Donkey-hide gelatin-oyster powder compound as well as preparation method and application thereof

Abstract

The invention relates to a colla corii asini-oyster powder compound as well as a preparation method and application thereof, and belongs to the technical field of medicines and health-care products. The colla corii asini-oyster powder compound provided by the invention comprises the following components in parts by weight: a) 38-42 parts of colla corii asini; b) 15-25 parts of oyster powder; and c) 1-5 parts of an auxiliary material, wherein the auxiliary material is selected from one or more of polyvinylpyrrolidone, copovidone, hydroxy propyl cellulose and hydroxyethyl cellulose. The donkey-hide gelatin and oyster powder compound is prepared by using a hot-melt extrusion technology, so that the properties such as smell, wettability and dispersity of donkey-hide gelatin are obviously improved, and the donkey-hide gelatin has a good effect in an osteoporosis rat model and a mouse calcium deficiency model. The preparation process is simple and controllable, and has a good market prospect.

Description

Technical Field

[0001] This application relates to a compound of donkey-hide gelatin and oyster powder, its preparation method and application, belonging to the field of pharmaceutical and health product technology. Background Technology

[0002] With the improvement of modern living standards and the enhancement of health awareness, health preservation is gradually showing a trend towards younger generations. In recent years, the health product industry has maintained a continuous growth trend, and the market size has continued to expand. At the same time, traditional Chinese medicine health foods have obvious theoretical advantages, because traditional Chinese medicine is a unique medical culture formed in my country over a long period of development. Against the backdrop of increasingly fierce industry competition and constantly upgrading consumer demands, the combined application of modern formulation technologies and traditional Chinese medicine has become a new trend in the development of health care products. Donkey-hide gelatin (Ejiao) is a solid gelatin made from the dried or fresh skin of donkeys. It has a sweet taste and mild properties, and has the effects of nourishing yin and moistening the lungs, replenishing blood, and stopping bleeding. It is known as one of the "Three Treasures of Nourishment" along with ginseng and deer antler, and has a medicinal history of more than two thousand years in China. Modern pharmacological research reveals that donkey-hide gelatin also has various pharmacological activities such as improving osteoporosis, anti-anemia, enhancing immunity, and anti-tumor effects, thus possessing enormous development potential. Oyster shells have a salty and astringent taste, are slightly cold in nature, and enter the liver, heart, and kidney meridians. They have various effects such as relieving seminal emission, frequent urination, acid reflux, stomach pain, irritability, palpitations, metrorrhagia, and leukorrhea, and can neutralize the rich and greasy nature of donkey-hide gelatin. Furthermore, oyster powder's main component is calcium carbonate, a calcium supplement that can replenish the body's calcium needs, beneficial for bone health and the normal function of the cardiovascular system. In summary, this invention utilizes hot-melt extrusion technology to prepare a compound of donkey-hide gelatin and oyster powder, greatly expanding the application market for new donkey-hide gelatin preparations across different genders, age groups, and indications. Hot-melt extrusion is a continuous production process that achieves special interactions between raw materials and excipients by controlling extrusion temperature, pressure, screw speed, and other conditions after mixing raw and excipient materials in a molten state. It has many advantages such as low cost, green production, simple process, and good reproducibility, and has been gradually applied in the field of new drug development in recent years.

[0003] Existing products involving donkey-hide gelatin and oyster powder generally suffer from poor dispersibility and solubility. Furthermore, the presence of donkey-hide gelatin gives the products a noticeable rusty smell, and they are prone to absorbing moisture and clumping together, resulting in a sticky texture. In addition, there are no reports in the existing technology of compounding donkey-hide gelatin and oyster powder to prepare compound products for relieving or treating osteoporosis symptoms. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides a compound of donkey-hide gelatin and oyster powder, its preparation method, and its applications. This method utilizes hot-melt extrusion technology to prepare the compound of donkey-hide gelatin and oyster powder, significantly improving the odor, wettability, and dispersibility of the donkey-hide gelatin, and demonstrating good efficacy in a rat model of osteoporosis. The preparation process of this invention is simple and controllable, and it has good market prospects.

[0005] This application provides a compound of donkey-hide gelatin and oyster powder, wherein the compound of donkey-hide gelatin and oyster powder comprises, by weight parts:

[0006] a) 38-42 parts of donkey-hide gelatin;

[0007] b) 15-25 parts oyster powder;

[0008] c) 1 to 5 parts of excipients, wherein the excipients are selected from one or more of polyvinylpyrrolidone, copovidone, hydroxypropyl cellulose, and hydroxyethyl cellulose.

[0009] Optionally, the compound of donkey-hide gelatin and oyster powder includes, by weight, 40 parts of donkey-hide gelatin; 17-23 parts of oyster powder; and 1-3 parts of excipients.

[0010] This application provides a method for preparing the above-mentioned donkey-hide gelatin-oyster powder compound, wherein the donkey-hide gelatin-oyster powder compound is prepared by using hot melt extrusion technology to prepare donkey-hide gelatin, oyster powder and excipients.

[0011] Optionally, the preparation method includes the following steps:

[0012] 1) Mix the sieved donkey-hide gelatin, oyster powder and excipients evenly to obtain a premix;

[0013] 2) The obtained premix is ​​sent to a hot melt extruder, and after hot melting, homogenization, cooling and extrusion, the extrudate is obtained;

[0014] 3) After sieving the obtained extrudate, the donkey-hide gelatin-oyster powder compound is obtained.

[0015] Optionally, the conditions for hot melt extrusion in step 2) include: extrusion temperature of 120-200℃ and rotation speed of 50-110 rpm.

[0016] Optionally, the conditions for hot melt extrusion in step 2) include: extrusion temperature of 140-180℃ and rotation speed of 60-100rpm.

[0017] Optionally, the sieve size in steps 1) and 3) is 30 to 100 mesh.

[0018] Optionally, the sieve size in steps 1) and 3) is 50 to 100 mesh.

[0019] This application provides the use of the above-mentioned donkey-hide gelatin-oyster powder compound in the preparation of drugs for the prevention and treatment of diseases caused by calcium deficiency.

[0020] This application also provides a medicament for preventing and / or treating diseases caused by calcium deficiency, the medicament comprising the above-mentioned donkey-hide gelatin-oyster powder compound.

[0021] Optionally, the diseases caused by calcium deficiency include osteoporosis, bone and joint pain, nephrotic syndrome, rickets, tetany, cardiovascular disease, or loose teeth.

[0022] The beneficial effects of this application include, but are not limited to:

[0023] 1. According to the donkey-hide gelatin-oyster powder compound of this application, its preparation method and application, the donkey-hide gelatin-oyster powder compound provided in this application is prepared by limiting the particle size of raw materials and excipients and using hot melt extrusion technology to compound donkey-hide gelatin, oyster powder and excipients. It significantly improves the odor, wettability and dispersibility of donkey-hide gelatin. Moreover, the donkey-hide gelatin-oyster powder compound shows good efficacy in an osteoporosis rat model, which greatly improves the innovation and market competitiveness of donkey-hide gelatin preparations and has good market prospects.

[0024] 2. According to the donkey-hide gelatin-oyster powder compound of this application, its preparation method and application, the donkey-hide gelatin-oyster powder compound is prepared by hot melt extrusion technology by controlling the extrusion temperature, pressure, screw speed and other conditions. The preparation process of this application is simple, green and controllable, which can reduce production costs and achieve green and continuous production, and is suitable for industrial application. Attached Figure Description

[0025] Figure 1 The sensory results of the donkey-hide gelatin raw material (left) and the donkey-hide gelatin-oyster powder compound (right) involved in Test Example 1 of this application are shown in the figure.

[0026] Figure 2 The scanning electron microscope (10000X) results of the donkey-hide gelatin raw material (left) and the donkey-hide gelatin-oyster powder compound (right) involved in Test Example 2 of this application are shown.

[0027] Figure 3 This is a polarized light thermal stage microscope image of the donkey-hide gelatin-oyster powder compound involved in Test Example 2 of this application;

[0028] Figure 4 This is a polarized light thermal stage microscope image of the physical mixture of donkey-hide gelatin-oyster powder-hydroxypropyl cellulose involved in Test Example 2 of this application;

[0029] Figure 5 This is a polarized light thermal stage microscope image of the donkey-hide gelatin raw material involved in Test Example 2 of this application;

[0030] Figure 6 This is a polarized light thermal stage microscope image of the oyster powder involved in Test Example 2 of this application;

[0031] Figure 7 This is a polarized light thermal stage microscope image of the hydroxypropyl cellulose involved in Test Example 2 of this application;

[0032] Figure 8 The effect of the donkey-hide gelatin-oyster powder compound on the wet weight of rat bones was as follows: compared with the blank group, **P<0.01; compared with the model group, #P<0.05.

[0033] Figure 9 The effects of the compound of donkey-hide gelatin and oyster powder on serum calcium, phosphorus, and ALP (alkaline phosphatase) in rats (compared with the blank group, *P<0.05, **P<0.01, ***P<0.001; compared with the model group, #P<0.05, ##P<0.01, ###P<0.001);

[0034] Figure 10 Two-dimensional images of the cross-section of the femur of each group of rats using Micro-CT.

[0035] Figure 11 Two-dimensional images of the coronal section of the femur of rats in each group using Micro-CT.

[0036] Figure 12 The effect of the compound of donkey-hide gelatin and oyster powder on bone mass changes in rats (compared with the blank group, *P<0.05, **P<0.01, ***P<0.001; compared with the model group, #P<0.05, ##P<0.01, ###P<0.001);

[0037] Figure 13 Results of a mouse gripping test;

[0038] Figure 14 HE staining images of the gastrocnemius muscle of mice in each group;

[0039] Figure 15 The effect of donkey-hide gelatin-oyster powder compound on serum-related factors in mice;

[0040] Figure 16 Image of a kidney tissue sample;

[0041] Figure 17 This is a light microscope image of a mouse kidney section stained with hematoxylin and eosin (HE). Detailed Implementation

[0042] The present application is described in detail below with reference to the embodiments, but the present application is not limited to these embodiments. Unless otherwise specified, the raw materials and catalysts in the embodiments of the present application are all purchased through commercial channels.

[0043] Example 1

[0044] The compound of donkey-hide gelatin and oyster powder in this embodiment includes, by weight, 40 parts of donkey-hide gelatin; 20 parts of oyster powder; and 2 parts of excipients, wherein the excipients are hydroxypropyl cellulose.

[0045] The preparation method is as follows:

[0046] 1) Weigh out the donkey-hide gelatin, oyster powder and excipients that have passed through a 75-mesh sieve according to the mass ratio, and vortex for 20 minutes to mix them evenly to obtain a mixture;

[0047] 2) Set the temperature of the hot melt extruder to 160℃ and the rotation speed to 80rpm. Feed, mix, melt, homogenize, cool, and extrude at a uniform speed to obtain the extrudate.

[0048] 3) Pass through a 50-mesh sieve to obtain the compound of donkey-hide gelatin and oyster powder.

[0049] Example 2

[0050] The compound of donkey-hide gelatin and oyster powder in this embodiment includes, by weight, 38 parts of donkey-hide gelatin; 15 parts of oyster powder; and 1 part of excipient, wherein the excipient is polyvinylpyrrolidone.

[0051] The preparation method is as follows:

[0052] 1) Weigh out the donkey-hide gelatin, oyster powder and excipients that have passed through a 75-mesh sieve according to the mass ratio, and vortex for 20 minutes to mix them evenly to obtain a mixture;

[0053] 2) Set the temperature of the hot melt extruder to 120℃ and the rotation speed to 50rpm. Feed, mix, melt, homogenize, cool, and extrude at a uniform speed to obtain the extrudate.

[0054] 3) Pass through a 50-mesh sieve to obtain the compound of donkey-hide gelatin and oyster powder.

[0055] Example 3

[0056] The compound of donkey-hide gelatin and oyster powder in this embodiment includes, by weight, 42 parts of donkey-hide gelatin; 25 parts of oyster powder; and 5 parts of excipients, wherein the excipients are copovidone.

[0057] The preparation method is as follows:

[0058] 1) Weigh out the donkey-hide gelatin, oyster powder and excipients that have passed through a 100-mesh sieve according to the mass ratio, and vortex for 20 minutes to mix them evenly to obtain a mixture;

[0059] 2) Set the temperature of the hot melt extruder to 200℃ and the rotation speed to 110rpm. Feed, mix, melt, homogenize, cool, and extrude at a uniform speed to obtain the extrudate.

[0060] 3) Pass through a 75-mesh sieve to obtain the compound of donkey-hide gelatin and oyster powder.

[0061] Example 4

[0062] This embodiment is basically the same as Embodiment 1, except that the excipient is hydroxyethyl cellulose.

[0063] Comparative Example 1

[0064] Weigh out the donkey-hide gelatin, oyster powder and excipients that have passed through a 100-mesh sieve according to the same compounding mass ratio as in Example 1, and mix them by vortexing for 20 minutes to obtain a mechanical mixture of donkey-hide gelatin, oyster powder and excipients.

[0065] Test Example 1 Sensory Evaluation

[0066] Sensory evaluation results of the texture characteristics of the raw material of donkey-hide gelatin, the donkey-hide gelatin-oyster powder compound in Example 1, and the mechanical mixture of donkey-hide gelatin-oyster powder-excipients in Comparative Example 1 are as follows: Figure 1 As shown in Table 1.

[0067] Table 1. Sensory evaluation of donkey-hide gelatin raw materials and donkey-hide gelatin-oyster powder compound.

[0068]

[0069] Experimental results show that the raw material of donkey-hide gelatin is light yellow in color, has a rusty smell, is loose powder, easily absorbs moisture and clumps together, and has a sticky feel. The donkey-hide gelatin-oyster powder compound provided in this application is brownish-yellow, odorless, loose in texture and non-sticky. Moreover, the mechanical mixing of donkey-hide gelatin-oyster powder-excipients did not improve the rusty smell of donkey-hide gelatin itself, nor did it improve the texture and stickiness. However, by adding oyster powder and excipients and using a hot melt extrusion process, this application can significantly improve the texture characteristics of donkey-hide gelatin, mask its odor, and improve its sticky feel.

[0070] Test Example 2 Microstructure

[0071] The microstructures of donkey-hide gelatin, a mechanical mixture of donkey-hide gelatin and oyster powder, and a compound of donkey-hide gelatin and oyster powder were observed at 10,000x magnification using a scanning electron microscope. The results are as follows: Figure 2 As shown.

[0072] Experimental results show that the raw donkey-hide gelatin has a dense, blocky structure, while the donkey-hide gelatin-oyster powder compound in Comparative Example 1 has an aggregated, spherical structure without sharp edges. This indicates that the donkey-hide gelatin-oyster powder compound provided in this application alters the microstructure of donkey-hide gelatin, and the difference in microstructure between the raw donkey-hide gelatin and the donkey-hide gelatin-oyster powder compound affects their in vitro physicochemical properties.

[0073] The decomposition temperature of the product was tested using a hot-stage polarized light microscope. The donkey-hide gelatin-oyster powder compound provided in this application began to decompose at 190℃. Figure 3As shown; however, the mechanical mixture of donkey-hide gelatin, oyster powder, and excipients begins to decompose at 175℃, as... Figure 4 As shown, and clearly distinguished from donkey-hide gelatin ( Figure 5 ), oyster powder ( Figure 6 ), hydroxypropyl cellulose ( Figure 7 Its own decomposition temperature.

[0074] It is evident that adding oyster powder and excipients to donkey-hide gelatin, along with a hot melt extrusion process, altered the microstructure of the donkey-hide gelatin-oyster powder compound, resulting in a significant improvement effect.

[0075] Test Example 3: Iron content of a new compound dosage form of donkey-hide gelatin

[0076] The compound of donkey-hide gelatin and oyster powder involved in this invention increases the calcium content of donkey-hide gelatin.

[0077] The calcium content in the donkey-hide gelatin-oyster powder compound was determined by inductively coupled plasma mass spectrometry (ICP-MS), as shown in Table 2.

[0078] Table 2. ICP-MS detection results of the donkey-hide gelatin-oyster powder compound.

[0079]

[0080] ICP-MS results showed that by using hot melt extrusion technology to combine donkey-hide gelatin raw materials with marine calcium sources and organic acid salt iron supplements, the calcium and iron content of the compound was significantly increased: the calcium content in the donkey-hide gelatin-oyster powder compound was 15.38%, which was about 130 times higher than that of the donkey-hide gelatin raw materials (0.079%~0.118%).

[0081] Test Example 4: Wettability Test

[0082] Accurately weigh 10g of the donkey-hide gelatin-oyster powder compound and disperse it in a beaker containing 250mL of 100℃ water. Measure the wetting time under static conditions. Start timing when the powder is placed in the beaker and record the time it takes for the powder to be completely wetted and immersed in the water; this is the wetting time. The same test method was used to test the donkey-hide gelatin raw material sample and the donkey-hide gelatin-oyster powder-excipient mechanical mixture in Comparative Example 1. The results are shown in Table 2.

[0083] Table 3. Wetting test data of donkey-hide gelatin-oyster powder compound and donkey-hide gelatin raw material.

[0084]

[0085] Experimental results show that the wettability of the donkey-hide gelatin-oyster powder compound of this application is significantly improved compared with that of the raw donkey-hide gelatin. The wettability of the donkey-hide gelatin-oyster powder compound is about 8 times higher than that of the donkey-hide gelatin itself. The donkey-hide gelatin-oyster powder compound provided by this application makes it easy for donkey-hide gelatin to dissolve quickly in water, which shows that it significantly improves the wettability of donkey-hide gelatin. However, the mechanical mixture of donkey-hide gelatin-oyster powder and excipients did not show any improvement effect.

[0086] Test Example 5: Dispersion Test

[0087] Accurately weigh 10g of the donkey-hide gelatin-oyster powder compound and disperse it in a beaker containing 250mL of 100℃ water placed on a magnetic stirrer. Stir quickly to make it evenly dispersed in the water and record the time it takes for the powder to be completely dispersed in the water, which is the dispersion time. Use the same test method to test the donkey-hide gelatin raw material sample and the donkey-hide gelatin-oyster powder-excipient mechanical mixture in Comparative Example 1. The results are shown in Table 3.

[0088] Table 4. Dispersibility test data of the donkey-hide gelatin-oyster powder compound and donkey-hide gelatin raw material.

[0089]

[0090]

[0091] Experimental results show that the dispersibility of the donkey-hide gelatin-oyster powder compound of this application is significantly improved compared with that of the donkey-hide gelatin raw material. The dispersibility of the donkey-hide gelatin-oyster powder compound is about 11 times higher than that of donkey-hide gelatin itself. The donkey-hide gelatin-oyster powder compound provided by this application facilitates the rapid release of donkey-hide gelatin in water, which shows a significant improvement in the dispersibility of donkey-hide gelatin. However, the mechanical mixture of donkey-hide gelatin-oyster powder and excipients did not show any improvement effect.

[0092] Test Case 6: Improvement in Osteoporosis

[0093] The reagents involved were: D-galactose (Sigma-Aldrich LLC., batch number V900922), chloral hydrate (purchased from Tianjin Damao Chemical Reagent Factory), calcium assay kit (Changchun Huili Biotechnology Co., Ltd., batch number 2024001), inorganic phosphorus assay kit (Changchun Huili Biotechnology Co., Ltd., batch number 2024001), and alkaline phosphatase assay kit (Redu Life Science Co., Ltd., batch number 20240412).

[0094] The instruments involved are: 0.0001% electronic balance (Mettler-Toledo ME204E / 02), balance (Huachi HC20002), fully automated biochemical analyzer (Redu Life Science Co., Ltd.), Venus Micro CT (Ping Sheng Medical Technology, VNC-102), and centrifuge (BECKMAN COVLTER Microfuge 20R).

[0095] The experimental animals involved were female SD rats, SPF grade, weighing 180–220 g, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0096] Sample preparation: Weigh an appropriate amount of the compound preparation powder of donkey-hide gelatin and oyster powder from Example 1, add an appropriate amount of purified water, and prepare a suspension of 0.1 g / mL. The final sample can be used for oral gavage in rats.

[0097] The test operation steps include:

[0098] 1) Animal grouping and model establishment: Female SD rats were acclimatized for 7 days and randomly divided into 4 groups of 10 rats each: control group (Concerntration, C), model group (Model, M), low-dose group (Low, 0.15 g / kg, L), and high-dose group (High, 0.3 g / kg, H).

[0099] The modeling method is as follows: Except for the control group, rats in all other groups were subcutaneously injected with 500 mg / kg of D-galactose daily, while the control group was injected with an equal volume of physiological saline daily for 10 consecutive weeks. After the 5th week, the combination of movement restriction factors was started, that is, the activity space of the rats was reduced. Rats were housed in cages of 5 (control group: volume 485×350×200mm). 3 Model group and drug-treated group: 320×215×170mm 3 ), maintained for 4 hours daily. At the same time, after the 5th week, each treatment group began gavage administration, while the control group and model group were given an equal volume of distilled water.

[0100] 2) Sample collection: Ten weeks later, rats were anesthetized by intraperitoneal injection of 10% chloral hydrate. Blood was collected from the heart, allowed to stand at room temperature for 30 minutes, and then centrifuged at 3000 r / min for 15 minutes. The supernatant was collected and stored in EP tubes at -80℃. At the same time, rat femurs were harvested, excess adhering muscle tissue was removed, and the wet weight of fresh femurs from each experimental group was accurately weighed using an electronic analytical balance.

[0101] 3) Detection method: Wet weight of femur was weighed using an electronic analytical balance; serum calcium, phosphorus and ALP content was measured using a fully automated biochemical analyzer; bone mineral density was measured using Venus Micro-CT with scanning conditions of 90kV voltage and 90μA current, and the images were analyzed based on parameters such as bone mineral density.

[0102] The experimental results are as follows:

[0103] 1) Effects of donkey-hide gelatin-oyster powder compound on basic physiological indicators and serum bone formation markers in experimental rats

[0104] In this experiment, rats underwent specific treatment after 10 weeks. They were first anesthetized by intraperitoneal injection of 10% chloral hydrate, followed by blood collection from the heart. Blood samples were allowed to stand at room temperature for 30 minutes, then centrifuged at 3000 rpm for 15 minutes. The supernatant was collected and stored in EP tubes at -80°C. Simultaneously, the rat femur was removed, excess adhering muscle tissue was carefully removed, and the wet weight of the fresh femur was accurately measured using an electronic analytical balance.

[0105] Analysis of femoral wet weight data (see...) Figure 8 Compared with the control group, the wet weight of the femur in the model group rats was significantly reduced (P<0.01). In comparison with the model group, the wet weight of the femur in the low-dose group of the donkey-hide gelatin-oyster powder compound rats increased, while the wet weight of the femur in the high-dose group rats showed a significant increase (P<0.05), revealing the positive effect of the compound on femur weight.

[0106] To investigate the effects on bone formation, an automated biochemical analyzer was used to detect the levels of serum calcium, phosphorus, and ALP (alkaline phosphatase)—indicators closely related to bone formation—in rats of each experimental group. The results are as follows: Figure 9 As shown in the figure, compared with the control group, the serum calcium (Ca) level in the model group was significantly decreased (P<0.01), the serum phosphorus (P) level was significantly decreased (P<0.05), and the serum ALP level was significantly increased (P<0.01). Compared with the model group, the high-dose group of the donkey-hide gelatin-oyster powder compound showed significantly increased serum Ca (P<0.05) and P (P<0.05) levels, while the low-dose and high-dose groups of the compound showed significantly decreased serum ALP levels (P<0.01). These results indicate that the donkey-hide gelatin-oyster powder compound can effectively regulate blood calcium and phosphorus levels and has a positive impact on related physiological processes of bone growth.

[0107] 2) Effects of donkey-hide gelatin-oyster powder compound on improving bone microstructure and bone mineral density in a D-galactose-induced osteoporosis model of female rats.

[0108] For bone mineral density measurement, Venus Micro-CT was used for precise determination. First, the muscles and attached tissues on the rat femur were completely removed, and the rat was placed in a Micro-CT scanner. X-ray scanning of the femoral metaphysis was performed using a 90kV scanning voltage and a 90μA current. After the scan, the image and data were analyzed using the instrument's built-in software based on key parameters such as bone mineral density, bone mineral content, bone volume fraction, bone surface area to tissue volume ratio, trabecular separation, and trabecular pattern factor.

[0109] Cross-sections of the femur of rats in each group ( Figure 10 ) and two-dimensional images of coronal micro-CT ( Figure 11In the study, it was clearly observed that the femoral trabeculae of the normal group rats were robust, with small gaps, numerous, and well-continuous. In the model group established by administering D-galactose to female rats, the femoral trabeculae showed fractures, shortening, degeneration of the reticular structure, and large gaps in the central region. In contrast, the femoral trabeculae of the donkey-hide gelatin-oyster powder compound group were densely and uniformly distributed, with small gaps and good continuity. This indicates that administration of D-galactose to female rats reduced femoral bone mass and disrupted bone microstructure, successfully establishing an animal model of osteoporosis. Furthermore, the donkey-hide gelatin-oyster powder compound group increased femoral bone mass and improved bone microstructure in the female rats of the D-galactose model group.

[0110] Further analysis was conducted on the changes in bone mass parameters of the femur in each group of rats using Micro-CT scans (see...). Figure 12 Compared with the control group, the model group showed significantly lower levels of bone mineral density (P<0.01), bone mineral content (P<0.01), bone volume fraction (P<0.01), and bone surface area to tissue volume ratio (P<0.05) in femoral Micro-CT indices, while significantly higher levels of trabecular separation (P<0.01) and trabecular pattern factor (P<0.01), fully demonstrating the successful establishment of the osteoporosis model. Compared with the model group, the high-dose intervention group of donkey-hide gelatin-oyster powder compound showed significantly increased levels of bone mineral density (P<0.05), bone mineral content (P<0.05), bone volume fraction (P<0.05), and bone surface area to tissue volume ratio (P<0.05) in femoral Micro-CT indices, while significantly decreased levels of trabecular separation (P<0.05) and trabecular pattern factor (P<0.05). Further evidence indicates that the donkey-hide gelatin-oyster powder compound can significantly improve D-galactose-induced bone loss in female rats, optimize bone mass data, and has a definite therapeutic effect on osteoporosis in this model. In summary, the donkey-hide gelatin-oyster powder compound provided in this application has a significant ameliorative effect on osteoporosis.

[0111] Test Case 7: Establishment of a calcium-deficient animal model and detection of drug efficacy

[0112] The reagents involved were: chloral hydrate purchased from Tianjin Damao Chemical Reagent Factory; calcium content detection kit (o-cresolphthalein complex copper colorimetric method) (Adison Biotechnology Co., Ltd., ADS-W-D010); mouse osteocalcin (OCN) ELISA research kit (Jingmei Biotechnology Co., Ltd., JM-11567M1); mouse parathyroid hormone (PTH) ELISA research kit (Jingmei Biotechnology Co., Ltd., JM-02819M1); and mouse type I collagen C-terminal peptide (CTX-1) ELISA research kit (Jingmei Biotechnology Co., Ltd., JM-03091M1).

[0113] The instruments involved are: a 0.01% electronic balance (Mettler-Toledo ME204E / 02), a balance (Huachi HC20002), a gravity measuring instrument (Beijing Zhongshi Dichuang Technology Development Co., Ltd.), a multi-functional microplate reader (MOLECULARDEVICES), and a centrifuge (BECKMAN COVLTER Microfuge20R).

[0114] The experimental animals involved were ICR mice, SPF grade, weighing 18–22 g, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0115] Sample preparation: Weigh an appropriate amount of the compound preparation powder of donkey-hide gelatin and oyster powder from Example 1, add an appropriate amount of purified water, and prepare a suspension of 0.1 g / mL. The final sample can be used for oral gavage in rats.

[0116] The test operation steps include:

[0117] 1) Animal modeling and drug administration: ICR mice were acclimatized for 7 days, and 8 mice were randomly selected as the normal group and fed maintenance diet (normal maintenance diet calcium content 12.2g / kg). The other 40 mice were used for modeling and fed low-calcium purified diet (calcium content <0.1%, the diet was purchased from Xiaoshuyoutai (Beijing) Biotechnology Co., Ltd.) for 4 weeks. They were given deionized water to avoid obtaining calcium from drinking water, thus establishing a low-calcium mouse model.

[0118] Four weeks later, the model mice were randomly divided into five groups according to body weight: a low-calcium model control group, a CaCl2 positive control group, a low-dose group, a medium-dose group, and a high-dose group. The normal control group was fed a normal calcium diet and then administered deionized water by gavage for four weeks. The other groups were fed a low-calcium diet and administered deionized water by gavage, respectively. The dosage for each treatment group was as follows: 277.5 mg / kg for the CaCl2 positive control group (equivalent to 600 mg of calcium per day in humans), 0.25 g / kg for the low-dose group, 0.5 g / kg for the medium-low-dose group, and 1 g / kg for the high-low-dose group, for a total of four weeks.

[0119] 2) Grasping force test: This test examines the mouse's ability to grasp objects with its limbs. The mouse's forelimbs grip the grid of the grip tester, and the mouse's tail is gently pulled. The readings on the grip tester are recorded.

[0120] 3) Sample collection: Ten weeks later, rats were anesthetized by intraperitoneal injection of 10% chloral hydrate. Blood was collected from the heart, allowed to stand at room temperature for 30 minutes, and then centrifuged at 3000 rpm for 15 minutes. The supernatant was collected and stored in EP tubes at -80℃. The kidneys and gastrocnemius muscles were completely dissected, and portions were placed in paraformaldehyde fixative for tissue sectioning.

[0121] 4) Biochemical index testing: Refer to the kit instructions to measure relevant indicators in serum.

[0122] The experimental results are as follows:

[0123] (1) Effects of donkey-hide gelatin-oyster powder compound on muscle function in mice

[0124] The results of the gripping force test are as follows Figure 13 As shown, compared with the blank group, the grip strength of the model group was significantly reduced (P < 0.001). Compared with the model group, the positive control group, the medium-dose group, and the high-dose group significantly increased the grip strength of mice (P < 0.05). These results indicate that the donkey-hide gelatin-oyster powder compound can improve muscle function in calcium-deficient model mice.

[0125] (2) Effect of donkey-hide gelatin-oyster powder compound on HE staining of mouse gastrocnemius muscle

[0126] The results of HE-stained mouse gastrocnemius muscle sections observed under a light microscope are as follows: Figure 14 As shown, the gastrocnemius muscle fibers of the control group mice were arranged more neatly, with uniform diameter and clear cell membranes, and the cross-section of the muscle cells showed polygonal shapes. The model group showed localized loosening of the skeletal muscle fiber structure and widening of the interstitial space between muscle fibers. The low-dose administration group showed slight improvement in the widening of the interstitial space between muscle fibers and the loosening of the arrangement. The medium-dose administration group, the high-dose administration group, and the positive control group showed more significant improvement than the model group.

[0127] (3) Effects of donkey-hide gelatin-oyster powder compound on serum-related factors in mice

[0128] Serum calcium is an important indicator for evaluating whether the body is deficient in calcium. Serum calcium test results are as follows: Figure 15 As shown, compared with the blank group, the serum calcium content in the model group was significantly decreased (P < 0.001). Compared with the model group, the positive control group, low-dose group, and high-dose group significantly increased the serum calcium content in mice (P < 0.001).

[0129] 0.01).

[0130] Parathyroid hormone (PTH) is a basic single-chain polypeptide hormone secreted by the chief cells of the parathyroid gland. Its main function is to regulate calcium and phosphorus metabolism in vertebrates, leading to increased blood calcium levels and decreased blood phosphorus levels. PTH test results are as follows... Figure 15 As shown, the PTH content in the model group was significantly increased compared with the blank group (P < 0.001). Compared with the model group, the positive control group, low-dose group, and medium-dose group significantly reduced the PTH content in mouse serum (P < 0.05).

[0131] The biochemical markers of bone metabolism in mouse serum were detected by enzyme-linked immunosorbent assay (ELISA), and the results are as follows: Figure 15As shown, compared with the control group, the CTX-1 content in the model group was significantly increased (P < 0.01). Compared with the model group, the positive control group and the high-dose group significantly reduced the CTX-1 content in mouse serum (P < 0.05). Compared with the control group, the OCN content in the model group was significantly increased (P < 0.05), and compared with the model group, the positive control group and the low-dose group significantly reduced the OCN content in mouse serum (P < 0.05).

[0132] (4) Effects of donkey-hide gelatin-oyster powder compound on the appearance and HE staining of mouse kidneys

[0133] Kidney tissue samples, such as Figure 16 As shown, compared with the control group, the kidneys in the model group were lighter in color, almost grayish-white, and although they still had the general outline of kidneys, their texture was atrophied. All treatment groups showed significant improvement in the superficial lesions of the kidneys caused by calcium deficiency.

[0134] The results of HE-stained mouse kidney sections observed under a light microscope are as follows: Figure 17 As shown, compared with the control group, the model group exhibited disordered kidney structure and units, abnormal glomerular morphology, compensatory dilation of some lumens, and thinning of the renal tubular walls. Compared with the model group, each treatment group showed significant improvement in kidney lesions caused by calcium deficiency.

[0135] In summary, the results show that the donkey-hide gelatin-oyster powder compound provided in this application has a significant effect on improving the calcium deficiency model.

[0136] Optimization of the preparation method in Experiment Example 1

[0137] Before determining the final experimental scheme, the researchers repeatedly explored the dosage of each raw material and excipient in the donkey-hide gelatin-oyster powder compound, as well as the preparation conditions. Some of the experimental samples are shown in Table 4.

[0138] Table 4 Optimization of preparation conditions for donkey-hide gelatin-oyster powder compound

[0139]

[0140] In the exploratory experiments on the preparation process, we found that Sample 1's product was loose, non-sticky, and odorless; Sample 2 had insufficient oyster powder extract, resulting in a sticky, moisture-absorbing product with a rusty taste; Sample 3 was odorless, loose, and non-sticky, but the amount of oyster powder added exceeded the currently recommended daily intake. Therefore, we ultimately chose oyster powder that met the recommended daily intake while ensuring the improvement effect on donkey-hide gelatin. Sample 4 had no added excipients, and due to the sticky nature of donkey-hide gelatin, the product adhered to the screw and could not be extruded smoothly. Sample 5 had good product properties, but compared to Sample 1, it had too many excipients, resulting in higher costs. Sample 6 had too low a temperature, preventing the excipients from melting and functioning properly, causing the product to adhere to the screw and preventing smooth extrusion. Sample 7 had too high a temperature, causing some donkey-hide gelatin to decompose and turn black under screw extrusion. Samples 8 and 9 had loose, non-sticky extruded products, with no blackening, and the extrusion process was smooth. Although Sample 10 had a loose and non-sticky texture, its production efficiency was affected by the excessively low screw speed. Sample 11 had an excessively high screw speed, resulting in uneven mixing of the donkey-hide gelatin, oyster extract, and excipients, leading to a sticky product and poor improvement. Samples 12 and 13 experienced increased screw torque load during extrusion due to excessively low sieve mesh size and excessively large feed particle size, affecting normal machine operation.

[0141] The above description is merely a part of the embodiments of this application, and its protection scope is not limited to these specific embodiments, but is defined by the claims. For those skilled in the art, this application can be modified, equivalently substituted, or improved in various ways. Any modifications, equivalent substitutions, or improvements made within the technical concept and principles of this application should be included within the protection scope of this application.

Claims

1. A compound of donkey-hide gelatin and oyster powder, characterized in that, The compound of donkey-hide gelatin and oyster powder comprises, by weight, the following: a) 38-42 parts of donkey-hide gelatin; b) 15-25 parts oyster powder; c) 1 to 5 parts of excipients, wherein the excipients are selected from one or more of polyvinylpyrrolidone, copovidone, hydroxypropyl cellulose, and hydroxyethyl cellulose.

2. The compound of donkey-hide gelatin and oyster powder according to claim 1, characterized in that, The compound of donkey-hide gelatin and oyster powder comprises, by weight, 40 parts of donkey-hide gelatin; 17-23 parts of oyster powder; and 1-3 parts of excipients.

3. A method for preparing the donkey-hide gelatin-oyster powder compound as described in claim 1 or 2, characterized in that, The compound of donkey-hide gelatin and oyster powder is prepared by using hot melt extrusion technology to make donkey-hide gelatin, oyster powder and excipients.

4. The preparation method of the donkey-hide gelatin-oyster powder compound according to claim 3, characterized in that, The preparation method includes the following steps: 1) Mix the sieved donkey-hide gelatin, oyster powder and excipients evenly to obtain a premix; 2) The obtained premix is ​​sent to a hot melt extruder, and after hot melting, homogenization, cooling and extrusion, the extrudate is obtained; 3) After sieving the obtained extrudate, the donkey-hide gelatin-oyster powder compound is obtained.

5. The preparation method of the donkey-hide gelatin-oyster powder compound according to claim 4, characterized in that, The conditions for hot melt extrusion in step 2) include: extrusion temperature of 120-200℃ and rotation speed of 50-100rpm.

6. The preparation method of the donkey-hide gelatin-oyster powder compound according to claim 5, characterized in that, The conditions for hot melt extrusion in step 2) include: extrusion temperature of 140-180℃ and rotation speed of 60-100rpm.

7. The preparation method of the donkey-hide gelatin-oyster powder compound according to claim 4, characterized in that, The sieve size in steps 1) and 3) is 30 to 100 mesh.

8. The preparation method of the donkey-hide gelatin-oyster powder compound according to claim 7, characterized in that, The sieve size in steps 1) and 3) is 50-100 mesh.

9. The use of the donkey-hide gelatin-oyster powder compound as described in claim 1 or 2 in the preparation of drugs for the prevention and treatment of diseases caused by calcium deficiency; Preferably, the diseases caused by calcium deficiency include osteoporosis, bone and joint pain, nephrotic syndrome, rickets, tetany, cardiovascular disease, or loose teeth.

10. A medicine for preventing and / or treating diseases caused by calcium deficiency, characterized in that, The drug includes the donkey-hide gelatin-oyster powder compound as described in claim 1 or 2; Preferably, the diseases caused by calcium deficiency include osteoporosis, bone and joint pain, nephrotic syndrome, rickets, tetany, cardiovascular disease, or loose teeth.

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