Traditional Chinese medicine composition for treating sepsis encephalopathy as well as preparation method and application thereof

Abstract

The invention discloses a traditional Chinese medicine composition for treating sepsis encephalopathy. The traditional Chinese medicine composition is prepared from the following raw material medicines in parts by weight: 20-40 parts of ginseng, 6-18 parts of raw rhubarb, 20-40 parts of sargentgloryvine stem, 20-40 parts of dandelion, 20-40 parts of monkshood and 3-9 parts of leech. The traditional Chinese medicine composition has the effects of tonifying qi and restoring yang, relaxing bowels and detoxifying, and promoting blood circulation and removing blood stasis, and clinical tests show that the traditional Chinese medicine composition can remarkably improve the clinical score, inflammatory response and brain dysfunction degree of sepsis-related encephalopathy patients, relieve nerve injury and improve 28-day prognosis, and shows good effectiveness and safety. In addition, the invention further discloses a preparation method and application of the traditional Chinese medicine composition.

Description

TECHNICAL FIELD

[0001] The present application relates to a traditional Chinese medicine composition for treating sepsis encephalopathy, and further relates to a preparation method and use of the traditional Chinese medicine composition. BACKGROUND

[0002] Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection. Worldwide, there are approximately 489 million sepsis cases and 110 million sepsis-related deaths each year. This disease is one of the most common acute and critical conditions in clinical practice and is a major cause of global health burden. Patients with sepsis are often accompanied by severe diffuse brain dysfunction, with an overall incidence of up to 70%, and are closely related to the increase in mortality of patients with sepsis and long-term cognitive impairment, including memory, attention, and speech communication, as well as psychological disorders, including anxiety, post-traumatic stress disorder, etc. It is currently commonly referred to as "sepsis-related encephalopathy". The pathological mechanism of this disease is complex, involving processes such as neuroinflammation, oxidative stress, and blood-brain barrier disruption.

[0003] Current research suggests that the pathological process of brain injury induced by sepsis mainly involves neuroinflammation, microcirculatory dysfunction, and metabolic disorders. When the host is severely infected, the body's innate immune system and adaptive immune system are activated, accompanied by a complex process characterized by simultaneous occurrence of pro-inflammatory and anti-inflammatory responses, and immune homeostasis impairment, and a large number of inflammatory factors and trigger cytokine storms are produced and released. In brain tissue, these cytokines produced and released by the body, such as TNF-α, IL-1β, and IL-6, can induce neuroinflammation-mediated neuronal dysfunction and ultimately lead to neuronal cell death. In this process, including activation of astrocytes and microglia, damage to vascular endothelial cells and blood-brain barrier, and increased permeability, further promote neuroinflammatory response, and neuroinflammation can exacerbate immune disorders and metabolic disorders, further promoting brain tissue damage.

[0004] It is of great significance to take timely and effective intervention measures for patients with sepsis-related encephalopathy, protect their brain function, and reduce neural damage to reverse the progression of the disease and improve prognosis. However, modern medicine lacks specific intervention measures and can only rely on the use of broad-spectrum antibiotics and symptomatic supportive treatment, including limited measures to ensure adequate fluid resuscitation, maintenance of blood pressure, electrolyte and acid-base balance, etc. Although some scholars have conducted relevant research to find effective specific treatment methods, none of them have achieved satisfactory results

[0005] In the process of inheritance and development over thousands of years, traditional Chinese medicine has formed a systematic theoretical understanding of infectious diseases and accumulated rich practical experience. Traditional Chinese medicine believes that sepsis-associated encephalopathy belongs to the category of "coma" in "warm febrile diseases" of traditional Chinese medicine. The pathogenesis of this disease is mostly caused by exogenous toxic pathogens entering the interior, the struggle between healthy qi and pathogenic factors, transforming into heat, generating phlegm and blood stasis, disturbing the clear orifices. In addition, the depletion of healthy qi makes it unable to expel pathogenic factors, and is unable to promote the operation of qi and blood, further leading to disharmony of zang-fu organs and loss of nourishment of the mental orifices. When such patients get sick, both excessive internal heat-toxic pathogens and deficiency of healthy qi exist, accompanied by unsmooth qi and blood circulation and blood stasis in the blood vessels. Its essence is the coexistence of toxic pathogens, deficiency and blood stasis, and the condition is often urgent, dangerous, complex and changeable. It is necessary to actively cut off the disease condition, reverse the disease trend, and always pay attention to strengthening healthy qi to expel pathogenic factors, and assist in promoting qi and blood so that the blood vessels can be unobstructed. Traditional Chinese medicine provides an all-round treatment idea for the treatment of sepsis-related encephalopathy, promoting the overall recovery of patients. Summary of the Invention

[0006] In view of the above-mentioned deficiencies of the prior art, according to the embodiments of the present invention, it is desired to provide a traditional Chinese medicine composition for treating sepsis encephalopathy, aiming to treat sepsis encephalopathy, improve the clinical score of patients, relieve inflammatory reactions, reduce nerve damage, improve prognosis, and save public medical resources. In addition, it is also desired to propose a preparation method and uses of the traditional Chinese medicine composition.

[0007] According to an embodiment, a traditional Chinese medicine composition for treating sepsis encephalopathy provided by the present invention is prepared from the following raw materials by weight: ginseng 20-40, raw rhubarb 6-18, caulis spatholobi 20-40, dandelion 20-40, aconite root 20-40, leech 3-9.

[0008] Preferably, in the traditional Chinese medicine composition for preventing and treating novel coronavirus infection described above in the present invention, the weights of the raw materials are respectively: ginseng 26-34, rhubarb 9-14, caulis spatholobi 26-34, dandelion 26-34, aconite root 26-34, leech 5-7; more preferably, the weights of the raw materials are respectively: ginseng 30, rhubarb 12, caulis spatholobi 30, dandelion 30, aconite root 30, leech 6.

[0009] Preferably, in the traditional Chinese medicine composition for treating sepsis encephalopathy described above in the present invention, the rhubarb is raw rhubarb.

[0010] The traditional Chinese medicine composition for treating sepsis encephalopathy described above in the present invention can be prepared into decoction, tablets, capsules, powders, granules, oral liquids, pills, tinctures, syrups, suppositories, gels, sprays or injections according to conventional methods.

[0011] According to an embodiment, the present invention provides a method for preparing a traditional Chinese medicine decoction for treating septic encephalopathy, comprising the following steps: weighing ginseng, rhubarb, red vine, dandelion, aconite and leech according to the weight parts, adding 4-10 times the amount of water and decocting twice, each time for 0.5-2 hours, combining the decoctions, filtering, and concentrating to 2-4 times the amount, thus obtaining the decoction.

[0012] According to an embodiment, the present invention provides a method for preparing a traditional Chinese medicine composition granule for treating septic encephalopathy, comprising the following steps:

[0013] A. Weigh the aconite root according to the weight, add 8-12 times the amount of water, bring to a boil over high heat, then simmer over low heat for 60-90 minutes, filter, and collect the filtrate A.

[0014] B. Weigh out the ginseng according to the weight, add 6-8 times the amount of water, decoct at 60-80℃ for 30-60 minutes, filter, and collect the filtrate B.

[0015] C. Weigh out the following ingredients by weight: red vine, dandelion, leech, and rhubarb. Add 5–8 times the amount of water and decoct twice, each time for 1–2 hours. Combine the extracts and filter. Collect the filtrate C.

[0016] D. Combine the filtrates from steps A, B, and C, concentrate under reduced pressure to a relative density of 1.10–1.25 at 60°C; allow to stand for precipitation for 8–12 hours, then filter to obtain a clear concentrate; spray dry and collect the spray-dried powder.

[0017] E. Add pharmaceutically acceptable excipients (such as dextrin / maltodextrin, etc.) to the spray-dried powder as needed, granulate by spray or wet granulation, dry at low temperature, granulate and sieve to obtain granules.

[0018] According to an embodiment, the present invention provides a method for preparing a capsule of a traditional Chinese medicine composition for treating septic encephalopathy, characterized by comprising the following steps:

[0019] A. Weigh the aconite root according to the weight, add 8-12 times the amount of water, decoct for 60-90 minutes, filter, and obtain filtrate A.

[0020] B. Weigh out the ginseng according to the weight proportions, add 6-8 times the amount of water, decoct at 60-80℃ for 30-60 minutes, filter, and obtain filtrate B;

[0021] C. Weigh out the following ingredients by weight: red vine, dandelion, leech, and rhubarb. Add 5–8 times the amount of water and decoct twice, each time for 1–2 hours. Combine the decoctions and filter to obtain filtrate C.

[0022] D. Combine the filtrates obtained in steps A, B, and C, concentrate them under reduced pressure to form a thick paste, and dry them at low temperature (vacuum / belt / spray drying are all acceptable) to obtain a dry powder;

[0023] E. Mix the dry powder with pharmaceutically acceptable excipients (such as microcrystalline cellulose, cross-linked polyvinylpyrrolidone, magnesium stearate, etc.), (optionally) granulate, dry at low temperature until the water content meets the standard, and fill into hollow capsules to obtain the capsule preparation.

[0024] Subsequent examples and test examples show that the traditional Chinese medicine composition of the present invention has the effects of replenishing qi and restoring yang, purging the fu-organs and detoxifying, activating blood circulation and removing blood stasis. For patients with septic encephalopathy, it can improve the overall clinical score, relieve the inflammatory response, reduce nerve damage, improve the prognosis, and has good safety and effectiveness.

[0025] Compared with the prior art, the traditional Chinese medicine composition of the present invention is based on the characteristics of patients with septic encephalopathy and is based on the classical formula "Shenfu Decoction" in "Yanshi Jisheng Fang" in the Song Dynasty, which has the effects of restoring yang, replenishing qi, and relieving collapse. It is mainly used for treating syndromes of true yang deficiency, shortness of breath, spontaneous sweating and night sweating, dizziness due to qi deficiency, and weakness of yang qi. On the basis of this formula, it is obtained through clinical trials.

[0026] According to the characteristics of patients with septic encephalopathy, especially the core pathogenesis of toxin damaging brain collaterals and qi deficiency and blood stasis obstruction, in the present invention, ginseng is used as the monarch drug, which can greatly replenish qi, restore pulse and arrest collapse, soothe the nerves and improve intelligence, invigorate the qi of the spleen and lungs internally, and support the healthy qi externally to resist the invasion of pathogenic factors; rhubarb is used to purge downward and promote defecation, clear heat and purge fire, activate blood circulation and remove blood stasis, Sargentodoxa cuneata is used to clear heat and detoxify, activate blood circulation and dredge collaterals, Taraxacum mongolicum is used to clear heat and promote diuresis, detoxify and dissipate carbuncles. The three drugs are used together to purge the fu-organs and clear heat, detoxify and remove blood stasis, and act as the ministerial drugs together; aconite root is used to restore yang and rescue from collapse, warm the channels and promote blood circulation, assist yang qi to promote blood circulation and remove blood stasis, leech is used to break blood and remove blood stasis, dredge the channels and collaterals, and penetrate deep into the blood aspect to search for and remove stagnant toxins. The two drugs together act as the adjuvant and guiding drugs. The whole formula together has the effects of replenishing qi and arresting collapse, clearing heat and detoxifying, activating blood circulation and dredging collaterals, can improve the clinical score, relieve the inflammatory response, reduce nerve damage, improve the prognosis, and has good safety and effectiveness. Description of the Drawings

[0027] Figure 1 Showing the comparison of the survival of three groups of mice: Sham group, sham operation group; CLP group, model group; SHG group, experimental group of the traditional Chinese medicine decoction in Example 7; **P < 0.01 compared with the sham operation group; ##P < 0.01 compared with the drug administration group

[0028] Figure 2 Showing the results of the neurological behavior score and open field test of mice: (a) Modified neurological severity score, (b) Neurological behavior score, (c) Total moving distance in the open field test, (d) Residence time in the central area of the open field test, (e) Display of the movement trajectories of the three groups. Sham group, sham operation group; CLP group, model group; SHG group, experimental group of the traditional Chinese medicine decoction in Example 7; *P < 0.05, ***P < 0.001, ns P > 0.05

[0029] Figure 3Morphological changes in mouse brain tissue (HE staining, ×200): (a) cortex, (b) hippocampus; Sham group, sham-operated group; CLP group, model group; SHG group, experimental group of traditional Chinese medicine decoction in Example 7.

[0030] Figure 4 The following images show the detection of apoptosis in mouse brain tissue (TUNEL staining, ×200): (a) cortex, (b) hippocampus; Shamgroup, sham-operated group; CLP group, model group; SHG group, experimental group of traditional Chinese medicine decoction in Example 7.

[0031] Figure 5 The changes in the levels of inflammatory and neurological damage factors in mice were shown: (ad) serum IL-6, TNF-α, S100-β, and NSE levels; (eh) brain tissue MDA, SOD, S100-β, and NSE levels; Sham group (sham-operated group); CLP group (model group); SHG group (experimental group of traditional Chinese medicine decoction in Example 7); ***P < 0.001, ns P > 0.05 Detailed Implementation

[0032] The present invention will be further illustrated below with reference to the accompanying drawings and specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. After reading this invention, any modifications of the invention in various equivalent forms by those skilled in the art will fall within the scope defined by the appended claims.

[0033] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0034] Examples 1-5 (Preparation of decoctions)

[0035] Weigh out the ginseng, rhubarb, red vine, dandelion, aconite, and leech listed in Table 1, decoct them twice, adding 1000ml of water each time, and decoct for 1 hour each time. Combine the decoctions, filter, and obtain 600ml of decoction.

[0036] Table 1. Weights of each active pharmaceutical ingredient in Examples 1-5

[0037] Raw material drug Example 1 (g) Example 2 (g) Example 3 (g) Example 4 (g) Example 5 (g) Ginseng 40 36 32 20 28 Rhubarb 18 16 14 10 8 Red vine 38 36 32 28 25 Dandelion 38 36 32 28 26 Monkshood 40 35 32 29 27 Leech 8 7.5 7 5.5 5

[0038] Example 6 (Preparation of Decoction)

[0039] Weigh out 28g of ginseng, 12g of rhubarb, 26g of red vine, 30g of dandelion, 32g of aconite, and 6g of leech. Decoct twice, adding 1000ml of water each time, and decocting for 1 hour each time. Combine the decoctions, filter, and obtain 400ml of soup.

[0040] Example 7 (Preparation of Decoction)

[0041] Weigh out 30g of ginseng, 12g of rhubarb, 30g of red vine, 30g of dandelion, 30g of aconite, and 6g of leech. Decoct twice with water, adding 1200ml of water each time, and decocting for 0.5 hours each time. Combine the decoctions, filter, and obtain 500ml of decoction.

[0042] Example 8 (Preparation of Granules)

[0043] A. Weigh 2800g of Aconitum carmichaelii according to the weight ratio, add 10 times the amount of water, bring to a boil over high heat, then simmer over low heat for 80 minutes, filter, and collect the filtrate A.

[0044] B. Weigh 2800g of ginseng according to the weight ratio, add 10 times the amount of water, decoct at 70℃ for 50 minutes, filter, and collect the filtrate B.

[0045] C. Weigh out 2800g of red vine, 2800g of dandelion, 700g of leech, and 1100g of rhubarb according to the weight ratio, add 6 times the amount of water, decoct twice, 2 hours each time, combine the extracts and filter, and collect the filtrate C.

[0046] D. Combine the filtrates from steps A, B, and C, concentrate under reduced pressure to a relative density of 1.10–1.25 at 60°C; allow to stand for 10 hours to settle, then filter to obtain a clear concentrate; spray dry and collect the spray-dried powder.

[0047] E. Add pharmaceutically acceptable excipients (such as dextrin / maltodextrin, etc.) to the spray-dried powder as needed, granulate by spray or wet granulation, dry at low temperature, granulate and sieve to obtain granules.

[0048] Example 9 (Preparation of Capsules)

[0049] A. Weigh 2800g of Aconitum carmichaelii according to the weight ratio, add 10 times the amount of water, decoct for 80 minutes, filter, and obtain filtrate A;

[0050] B. Weigh 2800g of ginseng according to the weight ratio, add 6-8 times the amount of water, decoct at 70℃ for 50 minutes, filter, and obtain filtrate B;

[0051] C. Weigh out 2800g of red vine, 2800g of dandelion, 700g of leech, and 1100g of rhubarb according to the weight ratio, add 6 times the amount of water, decoct twice, 2 hours each time, combine and filter to obtain filtrate C.

[0052] D. Combine the filtrates obtained in steps A, B, and C, concentrate them under reduced pressure to form a thick paste, and dry them at low temperature (vacuum / belt / spray drying are all acceptable) to obtain a dry powder;

[0053] E. Mix the dried powder with pharmaceutically acceptable excipients (such as microcrystalline cellulose, crospovidone, magnesium stearate, etc.), (optionally) granulate, dry at low temperature until the moisture content meets the standard, and fill into empty capsules to obtain capsules.

[0054] Experimental Example 1 (Clinical Therapeutic Effect - Clinical Efficacy of the Herbal Composition of the Invention on Patients with Septic Encephalopathy)

[0055] 1. Materials and Methods

[0056] 1.1 General Information

[0057] Sixty-five patients with septic encephalopathy were collected as research subjects.

[0058] 1.2 Inclusion criteria:

[0059] (1) The patient is 18 years of age or older;

[0060] (2) Meets the diagnostic criteria of the Third International Consensus Definition of Sepsis and Septic Shock (Sepsis-3);

[0061] (3) The infection site is clearly identified, such as the thoracic organs, abdominal organs, urinary system, etc.

[0062] (4) New-onset brain dysfunction following infection, meeting at least one or both of the following criteria:

[0063] ①GCS score <15 points, or GCS score decreased by ≥2 points from baseline in the past 48 hours;

[0064] ② Positive delirium assessment (CAM-ICU assessment), including acute onset or fluctuation of mental and consciousness status changes, attention deficit, and abnormal assessment using the Richmond agitation-sedation scale (RASS score), or accompanied by confusion [18,19];

[0065] (5) The patient voluntarily participates in this study and signs (or is signed by a family member or legal representative) the informed consent form.

[0066] 1.2 Experimental Design

[0067] After meeting the inclusion criteria, all patients were randomly assigned 1:1 to either the experimental group or the control group using a computer-generated random number table.

[0068] Experimental group: Only received the herbal decoction of Example 7, taken once in the morning and once in the evening (40 minutes after meals), warm, for 14 days as one course of treatment.

[0069] Control group: Received routine Western medicine treatment (referring to the 2016 International Guidelines for Severe Sepsis and Septic Shock).

[0070] 1.3 Test Methods

[0071] Baseline observational indicators and clinical data of patients were collected, including gender, age, vital signs (heart rate, systolic blood pressure, diastolic blood pressure, mean arterial pressure, body temperature), personal history, underlying diseases, source of infection, use of mechanical ventilation, and use of vasoactive drugs. The primary efficacy endpoint of this trial was the APACHE II score before and after treatment. Secondary endpoints included organ function-related scores: SOFA score and GCS score. Inflammatory markers included peripheral blood leukocyte count, neutrophil percentage, lymphocyte percentage, C-reactive protein, procalcitonin, TNF-α, and IL-6 before and after treatment. Coagulation markers included peripheral blood platelet count, fibrinogen, prothrombin time, partially activated prothrombin time, and D-dimer before and after treatment. Neurological injury markers included RASS score, peripheral blood neuron-specific enolase (NSE), central nervous system-specific protein (S100-β), and neurofilament light chain (NfL) values ​​before and after treatment. 28-day prognosis included the number of patients surviving at 28 days and the survival rate in both groups. Traditional Chinese Medicine (TCM) Syndrome Score: A TCM syndrome score scale was developed based on the "Guiding Principles for Clinical Research of New Traditional Chinese Medicines (Trial Implementation)" to assess the changes in the total score before and after treatment, and to determine the overall effective rate of syndrome treatment (the percentage of clinically cured, significantly effective, and effective cases): Clinically cured: TCM clinical symptoms and signs disappear or basically disappear, and the syndrome score decreases by ≥95%; Significantly effective: TCM clinical symptoms and signs improve significantly, and the syndrome score decreases by ≥70%; Effective: TCM clinical symptoms and signs improve, and the syndrome score decreases by ≥30%; Ineffective: TCM clinical symptoms and signs do not improve significantly, or even worsen, and the syndrome score decreases by less than 30%; The calculation formula adopts the nimodipine method, i.e., [(pre-treatment score - post-treatment score) ÷ pre-treatment score] × 100%. Safety indicators: Liver function (including alanine aminotransferase, aspartate aminotransferase, and total bilirubin), kidney function (including blood urea nitrogen and serum creatinine), and electrolytes (including serum potassium and serum sodium) before and after treatment. Adverse event monitoring: For any possible adverse reaction symptoms / adverse events, record them in a timely manner, including the time of occurrence, clinical manifestations, intervention measures, and outcomes.

[0072] 1.4 Data Analysis

[0073] Data analysis and calculations were performed using SPSS 26.0. The Shapiro-Wilk test was used to test normality. For normally distributed continuous variables, values ​​were expressed as mean ± standard deviation (x ± s), and independent samples t-tests were used for comparison. For non-normally distributed variables, values ​​were expressed as median and interquartile range (IQR: Q25, Q75), and Kruskal-Wallis tests were used for comparison. Categorical variables were expressed as percentages, and chi-square tests were used for comparison. A p-value < 0.05 was considered statistically significant.

[0074] After completing the primary analysis, all cases were re-divided into survival and death groups based on 28-day survival outcomes. Baseline data were first compared between the two groups. Statistically significant differences (P < 0.05) and important clinical confounding factors were included in univariate logistic regression analysis. Multicollinearity of the included variables was tested using the variance inflation factor (VIF). Variables with significant collinearity (VIF > 10) were discarded according to clinical or statistical principles. Then, stepwise regression was applied to multivariate logistic regression analysis of variables with P < 0.05 from the univariate analysis to further analyze the independent impact of drug treatment on 28-day survival rate and to correct for possible confounding factors. Clinically significant variables such as age and sex were also included in the multivariate analysis, and the Hosmer-Lemeshow test was used to assess the goodness of fit (P > 0.05 indicated a good fit).

[0075] 2. Test Results

[0076] 2.1 General Information

[0077] This study included 65 patients. Four patients were excluded due to failure to complete the randomization intervention (two patients did not complete 72 hours of treatment, and two patients withdrew midway). Ultimately, 61 patients completed the randomization intervention and were included in the study analysis, with 31 cases in the experimental group and 30 cases in the control group. The experimental group consisted of 21 males and 10 females, while the control group consisted of 20 males and 10 females. The median age of the two groups was 73.0 years and 76.0 years, respectively. Regarding underlying diseases, the experimental group had 17 (54.8%) patients with hypertension, 10 (32.3%) with coronary atherosclerotic heart disease, 6 (19.4%) with atrial fibrillation, 1 (3.2%) with chronic obstructive pulmonary disease, and 3 (9.7%) with chronic renal insufficiency, respectively. In the control group, the corresponding disease predators were 18 (60.0%), 7 (23.3%), 9 (30.0%), 2 (6.7%), 5 (16.7%), and 2 (6.7%), respectively. Regarding the source of infection, the experimental group had 26 (83.9%) patients with respiratory infections, 3 (9.7%) patients with abdominal infections, and 2 (6.5%) patients with urinary tract infections, respectively. In the control group, the corresponding infection sources were 27 (90.0%), 2 (6.7%), and 1 (3.3%), respectively. A comparative analysis of the general information of the two groups of patients revealed no significant differences between them in terms of age, sex ratio, vital signs (heart rate, body temperature, systolic blood pressure, diastolic blood pressure, mean arterial pressure), pre-existing underlying diseases, source of infection, use of mechanical ventilation, and use of vasoactive drugs (P > 0.05). Data are shown in Table 2.

[0078] Table 2 Comparison of basic information of the two groups of patients

[0079]

[0080]

[0081] Note: IQR: Interquartile Range

[0082] 2.2 Key Observation Indicators

[0083] The clinical scores of the two groups are shown in Table 3. Before treatment, the median APACHE II score in the experimental group was 18 points, while the median APACHE II score in the control group was 16 points. There was no statistically significant difference between the two groups (P > 0.05). After the treatment course, the median APACHE II score in the experimental group was 11 points, while the median APACHE II score in the control group was 14 points. The intergroup comparison showed a significant difference between the experimental and control groups (P < 0.05). Intra-group analyses before and after treatment in both the experimental and control groups showed that the score in the experimental group improved significantly after treatment compared to before treatment (P < 0.05); the score in the control group improved after treatment compared to before treatment, but the difference was not statistically significant. See Table 3 for details.

[0084] Table 3 Main Observation Indicators

[0085]

[0086] Note: IQR: Interquartile Range; P-value is the result of intergroup comparison; * indicates that P < 0.05 after treatment compared to before treatment.

[0087] 2.3 Secondary observation indicators

[0088] 2.3.1 Organ function-related scoring indicators

[0089] Table 4 shows the organ function-related scoring indicators for the two groups. There were no statistically significant differences in SOFA and GCS scores between the two groups before treatment (P > 0.05). After the treatment course, intergroup comparisons showed that the SOFA and GCS scores in the experimental group were significantly different from those in the control group (P < 0.05). Intragroup analyses before and after treatment in both the experimental and control groups showed that the scores in the experimental group improved significantly after treatment compared to before treatment (P < 0.05); while the scores in the control group improved after treatment compared to before treatment, but the difference was not statistically significant.

[0090] Table 4 Organ function-related scoring indicators

[0091]

[0092] Note: IQR: Interquartile Range; P-value is the result of intergroup comparison; * indicates that P < 0.05 after treatment compared to before treatment.

[0093] 2.3.2 Inflammatory markers

[0094] Statistical analysis of white blood cell count, neutrophil percentage, lymphocyte percentage, C-reactive protein, procalcitonin, TNF-α, and IL-6 levels before and after treatment in both groups revealed no statistically significant differences in these indicators before treatment (P > 0.05). After treatment, intergroup comparisons showed that the experimental group had significantly lower levels of white blood cell count, neutrophil percentage, C-reactive protein, procalcitonin, TNF-α, and IL-6 than the control group, with statistically significant differences (P < 0.05). Intragroup analyses within both the experimental and control groups showed that the above indicators in the experimental group significantly improved after treatment compared to before treatment (P < 0.05) (Table 5).

[0095] Table 5 Inflammatory markers

[0096]

[0097]

[0098] Note: IQR: Interquartile Range; P-value is the result of intergroup comparison; * indicates that P < 0.05 after treatment compared to before treatment.

[0099] 2.3.3 Coagulation Indicators

[0100] Before treatment, there were no statistically significant differences in platelet count, prothrombin time, activated partial thromboplastin time, D-dimer, and fibrinogen levels between the two groups (P > 0.05). After treatment, no statistically significant differences were found between the groups for these indicators (P > 0.05) (Table 6).

[0101] Table 6 Coagulation Indicators

[0102]

[0103] Note: IQR: Interquartile Range; P-value is the result of intergroup comparison.

[0104] 2.3.4 Neurological Injury Indicators

[0105] There were no statistically significant differences in RASS scores and the three indicators (NSE, S100-β, and NfL) between the two groups before treatment (P > 0.05). After treatment, the experimental group showed significant improvement in all the above indicators compared with the control group, with statistically significant differences (P < 0.05). Within each group, the above indicators were compared before and after treatment, and the results showed that all three indicators in both the experimental and control groups decreased significantly after treatment (P < 0.05) (Table 7).

[0106] Table 7 Indicators of Nerve Injury

[0107]

[0108] Note: IQR: Interquartile Range; P-value is the result of intergroup comparison; * indicates that P < 0.05 after treatment compared to before treatment.

[0109] 2.3.5 Prognosis at 28 Days

[0110] In the experimental group, 24 patients (77.4%) survived and 7 patients (33.6%) died after 28 days; in the control group, 15 patients (50.0%) survived and 15 patients (50.0%) died. The risk of all-cause mortality in the experimental group was 0.292 times that in the control group (95% CI: 0.097, 0.881). The 28-day survival rate in the experimental group was better than that in the control group, and the difference was statistically significant (P < 0.05) (Table 8).

[0111] Table 8. Prognosis after 28 days

[0112]

[0113] Note: The p-value is based on the chi-square test result, indicating the statistical significance of the difference in 28-day survival rate between the treatment group and the control group.

[0114] 2.3.6 Evaluation of Traditional Chinese Medicine Syndrome Scores

[0115] There was no statistically significant difference in TCM syndrome scores between the two groups before treatment (P > 0.05). After treatment, intergroup comparisons showed that the TCM syndrome scores in the experimental group were significantly lower than those in the control group, with statistically significant differences (P < 0.05). Intragroup comparisons of TCM syndrome scores before and after treatment in both groups showed a significant decrease after treatment (P < 0.05). According to the TCM syndrome efficacy evaluation criteria mentioned above, the total effective rate of syndrome efficacy in the experimental group (80.6%) was better than that in the control group (36.7%), with a statistically significant difference (P < 0.05) (Table 9).

[0116] Table 9 Evaluation of Traditional Chinese Medicine Syndrome Scores

[0117]

[0118] Note: P-values ​​are for intergroup comparisons; * indicates P < 0.05 after treatment compared to before treatment.

[0119] 2.4 Safety Indicators

[0120] The comparison of liver function, kidney function and electrolyte-related indicators between the two groups before and after treatment showed no statistically significant differences in the above indicators between the two groups before and after treatment (P>0.05) (Table 10).

[0121] Table 10 Safety Indicators

[0122]

[0123] Note: IQR: Interquartile Range; P-value is the result of intergroup comparison.

[0124] 2.5 Adverse Events

[0125] During the study, none of the included patients experienced any serious adverse reactions related to medication.

[0126] 2.6 Results of the secondary grouping analysis

[0127] This study included 22 patients with a total poor prognosis after 28 days, of whom 39 survived. They were then regrouped into a survival group (n=39) and a death group (n=22) based on their 28-day survival status.

[0128] 2.6.1 Comparison of baseline data based on outcome grouping

[0129] Baseline data of the survival group and the death group were compared between the two groups. The results showed that there were significant differences between the two groups in the following items: APACHE II score, SOFA score, GCS score, procalcitonin, IL-6, D-dimer, RASS score, alanine aminotransferase, blood urea nitrogen, blood sodium, use of vasoactive drugs, and whether or not the traditional Chinese medicine decoction in Example 7 was used for treatment (P < 0.05), as shown in Table 11.

[0130] Table 11 Comparison of data grouped according to outcome

[0131]

[0132]

[0133] Note: IQR: Interquartile Range

[0134] 2.6.2 Univariate Analysis

[0135] Indicators with statistically significant differences in baseline data between the survival group and the death group (P < 0.05) and important clinical confounding factors (age, sex) were included in univariate logistic regression analysis. The results showed that seven items, including baseline APACHE II score, SOFA score, GCS score, RASS score, serum sodium, use of vasoactive drugs, and use of traditional Chinese medicine decoction intervention in Example 7, were significantly correlated with 28-day survival (P < 0.05) (Table 12).

[0136] 2.6.3 Multifactor Analysis

[0137] Multivariate logistic regression analysis was performed on the indicators with P < 0.05 in the univariate analysis above, as well as important clinical confounding factors (age, sex). The results showed that after adjusting for factors such as age, sex, APACHE II score, SOFA score, GCS score, RASS score, serum sodium, and use of vasoactive drugs, "intervention with the traditional Chinese medicine decoction in Example 7" was still an independent factor affecting 28-day survival (OR: 5.558, 95% CI: 1.117, 27.655, P < 0.05), as shown in Table 1-11.

[0138] Table 12 List of univariate and multivariate analyses

[0139]

[0140] Experimental Example 2 (Clinical Therapeutic Effect - Basic Experiment on the Effect of the Herbal Composition of the Invention on Mice with Septic Encephalopathy)

[0141] 1. Materials and Methods

[0142] 1.1 Animals

[0143] In this study, male C57BL / 6J mice aged 8–12 weeks and weighing 20–25 g were used as experimental subjects. The mice were provided by Shanghai Silex Laboratory Animal Co., Ltd. Husbandry conditions were strictly controlled at a room temperature of 22±0.5℃, relative humidity of 50–60%, and a 12-hour light-dark cycle, while providing standard feed and free access to water. The mice were acclimatized to the experimental environment for 7 days and were fasted for 6 hours before the start of the formal experiment to minimize experimental interference.

[0144] 1.2 Construction of a sepsis encephalopathy model.

[0145] To establish a sepsis-associated encephalopathy model, the cecal ligation and puncture (CLP) method was used, following standard operating procedures described in existing literature. The specific procedures were as follows: Mice were anesthetized with isoflurane inhalation (induction concentration 4%, maintenance concentration 2%). After successful anesthesia, the mice were placed abdomen-up on the operating table with limbs immobilized. Hair on the lower abdomen was removed with a power shaver. After disinfection with 0.5% povidone-iodine solution, a 1 cm incision was made along the midline in the lower abdomen. After exposing and separating the cecum, the middle segment of the cecum was ligated with 3.0 silk suture to avoid completely blocking intestinal blood flow. A 22-gauge needle was then used to puncture the cecum, ensuring complete patency of the puncture site. A small amount of feces was squeezed out of the puncture site by gently pressing the cecum. The cecum was then returned to the abdominal cavity, and the abdominal incision was sutured layer by layer. Immediately after the surgery, the mice were resuscitated by subcutaneous injection of sterile saline (0.9%, 24 mL / kg), and the environment was kept warm to avoid causing a drop in body temperature.

[0146] 1.3 Animal experimental drugs, grouping and treatment protocols.

[0147] The mice were treated with the traditional Chinese medicine decoction in Example 7. Following the method of converting human and mouse equivalent doses in "Methodology of Pharmacological Research of Traditional Chinese Medicine" edited by Chen Qi, a solution with a concentration of 1.9 g / mL was prepared for later use.

[0148] The experimental mice were randomly divided into three groups: a sham-operated group, a model group, and a drug-treated group (n=24 in each group). The specific treatment protocols are as follows:

[0149] (1) Sham surgery group: Received the same anesthesia and laparotomy as CLP method, but without cecal ligation and puncture.

[0150] (2) Model group: A sepsis-related encephalopathy model was established using the CLP method.

[0151] (3) Drug administration group: A sepsis-related encephalopathy model was established using the CLP method.

[0152] Twenty-four hours after the model was established, observe the mice's fur, eye discharge, and activity level. Record and evaluate the modified neurologic severity score (mNSS). A lack of energy, reduced activity, frizzy and dull fur, purulent discharge from the corners of the eyes, or a significantly elevated mNSS score indicate successful model establishment.

[0153] After the evaluation, the three groups of mice were administered the corresponding medication regimens as follows:

[0154] (1) Sham surgery group: 0.3 mL of normal saline was administered by gavage once a day for 5 consecutive days.

[0155] (2) Model group: 0.3 mL of physiological saline was administered by gavage once a day for 5 consecutive days.

[0156] (3) Administration group: 0.3 mL of the traditional Chinese medicine decoction prepared in Example 7 was administered by gavage once a day for 5 consecutive days.

[0157] 1.4 Acquisition and preservation of experimental samples.

[0158] After the evaluation, mice were anesthetized using isoflurane inhalation at an induction concentration of 4% and a maintenance concentration of 2%. The mice were completely unconscious after anesthesia to ensure painless subsequent procedures. Using aseptic techniques, 0.2 mL of blood was collected from the mouse eyeballs and immediately stored at 4°C. The left ventricle was perfused with 50 mL of pre-cooled physiological saline (4°C), while the right atrium was opened to drain blood. Successful perfusion was indicated by the viscera (such as the liver and lungs) turning noticeably white. The mouse brain tissue was quickly removed and divided into left and right hemispheres. The left hemisphere was fixed in 4% paraformaldehyde solution for subsequent histological analysis. The right hemisphere was separated into the cortex and hippocampus, placed in cryovials, frozen in liquid nitrogen, and then transferred to a -80°C freezer. Blood samples were centrifuged at 3,500 rpm for 10 minutes at 4°C, and the supernatant serum was separated and stored at -80°C for subsequent experiments.

[0159] 1.5 Statistical Analysis.

[0160] The statistical analysis of the data in this study was performed using SPSS 26.0 statistical software platform, and GraphPadPrism 9.0 was used for data visualization and graphical presentation. All experimental data are expressed as mean ± standard error (x ± sx). Independent samples t-tests were mainly used for comparisons between groups, with a significance level set at P < 0.05. For comparisons among multiple groups, one-way ANOVA was used; if the differences were significant, non-normally distributed data were analyzed using non-parametric tests. All experiments were performed in at least three replicates to ensure the reliability and reproducibility of the results.

[0161] 2. Research Results

[0162] 2.1 Example 7: Traditional Chinese medicine decoction improves survival rate in mice with sepsis-associated encephalopathy.

[0163] The survival of mice in the model group, sham-operated group, and the group treated with the traditional Chinese medicine decoction in Example 7 was observed. The results showed that 24 hours after modeling, there was no significant difference in survival between the model group and the treatment group (P > 0.05). From the 3rd day after surgery, the survival rate of mice in the treatment group showed an increasing trend compared to the model group, and a significant difference appeared on the 5th day (P < 0.05). Figure 1 ).

[0164] 2.2 Example 7: Traditional Chinese medicine decoction improves neurobehavioral function in mice with sepsis-associated encephalopathy.

[0165] Animal behavioral scoring: The experiment assessed the mice’s motor, sensory, balance and reflex abilities using a modified neurological severity score. Figure 2As shown in (a), in the model group, mice exhibited significant decline in motor function, sensory loss, and decreased reflex ability, with a significantly higher total score than the model group. In Example 7, the scores of mice treated with the herbal decoction were significantly lower than those in the model group (P < 0.05), indicating that the motor and coordination abilities of the mice in the treatment group had recovered to some extent. Neurological reflex scores: Neurological reflex scores assessed the mice's neurological reflex abilities from five aspects: auricular reflex, corneal reflex, righting reflex, tail-flicking reflex, and escape reflex. On the first day after modeling, the neurological reflex scores of both the model group and the treatment group were significantly lower than those in the sham-operated group (P < 0.05). However, on the third and fifth days, the neurological reflex scores of the treatment group were still lower than those in the sham-operated group, but significantly higher than those in the model group (P < 0.05), suggesting that the neurological reflex abilities of the mice in the treatment group had recovered. Figure 2 (b) Open field test results: The open field test results showed that the total movement distance of the model group mice was significantly lower than that of the sham-operated group (P < 0.05), see [link to relevant documentation]. Figure 2 (c) The time spent in the central region was significantly shortened (P < 0.05), see Figure 2 (d) indicates that CLP-induced sepsis-associated encephalopathy mice exhibited a significant decline in motor function and cognitive status. The total distance traveled by mice in the drug-treated group was not significantly different from that in the model group, but it was already higher than that in the model group. Figure 2 (c) The time spent in the central region was significantly longer than that in the model group (P < 0.05), see [reference needed]. Figure 2 (d) Furthermore, the trajectory distribution of the mice in the drug-treated group was more uniform than that of the mice in the model group, see... Figure 2 (e) indicates that the activity level and cognitive status of the mice in the drug-treated group were improved.

[0166] 2.3 Example 7: Traditional Chinese medicine decoction improves brain tissue damage in mice with sepsis-associated encephalopathy.

[0167] HE staining showed that the brain tissue of mice in the sham-operated group was structurally intact, with tightly packed neurons, normal cell body morphology, and uniform and clear nuclear staining, without obvious inflammatory cell infiltration or pathological changes. In contrast, the CLP model group showed significant pathological damage in mouse brain tissue, including disordered neuronal arrangement, shrunken or deformed cell bodies, chromatin condensation, and vacuolar changes, indicating severe brain damage. The brain tissue of mice in the drug-treated group showed more regular neuronal arrangement and near-normal cell body morphology. Figure 3 ).

[0168] TUNEL staining showed virtually no positive cells in the brain tissue of the sham-operated group mice. In contrast, the number of positively stained cells was significantly increased in the model group, suggesting that CLP-induced sepsis triggered extensive apoptosis in the brain tissue. The number of positively stained cells in the mice treated with the traditional Chinese medicine decoction in Example 7 was significantly reduced compared to the model group. These results suggest that the pathological damage to the brain tissue of the mice treated with the traditional Chinese medicine decoction in Example 7 was protected.Figure 4 ).

[0169] 2.4 Example 7: Traditional Chinese medicine decoction improves the levels of inflammatory and neurological damage factors in mice with sepsis-associated encephalopathy.

[0170] Changes in serum inflammatory factors and neurological damage markers were detected by ELISA. Results showed that serum IL-6 and TNF-α levels in both the model group and the drug-treated group were significantly higher than in the sham-operated group (P < 0.001), indicating that sepsis triggered a significant systemic inflammatory response. Furthermore, serum concentrations of the neurological damage markers NSE and S100-β were also significantly higher in the drug-treated group than in the sham-operated group (P < 0.001), reflecting significant neurological damage in mice with sepsis-associated encephalopathy. In contrast, serum IL-6 and TNF-α levels in the drug-treated group were significantly decreased (P < 0.001), and the concentrations of NSE and S100-β were also significantly reduced (P < 0.001). Figure 5 ad).

[0171] The levels of MDA and SOD in brain tissue reflect oxidative stress. This experiment detected their content in brain tissue. The results showed that the MDA level in the brain tissue of mice in the model group and the drug-treated group was significantly higher than that in the sham-operated group (P < 0.001), suggesting that brain tissue underwent severe lipid peroxidation under sepsis-induced damage. Simultaneously, the activity of the antioxidant enzyme SOD was also significantly lower than that in the sham-operated group (P < 0.001), indicating that the tissue's antioxidant capacity was inhibited. In contrast, the MDA level in the brain tissue of mice in the drug-treated group was significantly lower than that in the model group (P < 0.001), and SOD activity was significantly restored compared to the model group (P < 0.001). Furthermore, in the model group, the levels of NSE and S100-β in the brain tissue of mice were significantly higher than those in the sham-operated group (P < 0.001), indicating significant neuronal and glial cell damage. In contrast, the concentrations of NSE and S100-β in the brain tissue of the drug-treated group were significantly lower (P < 0.001). Figure 5 eh).

Claims

1. A traditional Chinese medicine composition for treating sepsis encephalopathy, characterized in that, It is prepared from the following raw materials: ginseng 20-40, raw rhubarb 6-18, red vine 20-40, dandelion 20-40, aconite 20-40, and leech 3-9.

2. The traditional Chinese medicine composition for treating sepsis encephalopathy according to claim 1, characterized in that, The raw materials are weighed as follows: ginseng 26-34, rhubarb 9-14, red vine 26-34, dandelion 26-34, aconite 26-34, and leech 5-7. 3.The traditional Chinese medicine composition for preventing and treating novel coronavirus infection according to claim 1, characterized in that, The raw materials are weighed as follows: ginseng 30, rhubarb 12, red vine 30, dandelion 30, aconite 30, and leech 6.

4. The traditional Chinese medicine composition for treating sepsis encephalopathy according to any one of claims 1-3, characterized in that, The rhubarb is raw rhubarb.

5. The traditional Chinese medicine composition for treating sepsis encephalopathy according to any one of claims 1-3, characterized in that, The dosage form of the traditional Chinese medicine composition is a decoction, tablet, capsule, powder, granule, oral liquid, pill, tincture, syrup, suppository, gel, spray, or injection prepared according to conventional methods.

6. A method for preparing the traditional Chinese medicine composition decoction for treating sepsis encephalopathy according to claim 5, characterized in that, Weigh ginseng, rhubarb, red vine, dandelion, aconite, and leech by weight parts, and decoct twice with 4-10 times the amount of water, each time for 0.5-2 hours. Combine the decoctions, filter, and concentrate to 2-4 times the amount, and obtain the product.

7. A method for preparing the granules of the traditional Chinese medicine composition for treating sepsis encephalopathy according to claim 5, characterized in that, The method comprises the following steps: A. Weigh aconite by weight parts, add 8-12 times the amount of water, and decoct with a strong fire until boiling, then change to a weak fire and decoct for 60-90 minutes. Filter to obtain filtrate A; B. Weigh ginseng by weight parts, add 6-8 times the amount of water, and decoct at 60-80°C for 30-60 minutes. Filter to obtain filtrate B; C. Weigh red vine, dandelion, leech, and rhubarb by weight parts, add 5-8 times the amount of water, and decoct twice, each time for 1-2 hours. Combine the filtrates and filter to obtain filtrate C; D. Combine the filtrates obtained in steps A, B, and C, and concentrate under reduced pressure to a relative density of 1.10-1.25 at 60°C. Allow to stand for 8-12 hours, then filter to obtain a clear concentrated solution. Spray dry, and collect the spray-dried powder; E. Add a pharmaceutically acceptable excipient to the spray-dried powder as needed, spray or wet-granulate, dry at low temperature, sieve, and obtain granules.

8. A method for preparing the Chinese medicinal composition capsule for treating sepsis encephalopathy according to claim 5, characterized in that, The method comprises the following steps: A. Weigh aconite by weight parts, add 8-12 times the amount of water, and decoct for 60-90 minutes. Filter to obtain filtrate A; B. Weigh ginseng by weight parts, add 6-8 times the amount of water, and decoct at 60-80°C for 30-60 minutes. Filter to obtain filtrate B; C. Weigh red vine, dandelion, leech, and rhubarb by weight parts, add 5-8 times the amount of water, and decoct twice, each time for 1-2 hours. Combine the filtrates and filter to obtain filtrate C; D. Combine the filtrates obtained in steps A, B, and C, and concentrate under reduced pressure to a thick paste. Dry to obtain a dry powder; E. Mix the dry powder with a pharmaceutically acceptable excipient, dry at low temperature until the water content meets the standard, and fill into a hollow capsule to obtain a capsule.

9. Use of the traditional Chinese medicine composition of any one of claims 1-4 in the preparation of a medicament for treating sepsis encephalopathy.

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