A traditional Chinese medicine preparation composition for preventing brittle fracture, a preparation method and application thereof

Abstract

The application provides a traditional Chinese medicine preparation composition for preventing brittle fracture, a preparation method and application thereof, and belongs to the technical field of traditional Chinese medicines. The traditional Chinese medicine preparation composition is composed of the following components: (Z1) a scorching rhizoma drynariae preparation; (Z2) a herba epimedii preparation; and (Z3) a cistanche preparation. The traditional Chinese medicine preparation composition is verified by rich clinical data, and after a course of taking the Peiyuan Jian Gu granules, the bone density of patients with ΔCTX < 0 is significantly improved; on the one hand, it is suggested that CTX can be used as a marker for evaluating drug efficacy, and on the other hand, whether to continue taking the medicine can be prompted through CTX in the metabolic index, thereby providing a reliable theoretical basis for the application of the preparation of the application in the timing and effect judgment of the medicine for preventing brittle fracture.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine technology, specifically relating to a traditional Chinese medicine preparation composition for preventing fragility fractures, its preparation method, and its application. Background Technology

[0002] Fragility fractures, also known as osteoporotic fractures, are fractures caused by decreased bone strength and increased fragility due to osteoporosis. They commonly occur in the elderly or postmenopausal women. Fragility fractures not only affect patients' quality of life but can also lead to serious complications such as disability or death. Currently, the main clinical methods for preventing fragility fractures include calcium supplementation, vitamin D, bisphosphonates, and lifestyle interventions. However, these methods have certain limitations: Western medicines may cause side effects such as gastrointestinal discomfort and kidney damage; furthermore, existing traditional Chinese medicine preparations are mostly for the treatment of osteoporosis, with limited research on early interventions for preventing fragility fractures, and a lack of systematic clinical and basic evidence.

[0003] Traditional Chinese medicine believes that fragility fractures are closely related to factors such as kidney essence deficiency, kidney yang deficiency, and blood stasis. Based on the theory that "the kidney governs the bones," tonifying the kidney and strengthening the bones and muscles is a key approach to preventing fragility fractures. Although existing Chinese herbal preparations have the effect of tonifying the kidney and strengthening bones, their formulas are simple, their active ingredients are unclear, and clinical evidence is insufficient, which cannot meet the clinical needs for preventing fragility fractures.

[0004] Therefore, there is an urgent need in this field for a traditional Chinese medicine preparation that has a reasonable formulation, definite efficacy, high safety, and the ability to prevent fragility fractures. Summary of the Invention

[0005] This invention aims to provide a traditional Chinese medicine preparation with a reasonable formulation, definite efficacy, high safety, and the ability to prevent fragility fractures. Specifically, it relates to a traditional Chinese medicine preparation composition for preventing fragility fractures, its preparation method, and its application.

[0006] In a first aspect of the present invention, a traditional Chinese medicine preparation composition for preventing fragility fractures is provided, the composition comprising the following components:

[0007] (Z1) Preparation of bone-scalding and bone-strengthening herbal medicine;

[0008] (Z2) Epimedium preparations;

[0009] (Z3) Cistanche deserticola preparations;

[0010] The active ingredient in the prepared bone-scalding and purging preparation is 20-50 parts by weight, and the remainder is pharmaceutically acceptable excipients.

[0011] The proportion of active ingredients in the epimedium preparation is 20-50 parts by weight, and the remainder is pharmaceutically acceptable excipients.

[0012] The active ingredient in the Cistanche deserticola preparation is 30-60 parts by weight, and the remainder is pharmaceutically acceptable excipients.

[0013] In another preferred embodiment, the traditional Chinese medicine preparation composition is prepared by decocting Drynaria fortunei, Epimedium brevicornu, and Cistanche deserticola together.

[0014] In another preferred embodiment, the active ingredient in the prepared *Drynaria fortunei* is 50-98 parts by weight, with the remainder being pharmaceutically acceptable excipients.

[0015] In another preferred embodiment, the proportion of active ingredient in the epimedium preparation is 60-100 parts by weight, with the remainder being pharmaceutically acceptable excipients.

[0016] In another preferred embodiment, the proportion of active ingredients in the Cistanche deserticola preparation is 55-80 parts by weight, with the remainder being pharmaceutically acceptable excipients.

[0017] In another preferred embodiment, the fragility fracture is an osteoporotic fracture, including fractures caused by primary osteoporosis.

[0018] In another preferred embodiment, the dosage form is selected from the group consisting of granules, capsules, tablets, pills, or oral liquids.

[0019] In another preferred embodiment, the dosage form is granules.

[0020] In a second aspect of the present invention, a method for preparing a traditional Chinese medicine preparation composition as described in the first aspect is provided, the method comprising the steps of:

[0021] (1) Provide the sliced ​​Drynaria fortunei, decoct it with water 1-3 times, filter the filtrate, concentrate it to obtain Drynaria fortunei paste, add excipients to prepare Drynaria fortunei preparation;

[0022] (2) Provide Epimedium slices, decoct them in water 1-3 times, filter and collect the filtrate, concentrate to obtain Epimedium extract, add excipients to prepare Epimedium preparations;

[0023] (3) Provide Cistanche deserticola, decoct it 1-3 times with water, filter and collect the filtrate, concentrate it to obtain Cistanche deserticola extract, add excipients to prepare Cistanche deserticola preparation.

[0024] In another preferred embodiment, the volume ratio of the hot bone-strengthening herbal slices to water in step (1) is 1:8-10.

[0025] In another preferred embodiment, the volume ratio of Epimedium slices to water in step (2) is 1:8-10.

[0026] In another preferred embodiment, the volume ratio of the Cistanche deserticola slices to water in step (3) is 1:8-10.

[0027] In another preferred embodiment, the plaster of dried bone-setting herb accounts for 8 wt%-15 wt% of the raw material dried bone-setting herb slices, the plaster of epimedium accounts for 8 wt%-15 wt% of the raw material epimedium slices, and the plaster of cistanche accounts for 38 wt%-47 wt% of the raw material cistanche.

[0028] In another preferred embodiment, the simmering time is 30 minutes to 1 hour.

[0029] In another preferred embodiment, the concentration in step (1) is to concentrate the filtrate to a clear paste with a relative density of 1.1-1.5.

[0030] In a third aspect of the invention, a kit is provided, the kit comprising:

[0031] (1) The traditional Chinese medicine preparation composition as described in the first aspect;

[0032] (2) Detection reagent for type I collagen C-terminal peptide (CTX);

[0033] (3) Label or instruction manual;

[0034] The traditional Chinese medicine preparation composition is applied in batches;

[0035] The detection of the type I collagen C-terminal peptide was performed after the first batch of traditional Chinese medicine preparation composition was administered;

[0036] Furthermore, the kit is used to prepare a medicine box for preventing fragility fractures.

[0037] In another preferred embodiment, the traditional Chinese medicine preparation composition is administered at a dose of 30g of medicinal materials per day per person each time.

[0038] In another preferred embodiment, the first batch of the traditional Chinese medicine preparation composition is administered at a dose of 30g of medicinal materials per day per person for 2-4 months.

[0039] In another preferred embodiment, when a decrease in type I collagen C-terminal peptide (CTX) is detected, a second batch of the traditional Chinese medicine preparation composition is applied, wherein the decrease refers to a decrease compared to before the application of the first batch of the traditional Chinese medicine preparation composition, i.e., ΔCTX < 0; when the detection result of type I collagen C-terminal peptide (CTX) is ΔCTX > 0, the application of the traditional Chinese medicine preparation composition is terminated.

[0040] In another preferred embodiment, the label or instructions specify the method of administration:

[0041] (1) Oral administration of the aforementioned traditional Chinese medicine preparation composition;

[0042] (2) When the detection result of type I collagen C-terminal peptide (CTX) ΔCTX>0, the application of the traditional Chinese medicine preparation composition shall be terminated.

[0043] In another preferred embodiment, the detection reagent for type I collagen C-terminal peptide (CTX) is a type I collagen C-terminal peptide (CTX) kit.

[0044] In another preferred embodiment, the detection of type I collagen C-terminal peptide (CTX) kit is performed according to the instructions of the ELISA kit.

[0045] In a fourth aspect of the invention, the use of the kit described in the third aspect is provided for preparing a kit for preventing fragility fractures.

[0046] In another preferred embodiment, the kit inhibits bone resorption by downregulating the bone metabolism marker CTX (type I collagen C-terminal peptide).

[0047] In another preferred embodiment, the medicine box increases bone density.

[0048] In another preferred embodiment, the medicine box increases bone density in the lumbar region.

[0049] In another preferred embodiment, the medicine box increases the probability of fracture-free survival.

[0050] In another preferred embodiment, the kit reduces the number of SA-β-gal positive cells in bone marrow mesenchymal stem cells.

[0051] In another preferred embodiment, the medicine box has the effect of inhibiting cell aging.

[0052] In another preferred embodiment, the kit increases the number of mineralized nodules in the extracellular matrix of bone marrow mesenchymal stem cells.

[0053] In another preferred embodiment, the kit promotes the formation of a mineralized matrix for the maturation of bone marrow mesenchymal stem cells.

[0054] In another preferred embodiment, the cassette promotes calcium salt deposition in bone marrow mesenchymal stem cells.

[0055] In another preferred embodiment, the kit promotes osteogenic differentiation and exerts a osteoprotective effect by regulating the cell metabolism and key molecular functions of bone marrow mesenchymal stem cells (BMSCs).

[0056] In another preferred embodiment, the pharmaceutical composition promotes osteogenic differentiation and mineralization of bone marrow mesenchymal stem cells by regulating cytoskeleton remodeling, calcium ion homeostasis, and related molecular signaling pathways, thereby exerting a osteoprotective effect.

[0057] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0058] Figure 1 The changes in bone metabolism indicators were shown after 3 months of intervention.

[0059] Figure 2 The changes in bone mineral density were shown after 6 months of intervention.

[0060] Figure 3 The study showed the trend of bone mineral density changes after 6 months in the positive group (CTX decreased) and the negative group (CTX did not decrease).

[0061] Figure 4 The results show the percentage of experimental groups in the positive group (CTX decreased) and the negative group (CTX did not decrease).

[0062] Figure 5 The Kaplan-Meier survival curves for intention-to-treat (ITT) are shown.

[0063] Figure 6 The Kaplan-Meier survival curves for protocol compliance (PP) are shown.

[0064] Figure 7 The scheme compliance (PP) Weibull survival curve is shown.

[0065] Figure 8 The study demonstrated the effects of different concentrations of drug-containing serum on the viability of bone marrow mesenchymal stem cells.

[0066] Figure 9 The study demonstrated the effect of serum containing Peiyuan Jian Gu Granules on the aging of bone marrow mesenchymal stem cells.

[0067] Figure 10 The study demonstrated the effect of serum containing Peiyuan Jian Gu Granules on the mineralization capacity of osteoblasts.

[0068] Figure 11 The study showed the GO pathway enrichment analysis of differentially expressed genes after intervention with Peiyuan Granules.

[0069] Figure 12 The KEGG pathway enrichment analysis of differentially expressed genes after intervention with Peiyuan Granules is shown. Detailed Implementation

[0070] Through extensive and in-depth research and numerous experimental screenings, the inventors have developed a traditional Chinese medicine (TCM) preparation composition for preventing fragility fractures. This composition comprises a *Drynaria fortunei* preparation, an *Epimedium* preparation, and a *Cistanche deserticola* preparation. In this invention, rich clinical data verification shows that after one course of treatment with the Peiyuan Jian Gu granules of this invention, patients with ΔCTX < 0 exhibit significant improvement in bone mineral density. This suggests that CTX can serve as a biomarker for evaluating efficacy, and that CTX levels in metabolic indicators can indicate whether to continue medication, providing a reliable theoretical basis for the timing of drug administration and efficacy assessment of the preparation in preventing fragility fractures. Furthermore, through cell experiments using bone marrow mesenchymal stem cells, this invention further demonstrates that the TCM preparation exerts its effects through a multi-target mechanism (such as promoting osteoogenesis, inhibiting bone resorption, and regulating calcium pathways), providing in-depth and reliable theoretical support for the clinical application of the TCM preparation composition. Based on these findings, the inventors have completed this invention.

[0071] Terminology Explanation

[0072] The terms "traditional Chinese medicine preparation composition of the present invention", "composition of the present invention", "preparation of the present invention", "preparation granules of the present invention", and "peiyuan jiangu granules" mentioned in this invention are all interchangeable and refer to preparations obtained by the preparation method of the present invention.

[0073] The traditional Chinese medicine preparation composition of the present invention

[0074] Drynaria fortunei (Kunze) J.Sm., a plant in the Polypodiaceae family, is a dried rhizome. It is bitter and warm in nature, and enters the liver and kidney meridians. It has the effects of tonifying the kidneys and strengthening bones, promoting healing of injuries and relieving pain. It can be used for kidney deficiency causing lower back pain, tinnitus and deafness, loose teeth, falls and sprains, and fractures; externally, it can be used to treat alopecia areata and vitiligo. Hot Drynaria fortunei preparation: Heat sand in a pot, add the cleaned Drynaria fortunei, and stir-fry until it puffs up and the hairs turn yellowish-brown. Quickly remove it, sift out the sand, let it cool, and remove the hairs.

[0075] Epimedium: A plant of the Berberidaceae family, its dried leaves are used medicinally. Raw Epimedium (removed impurities), sprayed with water to slightly moisten, then shredded and dried. For roasted Epimedium, melt mutton fat (20 kg for every 100 kg of Epimedium), add the shredded Epimedium, and stir-fry over low heat until the surface is slightly yellow and the fat is absorbed. Remove and let cool.

[0076] Cistanche deserticola: A plant of the Orobanchaceae family, its dried, fleshy stem with scaly leaves is used medicinally. The pharmacopoeia lists two types of processed Cistanche deserticola: raw Cistanche deserticola slices (raw) and processed Cistanche deserticola (prepared by wine): Raw Cistanche deserticola slices are prepared by removing impurities, washing, soaking thoroughly, cutting into thick slices, and drying (the scaly leaves must be retained); processed Cistanche deserticola is prepared by taking Cistanche deserticola slices, adding rice wine (20 kg of rice wine per 100 kg of Cistanche deserticola), mixing well, allowing the wine to be absorbed, then stir-frying over low heat until the surface turns black, removing and cooling.

[0077] In this invention, the traditional Chinese medicine preparation composition consists of a preparation of *Dryopteris crassirhizoma*, a preparation of *Epimedium*, and a preparation of *Cistanche deserticola*.

[0078] The active ingredient in the *Drynaria fortunei* preparation is 20-50 parts by weight, with the remainder being pharmaceutically acceptable excipients; the active ingredient in the *Epimedium* preparation is 20-50 parts by weight, with the remainder being pharmaceutically acceptable excipients; and the active ingredient in the *Cistanche deserticola* preparation is 30-60 parts by weight, with the remainder being pharmaceutically acceptable excipients.

[0079] Preparation method of traditional Chinese medicine preparation composition of the present invention

[0080] In this invention, a method for preparing a traditional Chinese medicine preparation composition as described in the first aspect is provided, the method comprising the steps of:

[0081] (1) Provide the sliced ​​Drynaria fortunei, decoct it with water 1-3 times, filter the filtrate, concentrate it to obtain Drynaria fortunei paste, add excipients to prepare Drynaria fortunei preparation;

[0082] (2) Provide Epimedium slices, decoct them in water 1-3 times, filter and collect the filtrate, concentrate to obtain Epimedium extract, add excipients to prepare Epimedium preparations;

[0083] (3) Provide Cistanche deserticola, decoct it 1-3 times with water, filter and collect the filtrate, concentrate it to obtain Cistanche deserticola extract, add excipients to prepare Cistanche deserticola preparation.

[0084] Preferably, the above three types of granules are mixed and packaged in a ratio of 10-15g of Drynaria fortunei, 5-15g of Epimedium brevicornu, and 5-15g of Cistanche deserticola, i.e., 1-1.5g of Drynaria fortunei granules, 1.5-2g of Epimedium brevicornu granules, and 6-10g of Cistanche deserticola granules.

[0085] Preferably, the volume ratio of the boiled bone-strengthening herbal slices to water in step (1) is 1:8-10.

[0086] Preferably, the volume ratio of Epimedium slices to water in step (2) is 1:8-10.

[0087] Preferably, the volume ratio of the Cistanche deserticola slices to water in step (3) is 1:8-10.

[0088] Preferably, the hot bone-scraping paste accounts for 8wt%-15wt% of the raw material hot bone-scraping slices, the epimedium paste accounts for 8wt%-15wt% of the raw epimedium slices, and the cistanche paste accounts for 38wt%-47wt% of the raw cistanche.

[0089] The reagent kit of the present invention

[0090] In this invention, a kit is provided, the kit comprising: (1) the traditional Chinese medicine preparation composition of this invention; (2) a detection reagent for type I collagen C-terminal peptide (CTX); (3) a label or instruction manual; and the kit is used to prepare a medicine box for treating fragility fractures.

[0091] Preferably, the detection reagent for type I collagen C-terminal peptide (CTX) is a type I collagen C-terminal peptide (CTX-1) kit.

[0092] Preferably, the detection of type I collagen C-terminal peptide (CTX) kit is performed according to the ELISA kit instructions.

[0093] Preferably, when a decrease in type I collagen C-terminal peptide (CTX) is detected, a second batch of the traditional Chinese medicine preparation composition is applied, wherein the decrease refers to a decrease compared to before the application of the first batch of the traditional Chinese medicine preparation composition, i.e., ΔCTX < 0; when the detection result of type I collagen C-terminal peptide (CTX) is ΔCTX > 0, the application of the traditional Chinese medicine preparation composition is terminated.

[0094] Preferably, ΔCTX = CTX after application of the first batch of traditional Chinese medicine preparation composition - CTX before application of the first batch of traditional Chinese medicine preparation composition.

[0095] Preferably, the label or instructions specify the method of administration:

[0096] (1) Oral administration of the aforementioned traditional Chinese medicine preparation composition;

[0097] (2) When the detection result of type I collagen C-terminal peptide (CTX) ΔCTX>0, the application of the traditional Chinese medicine preparation composition shall be terminated.

[0098] Preferably, the detection reagent for type I collagen C-terminal peptide (CTX) is a type I collagen C-terminal peptide (CTX) kit.

[0099] Preferably, the detection of type I collagen C-terminal peptide (CTX) kit is performed according to the ELISA kit instructions.

[0100] Preferably, the ELISA detection method is as follows:

[0101] First, allow reagents to equilibrate at room temperature for 30 minutes. Prepare reagents, including washing buffer, standards, biotin antigen, and avidin-HRP dilution. Serum treatment: Incubate at room temperature for 10-20 minutes, centrifuge at 3000 rpm for 15 minutes, collect the supernatant, and store at -80℃. Thaw samples before use. Do not add samples to blank wells; add only colorimetric reagents A and B and stop solution for zeroing. Add 50 μL of diluted standards, standard diluent, and sample to standard wells, zero wells, and sample wells, respectively. After adding all samples, add 50 μL of biotin antigen working solution to each well. Gently shake in a cross shape, seal with the film, and incubate at 37℃ for 30 minutes. First wash: Remove the sealing film, shake off excess liquid, fill each well with washing buffer, let stand for 30 seconds, shake off excess liquid, and repeat this process 5 times. Add 50 μL of avidin-HRP to each standard well, zero well, and sample well, and repeat the above incubation and washing steps once. To begin color development, add 50 μL of colorimetric reagent A to each well, followed by colorimetric reagent B in sequence, and incubate for 10 min. If color development is successful, add 50 μL of stop solution to each well. Zero the microscope using a blank well, measure the absorbance at 450 nm, calculate the standard curve, and calculate the concentration.

[0102] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0103] (1) The active ingredients contained in the traditional Chinese medicine preparation composition of the present invention are refined, the principal, assistant, adjuvant and guide ingredients are reasonably matched, the efficacy is definite, the safety is high, and there are no obvious adverse reactions.

[0104] (2) The traditional Chinese medicine preparation composition of the present invention has a simple preparation process, is easy to operate and is easy to industrialize.

[0105] (3) The traditional Chinese medicine preparation composition of the present invention can downregulate the metabolic index CTX in the early stage and significantly improve the bone density of patients in the later stage, especially the bone density of the lumbar region, thereby reducing the incidence of fractures in the later stage and increasing the probability of fracture-free survival.

[0106] (4) In this invention, through extensive clinical data verification, patients with ΔCTX<0 after one course of treatment with Peiyuan Jian Gu Granules of this invention showed a significant improvement in bone density. This suggests that CTX can be used as a biomarker for evaluating drug efficacy, and that CTX in metabolic indicators can indicate whether to continue medication. This provides a reliable theoretical basis for the application of the formulation of this invention in the timing of drug administration and the judgment of its effects in the prevention of fragility fractures.

[0107] (5) The traditional Chinese medicine preparation composition of the present invention inhibits cell senescence by reducing the number of SA-β-gal positive cells in bone marrow mesenchymal stem cells, promotes the formation of mature mineralized matrix of bone marrow mesenchymal stem cells by increasing the number of mineralized nodules in the extracellular matrix of bone marrow mesenchymal stem cells, and promotes calcium salt deposition of bone marrow mesenchymal stem cells.

[0108] (6) In this invention, cell experiments with bone marrow mesenchymal stem cells further demonstrate that the traditional Chinese medicine preparation of this invention can exert its effects through a multi-target mechanism (such as promoting osteogenic formation, inhibiting bone resorption, and regulating calcium pathways). Specifically, it can promote osteogenic differentiation and exert osteoprotective effects by regulating the cell metabolism and key molecular functions of bone marrow mesenchymal stem cells (BMSCs); and promote osteogenic differentiation and mineralization functions of bone marrow mesenchymal stem cells by regulating cytoskeleton remodeling, calcium ion homeostasis, and related molecular signaling pathways, thereby exerting osteoprotective effects. This provides in-depth and reliable theoretical support for the clinical application of the traditional Chinese medicine preparation composition of this invention.

[0109] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated.

[0110] Example 1: Preparation of Granules

[0111] The bone-strengthening granules of the present invention are prepared by the following method:

[0112] (1) Take 6500g of dried bone-strengthening herb slices, add water and decoct, filter, concentrate the filtrate into a clear paste (dry extract yield is 8%-15%), add appropriate amount of excipients, dry (or dry and pulverize), add appropriate amount of excipients again, mix well, granulate, and make 1000g.

[0113] (2) Take 5000g of Epimedium slices, add water and decoct, filter, concentrate the filtrate into a clear extract (dry extract yield is 13%-20%), add appropriate amount of excipients, dry (or dry and pulverize), add appropriate amount of excipients again, mix well, granulate, and make 1000g.

[0114] (3) Take 1500g of Cistanche deserticola (cistanche wine) slices, add water and decoct, filter, concentrate the filtrate into a clear extract (dry extract yield is 38%-47%), add appropriate amount of excipients, mix well, dry (or dry and pulverize), add appropriate amount of excipients again, mix well, granulate, and make 1000g, which is the final product.

[0115] The above three types of granules are mixed and packaged according to the following ratio: 9g of Drynaria fortunei, 9g of Epimedium brevicornu, and 12g of Cistanche deserticola. That is, 1.38g of Drynaria fortunei granules, 1.8g of Epimedium brevicornu granules, and 8g of Cistanche deserticola granules.

[0116] Alternatively, the bone-strengthening granules of the present invention can also be prepared by the following method:

[0117] Take 10-15g of Cistanche deserticola (processed with wine), 5-15g of Epimedium, and 5-15g of Drynaria fortunei, and grind them into coarse powder. Add 8-10 times the amount of water, decoct twice, 1.5 hours each time, combine the filtrates, and concentrate to an extract with a relative density of 1.2. Add an appropriate amount of dextrin and sucrose, mix well, dry, and make into granules, 5g per bag.

[0118] Example 2: Clinical Application Validation

[0119] In the clinical trial, the dosage of the medicine was 30g / day / person. A previous study included 48 patients with osteoporosis (OP), who were randomly assigned to a treatment group and a control group. The treatment group received Peiyuan Jian Gu Granules for 3 consecutive months, while the control group received a placebo. Both groups received granules for 3 consecutive months. For basic treatment, both groups took Caltrate. Among the enrolled cases, there were no significant differences in gender, age, height, weight, lumbar spine and hip bone mineral density, or bone metabolism indicators (PINP, OST, ALP, CTX) between the control and treatment groups, and the overall baseline was relatively balanced (Table 1).

[0120] Table 1 Baseline Overview

[0121]

[0122] Note: BMD, bone mineral density; CTX, type I collagen C-terminal peptide; PINP, type I collagen N-terminal elongated peptide; PTH, parathyroid hormone; ALP, alkaline phosphatase; OST, osteocalcin.

[0123] 1. Changes in bone metabolism markers and bone mineral density

[0124] The 3-month follow-up results showed the following changes in bone metabolism parameters between the control group and the experimental group: ALP (U / L) had an average change of -3.86 (SD=15.6) in the control group and -8.05 (SD=15.7) in the experimental group; OPG (ng / mL) had an average change of -1.97 (SD=5.29) in the control group and -4.89 (SD=10.1) in the experimental group; PINP (ng / mL) had an average change of -3.35 (SD=24.1) in the control group and -5.72 (SD=24.3) in the experimental group. There were no statistically significant differences in these three parameters.

[0125] The mean change in CTX (ng / mL) in the control group was -0.0187, while that in the experimental group was -0.163, and the difference between the two groups was statistically significant (P = 0.0292).

[0126] In other words, during the first three months of treatment, the experimental group showed a greater decrease in CTX levels, while other indicators (ALP, OPG, PINP) showed no significant differences. This suggests that the treatment effect can be monitored by detecting CTX levels, and also indicates that Peiyuan Jian Gu Granules have a certain inhibitory effect on bone resorption. Figure 1 ).

[0127] The 6-month follow-up results showed that ( Figure 2 The changes in bone mineral density in the control group and the experimental group are as follows:

[0128] The mean change in lumbar bone mineral density (T-score) was -0.271 in the control group (SD = 0.399) and 0.093 in the experimental group (SD = 0.336), and the difference between the two groups was statistically significant (P = 0.015).

[0129] The mean change in lumbar bone mineral density (Z-score) in the control group was -0.207 (SD = 0.347), while that in the experimental group was 0.157 (SD = 0.399), and the difference was statistically significant (P = 0.016).

[0130] The mean change in hip bone mineral density (T-score) was -0.007 in the control group and 0.043 in the experimental group. The mean change in hip bone mineral density (Z-score) was 0 in the control group and -0.036 in the experimental group. The difference was not statistically significant.

[0131] As shown above, the experimental group exhibited greater positive changes in the T and Z values ​​of lumbar bone mineral density, suggesting that Peiyuan Jian Gu Granules can effectively improve the bone mineral density of patients.

[0132] Overall, the experimental group showed greater positive changes in lumbar bone mineral density T-scores and Z-scores, suggesting that Peiyuan Jian Gu Granules can effectively improve bone mineral density in patients. Data from some follow-up patients are shown in Tables 2 and 3 below.

[0133] Table 2 shows bone mineral density data of some follow-up patients before and after treatment.

[0134]

[0135]

[0136] Table 3 shows the CTX and bone mineral density changes in some follow-up patients.

[0137] in conclusion

[0138] The data in the table show that after the first dose of medication, some patients experienced a decrease in CTX levels. Patients with decreased CTX levels, compared to those with increased CTX levels, showed a significant increase in bone mineral density during the subsequent 6-month follow-up. This indicates that for patients whose CTX levels decreased after intervention, long-term use of the Peiyuan Jian Gu Granules of this invention can achieve greater bone mineral density benefits.

[0139] In other words, if the bone metabolite CTX value does not decrease after administering one batch of the Peiyuan Jian Gu Granules of the present invention (i.e., ΔCTX≥0), it should be recommended to stop taking the Peiyuan Jian Gu Granules of the present invention and change the treatment strategy; if the bone metabolite CTX value decreases after administering one batch of the Peiyuan Jian Gu Granules of the present invention (i.e., ΔCTX<0), it indicates that the Peiyuan Jian Gu Granules of the present invention are effective for the patient, and it can be recommended to continue taking the medication to consolidate the effect and achieve the goal of continuously increasing bone density and reducing the probability of fracture.

[0140] Further, based on 3-month CTX changes, subjects were divided into a positive group (CTX decreased) and a negative group (CTX did not decrease). The positive group showed a positive trend in bone mineral density changes over 6 months, especially in the hip T-score, with an average change of 0.2 (compared to -0.2 in the negative group). Cohen's d = 0.85 indicates a large effect size, suggesting a more significant improvement in bone mineral density in the positive group. The lumbar T-score (d = 0.59, moderate effect) and lumbar Z-score (d = 0.41, moderate effect) also showed better changes in the positive group (0 vs. -0.2 and 0 vs. -0.1, respectively). See [link to relevant documentation]. Figure 3 This indicates that patients with a more significant decrease in CTX after 3 months of intervention benefited more in bone mineral density after 6 months of intervention. Furthermore, the proportion of the experimental group (intervention group) in the positive CTX group was 55.6%, higher than the 40.0% in the negative group (see...). Figure 4 This distribution trend supports the possibility that Peiyuan Jian Gu Granules may promote an early decrease in CTX, i.e., inhibit osteoclasts, thereby increasing bone density.

[0141] Therefore, the 6-month follow-up results show that patients with ΔCTX < 0 after 3 months of taking the Peiyuan Jian Gu granules of this invention experienced a significant improvement in bone mineral density. This result suggests that CTX can serve as a biomarker for evaluating efficacy, and that CTX levels in metabolic indicators can suggest whether to continue medication.

[0142] 2. Follow-up results of fracture

[0143] Four-year fracture follow-up results showed that, in the intention-to-treat (ITT) analysis,

[0144] In the control group, 5 fractures occurred, with an incidence rate of 20.83% and a 4-year fracture-free survival probability of 0.83 (95% CI: 0.69-0.99). In the experimental group, no fractures occurred, with an incidence rate of 0% and a 4-year fracture-free survival probability of 1.00. Fisher's exact test showed a statistically significant difference between the groups (P = 0.049). Figure 5 .

[0145] In the protocol-specific (PP) analysis, 5 fractures occurred in the control group, with a 4-year fracture-free survival probability of 0.76 (95% CI: 0.58–0.99). No fractures occurred in the experimental group, with a 4-year fracture-free survival probability of 1.0000. (See [link to relevant data]). Figure 6 .

[0146] The Weibull model showed a significant difference between groups (Chisq = 7.2, P = 0.0073), with a hazard ratio (HR) of 1.07e-09 (experimental group vs. control group), indicating that the fracture risk in the experimental group was extremely low. Figure 7 .

[0147] Overall, the experimental group showed higher fracture-free survival rates in both ITT and PP analyses, suggesting that the intervention may significantly reduce the risk of fracture, especially in the protocol-compliant population.

[0148] In summary, clinical trial data demonstrate that the Peiyuan Jian Gu Granules of this invention can downregulate the metabolic index CTX in the early stage and significantly improve bone mineral density, especially lumbar bone mineral density, in the middle stage, thereby reducing the incidence of fractures and increasing the probability of fracture-free survival. These results suggest that after taking the Peiyuan Jian Gu Granules of this invention, the efficacy can be measured by monitoring the CTX level. Furthermore, after the CTX level decreases, continued use of the Peiyuan Jian Gu Granules can consolidate the therapeutic effect, further reducing the incidence of fractures and increasing the probability of fracture-free survival.

[0149] Example 3: Cellular Experiment of Peiyuan Jian Gu Granules Intervening in Bone Marrow Mesenchymal Stem Cells

[0150] In this experiment, rats were given Peiyuan Jian Gu Granules, and drug-containing serum containing the active pharmaceutical ingredients was collected from their blood. In vitro experiments were conducted on the intervention of primary mesenchymal stem cells of mice with Peiyuan Jian Gu Granules-containing serum, and cell senescence, early osteogenic differentiation capacity, and calcium salt deposition after osteogenic differentiation were detected. At the same time, the genes and pathways related to the efficacy of Peiyuan Jian Gu Granules were identified by transcriptome sequencing.

[0151] The effects of different concentrations of drug-containing / control serum on the viability of primary bone marrow mesenchymal stem cells were detected using a 24h / 48h CCK-8 assay. Differences between the drug-containing serum group and the control serum group were compared to screen for the optimal concentration of drug-containing serum—10%—that showed no significant inhibitory effect on cell viability, which was then used as the condition for subsequent experiments. Figure 8 ).

[0152] Peiyuan Jian Gu Granules contain serum that has a significant inhibitory effect on cell senescence. Figure 9 SA-β-gal staining results showed that, compared with the control serum group, the number of SA-β-gal positive cells in the drug-containing serum group was significantly reduced (P<0.05), suggesting that the drug-containing serum of Peiyuan Jian Gu Granules can effectively reduce the degree of cell senescence and play a role in protecting cell activity and delaying aging.

[0153] Von Kossa staining was used to detect calcium phosphate mineral deposition in the extracellular matrix, further supporting the claim that serum containing Peiyuan Jian Gu Granules effectively promotes the formation of mature mineralized matrix in bone marrow mesenchymal stem cells. Figure 10 As shown, compared with the control serum group, the number of mineralized nodules and the area of ​​black mineralized deposits were significantly increased in the drug-containing serum group. Further semi-quantitative analysis showed that the integrated density and the percentage of mineralized area in the drug-containing serum group were significantly higher than those in the control serum group, and the differences between the groups were statistically significant (P<0.05).

[0154] To further explore the potential mechanism by which serum containing Peiyuan Jian Gu Granules promotes calcium salt deposition in bone marrow mesenchymal stem cells, the inventors conducted GO functional classification analysis on the differentially expressed genes in bone marrow mesenchymal stem cells of the drug-containing serum group and the control serum group.

[0155] like Figure 11 The differentially expressed genes were mainly enriched in osteogenic biological processes such as cellular processes, metabolic processes, response to stimulus, and biological regulation, suggesting that drug-containing serum may effectively promote osteogenic differentiation and calcium salt deposition by regulating the cellular metabolism and stress response of bone marrow mesenchymal stem cells.

[0156] Furthermore, molecular function enrichment analysis showed significant enrichment in binding and catalytic activity, which are closely related to key molecular functions such as extracellular matrix protein synthesis, calcium ion binding, and enzyme activity regulation during osteoogenesis.

[0157] The above results suggest that serum containing Peiyuan Jian Gu Granules may exert an osteoprotective effect by influencing BMSC cell metabolism and key molecular functions, thereby promoting osteogenic differentiation.

[0158] Furthermore, KEGG enrichment analysis showed that differentially expressed genes were significantly enriched in biological processes and molecular functions related to actin-myosin filament sliding, myofibril assembly, and the negative regulation of cytosolic calcium ion concentration. Figure 12 These results suggest that serum containing Peiyuan Jian Gu granules may exert a osteoprotective effect by regulating cytoskeleton remodeling, calcium ion homeostasis, and related molecular signaling pathways, thereby promoting osteogenic differentiation and mineralization of bone marrow mesenchymal stem cells.

[0159] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A traditional Chinese medicine preparation composition for preventing fragility fractures, characterized in that, The composition comprises the following components: (Z1) Preparation of bone-scalding and bone-strengthening herbal medicine; (Z2) Epimedium preparations; and (Z3) Cistanche deserticola preparations; The active ingredient in the prepared bone-scalding and purging preparation is 20-50 parts by weight, and the remainder is pharmaceutically acceptable excipients. The proportion of active ingredients in the epimedium preparation is 20-50 parts by weight, and the remainder is pharmaceutically acceptable excipients. The active ingredient in the Cistanche deserticola preparation is 30-60 parts by weight, and the remainder is pharmaceutically acceptable excipients.

2. The traditional Chinese medicine preparation composition as described in claim 1, characterized in that, The fragility fracture is an osteoporotic fracture, including fractures caused by primary osteoporosis.

3. The traditional Chinese medicine preparation composition according to claim 1, wherein the dosage form is selected from the group consisting of granules, capsules, tablets, pills, or oral liquids.

4. A method for preparing the traditional Chinese medicine preparation composition as described in claim 1, characterized in that, The method includes the following steps: (1) Provide the sliced ​​Drynaria fortunei, decoct it with water 1-3 times, filter the filtrate, concentrate it to obtain Drynaria fortunei paste, add excipients to prepare Drynaria fortunei preparation; (2) Provide Epimedium slices, decoct them in water 1-3 times, filter and collect the filtrate, concentrate to obtain Epimedium extract, add excipients to prepare Epimedium preparations; (3) Provide Cistanche deserticola, decoct it 1-3 times with water, filter and collect the filtrate, concentrate it to obtain Cistanche deserticola extract, add excipients to prepare Cistanche deserticola preparation.

5. The method as described in claim 4, characterized in that, The hot bone-soothing paste accounts for 8wt%-15wt% of the raw material hot bone-soothing slices, the epimedium paste accounts for 8wt%-15wt% of the raw epimedium slices, and the cistanche paste accounts for 38wt%-47wt% of the raw cistanche.

6. A reagent kit, characterized in that, The kit includes: (1) The traditional Chinese medicine preparation composition as described in claim 1; (2) Detection reagent for type I collagen C-terminal peptide (CTX); (3) Label or instruction manual; The traditional Chinese medicine preparation composition is applied in batches; The detection of the type I collagen C-terminal peptide was performed after the first batch of traditional Chinese medicine preparation composition was administered; Furthermore, the kit is used to prepare a medicine box for preventing fragility fractures.

7. The kit according to claim 6, characterized in that, When a decrease in type I collagen C-terminal peptide (CTX) is detected, a second batch of the traditional Chinese medicine preparation composition is administered. The decrease refers to a decrease compared to before the administration of the first batch of the traditional Chinese medicine preparation composition, i.e., ΔCTX < 0. When the detection result of type I collagen C-terminal peptide (CTX) is ΔCTX > 0, the administration of the traditional Chinese medicine preparation composition is terminated.

8. Use of the kit according to claim 6, characterized in that, Used to prepare medicine boxes for preventing fragility fractures.

9. The use as described in claim 8, characterized in that, The medicine box inhibits bone resorption by downregulating the bone metabolism marker CTX.

10. The use as described in claim 8, characterized in that, The kit promotes osteogenic differentiation and exerts a bone-protective effect by regulating the cell metabolism and key molecular functions of bone marrow mesenchymal stem cells (BMSCs).

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