Application of herba epimedii extract in preparation of medicine for treating and / or preventing hyperammoemia-related diseases

Abstract

The invention relates to an application of a herba epimedii extract in preparation of a medicine for treating and / or preventing hyperammoemia related diseases, and belongs to the technical field of biological medicines. The invention provides an application of a herba epimedii extract in preparation of a medicine for treating and / or preventing hyperamminemia related diseases. In the research, the applicant discovers that the total flavonoids of herba epimedii can obviously reduce the blood ammonia and brain ammonia level of acute hepatic failure mice induced by lipopolysaccharide and D-galactosamine, and prompts that the total flavonoids of herba epimedii can obviously reduce the blood ammonia and brain ammonia level, and can be used for preparing medicines for treating and / or preventing diseases related to hyperammoemia.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine. Specifically, it relates to the medical use of total flavonoids of Epimedium: the extract of total flavonoids of Epimedium can significantly reduce the levels of blood ammonia and brain ammonia, and can be used to prepare drugs for treating and / or preventing diseases related to hyperammonemia. Background Art

[0002] Hyperammonemia refers to a pathological state in which the blood ammonia level rises beyond the normal range. Hyperammonemia has obvious toxic effects on the central nervous system and can cause manifestations of hepatic encephalopathy such as disturbance of consciousness, mental confusion, coma, etc. The causes of hyperammonemia include liver diseases, congenital metabolic diseases, portosystemic shunts, and other factors such as constipation. Among them, the typical pathological indicators of hepatic encephalopathy are significantly elevated blood ammonia and brain ammonia levels, accompanied by brain pathological features such as astrocyte swelling and neuroinflammation [Hepatobiliary Diseases Group, Chinese Society of Gastroenterology, Expert Consensus on the Clinical Diagnosis and Treatment of Latent Hepatic Encephalopathy in China. Chinese Journal of Gastroenterology, Vol. 28, No. 12, 2023, 722 - 738]. So far, there is still a lack of specific drugs for treating hyperammonemia, and the mortality rate is high. Currently, although lactulose and rifaximin are first-line drugs for treating hepatic encephalopathy, both are often accompanied by adverse gastrointestinal reactions, including abdominal distension, abdominal pain, nausea, etc., and there is a deficiency of easy recurrence after drug withdrawal. Therefore, there is an urgent need to develop new drugs with the effects of reducing blood ammonia and brain ammonia and being safe and effective.

[0003] Epimedium is a perennial herb with extensive medicinal value. Also known as Xianlingpi, it has a long medicinal history. Li Shizhen said in "Compendium of Materia Medica" that it has the effects of tonifying essence qi, strengthening bones and muscles, tonifying the waist and knees, and enhancing cardiac function. Modern chemical composition research shows that the main active components of Epimedium are total flavonoids of Epimedium and polysaccharides of Epimedium. Total flavonoids of Epimedium is a mixture mainly composed of various icariin and icariside. Modern pharmacological research shows that total flavonoids of Epimedium has functions such as promoting immune function, participating in bone metabolism, anti-tumor, and enhancing cardiovascular activity. Currently, there is no literature on the use of total flavonoids of Epimedium for treating and / or preventing diseases related to hyperammonemia. Summary of the Invention

[0004] Aiming at the problems existing in the above-mentioned prior art, the purpose of the present invention is to design and provide a safe and effective traditional Chinese medicine that uses total flavonoids of Epimedium to prepare drugs for reducing blood ammonia and brain ammonia levels and treating and / or preventing diseases related to hyperammonemia.

[0005] In order to achieve the above purpose, the present invention adopts the following technical solutions:

[0006] On the one hand, the present invention provides the application of Epimedium extract in the preparation of drugs for treating and / or preventing diseases related to hyperammonemia.

[0007] The application described above, wherein the epimedium extract is total flavonoids of epimedium.

[0008] The application described above, wherein the hyperammonemia-related disease is hepatic encephalopathy, congenital metabolic disease, portosystemic shunt or constipation.

[0009] The application described above, wherein the hyperammonemia-related disease is acute liver failure.

[0010] The application described above, wherein the epimedium extract can reduce the blood ammonia level.

[0011] The application described above, wherein the epimedium extract can reduce the brain ammonia level.

[0012] The application described above, wherein the epimedium extract can reduce the swelling of astrocytes in brain tissue.

[0013] In a second aspect, the present invention provides a pharmaceutical composition for treating and / or preventing hyperammonemia-related diseases, comprising an epimedium extract and a pharmaceutically acceptable excipient.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] The applicant found in the research that total flavonoids of epimedium can significantly reduce the blood ammonia and brain ammonia levels in mice with acute liver failure induced by lipopolysaccharide combined with D-galactosamine, suggesting that the total flavonoids extract of epimedium can significantly reduce the blood ammonia and brain ammonia levels and can be used to prepare drugs for treating and / or preventing diseases related to hyperammonemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is the standard curve of icariin solution;

[0017] Figure 2 It is the effect of total flavonoids extract of epimedium on reducing blood ammonia and brain ammonia levels in mice induced by LPS-D-GalN;

[0018] Figure 3 It is the result diagram of the morphology of astrocytes and the expression of GFAP protein in the hippocampal region of mouse brain;

[0019] Figure 4 It is the effect of total flavonoids of epimedium on reducing the swelling of astrocytes in the hippocampal region of mouse brain tissue induced by LPS-D-GalN. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0020] The present invention will be further described below with reference to the drawings and embodiments.

[0021] Example 1: Preparation of total flavonoids of epimedium

[0022] 1. Determination of the alcohol absorption amount of epimedium medicinal materials

[0023] Weigh 20 g of the crude powder of Epimedium leaves, add 10 times the volume of 65% ethanol, soak for 30 min, heat under reflux for 30 min, and then filter to obtain 7.25 times the EF extract. Alcohol absorption = (alcohol added - alcohol obtained after filtration) / weight of Epimedium herb = 2.75 times / 20 g. Taking the integer multiple, the alcohol absorption is approximately 3 times the raw material amount.

[0024] 2. Optimize the extraction process by orthogonal experiment

[0025] Taking the extraction rate of total flavonoids (EF) from Epimedium as the index, the ethanol concentration, solid-liquid ratio, and extraction time are the factors affecting the extraction rate. Each factor is set at 3 levels respectively, and an L9(3 4 ) orthogonal experiment is designed to screen the optimal process for EF extraction. The levels of different factors are shown in Table 1 below.

[0026] Weigh 20 g of the crude powder of Epimedium, extract according to the orthogonal experiment factor level table, shake well and filter, collect the two extraction liquids and combine them into a volumetric flask, and make up to the mark with ethanol to obtain the EF extract. The determination of EF in the extract is carried out according to the ultraviolet-visible spectrophotometry method (General Rule 0401) in Chinese Pharmacopoeia.

[0027] Table 1 Orthogonal experiment factor level table for EF extraction process

[0028]

[0029]

[0030] Perform variance analysis on the experimental results according to the orthogonal experiment results. The results are shown in Tables 2 and 3 below.

[0031] The results show that the order of the influence of each factor on the EF extraction effect is extraction time (C) > ethanol concentration (A) > solid-liquid ratio (B). Among them, extraction time (C) and ethanol concentration (A) are significant factors, and solid-liquid ratio (B) is an insignificant factor. The optimal extraction process for Epimedium is A2B2C3. Combining with the 3-fold alcohol absorption of Epimedium herb, that is, after weighing a certain amount of Epimedium herb, add 13 times the volume of 65% ethanol for reflux extraction for 2 hours for the first extraction, and add 8 times the volume of 65% ethanol for reflux extraction for 1.5 hours for the second extraction.

[0032] Table 2 Range analysis table

[0033]

[0034] Table 3 ANOVA table

[0035]

[0036] 2. Verification experiment

[0037] Weigh 50 g of the crude Epimedii Herba powder in three batches respectively, extract it according to the above optimal extraction process conditions, determine the content of EF, and verify the feasibility of the optimal extraction process.

[0038] The results of the verification experiment are shown in Table 4 below. The EF content is 47.05 mg / g of the medicinal material, and the RSD is 1.60%. It can be seen that the EF extraction process optimized in this experiment is stable and reliable, and the extraction rate is relatively high.

[0039] Table 4 Results of the verification experiment

[0040]

[0041] 3. Determination of the content of EF by ultraviolet spectrophotometry

[0042] (1) Preparation of icariin reference substance solution

[0043] Precisely weigh an appropriate amount of icariin reference substance and place it in a 10 mL volumetric flask. Add methanol to the scale of the volumetric flask to make the volume constant, and shake well to obtain the icariin standard stock solution. Pipette an appropriate amount of the icariin standard stock solution into a volumetric flask, and dilute it with methanol to obtain icariin standard solutions with concentrations of 3.06, 6.12, 12.24, 24.48, and 48.96 μg / mL respectively. Shake well to obtain a series of icariin reference substance solutions.

[0044] (2) Preparation of test solution

[0045] Pipette an appropriate amount of the EF extract into a volumetric flask, add methanol to the scale of the volumetric flask to make the volume constant, and shake well to obtain the test solution.

[0046] (3) Content determination

[0047] Take the series of icariin reference substance solutions and the test solution, use methanol as the blank control, and determine the content of EF in the extract according to the ultraviolet-visible spectrophotometry method (General Principles 0401) in the Chinese Pharmacopoeia. Take the concentration of the icariin reference substance solution as the abscissa X and the absorbance value as the ordinate Y to plot the standard curve.

[0048] The obtained standard curve of the icariin solution is as Figure 1 shown. The standard curve of icariin is A = 0.042x - 0.0106, R 2 = 0.9999. It shows that there is a good linear relationship between the icariin concentration in the range of 3.06 - 48.87 μg / mL and the absorbance. Substitute the absorbance value of the test solution into the icariin standard curve equation to calculate the content of EF in the test sample. The calculation formula for the content of Epimedium flavonoids is:

[0049]

[0050] 4. Determination of the content of 5 flavonoid monomers in the EF extract by HPLC

[0051] (1) Preparation of mixed reference substance solution

[0052] Accurately weigh appropriate amounts of reference substances of epimedin A, epimedin B, epimedin C, icariin, and baohuoside I and place them in a volumetric flask. Add methanol to volume to the scale of the volumetric flask and shake well to obtain a mixed reference substance stock solution with concentrations of 412, 404, 400, 394, and 192 μg / mL for epimedin A, epimedin B, epimedin C, icariin, and baohuoside I, respectively.

[0053] (2) Preparation of test solution

[0054] Accurately weigh 9.82 mg of EF freeze-dried powder, place it in a volumetric flask, add methanol to volume to the scale of the volumetric flask, shake well to obtain a 982 μg / mL test solution. Centrifuge at 12000 rpm / min for 10 min, take the supernatant and transfer it to a liquid phase vial, and store at 4°C.

[0055] (3) Chromatographic conditions

[0056] Use octadecylsilane chemically bonded silica gel as the filler (250 mm × 4.6 mm, 5 μm); use acetonitrile as mobile phase A and water as mobile phase B, and perform elution according to the mobile phase gradient in Table 5; the column temperature is 25°C; the flow rate is 1 mL / min, and the detection wavelength is 270 nm. The theoretical plate number calculated based on the icariin peak should be not less than 8000. Determine the contents of epimedin A, epimedin B, epimedin C, icariin, and baohuoside I in the test solution, and record the HPLC chromatogram and the spectral information of each component peak.

[0057] Table 5 Mobile phase gradient elution program

[0058]

[0059] Under these mobile phase conditions, the component peak shapes of epimedin A, epimedin B, epimedin C, icariin, and baohuoside I in the mixed standard substance solution are good and the response values are high. Calculate the contents of each target component according to the external standard single-point method. The contents of epimedin A, epimedin B, epimedin C, icariin, and baohuoside I are 14.57 ± 0.85 μg / mg, 27.86 ± 0.83 μg / mg, 149.74 ± 5.79 μg / mg, 40.95 ± 1.49 μg / mg, and 9.85 ± 0.61 μg / mg, respectively, accounting for 1.46 ± 0.09%, 2.79 ± 0.09%, 14.97 ± 0.58%, 4.10 ± 0.15%, and 0.98 ± 0.06% of the freeze-dried powder, respectively. The results are shown in Table 6 below.

[0060] Table 6 Determination of the contents of 5 monomers in the icariin flavonoid extract

[0061]

[0062] Example 2: Effect of Total Flavonoids from Epimedium on Reducing Blood Ammonia Level and Brain Ammonia Level in Mice Induced by LPS-D-GalN

[0063] 1. Materials and Methods

[0064] Experimental animals: C57BL / 6J male mice, 8 - 9 months old, weighing 28 ± 3 g, purchased from the Experimental Animal Management Center of Southern Medical University. The mice were placed at room temperature (20 ± 2°C) with a standard 12-hour light / dark cycle and allowed free access to food and water. They were adaptively fed for 7 days before the experiment began.

[0065] 2. Reagents and Drugs

[0066] Blood ammonia test kit (Nanjing Jiancheng Bioengineering Institute);

[0067] Lipopolysaccharides (LPS, Escherichia coli (O111:B4), Sigma), a 500 ng / mL solution was prepared with sterile normal saline and used for modeling by intraperitoneal injection at a dose of 5 μg / kg;

[0068] D-(+)-Galactosamine hydrochloride (D-GalN, ≥99%, Sigma), a 20 mg / mL solution was prepared with sterile normal saline and used for modeling by intraperitoneal injection at a dose of 200 mg / kg;

[0069] Silibinin capsules (Tianjin Tasly Saint Pharmaceutical Co., Ltd.), a 10 mg / mL solution was prepared with distilled water and used for gavage in mice at a dose of 100 mg / kg;

[0070] The total flavonoids from Epimedium obtained in Example 1 under the optimal extraction process conditions were dissolved separately with distilled water and used for gavage in mice at three low, medium, and high doses of 50 mg / kg, 100 mg / kg, and 800 mg / kg, respectively.

[0071] 3. Main Instruments

[0072] Full-wavelength microplate reader Multiskan GO, centrifuge, analytical balance, small vortex mixer, etc.

[0073] 4. Grouping, Drug Administration and Modeling Methods for Mice

[0074] C57BL / 6J male mice, 10 months old, weighing 20 ± 2 g. The mice were placed at room temperature (25 ± 2 °C) with a standard 12-h light / dark cycle and had free access to food and water. They were adaptively fed for 7 days before the experiment. They were grouped according to Table 7. The mice in the blank group were prophylactically gavaged with normal saline for 14 consecutive days, and the mice in the dosing groups were prophylactically gavaged with silybin or different doses of EF for 28 consecutive days, once a day. They were fasted for 10 h before modeling but allowed to drink water. One hour after the last dose, the Control group was intraperitoneally injected with sterile normal saline, and the remaining groups were intraperitoneally injected with LPS (5 μg / kg). After 15 min, D-GalN (200 mg / kg) was injected for ALI modeling. The status of the mice was closely observed. Five hours after modeling, the mice were weighed, blood was collected by eye enucleation, left to stand at room temperature for 30 min, then centrifuged at 4000 r / min at 4 °C for 10 min to separate the serum, which was stored at -80 °C for determination of serum indexes; the mice were sacrificed by cervical dislocation, and after dissection, the liver and kidneys were collected, washed with pre-cooled normal saline and then dried, weighed and recorded; the weighed liver tissue was divided into three parts, one part was fixed in 4% paraformaldehyde solution for pathological examination, and the other two parts were frozen at -80 °C for determination of liver indexes.

[0075] Table 7 Grouping of experimental animals

[0076]

[0077] (1) Detection of blood ammonia level

[0078] For the mice after the above different treatments, blood was collected by eye enucleation, placed in a centrifuge tube, centrifuged at 3000 rpm at 4 °C for 10 min, and the serum was taken. The NH3 level in the serum was detected using a kit and an enzyme-labeled instrument.

[0079] (2) Detection of brain ammonia level

[0080] Mouse brain tissue was taken, PBS was added at a ratio of 1:9 (w / v), homogenized, centrifuged at 12000 rpm at 4 °C for 10 min, and the supernatant was taken as the sample to be measured and determined according to the kit instructions.

[0081] The results of the effects of EF on blood ammonia and brain ammonia levels in ALI mice are shown in Table 8 below.

[0082] Table 8 Effects of EF on blood ammonia and brain ammonia levels in ALI mice

[0083]

[0084] Compared with the blank group, # P < 0.05, ## P < 0.01, ###P < 0.001; compared with the model group, *P < 0.05, **P < 0.01, ***P < 0.001

[0085] The results were as Figure 2 shown, indicating that total flavonoids of Epimedium can significantly reduce the levels of blood ammonia and brain ammonia, and have a significant improvement effect on the symptoms of diseases such as hyperammonemia.

[0086] Example 3: Effect of total flavonoids of Epimedium (EF) on reducing the swelling of astrocytes in the mouse brain tissue induced by LPS-D-GalN

[0087] Immunofluorescence staining of glial fibrillary acidic protein (GFAP) in the hippocampal region of mouse brain tissue:

[0088] Take the brain tissues of mice in different groups in Example 2 respectively, and analyze the hyperplasia reaction of astrocytes in the hippocampal region of the brain tissues of mice in different groups according to the steps of cryosectioning, fixing, antigen repair, blocking, primary antibody incubation, secondary antibody incubation, mounting, and observing and recording the immunofluorescence images of each tissue under an inverted fluorescence microscope.

[0089] As Figure 3 and 4 shown, GFAP is a characteristic intermediate filament protein of astrocytes, and its expression level is positively correlated with the degree of cell activation. Under pathological conditions, when astrocytes proliferate (increase in number) and hypertrophy (increase in volume), the expression of GFAP is significantly up-regulated. As Figure 3 and 4 shown, it is indicated that compared with the normal control group of mice, the fluorescence signal intensity of GFAP protein and the positive protein expression area in the hippocampal region of LPS / D-GalN group mice increased extremely significantly (P < 0.001), indicating that LPS / D-GalN induced excessive hyperplasia of astrocytes in mice, and there were pathological states such as obvious increase in cell volume, round cell body, and swelling and thickening of the processes of astrocytes. Compared with the LPS / D-GalN group of mice, the positive protein expression area of GFAP in the EF group of mice decreased extremely significantly (P < 0.001), indicating that EF significantly inhibited the hyperplasia reaction of astrocytes in mice induced by LPS / D-GalN, and astrocytes have a multi-process morphology, and their cell body and process structures are relatively regular. In addition, compared with the normal control group, there was no statistical difference in the positive protein expression area of GFAP in the EF administration group (P > 0.05), indicating that total flavonoids of Epimedium can inhibit the excessive activation of astrocytes in the hippocampal region of the mouse brain tissue induced by LPS / D-GalN, reduce the swelling of astrocytes, and restore it to near the normal level.

[0090] In summary, the epimedium extract can significantly reduce the levels of blood ammonia and brain ammonia, and alleviate the pathological states such as the swelling of astrocytes caused by hyperammonemia. The total flavonoid extract of epimedium has the effects of reducing blood ammonia and brain ammonia, and can be used for the treatment of hyperammonemia.

Claims

1. Use of epimedium extract in the preparation of a medicament for treating and / or preventing hyperammonemia-related diseases.

2. The application according to claim 1, wherein The epimedium extract is total flavonoids of epimedium.

3. The application according to claim 1, characterized in that The hyperammonemia-related diseases are hepatic encephalopathy, congenital metabolic diseases, portosystemic shunt or constipation.

4. The application according to claim 1, characterized in that, The hyperammonemia-related disease is acute liver failure.

5. The application according to claim 1, characterized in that, The epimedium extract can reduce the blood ammonia level.

6. The application according to claim 1, characterized in that, The epimedium extract can reduce the brain ammonia level.

7. The application according to claim 1, characterized in that, The epimedium extract can reduce the swelling effect of astrocytes in the brain tissue.

8. A pharmaceutical composition for treating and / or preventing hyperammonemia-related diseases, characterized in that, It contains epimedium extract and pharmaceutically acceptable excipients.

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