Preparation method of morinda officinalis polysaccharide and application thereof in anti-osteoporosis drugs

Abstract

The application belongs to the technical field of biology and particularly relates to a preparation method of polysaccharide in Millettia pinnata and application thereof in anti-osteoporosis drugs. The method comprises the following steps: defatting Millettia pinnata powder by reflux to obtain defatted Millettia pinnata powder; mixing the defatted Millettia pinnata powder with pure water, performing enzymolysis by ultrasonic-assisted enzyme method, and performing filtration to obtain a filtrate; evaporating and concentrating the filtrate, adding anhydrous ethanol for alcohol precipitation, and performing freeze-drying to obtain crude polysaccharide in Millettia pinnata; removing protein and decolorizing the crude polysaccharide, and taking supernatant; performing dialysis on the obtained supernatant, adding anhydrous ethanol for alcohol precipitation, and performing freeze-drying to obtain polysaccharide in Millettia pinnata. The preparation method realizes efficient extraction and separation of polysaccharide in Millettia pinnata, guarantees relatively high growth inhibition activity of osteoclasts and growth promotion activity of osteoblasts, and realizes bidirectional metabolic regulation of anti-osteoporosis.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a preparation method of millettia speciosa polysaccharide and its application in anti-osteoporosis drugs. Background Art

[0002] As a medicinal and edible Chinese herbal medicine, millettia speciosa has the effects of treating arthritis and bronchitis, protecting the liver, strengthening muscles and bones, tonifying deficiency and moistening the lungs, and these medicinal effects are closely related to active ingredients such as flavonoids and polysaccharides therein. Research shows that millettia speciosa polysaccharide has an immunomodulatory effect, can stimulate the activity of the immune system, and helps to enhance the resistance of the body; millettia speciosa polysaccharide has anti-inflammatory properties, which has certain benefits for relieving inflammatory symptoms and diseases; millettia speciosa polysaccharide is a natural antioxidant, which can help combat the damage of free radicals, thus contributing to maintaining cell health. To sum up, although there are some reports on the pharmacological effects of millettia speciosa polysaccharide, its pharmacological effect of inhibiting the growth of osteoclasts and promoting the growth of osteoblasts, thereby being able to prevent and treat osteoporosis, has not been reported yet.

[0003] At present, there are few research reports on the extraction, separation and purification of millettia speciosa polysaccharide. Therefore, preparing refined millettia speciosa polysaccharide with uniform molecular weight through a series of separation and purification techniques and evaluating its activity can provide a scientific basis and theoretical guidance for the in-depth development and utilization of millettia speciosa polysaccharide. Summary of the Invention

[0004] The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide a preparation method of millettia speciosa polysaccharide to achieve high extraction efficiency and purity of millettia speciosa polysaccharide, and ensure its high activity of inhibiting the growth of osteoclasts and promoting the growth of osteoblasts, and achieve the two-way metabolic regulation of anti-osteoporosis.

[0005] To achieve the above purpose, the technical solutions adopted by the present invention include:

[0006] In the first aspect, the present invention provides a preparation method of millettia speciosa polysaccharide, including the following steps:

[0007] (1) Degreasing: performing degreasing treatment on millettia speciosa powder;

[0008] (2) Ultrasonic-assisted enzymatic treatment: mixing degreased millettia speciosa powder with pure water, ultrasonicating, and simultaneously adding an enzyme for enzymatic hydrolysis, then filtering by suction to obtain a filtrate;

[0009] (3) Ethanol precipitation: concentrating the filtrate obtained in step (2), then adding anhydrous ethanol, centrifuging, and freeze-drying the precipitate obtained by centrifugation to obtain a crude product of millettia speciosa polysaccharide;

[0010] (4) Polysaccharide refining: The crude polysaccharide obtained in step (3) is subjected to protein removal, decolorization, and dialysis with alcohol precipitation to obtain polysaccharide.

[0011] The method for preparing *Achyranthes bidentata* polysaccharide described in this invention involves treating *Achyranthes bidentata* with ultrasound-assisted enzymatic hydrolysis, followed by a series of purification steps including alcohol precipitation, protein removal, decolorization, and dialysis, achieving high extraction efficiency. Due to the dense structure of *Achyranthes bidentata*, traditional physical methods are insufficient for efficiently separating and extracting the desired polysaccharide. Ultrasound-assisted enzymatic hydrolysis is a highly efficient and environmentally friendly polysaccharide extraction method. Enzyme treatment ensures specific hydrolysis of the polysaccharide, avoiding the influence of other impurities during purification. Ultrasound increases the contact area between the polysaccharide and the enzyme, promoting the hydrolysis process and significantly improving the extraction efficiency. Furthermore, the heat generated by ultrasound is relatively small and does not significantly damage the structure and activity of the polysaccharide, thus further enhancing the extraction efficiency of *Achyranthes bidentata* polysaccharide.

[0012] Preferably, in step (3), the ultrasonic treatment time is 20-40 min and the ultrasonic power is 300-400 W.

[0013] Preferably, in step (3), the enzymatic hydrolysis time is 40-80 min and the enzymatic hydrolysis temperature is 65-75℃.

[0014] In ultrasound-assisted enzymatic hydrolysis, the ultrasound time, ultrasound power, enzyme selection, hydrolysis temperature, and hydrolysis time all significantly affect the optimal extraction yield and polysaccharide quality. Longer ultrasound times generally lead to more thorough polysaccharide separation, but may also cause degradation or structural alterations. Higher ultrasound power can separate polysaccharides more quickly, but may also cause temperature increases, thus increasing the risk of polysaccharide degradation. Too short a hydrolysis time may result in incomplete hydrolysis, while too long a hydrolysis time may lead to polysaccharide degradation. Appropriate hydrolysis temperature helps maintain enzyme activity, but it is also important to avoid high temperatures that could lead to enzyme inactivation or the heat sensitivity of polysaccharides. Therefore, it is necessary to appropriately adjust and optimize the ultrasound time, ultrasound power, hydrolysis time, and hydrolysis temperature during ultrasound-assisted enzymatic hydrolysis to balance the polysaccharide purification effect and polysaccharide integrity. Through experimentation, the inventors of this application discovered that when amylase is selected for enzymatic hydrolysis of *Achyranthes bidentata*, and the ultrasonic time is 20-40 min, the ultrasonic power is 300-400 W, the hydrolysis time is 40-80 min, and the hydrolysis temperature is 65-75℃, the optimal hydrolysis effect can be achieved, thereby obtaining high-purity, high-quality *Achyranthes bidentata* polysaccharide, while minimizing polysaccharide loss.

[0015] Preferably, in step (3), the enzyme is an amylase, and the mass of the enzyme is 1%-4% of the mass of the defatted Niu Dali powder.

[0016] Preferably, in step (4), the specific steps for removing protein are as follows: dissolve the crude *Smilax glabra* polysaccharide obtained in step (3) in water, add Sevag reagent, stir and centrifuge to obtain supernatant; the mass ratio of the crude *Smilax glabra* polysaccharide to Sevag reagent is 5:1.

[0017] Sevag reagent consists of an organic phase and an aqueous phase. The Sevag method has a good protein removal effect on crude polysaccharide because proteins are generally more hydrophilic and easily soluble in the aqueous phase, while polysaccharides tend to dissolve in the organic phase, thus achieving effective separation of polysaccharides and proteins. Furthermore, the Sevag method has little impact on the stability of polysaccharides during protein removal, allowing the structure and properties of polysaccharides to be preserved. However, the mass ratio of crude polysaccharide to Sevag reagent affects the protein removal effect of the Sevag method, thereby affecting the purity of the final polysaccharide. Experimental studies have shown that when the mass ratio of crude *Smilax glabra* polysaccharide to Sevag reagent is 5:1, the purified *Smilax glabra* polysaccharide has higher purity and quality.

[0018] Preferably, in step (4), the specific steps of decolorization are as follows: add polyamide to the supernatant obtained after removing protein, stir to decolorize, centrifuge, and obtain supernatant; the volume ratio of supernatant to polyamide is 2:1-6:1.

[0019] Polyamides exhibit high adsorption selectivity, allowing for the selective adsorption of specific components in polysaccharides by adjusting pH and temperature. This selectively removes certain impurities and pigments without affecting the quality and properties of the polysaccharide itself. Furthermore, polyamides are easy to operate and, compared to other purification methods, are a relatively economical and efficient decolorizing agent. They effectively improve the purity of polysaccharide products without requiring expensive equipment or reagents. The volume ratio of supernatant to polyamide directly affects the decolorization effect, thus influencing the purity of the final polysaccharide. If the volume ratio is too low, the polyamide's adsorption capacity may be insufficient, failing to effectively remove all pigments and impurities. Conversely, a high volume ratio may dilute the polysaccharide components in the supernatant, wasting polyamide and increasing processing costs. The inventors discovered that a volume ratio of supernatant to polyamide of 2:1 to 6:1 achieves the best decolorization effect while maximizing the preservation of the quality and purity of *Euphorbia lathyris* polysaccharide.

[0020] Preferably, in step (4), the specific steps of dialysis and alcohol precipitation are as follows: the supernatant obtained after decolorization is dialyzed, the liquid in the dialysis bag is taken, anhydrous ethanol is added, centrifuged, and the resulting precipitate is freeze-dried to obtain *Euphorbia lactea* polysaccharide;

[0021] The dialysis bags used in the dialysis have a cutoff molecular weight of 8000-14000 Da, and the dialysis time is 40-50 hours.

[0022] Dialysis is a relatively gentle separation method based on molecular size, which is very effective in selectively removing small molecule impurities and does not require high temperatures or organic solvents, thus helping to maintain the stability and bioactivity of polysaccharides. During dialysis, the cutoff molecular weight of the dialysis bag and the dialysis time have a certain impact on the purity of polysaccharides. If the cutoff molecular weight is too low or the dialysis time is too short, small molecule impurities cannot be completely removed, affecting the purity of the final polysaccharide. On the other hand, if the cutoff molecular weight is too high or the dialysis time is too long, polysaccharide loss may occur. Therefore, it is necessary to balance the need to remove impurities and retain polysaccharides to the greatest extent. Through experimental research, the inventors found that when the cutoff molecular weight of the dialysis bag is 8000-14000 Da and the dialysis time is 40-50 h, it is possible to ensure the acquisition of high-quality and high-purity *Euphorbia lathyris* polysaccharides.

[0023] Secondly, the present invention provides the application of the *Smilax glabra* polysaccharide prepared by the above preparation method in the preparation of anti-osteoporosis drugs.

[0024] Preferably, the *Smilax glabra* polysaccharide is used to inhibit osteoclast growth and promote osteoblast growth.

[0025] Preferably, the *Smilax glabra* polysaccharide is used to inhibit TRAP activity and promote ALP activity.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0027] 1. The polysaccharide of *Smilax glabra* provided by this invention is prepared by using *Smilax glabra* as raw material through ultrasonic-assisted enzymatic hydrolysis, protein removal, decolorization, dialysis and other methods, which realizes the efficient separation and purification of *Smilax glabra* polysaccharide and ensures its high biological activity.

[0028] 2. The *Niu Dali* polysaccharide provided by this invention has been shown to inhibit osteoclast growth and promote osteoblast growth without any toxic side effects on macrophage cell lines differentiated into osteoclasts and osteoblasts. This enables the prevention and treatment of osteoporosis through bidirectional metabolic regulation, filling the gap in the field of anti-osteoporosis application of *Niu Dali* polysaccharide and expanding its application scope. Attached Figure Description

[0029] Figure 1 The glucose standard curve was plotted for the determination of the polysaccharide content of *Achyranthes bidentata* in Example 1.

[0030] Figure 2 The graph shows the toxic effects of different concentrations of *Smilax glabra* polysaccharide prepared in Example 1 on RAW264.7 macrophages.

[0031] Figure 3 The graph shows the toxic effects of *Smilax glabra* polysaccharide prepared in Example 1 on MC3T3-E1 cells at different concentrations.

[0032] Figure 4 Figure showing the effect of polysaccharides from Millettia speciosa Champ. prepared in Example 1 on the TRAP enzyme activity of osteoclasts at different concentrations;

[0033] Figure 5 Figure showing the effect of polysaccharides from Millettia speciosa Champ. prepared in Example 1 on the ALP enzyme activity of osteoblasts at different concentrations. Detailed implementation manners

[0034] To better illustrate the purpose, technical solution and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. Those skilled in the art should understand that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0035] Unless otherwise specified, the test methods used in the examples are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, can all be obtained from commercial channels.

[0036] Example 1

[0037] This example provides a preparation method of polysaccharides from Millettia speciosa Champ., and the specific steps of the method are as follows:

[0038] (1) Crushing: Crushing the dried Millettia speciosa Champ. and sieving to obtain Millettia speciosa Champ. powder;

[0039] (2) Defatting: Refluxing and defatting the Millettia speciosa Champ. powder with 10 times the amount of absolute ethanol, filtering, evaporating to remove the absolute ethanol, and drying to obtain defatted Millettia speciosa Champ. powder;

[0040] (3) Ultrasonic-assisted enzymatic treatment: Mixing the defatted Millettia speciosa Champ. powder with pure water at a solid-liquid ratio of 1:15 (w / v), ultrasonicating for 30 min with an ultrasonic power of 350 w, adding 2.5% of amylase based on the mass of the defatted Millettia speciosa Champ. powder, enzymolyzing at 70 °C for 60 min, and filtering to obtain a filtrate;

[0041] (4) Ethanol precipitation: Evaporating the filtrate, concentrating the volume to 20% of the original volume, then adding 4 times the amount of absolute ethanol, refrigerating overnight, centrifuging, and freeze-drying the obtained precipitate to obtain crude polysaccharides from Millettia speciosa Champ.;

[0042] (5) Protein removal: Dissolving the crude polysaccharides from Millettia speciosa Champ. in water, adding Sevag reagent, with a mass ratio of the crude polysaccharides from Millettia speciosa Champ. to Sevag reagent of 5:1, stirring and then centrifuging, and taking the supernatant;

[0043] (6) Decolorization: Adding polyamide to the supernatant obtained in step (5), with a volume ratio of the supernatant to polyamide of 4:1, stirring and decolorizing at 50 °C for 30 min, centrifuging, and taking the supernatant;

[0044] (7) Dialysis and ethanol precipitation: Place the supernatant obtained in step (6) into a dialysis bag with a cut-off molecular weight of 10,000 Da, dialyze for 48 h, take the liquid in the dialysis bag, add absolute ethanol, refrigerate overnight, centrifuge, and freeze-dry the obtained precipitate to obtain polysaccharide from Millettia speciosa Champ.

[0045] Example 2

[0046] This example provides a method for preparing polysaccharide from Millettia speciosa Champ., and the specific steps of this method are as follows:

[0047] (1) Crushing: Crush the dried Millettia speciosa Champ., sieve it, and obtain Millettia speciosa Champ. powder;

[0048] (2) Degreasing: Reflux and degrease the Millettia speciosa Champ. powder with 10 times the amount of absolute ethanol, filter, evaporate to remove absolute ethanol, and dry it to obtain degreased Millettia speciosa Champ. powder;

[0049] (3) Ultrasonic-assisted enzymatic treatment: Mix the degreased Millettia speciosa Champ. powder and pure water at a solid-liquid ratio of 1:15 (w / v), ultrasonicate for 20 min, the ultrasonic power is 400 w, add 4% of amylase based on the mass of the degreased Millettia speciosa Champ. powder, enzymatically hydrolyze at 65 °C for 80 min, and filter by suction to obtain a filtrate;

[0050] (4) Ethanol precipitation: Evaporate the filtrate, concentrate the volume to 20% of the original volume, then add 4 times the amount of absolute ethanol, refrigerate overnight, centrifuge, and freeze-dry the obtained precipitate to obtain crude polysaccharide from Millettia speciosa Champ.;

[0051] (5) Protein removal: Dissolve the crude polysaccharide from Millettia speciosa Champ. in water, add Sevag reagent, and the mass ratio of the crude polysaccharide to Sevag reagent is 5:1, stir and then centrifuge, and take the supernatant;

[0052] (6) Decolorization: Add polyamide to the supernatant obtained in step (5), and the volume ratio of the supernatant to polyamide is 2:1, stir and decolorize at 50 °C for 30 min, centrifuge, and take the supernatant;

[0053] (7) Dialysis and ethanol precipitation: Place the supernatant obtained in step (6) into a dialysis bag with a cut-off molecular weight of 8,000 Da, dialyze for 40 h, take the liquid in the dialysis bag, add absolute ethanol, refrigerate overnight, centrifuge, and freeze-dry the obtained precipitate to obtain polysaccharide from Millettia speciosa Champ..

[0054] Example 3

[0055] This example provides a method for preparing polysaccharide from Millettia speciosa Champ., and the specific steps of this method are as follows:

[0056] (1) Crushing: Crush the dried Millettia speciosa Champ., sieve it, and obtain Millettia speciosa Champ. powder;

[0057] (2) Defatting: Defat the Millettia speciosa Champ. powder with 10 times the amount of absolute ethanol by reflux, filter, evaporate to remove the absolute ethanol, dry, and obtain defatted Millettia speciosa Champ. powder;

[0058] (3) Ultrasonic-assisted enzymatic treatment: Mix the defatted Millettia speciosa Champ. powder and pure water at a solid-liquid ratio of 1:15 (w / v), ultrasonicate for 40 min with an ultrasonic power of 300 w, add 1% of amylase based on the mass of the defatted Millettia speciosa Champ. powder, enzymatically hydrolyze at 75 °C for 40 min, and filter by suction to obtain a filtrate;

[0059] (4) Ethanol precipitation: Evaporate the filtrate, concentrate the volume to 20% of the original volume, then add 4 times the amount of absolute ethanol, refrigerate overnight, centrifuge, and freeze-dry the obtained precipitate to obtain crude Millettia speciosa Champ. polysaccharide;

[0060] (5) Protein removal: Dissolve the crude Millettia speciosa Champ. polysaccharide in water, add Sevag reagent, with a mass ratio of the crude polysaccharide to Sevag reagent of 5:1, stir and then centrifuge, and take the supernatant;

[0061] (6) Decolorization: Add polyamide to the supernatant obtained in step (5), with a volume ratio of the supernatant to polyamide of 6:1, stir and decolorize at 50 °C for 30 min, centrifuge, and take the supernatant;

[0062] (7) Dialysis and ethanol precipitation: Place the supernatant obtained in step (6) in a dialysis bag with a cut-off molecular weight of 14000 Da, dialyze for 50 h, take the liquid in the dialysis bag, add absolute ethanol, refrigerate overnight, centrifuge, and freeze-dry the obtained precipitate to obtain Millettia speciosa Champ. polysaccharide.

[0063] Example 4

[0064] This example provides a method for preparing Millettia speciosa Champ. polysaccharide, and the specific steps of this method are as follows:

[0065] (1) Crushing: Crush the dried Millettia speciosa Champ., sieve, and obtain Millettia speciosa Champ. powder;

[0066] (2) Defatting: Defat the Millettia speciosa Champ. powder with 10 times the amount of absolute ethanol by reflux, filter, evaporate to remove the absolute ethanol, dry, and obtain defatted Millettia speciosa Champ. powder;

[0067] (3) Ultrasonic-assisted enzymatic treatment: Mix the defatted Millettia speciosa Champ. powder and pure water at a solid-liquid ratio of 1:15 (w / v), ultrasonicate for 30 min with an ultrasonic power of 350 w, add 1% of amylase based on the mass of the defatted Millettia speciosa Champ. powder, enzymatically hydrolyze at 65 °C for 80 min, and filter by suction to obtain a filtrate;

[0068] (4) Ethanol precipitation: Evaporate the filtrate, concentrate the volume to 20% of the original volume, then add 4 times the amount of absolute ethanol, refrigerate overnight, centrifuge, and freeze-dry the obtained precipitate to obtain crude Millettia speciosa Champ. polysaccharide;

[0069] (5) Protein removal: Dissolve the crude polysaccharide of Millettia speciosa Champ. in water, add Sevag reagent, and the mass ratio of the crude polysaccharide to Sevag reagent is 5:1. After stirring, centrifuge and take the supernatant;

[0070] (6) Decolorization: Add polyamide to the supernatant obtained in step (5), and the volume ratio of the supernatant to polyamide is 4:1. Stir and decolorize at 50 °C for 30 min, centrifuge, and take the supernatant;

[0071] (7) Dialysis and alcohol precipitation: Place the supernatant obtained in step (6) in a dialysis bag with a cut-off molecular weight of 10000 Da, dialyze for 48 h, take the liquid in the dialysis bag, add absolute ethanol, refrigerate overnight, centrifuge, and freeze-dry the obtained precipitate to obtain Millettia speciosa Champ. polysaccharide.

[0072] Example 5

[0073] This example provides a preparation method of Millettia speciosa Champ. polysaccharide, and the specific steps of this method are as follows:

[0074] (1) Crushing: Crush the dried Millettia speciosa Champ., sieve it, and obtain Millettia speciosa Champ. powder;

[0075] (2) Defatting: Reflux and defat the Millettia speciosa Champ. powder with 10 times the amount of absolute ethanol, filter, evaporate to remove absolute ethanol, and dry it to obtain defatted Millettia speciosa Champ. powder;

[0076] (3) Ultrasonic-assisted enzymatic treatment: Mix the defatted Millettia speciosa Champ. powder and pure water at a solid-liquid ratio of 1:15 (w / v), ultrasonic for 30 min, the ultrasonic power is 350 w, add 2.5% of amylase based on the mass of the defatted Millettia speciosa Champ. powder, enzymatically hydrolyze at 70 °C for 60 min, and filter to obtain the filtrate;

[0077] (4) Ethanol precipitation: Evaporate the filtrate, concentrate the volume to 20% of the original volume, then add 4 times the amount of absolute ethanol, refrigerate overnight, centrifuge, and freeze-dry the obtained precipitate to obtain the crude polysaccharide of Millettia speciosa Champ.;

[0078] (5) Protein removal: Dissolve the crude polysaccharide of Millettia speciosa Champ. in water, add Sevag reagent, and the mass ratio of the crude polysaccharide to Sevag reagent is 5:1. After stirring, centrifuge and take the supernatant;

[0079] (6) Decolorization: Add polyamide to the supernatant obtained in step (5), and the volume ratio of the supernatant to polyamide is 6:1. Stir and decolorize at 50 °C for 30 min, centrifuge, and take the supernatant;

[0080] (7) Dialysis and alcohol precipitation: Place the supernatant obtained in step (6) in a dialysis bag with a cut-off molecular weight of 8000 Da, dialyze for 40 h, take the liquid in the dialysis bag, add absolute ethanol, refrigerate overnight, centrifuge, and freeze-dry the obtained precipitate to obtain Millettia speciosa Champ. polysaccharide.

[0081] Comparative Example 1

[0082] This comparative example provides a method for preparing polysaccharide from Millettia speciosa Champ. The difference between this comparative example and Example 1 is as follows:

[0083] In step (3), only ultrasonic treatment is used: the ultrasonic time is 30 min and the ultrasonic power is 350 w.

[0084] Comparative Example 2

[0085] This comparative example provides a method for preparing polysaccharide from Millettia speciosa Champ. The difference between this comparative example and Example 1 is as follows:

[0086] In step (3), only enzymatic treatment is used: adding 2.5% of amylase based on the mass of defatted Millettia speciosa Champ. powder and carrying out enzymatic hydrolysis at 70 °C for 60 min.

[0087] Comparative Example 3

[0088] This comparative example provides a method for preparing polysaccharide from Millettia speciosa Champ. The difference between this comparative example and Example 1 is as follows:

[0089] In step (3), the specific conditions of ultrasonic-assisted enzymatic treatment are: the ultrasonic time is 10 min, the ultrasonic power is 450 w, adding 5% of amylase based on the mass of defatted Millettia speciosa Champ. powder, and carrying out enzymatic hydrolysis at 80 °C for 30 min.

[0090] Comparative Example 4

[0091] This comparative example provides a method for preparing polysaccharide from Millettia speciosa Champ. The difference between this comparative example and Example 1 is as follows:

[0092] In step (3), the specific conditions of ultrasonic-assisted enzymatic treatment are: the ultrasonic time is 50 min, the ultrasonic power is 250 w, adding 0.5% of amylase based on the mass of defatted Millettia speciosa Champ. powder, and carrying out enzymatic hydrolysis at 60 °C for 90 min.

[0093] Comparative Example 5

[0094] This comparative example provides a method for preparing polysaccharide from Millettia speciosa Champ. The difference between this comparative example and Example 1 is as follows:

[0095] In step (3), the enzyme used is cellulase.

[0096] Comparative Example 6

[0097] This comparative example provides a method for preparing polysaccharide from Millettia speciosa Champ. The specific steps of this method are as follows:

[0098] (1) Crushing: Crushing the dried Millettia speciosa Champ. and sieving it to obtain Millettia speciosa Champ. powder;

[0099] (2) Defatting: Defat the Millettia speciosa Champ. powder by refluxing it with 10 times the amount of absolute ethanol, filter, evaporate to remove the absolute ethanol, dry it, and obtain defatted Millettia speciosa Champ. powder;

[0100] (3) Ultrasonic-assisted enzymatic treatment: Mix the defatted Millettia speciosa Champ. powder with pure water at a solid-liquid ratio of 1:15 (w / v), ultrasonicate for 30 min with an ultrasonic power of 350 w, add 2.5% of amylase based on the mass of the defatted Millettia speciosa Champ. powder, enzymatically hydrolyze at 70 °C for 60 min, and filter by suction to obtain a filtrate;

[0101] (4) Ethanol precipitation: Evaporate the filtrate, concentrate the volume to 20% of the original volume, then add 4 times the amount of absolute ethanol, refrigerate overnight, centrifuge, and freeze-dry the obtained precipitate to obtain crude Millettia speciosa Champ. polysaccharide;

[0102] (5) Protein removal: Dissolve the crude Millettia speciosa Champ. polysaccharide in water, add Sevag reagent, with the mass ratio of the crude polysaccharide to Sevag reagent being 3:1, stir and then centrifuge, and take the supernatant;

[0103] (6) Decolorization: Add polyamide to the supernatant obtained in step (5), with the volume ratio of the supernatant to polyamide being 7:1, stir and decolorize at 50 °C for 30 min, centrifuge, and take the supernatant;

[0104] (7) Dialysis and ethanol precipitation: Place the supernatant obtained in step (6) in a dialysis bag with a cut-off molecular weight of 7000 Da, dialyze for 35 h, take the liquid in the dialysis bag, add absolute ethanol, refrigerate overnight, centrifuge, and freeze-dry the obtained precipitate to obtain Millettia speciosa Champ. polysaccharide.

[0105] Comparative Example 7

[0106] This comparative example provides a method for preparing Millettia speciosa Champ. polysaccharide, and the specific steps of this method are as follows:

[0107] (1) Crushing: Crush the dried Millettia speciosa Champ., sieve it, and obtain Millettia speciosa Champ. powder;

[0108] (2) Defatting: Defat the Millettia speciosa Champ. powder by refluxing it with 10 times the amount of absolute ethanol, filter, evaporate to remove the absolute ethanol, dry it, and obtain defatted Millettia speciosa Champ. powder;

[0109] (3) Ultrasonic-assisted enzymatic treatment: Mix the defatted Millettia speciosa Champ. powder with pure water at a solid-liquid ratio of 1:15 (w / v), ultrasonicate for 30 min with an ultrasonic power of 350 w, add 2.5% of amylase based on the mass of the defatted Millettia speciosa Champ. powder, enzymatically hydrolyze at 70 °C for 60 min, and filter by suction to obtain a filtrate;

[0110] (4) Ethanol precipitation: Evaporate the filtrate, concentrate the volume to 20% of the original volume, add 4 times the amount of anhydrous ethanol, refrigerate overnight, centrifuge, freeze dry the obtained precipitate to obtain crude Niu Dali polysaccharide.

[0111] (5) Protein removal: Dissolve crude polysaccharide of *Smilax glabra* in water, add Sevag reagent, the mass ratio of crude polysaccharide to Sevag reagent is 7:1, stir and centrifuge, and take the supernatant.

[0112] (6) Decolorization: Add polyamide to the supernatant obtained in step (5), with a volume ratio of supernatant to polyamide of 1:1. Stir at 50°C for 30 min to decolorize, centrifuge, and take the supernatant.

[0113] (7) Dialysis and alcohol precipitation: The supernatant obtained in step (6) was placed in a dialysis bag with a cutoff molecular weight of 15000 Da and dialyzed for 35 h. The liquid in the dialysis bag was taken, anhydrous ethanol was added, and the mixture was refrigerated overnight. After centrifugation, the precipitate was freeze-dried to obtain the polysaccharide of *Euphorbia hirta*.

[0114] Example 1

[0115] This example uses the *Niu Da Li* polysaccharides prepared in Examples 1-5 and Comparative Examples 1-7 as samples. The content of *Niu Da Li* polysaccharides was determined by the phenol-sulfuric acid method, and the purity of *Niu Da Li* polysaccharides was determined by high performance liquid chromatography. The experimental results are shown in Table 1.

[0116] 1. The specific steps for determining the polysaccharide content of *Achyranthes bidentata* using the phenol-sulfuric acid method are as follows:

[0117] (1) Standard curve preparation: Accurately weigh 10 mg of glucose standard into a 100 mL volumetric flask, and dilute to 100 mL with distilled water to prepare a 0.1 mg / mL glucose standard solution. Transfer the prepared glucose standard solution to a volume of 5 mL with distilled water to prepare 0.02 mg / mL, 0.04 mg / mL, 0.06 mg / mL, 0.08 mg / mL, 0.10 mg / mL, 0.12 mg / mL, and 0.14 mg / mL solutions respectively. Take 200 μL of each concentration dilution and place it in a 2 mL test tube. Add 100 μL of 6% phenol and 500 μL of concentrated sulfuric acid to each test tube, let stand for 10 min, shake well, and let stand at room temperature for 20 min. Apply 200 μL to each well of a 96-well plate, making 3 wells per test tube. Measure the absorbance at 490 nm using an ELISA reader. Use distilled water as a blank control to obtain the standard curve. The standard curve is shown below. Figure 1 As shown, its regression equation is y = 8.6042x + 0.0166.

[0118] (2) Determination of polysaccharide content: Take the total polysaccharide of *Smilax glabra* and prepare a 1 mg / ml solution with distilled water. Dilute it 10 times with distilled water, pipette 200 μL, and inject it into a 96-well plate with 3 wells. Add phenol and sulfuric acid in proportion, let stand for 10 min, shake well, and let stand at room temperature for 20 min. Measure the absorbance at a wavelength of 490 nm using an enzyme-linked immunosorbent assay (ELISA) reader. Substitute the absorbance into the standard curve to calculate the polysaccharide content.

[0119] Calculate using the following formula:

[0120] Polysaccharide content (%) = (C × V2 × D) / (W × V1 × 10) × 100%

[0121] Wherein, C—sugar content (mg) calculated from the standard curve, V2—total volume of extract (mL), V1—volume used for measurement (mL), D—dilution factor, and W—sample weight (g).

[0122] Table 1

[0123]

[0124] As shown in Table 1, the purification methods for *Achyranthes bidentata* polysaccharides in Examples 1-5 involved treating *Achyranthes bidentata* with ultrasound-assisted enzymatic methods, followed by a series of purification processes including alcohol precipitation, protein removal, decolorization, and dialysis. This achieved the separation and purification of high-purity, high-concentration *Achyranthes bidentata* polysaccharides. In contrast, the polysaccharides obtained in Comparative Examples 1-2, which used ultrasound and enzymatic methods alone respectively, had significantly lower purity and concentration than those in the examples. This indicates that in the *Achyranthes bidentata* extraction process described in this invention, the combination of ultrasound and enzymatic methods significantly improves the extraction efficiency of polysaccharides. The two methods work synergistically to enhance the separation and purification effect of high-purity polysaccharides, making the obtained polysaccharides suitable for further biomedical applications. Furthermore, in Comparative Examples 3-4, the differences in ultrasound time and power, enzymatic hydrolysis time, and other factors all contributed to the purification of high-purity polysaccharides. When the temperature range was outside the preferred range of this invention, the extraction efficiency and purity of the prepared *Achyranthes bidentata* polysaccharide were significantly lower than those in Example 1. This indicates that in the process of preparing *Achyranthes bidentata* polysaccharide using the ultrasound-assisted enzymatic method, only within a specific range of ultrasound time, power, enzymatic hydrolysis time, and temperature can the synergistic effect be better exerted to enhance the separation and purification effect of polysaccharide. In addition, the extraction efficiency and purity of the *Achyranthes bidentata* polysaccharide finally obtained by enzymatic hydrolysis of *Achyranthes bidentata* using cellulase in Comparative Example 5 were worse than those in Example 1. Furthermore, as can be seen from Comparative Examples 6-7, in the process of preparing *Achyranthes bidentata* polysaccharide in this invention, the Sevag reagent, the amount of polyamide added, and the molecular weight cutoff and dialysis time during dialysis all have different degrees of influence on the extraction efficiency and purity of polysaccharide.

[0125] Example 2

[0126] The *Smilax glabra* polysaccharide prepared in Example 1 was used as a sample to investigate its effect on the survival rate of RAW246.7 macrophages and MC3T3-E1 cells. The specific experimental methods are as follows.

[0127] The effect of *Smilax glabra* polysaccharide on the viability of mouse macrophages RAW246.7 and MC3T3-E1 cells was detected using the MTT assay. Cells in logarithmic growth phase were harvested and their concentration adjusted to 5 × 10⁻⁶ cells. 3 Cells were seeded per well in a 96-well plate with 5 replicates, 100 μL per well. The plates were incubated at 37°C and 5% CO2 for 24 h until the cells adhered. The original culture medium was discarded, and culture medium containing different concentrations of the drug was added as the drug treatment group. The plates were incubated for another 24 h. 10 μL of MTT (5 mg / mL) was added to each well. After 4 h, the cell culture medium was discarded, and 100 μL of DMSO was added to each well. The plates were shaken in the dark for 5 min, and the absorbance was measured at 550 nm.

[0128] The results are as follows Figure 2-3 As shown, *Smilax glabra* polysaccharide showed no significant cytotoxic effects at concentrations of 25 μg / mL, 50 μg / mL, and 100 μg / mL. Therefore, the effects of its inhibitory effect on the activity of tartrate-resistant acid phosphatase (TRAP), a marker enzyme of osteoclasts, and its effect on the activity of alkaline phosphatase (ALP), a marker enzyme of osteoblast formation, were examined at these three concentrations.

[0129] Example 3

[0130] The *Smilax glabra* polysaccharide prepared in Example 1 was used as a sample to investigate its effect on TRAP activity during RANKL-induced differentiation of RAW246.7 macrophages into osteoclasts. The specific experimental method is as follows.

[0131] The inhibitory activity of *Smilax glabra* polysaccharide on osteoclast differentiation was evaluated using a RANKL-induced RAW246.7 mouse macrophage osteoporosis model. The activity of osteoclast marker enzymes was detected using an anti-tartrate acid phosphatase (TRAP) assay kit. Logarithmic growth phase cells were harvested and the cell concentration was adjusted to 6 × 10⁻⁶ cells / year. 3Cells were seeded per well in 96-well plates, with three replicates per well, 100 μL per well. Cells were incubated at 37°C, 5% CO2 for 24 h until adherence. The original culture medium was discarded, and culture medium containing 50 ng / ml RANKL was added to induce osteoclast differentiation for 7 days, changing the medium every two days. After induction, the old induction medium was discarded, and induction medium containing different concentrations of the drug was added as the drug treatment group. After culturing for another 24 h, cell lysates were collected, and TRAP activity was measured according to the instructions of the tartrate-resistant acid phosphatase (TRAP) assay kit. The absorbance was measured at 405 nm using a microplate reader. Since the content of p-NP is positively correlated with TRAP enzyme activity, the content of this compound is used to represent the TRAP enzyme activity in osteoclasts.

[0132] The results are as follows Figure 4 As shown, the model group had a high TRAP enzyme activity, indicating that the model was successfully induced. When different concentrations of *Smilax glabra* polysaccharide were applied to osteoclasts, the TRAP enzyme activity was found to decrease significantly in a concentration-dependent manner, indicating that *Smilax glabra* polysaccharide can inhibit osteoclast formation.

[0133] Example of effect 4

[0134] The polysaccharide prepared in Example 1 was used as a sample to investigate its effect on ALP activity in osteoblasts differentiated from MC3T3-E1 cells. The specific experimental method is as follows.

[0135] MC3T3-E1 cells were induced to differentiate into osteoblasts using complete culture medium containing 10 nM dexamethasone, 10 mM β-glycerophosphate sodium pentahydrate, and 50 μg / mL vitamin C. The effect of *Smilax glabra* polysaccharide on promoting osteoblast differentiation was evaluated, and the activity of alkaline phosphatase (ALP) marker enzymes in osteoblasts was detected using an alkaline phosphatase (ALP) assay kit. Cells in logarithmic growth phase were harvested and the cell concentration was adjusted to 2.5 × 10⁻⁶ cells / mL. 5 The cells were seeded into 6-well plates at 1 ml per well and cultured at 37°C in a 5% CO2 incubator for 24 hours until the cells adhered. The original culture medium was discarded, and induction medium containing different concentrations of the drug was added to induce cell differentiation for 5 days. The medium was changed every two days. After the drug administration was completed, the old induction medium was discarded, and the cell lysate was taken. The ALP content was measured according to the instructions of the alkaline phosphatase (ALP) assay kit and normalized using the BCA protein quantification method.

[0136] The results are as follows Figure 5 As shown, when different concentrations of *Achyranthes bidentata* polysaccharide were applied to MC3T3-E1 cells, their ALP activity began to increase significantly at a concentration of 50 μg / mL, and this increase was concentration-dependent, indicating that *Achyranthes bidentata* polysaccharide can promote osteoblast formation.

[0137] In summary, the *Niu Dali* polysaccharide provided by this invention, without significant toxic side effects on macrophage cell lines differentiating into osteoclasts and osteoblasts, is the first to demonstrate that it inhibits osteoclast growth and promotes osteoblast growth. Furthermore, the *Niu Dali* polysaccharide inhibits osteoclast growth and promotes osteoblast growth by inhibiting TRAP activity during osteoclast differentiation and enhancing ALP activity during osteoblast differentiation, thus achieving the prevention and treatment of osteoporosis through bidirectional metabolic regulation. This provides a scientific basis for the application of *Niu Dali* polysaccharide in the fight against osteoporosis.

[0138] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. The application of *Smilax glabra* polysaccharide in the preparation of an anti-osteoporosis drug, characterized in that, The preparation method of the *Smilax glabra* polysaccharide includes the following steps: (1) Degreasing: The powder of *Niu Dali* is degreased to obtain defatted *Niu Dali* powder; (2) Ultrasonic-assisted enzymatic treatment: The defatted Niu Dali powder obtained in step (1) is mixed with pure water, ultrasonicated, and amylase is added for enzymatic hydrolysis. Then the mixture is filtered to obtain the filtrate. (3) Ethanol precipitation: The filtrate obtained in step (2) is concentrated, anhydrous ethanol is added, centrifuged, and the precipitate obtained by centrifugation is freeze-dried to obtain crude Niu Dali polysaccharide. (4) Purification: The crude polysaccharide obtained in step (3) is subjected to protein removal, decolorization, and dialysis with alcohol precipitation to obtain the polysaccharide.

2. Use according to claim 1, characterized in that, In step (2), the ultrasound time is 20-40 min and the ultrasound power is 300-400 W.

3. Use according to claim 1, characterized in that, In step (2), the mass of the enzyme is 1%-4% of the mass of the defatted Niu Dali powder.

4. Use according to claim 1, characterized in that, In step (2), the enzymatic hydrolysis time is 40-80 min and the enzymatic hydrolysis temperature is 65-75℃.

5. The use according to claim 1, characterized in that, In step (4), the specific steps for removing protein are as follows: dissolve the crude polysaccharide obtained in step (3) in water, add Sevag reagent, stir and centrifuge to obtain supernatant; The mass ratio of crude *Smilax glabra* polysaccharide to Sevag reagent is 5:

1.

6. Use according to claim 1, characterized in that, In step (4), the specific steps for decolorization are as follows: add polyamide to the supernatant obtained after removing protein, stir to decolorize, centrifuge, and obtain supernatant; The volume ratio of the supernatant obtained after protein removal to polyamide is 2:1 to 6:

1.

7. The use according to claim 1, characterized in that, In step (4), the specific steps of dialysis and alcohol precipitation are as follows: the supernatant obtained after decolorization is dialyzed, the liquid in the dialysis bag is taken, anhydrous ethanol is added, centrifuged, and the precipitate is freeze-dried to obtain Niu Dali polysaccharide; The dialysis bags used in the dialysis have a cutoff molecular weight of 8000-14000 Da, and the dialysis time is 40-50 h.

8. The use according to claim 1, characterized in that, The polysaccharide from *Smilax glabra* is used to inhibit the growth of osteoclasts and promote the growth of osteoblasts.

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