Application of rhizoma polygonati water extract in preparation of medicine for treating acute appendicitis

Abstract

The invention discloses an application of a rhizoma polygonati water extract in preparation of a medicine for treating acute appendicitis. The rhizoma polygonati water extract provided by the invention can be used for remarkably improving clinical symptoms of appendicitis, effectively protecting mechanical barrier integrity of intestinal mucosa and relieving inflammatory response. According to the invention, the treatment effect of the rhizoma polygonati water extract on acute appendicitis is defined for the first time, the application blank of traditional Chinese medicines in the field is filled, and a scientific basis is provided for subsequent development of similar medicines by revealing the action mechanism (such as regulation and control of tight junction proteins and inflammatory factors) of the rhizoma polygonati water extract; the method has important value in the theoretical research level and the application and popularization aspects.

Description

Technical Field

[0001] The invention relates to the field of biomedicine, in particular to application of a water extract of polygonatum sibiricum in preparing medicine for treating acute appendicitis. Background Art

[0002] Acute appendicitis is one of the most common acute abdominal conditions in general surgery, with a high morbidity rate. Statistics show that patients hospitalized for acute appendicitis account for approximately 10-15% of all gastrointestinal surgery inpatients during the same period. Young and middle-aged individuals are most susceptible, accounting for approximately 40% of cases. The main causative factors of acute appendicitis include obstruction of the appendix lumen, bacterial infection, and neuroreflexia. Typical clinical manifestations include persistent, migratory, or paroxysmal abdominal pain, primarily in the right lower abdomen, accompanied by nausea and vomiting. Laboratory tests in most patients reveal elevated white blood cell and neutrophil counts, C-reactive protein, calcitonin, and interleukins. Incorrect or delayed treatment can lead to infection and progression to perforation, acute suppurative peritonitis, and, in severe cases, panperitonitis, portal vein thrombosis, or septic shock, which can be life-threatening.

[0003] Traditionally, appendicitis requires immediate surgical resection upon diagnosis, as it can worsen and become life-threatening. However, some studies suggest that acute uncomplicated appendicitis and complicated appendicitis develop independently. Therefore, surgery remains the primary treatment for acute complicated appendicitis (perforated, gangrenous, and periappendiceal abscess), while controversy remains about whether surgical resection is the preferred treatment for acute uncomplicated appendicitis. This is driven by a shift in understanding of the appendix's function: the appendix is ​​not useless. Its mucosa is rich in lymphoid tissue and forms a crucial component of the intestinal mucosa-associated lymphoid tissue (GALT). It produces immunoglobulins (sIgA) that contribute to the formation of the gastrointestinal biofilm barrier and secretes digestive enzymes and hormones to maintain intestinal function. Furthermore, the appendix serves as a "safe reservoir" for intestinal flora, promoting post-colectomy bacterial repopulation. Loss of the appendix is ​​thought to increase the risk of colorectal cancer, autoimmune diseases, and Crohn's disease. On the other hand, the disadvantages of surgical treatment cannot be ignored, including surgical trauma, anesthesia and blood loss risks, postoperative adhesive intestinal obstruction, infection and other complications. Even for suitable complex cases (such as suppurative appendicitis or abscess), ultrasound or CT-guided abdominal puncture and drainage is not risk-free and is not effective for simple appendicitis. In addition, the surgical risk is significantly increased in elderly patients with cardiovascular or pulmonary diseases. Therefore, exploring effective conservative treatment options, especially for non-complex appendicitis and high-risk patients, is of great clinical significance.

[0004] Current conservative treatment typically involves antibiotics and anti-inflammatory drugs combined with fluid replacement and electrolyte balance maintenance. Studies have shown that currently recommended empirical antibiotic regimens remain effective for appendicitis, with most studies reporting efficacy rates of 75%-80%. However, conservative treatment alone has significant limitations: it generally cannot fully replace the effectiveness of surgery; longer treatment cycles result in prolonged pain for patients, increasing the medical burden; and prolonged antibiotic use can easily lead to intestinal flora disturbances and other clinical symptoms.

[0005] Based on the above-mentioned deficiencies in the prior art, the present invention explores, verifies and confirms the preventive, ameliorative and therapeutic effects of Polygonatum sibiricum on appendicitis through a series of scientific research, thereby developing a new technical direction for the prevention and treatment of appendicitis in economic animals and humans and laying a solid foundation for technical application. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide application of a water extract of polygonatum sibiricum in preparing medicine for treating acute appendicitis.

[0007] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows.

[0008] Application of polygonatum water extract in preparing medicine for preventing and / or treating acute appendicitis.

[0009] Further preferably, the drug is a drug that can upregulate the expression of intestinal mucosal barrier tight junction proteins ZO-1, Occludin and E-cadherin to prevent and / or treat acute appendicitis.

[0010] Further preferably, the drug is a drug that can reduce the level of inflammatory factors; the inflammatory factors include at least one of the serum inflammatory factors TNF-α, IL-1β and IL-17.

[0011] Further preferably, the drug is a drug that upregulates the expression of intestinal mucosal barrier tight junction proteins ZO-1, Occludin and E-cadherin and reduces the levels of serum inflammatory factors TNF-α, IL-1β and IL-17.

[0012] More preferably, the drug is a human drug or a veterinary drug.

[0013] A method for preparing a water extract of polygonatum sibiricum comprises the following steps:

[0014] (1) Wash the rhizome of Polygonatum sibiricum and dry it at 60°C, then weigh 100g;

[0015] (2) Add distilled water with a solid-liquid ratio of 1:6, soak for 30 minutes, boil, simmer for 40 minutes, and filter to obtain the first filtrate;

[0016] (3) The residue is added with distilled water, and the solid-liquid ratio is 1:4. After boiling, the residue is slowly fried for 40 minutes, and the second filtrate is obtained after filtration;

[0017] (4) The two filtrates are combined, concentrated to 100 mL by rotary evaporation, and stored at 4°C.

[0018] A pharmaceutical composition for treating acute appendicitis, comprising a therapeutically effective amount of a water extract of Rhizoma Polygonati as an active ingredient, and a pharmaceutically acceptable excipient, wherein the water extract of Rhizoma Polygonati is prepared according to the method of claim 2; the administration dose of the water extract of Rhizoma Polygonati in the pharmaceutical composition is 200-600 mg of crude drug amount / kg of body weight / day, preferably 400 mg of crude drug amount / kg of body weight / day.

[0019] The application of the water extract of Rhizoma Polygonati in up-regulating the expression of ZO-1, Occludin and E-cadherin proteins in animals.

[0020] The application of the water extract of Rhizoma Polygonati in reducing the levels of TNF-α, IL-1β and IL-17 in animals.

[0021] Further preferably, the water extract of Rhizoma Polygonati comprises a concentrated solution, a dried powder or a freeze-dried powder.

[0022] The beneficial effects produced by the above technical solution are as follows: through a rat experimental appendicitis model, the water extract of Rhizoma Polygonati can significantly reduce the colon pathological injury score of the experimental appendicitis rats within 2 weeks; immunofluorescence shows that the water extract of Rhizoma Polygonati treatment can significantly increase the expression amount of Occludin, ZO-1 and E-cadherin in the colon tissues of the experimental appendicitis rats, indicating that the water extract of Rhizoma Polygonati treatment can improve the intestinal mucosal barrier function; the detection of serum inflammatory factors TNF-α, IL-1β and IL-17 finds that there is a significant statistical difference (P<0.05) in the expression between the PSR group and the Model group. The water extract of Rhizoma Polygonati provided by the present application can significantly improve the clinical symptoms of the experimental appendicitis rats, effectively protect the integrity of the intestinal mucosal mechanical barrier, and reduce the inflammatory response. These results suggest that the water extract of Rhizoma Polygonati has a therapeutic effect on acute appendicitis, and provide preliminary experimental evidence for its use as an alternative or auxiliary treatment scheme for the disease. The present application first clearly defines the therapeutic effect of the water extract of Rhizoma Polygonati on acute appendicitis, fills the application gap of traditional Chinese medicine in this field, and provides a scientific basis for the subsequent development of similar drugs by revealing its action mechanism (such as regulating tight junction proteins and inflammatory factors). BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 It is a photograph of cecal ligation surgery.

[0024] Figure 2 It is a colon HE staining result graph of rats in the model group, the intervention group and the sham operation group.

[0025] Figure 3 This is a graph showing the difference in ZO-1 protein expression in the intestinal tissues of rats in the model group, intervention group, and sham operation group.

[0026] Figure 4 This is a graph showing the differences in E-Cadherin protein expression in the intestinal tissues of rats in the model group, intervention group, and sham operation group.

[0027] Figure 5 This is a graph showing the differences in Occludin-1 protein expression in the intestinal tissues of rats in the model group, intervention group, and sham operation group.

[0028] Figure 6 This is the result of immunofluorescence intensity analysis of ZO-1, Occludin-1 and E-Cadherin proteins in the intestinal tissues of rats in the model group, intervention group and sham operation group.

[0029] Figure 7 This is the expression result of inflammatory factors TNF-α, IL-1β, and IL-17 in the serum of rats in the model group, intervention group, and sham operation group. DETAILED DESCRIPTION

[0030] The following examples illustrate the present invention in detail. The various raw materials and equipment used in the present invention are conventional commercial products and can be directly obtained through commercial purchase. The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0031] It should be understood that when used in the present specification and the appended claims, the term "comprising" indicates the presence of described features, integers, steps, operations, elements and / or components, but does not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components and / or collections thereof.

[0032] It will also be understood that the term "and / or" used in this specification and the appended claims refers to and includes any and all possible combinations of one or more of the associated listed items.

[0033] References to "one embodiment" or "some embodiments" in this specification mean that a particular feature, structure, or characteristic described in conjunction with that embodiment is included in one or more embodiments of the present application. Thus, phrases such as "in one embodiment," "in some embodiments," "in other embodiments," and "in other embodiments" appearing in various places in this specification do not necessarily refer to the same embodiment, but rather mean "one or more but not all embodiments," unless otherwise specifically emphasized. The terms "including," "comprising," "having," and variations thereof all mean "including but not limited to," unless otherwise specifically emphasized.

[0034] In addition, in the description of the present application specification and the appended claims, the terms "first", "second", "third", etc. are only used to distinguish the descriptions and cannot be understood as indicating or implying relative importance.

[0035] The following will be combined with specific embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0036] Example 1. Experimental materials

[0037] 1.1 Medicinal materials and reagents

[0038] The Polygonatum sibiricum used in this experiment was collected from the Ziwuling forest area. This medicinal material is the rhizome of Polygonatum sibiricum, a plant of the Liliaceae family. The sample was stored at the Key Laboratory of Bioresource Conservation, Utilization, and Ecological Restoration in Longdong, Gansu Province. The required reagents are listed in Table 1.

[0039] Table 1 Experimental reagents

[0040]

[0041]

[0042] 1.2 Preparation of Polygonatum sibiricum Water Extract

[0043] The rhizome of Polygonatum sibiricum was cleaned and dried at 60℃. 100g was weighed and added with distilled water at a solid-liquid ratio of 1:6. The mixture was soaked for 30min, boiled, and simmered for 40min. The decoction was filtered through 4 layers of gauze to obtain the first filtrate. The residue was added with distilled water at a solid-liquid ratio of 1:4, boiled, and simmered for 40min to obtain the second filtrate. The two filtrates were combined and concentrated to 100mL by rotary evaporation. The mixture was stored in a refrigerator at 4℃ for later use.

[0044] Example 2, experimental method

[0045] 2.1 Establishment and evaluation of the experimental appendicitis model in rats

[0046] 2.1.1 Experimental animals

[0047] This experiment used 18 8-week-old female Sprague-Dawley rats weighing 250-300 g, SPF grade (specific pathogen-free) provided by the Animal Experimental Center of Lanzhou University. The experimental animals were kept at a temperature of 22 ± 2°C, a relative humidity of 55 ± 5%, and a 12-hour light-dark cycle. The rats had unrestricted access to food, water, and activity, and were fed an adaptive diet for 1 week. The relevant facilities and experimental procedures were carried out in accordance with the technical standards of the Ethics Committee of the Second Hospital of Lanzhou University (approval number: D2024-958).

[0048] 2.1.2 Processing Groups

[0049] Mice were randomly divided into three groups according to the random number table method: model group (Model group, n = 6), Polygonatum sibiricum water extract intervention group (PSR group, n = 6), and sham operation group (Sham group, n = 6). The Polygonatum sibiricum water extract group was given oral gavage at a concentration of 400 mg / kg per day for 14 days, and the Model group and Sham group were given the same dose of normal saline for 14 days. On the 15th day, cecal ligation surgery (Model group, PSR group) and sham operation (Sham group) were performed respectively.

[0050] 2.1.3 Modeling process

[0051] According to literature research, SD rats undergoing cecal ligation can be used as an experimental appendicitis model for scientific research. After cecal ligation, rats show pathological changes similar to appendicitis. It has two outstanding advantages. Not only is the breeding process easy, but it also provides convenience for the conduct of experiments. Therefore, this experiment constructed an experimental appendicitis model by performing cecal ligation (such as Figure 1 The specific operation process is as follows:

[0052] (1) Anesthetize the rats with 3.5% pentobarbital (40 mg / kg) by intraperitoneal injection. Stimulate the inner and outer canthi of the rat's eye. If the rat does not respond, confirm that the anesthesia is successful. Fix the rat's limbs on the surgical board in a supine position, and use ophthalmic scissors to cut the rat's lower abdominal hair and expose the skin. Use a sterile alcohol cotton swab to disinfect the rat's abdominal skin and perineum 3-4 times in a fixed direction. Prepare sterile gauze and cut a 1cm×1cm notch in the middle. Place it on the laparotomy area to fully expose the surgical site.

[0053] (2) Use toothless ophthalmic forceps to steadily lift the abdominal wall muscles to fully expose the abdominal cavity. According to the physiological structure and direction of the large intestine, explore and visualize the cecum in sequence. Then, use blunt dissection forceps to separate the cecum from the surrounding tissues, remove the cecum and accurately locate it. During the operation, the small intestine and other parts of the large intestine must be left in the peritoneal cavity, and special attention should be paid to avoid damaging the cecal branches of the ileocecal artery;

[0054] (3) Isolate the cecum, connect the distal end of the cecum to the beginning of the cecum, and tie the middle part (50%) with 2-0 non-absorbable silk suture. Use a 16G needle to puncture the distal cecum transversely and longitudinally (be careful not to damage the cecal artery), and ensure that a small amount of feces is squeezed out to prevent the wound from closing.

[0055] (4) Carefully reposition the cecum into the abdominal cavity. Do not allow feces to spread from the cecum to the edge of the abdominal wall wound during retraction. Continue to suture the muscle layer and skin layer layer by layer with two stitches each using 5-0 non-absorbable silk sutures.

[0056] (5) After recovery from anesthesia, the rat was placed back on fresh bedding and given food and water;

[0057] (6) The sham operation group was operated as follows: After the experimental animals were anesthetized, the abdominal cavity was opened according to the established procedure. After entering the abdominal cavity, the cecum was accurately located and separated. After confirming that the separated tissue was the cecum, it was gently returned to the abdominal cavity. Subsequent operations, including abdominal closure, were consistent with those of the other experimental groups.

[0058] 2.1.4 Observe the general condition of the rats (spiritual state, appetite, body weight, and stool).

[0059] 2.1.5 Collection of materials

[0060] (1) Rats were killed by overdose of anesthesia;

[0061] (2) Observation of gross specimens before sampling (inflammation, adhesion, suppuration, perforation, necrosis, etc.);

[0062] (3) Sampling: First, process the blood sample. The rat's eyeball was removed and the blood in the eye socket was dripped into a 1.5 ml EP tube. Then, the EP tube containing the blood was placed in a centrifuge at 4°C and centrifuged at 2500 rpm / min for 15 minutes. After the centrifugation, the rat serum in the upper layer was carefully collected and properly stored in a dry ice bucket. Then, tissue samples were collected. Cut open the rat's abdominal cavity, find the anus end, cut the intestine from this part, and gradually separate it to the cecum end. The tissue at the cecal ligation site, as well as the intestinal mucosa and intestinal contents at the distal end of the colon were obtained respectively. The obtained tissue samples were photographed and recorded. Afterwards, a portion of the colon tissue from each group of rats was selected and fixed with 4% paraformaldehyde. The remaining colon tissue and intestinal contents of each group of rats were stored in a refrigerator at -80°C for future use.

[0063] 2.2 HE staining

[0064] 2.2.1 Operation steps (1) Fixation: Immerse the intestinal tissue sample in 4% paraformaldehyde solution and fix it at room temperature for 24 hours to ensure the stability of tissue cell morphology;

[0065] (2) Dehydration: Place the fixed samples in different concentrations of alcohol solutions for dehydration. First, soak them in 70% alcohol for 30 minutes, then in 80% alcohol for 45 minutes, then in 95% alcohol for 30 minutes and 40 minutes respectively, and finally in 100% alcohol for 30 minutes and 40 minutes respectively, to gradually remove the water in the tissue;

[0066] (3) Transparency: Transfer the dehydrated sample to a xylene solution for transparency. First, place it in xylene I for 10 minutes, then transfer it to xylene II for 10 minutes to make the tissue transparent, which is convenient for subsequent wax immersion.

[0067] (4) Wax immersion: Place the transparent sample in a constant temperature environment of 65°C, soak it in soft paraffin for 2 hours, and then place it in hard paraffin for 3 hours to allow the paraffin to fully penetrate the tissue;

[0068] (5) Embedding: Place the wax-soaked specimen in the molten wax in the embedding frame, wait for the molten wax to cool and solidify naturally, and then embed the specimen in the paraffin block;

[0069] (6) Sectioning: Use a paraffin slicer to perform coronal serial sectioning on the embedded paraffin block to obtain tissue sections of appropriate thickness;

[0070] (7) Spreading: Spread the cut paraffin sections in warm water, then adhere them with a glass slide. Then, dry the slide in an oven at 37°C to ensure that the sections are firmly attached to the slide.

[0071] (8) Dewaxing and hydration: The dried slides were put into different reagents in turn for dewaxing and hydration. First, the slides were immersed in xylene I for 20 minutes, then moved to xylene II for 20 minutes, and then immersed in 100% alcohol, 95% alcohol, 80% alcohol, and 70% alcohol for 10 minutes each, and finally rinsed with distilled water for 2 minutes;

[0072] (9) Staining: The dewaxed and hydrated sections were stained. First, the sections were stained with hematoxylin staining solution for 3 minutes, washed with distilled water, differentiated with 1% hydrochloric acid alcohol for 15 seconds, and then rinsed with running water. Finally, the sections were re-stained with 5% eosin alcohol solution for 3-5 minutes to make the cell nucleus and cytoplasm show different colors;

[0073] (10) Dehydration, transparency, and mounting: The stained sections were dehydrated again with gradient alcohol, then made transparent in xylene, and finally mounted with neutral balsam for sample preparation to facilitate observation under a microscope.

[0074] 2.2.2 Pathological scoring criteria

[0075] (1) Three microscopic fields were randomly selected from each section. Then, according to the histochemistry score (HI) standard developed by Dieleman et al., the colon tissues of rats in each group were scored carefully, and the degree of inflammation, damage range, crypt destruction, and lesion range were observed under a microscope.

[0076] (2) Two pathologists performed double-blind reading and scoring of the sections, and the average value was taken for statistical analysis.

[0077] Table 2: Pathological damage score criteria for rat cecum

[0078]

[0079] 2.3 Immunofluorescence staining

[0080] (1) Dewaxing: The sections were placed in xylene I for 20 minutes, and then transferred to xylene II for 20 minutes to remove the paraffin on the sections;

[0081] (2) Gradient alcohol: The dewaxed sections were placed in different concentrations of alcohol solutions in turn, i.e., 100% alcohol for 3 minutes, 95% alcohol for 3 minutes, and 85% alcohol for 3 minutes, to complete the dehydration and rinsing steps;

[0082] (3) Initial washing: The sections were first washed in distilled water for 3 minutes, and then washed in PBS I, PBS II, and PBS III for 3 minutes each to remove residual alcohol and other impurities;

[0083] (4) Antigen repair: Add the prepared EDTA solution to the pressure cooker, heat it until the solution boils, and carefully place the sample slices in. Close the pressure cooker lid and start cooking. When the pressure cooker starts to steam, start the timer and continue for 3 minutes. After that, wait for the pressure cooker to cool naturally to room temperature. The entire process from placing the slices in to cooling and adding water is timed for 10 minutes, of which the steaming stage is 3 minutes;

[0084] (5) Secondary washing: Remove the antigen-retrieved sections and wash them in PBSI, PBSII, and PBSIII for 3 minutes each to further clean the sections and prepare for subsequent operations;

[0085] (6) Elimination of peroxidase: Use endogenous peroxidase blocker, add 2 drops on the surface of the slice sample, and incubate at room temperature for 10 minutes to block the activity of endogenous peroxidase;

[0086] (7) Three washes: Wash the incubated sections in PBSI, PBSII, and PBSIII for 3 minutes each to remove unreacted blocking agents and other impurities;

[0087] (8) Adding primary antibody: Add an appropriate amount of primary antibody to the slice and incubate the slice at 4°C overnight to allow the primary antibody to fully bind to the target antigen;

[0088] (9) Four washes: Wash the sections with PBSI for 8 minutes, then wash with PBSII and PBSIII for 8 minutes each to remove unbound primary antibody and other impurities;

[0089] (10) Adding secondary antibody: Gently wipe the liquid on the surface of the slice, add 2 drops of secondary antibody on the slice, keep it in a dark environment, and incubate for 2 hours to allow the secondary antibody to bind to the primary antibody;

[0090] (11) Five washes: Wash the sections in PBSI, PBSII, PBSIII, and PBSIV for 5 minutes each to remove unbound secondary antibodies and other residual substances;

[0091] (12) Sealing operation: Use anti-fluorescence attenuation mounting medium to directly seal the slices to protect the slices and prevent fluorescence attenuation;

[0092] (13) Observation and Analysis: The sealed sections were placed under a fluorescence microscope for observation and relevant images were collected. Subsequently, the fluorescence intensity of the collected images was analyzed using imageJ software and data statistics were completed.

[0093] 2.4 Enzyme-linked immunosorbent assay

[0094] 2.4.1 Serum TNF-α determination

[0095] (1) Kit preparation: Take the kit out of the storage environment and place it at room temperature for 20 minutes to allow the reagents in the kit to reach the appropriate reaction temperature.

[0096] (2) Dilution of washing solution: According to the instructions, distilled water is used to dilute the concentrated washing solution in the corresponding proportion to prepare a 1X working solution for subsequent plate washing.

[0097] (3) Preparation of standard substances: Use diluent to perform gradient dilution of the standard substance to accurately prepare standard working solutions of different concentrations in preparation for drawing the standard curve.

[0098] (4) Sample addition: Accurately add a mixture of rat serum sample and sample diluent to the sample wells to a total volume of 100 μL. Note that no sample is added to the blank wells.

[0099] (5) Incubation reaction 1: Add diluent to the blank well and add diluted sample or standard of different concentrations to the remaining corresponding wells (100 μL per well). After the sample addition is completed, seal the reaction wells with sealing tape and incubate the ELISA plate in a 37°C oven for 90 minutes to allow the target substance in the sample to fully react with the substance coated on the ELISA plate.

[0100] (6) First plate wash: After incubation, carefully discard the liquid in the wells and pat dry on clean absorbent paper. Then, add 400 μL of washing solution to each well, let it sit for 30 seconds, then discard the washing solution and pat dry on absorbent paper again. Repeat the above plate wash operation 5 times to completely remove unbound substances.

[0101] (7) Antibody addition and incubation: Add biotinylated antibody diluent to the blank wells and 100 μL of biotinylated antibody working solution to the remaining wells. After sealing the reaction wells, place the ELISA plate in a 37°C incubator and incubate for 60 minutes to allow the biotinylated antibody to specifically bind to the target substance.

[0102] (8) Second plate wash: After incubation, discard the liquid in the wells and pat dry on absorbent paper. Add 400 μL of washing solution to each well, let it stand for 30 seconds, then discard the washing solution and pat dry on absorbent paper. Repeat the plate wash five times to remove unbound biotinylated antibodies.

[0103] (9) Enzyme conjugate addition and incubation: Add enzyme conjugate diluent to the blank wells and 100 μL of enzyme conjugate working solution to the remaining wells. After sealing the reaction wells, place the ELISA plate in a 37°C incubator and incubate in the dark for 30 minutes to allow the enzyme conjugate to bind to the biotinylated antibody.

[0104] (10) Third plate wash: After incubation, discard the liquid in the wells and pat dry on clean absorbent paper. Add 400 μL of washing solution to each well, let it stand for 30 seconds, then discard the washing solution and pat dry on absorbent paper. Repeat the plate wash five times to remove unbound enzyme conjugates.

[0105] (11) Color development reaction: Add 100 μL of color development substrate to each well, place the ELISA plate in a 37°C constant temperature box, and incubate in the dark for 15 minutes to allow the enzyme to catalyze the substrate to develop color.

[0106] (12) Reaction termination: Add 100 μL of reaction termination solution to each well. The color of the solution changes from blue to yellow, and the enzymatic reaction is terminated.

[0107] (13) OD value determination: Use a microplate reader to measure the OD value of each well at a wavelength of 450 nm and record the data.

[0108] (14) Concentration calculation: A standard curve is established based on the measured OD value of the standard sample. The concentration of TNF-α in the sample is then calculated using the standard curve based on the OD value of the sample.

[0109] 2.4.2 Serum IL-17 determination

[0110] (1) Kit preparation: Take the kit out of the storage environment and place it at room temperature for 1 hour to equilibrate the reagents in the kit to the room temperature to ensure the accuracy of subsequent experiments.

[0111] (2) Standard dilution: According to the requirements of the kit instructions, the standard is accurately diluted to prepare a series of standard solutions with different concentration gradients for subsequent drawing of the standard curve.

[0112] (3) Sample addition: Set up blank wells, standard wells, and sample wells on the enzyme-labeled plate. Accurately add 50 μL of the diluted standard solution to the standard wells. For the sample wells, first add 40 μL of sample diluent, then add 10 μL of the sample to be tested, ensuring accurate sample addition and standardized operation.

[0113] (4) First incubation: After the sample is added, seal the ELISA plate with a sealing film and then incubate it at 37°C for 30 minutes to allow the target substance in the sample to fully react with the coating on the ELISA plate.

[0114] (5) First wash: Carefully remove the sealing film, discard the liquid in the plate, and shake dry any remaining liquid. Next, fill each well with wash solution, let the wash solution sit in the well for 30 seconds, and then discard it. Repeat this wash process five times. Finally, pat the plate dry to remove any unbound material.

[0115] (6) Enzyme addition operation: Except for the blank well, accurately add 50 μL of enzyme-labeled reagent to each well to allow the enzyme-labeled reagent to react specifically with the bound substances in the well.

[0116] (7) Second incubation: After the enzyme is added, seal the plate again with a sealing film and incubate the plate at 37°C for 30 minutes to promote the full combination of the enzyme-labeled reagent and the target substance.

[0117] (8) Second wash: Carefully remove the sealing film, discard the liquid in the plate and shake dry. Fill each well with washing solution, let it stand for 30 seconds and then discard. Repeat the wash five times and finally pat dry to remove unbound enzyme-labeled reagent.

[0118] (9) Color development: Add 50 μL of color developer A and 50 μL of color developer B to each well in sequence. Gently shake to mix thoroughly to allow the color developers to react. Then, place the plate in a dark environment at 37°C for 10 minutes to develop the color and observe the color change.

[0119] (10) Reaction termination: After the color development time is over, add 50 μL of stop solution to each well to terminate the enzymatic reaction. At this time, the color of the solution will change accordingly.

[0120] (11) Absorbance determination: Use a microplate reader at a wavelength of 450 nm to measure the absorbance (OD value) of each well in sequence and record the measurement results.

[0121] (12) Concentration calculation: A standard curve is established based on the measured OD value of the standard. The IL-17 concentration in the sample is then calculated using the standard curve based on the OD value of the sample to be tested.

[0122] 2.4.3 Serum IL-1β determination

[0123] (1) Kit pretreatment: Remove the kit from the storage environment and place it at room temperature for 30 minutes to equilibrate the reagent temperature in the kit to the room temperature to ensure the stability and accuracy of subsequent experiments.

[0124] (2) Sample addition: Set up zero well, standard wells, and sample wells on the ELISA plate. Accurately add 100 μL of sample diluent to the zero well, and add 100 μL of the graded diluted standard solution or sample to each of the remaining wells. Ensure that the sample addition volume is accurate and the operation process is standardized.

[0125] (3) First incubation: After the sample is added, cover the ELISA plate with a film and incubate it at 37°C for 2 hours to allow the target substance in the sample to fully combine with the coating on the ELISA plate.

[0126] (4) First wash: Carefully remove the plate seal, pour out the liquid in the plate, and pat the plate dry. Subsequently, wash the plate with washing solution. After each wash, pour out the liquid and pat the plate dry. Repeat this washing step 4 times to completely remove unbound substances.

[0127] (5) Addition and incubation of detection antibodies: Accurately add 100 μL of detection antibodies to each well. After addition, cover the plate with the sealing film again and incubate the ELISA plate at 37°C for 1 hour to allow the detection antibodies to specifically bind to the target substance.

[0128] (6) Second wash: Gently remove the plate seal, pour out the liquid in the plate and pat dry. Wash the plate with washing solution, pour out the liquid after washing and pat dry. Repeat this washing process 4 times to remove unbound detection antibody.

[0129] (7) Addition of HRP-labeled streptavidin: Accurately add 100 μL of HRP-labeled streptavidin to each well, ensuring that the amount added to each well is consistent to provide the necessary conditions for subsequent reactions.

[0130] (8) Second incubation: After adding HRP-labeled streptavidin, cover with a sealing film and incubate the ELISA plate at 37°C for 40 minutes to allow the HRP-labeled streptavidin to fully bind to the detection antibody.

[0131] (9) Third wash: Carefully remove the sealant, pour out the liquid in the plate, and pat dry. Wash the plate with washing solution, pour out the liquid, and pat dry. Repeat this washing step 4 times to remove unbound HRP-labeled streptavidin.

[0132] (10) Color development reaction: Add 100 μL of color development solution to each well. After addition, place the ELISA plate in a dark environment at 37°C for 15 minutes to develop the color. Observe the color change to ensure that the color development reaction proceeds normally.

[0133] (11) Reaction termination: After the color development time is over, 100 μL of stop solution is quickly added to each well to terminate the enzymatic reaction. At this time, the color of the solution will change accordingly.

[0134] (12) Optical density determination: Use a microplate reader at a wavelength of 450 nm to measure the optical density (OD) of each well and record it.

[0135] (13) Concentration calculation: Based on the measured OD value of the standard, a standard curve is established. Based on the OD value of the sample to be tested, the concentration of IL-1β in the sample is calculated using the standard curve.

[0136] 2.5 Statistical analysis

[0137] Data in this study are presented as mean ± standard error of variance (SEM). All statistical analyses and figures were performed using R4.0.3 software. For comparisons of multiple data sets, if the data were normally distributed and had homogeneous variances, one-way ANOVA was used, followed by a Tukey post hoc test to further compare group differences. If the data were normally distributed but had heterogeneous variances, the Brown-Forsythe test was used to analyze the data, with Dunnett T post hoc tests to confirm specific differences between the groups. If the data were not normally distributed, the Kruskal-Wallis test was used as a nonparametric test. For comparisons of two data sets, if the data met the relevant criteria of normal distribution and homogeneity of variance, the independent sample t-test was used to determine whether the two groups were significantly different. In all statistical analyses, a P value < 0.05 was considered statistically significant, indicating that the observed differences were not due to random chance but were real.

[0138] Example 3, Results

[0139] 3.1 Effect of Polygonatum sibiricum aqueous extract on intestinal pathology in rats with experimental appendicitis

[0140] 3.1.1 Effects of Polygonatum sibiricum Water Extract on Rat Gross Specimens

[0141] After successful modeling, rats in all three groups were observed for three consecutive days, with no mortality. The overall condition of rats in the PSR group was better than that in the Model group. Gross specimen evaluation revealed a significantly increased incidence of intestinal adhesion, suppuration, perforation, and necrosis in the Model group compared with the Sham and PSR groups. In contrast, PSR intervention significantly reduced intestinal adhesions and the incidence of suppuration and necrosis, with no cases of intestinal perforation observed. (See Table 3.)

[0142] Table 3 Evaluation of rat gross specimens

[0143] Intestinal adhesion Pyosis Intestinal perforation Intestinal necrosis Model group E1 Severe Yes Yes Yes E2 Severe Yes Yes Yes E3 Moderate No No No E4 Severe Yes Yes Yes E5 Severe Yes Yes Yes E6 Mild Yes No No 6 / 6 5 / 6 4 / 6 4 / 6 PSR group I1 Mild No No No I2 Mild No No No I3 Mild No No No I4 No No No No I5 Mild No No No I6 Mild No No No 5 / 6 0 / 6 0 / 6 0 / 6 Sham group S1 No No No No S2 No No No No S3 No No No No S4 No No No No S5 No No No No S6 No No No No 0 / 6 0 / 6 0 / 6 0 / 6

[0144] 3.1.2 Effects of Polygonatum sibiricum Water Extract on Intestinal Pathological Damage in Rats

[0145] In order to explore the effect of Polygonatum sibiricum water extract on intestinal pathology in rats, HE staining was performed on the colon of rats. Three samples were randomly selected from each group, and the intestinal mucosal damage of the three groups of rats was evaluated. The damage in the PSR group was significantly reduced compared with the Model group (magnifications were 40 times, 100 times, and 200 times, respectively). According to the rat colon pathology damage scoring standard, the results showed that the scores of the PSR group in the four aspects of inflammation degree, damage range, crypt destruction, and lesion range were significantly lower than those of the Model group, indicating that Polygonatum sibiricum water extract can effectively inhibit the intestinal damage and inflammatory response of experimental appendicitis in rats. Figure 2 , Table 4.

[0146] Table 4 Pathological injury scores of rat colon

[0147]

[0148]

[0149] 3.2 Effects of Polygonatum sibiricum Water Extract on Intestinal Mucosal Barrier Function in Rats

[0150] Through the observation of the gross specimens of the three groups of rats and the evaluation of HE staining, it is speculated that the water extract of Polygonatum sibiricum may have a protective effect on the intestinal mucosal barrier of experimental appendicitis. Therefore, this experiment used the immunofluorescence method to detect the expression of ZO-1, Occludin-1, and E-Cadherin proteins in the intestinal tissues of the three groups of rats. The results showed that the expression of ZO-1 in the PSR group was enhanced compared with the Model group (magnification was 10 times, 20 times, and 40 times, respectively). Figure 3 ; The expression of E-Cadherin in the PSR group was enhanced compared with that in the Model group (magnifications were 10 times, 20 times, and 40 times, respectively). Figure 4 ; The expression of Occludin-1 in the PSR group was enhanced compared with that in the Model group (magnifications were 10 times, 20 times, and 40 times, respectively). Figure 5 The expression intensities of ZO-1, Occludin and E-Cadherin proteins in the intestinal tissues of the three groups of rats were compared among the three groups (Table 5). Statistical analysis showed that there were significant differences in the immunofluorescence expression intensities of ZO-1, Occludin and E-Cadherin proteins between the PSR group and the Model group (P<0.05), and there were significant differences in the immunofluorescence expression intensities of ZO-1 and Occludin proteins between the PSR group and the Sham group (P<0.05). Figure 6 , as shown in Table 6.

[0151] Table 5 Expression intensity of ZO-1, Occludin and E-Cadherin proteins in intestinal tissues of three groups of rats

[0152]

[0153] Table 6 Correlation analysis results of ZO-1, Occludin and E-Cadherin protein expression in intestinal tissues of three groups of rats

[0154]

[0155] These results suggest that experimental appendicitis can cause a decrease in the expression of intestinal mucosal barrier-related proteins in the corresponding parts of the intestine, while the water extract of Polygonatum sibiricum can promote the expression of intestinal mucosal barrier-related proteins. The water extract of Polygonatum sibiricum may promote the repair of the intestinal mucosal barrier caused by experimental appendicitis.

[0156] 3.3 Effects of Polygonatum sibiricum aqueous extract on inflammatory factors in rat experimental appendicitis model

[0157] In order to clarify the inhibitory effect of Polygonatum sibiricum water extract on the inflammatory response of the experimental appendicitis model in rats, ELISA was performed to detect the serum inflammatory cytokines TNF-α, IL-1β, and IL-17 in the three groups of rats (Table 7). Figure 7 As shown in Table 8, there were significant statistical differences in the expression of inflammatory factors TNF-α, IL-1β, and IL-17 between the PSR group and the Model group (P < 0.05).

[0158] Table 7 ELISA detection of serum inflammatory cytokines TNF-α, IL-1β, IL-17 of three groups of rats

[0159]

[0160] Table 8 Correlation analysis results of ELISA of inflammatory cytokines TNF-α, IL-1β, and IL-17 in the serum of three groups of rats

[0161]

[0162] In this study, SD rats were used to construct an experimental appendicitis model by cecal ligation and puncture. This model successfully simulated the pathological changes of appendicitis and had the advantages of simple feeding and convenient operation. The results showed that continuous gavage of the water extract of Polygonatum for 2 weeks could significantly reduce the colon pathological injury score of the model rats. At the same time, the water extract of Polygonatum could significantly up-regulate the expression of tight junction proteins Occludin, ZO-1 and E-cadherin in the colon tissue, indicating that the treatment of Polygonatum water extract could improve the intestinal mucosal barrier function. The water extract of Polygonatum could significantly reduce the levels of serum inflammatory factors TNF-α, IL-1β and IL-17, indicating that the water extract of Polygonatum could effectively reduce inflammation. In summary, the water extract of Polygonatum can significantly improve the clinical symptoms of experimental appendicitis rats, effectively protect the integrity of the intestinal mucosal mechanical barrier, and reduce inflammation. These results suggest that the water extract of Polygonatum has a potential therapeutic effect on acute appendicitis, providing preliminary experimental evidence for its use as an alternative or adjunctive treatment for the disease.

[0163] Although embodiments of the present application have been shown and described, it is to be understood that various modifications, substitutions, replacements and changes can be made to these examples without departing from the principles and spirit of the present application, and the scope of the present application is defined by the appended claims and their equivalents.

[0164] In the above embodiments, the description of each embodiment has its own focus, and the parts not described or recorded in detail in a certain embodiment can be referred to the relevant description of other embodiments.

[0165] The above-described embodiments are only used to illustrate the technical solutions of the present application, and not to limit them; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or make equivalent substitutions for part of the technical features; and these modifications or substitutions do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application, and should be included in the protection scope of the present application.

Claims

1. Use of a water extract of Polygonatum sibiricum in preparing a medicine for preventing and / or treating acute appendicitis.

2. The use of the water extract of Polygonatum sibiricum according to claim 1 in preparing a medicament for preventing and / or treating acute appendicitis, characterized in that: The drug is a drug that can upregulate the expression of intestinal mucosal barrier tight junction proteins ZO-1, Occludin and E-cadherin to prevent and / or treat acute appendicitis.

3. The use of the water extract of Polygonatum sibiricum according to claim 1 in preparing a medicament for preventing and / or treating acute appendicitis, characterized in that: The drug is a drug that can reduce the level of inflammatory factors; the inflammatory factors include at least one of the serum inflammatory factors TNF-α, IL-1β and IL-17.

4. The use according to any one of claims 1 to 3, characterized in that: The drug is a drug that upregulates the expression of intestinal mucosal barrier tight junction proteins ZO-1, Occludin and E-cadherin and reduces the levels of serum inflammatory factors TNF-α, IL-1β and IL-17.

5. The use according to any one of claims 1 to 4, characterized in that: The medicine is for human or veterinary use.

6. A method for preparing a water extract of Polygonatum sibiricum, characterized in that: The following steps are involved: (1) Wash the rhizome of Polygonatum sibiricum and dry it at 60℃, then weigh 100g; (2) Add distilled water with a solid-liquid ratio of 1:6, soak for 30 minutes, boil, simmer for 40 minutes, and filter to obtain the first filtrate; (3) Add distilled water to the medicinal residue with a solid-liquid ratio of 1:4, boil, and then simmer for 40 minutes, and filter to obtain the second filtrate; (4) Combine the two filtrates, concentrate to 100 mL by rotary evaporation, and store at 4°C.

7. A pharmaceutical composition for treating acute appendicitis, characterized in that: The pharmaceutical composition comprises a therapeutically effective amount of a water extract of polygonatum as an active ingredient and pharmaceutically acceptable excipients, wherein the water extract of polygonatum is prepared according to the method of claim 6; the dosage of the water extract of polygonatum in the pharmaceutical composition is 200-600 mg of the raw drug / kg body weight / day, preferably 400 mg of the raw drug / kg body weight / day.

8. Application of Polygonatum sibiricum water extract in upregulating the expression of ZO-1, Occludin and E-cadherin proteins in animals.

9. Application of Polygonatum sibiricum water extract in reducing TNF-α, IL-1β and IL-17 levels in animals.

10. The use according to any one of claims 1 to 5, the method according to claim 6, the pharmaceutical composition according to claim 7, the use according to claim 8, or the use according to claim 9, characterized in that: The polygonatum water extract includes concentrated solution, dry powder or freeze-dried powder.

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