Application of composition containing butyric acid producing bacteria and vitamin K2 in preparation of medicine for treating osteoporosis

Abstract

The invention provides application of a composition containing butyric acid producing bacteria and vitamin K2 in preparation of a medicine for treating osteoporosis, and relates to the technical field of biological medicines, the composition comprises butyric acid producing bacteria and vitamin K2; wherein the butyric acid producing bacteria are selected from any one or more of clostridium butyricum, rosella and clostridium praeparatum; and the vitamin K2 is selected from any one or more of menadione-4 or menadione-7. The composition can play a synergistic effect through an intestine-bone axis, and compared with a single component, the composition can more remarkably improve the bone mineral density. The composition has comprehensive effects and high safety, provides a brand-new nutrition intervention strategy with a synergistic effect for prevention and treatment of osteoporosis, and provides a brand-new possibility for preparation of medicines related to osteoporosis.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more particularly to the use of a composition containing butyric acid-producing bacteria and vitamin K2 in the preparation of a medicament for treating osteoporosis. Background Technology

[0002] Osteoporosis is a systemic metabolic bone disease characterized by decreased bone mass and damage to bone microstructure, leading to increased bone fragility and susceptibility to fractures. With the accelerating aging of the population, its incidence has risen significantly, becoming a major public health issue. Currently, clinical treatment strategies for osteoporosis mainly include basic supplements (such as calcium and vitamin D) and anti-resorption drugs (such as bisphosphonates and RANKL inhibitors) or bone-forming drugs (such as teriparatide). However, existing drugs have certain limitations: long-term use of bisphosphonates may increase the risk of atypical femoral fractures or osteonecrosis of the jaw; hormone replacement therapy may bring potential cardiovascular and breast side effects; and the high cost of some drugs also limits their widespread use.

[0003] In recent years, the interaction between gut microbiota and bone health, known as the "gut-bone axis," has gradually become a research hotspot. Gut microbiota indirectly regulates bone metabolism through multiple pathways, including modulating the immune system, influencing the release of inflammatory factors, and participating in host metabolism. While some studies have shown that supplementing with probiotics or vitamin K2 alone can improve bone density, the effects are often limited or unstable. Current technologies lack precise formulations that combine probiotics with specific functions and bone nutrients, particularly mature protocols for synergistic intervention targeting key aspects of the "gut-bone axis" (such as microbial metabolites and vitamin activation). This leaves room for exploring novel treatment strategies. Therefore, developing safe, effective, and easily sustainable prevention and treatment strategies is of great significance.

[0004] Therefore, this invention is proposed. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides the application of a composition containing butyric acid-producing bacteria and vitamin K2 in the preparation of a medicament for treating osteoporosis. By combining butyric acid-producing bacteria with vitamin K2, and further incorporating other functional substances, this invention enables applications in osteoporosis-related medications, opening up possibilities for exploring novel treatment strategies.

[0006] In order to achieve the objective of this invention, the following technical solution is adopted: This invention provides the use of a composition containing butyric acid-producing bacteria and vitamin K2 in the preparation of a medicament for treating osteoporosis, said composition comprising: butyric acid-producing bacteria and vitamin K2; The butyric acid-producing bacteria are selected from any one or more of Clostridium butyricum, Rosneria, or Clostridium plasmidoides. The vitamin K2 is selected from one or more of menaquinone-4 or menaquinone-7.

[0007] Furthermore, the viable count of butyric acid-producing bacteria in the composition is 1 × 10⁻⁶. 6 CFU / g-1×10 12 CFU / g.

[0008] Furthermore, the viable count of butyric acid-producing bacteria in the composition is 1 × 10⁻⁶. 9 CFU / g-1×10 11 CFU / g.

[0009] Furthermore, the dosage of vitamin K2 in the composition is 10 μg / g to 500 μg / g.

[0010] Furthermore, the dosage of vitamin K2 in the composition is 45 μg / g to 200 μg / g.

[0011] Furthermore, the dosage of vitamin K2 in the composition is 100 μg / g or 200 μg / g.

[0012] Furthermore, the composition also includes any one or more of the following: prebiotics, calcium, vitamin D, and pharmaceutical carriers.

[0013] Furthermore, the prebiotic is fructooligosaccharide and / or resistant dextrin; The vitamin D mentioned is vitamin D3; The calcium agent is calcium citrate; The pharmaceutical carrier is any one or more of pregelatinized starch, microcrystalline cellulose, mannitol, skim milk powder, trehalose, ascorbyl palmitate, tocopherol, or glycerol.

[0014] Furthermore, the composition is any one of capsules, tablets, powders, or liquid formulations.

[0015] The present invention also provides a pharmaceutical composition comprising: The number of viable bacteria is 1×10 6 CFU / g-1×10 12 CFU / g of butyric acid-producing bacteria and vitamin K2 at doses of 10μg / g-500μg / g; The butyric acid-producing bacteria are selected from any one or more of Clostridium butyricum, Rosneria, or Clostridium plasmidoides. The vitamin K2 is selected from one or more of menaquinone-4 or menaquinone-7.

[0016] Furthermore, the pharmaceutical composition also includes any one or more of the following: prebiotics, calcium, vitamin D, and pharmaceutical carriers; The prebiotic is fructooligosaccharide and / or resistant dextrin; The vitamin D mentioned is vitamin D3; The calcium agent is calcium citrate; The pharmaceutical carrier is any one or more of pregelatinized starch, microcrystalline cellulose, mannitol, skim milk powder, trehalose, ascorbyl palmitate, tocopherol, or glycerol.

[0017] The present invention also provides a method for preparing the above-described pharmaceutical composition.

[0018] The present invention has the following technical effects: The butyric acid-producing bacteria and vitamin K2 composition provided by this invention exhibits significant technical effects in the treatment of osteoporosis. Experiments have confirmed that this composition exerts a synergistic effect through the gut-bone axis. The butyric acid-producing bacteria regulate the intestinal flora, inhibit inflammatory factors, and improve intestinal barrier function by producing butyrate, thereby creating a favorable intestinal microenvironment for the absorption and efficient utilization of vitamin K2. Simultaneously, vitamin K2 more fully activates osteocalcin, promoting calcium salt deposition in the bone matrix. Compared to using a single component, the combined use of these two components significantly increases bone mineral density. This composition offers comprehensive effects and high safety, providing a novel nutritional intervention strategy with synergistic effects for the prevention and treatment of osteoporosis. Attached Figure Description

[0019] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0020] Figure 1 Analysis of gut microbiota in stool showed that the abundance of Lactobacillus was reduced in the gut microbiota of patients with osteoporosis. Figure 2 The results of the analysis of gut microbiota in stool showed that the abundance of Lactobacillus was reduced in the gut microbiota of patients with osteoporosis. Figure 3 The results of the analysis of intestinal flora in stool specifically showed a decrease in the abundance of Bacillus species, to which Lactobacillus belongs, in the intestinal flora of patients with osteoporosis. Figure 4 The results of the analysis of gut microbiota in stool showed that the abundance of Butyrate-producing Rhodesella spp. was reduced in the gut microbiota of patients with osteoporosis. Figure 5The blood samples of osteoporosis patients and control groups were analyzed by high performance liquid chromatography. a) Butyric acid content in the serum of osteoporosis patients; b) Butyric acid content in the serum of control group patients; c) Butyric acid standard control. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0022] In a first aspect, the present invention provides the use of a composition containing butyric acid-producing bacteria and vitamin K2 in the preparation of a medicament for treating osteoporosis, said composition comprising: butyric acid-producing bacteria and vitamin K2; The butyric acid-producing bacteria are selected from any one or more of Clostridium butyricum, Rosneria, or Clostridium plasmidoides. The vitamin K2 is selected from one or more of menaquinone-4 or menaquinone-7.

[0023] In some embodiments, the viable count of butyric acid-producing bacteria in the composition is 1 × 10⁻⁶. 6 CFU / g-1×10 12 CFU / g.

[0024] In some embodiments, the viable count of butyric acid-producing bacteria in the composition is 1 × 10⁻⁶. 9 CFU / g-1×10 11 CFU / g.

[0025] In some embodiments, the dosage of vitamin K2 in the composition is 10 μg / g to 500 μg / g.

[0026] In some embodiments, the dosage of vitamin K2 in the composition is 45 μg / g to 200 μg / g.

[0027] In some embodiments, the dosage of vitamin K2 in the composition is 100 μg / g or 200 μg / g.

[0028] In some embodiments, the composition further includes any one or more of the following: prebiotics, calcium, vitamin D, and pharmaceutical carriers.

[0029] In some embodiments, the prebiotic is fructooligosaccharide and / or resistant dextrin; The vitamin D mentioned is vitamin D3; The calcium agent is calcium citrate; The pharmaceutical carrier is any one or more of pregelatinized starch, microcrystalline cellulose, mannitol, skim milk powder, trehalose, ascorbyl palmitate, tocopherol, or glycerol.

[0030] In some embodiments, the composition is any one of capsules, tablets, powders, or liquid formulations.

[0031] In a second aspect, the present invention also provides a pharmaceutical composition comprising: The number of viable bacteria is 1×10 6 CFU / g-1×10 12 CFU / g of butyric acid-producing bacteria and vitamin K2 at doses of 10μg / g-500μg / g; The butyric acid-producing bacteria are selected from any one or more of Clostridium butyricum, Rosneria, or Clostridium plasmidoides. The vitamin K2 is selected from one or more of menaquinone-4 or menaquinone-7.

[0032] In some embodiments, the pharmaceutical composition further includes any one or more of the following: prebiotics, calcium, vitamin D, and pharmaceutical carriers; The prebiotic is fructooligosaccharide and / or resistant dextrin; The vitamin D mentioned is vitamin D3; The calcium agent is calcium citrate; The pharmaceutical carrier is any one or more of pregelatinized starch, microcrystalline cellulose, mannitol, skim milk powder, trehalose, ascorbyl palmitate, tocopherol, or glycerol.

[0033] Thirdly, the present invention also provides a method for preparing the above-mentioned pharmaceutical composition.

[0034] The following is a detailed explanation using specific embodiments: Example 1 A. Preparation of freeze-dried butyric acid-producing bacteria powder Use reinforced clostridium medium (RCM) and adjust the pH of the medium to 6.8-7.2. Dispense the medium into test tubes or culture flasks and autoclave at 121°C for 15 minutes.

[0035] In an anaerobic workbench, Clostridium butyricum inoculum is inoculated into the culture medium and anaerobically cultured at 37°C for 24-48 hours until the culture medium becomes turbid (OD600 ≈ 1.0-1.5, indicating that the bacterial growth has entered the stationary phase).

[0036] Centrifuge the culture medium at 4°C (10,000 × g, 10 min) and collect the bacterial pellet. Wash the bacterial cells twice with sterile phosphate-buffered saline (PBS, pH 7.4) to remove residual culture medium components.

[0037] The washed bacterial cells were resuspended in a freeze-drying protectant, and the bacterial suspension concentration was adjusted to approximately 1 × 10⁻⁶. 10 The bacterial suspension was dispensed into freeze-drying bottles at CFU / mL and pre-frozen at -40°C for 2 hours.

[0038] The temperature was then gradually increased to -20°C under vacuum (<0.1 mbar) and maintained for 10 hours for a second drying. The temperature was then increased to 25°C and maintained for 4 hours for a second drying to obtain freeze-dried bacterial powder.

[0039] The viable count of the freeze-dried bacterial powder was verified using the plate count method to ensure it reached 1×10⁻⁶. 10 CFU / g or higher.

[0040] B. Preparation of a composition containing Clostridium butyricum and vitamin K2 Weigh out 1×10⁻⁶ lyophilized bacterial powder 10 CFU / g) 10 g (provides 1×10 11 (total live bacteria CFU).

[0041] Weigh out 10 mg of vitamin K2 (menaquinone-7 powder) (equivalent to 100 μg / g in the final composition).

[0042] Place the vitamin K2 powder and freeze-dried bacterial powder into a mixer under dry conditions (relative humidity <20%) and mix for 30 minutes to ensure uniform distribution.

[0043] C. Formulation The mixture was directly filled into empty capsules (each capsule containing 0.5 g of butyric acid-producing bacteria, 5 × 10⁶). 9 CFU and Vitamin K2 50μg).

[0044] Alternatively, it can be compressed into tablets (with a small amount of lubricant such as magnesium stearate added, and a compression pressure of 5-10 kN).

[0045] Note: Aseptic technique must be maintained throughout the entire preparation process, and vitamin K2 must be handled and stored away from light. Freeze-dried bacterial powder should be stored at -20°C. After mixing, the product can be stored at 4°C for a short period (e.g., 1 month), while long-term storage is recommended at -20°C.

[0046] Example 2 The preparation of Clostridium butyricum lyophilized powder was the same as in Example 1, using 2.0g of Clostridium butyricum lyophilized powder.

[0047] Take 2 mg of vitamin K2 powder (menaquinone-7) MK-7 and grind and mix it with 198 mg of pregelatinized starch to obtain 200 mg of 1% vitamin K2 premixed powder.

[0048] 1% Vitamin D3 spray powder: 1.6g Fructooligosaccharides: 4.0g Calcium citrate: 6.0g Pregelatinized starch: 6.2g For trace component premixing, 200 mg of premixed vitamin K2 powder and an equal amount of 1% vitamin D3 spray powder are carefully ground and mixed in a mortar. More 1% vitamin D3 spray powder is gradually added until it is completely mixed evenly, ensuring uniform distribution of trace components, to obtain vitamin mixed powder.

[0049] Place the fructooligosaccharides, calcium citrate, and remaining pregelatinized starch into a small mixer or resealable bag and mix manually for 5 minutes. Then add the vitamin mixture powder and mix for 10 minutes. Finally, add the lyophilized butyric acid bacteria powder and gently mix for 15-20 minutes until completely homogeneous.

[0050] The powder is filled into capsules or compressed into tablets.

[0051] Experimental Example 1: Butyric acid-producing bacteria may inhibit osteoporosis by activating the Wnt / β-catenin signaling pathway through their metabolite, butyrate. Patients diagnosed with osteoporosis were selected, and in collaboration with the Department of Gastroenterology and the Institute of Digestive Diseases of our hospital, further gene testing was performed on the feces of the selected patients. Intestinal flora analysis was conducted using 16S rRNA sequencing. Comparison with the control group revealed that osteoporosis patients exhibited dysbiosis, with a decrease in the abundance of Lactobacillus and some butyric acid-producing bacteria, while the abundance of pathogenic bacteria such as Clostridium perfringens increased. Figure 1 - Figure 4 Meanwhile, high-performance liquid chromatography (HPLC) analysis of blood samples from osteoporosis patients and a control group revealed that serum butyrate levels in osteoporosis patients were lower than those in the control group. Figure 5 a- Figure 5 c). Supplementing with short-chain fatty acids such as butyrate can reverse this process to some extent, which further proves the important role of the lack of butyrate-producing bacteria in the pathogenesis of osteoporosis.

[0052] Experimental Example 2: Effects of Butyric Acid-Producing Bacteria and Vitamin K2 on Bone Turning Markers in Mice 1. Animal selection: 1) Select female ICR mice, as both strains respond well to the ovariectomy-induced osteoporosis model.

[0053] 2) Age and weight: 10-12 weeks old, weight 20±2g.

[0054] 3) Each group should contain at least 10 animals to meet statistical requirements.

[0055] 1.1 Rearing environment: 1) SPF-grade animal room, with temperature controlled at 22±2℃, relative humidity at 50±10%, and 12-hour light / dark cycle.

[0056] 2) Keep 4-5 mice per cage, use sterile bedding, and change it regularly.

[0057] 1.2 Feed and Water: Mice were given a standard maintenance diet and free access to water. All mice were allowed to acclimatize to the environment for at least one week before the experiment began.

[0058] 2. Establishment of an osteoporosis model (oophorectomy) 2.1 Grouping and Surgery 2.1.1 Sham surgery group: Mice underwent the same surgical procedure (incision of skin and muscle to expose the ovary), but the ovary was not removed; only a small piece of adipose tissue surrounding the ovary was removed. This group served as a normal control.

[0059] 2.1.2 Model Group and Treatment Group Bilateral oophorectomy was performed to establish an osteoporosis model.

[0060] 2.2 Surgical Procedure a) Anesthesia was administered via inhalation of 1-3% isoflurane; b) Skin preparation and disinfection The skin on both sides of the lumbar spine on the back of the mouse was prepared and disinfected with iodine and 75% alcohol.

[0061] c) Make a longitudinal incision of about 1 cm on the back of each side.

[0062] d) Ligation and excision Locate the fat deposits around the ovary, gently pull them out to expose the ovary. Ligate the junction of the ovary and uterine horn, then remove the ovary.

[0063] e) The uterine horn is returned to the abdominal cavity, and the muscles and skin are sutured layer by layer.

[0064] 2.3 Postoperative care Keep the patient isolated until they regain consciousness, and keep them warm. You can inject an appropriate amount of saline solution subcutaneously to replenish fluids. Observe the wound healing within three days after the operation.

[0065] 3. Experimental grouping and dosing regimen 3.1 Experimental Groups (6 groups in total) Group 1 Sham surgery group + solvent / blank control Group 2 Ovarian removal model group + solvent / blank control Group 3 Ovarian removal + butyric acid-producing bacteria intervention group Group 4 Ovarian removal + Vitamin K2 intervention group Group 5 Ovarian removal + butyric acid-producing bacteria and vitamin K2 combination group Group 6 Ovarian removal + positive control group (alendronate sodium, 70 μg / kg, once a week) 3.2 Dosing regimen a) Administration start time: one week after surgery, after the mice have fully recovered.

[0066] b) Dosing cycle: 12 weeks of continuous administration.

[0067] 3.3 Route and frequency of administration Butyric acid-producing bacteria: Administer the bacterial solution orally via gavage once daily. The dosage is 1×10⁻⁶. 9 CFU / animal / day, butyric acid-producing bacteria suspended in 0.2 mL of sterile PBS or physiological saline.

[0068] Vitamin K2: Administered orally by gavage once daily. The dosage is 100 μg / animal / day, dissolved in 0.2 mL of corn oil containing a small amount of emulsifier (0.5% Tween-80).

[0069] Composition group: The above-mentioned dose of bacteria and vitamin K2 were mixed in 0.2 mL of the same carrier and administered by gavage.

[0070] Control group: The sham surgery group and the model group were given the same volume of solvent by gavage.

[0071] 4. Sample Collection and Processing 4.1 Blood Sample Collection a) Time point: After the last administration, fasting but not watering was allowed for 12 hours before collection.

[0072] b) Blood is drawn from the eye or abdominal aorta and collected in a vacuum blood collection tube that does not contain anticoagulants.

[0073] 4.2 Serum separation a) Let the blood collection tube stand at room temperature for 30-60 minutes until the blood clots naturally. Then centrifuge at 3000 rpm for 15 minutes. Carefully aspirate the supernatant using a pipette; this is the serum.

[0074] b) Aliquot the serum from each mouse into EP tubes and immediately store them in an ultra-low temperature freezer at -80°C to avoid repeated freeze-thaw cycles.

[0075] 5. Detection of bone metabolism markers (ELISA method) 5.1 Testing Indicators: a) Bone formation markers: P1NP b) Bone resorption marker: CTX-1 5.2 Detection Procedure (using a commercially available ELISA kit): a) Reagent preparation: Remove the kit from the 4°C freezer and allow it to reach room temperature. Prepare the washing buffer, standards, biotinylated antibody, and enzyme conjugate according to the instructions.

[0076] b) Sample addition: Different concentrations of standards and diluted serum samples to be tested were added sequentially to the wells of an antibody-coated ELISA plate.

[0077] c) Incubation and washing: Add biotinylated detection antibody and incubate at 37°C for 1 hour.

[0078] Wash the plate 3-5 times with detergent and pat dry.

[0079] Add horseradish peroxidase-labeled streptavidin and incubate at 37°C for 30 minutes.

[0080] Wash the board five more times and pat it dry completely.

[0081] d) Color development and termination: Add the substrate TMB and incubate at 37°C in the dark for 15-30 minutes.

[0082] When the standard solution shows a clear gradient of blue, add the stop solution, and the solution color will change from blue to yellow.

[0083] e) Detection and calculation: Immediately measure the absorbance of each well using an ELISA reader at a wavelength of 450 nm.

[0084] Plot a standard curve with the concentration of the standard on the x-axis and the OD value on the y-axis.

[0085] Substitute the OD value of the sample into the standard curve to calculate the concentrations of P1NP and CTX-1 in the sample.

[0086] 6. Data Analysis and Expected Results 6.1 Data Processing: a) All data are expressed as mean ± standard deviation.

[0087] b) One-way ANOVA was performed using statistical software (such as SPSS or GraphPad Prism), and Tukey post-hoc tests were used for intergroup comparisons.

[0088] c) P < 0.05 is considered statistically significant.

[0089] Experiment Example 3: Bioavailability and Activation Mechanism of Vitamin K2 1. Sample Collection After the last dose and collection of a blood sample for bone metabolism marker detection, the following procedures were performed simultaneously: Serum separation: The method was the same as in Experiment 2. The separated serum was aliquoted and stored at -80°C for subsequent detection of vitamin K2 function indicators and inflammatory factors.

[0090] 2. Experimental methods and procedures The level of incompletely carboxylated osteocalcin in serum was measured.

[0091] a) Following the kit instructions, add serum samples and standards of different concentrations to the wells of a pre-coated antibody-impregnated ELISA plate. b) Add biotinylated detection antibody and incubate at 37°C for 1 hour. c) After washing, add horseradish peroxidase-labeled streptavidin and incubate at 37°C for 30 minutes. d) After washing again, add TMB substrate solution for color development in the dark, then terminate the reaction with stop solution. e) Immediately measure the absorbance (OD value) of each well at 450 nm using an ELISA reader. f) Calculate the concentration of incompletely carboxylated osteocalcin (ucOC) in the sample based on the standard curve; assess the vitamin K2 activation level using the absolute concentration of ucOC.

[0092] Experiment Example 4: Detection of Systemic Inflammatory Factors The detection of tumor necrosis factor-α in serum using a mouse-specific ELISA kit includes steps such as sample addition, incubation, washing, color development, termination, and detection; only the kit used differs.

[0093] The experimental results of Experiment 2-4 are shown in Table 1. Table 1: Effects of butyric acid-producing bacteria and vitamin K2 on bone turnover markers in mice Note: ① All data are expressed as mean ± standard deviation; ② ** indicates P < 0.01 compared with the sham surgery group; ③# indicates that compared with the model group, P<0.05; ④## indicates that compared with the model group, P<0.01; ⑤&&: indicates that compared with the butyric acid-producing bacteria group and the vitamin K2 group, P<0.01.

[0094] Conclusion Analysis: I. Successful Model Construction and the Basic Effects of the Invention Model group vs. sham surgery group: All indicators in the model group were significantly different from those in the sham surgery group (**). Simultaneously, bone mineral density decreased significantly, indicating poor bone quality; P1NP (bone formation marker) was significantly decreased, while CTX-1 (bone resorption marker) was significantly increased, demonstrating an imbalance in bone metabolism, with bone resorption far exceeding bone formation, which is the core pathological characteristic of osteoporosis.

[0095] An elevated ucOC (incompletely carboxylated osteocalcin) level indicates a relative deficiency or activation disorder of vitamin K2 in the body, which prevents osteocalcin from being fully carboxylated and thus unable to perform its mineralization-promoting function.

[0096] The sharp increase in TNF-α (inflammatory factor) levels confirms the chronic inflammatory state in the process of osteoporosis, which is consistent with the view in the gut-bone axis theory that inflammation promotes bone resorption.

[0097] It is evident that the osteoporosis model was successfully established, providing a reliable disease model for evaluating the efficacy of this invention.

[0098] II. Effectiveness and Limitations of Individual Components Butyric acid producing bacteria group vs. model group All indicators in this group showed significant improvement (#), primarily through the gut-bone axis. Butyrate production led to anti-inflammatory effects (decreased TNF-α) and may indirectly inhibit bone resorption (decreased CTX-1) and slightly promote bone formation (increased P1NP) through immune regulation.

[0099] However, butyric acid-producing bacteria alone have limited effect on improving vitamin K2 activation (ucOC index) and are less effective than vitamin K2 alone.

[0100] Vitamin K2 group vs. model group All indicators in this group showed significant improvement (# / ##), with the effect directly impacting bone metabolism. By providing cofactors, the carboxylation efficiency of osteocalcin was significantly enhanced (ucOC was greatly reduced), thereby promoting bone formation (increased P1NP) and inhibiting bone resorption (decreased CTX-1).

[0101] However, the anti-inflammatory effect of vitamin K2 alone was weaker (the decrease in TNF-α was less than that of the butyric acid-producing bacteria group), indicating that it is difficult to improve bone metabolism by regulating immune inflammatory pathways.

[0102] It is evident that using butyric acid-producing bacteria or vitamin K2 alone is effective, but each has its own limitations in terms of mechanism, resulting in limited effectiveness.

[0103] III. Synergistic effect of butyric acid-producing bacteria and vitamin K2 Butyric acid-producing bacteria and vitamin K2 exhibit synergistic effects, which are comprehensively superior to either one used alone. Furthermore, the synergistic effect is not simply additive, as can be seen from the experimental data above. Bone mineral density: Composition group (0.115) > Vitamin K2 group (0.098) > Butyric acid-producing bacteria group (0.095).

[0104] P1NP (bone formation): Composition group (38.5) > Vitamin K2 group (30.1) > Butyric acid producing bacteria group (28.3).

[0105] CTX-1 (bone resorption): Combination group (1650) < Vitamin K2 group (2250) < Butyric acid-producing bacteria group (2350).

[0106] Across all core bone parameters, the combined composition significantly outperformed any single component, with improvements exceeding the arithmetic mean of the individual component effects. This clearly demonstrates a synergistic effect of "1+1>2," rather than a simple additive result.

[0107] Regarding vitamin K2 activation, the combined group showed the lowest ucOC level (6.2), which was significantly better than the vitamin K2 alone group (8.5). This is one of the strongest pieces of evidence demonstrating synergistic effects. It indicates that butyric acid-producing bacteria create better conditions for vitamin K2 absorption or function by improving the intestinal environment (such as anti-inflammatory and barrier repair), thereby maximizing its bioavailability and activation efficiency.

[0108] In terms of anti-inflammatory effects, the combined group had the lowest TNF-α level (15.8), which was significantly better than the butyric acid-producing bacteria alone group (25.2). This suggests that vitamin K2 may also have some positive interaction with butyric acid-producing bacteria in regulating immunity, jointly creating a low-inflammatory environment more conducive to bone health.

[0109] IV. Comparison with positive control drugs The combined group showed positive results in multiple parameters (such as bone mineral density, P1NP, CTX-1, ucOC, TNF-α). α In all cases, they showed a trend of being comparable to or better than the positive control drug (alendronate sodium).

[0110] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; 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 or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the technical solutions of the embodiments of the present invention.

Claims

1. The use of a composition containing butyric acid-producing bacteria and vitamin K2 in the preparation of a medicament for treating osteoporosis, characterized in that, The composition comprises: butyric acid-producing bacteria and vitamin K2; The butyric acid-producing bacteria are selected from any one or more of Clostridium butyricum, Rosneria, or Clostridium plasmidoides. The vitamin K2 is selected from one or more of menaquinone-4 or menaquinone-7.

2. The use of the composition containing butyric acid-producing bacteria and vitamin K2 according to claim 1 in the preparation of a medicament for treating osteoporosis, characterized in that, The viable count of butyric acid-producing bacteria in the composition is 1×10⁻⁶. 6 CFU / g-1×10 12 CFU / g.

3. The use of the composition containing butyric acid-producing bacteria and vitamin K2 according to claim 1 in the preparation of a medicament for treating osteoporosis, characterized in that, The dosage of vitamin K2 in the composition is 10 μg / g to 500 μg / g.

4. The use of the composition containing butyric acid-producing bacteria and vitamin K2 according to claim 1 in the preparation of a medicament for treating osteoporosis, characterized in that, The composition further includes any one or more of the following: prebiotics, calcium, vitamin D, and pharmaceutical carriers.

5. The use of the composition containing butyric acid-producing bacteria and vitamin K2 according to claim 4 in the preparation of a medicament for treating osteoporosis, characterized in that, The prebiotic is fructooligosaccharide and / or resistant dextrin; The vitamin D mentioned is vitamin D3; The calcium agent is calcium citrate; The pharmaceutical carrier is any one or more of pregelatinized starch, microcrystalline cellulose, mannitol, skim milk powder, trehalose, ascorbyl palmitate, tocopherol, or glycerol.

6. The use of the composition containing butyric acid-producing bacteria and vitamin K2 according to claim 1 in the preparation of a medicament for treating osteoporosis, characterized in that, The composition is any one of capsules, tablets, powders or liquid formulations.

7. A pharmaceutical composition applicable to any one of claims 1-6, characterized in that, The pharmaceutical composition comprises: The number of viable bacteria is 1×10 6 CFU / g-1×10 12 CFU / g of butyric acid-producing bacteria and vitamin K2 at doses of 10μg / g-500μg / g; The butyric acid-producing bacteria are selected from any one or more of Clostridium butyricum, Rosneria, or Clostridium plasmidoides. The vitamin K2 is selected from one or more of menaquinone-4 or menaquinone-7.

8. The pharmaceutical composition according to claim 7, characterized in that, The pharmaceutical composition further includes any one or more of the following: prebiotics, calcium, vitamin D, and pharmaceutical carriers; The prebiotic is fructooligosaccharide and / or resistant dextrin; The vitamin D mentioned is vitamin D3; The calcium agent is calcium citrate; The pharmaceutical carrier is any one or more of pregelatinized starch, microcrystalline cellulose, mannitol, skim milk powder, trehalose, ascorbyl palmitate, tocopherol, or glycerol.

9. A method for preparing the pharmaceutical composition according to any one of claims 7-8.

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