Radix codonopsis and radix astragali kidney-tonifying composition as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of traditional Chinese medicine preparation, and provides a radix codonopsis and radix astragali kidney-tonifying composition as well as a preparation method and application thereof, and the radix codonopsis and radix astragali kidney-tonifying composition is prepared from the following raw materials in parts by weight: 7 to 13 parts of radix codonopsis, 12 to 18 parts of radix astragali, 7 to 13 parts of fructus corni, 7 to 13 parts of fructus rubi, 7 to 13 parts of fructus lycii, 12 to 18 parts of rhizoma polygonati and 7 to 13 parts of folium cortex eucommiae. The prepared medicament has the advantages of being convenient to take, carry and store, stable and controllable in product quality, simple and convenient in production process and economical in cost, and has the remarkable effects of tonifying kidney, strengthening essence and strengthening tendons and bones. By deepening the development and utilization of medicinal and edible raw materials such as astragalus membranaceus and codonopsis pilosula, the uniform and stable traditional Chinese medicine granules are prepared, and the traditional Chinese medicine granules are suitable for conditioning the sub-health status caused by work fatigue and life pressure of modern people and can also meet the health-preserving requirements of middle-aged and elderly people for spleen-kidney deficiency and deficiency of qi and yin.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine preparation technology, and in particular to a ginseng and astragalus kidney-tonifying composition, its preparation method, and its application. Background Technology

[0002] Studies have shown that osteoporosis has become a major public health problem threatening the physical and mental health of middle-aged and elderly people worldwide. Traditional Chinese medicine (TCM) believes that in the elderly, the body's vital energy (qi) gradually declines, leading to kidney essence deficiency, yin-yang imbalance, yin deficiency and blood stasis, yang deficiency and blood coagulation, obstructing the bone vessels, preventing bone marrow production, and causing bone atrophy and bone loss, thus leading to osteoporosis. Based on the TCM theories of "the kidney stores essence, governs bone and produces marrow" and "the liver and kidney share the same origin," the fundamental pathogenesis of osteoporosis lies in "kidney essence deficiency and failure to transform essence and blood," resulting in insufficient nourishment of the bones. Therefore, the key to its prevention and treatment lies in regulating and tonifying the liver and kidneys to restore bone nourishment and reduce bone loss. This study, adhering to the TCM concept of "treating disease before it occurs," has developed a TCM formula granule with the effects of tonifying the liver and kidneys and strengthening tendons and bones, aiming to prevent bone loss from the source and block the progression of osteoporosis, which is of great significance for safeguarding public health. Summary of the Invention

[0003] The purpose of this invention is to provide a ginseng and astragalus kidney-tonifying composition, its preparation method and application. This ginseng and astragalus kidney-tonifying granule is very suitable for the sub-health state caused by modern work fatigue and life stress, as well as for the conditioning of spleen and kidney deficiency and qi and yin deficiency in middle-aged and elderly people.

[0004] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a composition for tonifying the kidney with ginseng and astragalus, comprising the following raw materials in parts by weight: 7-13 parts of Codonopsis pilosula, 12-18 parts of Astragalus membranaceus, 7-13 parts of Cornus officinalis, 7-13 parts of Rubus idaeus, 7-13 parts of Lycium barbarum, 12-18 parts of Polygonatum sibiricum, and 7-13 parts of Eucommia ulmoides leaves.

[0005] The present invention also provides a ginseng and astragalus kidney-tonifying granule containing the composition, comprising the following raw materials in parts by weight: 50-56 parts of the composition, 43-49 parts of diluent, and 0.5-2 parts of flavoring agent.

[0006] Preferably, the diluent comprises the following raw materials in parts by weight: 4-6 parts dextrin and 2-4 parts mannitol; the flavoring agent comprises the following raw materials in parts by weight: 4-6 parts mogroside and 2-4 parts anhydrous citric acid.

[0007] The present invention also provides a method for preparing the aforementioned ginseng and astragalus kidney-tonifying granules, comprising the following steps: (1) Weigh the raw materials according to the mass ratio of Codonopsis pilosula 7-13 parts, Astragalus membranaceus 12-18 parts, Cornus officinalis 7-13 parts, Rubus idaeus 7-13 parts, Lycium barbarum 7-13 parts, Polygonatum sibiricum 12-18 parts and Eucommia ulmoides leaf 7-13 parts, mix them to obtain a mixture for later use; (2) Mix the mixture with water, soak, reflux extract 2-4 times, combine the decoctions, vacuum filter to obtain a thick paste; (3) Dry the thick paste to obtain extract powder; (4) Mix the extract powder, diluent, flavoring agent and wetting agent, sieve and dry to obtain the final ginseng and astragalus kidney-tonifying granules.

[0008] Preferably, in step (2), the mass ratio of the mixture to water is 0.8~1.2:10~12; the soaking time is 20~40 min; the reflux extraction time is 60~80 min; and the reflux extraction temperature is 95~105℃.

[0009] Preferably, the drying temperature in step (3) is 60~80℃ and the drying time is 45~50h.

[0010] Preferably, the wetting agent in step (4) is 80-90% ethanol, and the amount of the wetting agent is 20-30% of the total mass of the extract powder, diluent and flavoring agent.

[0011] Preferably, the mesh size of the sieve in step (4) is 70~90 mesh, the drying temperature is 40~50℃, and the drying time is 3~5h.

[0012] The beneficial effects of this invention are as follows: Astragalus membranaceus, sweet and warm, tonifies qi and raises yang, strengthens the defensive qi and consolidates the exterior; Codonopsis pilosula, sweet and neutral, tonifies while also protecting, tonifies the middle jiao and benefits qi, generates fluids and nourishes blood. These two herbs complement each other, serving as the principal herbs, greatly tonifying primordial qi, nourishing qi and blood, laying the foundation for the entire formula's qi-tonifying effect. This ensures sufficient qi and blood, leading to the natural generation of essence and blood. Polygonatum sibiricum, sweet and neutral, nourishes yin and replenishes essence, invigorates qi and strengthens the spleen, and gently tonifies the yin of the spleen and kidneys; Lycium barbarum, sweet and neutral, nourishes the liver and kidneys, benefits essence and improves eyesight. Together, they assist the principal herbs in nourishing yin and replenishing essence, serving as the assistant herbs, targeting the root cause of kidney essence deficiency, ensuring sufficient kidney essence and nourished bone marrow. Cornus officinalis, sour and warm, tonifies the liver and kidneys while astringing and consolidating; Rubus idaeus, sweet, sour and slightly warm, excels at tonifying the kidneys, consolidating essence, and reducing urination. Both serve as adjuvant herbs, enhancing the astringent and consolidating effects, consolidating the body's essence and preventing the dissipation of essence and blood. Eucommia ulmoides leaves, sweet and warm, primarily enter the liver and kidney meridians, effectively tonifying the liver and kidneys, and strengthening tendons and bones. As an adjuvant, it guides the other herbs downwards to the liver and kidneys, while simultaneously strengthening muscles and bones, thus directing the overall tonifying effect of the formula to the bones and directly enhancing their resilience. The combined effects of the herbs are nourishing without being drying, moisturizing without being cloying, and employing both tonifying and astringent properties to collectively tonify the kidneys, replenish essence, and strengthen muscles and bones.

[0013] This invention utilizes thin-layer chromatography (TLC) for the qualitative identification of Shenqi Bushen granules. TLC analysis was performed on Astragalus membranaceus, Codonopsis pilosula, Rubus idaeus, Eucommia ulmoides leaves, and Lycium barbarum in the prescription. The specificity and robustness of the TLC conditions for the identified samples under different manufacturers, humidity levels, temperatures, and light sources were investigated. An analytical method with good specificity, ideal separation effect, and no interference from negative samples was established, providing important reference for the quality evaluation of Shenqi Bushen formula.

[0014] This invention investigates the effects of Shenqi Bushen Decoction on kidney and bone-related indicators in glucocorticoid-induced osteoporosis mice through pharmacological experiments. The experiments demonstrate that Shenqi Bushen Decoction can maintain the functional balance between osteoclasts and osteoblasts by inhibiting the release of inflammatory factors; improve osteoporosis by tonifying the kidneys by increasing estrogen levels in mice; inhibit adipogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and reduce bone loss; and increase bone mineralization deposition rate (MAR) and bone formation rate (BFR / BS) in mice, promoting osteogenic activity and directly promoting bone growth, leading to an increase in bone mass. Attached Figure Description

[0015] Figure 1 The effect of extraction times on the content of active ingredients; Figure 2 The effect of extraction time on the content of active ingredients; Figure 3 The effect of the material-to-liquid ratio on the content of active ingredients; Figure 4 Extraction time × material-to-liquid ratio; Figure 5 Extraction time × number of extractions; Figure 6 Extraction count × material-to-liquid ratio; Figure 7 Thin-layer chromatography for identification of Astragalus membranaceus, where 1: astragaloside A reference standard; 2: Astragalus membranaceus reference material; 3-5: test solution; 6: negative solution for Astragalus membranaceus deficiency; Figure 8 Thin-layer chromatography for identification of Codonopsis pilosula, including: 1: Codonopsis pilosula glycoside reference standard; 2: Codonopsis pilosula reference material; 3-5: test solution; 6: negative solution for Codonopsis pilosula deficiency; Figure 9 For the thin-layer chromatography identification of raspberries, 1: linalool reference standard; 2: raspberry reference material; 3-5: test solution; 6: negative solution for raspberries (without raspberry); Figure 10 Thin-layer chromatography for the identification of Eucommia ulmoides leaves, including: 1: chlorogenic acid reference standard; 2: Eucommia ulmoides leaf reference material; 3-5: test solution; 6: negative solution for Eucommia ulmoides leaves. Figure 11Thin-layer chromatography was used for the identification of wolfberry, where 1: wolfberry reference material; 2-4: test solution; 5: negative solution for wolfberry deficiency. Figure 12 The result of the experimental mouse body weight ( ±SD, n=12); Figure 13 Results of Micro CT scan; Figure 14 HE staining results of kidney tissue from osteoporotic mice; Figure 15 HE staining results of femoral tissue from osteoporotic mice. Detailed Implementation

[0016] This invention provides a composition for tonifying the kidney with ginseng and astragalus, comprising the following raw materials in parts by weight: 7-13 parts of Codonopsis pilosula, 12-18 parts of Astragalus membranaceus, 7-13 parts of Cornus officinalis, 7-13 parts of Rubus idaeus, 7-13 parts of Lycium barbarum, 12-18 parts of Polygonatum sibiricum, and 7-13 parts of Eucommia ulmoides leaves.

[0017] In this invention, the kidney-tonifying composition preferably contains the following raw materials in parts by weight: 10 parts Codonopsis pilosula, 15 parts Astragalus membranaceus, 10 parts Cornus officinalis, 10 parts Rubus idaeus, 10 parts Lycium barbarum, 15 parts Polygonatum sibiricum, and 10 parts Eucommia ulmoides leaves.

[0018] The present invention also provides a ginseng and astragalus kidney-tonifying granule containing the composition, comprising the following raw materials in parts by weight: 50-56 parts of the composition, 43-49 parts of diluent, and 0.5-2 parts of flavoring agent.

[0019] In this invention, the ginseng and astragalus kidney-tonifying granules preferably contain the following raw materials in parts by weight: 53 parts of the composition, 46 parts of diluent, and 1 part of flavoring agent.

[0020] In this invention, the diluent preferably contains the following raw materials in parts by weight: 4-6 parts dextrin and 2-4 parts mannitol, and more preferably contains the following raw materials in parts by weight: 5 parts dextrin and 3 parts mannitol; the flavoring agent preferably contains the following raw materials in parts by weight: 4-6 parts mogroside and 2-4 parts anhydrous citric acid, and more preferably contains the following raw materials in parts by weight: 5 parts mogroside and 3 parts anhydrous citric acid.

[0021] The present invention also provides a method for preparing the aforementioned ginseng and astragalus kidney-tonifying granules, comprising the following steps: (1) Weigh the raw materials according to the mass ratio of Codonopsis pilosula 7-13 parts, Astragalus membranaceus 12-18 parts, Cornus officinalis 7-13 parts, Rubus idaeus 7-13 parts, Lycium barbarum 7-13 parts, Polygonatum sibiricum 12-18 parts and Eucommia ulmoides leaf 7-13 parts, mix them to obtain a mixture for later use; (2) Mix the mixture with water, soak, reflux extract 2-4 times, combine the decoctions, vacuum filter to obtain a thick paste; (3) Dry the thick paste to obtain extract powder; (4) Mix the extract powder, diluent, flavoring agent and wetting agent, sieve and dry to obtain the final ginseng and astragalus kidney-tonifying granules.

[0022] In this invention, the mass ratio of the mixture to water in step (2) is preferably 0.8~1.2:10~12, more preferably 1:11; the soaking time is preferably 20~40 min, more preferably 25~35 min, and even more preferably 30 min; the reflux extraction time is preferably 60~80 min, more preferably 70 min, the reflux extraction temperature is preferably 95~105℃, more preferably 100℃, and the number of reflux extractions is preferably 2 times; the relative density of the thick paste is preferably 1.32~1.35 g / mL, more preferably 1.34 g / mL.

[0023] In this invention, the drying temperature in step (3) is preferably 60~80℃, more preferably 65~75℃, and even more preferably 70℃, the drying time is preferably 45~50h, more preferably 48h, and the moisture content of the extract powder is preferably <5wt%, more preferably 4wt%.

[0024] In this invention, the wetting agent in step (4) is preferably 80-90% ethanol, more preferably 85% ethanol, and the amount of the wetting agent is preferably 20-30% of the total mass of the extract powder, diluent and flavoring agent, more preferably 25% of the total mass of the extract powder, diluent and flavoring agent.

[0025] In this invention, the mesh size of the sieve in step (4) is preferably 70-90 mesh, more preferably 75-85 mesh, and even more preferably 80 mesh; the drying temperature is preferably 40-50℃, more preferably 45℃; and the drying time is preferably 3-5h, more preferably 3.5-4.5h, and even more preferably 4h.

[0026] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0027] Example 1: Optimization of the extraction process of Shenqi Bushen Granules by combining single-factor and response surface methodology.

[0028] 1. The contents of total saponins and total polysaccharides in this formula were determined by ultraviolet spectrophotometry. The effects of key process parameters such as extraction time, number of extractions, and solid-liquid ratio on the extraction rate of effective components were investigated through single-factor experiments. Furthermore, the extraction process was optimized by combining single-factor and response surface methodology to determine the optimal extraction conditions.

[0029] 1.2 Establishment of the standard curve

[0030] 1.2.1 Polysaccharide Standard Curve

[0031] Accurately weigh 1.0 g of sample powder, add 100 ml of 80% ethanol, and reflux in a water bath for 2 h. Centrifuge at 4000 rpm for 10 min, discard the supernatant, add 100 ml of 95% ethanol to the residue, centrifuge at 4000 rpm for 10 min, discard the supernatant, and dilute to 100 mL with distilled water. Take 10 mg of glucose standard and dilute to 100 mL with distilled water in a volumetric flask to obtain a 0.1 mg / mL reference solution. Pipette 0.0 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, and 1.0 mL into test tubes, respectively, add distilled water to 1 mL, add 1 mL of 5% phenol solution, slowly add 5 mL of concentrated sulfuric acid, shake well, heat in a 90℃ water bath for 30 min, cool to room temperature, and use distilled water as a blank control. Measure the absorbance at 490 nm and plot a standard curve with glucose concentration on the x-axis and absorbance on the y-axis. The regression equation is: A = 0.1779c + 0.0205, R0 2 =0.9997. The results show that the glucose injection concentration exhibits a good linear relationship in the range of 1.80 to 4.91.

[0032] 1.2.2 Saponin Standard Curve

[0033] Accurately weigh 2.5 mg of astragaloside A reference standard dried to constant weight, place it in a 10 ml volumetric flask, dissolve it in anhydrous ethanol, and dilute to the mark to prepare a reference solution with a mass concentration of 0.25 mg / ml. Accurately pipette 0, 0.2, 0.4, 0.6, 0.8, and 1.0 ml of the astragaloside A reference standard solution into 10 ml stoppered test tubes, evaporate to dryness in a water bath, add 0.2 ml of 5% vanillin-glacial acetic acid solution and 0.8 ml of perchloric acid to each, heat in a 65℃ water bath for 15 min, remove, cool to room temperature, add glacial acetic acid to each to a final volume of 5 ml, and use 0 ml of astragaloside A reference standard solution as a blank control within 30 min. Measure the absorbance at 560 nm. Plot a standard curve with absorbance as the ordinate and the mass concentration of the astragaloside A reference standard solution as the abscissa. The regression equation is: A = 0.1689x + 0.0131, R² = 0.9998. The results showed that astragaloside A exhibited a good linear relationship within the range of 3.14–8.31.

[0034] 1.3 Results of Single-Factor Experiment

[0035] Experiments revealed that the number of extractions significantly affected the dissolution of active ingredients. With increasing extraction frequency, the total polysaccharide and total flavonoid content of the extract showed a gradual decreasing trend. The first extraction yielded the highest dissolution rate of active ingredients, while subsequent extractions may have damaged the polysaccharide structure due to prolonged extraction time, leading to a decrease in content. Therefore, this study selected two extractions as the optimal number of extractions to ensure sufficient dissolution of the components while avoiding resource waste caused by multiple extractions. The results are shown below. Figure 1 .

[0036] The extraction was performed twice, with a solid-liquid ratio of 1:10. Five extraction times (40 min, 50 min, 60 min, 70 min, and 80 min) were established to investigate the effect of extraction temperature on the active ingredients. The results showed that the total polysaccharide and total saponin contents reached their peak values ​​at an extraction time of 60 min. The results are shown below. Figure 2 .

[0037] The extraction time was set at 60 min, and the extraction was performed twice. Different solid-liquid ratios were set to investigate the effect of the solid-liquid ratio on the content of active ingredients. The results showed that the contents of total polysaccharides and total saponins reached their peak at a solid-liquid ratio of 1:10. The results are shown below. Figure 3 .

[0038] 1.4 Response Surface Test

[0039] Through single-factor experiments, three-factor, three-level response surface optimization experiments were conducted by selecting extraction times of 50 min, 60 min, and 70 min, extraction times of 1, 2, and 3, and material-liquid ratios of 1:8, 1:10, and 1:12.

[0040] A response surface methodology (RSM) experiment was designed using the Box-Behnken design principle. Multiple quadratic regression analysis was performed on the response surface results, yielding the regression equation: OD = 4.60 + 0.4150A + 0.6075B + 0.4125C - 0.1850AB - 0.0300AC + 0.1800BC - 0.4145A 2 -1.17B 2 -0.6595C 2 (OD: Total polysaccharide content). Analysis of variance was performed on the model. The F-value was 29.21, P < 0.0001, indicating the model is significant. The F-value for the lack-of-fit term was 1.19, P = 0.4204, which was not significant, indicating a good model fit. The influence of each factor on the total polysaccharide content was in the order of B (number of extractions) > A (extraction time) > C (solid-liquid ratio). The dominant effects of the number of extractions and extraction time may be related to the solubility of polysaccharide components, while the secondary effect of the solid-liquid ratio reflects the balance requirement of solvent supply. The results are shown in Table 1 below.

[0041] Table 1. Response surface methodology analysis of variance

[0042] Note: P <0.05 indicates a significant effect. P <0.01 indicates a highly significant effect. P A value greater than 0.05 indicates that the effect is not significant.

[0043] Using total polysaccharide content as an indicator, a response surface methodology was established using Design-Expert 13.0.1.0 software to explore the effects of the interaction between extraction time, extraction times, and solid-liquid ratio on polysaccharide dissolution and the optimal combination thereof. (See...) Figures 4-6 .

[0044] Based on a regression model fitted using response surface methodology, the optimal number of extractions (2.204), extraction time (70 min), and material-to-liquid ratio (1:10.636) were predicted, with a theoretical total polysaccharide content of 14.125 mg / g. Considering practical production feasibility, the parameters were optimized to 2 extractions, 70 min extraction time, and a material-to-liquid ratio of 1:11. Three repeated experiments were conducted under these conditions, and the average total polysaccharide content was 14.112 mg / g, showing no significant difference from the model's predicted theoretical value (P<0.05), demonstrating the stability and reliability of the extraction process. Under the optimal extraction conditions, the total saponin content in the Shenqi Bushen Fang solid beverage was 7.495 mg / g, with a yield of 29.65%, indicating that the process effectively retains the active ingredients and solids of Astragalus membranaceus.

[0045] Example 2: Optimization of the molding process of Shenqi Bushen Granules by combining single-factor experiments and orthogonal experiments.

[0046] 2.1 Particle Preparation Method

[0047] After extracting and concentrating the drug under the above-mentioned optimal extraction conditions, it was dried in an oven at 70°C. The dried extract was then pulverized, passed through a 100-mesh sieve, sealed, and placed in a desiccator for later use. A diluent was added to the fine powder, and the mixture was wet-granulated into granules.

[0048] 2.1.1 Molding property test

[0049] The granule forming rate was determined according to the "Determination of Particle Size and Particle Size Distribution" method in the 2020 edition of the Chinese Pharmacopoeia (Part IV). The total percentage of granules that can pass through sieve No. 1 and those that cannot pass through sieve No. 5 should not be less than 85%.

[0050] 2.1.2 Hygroscopicity test

[0051] Take an appropriate amount of granules and dry them to constant weight in a 30℃ drying oven. Transfer the dried granules to a volumetric flask that has also been kept at constant weight. Accurately weigh the granules and place them in a desiccator (pre-equilibrated with supersaturated NaCl solution for 24 hours at a relative humidity of 75%). After 48 hours, accurately weigh them again. Repeat the weighing process three times and take the average value. Calculate the moisture absorption rate; the moisture absorption rate of the granules should be less than 30%.

[0052] 2.1.3 Angle of Repose Measurement

[0053] Take an appropriate amount of particles and place the graph paper horizontally on the table. Fix three funnels connected end to end, with the bottom of the funnels 2 cm above the table. Let the particles to be tested fall from top to bottom along the wall of the fixed funnels until the top of the particles stops rising. Record the diameter of the cone bottom and the height of the cone of the particle. Repeat 5 times and take the average value. The angle of repose of the particles should be less than 50°.

[0054] 2.1.4 Solubility test

[0055] The solubility of the granules was determined according to the "General Rules" of the 2020 edition of the Chinese Pharmacopoeia (Part IV), specifically the method for testing the solubility of soluble granules. 10g of granules was weighed and added to 200mL of water at 100℃. The mixture was stirred for 5 minutes, and the dissolution time was immediately observed and recorded.

[0056] 2.2 Single-factor investigation

[0057] 2.2.1 Investigation of Granule Diluents: Based on literature review and preliminary experiments, six diluents were initially selected for evaluation: dextrin, maltodextrin, mannitol, xylitol, soluble starch, and sodium carboxymethyl cellulose. Preliminary experiments showed that a 1:0.9 ratio of drug to diluent resulted in the best granulation effect, so this ratio was chosen as the starting point for this study. Experimental results showed that maltodextrin and sodium carboxymethyl cellulose could not form a soft mass, and while soluble starch could be granulated, its solubility was unsatisfactory. Therefore, dextrin, mannitol, and xylitol were initially selected as diluents, combined in a 1:1 ratio. Finally, dextrin + mannitol was determined as the diluent for the Shenqi Bushen Granules. The results are shown in Table 2 below.

[0058] Table 2. Examination of Diluent Types

[0059] 2.2.2 Granulation-Excipient Ratio Investigation: During the granulation process of dry extract, the amount of pharmaceutical excipients must strictly follow the pharmacopoeia regulations, and the maximum allowable amount shall not exceed twice the amount of drug. To determine the optimal excipient ratio, a series of ratio gradients (1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1.0, 1:1.1, 1:1.2) were set up for experiments. The results are shown in Table 3 below. The experimental results show that the granule properties are best when the excipient ratio is 1:0.9.

[0060] Table 3. Comparison of Drug-Excipient Ratio

[0061] 2.2.3 Investigation on the concentration of granule wetting agent: Under the condition of drug:diluent (1:0.9), granulation was carried out using different concentrations of ethanol (75%, 80%, 85%, 90%, 95%) to investigate the effect of wetting agent concentration on granule formation. The results are shown in Table 4 below.

[0062] Table 4 Ethanol Concentration Investigation Table

[0063] The results showed that when the ethanol concentration was 85%, the particles had a beautiful appearance, good solubility, and high molding rate. As the ethanol concentration increased, the water content decreased, the adhesion deteriorated, and the soft material gradually hardened.

[0064] 2.2.4 Investigation on the dosage of granule wetting agent: Under the conditions of drug:diluent (1:0.9) and 85% ethanol as wetting agent, 10%, 20%, 30%, 40% and 50% of the drug mass of ethanol were added to conduct experiments to investigate the dosage of ethanol. The results are shown in Table 5 below.

[0065] Table 5. Ethanol Usage Study

[0066] Insufficient ethanol content results in a loose texture and poor adhesion of the soft material, while excessive ethanol content causes it to clump together, making granulation impossible. This experiment found that while granules could be formed at 20% and 30% ethanol content, the forming rate was low. Subsequently, a 25% ethanol content was used in three repeated experiments, revealing better forming rate and particle uniformity. Therefore, 25% was selected as the optimal dosage for granulation.

[0067] 2.3 Orthogonal Experiment Optimization of Molding Process

[0068] Evaluation indicators for granules: The forming rate (A), hygroscopicity (B), angle of repose (C), granule properties (D) and solubility (E) of the ginseng and astragalus kidney-tonifying granules were measured, and the comprehensive score (V) was calculated, V=30A / Amax+20Bmin / B+20Cmin / C+20D / Dmax+10Emin / E.

[0069] Based on single-factor experiments, three factors were determined: the ratio of drug to excipient (drug: dextrin + mannitol) (X), the concentration of wetting agent ethanol (Y), and the dosage (Z). An L9(4³) orthogonal experiment was then conducted. The granule formability, solubility, angle of repose, and shape were comprehensively evaluated to select the optimal formulation. The factor levels and the analysis of variance for the orthogonal experiment are shown in Tables 6-8 below.

[0070] Table 6. Factors and Levels in Orthogonal Experiments

[0071] Table 7. Results of orthogonal experiments on molding process

[0072] Table 8 Results of Analysis of Variance for Molding Process

[0073] According to the intuitive analysis table and variance analysis table of the orthogonal experiment, among the three factors of drug-to-diluent ratio (X), wetting agent ethanol concentration (Y), and wetting agent dosage (Z), the influence on the overall granule score is X > Z > Y, from largest to smallest. None of the factors have a significant impact on the molding process. Finally, the optimal formulation for the diluent of Shenqi Bushen granules was determined to be X2Y3Z2 or X2Y2Z2, i.e., a drug-to-excipient ratio of 1:0.9, with 85% ethanol as the wetting agent at a dosage of 25%.

[0074] 2.4 Sensory evaluation to optimize granule flavor

[0075] Sensory evaluation ultimately determined that a 5:3 mixture of dextrin and mannitol, with the addition of 0.5% mogroside and 0.3% anhydrous citric acid, resulted in a Shenqi Bushen granule that was yellowish-brown with a uniform color, smooth texture, good dissolving properties, clear solution, rich aroma, no off-odor, and a sweet, smooth taste with the characteristic flavors of astragalus and codonopsis. Three batches of pilot-scale products were prepared according to the optimal process for Shenqi Bushen granules, and relevant quality checks were conducted. Moisture absorption rate, angle of repose, molding rate, granule morphology, and dissolution time were selected as core evaluation indicators. A comprehensive scoring system was implemented, and the relative standard deviation (RSD) of the indicator data was calculated. The results are shown in Table 9 below. The results show that Shenqi Bushen granules performed well in the repeatability experiment, therefore this design method is reasonable and reliable.

[0076] Table 9 Results of Repeatability Validation Tests

[0077] The results showed that the orthogonal experiment had good repeatability, and the preparation process of Shenqi Bushen Granules was reasonable, reliable, stable and feasible.

[0078] Example 3: Preparation of Ginseng and Astragalus Kidney-Tonifying Granules

[0079] 3.1 Raw material preparation

[0080] The composition consists of 53 parts by weight of ginseng and astragalus kidney-tonifying compound, 46 parts by weight of diluent, and 1 part by weight of flavoring agent.

[0081] The ginseng and astragalus kidney-tonifying composition is composed of the following raw materials in parts by weight: 10 parts of Codonopsis pilosula, 15 parts of Astragalus membranaceus, 10 parts of Cornus officinalis, 10 parts of Rubus idaeus, 10 parts of Lycium barbarum, 15 parts of Polygonatum sibiricum and 10 parts of Eucommia ulmoides leaves. The diluent is composed of the following raw materials in parts by weight: 5 parts dextrin and 3 parts mannitol; The flavoring agent is composed of the following raw materials in parts by weight: 5 parts of mogroside and 3 parts of anhydrous citric acid.

[0082] 3.2 Preparation of Ginseng and Astragalus Kidney-Tonifying Granules

[0083] (1) Weigh the raw materials according to the mass ratio of 10 parts Codonopsis pilosula, 15 parts Astragalus membranaceus, 10 parts Cornus officinalis, 10 parts Rubus idaeus, 10 parts Lycium barbarum, 15 parts Polygonatum sibiricum and 10 parts Eucommia ulmoides leaves, mix them, wash off the surface soil, and obtain the mixture for later use.

[0084] (2) Add water at 11 times the total mass of the mixture to the mixture, soak for 30 min, heat at 100℃ and reflux twice for 70 min each time, combine the two decoctions and concentrate to obtain a thick paste with a relative density of 1.34 g / ml.

[0085] (3) Then put the viscous paste into an oven and dry it until it is dry. The drying conditions are 70℃ and 48h to obtain an extract powder with a water content of 4%.

[0086] (4) Grind the extract powder into fine powder using a mortar and pestle, add diluent, flavoring agent and wetting agent, mix and granulate, and then pass through an 80-mesh sieve to obtain wet granules; the wetting agent is 85% ethanol, and the amount of wetting agent is 25% of the total mass of extract powder, diluent and flavoring agent.

[0087] (5) Place the wet granules in an oven at 45°C for 4 hours to dry them, and then prepare Ginseng and Astragalus Kidney-Tonifying Granules.

[0088] Example 4 Thin-layer chromatography study

[0089] 4.1 Thin-layer chromatography method for identifying Astragalus membranaceus

[0090] Preparation of test solution: Weigh 2 g of Shenqi Bushen Granules, add 40 ml of dichloromethane, extract ultrasonically for 60 min, filter, and discard the dichloromethane phase. After evaporating the residue with solvent, add 30 ml of water-saturated n-butanol and extract ultrasonically again for 30 min, then filter. Collect the filtrate, wash with three times its volume of ammonia solution, and discard the washings. Recover the n-butanol phase to dryness with solvent, and dissolve the residue in 2 ml of methanol. Reference herb solution: Weigh 1.0 g of Astragalus standard reference herb powder and prepare according to the method for preparing the test solution. Preparation method of reference solution: Accurately weigh an appropriate amount of astragaloside A reference standard, dissolve in methanol, and prepare a reference solution containing 1 mg of astragaloside A per 1 mL. Negative control solution: Prepare negative control sample granules lacking Astragalus using the preparation method of Shenqi Bushen Formula solid beverage, and prepare the negative control solution using the same method.

[0091] According to the thin-layer chromatography method (General Rule 0502), the above four solutions were applied separately to the same high-performance silica gel G thin-layer plate using a micro-spotting device. The plate was developed using a mixed solvent system of dichloromethane-anhydrous ethanol-water (70:45:6.5). After development, the plate was removed and allowed to air dry at room temperature. After drying, a 10% sulfuric acid ethanol solution was sprayed evenly as a colorimetric reagent, and the plate was heated at 105°C until the chromatographic spots were clearly visible. The spots were then examined under a 365nm ultraviolet light. Based on the above thin-layer chromatography identification method for Astragalus membranaceus, the fluorescence spots of the test sample and the reference sample migrated at the same position and had the same color, with Rf=0.60. The results are as follows. Figure 7 As shown.

[0092] 4.2 Thin-layer chromatography identification method for Codonopsis pilosula

[0093] Thin-layer chromatography (TLC) experiments were performed according to the 2020 edition of the Chinese Pharmacopoeia for Codonopsis pilosula. The fluorescent spots of the test sample and the reference sample migrated at the same positions and had the same color, with Rf=0.60. The results are as follows: Figure 8 As shown.

[0094] 4.3 Thin-layer chromatography method for identifying raspberries

[0095] Preparation of test sample solution: Weigh 2 g of test sample powder, place it in a 50 ml Erlenmeyer flask, add 30 ml of methanol, and sonicate (30 min) before filtering. Evaporate the filtrate to dryness under suitable conditions, and dissolve the residue in 2 ml of methanol. Preparation of raspberry reference material solution: Take 1.0 g of standard raspberry powder and follow the preparation method of the test sample solution. Preparation of reference solution: Accurately weigh an appropriate amount of linalool reference standard, dissolve it in methanol, and dilute to volume to prepare a 1.02 mg / ml linalool solution. Negative control solution: Based on the preparation process of the Shenqi Bushen Fang solid beverage, first prepare negative control granules lacking raspberry, and then prepare its solution using the same method.

[0096] According to the thin-layer chromatography method (General Rule 0502), 2 μL of linalool reference solution and 4 μL of each of the other three test solutions were spotted onto the same silica gel G thin-layer plate. Chromatography was performed using an ethyl acetate-methanol-water-formic acid (92:2:4:0.5) mixture as the developing solvent. After development, the plate was removed and allowed to dry naturally by solvent evaporation. Aluminum trichloride solution was sprayed evenly for color development, and the plate was heated in a 105°C oven for 5 minutes. After the color reaction, the plate was examined under a 365 nm UV lamp. Following the above-described thin-layer chromatography method for raspberry identification, the fluorescent spots on the test sample and the reference sample chromatograms migrated at the same position and had the same color, with Rf = 0.56. The results are as follows. Figure 9 As shown.

[0097] 4.4 Thin-layer chromatography method for identifying Eucommia ulmoides leaves

[0098] Thin-layer chromatography (TLC) was performed according to the method for identifying Eucommia ulmoides leaves in the Pharmacopoeia. The fluorescence spots of the test sample and the reference sample migrated at the same positions and had the same color, with Rf=0.32. The results are as follows. Figure 10 As shown.

[0099] 4.5 Thin-layer chromatography method for identifying wolfberry

[0100] Thin-layer chromatography (TLC) was performed according to the method for identifying Lycium barbarum in the Pharmacopoeia. The fluorescent spots of the test sample and the reference sample migrated at the same positions and had the same color, with Rf=0.70. The results are as follows. Figure 11 As shown.

[0101] 4.6 Methods for evaluating the durability of thin-layer coatings

[0102] Three different manufacturers from Qingdao, Nanjing, and Yantai were selected, along with three relative humidity (RH) levels of 32%, 65%, and 88%, room temperature (25℃) and low temperature (4℃) environments, and two different light sources of ultraviolet and sunlight. The durability was investigated by spotting samples and then unfolding them according to the above method.

[0103] This study optimized the chromatographic conditions for Astragalus membranaceus, Codonopsis pilosula, Rubus idaeus, Eucommia ulmoides leaves, and Lycium barbarum using thin-layer chromatography (TLC). The robustness of the method was systematically evaluated, focusing on the effects of different brands of TLC plates, fluctuations in ambient temperature and humidity, and differences in light intensity on chromatographic development. Results showed that in the TLC chromatogram of Shenqi Bushen Granules, the reference standard consistently displayed a consistent color spot at the corresponding position. These spots were clearly distinguishable, with good separation, and no interference was observed from the negative control, fully validating the reproducibility and specificity of the method. In conclusion, the TLC qualitative identification method for Shenqi Bushen Granules established in this study is convenient, specific, and reproducible, yielding accurate and reliable results, and the reagents used are safe and controllable.

[0104] Example 5: Pharmacological Study of Shenqi Bushen Granules

[0105] With the increasing aging of the population, osteoporosis (OP) is the most common skeletal disease among the elderly, characterized by low bone mass, damaged bone microstructure, increased bone fragility, and susceptibility to fractures. It significantly impacts people's quality of life and has become a pressing public health issue. Traditional Chinese medicine believes that the kidneys govern bones and produce marrow; the kidneys and bone growth and development are closely related. Insufficient kidney essence, liver stagnation and blood deficiency, and the inability of the liver and kidneys to mutually transform essence and blood lead to malnourishment of bones, easily causing osteoporosis. Therefore, the treatment of osteoporosis should focus on tonifying the kidneys. Based on this TCM theory, this study established a mouse model of osteoporosis to verify the efficacy of Shenqi Bushen Granules in tonifying the kidneys, replenishing essence, and strengthening tendons and bones.

[0106] 5.1 Establishment of a mouse model of osteoporosis induced by kidney deficiency: This experiment used 12-week-old female C57BL / 6 mice for model establishment, totaling 96 mice. After one week of acclimatization, they were randomly divided into 8 groups of 12 mice each. Seven groups received intraperitoneal injections of hydrocortisone acetate 15 mg / kg / day daily, while the remaining group received the same dose of physiological saline for one month. The mice's food and water intake, urine output, activity level, and general mental condition were observed. Successful model establishment was defined as mice exhibiting characteristics such as huddling together, lethargy, sluggishness, and dull, yellowish fur.

[0107] 5.2 Grouping and Dosing

[0108] Mice that successfully developed the model were randomly divided into four groups: a model group, low-, medium-, and high-dose groups of a kidney-tonifying formula (Low 0.65 g / kg / day, Mid 0.98 g / kg / day, High 1.3 g / kg / day), a positive control group of traditional Chinese medicine (CMP Liuwei Dihuang Wan 0.12 g / kg / day), a positive control group of Western medicine (WMP alendronate sodium 0.46 mg / kg / week), and a group of Astragalus decoction (HQY 0.025 g / kg / day), with 12 mice in each group. The treatment groups were administered the medication by gavage, while the control and model groups were administered physiological saline by gavage. After one month of continuous administration, blood samples were collected from the orbital rim.

[0109] 5.3 Indicator Testing

[0110] The mice's mental state, body posture, fur color, activity level, eye mucosal color, ear appearance, and overall condition, including feces, must be carefully monitored daily. Each group of mice should be weighed every seven days, and detailed records should be kept.

[0111] Serum levels of IL-6, TNF-α, E2, T, BALP, and CTX-1 in mice were detected using enzyme-linked immunosorbent assay (ELISA). HDL-C, TC, and TG levels were measured in the liver. Mouse kidneys were stained with hematoxylin and eosin (HE), and mouse femoral tissue was co-stained with Calcein-Alizarin and irradiated with MicroCT.

[0112] 5.4 Experimental Results

[0113] Statistical analysis

[0114] All data are expressed as mean ± standard deviation. Data are expressed as ±SD. Statistical analysis was performed using SPSS Statistics 23.0 software, and graphing was done using Graphpad Pism 9.5. Independent samples t-tests were used for comparisons between two groups, and one-way ANOVA was used for comparisons among multiple groups. Levene's statistic test for homogeneity of variance was performed. If the variances were homogeneous, the F-test was used to compare population means, followed by Duncan's method for pairwise comparisons between groups. If the variances were unequal, the Welch test was used to compare population means, and Tamhane's T² method was used for pairwise comparisons between groups. A p-value < 0.05 was considered statistically significant.

[0115] The changes in mouse body weight are shown in Table 10 below. Figure 12The results showed that there was no difference in body weight among the eight groups of mice during the first week of adaptive feeding, and they were then randomly assigned to other groups. The second and third weeks were the initial stage of model establishment, and there were no significant differences in body weight among the groups. By the fourth week, compared with the control group, the body weight of the other six groups (except the high-dose group) began to show differences. By the fifth week, the body weight of the model group mice was significantly different from that of the control group, indicating a significant decrease in body weight and successful model establishment. From the sixth week onwards, the mice were administered the drug. The high-dose group of Shenqi Bushen Granules began to show differences in body weight compared to the model group, which may be related to the drug's effect. By the ninth week, the body weight of the administered groups was significantly different from that of the model group, but not from the control group.

[0116] Table 10 Results of mouse body weight ( ±SD, n=12)

[0117] Note: The # symbol represents the comparison between the model group and the blank group. This means p < 0.05. # represents p < 0.01, # represents p < 0.05, and ## represents p < 0.01.

[0118] To investigate the effect of Shenqi Bushen Granules on bone content in a mouse model of osteoporosis, we first used MicroCT to analyze the changes in bone content in each group of mice. The results are shown in Table 11 below. Figure 13 The results showed that compared with the control group, the model group had significantly lower levels of bone tissue ratio (BV / TV) (p < 0.0001), trabecular thickness (Tb.Th) (p < 0.0001), trabecular number (Tb.N) (p < 0.05), and bone mineral density (BMD) (p < 0.0001), demonstrating the successful establishment of the mouse osteoporosis model. After intervention with Shenqi Bushen Granules, the bone tissue ratio (BV / TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), and bone mineral density (BMD) all increased. There were no significant differences in BV / TV, Tb.Th, and Tb.N content between the low, medium, and high dose groups of Shenqi Bushen Granules and the control group. For BMD, the low, medium, and high dose groups showed significant differences compared with the model group (p < 0.05).

[0119] Table 11 Micro CT analysis of bone mass changes in mice ( ±SD, n=8)

[0120] Note: There were no significant differences between groups labeled with the same letter, while there were significant differences between groups labeled with different letters.

[0121] Through the micro-CT analysis described above, we found that Shenqi Bushen Granules improved bone loss, significant trabecular fracture, and a significant reduction in the number of trabecular bones in the osteoporosis mouse model. However, its effect on bone density (BMD) did not reach the same level as the control group. Therefore, we speculate that it mainly promotes bone mass increase through its osteogenic effect. To verify whether the effect of Shenqi Bushen Granules on bone mass is due to its osteogenic effect, Calcein and Alizarin Complexon were injected into the mice one week and two days before sample collection to label new bone formation, respectively. The results are shown in Table 12 below. The results showed that the rate of new bone formation in osteoporotic mice was much lower than that in healthy mice. Compared with the control group, the mineralization deposition rate (MAR) (p<0.0001), bone formation rate (BFR / BS) (p<0.0001), and new bone formation length (p<0.05) of the model group mice were significantly reduced, while the mineralization deposition rate (p<0.0001), bone formation rate (p<0.0001), and new bone formation length (p<0.01) of new bone were significantly improved in mice that were given Shenqi Bushen Granules.

[0122] Table 12 Effects of Shenqi Bushen Decoction on New Bone Formation in Osteoporotic Mice ( ±SD, n=8)

[0123] Note: There were no significant differences between groups labeled with the same letter, while there were significant differences between groups labeled with different letters.

[0124] Based on Calcein-Alizarin double-label co-staining results, we found that Shenqi Bushen Granules promoted osteogenic activity in osteoporotic mice, thereby increasing bone mass. Therefore, we tested enzymes related to osteoblast activity, including bone-specific alkaline phosphatase (BALP) and type I collagen crosslinking C. Terminal peptide (type I collagen carboxy) Terminal peptide (CTX). Results are shown in Table 13 below. The results showed that compared with the control group, the levels of CTX-I (p<0.0001) and BALP (p<0.0001) in the model group mice were significantly increased, and both CTX-I and BALP decreased after administration. For CTX-I, there was no significant difference between the medium-dose group and the positive control group (p=0.9995), and no significant difference between the high-dose group and the positive control group (p=0.9998). For BALP, there was no significant difference between the high-dose group and the positive control group (p=0.6810), but a significant difference between the medium-dose group and the high-dose group (p=0.0001). The medium-dose group had a greater effect on BALP levels than the high-dose group, which may be related to the side effects of the drug.

[0125] Table 13 Effects of Shenqi Bushen Decoction on CTX-I and BALP in osteoporotic mice ( ±SD, n=8)

[0126] Note: There were no significant differences between groups labeled with the same letter, while there were significant differences between groups labeled with different letters.

[0127] The pathological changes in the kidneys of osteoporotic mice were observed by HE staining. The therapeutic effect of Shenqi Bushen Granules on the kidneys was verified by detecting estrogen levels (E2) and testosterone (T). Results are shown in Table 14 below. Figure 14 HE section analysis revealed that the kidneys of the model group mice showed sclerosis of most glomeruli and interstitial vascular hemorrhage; renal tubules were generally atrophied and disappeared, with dilated remaining lumens. The overall kidney volume was reduced, the texture was hardened, and the surface was granular. These irreversible tissue damages led to renal failure, hormonal imbalances, abnormal calcium and phosphorus metabolism, and ultimately pathological changes in the bones. Combined with ELISA results, compared with the control group, the hormone levels E2 (p<0.0001) and T (p<0.05) in the model group were lower than those in the control group. For E2, there was no significant difference between the medium-dose group (p>0.9999) and the Astragalus Decoction group (p>0.9999) and the positive traditional Chinese medicine group, while there was no significant difference between the high-dose group (p=0.9778) and the control group and the positive Western medicine group (p=0.8732). There were no significant differences in serum T levels in mice between the low-dose group (p>0.9999), the medium-dose group (p=0.969), and the positive control group (p>0.9999) and the blank control group.

[0128] Table 14. Effects of Shenqi Bushen Decoction on Hormone Levels in Osteoporotic Mice ( ±SD, n=8)

[0129] The levels of inflammatory factors in mice were measured, and the results are shown in Table 15 below. The results showed that the levels of inflammatory factors in mice decreased significantly after drug intervention. For IL-6, the medium (p < 0.01) and high (p < 0.01) dose groups were significantly different from the model group. For TNF-α, the low (p < 0.01), medium (p < 0.01), and high (p < 0.01) dose groups were significantly different from the model group. Moreover, there was no significant difference between the high-dose group and the positive traditional Chinese medicine group (p > 0.9999) and the positive Western medicine group (p = 0.118).

[0130] Table 15 Effects of Shenqi Bushen Decoction on Inflammatory Factors in Osteoporotic Mice ( ±SD, n=8)

[0131] Note: There were no significant differences between groups labeled with the same letter, while there were significant differences between groups labeled with different letters.

[0132] Bone marrow-derived mesenchymal stem cells (BMSCs) are highly plastic adult stem cells that can differentiate into osteoblasts, adipocytes, and chondrocytes. To investigate the effects of Shenqi Bushen granules on adipogenic differentiation, the levels of HDL-C, TC, and TG in the liver were measured. The results are shown in Table 16 below. Figure 15 The results showed that, compared with the control group, the model group mice had decreased high-density lipoprotein cholesterol (HDL-C) (p<0.0001) levels and increased total cholesterol (TC) (p<0.0001) and triglyceride (TG) (p<0.0001) levels. After administration, compared with the model group, the Shenqi Bushen granules group had increased HDL-C (p<0.0001) levels and decreased TC (p<0.0001) and TG (p<0.0001) levels.

[0133] Table 16 Effects of Shenqi Bushen Decoction on Adipogenic Differentiation in Osteoporotic Mice ( ±SD, n=8)

[0134] Note: There were no significant differences between groups labeled with the same letter, while there were significant differences between groups labeled with different letters.

[0135] 5.5 Discussion

[0136] 5.5.1 Effects of Shenqi Bushen Granules on Body Weight, Hormones, and Inflammatory Factors in a Mouse Model of Osteoporosis

[0137] Traditional Chinese medicine theory holds that kidney essence deficiency is one of the main causes of osteoporosis. The kidneys govern water and storage, serving as the foundation of innate essence. Maintaining the kidney's vital energy ("true fire") is essential for balancing Yin and Yang, harmonizing Qi and blood, and nourishing the marrow. When Qi and essence are deficient, the function of marrow production and bone formation is impaired, leading to weak and atrophied bones. Studies have shown that estrogen plays a crucial role in maintaining bone homeostasis. It promotes the proliferation and differentiation of osteoblasts and inhibits the activity of osteoclasts. As women enter menopause or experience progressive ovarian decline, ovarian function gradually declines, resulting in decreased estrogen levels. This leads to slower bone formation, while osteoclast activity relatively increases, causing bone loss to outpace bone formation, ultimately resulting in osteoporosis. In the pathogenesis of osteoporosis, decreased bone formation and increased bone resorption constitute the core links in disease progression. Maintaining bone homeostasis depends on the functional balance between osteoclasts and osteoblasts. Increased bone resorption is regulated by various cytokines and inflammatory factors, such as interleukin (IL) and tumor necrosis factor-α (TNF-α). In a state of chronic inflammation, the release of these inflammatory factors increases, directly stimulating the activity of osteoclasts and promoting bone resorption. At the same time, it also inhibits the function of osteoblasts, further aggravating bone loss and thus promoting the occurrence and development of osteoporosis.

[0138] The experimental results showed that the body weight and estrogen levels in the model group mice were significantly reduced, while the release of inflammatory factors increased. These results indicate that the mouse osteoporosis model was successfully established in this study. Intervention with Shenqi Bushen Granules can increase mouse body weight to a certain extent, increase estrogen (E2) and testosterone (T) levels in mice, and also exhibits a certain anti-inflammatory effect. By inhibiting the release of inflammatory factors such as IL-6 and TNF-α, it reduces the excessive activation of osteoclasts and the inhibition of osteogenic function, thereby intervening in the progression of osteoporosis.

[0139] 5.5.2 Effects of Shenqi Bushen Granules on Bone Microstructure in Osteoporotic Mice

[0140] Measuring bone mineral density and bone microstructure in laboratory animals is a crucial criterion for determining the success of model establishment. This experiment used Micro-CT to detect bone mineral density and bone microstructure parameters in experimental mice. Micro-CT technology uses X-rays from different directions to pass through the sample, utilizing the absorption and attenuation characteristics of the X-rays to generate images on a detector. Finally, the sample is reconstructed in three dimensions to obtain relevant parameters of its three-dimensional trabecular bone. Micro-CT offers a more detailed spatial resolution than medical CT, achieving accurate imaging at the micrometer level. Therefore, Micro-CT is a superior method for determining the success of model establishment.

[0141] Micro-CT scans showed that BV / TV, Tb.Th, Tb.N, and BMD were all significantly downregulated in the model group, proving the successful establishment of the experimental model. After intervention with Shenqi Bushen Granules, the bone microstructure indicators of mice were improved. The number and thickness of trabeculae in the femoral tissue of mice were restored to varying degrees, the connectivity of the reticular structure was significantly improved, and the bone microstructure showed a clear trend of improvement.

[0142] Biochemical indicators in the blood can reflect bone tissue metabolism. BALP, an extracellular enzyme of osteoblasts, directly reflects the activity and functional status of osteoblasts. BALP proliferation, differentiation, and maturation are closely related to normal bone growth and development. CTX-I is the most widely used marker of collagen degradation; CTX levels reflect the bone resorption activity of osteoclasts. When osteoporosis occurs, calcium levels decrease. To maintain stable blood calcium, the body mobilizes calcium into the blood, causing changes in bone metabolism and leading to compensatory proliferation of osteoblasts, resulting in elevated BALP and CTX-I levels.

[0143] Using Calcein-Alizarin dual-dye labeling to assess new bone formation in mice, the study showed that BALP and CTX-I levels in osteoporotic mice were both reduced after drug intervention compared to before treatment. MAR, BFR / BS, and Length were all increased after drug intervention compared to before treatment, and the rate of new bone formation in osteoporotic mice was significantly lower than in healthy mice. This indicates that Shenqi Bushen Granules can promote new bone formation in osteoporotic mice, thereby playing a therapeutic role in osteoporosis.

[0144] 5.5.3 Effects of Shenqi Bushen Granules on Adipogenic Differentiation in Osteoporotic Mice

[0145] Studies have shown that the differentiation function of bone marrow mesenchymal stem cells (BMSCs) typically exhibits an inverse relationship: adipogenic factors promote adipogenic differentiation of BMSCs while inhibiting the expression of osteogenic factors, thus hindering the differentiation of BMSCs into osteoblasts and disrupting the osteogenic-adipogenic differentiation balance of BMSCs, ultimately inducing osteoporosis and other bone metabolic diseases. Therefore, during the development of osteoporosis, the osteogenic-adipogenic differentiation balance of bone marrow mesenchymal stem cells may be disrupted, leading to changes in lipid content in mice.

[0146] In this study, imaging showed that BV / TV, Tb.Th, and Tb.N were significantly downregulated, and BFR / BS and MAR were significantly reduced in the model group mice. HE staining analysis showed a significant increase in intramedullary fat content in osteoporotic mice, while the intramedullary fat content decreased after intervention with Shenqi Bushen Granules. These results indicate that adipogenesis is increased in osteoporotic mice, and BMSCs differentiate into adipogenic cells, leading to osteoporosis. However, the osteogenic capacity of mice treated with Shenqi Bushen Granules was improved, while adipogenesis was inhibited. In summary, the histological and imaging results indicate that osteogenic capacity is decreased and adipogenesis is increased in osteoporotic mice, and this pathological change was significantly improved after treatment with Shenqi Bushen Granules.

[0147] 5.6 Summary

[0148] In this experiment, we successfully established an osteoporosis model in mice by intraperitoneal injection of hydrocortisone. The model group mice showed changes in the number and microstructure of bone trabeculae, decreased serum hormone levels, increased inflammatory factor levels, altered serum bone metabolism markers, and disruption of the osteogenic-adipogenic differentiation balance. These pathological changes were significantly improved after intervention with Shenqi Bushen Granules. The experimental results indicate that Shenqi Bushen Granules can maintain the functional balance between osteoclasts and osteoblasts by inhibiting the release of inflammatory factors; improve osteoporosis by increasing estrogen levels in mice through kidney tonification; inhibit adipogenic differentiation of bone mesenchymal stem cells (BMSCs) and reduce bone loss; and increase bone resorption ratio (MAR) and bone formation rate (BFR / BS) in mice, promoting osteoogenesis and directly increasing bone formation.

[0149] As can be seen from the above embodiments, the present invention provides a ginseng and astragalus kidney-tonifying composition, its preparation method, and its application. The ginseng and astragalus kidney-tonifying composition comprises the following raw materials in parts by weight: 7-13 parts Codonopsis pilosula, 12-18 parts Astragalus membranaceus, 7-13 parts Cornus officinalis, 7-13 parts Rubus idaeus, 7-13 parts Lycium barbarum, 12-18 parts Polygonatum sibiricum, and 7-13 parts Eucommia ulmoides leaves. The medicine prepared by the present invention has the advantages of convenient administration, carrying, and storage; stable and controllable product quality; simple production process; and economical cost. It also possesses significant effects in tonifying the kidneys and replenishing essence, strengthening muscles and bones. By deepening the development and utilization of medicinal and edible raw materials such as Astragalus membranaceus and Codonopsis pilosula, the present invention prepares uniform and stable Chinese medicine granules, which are suitable for conditioning the sub-health state caused by work fatigue and life stress in modern people, and can also meet the health needs of middle-aged and elderly people with spleen and kidney deficiency and insufficient qi and yin.

Claims

1. A composition for tonifying the kidneys with ginseng and astragalus, characterized in that, The ingredients contain the following parts by weight: Codonopsis pilosula 7-13 parts, Astragalus membranaceus 12-18 parts, Cornus officinalis 7-13 parts, Rubus idaeus 7-13 parts, Lycium barbarum 7-13 parts, Polygonatum sibiricum 12-18 parts, and Eucommia ulmoides leaves 7-13 parts.

2. A ginseng and astragalus kidney-tonifying granule containing the composition of claim 1, characterized in that, The raw materials comprise the following parts by weight: 50-56 parts of the composition according to claim 1, 43-49 parts of diluent, and 0.5-2 parts of flavoring agent.

3. The ginseng and astragalus kidney-tonifying granules according to claim 2, characterized in that, The diluent contains the following raw materials in parts by weight: 4-6 parts dextrin and 2-4 parts mannitol; the flavoring agent contains the following raw materials in parts by weight: 4-6 parts mogroside and 2-4 parts anhydrous citric acid.

4. A method for preparing the ginseng and astragalus kidney-tonifying granules of claim 2, characterized in that, Includes the following steps: (1) Weigh the raw materials according to the mass ratio of Codonopsis pilosula 7-13 parts, Astragalus membranaceus 12-18 parts, Cornus officinalis 7-13 parts, Rubus idaeus 7-13 parts, Lycium barbarum 7-13 parts, Polygonatum sibiricum 12-18 parts and Eucommia ulmoides leaf 7-13 parts, mix them to obtain a mixture for later use; (2) Mix the mixture with water, soak, reflux extract 2-4 times, combine the decoctions, vacuum filter to obtain a thick paste; (3) Dry the thick paste to obtain extract powder; (4) Mix the extract powder, diluent, flavoring agent and wetting agent, sieve and dry to obtain the final ginseng and astragalus kidney-tonifying granules.

5. The method according to claim 4, characterized in that, In step (2), the mass ratio of the mixture to water is 0.8~1.2:10~12; the soaking time is 20~40 min; the reflux extraction time is 60~80 min; and the reflux extraction temperature is 95~105℃.

6. The method according to claim 5, characterized in that, The drying temperature in step (3) is 60~80℃, and the drying time is 45~50h.

7. The method according to claim 6, characterized in that, The wetting agent in step (4) is 80-90% ethanol, and the amount of the wetting agent is 20-30% of the total mass of the extract powder, diluent and flavoring agent.

8. The method according to claim 7, characterized in that, In step (4), the sieve mesh size is 70-90 mesh, the drying temperature is 40-50℃, and the drying time is 3-5h.

Images