Application of nerve soothing and brain strengthening liquid in preparation of medicine for treating or preventing chronic fatigue syndrome

Abstract

The invention relates to the field of medical application, in particular to application of nerve-soothing and brain-strengthening liquid to preparation of medicine for treating or preventing chronic fatigue syndrome. According to the invention, the nerve-soothing and brain-strengthening liquid is used for research on treatment or prevention of chronic fatigue syndrome for the first time. By establishing an intraperitoneal injection polyinosinic-cytidylic acid composite swimming induced CFS mouse model and a multi-factor composite stress induced CFS rat model, and by means of an exhaustive swimming experiment, a tail suspension experiment, a mechanical pain threshold, a Bannes maze and a pentobarbital sodium sleep experiment, an endocrine function, an immunologic function, skeletal muscle energy metabolism and other modes are detected, and the skeletal muscle energy metabolism can be detected. It is found that the nerve soothing and brain strengthening liquid can improve the CFS symptoms shown by mice and rats of the two models.

Description

Technical Field

[0001] The invention relates to the field of medical applications, and in particular to the application of tranquilizing and brain-strengthening liquid in the preparation of medicines for treating or preventing chronic fatigue syndrome. Background Art

[0002] Chronic fatigue syndrome (CFS), also known as myalgic encephalomyelitis (ME), is defined as a chronic systemic syndrome characterized by long-term fatigue, which lasts for more than 6 months and is accompanied by four or more of the following symptoms: short-term memory loss, inattention, sore throat, lymphadenopathy, muscle aches, joint pain, headache, inability to recover after sleep, discomfort within 24 hours after physical or mental work, and fatigue that cannot be relieved after rest. In subsequent studies, other complications of CFS were discovered, such as anxiety, depression, loss of appetite or hyperphagia, sleep disorders, fever, and a variety of mental symptoms. It is estimated that millions of people worldwide are affected by CFS, and women are more susceptible than men. It is very important to study the cause of CFS and its treatment and management, because the disease not only affects the patient's body, but also damages their work, life, and family.

[0003] At present, there is no specific drug for the treatment of CFS. Western medicine treatment is mostly symptomatic treatment to relieve clinical symptoms, such as drug treatment including the use of analgesics, non-steroidal anti-inflammatory drugs, steroid drugs, antidepressants, antivirals, anticonvulsants, immunomodulatory drugs and nutritional supplements, etc. Non-drug therapies mainly include cognitive behavioral therapy, graded exercise therapy, adaptive pacing therapy, hyperbaric oxygen therapy, etc. However, the above methods can only relieve some clinical symptoms at present, and long-term treatment is very likely to produce adverse reactions.

[0004] There is no such disease name as "chronic fatigue syndrome" in traditional Chinese medicine. The word "fatigue" in traditional Chinese medicine literature was first seen in Zhang Zhongjing's "Golden Chamber" in the Han Dynasty. According to the patient's clinical symptoms, CFS can be classified into the categories of "fatigue", "asthenia", "laziness", "uselessness of limbs", "unretracted limbs", "soreness of limbs", "heaviness of limbs" and "exhaustion of limbs" in traditional medicine. Most of the methods of traditional Chinese medicine for treating CFS are mainly external treatments, such as acupuncture, moxibustion, acupoint application, massage and cupping, etc., in order to achieve the effects of dredging meridians, harmonizing yin and yang, and strengthening the body and eliminating evil, thereby improving the function of internal organs, harmonizing qi, blood, yin and yang, and treating chronic fatigue syndrome. There are few reports on the methods of traditional Chinese medicine for treating CFS. Traditional Chinese medicine has a long history in my country. Some active compounds in traditional Chinese medicine may have lower adverse reactions and side effects, and can be used as a substitute and supplement for Western medicine, reflecting the therapeutic advantages of traditional Chinese medicine. Therefore, it is particularly important to develop drugs with lower adverse reactions and side effects. Summary of the invention

[0005] Therefore, the object of the present invention is to provide a new use of the tranquilizing and brain-strengthening liquid, namely, the use of the tranquilizing and brain-strengthening liquid in the preparation of a medicine for treating or preventing chronic fatigue syndrome.

[0006] To this end, this application provides the following technical solutions.

[0007] The invention provides application of tranquilizing and brain-strengthening liquid in preparing medicine for treating, preventing or alleviating chronic fatigue syndrome.

[0008] Optionally, the raw material composition of the tranquilizing and brain-strengthening liquid includes ginseng, schisandra chinensis, ophiopogon japonicus, wolfberry fruit and salvia miltiorrhiza.

[0009] Optionally, in the raw materials of the tranquilizing and brain-strengthening liquid, the mass ratio of ginseng, schisandra chinensis, ophiopogon japonicus, wolfberry and salvia miltiorrhiza is 20-80:100-160:100-160:100-160:60-120.

[0010] Optionally, at least one of (1) to (9) is satisfied:

[0011] (1) The drug can regulate the dysfunction of the hypothalamus-pituitary-adrenal axis (HPA axis) caused by chronic fatigue syndrome;

[0012] (2) The drug can improve the thymus index of patients or animals with chronic fatigue syndrome;

[0013] (3) The drug can reduce the CD8 + T cell ratio;

[0014] (4) The drug can reduce the level of inflammatory factors in patients or animals with chronic fatigue syndrome;

[0015] (5) The drug can regulate energy metabolism in patients with chronic fatigue syndrome or animals;

[0016] (6) The drug can alleviate oxidative stress in patients with chronic fatigue syndrome or animals;

[0017] (7) The drug can increase the level of immunoglobulin in patients or animals with chronic fatigue syndrome;

[0018] (8) The drug can increase the complement content in patients or animals with chronic fatigue syndrome;

[0019] (9) The drug can regulate the intestinal flora of patients with chronic fatigue syndrome or animals.

[0020] Optionally, the inflammatory factors include at least one of C-reactive protein (CRP), interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-2 (IL-2), gamma interferon (IFN-γ), alpha interferon (IFN-α), and tumor necrosis factor-α (TNF-α).

[0021] Optionally, the immunoglobulin includes at least one of immunoglobulin G (IgG), immunoglobulin M (IgM), and immunoglobulin A (IgA).

[0022] Optionally, the complement includes at least one of C3 and C4.

[0023] Optionally, the energy metabolism includes amino acid metabolism.

[0024] Optionally, the drug can regulate the levels of serum cortisol (CORT) and adrenocorticotropic hormone (ACTH) in patients with chronic fatigue syndrome or animals, the levels of 5-hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), and 5-hydroxytryptamine transporter (5-HTT) in the hippocampus, prefrontal cortex, and hypothalamus, and the level of corticotropin-releasing hormone (CRH) in the hypothalamus.

[0025] Optionally, the drug is capable of activating the mitochondrial biogenesis signaling pathway in chronic fatigue syndrome patients or animals.

[0026] Optionally, the drug can improve the function of the mitochondrial electron transport chain in patients or animals with chronic fatigue syndrome.

[0027] Optionally, the drug can reduce the levels of serum lactic acid (LAC) and serum urea nitrogen (BUN) in skeletal muscle cells of patients with chronic fatigue syndrome or animals.

[0028] Optionally, the drug can increase the liver glycogen and muscle glycogen content in patients or animals with chronic fatigue syndrome.

[0029] Optionally, the drug can increase the number of mitochondria in skeletal muscle cells of patients with chronic fatigue syndrome or animals and reduce the mitochondrial vacuolation rate.

[0030] Optionally, the drug can increase the content of adenosine triphosphate (ATP) in skeletal muscle cells of patients or animals with chronic fatigue syndrome.

[0031] Optionally, the drug can enhance the activity of key enzymes of the tricarboxylic acid (TCA) cycle in skeletal muscle cells of patients or animals with chronic fatigue syndrome.

[0032] Optionally, the drug can increase the content of pigment C oxidase subunit I (COX I) and the activity of ATP synthase in skeletal muscle cells in patients with chronic fatigue syndrome or animals.

[0033] Optionally, the key enzymes of the tricarboxylic acid cycle include succinate dehydrogenase (SDH), malate dehydrogenase (MDH), and pyruvate dehydrogenase (PDH).

[0034] Optionally, the drug can reduce the malondialdehyde (MDA) content in patients or animals with chronic fatigue syndrome.

[0035] Optionally, the drug can increase superoxide dismutase (SOD) activity, glutathione peroxidase activity, and glutathione (GSH) content in patients with chronic fatigue syndrome or animals.

[0036] Optionally, at least one of A to G is satisfied: A: the drug can relieve the despair and / or depression and / or anxiety of patients with chronic fatigue syndrome or animals; B: the drug can improve the pain threshold of patients with chronic fatigue syndrome or animals; C: the drug can improve the learning and memory ability of patients with chronic fatigue syndrome or animals; D: the drug can improve the sleep quality of patients with chronic fatigue syndrome or animals; E: the drug can improve the immune function of patients with chronic fatigue syndrome or animals; F: the drug can relieve fatigue in patients with chronic fatigue syndrome or animals; G: the drug can relieve neuroendocrine disorders in patients with chronic fatigue syndrome or animals.

[0037] The technical solution of the present invention has the following advantages:

[0038] The present invention is the first to use tranquilizing and brain-strengthening liquid for the study of treating or preventing chronic fatigue syndrome. By establishing a CFS mouse model induced by intraperitoneal injection of polyinosinic acid combined with swimming, and a CFS rat model induced by multi-factor combined stress, and through exhaustive swimming test, tail suspension test, mechanical pain threshold, Barnes maze, sodium pentobarbital sleep test, endocrine function, immune function, skeletal muscle energy metabolism and other methods, it was found that tranquilizing and brain-strengthening liquid can improve the CFS symptoms exhibited by mice and rats in the above two models. Further analysis shows that tranquilizing and brain-strengthening liquid can regulate the dysfunction of the HPA axis caused by chronic fatigue syndrome; improve the thymus index of patients or animals with chronic fatigue syndrome; and reduce the CD8 + T cell ratio; reduce the level of inflammatory factors in patients with chronic fatigue syndrome or animals; activate the mitochondrial biogenesis signaling pathway in patients with chronic fatigue syndrome or animals, improve skeletal muscle mitochondrial function, and improve energy metabolism; reduce the LAC and BUN content in skeletal muscle cells in patients with chronic fatigue syndrome or animals; increase the liver glycogen and muscle glycogen content in patients with chronic fatigue syndrome or animals; relieve oxidative stress damage in patients with chronic fatigue syndrome or animals; increase the immunoglobulin content in patients with chronic fatigue syndrome or animals; increase the complement content in patients with chronic fatigue syndrome or animals; regulate the intestinal flora in patients with chronic fatigue syndrome or animals; regulate amino acid metabolism in patients with chronic fatigue syndrome or animals; and then relieve the symptoms of fatigue, depression, anxiety, poor learning and memory ability, reduced immune function, reduced pain threshold, sleep disorders, and neuroendocrine disorders caused by CFS, and ultimately achieve the effect of treating chronic fatigue syndrome.

[0039] Anshen Jiannao Liquid is composed of five herbs: ginseng, schisandra (fried with vinegar), ophiopogon, wolfberry, and salvia. It is used for dizziness, fatigue, forgetfulness, and insomnia caused by insufficient kidney essence and deficiency of both qi and blood. Among them, ginseng belongs to the traditional tonic and strengthening Chinese medicine. It has the effects of greatly replenishing vital energy, tonifying the spleen and lungs, consolidating the body and promoting the production of body fluids. It has a unique therapeutic effect in improving the body's immunity and is widely used to treat various deficiency syndromes. Ginseng polysaccharides also increase the levels of superoxide dismutase, catalase, and glutathione in renal tissue, as well as ATP and mitochondrial membrane potential. It can improve CFS symptoms and protect the body's health by reducing mitochondrial damage and ROS content. Schisandra is one of the commonly used Chinese herbal medicines for the treatment of CFS. Polysaccharides are the main active ingredients of schisandra, which have the effects of protecting the liver, anti-tumor, enhancing immunity, anti-aging, and anti-fatigue. Lycium barbarum nourishes blood and liver, and ophiopogon nourishes yin and strengthens water. The combination of the two nourishes the liver with yin and blood nourishing medicines to enable the liver to better perform its normal physiological functions. Lycium barbarum and Ophiopogon japonicus can nourish yin and soothe the liver, making the liver blood sufficient and the qi flow smoothly, thus promoting the transportation and transformation function of the spleen and stomach in the middle jiao and the circulation function of qi and blood. Modern experimental studies have shown that Lycium barbarum and Ophiopogon japonicus can improve the secretion of central nervous system transmitters and relieve brain fatigue. Salvia miltiorrhiza can promote blood circulation and dredge collaterals, cool blood and reduce swelling, relieve restlessness and clear the heart. When used together with Lycium barbarum, it can benefit the heart qi and dredge the meridians, so that the dissipated qi can be collected, the qi and blood can be nourished, the qi is strong and the blood is generated, the mind is calmed, and the body fatigue is relieved. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0041] Figure 1 This is the Beta diversity analysis image of the intestinal flora sequencing data of group B rats in Experimental Example 3; in the legend, K: NTB group; M: MB group; D: JNLB group; Z: JNMB group; G: JNHB group; B: BNB group;

[0042] Figure 2 This is the statistical analysis image of metabolites of rats in group B in Experimental Example 4; in the legend, K: NTB group; M: MB group; D: JNLB group; Z: JNMB group; G: JNHB group; B: BNB group;

[0043] Figure 3These are the ultrastructural images of skeletal muscle mitochondria of mice in group A of Experimental Example 5 (×12000, n=4); wherein, A: NTA group; B: MA group; C: JNHA group; D: JNMA group; E: JNLA group; F: BNA group; “1” represents Z line; “2” represents M line; “3” represents mitochondria of normal morphology; and “4” represents mitochondrial vacuoles. DETAILED DESCRIPTION

[0044] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0045] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

[0046] The Anshen Jiannao Liquid and Anshen Bu Nao Liquid used in the present invention are produced by the pharmaceutical factory of Beijing Tong Ren Tang Technology Development Co., Ltd.

[0047] Construction of CFS mouse model

[0048] ① Group A: Intramuscular injection of polyinosinic acid (PolyI:C) combined with swimming to induce CFS mouse model

[0049] Selection of mice: 252 SPF-grade C57BL / 6J mice, weighing 16-18g, both male and female, provided by Sibeifu (Beijing) Biotechnology Co., Ltd., production license number: SCXK (Beijing) 2019-0010, certificate number: 110324221106605663, 110324221107502428, 110324231103307132. The above animals were kept in the barrier environment animal laboratory of Beijing University of Chinese Medicine, and the experimental unit use license number: SYXK (Beijing) 2020-0033. During the experiment, the relative temperature of the animal breeding room was maintained at 23.0±1.0℃, the relative humidity was 55-65%, and the lighting conditions were 12h / 12h light and dark alternation. During the experiment, the animals were free to eat and drink. The experiment was approved by the Experimental Animal Ethics Subcommittee of the Academic Committee of Beijing University of Chinese Medicine, and the ethics numbers are: BUCM-4-2022100802-4046, BUCM-4-2022102201-4054, and BUCM-2023042303-2057.

[0050] Preparation of Poly I:C reagent: Accurately weigh 40 mg of Poly I:C powder, pour into a 20 mL volumetric flask, measure 14 mL of normal saline to dissolve, make up to 20 mL, fully dissolve to a concentration of 2 mg / mL, and prepare before use.

[0051] Construction scheme of CFS model mice: After the mice were adaptively raised, they were preliminarily screened according to their weight and status. Twelve mice were divided into a blank group according to the random number table method, and the rest of the mice were used as the modeling group to participate in the modeling. The modeling group was subjected to forced swimming test on the morning of the first day. The mice were forced to swim with a weight of 5% of their own body weight. The first sinking time and the 3s sinking latency were recorded as the initial value. After 2h of forced swimming, the modeling group mice were intraperitoneally injected with poly I:C (20mg / kg, 0.1mL / 10g). 2h after the injection, the second round of forced swimming test was performed. The 3s sinking latency of the second forced swimming was lower than 60% of the initial value as the standard for the occurrence of fatigue in mice. Screening of fatigued mice. After that, the blank group mice were intraperitoneally injected with normal saline (0.1mL / 10g) every 3 days. The remaining mice were intraperitoneally injected with Poly I:C (20 mg / kg, 0.1 mL / 10 g) every 3 days and simultaneously swam with a load (5% of their body weight) until their entire bodies were submerged in water for 3 seconds in the morning and afternoon every day. The first sinking time and the 3-second sinking latency were recorded.

[0052] Grouping of CFS model mice: The mice with fatigue symptoms were divided into 5 groups according to the stratified random grouping method: model group A (MA), high-dose group A of Anshen Jiannaoye (JNHA), medium-dose group A of Anshen Jiannaoye (JNMA), low-dose group A of Anshen Jiannaoye (JNLA), and group A of Anshen Bu Naoye (BNA), plus blank group A (NTA), a total of 6 groups of animals, 11 to 12 mice in each group, both male and female. Each drug group was given the corresponding drug by gavage once a day. At the same time as the drug administration, the model was continued for 14 days according to the above scheme.

[0053] Dosage of each group: The oral dose of mice in the drug-treated group was calculated according to the body surface area conversion method. Calculated based on the human body weight of 60 kg, the equivalent dose of mice is 9 times the clinical human dose. The clinical human dose of Anshen Jiannao Liquid is 30 mL / d, and the clinical human dose of Anshen Bu Nao Liquid is 20 mL / d. The doses of the medium-dose Anshen Jiannao Liquid (4.50 mL / kg) Group A and the Anshen Bu Nao Liquid (3.00 mL / kg) Group A are clinical equivalent doses; the low-dose Anshen Jiannao Liquid (2.25 mL / kg) Group A is 1 / 2 of the clinical equivalent dose; the high-dose Anshen Jiannao Liquid (9.00 mL / kg) Group A is 2 times the clinical equivalent dose; the NTA group and the MA group were given the same amount of deionized water as the high-dose Anshen Jiannao Liquid Group A.

[0054] ② Group B: CFS rat model induced by multi-factor combined stress

[0055] Selection of rats: 139 SPF SD rats, weighing 140-160g, both male and female, provided by Sibeifu (Beijing) Biotechnology Co., Ltd., production license number: SCXK (Beijing) 2019-0010, certificate number: 110324231102000858, 110324231102001038, 110324231105943568, 110324231105943638. The above animals were kept in the barrier environment animal laboratory of Beijing University of Chinese Medicine, and the experimental unit use license number is SYXK (Beijing) 2020-0033. During the experiment, the relative temperature of the animal breeding room was maintained at 23.0±1.0℃, the relative humidity was 55-65%, and the lighting conditions were 12h / 12h light and dark alternation. During the experiment, the animals were free to eat and drink. The experiment was approved by the Experimental Animal Ethics Subcommittee of the Academic Committee of Beijing University of Chinese Medicine, and the ethics numbers are: BUCM-2023031401-1035, BUCM-2023092703-3181.

[0056] Construction scheme of CFS model rats: After 7 days of adaptive feeding of rats, preliminary screening was carried out according to the weight, open field test and status of the animals. 10 to 12 rats were divided into the blank group according to the random number table method, and the rest of the rats were used as the modeling group to participate in the modeling. The rats in the blank group had a normal diet and were not given any stress intervention. The rats in the modeling group were given a variety of stresses, including: tail clamping, forced swimming, restraint, sleep deprivation, and crowding. Among them: Tail clamping: homemade tail clamp clamped 1 / 3 of the tail end of the rat, 2 minutes / time, 6 times / day, 3 times in the morning and afternoon. Restraint: The rats in the modeling group were placed in a homemade restraint device, the first restraint was 2 hours, and then each time was increased by 30 minutes, 2 times / day, once in the morning and afternoon. Crowding: After the other stresses were completed every day, the rats were pulled back to the feeding room, and the rats in the modeling group were caged together, 6 rats / cage, and 3 rats / cage in the blank group. Forced swimming: Forced swimming is performed on the second day after the stress of other factors ends. The rat is loaded with 4% weight. The standard is that the rat cannot return to the water surface after being immersed in water for 5 seconds. The sinking latency of the rat is recorded. Sleep deprivation: This is carried out for 2 consecutive days. The rat is placed in a homemade water platform at 10:00 on the first day, and is taken out at 8:00 on the second day to feed and water. It is put back in at 10:00 in the morning and taken out at 8:00 on the third day to swim with weight. The water in the sleep deprivation box is changed every day to keep the box clean. In each round, the stress factors such as tail clamping, forced swimming, restraint, sleep deprivation, and crowding are carried out in turn, and each round lasts for 7 days.

[0057] After 4 weeks of modeling, the rats in the modeling group were identified by exhaustive swimming test, tail suspension test and open field test. Compared with the blank group B, the latency time of sinking after exhaustive swimming was significantly reduced (P<0.01, indicating that the rats were in a state of fatigue, which was consistent with the symptoms of clinical CFS patients), the immobility time of tail suspension was significantly increased (P<0.01, indicating that the rats were in a state of despair and depression, which was consistent with the symptoms of clinical CFS patients), and the vertical score and horizontal score in the open field test were significantly reduced (P<0.01, indicating that the rats' autonomous activities and exploratory behavior abilities were weakened, and they showed anxiety and depression, which was consistent with the symptoms of clinical CFS patients). The rats with successful model replication were screened for subsequent experiments.

[0058] Grouping of CFS model rats: The rats with successful modeling were divided into 5 groups according to the stratified random grouping method: model group B (MB), high-dose group B of Anshen Jiannaoye (JNHB), medium-dose group B of Anshen Jiannaoye (JNMB), low-dose group B of Anshen Jiannaoye (JNLB), group B of Anshen Bu Naoye (BNB), plus blank group B (NTB), a total of 6 groups of rats, 10 to 12 rats in each group, both male and female. The corresponding drugs were given by gavage, and the blank control group was given an equal volume of deionized water by gavage. At the same time as the drug administration, the modeling was continued for 14 days according to the above scheme, and the drug administration was continued during the behavioral test.

[0059] Dosage of each group: The oral dose of rats in the drug-treated group was calculated according to the body surface area conversion method. Calculated based on a human body weight of 60 kg, the rat equivalent dose is 6.3 times the clinical human dose. The clinical human dose of Anshen Jiannao Liquid is 30 mL / d, and the clinical human dose of Anshen Bu Nao Liquid is 20 mL / d. The doses of the medium-dose Anshen Jiannao Liquid (3.15 mL / kg) group B and the Anshen Bu Nao Liquid (2.1 mL / kg) group B are clinical equivalent doses; the low-dose Anshen Jiannao Liquid (1.575 mL / kg) group B is 1 / 2 of the clinical equivalent dose; the high-dose Anshen Jiannao Liquid (6.3 mL / kg) group B is 2 times the clinical equivalent dose; the NTB group and the MB group were given the same amount of deionized water as the high-dose Anshen Jiannao Liquid group B.

[0060] In each experimental example, unless otherwise specified, the statistical method used was: IBM SPSS Statistics 25 software was used to perform statistical analysis on the experimental results. Data that met the normal distribution were subjected to a one-way ANOVA test. Indicates; non-parametric tests were performed on non-normally distributed data, and the data were expressed as Md(P 25 , P 75 ) indicates that P < 0.05 was considered to be significantly different.

[0061] Experimental Example 1 (Behavioral Measurement)

[0062] For mice in group A and rats in group B: After 14 days of drug administration, behavioral tests including exhaustive swimming, tail suspension, elevated plus maze, mechanical withdrawal threshold (MWT), Barnes maze and sleep induced by threshold hypnotic dose of sodium pentobarbital were performed 1 hour after drug administration every day.

[0063] ① Exhaustive swimming: The mice were placed in the behavioral laboratory to adapt to the environment for 1 hour. After weighing, a weight of 6% of their body weight was tied to the base of their tails. They were placed in a bucket for a weight-bearing exhaustive swimming test. The water depth exceeded the length of the mice and the water temperature was 21-23°C. The first sinking time and exhaustion time of the mice were recorded. The time from the start of the test to the first time the entire trunk of the mice was immersed in water was recorded as the first sinking time. The time from the start of the test to the time when the mouth and nose of the mice sank into the water for more than 6 seconds and still could not return to the surface of the water was recorded as the exhaustion time. The mice in group A were placed in the behavioral laboratory immediately after the end of the drug administration, and the rats in group B were placed in the behavioral laboratory on the day of the test. The exhaustion time can reflect the physical strength and fatigue resistance of the mice. It is an important indicator for measuring the degree of fatigue of the mice. The shortening of the exhaustion time indicates that its fatigue degree has deepened. The obtained data are shown in Table 1.

[0064] In all tables: Compared with the blank group, # P<0.05, ## P<0.01; compared with the model group, * P<0.05, ** P<0.01.

[0065] Table 1

[0066]

[0067] Mice in group A: Compared with the NTA group, the first sinking time and exhaustion time of mice in the MA group were significantly shortened (P<0.01), indicating that the physical strength of mice in the MA group decreased significantly and fatigue occurred. Compared with the MA group, the first sinking time of mice in the JNHA and JNMA groups was significantly prolonged (P<0.05), and the exhaustion time of mice in the JNHA group was significantly prolonged (P<0.01), indicating that Anshen Jiannao Liquid can improve the fatigue state of CFS mice.

[0068] Rats in group B: Compared with the NTB group, the exhaustion time of rats in group MB was significantly shortened (P<0.01). Compared with the MB group, the exhaustion time of rats in group B and group BNB was significantly prolonged (P<0.01), indicating that both Anshen Jiannao Liquid and Anshen Bu Nao Liquid can significantly improve the fatigue of CFS rats.

[0069] ② Tail suspension test: The tail of the experimental animal is fixed so that its head is suspended downward. The animal struggles in this environment and tries to escape from the predicament, thus providing an unavoidable oppressive environment. The experimental animal struggles desperately in this environment and tries to escape but cannot escape, and appears in an "immobile state", reflecting a state called "behavioral despair". This behavioral despair model is similar to depression, so tail suspension tests are often performed in animal experiments to evaluate animal despair and depression. The continuous struggle of the mouse during the experiment reflects the physical condition of the mouse, and the time the mouse hangs its tail and does not move reflects the severity of the mouse's despair. The data obtained are shown in Table 2.

[0070] Table 2

[0071]

[0072] Mice in group A: Compared with the NTA group, the tail suspension immobility time of mice in the MA group was significantly prolonged (P<0.01), and the despair was severe, showing depression. Compared with the MA group, the tail suspension immobility time of mice in the JNHA, JNMA and BNA groups was significantly shortened (P<0.01 or P<0.05), indicating that Anshen Jiannao Liquid and Anshen Bu Nao Liquid can relieve the despair and depression of mice and relieve psychological fatigue.

[0073] Rats in group B: Compared with the NTB group, the immobility time of the tail suspension of the rats in group MB was significantly prolonged (P<0.01), indicating that the rats in group MB showed behavioral despair model and depression. Compared with the MB group, the immobility time of the tail suspension of the rats in group B after administration of Anshen Jiannaoye was significantly shortened (P<0.01), and the immobility time of the rats in group BNB was significantly shortened (P<0.01), indicating that Anshen Jiannaoye and Anshen Bu Naoye can effectively improve the behavioral despair state and depression of the rats with depression.

[0074] ③ Elevated plus maze: The elevated plus maze is a maze with four choices, two of which are open spaces without wall protection, and the other two are closed spaces. Animals will seek shelter by nature, so they will stay in the closed arms with shelter for a longer time. The fear of open areas by nature of mice is used to observe their anxiety or fear-induced behaviors. The data are shown in Table 3.

[0075] Rats in group B: Compared with the NTB group, the percentage of time spent in the open arm (OT%) of the MB group was significantly reduced (P<0.05), indicating that their autonomous activities were reduced and anxiety was present. Compared with the MB group, the percentage of open arm entries (OE%) and the percentage of time spent in the JNMB group were significantly increased (P<0.01), indicating that Anshen Jiannaoye can relieve anxiety in CFS rats.

[0076] Table 3

[0077]

[0078] ④ Mechanical withdrawal reflex threshold: The pain test is an experimental method used to evaluate the pain sensitivity of animals. By using Von Frey fiber hairs or electronic Von Frey pressure analgesics to stimulate the soft part of the mouse's foot, their reaction threshold to mechanical pain stimulation is measured. This is used to evaluate the occurrence, duration and intensity of pain, and to test the effect of drugs. The A group of mice were stimulated with Von Frey fiber hairs at the thinner part of the foot pad, and the up-and-down method was used for measurement; the 50% positive reaction dose (ED) was obtained according to the following formula and the mechanical pain threshold calculation table. 50 );ED 50 =10 loga+k × b (a: the corresponding value of the cilia in the last test; b: the average value of the logarithmic difference of the cilia used; k: the corresponding parameter value in the table). The electronic Von Frey pressure analgesia was used to measure the skin on the plantar side of the left sole of the rats in group B until the leg was raised or the foot was actively retracted, and the mechanical foot retraction threshold (expressed in g) was measured. Each rat was measured 3 times, each time with an interval of 5 minutes, and the average value was taken. The measured data are shown in Table 4.

[0079] Mice in group A: Compared with the NTA group, the ED of mice in the MA group 50 The ED of mice in the JNHA, JNMA and BNA groups was significantly lower than that in the MA group (P<0.01), indicating that the pain threshold of mice in the MA group was lower, which is consistent with the symptoms of muscle soreness in clinical CFS patients. 50 The levels of CFS mice showed a significant increase (P<0.01), indicating that Anshen Jiannao Liquid and Anshen Bu Nao Liquid can increase the pain threshold of CFS mice and relieve the pain.

[0080] Rats in group B: Compared with the NTB group, the pain threshold of rats in the MB group was significantly reduced (P<0.01), indicating that CFS rats had symptoms of pain. Compared with the MB group, the pain threshold of rats in the JNHB and JNMB groups increased (P<0.05 or P<0.01), and the pain threshold of rats in the BNB group increased (P<0.05). This shows that Anshen Jiannao Liquid and Anshen Bu Nao Liquid can effectively relieve the symptoms of pain in CFS rats.

[0081] Table 4

[0082]

[0083] ⑤ Barnes maze: Impairment in learning and memory is a typical symptom of CFS. The Barnes maze was established by taking advantage of the characteristics of rodents that they avoid light, like darkness and love to explore. It can be used to detect the spatial memory ability of animals. During the test, the longer the escape latency, the lower the animal's learning and memory ability. This model has less stress stimulation on animals. It does not require fasting like the radial arm maze (eight-arm maze), nor is it as stressful as the water maze. Therefore, it is more commonly used in memory research, especially for stress-related memory research. The specific operation is: place the experimental animals in the behavioral laboratory for adaptation preparation for 1 hour, place the target box at a hole on the experimental table, and mark the surrounding of the experimental area. After that, the target box will be installed at the marked hole in all experiments. At the beginning of the experiment, the mouse was gently placed in the center of the platform. A light-proof barrel was used to hold the mouse so that it could adapt for 10 seconds, and then the barrel was picked up. The observer observed at least 1 meter away from the maze. If the mouse did not find the target box within 3 minutes, the experimenter would guide the mouse to the target box and stay there for 60 seconds. If the mouse found the target box within the specified time, it would be taken out after the mouse adapted to the target box for 60 seconds. This training was conducted twice a day, and the learning and memory ability test was conducted after 4 days. The test method was the same as the training method. The activity of the mouse was observed within 3 minutes. Regardless of whether the target box was found or not, the mouse was no longer guided to enter. The escape latency and number of errors of the mouse were recorded. The measured data are shown in Tables 5 to 8.

[0084] Mice in group A: During the learning period, the escape latency of mice in group MA was significantly prolonged on the 4th day compared with that in group NTA (P<0.05). Compared with group MA, the escape latency of mice in groups JNMA, JNLA and BNA was significantly shortened on the 4th day (P<0.05). During the test period, compared with group NTA, the escape latency of mice in group MA was significantly prolonged (P<0.01), and the number of errors was significantly increased (P<0.05). Compared with group MA, the escape latency of mice in groups JNMA, JNHA and BNA was significantly shortened (P<0.05); the number of errors of mice in group BNA was significantly reduced (P<0.05). This indicates that Poly I:C combined swimming can cause a decrease in the learning and memory ability of mice, which is consistent with the symptoms of memory impairment in clinical CFS patients. Anshen Jiannaoye and Anshen Bu Naoye can significantly improve their learning and memory impairment.

[0085] Rats in group B: During the learning period, the escape latency of rats in group MB was significantly prolonged on the 4th day compared with that in group NTB (P<0.01). Compared with group MB, the escape latency of rats in groups JNMB and JNHB was significantly shortened on the 4th day (P<0.05). During the test period, the escape latency of rats in group MB was significantly prolonged compared with that in group NTB (P<0.01), indicating that CFS rats had problems such as learning and memory disorders. Compared with group MB, the escape latency of rats in groups JNMB, JNHB and BNB was significantly shortened (P<0.05), indicating that Anshen Jiannaoye and Anshen Bu Naoye can effectively improve the learning and memory disorders of CFS rats.

[0086] Table 5 Escape latency of mice in group A (s)

[0087]

[0088] Table 6 Number of errors of mice in group A (times)

[0089]

[0090] Table 7 Escape latency of rats in group B (s)

[0091]

[0092] Table 8 Number of errors of rats in group B (times)

[0093]

[0094] ⑥ Sodium pentobarbital experiment: By injecting the same threshold hypnotic dose of sodium pentobarbital into each group of mice, we observed whether Anshen Jiannao Liquid and Anshen Bunao Liquid could prolong the sleep time of sodium pentobarbital and improve the sleep disorder of CFS mice. The data are shown in Tables 9 and 10.

[0095] Mice in group A: Compared with the NTA group, the sleep latency of mice in the MA group was significantly prolonged (P<0.05), indicating that CFS mice had difficulty falling asleep and sleep disorders, which was consistent with the clinical symptoms of CFS. Compared with the MA group, the sleep latency of the JNMA group was significantly reduced (P<0.05), indicating that Anshen Jiannao Liquid can improve the difficulty of falling asleep in CFS mice.

[0096] Rats in group B: Compared with the NTB group, the sleep duration of rats in the MB group was significantly shortened (P<0.05), and sleep disorders occurred. Compared with the MB group, the sleep duration of rats in the JNHB, JNMB and BNB groups was significantly prolonged (P<0.05 or P<0.01), indicating that Anshen Jiannaoye and Anshen Bu Naoye can improve the sleep disorders of CFS rats.

[0097] Table 9 Sleep of mice in group A

[0098]

[0099] Table 10 Sleep of rats in group B

[0100]

[0101]

[0102] Experimental Example 2 (Determination of neuroendocrine function and immune function)

[0103] Sample collection: After the behavioral test, mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital, and blood was collected from the retro-orbital venous plexus. Serum and plasma were collected. The mice were killed by dislocating the cervical vertebra after blood collection. The prefrontal cortex, hippocampus, hypothalamus and skeletal muscle were collected and packaged in cryopreservation tubes and stored in a -80℃ refrigerator. The abdominal cavity of the mouse was cut open, the thymus was taken out, and the weight of the thymus was weighed and recorded.

[0104] (1) Detection of HPA axis function (neuroendocrine): The HPA axis includes the hypothalamus, pituitary gland, and adrenal glands. It is an important part of the neuroendocrine system and is involved in the regulation of the body's stress response and immune system function. Some CFS patients, such as the constructed group A mice, have significantly reduced HPA axis activity and reduced CORT content. HPA axis dysfunction can cause abnormalities in multiple systems such as endocrine, immune, and nervous systems, leading to multiple symptoms such as fatigue, abnormal emotions, and poor learning and memory abilities.

[0105] For some CFS patients, such as the rats in group B, repeated activation of the HPA axis can produce damaging physiological effects that cause it to function abnormally, which will inevitably cause abnormal secretion of cortisol, melatonin, 5-HT, etc., which will produce clinical manifestations such as weakness and fatigue, sleep disorders, cognitive decline, and emotional abnormalities. These manifestations are basically consistent with the clinical symptom group of CFS. Under the action of stress, the negative feedback inhibition effect of the HPA axis is weakened, resulting in a peak in the secretion of ACTH and CORT, leading to the hyperfunction of the HPA axis.

[0106] ELISA (enzyme-linked immunosorbent assay) was used to detect the levels of CORT and ACTH in the mouse serum, the level of CRH in the hypothalamus, and the levels of 5-HIAA, IFN-α, 5-HT and 5-HTT in the prefrontal cortex, hippocampus and hypothalamus.

[0107] ①CORT in mice in group A, data are shown in Table 11: Compared with the NTA group, the CORT content of mice in group MA was significantly reduced (P<0.01), which is consistent with the symptoms of CFS patients. Compared with the MA group, the CORT content of JNHA, JNMA and BNA groups was significantly increased (P<0.01 or P<0.05). Anshen Jiannao Liquid and Anshen Bu Nao Liquid can activate the HPA axis, thereby improving the state of endocrine, immune and nervous system dysfunction in CFS mice in group A.

[0108] Table 11

[0109]

[0110] The content of IFN-α in the prefrontal cortex and hypothalamus of the MA group was significantly increased, the content of 5-HTT in the hypothalamus was significantly increased, and the content of 5-HT in the hippocampus and hypothalamus was significantly decreased, indicating that the neuroendocrine function of the mice in the MA group was disordered. Neuroendocrine function is closely related to CFS, and CFS patients show disorders of neurotransmitters in the brain. 5-HT plays an important role in the brain. The production of sleep is closely related to central 5-HT. Mental activity and emotions are also regulated by 5-HT. When the central 5-HT level decreases, symptoms of CFS such as poor mental state, slow reaction, decreased attention, laziness, decreased sleep quality, easy tension, easy panic, easy anger, loss of previous interests and hobbies will appear, and emotions such as depression and anxiety will be shown. The decrease in 5-HT content will also cause a decrease in pain threshold, affecting people's stress level to pain. 5-HT is also considered to be a key neurotransmitter involved in stress-induced anxiety. Accelerated 5-HT metabolism can occur in the brain tissue of animals under stress. 5-HIAA is a metabolite of 5-HT. Studies have shown that the 5-HIAA content in the brain of CFS rats decreases. Peripheral injection of PolyI:C triggers an inflammatory response, increases IFN-α, and the released inflammatory factors act on the central nervous system through the blood-brain barrier, leading to the expression of 5-HTT mRNA in astrocytes, thereby increasing their 5-HTT content. Anshen Jiannao Liquid can adjust the content of these factors, reduce central inflammatory response, reduce 5-HTT content, increase 5-HT content in the brain, regulate 5-HIAA content, and thus regulate the central nervous function of CFS mice.

[0111] ② In the prefrontal cortex of mice in group A: Compared with the NTA group, the IFN-α content of mice in the MA group was significantly increased (P<0.01). Compared with the MA group, the IFN-α content of mice in the JNHA group and the BNA group was significantly decreased (P<0.05). See Table 12 for details.

[0112] Table 12

[0113]

[0114] ③In the hippocampus of mice in group A: Compared with the NTA group, the 5-HIAA and 5-HT levels in mice in the MA group were significantly reduced (P<0.05). Compared with the MA group, the 5-HT levels in mice in the JNMA group and the BNA group were significantly increased (P<0.05). See Table 13 for data.

[0115] Table 13

[0116]

[0117] ④In the hypothalamus of mice in group A: Compared with the NTA group, the contents of 5-HIAA, IFN-α and 5-HTT in mice in group MA were significantly increased (P<0.01), and the content of 5-HT was significantly decreased (P<0.01). Compared with the MA group, the contents of 5-HIAA, IFN-α and 5-HTT in mice in groups JNHA and JNMA were significantly decreased (P<0.01 or P<0.05), the contents of IFN-α and 5-HTT in mice in group BNA were significantly decreased (P<0.01 or P<0.05), and the contents of 5-HT in mice in groups JNHA and BNA were significantly increased (P<0.05). The data are shown in Table 14.

[0118] Table 14

[0119]

[0120] ⑤ In the hypothalamus of rats in group B: Compared with the NTB group, the CRH content of rats in the MB group increased significantly (P<0.01). Compared with the MB group, the CRH content of rats in the JNHB group and the BNB group decreased significantly (P<0.01 or P<0.05). The data are shown in Table 15.

[0121] Table 15

[0122]

[0123] The main function of CRH is to promote the synthesis and release of ACTH by the pituitary gland. CRH is found in tissues outside the hypothalamus, such as the amygdala, hippocampus, midbrain, pineal gland, gastrointestinal tract, pancreas, adrenal gland, placenta, etc. The hypothalamus is a key link in initiating the regulation of HPA axis function. Detecting the CRH content in the hypothalamus can effectively measure the functional state of the HPA axis. Under stress, the secretion of ACTH and CORT increases, and CRH, as an upstream substance of the HPA axis, will also increase in content.

[0124] ⑥ ACTH in serum of rats in group B: Compared with the NTB group, the ACTH content of rats in the MB group increased significantly (P<0.05), indicating that the secretion peak of ACTH in CFS rats in the MB group led to the hyperfunction of the HPA axis, which caused the CFS rats in the MB group to suffer from asthenic fatigue, sleep disorders, decreased cognitive function, abnormal emotions, etc. Compared with the MB group, the ACTH content of the JNHB group and the BNB group decreased significantly (P<0.01 or P<0.05), indicating that Anshen Jiannaoye and Anshen Bu Naoye can alleviate the hyperfunction of the HPA axis in CFS rats. The data are shown in Table 15.

[0125] ⑦ CORT in serum of rats in group B: Under stress and other conditions, serum CORT increases. Long-term increase in CORT can lead to damage to hippocampal morphology and function and decreased learning and memory ability. Compared with the NTB group, the CORT content of rats in the MB group increased significantly (P<0.01), indicating that the HPA axis of CFS rats in the MB group was hyperactive. Compared with the MB group, the CORT content of the JNMB, JNHB and BNB groups decreased significantly (P<0.01 or P<0.05). The data are shown in Table 15.

[0126] (2) Detection of immune function: Long-term immune response disorder is considered to be one of the causes of CFS. The human immune system is composed of immune organs, immune cells and immune active substances, which have the functions of immune surveillance, defense and regulation. Among them, immune active substances include antibodies, lysozyme, complement, immunoglobulin, interferon, interleukin (IL), cytokines, etc.; one of the most important immune organs is the thymus.

[0127] ① Thymus index: As an important immune organ of the body, the size of the thymus index reflects the size of the immune organ and indirectly reflects the immune function of the body. As an important innate immune organ, the decrease in the thymus index indicates that the immune organ of the experimental mouse is atrophied, the function is reduced, and the immune function is reduced. The calculation formula is: mouse: thymus index = thymus mass (mg) / body mass (g); rat: thymus index = thymus mass (g) / body mass (100g). See Table 16 for data.

[0128] Thymus index of mice in group A: Compared with the NTA group, the thymus index of mice in group MA was significantly decreased (P<0.05), indicating that their immune function declined. Compared with the MA group, the thymus index of the JNMA group was significantly increased (P<0.05), indicating that it can improve the immune function of CFS mice in the MA group.

[0129] Thymus index of rats in group B: Compared with the NTB group, the thymus index of rats in group MB was significantly lower (P<0.01), indicating that the immune organs of CFS rats in group MB were atrophied and the immune function was decreased. Compared with the MB group, the thymus index of rats in group B and BNB groups at all doses of Anshen Jiannao Liquid was significantly increased (P<0.01 or P<0.05), indicating that Anshen Jiannao Liquid and Anshen Bu Nao Liquid can improve the immune function of CFS rats in group MB and enhance their body resistance.

[0130] Table 16

[0131]

[0132] ② Peripheral blood T lymphocyte surface antibody markers: CFS patients have immune dysfunction, which is manifested as reduced immunity. T lymphocytes are the most numerous and important type of immune active functional cells in the human body. They are an important part of the body's immune system. When affected by specific antigens or mitogens, they can transform themselves into lymphoblasts. The level of their transformation can reflect the proliferation level of T lymphocytes and the state of the body's cellular immune function. Mature T lymphocytes are divided into CD4 + Helper T cells and CD8 + Cytotoxic T cells, these two cells have different immune regulatory functions and can enhance or inhibit the activity of other immune cells. + T cells have the function of inhibiting immune cell activity and reducing immune response, while CD4 + T is a kind of auxiliary functional cell, which plays a key role in the human immune barrier. + 、CD4 + / CD8 + A decrease in the level means a decrease in the body's immunity. A decrease in immunity increases the risk of infection, which will aggravate the occurrence and development of CFS. + 、CD8 + The data are shown in Table 17.

[0133] Mice in group A: Compared with those in group NTA, mice in group MA had a higher CD4 + The proportion of T cells decreased significantly (P<0.01), and CD8 + The proportion of T cells in the JNHA group was significantly increased (P<0.05), indicating that the immune capacity of CFS mice in the MA group was reduced. + The proportion of T cells was significantly reduced (P<0.05). This indicates that Anshen Jiannao Liquid can improve the immune function of mice, enhance their resistance, and thus resist fatigue.

[0134] Table 17

[0135]

[0136] ③ Serum inflammatory factor content: IFN-γ and IL-2 are important cytokines mediated by Th1 immune response, which can increase the IL-6 level in CFS patients and aggravate immune disorders. IL-6 is an important proinflammatory cytokine in clinical practice. Its value increases significantly under the influence of inflammatory response, which in turn promotes the aggregation of neutrophils in the lesion and aggravates the inflammatory response. IL-1β, IL-6 and TNF-α are closely related. TNF-α is the upstream cytokine of IL-1β, and IL-1β and TNF-α are the main activators of IL-6. The three interact with each other, further promoting the production of proinflammatory cytokines, leading to the continuous strengthening of inflammatory response. The excessive presence of proinflammatory cytokines can seriously affect cognitive function. IL-1β, IL-6 and TNF-α can also cause neuronal damage by releasing a large number of inflammatory mediators such as oxygen free radicals and lysosomal enzymes outside the cells. IL-1β and TNF-α can also destroy the blood-brain barrier and allow peripheral inflammatory factors to enter the central nervous system. IL-1β and IL-6, as "endogenous pyrogens", mediate the interaction of the neuro-endocrine-immune network, regulate behaviors such as fever, eating and sleeping, and induce symptoms such as fatigue and depression. CRP is an inflammatory response marker secreted by the liver and has an immune regulatory function on the body. The more severe the fatigue of the patient, the higher the levels of IL-6 and CRP. Studies have shown that the levels of IL-10, IFN-γ and TNF-α in the peripheral blood of CFS patients are increased. The ELISA method was used to determine the levels of CRP, IL-1β, IL-6, IL-2, IFN-γ and TNF-α in mouse serum. The data are shown in Tables 18 and 19.

[0137] Mice in group A: Compared with the NTA group, the levels of CRP, IL-1β, IL-6, IL-2, IFN-γ and TNF-α in mice in group MA were significantly increased (P<0.01), indicating that CFS mice in group MA had peripheral inflammation, and at the same time, they passed through the blood-brain barrier to cause central inflammation, up-regulated the content of 5-HTT, and thus led to a decrease in the central 5-HT content, showing depression. Compared with the MA group, the levels of CRP, IFN-γ, IL-1β and IL-6 in group A and group BNA of each dose of Anshen Jiannaoye were significantly decreased (P<0.01). The levels of IL-2 in the JNHA, JNMA and BNA groups were significantly decreased (P<0.01 or P<0.05). The levels of TNF-α in the JNHA and BNA groups were significantly decreased (P<0.01 or P<0.05). This shows that Anshen Jiannaoye and Anshen Bu Naoye can significantly reduce the levels of inflammatory factors and alleviate the inflammatory response in CFS mice.

[0138] Table 18

[0139]

[0140] Table 19

[0141]

[0142] ④ Immunoglobulin content: Immunoglobulin (Ig) is a globulin similar to antibodies and plays an important role in the body's defense against diseases. IgG is the main component of serum immunoglobulins, accounting for 75% of total immunoglobulins. It is the most persistent and important antibody in the primary immune response and exists only in monomeric form. Most antibacterial, antitoxic and antiviral antibodies belong to IgG, which plays a leading role in anti-infection. Some researchers have found that CFS symptoms are significantly correlated with the content of IgG subclasses IgG3 and IgG4. The decrease in IgG content in CFS patients indicates that their body's anti-infection immunity is reduced. IgM is the first Ig produced in the immune response induced by antigen stimulation. It can bind to complement and is mainly distributed in serum. IgA can mediate antibody-dependent cell-mediated cytotoxicity (ADCC) of opsonized phagocytic antibodies. IgA and IgG are important lines of defense for humoral immunity and can activate complement after binding to the corresponding antigens. Clinical trials have found that compared with the immunological indicators of healthy people, the levels of serum immunoglobulins IgA, IgG and IgM in CFS patients are lower than those in healthy people. The ELISA method was used to determine the levels of serum IgA, IgG and IgM in rats. The data are shown in Table 20.

[0143] Rats in group B: Compared with the NTB group, the IgG, IgM and IgA levels of rats in the MB group were significantly decreased (P<0.01), which was consistent with the clinical indicators of CFS patients. Compared with the MB group, the IgG level of the JNMB group was significantly increased (P<0.01), the IgM level of the JNHB group and the BNB group was significantly increased (P<0.01), and the IgA level of the JNMB, JNHB group and the BNB group was significantly increased (P<0.01), indicating that Anshen Jiannaoye and Anshen Bu Naoye can improve the immune ability of CFS rats and stabilize the immune system.

[0144] Table 20

[0145]

[0146] ⑤C3 and C4 content: Complement is the main component of nonspecific immunity and plays an important role in guiding specific immunity and maintaining immune homeostasis. C3 is an important complement protein in the complement system. It plays a pivotal role in the activation process of the complement system and has the important function of regulating immunity. C4 has the functions of regulating complement activation, preventing immune complex deposition, neutralizing viruses, immune recognition, and maintaining immune homeostasis. The reduction of C3 and C4 content indicates that the body's immune function is reduced, causing immune system disorders and inducing CFS. The ELISA method was used to determine the C3 and C4 content in rat serum. The data are shown in Table 21.

[0147] Rats in group B: Compared with the NTB group, the C3 and C4 contents of rats in group MB were significantly decreased (P<0.01), indicating that the immunity of CFS rats in group MB was reduced. Compared with the MB group, the C3 contents of group B and group BNB increased significantly (P<0.01) after administration of Anshen Jiannaoye at different doses, and the C4 contents of groups JNMB, JNHB and BNB increased significantly (P<0.01), which improved the immune function of CFS rats.

[0148] Table 21

[0149]

[0150] Experimental Example 3 (Analysis of fecal intestinal flora)

[0151] Studies have found that typical symptoms of CFS, such as digestive disorders, depression, cognitive problems, and even fatigue itself, are believed to be closely related to intestinal microbiome disorders. In terms of treatment, clinical trials have demonstrated the efficacy of probiotics in reducing anxiety and inflammatory biomarkers in ME / CFS patients. Therefore, exploring the causal relationship or association between changes in the intestinal microbiome and CFS has become a new direction for exploring the pathogenesis of CFS.

[0152] Feces of experimental rats were collected as samples during behavioral testing. The third-generation full-length 16srRNA gene sequencing method was used. The third-generation microbial diversity is based on the PacBio sequencing platform, using the single-molecule real-time sequencing (SMRT Cell) method, and multiple rounds of sequencing of a single fragment based on the HIFI mode, which ensures the length of the sequencing fragment while ensuring the accuracy of the single fragment sequencing data.

[0153] The specific detection method is as follows: Total genomic DNA was extracted from rat fecal samples using the TGuide S96 Magnetic Soil / Feces DNA Kit. The quality and quantity of the extracted DNA were checked using electrophoresis on a 1.8% agarose gel, and the DNA concentration and purity were determined using a NanoDrop 2000 UV-Vis spectrophotometer. The full-length 16S rRNA gene was amplified with primer pair 27F: AGRGTTTGATYNTGGCTCAG and 1492R: TASGGHTACCTTGTTASGACTT. Both the forward and reverse 16S primers were tailed with a sample-specific PacBio barcode sequence to allow for multiplex sequencing. We chose to use barcoded primers because this reduced the formation of chimeras compared to the alternative of adding primers in a second PCR reaction. PCR amplification was performed for 25 cycles using KOD One PCR premix, with an initial denaturation at 95°C for 2 min, followed by 25 cycles of denaturation at 98°C for 10 s, annealing at 55°C for 30 s, extension at 72°C for 1.5 min, and a final step of 2 min at 72°C. The total number of PCR amplicons was purified with VAHTSTM DNA cleaning beads and quantified using the Qubit dsDNA HS Detection Kit and Qubit3.0 Fluorometer (Invitrogen, Thermo Fisher Scientific, Oregon, USA). After a single quantification step, the amplicons were combined in equal amounts. SMRTbell Express Template Prep Kit 2.0 prepared SMRTbell libraries from amplified DNA. Sequencing of purified SMRTbell libraries in mixed and barcoded samples was performed on the PacBio Sequel II platform using the Sequel II Binding Kit 2.0.

[0154] Statistical methods for intestinal flora: Qualified sequences with a similarity threshold of more than 97% were assigned to an operational taxonomic unit (OTU) using USEARCH (version 10.0). OTU / ASVs were classified and annotated based on the naive Bayes classifier in QIIME2 using the SILVA database (version 138.1) with a confidence threshold of 70%. Alpha identification of species diversity for each sample was performed using QIIME2 software. Beta diversity calculations were analyzed by the principal coordinates analysis (PCoA) method based on Euclidean distance to assess the diversity of species complexity in the samples. Bacterial abundance and diversity were compared using one-way analysis of variance. LDA and LEfSe were combined to evaluate differentially abundant taxa.

[0155] ① Beta diversity analysis of intestinal flora sequencing data

[0156] Rats in group B: Figure 1 As shown in the figure, the structure of the intestinal flora of each group of samples was analyzed based on the PCoA method. The Beta method was used for analysis. The distance between the sample points was negatively correlated with the similarity of the intestinal flora structure of the samples. The distance between the samples in each group was small, and the similarity within the group was large, indicating that the difference within the group was small. The distance between the NTB group and the MB group was large, and the similarity between the groups was small, indicating that there was a large difference in the intestinal flora structure of the two groups of rats. Comparing the distance between the MB group and the BNB group and the B group samples of the Anshen Jiannaoye, it was farthest from the JNHB group, followed by the BNB group, and finally the JNLB group and the JNMB group. The farther the distance between the MB group and each group, the smaller the similarity between the groups, and the more obvious the difference in the intestinal flora structure of rats between the groups. The relief of the symptoms of CFS rats by Anshen Jiannaoye and Anshen Bu Naoye may be related to the influence on the intestinal flora structure of CFS rats.

[0157] ② Species annotation and taxonomic analysis of intestinal flora sequencing

[0158] The intestinal flora describes the microbial community in the gastrointestinal tract, which is composed of a large number of bacteria, archaea, bacteriophages, yeasts, protozoa and fungal species, which have a symbiotic relationship with the human intestine. There are a large number of microorganisms in the intestine, with a wide variety and large numbers, most of which are anaerobic bacteria. At the phylum level, it is divided into four major categories: Bacteroidetes, Firmicutes, Proteobacteria and Actinobacteria. Functionally, it is divided into three categories: beneficial bacteria, symbiotic bacteria and harmful bacteria. The intestinal microbiome can affect the strength of the intestinal barrier, the activation of intestinal immune cells and inflammatory signaling pathways in many ways, participate in the body's protection, growth, metabolism and immune processes, and jointly maintain the body's health.

[0159] Comparison of stool samples from CFS patients and healthy controls revealed that the relative abundance of Firmicutes decreased in CFS patients, while the relative abundance of Bacteroidetes increased. Many members of Firmicutes are beneficial bacteria, such as Lactobacillus, Faecalibacterium, Eubacterium, and Roseburia, which produce acetate, butyrate, lactate, and antibacterial substances that prevent pathogens from interfering with health, stabilize colon cell function, reduce inflammation, protect the intestinal barrier, and promote intestinal microbial balance.

[0160] Rats in group B: Comparison of the differences in the phylum level among the rats in each group is shown in Table 22. In the rats in the NTB group, the combined proportion of Bacteroidetes and Firmicutes was as high as more than 95%, followed by Proteobacteria, etc., and also included other less abundant phyla, such as Cyanobacteria and Desulfobacterota. Compared with the intestinal flora in the rats in the NTB group, the proportion of Bacteroidetes, Proteobacteria, Cyanobacteria, and Desulfobacterota in the rats in the MB group increased, and the proportion of Firmicutes decreased. Compared with the rats in the MB group, the proportion of Bacteroidetes and Proteobacteria in the B group and the BNB group of each dose of Anshen Jiannaoye decreased, and the proportion of Firmicutes increased. Anshen Jiannaoye and Anshen Bu Naoye can promote the production of acetate, butyrate, lactate and antibacterial substances, protect the intestinal barrier and promote the balance of intestinal microorganisms, and resist inflammation.

[0161] Table 22

[0162]

[0163] ③ Analysis of differences between groups of intestinal flora sequencing data

[0164] Ruminococcus can produce butyrate. The abundance of Ruminococcus is reduced in CFS patients, and the abundance of Ruminococcus is negatively correlated with the severity of fatigue symptoms in CFS patients. The lower the abundance of Ruminococcus, the higher the fatigue level in CFS patients.

[0165] Rats in group B: See Table 23. At the family level, compared with the MB group, the abundance of unclassified_Izemoplasmatales, Anaplasmataceae, uncultured_Ruminococcaceae_bacterium and Deferribacteraceae in the JNMB group was significantly increased (P<0.05), indicating that the medium dose of Anshen Jiannao Liquid can reduce the fatigue level of CFS patients and improve the fatigue symptoms of patients.

[0166] Table 23

[0167]

[0168] Experimental Example 4 (Non-targeted metabolomics analysis of plasma)

[0169] The onset of CFS involves multiple systems of the body, and the pathogenesis is still unclear. Non-targeted metabolomics simultaneously conducts qualitative and quantitative analysis of almost all metabolites under certain physiological states or specific conditions to find and identify differential metabolites. Compared with genomics, transcriptomics and proteomics, as a systematic metabolite analysis platform, non-targeted metabolomics has unique advantages in the development and verification of metabolite-related disease diagnostic markers and the study of metabolite-related signaling pathway mechanisms.

[0170] Sample processing: Take 100 μL of plasma, add 500 μL of extract containing L-2-chlorophenylalanine internal standard (methanol: acetonitrile = 1:1, internal standard concentration 20 mg / L), vortex mix for 30 seconds, ultrasonicate in ice water bath for 10 minutes, stand at -20℃ for 1 hour, centrifuge at 4℃, 12000r / min for 15 minutes, take out 500 μL of supernatant in EP tube, and dry the extract in a vacuum concentrator. Add 160 μL of 50% acetonitrile aqueous solution to the dried metabolites for re-dissolution, vortex for 30 seconds, ultrasonicate in ice water bath for 10 minutes, centrifuge at 4℃, 12 000r / min for 15 minutes, take 120 μL of supernatant in 2mL injection bottle, and take 10 μL of each sample to mix into QC sample for injection and detection.

[0171] Chromatographic and mass spectrometric analysis: The samples were pre-separated by ACQUITY-UPLC-T3 column. Mobile phase A was 0.1% formic acid in water; mobile phase B was 0.1% formic acid in acetonitrile, gradient elution: 0-0.5 min, 5% B; 0.5-5.5 min, 5%-50% B; 5.5-9 min, 50%-95% B; 9-10.5 min, 95% B; 10.5-12.0 min, 95%-5% B. The pre-separated sample metabolites were collected by Xevo G2-XSQTof high-resolution mass spectrometry with dual-channel data acquisition of 2V low collision energy and 10-40V high collision energy.

[0172] ESI ion source parameters: capillary voltage 2000 V in positive ion mode, or -1500 V in negative ion mode, cone voltage 30 V, ion source temperature 150 °C, solvent gas temperature 500 °C, backwash gas flow rate 50 L / h, desolvation gas flow rate 800 L / h.

[0173] Statistical methods for plasma metabolomics: Principal component analysis and Spearman correlation analysis were used to determine the repeatability of samples within the group and quality control samples. The identified compounds were searched in the KEGG, HMDB and lipid map databases to obtain classification and pathway information. According to the grouping information, the difference fold was calculated and compared, and the difference significance P value of each compound was calculated using the T test. The R language package ropls was used for OPLS-DA modeling, and 200 permutation tests were performed to verify the reliability of the model. The difference fold, P value and VIP value of the OPLS-DA model were combined to screen differential metabolites with the screening criteria of FC>1, P<0.05 and VIP>1. Metabo Analyst software was used to perform enrichment analysis and topological analysis to screen the key differential metabolic pathways involved in the plasma of group B rats. FC: Fold change, which indicates the difference fold in the expression of a metabolite between the two groups. FC>1 indicates that the metabolite is a more significantly different metabolite. VIP: It is the variable weight value of the OPLS-DA model variable, which can be used to measure the influence and explanatory power of the difference in the accumulation of each metabolite on the classification and discrimination of each group of samples. VIP ≥ 1 is a common screening standard for differential metabolites.

[0174] ① Statistical analysis of metabolites: Figure 2 As shown in the figure, the unsupervised pattern recognition method PCA (principal component analysis) analysis can reflect the original state of the data and intuitively show the overall differences between different samples. The PCA results show that the MB group and the NTB group can be clearly distinguished, indicating that there are differences in the plasma metabolite composition between the MB group and the NTB group. There is a certain separation trend between the B group and the MB group, indicating that the material metabolism status of the rats has changed during the drug treatment process.

[0175] ②Classification of differential metabolites: The plasma of rats in each group was preliminarily analyzed using FC>1, P<0.05 and VIP>1 as the screening criteria for significant differences in metabolites. Compared with the NTB group, the MB group had 851 differential metabolites, of which 439 metabolites increased and 412 metabolites decreased. Compared with the JNLB group, the MB group had 763 differential metabolites, of which 476 metabolites increased and 287 metabolites decreased. Compared with the JNMB group, the MB group had 945 differential metabolites, of which 614 metabolites increased and 231 metabolites decreased. Compared with the JNHB group, the MB group had 1028 differential metabolites, of which 611 metabolites increased and 417 metabolites decreased. Compared with the BNB group, the MB group had 910 differential metabolites, of which 535 metabolites increased and 375 metabolites decreased.

[0176] The differential metabolites were then classified by searching in KEGG, HMDB and lipid map databases. The KEGG metabolic pathways of MB group compared with NTB group, MB group compared with JNLB group, MB group compared with JNMB group, MB group compared with JNHB group, and MB group compared with BNB group can be divided into 10 categories, including: amino acid metabolism, biosynthesis of other secondary metabolites, digestive system, lipid metabolism, nucleotide metabolism, etc.

[0177] ③ Analysis of non-targeted differential metabolites in plasma: OPLS-DA was used to identify differential compounds with FC>1, P<0.05 and VIP>1 as the screening criteria for significant differences in metabolites. Compared with the NTB group, xanthine, 4,8-dihydroxyquinoline, o-phospho-L-serine, N-carbamoylputrescine, cis-3-carboxyethyl-3,5-cyclohexadiene-1,2-diol and 3,4-dihydroxy-L-phenylalanine in the plasma of rats in the MB group were significantly downregulated, leading to disorders in purine metabolism, tryptophan, cysteine ​​and methionine metabolism, glycine, serine and threonine metabolism, arginine and proline metabolism and phenylalanine metabolism pathways. Most of the amino acid skeletons are intermediates of the TCA cycle and participate in the body's functions. Disorders in the amino acid metabolic pathway will greatly affect the energy supply of TCA and cause fatigue.

[0178] Compared with the MB group, the JNMB group could significantly adjust 19 metabolites such as palmitoyl aromatic acid-C, ganwu lactone, N-eicosapentaenoyl phenylalanine and aspartic acid. Compared with the MB group, the BNB group could significantly adjust 19 metabolites such as 6-thioguanine 5'-monophosphate and D-fructose 6-phosphate. Compared with the MB group, the JNHB group could significantly adjust 14 metabolites such as uridine 2',3'-cyclic phosphate, palmitoyl aromatic acid-C, xanthine and 13(S)-propylene diphenyl ether, adjust the body's amino acid metabolism, improve the body's TCA cycle in CFS rats, restore energy supply, and resist body fatigue.

[0179] ④ Analysis of non-targeted differential metabolic pathways in plasma: The hypergeometric distribution test was used to calculate the different metabolites with KEGG pathway enrichment significance, and to screen metabolic pathways related to CFS. Based on topological analysis, weight calculation was performed. Through comprehensive analysis of P value, Holm P, false positive rate (FDR) and pathway impact value, it was found that the MB group and the NTB group had significant correlations with CFS in pathways such as glyoxylate and dicarboxylate metabolism and purine metabolism. Glyoxylate and dicarboxylate metabolism are branches of the TCA cycle, which can lead to abnormal levels of various reactants and products in the tricarboxylic acid cycle by affecting the activity of key enzymes in the tricarboxylic acid cycle and the content of products. Therefore, the disorder of glyoxylate and dicarboxylate metabolism also represents the disorder of the body's TCA cycle, indicating that there is a problem with its energy metabolism. Purine metabolism participates in the body's energy supply and metabolic regulation. Therefore, the disorder of purine metabolism in CFS rats in the MB group will lead to abnormal energy metabolism and imbalance of related amino acids. Compared with the MB group, the JNMB and JNHB groups could adjust the TCA cycle pathway, and the BNB group could adjust the glyoxylate and dicarboxylic acid metabolism and purine metabolism pathways, thereby alleviating the energy metabolism disorder in CFS rats, increasing energy supply, and reducing body fatigue.

[0180] Experimental Example 5 (Study on the mechanism of action on skeletal muscle energy metabolism)

[0181] Sample processing: Mouse skeletal muscle samples were taken and the mitochondria of mouse skeletal muscle for the experiment were extracted by biochemical method according to the instructions of the kit. Kit used: Mitochondrial Extraction Kit (Beijing Solebold Technology Co., Ltd., batch number: 2309001).

[0182] (1) Observation of skeletal muscle mitochondrial ultrastructure: Mitochondria are the site of energy metabolism in the body. Damage to the structure and function of mitochondria and reduction in their number will lead to insufficient energy supply and fatigue. The ultrastructure of skeletal muscle mitochondria in group A mice was observed using an electron microscope. The results are as follows: Figure 3 shown.

[0183] Mice in group A: The skeletal muscle myofibrils in the NTA group were arranged neatly, the Z and M lines were clear and neatly arranged, and the mitochondrial structure was intact and neatly arranged. Compared with the NTA group, the skeletal muscle myofibrils in the MA group were unclear and irregularly arranged, the Z and M lines were blurred, the mitochondria were disordered, and most of them showed vacuolar changes. Compared with the MA group, the skeletal muscle myofibrils in the A group and the BNA group of each dose of Anshen Jiannaoye were arranged neatly, the Z and M lines were clearly visible, the mitochondrial structure was intact and neatly arranged, and a small number of vacuoles appeared.

[0184] The semi-quantitative results of skeletal muscle transmission electron microscopy are shown in Table 24. Compared with the NTA group, the number of skeletal muscle mitochondria in the MA group was significantly reduced (P<0.01), and the mitochondrial vacuolation rate was significantly increased (P<0.01), indicating that the mitochondrial structure of the skeletal muscle of the MA group was damaged and the mitochondrial function was affected. Compared with the MA group, the number of mitochondria in the JNHA, JNMA and BNA groups was significantly increased (P<0.01), and the mitochondrial vacuolation rate of the skeletal muscle of the mice in the A group and the BNA group at various doses of the Anshen Jiannao Liquid was significantly decreased (P<0.01).

[0185] Table 24

[0186]

[0187] (2) Glycogen content test: During long-term intense exercise, the main function of maintaining blood sugar levels in the body is liver glycogen. When entering the blood, liver glycogen exists in the form of glucose and reaches various parts of the body with blood circulation to provide continuous energy for the muscles. During exercise, glycogen needs to be broken down to provide energy for the body. Therefore, the more liver glycogen reserves, the stronger the body's ability to resist fatigue. Insufficient liver glycogen will cause fatigue in the body. The biochemical method was used to detect the muscle glycogen content and liver glycogen content of rats in Group B. The data are shown in Table 25.

[0188] Rats in group B: Compared with the NTB group, the muscle glycogen and liver glycogen content of the MB group rats were significantly reduced (P<0.01). Compared with the MB group, the liver glycogen content of the JNMB and JNHB groups was significantly increased (P<0.05), and the muscle glycogen of the JNMB and BNB groups was significantly increased (P<0.05), indicating that Anshen Jiannao Liquid and Anshen Bu Nao Liquid can restore the body's glycogen content and resist body fatigue.

[0189] Table 25

[0190]

[0191] (3) Test of skeletal muscle ATP content: ATP is a high-energy phosphate compound that participates in the body's energy metabolism and is the main source of energy in the body. In cells, it can be converted with ADP to achieve energy storage and release, thereby ensuring the energy supply for various life activities of the cell. Mitochondria are the main place where the body produces energy. When the body has sufficient oxygen, 1 molecule of glucose can eventually produce 30 to 32 molecules of ATP. When the body exercises vigorously, the muscle oxygen supply is insufficient and the cells perform anaerobic respiration. Glucose generates pyruvate through the glycolysis process and does not enter the TCA cycle. Therefore, 1 molecule of glucose only produces 2 molecules of ATP, the ATP content decreases, and the body's energy supply is insufficient, resulting in energy metabolism disorders, insufficient energy supply, and fatigue. The skeletal muscle ATP content of mice in group A and rats in group B was detected by biochemical method. The data are shown in Table 26.

[0192] Mice in group A: Compared with those in group NTA, the ATP content in skeletal muscle of mice in group MA was significantly decreased (P<0.01), indicating that Poly I:C combined swimming would cause energy metabolism disorders in skeletal muscle of CFS mice. Compared with those in group MA, the ATP content in skeletal muscle of mice in group JNHA and group BNA was significantly increased (P<0.05), indicating sufficient energy supply and eliminating fatigue in CFS mice.

[0193] Rats in Group B: Compared with those in the NTB group, the proportion of ATP concentration in skeletal muscle of rats in the MB group was significantly decreased (P<0.01), indicating that multi-factor combined stress can lead to disorders in skeletal muscle energy metabolism in CFS rats; compared with those in the MB group, the proportion of ATP concentration in skeletal muscle of rats in the JNMB group was significantly increased (P<0.05), indicating that Anshen Jiannao Liquid can effectively improve the disorders in skeletal muscle energy metabolism in rats.

[0194] Table 26

[0195]

[0196] (4) Testing of key enzymes in the TCA cycle

[0197] ① Pyruvate content: The conversion of pyruvate into acetyl-CoA is the first step in entering the TCA cycle. When the pyruvate content decreases, the TCA cycle will inevitably be affected, and the body's energy supply will be abnormal, resulting in fatigue symptoms. The skeletal muscle pyruvate content of rats in group B was detected using a biochemical method. The data are shown in Table 27. Rats in group B: Compared with the NTB group, the skeletal muscle pyruvate content of rats in the MB group was significantly reduced (P<0.01). Compared with the MB group, the skeletal muscle pyruvate content of rats in the JNHB group was significantly increased (P<0.05).

[0198] Table 27

[0199]

[0200] ②Testing of skeletal muscle PDH, SDH, and MDH activity: PDH is a key enzyme located in the mitochondrial matrix and is an important rate-limiting enzyme in mitochondrial energy metabolism. It is related to glycolysis, TCA cycle, and oxidative phosphorylation. It can maintain a dynamic balance between anaerobic oxidation of sugar and TCA cycle, and helps regulate energy metabolism and ATP generation. PDH activity is important for muscle function and movement. The lack of PDH enzyme activity will lead to abnormal metabolism of pyruvate and a lack of TCA cycle substrates, which is one of the most common causes of abnormal mitochondrial energy metabolism and cell function activity disorders. In rodent models injected with peroxisome proliferators-activated receptors (PPARs)-δ agonists, the reduction of PDH oxidative decarboxylation caused by upregulation of pyruvate dehydrogenase kinase 4 (PDK4) will impair muscle function. Mitochondrial pyruvate oxidation is impaired in CFS patients. SDH is a key enzyme in the synthesis of ATP in the process of aerobic respiration of cells and one of the key enzymes in the TCA cycle. It is located in the inner membrane of mitochondria and participates in the TCA cycle. It is also a component of the respiratory chain complex II. SDH connects oxidative phosphorylation and electron transfer, can provide electrons for mitochondria and the respiratory chain, and connects the TCA cycle with the respiratory chain. The generated FADH2 does not dissociate from the enzyme, but directly uses electrons to reduce coenzyme Q, and then transfers it to the respiratory chain complex III, thereby participating in the energy supply of the respiratory chain. When the activity of SDH decreases, the body reduces the transfer of FADH2 to the respiratory chain, hinders the progress of the TCA cycle, leads to a decrease in body energy, and induces the generation of CFS. MDH is the last key enzyme in the TCA cycle. The activity of SDH and MDH directly affects the speed of the TCA cycle, thereby affecting mitochondrial energy metabolism. The PDH activity of skeletal muscle of mice in group A and the activity of mitochondrial SDH and MDH were detected by biochemical method; the PDH and SDH activities of skeletal muscle of rats in group B were detected by biochemical method.

[0201] Mice in group A, data are shown in Table 28: Compared with the NTA group, the PDH activity in skeletal muscle of mice in the MA group was significantly decreased (P<0.01). Compared with the MA group, the PDH activity in skeletal muscle of mice in the JNHA group and the BNA group was significantly increased (P<0.01 or P<0.05). Compared with the NTA group, the mitochondrial SDH and MDH activities in skeletal muscle of mice in the MA group were significantly decreased (P<0.01). Compared with the MA group, the JNHA group could significantly increase the mitochondrial SDH activity in skeletal muscle of mice (P<0.05), and the BNA group could significantly increase the mitochondrial MDH activity in skeletal muscle of mice (P<0.05).

[0202] Table 28

[0203]

[0204] The data of rats in group B are shown in Table 29: Compared with the NTB group, the activities of PDH and SDH in skeletal muscle of rats in group MB were significantly decreased (P<0.01), the TCA cycle and mitochondrial energy supply were blocked, and energy metabolism was impaired. Compared with the MB group, the PDH activity in skeletal muscle of rats in group B and BNB groups at various doses of Anshen Jiannaoye was significantly increased (P<0.05 or P<0.01), and the SDH activity in skeletal muscle of rats in JNHB, JNMB and BNB groups was significantly increased (P<0.05). This shows that Anshen Jiannaoye and Anshen Bu Naoye can increase the activities of PDH and SDH in skeletal muscle of CFS rats, improve the problem of energy metabolism disorders, and reduce the fatigue degree of CFS rats.

[0205] Table 29

[0206]

[0207] (5) Test of mitochondrial electron transport chain: Mitochondrial electron transport chain (ETC) metabolites are key substances that affect mitochondrial function and ATP synthesis, and play a role in CFS pathology. It contains four enzyme complexes and ATP synthase. Changes in the activity of respiratory chain complexes caused by life activities can reflect changes in mitochondrial function. COXV (ATP synthase) is the last complex of the mitochondrial respiratory chain. It transfers protons through electrochemical gradients in mitochondria in the form of ADP, Pi and Mg2 + COXⅠis the fourth central enzyme complex on ETC and a key rate-limiting site, affecting mitochondrial functional morphology and energy metabolism. Lymphocytes of CFS patients have complex Complex V defects, accompanied by compensatory upregulation of respiratory capacity. The signal transduction and metabolic pathways involved in this compensatory upregulation may reduce the ability of cells to respond to further acute increases in ATP demand after long-term upregulation, making them unable to fully cope with the sharp increase in energy demand, causing mitochondrial dysfunction and decreased ATP synthesis capacity. The decrease in ATP synthase activity will affect the synthesis of ATP by the mitochondrial respiratory chain, causing mitochondrial dysfunction and reduced energy supply. A significant decrease in COXⅠ content will reduce the rate of electron transfer and ATP generation, hinder the body's normal energy supply, and cause fatigue in the body. The ELISA method was used to detect the content of COXⅠ in the skeletal muscle mitochondria of mice in group A, and the biochemical method was used to detect the activity of COX V in the skeletal muscle mitochondria of rats in group B. The data are shown in Table 30.

[0208] Mice in group A: Compared with the NTA group, the mitochondrial COXⅠ content in skeletal muscle of mice in group MA was significantly decreased (P<0.05), and the electron transfer and ATP generation rate were decreased. Compared with the MA group, the mitochondrial COXⅠ content in skeletal muscle of mice in group JNHA and BNA could significantly increase the mitochondrial COXⅠ content in skeletal muscle (P<0.05). Rats in group B: Compared with the NTB group, the mitochondrial ATP synthase activity in skeletal muscle of rats in group MB was significantly decreased (P<0.05), the mitochondrial respiratory chain synthesis of ATP was blocked, the energy supply was reduced, and the CFS rats in group MB showed fatigue. Compared with the MB group, the mitochondrial ATP synthase activity in skeletal muscle of rats in group JNMB and BNB could significantly increase the mitochondrial ATP synthase activity in skeletal muscle of rats (P<0.05 or P<0.01).

[0209] Table 30

[0210]

[0211] (6) Testing of energy metabolites: Serum LAC is a hallmark indicator of fatigue and can also indirectly reflect mitochondrial function. Improved skeletal muscle mitochondrial function can reduce serum LAC levels. When the number and activity of mitochondria decrease, the utilization rate of pyruvate by mitochondria decreases, and the body produces a large amount of LAC accumulation. The long-term accumulation of LAC will squeeze blood vessels, hinder the normal circulation of blood, and cause symptoms such as muscle soreness and pain. BUN is an indicator that can sensitively reflect the body's tolerance to high-intensity exercise loads. After long-term exercise, due to insufficient sugar and fat metabolism in the body, the body's protein and amino acid metabolism is enhanced, and the blood BUN content increases significantly. As the exercise load increases, the blood BUN concentration continues to increase. The weaker the body's ability to adapt to the load, the higher the BUN content in the body. The serum LAC and BUN levels of mice in Group A were detected using a biochemical method. The data are shown in Table 31.

[0212] Mice in group A: Compared with the NTA group, the serum LAC content of mice in group MA was significantly increased (P<0.01). Compared with the MA group, the serum LAC content of mice in groups JNHA, JNMA and BNA was significantly decreased (P<0.01). Compared with the NTA group, the serum BUN content of mice in group MA was significantly increased (P<0.01). Compared with the MA group, the serum BUN content of mice in groups A and BNA at all doses of Anshen Jiannao Liquid was significantly decreased (P<0.01).

[0213] Table 31

[0214]

[0215] (7) Testing of skeletal muscle oxidative stress levels: When exhausted, CFS patients have impaired oxidative metabolic function and mitochondrial dysfunction, which ultimately leads to fatigue and discomfort after exertion. When tissues are damaged, ROS in mitochondria increase dramatically, thereby damaging mitochondria and causing mitochondrial dysfunction. ROS is a class of single-electron reduction products of oxygen, including singlet oxygen, superoxide, peroxide, hydroxyl radicals, hypochlorous acid and nitric oxide. ROS is a key factor in inducing cell apoptosis. Excessive accumulation of ROS or reduction of antioxidant substances can cause apoptosis. The accumulation of ROS disrupts the body's metabolic balance and leads to fatigue symptoms. CFS patients also show clinical symptoms of enhanced oxidative stress, and biochemical markers related to oxidative stress also play a key role in skeletal muscle fatigue. Excessive free radicals attack fatty acids on the cell membrane and are eventually metabolized into MDA, which directly reflects the degree of lipid peroxidation. In long-term exhaustive exercise dominated by aerobic metabolism, the increase in MDA content in the body is an important indicator of tissue cell damage caused by accumulated free radicals in the body, which can reflect the body's lipid peroxidation level caused by free radical damage. The increase in MDA content indicates that the body's lipid peroxidation level is too high, leading to tissue cell damage. GSH and SOD, as antioxidants, can protect cells from oxidative stress damage. The decrease in GSH and SOD content is due to the fact that the antioxidant system in the human body can no longer effectively resist the oxidation of free radicals. Therefore, the change in this indicator can be used to measure the body's ability to scavenge free radicals. GSH-Px is also an important antioxidant and a natural scavenger of ROS in the body. The decrease in GSH-Px activity and GSH content indicates that the antioxidant system in the body can no longer effectively resist the oxidation of free radicals. Excessive free radicals will damage mitochondrial function, resulting in obstacles in its energy supply to the body. The GSH-Px enzyme activity in CFS rats is significantly reduced, the ability to scavenge free radicals is reduced, and the body's energy metabolism is disordered, causing fatigue symptoms in CFS rats. The biochemical method was used to detect the MDA, GSH content and SOD activity in the skeletal muscle of mice in group A, and the data are shown in Table 32; the biochemical method was used to detect the MDA, GSH content and GSH-Px activity in the skeletal muscle of rats in group B, and the data are shown in Table 33.

[0216] Mice in group A: Compared with the NTA group, the SOD activity in the skeletal muscle of mice in the MA group was significantly decreased (P<0.05). Compared with the MA group, the SOD activity in the skeletal muscle of mice in the JNHA group was significantly increased (P<0.05). Compared with the NTA group, the MDA content in the skeletal muscle of mice in the MA group was significantly increased (P<0.01). Compared with the MA group, the MDA content in the skeletal muscle of mice in the JNHA, JNMA and BNA groups was significantly decreased (P<0.05). Compared with the NTA group, the GSH content in the skeletal muscle of mice in the MA group was significantly decreased (P<0.01).

[0217] Table 32

[0218]

[0219] Rats in group B: Compared with the NTB group, the MDA content in skeletal muscle of rats in the MB group was significantly increased (P<0.01); compared with the MB group, the MDA content in skeletal muscle of rats in the JNMB group and the BNB group was significantly decreased (P<0.05 or P<0.01). Compared with the NTB group, the GSH content in skeletal muscle of rats in the MB group was significantly decreased (P<0.01). Compared with the MB group, the GSH content in skeletal muscle of rats in the JNMB and JNHB groups was significantly increased (P<0.05 or P<0.01). Compared with the NTB group, the GSH-Px enzyme activity in skeletal muscle of rats in the MB group was significantly decreased (P<0.01). Compared with the MB group, the GSH-Px enzyme activity in skeletal muscle of rats in the JNLB, JNHB and BNB groups was significantly increased (P<0.05 or P<0.01).

[0220] Table 33

[0221]

[0222] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. Application of tranquilizing and brain-strengthening liquid in the preparation of medicines for treating, preventing or alleviating chronic fatigue syndrome.

2. The use according to claim 1, characterized in that: The raw material medicine composition of the tranquilizing and brain-strengthening liquid includes ginseng, schisandra chinensis, ophiopogon japonicus, wolfberry fruit and salvia miltiorrhiza.

3. The use according to claim 2, characterized in that: In the raw materials of the tranquilizing and brain-strengthening liquid, the mass ratio of ginseng, schisandra chinensis, ophiopogon japonicus, wolfberry and salvia miltiorrhiza is 20-80:100-160:100-160:100-160:60-120.

4. The use according to any one of claims 1 to 3, characterized in that: Satisfy at least one of (1) to (9): (1) The drug can regulate the dysfunction of the hypothalamus-pituitary-adrenal axis caused by chronic fatigue syndrome; (2) The drug can improve the thymus index of patients or animals with chronic fatigue syndrome; (3) The drug can reduce the CD8 + T cell ratio; (4) The drug can reduce the level of inflammatory factors in patients or animals with chronic fatigue syndrome; (5) The drug can regulate energy metabolism in patients with chronic fatigue syndrome or animals; (6) The drug can alleviate oxidative stress in patients with chronic fatigue syndrome or animals; (7) The drug can increase the level of immunoglobulin in patients or animals with chronic fatigue syndrome; (8) The drug can increase the complement content in patients or animals with chronic fatigue syndrome; (9) The drug can regulate the intestinal flora of patients with chronic fatigue syndrome or animals.

5. The use according to claim 4, characterized in that: The inflammatory factors include at least one of C-reactive protein, interleukin-1β, interleukin-6, interleukin-2, interferon-γ, interferon-α, and tumor necrosis factor-α; And / or, the immunoglobulin includes at least one of immunoglobulin G, immunoglobulin M, and immunoglobulin A; And / or, the complement includes at least one of C3 and C4; And / or, the energy metabolism includes amino acid metabolism.

6. The use according to claim 4, characterized in that: The drug can regulate the levels of serum cortisol and adrenocorticotropic hormone in patients with chronic fatigue syndrome or animals, the levels of 5-hydroxytryptamine, 5-hydroxyindoleacetic acid, and 5-hydroxytryptamine transporter in the hippocampus, prefrontal cortex, and hypothalamus, and the level of corticotropin-releasing hormone in the hypothalamus; and / or, the drug is capable of activating the mitochondrial biogenesis signaling pathway in a chronic fatigue syndrome patient or animal; and / or, the drug can improve the function of the mitochondrial electron transport chain in patients or animals with chronic fatigue syndrome; and / or, the drug can reduce the levels of serum lactic acid and serum urea nitrogen in skeletal muscle cells in patients with chronic fatigue syndrome or animals; And / or, the drug can increase the liver glycogen and muscle glycogen content in patients or animals with chronic fatigue syndrome.

7. The use according to claim 6, characterized in that: The drug can increase the number of mitochondria in skeletal muscle cells in patients with chronic fatigue syndrome or animals and reduce the mitochondrial vacuolation rate; and / or, the drug can increase the content of adenosine triphosphate in skeletal muscle cells in patients with chronic fatigue syndrome or animals; and / or, the drug can enhance the activity of key enzymes of the tricarboxylic acid cycle in skeletal muscle cells in patients with chronic fatigue syndrome or animals; And / or, the drug can increase the content of pigment C oxidase subunit I and ATP synthase activity in skeletal muscle cells of patients with chronic fatigue syndrome or animals.

8. The use according to claim 7, characterized in that: The key enzymes of the tricarboxylic acid cycle include succinate dehydrogenase, malate dehydrogenase, and pyruvate dehydrogenase.

9. The use according to claim 4, characterized in that: The drug can reduce the malondialdehyde content in patients or animals with chronic fatigue syndrome; And / or, the drug can increase superoxide dismutase activity, glutathione peroxidase activity and glutathione content in patients with chronic fatigue syndrome or animals.

10. The use according to any one of claims 1 to 9, characterized in that: Satisfy at least one of A to G: A: The drug can relieve the despair and / or depression and / or anxiety of patients or animals with chronic fatigue syndrome; B: The drug can increase the pain threshold of patients or animals with chronic fatigue syndrome; C: The drug can improve the learning and memory abilities of patients or animals with chronic fatigue syndrome; D: The drug can improve the sleep quality of patients or animals with chronic fatigue syndrome; E: The drug can improve the immune function of patients or animals with chronic fatigue syndrome; F: The drug can relieve fatigue in patients with chronic fatigue syndrome or animals; G: The drug can alleviate neuroendocrine disorders in patients or animals with chronic fatigue syndrome.

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