Donkey-hide gelatin-concha haliotidis compound as well as preparation method and application thereof

Abstract

The invention relates to a colla corii asini-concha haliotidis 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-concha haliotidis 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 concha haliotidis; 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 concha haliotidis compound is prepared by using a hot melt extrusion technology, so that the smell, wettability, dispersity and other properties of the donkey-hide gelatin are obviously improved, and the donkey-hide gelatin compound shows a good effect in an osteoporosis rat model and a calcium-deficient mouse 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 abalone shell, its preparation method and application, belonging to the field of pharmaceutical and health product technology. Background Technology

[0002] In recent years, with the continuous improvement of modern living standards and the increasing awareness of health care, health preservation has gradually shown a trend of becoming more popular among younger people. Against this backdrop, the health supplement industry has maintained a growth momentum, and the market size is constantly expanding. It is worth noting that traditional Chinese medicine (TCM) health foods have significant advantages in terms of theory, after all, TCM is a unique medical culture formed in my country over a long period of development. Currently, industry competition is becoming increasingly fierce, and consumer demands are constantly upgrading. Under these circumstances, the combined application of modern formulation technologies and traditional Chinese medicine has become a new trend in the development of modern medical and health care products.

[0003] Donkey-hide gelatin (Ejiao) is a solid gelatin made from the dried or fresh hide of the donkey (Equus equina). It has a sweet taste and mild properties, possessing numerous health benefits including nourishing yin and lungs, replenishing blood, and stopping bleeding. Along with ginseng and deer antler, it is praised as one of the "Three Treasures of Tonics." In China, donkey-hide gelatin has a medicinal history spanning over two thousand years. Modern pharmacological research further reveals that it also exhibits various pharmacological activities, such as improving osteoporosis, combating anemia, enhancing immunity, and anti-tumor activity, demonstrating its enormous development potential and value.

[0004] Abalone shell (Shi Jue Ming) has a salty taste and slightly cold nature, and enters the liver meridian. It possesses various effects such as anti-infection, clearing the liver and improving eyesight, and can neutralize the rich and greasy nature of donkey-hide gelatin. Furthermore, abalone shell is high in calcium carbonate, which can effectively supplement the calcium needed by the human body and is beneficial to bone health. Hot melt extrusion, as a continuous production method, focuses on mixing raw and auxiliary materials in a molten state and promoting special interactions between them through precise control of key conditions such as extrusion temperature, pressure, and screw speed. This process is not only low-cost and in line with the concept of green production, but also simple and reproducible, thus it has been gradually applied and promoted in the field of new drug development in recent years.

[0005] Existing products involving donkey-hide gelatin and abalone shell 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 abalone shell to prepare compound products for relieving or treating osteoporosis symptoms. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides a compound of donkey-hide gelatin and abalone shell, its preparation method, and its application. This method utilizes hot-melt extrusion technology to prepare the compound of donkey-hide gelatin and abalone shell. On one hand, this masks the distinctive odor of donkey-hide gelatin, improves its sticky texture, and enhances its wettability and dispersibility. On the other hand, donkey-hide gelatin is rich and greasy, which can easily impair spleen and stomach function. Combining donkey-hide gelatin with abalone shell fully utilizes the neutralizing effect of abalone shell on the rich and greasy nature of donkey-hide gelatin, enhancing its moisturizing effect. Furthermore, the addition of abalone shell as a calcium supplement has good expected effects on alleviating symptoms such as osteoporosis in women and the elderly, rickets in children, excessive sweating, and irritability. This invention can greatly improve the innovation and market competitiveness of donkey-hide gelatin preparations. This application provides a compound of donkey-hide gelatin and abalone shell, which comprises, by weight, the following components:

[0007] a) 38-42 parts of donkey-hide gelatin;

[0008] b) 15-25 parts of abalone shell;

[0009] c) 1 to 5 parts of excipients, wherein the excipients are selected from one or more of polyvinylpyrrolidone, copovidone, hydroxypropyl cellulose, and hydroxyethyl cellulose.

[0010] Optionally, the compound of donkey-hide gelatin and abalone shell includes, by weight, 40 parts of donkey-hide gelatin; 18-22 parts of abalone shell; and 1-3 parts of excipients.

[0011] This application provides a method for preparing the above-mentioned donkey-hide gelatin-abalone shell compound, wherein the donkey-hide gelatin-abalone shell compound is prepared by using hot melt extrusion technology to prepare donkey-hide gelatin, abalone shell and excipients.

[0012] Optionally, the preparation method includes the following steps:

[0013] 1) Mix the sieved donkey-hide gelatin, abalone shell and excipients evenly to obtain a premix;

[0014] 2) The obtained premix is ​​sent to a hot melt extruder, and after hot melting, homogenization, cooling and extrusion, the extrudate is obtained;

[0015] 3) After sieving the obtained extrudate, the donkey-hide gelatin-abalone shell compound is obtained.

[0016] Optionally, the conditions for hot melt extrusion in step 2) include: extrusion temperature of 140-190℃ and rotation speed of 30-110 rpm.

[0017] Optionally, the conditions for hot melt extrusion in step 2) include: extrusion temperature of 160-180℃ and rotation speed of 60-100rpm.

[0018] Optionally, the sieve size in steps 1) and 3) is 30 to 100 mesh.

[0019] Optionally, the sieve size in steps 1) and 3) is 50 to 100 mesh.

[0020] This application provides the use of the above-mentioned donkey-hide gelatin-abalone shell compound in a drug for the prevention and / or treatment of diseases caused by calcium deficiency.

[0021] This application provides a medicament for the prevention and / or treatment of diseases caused by calcium deficiency, the medicament comprising the above-mentioned donkey-hide gelatin-abalone shell compound.

[0022] Optionally, the diseases caused by calcium deficiency include osteoporosis, bone and joint pain, nephrotic syndrome, rickets, tetany, cardiovascular disease, or loose teeth.

[0023] The beneficial effects of this application include, but are not limited to:

[0024] 1. According to the donkey-hide gelatin-abalone shell compound, its preparation method and application, the donkey-hide gelatin-abalone shell 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, abalone shell and excipients. It significantly improves the odor, wettability and dispersibility of donkey-hide gelatin. Moreover, the donkey-hide gelatin-abalone shell 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.

[0025] 2. According to the compound of donkey-hide gelatin and abalone shell of this application, its preparation method and application, the compound of donkey-hide gelatin and abalone shell of this application is prepared by hot melt extrusion technology and by controlling the extrusion temperature, pressure, screw speed and other conditions. The preparation process in 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

[0026] The accompanying drawings, which are provided to further illustrate this application and form part of this application, illustrate exemplary embodiments of this application and are used to explain this application, but do not constitute an undue limitation of this application.

[0027] Figure 1 The sensory results of the donkey-hide gelatin raw material (left) and the donkey-hide gelatin-abalone shell compound (right) involved in Test Example 1 of this application are shown in the figure.

[0028] Figure 2 The scanning electron microscope (10000X) results of the donkey-hide gelatin raw material (left) and the donkey-hide gelatin-abalone shell compound (right) involved in Test Example 2 of this application are shown.

[0029] Figure 3 This is a polarized light thermal stage microscope image of the donkey-hide gelatin-abalone shell compound involved in Test Example 2 of this application;

[0030] Figure 4 This is a polarized light thermal stage microscope image of the physical mixture of donkey-hide gelatin-abalone shell-hydroxypropyl cellulose involved in Test Example 2 of this application;

[0031] 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;

[0032] Figure 6 This is a polarized light thermal stage microscope image of Abalone shell involved in Test Example 2 of this application;

[0033] Figure 7 This is a polarized light thermal stage microscope image of the hydroxypropyl cellulose involved in Test Example 2 of this application;

[0034] Figure 8 The effect of the donkey-hide gelatin-abalone shell 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.

[0035] Figure 9 The effects of the donkey-hide gelatin-abalone shell compound 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);

[0036] Figure 10 Two-dimensional images of the cross-section of the femur of each group of rats using Micro-CT.

[0037] Figure 11 Two-dimensional images of the coronal section of the femur of rats in each group using Micro-CT.

[0038] Figure 12 The effect of the donkey-hide gelatin-abalone shell compound 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);

[0039] Figure 13 The image shows the results of the gripping force experiment.

[0040] Figure 14 HE staining images of the gastrocnemius muscle of mice in each group;

[0041] Figure 15 The effect of donkey-hide gelatin-abalone shell compound on relevant factors in mouse serum;

[0042] Figure 16 Image of a kidney tissue sample;

[0043] Figure 17 To observe the results of HE-stained mouse kidney sections under a light microscope. Detailed Implementation

[0044] 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.

[0045] Example 1

[0046] The compound of donkey-hide gelatin and abalone shell in this embodiment includes, by weight, the following: a) 40 parts of donkey-hide gelatin; b) 20 parts of abalone shell; c) 2 parts of excipients, wherein the excipients are hydroxypropyl cellulose.

[0047] The preparation method is as follows:

[0048] 1) Weigh out the donkey-hide gelatin, abalone shell 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;

[0049] 2) Set the temperature of the hot melt extruder to 165℃ and the rotation speed to 80rpm. Feed, mix, melt, homogenize, cool, and extrude at a uniform speed to obtain the extrudate.

[0050] 3) Pass through a 50-mesh sieve to obtain the compound of donkey-hide gelatin and abalone shell.

[0051] Example 2

[0052] The compound of donkey-hide gelatin and abalone shell in this embodiment includes, by weight, the following: a) 38 parts of donkey-hide gelatin; b) 15 parts of abalone shell; c) 1 part of excipient, wherein the excipient is polyvinylpyrrolidone.

[0053] The preparation method is as follows:

[0054] 1) Weigh out the donkey-hide gelatin, abalone shell 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;

[0055] 2) Set the temperature of the hot melt extruder to 140℃ and the rotation speed to 25rpm. Feed, mix, melt, homogenize, cool, and extrude at a uniform speed to obtain the extrudate.

[0056] 3) Pass through a 50-mesh sieve to obtain the compound of donkey-hide gelatin and abalone shell.

[0057] Example 3

[0058] The compound of donkey-hide gelatin and abalone shell in this embodiment includes, by weight, 42 parts of donkey-hide gelatin; 25 parts of abalone shell; and 5 parts of excipients, wherein the excipients are copovidone.

[0059] The preparation method is as follows:

[0060] 1) Weigh out the donkey-hide gelatin, abalone shell 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;

[0061] 2) Set the temperature of the hot melt extruder to 190℃ and the rotation speed to 120rpm. Feed, mix, melt, homogenize, cool, and extrude at a uniform speed to obtain the extrudate.

[0062] 3) Pass through a 75-mesh sieve to obtain the compound of donkey-hide gelatin and abalone shell.

[0063] Example 4

[0064] This embodiment is basically the same as Embodiment 1, except that the excipient is hydroxyethyl cellulose.

[0065] Comparative Example 1

[0066] According to the same compounding mass ratio in Example 1, weigh out the donkey-hide gelatin, abalone shell and excipients after passing through a 100-mesh sieve, and mix them by vortexing for 20 minutes to obtain a mechanical mixture of donkey-hide gelatin, abalone shell and excipients.

[0067] Test Example 1 Sensory Evaluation

[0068] Sensory evaluation results of the texture characteristics of the raw material of donkey-hide gelatin, the donkey-hide gelatin-abalone shell compound in Example 1, and the mechanical mixture of donkey-hide gelatin-abalone shell-excipients in Comparative Example 1 are as follows: Figure 1 As shown in Table 1.

[0069] Table 1 Sensory evaluation of donkey-hide gelatin raw materials and donkey-hide gelatin-abalone shell compound

[0070]

[0071] Experimental results showed that raw donkey-hide gelatin has a pale yellow appearance and a rusty smell. It is a loose powder that easily absorbs moisture and clumps, and has a sticky texture. The donkey-hide gelatin-abalone shell composite involved in this application, however, is brownish-yellow, odorless, and while also loose in texture, it is not sticky. It is noteworthy that simply mechanically mixing donkey-hide gelatin with abalone shell and excipients failed to eliminate the original rusty smell of the donkey-hide gelatin, nor did it significantly improve its texture and stickiness. However, in this application, by employing a specific process of adding abalone shell and excipients combined with hot-melt extrusion, the texture characteristics of donkey-hide gelatin can be significantly improved, its unpleasant odor effectively masked, and its sticky tactile experience optimized.

[0072] Test Example 2 Microstructure

[0073] The microstructures of donkey-hide gelatin and the donkey-hide gelatin-abalone shell compound were observed using a scanning electron microscope at 10,000x magnification. The results are as follows: Figure 2 As shown.

[0074] Experimental results show that the raw material of donkey-hide gelatin has a dense blocky structure, while the donkey-hide gelatin-auspicious herb compound in Comparative Example 1 has a dispersed spherical structure without sharp edges. It can be seen that the donkey-hide gelatin-auspicious herb compound prepared by the hot melt extrusion technology in this application destroys the original microstructure of donkey-hide gelatin, and the difference in microstructure between the raw material of donkey-hide gelatin and the donkey-hide gelatin-auspicious herb compound will affect its in vitro physicochemical properties.

[0075] The decomposition temperature of the product was tested using a hot-stage polarized light microscope. The donkey-hide gelatin-abalone shell compound provided in this application began to decompose at 205℃. Figure 3 As shown; however, the mechanical mixture of donkey-hide gelatin, abalone shell, and excipients begins to decompose at 150℃, as... Figure 4 As shown, and clearly distinguished from donkey-hide gelatin ( Figure 5 ), Shi Jue Ming ( Figure 6 ), hydroxypropyl cellulose ( Figure 7 Its own decomposition temperature.

[0076] It is evident that the addition of abalone shell and excipients to donkey-hide gelatin, along with a hot melt extrusion process, alters the microstructure of the donkey-hide gelatin-abalone shell compound, resulting in a significant improvement effect.

[0077] Test Example 3: Calcium content of a new compound formulation of donkey-hide gelatin

[0078] The compound of donkey-hide gelatin and abalone shell involved in this invention increases the calcium content of donkey-hide gelatin.

[0079] The calcium content in the donkey-hide gelatin-abalone shell compound was determined by inductively coupled plasma mass spectrometry (ICP-MS), as shown in Table 2.

[0080] Table 2. ICP-MS detection results of the donkey-hide gelatin-abalone shell compound.

[0081]

[0082] 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-abalone shell compound was 15.76%, which was about 134 times higher than that of the donkey-hide gelatin raw materials (0.079%~0.118%).

[0083] Test Example 4: Wettability Test

[0084] Accurately weigh 10g of the donkey-hide gelatin-abalone shell 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-abalone shell-excipient mechanical mixture in Comparative Example 1. The results are shown in Table 3.

[0085] Table 3. Wetting test data of donkey-hide gelatin-abalone shell compound and donkey-hide gelatin raw material.

[0086]

[0087] Experimental results show that the wettability of the donkey-hide gelatin-abalone shell compound in this application is significantly improved compared with that of donkey-hide gelatin raw material. The wettability of the donkey-hide gelatin-abalone shell compound is about 7 times higher than that of donkey-hide gelatin itself. The donkey-hide gelatin-abalone shell compound provided in this application facilitates the rapid dissolution of donkey-hide gelatin in water, which shows a significant improvement in the wettability of donkey-hide gelatin. However, the mechanical mixture of donkey-hide gelatin-abalone shell and excipients did not show any improvement effect.

[0088] Test Example 5: Dispersion Test

[0089] Accurately weigh 10g of the donkey-hide gelatin-abalone shell compound and disperse it in a beaker containing 250mL of 100℃ water placed on a magnetic stirrer. Stir quickly to disperse it evenly 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-abalone shell-excipient mechanical mixture in Comparative Example 1. The results are shown in Table 4.

[0090] Table 4. Dispersibility test data of donkey-hide gelatin-abalone shell compound and donkey-hide gelatin raw material.

[0091]

[0092] Experimental results show that the dispersibility of the donkey-hide gelatin-abalone shell compound of this application is significantly improved compared with that of donkey-hide gelatin raw material. The dispersibility of the donkey-hide gelatin-abalone shell compound is about 11 times higher than that of donkey-hide gelatin itself. The donkey-hide gelatin-abalone shell 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-abalone shell and excipients did not show any improvement effect.

[0093] Test Case 6: Improvement in Osteoporosis

[0094] 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).

[0095] 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).

[0096] The experimental animals involved were female SD rats, SPF grade, weighing 180–220 g, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0097] Sample preparation: Weigh an appropriate amount of the donkey-hide gelatin-abalone shell compound powder prepared in 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.

[0098] The test operation steps include:

[0099] 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).

[0100] 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.

[0101] 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.

[0102] 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.

[0103] The experimental results are as follows:

[0104] 1) Femoral wet weight analysis: Through analysis of femoral wet weight data (see...) Figure 8 Compared with the blank group, the wet weight of the femur in the model group rats was significantly reduced (P<0.01); compared with the model group, the wet weight of the femur in the low-dose group of donkey-hide gelatin-abalone shell compound rats was increased, and the wet weight of the femur in the high-dose group of donkey-hide gelatin-abalone shell compound rats was significantly increased (P<0.05), indicating that the compound has a positive effect on femur weight.

[0105] 2) Effects of donkey-hide gelatin-abalone shell compound on serum calcium, phosphorus, and ALP (alkaline phosphatase) levels in experimental rats.

[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 figures. Regarding serum calcium (Ca) levels, compared to the control group, the serum Ca levels in the model group were significantly lower (P<0.01); while compared to the model group, the high-dose group of the donkey-hide gelatin-abalone shell compound showed a significant increase in serum Ca levels (P<0.01). Regarding serum phosphorus (P) levels, compared to the control group, the serum P levels in the model group were significantly lower (P<0.05); compared to the model group, the high-dose group of the donkey-hide gelatin-abalone shell compound showed a significant increase in serum P levels (P<0.01). Regarding serum alanine lipid (ALP) levels, compared to the control group, the serum ALP levels in the model group were significantly increased (P<0.001); compared to the model group, the low-dose and high-dose groups of the donkey-hide gelatin-abalone shell compound showed significant decreases in serum ALP levels (P<0.01).

[0107] The above results indicate that the compound of donkey-hide gelatin and abalone shell can increase the levels of calcium and phosphorus in the blood and promote bone growth.

[0108] 3) Effects of donkey-hide gelatin-abalone shell compound on the bone microstructure of experimental rats

[0109] By analyzing the femoral cross-sections of each group of rats ( Figure 10 ) and coronal Micro-CT 2D image ( Figure 11 ) observation, and changes in bone mass parameters of the femur on Micro-CT scans ( Figure 12The analysis yielded the following results: In the normal group, the femoral trabeculae of rats were thicker, with smaller gaps, more numerous, and better continuity; In the model group, after administration of D-galactose, the femur showed trabecular fracture, shortening, and degeneration of the reticular structure, with increased central gaps. Simultaneously, the femoral Micro-CT indices showed significantly decreased 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), while significantly increased trabecular separation (P<0.01) and trabecular pattern factor (P<0.01). This indicates that female rats administered D-galactose experienced reduced bone mass and disruption of bone microstructure, successfully establishing an animal model of osteoporosis.

[0110] Compared with the model group, the femur trabeculae of rats in the donkey-hide gelatin-abalone shell compound group showed a more uniform and dense distribution with smaller gaps and better continuity. The high-dose donkey-hide gelatin-abalone shell compound intervention group showed significantly increased 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 femur Micro-CT parameters, while significantly decreased trabecular separation (P<0.05) and trabecular pattern factor (P<0.05). This indicates that the donkey-hide gelatin-abalone shell compound group can increase bone mass and improve bone microstructure in female rats in the D-galactose model group, and has a therapeutic effect on osteoporosis in this model. Specifically, the high-dose donkey-hide gelatin-abalone shell compound significantly improved D-galactose-induced bone loss and bone mass data in female rats.

[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 donkey-hide gelatin-abalone shell compound powder prepared in 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-abalone shell compound on muscle function in mice

[0124] The results of the gripping force test are as follows Figure 13As 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, low-dose group, and high-dose group significantly increased the grip strength of mice (P < 0.05). This indicates that the donkey-hide gelatin-abalone shell compound can improve muscle function in calcium-deficient model mice.

[0125] (2) Effects of donkey-hide gelatin-abalone shell 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-abalone shell 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, all treatment groups significantly increased the serum calcium content in mice (P < 0.01).

[0129] 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, compared with the blank group, the PTH content in the model group was significantly increased (P < 0.05). Compared with the model group, the positive control group, the medium-dose group, and the high-dose group significantly reduced the PTH content in mouse serum (P < 0.05).

[0130] The biochemical markers of bone metabolism in mouse serum were detected by enzyme-linked immunosorbent assay (ELISA), and the results are as follows: Figure 15 As 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 medium-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 < 0.01), and compared with the model group, the positive control group and the medium-dose group significantly reduced the OCN content in mouse serum (P < 0.05).

[0131] 4) Effects of donkey-hide gelatin-abalone shell compound on mouse kidney appearance and HE staining

[0132] 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.

[0133] 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.

[0134] In summary, the results show that the compound of donkey-hide gelatin and abalone shell provided in this application has a significant effect on improving the calcium deficiency model.

[0135] Optimization of the preparation method in Experiment Example 1

[0136] Before determining the final experimental scheme, the researchers repeatedly explored the dosage of each raw material and excipient in the donkey-hide gelatin-abalone shell compound, as well as the preparation conditions. Some of the experimental samples are shown in Table 5.

[0137] Table 5 Optimization of preparation conditions for donkey-hide gelatin-abalone shell compound

[0138]

[0139] During the exploration of the preparation process, the experimental results revealed several situations: Sample 1 had a loose texture, was non-sticky, and odorless; Sample 2, due to insufficient addition of abalone shell extract, remained sticky, easily absorbed moisture, and had a rusty smell; Sample 3, although odorless and of good texture, had excessive addition of abalone shell extract, exceeding the recommended daily intake, requiring a balance between the improvement effect and potential side effects; Sample 4, due to the lack of excipients, adhered to the screw and could not be extruded; Sample 5, although of good properties, had excessive excipients leading to increased costs; Sample 6, with excessively low temperature, prevented the excipients from melting, causing the product to adhere to the screw; Sample 7, with excessively high temperature, caused the donkey-hide gelatin to decompose and turn black; Samples 8 and 9 had a loose texture, were non-sticky, and prepared smoothly; Sample 10, although of acceptable texture, had low screw speed affecting efficiency; Sample 11, due to excessively high screw speed leading to uneven mixing, still had a sticky feel; Samples 12 and 13, due to low sieve mesh size and large feed particle size of the raw materials, increased screw torque load, affecting normal machine operation.

[0140] The above description is merely an example of embodiments of this application, and its scope of protection is not limited to these specific examples, but is explicitly defined by the claims. For those skilled in the art, this application allows for various modifications, equivalent substitutions, and improvements within the scope of the technical concept and principles, and these should all be considered to be included within the scope of protection of this application.

Claims

1. A compound of donkey-hide gelatin and abalone shell, characterized in that, The compound of donkey-hide gelatin and abalone shell comprises, by weight parts: a) 38-42 parts of donkey-hide gelatin; b) 15-25 parts of abalone shell; 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 abalone shell according to claim 1, characterized in that, The compound of donkey-hide gelatin and abalone shell comprises, by weight, 40 parts of donkey-hide gelatin; 18-22 parts of abalone shell; and 1-3 parts of excipients.

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

4. The preparation method of the donkey-hide gelatin-abalone shell compound according to claim 3, characterized in that, The preparation method includes the following steps: 1) Mix the sieved donkey-hide gelatin, abalone shell 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-abalone shell compound is obtained.

5. The preparation method of the donkey-hide gelatin-abalone shell compound according to claim 4, characterized in that, The conditions for hot melt extrusion in step 2) include: extrusion temperature of 140-190℃ and rotation speed of 30-110rpm.

6. The preparation method of the donkey-hide gelatin-abalone shell compound according to claim 5, characterized in that, The conditions for hot melt extrusion in step 2) include: extrusion temperature of 160-180℃ and rotation speed of 60-100rpm.

7. The preparation method of the donkey-hide gelatin-abalone shell 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-abalone shell 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-abalone shell 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-abalone shell 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.

Images