A multi-effect synergistic bone density enhancing composition enriched with euphausia superba oil and a preparation method thereof

Abstract

The application provides a multi-effect synergistic bone density enhancing composite composition rich in Antarctic krill oil and a preparation method thereof. The composition takes Antarctic krill oil as a main active ingredient, is matched with important extracts such as psoralea, radix lepidii, rhizoma drynariae and pueraria, and is compounded with calcium compounds, nutrients and suspending agents, so that a multi-effect synergistic bone health support system is formed through scientific proportioning. The phospholipid type Omega-3 fatty acid and astaxanthin in the Antarctic krill oil have antioxidant properties and are helpful to improve the bone metabolism microenvironment; the traditional Chinese medicine extracts can synergistically promote the activity of bone cells and increase bone calcium deposition; the nutrients and calcium sources jointly act, and are beneficial to the stability of the bone structure. The preparation method adopts the ultrafine grinding and colloid mill emulsification process, improves the dispersity, stability and bioavailability of the components, and prepares the finished product soft capsules through pill pressing, shaping and drying. The obtained product has stable formula, high absorption efficiency, and is suitable for the fields of bone health conditioning, functional food and health products.

Description

Technical Field

[0001] This invention relates to the field of functional food and health product technology, and in particular to a multi-effect synergistic bone density-enhancing composite composition rich in Antarctic krill oil and its preparation method. Background Technology

[0002] With the accelerating aging of the global population, the incidence of osteoporosis, osteoarthritis, and other degenerative bone diseases is rising year by year, becoming a major public health issue affecting the quality of life of middle-aged and elderly people. Related studies and clinical statistics show that bone loss, decreased bone density, and degeneration of articular cartilage not only significantly increase the risk of fractures but also cause a series of complications such as long-term pain and limited mobility, placing a heavy burden on individuals, families, and the healthcare system. Against this backdrop, the demand for nutritional supplements to maintain and improve bone health continues to grow, and the scientific nature of product formulations and their comprehensive intervention capabilities are gradually becoming a focus of industry attention.

[0003] Currently, products on the market for bone health maintenance mainly fall into the following categories: one category consists of mineral supplements primarily composed of calcium preparations, supplemented with vitamin D; another category comprises joint care products with glucosamine and chondroitin sulfate as the main ingredients; and a third category consists of functional extracts derived from plants or animals. While these products can improve bone or joint-related indicators to some extent, they generally have several shortcomings. For example, most products have relatively simple ingredients or are simply compounded, failing to systematically design for multiple aspects such as the bidirectional balance between bone formation and bone resorption in bone metabolism, and cartilage repair and increased bone density, resulting in limited synergistic effects. Furthermore, the bioavailability of some key ingredients is limited, especially traditional calcium preparations, which have low absorption efficiency in the absence of effective cofactors. In addition, existing products do not adequately address the chronic inflammatory microenvironment of bone tissue, while chronic low-grade inflammation is one of the important contributing factors to accelerated bone loss and cartilage degeneration.

[0004] In recent years, Antarctic krill oil has gained attention for its unique nutritional value and physiological functions due to its rich content of phospholipid-type Omega-3 polyunsaturated fatty acids (EPA and DHA), astaxanthin, vitamins A and E, animal flavonoids, chitin, and other essential trace elements. Studies have shown that Omega-3 fatty acids can stimulate the proliferation and differentiation of osteoblasts (responsible for bone formation), increase bone matrix synthesis, and reduce the activity of osteoclasts (responsible for bone resorption) by regulating the release of inflammatory factors (such as prostaglandin E2 and interleukin-6), thereby slowing bone loss and improving the bone microenvironment. The phospholipid-type Omega-3 fatty acids in krill oil are more easily absorbed and utilized by the human body. Chronic inflammation is one of the causes of osteoporosis, and the natural astaxanthin in krill oil is a powerful antioxidant that can scavenge free radicals and reduce the damage of inflammation to bone tissue. Some clinical trials have found that krill oil shows a trend of improvement in bone health indicators (such as osteocalcin and urinary deoxypyridinium) in patients with osteoporosis or those with low bone density.

[0005] On the other hand, traditional Chinese medicine has a long history and extensive experience in the field of bone health. Traditional Chinese medicines such as Psoralea corylifolia, Morinda officinalis, Drynaria fortunei, Pueraria lobata, and Epimedium have the effects of tonifying the kidneys and strengthening bones, as well as promoting blood circulation and unblocking meridians. Modern pharmacological studies have confirmed that these Chinese medicines contain a variety of active ingredients that promote bone formation and inhibit bone resorption.

[0006] Furthermore, modern nutritional and molecular pharmacological studies have shown that modern nutrients such as glucosamine, chondroitin sulfate, and vitamin D3 are not only components of articular cartilage, but also key factors that directly participate in and profoundly regulate bone metabolism.

[0007] Although the individual functions of the aforementioned components are clearly defined, the application of krill oil in bone health products is mostly limited to single-component addition, lacking systematic research on its compatibility with other active bone health ingredients. Furthermore, research on the application of traditional Chinese medicine extracts combined with modern marine bioactive components in bone health products is also insufficient. Combining the core anti-inflammatory and antioxidant advantages of Antarctic krill oil with traditional Chinese medicine extracts supported by the TCM theory of "tonifying the kidneys and strengthening bones" (such as Psoralea corylifolia and Morinda officinalis), and integrating them with the cartilage repair-oriented effects of glucosamine and chondroitin sulfate, as well as the calcium metabolism regulation led by vitamin D3, to construct a multi-dimensional, multi-target synergistic compound formula with significant innovative significance and application potential.

[0008] In terms of manufacturing processes, existing bone health products are mostly produced using simple mixing and traditional pulverization methods. This results in insufficient dispersibility and stability between components with different physicochemical properties, which can easily affect product consistency and bioavailability. Although technologies such as ultrafine pulverization and emulsification have been applied in other fields, their systematic application in composite bone health products remains relatively limited.

[0009] Therefore, how to develop a bone health product that can comprehensively leverage the synergistic effects of marine bioactive components, traditional Chinese medicine extracts, and modern nutrients based on existing technologies, and improve stability and bioavailability through reasonable preparation processes, has become an urgent problem to be solved. Summary of the Invention

[0010] To address the aforementioned technical problems of insufficient synergy among existing bone health products, low bioavailability, and poor stability of the preparation system, this invention provides a multi-effect synergistic bone density-enhancing composite composition rich in Antarctic krill oil and its preparation method. This invention primarily utilizes the fusion of Antarctic krill oil with traditional Chinese medicine extracts and modern nutrients. Through the scientific formulation and synergistic effects of multiple components, it achieves comprehensive and multi-target bone health support. Simultaneously, it provides a preparation process combining ultrafine grinding and colloid mill emulsification to improve the dispersion uniformity, stability, and bioavailability of each active ingredient in the system. This results in a multi-effect synergistic Antarctic krill oil composite composition product with high absorption efficiency, comprehensive effects, stable dosage form, and convenient use, meeting the bone health conditioning needs of different population groups.

[0011] The technical means employed in this invention are as follows: A multi-effect synergistic bone density-enhancing composite composition rich in Antarctic krill oil, wherein the components of the composition are, by weight, as follows: 50-90 parts Antarctic krill oil, 10-50 parts Chinese herbal extracts, 10-50 parts nutrients, 5-30 parts calcium compounds, and 0.5-3 parts suspending agent.

[0012] Furthermore, the herbal extract is one or more of the following: Psoralea corylifolia extract, Drynaria fortunei extract, Morinda officinalis extract, Pueraria lobata extract, and Epimedium extract.

[0013] Specifically, phospholipid-type Omega-3 fatty acids in krill oil can regulate the levels of hormones related to bone metabolism, promote osteoblast proliferation and differentiation, and inhibit osteoclast activity. Astaxanthin can reduce oxidative stress damage to osteoblasts and provide a favorable cellular environment for bone formation. Psoralea corylifolia directly increases bone mineralization by regulating calcium and phosphorus metabolism, inhibiting osteoclast activity, and promoting osteoblast proliferation. Its active ingredients (such as psoralen and isopsoralen) can activate vitamin D receptors, enhance intestinal calcium absorption, and regulate the Wnt / β-catenin signaling pathway to accelerate bone formation. Morinda officinalis contains various bone-forming compounds. Promoting factors (such as triterpenoids) can promote the expression of bone morphogenetic proteins, enhance bone density, and reduce bone destruction through anti-inflammatory effects (inhibiting prostaglandin E2 synthesis); Drynaria fortunei can stimulate osteoblast proliferation, accelerate fracture healing, regulate calcium and phosphorus metabolism, inhibit osteoclast activity, and increase bone density. Its polysaccharide components can also enhance immune function, indirectly supporting bone health; Pueraria lobata contains isoflavones (such as puerarin), which have phytoestrogenic effects, can bidirectionally regulate estrogen levels, reduce bone calcium loss, and enhance bone density; Epimedium brevicornu in Epimedium can upregulate osteocalcin expression and delay bone loss.

[0014] Furthermore, the nutrient is one or more of glucosamine hydrochloride, chondroitin sulfate, and vitamin D3.

[0015] Specifically, glucosamine is not only a raw material for cartilage synthesis, but it can also participate in bone metabolism regulation by affecting the activity of osteoblasts and osteoclasts; chondroitin sulfate provides cartilage elasticity and can also combine with the collagen fiber network in the bone matrix to enhance bone toughness; vitamin D3 is a core regulator of calcium metabolism, ensuring the effective absorption and utilization of calcium and phosphorus.

[0016] Furthermore, the calcium compound is one of calcium carbonate, calcium citrate, calcium lactate, calcium acetate, or amino acid chelated calcium.

[0017] Furthermore, the suspending agent is one of beeswax, gum arabic, sodium alginate, or sodium carboxymethyl cellulose.

[0018] Further, the components of the composition, by weight, are as follows: 60-90 parts Antarctic krill oil; within the total weight range of 10-50 parts of the herbal extracts, 10-30 parts of Psoralea corylifolia extract, 10-30 parts of Drynaria fortunei extract, 5-20 parts of Morinda officinalis extract, 3-15 parts of Pueraria lobata extract, and 3-15 parts of Epimedium brevicornu extract; within the total weight range of 10-50 parts of the nutrients, 10-30 parts of glucosamine hydrochloride, 10-30 parts of chondroitin sulfate, and 0.0005-0.0015 parts of vitamin D3; 5-20 parts of calcium carbonate; and 1-2 parts of beeswax as the suspending agent.

[0019] This invention also provides a method for preparing the above-mentioned multi-effect synergistic bone density-enhancing composite composition rich in Antarctic krill oil, comprising the following steps: (1) The solid components in the Chinese herbal extract, nutrients and calcium compounds are subjected to ultra-fine pulverization treatment, wherein the ultra-fine pulverization treatment is performed by an air jet mill or a vibratory mill, and the pulverization fineness is 100-150 mesh; (2) Dissolve vitamin D3 in a portion of Antarctic krill oil, mix well, and obtain a fortified krill oil solution; (3) Add the suspending agent to the remaining Antarctic krill oil, heat and stir to mix evenly to obtain an oil suspension; (4) The ultrafine pulverized solid powder, enhanced krill oil solution and oil suspension are stirred and mixed evenly, emulsified by colloid mill, defoamed and filtered to obtain the liquid; (5) Mix gelatin, glycerin and water in a preset ratio, heat to dissolve, degas and filter to obtain a gel solution; (6) The liquid obtained in step (4) and the adhesive obtained in step (5) are pressed into shape using a soft capsule pressing device; (7) After shaping, drying and pellet selection, the finished soft capsules are obtained.

[0020] The composite composition is preferably formulated into a soft capsule dosage form to improve the stability of the active ingredients and the convenience of administration. The composite composition can also be formulated into other formulation types according to the application scenario without affecting its multi-component synergistic effect.

[0021] Furthermore, the oil suspension is obtained by heating a mixture of suspending agent and Antarctic krill oil to 65-75°C, stirring for 20-40 minutes, and then cooling to room temperature; the colloid mill emulsification conditions are: rotation speed 2000-4000 rpm, processing time 10-20 minutes, and temperature controlled at 40-50°C; the liquid is obtained after vacuum degassing (-0.06~-0.08 MPa) and filtration through an 80-100 mesh sieve.

[0022] Further, the adhesive solution is prepared by adding gelatin, glycerin and water in a weight ratio of 2.5:1:2.5 into a dissolving tank, heating to about 70°C, stirring for 30 to 60 minutes, degassing under vacuum (-0.06 to -0.08 MPa) and filtering through an 80 to 100 mesh sieve, and then keeping warm at 50 to 60°C.

[0023] Furthermore, in the pelleting process, the thickness of the rubber sheet is controlled to be 0.6~0.9 mm, and the content of a single soft capsule is 400~800 mg; the shaping conditions are: temperature 18~25℃, relative humidity 30~45%, time 4~6 hours; the drying conditions are: temperature 24~32℃, relative humidity 25~35%, time 20~28 hours.

[0024] Compared with the prior art, the present invention has the following advantages: 1. This invention combines Antarctic krill oil with extracts of traditional Chinese medicines such as psoralea, morinda officinalis, drynaria fortunei, and kudzu root, as well as modern nutrients such as calcium compounds, glucosamine, chondroitin sulfate, and vitamin D3. Through a scientific formulation of multiple components, it achieves comprehensive, multi-target bone health support by working synergistically to regulate bone metabolism hormone levels, promote osteoblast activity, inhibit osteoclast function, and provide raw materials for bone building. This results in "cartilage protection, inflammation regulation, and bone mineral enhancement."

[0025] 2. The Omega-3 fatty acids in Antarctic krill oil used in this invention exist in the form of phospholipids, which can promote the transmembrane transport of glucosamine, psoralen, and other components, and improve their utilization in the body. The potent anti-inflammatory properties of Omega-3 fatty acids can also inhibit joint inflammation, relieve pain and stiffness, and complement the cartilage repair and osteocyte proliferation and differentiation effects of glucosamine, psoralen, etc., to jointly improve joint function and further reduce the risk of osteoarthritis. Astaxanthin can protect other active ingredients from oxidative degradation and prolong their half-life in the body, thereby indirectly improving bioavailability. At the same time, krill oil can also improve intestinal barrier function by reducing intestinal inflammation (such as inhibiting the NF-κB pathway), creating a favorable environment for the absorption of glucosamine, psoralen, and other components.

[0026] 3. The extracts of traditional Chinese medicines such as Psoralea corylifolia, Morinda officinalis, Drynaria fortunei, and Pueraria lobata selected in this invention have good synergistic effects in promoting osteoblast activity and supporting bone calcium deposition. Combined with the effects of glucosamine and chondroitin sulfate in supporting cartilage structure, and with the assistance of vitamin D3, they synergistically promote calcium absorption and utilization, so that the formula has a complementary and synergistic effect in supporting bone structure and regulating bone metabolism.

[0027] 4. This invention uses ultrafine grinding technology to refine solid components to a finer mesh size of 100 mesh or more, increasing their specific surface area and dispersibility and dissolution rate in the system; and uses colloid mill emulsification technology to achieve uniform mixing of oil phase, water phase and solid phase, forming a stable system, effectively avoiding component stratification and sedimentation, and significantly improving bioavailability.

[0028] 5. The soft capsules used in this invention are a preferred dosage form. Compared with commercially available tablets, granules or liquid dosage forms, they can effectively isolate oxygen and light, preventing the active ingredients from oxidizing and deteriorating. At the same time, they can mask the special odor of some ingredients and improve the palatability of the product. In addition, soft capsules disintegrate rapidly in the gastrointestinal tract, which is conducive to the release and absorption of active ingredients.

[0029] In summary, this invention scientifically combines Antarctic krill oil, a highly bioavailable marine active ingredient, with various traditional Chinese medicine extracts and modern bone health nutrients. By employing advanced preparation processes such as ultrafine grinding and colloid mill emulsification, it significantly improves the dispersion uniformity, bioavailability, and system stability of key active ingredients. The resulting composite composition possesses technical advantages such as synergistic effects, high absorption efficiency, and broad coverage, making it suitable for developing functional foods or health products for bone health conditioning, enhancing bone density, and preventing osteoporosis, with promising application prospects. Detailed Implementation

[0030] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0031] Example 1 This embodiment provides a multi-effect synergistic bone density enhancement composite composition rich in Antarctic krill oil, the specific formula of which is as follows: 60 parts Antarctic krill oil, 20 parts Psoralea corylifolia extract, 17.5 parts glucosamine hydrochloride, 5 parts calcium carbonate, 0.0005 parts vitamin D3, and 2 parts beeswax.

[0032] The preparation method of the above-mentioned multi-effect synergistic bone density-enhancing composite composition rich in Antarctic krill oil is described with soft capsule formulation as the preferred embodiment, as follows: (1) Psoralea extract, glucosamine hydrochloride and calcium carbonate were ultra-finely pulverized using an air jet mill and filtered through a 100-mesh sieve to obtain solid powder; (2) Dissolve vitamin D3 in 1 part Antarctic krill oil, mix well to form a fortified krill oil solution; (3) Add beeswax to the remaining Antarctic krill oil, heat to 70°C to melt, stir for 30 minutes, cool to room temperature, and obtain an oil suspension for later use; (4) The ultra-fine pulverized solid powder is mixed with the enhanced krill oil solution and oil suspension for 30 min, emulsified for 20 min by colloid mill at 3000 rpm and 40℃, then degassed under vacuum (-0.06~-0.08MPa) to remove foam, and filtered through an 80 mesh sieve to obtain the liquid for later use. (5) Add gelatin, glycerin and water in a ratio of 2.5:1:2.5 to the gelling tank, heat to 70°C, stir for 1 hour, degas under vacuum (-0.06~-0.08MPa), pass through an 80-mesh sieve, and keep the gelatin solution at 50°C for later use. (6) After the soft capsule pelleting machine is successfully debugged, the liquid material and the gel are pressed into soft capsules through the soft capsule pelleting machine. The thickness of the gel is 0.6~0.7mm and the contents are 500mg / capsule. (7) The soft capsules pressed by the pellet press are transported to the rotating drum, and the temperature is 20±2℃ and the humidity is 40-45% for 4 hours to set. The set soft capsules are placed in the drying tray, and the temperature is 26±2℃ and the humidity is 30-40% for 24 hours to dry. The dried soft capsules are placed on the light inspection machine, and unqualified capsules such as irregularly shaped capsules, bubble capsules, and sticky capsules are manually selected to obtain the finished composite soft capsules.

[0033] Example 2 This embodiment provides a multi-effect synergistic bone density enhancement composite composition rich in Antarctic krill oil, the specific formula of which is as follows: 90 parts Antarctic krill oil, 10 parts Epimedium extract, 20 parts glucosamine hydrochloride, 15 parts calcium carbonate, 0.001 parts vitamin D3, and 1 part beeswax.

[0034] Unlike Example 1, in the preparation method of the above-mentioned multi-effect synergistic bone density-enhancing composite composition rich in Antarctic krill oil, Step (1) involves pulverizing Epimedium extract, glucosamine hydrochloride, and calcium carbonate using an air jet mill and filtering them through a 100-mesh sieve to obtain solid powder. Step (4) involves mixing the ultra-fine pulverized solid powder with the enhanced krill oil solution and oil suspension for 30 minutes, emulsifying it for 10 minutes at 2000 rpm and 40°C using a colloid mill, then degassing it under vacuum (-0.06~-0.08 MPa) to remove foam, and filtering it through a 100-mesh sieve to obtain the liquid for later use. In step (6), after the soft capsule pelleting machine is successfully debugged, the liquid material and the gel are pressed into soft capsules by the soft capsule pelleting machine. The thickness of the gel shell is 0.7~0.85mm and the content is 800mg / capsule.

[0035] Example 3 This embodiment provides a multi-effect synergistic bone density enhancement composite composition rich in Antarctic krill oil, the specific formula of which is as follows: 70 parts Antarctic krill oil, 10 parts Drynaria fortunei extract, 10 parts Morinda officinalis extract, 10 parts chondroitin sulfate, 15 parts calcium citrate, 0.0015 parts vitamin D3, and 2 parts beeswax.

[0036] Unlike Example 1, in the preparation method of the above-mentioned multi-effect synergistic bone density enhancement composite composition rich in Antarctic krill oil, step (1) involves ultra-finely pulverizing the extracts of Drynaria fortunei, Morinda officinalis, chondroitin sulfate, and calcium citrate using an air jet mill and filtering them through a 120-mesh sieve to obtain solid powder. In step (5), gelatin, glycerin and water are added to the gelling tank in a ratio of 2.5:1:2.5, heated to 70°C, stirred for 1 hour, degassed under vacuum (-0.06~-0.08MPa), passed through a 100-mesh sieve, and the resulting gel solution is kept at 50°C for later use.

[0037] Example 4 This embodiment provides a multi-effect synergistic bone density enhancement composite composition rich in Antarctic krill oil, the specific formula of which is as follows: 80 parts Antarctic krill oil, 20 parts Drynaria fortunei extract, 10 parts glucosamine hydrochloride, 10 parts chondroitin sulfate, 10 parts amino acid chelated calcium, 0.001 parts vitamin D3, and 2 parts beeswax.

[0038] Unlike Example 1, in the preparation method of the above-mentioned multi-effect synergistic bone density enhancement composite composition rich in Antarctic krill oil, step (1) involves ultra-finely pulverizing the extract of Drynaria fortunei, glucosamine hydrochloride, chondroitin sulfate, and amino acid chelated calcium using an air jet mill and filtering them through a 100-mesh sieve to obtain solid powder. In step (6), after the soft capsule pelleting machine is successfully debugged, the liquid material and the gel are pressed into soft capsules by the soft capsule pelleting machine. The thickness of the gel is 0.65~0.75mm and the content is 500mg / capsule.

[0039] Example 5 This embodiment provides a multi-effect synergistic bone density enhancement composite composition rich in Antarctic krill oil, the specific formula of which is as follows: 80 parts Antarctic krill oil, 15 parts Psoralea corylifolia extract, 10 parts Drynaria fortunei extract, 7 parts Morinda officinalis extract, 5 parts Pueraria lobata extract, 5 parts Epimedium extract, 5 parts calcium carbonate, 0.0005 parts vitamin D3, and 2 parts beeswax.

[0040] Unlike Example 1, in the preparation method of the above-mentioned multi-effect synergistic bone density enhancement composite composition rich in Antarctic krill oil, step (1) involves ultra-finely pulverizing Psoralea corylifolia extract, Drynaria fortunei extract, Morinda officinalis extract, Pueraria lobata extract, Epimedium extract, and calcium carbonate using an air jet mill and filtering them through a 100-mesh sieve to obtain solid powder. In step (4), the ultra-fine pulverized solid powder is mixed with the enhanced krill oil solution and oil suspension for 30 min, emulsified for 20 min by a colloid mill at 4000 rpm and 50°C, then degassed under vacuum (-0.06~-0.08 MPa) to remove foam, and filtered through an 80-mesh sieve to obtain the liquid for later use. In step (6), after the soft capsule pelleting machine is successfully debugged, the liquid material and the gel are pressed into soft capsules by the soft capsule pelleting machine. The thickness of the gel is 0.65~0.75mm and the content is 500mg / capsule.

[0041] Example 6 This embodiment provides a multi-effect synergistic bone density enhancement composite composition rich in Antarctic krill oil, the specific formulation of which is as follows: 90 parts Antarctic krill oil, 10 parts Psoralea corylifolia extract, 10 parts Drynaria fortunei extract, 6 parts Morinda officinalis extract, 5 parts Pueraria lobata extract, 3 parts Epimedium extract, 7.5 parts glucosamine hydrochloride, 7.5 parts chondroitin sulfate, 5 parts calcium carbonate, 0.0005 parts vitamin D3, and 2 parts beeswax.

[0042] Unlike Example 1, in the preparation method of the above-mentioned multi-effect synergistic bone density enhancement composite composition rich in Antarctic krill oil, step (1) involves ultra-finely pulverizing the extracts of Psoralea corylifolia, Drynaria fortunei, Morinda officinalis, Pueraria lobata, Epimedium brevicornu, glucosamine hydrochloride, chondroitin sulfate, and calcium carbonate using an air jet mill and filtering them through a 100-mesh sieve to obtain solid powder. In step (3), beeswax is added to the remaining Antarctic krill oil, heated to 68°C to melt, stirred for 30 minutes, and cooled to room temperature to obtain an oil suspension for later use; Step (4): Mix the ultra-fine pulverized solid powder with the enhanced krill oil solution and oil suspension for 30 min, emulsify for 20 min by colloid milling at 4000 rpm and 50℃, then degas under vacuum (-0.06~-0.08MPa) to remove foam, and filter through an 80-mesh sieve to obtain the liquid for later use. Step (6): After the soft capsule pelleting machine is successfully debugged, the liquid material and the gel are pressed into soft capsules by the soft capsule pelleting machine. The thickness of the gel is 0.65~0.75mm and the content is 500mg / capsule.

[0043] Comparative Example 1 This comparative example provides an Antarctic krill oil soft capsule, specifically a 100% Antarctic krill oil single-ingredient soft capsule.

[0044] The method for preparing the above-mentioned Antarctic krill oil soft capsules is as follows: (1) Add gelatin, glycerin and water in a ratio of 2.5:1:2.5 to a gelling tank, heat to 70°C, stir for 1 hour, degas under vacuum (-0.06~-0.08MPa), pass through an 80-mesh sieve, and keep the gel at 50°C for later use. (2) After the soft capsule press is successfully adjusted, Antarctic krill oil and gelatin liquid are pressed into soft capsules by the soft capsule press. The thickness of the gelatin shell is 0.65~0.75mm and the contents are 500mg / capsule. (3) The soft capsules pressed by the pellet press are transported to the rotating drum, and the temperature is 20±2℃ and the humidity is 40-45% for 4 hours to set. The set soft capsules are placed in the drying tray, and the temperature is 26±2℃ and the humidity is 30-40% for 24 hours to dry. The dried soft capsules are placed on the light inspection machine, and unqualified capsules such as irregularly shaped capsules, bubble capsules, and sticky capsules are manually selected to obtain the finished Antarctic krill oil soft capsules.

[0045] Comparative Example 2 This comparative example provides a bone density-enhancing composite soft capsule, which differs from Example 5 only in its formulation. In this comparative example, Antarctic krill oil is replaced with soybean oil, and the preparation process is the same as in Example 5.

[0046] Comparative Example 3 This comparative example provides a bone density-enhancing powder with the following specific formula: 40 parts glucosamine hydrochloride, 40 parts chondroitin sulfate, 15 parts calcium carbonate, and 0.0015 parts vitamin D3.

[0047] The method for preparing the above-mentioned powder is as follows: (1) Glucosamine hydrochloride, chondroitin sulfate and calcium carbonate were ultra-finely pulverized using an air jet mill and filtered through a 100-mesh sieve to obtain solid powder; (2) Mix the solid powder with vitamin D3 microcapsule powder evenly to obtain the finished product of bone density enhancement powder.

[0048] Efficacy evaluation test case To verify the multi-effect synergistic advantages of the compound soft capsule composition of the present invention in promoting bone formation, this experimental example used mouse bone marrow mesenchymal stem cells (BMSCs) as an in vitro model and systematically evaluated the efficacy of each embodiment and comparative product in promoting BMSC proliferation and its differentiation into osteoblasts.

[0049] 1. Experimental Materials and Methods 1.1 Preparation of experimental samples The contents or powders of the finished soft capsules from Examples 1-6 and Comparative Examples 1-3 were subjected to simulated digestion: artificial gastric juice (containing pepsin, pH 1.2) was added and digested at 37°C with shaking for 2 hours. After adjusting the pH to neutral, artificial intestinal juice (containing pancreatic enzymes and bile salts) was added and digestion continued for 4 hours. The digestive solution was centrifuged, filtered, and freeze-dried to obtain the extract powder of each sample. In the experiment, the sample powder was dissolved in α-MEM basal culture medium containing 1% fetal bovine serum, sterilized through a 0.22 μm filter membrane, and prepared into a drug-containing culture medium with a final concentration of 200 μg / mL for later use.

[0050] 1.2 Isolation, culture and identification of mouse BMSCs Four-week-old C57BL / 6 mice were euthanized by cervical dislocation and disinfected by immersion in 75% ethanol for 5 minutes. The femur and tibia were aseptically separated in a laminar flow hood, and attached muscle was removed. The bones were rinsed with PBS containing antibiotics. Both ends of the bones were cut off, and the bone marrow cavity was repeatedly flushed with 1 mL of α-MEM complete culture medium (containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin) until the bone hairs turned white. The collected bone marrow cell suspension was filtered through a 70 μm cell sieve to remove tissue fragments, and then... 6 Cells were seeded at a density of cells / cm² in culture flasks. They were incubated in an incubator at 37°C, 5% CO2, and saturated humidity. After 48 hours, the medium was completely replaced for the first time, discarding any non-adherent cells. Subsequent medium replacements were performed every 3 days. When cells reached 80%-90% confluence, they were digested with 0.25% trypsin-EDTA and passaged at a 1:2 ratio. Flow cytometry was used to detect surface markers in the 5th generation cells (CD44 and CD90 positivity >95%, CD34 and CD45 positivity <2%). Multi-lineage differentiation capacity was assessed using osteogenic and adipogenic induction media to confirm the obtained cells as BMSCs.

[0051] 1.3 Experimental Grouping The cells were randomly divided into the following 11 groups: Blank control group (Control): Only α-MEM basal culture medium containing 1% fetal bovine serum was used.

[0052] Positive control group (Dex): Add 10⁻ to the basal culture medium 8 mol / L dexamethasone.

[0053] Comparative Example 1 (C1): Basal culture medium + Comparative Example 1 (pure krill oil) extract.

[0054] Comparative Example 2 (C2): Basal culture medium + Comparative Example 2 (soybean oil formulation) extract.

[0055] Comparative Example 3 (C3): basal culture medium + Comparative Example 3 (conventional powder) extract.

[0056] Example 1 group (S1): basal culture medium + extract from Example 1.

[0057] Example 2 group (S2): basal culture medium + extract from Example 2.

[0058] Example 3 group (S3): basal culture medium + extract from Example 3.

[0059] Example 4 group (S4): basal culture medium + extract from Example 4.

[0060] Example 5 group (S5): basal culture medium + extract from Example 5.

[0061] Example 6 group (S6): basal culture medium + extract from Example 6.

[0062] 1.4 Detection Indicators and Methods 1.4.1 Cell proliferation assay (MTT method) BMSCs were seeded at a density of 5 × 10³ cells / well in 96-well plates, with 100 μL of complete culture medium per well. After 24 hours of culture, the cells adhered, and the original culture medium was discarded. 100 μL of drug-containing culture medium was added to each experimental group and control group. Each group had 6 replicates. After 24, 48, and 72 hours of culture, 20 μL of MTT solution (5 mg / mL) was added to each well, and the cells were cultured for another 4 hours. The liquid in the wells was carefully aspirated, and 150 μL of DMSO was added to each well. The plates were then shaken slowly for 10 minutes to completely dissolve the formazan crystals. The absorbance (OD value) of each well was measured at 490 nm using a microplate reader. The relative proliferation rate of each group at each time point was calculated using the mean OD value of the blank control group at 72 hours as a baseline (set as 100%).

[0063] 1.4.2 Osteogenic Differentiation Capacity Detection (1) Alkaline phosphatase activity detection (early differentiation marker) BMSC at 2×10 4Cells were seeded at a density of [number] cells / well in 24-well plates. After adhesion, the medium was replaced with osteogenic induction medium (i.e., 50 μg / mL ascorbic acid and 10 mM β-glycerophosphate sodium were added to the drug-containing basal medium). The medium was completely changed every 3 days. After 7 days of culture, the culture medium was discarded, and the cells were gently washed twice with pre-chilled PBS. 200 μL of 0.1% Triton X-100 cell lysis buffer was added to each well, and the cells were lysed on ice for 30 minutes. The lysis buffer was collected, centrifuged at 12,000 rpm for 10 minutes at 4°C, and the supernatant was collected. The absorbance of the enzymatic reaction products was measured at 405 nm using the p-nitrophenyl phosphate (pNPP) method, strictly following the instructions of the kit (Nanjing Jiancheng, A059-2). Simultaneously, the total protein concentration of each sample was determined using a BCA protein concentration assay kit. ALP activity is defined as the amount of 1 nmol of p-nitrophenol produced per minute per milligram of protein at 37°C, and the final result is expressed as U / mg protein.

[0064] (2) Alizarin red staining and quantification of mineralized nodules (markers of late differentiation) Cell seeding and induction methods were the same as for ALP detection. After 21 days of culture, the culture medium was discarded, and the cells were washed twice with PBS. Each well was then fixed with 4% paraformaldehyde for 30 minutes. After fixation, the cells were washed three times with deionized water. 2% Alizarin Red S staining solution (pH 4.2) was added to cover the bottom of the wells, and staining was performed at room temperature for 15 minutes. The staining solution was discarded, and the cells were gently washed repeatedly with deionized water until the background was colorless, then air-dried at room temperature. Typical mineralized nodules (orange-red to red) formation were observed and photographed under an inverted microscope. For quantitative analysis, 500 μL of 10% cetylpyridine chloride (CPC) solution was added to each well, and the wells were shaken on a horizontal shaker at room temperature for 30 minutes to ensure complete dissolution of the bound dye. 200 μL of the supernatant was transferred to a 96-well plate, and the absorbance (OD value) was measured at 562 nm using a microplate reader to relatively quantify the amount of calcium salt deposition.

[0065] 1.5 Statistical Analysis All experiments were independently repeated three times. Data are expressed as mean ± standard deviation (Mean ± SD). Statistical analysis was performed using GraphPad Prism 9.0 software. One-way ANOVA was used for comparisons among multiple groups, and Tukey's post-hoc test was used for pairwise comparisons between groups. A p-value < 0.05 was considered statistically significant.

[0066] 2. Experimental Results 2.1 Effect on BMSC proliferation (MTT experiment) As shown in Table 1, after 72 hours of culture, all sample groups showed a promoting effect on BMSC proliferation. Among them, the proliferation-promoting effect of Example 6 (S6) was the most significant, with a relative proliferation rate of 165.3%, significantly higher than all other groups (P<0.01). The effect of Example 3 (S3) was the second most significant, at 158.7%. The proliferation rate of Example 2 (S2) was 140.2%, lower than that of Example 1 (152.6%), which is related to the fact that its formula only contained a single herb extract of Epimedium, resulting in relatively weak synergistic effects. Although the comparative groups showed some effect, their results were significantly lower than those of all example groups.

[0067] Table 1: Effects of each sample group on BMSC proliferation (relative proliferation rate, %, after 72 hours of culture) Grouping Relative proliferation rate (Mean ± SD) P-value compared to Control P-value compared to C1 (Comparative Example 1) Control 100.0 ± 3.8 - - Dex 130.4 ± 4.9 <0.01 <0.01 C1 142.1 ± 5.1 <0.01 - C2 135.6 ± 4.7 <0.01 >0.05 C3 128.9 ± 4.3 <0.01 <0.05 S1 152.6 ± 5.5 <0.01 <0.01 S2 140.2 ± 5.0 <0.01 >0.05 S3 158.7 ± 5.8 <0.01 <0.01 S4 155.2 ± 5.4 <0.01 <0.01 S5 155.9 ± 5.6 <0.01 <0.01 S6 165.3 ± 6.0 <0.01 <0.01 2.2 Effects on osteogenic differentiation of BMSCs 2.2.1 Alkaline phosphatase activity As shown in Table 2, 7 days after osteogenic induction, the ALP activity in all experimental groups was higher than that in the blank control group. The highest ALP activity was observed in Example 6 (S6), reaching 4.32 U / mg prot, which was 1.52 times that of Comparative Example 1 (2.85 U / mg prot) (P<0.01). The activity in Example 3 (S3) was 3.98 U / mg prot, second only to S6. The ALP activity in Example 2 (S2) was 2.95 U / mg prot, higher than Comparative Example 1, but significantly lower than the other example groups, consistent with its proliferative trend. The ALP activities in Examples 1, 4, and 5 were 3.45, 3.78, and 3.82 U / mg prot, respectively, all significantly higher than all comparative example groups (P<0.01).

[0068] Table 2: Effects of each sample group on BMSC alkaline phosphatase (ALP) activity Grouping ALP activity (U / mg prot, Mean ± SD) Compared with group C1, the p-value Control 0.85 ± 0.08 <0.01 Dex 2.10 ± 0.20 <0.05 C1 2.85 ± 0.27 - C2 2.30 ± 0.22 <0.05 C3 1.80 ± 0.17 <0.01 S1 3.45 ± 0.33 <0.01 S2 2.95 ± 0.28 >0.05 S3 3.98 ± 0.38 <0.01 S4 3.78 ± 0.36 <0.01 S5 3.82 ± 0.36 <0.01 S6 4.32 ± 0.41 <0.01 2.2.2 Formation of mineralized nodules The results of alizarin red staining quantification 21 days after osteogenic induction showed (Table 3) that the group in Example 6 (S6) induced the formation of the most mineralized nodules, with the highest OD. 562 The values ​​were significantly higher than all other groups. Group 3 (S3) ranked second in mineralization capacity. Group 2 (S2) had relatively weak mineralization capacity, with an OD value of 0.335, which was not statistically different from Comparative Example 1. Groups 1, 4, and 5 also formed obvious mineralized nodules, with OD values ​​significantly higher than the Comparative Example group.

[0069] Table 3: Quantitative analysis of mineralized nodules induced by BMSCs in each sample group (OD562) Grouping <![CDATA[OD 562 Value (Mean ± SD) Compared with group C1, the p-value Control 0.215 ± 0.022 <0.01 Dex 0.310 ± 0.030 <0.05 C1 0.348 ± 0.033 - C2 0.315 ± 0.030 <0.05 C3 0.285 ± 0.027 <0.05 S1 0.405 ± 0.039 <0.01 S2 0.335 ± 0.032 >0.05 S3 0.465 ± 0.044 <0.01 S4 0.442 ± 0.042 <0.01 S5 0.450 ± 0.043 <0.01 S6 0.510 ± 0.048 <0.01 3. Experimental Conclusions This efficacy evaluation study, through rigorous and detailed cellular experimental data, comprehensively demonstrates at the cellular level that the product of this invention can efficiently promote the proliferation and differentiation of osteoblast precursor cells through multi-target and multi-pathway synergistic effects. Simultaneously, it also confirms that Antarctic krill oil is an important active base and carrier, and that the formulation with Antarctic krill oil as the core, in conjunction with specific extracts of various traditional Chinese medicines and modern nutrients, possesses clear scientific validity and synergistic effects. These data collectively provide direct, powerful, and complete scientific evidence for the product's core efficacy of "enhancing bone density."

[0070] The above embodiments are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A multi-effect synergistic bone density-enhancing composite composition rich in Antarctic krill oil, characterized in that, The components of the composition are, by weight, as follows: 50-90 parts Antarctic krill oil, 10-50 parts Chinese herbal extracts, 10-50 parts nutrients, 5-30 parts calcium compounds, and 0.5-3 parts suspending agent.

2. The multi-effect synergistic bone density-enhancing composite composition rich in Antarctic krill oil according to claim 1, characterized in that, The herbal extracts are one or more of the following: Psoralea corylifolia extract, Drynaria fortunei extract, Morinda officinalis extract, Pueraria lobata extract, and Epimedium extract.

3. The multi-effect synergistic bone density-enhancing composite composition rich in Antarctic krill oil according to claim 2, characterized in that, The nutrients are one or more of glucosamine hydrochloride, chondroitin sulfate, and vitamin D3.

4. The multi-effect synergistic bone density-enhancing composite composition rich in Antarctic krill oil according to claim 3, characterized in that, The calcium compound is one of calcium carbonate, calcium citrate, calcium lactate, calcium acetate, or amino acid chelated calcium.

5. The multi-effect synergistic bone density-enhancing composite composition rich in Antarctic krill oil according to claim 4, characterized in that, The suspending agent is one of beeswax, gum arabic, sodium alginate, or sodium carboxymethyl cellulose.

6. The multi-effect synergistic composite composition rich in Antarctic krill oil according to claim 5, characterized in that, The components of the composition, by weight, are as follows: 60-90 parts Antarctic krill oil; within the total weight range of 10-50 parts of the herbal extracts, 10-30 parts of Psoralea corylifolia extract, 10-30 parts of Drynaria fortunei extract, 5-20 parts of Morinda officinalis extract, 3-15 parts of Pueraria lobata extract, and 3-15 parts of Epimedium brevicornu extract; within the total weight range of 10-50 parts of the nutrients, 10-30 parts of glucosamine hydrochloride, 10-30 parts of chondroitin sulfate, and 0.0005-0.0015 parts of vitamin D3; 5-20 parts of calcium carbonate; and 1-2 parts of beeswax as the suspending agent.

7. A method for preparing a multi-effect synergistic bone density-enhancing composite composition rich in Antarctic krill oil as described in any one of claims 1 to 6, characterized in that, Includes the following steps: (1) The solid components in the Chinese herbal extract, nutrients and calcium compounds are subjected to ultra-fine grinding treatment, and the grinding fineness is 100-150 mesh; (2) Dissolve vitamin D3 in a portion of Antarctic krill oil, mix well, and obtain a fortified krill oil solution; (3) Add the suspending agent to the remaining Antarctic krill oil, heat and stir to mix evenly to obtain an oil suspension; (4) The ultrafine pulverized solid powder, enhanced krill oil solution and oil suspension are stirred and mixed evenly, emulsified by colloid mill, defoamed and filtered to obtain the liquid; (5) Mix gelatin, glycerin and water in a preset ratio, heat to dissolve, degas and filter to obtain a gel solution; (6) The liquid obtained in step (4) and the adhesive obtained in step (5) are pressed into shape using a soft capsule pressing device; (7) After shaping, drying and pellet selection, the finished soft capsules are obtained.

8. The preparation method according to claim 7, characterized in that, The oil suspension is obtained by heating a mixture of suspending agent and Antarctic krill oil to 65-75°C, stirring for 20-40 minutes, and then cooling to room temperature; the colloid mill emulsification conditions are: rotation speed 2000-4000 rpm, processing time 10-20 minutes, and temperature controlled at 40-50°C; the liquid is obtained by vacuum degassing (-0.06~-0.08 MPa) and filtering through an 80-100 mesh sieve.

9. The preparation method according to claim 7, characterized in that, The adhesive solution is prepared by adding gelatin, glycerin and water in a weight ratio of 2.5:1:2.5 into a dissolving tank, heating to about 70°C, stirring for 30 to 60 minutes, degassing under vacuum (-0.06 to -0.08 MPa) and filtering through an 80 to 100 mesh sieve, and then keeping warm at 50 to 60°C.

10. The preparation method according to claim 7, characterized in that, In the pelleting process, the thickness of the rubber sheet is controlled to be 0.6~0.9 mm, and the content of a single soft capsule is 400~800 mg; the shaping conditions are: temperature 18~25℃, relative humidity 30~45%, time 4~6 hours; the drying conditions are: temperature 24~32℃, relative humidity 25~35%, time 20~28 hours.