Intestinal tract hemostasis composition as well as preparation method and application thereof

Abstract

The invention discloses an intestinal tract hemostatic composition as well as a preparation method and application thereof, and relates to the field of hemostatic drugs. Wherein the catechin, the hyperoside, the isoquercitrin, the quercetin-3-O-glucuronide, the gallocatechin, the myricetin-3-O-galactoside, the proanthocyanidin B1, the proanthocyanidin B3, the syringe-3-O-glucoside and the astragalus smicus glycoside are flavonoid compounds; nuciferine, higenamine, N-methyl-higenamine and N-trans-feruloyl tyramine are taken as alkaloid compounds, and the compound is selected from the group consisting of higenamine, nuciferine, higenamine The composition has the advantages of simple preparation process, good hemostatic effect and the like, and provides a new raw material choice for the development of intestinal bleeding treatment medicines.

Description

Technical Field

[0001] The present invention relates to the field of intestinal hemostatic drugs, and in particular to an intestinal hemostatic composition, a preparation method and application thereof. Background Art

[0002] Intestinal bleeding is one of the common clinical emergencies with high morbidity and mortality. It is caused by factors such as inflammation of the intestine itself, mechanical injury, vascular lesions, tumors, etc. It can also be caused by lesions of adjacent organs and systemic diseases involving the intestine. Intestinal bleeding has multiple hazards, mainly including acute hemorrhagic shock, anemia, impaired heart function, increased risk of infection and other complications. Acute hemorrhagic shock is one of the most serious complications of intestinal bleeding, which may lead to circulatory failure, manifested as dizziness, palpitations, fatigue, thirst, sweating, irritability, pale complexion, increased heart rate, decreased blood pressure, and even fainting and shock. Massive bleeding may also lead to anemia, and long-term chronic blood loss can cause iron deficiency anemia, manifested as fatigue, dizziness, pale complexion, etc. Intestinal bleeding not only has a serious impact on the patient's physical health, but may also be life-threatening.

[0003] Existing traditional drugs for treating intestinal bleeding, such as thrombin and tranexamic acid, are effective to a certain extent, but they have problems such as short duration of action, frequent administration, and poor effect on certain types of bleeding. In addition, long-term use of these drugs may cause adverse reactions such as thrombosis. In recent years, with the in-depth study of the mechanism of intestinal bleeding and the development of new materials technology, some new hemostatic drugs such as recombinant coagulation factors and hemostatic powders have been developed, but these drugs are usually expensive and the effect is still unsatisfactory in some cases. Therefore, the development of an efficient, safe and simple to prepare intestinal hemostatic drug is of great clinical significance. Summary of the Invention

[0004] The purpose of the present invention is to solve at least one of the technical problems existing in the prior art and to provide an intestinal hemostatic composition and a preparation method and application thereof.

[0005] The technical solutions of the present invention are as follows: The intestinal hemostatic composition comprises the following raw materials in parts by weight: 30-40 parts of catechins, 50-60 parts of hyperoside, 10-20 parts of isoquercetin, 20-30 parts of nuciferine, quercetin-3- O - 50-60 parts of glucuronide and 5-10 parts of gallic acid catechin.

[0006] Preferably, the following raw materials in parts by weight are also included: Myricetin-3- O -galactoside 1-5 parts, proanthocyanidin B1 5-10 parts, proanthocyanidin B3 5-10 parts, syringetin-3-O - 5-10 parts of glucoside.

[0007] Preferably, the following raw materials in parts by weight are also included: 1-5 parts of astragaloside, 5-10 parts of higenamine, N -1-5 parts of methylcoclaurine, N - 1-5 parts of trans-feruloyltyramine.

[0008] The present invention also discloses a method for preparing an intestinal hemostatic composition, comprising the following steps: weighing each raw material, dissolving each in 50% by volume DMSO to obtain a mother liquor of each component, then removing the mother liquor of each component, mixing them evenly, and then dissolving them in 50% by volume DMSO to adjust the volume to obtain the composition.

[0009] The present invention also discloses an application of the intestinal hemostatic composition in preparing a medicine for treating intestinal bleeding.

[0010] Preferably, the drug further comprises a pharmaceutically acceptable carrier.

[0011] Preferably, the dosage form of the drug is one of tablets, granules, capsules and injections.

[0012] The beneficial effects of the present invention are as follows: the present invention provides an intestinal hemostatic pharmaceutical composition and a preparation method thereof, which improves the hemostatic effect and reduces the rebleeding rate by regulating the formula and proportion of the composition, thereby providing a new option for the treatment of intestinal bleeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The incidence of intestinal bleeding in zebrafish after treatment with three different compound combinations; Figure 2 The number of platelets in the intestine after treatment with three different compound combinations. Compared with the normal control group, ### indicates that the group has a significant difference of p < 0.001; compared with the model group: Indicates that the group has a significant difference at p < 0.001; Figure 3 The 5-HT levels in zebrafish after treatment with three different compound combinations. Compared with the normal control group, ### indicates a significant difference of p < 0.001; compared with the model group: Indicates that the groups have significant differences at p < 0.01. Indicates that the groups have significant differences at p < 0.001. DETAILED DESCRIPTION

[0014] The following embodiments of the present invention are described in detail. The embodiments described below are exemplary and are only used to explain the present invention, and are not to be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, the techniques or conditions described in the literature in this field or the product specifications are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be obtained commercially.

[0015] The raw materials used in the following examples are as follows: Catechin (CAS: 154-23-4), hyperoside (CAS: 482-36-0), isoquercetin (CAS: 482-35-9), nuciferine (CAS: 475-83-2), quercetin-3- O -Glucuronide (CAS: 22688-79-5), Gallicatechin (CAS: 970-73-0), Myricetin-3- O -galactoside (CAS: 15648-86-9), proanthocyanidin B1 (CAS: 20315-25-7), proanthocyanidin B3 (CAS: 23567-23-9), syringetin-3- O -Glucoside (CAS: 40039-49-4), Astragaloside (CAS: 480-10-4), Higenamine (CAS: 5843-65-2), N -Methylcoclaurine (CAS: 5096-70-8), N -trans-feruloyltyramine (CAS: 66648-43-9).

[0016] Example 1 This embodiment provides a compound combination for treating intestinal bleeding, which is composed of 6 monomeric compounds, including: catechin 35 parts, hyperoside 54 parts, isoquercetin 19 parts, nuciferine 29 parts, quercetin-3- O 54 parts of catechin-3-glucuronide and 7 parts of gallic acid catechin (each equivalent to 5 μg). Preparation method: Accurately weigh 10 mg of each of the above 6 reference substances and dissolve them in 10 mL of 50% DMSO to prepare a stock solution of each reference substance at a concentration of 1 mg / mL. From the stock solution, 174.0 μL of catechin, 270.5 μL of hyperoside, 94.0 μL of isoquercetin, 146.0 μL of nuciferine, and quercetin-3- O 273.5 μL of catechin-glucuronide and 36.5 μL of gallic acid were mixed evenly, dissolved in 50% DMSO and diluted to 50 mL to obtain compound combination 1.

[0017] Example 2 This embodiment provides a compound combination for treating intestinal bleeding, which is composed of 10 monomer compounds, including: catechin 35 parts, hyperoside 54 parts, isoquercetin 19 parts, nuciferine 29 parts, quercetin-3- O - 54 parts of glucuronide, 7 parts of gallic acid catechin, myricetin-3- O -galactoside 3 parts, proanthocyanidin B1 10 parts, proanthocyanidin B3 6 parts, syringetin-3- O 5 portions of 5 μg of catechin-3-glucoside (each portion is equivalent to 5 μg). Preparation method: Accurately weigh 10 mg of each of the above 10 reference substances and dissolve them in 10 mL of 50% DMSO to prepare a stock solution of each reference substance with a concentration of 1 mg / mL. From the stock solution, 174.0 μL of catechin, 270.5 μL of hyperoside, 94.0 μL of isoquercetin, 146.0 μL of nuciferine, and quercetin-3- O -glucuronide 273.5 μL, gallic acid catechin 36.5 μL, myricetin-3- O -galactoside 14.5 μL, proanthocyanidin B1 49.5 μL, proanthocyanidin B3 27.5 μL and syringetin-3- O 25.0 μL of -glucoside was mixed evenly, dissolved in 50% DMSO and the volume was adjusted to 50 mL to obtain compound combination 2.

[0018] Example 3 This embodiment provides a compound combination for treating intestinal bleeding, which is composed of 10 monomer compounds, including: catechin 35 parts, hyperoside 54 parts, isoquercetin 19 parts, nuciferine 29 parts, quercetin-3- O - 54 parts of glucuronide, 7 parts of gallic acid tea, 4 parts of astragaloside, 10 parts of higenamine, N -2 parts of methyl hengzhoulin, N 2 portions of -trans-feruloyltyramine (each equivalent to 5 μg). Preparation method: Accurately weigh 10 mg of each of the above 10 reference substances and dissolve them in 10 mL of 50% DMSO to prepare a stock solution of each reference substance at a concentration of 1 mg / mL. From the stock solution, pipette 174.0 μL of catechin, 270.5 μL of hyperoside, 94.0 μL of isoquercetin, 146.0 μL of nuciferine, 100.0 μL of quercetin-3- O -glucuronide 273.5 μL, gallocatechin 36.5 μL, astragaloside 21.0 μL, higenamine 48.0 μL, N -Methylcoclaurine 10.0 μL and N 11.5 μL of -trans-feruloyltyramine was mixed evenly, dissolved with 50% DMSO and the volume was adjusted to 50 mL to obtain compound combination 3.

[0019] The zebrafish experimental model was used to evaluate the efficacy of the compound combination prepared in Examples 1-3 in treating intestinal bleeding.

[0020] 1. Experimental Materials Experimental group drugs: compound combinations prepared in Example 1, Example 2, and Example 3, namely, compound combination 1, compound combination 2, and compound combination 3.

[0021] Control positive drug: icariin.

[0022] Simvastatin is an inducer of the zebrafish intestinal bleeding model.

[0023] Experimental animals: Wild-type AB strain zebrafish were bred by natural pair mating.

[0024] 2. Experimental methods and results (1) Toxicity test Four-day-post-fertilization zebrafish were randomly selected and placed in 6-well plates (3 mL well volume), with 30 per well (experimental group). Each compound combination was added at a volume ratio of 0.781% (drug solution / culture medium, v / v). A positive control group with a final concentration of 20.0 μg / mL was established, along with a normal control group and a model group. Simvastatin solution was added to all groups except the normal control group to a final concentration of 7 μM to establish a zebrafish intestinal bleeding model. After incubation at 28°C for one day, the number of surviving zebrafish was counted, and the survival rate was used to evaluate the toxicity of each compound combination. The results are shown in Table 1. The survival rate of zebrafish in each group was 100%, indicating that none of the three compound combinations exhibited toxicity in the zebrafish intestinal bleeding model.

[0025] Table 1 Toxicity evaluation test results

[0026] (2) Incidence of intestinal bleeding Zebrafish 4 days after fertilization were randomly selected and placed in a 6-well plate (well volume of 3 mL), with 30 fish in each well (experimental group). Compound combinations were added at a volume ratio of 0.781% (drug solution / culture solution, V / V). A positive drug control group was set up with a final concentration of 20.0 μg / mL. A normal control group and a model group were also set up. Except for the normal control group, simvastatin solution was added to the remaining experimental groups at the same time to a final concentration of 7 μM to establish a zebrafish intestinal bleeding model. After continuing to culture at 28°C for 1 day, zebrafish in each group were photographed under a microscope, and the incidence of intestinal bleeding was used as an indicator to evaluate the effect of each compound combination in inhibiting intestinal bleeding. The results are shown in Figure 1The incidence of intestinal bleeding in the model group was 67%, indicating that the model was successfully established. Compared with the model group, the incidence of intestinal bleeding in the experimental group was reduced to 37%, 30%, and 37% after intervention with compound combinations 1, 2, and 3, respectively, which is comparable to the positive drug control group, indicating that all three compound combinations have a significant effect in inhibiting intestinal bleeding.

[0027] (3) Intestinal platelet count Transgenic platelet green fluorescent zebrafish 4 days after fertilization were randomly selected and placed in a 6-well plate (well volume of 3 mL), with 30 fish in each well (experimental group). Compound combinations were added at a volume ratio of 0.781% (drug solution / culture solution, V / V). A positive drug control group was set up with a final concentration of 20.0 μg / mL, and a normal control group and a model group were also set up. Except for the normal control group, simvastatin solution was added to the remaining groups at the same time to a final concentration of 7 μM to establish a zebrafish intestinal bleeding model. After continuing to culture at 28°C for 1 day, zebrafish from each group were photographed under a fluorescence microscope, and data were collected and analyzed using NIS-Elements Dv3.20 advanced image processing software. The number of zebrafish intestinal platelets was counted, and this indicator was used to evaluate the effect of the sample in inhibiting intestinal bleeding. The results were expressed as "mean ± standard error" and the t-test was used for difference analysis. The results are shown in Figure 2. Figure 2 As shown, the average number of platelets in the zebrafish intestine in the model group was 10.80, which was significantly different from the normal control group (p < 0.001), indicating that the model was successfully established. Compared with the model group, the platelet count in the zebrafish intestine in the experimental group after intervention with compound combinations 1, 2, and 3 decreased to an average of 6.90, 7.13, and 7.67, respectively (p < 0.001), comparable to the positive drug group, indicating that all three compound combinations have a significant effect in inhibiting intestinal bleeding.

[0028] (4) 5-HT content Transgenic platelet green fluorescent zebrafish 4 days after fertilization were randomly selected and placed in a 6-well plate (well volume of 3 mL), with 30 fish in each well (experimental group). Compound combinations were added at a volume ratio of 0.781% (drug solution / culture solution, V / V). A positive drug control group was set up with a final concentration of 20.0 μg / mL, and a normal control group and a model group were also set up. Except for the normal control group, simvastatin solution was added to the remaining groups at the same time to a final concentration of 7 μM to establish a zebrafish intestinal bleeding model. After continuing to culture at 28°C for 1 day, the zebrafish were taken and extracted according to the instructions of the zebrafish 5-hydroxytryptamine quantitative detection kit (Shanghai Enzyme-Linked Biotechnology Co., Ltd.), and the content of serotonin in the zebrafish of each group was compared, and this indicator was used to evaluate the effect of the sample in inhibiting intestinal bleeding. The results are expressed as "mean ± standard error" and the difference analysis was performed using the t-test. The results are shown in Figure 3 As shown, the serotonin content in the model group was 1.04 ng / mg protein, significantly lower than the 1.39 ng / mg protein in the normal control group (p < 0.01), indicating that the model was successfully established. Compared with the model group, the serotonin content in the zebrafish treated with compound combinations 1, 2, and 3 increased to 1.28, 1.22, and 1.27 ng / mg protein (p < 0.01), respectively, comparable to the positive drug group, indicating that all three compound combinations have a significant inhibitory effect on intestinal bleeding.

[0029] In summary, catechin, hyperoside, isoquercetin, quercetin-3-O-glucuronide, gallocatechin, myricetin-3- O -galactoside, proanthocyanidin B1, proanthocyanidin B3, syringetin-3- O Glucoside and astragalin are flavonoids. Catechin and isoquercetin activate platelet surface receptors (such as GP IIb / IIIa), promoting platelet aggregation and coagulation factor activation / expression, thereby activating the coagulation pathway. They protect the vascular endothelium through antioxidant and free radical scavenging, inhibit the release of inflammatory factors, and maintain coagulation balance, exerting a hemostatic effect. Nuciferine, norcoclaurine, N-methylcoclaurine, and N-trans-feruloyltyramine are alkaloids. Nuciferine, a 5-hydroxytryptamine receptor antagonist, exerts antiplatelet and anticoagulant effects. Its antioxidant and anti-inflammatory properties help maintain endothelial homeostasis and coagulation balance, aiding hemostasis. When used in combination with flavonoids, it achieves hemostasis without bruising.

[0030] Therefore, the three compound combinations with hemostatic function provided by the present invention are composed of flavonoids and alkaloid compounds, which synergistically exert hemostatic effects, have the advantages of simple preparation process and good hemostatic effect, and provide new raw material options for the development of drugs for the treatment of intestinal bleeding.

[0031] The above-described embodiments merely represent preferred implementations of the present invention. While the descriptions thereof are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make various other corresponding changes and modifications based on the technical solutions and concepts described above, and all such changes and modifications should fall within the scope of protection of the claims of the present invention.

Claims

1. An intestinal hemostatic composition, characterized in that: It includes the following raw materials in parts by weight: 30-40 parts of catechins, 50-60 parts of hyperoside, 10-20 parts of isoquercetin, 20-30 parts of nuciferine, quercetin-3- O - 50-60 parts of glucuronide and 5-10 parts of gallic acid catechin.

2. The intestinal hemostatic composition according to claim 1, characterized in that Also includes the following raw materials by weight: Myricetin-3- O -galactoside 1-5 parts, proanthocyanidin B1 5-10 parts, proanthocyanidin B3 5-10 parts, syringetin-3- O - 5-10 parts of glucoside.

3. The intestinal hemostatic composition according to claim 1, characterized in that Also includes the following raw materials by weight: 1-5 parts of astragaloside, 5-10 parts of higenamine, N -1-5 parts of methylcoclaurine, N - 1-5 parts of trans-feruloyltyramine.

4. The method for preparing the intestinal hemostatic composition according to any one of claims 1 to 3, characterized in that: The method comprises the following steps: weighing each raw material, dissolving each raw material in 50% by volume DMSO to obtain a mother liquor of each component, then removing the mother liquor of each component, mixing them evenly, and then dissolving them in 50% by volume DMSO to make the volume constant.

5. Use of the intestinal hemostatic composition according to any one of claims 1 to 3 in the preparation of a drug for treating intestinal bleeding.

6. The use according to claim 5, characterized in that The drug further includes a pharmaceutically acceptable carrier.

7. The use according to claim 5, characterized in that The dosage form of the medicine is one of tablets, granules, capsules and injections.

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