Application of baicalein in preparation of medicine for antagonizing jellyfish toxin lethal

Abstract

The invention provides application of baicalein in preparation of a medicine for antagonizing jellyfish toxin lethal, and belongs to the technical field of medicine. Experiments prove that baicalein can relieve multi-organ function damage caused by jellyfish stings. Cell experiments show that baicalein can significantly improve the proliferation rate of RAW 264.7 cells and antagonize the apoptosis level induced by jellyfish toxin; mouse experiments show that the baicalein can obviously improve the survival rate of mice and relieve damage of jellyfish toxin to the heart, the liver and the kidney.

Description

Technical Field

[0001] The invention relates to the technical field of medicine, and in particular to the application of baicalin in preparing medicines for antagonizing the lethality of jellyfish toxins. Background Art

[0002] With the explosive growth of jellyfish, the number of jellyfish stings is increasing, and tens of thousands of people are injured every year. Mild cases may cause allergic reactions, while severe cases may inhibit the central nervous system and respiratory system, cause multiple organ damage, and thus cause death. Jellyfish toxins are complex in composition, with diverse toxicity and varying strengths, which brings great troubles to the diagnosis and treatment of jellyfish stings, and also causes loss of life and property to my country's offshore workers and coastal residents, affecting the level of economic development. Therefore, how to antagonize the problem of death caused by jellyfish toxins has always been the focus and hot spot of current research.

[0003] Scutellaria baicalensis is the dried root of the Lamiaceae plant Scutellaria baicalensis. It has a long history of medicinal use. It tastes bitter and cold in nature. It enters the lung, gallbladder, small intestine, large intestine and spleen meridians. It has the effects of clearing away heat and dampness, purging fire and detoxifying, stopping bleeding and calming the fetus. It is commonly used in clinic for a variety of diseases such as damp-heat, jaundice, lung heat cough, blood heat vomiting and bleeding. The traditional Chinese medicine Scutellaria baicalensis is widely distributed in Hubei, Henan, Shandong, Jiangsu and other places, and its planting areas provide a sufficient drug basis. Baicalein is one of the flavonoid components with the highest content in the traditional Chinese medicine Scutellaria baicalensis. Studies have shown that Scutellaria baicalensis has the effect of protecting cardiovascular and cerebrovascular vessels. It can protect myocardial cells from multiple pathways and multiple targets, and protect cardiovascular and cerebrovascular vessels by reducing pulmonary artery pressure and resisting arteriosclerosis. Patent CN103301268A discloses an ointment containing baicalin that can be used for skin damage caused by jellyfish stings, but skin damage and organ function damage are two completely different symptoms of poisoning, and baicalin and baicalein are two different compounds, and there are also certain differences in their efficacy. Therefore, it is impossible to determine whether baicalin has the effect of improving organ function damage caused by jellyfish toxins.

[0004] Baicalein can improve heart rate, ST segment and heart coefficient, reduce oxidative stress, inflammation and apoptosis levels, thereby improving myocardial ischemia, and can inhibit inflammatory cytokines and chemokines, remove reactive oxygen and improve antioxidant status. In addition, baicalein can fight against influenza virus, HIV, hepatitis B virus, measles virus and other viruses. In other words, baicalein has good pharmacological activity, almost no serious toxic and side effects, and has broad application prospects. Therefore, the present invention attempts to use baicalein to antagonize jellyfish toxins, in order to provide an effective treatment plan for the prevention and / or treatment of jellyfish stings, and also provides new ideas for the clinical application of baicalein, actively responds to the country's revitalization and development of traditional Chinese medicine, and provides a new research direction for the application of traditional Chinese medicine. Summary of the invention

[0005] The purpose of the present invention is to provide the application of baicalin in preparing medicine for antagonizing the lethality of jellyfish toxin.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The invention provides application of baicalin in preparing medicine for antagonizing lethality caused by jellyfish toxins.

[0008] Preferably, the lethal aspects of the jellyfish toxin include multi-organ damage caused by jellyfish stings.

[0009] Preferably, the multi-organ function damage includes myocardial fiber deformation, liver tissue cord structure rupture, vacuolar deformation accompanied by diffuse bleeding, dermal capillary dilation, collagen fiber separation and rupture, massive inflammatory cell infiltration and glomerular hemorrhage.

[0010] Preferably, the drug is a drug composition with baicalein as the sole active ingredient or contains baicalein.

[0011] Preferably, the medicine is a decoction, powder, pill, injection, mixture, oral ampoule, tablet, capsule, pellet or emulsion.

[0012] The invention also provides the use of baicalin in preparing medicine for preventing and or treating jellyfish stings.

[0013] Beneficial effects:

[0014] Through cell experiments, it was verified that baicalin can significantly increase the proliferation rate of RAW 264.7 cells and antagonize the level of apoptosis induced by jellyfish toxins; through mouse experiments, it was verified that jellyfish toxins were injected into the tail vein 30 minutes after baicalin administration, and the results showed that the survival rate of mice was significantly increased. Two weeks after oral administration of baicalin, pathological examination showed that the heart, liver and kidney of the baicalin intervention group showed significant relief and recovery compared with the TE group alone. The results of in vivo and in vitro experiments showed that baicalin provides a new theoretical basis for preventing or alleviating the poisoning symptoms and cell damage caused by jellyfish stings. In other words, the present invention also provides a new clinical treatment method for preventing or alleviating systemic poisoning reactions and cell damage caused by jellyfish stings, and provides a new research direction and idea for the development and application of traditional Chinese medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The results of the activity evaluation of 20 natural Chinese medicinal compounds on phospholipase A2 in Example 2;

[0016] Figure 2 The results of the evaluation of the cytotoxicity of 20 natural Chinese medicinal compounds on TE-induced RAW264.7 cells in Example 3;

[0017] Figure 3 It is the correlation analysis between PLA2 (phospholipase A2) activity and CCK8 cell activity;

[0018] Figure 4 The level of cell apoptosis induced by jellyfish toxin antagonized by baicalein in Example 4;

[0019] Figure 5 is the LD50 of TE of jellyfish causing death in mice 50 concentration;

[0020] Figure 6 is a graph showing the mortality rate of mice in Example 5;

[0021] Figure 7 Evaluation of blood biochemical indexes of mice in Example 6, where * is compared with the control group, # is compared with the TE group, * / #, P<0.05, ** / ##, P<0.01, *** / ###, P<0.005, **** / ####, P<0.001;

[0022] Figure 8 These are histopathological sections of the mouse heart, liver and kidney in Example 6. DETAILED DESCRIPTION

[0023] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0024] The instruments used in the embodiments of the present invention are as follows:

[0025] High-speed centrifuge, pipette, and Vortex-5 vortex vortexer were purchased from Eppendorf, Germany;

[0026] HH-501(A) super constant temperature water bath was purchased from Baita Xinbao Instrument Factory, Jintan District;

[0027] The carbon dioxide incubator (CLM-170B-8-CN) was purchased from Jiapo Yisi Technology Co., Ltd.;

[0028] The clean bench (W-CJ-D) was purchased from Zhengzhou North-South Instrument Equipment Co., Ltd.

[0029] The constant temperature water bath (DK-8B) was purchased from Shanghai Jinghong Experimental Equipment Co., Ltd.;

[0030] Analytical electronic balance FA1004 was purchased from Shanghai Hengping Scientific Instrument Co., Ltd.;

[0031] Fluorescence microscope Axio Vert.A1 was purchased from Zeiss Technology Co., Ltd.;

[0032] The flake ice machine SZB-20 was purchased from Shanghai Kuncheng Scientific Instrument Co., Ltd.

[0033] Example 1 Preparation of jellyfish toxin

[0034] Jellyfish were purchased from Shanghai Oceanarium, and the tentacle tissue of the jellyfish was cut off. The tentacle tissue was stirred and autolyzed at 4°C for 72 hours using a magnetic stirrer (speed of 3000g) until there was no obvious tissue block, and an autolysis mixture was obtained. The autolysis mixture was double-filtered through a 200-mesh sieve and centrifuged at 4°C, 1000g for 15 minutes; the supernatant was dialyzed in 1×PBS overnight, and the permeate was divided into 15mL centrifuge tubes and stored at -80°C for use to obtain jellyfish toxin (TE).

[0035] Example 220 Inhibitory Effects of 20 Natural Chinese Medicine Compounds on Jellyfish TE Phospholipase A2

[0036] Twenty natural Chinese medicine compounds were provided by the School of Pharmacy of Naval Medical University, and their names are shown in Table 1.

[0037] Table 1 Names of natural Chinese medicine compounds

[0038] serial number Chinese name English name 207 Morroniside Morroniside 210 Dehydrocostus lactone Dehydrocostus Lactone 212 Phytolaccaepa saponin A Esculentoside A 215 Cimicifuga glycosides Cimicifugoside 217 Carnosic acid Carnosicacid 219 Glycyrrhizin Tabersonine 222 Garcinia A Gambogicacid 225 Corydalis A Corydaline 232 Sesamolin Sesamolin 234 10-Deacetylpaclitaxel 10-Deacetyltaxol 237 Angloside C Angoroside C 244 Docetaxel Docetaxel 249 Cinobufacini Cinobufotalin 259 Junggar aconitine Songorine 264 Baicalin Baicalein 270 Rafenolactone Triptophenolide 284 Diacerein Diacerein 286 Ilex glabra Pedunculoside 295 Rebaudioside A RebaudiosideA 297 Benzoylpaeoniflorin Benzoylpaeoniflorin

[0039] Pipette 1 mL Tris-HCl (PH = 8), 19 mL distilled water, 117 mg NaCl and 22.2 mg CaCl 2 Prepare a 50mmol / L Tris-HCl solution; weigh 1mg NOBA and 1mL acetonitrile to prepare a 1mg / mL solution, where NOBA is used as a substrate to detect PLA2 activity. Set up NCl group, TE group, and drug administration group, and administer the drugs as shown in Table 2. Place the above mixture in a 37℃ incubator and incubate for 60min, and measure the absorbance of the sample at 405nm using an enzyme reader (n=3).

[0040] Table 2 Dosing table

[0041] Grouping Blank group (NCL group) TE group (model group) 0.3mM (administered group) TE(μL) 0 15 15 PBS (μL) 35 20 0 Natural compound (μL) 0 0 20 Tris-HCl (μL) 200 200 200 NOBA(μL) 15 15 15

[0042] Through the screening experiment of phospholipase A2 activity of 20 natural Chinese medicine compounds ( Figure 1 ), where the absorbance can indicate the activity of phospholipase A2, and it was found that 20 natural compounds all have an inhibitory effect on phospholipase A2.

[0043] Example 3 Baicalein improves the survival rate of TE-induced cells in jellyfish

[0044] Well-growing Raw264.7 cells were selected for cell experiments. Twenty natural Chinese medicine compounds were graded diluted to 100 μM, 60 μM, and 30 μM using serum-free DMEM. At the same time, jellyfish TE was diluted to an IC value of 12.46 μg / mL. 50 Concentration: 15 μL / well TE, 20 μL / well natural Chinese medicine compound monomers were mixed with DMEM to make 100 μL and added to each well, and incubated for 2 hours.

[0045] The CCK8 method was used to analyze the effects of natural compounds on the cytotoxicity caused by jellyfish TE. The group without venom was used as a negative control to eliminate the effect of DMEM. The sample addition table and culture process are shown in Table 3.

[0046] Table 3. Sample addition table for the determination of cytotoxicity of natural compounds against jellyfish toxins on Raw 264.7 cells

[0047]

[0048]

[0049] The calculation formula of cell viability is: cell viability (%) = (A experimental group - A blank group) / (A control group - A blank group) * 100%. The data were analyzed and processed using GraphPad Prism 8.0.2 software, and the drug administration group and TE group were normalized to draw a bar graph.

[0050] The CCK8 assay was used to further test 20 traditional Chinese medicine compounds to compare the effects of different natural traditional Chinese medicine compounds on the survival rate of macrophages induced by jellyfish toxins. Figure 2 It can be seen that the effect of baicalin Chinese medicine compound on macrophage survival rate is the most significant. The relationship between PLA2 (phospholipase A2) activity and CCK8 cell activity shows that ( Figure 3 ), baicalein can both inhibit PLA2 (phospholipase A2) activity and enhance cell activity, and it has the best effect among 20 natural Chinese medicine compounds. Therefore, baicalein was selected for subsequent experiments.

[0051] Example 4 Evaluation of the effect of baicalin on antagonizing apoptosis of jellyfish TE cells

[0052] RAW264.7 cells were seeded in 6-well plates and cultured for 24 hours until the cells evenly covered the culture plate. Subsequently, the cells were subjected to the following operations: the Control group was treated with DMEM, the TE group was treated with 16.24 μg / mL for 2 hours, the Baicalein group was administered with 60 μM baicalein for 2 hours, and the combined treatment group (TE-Baicalein group) was administered with 60 μM baicalein and TE 16.24 μg / mL for 2 hours. Cells were collected using PBS, and 5 μL AnnexinV-FITC and 10 μL PI were added to the cell suspension and mixed thoroughly. Flow cytometry was used to detect the apoptosis using the Yisheng Bio AnnexinV-FITC / PI apoptosis detection kit.

[0053] Depend on Figure 4 It can be seen that the level of cell apoptosis in the TE group was significantly higher than that in the Control group; there was no significant change in the level of cell apoptosis in the Baicalein group compared with the Control group, indicating that baicalein had no effect on cells; the level of cell apoptosis in the treatment group was significantly lower than that in the TE group, indicating that baicalein had an inhibitory effect on cell apoptosis induced by jellyfish toxins.

[0054] Example 5 Evaluation of the effect of baicalin on antagonizing jellyfish toxin-induced death in mice

[0055] ICR mice were purchased from Shanghai JESJ Biotechnology Co., Ltd. All were ICR male mice with an average weight of 20±2g. The mice were kept at 22-23°C, relative humidity of 55-60%, and a light / dark cycle of 12h with adequate feed and water.

[0056] All animal care and experiments were performed in strict accordance with the guidelines of the Animal Care and Use Committee of the Chinese Institute of Nutritional Sciences and approved by the Animal Experiment Ethics Committee of the Naval Medical University.

[0057] Baicalein was prepared into a 250mM stock solution using DMSO, and the same volume of baicalein stock solution and Tween-80 were diluted into 6, 10, and 30mg / kg dosing mixed groups using normal saline. The mice were injected with different concentrations of jellyfish toxin (0, 4.2, 6.3, 8.3, and 13.9mg / kg), and the time of death of the mice was recorded to plot the LD of the mice. 50 The concentration of 7 mg / kg was used in subsequent experiments, such as Figure 5 shown.

[0058] The control group, TE group, TE-BAI (6, 10, 30 mg / kg) group, and BAI (6, 10, 30 mg / kg) group were set up. The mice were randomly divided into 8 groups, with 12 mice in each group. The drugs were injected by tail vein injection, the time of death of the mice was recorded, and the survival curve of the mice was drawn, as shown in Figure 2. Figure 6As shown in the figure, all mice in the BAI (6, 10, 30 mg / kg) groups survived, and the curve overlapped with that of the Control group, and then did not appear in the figure. Figure 6 It can be seen that the antagonistic effect of 30 mg / kg baicalein injected into the tail vein can reach 75% compared with 6 mg / kg and 10 mg / kg. Baicalein can effectively improve the survival rate of mice after stinging.

[0059] Example 6 Determination of mouse blood biochemical indexes and analysis of heart, liver and kidney tissue pathological sections

[0060] The mice were randomly divided into 4 groups (Control group, TE group, TE-Baicalein (100 mg / kg) group and Baicalein group), with 6 mice in each group. Baicalein (100 mg / kg) was intragastrically administered to the mice in the TE-Baicalein (100 mg / kg) and Baicalein groups for two consecutive weeks. TE was injected into the tail vein of the TE group and TE-Baicalein (100 mg / kg) group, and the rest of the groups were injected with normal saline instead, and observed for 4-6 hours. 4-6 hours after the injection, whole blood was collected from the mouse orbit using a capillary tube soaked in sodium heparin and placed in a 1.5 mL centrifuge tube. The blood was immediately centrifuged at 4°C, 10,000 rpm for 15 min. The supernatant was collected and stored in a 4°C refrigerator and sent to Bolf Biotech for testing of blood biochemical indicators, including cardiac indicators: lactate dehydrogenase (LDH), aspartate aminotransferase (AST); liver indicators: creatine kinase isoenzyme MB (CK-MB), total bilirubin (TBIL), alanine aminotransferase (ALT), direct bilirubin (DBIL-Z); kidney indicators: urea nitrogen (BUN), creatinine (CR). Normal saline was the control group (n=4).

[0061] After whole blood was collected, mice in each group were deeply anesthetized with isoflurane and then euthanized. Heart, liver, and kidney tissues were preserved using 4% paraformaldehyde fixative (biological sharp agent, USA), and pathological section analysis was performed according to the following experimental method:

[0062] (1) Sampling and fixation: The collected heart, liver and kidney tissues were fixed in 4% paraformaldehyde fixative and stored at 4°C until use.

[0063] (2) Dehydration: Tissue blocks were removed from the 4% paraformaldehyde fixative, washed three times with PBS, and then dehydrated in 80%, 90%, 95%, and 100% ethanol solutions for 2 h.

[0064] (3) Transparency: After dehydration, absorb the alcohol on the surface of the tissue and then place the tissue block in pure xylene I and pure xylene II for 35 min until the tissue becomes transparent.

[0065] (4) Embedding: The processed tissue blocks are suspended in liquid paraffin and cut into thin slices after the paraffin is fully cooled and fixed. The slice thickness is set to 5 microns, and then the slices are placed in a 40°C water bath to unfold and attach to a slide, and finally placed in a 60°C oven to dry for 3 to 5 hours for later use.

[0066] (5) Dewaxing and dehydrating: Take out the slices and place the tissue blocks in pure xylene solution for 30 min each, then place them in anhydrous ethanol and 95%, 90%, 80% and 70% ethanol for dewaxing. The treatment time for each concentration is 5 min, and then rinse with double distilled water.

[0067] (6) Staining: This method uses the hematoxylin-eosin (HE) staining method. First, the treated slice samples are immersed in a hematoxylin solution for 5 minutes, purified in distilled water for 20 minutes, differentiated with 1% hydrochloric acid ethanol for 3 to 5 seconds, then washed in tap water for 20 minutes, rinsed with double distilled water to return to blue treatment, and then immersed in eosin solution for another 3 minutes. The samples are then immersed in 95% ethanol for 3 seconds and anhydrous ethanol for 3 minutes, and repeated twice to achieve the dehydration effect. Finally, they are treated with pure xylene I and pure xylene II solutions for 5 minutes each to make them transparent.

[0068] (7) Sealing: Seal the sections with neutral gum, observe under an electron microscope, and collect and analyze images.

[0069] Depend on Figure 7 As shown in the figure, after baicalein intervention, the cardiac function indexes LDH and AST, the liver function indexes CK-MB and TBIL, and the renal function indexes CR and BUN all showed a downward trend compared with the TE group alone, and had a certain statistical significance.

[0070] Depend on Figure 8 It can be seen that H&E staining of the heart, liver and kidney showed that histopathology showed that TE of jellyfish caused blurred myocardial cell structure, deformed myocardial fibers, disordered arrangement, obvious edema of myocardial cells, disappearance of some nuclei, myocardial dissolution and inflammatory cell infiltration. After antagonistic intervention by baicalin, the myocardial cell structure was arranged in an orderly manner and the myocardial structure was normal; the liver cells in the control group were arranged neatly, the liver cells were arranged radially along the central vein, the nuclei were intact and the boundaries were clear, and clear hepatic cords were visible. TE intervention caused blurred hepatocyte boundaries and broken liver tissue cord-like structures, while baicalin intervention made the liver cells arranged neatly and the nuclei were clearly visible; compared with the normal group of kidney cells, the TE group caused abnormal glomerular structure and vacuoles, while baicalin intervention made the glomerular structure intact and reduced TE-induced organ lesions. It can be seen that due to the complex composition of jellyfish toxin TE, it can cause multiple organ damage in mice, and baicalin can have an antagonistic effect on this damage, thereby improving the survival rate of mice.

[0071] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. The application of baicalin in the preparation of drugs for antagonizing the lethality of jellyfish toxins.

2. The use according to claim 1, characterized in that The lethal aspects of the jellyfish toxin include multiple organ damage caused by jellyfish stings.

3. The use according to claim 2, characterized in that The multiple organ function damage includes myocardial fiber deformation, liver tissue cord structure rupture, vacuolar deformation accompanied by diffuse bleeding, dermal capillary dilation, collagen fiber separation and rupture, a large number of inflammatory cell infiltration and glomerular hemorrhage.

4. The use according to claim 1, characterized in that The medicine is a medicine composition with baicalein as the sole active ingredient or containing baicalein.

5. The use according to claim 4, characterized in that The medicine is a decoction, powder, pill, injection, mixture, oral ampoule, tablet, capsule, pill or emulsion.

6. Use of baicalin in the preparation of medicines for preventing and or treating jellyfish stings.

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