Hippocampus extract with activity of improving renal anemia as well as preparation method and application of hippocampus extract

Abstract

The invention discloses a hippocampus extract with activity of improving renal anemia as well as a preparation method and application of the hippocampus extract, and belongs to the technical field of biological medicines. The hippocampus extract with different molecular magnitudes is prepared through optimization of a membrane separation process, experiments prove that the hippocampus extract can remarkably improve the activity of fluorescein enzyme and promote generation of EPO of a human embryonic kidney cell HEK293 cell line, and particularly, the blood enriching activity of the hippocampus extract with the molecular weight smaller than 20 kDa is obviously higher than that of a hippocampus total extract, so that the hippocampus extract with the molecular weight smaller than 20 kDa has a good application prospect. The traditional Chinese medicine composition can effectively reduce the inosine and urea nitrogen levels of rats suffering from renal injury type anemia and improve renal failure, and has the effect of remarkably improving renal anemia. The hippocampus extract obtained through optimization of the preparation method relieves the symptoms of renal anemia from multiple aspects, improves renal injury, provides a scientific basis for clinical research of drugs for improving renal anemia and development of functional foods, and has great application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to a hippocampus extract having the activity of improving renal anemia, and a preparation method and application thereof. Background Art

[0002] With the advent of an aging society, the incidence and age of death from chronic kidney disease (CKD) are increasing annually, making it a global public health issue. It is projected to become the fifth leading cause of death worldwide by 2040. Renal anemia is a common complication of CKD, which not only exacerbates the disease's progression but also causes heart failure and increases mortality. The pathogenesis of renal anemia is complex, associated with decreased bone marrow hematopoietic function, inhibition of erythroid proliferation by inflammatory cytokines, inhibition of erythropoietin synthesis in the kidneys, stimulation of hepcidin gene expression, and consequently, impaired iron release, as well as shortened erythrocyte lifespan. This is typically manifested by decreased peripheral blood erythrocyte and reticulocyte counts, as well as the percentage of immature reticulocytes. The incidence of anemia increases with declining renal function.

[0003] Currently, the primary treatment for renal anemia is injections of erythropoiesis-stimulating agents, but these carry the risk of functional iron deficiency or adverse cardiovascular reactions. Rosatom is a novel oral medication for the treatment of renal anemia. It primarily acts by targeting the hypoxia-inducible factor pathway, promoting endogenous erythropoietin production and receptor expression, and optimizing iron utilization, thereby increasing hemoglobin. However, this medication may increase the risk of adverse reactions such as deep vein thrombosis, arteriovenous fistula thrombosis, hypertension, and hyperkalemia.

[0004] Seahorse has a long history of medicinal use in my country, first mentioned in Chen Cangqi's "Supplement to Materia Medica" during the Tang Dynasty. It is also mentioned in "Compendium of Materia Medica" and "Illustrated Classic of Materia Medica." Seahorse has a sweet, salty, and warming nature. It enters the liver and kidney meridians. It has the effects of warming the kidneys and strengthening yang, dispersing nodules and relieving swelling. It is used to treat impotence, enuresis, asthma due to kidney deficiency, accumulation of masses, injuries from falls, and external treatment of carbuncles and furuncles. Seahorse was first used as a medicine in my country and has been included in all pharmacopoeias. Modern research on its pharmacological effects primarily focuses on sex hormone-like effects, antioxidant properties, and anti-tumor properties. Numerous prescriptions containing seahorse are available, but research on its efficacy in improving renal anemia is lacking. Summary of the Invention

[0005] The purpose of the present invention is to provide a hippocampus extract having the activity of improving renal anemia, and a preparation method and application thereof.

[0006] In order to achieve the above-mentioned purpose, the present invention adopts the following technical solutions: The present invention provides a method for preparing a hippocampus extract having the activity of improving renal anemia, comprising the following steps: S1: Cut the hippocampus into pieces, soak it in water, and homogenize it to obtain hippocampal homogenate; S2: Adjust the pH of the hippocampus homogenate, add biological protease for extraction, and obtain the hippocampus extract.

[0007] S3: heating the hippocampus extract to 100°C to inactivate the enzyme, cooling to room temperature, centrifuging to remove precipitated impurities, and separating the supernatant; S4: performing membrane separation on the supernatant to obtain a desalted solution; S5: concentrating the desalted solution and drying it to obtain a hippocampus extract having activity in improving renal anemia.

[0008] Furthermore, the seahorse is at least one of the three-spotted seahorse, the lined seahorse, the large seahorse, the spiny seahorse, the small seahorse and the distended-belly seahorse.

[0009] Furthermore, in step S1, the material-liquid ratio of seahorse to water is 1:5-40.

[0010] Furthermore, in step S1, the soaking time is 0.5-3 hours, and the homogenization time is 5-10 minutes.

[0011] Furthermore, the biological protease in step S2 is at least one of pepsin, bromelain, papain, neutral protease, trypsin, pancreatin and alkaline protease.

[0012] Furthermore, the amount of the biological protease used is 0-8000U per gram of hippocampus mass.

[0013] Furthermore, the pH value in step S2 is 1-9.

[0014] Furthermore, the extraction conditions in step S2 are a temperature of 30-100° C., a pressure of 0-0.4 MPa, a stirring speed of 0-400 rpm, and an extraction time of 0.5-5 hours.

[0015] Furthermore, the enzyme inactivation time in step S3 is 0-20 minutes; the centrifugation operation is 3000-12000 rpm, and the centrifugation is 5-10 minutes.

[0016] Furthermore, when the dosage of the biological protease is 0 U, the preparation method of the hippocampus extract is water extraction, specifically high temperature and high pressure water extraction and low temperature and normal pressure water extraction.

[0017] Furthermore, the yield of the product with a molecular weight less than 20 kDa obtained by the water extraction method is 8%-30%.

[0018] Furthermore, when the amount of the biological protease is greater than 0 U, the preparation method of the hippocampus extract is enzymatic hydrolysis.

[0019] Furthermore, the yield of the product with a molecular weight less than 20 kDa obtained by the enzymatic hydrolysis method is 60%-90%.

[0020] Furthermore, the yield of the product with a molecular weight less than 20 kDa obtained by the water extraction method is lower than the yield of the product with a molecular weight less than 20 kDa obtained by the enzymatic hydrolysis method.

[0021] Furthermore, the membrane separation operation in step S4 is specifically to first filter with an ultrafiltration membrane and then perform nanofiltration desalination, the molecular weight cutoff of the ultrafiltration membrane is 20 kDa, and the molecular weight cutoff of the nanofiltration membrane is 150-300 Da.

[0022] Furthermore, the specific separation operation in step S4 is: adding an equal volume of double-distilled water to the supernatant in step S3, and replenishing the liquid during the separation process while keeping the volume unchanged until the conductivity value of the outflowing liquid is close to that of double-distilled water.

[0023] Furthermore, the drying method in step S5 is at least one of reduced pressure drying, freeze drying, and spray drying.

[0024] Furthermore, the reduced pressure drying conditions are -0.1-0 MPa, 30-60° C., and drying for 2-3 days.

[0025] Furthermore, the freeze-drying conditions are 20-40 Pa, -60°C to -40°C, and freeze-drying for 2-3 days.

[0026] The present invention also provides a hippocampus extract obtained by the above preparation method, wherein the hippocampus extract has Molecular weight <20kDa.

[0027] The present invention also provides the use of the seahorse extract in preparing food and / or medicine for improving renal anemia.

[0028] Furthermore, the improvement of renal anemia specifically includes at least one of improving renal damage, improving renal functional indicators, improving renal anemia, improving blood counts, erythropoietin, increasing hemoglobin, increasing red blood cells, promoting reticulocyte production, increasing the percentage of immature reticulocytes, reducing the percentage of reticulocytes with low fluorescence intensity, and improving hematopoietic capacity.

[0029] Furthermore, the indicators for improving kidney function are specifically inosine and urea nitrogen.

[0030] Compared with the prior art, the present invention has the following advantages and beneficial effects: (1) The present invention provides a simple, efficient, and highly operable method for preparing a hippocampus extract for improving renal anemia. The present invention provides three different methods for preparing hippocampus extracts, including a high-temperature and high-pressure water extraction method, a low-temperature and normal-pressure water extraction method, and an enzymatic hydrolysis method, which provide data support for subsequent related research and have reference significance; (2) The seahorse extract prepared by the enzymatic hydrolysis method provided by the present invention has a product yield of 60%-90% with a molecular weight of less than 20 kDa, and the enzymatic hydrolysis process is more efficient; (3) The present invention discovered for the first time that seahorse extract can significantly increase the activity of luciferase, promote the production of EPO, and has significant blood-tonifying activity; and the blood-tonifying activity of seahorse extract with a molecular weight of less than 20 kDa is significantly higher than that of total seahorse extract. It is also confirmed that seahorse extract with a molecular weight of less than 20 kDa can reduce the levels of inosine and urea nitrogen in rats with anemia caused by renal damage, increase the levels of hemoglobin, red blood cells and reticulocytes, increase the percentage of immature reticulocytes, and promote the recovery of bone marrow hematopoietic function, thereby improving renal function and the symptoms of renal anemia. It can be used in the subsequent development of drugs and functional foods to improve renal anemia, and has a broad development and application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a graph showing the in vitro blood-tonifying activity results of the total extract of hippocampus and products of different molecular weights in Example 1; Figure 2 This is a graph showing the in vitro blood-tonifying activity results of the total extract of hippocampus and products of different molecular weights in Example 2; Figure 3 This is a graph showing the in vitro blood-tonifying activity results of the total extract of hippocampus and products of different molecular weights in Example 3; Figure 4 This is a graph showing the weight growth rate of rats in the renal injury anemia model treated with the hippocampal product having a molecular weight less than 20 kDa according to Example 3 for 4 weeks. DETAILED DESCRIPTION

[0032] The present invention will be further described in detail below with reference to the accompanying drawings and through specific embodiments. It should be noted that the following embodiments should not be construed as limiting the present invention.

[0033] The material-liquid ratio of the seahorse and water described in the present invention is the mass-to-volume ratio of the seahorse and water, with the mass unit being g and the volume unit being mL.

[0034] The instruments and reagents used in the embodiments of the present invention are: 1. Sample information Three-spot seahorse (batch number 202312072), distended seahorse (batch number 2401075), lined seahorse (batch number 20230218), spiny seahorse (batch number 20230124), and large seahorse (batch number 20230328) were all purchased from Zhengtong Pharmacy, and the manufacturer was Bozhou Huikangtang Chinese Medicine Technology Co., Ltd.

[0035] 2. Instruments and test drugs YXQ-LB-100SⅡ vertical pressure steam sterilizer (Boxun Technology Co., Ltd.), H1650-W high-speed centrifuge (Hunan Xiangyi Company), EYELA N-3010 rotary evaporator, SCIENTZ-18N freeze dryer, SpectraMax L microplate reader (Molecular Devices, USA), clean bench, CO2 cell culture incubator (Shanghai Puhexi Health Medical Equipment Co., Ltd.), 150-300Da nanofiltration membrane (GE), 20kDa cutoff ultrafiltration membrane (Millipore), pepsin, trypsin, and pancreatic enzyme (Sinopharm Chemical Reagent Co., Ltd.), papain, bromelain, and alkaline protease (Beijing Solaibao Biotechnology Co., Ltd.), human embryonic kidney HEK293 cells (Wuhan Punosai Life Science Technology Co., Ltd.), rosastat (Aladdin), luciferase (Tropix Biotechnology Co., Ltd., USA) Inc., Bedford, MA), FlexA-200 enzyme-linked immunosorbent assay (Hangzhou Aosheng Instrument Co., Ltd.), gentamicin sulfate (Selleckchem), inosine assay kit (Nanjing Jiancheng Bioengineering Institute), urea nitrogen test kit (Nanjing Jiancheng Bioengineering Institute), animal blood cell analysis and detection PDX kit (IDEXX Laboratories, USA), sodium chloride injection (Chenxin Pharmaceutical Co., Ltd.), etc.

[0036] 3. Experimental Animals SPF-grade, healthy, virgin Wistar male rats weighing 170–190 g were used (Wei Tong Lihua Laboratory Animal Technology Co., Ltd.).

[0037] Example 1: Preparation of hippocampus extract S1: Cut the hippocampus trispotus into pieces, add water at a material-liquid ratio of 1:15, soak for 2 hours, and homogenize for 5 minutes to obtain hippocampus homogenate.

[0038] S2: The pH of the hippocampus homogenate was adjusted to 2, and pepsin was added at a concentration of 60 U per gram of hippocampus. The hippocampus was extracted at a temperature of 37° C. and a stirring speed of 150 rpm for 2 hours to obtain a hippocampus extract.

[0039] S3: The hippocampus extract was heated to 100°C, the enzyme was inactivated for 10 minutes, the extract was cooled to room temperature, and the extract was centrifuged at 10,000 rpm for 10 minutes to remove precipitated impurities and separate the supernatant.

[0040] S4: Parallel preparation of desalination solutions after three different membrane separation operations: (1) Desalting the supernatant in step S3 by passing it through a nanofiltration membrane with a molecular weight cutoff of 150-300 Da; (2) The supernatant in step S3 is separated using an ultrafiltration membrane with a molecular weight cutoff of 20 kDa, and the ultrafiltration solution with a molecular weight less than 20 kDa is collected and then desalted by a nanofiltration membrane with a molecular weight cutoff of 150-300 Da; (3) The supernatant in step S3 is separated using an ultrafiltration membrane with a molecular weight cutoff of 20 kDa, and the ultrafiltration solution with a molecular weight greater than 20 kDa is collected and then desalted by a nanofiltration membrane with a molecular weight cutoff of 150-300 Da.

[0041] The specific process of ultrafiltration is as follows: add 3 times the volume of double distilled water to the solution to be ultrafiltered, ultrafilter to half the volume, then add water to the original volume, and repeat 2 times.

[0042] The specific process of nanofiltration desalination is: add equal volumes of double-distilled water to the solutions that need to be nanofiltered respectively, replenish the liquid at the same time during the desalination process, keep the volume unchanged, until the conductivity value of the outflowing liquid is close to that of double-distilled water, and obtain three desalted solutions.

[0043] S5: The three desalted solutions in step S4 are concentrated under reduced pressure to 100-200 mL at 50° C. and 30 hPa, and then freeze-dried at 20-40 Pa and -60° C. to -40° C. for 2-3 days to obtain a total hippocampal extract, a product with a molecular weight of less than 20 kDa, and a product with a molecular weight of greater than 20 kDa, respectively.

[0044] Example 2: Preparation of hippocampus extract Except for the following steps, the remaining steps of this embodiment are the same as those of embodiment 1.

[0045] S2: Adjust the pH of the hippocampus homogenate to 2.0, add pepsin at a concentration of 25 U per gram of hippocampus, and extract at a temperature of 37°C and a stirring speed of 150 rpm for 2 hours; adjust the pH to 7.5, add trypsin at a concentration of 1000 U per gram of hippocampus, and extract at a temperature of 37°C and a stirring speed of 150 rpm for 2 hours to obtain a hippocampus extract.

[0046] S3: The hippocampus extract was heated to 100°C, the enzyme was inactivated for 20 minutes, the extract was cooled to room temperature, and the extract was centrifuged at 8000 rpm for 15 minutes to remove precipitated impurities and separate the supernatant.

[0047] Example 3: Preparation of hippocampus extract components This example adopts a high-temperature and high-pressure water extraction process to prepare the hippocampus extract. The specific operation is the same as that in Example 1 except for the following steps.

[0048] S2: The hippocampus homogenate was extracted at 100°C and 0.4 MPa for 4 h to obtain the hippocampus extract.

[0049] S3: The hippocampus extract was cooled to room temperature, centrifuged at 3000 rpm for 20 minutes to remove precipitated impurities, and the supernatant was separated.

[0050] Example 4: Preparation of Hippocampus Extract In this embodiment, a low-temperature, normal-pressure water extraction process is used to prepare the hippocampus extract. The specific operation is the same as that in Example 1 except for the following steps.

[0051] S1: Cut the distended hippocampus into pieces, soak them in a material-liquid ratio of 1:20 for 1 hour, and homogenize them for 5 minutes to obtain hippocampal homogenate.

[0052] S2: The hippocampus homogenate was extracted at 40°C for 5 hours to obtain the hippocampus extract.

[0053] S3: The hippocampus extract was heated to 100°C, cooled to room temperature, and centrifuged at 5000 rpm for 20 minutes to remove precipitated impurities and separate the supernatant.

[0054] S5: The three desalted solutions in step S4 are concentrated under reduced pressure to 100-200 mL at 45°C and 25 hPa, and then dried under reduced pressure at -0.1 MPa to 0 MPa and 30°C to 60°C for 2-3 days to obtain a total hippocampal extract, a product with a molecular weight of less than 20 kDa, and a product with a molecular weight of greater than 20 kDa.

[0055] Example 5: Preparation of hippocampus extract Except for the following steps, the remaining steps of this embodiment are the same as those of embodiment 1.

[0056] S1: Cut the spiny hippocampus into pieces, soak them in a solid-liquid ratio of 1:10 for 1 hour, and homogenize them for 5 minutes to obtain hippocampus homogenate.

[0057] S2: Adjust the pH of the hippocampus homogenate to 7.0, add 5000 U of papain per gram of hippocampus, extract at 55° C. and 200 rpm for 4 hours to obtain a hippocampus extract.

[0058] Example 6: Preparation of Hippocampus Extract Except for the following steps, the remaining steps of this embodiment are the same as those of embodiment 1.

[0059] S1: Cut the hippocampus linearis into pieces, add water at a material-liquid ratio of 1:10, soak for 0.5 h, and homogenize for 5 minutes to obtain hippocampus homogenate.

[0060] S2: Adjust the pH of the hippocampus homogenate to 7.5, add bromelain, add 6000 U of bromelain per gram of hippocampus, extract at 40° C. and stirring at 250 rpm for 5 hours to obtain a hippocampus extract.

[0061] S3: The hippocampus extract was heated to 100°C, the enzyme was inactivated for 20 minutes, the extract was cooled to room temperature, and the extract was centrifuged at 12,000 rpm for 20 minutes to remove precipitated impurities and separate the supernatant.

[0062] Example 7: Preparation of Hippocampus Extract Except for the following steps, the remaining steps of this embodiment are the same as those of embodiment 1.

[0063] S1: Cut the distended hippocampus into pieces, add water at a material-liquid ratio of 1:5, soak for 2 hours, and homogenize for 5 minutes to obtain hippocampus homogenate.

[0064] S2: The pH of the hippocampus homogenate was adjusted to 8.5, and pancreatin was added at a concentration of 200 U per gram of hippocampus. The hippocampus was extracted at a temperature of 45° C. and a stirring speed of 150 rpm for 3 hours to obtain a hippocampus extract.

[0065] S3: The hippocampus extract was heated to 100°C, the enzyme was inactivated for 15 minutes, cooled to room temperature, centrifuged at 4000 rpm for 20 minutes to remove precipitated impurities, and the supernatant was separated.

[0066] Example 8: Preparation of Hippocampus Extract Except for the following steps, the remaining steps of this embodiment are the same as those of embodiment 1.

[0067] S1: Cut the seahorse into pieces, add water at a material-liquid ratio of 1:5, soak for 2 hours, and homogenize for 5 minutes to obtain seahorse homogenate.

[0068] S2: Adjust the pH of the hippocampus homogenate to 9, add alkaline protease, add 2000 U of alkaline protease per gram of hippocampus, extract at 55° C. and stirring speed of 300 rpm for 5 hours to obtain hippocampus extract.

[0069] In combination with Examples 1-8, it can be seen that Example 3 is a high-temperature and high-pressure water extraction process for preparing seahorse extract, Example 4 is a low-temperature and normal-pressure water extraction process for preparing seahorse extract, and Examples 1-2 and Examples 5-8 are seahorse extracts prepared by different enzymatic hydrolysis processes. The yields of different products in Examples 1 to 8 were measured, and the statistical results are shown in Table 1 for details.

[0070] Table 1. Yields of different products in Examples 1 to 8

[0071] As can be seen from Table 1, in Examples 1 to 8, the yield of the total extract of hippocampus is relatively high, ranging from 16.83% to 91.73%, and the yield of the portion with a molecular weight of less than 20 kDa is 8.02% to 87.15%; and the proportion of the portion with a molecular weight of less than 20 kDa in the total extract of hippocampus is greater than 50%, indicating that most of the total extract of hippocampus is the portion with a molecular weight of less than 20 kDa; at the same time, combined with Table 3, it can be seen that the yield of the product with a molecular weight of less than 20 kDa obtained by the water extraction process is less than the yield of the product with a molecular weight of less than 20 kDa obtained by the enzymatic hydrolysis process, among which the yield of the product with a molecular weight of less than 20 kDa obtained by the enzymatic hydrolysis method is 60%-90%, which also provides reference significance for subsequent related research.

[0072] Example 9: In vitro evaluation of the blood-tonifying effect of seahorse extract In this example, the total extract of hippocampus obtained in Examples 1 to 3, the product with a molecular weight less than 20 kDa, and the product with a molecular weight greater than 20 kDa were selected as the test sample group. A human embryonic kidney cell line, HEK293, was used to construct a research model for detecting the expression regulation of erythropoietin (EPO). The cells were transfected with a hypoxia response element-luciferase vector, and the in vitro blood-enriching efficacy of the hippocampus extract was evaluated. The hypoxia response element is located upstream of the EPO channel. In an oxygen-deficient environment, the hypoxia response component can be stimulated to promote EPO production. A firefly luciferase reporter gene was inserted into the downstream sequence of the hypoxia response element. If the test sample can stabilize the hypoxia-inducible element HIF- α It can activate the hypoxia response element and then express the firefly fluorescein reporter gene. After adding the drug, the level of drug regulation on EPO can be tested by detecting the intensity of fluorescein luminescence. The details are as follows: HEK293 cells were seeded in 96-well plates at 2 × 10 cells per well. 4 The total extract of hippocampus prepared in Examples 1 to 3 and the product with a molecular weight of less than 20 kDa and the product with a molecular weight of greater than 20 kDa were added to the cells using a high-throughput sampling needle (the final concentration of the drug was 1000 μ g / mL), with 40 μRosastat was used as a positive drug at a final concentration of 37 M, and incubated for 24 hours at 37°C and 5% CO2. Luciferin enzyme assay substrate was added to each well and incubated in a dark incubator for 5 minutes. Fluorescein activity was measured using a SpectraMax L microplate reader. Relative luciferin activity represented blood-enriching activity. Relative luciferase activity = luciferin activity in the sample group / luciferin activity in the blank control group × 100%. The experimental results showed that compared with the blank control group (fluorescent mycin activity was 100%), the total extract of hippocampus and the product with a molecular weight of less than 20 kDa in the positive control group and Examples 1 to 3 were significantly higher than those in the blank control group (fluorescent mycin activity was 100%). μ The groups with a molecular weight of 20 kDa and 20 kDa can significantly increase the relative luciferase activity, while the products with a molecular weight greater than 20 kDa cannot effectively increase the luciferase activity. This indicates that the products with a molecular weight less than 20 kDa obtained after ultrafiltration membrane interception are the main components of the hippocampus that play a role in blood-tonifying activity. Figures 1-3 .

[0073] Example 10: Evaluation and Analysis of the Hematogenic Activity of Seahorse Extract In this example, the total extracts of hippocampus obtained in Examples 1 to 8 and products with a molecular weight of less than 20 kDa were selected as the test sample group. A human embryonic kidney cell line HEK293 was used to construct a research model for detecting the expression regulation of erythropoietin (EPO) and to evaluate its blood-tonifying activity (the final concentration of the drug was 250 and 1000 μg / mL). μ g / mL).

[0074] The experimental results are shown in Table 2. The experimental results show that compared with the blank control group (fluorescein activity is 100%), the positive control group and the hippocampus extracts 250, 1000 μ g / mL administration groups could significantly increase the relative luciferase activity, and the relative luciferase activity of the rosastat positive control group was 538.94%±70.10% ( p <0.001). The relative luciferase activity of the hippocampus extract-treated groups in Examples 1-8 ranged from 133.78% to 1404.93%, indicating that the hippocampus extracts of Examples 1-8 significantly increased luciferase activity, promoted EPO production, and exhibited significant blood-tonifying activity. Furthermore, the blood-tonifying activity of the hippocampus extract with a molecular weight less than 20 kDa was significantly higher than that of the total hippocampus extract, indicating that the blood-tonifying activity of the hippocampus was further enhanced after further separation and purification. The statistical results are detailed in Table 2.

[0075] Table 2. In vitro blood-enriching activity results of different products from Examples 1 to 8 (Note: Compared with the blank control group, *** represents p <0.001, ** represents p <0.01, * representsp <0.05)

[0076] Example 11: Analysis of the therapeutic effect of hippocampus extract on renal anemia In this example, a rat model of renal injury anemia was established to evaluate the therapeutic effect of the hippocampus extract with a molecular weight less than 20 kDa prepared in Example 3 on renal anemia, as follows: Modeling: Wistar male rats were acclimated to feeding for one week and then randomly divided into a blank group and a model group based on body weight. The model group received an intraperitoneal injection of 100 mg / kg of gentamicin sulfate (1 mL / 100 g rat body weight) dissolved in normal saline. The blank group received an equal volume of normal saline. Treatment continued for two weeks. After the last dose, orbital blood was drawn for measurement of inosine, urea nitrogen, and routine blood tests. Compared with the blank group, the model group showed significantly elevated inosine and urea nitrogen levels, indicating the development of renal damage. Routine blood tests revealed significantly lower hemoglobin, red blood cell count, reticulocyte, and immature reticulocyte levels in the model group compared to the blank control group, indicating successful establishment of a renal injury anemia model. Statistical results are shown in Table 3.

[0077] Table 3. Results of kidney function and blood routine indexes after two weeks of modeling (Note: Compared with the blank control group, *** represents p <0.001, ** represents p <0.01, * represents p <0.05)

[0078] Grouping and Dosing: Model rats were sorted according to reticulocyte count and divided into a serpentine pattern into a model group, a positive drug group, and a hippocampus extract group. After grouping, all groups except the blank group continued to receive intraperitoneal injections of gentamicin sulfate. Each group was gavaged daily according to body weight. The blank and model groups received purified water, the positive drug group received 30 mg / (kg·d) of rosastat, and the hippocampus extract group received the extract (with a molecular weight less than 20 kDa) prepared in Example 3. The dose was twice the human clinical equivalent dose (0.45 g / (kg·d) of hippocampus extract (with a molecular weight less than 20 kDa)). Administration was continued by gavage for 4 weeks.

[0079] Index detection: The rats' condition was observed and their body weight was recorded during the administration period. After the experiment, blood was collected from the eye sockets to measure blood routine, inosine, and urea nitrogen levels to evaluate the efficacy in improving renal anemia.

[0080] The experimental results showed that compared with the model group, the rats in the hippocampus extract group had a good mental state, shiny hair, and rapid weight gain. After 5 days of administration, their weight reached the level of the blank group, and then their weight was always higher than that of the blank group. In addition, the weight growth rate of the rats in the hippocampus extract group during the administration period was higher than that of the blank group and the positive control group. See Table 4 and Figure 4 After the administration, the levels of inosine and urea nitrogen were measured. Compared with the blank group, the levels of inosine and urea nitrogen in the model group were significantly increased; while compared with the model group, the levels of inosine and urea nitrogen in the hippocampus extract group and the positive drug group were significantly decreased ( P <0.05), indicating that hippocampus can improve renal damage and restore renal function; blood item analysis found that compared with the blank group, the hemoglobin, red blood cell count, reticulocytes, immature reticulocyte ratio, high fluorescence intensity reticulocyte ratio, and medium fluorescence intensity reticulocyte ratio of the model group rats were significantly reduced, and the ratio of low fluorescence intensity reticulocytes was significantly increased, indicating that the bone marrow hematopoietic function of the model group rats was not restored; compared with the model group, the hemoglobin, red blood cell count, reticulocytes, immature reticulocyte ratio, high fluorescence intensity reticulocyte ratio, and medium fluorescence intensity reticulocyte ratio of the hippocampus extract group were significantly increased, and the ratio of low fluorescence intensity reticulocytes was significantly decreased, see Table 5 for details; the above shows that hippocampus can promote the formation of immature reticulocytes and promote the transformation of reticulocytes into mature red blood cells, thereby promoting the recovery of bone marrow hematopoietic function and having the effect of improving renal anemia.

[0081] Table 4. Body weight changes of rats after 4 weeks of drug administration (g)

[0082] Table 5. Results of kidney function and blood routine indexes after two weeks of modeling (Note: Compared with the blank control group, *** represents p <0.001, ** represents p <0.01, * represents p <0.05; compared with the model group, ### represent p <0.001, ## represent p <0.01, # represent p <0.05;)

[0083] As shown above, in the human embryonic kidney cell HEK293 cell line erythropoietin expression regulation model, both the total hippocampus extract and the product with a molecular weight less than 20kDa can significantly increase the activity of luciferase and promote the production of EPO, showing significant blood-tonifying activity. The blood-tonifying activity of the hippocampus extract with a molecular weight less than 20kDa is significantly higher than that of the total hippocampus extract. Further verification of the efficacy of the hippocampus extract with a molecular weight less than 20kDa in improving renal anemia in vivo found that it can improve renal function indicators in rats with renal damage anemia model, restore renal function, promote the production of immature reticulocytes, and promote the transformation of reticulocytes into mature red blood cells, showing significant efficacy in improving renal anemia.

[0084] Therefore, seahorse extract can be widely used in the preparation of drugs and foods for improving kidney damage, improving renal anemia, improving blood counts, stimulating erythropoietin, increasing hemoglobin, promoting reticulocyte production, increasing the percentage of immature reticulocytes, reducing the percentage of reticulocytes with low fluorescence intensity, and improving hematopoietic capacity, and has broad application prospects.

[0085] The embodiments described above are only part of the embodiments of the present invention, rather than all the embodiments, and are not intended to limit the present invention. Based on the embodiments of the present invention, those skilled in the art may make several improvements and supplements without departing from the principles of the present invention, and these improvements and supplements should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a hippocampus extract having the activity of improving renal anemia, characterized in that: The following steps are involved: S1: Cut the hippocampus into pieces, soak it in water, and homogenize it to obtain hippocampal homogenate; S2: adjusting the pH of the hippocampus homogenate, adding biological protease for extraction, and obtaining the hippocampus extract; S3: heating the hippocampus extract to inactivate enzymes, cooling to room temperature, and centrifuging to remove precipitated impurities, thereby separating the supernatant; S4: performing membrane separation on the supernatant to obtain a desalted solution; S5: concentrating the desalted solution and drying it to obtain a hippocampus extract having activity in improving renal anemia.

2. The method for preparing a hippocampus extract having the activity of improving renal anemia according to claim 1, characterized in that: The seahorse is at least one of the three-spotted seahorse, the lined seahorse, the large seahorse, the spiny seahorse, the small seahorse and the distended-belly seahorse.

3. The method for preparing a hippocampus extract having the activity of improving renal anemia according to claim 1, characterized in that: In step S1, the material-liquid ratio of seahorse to water is 1:5-40.

4. The method for preparing a hippocampus extract having the activity of improving renal anemia according to claim 1, characterized in that: In step S2, the biological protease is at least one of pepsin, bromelain, papain, neutral protease, trypsin, pancreatin and alkaline protease.

5. The method for preparing a hippocampus extract having the activity of improving renal anemia according to claim 1, characterized in that: The amount of the biological protease used in step S2 is 0-8000 U per gram of hippocampus mass; the pH value is 1-9; and the extraction conditions are a temperature of 30-100° C. and a pressure of 0-0.4 MPa.

6. The method for preparing a hippocampus extract having the activity of improving renal anemia according to claim 1, characterized in that: The membrane separation operation in step S4 is specifically to first filter with an ultrafiltration membrane and then perform nanofiltration to remove salt. The molecular weight cutoff of the ultrafiltration membrane is 20 kDa, and the molecular weight cutoff of the nanofiltration membrane is 150-300 Da.

7. The hippocampus extract prepared by the preparation method according to any one of claims 1 to 6, characterized in that: The molecular weight of the hippocampus extract is less than 20 kDa.

8. Use of the hippocampus extract according to claim 7 in preparing food and / or medicine for improving renal anemia.

9. The use according to claim 8, characterized in that The improvement of renal anemia specifically includes at least one of improving renal damage, improving renal functional indicators, improving renal anemia, improving blood counts, increasing erythropoietin, increasing hemoglobin, increasing red blood cells, promoting reticulocyte production, increasing the percentage of immature reticulocytes, reducing the percentage of reticulocytes with low fluorescence intensity, and improving hematopoietic capacity.

10. The use according to claim 9, characterized in that The indicators for improving kidney function are specifically inosine and urea nitrogen.

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