A composition with combined adjuvant therapeutic effects against diabetes and arthritis and its preparation method

Abstract

This invention provides a composition with combined adjuvant therapeutic effects against diabetes and arthritis, and its preparation method, belonging to the field of health product development technology. The composition is prepared by mixing fucoidan, green-lipped mussel extract, and prickly pear fermented extract. The green-lipped mussel extract is obtained by extracting green-lipped mussels with different concentrations of ethanol-sodium chloride solution; the prickly pear fermented extract is obtained by fermenting prickly pear with a combination of *Lactobacillus plantarum* and *Bacillus tekirae*. This invention overcomes the shortcomings of existing technologies, effectively enhancing blood sugar regulation through the synergistic effect of multiple components while also exhibiting good anti-inflammatory effects, thus providing a good adjuvant therapeutic effect for diabetes and arthritis.

Description

Technical Field

[0001] This invention relates to the field of health product development technology, specifically to a composition with combined adjuvant therapeutic effects against diabetes and arthritis, and its preparation method. Background Technology

[0002] Osteoarthritis (OA) is a chronic degenerative joint disease characterized by cartilage degeneration, bone remodeling, osteophyte formation, and subchondral sclerosis, affecting approximately 30% of middle-aged people (45-65 years old) and up to 85% of people over 65 years old worldwide. Pathogenesis involves chondrocyte hypertrophy, proteoglycan depletion, cartilage clefts, and structural bone changes, driven by pro-inflammatory cytokines (CK), such as tumor necrosis factor-α (TNF-α), interleukins (IL), matrix metalloproteinases (MMPs), and reactive oxygen species (ROS), which accelerate cartilage degradation and impair matrix synthesis. Chronic OA-related pain, classified as nonprotective pain, is triggered by sensory nerve stimulation and exacerbated by neural and inflammatory pathways, including the COX and LOX-mediated arachidonic acid cascade. Similarly, diabetes mellitus (DM) is a chronic metabolic disorder characterized by impaired glucose metabolism due to insulin deficiency or resistance. Prediabetes, characterized by elevated fasting blood glucose or impaired glucose tolerance, significantly increases the risk of type 2 diabetes and related complications. Insulin resistance (IR) is a hallmark of type 2 diabetes mellitus, caused by defects in insulin signaling, particularly in the PI3K-AKT pathway, impairing glucose uptake and glycogen synthesis. IR is influenced by age, obesity, inflammation, and genetic susceptibility, with chronic low-grade inflammation playing a crucial role in metabolic dysfunction. Current treatments for osteoarthritis (OA), including glucosamine, hyaluronic acid, nonsteroidal anti-inflammatory drugs (NSAIDs), and glucocorticoids, primarily relieve symptoms but do not halt disease progression. Similarly, the management of type 2 diabetes relies on glucose-lowering therapies, including insulin, biguanides (such as metformin), insulin secretagogues, and alpha-glucosidase inhibitors, which can improve insulin sensitivity but may have significant side effects. Given the shared inflammatory mechanisms of OA and T2DM, there is an urgent need for safer and more targeted treatment strategies to address metabolic dysfunction and joint degeneration.

[0003] Green-lipped mussel (GLM) is a marine bivalve mollusc. GLM extract has been shown to be effective in managing osteoarthritis (OA), inhibiting COX and LOX pathways, regulating CK, inhibiting osteoclast activity, and exerting antioxidant effects through furanyl acid extract. However, the overall effects of products obtained by different extraction methods vary. Fucoidan is a sulfated polysaccharide that has potential in the treatment of diabetes and also has anti-inflammatory effects. Compared with traditional drug treatments, GLM and fucoidan, as natural extracts, are relatively safe. However, in existing studies, the therapeutic effects of these substances on diabetes and arthritis are not ideal. Therefore, designing a product with GLM and fucoidan as the main materials to comprehensively improve the adjuvant therapeutic effect has broad application prospects. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a composition and its preparation method that have a combined adjuvant therapeutic effect on diabetes and arthritis. Through the synergistic effect of multiple components, it effectively enhances the blood glucose regulation effect while also exhibiting a good anti-inflammatory effect, thus providing a good adjuvant therapeutic effect on diabetes and arthritis.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] A composition with combined adjuvant therapeutic effects against diabetes and arthritis, the composition comprising the following components by weight: 3-5 parts fucoidan, 1-2 parts green-lipped mussel extract, and 1-3 parts prickly pear fermented extract; wherein the green-lipped mussel extract is obtained by extracting green-lipped mussels with ethanol-sodium chloride solution of different concentrations; and the prickly pear fermented extract is obtained by extracting prickly pears by co-fermentation with Lactobacillus plantarum and Bacillus tekirae.

[0007] Preferably, the preparation method of the green-lipped mussel extract includes the following steps:

[0008] S1-1. After removing the shells from the green-lipped mussels, clean them, freeze them, grind them into powder, and keep them warm in an ice water bath to obtain the ground material for later use.

[0009] S1-2. Prepare a high-concentration sodium chloride solution using 60% ethanol solution and a low-concentration sodium chloride solution using 80% ethanol solution; the sodium chloride content in the high-concentration sodium chloride solution is 12-15 g / L and the sodium chloride content in the low-concentration sodium chloride solution is 6-8 g / L.

[0010] S1-3. The grinding material is mixed with a high-concentration sodium chloride solution and extracted by ultrasonic vibration in an ice-water bath. Then, it is pressed and filtered. The filter residue is then added to a low-concentration sodium chloride solution and extracted by ultrasonic vibration in a water bath at 40-50℃. After pressing and filtering, the two filtrates are combined, concentrated under vacuum, and then freeze-dried to obtain green-lipped mussel extract.

[0011] Preferably, the freezing temperature in step S1-1 is -8°C to -10°C, and the freezing time is 4-6 hours.

[0012] Preferably, the ultrasonic oscillation extraction power in steps S1-3 is 400-600W, and the extraction time is 20-30min.

[0013] Preferably, the specific preparation method of the prickly pear fermented extract includes the following steps:

[0014] S2-1. Select fresh prickly pears, add water and grind them into a paste to obtain prickly pear paste for later use;

[0015] S2-2. Inoculate Lactobacillus plantarum into the prickly pear pulp and ferment for 6-8 hours to obtain the first fermented material for later use.

[0016] S2-3. Inoculate Bacillus tekirae into the first fermentation material and continue fermentation for 4-8 hours to obtain the second fermentation material for later use.

[0017] S2-4. Place the second fermentation material in a high-pressure environment of 4-6 MPa, extract it in a water bath at 50-60℃ for 2-3 hours, then press, filter, and freeze dry to obtain the prickly pear fermentation extract.

[0018] Preferably, the inoculation amount of Lactobacillus plantarum in step S2-2 is 0.2%-0.3% of the total mass of the prickly pear pulp.

[0019] Preferably, the inoculation amount of Bacillus tekirii in steps S2-3 is 0.1%-0.2% of the total mass of the first fermentation material.

[0020] Preferably, the fucoidan is a small molecule fucoidan with a molecular weight of less than 10 kDa.

[0021] The preparation method of the composition with combined adjuvant therapeutic effects against diabetes and arthritis is as follows: after mixing and stirring the fucoidan, green-lipped mussel extract and prickly pear fermented extract evenly, the mixture is sealed with nitrogen and stored in the dark.

[0022] This invention provides a composition with combined adjuvant therapeutic effects against diabetes and arthritis, and its preparation method, which has the following advantages compared with the prior art:

[0023] This invention uses fucoidan, green-lipped mussel extract, and prickly pear fermented extract as the main active ingredients. The green-lipped mussel extract is extracted with different concentrations of sodium chloride ethanol solution at different temperatures and then mixed. It has higher activity in anti-inflammatory and blood sugar regulation effects. The addition of prickly pear fermented extract can further enhance the overall adjuvant therapeutic effect. Compared with the effects of a single substance, it can play a good synergistic role and comprehensively improve the effects of anti-diabetes and arthritis. Attached Figure Description

[0024] Figure 1 This is a schematic diagram comparing the levels of FBG and FINS in T2DM rats after treatment with different oral medications in an embodiment of the present invention.

[0025] Figure 2 This is a schematic diagram comparing the levels of TC and TG in T2DM rats after treatment with different oral medications in an embodiment of the present invention.

[0026] Figure 3 This is a schematic diagram showing the comparison of LDL-C and HDL-C levels in T2DM rats after treatment with different oral medications in this embodiment of the invention.

[0027] Figure 4 This is a schematic diagram showing the comparison of serum inflammatory factors IL-6 and TNF-α levels in arthritic rats after treatment with different oral gavage drugs in an embodiment of the present invention. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with 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 skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] The *Lactobacillus plantarum* used in the following examples was purchased from Xi'an Rongzhen Biotechnology Co., Ltd., with a viable count of 10 billion CFU / g; *Bacillus tekirae* was purchased from Nanjing Agricultural University, with accession number CGMCC No. 15973, a viable count of 10 billion CFU / g, and this strain was disclosed in patent application number "CN202011412682.9" filed on December 3, 2020; and fucoidan was purchased from Shaanxi Haibo Biotechnology Co., Ltd., with a molecular weight of less than 10 kDa.

[0030] Example 1:

[0031] Preparation of raw materials:

[0032] 1. Preparation of green-lipped mussel extract:

[0033] 1.1 Preparation of GLM-1:

[0034] (1) After removing the shells from the green-lipped mussels, clean them and freeze them at -10°C for 5 hours. Then grind and crush them, keep them warm in an ice water bath, and use the ground material for later use.

[0035] (2) Prepare a high-concentration sodium chloride solution with a sodium chloride content of 14 g / L using 60% ethanol solution, and prepare a low-concentration sodium chloride solution with a sodium chloride content of 7 g / L using 80% ethanol solution;

[0036] (3) Add 5 times the volume of high-concentration sodium chloride solution to the grinding material and extract it with ultrasonic vibration at 400W for 25 minutes in an ice-water bath. Then press and filter. Add 5 times the volume of low-concentration sodium chloride solution to the filter residue and extract it with ultrasonic vibration at 400W for 25 minutes in a water bath at 45℃. Then press and filter. Combine the two filtrates, concentrate them under vacuum, and freeze dry to obtain GLM-1.

[0037] 1.2 Preparation of GLM-2:

[0038] (1) After removing the shells from the green-lipped mussels, clean them and freeze them at -10°C for 5 hours. Then grind and crush them, keep them warm in an ice water bath, and use the ground material for later use.

[0039] (2) Prepare a high-concentration sodium chloride solution with a sodium chloride content of 14 g / L using a 60% ethanol solution;

[0040] (3) Add 5 times the volume of high-concentration sodium chloride solution to the grinding material, extract with ultrasonic vibration at 400W for 25 minutes in an ice-water bath, then press and filter. Add 5 times the volume of high-concentration sodium chloride solution to the filter residue, extract with ultrasonic vibration at 400W for 25 minutes in a water bath at 45℃, then press and filter. Combine the two filtrates, concentrate under vacuum and freeze dry to obtain GLM-2.

[0041] 1.3 Preparation of GLM-3:

[0042] (1) After removing the shells from the green-lipped mussels, clean them and freeze them at -10°C for 5 hours. Then grind and crush them, keep them warm in an ice water bath, and use the ground material for later use.

[0043] (2) Prepare a low-concentration sodium chloride solution with a sodium chloride content of 7 g / L using 80% ethanol solution;

[0044] (3) Add 5 times the volume of low-concentration sodium chloride solution to the grinding material, extract with ultrasonic vibration at 400W for 25 minutes in an ice-water bath, then press and filter. Add 5 times the volume of low-concentration sodium chloride solution to the filter residue, extract with ultrasonic vibration at 400W for 25 minutes in a water bath at 45℃, then press and filter. Combine the two filtrates, concentrate under vacuum and freeze dry to obtain GLM-3.

[0045] 2. Preparation of Rosa rugosa (CR) extract:

[0046] 2.1 Preparation of CR-1:

[0047] (1) Select fresh prickly pears, add twice the volume of water and grind them into a paste to obtain prickly pear paste for later use;

[0048] (2) Inoculate the prickly pear pulp with 0.2% of the total mass of Lactobacillus plantarum and ferment for 7 hours to obtain the first fermented material for later use;

[0049] (3) Inoculate the first fermentation material with 0.1% of the total mass of the first fermentation material with Bacillus tekirae and continue fermentation for 6 hours to obtain the second fermentation material for later use;

[0050] (4) The second fermentation material was placed in a high-pressure environment of 5MPa, extracted in a water bath at 55℃ for 2.5h, then pressed, filtered and freeze-dried to obtain CR-1.

[0051] 2.2 Preparation of CR-2:

[0052] (1) Select fresh prickly pears, add twice the volume of water and grind them into a paste to obtain prickly pear paste for later use;

[0053] (2) Inoculate the prickly pear pulp with 0.2% of the total mass of Lactobacillus plantarum and ferment for 13 hours to obtain fermented material for later use;

[0054] (3) The fermentation material was placed in a high-pressure environment of 5MPa, extracted in a water bath at 55℃ for 2.5h, then pressed, filtered and freeze-dried to obtain CR-2.

[0055] 2.3 Preparation of CR-3:

[0056] (1) Select fresh prickly pears, add twice the volume of water and grind them into a paste to obtain prickly pear paste for later use;

[0057] (2) Inoculate the prickly pear pulp with 0.1% of the total mass of Bacillus tekirae and continue fermentation for 13 hours to obtain fermented material for later use;

[0058] (3) The fermentation material was placed in a high-pressure environment of 5MPa, extracted in a water bath at 55℃ for 2.5h, then pressed, filtered and freeze-dried to obtain CR-3.

[0059] 2.4 Preparation of CR-4:

[0060] (1) Select fresh prickly pears, add twice the volume of water and grind them into a paste to obtain prickly pear paste for later use;

[0061] (2) Inoculate the prickly pear pulp with 0.1% of the total mass of Bacillus tekirae and ferment for 6 hours to obtain the first fermented material for later use;

[0062] (3) Inoculate the first fermentation material with 0.2% of the total mass of the first fermentation material and continue fermentation for 7 hours to obtain the second fermentation material for later use;

[0063] (4) The second fermentation material was placed in a high-pressure environment of 5MPa, extracted in a water bath at 55℃ for 2.5h, then pressed, filtered and freeze-dried to obtain CR-4.

[0064] Example 2:

[0065] Preparation of the composition:

[0066] Referring to Example 1 above and Table 1 below, the raw materials were prepared (in parts by weight), and the raw materials were mixed and stirred evenly to obtain different compositions:

[0067] Table 1

[0068]

[0069] Detection:

[0070] 1. To test the preventive and therapeutic effects of the above-mentioned compositions on type 2 diabetes:

[0071] Seventy-eight six-week-old male SD rats were selected. Six rats were randomly chosen as the blank control group and fed a normal diet. The remaining 72 rats were fed a high-fat, high-sugar diet for four weeks. After four weeks, they were given a single intraperitoneal injection of streptozotocin at a dose of 70 mg / kg. Two weeks later, a fasting blood glucose level ≥11.1 nmol / L was considered sufficient to establish a type 2 diabetes mellitus (T2DM) model in rats. The established model rats were then divided into 12 groups, all fed a normal diet. One group served as the T2DM model group, and the other 11 groups served as the experimental groups. The experimental groups were further divided according to the following... Rats were administered 400 mg / kg of the following compositions by gavage: Composition 1, Composition 2, Composition 3, Composition 4, Composition 5, Composition 6, Composition 7, Composition 8, fucoidan, GLM-1, and CR-1. The T2DM model group was administered the same volume of physiological saline by gavage. After four weeks, the fasting blood glucose (FBG), fasting serum insulin (FINS), total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) levels were measured in each group. Specific results are as follows: Figure 1 , Figure 2 and Figure 3 And as shown in Tables 2 and 3 below:

[0072] Table 2

[0073]

[0074] Table 3

[0075]

[0076] The above tests show that the gavage drugs used in experimental groups 1-11 can reduce blood glucose levels and fasting serum insulin levels to a certain extent. Among them, experimental groups 1 and 2 have the best effects. Furthermore, the drugs used in experimental groups 1-11 can counteract lipid metabolism disorders in rats, resulting in a decrease in TC, TG, and LDL-C levels and an increase in HDL-C levels.

[0077] 2. To test the therapeutic effects of the above-mentioned compositions on arthritis.

[0078] Seventy-eight 6-week-old male SD rats were selected. Six rats were randomly selected as the blank control group and fed with a normal diet. The remaining 72 rats were used to establish a rat model of arthritis using the collagen-induced method: bovine type II collagen was dissolved in 0.05M acetic acid to a final concentration of 3 mg / mL. Then, an equal volume of complete Freund's adjuvant (CFA) was mixed and stirred at high speed under ice bath conditions to form an emulsion. After anesthetizing the rats, 0.2 mL of the emulsion was injected intradermally into the plantar part of the rat's paw. On the 10th day after the initial immunization, the same dose of the emulsion was injected at multiple points at the base of the tail for booster immunization (the rats were fed with a normal diet during the initial immunization).

[0079] On day 8 after booster immunization, 72 rats were randomly divided into 12 groups. One group served as the arthritis model group, and the other 11 groups served as the experimental groups. The experimental groups were administered 300 mg / kg of each of the following compositions by gavage: composition 1, composition 2, composition 3, composition 4, composition 5, composition 6, composition 7, composition 8, fucoidan, GLM-1, and CR-1. The arthritis model group was administered the same volume of physiological saline by gavage. After 4 weeks of administration and 1 week of drug withdrawal, the serum levels of inflammatory factors IL-6 and TNF-α in each group were measured. The specific results are shown in Table 4 below.

[0080] Table 4

[0081]

[0082] As shown in the table above, both Composition 1 and Composition 2 can effectively reduce the content of verification factor in rat blood, thereby alleviating arthritis symptoms.

[0083] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A composition having anti-diabetic and combined adjuvant treatment of arthritis effects, characterized in that, The composition is composed of the following mass parts of substances: fucoidan 3-5 parts, green-lipped mussel extract 1-2 parts, and fermented roselle extract 1-3 parts. ​ The preparation method of the green-lipped mussel extract comprises the following steps: S1-1, after the green-lipped mussel is shelled, it is washed, then frozen and ground, and ice water bath is used for preservation to obtain a grinding material for standby use; S1-2, 60% ethanol solution is used to prepare a high-concentration sodium chloride solution, and 80% ethanol solution is used to prepare a low-concentration sodium chloride solution; the content of sodium chloride in the high-concentration sodium chloride solution is 12-15 g / L, and the content of sodium chloride in the low-concentration sodium chloride solution is 6-8 g / L; S1-3, the grinding material is mixed with the high-concentration sodium chloride solution, ultrasonic oscillation extraction is carried out in an ice water bath, then squeezing and filtration are carried out, the filter residue is added with the low-concentration sodium chloride solution, ultrasonic oscillation extraction is carried out under the condition of a 40-50 ℃ water bath, then squeezing and filtration are carried out, the two filtrates are combined, vacuum concentration is carried out, and then freeze-drying is carried out to obtain the green-lipped mussel extract; The specific preparation method of the fermented roselle extract comprises the following steps: S2-1, fresh roselle is selected, and water is used for grinding to obtain roselle slurry for standby use; S2-2, Lactobacillus plantarum is inoculated into the roselle slurry for fermentation treatment for 6-8 h to obtain first fermentation material for standby use; S2-3, Bacillus coagulans is inoculated into the first fermentation material for continuous fermentation treatment for 4-8 h to obtain second fermentation material for standby use; S2-4, the second fermentation material is placed in a high-pressure environment of 4-6 MPa, 50-60 ℃ water bath extraction is carried out for 2-3 h, then squeezing and filtration are carried out, and then freeze-drying is carried out to obtain the fermented roselle extract.

2. The composition of claim 1, wherein: In the step S1-1, the freezing temperature is -8 ℃ to -10 ℃, and the freezing time is 4-6 h.

3. The composition of claim 1, wherein: In the step S1-3, the ultrasonic oscillation extraction power is 400-600 W, and the extraction time is 20-30 min.

4. The composition of claim 1, wherein: In the step S2-2, the inoculation amount of Lactobacillus plantarum is 0.2%-0.3% of the total mass of the roselle slurry.

5. The composition of claim 1, wherein: In the step S2-3, the inoculation amount of Bacillus coagulans is 0.1%-0.2% of the total mass of the first fermentation material.

6. The composition of claim 1, wherein: The fucoidan is small-molecule fucoidan, and the molecular weight is less than 10 KDa.

7. A process for the preparation of a composition having anti-diabetic and co-adjuvant therapy for arthritis as claimed in any one of claims 1 to 6, wherein the process comprises: a) mixing the ingredients of the composition; b) filling the composition in a suitable container; and c) sealing the container. The preparation method is that the fucoidan, the green-lipped mussel extract and the fermented roselle extract are uniformly mixed, then nitrogen sealing and light shielding preservation are carried out.

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