Traditional Chinese medicine composition for preventing and / or treating radioactive intestinal injury and application thereof

Abstract

The invention provides a traditional Chinese medicine composition for preventing and / or treating radioactive intestinal injury and application thereof, and the traditional Chinese medicine composition comprises the following components in parts by weight: 15-45 parts of ginseng, 15-45 parts of bighead atractylodes rhizome, 15-45 parts of poria, 15-45 parts of honey-fried licorice root, 15-30 parts of dogwood and 5-10 parts of vinegar schisandra chinensis. The traditional Chinese medicine composition can effectively prevent and / or treat acute and chronic radiation intestinal injury through anti-inflammatory, antibacterial and anti-apoptosis, and has a huge application prospect in preparation of drugs for preventing and / or treating acute and chronic radiation intestinal injury.

Description

Technical Field

[0001] The invention belongs to the field of traditional Chinese medicine, and particularly relates to a traditional Chinese medicine composition for preventing and / or treating radiation intestinal injury and application thereof. Background Art

[0002] Radiotherapy is one of the three pillars of cancer treatment and plays a vital role in inhibiting the growth of malignant tumor cells. However, normal intestinal tissue will suffer varying degrees of radiation damage due to the massive production of reactive oxygen species (ROS) during pelvic radiotherapy, and unirradiated intestinal tissue will also show similar radiation damage due to radiation-induced bystander effects. The incidence of acute and chronic radiation intestinal injury is as high as 75% to 81%, and the main symptoms are nausea, vomiting, abdominal pain, mucus, purulent stools or fresh blood in stools. More serious cases can lead to serious complications such as intestinal stenosis, intestinal obstruction and intestinal fistula. The occurrence of radiation intestinal injury during treatment often leads to a decrease in the patient's quality of life, poor treatment compliance, and prolonged treatment courses, affecting the patient's overall survival and prognosis. However, the current treatment for acute and chronic radiation intestinal injury still remains at the level of surgery and experimental drugs, and there is no effective treatment plan. Therefore, the prevention and treatment of acute and chronic radiation intestinal injury is an important clinical problem that needs to be solved in the field of tumor treatment.

[0003] Many TCM experts have classified radiation-induced intestinal injury into the categories of "intestinal diarrhea", "diarrhea" and "dysentery" according to the clinical symptoms, and have implemented syndrome differentiation and treatment. In the category of TCM etiology, radiation belongs to the category of "fire, heat and toxic evil" caused by external factors, and the disease is located in the intestine. The pathological elements are mainly "dampness, heat and toxicity", and the pathological nature is deficiency in the root and excess in the superficial, with spleen deficiency as the root and dampness and heat as the superficial. Professor Li Renting believes that because cancer toxins and radioactive toxins damage the body's positive energy together, the pathogenesis of radiation-induced intestinal injury is essentially deficiency in the body's positive energy, which is always deficiency in the root and excess in the superficial, and summarizes the pathological factors of this injury as "toxicity, heat, stasis and deficiency". The pathogenesis of TCM is: Tumor patients have the deficiency of vital energy, and the accumulation of cancer toxins. The heat and toxicity of radiation directly damage the spleen and stomach, causing dysfunction of transportation and transformation, endogenous dampness, dampness and heat fighting each other, and accumulating in the intestines and viscera. Dampness, heat and toxicity are entangled, causing stagnation of visceral qi, poor qi movement, dysfunction of the small intestine, and dysfunction of the large intestine. Therefore, abdominal pain, diarrhea, tenesmus, bloody stools, and changes in the frequency of bowel movements are seen; at the same time, the heat fumigates the intestines, damaging the blood vessels, causing blood to overflow outside the veins, causing the flesh to rot and become pus, causing burning pain in the anus, and bloody stools with mucus and pus. If the above symptoms are prolonged and unhealed, the body is weak, and long-term deficiency will lead to stasis. In addition, the qi and blood are damaged, and the yang of the spleen and kidneys are damaged over time. Then, excessive diarrhea, indigestion, sore waist and weak knees, accompanied by weight loss, dizziness, tinnitus, fever in the five hearts, fatigue, shortness of breath and other symptoms of deficiency of yin and yang, qi and blood are seen. In summary, radiation-induced intestinal injury is a dynamic pathological evolution process in which deficiency, dampness, heat, stasis and toxicity are mixed.

[0004] The TCM syndromes of patients with radiation-induced intestinal injury are mainly manifested as abdominal pain, diarrhea, frequent bowel movements, tenesmus, mucus or purulent blood in the stool, burning pain and swelling in the anus, mucus and purulent blood in the stool, pale red or congested tongue, yellow and greasy tongue coating, and soft and rapid or slippery and rapid pulse. The pathogenesis is deficiency in origin and excess in superficiality, and the combination of deficiency and excess is the basic pathogenesis. The disease location is in the large intestine, and the main diseased viscera is the spleen. The heat toxin accumulates, the spleen and stomach are damaged, resulting in the loss of functions of the intestine in receiving, digesting, separating the clear from the turbid, and conduction. The TCM syndrome differentiation of this disease is mainly intestinal damp-heat syndrome. Combining with the TCM pathogenesis and syndrome differentiation types of radiation-induced intestinal injury, the "Expert Consensus on the Integrated Chinese and Western Medicine Diagnosis and Treatment of Radiation-induced Intestinal Injury" issued in 2023 agrees that the damp-heat constitution, yang-deficiency constitution, and stasis constitution are more common in this part of patients.

[0005] In recent years, many scholars have found through basic research and large-scale clinical trials that traditional Chinese medicine prescriptions can relieve radiation-induced intestinal injury, which provides a new strategy for the treatment of radiation-induced intestinal injury. Previous animal experiments and human trials have shown that previous literature reports that Bazhen Decoction combined with Gegen Qinlian Decoction, Huangqin Decoction combined with Huaihua Pills, Shenling Baizhu Powder, Huaihua Powder, and Baitouweng Decoction, Qingre Liangxue Decoction, Jiawei Changfeng Decoction, etc. can significantly improve radiation-induced intestinal injury. However, the current research on traditional Chinese medicine prescriptions has only scratched the surface, and the research scope still needs to be expanded to find more suitable prescriptions. Summary of the Invention

[0006] To solve the problems and deficiencies in the prior art, the present invention provides a traditional Chinese medicine composition for preventing and / or treating radiation-induced intestinal injury and its application. This traditional Chinese medicine composition can effectively prevent and / or treat acute and chronic radiation-induced intestinal injury problems through anti-inflammatory, antibacterial, and anti-apoptotic effects, and has great application prospects in the preparation of drugs for preventing and / or treating acute and chronic radiation-induced intestinal injury.

[0007] According to the first aspect of the present invention, there is provided a traditional Chinese medicine composition for preventing and / or treating radiation-induced intestinal injury, which, calculated by weight, comprises the following components: 15-45 parts of ginseng, 15-45 parts of atractylodes macrocephala, 15-45 parts of poria cocos, 15-45 parts of roasted licorice, 15-30 parts of cornel, and 5-10 parts of vinegar-processed schisandra chinensis.

[0008] Ginseng belongs to traditional precious Chinese medicine. Taking it for a long time does no harm, and it has the effects of prolonging life and greatly tonifying primordial qi, and has been widely concerned in clinical applications. Modern pharmacological research shows that its effective active ingredients mainly include saponins, volatile oils, sugars, amino acids, etc., and have various pharmacological effects such as antioxidant, improving immunity, and anti-fatigue, and are widely used in clinical applications for anti-tumor, nervous system, and immune regulation, etc., and have good improvement or regulatory effects.

[0009] Atractylodes macrocephala is also known as Yuzhu, Zheshu, Wushu, etc. It is a herbaceous plant in tropical and subtropical regions. It is mostly used as medicine with its rhizome. Clinically, it has the effects of strengthening the spleen and replenishing qi, drying dampness and stopping sweating, etc. Modern pharmacological research shows that Atractylodes macrocephala contains various effective chemical components such as volatile oils, lactones, polysaccharides, etc., and plays an important role in the immune system, nervous system, gastrointestinal tract, etc. It may exert pharmacological effects by improving the body's oxidative stress response, inhibiting the expression of inflammatory mediators and inflammatory factors.

[0010] Poria cocos, also known as Fuling, is the dried sclerotium of the plant Poria cocos in the family Polyporaceae. It is mainly produced in Yunnan, Anhui, Hubei, Henan, Sichuan and other places. The product from Yunnan is called "Yunling". Traditional Chinese medicine is used for edema with scanty urine, phlegm retention with dizziness and palpitation, weakness of the spleen with poor appetite, loose stools and diarrhea, restlessness of the mind, palpitations and insomnia, etc. Poria cocos is widely used clinically alone or in combination with other drugs. It is a commonly used diuretic and dampness-resolving medicine in Chinese herbal medicine. Modern pharmacological research shows that Poria cocos has the effects of inhibiting tumors, enhancing immunity, anti-aging, sedation, etc. At present, the research on its chemical components mainly focuses on the triterpene part extracted by organic solvents and the polysaccharide part of the water extract.

[0011] Prepared Licorice Root is the processed product of Licorice Root after being stir-fried with honey. It tastes sweet and neutral, and belongs to the heart, lung, spleen and stomach meridians. It has the effects of invigorating the spleen and stomach, replenishing qi and restoring the pulse. Clinically, it can be used for the treatment of weakness of the spleen and stomach, lassitude and fatigue, palpitation with rapid heart rate, and intermittent pulse. Research shows that the chemical components of Prepared Licorice Root mainly include triterpenoid saponins, flavonoids, polysaccharides, coumarins, alkaloids, etc. The contents of liquiritin and glycyrrhizic acid are important quality indicators. Pharmacological research has clarified that Prepared Licorice Root has the effects of anti-tumor, anti-depression, anti-inflammatory, anti-arrhythmia, liver protection, antioxidant and immune regulation.

[0012] Cornus officinalis is also known as Fructus Corni, Shuzao, Shizao, Rouzao, Yurou, Zaopi, Yaozao, etc. It is a plant of the genus Cornus in the family Cornaceae. Traditional Chinese medicine pharmacology believes that Cornus officinalis is moist and slightly warm in nature, warm but not dry, tonifying but not excessive. It belongs to the liver and kidney meridians, can enter the lower energizer, and consolidate the thoroughfare and conception vessels. It has the effects of tonifying the liver and kidney, astringing essence and arresting seminal emission, etc., and has good therapeutic effects on diseases such as liver and kidney deficiency, spermatorrhea and spermatorrhea, metrorrhagia and leukorrhea, internal heat and diabetes. The main pharmacodynamic components in Cornus officinalis are iridoids and their glycosides, triterpenoids, flavonoids, tannins, organic acids, polysaccharides, etc. At present, the research on the chemical components of Cornus officinalis mostly focuses on iridoids and their glycosides, triterpenoids and polysaccharides. Cornus officinalis can act on multiple aspects of the body, but the main effects are concentrated in the immune system, circulatory system, nervous system and urinary system, and it has various pharmacological effects such as anti-tumor, protecting the myocardium, lowering blood sugar, regulating bone metabolism, protecting neurons, antioxidant, protecting the liver, regulating retinol, anti-aging, anti-inflammatory, etc.

[0013] Schisandra chinensis (Turcz.) Baill. var. chinensis refers to Schisandra chinensis (Turcz.) Baill. processed with vinegar. It tastes sour and sweet, is warm in nature, and belongs to the lung, heart, and kidney meridians. The fruits of Schisandra chinensis are basically spherical, irregular or flattened, with a relatively rough and wrinkled epidermis, not smooth. The seeds are kidney-shaped. It was first recorded in "Shennong Ben Cao Jing". Its medicinal properties are relatively mild, belonging to the heart, lung, and kidney meridians, and it has the effects of astringing and arresting sweating, promoting the production of body fluids to quench thirst, tonifying the kidney and calming the mind. Clinically, it is used to treat cough with deficiency asthma, spermatorrhea, spontaneous sweating, thirst, and insomnia with dreaminess, etc. The chemical constituents of Schisandra chinensis mainly include lignans, volatile oils, organic acids, terpenoids, flavonoids, polysaccharides, and inorganic elements, etc. Among them, lignans are the main components of Schisandra chinensis and are the basic substances for various pharmacological activities. In addition, Schisandra chinensis also has functions such as preventing liver damage, anti-cancer, anti-AIDS, anti-Alzheimer's disease, and regulating the central nervous system, and has extremely high medicinal and nutritional values.

[0014] The above-mentioned several traditional Chinese medicine components are similar to most traditional Chinese medicine components and all have certain tonifying effects. They can all regulate the physiological functions of the human body to a certain extent and help the body restore a balanced state. Whether it is due to diseases, fatigue or other reasons leading to a decline in body functions, they all have a certain conditioning and improvement effect, which helps to improve various functions of the body and promote the recovery of health.

[0015] However, the efficacy focuses of different traditional Chinese medicines are different, and traditional Chinese medicine emphasizes how to make reasonable compatibility. Reasonable compatibility can adjust its biases, control its toxicity, enhance or change the original functions, eliminate or relieve the adverse factors to the human body, and exert its comprehensive effects of complementing each other or opposing and yet complementary, with the advantages of multiple pathways, multiple targets, multiple systems and low side effects, and has a better overall regulatory function than single herbs. At the same time, different total amounts and ratios of drugs will lead to the interaction and content changes among various chemical components, resulting in differences in efficacy and having an important impact on clinical efficacy.

[0016] At present, many studies have shown that the use of traditional Chinese medicine in combination with radiotherapy and chemotherapy for the treatment of tumors can increase the body's resistance, reduce the recurrence rate of diseases, reduce the side effects after radiotherapy and chemotherapy, etc. Radiation-induced intestinal injury is one of the biggest problems encountered after radiotherapy and chemotherapy. Therefore, how to find a suitable traditional Chinese medicine composition to prevent and / or treat acute and chronic radiation-induced intestinal injury is of great significance for reducing the pain of patients after radiotherapy and chemotherapy, enhancing the immunity of patients after radiotherapy and chemotherapy, and prolonging life.

[0017] During the long-term research process of this application, it was found that mixing the above-mentioned traditional Chinese medicines, namely ginseng, atractylodes macrocephala, poria cocos, roasted licorice root, cornel fruit, and schisandra chinensis vinegar, in a specific proportion can effectively prevent and / or treat acute and chronic radiation-induced intestinal injury. Moreover, the long-term curative effect is definite, it is suitable for long-term use, and has the value of popularization and application. Specifically, among them, ginseng replenishes qi and promotes the production of body fluids as the monarch drug, atractylodes macrocephala strengthens the spleen and eliminates dampness as the minister drug, poria cocos is sweet and bland, promoting diuresis and clearing heat as the assistant drug, roasted licorice root harmonizes the middle-jiao and benefits the earth as the envoy drug, supplemented by cornel fruit to nourish the liver and yin-blood, and schisandra chinensis to absorb the qi of the five flavors and return it to the kidney. This prescription altogether promotes the replenishment of qi and production of body fluids, strengthens the spleen and stops diarrhea, and tonifies the liver and kidney. And through relevant experiments, it is verified that the above-mentioned traditional Chinese medicine composition can enhance the radiation resistance and anti-apoptosis ability of normal intestinal epithelial cells in mice with radiation-induced intestinal injury, improve the body weight, survival, and intestinal injury of mice with radiation-induced intestinal injury, inhibit pathogenic bacteria to maintain the balance of intestinal microecology, regulate the over-activation of immunosuppressive inflammation, and finally treat acute and chronic radiation-induced intestinal injury.

[0018] It should be noted here that if the above-mentioned traditional Chinese medicine materials combination or ratio is changed, effective treatment effects for preventing and / or treating acute and chronic radiation-induced intestinal injury cannot be obtained. Therefore, there is a specific interaction relationship among various materials of the traditional Chinese medicine composition provided by this application, and it needs to be mixed and prepared in a specific proportion to achieve obvious synergistic effects, and will not produce obvious toxicity, so as to achieve obvious treatment effects on preventing and / or treating acute and chronic radiation-induced intestinal injury.

[0019] Preferably, the above-mentioned traditional Chinese medicine composition, calculated by weight, includes the following components: 30 parts of ginseng, 30 parts of atractylodes macrocephala, 30 parts of poria cocos, 30 parts of roasted licorice root, 22.5 parts of cornel fruit, and 7.5 parts of schisandra chinensis vinegar. Further, under the above ratio, the synergistic effect of these traditional Chinese medicines is better and the side effects are smaller. Therefore, it is more beneficial to prevent and / or treat acute and chronic radiation-induced intestinal injury, that is, it is more beneficial to relieve the pain of patients and extend the lifespan of patients.

[0020] Preferably, the above-mentioned traditional Chinese medicine composition, calculated by weight, consists of the following components: 15 - 45 parts of ginseng, 15 - 45 parts of atractylodes macrocephala, 15 - 45 parts of poria cocos, 15 - 45 parts of roasted licorice root, 15 - 30 parts of cornel fruit, and 5 - 10 parts of schisandra chinensis vinegar.

[0021] Preferably, the above-mentioned traditional Chinese medicine composition, calculated by weight, consists of the following components: 30 parts of ginseng, 30 parts of atractylodes macrocephala, 30 parts of poria cocos, 30 parts of roasted licorice root, 22.5 parts of cornel fruit, and 7.5 parts of schisandra chinensis vinegar.

[0022] Preferably, the pharmaceutical dosage form of the above-mentioned traditional Chinese medicine composition includes at least one of decoction, granule, powder, tablet, pill, and capsule.

[0023] According to the second aspect of the present invention, there is provided a preparation method of the above-mentioned traditional Chinese medicine composition, and the specific operation is as follows: Ginseng, Atractylodes macrocephala, Poria cocos, roasted licorice root, Cornus officinalis, and Schisandra chinensis vinegar are soaked in water for 0.4 - 2 hours, then brought to a boil over high heat and then simmered for 0.5 - 2 hours, after which the residue is removed and the juice is taken. Then, the medicinal juice is made into a medicament according to actual needs to obtain the traditional Chinese medicine composition.

[0024] Preferably, the water addition is controlled such that the water surface exceeds the medicinal material surface by 2 - 5 cm.

[0025] Preferably, the water addition is controlled such that the water surface exceeds the medicinal material surface by 2 - 3 cm.

[0026] Preferably, the traditional Chinese medicine composition further comprises a pharmaceutically acceptable excipient. Specifically, the excipients used are commonly used carriers or excipients in the art, including but not limited to starch, lactose, glucose, sodium carboxymethylcellulose, ethylcellulose, and methylcellulose, malt, gelatin, polyols (such as propylene glycol, glycerol, mannitol), tabletting agents, etc., to help improve the stability of the drug or enhance its activity, or to produce an acceptable taste or odor in the case of oral administration.

[0027] According to the third aspect of the present invention, there is provided an application of the above-mentioned traditional Chinese medicine composition or the traditional Chinese medicine composition prepared by the above-mentioned preparation method of the traditional Chinese medicine composition in the preparation of a drug for preventing and / or treating radiation-induced intestinal injury.

[0028] According to the fourth aspect of the present invention, there is provided an application of the above-mentioned traditional Chinese medicine composition or the traditional Chinese medicine composition prepared by the above-mentioned preparation method of the traditional Chinese medicine composition in the preparation of a drug for preventing and / or treating acute radiation-induced intestinal injury.

[0029] According to the fifth aspect of the present invention, there is provided an application of the above-mentioned traditional Chinese medicine composition or the traditional Chinese medicine composition prepared by the above-mentioned preparation method of the traditional Chinese medicine composition in the preparation of a drug for preventing and / or treating chronic radiation-induced intestinal injury.

[0030] According to the sixth aspect of the present invention, there is provided an application of the above-mentioned traditional Chinese medicine composition or the traditional Chinese medicine composition prepared by the above-mentioned preparation method of the traditional Chinese medicine composition in the preparation of a drug for preventing and / or treating excessive proliferation of intestinal pro-inflammatory immune cells, intestinal flora disorder, and intestinal epithelial cell apoptosis in patients with radiation-induced intestinal injury.

[0031] According to the seventh aspect of the present invention, there is provided an application of the above-mentioned traditional Chinese medicine composition or the traditional Chinese medicine composition prepared by the above-mentioned preparation method of the traditional Chinese medicine composition in the preparation of a drug for preventing and / or treating excessive proliferation of intestinal pro-inflammatory immune cells, intestinal flora disorder, and intestinal epithelial cell apoptosis in patients with acute radiation-induced intestinal injury.

[0032] According to the eighth aspect of the present invention, there is provided an application of a traditional Chinese medicine composition prepared by the above traditional Chinese medicine composition or the preparation method of the above traditional Chinese medicine composition in the preparation of a drug for preventing and / or treating excessive proliferation of intestinal pro-inflammatory immune cells, intestinal flora disorder, and apoptosis of intestinal epithelial cells in patients with chronic radiation-induced intestinal injury.

[0033] In summary, compared with the prior art, the present invention has the following beneficial effects:

[0034] All the constituent raw materials of the traditional Chinese medicine composition provided by the present invention can be commercially purchased, are easy to implement, and have a low cost; it is a pure traditional Chinese medicine preparation without side effects and is suitable for long-term use; the test results show that the traditional Chinese medicine composition provided by the present invention can effectively treat, prevent and / or treat acute and chronic radiation-induced intestinal injury, and has a definite long-term curative effect, is suitable for long-term use, and has the value of popularization and application. Description of the Drawings

[0035] Figure 1 Test results of the radiation resistance ability of normal intestinal epithelial cells in NCM460 (control group) and traditional Chinese medicine composition group (experimental group) in Example 2. Among them, A is the result of the plate cloning experiment; B is the result of the multi-target single-hit model construction experiment; C is the result of the comet experiment; D is the statistical analysis result of the comet experiment.

[0036] Figure 2 Test results of the anti-apoptosis ability of normal intestinal epithelial cells in NCM460 (control group) and traditional Chinese medicine composition group (experimental group) in Example 2.

[0037] Figure 3 Results of the body weight changes of mice with radiation-induced intestinal injury in the Control group, IR group, and traditional Chinese medicine composition + IR group in Example 3.

[0038] Figure 4 Results of the survival changes of mice with radiation-induced intestinal injury in the Control group, IR group, and traditional Chinese medicine composition + IR group in Example 3.

[0039] Figure 5 HE staining pictures (40×) and statistical analysis of intestinal tissues of mice with radiation-induced intestinal injury in the Control group, IR group, and traditional Chinese medicine composition + IR group in Example 3. Among them, A is the HE picture; B is the result of the villus length; C is the result of the crypt and villus ratio; D is the result of the immune score.

[0040] Figure 6 Results of intestinal immune cells of mice with radiation-induced intestinal injury in the Control group, IR group, and traditional Chinese medicine composition + IR group in Example 3.

[0041] Figure 7Results of intestinal flora species diversity in mice with radiation-induced intestinal injury in the Control group, IR group, and traditional Chinese medicine composition + IR group in Example 3. Among them, A is the Shannon index result, and B is the PcoA composition analysis result.

[0042] Figure 8 Results of intestinal flora subtypes in mice with radiation-induced intestinal injury in the Control group, IR group, and traditional Chinese medicine composition + IR group in Example 3. Detailed implementation manners

[0043] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments.

[0044] The raw materials of ginseng, atractylodes macrocephala, poria cocos, roasted licorice root, cornel fruit, and schisandra chinensis vinegar used in the following embodiments all comply with the relevant regulations under each medicinal material item in Part I of the Chinese Pharmacopoeia 2005 Edition. Each medicinal material has been processed through cleaning, cutting, processing, pulverization, etc. Before feeding, through identification, the physical objects of each medicinal material are consistent with the names, and the quality meets the national pharmacopoeia standards (the specific medicinal material identification method refers to the pharmacopoeia standards for implementation).

[0045] Example 1

[0046] Preparation of traditional Chinese medicine composition:

[0047] Take 30 parts of ginseng, 30 parts of atractylodes macrocephala, 30 parts of poria cocos, 30 parts of roasted licorice root, 22.5 parts of cornel fruit, and 7.5 parts of schisandra chinensis vinegar. Soak all the medicinal materials in clean water for 0.5 hour, control the water addition amount so that the water surface exceeds the medicinal material surface by 2 - 3 cm, then bring to a boil over high heat and turn to low heat for decoction for 1 hour, then remove the residue and take the juice, or concentrate to a thick extract by conventional methods, add appropriate pharmaceutical excipients, granulate, dry, size the granules, and make into granules or vacuum dry, pulverize and granulate for filling into capsules; or add appropriate pharmaceutical excipients and directly make into pills, powders, granules, oral liquids, or syrups.

[0048] Effect of traditional Chinese medicine composition on radiation resistance and anti-apoptosis ability of normal intestinal epithelial cells in Example 2

[0049] 1.1 Drug preparation

[0050] Take 30 parts of ginseng, 30 parts of atractylodes macrocephala, 30 parts of poria cocos, 30 parts of roasted licorice root, 22.5 parts of cornel fruit, and 7.5 parts of schisandra chinensis vinegar. Soak all the medicinal materials in clean water for 0.5 hour, control the water addition amount so that the water surface exceeds the medicinal material surface by 2 - 3 cm, then bring to a boil over high heat and turn to low heat for decoction for 1 hour, then remove the residue and take the juice, and then heat and concentrate the juice to a dose of 4 g / mL. The juice is used as the traditional Chinese medicine composition for administration.

[0051] 1.2 Cell experiments

[0052] Normal small intestinal epithelial cells NCM460 were cultured in RPMI-1640 medium containing 10% fetal bovine serum and incubated in a cell incubator with 5% CO 2 at 37°C.

[0053] 1.3 Experimental methods

[0054] The cells were irradiated with 6 MeV electron beam to establish a cell radiation damage model. In this experiment, a total of one experimental group and one control group were set up. Among them, the experimental group was the drug administration group, and the drug administration group was given the traditional Chinese medicine composition diluted to 1 μg / mL. The difference between the control group and the experimental group was that no drug was given.

[0055] 1.4 Experimental results

[0056] (1) Colony formation assay

[0057] Before irradiation, NCM460 cells were digested to obtain a cell suspension and diluted in a 6-well plate at 400, 800, 1200, and 1600 cells / well. The next day, after the cells adhered, the traditional Chinese medicine composition was added or not added. Subsequently, the cells were irradiated with doses of 2, 4, 6, 8, and 10 Gy (3 replicate sub-wells for each dose), and the culture plates were incubated for 7 days. After washing with PBS, the cells were fixed in methanol for 30 minutes and stained with 0.1% crystal violet for 15 minutes. Using the double-blind method, the number of monoclonal colonies was calculated. The survival curve was plotted based on the multi-target single-hit model.

[0058] The results are shown in Figure 1 A, 1B (*P<0.05), indicating that the addition of the traditional Chinese medicine composition can increase the number of colony-forming points in the colony formation assay and enhance the radiation tolerance of the cells.

[0059] (2) Comet assay

[0060] Four hours after irradiating the two groups of cells with 6 Gy, the cells were digested with trypsin, washed once with PBS, and resuspended at 1×10 6Resuspend at a density of cells / mL. Perform the comet assay according to the instructions of the Kaiji Comet Assay Kit. Spread the first layer of 1.5% normal melting point agarose on the glass slide, then spread the second layer of 0.7% low melting point agarose, which contains 10 μL of cells, and finally the third layer of 1.5% normal boiling point agarose. Immerse the glass slide in pre-cooled lysis buffer at 4 °C for 2 hours, wash it with PBS for 5 minutes, and then place it in a horizontal electrophoresis tank containing 1 L of TAE buffer for 30 minutes. After the DNA is completely unwound, electrophorese the glass slide at 100 V for 15 minutes in the dark. After electrophoresis, add 10 μL of PI staining solution to the agarose gel. Use a coverslip to observe and photograph the glass slide under a fluorescence microscope, and randomly select 50 comets. The percentage of comet tail DNA is analyzed using CASP 1.2.3 software.

[0061] The results are as Figure 1 shown in C, 1D (***P < 0.001), which indicates that adding the traditional Chinese medicine composition can shorten the length of the comet tail that reflects DNA damage in the comet assay.

[0062] (3) Experiment on the anti-apoptotic ability of cells

[0063] After irradiating the two groups of cells with 6 Gy, obtain cell suspensions by digesting with trypsin without EDTA. Wash the cells twice with PBS (300×g, centrifuge for 5 min) to collect 1 - 5×10 5 cells, then add 500 μL of Binding Buffer and gently blow to make a single-cell suspension, and then add 5 μL of Annexin V-FITC and 5 μL of Propidium Iodide, and gently mix. React for 5 - 10 min at room temperature in the dark, and detect using a BD flow cytometer. Statistically analyze the cells positive for Annexin V-FITC as the apoptotic cells.

[0064] The results are as Figure 2 shown (***P < 0.001), and adding the traditional Chinese medicine composition can significantly enhance the anti-apoptotic ability of normal intestinal epithelial cells.

[0065] The above results show that the traditional Chinese medicine composition provided by the present invention can significantly enhance the radiation resistance and anti-apoptotic ability of normal intestinal epithelial cells.

[0066] Example 3 Effects of the traditional Chinese medicine composition on the body weight, survival, and intestinal injury of mice with radiation-induced intestinal injury

[0067] 1.1 Drug preparation

[0068] Take 30 parts of ginseng, 30 parts of Atractylodes macrocephala, 30 parts of Poria cocos, 30 parts of roasted licorice root, 22.5 parts of Cornus officinalis, and 7.5 parts of Schisandra chinensis processed with vinegar. Soak all the medicinal materials in purified water for 0.5 hour, control the water addition so that the water surface is 2 - 3 cm higher than the medicinal materials surface, then bring to a boil over high heat and turn to low heat for decocting for 1 hour, remove the residue and take the juice, and then heat - concentrate the medicinal juice to a dose of 4 g / mL. The medicinal juice is administered as a traditional Chinese medicine composition.

[0069] 1.2 Animal experiments

[0070] According to the conversion of drug doses based on the body weight of humans and mice, the total amount of traditional Chinese medicine for each plaster is 120 g. Calculated based on an adult body weight of 60 kg, the clinical drug dosage for humans is 2 g / kg. The mouse dosage is 12.33 times that of humans, that is, 24.66 g / kg. Given that the concentration of the water - decocted medicinal juice of this prescription (traditional Chinese medicine composition) is 4 g / mL, that is, the clinical drug dosage for a 20 - g - body - weight mouse is 0.375 mL.

[0071] 1.3 Experimental methods

[0072] Fifteen 5 - 6 - week - old C57BL / 6 mice were randomly divided into 4 groups after 7 days of normal feeding: Control group (n = 5) (control group), IR group (simple irradiation group) (n = 5), traditional Chinese medicine composition group + IR (n = 5).

[0073] From day 0 to day 5, the mice in the Control group and the IR group were gavaged with 0.375 mL of PBS, and the traditional Chinese medicine composition + IR group was gavaged with 0.375 mL of the traditional Chinese medicine composition.

[0074] On day 6, after fasting and water - deprivation for 2 h in each group of mice, the mice were anesthetized and whole - abdomen irradiated with 6 MeV X - rays, with an irradiation dose of 14 Gy. After the irradiation was completed, fasting and water - deprivation were lifted.

[0075] On day 12, dissect and detect various indicators.

[0076] 1.4 Experimental results

[0077] (1) Body weight changes

[0078] Perform dynamic statistical analysis on the body weights of mice in each group, and record the body weight changes of mice on the 3rd, 6th, 7th, 8th, 9th, 10th, 11th, and 12th days after drug administration respectively. Use the body weight before drug administration as the calculation benchmark to calculate the body weight changes of mice on the 3rd, 6th, 7th, 8th, 9th, 10th, 11th, and 12th days relative to the body weight on day 0.

[0079] Results Figure 3As shown, the results showed that the body weights of the mice in the Control group were stable without significant fluctuations and all survived. The body weights of the mice in the IR group decreased rapidly until they reached the state of euthanasia according to animal ethics. The decrease in body weight of the mice in the Traditional Chinese Medicine Composition + IR group was significantly less than that in the IR group. This indicates that the Traditional Chinese Medicine Composition can significantly improve body weight in mice with radiation injury.

[0080] (2) Survival changes

[0081] Kaplan Meier survival analysis was performed on the survival time of mice in each group. The Survival Cruve statistical module in GraphPad Prism software was used for Kaplan Meier survival analysis and statistical difference analysis between groups.

[0082] The results showed that all the mice in the Control group survived. All the mice in the IR group died before the end of the experiment. Only 1 mouse died in the Traditional Chinese Medicine Composition + IR group, and the number of surviving mice was more and the survival time was longer. Among them, the survival rate of the mice in the Traditional Chinese Medicine Composition + IR group reached 80%. This indicates that the Traditional Chinese Medicine Composition can significantly improve survival in mice with radiation injury.

[0083] (3) HE staining and statistical analysis

[0084] HE staining and statistical analysis were performed on the mice in each group. The intestinal tissues of the mice in each group were sacrificed and collected, fixed overnight in 4% paraformaldehyde. The tissues were dehydrated using an automated system and embedded in paraffin. Four-micron sections were cut for H&E staining. The slides were baked at 65 °C for 4 hours and dewaxed in xylene (15 minutes each time, three times). The tissues were rehydrated using ethanol solutions (95%, 90%, 80%, and 70% concentrations), 5 minutes each time. The tissues were washed with PBS, stained with hematoxylin for 5 minutes, differentiated in 1% acidic ethanol for 5 seconds, rinsed with water, and counterstained with eosin for 3 minutes. The samples were dehydrated in an ethanol series (70%, 80%, 90%, and 95% concentrations, 30 seconds each), cleared with xylene twice (2 minutes each time). The tissues were fixed with neutral gum and photographed under a microscope. Using ImageJ software, the villus length (from the tip of the villus to the crypt opening) and crypt length (measured from the crypt opening to the bottom) were statistically analyzed. A total of at least 50 villi in five fields of view were counted. The pathological damage assessment criteria are as follows: 0 (normal), no damage; 1 (mild), mild submucosal and / or lamina propria separation; 2 (moderate), moderate submucosal and / or lamina propria separation and / or submucosal and muscular layer edema; 3 (severe), severe submucosal and / or lamina propria separation and / or severe submucosal and muscular layer edema, accompanied by villus shedding; 4 (necrosis), villus loss and necrosis.

[0085] The results are as Figure 5As shown, it shows that the IR group has glandular tissue damage, mucosal congestion and edema, shortening of small intestinal villi, shortening of crypts and other pathological injuries of intestinal tissues, and a significant increase in the immune score. In the traditional Chinese medicine composition + IR group, the damage to the glandular structure is reduced, the mucosal congestion and edema are significantly improved, the villus length increases, the ratio of crypts to villi decreases, and the immune score is significantly reduced. This indicates that the traditional Chinese medicine composition provided by the present invention can alleviate radiation-induced intestinal injury.

[0086] (4) Immune cell analysis

[0087] The numbers of immune cells Th17 and Treg cells in the intestinal tissues of mice in each group were detected by Western blotting (protein immunoblotting detection) (FOXP3 is a key molecular index of Treg cells, RORγ is a key molecular index of Th17 cells, and β-actin is an internal reference used to calibrate the protein content of each group of cells). The results are as Figure 6 shown. The number of Th17 cells in the intestinal tissues of mice in the IR group increased significantly, and the number of Treg cells decreased significantly. The number of Th17 cells in the intestine of the traditional Chinese medicine composition + IR group decreased significantly, and the number of Treg cells increased significantly. This indicates that the traditional Chinese medicine composition can regulate the immune state in the intestine.

[0088] (5) Analysis of intestinal flora species diversity

[0089] The composition and typing of intestinal flora in mice in each group were detected by 16S rRNA sequencing. The results are as Figure 7 shown. The α species diversity of intestinal flora in mice in the IR group was significantly lower than that in the Control group, while the α species diversity of intestinal flora in mice in the traditional Chinese medicine composition + IR group was close to that in the Control group, and there were differences in the β diversity among the three groups of mice. This indicates that the traditional Chinese medicine composition can restore the intestinal flora homeostasis (see Figure 7 ).

[0090] Furthermore, bacterial typing was performed using the Bugbase database. The results are as Figure 8 shown, which indicates that the abundance of conditional pathogenic bacteria in the intestinal flora of mice in the IR group is significantly higher than that in the Control group, and the abundance of conditional pathogenic bacteria in the intestinal flora of mice in the traditional Chinese medicine composition + IR group is significantly lower than that in the IR group. This indicates that the traditional Chinese medicine composition can inhibit pathogenic bacteria in the intestine.

[0091] The above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced, but these modifications or replacements are all within the protection scope of the present invention.

Claims

1. A Chinese medicine composition for preventing and / or treating radiation-induced intestinal injury, characterized in that: Calculated by weight, the composition includes: 15-45 parts of ginseng, 15-45 parts of atractylodes macrocephala, 15-45 parts of Poria cocos, 15-45 parts of roasted licorice root, 15-30 parts of cornus officinalis and 5-10 parts of vinegar schisandra chinensis.

2. The Chinese medicine composition for preventing and / or treating radiation-induced intestinal injury according to claim 1, characterized in that: Calculated by weight, it includes the following components: 30 parts of the ginseng, 30 parts of the atractylodes, 30 parts of the Poria, 30 parts of the roasted liquorice, 22.5 parts of the cornus officinalis and 7.5 parts of the vinegar schisandra chinensis.

3. The Chinese medicine composition for preventing and / or treating radiation-induced intestinal injury according to claim 1, characterized in that: Calculated by weight, it is composed of the following components: 15-45 parts of the ginseng, 15-45 parts of the atractylodes, 15-45 parts of the Poria, 15-45 parts of the roasted liquorice, 15-30 parts of the cornus officinalis and 5-10 parts of the vinegar schisandra.

4. The Chinese medicine composition for preventing and / or treating radiation-induced intestinal injury according to claim 3, characterized in that: Calculated by weight, it is composed of the following components: 30 parts of the ginseng, 30 parts of the atractylodes, 30 parts of the Poria, 30 parts of the roasted liquorice, 22.5 parts of the cornus officinalis and 7.5 parts of the vinegar schisandra chinensis.

5. The Chinese medicinal composition for preventing and / or treating radiation-induced intestinal injury according to claim 1, characterized in that: The pharmaceutical dosage form includes at least one of decoction, granules, powder, tablets, pills, and capsules.

6. A method for preparing a Chinese medicine composition for preventing and / or treating radiation-induced intestinal injury as claimed in any one of claims 1 to 5, characterized in that: The specific operations are as follows: The ginseng, the atractylodes, the Poria, the roasted liquorice, the cornus and the vinegar schisandra are placed in water and soaked for 0.4 to 2 hours, then boiled over high heat and then simmered over low heat for 0.5 to 2 hours, after which the residue is removed and the juice is taken out, and then the juice is made into a medicine according to actual needs to obtain the traditional Chinese medicine composition.

7. Use of a Chinese medicine composition for preventing and / or treating radiation-induced intestinal injury as described in claims 1 to 5 or a Chinese medicine composition prepared by the method for preparing a Chinese medicine composition for preventing and / or treating radiation-induced intestinal injury as described in claim 6 in the preparation of medicines for preventing and / or treating acute radiation-induced intestinal injury.

8. Use of a Chinese medicine composition for preventing and / or treating radiation-induced intestinal injury as described in claims 1 to 5 or a Chinese medicine composition prepared by the method for preparing a Chinese medicine composition for preventing and / or treating radiation-induced intestinal injury as described in claim 6 in the preparation of medicines for preventing and / or treating chronic radiation-induced intestinal injury.

9. Use of a Chinese medicine composition for preventing and / or treating radiation-induced intestinal injury as described in claims 1 to 5 or a Chinese medicine composition prepared by the method for preparing a Chinese medicine composition for preventing and / or treating radiation-induced intestinal injury as described in claim 6 in the preparation of a drug for preventing and / or treating excessive proliferation of intestinal proinflammatory immune cells, intestinal flora disorder, and intestinal epithelial cell apoptosis in patients with acute radiation-induced intestinal injury.

10. Use of a Chinese medicine composition for preventing and / or treating radiation-induced intestinal injury as described in claims 1 to 5 or a Chinese medicine composition prepared by the method for preparing a Chinese medicine composition for preventing and / or treating radiation-induced intestinal injury as described in claim 6 in the preparation of a drug for preventing and / or treating excessive proliferation of intestinal proinflammatory immune cells, intestinal flora disorders, and intestinal epithelial cell apoptosis in patients with chronic radiation-induced intestinal injury.

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