Application of baicalin in the preparation of drugs that antagonize lethality from jellyfish toxins

Abstract

This invention provides the application of baicalin in the preparation of drugs antagonizing jellyfish toxin lethality, belonging to the field of pharmaceutical technology. Experiments have verified that baicalin can alleviate multi-organ dysfunction caused by jellyfish stings. Cell experiments show that baicalin can significantly increase the proliferation rate of RAW 264.7 cells and antagonize jellyfish toxin-induced apoptosis; mouse experiments show that baicalin can significantly improve mouse survival rate and alleviate the damage to the heart, liver, and kidneys caused by jellyfish toxin.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to the application of baicalin in the preparation of drugs that antagonize lethality from jellyfish toxins. Background Technology

[0002] With the explosive growth of jellyfish populations, jellyfish stings are becoming increasingly common, affecting tens of thousands of people annually. Mild stings may cause allergic reactions, while severe stings can suppress the central nervous and respiratory systems, leading to multiple organ dysfunction and death. Jellyfish venom is complex in composition, diverse in toxicity, and varies in potency, posing significant challenges to the diagnosis and treatment of jellyfish stings. Therefore, how to counteract the lethal effects of jellyfish venom remains a key research focus and hot topic.

[0003] Scutellaria baicalensis, the dried root of the plant Scutellaria baicalensis (family Lamiaceae), has a long history of medicinal use. It is bitter and cold in nature, and enters the lung, gallbladder, small intestine, large intestine, and spleen meridians. It has the effects of clearing heat and drying dampness, purging fire and detoxifying, stopping bleeding and calming the fetus. Clinically, it is often used for various conditions such as damp-heat syndrome, jaundice, cough due to lung heat, and hematemesis due to blood heat. Scutellaria baicalensis is widely distributed in Hubei, Henan, Shandong, Jiangsu, and other regions, providing a sufficient medicinal base. Baicalein is one of the most abundant flavonoid components in Scutellaria baicalensis. Studies have shown that Scutellaria baicalensis has a protective effect on the cardiovascular system, protecting cardiomyocytes through multiple pathways and targets, and protecting the cardiovascular system 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. However, skin damage and organ dysfunction are two completely different symptoms of poisoning, and baicalin and baicalein are two different compounds with certain differences in their efficacy. Therefore, it cannot be determined whether baicalin has the effect of improving organ dysfunction caused by jellyfish toxins.

[0004] Baicalein can improve heart rate, ST segment, and cardiac coefficient, and reduce oxidative stress, inflammation, and apoptosis levels, thereby improving myocardial ischemia. It can also inhibit inflammatory cytokines and chemokines, scavenging reactive oxygen species and improving antioxidant status. Furthermore, baicalein can combat various viruses, including influenza virus, HIV, hepatitis B virus, and measles virus. In other words, baicalein possesses good pharmacological activity with almost no serious toxic side effects, showing broad application prospects. Therefore, this invention attempts to use baicalein to antagonize jellyfish toxins, aiming to provide an effective treatment for the prevention and / or treatment of jellyfish stings, offering new insights into the clinical application of baicalein, and providing a new research direction for the application of traditional Chinese medicine. Summary of the Invention

[0005] The purpose of this invention is to provide the application of baicalin in the preparation of drugs that antagonize lethality from jellyfish toxins.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides the application of baicalin in the preparation of drugs that antagonize lethality from jellyfish toxins.

[0008] Preferably, the lethal aspect of the jellyfish toxin includes multi-organ dysfunction caused by jellyfish stings.

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

[0010] Preferably, the drug is a pharmaceutical composition containing baicalein as the sole active ingredient.

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

[0012] The present invention also provides the use of baicalin in the preparation of drugs for the prevention and / or treatment of jellyfish stings.

[0013] Beneficial effects:

[0014] Cellular experiments verified that baicalin significantly increased the proliferation rate of RAW 264.7 cells and antagonized jellyfish toxin-induced apoptosis. Mouse experiments confirmed that tail vein injection of jellyfish toxin 30 minutes after baicalin administration significantly increased mouse survival rates. Two weeks after gavage administration of baicalin, pathological examination showed that the baicalin intervention group exhibited significant relief and recovery in the heart, liver, and kidneys compared to the TE group alone. These in vitro and in vivo results provide new theoretical basis for the prevention or alleviation of jellyfish sting-induced poisoning symptoms and cell damage. In other words, this invention provides a new clinical treatment method for preventing or alleviating systemic poisoning and cell damage caused by jellyfish stings, offering new research directions and ideas for the development and application of traditional Chinese medicine. Attached Figure Description

[0015] Figure 1 The results of the activity evaluation of phospholipase A2 by 20 natural Chinese herbal medicine compounds in Example 2 are shown.

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

[0017] Figure 3Correlation analysis of PLA2 (phospholipase A2) activity and CCK8 cell activity;

[0018] Figure 4 The level of apoptosis induced by baicalin antagonizing jellyfish toxin in Example 4;

[0019] Figure 5 LD50 of jellyfish TE-induced death in mice 50 concentration;

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

[0021] Figure 7 For the evaluation of mouse blood biochemical indicators in Example 6, among which This is a comparison with the control group; # indicates a comparison with the TE group. / #,P<0.05, / ##,P<0.01, / ###,P<0.005, / ####,P<0.001;

[0022] Figure 8 The images show pathological sections of mouse heart, liver, and kidney tissues from Example 6. Detailed Implementation

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

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

[0025] High-speed centrifuges, pipettes, and Vortex-5 vortex mixers were purchased from Eppendorf GmbH, Germany.

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

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

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

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

[0030] The analytical electronic balance FA1004 was purchased from Shanghai Hengping Scientific Instruments Co., Ltd.

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

[0032] The SZB-20 snowflake ice maker was purchased from Shanghai Kuncheng Scientific Instruments Co., Ltd.

[0033] Example 1: Preparation of jellyfish toxin

[0034] Jellyfish were purchased from the Shanghai Ocean Aquarium. Tentacle tissue was cut off and autolyzed using a magnetic stirrer (3000g) at 4°C for 72 hours until no obvious tissue fragments remained, yielding an autolysate mixture. The autolysate mixture was double-filtered through a 200-mesh sieve and centrifuged at 1000g for 15 minutes at 4°C. The supernatant was dialyzed overnight in 1×PBS. The permeate was aliquoted into 15mL centrifuge tubes and stored at -80°C for later use, yielding jellyfish toxin (TE).

[0035] Example 2: Inhibitory effect of 20 natural Chinese herbal medicine compounds on TE phospholipase A2 in jellyfish

[0036] The 20 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 medicinal compounds

[0038]

[0039] Prepare a 50 mmol / L Tris-HCl solution by mixing 1 mL Tris-HCl (pH=8), 19 mL distilled water, 117 mg NaCl, and 22.2 mg CaCl2. Prepare a 1 mg / mL solution by mixing 1 mg NOBA with 1 mL acetonitrile, where NOBA is used as a substrate for detecting PLA2 activity. Set up NCl group, TE group, and drug administration group, and administer the drugs as shown in Table 2. Incubate the above mixtures in a 37℃ incubator for 60 min, and measure the absorbance of the samples at 405 nm using a microplate reader (n=3).

[0040] Table 2 Dosing Table

[0041]

[0042] Screening experiments were conducted on the phospholipase A2 activity of 20 natural Chinese herbal medicine compounds. Figure 1 The absorbance level can indicate the activity of phospholipase A2, and 20 natural compounds were found to inhibit phospholipase A2.

[0043] Example 3: Baicalin enhances TE-induced cell survival rate in jellyfish.

[0044] Raw264.7 cells with good growth were selected for cell experiments. Twenty natural Chinese herbal medicine compounds were serially diluted to 100 μM, 60 μM, and 30 μM using serum-free DMEM. Meanwhile, jellyfish TE was diluted to an IC50 concentration of 12.46 μg / mL. 50 Concentration. Natural Chinese herbal compound monomers were mixed with DMEM at a concentration of 15 μL / well TE and 20 μL / well to make 100 μL, and added to each well. The mixture was incubated for 2 h.

[0045] The CCK8 assay was used to analyze the effect of natural compounds on TE-induced cytotoxicity in jellyfish. The group without venom served as a negative control to eliminate the influence of DMEM. The sample loading table and culture process are shown in Table 3.

[0046] Table 3. Sample loading table for the assay of natural compound antagonism of jellyfish toxin against Raw 264.7 cytotoxicity.

[0047]

[0048] The formula for calculating cell viability is: Cell viability (%) = (Experimental group A - Blank group A) / (Control group A - Blank group A) 100%. Data were analyzed and processed using GraphPad Prism 8.0.2 software, and bar charts were plotted after normalization of the drug-treated group and the TE group.

[0049] Further experiments were conducted using the CCK8 assay to investigate the effects of 20 traditional Chinese medicine compounds on the survival rate of macrophages induced by jellyfish toxin. Figure 2 It can be seen that baicalin, a traditional Chinese medicine compound, has the most significant effect on macrophage survival. 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, making it the most effective among 20 natural Chinese herbal compounds. Therefore, baicalein was selected for subsequent experiments.

[0050] Example 4 Evaluation of the antagonistic effect of baicalin on apoptosis in jellyfish TE cells

[0051] RAW264.7 cells were seeded in 6-well plates and cultured for 24 h until the cells uniformly confined the culture plates. Subsequently, the cells underwent the following treatments: the Control group was treated with DMEM, the TE group was treated with 16.24 μg / mL for 2 h, the Baicalein group was treated with 60 μM baicalein for 2 h, and the combined treatment group (TE-Baicalein group) received both 60 μM baicalein and TE 16.24 μg / mL for 2 h. Cells were collected using PBS, and 5 μL of Annexin V-FITC and 10 μL of PI were added to the cell suspension and mixed thoroughly. Flow cytometry was performed using the Annexin V-FITC / PI apoptosis detection kit from Yisheng Biotechnology.

[0052] Depend on Figure 4 It can be seen that the apoptosis level in the TE group was significantly higher than that in the Control group; the apoptosis level in the Baicalein group was not significantly different from that in the Control group, indicating that baicalein does not affect cells; the apoptosis level in the treatment group was significantly lower than that in the TE group, indicating that baicalein has an inhibitory effect on apoptosis induced by jellyfish toxin.

[0053] Example 5 Evaluation of the mortality induced in mice by baicalin antagonizing jellyfish toxin

[0054] ICR mice were purchased from Shanghai Jiesijie Biotechnology Co., Ltd. All were male ICR mice with an average weight of 20±2g. The mice were kept at 22-23℃ with a relative humidity of 55-60% and a 12-hour light / dark cycle, and were provided with sufficient food and water.

[0055] All animal care and experiments were conducted 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.

[0056] Baicalein was prepared into a 250 mM stock solution using DMSO. An equal volume of the baicalein stock solution and Tween-80 was diluted with physiological saline to create dosing mixtures of 6, 10, and 30 mg / kg. Mice were injected with different concentrations of jellyfish toxin (0, 4.2, 6.3, 8.3, and 13.9 mg / kg), and the time of death was recorded to obtain the LD50 for mice. 50 A concentration of 7 mg / kg was used for subsequent experiments, such as... Figure 5 As shown.

[0057] The following groups were set up: Control group, TE group, TE-BAI (6, 10, 30 mg / kg) group, and BAI (6, 10, 30 mg / kg) group. Mice were randomly divided into 8 groups of 12 each. Administered the drugs via tail vein injection. Mice mortality times were recorded, and mouse survival curves were plotted. Figure 6As shown, all mice in the BAI (6, 10, 30 mg / kg) groups survived, and their curves overlapped with those of the Control group, thus not appearing in the graph. Figure 6 It is known that baicalein administered via tail vein at a dose of 30 mg / kg has the best antagonistic effect, reaching 75%, compared to 6 mg / kg and 10 mg / kg. Baicalein can effectively improve the survival rate of mice after stings.

[0058] Example 6: Determination of blood biochemical indicators and histopathological analysis of heart, liver and kidney tissue sections in mice.

[0059] Mice were randomly divided into four groups (Control group, TE group, TE-Baicalein (100 mg / kg) group, and Baicalein group), with six mice in each group. Mice in the TE-Baicalein (100 mg / kg) group and the Baicalein group were administered baicalein (100 mg / kg) by gavage for two consecutive weeks. Mice in the TE group and the TE-Baicalein (100 mg / kg) group were injected with TE via tail vein. The other groups were administered physiological saline instead. Mice were observed for 4–6 hours. Four to six hours after injection, whole blood was collected from the orbital region of mice using capillary tubes soaked in heparin sodium and placed in 1.5 mL centrifuge tubes. The tubes were immediately centrifuged at 10,000 rpm for 15 min at 4°C. The supernatant was collected, stored at 4°C, and sent to Bolf Biotechnology Co., Ltd. for blood biochemical analysis, including cardiac indicators: lactate dehydrogenase (LDH) and aspartate aminotransferase (AST); liver indicators: creatine kinase isoenzyme MB (CK-MB), total bilirubin (TBIL), alanine aminotransferase (ALT), and direct bilirubin (DBIL-Z); and kidney indicators: blood urea nitrogen (BUN) and creatinine (CR). Physiological saline served as the control group (n=4).

[0060] After whole blood collection, 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 methods:

[0061] (1) Sampling and fixation: The heart, liver and kidney tissues collected were fixed in 4% paraformaldehyde fixative and stored at 4°C for later use.

[0062] (2) Dehydration: The tissue blocks were removed from the 4% paraformaldehyde fixative, washed three times with PBS, and then dehydrated in ethanol solutions of 80%, 90%, 95% and 100% concentrations for 2 hours.

[0063] (3) Transparency: After dehydration, the alcohol on the surface of the tissue is absorbed, and then the tissue block is placed in pure xylene I and pure xylene II for 35 minutes until the tissue becomes transparent.

[0064] (4) Embedding: The prepared tissue block is suspended in liquid paraffin. After the paraffin has cooled and solidified, it is cut into thin sections. The section thickness is set to 5 micrometers. Then, the sections are placed in a 40°C water bath to unfold and attach to a glass slide. Finally, they are placed in a 60°C oven to dry for 3-5 hours for later use.

[0065] (5) Dewaxing and dehydration: Take out the sections and put the tissue blocks into pure xylene solution for 30 min each. Then, put them into anhydrous ethanol and ethanol with concentrations of 95%, 90%, 80% and 70% for dewaxing. The treatment time for each concentration is 5 min. Then rinse with double distilled water.

[0066] (6) Staining: The hematoxylin-eosin (HE) staining method was used. First, the treated section sample was immersed in hematoxylin solution for 5 min, purified in distilled water for 20 min, differentiated with 1% hydrochloric acid ethanol for 3-5 s, then washed in tap water for 20 min, rinsed with double distilled water for blueing treatment, then immersed in eosin solution for 3 min, then immersed in 95% ethanol for 3 s and anhydrous ethanol for 3 min in sequence, repeated twice to achieve dehydration effect, and finally treated with pure xylene I and pure xylene II solutions for 5 min each for clearing treatment.

[0067] (7) Mounting: Mount the slides with neutral resin, observe them under an electron microscope, and collect and analyze the images.

[0068] Depend on Figure 7 As shown, the cardiac function indicators LDH and AST, liver function indicators CK-MB and TBIL, and kidney function indicators CR and BUN after baicalein intervention all showed a downward trend compared with the TE group alone, and these results were statistically significant.

[0069] Depend on Figure 8 H&E staining of the heart, liver, and kidneys revealed that histopathological examination showed that jellyfish TE caused blurred cardiomyocyte structure, deformed and disordered myocardial fibers, significant myocardial cell edema, loss of some cell nuclei, myocardial lysis, and inflammatory cell infiltration. After intervention with baicalin antagonism, the cardiomyocyte structure was restored to an orderly arrangement, and the myocardial structure returned to normal. In the control group, hepatocytes were neatly arranged, radiating along the central vein, with intact nuclei and clear boundaries, and clearly visible hepatic cords. TE intervention caused blurred hepatocyte boundaries and broken cord-like structures in the liver tissue, while baicalin intervention restored neat hepatocyte arrangement and clearly visible nuclei. Compared to the normal group, the TE group caused abnormal glomerular structure and vacuolation, while baicalin intervention restored the glomerular structure and reduced TE-induced organ damage. Therefore, because jellyfish toxicity (TE) has complex components that can cause multi-organ damage in mice, and baicalin can antagonize this damage, thereby improving the survival rate of mice.

[0070] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of baicalein as the sole active ingredient in the preparation of drugs that antagonize lethality from jellyfish toxins.

2. The application as described in claim 1, characterized in that, The lethality of jellyfish toxins includes multi-organ dysfunction caused by jellyfish stings.

3. The application as described in claim 2, characterized in that, The multi-organ dysfunction includes myocardial fiber deformation, rupture of the cord-like structure of liver tissue, and glomerular hemorrhage.

4. The application as described in claim 3, characterized in that, The drug is in the form of decoction, powder, pill, injection, mixture, tablet, capsule or emulsion.

5. Application of baicalin as the sole active ingredient in the preparation of drugs for the prevention and / or treatment of jellyfish stings.

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