

[0055] The open field was an open box structure measuring 40 cm × 40 cm × 30 cm with a black bottom. The camera equipment was placed in the center of the area. Mice were placed in the experimental room 60 min before the start of the experiment to allow for acclimatization. Uniform lighting and a quiet environment were ensured throughout the experiment. Mice were gently placed in the center of the square and allowed to move freely in the open field for 10 min. The center distance and time were recorded. Between tests, the area was cleaned with 20% alcohol. After the alcohol odor had dissipated and there were no obvious signs of moisture on the bottom of the box, the next mouse was tested.

[0056] 5.2 Elevated plus maze test (EPM)

[0057] The elevated maze consisted of 70 cm × 5 cm × 20 cm black plexiglass with a white surface to provide contrast. Testing was performed under red light (<10 lux) and was soundproofed from the outside. Between tests, the EPM apparatus was manually cleaned with a 20% ethanol solution. Mice were placed in the center of the maze, and the open arm time and open arm entrances were recorded for 5 min using a video tracking system (Ethovision).

[0058] 5.3 Light and dark box experiment

[0059] The acclimatized mice were placed in the center of a light-dark box (44 cm × 21 cm × 21 cm, with the light box occupying 1 / 3 and the dark box occupying 2 / 3), with their heads facing the dark box. The number of times the mice entered the light box and the activity time in the light box were recorded within 5 minutes.

[0060] 6.ELISA method to detect oxidative stress related indicators

[0061] After the behavioral test, the mice in each group were decapitated to remove the brain, and the hippocampal tissue of the mice was quickly removed. The supernatant was taken after homogenization and centrifugation to determine the MDA content and SOD and GSH-Px activities in the hippocampal tissue. All operations were carried out strictly according to the instructions of the kit.

[0062] 7. Western Blot analysis of Bcl-2 and Bax expression in mouse hippocampus

[0063] After the behavioral test, the brain was removed by decapitation and the hippocampal tissue of the mouse was quickly isolated, and the protein content of each group was determined by the BCA method. SDS-PAGE gel electrophoresis, transfer membrane for 1 hour, blocking, Bcl-primary antibody (1:500), Bax-antibody (1:500), β-actin primary antibody (1:1000) incubated at room temperature for 1 hour, secondary antibody (1:5000) incubated at room temperature for 1 hour, the membrane was placed in a gel imager, and ECL luminescent agent was added for development.

[0064] 8. Results

[0065] 8.1 Effects of pellitoryline on the behavior of CRS mice

[0066] 8.1.1 Open field test results

[0067] The results of the open field test showed that compared with the Control group, the total distance, central distance percentage, and central time percentage of mice in the CRS model group were significantly reduced, indicating that the exploratory behavior and spontaneous activity of mice in the CRS model group in the new environment were significantly reduced, while the tension was significantly increased. Therefore, CRS successfully induced anxiety-like behavior in mice. Compared with the CRS model group, the total distance, central distance percentage, and central time percentage of mice in the DIA group and each dose group of PEL changed, and the PEL (10 mg / kg) group had a better effect, with significant statistical differences. PEL can improve the anxiety-like behavior of mice. The results are shown in Figure 1 , Figure 1 The results of the open field test of each group of mice, n = 6, compared with the Control group, ## P<0.01, ### P<0.001; compared with the CRS group, *P<0.05, **P<0.01, ***P<0.001.

[0068] 8.1.2 Elevated plus maze test results

[0069] The results of the elevated plus maze test showed that compared with the Control group, the total number of arm entries and the percentage of open arm entry time in the CRS group were significantly reduced, while the percentage of closed arm entry time was significantly increased, indicating that the mice were in an anxious state caused by CRS. Compared with the CRS stress group mice, after DIA and PEL (high and low doses) intervention, the total number of arm entries and the percentage of open arm entry time increased, while the percentage of closed arm entry time decreased, especially the high dose of PEL was statistically significant. PEL can improve the anxiety-like behavior of mice. The results are shown in Figure 2 , Figure 2 The results of the elevated plus maze test of mice in each group, n = 6, compared with the Control group, ## P<0.01, ### P<0.001; compared with the CRS group, *P<0.05, **P<0.01, ***P<0.001.

[0070] 8.1.3 Results of light and dark box experiments

[0071] The results of the light-dark box experiment showed that compared with the Control group, the number of times the mice in the CRS group entered the light box was less than that of the Control group, and the activity time in the light box was shorter than that of the blank group, and the difference was statistically significant. Compared with the CRS group, the number of times the mice in the DIA group and the high-dose PEL group entered the light box increased, and the difference was statistically significant, indicating that PEL can improve the anxiety-like behavior of mice. There was no statistically significant difference between the low-dose PEL group and the CRS group. Figure 3 . Figure 3 Results of the light-dark box experiment of each group of mice, n=6, compared with the Control group, ### P<0.001; compared with the CRS group, **P<0.01, ***P<0.001.

[0072] 8.2 Effects of pellitoryline on MDA content and SOD and GSH-Px activities in hippocampus of CRS mice

[0073] The results of Elisa experiments showed that compared with the Control group, the MDA content in the hippocampus tissue of mice in the CRS group was significantly increased, and the activities of SOD and GSH-Px were significantly decreased. Compared with the CRS group, the MDA content in the hippocampus tissue of mice in the DIA group and the high-dose PEL group was significantly decreased, and the activities of SOD and GSH-Px were significantly increased, which was statistically significant. Figure 4 , Figure 4 MDA content and SOD and GSH-Px activities in hippocampal tissue of mice in each group, n=6, compared with the Control group, # P < 0.001, ### P<0.001; compared with the CRS group, *P<0.05, **P<0.01, ***P<0.001. There was no statistically significant difference between the low-dose PEL group and the CRS group. The results suggest that high-dose PEL (10 mg / kg) can reduce CRS-induced oxidative stress in the mouse hippocampus.

[0074] Table 1 Determination results of oxidative stress indexes in hippocampal tissue of mice in each group

[0075]

[0076] 8.3 Effects of pellitorycin on the expression of Bcl-2 and Bax in the hippocampus of CRS mice

[0077] The results of Western blot experiments showed that compared with the Control group, the expression of Bcl-2 in the hippocampus of mice in the CRS group was significantly decreased, the expression of Bax was significantly increased, and the ratio of Bcl-2 / Bax was significantly reduced. Compared with the CRS group, the expression of Bcl-2 in the hippocampus of mice in the DIA group, the low-dose PEL group, and the high-dose PEL group was significantly increased, and the expression of Bax was significantly decreased, which was statistically significant. Figure 5 , Figure 5 The expression of Bcl-2 and Bax in the hippocampus tissue of mice in each group, n = 4. The results suggest that PEL can significantly inhibit CRS-induced apoptosis of hippocampal cells in mice.

[0078] In summary, pellitoryline (PEL) can significantly improve CRS-induced anxiety-like behavior in mice, and its main mechanism is related to PEL's inhibition of oxidative stress and neuronal apoptosis in the hippocampus.

[0079] The above contents are further detailed descriptions of the present invention in combination with specific preferred embodiments, and the specific implementation of the present invention cannot be limited to these descriptions. For ordinary technicians in the field to which the present invention belongs, several simple deductions or substitutions can be made without departing from the concept of the present invention, which should be regarded as falling within the protection scope of the present invention.

Claims

1. Application of pellitoryline in the preparation of anti-anxiety drugs.

2. The application according to claim 1, characterized in that: The antianxiety drug comprises pellitorine and a drug carrier, and the dosage form of the antianxiety drug is tablets, capsules, pills, injections, sustained-release preparations, controlled-release preparations or microparticle delivery systems.

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