Anti-osteoporosis processed isinglass as well as preparation and application thereof

Abstract

The invention relates to the technical field of traditional Chinese medicines, in particular to anti-osteoporosis processed isinglass as well as preparation and application thereof. According to the prepared isinglass provided by the invention, the component M is less than or equal to 5kDa, and the proportion is 63.71%, so that the intestinal absorption efficiency and the bioavailability can be remarkably improved. The invention also provides an application of the processed isinglass in preparation of medicines. The medicines can be used for treating and / or preventing osteoporosis caused by estrogen deficiency, intestinal flora imbalance caused by estrogen deficiency or bone diseases caused by estrogen deficiency. Compared with traditional donkey-hide gelatin, the prepared isinglass is better in molecular weight distribution and richer in Maillard reaction products; compared with swim bladder collagen which is not processed by the preparation method disclosed by the invention, the processed swim bladder collagen disclosed by the invention generates active ingredients such as melanoidins through a Maillard reaction, and the active ingredients are cooperated with amino acids, so that the limitation of traditional glues in anti-osteoporosis application is broken through, and a scientific basis is provided for development of functional bone health products.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine technology, specifically to a processed fish maw glue for combating osteoporosis and its preparation and application. Background Technology

[0002] Osteoporosis is a metabolic bone disease characterized by rapid bone loss and microstructural deterioration. Osteoporosis-related fractures show an age-dependent increase, particularly in postmenopausal women. Estrogen deficiency in this population leads to bone resorption exceeding bone formation, inducing postmenopausal osteoporosis.

[0003] The Maillard reaction is widespread in food processing and storage, as well as tobacco development, traditional Chinese medicine processing, and winemaking. 5-HMF, a representative intermediate product of the Maillard reaction, is a common reaction product in the heating and processing of various food and traditional Chinese medicine preparations. Studies have shown that 5-HMF possesses various biological activities, including anti-inflammatory, antibacterial, antioxidant stress, anti-hypoxic, immunomodulatory, and antitumor functions. Melanoidins are highly polymerized, brown, nitrogen-containing final products of the Maillard reaction; their complex polymerization structure endows them with diverse biological activities. Many studies have reported that melanoidins possess a variety of biological activities, including antioxidant, antibacterial, anti-inflammatory, antihypertensive, and prebiotic activities.

[0004] Mammalian gelatinous traditional Chinese medicines have the potential to enhance bone formation, inhibit bone resorption, and improve bone metabolism indicators in osteoporosis models. For example, deer antler gelatin and tortoise shell gelatin can both promote bone formation or inhibit osteoclast activity. However, mammalian gelatinous traditional Chinese medicines carry the risk of prion-causing diseases, such as bovine spongiform encephalopathy (BSE) and zoonotic pathogens. Furthermore, the high cost of mammalian gelatinous traditional Chinese medicines and the limited supply of deer and donkey hides restrict their market application.

[0005] Fish maw glue, also known as fish bladder, fish maw, or white flower glue, is the dried swim bladder of fish such as the large yellow croaker (Sciaenidae), small yellow croaker (Sciaenidae), or the Chinese sturgeon (Stachys chinensis), grass carp (Stachys acutus), etc. It is typically pressed into oblong thin sheets, pale yellow in color, horny in texture, and slightly glossy. It dissolves upon prolonged boiling, and the thick solution solidifies into a gel-like substance upon cooling, exhibiting strong viscosity. It is one of the "Eight Treasures of the Sea." Traditional Chinese medicine believes that fish maw is sweet and neutral in nature, entering the kidney meridian, and has the functions of replenishing essence and blood, nourishing tendons and veins, nourishing the liver and kidneys, and nourishing blood and stopping bleeding. It is suitable for kidney deficiency with spermatorrhea, hematemesis, metrorrhagia, and weakness of the lower back and knees. The *Compendium of Materia Medica* states that it "stops vomiting blood, disperses blood stasis, and reduces swelling and toxins." Consuming it in porridge can strengthen the spleen and replenish blood. Currently, commercial fish maw products are mainly based on primary processing. Research on the formulation design and efficacy mechanisms of high-value-added products based on fish maw collagen, such as nutritional supplements, is still immature. Summary of the Invention

[0006] This invention aims to provide a processed fish maw gelatin based on aquatic biological resources, which is safer, lower in cost, and can inhibit osteoclast differentiation while promoting osteogenic activity, significantly improve bone density and trabecular microstructure, and simultaneously reduce pathological indicators such as anti-tartrate acid phosphatase (TRACP-5b) and TNF-α in serum.

[0007] To achieve the above objectives, the present invention first provides a method for preparing processed fish swim bladder glue, comprising the following steps:

[0008] Step 1: Obtain the primary adhesive solution, including the following steps:

[0009] Step 101: Pulverize the dried fish bladder, place it in a container, add water at a material-to-liquid ratio of 1:30, and record the initial liquid level.

[0010] Step 102: After boiling at 100℃, cool down to 80-90℃ and continue steaming.

[0011] Step 103: Continue steaming for 192-216 hours, adding water every 4 hours during the steaming process to the initial liquid level line to obtain the primary adhesive solution;

[0012] Step 2: Concentrate the primary adhesive solution obtained in step 103, including the following steps:

[0013] Step 201: Separate the primary adhesive solution through a 400-mesh sieve to obtain an extract.

[0014] Step 202: Evaporate the extract obtained in step 201 until the viscosity is such that it flows down in a bead-like manner; then add rock sugar and continue to concentrate until the paste becomes a semi-transparent gel-like state, which can be hung into a flag. Add alcoholic food and stir vigorously, heat over low heat, and concentrate the gel until it falls slowly in a sheet-like state to obtain the concentrated gel.

[0015] Step 3: The concentrated adhesive solution obtained in step 202 is subjected to a setting process, including the following steps:

[0016] Step 301: Freeze the concentrated adhesive solution to obtain a frozen adhesive solution;

[0017] Step 302: The frozen liquid obtained in step 301 is vacuum freeze-dried at a pressure of 0.1 mbar using a vacuum freeze dryer to obtain processed fish maw glue.

[0018] In the above preparation method, the mass ratio of rock sugar to fish maw glue in step 202 is 1:50; the alcoholic food is rice wine, the mass ratio of rice wine to fish maw glue is 1:50, and the density of the concentrated glue solution is 1.35-1.40 g / cm³. 3; The freezing temperature in Step 301 is -80°C and the freezing time is 4 hours. The vacuum freeze-drying time in Step 302 is 48 hours.

[0019] On the other hand, the present invention provides a processed fish swim bladder glue obtained by the above preparation method. The processed fish swim bladder glue contains 5-hydroxymethylfurfural and melanoidins.

[0020] For the above-mentioned processed fish swim bladder glue, the molecular weight distribution of the processed fish swim bladder glue is as follows: the proportion of 400 Da < M ≤ 5 kDa is 63.71%, the proportion of 5 kDa < M ≤ 10 kDa is 12.87%, and the proportion of M > 10 kDa is 23.46%.

[0021] For the above-mentioned processed fish swim bladder glue, the processed fish swim bladder glue contains free amino acids, and the free amino acids include glycine, glutamic acid, proline, alanine, proline, arginine, lysine and leucine.

[0022] On the other hand, the present invention provides the application of the above-mentioned processed fish swim bladder glue in the preparation of a drug, and the drug is used for intestinal flora imbalance caused by estrogen deficiency or bone disease caused by estrogen deficiency.

[0023] In the above application, the drug is used to inhibit the loss of bone mineral density caused by estrogen deficiency in mammals to treat and / or prevent osteoporosis; and / or to reduce the reduction of trabecular bone number and / or the increase of trabecular bone spacing caused by estrogen deficiency in mammals to treat and / or prevent osteoporosis.

[0024] In the above application, the drug is used to regulate the bone metabolism balance to treat and / or prevent osteoporosis; the regulation of the bone metabolism balance is to inhibit the increase of type I collagen amino-terminal propeptide and osteocalcin in the serum of mammals; reduce alkaline phosphatase and tartrate-resistant acid phosphatase in the serum; reduce deoxypyridinoline, calcium and phosphorus in urine; reduce the level of inflammatory factors in mammals, and the inflammatory factors include TNF-α and IL-17A.

[0025] In the above application, the drug is used to inhibit the expression of osteoclast differentiation factor in mammals to treat and / or prevent osteoporosis, and / or to increase the expression of transcription factor Runx2 in mammals to treat and / or prevent osteoporosis.

[0026] Finally, the present invention also provides the application of the processed fish swim bladder glue in the preparation of a nutritional supplement or a dietary supplement.

[0027] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages:

[0028] (1) The molecular weight distribution of the processed fish maw collagen obtained by the preparation method provided in this invention shows that the proportion of small molecule peptides <5kDa in SBG reaches 63.71%, which is significantly higher than that of donkey-hide gelatin (5.92%) and fish maw collagen (0.35%). Small molecule peptides are more easily absorbed by the intestines, and the 1-3kDa peptides have been proven to have anti-osteoporosis activity, giving SBG higher bioavailability.

[0029] (2) The processed fish maw gel provided by this invention inhibits the abnormal increase of type I collagen amino-terminal elongated peptide (P1NP) and osteocalcin (OCN), which are key markers of bone formation in serum, and simultaneously reduces key indicators of bone resorption such as alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRACP-5b), thereby effectively balancing the rate of bone turnover; at the same time, it significantly reduces the excretion of bone collagen degradation products such as deoxypyridinium phosphate (DPD) and calcium and phosphorus in urine, thereby reducing the risk of bone loss from a biomechanical perspective; further, it blocks the inflammation-mediated osteoclast activation pathway by inhibiting the overexpression of pro-inflammatory factors TNF-α and IL-17A.

[0030] (3) The processed fish maw gelatin provided by this invention targets the core pathological mechanism of osteoporosis induced by estrogen deficiency in postmenopausal women, namely, rapid bone loss, degradation of trabecular microstructure, and a sharp increase in fracture risk. By processing the fish maw gelatin, it inhibits TRACP-5b and urinary calcium and phosphorus excretion to slow bone loss, and regulates the bone formation index P1NP / OCN to physiological levels to promote bone remodeling. Simultaneously, it inhibits osteoclast activation mediated by inflammatory factors such as TNF-α and IL-17A, thus fundamentally blocking the deterioration of the bone microenvironment. Compared to traditional therapies, this invention combines the advantages of zero disease risk from marine sources, synergistic effects across multiple targets, and high bioavailability. Attached Figure Description

[0031] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0032] Figure 1 The elution profiles are for SBG fractionated samples; where a represents SBC; b represents 24h; c represents 48h; d represents 72h; e represents 96h; f represents 120h; g represents 144h; h represents 168h; i represents 192h; j represents 216h; k represents SBG; and l represents EJ.

[0033] Figure 2 The image shows the results of Maillard fluorescence product determination.

[0034] Figure 3The graph shows the results of 5-hydroxymethylfurfural determination.

[0035] Figure 4 The graph shows the results of melanoidin content.

[0036] Figure 5 Micro-CT scans of the femurs of mice in each group.

[0037] Figure 6 Three-dimensional reconstructed images of the trabecular bone structure in the distal femur of mice in each group.

[0038] Figure 7 The graph shows the quantitative analysis data of femoral BMD in each group of mice.

[0039] Figure 8 The graph shows the quantitative analysis data of femoral BV / TV in each group of mice.

[0040] Figure 9 The graph shows the quantitative analysis data of Tb.N in the femur of mice in each group.

[0041] Figure 10 The graph shows the quantitative analysis data of Tb.Sp in the femur of mice in each group.

[0042] Figure 11 Image showing HE staining results of mouse femur.

[0043] Figure 12 This is a diagram showing the results of TRAP staining in the femur of a mouse.

[0044] Figure 13 This image shows representative immunohistochemical staining results of RUNX2 and RANKL proteins in the femur of mice.

[0045] Figure 14 A graph showing the quantitative analysis data of the RUNX2 positive expression region on the bone surface of the femur.

[0046] Figure 15 This is a graph showing the quantitative analysis data of RANKL-positive expression regions on the bone surface of the femur.

[0047] Figure 16 The graph shows the serum and urine related indicators of mice; in order, they are E2, OCN, PINP, b-ALP, TRAP, DPD, Ca, P, TNF-α, and IL-17A. Detailed Implementation

[0048] The technical solution of the present invention will be described below with reference to embodiments; however, the present invention is not limited to the following embodiments. Unless otherwise specified, the experimental and detection methods described in each embodiment are conventional methods; the reagents and materials described are commercially available unless otherwise specified. Unless otherwise specified, all percentages in the following embodiments refer to mass percentages. Unless otherwise specified, all proportions in the following embodiments refer to mass ratios.

[0049] The abbreviations used in this invention are explained in Table 1:

[0050] Table 1: Explanation of Abbreviations

[0051]

[0052] Example 1

[0053] This embodiment is intended to illustrate a method for preparing fish swim bladder gelatin (SBG).

[0054] This invention provides a method for preparing processed fish swim bladder glue, comprising the following steps:

[0055] Step 1: Obtain the primary adhesive solution, including the following steps:

[0056] Step 101: Powder the dried fish maw for pretreatment, place it in a non-stick pan, add water at a material-to-liquid ratio of 1:30, and record the initial liquid level.

[0057] Step 102: Heat to 100℃ and bring to a boil, then cool to 80℃ and continue steaming.

[0058] Step 103: Continue steaming for 216 hours, adding water every 4 hours during the steaming process to the initial liquid level line to obtain the primary adhesive solution;

[0059] Step 2: Concentrate the primary adhesive solution obtained in step 103, including the following steps:

[0060] Step 201: Separate the primary adhesive solution through a 400-mesh sieve to obtain an extract.

[0061] Step 202: Evaporate the extract obtained in step 201 until the viscosity is such that it flows down in a bead-like manner; then add rock sugar and continue to concentrate until the paste becomes a semi-transparent gel-like state, which can be hung into a flag. Add alcoholic food and stir vigorously, heat over low heat, and concentrate the gel until it falls slowly in a sheet-like state to obtain the concentrated gel.

[0062] Step 3: The concentrated adhesive solution obtained in step 202 is subjected to a setting process, including the following steps:

[0063] Step 301: Freeze the concentrated adhesive solution to obtain a frozen adhesive solution;

[0064] Step 302: The frozen liquid obtained in step 301 is vacuum freeze-dried at a pressure of 0.1 mbar using a vacuum freeze dryer to obtain processed fish maw glue.

[0065] In step 202, the mass ratio of rock sugar to fish maw glue is 1:50; the alcoholic food is Shaoxing rice wine, the mass ratio of rice wine to fish maw glue is 1:50, and the density of the concentrated glue solution is 1.40 g / cm³. 3 .

[0066] Molecular weight analysis was performed on the processed fish maw gelatin obtained in this embodiment, and a control group was set up to show that the processed fish maw gelatin in this embodiment has higher intestinal absorption efficiency and biological activity.

[0067] The donkey-hide gelatin (EJ) used in this embodiment was purchased from Shandong Dong-E-E-Jiao Company.

[0068] Fish swim bladder collagen (SBC): The pulverized fish swim bladder sample was mixed with deionized water at a weight-to-volume ratio of 1:10 (w / v). This aqueous suspension was subjected to thermal extraction at 80°C with constant stirring for 2 h. The digested mixture was filtered sequentially through two layers of sterile gauze to remove insoluble residues. The clarified extract was concentrated under vacuum by rotary evaporation to reduce solvent volume, and then freeze-dried for 48 h to obtain the final fish swim bladder collagen (SBC).

[0069] During the 216-hour SBG cooking process, samples were taken and tested at cooking times of 24h, 48h, 72h, 96h, 120h, 144h, 168h, 192h, and 216h. After pre-freezing at -80℃ for 24h, the samples were vacuum freeze-dried for 48h and stored at -20℃ for later use. The resulting SBG fractionated samples were named 24h, 48h, 72h, 96h, 120h, 144h, 168h, 192h, and 216h, respectively.

[0070] The molecular weight distributions of SBC, SBG, and donkey-hide gelatin were analyzed using high-performance liquid chromatography (Agilent 1260 Infinity III) and a TSKgel G2000SWXL size exclusion column (7.8 mm × 300 mm, Tosoh Bioscience). The determination method followed GB / T22492-2008, with a mobile phase of 0.1% (v / v) trifluoroacetic acid and 20% acetonitrile at a flow rate of 0.5 mL / min. The elution curves were detected at 220 nm using UV-Vis spectrophotometry. The chromatograms used a molecular weight standard curve (y = -2.4942x + 24.464, y: lgMW, x: time, R² = 0.9863), established using protein and peptide standards with specified molecular weights. The molecular weights of these standards are as follows: insulin (5754 Da), bacitracin (1412 Da), glycine-glycine-tyrosine-arginine (354 Da), and glycine-glycine-glycine-glycine (75 Da). The results are as follows... Figure 1 As shown, size exclusion chromatography analysis revealed that 40.9% of the components in SBG were distributed in the <1kDa range, and 32.2% were distributed in the 1–5kDa range, while the molecular weights of SBC and EJ were mainly distributed in the >10kDa range. See Table 2 for details.

[0071] Table 2: Molecular weight distribution of SBC, SBG, and EJ

[0072]

[0073]

[0074] As the cooking time increased, the overall molecular weight of fish swim bladder collagen continuously decreased, while the proportion of low molecular weight regions (MW < 5 kDa) steadily increased. This indicates that prolonged cooking gradually destroys the molecular structure of collagen, breaking it down into smaller molecules. Compared to the concentrated collagen solution after 216 hours of cooking without any additives, SBG showed a lower proportion of low molecular weight regions (MW < 5 kDa) and a larger proportion of high molecular weight regions (MW > 10 kDa). This may be because during the concentration process with additives, a large amount of high molecular weight polymers such as melanoidins are generated in the late Maillard reaction, thus affecting the molecular weight distribution of SBG. Although there is a certain proportion in the high molecular weight region, the composition of SBG is still mainly low molecular weight peptides (MW < 5 kDa). This compositional characteristic suggests that it may possess certain biological activities.

[0075] In addition, compared to EJ and SBC, whose molecular weights are concentrated in the MW > 10 kDa range, SBG exhibits a more dispersed molecular weight distribution, with a certain proportion in the low molecular weight region. Low molecular weight peptides (MW < 5 kDa) have better intestinal permeability and higher biological activity. Collagen hydrolysates with molecular weights between 1 kDa and 3 kDa showed the highest anti-osteoporosis activity. Therefore, SBG may possess stronger biological activity.

[0076] In summary, SBG's molecular weight gradually decreases during the cooking process. Subsequent concentration and excipient addition steps generate high-molecular-weight polymers via the Maillard reaction, leading to an increase in the proportion of high-molecular-weight regions, but it still primarily consists of low-molecular-weight peptides. In contrast, EJ and SBC have their molecular weights concentrated in the high-molecular-weight region.

[0077] Example 2

[0078] This embodiment is intended to illustrate that the SBG prepared in Example 1 contains a large amount of amino acids and can have an effect on osteoporosis.

[0079] In this embodiment, ninhydrin post-column derivatization and ion-exchange chromatography were used to determine the amino acid composition of SBC, donkey-hide gelatin, and SBG. 100 mg of the test powder was hydrolyzed for 22 h in a sealed vacuum hydrolysis tube at 110 ± 1 °C with 15 mL of 6M HCl (analytical grade) under nitrogen atmosphere. The hydrolysis product was filtered through quantitative filter paper and then diluted to 50 mL with deionized water. A 1.0 mL aliquot was then evaporated to dryness under reduced pressure at 45 °C. The residue was then recombine in 2.0 mL of pH 2.2 sodium citrate buffer and filtered through a 0.22 μm nylon membrane before injection. Retention time and peak area were used for qualitative and quantitative analysis compared to amino acid standards.

[0080] To clarify the amino acid composition of EJ, SBC, and SBG, acid hydrolysis was used to determine their amino acid composition. The results are shown in Table 3. All samples were rich in 17 essential and non-essential amino acids, with glycine, proline, glutamic acid, alanine, arginine, aspartic acid, and lysine being the most abundant amino acids. Glycine and proline are the most abundant amino acids in collagen, indicating that EJ, SBC, and SBG conform to the characteristics of collagen and its hydrolysates. The amino acid compositions of SBC and SBG were basically similar. Possibly due to the different sources of donkey skin and fish swim bladder, the total amino acid content of SBC and SBG was 1.24 and 1.26 times that of EJ, respectively.

[0081] Table 3: SBG Amino Acid Content Determination Table

[0082]

[0083]

[0084] Next, the free amino acids in SBG were determined: 0.5g of the sample was rapidly frozen in liquid nitrogen, then ground and transferred to a 10mL centrifuge tube. 5mL of 0.01M hydrochloric acid was added, vortexed for 1 min, and then heated in a boiling water bath for 30 min. The mixture was centrifuged at 10000×g for 10 min (4℃), and the supernatant was collected. The precipitate was resuspended in 4mL of 0.01M hydrochloric acid and extracted by sonication for 5 min (40kHz, 100W). After centrifugation under the same conditions, the supernatants were combined and brought to a final volume of 10mL. The mixture was filtered through a 0.22μm aqueous filter membrane, and the filtrate was collected for analysis.

[0085] The results are shown in Tables 4-7. SBG contains a large amount of free amino acids, with the most abundant free amino acids being aspartic acid, glycine, isoleucine, tryptophan, and alanine. In contrast, the contents of all free amino acids in EJ and SBC are significantly lower than those in SBG.

[0086] Table 4: Composition of essential amino acids in free amino acids

[0087]

[0088] Note: Essential amino acids: threonine, leucine, phenylalanine, valine, isoleucine, methionine, lysine; values ​​with different superscript letters indicate significant differences at the p<0.05 level.

[0089] Table 5: Composition of acidic amino acids in free amino acids

[0090]

[0091] Note: Acidic amino acids: glutamic acid, aspartic acid; values ​​with different superscript letters indicate significant differences at the p<0.05 level.

[0092] Table 6: Composition of Polar and Neutral Amino Acids in Free Amino Acids

[0093]

[0094]

[0095] Note: Polar and neutral amino acids: glycine, serine, cysteine, tyrosine, alanine, proline; values ​​with different superscript letters indicate significant differences at the p < 0.05 level.

[0096] Table 7: Composition of basic amino acids in free amino acids

[0097]

[0098] Note: Basic amino acids: arginine, histidine; values ​​with different superscript letters indicate significant differences at the p < 0.05 level.

[0099] During SBG preparation, the content of almost all amino acids showed an increasing trend. This indicates that amino acids are continuously generated or accumulated over time during SBG preparation, as the preparation process promotes the breakdown of collagen, producing more amino acids. The Maillard reaction products of amino acids are biologically active. The content of valine, tyrosine, and phenylalanine showed a trend of first increasing, then decreasing, and then increasing again. Glycine, glutamate, and proline exert estrogen-mimicking osteoprotective effects through the ERα-mediated signaling pathway, while alanine, proline, arginine, lysine, and leucine promote insulin secretion, thereby promoting osteoblast proliferation and differentiation. Arginine promotes the secretion of growth hormone and insulin-like growth factor-1, which can inhibit the progression of osteoporosis. Serine enhances osteoblast activity, while lysine, threonine, methionine, and isoleucine synergistically inhibit osteoclast activity while enhancing osteoblast function. Arginine, lysine, and glycine are associated with improved collagen formation or synthesis, thus affecting the mechanical strength of bone tissue. Given that muscle loss is one of the causes of bone loss, glycine, lysine, and leucine can mitigate musculoskeletal degeneration. SBG contains a large amount of free amino acids, which may endow it with unique biological activities.

[0100] Example 3

[0101] This embodiment is intended to illustrate that the SBG prepared in Example 1 undergoes the Maillard reaction, and that the SBG contains melanoidins and 5-hydroxymethylfurfural, thus having an effect on osteoporosis.

[0102] Intermediate products generated in the initial stage of the Maillard reaction typically lack fluorescent properties. However, before the formation of browning products in the advanced stage, these intermediate products can undergo cross-linking reactions with neighboring protein or amino acid molecules to form fluorescent polymers; some final products also exhibit fluorescence. For example... Figure 2As shown, during the preparation of SBG, the content of Maillard reaction fluorescent products continuously increased, especially in the concentration step, which significantly increased the content of Maillard intermediates. This indicates that the amount of Maillard intermediates in fish maw glue continuously increases during preparation. Simultaneously, the peak height wavelength shifts towards longer wavelengths, indicating that the composition of the fluorescent products is constantly changing during preparation. Comparing EJ and SBG, the peak height wavelength of EJ shifts towards longer wavelengths, indicating a difference in the composition of their fluorescent products. Furthermore, the content of fluorescent intermediates in the SBG product is higher than that in EJ, suggesting that the Maillard reaction in donkey-hide gelatin may be mainly concentrated in the late stage or in the early stage of the Maillard reaction. The content of fluorescent intermediates in SBC is much lower than that in SBG. In summary, the content of Maillard reaction intermediates in fish maw glue continuously increases during preparation, and the composition of fluorescent products continuously changes; compared with SBG, the reaction degree of SBC and EJ in the middle stage of the Maillard reaction is significantly lower.

[0103] 1. Determination of 5-hydroxymethylfurfural:

[0104] An Agilent C18 column (250 mm × 4.6 mm, 5 μm) was used as the separation medium. The mobile phase was acetonitrile-water (5:95, v / v). The column temperature was controlled at 30 °C, the flow rate was set at 1.0 mL / min, the detection wavelength was selected at 284 nm, and the injection volume was 10 μL. The standard curve was y = 59.415x - 8.1843, where y is the peak area, x is the concentration (μg / mL), and R² = 0.9948. 1 g of sample was accurately weighed and placed in a suitable container. 5% acetonitrile solution was added, and the volume was adjusted to 10 mL to obtain a sample solution with a concentration of 100 mg / mL. The solution was then filtered through a 0.22 μm microporous membrane, and the filtrate was used for subsequent analysis.

[0105] In this embodiment, during the SBG cooking process, as follows: Figure 3As shown, 5-hydroxymethylfurfural was not detected until the 144th hour. With further extension of the cooking time, the content of 5-hydroxymethylfurfural continued to rise, especially during the concentration stage, where its content increased significantly. The absence of 5-hydroxymethylfurfural during the preparation process was due to the short heating time, low content of free amino acids and small molecule peptides, and a low degree of Maillard reaction, corresponding to the previously determined low content of Maillard intermediates. In contrast, 5-hydroxymethylfurfural was not detected in either SBC or EJ. The fact that 5-hydroxymethylfurfural, as an intermediate product of the Maillard reaction, was not detected in EJ is likely because the Maillard reaction in EJ had progressed to a late stage. 5-Hydroxymethylfurfural exhibits anti-catabolism and anti-inflammatory effects on the inflammatory response of chondrocytes in human osteoarthritis. Overall, among the comparisons of SBC, EJ, and SBG, only SBG produces 5-hydroxymethylfurfural, suggesting that it may have unique advantages in terms of biological activity. Furthermore, during the boiling process, the 5-hydroxymethylfurfural content of SBG changes significantly with boiling time and concentration stage.

[0106] 2. Determination of melanoidins:

[0107] Weigh 1g of sample, dissolve it in 50mL of deionized water, centrifuge at 4000r / min for 5min, take the supernatant and measure the absorbance at a wavelength of 420nm, and then calculate the melanoidin content in the sample according to formula 2-1.

[0108] Melanoid content (mg / mL) = A × 0.1 / 0.269 Equation (1)

[0109] In formula (1): A is the absorbance of the sample at a wavelength of 420 nm; 0.269 is the absorbance of the solution containing 0.1 mg of melanoidin.

[0110] In this embodiment, as Figure 4 As shown, the melanoidin concentration exhibited a significant time-dependent increasing trend during SBG preparation, indicating that the late stage of the Maillard reaction continued to advance with prolonged cooking time. Notably, the melanoidin content increased significantly during the concentration stage, possibly related to the addition of rice wine and rock sugar. Rice wine naturally contains a large amount of melanoidin pigments, and the addition of rock sugar provided reducing sugars, thus promoting the Maillard reaction process. Current research indicates that melanoidins possess various biological activities, including anti-inflammatory and antioxidant effects.

[0111] However, the melanoidin content of EJ was significantly higher than that of SBG (p<0.05). Combined with the previously determined results of Maillard intermediates and 5-hydroxymethylfurfural content, this indicates that the late-stage Maillard reaction process of EJ was more advanced. The melanoidin content of SBG was 10.51 times that of SBC (p<0.001). In summary, the melanoidin content of SBG continuously increased during the preparation process, and rose sharply during the concentration stage. In contrast, EJ exhibited a higher concentration of melanoidin during the cooking process, indicating a more advanced late-stage Maillard reaction process. SBC, on the other hand, had extremely low levels of melanoidin.

[0112] Example 4

[0113] This embodiment is intended to illustrate that the SBG prepared in Example 1 has a good effect on osteoporosis caused by estrogen deficiency.

[0114] Female mice: purchased from Zhuhai Bestone Company, animal ethics number: No. GDOU-LAE-2023-041.

[0115] Forty-two eight-week-old female C57BL / 6 mice were randomly divided into two groups according to their body weight: a sham operation group (n=6) and a model group (n=36).

[0116] Mice in both the model group and the sham-operated group were anesthetized via intraperitoneal injection of 1.25% tribromoethanol solution at a dose calculated at 0.2 ml / 10 g body weight. They were placed in a lateral decubitus position and fixed on a sterile operating table. The surgical area was prepared by thoroughly removing hair from the back using an electric epilator, followed by three applications of routine povidone-iodine solution disinfection, and a sterile drape was applied. Povidone-iodine was applied to the surgical area, and a longitudinal incision of approximately 1 cm was made along the midline of the back at the vertebral level using ophthalmic scissors. Subcutaneous connective tissue, muscle layer, and peritoneum were dissected layer by layer using microsurgical forceps. Sterile cotton swabs were used for hemostasis during the operation to maintain a clear surgical field. The abdominal fat pad was gently retracted with microsurgical forceps to expose the pink ovarian tissue and connected fallopian tube structures. The ovarian vessels and proximal fallopian tubes were double-ligated using 4-0 non-absorbable sutures. Both ovaries were completely removed 0.5 mm distal to the ligation sutures, and the stumps were disinfected with povidone-iodine cotton balls and returned to the abdominal cavity. The peritoneum, muscle layer, and subcutaneous tissue were sutured in layers using 5-0 absorbable sutures, while the epidermis was sutured intermittently using 6-0 nylon sutures. After surgery, povidone-iodine disinfectant and erythromycin ointment were applied alternately to the surgical site. Mice were given intramuscular injections of penicillin (32,000 units per mouse) daily for three consecutive days post-surgery to prevent infection, and were observed individually until fully recovered. For the sham-operated group, anesthesia and incision methods were the same as above, except that only part of the fat was removed; subsequent procedures were the same.

[0117] After one week of postoperative recovery with no abnormalities in the wound, the mice were divided into four groups: a model group (OVX group, n=6), a donkey-hide gelatin group (n=6), a fish maw collagen group (n=6), a low-dose fish maw collagen group (n=6), a medium-dose fish maw collagen group (n=6), and a high-dose fish maw collagen group (n=6) (n=6). Mice were administered the drugs via gavage. The low-, medium-, and high-dose fish maw collagen groups were given concentrations of 0.75 g / kg, 1.5 g / kg, and 3 g / kg, respectively. The donkey-hide gelatin and fish maw collagen groups were given a concentration of 1.5 g / kg. The sham-operated group and the OVX model group were given an equal volume of distilled water via gavage. All mice were administered the drugs once daily for 12 weeks, with body weight recorded weekly. Three days before the end of the treatment period, urine and feces were collected from each group and stored at -80°C. After drug administration, mice in each group were euthanized by enucleation to collect blood. Bilateral femurs were harvested, and the soft tissue surrounding the bone was removed. The bone tissue was fixed with 4% paraformaldehyde and used for decalcified paraffin sections and three-dimensional reconstruction analysis. Liver tissue was fixed with 4% paraformaldehyde.

[0118] Morphological parameters of mouse femoral tissue were assessed using Mirco-CT to measure bone mineral density in the proximal femur of the right femur. CT-AN software was used for three-dimensional analysis of trabecular bone parameters, and CTVOX was used for three-dimensional reconstruction. The instrument scanning parameters were: 55 kV tube potential, 148 μA tube current, 8 W power, and 10.4 μm resolution. Relevant detection parameters included BMD (g / cm³). 3 Bone volume fraction (BV / TV, %), number of trabeculae (Tb.N, 1 / mm), and trabecular separation (Tb.Sp, mm).

[0119] Analysis using micro-CT, such as Figures 5-6 As shown, three-dimensional structural changes in the mouse femur were observed, with key indicators including BMD, BV / TV, Tb.N, and Tb.Sp. We used micro-CT to reconstruct the three-dimensional microstructure of the mouse femur. Compared to the sham-operated group, the model group showed a significant reduction in trabeculae and a looser internal structure. Compared to the model group, the medium- and high-dose fish maw collagen groups showed denser, more uniformly distributed, and more complete trabeculae in terms of morphology and structure. There was no significant difference in trabeculae between the donkey-hide gelatin group and the fish maw collagen group. Compared to the Sham group, the OVX group showed significant loss and loose arrangement of trabeculae. Compared to the OVX group, the medium- and high-dose SBG groups showed higher trabeculae density, more uniform distribution, and more complete morphology and structure, while there was no significant difference in trabeculae between the EJ and SBC groups.

[0120] like Figures 7-10As shown, the bone density (BMD) of the distal femur in the OVX group was significantly lower than that in the sham-operated group (p<0.001), while the bone volume (BV) / tear volume (TV) and total bone density (Tb.N) were significantly reduced (p<0.01), and the total bone density (Tb.Sp) was significantly increased (p<0.001), confirming the successful establishment of the osteoporosis model. Compared with the OVX group, BMD and Tb.N were significantly increased in all SBG dose groups (p<0.05), while BV / tear volume (BV / TV) was significantly increased (p<0.05) and Tb.Sp was significantly decreased (p<0.05) in the SBGM and SBGH groups. The SBGH group showed the most significant effect, with BMD, BV / TV, and Tb.N reaching (206.44±17.65) mg / cm². 3 The percentages of β-carotene (BMD), BV / TV, and Tb.N decreased to (7.45±1.21)% and (3.19±0.50) L / mm (p<0.001), respectively, while Tb.Sp decreased to (0.26±0.04) mm (p<0.01). There were no statistically significant differences in BMD, BV / TV, and Tb.N between the EJ and SBC groups and the OVX group. These results indicate that SBG has the potential to alleviate osteoporosis in ovariectomized mice, while EJ and SBC did not show significant efficacy.

[0121] Example 5

[0122] This embodiment aims to stain the mouse femoral tissue in Example 2 to further demonstrate that SBG can increase the number of trabeculae, reduce the spacing between trabeculae, and cure osteoporosis caused by estrogen deficiency.

[0123] Preparation of decalcified bone paraffin sections: Femoral bone specimens were fixed in a gradient of 4% paraformaldehyde solution (pH 7.4) at 4°C for 24 hours; pulsed rinsing with distilled water (5 times × 3 minutes); dynamic decalcification with 15% EDTA decalcification solution (pH 7.2) (changing the solution daily) until the needle could easily penetrate the tissue block, indicating decalcification was complete. Gradient ethanol dehydration (70%–100% ethanol, 2 hours per grade); xylene clearing (30 minutes each for grades I and II); paraffin impregnation (56°C low-melting-point paraffin, 3 times × 1 hour); sagittal oriented embedding. 4.5 μm continuous sections were prepared using low-temperature trimming techniques, and glass slides were pre-coated with poly-L-lysine to prevent delamination.

[0124] Immerse the tissue sections vertically in fresh xylene solution (analytical grade, water content ≤0.1%) and let stand at room temperature (25±2℃) for 10 minutes. Repeat the immersion in xylene for 10 minutes, ensuring the liquid completely covers the tissue sections throughout the process. Transfer sequentially to: 100% ethanol I: 3 minutes; 100% ethanol II: 3 minutes; 95% ethanol: 2 minutes; 85% ethanol: 2 minutes; 70% ethanol: 1 minute. Replace with fresh ethanol at each stage, keeping the container sealed to prevent evaporation during the immersion process. Dehydration with a gradient of ethanol: 70% ethanol: 30 seconds; 85% ethanol: 1 minute; 95% ethanol: 2 minutes; 100% ethanol I: 3 minutes; 100% ethanol II: 3 minutes; then immerse in xylene: Xylene I: 5 minutes; Xylene II: 5 minutes.

[0125] The entire procedure was performed in a fume hood, with fresh xylene added hourly to ensure transparency. While the sections were still wet, a suitable amount of neutral resin (refractive index 1.52±0.02) was added; a coverslip was slowly added to avoid air bubbles, and the slide was then mounted and cured in a 56℃ oven for 2 hours. The slides were then photographed under a microscope.

[0126] Figure 11 This image shows the HE staining results of the femurs of mice in this study. The Sham group maintained a dense trabecular network with uniform spatial distribution and strong connections, while the OVX, EJ, and SBC groups showed characteristic osteoporotic changes, including trabecular fragmentation, reduced connectivity, and increased intertrabecular spacing within the medullary cavity. Notably, SBG intervention (SBGM and SBGH groups) significantly restored trabecular integrity, manifested as an increase in the number of trabeculae and a decrease in intertrabecular spacing. The HE staining results were largely consistent with the Micro-CT results in this study, jointly demonstrating the efficacy of SBG in reversing bone loss and microstructural deterioration caused by ovariectomy.

[0127] Example 6

[0128] This embodiment is intended to illustrate that SBG can be used to cure osteoporosis by inhibiting the expression of osteoclast differentiation factor in mammals.

[0129] In this embodiment, TRAP staining of the femur of the mice in Example 2 was performed.

[0130] An acidic buffer system (50 mM potassium sodium tartrate, pH 5.0) containing 1.0 mg / mL naphthol AS-BI phosphate and 0.6 mg / mL Solid Red TR salt was precisely prepared and used immediately after sterilization via a 0.22 μm microporous membrane. For pre-staining, a xylene gradient dewaxing process (10 minutes each for stages I and II) was used in conjunction with an ethanol gradient hydration process (3 minutes per stage from 100% to 70%). After rinsing with deionized water, the tissue sections were pre-equilibrated at 37°C for 20 minutes to activate enzyme activity. During the critical staining stage, fresh TRAP incubation solution was evenly applied to the tissue sections, and the sections were incubated in the dark for 45 minutes under strictly controlled temperature (37°C ± 0.5°C) and humidity (>80%) conditions. This allowed the enzyme-substrate reaction to catalyze the formation of a wine-red, insoluble precipitate by acid phosphatase in the osteoclast cytoplasm. After the reaction was terminated, the cell nuclei were counterstained with Mayer hematoxylin (1.5 minutes), differentiated with hydrochloric acid and ethanol and then blued with running water. Finally, the neutral resin was mounted by gradient dehydration with ethanol (1 minute per 70% to 100% step) and clearing with xylene (5 minutes each for I and II), and photographed under a microscope.

[0131] The results are as follows Figure 12 As shown, TRAP staining revealed a significant increase in osteoclast surface area in the model group. However, dose-dependent treatment with fish maw glue resulted in a substantial reduction in osteoclast surface area in all dose groups. These findings from femoral TRAP staining further confirm that the intervention with processed fish maw glue effectively alleviates excessive bone resorption in ovariectomized mice, indicating its potential inhibitory effect on excessive activation of osteoclasts in vivo.

[0132] Example 7

[0133] This example aims to illustrate that SBG can alleviate osteoporosis by promoting bone formation and inhibiting bone resorption activity. This example demonstrates chemical staining of mouse femoral tissue from Example 2.

[0134] First, paraffin sections were dewaxed using a xylene gradient (10 minutes each for stages I and II), followed by gradient ethanol hydration (3 minutes per stage from 100% to 70%). Then, autoclaving with EDTA buffer (pH 9.0) at 121°C for 3 minutes was performed to fully expose the antigenic epitopes. Subsequently, incubation with 3% hydrogen peroxide solution at room temperature in the dark for 20 minutes was used to block endogenous peroxidase activity, followed by blocking non-specific binding sites with 5% bovine serum albumin (BSA) for 30 minutes. For the primary antibody incubation stage, diluted antibody (typically 1:100–1:200 titer) was evenly applied to the tissue area and incubated overnight in a humidified chamber at 4°C to ensure adequate binding. The next day, the tissue was washed with PBS buffer (3 times × 5 minutes) to remove unbound antibody. The secondary antibody reaction used an HRP-labeled polymer system (such as EnVision™), incubated at room temperature for 1 hour to achieve signal cascade amplification. The DAB staining process was strictly monitored under a microscope until a clear brown-yellow signal appeared, at which point the reaction was immediately terminated. After counterstaining cell nuclei with hematoxylin (2 minutes), the cells were dehydrated with a gradient of ethanol (70%–100%, 1 minute per grade), cleared with xylene (5 minutes each for grades I and II), and mounted with neutral resin. The staining results were observed under a light microscope, and the image data were analyzed using ImageJ.

[0135] The results are as follows Figures 13-15 As shown, immunohistochemical analysis of femoral tissue revealed significant changes in these key regulatory factors after ovariectomy. Compared to the Sham group, the OVX group showed a 65.41% decrease in RUNX2 expression and a 4.84-fold increase in RANKL levels (p<0.001) in the femur. Medium- and high-dose SBG intervention reversed these pathological changes, with RUNX2 expression increasing by 1.76-fold and 1.78-fold, respectively, and RANKL levels decreasing by 52% and 58%, respectively, compared to the OVX group (p<0.001). Notably, there were no significant differences in RUNX2 and RANKL expression among the EJ, SBC, and OVX groups (p>0.05). These results suggest that SBG may exert its anti-osteoporosis effect through a dual mechanism of promoting bone formation and inhibiting bone resorption, while EJ and SBC did not show significant therapeutic effects.

[0136] Example 8

[0137] This embodiment demonstrates that SBG can regulate bone metabolism balance to cure osteoporosis by detecting indicators in the urine and serum of mice collected in Example 2.

[0138] Urine samples: After centrifugation, urine samples were centrifuged at 3000g for 15 min at 4℃ and stored at -80℃. Whole blood samples were collected from the eyes of mice in each group, incubated at 4℃ for 4 hours, and then centrifuged at 3000g for 15 min at 4℃. The supernatant serum was aliquoted into 200μL EP tubes and stored at -80℃. Before testing, the thawed samples were centrifuged again.

[0139] Serum and urine related indicators: estradiol (E2), tartrate-resistant acid phosphatase (TRAP), osteocalcin (OCN), bone-specific alkaline phosphatase (BALP), type I collagen N-terminal elongated peptide (P1NP), deoxypyridinoline (DPD), tumor necrosis factor (TNF-α), and interleukin (IL-17A) were detected using ELISA kits according to the manufacturer's instructions. Urinary calcium and phosphorus ions were detected using a calcium (Ca) and phosphorus (P) assay kit.

[0140] like Figure 16 As shown, compared with the Sham group, the serum estrogen level in the OVX group mice was significantly lower (p<0.05), confirming the successful establishment of the model. The serum levels of TRAP, β-ALP, TNF-α, and IL-17A, as well as urinary DPD, Ca, and P levels in the OVX group were significantly higher than those in the Sham group, while serum OCN and PINP levels were lower. To evaluate the anti-osteoporosis effect of SBG, we compared SBG-treated mice with the OVX group. The high-dose SBG group showed the most significant bone-forming effect, with serum OCN and PINP levels increasing by 25% and 16%, respectively (p<0.001), consistent with the upregulation of RUNX2 expression in immunohistochemical staining images (p<0.001), indicating that SBG has bone-forming activity. Meanwhile, high-dose SBG treatment reduced serum β-ALP and TRAP levels, as well as urinary DPD, P, and Ca levels by 21.37%, 8.94%, 17.03%, 47.13%, and 28.89%, respectively. This, consistent with downregulation of RANKL expression in immunohistochemical staining images (p<0.001) and a reduction in osteoclast staining area in TRAP staining, further supports its anti-bone resorption effect. In addition, SBG significantly reduced TNF-α and IL-17A levels by 44.74% and 49.26%, respectively (p<0.001), further confirming its anti-bone resorption effect, as these inflammatory cytokines promote osteoclast formation. However, there were no significant changes in the donkey-hide gelatin group and the SBC group compared to the OVX group (p>0.05). In conclusion, SBG exerts a dose-dependent improvement effect on estrogen deficiency-induced osteoporosis through a dual action of promoting bone formation and inhibiting bone resorption, as well as its anti-inflammatory effect.

[0141] Example 9

[0142] This embodiment is intended to illustrate that SBG can regulate osteoporosis induced by oophorectomy-induced gut microbiota dysbiosis.

[0143] In this study, 16S rRNA gene sequencing was performed on fecal samples from the sham surgery, OVX, and SBG groups to comprehensively analyze the bacterial community structure. Principal component analysis (PCA) and principal coordinate analysis (PCoA) of β-diversity showed a clear clustering pattern in the gut microbiota composition. The OVX group and the sham surgery control group were significantly separated along the principal coordinate, indicating estrogen deficiency-induced changes in microbial structure. Furthermore, the gut microbiota structure of the SBG group was highly similar to that of the sham surgery group. These results suggest that SBG improves ovariectomy-induced osteoporosis by modulating the gut microbiota.

[0144] As described above, the basic principles, main features, and advantages of the present invention have been well described. The above embodiments and specifications are merely descriptions of preferred embodiments of the present invention, and the present invention is not limited to the above embodiments. Various changes and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit and scope of the present invention should fall within the protection scope defined by the present invention.

Claims

1. A method for preparing processed fish swim bladder glue, characterized in that, It includes the following steps: Step 1: Obtain the primary glue solution, including the following steps: Step 101: Crush and pre-treat the dried fish swim bladder, place it in a container, add clear water according to a material-liquid ratio of 1:30, and record the initial liquid level line; Step 102: Heat to 100 °C, boil, and then cool to 80 - 90 °C for continuous steaming; Step 103: Continuously steam for 192 - 216 h, and replenish water to the initial liquid level line every 4 hours during the steaming process to obtain the primary glue solution; Step 2: Concentrate the primary glue solution obtained in Step 103, including the following steps: Step 201: Separate the solid and liquid of the primary glue solution through a 400-mesh sieve to obtain the extract; Step 202: Evaporate the extract obtained in Step 201 until it has a viscosity of flowing down in a bead-like form; then add rock sugar and continuously concentrate until the paste is in a semi-transparent jelly-like state. When it can be hung into a flag, add alcohol-containing food, stir vigorously, heat with a small fire, and concentrate the glue solution until it has a form of slowly falling in flakes to obtain the concentrated glue solution; Step 3: Subject the concentrated glue solution obtained in Step 202 to shaping treatment, including the following steps: Step 301: Freeze the concentrated glue solution to obtain a frozen glue solution; Step 302: Use a vacuum freeze dryer to conduct vacuum freeze drying treatment on the frozen glue solution obtained in Step 301 under a pressure of 0.1 mbar to obtain the processed fish swim bladder glue.

2. The preparation method according to claim 1, characterized in that, In step 202, the mass ratio of rock sugar to fish maw glue is 1:50; the alcoholic food is rice wine, the mass ratio of rice wine to fish maw glue is 1:50, and the density of the concentrated glue solution is 1.35-1.40 g / cm³. 3 The freezing temperature in step 301 is -80℃ and the freezing time is 4 hours; the vacuum freeze-drying time in step 302 is 48 hours.

3. A processed fish maw glue obtained by the preparation method of claim 1, characterized in that, The processed fish swim bladder glue contains 5-hydroxymethylfurfural and melanoidins.

4. The prepared fish maw glue according to claim 2, characterized in that, The molecular weight distribution of the processed fish swim bladder glue is as follows: the proportion of 400 Da < M ≤ 5 kDa is 63.71%, the proportion of 5 kDa < M ≤ 10 kDa is 12.87%, and the proportion of M > 10 kDa is 23.46%.

5. The prepared fish maw glue according to claim 1, characterized in that, The processed fish swim bladder glue contains free amino acids, and the free amino acids include glycine, glutamic acid, proline, alanine, proline, arginine, lysine, and leucine.

6. The application of the processed fish maw glue according to claim 3 in the preparation of medicines, characterized in that, The drug is used for intestinal flora imbalance caused by estrogen deficiency or bone diseases caused by estrogen deficiency.

7. The application according to claim 6, characterized in that, The drug is used to inhibit bone mineral density loss caused by estrogen deficiency in mammals to treat and / or prevent osteoporosis; and / or to reduce the reduction of trabecular bone number and / or the increase of trabecular bone spacing caused by estrogen deficiency in mammals to treat and / or prevent osteoporosis.

8. The application according to claim 6, characterized in that, The drug is used to regulate bone metabolism balance to treat and / or prevent osteoporosis; the regulation of bone metabolism balance is to inhibit the increase of type I collagen amino-terminal extension peptide and osteocalcin in the serum of mammals; reduce alkaline phosphatase and tartrate-resistant acid phosphatase in the serum; reduce deoxypyridinoline, calcium, and phosphorus in urine; reduce the level of inflammatory factors in mammals, and the inflammatory factors include TNF-α and IL-17A.

9. The application according to claim 6, characterized in that, The drug is used to inhibit the expression of osteoclast differentiation factor in mammals to treat and / or prevent osteoporosis, and / or to increase the expression of transcription factor Runx2 in mammals to treat and / or prevent osteoporosis.

10. Use of the processed fish swim bladder glue according to claim 3 in the preparation of a nutritional supplement or a dietary supplement.

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