Application of xylotetraose or composition of xylotetraose and nystose in preparation of anti-osteoporosis product

Abstract

The invention provides application of xylotetraose or a composition of xylotetraose and nystose in preparation of an anti-osteoporosis product. The invention discloses the synergistic effect of the xylotetraose and the nystose in the aspect of osteoporosis resistance for the first time. When the zebrafish bone mineral composition and the zebrafish bone mineral composition are combined for use, the bone mineralization degree of zebrafish can be remarkably enhanced, the mineralization rate is increased, osteoblast activity is improved, bone metabolic disorder is effectively reversed, and the synergistic anti-osteoporosis potential of the zebrafish bone mineral composition is fully proved. Meanwhile, the invention also discloses the anti-osteoporosis effect of the xylotetraose for the first time, and the xylotetraose can promote bone mineralization, promote bone formation, inhibit bone resorption and regulate bone metabolism balance to a certain extent.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medicine, and relates to application of xylotetraose or a composition of xylotetraose and nigerose in preparation of an anti-osteoporosis product. BACKGROUND

[0002] The information disclosed in this Background section is only for the purpose of increasing the understanding of the background of the present application and should not be taken as an acknowledgement or any form of suggestion that this information forms prior art that is already commonly known to a person of ordinary skill in the art.

[0003] Osteoporosis is a systemic bone disease characterized by low bone mass, damage to bone tissue microstructure, increased bone fragility, and easy fracture, and has become a global major public health problem. Current treatment drugs mainly include bisphosphonates, RANKL inhibitors, and parathyroid hormone analogs. However, these drugs often have the shortcomings of high price, long-term use possibly leading to mandibular necrosis, atypical femoral fractures, or cardiovascular risks, which limit their wide application. Therefore, there is a great clinical demand and social value for developing safe, effective, and natural product-derived anti-osteoporosis drugs. SUMMARY

[0004] To solve the above problems, the present application provides application of xylotetraose or a composition of xylotetraose and nigerose in preparation of an anti-osteoporosis product. The present application is based on a zebrafish osteoporosis model, uses the SynergyFinder website to analyze the synergistic efficacy of xylotetraose and nigerose, optimizes the synergistic concentration and ratio of xylotetraose and nigerose by using a central composite design-response surface method, and performs zebrafish experiments for verification. The results first reveal the synergistic effect of xylotetraose and nigerose in anti-osteoporosis. When used in combination, they can significantly enhance the degree of bone mineralization, accelerate the mineralization rate, improve the activity of osteoblasts, and effectively reverse bone metabolic disorders of zebrafish, fully confirming their synergistic anti-osteoporosis potential. The present application provides a new idea and reliable experimental basis for developing drugs with xylotetraose and nigerose as effective ingredients, and has good development prospects and application value.

[0005] To achieve the above-mentioned purposes, the present application adopts the following technical solutions: In a first aspect of the present application, application of xylotetraose or a composition of xylotetraose and nigerose in preparation of an anti-osteoporosis product is provided.

[0006] In a second aspect of the present application, an anti-osteoporosis drug is provided, and the active ingredients include xylotetraose or a composition of xylotetraose and nigerose.

[0007] Advantages of the present application (1) The present application first discloses the synergistic effect of xylotetraose and nigerose in anti-osteoporosis. When the two are used in combination, they can significantly enhance the degree of bone mineralization in zebrafish, accelerate the mineralization rate, improve the activity of osteoblasts and effectively reverse bone metabolic disorders, fully demonstrating their synergistic anti-osteoporosis potential.

[0008] (2) The present application first discloses that xylotetraose has an anti-osteoporosis effect, can promote bone mineralization to some extent, promote bone formation and inhibit bone resorption, and regulate bone metabolism balance. BRIEF DESCRIPTION OF DRAWINGS

[0009] The drawings accompanying the specification of the present application serve to provide further understanding of the present application, and the exemplary embodiments of the present application and the description thereof serve to explain the present application, and do not constitute an improper limitation on the present application.

[0010] Figure 1 Synergistic effect of xylotetraose and nigerose combination administration, A. Dose-effect matrix diagram of combination administration; B. HSA synergistic effect diagram; Figure 2 Effect surface 3D diagram and contour diagram of xylotetraose and nigerose synergistic effect on fluorescence area and fluorescence intensity, A. Effect surface 3D diagram of fluorescence area; B. Contour diagram of fluorescence area; C. Effect surface 3D diagram of fluorescence intensity; D. Contour diagram of fluorescence intensity; Figure 3 Effect of xylotetraose and nigerose on the cumulative degree of bone mineralization in zebrafish, A. Representative image of zebrafish alizarin red staining; B. Alizarin red relative staining area histogram; C. Alizarin red relative staining optical density histogram; Figure 4 Effect of xylotetraose and nigerose on the bone mineralization rate of zebrafish, A. Zebrafish spine site calcein staining diagram of each group; B. Spine site fluorescence intensity histogram; Figure 5 Effect of xylotetraose and nigerose on bone metabolism related biochemical indicators of zebrafish; A. Relative activity of ALP, B. Relative activity of TRAP; Figure 6 Effect of xylotetraose and nigerose on osteoblast differentiation in zebrafish, A. Osteoblast fluorescence diagram of each group of zebrafish skull region; B. Relative fluorescence area histogram; C. Relative fluorescence intensity histogram.

[0011] Among them, *: p value <0.05, **: p value <0.01, ***: p value <0.001. DETAILED DESCRIPTION

[0012] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0013] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the art. The reagents and raw materials used in this invention are readily available through conventional means, and unless otherwise specified, they are used in accordance with conventional methods in the art or product instructions. Similarly, unless otherwise specified, the test methods of this invention are performed in accordance with conventional methods in the art or industry-standard methods or practices. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0014] Regarding osteoporosis, studies have reported that nysose can increase alkaline phosphatase (ALP) activity by activating the Wnt / β-catenin signaling pathway, thereby promoting the proliferation of bone marrow mesenchymal stem cells (BMSCs) and bone mineralization. Xylotetrasaccharide is also a naturally occurring oligosaccharide, but research on its application in bone health, particularly in combating osteoporosis, is currently lacking, and there are no reports revealing a synergistic effect of nysose in anti-osteoporosis efforts. Clarifying whether xylotetrasaccharide possesses anti-osteoporosis activity and whether there is a synergistic effect between xylotetrasaccharide and nysose is crucial.

[0015] Therefore, the present invention provides the use of xylo-tetraose or a combination thereof with nisose in the preparation of products for treating osteoporosis.

[0016] The concentration of xylo-tetrasaccharide can affect its anti-osteoporosis effect. Therefore, this invention studies the concentration of xylo-tetrasaccharide. Preferably, the concentration of xylo-tetrasaccharide is 6.25~50 μM to obtain a better anti-osteoporosis effect.

[0017] The ratio of xylotetraose and nisose affects the anti-osteoporosis effect of the composition. Therefore, the present invention has studied the ratio of the two in the composition. Preferably, the concentration of xylotetraose in the composition is 6.25~50 μM and the concentration of nisose is 6.25~50 μM. More preferably, the concentration of xylotetraose in the composition is 30 μM and the concentration of nisose is 29 μM to obtain a better anti-osteoporosis effect.

[0018] This invention reveals for the first time the anti-osteoporosis effect of xylo-tetrasaccharide, and the synergistic effect of xylo-tetrasaccharide and nysose in anti-osteoporosis. Preferably, the pharmacological effects include at least one of the following: a) Enhances bone mineralization; b) Accelerates the mineralization rate; c) Inhibits bone resorption; d) Promotes osteoblast differentiation; e) Enhance osteoblast activity.

[0019] This invention does not impose any special limitation on the type of product. Preferably, the product is a pharmaceutical or experimental reagent to meet different application needs.

[0020] The present invention also provides an anti-osteoporosis drug, the active ingredient of which includes xylotetraose or a combination thereof with nysose.

[0021] The concentration of xylo-tetrasaccharide can affect its anti-osteoporosis effect. Therefore, this invention studies the concentration of xylo-tetrasaccharide. Preferably, the concentration of xylo-tetrasaccharide is 6.25~50 μM to obtain a better anti-osteoporosis effect.

[0022] The ratio of xylotetraose and nisose affects the anti-osteoporosis effect of the composition. Therefore, the present invention has studied the ratio of the two in the composition. Preferably, the dosage concentration of xylotetraose in the composition is 6.25~50 μM and the dosage concentration of nisose is 6.25~50 μM. More preferably, the dosage concentration of xylotetraose in the composition is 30 μM and the dosage concentration of nisose is 29 μM to obtain a better anti-osteoporosis effect.

[0023] Preferably, it also includes excipients to better improve the stability, safety and efficacy of the drug.

[0024] The present invention will be further described in detail below with reference to specific embodiments. It should be noted that the specific embodiments are explanations of the present invention and not limitations thereof.

[0025] Example 1: Zebrafish rearing Wild-type AB strain and transgenic osteoblast green fluorescent Tg (0la.Sp7;nlsGEP) strain male and female zebrafish were isolated and fed with alternating light (14 h) and dark (10 h) conditions, and were fed newly hatched brine shrimp larvae daily. When collecting fertilized eggs, sexually mature healthy zebrafish were placed in a partitioned tank at a female-to-male ratio of 1:2. The partition was removed the following day while providing illumination, and the fertilized eggs were harvested between 9:00 and 11:00. After disinfection and washing, the fertilized eggs were transferred to zebrafish embryo culture water (1L of purified water containing 0.0128g KCl, 0.0484g CaCl2, 0.2932g NaCl, and 0.08g MgSO4·7H2O) and cultured under controlled light at 28±0.5℃.

[0026] Example 2 Screening of xylotetraose and nisose intervention concentrations Wild-type AB strain zebrafish juveniles were fertilized and developed for 72 hpf. Normally developed juveniles were selected under a stereomicroscope and transferred to 24-well plates (10 juveniles per well). Each well was exposed to 2 mL of drug solutions containing different concentrations (6.25–200 μM) of xylotetraose and nisose. A normal control group (zebrafish embryo culture water) was also included, with three replicates per group. All juveniles were immersed in a constant temperature incubator at 28 ± 0.5 °C for 72 h. The drug solution was changed daily to maintain drug concentration and water quality stability. Mortality and deformity were observed and recorded daily. The optimal drug concentration with lower mortality and deformity rates was selected for further investigation of its anti-osteoporosis efficacy and the synergistic effect of the two drugs.

[0027] As shown in Table 1, when the concentration reached 200 μM, the mortality rates of both the xylotetraose and nisose groups exceeded 15%; at a concentration of 100 μM, although the mortality rates of both groups were controlled below 15%, the deformity rates were high, exceeding 50%. Therefore, this invention selected 6.25–50 μM as the drug concentration for subsequent experiments to investigate the anti-osteoporosis efficacy of xylotetraose and nisose and their synergistic effect.

[0028] Table 1. Effects of different concentrations of xylotetraose and nisose on mortality and deformity rate in zebrafish ( ± s (n=10)

[0029] Compared with the normal group: * P < 0.05 ** P < 0.01, *** P < 0.001.

[0030] Example 3: Synergistic effect of xylan and nisose analyzed using the SynergyFinder website. After 72 hpf of fertilization and development, zebrafish larvae of the transgenic osteoblast green fluorescent Tg (0la.Sp7;nlsGEP) strain were selected under a stereomicroscope and transferred into 24-well plates with 10 larvae per well. The plates included a normal group (zebrafish embryo culture water), a model group (30 μM prednisolone), xylotetrasaccharide single-drug groups (6.25, 12.5, 25, 50 μM), nisulphurose single-drug groups (6.25, 12.5, 25, 50 μM), and various combination drug groups (each concentration of xylotetrasaccharide combined with each concentration of nisulphurose). Each group had 3 replicates. 2 mL of the corresponding drug solution was added to each well, and the plates were incubated at 28±0.5℃ for 144 h, with half of the drug solution replaced daily. After drug treatment, six zebrafish from each group were randomly selected and anesthetized in 0.02% tricaine solution. They were fixed on slides using 4% methylcellulose solution, and their green fluorescence images of osteoblasts in the skull region were systematically observed and acquired under a fluorescence stereomicroscope. The fluorescence area was quantitatively analyzed using ImageJ 1.8.0 software. The bone activity recovery rate of each group was calculated using the following formula. The fluorescence inhibition rates of each group were imported into the SynergyFinder website (https: / / synergyfinder.fimm.fi) to analyze whether the two had a synergistic anti-osteoporosis effect.

[0031]

[0032] To evaluate the synergistic anti-osteoporosis efficacy of xylan and nisose, this invention utilized the SynergyFinder website to analyze the osteogenic fluorescence recovery rate of zebrafish in both the single-drug and combined-drug groups. The results are shown below. Figure 1 The dose-efficacy matrix of the combined administration showed that the osteogenic fluorescence recovery rate of each concentration combined administration group was higher than that of the xylitol or nesusose single administration groups. The synergistic effect diagram of the HSA model analysis showed that the overall synergy score was positive (HSA synergy score=5.752), indicating that there was a good synergistic effect of combined administration, indicating that xylitol and nesusose have a good anti-osteoporosis efficacy.

[0033] Example 4: Optimization of the synergistic concentration of xylan and nisose using a star-shaped design-response surface methodology. Xylitol concentration (X1) and nisose concentration (X2) were used as independent variables. Based on preliminary experiments, the level ranges of each factor were determined as follows: xylitol 0–50 μM, nisose 0–50 μM, and the center point concentration 25 μM. The fluorescence area and fluorescence intensity measured by the zebrafish osteoblast fluorescence model were used as comprehensive evaluation indicators. The Box-Behnken response surface methodology was used to investigate and optimize the synergistic concentrations of xylitol and nisose. The settings of each factor and level are shown in Table 2. Based on the star point design principle, a total of 13 experimental points were set, including 4 factorial points, 4 axial points, and 5 center points to evaluate experimental error.

[0034] Table 2 Box-Behnken response surface factors and levels

[0035] This invention utilizes a star-point design-response surface methodology to optimize the optimal synergistic concentrations of xylane and nisose, with results shown in Tables 3 and 4. Multiple nonlinear regression fitting was performed using fluorescence area and fluorescence intensity as indicators, yielding the following fitting equation: Fluorescence area Y1 = 34423.50 + 1764.35X1 + 1240.50X2 - 456.92X1X2 - 2540.71X1 2 -2967.64X2 2 The fit is good (R). 2 =0.9392), the model was significant (P=0.0004), and the lack-of-fit term was not significant (P=0.2664); fluorescence intensity Y2=650945+22513.63X1+23721.67X2-6711.75X1X2-46796.54X1 2 -45454.90X2 2 The fit is good (R). 2 The model was highly significant (P < 0.0001) with a non-significant lack-of-fit term (P = 0.0770), indicating that the model is reliable and can accurately interpret and predict experimental results.

[0036] Using Design-Expert 13 software, 3D effect surface plots and contour plots of the interactions between various factors on fluorescence area and fluorescence intensity were generated, such as... Figure 2 As shown. The interaction strength of relevant factors was confirmed, and considering all relevant factors, the optimal synergistic concentrations were predicted to be xylitol 29.803 μM and nisose 28.780 μM, with a synergistic ratio of approximately 1:1. Based on the actual process and considering the ease of operation in drug weighing and preparing the stock solution, this invention appropriately rounded down the theoretically optimal concentrations, determining the concentrations for subsequent osteoporosis verification experiments to be xylitol 30 μM and nisose 29 μM.

[0037] Table 3. Results of analysis of variance for fluorescence area

[0038] Table 4. Results of analysis of variance for fluorescence intensity

[0039] Example 5: Verification of the optimal synergistic concentration of xylotetraose and nisose Wild-type AB strain and transgenic osteoblast green fluorescent Tg (0la.Sp7;nlsGEP) strain zebrafish larvae were fertilized and developed for 72 hpf. Normally developing larvae were selected under a stereomicroscope and transferred into 6-well plates, 30 larvae per well. The following groups were established: normal group (zebrafish embryo culture water), model group (30 μM prednisolone), etidronate disodium group (30 μM prednisolone + 120 μM etidronate disodium), xylooligosaccharide control group [30 μM prednisolone + 27.31 μg / mL (25.99 μM ~ 90.93 μM) xylooligosaccharide], xylotetrasaccharide group (30 μM prednisolone + 50 μM xylotetrasaccharide), nysaccharide group (30 μM prednisolone + 50 μM nysaccharide), and disaccharide synergistic group (30 μM prednisolone + 50 μM nysaccharide). (μM prednisolone + 30μM xylotetraose + 29μM nisose), with 3 replicate wells per group. Add 5 mL of the corresponding drug solution to each well and immerse in a constant temperature incubator at 28±0.5℃ for 144 h. Replace half of the drug solution every day during this period.

[0040] It should be noted that xylooligosaccharides are a mixture, and this invention uses mass concentration as the expression form. The range of molar concentration is indicated according to the range of molar mass. The average molar concentration in the xylooligosaccharide control group is about 50 μM.

[0041] 1. Alizarin staining in zebrafish (1) Fixation: After the wild-type AB strain zebrafish were treated with drugs, 6 zebrafish were randomly selected from each group, anesthetized with 0.02% tricaine solution, and then fixed with 4% paraformaldehyde at room temperature for 2 hours.

[0042] (2) Bleaching: After discarding the fixative, wash three times with PBST and immerse in a bleaching agent containing 0.5% KOH and 3% H2O2 for 30 min to clear.

[0043] (3) Refixation: After discarding the bleach, wash 3 times with PBST and fix overnight at 4°C with 4% paraformaldehyde.

[0044] (4) Dehydration: The next day, discard the fixative, wash 3 times with PBST, and dehydrate in 50% methanol (prepared with PBS) and 100% methanol for 5 min each time. Finally, replace with fresh 100% methanol and place at -20℃ for 2 h.

[0045] (5) Rehydration: Discard the methanol and wash and rehydrate with a gradient of 100% - 75% - 50% - 25% methanol for 5 minutes each time. After the washing is completed, wash with PBST 3 times.

[0046] (6) Staining: After cleaning, place the zebrafish in 0.01% alizarin red staining solution (prepared with 0.5% KOH) and stain at room temperature in the dark for 1 hour. Observe whether the zebrafish are stained.

[0047] (7) Wash away the excess stain: Discard the staining solution and wash three times with PBST.

[0048] (8) Storage: After cleaning, place in 80% glycerin and store at -20℃ until photographed.

[0049] (9) Photographs: Observe and collect stained images of the head bones under white light using a fluorescence stereomicroscope.

[0050] (10) Data statistics: Image J 1.8.0 software was used to quantitatively analyze the staining area and staining intensity of the bone regions in each group.

[0051] 2. Calcein staining of zebrafish (1) Staining: After the wild-type AB strain zebrafish were treated with drugs, 6 zebrafish were randomly selected from each group and transferred to zebrafish embryo culture water containing 0.1% calcein. They were then immersed in the water for 30 minutes in the dark for staining.

[0052] (2) Cleaning and anesthesia: After staining, the zebrafish were cleaned three times with embryo culture water and anesthetized with 0.02% tricaine solution.

[0053] (3) Photographs: Observe and collect green fluorescent images of the skeletal system of the spine under the fluorescence of a fluorescence stereomicroscope.

[0054] (4) Data statistics: The fluorescence intensity of the bone region was quantitatively analyzed using Image J 1.8.0 software.

[0055] 3. Determination of biochemical indicators related to zebrafish bone metabolism After drug treatment of wild-type AB strain zebrafish, 30 zebrafish from each group were anesthetized in an appropriate amount of 0.02% tricaine solution, rinsed three times with purified water, and the water was removed. The zebrafish from each group were added to PBS at a weight (g):volume (mL) ratio of 1:9, homogenized at 4℃, and then centrifuged at 2500 r / min for 10 min. The supernatant was collected. A portion of the supernatant was diluted 10-fold, and protein concentration was determined using the Beyotime BCA protein assay kit for subsequent protein concentration correction. The remaining supernatant was used according to the kit instructions to determine the activities of alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP).

[0056] 4. Analysis of osteoblast differentiation in zebrafish After drug treatment of zebrafish with transgenic osteoblast green fluorescent Tg (0la.Sp7;nlsGEP) strains, six zebrafish from each group were randomly selected and anesthetized in 0.02% tricaine solution, then fixed on glass slides using 4% methylcellulose solution. Green fluorescent images of osteoblasts in the zebrafish skull region were systematically observed and acquired under a fluorescence stereomicroscope. ImageJ 1.8.0 software was used for quantitative analysis of the fluorescence area and intensity.

[0057] 5. Validation of the synergistic anti-osteoporosis efficacy of xylotetraose and nisose. 5.1 Effects of xylotetrasaccharide synergistic with nyssose on cumulative bone mineralization in zebrafish To evaluate the anti-osteoporosis effects of xylotetraose and its synergistic effect with nyssose, this invention analyzed the cumulative bone mineralization in zebrafish using alizarin red staining. The results are as follows: Figure 3 As shown, compared with the normal group, the alizarin red staining area and intensity of zebrafish in the model group were significantly reduced, indicating that prednisolone successfully induced the zebrafish osteoporosis model. In the drug intervention groups, the bone staining area and intensity of the etidronate disodium group were significantly increased compared with the model group, verifying the reliability of the experimental system. The staining degree of zebrafish in the xylooligosaccharide group did not change significantly compared with the model group, indicating that its bone mineralization was not significantly improved; the staining area and intensity of zebrafish in the xylo-tetrasaccharide and nisose groups were both increased compared with the model group, indicating that xylo-tetrasaccharide and nisose can significantly improve the bone mineralization of zebrafish. It is noteworthy that the synergistic effect of the disaccharide combination of xylo-tetrasaccharide and nisose showed significantly higher alizarin red staining area and intensity than the model group, and was also significantly better than the groups treated with each sugar alone, demonstrating the most significant effect in promoting bone mineralization. The above results indicate that both xylo-tetraose and nisose have certain effects on promoting bone mineralization, and when used in combination, they show a synergistic enhancement effect in promoting bone mineralization, with significantly better efficacy than when used alone, proving that the combined administration of the two can effectively enhance anti-osteoporosis activity.

[0058] 5.2 Effects of xylotetrasaccharide and nysaccharide on the mineralization rate of zebrafish skeleton To investigate the synergistic effect of xylotetraose and nysose on bone formation dynamics, this invention uses calcein staining to evaluate the rate of new bone mineralization in zebrafish. The results are as follows: Figure 4 As shown, compared with the normal group, the fluorescence intensity of calcein in the spinal region of zebrafish in the model group was significantly reduced, indicating that prednisolone successfully inhibited the rate of new bone deposition. In contrast, the etidronate disodium group showed a significant reversal effect, with its fluorescence intensity significantly enhanced compared with the model group, indicating that it promotes bone mineralization and accelerates the mineralization rate. In the drug intervention groups, the fluorescence intensity of zebrafish treated with xylooligosaccharides did not change significantly compared with the model group, while xylo-tetrasaccharide or nisose treatment alone could increase the fluorescence intensity to some extent. However, the synergistic effect of xylo-tetrasaccharide and nisose combined showed the strongest mineralization-promoting effect, with its fluorescence intensity not only significantly higher than the model group but also significantly better than the individual sugar treatment groups. This indicates that the synergistic effect of the two can most effectively accelerate the bone mineralization process. The above results demonstrate that both xylo-tetrasaccharide and nisose can promote the rate of bone mineralization, and when used in combination, they show a clear synergistic effect in promoting the rate of bone mineralization, with a significantly better effect than single administration.

[0059] 5.3 Effects of xylotetrasaccharide and nysaccharide on biochemical indicators related to bone metabolism in zebrafish ALP is an important marker of osteoblast differentiation and activity, and its activity level directly reflects bone formation capacity; while TRAP is a characteristic enzyme of osteoclasts, and its activity level reflects bone resorption status. Together, they constitute key indicators for evaluating bone metabolic homeostasis. Figure 5 As shown, compared with the normal group, the ALP activity in the model group of zebrafish was significantly reduced, while the TRAP activity was significantly increased, indicating that prednisolone successfully induced bone metabolism disorder, resulting in a pathological state of obvious bone formation inhibition and enhanced bone resorption. The etidronate disodium group showed a significant regulatory effect, with ALP activity significantly increased compared to the model group, while TRAP activity was significantly reduced. Except for the xylooligosaccharide group, the ALP and TRAP activities of all drug treatment groups were improved compared to the model group. Among them, the synergistic disaccharide group showed significantly higher ALP activity than the model group and significantly reduced TRAP activity, which was significantly better than the xylo-tetrasaccharide group and the nisose group. These results indicate that the synergistic administration of xylo-tetrasaccharide and nisose most effectively promotes bone formation and inhibits bone resorption, and is superior to the individual sugar administration groups in regulating bone metabolism balance, showing the best effect.

[0060] 5.4. Effects of xylotetrasaccharide and nysaccharide on osteoblast differentiation in zebrafish This invention utilizes a transgenic osteoblast fluorescent zebrafish model to assess osteoblast activity by quantitatively analyzing fluorescence signals in the skull region. Results are as follows: Figure 6As shown, compared with the normal group, the fluorescence area and intensity of the zebrafish skull region in the model group were significantly reduced, indicating that prednisolone significantly inhibited osteoblast differentiation activity. The fluorescence area and intensity of the etidronate disodium group were significantly enhanced compared with the model group, indicating that it promotes osteoblast differentiation. Except for the xylooligosaccharide group, whose fluorescence indicators showed no significant change compared with the model group, the fluorescence indicators of all other treatment groups were improved compared with the model group. Among them, the fluorescence area and intensity of the disaccharide synergistic group were significantly higher than those of the model group, and significantly better than those of the xylotetraose and nisose groups, suggesting that it had the most significant effect in promoting osteoblast differentiation. These results indicate that both xylotetraose and nisose promote osteoblast differentiation, and the disaccharide synergistic group has the best effect in promoting osteoblast differentiation, proving that the synergistic administration of xylotetraose and nisose has a better effect on promoting bone formation.

[0061] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The use of xylo-tetraose or a combination thereof with nisose in the preparation of products for treating osteoporosis.

2. The application as described in claim 1, characterized in that, The dosage of xylotetraose is 6.25~50 μM.

3. The application as described in claim 1, characterized in that, In the composition, the concentration of xylotetraose is 6.25~50 μM, and the concentration of nysose is 6.25~50 μM.

4. The application as described in claim 1, characterized in that, In the composition, the dosage concentration of xylotetraose is 30 μM and the dosage concentration of nisose is 29 μM.

5. The application as described in claim 1, characterized in that, The medicinal effects include at least one of the following: a) Enhances bone mineralization; b) Accelerates the mineralization rate; c) Inhibits bone resorption; d) Promotes osteoblast differentiation; e) Enhance osteoblast activity.

6. The application as described in claim 1, characterized in that, The product is a pharmaceutical or laboratory reagent.

7. A drug for treating osteoporosis, characterized in that, The active ingredients include xylo-tetraose or a combination thereof with nysose.

8. The anti-osteoporosis drug as described in claim 7, characterized in that, The dosage of xylotetraose is 6.25~50 μM.

9. The anti-osteoporosis drug as described in claim 7, characterized in that, In the composition, the concentration of xylitol is 6.25-50 μM and the concentration of nyssose is 6.25-50 μM. Alternatively, the dosage of xylotetraose is 30 μM, and the dosage of nisose is 29 μM.

10. The anti-osteoporosis drug as described in claim 7, characterized in that, Also includes: Auxiliary materials.

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