A medicine for treating osteoporosis and its preparation method

Abstract

The application discloses a medicine for treating osteoporosis and a preparation method thereof, and belongs to the field of traditional Chinese medicine. The medicine is mainly composed of Millettia pachycarpa Benth. and is prepared by using effective components in the Millettia pachycarpa Benth. to inhibit osteoclasts, and is additionally composed of Spatholobus suberectus Dunn, Fissistigma cavaleriei, Longan arillus and Achyranthes bidentata Bl. The Millettia pachycarpa Benth. and the Fissistigma cavaleriei are used to achieve the effects of strengthening dampness, relaxing muscles and collaterals, and the Millettia pachycarpa Benth. and the Spatholobus suberectus Dunn are used to achieve the effects of tonifying blood and qi, relaxing muscles and collaterals, regulating menstruation and relieving pain. The Millettia pachycarpa Benth. and the Achyranthes bidentata Bl. are used to achieve the effects of tonifying liver and kidney, strengthening tendons and bones, promoting water and relieving stranguria, and guiding blood downward. The Longan arillus is mainly used to achieve the effects of invigorating the spleen and heart, tonifying qi and blood, and soothing the nerves. The problems that patients with weak spleen and stomach cannot eat the combination of the Millettia pachycarpa Benth. and the Spatholobus suberectus Dunn are solved to a certain extent, and the medicine has better therapeutic effect.

Description

Technical Field

[0001] This invention relates to the field of bone disease treatment technology, and in particular to a drug for treating osteoporosis and its preparation method. Background Technology

[0002] Bone tissue is a special connective tissue formed by the mineralization of an organic matrix, possessing elasticity and strength. Simultaneously, bone tissue is a metabolically active and self-regulating dynamic organ, primarily maintaining bone homeostasis through a dynamic balance between bone formation and bone resorption. As the only cells in the body with bone resorption function, osteoclasts are crucial for maintaining bone metabolic balance. Inflammation is defined as the defensive response of living tissues with a vascular system to damaging factors. Inflammation can affect physiological activities such as cell proliferation and metabolism, leading to pathological changes in human tissues. Inflammation often causes abnormalities in the number and function of osteoclasts; inflammation and osteoclasts influence each other, resulting in excessive bone resorption and promoting osteolysis, ultimately leading to many inflammatory osteolytic diseases, including rheumatoid arthritis, infectious osteolysis, aseptic loosening of joint prostheses, and periodontitis. Histologically, these inflammatory osteolytic diseases are characterized by bone loss and a reduction or disappearance of bone trabeculae. Based on the mechanism of inflammatory osteolysis, anti-bone resorption drugs targeting osteoclasts are currently the first-line drugs for the prevention and treatment of inflammatory osteolysis, such as bisphosphonates and denosumab. However, long-term use of bisphosphonates can lead to complications such as osteonecrosis of the mandible and nonspecific fractures of the femur. Denosumab has been found to have the potential risk of inducing malignant tumors in clinical trials.

[0003] Niu Dali is the dried tuberous root of *Millettia speciosa* Champ., a legume. According to Zhuang medicine theory, Niu Dali is neutral in nature and sweet in taste, capable of regulating qi, blood circulation, and replenishing qi deficiency. It is commonly used by the Zhuang people to treat lower back and leg pain, fever, tuberculosis, chronic hepatitis, and chronic gastritis. As a food and medicine of the same origin, Niu Dali is also used in health preservation. Currently, it is used clinically to treat lumbar muscle strain, cough due to lung deficiency, rheumatoid arthritis, and injuries from falls and blows. Modern pharmacological studies have shown that Niu Dali has anti-inflammatory, analgesic, antioxidant, anti-liver fibrosis, anti-tumor, and hypoglycemic effects. Research reports that the water extract of Niu Dali also has anti-inflammatory, analgesic, antioxidant, anti-fatigue, and immunomodulatory effects. Summary of the Invention

[0004] The purpose of this invention is to provide a drug for treating osteoporosis and its preparation method. It utilizes *Smilax glabra* water extract as the main component, combined with other traditional Chinese medicinal herbs. Based on the modern TCM theory that treating osteoporosis often involves strengthening the spleen and replenishing qi, dispelling wind and dampness, and strengthening muscles and bones, multiple Chinese medicinal herbs are added to complement each other. This allows the drug to inhibit osteoclasts while enhancing the patient's constitution, thereby achieving the treatment of inflammatory osteolysis and avoiding the drawbacks of significant side effects associated with chemically synthesized drugs.

[0005] The medicine for treating osteoporosis comprises the following raw materials in the following weight ratios: 20-30 parts of *Achyranthes bidentata*, 10-15 parts of *Smilax china*, 10-15 parts of *Spatholobus suberectus*, 8-12 parts of *Achyranthes bidentata*, and 5-10 parts of longan pulp.

[0006] To realize a drug for treating osteoporosis, the preparation method of the drug for treating osteoporosis of the present invention includes the following steps:

[0007] (1) Clean the following herbs: *Smilax glabra*, *Smilax china*, *Achyranthes bidentata*, and longan pulp; remove impurities and dry them; weigh each herb according to the weight fraction.

[0008] (2) After weighing the *Smilax glabra*, crush it, add water, cellulase and pectinase, enzymatically hydrolyze for 2-2.5 hours, filter, and obtain *Smilax glabra* filtrate.

[0009] (3) Reflux extract the filtrate of *Smilax glabra* at 100℃ for 2 hours, collect the filtrate, add distilled water to the remaining residue, reflux at 100℃ for 1 hour, collect all the filtrates, combine and concentrate to obtain the water extract of *Smilax glabra* for later use.

[0010] (4) Weigh the following herbs: Smilax glabra, Spatholobus suberectus, Achyranthes bidentata and Longan pulp, and grind them to 250-300 mesh using an ultra-micro pulverizer to obtain a mixed powder of Chinese medicinal materials;

[0011] (5) Soak the mixed powder of Chinese medicinal materials in water at a ratio of 5 to 10 times the total weight of the Chinese medicinal materials for 1 to 1.5 hours, heat to boiling, then simmer over low heat for 2 to 3 hours, and filter with a 350-mesh filter to obtain the mixed filtrate.

[0012] (6) Mix the mixed filtrate with the water extract of *Smilax glabra*, heat and concentrate it to a paste state, and add excipients to make a medicine.

[0013] Furthermore, the excipients are any one or a combination of several of the following: starch, sucrose, lactose, powdered sugar, glucose, mannitol, xylitol, polyethylene glycol, isopropanol, Tween-80, glycerin, propylene glycol, sodium microcrystalline cellulose, dextrin, cyclodextrin, or gelatin.

[0014] Furthermore, the medicine is in the form of capsules, granules, tablets, or oral liquid.

[0015] The effects of various Chinese medicinal herbs in medications for treating osteoporosis:

[0016] Niu Dali contains a variety of components such as flavonoids, triglycerides, diethyl alcohol, alkaloids, and organic acids. It has a variety of effects such as antitussive and antiasthmatic effects, liver protection, antioxidant, anti-inflammatory, and immune enhancement. Among them, the ethanol extract of Niu Dali contains a variety of compounds that can inhibit the activity of osteoclasts.

[0017] The main functions of *Smilax glabra* are to dispel wind and dampness, eliminate blood stasis and detoxify. When combined with *Smilax glabra*, it can enhance the effects of dispelling wind and dampness and relaxing muscles and tendons. It has certain therapeutic effects on symptoms such as soreness and weakness of the waist and knees, numbness of limbs, dizziness, cough, hemoptysis, traumatic injuries, rheumatic pain, seminal emission, and increased leukorrhea. Modern TCM theory believes that insufficient kidney qi and kidney yang are unable to support the body's vital energy, and insufficient kidney yin and kidney essence are unable to nourish the body, ultimately aggravating osteoporosis and causing symptoms such as bone and joint pain. Pain symptoms in osteoporosis patients can be treated with drugs that tonify the liver and kidneys and dispel wind and dampness. Existing literature also shows that the active ingredients in *Smilax glabra* have the effect of regulating the relevant signaling pathways of knee osteoarthritis progression.

[0018] Chicken blood vine can invigorate blood, replenish blood, regulate menstruation, relieve pain, and relax muscles and tendons. When combined with oxtail, it can help replenish blood and qi, improve human immunity, and relax muscles and tendons. It has a certain curative effect on joint pain, numbness, and difficulty in flexion and extension. Modern TCM theory believes that osteoporosis is caused by a variety of pathogenesis, with kidney essence deficiency as the root cause. Kidney essence deficiency leads to insufficient qi and blood throughout the body. Insufficient qi is unable to propel blood circulation, which easily leads to blood stasis. That is, deficiency over time will inevitably lead to stasis. Existing literature also shows that multiple compounds in chicken blood vine can synergistically target two key targets in the KEGG signaling pathway and have an anti-osteoporosis effect.

[0019] Achyranthes bidentata, a traditional Chinese medicine, was first recorded in the classic Chinese medicine book *Shennong Bencao Jing*. The book describes its effects as clearing the meridians, nourishing the liver and kidneys, strengthening tendons and bones, and guiding blood downwards. Osteoporosis is mainly caused by liver and kidney deficiency; therefore, Achyranthes bidentata has good clinical efficacy for patients with liver and kidney deficiency-type osteoporosis. Existing pharmacological studies have also found that substances such as achyranthesol contained in Achyranthes bidentata can promote osteoblast proliferation and effectively increase bone mass. Studies on the extraction of drugs affecting bone metabolism have found that chemical components in raw Achyranthes bidentata, such as ketones, trichloroacetins, and polysaccharides, have effects on glucose metabolism, hemorheology, and anti-osteoporosis.

[0020] Longan pulp has the effects of nourishing the heart and spleen, nourishing blood and calming the mind. It is often used for symptoms such as insufficient qi and blood, palpitations, forgetfulness and insomnia, and blood deficiency and chlorosis. It can help improve adverse reactions such as nausea, vomiting, abdominal pain and diarrhea that occur when patients with weak spleen and stomach take medicines containing Niu Dali and Ji Xue Teng.

[0021] The significant beneficial effects of this invention are:

[0022] (1) The main component of this invention is the water extract of *Smilax glabra*, which contains compounds that can effectively inhibit osteoclast activity, thereby helping to increase bone density in the patient's bone tissue. It has a good therapeutic effect on inflammatory osteolytic diseases such as rheumatoid arthritis, infectious osteolysis, aseptic loosening of joint prostheses, and periodontitis caused by abnormal number and function of osteoclasts. It has no toxic side effects, high safety, and avoids the disadvantages of large side effects of chemically synthesized drugs, providing an effective way to treat osteoporosis.

[0023] (2) This invention combines Niu Dali with several other medicinal materials to enhance each other's effects. In addition to having the therapeutic effect on osteoporosis, it also has the effects of dispelling wind and dampness, eliminating blood stasis and detoxifying, promoting blood circulation and replenishing blood, relaxing muscles and tendons, and tonifying the heart and spleen, thus further improving the therapeutic effect. Attached Figure Description

[0024] Figure 1 A diagram showing MS inhibiting RANKL-induced osteoclast formation in vitro;

[0025] Figure 2 A diagram showing how MS suppresses the expression of osteoclast marker genes;

[0026] Figure 3 A diagram showing that MS can reduce bone loss caused by LPS-induced inflammation.

[0027] The meanings of the symbols in the image are as follows:

[0028] Figure 1 (A) shows the results of MS toxicity test on cells, (B) shows the results of tartrate-resistant acid phosphatase (TRAcP) staining of osteoclasts, and (C) shows the statistical results of the number and area of ​​mature osteoclasts.

[0029] Figure 2 In the table, (A) is the expression rate of Tracp gene relative to β-actin in osteoclasts formed under MS intervention, (B) is the expression rate of Dcstamp gene relative to β-actin, (C) is the expression rate of c-Fos gene relative to β-actin, (D) is the expression rate of Nfatc1 gene relative to β-actin, (E) is the expression rate of Ctsk gene relative to β-actin, and (F) is the expression rate of Atp6vod2 gene relative to β-actin.

[0030] Figure 3(A) is a micro-CT analysis image; (B) are the quantitative analysis results of trabecular volume (BV / TV), trabecular number (Tb.N), and trabecular separation (Tb.Sp); (C) is a representative HE-stained image of bone tissue; (D) is the quantitative analysis result of changes in trabecular structure; (E) is a representative TRAcP-stained image of bone tissue; and (F) is the qualitative analysis result of osteoclast number and bone resorption area. Detailed Implementation

[0031] The present invention will be further described in detail below with reference to specific embodiments, but the implementation of the present invention is not limited to the scope shown in the embodiments. These embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, after reading the contents of this invention, those skilled in the art can make various modifications to the present invention, and these equivalent changes also fall within the scope defined by the appended claims. Detailed Implementation Method 1

[0033] The medicine for treating osteoporosis comprises the following ingredients in the following weight ratios: 20 parts of *Achyranthes bidentata*, 10 parts of *Smilax china*, 10 parts of *Spatholobus suberectus*, 8 parts of *Achyranthes bidentata*, and 5 parts of longan pulp.

[0034] To realize a drug for treating osteoporosis, the preparation method of the drug for treating osteoporosis of the present invention includes the following steps:

[0035] (1) Clean the following herbs: *Smilax glabra*, *Smilax china*, *Achyranthes bidentata*, and longan pulp; remove impurities and dry them; weigh each herb according to the weight fraction.

[0036] (2) After weighing the ox-dali, crush it, add water, cellulase and pectinase, enzymatically hydrolyze for 2 hours, filter, and obtain ox-dali filtrate;

[0037] (3) Reflux extract the filtrate of *Smilax glabra* at 100℃ for 2 hours, collect the filtrate, add distilled water to the remaining residue, reflux at 100℃ for 1 hour, collect all the filtrates, combine and concentrate to obtain the water extract of *Smilax glabra* for later use.

[0038] (4) Weigh the herbs, including *Smilax china*, *Spatholobus suberectus*, *Achyranthes bidentata*, and longan pulp, and grind them to 250 mesh using an ultra-micro pulverizer to obtain a mixed powder of Chinese medicinal materials.

[0039] (5) Soak the mixed powder of Chinese medicinal materials in water equal to 5 times the total weight of Chinese medicinal materials for 1 hour, heat to boiling, then simmer for 2 hours, filter with a 350 mesh filter to obtain the mixed filtrate.

[0040] (6) Mix the mixed filtrate with the water extract of *Smilax glabra*, heat and concentrate it to a paste state, and add excipients to make a medicine.

[0041] Furthermore, the excipients are starch, powdered sugar, glycerin, propylene glycol, dextrin, and gelatin.

[0042] Furthermore, the medicine is in the form of capsules. Detailed Implementation Method 2

[0044] The medicine for treating osteoporosis comprises the following ingredients in the following weight ratios: 25 parts of *Achyranthes bidentata*, 12 parts of *Smilax china*, 12 parts of *Spatholobus suberectus*, 10 parts of *Achyranthes bidentata*, and 7 parts of longan pulp.

[0045] To realize a drug for treating osteoporosis, the preparation method of the drug for treating osteoporosis of the present invention includes the following steps:

[0046] (1) Clean the following herbs: *Smilax glabra*, *Smilax china*, *Achyranthes bidentata*, and longan pulp; remove impurities and dry them; weigh each herb according to the weight fraction.

[0047] (2) After weighing the ox-dali, crush it, add water, cellulase and pectinase, enzymatically hydrolyze for 2 hours, filter, and obtain ox-dali filtrate;

[0048] (3) Reflux extract the filtrate of *Smilax glabra* at 100℃ for 2 hours, collect the filtrate, add distilled water to the remaining residue, reflux at 100℃ for 1 hour, collect all the filtrates, combine and concentrate to obtain the water extract of *Smilax glabra* for later use.

[0049] (4) Weigh the herbs, including *Smilax china*, *Spatholobus suberectus*, *Achyranthes bidentata*, and longan pulp, and grind them to 250 mesh using an ultra-micro pulverizer to obtain a mixed powder of Chinese medicinal materials.

[0050] (5) Soak the mixed powder of Chinese medicinal materials in water equal to 7 times the total weight of Chinese medicinal materials for 1 hour, heat to boiling, then simmer for 2.5 hours, filter with a 350 mesh screen to obtain the mixed filtrate;

[0051] (6) Mix the mixed filtrate with the water extract of *Smilax glabra*, heat and concentrate it to a paste state, and add excipients to make a medicine.

[0052] Furthermore, the excipients are starch, powdered sugar, dextrin, and gelatin.

[0053] Furthermore, the medicine is a tablet. Detailed Implementation Method 3

[0055] The medicine for treating osteoporosis comprises the following ingredients in the following weight ratios: 30 parts of *Achyranthes bidentata*, 15 parts of *Smilax china*, 15 parts of *Spatholobus suberectus*, 12 parts of *Achyranthes bidentata*, and 10 parts of longan pulp.

[0056] To realize a drug for treating osteoporosis, the preparation method of the drug for treating osteoporosis of the present invention includes the following steps:

[0057] (1) Clean the following herbs: *Smilax glabra*, *Smilax china*, *Achyranthes bidentata*, and longan pulp; remove impurities and dry them; weigh each herb according to the weight fraction.

[0058] (2) After weighing the *Smilax glabra*, crush it, add water, cellulase and pectinase, enzymatically hydrolyze for 2.5 h, filter, and obtain *Smilax glabra* filtrate;

[0059] (3) Reflux extract the filtrate of *Smilax glabra* at 100℃ for 2 hours, collect the filtrate, add distilled water to the remaining residue, reflux at 100℃ for 1 hour, collect all the filtrates, combine and concentrate to obtain the water extract of *Smilax glabra* for later use.

[0060] (4) Weigh the following herbs: Smilax glabra, Spatholobus suberectus, Achyranthes bidentata and longan pulp, and grind them to 300 mesh using an ultra-micro pulverizer to obtain a mixed powder of Chinese medicinal materials;

[0061] (5) Soak the mixed powder of Chinese medicinal materials in water at a ratio of 10 times the total weight of the Chinese medicinal materials for 1.5 hours, heat to boiling, then simmer over low heat for 3 hours, and filter with a 350-mesh filter to obtain the mixed filtrate.

[0062] (6) Mix the mixed filtrate with the water extract of *Smilax glabra*, heat and concentrate it to a paste state, and add excipients to make a medicine.

[0063] In a preferred embodiment, the excipients are glucose, Tween-80, glycerol, and propylene glycol.

[0064] Preferably, the medicine is an oral liquid.

[0065] Experiment on the inhibitory effect of *Smilax glabra* water extract on osteoclast activity:

[0066] 1. Materials and reagents

[0067] 1.1 *Achyranthes bidentata* was purchased from Nanning Shengyuantang Traditional Chinese Medicine Co., Ltd. (Nanning, China). α-MEM, fetal bovine serum, and penicillin-streptomycin reagent were purchased from Gibco, USA. RANKL and M-CSF were purchased from R&D, USA. CellCounting Kit-8 was purchased from Invitrogen, USA, and reverse transcription kit was purchased from Thermo Fisher Scientific.

[0068] 1.2 Aqueous extract of *Smilax glabra*

[0069] Pulverize approximately 100g of dried *Achyranthes bidentata*, add water, cellulase, and pectinase, and enzymatically hydrolyze for 2.5h. Filter and reflux extract at 100℃ for 2h. Collect the filtrate, add distilled water (v / w = 6:1) to the remaining residue, reflux at 100℃ for 1h, collect all filtrates, combine and concentrate to 1g / ml (calculated based on herbal value).

[0070] 2. CCK8 cytotoxicity test

[0071] Freshly isolated bone marrow macrophages (BMMs) from C57BL / 6J mice were seeded at 6 × 10³ cells / well in 96-well plates and incubated overnight to allow cell adhesion. The following day, MS solutions were added to the cell culture medium at concentration gradients of 0, 12.5, 25, 50, 100, and 200 μg / mL and incubated for 96 h. After incubation, 10 μL / well of CCK8 solution was added, and the cells were co-incubated for 2 h. The absorbance was then read at 450 nm using an automated quantitative microplate reader (BMG, Germany). The absorbance values ​​were compared to analyze the toxicity of the *Smilax glabra* extract to the cells.

[0072] 3. MS inhibition of osteoclast differentiation experiment

[0073] BMMs were seeded at a density of 6 × 10³ cells / well in 96-well plates. Cells were cultured in complete medium (containing α-MEM and 10% fetal bovine serum) containing M-CSF (25 ng / mL) and RANKL (50 ng / mL), and treated with MS at concentration gradients of 0, 12.5, 25, 50, 100, and 200 μg / mL. The cell culture medium was changed every 2 days until osteoclast formation. Cells were fixed with 4% paraformaldehyde for 30 min, washed three times with PBS, and stained for tartrate-resistant acid phosphatase (TRAcP) (incubated at 37°C for 30 min). TRAcP-positive multinucleated cells (>3 nuclei) were identified as osteoclasts, and the number of osteoclasts treated at different concentration gradients was counted and compared.

[0074] 4. Real-time quantitative polymerase chain reaction (PCR) analysis

[0075] BMMs were seeded at 1×10⁵ cells / well in 6-well plates. After cell adhesion, cells were stimulated with 50 ng / mL RANKL and 25 ng / mL LM-CSF, followed by MS intervention at 100 and 200 μg / mL until mature osteoclasts formed. Total RNA was isolated from the cells using Trizol reagent and reverse transcribed into single-stranded cDNA using 1 μg RNA template and reverse transcriptase with oligonucleotide primers. Polymerase chain reaction (PCR) was used to amplify specific sequences using the following cycle: 94℃ for 5 mins, 94℃ for 30 cycles, 40 s, 60℃ for 40 s, 72℃ for 40 s, and a final extension of 5 mins at 72℃.

[0076] The specific primer sequences for osteoclast-related genes, using β-actin as an internal control, are as follows:

[0077]

[0078] Using a 96-well PCR plate, set the genes to be measured and the primers. Under light-protected conditions, add 200 ng of cDNA, 1 μL each of the front and back primers, and 5 μL of SYBR Green fluorescent reagent to each well. Add DEPC water to make a 10 μL system per well. After sample addition, cover the 96-well plate with sealing paper to prevent sample evaporation during heating, and then briefly centrifuge the PCR plate using a plate centrifuge. Place the PCR plate in a LightCycer 96PCR instrument for amplification. The program is set as follows: 95℃ for 10 mins, 55 cycles, then reduce to 60℃ for 15 s, followed by 72℃ for 40 s. The concentration of the target gene is calculated using the 2-ΔΔCT method with β-actin as an internal control.

[0079] 5. Establishment and analysis of a mouse model of systemic osteolysis induced by LPS inflammation.

[0080] The establishment of a mouse model of systemic osteolysis induced by LPS was conducted according to the guidelines of the Animal Ethics Committee of Guangxi Medical University. After fasting and depriving mice of water for 6–8 hours, 28 9-week-old male C57BL / 6J mice were randomly divided into four groups: Sham group, LPS group, and LPS+MS (2 g / kg and 4 g / kg) group. The Sham group received only intraperitoneal injection of saline, while the remaining groups received intraperitoneal injections of 5 mg / kg LPS on days 1 and 4. The LPS+MS groups also received oral administration of 2 g / kg and 4 g / kg MS, respectively, every other day. After administration, the animals were anesthetized, blood was collected from the eyeballs, and the mice were euthanized by cervical dislocation and bilateral tibias were separated. The mice were then placed in 4% paraformaldehyde at room temperature for 48 hours, followed by replacement with 75% alcohol at room temperature for subsequent sectioning and CT scans.

[0081] 6. Microscopic CT analysis and histological analysis of laboratory animal samples

[0082] First, the fully fixed specimen was removed, and any remaining tissue from both hind limbs was cleared. The bones were then temporarily placed in alcohol to ensure they remained moist. The left tibia specimen from the experimental mice was scanned and analyzed using a high-resolution micro-CT scanner (Sky Scan; Bruker, USA) with a micro-voltage of 70 kV, a scanning slice interval of 10 μm, a current of 200 μA, and a resolution of 1024 × 1024 pixels. We selected the region of interest at the distal end of the tibia for three-dimensional reconstruction. The reconstructed 3D images were used to determine bone-related parameters such as percentage bone volume per tissue volume (BV / TV), trabecular number (Tb.N), and trabecular separation (Tb.Sp).

[0083] Changes in bone microstructure were analyzed using histomorphological methods, and the number of osteoclasts in bone tissue after drug treatment was compared and analyzed. The left tibia of mice was removed, soft tissue was removed, and the tissue was fixed in 4% paraformaldehyde for 24-48 hours. Then, the tissue was soaked in 10% EDTA and decalcified in a decalcifier for approximately 5-7 days. After decalcification, the tissue was embedded, sectioned, and stained with hematoxylin and eosin (HE) and cyclophosphamide (TRAcP).

[0084] 7. Statistics

[0085] Data in this study are expressed as mean ± standard deviation (SD). One-way ANOVA and pairwise comparison tests were used to determine the significance of differences between results. A p-value less than 0.05 was considered statistically significant. The statistical results for each group are as follows.

[0086] Group BV / TV Tb.N Tb.Sp sham1 13.98857 0.03457 18.31584 sham2 16.61421 0.02983 18.67273 sham3 13.26049 0.0342 19.34669 sham4 14.80647 0.02506 20.42138 sham5 16.56072 0.02514 19.48701 sham6 16.55295 0.02902 17.65567 LPS1 7.13387 0.01341 25.32863 LPS2 9.07504 0.01402 30.55722 LPS3 9.4557 0.01601 25.83882 LPS4 8.74524 0.01995 23.34423 LPS5 10.77997 0.01987 21.54712 LPS6 10.029 0.02046 21.46268 low1 14.62361 0.02543 12.0661 low2 10.93688 0.03478 12.47509 low3 11.66986 0.03099 12.74305 low4 11.16038 0.02072 20.97128 low5 10.89425 0.02992 21.48353 low6 11.4025 0.02934 22.54807 high1 11.71559 0.0377 12.97705 high2 14.83787 0.03972 11.02396 high3 14.61267 0.02543 20.02403 high4 16.01745 0.02753 11.56942 high5 16.11385 0.0217 12.74615 high6 17.3975 0.0323 9.8727

[0087] Experimental results

[0088] 1. MS inhibits RANKL-induced osteoclast formation in vitro.

[0089] First, cell viability under different concentrations of MS was detected using a CCK8 assay. The results showed that MS concentrations below 200 μg / mL had no effect on the proliferation of bone marrow cytokines (BMMs). To further investigate the effect of MS on osteoclast formation, BMMs were treated with different concentrations of MS in the presence of 25 ng / mL M-CSF and 50 ng / mL RANKL. Tartrate-resistant acid phosphatase (TRAP) staining results indicated that MS could inhibit osteoclast formation in a concentration-dependent manner. Figure 1 (As shown).

[0090] 2. MS inhibits the expression of osteoclast marker genes.

[0091] The inventors examined the expression of osteoclast-specific genes Tracp, Dcstamp, c-Fos, Nfatc1, Ctsk, and Atp6v0d2 in osteoclast-specific mesenchymal stem cells (BMMs) induced by 50 ng / ml RANKL. RT-PCR results showed that RANKL stimulated the expression of these osteoclast-specific genes. 200 ng / ml MS reduced the expression of these genes induced by RANKL. Figure 2 (As shown).

[0092] 3. MS reduces bone loss caused by LPS-induced inflammation.

[0093] LPS-treated mice were selected to observe the effect of MS on LPS-induced osteoporosis. The inventors performed micro-CT analysis on the dissected left tibia. The results showed that in the positive control group, LPS significantly reduced bone mineral density in mice. Compared with the LPS model group, the MS-treated group showed significantly increased trabecular bone density, significantly smaller trabecular spacing, and significantly increased bone mass. Further immunohistochemical analysis of bone tissue showed that, compared with the LPS model group, the MS-treated group significantly inhibited LPS-induced bone loss.

[0094] discuss

[0095] In this study, the inventors observed that the aqueous extract of *Smilax glabra* could improve bone loss caused by LPS-induced inflammation in vivo. Based on this, the inventors believe that the aqueous extract of *Smilax glabra* may have great potential and clinical value in the prevention and treatment of inflammatory osteolysis.

[0096] Osteoclasts resorb bone and cartilage, thereby regulating bone remodeling and mineral balance. However, significantly increased osteoclast function has been observed in bone loss diseases such as osteoporosis and rheumatoid arthritis. Therefore, reducing osteoclast activity is a typical anti-resorption treatment strategy for these osteolytic diseases. This invention demonstrates for the first time that *Smilax glabra* aqueous extract inhibits osteoclast differentiation and bone resorption, and also shows that treatment with *Smilax glabra* aqueous extract can reduce bone loss in LPS-inflamed mice. The inventors' data indicate that this natural active ingredient, *Smilax glabra* aqueous extract, has potential applications in the treatment of osteolytic diseases.

[0097] Experimental studies on the treatment of osteoporosis using the drug described in this invention:

[0098] 1. Establishment of an ovariectomy-induced osteoporosis model in mice

[0099] The establishment of the ovariectomy-induced osteoporosis mouse model was carried out in accordance with the guidelines of the Animal Ethics Committee of Guangxi Medical University. First, 28 12-week-old female C57BL / 6J mice were randomly divided into 4 groups: sham operation group (injected with PBS), operation group (bilateral ovariectomy), Niu Da Li water extract group, and Niu Da Li prescription group.

[0100] The specific modeling process is as follows: Mice in the surgical group, *Niu Da Li* water extract group, and *Niu Da Li* herbal formula group were anesthetized with chloral hydrate. Surgical incisions were made along both sides of the lumbar spine to access the abdominal cavity. Both ovaries were removed, and the wounds were sutured. In the sham surgery group, the ovaries were located but not removed; a small amount of nearby fat was removed, and the rest of the procedure was the same as in the surgical group. All surgeries were performed under strict aseptic conditions. Post-surgery, mice were placed in cages and allowed to move freely. One week after surgery, the *Niu Da Li* water extract group and the *Niu Da Li* herbal formula group were administered *Niu Da Li* water extract (50ug / ml) and *Niu Da Li* herbal formula water extract, respectively, via gavage. The *Niu Da Li* herbal formula water extract was an oral preparation made from *Niu Da Li* water extract (50ug / ml) and corresponding proportions of *Qian Jin Ba*, *Ji Xue Teng*, *Niu Xi*, and *Long Yan Rou*, with a dosage of 0.4g / kg, administered daily for 6 consecutive weeks. The surgical and sham surgery groups received the same amount of PBS. After the administration period, the animals were anesthetized, blood was collected from the eyeballs, and the animals were euthanized by cervical dislocation, followed by separation of the bilateral tibia and lumbar vertebrae.

[0101] 2. Data Detection

[0102] First, the fully fixed specimen was removed, and any remaining tissue was cleaned. The bone was then temporarily placed in alcohol to keep it moist. High-resolution micro-CT (Sky Scan; Bruker, USA) was used to scan and analyze the mouse bone specimens at a micro-voltage of 70 kV, a scanning slice interval of 10 μm, a current of 200 μA, and a resolution of 1024 × 1024 pixels. The measurement results were obtained, and the average value was taken. The results are as follows.

[0103] Group <![CDATA[Tibia (g / cm 3 )]]> <![CDATA[Lumbar vertebra (g / cm 3 )]]> Sham group 0.131±0.003 0.105±0.018 Surgery group 0.111±0.020 0.084±0.011 Aqueous extract of Millettia speciosa Champ. group 0.125±0.015 0.117±0.019 Aqueous extract of formula of Millettia speciosa Champ. group 0.134±0.011 0.121±0.007

[0104] The data above show that after ovariectomy, mice without corresponding treatment will experience a decrease in bone density. However, treatment with *Achyranthes bidentata* extract will increase bone density to some extent. Furthermore, treatment with the osteoporosis drug described in this invention will result in a significant increase in bone density. This indicates that the osteoporosis drug described in this invention has a better therapeutic effect on osteoporosis than *Achyranthes bidentata* extract alone.

Claims

1. A drug for treating osteoporosis, characterized in that, The medicine is composed of the following raw materials in the following weight ratios: 20-30 parts of *Achyranthes bidentata*, 10-15 parts of *Smilax china*, 10-15 parts of *Spatholobus suberectus*, 8-12 parts of *Achyranthes bidentata*, and 5-10 parts of longan pulp.

2. A method for preparing the medicament for treating osteoporosis according to claim 1, comprising the following steps: (1) Clean the following herbs: *Smilax glabra*, *Smilax china*, *Achyranthes bidentata*, and longan pulp; remove impurities and dry them; weigh each herb according to the weight fraction. (2) After weighing the ox-dali, crush it, add water, cellulase and pectinase, enzymatically hydrolyze for 2-2.5 hours, filter, and obtain ox-dali filtrate; (3) Reflux extract the filtrate of *Smilax glabra* at 100°C for 2 hours, collect the filtrate, add distilled water to the remaining residue, reflux at 100°C for 1 hour, collect all the filtrates, combine and concentrate to obtain the water extract of *Smilax glabra* for later use; (4) Weigh the following herbs: Smilax glabra, Spatholobus suberectus, Achyranthes bidentata and Longan pulp, and grind them to 250-300 mesh using an ultra-micro pulverizer to obtain a mixed powder of Chinese medicinal materials; (5) Soak the mixed powder of Chinese medicinal materials in water at a ratio of 5 to 10 times the total weight of the Chinese medicinal materials for 1 to 1.5 hours, heat to boiling, then simmer over low heat for 2 to 3 hours, and filter with a 350-mesh filter to obtain the mixed filtrate; (6) Mix the mixed filtrate with the water extract of *Smilax glabra*, heat and concentrate it to a paste state, and add excipients to make a medicine.

3. The method for preparing the medicament for treating osteoporosis according to claim 2, characterized in that: The excipients are any one or a combination of several of the following: starch, sucrose, lactose, powdered sugar, glucose, mannitol, xylitol, polyethylene glycol, isopropanol, Tween-80, glycerin, propylene glycol, sodium microcrystalline cellulose, dextrin, cyclodextrin, or gelatin.

4. The method for preparing the medicament for treating osteoporosis according to claim 2, characterized in that: The medicine is in the form of capsules, granules, tablets, or oral liquid.

Images