Application of echinacoside in preparation of medicine for preventing or treating radiation damage

Abstract

The invention belongs to the technical field of medical biology, and particularly relates to application of echinacoside in preparation of a medicine for preventing or treating radiation damage. The radiation injury comprises skin injury, lung injury, intestinal injury, hematopoietic injury, reproductive function injury and delaying of aging caused by radiation. The radiation includes non-ionizing radiation and ionizing radiation. The medicine also comprises other medicines for preventing or treating radiation damage. In anti-radiation drug screening based on multiple cell models, echinacoside is found to have a repair promoting effect on small intestine mucosa epithelial cell injury caused by radiation; a mouse radiation injury model shows that echinacoside can effectively protect an organism, reduce death caused by a lethal dose of cobalt-60 gamma rays and delay aging caused by radiation. The echinacoside has a good clinical application prospect in the aspect of preventing or treating radiation damage, and has no obvious toxic or side effect.

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutical biotechnology, and specifically relates to the use of echinacoside in preparing medicines for preventing or treating radiation damage. Background Art

[0002] With the continuous development of economy and science and technology, radiation has been widely used in various fields. While it brings convenience to human beings, it also causes considerable harm to human health. In particular, the rapid development of the nuclear industry has exposed more and more people to the threat of nuclear radiation. In addition, the incidence of malignant tumors continues to increase. Radiotherapy, as a conventional anti-tumor treatment method, kills tumor cells while also damaging normal cells.

[0003] There is currently a lack of effective radiation protection agents in clinical practice. Although traditional chemical anti-radiation drugs such as amifostine are effective and approved by the FDA for marketing, they cause nausea, vomiting, hypotension and other toxic side effects to patients. Therefore, it is necessary to find safer and more effective anti-radiation drugs to meet clinical needs. In addition, it is also necessary to protect against radiation-related diseases such as aging and other diseases. In recent years, scientific researchers have turned their attention to effective anti-radiation active ingredients in traditional Chinese medicine, which have the characteristics of high safety, low toxicity, few side effects, and patient tolerance, and are expected to become a new way to discover anti-radiation drugs.

[0004] Echinacoside was first discovered in the extract of Echinacea, a plant of the genus Echinacea in the Asteraceae family native to North America. It is also one of the main components of the desert plant Cistanche deserticola, a plant of the genus Cistanche in the Orobanchaceae family that nourishes the kidney yang, improves the essence and blood, and moisturizes the intestines. Previous studies have found that echinacoside has antioxidant, anti-inflammatory, anti-tumor, liver protection, nerve protection, wound healing, and learning and memory improvement effects.

[0005] However, there are no reports on the anti-radiation effect of echinacoside. Summary of the invention

[0006] In order to solve the problem of lack of effective radiation protection agents in clinical practice, the purpose of the present invention is to provide a safe, effective, convenient and economical drug for preventing or treating radiation damage, providing a new idea for preventing or treating radiation damage in clinical practice.

[0007] The technical solution for achieving the purpose of the present invention is as follows:

[0008] The present invention provides the use of echinacoside in preparing a medicine for preventing or treating radiation damage to a subject.

[0009] Preferably, the echinacoside can be extracted and separated from the flowers, leaves and fruits of plants containing echinacoside by a biological purification method, or can be purchased from commercially available natural product monomers.

[0010] As an alternative, in the above use, the radiation damage is skin damage, lung damage, intestinal damage, hematopoietic damage, reproductive function damage or aging caused by radiation.

[0011] Additionally, the drug prolongs the life span of radiation damaged subjects.

[0012] Preferably, the intestinal damage is small intestinal mucosal epithelial cell damage.

[0013] More preferably, the echinacoside can upregulate the relative abundance of microorganisms of the genera Lachnoclostridium, Roseburia, Prevotella and Tyzzerella.

[0014] Preferably, the subject is a mammal.

[0015] More preferably, the subject is a human, a companion animal, a farm animal or a laboratory animal.

[0016] As an option, in the above use, the radiation is non-ionizing radiation or ionizing radiation.

[0017] As an option, in the above use, the radiation is ultraviolet radiation, X-ray radiation, gamma ray radiation, alpha ray radiation, beta ray radiation, neutron ray radiation or laser radiation.

[0018] Preferably, the radiation is X-ray radiation, gamma-ray radiation, alpha-ray radiation or beta-ray radiation.

[0019] As an option, in the above uses, the radiation is caused by cancer treatment, diagnostic procedures, occupational exposure, nuclear and radiation emergencies, or terrorist attacks.

[0020] Preferably, the diagnostic procedure is a dental or bone X-ray.

[0021] Also preferably, the diagnostic procedure comprises a PET or CT scan.

[0022] Preferably, the occupational exposure is the occupational exposure of laboratory technicians or the occupational exposure of nuclear power plant workers.

[0023] As an option, in the above use, the medicine also contains other drugs for preventing or treating radiation damage.

[0024] As an alternative, in the above use, the drug is a chemical anti-radiation drug or an anti-radiation drug of natural origin.

[0025] As an optional mode, in the above-mentioned use, the chemical anti-radiation drug is amifostine, and the anti-radiation drug of natural origin is astragaloside IV, ginsenoside Rg1, gypenosyl saponin, angelica polysaccharide, wolfberry polysaccharide, astragalus polysaccharide, ganoderma lucidum polysaccharide, polygonatum polysaccharide, silymarin, quercetin, baicalin or hesperidin.

[0026] Preferably, the medicament comprises a pharmaceutically active ingredient and a pharmaceutically acceptable carrier.

[0027] In addition, preferably, the pharmaceutically acceptable carrier refers to a conventional pharmaceutical carrier in the field of pharmaceutical preparations, selected from one or more of fillers, binders, disintegrants, lubricants, suspending agents, wetting agents, pigments, flavoring agents, solvents, and surfactants.

[0028] More preferably, the pharmaceutically active ingredient is echinacoside as the sole active ingredient or consists of echinacoside and polygonatum sibiricum polysaccharide.

[0029] More preferably, in the pharmaceutical active ingredient, the weight ratio of the echinacoside to the polygonatum polysaccharide is 10:1-1:1.

[0030] For example, the weight ratio of the echinacoside to the polygonatum polysaccharide is 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1 or 1:1.

[0031] As an option, in the above use, the drug is an oral preparation or an external preparation.

[0032] As an optional mode, in the above-mentioned use, the oral preparation is a tablet, a capsule, a granule or an oral liquid, and the external preparation is a gel, an ointment, an aerosol or a spray.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] (1) The present invention provides a safe, effective, convenient and economical drug for preventing or treating radiation damage, providing a new idea for preventing or treating radiation damage in clinical practice.

[0035] (2) Pharmacological experiments have shown that, in the screening of anti-radiation drugs based on multiple cell models, echinacoside has a pro-repair effect on radiation-induced damage to small intestinal mucosal epithelial cells. Through the mouse radiation damage model, it was found that echinacoside can effectively protect the body, reduce the death caused by lethal doses of cobalt-60 gamma rays, alleviate radiation intestinal damage, and reduce radiation-induced damage to the reproductive system of male mice, while delaying radiation-induced aging.

[0036] (3) The natural product monomer echinacoside used in the present invention has a wide range of sources and can be produced by mature industrial methods, so that the drug of the present invention can be industrially produced on a large scale. In addition, by adding a pharmaceutically acceptable carrier, the drug of the present invention can be made into a conventional oral preparation or external preparation, so that the drug treatment is stable, the drug effect is improved, and it is easy to use. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 :Effects of echinacoside on human small intestinal epithelial cells HIEC-6 irradiated with 4Gy Cobalt-60γ-rays.

[0038] Figure 2 : Effects of echinacoside on human lymphoblastoid cells AHH-1 irradiated with 4Gy Cobalt-60 gamma rays.

[0039] Figure 3 :Effects of echinacoside on human vascular endothelial cells (HUVEC) irradiated with 3Gy Cobalt-60γ-rays.

[0040] Figure 4 : The dose range of echinacoside in alleviating radiation damage to small intestinal mucosal epithelial cells.

[0041] Figure 5 : Echinacoside increases the 30-day survival rate of mice irradiated with a single dose of 6.5 Gy whole body irradiation.

[0042] Figure 6 : Echinacoside increases the 30-day survival rate of mice irradiated with 7.0 Gy single whole-body irradiation.

[0043] Figure 7 : Echinacoside increases the 30-day survival rate of mice irradiated with a single dose of 7.5 Gy whole body irradiation.

[0044] Figure 8 : Species diversity of samples in each group.

[0045] Fig. 9 :Differences and changes in abundance of fecal microorganisms among different mouse species.

[0046] Fig.10 :Echinacoside inhibits the decrease of peripheral blood leukocyte count in irradiated mice.

[0047] Fig.11 :The combination of echinacoside and polysaccharides inhibits the decrease of peripheral blood leukocyte count in irradiated mice.

[0048] Fig.12 : The combination of echinacoside and polysaccharides reduces the risk of death in mice irradiated with 8Gy cobalt-60.

[0049] Fig.13:The combination of echinacea glycoside and polysaccharides increases the median survival time of mice irradiated with 8.5Gy cobalt-60. DETAILED DESCRIPTION

[0050] In order to facilitate those skilled in the art to understand the present invention, the main terms used in the present invention are explained below:

[0051] The drugs described herein can be used in a "therapeutically effective amount." A "therapeutically effective amount" refers to an amount effective to achieve the desired therapeutic result at the dosage and time period necessary. The therapeutically effective amount can vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the composition to elicit a desired response in the individual. A therapeutically effective amount is also an amount in which the therapeutically beneficial effects outweigh any toxic or deleterious effects of the molecule.

[0052] As used herein, the phrase "pharmaceutically acceptable" refers to those compounds, materials, compositions, carriers and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, and are commensurate with a reasonable benefit / risk ratio.

[0053] "Pharmaceutically acceptable carrier" refers to an excipient that can be used to prepare a pharmaceutical composition that is generally safe, non-toxic, and biologically or otherwise not adverse, and includes carriers that are acceptable for veterinary and human pharmaceutical use. As used herein, "pharmaceutically acceptable carrier" includes one and more than one such carrier.

[0054] The medicament of the present invention can be administered to a subject by any suitable method known to those skilled in the art, such as orally, parenterally, transmucosally, transdermally, intramuscularly, intravenously, intradermally, subcutaneously, intraperitoneally, intraventricularly, intracranially, intravaginally, intratumorally or intraorally. Controlled release can also be used by embedding the active ingredient in a suitable polymer, which can then be inserted subcutaneously, intratumorally, intraorally, as a patch on the skin or intravaginally. Coating medical devices with the active ingredient is also included.

[0055] In some embodiments, the medicament of the present invention is preferably administered orally, and is therefore formulated in a form suitable for oral administration, i.e., as a solid or liquid preparation. Suitable solid oral preparations include tablets, capsules, pills, granules and pellets, etc. Suitable liquid oral preparations include solutions, suspensions, dispersants, emulsions and oils, etc. In some embodiments, the active ingredient is formulated into capsules. According to this embodiment, the composition of the present invention also includes a desiccant in addition to the active compound and an inert carrier or diluent, and a gelatin capsule in addition to other excipients.

[0056] In some embodiments, the medicament of the present invention is topically applied to the body surface and is therefore formulated in a form suitable for topical administration. Suitable topical preparations include gels, ointments, creams, lotions, drops, and controlled release polymers, etc. For topical administration, the echinacoside active ingredient is prepared and applied as a solution, suspension, or emulsion in a physiologically acceptable diluent with or without a pharmaceutical carrier.

[0057] Diseases or conditions that may be treated or prevented by the medicaments of the present invention include, for example, but are not limited to, radiation damage, aging phenotypes, aging-related diseases or conditions of aging, radiation-related diseases or conditions of aging, and signs of aging.

[0058] In one example, a subject in need of radiation protection or radiation mitigation according to the methods provided herein is a subject who will, has, or has been exposed to a potentially harmful amount of radiation. It should be understood that such exposure can be a single exposure, periodic exposure, sporadic exposure, or continuous exposure to radiation. It should also be understood that such radiation exposure includes accidental exposure, accidental or intentional exposure.

[0059] Examples of subjects who may need radiation protection or radiation mitigation according to the methods of the present invention include, but are not limited to, patients exposed to radiation (e.g., proton radiation, photon radiation) as part of a treatment regimen (e.g., cancer patients requiring radiation therapy), subjects exposed to radiation to diagnose a disease or condition (e.g., subjects receiving dental or bone X-rays, patients receiving PET scans, CT scans, etc.). Examples of subjects who may need radiation protection or radiation mitigation according to the methods of the present invention also include people who may be exposed to radiation due to their occupations or lifestyle choices (e.g., aircraft crews or other frequent air travelers, even space travelers, laboratory technicians and other workers exposed to higher than average radiation levels, or people exposed through the use of electronic devices) or people exposed to radon accumulation (e.g., accumulation in homes or mines) or outdoor workers or sunbathers exposed to natural radiation from the sun. Other subjects who may need radiation protection according to the methods of the present invention include people who are accidentally exposed to radiation (e.g., leaks or spills) (e.g., nuclear reactor leaks or accidents or laboratory spills). Those exposed to radiation from exploding nuclear warheads are also considered due to war or terrorism. Other subjects include those exposed to conventional explosives where terrorists detonate radioactive materials.

[0060] The term "subject" includes mammals, such as humans, companion animals (e.g., dogs, cats, birds, etc.), farm animals (e.g., cattle, sheep, pigs, horses, poultry, etc.), and laboratory animals (e.g., rats, mice, guinea pigs, birds, etc.). In addition to humans, subjects may include dogs, cats, pigs, cattle, sheep, goats, horses, buffaloes, ostriches, guinea pigs, rats, mice, birds (e.g., parakeets), and other wild, domestic, or commercially useful animals (e.g., chickens, geese, turkeys, fish). The term "subject" does not exclude individuals who are normal in all respects. The term "subject" includes, but is not limited to, a person who needs treatment or is susceptible to a disease or its sequelae.

[0061] Any patent, patent application publication, or scientific publication cited herein is hereby incorporated by reference in its entirety.

[0062] In the following examples, many specific details are set forth in order to provide a thorough understanding of the present invention. However, it will be appreciated by those skilled in the art that the present invention can be practiced without these specific details. In other cases, well-known methods, procedures and components are not described in detail in order to avoid confusing the present invention. Therefore, these embodiments should never be interpreted as limiting the broad scope of the present invention.

[0063] Example 1: Echinacoside has a protective effect on radiation-induced damage to human small intestinal epithelial cells

[0064] The hematopoietic system, circulatory system and digestive system are very sensitive to ionizing radiation. In patients exposed to medium and low doses of ionizing radiation, a decrease in peripheral blood leukocytes, lymphocytes and platelets can be detected, and punctate hemorrhages, as well as gastrointestinal reactions such as abdominal pain, diarrhea, and bloody stools can be observed.

[0065] In this example, human peripheral blood lymphoblastoid cell AHH-1 cell line, human umbilical vein endothelial cell HUVEC cell line, and human small intestinal mucosal epithelial cell HIEC-6 cell line were selected to conduct in vitro evaluation studies on the radiation damage protection effect of echinacoside.

[0066] Human peripheral blood lymphoblastoid cell line AHH-1 was purchased from ATCC. One hour before irradiation, echinacoside solution (China Food and Drug Inspection Institute) was prepared with 1640 culture medium (Hyclone) containing 10% fetal bovine serum (Gibco), and added to 96-well plates after multiple dilution for preventive intervention on AHH-1 cells. The final concentrations of echinacoside were 0.39, 0.78, 1.56, 3.12, 6.25, 12.5, 25, 50, and 100 μg / ml, respectively; 1640 culture medium containing 10% fetal bovine serum was added to each well of the sham group, irradiation model group (IR), and blank culture medium group without cells. AHH-1 was irradiated with 4 Gy of cobalt-60 γ-rays to cause radiation damage, and the sham group was shielded with 6 cm thick lead bricks. 48 hours after irradiation, 10 μL of CCK-8 was added to each well, and the absorbance of each well was read at a wavelength of 450 nm using an enzyme reader to calculate the cell viability of each group.

[0067] Human umbilical vein endothelial cell (HUVEC) cell line was purchased from ScienCell. HUVEC cells in the logarithmic growth phase within the sixth generation were trypsinized and resuspended, and the cell concentration was adjusted to 2×10 4 / mL, 100μL of HUVEC cell suspension was added to each well of the 96-well plate. After 12 hours, HUVECs were completely attached to the wall, and echinacoside intervention and irradiation began. One hour before irradiation, the echinacoside solution was prepared with ECM culture medium and diluted in multiples to make the final concentration of echinacoside 0.39, 0.78, 1.56, 3.12, 6.25, 12.5, 25, 50, and 100μg / mL. The culture medium in each well of the 96-well plate was replaced according to the group for preventive intervention; the sham group, irradiation model group (IR) and cell-free blank culture medium group were added with an equal volume of ECM culture medium to each well. HUVEC radiation damage was caused by 3Gy cobalt-60γ-ray irradiation, and the sham irradiation group was shielded with a 6cm thick lead brick. Five days after irradiation, the supernatant was removed, 100 μL of serum-free and cytokine-free culture medium containing 10% CCK-8 was added to each well, and the absorbance value of each well was read at a wavelength of 450 nm using an ELISA reader to calculate the cell viability of each group.

[0068] The human intestinal epithelial cell line HIEC-6 was purchased from Shanghai Jining Industrial Co., Ltd. HIEC-6 cells in the logarithmic growth phase were trypsinized and resuspended, and the cell concentration was adjusted to 1×10 4 / mL, 100μL of HIEC-6 cell suspension was added to each well of the 96-well plate. After 12 hours, HIEC-6 was completely attached to the wall, and echinacoside intervention and irradiation began. One hour before irradiation, echinacoside solution was prepared with Opti-MEM culture medium containing 5% fetal bovine serum and diluted in multiples to make the final concentration of echinacoside 6.25, 12.5, 25, 50, 100, and 200μg / mL. The culture medium in each well of the 96-well plate was replaced according to the group for preventive intervention; Opti-MEM culture medium containing 5% fetal bovine serum was added to each well of the sham group, irradiation model group (IR) and cell-free blank culture medium group. HIEC-6 was irradiated with 4Gy of cobalt-60γ rays to cause radiation damage, and the sham irradiation group was shielded with 6cm thick lead bricks. Five days after irradiation, the supernatant was removed, 100 μL of serum-free Opti-MEM culture medium containing 10% CCK-8 was added to each well, and the absorbance value of each well was read at a wavelength of 450 nm using an ELISA reader to calculate the cell viability of each group.

[0069] Cell viability = (OD450 of the test group - OD450 of the blank culture medium group) ÷ (OD450 of the sham irradiation group - OD450 of the blank culture medium group)

[0070] The cell viability test was performed with 6 replicates in each group. The cell viability of the echinacea glycoside intervention groups at different concentrations was compared with that of the irradiation model group using Student's T test, and the difference was statistically significant when P < 0.05.

[0071] like Figures 1 to 3 As shown in the results, it can be seen that echinacoside was given for prevention 1 hour before irradiation. The cell viability of the 100 and 200 μg / mL echinacoside intervention groups on HIEC-6 radiation-damaged cells was significantly higher than that of the irradiation model group and was statistically different (P < 0.05); on AHH-1 and HUVEC cells, the cell viability of the 0.39-100 μg / mL echinacoside intervention group did not increase, and there was a downward trend. This suggests that echinacoside can effectively alleviate the damage to the small intestinal mucosal epithelial cells caused by ionizing radiation damage; while echinacoside prevention did not have a protective effect on the peripheral blood lymphoblasts AHH-1 and vascular endothelial cells HUVEC at the cell viability level.

[0072] Example 2: Dose range of echinacoside in alleviating radiation damage to small intestinal mucosal epithelial cells

[0073] The human intestinal epithelial cell line HIEC-6 was purchased from Shanghai Jining Industrial Co., Ltd. HIEC-6 cells in the logarithmic growth phase were trypsinized and resuspended, and the cell concentration was adjusted to 1×10 4 / mL, 100μL of HIEC-6 cell suspension was added to each well of the 96-well plate. After 12 hours, HIEC-6 was completely attached to the wall, and echinacoside intervention and irradiation began. One hour before irradiation, echinacoside solution was prepared with Opti-MEM culture medium containing 5% fetal bovine serum and diluted in multiples to make the final concentration of echinacoside 50, 60, 70, 80, 90, and 100μg / mL. The culture medium in each well of the 96-well plate was replaced according to the group for preventive intervention; Opti-MEM culture medium containing 5% fetal bovine serum was added to each well of the sham group, irradiation model group (IR) and cell-free blank culture medium group. HIEC-6 was irradiated with 4Gy of cobalt-60γ rays to cause radiation damage, and the sham irradiation group was shielded with 6cm thick lead bricks. Five days after irradiation, the supernatant was removed and 100 μL of serum-free Opti-MEM culture medium containing 10% CCK-8 was added to each well. Two hours later, the absorbance of each well was read at a wavelength of 450 nm using an ELISA reader to calculate the cell viability of each group.

[0074] Cell viability = (OD450 of the test group - OD450 of the blank culture medium group) ÷ (OD450 of the sham irradiation group - OD450 of the blank culture medium group)

[0075] The cell viability test was performed with 6 replicates in each group. The cell viability of the echinacea glycoside intervention groups at different concentrations was compared with that of the irradiation model group using Student's T test, and the difference was statistically significant when P < 0.05.

[0076] like Figure 4 As shown, the experimental results showed that 60-100 μg / mL of echinacea glycosides can effectively alleviate the damage of the small intestinal mucosal epithelium irradiated by 4Gy cobalt-60 γ-rays, and significantly improve its cell viability (P<0.0001), so that its cell viability is improved to the level of normal small intestinal mucosal epithelial cells.

[0077] Example 3: Echinacoside improves the 30-day survival rate of mice irradiated with a lethal dose of cobalt-60 gamma rays

[0078] The 30-day survival rate is the most intuitive indicator for evaluating the efficacy of anti-radiation drugs. SPF-grade Balb / c male mice, 6 weeks old, weighing 20±1g, were purchased from Sibeifu (Beijing) Biotechnology Co., Ltd. After being raised in IVC cages in an SPF-grade breeding environment for 3 days, cobalt-60 gamma ray irradiation induced hematopoietic, immune, intestinal and other damages, and induced death. It can be seen that: after a single 6.0Gy whole-body irradiation, 50% of the mice died within 30 days, after a single 6.5Gy whole-body irradiation, 70% of the mice died within 30 days, and a single whole-body irradiation of more than 6.8Gy can cause all mice to die within 30 days after irradiation.

[0079] In this example, mice were irradiated with a 70% lethal dose of 6.5 Gy ( Figure 5 ) and 100% lethal dose of 7.0 Gy irradiated mice ( Figure 6 ) to observe the effect of preventive oral administration of echinacoside on radiation-induced lethality in mice. From the 30-day survival record results, it can be seen that oral administration of 60-120 mg / kg of echinacoside 1 hour before irradiation can effectively protect mice irradiated with a lethal dose, prolong their life time, and increase the chance of receiving treatment; and oral administration of 60-120 mg / kg of echinacoside 1 hour before irradiation can also effectively increase the survival rate of mice irradiated with a lethal dose by 70%, and increase the 30-day survival rate of Balb / c mice irradiated with 6.5Gy from 30% to more than 80%. In particular, 60 mg / kg of echinacoside is better than the only FDA-approved anti-radiation drug amifostine in prolonging the life time and improving the survival rate of mice irradiated with 6.5-7.0Gy. When mice were irradiated with a higher completely lethal irradiation dose of 7.5Gy, there was no effect of a single preventive oral administration of echinacoside before irradiation on the survival time and survival rate of mice, but a single therapeutic oral administration after irradiation increased the survival rate of mice by 40% ( Figure 7 ). The above examples suggest that in radiation-induced fatal emergencies, echinacoside has good effects in both prevention and treatment. Moreover, echinacoside is preferably taken orally and can be stored at room temperature away from light; while WR2721 (amifostine) is administered by intravenous injection, and its lyophilized powder must be stored at 4°C and used immediately after preparation. Based on the above results and analysis, it can be seen that echinacoside, as an anti-radiation drug, can be made into tablets and granules, which are easier to store, carry, distribute, and use, and can better improve patient compliance.

[0080] Example 4: Echinacoside modulates the intestinal microbiome of mice irradiated with a lethal dose of cobalt-60 gamma rays

[0081] Intestinal microbes are the largest and most complex micro-ecological system in the human body, with a total volume of about 10 times the number of somatic cells. Their impact on metabolic capacity and body health cannot be underestimated. Existing literature reports suggest that intestinal microbes have a good effect on alleviating intestinal radiation damage and improving the survival rate of mice irradiated with a lethal dose, and can become a reliable target for the prevention and treatment of radiation damage.

[0082] In this example, Balb / c mice were treated with 6.5 Gy 60 After a single whole-body irradiation with Coγ-rays, the mice were sterilely sampled 7 days after irradiation. The effects of the anti-radiation drug echinacoside (ECH) on the species and relative abundance of intestinal microorganisms in mice were analyzed using the intestinal microbiome 16S detection technology. By analyzing the 16S rRNA detection results using bioinformatics technology, it can be seen that the species diversity of fecal microorganisms in mice killed by ionizing radiation is significantly different from that in the other four groups, and the irradiated mice that received echinacoside intervention are close to the microbial diversity of normal mice ( Figure 8); Further analysis revealed that echinacea glycosides could alleviate radiation-induced damage and enable irradiated mice to survive by regulating the relative abundance of microorganisms of the genera Lachnoclostridium, Roseburia, Prevotella, and Tyzzerella ( Fig. 9 ).

[0083] Example 5: Echinacoside effectively delays aging and prolongs the life span of mice surviving high-dose cobalt-60 gamma irradiation

[0084] Balb / c male mice were divided into sham, irradiation model, and echinacoside 30, 60, and 90 mg / kg intervention groups, with 10 mice in each group. After being housed in IVC cages in an SPF animal room for 3 days, mice were given a single oral gavage 1 hour before irradiation. The sham and irradiation model groups were given 0.2 mL of distilled water each, and the drug intervention groups were gavaged with 0.2 mL of echinacoside solution of different concentrations to make the oral intervention dose of mice reach 30, 60, and 90 mg / kg echinacoside, respectively. The mice were transported to the cobalt source of Beijing Normal University in SPF irradiation cages, and irradiated at a radiation dose rate of 1 Gy / min for 6 minutes. After irradiation, they were immediately transported back to the SPF animal room and housed in IVC cages as usual. After receiving 6 Gy 60 At 52 weeks after single whole-body irradiation with Coγ-rays, among the 5 surviving mice in the irradiation model group, 1 died, and the remaining 4 mice had significantly higher senility symptoms than the mice in the 60mg / kg echinacoside prevention group. All 4 mice showed symptoms of hair loss, dermatitis, and diarrhea, 3 mice (3 / 4, 75%) showed crystalline opacity, and 2 mice (2 / 4, 50%) showed anal prolapse. These 4 surviving mice died successively from then until 75 weeks after irradiation. The hair loss of mice orally taking 60mg / kg was milder than that of mice surviving simple irradiation at 52 weeks after irradiation, and only 1 mouse showed mild cataract symptoms. 5 mice survived 75 weeks after irradiation, and 4 mice were still alive at 85 weeks after irradiation. It can be seen that preventive oral administration of echinacoside can effectively delay the onset of aging symptoms and prolong the life cycle of mice surviving high-dose Co-60γ-ray irradiation.

[0085] Example 6: Protective effect of echinacoside on peripheral blood count of mice irradiated with cobalt-60 gamma rays

[0086] Balb / c male mice were housed in IVC cages in an SPF animal room for 3 days and then given a single oral gavage of 0.2 mL / mouse 1 hour before irradiation. The sham irradiation group and irradiation model group were given distilled water, and the 30, 60, and 90 mg / kg echinacoside intervention groups were given an oral gavage of the corresponding concentrations of echinacoside aqueous solution. 60 On the 7th day after a single whole-body irradiation with Coγ-rays, the number of peripheral blood leukocytes dropped to the lowest level. Fig.10As shown in the results, a single preventive oral gavage of 60 mg / kg echinacoside 1 hour before irradiation can increase the peripheral blood leukocyte count of irradiated mice. The average values ​​of peripheral blood leukocyte counts of mice in the 30, 60 and 90 mg / kg echinacoside groups were increased compared with those in the irradiation model group. The Student T test showed that the leukocyte count in the 60 mg / kg echinacoside group was increased compared with that in the irradiation control group, and the difference was statistically significant (P<0.05).

[0087] Example 7: Echinacea glycoside combined with plant polysaccharides enhances the anti-radiation effect

[0088] Balb / c male mice received 3.5 Gy and 8 Gy 60 The corresponding solution was orally administered orally 1 hour before single whole-body irradiation with Coγ-rays (0.2 mL / mouse). The mice in the normal control group were administered with distilled water, the mice in the echinacoside group were administered with an aqueous solution of echinacoside (50 mg / kg), the mice in the polygonatum polysaccharide group were administered with an aqueous solution of polygonatum polysaccharide (12.5 mg / kg), and the mice in the combined use group of echinacoside + polygonatum polysaccharide were administered with a mixed aqueous solution of the two (doses of echinacoside 50 mg / kg + polygonatum polysaccharide 12.5 mg / kg).

[0089] like Fig.11 As shown, the number of peripheral blood leukocytes in mice decreased significantly on the third day after 3.5 Gy irradiation. Both echinacea glycoside and polygonatum polysaccharide alone could effectively increase the number of peripheral blood leukocytes (P<0.05). The number of peripheral blood leukocytes in the mice in the echinacea glycoside + polygonatum polysaccharide combination group was further increased compared with that in the groups using either alone, and there were statistical differences compared with the peripheral blood leukocytes in the echinacea glycoside group and polygonatum polysaccharide group (P<0.05).

[0090] like Fig.12 As shown in the research results, we can see that: after receiving a completely lethal dose of 8Gy 60 Under the condition of single whole-body irradiation with Coγ-rays, the median survival time of mice was 4.5 days, and the 30-day survival rate was zero; after a single oral administration of echinacea glycoside or polygonatum polysaccharide 1 hour before irradiation, although the 30-day survival rate of the irradiated mice was still zero, both of them could effectively prolong the median survival time and average survival time of the irradiated mice (P<0.05), and the median survival time of mice in the echinacea glycoside group and the polygonatum polysaccharide group was greater than or equal to 10 days; and the median survival time of mice that were co-administered with echinacea glycoside and polygonatum polysaccharide 1 hour before irradiation was significantly increased compared with the 8Gy irradiation group (P<0.05), from 4.5 days to 10.5 days. Although there was no statistical difference in the median survival time of the combination group of echinacea glycoside and polygonatum polysaccharide compared with the group with either of the two intervention groups alone, its 30-day survival rate increased from zero to 20%.

[0091] like Fig.13 As shown in the survival curve of the research results, we can see that:60 When the single whole-body irradiation dose of Coγ-rays was increased to 8.5Gy, there was no difference in the survival rate among the groups. The log-rank test found that the median survival time of mice in the echinacoside and polygonatum polysaccharide intervention groups alone was no different from that in the irradiation model group, while the median survival time of mice in the combined intervention group was significantly increased compared with the irradiation model group, the echinacoside intervention group, and the polygonatum polysaccharide intervention group (P<0.05).

[0092] The results suggest that echinacoside alone has an anti-radiation effect, which can alleviate hematopoietic damage in irradiated subjects, inhibit peripheral leukocytopenia, and improve the survival of mice. However, the risk of radiation-induced death cannot be reduced when the irradiated subjects (such as mice) are exposed to a radiation dose of 8.5 Gy or above. The anti-radiation effect of echinacoside combined with Polygonatum sibiricum polysaccharide is increased, which further inhibits the leukocytopenia in peripheral blood of irradiated subjects, reduces the risk of death caused by higher irradiation doses, or effectively increases the median survival time of subjects irradiated with higher lethal doses.

[0093] In addition, in the above-mentioned acute and long-term experiments involving animals, no obvious adverse reactions were observed for the drug of the present invention, and the experimental animals had good tolerance to it.

[0094] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. Use of echinacoside in the preparation of a medicament for preventing or treating radiation damage in a subject.

2. The use according to claim 1, characterized in that: The radiation damage is skin damage, lung damage, intestinal damage, hematopoietic damage, reproductive function damage or aging caused by radiation, and / or the drug extends the life span of the radiation damaged subject, the subject is a mammal, preferably, the subject is a human, a companion animal, a farm animal or a laboratory animal.

3. The use according to claim 1 or claim 2, characterized in that: The radiation is non-ionizing radiation or ionizing radiation.

4. The use according to any one of claims 1 to 3, characterized in that: The radiation is ultraviolet radiation, X-ray radiation, gamma ray radiation, alpha ray radiation, beta ray radiation, neutron ray radiation or laser radiation.

5. The use according to any one of claims 1 to 4, characterized in that: The radiation is caused by cancer treatment, diagnostic procedures, occupational exposure or terrorist attacks.

6. The use according to any one of claims 1 to 4, characterized in that: The medicine also contains other medicines for preventing or treating radiation damage.

7. The use according to claim 6, characterized in that: The drug is a chemical anti-radiation drug or an anti-radiation drug of natural origin.

8. The use according to claim 7, characterized in that: The chemical anti-radiation drug is amifostine, and the natural anti-radiation drug is astragaloside IV, ginsenoside Rg1, gypenosyl saponin, angelica polysaccharide, wolfberry polysaccharide, astragalus polysaccharide, ganoderma lucidum polysaccharide, polygonatum polysaccharide, silymarin, quercetin, baicalin or hesperidin.

9. The use according to any one of claims 1 to 8, characterized in that: The medicine is an oral preparation or an external preparation.

10. The use according to any one of claims 1 to 9, characterized in that: The oral preparation is a tablet, a capsule, a granule or an oral liquid, and the external preparation is a gel, an ointment, an aerosol or a spray.

Images