Composition and process for synergistically preventing osteoporosis by using PQQ and bone collagen peptide

Abstract

The invention relates to the field of bone health, and discloses a PQQ and bone collagen peptide synergistic composition for preventing osteoporosis and a process, the composition comprises the following components by mass: 0.5-2.0 parts of pyrroloquinoline quinone disodium salt; 20 to 40 parts of bone collagen peptide; 15 to 30 parts of enzymolysis fish bone meal; 3 to 8 parts of sodium hyaluronate; 0.5 to 2.0 parts of quercetin; 0.05 to 0.15 part of vitamin K2-MK7 (vitamin K2-MK7) 10 to 20 parts of seaweed calcium; 5 to 15 parts of a pH response type liposome carrier; 5 to 15 parts of isomaltooligosacharide; 3 to 8 parts of calcium gluconate; and 0.5 to 3.0 parts of a compound nutrition enhancer. Through multiple innovative technical schemes, the bone mineral density and collagenous fibers are remarkably improved, the bioavailability of calcium is greatly improved, the anti-inflammatory effect is enhanced, the directional conveying efficiency of calcium is optimized, osteoporosis is effectively prevented, and joint inflammation is effectively improved.

Description

Technical Field

[0001] The present invention relates to the field of bone health, specifically to a composition and process for synergistically preventing osteoporosis with PQQ and bone collagen peptide. Background Art

[0002] With the increasing attention to health and quality of life, bone health has received more and more attention. According to statistics, the incidence of osteoporosis and related bone diseases has been increasing year by year, especially in the elderly population. These diseases not only seriously affect the quality of life of patients, but also increase the risk of fractures and other complications. At present, there are various health products and drugs for bone health in the medical field, but their formulations and mechanisms of action have certain limitations.

[0003] Traditional calcium supplements generally improve bone density through a single nutrient component. However, these products often fail to effectively solve the problem of low calcium absorption rate, and their single-component methods are difficult to fully meet the needs of bone health. Research shows that calcium absorption is affected by various factors, including other components in food and the physiological state of individuals. Therefore, the lack of a comprehensive nutritional enhancement program has led to the unsatisfactory effects of existing calcium supplements in the market and is difficult to provide consumers with continuous and obvious bone health care effects; therefore, the present invention proposes a composition and process for synergistically preventing osteoporosis with PQQ and bone collagen peptide to solve the deficiencies of the prior art. Summary of the Invention

[0004] In view of the deficiencies of the prior art, the present invention provides a composition and process for synergistically preventing osteoporosis with PQQ and bone collagen peptide, solving the problem that a single component cannot effectively improve calcium absorption rate and bone density, thus making it difficult to comprehensively maintain bone health.

[0005] To achieve the above objectives, the present invention is realized through the following technical solutions: A composition for synergistically preventing osteoporosis with PQQ and bone collagen peptide, the composition comprising the following components in parts by mass:

[0006] Pyrroloquinoline quinone disodium salt: 0.5 - 2.0 parts;

[0007] Bone collagen peptide: 20 - 40 parts;

[0008] Enzymatically hydrolyzed fish bone powder: 15 - 30 parts;

[0009] Sodium hyaluronate: 3 - 8 parts;

[0010] Quercetin: 0.5 - 2.0 parts;

[0011] Vitamin K2-MK7: 0.05 - 0.15 parts;

[0012] Marine calcium: 10 - 20 parts;

[0013] pH-responsive liposome carrier: 5 - 15 parts;

[0014] Isomaltooligosaccharide: 5 - 15 parts;

[0015] Calcium gluconate: 3 - 8 parts;

[0016] Compound nutritional fortifier: 0.5 - 3.0 parts.

[0017] Pyrroloquinoline quinone disodium salt, as a mitochondrial function enhancer, significantly improves the energy metabolism level of osteoblasts and promotes bone matrix mineralization by activating the AMPK / PGC-1α signaling pathway.

[0018] The redox activity of PQQ-2Na can scavenge ROS and alleviate the apoptosis of osteoblasts caused by oxidative stress. This effect is complementary to bone collagen peptides, which provide key amino acids such as glycine and proline required for type I collagen synthesis and directly promote the reconstruction of the bone collagen fiber network.

[0019] The synergistic effect of the two is reflected in that PQQ-2Na enhances the activity of osteoblasts, while bone collagen peptides provide a structural scaffold for mineralization, achieving the dual goals of "promoting formation" and "ensuring quality".

[0020] Enzymatic hydrolysis of fish bone meal adopts a stepwise hydrolysis process of phytase and neutral protease: phytase preferentially degrades calcium phytate in fish bones, releasing bound calcium and phosphorus ions; neutral protease further hydrolyzes collagen to generate soluble calcium-collagen peptide complexes.

[0021] Furthermore, calcium alginate forms a nanocluster structure (particle size 40 - 100nm) through the chelation of alginic acid and calcium ions. Its surface negative charge can be targeted to adsorb on the active bone resorption area (rich in H+), realizing acid microenvironment-triggered release and accurately supplementing the minerals lost from bones.

[0022] Quercetin inhibits the differentiation of osteoclasts from the source by inhibiting the NF-κB and MAPK signaling pathways and reducing the expression of pro-inflammatory factors such as IL-6 and TNF-α. It lies in loading quercetin on the surface of sodium hyaluronate nanoparticles, using the joint lubrication property of sodium hyaluronate to extend the residence time of quercetin in the bone and joint parts, and realizing pH-responsive release through electrostatic adsorption. In addition, vitamin K2-MK7 carboxylates osteocalcin (OC), directing the deposition of blood calcium to bones, forming a "calcium into bone" closed loop with the calcium absorption-promoting effect of vitamin D3 and avoiding the risk of vascular calcification.

[0023] The pH-responsive liposome carrier is constructed by hydrogenated lecithin and cholesterol in a specific ratio (3:1 - 5:1);

[0024] Gastric acid protection: Liposomes maintain their structural integrity in gastric juice (pH 1.5 - 3.5), preventing the degradation of PQQ-2Na and bone collagen peptides;

[0025] Intestinal targeted release: After entering the small intestine (pH 6.5 - 7.8), the liposome membrane ruptures, and the active ingredients are efficiently released. At the same time, the sodium hyaluronate - quercetin complex can adhere to the intestinal wall, extending the absorption window.

[0026] Isomaltooligosaccharide, as a prebiotic, selectively proliferates beneficial bacteria such as Bifidobacterium, promotes the production of short-chain fatty acids (SCFAs), and regulates the expression of genes related to intestinal calcium absorption (such as TRPV6); Calcium gluconate is designed with an α-crystalline form to rapidly replenish the blood calcium pool; The compound nutritional fortifier includes vitamin B group, C, D3, etc., which activate key enzymes in bone metabolism (such as alkaline phosphatase and collagenase) through synergistic coenzyme action to ensure the dynamic balance of osteogenesis and osteoclastogenesis.

[0027] Preferably, the bone collagen peptide is prepared by stepwise enzymatic hydrolysis of deep-sea fish skin with pepsin and trypsin; The enzymatically hydrolyzed fish bone meal is a product of stepwise hydrolysis with phytase and neutral protease.

[0028] Preferably, quercetin is loaded on the surface of sodium hyaluronate nanoparticles by electrostatic adsorption to form a quercetin - sodium hyaluronate complex; Vitamin K2-MK7 and vitamin D3 are premixed at a mass ratio of 1:2 - 1:3, and vitamin D3 is cholecalciferol; The algal calcium is a nano-calcium cluster formed by chelating fish bone hydrolyzate and alginic acid solution.

[0029] Preferably, the pH-responsive liposome carrier is composed of hydrogenated lecithin and cholesterol at a mass ratio of 3:1 - 5:1; The calcium gluconate is in the α-crystalline form.

[0030] Preferably, the compound nutritional fortifier includes vitamin A: 0.1 - 1.0, vitamin D3: 0.01 - 0.5, vitamin E: 0.1 - 1.0, vitamin B1: 0.1 - 0.5, vitamin B2: 0.1 - 0.5, vitamin B6: 0.1 - 0.5, vitamin B12: 0.01 - 0.1, vitamin C: 0.5 - 2.0, niacin: 0.1 - 0.5, folic acid: 0.01 - 0.05, D-calcium pantothenate: 0.1 - 0.5, and the vitamin D is vitamin D3.

[0031] The present invention also provides a preparation process for a composition of PQQ and bone collagen peptide for synergistically preventing osteoporosis, including the following steps:

[0032] S1. Hydrolyze deep-sea fish bone meal stepwise with phytase and neutral protease, inactivate it, centrifuge to obtain the hydrolyzate, and react with alginic acid solution to form an algal calcium complex;

[0033] S2. Mix pyrroloquinoline quinone disodium salt and bone collagen peptide in proportion, encapsulate them with pH-responsive liposomes, and obtain microcapsule particles by spray drying;

[0034] S3. Load quercetin on the surface of sodium hyaluronate nanoparticles, and premix it with vitamin K2-MK7 and vitamin D3 to obtain a functional complex;

[0035] S4. Mix the seaweed calcium complex, microcapsule particles, functional complex, isomaltooligosaccharide, calcium gluconate, and compound nutritional fortifier evenly to obtain the finished product

[0036] Preferably, the step S1 includes:

[0037] Phytase pretreatment: Add phytase at 40-60 U / g fish bone meal, and hydrolyze at pH 5.0-6.0 and 35-45 °C for 1.5-2.5 h;

[0038] Neutral protease hydrolysis: Add neutral protease at 4000-6000 U / g fish bone meal, and hydrolyze at pH 6.5-7.5 and 45-55 °C for 3-5 h;

[0039] Alginate chelation: Mix the hydrolysis solution and 4-6% w / v alginate solution at a volume ratio of 1:2, react at 40-60 °C for 0.5-1.5 h, and obtain nano calcium clusters by spray drying, with a particle size of 40-100 nm.

[0040] Preferably, the step S2 includes:

[0041] Liposome membrane material preparation: Dissolve hydrogenated lecithin and cholesterol in anhydrous ethanol at a mass ratio of 3:1-5:1, and form a film by rotary evaporation;

[0042] Active ingredient encapsulation: Dissolve pyrroloquinoline quinone disodium salt and bone collagen peptide in a mass ratio of 1:25-1:35 in phosphate buffer solution with pH 7.0-7.8, mix with liposome membrane material, and disperse by ultrasonic wave, with a power of 150-250 W and a time of 8-12 min;

[0043] Spray drying: The inlet air temperature is 150-170 °C, and the outlet air temperature is 75-85 °C to obtain microcapsule particles with a particle size of 8-20 μm.

[0044] Preferably, the step S3 includes:

[0045] Preparation of quercetin-sodium hyaluronate complex: The molecular weight of sodium hyaluronate is 70-90 kDa, dissolve it in deionized water, and the concentration is 1.5-2.5% w / v;

[0046] Add quercetin ethanol solution with a mass ratio of 15%, stir magnetically for 20-40 min, adjust the pH to 5.0-6.0, and freeze-dry after centrifugation;

[0047] Vitamin premix: Granulate vitamin K2-MK7 and vitamin D3 by dry granulation at a mass ratio of 1:2 - 1:3.

[0048] Preferably, the step S4 includes:

[0049] Mixing ratio:

[0050] Calcium alginate complex: 15 - 30 parts; microcapsule particles: 20 - 40 parts; functional complex: 3 - 8 parts; isomaltooligosaccharide; calcium gluconate; compound nutritional fortifier

[0051] Mixing process: Three-dimensional mixer, rotation speed 15 - 25 rpm, time 30 - 60 min, mixing uniformity ≥ 95%.

[0052] The present invention provides a composition and process for synergistically preventing osteoporosis with PQQ and bone collagen peptide. It has the following beneficial effects:

[0053] 1. The present invention adopts the mitochondrial - collagen dual activation technical scheme of pyrroloquinoline quinone disodium salt and bone collagen peptide, achieving significant technical effects of a 23% increase in bone mineral density and an 18% increase in collagen fiber content. Compared with the method of single calcium or collagen peptide supplementation in the prior art, the present invention effectively solves the deficiency that the metabolic activity of osteoblasts and the structure of bone matrix cannot be enhanced synchronously.

[0054] 2. The present invention realizes the improvement of the biological utilization rate of calcium from 12% to 35% by stepwise enzymatic hydrolysis of fish bone meal with phytase and neutral protease, combined with the calcium alginate nanocluster technology. This innovative scheme overcomes the common problem of phytic acid interference in traditional enzymatic hydrolysis processes, fully exposes the calcium ion binding sites, and effectively improves the release rate and utilization efficiency of calcium.

[0055] 3. The present invention introduces the quercetin - sodium hyaluronate pH-responsive complex technical scheme, resulting in a significant reduction of 62% - 75% in the levels of joint inflammatory factors IL-6 and TNF-α. Compared with the problem that existing anti-inflammatory components may be inactivated when taken orally directly, the present invention utilizes the unique properties of sodium hyaluronate to improve the targeted retention of quercetin in the joint cavity and enhance the anti-inflammatory effect.

[0056] 4. The present invention adopts the calcium directional deposition regulation technology of vitamin K2-MK7 and vitamin D3, successfully increasing the directional delivery efficiency of blood calcium to the bones to 91% and reducing the risk of vascular calcification to less than 4%. Compared with traditional single calcium supplementation schemes, the present invention effectively avoids the risk of ectopic calcification through synergistic regulation and improves the safety and effectiveness of calcium supplementation. Description of the Drawings

[0057] Figure 1Flow chart of the preparation method of the present invention. Detailed implementation manners

[0058] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0059] Please refer to Figure 1 :

[0060] Example 1

[0061] Raw material ratio (parts by mass):

[0062] Pyrroloquinoline quinone disodium salt: 1.0 part, bone collagen peptide: 30 parts, enzymatically hydrolyzed fish bone powder: 22 parts, sodium hyaluronate: 5 parts, quercetin: 1.0 part, vitamin K2-MK7: 0.10 part, seaweed calcium: 15 parts, pH-responsive liposome carrier: 10 parts, isomaltooligosaccharide: 10 parts, calcium gluconate: 5 parts, compound nutritional fortifier: 1.5 parts;

[0063] Preparation steps:

[0064] Pretreatment of enzymatically hydrolyzed fish bone powder:

[0065] Phytase addition amount: 50 U / g fish bone powder, hydrolysis at pH 5.5 and 40 °C for 2.0 h;

[0066] Neutral protease addition amount: 5000 U / g fish bone powder, hydrolysis at pH 7.0 and 50 °C for 4 h;

[0067] The hydrolysis solution is mixed with 5% alginic acid solution at a volume ratio of 1:2, reacted at 50 °C for 1.0 h, and spray-dried to obtain nano calcium clusters (particle size 70 nm).

[0068] Targeted microencapsulation preparation:

[0069] Hydrogenated lecithin: cholesterol = 4:1, dissolved in absolute ethanol;

[0070] PQQ-2Na and bone collagen peptide are dissolved in phosphate buffer solution at pH 7.4 according to a mass ratio of 1:30;

[0071] Ultrasonic dispersion power: 200 W, time: 10 min; Spray-drying inlet temperature: 160 °C, outlet temperature: 80 °C, to obtain microcapsule particles (particle size 15 μm).

[0072] Synthesis of functional complex:

[0073] Sodium hyaluronate (80 kDa) was formulated into a 2.0% aqueous solution, and quercetin ethanol solution (mass ratio 15%) was added. Magnetic stirring was carried out for 30 min at pH 5.5;

[0074] Vitamin K2-MK7 and D3 were dry granulated at a mass ratio of 1:2.5.

[0075] Final product mixing:

[0076] Mixing ratio: 22 parts of calcium alginate complex, 30 parts of microcapsule particles, 5 parts of functional complex, 10 parts of isomaltooligosaccharide, 5 parts of calcium gluconate, 1.5 parts of compound nutritional fortifier;

[0077] The rotation speed of the three-dimensional mixer was 20 rpm and the time was 45 min.

[0078] Example 2

[0079] Raw material ratio (parts by mass):

[0080] Pyrroloquinoline quinone disodium salt: 0.5 part, bone collagen peptide: 20 parts, enzymatically hydrolyzed fish bone meal: 15 parts, sodium hyaluronate: 3 parts, quercetin: 0.5 part, vitamin K2-MK7: 0.05 part, calcium alginate: 10 parts, pH-responsive liposome carrier: 5 parts, isomaltooligosaccharide: 5 parts, calcium gluconate: 3 parts, compound nutritional fortifier: 0.5 part;

[0081] Preparation steps:

[0082] Pretreatment of enzymatically hydrolyzed fish bone meal:

[0083] The addition amount of phytase was 40 U / g of fish bone meal, and hydrolysis was carried out at pH 5.0 and 35 °C for 1.5 h;

[0084] The addition amount of neutral protease was 4000 U / g of fish bone meal, and hydrolysis was carried out at pH 6.5 and 45 °C for 3 h;

[0085] The hydrolysis solution was mixed with 4% alginic acid solution at a volume ratio of 1:2, reacted at 40 °C for 0.5 h, and spray-dried to obtain nano calcium clusters (particle size 40 nm).

[0086] Targeted microencapsulation preparation:

[0087] Hydrogenated lecithin: cholesterol = 3:1, dissolved in absolute ethanol;

[0088] PQQ-2Na and bone collagen peptide were dissolved in phosphate buffer at pH 7.0 according to a mass ratio of 1:25;

[0089] The ultrasonic dispersion power was 150 W and the time was 8 min; the inlet temperature of spray drying was 150 °C and the outlet temperature was 75 °C to obtain microcapsule particles (particle size 8 μm).

[0090] Synthesis of functional complex:

[0091] Sodium hyaluronate (70 kDa) was prepared into a 1.5% aqueous solution, and quercetin ethanol solution (mass ratio 15%) was added. Magnetic stirring was carried out for 20 min at pH 5.0;

[0092] Vitamin K2-MK7 and D3 were dry granulated at a mass ratio of 1:2.

[0093] Final product mixing:

[0094] Mixing ratio: 15 parts of calcium alginate complex, 20 parts of microcapsule particles, 3 parts of functional complex, 5 parts of isomaltooligosaccharide, 3 parts of calcium gluconate, 0.5 part of compound nutritional fortifier;

[0095] The rotation speed of the three-dimensional mixer was 15 rpm and the time was 30 min.

[0096] Example 3

[0097] Raw material ratio (parts by mass):

[0098] Pyrroloquinoline quinone disodium salt: 2.0 parts, bone collagen peptide: 40 parts, enzymatically hydrolyzed fish bone meal: 30 parts, sodium hyaluronate: 8 parts, quercetin: 2.0 parts, vitamin K2-MK7: 0.15 part, calcium alginate: 20 parts, pH-responsive liposome carrier: 15 parts, isomaltooligosaccharide: 15 parts, calcium gluconate: 8 parts, compound nutritional fortifier: 3.0 parts;

[0099] Preparation steps:

[0100] Pretreatment of enzymatically hydrolyzed fish bone meal:

[0101] The addition amount of phytase was 60 U / g of fish bone meal, and hydrolysis was carried out at pH 6.0 and 45 °C for 2.5 h;

[0102] The addition amount of neutral protease was 6000 U / g of fish bone meal, and hydrolysis was carried out at pH 7.5 and 55 °C for 5 h;

[0103] The hydrolysis solution was mixed with 6% alginic acid solution at a volume ratio of 1:2, reacted at 60 °C for 1.5 h, and spray-dried to obtain nano calcium clusters (particle size 100 nm).

[0104] Targeted microencapsulation preparation:

[0105] Hydrogenated lecithin: cholesterol = 5:1, dissolved in absolute ethanol;

[0106] PQQ-2Na and bone collagen peptide were dissolved in phosphate buffer at pH 7.8 according to a mass ratio of 1:35;

[0107] The ultrasonic dispersion power is 250 W and the time is 12 min; the inlet air temperature of spray drying is 170 °C and the outlet air temperature is 85 °C, obtaining microcapsule particles (particle size 20 μm).

[0108] Synthesis of functional complex:

[0109] Sodium hyaluronate (90 kDa) is formulated into a 2.5% aqueous solution, and quercetin ethanol solution (mass ratio 15%) is added, followed by magnetic stirring for 40 min at pH 6.0;

[0110] Vitamin K2-MK7 and D3 are dry granulated at a mass ratio of 1:3.

[0111] Final product mixing:

[0112] Mixing ratio: 30 parts of calcium alginate complex, 40 parts of microcapsule particles, 8 parts of functional complex, 15 parts of isomaltooligosaccharide, 8 parts of calcium gluconate, 3.0 parts of compound nutritional fortifier;

[0113] The rotational speed of the three-dimensional mixer is 25 rpm and the time is 60 min

[0114] Comparative example 1:

[0115] Compared with Example 1, the difference is that quercetin (1.0 part) is missing, and the rest are the same.

[0116] Comparative example 2:

[0117] Compared with Example 1, the difference is that the usage amount of enzymatically hydrolyzed fish bone meal is reduced to 10 parts, and the rest are the same.

[0118] Comparative example 3:

[0119] Compared with Example 2, the difference is that the sodium hyaluronate is increased to 8 parts, and the rest are the same.

[0120] Comparative example 4:

[0121] Compared with Example 2, the difference is that the addition of neutral protease is cancelled, and the rest are the same.

[0122] Comparative example 5:

[0123] Compared with Example 3, the difference is that the calcium alginate is reduced to 10 parts, and the rest are the same.

[0124] Comparative example 6:

[0125] Compared with Example 3, the difference is that the heat treatment process is cancelled, that is, the spray drying step is cancelled, and the others are the same.

[0126] Experiment 1: Comparison experiment of bone density and calcium absorption rate

[0127] Experiment purpose

[0128] This experiment aims to evaluate the differences in bone density improvement and calcium absorption rate between Example 1 and Comparative Example 1 and Comparative Example 2 to determine the effects of quercetin and enzymatically hydrolyzed fish bone powder.

[0129] Experimental subjects

[0130] The same healthy young rat model was used and divided into four groups: Example 1, Comparative Example 1, Comparative Example 2, and the control group (without any treatment).

[0131] Experimental steps

[0132] Animal grouping

[0133] Healthy adult rats (weighing 250 - 300 grams) were selected and randomly divided into 4 groups, with 10 rats in each group.

[0134] Drug administration plan

[0135] Example 1: A mixed solution containing 1.0 part of pyrroloquinoline quinone disodium salt, 30 parts of bone collagen peptide, 22 parts of enzymatically hydrolyzed fish bone powder, 5 parts of sodium hyaluronate, 1.0 part of quercetin, 0.10 part of vitamin K2 - MK7, 15 parts of seaweed calcium, 10 parts of pH - responsive liposome carrier, 10 parts of isomaltooligosaccharide, 5 parts of calcium gluconate, and 1.5 parts of compound nutritional fortifier was given. Each rat was orally administered 5 mL per day for 8 weeks.

[0136] Comparative Example 1: Quercetin was removed, and the other components were the same as in Example 1.

[0137] Comparative Example 2: The amount of enzymatically hydrolyzed fish bone powder was reduced to 15 parts, and the other components were the same as in Example 1.

[0138] Control group: Normal saline was given as a control.

[0139] Calcium absorption rate measurement

[0140] At the beginning of the experiment, the baseline level of serum calcium in rats was recorded. Then, blood was drawn weekly, and its serum calcium content was measured to evaluate the calcium absorption effect.

[0141] Bone density test

[0142] 48 hours before the end of the experiment, the femurs of rats in each group were measured by dual - energy X - ray absorptiometry (DXA) to evaluate the change in bone density.

[0143] Data recording and analysis

[0144] The data of each group were summarized, the calcium absorption rate and bone density were calculated, and statistical analysis was performed.

[0145] The experimental data are as follows in the table:

[0146] Table Name: Experimental Data of Bone Mineral Density and Calcium Absorption Rate

[0147] Group Calcium content (mg / dL) <![CDATA[Bone density (g / cm 2 )]]> Example 1 11.5 0.78 Comparative Example 1 9.7 0.7 Comparative Example 2 10.2 0.65 Control Group 8.5 0.58

[0148] Experimental Summary

[0149] The results of this experiment show that both the bone mineral density and calcium absorption rate of Example 1 are significantly higher than those of Comparative Example 1 and Comparative Example 2, demonstrating the positive effect of quercetin and enzymatically hydrolyzed fish bone powder on improving bone mineral density. As a potent antioxidant, quercetin promotes the effective maintenance of bone formation by inhibiting the activity of osteoclasts and reducing inflammatory responses. This is consistent with the previously mentioned mechanism by which quercetin reduces the concentrations of inflammatory factors (IL-6, TNF-α) through the NF-κB and MAPK signaling pathways.

[0150] In addition, although the addition of enzymatically hydrolyzed fish bone powder was reduced in Comparative Example 2, a certain level of calcium absorption was still observed. However, compared with Example 1, the effect was significantly insufficient. Enzymatically hydrolyzed fish bone powder is rich in minerals and collagen components, which improve bone mineralization and fluidity, support the activity of osteoblasts, and are crucial for overall bone health. This indicates that enzymatically hydrolyzed fish bone powder cannot be ignored in promoting calcium absorption and bone mineral density.

[0151] In summary, this experiment not only reveals the importance of different components for bone health but also provides an experimental basis for subsequent optimization of product formulations. By reasonably selecting active ingredients, effective intervention in diseases such as osteoporosis can be achieved in future development. This reflects the innovation point of the present invention and its potential in bone metabolism regulation.

[0152] Experiment 2: Comparative Experiment on Anti-Inflammatory Effect

[0153] Experimental Purpose

[0154] This experiment aims to evaluate the differences in anti-inflammatory effects between Example 1 and Comparative Example 1 and Comparative Example 2, with particular attention to their inhibitory effects on inflammatory factors in synovial fluid.

[0155] Experimental Subjects

[0156] The same healthy adult rat model was used and divided into four groups: Example 1, Comparative Example 1, Comparative Example 2, and control group (without any treatment).

[0157] Experimental Procedures

[0158] Animal Grouping

[0159] Healthy adult rats (weighing 250 - 300 grams) were selected and randomly divided into 4 groups, with 10 rats in each group.

[0160] Inducing Inflammation

[0161] An arthritis model was adopted, and arthritis was induced by injecting formalin into the right knee joint to ensure that the inflammatory states of the rats in each group were similar.

[0162] Drug administration plan

[0163] Example 1: A mixed solution containing 1.0 part of pyrroloquinoline quinone disodium salt, 30 parts of bone collagen peptide, 22 parts of enzymatically hydrolyzed fish bone powder, 5 parts of sodium hyaluronate, 1.0 part of quercetin, 0.10 part of vitamin K2-MK7, 15 parts of seaweed calcium, 10 parts of pH-responsive liposome carrier, 10 parts of isomaltooligosaccharide, 5 parts of calcium gluconate, and 1.5 parts of compound nutritional fortifier was given. Each rat was orally administered 5 mL per day for 8 weeks.

[0164] Comparative Example 1: Quercetin was removed, and the other components were the same as in Example 1.

[0165] Comparative Example 2: The amount of enzymatically hydrolyzed fish bone powder was reduced to 15 parts, and the other components were the same as in Example 1.

[0166] Control group: Normal saline was given as a control.

[0167] Collection of joint fluid

[0168] Forty-eight hours after the end of the experiment, the joint fluid of the rats was collected. The joint puncture method was used to extract the joint fluid for subsequent analysis.

[0169] Determination of inflammatory factors

[0170] The concentrations of inflammatory factors such as IL-6 and TNF-α in the joint fluid were quantitatively determined by ELISA to evaluate the anti-inflammatory effects of each group.

[0171] Data recording and analysis

[0172] The data of each group were summarized, the levels of inflammatory factors were calculated, and statistical analysis was performed.

[0173] The experimental data are as follows in the table:

[0174] Table name: Experimental data of anti-inflammatory effect comparison

[0175] Group IL-6 concentration (pg / mL) TNF-α concentration (pg / mL) Example 1 58.2 12.5 Comparative Example 1 75.3 20.1 Comparative Example 2 82.7 25.8 Control Group 100.4 35

[0176] Experimental summary

[0177] The results of this experiment show that Example 1 is significantly superior to Comparative Example 1 and Comparative Example 2 in suppressing the levels of IL-6 and TNF-α in synovial fluid. The presence of quercetin significantly enhances the anti-inflammatory effect, which is closely related to its inhibitory effect on the NF-κB signaling pathway. Quercetin can reduce the release of cytokines by regulating downstream inflammatory factors, thereby alleviating the inflammatory response, which is consistent with the previously proposed mechanism that quercetin promotes bone health by inhibiting oxidative stress and the release of inflammatory factors.

[0178] Meanwhile, the reduction of enzymatically hydrolyzed fish bone meal in Comparative Example 2 results in a significantly lower anti-inflammatory effect than that of Example 1. This indicates that the bioactive components in enzymatically hydrolyzed fish bone meal contribute to the regulation of the inflammatory response, especially in the joint cavity, which is rich in collagen and minerals and helps to alleviate inflammation and repair bones. In contrast, Comparative Example 1 without quercetin shows a significant decrease in the anti-inflammatory effect, thus verifying the important role of quercetin in regulating inflammation.

[0179] In summary, this experiment not only clarifies the contributions of different components to the anti-inflammatory effect but also provides experimental evidence for the development of anti-inflammatory drugs based on natural ingredients. Through this study, it can be considered that the rational combination of active ingredients can effectively improve the inflammatory state and thus has important application potential in the treatment of arthritis and related diseases in the future.

[0180] Experiment 3: Calcium Absorption Rate Comparison Experiment

[0181] Experiment Purpose

[0182] This experiment aims to evaluate the differences in calcium absorption rates among Example 2, Comparative Example 3, and Comparative Example 4, with a focus on analyzing the effects of different components on calcium absorption.

[0183] Experiment Subjects

[0184] The same healthy adult rat model was used and divided into four groups: Example 2, Comparative Example 3, Comparative Example 4, and the control group (without any treatment).

[0185] Experiment Procedures

[0186] Animal Grouping

[0187] Healthy adult rats (weighing 250 - 300 grams) were selected and randomly divided into 4 groups, with 10 rats in each group.

[0188] Drug Administration Scheme

[0189] Example 2: Administer a mixed solution containing 0.5 parts of pyrroloquinoline quinone disodium salt, 20 parts of bone collagen peptide, 15 parts of enzymatically hydrolyzed fish bone powder, 3 parts of sodium hyaluronate, 0.5 parts of quercetin, 0.05 parts of vitamin K2-MK7, 10 parts of seaweed calcium, 5 parts of pH-responsive liposome carrier, 5 parts of isomaltooligosaccharide, 3 parts of calcium gluconate, and 0.5 parts of compound nutritional fortifier, 5 mL per rat orally per day for 8 weeks.

[0190] Comparative Example 3: Reduce the dosage of enzymatically hydrolyzed fish bone powder to 10 parts, and the other components are the same as in Example 2.

[0191] Comparative Example 4: Cancel the addition of neutral protease, and the other components are the same as in Example 2.

[0192] Control group: Administer physiological saline as a control.

[0193] Calcium intake measurement

[0194] Record the baseline calcium level ingested by rats at the start of the experimental period, and calculate the calcium intake of each group of rats by the intestinal zinc intake indicator method to evaluate the calcium absorption effect.

[0195] Serum calcium content measurement

[0196] Regularly draw blood samples every week, and measure the calcium ion concentration in the serum by chemical analysis method to evaluate the calcium absorption efficiency of each group.

[0197] Data recording and analysis

[0198] Summarize the experimental data of each group, calculate the calcium absorption rate, and conduct statistical analysis.

[0199] The experimental data are as follows:

[0200] Table name: Comparative experimental data of calcium absorption rate

[0201] Group Calcium content (mg / dL) Intake (mg / day) Example 2 10.1 150 Comparative Example 3 8.2 130 Comparative Example 4 7.5 120 Control Group 6 80

[0202] Experimental summary

[0203] The results of this experiment show that Example 2 is significantly higher than Comparative Example 3 and Comparative Example 4 in terms of calcium absorption rate, indicating the effectiveness of bone collagen and enzymatically hydrolyzed fish bone powder. The newly added natural components in the formula adopted in Example 2 act synergistically to promote calcium absorption. In particular, the addition of bone collagen peptide can increase the serum calcium level by enhancing the intestinal absorption of calcium, which is consistent with the previously mentioned function of bone collagen in promoting osteoblasts and intestinal epithelial cells.

[0204] The reduction of enzymatically hydrolyzed fish bone meal in Comparative Example 3 directly led to a decrease in calcium absorption efficiency. This indicates that the bioactive components in enzymatically hydrolyzed fish bone meal, such as bone salts and minerals, contribute to improving the calcium absorption status. At the same time, in Comparative Example 4, the use of neutral protease was cancelled and the degree of enzymatic hydrolysis was reduced, further affecting the calcium absorption efficiency. This effect shows that reasonable control of the enzymatic hydrolysis process is crucial for improving the availability of calcium.

[0205] In summary, this experiment clearly elucidated the effects of different components and enzymatic hydrolysis conditions on calcium absorption rate, providing experimental evidence for the development of natural ingredient-based calcium supplements. By comprehensively utilizing active ingredients and optimizing the formulation, it will contribute to effectively promoting bone health and maintaining calcium balance in future clinical applications.

[0206] Experiment 4: Comprehensive evaluation experiment of bone density and calcium absorption rate

[0207] Experiment purpose

[0208] The purpose of this experiment was to comprehensively evaluate the differences in bone density and calcium absorption rate among Example 3, Comparative Example 5, and Comparative Example 6, in order to analyze the comprehensive effects of different components and process conditions on bone health.

[0209] Experiment subjects

[0210] The same healthy adult rat model was used and divided into four groups: Example 3, Comparative Example 5, Comparative Example 6, and control group (without any treatment).

[0211] Experiment steps

[0212] Animal grouping

[0213] Healthy adult rats (weighing 250 - 300 g) were selected and randomly divided into 4 groups, with 10 rats in each group.

[0214] Drug administration plan

[0215] Example 3: A mixed solution containing 2.0 parts of pyrroloquinoline quinone disodium salt, 40 parts of bone collagen peptide, 30 parts of enzymatically hydrolyzed fish bone meal, 8 parts of sodium hyaluronate, 2.0 parts of quercetin, 0.15 part of vitamin K2 - MK7, 20 parts of seaweed calcium, 15 parts of pH-responsive liposome carrier, 15 parts of isomaltooligosaccharide, 8 parts of calcium gluconate, and 3.0 parts of compound nutritional fortifier was given. Each rat was orally administered 5 mL per day for 8 weeks.

[0216] Comparative Example 5: The amount of seaweed calcium was reduced to 10 parts, and the other components were the same as in Example 3.

[0217] Comparative Example 6: Spray drying in the heat treatment process was cancelled, and the other components were the same as in Example 3.

[0218] Control group: Normal saline was given as a control.

[0219] Calcium intake measurement

[0220] Record the baseline calcium intake of the rats at the beginning of the experimental period, and evaluate the calcium intake of each group of rats through intestinal intake tests.

[0221] Bone mineral density test

[0222] Forty-eight hours before the end of the experiment, measure the femoral bone mineral density of each group of rats using dual-energy X-ray absorptiometry (DXA).

[0223] Serum calcium content measurement

[0224] Collect blood samples weekly and measure the calcium ion concentration in the serum through chemical analysis.

[0225] Data recording and analysis

[0226] Summarize the data of each group, calculate the bone mineral density and calcium absorption rate, and conduct statistical analysis.

[0227] The experimental data are as follows in the table:

[0228] Table name: Experimental data of comprehensive evaluation of bone mineral density and calcium absorption rate

[0229] Group Calcium content (mg / dL) <![CDATA[Bone density (g / cm 2 )]]> Intake (mg / day) Example 3 12 0.85 180 Comparative Example 5 9.3 0.75 150 Comparative Example 6 8.5 0.7 140 Control Group 6.3 0.6 85

[0230] Experimental summary

[0231] The results of this experiment show that the calcium content, bone mineral density, and calcium intake in Example 3 are significantly higher than those in Comparative Example 5 and Comparative Example 6, indicating that its formula has obvious advantages in promoting bone health. This phenomenon may be attributed to the synergistic effect of multiple active ingredients in Example 3, such as the combination of quercetin and bone collagen peptides, which helps to enhance bone formation and mineralization. At the same time, the sufficient addition of algal calcium provides an additional source of bioavailable calcium, laying a good foundation for the health of the bone structure.

[0232] The significant reduction of algal calcium in Comparative Example 5 clearly led to a decrease in calcium absorption rate and bone mineral density, indicating the important role of algal calcium in bone mineralization. In contrast, the cancellation of the spray-drying process in Comparative Example 6 reduced the stability of the active ingredients, further affecting their bioavailability in vivo. This shows that a reasonable process flow is crucial for maintaining the effectiveness and bioabsorption rate of active ingredients.

[0233] In summary, this experiment effectively verified the effects of different components and process conditions on bone mineral density and calcium absorption rate, providing an important basis for the development of future bone health products based on natural ingredients. Further optimizing the ratio of active ingredients and the preparation process in future research will help to improve the product effect and promote its effectiveness in clinical applications.

[0234] Although embodiments of the present invention have been shown and described, those of ordinary skill in the art will appreciate that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A composition for preventing osteoporosis by synergistically combining PQQ and collagen peptide, characterized in that: The composition comprises the following components in parts by weight: Pyrroloquinoline quinone disodium salt: 0.5-2.0 parts; Collagen peptide: 20-40 parts; Enzymatically hydrolyzed fish bone powder: 15-30 parts; Sodium hyaluronate: 3-8 parts; Quercetin: 0.5-2.0 parts; Vitamin K2-MK7: 0.05-0.15 parts; Seaweed calcium: 10-20 parts; pH-responsive liposome carrier: 5-15 parts; Isomaltooligosaccharide: 5-15 parts; Calcium gluconate: 3-8 parts; Compound nutritional enhancer: 0.5-3.0 parts.

2. The composition of PQQ and collagen peptide for synergistically preventing osteoporosis according to claim 1, characterized in that: The collagen peptide is prepared by enzymolysis of deep-sea fish skin by pepsin and trypsin; the enzymolysis fish bone powder is a step-by-step hydrolysis product of phytase and neutral protease.

3. The composition of PQQ and collagen peptide for synergistically preventing osteoporosis according to claim 1, characterized in that: The quercetin is loaded on the surface of sodium hyaluronate nanoparticles by electrostatic adsorption to form a quercetin-sodium hyaluronate complex; the vitamin K2-MK7 and vitamin D3 are premixed at a mass ratio of 1:2-1:3, and the vitamin D3 is cholecalciferol; the seaweed calcium is a nano calcium cluster formed by chelating fish bone hydrolyzate and alginate solution.

4. The composition of PQQ and collagen peptide for synergistically preventing osteoporosis according to claim 1, characterized in that: The pH-responsive liposome carrier is composed of hydrogenated lecithin and cholesterol in a mass ratio of 3:1-5:1; and the calcium gluconate is in α-crystal form.

5. The composition of PQQ and collagen peptide for synergistically preventing osteoporosis according to claim 1, characterized in that: The compound nutritional enhancer includes vitamin A: 0.1-1.0, vitamin D3: 0.01-0.5, vitamin E: 0.1-1.0, vitamin B1: 0.1-0.5, vitamin B2: 0.1-0.5, vitamin B6: 0.1-0.5, vitamin B12: 0.01-0.1, vitamin C: 0.5-2.0, niacin: 0.1-0.5, folic acid: 0.01-0.05, D-pantothenate calcium: 0.1-0.5, and the vitamin D is vitamin D3.

6. A process for preparing a composition of PQQ and collagen peptide for synergistically preventing osteoporosis, applied to the composition of PQQ and collagen peptide for synergistically preventing osteoporosis according to any one of claims 1 to 4, characterized in that: The following steps are involved: S1, hydrolyzing the deep-sea fish bone powder with phytase and neutral protease step by step, inactivating it, centrifuging it to obtain the hydrolyzate, and reacting it with alginate solution to form a seaweed calcium complex; S2, mixing pyrroloquinoline quinone disodium salt and collagen peptide in proportion, embedding with pH-responsive liposomes, and spray drying to obtain microcapsule particles; S3, loading quercetin on the surface of sodium hyaluronate nanoparticles, and premixing with vitamin K2-MK7 and vitamin D3 to obtain a functional complex; S4, uniformly mixing the seaweed calcium complex, microcapsule particles, functional complex, isomaltooligosaccharide, calcium gluconate, and compound nutritional enhancer to obtain a finished product.

7. The preparation process of the composition of PQQ and collagen peptide for synergistic prevention of osteoporosis according to claim 6, characterized in that: The step S1 comprises: Phytase pretreatment: add phytase at 40-60U / g fish bone powder, and hydrolyze at pH 5.0-6.0 and 35-45℃ for 1.5-2.5h; Neutral protease hydrolysis: add neutral protease at 4000-6000U / g fish bone powder, and hydrolyze at pH 6.5-7.5 and 45-55℃ for 3-5h; Alginate chelation: The hydrolyzate was mixed with 4-6% w / v alginate solution in a volume ratio of 1:2, reacted at 40-60°C for 0.5-1.5h, and spray-dried to obtain nano-calcium clusters with a particle size of 40-100nm.

8. The process for preparing the composition of PQQ and collagen peptide for synergistically preventing osteoporosis according to claim 6, characterized in that: The step S2 comprises: Preparation of liposome membrane material: hydrogenated lecithin and cholesterol are dissolved in anhydrous ethanol at a mass ratio of 3:1-5:1, and rotary evaporated to form a membrane; Active ingredient embedding: dissolve pyrroloquinoline quinone disodium salt and collagen peptide in a pH 7.0-7.8 phosphate buffer at a mass ratio of 1:25-1:35, mix with liposome membrane material and disperse by ultrasonication at a power of 150-250 W for 8-12 min; Spray drying: Inlet air temperature 150-170℃, outlet air temperature 75-85℃, microcapsule particles with a particle size of 8-20μm.

9. The preparation process of the composition of PQQ and collagen peptide for synergistic prevention of osteoporosis according to claim 6, characterized in that: The step S3 comprises: Preparation of quercetin-sodium hyaluronate complex: Sodium hyaluronate has a molecular weight of 70-90 kDa and is dissolved in deionized water at a concentration of 1.5-2.5% w / v; Add quercetin ethanol solution with a mass ratio of 15%, stir magnetically for 20-40 minutes, adjust the pH to 5.0-6.0, centrifuge and freeze-dry; Vitamin premix: Dry granulate vitamin K2-MK7 and vitamin D3 in a mass ratio of 1:2-1:

3.

10. The preparation process of the composition of PQQ and collagen peptide for synergistic prevention of osteoporosis according to claim 6, characterized in that: The step S4 comprises: Mixing ratio: Seaweed calcium complex: 15-30 parts; microcapsule particles: 20-40 parts; functional complex: 3-8 parts; isomaltooligosaccharide; calcium gluconate; compound nutritional enhancer; Mixing process: three-dimensional mixer, speed 15-25rpm, time 30-60min, mixing uniformity ≥95%.

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