Application of Epihevetide in preparation of related products for treating cerebral hemorrhage
By using Epiheveadride as a main ingredient, the problem of uncertain efficacy and thromboembolic risk of existing drugs for treating cerebral hemorrhage is solved, significantly alleviating atorvastatin-induced cerebral hemorrhage damage, providing a new drug candidate for treating cerebral hemorrhage , has broad market prospects.
Patent Information
- Application Number
- CN202411502658.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-25
- Publication Date
- 2025-01-10
AI Technical Summary
The efficacy of existing drugs for treating cerebral hemorrhage is uncertain and may lead to the risk of thromboembolic, and there is a lack of effective novel therapeutic drugs.
Products that use Epiheveadride as the main ingredient for the prevention or treatment of cerebral hemorrhage, verify their therapeutic effect on atorvastatin-induced spontaneous cerebral hemorrhage in zebrafish models, combined with pharmaceutically acceptable carriers or adjuvants , form health food, special medical food or medicine.
Epiheveadride significantly alleviates brain vascular damage induced by the HMG-CoA reductase inhibitor atorvastatin, has a significant effect on treating cerebral hemorrhage and has few toxic side effects, providing a new drug candidate for the treatment of cerebral hemorrhage.
Smart Images
Figure CN119280222A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to application of Epiheveadride in preparing products related to treating cerebral hemorrhage, and belongs to the technical field of biomedicine. Background Art
[0002] Intracerebral hemorrhage (ICH) refers to bleeding caused by vascular lesions, necrosis, and rupture in the brain, and is one of the common clinical diseases. Spontaneous cerebral hemorrhage is bleeding in the brain parenchyma caused by non-traumatic reasons. As a common disease, spontaneous cerebral hemorrhage has the characteristics of rapid onset, rapid changes, high disability rate, and high mortality rate. It is one of the main diseases that endanger human health. Relevant data show that spontaneous cerebral hemorrhage accounts for 10%-30% of all strokes, with a mortality rate as high as 30%-50%, and survivors often have severe neurological dysfunction. At present, the drugs for the treatment of cerebral hemorrhage mainly include recombinant VIIa, aminocaproic acid or tranexamic acid. However, since the clinical efficacy of the above-mentioned hemostatic drugs in the treatment of cerebral hemorrhage is not yet very certain, they often have the risk of causing thromboembolism. It is very necessary to develop new drugs or products related to the treatment of cerebral hemorrhage.
[0003] Zebrafish is a new vertebrate model organism that has emerged in recent years. It has many characteristics, such as small size, in vitro fertilization, in vitro development, and full body transparency in early development. At the genetic level, zebrafish has up to 87% homology with humans, and its cerebrovascular and nervous systems are also very similar to humans in physiological structure and function. Because the vascular structure of zebrafish is easy to observe and the genes and drug targets are relatively conservative, it has become an ideal animal model in the field of cardiovascular and cerebrovascular diseases and related drug research and development.
[0004] Statins such as atorvastatin (ATV) are inhibitors of HMG-CoA reductase, which can interfere with the expression levels of VE-cadherin and occludin proteins, thereby reducing the number of intercellular junctions between vascular endothelial cells (VECs), destroying the integrity of the vascular wall, and leading to non-traumatic bleeding. ATV-induced spontaneous cerebral hemorrhage in zebrafish embryos is a well-established model of spontaneous cerebral hemorrhage for studying cerebrovascular protection and vascular stability.
[0005] Epiheveadride is an oxidized derivative of the nonsteroidal compound dihydroepiheveadride. Studies have found that it has certain antifungal activity and has excellent medicinal potential. However, the current research on Epiheveadride in cardiovascular and cerebrovascular aspects is still very limited, especially the research using the treatment of cerebral hemorrhage as an evaluation indicator, which has not yet been reported. Summary of the invention
[0006] In view of the deficiencies in the prior art, the present invention provides an application of Epiheveadride in the preparation of products related to the treatment of cerebral hemorrhage.
[0007] The technical solution of the present invention is as follows:
[0008] The use of Epiheveadride in the preparation of products related to the prevention or treatment of cerebral hemorrhage, wherein the chemical structure of Epiheveadride is shown in Formula I:
[0009]
[0010] Preferably according to the present invention, the products related to the prevention or treatment of cerebral hemorrhage include health foods, special medical foods, and medicines.
[0011] Further preferably, the drug contains one or more pharmaceutically acceptable carriers or adjuvants.
[0012] Further preferably, the auxiliary agent is at least one of a sustained-release agent, a filler, a binder, a wetting agent, a disintegrant, an absorption enhancer, a surfactant or a lubricant.
[0013] Preferably according to the present invention, the cerebral hemorrhage is spontaneous cerebral hemorrhage.
[0014] Further preferably, the cerebral hemorrhage is spontaneous cerebral hemorrhage induced by statins.
[0015] More preferably, the statin is atorvastatin.
[0016] A product for preventing or treating cerebral hemorrhage, wherein the active ingredient comprises Epiheveadride, and the chemical structural formula of the Epiheveadride is shown in Formula I.
[0017] Preferably according to the present invention, the products related to the prevention or treatment of cerebral hemorrhage include health foods, special medical foods, and medicines.
[0018] Further preferably, the drug contains one or more pharmaceutically acceptable carriers or adjuvants.
[0019] Further preferably, the auxiliary agent is at least one of a sustained-release agent, a filler, a binder, a wetting agent, a disintegrant, an absorption enhancer, a surfactant or a lubricant.
[0020] Preferably according to the present invention, the cerebral hemorrhage is spontaneous cerebral hemorrhage.
[0021] Further preferably, the cerebral hemorrhage is spontaneous cerebral hemorrhage induced by statins.
[0022] More preferably, the statin is atorvastatin.
[0023] The beneficial effects of the present invention are as follows:
[0024] The present invention first discovered that Epiheveadride has the effect of treating cerebral hemorrhage. The present invention uses zebrafish as experimental animals for detection and finds that Epiheveadride can effectively reduce brain vascular damage caused by the HMG-CoA reductase inhibitor atorvastatin (ATV), has a significant effect of treating cerebral hemorrhage in the body, and has little toxic and side effects. The present invention can provide a candidate for drugs for treating cerebral hemorrhage and has broad market prospects. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] Figure 1 Testing photos for Epiheveadride safety evaluation;
[0026] In the figure: 0 represents the blank control group, 10 represents the 10 μM concentration of Epiheveadride treated group; 20 represents the 20 μM concentration of Epiheveadride treated group; 40 represents the 40 μM concentration of Epiheveadride treated group.
[0027] Figure 2 These are photos of brain hemorrhage in zebrafish treated with Epiheveadride (low-dose group, medium-dose group, and high-dose group);
[0028] In the figure: 0 represents the blank control group, ATV represents the modeling group, 10 represents the 10 μM concentration Epiheveadride treatment group; 20 represents the 20 μM concentration Epiheveadride treatment group; 40 represents the 40 μM concentration Epiheveadride treatment group.
[0029] Figure 3 This is a statistical bar graph of brain hemorrhage area after zebrafish were treated with Epiheveadride (low-dose group, medium-dose group, and high-dose group);
[0030] In the figure: 0 represents the blank control group, ATV represents the modeling group, 10 represents the 10 μM concentration Epiheveadride treatment group; 20 represents the 20 μM concentration Epiheveadride treatment group; 40 represents the 40 μM concentration Epiheveadride treatment group;
[0031] ### indicates P<0.001 compared with the blank control group; *** indicates P<0.001 compared with ATV.
[0032] Figure 4 The photos of brain hemorrhage in zebrafish treated with 40μM concentration of Epiheveadride and 10μM concentration of valsartan (VST) were shown;
[0033] In the figure: 0 represents the blank control group, ATV represents the modeling group, VST represents the 10 μM valsartan treatment group, and 40 represents the 40 μM Epiheveadride treatment group.
[0034] Figure 5 This is a statistical bar graph of brain hemorrhage area after zebrafish were treated with 40μM concentration of Epiheveadride and 10μM concentration of valsartan;
[0035] In the figure: 0 represents the blank control group, ATV represents the modeling group, VST represents the 10 μM valsartan treatment group, 40 represents the 40 μM Epiheveadride treatment group; aaa represents P<0.001 compared with the blank control group; bbb represents P<0.001 compared with the ATV group, bbb represents P<0.001 compared with the ATV group, and c represents P<0.05 compared with the VST group. DETAILED DESCRIPTION
[0036] The technical solution of the present invention is further described below in conjunction with the embodiments and the accompanying drawings of the specification, but the protection scope of the present invention is not limited thereto.
[0037] The contents without specific conditions in the examples were carried out under conventional conditions; the reagents or instruments used without indicating the manufacturer were all common commercially available products.
[0038] Epiheveadride can be prepared according to existing methods, such as referring to the method provided in the document "A Pair of New SpirocyclicAlkaloid Enantiomers with TrxR Inhibitory Activities Were Isolated from Marine-Derived Aspergillus ruber TX-M4-1" (WANG Cong, WANG Yufei, etc., 2023).
[0039] Reagent preparation:
[0040] The composition of embryo culture water is as follows:
[0041] CaCl 2 0.4 mM, NaCl 5 mM, MgSO 4 0.16mM, KCl 0.17mM, prepared in deionized water.
[0042] Experimental Example 1
[0043] Safety evaluation of Epiheveadride
[0044] 1. Zebrafish sample pretreatment:
[0045] Healthy and sexually mature AB zebrafish were placed in a mating tank at a ratio of 1:1 between male and female, with a partition placed in the middle, and placed in a dark environment. The partition was removed before the lights were turned on the next day, and light stimulation was used to stimulate ovulation. The adult fish were removed half an hour after ovulation, and the ovulation time was controlled within half an hour to reduce the difference in development time between embryos. Fertilized eggs were collected, disinfected and cleaned; then the fertilized eggs were moved into clean zebrafish embryo culture water, and 0.2ppm methylene blue was added. The culture was controlled for 12 hours at 28°C and a 14h light / 10h dark cycle.
[0046] The composition of embryo culture water is as follows:
[0047] CaCl2 0.4mM, NaCl 5mM, MgSO 4 0.16mM, KCl 0.17mM, prepared in deionized water.
[0048] 2. Epiheveadride treatment:
[0049] The zebrafish larvae described in step 1 were randomly divided into a blank control group and an Epiheveadride treatment group (the treatment groups were divided into a low-dose group, a medium-dose group, and a high-dose group), with 3 parallel wells in each group and 15 fish in each well. The following steps were performed: the embryo culture water in the 12-well plate was removed, 2 mL of embryo culture water was added to the control group, 2 mL of embryo culture water containing 10 μM Epiheveadride was added to the Epiheveadride treatment group (low-dose group), 2 mL of embryo culture water containing 20 μM Epiheveadride was added to the Epiheveadride treatment group (medium-dose group), and 2 mL of embryo culture water containing 40 μM Epiheveadride was added to the Epiheveadride treatment group (high-dose group), and the culture was carried out in a 28°C constant temperature incubator for 72 hours.
[0050] 3. Epiheveadride safety evaluation test:
[0051] The two groups of zebrafish were placed in a tricaine solution with a mass concentration of 0.02% to anesthetize them. Images of 24hpf, 48hpf, and 72hpf zebrafish were collected under a microscope, and the hatching rate and mortality rate were calculated.
[0052] The experimental results are as follows Figure 1As shown, compared with the blank control group, the Epiheveadride-treated groups (low-dose group, medium-dose group, and high-dose group) had not hatched at 24hpf, the mortality rate was 0%, and there was no deformity; the Epiheveadride-treated groups (low-dose group, medium-dose group, and high-dose group) had a hatching rate of 100% at 48hpf, a mortality rate of 0%, and no deformity; the Epiheveadride-treated groups (low-dose group, medium-dose group, and high-dose group) had a hatching rate of 100% at 72hpf, a mortality rate of 0%, and no deformity, which indicates that 10, 20, and 40 μM Epiheveadride have no developmental toxicity to zebrafish embryos and are all within the safe concentration range.
[0053] Example 1
[0054] The therapeutic effect of different concentrations of Epiheveadride on cerebral hemorrhage in zebrafish
[0055] 1. Zebrafish sample pretreatment:
[0056] Healthy and sexually mature AB zebrafish were placed in a mating tank at a ratio of 1:1 between male and female, with a partition placed in the middle, and placed in a dark environment. The partition was removed before the lights were turned on the next day, and light stimulation was used to stimulate ovulation. The adult fish were removed half an hour after ovulation, and the ovulation time was controlled within half an hour to reduce the difference in development time between embryos. Fertilized eggs were collected, disinfected and cleaned; then the fertilized eggs were moved into clean zebrafish embryo culture water, and 0.2ppm methylene blue was added. The zebrafish embryos were cultured under light control for 24 hours at 28℃ and a 14h light / 10h dark cycle. The zebrafish embryos 24 hours after birth were de-egged with a 1mg / mL concentration of pronase E solution, and the residual pronase E in the zebrafish embryos was rinsed with fish water.
[0057] 2. Epiheveadride treatment:
[0058] The zebrafish embryos described in step 1 were randomly divided into a blank control, an ATV modeling group, and an Epiheveadride treatment group (the treatment groups were divided into a low-dose group, a medium-dose group, and a high-dose group), with 3 parallel wells in each group and 10 fish in each well. The embryo culture water in the 12-well plate was removed, and 2 mL of embryo culture water was added to the control group and cultured in a 28°C constant temperature incubator for 24 hours; ATV with a final concentration of 1.25 μM was added to the 2 mL of embryo culture water added to the modeling group, and cultured in a 28°C constant temperature incubator for 24 hours; 2 mL of embryo culture water was added to the Epiheveadride treatment group (low-dose group) ATV and 10 μM Epiheveadride were added to the culture water at a final concentration of 1.25 μM. ATV and 20 μM Epiheveadride were added to 2 mL of embryo culture water in the Epiheveadride treatment group (medium-dose group). ATV and 40 μM Epiheveadride were added to 2 mL of embryo culture water in the Epiheveadride treatment group (high-dose group). Culture was continued in a constant temperature incubator at 28°C for 24 hours.
[0059] 3. Statistics of zebrafish brain hemorrhage area:
[0060] The five groups of zebrafish were placed in a tricaine solution with a mass concentration of 0.02% to anesthetize them. Zebrafish brain hemorrhage images were collected under a microscope. Based on the collected images, Image-Pro Plus6.0 software was used to count the area of brain hemorrhage in each group of zebrafish. GraphPad Prism 8.0.2 software was used to process the data.
[0061] The experimental results are as follows Figure 2 , Figure 3 As shown in the figure, compared with the blank control group, the zebrafish brain in the modeling group had obvious hemorrhage, and it was extremely statistically significant, which indicated that ATV caused zebrafish cerebral hemorrhage and the modeling was successful. However, compared with the modeling group, after treatment with Epiheveadride (low-dose group, medium-dose group, and high-dose group), the hemorrhage area in the zebrafish brain was significantly reduced, and it was extremely statistically significant; after treatment with the medium-dose group of Epiheveadride, the hemorrhage area in the zebrafish brain was significantly smaller than that in the low-dose treatment group of Epiheveadride, and after treatment with the high-dose group of Epiheveadride, the hemorrhage area in the zebrafish brain was significantly smaller than that in the medium-dose treatment group of Epiheveadride, which indicated that different concentrations of Epiheveadride could effectively reduce ATV-induced zebrafish cerebral hemorrhage, and the therapeutic effect showed dose-dependency, and within a certain range, it increased with the increase of dose.
[0062] Comparative Example 1
[0063] Comparison of the protective activities of Epiheveadride and valsartan (VST) against brain hemorrhage in zebrafish
[0064] 1. The pretreatment of zebrafish samples was the same as in Example 1.
[0065] 2. Epiheveadride and valsartan (VST) treatment of zebrafish:
[0066] The zebrafish embryos described in step 1 were randomly divided into blank control, modeling, valsartan (VST) and Epiheveadride treatment groups, with 3 parallel wells in each group and 10 strips in each well. The embryo culture water in the 12-well plate was removed, and 2 mL of embryo culture water was added to the blank control group and cultured in a 28°C constant temperature incubator for 24 hours; ATV with a final concentration of 1.25 μM was added to the 2 mL of embryo culture water added to the modeling group; VST with a final concentration of 10 μM and ATV with a final concentration of 1.25 μM were added to the 2 mL of embryo culture water added to the VST group, and cultured in a 28°C constant temperature incubator for 24 hours; Epiheveadride with a final concentration of 40 μM and ATV with a final concentration of 1.25 μM were added to the 2 mL of embryo culture water added to the Epiheveadride treatment group, and cultured in a 28°C constant temperature incubator for 24 hours.
[0067] 3. The statistics of zebrafish brain hemorrhage area are the same as those in Example 1.
[0068] The experimental results are as follows Figure 4 , Figure 5 As shown, compared with the blank control group, the zebrafish brain in the modeling group had obvious hemorrhage, and it was extremely statistically significant. This shows that ATV caused cerebral hemorrhage in zebrafish and the modeling was successful. Compared with the modeling group, the hemorrhage area of the zebrafish brain was significantly reduced after treatment with VST and Epiheveadride, and it was statistically significant. Among them, the hemorrhage area of the zebrafish brain in the Epiheveadride treatment group was significantly smaller than that in the VST treatment group, and it was statistically significant. This shows that compared with VST, which is known to have the activity of treating cerebral hemorrhage, Epiheveadride has stronger activity in treating cerebral hemorrhage.
[0069] The present invention discovers for the first time that Epiheveadride has the effect of treating cerebral hemorrhage, and compared with the known valsartan (VST) with the activity of treating cerebral hemorrhage, Epiheveadride has stronger activity of treating cerebral hemorrhage. The present invention can provide a candidate for the drug of treating cerebral hemorrhage, and has broad market prospects.
Claims
1. Application of Epiheveadride in the preparation of products related to the prevention or treatment of cerebral hemorrhage, wherein the chemical structural formula of Epiheveadride is shown in Formula I:
2. The use according to claim 1, characterized in that The products related to the prevention or treatment of cerebral hemorrhage include health foods, special medical foods, and medicines.
3. The use according to claim 2, characterized in that The medicine contains one or more pharmaceutically acceptable carriers or adjuvants.
4. The use according to claim 3, characterized in that The auxiliary agent is at least one of a sustained-release agent, a filler, a binder, a wetting agent, a disintegrant, an absorption promoter, a surfactant or a lubricant.
5. The use according to claim 1, characterized in that The cerebral hemorrhage is spontaneous cerebral hemorrhage.
6. The use according to claim 5, characterized in that The cerebral hemorrhage is spontaneous cerebral hemorrhage induced by statins.
7. The use according to claim 6, characterized in that The statin is atorvastatin.
8. A product for preventing or treating cerebral hemorrhage, characterized in that: The active ingredient includes Epiheveadride, and the chemical structural formula of the Epiheveadride is shown as Formula I in claim 1.
9. The related product according to claim 8, characterized in that: The products related to the prevention or treatment of cerebral hemorrhage include health foods, special medical foods, and medicines; Preferably, the drug contains one or more pharmaceutically acceptable carriers or adjuvants; Preferably, the auxiliary agent is at least one of a sustained-release agent, a filler, a binder, a wetting agent, a disintegrant, an absorption enhancer, a surfactant or a lubricant.
10. The related product according to claim 8, characterized in that: The cerebral hemorrhage is spontaneous cerebral hemorrhage; Preferably, the cerebral hemorrhage is spontaneous cerebral hemorrhage induced by statins; Preferably, the statin is atorvastatin.