A traditional Chinese medicine composition for strengthening body resistance and increasing white blood cells, and a preparation method and application thereof

Abstract

This invention belongs to the field of traditional Chinese medicine technology, specifically relating to a traditional Chinese medicine composition for tonifying vital energy and promoting blood circulation, its preparation method, and its application. The traditional Chinese medicine composition of this invention, by weight, comprises: 6-15 parts of raw ginseng, 10-15 parts of epimedium, 10-20 parts of atractylodes macrocephala, and 6-15 parts of prepared licorice root. This traditional Chinese medicine composition has the effects of greatly tonifying vital energy, strengthening the spleen, and warming the kidneys. It can be used to treat fatigue and susceptibility to COVID-19, and also to treat symptoms such as fatigue, poor appetite, abdominal distension, and belching caused by spleen and stomach qi deficiency. Clinical studies have found that this traditional Chinese medicine composition can significantly increase the lymphocyte count in patients, improve their immunosuppressive state, thereby significantly improving the fatigue symptoms in patients with COVID-19 and achieving the goal of effectively treating COVID-19.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine technology, specifically relating to a traditional Chinese medicine composition for strengthening the body's resistance and promoting the white blood cell count, its preparation method, and its application. Background Technology

[0002] There is mounting evidence that a significant proportion of patients who have “recovered” from hospitalization due to COVID-19 infection suffer from long-term complications and sequelae affecting multiple organs and systems, including various physical and neuropsychiatric symptoms, which generally last for more than 12 weeks.

[0003] According to the World Health Organization, prolonged COVID-19 refers to the presence of symptoms three months after infection with the novel coronavirus, with symptoms and effects lasting for at least two months and not explainable by other diagnoses.

[0004] Symptoms of prolonged COVID-19 can be categorized into three types: residual symptoms after recovery, organ dysfunction persisting after recovery, and new symptoms in asymptomatic or mild cases. It may involve multiple systems, including the circulatory, respiratory, urinary, digestive, endocrine, nervous, and mental systems, and includes a variety of symptoms such as fatigue, chest tightness, shortness of breath, chest pain, palpitations, cognitive impairment (brain fog), sleep disturbances, gastrointestinal symptoms, anxiety, and depression. The immune response after COVID-19 infection exhibits an initial surge followed by a decline. Initially, pro-inflammatory responses dominate, followed by a chronic immunosuppressive phase, with persistent inflammation, immunosuppression, and catabolism syndrome (PICS) predominating during the recovery period. Studies have shown that in prolonged COVID-19 patients, a simultaneous reduction in lymphocytes and the production of autoantibodies can be detected.

[0005] While some researchers have proposed using immunomodulatory drugs, such as intravenous immunoglobulin, to treat the immunosuppressive state of long-term COVID-19 patients, there is currently insufficient evidence to prove their effectiveness. Furthermore, no corresponding clinical studies have demonstrated their ability to regulate immunosuppression or improve clinical symptoms. In addition, several studies on the treatment of long-term COVID-19 are underway, such as using dietary supplementation with nicotinamide nucleoside to regulate pro-inflammatory factors and reduce cognitive symptoms and fatigue, supplementing with probiotics to reduce inflammation in long-term COVID-19 patients, and utilizing the antioxidant and anti-inflammatory effects of melatonin. However, the effectiveness and safety of these methods in improving long-term COVID-19 require further evaluation.

[0006] Traditional Chinese medicine (TCM) has played an important role in the treatment of COVID-19 infection due to its multi-target efficacy and minimal side effects. Currently, there is still a lack of effective TCM compositions for treating chronic COVID-19, therefore it is necessary to develop such a composition. Summary of the Invention

[0007] The first objective of this invention is to provide a traditional Chinese medicine composition that strengthens the body and promotes the white blood cell count; the second objective of this invention is to provide a method for preparing the traditional Chinese medicine composition; and the third objective of this invention is to provide an application of the traditional Chinese medicine composition.

[0008] According to a first aspect of the present invention, a traditional Chinese medicine composition for strengthening the body and promoting blood circulation is provided, wherein the raw materials comprise, by weight, 6-15 parts of raw ginseng, 10-15 parts of epimedium, 10-20 parts of atractylodes macrocephala, and 6-15 parts of prepared licorice.

[0009] This invention believes that the most common clinical manifestation of prolonged COVID-19 is persistent fatigue, and that the pathogenesis of prolonged COVID-19 is immunosuppression. Therefore, this invention proposes a formulation targeting its pathogenesis and clinical manifestations.

[0010] This invention posits that Yang Qi is the foundation of Qi transformation in the body. Weakness of Yang Qi can lead to impaired circulation of Qi, blood, and body fluids, resulting in insufficient nourishment of the five internal organs, muscles, and bones. The spleen is the foundation of acquired constitution, while the kidney is the foundation of innate constitution. The spleen governs the transformation and transportation of the essence of food and water, requiring the warming effect of kidney Yang. Similarly, the essence of the kidneys also depends on the continuous replenishment and generation of the essence of food and water. The relationship between the spleen and kidneys is one of mutual support and influence between acquired and innate constitution. This highlights the importance of spleen and kidney function in the human body, and its significance in enhancing the immunity of long-term COVID-19 patients. This invention, through the principle of tonifying the spleen and kidneys (the foundations of both acquired and innate constitution) and supporting the body's vital energy, formulates a traditional Chinese medicine composition that can increase lymphocyte count, enhance immunity, and alleviate fatigue and weakness symptoms in long-term COVID-19 patients.

[0011] In this formula, sun-dried ginseng, a powerful tonic for vital energy, serves as the principal ingredient. Epimedium, which tonifies the kidneys, strengthens yang, fortifies muscles and bones, and dispels wind and dampness, serves as the assistant ingredient. The combination of these two ingredients greatly tonifies vital energy and promotes yang circulation, thus alleviating fatigue. Furthermore, it strengthens the spleen and kidneys, warms and nourishes the limbs, allowing them to move freely. Atractylodes macrocephala tonifies the spleen and stomach, dries dampness, and harmonizes the middle jiao, specifically treating weak spleen and stomach qi, loss of appetite, fatigue, and shortness of breath; it serves as the adjuvant ingredient. Prepared licorice root tonifies the spleen and qi, harmonizes the other ingredients, and is specifically used for weak spleen and stomach, fatigue, and weakness; it serves as the guiding ingredient. The combination of Atractylodes macrocephala and prepared licorice root is specifically for strengthening the spleen and qi. The addition of Atractylodes macrocephala to dry dampness and harmonize the middle jiao can dispel wind-dampness obstruction and enhance the tonic effect of sun-dried ginseng. All the ingredients work together to greatly tonify vital energy, strengthen the spleen, and warm the kidneys.

[0012] The raw materials for this formula come from the following sources:

[0013] Sun-dried ginseng: The dried root of Panax ginseng CAMey., a plant in the Araliaceae family. Cultivated ginseng is called "garden ginseng," and garden ginseng that has been sun-dried or oven-dried is called "sun-dried ginseng." It enters the spleen, lung, and heart meridians. It is sweet and slightly bitter, and neutral in nature.

[0014] Epimedium: The whole herb of Epimedium brevicornu, a plant in the Berberidaceae family, and other plants in the same genus. Pungent and warm in nature. Enters the liver and kidney meridians.

[0015] Atractylodes macrocephala: The dried rhizome of Atractylodes macrocephala Koidz., a plant in the Asteraceae family. It enters the spleen and stomach meridians. It is bitter, sweet, and warm in nature.

[0016] Licorice: The dried root of *Glycyrrhiza uralensis* Fisch., *Glycyrrhiza inflata* Bat., or *Glycyrrhiza glabra* L. (all belonging to the legume family). It enters the heart, lung, spleen, and stomach meridians. It is sweet and neutral in nature.

[0017] In some embodiments, the raw material composition by weight includes: 9 parts of sun-dried ginseng, 10 parts of epimedium, 10 parts of atractylodes macrocephala, and 10 parts of roasted licorice.

[0018] In some embodiments, the dosage form of the traditional Chinese medicine composition is an oral solution, powder, or granule.

[0019] In some implementations, pharmaceutically acceptable excipients are also included.

[0020] In some embodiments, the excipient is at least one selected from the following: sustained-release agent, excipient, filler, binder, wetting agent, disintegrant, absorption promoter, adsorbent carrier, surfactant, and lubricant.

[0021] According to a second aspect of the present invention, a method for preparing the above-mentioned tonifying and purifying traditional Chinese medicine composition is provided, wherein when the dosage form of the traditional Chinese medicine composition is an oral solution, the preparation method includes the following steps:

[0022] Soak the raw materials in 8-12 times their weight of water for 20-40 minutes, then heat to boiling and maintain a gentle boil for 1-2 hours. Filter, and repeat the decoction process 1-2 times on the residue. Combine the filtrates to obtain the final product. The oral solution is further packaged after cooling, making it easy to carry and distribute.

[0023] According to a third aspect of the present invention, a method for preparing the above-mentioned tonifying and purifying traditional Chinese medicine composition is provided, wherein when the dosage form of the traditional Chinese medicine composition is a powder, the preparation method includes the following steps:

[0024] Crush or chop each raw material separately, then pass the crushed or chopped raw materials through 10-mesh and 50-mesh sieves respectively. Continue to crush or chop the coarse particles that cannot pass through the 10-mesh sieve, and take the powder that is between the 10-mesh and 50-mesh sieves. Then mix the powders of each raw material according to the formula, put them into a packaging bag, and seal it.

[0025] According to a fourth aspect of the present invention, the use of the above-described tonifying and invigorating traditional Chinese medicine composition in the preparation of a medicament for treating sepsis and / or secondary infections of sepsis is provided.

[0026] According to a fifth aspect of the present invention, the use of the above-described tonifying and invigorating traditional Chinese medicine composition in the preparation of a medicament for treating COVID-19 is provided.

[0027] In some implementations, symptoms of long-term COVID-19 include fatigue and weakness.

[0028] According to a sixth aspect of the present invention, the above-described tonifying and invigorating traditional Chinese medicine composition is provided for use in the preparation of a medicament for treating fatigue and / or poor appetite and / or abdominal distension and / or belching caused by spleen and stomach qi deficiency.

[0029] According to a seventh aspect of the present invention, the application of the above-described tonifying and invigorating traditional Chinese medicine composition in the preparation of a medicinal and edible substitute tea is provided.

[0030] In the traditional Chinese medicine composition of the present invention, ginseng, epimedium, and atractylodes macrocephala are traditional Chinese medicinal materials listed in the "List of Items that can be used in health food", and licorice root is a traditional Chinese medicinal material listed in the "List of Items that are both food and medicine". The raw materials have the characteristics of being both food and medicine and being safe to use, and can be used to prepare a substitute tea that is both food and medicine.

[0031] The preparation method of the medicinal and edible substitute tea of ​​the present invention includes the following steps: drying raw ginseng, epimedium, atractylodes macrocephala and roasted licorice to a moisture content of <3.0%, removing impurities and inferior raw materials, sterilizing with ultraviolet light, weighing each raw material according to the raw material ratio, mixing evenly, and then obtaining the product.

[0032] The resulting medicinal and edible substitute tea is packaged in a gauze tea bag and brewed with 100mL of warm water before use. This medicinal and edible substitute tea can be used to treat fatigue and susceptibility associated with COVID-19; it can also be used to treat symptoms such as fatigue, poor appetite, abdominal distension, and belching caused by spleen and stomach qi deficiency.

[0033] Compared with the prior art, the beneficial effects of the present invention include:

[0034] (1) Animal experiments show that the traditional Chinese medicine composition of the present invention can effectively slow down the weight loss of mice with secondary infection and improve the survival rate of mice with secondary infection; reduce the inflammation of lungs, liver and kidneys of mice with secondary infection; improve the edema of lungs, liver and kidneys caused by secondary infection and have a protective effect on thymus; increase the secretion level of anti-inflammatory factors in the body after secondary infection and reduce the content of inflammatory factors in serum, thus having a good anti-inflammatory effect; have a certain protective effect on T cells and thymus, and at the same time, can increase the level of peripheral blood leukocytes under the immunosuppressed state after sepsis, improve the body's immune status and enhance the ability to fight infection.

[0035] (2) The traditional Chinese medicine composition of this invention has a simple formulation, consisting of only four herbs, but its effects are concentrated and specifically designed to support the body's vital energy and enhance immunity. Clinical studies have found that the traditional Chinese medicine composition of this invention can significantly improve the fatigue and weakness symptoms of long-term COVID-19 patients by supporting their vital energy, and significantly increase the lymphocyte count, thereby improving their immunosuppressive state and achieving the goal of effectively treating long-term COVID-19. The traditional Chinese medicine composition of this invention can improve the SF-36 health questionnaire score and EQ-5D-5L score of long-term COVID-19 patients.

[0036] (3) The traditional Chinese medicine composition of the present invention can be used to treat fatigue and weakness and susceptibility to COVID-19, and can also be used to treat symptoms such as weakness, poor appetite, abdominal distension and belching caused by spleen and stomach qi deficiency. Attached Figure Description

[0037] Figure 1 This is a comparison of the survival time of mice in each group in Experiment 1.

[0038] Figure 2 This is a graph showing the changes in body weight of mice in each group during Experiment 2.

[0039] Figure 3 These are HE staining images of the lungs of mice in each group during Experiment 2.

[0040] Figure 4 These are HE staining images of the livers of mice in each group during Experiment 2.

[0041] Figure 5 These are HE staining images of the kidneys of mice in each group during Experiment 2.

[0042] Figure 6 The spleen index is the value of each group of mice in Experiment 2.

[0043] Figure 7 These are the lung indices of mice in each group during Experiment 2.

[0044] Figure 8 These are the liver indices of mice in each group during Experiment 2.

[0045] Figure 9 These are the kidney indices of mice in each group during Experiment 2.

[0046] Figure 10 It is the thymus index of mice in each group in Experiment 2.

[0047] Figure 11 These are the ELISA results of serum IL-1β in mice from each group in Experiment 2.

[0048] Figure 12 These are the ELISA results of serum TNF-α in mice from each group in Experiment 2.

[0049] Figure 13These are the ELISA results of serum IL-6 in mice from each group in Experiment 2.

[0050] Figure 14 These are the ELISA results of serum IL-4 in mice from each group in Experiment 2.

[0051] Figure 15 These are the ELISA results of serum IL-10 in mice from each group in Experiment 2.

[0052] Figure 16 The results are flow cytometry findings on CD3 (T cell) expression in the spleens of mice in each group during Experiment 2.

[0053] Figure 17 The results are obtained by flow cytometry of NK1.1 expression in the spleen of mice in each group during Experiment 2.

[0054] Figure 18 The results are flow cytometry findings on the expression of CD19 and B220 (B cells) in the bone marrow of mice in each group during Experiment 2.

[0055] Figure 19 The results are flow cytometry findings on the expression of CD11b (monocytes, macrophages) and F4 / 80 in the bone marrow of mice in each group of Experiment 2.

[0056] Figure 20 The results are flow cytometry findings of CD3 expression in peripheral blood of mice in Experiment 2.

[0057] Figure 21 The results are flow cytometry findings of CD19 (B cell) expression in peripheral blood of mice in Experiment 2.

[0058] Figure 22 The results are flow cytometry findings of NK1.1 expression in peripheral blood of mice in Experiment 2.

[0059] Figure 23 The results are flow cytometry findings of CD11b and F4 / 80 expression in peripheral blood of mice in Experiment 2. Detailed Implementation

[0060] The present invention will be further described in detail below with reference to specific embodiments. It is worth noting that the following embodiments are only for better explaining the content of the present invention and do not limit the scope of protection of the present invention. Process steps not disclosed in the embodiments are prior art. Unless otherwise specified, all raw materials are commercially available.

[0061] Example 1

[0062] The oral solution for strengthening the body and promoting white blood cell count in this embodiment is composed of the following ingredients: 9g of raw ginseng, 10g of epimedium, 10g of atractylodes macrocephala, and 10g of prepared licorice root.

[0063] Its preparation method includes the following steps:

[0064] (1) Soak the raw materials in 10 times their weight of water for 30 minutes, then heat to boiling and keep simmering for 1 hour. After separation, obtain the first decoction and the dregs. Take the dregs obtained after the first decoction, add water equivalent to 8 times the total weight of the raw materials, heat to boiling and keep simmering for 1 hour. After separation, obtain the second decoction and the dregs. Combine the first decoction and the second decoction to obtain the Fuzheng Shengbai oral solution.

[0065] (2) Cool the obtained Fuzheng Shengbai oral solution and then dispense it into individual containers. This ratio is for one dose, which is approximately 150 mL.

[0066] Example 2

[0067] The Fuzheng Shengbai powder of this embodiment is composed of the following raw materials: 9g of raw ginseng, 10g of epimedium, 10g of atractylodes macrocephala, and 10g of prepared licorice.

[0068] Its preparation method includes the following steps:

[0069] (1) Take raw ginseng, crush it, and pass it through a 10-mesh and a 50-mesh sieve at the same time. Continue to crush the coarse particles that cannot pass through the 10-mesh sieve, and take the powder that is between the 10-mesh and 50-mesh sieves.

[0070] (2) Powder the Epimedium, Atractylodes macrocephala, and Glycyrrhiza uralensis into powder according to the method in step (1);

[0071] (3) Mix all the raw material powders evenly, pack them into a sealed bag, and seal it. When taking it, dissolve it in 150mL of warm water.

[0072] Example 3

[0073] The oral solution for strengthening the body and promoting white blood cell count in this embodiment is composed of the following ingredients: 15g of raw ginseng, 15g of epimedium, 20g of atractylodes macrocephala, and 15g of prepared licorice root.

[0074] Its preparation method is the same as that of Example 1.

[0075] Example 4

[0076] The Fuzheng Shengbai powder of this embodiment is composed of the following raw materials: 6g of raw ginseng, 12g of epimedium, 15g of atractylodes macrocephala, and 6g of prepared licorice.

[0077] Its preparation method is the same as that in Example 2.

[0078] To illustrate the efficacy of the traditional Chinese medicine composition for strengthening the body and promoting white blood cell count of the present invention, the following animal experiments were conducted using the oral solution for strengthening the body and promoting white blood cell count prepared in Example 1.

[0079] Experiment 1 Death Protection Experiment

[0080] Fifty male SPF-grade C57BL / 6 mice, weighing 20-22g (8-10 weeks old), were randomly divided into five groups of ten mice each after 3-4 days of acclimatization: sham-operated group (sham group), model group (PA group), thymosin group (Th group), low-dose Fuzheng Shengbai Fang group (FZSB 2.96g / kg group), and medium-dose Fuzheng Shengbai Fang group (FZSB 5.92g / kg group). Except for the sham-operated group (which underwent the same surgery but without ligation and puncture), all other groups underwent cecal ligation and puncture (CLP) surgery. Approximately two-thirds of the cecum was ligated and punctured twice with a 20-gauge needle, leading to sepsis. Immediately after abdominal closure, 1 ml of 0.9% saline was injected intraperitoneally for resuscitation. A secondary infection model was established on day 4 after CLP. Mice surviving after CLP were administered 20 μL (2 × 10⁻⁶ g / kg) of cecal ligation and puncture. 8 CFU (intranasal) was used to inoculate Pseudomonas aeruginosa (ATCC: 27853). In the sham-operated group, an equal volume of PBS (Beijing Solarbio Science & Technology Co., Ltd., batch number 2308001, diluted with sterile water to a 1:1 concentration) was administered as nasal drops. Broad-spectrum carbapenem antibiotics (imipenem / cilastatin, Zhuhai Federal Pharmaceutical Co., Ltd., batch number 230220809, 25 mg / kg) were administered twice: 1 hour after CLP and 2 hours after Pseudomonas aeruginosa infection.

[0081] The medication administration details for each group are as follows:

[0082] Thymopeptide group: Thymopeptide was injected subcutaneously every other day starting 1 hour after CLP, with an injection volume of 50 μl (diluted with sterile water), for a total of 6 injections;

[0083] Low-dose group of Fuzheng Shengbai formula: Gavage administration was started 2 hours after CLP. The drug was the Fuzheng Shengbai oral solution prepared in Example 1, administered once a day at a dose of 2.96 g / kg for 12 consecutive days.

[0084] Medium-dose group of Fuzheng Shengbai formula: Gavage administration was started 2 hours after CLP. The drug was the Fuzheng Shengbai oral solution prepared in Example 1, administered once a day at a dose of 5.92 g / kg for 12 consecutive days.

[0085] The sham surgery group and the model group were administered an equal volume of sterile water by gavage once a day for 12 consecutive days.

[0086] Mice were monitored for 11 days starting from CLP surgery. Mice mortality was recorded (a weight loss of >20% was considered death).

[0087] The log-rank test was used to compare the survival time of different groups. The results of the survival time comparison of mice in each group are as follows: Figure 1 As shown.

[0088] from Figure 1 It can be seen that the survival curves of the model group and the medium-dose group of Fuzheng Shengbai Formula are significantly different (P<0.01), suggesting that the traditional Chinese medicine composition of the present invention can improve the survival rate of mice with secondary infection.

[0089] Experiment 2: Drug Efficacy Test

[0090] 1. Grouping, drug administration, and modeling

[0091] Sixty male SPF-grade C57BL / 6 mice, weighing 20-22g (8-10 weeks old), were randomly divided into 5 groups of 12 mice each after 3-4 days of acclimatization: sham group, model group (PA group), thymosin group (Th group), low-dose Fuzheng Shengbaifang group (FZSB 2.96g / kg group), and high-dose Fuzheng Shengbaifang group (FZSB 5.92g / kg group).

[0092] Low-dose group of Fuzheng Shengbai formula: The drug was administered 7 days before modeling. The drug was the Fuzheng Shengbai oral solution prepared in Example 1, once a day, at a dose of 2.96 g / kg, for 14 consecutive days.

[0093] High-dose group of Fuzheng Shengbai formula: The drug was administered 7 days before modeling. The drug was the Fuzheng Shengbai oral solution prepared in Example 1, once a day, at a dose of 5.92 g / kg, for 14 consecutive days.

[0094] Thymopeptide group: Thymopeptide was injected subcutaneously every other day starting 1 hour after CLP, with an injection volume of 50 μl (diluted with sterile water), for a total of 4 injections;

[0095] Sham surgery group and model group: Seven days before modeling, the same amount of sterile water was administered by gavage once a day for 14 consecutive days.

[0096] The model was established as follows: Except for the sham-operated group (which underwent the same surgery but without ligation and puncture), all other groups of mice underwent cecal ligation and puncture (CLP) surgery. Approximately two-thirds of the cecum was ligated and punctured twice with a 20-gauge needle, leading to sepsis. Immediately after abdominal closure, 1 ml of 0.9% saline was injected intraperitoneally for resuscitation. A secondary infection model was established on day 5 after CLP. Mice that survived CLP were administered 20 μL (2 × 10⁻⁶) of saline solution. 8CFU (intranasal) was used to inoculate Pseudomonas aeruginosa (ATCC: 27853). In the sham-operated group, an equal volume of PBS (Beijing Solarbio Science & Technology Co., Ltd., batch number 2308001, diluted with sterile water to a 1:1 concentration) was administered as nasal drops. Broad-spectrum carbapenem antibiotics (imipenem / cilastatin, Zhuhai Federal Pharmaceutical Co., Ltd., batch number 230220809, 25 mg / kg) were administered twice: 1 hour after CLP and 2 hours after Pseudomonas aeruginosa infection.

[0097] Peripheral blood and organ tissues were collected 24 hours after secondary infection. Due to anesthesia during nasal instillation causing the death of some mice, the final number of mice in each group was 9.

[0098] 2. Observation indicators

[0099] (1) Mice were monitored for 11 days after CLP surgery. Changes in mouse body weight were recorded (a decrease in body weight of >20% was considered death).

[0100] (2) Lung, liver, and kidney tissues from 3 mice in each group were fixed with paraformaldehyde and stained with hematoxylin and eosin (HE). Organ tissues fixed in 4% paraformaldehyde solution were removed and rinsed three times with running water for 5 minutes each time. Gradient ethanol dehydration was performed: 30% ethanol, 50% ethanol, 70% ethanol, 95% ethanol 1, 95% ethanol 2, and anhydrous ethanol were added sequentially, adjusting the dehydration time according to the tissue type and size. Clearing was performed: 50% ethanol and 50% xylene mixed solution, xylene 1, xylene 2, and xylene 3 were added sequentially in a fume hood. Paraffin embedding and sectioning were performed, with liver and lung sections at 5 μm thickness and kidney sections at 2 μm thickness. The sections were baked overnight in a 60℃ oven. The next day, the baked paraffin sections were removed and sequentially immersed in xylene 1, xylene 2, anhydrous ethanol 1, and anhydrous ethanol 2, then rinsed with running water to remove the alcohol from the samples, completing the dewaxing process. Immerse the slides in a staining solution containing hematoxylin for 3-5 minutes, wash with water until colorless, immerse in differentiation solution for 3-5 seconds, then wash quickly with water. Immerse in blueing solution for 3-5 seconds, then wash quickly with water. Immerse the slides sequentially in 85% ethanol, 95% ethanol, eosin dye, anhydrous ethanol 1, anhydrous ethanol 2, anhydrous ethanol 3, n-butanol, xylene 1, xylene 2 for 3-5 minutes, then remove and air dry quickly in a ventilated area. Mount with neutral resin. Scan and save. Use SlideViewer software to examine the slides and observe the pathological damage.

[0101] (3) Take lungs, liver, kidneys, spleen, and thymus from 3 mice in each group to calculate the organ index. Calculation formula: Organ index = organ weight / mouse body weight * 100%.

[0102] (4) Serum from 3 mice in each group was collected and the levels of IL-4, IL-10, IL-1β, IL-6 and TNF-α were detected by ELISA.

[0103] ① Mouse IL-4, IL-10, IL-1β ELISA (Neobioscience / Xinbosheng)

[0104] Take out the strips required for the experiment from the sealed bag that has been equilibrated to room temperature. Add standard and specimen universal diluent to the blank wells, and add specimens or standard products of different concentrations (100 μl / well) to the remaining corresponding wells. Seal the reaction wells with sealing tape, incubate in a 37 °C incubator in the dark for 90 min. Wash the plate 5 times. After adding the washing solution each time, wait for 30 - 60 s and then pour out the washing solution and pat dry. Add biotinylated antibody diluent to the blank wells, and add biotinylated antibody working solution (100 μl / well) to the remaining wells. Seal the reaction wells with new sealing tape, incubate in a 37 °C incubator in the dark for 60 min. Wash the plate 5 times, and the operation is the same as before. Add enzyme conjugate diluent to the blank wells, and add enzyme conjugate working solution (100 μl / well) to the remaining wells. Seal the reaction wells with new sealing tape, incubate in a 37 °C incubator in the dark for 30 min. Turn on the power of the microplate reader, preheat the instrument, and set up the detection program. Wash the plate 5 times. Add 100 μl of chromogenic substrate (TMB) to each well, incubate in a 37 °C incubator in the dark for 15 min. Add 100 μl of reaction termination solution to each well, mix well and immediately measure the absorbance at 450 nm (within 3 min), and calculate the sample concentration according to the standard curve.

[0105] ② Mouse IL-6 ELISA (B&D Company)

[0106] Coating buffer preparation: Mix 7.13 g of sodium bicarbonate and 1.59 g of sodium carbonate, add pure water to 1 L, and adjust the pH value to 9.5. Dilute the capture antibody 1:250 in the coating buffer, coat 100 μl per well in the microplate, and incubate overnight in a 4 °C refrigerator. Wash 3 times with 300 μl of washing solution. After the last washing, invert the plate and blot the residual buffer on absorbent paper. Block the plate with 200 μl / well of assay diluent. Incubate at room temperature for 1 h. Wash 3 times. Prepare the standard and dilute the samples with assay diluent. Add 100 μl of standard and samples, and add the control to the designated wells. Seal the plate and incubate at room temperature for 2 h. Wash the plate 5 times. Add 100 μl of detection solution (detection antibody + SAV-HRP reagent) to each well. Seal the plate and incubate at room temperature for 1 h. Wash the plate 7 times. After adding the washing solution each time, wait for 30 - 60 s and then pour out the washing solution and dry. Add 100 μl of TMB substrate solution to each well. Incubate at room temperature in the dark (without adding a sealing agent) for 30 min. Add 50 μl of termination solution to each well. Read the absorbance at 450 nm within 30 min after stopping the reaction. Calculate the sample concentration according to the standard curve.

[0107] ③ Mouse TNF-α ELISA (R&D Company)

[0108] Washing buffer: 0.05% Tween-20 with PBS; Standard / sample dilution buffer: 1% BSA with PBS. Capture antibody was diluted 1:125 in coating buffer, 100 μl per well was used to coat microwells, and incubated overnight at room temperature. The plate was washed 3 times with 300 μl of washing buffer per well, patting dry after each wash. 300 μl of standard / sample dilution buffer was used to block the microwells at room temperature for 1 h, followed by 3 washes. Standards / samples were diluted to a specific ratio using dilution buffer, 100 μl per well, incubated at room temperature for 2 h, followed by 3 washes. Detection antibodies were diluted 1:60 using dilution buffer, 100 μl per well, incubated at room temperature for 2 h, followed by 3 washes. SAV-HRP reagent was diluted 1:40 using dilution buffer, 100 μl per well, incubated at room temperature in the dark for 20 min, followed by 3 washes. Add 100 μl of colorimetric solution to each well and incubate at room temperature in the dark for 20 min. Then add 50 μl of stop solution to each well and measure the absorbance at 450 nm. Calculate the sample concentration based on the standard curve.

[0109] (5) The spleen and bone marrow of 6 mice in each group were taken for flow cytometry to detect the number and classification of immune cell subsets.

[0110] ① Spleen cell suspension preparation steps: Remove mouse spleens and immerse them in clean PBS solution. Place the spleen in a 200-mesh sieve and gently grind it with a tissue homogenizer until no obvious red lumps remain. Rinse the sieve with 15 mL of PBS and collect the rinsing solution in a 15 mL centrifuge tube. Centrifuge at 300 g for 5 min and discard the supernatant. Add 2 mL of 1× erythrocyte lysis buffer to resuspend the cells. Lyse at room temperature for 2–3 min, then immediately add 10 mL of PBS and centrifuge at 300 g for 5 min. Discard the supernatant. Resuspend the spleen cells in cell staining buffer. Filter the cell suspension again through a 200-mesh sieve, count the cells, and adjust the cell concentration to 1×10⁻⁶. 7 Cells / mL. Add 100 μL of single-cell suspension to a flow cytometry tube, then add CD3-FITC and NK1.1-BV421 antibodies, mix well, and incubate at room temperature for 30 min in the dark. Wash once with 2 mL PBS, centrifuge at 1000 rpm for 5 min at 4°C. Add 400 μL PBS and filter into a flow cytometry tube. Analyze the data using a flow cytometer and analyze with NovoExpress to determine antibody expression.

[0111] ② Preparation of bone marrow single-cell suspension: Carefully pinch the abdominal skin between the mouse's hip joints with ophthalmic forceps, carefully cut open with ophthalmic scissors, and separate the skin of the hind limbs. Cut the skin downwards at the ankle and upwards at the hip joint to free the mouse's two hind limbs. Carefully dissect the muscles, cut off the femur and tibia, remove the cartilage at both ends, and expose the red bone marrow cavity. Note that as much bone marrow cavity as possible should be preserved in this process. Using a 1mL sterile syringe, draw 1mL of PBS solution and gently insert it into the bone marrow cavity to flush out the bone marrow. Repeat 2-3 times to flush out most of the cells. After flushing, gently pipette the cells to disperse the cell clumps. Filter the bone marrow flushing solution through a 200-mesh filter, collect the filtrate in a 15mL centrifuge tube, centrifuge at 300g for 5min, and discard the supernatant. Resuspend the cells in cell staining buffer and count them, adjusting the cell concentration to 1×10⁻⁶. 7 Cells / mL. Add 100 μL of single-cell suspension to a flow cytometry tube, then add CD19-APC, B220-FITC, F4 / 80-PE, and CD11b-PE / Cy5.5 antibodies. Mix well and incubate at room temperature for 30 min in the dark. Wash once with 2 mL PBS, centrifuge at 1000 rpm for 5 min at 4°C. Add 400 μL PBS and filter into a flow cytometry tube. Analyze the data using a NovoExpress analyzer to determine antibody expression.

[0112] (6) Peripheral anticoagulated blood was collected from 6 mice in each group and flow cytometry was used for immunophenotyping.

[0113] Peripheral blood cell suspension preparation steps: Take 100 μL of each blood sample, add 1 mL of 1× erythrocyte lysis buffer, and lyse for 8 min at room temperature (mix twice during lysis). Centrifuge at 1000-1500 rpm for 5 min at 4℃. Discard the supernatant, maintaining a 100 μL system per sample. Add 4.5 μL of peripheral blood mixed antibody (adjust according to the actual antibody's instructions), and incubate on ice in the dark for 30 min. Wash once with 2 mL of PBS, maintaining the same centrifugation conditions. Add 300 μL of PBS and filter into a flow cytometry tube. Analyze the data using a flow cytometer and analyze with NovoExpress to determine antibody expression.

[0114] 3. Experimental Results

[0115] The changes in body weight of mice in each group are as follows: Figure 2 As shown. From Figure 2It can be seen that the weight of mice in the sham-operated group showed negative growth before day 6 and positive growth after day 6. The weight of mice in the other groups showed negative growth within 12 days. Among them, the weight loss of mice in the high-dose group and the low-dose group of Fuzheng Shengbai formula was less than that in the model group, indicating that the Fuzheng Shengbai traditional Chinese medicine composition of the present invention can effectively slow down the weight loss of mice with secondary infection and improve the survival rate of mice.

[0116] HE staining images of mouse lungs in each group are shown below. Figure 3 As shown. From Figure 3 It can be seen that the lungs of the model group mice showed inflammatory cell infiltration, significant alveolar wall thickening, significant interstitial widening, and hemorrhage. Lung damage was significantly reduced in all treatment groups, with the low-dose Fuzheng Shengbai formula group showing the most significant improvement.

[0117] HE staining images of mouse livers in each group are shown below. Figure 4 As shown. From Figure 4 It can be seen that the livers of mice in the model group showed obvious inflammatory response and lymphocyte aggregation, the livers of mice in the thymopeptide group showed diffuse inflammatory infiltration, and the liver inflammatory response of mice in the low-dose and high-dose groups of Fuzheng Shengbaifang was significantly reduced.

[0118] HE staining images of mouse kidneys in each group are shown below. Figure 5 As shown, from Figure 5 It can be seen that the kidneys of mice in the model group showed obvious inflammatory infiltration and hemorrhage. The kidneys of mice in the thymopeptide group and the high-dose group of Fuzheng Shengbaifang also showed a certain degree of inflammatory infiltration. The kidney inflammation of mice in the low-dose group of Fuzheng Shengbaifang was significantly reduced.

[0119] Figure 6-10 The figures show the organ indices of mice in each group. *P<0.05, **P<0.01 in the figure.

[0120] ***P<0.001, where, Figure 6 These are the spleen indices of mice in each group. Figure 7 These are the lung indices of each group of mice. Figure 8 These are the liver indices of each group of mice. Figure 9 These are the kidney indices of each group of mice. Figure 10 These are the thymus indices of mice in each group. From... Figure 6-10As can be seen, compared with the sham-operated group mice, the spleen, lung, liver, and thymus indices of the model group mice were significantly different. Specifically, compared with the model group, the lung index of the low-dose Fuzheng Shengbai formula group mice was significantly lower (P<0.05), indicating that the Fuzheng Shengbai herbal composition of the present invention can improve pulmonary edema caused by secondary infection. Compared with the model group, the liver index of the low-dose Fuzheng Shengbai formula group mice was lower, the kidney index of the low-dose and high-dose Fuzheng Shengbai formula groups mice was lower, and the thymus index of the low-dose and high-dose Fuzheng Shengbai formula groups mice was higher, but the difference was not significant. This indicates that the Fuzheng Shengbai herbal composition of the present invention can improve liver and kidney edema caused by secondary infection and has a protective effect on the thymus.

[0121] Figure 11-15 The figures show the serum ELISA results for each group of mice. In the figure, *P<0.05, **P<0.01, and ****P<0.0001. Figure 11 This is the result of an ELISA test for serum IL-1β. Figure 12 This is the result of an ELISA test for serum TNF-α. Figure 13 This is the result of an ELISA test for serum IL-6. Figure 14 This is the result of an ELISA test for serum IL-4. Figure 15 This is the result of an ELISA test for serum IL-10. From... Figure 11-15 It can be seen that, in terms of serum pro-inflammatory factor levels, compared with the sham-operated group, the model group showed increased levels of IL-1β, TNF-α, and IL-6, with IL-1β and IL-6 showing significant increases. However, compared with the model group, both the low-dose and high-dose groups of the Fuzheng Shengbai formula showed some degree of downregulation of IL-1β and IL-6, and the low-dose group showed some degree of downregulation of TNF-α. The high-dose group showed a significant downregulation of serum IL-6, showing statistically significant differences compared with the model group. In terms of serum anti-inflammatory factor levels, compared with the sham-operated group, the model group showed decreased levels of IL-4 and IL-10, with IL-10 showing a significant decrease. Compared with the model group, both the low-dose and high-dose groups of the Fuzheng Shengbai formula showed some degree of upregulation of IL-4, and the low-dose group showed a significant upregulation of IL-10, with the low-dose group showing a significant upregulation of serum IL-4. This indicates that the traditional Chinese medicine composition for strengthening the body and promoting blood circulation can increase the secretion level of anti-inflammatory factors in the body and reduce the content of inflammatory factors in the serum, thus having a good anti-inflammatory effect.

[0122] Figure 16 The results of flow cytometry show the expression of CD3 (T cells) in the spleen. Figure 17 Flow cytometry results show the expression of NK1.1 in the spleen. From... Figure 16-17It can be seen that, compared with the sham-operated group, CD3 expression in all groups decreased significantly, with the decrease being more pronounced in the low-dose and high-dose groups of the Fuzheng Shengbai formula. Compared with the sham-operated group, NK1.1 expression in all groups increased significantly, with the increase being more pronounced in the low-dose and high-dose groups of the Fuzheng Shengbai formula. There was no statistically significant difference between the low-dose and high-dose groups of the Fuzheng Shengbai formula and the model group.

[0123] Figure 18 Flow cytometry results showing the expression of CD19 and B220 (B cells) in bone marrow. Figure 19 Flow cytometry results showing the expression of CD11b (monocytes, macrophages) and F4 / 80 in bone marrow. Figure 18-19 It can be seen that, compared with the sham surgery group, B220 expression decreased significantly in all groups, with the decrease being more pronounced in the low-dose group of Fuzheng Shengbaifang; compared with the sham surgery group, CD11b expression increased in all groups, with the increase being most significant in the low-dose group of Fuzheng Shengbaifang. However, there was no statistically significant difference between the low-dose and high-dose groups of Fuzheng Shengbaifang and the model group.

[0124] Figure 20 The results of flow cytometry show the expression of CD3 in peripheral blood. Figure 21 The results of flow cytometry show the expression of CD19 (B cells) in peripheral blood. Figure 22 The results of flow cytometry analysis show the expression of NK1.1 in peripheral blood. Figure 23 Flow cytometry results showing the expression of CD11b and F4 / 80 in peripheral blood. Figure 20-23 It can be seen that, compared with the sham surgery group, the levels of each indicator in the model group did not change significantly, while the CD3 level in the drug administration group decreased to varying degrees, with a significant decrease in the high-dose group of Fuzheng Shengbaifang; CD19, NK1.1, and CD11b in the drug administration group all showed significant increases.

[0125] The flow cytometry results showed that T cells were not mobilized in either the low-dose or high-dose group of Fuzheng Shengbaifang, suggesting that the traditional Chinese medicine composition of the present invention has a certain protective effect on T cells and thymus. At the same time, the levels of B lymphocytes, NK cells, and monocytes in peripheral blood were significantly increased in both the low-dose and high-dose groups of Fuzheng Shengbaifang, which can better clear pathogens, suggesting that the traditional Chinese medicine composition of the present invention can increase the level of peripheral blood leukocytes in the immunosuppressed state after sepsis, improve the body's immune status, and enhance anti-infection ability.

[0126] The above descriptions are merely some specific embodiments of the present invention. For those skilled in the art, various modifications and improvements can be made without departing from the inventive concept of the present invention, and all such modifications and improvements fall within the scope of protection of the present invention.

Claims

1. A traditional Chinese medicine composition for treating sepsis and / or secondary sepsis infections, characterized in that, The raw material composition by weight is as follows: 6-15 parts of sun-dried ginseng, 10-15 parts of epimedium, 10-20 parts of atractylodes macrocephala, and 6-15 parts of roasted licorice.

2. The traditional Chinese medicine composition for treating sepsis and / or secondary sepsis infection according to claim 1, characterized in that, The raw material composition by weight is: 9 parts of sun-dried ginseng, 10 parts of epimedium, 10 parts of atractylodes macrocephala, and 10 parts of roasted licorice.

3. The traditional Chinese medicine composition for treating sepsis and / or secondary sepsis infection according to claim 1 or 2, characterized in that, The dosage form of the traditional Chinese medicine composition is an oral solution, powder, or granule.

4. The traditional Chinese medicine composition for treating sepsis and / or secondary sepsis infection according to claim 1 or 2, characterized in that, It also includes pharmaceutically acceptable excipients.

5. A method for preparing the traditional Chinese medicine composition for treating sepsis and / or secondary sepsis infection according to any one of claims 1-4, characterized in that, When the dosage form of the traditional Chinese medicine composition is an oral solution, its preparation method includes the following steps: Soak the raw materials in 8-12 times their weight of water for 20-40 minutes, then heat to boiling and maintain a gentle boil for 1-2 hours. Filter the mixture and boil the residue 1-2 times. Combine the filtrates to obtain the final product.

6. A method for preparing the traditional Chinese medicine composition for treating sepsis and / or secondary sepsis infection according to any one of claims 1-4, characterized in that, When the dosage form of the traditional Chinese medicine composition is a powder, its preparation method includes the following steps: Crush or chop each raw material separately, then pass the crushed or chopped raw materials through 10-mesh and 50-mesh sieves respectively. Continue to crush or chop the coarse particles that cannot pass through the 10-mesh sieve, and take the powder that is between the 10-mesh and 50-mesh sieves. Then mix the powders of each raw material according to the formula, put them into a packaging bag, and seal it.

7. The use of the traditional Chinese medicine composition for treating sepsis and / or secondary sepsis infection as described in any one of claims 1-4 in the preparation of a medicament for treating sepsis and / or secondary sepsis infection.

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