Donkey-hide gelatin sustained-release granules, preparation method and application

Abstract

The invention discloses colla corii asini sustained-release granules as well as a preparation method and application thereof, and relates to the technical field of functional foods and medicines. A prescription of the colla corii asini sustained-release granules comprises the following components in parts by weight: 100 parts of colla corii asini blocks, 8-15 parts of ethyl cellulose, 0.5-2 parts of a plasticizer and 0.5-3 parts of an anti-sticking agent. The traditional colla corii asini powder is subjected to sustained-release film coating, so that the stability of the colla corii asini can be improved, and the sustained release of the colla corii asini can be realized and the in-vivo action duration can be prolonged according to the release rate measurement, thereby improving the bioavailability; the invention provides a novel colla corii asini dosage form for the development of functional foods and medicines for improving blood stasis syndrome and microcirculation disturbance.

Description

Technical Field

[0001] The present invention relates to the technical fields of functional foods and medicines, and particularly relates to a donkey-hide gelatin sustained-release granule, a preparation method and an application thereof. Background Art

[0002] Since ancient times, donkey-hide gelatin has been a relatively precious herb and also a famous traditional Chinese medicine in China. It has a medicinal history of more than 2,500 years. It first appeared in "Shennong's Herbal Classic" and is known as the "sacred medicine for nourishing blood". As a top-grade tonic, donkey-hide gelatin has the effects of treating limb pain and soreness, preventing miscarriage, replenishing qi and nourishing yin, expelling wind and moistening dryness, resolving phlegm and clearing the lungs, etc. In addition, it has outstanding effects in nourishing blood, tonifying blood and stopping bleeding. Donkey-hide gelatin is one of the first batch of Chinese medicinal materials that are both medicine and food. Modern research shows that the main components of donkey-hide gelatin are proteins and their decomposition products, with a molecular weight distribution of 10 - 250KDa, and contain a variety of active components, such as collagen, immunoglobulin, biglycan, lumican and other small leucine-rich proteoglycans, glycosaminoglycans, etc. The sustained-release film coating of drugs enables the drugs to be slowly released in the gastric juice according to a certain rule, and the drugs maintain an effective drug concentration for a long time, so as to reduce the drug dosage, improve the bioavailability and drug efficacy, and prolong the drug action time. At present, the research on new dosage forms of donkey-hide gelatin, especially sustained-release donkey-hide gelatin, is still blank.

[0003] Microcirculation is the blood circulation in the capillaries between the arterioles and venules. It is the basic structural and functional unit of the circulatory system, including the blood circulation in arterioles, venules, lymphatic capillaries and tissue ducts. The microcirculation capillary network is an aggregate with a diameter less than 100μm, responsible for delivering oxygen to tissues, exchanging nutrients and wastes, and regulating inflammation and coagulation. Blood microcirculation disorders can lead to local ischemia, hypoxia and perfusion injury by affecting local tissue blood perfusion and changing blood flow resistance, and further cause organ function damage. Microcirculation disorders often occur under pathological conditions such as inflammatory reactions and metabolic disorders, and are mostly manifested as microvascular varicose, deformed, spastic, atrophic or occluded, damaged inner walls of blood vessels, increased permeability, red blood cell exudation and bleeding around microvessels, slowed blood flow velocity, blood stasis, microthrombus formation, vascular embolism, reduced deformability of red blood cells, etc.

[0004] In traditional Chinese medicine theory, blood stasis syndrome is caused by "abnormal blood circulation" or "blocked blood vessels", manifested as "blood coagulation without stasis" and "stagnation without flow". Its pathological basis is blood circulation disorders, especially microcirculation disorders and hemorheological changes. Microcirculation disorders in various organs of the human body can cause irreversible damage. For example, microcirculation disorders in the brain can lead to the destruction of the blood-brain barrier, causing cerebral edema and neuron death, and then leading to cognitive decline and neurodegenerative diseases; pulmonary microcirculation disorders can affect the body's hematopoiesis and hemostasis functions, and cause pulmonary edema and lung injury; intestinal microcirculation disorders can cause damage to the intestinal barrier function by stimulating inflammatory cell reactions, resulting in the transfer of bacteria, proteases, and toxins from the intestine to the systemic circulation. Commonly used drugs for improving microcirculation mainly include vasoprotective drugs, anticoagulants, thrombolytic and defibrinating drugs, antiplatelet drugs, vasodilators, and drugs for reducing blood viscosity. Although they can improve blood stasis and microcirculation disorders to a certain extent, they have certain toxic and side effects on the human body. Studies have confirmed that donkey-hide gelatin or its compound has the effects of improving nailfold microcirculation, cardiac microcirculation, and cerebrovascular microcirculation, but the improvement effect of donkey-hide gelatin sustained-release granules on systemic microcirculation disorders has not been confirmed. Currently, donkey-hide gelatin powder is inconvenient to consume. It needs to be dissolved in warm water and taken orally, and the dissolution time is relatively long. Summary of the Invention

[0005] To overcome the above defects, the present invention provides a donkey-hide gelatin sustained-release granule, a preparation method, and an application. In this study, the sustained-release technology is applied to the donkey-hide gelatin production process to prepare a sustained-release donkey-hide gelatin granule, which can achieve the slow release of donkey-hide gelatin, improve the bioavailability and biological activity.

[0006] The object of the present invention is achieved by the following technical solutions: A donkey-hide gelatin sustained-release granule, the components and weight parts of its prescription include: 100 parts of donkey-hide gelatin block, 8-15 parts of ethyl cellulose, 0.5-2 parts of plasticizer, and 0.5-3 parts of anti-adhesive.

[0007] Preferably, the components and weight parts of its prescription include: 100 parts of donkey-hide gelatin block, 8 parts of ethyl cellulose, 0.5 part of plasticizer, and 0.5 part of anti-adhesive.

[0008] Preferably, the components and weight parts of its prescription include: 100 parts of donkey-hide gelatin block, 15 parts of ethyl cellulose, 2 parts of plasticizer, and 3 parts of anti-adhesive.

[0009] Preferably, the components and weight parts of its prescription include: 100 parts of donkey-hide gelatin block, 12 parts of ethyl cellulose, 1 part of plasticizer, and 2 parts of anti-adhesive.

[0010] Preferably, the plasticizer is triethyl citrate, and the anti-adhesive is talcum powder.

[0011] Preferably, the preparation method of the Ejiao block includes: drying the skin or fresh skin of Equus asinus L. of the Equidae family, followed by decocting, concentrating, adding excipients, forming, and drying.

[0012] The present invention also provides a preparation method of Ejiao sustained-release granules, which includes the following steps: 1) Preparing materials: Prepare materials according to the components and weight portions of the above prescription. 2) Adding ethyl cellulose to an ethanol aqueous solution with a volume ratio of 85% and stirring until the ethyl cellulose is completely dissolved to form a uniform and transparent solution. 3) Adding plasticizers and anti-adhesives: Add the prescribed amounts of plasticizers and anti-adhesives to the ethyl cellulose solution, and continue stirring to dissolve the plasticizers and make the anti-adhesives uniformly suspended in the solution. 4) Filtering the coating solution: Filter the prepared coating solution through a 120-mesh sieve to remove possible lumps. 5) Pretreating Ejiao: Crush the Ejiao block with a pulverizer and pass it through an 80-mesh sieve. 6) Putting the crushed Ejiao powder into a fluidized bed coater, turning on the blower, and adjusting the inlet air temperature to 40 - 50 °C to preheat the powder in the air stream for 10 - 15 minutes. 7) Setting fluidized bed coating parameters: Control the inlet air temperature at 40 - 50 °C, the outlet air temperature at 30 - 40 °C, adjust the blower frequency according to the fluidization state of the powder to keep the powder in a good fluidization state, control the spray pressure of the spray gun at 0.15 MPa, and adjust the spray speed according to the concentration of the coating solution and the coating progress. 8) When the Ejiao powder reaches the preheating temperature and is in a stable fluidization state, turn on the spray gun and evenly spray the coating solution into the fluidized bed. Closely observe the fluidization state and coating situation of the powder, and timely adjust various parameters to ensure uniform coating. 9) After spraying the coating solution, continue to fluidize the powder in the fluidized bed for 15 minutes to further dry and solidify the coating film to obtain Ejiao sustained-release granules. 10) Take out the coated Ejiao sustained-release granules from the fluidized bed coater, put them into a drying oven, and age them at 50 °C for 3 hours to further remove residual solvents and moisture and make the coating film dense and reliable. 11) Pass the dried Ejiao sustained-release granules through a 60 - 80-mesh sieve to remove large particles and obtain uniform Ejiao sustained-release granules.

[0013] Preferably, the stirring speed in step 1) is controlled at 300 - 500 r / min.

[0014] The present invention also provides an application of the Ejiao sustained-release granules. The above-mentioned Ejiao sustained-release granules can be used to prepare traditional Chinese medicine preparations or other functional foods.

[0015] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows: (1) By performing sustained-release film coating on traditional donkey-hide gelatin powder, the stability of donkey-hide gelatin can be improved. Through dissolution rate determination, it is found that the slow release of donkey-hide gelatin can be achieved, prolonging the action duration in the body, thereby improving the bioavailability. The wetting agent used in the coating solution of the present invention is 85% anhydrous ethanol, which can be removed by volatilization later and does not contain toxic and harmful solvents, and will not cause adverse effects on the product. The content of donkey-hide gelatin in the finally prepared sustained-release film-coated donkey-hide gelatin powder of the present invention is as high as about 90%. Compared with general sustained-release coating methods, the contents of sustained-release agents, plasticizers, and anti-adhesion agents used are lower, ensuring the donkey-hide gelatin content to the greatest extent.

[0016] (2) It is verified by animal experiments that the sustained-release film-coated donkey-hide gelatin powder prepared by the present invention can significantly increase the blood perfusion volume of mice with microcirculation disorders, increase the number of open capillaries, improve blood rheology (whole blood viscosity, plasma viscosity, hematocrit, fibrinogen, erythrocyte sedimentation rate, etc.), inhibit the inflammatory response, and improve the blood flow grade of mice with microcirculation disorders compared with the donkey-hide gelatin powder without sustained-release film coating, thus having better biological pharmacodynamic effects. The present invention provides a new donkey-hide gelatin dosage form for the development of functional foods and drugs related to improving blood stasis syndrome and microcirculation disorders. Description of the Drawings

[0017] In order to more clearly illustrate the content of the present invention, the following further describes the present invention in detail according to specific embodiments in combination with the drawings, where: Figure 1 is the in vitro release curve of Effect Example 1; Figure 2 is the effect of Effect Example 3 on the body weight of mice with microcirculation disorders; Figure 3 is the effect of Effect Example 3 on the blood perfusion volume of mice with microcirculation disorders; among them, A is the average blood perfusion volume of the auricles of mice in each group; B is the difference in PU of the blood perfusion volume of the auricles of mice in each group; C is the representative image of blood flow imaging of the auricles of mice in each group; * represents the significant difference compared with the model group in each group, * is P < 0.05, and ** is P < 0.01; Figure 4 is the real-time blood perfusion volume of the auricles of mice with microcirculation disorders in each group in Effect Example 3; among them, the black broken line represents the real-time blood perfusion volume of the whole ear, and the blue broken line represents the real-time blood perfusion volume of the ROI area in the circle; Figure 5 is the effect of Effect Example 3 on the number of open capillaries in the marginal ear veins of mice with microcirculation disorders; among them, A is the number of open capillaries in mice in each group; B is the image of the marginal ear vein capillaries of mice; * represents the significant difference compared with the model group in each group, * is P < 0.05; Figure 6 Effect of Example 3 of the efficacy on hemorheology in mice with microcirculation disturbance; wherein, A is low shear whole blood viscosity, B is medium shear whole blood viscosity, C is high shear whole blood viscosity, D is plasma viscosity, E is hematocrit, F is erythrocyte sedimentation rate, G is fibrinogen, * indicates P < 0.05, ** indicates P < 0.01; Figure 7 Effect of Example 3 of the efficacy on vascular active factors and inflammatory levels in mice with microcirculation disturbance; wherein, A is plasminogen activator inhibitor-1 (PAI-1), B is stable metabolite of prostacyclin (6-keto-PGF), C is tumor necrosis factor-α (TNF-α), D is interleukin-1β (IL-1β), * indicates P < 0.05, ** indicates P < 0.01; Figure 8 Effect of Example 3 of the efficacy on histopathology of liver and kidney in mice with microcirculation disturbance. Detailed implementation mode

[0018] Example 1: A donkey-hide gelatin sustained-release granule, the components and weight parts of its prescription include: 100 parts of donkey-hide gelatin block, 10 parts of ethyl cellulose, 1 part of triethyl citrate, and 1 part of talcum powder.

[0019] A preparation method of the donkey-hide gelatin sustained-release granule includes the following steps: 1) Prepare materials according to the components and weight parts of the above prescription; 2) Add ethyl cellulose to an ethanol aqueous solution with a volume ratio of 85% and stir, controlling the stirring speed at 300 r / min until the ethyl cellulose is completely dissolved to form a uniform and transparent solution; 3) Add plasticizer and anti-adhesive: Add the prescribed amount of triethyl citrate and talcum powder to the ethyl cellulose solution, continue to stir to dissolve the triethyl citrate, and make the talcum powder evenly suspended in the solution; 4) Filter the coating solution: Filter the prepared coating solution through a 120-mesh sieve to remove possible lumps; 5) Pretreat donkey-hide gelatin: Crush the donkey-hide gelatin block with a pulverizer and pass through an 80-mesh sieve; 6) Put the pulverized donkey-hide gelatin powder into a fluidized bed coater, turn on the blower, adjust the inlet air temperature to 45 °C, and preheat the powder in the air stream for 15 minutes; 7) Set the fluidized bed coating parameters: Control the inlet air temperature at 40 °C, the outlet air temperature at 30 °C, adjust the blower frequency according to the fluidization state of the powder to make the powder in a good fluidization state, control the spray pressure of the spray gun at 0.15 MPa, and the spray speed at 7 mL / min; 8) After the donkey-hide gelatin powder reaches the preheating temperature and is in a stable fluidized state, turn on the spray gun and evenly spray the coating solution into the fluidized bed. Closely observe the fluidization state of the powder and the coating situation, and adjust various parameters in a timely manner to ensure uniform coating. 9) After the coating solution is sprayed out, continue to fluidize the powder in the fluidized bed for 15 minutes to further dry and solidify the coating film to obtain the donkey-hide gelatin sustained-release granules. 10) Take out the coated donkey-hide gelatin sustained-release granules from the fluidized bed coater and put them into a drying oven. Age them at 50°C for 3 hours to further remove residual solvents and moisture, and make the coating film dense and reliable. 11) Pass the dried donkey-hide gelatin sustained-release granules through a 60-80 mesh sieve to remove large particles and obtain uniform donkey-hide gelatin sustained-release granules.

[0020] Perform quality control on the prepared sustained-release film-coated donkey-hide gelatin powder. Observe the appearance of the coated powder and find that the color is uniform, without obvious adhesion and breakage. Use the sieving method to measure the particle size distribution of the coated powder, which meets the specified range. Observe the thickness of the coating film through a microscope and find that the coating thickness is uniform. Therefore, the sustained-release film-coated donkey-hide gelatin powder obtained in this example meets the quality requirements.

[0021] The usage frequency of the donkey-hide gelatin sustained-release granules of the present invention is 1-3 times a day, 3-6 grams each time, and the usage days are more than 30 days.

[0022] Example 2: A kind of donkey-hide gelatin sustained-release granules, the components and weight parts of its prescription include: 100 parts of donkey-hide gelatin blocks, 12 parts of ethyl cellulose, 0.5 part of triethyl citrate, and 0.5 part of talcum powder.

[0023] A preparation method of a kind of donkey-hide gelatin sustained-release granules, comprising the following steps: 1) Prepare materials, and prepare materials according to the components and weight parts of the above prescription. 2) Add ethyl cellulose to an ethanol aqueous solution with a volume ratio of 85% and stir. Control the stirring speed at 500 r / min until the ethyl cellulose is completely dissolved to form a uniform and transparent solution. 3) Add plasticizer and anti-adhesive: Add the prescribed amount of triethyl citrate and talcum powder to the ethyl cellulose solution, and continue to stir to dissolve the triethyl citrate and make the talcum powder evenly suspended in the solution. 4) Filter the coating solution: Filter the prepared coating solution through a 120-mesh sieve to remove possible lumps. 5) Pretreatment of donkey-hide gelatin: Crush the donkey-hide gelatin blocks with a pulverizer and pass through an 80-mesh sieve. 6) Put the crushed donkey-hide gelatin powder into a fluidized bed coater, turn on the fan, and adjust the inlet air temperature to 45°C to preheat the powder in the air stream for 10 minutes. 7) Fluidized bed coating parameter setting: The inlet air temperature is controlled at 45°C, the outlet air temperature is controlled at 40°C, the fan frequency is adjusted according to the fluidization state of the powder to keep the powder in a good fluidization state, the spray pressure of the spray gun is controlled at 0.15 MPa, and the spray speed is 7 mL / min; 8) After the donkey-hide gelatin powder reaches the preheating temperature and is in a stable fluidization state, turn on the spray gun and evenly spray the coating solution into the fluidized bed. Closely observe the fluidization state of the powder and the coating situation, and adjust various parameters in a timely manner to ensure uniform coating; 9) After the coating solution is sprayed out, continue to fluidize the powder in the fluidized bed for 15 minutes to further dry and cure the coating film to obtain slow-release donkey-hide gelatin granules; 10) Take out the coated slow-release donkey-hide gelatin granules from the fluidized bed coater, put them into a drying oven, and age them at 50°C for 3 hours to further remove residual solvents and moisture and make the coating film dense and reliable; 11) Pass the dried slow-release donkey-hide gelatin granules through a 60-mesh sieve to remove large particles and obtain uniform slow-release donkey-hide gelatin granules.

[0024] Quality control was carried out on the slow-release film-coated donkey-hide gelatin powder prepared above. Observe the appearance of the coated powder and find that the color is uniform and there are no obvious adhesions and damages; the particle size distribution of the coated powder was determined by the sieving method and meets the specified range; the thickness of the coating film was observed through a microscope and found to be uniform. Therefore, the slow-release film-coated donkey-hide gelatin powder obtained in this example meets the quality requirements.

[0025] The usage frequency of the slow-release donkey-hide gelatin granules of the present invention is 1-3 times a day, 3-6 grams each time, and the usage days are more than 30 days.

[0026] Comparative example: Take donkey-hide gelatin pieces of the same source and the same batch as in Example 1 and Example 2, crush them with a pulverizer and pass them through an 80-mesh sieve without performing the slow-release coating technology treatment.

[0027] Effect Example 1: In vitro release experiment According to the dissolution and release determination method of Part IV of the Chinese Pharmacopoeia 2020 Edition, using purified water as the release medium, measure the total amount of protein released, at a temperature of 37°C and a rotation speed of 100 rpm. Draw a release curve with the drug release time as the abscissa and the drug release concentration as the ordinate to evaluate the slow-release effect. The results are as Figure 1As shown, the release rates of Example 1 at 2h, 4h, 8h, 12h, 18h, and 24h were 14.51%, 22.74%, 38.17%, 61.99%, 82.38%, and 95.62% respectively, achieving slow dissolution within 24h. The release rates of Example 2 at 2h, 4h, 8h, 12h, 18h, and 24h were 17.85%, 25.43%, 48.29%, 68.38%, 90.12%, and 95.26% respectively. The release speed was slightly faster than that of Example 1, but still achieved slow dissolution within 24h. The release rate of the comparative example was as high as 90.13% at 2h and almost complete release at 4h. The above results indicate that the donkey-hide gelatin sustained-release granules of the present invention have a 24h sustained-release function, can effectively control the release amount and release rate of donkey-hide gelatin in the body, and maintain a stable and long-term effective blood drug concentration.

[0028] Effect Example 2: Stability Experiment Take the same mass of the donkey-hide gelatin sustained-release granules prepared in Examples 1-2 and the donkey-hide gelatin powder of the comparative example. After packaging with aluminum foil, place them under the accelerated experiment conditions of a temperature of 37±2°C and a relative humidity of 75%±5%. Sample at 0, 1, 2, and 3 months respectively. Detect the properties of the test substances through sensory evaluation, and detect the cumulative release rates at 2h, 8h, 12h, and 24h respectively according to the method of Effect Example 1. The results of the stability test are shown in Table 1. From the perspective of sensory properties, at the 0th month, there was no obvious difference between Examples 1-2 and the comparative example, but the structures of Examples 1-2 were relatively loose compared to the comparative example, and the structure of the comparative example was relatively compact. This may be because the donkey-hide gelatin powder of the comparative example was not coated and had a smaller particle size. At the 1st month, there was no obvious change in the sensory properties of Examples 1-2 and the comparative example. At the 2nd and 3rd months, there was no obvious change in the properties of Examples 1-2, but there was an obvious caking phenomenon in the donkey-hide gelatin powder of the comparative example. This may be because the sustained-release coating formed a physical barrier on the surface of the donkey-hide gelatin powder, reducing the contact between the donkey-hide gelatin powder and moisture in the air, thus avoiding caking caused by moisture absorption. In addition, the coating material was evenly distributed on the surface of the donkey-hide gelatin powder, making it difficult for the particles to aggregate and bond with each other. The experimental results of this part prove that the sustained-release film coating technology of donkey-hide gelatin powder designed by the present invention can maintain the uniform morphology of donkey-hide gelatin powder, avoid moisture absorption and caking phenomena, and thus improve the stability of the product.

[0029] From the results of the cumulative release rate, as the storage time of the accelerated experiment extended, the cumulative release percentage of the sustained-release film-coated donkey-hide gelatin powder prepared in Examples 1-2 did not change significantly and was significantly better than that of the comparative example, indicating that the sustained-release film-coated donkey-hide gelatin powder prepared by the present invention has good stability and can effectively prevent phase separation in the system.

[0030] Table 1 Effect Example 3: Animal Experiment on Improving Microcirculation Disorders Experimental materials 1.1 Experimental animals: Female ICR mice at the SPF level, weighing approximately 20 g.

[0031] 1.2 Test drugs: Dextran (MW500000).

[0032] 1.3 Reagents and consumables: HT series hemorheology cleaning solutions B and C; Plasminogen activator inhibitor-1 (PAI-1), Prostacyclin stable metabolite (6-keto-PGF), Tumor necrosis factor-α (TNF-α), Interleukin IL-1β detection kits; Sodium pentobarbital; HE staining solution (BH0001); Other reagents are of analytical purity.

[0033] Experimental methods 2.1 Animal grouping After 3 days of adaptive feeding of female ICR mice at the SPF level, they were randomly divided into a normal group, a model group, an Example 1 group, an Example 2 group, and a comparative example group, with 10 mice in each group. The mice were administered drugs by gavage. The normal group and the model group were gavaged with normal saline daily. The Ejiao intervention group was administered at 10 times the human recommended dose, that is, 1.5 g / kg / day, and continuously gavaged for no less than 30 days. Two weeks after drug intervention, the model group, the Example 1 group, the Example 2 group, and the comparative example group were injected with 10% dextran (MW500000) (10 ml / kg) via the tail vein to establish a microcirculation disorder model, and injected every other day. The normal group was injected with the same dose of normal saline via the tail vein for two weeks.

[0034] 2.2 Detection indicators 2.2.1 Body weight and organ index The mice were weighed weekly and the body weight was recorded. After the mice were sacrificed, the liver, spleen, kidney, heart, and lung were taken, weighed, and the organ index was calculated.

[0035] 2.2.2 Blood perfusion volume monitoring Approximately 1 h after the last administration, the mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (at a dose of 0.05 ml / 10 g), so that the auricles of the mice were flattened on the ear holder. After anesthesia, 10% dextran was injected via the tail vein, and a laser speckle imager was used to monitor and record the microcirculation blood perfusion volume of the mouse auricle at 0 min (before dextran injection), 5 min, 15 min, and 30 min after dextran injection. The sampling frequency was 1 image / s, and the monitoring time was 1 min.

[0036] 2.2.3 Number of open capillaries Approximately 1 hour after the last administration, mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (0.05 ml / 10 g dose) prepared in advance, and the auricles of the mice were flattened on the ear holder. After anesthesia, 10% dextran was injected via the tail vein, and the number of open capillaries in the mice was monitored and recorded at 0 min and 30 min after dextran injection using a stereomicroscope.

[0037] 2.2.4 Determination of hemorheological indexes After the experiment, whole blood was collected from the mice and placed in a blood collection tube containing EDTA (ethylenediaminetetraacetate) anticoagulant. Hemorheological indexes, including high shear viscosity of whole blood, low shear viscosity of whole blood, plasma viscosity, hematocrit, etc., were measured using a blood rheometer. The whole blood sample was placed at room temperature and inverted 4 - 5 times to mix it thoroughly, and the presence of coagulation, hemolysis, and lipemia was observed. After the sample state was qualified, it could be tested on the machine.

[0038] 2.2.5 Determination of vasoactive related factors and inflammatory factors After the experiment, whole blood was collected from the mice and serum was separated. The contents of inflammatory related factors PAI - 1, 6 - keto - PGF, TNF - α, and IL - 1β in the serum were measured with reference to the ELISA kit method.

[0039] 2.2.6 Histopathological observation After the experiment, the mice were sacrificed and various tissues were fixed with 4% paraformaldehyde. 1) Macroscopically observe whether there are bleeding points in the visceral tissues such as the liver, kidney, spleen, and skin of the mice; 2) Perform HE staining on the liver and kidney, observe the tissue sections under a light microscope, and compare the histopathological morphology of the mice in each group.

[0040] 2.2.7 Statistical analysis SPSS 19.0 software was used for statistical analysis. The data were expressed as mean ± standard deviation; t - test was used for comparison between two groups; two - way ANOVA analysis was used for comparison between three groups or more groups; P < 0.05 was considered statistically significant.

[0041] 2.2 Experimental results 2.2.1 Body weight and organ index The results of body weight are shown in Figure 2 , from the first week to the sixth week, the body weight of the mice in each group showed a gradually increasing trend. In the later stage, the body weight of the mice with microcirculation disorders increased more slowly than that of the normal mice, but the body weight growth rate of the mice in Examples 1 - 2 was higher than that of the model group and the comparative example group, with no significant difference (P > 0.05).

[0042] The organ indices of the mice are shown in Table 2. The results show that: compared with the normal group, the organ indices of the model group all increased to some extent. Among them, the spleen index was significant (P<0.05), indicating organ enlargement, which may be related to microcirculation disorders; compared with the model group, Examples 1-2 could significantly improve the spleen index of the mice (P<0.05), and the change in the spleen index of the comparative example group was not significant (P>0.05); compared with the comparative example group, the example group had a better improvement effect on the liver, kidney, and heart, but it did not show significance. Research shows that when there is blood stasis or microcirculation disorders, the local blood perfusion of the spleen is insufficient, which will activate the inflammatory response and the proliferation of immune cells, and then lead to spleen enlargement. The sustained-release film-coated Ejiao powder prepared in the examples of the present invention may reduce the spleen index by improving blood stasis.

[0043] Table 2 *Compared with the normal group, P<0.05, #compared with the model group, P<0.05.

[0044] 2.2.2 Blood perfusion volume of mice Blood perfusion volume is a necessary condition for maintaining the normal physiological activities of body cells and tissues. When there is microcirculation disorder, it is easy to cause a decrease in blood perfusion volume, a reduction in the oxygen and nutrients required by cells and the body, and tissue damage. Auricular microcirculation, as a manifestation of systemic microcirculation, has a certain representative significance. The skin of the mouse auricle is thin, the microvessels are superficial, distributed in a plane and fully unfolded, and a complete terminal vascular network is formed from arterioles and venules to capillaries. The morphology of the microvessels is clear and easy to observe. The experimental results are as Figure 3 shown, Figure 3 A shows the average blood perfusion volume of each group of mice at different time points. There was no significant change in the blood perfusion volume of the normal group at each time point (P>0.05); after the model group of mice was injected with dextran through the tail vein for 5 minutes, the blood perfusion volume decreased significantly compared with that before injection (P<0.01), and it did not return to the pre-injection level at the 15-minute and 30-minute time points (P<0.01). In the group of Example 1, the blood perfusion volume not only did not show a downward trend but increased at the 5-minute, 15-minute, and 30-minute time points after injecting dextran (P<0.01); in the group of Example 2, the blood perfusion volume decreased significantly 5 minutes after injecting dextran (P<0.05), and returned to the normal level at the 15-minute and 30-minute time points; in the comparative example group, the blood perfusion volume decreased significantly 5 minutes after injecting dextran (P<0.05), did not return to the normal level at the 15-minute time point, and returned to the normal level at the 30-minute time point.

[0045] Figure 3B is the comparison of the difference in the blood perfusion volume (PU) of mice. In the model group, the blood perfusion volume was at a negative level within 30 minutes after the tail vein injection of dextran. The average blood perfusion volume of the Example 1 group was at a positive level at each time point. The blood perfusion volume of the mice in the Example 2 group was at a positive level at 15 minutes and 30 minutes after the injection of dextran. The blood perfusion volume of the mice in the control group was close to the normal level at 30 minutes. Figure 3 C is the representative diagram of laser speckle flow imaging of the auricles of mice in each group. In the normal group, there were no significant changes in the blood flow of the marginal veins of the ears of mice at each time point, and the color was bluish. In the model group, the blood flow was abundant before the injection of dextran (the color was bright red-yellow), and the blood flow decreased significantly 5 minutes after the injection (the color was bluish), and did not recover at 15 minutes and 30 minutes. In the Example 1 group, there was no decrease in the blood flow from 5 to 30 minutes after the injection of dextran, the color was bright red-yellow, and the blood flow was abundant. In the Example 2 group, the blood flow decreased 5 minutes after the injection of dextran, the color became bluish, and the blood flow recovered 15 minutes later (the color became bright yellow). In the control group, the blood flow decreased within 5 to 15 minutes after the injection and recovered at 30 minutes (the color became bright yellow). Figure 4 It is a line graph of the real-time blood perfusion volume of the auricles of mice. The above results show that compared with the control group, the Ejiao sustained-release granules can prevent the blood perfusion from being affected by dextran, or even if it is affected, it can quickly return to the normal level. Therefore, it can more significantly improve microcirculation disorders. It is speculated that the reason may be that the Ejiao sustained-release granules can maintain an effective blood drug concentration for a longer time compared with ordinary Ejiao powder. In addition, the dissolution and absorption methods of Ejiao in the gastrointestinal tract have changed, which can enable the active substances in Ejiao to enter the blood circulation more fully, providing a sufficient material basis for improving microcirculation disorders.

[0046] 2.2.3 Number of open capillaries in mice The microcirculation capillary network is responsible for delivering oxygen to tissues, exchanging nutrients and wastes. The number of open capillaries in mice before and 30 minutes after the injection of dextran was monitored and recorded using a stereomicroscope. The results are as Figure 5 A- Figure 5 shown in Figure 5 B. As can be seen from Figure 5 A, there was no significant difference in the number of open marginal veins of the ears of mice in each group before the injection of dextran. The number of open capillaries in the marginal veins of the ears of mice in the model group decreased significantly at 30 minutes compared with 0 minutes (P<0.05). There were no obvious changes in the normal group of mice at 0 minutes and 30 minutes. The number of open capillaries in the ears of mice in the Example 1 group and the Example 2 group not only did not decrease at 30 minutes, but also showed a slight increasing trend (P>0.05). The number of open capillaries in the control group of mice decreased at 30 minutes compared with 0 minutes (P>0.05). Figure 5B is the real-time image of the marginal vein of the mouse ear. It can be seen from the figure that 30 minutes after the injection of dextran in the model group of mice, the overall outline of the ear margin turned white, the blood vessels became thinner, the redness decreased, and the number of capillaries decreased. 30 minutes after the injection of dextran in the mice of Example 1-2 groups, the ear margin contour was almost the same as before injection, showing a rosy ear margin, obvious blood vessel bifurcations, and clear venation. The above results indicate that the Ejiao sustained-release granules can improve the number of open capillaries in the ear margin of mice with microcirculation disorders.

[0047] 2.2.4 Blood rheology of mice The discipline that studies the laws of the flow and deformation of blood and its components is called hemorheology. From a modern scientific perspective, there is a certain correlation between the changes in hemorheology and the pathogenesis of blood stasis. Abnormal rheological manifestations such as thickening, viscosity, and easy aggregation of blood are commonly seen in patients with blood stasis. Blood viscosity is crucial for maintaining the normal perfusion of capillary blood in microcirculation. Once the blood becomes viscous, it is easy to form thrombi, increasing the incidence of cardiovascular and cerebrovascular diseases, and further leading to reduced tissue perfusion, resulting in hypoxia and acidosis, increasing the rigidity of red blood cells, promoting a further increase in blood viscosity, platelet aggregation, thrombus formation, blocking blood flow, and aggravating microcirculation disorders, forming a "vicious cycle". The experimental results are shown in Table 3 and Figure 6 As shown, compared with the normal group, the low shear rate of whole blood viscosity, medium shear rate of whole blood viscosity, high shear rate of whole blood viscosity, plasma viscosity, hematocrit, and fibrinogen in the model group were significantly increased (P < 0.05 or P < 0.01), and the erythrocyte sedimentation rate was significantly decreased (P < 0.05). Compared with the model group, the low shear rate of whole blood viscosity, medium shear rate of whole blood viscosity, high shear rate of whole blood viscosity, plasma viscosity, hematocrit, and fibrinogen levels in Example 1-2 groups could be significantly reduced (P < 0.05 or P < 0.01), while the erythrocyte sedimentation rate level was significantly increased (P < 0.05); the control group could reduce the whole blood viscosity, plasma viscosity, hematocrit, and fibrinogen levels to a certain extent, but there was no significant difference (P > 0.05). The above results indicate that the Ejiao sustained-release granules of the present invention can improve the blood rheology of mice with microcirculation disorders by reducing the whole blood viscosity, plasma viscosity, hematocrit, fibrinogen, and increasing the erythrocyte sedimentation rate.

[0048] Table 3 *: Compared with normal, P < 0.05; **: Compared with normal, P < 0.01; #: Compared with the model group, P < 0.05; ##: Compared with the model group, P < 0.01.

[0049] 2.2.5 Vascular activity-related factors and inflammation levels PAI-1, also known as plasminogen activator inhibitor-1, is the main substance inhibiting fibrinolysis activity in the blood circulation. It is the main inhibitor of tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA), and is a rapid inhibitor of t-PA. The dynamic balance between t-PA and PAI-1 plays a decisive role in maintaining the homeostasis of the plasma fibrinolytic system (simply referred to as the fibrinolytic system). An increase in PAI-1 level can lead to a decrease in fibrinolysis activity, thereby increasing the formation of thrombi, and is an independent risk factor for thrombotic diseases. Prostacyclin (PGF) is usually an important vasodilator, synthesized by vascular endothelial cells. It inhibits platelet aggregation by increasing cAMP in platelets and plays a powerful role in vasodilation. Prostacyclin also participates in regulating physiological processes such as vascular permeability and inhibiting the proliferation of vascular smooth muscle cells, and plays a crucial role in maintaining vascular homeostasis and physiological functions. The test results are as Figure 7 shown in A - B. Compared with the normal group, the PAI-1 level in the model group was significantly increased (P < 0.05). The PAI-1 levels in the Example 1 - 2 groups were extremely significantly decreased compared with the model group (P < 0.05), and the PAI-1 level in the Comparative Example 1 group was significantly decreased compared with the model group (P < 0.01). Therefore, the Ejiao sustained-release granules can more effectively reduce the PAI-1 level in mice with microcirculation disorders. The content of 6-keto-F1α in the serum of mice in the model group was significantly decreased compared with the normal group (P < 0.05). The 6-keto-F1α levels in the Example 1 - 2 groups were significantly increased compared with the model group (P < 0.05). The 6-keto-F1α level in the Comparative Example group increased to a certain extent but not significantly. The above results indicate that the Ejiao sustained-release granules may play a role in promoting microcirculation by improving the levels of PAI-1 and 6-keto-F1α.

[0050] The essence of microcirculation disorder is the lack of nutrient supply required for cell replication and the obstruction of the transport of the cell's own metabolic wastes. After the occurrence of local microcirculation disorder caused by the obstruction of a certain number of arterial capillaries, when invaded by pathogenic microorganisms, it will lead to the occurrence and development of inflammation in the body's tissues and organs, and then cause the decline or death of normal tissue cells, stimulating the nerve sensory endings to produce feelings of cold, soreness, and numbness. TNF-α is secreted by macrophages, monocytes, neutrophils, CD4 + T cells, and NK cells, and belongs to pro-inflammatory cytokines. It participates in normal inflammatory reactions and immune reactions, and can coordinately regulate the production of other cytokines, cell survival and death to coordinate tissue homeostasis. The precursor of IL-1β has no biological activity and needs to be cleaved by Caspase-1 into an active form, and then released extracellularly and binds to the pro-inflammatory receptors of adjacent cells, thereby triggering a strong inflammatory reaction. The effect of Ejiao powder on the inflammation level of mice with microcirculation disorders is as Figure 7As shown in Figures C-D, the serum TNF-α and IL-1β levels in the model group were significantly higher than those in the normal group (P<0.05, P<0.01), suggesting that inflammatory responses occurred in mice under the microcirculation disorder model. Compared with the model group, the Ejiao sustained-release granules could significantly reduce the levels of TNF-α and IL-1β (P<0.01 or P<0.05), indicating that it could improve the inflammatory response caused by microcirculation disorder, increase the blood flow grade, thereby inhibiting the occurrence and development of inflammation in the body's tissues and organs and maintaining the normal functions of organs.

[0051] 2.2.6 Histopathological observation After the experiment, the organs were observed macroscopically. No obvious bleeding points were found in the liver, kidney, spleen, skin, heart, etc. of each group of animals. The occurrence of microcirculation disorder in the liver is the pathological basis of viral hepatitis, liver fibrosis, and cirrhosis. Microcirculation disorder seriously affects the material exchange between hepatocytes and blood, resulting in energy metabolism disorder and toxin accumulation in hepatocytes, and aggravating hepatocyte damage. The liver was observed by histological section, and the results are as Figure 8 shown. The histological structure of the hepatic lobules in the normal group of mice was still intact, the cell boundaries were clear, the cell nuclei were intact, and no changes in the lobular structure or obvious intrahepatic hemorrhage were seen; the model group showed moderate ballooning degeneration, disordered cell structure, unclear boundaries, and mild inflammatory cell infiltration; the morphology of the Example 1 group was close to that of the normal group, the hepatic lobular structure was intact, no intrahepatic hemorrhage was seen, and the ballooning degeneration was significantly improved; the cell structure of the Example 2 group was relatively complete, but there was mild ballooning degeneration, the boundaries were relatively blurred, and no intrahepatic hemorrhage was seen; the Comparative Example group was improved to a certain extent compared with the model group, showing a reduction in ballooning degeneration, but the improvement degree was lower than that of the Example 1-2 groups. It can be seen that under microcirculation disorder, the liver of mice showed parenchymal damage and changes, and the Ejiao sustained-release granules played a certain protective role in the liver damage caused by microcirculation disorder.

[0052] The renal microcirculation is a complex microvascular network composed of parts such as arterioles, venules, and microvessels. More and more studies have shown that the occurrence and development of various kidney diseases are related to renal microcirculation disorder. The pathological changes in renal microcirculation function can cause a decrease in the blood perfusion level of renal microvessels and microhemodynamic disorders, local ischemia, hypoxia, and a decline in renal function in the kidneys, promoting the progression of kidney diseases. The pathological results of the kidneys of each group of mice are as Figure 8As shown, the glomerular tissue structure of the mice in the normal group was normal, the glomeruli and renal tubules were arranged neatly, the capillaries were not significantly dilated, there was no obvious punctate hemorrhage in the interstitium, and there was no degeneration or necrosis. In the model group, the mesangial matrix in the renal cortex was significantly thickened and showed vacuolar degeneration. In addition, inflammatory cell infiltration occurred in some interstitial regions. In the groups of Examples 1-2, the glomerular structure was relatively intact, with mild vacuolar degeneration and interstitial inflammatory cell infiltration, the capillaries were not significantly dilated, there was no obvious punctate hemorrhage in the interstitium, and there was no degeneration or necrosis. The control group showed partial vacuolar degeneration and interstitial inflammatory cell infiltration. It can be seen that under microcirculation disorders, the kidneys of mice showed substantial damage and changes, and the Ejiao sustained-release granules played a certain degree of protective role in the kidney damage caused by microcirculation disorders.

[0053] The above results indicate that the Ejiao sustained-release granules of the present invention can improve blood stasis syndrome and microcirculation disorders by increasing the blood perfusion volume of mice, increasing the number of open capillaries, improving blood rheology, inhibiting inflammatory reactions, and changing tissue pathological morphology.

[0054] Example 3 An application of Ejiao sustained-release granules. The Ejiao sustained-release granules prepared in Example 1 or 2 can be used to prepare traditional Chinese medicine preparations or other functional foods.

[0055] Obviously, the above examples are only for clear illustration and are not limitations on the implementation modes. For those of ordinary skill in the art, other different forms of changes or variations can be made based on the above description. It is not necessary and impossible to list all the implementation modes here. And the obvious changes or variations derived therefrom are still within the protection scope of the present invention.

Claims

1. A sustained-release donkey-hide gelatin granule, characterized in that: The components and weight proportions of the prescription include: 100 parts of donkey-hide gelatin blocks, 8-15 parts of ethyl cellulose, 0.5-2 parts of plasticizer, and 0.5-3 parts of anti-adhesive agent.

2. The sustained-release donkey-hide gelatin granules according to claim 1, characterized in that: The components and weight proportions of the prescription include: 100 parts of donkey-hide gelatin blocks, 8 parts of ethyl cellulose, 0.5 parts of plasticizer, and 0.5 parts of anti-adhesive agent.

3. The sustained-release donkey-hide gelatin granules according to claim, characterized in that: The components and weight proportions of the prescription include: 100 parts of donkey-hide gelatin blocks, 15 parts of ethyl cellulose, 2 parts of plasticizer, and 3 parts of anti-adhesive agent.

4. The sustained-release donkey-hide gelatin granules according to claim, characterized in that: The components and weight proportions of the prescription include: 100 parts of donkey-hide gelatin blocks, 10 parts of ethyl cellulose, 1 part of plasticizer, and 1 part of anti-adhesive agent.

5. The sustained-release donkey-hide gelatin granules according to any one of claims 1 to 4, characterized in that: The plasticizer is triethyl citrate, and the anti-sticking agent is talcum powder.

6. The sustained-release donkey-hide gelatin granules according to any one of claims 1 to 4, characterized in that: The preparation method of the donkey-hide gelatin block comprises the following steps: decocting, concentrating, adding auxiliary materials, molding and drying the dried or fresh skin of the donkey Equus asinus L. of the equine family.

7. A method for preparing sustained-release donkey-hide gelatin granules, characterized in that: The steps include: 1) preparing materials according to the components and weight proportions of the prescription according to any one of claims 1 to 6; 2) Add ethyl cellulose to 85% by volume ethanol-water solution and stir until the ethyl cellulose is completely dissolved to form a uniform and transparent solution; 3) Add plasticizer and anti-adhesive agent: Add the prescribed amount of plasticizer and anti-adhesive agent to the ethyl cellulose solution, and continue stirring to dissolve the plasticizer and evenly suspend the anti-adhesive agent in the solution; 4) Filter the coating solution: Filter the prepared coating solution through a 120-mesh sieve to remove any lumps that may exist; 5) Pretreatment of donkey-hide gelatin: crush the donkey-hide gelatin blocks with a grinder and pass through an 80-mesh sieve; 6) Put the crushed donkey-hide gelatin powder into the fluidized bed coating machine, turn on the fan, adjust the air inlet temperature to 40-50°C, and preheat the powder in the air flow for 10-15 minutes; 7) Fluidized bed coating parameter setting: the inlet air temperature is controlled at 40-50℃, the outlet air temperature is controlled at 30-40℃, the fan frequency is adjusted according to the fluidization state of the powder to keep the powder in a good fluidization state, the spray pressure of the spray gun is controlled at 0.15MPa, and the spray speed is adjusted according to the concentration of the coating solution and the coating progress; 8) When the donkey-hide gelatin powder reaches the preheating temperature and is in a stable fluidized state, turn on the spray gun and spray the coating liquid evenly into the fluidized bed. Closely observe the fluidization state and coating condition of the powder, and adjust various parameters in time to ensure uniform coating; 9) After the coating liquid is sprayed, the powder is allowed to continue to fluidize in the fluidized bed for 15 minutes to further dry and solidify the coating film to obtain the sustained-release donkey-hide gelatin particles; 10) Take the coated donkey-hide gelatin sustained-release granules out of the fluidized bed coater, put them into a drying oven, and age them at 50°C for 3 hours to further remove residual solvent and moisture and make the coating film dense and reliable; 11) The dried sustained-release donkey-hide gelatin granules are sieved through a 60-80 mesh sieve to remove large particles to obtain uniform sustained-release donkey-hide gelatin granules.

8. The method for preparing the sustained-release donkey-hide gelatin granules according to claim 7, characterized in that: The stirring speed of step 1) is controlled at 300-500 r / min.

9. An application of donkey-hide gelatin sustained-release granules, characterized in that: The sustained-release donkey-hide gelatin granules described in any one of claims 1 to 6 can be used to prepare traditional Chinese medicines or other functional foods.

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